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Postgraduate Program in Experimental Pathology

ANNALS

II International Symposium of Experimental Pathology

VII Simpósio de Patologia Experimental da UEL

Editorial board

Eduardo José de Almeida Araújo – Chair

Aedra Carla Bufalo Kawassaki

Glauco Akelinghton Freire Vitiello

Jackson Gabriel Miyamoto

João Paulo Assolini

Kleber Paiva Trugilo

Renato Daniel Ramalho Cardoso

Suellen Ribeiro da Silva Scarton

Responsible for publishing:

UNIVERSIDADE ESTADUAL DE LONDRINA

PROGRAMA DE PÓS-GRADUAÇÃO EM PATOLOGIA EXPERIMENTAL

Rodovia Celso Garcia Cid. PR445 Km 380, Campus Universitário / CEP: 86057-970

LONDRINA / PR / BRASIL

Contact: Eduardo José de Almeida Araújo

Telephone: +55 43 3371-5498

Postgraduate Program in Experimental Pathology Londrina-Brazil 1st Edition p.1-241 2017

The authors of the abstracts are totally responsible for the veracity of the information. The Scientific Committee is not responsible for 'spell-checking' and 'grammar-checking' of the abstracts.

All authors agreed with the publication of the accepted abstracts in the Annals of II International

Symposium of Experimental Pathology and VII Simpósio de Patologia Experimental, without any

consideration regarding copyrights.

Catalogação na publicação elaborada pela Divisão de Processos Técnicos da

Biblioteca Central da Universidade Estadual de Londrina.

Dados Internacionais de Catalogação-na-Publicação (CIP)

I61a International Symposium of Experimental Pathology (2. : 2017 : Londrina, PR)

Annals [of the] II International Symposium of Experimental Pathology [e do] VII

Simpósio de Patologia Experimental da UEL [livro eletrônico] / Editorial board:

Eduardo José de Almeida Araújo ...[et al.]. – Londrina : UEL, 2017.

1 Livro digital.

Vários autores.

Inclui bibliografia.

Disponível em: http://www.uel.br/eventos/simposiopato/

ISBN 978-85-7846-449-3

1. Patologia experimental – Congressos. I. Araújo, Eduardo José de Almeida.

II. Universidade Estadual de Londrina. Centro de Ciências Biológicas. Programa de

Pós-Graduação em Patologia Experimental. III. Queen Mary University of London.

IV. Simpósio de Patologia Experimental da UEL (7. : 2017 : Londrina, PR). V. Título. VI.

Annals [do] VII Simpósio de Patologia Experimental da UEL.

CDU 616-092

ANNALS

II International Symposium of Experimental Pathology

VII Simpósio de Patologia Experimental da UEL

Organizing Committees

Chair: Eduardo José de Almeida Araújo (UEL) Scientific Committee: L. Ashley Blackshaw (QMUL/UK) Qasim Aziz (QMUL/UK) Maria Angelica Ehara Watanabe (UEL) Waldiceu Aparecido Verri Junior (UEL) Wander Rogério Pavanelli (UEL) Aedra Carla Bufalo Kawassaki (UEL) Glauco Akelinghton Freire Vitiello (UEL) Jackson Gabriel Miyamoto (UEL) João Paulo Assolini (UEL) Kleber Paiva Trugilo (UEL) Renato Daniel Ramalho Cardoso (UEL) Suellen Ribeiro da Silva Scarton (UEL)

Financial Committee: Phileno Pinge Filho (UEL) Diego Lima Petenuci (UEL) Marilia Fernandes Manchope (UEL)

Disclosure Committee: Marli Cardoso Martins Pinge (UEL) Alberto Yoichi Sakaguchi (UEL) Daniele Sapede Alvarenga (UEL) Nadia Calvo Martins Okuyama (UEL) Natália Medeiros Dias Lopes (UEL) Renata Streck Fernandes (UEL)

Sponsorship Committee: Mario Augusto Ono (UEL) Anelise Franciosi (UEL) Camila Regina Basso (UEL) Giovana Gomes de Carvalho (UEL) Paola Singi (UEL)

Social Committee: Eiko Nakagawa Itano (UEL) Brunna Emanuella França Robles (UEL) Lorena Flor da Rosa Franchi Santos (UEL) Rafaela Macagnan (UEL)

Graphic Designer: Érica Romão Pereira (UEL)

CONTENTS

PROGRAM ...................................................................................................................................................... 14

SHORT-COURSES ......................................................................................................................................... 17

EXPANDED ABSTRACTS.............................................................................................................................. 20

ACTIVITY OF SILVER BIOGENIC NANOPARTICLES ON RH AND ME-49 STRAINS OF Toxoplasma

gondii IN HeLa CELLS ................................................................................................................................. 21

ANALYSIS OF IL-10 rs1800872 (c.-592 C>A) POLYMORPHISM IN THE HPV INFECTION AND

CERVICAL CANCER DEVELOPMENT ...................................................................................................... 25

ANTITUMORAL ACTION OF THIOHYDANTOINS ON CELLS OF CARCINOMA LUNG ......................... 28

DETECTION OF INFECTION BY Paracoccidioides brasiliensis IN NILE TILAPIA ................................... 31

EFFECT OF ASPIRIN-TRIGGERED LIPOXIN IN T. cruzi CELL INVASION ............................................. 34

EFFECTS OF PHOTOTHERAPY DURING CHRONIC EXPERIMENTAL COLITIS INDUCED BY

DEXTRAN SULFATE SODIUM (DSS) IN MICE ......................................................................................... 38

EVALUATION OF PHAGOCYTIC ACTIVITY AND IN VITRO CANDIDACY OF PERITONEAL

MACROPHAGES INFECTED WITH DIFFERENT MORPHOTYPES OF Candida tropicalis .................... 41

HISTOLOGICAL ANALYSIS OF GUT-ASSOCIATED LYMPHOID TISSUE OF HEALTHY RATS

SUPPLEMENTED WITH IGY ...................................................................................................................... 44

IDENTIFICATION OF THE GUT-VASCULAR BARRIER IN WISTAR RATS ............................................ 48

INVOLVEMENT OF THE POLYMORPHISMS OF NFKB1 (rs28362491) AND NFKBIA (rs696) IN THE

DEVELOPMENT OF CERVICAL CANCER ................................................................................................ 52

OXIDATIVE STRESS AND INFLAMMATORY RESPONSE DURING CARDIAC SURGEY WITH

CARDIOPULMONARY BYPASS ................................................................................................................. 56

PHYSICAL EXERCISE EFFECT ON DAMAGING ACTION OF HYPERHOMOCYSTEINEMIA TO THE

MALE REPRODUCTIVE SYSTEM .............................................................................................................. 58

PREVALENCE OF MOUSE MAMMARY TUMOR VIRUS-LIKE (MMTV-LIKE) AND ITS IMPLICATION

ON THE PATHOGENESIS OF BREAST CANCER .................................................................................... 62

ABSTRACTS SELECTED FOR ORAL PRESENTATION ......................................................................................... 66

ASPIRIN ANTI-INFLAMMATORY EFFECT ON MUSCLE TISSUE DURING MURINE Trypanosoma cruzi

EXPERIMENTAL INFECTION ..................................................................................................................... 67

BUDLEIN A INHIBITS EXPERIMENTAL GOUT ARTHIRITS BY TARGETING NF-ΚB ACTIVATION

IN MICE ........................................................................................................................................................ 68

DIOSMIN AMELIORATES ZYMOSAN-INDUCED ARTHRITIS AND PERITONITIS BY REDUCING

LEUKOCYTE RECRUITMENT AND PRO-INFLAMMATORY CYTOKINES PRODUCTION IN MICE ..... 69

EFFECTS OF CREATINE SUPPLEMENTATION ON DIFFERENT MUSCLES IN WALKER-256 TUMOR-

BEARING RATS........................................................................................................................................... 70

EPIDEMIOLOGICAL ASPECTS OF SCHISTOSOMIASIS IN NORTH OF PARANÁ ................................ 71

THE EFFECTS OF TRICLOSAN EXPOSURE ON ESTROUS CYCLE AND SEXUAL BEHAVIOR IN

TWO GENERATIONS OF FEMALE WISTAR RATS .................................................................................. 72

ABSTRACTS SELECTED FOR POSTER PRESENTATION ..................................................................................... 73

CLINICAL PATHOLOGY

ANALYSIS OF HEPARINE ACTION IN OXIDATIVE STRESS BIOMARKERS IN CHRONIC RENAL

PATIENTS SUBMITTED TO HEMODIALYSIS ........................................................................................... 74

BREAST CANCER AND PHOSPHOLIPASE A2 FROM SNAKE VENOM, AS ANTITUMORAL AGENT: A

REVIEW ....................................................................................................................................................... 75

CCR5-DELTA32 ALLELIC VARIANT: CORRELATION WITH METASTASIS PARAMETER IN

BREAST CANCER ....................................................................................................................................... 76

DIFFERENCES BETWEEN TWO MEMBRANES TYPES USED IN HEMODIALYSIS SESSIONS

RELATING THE BIOMARKERS OF OXIDATIVE STRESS PRE- AND THE POSTDIALYSIS ................. 77

EFFECT OF RESISTANCE TRAINING ON MUSCLE STRENGTH AND BODY COMPOSITION IN

BREAST AND PROSTATE CANCER PATIENTS: A META-ANALYSIS. .................................................. 78

ERYTHROCYTE ANTIOXIDANTS INCREASE IN PATIENTS WITH MULTIPLE BASAL CELL

CARCINOMA, SQUAMOUS CELL CARCINOMA AND ACTINIC KERATOSIS LESIONS ....................... 79

EVALUATION OF POLYMORPHISM rs3591676 OF THE AMACR GENE IN PATIENTS WITH

PROSTATE CANCER .................................................................................................................................. 80

EVALUATION OF SYSTEMIC OXIDATIVE PROFILE IN PATIENTS WITH NON-MELANOMA SKIN

CANCER AND ACTINIC KERATOSIS ........................................................................................................ 81

EVALUATION OF THE EFFECTIVENESS OF OLFACTORY TRAINING AS A TREATMENT FOR

PERSISTENT OLFACTORY LOSS ............................................................................................................. 82

EVALUATION OF THE PROLONGED USE OF DIETARY SUPPLEMENTS BY SPORTSMEN .............. 83

EVALUATION OF THE REDOX STATE AND RESISTANCE INSULIN IN ADOLESCENTS WITH

METABOLIC SYNDROME .......................................................................................................................... 84

EVALUATION OF THE REDOX STATE DURING THE INDUCTION PHASE OF CHILDHOOD B-CELL

ACUTE LYMPHOCYTIC LEUKEMIA TREATMENT ................................................................................... 85

HEPATIC RESPONSE TO GLUCAGON, cAMP AND ADRENERGIC AGONISTS IN WALKER-256

TUMOR-BEARING RATS ............................................................................................................................ 86

IMMUNE-INFLAMMATORY, METABOLIC, OXIDATIVE AND NITROSATIVE STRESS SET OF

BIOMARKERS PREDICTS ACUTE ISCHEMIC STROKE AND SHORT-TERM OUTCOME ................... 87

IMPACT OF TWO DIFFERENT RESISTANCE EXERCISE PROGRAMS IN OXIDATIVE STATUS AND

CARDIOVASCULAR RISK FACTORS OF PATIENTS WITH METABOLIC SYNDROME ........................ 88

INFLUENCE OF HOMOCYSTEINE LEVELS ON ADHESION MOLECULES AND OXIDATIVE STRESS

IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS .................................................................. 89

INFLUENCE OF LACTATE CIRCULATING LEVELS IN HEPATIC GLUCONEOGENESIS IN WALKER-

256 TUMOR BEARING-RATS ..................................................................................................................... 90

LIPIDOMICS ANALYSIS OF THE VAGINAL DISCHARGE IN WOMEN WITH VULVOVAGINAL

CANDIDIASIS AND CYTOLYTIC VAGINOSIS ........................................................................................... 91

PATHOPHYSIOLOGY OF AMYOTROPHIC LATERAL SCLEROSIS ....................................................... 92

PYRROLOQUINOLINE QUINONE REDUCES HEPATOCELLULAR DAMAGE IN MICE MODEL OF

PARTIAL HEPATECTOMY AFTER ISCHEMIA AND REPERFUSION INJURY ....................................... 93

RELATIONSHIP BETWEEN THE WAIST-TO-HEIGHT RATIO AND CARDIOMETABOLIC RISK IN

ADOLESCENTS........................................................................................................................................... 94

ROLE OF THE INTESTINAL MICROBIOTA IN THE PATHOPHYSIOLOGY OF OBESITY ..................... 95

TGFB1 HAPLOTYPE STRUCTURES HAVE SUBTYPE-SPECIFIC EFFECTS ON BREAST CANCER

SUSCEPTIBILITY AND CLINICAL PRESENTATION................................................................................. 96

THE EFFECT OF CHRONIC SUPPLEMENTATION WITH VITAMIN D ON HYPOTHALAMIC OBESITY

...................................................................................................................................................................... 97

THE INFLUENCE OF INTERLEUKIN-6 ON INSULIN RESISTANCE ........................................................ 98

THE ROLE OF ANNEXIN A1 AND FPR1 IN BREAST CANCER: A REVIEW.......................................... 99

TP53 GENE POLYMORPHISM: RELATION TO SUSCEPTIBILITY AND CLINICAL OUTCOME IN A

LUMINAL/HER2- BREAST CANCER SAMPLE ........................................................................................ 100

TREATMENT WITH METFORMIN AND INSULIN IN WALKER-256 TUMOR BEARING RATS:

EFFECTS ON PANCREATIC ISLETS ...................................................................................................... 101

ZIKA VIRUS DETECTION IN VISCERAL SAMPLES FROM A STILLBORN FETUS ............................. 102

EPIDEMIOLOGY AND PUBLIC HEALTH

COMORBIDITIES ASSOCIATED WITH OVERWEIGHT IN ADOLESCENTS ........................................ 103

DENGUE, CHIKUNGUNYA AND ZIKA: Aedes aegypti AND EMERGING DISEASES IN BRAZIL ........ 104

DESCRIPTIVE EPIDEMIOLOGICAL STUDY OF AMERICAN TEGUMENTARY LEISHMANIASIS

CASES IN NORTHERN PARANA FROM 2010 TO 2015......................................................................... 105

ECO-EPIDEMIOLOGICAL ASPECTS OF PARACOCCIDIOIDOMYCOSIS ............................................ 106

EPIDEMIOLOGY OF Aedes aegypti IN THE MUNICIPALITY OF CAMBÉ - PARANÁ ........................... 107

EVALUATION OF THE EFFECTIVENESS OF GENEXPERT® MTB/RIF EQUIPMENT IN

TUBERCULOSIS DIAGNOSIS IN WEST PAULISTA PRISON UNITS .................................................... 108

FAMILY INCOME AND AGE AS INDEPENDENT FACTORS FOR THE DEVELOPMENT OF

CERVICAL CANCER ................................................................................................................................. 109

INFLUENCE OF TGFB1 rs1800468 POLYMORPHISM IN HPV INFECTION ......................................... 110

POTENTIAL BACTERICIDAL EFFECT OF IRON DOPED ZINC OXIDE NANOCRYSTALS ................. 111

PREVALENCE OF METABOLIC SYNDROME IN ADOLESCENTS OF DIFFERENT SOCIOECONOMIC

STATUS ..................................................................................................................................................... 112

QUALITY CONTROL OF THE CANINE VISCERAL LEISHMANIASIS DIAGNOSIS OF THE

LEISHMANIOSES NETWORK OF THE ADOLFO LUTZ INSTITUTE (ALI) - REGIONAL LABORATORY

CENTER OF PRESIDENTE PRUDENTE V.............................................................................................. 113

Staphylococcus aureus OF LESIONS IN INFERIOR MEMBERS: ISOLATION AND PROFILE OF

SUSCETIBILITY TO ANTIMICROBIALS OFTITLE ................................................................................... 114

GENERAL PATHOLOGY

A COMPARATIVE STUDY OF PATHOPHYSIOLOGICAL ALTERATIONS IN SCORPIONISM INDUCED

BY Tityus serrulatus AND Tityus bahiensis VENOMS .............................................................................. 115

AGGRECAN IS INVOLVED WITH THE EXTRINSIC AND INTRINSIC INNVERVATION OF

GASTROINTESTINAL TRACT OF HUMANS AND MICE ........................................................................ 116

AIRWAYS INFLAMMATION INDUCED BY Rhopalurus rochai VENOM ................................................. 117

ALTERATIONS OF THE COLONIC EPITHELIUM OF RATS DURING THE COURSE OF

TOXOPLASMIC INFECTION ..................................................................................................................... 118

AN EVALUATION OF AN ANIMAL MODEL OF CEREBRAL PALSY: THE EFFECTS ON THE

INTRAMUSCULAR COLLAGEN OF THE EXTENSOR DIGITORUM LONGUS MUSCLE ..................... 119

ANALYSIS OF CELL DEATH PATTERNS INDUCED BY METFORMIN IN HUMAN MCF-7 BREAST

CANCER CELLS ........................................................................................................................................ 120

ANTIOXIDANT ACTIVITY OF YELLOW PITAYA (Selenicereus megalanthus)....................................... 121

ANTIOXIDANT PROTECTION OF YELLOW PITAYA (Selenicereus megalanthus)

TO BIOLOGICAL MEMBRANES ............................................................................................................... 122

Bombyx mori SERICIN IMPROVES TESTICULAR ANTIOXIDANT SYSTEM AND EPIDIDYMIS OF

C57BL/6 MICE OBESE BY HIGH FAT DIET ............................................................................................ 123

BENZNIDAZOLE AND ASPIRIN ASSOCIATION FOR THE TREATMENT OF CHRONIC

EXPERIMENTAL Trypanosoma cruzi INFECTION................................................................................... 124

CHARACTERIZATION OF EXPERIMENTAL ACUTE COLITIS INDUCED BY CONCENTRATIONS OF

SODIUM DEXTRAN SULFATE IN MICE .................................................................................................. 125

CONSEQUENCE OF MATERNAL PROTEIN RESTRICTION ON MUSCLE SPINDLES IN RATS AT 21

DAYS OF AGE ........................................................................................................................................... 126

CREATINE SUPPLEMENTATION ANTICIPATES ETHANOL- INDUCED HEPATIC OXIDATIVE

STRESS AND FATTY LIVER IN MICE ..................................................................................................... 127

EFFECT OF RESISTANCE TRAINING ON CANCER CACHEXIA: A REVIEW OF THE LITERATURE 128

EFFICACY, SAFETY AND METHOD FOR CONFIRMATION OF THE PRESENCE OF OLFACTORY

EPITHELIUM IN BIOPSIES FROM THE SUPERIOR TURBINATE ......................................................... 129

EVALUATION OF POTENTIAL ANTINEOPLASTIC ACTION OF RESVERATROL, ISOLATED AND

ASSOCIATED TO ETHANOL, IN N-METHYL-N’-NITRO-N-NITROSOGUANIDINE (MNNG)-INDUCED

CARCINOGENESIS IN THE COLON OF RATS ....................................................................................... 130

FUNCTIONAL EVALUATION OF RATS SUBMITTED TO AN EXPERIMENTAL MODEL OF

CEREBRAL PALSY ................................................................................................................................... 131

GENETIC MECHANISMS IN THE DEVELOPMENT OF BREAST CANCER .......................................... 132

GLYCOLYTIC MUSCLE WASTING PROMOTES PRECOCIUOS MITOCHONDRIAL AND

SARCOPLASMIC RETICULUM DYNAMIC ALTERATIONS IN AN EXPERIMENTAL MODEL OF

PRECACHEXIA .......................................................................................................................................... 133

HISTOPHATOLOGICAL AND IMMUNOHISTOCHEMICAL FINDINGS OBSERVED IN COATIS (Nasua

nasua) MANTEINED AT THE BELA VISTA SANCTUARY, PARANA, SOUTHERN BRAZIL ................. 134

HORMONAL THYROID DISORDER IN MICE FED CONTROL DIET AND HIGH-FAT DIET IMPAIRED

SPERM COUNTS AND INFLAMMATION PROFILE IN TESTIS AND EPIDIDYMIS ............................... 135

IMPACT OF MATERNAL PROTEIN RESTRICTION DURING RAT PREGNANCY AND LACTATION ON

INTRAMUSCULAR CONJUNCTIVE TISSUE OF OFFSPRINGS ............................................................ 136

INTERACTION BETWEEN OBESITY AND ACUTE INFECTION BY Trypanosoma cruzi ON

CARDIOVASCULAR PARAMETERS........................................................................................................ 137

IPA/NO: EFFECTS AND ACTION MECHANISM IN CHRONIC CONSTRICTION INJURY-INDUCED

NEUROPATHIC PAIN IN MICE. ................................................................................................................ 138

Lactobacillus plantarum REDUCES INSULIN RESISTANCE AND YACON OR SYMBIOTIC REDUCES

OXIDATIVE STRESS IN RATS WITH METABOLIC SYNDROME .......................................................... 139

LOW NITRIC OXIDE CONCENTRATION IN PLASMA AS AN IMPORTANT FACTOR IN

DEVELOPMENT OF HYPERTENSION IN OBESE MICE ........................................................................ 140

MELANOMA THERAPY: WHAT IS IT FOR NOW AND FOR THE FUTURE? ........................................ 141

METABOLIC RISK FACTORS CORRELATE WITH OXIDATIVE STRESS AND INFLAMMATORY

BIOMARKERS IN APPARENTLY HEALTHY OLDER SUBJECTS .......................................................... 142

MORPHOLOGICAL CHANGES IN THE SOLEUS MUSCLE DUE TO PERIODONTAL DISEASE ........ 143

NUMBER OF ASTROCYTES IN THE DORSAL STRIATUM OF RATS SUBMITTED TO A CEREBRAL

PALSY LIKE-MODEL ................................................................................................................................. 144

PARASITIC PNEUMONIA BY Angiostrongylus spp EM COATI (Nasua nasua) MAINTENED AT THE

BELA VISTA SANCTUARY, PARANÁ, SOUTHERN BRAZIL .................................................................. 145

PARTICIPATION OF IL-1Β AND IL-33/ST2 SIGNALING IN EHRLICH TUMOR-INDUCED PAIN IN MICE

.................................................................................................................................................................... 146

PESTICIDE AND BREAST CANCER........................................................................................................ 147

PRENATAL INJECTIONS OF LIPOPOLYSACCHARIDE, PERINATAL ANOXIA AND SENSORIMOTOR

RESTRICTION: IMPLICATIONS FOR A MODEL OF CEREBRAL PALSY-LIKE .................................... 148

PRE-NEOPLASTIC AND NEOPLASTIC HISTOPATHOLOGICAL LESIONS OBSERVED IN GROSSLY

NORMAL MAMMARY GLANDS OF ADULT FEMALE DOGS ................................................................. 149

PTPRΖ/PHOSPHACAN IS EXPRESSED IN SENSORY AND MOTOR NERVES OF THE

GASTROINTESTINAL TRACT OF HUMANS AND MICE ........................................................................ 151

RESISTANCE EXERCISE TRAINING ATTENUATES CANCER-INDUCED MUSCLE ATROPHY AND

STRENGTH LOSS BY DECREASING SYSTEMIC INFLAMMATION AND PROTEOLYSIS SIGNALING

IN WALKER-256 TUMOR-BEARING RATS. ............................................................................................ 152

RESISTANCE TRAINING AS PREVENTION AND TREATMENT OF CANCER CACHEXIA IN TWO

EXPERIMENTAL MODELS OF SOLID TUMORS .................................................................................... 153

Senecavirus A ENTERS THE CENTRAL NERVOUS SYSTEM VIA THE BLOOD-CEREBROSPINAL

FLUID BARRIER IN NATURALLY INFECTED PIGLETS ......................................................................... 154

TACTILE AND KINESTHETIC STIMULATION REVERSES THE EFFECTS OF PRENATAL STRESS IN

THE CA3 REGION OF THE HIPPOCAMPUS IN RATS: STEREOLOGICAL STUDY. ........................... 155

STUDY OF THE GENETIC POLYMORPHISM rs3087465 OF TGFB RECEPTOR II: POSSIBLE ROLE

AS A PROGNOSTIC MARKER FOR WILMS TUMOR ............................................................................. 156

IMMUNOLOGY

ANALYSIS OF FOXP3 TRANSCRIPTION FACTOR rs2232365 POLYMORPHISM IN HPV INFECTION:

A CASE-CONTROL STUDY ...................................................................................................................... 157

APPLICATIONS OF IGY ANTIBODIES TO INFLUENZA A VIRUS DETECTION IN INFECTED CELLS

BY IMMUNOCYTOCHEMISTRY ............................................................................................................... 158

CXCL12 RS1801157 POLYMORPHISM CONTRIBUTION TO HPV INFECTION AND CERVICAL

LESIONS DEVELOPMENT. ...................................................................................................................... 159

DENGUE VACCINE AND THE IMMUNE RESPONSE ............................................................................ 160

EFFECT OF GREEN TEA (EGCG) ON THE CXCR4 EXPRESSION IN PERIPHERAL BLOOD

MONONUCLEAR CELLS .......................................................................................................................... 161

E-SELECTIN IS A LINK BETWEEN METABOLIC SYNDROME AND DISEASE ACTIVITY IN

RHEUMATOID ARTHRITIS ....................................................................................................................... 162

EVALUATION OF THE REACTIVITY AGAINST NATIVE AND RECOMBINANT GP43 OF

Paracoccidioides brasiliensis IN SERUM SAMPLES POSITIVE FOR CANDIDEMIA ............................. 163

EXPERIMENTAL CUTANEOUS CANDIDIASIS IN DIABETIC MICE AND EVALUATION OF TISSUE

REPAIR ...................................................................................................................................................... 164

EXPRESSION OF INDOLEAMINE 2,3-DIOXYGENASE IN CANCERS .................................................. 165

FOXP3 GENE POLYMORPHISMS DO NOT AFFECT SUSCEPTIBILITY NOR PROGNOSIS FOR

CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA IN BRAZILIAN PATIENTS ................................. 166

FUNCTIONAL CAPACITY OF OLDER WOMEN WITH DIFFERENT LEVELS OF INFLAMMATION .... 167

LUPUS PATIENTS WITH METABOLIC SYNDROME HAVE A DIFFERENT PROINFLAMMATORY

PROFILE THAN THOSE WITHOUT ......................................................................................................... 168

MOLECULAR CHARACTERIZATION OF HIV-1 AND MECHANISMS OF VIRAL RESISTANCE ......... 169

PREDICTIVE USE OF GENETIC POLYMORPHISMS AS A TOOL FOR CLINICAL PRACTICE IN

LEISHMANIASIS INFECTIONS................................................................................................................. 170

PREDICTIVE USE OF GENETIC POLYMORPHYSMS IN CROHN’S DISEASE .................................... 171

PRO-INFLAMMATORY PROFILE OF OLDER WOMEN WITH DIFFERENT LEVELS OF OBESITY .... 172

ROLE OF TOLL-LIKE RECEPTORS SIGNALING AND MYD88 ADAPTER PROTEIN IN THE

IMMUNOPROTECTION AGAINST Paracoccidioides brasiliensis: AN UPDATE..................................... 173

SLEEP RESTRICTION ALTERS TESTICULAR INFLAMMATORY PROFILE IN PERIPUBERTAL RATS

.................................................................................................................................................................... 174

THE USE OF ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) TO DIAGNOSE

PARACOCCIDIOIDOMYCOSIS: A SYSTEMATIC REVIEW .................................................................... 175

TRANSFORMING GROWTH FACTOR BETA 1 GENE HAPLOTYPE INFLUENCES HUMAN

PAPILLOMAVIRUS INFECTION ............................................................................................................... 176

MICROBIOLOGY AND PARASITOLOGY

ANALYSIS OF THE VIRULENCE OF GEOGRAPHICAL ISOLATES OF THE Bombyx Mori

Nucleopolyhedrovirus (NPV). ..................................................................................................................... 177

ANTIMICROBIAL ACTIVITY OF THE METHANOL EXTRACT Eugenia involucrata FRONT OF THE

DIFFERENT SEROTYPES OF Salmonella spp. ....................................................................................... 178

ANTIMICROBIAL RESISTANCE USE OF ANTIBIOTICS AND THE INDISCRIMINATED USE OF

ANTIBIOTICS ............................................................................................................................................. 179

ANTIVIRAL ACTIVITY OF METHANOL, ETHANOL AND ACETONE EXTRACTS FROM BARK OF

Trichilia catigua IN THE REPLICATION OF HERPES VIRUS RESISTANT TO ACYCLOVIR................ 180

CAFFEIC ACID HAS ANTI-AMASTIGOTE EFFECT IN L. amazonensis-INFECTED MACROPHAGES

.................................................................................................................................................................... 181

CONCANAVALIN-A INDUCE M1 MACROPHAGE POLARIZATION IN INFECTION BY

Leishmania (L) amazonensis ..................................................................................................................... 182

DETECTION OF MYCOPLASMA SPECIES IN MAMMAL CELL CULTURES USING PCR ................... 183

EVALUATION OF ANTIVIRAL ACTIVITY OF PROPOLIS FROM Melipona quadrifasciata AGAINST

HERPES SIMPLEX VIRUS, IN VITRO. ..................................................................................................... 184

EVOLUTION OF THE EXPERIMENTAL ARTRITE INDUCED BY Paracoccidioides brasiliensis IN

WISTAR RATS ........................................................................................................................................... 185

FIRST REPORT OF NEW DELHI METALLO-β-LACTAMASE-PRODUCING Klebsiella pneumoniae IN

CLINICAL ISOLATES FROM UNIVERSITY HOSPITAL .......................................................................... 186

FUSARIOSIS IN IMMUNOCOMPETENT MICE ....................................................................................... 187

Galleria mellonella AS AN IN VIVO MODEL FOR THE EVALUATION OF THE VIRULENCE POTENTIAL

OF Enterococcus faecium AND Enterococcus faecalis ............................................................................ 188

IN VITRO ACTIVITY OF CAFFEIC ACID ON PROMASTIGOTE FORMS OF L. amazonensis .............. 189

INCIDENCE OF CASES OF CRYPTOSPORIDIOSIS- Cryptosporidium spp- IN PATIENTS ATTENDED

AT THE UNIVERSITY HOSPITAL OF LONDRINA................................................................................... 190

INCIDENCE OF MULTI-DRUG RESISTANT PATHOGENS FROM ―ESKAPE‖ GROUP IN THE

INTENSIVE THERAPY UNIT OF A UNIVERSITY HOSPITAL OF THE SOUTH REGION OF BRAZIL. 191

INSULIN AND GLUCOSE PROMOTES PROLIFERATION IN VITRO OF

Leishmania (Leishmania) amazonensis .................................................................................................... 192

MANGIFERIN INHIBITS ACYCLOVIR-RESISTANT HERPES SIMPLEX VIRUS TYPE 1, IN VITRO.... 193

MCR-1 COLISTIN RESISTANCE IN ESBL PRODUCING-Escherichia coli, IN BRAZIL ......................... 194

MORPHOLOGICAL ALTERATIONS IN THE ENTERIC NERVOUS SYSTEM OF RATS INFECTED

WITH Toxoplasma gondii ........................................................................................................................... 195

MORPHOLOGICAL ALTERATIONS OF HAMSTERS ANKLE JOINT CAUSED BY

Leishmania (Viannia) braziliensis EXPERIMENTAL INFECTION ........................................................... 196

MORPHOLOGICAL ASPECTS OF ANKLE JOINT’S SYNOVIAL MEMBRANE IN HAMSTERS

EXPERIMENTALLY INFECTED BY Leishmania (Viannia) braziliensis ................................................... 197

OUTBREAK OF VERMINOTIC PNEUMONIA IN CATTLE FROM NORTHERN PARANÁ ..................... 198

PROSPECTING AND CHARACTERIZATION OF INTRINSIC DISORDER IN PROTEINS FROM VIRUS

ASSOCIATED WITH DISEASES .............................................................................................................. 199

RECOMBINANT PROTEIN P21 FROM Trypanosoma cruzi REDUCES BREAST CANCER CELL

MIGRATION IN VITRO .............................................................................................................................. 200

Salmonella spp. IN FISH AND FISHERY PRODUCTS: A REVIEW ........................................................ 201

SCHISTOSOMIASIS MANSONI: EPIDEMIOLOGICAL SURVEY OF PARASITOSIS IN THE

MUNICIPALITY OF LONDRINA - PARANÁ. ............................................................................................. 202

SENSITIVITY PROFILE OF POSITIVE GRAM COCCI ISOLATED FROM BIOLOGICAL SAMPLES OF

HOSPITALIZED PATIENTS FROM UNIVERSITY HOSPITAL OF LONDRINA DURING THE YEARS OF

2012 TO 2016. ........................................................................................................................................... 203

STAR TICK AND ROCKY MOUNTAIN SPOTTED FEVER ...................................................................... 204

SUSCEPTIBILITY EVALUATION OF THE ANTIMICROBIAL AGENTS USED IN THE EMPIRICAL

THERAPY OF URINARY TRACT INFECTIONS CAUSED BY GRAM-NEGATIVE BACILLI AT THE

UNIVERSITY HOSPITAL OF LONDRINA 2012-2016 .............................................................................. 205

ULTRASTRUCTURE OF BOMBYXMORI (LEPIDOPTERA: BOMBYCIDAE) INTEGUMENT INFECTED

BY BOMBYXMORINUCLEOPOLYHEDROVIRUS ................................................................................... 206

VALIDATION OF POTENTIAL INHIBITORS AGAINST MALARIA .......................................................... 207

PHARMACOLOGY AND TOXICOLOGY

ANTICARCINOGENIC EFFECT OF THE LECTIN ARTINM ON AFB1-INDUCED

HEPATOCARCINOGENESIS IN RATS .................................................................................................... 208

BALANCE DISORDER AND HEARING LOSS AFTER ANTINEOPLASTIC AGENTS AND

AMINOGLYCOSIDES USE ....................................................................................................................... 209

BENEFICIAL EFFECTS OF BRAZIL NUTS (Bertholettia excelsea) CONSUMPTION ON

CARDIOVASCULAR RISK. ....................................................................................................................... 210

CHANGES IN THE REPRODUCTIVE PERFORMANCE OF MICE EXPOSED TO BUPROPIONA

CHLORIDRATE DURING SPERMATOGENESIS .................................................................................... 211

CONGENITAL ABNORMALITIES DUE TO CAPTOPRIL ADMINISTRATION DURING PREGNANCY

OVER MICE OFFSPRING ......................................................................................................................... 212

CONGENITAL MALFORMATION AND INTRAUTERIAL GROWTH DELAY IN FETUSES OF MICE

TREATED WITH IBUPROFEN DURING PREGNANCY .......................................................................... 213

CONGENITAL MALFORMATION IN OFFSPRING OF MICE EXPOSED TO BUPROPIONA

CHLORIDRATE DURING SPERMATOGENESIS .................................................................................... 214

DETECTION OF ALDICARB AND CARBARYL IN THE GASTRIC CONTENT OF A DOG FOUND DEAD,

SUSPECTED OF CRIMINAL POISONING ............................................................................................... 215

DRUGS USED TO TREAT BIPOLAR DISORDER AND TOBACCO USE DISORDER REDUCE

REACTIVE OXYGEN AND NITROGEN SPECIES PRODUCED BY NEUTROPHILS IN VITRO ........... 216

EFFECT OF THE ACUTE ETHANOL CONSUMPTION IN NEUTROPHIL RECRUITMENT TO THE

INFLAMMATORY FOCUS. ........................................................................................................................ 217

EFFECTS OF INOS INHIBITION ON CARDIOVASCULAR RISK OF ENDOTOXEMIC FEMALE RATS:

ROLE OF ESTROGEN .............................................................................................................................. 218

EVALUATION OF METABOLIC PARAMETERS IN ADULT RATS EXPOSED TO FLUOXETINE DURING

GESTATION AND LACTATION ................................................................................................................ 219

EVALUATION OF REPRODUCTIVE PERFORMANCE AND TOXICITY IN MALE MICE TREATED WITH

ISOTRETINOIN .......................................................................................................................................... 220

EVALUATION OF THE EFFECTS OF LIXISENATIDE ON SOME METABOLIC PARAMETERS

AFFECTED BY WALKER-256 TUMOR. ................................................................................................... 221

EVALUATION OF THE HEPATOTOXIC POTENTIAL OF ACETAMIFENE (PARACETAMOL®) IN

RATTUS NORVEGICUS ALBINUS MODEL DURING THE BREASTFEEDING PERIOD ...................... 222

EXPOSURE OF ADULT RATS TO COMMERCIAL GASOLINE IMPAIRS SPERMATOGENESIS AND

SPERM QUALITY ...................................................................................................................................... 223

HEATH LEGDGER’S DEATH: A DRUG INTERACTION CASE STUDY ................................................. 224

HESPERIDIN METHYL CHALCONE AMELIORATES MONOSODIUM URATE CRYSTALS-INDUCED

EXPERIMENTAL GOUTY ARTHRITIS BY INHIBITING NFB ACTIVATION IN MICE .......................... 225

INFLUENCE OF MATERNAL TREATMENT WITH METFORMIN ON OFFSPRING’S METABOLIC AND

CARDIOVASCULAR PARAMETERS OF RATS ....................................................................................... 226

INTRAUTERINE EXPOSURE TO METFORMIN DOES NOT LEAD TO CHANGES IN THE VASCULAR

SYSTEM OF ADULTHOOD OFFSPRING ................................................................................................ 227

KAURENOIC ACID ATTENUATES ZYMOSAN-INUCED ARTHRITIS AND PERITONITIS IN MICE ..... 228

MALFORMATION OF THE DENTAL GERM IN MICE EXPOSET TO METHYLPHENIDATE DURING

PREGNANCY ............................................................................................................................................. 229

MATERNAL EXPOSURE TO FLUOXETINE DURING GESTATION AND LACTATION UP-REGULATES

NEURONAL NITRIC OXIDE SYNTHASE EXPRESSION LEADING TO DECREASED AORTIC

CONTRACTION IN ADULT FEMALE PROGENY .................................................................................... 230

MATERNAL TOXICITY OF TRICLOSAN IN A TWO-GENERATION STUDY IN WISTAR RATS .......... 231

NITRIC OXIDE INVOLVEMENT IN ACUTE LUNG INJURY CAUSED BY Tityus bahiensis VENOM .... 232

PERIPUBERAL EXPOSURE OF MALE RATS TO LOW DOSES OF MALATHION LEADS TO

HISTOPATHOLOGICAL AND INFLAMMATORY PROFILE ALTERATIONS IN TESTIS AND

EPIDIDYMIS ............................................................................................................................................... 233

PROTECTIVE EFFECT OF TRANS-CHALCONE IN A MODEL OF ACETIC ACID-INDUCED COLITIS IN

MICE: REDUCTION OF NEUTROPHIL RECRUITMENT, PRO-INFLAMMATORY CYTOKINES

PRODUCTION, AND NF-κB ACTIVATION ............................................................................................... 234

QUERCETIN LOADED MICROCAPSULES REESTABLISH LIVER GLYCOGEN LEVELS IN A MODEL

OF HYPERGLYCEMIA INDUCED BY 66% FRUCTOSE IN RATS.......................................................... 235

SUPPRESSIVE EFFECT OF INSULIN ON HEPATIC GLUCOSE PRODUCTION AND

GLYCOGENOLYSIS STIMULATED BY CAMP. ....................................................................................... 236

THE FLAVONOID HESPERIDIN METHYL CHALCONE AMELIORATES ACETIC ACID-INDUCED

COLITIS BY REDUCING NEUTROPHIL RECRUITMENT, PRO-INFLAMMATORY CYTOKINES

PRODUCTION, AND NF-κB ACTIVATION IN MICE ................................................................................ 237

VINPOCETINE ATTENUATES SUPEROXIDE ANION-INDUCED PAIN BY INHIBITING PRO-

INFLAMMATORY CYTOKINE PRODUCTION AND BY INCREASING NRF2 EXPRESSION IN MICE 239

VITAMIN D DECREASES RADICAL COMPOUNDS PRODUCED BY NEUTROPHILS IN VITRO ........ 240

13

The Postraduate Program in Experimental Pathology of the Universidade Estadual de Londrina (Brazil) promoted the II International Symposium of Experimental Pathology (ISEP 2017) and VII Simpósio de Patologia Experimental da UEL, in Londrina, PR, Brazil, August 2nd to 4th, 2017.

The first five editions of this event were promoted in order to reach junior and senior researchers in Experimental Pathology from Londrina. After becoming a local well-established event we decided to expand to a larger conference (ISEP 2016) to join more researchers interested in Pathology, Immunology and Parasitology.

The ISEP 2017 provided a program with international and Brazilian speakers including lectures, round table and short-courses. Besides, we did encourage participants to present their work during the Symposium as poster or oral presentation. We are sure that was an excellent opportunity to discuss basic and clinical research with numerous colleagues from Brazil and other countries.

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PROGRAM

WEDNESDAY, 02.08.2017

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10:00 – 16:00 Registration

13:00 – 15:30 Oral presentation from MSc students – Research Projects

15:30 – 16:00 Coffee Break

16:00 – 17:30 Oral presentation from MSc students – Research Projects

17:30 – 18:30 Registration and putting up of posters and photos for exhibition

18:30 – 19:00 Opening ceremony

19:00 – 20:00

Opening lecture: NEW DEVELOPMENTS IN GASTROINTESTINAL SENSORY FUNCTION

Speaker: L. Ashley Blackshaw, PhD

Queen Mary University of London – UK

20:00 – 22:00 Poster session and reception cocktail

THURSDAY, 03.08.2017

UEL

8:00 – 10:00 Short-courses I to IX


10:30 – 12:00 Short-courses I to IX

12:00 – 13:30 Lunch Time

13:30 – 14:30

Lecture II: NEUROPARASITOLOGY: CONSEQUENCES FOR THE NEURAL SYSTEMS

DUE INFECTION WITH Toxoplasma AND Trypanosoma cruzi

Speaker: Rosa Maria Esteves Arantes, PhD

Federal University of Minas Gerais (UFMG)

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14:30 – 15:30

Round table 1: INVOLVEMENT OF OXIDATIVE STRESS IN CANCER DEVELOPMENT

AND COMORBIDITIES

Session Chair: Flávia A. Guarnier, PhD

Department of General Pathology – State University of Londrina

Lecture I: “Influence of oxidative stress on muscle adaptation on cancer-induced cachexia:

are we moving towards survival improvement?”

Speaker: Flávia A. Guarnier, PhD


Lecture II: “Oxidative modifications in cancer: initiation, promotion and progression”

Speaker: Rodrigo Cabral Luiz, PhD


Lecture III: “Oxidative alterations on enteric neurons promoted by Walker Tumor-256

solid tumor: an experience of interaction between UEL / UEM / Mayo Clinic”

Speaker: Jacqueline Nelisis Zanoni, PhD

Department of Morphology - State University of Maringá


16:00 – 17:00

Lecture III: ROLE OF T CELLS ON DENGUE VIRUS PATHOGENESIS

Speaker: Juliano Bordignon, PhD

Carlos Chagas Institute (ICC) - Oswaldo Cruz Foundation (Fiocruz)

17:00 – 18:00

Round Table 2: EMERGING AND NEGLECTED INFECTIOUS DISEASES: INSIGHTS,

ADVANCES AND CHALLENGES

Session Chairs:

Phileno Pinge Filho, PhD

Waldiceu Aparecido Verri Junior, PhD

Wander Rogério Pavanelli, PhD


Lecture I: “Leishmania infection: painful or painless?”

Speaker: Sergio M. Borghi, PhD


Lecture II: “Emerging viruses”

Speaker: Juliano Bordignon, PhD:

Carlos Chagas Institute (ICC) - Oswaldo Cruz Foundation (Fiocruz)

Lecture III: “Nanotechnological approach on the development of new therapeutical

alternatives for the treatment of Chagas disease"

Speaker: Danielle Lazarin Bidóia, PhD

Department of Basic Health Sciences – State University of Maringá

18:00 – 19:00 Oral presentations

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FRIDAY, 04.08.2017

UEL

8:00 – 10:00 Short-courses VIII to XVII


10:30 – 12:00 Short-courses VIII to XVII

12:00 – 13:30 Lunch Time

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13:30 – 14:30

Round table 3: INTEGRATING KNOWLEDGE ON MOLECULAR MARKERS IN HUMAN

CANCER

Session Chair: Maria Angelica Ehara Watanabe, PhD


Lecture I: “Emerging approaches involved in pediatric tumors: advances in acute

lymphoblastic leukemia and Wilms tumor”

Speaker: Carlos Eduardo Coral de Oliveira, PhD

Professor of Pharmacology, Pontifical Catholic University of Paraná

Postdoctoral Fellow of Experimental Pathology Postgraduate Program, State University of

Londrina

Lecture II: “Markers of risk stratification and prognostic indicators in human breast

cancer”

Speaker: Roberta Losi Guembarovski, PhD

Professor of Cellular Biology, State University of Londrina

14:30 – 15:30

Round Table 4: HOST-MICROORGANISM INTERACTION: IMMUNE RESPONSE, ECO-

EPIDEMIOLOGY AND MOLECULAR DIAGNOSIS OF PARACOCCIDIOIDOMYCOSIS

AND OTHER SYSTEMIC MYCOSES

Session Chair: Eiko N. Itano, PhD.


Lecture I: “Paracoccidioides brasiliensis and P. lutzii and their impact on immune

response”

Speaker: Eiko N. Itano, PhD.


Lecture II: “Eco-epidemiology of Paracoccidioidomycosis”

Speaker: Mario Augusto Ono, PhD / Rafaela Macagnan, MSc


Lecture III: “Molecular diagnosis of Paracoccidioidomycosis and other systemic mycoses”

Speaker: Emerson José Venâncio, PhD



16:00 – 17:00

Lecture IV: NEURONAL EXTRACELLULAR MATRIX AND GASTROINTESTINAL

FUNCTION

Speaker: L. Ashley Blackshaw, PhD

Queen Mary University of London – UK

17:00 – 18:00 Awards, Closing Ceremony and Closing Cocktail

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SHORT-COURSES

Short-course I: “Invertebrate models to study host-pathogen interactions: Galleria mellonella and Tenebrio

molitor”

Coordinator/Speaker: Ricardo Sérgio Couto Almeida, PhD

Laboratory of Alternative Methods to the Use of Animal Models – LAMEA – Department of Microbiology – State University of Londrina

Short-course II: “Immunological and molecular diagnosis of systemic mycoses”

Coordinators/ Speakers:

Mario Augusto Ono, PhD

Laboratory of Animal Immunology – Department of General Pathology – State University of Londrina

Emerson José Venâncio, PhD

Laboratory of Immunology IV – Department of General Pathology – State University of Londrina

Eiko Nakagawa Itano, PhD

Laboratory of Applied Immunology – Department of General Pathology – State University of Londrina

Short-course III: “Editing and production of photographic images for scientific publishing”

Coordinator/Speaker: Thiago Fernandes, PhD

State University of Londrina

Short-course IV: “Genomic DNA extraction from peripheral blood: a salting out procedure”

Coordinator: Karen Brajão de Oliveira, PhD

Speaker: Fernando Cezar dos Santos, MSc – PhD student in Experimental Pathology Postgraduate Program

Laboratory of Molecular Genetics and Immunology - Department of General Pathology – State University of Londrina

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Short-course V: “Methodology of the popularization of scientific knowledge in health”

Coordinator/Speaker: Débora de Mello Gonçales Sant’ana, PhD

Department of Morphological Sciences – State University of Maringá

Short-course VI: “Cell culture in the study of cancer”

Coordinator/Speaker: Poliana Camila Marinello, PhD

Laboratory of Molecular Pathology - Department of General Pathology – State University of Londrina

Short-course VII: “Clinical-laboratory approach of leukemia by case reports”

Coordinator/Speaker: Newton Hashimoto, MSc

Philadelphia University Center (UNIFIL)

Short-course VIII: “The use of flow cytometry in the study of anticancer drugs in human cell lines”

Coordinator: Mário Sérgio Mantovani, PhD

Speakers:

Adrivanio Baranoski, MSc – PhD student in Genetics and Molecular Biology Postgraduate Program

Bruna Isabela Biazi, MSc - PhD student in Genetics and Molecular Biology Postgraduate Program

Lilian Areal Marques, MSc - PhD student in Genetics and Molecular Biology Postgraduate Program

Thalita Alves Zanetti, MSc - PhD student in Genetics and Molecular Biology Postgraduate Program

Laboratory of Toxicological Genetics – State University of Londrina

Short-course IX: “Methods of indirect diagnosis for parasitic zoonoses”

Coordinator: Regina Mitsuka Breganó, PhD

Speakers:

Eloiza Teles Caldart, MSc - PhD Student in Animal Science Postgraduate Program

Fernanda Pinto Ferreira, MSc - PhD Student in Animal Science Postgraduate Program

Laboratory of Zoonoses and Public Health – State University of Londrina

Short-course X: “miRNAs: prediction and analysis through bioinformatics”

Coordinator: Laurival Vilas Boas, PhD

Speakers:

Renan José Casarotto Appel, BSc - MSc student in Genetics and Molecular Biology Postgraduate Program

Kátia Brumatti Gonçalves, MSc – PhD student in Genetics and Molecular Biology Postgraduate Program

Department of General Biology - State University of Londrina

Short-course XI: “Implementing molecular biology in the investigation of molecular markers in human diseases”

Coordinator: Maria Angelica Ehara Watanabe, PhD

Speakers:

Glauco Akelinghton Freire Vitiello, MSc – PhD student in Experimental Pathology Postgraduate Program

Marla Karine Amarante, PhD


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Short-course XII: “Introduction to Mendeley”

Coordinator/Speaker: Thiago Viana Camata, MSc

Northern University of Paraná (UNOPAR)

Short-course XIII: “Imunohistochemistry: general concepts”

Coordinator/Speaker: Eduardo José de Almeida Araújo, PhD

Department of Histology – State University of Londrina

Short-course XIV: “Practical foundations of parasitology”

Coordinator/Speaker: Fabiana M. R. Lopes Mori, PhD

Philadelphia University Center (UNIFIL)

Short-course XV: “Mechanisms of bacterial invasion: techniques for detection”

Coordinator: Renata Katsuko Takayama Kobayashi, PhD

Speaker: Marcelly Chue Gonçalves, MSc

Laboratory of Basic and Applied Bacteriology - Department of Microbiology – State University of Londrina

Short-course XVI: “Introduction to biological electron microscopy”

Coordinator/Speaker: Admilton Gonçalves de Oliveira Junior, PhD

Collaborators: Thiago Fernandes, PhD and Osvaldo Capelo

Laboratory of Microbial Biotechnology – LABIM/ Laboratory of Electron Microscopy and Microanalysis – LMEM - Department of Microbiology – State University of Londrina

Short-course XVII: “Free radicals and diseases”

Coordinator/Speaker: Décio Sabbatini Barbosa, PhD

Department of Pathology, Clinical and Toxicological Analysis – State University of Londrina

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EXPANDED ABSTRACTS ORAL PRESENTATION FROM MSc STUDENTS

Research projects

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ACTIVITY OF SILVER BIOGENIC NANOPARTICLES ON RH AND ME-49 STRAINS OF Toxoplasma

gondii IN HeLa CELLS

Machado, L. F. ¹, Costa, I. N. ¹

¹ State University of Londrina – Department of Pathological Sciences – Laboratory of Experimental Protozoology,

Londrina, PR, Brazil.

E-mail: [email protected]

Keywords: Silver nanopaticles, toxoplasmosis, HeLa cells, cytokines.

Abstract

Toxoplasmosis is the infection caused by Toxoplasma gondii, an obligate intracellular parasite capable of infecting

nucleated cells of homeothermic animals. It is often asymptomatic or with nonspecific symptoms; However, it may

become clinically evident in immunocompromised individuals, congenital infections and cases of ocular

toxoplasmosis. Conventional treatment is done with the combined use of pyrimethamine and sulfadiazine, however,

they present high toxicity and many adverse effects by interacting indistinctly with the biochemical processes of both

the parasite and the host. This encourages research into alternative treatments for this infection. In this context,

nanotechnology presents promising advancements by providing diagnostic, treatment and immunization advantages

of various infectious diseases. As an example of the use of nanotechnology has been used the biogenic silver

nanoparticles as an alternative tool in the treatment of toxoplasmosis, since they present antimicrobial, antiviral and

antiparasitic activities. Therefore, the objective is to investigate the biological action of silver nanoparticles on HeLa

cells infected with RH and ME-49 strain of T. gondii. For this, first, the cytotoxicity of treatment with silver

nanoparticles compared to conventional treatment (sulfadiazine and pyrimethamine) for toxoplasmosis will be

evaluated. Then the intracellular adhesion, infection and proliferation rates of the parasite will be determined, as well

as the production of Th1, Th2 and Th17 standard cytokines from the HeLa cell supernatant. Knowing the

toxoplasmosis severity worldwide, and considering the difficulties of conventional treatment, promising results are

expected with the insertion of silver nanoparticles as a therapeutic strategy, thus contributing to scientific, economic

and social development.

Hypothesis

Silver nanoparticles have anti-Toxoplasma activity, reducing the adhesion, infection and proliferation rates of this

parasite in HeLa cells. Besides that, silver nanoparticles have immunological effects such as increased Th1 standard

cytochrome levels (characteristic cellular immune response to Toxoplasma gondii).

Introduction

Toxoplasma gondii infection is one of the zoonoses most known worldwide (1) and is considered a public

health problem because it develops a worrying aspect in immunocompromised patients in congenital infection and in

ocular toxoplasmosis. The factors that control the pathogenicity of T. gondii are mainly related to the host immune

system, the parasite load and the virulence of the parasite strain. The strains are widely distributed in nature and,

according to molecular analyzes, can present three different genotypes: type I, II and III (2). Type I strains are related

to high virulence in mice, such as the RH strain (3,4), while type II strains are cytogenic or of low virulence, such as

the ME-49 strain (5).

The treatment of toxoplasmosis has been done since 1950 with the combined use of pyrimethamine and

sulfadiazine, which are drugs responsible for the blocking of enzymes essential for the survival and replication of the

parasite (6). However, they are not well tolerated by the organism, presenting many adverse effects, such as the

suppression of bone marrow activity, besides interacting indistinctly with the biochemical processes of both the

parasite and the host (7,8,9). Due to the difficulty in treating toxoplasmosis and toxicity presented by conventional

drugs, as well as the emergence of strains with different sensitivities and resistance to drugs available, other

compounds have been researched as an alternative treatment for this infection, especially the nanoparticles.

The nanoparticles are characterized by their size smaller than 1000 nm and may be metallic, lipid or polymeric

(10). Some of them, especially those of metallic origin, are being used in the treatment of several infections (11),

including toxoplasmosis (12). In this sense, they can act as drug carriers, reducing toxicity, modulating

pharmacokinetics, increasing bioavailability, as well as targeted delivery of the drug to the specific target (13,14).

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In this context, the success of the use of nanotechnology involves strategies possibly able to associate

therapeutic advantages since they promote the vectorization of drugs already used in the pharmaceutical area, also

because they have the capacity to efficiently compartmentalize several groups of therapeutic agents and to modify the

properties and the behavior of active substances in biological environment. Furthermore, increasing advances in the

study of nanotechnology as additional or alternative forms of treatment for many infections are promising sources of

research in the scientific field.

In this aspect, nanotechnology stands out for the advantages presented when compared to the drugs currently

used in toxoplasmosis which are, in turn, highly toxic.

Objective

To assess the anti-proliferative and immune action of silver nanoparticles in cells HeLa against experimental

infection by the Toxoplasma gondii RH and ME49 strains.

Materials and Methods

HeLa Cell Culture

There was no need to submit the project to the Ethics Committees in Human or Animal research. HeLa cells,

from a human uterine cervix tumor, were purchased from the American Type Culture Collection (Manassas, VA, USA)

and maintained in culture at the Parasitology Laboratory of the State University of Londrina. Cultivation will be

performed in culture flasks with RPMI 1640 medium (Sigma Chemical CO, Brazil) supplemented with 10% inactivated

fetal bovine serum (FBS) (Sigma-Aldrich), 1% antibiotics (10,000 U/ml penicillin and 10mg/mL of streptomycin)

(Cultilab, Brazil), L-glutamine, sodium pyruvate and 2β-mercaptoethanol (complete medium for HeLa-MCH). The cell

culture will be kept in a 5% CO2 oven at 10-37°C and used for in vitro experimental infection trials as well as for

maintenance of T. gondii strains.

Maintenance of T. gondii strains

Tachyzoites of T. gondii RH and ME-49 strains will be kindly provided by professor Italmar Teodorico Navarro

and professor João Luiz Garcia of the State University of Londrina. The parasites will be maintained in culture of HeLa

cells with MCH, changing only the percentage of fetal bovine serum to 2% in a 5% CO2 oven at 37°C where

successive passages will be performed in order to maintain the parasite in vitro (15).

Biogenic production of silver nanoparticles

The silver nanoparticles will be supplied by professor Gerson Nakazato of the Laboratory of Microbiology of

the State University of Londrina and produced by Professor Duran et al. (16). Briefly, the filamentous fungus Fusarium

oxysporum was cultivated in medium containing malt extract (2%), and yeast extract (0.5%) at 28°C for 6 consecutive

days. The biomass was resuspended and filtered in sterile distilled water. Approximately 10g of the fungal suspension

was transferred to an Erlenmeyer flask containing 100ml of distilled water, stored for 72 hours at 28°C. After filtration

of the biomass AgNO3 (Sigma®) was added and the final filtrate was again 10mM and maintained for 24 hours at

28°C. The X-ray of the silver nanoparticles were characterized by scanning electron microscopy (SEM), diffraction

analysis (DRX), energy dispersive spectroscopy (EDS), demonstrating nanoparticle scanning analysis (NTA) (17). The

obtained compound will be diluted to the concentration of 10 mM in 500μL of RPMI medium (volume 4000μl and a

concentration of 5mM) for subsequent dilutions.

Viability of the HeLa cell by MTT Test

The viability of the HeLa cells after treatment with the silver nanoparticles will be evaluated based on

mitochondrial oxidation by means of the colorimetric tetrazolium salt MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-

diphenyltetrazolium Thiazole blue) (Sigma Chemical Co., Brazil) (18). HeLa cells will be cultured in 96-well plates

(3x104 cells/ well/200 μl) for 24 hours in MCH medium at 37°C and 5% CO2. After this period, the cells will receive

treatment for 24 hours with the silver nanoparticles. Cells receiving only RPMI will be used as a negative control. After

the treatments, the supernatants will be removed and the cells received MTT solution (5mg/mL) for 3 hours under the

same culture conditions in the oven at 37°C and 5% CO2. Formazan crystals will be solubilized in 10% Sodium

Dodecyl Sulfate (SDS) and 50% Dimethyl Formamide (DMF), and after 30 minutes the absorbance will be measured

at 570 nm on a plate reader (Thermo-TP reader Thermo Plate Plate - TP-Reader). The results will be expressed as a

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relative percentage reduction of MTT for the control group calculated with the following formula: Viable cells (%) =

(Optical density treated cell / Optical density of untreated cell) X 100.

Viability of T. gondii Tachyzoites by Tripan Blue

Tripan blue staining will be used to verify the viability of T. gondii tachyzoites (both types of strains) treated or

not treated with silver nanoparticles for 30 minutes. After the treatments, the parasites will be stained with tripan blue

and counted in a Neubauer chamber with an optical microscope (e100, nikon - led). Parasites presenting clear

cytoplasm will be considered viable, and those that present a blue staining was not feasible(8). The percentage of

viable tachyzoites will be calculated with the following formula: viable cells (%) = (total viable cells / total cells (viable +

dead) X100.

Experimental Infection

To evaluate the effect of silver nanoparticles on adhesion, invasion and proliferation of T. gondii in HeLa cells,

we will use experimental models of infection with 3 cells subsequently treated. Treatments as follows: cells not

infected with T. gondii, cells infected with T. gondii and treated with RPMI medium (negative control), cells infected

with T. gondii and treated with pyrimethamine and sulfadiazine (positive control), cells infected with T. gondii and

treated with silver nanoparticles alone or in combination with sulfadiazine and pyrimethamine. Treatment will always

occur with the two types of strains proposed in this study (RH and ME-49).

Briefly, HeLa cells (1x105) will be kept in 24-well plates containing 13mm round coverslips (Ciencor Scientific,

Brazil) and will be infected with 5x105 tachyzoites of the T. gondii RH and ME-49 strains. After 3 hours of infection,

cells will be washed and treated with sulfadiazine and pyrimethamine used as a positive control, and silver

nanoparticles alone or associated with sulfadiazine plus pyrimethamine for 24 hours. Cells infected with the parasite

and treated with RPMI will be used as a negative control. After 24 hours of treatment, the cells will be fixed with 10%

paraformaldehyde and PBS for 24 hours, and washed with PBS to remove unfixed material. Cells will be stained with

1% toluidine blue (Sigma Chemical Co.) for 5 minutes and mounted on glass slides for optical microscope (e100,

Nikon-led) analysis. Cells will be analyzed by increasing light immersion microscopy to check adhesion rates, infection

(number of infected cells per 200 cells examined); and intracellular proliferation of the parasite (total number of

parasites per 200 cells examined). Then percentages of inhibition of adhesion, infection rate and inhibition of T. gondii

intracellular proliferation in the presence of treatments will be calculated as follows: mean of intracellular adhesion,

infection or proliferation rates analyzed in untreated cells will correspond to 100% of the indexes (17). The percent

inhibition of these parameters under treatments with silver nanoparticles will be calculated by subtracting the

percentage values obtained in cells treated with each silver nanoparticle obtained with the untreated cells. The

supernatant from the cells will be collected and stored at -80 ° C for further cytokine dosing.

Determination of cytokine levels

In order to ascertain whether the treatment is capable of altering the production of cytokines these will be

analyzed. The level of the cytokines present in the supernatant of the infected cells will be analyzed by citometric bead

array (CBA). Cytokine levels will be determined according to kit instructions (BD CBA Human Th1/Th2/Th17) using

Becton Dickinson's FACS Canto II. The cytometry data will be analyzed in the FCAP Array program, v1.0.1, from Soft

Flow Hungary Ltd. thus obtaining the concentrations of the cytokines in pg/ mL.

Expected Results

We expect promising results with the insertion of silver nanoparticles as a therapeutic strategy, thus

contributing to scientific, economic and social development.

References

1. Elsheika HM. Congenital toxoplasmosis: priorities for further health promotion action. Public Health. 2008:

122(4):335-353.

2. Sibley LD, Ajioka JW. Population structure of Toxoplasma gondii: clonal expansion driven by infrequent

recombination and selective sweeps. Annual review of Microbiology. 2008:62;329-351.

3. Dubey JP, Frenkel JK, Shen SK, Kwok CH. Infection and immunity with the RH strain of Toxoplasma gondii in rats

and mice. J. Parasitol. 1999:85(4):657-662.

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25

ANALYSIS OF IL-10 rs1800872 (c.-592 C>A) POLYMORPHISM IN THE HPV INFECTION AND

CERVICAL CANCER DEVELOPMENT

Pereira, A. P. L.1, Oliveira, K. B.

1

1State University of Londrina – Department of Pathological Sciences.


Keywords: Cervical cancer, Virus Diseases, Interleukin, Single Nucleotide Polymorphism.

Abstract

Cervical cancer (CC) is the third malignant tumor that affects women in Brazil and has one of the highest

incidences worldwide. Cervix squamous intraepithelial lesion (SIL) is caused by Human papillomavirus (HPV),

transmitted by sexual manner and it presents the initial bases for tumor progression. HPV’s genome can show many

genetics variations that leads to the formation of several types of virus. Therefore, HPV viruses are divided

considering its carcinogenic potential, into two categories, low-risk and high-risk. The vast majority of women will have

contact with the virus at some point of their sexually active life, the infection is a crucial factor that determines the

formation of low-grade squamous intraepithelial lesions (LSIL) and high-grade squamous intraepithelial lesions (HSIL)

if the lesions are left untreated, they can progress to cervical cancer (CC). The immune response for the viral infection

is critical to determine the characteristics of the infection and the persistence of the lesion. Interleukin-10 is described

as an immunoregulatory cytokine and has an important role in the inflammatory response triggered by the virus, with

an ambiguous profile playing both anti-inflammatory and pro-inflammatory roles. Since the polymorphism (c.-592 C>A)

modifies the IL-10 production, it can affect the immune response and determines cancer disclosure. Thereby, the aim

of this work is to understand better the role of IL-10 on CC, associating the presence of the rs1800872 polymorphism

(c.-592 C>A), detected by PCR-RFLP to CC development.

Hypothesis

The IL-10 (c.-592 C>A) polymorphism is associated with CC development.

Introduction

CC is the third among the neoplasias in Brazil that affects women and the latest estimate of CC for the year

2016, it estimated 16,000 new cases according to the National Cancer Institute (NICA).

Cervix squamous intraepithelial lesion (SIL) are the precursor of CC and it is classified as low-grade

squamous intraepithelial lesions (LSIL) and high-grade squamous intraepithelial lesions (HSIL), about 70% of LSIL

has a transitory profile and can spontaneous regress clearing the lesion [1–3].

The infection of human papillomavirus (HPV) is described by the literature as the primary factor that leads to

CC, being responsible for the development of LSIL and HSIL. HPV is the most common sexually transmitted disease

(STD) worldwide and about 80% of sexually active women will be exposed to HPV [4–6].

More than 200 types of HPV have been identified and approximately 150 HPV types have been sequenced

and they are classified by their carcinogenic potential, low risk (LR-HPV) and high risk (HR-HPV). The vast majority of

LR-HPV infections are caused by types 6 and 11, responsible for 90% of genital warts and for the HR-HPV types 16 e

18 they are related to 70% of the CC cases [7–9].

Although the infection by HPV shows a high incidence, only 5% of HSIL cases will lead to CC, thus only the

presence of HPV is not enough for the infection persistence and cancer development [10].

The profile of HPV infection and its outcome are correlated with a number of behavioral factors, such as age

at onset of sexual life, number of sexual partners during lifetime, that may increase exposure to the virus and non-use

of barrier contraceptives, such as male condom that prevents the contact with the infected region. Individual habits like

smoking and sedentary lifestyle, stress, viral type and also genetic factors act together by modeling the immune

response [11,12].

Therefore, the immune response is a determining factor for the establishment of the disease, its regression or

persistence. In this context it is known that Interleukine-10 (IL-10) is important for the inflammatory response triggered

by the virus and has an ambiguous profile playing both anti-inflammatory and pro-inflammatory roles, featuring an

immunoregulatory cytokine [13].

IL-10 is produced and secreted by several cell types including macrophages, dendritic cells (DC), B

lymphocytes, regulatory T lymphocytes (Treg), T lymphocytes CD8+ and CD4+ and natural killer (NK). It was first

26

isolated in the 90’s and was described as a synthesis factor inhibitory cytokine (CSIF), because of its ability to inhibit

Th1 profile lymphocyte response, decreasing T cell proliferation, and also inhibiting antigen presenting cells (APC's)

by regulating various cytokines involved in the defense signaling cascade [14,15].

The immune response in viral infections involves both cellular (Th1) and humoral (Th2) immune responses,

studies on the HPV immune response demonstrate that the infection leads to a Th1 response followed by soon

diminished and the Th2 response prevails, consequently, this way IL-10 can be associated with an evasion

mechanism for the virus by shutting the response to Th2 [16,17].

The polymorphism rs1800872 (c.-592 C>A) is the object of study of the present work, consists of the

exchange of nucleotide cytosine (C) by adenine (A) at the position -592 of the IL-10 gene. This polymorphism has

been associated with a higher serum concentration of IL-10 and may be a key factor for the progression of tumors or

even playing protective function [11,18,19].

Objectives

The main objective of this work is to evaluate the single nucleotide polymorphism of IL-10 rs1800872 (c.-592

C>A), localized on the promoter region of IL-10’s gene, influence in CC. As well as to analyze sociodemographic data,

clinical diagnosis and sexual/reproductive features that could be interacting with CC.


This study was approved by the Research Ethics Committee Involving Human Beings – State University of

Londrina, number 133/2012, which is in agreement with the National Commission of Ethics in Research, resolution

196/96 - National Health Council (CNS), as authorized by the Municipal Health Authority of the State of Paraná, CP

14/2012/GES/MAS/PML.

A sociodemographic questionnaire will be applied for those women who agree to participate in this project, in

order to gather data about individual habits such as age, marital status, schooling level, smoking status and

reproductive life style.

The samples will be obtained from Cancer Hospital of Londrina, Basic Unit Health Dr. Justiniano Clímaco da

Silva and Dr. Paulo Roberto Moita da Silva. The DNA will be extracted from cancer biopsy samples and mononuclear

cells in the peripheral blood.

The IL-10 gene polymorphism analysis will be performed by Polymerase Chain Reaction (PCR) followed by

Restriction Fragment Length Polymorphism (RFLP) technique using the restriction enzyme RsaI.

The frequency of allele A will be calculated as: [1x(h+2H)]/2N, where h represents the heterozygous genotype,

H the homozygous genotype and N the sample size for each population. The Hardy-Weinberg equilibrium will be

determined by the Chi-Square Test ( 2). The genotypic distribution between the groups studied will be analyzed by

the 2 test and the Odds Ratio (OR) with a 95% confidence interval (CI).

For the correlation analyzes between clinicopathological data, the Spearman Rho or Kendall tau correlation

tests will be used. Binary logistic regression analysis, adjusted for several confounders will be used to estimate the

association between the polymorphism and the CC development. All statistical analyzes will be performed using

SPSS Statistics 22.0 (SPSS inc., Chicago, Illinois, USA) with significance level set as p<0.05.

Expected Results

The results are expected to provide a better understanding of the role of IL-10 polymorphism rs1800872 (c.-

592 C>A) in the immunopathogenesis of human papillomavirus infection disclosing in CC.

Acknowledgments

To the State University of Londrina and the financial support from Fundação Capes, Fundação Araucária,

CNPq and the Basic Unit Health Care of Dr. Justiniano Clímaco da Silva and Dr. Paulo Roberto Moita da Silva and

Cancer Hospital for provide the samples.

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cancer, Lancet. 56370 (2007) 890–970. doi:10.2222/jsv.56.219.

[2] S. Shrestha, C. Wang, B. Aissani, C.M. Wilson, J. Tang, R.A. Kaslow, Interleukin-10 gene (IL10)

27

polymorphisms and human papillomavirus clearance among immunosuppressed adolescents, Cancer

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polyadenylation., Nat. Rev. Microbiol. 11 (2013) 239–51. doi:10.1038/nrmicro2984.

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E.M.B. de Albuquerque, M.T.C. Muniz, A.C. de Freitas, An interleukin-10 gene polymorphism associated with

the development of cervical lesions in women infected with Human Papillomavirus and using oral

contraceptives, Infect. Genet. Evol. 19 (2013) 32–37. doi:10.1016/j.meegid.2013.06.016.

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[13] K. Zhang, L. Zhang, X. Wang, L. Zhang, The IL-10 promoter haplotype and cancer risk: Evidence from a meta-

analysis, Fam. Cancer. 11 (2012) 313–319. doi:10.1007/s10689-012-9533-7.

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cancer, Cancer Lett. 367 (2015) 103–107. doi:10.1016/j.canlet.2015.07.009.

[15] S. Pestka, C.D. Krause, D. Sarkar, M.R. Walter, Y. Shi, P.B. Fisher, Interleukine-10 and Related Cytokines and

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[16] A.D. Santin, P.L. Hermonat, A. Ravaggi, S. Bellone, S. Pecorelli, J.J. Roman, G.P. Parham, M.J. Cannon,

Interleukin-10 increases Th1 cytokine production and cytotoxic potential in human papillomavirus-specific

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[17] S. Syrjänen, P. Naud, L. Sarian, S. Derchain, C. Roteli-Martins, A. Longatto-Filho, S. Tatti, M. Branca, M.

Eržen, L.S. Hammes, S. Costa, K. Syrjänen, Immunosuppressive cytokine Interleukin-10 (IL-10) is up-

regulated in high-grade CIN but not associated with high-risk human papillomavirus (HPV) at baseline,

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doi:10.1007/s00428-009-0850-7.

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doi:10.1073/pnas.95.16.9465.

28

ANTITUMORAL ACTION OF THIOHYDANTOINS ON CELLS OF CARCINOMA LUNG

Concato, V. M.

1, Pavanelli, W. R.

1

1State University of Londrina / Department of Pathological Sciences.


Keywords: lung cancer, carcinoma, A549 cell, thiohydantoin, cytotoxicity.

Abstract

Lung cancer is considered a malignant disease whose onset is through the airways or in lung parenchyma.

The treatment of lung cancer is based on the use of gemcitabine, carboplatin, cisplatin, irinotecan, or etoposide,

however they present high toxicity and difficulty in administration. In the face of these adversities, it is evident that the

treatment for this disease requires the discovery of new therapies that are less toxic to the host and which have

antitumor activity. Thiohydantoins are synthetic compounds, sulfur analogues of the hydantoin group with one or both

carbonyl groups. They are characterized by antimutagenic, anticarcinogenic, antioxidant and antiviral activity. Thus,

this work aims to evaluate the anticarcinogenic activity on cells of lung carcinoma, as well as to elucidate the

mechanisms of death involved.

Hypothesis

Thiohidantoins exhibit antitumor activity and induce death in the human lung epithelial carcinoma cell line,

A549.

Introduction

Cancer is a complex chronic disease characterized by abnormal cell growth, with the ability to invade adjacent

and distant tissues. This disease is related to a variety of cellular and molecular mechanisms associated with initiation

and progression (1,2).

Lung cancer is one of the most common causes of mortality among all cancers(3) represented by

malignancies of origin in the airways or pulmonary parenchyma (4) and in the literature is divided into two major

subtypes: Non-Small Cell Lung Cancer (NSCLC) and Small Cell Lung cancer (SCLC) (3, 5, 6).

The current chemotherapy treatment is done by the administration of drug combination, the most commonly

used are gemcitabine, carboplatin, cisplatin, irinotecan and etoposide. However, these drugs cause serious side

effects, which can lead to death and have a low response, hindering the effectiveness of current treatments (7,8,9).

Therefore, new compounds for the treatment of lung cancer are still required research target.

In this context, thiohidantoins, which are synthetic compounds, are sulfur analogs of the hydantoin group with

one or both carbonyl groups substituted by thiocarbonyl groups. They can be synthesized through amino acid

molecules or coupled molecules such as amino esters and thiocyanate.

The yield may be variable depending on the amino acid used, ranging from 20 to 90% (10). The thiohidantoins

are characterized by their diverse biological action, such as anti-viral activity (11), antiviral against herpes simplex

virus (HSV) (12), antimutagenic (13), anticarcinogenic (14), antimalarial (15) and leishmanicide ( L. donovani) (16),

showing no cytotoxicity on mammalian cells J774 (16) and HepG2 (17) and Vero (18) cell lines. However, the

anticarcinogenic effects in lung epithelial carcinoma are not known yet.

Objectives

To identify among 12 thiohidantoins which are most effective in relation to the antineoplastic activity and to

evaluate the mechanisms involved in death of human lung epithelial carcinoma cell line.


Cell culture

The lung carcinoma cell line A549 will be cultivated in culture flasks with Dulbeco's Modified Eagle's Medium

(DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% mixture of penicillin/ streptomycin

(St. Cruz). The culture will be mantained in the incubator (Sanyo, Japan) humidified at 37 °C with 5% CO2. Tests for

cytotoxicity and genotoxicity proliferation were performed in 24 well plates with 2x105 cells / well and maintained in the

incubator for 24 hours for adhesion and cell stabilization.

mailto:[email protected]

29

Activity of the thiohidantoin compounds on A549 cells

The A549 cell line will be treated with the thiohidantoin synthetic compounds for 24, 48 and 72 hours. The cell

viability of these cells will be evaluated by means of the MTT (3-(4,5-dimethilthiazol-2yl)-2,5-diphenyltetrazólium

bromide) (Sigma, ST, Louis, MO,USA) assay, using DMEN-F12 (Eagle Modificado de Dulbeco/ Nutrient Mixture F-12)

as a control, as a control of the vehicle, 0.01% DMSO (dimethil sulfoxide) (Sigma, ST.Louis, MO,USA) and as a

negative control the hydrogen peroxide (H2O2) will be used. For the chemotherapeutic control, we will use the

lyophilized drug Cisplatin (5 to 50 μM).

Cell proliferation and cell viability

After treatment, the cells will be washed with phosphate-saline solution (PBS), trypsinized, suspended in

Trypan blue (0.05%) and counted in Neubauer's chamber to verify the increase or decrease of cell proliferation. For

cell viability, a total of 300 cells will be counted, distinguishing non-viable cells (stained in blue).

Determination of the mechanism of death

A549 cells will be treated with thiohidantoins, washed with PBS and then incubated with specific markers for

cell membrane integrity analysis (propidium iodide, 0.50 μg / mL); Phosphatidylserine exposure (Annexin V FITC, 5 μl)

and membrane potential analysis (tetramethylrhodamine-ethyl ester, 25 nM). The readings will be performed through

a microplate fluorescence reader.

Kite Assay

The comet test evaluates the damage to the genetic material. After the treatments, the trypsinized cells will be

transferred to agarose slides, submitted to the lysis solution, to alkaline pH electrophoresis and analyzed by Gel Red

staining.

Measurement of malondialdehyde (MDA) levels

The MDA is the final product of lipid peroxidation and will be evaluated to determine the involvement of

oxidative stress. After treatment, the cells will be washed and trypsinized, centrifuged and the determination of the

levels will be done by the HPLC (High performance liquid chromatography) system equipped with an LC20AT pump,

an absorbance detector (SPM20A series of UV diodes) and the reverse phase C18 column. The results will be

expressed as MDA nM / g of the total protein.

Quantification of lactic dehydrogenase (LDH)

After treatment, the culture medium will be collected for the LDH assay. The LDH quantification kit will be to

measure the release of LDH by the damaged cell cytosol in the culture medium and to estimate membrane damage

associated with necrosis. The test is based on the reduction of NAD (nicotinamide adenine dinucleotide) by LDH.

Immunocytochemistry

The immunocytochemistry technique will be used to study signaling pathways involved in thiohidantoins

treatments with labeling molecules using the biotin-streptavidin method with the LSAB kit. The intensity will be

measured in pixels, which will be categorized as strong positive (3+), positive (2+), weak positive (1+) and negative

(0).

Flow cytometry

For this analysis, the cells treated with thiohidantoins will be centrifuged, washed with PBS and incubated with

ice cold ethyl alcohol (70%) for 24 h at -20 ° C. After incubation the cells will be washed again with PBS and incubated

with solutions containing ribonucleases. The analysis of the percentage of cells in sub-G1, G0 / G1, S, G2 / M and will

be performed in flow cytometry.

Autophagy test (Monodanzilcadaverina - MDC)

For this test, cells will be cultured on a circular slide, treated and incubated with MDC. The photomicrographs

will be used to calculate the percentage of cells containing cytoplasmic autophagic vacuoles.

30

Statistical analysis

For the comparison between the treated and control groups, the Student T-Test and Analysis of Variance

(Anova) will be used, followed by Bonferroni post-test (GraphPad-Prism V4 software). Therefore, values of p less than

0.05 (p <0.05) will be considered as significant difference.

Expected results

With this work thiohidantoins are expected to reduce cell proliferation through the induction of apoptotic

mechanisms.

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Molecular Biology. 2011; 106: 380-390.

2- White AC, Lowry WE. Defining the role for adult stem cells as cancer cells of origin. Trends Cell Biol. 2015; 25(1):

11–20.

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one. B. Int. J. Pha. Chem. Bio. Sci. 2014; 4(3), 447-452.

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and bis-imidazolidineiminothiones and imidazolidineiminodithiones with antitumor, antiviral, antibacterial and

antifungal activities – part I. Eur. J. Med. Chem. 2009; 44: 4315–4334,.

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class of antitumor agents. Anticancer Drugs, 1996; 7: 873-878,.

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Kumar, V. Discovery of highly selective 7-chloroquinoline-thiohydantoins with potent antimalarial activity. Eur. J.

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Farmaco. 2002; 57: 355–362.

31

DETECTION OF INFECTION BY Paracoccidioides brasiliensis IN NILE TILAPIA

Suguiura, I.M.S1, Ono, M. A.

1 1State University of Londrina / Department of Experimental Pathology.

E-mail [email protected]

Keywords: Paracoccidioidomycosis, epidemiology, fish, tilapia

Abstract

The thermo-dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccioidomycosis a deep

mycosis with high prevalence in Latin American countries. The infection by P. brasiliensis has been described in

several homeothermic species; however, the infection in ectothermic animals such as fish has never been reported.

The objective of this study was to evaluate the infection by P. brasiliensis in Nile tilapia (Oreochromis niloticus) raised

in cages from the Northern Paraná state, Brazil. In a previous (unpublished) study, we detected antibodies against P.

brasiliensis gp43 in tilapias from Northern Paraná state, suggesting the infection. Tissue samples (encephalon, liver

and kidney samples) from caged fish will be analyzed by PCR for P. brasiliensis (ITS region). In addition, juvenile

tilapias will be will be inoculated with P. brasiliensis (1x107 yeast cells) by intraperitoneal route and the course of

infection will be evaluated by molecular and immunological methods.

Hypothesis

Fish is susceptible to infection by Paracoccidioides brasiliensis.

Introduction

Paracoccidioides brasiliensis is a thermo-dimorphic that causes paracoccidioidomycosis (PCM), the most

prevalent systemic mycosis in Latin America. Argentina, Venezuela, Colombia and Brazil are endemic areas of PCM

showing a high prevalence of the disease (18).The fungus infects a wide variety of warm-blooded organisms ranging

from humans to domestic and wild animals, (2,3,4,7,8,9,10,13,14).

Since the first description of the fungus by Adolph Lutz, P. brasiliensis was isolated from soil in Brazil,

Argentina and Venezuela (19,12,1,20). The soil is the probable spot habitat of P. brasiliensis, however, some

researchers (5, 15) introduced the hypothesis of an aquatic habitat for the fungus.

Epidemiological data suggest that the fungus lives in very humid areas such as close to water courses and

regions with significant rainfall. In addition, the optimal ―in vitro‖ growth in high humid conditions suggests the

preference for a very humid microniche (16). In this way, the detection of the fungus in aquatic species and their

possible role in the P. brasiliensis eco-epidemiology may show new pieces of evidence about the fungus habitat.

Nile tilapia (Oreochromis niloticus) is the fish most farmed in Paraná state (11), an endemic area for PCM. Tilapia

farmers raise the fish basically in two different systems, in ponds that is a prepared hole in the ground and in cages

where the fish are kept in a wire or plastic cage in a water body, usually in a river or lake (17).

Objectives

To detect the infection by P. brasiliensis in Nile tilapia (Oreochromis niloticus).

To evaluate the susceptibility of Nile tilapia (Oreochromis niloticus) to P. brasiliensis experimental infection.


DNA Samples

100 DNA samples (liver n=33, encephalon n=32, kidney=35) were collected from caged tilapia from fish farms

located in the Paranapanema river basin in 2013, Paraná, Brazil; Approved by Animal Ethics Committee of the State

University of Londrina (CEUA nº 294/12). The samples will be processed according to Corredor et al. (6) with

modifications, using phenol and proteinase K (Sigma-Aldrich, St Louis, MO, USA).

Fish experimental infection

Juvenile tilapias (n=12) from a commercial fish farmer weighting 20 - 35 g will be sort in two groups of 6

individuals each, the first group will be infected with P. brasiliensis and the second will be inoculated with sterile PBS

(control). They will be kept in 50 L glass tanks under constant aeration and fed daily to satiation with commercial


32

tilapia feed. The fish will be anesthetized with eugenol (Biodinâmica, Ibiporã, Pr, Brazil) 75 mg L-1 by immersion and

inoculated by intraperitoneal route with P. brasiliensis B-339 (1x107) or sterile PBS on day 0. Both groups, P.

brasiliensis and PBS, will be observed daily to check any clinical signs of disease, such as anorexia, coloration, skin

lesions, abnormal swimming and death. On day 30 the fish will be euthanized and blood samples will be collected and

assayed by ELISA. Also, encephalon, liver and kidney will be collected and analyzed by PCR, histopathological exam

and culture.

Indirect ELISA with gp43 antigen

Polystyrene flat-bottom microtiter plates (Costar Corporation, Corning, NY, USA) will be coated with purified

P. brasiliensis gp43, in 0.1 M carbonate buffer, pH 9.6 (250 ng well-1) at 4 ºC overnight. The plates will be washed

PBS containing 0.1 % Tween (PBS-T) and blocked with PBS-T 5 % skim milk (PBS-T-M). After washing with PBS-T,

the serum samples will be diluted 1:100 in PBS 1 % skim milk (PBS-T-M) and incubated at 25 °C for 1 h. The plates

will be washed and incubated at 25 °C for 1 h with rabbit antibody anti-tilapia IgM. After washing will be incubated at

25 ºC for 1 hour with anti-rabbit IgG peroxidase conjugate (Sigma-Aldrich, St Louis, MO, USA). After washing with

PBS-T, a solution of substrate/chromogen (H2O2/ tetramethylbenzidine) will be added. The reaction will be stopped

with 4 N H2SO4. Absorbance will be measured at 450 nm. The serum samples with two and half times the

absorbance of the well without serum will be considered positive. The positive control will be the serum from a tilapia

immunized with P. brasiliensis.The statistical analysis will be performed with IBM SPSS Statistics 20 program and

data were analyzed by the Pearson chi-square test. The values of P ≤ 0.05 will be considered statistically significant.

Molecular analysis

PCR will be performed according to Theodoro et al. (21) with the primers Pb-ITSR (5’-

AAGGGTGTCGATCGAGAGAG-3’) and Pb-ITSE (5’-GAGCTTTGACGTCTGAGACC-3’) under the annealing

temperature of 62º C. The set of primers were designed to amplify a 387-bp region of the P. brasilienses DNA. The

reaction will be performed with GoTaq (Promega, Madison, WI, USA), using DNA from P. brasiliensis B-339 as a

positive control and ultrapure water as negative control. Electrophoresis will be performed in polyacrylamide gel with

silver nitrate staining. The products of positive samples will be purified with ammonium acetate and sequenced by the

Sanger method using Big Dye Terminator Sequencing Kits (Thermo Fisher Scientific, Waltham, MA, USA). The

sequencing analysis will be performed using the BioEdit program [31], and the sequence of nucleotides obtained will

be compared with the National Center database for Biotechnology Information (www.ncbi.nlm.nih.gov) [32] searched

through BLAST (http://blast.ncbi.nlm.nih.gov/).

Preliminary Results/ Expected Results

Unpublished data, from previous studies at our laboratory, showed presence of antibodies against P.

brasiliensis in tilapias from the two main farming conditions in the Northern Paraná state. Serum samples from tilapias

raised in ponds (n=105) and in cages (n=109) were analyzed by indirect ELISA using gp43 as antigen and a positivity

of 17.4% and 5.7% was observed in tilapias raised in cages and ponds, respectively. This result suggests that tilapias

can be infected by P. brasiliensis. A next step to corroborate this finding is to detect the fungus in fish tissues and to

evaluate the susceptibility of tilapia to development of paracoccidioidomycosis disease.

Acknowledgments

The authors thank the CNPq and Araucaria Foundation for financial support and CNPq for the productivity

fellowship granted to M. A. Ono.

References

1. Albornoz MB. Isolation of Paracoccidioides brasiliensis from rural soil in Venezuela. Sabouradia.1971; v. 9: 248-

253.

2. Belitardo DR, Calefi AS, Sbeghen MR, Oliveira GG, Watanabe MAE, Camargo ZP, et al. Paracoccidioides

brasiliensis infection in domestic rabbits (Oryctolagus cuniculus). Mycoses. 2014; v.57, no.4: 222-227.

3. Belitardo DR, Calefi AS, Borges IK, Oliveira GG, Sbeghen MR, Itano EN, et al. Detection of antibodies against

Paracoccidioides brasiliensis in free-range domestic pigs (Sus scrofa). Mycopathologia. 2014; v.177, no. 1-2: 91-95.

33

4. Bosco SMG, Theodoro RC, Macoris SAG, Farias MR, Muro M, Ribeiro MG, et al. Morphological and molecular

characterization of the first isolate of Paracoccidioides brasiliensis from dog (Canis familiaris). Rev Inst Med Trop

São Paulo. 2005; v.47(Suppl.14): 62–63.

5. Conti Días IA, Rilla FD. Hipótesis sobre el nicho ecológico de Paracoccidioides brasiliensis. Rev Méd Urug. 1989;

v.5: 97-103.

6. Corredor GG, Peralta LA, Castaño JH, Zuluaga JS, Henao B, Arango M, et al. The naked-tailed armadillo

Cabassous centralis (Miller 1899): a new host to Paracoccidioides brasiliensis. Molecular identification of the

isolate. Med Mycol. 2005; v.43, n. 3: 275-80.

7. Corte AC, Svoboda WK, Navarro IT, Freire RL, Malanski LS, Shiozawa MM, et al. Paracoccidioidomycosis in wild

monkeys from Paraná State, Brazil. Mycopathologia. 2007; v.164, n.5: 225–228.

8. Corte AC, Itano EN, Freire RL, Camargo ZP, Ono MA. Detection of antibodies to Paracoccidioides brasiliensis in

horses from northern Region of Paraná State. Semi Ciê Agrá. 2009; v.30, n.2: 441-446.

9. Ferreira JB, Navarro IT, Freire RL, Oliveira GG, Omori AM, Belitardo DR, et al. Evaluation of Paracoccidioides

brasiliensis Infection in Dairy Goats. Mycopathologia. 2013; v.176, n.1-2: 95-99.

10. Fontana FF, Santos CTB, Esteves FM, Rocha A, Fernandes GF, Amaral CC, et al. Seroepidemiological

survey of paracoccidioidomycosis infection among urban and rural dogs from Uberaba, Minas Gerais, Brazil.

Mycopathologia. 2010; v.169, no. 3: 159-165.

11. Lopera-Barrero NB, Ribeiro RP, Povh JA, Mendez LDV, Poveda-Parra AR, Digmayer M. Produção de

Organismos Aquáticos: Uma visão geral no Brasil e no Mundo. 1. ed. Guaíba: Agrolivros. 2011; 179-182.

12. NegroniPE. El Paracoccidioides brasiliensis vive saprofiticamente enel suelo Argentino. Pre Med Arge. 1966;

v. 53: 2831-2832.

13. Oliveira GG, Navarro IT, Freire RL, Belitardo DR, Silveira LH, Camargo ZP, et al.Serological Survey of

Paracoccidioidomycosis in Sheep. Mycopathologia. 2012; v.173, n.1: 63-68.

14. Oliveira GG, Silveira LH, Itano EN, Soares RM, Freire RL, Watanabe M. A, et al.Serological Evidence of

Paracoccidioides brasiliensis Infection in Chickens from Paraná and Mato Grosso do Sul States, Brazil.

Mycopathologia. 2011; v.171, n.3: 197-202.

15. Restrepo A. The ecology of Paracoccidioides brasiliensis: A puzzle still unsolved. Sabouraudia. 1985; v.23:

323–334.

16. Restrepo A, Mcewen JG, Castaneda E. The habitat of Paracoccidioides brasiliensis: how far from solving the

riddle? Med mycol. 2001; v.39, no.3: 233-241.

17. Rodrigues AP, Lima AF, Alves AL, Rosa DK, Torati LS, Dos Santos VRV.Psicultura de água doce -

multiplicando conhecimentos, Brasilia, DF, Brazil. 1ª ed. EMBRAPA. 2013.

18. San-BlasG, Niño-Vega G, Iturriaga T. Paracoccidioides brasiliensis and paracoccidioidomycosis: molecular

approaches to morphogenesis, diagnosis, epidemiology, taxonomy and genetics."Med mycol. 2002; 40.3: 225-242.

19. ShomeSK, Batista AC. Occurrence of Paracoccidioides brasiliensis in the soil of Recife, Brazil. Rev Fac Med

UniCeará. 1963; v. 3: 90 – 94.

20. Silva-Vergara ML,Martinez R, Chad A, Madeira M, Freitas-Silva G, Leite Maffei CM. Isolation of a

Paracoccidioides brasiliensis strain from the soil of a coffee plantation in Ibiá, State of Minas Gerais. Med Mycol.

1998; v. 36: p. 37-42.

21. Theodoro RC, Candeias JMG, Araújo JrJP, Bosco SDMG, Macoris SADG, Junior LOP et al. Molecular

detection of Paracoccidioides brasiliensis in soil.Med Mycol. 2005; v. 43: p 725 – 729.

34

EFFECT OF ASPIRIN-TRIGGERED LIPOXIN IN T. cruzi CELL INVASION

Suzukawa, H. T1, Bordignon, J², Pinge-Filho, P¹.

1Universidade Estadual de Londrina/Departamento de Ciências Patológicas

² Instituto Carlos Chagas, Fundação Oswaldo Cruz


Keywords: Trypanosoma cruzi, 15-epi-LXA4, THP-1 cells, Nitric oxide, Cytokines

Abstract

Chagas disease (CD) is an infection caused by the parasite Trypanosoma cruzi. This pathology is endemic in

Latin America and has become a public health issue in some non-endemic countries like the USA, Spain and

Australia. There is no curative treatment against CD. Non-steroidal anti-inflammatory drugs (NSAIDs), like aspirin

(ASA), have been assayed as drugs with therapeutic potential in CD, but the studies about this issue show

contradictory results; also, the mechanism involved in ASA effect is not yet clear. Therefore, we aimed to analyze the

effect of 15-epi-LXA4 (an ASA-triggered lipoxin) on T. cruzi entry into THP-1 monocytes differentiated in

macrophages.

Hypothesis

The effect of ASA has been associated, at least in part, to a metabolic switch towards a pathway linked to the

acetylation of the COX-2 isoenzyme. This acetylation enables COX-2 to synthetize other lipid products derived from

arachidonic acid (AA), some of them with anti-inflammatory properties. These metabolic products have been called

―ASA-triggered lipoxins‖ (ATLs). Correspondingly, ASA-triggered 15-epi-Lipoxin-A4 (15-epi-LXA4) has been described

as an anti-inflammatory lipid able to inhibit IL-6, TNF-α and IL-8 production, as well as NFκB, ERK1/2 and p38

activation. Our hypothesis is that ATLS are responsible for inhibiting the entry of T. cruzi into host cells that were

previously treated with ASA, as described by our group (1, 2).

Introduction

Chagas disease, caused by the protozoan Trypanosoma cruzi, affects around 7 million people worldwide and

has become a global health problem (3, 4). The innate immune response in the acute phase of infection is important

for the control of the para site load and survival’s host, especially by the action of phagocytes such as macrophages

(5).

Despite of activation of a potent immune response by the host, the acute phase of CD is characterized by

immunosuppression induced by T. cruzi to evade the host immune response. This immunosuppressive state is

mediated by prostaglandins (6-8), nitric oxide and cytokines (9), such as transforming growth factor-β (TGF-β) (10).

Increased circulating levels of prostaglandin E2 (PGE2) (11), thromboxane A2 (TXA2), and prostaglandin F2α (PGF2α)

have been reported in mice infected with T. cruzi (12), and during the acute phase, macrophages and spleen cells

from T. cruzi-infected mice produce high levels of PGE2 (11). Thus, the inhibition of the host cyclooxygenase (COX)

enzyme emerges as a potential therapeutic target. In fact, in infected BALB/c mice, treatment with ASA, indomethacin

decreases parasitemia and delays mortality(13) However, some gaps remains in the literature data (14), revised by

Machado and collaborators(15).

Lipids are involved in the initiation and resolution of inflammation. Pro-inflammatory lipid species originate

mostly from the omega-6 polyunsaturated fatty acid arachidonic acid which is released from the membrane by the

activity of phospholipases. COX-1 and 2 use polyunsaturated fatty acids (PUFAs) released from the membrane by the

enzyme phospholipase A as a substrate for the production of prostanoids, such as thromboxanes and prostaglandins.

ASA and nimesulide are drugs that inhibits COX to synthetize lipid products derived from arachidonic acid

(AA), some of them with anti-inflammatory properties. Nimesulide acts by binding to the catalytic site of COX-2,

preventing the arachidonic acid from enzyme action and production of lipid mediators. The effect of ASA has been

associated, at least in part, to a metabolic switch towards a pathway linked to the acetylation of the COX-2 isoenzyme.

This acetylation enables COX-2 to synthetize other lipid products derived from AA, some of them with anti-

inflammatory properties. These metabolic products have been called ―ASA-triggered lipoxins‖ (ATLs).

Correspondingly, ASA-triggered 15-epi-Lipoxin-A4 (15-epi-LXA4) has been described as an anti-inflammatory lipid

able to inhibit IL-6, TNF-α and IL-8 production, as well as NFκB, ERK1/2 and p38 activation. However, lipoxin A4

(LXA4) was originally found as an anti-inflammatory and pro-resolution eicosanoid, and the anti-inflammatory effects of


35

LXA4 have been observed in various types of inflammatory diseases. However, it has not been analyzed the

protective role of ASA in human macrophages relating to the production of 15-epi-LXA4.

Objectives

The objective of this work will be to investigate the effect of ATLs, PUFAs and the synergism of treatment of

nimesulide on the entry of T. cruzi into THP-1 cells differentiated in macrophages at 2h, 6h, 12h, 24h and 48h post

infection. We will do also the quantification of the production of prostaglandin E2, IL-1β and nitric oxide (NO) and 15-

epi-LXA4 in THP-1 macrophages treated or not with ATLS, PUFAs, ASA and nimesulide infected or not with T. cruzi (Y

strain)


Ethics statement:

Ethical clearance for the maintenance and experimentation with the parasite was obtained from the Internal

Scientific Commission and the Ethics in Animal Experimentation Committee of State University of Londrina (Process

number: 24841.2016.41).

Generation of human macrophages

THP-1 monocytes (ATCC TIB-202) (a kind gift from Juliano Bordignon at Fiocruz-ICC-Pr) will be propagated

in RPMI media (Gibco) containing 10% FBS (Gibco) and 100 U/ml penicillin (Gibco) and 100 mg/ml streptomycin

(Gibco). To generate THP-1 macrophages, monocyte cultures will be treated with of phorbol 12-myristate 13-acetate

(Sigma-Aldrich) (1 µg/ml PMA per 106 cells) in complete RPMI media for 72h, will wash, and reste for 5 days before

their use in experiments (16). The phenotypic identity will be confirmed using cell markers: CD11b, CD14, HLA-DR,

CD206, CD86, CD80 and isotypic controls.

Parasites

Trypomastigotes of the T. cruzi (Tc II genotype, Y strain) will be obtained from the supernatant of T. cruzi-

infected Rhesus Monkey Kidney Epithelial Cells (LLC-MK2) culture cells (ATCC CCL-7; American Type Culture

Collection, Rockville, MD) grown in RPMI 1640 medium containing 10% inactivated fetal bovine serum (FBS), 40 µg

ml-1 gentamicin, 100 units penicillin ml-1, 100 µg ml-1 streptomycin. Subconfluent cultures of LLC-MK2 will be

infected with 5 × 106 trypomastigotes. Free parasites will be removed after 24 h and cultures were maintained in 10%

FBS-RPMI 1640. Five days following infection, free trypomastigote forms could be found in the cell supernatants for

use.

Treatments and infection

THP-1 macrophages (1×106/mL) will be incubated in RPMI 1640 (Gibco, BRL) medium supplemented with

10% inactivated fetal bovine serum (FBS, Gibco, BRL), 2 mM L-glutamine and antibiotics (penicillin [100 U/mL] and

streptomycin [100 µg/mL]) incubator overnight at 37°C and 5% CO2. The non-adherent cells were then discarded and

the remaining adherent cells will be washed extensively with warm phosphate-buffered saline (PBS) before the

interaction assays. In addition, 2.0 × 105 cells will be cultured on 48-well dishes. One set of plates was used to

quantify, PGE2, IL-1β and 15-epi-LXA4 and the other set for NO detection. Before T. cruzi infection, THP-1

macrophages will be incubated for 1 h at 37°C in a 5% CO2 atmosphere in the presence of ASA (1.25 mM) (2), 15-

epi-LXA4 (0-300nM) (17), PUFAS (Cayman Chemicals) (0-100µM) and nimesulide (0 to 1.27 µM). After incubation, the

medium containing drugs will be removed and cells will be allowed to interact with trypomastigote forms added in a

ratio of 5 parasites per cell for 48h.

Cell viability assay

The viability of the cells obtained from the cultures before and after incubation, was determined using MTT

(Sigma-Aldrich) assay, showing the mitochondrial activity of living cells. Cells will be incubated with MTT (final

concentration 0.5 mg/mL, 0.1%) at 37°C for 4 h. The supernatant will be aspirated and DMSO (Cayman Chemicals)

will be added. Insoluble crystals were dissolved by mixing and the plates will be read using a BioRad multiplate reader

(Hercules, CA), at a test wavelength of 570 nm and a reference wavelength of 630 nm. The cell viability percentage

will be calculated using the formula: % cell viability = (mean absorbency in test wells/mean absorbency in control

wells) × 100.

36

Elisa

Cells that were either uninfected or infected, treated or not with different drugs, will be incubated at 37°C for 2-

48h. PGE2 and 15-epi-LXA4 concentration in the supernatant will be assessed by competitive ELISA (Cayman,

Chemical, USA), according to the manufacturer protocol. Levels of IL-1β in 100 µL medium will be measured by

commercial ELISA kits (Ready-SET-Go! eBioscience, San Diego, CA), according to the manufacturer instructions.

Nitric oxide

Production of NO will be determined by measuring the level of accumulated nitrite, a metaboli te of NO, in the

culture supernatant using Griess reagent (Sigma-Aldrich). After 24-48h treatment with drugs, the culture supernatant

will be collected and mixed with an equal volume of Griess reagent in 96-well culture plates, and incubated at room

temperature for 10 min. The absorbance will be measured at 540 nm and nitrite concentrations will be calculated

using a standard curve for sodium nitrite.


Statistical analysis will be performed using analysis of variance (ANOVA) followed by a Bonferroni’s multiple

comparison test. Data will be expressed as mean ± standard error of the mean. The results will be considered

significant when p < 0.05. Statistical analysis will be performed using the GraphPad Prism 5.0 software (GraphPad

Software, San Diego, CA, USA).

Figure 1 – Summary of material and methods

Figure 1 – Summary of material and methods. Macrophage differentiate from THP1 cells

will be the subject of cytotoxicity assay (MTT), internalization assay (2h, 6h, 12h, 24h, and

48h infection) and supernatant measurement of IL-1β, PGE2 and 15-epi-LXA4 by ELISA.

Expected Results

Cell invasion by T. cruzi and its intracellular replication are essential for progression of the parasite life cycle

and development of Chagas disease. Prostaglandin E2 (PGE₂) and other eicosanoids potently modulate host

response and contribute to Chagas disease progression. In this study, we wil be evaluated the effect of ATLs and

nimesulide, on the T. cruzi invasion and its influence on nitric oxide and cytokine production in human macrophages.

We hope to demonstrate that 15-epi-LXA4 plays a fundamental role in the process of parasite invasion in an in vitro

model of T. cruzi infection.

References

1. Malvezi AD, Panis C, da Silva RV, Carvalho de Freitas R, Martins MI, Tatakihara VL, et al. Inhibition of cyclooxygenase-1

and cyclooxygenase-2 impairs Trypanosoma cruzi entry in cardiac cell and promotes differential modulation of inflammatory

response. Antimicrobial agents and chemotherapy. 2014.

2. Malvezi AD, da Silva RV, Panis C, Yamauchi LM, Lovo-Martins MI, Zanluqui NG, et al. Aspirin modulates innate

inflammatory response and inhibits the entry of Trypanosoma cruzi in mouse peritoneal macrophages. Mediators of inflammation.

2014;2014:580919.

3. Bern C. Chagas' Disease. The New England journal of medicine. 2015;373(19):1882.

37

4. World Health Organization . World Health Organization: Chagas Disease. Available at

http://www.who.int/mediacentre/factsheets/fs340/en/. 2017.

5. Aliberti JC, Cardoso MA, Martins GA, Gazzinelli RT, Vieira LQ, Silva JS. Interleukin-12 mediates resistance to

Trypanosoma cruzi in mice and is produced by murine macrophages in response to live trypomastigotes. Infection and immunity.

1996;64(6):1961-7.

6. Abdalla GK, Faria GE, Silva KT, Castro EC, Reis MA, Michelin MA. Trypanosoma cruzi: the role of PGE2 in immune

response during the acute phase of experimental infection. Experimental parasitology. 2008;118(4):514-21.

7. Pinge-Filho P, Tadokoro CE, Abrahamsohn IA. Prostaglandins mediate suppression of lymphocyte proliferation and

cytokine synthesis in acute Trypanosoma cruzi infection. Cellular immunology. 1999;193(1):90-8.

8. Michelin MA, Silva JS, Cunha FQ. Inducible cyclooxygenase released prostaglandin mediates immunosuppression in

acute phase of experimental Trypanosoma cruzi infection. Experimental parasitology. 2005;111(2):71-9.

9. Abrahamsohn IA, Coffman RL. Cytokine and nitric oxide regulation of the immunosuppression in Trypanosoma cruzi

infection. Journal of immunology. 1995;155(8):3955-63.

10. Li MO, Wan YY, Sanjabi S, Robertson AK, Flavell RA. Transforming growth factor-beta regulation of immune responses.

Annu Rev Immunol. 2006;24:99-146.

11. Borges MM, Kloetzel JK, Andrade HF, Jr., Tadokoro CE, Pinge-Filho P, Abrahamsohn I. Prostaglandin and nitric oxide

regulate TNF-alpha production during Trypanosoma cruzi infection. Immunology letters. 1998;63(1):1-8.

12. Cardoni RL, Antunez MI. Circulating levels of cyclooxygenase metabolites in experimental Trypanosoma cruzi infections.

Mediators of inflammation. 2004;13(4):235-40.

13. Freire-de-Lima CG, Nascimento DO, Soares MB, Bozza PT, Castro-Faria-Neto HC, de Mello FG, et al. Uptake of

apoptotic cells drives the growth of a pathogenic trypanosome in macrophages. Nature. 2000;403(6766):199-203.

14. Hideko Tatakihara VL, Cecchini R, Borges CL, Malvezi AD, Graca-de Souza VK, Yamada-Ogatta SF, et al. Effects of

cyclooxygenase inhibitors on parasite burden, anemia and oxidative stress in murine Trypanosoma cruzi infection. FEMS

immunology and medical microbiology. 2008;52(1):47-58.

15. Machado FS, Mukherjee S, Weiss LM, Tanowitz HB, Ashton AW. Bioactive lipids in Trypanosoma cruzi infection.

Advances in parasitology. 2011;76:1-31.

16. Castaneda-Sanchez JI, Dominguez-Martinez DA, Olivar-Espinosa N, Garcia-Perez BE, Lorono-Pino MA, Luna-Herrera J,

et al. Expression of Antimicrobial Peptides in Human Monocytic Cells and Neutrophils in Response to Dengue Virus Type 2.

Intervirology. 2016;59(1):8-19.

17. Kure I, Nishiumi S, Nishitani Y, Tanoue T, Ishida T, Mizuno M, et al. Lipoxin A(4) reduces lipopolysaccharide-induced

inflammation in macrophages and intestinal epithelial cells through inhibition of nuclear factor-kappaB activation. The Journal of

pharmacology and experimental therapeutics. 2010;332(2):541-8.

http://www.who.int/mediacentre/factsheets/fs340/en/

38

EFFECTS OF PHOTOTHERAPY DURING CHRONIC EXPERIMENTAL COLITIS INDUCED BY

DEXTRAN SULFATE SODIUM (DSS) IN MICE

Santos, T. S 1, Araújo, E. J. A.1

1State University of Londrina/ Department of Histology, Brazil


Keywords: Phototherapy, Colitis, Enteric Nervous System.

Abstract

Inflammatory bowel Diseases (IBD) represent one of the main concerns the gastroenterology currently. Colitis is one

of the forms of IBD, when patients demonstrate diarrhea and rectal bleeding, abdominal pain, severe loss of appetite

and weight, and risk of colorectal carcinoma. Only palliative treatments by means of nutritional therapy, medical and/or

surgical are available. Phototherapy (above 800 nm) has been presenting relevant results in the treatment of injured

and inflamed tissues, because of its potential anti-inflammatory and healing. Therefore, this project aims to deepen

the knowledge about the therapeutic potential of the use of light emitting diodes (LED) in the infrared range (940 nm)

on an experimental model of development of chronic colitis. For that, 156 C57bl/6 mice will be distributed into six

groups: control, colitis with no treatment and colitis treated with LED therapy and/or mesalamine. Induction of chronic

colitis will be done by providing 3% dextran sulfate sodium (DSS) dissolved in the drinking water for seven days, then

a recovery during 28 days, when the mice will be treated. The application of light will occur once a week, performed

with LED equipment during 5 minutes per application. The distal colon of each mouse will be evaluated considering

molecular, morphological and functional parameters. It will expected that the LED therapy promote mitigation motility

changes usually caused by DSS-induced colitis of mice. Besides, the molecular, morphological, and functional

analysis will provide evidence of the mechanism of action of LED therapy.

Hypothesis

The phototherapy at 940nm reduces DSS-induced intestinal inflammation in mice and restores the intestinal motility.

Introduction

Inflammatory bowel Diseases (IBD) represent one of the main concerns of gastroenterology, because the

number of patients has increased. Besides, IBD reach individuals of different ages and gender, with frequent

recurrences and can manifest clinical forms of high gravity (1, 2).

IBD are a group of chronic inflammatory diseases of unknown cause, which affect the gut. They can be

divided into two main groups: Crohn's disease and colitis (2). Colitis presents an incidence of 3-20 new cases per year

for every 100,000 inhabitants in the world, showing greater predominance in Europe and North America. It is

considered that the lower incidence in other parts of the world might be related to technical problems of diagnosis of

disease (3, 4).

Colitis patients demonstrate diarrhea and rectal bleeding, abdominal pain, severe loss of appetite and weight,

and risk of colorectal carcinoma (5). There are only palliative treatments currently by means of nutritional therapy,

medical and/or surgical (6). Besides, all drugs available have several side effects. Therefore, it is still required the

development of new effective treatment with less side effects (7).

In this sense, phototherapy has been studied. Light with wavelength under 800 nm has the tendency to trigger

pro-inflammatory stimuli and the opposite occurs using light with wavelength over 800 nm. It has been suggested that

light close to limit of the infrared (1000 nm) presents greater tendency to produce anti-inflammatory stimuli (8).

LASERS or light emitting diodes (LED) can produce light. Both can be used, but in specific situations. For the

treatment of injuries, it is given preference to the use of LED (LED therapy) due two factors: the lower cost of

manufacturing of light-emitting equipment and also because the light produced by LED does not stimulate the

agitation of water molecules (cold light). Therefore, does not damage the tissue by increasing temperature (8, 9).

The LED therapy with wavelengths over 800 nm has been studied in different experimental models and

demonstrated effectiveness in reducing edema, migration of inflammatory cells, decreased production of cytokines

pro-inflammatory and improvement of the tissue regeneration (10, 11, 12). Because of this, the LED therapy has been

employed in the treatment of injured and inflamed tissues and offers great results towards to be used as anti -

inflammatory and healing (13, 8, 12).


39

A recent study published by the research group showed that the use of LED therapy in acetic acid-induced

colitis mice was effective to control the inflammatory process, reducing edema, pro-Inflammatory Cytokines (IL-1β,

TNF-α and IL-6), inflammatory infiltrate and a score of macroscopic and microscopic lesions. In addition, this same

study showed that colitis mice had delayed gastrointestinal transit, which was mitigated by the LED therapy (14).

Although the experimental model of colitis induction by administration of acetic acid is effective to produce an

acute inflammatory process, the colonic tissue demonstrates relatively distant histological characteristics of the

findings in colitis patients (14). Therefore, new phototherapy tests have to be carried out in other experimental models

more similar to what occurs in humans (15). In this sense, the literature consider that dextran sulfate sodium (DSS)-

induced chronic colitis is the experimental model closer to the colitis of humans (16).

Objectives

The goal of this project is to evaluate the anti-inflammatory potential of light with wavelengths in the infrared

range (near 940 nm) on DSS-induced chronic colitis in mice.


All the procedures detailed below have been approved by the Ethical Committee on use of Animals at the

State University of Londrina (UEL/CEUA), protocol number 4023.2017.31.

It will be used 156 C57Bl/6 60 days old male mice (Mus musculus) distributed into six groups: 1) control -

healthy mice with no treatment, 2) control LED - healthy mice treated with LED, 3) colitis – colitis mice with RCU no

treatment, 4) colitis LED - colitis mice treated with LED, 5) colitis mesalamine – colitis mice treated with mesalamine;

6) colitis LED+ mesalamine - colitis mice treated with LED and mesalamine. Colitis is induced by the administration of

3% (w/v) DSS (MW 36-45 kDa) dissolved in the drinking water during the first 7 days of the experiment, then the

animals will receive pure water for 28 days. The light treatment will be done with LED producing light in the infrared

(940nm), 45 nm bandwidth, density of 9.5 mW/cm2 power and intensity of 4.0 energy J/cm

2 for 5 minutes per

application once a week after DSS administration.

The animals will be subjected to euthanasia (ketamine + xylazine) followed by decapitation after 35 days of

experiment.

It will be assessed the disease activity index (DAI), based on weight loss, diarrhea accompanied by blood and

shortening of the colon. The DAI will be calculated per score of loss of weigh, diarrhea and rectal bleeding. Weight

loss will be defined as the difference between initial and final weight; diarrhea as the absence of formation of pellets

fecal and the presence of fecal material continuous fluid in the colon; and the rectal bleeding will be evaluated based

on the presence of diarrhea containing blood visible and in the presence of gross rectal bleeding. The values of DAI

will be calculated as {(weight loss score) + (diarrhea index) + (rectal bleeding index)}/4 (17, 4).

The intestinal transit will be evaluated during the 48 hours before the euthanasia of eight mice from each

group. It will be administered 0.5 mL of a non-absorbable marker (6% carmine red + 0.5% methylcellulose) orally. The

mice will be placed in individual cages, with access to food and water ad libitum. The gastrointestinal transit will be

expressed as the time of the appearance of first fecal pellet red.

Macroscopic lesions will be evaluated after euthanasia. The colon will be exposed, open lengthwise and

scores of injury will be determined in accordance with the findings described by Morris et al. (18).

The profile inflammatory will be evaluated by means of determination of cytokines TNF-α, IL-1β, IL-6, IL-10

and IL-12. The colonic tissue samples will be collected and homogenized in sterile saline, centrifuged (3000 rpm, 4° C,

10 minutes) and, then, the supernatant will be used to assess the levels of cytokines by ELISA (enzyme-linked

immunosorbent assay), using commercial kits and performed according to the manufacturer's instructions.

In addition, the analysis of the morphology of the intestinal wall, including the myenteric plexus, and the

colonic motility will be evaluated based on protocols used by the Neurogastroenterology research group at the UEL.

Microscopic lesions are will be evaluated in HE sections, considering microscopic parameters as suggested by

Appleyard et all. (19).

For the morphological analysis of the myenteric plexus, colons will be washed in PBS 0.1M pH7 .4. Then, will

be fixed at 4% parafolmaldehyde buffered with PBS for 3 hours at room temperature. After removing the fixative

solution, colon rings will open at the edge mesocolic to be microdissected aiming to remove the mucosa and

submucosa. Whole mounts containing the muscle layer and the myenteric plexus will be submitted to

immunofluorescence technique for analysis of cell body of myenteric neurons (PGP 9.5, ChAT and nNOS), neuronal

extensions (substance P and VIP) and evaluation of Interstitial Cells of Cajal (ICC).

40

Expected results

The phototherapy possibly acts as an anti-inflammatory, decreasing inflammatory parameters such as

macroscopic, microscopic lesions and concentration of pro-inflammatory cytokines, as well as colonic intestinal

motility recovering, preserving the enteric nervous system in a model of chronic colitis induced by DSS.

References

1- Hanauer SB. Inflammatory bowel disease revisited SB.: newer drugs. Scandinavian journal of gastroenterology.

1990: 25 (175): 97-106.

2- Kwon HK, Murakami, Tanaka T, Ohigashi H. Dietary rutin, but not its aglycone form quercetin, ameliorates dextran

sulfate sodium-induced experimental colitis in mice: attenuation of pro-inflammatory gene expression. Biochemical

pharmacology. 2005: 69 (3): 395-406.

3- Baumgart DC. The diagnosis and treatment of crohn's disease and ulcerative colitis. Deutsches ärzteblatt

international. 2009: 106 (8): 123-133.

4- Hendrickson BA, Gokhale R, Cho, JH. Clinical aspects and pathophysiology of inflammatory bowel disease. Clin

microbiol Rev. 2002: 15 (1): 79-94.

5- Dubeau MF, Lacucci H, Beck PL, Moran GW, Kaplan GG, Ghosh S, Panaccione R. Drug-induced inflammatory

bowel disease and ibd-like conditions. Inflamm bowel dis. 2013: 19 (2): 445-56.

6- Burger D, Travis S. Conventional medical management of inflammatory bowel disease. Gastroenterology. 2011:

140 (6): 1827-1837.

7- Guazelli CFS, Fattori V, Colombo BB, Georgetti SR, Vicentini FTMC, Casagrande R, Baracat MM, Verri Jr EWA.

Quercetin-loaded microcapsules ameliorate experimental colitis in mice by anti-inflammatory and antioxidant

mechanisms. Journal of natural products. 2013: 76 (1): 200-208.

8- Desmet KD, Paz DA , Corry JJ , Eells JT , Wong-Riley MT , Henry MM , Buchmann EV, Connelly MP , Dovi JV ,

Liang HL, Henshel DS , Yeager RL , Millsap DS , Lim J , Gould LJ , Das R , Jett M , Hodgson BD , Margolis D ,

Whelan HT. Clinical and experimental applications of nir-led photobiomodulation. Photomedicine and laser surgery.

2006: 24 (2): 121-128.

9- Schubert EF. Light emitting diodes. 2. Ed. Cambridge: cambridge univestity press, 2006.

10- Choi H, Lim W, Kim I, Kim J, Ko Y, Kwon H, Kim S, Kabir KMA, Li X, Kim O, Lee Y, Kim S, Kim O. Inflammatory

cytokines are suppressed by light-emitting diode irradiation of p. Gingivalis lps-treated human gingival fibroblasts:

inflammatory cytokine changes by led irradiation. Lasers in medical science. 2011: 27 (2): 1-9.

11- Camargo MZ, Siqueira CPCM, Preti MCPP, Nakamura FY, Lima FM, Dias IFL, Filho DOT, Ramos SP. Effects of

light emitting diode (LED) therapy and cold water immersion therapy on exercise-induced muscle damage in rats.

Lasers in medical science. 2012: 27 (5): 1051-1058.

12- Fonseca PD, Lima FM, Higashi DT, Koyama DFV, Toginho Filho DO, Dias IFL, Ramos SP. Effects of light emitting

diode (led) therapy at 940 nm on inflammatory root resorption in rats. Lasers in medical science. 2012: 28 (1): 3-12.

13- Eells JT, Wong-Riley MTT, VerHoeve J, Henry M, Buchman EV, Kane MP, Gould LJ, Das R, Jett M, Hodgson

BD, Margolis D, Whelan HT. Mitochondrial signal transduction in accelerated wound healing by retinal and near-

infrared light theray. Mitochondrion. 2004: 4 (5-6): 559-567.

14- Belém M, Andrade GMM, Carlos TM, Gyazelli CFS, Fattori V, Toginho-Filho DO, Dias IFL, Verri Jr WA, Araújo

EJA . Light-emitting diodes at 940 nm attenuate colitis-inflammatory process induced in mice. Journal of

photochemistry & photobiology, b: biology. 2016: 162: 367-373.

15- Melgar H, Karlsson l, Rehnstrom E, Karlsson A, Utkovic H, Jansson L, Michaelsson E. Validation of dextran

sulfate sodium-induced murine colitis using four therapeutic agents for human inflammatory bowel disease.

International immunopharmacology. 2008: 8 (6): 836-844.

16- Neurath M, Fuss I, Strober W. TNBS-colitis. International reviews of immunology. 2000: 19 (1): 51-62.

17- Jeon YD, Bang KS, Shin MK, Lee JH, Chang YN, Jin JS. Regulatory Effects of the extract of glicirrizas on

radiation-induced ulcerative colitis by dss. Bmc complementary and alternative medicine. 2016.

18- Morris GP, Beck PL , Herridge MS , Depew WT , Szewczuk MR , Wallace JL . Hapten-induced model of chronic

inflammation and ulceration in the rat colon. Gastroenterology. 1989: 795-803.

19- Appleyard CB, Wallace JL. Reactivation of hapten-induced colitis and its prevention by anti-inflammatory drugs.

American journal of physiology. 1995: 269 (1): 119-125.

41

EVALUATION OF PHAGOCYTIC ACTIVITY AND IN VITRO CANDIDACY OF PERITONEAL

MACROPHAGES INFECTED WITH DIFFERENT MORPHOTYPES OF Candida tropicalis

Cajuca, R.D.; Conchon-Costa, I.

State University of Londrina/Laboratory of Experimental Protozoology


Keywords: Candida tropicalis, candidemia, switching, phenotypic.

Abstract

The specie Candida tropicalis is considered the second most commonlyisolated in the hospital areas,

frequently associated as a cause ofcandidemia in Brazilian hospitals. One of the factors of virulence of thispathogen is

the phenotypic change, resulting in alteration of themorphology of the same one.It is known that the recognition ofthe

fungus by the innate immune system is the first step to activate theimmune response and take survival after infection.

This recognition occursthrough the identification of molecular patterns present on the surface ofthe pathogen by

phagocytic cells. Therefore, phenotypic modification mayalter recognition and, consequently, impair the protective

response tofungal infection by altering fungal resistance. Objective this work is evaluates the different morphotypes of

the isolate of Candida tropicalis 49.07 in concern the parasite/host relationship.For this, the phagocytic and

candidacidal assay will be performed on peritoneal macrophages co-incubated with different morphotypes of Candida

tropicalis. In order to elucidate the microbicidal mechanisms and interaction of the macrophages with the fungus C.

tropicalis will be performed dosage of cytokines, reactive oxygen species and the mannose receptor activity. It is

expected with this study to elucidate the host parasite relation of fungi that present the ability to perform phenotypic

switching changing its virulence.

Hypothesis

Our hypothesis is that different morphotypes of the same isolate of Candida tropicalis may havediferents

levels of virulence.

Introduction

The Candida Tropicalis fungus is frequently (80%) the cause of candidemiaof fungal infections in brazilian

hospitals, being the second Candida species more commonly isolated in these enviroments. Infections of thisfungus

reach patients of all ages, but it more prevalent in adultand elderly patients. Yeasts of the genus Candida are

commensal,they can colonize the gastrointestinal tract in 80% of the healthy adultpopulation and the vagina in 20 to

30% of women (1-2).

In the process of infection, the recognition and internalization occursthrough the receptors present in the cells

(PRRs), resulting in theproduction of proinflammatory cytokines, such as Interleukin-12 (IL-12)and Tumor Necrosis

Factor-α (TNF-α), increased expression of costimulatorymolecules (CD28,CD80 e CD86) and MHC class II (3,4). Also

someanti-inflammatory cytokines are modulated by this recognition, such asIL-4 and IL-10,triggering an

immunosuppressive effect (5). Several factors contribute for the susceptibility of infectionscaused by Candida

tropicalis. This fungus has high pathogenic potential inneutropenic patients, when there is suppression of the bacterial

flora bythe use of antimicrobials and in gastrointestinal mucosa with damage (6).

Although certain aspects of virulence are genetically determined, they are expressed by microorganisms only

when there are favorable environmental conditions, such as nutritional content, oxygen atmosphere and temperature.

The main virulence factors for Candida species are adherence, cell surface hydrophobicity, germ tube production,

exoenzyme production, tigmotropism, and phenotypic switching (7-9,2).Phenotypic switching is the adaptation of the

fungus to environmentalvariations in the host (10). The process consists of changes in thesurface of the pathogen and

consequently of the colonies, includingchanges in shape and adhesion. These alterations making them more

virulentand consequently resistant to antifungal and neutrophil phagocytosis(11,12).

Three variants of of the isolate of Candida tropicalis 49.07 were identified: smooth (parental phenotype) and

two variants(ring and rugose) and their revertants (strains that changed from thevariant back to the parental

phenotype) which are associated with changes in virulence, including biofilm formation, morphogenesis, hemolysis, as

well as the change in susceptibility to itraconazole(16). Therefore, it is of great interest to study the infection of

different morphotypes of the same isolate, since the virulence can be altered and consequently the infectious process.


42

Objectives

To evaluate the different morphotypes of the isolate of Candida tropicalis 49.07 in concern the parasite/host

relationship.


This project was submitted to the Ethics Committee of the StateUniversity of Londrina for Animal

Experimentation and is awaiting approval.

C. tropicalisculture

Will be studied five morphotypes from Candida tropicalis isolate 49.07: smooth morphologie (parental

morphotype), crepe and rugose (variant morphotypes), smooth (revertant of crepe) and rough colonies (revertant of

rugose) as described by MORALEZ et al. (2014). Those isolates are part of the yeast collection of the Laboratory of

Genetics and Molecular Biology of Fungi of the State University of Londrina. Cells of each morphotype (parental,

variants and revertants) will be inoculated into 3 mL of liquid YPD medium (1% yeast extract, 2% peptone and 2%

glucose) and cultured for 24 hours at 28°C under 180 RPM shaking. The cells will be adjusted to a concentration of

6x103 cells/mL in 50μl, distributed in Petri dishes containing solidified YPD medium and incubated in B.O.D.for 96

hours at 28°C.

Phagocytosis assays

Macrophages (4x105) will be recovered from peritoneal cavity of Balb/c mice with buffered saline (PBS)

supplemented with 3% of fetal bovine serum (FBS), cultured in 24-wells plates with RPMI 1640 medium (10% FBS) to

24 h (37C, 5% CO2). After that 4x106 of yeast cells will be then co-incubated with phagocytes for 2 hr at 37°C. Cells

will be stained by May-Grumwald-Giemsa and analyze by optical microscopy counting 10 fields to evaluate the

percentage of phagocytosis, and the supernatant will be stored for future experiments.

Candidacidal assay

The candidacidalactivity will be performed according to the method described by Cenci et al. 1991. Briefly,

(4x105) macrophages from peritoneal cavity will be plated (0.2 ml/well) in 24-well plates (Corning Costar, New York,

NY, USA) and incubated with 4x106 yeast in 1 mL of PBS 24 hours at 37°C. Will be added Sterilized double-distilled

water (1 ml) to the wells for 10 min. Serial dilutions (10-fold) from each well will be made in PBS. Aliquots (50µl) will

be plated on Sabouraud agar and the number of colony forming units (CFU) will be determined after incubation for 48

h at 37°C.

Cytokine assay

The supernatant obtained in the phagocytic assay will be used to determine cytokine levels by the ELISA

technique using commercial kits from eBioscience (San Diego, CA, USA) according to the manufacturer's instructions.

The absorbance will be read at 450 nm using a spectrophotometer.

Determination of reactive oxygen species (ROS) by fluorescence

To evaluate ROS production, cells of peritoneal exudate will be plaque in a black 96-well plate (20 μl/well) and

incubated in the dark for 30 minutes at 37°C, 5% CO2 with 2 μM H2DCFDA (Diacetate 27-dichlorofluorescein)

(Sigma-Aldrich) diluted in DMSO adjusted to 200 μL. Hydrogen peroxide (200 uM) will be used as a positive control.

The reactive oxygen species (ROS) will be measured as a result of the conversion of non-fluorescent dye into highly

fluorescent 20,70-dichlorofluorescein in a reader of Fluorescence microplates (Victor X3, PerkinElmer, Finland) in 530

nm.

Mannose receptor activity

To evaluate the participation of mannose receptors in the internalization of fungi, phagocytes (4x105) will be

incubated with 10µg/mL of mannose and 1µg/mL of mannan (Sigma) before incubation with 1x106 yeasts for 2 hr at

37°C. Cells will be stained by May-Grumwald-Giemsa and analyzed by optical microscopy counting 10 fields to

evaluate the percentage of phagocytosis.

Expected Results

43

It is expected with this study to elucidate the host / parasite relation of fungi that present the ability to perform

phenotypic switching

Acknowledgments

First of all I would like to thank Professor Wagner Loyola for the opportunity to continue in the scientific area.

The teacher Ivete Conchon Costa who welcomed me in her laboratory.

To prof. Marcia Cristina Furlaneto for "presenting" to me Candia tropicalis.

To all the personnel of the laboratory of parasitology and microbiology, of the State University of Londrina.

CAPES

References

1. AVRELLA, D.; GOULART, L. S. Isolamento de Candida spp. da mucosa oral de pacientes submetidos ao

tratamento quimioterápico. RBAC, jul. 2008,v. 40, n. 1, p. 205-207.

2. NUCCI M, COLOMBO A.L. Candidemia due to Candida tropicalis: clinical, epidemiologic, and microbiologic

characteristics of 188 episodes occurring in tertiary care hospitals. DiagMicrobiol Infect Dis. 2007; 58:77-82.

3. SYME, R. M.; SPURRELL, J. C.; AMANKWAH, E. K.; GREEN, F. H.; MODY, C. H. Primary dendritic cells

phagocytose Cryptococcus neoformans via mannose receptors and Fcγ receptor II for presentation to T

lymphocytes. Infection and Immunity, 2002, v. 70, p. 5972-5981.

4. MANSOUR, M. K.; SCHLESINGER, L. S.; LEVITZ, S. M. Optimal T cell responses to Cryptococcus

neoformansmannoprotein are dependent on recognition of conjugated carbohydrates by mannose receptors.

Journal of Immunology, Baltimore, v. 168, n. 6, p. 2872– 2879, 2002.

5. TONNETII, L.; SPACCAPELO, R.; CENCEI, E.; MENCACCI, A.; PUCCETTI, P.; COFFMAN, R. L.; BISTONI, F.;

ROMANI, L. Interleukin-4 and 10 exacerbat candidiasis in mice. European Journal of Immunology,

Weinheim,1995, v. 25, p.1559-1565.

6. WINGAR JR. Importance of Candida species other than C. albicans as pathogens in oncology patients. Clinical

Infectious Diseases. 20: 115-125, 1995.

7. LAMFON H.; PORTER S. R.; MCCULLOUGH M.; JONATHAN PRATTEN J. Susceptibility of Candida albicans

biofilms grown in a constant depth film fermentor to chlorhecidine, fluconazole and miconazole: a longitudinal

study. J AntimicrobChemoth, 53: 383-385, 2004.

8. DE BERNARDIS, F.; SULLIVAN, P. A., CASSONE, A. Aspartys proteinases of Candida albicans and their role in

pathogenicity. Med Mycol, 39(4): 303-13, 2001

9. SOLL, D.R. Candida commensalism and virulence: the evolution of phenotypic plasticity. ActaTropica, 81: 101-10,

2002.

10. KULETA, J. K.; KOZIK, M. R.; KOZIK, A. Fungi pathogenic to humans: molecular bases of virulence of Candida

albicans, Cryptococcus neoformans and Aspergillusfumigatus. ActaBiochimicaPolonica, 2009, v. 56, p. 211-224,

2009.

11. MILLER, M. JOHNSON, A. White-opaque switching ih C. albicans in controlled by

mating-type locus homodomain proteins and allows afficient mating. Cell, 3: 293-2002.

12. NAGLIK, J. R.; CHALLACOMBE, J.; HUBE, B. Candida albicans secreted aspartil

proteinases in virulence and pathogenesis. Microbiol. Mol. Biol. Rev., Washington, v.67, n. 3, p. 400-28, 2003.

13. BACELO, K. L. Avaliação da variabilidade fenotípica e molecular de isolados de Candidaalbicans após período

de armazenamento das culturas e em duas ocasiões de coleta [dissertação]. Ribeirão Preto: Universidade de São

Paulo, Faculdade de Ciências Farmacêuticas de Ribeirão Preto; 2008

14. BARBEDO L. S; SGARBI, D. B. G. Candidíase. Jornal Brasileiro de Doenças Sexualmente Transmissíveis. Rio

de Janeiro, p. 22 – 38, abril. 2010.

15. BERMAN, J. SUDBERY, P.E. Candida albicans: a molecular revolution built on lessons from budding yest. Nat.

Ver. Genetics, dec. 2002, v.3,p.918-302.

16. MORALEZ A. T. P.; FRANÇA E. J. G.; FURLANETO-MAIA L.; QUESADA M. B.; FURLANETO M. C. Phenotypic

switching in Candida tropicalis: association with modification of putative virulence attributes and antifungal drug

sensitivity. Med Myc.

44

HISTOLOGICAL ANALYSIS OF GUT-ASSOCIATED LYMPHOID TISSUE OF HEALTHY RATS

SUPPLEMENTED WITH IGY

Damasco, K.F.1, Venancio, E.J.

1

1State University of Londrina/Department of General Pathology.


Keywords: IgY, intestinal mucosa, influence, rats.

Abstract

The gut-associated lymphoid tissue (GALT) comprises one of the constituents of the immune system. It is formed by

the intestinal mucosa, Peyer's patches, isolated lymphoid follicles and mesenteric lymph nodes. Despite this, the

intestinal mucosa is an important port of entry of pathogens. In this context, IgY antibodies have been investigated to

prevent adhesion and microbial colonization in the intestinal mucosa and, in addition, neutralizing the toxins.

Furthermore, some studies suggest that, IgY may influence the functioning of GALT, reducing the levels of secretory

IgA in the jejunum, the amount of goblet cells, intraepithelial lymphocytes and CD8+ T lymphocytes in the Peyer's

patches, post-infections. Thus, the present study aims to analyze the effects of in vivo supplementation of healthy

Wistar rats with IgY (IgY i26 and Max GI control) on GALT. Twelve Wistar rats will be supplemented with 140 mg IgY

i26, 12 with 140mg IgY Max Control, and 12 without treatment for 14 days and then induced to euthanasia for the

collection of 5 cm of the jejunum and ileum, 4 Peyer's patches and 4 mesenteric lymph nodes. All samples will be

fixed, included in paraffin and hematoxylin-eosin staining. In the mucosa will be measured the height and area of the

villi beyond the depth of the intestinal crypt. In the lymph nodes and in the Peyer's patches, the lymphoid follicles and

germinal centers will be quantified and measured as area and perimeter. It is expected that supplementation of the

animals with IgY will influence the parameters analyzed.

Hypothesis

IgY antibodies administered by oral route have immunodulatory activity in the gut-associated lymphoid tissue.

Introduction

The immune system consists of organs, tissues, cells and molecules. The organs are responsible for the

production and maturation of immune cells. In addition, they are sites where the immune response occurs. On the

other hand, lymphoid tissues are aggregates of cells of the immune system that are extremely important for triggering

the immune response (1).

In this context, the gut-associated lymphoid tissue (GALT) is formed by intestinal mucosa, Peyer's patches,

isolated lymphoid follicles and mesenteric lymph nodes (2,3).

The intestinal mucosa is the first line of defense to oral pathogens. It is composed of an epithelium, the lamina

propria and the muscular layer of the mucosa. The epithelium is composed of enterocytes, intraepithelial lymphocytes

(IEL), M-cells, goblets and Paneth, and mucus-secreting glands. The lamina propria is formed by a loose connective

tissue containing tubular glands, lymphocytes, Peyer's patches, isolated follicles and lymphatic vessels (1,4).

The Peyer's patches and mesenteric lymph nodes comprise clusters of lymphoid follicles which, despite their

peculiar locations, structures and functions, generally provide contact with the naive lymphoid cells and, respectively,

the development and establishment of local and systemic immune response. A similar function is observed in isolated

lymphoid follicles (1,4).

Therefore, GALT has all the elements necessary for the development of the immune response. However,

even the intestinal mucosa is an important gateway for pathogens (5).

In this context, oral administration of IgY antibodies has been investigated against various intestinal infections

(5,6). IgY is an acquired immunoglobulin from bird eggs that is phenotypically similar to IgG produced in mammals.

When administered orally, it may inhibit adhesion and subsequent microbial colonization in the intestinal mucosa

preventing its spread, in addition to neutralizing toxins. Besides that, IgY antibodies do not activate the complement

system and do not bind to rheumatoid factors and Fc receptors present in mammals, therefore, they do not induce an

inflammatory response when applied in mammals (5,6).

Some studies also suggest that oral administration of IgY antibodies may influence the functioning of GALT

and contribute to the establishment of intestinal and consequently systemic health (5).

45

One study reports, for example, that ingestion of specific IgY by chickens infected with E. coli reduces the

proportion of heterophiles / lymphocytes in the bloodstream, levels of secretory IgA in the jejunum beyond the amount

of goblet cells and lymphatic follicles, induced Infection (7).

IgY minimizes basement membrane detachment and epithelial vacuolization, reduces intestinal damage in

mice infected with enteropathogenic E. coli, and stimulates the increase of IL-10 and the reduction of TNF-α and INF-γ

levels in the intestinal mucosa of mice infected with Salmonella typhimurium (6,8). IgY also reduces the number of

intraepithelial lymphocytes (IEL) and TCD4 + and TCD8 + lymphocytes in the lamina propria in addition to TCD8 + in

the Peyer's patches in order to attenuate the inflammatory response induced and subsequent epithelial damage (6).

Although one study reports that administration of specific IgY to human rotavirus-infected piglets reduces the

number of IgA-secreting cells, in addition to inducing the production of IgA and IgG anti-IgY (9), oral administration of

IgY anti-rotavirus Calves infected with bovine rotavirus induce the increase of IgA secreting cells in the intestinal

mucosa mainly of the duodenum (10).

From this and the few findings in the literature directed to the influence of IgY on GALT, the present study

aims to analyze the effects of oral administration of IgY on the intestinal mucosa, Peyer's patches and mesenteric

lymph nodes.

Objectives

General:

To analyze the possible immunomodulatory effects of in vivo supplementation of healthy Wistar rats with IgY

i26 and Max GI Control on GALT.


Experimental design:

The use of 36 male Wistar rats of approximately 5 months of age will be submitted to approval by the

Commission of Ethics in the Use of Animals of the State University of Londrina - CEUA / UEL.

The animals will be obtained from the animal facility from UEL and kept in the experimental room of the

department of general psychology and behavioral analysis (PGAC) from the UEL where they will be accommodated in

an acclimatized environment at approximately 22ºC with a variation of +/- 1ºC, conditioned at the light/dark cycles of

12:00 hours and fed with feed and water ad libitum.

They will be anesthetized with xylazine and ketamine and subjected to cardiac puncture to obtain blood and

serum that will be used for hemogram and c-reactive protein analysis. The hematological and biochemical

examinations aim to certify the health condition of the animals.

The animals will be divided into three groups with 4 animals each where the animals oh the control group will

receive 1ml of 1X PBS saline solution (phosphate-buffered saline) and the animals in the tests 1 and 2 groups will be

inoculated orally with 140mg of IgY (IgY i26 and Max GI Control, respectively) diluted in 1X PBS for 14 days. Then

the animals will be induced to euthanasia in the CO2 chamber to obtain the tissue samples. The experiment will be

repeated 3 times under the same conditions.

IgY hyperimmune i26 and Max GI Control used for animal supplementation are dietary supplements made

with egg powder obtained from chickens immunized against 26 bacterial strains which are relevant for imbalance of

the human intestinal tract and will be purchased from the U.S.A. (11,12).

The figure below illustrates the chronological order of the procedures described above.

46

Morphometric analysis:

After the euthanasia, will be collected from each animal 5cm randomly from the medial and distal regions of

the small intestine, 4 peyer's plaques and 4 mesenteric lymph nodes, which will be conditioned to the Bouin fixative

solution for 24 hours as determined by Beçak and Paulete (13).

Subsequently, both pieces will be dehydrated with 70% alcohol and subjected to the dehydration and inclusion

process with impregnation by paraffin wax to remove water from the tissue, the replacement of the alcohol with a

substance miscible with the inclusion medium such as xylol and the stiffening of the material to perform the microtomy

(14).

The cuts will be performed on the 7μm thick microtome, packed in a water bath at 45ºC and then collected on

a histological slide and stored in the oven at 37ºC for 24 hours (14).

After the procedure of fixing and distending the cuts, the slides will be stained by hematoxylin-eosin (HE), as

determined by Junqueira and Carneiro (14), whose hematoxylin stains blue or violet acidic structures such as the

nucleus and eosin, basic structures as the cytoplasm.

Finally, the slides will be analyzed under light microscopy with photographically recorded documentation in a

Motic Plus 2.0 image capture system coupled to the optical microscope.

During the analysis of the intestinal mucosa, the area and height of the intestinal villus will be measured

beyond the depth of the crypt, and the goblet cells and IEL will be quantified, whereas in the analysis of Peyer's

patches and lymph nodes will be quantified the lymphoid follicles and germ centers, besides measured their area and

perimeter.

The results obtained will be submitted to the normality test and homogeneity of the variances and, if the

assumptions are met, the analysis of variance (ANOVA) with p <0.05 will be used.

Expected Results

Based on the available knowledge effects of oral IgY intake on the performance and effectiveness of the

immune system, it is expected that supplementation of healthy Wistar rats with IgY i26 and Max GI Control has

influence on the intestinal mucosa, Peyer's patches and/or mesenteric lymph nodes in relation to the parameters

analyzed.

Acknowledgments

Damasco, K.F. Received a scholarship from CAPES.

References

[1] Abbas AK, Lichtman AH, Pillai S. Células e tecidos do sistema imune. In: Abbas AK, Lichtman AH, Pillai S.

Imunologia celular e molecular.8. ed. Rio de janeiro: Elsevier; 2015. p.13-33.

[2] Li X, Yao Y, Wang X, Zhen Y, Thacker PA, Wang L, et al. Chicken egg yolk antibodies (IgY) modulate the intestinal

mucosal immune response in a mouse model of Salmonella typhimurium infection. Int Immunopharmacol. 2016 jul;

36: 305-314.

[3] Torché AM, Dimna ML, Corre PL, Mespléde A, Gal SL, Cariolet R, et al. Immune responses after local

administration of IgY loaded-PLGA microspheres in gut-associated lymphoid tissue in pigs. Vet Immunol

Immunopathol. 2006 feb; 109(3-4): 209-217.

[4] Ovalle WK, Nahirney PC. Sistema linfoide. In: Ovalle WK, Nahirney PC. Netter – bases da histologia. 2. ed. Rio de

janeiro: Elsevier; 2014. p. 195-212.

[5] Li X, Wang L, Zhen Y, Li S, Xu Y. Chicken egg yolk antibodies (IgY) as non-antibiotic production enhancers for use

in swine production: a review. J Anim Sci Biotechnol. 2015 aug; 6(1): 1-10.

[6] Thu HM, Myat TW, Win MM, Thant KZ, Rahman S, Umeda K, et al. Chicken egg yolk antibodies (IgY) for

prophylaxis and treatment of rotavirus diarrhea in human and animal neonates: A concise review. Korean J Food Sci

Anim Resour. 2017 feb; 37(1): 1-9.

[7] Mahdavi AH, Rahmani HR, Nili N, Samie AH, Soleimanian-Zad S, Jahanian R. Effects of dietary egg yolk antibody

powder on growth performance, intestinal Escherichia coli colonization, and immunocompetence of challenged broiler

chicks. Poult Sci. 2010 mar; 89(3): 484-494.

[8] Vulcano AB, Tino‐De‐Franco, Amaral JA, Ribeiro OG, Cabrera WHK, Bordenalli MA, et al. Oral infection with

enteropathogenic Escherichia coli triggers immune response and intestinal histological alterations in mice selected for

their minimal acute inflammatory responses. Microbiol Immunol. 2014 jun; 58(6): 352-359.

https://www.ncbi.nlm.nih.gov/pubmed/?term=Mahdavi%20AH%5BAuthor%5D&cauthor=true&cauthor_uid=20181864

https://www.ncbi.nlm.nih.gov/pubmed/?term=Rahmani%20HR%5BAuthor%5D&cauthor=true&cauthor_uid=20181864

https://www.ncbi.nlm.nih.gov/pubmed/?term=Nili%20N%5BAuthor%5D&cauthor=true&cauthor_uid=20181864

https://www.ncbi.nlm.nih.gov/pubmed/?term=Samie%20AH%5BAuthor%5D&cauthor=true&cauthor_uid=20181864

https://www.ncbi.nlm.nih.gov/pubmed/?term=Soleimanian-Zad%20S%5BAuthor%5D&cauthor=true&cauthor_uid=20181864

https://www.ncbi.nlm.nih.gov/pubmed/?term=Jahanian%20R%5BAuthor%5D&cauthor=true&cauthor_uid=20181864

47

[9] Vega CG, Bok M, Vlasova AN, Chattha KS, Fernández FM, Wigdorovitz A, et al. IgY antibodies protect against

human rotavirus induced diarrhea in the neonatal gnotobiotic piglet disease model. PLoS One. 2012 aug; 7(8): 1-16.

[10] Vega C, Bok M, Chacana P, Saif L, Fernandez F, Parreño V. Egg yolk IgY: protection against rotavirus induced

diarrhea and modulatory effect on the systemic and mucosal antibody responses in newborn calves. Vet Immunol

Immunopathol. 2011 aug; 142(3-4): 156-169.

[11] Ergomax [Internet]. Hyperimmune egg – Egg complex powder [acesso em 10 jun 2017]. Disponível em:

https://www.ergomaxsupplements.com/hyperimmune-egg-ei-complex-poeder.

[12] My Wellness Journey [Internet]. Max GI Control [acesso em 10 jun 2017]. Disponível em:

http://mwjourney.com/wp-content/uploads/2016/06/Max-GI-Control-DRS.pdf.

[13] Beçak W, Paulete J. Técnicas de citologia e histologia. São Paulo: Nobel; 1970.

[14] Junqueira LCU, Carneiro J. Métodos de estudo em histologia. In: Junqueira LCU, Carneiro J. Histologia básica.

10.ed. Guanabara Koogan; 2004. p. 1-22.

https://www.ncbi.nlm.nih.gov/pubmed/?term=Vega%20C%5BAuthor%5D&cauthor=true&cauthor_uid=21652087

https://www.ncbi.nlm.nih.gov/pubmed/?term=Bok%20M%5BAuthor%5D&cauthor=true&cauthor_uid=21652087

https://www.ncbi.nlm.nih.gov/pubmed/?term=Chacana%20P%5BAuthor%5D&cauthor=true&cauthor_uid=21652087

https://www.ncbi.nlm.nih.gov/pubmed/?term=Saif%20L%5BAuthor%5D&cauthor=true&cauthor_uid=21652087

https://www.ncbi.nlm.nih.gov/pubmed/?term=Fernandez%20F%5BAuthor%5D&cauthor=true&cauthor_uid=21652087

https://www.ncbi.nlm.nih.gov/pubmed/?term=Parre%C3%B1o%20V%5BAuthor%5D&cauthor=true&cauthor_uid=21652087

48

IDENTIFICATION OF THE GUT-VASCULAR BARRIER IN WISTAR RATS

Nascimento, A. M.1, Venancio, E. J.

1

1State University of Londrina/Department of Pathological Sciences.


KEYWORDS: endothelial cells, glial cells, Salmonella, obesity.

Abstract

Recently the existence of a gut-vascular barrier in mice and humans with morphological and functional

characteristics similar to those of the blood-brain barrier has been identified. This prevents the entry of the microbiota

and controls the passage of antigens into the bloodstream. As Wistar rats are an animal model widely used in the

research, the objective of this work is to identify the presence of the gut-vascular barrier in Wistar rats. Rats will be

infected orally with 109 of S. enterica subsp. enterica serovar Typhimurium, 24 hours after infection the animals will be

anesthetized, their intestinal loops will be exteriorized and tethered. 2 mg FITC-dextran 70 kDa will be injected into the

loop in the portion relating to the ileum. After 1h, 2 ml of blood will be collected via cardiac puncture and the presence

of the fluorophore will be measured. The amount of dye will also be measured in the liver and spleen by

histofluorescence. 2/3 of the liver will be used for counting bacterial colonies. Blood samples will be collected prior to

the permeability assay to assess the immune status of the animals by blood counts and the dose levels of protein C

reatine. The statistical analysis will be performed using the t test for independent samples or Mann Whitney,

considering P <0.05 as significant. From the answers obtained in this study it is expected to perform a systematic

review and a second experimental trial relating the intestine-vascular barrier and the obesity picture.

Hypothesis

There is a gut-vascular barrier in rats.

Introduction

The intestinal microbiota is actively excluded from the bloodstream and the factors contributing to this

exclusion are largely unknown (1). Recently, Spadoni et al. (2) identified the existence of a gut-vascular barrier in mice

and humans that prevents the entry of the microbiota and controls the passage of antigens into the bloodstream. This

barrier presents morphological and functional characteristics similar to those of the blood-brain barrier, consisting of

endothelial cells, pericytes and glial cells (2).

The vascular endothelium consists of a layer of narrowly juxtaposed endothelial cells lining the inner surface

of the blood vessels (3). In the microcirculation, the endothelium comprises the wall of capillaries and post-capillary

venules resting on a basal lamina and functions as an endothelial barrier characterized by the presence of junctional

complexes that include occlusive joints and adherent joints (3,4). The barrier function depends on the integrity of the

endothelial structure (cytoskeleton and cell-cell junctions, extracellular matrix and basement membrane), therefore,

malfunctions in this barrier, result in increased permeability. This change in permeability may occur in response to

inflammatory agents, pathogens or disease states (3).

Enteric glial cells, one of the components of the enteric nervous system (SNE), are homologous to astrocytes,

cells found in the central nervous system (5). These cells are found in the enteric (intraganglionic) ganglia, in the

myenteric and submucosal (interganglionic) plexuses of the SNE, and are also found in the muscular layers of the

intestine (intramuscular) and the lamina propria of the mucosa (subepithelial) (6). Derivatives of neural crest

progenitor cells, the glial cells of the intestine present a final phenotype according to the microenvironment of the

intestinal wall (7,8). Hanani and Reichenbach9 classified the enteric glial cells into type I, II, III and IV, based on the

morphological differences of these cell populations. Type III glial cells or enteric glial cells of the mucosa are located

near the epithelial border and their extensions have already been seen in direct contact with the basal membrane of

the epithelial cells and lamina propria of the blood capillaries, similar to the astrocytes that control adhesion Of the

endothelial cells of the blood vessels in the CNS forming the blood-brain barrier (10,11).

Modulation of the expression of molecules involved in the formation of junctional complexes can be influenced

by pathogens such as Salmonella. Spadoni et al. (2) showed that mice exhibit vesicle-associated plasma membrane

protein (PV1), a marker of endothelial cell permeability, in intestinal endothelial cells and correlate this finding with

increased vessel permeability, as a result of the dissemination of Salmonella to the liver and spleen, plus liver

damage.


49

When investigating enteric glial cell populations in mice submitted to a high-fat diet inducing obesity,

hyperglycemia and insulin resistance, Stenkamp-Strahm et al. (12) observed that there is a reduction of glial cell

density in the duodenum mucosa, however, the density in the myoenteric plexus It does not change.

These findings indicate the importance of the gut-vascular barrier in maintaining the homeostasis of the

organism and how this barrier can be affected by pathogens, such as Salmonella, and that metabolic disorders can

also induce modifications in the structure of the barrier unit, since it affects one of the cell types that characterize this

structure.

The recent description of the gut-vascular barrier in humans and mice (2) suggests that it is present also in

other mammals. Since Wistar rats are a widely used animal model, the characterization of this structure in this model

becomes important so that it can be used to obtain more information about the functions and pathologies related to

the gut-vascular barrier.

Objectives

To identify the presence of the gut-vascular barrier in Wistar rats.


This project was approved by the Committee on Ethics in the Use of Animals of the State University of

Londrina (CEUA / UEL), protocol CEUA nº 11501.2016.78.

1. Animals

14 male Wistar rats with a mean weight of 270 g will be divided into 2 groups (control and infected). During the

experimental period the animals will be kept in collective cages, with 4 animals per cage, lined with wood shavings,

with commercial (normocaloric) and water ad libitum, controlled temperature (24 ± 2 ºC) and dark light cycle of 12/12

hours.

2. Bacterial samples

The strain of Salmonella enterica subsp. Enterica serovar Typhimurium (UK 1) will be kindly provided by Prof.

Dr. Gerson Nakazako of the Laboratory of Basic and Applied Bacteriology of the Department of Microbiology of the

State University of Londrina.

The bacterial samples will be cultured in MacConkey selective medium for 24 hours and kept in an oven at

37 ºC. After growth, they will be stored in 1.5 mL microtubes containing nutrient agar and kept at room temperature.

3. Evaluation of permeability of the gut-vascular barrier

3.1 Salmonella infection

Rats will be infected orally with 109 of S. enterica subsp. enterica serovar Typhimurium (UK 1). The bacteria

will be grown in nutrient agar, subsequently 5 colonies will be passed into tubes containing 5 mL of Luria-Bertani broth

(LB) and incubated for 18 hours static at 37 ºC, after 18 hours growth the falcon content will be poured into an

erlenmeyer containing 45 mL of LB, that flask will be maintained for 3h30 under agitation (150 rpm) at 37 °C, after

which a 5 mL aliquot will be centrifuged for 5 minutes at 5000 rpm (2688 g) at 4 °C, the supernatant will be discarded

and The cells resuspended in 5 ml of PBS. 24 hours after infection, the animals will be anesthetized with ketamine

and xylazine (75 mg/kg and 10 mg/kg, respectively) intramuscularly, their intestinal loops will be externalized and

lashed. 2 mg of 70 kDa FITC-dextran (Sigma-Aldrich) will be injected into the loop in the ileum portion. After 1h, 2 mL

of blood will be collected via cardiac puncture and the presence of the fluorophore will be measured with the Victor

TM3 fluorometer (Perkin Elmer) with excitation at 490 nm and emission 530 nm. The amount of dye will be measured

in the liver and spleen by histofluorescence as described below.

3.2 Histofluorescence

After collection of blood the liver and spleen will be excised, both will be used to evaluate the amount of dye

present in the organ by microscopy, the liver will be divided and 1/3 of that organ will be used for this purpose. The

organs will be fixed for 3 hours in 4% paraformaldehyde. They will then be washed with PBS and packed in PBS/30%

Sucrose for 24 hours at 4 °C. The solution will then be exchanged for PBS/30% Sucrose and OCT in the ratio of 1: 1

for 24 hours at 4 °C, followed by freezing in liquid nitrogen. Subsequently, cryoprests will be performed, they will be

50

washed with PBS and then incubated with anti-fade/DAPI (4 ', 6-diamidin-2-phenylindole) for labeling the nuclei.

Images will be analyzed for brightness in ImageJ software.

3.3 Colony count

The remaining 2/3 of the liver will be used for counting colonies. The organ will be macerated using a

TURRAX type disintegrator and plated in MacConkey selective medium and in nutrient agar for colonies counting after

culture of 24 hours at 37 ºC.

4. Analysis of the immunological state: C reative protein and hemogram

Samples of blood will be collected by cardiac puncture after the period and acclimatization of the animals to

the vivarium (7 days) for evaluation of C reactive protein and blood count. C reactive protein will be evaluated in

plasma samples using commercial kit (Siemens, Munich, Germany) on Dimension biochemical routine equipment

(Siemens, Munich, Germany), as recommended by the manufacturer. For blood counts, blood samples will be stored

in tubes containing 5% EDTA. These samples will be analyzed the same day on an automated hematology analyzer

(BC-2800 VET, Mindray, Shenzhen, China).

5. Statistical analysis

Homogeneity tests (Levene test) and normality (Shapiro-Wilk test) will be applied followed by the t test for

independent samples or Mann Whitney, considering P <0.05 as significant.

Expected Results

The objective of this study was to establish the presence of the gut-vascular barrier in Wistar rats, if confirmed

the presence of this structure in these animals is intended to investigate the effect of obesity on Wistar rats induced by

a cafeteria diet on the barrier. In advance of this investigation it is desired to carry out a systematic review to answer

the following question: Are obese individuals more affected by bacterial infections that have as a gateway the

gastrointestinal tract compared to eutrophic?

Acknowledgments

I thank Professor Eduardo José de Almeida Araújo for giving his time and experience in histological analysis,

to Professor Emerson José Venancio for his orientation, to the postgraduate program in Experimental Pathology by

the opportunity, the State University of Londrina and CAPES, for the structure and development.

References

1- Macpherson AJ, Smith K. Mesenteric lymph nodes at the center of immune anatomy. Exp Med. 2006; 203 (3):

497-500.

2- Spadoni I, Zagato E, Bertocchi A, Paolinelli R, Hot E, Sabatino AD, et al. A gut-vascular barrier controls the

systemic dissemination of bacteria. Science. 2015; 350: 830-834.

3- Yuan SY, Rigor RR. Regulation of Endothelial Barrier Function. 1. ed. San Rafael: Morgan & Claypool Life

Sciences; 2010.

4- Junqueira LC, Carneiro J. Histologia básica. 12. ed. Rio de Janeiro: Guanabara Koogan; 2013.

5- Rühl A, Nasser Y, Sharke KA. Enteric glia. Neurogastroent Motil. 2004; 16: 44-49.

6- Gulbransen BD, Sharkey KA. Novel functional roles for enteric glia in the gastrointestinal tract. Nature Rev:

Gastroenterol Hepatol. 2012; 9: 625-632.

7- Rühl A. Glial cells in the gut. Neurogastroent Motil, 2005; 17: 777-790.

8- Dulac C, Douarin NML. Phenotypic plasticity of Schwann cells and enteric glial cells in response to the

microenvironment. P Natl Acad Sci. 1991; 88: 6358-6362.

9- Hanani M, Reichenbach A. Morphology of horseradish peroxidase (HRP)-injected glial cells in the myenteric

plexus of the guinea-pig. Cell Tissue Res. 1994; 278 (1): 153-160.

10- Abbott NJ, Rönnbäck L, Hansson E. Astrocyte–endothelial interactions at the blood–brain barrier. Nature Rev:

Neurosci. 2006; 7: 41-53.

11- Savidge TC, Newman P, Pothoulakis C, Ruhl A, Neunlist M et al. Enteric Glia Regulate Intestinal Barrier Function

and Inflammation Via Release of S-Nitrosoglutathione. Gastroenterology. 2007; 132 (4): 1344-1358.

http://www.morganclaypool.com/

http://www.morganclaypool.com/

51

12- Stenkamp-Strahm C, Patterson S, Boren J, Gericke M, Balemba O. High-fat diet and age-dependent effects on

enteric glial cell populations of mouse small intestine. Auton Neurosci. 2013; 177 (2): 199-210.

52

INVOLVEMENT OF THE POLYMORPHISMS OF NFKB1 (rs28362491) AND NFKBIA (rs696) IN THE

DEVELOPMENT OF CERVICAL CANCER

Sena, M. M.1, De Oliveira, K. B.

1

1 State University of Londrina/ Department of Pathological Sciences, Londrina, Brazil.


Keywords: polymorphism, NF-kappa B, HPV, cervical cancer.

Abstract

Cervical cancer is the fourth most common cancer type in women, and its etiology is multifactorial. It can be

caused by several environmental and hereditary factors. Among the first, HPV infection is the most common, being

related to up to 70% of cervical cancers. In relation to hereditary factors, the polymorphisms that affect NFƙB genes

play an important role, increasing susceptibility to the development of cervical cancer. Thus, the objective of this study

will be analyze the polymorphisms of NFKB1 (rs28362491) and NFKBIA (rs696) in women positive for the presence of

HPV DNA and to associate the infection with socioepidemiological, reproductive and sexual behavior data, as well as

to the development of pre-malignant and malignant lesions. PCR-RFLP will be used for viral detection and genotyping

and for identification of the polymorphic alleles. All statistical analyses will be carried out using the SPSS Statistics

22.0 software, all tests will be two-tailed, considering a p-value <0.05 for statistical significance. Categorical data will

be analyzed by χ2 test and continuous data by parametric tests when normality is assumed or by non-parametric

among non-normal data. Binary logistic regression analysis, adjusted for several confounders will be used to estimate

the association between polymorphism genotypes and HPV infection. The results obtained will contribute to a better

understanding of both polymorphisms role associated with the presence of HPV infection in the pathogenesis of

cervical carcinoma and in the development of premalignant and malignant lesions.

Hypothesis

The polymorphisms of NFKB1 (rs28362491) and NFKBIA (rs696) are associated to the development of

cervical malignancies increased susceptibility.

Introduction

Cervical cancer is the fourth most common cancer in women, occurring approximately 500,000 new cases

and 270,000 deaths annually around the world due this disease, according to Graham (1). In Brazil, it is the third most

common type of cancer in women, with a little more than 16,000 cases estimated by the National Cancer Institute for

2016 (2). Although etiology of carcinogenesis has not been fully elucidated yet, many evidences suggest that it is a

multifactorial process caused by interactions between several environmental and hereditary factors (3).

In this context, Human Papillomavirus (HPV) is the etiological agent of the most common viral infection of the

anogenital tract, affecting vulva, vagina, penis and anus, besides the cervix (4). Infection by some viral types is

established as a prerequisite for most of the malignant cervical lesions (5). Viral types can be classified according to

their association with cervical cancer in high carcinogenic risk (HR-HPV), low carcinogenic risk (LR-HPV) and

undetermined risk (UR-HPV) (6). HPV16 and HPV18 are associated with the highest carcinogenic potential, being

associated with 50% of the high-grade intraepithelial lesions (LIEAG) (7) and accounting for approximately 50% and

20% of cervical cancers, respectively (8).

Although HPV infection is a necessary etiological factor (9) and well-recognized in the development of cervical

carcinoma, itself is insufficient to trigger such process, so it is evident that other factors, such as smoking, alcohol

consumption, immunological status, number of sexual partners, host genetic predisposition, among others, play an

important role in cervical carcinogenesis (10). Among the genetic factors that may play a role in the pathogenesis and

progression of this malignancy are mutations in genes of essential inflammatory mediators, such as those found in

genes responsible for encoding the Nuclear Factor kappa B (NF-ƙB) family proteins (11).

NF-ƙB, discovered by Sen and Baltimore in 1986, consists of a pleiotropic transcription factor which in

mammals is composed for five members: c-Rel (REL), Rel-B (RELB), p65 (RELA), p50/105 (NFKB1), and p52/p100

(NFKB2) (3). NF-ƙB-regulated genes are related to immune response, cell adhesion, proliferation, differentiation,

apoptosis, metastasis and angiogenesis, according to Eskandari-Nasab et al. and Li et. al. (12,13).

In the cytoplasm of resting cells, NF-ƙB remains in its inactivated form when in association with the inhibitory

protein IƙBα, β and γ, the first being encoded by the NFKBIA gene, the most common protein in this family (14).


53

However, due to a wide range of stimuli, there is the phosphorylation of NF-ƙBIA, allowing the release and nuclear

translocation of NF-ƙB. Thus, in the nucleus, NF-ƙB is capable of binding to DNA promoter regions, inducing cell

proliferation and transformation, and preventing the elimination of pre-malignant and malignant cells by upregulation of

anti-apoptotic proteins (10).

Currently, different alterations on genes encoding components from NFƙB/IƙB pathway have been studied

(10,12–14), among which the most searched are the insertion/deletion polymorphism (-94 insertion/deletion ATTG,

rs28362491) in NFKB1, that is located between two important promoter regulatory elements; and the exchange of a

nucleotide on the 3’UTR region of NFKBIA (2758A > G, rs696) (10). This polymorphic variations were associated with

an increased risk for gastric cancer (15), colorectal cancer (16), melanoma (17), nasopharyngeal carcinoma (18) and

cervical squamous cell carcinoma (19), although discrepant effects are suggested in different populations (12) and

many studies reported conflicting results (20).

Then, given the importance of NF-ƙB/IƙB in regulating the apoptotic pathway, it has become clear that

variations in the NFKB1 and NFKBIA genes are decisive for their dysregulation (10) and, consequently, there is a

breakdown of the cell cycle, lack of proliferation control and inhibition of apoptosis. Based on this information, it is

possible to associate that the combined effects of NFKB1 and NFKBIA polymorphisms may result in increased

susceptibility to cervical tumors development.

Objectives

To analyze the polymorphisms of NFKB1 (rs28362491) and NFKBIA (rs696) in women positive for the

presence of HPV virus DNA treated by the cervical cancer prevention programs of the public health sector of the

Northern region of Paraná, according to sociodemographic/epidemiological, reproductive and sexual behavior

characteristics, as well as to the development of pre-malignant and malignant lesions.


This study was evaluated and approved by the Institutional Ethics Committee Involving Humans at State

University of Londrina, Londrina – Paraná (PR), registered under the CONEP 5231 process and obtaining the opinion

CEP/UEL 133/2012 (CAAE 05505912.0.0000.5231), which is in accordance with Resolution 196/96 of the National

Health Council/MS and Supplementary Resolutions. The study purpose and procedures will be explained to all

patients and written informed consent will be obtained prior sample collection.

This work is contained in the observational category, consisting of a prevalence/transverse study, in which a

total of 300 samples will be obtained, of which 100 will be positives for HPV, 150 negatives for the virus and 50

samples from cervical tumors, all collected in the city of Londrina-PR. The samples will be collected in the moment of

cervical cancer screening by Papanicolau’s test during gynecological routine.

Genomic DNA will be obtained from cytobrushes containing cervical secretion by using the DNAzol Reagent

(Invitrogen Inc., Carlsbad, CA, USA), according to the manufacturer’s instructions and from the peripheral blood by the

salting-out procedure.

Molecular detection of HPV will occur through the Polymerase Chain Reaction (PCR), under the conditions:

Buffer Invitrogen concentration 1x; 2 mM MgCl2; 0.19 mM deoxynucleotide triphosphate (dNTP); 750 nM of each

primer and 1.25 U of Taq DNA polymerase; initial cycle of 94.0ºC for 5 minutes, followed by 37 cycles of 30 seconds

at 94.0ºC, 1 minute at 55.0ºC, 1 minute at 72.0ºC and final extension step at 72.0ºC for 10 minutes, which will be

performed using the primers MY09 (5’-CGTCCMAARGGAWACTGATC-3’) and MY11 (5’-

GCMCAGGGWCATAAYAATGG-3’), that amplify a conserved region of approximately 450 base pairs (bp) of the HPV

L1 protein gene.

The virus-positive samples will be subjected to restriction enzyme cleavage to identify the various types of

HPV according to the technique described by Santiago et al. (2006) (21). Briefly, the restriction enzyme HpyCH4V will

be employed following the manufacturer’s instructions (overnight incubation at 37ºC).

Polymorphisms analysis will be performed using PCR followed by restriction fragment length polymorphism

analysis (RFLP) from DNA samples obtained from peripheral blood. The PCR for the two polymorphisms will be

performed under the conditions: Buffer Invitrogen concentration 1x; 1.5 mM MgCl2; 0.10 mM dNTP; 0.20 µM of each

primer and 1 U of Taq polymerase. The cycling for NFKB1 will consist of: initial cycle of 95.0ºC for 5 minutes, followed

by 35 cycles of 30 seconds at 95.0ºC, 30 seconds at 65ºC, 30 seconds at 72ºC and final extension step at 72.0 ºC for

5 minutes; while for NFKBIA it will be: initial cycle of 95.0ºC for 5 minutes, followed by 35 cycles of 30 seconds at

95.0ºC, 30 seconds at 61.0ºC, 40 seconds at 72.0ºC and final extension step at 72.0ºC for 5 minutes. The primers

54

used for the NFKB1 polymorphism are: foward (5’-TGGGCACAAGTCGTTTATGA-3’) and reverse (5’-

CTGGAGCCGGTAGGGAAG-3’), which amplify a 281 or 285 bp fragment, depending on the insertion/deletion; while

the primers used for NFKBIA are: foward (5’- GGCTGAAAGAACATGGACTTG-3’) and reverse (5’-

GTACACCATTTACAGGAGGG-3’), which amplify a fragment of 424 bp.

The amplified fragments of NFKB1 and NFKBIA will undergo enzymatic digestion by the enzymes PfIMI and

Haelll, respectively, and both will be incubated for 15 minutes at 37.0ºC. Fragments generated by polymorphic alleles

of NFKB1 have 240 and 45 bp and of NFKBIA, 306 and 118 bp.

PCR products of HPV molecular detection and NF-ƙB amplification, as well as of viral typing and

polymorphisms analysis, will be subjected to 10% polyacrylamide gel electrophoresis subsequently stained with silver

nitrate (AgNO3).

All statistical analyses will be carried out using the SPSS Statistics 22.0 software, all tests will be two-tailed,

considering a p-value <0.05 for statistical significance. Categorical data will be analyzed by χ2 test and continuous

data by parametric tests when normality is assumed or by non-parametric among non-normal data. Binary logistic

regression analysis, adjusted for several confounders will be used to estimate the association between polymorphism

genotypes and HPV infection.


This project will allow a better understanding of the polymorphisms role in the NFKB1 and NFKBIA genes

associated to the presence of HPV infection in the pathogenesis of premalignant and malignant cervical lesions

development. In addition, it will be possible to evaluate the frequency of HPV infection in our region and to correlate it

with socioeconomic, epidemiological and sexual behavior patients features, finally allowing the development of

effective prevention and promotion health measures among women attended by the public health network.

Acknowledgments

Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná and MCTI/CNPq.

References

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cancer risk: Evidence from 50 case-control studies. Oncotarget. 2017;8(6):9806–22.

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Samples in a Mexican Population. J Med Virol. 2015;87:2098–105.

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2006;37:38–46.

56

OXIDATIVE STRESS AND INFLAMMATORY RESPONSE DURING CARDIAC SURGEY WITH

CARDIOPULMONARY BYPASS

Karigyo, C.1, Murakami, A.

1, Cecchini, C.

2

1Department of Cardiac Surgery, Hospital Norte Paranaense, Arapongas (PR)

2Laboratory of Pathophysiology of Free Radicals, Department of Pathological Sciences, State University of Londrina

(PR)


Keywords: Oxidative stress, systemic inflammatory response, cardiopulmonary bypass, myocardial protection,

ischemia-reperfusion injury.

Abstract

Cardiopulmonary bypass, commonly used during cardiac operations, has been associated with a systemic

inflammatory response that can lead to several organic alterations and postoperative complications. The inflammation

added to the ischemia-reperfusion injury promotes the formation of free radicals that lead to oxidative stress damage.

The purpose of this work is to measure inflammatory and oxidative stress markers during different periods of the

operations and to compare the findings between distinct groups of patients in respect to the kind of heart disease

(ischemic or valvular) and left ventricle function (preserved or not).

Hypothesis

One: Inflammation and oxidative stress markers increase during cardiopulmonary bypass, but with more

intensity in coronary bypass group compared with heart valve operations (aortic, mitral) in preserved left heart

function.

Two: The presence of left heart dysfunction leads to a more pronounced degree of inflammation and oxidative

stress during cardiopulmonary bypass independently of the type of operation.

Introduction

Extracorporeal circulation is a fundamental part of many cardiac operations because it can provide a dry and

clean surgical field during cardiovascular procedures. The circuit replaces the heart and lung natural functions.

However, during cardiopulmonary bypass the patient is submitted to a number of non physiological conditions, and

these alterations can promote a systemic inflammatory response that may affect postoperative outcomes. The contact

of blood with the artificial circuit components activates the complement system, leading to the production of

chemotactic and vasoative substances and activation of leukocytes and production of inflammatory mediators and free

radicals. Besides the reactions provoked by the cardiopulmonary bypass, the aortic cross-clamp is a critical event

frequently performed during cardiac surgeries and it is tightly related to free radicals release and oxidative stress and

damage associated with these operations [1-3].

The myocardium submitted to ischemia-reperfusion is an important source of tumor necrosis factor α (TNF-α)

and interleukins IL-6 and IL-8, mainly when the left ventricle in dysfunctioning [3,4].

Oxidative stress markers are increased in cardiac operations utilizing cardiopulmonary bypass compared to

that without extracorporeal circulation (off-pump). Some studies found a more pronounced oxidative stress profile in

patients with dilated cardiomyopathy (with left ventricular ejection fraction <45%) [5].

Objectives

Compare quantitatively the concentrations of inflammatory mediators (IL-6, IL-8, TNF-α and C-reactive

protein) and oxidative stress markers (TBARS - thiobarbituric acid-reactive substance, superoxide dismutase (SOD),

catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPX) during different times of each cardiac

operation (T0=before cardiopulmonary bypass, T1=before aortic cross-clamping, T2=after aortic cross-clamping,

T4=after the end of cardiopulmonary bypass).


This project will be submitted to the State University of Londrina Ethics Committee and to the Hospital Norte

Paranaense Ethics Committee for approval before initiation.

57

This work is a prospective and observational study that will be composed by human cases/patients affected by

cardiovascular diseases and candidates for surgical treatment admitted to the Hospital Norte Paranaense of

Arapongas, Paraná, Brazil.

Inclusion criteria: three-vessel coronary artery disease, mitral valve disease (stenosis or regurgitation), aortic

valve disease (stenosis or regurgitation) – diagnostic parameters from coronary angiography and echocardiography.

Exclusion criteria: combined surgeries, reoperative procedures, previous myocardial infarction, infection, renal

failure, rheumatic disease or previous pulmonary embolism.

Surgical technique will be composed by median sternotomy, cannulation of both superior and inferior vena

cava and ascending aorta, placement of antegrade and retrograde cardioplegia cannulas for cold blood cardioplegia

for myocardial protection (Braile Biomédica). Blood samples will be collected from arterial lines in the pre-established

time periods (T0=before cardiopulmonary bypass, T1=before aortic cross-clamping, T2=after aortic cross-clamping,

T4=after the end of cardiopulmonary bypass).

In the statistical analysis, the categorical variables will be presented as percentage while the continuous

variables, as mean ± standard deviation, being applied the interquartile mean, when necessary. The general

characteristics, biochemical characteristics and frequency of cardiovascular events will be analyzed by Student's t test

for continuous variables and the chi-square test or Fisher's test for categorical variables. A p ≤ 0.05 value will be

considered significant for comparisons.


Expected results in accordance with our hypothesis:

One: elevated inflammatory markers concentrations in the group of preserved left ventricle ejection fraction

markedly in the coronary artery bypass grafting patients;

Two: in both coronary and valvular patients, the reduced left ventricle ejection fraction will be associated with

increased inflammatory and oxidative stress markers specially after the aortic cross-clamp time.

Acknowledgments

We thank the Hospital Norte Paranaense (Honpar) for supporting this project and allowing us to perform data

collection from its patients and the State University of Londrina for supporting this project in all of its phases.

References

1. Molyneux V, Klein AA. Equipment and monitoring for cardiopulmonary bypass. In: Ghosh S, Falter F, Perrino AC

Jr, eds. Cardiopulmonary bypass. 2nd

ed. Cambridge University Press; 2015. p. 1-23.

2. Paparella D, Yau TM, Young E. Cardiopulmonary bypass induced inflammation: pathophysiology and treatment.

An update. Eur J Cardiothorac Surg. 2002 Feb;21(2):232-44.

3. Biglioli P, Cannata A, Alamanni F, Naliato M, Porqueddu M, Zanobini M, et al. Biological effects of off-pump vs.

on-pump coronary artery surgery: focus on inflammation, hemostasis and oxidative stress. Eur J

CardiothoracSurg. 2003 Aug;24(2):260-9.

4. Wan S, DeSmet JM, Barvais L, Goldstein M, Vincent JL, LeClerc JL. Myocardium is a major source

of proinflammatory cytokines in patients undergoing cardiopulmonary bypass. J ThoracCardiovascSurg. 1996

Sep;112(3):806-11.

5. Simeunovic D, Seferovic PM, Ristic AD, Nikolic D, Risimic D, Seferovic J, et al. Evaluation

of Oxidative Stress Markers and Catecholamine Changes in Patients with Dilated Cardiomyopathy Before and

After Cardiopulmonary Exercise Testing. Hellenic J Cardiol. 2015 Sep-Oct;56(5):394-401.

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https://www.ncbi.nlm.nih.gov/pubmed/?term=Young%20E%5BAuthor%5D&cauthor=true&cauthor_uid=11825729

https://www.ncbi.nlm.nih.gov/pubmed?term=Paparella+D%5Bauthor%5D+AND+update&TransSchema=title&cmd=detailssearch

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https://www.ncbi.nlm.nih.gov/pubmed/?term=Cannata%20A%5BAuthor%5D&cauthor=true&cauthor_uid=12895618

https://www.ncbi.nlm.nih.gov/pubmed/?term=Alamanni%20F%5BAuthor%5D&cauthor=true&cauthor_uid=12895618

https://www.ncbi.nlm.nih.gov/pubmed/?term=Naliato%20M%5BAuthor%5D&cauthor=true&cauthor_uid=12895618

https://www.ncbi.nlm.nih.gov/pubmed/?term=Porqueddu%20M%5BAuthor%5D&cauthor=true&cauthor_uid=12895618

https://www.ncbi.nlm.nih.gov/pubmed/?term=Zanobini%20M%5BAuthor%5D&cauthor=true&cauthor_uid=12895618

https://www.ncbi.nlm.nih.gov/pubmed/12895618


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https://www.ncbi.nlm.nih.gov/pubmed/?term=Barvais%20L%5BAuthor%5D&cauthor=true&cauthor_uid=8800171

https://www.ncbi.nlm.nih.gov/pubmed/?term=Goldstein%20M%5BAuthor%5D&cauthor=true&cauthor_uid=8800171

https://www.ncbi.nlm.nih.gov/pubmed/?term=Vincent%20JL%5BAuthor%5D&cauthor=true&cauthor_uid=8800171

https://www.ncbi.nlm.nih.gov/pubmed/?term=LeClerc%20JL%5BAuthor%5D&cauthor=true&cauthor_uid=8800171

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https://www.ncbi.nlm.nih.gov/pubmed/?term=Simeunovic%20D%5BAuthor%5D&cauthor=true&cauthor_uid=26429367

https://www.ncbi.nlm.nih.gov/pubmed/?term=Seferovic%20PM%5BAuthor%5D&cauthor=true&cauthor_uid=26429367

https://www.ncbi.nlm.nih.gov/pubmed/?term=Ristic%20AD%5BAuthor%5D&cauthor=true&cauthor_uid=26429367

https://www.ncbi.nlm.nih.gov/pubmed/?term=Nikolic%20D%5BAuthor%5D&cauthor=true&cauthor_uid=26429367

https://www.ncbi.nlm.nih.gov/pubmed/?term=Risimic%20D%5BAuthor%5D&cauthor=true&cauthor_uid=26429367

https://www.ncbi.nlm.nih.gov/pubmed/?term=Seferovic%20J%5BAuthor%5D&cauthor=true&cauthor_uid=26429367


58

PHYSICAL EXERCISE EFFECT ON DAMAGING ACTION OF HYPERHOMOCYSTEINEMIA TO THE

MALE REPRODUCTIVE SYSTEM

Santos, D. P.1, Deminice, R.

2, Fernandes, G. S. A.

1

1 State University of Londrina /Department of Pathological Sciences

2State University of Londrina/Department of Physical Education


Keywords: Hyperhomocysteinemia, spermatogenesis, oxidative damage, physical exercise.

Abstract

Homocysteine is an amino acid formed from the demethylation of methionine. Unbalance or impairment in the

metabolism of homocysteine may generate elevation on its levels, characterizing the hyperhomocysteinemia. This

state might promote the creation of reactive oxygen species, lipid peroxidation and cell damage, additionally important

disturbances in the antioxidant defense system. Hyperhomocysteinemia correlates with difficulty in sperm function by

inducing oxidative stress, wich is considered one of the main contributors to this situation. High levels of homocysteine

into plasma and ejaculate has been related with dysfunctions in spermatogenesis and therefore male infertility.

Physical exercise has exhibited changes in the plasma concentrations of homocysteine, nonetheless its effect on this

amino acid has not been settled. The objective of this project is to assess the influence of physical exercise on

damaging action of hyperhomocysteinemia to the male reproductive system, experimentally induced by overload of dl-

Homocysteine thiolactone in Swiss mice.

Hypothesis

Physical exercise modulates the plasma homocysteine concentrations that will reflect positive changes,

turning up a compensatory behavior to the damaging action of hyperhomocysteinemia to the male reproductive

system.

Introduction

Homocysteine (Hcy) is an amino acid shaped from the demethylation of methionine derived from diet or its

catabolism, it has as aim the interaction of two metabolic pathways: remethylation and transsulfuration. In the

synthesis of homocysteine, a considerable proportion of methionine is activated by ATP to form S-adenosylmethionine

(SAM) in a reaction catalyzed by the enzyme S-adenosylmethionine synthase. The SAM primarily acts as an universal

methyl donor, involving a series of acceptors. The subproduct of this methylation reaction is S-adenosylhomocysteine

(SAH), it is hydrolyzed to generate adenosine and homocysteine (1). These reactions known as transmethylation took

place in all cells of the body. Hcy may be remethylated for methionine by enzymes betaine homocysteine

methyltransferase and methionine synthase, or undergo transsulfuration where homocysteine reacts with serine to

form cystathionine in an irreversible reaction catalyzed by cystathione-β-synthase enzymes. These reactions convert

Hcy to cysteine (2). Besides the importance of cysteine synthesis, the transsulfuration participates by catabolizing

excess homocysteine that was not required by methylation (1).

Unbalance or impairment in the metabolism of Hcy is able to generate elevation in its levels characterizing the

hyperhomocysteinemia (HHcy). Studies using experimental models of HHcy and endothelial cells incubated with Hcy

indicates wich high levels of this amino acid can promote the shaping of reactive oxygen species (ROS), especially

superoxide anion by the auto-oxidation of Hcy and/or cysteine, it can cause lipid peroxidation and cellular damage

(3,4). Authors have reported that Hcy can also cause important disorders in the antioxidant defense system (5). Aitken

et al. (6) suggest that HHcy correlates with defective sperm function because of Hcy induces oxidative stress which is

considered one of the main contributors to this situation. The presence of high levels of Hcy in plasma and ejaculate

has been related with dysfunctions in spermatogenesis and consequently male infertility (7,8). In a recent study,

Aitken et al. (6) have observed that the exposure of spermatozoa to Hcy is damaging to their motility and ferti lization

capacity, once the spermatozoa after exposure shows strong signals of generation of ROS by mitochondria, besides

indicating evidence of decreased activity of the paraoxonase type 1 enzyme (PON-1). PON-1 is an enzyme with

antioxidant activity whose low levels have also been associated with impaired parameters of concentration, motility

and sperm morphology (9).

Studies have shown changes in plasma Hcy concentrations when humans and rodents were subjected to high

intensity acute physical exercise (10,11,12,13). This change is related, in part, to the change in energy demand


59

caused by exercise that may reflect a modulation of methylation reactions and subsequent changes in plasma Hcy

concentrations. In this sense, studies have shown that the concentration of plasma Hcy is dependent oF the type,

volume and intensity of exercise, as well as age and level of physical conditioning (14,15). Deminice et.al. (16) in their

meta-analysis on the physical training approach and the behavior of Hcy did not obtained a consensus on the effects

that the physical training exerts on this amino acid. Although physical exercise is considered an adjuvant in the

therapy of various chronic-degenerative diseases, it is little is known about the effect physical exercise on HHcy-

induced damage.

Objectives

To evaluate the influence of physical exercise and HHcy, isolated or associated, on the male reproductive

system in Swiss mice.


Experimental Delimitation

We will use 80 Swiss male mice weighing aproximately 20 grams (g), they are from the Central Bioterium of

the State University of Londrina (UEL) and they will be kept in the Bioterium of the Department of Physical Education

and Sports of UEL. The animals will be used to perform two experiments as described below.

Experiment 1: It will be performed with 40 mice to determine the time of exposure to dl-homocysteine

thiolactone to generate HHcy, the mice will be divided randomly into 2 groups: control group (C, n = 20), HHcy group

(HHcy, n = 20). The animals will be accommodated in collective cages with a limit of 5 animals. Half of the animals in

each group (n = 10) will be euthanized after 30 days, and the remainder after 60 days. The access to water and food

will be free.

Experiment 2: To verify/investigate the effect of physical exercise on the damage induced by HHcy, 40 mice

will be randomly divided into 4 groups: control group (C, n = 10), group HHcy (HHcy, n = 10), exercised group (Ex, n =

10) and group HHcy Exercised (HHcyEx, n = 10). The animals will remain in individual boxes and the access to water

and food will be free. The time for animal euthanasia will be based on the previous experiment.

The diet will be with ration (Nuvilab CR-1, Nuvital) and water ad libitum. The cycle of 12 hours of light and dark

will be respected. All procedures will be performed after the approval of the project by the Animal Experimentation

Ethics Committee of the State University of Londrina.

HHcy Induction

The animals will receive 1 g / kg dl-homocysteine thiolactone in drinking water to induce HHcy. The solution

will be prepared each two days and the consumption will be measured by the difference between the quantity placed

and the remaining liquid in the drinking fountain (17). The animals will be weighed weekly for adjust in the dosage of

dl-homocysteine thiolactone.

Exercise

It will occur voluntarily in race on specific exercise wheels for mice adapted to conventional box. The distance

traveled will be recorded daily using rotation counters adapted to the exercise wheels.

Euthanasia and Collection of Biological Materials

For euthanasia, all animals will be anesthetized with ketamine 0.02g and xylazine 0.004g / kg body weight.

Subsequently, the abdominal cavity will be opened and blood will be collected from the inferior vein cava in its

abdominal portion in heparinized tubes. The right testicles and epididymis will be removed, weighed on a precision

analytical balance and frozen at -20ºC for further determination of the number of mature spermatids. The right ductus

deferens will be weighted and the spermatozoa will be obtained for analysis of sperm morphology. The spermatozoa

of the left ductus deferens will be used to evaluate sperm motility. The seminal vesicles (full and empty, without the

coagulating gland) and the ventral prostates will be weighed and discarded. The left testicles and epididymis (n = 5)

will be used for histopathological and morphometric analysis, and the other lefts testicles and epididymis (n = 5) will be

used to determine the inflammatory profile.

60

Analysis in the Plasma

Plasma concentrations of Hcy, methionine and cysteine will be determined by Gas Chromatography (GC-17A

Shimadzu®, Kyoto, Japan) by derivation of amino acids from the EZ: Faast Amino Acid Analysis kit (Phenomenex®).

It will be determined the concentration of SAM and SAH by High Performance Liquid Chromatography (HPLC) using

Phenomenex® C18 column. In addition, the testosterone hormone will be measured by radioimmunoassay (RIE) of

the double antibody using the TESTOSTERONE MAIA® kit (Biochem Immuno System).

Reproductive System Analysis

The daily sperm production per testicles and sperm transit through the epididymis will be calculated through

the right testicles and epididymis samples (18,19). The analysis of sperm morphology will be performed through of the

washed from the right ductus deferent, 200 sperm per animal will be analyzed and the morphological abnormalities

found will be classified into head abnormalities and tail abnormalities (19,20,21).

The left testicles and epididymis (n = 5) will undergo histological processing for morphometric and

histopathological analyzes. For histopathological analysis of the testicles, 3 non-consecutive transverse sections per

animal will be used, analyzing 100 cuts of seminiferous tubules per animal. The diameter of the seminiferous tubules

and height of the germinal epithelium will be evaluated through 10 sections of seminiferous tubules per animal in

stage IX of spermatogenesis. For the evaluation of the spermatogenic process, 100 transverse sections of

seminiferous tubules per animal will be analyzed. It will also be performed the count of Sertoli cell numbers in 20

seminiferous tubules per mice in stage VII of spermatogenesis (22). For the histopathological analysis of the

epididymis, 3 non-consecutive transverse sections of the organ per animal will be used and the epithelium and

interstitial aspect of the epididymis will be analyzed in a qualitative mode and for stereological analysis it will be

performed according to Weibel's method (23).

The measure of the presence of mast cells in the testicles and epididymis will be ocorred by 1% toluidine blue

staining. For the evaluation of myeloperoxidase and N-acetylglicosaminidase activity, 5 samples of the left testicles

and epididymis of the mice will be used (24).


The results will be presented in mean values ± standard deviation. One-way analysis of variance (ANOVA)

followed by Tukey post-hoc to establish possible differences between the experimental groups. The results will be

considered significant when p <0.05. The Origin 8.6® and GraphPad Prisma 5® programs will be used for proper

statistical analysis.

Expected Results

Although the relationship between exercise and homocysteine levels are not well settled, it is expected that

physical exercise will positively modulate the circulating homocysteine concentration and consequently bring down the

damaging effects of HHcy to the male reproductive system.

Acknowledgments

We gratefully acknowledge to financial support came from Coordination of Improvement of Higher Level

Personnel (CAPES).

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2- Brosnan JT, Jacobs RL, Stead LM, Brosnan ME. Methylation demand: a key determinant of homocysteine

metabolism. Acta Biochim. Pol., 2004:51, 405-413.

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some parameters of oxidative stress in rat hippocampus. Metab. Brain Dis. 2003:18, 147-154.

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Chapin RE, Heindel JH. 1993, San Diego, California: Academic Press, Inc, 334-343.

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male offspring in rats. Reprod. Toxic. 2011:32, 425–433.

23- Weibel ER. Principles and methods for the morphometric study of the lung and other organs. Lab. Invest. 1963:12,

131.

24- Casagrande R, Georgetti SR, Verri WA Jr, Dorta DJ, dos Santos AC, Fonseca MJ. Protective effect of topical

formulations containing quercetin against UVB-induced oxidative stress in hairless mice. J Photochem Photobiol B.,

2006:84, 21-27.

62

PREVALENCE OF MOUSE MAMMARY TUMOR VIRUS-LIKE (MMTV-LIKE) AND ITS IMPLICATION ON

THE PATHOGENESIS OF BREAST CANCER

Pereira, N. S.1, Amarante, M. K.

1, Watanabe, M. A. E.

1

1 Londrina State University, Laboratory of DNA Polymorphisms and Immunology, Department of Pathological

Sciences, Biological Sciences Center, Londrina, Parana, Brazil.


Keywords: breast tumor, mammary cancer virus, cytokines.

Abstract

Breast cancer (BC) is a complex heterogeneous disease whose evolution depends on the tumor-host

interaction. This type of cancer occurs when breast cells grow uncontrollably, and could invade nearby tissues or

promote metastasis. Recently, the identification of the sequence of mice mammary tumor virus (MMTV) and human

mammary tumor virus (MMTV-like) has supported the theory of viral etiology. It is known that virus associated tumor

cells can show viral antigens, representing a potential antigenic target, indicating a promising treatment for BC. It is

known that viruses can activate immune response and drive the production of some cytokines, like interferons. In this

context, the present project aims to investigate the presence of MMTV-like virus, as well as gene expression and

cytokines levels, like IFN-alpha, IFN-beta, IFN-gamma and IL-18, in BC patients from southern Brazil. This project was

approved by Ethic Committee from UEL (CAAE 47709015.2.0000.5231). The 350 BC patients, will be grouped based

on tumor positivity for hormonal receptors (ER/PR) and HER2. DNA will be obtained from the surgical excision tumor

and normal tissue. For viral DNA, sequence referred to env gene will be determinate by nested and viral RNA

detection will be performed by semi-nested PCR. All positive samples will be sequenced. Cytokines will be analyzed

by flow cytometry. Therefore, at the end of this project, we intend to obtain relevant data about MMTV-like virus

association in breast cancer increasing the knowing on tumor microenvironment.

Hypothesis

Preliminary laboratory data indicate the presence of the viral DNA fragment in some breast cancer tissues.

Thus, the presence of virus in the breast tumor and normal tissue, which can have great impact and implications in

prevention, diagnosis and also in the tumor therapy and in the development of BC.

Introduction

Breast cancer (BC) is a serious public health problem, considering the number of women who are diagnosed

and deaths that occur annually from this disease. In Brazil, the estimate is about 57.960 new cases of breast cancer

between 2016-2017 (1). The clinical course and survival of BC vary for each patient and depend on a complex series

of factors. Risk factors for this disease include age, parity, age of first gestation, breastfeeding, age of menarche and

menopause, treatment with estrogen, environment, stress, immune status and nutrition. Family history is another

important risk factor, emphasizing the genetic aspect of this disease (2, 3). Metastatic disease accounts for the

majority of these patients death (4).

It is generally accepted that environmental factors play a role in the etiology of various types of cancer. Some

viruses, due to their complexity, are considered etiological agents of some neoplasia, such as cervical cancer (Human

Papilloma Virus), lymphoma (Epstein-Barr Virus) and leukemias (Human T-Cell Lymphotropic Virus Type I).

Retroviruses are good candidates to participate in the etiology of some diseases that, like breast cancer, appear

sporadically or hereditarily. The life cycle of these viruses involves a mandatory stage in which the provirus is inserted

into the host genome, and this process is permanent (5).

Mouse mammary tumor virus (MMTV), from Betaretroviridae family, was discovered by Bittner (6) in 1936.

This virus is the most common cause of BC and T-cell lymphomas in mice (7, 8). MMTV is a retrovirus that has a long

terminal repeat sequence (LTR), with approximately 1.3kb. This region encodes a protein, a superantigen (Sag) (9),

that activate specific T lymphocyte Vβ subsets.

Since MMTV was discovered, several researchers have investigated for a similar virus in human BC. Some

studies have indicated that a virus similar to MMTV, the MMTV-like virus, which is also known as human mammary

tumor virus (HMTV), may associated with human BC (10, 11).

The possible role of MMTV-like sequences in human BC remains controversial. The env gene sequence is

represented by an open reading frame (ORF) of approximately 1.6 kb, being 94-99% identical to the env gene of


63

MMTV and, when present in BC samples, can indicate the presence of the virus (12). This sequence was found in

varying proportions in breast tumor tissues, ranging from 0 to 74% of cases, but rarely in normal tissues (13).

Some viruses can activate immune cells, like dendritic cells, macrophage and lymphocytes, and drives the

production of pro-inflammatory cytokines. Interferons (IFNs) are secreted cytokines that have antiviral effects in an

innate or adaptive response. In class I, IFN-alpha and IFN-beta, which can upregulate the expression of several genes

and, in combination, create a state of antiviral activity. Type II IFN, IFN-gamma, that is secreted by activated T cell

and natural killer cells promoting antiviral cellular immune response (14). IFN-gamma production is controlled by

cytokines secreted by professional antigen-presenting cells (APCs), like interleukin (IL) 12 and 18. Although both

types of IFN (type I and II) are crucial in the immediate cellular response to viral infection, the activities of IFN-gamma

become important later in coordinating the immune response and establishing an antiviral state for longer term (15).

Since inflammation is an important component of many, if not all, diseases, we consider of great importance to

determine whether the MMTV-like genome should represent a stimulus to produce antiviral cytokines and

chemokines.

The involvement of MMTV-like in the BC pathogenesis has been investigated in many studies. However,

additional studies should be performed to clarify its role as a possible etiologic agent related to BC and its role in the

treatment and prognosis of patients diagnosed with BC.

Objectives

To investigate the presence of MMTV-like virus, as well as genic expression and levels of circulating

cytokines, like IFN-alpha, IFN-beta, IFN-gamma and IL-18, in BC patients from southern Brazil.


Ethical aspects: this project was approved by Ethic Committee from UEL (CAAE 47709015.2.0000.5231).

Women with diagnosis of BC was invited to participate in this research project during the clinical care in the

specialized services and in Londrina Cancer Hospital. The form of free-informed consent was signed by all donors

before the collections. The 350 BC patients, will be grouped based on tumor positivity for hormonal receptors (ER/PR)

and HER2.

DNA extraction: DNA will be obtained from the surgical excision tumor and normal tissue, through Biopur

MiniSpin Plus kit (Biometrix, Curitiba, Brazil). The DNA samples will be quantified by spectrophotometry in the Nano

Drop ND-2000c (Uniscience, EUA).

Detection of the env gene sequence from MMTV-like: sequence referred to env gene from MMTV-like will be

determinate by nested PCR, using four oligonucleotides described by Nartey, Moran (16). The reaction will take place

on thermocycle Biocycler (Biosystems, Brazil), on the following conditions: 95ºC for 15 minutes; followed by 35 cycles

of 95ºC for 30 seconds, 55ºC for 30 seconds and 72ºC for 1 minute; and a final extension of 72ºC for 10 minutes. The

amplified products will be visualized in 10% polyacrylamide gel stained with silver nitrate.

RNA extraction: RNA extraction will be performed from the breast tissue from patients diagnosed with BC and

from normal tissue, using TRIzol-LS (Invitrogen, EUA), according with instructions from the fabricant. The RNA

samples will be quantified by spectrophotometry in the Nano Drop ND-2000c (Uniscience, EUA).

cDNA synthesis: reverse transcription will be performed from 500ng of RNA with 2,5M of primer oligo [dT]

and 50U of Mulv reverse transcriptase (GeneAmp RNA PCR kit, Perkin Elmer). The reaction will be carried out in

specific buffer (50mM Tris-HCL pH 8.3, 75mM KCl, 3mM MgCl2, 200M dNTP) and submitted in thermocycler, at 45ºC

for 60 minutes.

Detection of MMTV-env/LTR: the junction sequence MMTV-env/LTR will be detected using 250ng of LTR3

cDNA and primers LTR-5MR (5′-ATAAGTCCCTGGTTGCCACC-3′) and ENV-3LR’ (5′-CATATGTGCTGCTACCTGTA-

3′), on the following conditions: 95ºC for 1 minute, 35 cycles of 95ºC for 30 seconds, 62ºC for 1 minute and 68ºC for

10 minutes. The expected product will be 1378pb. The semi-nested PCR reaction will use the ENV-3LR’ and LTR-1R’

(5′-TCAGGAGGAAGGTCGAGTTCT-3′) primers also following the above protocol. The expected product of 1018pb

will be visualized in agarose gel attained with blue green.

64

Sequencing: nested-PCR products for MMTV-like will be purified using PureLink™ PCR Purification Kit

(Invitrogen, Carlsbad, CA, USA), following the manufacturer's instructions. Sequencing reaction will be performed

using the BigDye® Terminator v3.1 kit (Applied Biosystems®, Foster City, CA, USA), 50ng of template and 5 pM

primer in a final volume of 10 μl. PCR conditions will be as follows: 10 seconds at 95ºC, 30 cycles of 20 seconds at

95ºC, 20 seconds at 50ºC and 1 minute at 60ºC. The amplified fragments will be sequenced on Genetic Analyzer

3500XL 24 capillaries (Applied Biosystems®, Foster City, CA, USA).

Flow cytometry analysis: soluble cytokines in tumor and normal adjacent tissue from individuals analyzed for

MMTV-like will be quantified using immunoassay based on beads (Cytometric Bead Array method – CBA, BD

Pharmingen CA, USA) The assay will be conducted using 70μL of the sample, and a standard 10-point curve (ranging

from 0 to 5000pg/mL) will be included for each quantified cytokine. The cytokines that will be evaluated are: IFNα,

IFNβ, IFNγ and IL-18. The Accuri C6 flow cytometer (BD Pharmingen) will be used. The FCAP Array software (version

3.1, BD Pharmingen) will be used to create the standard curves of each cytokine and convert the fluorescence

intensity (MFI) values to cytokine concentration.

Statistical analysis: the statistical analyses will be realized using SPSS Statistics 17.0 programme (SPSS Inc.,

Chicago, Illinois, EUA) and GraphPad Prism 5 (GraphPad, EUA), using descriptive statistics and association analysis

by Odds Ratio (OR) and Relative Risk (RR) and hypothesis test (Mann-Whitney, t-Student). For all data, the

significance level adopted will be p <0.05.


Immunology and Virology can greatly contribute to clarify the etiology of multifactor diseases, such as BC.

Preliminary results from our laboratory show the presence of MMTV-like env gene in some samples of breast tumor

tissue, corresponding to 18,8% of samples analyzed. Therefore, at the end of this project, we intend to obtain relevant

data about MMTV-like virus association in BC, increasing the knowing on tumor microenvironment.

Acknowledgments

CNPq, CAPES and Cancer Hospital of Londrina.

References

1. INCA INdC-. Estimativa 2016: incidência de câncer no Brasil. Rio de Janeiro2015.

2. Clark GM. Prognostic and Predictive Factors for Breast Cancer. Breast Cancer. 1995;2(2):79-89.

3. Hondermarck HV-E, A S.; Révillion, F.; Lemoine, J.; El-Yazidi-Belkoura, I.; Nurcombe, V.; Peyrat, J. P.

Proteomics of breast cancer for marker discovery and signal pathway profiling. Proteomics. 2001;1(10):1216-32.

4. Redig AJ, McAllister SS. Breast cancer as a systemic disease: a view of metastasis. Journal of Internal

Medicine. 2013.

5. Labat ML. Possible retroviral etiology of human breast cancer. Biomedicine & Pharmacotherary. 1998;52(1):6-

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6. Bittner JJ. Some Possible Effects of Nursing on the Mammary Gland Tumor Incidence in Mice. Science.

1936;84(2172):162.

7. Lawson JST, D. D.; Carpenter, E.; Ford, C. E.; Rawlinson, W. D.; Whitaker, N. J.; Delprado, W. Presence of

mouse mammary tumour-like virus gene sequences may be associated with morphology of specific human breast

cancer. Journal of Clinical Pathology. 2006;59(12):1287-92.

8. Lawson JSG, W. H.; Whitaker, N. J. Viruses and human breast cancer. Future Microbiology. 2006;1(1):33-51.

9. Ross SR. Mouse Mammary Tumor Virus Molecular Biology and Oncogenesis. Viruses. 2010;2:2000-12.

10. Baggiolini M, Loetscher P. Chemokines in inflammation and immunity. Immunology today. 2000;21(9):418-20.

11. Fridman WH, Galon J, Pages F, Tartour E, Sautes-Fridman C, Kroemer G. Prognostic and predictive impact

of intra- and peritumoral immune infiltrates. Cancer research. 2011;71(17):5601-5.

12. Mok MTSL, J. S.; Iacopetta, B. J.; Whitaker, N. J. Mouse mammary tumor virus-like env sequences in human

breast cancer. International journal of cancer. 2008;122(12):2864-70.

13. Hachana MT, M.; Ziadi, S.; Amara, K.; Gaddas, N.; Mokni, M.; Korbi, S. Prevalence and characteristics of the

MMTV-like associated breast carcinomas in Tunisia. Cancer Letters. 2008;271(2):222-30.

65

14. Randall RE, Goodbourn S. Interferons and viruses: an interplay between induction, signalling, antiviral

responses and virus countermeasures. Journal of General Virology. 2008;89(1):1-47.

15. Schroder K, Hertzog PJ, Ravasi T, Hume DA. Interferon-gamma: an overview of signals, mechanisms and

functions. Journal of leukocyte biology. 2004;75(2):163-89.

16. Nartey T, Moran H, Marin T, Arcaro KF, Anderton DL, Etkind P, et al. Human Mammary Tumor Virus (HMTV) sequences in human milk. Infectious agents and cancer. 2014;9:20.

66

ABSTRACTS SELECTED FOR

ORAL PRESENTATION

67

ASPIRIN ANTI-INFLAMMATORY EFFECT ON MUSCLE TISSUE DURING MURINE Trypanosoma cruzi

EXPERIMENTAL INFECTION

Oda, J.Y.

1; Belém, M.O.

2; Silva, A.V.

1; Uliana, C.H.

1; Sant´Ana, D.M.G.

3; Pinge-Filho, P.

4; Araújo, E. J. A

5 1Universidade Federal de Mato Grosso do Sul, School of Medicine, Três Lagoas, MS, Brazil

2Universidade Federal do

Ceará, Department of Physiology and Pharmacology, Fortaleza, CE, Brazil 3Universidade Estadual de Maringá, Department of Morphological Sciences, Maringá, PR, Brazil

4Universidade Estadual de Londrina, Department of Pathology, Londrina, PR, Brazil

5Universidade Estadual de Londrina, Department of Histology, Londrina, PR, Brazil


Keywords: Chagas Disease, Aspirin, muscular tissue, experimental infection by T. cruzi.

Introduction and objectives: Chagas' disease (CD) represents an important public health problem in Brazil due to

arising of new cases every year and also because there is no cure. CD infection has two well-defined phases. The

acute phase lasts approximately two to three months with non-specific symptoms. Most of the patients keep

asymptomatic during the chronic phase, however 30 to 40% develop the symptomatic form of CD after 10-20 years

post-infection. Intense inflammatory process with secondary lesions happens in various tissues throughout the

infection. The aim of this study was to evaluate whether the use of low doses of aspirin (ASA) during the acute (20 mg

/ kg) or chronic (50 mg / kg) phase of the murine T. cruzi experimental infection would reduce the inflammatory

process in skeletal, cardiac and smooth muscle tissues.

Material and methods: All procedures were approved by the Ethical Committee on the Use of Animals of the State

University of Londrina (UEL, protocol number: 156/2012). Young male Swiss mice were distributed into groups

receiving PBS or treated with low doses of aspirin during the acute (20 mg / kg, ASAEarly) or chronic (50 mg / kg,

ASADelayed) Y strain T. cruzi infection phases. After 75 days of infection, the skeletal muscle (biceps femoris), cardiac

(heart) and smooth (colon) were collected in order to quantify inflammatory foci in histological sections. Four semi-

serial 5-micrometer-sections from each tissue/mice were stained with HE. Ten microscopic fields (2 mm2) with 400x

magnification per tissue/animal were evaluated. It was counted the inflammatory cells found in the three tissues

evaluated. Groups were compared using two-way ANOVA followed by Tukey's post-test, considering p <0.05.

Results: Untreated infected mice showed an intense inflammatory infiltrate (p <0.01) in all muscle tissues evaluated.

Both treatments reduced the number of inflammatory foci in the three types of muscle tissue (p <0.05). ASAEarly

treatment reduced the inflammatory process in the skeletal muscle in 85% (p <0.01), cardiac muscle in 80% (p <0.01)

and smooth muscle in 60% (p<0.05). Meanwhile, ASADelayed treatment reduced inflammatory process in 55% (p <0.05)

in all muscle tissues evaluated.

Conclusion: It is concluded that the treatment with low doses of aspirin (ASA) reduced the inflammatory response in

the three types of muscle tissue of mice infected with T. cruzi.

Grant: Fundação Araucária and UFMS – Universidade Federal de Mato Grosso do Sul

68

BUDLEIN A INHIBITS EXPERIMENTAL GOUT ARTHIRITS BY TARGETING NF-ΚB ACTIVATION

IN MICE

Fattori, V.1; Zapelon, A.C.

1; Ruiz-Miyazawa, K.W.

1; Casagrande, R.

2; Arakawa, N.S.

2; Verri, W.A.

1 1Universidade Estadual de Londrina, Departamento de Ciências Patológicas, Londrina, PR, Brasil

2Universidade Estadual de Londrina, Departamento de Ciências Farmacêuticas, Londrina, PR, Brasil

[email protected]

Keywords: synovitis, NLPR3 inflammasome, sesquiterpene lactone, experimental arthritis

Introduction and objectives: Gout is the most common inflammatory arthritis worldwide. Gout is a painful

inflammatory disease induced by the deposition of monosodium urate (MSU) crystals in the joints and peri-articular

tissues. In general, gout arthritis is well controlled if properly managed, but, in spite of that, gout can trigger acute

flares that are usually the reason patients first seek medical attention. In fact, acute gout flares are recognized as one

of the most painful experiences known, on the same level as childbirth or visceral colic. If left untreated, continuing

urate crystal deposition not only causes further flares but also, and importantly, could cause eventual irreversible joint

damage with chronic symptoms and disability. Budlein A (BudA) is a sesquiterpene lactone with analgesic and anti-

inflammatory properties related to the inhibition of pro-inflammatory cytokines and leukocytes recruitment. Therefore,

we aimed at evaluating the effect of budlein A in a model of MSU-induced gout arthritis in mice.

Material and methods: Experiments were conducted in male Swiss mice or male C57BL/6 and LysM-GFP+ mice

with State University Ethics Committee on Animal Research and Welfare approval under process number

14544.2013.44. Mice (n=6 per group) were treated with BudA (1 or 10 mg/kg, per oral) or vehicle 1h before stimulus

with MSU (100µg/10µL, intra-articular). Mechanical hyperalgesia (electronic aesthesiometer) and knee joint edema

(caliper) were evaluated 1-15h after MSU injection. These data were analyzed using two-way ANOVA followed by

Tukey post-test. MSU-induced leukocyte migration in the joint wash was evaluated 15h after stimulus by counting

total leukocytes in Neubauer chamber, in the histopathological analysis (by HE staining), and in the joint wash of

MSU-stimulated LysM-GFP+ mice by confocal microscopy. The mRNA expression of the proinflammatory cytokines

IL-1β and TNF-α and the components of the inflammasome platform NLRP3, ASC, and caspase-1 were evaluated by

RT-qPCR. NF-κB activation was evaluated by ELISA. In vitro analysis using LPS-primed bone marrow-derived

macrophages (BMDMs) was performed 5h after stimulation with MSU crystals to determine mature IL-1β secretion by

ELISA. For these experiments, BMDMs were incubated with BudA at concentrations 1, 3, or 10μg/mL. These data

were analyzed using on-way ANOVA followed by the Tukey post-test

Results: BudA at 10 mg/kg reduced mechanical hyperalgesia and edema. Moreover, BudA decreased leukocytes

recruitment to the knee joint as evaluated by total leukocytes count in Neubauer chamber in 57%, reduced

inflammatory infiltrate as observed in HE staining in 60%, and reduced the intensity of fluorescence in MSU-stimulated

LysM-GFP+ mice in the confocal microscopy. Furthermore, BudA reduced the production of IL-1β and TNF-α both in

vivo (50% and 60%, respectively) and in vitro (41% and 65%, respectively). Also, BudA inhibited the expression of the

inflammasome components NLRP3 in 50%, ASC in 44%, and caspase-1 in 52% and reduced NF-κB activation in

40%.

Conclusion: Therefore, the sesquiterpene lactone BudA ameliorates MSU-induced gout arthritis by targeting NF-κB

activation and downstream TNF-α and IL-1β production. Also, budA reduced IL-1β maturation by inhibiting the mRNA

expression inflammasome components NLRP3, ASC, and Caspase-1. Thus, budA certainly deserves further studies

on its pre-clinical and clinical applicability.

Grants: Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de

Desenvolvimento Científico e Tecnológico (CNPq), Ministério da Ciência, Tecnologia e Inovação (MCTI), Secretaria

da Ciência, Tecnologia e Inovação (SETI), Fundação Araucária, and Paraná State Government.

69

DIOSMIN AMELIORATES ZYMOSAN-INDUCED ARTHRITIS AND PERITONITIS BY REDUCING

LEUKOCYTE RECRUITMENT AND PRO-INFLAMMATORY CYTOKINES PRODUCTION IN MICE

Ambrosio, F.1; Fattori, V.

1; Borghi, S.M.

1; Lourenco-Gonzalez, Y.

1; Casagrande, R.

2; Verri, Waldiceu A, Jr.

1

1 Universidade Estadual de Londrina, Departmento de Ciências Patológicas, Londrina, PR, Brazil.

2 Universidade Estadual de Londrina, Hospital Universitário, Departmento de Ciências Farmacêuticas, Londrina, PR,

Brazil.

Email: [email protected]

Keywords: zymosan, arthritis, inflammation, diosmin, flavonoids, synovitis.

Introduction and objectives: Rheumatic diseases, represented by varied forms of arthritis and other musculoskeletal

disorders, affect millions of people around the word. Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory

disease associated with debilitating pain, cartilage destruction, and loss of joint function. Although remarkable

progress has been made in the clinical treatment of RA, long-term administration of anti-rheumatic drugs still

possesses significant drawbacks, including high dose and high frequency of drug administration, high cost as well as

heart, liver, and kidney problems. Diosmin is a flavonoid present in citrus fruits and presents potent antioxidant and

anti-inflammatory activity, which can be partially attributed to its capacity of inhibiting NF-κB activation. Importantly,

flavonoids such as diosmin, usually present low toxicity, which allows their use in high doses. Therefore, in this study,

we investigated the efficacy of diosmin in two different models: zymosan-induced arthritis and peritonitis.

Material and Methods: Experiments were conducted on male Swiss mice. Animal care and handling procedures

were conducted according to International Association for the Study of Pain (IASP) guidelines and with Londrina

State University Ethics Committee on Animal Research and Welfare approval under process number 678.2017.63.

For the arthritis model, mice were treated with diosmin (10, 30, or 100 mg/kg, per oral) 60 min prior to the

administration of zymosan (500 μg/knee joint, intra-articular). Mechanical hyperalgesia and edema were evaluated 1-

7h after the stimulus, by the electronic version of von Frey filaments and caliper, respectively. Knee joint tissue was

collected 7h after intra-articular stimulus for determination neutrophil recruitment by measuring myeloperoxidase

(MPO) and for the determination the peripheral production of cytokines: TNF-α, IL-33, IL-6, and IL-1β by ELISA. In the

peritonitis model, mice were treated with diosmin (10, 30, or 100 mg/kg, per oral) 60 min prior to the administration of

zymosan (1 mg/cavity, intraperitoneal). At the end of 7h, the peritoneal wash was collected for evaluation of

leukocytes recruitment and cytokine production (TNF-α, IL-1β, and IL-6) by ELISA.

Results: Treatment with diosmin decreased zymosan-induced mechanical hyperalgesia, edema, and MPO activity in

the knee joint in a dose-dependent manner. Given that the dose of 100mg/kg was the most effective, this dose was

chosen for the evaluation of cytokines production. Diosmin at 100 mg/kg reduced zymosan-induced TNF-α in 43.5%,

IL-33 in 41.3%, IL-6 in 39.5%, and IL-1β in 38.3% in the knee joint. In the peritonitis model, diosmin decreased

zymosan-induced leukocyte recruitment (total leukocytes, polymorphonuclear, and mononuclear cells) to the

peritoneal cavity in a dose-dependent manner. Furthermore, diosmin also reduced zymosan-induced TNF-α in 27.6%,

IL-1β in 20.4%, and IL-6 in 30% in the peritoneal wash.

Conclusion: This study demonstrates that diosmin improves zymosan-induced arthritis and peritonitis by inhibiting

leukocytes recruitment and pro-inflammatory cytokine production, suggesting this flavonoid as a potential therapeutic

molecule for the treatment of inflammatory conditions.





70

EFFECTS OF CREATINE SUPPLEMENTATION ON DIFFERENT MUSCLES IN WALKER-256 TUMOR-

BEARING RATS

Cella P.S.1; Marinello P.C.

1-2; Borges F. H.

2; Ribeiro D. F.

1; Testa M. T. J.

1; Padilha C. S.

1; Duarte J.A.; Cecchini R.

2;

Guarnier F. A.,2; Deminice, R.

1 1 State University of Londrina, Department of physical education, Londrina, PR, Brazil.

2 State University of Londrina, Department of General Pathology, Londrina, PR, Brazil.

University of Porto, CIAFEL, faculty of sport, Porto, Portugal.


Keywords: Creatine supplementation, cancer cachexia, skeletal muscle, muscle wasting

Introduction and objectives: Many evidences of the potential therapeutic application of creatine supplementation in

reducing or even reversing body mass loss in muscle wasting diseases such as Duchenne dystrophy, myasthenia

gravis and amyotrophic lateral sclerosis have been reported. However, little is known about the effects of creatine

supplementation on muscle wasting induced by cancer cachexia. The aim of this study was to investigate the creatine

supplementation ability to protect against muscle loss in Walker-256 tumor-bearing rats.

Material and methods: Wistar rats were weighed and randomly assigned in four groups (n=8/group): control (C),

tumor-bearing (T), control supplemented with creatine (Cr) and tumor-bearing supplemented with creatine (TCr).

Creatine was added in water (8g/L) for 21 days. After 11 days of supplementation, tumor cells were inoculated in

animals from the T and TCr groups. Ten days after tumor implantation, animals were weighed and euthanized. Blood

samples were collected to leukocyte count. Water and food intake were daily monitored. The muscles soleus,

gastrocnemius, plantar, anterior tibial and extensor hallucis longus (EDL), spleen and tumor were dissected and

weighed for further analysis. Creatine and phosphocreatine were quantified in the white part of the gastrocnemius and

in tumor (central and peripheral regions). For statistical analysis, it was used student t test to compare two groups,

one-way ANOVA with Tukey post-test to identify changes between treatments and two-way ANOVA for repeated

measure to analyze the curves of water and food intake p < 0.05 was considered statistically significant. All

experimental procedures were approved by the Animal Experimentation Ethics Committee of State University of

Londrina (CEUA nº 17628.2012.6).

Results: The T group presented reduced body weight (-21%), loss of plantar mass (-13%), soleus muscle atrophy

(cross sectional area -34%), increased spleen size (181%) and leukocytosis (115%) (P<0,05). However, creatine

supplementation (TCr) prevented the reduction in body weight, reduced cachexia index, prevented plantar loss and

protect against muscle atrophy (23%) (TCr vs Cr), increased gastrocnemius mass and reduced tumor size (46%). In

food and water intake, muscles soleus, anterior tibial and EDL no differences were found between groups.

Phosphocreatine was found higher (P<0.05) in the white part of the gastrocnemius of T, Cr and TCr in relation C

group. In the central region of the tumor, creatine and phosphocreatine were increased in the supplemented group.

Leukocytosis and spleen size did not change with supplementation. Parameters of tumor aggressiveness, such as

proliferation and apoptosis of tumor cells, capsule size and the presence of fibrotic tissue in tumor were assessed but

no statistical difference were found.

Conclusion: The Walker-256 tumor cells promotes cachexia characterized by muscle atrophy and body weight loss,

splenomegaly and leukocytosis. Creatine supplementation partially reduced the cachexia promoted by tumor

development, preventing body weight loss, muscle atrophy, reducing tumor growth and promoting increases in

gastrocnemius mass.

Grants: CAPES (processo nº 88881.068035/2014-01).

71

EPIDEMIOLOGICAL ASPECTS OF SCHISTOSOMIASIS IN NORTH OF PARANÁ

Camargo, L. C.1; Guidugli, G.S.

1; Tomiotto-Pellissier, F. M.

1; Oliveira, F. J. A.

1; Pavanelli, W.R.

1; Costa, I.N.

1;

Conchon-Costa, I.1; Murad, W,. A.

2; Melanda, F. N.

1

1State University of Londrina, Department of Pathology Sciences, Londrina, PR, Brazil

2State University of Londrina, Department of Clinical and Toxicological Analysis, Londrina, PR, Brazil

e-mail: [email protected]

Keywords: Schistosomiasis, neglected disease, prevalence, descriptive epidemiological study.

Introduction and objectives: Schistosomiasis is a parasitic disease caused by flatworms of the genus Schistosoma.

About human parasites, only S. mansoni occurs in South and Central America. The introduction of schistosomiasis in

Brazil occurred due to the slave trade, which is why the disease spread through the Brazilian northeast, and later, to

the region of Minas Gerais. Over the years, there has been dispersion to other regions, including northern Paraná,

and is now considered an important public health problem in the country. It is estimated that about 1.5 million people

live in areas at risk in Brazil. Objective of this study was to describe the occurrence of schistosomiasis in the city of

Londrina, according to the prevalence and main epidemiological characteristics of the registered cases.

Material and methods: This is a descriptive epidemiological study. Fecal samples were collected from residents of

the city of Londrina, regions urban and rural, between January 2010 and December 2015. The samples were sent to

the parasitology laboratory of the University Hospital of Londrina / PR and the tests Parasitological findings of

Hoffmann, Pons & Janer (1934), Faust & Cols (1939) and Kato & Katz (1972). The descriptive analysis was performed

by means of absolute and relative frequencies, measures of central tendency and dispersion.

Results: Between 2010 and 2015, stool sample tests were performed on 12,904 people, of whom 29 (0.22%) were

positive for the presence of S. mansoni eggs. In all years cases were registered, with 2013 being the year of greatest

occurrence (10 cases). Among the positive cases, 20 (68.95%) were men and nine (31.05%) were women. Therefore,

the prevalence of patients diagnosed with schistosomiasis was higher among males. The mean age of the positive

cases was 37.7 (±17.69), occurring mostly in people over 15 years old (93.10%). The involvement of the adult

population may be related to the occupational exposure of these individuals.

Conclusion: Together, the data show that in the Londrina region, between 2010 and 2015, there was a low

prevalence of schistosomiasis. However, the disease occurred persistently, affecting mainly adults. Therefore, it is

emphasized the importance of knowing the epidemiological aspects of the disease so that preventive and control

actions are more effective.

72

THE EFFECTS OF TRICLOSAN EXPOSURE ON ESTROUS CYCLE AND SEXUAL BEHAVIOR IN TWO

GENERATIONS OF FEMALE WISTAR RATS

Montagnini, B. G1; Aleixo, J. F

1; Pereira, M. R. F

1; Costa, N. O

1; Borges, L. I

1; Gerardin, D. C. C

1

1 Universidade Estadual de Londrina, Department of Physiological Sciences, Londrina, PR, Brazil

[email protected]

Keywords: endocrine disruptor, triclosan, estrous cycle, sexual behavior.

Introduction and objectives: Triclosan (TCS) is a trichlorinated phenoxyphenol with broad-spectrum antimicrobial

action widely used in cosmetics and other personal care products such as shampoos, toothpaste, soaps and lotions.

The structural similarity to other known hormonal disrupters, such as bisphenol-A, may predict the potential

(anti)androgenic or (anti)estrogenic effect for TSC. Therefore, the present study aims to evaluate the TCS effects on

the estrous cyclicity and sexual behavior in a two-generation study.

Material and methods: Female and male Wistar rats (n = 17 animals/group/gender) of the parental (F0) generation

were daily treated with TSC at doses of 0.8, 2.4 and 8.0 mg/kg or corn oil (control), by gavage, from the postnatal day

(PND) 49, during mating (70 days after the beginning of the treatment), until weaning of F1 offspring. Treatment

continued in F1 animals from PND 22 the same way as in F0 generation. Vaginal smears were obtained daily over a

period of 21 days (PND 86 to 106). The female sexual behavior (starting on PND 120) was performed, lordosis

quotient ([number of lordosis/total mounts] × 100) and lordosis scores (total number of lordosis points/total number of

lordosis responses) were assigned for each test. Data ± SEM were compared by ANOVA (*p < 0.05) (CEUA:

283.2015.27).

Results: The coefficients of estrus, metaestrus/diestrus and proestrus, as well as the estrous cycle length were

similar between groups in both F0 and F1 generations (p > 0.05). No difference in the lordosis quotient (F (3,39) = 1.378,

p = 0.256) was observed in F0 females, however, a significant statistical decrease was detected in the lordosis score

(CTR group: 2.21 ± 0.17/ TCS 0.8 group: 2.40 ± 0.15/ TCS 2.4 group: 1.56 ± 0.14/ TCS 8.0 group: 2.27 ± 0.14) of

TCS 2.4, compared to their control group. In the F1 generation, the lordosis coefficient decreased in the females of

TCS 2.4 group (CTR group: 89.00 ± 3.79/ TCS 0.8 group: 75.00 ± 6.01/ TCS 2.4 group: 67.00 ± 5.78/ TCS 8.0 group:

83.00 ± 5.78) compared to CTR group (F(3,39) = 3.125, p = 0.038), although no effects in the lordosis score (F(3,39) =

0.608, p = 0.614) (n = 10 animals/group/generation).

Conclusion: Although TCS did not interfere in the estrous cycle, which is highly dependent of sexual steroidal

hormones, the repeated treatment compromised female sexual receptivity to the male.

Grants: CAPES and CNPq (454499/2014-0)

73

ABSTRACTS SELECTED FOR

POSTER PRESENTATION

CLINICAL PATHOLOGY 74

ANALYSIS OF HEPARINE ACTION IN OXIDATIVE STRESS BIOMARKERS IN CHRONIC RENAL

PATIENTS SUBMITTED TO HEMODIALYSIS

Matsumoto A.K.1, Bonifácio K.L.

1, Farias C.C.

1, Higachi L.

1, Junior R.M.

1, Martins R.F.D.B.

1, Michelin A.P.

1, Semeão

L.O.1, Barbosa D.S.

2, Delfino V.D.A.

3

1State University of Londrina, Postgraduate Laboratory, Londrina, PR, Brazil;

2 State University of Londrina, Department of Pathology, Clinical and Toxicological Analysis, Londrina, PR, Brazil;

3

Medical Clinic– CCS


Keywords: anticoagulation, dialysis, heparin, oxidative stress

Introduction and objectives: Patients on chronic hemodialysis (HD) are exposed to oxidative stress (OS) caused

partially by activated peripheral blood polymorphonuclear leukocytes (PMNLs) during a HD session. Unfractionated

heparin has remained the anticoagulant of choice in patients undergoing hemodialysis. Anticoagulation prevents

thrombosis in the circulating consists of a dose of heparin given as a bolus at the beginning of dialysis with a second

dose in the middle of the treatment to maintain appropriate anticoagulation.

Material and methods: The antioxidant potential in vitro was evaluated for the heparin ROS production by neutrophils

(respiratory burst) by chemiluminescence. The antioxidant potential in vitro was evaluated for the heparin by

respiratory burst. The neutrophils ROS production was evaluated by chemiluminescence in a microplate reader (Victor

X-3, PerkinElmer ™, USA. Human neutrophils were isolated from blood by Histopaque ficoll concentration gradient

and centrifugation. The respiratory burst was induced by phorbolmyristate acetate (PMA) and the results are

expressed in counts per minute (CPM). Blood samples were collected from 30 chronic renal pre- and the postdialysis

samples collected from these patients. Biomarkers of OS were performed as lipid peroxidation (LOOH), reduced

glutathione (GSH) catalase and paraoxonase 1 (PON 1). Initially, comparisons were perfomed according to the normal

distribution of data in the Shapiro-Wilk test, as well as the variance analysis by Levene`s test. If these criteria were

reached (p>0,05), variables were evaluated independent T test or Kruskall-Wallis if did not homogeneity. Data that did

not reach normal distribution and homogeneity of variance criteria were analyzed by the non-parametric test of

Wilcoxon. Antioxidant activity results are presented as means _ standard error mean (SEM). Results were considered

statistically significant if p < 0.05. In addition, all analyzes will be done with its own resources obtained in projects

approved by research promotion bodies such as Araucaria Foundation. This study was approved by the Institutional

Ethics Board (1.585.869).

Results: Respiratory burst the results are expressed as count per minute (c.p.m.). We found significant associations

between analysis pre and post dialysis in LOOH (p<0.01, n=30). Others OS parameters did not show difference

significant. The heparin (2.38 UI) analysis in respiratory burst each curve was plotted with the median of at least 14

replicates. Kruskall-Wallis indicated a significant difference in the peak values of the kinetic curves (p<0.05), the

median c.p.m. (min-max) values for control neutrophils were 60926 (51809–66243), the for heparin were 46711

(40290– 59253),

Conclusion: In vitro, 2.38 IU/mL heparin reduced neutrophils superoxide release rate stimulated with PMA, the

concentration at which heparin exhibited antioxidant activity is greater than the serum concentration found in patients

after HD (0.53 IU/mL). In vivo, LOOH increased after the dialysis session due to the activation of neutrophils by the

dialysis membrane itself, but the other parameters of the operating system did not present a difference probably due

to the presence of heparin. Based on our results, heparin, in vivo, influenced the results by the dialysis process,

heparin should be the best anticoagulant of choice.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC425058/


BREAST CANCER AND PHOSPHOLIPASE A2 FROM SNAKE VENOM, AS ANTITUMORAL AGENT: A

REVIEW

Lonardoni, L. R.1; Azevedo, F. V. P.

2; Zóia, M. A. P.

1; Tavernelli, N. L.

1; Ávila, V. R. M

2; Dantas, N. O.

3; Filho, L. R. G.

1

and Silva, A. C. A.3.

1 Laboratory of Nanobiotechnology, Institute of Genetics and Biochemistry, Federal University of Uberlandia, UFU-MG,

Brazil 2Laboratory of Biochemistry and Animal Toxins, Institute of Genetics and Biochemistry, Federal University of

Uberlandia, UFU-MG, Brazil. 3Laboratory of New Insulation and Semiconductor Materials, Institute of Physics, Federal University of Uberlandia,

UFU-MG, Brazil


Keywords: Antitumor, breast cancer, cancer, phospholipases A2.

Introduction: According to INCA, breast cancer is the second type of cancer that affects women around the world. It

is considered a heterogeneous disease for its pathological characteristics, hence, the disease monitoring is quite

complex, especially due to the existence of many tumor subtypes, to which has its own expression profile, therapeutic

response and clinical behavior. The treatment for breast cancer aims to reduce the process of cell division,

proliferation and metastasis. Snake venoms, which contains many bioactive substances, amongst them the

phospholipases A2 (PLA2s) that are enzymes capable of breaking phospholipids in the position sn-2, generating free

fatty acids and phospholipids. The PLA2s are related to many biological effects and shows effective in the action by

the activation medium of cellular signaling pathways, as destabilization of biological membranes. Still, the PLA2s are

associated with antibacterial and antiparasitic activities, being, therefore, of big interest medical-scientific.

Review: The isolated PLA2s from snake venom exhibit many biological activities and nowadays, it has been related to

many antitumor, antiangiogenic and antimetastasis proprieties. A study conducted from our group, demonstrated the

effects of phospholipase A2 (BnSp-6) in MDA-MB-231 cells of human triple negative breast cancer, where the enzyme

presented cytotoxicity, inhibit the cell adhesion and suggested a preference of PLA2s for tumor cells. Yan and

collaborators, showed that crotoxin (CrTx), a PLA2 of Crotalus durissus terrificus, was capable of induce cellular death

in leukemia lineages (k562) by apoptosis and autophagy, still, the same group observed a reduction in the cellular

viability and stimulus in the autophagic process in breast cancer cells (MCF-7).

Conclusion: PLA2 showed innumerous biologic activities related of the destabilization of biological membranes,

antitumor, antiangiogenic and antibacterial actions. Showed to be a possible target for investigation for triple negative

breast cancer, being considered a new tool to act in the signaling pathway or on the development of new drugs for

treatment.


CCR5-DELTA32 ALLELIC VARIANT: CORRELATION WITH METASTASIS PARAMETER IN

BREAST CANCER

Pasquini, J. F. G.1; Derossi, D. R.

2, Amarante, M. K.

1; Losi-Guembarovski R.

1, Vitiello, G. A. F.

1; Oliveira, C. E. C.

1;

Sakaguchi, A. Y.1; Campos, C. Z.

2, Carmelo, E. C. B.

2, Banin-Hirata, B. K.

1, Pereira, N. S.

1; Castro, V. D.

1; Motoori-

Fernandes, C. Y.1; Cebinelli, G. M.

1; Watanabe, M. A. E.

1.

1 Department of Pathological Sciences, Biological Science Center, Londrina State University, Londrina, Parana, Brazil.

2 Department of Clinical Research, Cancer Hospital of Londrina, Londrina, Parana, Brazil.


Keywords: breast cancer, CCR5, prognostic parameters.

Introduction and objectives: Breast cancer (BC) is the type that has the highest incidence and mortality in female

population in the world, both in developing and developed countries. Chemokines can act as pro-tumoral or anti-

tumoral regulators of malignancy by affecting cells of the tumor microenvironment and the tumor cells themselves.

The activation of the C-C motif chemokine receptor 5 (CCR5) in BC cells controls their invasiveness serving as a

driver for metastasis. CCR5 is the major receptor for the CC chemokine and is capable to bind to CCL3 (MIP1α),

CCL4 (MIP1β), and CCL5 (RANTES). Polymorphic variations in CCR5 gene, in particular the delta-32 (Delta32) leads

to decreased expression and dysfunction of the receptor. CCR5-Delta32 consists is a 32-bp deletion in the coding

region, encodes a truncated inactive receptor that is not expressed on the cell membrane. Inside this context, the aim

of the present study was to determine whether a polymorphism in CCR5 gene (rs333/delta32) was associated with BC

susceptibility and prognostic parameters.

Material and methods: Human Ethics Committee of the State University of Londrina, Paraná, Brazil, approved the

present study (CEP/UEL 189/2013-CAAE 17123113400005231). Patients and controls were informed in detail

regarding the research, and the consent term was obtained. Peripheral blood (5 mL) was collected with anticoagulant

(EDTA) from 167 BC patients attended in the Cancer Hospital of Londrina (CHL), Londrina, Paraná, Brazil, and from

180 control women. Control group, taken from the same patients’ Parana, Brazil, geographic region, had no BC

confirmed by clinical and imaging examination, no self-declared family history of BC and personal history of any

malignant disease. Data relating to clinicopathologic parameters of BC were kindly provided by CHL. The parameters

included: tumor size, lymph nodes involvement and/or distant metastasis, nuclear grade and tumoral staging (TNM

classification), which were determined according to the Union of International Control of Cancer classification criteria.

Genomic DNA was obtained from peripheral blood cells using Biopur Mini Spin Plus Kit (Biometrix Diagnostica,

Curitiba, Parana, Brazil), according to manufacturer instructions. Genotyping of CCR5-delta32 was determined by

conventional PCR, using specific primers.

Results: The mean age of BC patients was 55 (±13) years. Although some clinicopathologic characteristics were not

available, it was observed that 90.65% of patients presented ductal carcinoma, 2.92% presented lobular carcinoma,

5.26% presented in situ carcinoma and 1.17% presented rare subtypes of BC. In the present study the allele

frequency of CCR5-delta32 in breast cancer was 9.58% and 3.91% in control group. We did not find any association

between delta-32 genetic variant and BC susceptibility (CCR5-delta32: OR=1.24; CI 95%=0.59-2.58). Analysis in

relation to prognostic parameters found no significant correlation in relation to tumor size, tumor staging and neoplasia

subtypes. However, analysis in relation to prognostic parameters found a significant correlation between delta32 and

metastasis parameter (p=0.02).

Conclusion: The CCR5-delta32 allelic variant presented correlation with metastasis parameter in breast cancer

suggesting this receptor as candidate marker for prognosis in breast carcinogenesis.



DIFFERENCES BETWEEN TWO MEMBRANES TYPES USED IN HEMODIALYSIS SESSIONS

RELATING THE BIOMARKERS OF OXIDATIVE STRESS PRE- AND THE POSTDIALYSIS

Matsumoto A.K.1, Bonifácio K.L.

1, Farias C.C.

1, Higachi L.

1, Junior R.M.

1, Martins R.F.D.B.

1, Michelin A.P.

1, Semeão

L.O.1, Barbosa D.S.

2, Delfino V.D.A.

3



3

Medical Clinic– CCS


Keywords: chronic kidney disease, hemodialysis, membrane, oxidative stress

Introduction and objectives: Patients with chronic kidney disease (CKD) have high cardiovascular mortality and

morbidity and a high risk for developing malignancy. Excessive oxidative stress (OS) is thought to play a major role in

elevating these risks by increasing oxidative nucleic acid damage. The OS is defined as an imbalance between

oxidant and antioxidant agents and these processes may result in toxic effects on cells and tissues by increasing the

oxidation of lipids, proteins, carbohydrates, and DNA. Patients submitted to Hemodialysis (HD) may induce repetitive

bouts of OS, primarily through membrane bio-incompatibility and endotoxin challenge. With the different types of

membranes for dialyses found on the market and the high cost, the objective this study is was evaluate the difference

between these membranes in causing greater benefits to the patient and allowing the reuse in order to reduce costs.

Material and methods: Blood samples were collected from 30 chronic renal patients pre and post dialysis in 2

different times: collection 1 using the Polinefron membrane and collection 2 used of the Polysulfone membrane.

Biomarkers of OS were performed as lipid peroxidation (LOOH), superoxide dismutase (SOD, catalase and activity of

paraoxonase 1 (PON 1). Initially, comparisons were performed according to the normal distribution of data in the

Shapiro-Wilk test, as well as the variance analysis by Levene`s test. Difference among groups were analysed by the

independent T test (parametric data) or test of Wilcoxon test (non-parametric data). Antioxidant activity results are

presented as means _ standard error mean (SEM). Results were considered statistically significant if p < 0.05. This

study was approved by the Institutional Ethics Board (1.585.869)

Results: The results demonstrated significant associations LOOH and PON1 analysis between pre and the post

dialysis (p<0.001, n=30), but we did not significant in SOD and catalase test analysis pre and post dialysis and

between collection 1 using the Polinefron membrane and collection 2 used of the Polysulfone membrane.

Conclusion: The hemodialysis membrane bioincompatibility can contribute to increased OS and prevalence of

inflammation as the three major causes of OS in HD patients. The dialysis membranes seem to play a central role in

the increased production of ROS in these patients. Evidence there is no difference in the use of Polysulfone and

Polinefron membrane in relation to increase of reactive species of oxygen (RSO), nitrogen (RSN) and antioxidant

properties present in blood after dialysis. This is important when choosing the membrane for dialyses session.


EFFECT OF RESISTANCE TRAINING ON MUSCLE STRENGTH AND BODY COMPOSITION IN

BREAST AND PROSTATE CANCER PATIENTS: A META-ANALYSIS.

Padilha, C. S1, Marinello, P. C 1,2, Borges F. H3, Testa M. T. J1, Cella P. S1, Iarosz, K. C1, Guirro, P. B1,

Duarte J. A4, Deminice, R1.

1 Laboratory of Biochemistry of Exercise, State University of Londrina, Londrina, PR, Brazil. 2 Molecular Pathology

Laboratory, Department of Pathology Science, State University of Londrina, Londrina-PR, Brazil. 3 Laboratory of Free

Radicals and Pathophysiology State University of Londrina, Londrina-PR, Brazil. 4 CIAFEL, Faculty of Sport,

University of Porto, Porto, Portugal.

[email protected]

Key words: Exercise training, body fat, skeletal muscle, oncology.

Introduction: Breast cancer is the most common type of cancer diagnosed in women worldwide, and in males,

prostate cancer is most prevalent. Given the increasing number of studies reporting resistance training (RT) benefits

on muscle wasting, muscle strength and body composition can be viewed as clinically relevant. The participation of

cancer patients in RT has been associated with improved survival. For this reason, the purpose of present meta-

analysis was to determine and quantify the effects of RT on lower-limb muscular strength, lean body mass (LBM), and

body fat (BF) in breast and prostate cancer patients.

Review: This research was conducted using the following online database: Clinical Trial Register, Cochrane Trial

Register, PubMed, SPORT Discus, and SciELO, from September 2014 until May 2017. We used the following

keywords in various combinations with a systematic search: ―Cancer patients‖, ―Breast cancer‖, ―Prostate cancer‖,

―Wasting muscle‖, ―Muscle loss‖, ―Muscle function‖, ―Body fat‖, ―Lean body mass‖, ―Body composition‖, ―Resistance

training‖, ―Weight training‖, and ―Exercise‖. After selection of 272 full-text articles, 14 publications were included in this

meta-analysis. Overall, muscular strength increased significantly when compared to controls over time (mean:

38.19Kg, 95% CI [25.92-40.75], I2 = 100%, P=<0.0001) and when RT response was analyzed separately by prostate

and breast cancer, we observed similar increases in prostate cancer patients (mean: 33.03Kg, 95% CI [25.32-40.75],

I2 =99%, P=<0.0001) and breast cancer patients (mean: 42.64Kg, 95% CI [24.59-60.68], I

2 =100%, P=<0.0001)

compared to controls over time. Furthermore, we found a significant decrease over time for BF over time in RT group

compared to controls (mean: -0.94Kg, 95% CI [-1.29, -0.59], I2 =62%, P=<0.0001) and when RT response was

analyzed separately by prostate and breast cancer we observed similar decrease in prostate cancer patients (mean: -

1.05Kg, 95% CI [-1.59, -0.50], I2=82%, P=0.0002) and breast cancer patients (mean: -0.87Kg, 95% CI [-1.32, -0.41],

I2=0%, P=0.0002). However, there was no significant increase on LBM over time induced by RT.

Conclusion: RT is effective to increase lower-limb muscular strength and reduce BF in prostate and breast cancer

patients. These observations indicate that patients engaged in RT may have greater protection against side effects of

pharmaceutical interventions and improving patient outcomes.


ERYTHROCYTE ANTIOXIDANTS INCREASE IN PATIENTS WITH MULTIPLE BASAL CELL

CARCINOMA, SQUAMOUS CELL CARCINOMA AND ACTINIC KERATOSIS LESIONS

Freitas, L. B.

1; Marinello, P.C.

1; Brito, W. A.S

1; Silva, M.H.C.

1; Garcia, M.L.

1; Lopes, N.M.D.

1; Sanches, L. J.

1; Taguti,

P. S. 2; Kippert, J. P.

2; Lena, C. P.

2; Armani, A.

3; Luiz, R. C.

1; Gon, A.S.

2; Cecchini, R.

4; Cecchini, A.L.

1.

1 State University of Londrina, Laboratory of Molecular Pathology, Londrina, PR, Brazil. 2 State University of Londrina, Department of Internal Medicine, Londrina, PR, Brazil. 3 State University of Londrina, Department of Surgical Clinic, Londrina, PR, Brazil.

4 State University of Londrina, Laboratory of Physiopathology and Free Radicals, Londrina, PR, Brazil.


Keywords: Basal cell carcinoma; skin cancer; oxidative stress.

Introduction and objectives: Oxidative stress (OS) is closely related to skin carcinogenesis, mainly due to ultraviolet

radiation. Studies suggest the presence of particularities regarding oxidative stress among different types of non-

melanoma skin cancer (NMSC). However, the works are scarce and the difference in the systemic OS among patients

with one or more than one type of NMSC was never investigated. For this reason, this work aims to evaluate the

erythrocytic antioxidant defenses and lipoperoxidation in patients with basal cell carcinoma (BCC), the most commom

type of NMSC, and with BCC associated with others skin neoplasias (squamous cell carcinoma and actinic keratosis).

Material and methods: Participants were categorized in 3 groups: Control (without skin cancer history; n=38); BCC

(n= 50) and BCC associated with others skin neoplasias (BCC-ML; n=12) (CEP-UEL process n. 1.077.557). Blood

samples were collected and erythrocytic catalase and superoxide dismutase activity, oxidized and reduced glutathione

levels (GSSG and GSH, respectively) was determined. Lipoperoxidation was assessed by chemiluminescence

stimulated by tert-butyl-hydroperoxide. The statistical analysis was performed using one-way ANOVA (one-way or

two-way, when applicable) or Kruskal-Wallis after the verification of the normality of the data, p<0.05 was considered

significant.

Results: Patients from BCC presented reduced levels of GSH (684.3 ± 122.3) when compared with the control group

(787.3 ± 83.8) and BCC-ML (798.3 ± 159.3) presented increased levels of GSH when compared with the BCC group

(p<0.05). GSSG and the stress index did not change. Catalase activity did not alter in BCC, but significantly increased

in BCC-ML (47.01 ± 14.72) when compared to control (37.23 ± 8.04; p<0.05). Superoxide Dismutase activity

significantly decreased in BCC (0.103 ± 0.02) and BCC-ML (0.094 ± 0.01) groups when compared to control (0.113 ±

0.01; p<0.05). Chemiluminescence curves show higher lipoperoxidation in BCC group in comparison with control

(p<0.0001).

Conclusion: Patients with basal cell carcinoma associated with others skin neoplasias present elevation in

erythrocyte antioxidants when compared to patients with only basal cell carcinoma. This elevation is probably related

to the absence of increased lipoperoxidation observed in this group. These results indicates the presence of

differences in relation to systemic OS in patients with different clinical outcomes and suggest the importance of OS in

the progression of NMSC.

Grants: Araucaria Foundation (001/2017) and National Council for Scientific and Technological Development (CNPq).


EVALUATION OF POLYMORPHISM rs3591676 OF THE AMACR GENE IN PATIENTS WITH

PROSTATE CANCER

Nóbrega, M.¹; Souza, M. F.¹; Souza, M. R.¹; Serpeloni, J. M.¹; Fuganti, P. E.²; Cólus, I. M. S.¹. 1 Department of General Biology, State University of Londrina, Londrina, Paraná, Brazil.

2 Londrina Cancer Hospital, Londrina, Paraná, Brazil.


Keywords: Prostate cancer, SNP, AMACR, biomarkers

Introduction: Prostate cancer (PCa) is the second most commun type of cancer in men. In Brazil, were estimated

60.180 cases in 2017. AMACR (alpha-methyl coenzyme A recemase) is an enzyme that plays an important role in bile

acid biosynthesis and beta-oxidation of branched-chain fatty acids. Studies have shown an increased expression of

the gene AMACR in prostate malignant cells, acting as a sensitive and specific marker for PCa cancer detection.

Objective: The aim of this study was evaluating the polymorphism rs3591676 of the AMACR (P504S) gene as

diagnostic and prognostic marker for PCa. Material and Methods: This study was approved by Human Research

Ethics Committee of the State University of Londrina, Brazil (CAAE19769913.0.0000.5231). A case-control study was

performed with 281 patients with histopathological confirmation of PCa and 281 healthy controls, matched by age,

race, smoking and alcohol consumption habits. DNA was extracted from peripheral blood using PCR kit High Pure

Template Preparation Kit (Roche). Polymorphism analysis was performed by real time qPCR using TaqMan probes.

Odds Ratio analysis (OR) with a confidence interval (IC) of 95% was obtained by univariate logistic regression using

the statistical software BioEstat 5.0 and SPSS 20.0. Results: Evaluation of risk alleles of the rs3591676

polymorphism showed no association with prostate cancer [TC = 0.95 (0.66 -1.38) and TT = 0.67(0.42-1.08)]. Clinical

and histopathological parameters such as PSA, Gleason score, extraprostatic invasion, intraperineural invasion, bone

metastasis and biochemical recurrence also were evaluated and showed no association. Some autors have been

studying this polymorphism, but the resusts are controversial, however, the majority of studies showed no association

between this polymorphism and PCa, as we observed in our findings. Conclusion: Our results showed that the

polymorphism rs3591676in the AMACR gene has no association with PCa Risk and cannot be used as prognostic or

diagnostic marker.

Grants: Fundação Araucária; CAPES; CNPq-PQ.


EVALUATION OF SYSTEMIC OXIDATIVE PROFILE IN PATIENTS WITH NON-MELANOMA SKIN

CANCER AND ACTINIC KERATOSIS

Brito, W. A. S1; Marinello, P. C.

1; Garcia, M. L.

1; Cunha, M. H.

1; Freitas, L. B.

1; Lopes, N. M. D.

1; Sanches, L. J.

1;

Taguti, P. S. 2; Kippert, J. P.

2; Lena, C. P.

2; Armani, A.

3; Luiz, R. C.

1; Gon, A. S.

2; Cecchini, R.

4; Cecchini, A. L.

1.


2 State University of Londrina, Department of Internal Medicine, Londrina, PR, Brazil. 3 State University of Londrina, Department of Surgical Clinic, Londrina, PR, Brazil.



Keywords: Basal cell carcinoma, Squamous cell carcinoma, Actinic keratosis, Oxidative stress, Antioxidants.

Introduction and objectives: Non-melanoma skin cancer (NMSC) is the neoplasia with the highest incidence in the

world. The cure of NMSC is the surgical removal of the lesions, which can be often mutilating, causing social and

psychological impact on patients. Basal cell carcinoma (BCC) is the most common type of NMSC. Actinic keratosis

(AK) is a pre-malignant neoplasia that has potential to progress to squamous cell carcinoma (SCC), another type of

NMSC. It is known that oxidative stress (OS) has an important role in skin carcinogenesis, which occurs mainly due to

ultraviolet radiation. However, systemic OS in patients with different types of NMSC was never investigated. Thus, we

evaluated the oxidative profile in plasma and erythrocytes of patients with BCC, SCC and AK.

Material and methods: Participants were categorized in 4 groups: Control (without skin cancer history; n=45); AK

(n=11); BCC (n=48) and SCC (n=9) (CEP-UEL process n. 1.077.557). Blood samples were collected, erythrocytic

catalase and superoxide dismutase (SOD) activity, oxidized and reduced glutathione levels (GSSG and GSH,

respectively) and lipid peroxidation was assessed by chemiluminescence stimulated by tert-butyl-hydroperoxide.

Malondialdehyde (MDA) and total thiols concentrations in plasma were determined. The statistical analysis was

performed using ANOVA (one-way and two-way, when applicable) or Kruskal-Wallis after the verification of the

normality of the data; p<0.05 was considered significant.

Results: Patients from BCC presented reduced levels of GSH and reduced SOD activity when compared to control,

which were not observed in AK and SCC. Increased lipid peroxidation were observed in AK and BCC groups when

compared to control. There were not significant differences observed in catalase activity, GSSG levels, plasma MDA

and total thiols between groups.

Conclusion: The results indicate that BCC patients presents lower levels of erythrocytic antioxidant defenses,

observed by reduced GSH and SOD activity, and increased lipoperoxidation than the other groups. These

preliminaries results suggest that patients with BCC present increased systemic oxidative stress, what supports the

research of new biomarkers to aid in prognosis of patients with different types of NMSC.

Grant: National Council for Scientific and Technological Development (CNPq).


EVALUATION OF THE EFFECTIVENESS OF OLFACTORY TRAINING AS A TREATMENT FOR

PERSISTENT OLFACTORY LOSS

Miyazawa, I. N.

1; Silva, G. S.

1; Garcia, E. C. D.

2; Fornazieri, M. A.

3

1 Universidade Estadual de Londrina, Department of Medical Clinic, Londrina, PR, Brazil

2Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil

3 Universidade Estadual de Londrina, Department of Clinical Surgery, Otolaryngology Division, Londrina, PR, Brazil


Keywords: Smell, Anosmia, Olfaction Disorders.

Introduction and objectives: Olfaction is a human chemical sense which dysfunction can lead to severe commitment

of quality of life. There are several disturbances related to olfactory loss, including: neurological diseases such as

Parkinson and Alzheimer, nasal and obstructive sinus diseases, such as chronic rhinosinusitis, in addition to traumatic

brain injury, aging, congenital causes, psychiatric disorders and upper respiratory infections. Recently, olfactory

training has been proposed as a form of treatment for olfactory disorders and seemed to obtain satisfactory results in

patients with olfactory loss post traumatic, post infectious, idiopathic, and even Parkinson's disease. The objective of

this study was to verify the effect of olfactory training in patients with persistent olfactory loss of diverse causes and to

evaluate possible prognostic factors of its efficacy.

Material and methods: Retrospective study approved by the Research Ethics Committee Involving Human Beings

(approval number: 1.073.331), conducted at the University Hospital of the State University of Londrina through the

analysis of medical charts of 30 patients (22 females, 8 males) between 17 and 82 years (mean 51.9 years) who

underwent olfactory training as a treatment for persistent olfactory loss. Olfactory dysfunctions etiologies were upper

respiratory tract infections, head trauma and idiopathic and the mean duration of olfactory loss was 27.3 months (from

1 month to 12 years). Two types of olfactory training were performed: a group of 15 patients trained with a

standardized dose of commercially available products (coffee powder, toothpaste, vinegar, tangerine juice, honey,

vanilla essence and cloves) and 15 other patients used bottles with cotton balls soaked in neat essences of phenyl-

ethyl alcohol, eugenol, citronellal and eucalyptol. In both cases, training consisted of sniffing the odors for 10 seconds

twice a day for 3 months. Adherence was checked by monthly telephone calls. Patients were tested with the

University of Pennsylvania Smell Identification Test (UPSIT) before and after the treatment. Six points increase in the

test score was considered a significant improvement. Fisher's Exact Test was used for statistical analysis, with a

significance level of 5%.

Results: Eighteen out of the thirty patients who underwent treatment returned after 3 months of training. Causes of

olfactory deficit were respectively olfactory loss post-infection (n=9), post-traumatic (n=6) and idiopathic (n=3). Five of

them showed significant improvement (27.8%). Of these five patients, three had post-infection loss and two post-

traumatic loss. Treatment efficacy was not associated with the olfactory loss etiology (p = 0.634) or type of training

performed (p =1.0). Olfactory loss duration at the time of diagnosis also did not influence the recovery of patients'

olfactory function (p = 0.580).

Conclusion: Olfactory training achieved satisfactory efficacy in one third of patients with persistent olfactory loss.

More controlled studies are needed with a greater number of patients to prove that its efficacy is related to the

treatment and not to spontaneous recovery.

Grants: Fundação Araucária



EVALUATION OF THE PROLONGED USE OF DIETARY SUPPLEMENTS BY SPORTSMEN

Silva, L.C.1; Rickli-Prado, K.L.

1; Lenhard-Vidal, A.

1,2

1Faculdade Campo Real, Graduação em Biomedicina, Guarapuava, PR, Brazil.

2State University of Londrina, Department of Pathological Sciences, Londrina, PR, Brazil

[email protected]

Keywords: Dietary supplement, Exercise, Liver function tests, Performance-enhancing substances, Sports nutritional

sciences.

Introduction and objective: Nowadays there is an increased concern with looks and aesthetics. In order to quickly

achieve the perfect body, the inadequate use of dietary supplements, combined with strenuous physical activity, has

become increasingly more common. There are few studies on their effects and consequences, which may include

kidney/liver overloads. Therefore, current research aimed to evaluate liver enzymes, urine and the profile of

sportsmen from Guarapuava, Paraná, Brazil. Material and Methods: This was an experimental research conducted

in Faculdade Campo Real. Sportsmen were over 18 years-old and exercised in local gymnasiums at least three times

a week, regardless of activity. Control group: 16 non-consumers of dietary supplements; Test group: 20 consumers of

any amount and type of dietary supplements for over 6 months. Questionnaire: personal questions (schooling, age,

weight, kind and frequency of exercises), about supplements [type, frequency, purpose, prescription by whom, side

effects, use of other ergogenic aids (anabolic steroids)] or factors that may hinder the results (pre-existent conditions,

medicines, use of alcoholic beverages). Tests: dosage of Alanine transaminase (ALT), Aspartate transaminase (AST),

Gamma-glutamyltransferase (GGT) in the Bs-200e MINDRAY; Urinalysis. Project approval: Research Ethics

Committee from Unicentro from Guarapuava (n.1.571.986). Each participant gave written consent. Statistics: T test for

unpaired data sets, in GraphPad Prism 6.01, assuming as significant p<0.05; data expressed as mean±SD. Results:

Participants were from 18 to 51 years-old (mean age 25.3±7.4) and half were under 22 years-old. Half of the

interviewed sportsmen were undergraduates in both groups, while 40 and 12.5% were still enrolled in college and 10

and 37.5% only finished high school in the test and control groups, respectively. The test group consumed mostly

proteins and aimed at maximizing athletic effects (muscular mass gain, conditioning improvement). Sportsmen

consumed more than one type of supplement, usually between 8-24 months, while 40% consumed over 24 months.

Most didn’t have supervision of a nutritionist or physician. One user mentioned side effects (acne) and 3 (15%) used

anabolic steroids. The mean of the hepatic enzymes was within the normal range for both groups, but even though

there were higher ALT levels at the test group (p<0.05). The results of urinalysis were within normal limits.

Conclusion: The sportsmen consumed many different types of dietary supplements, especially proteins, generally

without medical prescription, in order to gain muscular mass and enhance performance. The use of anabolic steroids,

intramuscular injections and strenuous exercise are known to interfere with ALT results, because the enzyme is also

found in skeletal muscles. Therefore, two factors need to be considered as interferers: (1) the use of anabolic steroids

and (2) participants that may not have avoided exercises in the 24 hours prior to the tests. Although only ALT results

differed between groups, this subject still has much to be researched, as many people indiscriminately consume

dietary supplements, often exceeding the recommended daily intake of these nutrients.


EVALUATION OF THE REDOX STATE AND RESISTANCE INSULIN IN ADOLESCENTS WITH

METABOLIC SYNDROME

Martins, R.F.D.B.1, Bonifácio, K.L.

1,2, Farias, C.C.

1,2, Higachi L.

1,2, Michelin, A.P.

1,2, Matsumoto, A.K.

1, Semeão, L.O.

1,

Cyrino, E.S.3, Venturini, D.

1,2,4, Barbosa, D.S.

1,2,4

1Laboratory of Graduation Research, State University of Londrina, University Hospital, Londrina, PR, Brazil

2Graduation Program in Health Sciences, State University of Londrina, Londrina, PR, Brazil

3Metabolism, Nutrition, and Exercise Research Group, Sport and Physical Education Center, State University of

Londrina, Londrina, PR, Brazil 4Department of Clinical Analysis and Toxicological, State University of Londrina, Londrina, PR, Brazil


Keywords: Insulin Resistance, Metabolic Syndrome, Adolescents, Oxidative Stress

Introduction and Objectives: Key characteristics of the metabolic syndrome (MetS) are central obesity, insulin

resistance (IR) and dyslipidemia as evidenced by increased levels of insulin, glucose and triglycerides, but lowered

levels of high-density lipoprotein cholesterol (HDL-C). The MetS is accompanied by increased reactive oxygen (ROS)

and nitrogen (RNS) species and decreases in antioxidant defenses creating an environment that promotes oxidative

and nitrosative stress (O&NS). MetS and its major components, namely atherogenicity and IR, are strongly related to

an increased risk of diabetes type 2 and cardio-vascular disease. Moreover, IR is the most common metabolic

alteration associated with obesity and may contribute to atherosclerosis. The homeostatic model assessment (HOMA)

is a validated method to measure IR based on fasting serum glucose and insulin levels. This study aimed to examine

socio-demographic, clinical, malondialdehyde (MDA) levels, and antioxidant defenses (total radical-trapping

antioxidant parameter (TRAP)/uric acid ratio), in relation to IR in adolescents with and without MetS.

Material and methods: All participants (n=886) underwent a physical examination, anthropometric measurements

and a structured interview, which collected data on medical history. Exclusion criteria for the subjects were clinical or

laboratory signs of medical disease, being under treatment for any disease, use of illicit drugs, and not being regularly

enrolled in schools. After considering inclusion and exclusion criteria, all adolescents who met with the MetS criteria

(n=43) were included in the study and they were matched to 89 subjects without the MetS (ratio 1:2) as diagnosed

with International Diabetes Federation (IDF) criteria. We measured MDA, TRAP/uric acid, computed IR using the

updated homeostasis model assessment (HOMA-IR), body mass index (BMI), waist circumference (WC), insulin,

glucose, total cholesterol, HDL-C and triglyceride. We used analyses of contingency Tables (Χ2

-test) to check the

associations between sets of categorical variables, general linear model analyses to examine differences in

continuous socio-demographic, metabolic and O&NS biomarker data between categories, and the correlations were

assessed using Pearson’s product moment correlation coefficients. Parents and adolescents gave their signed

informed consent to participate in the study, according to the protocol (n°238/2010) which was approved by the Ethics

Committee on Research Involving Human Subjects of the State University of Londrina.

Results: The results showed the socio-demographic, metabolic and O&NS data in subjects with and without MetS.

There were no significant differences in age or sex between the two categories. Subjects with MetS showed higher

BMI, WC, insulin, glucose, triglyceride and HOMA-IR values than those without MetS. There were no significant

differences in total cholesterol between both groups, while HDL-C was significantly lower in adolescents with the

MetS. MDA levels were significantly increased in adolescents with MetS and TRAP/uric acid ratio was significantly

lower in subjects with MetS compared to those without. There were significant and positive correlations between the

HOMA-IR index and WC (r=0.434, p<0.001) and BMI (r=0.439, p<0.001).

Conclusion: In summary, adolescents with the MetS have a redox imbalance, characterized by increased lipid

oxidation and as well as lowered antioxidant levels, and this suggest that O&NS may play a role in the

pathophysiology of MetS..

Grants: CNPq and Capes.


EVALUATION OF THE REDOX STATE DURING THE INDUCTION PHASE OF CHILDHOOD B-CELL

ACUTE LYMPHOCYTIC LEUKEMIA TREATMENT

Semeão, L.O1, Bonifácio, K.L

1,2, Oliveira, G.B

1, Michelin, A.P.

1,2, Matsumoto, A.K

1, Martins, R.F.D.B

1, Trigo, F.C

3,

Victorino, V.J4, Panis, C

5, Barbosa, D.S

1,2,6



3Department of Medical Clinic, State University of Londrina, Londrina, PR, Brazil

4Departament of Pathological Sciences, State University of Londrina, Londrina, PR, Brazil

5Laboratory of Inflammatory Mediators, State University of West Paraná, UNIOESTE, Campus Francisco Beltrão, PR,

Brazil 6Department of Clinical Analysis and Toxicological, State University of Londrina, Londrina, PR, Brazil


Keywords: Acute lymphoblastic leukemia, Oxidative stress, Treatment

Introduction and Objectives: Studies have documented the participation of Oxidative stress (OS) in some aspects of

hematological neoplasias, including leukemia, all in all, it is known that cancer cells are under higher OS than normal

cells, which requires its adaptation to counterpart the continuous prooxidant impairment. B-cell acute lymphoblastic

leukemia (ALL) is the most common malignant neoplasia that affects child worldwide. Therefore, understand the

molecular mechanisms that underly the initial phases of treatment seems to be crucial. In spite of this, studies have

not focused on establishing the relationship between OS and disease prognosis in ALL patients undergoing the initial

phases of treatment, like the induction. Induction phase is characterized by a set of combined drugs know by

generating OS. The purpose of this study was to evaluate biomarkers of OS in patients with ALL in different phases of

induction.

Material and methods: Pediatric patients (0-18 years) diagnosed with ALL (B type) were from Instituto do Câncer de

Londrina, Paraná-Brazil, and were ongoing the chemotherapic schedules recommended by the Brazilian Childhood

Leukemia Treatment Group (GBTLI). Whole blood samples were obtained at diagnosis (D0), and sequentially in the

eighth (D8), fifteenth (D15), twenty-second (D22) and twenty-eighth (D28) days after starting the induction phase of

treatment. Following the routine sample collection employed by the GBLTI protocol, for peripheral plasma analysis, we

collected samples from all treatment intervals. The pro-oxidative parameters evaluated were estimated from the level

of nitric oxide by-products (NOx in μM) and hydroperoxides (LOOH in RLU); in addition, the antioxidants were the

sulphydryl groups (SH in μM) and the total plasmatic antioxidant capacity (TRAP in μM). Comparisons were

performed according to the normal distribution of data in the Shapiro-Wilk test, and the homogeneity analysis by

Levene`s test. The difference among groups was analyzed by ANOVA followed by Bonferroni`s test or Kruskal-Wallis

followed by Dunn´s test. All analyses were performed in the software GraphPad Prism 5.0 and SPSS 20, p<0.05 value

was considered as significant. This study was approved by the Institutional Ethics Board (24498213.0.0000.5231).

Results: A total of 17 patients was included in this study. The mean age of patients was 7.8 years, and most of the

patients were male (52.95%) and Caucasian (88.23%). We showed significant augment in NOx levels when

comparing D8 4.64(2.13–9.15) versus D22 6.60(3.87–23.45). We further observed increased LOOH at D22

1,560x103(800x10

3 – 4,900x10

3) when compared to D0 966x10

3 (690x10

3–3,980x10

3). At D28 we detected increased

NOx 8.54 (5.19-24.73) and LOOH 1,740 x 103(799 x 10

3 – 4,670 x 10

3) in relation to D8 4.64(2.13 – 9.15);

1,130x103(880 x10

3–2,230 x 10

3, respectively. Significant reduced SH levels were also observed when comparing D8

406.70(144.27– 604.89) versus D28 308.56 (205.32- 403.68).

Conclusion: The induction treatment is known to generate oxidative stress and is used in high doses to achieve rapid

tumor cells eradication. These findings could lead to new advances in the future and make in-roads for new studies

leading to a better management of this disease with the utilization of new biomarkers regarding the effectiveness of

treatment.

Grants: CNPq and Capes.


HEPATIC RESPONSE TO GLUCAGON, cAMP AND ADRENERGIC AGONISTS IN WALKER-256

TUMOR-BEARING RATS

Biazi, G. R.1; Frasson, I. G.

1; Miksza, D. R.

1, Bertolini, G. L.

1; Souza, H. M.

1

1Universidade Estadual de Londrina, Department of Physiological Sciences, Londrina, PR, Brazil


Keywords: Cachexia, tumor Walker-256, hepatic response to hormones.

Introduction and objectives: Cancer patients often present cachexia, a syndrome characterized by severe

catabolism of proteins and lipids, resulting in marked loss of muscle mass, adipose mass, and body weight. Cancer

cachexia is promoted by several factors, known as mediators of cachexia, produced by the tumor and the immune

system. However, it is possible that the altered response to hormones, such as insulin (insulin resistance), glucagon

and adrenaline, may also contribute to cachexia and to the metabolic abnormalities found in the liver of cancer

carriers, such as glycogen depletion, increased gluconeogenesis and inhibition of glycolysis from exogenous glucose.

Although insulin resistance in cancer carriers is well established, few studies have investigated the response to

glucagon and adrenaline in this pathological state. The main purpose of this study was to evaluate the hepatic

response to glucagon, cAMP and to the adrenergic agonists isoproterenol (β-agonist) and phenylephrine (α-agonist)

on glycogenolysis, glycolysis and gluconeogenesis in Walker-256 tumor-bearing rats.

Material and methods: Male Wistar rats (230-240 g), Walker-256 tumor-bearing with 12 days of tumor development,

and healthy (controls) were used. For tumor implantation, 8x107 Walker-256 cells were inoculated subcutaneously into

the right rear flank of the rats. Healthy rats were inoculated with PBS. The hepatic response to glucagon (1 nM), cAMP

(9 μM), isoproterenol (20 μM) and phenylephrine (2 μM) on glycogenolysis, gluconeogenesis and glycolysis was

evaluated in liver perfusion in situ. The ATP content in the liver was quantified by enzymatic method. The

experimental protocols were approved by Animal Experimentation Ethics Committee of the State University of

Londrina (register nº 10412).

Results: Glucagon and cAMP stimulated glycogenolysis and gluconeogenesis and inhibited hepatic glycolysis in

healthy and tumor-bearing rats. However, tumor-bearing rats showed a lower glycogenolytic, gluconeogenic and

glycolytic response to glucagon and cAMP, compared to healthy. The isoproterenol stimulated glycogenolysis,

gluconeogenesis and glycolysis in healthy rats, but practically had no effect on tumor-bearing rats. Phenylephrine

stimulated glycogenolysis and gluconeogenesis in healthy and tumor-bearing rats, with these responses being lower

in tumor-bearing rats. In addition, phenylephrine inhibited glycolysis in healthy rats and stimulated it in tumor-bearing

rats. Finally, the ATP content in the liver of tumor-bearing rats was smaller compared to the healthy rats.

Conclusion: The hepatic response to glucagon, cAMP, β-adrenergic agonist (isoproterenol) and α-adrenergic agonist

(phenylephrine) in glycogenolysis, gluconeogenesis and glycolysis is decreased in Walker-256 tumor-bearing rats,

except for phenylephrine in glycolysis. It is possible that this decreased response is due to the lower hepatic ATP

content, which is involved in the phosphorylation (activity) of key enzymes of these metabolic pathways.


IMMUNE-INFLAMMATORY, METABOLIC, OXIDATIVE AND NITROSATIVE STRESS SET OF

BIOMARKERS PREDICTS ACUTE ISCHEMIC STROKE AND SHORT-TERM OUTCOME

Lehmann, M. F.1,2

; Stadtlober, N.P. 3; Santos, L.F.R.F.

3; Alfieri, D.F.

2; Flauzino, T.

2; Cossentini, L.A.

3; Robles, B.E.F.

3;

Iriyoda, T.M.V.I.

3; Costa, N.T.

2; Micheletti, P.L.

2; Alcantara, C.C.; Medeiros, F.A.; Sá, M.C.; Kallaur, A.P.

2; Oliveira, S.R.

2;

Araújo, M.C.M.4; Bragato, E.F

4; Simão, A.N.C.

5; Maes, M.

6; Reiche, E.M.V.

5

1 Department of Clinical Surgery, Health Sciences Center, and Neurosurgery Service of the University Hospital, State

University of Londrina, Londrina, Paraná, Brazil;

2 Health Sciences Postgraduate Program, Health Sciences Center, State University of Londrina, Londrina, Paraná, Brazil

3 Experimental Pathological Postgraduate Program, Biological

Sciences Center, State University of Londrina, Londrina, Paraná, Brazil

4 Medicine Faculty, Health Sciences Center, State University of Londrina, Paraná, Brazil;

5 Department of Pathology, Clinical Analysis, and Toxicology, Health Sciences Center, State University of Londrina,

Paraná, Brazil;

6 IMPACT Strategic Research Centre, School of Medicine, Deakin University, Geelong, Victoria, Australia. [email protected]

Keywords: Oxidative stress, Nitrosative stress, Ischemic stroke, Inflammatory biomarkers

Introduction and objectives: Stroke is sudden onset of focal neurological deficit and a major cause of disability and

mortality worldwide. Acute ischemic stroke has a heterogeneous etiology caused by unmodifiable risk factors, such as

genetic, age, and sex, as well as by modifiable risk factors including hypertension, diabetes mellitus, dyslipidemia,

sedentary lifestyle and smoking. Inflammatory, metabolic, oxidative and nitrosative pathways have an important role

during the ischemic event. The main objective of this study was to identify biomarkers to the early prediction of AIS

and short-term outcome.

Material and methods: The protocol was approved by the Institutional Research Ethic Committee of the State

University of Londrina, (CAAE 0250.0.268.000-11) and a written consent form was obtained from all of the individuals.

A case-control study was conducted in 176 patients with AIS and 176 controls subjects. The modified Rankin Scale

(mRS) was applied up to eight hours of AIS (baseline) and after three-month follow-up, and blood samples were

obtained up to 24 hours of stroke. Inflammatory, metabolic, oxidative and nitrosative stress biomarkers (IMO&NS)

were determined. Analyses of variance (ANOVA) were employed to assess between-group differences in continuous

variables. Analyses of contingency tables (Χ2-tests) were used to assess the associations between sets of categorical

variables. Automatic stepwise binary logistic regression analysis was employed to delineate the most significant

biomarkers that are associated with stroke. Automatic stepwise regression analysis was employed to assess the most

significant IMO&NS and metabolic biomarkers.

Results: Automatic regression with O&N markers, metabolic biomarkers and demographic/clinical data showed that

the best set of predictors for AIS were sex (male), systolic blood pressure (SBP), glucose, nitric oxide metabolites

(NOx), hydroperoxides (LOOH), interleukin (IL)-6 and white blood cell (WBC); all positively associated. On other hand,

25-hydroxivitamin D [25(OH)D] was inversely correlated with AIS (p<0.001). Moreover, 89.4% of all AIS were correctly

classified with a sensitivity of 86.2% and a specificity of 93.0%. Values of ferritin, IL-6, C-reactive protein (CRP),

erythrocyte sedimentation rate (ESR), glucose were higher among those patients with baseline mRS>3 (p<0.001 for

all parameters) versus those with baseline mRS<3. While 25(OH)D is lower in patients with an increased mRS.

Conclusion: These results suggest that a set of IMO&NS biomarkers may be useful to the early prediction of AIS and

short-term outcome.

Grants: The study was supported by grants from the Coordination for the Improvement of Higher Level of Education

Personnel (CAPES) of Brazilian Ministry of Education; Institutional Program for Scientific Initiation Scholarship (PIBIC)

of the National Council for Scientific and Technological Development (CNPq); and State University of Londrina

(PROPPG).


IMPACT OF TWO DIFFERENT RESISTANCE EXERCISE PROGRAMS IN OXIDATIVE STATUS AND

CARDIOVASCULAR RISK FACTORS OF PATIENTS WITH METABOLIC SYNDROME

Alves, T.1; Barros, N. A.

1, Lemes, I. R.

2; Blegniski, F. P.

1; Silva, T. N. X.

1; Pastre, C. M.

2; Cecchini, R.

1; Júnior, J. N.

2;

Guarnier, F. A.1.

1 – State University of Londrina, Department of General Pathology, Londrina, Brazil.

2 – São Paulo State University (UNESP), Faculty of Sciences and Technology, Department of Physical Therapy,

PresidentePrudente, Brazil.

[email protected]

Keywords: Oxidative stress; exercise; metabolic syndrome

Introduction and objectives: Metabolic syndrome (MS) is considered a serious condition that comprises a number of

cardiovascular risk factors. Physical exercise practicing is one of the best treatment and prevention options, although

there is a challenge in prescription of exercises for patients with metabolic syndrome: reducing mass without affecting

lean body mass. The purpose of this study was to investigate the impact of two resistance exercise programs on

inflammatory and oxidativestatus and cardiovascular risk factors of volunteers diagnosed with MS.

Material and methods: The study included 54 adults, both sex, (50.87±5.67 years, 83.14±14.19kg, 166±10,4cm),

randomized to exercise groups or to a sedentary group (control). Functional (FT) and conventional (CT) groups

performed 12 weeks of periodized training, 3 times per week and control (CTRL) was instructed to not change habitual

activities. Cardiovascular risk factors, muscular strength, inflammatory and stress oxidative markers were analyzed

before and after training.

Results: Muscular strength was improved in both training groups when compared to CTRL (p<0.05).CT presented an

increase of SOD and catalase at the end of training and a significant decreasein tert-butyl hydroperoxide-initiated

chemiluminescence(p<0.05) after intervention when compared to CTRL. FTpresented significantly reduction in

systolic blood pressure and increase in catalase activity (p<0.05). CTRL presented worsening HDL levels at the end of

the period. Conclusion: Both programs improved muscular strength, antioxidant defense and maintained HDL levels. Therefore,

resistance training can be considered an effective long-term non-pharmacological therapy for abnormalities that

comprise metabolic syndrome.


INFLUENCE OF HOMOCYSTEINE LEVELS ON ADHESION MOLECULES AND OXIDATIVE STRESS IN

PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS

Requena, R. G.1; Robles, B. E.F.

2; Alcântra, C. C.

1; Stadtlober, N. P.

2; Santos, L. F. R. F.

2; Alfieri, D. F.

1; Flauzino, T.

1;

Iriyoda, T. M. V.4; Cossentini, L. A.

2; Sekiguchi, B.

1; Scavuzzi, B. M.

1; Lozovoy, M. A. B.

3; Vissoci Reiche, E. M. V.

3;

Dichi, I.5; Simão, A. N. C.

3.

1 Post Graduate Program in Health Sciences – University of Londrina, Brazil.

2 Post Graduate Program in Experimental Pathology – University of Londrina, Brazil.

3 Department of Pathology, Clinical Analysis and Toxicology – University of Londrina, Brazil.

4 Department of Rheumatology – University of Londrina, Brazil.

5 Department of Internal Medicine – University of Londrina, Brazil.

Email: [email protected], [email protected], [email protected], [email protected],

[email protected], [email protected], [email protected], [email protected],

[email protected], [email protected], [email protected],

[email protected], [email protected] [email protected], [email protected]

Keywords: Systemic lupus erythematosus; homocysteine; adhesion molecules; oxidative stress.

Introduction and objectives: Systemic Lupus Erythematosus (SLE) is a chronic inflammatory autoimmune disease

characterized by the production of autoantibodies and chronic inflammation. Increased homocysteine levels, adhesion

molecules and oxidative stress are considered important mechanisms contributing to the complex pathophysiological

network that characterizes SLE. Therefore, the aim of the present study was to analyze the association between

oxidative stress, adhesion molecules and homocysteine levels in SLE. Material and methods: The study included

176 subjects; 50 healthy individuals (control group) and 126 patients with SLE. Homocysteine levels was determined

by chemiluminescence microparticule immunoassay. Hyperhomocysteinemia was categorizes using the median of

SLE patients (10.59 ng/mL). Oxidative stress was assessed by lipid hydroperoxide by tert-butyl hydroperoxide-initiated

chemiluminescence (CL-LOOH), advanced products of protein oxidation (AOPP) by spectrophotometry, nitric oxide

metabolites (NOx) by the method of Griess and total plasma antioxidant capacity by TRAP methodology (Total

Radical-Trapping Antioxidant Parameter). Adhesion molecules, platelet endothelial cell adhesion molecule 1 (PECAM-

1), vascular cell adhesion molecule 1 (VCAM-1), endothelial-selectin (E-selectin), platelet-selectin (P-selectin), were

evaluated by Luminex Plataform. The study protocol was fully approved by the Ethical Committee of the University of

Londrina (Paraná, Brazil) (CAAE 01865212.0.0000.5231, CEP/UEL 205.328). Results: Patients with increased

homocysteine levels (≥10.59 ng/mL) presented higher hydroperoxides (p = 0.015), AOPP (p = 0.022), NOx (p =

0.011), PECAM-1 (p = 0.037), VCAM-1 (p = 0.026), E-Selectin (p = 0.017), P-Selectin (p = 0.047), and lower TRAP

values (p = 0.015) than patients with lower homocysteine levels (<10.59). In a binary logistic regression that included

the markers and parameters with statistical relevance between the groups with homocysteine <10.59 ng/mL, as a

reference group, and homocysteine ≥ 10.59 ng/mL, TRAP (OR= 0.976; 95% CI: 0.945 – 0.990; p =0.006) was found

as inversely associated, whereas hydroperoxides (OR= 1.000; 95% CI: 1.000-1.000; p=0.012), NOx (OR= 1.122; 95%

CI: 1.013 – 1.242; p =0.027), and PECAM-1 (OR=1.000; 95% CI: 1.000-1.000; p=0.004) were found to be directly

associated. Conclusion: The behavior of some adhesion molecules and oxidative stress markers depends in a large

scale of homocysteine levels. The data obtained in the present study allow suggesting that special efforts may be

directed to decrease homocysteine levels in SLE.














INFLUENCE OF LACTATE CIRCULATING LEVELS IN HEPATIC GLUCONEOGENESIS IN WALKER-

256 TUMOR BEARING-RATS

Frasson, I. G.1; Biazi, G. R.

2; Miksza, D. R.

3, Bertolini, G. L.4; Souza, H. M.

4.



Key words: cachexia, Walker-256 tumor, hepatic gluconeogenesis.

Introduction and objectives: Cancer patients often have severe weight loss (cachexia). It is suggested that

increased activity of the Cori cycle (glucose-lactate cycle), a futile cycle that converts the lactate, originated in the

tumor, into glucose by hepatic gluconeogenesis, contribute to cancer cachexia. Patients with cancer cachexia present

increased hepatic gluconeogenesis from lactate. However, our preliminary studies in liver perfusion in situ of rats with

Walker-256 tumor-induced cachexia show inhibition of gluconeogenesis from low lactate concentrations, similar to

those in healthy rats (2 mM). Walker-256 tumor bearing-rats show elevated blood lactate concentrations, respectively

3, 5.5 and 8 mM on days 5, 8 and 12 of tumor development. The aim of this study was to investigate, in Walker-256

tumor bearing-rats, the influence of these circulating concentrations of lactate on the hepatic gluconeogenesis and to

evaluate possible mechanisms involved in effects.

Material and methods: Walker-256 cells (0,1x106) were inoculated (sc) on the right rear flank of male Wistar rats

(230-240 g) (tumor-bearing rats). Healthy rats (controls) were inoculated with PBS. Gluconeogenesis, from several

glucose precursors (lactate, pyruvate and glutamine), associated or not to NADH, was evaluated in liver perfusion in

situ on days 5, 8 and/or 12 of tumor development. Hepatic gluconeogenesis was also evaluated in vivo by

intraperitoneal administration of gluconeogenic substrates (pyruvate or glutamine) on day 12 of the tumor. The

experimental protocols were approved by Animal Experimentation Ethics Committee of the State University of

Londrina (register nº 10399).

Results: In liver perfusion studies in situ, tumor-bearing rats with 5, 8 and 12 days of tumor development had lower

hepatic glucose production from 2 mM lactate, compared to healthy rats. However, hepatic glucose production in rats

with 5, 8 and 12 days of tumor, respectively, from lactate 3, 5.5 or 8 mM, was similar to that of healthy rats. The

hepatic glucose production from 2 mM lactate, associated with 50 or 75 μM NADH, but not with 25 μM NADH,

promoted a greater increase in the glucose production of rats with 12 days of tumor, compared to healthy. Rats with

12 days of tumor also had lower hepatic glucose production from pyruvate 2, 5 and 8 mM, compared to healthy.

However, hepatic glucose production from 2 mM pyruvate, associated with 50 or 75 μM NADH, but not with 25 μM

NADH, of rats with 12 days of tumor, was restored to values similar to healthy rats. Finally, rats with 12 days of tumor

had lower hepatic glucose production from 1 mM glutamine, but not from 2.5 mM glutamine or 1 mM glutamine

associated with 50 μM NADH, compared to healthy rats. In in vivo studies, intraperitoneal administration of pyruvate

(1.0 mg / kg) or glutamine (0.5 mg / kg) increased the glycemia of tumor-bearing (day 12) and healthy rats in a similar

manner.

Conclusion: High concentrations of lactate, similar to those found in the blood of Walker-256 tumor bearing-rats,

restored the inhibition of hepatic gluconeogenesis observed from low concentrations of lactate, by mechanisms that

probably involved increased redox potential (NADH).


LIPIDOMICS ANALYSIS OF THE VAGINAL DISCHARGE IN WOMEN WITH VULVOVAGINAL

CANDIDIASIS AND CYTOLYTIC VAGINOSIS

Sanches, J. M.1; Giraldo, P. C.

1; Duarte, G. H. B

2; Marques, L. A.

2; Amaral, R.

1; Nakahira, M.

2; Bieleveld, M. J. M.

3;

Silva, F. N.2; Tamarindo, G. H.

4; Eberlin, M. N.

2

1Campinas State University, Department of Tocoginecology, Campinas – São Paulo/Brazil

2Campinas State University, Institute of Chemistry, Campinas – São Paulo/Brazil

3University of São Paulo, Polytechnic school, São Paulo – São Paulo/Brazil

4Campinas State University, Institute of Biology, Campinas – São Paulo/Brazil


Keywords: Vaginal discharge; vaginitis; lipidomics; candidiasis; mass spectrometry.

Introduction: Vaginal discharge is a very common condition that affects women. Vulvovaginal candidiasis (VVC) and

cytolytic vaginosis (CV) are gynecological condition with symptoms that include vaginal itching, burning, abnormal

discharge, dysuria and dyspareunia. Although the symptoms are quite similar, the vaginal microenvironment and

amount of lactobacillus, inflammatory response, and treatment are completely different. The advances in mass

spectrometry techniques and bioinformatics have been improved with the need for the understanding the health and

disease process, using the lipidomics as a great tool to characterize potential biomarkers for many diseases.

Objective: Characterize the lipids in the vaginal discharge in women with VVC and CV as biomarkers, relating with

the physiology, physiopathology and metabolism of these conditions is the aim of this work. Material and methods: A

cross-sectional study with 24 women, non-pregnant, pre-menopausal, aged between 18 to 44 years was conducted in

the Outpatient Clinic of Genital Tract Infections of the Campinas State University Women’s Hospital, Campinas, Brazil.

All women provided written informed consent, approved by the Ethics and Research Committee,

CAAE:60648016.8.0000.5404. All women were interviewed, had an anamnesis and underwent a speculum-based

gynecological examination. After properly diagnosis, the patients were placed in three groups: VVC, CV or health

women. For the sample preparation was used deionized water following the addition of chloroform HPLC grade and of

methanol, vortex and centrifuged. All samples were dried using SpeedVac and kept frozen at -80oC until analysis.

Chromatographic separation was performed on an Agilent1290-Infinity ultra-high performance liquid chromatography

system, and a C-18 column was also utilized. The data were obtained on the positive and negative mode, by the

software MassHunter Qualitative (Agilent). All metabolites were evaluated for mass error (≤5 ppm), conclusive isotopic

and fragmentation pattern and plausible retention time, characterized as description by The Human Metabolome

Database, XCMS and Lipid Maps. Multivariate data analysis techniques were applied to process the acquired data.

The segregation between groups was analyzed by Principal Component Analysis (PCA). Results: The PCA analysis

showed an important separation which characterized the metabolites difference between the three groups. Selection

of the potential biomarkers provided by the Partial Least Square Regression (PLS-DA) is the result of the VIP

(Variable Influence on Projection), finding a total of 38 lipids. The main biomarkers for VVC associated with

inflammatory process were prostaglandins and O-Adipoylcarnitine. Phosphatidic acids and 13S-HpOTrE fatty acid

could increase the signals for inflammation and pain in VCC. The physiopathology of CV is described for the lyse of

the vaginal epithelium and lactobacilli overgrowing which have 1-(11Z-docosenoyl)-glycero-3-phosphate, and 5-

Aminopentanoic acid as biomarkers, respectively. Also, 1-oleoyl-cyclic phosphatidic acid and Palmitoleic acid are

present in injured tissue, possibly playing a role of recovering the vaginal epithelium in CV. Some lipids are also

related with oxidative stress and apoptosis, especially in CV that include phosphatidylserines, N-(tetradecanoyl)-

sphinganine and Glycochenodeoxycholic acid 7-sulfate. Conclusion: This work has a very important role to elucidate

the metabolic mechanisms of the lipids in VVC and CV that could relate with their biofunction and pathways in

physiopathology.

Grants: FAPESP


PATHOPHYSIOLOGY OF AMYOTROPHIC LATERAL SCLEROSIS

Giroldo, I.R¹; da Silva, C.F¹; Campois, T.G¹

¹Centro Universitário Filadélfia, Department of Biomedicine, Londrina, PR, Brasil

[email protected]

Key words: Sclerosis, Amyotrophic lateral sclerosis, degenerative diseases, amyotrophic lateral sclerosis,

pathophysiology.

Introduction: Amyotrophic Lateral Sclerosis (ALS) corresponds to a rare neurodegenerative disease that affects

motorneurons causing a progressive degeneration of the motoneurons and consequent muscular compromise. The

disease may be associated with molecular genetics, in which there is a mutation of the gene encoding the copper-zinc

superoxide dismutase of chromosome 21. According to the Brazilian Association of Amyotrophic Lateral Sclerosis, it's

incidence in the population is 0.6 - 2.6 per 100,000 inhabitants. Since age is the main factor for its occurrence, ALS

prevails in patients between the ages of 55 and 75 years. In addition, it can also have family causes, which

correspond to 5% to 10% of cases. Already the average survival of the affected individuals is of 3 to 5 years, due to

the respiratory insufficiency that they acquire. However, with the aid of mechanical ventilation, this can incrise 15

years or more.

Review: Since 1830, Amyotrophic Lateral Sclerosis (ALS) has been verified. However, only in 1969, Brain and Walton

associated the true symptoms and causes to ALS. The disease arises as consequence to several pathophysiological

mechanisms and cellular dysfunctions this affects motorneurons in the brain, such as upper and lower motoneurons

compromising voluntary muscle contraction among other neural functions.

It is the degeneration of the inferior motoneurons, which prodruce into the skeletal muscles, which causes most of the

neurological deficiencies of the disease. When the main symptoms of Amyotrophic Lateral Sclerosis can be divided

into: direct symptoms of motoneural degeneration, where there is weakness, atrophy, fasciculations, cramps,

dyspepsia, dysphagia, dyspnea, dyspnea and emotional lability and resulting indirect symptoms of primary symptoms,

in which there are psychological disorders, sleep disturbances, constipation, sialorrhea, thickening of mucous

secretions, symptoms of chronic hypoventilation and pain. Patients present progressive physical failure, respiratory

failure being the main cause of death.

As the disease progresses ventilatory assistance is required. In most cases, ALS is sporadic, which arises from the

axon transport dysfunction. This results in less glutamate reclaimed by the axon, with constant neuron stimulation and

excessive release of degrative enzymes, the process called excitotoxicity. On the other hand, there are cases of

Amyotrophic Lateral Sclerosis of genetic cause, in which there are abnormalities in chromosome 21.

In young people, the change is on chromosome 2. Therefore, according to the sporadic condition, ALS is treatable

with a drug, such as Riluzole, which inhibits the release of glutamate in the synaptic cleft. Still, motor and respiratory

physiotherapy assists against the loss of motor controls, as well as healthy nutrition and psychological support.

Conclusion: Amyotrophic Lateral Sclerosis is part of an important group of neurodegenerative diseases, because it is

caused by the loss of lower and upper motor neurons and affects the motor system, compromising voluntary

contraction of muscles. Justifying, therefore, weakness, atrophy, atony, arreflexia and other symptoms as well as

respiratory insufficiency, symptom that can lead the patient to death.


PYRROLOQUINOLINE QUINONE REDUCES HEPATOCELLULAR DAMAGE IN MICE MODEL OF

PARTIAL HEPATECTOMY AFTER ISCHEMIA AND REPERFUSION INJURY

Gomes, M. S.1; Apolinario, L. A.

1; Augusto, M. J.

1 ; Forti, D.

1; Silva, D. M.

1; Ramalho, F. S.

1

1 University of São Paulo, Departament of Pathology, Ribeirão Preto Medical School, , Ribeirão Preto, SP, Brazil.

Autor address: [email protected]

Keywords: Ischemia and Reperfusion, Oxidative Stress, PQQ Cofactor, Liver transplantation, Antioxidants.

Introduction and objectives: The hepatic ischemia and reperfusion (I/R) injury occurs in several clinical conditions,

as well as in the major hepatectomies and the liver transplantation. The pathophysiology of this disease is mainly

composed by an acute inflammatory response and tissue oxidative stress in response to excessive oxygen reactive

species generation in the liver. As result of I/R injury, cell proliferation is impaired. On the other hand, pyrroloquinoline

quinone (PQQ) has been studied as a substance that can confer protection against I/R injury in different organs. PQQ

shows diverse roles in physiological aspects involved in protection against oxidative stress, induction of cell

proliferation and tissue fibrosis inhibition. The aim of this study was to evaluate the role of the PQQ treatment in partial

hepatectomy after hepatic I/R injury in mice.

Material and methods: Balb-c mice were underwent to hepatic injury by normothermic I/R followed by partial

hepatectomy, and treated with PQQ (10mg/kg body weight) or saline solution. The degree of hepatocellular damage

was evaluated by the serum level of aminotransferases and histological score of tissue damage. The intensity of liver

oxidative stress was quantified by the measurement of malondialdehyde (MDA) and glutathione reduced (GSH) levels

on the liver parenchyma. This experimental protocol was approved by the Animal Research Ethical Committee of the

Ribeirão Preto Medical School, University of São Paulo (process no. 103/2016).

Results: PQQ significantly reduced serum and histological parameters of liver injury. Additionally, the PQQ-treated

animals exhibited reduced hepatic oxidative stress due to the decrease in liver levels of MDA and the preservation of

the antioxidant reserve of hepatic GSH.

Conclusion: The results obtained so far indicate that PQQ can protect the liver parenchyma against normothermic I/R

followed by partial hepatectomy. This effect seems to be related to its high antioxidant capacity. We expected that

these results improve hepatocellular proliferation after partial hepatectomy.

Grants: CAPES


RELATIONSHIP BETWEEN THE WAIST-TO-HEIGHT RATIO AND CARDIOMETABOLIC RISK IN

ADOLESCENTS

Martins, R. F.D.B1, Bonifácio, K.L

1,2, Semeão, L.O

1, Michelin, A.P.

1,2, Matsumoto, A.K

1, Bonacini, A.G.S

1, Barbosa,

D.S1,2,3

, Cyrino, E.S4, Venturini, D.

1,2,3



3Department of Clinical Analysis and Toxicological, State University of Londrina, Londrina, PR, Brazil

4Metabolism, Nutrition, and Exercise Research Group, Sport and Physical Education Center, State University of

Londrina, Londrina, PR, Brazil


Keywords: adolescents, metabolic risk, waist-to-height ratio

Introduction and Objectives: Overweight and obesity have increased significantly in younger and younger

populations, especially during adolescence. Precise and accurate diagnosis is of fundamental importance for the

evaluation of this population. Because of the limitations of body mass index (BMI), waist circumference (WC) and

abdominal circumference (AC), waist-to-height ratio (WHR) has been suggested to assess adiposity. The WHR

incorporates AC as a measure of abdominal adiposity, but also adjusts it to the size of the individual by dividing by its

stature. Thus, the objective of the present study was to evaluate the presence of metabolic risk through WHR ratio

and an expressive number of adolescent school children in the city of Londrina.

Material and methods: The present study evaluated the athropometric (body mass index, waist circumference,

abdominal circumference and waist-to-heigth ratio) and metabolic parameters (glucose, total cholesterol, high-density

lipoprotein, and triglycerides) of 977 adolescents. Of the 977 adolescents studied, 10.64% had a WHR greater than

0.5, that is, presenting a cardiometabolic risk. Comparisons between groups were performed using the nonparametric

Kruskal–Wallis test with the post-hoc Dunn test. The results were considered significant when P < 0.05. A statistical

analysis program Graph Pad Prism version 3.0 was used for evaluations. . The original project was approved by the

Research Ethics Committee of the State University of Londrina (protocol: 238/2010). Results The group with WHR >

0.5 presented a significant increase in plasma levels of total cholesterol (p <0.0001), LDL-C (P <0.0001), triglycerides

(p <0.0001), TC/HDL-C (p <0.0001) and LDL-C/HDL-C (p <0.0001) ratios, as well as other anthropometric indexes:

BMI (p<0.0001), waist circumference (p <0.0001) and abdominal circumference (p <0.0001); and a significant

decrease in HDL-C levels (p <0.0001), relative to the group with WHR 0.5. In the group with WHR> 0.5, BMI, WC

and AC correlated negatively with HDL-C (p = 0.043), (p = 0.016), (p = 0.004), and positively with plasma triglyceride

levels (p = 0.044), (p = 0.0016), (p = 0.004), respectively. The WC still correlated positively with the TC/HDL-C index

(p = 0.029).

Conclusion: This study concluded that, in Brazilian adolescents, WHR is a good predictor of cardiovascular risk and,

since it is an easily available and non-invasive parameter, it should be used in populations for stratification of

cardiometabolic risk as early as possible.

Grants: CNPq


ROLE OF THE INTESTINAL MICROBIOTA IN THE PATHOPHYSIOLOGY OF OBESITY

Da Silva, W. L. 1; Silva, A. V.

2; Oda, J. Y.

2

1 School of Medicine, Federal University of Mato Grosso do Sul, Três Lagoas, MS, Brazil.

2 School of Medicine, Federal University of Mato Grosso do Sul, Três Lagoas, MS, Brazil.


Key words: gut, microbiota, obesity, diet.

Introduction: Obesity currently stands out as a major public health problem, since the changes in lifestyle and an

increase in high calorie food intake has become more frequent, however, different factors of the types of food are

important. Recent evidence suggests that the intestinal microbiota plays an important role in energy metabolism, so

the microorganisms that constitute the human intestinal microbiota (MI) participate in the caloric extraction of

indigestible polysaccharides and their incorporation into adipocytes. The two main bacterial divisions present are

Firmicutes (60% -80%) and Bacteroidetes (20% -40%). In a way not understood, these bacterial divisions undergo

changes (dysbiosis) related to the type of feeding generating disturbances in the energy metabolism. Thus, several

studies seek to relate the food pattern to the modulation of the IM, a factor that could favor the condition of obesity of

the individual. Therefore, the objective of this study was to study the modulatory role of feeding in IM and its role in the

pathophysiology of obesity. For this, a search was performed on the database PubMed, MEDLINE, LILACS, IBECS,

using the keywords "gut microbial" AND "obesity", AND "diet", using as filters, articles published between 1997 To

2017, english language.

Review: The search produced 623 articles, 23 of these were included after screening by title and summary

evaluation, of which eight were selected for full reading and framed for this study review. According to the data

collected, the intestine of the obese individual has properties not yet characterized that favor the bacterial phylum

Firmicutes, which induce weight gain increasing the absorption of monosaccharides and the storage of triglycerides in

the adipocytes. Changes in the Firmicutes / Bacteroidetes ratio were associated with obesity and insulin

resistance. Tests with inoculation of microbiota in mice showed that hyperglycemia appears to be the main metabolic

parameter affected in the condition of dysbiosis. However, changes in intestinal permeability also influenced weight

gain and adiposity, since the high fat diet causes increased intestinal permeability favoring obesity. In contrast, the

intake of oligosaccharides of animal origin has been shown to reduce intestinal permeability, increasing the

proliferation of probiotics (Bifidobacterium and Lactobacillus) and glucose homeostasis, leading to weight

reduction. The transplantation of IM from normal mice to germ-free recipients evidenced an increase in body fat

without increasing food consumption, proving the microbiota's participation in energy metabolism. It is believed that

the development of obesity is related to the individual's systemic exposure to lipopolysaccharide derived from MI,

generating a chronic inflammatory response.

Conclusion: The diet rich in fat and polysaccharides modifies MI resulting in the dysbiosis through the predominance

of Firmicutes and / or increasing the permeability of the intestinal cells. Both alter the energetic metabolism favoring

the elevation of the weight, predisposing to obesity. This work contributes to the understanding of the modulatory role

of feeding in IM, providing future perspectives of therapies for the control of obesity, through the modification of IM

using probiotics, taking into account the needs of dietary adjustments.



TGFB1 HAPLOTYPE STRUCTURES HAVE SUBTYPE-SPECIFIC EFFECTS ON BREAST CANCER

SUSCEPTIBILITY AND CLINICAL PRESENTATION

Vitiello, G. A. F.1; Losi-Guembarovski, R.

2; Ishibashi, C. M.

1; Amarante, M. K.

1; Campos, C. Z.

3; Rosa, M. H.

1; Motoori-

Fernandes, C. Y.1; Carmelo, E. C. B.

3; Ceribelli, J. R.


1

1Department of Pathological Sciences, Londrina State University, Londrina, PR, Brazil

2 Department of General Biology, Londrina State University, Londrina, PR, Brazil

3 Department of Clinical Research, Londrina Cancer Hospital, Londrina, PR, Brazil

[email protected]

Keywords: Breast cancer; transforming growth factor beta; polymorphism; susceptibility; prognosis

Introduction and objectives: Breast cancer (BC) is a complex disease presenting different subtypes and high

degree of interindividual variation. Transforming growth factor beta 1 (TGFβ1) is a pleiotropic cytokine, exerting its’

function in a cell- and context-dependent manner. These effects have been documented in BC, in which it inhibits

growth of less aggressive tumors, such as luminal A subtype, while promotes growth and invasiveness of highly

aggressive subgroups, such as HER2+

and triple negative (TN). Polymorphisms in TGFβ1 gene (TGFB1) have been

described and associated with cancers, including BC; however, the potential subtype-specific effects of these

polymorphisms have not been assessed in previous literature. Therefore, the aim of this study was to evaluate the

effect of a haplotype structures comprising four TGFB1 polymorphisms, two on promoter region, G-800A (rs1800468)

and C-509T (rs1800469), and two on signal peptide, C29T (Pro10Leu; rs1800470) and G74C (Arg25Pro; rs1800471),

on the susceptibility and clinical presentation of BC subtypes.

Material and methods: DNA was extracted from peripheral blood or surgical excision tissue from 323 BC patients,

which were grouped based on tumor positivity for hormonal receptors (ER/PR) and HER2, and from peripheral blood

of 405 women without evidence of breast neoplasm, no personal history of malignancy and no familial history of BC.

All procedures were approved by institutional ethics committee for research involving human subjects (CEP/UEL

189/2013 – CAAE 17123113400005231) and all individuals signed free-informed consent. SNPs genotyping was

performed PCR-RFPL, and haplotype inference was made by PHASE 2.1 software. For association study, the odds

ratio (OR) calculus was made through logistic regression adjusted by age, and correlation with clinicopathological

parameters was assessed through Kendall’s Tau-b correlation coefficient. All analysis had 95% confidence interval

(CI) and were two tailed.

Results: GCTG haplotype, which is associated with decreased TGFβ1 production, was associated with decreased

risk for HER2+ BCs both in recessive (OR = 0.35; CI = 0.13 – 0.9; p = 0.03) and dominant (OR = 0.51; CI = 0.29 – 0.9;

p = 0.02), while GTCG, which is associated with increased TGFβ1 production, was positively associated with

increased risk for this cancer subtype (OR = 1.89; CI=1.07 – 3.35; p = 0.03). Furthermore, GCCG haplotype was

associated with decreased risk for ER/PR+HER2

- BC in dominant model (OR = 0.49; CI = 0.24 – 0.97; p = 0.04).

GCTG haplotype negatively correlated with tumor size (Tau-b = -0.25) and stage (Tau-b = -0.24) in HER2+ BCs and

with lymph node metastasis (LNM) in ER-PR

-HER2

- BCs (Tau-b = -0.32), while GTCG positively correlated with

histopathological grade (Tau-b = 0.31) and with LNM (Tau-b = 0.38) in HER2+ BCs and with tumor size in ER

-PR

-

HER2- BCs (Tau-b = 0.33).

Conclusion: These results indicate that the effects of TGFB1 haplotypes in BC subtypes reflects that of TGFβ1, with

high-producer variants increasing the risk and correlating with worst prognosis parameters in aggressive subtypes and

low-producer variants exerting the opposite effect. These data further highlight TGFβ1 roles in BC and may assist in

screening for poor-prognosis patients that may benefit of recently proposed treatments targeting TGFβ1.

Grants: CNPq, CAPES, Fundação Araucária


THE EFFECT OF CHRONIC SUPPLEMENTATION WITH VITAMIN D ON HYPOTHALAMIC OBESITY

Amaral, B. K.1; Guareschi, Z. M

.1; Valcanaia, A. C.

1; Hotz, P. G

.1; Ceglarek, V. M.

1; Grassiolli, S.

1

1 Universidade Estadual do Oeste do Paraná, LAFEM Department, Cascavel, PR, Brazil.


Keywords: Obesity, Vitamin D, Insulins.

Introduction and objectives: The World Health Organization (WHO) classifies obesity as a global epidemic, an

event intimately related to comorbidities, such as diabetes and cardiovascular diseases. Hypovitaminosis is frequently

observed in obese people. Thus, high adipose tissue content promotes low biodisponibility of Vitamin D (VD)

aggravating insulin resistance and dyslipidemia. This study evaluate the effect of chronic VD supplementation in

hypothalamic obesity induced by neonatal treatment with glutamate monosodium (MSG).

Material and methods: This is an experimental study, it was done at the laboratory of physiology and endocrinology

metabolism from Universidade Estadual do Oeste do Paraná, previously approved by the ethics committee at

13/11/2015 by the ordinance 2729/2014.

During five first days after birth male Wistar rats received subcutaneous injection of MSG (4g/Kg) and the group

Control (CON) received saline. After weaning the MSG rats were randomically subdivided in supplemented with VD or

non supplemented (ns) forming three experimental groups (n=6/group): CON-ns; MSG-ns and MSG-VD.

Supplemented rats received VD (12µg/Kg) 3 times/week during 72 days. At 92 days of life the animals were

euthanized and the adipose tissue content and blood metabolic biomarkers (glycaemia, triglycerides and total

cholesterol) evaluated. Pancreatic islets were isolated by collagenase technique and glucose induced insulin secretion

analyzed by radioimmunoassay method. Values were expressed by mean±stander error mean (SEM). T test was

used to compare CON-ns versus MSG-ns and MSG-ns versus MSG-VD; p<0.05.

Results: The MSG-ns rats show high adipose tissue content associated with hyperglycemia and hyperinsulinemia in

relation to CON-ns animals (p<0.05). The VD supplementation did not affect significantly adipose tissue content and

glycemia in relation to MSG-ns group. However, MSG-VD rats presented reduction of 70.14% and 45.51% in insulin

and tryglicerides, respectivelly compared with MSG-ns (p<0.05). In contrast, MSG-VD rats show high cholesterol

29.01% in relation to MSG-ns group (p<0.05). Glucose-induced insulin secretion was 43.09% lower in islets from

MSG-VD in relation to islets from MSG-ns (p<0.05).

Conclusion: The chronic VD supplementation in MSG obese rats avoids insulin hypersecretion as well, disrupting in

triglycerides plasmatic levels, without affect adipose tissue content.

Grants: Fundação Araucária for the scholarship.


THE INFLUENCE OF INTERLEUKIN-6 ON INSULIN RESISTANCE

GALIA, W.B.S.1; DIAZ, B.F.

1; FERRAZ, L.S.

1; SILVA, F.F.

2; SOUZA, H.M.

1; BERTOLINI, G.L

1.

1Department of Physiological Sciences, State University of Londrina, Londrina, PR, Brazil.

² Department of Physiology and Biophysics, São Paulo University, São Paulo, SP, Brazil.


Key words: cancer, cytokines, diabetes mellitus, insulin, obesity.

Introduction: The insulin resistance (IR) is characterized by the reduction of the metabolic effects of insulin. It is

present in approximately 20 to 25% of the people and is commonly associated with pathologies as obesity, diabetes

mellitus 2 (DM2) and cancer. Patients with these pathologies present several metabolic alterations, often due to IR. IR

causes an increase in catabolism, degrading the energy reserves. The related mechanisms associated with the

development of IR in diabetic patients seem to involve decreased synthesis of insulin receptors and translocation of

glucose transporter proteins (GLUT4) to the plasma membrane, as well as decreased insulin receptor affinity and

autophosphorylation of the β-tyrosine kinase subunit. It is likely that pro-inflammatory cytokines produced by immune

system, as interleukin-6 (IL-6), can contribute to the development of IR.

Review: Obesity is directly associated with DM2, and pro-inflammatory factors produced by adipose tissue perform an

important role in this association between obesity and diabetes. Several animal models suggest that inflammatory

processes have a causal relationship with obesity and its comorbidities like IR, DM2 and cardiovascular diseases. IL-6

seems to be related to IR by mechanisms such as activation of tyrosine phosphatase or interaction with the

suppressor of the cytokine signaling proteins (SOCS) and the insulin receptor. IL-6 can affect the insulin action on

liver, muscle and adipose tissue. In vivo studies in rats demonstrated induction of hepatic and skeletal muscle insulin

resistance after infusion of IL-6. These results were associated with deficiency in insulin signaling and suggest direct

effect of IL-6 on muscle insulin resistance. It has also been observed that IL-6 inhibits the signal transduction

mediated by insulin receptor activation in adipocytes and hepatocytes, and that IL-6 depletion increases hepatic

responsiveness to insulin. In vitro, IL-6 does not have acute effect on insulin action in adipocytes, but decreases the

expression of insulin receptor substrate 1 (IRS-1), GLUT4 and peroxisome proliferator-activated gamma (PPAR-).

Also in vitro, IL-6 decreases the insulin signaling in hepatocytes via increased SOCS-3 expression.

Conclusion: Researches related to the influence of IL-6 on IR are important for the academic community. Thus, more

studies should be conducted to elucidate the whole mechanism related to it, with the aim to find new strategies for the

treatment of insulin resistant patients.


THE ROLE OF ANNEXIN A1 AND FPR1 IN BREAST CANCER: A REVIEW

Tavernelli, N. L.1; Zóia, M. A. P.

1; Azevedo, F. V. P.

2; Lonardoni, L. R.

1; Ávila, V. R. M.

2; Dantas, N. O.

3; Goulart Filho,

L. R.1; Silva, A. C. A.

3.

1 Laboratory of Nanobiotechnology, Institute of Genetics and Biochemistry, Federal University of Uberlandia, UFU-MG,

Brazil 2Laboratory of Biochemistry and Animal Toxins, Institute of Genetics and Biochemistry, Federal University of

Uberlandia, UFU-MG, Brazil. 3Laboratory of New Insulation and Semiconductor Materials, Institute of Physics, Federal University of Uberlandia,

UFU-MG, Brazil


Key words: Breast Cancer, Annexin A1, treatment, FPR1

Introduction: The Annexin A1 (ANXA1) is an anti-inflammatory protein that belongs to the Annexin family, which is

characterized for calcium dependent proteins that bonds to phospholipids. These proteins are part of many

mechanism including cellular differentiation, cellular growth control, signal transduction and cytoskeleton formation.

Particularly in anti-inflammatory process, ANXA1 bounds to its receptor formil peptide receptor 1 (FPR1), a

transmembrane receptor bound to protein G. This receptor is associated to many mechanisms that include host

defense, promoting leucocyte migration, cytokine production and degranulation of phagocytic cells. In this context, the

bond between ANXA1 and FPR1 inhibit the process of diapedesis from inflammatory cells such as neutrophils and

leucocytes characterizing, therefore, an anti-inflammatory mechanism. However, the signaling of ANXA1 by FRPR1 is

also important in the development and progression of many types of cancer, between them, breast cancer, that is the

second most incident in Brazil’s female population. Breast cancer is a heterogenous disease and have many

molecular markers that comprehend 3 main receptor types: estrogen (ER), progesterone (PR) and type 2 receptor of

human epidermal growth factor (HER2). Thus, by this receptors, the breast cancer is classified in 4 types: luminal A

(ER+, PR+/-, HER2-), luminal B (ER+, PR+/-, HER2+), HER2 (ER-, PR-, HER2+) and triple negative (ER-, PR-,

HER2-). Triple negative breast cancer is the most aggressive and lacks specific treatment. Thus, the search for new

molecular biomarkers for this type of cancer is essential.

Review: The role of ANXA1 on cancer is very controversial, however, evidences indicate that the signaling of this

protein has a correlation with the growth of the breast cancer aggressiveness. The ANXA1 signals in a autocrine way

the triple negative breast cancer cells. When localized in the membrane, ANXA1 stimulates FPR1 to activate

oncogenesis pathways. In addition, in triple negative breast cancer was observed the growth of expression of FPR1,

that relates with secretion of ANXA1 and this active signaling activates many molecules that culminates in the growth

of aggressiveness of cancer cells, promoting growth in the invasion potentials, migration and proliferation.

Conclusion: Against the foregoing, the ANXA1 protein has correlation with the tumor aggressiveness, and, therefore,

the propose of this paper is that it is used as a tumor biomarker for triple negative breast cancer treatment. Still, the

detection of ANXA1 could be used as a prognostic factor independent.


TP53 GENE POLYMORPHISM: RELATION TO SUSCEPTIBILITY AND CLINICAL OUTCOME IN A

LUMINAL/HER2- BREAST CANCER SAMPLE

Motoori-Fernandes, C. Y.1; Kishima, M. O.

2; Losi-Guembarovski, R.

1; Vitiello, G. A. F.


1; Oliveira, K.

B.1; Campos, C. Z.

3; Losi-Guembarovski, A.

2; Amarante, M. K.

1; Banin-Hirata, B. K.

1; Pasquini, J. F. G.

1;

Watanabe, M. A. E.1

1Department of General Pathology, Londrina State University, Londrina, PR, Brazil

2Department of Pathology, Clinical Analysis and Toxicology, Londrina State University, Londrina, PR, Brazil;

3Department of Clinical Research, Londrina Cancer Hospital, Londrina, PR, Brazil

[email protected]

Keywords: TP53, breast cancer, clinicopathological features, luminal, HER2

Introduction and objectives: Breast cancer (BC) is a heterogeneous neoplasia comprising distinct diseases,

histological characteristics and clinical outcomes, involving a progression through a range of processes, since ductal

hyperproliferation, followed development of carcinoma in situ, invasive carcinoma, and metastatic disease. TP53

suppressor tumoral that encodes the p53 protein is the most commonly mutated gene in many human cancers,

including BC. These mutations could disrupt its’ tumor suppressor functions and promote tumorigenesis. A

polymorphism (rs1042522) at codon 72 of TP53 characterized by the switch from a guanine to a cytosine (G>C)

changing an arginine to a proline (Arg>Pro) in amino acid chain, was suggested to be involved in many cancers.

However, the biological significance of this genetic variant in BC remains unclear, especially in defined BC subtypes.

In this context, the present study aimed to investigate the possible association of this polymorphism in a luminal

HER2- BC sample (ER/PR

+HER2

-), in relation to susceptibility and prognostic parameters.

Material and methods: DNA was extracted from tumor tissue and peripheral blood, respectively collected from 33

luminal HER2- BC patients with clinicopathological data available (tumor size, metastasis, histopathological grade,

Ki67 proliferation index and p53 immunostaining) and 146 women with no evidence of mammary alterations proved by

recent mammograph examination, no family history of BC and no personal history of any malignance. TP53

polymorphism was evaluated through allele-specific PCR. Case-control study was performed through Odds Ratio

(OR) calculus with 95% confidence interval (CI) and correlations between polymorphism and clinicopathological

parameters was assessed by Kendall’s tau-b rank correlation coefficient.

Results: TP53 polymorphism was associated with increased BC susceptibility both in heterozygote (OR = 2.59; CI =

1.16 - 5.81; p = 0.032) and dominant model (OR = 2.38; CI = 1.1 – 5.13; p = 0.026). Correlation analyses showed that

patients carrying the variant allele (Pro) tended to have smaller tumors (tau-b = -0.37; p = 0.04) and lowest

proliferation indexes (tau-b = -0.42; p = 0.02).

Conclusion: These results indicate that TP53 Pro allele increase susceptibility to luminal HER2- BCs, but indicate

better prognosis parameters among patients, suggesting this polymorphism as a candidate marker in this BC sample. Grants: CAPES, CNPq, Fundação Araucária.


TREATMENT WITH METFORMIN AND INSULIN IN WALKER-256 TUMOR BEARING RATS: EFFECTS

ON PANCREATIC ISLETS

Ramalho, M.C1; Galia, W.B.S.

1; De Fatima Silva, F

1,2; Carpinelli, A. R.

2; De Souza, H. M

1; Graciano, M. F.

1.

1Departamento de Ciências Fisiológicas, Centro de Ciências Biológicas, Universidade Estadual de Londrina,

Londrina/PR; 2Departamento de Fisiologia e Biofísica, Instituto de Ciências Biomédicas I, Universidade de São Paulo, São Paulo/

SP.

[email protected]

Keywords: cachexia, insulin resistance, insulin secretion

Introduction and objectives: Cancer-induced cachexia remains a significant cause of morbidity and mortality and, to

date, has no effective treatment. This metabolic process is characterized by progressive weight loss, caused by

depletion of muscle and adipose tissues associated with reduced food intake and insulin resistance. The model of

cachexia induced by Walker-256 tumor showed hypoinsulinemia and increased insulin resistance in peripheral

tissues. There are evidences that insulin sensitizers, such as metformin, may improve peripheral insulin sensitivity in

patients with cancer, which could prevent the dysfunction of insulin secretion. Our aim was to evaluate the effect of

metformin therapy with or without combination with insulin on insulin secretory capacity of pancreatic islets and insulin

content from Walker-256 tumor-bearing rats.

Material and methods: The experimental procedures were approved by Ethics Committee of State University of

Londrina (nº 11536.2014.18). Male Wistar rats (230g) inoculated with tumor cells were treated with oral administration

of metformin (500 mg/kg) daily during 12 days, or oral administration of metformin associated with subcutaneous NPH

insulin (1,0 U.kg-1

) daily. Treatments started on the day of tumor cell inoculation. After treatment, pancreatic islets

were isolated from pancreas. Insulin secretion and intracellular insulin content of pancreatic islets were analyzed in

response to glucose and measured by radioimmunoassay. The results were expressed as mean ± standard error and

evaluated by variance analysis (ANOVA) with Bonferroni post-test.

Results: Insulin secretion from isolated islets of tumor-bearing animals incubated at 16.7 mM glucose was reduced by

50% compared with healthy rats. This effect was not improved by the treatments (n ≥4). Intracellular insulin content of

islets from tumor bearing-rats was reduced by 35% compared with healthy rats, with no improvement by the

treatments (n ≥4).

Conclusion: Cachexia induced by Walker-256 tumor affects both insulin secretion stimulated by glucose and insulin

synthesis by pancreatic beta cells. Treatments with metformin and insulin did not improve secretory function

stimulated by glucose and the synthesis of insulin by islets.


ZIKA VIRUS DETECTION IN VISCERAL SAMPLES FROM A STILLBORN FETUS

Leme, J. C. 1; Gomes, M. S.

1; Yamamoto, A. Y.

2; Silva, D. M.

1; Apolinario, L. A.

1; Augusto, M. J.

1; Duarte, G.

3;

Mussi-Pinhata, M. M. 2; Ramalho, F. S.

1

University of São Paulo, Ribeirão Preto Medical School, Departaments of 1Pathology,

2Pediatrics and

3Gynecology and Obstetrics, Ribeirão Preto, SP, Brazil.

Author address: [email protected]

Keywords: Zika vírus, flavivirus, microcephaly, congenital infection, stillborn fetus.

Introduction: Zika virus (ZIKV) is a Flavivirus belonging to the same family as the Dengue, Chikungunya and Yellow

Fever viruses, which are transmitted to humans through bites from Aedes mosquitos. During the 2015 outbreak in

Brazil, vertical transmission of ZIKV was confirmed by the detection and sequencing of the ZIKV genome from the

brain of microcephalic fetuses. Due to strong neurotropism of ZIKV by the nervous tissue, ZIKV RNA had not yet been

detected in fetal organs other than the central nervous system. Here, we describe the autopsy findings from a stillborn

fetus that developed intrauterine ZIKV infection. ZIKV RNA was detected in samples from the brain, lungs, kidneys,

spleen and thymus.

Case report: This case was a stillborn baby girl, whose mother was 25 years old and had developed a rash and fever

at the 10th week of pregnancy, at which point real-time PCR analysis of the mother’s serum confirmed a ZIKV

infection. At 23 weeks of gestation, a fetal magnetic resonance imaging showed microcephaly, diffuse brain atrophy,

intense thinning of the cerebral mantle, ventriculomegaly, cerebellar hypoplasia, and atrophy of the brainstem and

spinal cord. Micrognathia and malformations of the lower limbs were also observed. Fetal death was diagnosed at the

34th gestational week. A stillborn baby girl was delivered vaginally two days after death, and she weighed 1135 g and

had a head circumference of 22.4 cm.

An external body inspection showed diffuse and intense skin maceration, microcephaly and arthrogryposis. At gross

examination, the brain parenchyma exhibited severe liquefactive necrosis and numerous calcifications. The thoracic

and abdominal viscera also exhibited advanced autolysis.

The histopathological examination of the lungs, heart, thymus, kidneys, liver and spleen evidenced diffuse coagulative

necrosis of the parenchymal cells as well as moderate macrophage infiltration. Immunohistochemical labeling with the

anti-flavivirus monoclonal antibody 4G2 revealed strong cytoplasmic staining in macrophages in the brain, lungs,

kidneys and spleen. However, the flavivirus envelope protein was not detected in the parenchymal cells of these

organs. Real-time PCR assays revealed high levels of the ZIKV RNA in samples from the brain, lungs, kidneys,

thymus and spleen.

Final consideration: Several authors have reported difficulty in detecting ZIKV outside the central nervous system.

However, we were able to detect relatively high levels of ZIKV RNA in the lungs, kidneys, spleen and thymus samples

from the stillborn fetus. Although ZIKV proteins were not detected in the cellular remnants of these organs, high levels

of these proteins were detected in the macrophages that had infiltrated the autolyzed parenchyma of the lungs,

kidneys and spleen. These mononuclear phagocytes persist in tissue microenvironments with low oxygen content,

and they are promptly activated under these conditions. The detection of the ZIKV proteins in macrophages in the

autolyzed viscera of a macerated stillborn fetus suggests that ZIKV may be phagocytized from fetal fluids by activated

macrophages following fetal death.

This research protocol was approved by the Research Ethical Committee of the University Hospital of the Ribeirão

Preto Medical School (process nº 7404/2016).

EPIDEMIOLOGY AND PUBLIC HEALTH 103

COMORBIDITIES ASSOCIATED WITH OVERWEIGHT IN ADOLESCENTS

Silva, F. J. A.1; Tomeleri, C. M.

1; Souza, M. F.

2; Silva, D. R. P.

1; Cavalcante, E. F.

1; Antunes, M.

1; Polvani, A. C. T.

1;

Nabuco, H. C. G.1; Schiavoni, D.

1; Venturini, D.

3; Barbosa, D. S.

3, Okino, A.

3; Cyrino, E. S.

1.

1Londrina State University, Physical Education, Londrina, PR, Brazil.

2Federal University of Vale do São Francisco, Physical Education, Petrolina, PE, Brazil.

3 Londrina State University, Clinical Analyses Laboratory, Londrina, PR, Brazil.


Keywords: Obesity, comorbities, adolescents.

Introduction and objectives: Obesity is a condition of excess body fat often associated with a large number of

debilitating and life-threatening disorders. Among the major public health problems in childhood and adolescence,

excess body weight has received great attention in recent decades. This is due to the high prevalence of excess body

weight observed in several countries and its negative health consequences. Moreover, overweight may be associated

with a number of comorbidities in adolescents. Objective: To verify the comorbidities associated with overweight in

adolescent males.

Material and methods: This is a cross-sectional study involving 431 males adolescents aged 10–16 years. This

investigation was conducted according to the Declaration of Helsinki and was approved by the local University Ethics

Committee (238/2010). Fasting glucose, triglycerides, high-density lipoprotein cholesterol (HDL-C), and blood

pressure were measured. Blood samples were collected after a 12-hour fast. Analyzes were performed using a

biochemical Auto-Analyzer (Dimension RxL Max—Siemens Dade-Behring). Blood pressure measurements were

performed on the right arm, with a suitably sized cuff after a rest period of 10 minutes. The presence of comorbidities

was established according to cut-off points by International Diabetes Federation (metabolic syndrome criteria). Body

mass index (BMI, kg/m2) was calculated from body weight and height and adolescents were classified as eutrophic

(Group 1), overweight/ obese (Group 2) based on BMI percentile sets by age and sex proposed by Colle et al 2000.

Categorical variables were described using frequency distributions and presented as frequency (%) and chi-square

test were used to comparisons. Statistical significance was set at P<0.05.

Results: 22% of adolescents evaluated were overweight. Except elevated glucose (P>0.05) (Group1= 15% vs.

Group 2= 7%), the other comorbidities evaluated were associated (P<0.05) with overweight in the adolescent:

Elevated blood pressure (Group 1 = 16% vs. Group 2 = 32%), low HDL-C (Group 1 = 12% vs. Group 2 = 42%),

elevated triglycerides (Group 1 = 2% vs. Group 2 = 10%).

Conclusion: Elevated blood pressure, low HDL-C, and elevated triglycerides were the comorbidities associated with

overweight in adolescents males.

Grants: Capes, CNPq.


DENGUE, CHIKUNGUNYA AND ZIKA: Aedes aegypti AND EMERGING DISEASES IN BRAZIL

Terra, M.R.1; da Silva, R.S.

2; Lima, A.F.

3; Pereira, M.G.N.

4.

1 State University of Londrina, Department of Microbiology, Londrina, Brazil. 2

Universidade Estadual Paulista ―Júlio Mesquita‖, Botucatu, Brasil. 3 Instituto de Ensino Superior de Londrina - INESUL, Londrina, PR, Brasil.

4 Centro Universitário Filadélfia – UNIFIL, Londrina, PR, Brasil.


Keywords: Aedes aegypti, arboviruses, Chikungunya, Zika Virus.

Introduction: Epidemiological bulletins demonstrate the emergence of arboviruses Dengue, Chikungunya and Zika in

the national territory. In addition to the impact on morbidity and mortality rates. This emergency is due to the challenge

that is to control the vector of these arboviruses the mosquito Aedes aegypti that has worldwide distribution and its

breeding sites. Due to the exposed problem the present study had as objective to carry out a bibliographical review

about arboviruses in emergency in Brazil and Ae. aegypti. The present study is an exploratory-descriptive

bibliographical review using the electronic databases of LILACS and BDENF. Crossing the descriptors described in

the Descriptors in Health Sciences.

Review: Considered an acute febrile disease, dengue is caused by five serotypes of the genus Flavivirus, family

Flaviviridae (DENV I-V), and serotypes I, II, III and IV are present in Brazil. The incubation period is from 3 to 7 days

and clinical symptoms of Dengue infection begin later. Chikungunya (CHIKV) is an RNA virus of the Togaviridae

family. The first autochthonous transmission in Brazil of CHIK was detected in 2014 in Amapá. It is a febrile illness,

accompanied by a discreet occurrence of other general symptoms, such as headache, rash, malaise, edema and joint

pains. The Zika virus is a flavivirus (family Flaviviridae) originally isolated from a female Rhesus monkey in the Zika

Forest in Uganda in 1978. As symptomatology we can mention arthralgia, edema of extremities, low fever,

maculopapular rash Often pruritic, headaches, retroorbital pain, no purulent conjunctivitis, vertigo, myalgia, and

digestive disorder. The Zika virus has been correlated with Guillain-Barré syndrome as well as with maternal-fetal

transmission of the Zika virus which results in cases of microcephaly in the newborn causing a binge on the national

public health and International In addition to being arboviruses these diseases have as vector the Aedes (Stegomyia)

aegypti (Linnaeus, 1762) has anthropophilic habits being an urban species commonly found at rest in dwellings and

occasionally in the peri-domicile. In addition, the female is hematophagic periodically requiring the repotting of blood

from vertebrates to mature the eggs that are placed in natural and artificial reservoirs of clean water. When obtaining

the blood repast the female can contaminate the vertebrate with the arbovírus. Thus, the control of this vector is

fundamental for the eradication of these arboviruses and for this to happen not only must research be done to develop

vaccines that generate immunity to these viruses, but also effective measures must be taken for the prevention and

eradication of the breeding sites Of Ae. aegypti.

Conclusion: Arboviruses are considered of medical importance due to high morbidity and mortality rates and their

sequelae for human health. As a consequence Ae. aegypti has been considered a public health problem since the

discovery of this Diptero as vectors of diseases such as arbovirus. Therefore, it is necessary to implement strategies

in order to favor surveillance actions with the adoption of specific strategies in the fight against mosquitoes and their

breeding grounds to eradicate these diseases.


DESCRIPTIVE EPIDEMIOLOGICAL STUDY OF AMERICAN TEGUMENTARY LEISHMANIASIS CASES

IN NORTHERN PARANA FROM 2010 TO 2015

Da Silva, T. P.1; Detoni, M. B.

1; Lima, D. M.

1; Machado, L. F.

1 Tomiotto-Pellissier, F.

1; Oliveira, F. J. A.

1; Costa, I.

N. 1; Pavanelli, W. R.

1; Conchon-Costa, I.

1; Melanda, F. N.

1*

State University of Londrina, Department of Pathological Sciences, Londrina, Parana, Brazil.

*E-mail: [email protected]

Keywords: Leishmania, patients, Glucantime.

Introduction and objectives: American Tegumentary Leishmaniasis (ATL) is a zoonosis widely distributed in Brazil.

It is considered as one of the six most important infectious and parasitic diseases by the World Health Organization,

and therefore, constitutes a very serious public health problem registered in most Brazilian states. On grounds of the

relevance of this parasitosis, the following study evaluated the epidemiological, clinical, laboratory, therapeutical and

evolutionary aspects of ATL patients treated at the Universitary Hospital of the State University of Londrina and at its

attached Multi-Specialty Outpatient Clinic, from 2010 to 2015.

Material and methods: It is a retrospective and descriptive epidemiological study based on the analysis of the

records of patients who were diagnosed with ATL by presenting positive results either to a serology test, to the

intradermal Leishmanin Skin Testing (LST, or Montenegro test) or to a biopsy, from 2010 to 2015, including new and

prevalent cases. ODK Collect interface was used to collect the data that were sent to a spreadsheet and, then

analyzed by using the SPSS program, version 22.0. The study was approved by the Ethics Commission (CEP nº

1437269.2017) of the State University of Londrina.

Results: One hundred and eight (108) cases were identified during the studied period, most of them in 2014 (28.7%).

Among the positive cases, 59 patients (55.2%) were diagnosed by both serology test and LST method. Regarding

epidemiological aspects, 60.2% of the patients were male. The median age was 56.79 years. Rural worker was the

most common professional occupation (17.6%). It was clinically observed that, among the studied cases, single

lesions (84.3%) and ulcerated lesions (59.9%) prevailed, and that they were mainly located in the lower limbs (37.2%).

Glucantime® was the most used therapy (81.5%). Within clinical developments, 75% of the patients evolved to lesion

healing.

Conclusion: From the results obtained in the analyzed region, we can conclude that ATL mainly affect working-age

men and, mostly, rural area residents. Knowing the ATL epidemiological aspects in Londrina’s region allow preventive

and health promotion actions to be properly guided through the identification of the groups of people the more

vulnerable to the disease.


ECO-EPIDEMIOLOGICAL ASPECTS OF PARACOCCIDIOIDOMYCOSIS

Carvalho, G. G.1; Ono, M. A.

2.

1, 2 Universidade Estadual de Londrina, Department of Pathological Sciences, Londrina, PR, Brazil

[email protected]

Key words: paracoccidioidomycosis, Paracoccidioides brasiliensis, epidemiology, soil.

Introduction: The thermally dimorphic fungi Paracoccidioides brasiliensis and P. lutzii are the etiologic agents of

paracoccidioidomycosis (PCM), a systemic mycosis prevalent in Latin America. Paracoccidioides spp. belongs to the

Family Onygenaceae, such as Histoplasma capsulatum, Blastomyces dermatitidis and Coccidioides immitis, that also

present thermo-dimorphism and whose association with soil has already been determined. Clinical presentations of

PCM include asymptomatic infection and symptomatic manifested disease, which can be classified into acute or

chronic form. The objective of this review is to summarize eco-epidemiological aspects of PCM and factors that could

affect the isolation of Paracoccidioides spp. from nature.

Review: 1. Eco-epidemiology: PCM has an incidence rate of 1–3 cases/100,000 inhabitants and mortality rate of

1,4/million in Latin America. The mycosis affects mainly male agricultural workers. Soil is the probable habitat of

Paracoccidioides spp. and infection may occur by inhalation of propagules from this source. Epidemiological data

suggest that Paracoccidioides spp. are associated with fertile soils, near to watercourses, high precipitation rates, high

elevation, tropical and subtropical forests and mild temperatures. Paracoccidioides spp. present low growth rate, high

nutritional demand. The growth of P. brasiliensis was inhibited by several agricultural pesticides and this could be a

possible explanation for the difficult in isolation this fungus from agricultural soil samples.

2. Isolation of P. brasiliensis from soil: since its discovery in 1908, only four studies have demonstrated the isolation of

P. brasiliensis from soil. The first occurred in 1963 from soil samples of a cattle farm in Recife, Brazil, using direct

isolation method. The second one was in 1966 from a rural zone in Chaco, Argentina, also using direct isolation. In

1971, three isolates were recovered from soil samples of Miranda, Venezuela, by indirect method using mice. The last

report occurred also in Brazil, in 1998, from a coffee farm in Minas Gerais, by indirect method. Until now, no isolation

of P. lutzii from nature has been reported.

3. Isolation from other sources: this pathogen was also isolated from penguin feces in 1989 (Antarctic zone, Uruguay)

and a dog feed sample contaminated with soil, in 1990 (São Paulo, Brazil). Some isolates of P. brasiliensis were

obtained from different animal species such as dogs and armadillos, which have frequent contact with soil. Most of

Paracoccidioides isolates are from human clinical samples; however, factors such as long latency period of PCM and

migration of patients make it extremely difficult to identify the exact site and period of infection.

Conclusion: the difficulty in identifying the habitat of P. brasiliensis and P. lutzii may be due to several factors.

Molecular methods could help to find the habitat of these fungi. Soil is a very complex environment with presence of

several species of macro and microorganisms that could interfere in Paracoccidioides spp. growth. Further studies on

these interactions are necessary to better understanding the eco-epidemiology of PCM.

Grants: The work was supported by grants from CNPq and CAPES.


EPIDEMIOLOGY OF Aedes aegypti IN THE MUNICIPALITY OF CAMBÉ - PARANÁ


2; Lima, A.F.

3; Pereira, M.G.N.

4, Souza, R.S

5.

1 State University of Londrina, Department of Microbiology, Londrina, Brazil. 2

Universidade Estadual Paulista ―Júlio Mesquita‖, Botucatu, Brasil. 3 Instituto de Ensino Superior de Londrina - INESUL, Londrina, PR, Brasil.

4 Centro Universitário Filadélfia – UNIFIL, Londrina, PR, Brasil.

5Secretária de Educação do Paraná, Londrina, Brasil.


Keywords: Aedes aegypti; Breeding sites; epidemiological surveillance.

Introduction and objectives: Mosquitoes of the species Aedes (Stegomyia) aegypti are of great public health

interest, due to their ability to transmit important arboviruses such as Dengue and more recently Chikungunya and

Zika. Being found inhabiting rural, suburban and urban areas. This mosquito has diurnal and anthropophilic habits,

where the arboviruses are transmitted by the bite of the infected female that exhibits hematophagous habits during.

These breeding grounds can be classified as natural or artificial breeding grounds. Due to the exposed problem the

present study had the objective of surveying the larval density of Ae.aegypti and Which are its preferential breeding

sites in the municipality of Cambé - Paraná in order to improve the vector control strategy, and the study can subsume

control actions.

Material and methods: The research was carried out in the municipality of Cambé, Paraná. Where its 149 districts

are located in the Urban Zone and 10 glebas in the Rural Zone.The present study has a quantitative, retrospective

character of the density and distribution of Ae. aegyptii and their breeding preferences, whose data were obtained

between the years 2010 and 2016 through data recorded in the National Dengue Control Program System version

1.07. For the Urban Zone the neighborhoods were grouped into 5 strata divided by localities referring. For the Zona

Rural the neighborhoods were Bratislava, Rural Village, Rancho Ringo, Green Village, Rodovia Village and Warta.

The breeding sites were divided into: A1: Water tank (raised); A2: Other water storage tanks (low); B: Small mobile

deposits; C: Fixed deposits (pools, cisterns); D1: Tires and other rolling stock; D2: Garbage (plastic containers, cans)

scraps, debris; E: Natural deposits. The results were analyzed according to the following indicators: i) Breteau index

(number of positive containers for every 100 researched properties) And ii) relative frequency of researched and

positive containers according to type. For the application of the research all ethical aspects will be respected

according to Resolution 466/12 of the National Health Council and the research was approved by the Committee of

Ethics and Research with Humans under Consolidated opinion no. 2,020,340.

Results: The highest index of mean infestation by Ae. aegypti observed, considering all strata was the year of 2010

with 14.1%, the lowest observed in the year of 2013 with 0.6%. The stratum with the highest index of infestation was 4

with 6.27% and the lowest one corresponded to stratum 3 with 5.63, so there was not much variation among strata.

The type of breeder that presented the highest density of larvae over the years was the D2 corresponding to Garbage

(plastic containers, cans) scraps, debris. The positivity of the containers (container index) according to type was

similar in the different strata evaluated, especially for the most frequent types.

Conclusion: The finding of Ae. aegypti on a more frequent basis in recyclable materials such as plastic containers,

cans, scrap and debris demonstrates the responsibility of anthropic action as a multiplier of potential artificial breeding

sites of mosquito larvae with high potential to transmit arboviruses to humans.


EVALUATION OF THE EFFECTIVENESS OF GENEXPERT® MTB/RIF EQUIPMENT IN

TUBERCULOSIS DIAGNOSIS IN WEST PAULISTA PRISON UNITS

Monteiro, N. R. 1; D’ Andrea, L. A. Z.

1 ; Lima, P. E. S.

1 ; Romão, M. M.

1 1

Regional Laboratory Center of the Adolfo Lutz Institute of Presidente Prudente V

[email protected]

Keywords: Tuberculosis, diagnosis, molecular rapid test, culture.

Introduction and objectives: Tuberculosis (TB) is an infectious disease caused by a bacterium of the genus

Mycobacterium. TB is a serious public health problem, affecting mainly the prison population, in terms of incidence

and prevalence. Health Surveillance has as its premise to monitor the health situation of the population. For the

diagnosis, alternatives are presented to the traditional approach (smear microscopy), as is the case with the

automated system culture BACTEC MGIT® 960 ("gold standard") and GeneXpert® MTB/RIF (Rapid Molecular Test -

RMT) which detects the DNA from M. tuberculosis complex mycobacteria and strains resistant to rifampicin. In 2014,

the Regional Laboratory Center of the Adolfo Lutz Institute of Presidente Prudente V (RLC–ALI-PP V), in response to

the National Tuberculosis Control Program of Health Ministry (NTCP/HM), received the GeneXpert® MTB/RIF

equipment. Data of the field performance of this new technology are scarce, which enabled the evaluation of the

GeneXpert® MTB/RIF equipment against the culture in MGIT® 960, both performed in the RLC –ALI-PP V. Seeking

the improvement of the diagnosis of this aggravation. The present study had the objective of evaluating of the

effectiveness of the GeneXpert® MTB/RIF system, against MGIT® 960 culture, in biological samples for the diagnosis

of TB, from prison units in the area covered by RLC – ALI -PP V, in the period between October/2015 and

October/2016.

Material and methods: The study is an original retrospective work. Registered under CAAE protocol No.

50577015.6.0000.0059 and CEPIAL Opinion No. 1,387,145. It had as main sponsor Instituto Adolfo Lutz (Ministry of

Health) and was carried out in RLC-ALI-PP V. For this, the results of examinations of patients (4,509 samples)

attended in five prison units (Caiuá, Martinópolis, Marabá Paulista, Presidente Bernardes and Presidente Prudente)

were analyzed, obtained by System Gerenciador de Ambiente Laboratorial (GAL) and organized in an Excel

worksheet. Assessment of sensitivity and specificity were estimated according to Ferreira and Ávila (2001), using a

double entry table in which the diagnosis of the disease and the test result are related. The criterion of patient

selection was to consider only those that were sent two samples, whose quantities were sufficient to process in

parallel in GeneXpert® MTB/RIF and MGIT® 960 culture.

Results: Of 4,509 samples analyzed, 8.6%(388) belonged to the Caiuá Detention Center; 30.2% (1,361) of

Martinópolis Penitentiary; 25.8%(1,164) of Presidente Bernardes Penitentiary; 11.6%(524) of Marabá Paulista

Penitentiary; and 23.8%(1,072) of Presidente Prudente Penitentiary. Of this total, 26.6%(1,649) samples were

processed in parallel, and of these 95.2% (1,570) it was possible to obtain results in both tests, since there was

contamination of 4.8% (79) of the cultures. The culture in Bactec MGIT® 960 identified 7.34% (121) TB patients and

the RMT in GeneXpert® MTB/RIF identified 6.25% (103). Comparing, we obtained a specificity of 99.03%, sensitivity

of 72.73% and concordance of 97% between the tests.

Conclusion: Allowing to conclude that the GeneXpert® MTB/RIF system is very useful for the NTCP/HM, allowing

agility to the diagnosis and early treatment, contributing to the break in the chain of transmission of this disease.


FAMILY INCOME AND AGE AS INDEPENDENT FACTORS FOR THE DEVELOPMENT OF

CERVICAL CANCER

Aranome, A. M. F.1; Trugilo, K. P.

1; Maria, G. C. Q.

1; Okuyama, N. C. M.

1; Santos, F. C.

1; Sena, M. M.

1; Lombardi, A.

P. P.1; Pereira, E. R.

1; Ferreira, R. S.

1; Esposito, A.

1; Nishimura, A. M.

1; Mangieri, L. F. L.

2, Couto Filho, J. d’O.

3;

Oliveira, K. B.1.

1 Laboratory of Molecular Genetics and Immunology, Department of Pathological Sciences, State University of

Londrina, Londrina – PR, Brazil. 2 Department of Ginecology and Obstetrics, State University of Londrina, Londrina – PR, Brazil.

3 Cancer Hospital of Londrina, Londrina – PR, Brazil.

[email protected]

Keywords: Cervical intraepithelial neoplasia, Risk factors, Human papillomavirus

Introduction and objectives: Cervical cancer is among the most frequent cancer in women worldwide, resulting in

approximately 265.000 deaths per year and it is associated with the presence of human papillomavirus (HPV).

Furthermore, sexual behavior and environmental or exogenous cofactors, including hormonal contraceptives, tobacco

smoking, parity are linked with the disease process. However, epidemiological data about cervical cancer in Paraná

State are scarce. Therefore, this study aims to analyze the socio-demographic profile and the reproductive and sexual

behavioral characteristics of women in the cervical cancer development.

Material and methods: This study was approved by the Institutional Ethics Committee Involving Humans of the State

University of Londrina, which is in accordance with resolution 466/12, CAAE 56738316.3.0000.5231. For this purpose,

we collected data of 35 patients attended at the Cancer Hospital of Londrina, and 155 controls from Basic Health Units

of Londrina. Patients with cervical cancer were diagnosed by histopathology, while the absence of cervical lesions and

HPV DNA in controls was confirmed by oncotic cytology and by PCR technique, respectively. Socio-demographic

characteristics and clinical data were obtained from patients and controls through medical records and semi-structured

interviews. Association between socio-demographic characteristics and cervical cancer presence was assessed by

Chi-square test and binary logistic regression analysis, and p < 0.05 was considered as significant.

Results: Among patients with cervical cancer, 8.6% were in stage I, 42.9% in stage II, 40% in stage III and 8.6% in

stage IV according to FIGO classification. We observed that women who did not know about HPV (p = 0.003) and its

transmission (0.001), with age above 55 years (p <0.001), with low schooling level (p <0.001), widows (p = 0.003) and

with family income of 1 to 3 wages (p = 0.001) were associated with cervical cancer. When performing the logistic

regression test to these same data, the age greater than 55 years presented a higher risk greater than 6 times for the

cancer [OR = 6.39 CI95% (1.81-22.50), p = 0.004], while income of 1 to 3 wages offered cancer protection [OR = 0.30

CI95% (0.121-0.744), p = 0.009] when compared to a monthly income of ≤ 1 wage.

Conclusion: According to these results, the present study demonstrated a description of risk and protective factors

for the development of cervical cancer, helping to understand the disease and contributing to prevention programs

aimed at reducing the incidence rate of cervical cancer.


INFLUENCE OF TGFB1 rs1800468 POLYMORPHISM IN HPV INFECTION

Pereira, E. R.1; Cebinelli, G. C. M.

2; Trugilo, K. P.

1; Aranome, A. M. F

1; Sena, M. M.

1; Okuyama, N. C. M

1; Pereira,

A. P. L. 1; Santos, F. C.

1; Ferreira, R. S.

1; Maria, G.C.Q

1; Nishimura, A. M.

1; Esposito, A.

1; Oliveira, K.B.

1 1Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil

2Faculdade de Medicina de Ribeirão Preto, Department of pharmacology, Ribeirão Preto, SP, Brazil

Email: [email protected] / [email protected]

Keywords: Single nucleotide polymorphism. PCR. Infection. Case-control study.

Introduction and objectives: Human Papillomavirus (HPV) infection may cause cervical lesions which might

progress to cervical cancer. Many cytokines are produced in this microenvironment, and may contribute to infection

resolution or to virus persistence. In this context we highlight the transforming transformer β1 (TGFB1), a

multifunctional cytokine, that regulates a number of important cellular responses, participating in cell differentiation,

apoptosis, cell migration, immune responses, angiogenesis and formation of extracellular matrix. Several

polymorphisms on TGFB1 gene have been described, and since TGF-β1 production and expression can be

influenced by the polymorphism, the present study aimed to evaluate the influence of TGFB1rs1800468

polymorphism, located in a regulatory genic region, in HPV infection in women from the northern region of Paraná

attended by SUS.

Material and methods: This project was approved by the Ethics Committee in Research Involving Human Beings of

the State University of Londrina, Londrina – Paraná (PR), Brazil (CAAE 56738316.3.0000.5231) statement number

1.590.141, which is in accordance with 466/12 resolution of the National Commission of Ethics in Research. DNA was

extracted from cervical secretion to detect HPV, which was performed by polymerase chain reaction (PCR) and for the

polymorphism, peripheral blood DNA was extracted and evaluated by restriction fragment length polymorphism

(RFLP), while epidemiological data were obtained through a structured questionnaire. Genotype distribution was

association between the presence of HPV and the TGFB1 polymorphism.

Results: This case-control study enrolled 429 patients, 219 (51%) were included in HPV non-infected group (control)

and 210 (49%) in HPV infected group (cases). The epidemiological analysis showed that the presence of the virus

was more frequent in women less than 25 years of age (p <0.001), smoking (p = 0.014), single (p <0.001), that

declared the first sexual intercourse before 17 years (p = 0.012), who declared more than 4 sexual partners during

lifetime (p <0.001). Using a binary logistic regression, adjusted for several confounding factors, no association

between the polymorphism and HPV infection was observed (p = 0.252).

Conclusion: The TGFB1 rs1800468 polymorphism is not associated with HPV infection, however, further studies

should be performed to clarify the role of the TGFB1 in HPV infection.




POTENTIAL BACTERICIDAL EFFECT OF IRON DOPED ZINC OXIDE NANOCRYSTALS

Pereira Lima,G.Y.1,2

; Borges, A.L.S. 1,2

; Camargo, M.P.C.1,2

; Paula, A.T.2;Goulart, L.R

2;

Dantas, N.O.1; Silva, A.C.A.

1

1 Universidade Federal de Uberlândia, Laboratory of New Insulating Materials and Semiconductors (LNMIS),

Uberlandia, Brazil

² Universidade Federal de Uberlândia, Laboratory of Nanobiotechnology, Uberlândia, Brazil

[email protected]

Keywords: Fe doped ZnO NCs, nanocrystals, potential bactericidal.

Introduction and objectives: With the discovery of antibiotics plus their continuous and unrestrained use, resistant

bacteria’s were naturally select. Researchers all over the world started to look for new classes of antibiotics and new

treatment methods. The advancement of nanoscience provide new potential nanomaterials called nanocrystals (NCs)

that shown a satisfactory bactericidal effect, mainly zinc Oxide (ZnO) nanocrystals bactericidal effect depends on: NCs

stability, size, concentration and type and method of synthesis, The aim of this work was investigated the potential

bactericidal of the ratio of iron (Fe) doped ZnO NCs to FeO increasing Fe concentration (0,05 ;1; 5; 11 %wt to Zn) and

FeO NCs.

Material and methods: The bactericidal effect was researched against seven bacteria’s (in duplicate): Pseudomonas

aeruginosa, Enterococcus faecalis, Staphylococcus aureus, Proteus vulgaris, Escherichia coli, Acinetobacter

baumannii and Klebsiella pneumoniae with the same concentration of the NCs. The antimicrobials test were made

using the disk diffusion test methodology according Clinical Laboratory Standards Institute (CLSI-2017). The

bactericidal effect was quantified by the measure of inhibition zones presented by NCs. Ampicilin was used as positive

control and sterile ultrapure water as negative control .

Results: Pseudomonas aeruginosa was the most sensitive bacteria against NCs , Escherichia coli was sensitive

against all concentrations except FeO NCs and ZnO NCs doped with 11% Fe, Klebsiella pneumoniae show inhibition

zone in ZnO NCs doped with 1% Fe and 5% Fe and Acinetobacter baumannii show inhibition only in ZnO NCs doped

with 1% Fe.The others didn’t show inhibition zone.

Conclusion: The bactericidal effect occurs in four of the seven tested bacteria, all gram negative it may occurs due to

NCs penetration into the membrane, denaturing proteins and damages DNA causing bacteria death, may if with the

increase of the concentration the result will be better.

Grants: FAPEMIG, CNPq, CAPES and UFU.


PREVALENCE OF METABOLIC SYNDROME IN ADOLESCENTS OF DIFFERENT SOCIOECONOMIC

STATUS

Polvani, A. C. T.1;Tomeleri, C. M.

1; Souza, M. F.

2; Silva, D. R. P.

1; Cavalcante, E. F.

1; Antunes, M.

1; Nabuco, H. C.

G.1; Schiavoni, D.

1; Venturini, D.

3; Barbosa, D. S.

3; Okino, A.

3; Cyrino, E. S.

1.

1 Londrina State University, Department of Physical Education, Londrina, PR, Brazil.

2 Federal University of Vale do São Francisco, Department of Physical Education, Petrolina, PE, Brazil.


Email:[email protected]

Keywords: Metabolic syndrome X, youth, socioeconomic status.

Introduction and objectives: Epidemiological studies have linked socioeconomic status with the incidence of chronic

diseases and there are evidences that show that children from families of low socioeconomic status may have a

higher risk of developing cardiovascular disease (CVD). Metabolic syndrome (MetS) is a multicomponent disorder and

is closely linked to CVD. Thus, it is possible that the prevalence of MetS may be influenced by socioeconomic status...

Objective: To compare the prevalence of MetS and its individual components in adolescents of different

socioeconomic status.

Material and methods: This cross-sectional study involved 789 adolescents aged 10–16 years. This investigation

was conducted according to the Declaration of Helsinki and was approved by the local University Ethics Committee

(238/2010). The socioeconomic status was assessed using the instrument of the Brazilian Association of Research

Companies (BARC, 2008) which allows stratification by eight categories (A1, A2, B1, B2, C1, C2, D, and E [lowest]).

Subsequent, the adolescents were dichotomized (cut-off below B2) in low socioeconomic status (LSES) and moderate

socioeconomic status (MSES; cut-off above B2). Measurements of waist circumference, blood pressure, blood

glucose, HDL-C, and triglycerides were evaluated. The prevalence of MetS was defined according to the International

Diabetes Federation. Categorical variables were described using frequency distributions and presented as frequency

(%) and chi-square test were used to comparisons. Statistical significance was set at P<0.05.

Results: There was no difference (P>0.05) in the MetS prevalence rate between groups (LSES = 3.3% vs. MSES =

3.9%). There was also no difference in the prevalence of MetS components between the groups (P>0.05). Waist

circumference elevated was the most prevalent component in both groups (LSES = 20.7% vs. MSES = 23.1%),

followed by low HDL-C (LSES = 16.5% vs. MSES = 13.9%). The glucose and triglyceride components presented the

lowest prevalence (LSES = 5.3% vs. MSES = 4.6%) and (LSES = 2.0% vs. MSES = 3.9%), respectively. The rate

prevalence of blood pressure elevated was LSES = 15.3% vs. MSES = 20.3%.

Conclusion: The MetS prevalence rates did not differ among the different socioeconomic status evaluated.

Grants: Capes, CNPq.


QUALITY CONTROL OF THE CANINE VISCERAL LEISHMANIASIS DIAGNOSIS OF THE

LEISHMANIOSES NETWORK OF THE ADOLFO LUTZ INSTITUTE (ALI) - REGIONAL LABORATORY

CENTER OF PRESIDENTE PRUDENTE V

Oliveira, A. C. F.1; D’ Andrea, L. A. Z.

1,2 ; Urias, G.

1; Menezes, C. B.

1; Silva, R. R.P.

1; Romão, M. M.

1

1 Center Regional Laboratory, Adolfo Lutz Institute, Presidente Prudente;

2 Health and Geography Laboratory, Faculty

of Science and Technology, Universidade Estadual Paulista, Presidente Prudente

[email protected]

Keywords: Visceral Canine Leishmaniasis, Quality Control, Diagnosis, Leishmaniasis Network.

Introduction and objectives: Visceral leishmaniasis (VL) is a zoonosis caused by the protozoan Leishmania

infantum (sinonímia-Leishmania chagasi), transmitted mainly by the bite of Lutzomyia longipalpis. VL is a serious

public health problem, it is in frank expansion in São Paulo territory. In the National Health System (NHS), it is at the

national level that are taken the decisions and established the guidelines for the VL Surveillance and Control Program,

whose actions must be performed in all States and Municipalities. Currently, it has been used a diagnostic arsenal in

the confrontation of VL, being of great importance that there is the quality control program. The National Reference

Laboratory carries out the quality control of the Central Laboratories (CENLAs), and they make the quality control of

the Regional CENLAs, which in turn operates in the municipal sphere. The objective of this study was to evaluate the

quality of canine VL diagnosis in the area of the Leishmaniosis network of the Regional Laboratory Center of the

Adolfo Lutz Institute of Presidente Prudente V (RLC ALI PP V), Regional CENLA.

Material and methods: This is a retrospective study, registered and approved by CAAE No. 53247716.8.0000.0059,

opinion No. 1,934,175. Since 2012, the canine serological screening has become the responsibility of the

municipalities, using the rapid immunochromatographic test (RT) DPP for canine VL and the ELISA, both of Bio-

Manguinhos, as a confirmatory test, which it should has been performed in the CENLAs or in laboratories of the

Zoonoses Control Centers (ZCCs). For this, the municipalities needed to structure themselves, train their teams of

zoonoses, register and undergo supervision. Direct supervision aims to evaluate infrastructure requirements,

biosafety, equipment, inputs, human resources, specific technical practices and documentation, in which is produced

a technical report, used as an instrument for decision-making.

Results: In the area covered by RLC ALI PP V, Regional reference laboratory for VL, from 04/2012 to 04/2017, 26

training courses were carried out for a total of 208 hours, at 166 RH/employees, which belonged to 28 municipalities,

to use the RT DPP BioManguinhos as a screening test. In the period from 04/2014 to 04/2017, 60 technical visits

(direct supervision) were carried out in 25 municipalities of the Municipal Leishmaniasis network of the RLC ALI PP V,

with serological screening activities for canine VL, seven in 2013, 19 in 2014 , 11 in 2015 and four until 04/2017. In the

same period, the Regional CENLA received 1,126 serum samples sent by the municipal zoonoses services or ZCCs

for quality control (indirect supervision), 752 of which were non-reactive and 374 with a reagent result in the RT DDP

BioManguinhos, in their respective municipalities source. The concordance was 89.3% among the results of the

samples performed in the indirect supervision.

Conclusion: It is of great importance in Public Health the performance of the Leishmaniasis Network in the quality

control of the diagnosis, supporting the activities developed in the municipal scale, aiming effectiveness in the health

surveillance of this disease.


Staphylococcus aureus OF LESIONS IN INFERIOR MEMBERS: ISOLATION AND PROFILE OF

SUSCETIBILITY TO ANTIMICROBIALS OFTITLE


2; Souza, R.S

3; Pereira, J.A.R.

4; Alves, P.

4 1 State University of Londrina, Department of Microbiology, Londrina, Brazil.

2 Universidade Estadual Paulista ―Júlio Mesquita‖, Botucatu, Brasil.

3 Secretária de Educação do Paraná, Londrina, Brasil.

4Instituto de Ensino Superior de Londrina - INESUL, Londrina, PR, Brasil.


Keywords: Staphylococcus aureus; leg ulcer; antimicrobial resistance.

Introduction and objectives Injuries to lower limbs are considered a public health problem, especially when it comes

to lesions infected by pathogenic microorganisms such as Staphylococcus aureus due to their virulence and

resistance factors. Due to the problem, the study aimed to analyze the phenotypic profile of S. aureus isolates isolated

from lesions in the lower limbs of patients from the CISMEPAR wounds outpatient clinic in the city of Londrina, State

of Paraná, from 2001 to 2017.

Material and methods: For the application of the research, all ethical aspects were respected according to

Resolution 466/12, of the National Health Council, being carried out before approval by the Committee of Ethics and

Research with Human Beings, under constituted opinion No. 1,434,527. The population of this study was composed of

volunteers, adults of both sexes, attended by the Intermunicipal Health Consortium of the Paranapanema Middle

(Cismepar) located in Londrina, Paraná. All individuals or individuals signed the informed consent form. The diagnosis

of infection was based on clinical criteria. The inclusion criterion was the presence of clinically infected / infected

spleen in patients treated at the Cismepar wound clinic. The methodology used in this study was experimental

quantitative. After degermation with subsequent debridement of the wound, the sample of lesions in the lower limbs

(venous ulcer, arterial ulcer and diabetic foot injury) were collected by means of a sterile swab moistened with saline

solution and deposited in a test tube containing medium Stuart and transported in an isothermal box to the laboratory.

Isolation of S. aureus was performed by plaque inoculation of sheep blood agar which was incubated at 5% CO 2

tension. Characteristic colonies were selected and grown on Mullher Hinton Agar (MHA) at 37 ° C for 24-48 hrs and

were seeded on plates containing Baird Parker (BP) medium and incubated under the same conditions. Characteristic

colonies were identified as S. aureus and submitted to the morpho-tinturial test and the catalase test, where Gram-

negative cocci arranged in the form of catalase-positive staphylococci (grape clusters) were identified as S. aureus.

Results: We studied 41 patients, and 54 samples were collected predominantly in venous ulcer, from which 13 S.

aureus were isolated. The isolates of S. aureus in their vast majority were multiresistant antimicrobials tested:

Amicacin 11 (85%), Oxacillin 7 (54%), Ciprofloxacin 2 (15%), Gentamicin 8 (62%), Linezolide (37%), Tricycline 5

(38%), Teicoplanin 10 (77%), Clindamycin 4 (31%) and Erythromycin 4 (31%). Only 1 isolate was not resistant to

Amikacin.

Conclusion: Often S. aureus is isolated from lower limb lesions, however antimicrobial multiresistance is of concern

because it is a reservoir of antimicrobial resistance genes that can be transferred to bacteria from the normal

microbiota, hinder therapy and prolong treatment. In addition, it results in the loss of quality of life of the patient and in

the increase of the costs of the treatment.

GENERAL PATHOLOGY 115

A COMPARATIVE STUDY OF PATHOPHYSIOLOGICAL ALTERATIONS IN SCORPIONISM INDUCED

BY Tityus serrulatus AND Tityus bahiensis VENOMS

Miyamoto, J.G.1; Andrade, F.B.

1; Ferraz, C.R.

1; Cândido, D.M.

2; Venâncio, E.J.

1; Verri Jr, W. A.

1; Landgraf, M.A.V.

3;

Landgraf, R.G4; Kwasniewski, F.H.

1

1Universidade Estadual de Londrina, Departamento de Ciências Patológicas, Londrina, PR, Brazil.

2Instituto Butantan, Laboratório de Artrópodes, São Paulo, SP, Brazil.

3Universidade de São Paulo, Departamento de Farmacologia, São Paulo, SP, Brazil.

4Universidade Federal de São Paulo, Departamento de Ciências Farmacêuticas, Diadema, SP, Brazil.

[email protected]

Keywords: Tityus serrulatus, Tityus bahiensis, airways, leukocytes, inflammation.

Introduction and objectives: Severe manifestations in scorpionism may include leukocytosis and acute lung injury.

Clinical data from accidents caused by T. serrulatus and T. bahiensis show that T. serrulatus sting induces a worse

prognosis to the victims, but the reason for such finding is unknown. Since the knowledge of biological effects induced

by Brazilian scorpions is related in great part to T. serrulatus venom (Tsv), we studied the pathophysiological changes

in airways and blood leukocyte counts of rats envenomed by Tsv or T. bahiensis venom (Tbv).

Material and methods: After intravenous injection of Tsv or Tbv (200 µg/kg, diluted in apirogenic NaCl 0,9%) into

rats, following the indicated times the animals were killed by CO2 inhalation, and edema (in 30 minutes, n=6 to 14) and

hemorrhage (in 60 minutes, n=10 to 15) were investigated in the airways by Evans blue (EB) dye extravasation and

cyanometahemoglobin concentration using Drabkin's solution, respectively. TNF-α, IL-1β and IL-6 were measured by

Milliplex assay after 1 and 4 hours of envenomation (n=5 to 6). The protein content (by Bradford method, n=4 to 7)

and cellularity in brochoalveolar lavage (BAL, n=6 to 13) were analyzed in 4 and 24 hours, as well as the activity of

myeloperoxidase (MPO, n=5 to 7) in lung homogenates and leukocyte counts in blood (n=4 to 9). Control data were

obtained with rats injected with apirogenic NaCl 0,9%. The procedures were approved by the institutional ethics

committee (CEUA nº 16583.2013.29). Data were compared by one-way ANOVA and Tuckey’s post-hoc, or Kruskal-

Wallis and Dunn’s post-hoc. All analysis were performed in the GraphPad Prism (5.0) software (San Diego, CA, USA)

considering a significance level of α = 0.05. Results were expressed as mean ± standard error of means (SEM).

Results: Despite both venoms were able to induce EB extravasation into airways, higher extravasation was observed

in Tsv group; a similar pattern of response was found in protein content in BAL. Hemorrhage was restricted to the

inner bronchi and lungs, and it was higher in the lungs of Tbv group. Envenomation by Tsv or Tbv did not induce TNF-

α production, while IL-1β (at 1 hour) and IL-6 (at 1 and 4 hours) were increased only in Tsv group. Four and 24 hours

after envenomation MPO activity was increased, concomitantly with the number of polimorphonuclear (PMN) cells in

BAL of both groups. After 24 hours total leukocyte and PMN counts in BAL were increased in both groups. In blood,

Tsv induced leukocytosis, lymphopenia and neutrophilia after 4 hours, whereas Tbv induced lymphopenia and

neutrophilia, the last one in smaller degree. At 24 hours neutrophilia was found in both groups; although leuko and

lymphocytosis were also found, the values were higher in Tsv group; monocytosis was found only in Tsv group.

Conclusions: Both venoms induced pathophysiological changes in airways and blood leukocyte mobilization, but

most of the evaluated parameters were more affected by the Tsv. These results could help to explain the clinical data

arguing toward the pronounced severity associated with T. serrulatus stings.

Grants: Financial support from Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPQ (Process

457512/2014-8). JGM received scholarship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

(CAPES).


AGGRECAN IS INVOLVED WITH THE EXTRINSIC AND INTRINSIC INNVERVATION OF

GASTROINTESTINAL TRACT OF HUMANS AND MICE

Franciosi, A.1; Bacarin, C.C.

1; Mali-Junior, J

2.; Miqueloto, C.A.

3; Blackshaw, L.A.

4; Araújo, E.J.A.

1

1Londrina State University/Department of Histology, Londrina, Brazil.

2Cancer Hospital of Londrina – Sector of Gastroenterology, Londrina, Brazil.

3Londrina State University/Department of General Biology, Londrina, Brazil.

4Queen Mary University of London/Wingate Institute for Neurogastroenterology, London, UK.


Keywords: ACAN Proteoglycan, Extracellular Matrix, Enteric Nervous System.

Introduction: Aggrecan (ACAN) is an extracellular matrix (ECM) proteoglycan that belongs to the hyalectan family.

ACAN gives neuronal protection to the individuals susceptible to developing Alzheimer's disease when it is present in

perineuronal nets. However, there are no reports of this association in the peripheral nervous system, although ECM

disorders involve dysfunction of the gastrointestinal sensory and motor neurons. Objective: The objective of this study

was to verify whether ACAN is associated with structures that innervate the gastrointestinal tract (GIT) of humans and

mice. Methods: All procedures were approved by the Ethical Committee in Research Involving Humans at State

University of Londrina - UEL (1,073,326) and by the Ethical Committee for Use of Animals at UEL (032/2015), Brazil.

Samples of C57Bl/6 mice GIT were dissected to separate the gut wall into two parts: (1) mucosa+submucosa and (2)

muscle+serosa, which were stored in trizol. We extracted the total RNA from each part, which was converted to cDNA

for quantification. For morphological analysis, samples of GIT, sensory ganglia (Nodose and dorsal root ganglia) and

the spinal cord of mice and human GIT (stomach, large and small intestinal) were fixed in 4% paraformaldehyde for 3

hours, which were frozen later on OCT. GIT sections were submitted to immunofluorescence for marking enteric

neurons (anti-PGP9.5), cholinergic (anti-ChAT) and nitrergic (anti-nNOS) in double stain with ACAN (anti-ACAN). We

also evaluated the presence of ACAN in the sections of nodose ganglia (aggrecan/calrretinin), DRG and spinal cord

(aggrecan/CGRP). Results: mRNA for ACAN was more expressed in all layers of the stomach. Regarding the

intestine, it was weaker expressed in the mucosa+submucosa of the small intestinal and it was similarly expressed in

all layers of the large intestine. ACAN was observed immunohistochemically in structures that innervate the GIT of

both human and mouse. In sensory ganglia innervating the GIT (nodose and DRG), ACAN was present in afferent

neuronal cell bodies. Neurons marked with anti-ACAN had different intensity of labelling, and the brightest ones

tended to be positive for calretinin (nodose) and CGRP (DRG). In intrinsic enteric neurons of the GIT, we observed

intense co-location with submucosal and myenteric cell bodies evidenced with PGP9.5. Apparently, most positive

neurons to ACAN were also cholinergic and nitrergic. Intense labeling of ACAN was observed also in neuronal

varicosities. In addition, there was labeling of ACAN at the base of the mucosal epithelium, and diffusely in the

connective tissue of the submucosa and muscle layers. Conclusion: ACAN is expressed in the GIT of mice and this

proteoglycan is located in several cellular and tissue types, including structures that belong to the motor and sensory

innervation of the GIT of humans and mice.

Grant: CAPES (process number 88881.068190/2014-01)


AIRWAYS INFLAMMATION INDUCED BY Rhopalurus rochai VENOM

Miyamoto, J.G.1; Andrade, F.B.

1; Ferraz, C.R.

1; Cândido, D.M.

2; Venâncio, E.J.

1; Verri Jr, W.

1; Kwasniewski, F.H.

1


2Instituto Butantan, Laboratório de Artrópodes, São Paulo, SP, Brazil.

[email protected]

Keywords: scorpionism, airways, inflammation.

Introduction and objectives: In Brazil, scorpion sting is the most common accident with venomous animals. The

Tityus genus is related to the majority of cases, although any scorpion genus belonging to the Buthidae family should

be considered an animal of medical importance, which may induce acute lung injury in severe envenomation. In this

context, the Rhopalurus rochai is a scorpion found in the Northeast region that have been related to mild-moderate

cases; since there is a lack of epidemiological and experimental data, severe envenoming cannot be excluded. In this

study, we evaluated the inflammatory potential of the R. rochai venom (Rrv) in the rat airways.

Material and methods: After intravenous injection of Rrv (200 µg/kg, diluted in apirogenic NaCl 0,9%) into male

wistar rats, following the indicated times, the animals were killed by CO2 inhalation, edema (in 30 minutes, n = 5 to 7)

and hemorrhage (in 60 minutes, n = 4 to 6) were investigated in the airways by Evans blue (EB) dye extravasation

and cyanometahemoglobin concentration using Drabkin's solution, respectively. The protein content in the broncho-

alveolar lavage (by Bradford method, n = 5) was analyzed in 4 and 24 hours. The activity of myeloperoxidase (MPO, n

= 5) was accessed in lung homogenates at 4 hours. Control data were obtained with rats injected with apirogenic

NaCl 0,9%. The procedures were approved by the institutional ethics committee (CEUA nº 19958.2016.54). Data were

compared by student’s t-test or Mann-Whitney U test. All analysis were performed in the GraphPad Prism (5.0)

software (San Diego, CA, USA) considering a significance level of α = 0.05. Results were expressed as mean ±

standard error of means (SEM).

Results: Rrv induced EB extravasation in upper segments of the airways (trachea and upper bronchis), but no

hemorrhage was found in any segment. The protein content was increased only at 24 hours, while MPO activity was

heightened at 4 hours.

Conclusion: Rrv is capable of inducing airways inflammation in rats, although there were mild alterations, this venom

cannot be considered innocuous.

Grants: JGM received scholarship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).


ALTERATIONS OF THE COLONIC EPITHELIUM OF RATS DURING THE COURSE OF

TOXOPLASMIC INFECTION

Ortigoza, S.M.¹ *; Bergoc, H.G¹ *; Góis, M.B.2; Sant’Ana, D.M.G.²; Araújo, E.J.A.

1

1State University of Londrina, Department of Histology, Londrina, Brazil

²State University of Maringá, Department of Morphology, Maringá, Brazil

*Authors who shared the authorship of the work.

[email protected]; [email protected]

Keywords: Toxoplasmosis, colon, histology, mucins, lymphocytes.

Introduction and objectives: Toxoplasmosis is a zoonosis caused by the protozoan Toxoplasma gondii. Upon

invading the host, this parasite triggers an inflammatory bowel process that causes an increase in lymphocytes of the

intestinal epithelium (intraepithelial lymphocytes). This inflammatory process can also lead to morphometric and

functional alterations, one of which is alteration in the number of goblet cells, thus altering the mucin secretion profile.

Although there are several studies evaluating the small intestine of animals infected with T. gondii, little is known

about the changes that occur in the colon. Therefore, the objective of this work was to quantitatively analyze the

goblet cells and intraepithelial lymphocytes (IEL) present in the epithelium of the proximal colon of rats during the

course of toxoplasmic infection.

Material and methods: The procedures described were approved by the Committee of Ethical Conduct on the Use of

Animals in Experimentation of the State University of Maringá, number 079/2013. Male Wistar rats were distributed

into Control Group (CG) and Infected Groups: 6 hours (IG 6h), 12 hours (IG 12h), 24 hours (IG 24h), 48 hours (IG

48h), 72 hours (IG 72h), 7 days (IG 7d), 10 days (IG 10d) e 30 days (IG 30d), each group with 10 rats, exception the

IG 30d (n=5). The infected rats were orally inoculated with 5000 sporulated oocysts of the T. gondii strain ME-49. The

CG rats received sterile saline. After euthanasia, the proximal colon was submitted to routine histological procedure

with fixation in Bouin and inclusion in paraffin. Sections (thickness: 4 μm) were stained with Periodic Acid Schiff (PAS),

Alcian Blue (AB) pH 2.5), AB pH 1.0 and HE (Hematoxylin and Eosin). Goblet cells positive for the first three staining

were counted in 10 crypts per rat. IEL stained with HE were counted among 500 consecutive enterocytes in each rat.

All countings were performed under a light microscope. The comparison between the groups was done using one-way

ANOVA test, with Tukey post-test, considering 5% as significance level.

Results: A significant increase of goblet cells producing neutral mucin (PAS+) was observed from 6h up to 30 days

post infection (dpi). Acid mucin-producing goblet cells (AB pH 2.5+) increased from 12h up to 10 dpi. Sulphated acid

mucin-producing goblet cells (AB pH 1.0+) increased in all the animals of infected groups. Concerning the IEL, they

were increased at 24h, 48h and 72h post infection.

Conclusion: It is concluded that infection with ME-49 strain oocysts of T.gondii causes an increase of goblet cells and

intraepithelial lymphocytes in the colon of rats during the course of toxoplasmic infection.

Grants: CNPq, Fundação Araucária.


AN EVALUATION OF AN ANIMAL MODEL OF CEREBRAL PALSY: THE EFFECTS ON THE

INTRAMUSCULAR COLLAGEN OF THE EXTENSOR DIGITORUM LONGUS MUSCLE

Prado-Filho, D.1; Covatti, C.

1; Buratti, P.

1; Centenaro, L.A.

2; Torrejais. M.M.

1

1Universidade Estadual do Oeste do Paraná - UNIOESTE, Mestrado em Biociências e Saúde, Cascavel - PR, Brazil.

2Universidade Estadual do Oeste do Paraná - UNIOESTE, Centro de Ciências Médicas e Farmacêuticas, Cascavel,

PR, Brazil

[email protected]

Keywords: cerebral palsy, lipopolysaccharide, perinatal anoxia, sensorimotor restriction, extensor digitorum longus

muscle.

Introduction and objectives: Cerebral palsy (CP) is defined as a chronic encephalopathy that causes sensorimotor

disorders at different degrees of severity, and is considered the most common cause of physical deficiency in

childhood. The present study aimed to investigate the implications of an animal model of CP on the intramuscular

collagen of extensor digitorum longus muscle (EDL) of Wistar rats.

Material and methods: This research was a original work, approved by the Ethics in the Use of Animals Committee

(CEUA - nº 24/16) of UNIOESTE. To obtain the litters, pregnant Wistar rats were injected intraperitoneally with

vehicle (100 μl of sterile saline); and the others with LPS (200 μg/kg LPS in 100 μl of sterile saline). Both injections

were performed every 12 hours, as from the 17th gestational day (G17) until the end of gestation (G21). The day of

birth of the males offspring were separated into two groups: 1) the Control group - offsprings of rats injected with

saline during pregnancy (CTL) and 2) the CP group - offsprings of rats injected with LPS during pregnancy, submitted

to perinatal anoxia and sensorimotor restriction (CP). For perinatal anoxia the offsprings were placed in a closed

chamber, partially immersed in water at 37° C, with a flow of 9 L/min of nitrogen (100%) for 20 minutes on day of birth

(P0). The offsprings from the CP group underwent sensorimotor performed from P1 to P30, by immobilizing the hind

limbs of the animals for 16 h / day. At 48 days of age, the proximal portions of the right antimere EDL muscle were

maintained at room temperature for 30 to 40 minutes. After which, the samples were covered with neutral talc and

frozen in liquid nitrogen for storage in a Biofreezer at -80ºC. Subsequently, cross sections (7μm thick) of these

samples were obtained using a cryostat. To quantify the percentage of intramuscular collagen, the remainder of the

histological sections of the EDL muscle was stained with Masson’s Trichrome. Three microscopic images from each

animal were used in the analysis (magnification 200X). Once the images were captured, the percentage of

collagen/total area was calculated.

Results: The fibers were arranged in fascicles surrounded by the perimysium, with each fiber being surrounded by

endomysium. Intramuscular collagen showed a 34% increase in the CP group compared to the CTL group (p =

0.009).

Conclusion: The increase of intramuscular collagen is a finding commonly found in patients with spastic CP. This

animal model proved to be a useful mean to understand the pathophysiology of this clinical condition.

Grants: Coordination of Improvement of Higher Education Personnel (CAPES).



ANALYSIS OF CELL DEATH PATTERNS INDUCED BY METFORMIN IN HUMAN MCF-7 BREAST

CANCER CELLS

Lopes, N. M. D. 1; Sanches,

L. J.

1; Marinello,

P. C.

1; Brito, W.A.S.

1; Martins, M. I. L.

2; Pinge Filho, P.

2; Luiz, R. C.

1;

Cecchini, R. 3; Cecchini, A. L.

1.

1Universidade Estadual de Londrina, Laboratory of Molecular Pathology, Londrina, PR, Brazil

2 Universidade Estadual de Londrina, Laboratory of Experimental Immunopathology, Londrina, PR, Brazil

3 Universidade Estadual de Londrina, Laboratory of Pathophysiology and Free Radicals, Londrina, PR, Brazil

[email protected]

Keywords: Metformin 1, Cell death 2, Breast cancer 3, Oxidative stress 4.

Introduction and objectives: Worldwide, breast cancer is the neoplasia with the highest incidence and mortality

among women. For this reason, new drugs have been studied for treatment or use as adjuvants in the treatment of

this type of cancer. Among the drugs, metformin has shown promising results. Metformin is a drug widely used in the

treatment of type 2 diabetes and for several types of cancer has shown cytotoxic and antiproliferative effect. For

breast cancer, in vitro studies have shown that metformin induces death through the induction of oxidative stress,

however the specific type of cell death is unclear. Thus, the present study studied the types of cell death metformin is

able to induce on human breast cancer cells, MCF-7. For this evaluation the following inhibitors were used: a) Z-vad: a

pan-caspase inhibitor, capable of blocking apoptosis; b) Necrostatin-1 (Nec-1): an inhibitor of RIPK1 activity, capable

of blocking necroptosis c) Deferoxamine (DFO): an iron ion chelator, capable of blocking ferroptosis.

Material and methods: MCF-7 cells were treated with two concentrations of metformin (1mM and 5mM)

concomitantly with the death inhibitors, Z-vad (10μM), Necrostatin-1 (50μM) and Deferoxamine (100μM). After 24

hours of treatment, cell viability, proliferation and death were analyzed. In relation to the oxidative stress parameters, it

was evaluated the total thiol levels, reduced and oxidized glutathione (GSH and GSSG, respectively) and

malondialdehyde (MDA), a marker of lipoperoxidation.

Results: We observed that treatment with metformin reduced the viability of MCF-7 cells in the two concentrations

used (p<0.05), and that each inhibitor was able to restore cell viability. We have also confirmed that metformin is able

to generate oxidative stress by reducing antioxidant levels (total thiol, GSH and GSSG) in the two concentrations

(p<0.05). When cells were treated with the inhibitors, the antioxidants were preserved. MDA levels increased when

cells were treated with metformin (1mM and 5mM) and inhibitors. We observed a higher increase in MDA when the

cells were treated with metformin and Z-vad or DFO. In contrast, we observed a reduction in MDA levels when the cell

was treated with Nec-1 and Metformin 5mM.

Conclusion: Our results demonstrate that the generation of oxidative stress is an important mechanism of activation

of cell death in breast cancer cells treated with metformin, independent of the concentration used. In addition, the use

of inhibitors has enabled us to identify that metformin induces cell death through at least three different pathways

(apoptosis, necroptosis, and ferroptosis) and this effect was also independent of the concentration of metformin used.

This is a promising result, since multidrug resistance in the treatment of breast cancer can be dribbled through the

activation of multiple cell death pathways.


ANTIOXIDANT ACTIVITY OF YELLOW PITAYA (Selenicereus megalanthus)

Lens, H.H.M.1; Lone, A.B.

2; Blegniski, F.P.

1; Panis, C.

3; Takahashi, L.S.A.

4; Faria, R.T.

4; Cecchini, A.L.

1; Victorino,

V.J.1; Cecchini, R.

1 1 Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil

2 Empresa de Pesquisa e Extensão Rural de Santa Catarina (EPAGRI) – Estação Experimental de Itajaí, Itajaí, SC,

Brazil 3 Universidade do Oeste do Paraná, Laboratório de Mediadores Inflamatórios, Francisco Beltrão, PR, Brazil

4 Universidade Estadual de Londrina, Departament of Agronomy, Londrina, PR, Brazil


Keywords: Antioxidant, fruits (Selenicereus magalanthus), oxidative stress.

Introduction and objectives: The involvement of oxidative stress in several diseases is well established in the

literature. Compounds ingested in the diet may exert antioxidant protection against oxidative stress. Considering the

high potential of pitaya fruits for industrial use and its nutritional value, the goal of this study was to evaluate the

antioxidant capacity of yellow pitaya (Selenicereus megalanthus).

Material and methods: This is an original work. Selenicereus megalanthus fruits (N= 7) were obtained from the

Department of Agronomy, State University of Londrina, Paraná – Brazil. Fruits were sanitized and separately into

peels, pulp and seeds. Fruits samples were stored at -20° C until analyzes. We determined the antioxidant capacity of

S. megalanthus peels, pulp and seeds applying total radical antioxidant parameter (TRAP) assay by a highly sensitive

chemiluminescence (QL) method using Trolox, an analog of vitamin E as control. The antioxidant capacity of pitaya

samples were analyzed as its capacity of scavenging DPPH* (2,2-diphenyl-1-picryl-hydrazyl) radical by a

spectrophotometric assay. Results are expressed as mean ± standard error of means. Differences were assessed by

One-way analysis of variance (ANOVA) with Tukey as a post-hoc. p<0.05 was considered statistically significant.

Results: Our data shows antioxidant capacity of S. megalanthus fruits (peels= 166.5 ± 57.04 µM of Trolox; pulp=

47.91 ± 4.824 µM of Trolox; seeds = 3010 ± 579.7 µM of Trolox) by TRAP and significantly greater antioxidant activity

was observed in seeds of yellow pitaya as compared to peel and pulp (p< 0.05). Pitaya fruits also able to significantly

scavenge the DPPH* radical (peels= 66.25 ± 5.330 % DPPH*; pulp= 83.38 ± 1.890 % DPPH*; seeds= 59.44 ± 2.442

% DPPH*; p< 0.05) and we observed greater capacity of DPPH* scavenging for peels and seeds as compared to pulp

of yellow pitaya.

Conclusion: Our data shows for the first time in vitro antioxidant capacity of peels, pulp and seed (separately) from

yellow pitaya fruits (S. megalanthus).

Grants: CAPES, CNPq, Fundação Araucária.


ANTIOXIDANT PROTECTION OF YELLOW PITAYA (Selenicereus megalanthus)

TO BIOLOGICAL MEMBRANES

Lens, H.H.M. 1; Lone, A.B.

2; Blegniski, F.P.

1; Panis, C.

3; Takahashi, L.S.A.

4; Faria, R.T.

4; Cecchini, A.L.

1; Victorino,

V.J.1; Cecchini, R.

1 1 Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil

2 Empresa de Pesquisa e Extensão Rural de Santa Catarina (EPAGRI) – Estação Experimental de Itajaí, Itajaí, SC,

Brazil 3 Universidade do Oeste do Paraná, Laboratório de Mediadores Inflamatórios, Francisco Beltrão, PR, Brazil

4 Universidade Estadual de Londrina, Departament of Agronomy, Londrina, PR, Brazil


Keywords: Antioxidant, fruits (Selenicereus megalanthus), microsomes, oxidative stress.

Introduction and objectives: Studies regarding yellow pitaya (Selenicereus megalanthus) are still scarce.

Considering that oxidative stress is a condition observed in several diseases and that compounds ingested in the diet

may exert antioxidant protection, the goal of this study was to evaluate the antioxidant protection of yellow pitaya (S.

megalanthus) to membrane lipid peroxidation.

Material and methods: This is an original work. Selenicereus megalanthus fruits (N= 7) were obtained from the

Department of Agronomy, State University of Londrina, Paraná – Brazil. Fruits were sanitized and separately into

peels, pulp and seeds. Fruits samples were stored at -20° C until analyzes. To investigate the capacity of S.

megalanthus to protect biological membranes from lipid peroxidation, microsomes were oxidized with iron/ascorbic

acid system and compared to non-oxidized microsomes and evaluated by tert-butyl induced lipoperoxidation. Next,

microsomes were incubated with pitaya peel, pulp and seeds prior oxidation. As control groups, we evaluated the

chemiluminescence of pitaya samples alone and pitaya samples plus microsome. Lipid peroxidation was evaluated by

a highly sensitive chemiluminescence method (QL) and results were expressed as area under the curve (AUC).

Results are expressed as mean ± standard error of means. Differences were assessed by One-way analysis of

variance (ANOVA) with Tukey as a post-hoc. p<0.05 was considered statistically significant.

Results: Our data shows that oxidized system increased microsome lipoperoxidation as compared to control group

(control= 352300 ± 44700 AUC, oxidized= 919700 ± 60170 AUC; p<0.0001). We show for the first time that peels from

yellow pitaya are capable of protect biological membranes from oxidation (control= 352300 ± 44700 AUC, oxidized=

919700 ± 60170 AUC, peel= 276000 ± 2337 AUC, peel+ microsome= 343500 ± 38460 AUC, peel+ oxidized

microsome= 647500 ± 41750 AUC; p< 0.05). Pulp of yellow pitaya did not interfere in membrane oxidation (control=

352300 ± 44700 AUC, oxidized= 919700 ± 60170 AUC, pulp= 195400 ± 2628 AUC, pulp+ microsome= 304200 ±

20640 AUC, pulp+ oxidized microsome= 787700 ± 66750 AUC). Our results also show that seeds from yellow pitaya

protected microsome from oxidation (control= 352300 ± 44700 AUC, oxidized= 919700 ± 60170 AUC, seeds= 137600

± 2378 AUC, seeds+ microsome= 198500 ± 5522 AUC, seeds+ oxidized microsome= 292400 ± 33520 AUC; p< 0.05).

Conclusion: Our data shows for the first time in vitro antioxidant protection of biological membranes by peels, pulp

and seed (separately) from yellow pitaya fruits (S. megalanthus).

Grants: CAPES, CNPq, Fundação Araucária.


Bombyx mori SERICIN IMPROVES TESTICULAR ANTIOXIDANT SYSTEM AND EPIDIDYMIS OF

C57BL/6 MICE OBESE BY HIGH FAT DIET

Scarton, S. R. S.1; Retameiro, A. C. B.

2; Brancalhão, R. M. C.

2; Ribeiro, L. F. C.

2; Fernandes, G. S. A.

1; Beu, C. C. L.

2 1 State University of Londrina, Department of General Pathology, Londrina, PR, Brazil

2 Paraná West State University, Center for Biomedical and Health Sciences, Cascavel, PR, Brazil


Keywords: Silk protein, oxidative stress, Sertoli cell, cell injury, male reproduction.

Introduction: Decreased quality of human semen can be related to lifestyle, such as high energy consumption

and accumulation of fat in the scrotum, such as testicular lipomatosis, where there is elevation of the local

temperature, activation of proinflammatory cytokines and metabolic disturbance of the Sertoli cells. These cells

are responsible for the physical, nutritional support and development of germ cells. Together, these changes cause

increase of reactive oxygen species (ROS), which exert negative effects on the main groups of biomolecules such as

DNA, lipids and proteins. Enzymatic and non-enzymatic antioxidant cellular pathways act to maintain low

concentrations of ROS, allowing the normal occurrence of signaling processes that use oxidation without causing

cellular damage, in addition, one can find natural antioxidant substances, such as sericin, a protein extracted from the

cocoon Bombyx mori, which is biocompatible with potential for use as a food supplement. This research aimed to

evaluate whether sericin exerts antioxidant effects on testis and epididimys of C57BL/6 mice obese by high fat diet.

Material and methods: After approval by the ethics committee (CEUA 04/2014), 20 mice (76 days old) were

distributed in groups with n=5: Control (C); Sericin (S); High fat (HF); High fat and Sericin (HFS). The high fat diet was

offered to the HF and HFS animals for 14 weeks while C and S received a standard diet during the same period. In

the last 4 weeks the animals S and HFS were treated with 1,000 mg/kg body weight of sericin by gavage, while

animals C and HF received 0.8% saline. At 176 days the animals were killed by anesthetic saturation, testicles were

collected, submitted to histological processing and included in paraffin. Sections of 5 μm (3 slides/3 animals per

group) stained with HE were analyzed and photodocumented (n=40/slide) (Bx60®/DP71 Olympus Corporation, Tokyo,

Japan) 40x. For the analysis of biomarkers of the antioxidant system, the testis and the initial segment and cauda

regions of the epididymis were used, the tissues were homogenized in Tris-HCL buffer (pH 7.4) and centrifuged for 10

minutes (12000 rpm, 4°C), the supernatants were stored in -80°C freezer until analysis. The results were expressed

as mean ± sd. ANOVA double factor (α=5%) and compared by the LSD follow-up test, p<0.05.

Results: Morphological analyzes showed HF epithelial changes such as vacuolization of Sertoli cells and tubular

atrophy. These observations were less frequent in HFS. The indirect quantification of lipid peroxidation (LPO) in the

testis was significantly increased in HF and attenuated in HFS, with mean values returning to the C standard (p<0.05).

CAT activity was higher in C and S than in HF and HFS (p<0.05) in the initial segment of the epididymis; While there

was no significant difference in GST activity between groups in this segment. In the cauda of the epididymis, GST

activity increased significantly in HF (p<0.01) and HFS (p<0.05).

Conclusion: It can be concluded that the sericin associated with the hyperlipidic diet exerted antioxidant activity,

reducing the lipid peroxidation in the testis, activating the epididymal antioxidant systems and improving the

regeneration of the seminiferous epithelium.

Grants: Unioeste, Capes e Fundação Araucária


BENZNIDAZOLE AND ASPIRIN ASSOCIATION FOR THE TREATMENT OF CHRONIC

EXPERIMENTAL Trypanosoma cruzi INFECTION

Pereira, R.S1; Malvezi, A.D

1; Lucchetti, B.F.C

1; Tatakihara, V.L¹; Suzukawa, H.T

1; Lovo-Martins, M.I

2; Araújo, E.J.A

3;

Yamada-Ogatta, S.F4; Yamauchi, L.M

4; Martins-Pinge, M.C

5; Pinge-Filho, P¹


2 Carlos Chagas Institute - Fiocruz, Curitiba, PR, Brazil 3 State University of Londrina, Department of Histology, Londrina, PR, Brazil

4State University of Londrina, Department of Microbiology, Londrina, PR, Brazil

5State University of Londrina, Department of Physiological Sciences, Londrina, PR, Brazil


Keywords: Benznidazole, Aspirin, Chagas Disease

Introduction and objective: The drugs nifurtimox and benznidazole (BZN) have been used for the treatment of

Chagas disease since the 1970s. Alternative strategies are being designed to identify candidates among drugs

already available on the market that could be used in combination to improve the efficacy of Chagas disease

treatment. This work evaluates the effect of the association benznidazole (BZN) with aspirin (ASA) for the treatment of

experimental Chagas disease in the chronic stage, in Balb/c mice infected with Trypanosoma cruzi (Y strain).

Material and methods: This study was carried out in strict accordance with the recommendations in the Guide for the

Care and Use of Laboratory Animals of the Brazilian National Council of Animal Experimentation

(http://www.cobea.org.br/). The protocol was approved by the Committee on the Ethics of Animal Experiments at

Londrina State University (CEEA, process number 4628.2016.40). To date, experiments were performed using 40

mice with 8 to 12 weeks old. Infected mice with 5x103 by an intra-peritoneal route were treated by gavage with ASA

25mg/kg/day and BZN 25mg/kg/day, each separately or together diluted in the drinking water. The drinking water was

administered by gavage as a control for the potential impact of the stress associated with the daily handling

associated with gavaging mice. The gavaging was initiated two days post-infection (dpi) and parasitemia was

performed using the Brener method on alternate days for 30 days from the third dpi. Efficacy of the treatment was

evaluated through cardiovascular parameters, nitric oxide (NO) quantification in the plasma, cardiac tissue and spleen

at 180 dpi estimated by measuring nitrite, as described previously by Panis and collaborators in 2011.

Results: The treatments with BZN (25mg/kg/day) alone or in association with ASA (25mg/kg/day) significantly

(P<0.05) reduced mortality and decreased parasitemia. However, the mice treated with BZN and the ASA combination

maintained the levels of mean arterial pressure (MAP), systolic blood pressure (SBP) and heart rate (HR) closer to the

levels observed in control animals. While those of BZN group, had these parameters statistically increased. BZN

combined with ASA showed an increase in NO production than mice treated only with BZN. Interesting, the treated

with BZN and the ASA combination prevented heart enlargement characteristically observed in infected animals.

Conclusion: The therapeutic results from the combination of BZN with ASA presented lesser side effects than the

treatment with BZN.

Grants: Fundação Araucária; CAPES, CNPq, UniZambeze and MCTESTP Mozambique


CHARACTERIZATION OF EXPERIMENTAL ACUTE COLITIS INDUCED BY CONCENTRATIONS OF

SODIUM DEXTRAN SULFATE IN MICE

Fernandes, R.S. 1*

; Santos, T.S. 1*

; Sanches, L.J. 2; Cechinni, A.L.

2;

Araújo, E.J.A. 1

1Universidade Estadual de Londrina, Department of Histology, Londrina, PR, Brazil

2Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil [email protected]

*Authors who shared the main authorship of this work.

Keywords: Inflammatory bowel disease, colitis, dextrans, pathology.

Introduction and objectives: Inflammatory bowel disease is characterized by chronic, remitting or progressive

inflammation of the gastrointestinal tract, with two main forms - Crohn's disease and ulcerative colitis. The most

common intestinal manifestations include severe diarrhea, bloody stools, and weight loss. Among many models

available for studying colitis, dextran sodium sulfate (DSS) induction is the most widely used. In addition,

administration of DSS in the drinking water of mice during a short period of time is consistent with the symptoms of the

disease in humans. However, the severity of the induction depends on several characteristics of DSS, such as

molecular weight, concentration and duration of exposure. Because of this, it is very important to standardize the

protocol at dosage and optimal duration so that colitis develops slowly and steadily. Thus, the objective of this study

was to analyze the characteristics of induced colitis in mice using different concentrations of DSS.

Material and methods: All procedures were approved by CEUA/UEL nº 4023.2017.31. Male 12 weeks old C57BL/6

mice were distributed into 5 groups (n=3): Control (pure water), DSS 2%, DSS 3%, DSS 4% e DSS 5%. All groups

had free access to water for 7 days. The animals were submitted to clinical evaluation daily considering feces

(quantity, weight, consistency and presence of blood), weight loss and water consumption. The disease activity index

(DAI) evaluations were determined by previously established scoring system. After euthanasia, reduced glutathione

(GSH) was analyze as an oxidative stress indicator.

Results: Visible signs of disease, including reduced mobility and raised fur were evidenced in all mice. Results show

that all DSS groups, in comparison with the control group, presented a DAI (score of 0-8). Groups DSS 3% and DSS

4% presented clinical signs (diarrhea and hemorrhage) from the 5th day of induction (score 8), which were not

detected in the DSS 2% and DSS 5% (score 4 and 6, respectively). It was observed reduction of the body weight of

the mice from 5th day of exposure to DSS. A reduction of 32.8% in water consumption was observed in the 5% DSS

group, which were attributed probably to a change on the water taste. The levels of GSH at concentrations of 2, 3 and

5% of DSS remained close to the control level. The 4% DSS presented consumption of the intestinal antioxidant

defenses.

Conclusion: We conclude that the concentration of DSS is an important factor for the development of a murine model

of experimental colitis. DSS concentration of 3% was adequate to induce acute experimental colitis.

Grant: CAPES (scholarship)


CONSEQUENCE OF MATERNAL PROTEIN RESTRICTION ON MUSCLE SPINDLES IN RATS AT 21

DAYS OF AGE

Jeronimo, L.C.1; Buratti, P.

1; Confortim, H. D.

1; Matheus, S.M.M.

3; Pinheiro, P.F.F.

3; Brancalhão, R.M.C.

4; Torrejais,

M.M.2

1Universidade Estadual do Oeste do Paraná - UNIOESTE, Mestrado em Biociências e Saúde, Cascavel - PR, Brazil.

2Universidade Estadual do Oeste do Paraná - UNIOESTE, Centro de Ciências Médicas e Farmacêuticas, Cascavel -

PR, Brazil. 3Universidade Estadual Paulista ―Júlio de Mesquita Filho‖ - UNESP, Departamento de Anatomia, Botucatu - SP,

Brazil. 4Universidade Estadual do Oeste do Paraná - UNIOESTE, Centro de Ciências Biológicas e da Saúde, Cascavel - PR,

Brazil.

[email protected]

Keywords: protein restriction, muscle spindle, skeletal striated muscle.

Introduction and objectives: Skeletal muscle is the most abundant tissue in the body and one of the most

susceptible to adaptations, being mainly sensitive to protein malnutrition because it is a reservoir of protein in the

body. Therefore, when there is a protein deficit in the diet this tissue becomes depleted, causing changes in the

phases of growth, function and differentiation of muscle fibers. The aim of this study was to perform a morphological

and morphometric analysis of the muscle spindles of the extensor digitorum longus (EDL) muscle of rats at 21 days of

age submitted to maternal protein restriction during gestation and lactation periods.

Material and methods: The present study was approved by the Ethics Comission in Animal Experimentation (CEEA)

of Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP) with protocol certificate nº 264-CEEA. Is classified

as an experimental and quantitative study. Wistar rats were divided into two groups: control group – mothers fed a

normal protein diet (17%) during pregnancy and lactation and restricted group - mothers fed a low protein diet (6%)

during pregnancy and lactation. The offspring were maintained with mother during lactation (21 days). After weaning,

six male rats from each group were euthanized and the pelvic limb EDL muscle was dissected. These samples were

embedded in paraplast, transversely sectioned (five μm thick) into a microtome and subsequently stained with

Hematoxylin-Eosin. The prepared slides were photodocumented in 100X objective, with six images captured per

animal. Statistical analysis was performed using Student's t-test.

Results: Each muscle spindle had on average five intrafusal muscle fibers in the control and restricted groups. The

cross-sectional area of the muscle spindle presented a 43% reduction in the restricted group when compared to the

control group. There were no significant differences between the groups in relation to the largest diameter of the

spindles, intrafusal fiber numbers and the cross-sectional area of the intrafusal fibers. The muscle spindles are

arranged parallel to the extrafusal muscle fibers and are mechanical receptors capable of detecting the variation of

muscle length as well as the speed of this variation.

Conclusion: The protein restriction imposed in the gestation and lactation period probably produced a delay in

maturation of the EDL muscle spindles of offspring.


CREATINE SUPPLEMENTATION ANTICIPATES ETHANOL- INDUCED HEPATIC OXIDATIVE STRESS

AND FATTY LIVER IN MICE

Marinello, PC

1,2; Cella, PS

1; Testa, MTJ

1; Padilha, CS

1; Borges, FH

2; Guirro, PB

1; Souza-Neto, FP

2; Brito, WAS

2;

Iarosz, KC1; Voltarelli, FA

3; Cecchini, R

2; Cecchini, AL

2; Duarte, JAR

4; Deminice, R

1.

1 State University of Londrina, Department of Physical Education, Londrina-PR, Brazil.


3 Federal University of Mato Grosso, Faculty of Physical Education, Cuiabá, MT, Brazil.

4 University of Porto, CIAFEL, Faculty of Sport, Porto, Portugal.


Keywords: Creatine supplementation, alcoholic steatosis, oxidative stress

Introduction and objectives: Creatine has demonstrated protective effect on the liver fat accumulation in different

experimental models of non-alcoolic steatosis by regulating inflammation and β-oxidation involved genes. However, its

action in models of alcoholic hepatic steatosis (AHS) is poorly described. Thus, the objective of this study was to

investigate the effects of creatine supplementation (CrS) in the AHS pathophysiology, in two experimental periods, by

evaluating liver oxidative stress, fat accumulation, and tissue morphological changes.

Material and methods: Male Swiss mice (35 - 40g) were divided into 4 groups (n = 8/group): control (C), control

creatine (CC), ethanol (E) and ethanol creatine (EC) (CEUA-UEL: Process n. 21179.2016.78). Animals were fed for

14 or 28 days with Lieber-DeCarli diet (5% ethanol; groups E and EC); C and CC received an isocaloric diet without

ethanol. Creatine (1%) was added to the diet of CC and EC. After euthanasia, the liver was removed and weighed,

and a tissue fragment was fixed in 4% paraformaldehyde for histological processing; the remaining tissue was frozen

for biochemical analysis. Liver architecture was blindly evaluated after hematoxilin/eosine (H&E) staining by crosses

method and collagen deposition was investigated after Sirius red staining, using the ImageJ software. Oxidative stress

(malondialdehyde-MDA; carbonilated proteins; reduced and oxidized glutathione -GSH and GSSG, respectively) was

also assessed on liver.

Results: In the 14-day model, although histological analysis demonstrates liver steatosis in H&E staining, E group did

not present significantly alterations in liver weight, hepatic lipids and oxidative stress parameters. However, CrS

significantly intensified steatosis as well as increased liver weight, hepatic lipids, carbonilated proteins, MDA and GSH

when compared to E group. Tissue collagen deposition did not alter in any group. On the other hand, in the 28-day

model, it was possible to observe a significantly increase in hepatic lipids and oxidative stress (consumption of GSH

and raised levels of MDA and carbonilated proteins) in E group. In this experimental time, CrS only intensified hepatic

MDA (EC versus E), the other parameters remained equal to E group. The collagen deposition did not change

between groups.

Conclusion: The analysis of the results indicates that CrS did not revert ethanol-induced hepatic steatosis.

Furthermore, it is also possible to conclude that CrS anticipates hepatic ethanol toxicity, since 14 days of ethanol

consumption did not induce significative lesions in animals that did not receive supplementation, but increased several

indicators of AHS in the supplemented group.

Grant: Capes-PVE (Process nº 88881.068035/2014-01)


EFFECT OF RESISTANCE TRAINING ON CANCER CACHEXIA: A REVIEW OF THE LITERATURE

Testa, M. T. J. 1 , Cella, P. S.

1, Padilha, C. S.

1, Marinello, P. C.

1,2 , Deminice, R.

1

1 State University of Londrina, Laboratory of Biochemistry of Exercise, Londrina, PR, Brazil

2 State University of Londrina, Department of General Pathology, Londrina, PR, Brazil

[email protected]

Key words: Resistance training, Cancer and Cachexia.

Introduction: Cancer is a worldwide public health problem and the second leading cause of death globally. Cancer-

associated cachexia can be defined as a multifactorial syndrome characterized by severe and involuntary loss of body

weight, muscle mass, and frequently adipose tissue commonly promoted by metabolic disorders. Increased chronic

inflammation, muscle proteolysis, altered carbohydrate, protein and lipids metabolism and anorexia could be present

in this syndrome. Among the non-pharmacological interventions that may attenuate cancer-related skeletal muscle

wasting, chronic physical activity has been pointed as a good alternative, exerting an important role in improving the

quality of life. The resistance training (RT) has the capacity to promote morphological and functional modifications in

skeletal muscle. This work aims to review systematically the literature about the effects of RT in cancer cachexia.

Review: The review was conducted using the database available on PubMed, using the following key words:

―Resistance training‖ and ―cancer cachexia‖. The search resulted in 21 articles, in which 9 were selected according to

the main objective of this review (5 reviews and 4 original articles). The articles included in this review were published

between 2001 and 2016. The studies indicate that the tumor growth promotes the increase pro-inflammatory cytokines

levels, release of proteolysis induction factor and other products that are associated to the symptoms of fatigue, poor

sleep and mood disturbance. The skeletal muscle wasting occurs due to an imbalance of synthesis and degradation of

muscle proteins. RT is a potent stimulator of muscular protein synthesis and also stimulates muscular liberation of IL-

6, that increases the insulin sensibility and decreases the production of pro-inflammatory cytokines. Therefore, the

literature suggests that RT is a good strategy to improves muscle mass and strength in cancer patients.

Conclusion: Although the literature reports the benefits of RT on cancer cachexia, this field has been poorly

investigated. Furthermore, there are lacks about the mechanisms that this type of exercise could be a good

intervention to be used in cancer cachexia.


EFFICACY, SAFETY AND METHOD FOR CONFIRMATION OF THE PRESENCE OF OLFACTORY

EPITHELIUM IN BIOPSIES FROM THE SUPERIOR TURBINATE

Garcia, E. C. D.1; Rossaneis, A. C.

1; Pipino, A. S.

2; Gomes, G. V.

2; Verri, W. A., Jr

1 ; Fornazieri, M. A.

3

1Universidade Estadual de Londrina, Department of Experimental Pathology, Londrina, PR, Brazil

2 Universidade Estadual de Londrina, Department of Clinical Surgery, Londrina, PR, Brazil

3 Universidade Estadual de Londrina, Department of Clinical Surgery, Otolaryngology Division, Londrina, PR, Brazil


Keywords: smell, immunohistochemistry, histology.

Introduction and objectives: Olfactory epithelium biopsy has been shown to be a promising procedure for studying

various diseases and obtaining stem cells, but the procedure in office could cause discomfort to the patient and there

is the possibility that this removal of tissue could impair the olfactory capacity of the patient. Furthermore, is not a

standard to the best site of collection, method of confirming the presence of the olfactory epithelium and safety of the

procedure regarding the olfactory function of the patient. This study aimed to determine the efficacy of obtaining

integral olfactory epithelium proper for pathological analysis from the medial surface of the superior turbinate, evaluate

safety of this procedure for the total and specific olfactory function and calculate accuracy of morphologic criterion

(absence of goblet cells) to confirm olfactory epithelium presence.

Material and methods: This clinical study was conducted at the State University of Londrina and approved by the

Research Ethics Committee involving human beings (approval number: 1.024.603). All patients were informed about

the study and signed informed consent form. Biopsy of the nasal mucosa was performed in twenty-one individuals

without olfactory complaints during septoplasty. Olfactory function was assessed before and one month after the

biopsy using University of Pennsylvania Smell Identification Test (UPSIT), a test containing 40 microencapsulated

odors allocated in strips within each page of its four booklets. Specimens were analyzed with hematoxylin and eosin

stained slices for morphological criterion analysis (considered olfactory epithelium when found region of integral

epithelium without goblet cells) and immunohistochemistry using the anti-Olfactory Marker Protein antibody (specific

marker for olfactory epithelium). Student-T test and confidence intervals were used for the comparison between the

answers for UPSIT before and after of biopsy. Accuracy of morphologic criterion as method of confirm olfactory

epithelium presence was calculated.

Results: Integral olfactory epithelium was obtained in 91% of the patients in the medial surface of the superior

turbinate. There was no deterioration of olfactory function in any of the 40 tested odors after using this technique.

Accuracy of morphological criterion without immunohistochemistry was 94.7%.

Conclusion: Superior turbinate biopsy is a safe and efficient procedure to obtain olfactory epithelium. Morphological

analysis of specimens from human nasal cavities not showing goblet cells can be used to confirm the presence of

olfactory epithelium.

Grants:

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação Araucária.


EVALUATION OF POTENTIAL ANTINEOPLASTIC ACTION OF RESVERATROL, ISOLATED AND

ASSOCIATED TO ETHANOL, IN N-METHYL-N’-NITRO-N-NITROSOGUANIDINE (MNNG)-INDUCED

CARCINOGENESIS IN THE COLON OF RATS

Cesar, E.F 1; Minto, S. B.

1; Garcia, S.B.

1 1 Department of Pathology, Faculty of Medicine of Ribeirão Preto, University of São Paulo

(USP), Ribeirão Preto, São Paulo, Brazil.


Keywords: Resveratrol, Colorectal Cancer, MNNG, Apoptosis

Introduction: Resveratrol (RESV), a polyphenol present in several natural compounds, including wine, has been

associated with a chemopreventive effect in different types of cancer, including colorectal cancer (CRC). This

beneficial effect observed in several studies has been attributed especially due to its anti-oxidant action; Moreover,

RESV also is involved in several other cellular mechanisms, including apoptosis. Despite of it, a more accurate

analysis of a potential anti-neoplastic action of RESV in the promotion phase of CRC is still ongoing. CRC is one of

the most frequent types of cancer in the Western world, with incidence increasing in developing countries and despite

the efforts of scientists and the development of new drugs, this type of cancer still has high rates of morbidity and

mortality. Objectives: The aim of this study was evaluate a potential anti-neoplastic action of RESV, pure and

associated with ethanol, on the carcinogenesis of colorectal cancer induced by N-methyl-N'-nitro-N-nitrosoguanidine

(MNNG) in rats through the analysis of oxidative stress with malondialdehyde (MDA) and immunoexpression of

Caspase-3 (Casp-3), gamma-H2AX (H2AX), iPCNA (PCNA) and Caveolina-1 (CAV-1).

Material and methods: This project was approved by the Ethics Committee on the use of animals (CEUA) under

protocol number 008473/17. Our experiments were performed with 48 wistar rats, which were subjected to

carcinogenesis (0.5 ml of MNNG solution (5 mg / ml) and treated with placebo, RESV (1 mg / kg / day) and RESV (1

mg / kg / (G1), RESV + EtOH (G5), RESV + MNNG (G4), RESV + EtOH (G5) and RESV + EtOH + MNNG (G6). The

results were submitted to statistical analysis using Graph Pad Prism 5.0 software (San Diego, California). To verify the

differences we used the One-way Analysis of Variance (ANOVA) adopting a significance level of 0.05. There was

adopted the Tukey's (One-way) post-test when applicable.

Results: RESV decreases the production of MDA in all treated groups, evidencing its systemic antioxidant effect.

RESV increased the number of apoptotic corpuscles and apoptosis in dysplastic crypts, leading to the appearance of

acellular cryptic spaces (ACE) and, consequently, to a statistically significant reduction in the number of dysplastic

crypts in the treated groups. By decreasing expression of H2AX, PCNA and CAV1, we observed that RESV had a

protective effect on the development of pre-neoplastic lesions in MNNG-induced carcinogenesis. We attributed this

effect to its pro-apoptotic action, which has been shown to be an effective mechanism in colonic carcinogenesis in

rats, decreasing the number of dysplastic crypts and leading to the formation of ACE's, a phenomenon not yet

described in the literature. Unlike the initial hypothesis, EtOH mitigated the pro-apoptotic effects of RESV, but did not

diminish its antioxidant action.

Conclusion: Our findings have shown that the anti-neoplastic and chemopreventive action of RESV is effective in the

promotion phase of MNNG-induced colonic carcinogenesis and that the association between RESV + EtOh, the same

as that found in wines, has not been shown to be as effective as isolated RESV.

Grants: National Council for Scientific and Technological Development (CNPq)


FUNCTIONAL EVALUATION OF RATS SUBMITTED TO AN EXPERIMENTAL MODEL OF

CEREBRAL PALSY

Prado-Filho, D.1; Buratti, P.

1; Covatti, C.

1; Kuhn, C.

1; Centenaro, L. A.

2; Torrejais, M. M.

1

1 Universidade Estadual do Oeste do Paraná, Mestrado em Biociências e Saúde, Cascavel, PR, Brazil

2 Universidade Estadual do Oeste do Paraná, Centro de Ciências Médicas e Farmacêuticas, Cascavel, PR, Brazil

[email protected]

Keywords: lipopolysaccharide, perinatal anoxia, sensorimotor restriction, open field test.

Introduction and objectives: Cerebral palsy (CP) is a sensorimotor disorder caused by nerve damage during brain

development, the main symptoms of which are motor disorders. This study aimed to evaluate the motor activity of

Wistar rats in an experimental CP model.

Material and methods: This study is classified as experimental and quantitative. It was developed at the

Universidade Estadual do Oeste do Paraná (Unioeste) - Cascavel, under the approval of the Ethics Committee in the

Use of Animals (CEUA) (number 24/16). For the litters were used adult Wistar rats (11 females and 06 males).

Pregnant females were subjected to intraperitoneal injections of sterile saline solution (100 μL) or lipopolysaccharide

(LPS; 200 μg/kg LPS in 100 μL sterile saline) every 12 hours from the 17th gestational day to the end of the

pregnancy (21st gestational day). The male offspring were separated into two groups: Control group (CG, n = 8) -

offspring of rats injected with saline during pregnancy, and CP group (CPG, n = 8) - offspring of rats injected with LPS

during pregnancy, submitted to perinatal anoxia and sensorimotor restriction. For perinatal anoxia, the offsprings were

placed in a closed chamber, partially immersed in water at 37 °C ± 1, with a flow of 9 l/min of nitrogen (100%) for 20

minutes, on the day of birth (postnatal day 0, P0). From the 1st to the 30th postnatal day (P1 to P30), CPG animals

were submitted to sensorimotor restriction (16 hours/day). The locomotor activity was evaluated at the 29th and 45th

postnatal days. The animals' activity was filmed (five minutes) in the open field test, which consists of a wooden box

with the floor subdivided into 12 squares. The videos were used to collect data on locomotion time, crossing, rearing

and grooming frequencies. The normality of the data was verified with the Kolmogorov-Smirnov test and after the

Student's t-test was used. Values of p < 0.05 were considered significant.

Results: It was verified that at the 29 day of age, the animals of the CPG presented reduction of 23% in the

locomotion time in seconds (CG: 165.4 ± 22.7; CPG: 126.3 ± 15.1; p = 0.0012), of 42% in number of crossing (CG:

119.8 ± 15.1; CPG: 69.4 ± 26.0; p = 0.0003) and of 57% in rearing frequency (CG: 46.2 ± 11.2; CPG: 19.9 ± 13.5; p =

0.0008) when compared to CG. At 45 postnatal day, the animals had a reduction of 32% in the locomotion time in

seconds (CG: 99.9 ± 20.3; CPG: 67.2 ± 33.7; p = 0.0344) and 41% in the rearing frequency (CG: 29.9 ± 11.3; CPG:

17.7 ± 8.7; p = 0.0310) in relation to CG. The other parameters evaluated at 29 and 45 days of age remained similar

among the studied groups.

Conclusion: The animals in this study presented motor deficits, which may have resulted from the abnormal gait

pattern, nervous disorganization and muscular alterations caused by the insults used, serving as a basis for new

studies aimed at investigating CP.

Grants: Coordination of Improvement of Higher Level Personnel (CAPES) through a Master's degree scholarship.



GENETIC MECHANISMS IN THE DEVELOPMENT OF BREAST CANCER

Silva, I. C.1; Niero, A. P.

2; Hashimoto, N.

3.

¹ Londrina State University, postgraduate student, Londrina, PR, Brazil.

2Filadélfia University Center, postgraduate student, Londrina, PR, Brazil.

³Filadélfia University Center, Londrina, PR, Brazil

[email protected]

Key words: Breast cancer, genetics of cancer, molecular markers.

Introduction: The Cancer, one of the major causes of death in the world, is known as a genetic disease because it

results from genetic changes in normal cells that are capable of transforming them into malignant cells and are

transmitted from tumor cells to their daughter cells. With the aim of describe the classes of altered genes in the

pathogenesis of the Breast cancer, as well as to highlight the main tumor markers in use in clinical practice, a

bibliographic review was carried out, based on studies and researches available in the database Bireme through the

services of Medline, Scielo, Lilacs and Pubmed, selecting articles in Portuguese and English from the last 7 years.

Review: The Breast cancer occurs when the mammary cells escape the mechanisms of control of the cell cycle,

causing a disordered proliferation called neoplasia. The onset of the tumor results from mutations in three major

classes of genes: Tumor suppressors, Proto oncogenes and DNA repair genes. In the case of breast cancer, 5 to 10%

of the cases are inherited. The p53 gene, correlated to several types of neoplasms, BCRA1, BCRA2, CHECK2 and

PTEN genes are associated with breast cancer in studies available in the literature. The Knowledge of the genetic

mechanisms involved in breast cancer is necessary for a better understanding of the etiology of the illness, making

possible the development of new tumor markers for clinical practice and adoption of effective preventive measures.

Conclusion: Although monitoring and genetic counseling of women with mutations in BCRA1 and BCRA2 genes is

already being performed in clinical practice, further genetic markers are required. Because it is a multifactorial

disease, with relation to several genes, besides environmental factors, it is necessary a better understanding of its

genetic mechanisms to improve the methods of diagnosis, control and prevention of the disease.

EPIDEMIOLOGY AND PUBLIC HEALTH


GLYCOLYTIC MUSCLE WASTING PROMOTES PRECOCIUOS MITOCHONDRIAL AND

SARCOPLASMIC RETICULUM DYNAMIC ALTERATIONS IN AN EXPERIMENTAL MODEL OF

PRECACHEXIA

Blegniski F. P.1; Boncompagni S.

2; Pecorai C

2; Cecchini

R.

3; Protasi F

2; Guarnier F. A.

1 1 Pathophysiology Laboratory of Muscular Adaptations, Department of Pathological Sciences, Universidade Estadual

de Londrina, Londrina, Brazil 2 Center for Research on Ageing and Department of Neuroscience and Imaging, Università G. d’Annunzio, Chieti, Italy

3 Pathophysiology Laboratory of Free Radicals, Department of Pathological Sciences, Universidade Estadual de

Londrina, Londrina, Brazil

[email protected]

Keywords: EDL, mitochondria, sarcoplasmic reticulum, precachexia, N-acetylcysteine.

Introduction and objectives: Precachexia, the initial and often barely perceptible beginning of cancer cachexia

trajectory, is classified as an involuntary weight loss (i.e., ≤ 5%). Muscle wasting, the preeminent characteristic of this

syndrome, does not occur similarly in all types of muscle fibers, with glycolytic fibers more prone to atrophy than

oxidative fibers. Cancer progression promotes mitochondrial functional and morphological abnormalities in the skeletal

muscle. Oxidative stress in moderate level has been related to muscle loss. So far, no data have related mitochondrial

dynamics associated with sarcoplasmic reticulum ultrastructure changes and oxidative stress in specific fiber type in

the early stages of cachexia syndrome. The objective of the present study was to determine extensor digitorum longus

(EDL), a glycolytic muscle, ultrastructural alterations related to mitochondrial and sarcoplasmic reticulum, and

correlation with oxidative stress profile in a very early stage of cachexia induced by Walker-256 solid tumor.

Material and methods: Male Wistar rats (200-230g) were divided into three groups: Control (C), Walker-256 tumor

bearing rats (T) and Walker-256 tumor bearing rats treated with N-acetylcysteine 1% (TNAC). Animals were

euthanized (approved by Ethics Committee on Animal Experimentation from the Universidade Estadual de Londrina,

Brazil (ref.9775)). Body, tumor and EDL weights were registered. On skeletal muscle homogenates, lipid peroxidation

was determined by chemiluminescence (CL), oxidized (GSSG) and reduced (GSH) glutathione and carbonyl protein

content was determined. A histological parameter of muscle adaptation was determined by EDL cross-sectional area

(CSA). Sarcoplasmic reticulum (SR) volume and mitochondrial volume and surface were measured using a stereology

point-counting technique through electronic microscopy (ME) transversal sections micrographs. Extent of

mitochondrial network was estimated in EDL, by measuring the length of each individual mitochondria in longitudinal

sections. When appropriate, data were compared by One-way ANOVA followed by Tukey post-test or Kruskall–Wallis

followed by Dunn’s test. Mitochondrial quantitative data was compared by Student’s t-test. Differences were

considered statistically significant when P<0.05.

Results: Protein carbonyl content, GSH and GSSG showed no significant difference in all groups. In contrast, T CL

curves demonstrated to be different when compared to C and TNAC. The CSA frequency distribution of fibers showed

qualitative differences between groups. The 50th, 75

th and 90

th percentile of fiber area in T group were reduced when

compared to C group. The treatment was efficient to revert fiber area to control levels in all analyzed percentiles. The

relative fibers volume occupied by SR was significant altered in T group compared to C (-18.89%, P<0.05).

Mitochondrial volume and surface in T group was significantly reduced compared to C (22.41% and 16.11%,

respectively; P<0.01). Analysis of mitochondrial fusion/fission revealed increased fragmented mitochondria in T, with

significant increased fused organelles found in TNAC when compared with C and T. Treatment with N-acetylcysteine

1% was able to restore all mitochondrial features.

Conclusion: The present study demonstrated that in precachexia stage, where body mass waste is still not very well

established, EDL are already sensitive to mitochondrial and SR dynamics alterations correspondingly to oxidative

stress parameters forward to challenges that modulate muscle mass.

Grants: Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Estado do Paraná.


HISTOPHATOLOGICAL AND IMMUNOHISTOCHEMICAL FINDINGS OBSERVED IN COATIS (Nasua

nasua) MANTEINED AT THE BELA VISTA SANCTUARY, PARANA, SOUTHERN BRAZIL

Michelazzo, M. M. Z1; Oliveira, T. E. S.

1; Stolf, R.

1; Cubas, Z. S.

2; Moraes, W.

2;

Arimatea, R.2;Silva, E.Q.

3; Headley, S. A.

1,3

1Universidade Estadual de Londrina, Department of Preventive Veterinary Medicine, Londrina, PR, Brasil.

2Refúgio Biológico Bela Vista, Itaipu Binacional, Foz do Iguaçu, PR, Brasil

3Faculty of Veterinary, Universidade de Cuiabá, Mato Grosso, Brasil.


Keywords: co-infections, wild carnivore, Procyonidae, pneumonia, enteritis.

Introduction and objectives: Coatis (Nasua nasua), family Procyonidae are widely distributed in South America,

being common to the Atlantic and Amazon Rainforests, are omnivorous animals with food plasticity, the diet

composed of fruits and invertebrates, as well as small mammals, fish and birds. Coatis that inhabit anthropic areas are

known to be very close to humans and are frequently attacked by domestic dogs when they invade homes in search

of food. The objectives of this study were to evaluate the pathological findings and detect the possible occurrence of

antigens of infectious agents by immunohistochemistry in coatis from the Bela Vista Sanctuary.

Material and methods: Tissue fragments from 13 coatis (N. nasua) were collected from the Bela Vista Sanctuary,

located in the city of Foz de Iguaçu, Paraná. These tissues were maintained in 10% buffered formalin solution since

1993 and were cleaved for routine histopathological diagnosis at the Laboratory of Animal Pathology. Tissues from

these coatis were then analysed and diagnosed based on the predominant histopathological pattern. In addition,

selected formalin-fixed paraffin embedded tissues sections were submitted to immunohistochemistry (IHC) assays to

detect antigens of canine distemper virus (CDV; 1:1000), canine parvovirus-2 (CPV-2; 1:300), canine Adenovirus type

1 and 2 (CAdV-1 and 2; 1:200). These infectious agents were selected since they are the most frequently diagnosed

in canids. Permission was granted from Itaipu Binacional to use these tissues for scientific investigations.

Results: The most frequent lesion diagnosed in these coatis was pneumonia (92.3%; 12/13), with interstitial (75%;

9/13), parasitic pneumonia (17%; 2/13), and bronchointerstitial (8%; 1/13) pneumonia being the patterns of pulmonary

disease observed. Nephritis (54%; 7/13) was the second most frequently occurring lesion diagnosed, and was

subdivided in interstitial (86%; 6/13) and granulomatous (14%; 1/13) nephritis. Enteritis was also frequently diagnosed

and occurred in 46% (6/13) of all coatis evaluated, with granulomatous (67%; 4/13), and lymphoplasmacytic (33%;

2/13) enteritis being the patterns of intestinal disease. By IHC, only antigens of CDV, CAdV-1, and CAdV-2 were

observed in tissues from these coatis; positive immunoreactivity to CPV-2 was not identified in any coati. The principal

IHC findings were: positive immunoreactivity to CDV in 23% (3/13) of all coatis with immunoreactivity observed in the

epithelial cells of the lungs, urinary bladder, and small intestine; CAdV-2 (15%; 2/13) were positive in the epithelial

cells of the lungs and urinary bladder, and 8% (1/13) were positive to CAd-1 in the transitional epithelium of the urinary

bladder. Concomitant infections were identified: triple infection due to CDV, CAdV-1 and CAdV-2 in one coati, with

dual infection associated with CDV and CAdV-2 in another. These findings suggest that co-infections in wild

carnivores may be more frequent than previously estimated and that these coatis were infected by diseases that are

common to the domestic dog.

Conclusion: the fact that the Bela Vista Sanctuary is very close to a residential neighborhood in the city, suggests

that the interaction between domestic dogs and wild carnivores may be the most frequent form of transmission of the

viral agents diagnosed during this investigation.

Grants: MEC, CNPq.


HORMONAL THYROID DISORDER IN MICE FED CONTROL DIET AND HIGH-FAT DIET IMPAIRED

SPERM COUNTS AND INFLAMMATION PROFILE IN TESTIS AND EPIDIDYMIS

Punhagui, A. P. F.1,2

; Staurengo-Ferrari, L.2; Verri Jr, W. A.

2; Goulart-Silva, F.


1.

1 State University of Londrina, Department of General Biology, Londrina, PR, Brazil


3 University of São Paulo, Department of Physiology and Biophysics, São Paulo, SP, Brazil


Keywords: obesity, hypothyroidism, hyperthyroidism, male reproductive tract

Introduction and aim: Thyroid disorders and obesity are very common in the population and both are considered a

public health problem, since these may aggravate and induces several diseases like as the risk of infertility. This may

be characterized by alteration in the hormonal profile and abnormalities in the functional parameters of reproductive

tract. Therefore, we were attempted to evaluate the impact of thyroid hormone disorders in mice fed with standard diet

or high-fat diet on the testis and epididymis morphology and function.

Material and methods: For this study, adult male C57BL/6 mice were distributed in 6 experimental groups and fed

standard diet (LFD) or high-fat diet (HFD) for 12 weeks. Afterwards, both mice were treated with saline (LFD and

HFD), or PTU (antithyroid drug – LFD+PTU and HFD+PTU) or T3 (LFD+T3 and HFD+T3) for 30 days. At the end of

treatment, mice were anesthetized and submitted to euthanasia. The following organs were removed and weighted:

testis, epididymis, vas deferens, prostate, liver and seminal vesicle (full and empty). The vas deferens was collected

for evaluation of spermatic morphology. The testis and epididymis were collected for sperm count and NAG (N-acetyl-

β-D-glucosaminidase) and MPO (Myeloperoxidase) activity. All protocols were approved by the Ethics Committee on

Animal Use of State University of São Paulo - USP (CEUA / USP nº 106/23).

Results: The effectiveness induction of hypothyroidism and hyperthyroidism was confirmed by pituitary TSHβ mRNA

expression, which increased in hypothyroid and decreased in hyperthyroid mice fed standard diet or high-fat diet. In

respect to male reproductive tract of mice, we observed the following alterations: the prostate weight increased in the

HFD and HFD+PTU groups compare to LFD group. The empty vesicle weight increased in the HFD+PTU group, while

the empty as well as the full vesicle decreased in the HFD+T3 group, both compared with LFD group. When

compared to the LFD group, the sperm concentration and DSP (daily sperm production) decreased in the testis of the

LFD+T3 group, unlike the same parameters that increased in the HFD group. The number of neutrophils (MPO

activity) increased in the testis of HFD and HFD+PTU groups, as opposed to the caput of epididymis in which this

number decreased in the HFD+T3 group, both when compared to the LFD group. The number of macrophages (NAG

activity) increased in the testis of HFD+T3 group, as well as in the epididymis tail of LFD+PTU and HFD+T3 groups

when compared to the LFD. The analysis of sperm morphology did not show any significant difference among the

groups.

Conclusion: We partially conclude that obesity associated with thyroid disorders induces more damage to the male

reproductive system of mice than obese animals without thyroid disorders.

Grants: CAPES, FAPESP process number: 2014/12871-9.


IMPACT OF MATERNAL PROTEIN RESTRICTION DURING RAT PREGNANCY AND LACTATION ON

INTRAMUSCULAR CONJUNCTIVE TISSUE OF OFFSPRINGS

Jeronimo, L.C.1; Buratti, P.

1; Rodrigues, G.

3; Pinheiro, P.F.F.

3; Ribeiro, L.F.C.

2; Dal'Osto, V.L.C.

4; Torrejais, M.M.

2 1Universidade Estadual do Oeste do Paraná - UNIOESTE, Mestrado em Biociências e Saúde, Cascavel - PR, Brazil.

2Universidade Estadual do Oeste do Paraná - UNIOESTE, Centro de Ciências Médicas e Farmacêuticas, Cascavel -

PR, Brazil. 3Universidade Estadual Paulista ―Júlio de Mesquita Filho‖ - UNESP, Departamento de Anatomia, Botucatu - SP,

Brazil. 4Universidade Estadual do Oeste do Paraná - UNIOESTE, Centro de Ciências Biológicas e da Saúde, Cascavel - PR,

Brazil.

[email protected]

Keywords: protein restriction, collagen, skeletal striated muscle.

Introduction and objectives: The fetal period is essential for skeletal muscle developmental and may be influenced

by maternal treatments during pregnancy, affecting the postnatal muscular growth of the offspring. The objective of

this study was to evaluate the intramuscular collagen of the extensor digitorum longus (EDL) muscle of rats at 21 days

of age submitted to maternal protein restriction during gestation and lactation periods.

Material and methods: The present study was approved by the ethics comission in animal experimentation (ceea) of

Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP) with protocol certificate nº 264-ceea. Is classified as

an experimental and quantitative study. Wistar rats were divided into two groups: control group – mothers fed a

normal protein diet (17%) during pregnancy and lactation and restricted group - mothers fed a low protein diet (6%)

during pregnancy and lactation. The offspring were maintained with mother during lactation (21 days). After weaning,

six male rats from each group were euthanized and the pelvic limb edl muscle was dissected. These samples were

embedded in paraplast, transversely sectioned (five μm thick) into a microtome and later submitted to the masson

trichrome technique to evidence and study intramuscular collagen. The prepared slides were photodocumented in 40x

objective and six images were captured per animal and analyzed using image-pro plus 6.0 software. Statistical

analysis was performed using student's t-test.

Results: Morphological analysis of the EDL muscle revealed that in the two groups, the connective tissue was present

in the perimysium, involving the fascicles and in the endomysium, involving the muscle fibers. The analysis of the

percentage of connective tissue showed an increase of 18% in the offspring of the restricted group relative to the

offspring of the control group. The composition and arrangement of the surrounding connective tissue are important

for muscle function, and may vary in muscle disorders as observed in this study. Conclusion: Maternal protein restriction during gestation and lactation produced in the offspring an increase in

intramuscular connective tissue in the EDL muscle, which may cause changes in maintenance and muscle structure.


INTERACTION BETWEEN OBESITY AND ACUTE INFECTION BY Trypanosoma cruzi ON

CARDIOVASCULAR PARAMETERS

Lucchetti, B.F.C.1; Boaretto, N.

1; Lopes, F.N.C.¹; Malvezi, A.²; Lovo-Martins, M.I.²; Tatakihara, V.L.H.²; Pereira, R.S.²;

Pinge-Filho, P.²; Martins-Pinge, M.C.¹

¹Universidade Estadual de Londrina, Department of Physiological Sciences, Londrina, PR, Brazil

²Universidade Estadual de Londrina, Department of Pathological Sciences, Londrina, PR, Brazil


Keywords: Obesity; T. cruzi; Cardiovascular system; Nitric oxide.

Introduction and objectives: Chagas disease(CD), caused by the protozoan parasite, Trypanosoma cruzi, is

classified by the WHO as one of the major infectious diseases of the world. Infection with T. cruzi results in an intense

inflammatory reaction in many tissues. Because to the increased incidence of obesity worldwide, mainly in the areas

considered endemic for CD, it is necessary to investigate the effects of obesity and its consequences on acute CD.

Lipid and glucose metabolism are interrelated and are deregulated during CD, furthermore adipocytes represent an

important target for infection. The aim of this study is to evaluate the cardiovascular effects of obesity during acute

infection by T. cruzi.

Material and methods: Work previously approved by the CEUA (19665.2016.03). To induce obesity, the newborn

male mice will undergo subcutaneous injection of monosodium glutamate (4 mg/g body weight) from day 2 to day 6

after birth. Obesity was determined by the Lee index and adipose tissue weighing of the retroperitoneal and

perigonadal regions. At 70 days of age, the infected groups were submitted to an intraperitoneal inoculum of 5x10²

forms of trypomastigotes of the strain Y of T. cruzi. The parasitaemia and mortality was performed by the method of

Brener. Nitric oxide (NO) dosage in plasma and adipose tissue was performed by the cadmium and Griess technique.

The cardiovascular parameters were measured through the CODA platform, during the development of obesity and

after infection.

Results: The Lee index (0.350 vs 0.308), abdominal circumference (10.82 vs 9.39cm) and weight of perigonadal

(942.41 vs 190.14g) and retroperitoneal (623.76 vs 150.17g) adipose tissue was higher in the obese group (OG)

compared with control group (CG). OG had a higher mean arterial pressure (MAP) before infection compared to the

CG (118.12 vs 88.09 mmHg). Higher parasitaemia was observed in the animals of the obese group infected (OGI) on

days 13 (1.29x106

vs 2,2x104parasites/mL) and 15 (1.8x10

6 vs 3,5x10

4parasites/mL) post infection compared to the

animals of the control group infected (CGI). All animals in the OGI died by the end of the day 19 after infection, while

87.5% of CGI survived until day 30 post infection. There was a sudden drop in MAP in the OGI from day 9 post

infection (97.66 vs 124,29mmHg) p<0.05 that remained on day 13 (75.78 vs 110.29mmHg) p<0.05, 15(64.24 vs

105.07mmHg) and 19 post infection (65.6 vs115.92mmHg) when compared to the CGI. The CGI did not change their

MAP throughout the acute phase of infection on all evaluated days.

It was found a higher production of nitric oxide in the OGI compared to the CGI (120.95 vs 64.12nitrite/ml). An

increased NO in the adipose tissue of OGI was also found compared to the CGI (9.31 vs 5.20nitrite/mg).

Conclusion: The effects of obesity appear to have exacerbated the effects during the acute phase of CD. A large

increase of parasitaemia in these animals leads to a greater production of NO in the plasma and adipose tissue that

would be related to the abrupt fall of the blood pressure observed during infection.

Grants: CAPES/CNPq


IPA/NO: EFFECTS AND ACTION MECHANISM IN CHRONIC CONSTRICTION INJURY-INDUCED

NEUROPATHIC PAIN IN MICE.

Bertozzi, M. M. 1; Staurengo-Ferrari, L.

1; Zucoloto, A. Z.

1; Rossaneis, A. C.

1; Pinho-Ribeiro, F. A.

1; Fattori, V.

1; Borghi,

S. M.1; Casagrande, R.

2; Verri Jr, W. A.

1.

1 Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil

2 Universidade Estadual de Londrina, Department of Pharmaceutical Sciences, University Hospital (Health Science

Centre), PR, Brazil

[email protected]

Keywords: HNO; IPA/NO; pain; neuropathy.

Introduction and objectives: Chronic pain is a major health problem worldwide. Neuropathic pain is a complex

syndrome resultant of injury or dysfunction of somatosensory system, which still lacks adequate pharmacological

control. Nitroxyl (HNO), is the one-electron reduced form of nitric oxide (NO), and shows a distinct chemical and

pharmacological profile from that of NO.IPA/NO is a nitroxyl donor. Herein, the objective was to evaluate the effect

and mechanisms of action of IPA/NO on the neuropathic pain model induced by chronic constriction of the sciatic

nerve (CCI) in mice.

Material and methods: Male Swiss mice (n=6/group, CEUA n°22588.2015.19 and nº1305.2015.25) were submitted

to CCI and 7 days after, IPA/NO was administrated by subcutaneous or intrathecal route. In another experiment, mice

were chronically treated with IPA/NO between 7-14 days after CCI surgery, and mechanical and thermal hyperalgesia

were evaluated daily. At 3h chronic treatment with CCI, spinal cord samples were collected to analyze the cytokine

production by ELISA and activation of spinal glial cells by qPCR and immunofluorescence. Western blotting was also

used to evaluate the fosforilation levels of MAPKs and NF-B. IPA/NO treatment was also tested in capsaicin-model

of inflammatory pain and the dynamics of calcium in DRG cell culture. Data were analyzed by ANOVA followed by

Tukey’s post-hoc (p<0.05).

Results: IPA/NO inhibited in a single or continuous treatment CCI-induced mechanical and thermal hyperalgesia by

activating the cGMP/PKG/K+ATP channel signaling pathway. Daily treatment during 7 days with IPA/NO inhibited CCI-

induced mechanical and thermal hyperalgesia by reducing spinal cord cytokines IL-33, TNF-α, IL-1β levels and

increased IL-10 (ELISA) and reducing glial cells activation (Gfap, Iba-1, and Olig2) markers mRNA expression. In

addition, IPA/NO treatment also prevents the activation of MAPKs and NF-B (Western Blot) induced by CCI.

Regarding to inflammatory stimulus, the treatment with IPA/NO reduced the mechanical and thermical hyperalgesia

andovert pain-like behavior induced by capsaicin. In neurons cells culture derivate from DGR IPA/NO treatment

reduces Ca2+

influx capsaicin-induced.

Conclusion: These results demonstrate the efficacy of IPA/NO as a new analgesic drug.



Lactobacillus plantarum REDUCES INSULIN RESISTANCE AND YACON OR SYMBIOTIC REDUCES

OXIDATIVE STRESS IN RATS WITH METABOLIC SYNDROME

Mari,N.L.

1, Bregano, J.W.

2, Simão A.N.C.

2, Lozovoy M.A.B.

2, Bonifacio K.L.

1, Alfieri D.F.

1, Dichi I.

3,

Miglioranza L.H.S.4

1 M.Sc., Post Graduate Program in Health Science/University of Londrina, Parana, Brazil

2 Ph.D., Department of Pathology, Clinical Analysis and Toxicology/University of Londrina, Parana, Brazil

3 MD, Ph.D., Department of Internal Medicine/University of Londrina, Parana, Brazil

4 Ph.D.,Department of Food Science/University of Londrina, Parana, Brazil

[email protected]

Key words: metabolic parameters, probiotic, prebiotic, symbiotic, oxidative stress.

Introduction and objectives: There is an increasing interest in exploring the effects of probiotics, prebiotics and

symbiotic on Metabolic Syndrome (MetS) and oxidative stress has been implicated in its physiopathology. The aim of

this study was to evaluate metabolic parameters and oxidative stress in metabolic syndrome treated with prebiotic,

probiotic or symbiotic.

Material and methods: MetS was induced by diet supplemented with 66% fructose in male wistar rats. The animals

were divided into five groups (n=10 each): G1 received a standard diet without inducing MetS. Animal’s from G2, G3,

G4 e G5 were fed with 66% fructose supplement. G2 had no therapeutic interventions; G3 received treatment with

probiotic Lactobacillus plantarum Lp 115 (109 CFU); G4 received prebiotic yacon powder (10%) and G5 (symbiotic

group) was treated with a beverage containing L. plantarum Lp 115 and yacon (1011

CFU). All diets were administered

for eight weeks. The animals underwent the following laboratory blood analysis: glucose, high density lipoprotein

cholesterol (HDL-C), triacylglycerol and uric acid, which were evaluated by a biochemical auto-analyzer using Dade

Behring® kits. Plasma insulin was determined with a rat enzyme-linked immunosorbent assay kit. Insulin resistance

was assessed by homeostasis model of assessment insulin resistance (HOMA-IR). In relation to oxidative stress and

antioxidant capacity the following analyzes were performed, Tert-butyl hydroperoxide-initiated chemiluminescence

(CL-LOOH), evaluation of Nitric Oxide metabolites (NOx), Total Radical-Trapping Antioxidant parameter (TRAP),

determination of sulfhydryl (SH) groups of proteins.

Results: In relation to G1, rats fed high-fructose diet (G2) showed laboratorial features compatible with MetS; G2

showed reduced nitric oxide metabolites (NOx) levels (p = 0.012) and increased levels of sulfhydryl (SH) (p <0.0001)

and total radical-trapping antioxidant parameter/uric acid (TRAP/UA) (p = 0.044). In relation to G2, probiotic

decreased insulin and HOMA-IR (p = 0.015 and p = 0.004, respectively), whereas prebiotic reduced hydroperoxides

levels (p = 0.002), and increased SH (p = 0.049) and TRAP/UA (p = 0.034). Symbiotic resulted in increased HOMA-IR

(p = 0.034), reduced hydroperoxides (p = 0.015) and increased levels of NOx (p = 0.002) and SH (p = 0.031)

compared to G3.

Conclusion: In conclusion, Lactobacillus plantarum Lp 115 reduces insulin resistance and yacon or symbiotic

reduces lipid oxidation and increases antioxidant defenses in male wistar rats with high-fructose diet-induced MetS.


LOW NITRIC OXIDE CONCENTRATION IN PLASMA AS AN IMPORTANT FACTOR IN DEVELOPMENT

OF HYPERTENSION IN OBESE MICE

Boaretto N.1; Lucchetti, B. F. C.

2; Lopes, F. N. C

1; Tatakihara, V. L. H.

2; Pinge-Filho, P.

2; Martins-Pinge, M. C

1

1Universidade Estadual de Londrina, Department of Physiological Sciences, Londrina, PR, Brazil 2Universidade Estadual de Londrina, Department of Pathological Sciences, Londrina, PR, Brazil

[email protected]

Keywords: Obesity; oxide nitric, blood pressure

Introduction and objectives: Affecting almost 2 billion people in the world, obesity is considered a health problem

characterized by an imbalance in energy metabolism, leading to an excessive accumulation of adipose tissue. One

model used for the study of obesity is the model of obesity induced by monosodium glutamate (MSG). MSG in

newborn mice crosses the non-fully developed blood-brain barrier, causing lesions in the arcuate nucleus of the

hypothalamus that promote an imbalance in energy metabolism, thus inducing obesity in adult mice. High blood

pressure, one of the major consequences of obesity, is an important risk factor for a number of cardiovascular

diseases. One of the proposed reasons for the increased blood pressure in obese individuals is the excessive

accumulation of adipose tissue. In addition, nitric oxide (NO) plays an important role in blood pressure control because

of its vasodilator effect; witch appears to be decreased in obese animals. The aim of the study is to evaluate nitric

oxide levels in plasma and blood pressure alterations during obesity development in mice induced by MSG.

Material and methods: Work previously approved by the CEUA 19665.2016.03. To induce obesity, the newborn

male mice will undergo subcutaneous injection of MSG (4 mg/g body weight) from day 2 to day 6 after birth. The

cardiovascular parameters were performed through the CODA platform, during the development of obesity, every 10

days, from weaning at 30 days to 70 days of age. The mice were individually weighed always before the measurement

of the cardiovascular parameters. After 70 days of age the obesity was determined by the Lee index and adipose

tissue weighing of the retroperitoneal and perigonadal regions. And the NO dosage in plasma was performed by the

cadmium and Griess technique.

Results: During the development of the mice in the control group (CG) they had a greater body weight than the mice

of the obese group (OG) from day 30 to day 60 post birht, but on day 70 both groups had the same body weight. Soon

after weaning when the mice had 30 days, no difference was found in the mean arterial pressure between the CG and

OG, although from day 40 of life an increase in mean arterial pressure was found in the OG when compared to the

CG. In OG the MAP remaining higher until the end of the evaluation at 70 days post birth (118,12 vs 88,09mmHg). No

statistical difference was found in heart rate throughout the evaluation. The Lee index, abdominal circumference and

weight of perigonadal and retroperitoneal adipose tissue were higher in the mice of the OG compared with CG. We

also observed a higher production of nitric oxide in the plasma of the CG when compared to the animals of OG at day

70 (57,25 vs 38,63nitrite/mg).

Conclusion: Obese mice exhibited decreased levels of NO, along with increased adipose tissue, and both contribute

to the development of hypertension in these animals.

Grants: CAPES/CNPq


MELANOMA THERAPY: WHAT IS IT FOR NOW AND FOR THE FUTURE?

Sanches, L. J1; Lopes, N. M. D

1; Marinello. P. C

1; Cechinni, A. A

1 1 Universidade Estadual de Londrina, Department of Pathology, Londrina, PR, Brazil

[email protected]

Key words: melanoma1, oxidative stress 2, dacarbazine 3, metformin4, Interleukin-2 5.

Introduction: Melanoma is the most aggressive type of skin cancer, it originates in melanocytes, cells that produce

melanin, and has as its main risk factor sun exposure. According to the World Health Organization about 132,000 new

cases are diagnosed worldwide each year and this number appears to be increasing due to increased exposure to

ultraviolet radiation. Melanoma is refractory to therapies and presents chemoresistance as well as pronounced side

effects. When metastasis is present, the life expectancy is of 2 to 8 months and no therapy is effective in increasing

lifespan. Therefore, a new approaches with combined therapies are being tested.

Review: In melanoma chemoresistance deserves to be highlighted because it is a malignant neoplasm characterized

by almost universal resistance to therapy. Patients with untreated melanoma have demonstrated an increase in

systemic oxidative stress and impairment of the antioxidant system, which could favor the accumulation of reactive

oxygen species (ROS) and promote carcinogenesis in neighboring tissues. Increased resistance of melanoma cells to

oxidative stress may be one of the factors responsible for their poor response to treatment with chemotherapeutics,

which mostly work by increasing the levels of ROS in these cells as an inducer of apoptosis. Some drugs used to treat

cancer can increase the production of ROS. Agents used in the treatment of melanoma include dacarbazine (DTIC),

lomustine (CCNU), interleukin-2 and interferon-alpha, and the former two exert toxicity through ROS. Dacarbazine has

significant effects on disease progression in only 10-15% of treated patients. Bolus and high-dose interleukin-2 was

the last treatment approved by the US Food and Drug Administration for metastatic melanoma and has been the

treatment of choice in recent years. This cytokine induces T cell growth, which would increase the host's immune

response against the tumor. In 2005 a study in the UK, which tracked type 2 diabetic patients, found a lower incidence

of cancer in patients taking metformin. Since then studies have confirmed that diabetic patients treated with metformin

are 25 to 40% less likely to have cancer, compared to patients who take insulin or who use sulfonylureas. The

antineoplastic activity of metformin via AMPK activation is mediated through inhibition of mTORC1 (mTOR complex-1)

signaling, leading to inhibition of protein synthesis and cell proliferation.

Conclusion: Taking into account that the incidence of melanoma is increasing worldwide, which is a highly

aggressive cancer, resistant to available chemotherapies, it needs to study it deeply, and seek new therapies to

improve the quality of life and survival of patients.


METABOLIC RISK FACTORS CORRELATE WITH OXIDATIVE STRESS AND INFLAMMATORY

BIOMARKERS IN APPARENTLY HEALTHY OLDER SUBJECTS

Loyola, W. S.1; Farias, C. C.

1; Bonifácio, K. L.

1; Matsumoto, A. K.

1; Barbolasci, C. C.

1,2; Nogueira, A. S.

1; Nascimento,

M. A.3; Barbosa, D. S.

1, Probst, V. S.

1

1 State Londrina University, Center of Health Sciences, Londrina, Brazil.

2Deakin University, Metabolic Research Unit, School of Medicine, Geelong, Australia

3 State University of Paraná (UNESPAR), Physical Education Department, Paranavaí, Paraná, Brazil

[email protected]

Keywords: Metabolism, oxidative stress, inflammation, elderly

Introduction and objective: Metabolic risk factors (MRFs) are biochemical markers of metabolic disorders that may

be associated with the development of various comorbidities (e.g., metabolic syndrome, cardiovascular disease, and

stroke). In the aging process normally occurring physiological changes that favor the appearance of metabolic

disorders. The oxidative stress (OS) and inflammation have an important role in the pathophysiology of metabolic

disorders and in the aging process. Therefore, the aim of this study was to analyze MRFs, OS and inflammation in

apparently healthy older subjects.

Materials and Methods: All procedures employed during this study were approved by the Ethic Committee at State

Londrina University (CAAE:263225814.6.0000.5231). Blood was collected after an overnight fast (12h) from 56

subjects (age= 69.7±8.7; male= 44%) to obtain the values of laboratory exams of MRFs (glucose, high-density

lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), cholesterol (CT) and triglycerides (TG)),

OS biomarkers, advanced oxidation protein products (AOPP), paraoxonase 1 (PON-1), malondialdehyde (MDA) and

total radical trapping antioxidant parameter (TRAP) and inflammation biomarkers C-reactive protein (CRP), interleukin

6 receptor (IL-6R). Body mass index (BMI) and abdominal girth were also measured. Statistical analyses were

performed using SPSS ver. 19.0 (IBM Co., Armonk, NY, USA). All data were expressed as mean ± SD or median with

interquartile ranges. The Kolmogorov–Smirnov test was used to analyze normality of date distribution. Categorical

variables were analyzed using χ2 and t-tests with 95% confidence intervals (CIs). Pearson’s correlation coefficient

was used to assess relationships among metabolic risk factors, OS and inflammatory biomarkers. Binomial logistic

regression analysis was performed to find significant determinants between variables. Receiver Operating

Characteristic (ROC) was used to assess the utility of MRFs for discriminating a higher level of OS and inflammatory

biomarkers. Statistical significance was set at P<0.05.

Results: TG showed correlations with AOPP (r=0.71; p=0.0004) and with TRAP (r=0.28; p=0.0039); LDL-C with PON

1 (r=0.32; p=0.0018); CT with PON 1 (r=0.42; p=0.001); glucose with MDA (r=0.37; p=0.007); HDL-C with AOPP (r=-

0.29; p=0.03); BMI with AOPP (r=0.31; p=0.03) and with CRP (r=0.38; p=0.007); Abdominal girth with CRP (r=0.32;

p=0.022) and with IL6-R (r=0.34; p=0.0046). The areas under the ROC curves of TG for AOPP was 0.86 (95% CI

0.752-0.962; P =0 .001), of CT for PON-1 was 0.71 (95% CI 0.568-0.85; P =0 .01) and of LDL-C for PON-1 was 0.694

(95% CI 0.55-0.838; P =0 .025). Subjects with a higher TG level ( > 150mg/dl) had an odds ratio of 15.0 to have an

increased AOPP (CI=2,859-78.691; P=0.001); with higher glucose level ( > 99mg/dl) have an odds ratio of 2.4 to have

an increased MDA (CI=0.725-7.946; P=0.007); with higher CT level ( > 200mg/dl) had an odds ratio of 5.67 to have a

raised PON-1 and of 1.4 with higher LDL-C level ( > 130mg/dl); with higher BMI had an odds ratio of 7.3 to have an

elevated CRP (CI=2.009-25.479; P=0.002).

Conclusions: MRFs were highly associated with OS and inflammatory biomarkers. In addition, higher level of MRFs

may be useful to identify increasing in some OS biomarkers in apparently healthy older subjects.


MORPHOLOGICAL CHANGES IN THE SOLEUS MUSCLE DUE TO PERIODONTAL DISEASE

Zazula, M. F. 1, Leite, M. A.

1, Ribeiro, L. F. C.

1, Bertolini, G. R. F.

1, Nassar, C. A.

1, Nassar, P. O.

1

1 Universidade Estadual do Oeste do Paraná, Cascavel, PR, Brazil.


Key Words: Periodontal disease, inflammation, skeletal muscle

Introduction: Periodontal disease is closely related to oral hygiene, so excessive bacterial buildup on the dental

surface favors plaque development and an inflammatory reaction associated with risk factors such as obesity,

smoking, diabetes and mental stress. Periodontal disease is characterized by an inflammation that affects the tissues

that surround and support the teeth, the periodontium, and there is production of inflammatory mediators. Activation of

these inflammatory mediators allows periodontal disease to potentially contribute significantly to the systemic

inflammatory response of the host. One of the effects of systemic inflammation that has been studied is the action of

inflammatory cytokines on loss of muscle mass that can affect microcirculation, causing neuronal hypoxia, axonal

degeneration and muscle damage. Objective: To evaluate whether periodontal disease, with systemic effects, can

promote alterations in the soleus muscle.

Methods: Adult Wistar rats were divided into two groups: 1) Control Group (CG) and 2) Periodontal Disease Group

(PDG). Periodontal disease was induced with ligature method using a modified forceps and an exploratory probe, and

a 4.0 cotton thread ligature was placed around the right and left lower first molar. This ligature acted as a gingival

irritant for 30 days, promoting the accumulation of plaque and consequent development of periodontal disease,

characterizing periodontitis, confirmed by radiographic analysis. After the euthanasia, the soleus muscle was

dissected and processed for the histological analysis and stained with hematoxylin and eosin for morphometric (cross-

sectional area, smaller diameter of the muscle fiber, nucleus/fiber and capillary/fiber ratio) and morphologic analysis,

and stained with Masson's trichrome for connective tissue analysis. For data analysis, was used the one-way ANOVA

and post-test Tukey (p < 0.05). All procedures performed were in accordance with the ethical standards of the

institution.

Results: About morphometric measures, no differences were found between PDG and CG for the cross-sectional

area, smaller diameter, nucleus/fiber ratio and capillary/fiber ratio. However, the PDG showed a significant increase in

connective tissue compared to CG (p < 0.05). About morphologic analysis, CG showed normal morphology of

polygonal shape, multinucleated with nuclei in peripheral position, well-defined fascicles with preserved structure and

organization. In the PDG the fibers also presented, in most cases, normal appearance similar to the CG, however,

there was an apparent increase in the number of nuclei, many presented basophilic halo and some nuclei with central

position, and also presented some irregularly shaped fibers. In connective tissue morphologic analysis, CG and PDG

presented similar results, with typical features and arrangement in epimysium, endomysium and the perimysium.

Conclusion: Periodontal disease promoted deleterious effect with a significant increase of connective tissue in

morphometric analysis, and morphologic alterations, with degeneration of soleus muscle.



NUMBER OF ASTROCYTES IN THE DORSAL STRIATUM OF RATS SUBMITTED TO A CEREBRAL

PALSY LIKE-MODEL

Santos, A. S.1; Almeida, W.

2; Sbardelotto, B. M.

3; Muraro, E. N.

3; Rocha, L. C.

1; Souza, M. A.

4; Centenaro, L. A.

5.

¹ Universidade Estadual do Oeste do Paraná-UNIOESTE, Centro de Ciências Biológicas e Saúde, Cascavel, PR,

Brazil

² Universidade Federal do Rio Grande do Sul, Instituto de Ciências Básicas e Saúde, Porto Alegre, RS, Brazil

³ UNIOESTE, Centro de Ciências Médicas e Farmacêuticas, Cascavel, PR, Brazil 4 Universidade Federal do Paraná, Toledo, PR, Brazil

5 UNIOESTE, Centro de Ciências Médicas e Farmacêuticas, Cascavel, PR, Brazil


Keywords: Lipopolysaccharide, Perinatal Anoxia, Sensorimotor Restriction, Animal Model.

Introduction: Maternal infections, prematurity and asphyxia are currently considered risk factors for the development

of cerebral palsy (CP), a complex locomotion and posture disorder which results from insults to the developing brain

(Coq et al., 2008). Due to this multifactorial etiology, some of the proposed animal models of this clinical condition are

based on the combination of factors during different developmental periods. However, an animal model in rats that

reproduces completely the characteristics of CP still needs to be developed. Thus, the present study evaluated the

number of astrocytes in the striatum of rats submitted to injections of lipopolysaccharide (LPS), perinatal anoxia and

sensorimotor restriction, trying to reproduce the characteristics of CP in these animals.

Material and methods: Experimental procedures were approved by the Research Ethics Committee of the

Universidade Estadual do Oeste do Paraná (UNIOESTE, Nr. 24/16). Two experimental groups were used: Control

Group (CT, n=6) − offspring of rats injected with saline during pregnancy and Cerebral Palsy Group (CP, n=6) −

offspring of rats injected with LPS during pregnancy, submitted to perinatal asphyxia and sensorimotor restriction

during 30 days. At 48 days of life, the animals were euthanized. The brains were dissected, coronally sectioned and

stained using the silver nitrate method. Subsequently, images of the dorsal striatum were obtained and the number of

astrocytes was counted in an area of interest.

Results: An increase in the number of astrocytes was observed in the dorsal striatum of rats from the CP group in

comparison with the CT group (150.5 ± 22.00 and 78.33 ± 7.549, respectively). Conclusion: Astrocytes respond to all

forms of central nervous system insults by a process commonly referred as reactive astrogliosis, which involves

changes in their molecular expression and morphology (Eddleston and Mucke, 1993; Correa-Cerro and Mandell,

2007). In response to severe tissue damages, reactive astrogliosis can lead to the appearance of newly proliferated

astrocytes and scar formation (Faulkner et al., 2004; Drögemüller et al., 2008; Voskuhl et al., 2009). The CP model

adopted in the present study caused reactive astrogliosis in the dorsal striatum, as observed by the increase in the

number of astrocytes in the CP group. Animals submitted to the same interventions also presented motor deficits,

which could be due to the astrocytic scar formation in this important motor area (DOS SANTOS et al., 2017). Thus,

the combination of insults in the pre, peri and postnatal periods reproduces in rats part of the characteristics observed

in human patients with CP, being a valuable strategy for the future study of possible therapeutic interventions.

Grants: The authors would like to thank the Fundação Araucária de Apoio ao Desenvolvimento Científico e

Tecnológico do Paraná (Brazil) by granting the scholarship that supported this study.


PARASITIC PNEUMONIA BY Angiostrongylus spp EM COATI (Nasua nasua) MAINTENED AT THE

BELA VISTA SANCTUARY, PARANÁ, SOUTHERN BRAZIL

Michelazzo, M. M. Z1; Oliveira, T. E. S.

1; Medeiros, H.

1; Cubas, Z. S.

2; Moraes, W.

2;

Braun, R. A.2; Silva, E.Q.

3; Headley, S. A.

1,3

1Universidade Estadual de Londrina, Department of Preventive Veterinary Medicine, Londrina, PR, Brasil.

2Refúgio Biológico Bela Vista, Itaipu Binacional, Foz do Iguaçu, PR, Brasil

3Faculty of Veterinary, Universidade de Cuiabá, Mato Grosso, Brasil.


Keywords: lung worms, immunosuppression, wild carnivore, procionids

Introduction: Coatis (Nasua nasua), family Procyonidae are widely distributed in South America, being common to

the Atlantic and Amazon Rainforests, are omnivorous animals with food plasticity, and the diet is composed of fruits

and invertebrates, as well as small mammals, fish and birds. Like any wild species, coatis may suffer from parasitic

infestations that cause disease or not. Worms of the Angiostrongylidae family infest the airways of various domestic

and wild species. The objective of this work is to report the occurrence of natural pulmonary infestation by

Angiostrongylus spp in coatis.

Case report: Tissue fragments of the coati (N. nasua) was collected from the Bela Vista Sanctuary, located in the city

of Foz de Iguaçu, Paraná. These tissues were maintained in 10% buffered formalin solution and were cleaved for

routine histopathological diagnosis at the Laboratory of Animal Pathology, UEL. Histopathological evaluation revealed

parasitic pneumonia characterized by the marked multifocal presence of adult nematode parasites in the lumen of

bronchi and alveoli. Associated with the parasitic infestation, there was marked mixed inflammatory infiltrate

composed of lymphocytes, histiocytes and plasma cells in the alveolar septa. In addition, there were foci of smooth

muscle hyperplasia of the pulmonary arteries. The intralesional parasites in the bronchi are compatible with

nematodes of the genus Angiostrongylus belonging to the family Angiostrongylidae and superfamily

Metastrongyloidea, commonly known as lung worms. The histopathological lesions found in the lung of this coati are

consistent with those described in the literature in cases of natural infestation by Angiostrongylus spp in domestic

carnivores. These parasites naturally infect many species, including procyonids, birds, marsupials, and humans.

Many species of parasites coexist in harmony with the wild host without causing serious illness. However, in possibly

debilitated or immunosuppressed animals, infestation can lead to death. There are 21 species of Angiostrongylus

currently known in wild animals, these parasites are differentiated due to the location of predilection to complete the

biological cycle. Larvae can be found in the pulmonary arteries, right ventricle, mesenteric artery, encephalon, or

pulmonary bronchi and bronchioles. All species of Angiostrongylus depend on the passage through the

gastrointestinal tract to follow in the cycle and use slugs and snails as intermediate hosts. Additionally, this coati

presented granulomatous nephritis, lymphohistiocytic cholangiohepatitis, lymphohistiocytic enteritis and

lymphohistiocytic cystitis.

Final consideration: The most causa mortis in this case was related to severe respiratory failure caused by parasitic

pneumonia. The microscopic lesions observed in the kidneys, liver, intestines and urinary vesicles are suggestive of a

viral infection that would justify immunosuppression and predisposition to excessive proliferation of pulmonary worms.

Further studies of parasitic morphology and/or molecular examinations are required to confirm the microscopic

diagnosis and definitive identification of the nematode species involved in this case.

Grants: MEC, CNPq.


PARTICIPATION OF IL-1Β AND IL-33/ST2 SIGNALING IN EHRLICH TUMOR-INDUCED PAIN IN MICE

Talita P. Domiciano1*

, Ana C. Zarpelon2, Larissa Staurengo-Ferrari

1, Cássia Calixto-Campos

1, Victor Fattori

1,

Sergio Borghi1, José C. Alves-Filho

3, Fernando Q. Cunha

3, Thiago M. Cunha

3, Waldiceu A. Verri Jr

1

1 Universidade Estadual de Londrina, Department of Pathology, Londrina, PR, Brazil;

2 Universidade Federal do

Paraná, Campus Toledo; PR, Brazil; 3 Faculdade de Medicina de Ribeirão Preto – FMRP/USP, Ribeirão Preto,

SP

*[email protected]

Keywords: Pain, Cancer, Ehrlich tumor, IL-1β and IL-33/ST2.

Introduction and objectives: Signaling related to IL-1β/IL-1R and IL-33/ST2 are well-known pathways related to

inflammation and pain. However, the understanding about its roles in cancer-induced pain and inflammation needs to

be further elucidated. Thus, the present study aims to evaluate the role IL-1β, and IL-33/ST2 in Ehrlich tumor- induced

pain model in mice.

Material and methods: (CEUA #14543.2013.03). Male IL-1R-/-

and ST2-/-

mice together with their backgrounds

(C57BL/6 and BALB/c mice, respectively) were used in this study. Mice were inoculated with 1x106 cells/25μl of

Ehrlich tumor cell suspension the right hind paw. IL-1β protein levels were determined in paw tissue, tumor mass and

spinal cord (ELISA) and its mRNA expression together protein levels also in spinal cord (ELISA and western blot,

respectively). IL-33 mRNA expression and protein levels were also evaluated in spinal cord (RT-qPCR and ELISA,

respectively). IL-1R-/-

, ST2-/-

or respectively wild type mice were used for the evaluation of mechanical and thermal

hyperalgesia (digital analgesimeter and hot plate test respectively) and paw edema (caliper) prior to inoculation and

after every 2 days, until the day 12. All data were analyzed using Prism 5.0 statistical program (GraphPad software,

Inc.). We used one-way and two-way ANOVA with Tukey’s post hoc test for analysis with three or greater groups.

A p value equal or less than 0.05 was considered statistically significant.

Results: Initially, it was investigated the temporal profile of IL-1β protein levels and we observed significant increases

in paw tissue and tumor mass samples from 8 and 2 days respectively, which remains until the day 12 after tumor

inoculation. By your turn, spinal cord IL-1β mRNA expression increased in the 12th post inoculation, which was

confirmed at protein level by western blot analysis. Moreover, tumor inoculation induces mechanical and thermal

hyperalgesia, which were reverted by IL-1R deficient mice from 2-12 and 8 days, respectively. However, paw edema

was not affected in IL-1R-/-

mice. Additionally, we also detected increased mRNA expression and protein levels of IL-

33 in spinal cord samples in day 12 after tumor inoculation. The participation of IL-33/ST2 signaling in Ehrlich tumor-

induced pain was also evaluated using ST2 deficient mice. Mechanical and thermal hyperalgesia were significantly

inhibited in ST2-/-

mice in 6, 10 and 12 and 10 days post-tumor inoculation. Finally, ST2 deficiency does not affect paw

edema.

Conclusion: Thus, our study shows the participation of IL-1β, and IL-33/ST2 in the Ehrlich tumor induced pain model

providing a possible therapeutic target for cancer induced pain treatments.

Grants: Fundação Araucária (FA) – Apoio ao Desenvolvimento Científico e Tecnológico do Paraná and Conselho

Nacional de Desenvolvimento Científico e Tecnológico (CNPq).


PESTICIDE AND BREAST CANCER

Bufalo, A. C.1; Candiotto, L. Z.P

2; Rech, D.

2, 3; Panis, C.

2; Pavanelli, W. R.

1


2Universidade Estadual do Oeste do Paraná, Health Sciences Center, Francisco Beltrão, PR, Brazil

3Centro de Oncologia (CEONC), Francisco Beltrão, PR, Brazil

[email protected]

Key words: pesticide, breast cancer, endocrine disrupters.

Introduction: Breast cancer is the most malignant neoplasm of women in the world and several factors affect the

development and prognosis of this disease. Studies indicate that pesticides present in the environment are capable of

generating important endocrine changes, including cortisol synthesis, one of the main hormonal regulatory axes of the

immune response. Considering the importance of immune response as determinant of outcome in breast cancer, it is

necessary to understand in what way the continued exposure to pesticides would be able to deregulate the cortisol

secretion the point of modifying the immune response, and consequently to affect the developmentof this pathology.

Review: Brazil is one of largest consumers of pesticides in the world, being that the South and Southwest region

consuming around 70% of the pesticides sold in the country. It is estimated that in developing countries, such as

Brazil, approximately 25 million workers per year are contaminated by pesticides. The generated effects can be acute

which promote cases of intoxication that can be fatal or chronic that cause disturbances in different systems of the

human organism. In this sense, several studies describe the pesticides as endocrine disrupters, that is, they change

from the synthesis to the elimination of several hormones. Much of this work is closely related to changes in the

human reproductive system, however, some studies have highlighted its role in the cortisol axis as well. The

glucocorticoid receptors are targets of some pesticides, which act as antagonists or agonists, altering the action of

cortisol and, consequently, its effect on several systems. Occurs that the hypothalamic-pituitary-adrenal axis acts as a

cascade through which a small concentration of corticotropin secreted by the hypothalamus results in increased

secretion of adrenocorticotrophic hormone, leading to release by the adrenal gland of cortisol. This substance has a

potential deregulatory effect on the immune system, which can lead to immunosuppression for example by altering the

production of cytokines and chemokines which can lead to a higher incidence of cancer. Thus, the cumulative effects

can alter cortisol secretion and promote disturbance of the immune system. However, this relationship in the context

of pesticides has not yet been fully elucidated in humans. Some works with on occupational exposure of women to

pesticides show that there is a greater risk of malignant or pre-malignant findings, as well as increased risk of

precursor lesions. Although a vast dataset demonstrates the ability of immune system to recognize and control tumor

growth, chronic exposure to endocrine disrupters seems to intensify the production of pro-inflammatory factors, which

facilitates the growth of tumor cells. Exposure to pyrethroid compounds, for example, reduces host defenses, which

increases the chances of non-recognition of neoplastic cells.

Conclusion: Thus, more studies on the relationship of pesticides as potential deregulators of immunoendocrine

pathways in cancer may contribute significantly to the understanding of severe clinical conditions observed in patients

with neoplasia.

Grants: PPSUS - Fundação Araucária e Ministérios da Saúde; UNIOESTE; UEL.



PRENATAL INJECTIONS OF LIPOPOLYSACCHARIDE, PERINATAL ANOXIA AND SENSORIMOTOR

RESTRICTION: IMPLICATIONS FOR A MODEL OF CEREBRAL PALSY-LIKE

Santos, A. S.1; Almeida, W.

2; Sbardelotto, B. M.

3; Muraro, E. N.

3; Rocha, L. C.

1; Souza, M. A.

4; Centenaro, L. A.

5. 1 Universidade Estadual do Oeste do Paraná-UNIOESTE, Centro de Ciências Biológicas e Saúde, Cascavel, PR,

Brazil 2 Universidade Federal do Rio Grande do Sul, Instituto de Ciências Básicas e Saúde, Porto Alegre, RS, Brazil


4 Universidade Federal do Paraná, Toledo, PR, Brazil



Keywords: Animal Model, Gait Analysis, Spinal Cord, White Matter

Introduction: The term Cerebral Palsy (CP) refers not to a specific disease, but to a group of conditions with variable

severity, which has certain developmental features in common (GRAHAM et al., 2016). The development of an

experimental model of CP with a phenotype similar to that observed in humans is still a challenge. Thus, the objective

of this study was to evaluate the motor performance and the white matter of spinal cord of rats submitted to a CP

model based on prenatal injections of lipopolysaccharide (LPS), perinatal anoxia and sensorimotor restriction.

Material and methods: The present study was approved by the Ethics Committee of the Universidade Estadual do

Oeste do Paraná, (Nr. 24/16). Pregnant Wistar female rats received injections of vehicle (sterile saline) or LPS from

the 17th gestational day until the end of gestation. On the day of birth, the offspring of mothers injected with LPS were

submitted to asphyxia. The same animals had their hindlimbs movements restricted for 16 hours/day, during 30 days.

Then, the male pups were divided into two experimental groups: Control Group (CTG, n=6) − offspring of rats injected

with saline during pregnancy; and Cerebral Palsy Group (CPG, n=6) − offspring of rats injected with LPS during

pregnancy, submitted to perinatal asphyxia and sensorimotor restriction. At 29 and 45 days of age, the animals

performed motor tests. After this, rats were euthanized and spinal cord samples were dissected, sectioned and

stained for the white matter area analysis.

Results: On the 29th postnatal day there was a reduction in the base of support in the CPG group (3.63 ± 0.13) in

relation to the CTG (4.47 ± 0.26) (p = 0.009). On the 45th postnatal day, a reduction in the stride length of the CPG

group was observed in comparison to the CTG (10.70 ± 0.44 and 12.63 ± 0.65, respectively, p = 0.0007). Regarding

the analysis of the spinal cord white matter, there was no difference between the CPG and CTG groups (60.61 ±

38.79 and 56.81 ± 38.64 respectively, p = 0.509).

Conclusion: The CP model proposed in this study produced only subtle changes in the gait of the animals. This fact

can be related to the early removal of the sensorimotor restriction (15 days prior to euthanasia), which may have

allowed the partial recovery of motor skills from CPG animals. Despite changes in the white matter of the spinal cord

at the lumbar level were not observed, prenatal injections of LPS, perinatal asphyxia and the abnormal somatosensory

entries caused by the sensorimotor restriction may have contributed to the development of the motor disabilities

observed (PAPADELIS et al., 2014).

Grants: The authors would like to thank the Fundação Araucária de Apoio ao Desenvolvimento Científico e

Tecnológico do Paraná (Brazil) by granting the scholarship that supported this study.


PRE-NEOPLASTIC AND NEOPLASTIC HISTOPATHOLOGICAL LESIONS OBSERVED IN GROSSLY

NORMAL MAMMARY GLANDS OF ADULT FEMALE DOGS

Oliveira, T. E. S.1; Headley, S. A.

1, Martins, M. I. M.

2; Ferreira, E.

3, Cassali, G. D.

3, Di Santis, G.W.

1

1Universidade Estadual de Londrina, Department of Preventive Veterinary Medicine, Londrina, PR, Brazil

2Universidade Estadual de Londrina, Department of Veterinary Clinic, Londrina, PR, Brazil

3Universidade Federal de Minas Gerais, Department of General Pathology, Belo Horizonte, MG, Brazil


Keywords: carcinoma, hyperplasia, atypia ductal, benign neoplasms, intraepithelial lesions.

Introduction and objectives: Many studies use female dogs as comparative models to study breast cancer in

women due to the high incidence and prevalence of spontaneous intraepithelial lesions, even in the absence of

tumors in the mammary glands of female dogs. The objective of this study was to search pre-neoplastic and

neoplastic lesions in mammary glands of female dogs that were grossly normal but with previous mammary neoplasia.

Material and methods: Fragments of mammary chain tissue from 33 adult female dogs with suspected mammary

neoplasia, diagnosed by cytological examination, submitted for routine unilateral radical mastectomy at the

Theriogenology Sector of Company Animals, Veterinary Teaching Hospital, Universidade Estadual de Londrina were

selected and used in this study. The entire mammary chain was fixed in 10% buffered formalin solution. All mammary

glands without visible and palpable elevations were sectioned immediately caudal to the nipple, composing the Group

1 (G1), and then routinely processed for histopathological evaluation with the Hematoxylin and Eosin stain. The

classification of mammary lesions was based on Brazilian consensus. G1 was divided into 3 subgroups - A)

hyperplasia; B) benign neoplasms; C) malignant neoplasms.

Results: Eighty mammary glands were collected without macroscopic alterations, resulting in 70 histopathological

diagnoses, since in 10 glands there was absence of the mammary tissue. Lesions characteristic of G1A consisted

hyperplasia (54%; 38/70), followed by G1B benign neoplasia (30%; 21/70), and G1C malignant neoplasia (16%;

11/70). In G1A the histopathological patterns diagnosed were: ductal hyperplasia (45%; 17/38), atypical ductal

hyperplasia (42%; 16/38), alterations of columnar cells (8%; 3/38), and adenoses (5%; 2/38). The pre-neoplastic and

neoplastic lesions of this study were frequently observed in the thoracic (cranial and caudal) and cranial abdominal

mammary glands (28/38; 74%). Lesions of G1B included: adenomyoepiteliomas (34%; 7/21), adenomas (24%; 5/21),

ductal papillomas (19%; 4/21), benign mixed tumors (9%; 2/21), myoepitheliomas (9%; 2/21), and basaloid adenoma

(5%; 1/21). While G1C lesions were: high grade ductal in situ carcinomas (45%; 5/11), low grade ductal in situ

carcinomas (18%; 2/11), carcinoma in mixed tumors with intratumoral stromal invasion (18%; 2/11), and in situ

micropapillary carcinomas (18%; 2/11).

Conclusion: Hyperplasias was the most frequent lesions observed in this study, evidencing that there is high

frequency of spontaneous intraepithelial lesions, even in the absence of tumors, in the mammary glands of female

dogs. The involvement of 11 mammary glands with malignant neoplasms supports the theory that malignant tumors

can develop from hyperplastic or benign lesions. The results of this study demonstrated that due to the hormonal

influence, there are proliferative lesions throughout the mammary chain, even if it is not grossly visible or palpable. In

addition, after nodulectomy of preexisting tumorous mass, new primary malignant tumors may occur in other

mammary glands, which will continue under hormonal influence and a more aggressive immunophenotype may occur.

Grants: CNPq.


PROFILE OF THE SYSTEMIC AND TUMOR MICROENVIRONMENT ANTIOXIDANT RESPONSE IN A

MURINE METASTATIC MELANOMA MODEL

Melo, G. P1; Sanches, L. J

1; Bernardes, S.P

2; Souza-Neto, F.P

1; Luiz, R. C

1; Cecchini, R

1; Cecchini, A. A

1


2Federal University of Grande Dourados, Dourados, MS, Brazil

[email protected]

Keywords: Melanoma 1, antioxidante 2, metastasis 3, B16F10 4, C57BL/6 5.

Introduction and objectives: Melanoma is the most serious of skin cancers due to its high invasive ability. Since

redox imbalance can modulate cellular behavior, influencing host response as well as tumor growth, therefore

knowing that melanoma is a cancer highly sensitive to these changes at the systemic level and in the tumor

microenvironment, it becomes important to know if there are changes in antioxidant levels during the installation and

progression of melanoma metastasis.

Material and methods: (nº CEUA 6738.2015.40). B16F10 cells were grown in DMEM 10% FBS and inoculated 105

viable cells in 50µl of serum-free DMEM in male C57BL/6 mice (8-12 weeks), intravenous through ophthalmic plexus.

Control group received 50 µL DMEM serum-free. The mice were submitted to euthanasia after 3, 7, 14 or 21 days,

and the groups named M3, M7, M14 and M21 respectively, with n=8. The blood was collected by cardiac puncture

and plasma and erythrocytes separated, the first one stored at -20°C and the second one in alsever buffer at 8°C. The

lung was removed and stored at -80ºC. Antioxidants analyses were performed in erythrocytes (Catalase activity and

GSH content), plasma (Total radical-trapping antioxidant parameter-TRAP) and lung (Catalase activity, GSH content

and TRAP). Groups were compared to control group by One-Way ANOVA following Tukey’s post-hoc testing.

Results: Plasmatic TRAP showed an increase in the M14 group (9.9x10-1

± 1.5x10-1

vs 1.8 ± 2.2x10-1

µM trolox,

control group and M14, respectively; p<0.05) and M21 (9.9x10-1

± 1.5x10-1

vs 1.9 ± 2.6x10-2

µM trolox, control group

and M14, respectively; p<0.05). The erythrocyte catalase enzyme activity was higher in the groups M7 (1.0x101

± 1.3

vs 2.6x101

± 3.4 abs/min/mL, control group and M7, respectively; p <0.001), M14 (1.0x10

1 ± 1.3

vs 3.2x10

1 ± 1.3

abs/min/mL, control group and M14, respectively <0.0001) and M21 (1.0x101

± 1.3 vs 2.4x10

1 ± 3.4

abs/min/mL,

control group and M17, respectively; p<0.01). The erythrocyte GSH content was increase in group 7 days (5.1x103

±

3.6x101 vs 8.5x10

3 ± 8.2x10

1 nM GSH/mg protein, control group and M7, respectively; p <0.01) and 14 days (5.1x10

3

± 3.6x101 vs 7.9x10

2 ± 4.7x10

1 nM GSH/mg protein, control group and M14, respectively; p <0.05). Pulmonary TRAP

was decreased at 21 days (1.1 ± 0.1 vs 0.4 ± 0.1 µM trolox, control group and M21, respectively; p <0.05) and GSH

was also decreased at 14 days (0.4±0.1 vs 0.2 ± 0.0 nM GSH/mg protein, control group and M14, respectively; p

<0.05) and 21 days (0.4 ± 0.1 vs 0.1 ± 0.0 nM GSH/mg protein, control group and M21, respectively; p<0.01) both

compared to the control group. Catalase showed no significant change in pulmonary activity.

Conclusion: We can observe that with the progression of melanoma metastasis, there is an increase of systemic

antioxidants and a decreased of antioxidants in the tumor microenvironment, suggesting that they play a strong role in

the success of the implantation of the tumor cell in the lung and its proliferation.


PTPRΖ/PHOSPHACAN IS EXPRESSED IN SENSORY AND MOTOR NERVES OF THE

GASTROINTESTINAL TRACT OF HUMANS AND MICE

Mendes, J. D. L.1; Bacarin, C. C.

1; Mali-Júnior, J

2; Miqueloto, C. A

3; Blackshaw, L. A.

4; Araújo, E. J. A

1 1Universidade Estadual de Londrina, Department of Histology, Londrina, PR, Brazil

2 Hospital do câncer de Londrina- Department of Gastroenterology, Londrina, PR, Brazil

3 Universidade Estadual de Londrina- Department of General Biology, Londrina, PR, Brazil

4 Queen Mary University of London/Wingate Institute for Neurogastroenterology, United Kingdom.


Keywords: Receptor-Like Protein Tyrosine Phosphatases - Class 5, RPTPδ/phosphacan, Gastrointestinal Tract,

Extracellular Matrix.

Introduction and objectives: RPTPδ/phosphacan is among the molecules of the extracellular matrix (ECM) which

plays role in cells of the nervous system. During the development of the central nervous system RPTPδ/phosphacan is

involved in the fasciculation, growth and myelination of axons. Little is known about the presence and function of

RPTPδ/phosphacan in structures that innervate the gastrointestinal tract (GIT). Recently it was reported that the

absence of a molecule of ECM (tenascin x) from sensory and motor neurons in the human GIT can cause

gastrointestinal disorders due to functional impairment of its innervation. Therefore, studies are necessary that

evaluate the involvement of ECM in gastrointestinal disorders. This study was conducted with the aim of assessing

gene expression and the location of RPTPδ/phosphacan in structures that innervate the GIT of humans and mice.

Material and methods: All procedures were approved by the Ethical Committee in Research Involving Humans at

State University of Londrina - UEL (1,073,326) and by the Ethical Committee for Use of Animals at UEL (032/2015),

Brazil. Samples of C57Bl/6 mice GIT were dissected to separate the wall into two parts: (1) mucosa+submucosa and

(2) muscle+serosa, which were stored in trizol. We extracted the total RNA from each part, which was converted to

cDNA for quantification. For morphological analysis, samples of GIT, sensory ganglia (Nodose and dorsal root ganglia

- DRG) and the spinal cord of mice and human GIT were fixed in 4% paraformaldehyde for 3 hours, which were frozen

later on OCT. GIT sections were submitted to immunofluorescence for marking enteric neurons (anti -PGP9.5),

cholinergic (anti-ChAT) and nitrergic (anti-nNOS) in double stain with RPTPδ/phosphacan (anti- RPTPδ/phosphacan).

We also evaluated the presence of RPTPδ/phosphacan in sensory neurons within sections of nodose ganglia

(RPTPδ/phosphacan /calretinin), DRG and spinal cord (RPTPδ/phosphacan /CGRP).

Results: The RTq-PCR demonstrated that RPTPδ/phosphacan is expressed weakly in the small intestine, while in

the stomach and large intestine the RPTPδ/phosphacan is expressed similarly in the mucosa+submucosa. In the

muscle+serosa, RPTPδ/phosphacan is more expressed in the stomach than in the large intestine. The

immunofluorescence results confirmed the immunolocalization of RPTPδ/phosphacan in the GIT of mice and humans.

RPTPδ/phosphacan was observed in myenteric and submucosal neurons, especially in cholinergic neurons. In

sensory ganglia, we observed that the presence of RPTPδ/phosphacan in part of the cell bodies of the nodose ganglia

in colocalization with calretinin. On the other hand, RPTPδ/phosphacan was not colocalized with CGRP in spinal

ganglia. RPTPδ/phosphacan was observed in association with the layer of myelin around thicker axons.

Conclusion: It is concluded that RPTPδ/phosphacan mRNA is expressed in the GIT of mice and is associated with

the structures of sensitive and motor nerves of the GIT of mice and humans. Further studies will reveal how this

receptor/proteoglycan is involved in gut function.

Grant: CAPES (process number 88881.068190/2014-01)


RESISTANCE EXERCISE TRAINING ATTENUATES CANCER-INDUCED MUSCLE ATROPHY AND

STRENGTH LOSS BY DECREASING SYSTEMIC INFLAMMATION AND PROTEOLYSIS SIGNALING IN

WALKER-256 TUMOR-BEARING RATS.

Padilha, C. S1, Marinello, P. C

1,2, Testa M. T. J

1, Cella P. S

1, Guirro, P. B

1, Iarosz, K. C

1, Voltarelli, F. A

3, Duarte J. A

4,

Cecchini, R5, Guarnier, F. A

6, Deminice, R

1.

1 Laboratory of Biochemistry of Exercise, State University of Londrina, Londrina, PR, Brazil. 2 Molecular Pathology

Laboratory, Department of Pathology Science, State University of Londrina, Londrina-PR, Brazil. 3 Graduate Program

of Health Sciences, Federal University of Mato Grosso, Cuiabá-MT, Brazil. 4 CIAFEL, Faculty of Sport, University of

Porto, Porto, Portugal. 5 Laboratory of Free Radicals and Pathophysiology State University of Londrina, Londrina-PR,

Brazil. 6 Laboratory of Pathophysiology of Skeletal Muscle Adaptations, State University of Londrina, Londrina, PR,

Brazil.

[email protected]

Key words: Exercise training, functional performance, inflammation.

Introduction and objectives: Cancer-induced cachexia represents a multifactorial metabolic disorder characterized

by a progressive loss of body weight, mainly resulting from loss of skeletal muscle and frequently adipose tissue, in a

short period of time. The aim of our study was to investigate the effects of resistance exercise training protocol (RT)

on attenuation of muscle atrophy, strength loss, systemic inflammatory markers and proteolysis signaling in Walker-

256 tumor-bearing Wistar rats.

Material and methods: Thirty-Seven Wistar rats were divided into 4 groups: control (C,n=9), tumor-bearing (T,n=9),

exercised (E,n=9), and tumor-bearing exercised (TE,n=10). Walker-256 tumor cells were implanted in the right flank.

Forty-eight hours after tumor implantation the animals started a four weeks-period of RT. The training intensity was

determined based on the maximal load test (ML) that the animal was able to carry climbing a ladder apparatus, for the

first time. The training intensity was gradually increased over time (1-2 week =70% ML test, 3-week =80% ML test,

and 4-week =85% ML test). RT protocol consisted of climbing a ladder apparatus with weights tied to the animal's tail

in E and TE groups, while the physical activity of C and T rats was confined to the space of the cage. All experimental

procedures were approved by the Animal Experimentation Ethics Committee of State University of Londrina CEUA n

10670.2013.46.

Results: The Walker-256 tumor cells caused a reduction body weight gain (-9.85%), muscle weight (-7.84%), and fat

content (-41%) besides increasing in cachexia index (15.48%). Tumor growth also promoted decreased muscle

strength (-11.09%), increased muscle atrophy (P75=-24%), systemic leukocytes (+150%), and expression levels of

Atrogin-1 (+204%) were elevated in T animals compared to C. In contrast, RT was able to attenuate body weight loss

(+6,35%), muscle atrophy (P75= -1.12%), and muscle strength loss (+67.54%) caused by tumor growth. Exercised

tumor-bearing animals also presented attenuated systemic inflammation (leukocytes count -31.5% and mRNA

Atrogin-1 levels -44.6%).

Conclusion: In conclusion, resistance exercise training attenuates cachexia development, muscle wasting and

muscle strength loss, by interfering on tumor-induced systemic inflammation and proteolysis signaling.

Grant: We would like to express thanks to Coordination of Improvement of Higher Education Personnel

(CAPES/Brazil Proc: Capes_PVE 88881.068035/2014-01) and the National Council of Technological and Scientific

Development (CNPq/Brazil Proc. 477114/2013-0).


RESISTANCE TRAINING AS PREVENTION AND TREATMENT OF CANCER CACHEXIA IN TWO

EXPERIMENTAL MODELS OF SOLID TUMORS

Testa, M. T. J.1, Padilha, C. S.

1, Marinello, P. C.

1,2, Cella, P. S.

1, Jordão, A. A.

3, Duarte, J. A.

4, Cecchini, R.

2,

Guarnier, F. A.2, Deminice, R.

1.

1 State University of Londrina, Department of Physical Education, Londrina-PR, Brazil.


3 University of São Paulo, FMRP, São Paulo - SP, Brazil.

4 University of Porto, CIAFEL, Faculty of Sport, Porto, Portugal.


Keywords: Resistance training, muscle mass loss, cancer.

Introduction and objectives: The cachexia induced by cancer is a metabolic disorder characterized by unintended

and progressive body weight loss. Studies show that weight loss occurs through unbalance between synthesis and

degradation protein. The exercise has been showing an important role in this regard in order to prevent, slow or

reverse cachexia induced by cancer. Resistance training (RT) is a potent stimulator of protein synthesis. For this

reason, its action on prevention, decrease or reversal of cancer cachexia should be investigated. Based on this, the

objective of this study was to evaluate the responses of RT on cancer cachexia development before and after tumor

cells inoculation.

Material and methods: Two different models of cancer-induced cachexia were used: 1) Male Wistar rats (250g ± 30;

n=44) were inoculated with Walker-256 tumor cells (11x107) and 2) Male Swiss mice (30g ± 2,8; n=40) were

inoculated with Ehrlich tumor cells (1x106). In both experiments, the animals were divided into 4 groups with the same

number of animals: Control (C), Tumor (T), Exercised (E) e Tumor-bearing exercised (TE). In the Walker-256 model,

the rats were inoculated 6 weeks after RT, so the preventive action of RT was investigated. The tumor and RT were

kept for 12 days after inoculation. In Ehrlich model, the mices were trained during four-weeks after tumor inoculation,

investigating the treatment effect of the exercise. RT protocol consisted in climbing a ladder with gradual loads tied to

the animal’s tail. C and T groups remained inactive during all experiment, in both cases. Plasma were collected for

ELISA, gastrocnemius muscle for H&E staining and malondialdehyde, advanced oxidized proteins products, reduced

and oxidized glutathione and mRNA expression, besides tumoral immunohistochemistry for Ki67. For differences

between groups, one-way ANOVA was used, followed by Tukey post-test or student t-test (Mean ± SD; P<0.05). All

experimental procedures were approved by the Animal Experimentation Ethics Committee of State University of

Londrina (CEUA nº 28336.2014.38).

Results: Walker and Ehrlich tumor grew progressively, reaching 9% and 15% (P<0.05) of body weight, respectively.

The tumor growth decreased fiber cross-sectional area (Walker -32% and Ehrlich -28%, P<0.05), body weight (Walker

-10% and Ehrlich -15%, P<0.05) and reduced retroperitoneal fat (Walker -41% and Ehrlich -61%, P<0.05). In addition,

we observed increased systemic inflammation, represented by TNF-α and IL-6 in T groups of both experimental

models. Leukocytosis, muscle oxidative stress and decreased mRNA of mTOR were also found in animals from the T

group (P <0.05), on Walker model. In Ehrlich model, RT was able to attenuate body weight reduction, muscle waste

and the increase in systemic inflammation, besides preventing muscle strenght loss. However, RT did not reverse the

signaling of muscle proteolysis through the expression of FBXO32 mRNA in the Walker model. Furthermore, the

analysis of the Ehrlich tumor microenvironment demonstrated reduction in cell proliferation and increase in the

necrotic area.

Conclusion: RT attenuated cachexia development and muscle wasting by decreasing tumor-induced systemic

inflammation when performed before and after tumor cells inoculation, although the phenomenae were presented in

two different experimental models.

Grants: Capes-PVE (process nº 88881.068035/2014-01)


Senecavirus A ENTERS THE CENTRAL NERVOUS SYSTEM VIA THE BLOOD-CEREBROSPINAL

FLUID BARRIER IN NATURALLY INFECTED PIGLETS

Oliveira, T. E. S.

1; Michelazzo, M. M. Z.

1; Fernandes, T.

2, De Oliveira, A. G.

2, Leme, R. A.

1, Alfieri, A. A.

1, Headley, S.

A.1

1Universidade Estadual de Londrina, Department of Preventive Veterinary Medicine, Londrina, PR, Brazil

2Universidade Estadual de Londrina, Department of Electronic Microscopy and Microanalysis, Londrina, PR, Brazil


Keywords: Epidemic transient neonatal losses, plexus choroid, meningoencephalitis, SVA, Picornavirus.

Introduction and objectives: Senecavirus A (SVA) family Picornaviridae, is associated a syndrome known as the

Epidemic Transient Neonatal Losses (ETNL), which is characterized by diarrhea, neurological signs and/or sudden

death in neonatal piglets from Brazil, North America, and China. Some members of the Picornaviridae family, such as

the Cardiovirus and Enterovirus, enter the brain through the blood-cerebrospinal fluid barrier and are responsible for

cases of non-suppurative meningitis and meningoencephalitis in neonates and young children. However, the

pathogenesis associated with SVA in piglets is poorly understood. This study investigated via immunohistochemistry

(IHC) and transmission electron microscopy (TEM) the possible method of entry of SVA into the brain in naturally

infected piglets.

Material and methods: Six piglets, between 3 to 7 days of age, from different farms in southern Brazil, that died

suddenly with neurological signs were received at the Laboratory of Animal Pathology, UEL. Necropsy was done soon

after death; all tissue fragments were fixed in 10% buffered formalin solution and then routinely processed for

histopathological evaluation. Duplicate fragments were processed for immunohistochemistry (IHC) using a monoclonal

antibody (1:50 dilution) with overnight incubation at 4ºC. Tissue fragments of the brain with the choroid plexus fixed in

70% alcohol were processed for TEM. This sample was washed in phosphate buffer, post-fixed in osmium tetroxide,

and then embedded in Araldite. Ultrathin sections were performed using Leica Ultracut UCT, transferred to 200 mesh

grids, contrasted and visualized in a FEI Tecnai G2 TEM.

Results: Histopathology in all piglets revealed cortical necrosis, ballooning degeneration and hyperplasia of the

urothelium of the renal pelvis, ureter and urinary bladder, and atrophy of enterocytes; non-suppurative

meningoencephalitis was diagnosed in three of these. IHC revealed strong immunostaining for SVA antigens in the

apical enterocytes of the small intestine, endothelium of capillaries and epithelial cells of the choroid plexus, oral

mucosa and uroepithelium of the renal pelvis and urinary bladder. The ultrathin evaluation demonstrated multifocal

balloon degeneration of the endothelium of capillaries within the choroid plexus, lymphohistiocytic inflammation and

focally extensive necrosis of to the endothelium of capillaries. TEM demonstrated negative-stained nonenveloped

intracytoplasmic virus (30 of 50nn in diameter) predominantly close to capillaries of the choroid plexus, necrosis of

epithelial cells of the choroid plexus, and monocytes and macrophages containing innumerable phagolysosomes.

These findings suggest that SVA enters the brain by degeneration and necrosis to the blood-cerebrospinal fluid barrier

with subsequent dissemination into the brain. The clinical manifestations are consequences of neurological

dysfunction.

Conclusion: The fixation in alcohol 70%, although it was not the ideal method, allowed the visualization of the SVA by

TEM in the choroid plexus. This virus probably enters the nervous system of piglets via the choroid plexus producing

disruption of the blood-cerebrospinal fluid barrier with subsequent encephalitic dissemination. This virus produces

balloon degeneration and necrosis to the endothelium of capillaries within the choroid plexus.

Grants: CNPq.


TACTILE AND KINESTHETIC STIMULATION REVERSES THE EFFECTS OF PRENATAL STRESS IN

THE CA3 REGION OF THE HIPPOCAMPUS IN RATS: STEREOLOGICAL STUDY.

Loyola, W. S.1; Osorio, D. M.

1; Caro, H. G.

1

1 Catholic University of Maule, Department of Kinesiology, Talca, Chile.

[email protected]

Keywords: Prenatal Stress, Hippocampus, Tactile Stimulation

Introduction and objective: The hippocampus is a brain structure with cognitive functions, which is sensible to the

perinatal environment conditions, such as prenatal stress. Prenatal stress causes an overactivation in the

hypothalamic pituitary adrenal axis (HPA), producing modifications in the hippocampal cytoarchitecture. Postnatal

tactile and kinesthetic stimulation decrease the cortisol level decreasing the overactivation of HPT axis. Currently,

there are few studies about the effects of tactile/kinesthetic stimulation in hippocampus of rats with prenatal stress.

The study aimed to analyze the effect of postnatal tactile and kinesthetic stimulation in the CA3 region of the

hippocampus, in offspring rats exposed to prenatal stress.

Materials and Methods: It is a stereological study with 16 offspring rats from 6 pregnant female rats of Sprague-

Dawley strain, distributed in three groups: Control Group (CG), the Prenatal Maternal Stress by restriction group

(PMS), and Perinatal Maternal Stress with postnatal tactile and kinesthetic stimulation Group (PMS-TKS). CG did not

receive intervention. PMS group were exposed to stress by movement restriction of the pregnant female rats.

Stressed pregnant rats were stressed for three times per day for 45 min with interval > 2 h from the 11th

to 21st

gestational day. The PMS-TKS group received the same stress as PMS and, additionally, postnatal tactile and

kinesthetic stimulation between the 3rd

to 12th day (3 times a day for 10 minutes each). All offspring rats were

sacrificed 22 days after birth. The brains were extracted and cut in the horizontal plane with a thickness of 100 µm.

Nissl staining was used for the neuronal density measurement in stereological counting. Microscopic images of

pyramidal cell were obtained from the left hippocampus (CA3a and CA3b) between Bregma -2,30 mm and Bregma -

3,8mm according to Paxinos mouse brain atlas. The software used for the statistical analysis was Statistical Package

for the Social Sciences (SPSS) version 19.0 (IBM Co., Armonk, NY, USA) GraphPad PRISM 5.00 (GraphPad

Software, Inc., San Diego, CA, USA). The analysis of variance and Tukey post hoc comparisons were used to find a

significant difference between groups. Values were represented as mean ± SD. The level of significance was set at

p< 0.05. All procedures employed during this study were approved by the Institutional Animals Care and Use

Committee at Catholic University of Maule (N° 20.380).

Results: The Perinatal Maternal Stress in female offspring rats increased neuronal density (105cel/mm

3) only in CA3b

area (CG: 30.3± 4.2; PMS: 38.8±2.5; p<0.05). In comparison with Prenatal Maternal Stress, rats which received early

tactile and kinesthetic stimulation showed a decreasing in neuronal density in CA3b (PMS: 38.8±2.5; PMS-TKS:

30,6±3.2; p<0.05) and CA3c (PMS: 40,7.8±5.9; PMS-TKS: 31±0.66; p<0.05).

Conclusions: Prenatal stress produces modifications in the hippocampus, increasing the neuronal density in CA3b

area. In addition, Postnatal tactile and kinesthetic stimulation was able to reverse the Prenatal Maternal Stress effects

by decreasing neuronal density in CA3b and CA3c hippocampal areas. Decreases in neuronal density have been

associated with increases in dendritic branching, important to memory and learning.



STUDY OF THE GENETIC POLYMORPHISM rs3087465 OF TGFB RECEPTOR II: POSSIBLE ROLE AS

A PROGNOSTIC MARKER FOR WILMS TUMOR

Fujita T.C.1, Ishibashi, C.M.

1, Ariza, C.B.

1, Sakaguchi A. Y., Motoori-Fernandes, C.Y.

1 Kishima M.O.

2, Losi-

Guembarovski R.1, Vitiello, G.A.F.

1, Oliveira C.E.C.

1, Amarante M.k.

1, Banin-Hirata B.

1, Pasquini, J.F.G

1, Rosa, M.H.

1,

Watanabe M.A.E.1

1 Department of Pathological Sciences, Biological Science Center, State University of Londrina, Londrina, Parana, Brazil.

2 Department of Pathology, Clinical Analysis and Toxicology, Health Sciences Center, State University of Londrina,

Londrina, Parana, Brazil, 3 Cancer Hospital of Londrina, Londrina, Parana, Brazil.

[email protected]

Keywords: Wilms Tumor, genetic polymorphism, TGFBRII.

Introduction and objectives: Wilms’ tumor (WT) or nephroblastoma is one of the most common pediatric malignant

diseases. However, the biologic behavior of WT in tumor microenvironment is dependent on stromal cells as well as

molecules that somehow act in the development of malignancy. Transforming Growth Factor beta 1 (TGFβ1), the

most abundant member of the TGFβ subfamily of growth factors, a cytokine, exert important functions during

embryogenesis and in diverse physiological and disease systems. The TGFBR2 gene provides instructions for making

a protein called transforming growth factor-beta (TGF-β) receptor type 2. This receptor/ligand complex phosphorylates

proteins, which then enter the nucleus and regulate the transcription of a subset of genes related to cell proliferation.

Because TGF-β receptor type 2 helps prevent cells from growing and dividing too rapidly or in an uncontrolled way, it

can suppress the formation of tumors.

Many genetic polymorphisms in this gene have been reported in association with cancer development. It has

been describing the association between rs3087465 A allele and increased TGFBR2 transcription activity. Studies

regarding WT and TGFBRII are absent in the literature, however, studies with other tumors have demonstrated

association of allele A of rs3087465 polymorphism with risk reduction to various neoplasms, such as gastric cancer.

The present study investigated the TGFβ receptor 2 (TGFBRII) rs3087465 (G-875A) polymorphism in the

case-control study.

Material and methods: The present study was approved by Institutional Human Research Ethics Committee of the

State University of Londrina, Paraná, Brazil (CEP/UEL 189/2013 – CAAE 17123113400005231) and a term of free

and informed consent was signed by parents of the children donors to biological material collection. Clinical,

histological and volumetric data were collected from the medical records. The samples consisted of 35 paraffin

embedded tissue from children patients from Laboratory of Pathology of University Hospital of State University of

Londrina, Parana State, Brazil, and 128 childhood blood samples free of neoplasia as control group. In the neoplasia-

free control group, the DNA from blood samples was extracted from peripheral white blood cells using extraction kit

Mini Spin (Biometrix, Curitiba, PR, Brazil), according to manufacturer’s instructions and in some cases the DNA was

obtained from buccal cells using a protocol based on the use of ammonium acetate to proteins elimination. Genetic

polymorphisms were analyzed by polymerase chain reaction (PCR) followed by Restriction Fragment Length

Polymorphism (RFLP) analysis.

Results: No association for the rs3087465 polymorphism was verified. Control group presented GG (32,8%), GA

(57%), AA (10,2%) and patients GG(40%), GA (16%) and AA (14,3%). However it was verified that allele A carrier

presented protection for lymph node commitment (p<0.022).

Conclusion: Although more studies are needed to clarify the role of TGFβRII due to the lack of studies of this

cytokine in WT, we could speculate that the TGFβII promoter polymorphism in some way may be associated with

prognosis in WT.

Grants: CAPES, Fundação Araucária, CNPq


IMMUNOLOGY 157

ANALYSIS OF FOXP3 TRANSCRIPTION FACTOR rs2232365 POLYMORPHISM IN HPV INFECTION:

A CASE-CONTROL STUDY

Ferreira, R. S.1; Santos, F. C

1; Cebinelli, G.C .M.

1; Trugilo, K. P.

1; Okuyama, N. C. M.

1; Sena, M. M.

1; Pereira, A. P.

L. 1; Aranome, A. M. F.

1; Pereira, E. R.

1; Maria, G. C. Q.

1; Esposito, A.

1; Nishimura, A. M.

1; Mangieri, L. F. L.

1;

Oliveira, K. B. 1

1Universidade Estadual de Londrina, Department of Pathological Sciences, Londrina, PR, Brazil

[email protected]

Keywords: Cervical cancer, Immunogenetics, Polymerase Chain Reaction, SNP.

Introduction and objectives: Cervical cancer is the fourth cause of death in women worldwide, affecting more than

520,000 yearly and killing over 250,000. Infection by certain Human Papillomavirus (HPV) types is known to be the

main cause of cervical carcinoma (CC) and cervical intraepithelial neoplasia (CIN). The immune response has an

important role in the resolution of HPV infection. Some infections may not be eliminated and persist for many years. In

this context, regulatory T cells (Treg), due to their immunosuppressive role, are related to unfavorable prognosis in

HPV-related cancers. The FOXP3 transcription factor plays an important role in Treg cells development and is

involved in its function and regulation. The presence of SNP in the intronic region of this gene could alter expression

of FOXP3 protein, influencing in Treg cells function and cancer progression. Given the CC epidemiologic importance,

and the relation between FOXP3 expression and HPV oncogenic process, this study aimed to evaluate the presence

of the virus in women treated in CC prevention programs in the Public Health System (SUS) of the northern region of

Paraná state and to determine the frequency of the rs2232365 polymorphism of the FOXP3 gene in these women,

associating the genotypes with HPV infection.

Material and methods: Research Ethics Committee Involving Human Beings - State University of Londrina (nº133 /

2012, CAAE 05505912.0.0000.5231) approved this study, which is in accordance to CNS resolution 196/96. Those

patients who voluntarily agreed to participate read and signed the Term of Free and Informed Consent (TCLE). HPV

was detected by Polymerase Chain Reaction (PCR) in cervical cytobrushes samples and the polymorphism was

assessed through restriction fragment length polymorphism analysis (PCR-RFLP) in peripheral blood samples.

Statistical analyzes were performed using SPSS Statistics 22.0 (SPSS inc., Chicago, Illinois, USA). For all data, the

significance level adopted was p <0.05.

Results: In this study, of the 426 women investigated, HPV was detected in 48.8% (n = 208) of the patients and

51.2% (n=218) had a negative result in the test, being allocated in the control group. Regarding the genotypic

distribution of the rs2232365 polymorphism among HPV-positive women, 17.5% of the women presented a GG

genotype, 67.77% AG and 15.3% AA, while among the controls, 17% had the GG genotype, 63.1% AG and 19.9%

AA.

We used bivariate analysis to evaluate the association between the genotypes of FOXP3 rs2232365 polymorphism

and HPV infection. We found no association of the mutated allele (AG) presence in heterozygosity and HPV infection

when the AA genotype was used as reference (OR = 0.723 95% CI: 0.391-1.325; p = 0.296). When the mutated allele

in homozygosity was tested (GG), there was also no association with HPV infection (OR = 0.75 95% CI: 0.347-1.592;

p = 0.459); the same occurred when the genotypes containing the mutated allele were combined (AG+GG) (OR =

1.371 95% CI: 0.757-2.484; p = 0.373).

Conclusion: No significant association was found between FOXP3 rs2232365 polymorphism and HPV infection in

this study. Comprehensive studies in randomized populations are required to support these findings.

IMMUNOLOGY 158

APPLICATIONS OF IGY ANTIBODIES TO INFLUENZA A VIRUS DETECTION IN INFECTED CELLS BY

IMMUNOCYTOCHEMISTRY

Silva, M. C.1; Schaefer, R.

2; Gava, D.

2; Souza, C. K.

3; Venancio, E. J.

1 1Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil

2Embrapa Suínos e Aves , Concórdia, SC, Brazil

3Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil


Keywords: IgY, influenza, nucleoprotein, immunocytochemistry.

Introduction and objectives: Influenza is a disease caused by infection of the host respiratory tract by influenza type

A virus that affects a wide variety of species, including human, swine, equine, poultry and sea mammals. The viral

RNA is encapsidated by the nucleoprotein (NP), which is a highly conserved protein among influenza A viruses.

Detection of influenza virus can be accomplished by assessing specific immune response to the virus, by direct

detection of viral RNA or virus antigen in infected tissues. Some diagnostic tests use antibodies produced in mammals

to influenza virus detection. However, due to high cost and concerns related to animal welfare, alternative production

of antibodies in mammals has been described. The immunoglobulin Y (IgY) is a class of antibodies in poultry that is

transferred to the egg yolk. These antibodies are easy to obtain from eggs and their use has been described for

immunotherapy, immunodiagnosis and research. The objective of this study was to produce IgY antibodies anti -NP in

laying hens immunized with influenza A virus NP, and to evaluate the purified IgY antibodies in influenza virus

detection.

Material and methods: NP protein was supplied by EMBRAPA. For its production was performed by the cloning of

NP gene (from virus isolate A/swine/Brazil/12A/2010, H1N1) in pET23d vector and expression in E.coli BL21. Three

laying hens of White Leghorn line were immunized with 20 ug of NP via intramuscular. Booster doses were

administered on days 14, 28, 42, 84, 126 and 168 of the experiment. Seven days after the seventh dose of the

antigen, the produced eggs were collected and IgY antibodies were extracted from yolk by precipitation with

ammonium sulfate. Antibody levels were determined by ELISA assay. The specificity of anti-NP IgY was verified by

Western blotting. For immunocytochemistry, Madin-Darby Canine Kidney cells (MDCK) were infected with influenza A

virus. After 48 hours, the cells were fixed and washed with wash solution. Following, the monolayer cells were

incubated with either primary antibody, IgY or monoclonal antibody IgG (1:400). After incubation and washing steps,

conjugated antibody anti-IgY or anti-IgG was added to the cells. After the washing step the substrate was added,

incubated for 10 minutes and then the plate was washed with water. The cells monolayer was observed under a light

microscope.

Results: The produced IgY was analyzed by ELISA assay and the results showed that the laying hens immunized

with NP protein produced high levels of antibodies when compared to the non-immunized poultry. It was also

observed by Western blotting that anti-NP IgY antibodies was specific to NP protein of influenza A virus. Furthermore,

MDCK cells infected with influenza A virus showed positive labeling when incubated with either monoclonal antibody

IgG or IgY antibody anti-NP in immunocytochemistry test.

Conclusion: IgY antibodies specific to NP protein of influenza virus were obtained from egg yolk from laying hens

immunized with the NP protein. These antibodies were able to label influenza A virus infected cells, suggesting that

anti-NP IgY can be used in the diagnosis of influenza A virus.

Grants: CAPES and EMBRAPA project no. 02.11.01.006.00

IMMUNOLOGY 159

CXCL12 RS1801157 POLYMORPHISM CONTRIBUTION TO HPV INFECTION AND CERVICAL

LESIONS DEVELOPMENT.

Okuyama, N. C. M.1; Pereira, E. R.

1; Dos Santos, F. C.

1; Trugilo, K. P.

1; Sena, M. M.

1; Pereira, A. P. L.

1; Aranome, A.

M. F.1; Esposito, A.

1; De Oliveira, K. B.

1

1Universidade Estadual de Londrina, Department of Pathological Science, Londrina, Brazil


Keywords: CXCL12, polymorphism, HPV, cervical lesion

Introduction and objectives: Human papillomavirus (HPV) is the most common sexually transmitted virus in women

worldwide. The persistence of the virus may cause warts that are considered benign lesions, and low- or high-grade

squamous intraepithelial lesions (LSIL/HSIL). Immunological system plays an important role in the infections’

resolution. In this context, we highlight the chemokines, which are important regulators in the development of viral

infections and inflammation. Among which CXCL12 stands out, due to its pro-inflammatory features, acting as

chemoattractant and recruiting immune cells. Several polymorphisms were identified in CXCL12 gene including

rs1801157 in the 3’-untranslated region, which is characterized by a substitution of a guanine for an adenine. We

assessed the influence of this polymorphism in HPV infection and lesion development.

Material and methods: Institutional Ethics Committee Involving Humans at State University of Londrina, Londrina –

Paraná (PR), Brazil (CEP/UEL 133/2012; CAAE 05505912.0.0000.5231), approved this study. The study purpose and

procedures were explained to all patients and written informed consent was obtained. Polymerase Chain Reaction

(PCR) detected HPV DNA and the polymorphism was assessed through restriction fragment length polymorphism

analysis. ᵡ2 test was performed to estimate association between HPV presence and sociodemographic, reproductive

and sexual features. Binary logistic regression model adjusted for confounding factors was performed to establish the

association between HPV presence and CXCL12 polymorphism. A p value <0.05 was considered statistically

significant.

Results: In the present study, 364 women were included and categorized as HPV non-infected patients (195) and as

HPV-infected (169). HPV infection was more incident in women under 24 years old (p<0.001), single (p=0.002),

tobacco users (p<0.001), who had more than 4 sexual partners during lifetime (p=0.007), among those who presented

lower number of pregnancies (p=0.017). HPV was more frequent among patients genotype GA [OR=3.98; CI95%

(2.131-7.422), p<0.001], AA [OR=54.194; CI95% (10.779-272.471), p<0.001] and in allele A carriers [ORADJ=4.985;

CI95% (2.85-8.58), p<0.001], as observed by logistic regression analysis adjusted for several confounding factors. An

association between allele A carriers and HSIL development (p=0.003) was also observed.

Conclusion: Nonetheless, further analyses are necessary to elucidate the mechanism by which the polymorphism

may be responsible for HPV infection, its influence on chemokine expression in cervical microenvironment and its role

in lesions development. In the present study, we demonstrated that CXCL12 rs1801157 is independently associated

with HPV infection and exerts influence over HSIL development, suggesting it as a promising susceptibility biomarker

for HPV infection and lesions development

IMMUNOLOGY 160

DENGUE VACCINE AND THE IMMUNE RESPONSE

Sahd, C. S.1; Costa Júnior, W. L.

1; Concato, V. M.

1; Mendes, L. A. H.

1;Pavanelli, W. R.

1.

1Universidade Estadual de Londrina, Department of Experimental Pathology, Londrina, PR, Brazil


Key words: Dengue; dengue vaccine; vaccine immunogenicity; Sanofi Pasteur; Butantan.

Introduction: Dengue is an arboviral disease caused by 4 different viral serotypes in which all have the capacity to

cause serious infections. It may be asymptomatic or present a broad clinical spectrum, ranging from febrile

autoimmune disease to severe forms, which may evolve with circulatory shock and death. Since the last two decades

Brazil has become the country with the largest number of dengue cases in the world, representing 78% of reported

cases in the Americas and 61% of cases reported to the World Health Organization in that period. The availability of a

vaccine to control the spread of four virus serotypes and to reduce the severe forms. After years of research, the first

vaccine for mass use, produced by the Sanofi-Pasteur Laboratory, was approved, which proved effective in reducing

contamination by the dengue virus and mainly, reducing the occurrence of severe cases. Also in the final phase of

clinical study is a vaccine of different technology produced by the partnership between the Butantan Institute and the

National Institute of Health of the United States. A live attenuated virus vaccine developed by the Sanofi Pasteur

laboratory was launched in 2016 to be used in areas endemic for dengue fever, which has shown efficacy in reducing

the spread of dengue and especially the complications of dengue. In Brazil, another attenuated vaccine with different

composition is being developed by the Butantan Institute.

Review: In 2016 , a study using patient samples from a hyperendemic region in Sri Lanka were performed

demonstrating that a inadequate cellular response was associated with increased susceptibility to the disease and a

strong, multifunctional CD8 response was associated with protection against severe forms of the disease. Thus, it is

concluded that there are still few results regarding the effects of vaccines against dengue and so further studies

should be devoted to research on the benefits and harms of vaccines against dengue.

IMMUNOLOGY 161

EFFECT OF GREEN TEA (EGCG) ON THE CXCR4 EXPRESSION IN PERIPHERAL BLOOD

MONONUCLEAR CELLS

Sakaguchi, A. Y,1; Amarante, M. K.


1; Pereira, N. S.

1; Motoori-Fernandes, C. Y.

1; Vitiello, G. A. F.

1;

Pasquini, J. F. G.1; Rosa, M. H.

1; Ariza, C. B.

2; Ishibashi, C. M.

1; Banin-Hirata, B. K.

1; Petenuci, D. L.

1; Fujita, T. C.

1;

Castro, V. D.1; Watanabe, M. A. E.

1 1 Londrina State University, Laboratory of DNA Polymorphisms and Immunology, Department of Pathological

Sciences, Biological Sciences Center, Londrina, Parana, Brazil. 2 Philadelphia University Center, Londrina, Parana, Brazil.


Keywords: CXCR4 expression, catechin, epigallocatechin 3 gallate, flow cytometry

Introduction and objectives: Epigallocatechin 3 gallate (EGCG) is a tea polyphenol flavonoid, abounding in green

tea, and displays several beneficial health properties. Previous studies have linked EGCG with immunomodulatory

functions, particularly mediating expression of cytokines and chemokines in homeostasis and pathological conditions.

EGCG appears to have an immunosuppressive effect on the proliferation of peripheral blood mononuclear cells

(PBMC), indicating that EGCG is worth exploring for immunomodulatory effects in diseases and tissue transplantation.

In this regard, CXCR4 is a seven-span transmembrane G-coupled protein receptor that binds exclusively to the

chemokine CXCL12. In the immune system, CXCR4 is highly expressed by monocytes, B cells, and naive T cells in

peripheral blood, as well as early hematopoietic progenitor cells in bone marrow. This chemokine receptor has several

important functions in addition to inducing leukocyte chemotaxis. CXC receptor 4 (CXCR4) plays a key role in

migration and homing of hematopoietic progenitor stem cells to the docking sites, and natural compounds were shown

to modulate its expression. In this study, human PBMC were used to investigate the effects of EGCG on CXCR4

expression.

Material and methods: The Human Ethics Committee of the Londrina State University approved the present study

(CAEE 54639716.8.0000.523100), and voluntary written consent was obtained from each of the patients enrolled.

Peripheral blood in EDTA was obtained from healthy volunteers and PBMC were separated by Ficoll-Hypaque 1077

and maintained in culture in the presence or absence of EGCG for 24 h (0, 20, 50 and 100 ug/uL). After 24, 48 and 72

h, cell culture was submitted to different protocols. Lactate dehydrogenase (LDH) assay was used for cytotoxicity

effect. Flow cytometry and mRNA expression assays were performed in cultured PBMCs from blood donors.

Results: For cytotoxity assay, it was used the supernatant of PBMC cultures without and with EGCG in different

concentrations (20ug/mL, 50ug/mL and 100ug/mL). No significant differences were observed in LDH activity in

cultured PBMC supernatant at different concentrations (p>0.05), indicating that EGCG does not present toxicity or lytic

activity in PBMC. The CXCR4 mRNA expression was compared in PBMC treated with EGCG. An increased of

CXCR4 expression was verified at 2.4 and 1.8-fold for 100ug EGCG treatment group, after 24 and 48h, respectively.

There was no significant in CXCR4 expression after 72h (p>0.05). The CXCR4 surface expression increases with

higher EGCG concentrations (100ug/ml) after 24h (p<0,05); however CXCR4 surface expression did not alter with

different EGCG concentrations after 48 and 72h.

Conclusion: These results showed that EGCG treatment of human PBMC may promote CXCR4 expression, dose

and time dependent, which modifies cell behavior, depending on the biological system. Hopefully, this or similar

approaches will be further exploited to unravel the potential of EGCG for cell mobilization.

Grants: CAPES, Fundação Araucária, CNPq

IMMUNOLOGY 162

E-SELECTIN IS A LINK BETWEEN METABOLIC SYNDROME AND DISEASE ACTIVITY IN

RHEUMATOID ARTHRITIS

Amorim, A. H. C. G.1; Santos, L. F. R. F.

2; Stadlober, N. P.

2; Cossentini, L. A.

2; Robles, B. E. F.

2; Iriyoda, T. V. M.

3;

Costa, N. T.1; Medeiros, F. A.

4; Sá, M. C.

4; Micheletti, P. L. M.

5; Alfieri, D. F.

5; Flauzino, T.


6;

Reiche, E. M. V.6; Simão, A. N. C. S.

6; Dichi, I.

7.

1. Department of Rheumatology, University of Londrina (UEL), Londrina, PR, Brazil

2. Pos-Graduate Program in Experimental Pathology, University of Londrina, PR, Brazil.

3. Department of Rheumatology, Pontifical Catholic University of Paraná (PUC/PR), Londrina, PR, Brazil.

4. Pos-Graduate Program in Clinical and Laboratory Pathophysiology, University of Londrina, PR, Brazil.

5. Pos-Graduate Program in Health Sciences, University of Londrina, PR, Brazil.

6. Department of Pathology, Clinical Analysis and Toxicology – University of Londrina, Brazil.

7. Department of Internal Medicine – University of Londrina, Brazil.

[email protected]

Keywords: Rheumatoid arthritis, inflammation, biomarkers.

Introduction and objectives: Rheumatoid arthritis (RA) is a chronic inflammatory and systemic autoimmune disease.

Tumor necrosis factor alfa (TNF-α) plays crucial role in joint and end organ damage, increases adhesion molecules,

and promotes endothelial dysfunction in RA patients. TNF-α is also considered as one of the factors responsible for

favoring insulin resistance and dyslipidemia, which are important features of the Metabolic Syndrome (MetS). The

main objective of the present study was to evaluate the influence of MetS on the inflammatory process and disease

activity in RA patients.

Material and methods: 172 RA patients, of both sexes, aged from 18 to 69 years, were enrolled and divided into two

groups: the first group with MetS (RA MetS+, n= 57) and the second group without MetS (RA MetS−, n= 115).

Disease activity was determined by DAS28 (Disease Activity Score 28) score. Adhesion molecules, Plasminogen

Activator Inhibitor Type-1 (PAI-1), TNF-α and TNF-α receptors 1 (TNFR1) and 2 (TNFR2) were evaluated by Luminex

Plataform. The study protocol was fully approved by the Ethical Committee of the University of Londrina (Paraná,

Brazil) (CAAE: 06405812.1.0000.5231)

Results: E-Selectin (p=0.015) and PAI-1 (p=0.002) levels were significantly higher in RA patients with MetS than in

RA patients without MetS. TNFR1 (p=0.014) was significantly lower and TNFR2 significantly higher (p=0.011) in RA

patients with MetS, while TNF-α levels (p=0.523) did not differ between the groups. E-Selectin (OR=1.000, 95%CI=

1.000-1.000, p=0.017) and TNFR2 (OR=0.999, 95%CI=0.998-1.000, p=0.030) were positively associated with

presence of MetS in RA patients. There was significant and positive correlation between DAS28 calculated with

Erythrocyte Sedimentation Rate (ESR) and intercellular adhesion molecule 1 (ICAM-1) (r=0.211, p=0.036), E-Selectin

(r=0.221, p=0.028), and PAI-1 (r=0.204, p=0.043). Finally, there was also significant and positive correlation between

DAS28 calculated with C-Reactive Protein (CRP) and E-Selectin (r=0.210, p=0.037) and PAI-1 (r=0.197, p=0.051).

Conclusion: The presence of MetS contribute to inflammatory process and indirectly to disease activity evaluated by

DAS-28. E-selectin is a direct link between the presence of MetS and the clinical activity in RA. Our findings suggest

that physicians should screen RA patients for MetS to check disease activity and reduce risk of cardiovascular

diseases.

IMMUNOLOGY 163

EVALUATION OF THE REACTIVITY AGAINST NATIVE AND RECOMBINANT GP43 OF

Paracoccidioides brasiliensis IN SERUM SAMPLES POSITIVE FOR CANDIDEMIA

Robles, B. E. F.1; Macagnan, R.

1; Suguiura, I. M. S

1; Ono, M. A.¹

¹Universidade Estadual de Londrina, Department of Experimental Pathology, Londrina, PR, Brazil

Email: [email protected], [email protected], [email protected], [email protected]

Keywords: Paracoccidioides brasiliensis, systemic mycosis, immunodiagnostics, recombinant gp43, crossreactivity.

Introduction and objectives: Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the fungi

Paracoccidioides brasiliensis and P. lutzii. It is the most important systemic mycosis in Latin America, and affects

mainly male rural between 30 and 60 years of age. The glycoprotein with a molecular mass of 43 kDa (gp43), present

in the P. brasiliensis exoantigen, is the antigen most widely used in serological tests for the diagnosis and

epidemiological studies of PCM, although cross-reactivity may occur with antigens of other pathogens, due to

carbohydrate epitopes. The objective of this study was to evaluate the reactivity against native and recombinant Gp43

(rGp43) in serum samples of individuals positive and negative for systemic candidiasis.

Material and methods: The rGp43 was produced in Escherichia coli BL21 and purified by affinity chromatography.

The reactivity of P. brasiliensis native Gp43 and rGp43 was evaluated by Immunodiffusion Test, indirect ELISA and

Western blot, using serum samples from individuals seropositive and seronegative for systemic candidiasis. The study

was approved by the Research Ethics Committee of UEL (Opinion No.114/10).

Results: The seropositive and seronegative samples for systemic candidiasis were analyzed by immunodiffusion test,

and showed no reactivity with exoantigen of P. brasiliensis. When analyzed by indirect ELISA none of the seropositive

and seronegative samples for candidiasis showed cross reactivity with exoantigen of P. brasiliensis and with rgp43.

However, when reactivity against native gp43 was assayed by Western blot, we found that native Gp43 was

recognized by 72.7% of seropositive samples and 10% of seronegative samples for candidiasis, suggesting that the

antibodies present in seropositive samples for systemic candidiasis recognize carbohydrate epitopes present in native

gp43. Conclusion: These results suggest that the use of native Gp43 by the Western blot technique can lead to false-

positive reactions with seropositive samples for systemic candidiasis, which did not occur when using rgp43,

demonstrating its sensitivity and specificity for PCM.

Grants: The Araucária Foundation for financial support and to CAPES for granting the scholarship.




IMMUNOLOGY 164

EXPERIMENTAL CUTANEOUS CANDIDIASIS IN DIABETIC MICE AND EVALUATION OF TISSUE

REPAIR

Pupim, A.C. E.

1,2; Campois, T. G.

1; Araújo, E. J. A.

2; Svidizinski, T. I. E.

3; Felipe, I.

1 1State University of Londrina, Department of General Pathology, Londrina, PR, Brazil

2State University of Londrina, Department of Histology, Londrina, PR, Brazil;

3State University of Maringa, Department of Clinical Analysis 5790, Maringá, PR, Brazil

Email [email protected]

Keywords: cutaneous candidiasis, alloxan, macrophages, wound healing, cytokines.

Introduction and objectives: Diabetic patients seem to be predisposed to cutaneous candidiasis. In this study, we

evaluated the interference of diabetic conditions in alloxan-induced diabetic mice in relation to the development of

Candida albicans infection, the density of M1 and M2 macrophages, distribution of collagen type I and III and anti-

inflamamatory cytokines involved in tissue repair.

Material and methods: The experimental protocols were approved by the Animal Research Ethics Committee of

State University of Londrina, Brazil (Approval number 188/12). All procedures performed in studies involving animals

were in accordance with the ethical standards of the institution or practice at which the studies were conducted. The

mice were treated with intravenous alloxan, and all animals with blood glucose levels > 250 mg.dL-1

were inoculate

with C. albicans intradermally in the hind paw and were studied for up to 21 days. Control groups without alloxan were

used. The fungal burden was evaluated by periodic acid-Schiff (PAS) and by counting the colony forming units. Total

population of macrophages were targeted with antibody to F4/80 antigen and M2 macrophages with anti -arginase

antibody. Anti-inflammatory cytokines from popliteal lymph nodes were determined by capture ELISA procedures.

Picrosirius red staining allowed quantification of collagen types I and III in the infected skin by using a polarized light

microscope.

Results: Diabetic mice, versus non-diabetic mice, showed a significant lower density of F4/80 and M2 macrophages

(P<0,001), higher fungal burden (P<0,001), and deficiency in interleukin (IL)-4 production (P<0,001), and delayed IL-

13 responses (P<0,001). The later clearance of C. albicans enhanced tissue injury, leading to a decrease in collagen

type I (P<0,01). Moreover, collagen type III was increased by interference of IL-13 (P<0,01) and transforming growth

factor-b cytokines (P<0,001).

Conclusion: These findings highlight some important changes in diabetic animal responses to C. albicans infection

that may be important to the pathophysiological processes underpinning cutaneous candidiasis in diabetic patients.

Grants: This study was supported by the Fundação Araucaria (298/2012), Paraná, Brazil and Coordination for the

improvement of Higher Education Personnel (CAPES).


IMMUNOLOGY 165

EXPRESSION OF INDOLEAMINE 2,3-DIOXYGENASE IN CANCERS

Gomes, F.G.F.L.R.1; Mariano, C.F.A.

1; Trevisan, G.L.

1; Chahud, F.

1 1 Universidade de São Paulo, School of Medicine of Ribeirão Preto, Department of Pathology, Ribeirão Preto - SP,

Brazil


Keywords: Cancer, immunology, Indoleamine-Pyrrole 2,3,-Dioxygenase, 1-methyl-DL-tryptophan

Introduction: The indoleamine 2,3-dioxygenase (IDO1) enzyme is expressed by some cell types and also by tumor

cells. This enzyme inhibits the proliferation of T lymphocytes, compromising their cytotoxic activity.

Review: The IDO1 provides the initial barrier for the catabolism of tryptophan and kynuremine. T-cells need

tryptophan for clonal expansion and proliferation, which means depletion of this aminoacid reduces the T-cell

mediated immune response. The positive regulation of the IDO1 expression is mediated by pro-inflammatory signals

coming from microbial endotoxins and interferon gamma (IFN-γ). Multiple mechanisms have been considered to

explain how the expression of IDO1 can favor tumor growth and survival. The immunosuppressive properties

described previously can protect the tumor from the T-cell mediated response destruction in the tumor

microenvironment, or draining lymph nodes. Besides that, IDO1 can act favoring the tumor with non-immune

mechanisms, like the kynuremine. Some studies have already demonstrated that kynuremine promotes tumor survival

and cell motility by the activation of aromatic hidrocarbonates receptors. Another recent study has demonstrated the

role of IDO1 as an inflammatory mediator and in neovascularization. Mouse models suggest that developing tumors

actively recruit IDO1-expressing cells and induce IDO1 upregulation by dendritic cells in tumor-draining lymph nodes.

The expression of IDO1 has been related in several tumors, like: acute myeloid leukemia, breast cancer, cervical

cancer, colorectal cancer, cutaneous melanoma, diffuse large b-cell lymphoma, endometrial cancer, gastric cancer,

gliomas, hepatocellular carcinoma, multiple myeloma non-Hodgkin’s lymphoma, esophageal squamous cell

carcinoma, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic ductal carcinoma, renal cell

carcinoma, t-cell leukemia/lymphoma (adult), thyroid carcinoma, uterine cervical cancer, in various levels. High IDO1

expression in tumors has been associated with poor prognosis, and several studies have demonstrated such

association in acute myeloid leukemia, cutaneous melanoma, gliomas and other neoplasias. Several IDO1 inhibitor

drugs are now in phase I clinical trials as immunomodulators against cancer. Based on preclinical animal models

IDO1 inhibitors might be useful if used in combination with chemotherapy or vaccines. As mentioned in the literature,

the IDO1 pathway is linked to the CTLA-4 pathway. In a recent study patients with higher IDO1 expression in tumor

stromal cells were more likely to respond to therapy with the CTLA-4 blocking antibody ipilimumab suggesting that the

interactions between these two checkpoint pathways may be clinically relevant. Conclusion: These findings provide a good reason for the development of clinical studies using drugs that act as

IDO1 inhibitors. These recent developments identify potential new targets for cancer therapy as well as a range of

other clinical settings. Grants: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

IMMUNOLOGY 166

FOXP3 GENE POLYMORPHISMS DO NOT AFFECT SUSCEPTIBILITY NOR PROGNOSIS FOR

CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA IN BRAZILIAN PATIENTS

Petenuci, D. L.1; Oliveira, C. E. C.

1; Amarante, M. K.


1.

1 Laboratory of Molecular Genetic and Immunology, Department of Pathological Sciences, Biological Science Center,

University of Londrina, PR, Brazil

Keywords: ALL, polymorphism, FOXP3.

Introduction: Acute lymphoblastic leukemia (ALL) is a malignant disorder that originates from one single

hematopoietic precursor, accounting for approximately 80% of leukemia in the pediatric group. In disease

development, there is an initial accumulation of malignant leukocytes in the bone marrow with evasion tendency into

the bloodstream and invasion to other organs. Studies indicate a high percentage of regulatory T cells (Treg cells) in

several types of cancer, including childhood acute lymphoblastic leukemia (ALL), and it was usually associated with a

poor prognosis and advanced stages. The identification of single nucleotide polymorphisms in genes related to the

immune system in ALL patients is important as it may help to predict individual and population risk and clarify

pathophysiologic mechanisms relevant to this disease.

Objective: This study was undertaken to investigate the association of rs3761548 and rs2232365 FOXP3

polymorphisms in ALL.

Methods: We performed a case-control study involving 79 Brazilian children and adolescents with ALL and 91

unrelated children with no evidence of leukemias or other cancer. All subjects were genotyped for C/A rs3761548 and

A/G rs2232365 FOXP3 polymorphisms using allele specific-polymerase chain reaction (AS-PCR) assay. Odds ratio

was performed to obtain association study for risk of ALL development, at the 95% confidence intervals. The study

was approved by the Ethics Committee for Medical Experiment on Human Subjects, State University of Londrina,

Paraná, Brazil (CAAE N° 0164.0.268.000-09). Informed consent was obtained from all individuals (children guardians)

included in the study.

Results: Genotype distribution and allele frequency were not associated with ALL in male nor in female subjects (p

values ranged from 0.36 to 1.00). There was no association between alleles or genotypes of FOXP3 polymorphisms

and ALL development risk nor clinical outcome (p>0.05). Conclusions: Our results demonstrate that these FOXP3

gene polymorphisms are not a genetic susceptibility factor for ALL in the Brazilian population nor might be related with

clinical outcome. Analysis of other polymorphisms present in the FOXP3 gene associated with haplotype analysis in

patients with ALL are still necessary to validate our findings. We consider relevant a study with a larger group, as well

as an inclusion of patients of different ethnic origins.

IMMUNOLOGY 167

FUNCTIONAL CAPACITY OF OLDER WOMEN WITH DIFFERENT LEVELS OF INFLAMMATION

Tomeleri, C. M.1,2

; Clavaglieri, C. R.2; Cavalcante, E. F.

1; Souza, M. F.

3; Ribeiro, A. S.

4; Nunes, J. P. A.

1; Fabro, P. M.

C.1; Antunes, M.

1; Nabuco, H. C. G.

1; Venturini, D.

5; Barbosa, D. S.

5, Cyrino, E. S.

1

1Londrina State University, Physical Education, Londrina, PR, Brazil.

2University of Campinas – Unicamp, Physical Education, Campinas, SP, Brazil. 3 Federal University of Vale do São Francisco, Physical Education, Petrolina, PE, Brazil.

4University of Northern Parana, Center for Research in Health Sciences. Londrina, Brazil.



Keywords: C-reactive protein, inflammation, aged, sarcopenia.

Introduction and objectives: The decline in the functional capacity is considered one the worst consequences of

aging. Several factors may contribute for this decline, which include modifications in the different systems that exhibit

deterioration with the aging process. The ―Inflammaging‖ describes the low-grade chronic inflammation in aging and is

a highly significant risk factor for both morbidity and mortality in the elderly people and appears to aggravate the

deleterious effects of aging. Objective: To compare the functional capacity of older women with different levels of

inflammation.

Material and methods: Cross-sectional study involving 131 Brazilian older women; ≥ 60 years old. Written informed

consent was obtained from all participants. This investigation was conducted according to the Declaration of Helsinki

and was approved by the University Ethics Committee (048/2012). Functional capacity was assessed using the 10-m

walk test (10MW), and walking speed was calculated expressed in m/s. Body mass and height were measured and

body mass index (BMI) was calculated. Serum levels of C-reactive protein (CRP) by venous blood samples (after a 12

h fast) were evaluated, using a biochemical auto-analyzer system (Dimension RxL Max - Siemens Dade Behring).

Different levels of inflammation were defined according by Pearson et al (2003). Older women with CRP < 3.0 mg/L,

Group 1 (n= 68) and older women with CRP ≥ 3.0 mg/L (high inflammation), Group 2 (n= 63). Independent sample T-

tests was used to compare the means between groups.

Results: No difference (P>0.05) between groups for general characteristics (Group 1: 67.7 ± 4.3 years, 64.2 ± 12.8

kg, 155.4 ± 5.7 cm, 26.5 ± 4.7 Kg/m2,

and Group 2: 67.2 ± 4.2 years, 68.8 ± 13.7 kg, 156.1 ± 5.9 cm, 27.4 ± 7.0

Kg/m2). For the functional capacity Group 1 presented lower values for 10WM (Group 1: 7.3 ± 0.8s vs. Group 2: 8.0 ±

0.8s; P<0.05) and higher for walking speed (Group 1: 1.4 ± 0.1m/s vs. Group 2: 1.3 ± 0.1m/s; P<0.05) than the Group

2.

Conclusion: The functional capacity of older women is different between levels of inflammation. Older Women with a

higher degree of inflammation exhibit the worst functional capacity.

Grants: Capes, CNPq e Fundação Araucária.


IMMUNOLOGY 168

LUPUS PATIENTS WITH METABOLIC SYNDROME HAVE A DIFFERENT PROINFLAMMATORY

PROFILE THAN THOSE WITHOUT

Dall'Aqua, L. G. C.1; Alcântara, C. C.

2; Robles, B. E. F.

1; Stadtlober, N. P.

1, Silva, L. F. R. S.

1; Medeiros, F. A.

2; Sá, M.

C.2; Iriyoda, T. M. V.

1; Scavuzzi, B. M.

3; Cossentini, L. A.


4; Reiche, E. M. V.

4; Dichi, I.

5; Simão, A.

N. C.4.

1 Universidade Estadual de Londrina, Pós-Graduação em Patologia Experimental, Departamento de Ciências

Patológicas, Londrina, PR, Brazil 2Universidade Estadual de Londrina, Pós-Graduação em Fisiopatologia Clínica e Experimental, Departamento de

Patologia, Análises Clínicas e Toxicológicas, Londrina, PR, Brazil 3Universidade Estadual de Londrina, Pós-Graduação em Ciências da Saúde, Centro de Ciências da Saúde, Londrina,

PR, Brazil 4Universidade Estadual de Londrina, Departamento de Patologia, Análises Clínicas e Toxicológicas, Londrina,PR,

Brazil 5Universidade Estadual de Londrina, Departamento de Clínica Médica, Londrina, PR, Brazil


Keywords: Adhesion Molecule, Adipokines, Systemic Lupus Erythematosus, Metabolic Syndrome

Introduction and objectives: Systemic Lupus Erythematosus (SLE) is a chronic inflammatory autoimmune disease

with premature and accelerated atherosclerosis. The etiology of the increased risk of atherosclerosis has been

attributed to a combination of predisponent factors such as chronically elevated inflammatory markers, endothelial

dysfunction and, Metabolic Syndrome (MetS). Given the important role of cell adhesion molecules (CAMs) and

adipokines in the pathophysiology of SLE, premature atherosclerosis and disease activity, it is crucial to elucidate the

possible components that could modulate these levels and profiles. The objective of this study was to assess whether

the imbalance in adipokines and CAMs determined by the presence of Metabolic Syndrome (MetS) could affect

disease activity in Systemic Lupus Erythematosus (SLE) patients.

Material and methods: One-hundred and twenty-six SLE patients were divided in two groups, with (n=64) and

without MetS (n=62). Disease activity was determined using SLEDAI (SLE Disease Activity Index) considering the

SLEDAI ≥10, which is indicative of severe disease. Plasma levels of CAMs PECAM-1, VCAM-1, ICAM-1, E-selectin

and P-selectin and the adipokines MCP-1, hepatocyte growth factor (HGF), Lipocalin 2 (LCN2), leptin, resistin, TNF-α,

IL-1, IL-6, IL-8, IL-10, PAI-1 and serum amyloid A protein (SAA) were determined by Luminex multiplex analyses.

Plasma levels of adiponectin were measured using sandwich enzyme-linked immunosorbent assay (ELISA). The

study protocol was fully approved by the Ethical Committee of the University of Londrina (Paraná, Brazil) (CAAE

01865212.0.0000.5231, CEP/UEL 205.328).

Results: MetS patients had higher IL-8 (p=0.016), IL-10 (p=0.008), MCP-1 (p=0.002) and HGF (p=0.004). E-selectin

(p=0.020) and P-selectin (p=0.049) were associated with the presence of MetS. MetS was not directly associated with

disease activity measured by SLEDAI. ICAM-1 and LCN2 were lower and IL-6 and MCP-1 were higher in patients with

SLEDAI ≥10. After binary logistic regression analyses, IL-6 (p=0.024), MCP-1 (p=0.027) and LCN2 (p=0.009) were

associated with SLEDAI.

Conclusion: In conclusion, there are different levels of adipokines and cell adhesion molecules between SLE patients

with and without MetS, which indirectly interferes in the inflammatory network and could favor the modification of

disease activity.

IMMUNOLOGY 169

MOLECULAR CHARACTERIZATION OF HIV-1 AND MECHANISMS OF VIRAL RESISTANCE

Silva, T. F.1; Baronceli, D. C.

2; Ferreira, A.

1; Silva, L. R. C.


1; Pavanelli, W. R.

1; Costa, I. N.

1;

Melanda, F. N.1

1 Universidade Estadual de Londrina, Department of General Pathology, Londrina, PR, Brazil 2 Faculdade Intermunicipal do Noroeste do Paraná, College of Nursing, Loanda, PR, Brazil


Key words: HIV, AIDS, infection, viremia

Introduction: The HIV is a virus of the Retrovirinae Family, Orthoretrovirinae subfamily and Lentivirus genus. Two

types of HIV viruses have been identified, type 1 and type 2, the first being respectively the most common. The

epidemic caused by HIV-1 undergoes major changes, both in epidemiological level as at the molecular level, a greater

knowledge of the molecular mechanisms and immune response characteristics of our population are fundamental to

understanding the epidemic. According to estimates made by the Department of STD, AIDS and Viral Hepatitis

approximately 718 000 people living with HIV/AIDS in Brazil. This integrative review aimed to analyze key molecular

genetic markers associated with the virus and the host, relating them to the induction of resistance of HIV-1.

Review: For data collection was used the online databases PubMed and LILACS, resulting in the selection of 33

articles published between the years 2005 and 2014 in English and Portuguese, which formed the sample of this

research. The study allowed to describe the main molecular factors of both virus and host and their mutual

relationship and significance regarding the maintenance or combat of HIV-1 infection. Host and viral factors may

influence the course of HIV-1 infection. The variation patterns of HIV-1 genomic sequences can be used as genetic

markers to evaluate the trend in the evolution of infection and therapeutic effectiveness, such variations are caused by

mutations resulting from the absence of a checkpoint in the transcription of the viral genome by the exonuclease

reverse transcriptase, serving as an escape from the host's immune system, as well as genetic changes in surface

glycoproteins that support a strong genetic variation without losing its function, altering viral tropism and favor the

evolution of infection.

Conclusion: For many authors, the virus/host interaction is still surrounded by blind spots that, when unraveled, may

change the current treatment paradigm. Therefore, the mechanisms of immune response for containing viremia

through lymphocyte pathways, especially with regard to viral tropism and humoral response, are fundamental to the

understanding of HIV-1 immunopathogenesis and the control of viral resistance.

IMMUNOLOGY 170

PREDICTIVE USE OF GENETIC POLYMORPHISMS AS A TOOL FOR CLINICAL PRACTICE IN

LEISHMANIASIS INFECTIONS

Bertim, G. M. M; Santin, L. V.; Moreira, S. T.

Human Molecular Genetics Laboratory,

Parana Federal University of Technology, UTFPR, Santa Helena, Parana, Brazil

[email protected] Key words: Leishmaniasis, genetic polymorphisms, association study.

Introduction: Leishmaniasis is an endemic disease in geographic expansion, which has expanded to medium and

large urban areas, becoming a public health problem in Brazil and in other areas of the American continent. The

etiological agent of the disease, Leishmania sp., has a complex life cycle that presents an ideal immunopathological

characteristics to sustain the interaction maintained between its mammalian hosts. Its occurrence occurrence requires

only the presence of a susceptible vector and in a given area and an equally susceptible host/reservoir. Recent

studies have shown associations between genetic polymorphisms and leishmaniasis. Therefore, the objective of this

study was to do a survey about genetic polymorphisms which may be associated with the susceptibility or resistance

of the individual to leishmaniasis.

Review: By searching in the PubMed database, a bibliographic survey was carried out to highlight information about a

relation between non-HLA polymorphic variants and susceptibility or resistance to leishmaniasis. Some genes are

known to interfere with the infection caused by Leishmania sp., however, there are few association studies involving

polymorphisms performed up to the present moment. The presence of the TLR4896G (Asp299Gly) allele as well as

TLR41196T (Thr399Ileu) allele significantly increased the risk of infection and development of cutaneous

leishmaniasis. . The SLC11A1274

C allele provided protection to the development of leishmaniasis; and the 823

TT

genotype was associated with susceptibility to disease. Genotype TT of IL4RA gene confered susceptibility to visceral

leishmaniasis, as well as IFNGR1-470/-270/-56/+95

TTTT haplotype. About the cytokine genes, the presence of TNF-

308G allele that, despite controversy, upregulates the TNF production, was associated with susceptibility to

leishmaniasis.

Conclusion: The results showed that not-HLA polymorphic variants are associated with leishmaniasis. According to

the nitrogen-based pattern, the genes mentioned may make the host conducive to infection or resistant to it. Finding

predictive diagnosis markers for determination of susceptibility to leishmaniasis or even to the development of a more

severe form of the disease, may become an important tool to support the clinical conduct, once it provides individual

information about patient response to the infection which may increase the rate of cure.

IMMUNOLOGY 171

PREDICTIVE USE OF GENETIC POLYMORPHYSMS IN CROHN’S DISEASE

Santin, L. V.; Bertim, G. M. M.; Moreira, S. T.

Human Molecular Genetics Laboratory, Parana Federal University of Technology, UTFPR, Santa Helena, Parana,

Brazil


Key words: Genetic polymorphism, Crohn’s disease, Susceptibility.

Introduction: Crohn's Disease (CD) is a challenge for medicine and appears to show a tendency to increase in its

incidence worldwide. It is described as a subcategory of inflammatory bowel diseases that can affect the full digestive

system and is more frequent among the Caucasian population. Studies show that CD is caused by a combination of

factors that imply deregulation of the immune system, genetic and environmental factors, including the infectious one.

Intestinal microbiota has also been cited as a possible cause. Therefore, the objective of this work was to perform a

survey about genetic polymorphisms which may be associated with the susceptibility or resistance of the individual to

CD.

Review: Through research in the PubMed database, a bibliographic survey was carried out to highlight information

about a relation between non-HLA polymorphic variants and susceptibility or resistance to DC. Recent studies have

shown significant positive associations with DC. In a meta-analysis study, association was found between allele C of

rs1946518, rs187238 and rs360718, as well as CC genotype of rs360718 of IL-18 and DC. Polymorphic variants in

CD24 gene such as C170T (rs8734) and TG1527del (rs3838646) was also associated with the disease. A study

addressing the rs7517847 polymorphism of the IL-23R gene revealed the T allele also conferring susceptibility to DC.

The T allele for of the rs11235604 polymorphism at ATG16L2 gene has also been identified as a risk factor for DC.

Conclusion: The results obtained demonstrate that polymorphic variants are associated with DC. Seeing the future

establishment of personalized medicine, the genetic polymorphisms associated with DC will be an invaluable tool to

assist the doctor's clinical conduct. considerable advances in the area of pharmacogenetics, brought new strategies

for the treatment of the disease, since it provides individual information about the risk of developing the disease,

allowing the adoption of prophylactic behaviors and consequently survival of the individual.


IMMUNOLOGY 172

PRO-INFLAMMATORY PROFILE OF OLDER WOMEN WITH DIFFERENT LEVELS OF OBESITY

Tomeleri, C. M.1,2

; Clavaglieri, C. R.2; Polvani, A. C. T.

1; Nunes, J. P. A.

1; Fabro, P. M. C.

1; Antunes, M.

1; Souza, M.

F.1, Silva, F. J. A.

1; Ribeiro, A. S.

3; Venturini, D.

4; Barbosa, D. S.

4, Landucci, K.

4; Cyrino, E. S.

1 1Londrina State University, Department of Physical Education, Londrina, PR, Brazil.

2University of Campinas – Unicamp, Faculty of Physical Education, Campinas, SP, Brazil.

3 University of Northern Parana, Center for Research in Health Sciences. Londrina, Brazil.


Email:[email protected]

Keywords: Cytokines, inflammation, aged, obesity.

Introduction and objectives: Obesity and aging are related to chronically increased levels of inflammatory

biomarkers, a phenomenon described as low-grade inflammation. In this sense, it can be hypothesized that obese

older individuals have a higher risk of developing this condition. Objective: To compare pro-inflammatory profile of

older women with different levels of obesity.

Material and methods: This is a cross-sectional study involving 161 older women, ≥ 60 years old. Written informed

consent was obtained from all participants. This investigation was conducted according to the Declaration of Helsinki

and was approved by the local University Ethics Committee (048/2012). Body composition by whole-body dual-energy

X-ray absorptiometry (DXA), and Inflammatory markers: tumor necrosis factor alpha (TNF-α), Interleukin-6 (IL-6) and

C-reactive protein (CRP) by venous blood samples (after a 12 h fast) were evaluated. The serum levels of high-

sensitivity CRP were carried out using a biochemical auto-analyzer system (Dimension RxL Max - Siemens Dade

Behring), and TNF-α and IL-6 were determined by ELISA. The different levels of obesity were defined according to

percentage of body fat evaluated by DXA. The sample was separate into tertiles: Obesity 1 (body fat ≤ 32%, n=57),

Obesity 2 (body fat > 32% and ≤ 42.5%, n=51) and Obesity 3 (body fat > 42.5%, n=53). One-way analysis of variance

was applied, and Bonferroni’s post hoc test. The statistical significance was accepted at P < 0.05.

Results: The women in Group 1 (69.5 ± 6.9 years) were older than the women in group 2 (67.2 ± 5.2 years) and

group 3 (66.3 ± 4.2 years) (P<0.05). However, the Group 3, who were the most obese (Body fat > 42.5%), presented

the significantly highest values (P<0.05) for: TNF-α (Group 1 = 2.7 ± 0.7 pg/mL vs. Group 2 = 2.8 ± 0.7 pg/mL vs.

Group 3 = 3.8 ± 0.9 pg/mL); IL-6 (Group 1 = 2.6 ± 0.5 pg/mL vs. Group 2 = 3.0 ± 0.9 pg/mL vs. Group 3 = 4.0 ± 1.4

pg/mL) CRP (Group 1 = 2.4 ± 1.0 pg/mL vs. Group 2 = 2.7 ± 1.0 pg/mL vs. Group 3 = 3.3 ± 1.4 mg/L). The Group 2

showed higher values for IL-6 compared to group 1 (P<0.05).

Conclusion: The pro-inflammatory profile: TNF-α, IL-6, and CRP of older women is different between different levels

of obesity. Older Women with a higher degree of obesity exhibit the worst inflammatory profile with higher values for

TNF-α, IL-6, and CRP.

Grants: Capes, CNPq e Fundação Araucária.

IMMUNOLOGY 173

ROLE OF TOLL-LIKE RECEPTORS SIGNALING AND MYD88 ADAPTER PROTEIN IN THE

IMMUNOPROTECTION AGAINST Paracoccidioides brasiliensis: AN UPDATE

Robles, B. E. F. 1; Santos, F. C.

1; Macagnan, R.

1; Carvalho, G. G.

1 1 Universidade Estadual de Londrina, Department of Pathological Sciences, Londrina, PR, Brazil

[email protected]

Key words: Toll-Like receptor, MyD88 protein, paracoccidioidomycosis, immunity.

Introduction: Paracoccidioides brasiliensis, a thermo-dymorphic fungus, is the main causative agent of

paracoccidioidomycosis, a systemic mycosis endemic in Latin America. Myeloid differentiation factor 88 (Myd88), an

adapter protein, participates in signaling triggered by the recognition of pathogen-associated molecular patterns

(PAMPs) by Toll-like receptors (TLRs), a family of type 1 transmembrane proteins present in many cells that

participate in innate and adaptive responses, leading to the activation of protein kinases and transcription of cytokines

involved in proinflammatory response. The present work aimed to carry out a literature review, consulting current

scientific articles on the involvement of signaling via TLRs and MyD88 in defense against P. brasiliensis.

Review: Studies have shown that TLR2 deficiency increases the T helper (Th) 17 cells response associated with a

decrease in regulatory T cells (Treg) and increased lung injury due to the exacerbated inflammatory response.

Another study, using a more severe infection model, demonstrated that the increase in Th17 and decrease in Treg

was regulated by the expression of TLR4. In addition, TLR2 and TLR4 appear to participate in the recognition and

internalization of P. brasiliensis by neutrophils and monocytes. Researchers have pointed out that Myd88 is not

essential for the defense against systemic infection by P. brasiliensis. In contrast, it was found that the absence of

Myd88 signaling in peritoneal macrophages resulted in decreased phagocytic capacity and decreased levels of

interleukin 12 (IL-12) and nitric oxide. Myd88 knockout mice infected with P. brasiliensis developed severe infection

with decreased levels of Th1, Th2, Th17 and Treg cytokines and a high mortality rate associated with extensive fungal

lesions in several organs.

Conclusion: These data, taken together, demonstrate that signaling via TLRs and Myd88 plays an important role

both in innate and adaptive immune responses, conferring host resistance and increased phagocytic and fungicidal

capacity to immune system cells.

IMMUNOLOGY 174

SLEEP RESTRICTION ALTERS TESTICULAR INFLAMMATORY PROFILE IN PERIPUBERTAL RATS

Siervo, G. E. M. L.1,2

; Ogo, F. M.1,2

; Staurengo-Ferrari, L.2; Cunha, F. Q.

3; Verri Jr, W. A.


1 1 Universidade Estadual de Londrina, Department of General Biology, Londrina, PR, Brazil

2 Universidade Estadual de Londrina, Department of Pathological Sciences, Londrina, PR, Brazil

3 Universidade de São Paulo, Department of Pharmacology, Ribeirão Preto, SP, Brazil

[email protected] / [email protected]

Keywords: Sleep restriction, testis, puberty

Introduction and objectives: Puberty represents a period of complex sexual development, which leads to sexual

maturation and reaching reproductive capability. Sleep plays a critical role in immune system maintenance and

influences the cytokines under normal and pathological conditions. Testicular cytokines, under physiological

conditions, are involved in the spermatogenesis process. The aim of this study is to evaluate whether sleep restriction

during the peripubertal period could modulates the testicular inflammatory profile.

Material and methods: (CEUA/UEL protocol number 3467.2014.86). Thirty male Wistar rats (postnatal day - PND 40)

were used. The Sleep Restriction (SR) group was exposed to 21 days of sleep restriction by the modified multiple-

platform method (18h of SR and 6h of sleep, per day). The Control group (C) was maintained in their home-cages

during all the experiment. At PND 62, the rats were weighed, anesthetized and euthanized. Testis was removed,

weighted and used to inflammatory profile evaluation – IL-1β, IL-6, TNFα and IL-10 levels as well as NAG and MPO

activity.

Results: The testicular IL-1β, IL-6 and TNFα levels were decreased in SR animals, whereas IL-10 level was not

altered. Neutrophil recruitment to the testicular tissue and macrophage migration was not affected by SR as

demonstrated by the MPO and NAG assay, respectively.

Conclusion: Sleep restriction during the peripubertal period affects the cytokine concentrations in testis. More studies

are necessary to evaluate whether this condition can impair spermatogenesis process and testicular development.

IMMUNOLOGY 175

THE USE OF ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) TO DIAGNOSE

PARACOCCIDIOIDOMYCOSIS: A SYSTEMATIC REVIEW

Ribeiro, L.M.

1; Itano, E.I.

2; Lenhard-Vidal, A.

1,2

1Faculdade Campo Real, Graduação em Biomedicina, Guarapuava, PR, Brazil.


[email protected]

Keywords: Paracoccidioides brasiliensis, Paracoccidioides lutzii, Serologic tests, Systemic mycosis.

Introduction: Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the thermodimorphic fungi

Paracoccidioides brasiliensis and P. lutzii. Most cases occur in men over 40 years-old, through primary infection of the

lungs by inhalation of propagules found in nature and later dissemination to any organ. The gold standard diagnosis of

PCM is the identification of the fungus by direct examination or culture, which demands trained personnel and a good

sample. On the other hand, the enzyme-linked immunosorbent assay (ELISA) is a usually simple and fast test, which

can detect both antigens and antibodies in many kinds of samples.

Review: Current research is a systematic review of 61 articles obtained at the Virtual Health Library (Brazilian Health

Ministry) using the words "Paracoccidioidomycosis" and "ELISA". Articles were selected by: type (only original

articles); language (English or Portuguese); subject (diagnosis of human PCM by ELISA); and publication date (2000-

September 2016). Only 17 articles met the criteria and were published between 2003 and 2014. Just one is from

British/Colombian institutions, while the others are Brazilian (9 São Paulo, 4 Paraná, 2 Minas Gerais, 1 Pará). The

most used sample was serum and 3 researches also employed cerebrospinal fluid and/or bronchoalveolar lavage

fluid. The most used method was indirect ELISA (15) and to detect antibodies (mostly IgG): 7 to total antigens (cell

free antigen or exoantigen), 5 to native purified antigens, 3 to recombinant antigens and 1 to synthetic peptide. Solely

3 researches employed inhibition ELISA to detect specific antigens (gp43, gp70). Strains were mainly P. brasiliensis

(9 B339, 4 Pb18, 3 Pb113), 3 also employed environmental/clinical isolates, while only 2 investigated antibodies to cell

free antigens of P. lutzii. Conclusion: Between 1980 and 1995, PCM had the highest mortality rate among the

systemic mycoses and was the 8th most common cause of death from predominantly chronic or recurrent types of

infectious and parasitic diseases. Even though, its diagnosis is usually late because PCM remains not well known

among health professionals, coupled with the difficulty of the direct examination or culture. Therefore, sensitive

diagnostic techniques such as ELISA become very important. Despite the suspicion about a new species of

Paracoccidioides since 2006, current review demonstrates the subject is insufficiently researched concerning PCM

diagnosis. Gp43 or its specific antibodies were investigated until that year, which is expected, but as this antigen is

produced in small amounts or not at all in some strains of P. lutzii, the use of gp43 or its specific antibodies can

compromise the diagnosis of PCM in certain regions, as in the Central-West of Brazil, where P. lutzii predominates.

Even though ELISA has been proven highly efficient and specific, not enough research has been published in order to

use it as the new gold standard test throughout Brazil, in order to make PCM diagnosis faster and easier.

IMMUNOLOGY 176

TRANSFORMING GROWTH FACTOR BETA 1 GENE HAPLOTYPE INFLUENCES

HUMAN PAPILLOMAVIRUS INFECTION

Trugilo, K. P.1; Cebinelli, G. C. M.

1; Pereira, E. R.

1; Aranome, A. M. F.

1, Okuyama, N. C. M.

1; dos Santos, F. C.

1;

Pereira, A. P. L.1; Sena, M. M.

1; Ferreira, R. S.

1; Maria, G. C. Q

1;

Berti, F. C. B.1; Esposito, A.

1; Nishimura, A.M.

1; Oliveira, K. B.

1 1State University of Londrina, Department of Pathological Sciences, Londrina, PR, Brazil


Keywords: single nucleotide polymorphism, genetic association studies, restriction fragment length polymorphism

Introduction and objectives: Persistent Human Papillomavirus (HPV) infection presents a close association with

high-grade squamous intraepithelial lesion (HSIL) and cervical cancer development. However, HPV alone is not

sufficient for malignant progression. Exogenous and endogenous factors may influence the risk of cervical lesions.

Transforming Growth Factor β (TGFB), a cytokine that is known for regulation of cell growth, maturation, and

differentiation, has an important role in carcinogenesis and is associated with the progression of HPV infection to HSIL

and cervical cancer. Therefore, this study aimed to assess the association of genetic variability in TGFB1 gene with

HPV infection and development of cervical lesions.

Material and methods: In this case-control study (CAAE 56738316.3.0000.5231, resolution 466/12), cervical swabs

and blood samples were obtained from 358 outpatient women, along with socio-demographic and sexual behavioral

data. The study population was stratified by absence (non-infected, n=179) or presence (infected, n=179) of HPV

DNA, as tested by PCR, as well as by normal cytology or presence of cervical lesion according to cervical cytology.

Four single nucleotide polymorphisms (SNPs), c.−1638G>A and c.−1347C>T in the TGFB1 regulatory region, and

c.29C>T and c.74G>C in the TGFB1 signal peptide were genotyped using PCR-restriction fragment length

polymorphism. Haplotypes were estimated by PHASE software (version 2.1) and the linkage disequilibrium between

SNPs was analyzed by HaploView software (version 4.2). Statistical analyses were performed by SPSS software

(version 22.0) with P < 0.05 as significant.

Results: Each polymorphism had the minor allele frequency higher than 5.0% and the linkage disequilibrium were

higher between SNPs c.−1347C>T and c.29C>T (r2=0.93). From twelve estimated haplotypes only four (GCTG,

GTCG, ACTG, and GCCC) were more frequent than 5.0% (43.9%, 32.0%, 10.2%, and 5.1%, respectively). In a binary

logistic regression analysis adjusted to age, tobacco status, and number of sexual partners during lifetime, only GCTG

haplotype allele was associated with a lower susceptibility of HPV infection, both in heterozygosity (OR=0.57 and

IC95%=0.34-0.93, P=0.02) and homozygosity (OR=0.52 and IC95%=0.30-0.93, P=0.02). Among infected women,

TGFB1 genotypes and haplotypes were not associated with cervical lesions development.

Conclusion: Although further studies are necessary, the presented data demonstrate that TGFB1 GCTG haplotype is

significantly associated with protection against HPV infection.

Grants: Capes, PPSUS, Fundação Araucária, CNPq


MICROBIOLOGY AND PARASITOLOGY 177

ANALYSIS OF THE VIRULENCE OF GEOGRAPHICAL ISOLATES OF THE Bombyx Mori

Nucleopolyhedrovirus (NPV).

Poncio, A. C. F.1; C. Pedroso, O. C.

1; Santos, J. S.

1; Machado, L. G. V.

¹; Souza, P. A. L.

¹; Ribeiro, L. F. C.

¹;

Brancalhão, R. M. C.1.

1 Laboratory of Structural and Functional Biology, State University of Western Paraná – Cascavel, Paraná, Brazil

[email protected]

Keywords: Viral isolate, baculovirus, silkworm

Introduction and objectives: Bombyx Mori Nucleopolyhedrovirus (NPV) is an entomopathogenic virus of the

Baculoviridae family, which infects the Silkworm (Bombyx mori) and causes nuclear polyhedrosis disease. This virus

is polyorganotrophic and a 1996 viral isolate with multiple nucleocapsids was identified in the state of Paraná, Brazil

(BmNPV-01). This inoculum has been used in the cytopathological studies, aimed at establishing the target tissues

and infectious cycle in the body of the insect, presenting several passages (50) in the host. However, due to the

innumerable experiments and several cycles of replication it is possible the appearance of viral mutations that can

affect morphological characteristics and the virulence of the isolate. To analyze the morphology and symptoms of

BmNPV-01 and compare it with a 2014 isolate (BmNPV-02) , obtained directly from B. mori caterpillar breeding sites

and presented 2 passages in the host.

Material and methods: In the experimental research the caterpillars were organized into three groups: inoculated

with BmNPV-01; with BmNPV-02; and control group, not inoculated. The inoculation was performed at a concentration

of 2.4x10 7 COPs / mL and the greasy tissue was obtained and processed for light microscopy and scanning electron

microscopy. The virulence of the isolates was analyzed through the signs and symptoms of the infection using the

Pearson correlation coefficient and its significance assessed by the correlation t test. In the case of the symptoms, a

linear model was created and evaluated by the Akaike criterion (AIC), by the coefficient of determination (r2) and

followed by the models of the geographic isolates by means of the analysis of the variance assuming a distribution X2

and a = 0.05. The number of deaths was performed from c2 to k proportions, program R (R Core Team, 2016).

Nuclear volume, the Kruskal-Wallis test was applied.

Results: The variance analysis showed a significant difference between the two models (p = 0.0547). Therefore, the

BmNPV-02 inoculum promoted symptoms faster than the BmNPV-01. Regarding the volume of the fatty tissue cores

of the groups, a significant difference was observed between control and inocula, but little difference in morphometry

between BmNPV-01 and BmNPV-02. There was a statistical difference between the groups (H = 7.21, p = 0.02).

Comparing the control group (md = 56.73) with the inoculum1 (md = 268,44) we observed that the medians were

considered statistically different (p = 0.03). Also comparing the control with the inoculum2 (md = 300,64) the medians

are also statistically different (p = 0.01). However, comparing the two inoculants to each other, the medians were

considered statistically equal (p = 0.64).

Conclusion: The BmNPV-02 isolate is the most virulent, with no changes in nuclear volume and cytopathology.

Grants: (Cnpq).



ANTIMICROBIAL ACTIVITY OF THE METHANOL EXTRACT Eugenia involucrata FRONT OF THE

DIFFERENT SEROTYPES OF Salmonella spp.

Toledo, A. G. 1; Souza, J. G. L.

1; Santos, C. V.

1; Mallmann, A. P.; Luft, T. S.; Silva, K. C.; Pinto, F. G. S

1

1 State University of the West of Paraná, Department of Agricultural Biotechnology, Cascavel, PR, Brazil

[email protected]

Keywords: Biological products, anti-infective agents, gastroenteritis, microbial sensitivity tests, Salmonella enterica.

Introduction and objectives: Since 2011, Brazilian poultry has been the leader in the export of chicken meat, which

shows the growing importance of this sector. This factor, which is linked to increased production and the possible

transmission of diseases transmitted in food of poultry origin, among them gastroenteritis caused by Salmonella To try

to alleviate this problem, it is intended to use natural products as potential antimicrobials. In this context, the objective

of this work was being to verify the antimicrobial activity of the methanolic extract of Eugenia involucrata in front of five

serotypes of Salmonella enterica isolated from aviaries in the western region of Paraná: Rissen, Idikan, Livingston,

Havana and Grumpensis.

Material and methods: To obtain the methanol extract, the leaves were dried at 40º C and milled with a knife mill.

Methanol P.A. was added to the powder of the plant leaf in the ratio of 1:10 (w/v). After, it was placed on a rotary

shaker for 24 hours. Finally, the blend was centrifuged at 5000 rpm (revolutions per minute) for 15 minutes, then

vacuum pump filtered and rotary evaporator to remove the solvent completely. The minimum inhibitory concentration

(MIC) and minimum bacterial concentration (MBC) were determined by the broth microdilution method, with serial

concentrations ranging from 200 to 0.09 mg/mL of the extract. As a positive control was use gentamicin 200 mg/mL.

The bacterial metabolism was verified using 0,5% triphenyltetrazolium chloride (TTC) by the colorimetric method.

Results: The extract presented activity against all the serotypes tested, being: S. Idikan and S. Livingston with 3,12

mg/mL of MIC and 25 mg/mL of MBC, for both serotypes, followed by S. Rissen wih 3,12 mg/mL of MIC and 50

mg/mL of MBC and posteriorly, S. Havana and S. Grumpensis, both with values of 6,12 mg/mL of MIC and 25 mg/mL

of MBC.

Conclusion: From this work, it is concluded that the methanolic extract of Eugenia involucrata presented satisfactory

antimicrobial results on the tested strains, presenting efficiency in the fight against these microorganisms. Besides

that, it is important to emphasize the importance of alternative products of natural origin, such as plant extracts to

minimize resistance factors, as well as avoid problems caused by diseases of poultry origin.

Grants: CAPES, CNPq, PPRN.


ANTIMICROBIAL RESISTANCE USE OF ANTIBIOTICS AND THE INDISCRIMINATED USE OF

ANTIBIOTICS

da Silva, R. S1; Terra, M. R.

2; Nascimento, F. L. I.

3; Jeremias, J.T.Z.

3

1 Faculdade de Medicina de Botucatu, Botucatu, SP, Brazil.

2 Universidade Estadual de Londrina, Londrina, PR, Brazil

3 Instituto de Ensino Superior de Londrina, Londrina, PR, Brazil

[email protected]

Keywords: Resistant antimicrobials, antibiotic, self-medication.

Introduction and objectives: Antimicrobials correspond to a class of drugs that are often consumed in hospitals and

in the community. However, they are the only pharmacological agents that do not only affect patients who use them,

but also significantly interfere in the hospital environment due to altered microbial ecology. In this sense, the excessive

use of these drugs is associated with the emergence and selection of strains of resistant bacteria. The present study

aims to evaluate the use of antimicrobials by the population regarding dosage, mode of storage, adverse reactions,

prescription, disposal and self-medication.

Material and methods: The present study is cross - sectional observational and was approved by the Research

Ethics Committee involving Human Subjects in the. 1,434,495. A semi-structured questionnaire with open and

objective questions was applied to 175 participants in the City Center of Londrina - PR from May to June 2016 to

evaluate the use of antimicrobials by the population regarding posology, storage, prescription and reuse .

Results: Of the 175 respondents, 96% have been using antimicrobials at least once in the past 5 years. Amoxicillin

was prescribed in 27.9% and was prescribed by the physician in 75.7% of the cases, without any type of examination

in 34.6% of the prescriptions. In 63.4% of the prescriptions the patients received medical advice regarding dosage. A

worrying factor concerns the dosage of medication where 15.4% of the interviewees discontinued the use of the

medication due to the improvement of the symptoms in 35.4%. Medication leftovers were indicated in 36.6% of the

cases, where 60.6% reported reusing these leftovers by self-medicating in the case of colds (51.4%) and 9.9% of the

medication was donated to other people without a prescription.

Conclusion: Self-medication is a common practice, even to the present day even with the controlled purchase of

antimicrobials, where inappropriate self-medication can have undesirable effects, iatrogenic diseases and masking of

diseases, representing a problem to be prevented . The importance of antimicrobials in the expansion of the

resistance phenomenon lies in its selective role of resistant specimens, through selective pressure.


ANTIVIRAL ACTIVITY OF METHANOL, ETHANOL AND ACETONE EXTRACTS FROM BARK OF

Trichilia catigua IN THE REPLICATION OF HERPES VIRUS RESISTANT TO ACYCLOVIR

Botura, T. J.1; Faccin-Galhardi, L. C.

1; Lonni, A. G. S.

2; Linhares, R. E. C.

1; Nozawa, C.

1

1 State University of Londrina, Department of Microbiology, Londrina, PR, Brazil

2 State University of Londrina, Department of Pharmaceutical Sciences, Londrina, PR, Brazil

[email protected]

Keywords: Antiviral, T. catigua, herpesvirus, cell culture.

Introduction and objectives: Viral diseases continue to be responsible for significant rates of mobility and mortality.

In addition, the emergence of resistant strains of available antivirals is also noteworthy. The interest in the

development of antivirals of natural origin is mainly due to the search for more effective substances, with less

collateral effect and have different mechanisms of action. Thus, this study evaluated the antiviral activity of Trichilia

catigua extracts in the replication of Herpes Simplex Virus type 1 (HSV-1, cepa AR), a resistant strains to Acyclovir

(reference drug), in VERO cells.

Material and methods: Five extracts obtained from bark of T. catigua were tested in this work: EB2 (Methanol

extract), EB3 (acetone extract) and EB4 (ethanol extract) and EB9 (methanol + ethanol) and EB14 (methanol +

acetone + ethanol), a mixture of two and three solvents, respectively. The toxicity and antiviral activity of the extracts

in VERO cells was evaluated by the MTT colorimetric assay (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium

bromide) (Sigma Chem. Co.). The cytotoxic concentration of 50% (CC50%) and the 50% concentration inhibitory

(CI50%) were determined with linear regression and the selective index (SI) calculated at CC50%/CI50%.

Results: Of the five tested compounds, EB3 showed high toxicity in VERO cells, until the less concentration

investigated (3 µg/mL) and was not used in antiviral assays. The CC50% value found for EB2 and EB4 were of 200

µg/mL and 280 µg/mL, respectively. The EB14 presented CC50% similar to the two previous extracts, 240 µg/mL.

However, EB9 showed higher cytotoxicity, with CC50% of 143 µg/mL. When the compounds were evaluated for the

antiviral activity, the single solvent extracts (EB2 and EB4) inhibited about 35% of HSV-1 replication, at the lowest

tested concentration (3 µg/mL). The best antiviral activity was found for EB9, with dose-dependent inhibition (DP) of

100% to 3 µg/mL, 93% - 1.5 µg/mL, 75% - 0.75 µg/mL and 73.6% - 0,35 µg/mL. The CI50% was lower than 0,35

µg/mL and SI of 408,6. EB14 also showed inhibition of viral replication, with DP form, however with VI% lower when

compared to EB9, being 66.4% - 3 µg/mL, 37% - 1.5 µg/mL, 38.4% 0.75 µg/mL and 16.8% - 0.35 µg/mL, with IC50%

of 2 µg/mL and SI of 120.

Conclusion: Our results demonstrated that for the tested extracts, obtained from T. catigua, a better anti-herpetic

activity was found to extracts produced with mixtures of solvents and not with pure solvents. Previous studies of our

group, have shown that the T. catigua bark has a high content of phenolic compounds, when extracted from solvent

mixtures. In addition, an antiherpetic activity of the phenolic compounds has already been scientifically described.

Thus, our results suggest a relationship between a greater amount of phenolic compounds present in EB9 and EB14

and anti-herpetic activity, especially when evaluated in a sample resistant to the reference antiviral.

Grants: CAPES/CNPq/UEL


CAFFEIC ACID HAS ANTI-AMASTIGOTE EFFECT IN L. amazonensis-INFECTED MACROPHAGES

Bortoleti, B. T. S.1; Carloto, A. C. M

1.; Tomitto-Pellissier, F.

1; Gonçalves, M. D.

2; Miranda-Sapla, M. M.

1; Assolini, J.

P.1; Costa, I. N.


1; Pavanelli, W. R.

1


2Universidade Estadual de Londrina, Department of Chemistry, Londrina, PR, Brazil

[email protected]

Keywords: Macrophages, leishmaniasis, amastigote.

Introduction and objectives: Protozoans of the genus Leishmania are obligate parasites of phagocytic cells

responsible for American Cutaneous Leishmaniasis (ACL), a neglected tropical disease that affects the skin and

mucous membranes. The conventional treatment presents high toxicity, high cost and several effects, being

necessary for the search for new compounds. Caffeic acid, present in several natural extracts, has many biological

properties, such as antioxidant, anti-inflammatory, immunomodulatory and scavenning of free radicals. Thus, this work

aimed to identify the non-toxic concentrations for peritoneal macrophages of BALB/c mice and human erythrocytes, as

well as its leishmanical action on the amastigote forms of L. amazonensis

Material and methods: Promastigotes forms of L. amazonensis was maintained in 199 medium and used in

stacionary phase. The cytotoxicity of caffeic acid on peritoneal macrophages of BALB/c mice (ethics committee nº

3715.2015.22) was analyzed through the detection of LDH (lactate dehydrogenase) by the CytoTox 96 Non-

Radioactive Cytotoxicity kit. In this study, L. amazonensis-infected peritoneal BALB/c macrophages were treated with

caffeic acid and analyzed by the anti-amastigote assay, where the percentage of infected macrophages and the

number of amastigotes by macrophages were verified. The recovery assay of the promastigote forms was used as

confirmation of the anti-amastigote assay. Data were expressed as mean ± standard error of the mean. At least three

independent experiments were performed, each with duplicate datasets. Significant differences between the groups

were determined by one-way ANOVA, followed by Tukey’s test.

Results: The results showed that none of the concentrations used were toxic to the peritoneal macrophages. Were

observed that 12.5, 25 and 50 µg/mL of caffeic acid reduced the percentage of infected macrophages by at least 90%

when compared to the control. In addition, the treatment was able to reduce the number of amastigotes by

macrophage. Confirming these results, the recovery assay demonstrated that all tested concentrations were able to

reduce the percentage of promastigote forms recovered after 48, 72, 96 and 120 hours.

Conclusion: Together our data showed that caffeic acid has antiamastigote action on L. amazonesis- infected murine

macrophages, without causing toxicity to the host cell.

Grants: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) e Conselho Nacional de

Desenvolvimento Científico e Tecnológico (CNPq).



CONCANAVALIN-A INDUCE M1 MACROPHAGE POLARIZATION IN INFECTION BY

Leishmania (L) amazonensis

Alvarenga, D. S.1; Miranda-Sapla, M. M.

1; Tomiotto-Pellissier, F.

1; Mendes, L.

1; Silva, P. R. B.

1; Melanda, F. N.

1;

Costa, I. N.¹; Pavanelli, W. R.¹; Conchon-Costa, I.1

1 Department of Experimental Pathology – Laboratory of Experimental Protozoology, State University of Londrina,

Londrina, Brazil.

[email protected]

Keywords: Concanavalin-A, M1 macrophages, Leishmania amazonensis.

Introduction and objectives: Macrophages are professional phagocytes serving as a first line of defense being

inducers, regulators and effectors in the innate and acquired immunity. These cells have dual role in Leishmania

infections. The protozoan uses macrophages as host cells, where it survives and multiplies, and at the same time, the

macrophage, upon stimulation, can kill the parasites. Macrophages may undergo polarization according to the

stimulus received, macrophages referred as M1 phenotype acquire an effector function against intracellular

pathogens, increasing the activity of inducible nitric oxide synthase (iNOS) and NADPH oxidase, with consequent

increase in the production of nitric oxide and reactive oxygen species. Several studies have shown the

immunomodulatory potential of Concanavalin-A (Con-A) in experimental infections, inducing the cellular activation and

the production of proinflammatory mediators. Thus, the aim of this study was to evaluate the polarization of

intraperitoneal macrophages pretreated with Con-A and infected with Leishmania (L.) amazonensis (LLa).

Materials and methods: BALB/c mice (n=6) were pretreated intraperitoneally with Con-A (250μg mL-1

) or PBS 72h

before the infection with 107 promastigotes forms. Exudate cells were collected by rinsing the peritoneal cavity with

3mL of PBS. Then, the samples were centrifugated and the pellet was resuspended in 1mL of RPMI-albumin medium,

distributed on coverlips. Immunocytochemistry for macrophages M1 and M2 were performed by labeling of inducible

nitric oxide synthase (iNOS), MHC class II, arginase and CD206. The results were expressed as the mean ± standard

error and analyzed with the Prism 5.0 statistical program using the one-way ANOVA, followed by Tukey's test. This

study was approved by Londrina State University Ethics Committee for Animal Experimentation No. 056/2013.

Results: We observed that murine macrophages pretreated with Con-A and infected with L. amazonensis showed

increased expression of iNOS and MHC class II and decreased CD206 expression when compared to the PBS group.

Conclusion: In our model, we demonstrated that Con-A was able to promote M1 macrophage polarization when

infected with L. amazonensis.

Grants: I would like to thank CAPES for the scholarship.


DETECTION OF MYCOPLASMA SPECIES IN MAMMAL CELL CULTURES USING PCR

Mattos, B. B.1; Cilião, H. L.

1; Cólus, I. M. S.

1; Serpeloni, J. M.

1

1Universidade Estadual de Londrina, Departamento de Biologia Geral, Londrina, PR, Brasil

[email protected]

Keywords: Mycoplasma, cell culture, PCR.

Introduction and objectives: Studies using cell cultures began in the twentieth century with Ross Granvielle Harrison

and, since then, this method has been widely used in research laboratories. However, it is known that cell cultures can

be contaminated with microorganisms that can impair experiments. The most common contaminant are mycoplasmas,

characterized by lack of cell wall and 'fried eggs' shaped colonies. These prokaryotes alter cellular metabolism by

competing for the substrate and may invalidate the results obtained in in vitro experiments with cell cultures. This

study aims to analyze 15 normal and tumor cell lines from the Mutagenesis and Oncogenetics Laboratory of the State

University of Londrina (UEL), in order to evaluate their quality, since they are widely used in experiments.

Material and methods: Fifteen normal (CHO, HB4, NHA, PNT2, Wi, MRC-5 and GAS) and tumor (HTC, HeLa,

HepG2, PC3, LNCAP, Jurkat, ACP02 and ACP03) cell lines were used. All samples were cultured in their specific

medium and used to obtain the total DNA which extracted using commercial kit, and to performs PCR was using

Mollicutes primers, capable of amplifying conserved regions in different species of mycoplasmas. Finally, an agarose

gel electrophoresis was performed using specific primers for three species: M. orale, M. salivarium and M. arginine.

Results: The results obtained with primer for Mollicutes showed that, in a total of fifteen cultures, eleven were

contaminated. Only two cell lines showed contamination with mycoplasmas of M. orale. The most cultures were

contaminated with M. salivarium (nine). In the analysis of M. arginini, the band corresponding to the amplified DNA

was not observed even after tests with different conditions of PCR, suggesting that none of the strains had this

contaminant.

Conclusion: Based on the results obtained, cultures decontamination techniques will be employed with the use of

specific antibiotics and the analyzes will be redone in order to guarantee the quality of the cultures of the laboratory

cell bank.


EVALUATION OF ANTIVIRAL ACTIVITY OF PROPOLIS FROM Melipona quadrifasciata AGAINST

HERPES SIMPLEX VIRUS, IN VITRO.

Bertoli, A. M.1, Rechenchoski, D. Z.

1; Hochheim, S.

2; Cordova, C. M. M.

2; Galhardi, L. C. F.


1;

Nozawa, C.1

1 State University of Londrina, Department of Microbiology, Londrina, PR, Brazil

2 Regional University of Blumenau, Department of Chemistry, Blumenau, SC, Brazil

[email protected]

Keywords: Antiviral, propolis, HSV.

Introduction and objectives: Propolis has a resinous constitution collected by bees from different sources and its

chemical composition is associated to the plant diversity existing around the hive. The antimicrobial activity of propolis,

including the antiviral, is widely investigated. In this work, we investigated the anti-herpetic activity (HSV-1, Kos strain)

of propolis, produced by the bee M. quadrifasciata, in vitro.

Material and methods: The tested compounds were crude extract hydroalcoholic (EBH) obtained by exhaustive

maceration and partitioning generating 4 fractions: Ethyl acetate (FAc), Butanol (FBu), dichloromethane (FDi) and

aqueous (FAq). FDi was purified by silica gel column chromatography obtained the subfractions: F6, F9, F14, F24,

F34 and F45. The compound’s cytotoxicity and the antiviral activity were determined by MTT assay (dimethyl thiazole-

diphenyl tetrazolium bromide) and the selectivity index (IS) was calculated by the ratio between the cytotoxic and

inhibitory concentration of 50% (CC50%/IC50%).

Results: The least cytotoxic compound was the FAq, with CC50% of 1230 μg/mL and the most cytotoxic was found

for F9, with CC50% of 88.4 μg/mL. For the others compounds, the CC50% values, in μg/mL, were 143.7 (EBH), 420

(FAc), 350 (FBu), 272.5 (FDi), 380 (F6), 225 (F14), 472.5 (F24), 300 (F34) and 290 μg/mL (F45). After the

determination of cytotoxicity, the antiviral assay was performed, using concentrations ≤CC50% of tested compounds.

The highest anti-HSV-1 activity was found for F45, with a viral percentage of inhibition (%VI) of 70.6%, at 100 μg/mL,

followed by F14 (69% - 225 μg/mL), FAc 64.3% - 200 μg/mL) and FBu (57.7% - 300 μg/mL). The other compounds

presented %VI lower than 50%, at CC50%. Therefore, the IC50% was determined only for F14, F45, FAc and FBu

and presented the following values: 225 μg/mL, 58.5 μg/mL, 90.5 μg/mL and 294 μg/mL, with SI of 1.2, 4.6, 4.9 e 1.2,

respectively. Our study demonstrated that among the 11 tested compounds, only FAc and F45 inhibited about 70% of

HSV-1 replication, in VERO cells, with IS greater than 4.

Conclusion: The antimicrobial activity of propolis obtained by species of M. quadrifasciata bees has been

demonstrated against bacterial strains of S. aureus and E. coli, with weak inhibitory activity. However, the antiherpetic

activity of propolis from this bee species had not been reported. Further studies to improve the extraction of these two

compounds, as well as the identification of the chemical constituents, may be executed to continue the investigation of

the antiherpetic activity.




EVOLUTION OF THE EXPERIMENTAL ARTRITE INDUCED BY Paracoccidioides brasiliensis IN

WISTAR RATS

Zazula, M. F.1; Silva, C. F.

1; Dierings, L. C.

1; Eduardo, J. L.



1; Loth, A.

1.

1 Universidade Estadual do Oeste do Paraná, Cascavel, PR, Brazil.

[email protected]

Keywords: Experimental arthritis, paracoccidioidomycosis, Paracoccidioides brasiliensis.

Introduction and objectives: Paracoccidioidomycosis (PCM) is caused by the fungus Paracoccidioides brasiliensis

(Pb), and represents the most prevalent systemic mycosis in Latin America. It is considered a serious public health

problem, as it presents difficulties in the treatment and mainly affects economically active young adults. In systemic

infection, osteoarticular involvement may occur in about 60% of the chronic form. The joint manifestation of PCM is

accompanied by intense signs of inflammation and functional loss of the joint. However, few studies report details of

the infection, where the understanding of its evolution is based on assumptions found in the literature.

Material and methods: CEUA nº 05/2013. Thus, the present study evaluated the evolution of Pb-induced

experimental arthritis in the knee of Wistar rats. To that end, 24 Wistars rats with 45-60 days of life were distributed in

4 groups: control group; group 15 days; group 45 days; group 90 days, that were sacrificed on the respective dates. At

arthritis induction, the animals were anesthetized and the Pb18 suspension (1x105 concentration) was inoculated into

the medial region of the knee of each animal. The control group underwent the same procedure being inoculated with

PBS only. In order to analyze the evolution of edema and the perimeter of the joint were performed. After completion

of the exposure of each group, the animals were sacrificed the blood collected for analysis of the anti-Gp43 Pb

antibody, the joints were processed for histological analysis, stained in Hematoxylin and Eosin, and Grocott.

Results: Analyzes revealed classic anatomopathological signs of joint PMC, such as edema formation, synovitis and

granulomatous inflammation in all groups, except control. At 15 days of inoculation of the fungus, the anti-gp43 titre

was increased up to 45 days, followed by reduction of edema and titration of antibodies up to 90 days.

Conclusion: It was reported that there was progression of the joint lesion correlated with the exposure times during

the study, and with the presence of anatomopathological signs that indicated severe joint disease and infection

dissemination to the adjacent tissues.


FIRST REPORT OF NEW DELHI METALLO-β-LACTAMASE-PRODUCING Klebsiella pneumoniae IN

CLINICAL ISOLATES FROM UNIVERSITY HOSPITAL

Nava, R. G.1; Soncini, J. G. M.

1; Caraski, L.

1; Miranda, I. R.

1; Candido, E. P.

1; Mendes, E. C.

1; Buck, J. B.

1; Callado, G.

T.1; Magalhães, G. L.

1; Daga, A. P.

1; Carrara-Marroni, F. E.

1; Perugini, M. R. E.

1; Pelisson, M.

1; Vespero, E. C.

1.

1Universidade Estadual de Londrina, Laboratory of Clinical Microbiology of the University Hospital of Londrina,

Londrina, PR, Brazil

[email protected]

Keywords: Carbapenemases, enterobacteriaceae, resistance.

Introduction: The emergence of carbapenemase-producing Enterobacteriaceae has become a major public health

issue worldwide due to a high dissemination capacity and limited treatment options.According to Bush's classification,

the most prevalent and important carbapenemases in Enterobacteriaceae are the Klebsiella pneumoniae

carbapenemase — KPC (Ambler class A and Group 2f), metallo-β-lactamases (Ambler class B and Group 3) and

OXA type carbapenemases (Ambler class D and Group 2df). NDM-1 mediated by carbapenemases is the most

common class B carbapenemase in Enterobacteriaceae and has been detected increasingly in several countries. In

Brazil, this carbapenemase was first described in 2013, in Providencia rettgeri and Enterobacter hormaechei isolated

from the same hospital. In 2014, a NDM-1-producing Acinetobacter baumannii was also detected in Londrina, state of

Paraná. However, after this case, no further NDM-producing bacteria were reported in the Northern Paraná. In this

work, we have detected the first case of NDM-1-producing K. pneumoniae in University Hospital of Londrina (HU-

UEL).

Case report: On February 15, 2017, a 77-year-old female, diabetic, hypertensive, ex smoker, with previous stroke,

was admitted to the Hospital (HU-UEL), after falling from her own height, suffering a left femur transtrochanteric

fracture. On 27 February 2017, she underwent osteosynthesis and the multiple sepsis of pulmonary and urinary focus

evolved, which was treated. On day 34 (March 21, 2017), febrile and septic, blood cultures were performed. K.

pneumoniae was isolated from peripheral blood and blood via catheter. Susceptibility tests were conducted using the

disk diffusion method, automated testing (Vitek® 2 bioMérieux system). K. pneumoniae was resistant to meropenem

(MIC of ≥ 16mg/L), imipenem (MIC of 8mg/L), ertapenem (MIC of ≥ 8mg/L) and all cephalosporins were resistant, but

succeptible to aztreonam, colistin and polymyxin, according as CLSI breakpoints. The screening for NDM was

performed method using meropenem and imipenem with and without EDTA 0.1M which showed increase in > 5 mm in

diameter, suggesting the presence of a metallo-β-lactamase. The blaNDM-1 gene was detected by multiplex

quantitative PCR (qPCR). NDM was also confirmed by sequencing and was notified. On day 74 (April 30, 2017), the

patient presented urinary tract infection and hospital pneumonia. On day 83, without improvement, the patient was

treated only with palliative treatment. The patient died on day 102 of hospitalization. After laboratory confirmation of

infection by NDM-producing K. pneumoniae, it was reported and implementation of prompt infection control measures,

including isolation of the patient from the time of admission and surveillance cultures likely contributed to containing

the spread among our patient population. The study was approved by the research ethics committee of the State

University of Londrina CAAE0015.0.268.000-11.

Final consideration: In this work, the first case infection by NDM-producing K. pneumoniae in in the Hospital (HU-

UEL), was reported. Considering that carbapenemases-producing isolates are highly endemic in Brazil, our finding is

of major concern, since the emergence of NDM-1 is a serious threat to antimicrobial therapy against

Enterobacteriaceae in this country. It also shows the importance of epidemiological surveillance studies necessary to

know the dissemination of resistance mechanisms and control of hospital infections.


FUSARIOSIS IN IMMUNOCOMPETENT MICE

Costa, M. I.1; Rodrigues, F. A. V.

1; Jarros, I. C.

1; Veiga F. F.

1; Kischkel, B.

1; Negri M.

1; Becker T. C. A.

2; Svidzinski,

T. I. E. 1

1 Universidade Estadual de Maringá, Department of Clinical Analysis & Biomedicine, Maringá, PR, Brazil

2 Universidade Estadual de Maringá, Department of Basic Health Sciences, Maringá, PR, Brazil

[email protected]

Keywords: Disseminated fusariosis, Fusarium infection, Fusarium.

Introduction and objectives: Fusarium is the second mold most frequently associated with human fungal infections.

Due to the fact that this mold is easily found in the environment, the respiratory tract is one of the main gateways for

the development of the disease. The aim of this work was to assess by CFU (Colony Forming Unit) and

histopathology of the organ distribution of the fungus in immunocompetent mice infected with Fusarium solani by

intratracheal injection.

Material and methods: All procedures involving animals were approved by the Ethics Committee in Animal

Experimentation (6975260716/2016). Fifteen animals were inoculated with a suspension of 50 µl of 1x108 cells of

Fusarium solani. One animal represented the control of the procedure, consisting of an injection of PBS (Phosphate

Buffered Saline). The animals were culled 24 hours after the infection and the heart, kidneys, lungs, eyes, brain,

spleen, liver were aseptically removed. Half of each organ was weighed and homogenized in PBS and serial dilutions

of this homogenate were spread onto Potato Dextrose Agar, to determine CFU. The remaining halves of the organs

were HE and Grocott-gomori staining analysis.

Results: Intratracheal inoculation led to rapid development of systemic infection, with all mice becoming severely ill

within 24 hours. According to the CFU counts, fungal burdens in the lungs declined by 1-2 orders of magnitude

compared to the initial inoculum and no count were obtained from other organs. In the tissue analysis, it was possible

to observe in the lungs hyphae, severe tissue damage and massive necrosis of the alveolus without clear structures.

In the other organs, only the presence of fungal cells was observed.

Conclusion: It was possible to infer that even in immunocompetent mice, F. solani was able to disseminate over

different organs through intratracheal infection. This way, intratracheal infection was proven to be efficient for the

Fusarium infection.

Grants: We thank CAPES for the financial support of this project.


Galleria mellonella AS AN IN VIVO MODEL FOR THE EVALUATION OF THE VIRULENCE POTENTIAL

OF Enterococcus faecium AND Enterococcus faecalis

Terra, M. R.1; Furlaneto, M.C.

1; Furlaneto-Maia, L.

2

1 State University of Londrina, Department of Microbiology, Londrina, Brazil.

2 Federal Technological University of Paraná, Department of Food Technology, Londrina, Brazil.

[email protected]

Keywords: Enterococcus, virulence, Galleria mellonella.

Introduction and objectives: The genus Enterococcus is widely studied as to its genotypic and phenotypic profile of

virulence determinants, being attributed to the presence and expression of these determinants its emergence as a

pathogen of nosocomial infections. However, these virulence studies are limited to in vitro analysis, with few studies

being conducted in vivo. In view of this gap in science, our work proposed to evaluate the virulence potential of

Enterococcus sp. employing Galleria mellonella as a biological model.

Material and methods: For this, 14 isolates of Enterococcus were selected. Of these 3 are avirulent and 11 are

virulent, among them 7 containing the gene gelE (gelatinase), 2 containing the ace (adhesin), cpd (sex hormone) and

gelE and 2 genes containing the genes ace, asa (aggregation substance) gelE and cpd. As control, the strain ATCC

E. faecalis 29212 was used. For the survival assays of G. mellonella, groups of 10 larvae / isolates were used,

receiving an inoculum of 0.5 μL in the final concentration of 7.5x105 and 1.5x10

6. The larvae were incubated at 37ºC

with monitored survival every 24 hours for 120 hours, being considered dead when in the absence of response to

physical stimulation and melanization. The experiment was carried out in triplicate. As a control group, untreated

caterpillars and caterpillars were inoculated with 0.5 μL of 1X PBS, kept under the same conditions as in the test

group. The GraphPad Prism 5 program (La Jolla CA, USA) was used to analyze the results, arranged on a survival

curve using the Kaplan-Meier method and statistical analysis was performed using the log-rank test.

Results: Under the conditions tested, larvae inoculated with avirulent and virulent strains resulted in survival rates

between 93-100% and 60-93% for (P value <0.001), respectively. The survival rate of the G. mellonella group infected

with Enterococcus gelatinase positive isolates was 92-99%. In contrast, the group of larvae infected with

Enterococcus gelatinase negative presented 97-100% survival. After 48 hours of infection the initial bacterial

concentration differed according to the virulence profile, remaining stable for avirulent isolates, with 28 x 10-4

CFU /

larvae averaging 98% survival after 120 hours of infection. In the larvae infected with virulent isolates, there was a

decrease reaching a mean of 79% survival in 120 hours of infection.

Conclusion: The present study demonstrates that G. mellonella is a promising model for studying the survival of E.

faecalis and E. faecium isolates. Both species are able to kill G. mellonella larvae, leading to high mortality rates when

in higher concentrations. In the tested virulent isolated conditions were more lethal to the host when compared to the

avirulent isolates that present higher survival rates of G. mellonella being more persistent in the host when compared

to the virulent isolates that causes greater number of deaths in less time.

Grants: CNPq, Fundação Araucária


IN VITRO ACTIVITY OF CAFFEIC ACID ON PROMASTIGOTE FORMS OF L. amazonensis

Bortoleti, B. T. S.1; Carloto, A. C. M.

1; Tomitto-Pellissier, F.

1; Gonçalves, M. D.

2; Miranda-Sapla, M. M.

1; Assolini, J.

P.1; Costa, I. N.


1; Pavanelli, W. R.

1


2Universidade Estadual de Londrina, Department of Chemistry, Londrina, PR, Brazil

[email protected]

Keywords: Flow cytometer, leishmaniasis, treatment, annexin V, oxigen-reactive species.

Introduction and objectives: American Cutaneous Leishmaniasis (ACL) is an anthropozoonosis caused by protozoa

of the Leishmania genus transmitted to humans through the bite of the sandfly insect. It is present in several

manifestations such as ulcerative lesions, destructive mucosal inflammation and diffuse non-ulcerated lesions. The

high toxicity, high costs and resistance of some strains to current medications increase the search for more effective

therapeutic alternatives. In this context, caffeic acid presents as a potential drug, possessing several biological

activities as antioxidant, antihypertensive, anti-fibrotic and radical scavenging. In addition, extracts containing caffeic

acid, has leishmanicidal activity on L. major, L. donovani and L. braziliensis species. Based on these studies, the

objective this work was to verify the leishmanicidal activity of caffeic acid on the promastigotes forms of L.

amazonensis, as well the morphological alterations and mechanisms involved with parasite’s death.

Material and methods: Promastigotes forms of L. amazonensis was maintained in 199 medium and used in

stacionary phase. The antipromastigotes assay was performed through neubauer chamber counting. This assay was

confirmed by scanning electron microscopy and flow cytometer, using a BD Accuri™ C6 Plus personal flow cytometer.

The death mechanism was performed by fluorescence techniques using a H2DCFDA probe, propidium iodide and

annexin V labeling, assay confirmed by double labeling of propidium iodide and annexin V analyzed by flow

cytometer. Data were expressed as mean ± standard error of the mean. At least three independent experiments were

performed, each with duplicate datasets. Significant differences between the groups were determined by one-way

ANOVA, followed by Tukey’s test.

Results: The results showed that from 6.25 µg/mL all concentrations had direct action on promastigote forms after 24,

48 and 72 hours of treatment. The determined IC50 (12.5 µg/Ml) was used to observe the morphological changes

occurring in the parasites. The rounding and reduction of parasite body size were, and this result was complemented

by flow cytometer, where was observed a reduction of cell volume. Knowing that the compound induces morphological

changes, we investigate biochemistry and physiological alterations induced by the treatment. It was that the caffeic

acid induced production of reactive oxygen species (ROS), as well as exposure of phosphatidylserine and membrane

damage in the parasite. In addition, it was observed that the majority of the population was in late apoptosis-like

process.

Conclusion: It is concluded that caffeic acid is able to act directly on L. amazonensis promastigotes, altering its

morphology and being able to induce death by apoptosis-like mechanism.

Grants: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) e Conselho Nacional de

Desenvolvimento Científico e Tecnológico (CNPq).



INCIDENCE OF CASES OF CRYPTOSPORIDIOSIS- Cryptosporidium spp- IN PATIENTS ATTENDED

AT THE UNIVERSITY HOSPITAL OF LONDRINA.

Assis, E. F.1; Martins, C. M.

1; Moreira, D.

1.

1 Universidade Estadual De Londrina, Department Of Parasitology, Londrina, Pr, Brazil

[email protected]

Keywords: Cryptosporidiosis, parasites, opportunistic and human immunodeficiency virus.

Introduction and objectives: Human cryptosporidiosis is an infection caused by Cryptosporidium spp, an

opportunistic, emerging protozoan that has habitat portions of the gastrointestinal tract and is responsible for the

occurrence of episodes of severe watery diarrhea in humans, especially in immunocompromised individuals.

However, it has been noted that the number of cases in immunocompetent individuals has become increasingly

frequent. Diseases caused by opportunistic parasites are closely related to deficient immune states, such as in the

cases of the human immunodeficiency syndrome (AIDS) and also in transplanted individuals. However, lower

socioeconomic conditions, undernutrition and / or malnutrition, untreated water consumption, lack of basic sanitation,

immune system immaturity and inadequate hygienic habits are factors that can Cases of cryptosporidiosis in children,

as well as, has been associated with chronic diarrhea and reduced survival in HIV-positive patients.

Material and methods: Fecal samples from patients were collected in the sterile vial, preserved in S.A.F. (ACETO-

FORMALDEIDO SOLUTION), and with the reception of the samples in the crop-cultivation laboratory, at the Clinicas

Hospital of Londrina, were made by the methodology of Safranin Coloration, in which the microscopic reading, by

studying Cryptosporidium cysts . To diarrheic samples or not, they were collected from patients attended at the

outpatient clinic of Infectious Diseases, emergency room and wards of the University Hospital of Londrina between

June 2016 and June 2017.

Results: In consideration of the results obtained, 111 samples of patients attended at the Specialist Ambulatory of the

University Hospital of Londrina, from June 2016 to June 2017, of which 45.94% were positive samples for

Cryptosporidium, 26 (51%) were diagnosed as HIV positive. The distribution of positive cases for cryptosporidiosis in

HIV patients was as follows: 0 to 10 years -11 cases; From 11 to 20 years - 8 cases; 21 to 30 years- 1 case; 31 to 40

years - 6 cases; 41 to 50 years - 10 cases; 51 to 60 years - 6 cases; 61 to 70 years - 5 cases and 71 to 81 years - 4

cases. The study of Cryptosporidium spp in patients with HIV / AIDS showed that its presence is closely linked to the

occurrence of diarrheal syndrome, including the reduction in CD4 + T lymphocytes count.

Conclusion: Our work corroborates the researched literature, in which Cryptosporidium spp is a prevalent parasite in

HIV-positive patients. Regarding the samples surveyed in this time period, the highest incidence was in children aged

0 (zero) to 10 years. Some factors may be related to the results found: the possibility of vertical transmission,

breastfeeding by HIV positive mothers, lack of interest in the treatment of the parents with this syndrome,

unsuccessful prenatal care and / or lack of family counseling.


INCIDENCE OF MULTI-DRUG RESISTANT PATHOGENS FROM “ESKAPE” GROUP IN THE

INTENSIVE THERAPY UNIT OF A UNIVERSITY HOSPITAL OF THE SOUTH REGION OF BRAZIL.

Danelli, T.1; Aoki, G. H.

1; Duarte, F. C.

1; Ribeiro, M. A. G.

1; Tizura, A. T.

1; Gasparotto, L. C.

1; Vespero, E. C.

1; Carrara-

Marroni, F. E.1; Perugini, M. R. E.

1.

1Departamento de Patologia, Análises Clínicas e Toxicológicas, Universidade Estadual de Londrina, Paraná, Brasil.

[email protected]

Keywords: multiresistant microorganisms, incidence, antimicrobials, eskape.

Introduction and objectives: Infections caused by multidrug-resistant microorganisms have become prevalent in

recent decades and constitute a serious public health problem. The treatment of these infections is difficult and,

consequently, associated with high rates of morbidity and mortality. In 2008, Infectious Diseases Society of America

(IDSA) classified five resistant pathogens that cause the majority of hospital infections by the acronym ESKAPE

(Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas

aeruginosa, and Enterobacter spp.). The present study aims to evaluate the incidence of these pathogens in an

intensive care unit of a university hospital in the southern region of Brazil, from January to December 2014.

Material and methods: The results of antimicrobial susceptibility tests performed by automated microdilution by the

Vitek2® System (bioMérieux), for 814 clinical isolates, including blood, urine, respiratory secretions, cavity liquids,

bone fragments and tissue fragments, among others. This is a retrospective descriptive horizontal study, which was

approved by the Research Ethics Committee involving humans under the number CAE: 43013315.8.0000.5231.

Results: Resistance to carbapenems was observed with higher incidence in A. baumannii, followed by K.

pneumoniae and P. aeruginosa, respectively, with a mean of 14.57, 5.58 and 5.11 per 1,000 patients/day,

respectively. On the other hand, methicillin resistant S. aureus isolates had an average incidence of 2.98 per 1,000

patients/day, whereas vancomycin-resistant E. faecium presented 1.57 per 1,000 patients/day and carbapenem-

resistant E. cloacae 0,48 per 1,000 patients/day.

Conclusion: Hospital infections caused by ESKAPE group of microorganisms cause serious problems related to

health care in intensive care units, increasing the length of stay in these units, antimicrobial expenses and the

chances of patients evolving to death. It is very important value to point out that the conscious use of antimicrobials,

de-escalation of therapy and microbiological tests adequate to the identification of multidrug-resistant microorganisms,

favor the reduction of rates of these infections.


INSULIN AND GLUCOSE PROMOTES PROLIFERATION IN VITRO OF

Leishmania (Leishmania) amazonensis

Silva, P. R. B.1; Alvarenga, D. S.

1; Melanda, F. N.

1; Costa, I. N.¹; Pavanelli, W. R.¹; Conchon-Costa, I.

1 1 Department of Experimental Pathology – Laboratory of Experimental Protozoology, State University of Londrina,

Londrina, Brazil.

[email protected]

Keywords: Insulin, glucose, Leishmania amazonensis.

Introduction and objectives: Leishmaniasis is a zoonosis caused by protozoans of genus Leishmania spp.,

considered the sixth disease of importance by World Health Organization (WHO). Some studies have identified in

promastigotes of Leishmania the insulin receptor like insulin substrate-1 (IRS-1) receptor in human. The hormone

Insulin growth factor-1 (IGF-1) is capable of binding to this insulin receptor in Leishmania spp. parasites and lead to

the proliferation of promastigotes and amastigotes within the macrophages, exacerbating the lesion in mice. However,

there are no studies evidencing the action of insulin in Leishmania spp. Furthermore, we do not know too the effects of

glycemia in promastigotes forms once these are directly associated. Therefore the objective of this study is to evaluate

in vitro whether insulin or different glucose concentrations can affect the proliferation of L. amazonensis.

Material and methods: Promastigotes forms of L. amazonensis (105) were cultivated in 24-well plate and stimulated

with glucose (25, 12,5 and 6,25mM), and/or insulin (10mU/mL) in 1mL of 199 culture medium and incubated in a

B.O.D. at 24°C. The number of promastigotes were evaluated by counting in Neubauer chamber in 24, 48 and 72

hours. The results were expressed as the mean ± standard error (SEM) and analyzed with the Prism 5.0 statistical

program using the one-way ANOVA, followed by Tukey's test. This experiment does not require approval from the

ethics committee.

Results: We observed that the treated with glucose (25, 12,5 and 6,25mM) increased the proliferation significantly in

48 and 72h but, this event was not dose dependent. The treatment with insulin increased four or five times the

promastigotes proliferation in 48 and 72h respectively. Although increased the promastigotes forms proliferation in 48

and 72 h with insulin and glucose, together we did not observe an exacerbated effect.

Conclusion: Our data demonstrated that both insulin or glucose can affect the growth of promastigotes of L.

amazonensis. However, the stimulation with insulin and glucose concomitant did not intensify proliferation.


MANGIFERIN INHIBITS ACYCLOVIR-RESISTANT HERPES SIMPLEX VIRUS TYPE 1, IN VITRO

Rechenchoski, D. Z.1; Agostinho, K. F.

1; Faccin-Galhardi, L. C.

1; Ricardo, N. M. P. S.


1; Nozawa,

C.1

1 State University of Londrina, Department of Microbiology, Londrina, PR, Brazil.

2 Federal University of Ceara, Department of Organic and Inorganic Chemistry, CE, Brazil.

[email protected]

Keywords: Antiviral, mangiferin, HSV, resistance

Introduction and objective: Herpes simplex virus (HSV) infections are common; nevertheless, their outcome can be

of unpredictable prognosis in neonates and in immunosuppressed patients. Acyclovir (ACV) is the antiviral of choice

for treatment of the disease, however, its widespread use has shown that HSV develops resistance through mutations

in genes coding for thymidine kinase or for DNA polymerase. Therefore, it is important to develop new antiviral drugs

with different mechanisms of action which could be a substitute or a complement for ACV. In this context, the search

for natural substances has been encouraged. Mangiferin is a xanthone glucoside obtained from various plants, such

as Mangifera indica L. and empirically is used against diarrhea, vomiting, rheumatism, itch, asthma and gastric

disorders. Multiple pharmacological activities have already been scientifically demonstrated, such as: antitumor,

immunomodulatory, antidiabetic, antioxidant, anti-inflammatory, antiallergic, antibacterial, antifungal and antiviral. This

study aimed to investigate the antiviral activity of mangiferin against acyclovir-resistant strain of HSV-1 (AR-29).

Material and methods: The cytotoxicity of compound, in Vero cell culture, was evaluated by the dimethylthiazolyl-

diphenyltetrazolium bromide method (MTT) and the antiviral action by the plaque reduction assay, using the following

protocols: simultaneous treatment with the viral infection, virucidal effect and inhibition of adsorption. The 50%

citotoxic concentration (CC50) and the 50% inhibitory concentration (IC50) were determinated by linear regression and

the selectivity index (SI) expressed as the ratio of CC50/IC50. Additionally, indirect immunofluorescence assay was

performed for to evaluate the interference of the mangiferin in viral protein synthesis.

Results: The CC50 found for mangiferin was >500 μg/mL and the IC50 of 2.9 μg/mL, with SI >172.4. Preliminarly,

mangiferin was tested at concentrations of 12.5, 6, 3, 1.5 μg/mL, and showed the percent of viral inhibition (%VI) of

100%, 94.4%, 57.6% and 10.4%, at the simultaneous treatment with the infection, respectively. For virucidal assay,

the mangiferin demonstrated an inhibition of 100% for the two highest concentrations and 98.9%, 72.2% and 30.5% at

concentrations of 3, 1.5 and 0.7 μg/mL, respectively. While the inhibition of adsorption assay showed a %VI of 71.3%,

62.2% and 30.8%, at the concentrations of 12.5, 6 and 3 μg/mL, respectively. The inhibition of HSV-1 by mangiferin

was also demonstrated by the decrease of fluorescent cells, showing a percent of viral inhibition (%VI) of 54.1% and

35.2% at concentrations of 12.5 and 6 μg/mL, respectively.

Conclusion: We demonstrated that mangiferin showed a low toxicity, associated with high selectivity index, which

makes the substance potentially promising. Finally, based on the initial results in vitro of the mechanism of action of

this compound, in vivo experimentation can be conducted and qualify this product for the development of new drugs.



MCR-1 COLISTIN RESISTANCE IN ESBL PRODUCING-Escherichia coli, IN BRAZIL

Soncini, J. G. M.1; Nava, R. G.

1; Caraski, L.

1; Miranda, I. R.

1; Magalhães, G. L

1.; Perugini, M. R. E.

1; Pelisson, M.

1; De

Paula, S. B.1; Carrara-Marroni,F. E.

1; Koga, V. L.

2; Kobayashi, R. K. T.

2; Vespero, E. C.

1

1Universidade Estadual de Londrina, Laboratory of Clinical Microbiology of the University Hospital of Londrina,

Londrina, PR, Brazil 2Universidade Estadual de Londrina - Departamento de Microbiologia - CCB/UEL

[email protected]

Keywords: Enterobacteriaceae, colistin, mcr-1 gene.

Introduction: The plasmid-mediated colistin resistance mechanism MCR-1 as become a great challenge to public

health worldwide. In fact, since its initial identification in Enterobacteriaceae strains (mostly Escherichia coli) isolated

from animals, food, and humans in China, MCR-1 has also been reported in other countries in Asia, Africa, Europe,

and North America. In South America, E. coli harboring the mcr-1 gene has been present in food-producing animals

since at least 2012, being recently identified in human clinical samples from Argentina. In Brazil, the first case the

colistin-resistant Escherichia coli strain was recovered from a patient with a diabetic foot infection in, June 2016.

Case report: On February 10, 2017, a 90-year-old female, and was admitted to a public hospital in the north of

Parana, Brazil, with a previous history of recurrent intestinal constipation, due to Chagasic cardiopathy, megacolon

and megaesophagus. Previously, the patient was admitted to a secondary hospital for intestinal lavage, where she

was hospitalized for a day. After this procedure, she drained secretion in the stool. In the public tertiary hospital, she

was admitted to the emergency room and underwent laparotomy at the surgical center and collected secretion culture

during the procedure. Empiric therapy with ertapenem, ciprofloxacin and metronidazole was initiated. The culture

yielded growth of colistin-resistant extended-spectrum-lactamase (ESBL)-producing E. coli (EC-1 strain) and

Streptococcus anginosus. Identification initial of EC-1strain were performed with the VITEK 2 Systems (bioMe´rieux,

Durham, NC, USA). Screening for ESBL was performed in accordance with Clinical and Laboratory Standards

Institute (CLSI), 2017, recommendations using the double-disk synergy test. The blaTEM, blaSHV, blaCTX-M gene were

avaliable using specific primers and showing positive result for blaCTX-M. Amplicon was sequenced and compared to

sequences available in the GenBank database and showed belong to blaCTX-M-8 subtype. EC-1strain was showed

Minimal Inhibitory Concentration (MIC) for colistin (4 mg/L), realized by Etest method. The presence of the blaMCR-1

gene in EC-1 strainwas confirmed by Polymerase Chain Reaction (PCR) and confirmed by sequencing. A culture of

surveillance of all patient contacts was collected, but all the results were negative. After the 14-postoperative day, the

patient evolves to obit due to pneumonia and sepsis. This study was approved by the research ethics committee of

the State University of Londrina CAAE0015.0.268.000-11.

Final consideration: In this work, the first case infection by colistin-resistant microorganism in the public tertiary

hospital in North Parana state, was reported. Our findings emphasize the urgent need for coordinated global action in

the fight against pan-drug-resistant Gram-negative bacteria.


MORPHOLOGICAL ALTERATIONS IN THE ENTERIC NERVOUS SYSTEM OF RATS INFECTED WITH

Toxoplasma gondii

Machado, C. C. A.1; Sant’Ana, D.M.G.

2; Araújo, E. J. A.

1 1State University of Londrina, Department of Histology, Londrina, PR - Brazil

2State University of Maringa, Department of Morphological Science, Maringa, PR - Brazil

[email protected]

Key words: toxoplasmosis, enteric nervous system, neuronal plasticity.

Introduction: Toxoplasmosis is a worldwide disease caused by Toxoplasma gondii. During its biological cycle, the T.

gondii has to cross the intestinal wall to reach systemic circulation. The parasite triggers a local inflammatory process

due its intense proliferation and cellular invasion. Inflammation in the intestinal wall can cause damage in the enteric

nervous system (ENS). This review has the objective of presenting the main morphologic findings observed in the

ENS of rats infected with T. gondii.

Review: The alterations caused by T. gondii on the ENS of the small intestine vary according to infection time (acute

or chronic) and with genotype of T. gondii strain used (I, II or III). Rats inoculated intraperitoneally with genotype I

strain present death of jejunal myenteric neurons accompanied of morphometric alterations in all segments of the

small intestine during the chronic phase. The genotype II strains do not cause death myenteric neurons in the ileum,

however they provoke hypertrophy of the cell body during acute and chronic phases. Besides, hyperplasia of nitrergic

myenteric neurons was also observed. Rats infected with different T. gondii inoculum loads present intense jejunal

alterations when exposed to greater parasite doses. Besides, these rats present a reduction in the number of

metabolically active myenteric neurons, increasing of cholinergic neurons, and cell body atrophy. Regarding to

submucosal plexus, the infection with genotype II strains decreased the number of jejunal VIPergic neurons during the

chronic phase. Rats infected with genotype III strain present atrophy of total myenteric neurons in the duodenum and

ileum during the acute phase. In the chronic phase, this strain triggers hypertrophy of total myenteric neurons in

duodenum and atrophy of jejunal nitrergic myenteric neurons. Regarding the large intestine, rats infected with

genotype I strain present increase in density of ceacal total myenteric neurons during the infection chronic stage.

However, no changing was observed in the number of colonic myenteric neurons, but cells were hypertrophied. Rats

infected with genotype II strain present decrease in number of total myenteric neurons and increase of nitrergic

neurons. Larger parasite inoculum doses cause more significant alterations in the colonic neurons. In the submucosal

plexus, infections caused by this genotype II strain caused decrease in number of total neurons, hypertrophy and

increase in VIPergic neurons. Rats infected with genotype III strain show myenteric neurons atrophy in the descending

colon during the acute phase followed hypertrophy during the chronic phase.

Conclusion: The published data so far demonstrated that T. gondii is a parasite capable of provoke alterations in the

ENS. Further studies should be carried out mainly evaluating whether the alterations may be related to gastrointestinal

disturbs.

Grants: CAPES (Scholarship of the first author).


MORPHOLOGICAL ALTERATIONS OF HAMSTERS ANKLE JOINT CAUSED BY

Leishmania (Viannia) braziliensis EXPERIMENTAL INFECTION

Retameiro, A. C. B.1; Zanardini, B.


1; Ribeiro, L. F. C. 1, Lima, L. L.

2, Santos, A. G. A.

2, Silveira,

T. G. V. 2, Nogueira-Melo, G. A.

2 1Universidade Estadual do Oeste do Paraná, Cascavel, PR, Brazil

2Universidade Estadual de Maringá, Maringá, PR,Brazil

[email protected]

Keywords: Leishmaniasis, ankle Joint, ankle Injuries.

Introduction and objectives: Leishmaniasis is characterized as an infection sickness caused by parasites from

Leishmania gender, which ones multiply themselves inside of macrophages. American tegumentary leishmaniasis

(ATL) is a denomination that includes the cutaneous and mucosal form of this sickness that affects the entire Brazilian

territory. In south region, from the 296 cases registered in 2013, 273 were in Paraná. There are several clinical

manifestations, one of them, as shown in histopathological studies, are bone and joint diseases. In this way, the aim of

this study was analyze the effect of ATL in ankle joints of female hamsters.

Material and methods: All the procedures that involved animal’s use were approved by Ethic Commission In

Animal’s use from Universidade Estadual de Maringá (CEUA/UEM nº 7587260416). Ankle joints samp les were

collected from 8 left pelvic female hamsters (Mesocricetus auratus) members, which ones stayed in controlled

temperature (22º C ± 1) during 12 weeks. After this period, they were randomly separated into two groups: 1) Control

Group (CG, n=4) and 2) Infected Group (IG, n=4). For IG infection, the animals were anesthetized with a 10 mg/kg of

xilazin (Calmiun Agener-Union Animal Health) and 50 mg/kg of cetamin (Francotar® – Virbac Animal Health)

combination and suffered intradermal infection in left foot’s back with a 50 μl of promastigote suspension (2X107 -

MHOM/BR/2003/2311) and CG received 50 μl of PBS. After 90 days of parasite inoculation, the animals underwent

euthanasia and the samples were collected.

The ankles joint samples were descalcified by 5% trichloroacetic acid, submitted to histological routine, to obtain

sections (7 μm), stained with hematoxylin/eosin and photodocumented in to detailed morphological analysis.

Results: The left ankle joints of CG showed characteristic morphology, with a smooth cartilage surface and organized

in four normal cells layers. The chondrocytes were classified into gaps, corresponding to the deep zone separated

from the calcified zone by a basophilic line, the tidemark. In IG, the cartilage presents irregular characteristics in

relation to CG, like floculations in the surface, bigger chondrocytes, which ones were not organized in layers and the

tidemark could not be visualized. In addition, it was observed the formation of pannus recovering to articular cartilage,

being indicative of injury.

Conclusion: The ankle joint is a distal syndesmosis between tibia and fibula and a fitting formed by the distal third of

tibia and fibula bones with talus that provides moves like flection (plantar flection), extension (dorsal flexion), inversion

and eversion. With the injuries funded in IG we believe this moves will be negatively affected, what could compromise

them, justifying the alterations found in the animals of this group. What leads us to conclude that, in the studied

parameters, ATL has a negative effect under female hamster ankle joint.


MORPHOLOGICAL ASPECTS OF ANKLE JOINT’S SYNOVIAL MEMBRANE IN HAMSTERS

EXPERIMENTALLY INFECTED BY Leishmania (Viannia) braziliensis

Retameiro, A. C. B. 1; Zanardini, B.


1 Brancalhão, R. M. C.

1; Lima, L. L.

2, Santos, A. G. A.

2, Silveira,

T. G. V. 2, Nogueira-Melo, G. A.

2

1Universidade Estadual do Oeste do Paraná, Cascavel, PR, Brazil

2Universidade Estadual de Maringá, Maringá, PR, Brazil

[email protected]

Keywords: Synovial membrane, leishmaniasis, ankle Injuries

Introduction and objectives: American tegumentary leishmaniasis (ATL) is a patology causes by different

Leishmania species, parasite protozoa that, in amastigote stage assaults, mainly, macrophages, which ones are

responsible for part of the host’s immunological answer. The parasite can reach, in addition to the skin, many other

organs. In this way, the synovial membrane, highly vascularized structure, which is responsible to produce de synovial

liquid, can also be affected by Leishmania infection. ATL is a sickness with epidemiologic matter and widespread

through all the Brazilian territory, what makes it effects deserves attention. Thus, the aim of this study was to evaluate

the effect of the infection by Leishmania (Viannia) braziliensis, under the ankle joint synovial membrane of hamsters.

Material and methods: Female hamsters (Mesocricetus auratus) with nearly 90 days old were anesthetized with 10

mg/kg of xylazine and 50 mg/kg of cetamine. On control group (CG n=4) the animals were inoculated with 50 μL of

PBS by intradermal path on the left foot back. The infected group (IG, n=4) was inoculated in the same way with 50 μL

of L. (V) braziliensis promastigote suspension (2x107- MHOM/BR/2003/2311). The animals were kept in controlled

conditions with water and food at will and euthanized after 90 days of infection. The left ankle’s joint was collected,

processed with paraffin and the slides were stained with hematoxylin and eosin for morphological analyzes. The

experiment was approved by Ethic Comission In Animal’s Use from Universidade Estadual de Maringá (CEUA/UEM

nº7587260416).

Results: In CG, as expected the synovial membrane presented regular morphological characteristics, where intimate

synovial layer present two or three synoviocytes layers and the sub intima layer showed predominance of adipose

cells. In general, the IC group synovial membrane showed distinctive characteristics than those described for CG,

being observed inflammatory infiltrate and blood vassels increase as well. Furthermore, in some animals was possible

to observe a thicker and fibrous membrane. The lesions caused in animal’s pelvic member probably decrease moves

amplitude, what can promote alterations in ankle’s synovial membrane, as the presence of the pathogen itself.

Conclusion: Against the morphological alterations found on this study, we conclude that the L. (V) braziliensis

experimental infection promoted lesions on the female (Mesocricetus auratus) hamsters ankle’s joint synovial

membrane.


OUTBREAK OF VERMINOTIC PNEUMONIA IN CATTLE FROM NORTHERN PARANÁ

Michelazzo, M. M. Z.1; Oliveira, T. E. S.

1; Pelaquim, I. F.

1; Sasse, J. P.

1; Garcia, J. L.

1; Headley, S. A.

1

1Universidade Estadual de Londrina, Department of Preventive Veterinary Medicine, Londrina, PR, Brazil.

[email protected]

Keywords: Dictyocaulus, bovine diseases, lungworms, ruminants, verminous pneumonia.

Introduction: Dictyocaulus sp. is a nematode that parasitizes the respiratory tract of ruminants, equids and mules. D.

viviparus specie is the lungworm most commonly associated with disease in cattle. Adult worms can measure up to

eight centimeters in length and usually inhabit the caudal lobes of the lungs, resulting in verminotic pneumonia and/or

bronchitis. Clinical signs vary depending on the condition of animal and the severity of the infection, and may include

anorexia, weight loss, dry or productive cough, and tachypnea with forced expiratory movements. In Brazil, there are

few studies on the prevalence of Dictyocaulus sp. in herds. This report aims describes the occurrence of verminotic

pneumonia in a property with a complete cycle system from northern of Paraná, where four cattle died.

Case report: A 24 months old, female, Nelore cow, with clinical respiratory signs was received for necropsy at the

Laboratory of Animal Pathology, UEL. Stool samples of this animal and other cattle from the herd were sent to the

Laboratory of Parasitology, UEL for coproparasitological evaluation. Gross findings included atelectasis, severe

edema, ventro-caudal consolidation, and the presence of a marked number of filiform nematodes in the lumen of the

bronchi. Microscopically, there was observed chronic multifocal to coalescent, parasitic bronchopneumonia

characterized by a severe inflammatory infiltrate composed predominantly of eosinophils and neutrophils with few

lymphocytes and histiocytes in the epithelium of the alveoli, bronchioles and bronchi associated with intralesional

larvae and eggs of a nematode. In addition, there was severe pulmonary edema. Coproparasitological examination by

the Baerman technique revealed the first larval stage (L1) consistent with Dictyocaulus, due to characteristic

morphological features. A diagnosis of verminotic pneumonia was based on the clinical signs, morphological

identification of the nematodes collected at necropsy, pathological findings and coproparasitological examinations

which are consistent with the descriptions for this condition in the literature. In cases of infections with low parasitic

load, cattle usually recover and show no clinical signs of the disease. However, when there is a high parasitic load in

the pasture and favorable climatic conditions for the development of D. viviparus larvae, cattle animals are severely

infected and the disease progresses to more severe stages, with clinical signs and the death of animals as occurred

during this outbreak.

Final consideration: Due to the findings in the animal mentioned in this report, it is likely that the sanitary conditions

at the farm in question were compromised and the pasture probably had an elevated load of D. viviparus larvae which

facilitated the infection. In addition, a possible parasitic resistance to the active principle used in the deworming

protocol at that herd cannot be totally ignored as being contributory towards the development of the outbreak herein

described.

Grants: CAPES, CNPq, Fundação Araucária, MEC.


PROSPECTING AND CHARACTERIZATION OF INTRINSIC DISORDER IN PROTEINS FROM VIRUS

ASSOCIATED WITH DISEASES

Resende, M. A.1; Pereira Lima. G. Y.

1; Nicolau Junior, N.

1

1 Universidade Federal de Uberlândia, Instituto de Genética e Bioquímica, Uberlândia, MG, Brazil

[email protected]

Keywords: virus, intrinsically disorder, protein, disease.

Introduction and objectives: Intrinsically disordered proteins (IDPs) are part of proteins or the whole proteins which

do not have 3D structure in solution, dealing with natural and physiological conditions. But even though, these proteins

have not any stable conformation, they still keep their respective biological activities. It is important to emphasize that

viral proteins interact with many kind of components of the host, like membranes, nucleic acids, and proteins.

Furthermore, the lack of a rigid 3D structure allows disordered proteins to do various interactions at once. IDPs can

become flexible linkers from viral proteins and can hoodwink the human cell’s immune system, once, interactions with

host proteins make viral proteins harder to be recognized by the host’s immune system. For this reason, IDPs have

been associated with human diseases. The association between IDPs and diseases suggests that these can occur

not only by poor folding of proteins, but also by mistakes in identification and signalling of targets. The objectives of

this study consists in determine and quantify the presence of intrinsic disorder in proteins from viral strains which

triggers diseases in humans and predict possible binding sites in them.

Material and methods: The most common type and subtype of viruses that trigger human diseases such as Yellow

Fever Virus, Influenza A Virus, Hepatitis B Virus, JC polyomavirus, Mumps virus, Nyong nyong, Rubella virus and Zika

virus were selected through a literature review. After, sequences of protein from each viral subtype of virus were

selected on UNIPROT database (www.uniprot.org). Then, these sequences were submitted to the web server DisCon

(biomine.cs.vcu.edu/servers/DisCon/), which performs a prediction of disorder in the protein sequence. And then,

there was a submission of the sequences to the PONDR-FIT website (disorder.compbio.iupui.edu/metapredictor.php),

which detects the contribution of each residue to the disorder, generating easy-to-view graphic files. The sequences

were also submitted to the ANCHOR program which predicts binding sites with high potential in proteins with intrinsic

disorder. The data obtained were analysed by a comparative way.

Results: The results demonstrated an average disorder approximately corresponding to about 25.525% in each viral

subtype, with the exception of the Rubella virus that presented a discrepant average disorder, corresponding to

51.371%. Through the graphs a great predominance of disorder was observed in the extremities of the chains, and

although there was disorder in the middle of the sequences, they were few or non-existent. The data concerning the

potentials of binding sites consisted of variable numbers, not having constancy between them.

Conclusion: The results showed a presence of variable disorder for each viral type. However, more viral types will be

added to subsequent studies.

http://www.uniprot.org/


RECOMBINANT PROTEIN P21 FROM Trypanosoma cruzi REDUCES BREAST CANCER CELL

MIGRATION IN VITRO

Alves, S. N. 1; Uehara, I. A.

1; Borges, B. C.

1; Silva, M. J. B.

1

1 Universidade Federal de Uberlândia, Instututo of Biomedical Sciences, Uberlândia, Brazil

[email protected]

Keywords: Trypanosoma cruzi; rp21; breast cancer.

Introduction and objectives: The P21 is a surface protein that belongs to Trypanosoma cruzi and is secreted by it. In

macrophage this recombinant protein improves phagocytic capacity by a mechanism that depends on binding to the

chemokines receptor CXCR4, inducing actin filaments polymerization. In literature was verified some studies that

relate T. cruzi chronic infection with decrease of some types of cancer in mice. This antitumoral effect caused by T.

cruzi can be related with some of its secreted proteins. According to the literature and biological activities of rP21, the

purpose of this study is to identify whether this protein has an antimetastatic potencial.

Material and methods: Breast cancer cell line MDA-MB-231 and non-tumorigenic epithelial cell line MCF-10A were

treated with different concentrations of rP21 (100, 50, 25, 12.5 and 6.25 μg/mL) for 72 hours. After this, was made

evaluation of cell viability by MTT which consists in verify mitochondrial viability. The viability was measured by

spectrophotometry at 560 nm.

The invasion assay was performed with transwell and matrigel. The cell lines were submitted to the following

treatments: (1) cells plated in transwell exposed to SDF-1α 10ng/mL, as positive control for invasion, because SDF-1α

is chemotactic; (2) cells plated in transwell exposed to 100 μg/mL of rP21; (3) cells were pre-treated with 100 μg/mL of

rP21 for one hour and exposed to 10ng/mL of SDF-1α and (4) cells plated in transwell and exposed to RPMI without

bovine fetal serum. For statistical analysis was used Test T and One-Way ANOVA.

Results: The optical density obtained by spectrophotometry was converted to cell viability and the data was

compared. The statistical test T indicated no significant difference among the different concentrations and the control

(for p<0.01). In the invasion assay, the pre-treated cells with rP21 in well with SDF-1α showed lower migration than

positive control. Probably because rP21 bound to CXCR4 and induced actin polymerization increasing cell stiffness,

which contributes to low migration.

Conclusion: The rP21 shows to reduce cell migration in vitro in breast cancer cell line, probably due to actin

polymerization induction by CXCR4 binding. More assays need to be performed to conclude and confirm our

hypothesis.

Grants: Fapemig, Capes and CNPq.


Salmonella spp. IN FISH AND FISHERY PRODUCTS: A REVIEW

Pereira, C. H. Y.1; Saeki, E. K.

1 1 Instituto Adolfo Lutz – Centro de Laboratório Regional de Presidente Prudente-SP, São Paulo, Brazil

[email protected]

Key words: Foodborne diseases, Salmonella enterica serovar Typhi, Salmonella infections

Introduction: The consumption of fish has increased in the last decades, mainly due to the healthy diet. The safety of

the fish as to the standard microbiological is very important, since there is a concern about the quality of the food and

to the knowledge of the conditions hygienic and sanitary during its production, as is increasing the number of cases of

foodborne diseases. These represent an important public health problem, for causing damage to millions of people

around the world. Among the major pathogens associated with fish, which have emerged in the last twenty years, we

highlight the Salmonella spp.

Review: The bacteria of the genus Salmonella are Gram negative rod, mobile, facultative anaerobic, non-trainers

spores, belonging to the family Enterobacteriaceae. It is noteworthy that there are more than 2,500 serovars

considered potential pathogens to animals and humans. In general, the salmonellosis are self-limiting in healthily

individuals, however these may develop the chronic form, and asymptomatic of the disease, allowing the framework to

evolve to a fibrosis of the intestinal persistent. In the cases of immunocompromised individuals and physiologically

special as children, pregnant women and the elderly, may prove to be fatal from infection or associated complications.

In a pathological way, Salmonella spp. to invade the intestinal mucosa multiplies and therefore, can penetrate into the

lymphatic systems and cardiovascular, and thence may spread, eventually, to affect other organs. They replicate

quickly inside of macrophages. Salmonellosis has an incubation period of 6 hours after ingestion of contaminated

food, may have symptoms that last up to 72 hours, which varies in each case. There usually are a moderate fever,

accompanied by nausea, abdominal pain, cramps, and diarrhea. The serovars Typhi and Paratyphi can be

responsible for typhoid fever. They can spread in multiple organs, especially the spleen and in the liver, in severe

case, which may lead to death. In turn, the current legislation (ANVISA, Resolution number 12 of January 2nd, 2001)

stipulates the absence of Salmonella spp. in fish. An epidemiological study determined Salmonella spp. as being one

of the microorganisms most frequently involved in cases of foodborne diseases throughout the world. Necessary

actions to reverse this situation would be monitoring during all the steps of the process, from capture of the fish until

you reach the consumer's table, keeping a proper hygiene and awareness of the population regarding handling and

better choice in the form of consumption of this type of food.

Conclusion: It is inferred, that there is a low occurrence of Salmonella spp. in fish, however, is a result still of concern

from the point of view of public health. Therefore, the need of the action of the health surveillance is the inspection and

prevention of this foodborne infection to the consumer.



SCHISTOSOMIASIS MANSONI: EPIDEMIOLOGICAL SURVEY OF PARASITOSIS IN THE

MUNICIPALITY OF LONDRINA - PARANÁ.

Terra, M. R.1; da Silva, R. S.

2; Pereira , J. A. R.

3; Gonçalves, C. S. F.

4.

1 State University of Londrina, Department of Microbiology, Londrina, Brazil. 2 Universidade Estadual Paulista Júlio Mesquita - UNESP, Botucatu, Brasil. 3Instituto de Ensino Superior de Londrina - INESUL, Londrina, PR, Brasil.

4 Universidade Norte do Paraná-UNOPAR,Londrina, PR, Brasil.

[email protected]

Keywords: Schistosomiasis mansoni, intestinal schistosomiasis, epidemiology.

Introduction and Objectives: Schistosomiasis (EM) is also known as "water-belly" and "xistose" is an infectious-

parasitic disease of an acute and chronic character that has the etiological agent Schistosoma mansoni. . EM

transmission occurs through contact with bodies of water contaminated with mollusks of the genus Biomphalaria,

which are the intermediate hosts that release into the surrounding water that actively penetrates the skin and mucosa.

The human being is the definitive host of S. mansoni. The main focal points are the peridomiciliary sites polluted by

human excreta and abundant organic matter, providing an environment conducive to mollusc. Treatment of EM is

based on reducing parasite burden of the host by drugs such as praziquantel and oxaminiquina. Due to the above

problem, the present study had as objective to carry out the epidemiological survey of EM in the municipality of

Londrina, Paraná, between the periods of 2007 and 2015.

Material and methods: descriptive, retrospective epidemiological survey using secondary data obtained from the

Ministry of Health DATA / SUS related to the Program of Control of Schistosomiasis (PCE) with focus in the city of

Londrina, Paraná. For the research the number of positive cases for MS in the municipality of Londrina between the

periods 2007-2015 was raised. The rates of hospitalization and mortality related to EM in the municipality of Londrina

were surveyed in the Hospital Information System (SIH) for the period of. The descriptors used in the study were:

schistosomiasis mansoni, epidemiology, diagnosis, prophylaxis, treatment and their respective correspondents in the

English language.

Results: The total number of cases of EM in Londrina in this period was 66 cases. In the state of Paraná, it reached

784 cases in the same period. In the years 2007-2008 in Londrina 13 and 12 cases were reported, respectively.

Subsequently, there was a decline in the number of cases, with little variation (01-07 cases), except for the year of

2013, where there was a significant increase with the notification of 16 cases. Similar results were observed in the

state of Paraná during the period with a decline in the number of cases from 270 cases reported in 2007 to 6 cases in

2015. Regarding hospitalization and mortality rates, data were obtained from the period 2000 to 2012, where the rate

of hospitalization for EM in Londrina was 0.0-0.4 admissions per 100,000 inhabitants. In contrast, Paraná did not

exceed 0.1 hospitalization per 100,000 inhabitants in the same period. There was little variation in the mortality rate

(0.0 to 0.6) in Londrina and the state of Paraná (0.0 to 0.1) per 100,000 inhabitants in the period from 2000 to 2011. In

2007, there was an increase in the number of cases, By higher hospitalization rates and mortality.

Conclusion: Over the years surveyed, the city of Londrina and the state of Paraná show a decline in the number of

reported cases, as well as in the number of hospitalizations and the mortality rate by EM.


SENSITIVITY PROFILE OF POSITIVE GRAM COCCI ISOLATED FROM BIOLOGICAL SAMPLES OF

HOSPITALIZED PATIENTS FROM UNIVERSITY HOSPITAL OF LONDRINA DURING THE YEARS OF

2012 TO 2016.

Danelli, T.1; Tizura, A. T.

1; Gasparotto, L. C.

1; Duarte, F. C.

1; Vespero, E. C.


2; Lopes, G. K.

2;

Perugini, M. R. E.2.

1Health Sciences Center, State University of Londrina, Paraná, Brazil.

2Department of Pathology, Clinical and Toxicological Analysis, State University of Londrina, Paraná, Brazil.

[email protected]

Keywords: antimicrobials, sensitivity, gram positive cocci.

Introduction and objectives: Gram-positive cocci are among the most frequent infection agents, both in hospital and

community infection. Over the years, these microorganisms have developed resistance to antimicrobials,

compromising the therapeutic efficacy of patients affected by these infections. The objective of this study was to

evaluate the sensitivity of Gram positive cocci in the hospital setting.

Material and methods: This is a retrospective descriptive horizontal study. The minimum inhibitory concentrations

(MIC) to the antimicrobials of Staphylococcus aureus, S. epidermidis, S. haemolyticus, Enterococcus faecium, E.

faecalis, isolated from different biological samples of patients attended and hospitalized at the University Hospital of

Londrina - UEL, were analyzed from January 2012 to December 2016. Data were obtained from the epidemiological

database of the Vitek 2 automated system (bioMerieux USA). The present study was approved by the Research

Ethics Committee Involving Human Beings under the number CAE: 43013315.8.0000.5231.

Results: We analyzed 254 samples of E. faecium, 971 of Staphylococcus epidermidis, 354 of Staphylococcus

haemolyticus, 1632 of Staphylococcus aureus and 1209 of E. faecalis. E. faecalis showed greater sensitivity to some

antimicrobials than E. faecium as vancomycin, with a rate of 89% and 24%, teicoplanin with 91% and 26%, ampicillin

with 94% and 6%, benzilpenicillin with 89% and 4% and ciprofloxacin with 67% and 3%, respectively. However, they

have similar sensitivity to linezolid (E. faecium: 93% and E. faecalis: 93%), tigecycline (E. faecium: 100% and E.

faecalis: 100%), gentamicin at high concentration (E. faecium: 85% and E. faecalis: 63%), and streptomycin at high

concentration (E. faecium: 75% and E. faecalis: 57%). As for Staphylococcus spp., we found that S. aureus had

higher sensitivity to oxacillin (53%) than S. epidermidis (19%) and S. haemolyticus (7%) and all of them presented

100% of sensitivity to tigecycline and 98%, 97% and 91% respectively to linezolid.

Conclusion: This study showed different sensitivity profiles, using the MIC values, of Gram positive cocci from the

University Hospital of Londrina - UEL during the years 2012 to 2016, being this information of great importance in

helping the therapeutic choice of patients infected by these microorganisms and also for the institution's surveillance

program.


STAR TICK AND ROCKY MOUNTAIN SPOTTED FEVER

Castilha, E.P.1; Franciosi, A.

1; Mori, F. M. R. L.

1.

1Filadélfia University Center, Londrina, PR, Brazil.

[email protected]

Keywords: Amblyomma cajennense, rocky mountain spotted fever, tick.

Introduction: The genus Amblyomma presents around 30 species found in Brazil. The species of Amblyomma

cajennense, known in Brazil as "star tick", is the main vector of Rocky Mountain Spotted Fever (RMSF) or Brazilian

Spotted Fever (BSF), an infectious disease caused by Rickettsia rickettsii, spread by North, South America and

Central. The objective of the present review was to gather different pathways regarding spotted fever and its causative

agent, as well as the life cycle of this parasite, and the symptomatology caused by it.

Review: Rocky Mountain Spotted Fever (RMSF) is a reemerging zoonosis in Brazil, mainly in the Southeast and

Midwest. It is transmitted to humans during the blood repast of arthropods infected with Rickettsia rickettsii and

characterizes a serious rickettsiosis. The wide demographic distribution, trioxenic cycle and anthropophilic

characteristics associated with weather, vegetation and domestic or wild animals, maintain the pathogenic potential of

the vector Amblyomma cajennense. The transovarian transmission in the tick amplifies the presence of the etiologic

agent in this population and characterizes the tick as reservoir in nature. The Amblyomma sp., has three forms of life,

larva, nymph and adult, on its dorsal face presents a shield that covers the back. The tick performs an annual cycle,

one generation per cycle, with a behavior diapause of the larvae during the summer. The incubation period of the

disease is 2 to 14 days. The pathogenic potential of Rickesttsia rickettsii is to carry out changes in the proteins of the

endothelial cell membranes, with the vascular endothelium as its main target. It leads to cutaneous lesions (macules),

thrombus formation, focal necrosis, hemorrhagic rash, hemorrhage, microinfarction and ischemic demyelination. With

adequate treatment, the disease presents low death rates. The most effective preventive measure is the control of tick

populations at the lowest levels, substantially reducing the risks of human infestation. Larvae and nymphs are more

sensitive to carrapaticidal products than adult ticks.

Conclusion: Amblyomma cajennense represents an important vector of infectious diseases that affect humans.

Because it presents low host specificity and a favorable habitat, it presents a continuous pathogenic and

epidemiological potential for Brazilian Spotted Fever. The medium- and long-term combat of the simpler forms of tick

life prevents possible outbreaks of rickettsiosis.


SUSCEPTIBILITY EVALUATION OF THE ANTIMICROBIAL AGENTS USED IN THE EMPIRICAL

THERAPY OF URINARY TRACT INFECTIONS CAUSED BY GRAM-NEGATIVE BACILLI AT THE

UNIVERSITY HOSPITAL OF LONDRINA 2012-2016

Sant’Ana, T. C.1; Magalhães, G. L. G.

1; Kerbauy, G.

2; Vespero, E. C.


3; Pelisson, M.

3;

Perugini, M. R. E. 3.

1 Health Sciences Center, Universidade Estadual de Londrina, Londrina, Paraná, Brazil. 2 Universidade Estadual de Londrina, Nursing department, Londrina, Paraná, Brazil.

3 Universidade Estadual de Londrina, Pathology, Clinical and Toxicological analyzes Department, Londrina, Paraná,

Brazil.

[email protected]

Keywords: Antimicrobial, gram-negative bacteria, susceptibility.

Introduction and objectives: Urinary tract infections (UTI) are the most frequent infections diagnosed ambulatorily

and one of the most common causes of infection in the general population. The most used empirical antimicrobial

therapies for the treatment of UTIs in Brazil according to ANVISA are sulfamethoxazole-trimethoprim, quinolones,

cephalosporins of third generation, amoxicillin/clavulanic acid and nitrofurantoin. Knowing the first choice

antimicrobials for the treatment of UTIs, the study aimed to evaluate the susceptibility of gram-negative bacilli to these

drugs.

Material and methods: A retrospective descriptive horizontal study was conducted by analyzing the minimum

inhibitory concentration (MIC) of antimicrobials for Gram-negative bacilli bacteria from isolates of urine samples from

patients treated and hospitalized at HU-UEL from January 2012 to December 2016. Data were obtained from the

Vitek®2 (bioMérieux, USA) automated system epidemiological database. Approval number from the institutional ethics

committee is 43013315.8.0000.5231.

Results: 10.756 cultures of Gram-negative bacilli from isolates of urine were analyzed, in which 6.020 (55,97%) were

Escherichia coli, 1.936 (18%) Klebsiella pneumoniae, 911 (8,47%) Proteus mirabilis, 465 (4,32%) Enterobacter

cloacae, 364 (3,38%) Pseudomonas aeruginosa, 323 (3%) Acinetobacter baumannii, 303 (2,82%) Morganella

morganii, 218 (2,03%) Providencia stuartii and 216 (2%) Serratia marcescens. Among the drugs used for the empiric

treatment according to ANVISA, for sulfamethoxazole/trimethoprim was found 78% susceptibility for S. marcescens,

61% for E. coli, 58% for P.mirabilis, 51% for K. pneumoniae and 44% for A. baumanii, although P. aeruginosa showed

no susceptibility to this drug. For quinolones, results were 77% susceptibility for E.coli, 45% for K. pneumoniae, 45%

for P. aeruginosa and 24% for A. baumannii. For nitrofurantoin, E.coli showed 88%susceptibility, while the others

enterobacterias (except the P. mirabilis, P. stuartii and M. morganii, which are intrinsically resistant to this drug)

showed 13-15% susceptibility. A. baumannii and P. aeruginosa were 3-4% susceptible. For cephalosporins of third

generation (cefotaxime, ceftriaxone and ceftazidime), results were 90-95% susceptibility for E.coli, 41-46% for K.

pneumoniae, 20-37% for A. baumanii, and P.aeruginosa showed 55% susceptibility to ceftazidime, while to

cefotaxime and ceftriaxone, showed no susceptibility. And for amoxicillin-clavulanic acid, was found 83% susceptibility

for E.coli, and 47% for K. pneumoniae. A. baumanii and P.aeruginosa, were poorly tested for this drug, and those that

were tested, showed 0% susceptibility.

Conclusion: This study showed the existence of high rates of resistance to some antibiotics used in UTIs and is

therefore a concern because they are some of the first choice drugs used for empiric therapy of urinary tract

infections.


ULTRASTRUCTURE OF BOMBYXMORI (LEPIDOPTERA: BOMBYCIDAE) INTEGUMENT INFECTED

BY BOMBYXMORINUCLEOPOLYHEDROVIRUS

Poncio, A. C. F.3; Oro, A. L.

1; Britta, E. A. B.

2; Nakamura, C. V.


3; Torrejais, M. M.

3; Ribeiro, L.

F. C.3; Fernandez, M. A.

1.

1 Center for Biological Sciences, Department of Cell Biology and Genetics, State University of Maringá – Maringá,

Paraná, Brazil 2 Health Sciences Center, Department of Basic Health Sciences, State University of Maringá – Maringá, Paraná,

Brazil 3 Laboratory of Structural and Functional Biology, State University of Western Paraná - Cascavel, Paraná, Brazil

[email protected]

Keywords: Baculovirus, cytopathology, cuticle, ultrastructure.

Introduction and objectives: The entomopathogenic virus BombyxmoriNucleopolyhedrovirus (BmNPV) belongs to

Baculoviridae family and infects Bombyxmori (Linneau). This virus act causing a series of metabolic disorders that

may result in defective cocoons, leading to losses in the silk yarn industries. The major sign of infection are observed

in the integument of infected larvae.

Material and methods: Analyze ultrastructurally the integument cytopathology of B. mori hybrid larvae infected by

BmNPV. 5th instar larvae were inoculated with a viral suspension containing 1.70x10

7 polyhedral occlusion bodies/mL

and the symptomatology accompanied until cocoon spinning. On the 17th day after inoculation, integument segments

were processed to transmission electronic microscopy.

Results: Infection was confirmed by the presence of viral polyhedra in the nucleus of epidermal cell, showing similar

cytopathology described in other tissues infected by BmNPV. In the apical region of these cells it was observed an

alteration of microvilli and loss of contact of the plasma membrane plates with endocuticle.These structures,

synthesizers of chitin, lose their functionality due to metabolic changes that occur during viral infection, causing

endocuticle disorganization.

Conclusion: All insect integument was damaged, resulting in fragility and rupture, releasing BmNPV virus polyhedra

to the external environment. This is the first study that presents ultrastructural histopathological aspects of BmNPV

infection on integument and maintenance of structural cuticle.


VALIDATION OF POTENTIAL INHIBITORS AGAINST MALARIA

Pereira Lima, G. Y.1; Marques, P. S.

1; Resende, M. A.

1; Nicolau Junior, N.

1

1 Universidade Federal de Uberlândia, Instituto de Genética e Bioquímica, Uberlândia, MG Brazil

[email protected]

Keywords: bioinformatics, malaria, inhibitors.

Introduction and objectives: Malaria is caused by Plasmodium protozoa parasites more common in tropical areas

because the transmission of the parasites depends on anopheline mosquitoes, which proliferate better in a higher

temperature. In 2015 it was estimated a total of 205-316 million cases worldwide. Once infected with the malaria

parasite, it travels to the liver where occur the maturation.

After several days the mature parasites enter the bloodstream and start to infect red blood cells leading to coma, high

fever, anemia, muscle pain and vomit. New researches, aiming malaria prevention and treatment are dedicated to find

new potential inhibitors to specific malarial targets. These include N-myristoyltransferase (NMT), which is the enzyme

that catalyzes the transfer of myristate from myristoyl-CoA to the N-terminal glycine of the target protein and results in

the fatty being amide-bonded to the alfa-amino group of the amino acid. Because this, the enzyme is susceptible to

protein synthesis inhibitors. The objectives of this study consists in research potential ligands for three-dimensional

structure of Plasmodium vivax NMT through the construction of a pharmacophore model, virtual screening and

anchoring.

Material and methods: Initially known inhibitors were extracted from protein structures obtained from Protein Data

Bank(PDB): 4B12, 4B13, 4B14, 4BBH, 4CAE, 4CAF. These compounds were compressed into a mol2 file and

submitted to PharmaGist webserver, which predicts align ligands and generate pharmacophore points. The

pharmacophore models were created. The next step was the validation. For this, DUDE-E webserver was used to

created decoys using known inhibitors of NMT, and known inhibitors were extracted from Pfizer database, a both were

submitted to QUACPAC toll to Fix pKa and generate tautomers, lastly OMEGA2 toll was used to generated 3D model.

Several templates with five or six pharmacophore have been created but only one was selected, the one that has the

higher AUC value. AUC was generated when actives and inactives compounds were tested against the

pharmacophore models. Then, the vROCS tool was used to validated the model and to made a virtual screening

against two libraries using Tanimoto Combo score.

Results: The best model showed AUC: 0,880, it indicated that 88% the actives showed higher complex bound then

the inactives(decoys) and the top 500 hits were generated.

Conclusion: The work will continue yet using docking and molecular dynamics tools to refine the protein complex

interactions using force field.

Grants: Openeye.

PHARMACOLOGY AND TOXICOLOGY 208

ANTICARCINOGENIC EFFECT OF THE LECTIN ARTINM ON AFB1-INDUCED

HEPATOCARCINOGENESIS IN RATS

Gomes, M. S.1, Apolinario, L. A.

1, Oliveira, C. A. F.

2, Roque-Barreira, M. C.

3, Ramalho, L. N. Z.

1, Augusto, M. J.

1,

Silva, D. M.1, Ramalho, F. S.

1

1University of São Paulo, Department of Pathology, Ribeirão Preto, SP, Brazil

2University of São Paulo, Department of Food Engineering, Pirassununga, SP, Brazil 3 University of São Paulo, Department of Cellular Biology and Molecular and Bioagentes Pathogens, Ribeirão Preto,

SP, Brazil


Keywords: carcinoma hepatocellular, manose-binding lectins, aflatoxins, rats

Introduction and objectives: ArtinM is a D-mannose-binding lectin that interacts with phagocytic cell receptors

inducing the release of pro-inflammatory mediators related to the antitumor immune response. Aflatoxin B1 (AFB1) is

the mycotoxin with the highest toxigenic power, being able to induce hepatocellular carcinoma (HCC) in humans. The

aim of this research was to investigate the role of ArtinM in AFB1-induced hepatocarcinogenesis in rats.

Material and methods: Seventy-two rats were divided into three groups: Control- vehicle-treated animals; AFB1-

animals poisoned with AFB1; AFB1+ArtinM- animals treated with AFB1 and ArtinM. Rats were poisoned by gavage

daily with 400μg AFB1 per kilogram food during three months, while the AFB1+ArtinM group received additionally

three subcutaneous doses of the lectin (50μg per kg body weight per dose). The incidence of preneoplastic lesions

and liver tumors was measured 3 and 12 months after the start of treatments, respectively. Hepatic prohibitin and

cyclin D1 mRNA were assessed by real-time PCR, and liver expression of prohibitin and cyclin-dependent kinase

inhibitor (CDKI) p27Kip1

were evaluated by immunoblotting and immunohistochemistry, respectively. This experimental

protocol was approved by the Animal Research Ethical Committee of the School of Medicine of Ribeirao Preto

(process n° 127/2013).

Results: ArtinM-treated animals had fewer preneoplastic foci and hepatic tumors than the AFB1-poisened animals.

ArtinM increased hepatic mRNA and protein level of tumor suppressor prohibitin as well as augmented liver

expression of CDKI p27Kip1

. Furthermore, ArtinM treatment decreased hepatic cyclin D1 mRNA.

Conclusion: These data indicate that ArtinM was able to restrict the formation of pre-neoplastic foci and reduce the

incidence of tumor lesions in the liver rat. The mechanism underlying these effects appears to be related to inhibition

of cell cycle progression through overexpression of CDKI p27Kip1

and tumor suppressor prohibitin. Thus, the lectin

ArtinM exerts protective effect against hepatocarcinogenesis induced by subchronic exposure to the genotoxic agent

AFB1.

Grants: The authors thank the ―Conselho Nacional de Desenvolvimento Científico e Tecnológico‖ - "National Counsel

of Technological and Scientific Development" (CNPQ – n°142270/2013-0), for financial support and fellowships.

There are no conflicts of interest.


BALANCE DISORDER AND HEARING LOSS AFTER ANTINEOPLASTIC AGENTS AND

AMINOGLYCOSIDES USE

Bacchi, A. D.1; Maia Filho, F. C. C.

2.


2 Universidade do Oeste Paulista, Post-graduate Professor of Geriatrics, Presidente Prudente, SP, Brazil

[email protected]

Keywords: breast cancer, ototoxicity, antineoplastic drugs, hearing loss

Introduction: A 56-year-old woman has reported imbalance for four years and hearing loss for 10 years, associated

with a non-pulsatile tinnitus in her right ear.

Case report: Personal history: cancer in the right breast 15 years ago, having performed mastectomy and

chemotherapy with epirubicin and cyclophosphamide associated with external radiotherapy. It had recurrence 5 years

ago, when a new cycle with the same drugs was carried out. In both cycles, she presented nausea and vomiting,

alopecia and thrombocytopenia. She was also hospitalized 4 years ago due to pyelonephritis, for which amikacin was

prescribed for 4 days followed by ciprofloxacin, according to the urine culture.

Balance tests showed Romberg positive, with tendency to fall after closing the eyes, without preferential side.

Complementary exams: Recent audiometry with bilaterally moderate sensorineural hearing loss and

electronystagmography with bilateral labyrinthic hyporreflexia. The chemotherapy was adjuvant to mastectomy.

Epirubicin is an anthracycline, which belongs to so-called natural products, and acts in three ways:

• Cell membrane fluidity and ion transport alteration;

• Oxigen-reactive species formation;

• DNA and RNA synthesis blockade and decrease of topoisomerase II activity, promoting DNA rupture.

On the other hand, cyclophosphamide - from the group of nitrogen mustards - is an alkylating agent, capable of

replacing a hydrogen atom in another molecule with an alkyl radical. These drugs bind to the DNA, preventing the

separation of the two filaments in the spiral double helix, a vital phenomenon for cell replication. The therapeutic plan

using these two chemotherapeutics was chosen due to studies showing that this combination increases the survival

rate of patients with breast cancer.

Most chemotherapeutic agents act in a non-specific manner, injuring both normal and malignant cells, but especially

the fast growing cells (such as the gastrointestinal, capillary and bone marrow cells). This feature explains the most

common side effects such as nausea with vomiting, hair loss and the reduced platelet count. It is also known that the

chemotherapeutic agents prescribed in the case are potential ototoxic agents. They can be harmful to the inner ear,

affecting the hair cells (the sensory receptors of the auditory system) justifying the complaint of difficulty to listen

(documented by audiometry) and the tinnitus. Ototoxicity is also a potential effect of amikacin (an aminoglycoside

antimicrobial). The prescribed drugs, therefore, impair the Organ of Corti (which is located in the cochlea and is

responsible for hearing) and the posterior labyrinth neurosensory epithelia (responsible for part of the body balance).

Final consideration: In spite of being located in the same organ (inner ear), the part responsible for hearing is not

able to perform adequate compensation, different from the body balance, which has greater neuroplasticity, since it

also uses other mechanisms such as vision and proprioception. This difference explains the auditory symptom

(hearing loss and tinnitus) for 10 years and the unbalance complaint, that started 4 years ago, after the use of

aminoglycosides for severe renal infection. The authors attest the free informed consent from the patient.


BENEFICIAL EFFECTS OF BRAZIL NUTS (Bertholettia excelsea) CONSUMPTION ON

CARDIOVASCULAR RISK.

Marques, L. A. C1; Pelosi, G. G.

1 1 State University of Londrina, Department of Physiological Sciences, Londrina, PR, Brazil

[email protected]

Key words: Cardiovascular Diseases, Therapeutics, Bertholletia.

Introduction: Eating disorders and sedentary lifestyle play a major role in morbidity causes among cardiovascular

diseases (CD) and are frequently associated with obesity, diabetes and chronic kidney insufficiency. Brazil Nuts

(Bertholettia excelsea) has important nutritional characteristics to promote a healthy diet – essentially an excellent

source of unsaturated fatty acids, fibers, selenium (Se), B6 and E vitamins and electrolytes. Thus, the consumption of

the Brazil Nut may help the treatment of such patients due its antioxidant function and cholesterol reduction. The

objective of the review was elucidate the effects of Brazil nut consumption in patients with cardiovascular or other

related diseases.

Review: Se role Low Se plasmatic concentration is present in chronic kidney diseases (CKD), hypertension and

dyslipidemia, and affect erithrocitic glutathione peroxidase activity. Minor Se concentration plasmatic correlates

directly to higher heart attack odds leading to cardiovascular complications. The consumption of Brazil Nut shows a

capacity to increases Se plasmatic level and improve antioxidant profile in some related diseases.

Thyroid function and Cardiovascular diseases There are data indicating a relation of thyroid dysfunction and

cardiovascular diseases. Considering the high percentage of CKD patients with hypothyroidism, the Brazil nut

consumption in such patients may improve thyroid hormones profile, decreasing cardiovascular risk.

Lipid profile Cholesterol levels increase heart attack and atherosclerosis incidences. Higher triglyceride and LDL

levels usually relates to higher CD odds, besides lipoprotein A, which has an atherosclerogenic profile. Lowering

cholesterol levels by 10% reduce 15% in mortality by CD and Brazil nut antioxidant components actions over

cholesterol levels may decreases odds to coronaries diseases and atherosclerosis. Due its structural similarity,

phytosterols contained in Brazil nut also contributes to reduced cholesterol plasmatic levels.

Cardiovascular profile The Brazil nut consumption has a preventive role decreasing cardiovascular risk, considering

all the beneficial effects previous described. Heart attack, cerebral stroke, arrhythmia and angina are all associated

with atherosclerosis and others risk factors as hypertension, sedentary lifestyle and obesity that could be associated

with a preventive agent to reduce CD odds. Some Brazil nut components promote better blood viscosity besides minor

infarct and coronaries diseases odds. The antioxidant components play a major role in cardiovascular as previous

described, and immunologic systems, like minor cytokines plasmatic levels (TNF-α and IL-6) in CKD patients under

Brazil nut diet being related to a minor atherosclerogenic status and CD odds.

Conclusion: Being essentially an unsaturated fatty acids, fibers, micronutrients and Se source, Brazil nuts plays an

important role in antioxidant body components maintenance, hormone flotation, serum lipids control decreasing

cardiovascular risk. By the current literature, it is not possible to determine precisely the diet characteristics necessary

to maximize the beneficial effects related to Brazil nut consumption, but the literature suggest that as a preventive

agent of CD.


CHANGES IN THE REPRODUCTIVE PERFORMANCE OF MICE EXPOSED TO BUPROPIONA

CHLORIDRATE DURING SPERMATOGENESIS

Sanches, E. S. A. M.1,2

; Tsuzuki, F. 1; Joinhas, F.


1; Salles, M. J. S.

1

1 Universidade Estadual de Londrina, Department of General Biology, Londrina, PR, Brazil



Keywords: Bup, paternal toxicity, reproductive performance.

Introduction and objectives: Bupropion Hydrochloride (BUP) is an atypical non-tricyclic antidepressant, belonging to

the class of aminoketones, widely used in smoking cessation therapy, methamphetamine addiction treatment, and

attention deficit hyperactivity disorder (ADHD) in adults, among others. Its mechanism of action is the inhibition of

dopamine and noradrenaline recapture and studies have suggested that dopamine has the potential to impair

testicular function and sperm function. Thus the objective of this study was to evaluate the effects of BUP under the

reproductive performance of adult male mice treated during spermatogenesis.

Material and methods: Approval CEUA-UEL process n. 8722.2016.33. Adult male mice were divided into 2

experimental groups each containing 13 animals: treated group receiving daily BUP at a dose of 40 mg/kg diluted in

saline, via gavage for a period of 45 days and control group receiving saline solution under the same design

experimental. On the 35th day of treatment, the animals were placed to mate with females that did not undergo any

treatment, continued to be treated for another 10 days. On the 46th day the animals were submitted to euthanasia and

blood collected for evaluation of plasma testosterone levels. The testicles and epididymis were removed and used to

evaluate the sperm morphology, sperm count and histological analysis of the testicles (Johnsen protocol). The

absolute data with normal distribution were analyzed by Test T and those with non-normal distribution by Mann

Whitney and the level of significance considered was 5%.

Results: Treatment with BUP led to a decrease in the Johnsen score (Control: 9,354 ± 0,092, BUP: 7,615 ± 0,147)

and also the number of Sertoli cells (Control: 5,623 ± 0,184; BUP: 4,215 ± 0,097) and Leydig: 11.430 ± 0.817: BUP:

7.531 ± 0.213), all significantly. There was a decrease in testosterone levels (Control: 783.5 ± 154.2, BUP: 201.4 ±

54.8), changes in morphology of both head (Control: 8.134%, BUP: 10.423%) and tail (Control: 4,961%, BUP:

16,211%) and in addition there was a significant decrease in the daily production of spermatozoa in the testicles

(Control: 2,852 ± 0,211, BUP: 1,988 ± 0,116).

Conclusion: The drug tested had a potential toxic effect on the male reproductive system, altering spermatogenesis

and with probable impairment of fertility. The results obtained signal an alert for greater attention and follow-up of men

of childbearing age who use bupropion hydrochloride continuously.



CONGENITAL ABNORMALITIES DUE TO CAPTOPRIL ADMINISTRATION DURING PREGNANCY

OVER MICE OFFSPRING

Frítola, M.1; Sanches, E. S.

1; Martins, C. C. N.

1; Salles, M. J. S.

1 1Londrina State University, General Biology Department, Londrina, PR, Brazil

[email protected]

Keywords: Captopril, Hypertension, Pregnancy,Teratogenesis, Mice

Introduction and objectives: Systemic arterial hypertension (SAH) is a multifactorial, multicausal and multisystemic

syndrome, in which pregnant women are one of the affected profiles. Captopril is one of the most used medications to

control SAH and its administration during pregnancy is not recommended because it is capable to cross placental

barrier, with the possibility of affecting fetal development. Therefore, this study aimed to assess damage due to

Captopril administration during pregnancy in mice offspring.

Material and methods: This experimental study was approved by Ethics Committee on Animal research

(CEUA/UEL), under number17155.2016.07.A total of 45 pregnant female Swiss mice were arranged in three

experimental groups, in which they were gavaged with: captopril at doses of 12,5mg/Kg (G1) and 25mg/Kg (G2) in

treated groups, and destiled water in control group (G0). Gavage was performed after 3 hours fasting to improve

medication absorption. Treatment was started after implantation and lasted between the 5th and the 17

thdays of

pregnancy. On the 18th day, females were euthanized, and then laparotomy and hysterectomy were performed to

enable fetal removing and congenital malformations analysis. External, visceral and skeletal malformations were

analyzed on the offspring under stereoscopic microscope.Bones development degree and skeletal changes were the

parameter considered to skeletal analysis, and visceral analysis was performed by strategic cuts and microdissection.

Data were analyzed by Student t test, ANOVA, Fisher and Chi-square test, on GraphPad Prism, with significance set

at 5%.

Results:A total of 563 fetuses were examined for diagnosis of external malformations, of which 280 were designated

to skeletal analysis, and 283 to visceral analysis. Among visceral malformations, cleft palate was present, but without

statistical significance. In skeletal analysis, the occurrence of malformations on palate and axial skeleton were not

significant between treated groups, when compared to control group. However, cranial malformations occurrence was

statistically significant to both treated groups(G1 = 61.3% e G2 = 62.5%), in comparison to control group (8.5%), with

occipital incomplete ossification as the most common defect. The presence of clavicle alterations was also significant

between treated groups (G1 = 12.0% e G2 = 7.5%) versus control group (0%). Exposed groups showed significantly

more incomplete ossification and/or absent on limbs (G1 = 16.3% G2 = 13.8%), especially on phalanx, metatarsus

and metacarpus, when compared to non-exposed group (0%). Visceral analysis was performed by examination of

cerebral hemisphere, ventricles, palate, nasal cavity, organs of respiratory, digestive and urogenital system, and none

of these structures showed significant defects.

Conclusion: Based on data analyzed it is inferred that Captopril can cause congenital malformations, mainly skeletal

ones. It is suggested the change of this medication by another one with the same level of accuracy on arterial pressure

control, and which presents less risks to fetal development.


CONGENITAL MALFORMATION AND INTRAUTERIAL GROWTH DELAY IN FETUSES OF MICE

TREATED WITH IBUPROFEN DURING PREGNANCY

Tsuzuki, F.1; Silva, B. F.

1, Menezes, E. V.

1, Salles, M. J. S.

1.

1State University of Londrina, Department of General Biology, Londrina, PR, Brazil


Keywords Ibuprofen, Teratogenesis, Pregnancy, Nonsteroidal anti-inflammatory drugs, Malformation.

Introduction and objectives: Ibuprofen is a non-selective nonsteroidal anti-inflammatory drug (NSAID) derived from

propionic acid, an active COX-1 inhibitor which maintains stable physiology in many tissues and induces the

production of prostaglandins that are involved in regulating functions such as cytoprotection of the gastric mucosa,

renal homeostasis and platelet function. Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most widely

prescribed medications worldwide, although prescription is not required to get them. Easily obtained this drug provides

the use more and more frequent and without professional orientation, not only as anti-inflammatory, but also as

analgesic. It is known that the use of medications during the gestational period can cause morphological changes in

the fetus. Therefore, the present study aimed to investigate the toxicity and possible teratogenic effects of ibuprofen

exposure during the gestational period of mice.

Material and methods: Ethics Committee on Animal Experimentation of the State University of Londrina number:

19078.2016.13. Swiss pregnant female mice were divided into four experimental groups and received: 300 mg / kg

treated group (G01), 400 mg / kg treated group (G02), 500 mg / kg treated group (G03) of Ibuprofen and the control

group (G03) G0) sterile saline. The route of administration was the gavage and the treatment was started after

implantation in the 5th to the 17th of gestation. On the 18th day of pregnancy, the females were submitted to

euthanasia and laparotomy, for fetal removal, evaluation of intrauterine development and congenital malformations.

The data collected were analyzed by the GraphPad Prism 5 program, considering the significance of 5%, by the

Analysis of Variance (One-way ANOVA / Tukey) and the Chi-square / Fischer test.

Results: There was a statistically significant reduction in fetal weight of G3 and G2 compared to G0 (G0: 1.348 ±

0.03816, G1: 1.040 ± 0.04054, G2: 0.8835 ± 0.1480 **, G3: 0.9655 ± 0.08295 *) and fetal length of 400 mg / kg dose

relative to the control (G0: 2.502 ± 0.03820; G1: 2.324 ± 0.04434; G2: 1.653 ± 0.3137 *; G3: 2.279 ± 0.11678). In the

skeletal analyzes, sternum with incomplete ossification, irregular shape and absent sternum were found (G0: 23%,

G1: 44.26% *, G2: 50% **, G3: 38.64%). In the carpal bones was noted absence of distal phalanges of Carpi (G0: 0%;

G1: 0%; G2: 0%; G3: 9.09% *) and found a split or incomplete ossification and the bone above exoccipital (G0: 6.98%,

G1: 22.95% *, G2: 6.25%, G3: 34.09% **). Other skeletal malformations found such as the bones of the face (frontal,

nasal and zygomatic), the parietal and the interparietal that presented incomplete ossification or irregular shape.

Visceral malformations such as cerebral hemisphere, inner ear, medulla, nasal cavity, diencephalon, thyroid and

spinal cord were analyzed, but none of these parameters were statistically significant.

Conclusion: Ibuprofen slowed intrauterine growth of fetuses in the 400 and 500 mg / kg dose groups and promoted

some type of congenital skeletal malformation in all treated groups. It is imperative to use ibuprofen during pregnancy,

it is suggested to use the lowest effective doses and a short duration of treatment.


CONGENITAL MALFORMATION IN OFFSPRING OF MICE EXPOSED TO BUPROPIONA

CHLORIDRATE DURING SPERMATOGENESIS

Sanches, E. S. A. M.1,2

; Tsuzuki, F. 1; Joinhas, F.

1; Salles, M. J. S.

1




Keywords: Bup, teratogenesis, intrauterine development.

Introduction and objectives: Bupropion Hydrochloride (BUP) is an atypical antidepressant widely used in smoking

cessation therapies, in the treatment of mood bipolar disorder, and in treating attention deficit hyperactivity disorder

(ADHD) in adults. The use without medical indication of BUP among the population of childbearing age has been

increasing, in addition cases of accidental overdose with the use of this drug have also been increasing. Thus, due to

the lack of information about the paternal exposure, the objective of this study was to evaluate the intrauterine

development and teratogenicity of offspring of male mice treated with BUP during spermatogenesis.

Material and methods: The study was approved by CEUA-UEL process no. 8722.2016.33. Male mice were divided

into 2 experimental groups with 10 animals each: the treated group received daily BUP at the dose of 40mg/kg, diluted

in saline, via gavage and control group received saline solution under the same experimental design. On the 35th day

they were placed to mate with females that were not submitted to any treatment, in the proportion of 1 male for each

female. The female’s vagina was examined daily for confirmation of pregnancy. On the 18th day of pregnancy,

females were submitted to euthanasia for evaluation of intrauterine development with the presence of resorption,

number of live and dead fetuses, fetal and placental weight and fetal length. With these data were calculated:

resorption rate, rate of post-implantation losses and rate of fetal viability. To identify the presence of congenital

malformations, external, visceral and skeletal analyzes were performed in the fetuses. The absolute data with normal

distribution were analyzed by Test T or Fisher's Exact Test and those with non-normal distribution by Mann Whitney

and the level of significance considered was 5%.

Results: Parental exposure to BUP did not significantly affect intrauterine development or fetal weight adjusted to the

age of pregnancy. However, analyzes of congenital malformations were statistically significant for external evaluation:

kyphosis (Control: 0.00%, BUP: 5.26%) and presence of retroversed lower limbs (Control: 14.43%, BUP: 53.68%). In

the skeletal analysis the changes were in the bones of the skull: incomplete ossification of supra/exocipital (Control:

21.82%, BUP: 86.00%) and bone of the sternum: abnormal sternebio (Control: 25.45%, BUP: 82.00%), also

statistically significant. No significant changes were identified for the visceral analysis.

Conclusion: The results demonstrate a teratogenic potential of BUP. Thus, until prospective studies emerge, greater

care is required in prescribing the drug for men of reproductive age.



DETECTION OF ALDICARB AND CARBARYL IN THE GASTRIC CONTENT OF A DOG FOUND DEAD,

SUSPECTED OF CRIMINAL POISONING

B. H. de Souza1; V. A. S. Weigert

1; A. L. Chryssafidis

1 1 Laboratory of Veterinary Toxicology, Department of Preventive Veterinary Medicine, Universidade Estadual de

Londrina, Londrina, Paraná, Brazil.

[email protected]

Keywords: Pesticides, poisoning, chromatography

Introduction: Poisoning by acetylcholinesterase inhibitors represent the majority of clinical cases of acute intoxication

in small animal clinics in Brazil. Inseticides containing aldicarb were restricted in the country decades ago, but they

can still be found in the black market, being incorrectly used as rodenticides, because of its great toxicity. These

compounds are also used in criminal poisoning of companion animals. These illegal products, popularly called

―chumbinho‖, which roughly means ―small pieces of lead‖ in Portuguese, because of its characteristic granular

appearance. Chumbinho may contain aldicarb and other carbamate and organophosphorus compounds, in unknown

concentrations. The severity of clinical signs and treatment applied in cases of poisoning caused by carbamate have

some differences from cases involving organophosphorus compounds and hence the quick identification between

them is a major factor for the therapeutic success and survival of the patient. Additionally, the laboratory confirmation

of criminal poisoning is fundamental for a pet owner to pursue legal action against the person who committed the act.

Case report: A male French Bulldog, three months old, previously healthy, was found dead by his guardian at home,

with vomit next to his body. The animal was taken to a veterinary clinic in the same day and it was submitted to post

mortem analysis. No macroscopic alteration was found, but the stomach was filled with feed, crowded with black

granules, suggesting that the animal had been poisoned by chumbinho. The stomach content was submitted to the

Laboratory of Veterinary Toxicology of Universidade Estadual de Londrina, for the detection and quantification of

acetylcholinesterase inhibitors using a fast extraction of multiresidue pesticides and analysis by thin layer

chromatography (TLC) and high performance liquid chromatography with photodiode array detector (HPLC-PDA). The

TLC was positive for carbamate compounds, whilst the HPLC detected 2.77 mg of aldicarb and 3.95 mg of carbaryl in

5 g of gastric content, confirming the poisoning by chumbinho.

Final consideration: Because of the major importance of acetylcholinesterase inhibitors poisoning in small animal

clinics in Brazil and the therapeutic difference between carbamate and organophosphorus compounds, which may be

both present in chumbinho, a simple method for determination such compounds is of great value to help in the

diagnostic and treatment of poisoned animals. Additionally, the identification of these pesticides and determination of

the causa mortis is fundamental for the investigation of criminal poisoning of animals.


DRUGS USED TO TREAT BIPOLAR DISORDER AND TOBACCO USE DISORDER REDUCE

REACTIVE OXYGEN AND NITROGEN SPECIES PRODUCED BY NEUTROPHILS IN VITRO

Michelin, A.P.1; Bonifácio, K.L.

1; Matsumoto A.K.

1 Semeão, L.O.

1, Martins, R.F.D.

1, Moreira, E.G.

2 Barbosa, D.S.

3 1State University of Londrina, Postgraduate Laboratory, Londrina, PR, Brazil;

2 State University of Londrina, Department of Physiological Sciences

3 State University of Londrina, Department of Pathology, Clinical and Toxicological Analysis, Londrina, PR, Brazil


Keywords: bipolar disorder, antioxidant activity, medicines;

Introduction and objectives: Bipolar disorder (BD) is a recurrent chronic disease characterized by alterations in

mood. It affects more than 1% of the world's population, regardless of nationality, ethnicity or socioeconomic status.

High smoking rates have been described when BD, among the patients with mental disorder, 50-80% have tobacco

use disorder (TUD) compared to the general population and are associated with worse improvement results in these

patients. The production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is increased in

pathological conditions such as Parkinson, schizophrenia, and BD because of endogenous and exogenous stimuli.

The aim of this study was to evaluate whether drugs commonly used in the treatment these conditions - BD

associated with TUD- Lithium Carbonate (LC), Bupropion Hydrochloride (BH) and the adjuvant N-acetyl-cysteine

(NAC) have some antioxidant effect in the sense of identifying some mechanism of action beyond the already known.

Material and methods: The antioxidant potential in vitro was evaluated for the drugs LC (Carbolitium®), BH (Bup®)

and the manipulated formulation of NAC by respiratory burst. The neutrophils ROS production was evaluated by

chemiluminescence in a microplate reader (Victor X-3, PerkinElmer ™, USA. Human neutrophils were isolated from

blood by Histopaque ficoll concentration gradient and centrifugation. The respiratory burst was induced by

phorbolmyristate acetate (PMA) in the presence of DMSO (control group) and 10-2

M NAC, LC, BH and the results are

expressed in counts per minute (CPM) ± standard error mean (SEM). All medications were diluted with

dimethylsulfoxide (DMSO). The data were first transformed into logarithms (Logn), and thus, the analysis of variance

was performed for the comparison of one-way means (ANOVA) followed by the Tukey-Kramer test). The experiment

for each drug was composed of 10 replicates and the results were considered statistically significant when p-value

<0.05. This study was approved by the Ethics Research Committee at UEL (number CAAE 34935814.2.0000.5231). Results: The mean in CPM of neutrophil control was 10.69 (0.02). The mean values of NAC were 4.62 (0.03), LC

10.40 (0.03) and BH 7.51 (0.17). NAC and BH showed an antioxidant effect on the respiratory burst compared to

neutrophils control and LC, while NAC presented a higher antioxidant potential than BH.

Conclusion: According to the results it is possible to conclude that NAC and BH drugs have antioxidant activity in

vitro in the respiratory burst assay. More in-depth studies with these drugs should be performed in the future to verify if

such drugs can cause any other effect (antioxidant, for example) in a biological environment, besides those already

established.

Grants: CNPq


EFFECT OF THE ACUTE ETHANOL CONSUMPTION IN NEUTROPHIL RECRUITMENT TO THE

INFLAMMATORY FOCUS.

Oliveira, C.H.B.1; Fioravante, A.

1, Machado, L.P.

1, Piedade, B.P.

1; De Freitas, A.

1, 2 ;

1 Scientific initiation at Laboratory of Pharmacology of Inflammation, Department of Physiological Sciences, State

University of Londrina, Londrina, Brazil. 2 Coordinator of the Laboratory of Pharmacology of Inflammation, Department of Physiological Sciences, State

University of Londrina, Londrina, PR, Brazil.


Keywords: Inflammation, Neutrophils, Ethanol.

Introduction and objectives: According to the Center for Disease Control and Prevention (CDC) and Substance

Abuse and Mental Health Administration Service (SAMHSA) the consumption of ethanol, called as "binge drinking"

(defined by the CDC as four or more doses for women or five or more doses for men in a short period of time), is the

third leading cause of preventable death in the United States of America. Recently, was demonstrated that acute

ethanol exposure alters the innate immune system. However, the interrelationship between the acute use of ethanol

on neutrophil migration during an acute inflammatory response remains uncertain. Thus, in the present study we

addressed the role acute ethanol intake in neutrophil migration to the inflammatory site.

Material and methods: Adult female Swiss mice were used in this study and the experiments were conducted in

accordance with the ethical guidelines (CEUA: 11905.2016.46). Animals were randomly divided into four groups:

Control Group (n: 5), mice were pretreated with water via gavage and 30 minutes later saline was injected into the

peritoneal cavity; Group Ethanol (n: 4) animals were pretreated with 32% ethanol via gavage and 30 minutes later

received saline intraperitoneally (i.p.); Carrageenan Group (n: 5) mice were pretreated with water via gavage and 30

minutes later carrageenan was injected i.p.; Group Ethanol + Carrageenan (n: 4) animals were pretreated with 32%

ethanol via gavage and carrageenan was injected i.p. 30 minutes later. After 4 hours of i.p. administration of saline or

carrageenan, the blood was collected via cardiac puncture as well as peritoneal lavage. Total and differential

leukocyte counts were made. The data were analyzed statistically using the one-way ANOVA method, followed by

Bonferroni as a post-test.

Results: Our results demonstrate that the acute administration of ethanol promoted a reduction in the migration of

total leukocytes and neutrophils to the peritoneal cavity induced by administration of carrageenan. In addition, a

previous administration of ethanol did not alter the number of circulating leukocytes in blood. Conclusion: Thus, the

results suggest that the acute ethanol intake reduces the neutrophil migration to the inflammatory site, indicating that

the changes observed were not due the reduction in the number of circulating leukocytes.

Grants: Araucaria Foundation provides a social inclusion scholarship to Alaina Fioravante.


EFFECTS OF INOS INHIBITION ON CARDIOVASCULAR RISK OF ENDOTOXEMIC FEMALE RATS:

ROLE OF ESTROGEN

Castardo-de Paula, J.C.1, de Campos, B.H.

1, de Jager, L.

1, Zalunqui, N.G.

2, Pinge-Filho, P.

2, de Farias, C.C.

3,

Higachi, L.3, Barbosa, D.S.

3, Martins-Pinge, M.C.

1.

1State University of Londrina, Department of Physiological Sciences, Londrina, PR, Brazil 2State University of Londrina, Department of Pathological Sciences, Londrina, PR, Brazil

3State University of Londrina, Department of Clinical Pathology and Toxicological Analysis, Londrina, PR, Brazil

Email ([email protected])

Keywords: ovariectomy, nitric oxide, endotoxemia, blood pressure, heart rate.

Introduction and objectives: It is recognized that gender differences exist in cardiovascular function and also in

cardiovascular disease, and also, estrogen increases the bioavailability of nitric oxide (NO). However, no study has

evaluated the interaction of both on cardiovascular and oxidative parameters during endotoxemia.

Material and methods: Institucional Animal Care Committe Protocol number: 276.2013.81. Female wistar rats (220

to 270g) were subjected to ovariectomy or false surgery and divided into 3 groups: OVX (ovariectomized rats, n=17),

OVX+E (ovariectomized plus estradiol treatment, 1mg/Kg/day, p.o., n=16) and SHAM (false surgery, n=17). After 8

weeks animals were submitted to artery and vein catheterization for blood pressure and heart rate recordings, while

awake and freely moving, before and after LPS injection (5mg/Kg, IV) preceded by the injection of S-methylisothiourea

sulphate (SMT; 3 mg/kg) or sterile saline (0,9%). Cardiovascular recordings underwent spectral analysis for evaluation

of autonomic modulation. 2 hours after LPS injection, blood samples were collected for plasmatic measurements of

the total radical-trapping antioxidant (TRAP), nitrite levels (NO2), liperoxidation (LOOH) and Paraoxonase 1 (PON1)

activity. Statistical analysis: RM-ANOVA or 1way-ANOVA, post-hoc Bonferroni’s test. Mean ± SEM, significance level

of 95%, sample size at the end of protocols: 5-15.

Results: 2 hours after LPS, OVX+E treated with SMT presented a decrease of MAP, when compared to SHAM and to

MAP at time zero (ΔMAP, mmHg: SHAM= 0,8±3, OVX= -8,3±3, OVX+E= -13,3±4, n=8-9 per group). At this same

time, all SMT+LPS groups presented, in percentage, an increase of sympathetic modulation and a decrease of

parasympathetic modulation of HR, showed respectively by LFnu, LF/HF and HFnu parameters of HR variability. 2

hours after saline+LPS, OVX presented decreased TRAP when compared to SHAM (TRAP, uM trolox: SHAM=

379±24, OVX= 153±8, OVX+E= 312±34, , n=8-11 per group). When treated with SMT+LPS, OVX did not presented

altered TRAP after 2 hours, while estradiol presented reduced LOOH levels (x103 RLU: SHAM SMT+LPS= 4714±563,

OVX+E SMT+LPS= 1991±310, n=5-7 per group).

Conclusion: Since sympathetic activity is important to cardiac recovery in sepsis, our findings suggest that, in the

cardiovascular system, treatment with estradiol could be protective in inflammatory challenges, since iNOS is

inhibited.

Grants: Financial support: CNPq


EVALUATION OF METABOLIC PARAMETERS IN ADULT RATS EXPOSED TO FLUOXETINE DURING

GESTATION AND LACTATION

Silva, W. A. M.1; Marques, B. V. D.

1; Novi, D. R. B. S

1; Higashi, C. M.

1; Serafim, A. F. L.

1; Gregório, T. G.

1; Ceravolo

G. S.1

1 State University of Londrina, Department of Physiological Sciences, Londrina, PR, Brazil

[email protected]

Keywords: Antidepressants; Fluoxetine exposure; Intrauterine; Insulin.

Introduction and objectives: Fluoxetine (FLX) is one of the antidepressants most used during pregnancy to treat

affective disorders and anxiety because its side effects are reduced [1]. It crosses the placenta and is excreted in

breast milk exposing fetuses and neonates at important stages of development to this drug [2]. But even so in some

cases the treatment is still indicated [3][4]. Events such as illness or exposure to drugs, which occur during uterine

development, may influence late in the individual's health [5]. The objective of the study was to assess whether

intrauterine and lactational exposure to fluoxetine causes metabolic changes related to diabetes and obesity in adult

offspring.

Material and methods: Wistar rats (n=32), kept under standard conditions (22±2°C, 12h light-dark cycle), water and

feed at will, mated at night. From the gestational day 0 (GD0), diagnosed by vaginal smear, the rats were

individualized and divided into 2 groups, receiving daily treatments by gavage:

-Control: water;

-Fluoxetine: 5mg/kg/day (Daforin®).

They were weighed every three days to adjust dosage and gestational follow-up. On postnatal day 4 (PND 4), the

litters were reduced to 10 animals (5 males and 5 females) and weaned in PND 21. In PND 75, they were fed fasting

(4h), weighed and anesthetized with sodium thiopental (40mg/kg, intraperitoneally), and the tail was cut to measure

baseline glycemia using a glucometer. Blood was collected before and 4, 8, 12 and 16min after a regular

intraperitoneal injection of insulin (0.75U/kg, Humulin®).

The constant rate of glycemia disappearance during the insulin tolerance test (KITT) was calculated based on the

linear regression of the neperian logarithm of the glucose concentrations obtained. The naso-anal length was

measured. The Lee Index was calculated as follows: body weight 1/3(g) / nasal-anal length(cm) × 100. The

experimental protocols were approved by the UEL Ethics Committee for Animal Research (CEUA nº16166-2012.12).

The results are shown as mean±S.E.M. Statistical analysis was performed using the T-student test, using the Prism 6

software package (Graph Pad Software, Inc., San Diego, CA). Values were considered statistically significant when

P<0.05.

Results: In both groups we found that: The maternal body weight in the early stages of gestation was around 210g,

reaching 340g in the final stages; In lactation the weight remained between 220g and 270g; The size of the offspring

and the birth weight so many.

One adult offspring obtained for the control group: Glycemia = 114.5±5 (mg/dL); Weight = 259.5±10.86 (g); Lee index

= 30.27±0.36; KITT = 2.02±0.27. And for the FLX group: Glycemia = 114.65±7.55 (mg/dL); Weight = 272.1±11.64 (g);

Lee Index: 30.76±0.35; KITT = 2.35±0.34.

Conclusion: Maternal exposure to FLX during gestation and lactation does not interfere with maternal body weight,

birth size and birth weight. The fetal and neonatal exposure to FLX did not interfere in the analyzed parameters of the

adult offspring.


EVALUATION OF REPRODUCTIVE PERFORMANCE AND TOXICITY IN MALE MICE TREATED WITH

ISOTRETINOIN

Bispo, A.C.C1; Galvão, T.C

1; Tamoyese, V.M

1; Costa, G.A

1; Ramos, S.D.P

1; Salles, M.J.S

1.

1 State

University of Londrina, Department of General Biology, Londrina, PR, Brazil


Keywords: Isotretinoin, reproduction, spermatogenesis.

Introduction: Isotretinoin (Roacutan®), a derivative of vitamin A, is used to treat severe acne, principally affecting

adolescents. Objectives: Analyze reproductive performance in pubertal mice treated with isotretinoin during

spermatogenesis, and verify possible effects on offspring.

Material and methods: This study was approved by the Ethics Committee on Animal Experimentation of UEL, nº

2919201779/2017. Pubertal male mice were divided into 2 experimental groups: The animals of the treated group (G1)

received isotretinoin at a concentration of 1 mg/kg/day via gavage for 35 days; the control group (G0) received the

equivalent of the excipient in vegetable oil. On day 35 of treatment, the male animals were mated with females that did

not undergo any treatment, after which they continued to be treated for another 10 days, to guarantee at least two

estrus cycles in the females. On day 45 the animals were euthanized. Organs such as the epididymis, testicles, and

seminal vesicles were weighed and the testosterone dosed. The morphology of spermatozoa was analyzed, and

Johnsen's protocol (1970) used for histological analysis of the testicles. On the 18th day of gestation, the females were

euthanized, and the uterine contents analyzed. Visceral and skeletal congenital malformations were investigated. The

Student's t/ANOVA, Fischer and Chi-Square tests of the GraphPad Prism program were used, with a significance of

5% for statistical analysis of the results.

Results: The treated group presented statistically significant results, such as weight reduction of the epididymis (G0 =

0.091g ± 0.002 and G1 =0.080g ± 0.002), testicles (G0=0.211g ± 0.009 and G1= 0.199g ± 0.010), seminal vesicle (G0 =

0.222g ± 0.015 and G1 =0.176g ± 0.013), and low testosterone (G0= 2.423ng/mL ± 0.592 and G1 = 2.162ng/mL ±

0.464). The Johnsen score was lower when compared to the controls, as there was a higher prevalence of immature

seminiferous tubules in the treated mice, with incomplete germ cell lineage (G0= 9.28 and G1= 8.47). Analysis of

sperm morphology demonstrated alterations in the tails (G0= 21.17% and G1=22.57%) and heads (G0 =4.94% and G1

= 6.60%). Regarding the offspring development, the treated group presented a decreased number of live fetuses (G0=

10.220 ± 0.641 and G1= 8.296 ± 0.736), an increased percentage of resorptions (G0 = 21.020 ± 3.392 and G1 = 29.

330 ± 4.417), and post-implantation losses (G0 = 21.510 ± 3.347 and G1 = 29.540 ± 4.414). The offspring presented

skeletal malformations in the sternum (G0 = 9.09% and G1= 26.90%), palate (G0 = 0.70% and G1 = 10.07%), and

supraoccipital (G0 = 9.79% and G1=31.65%). The visceral malformations were irregularities of the olfactory bulbs (G0 =

0% and G1 = 5.18%), lungs (G0 = 2.26% and G1 = 8.15%), nasal septum (G0 = 1.50 % and G1 = 8.88%), and enlarged

heart (G0= 3.01% and G1 = 11.85%). Conclusion: Exposure to the drug caused damage to reproductive performance

and was teratogenic in the offspring. The information obtained could contribute to healthcare programs in the

population of reproductive age, making possible prevention strategies against possible damage to offspring.

Grants: CNPq


EVALUATION OF THE EFFECTS OF LIXISENATIDE ON SOME METABOLIC PARAMETERS

AFFECTED BY WALKER-256 TUMOR.

Quintilhano, D. L1; Biazi, G. R.

1; Frasson, I. G.

1; Miksza, D. R.

1; Souza, H. M.

1; Bertolini, G. L

1.



Keywords: cancer, cachexia, agonist of glucagon-like peptide.

Introduction and objectives: Cachexia occurs in many chronic diseases and it is present in approximately 80% of

patients with cancer being the main cause of mortality among these individuals. Walker-256 tumor, a carcinosarcoma

of rapid growth, causes severe loss of muscle and fat mass, insulin resistance, hypoinsulinemia and various metabolic

changes characteristic of cachexia. Since lixisenatide, an agonist of GLP-1 receptors, shows insulinotropic effect, the

aim of this study was to evaluate the effects of the treatment with lixisenatide on tumor growth and several metabolic

parameters affected by Walker-256 tumor.

Material and methods: Wistar male rats (250 g), healthy (H) or Walker-256 tumor-bearing (WK) treated with saline

(S) or lixisenatide (L), were used. For tumor implantation, 8x107 Walker-256 cells were inoculated subcutaneously in

the right rear flank of the animals. In the healthy group, PBS was inoculated at the same place. H and WK animals

were submitted to daily intraperitoneal injection of saline or lixisenatide (50 µg/kg) once a day during 6 days, starting

on tumor cells inoculation day. During this period, food intake was assessed. On the 6th day after treatment with

lixisenatide or saline, plasma concentrations of glucose, triacylglycerol and lactate, tumor growth, change in body

mass, and retroperitoneal and epididimal adipose tissues, gastrocnemius and long digital extensor muscles mass

were assessed. The peripheral response to insulin was evaluated by the insulin tolerance test. The experimental

protocols were approved by Animal Experimentation Ethics Committee of the State University of Londrina (protocol nº

12199.2015.14). The results were analyzed by appropriate statistical test adopting a significance level of 5% (p<0.05)

and were expressed as the mean±standard error of the mean of 9-11 animals.

Results: Tumor mass (g) was not different between WKS and WKL groups. Change in body mass (g%) was

significantly different (p<0.05) among all groups (HS=+28±3,4; HL=+7,5±1,8; WKS=-6,9±2,9; WKL=-22,5±2,4).

Retroperitoneal and epididimal adipose tissues mass (g%) were lower (p<0.05) in HL, WKS and WKL than HS group.

No significant differences on gastrocnemius and long digital extensor muscle mass (g%) were observed between

groups. Food intake was lower in HL than HS group, and no significant differences were observed between the other

groups. There were no differences in plasma concentrations of glucose, triacylglycerol and lactate between HS and

HL groups and between WKS and WKL groups. However, glycemia were lower and plasma concentrations of

triacylglycerol and lactate were higher in tumor-bearing animals than in healthy animals. Intravenous administration of

insulin did not promote blood glucose lowering in the WKS and WKL groups as observed in the HS and HL groups.

Conclusion: The lixisenatide treatment promoted less gain and greater loss of body mass in healthy and in tumor-

bearing animals, respectively, when compared with animals treated with saline and it was not able to improve the

peripheral insulin response of the tumor bearing animals. The change in body mass was accompanied by the change

in the retroperitoneal and epididimal adipose tissue mass. In HL group, these effects may be due to lower food intake

and delayed gastric emptying.


EVALUATION OF THE HEPATOTOXIC POTENTIAL OF ACETAMIFENE (PARACETAMOL®) IN

RATTUS NORVEGICUS ALBINUS MODEL DURING THE BREASTFEEDING PERIOD

Miguel, F. H.1; Machado, A. R. S. R.

2; Oda, J. Y.

2; Machado, A. M.

2 1 Faculdades Integradas de Três Lagoas, Três Lagoas – MS, Brazil.

2Universidade Federal de Mato Grosso do Sul – UFMS, Três Lagoas, MS, Brazil


Keywords: Acute liver failure, Acetamifene, Paracetamol, Hepatotoxic potential

Introduction and objectives: Paracetamol is a widely used medication because of the low cost and pharmacological

action however high doses can lead to acute liver failure, cardiovascular, gastrointestinal and neurological disorders.

Little is known about its effects on females in the breastfeeding period, and their infants who passively receive the

drug via breast milk. The objective of this study was to evaluate the hematological, biochemical (liver and renal

function) and histopathological (liver) alterations in Rattus norvergicus albinus lactating, treated with high doses of

Paracetamol and in their infants.

Material and methods: Initially, the equivalence calculation was performed to determine the dose of the drug, using

the equivalent of 50% of the maximum human dose (2000 mg) in relation to dose/weight, fractionated in 3 doses per

day. Four females (2 test females and 2 control females) were used, with ten infants each, who received via gavage,

Paracetamol or sterile saline for the entire period of breastfeeding (18 days) (Ethics Committee: 02/2016). After this

period, peripheral blood was collected from all animals for hematological and biochemical analyzes, and after

euthanasia, liver samples were collected for histopathological analysis.

Results: The hematological analysis of treated females showed: oligocythemia (treaties: 3,2x106/mm

3 - control:

5,7x106/mm

3), hypochromia (treaties: 10.1 g/dL - control: 15.2 g/dL), anisocytosis with a tendency to macrocytosis and

different poikilocytes, as spherocytes, codocytes and stomatocytes (related to cases of hepatopathies and hemolytic

anemia), echinocytes (related to cases of hyperuremia), and schizocytes (related to chemical aggressions by oxidative

drugs). In addition, it was possible to observe reticulocytes (15%), decreased platelet counts (treaties: 183,2x103/mm

3

- control: 543,6x103/mm

3), predominance of typical and atypical lymphocytes (77%) and basophilia (10%). In the

infants, similar parameters were observed, with an overall anemia, however with less intensity when compared to

mothers. The biochemical analyzes of liver markers showed a strong increase in the liver enzymes, AST and ALT

(treaties: 732 IU/mL and 222 IU/mL - control: 19 IU/mL and 14 IU/mL), with moderate alteration of markers such as:

Phosphatase Alkaline, Total Proteins and Albumin, and slight change in Gamma-GT and Bilirubin. Analysis of renal

markers showed a strong Urea increase and a moderate increase in Creatinine (treaties: 129 mg/dL and 2.7 mg/dL -

Control: 19 mg/dL and 0.9 mg/dL). It was also observed a strong increase of Serum Iron, consistent with hemolytic

anemia, and increase of amylases and Creatine Phosphokinase, associated with pancreatic and cardiac injury,

respectively. In the infants were observed similar parameters, however with less intensity. The macroscopic evaluation

of the livers, show, in treated female, vascularized cellular masses in different hepatic regions. Histopathological

analyzes showed extensive areas of centrilobular necrosis, areas of intense hepatic parenchymal degeneration,

preceding the liver fibrosis process. In the infants was also observed cell swelling and centrilobular necrosis.

Conclusion: Analyzing all these parameters, it was possible to conclude that the use of Paracetamol in high dosages

in short periods is capable of causing serious hepatic lesions, anemia and renal impairment, being excreted in breast

milk and causing similar lesions in newborns.


EXPOSURE OF ADULT RATS TO COMMERCIAL GASOLINE IMPAIRS SPERMATOGENESIS AND

SPERM QUALITY

Vieira, K. C. M. T.1; Tamião, A. A. F.

2; Silva, K. M.

2; Pereira, V. R.

1; Lima, T. V.

2; Ribas, V. P.

2; Lopes, L. M.

2;

Favareto, A. P. A.1,2

1Graduate Program in Environment and Regional Development, University of Western São Paulo – UNOESTE,

Presidente Prudente, SP, Brazil 2College of Science, Letters and Education from Presidente Prudente – FACLEPP, University of Western São Paulo –

UNOESTE, Presidente Prudente, SP, Brazil.

[email protected]

Keywords: Benzene, toluene, xylene, reproduction, rat

Introduction and objectives: Leaks at petrol stations cause major problems with contamination of groundwater and

soils. The major problems of petroleum by-products contamination are attributed to monoaromatic hydrocarbons

(benzene, toluene and xylene). Thesecompounds are the most soluble and mobile constituents of the gasoline

fraction, being the first to reach the water table. Contamination caused by leakage leads to possible exposure of the

general population when in contact with contaminated water and soil. Despite concerns about potential health risks

caused by exposure to gasoline components, most reproductive toxicology studies address constituents separately.

The objective of the present study was to evaluate the effects of exposure of adult male rats to the commercial mixture

of gasoline/ethanol on sperm parameters and spermatogenesis.

Material and methods: The study was approved by the Committee on Ethics in the Use of Animals (CEUA,protocol #

3183). Adult Wistar rats (n=8/group) were orally exposed to 0 (control, corn oil), 16 (Low dose group - LG), 160

(Medium dose group - MG) or 800mg/kg/day (High dose group - HG) of commercial gasoline, for 30 days. After the

end of exposure, animals were weighed and euthanized. Sperm were collected from the left vas deferens and

dispersed in modified HTF medium (34°C). Sperm parameters evaluated were motility (progressive, non-progressive

movement and immotile), plasma membrane integrity (eosin/nigrosin staining), acrosome integrity (Fast Green/Rose

Bengal staining) and mitochondrial activity (cytochrome C oxidase activity). Sperm were collected from the right vas

deferensand and fixed in 10 % formalin to morphology analysis. The right epididymis and testis were collected for the

sperm counts and determination of the sperm transit time. Left testes were collected, fixed and submitted to the

paraffin wax inclusion routine. H&E stained testicular sections were submitted to analysis of the Sertoli cell and germ

cells number. ANOVA with a posteriori Tukey test was used for statistical analysis.

Results: Sperm parameters were decreased in a dose-dependent manner. The mitochondrial activity and acrosomal

integrity were decreased in MG and HG when compared to the control group. Sperm vitality was decreased in HG

when compared to control group, but the other groups were similar to each other. The daily sperm production was

decreased in MG and HG when compared to control group. In addition, this parameter was lower in HG than in LG.

There was a significant delay in transit time in epididymal caudas in GM and HG when compared to control group.

Number of sperm with progressive movement was decreased in MG and HG when compared to control group There

was an increase of the number of isolated sperm heads in MG and HG when compared to control group, but LG was

similar to MG and control group for ths endpoint. Number of Sertoli cells, spermatogonia type A, spermatocytes in

pachytene and round spermatids were reduced in the three exposed groups when compared to control group.

Conclusion: In conclusion, the results show that the exposure to commercial gasoline/ethanol during adulthood, in

these experimental conditions impairs spermatogenesis and sperm quality.

Grants: CAPES



HEATH LEGDGER’S DEATH: A DRUG INTERACTION CASE STUDY

Bacchi, A. D.1


[email protected]

Keywords: acute intoxication, toxicology, pharmacology, prescription medications, drug interaction

Introduction: Heath Legder, famous Hollywood actor for the films "Brokeback Mountain" and "Batman The Dark

Knight", was found dead lying in his bed in the afternoon of January 22, 2008.

Case report: On the day of the actor's death, no signs of physical aggression, attempted murder or even suicide were

found. The death certificate reported inconclusive death, pending further studies.

On February 6, after a thorough toxicological examination, it was concluded that the manner of death was an

accident, resulting from the abuse of prescription medications.

It is publicly known that Ledger had suffered from depression in the past, for which sertraline (Zoloft), a

selective serotonin reuptake inhibitor, had been prescribed. However, this drug was not found in the post mortem

toxicological test, indicating that this drug was not involved in actor’s intoxication.

Another known fact was that Legder had some involvement with illegal drugs, such as cocaine. However, like

Zoloft, the toxicology test was negative for this drug.

Heath Ledger suffered from chronic and intense insomnia. This disorder worsened after the end of his

marriage. To control anxiety and to induce sleep, Ledger used benzodiazepines. On the toxicological examination,

diazepam, temazepam and alprazolam were found. These drugs are controlled because they can, with chronic use,

lead to tolerance and dependence. The benzodiazepines' mechanism of action consists in increasing the affinity of the

GABAA receptor for GABA. Thus, GABA binds more frequently to its receptor, increasing the opening frequency of the

chloride channel. The higher chloride influx leads to post synaptic neuron hyperpolarization and, consequently, central

depression. However, since this mechanism depends on the GABA binding to its receptor, it is unlikely that, in the

absence of mixed intake, benzodiazepine intoxication could cause death. It is worth mentioning that the temazepam

found may have been the result of hepatic biotransformation of diazepam.

Benzodiazepines were not the only drugs found in Heath Ledger’s drug test. The test was also positive for

doxylamine. Doxylamine is an antihistamine with strong sedative action. The sedative effect occurs by antagonizing

histamine H1 receptors in the Central Nervous System, reducing the maintenance of wakefulness. Doxylamine is not

a controlled drug and may act synergistically with benzodiazepines’ effects.

However, in addition to these drugs, two opioids were found in the actor's blood: oxycodone and

hydrocodone. Opioids act on pre- and post-synaptic μ receptors. In post-synaptic neuron, the activation of μ receptors

reduces glutamate exocytosis by closing calcium channels. In the post-synaptic cell, this interaction causes the

opening of potassium channels. Potassium efflux leads to neuronal hyperpolarization, causing dose-dependent central

depression. These drugs were probably responsible for the Ledger's death, potentiated by the other drugs mentioned

above.

Final consideration: It is possible to conclude that Ledger's death was probably due to a cardiorespiratory arrest

resulting from acute intoxication by the combined effects of opioids, benzodiazepines and antihistaminic. It is therefore

an interesting case study for learning pharmacology and drug interactions concepts. All information used in this article

was taken from public documents of free access.


HESPERIDIN METHYL CHALCONE AMELIORATES MONOSODIUM URATE CRYSTALS-INDUCED

EXPERIMENTAL GOUTY ARTHRITIS BY INHIBITING NFB ACTIVATION IN MICE

Badaro-Garcia, S1; Ruiz-Miyazawa, K.W.

1; Pinho-Ribeiro, F.A.

1; Borghi, S.M.

1; Staurengo-Ferrari, L.

1; Fattori, V.

1;

Casagrande, R.2; Verri, W.A.

1

1Universidade Estadual de Londrina, Department of Pathological Sciences, Londrina, PR, Brazil

2Universidade Estadual de Londrina, Department of Pharmaceutical Sciences, Londrina, PR, Brazil


Keywords: experimental arthritis, cytokines, NFκB; oxidative stress; synovitis; flavonoids

Introduction and objectives: Gout arthritis (GA) is a painful inflammatory disease dependent on the deposition of

monosodium urate (MSU) crystals in joints and peri-articular tissues. If properly pharmacological managed, GA is well-

controlled. Nevertheless, GA can trigger acute attacks that are one of the most painful experiences known. In this

context, flavonoids possess analgesic, anti-inflammatory, and antioxidants effects. Hesperidin methyl-chalcone (HMC)

is a bi-flavonoid which presents antioxidant, analgesic, and anti-inflammatory properties partly through inhibition of

NF-κB activation. Thus, we aimed at evaluating of the effect and mechanisms of HMC in a mouse model of MSU-

induced GA.

Material and methods: Male Swiss or LysM-GFP mice care and handling procedures were approved by the Ethics

Committee of the Universidade Estadual de Londrina (process number 14600.2013.73). Experimental gout was

induced by an intra-articular injection of MSU (100μg/10µL). Mice were treated with HMC (3, 10, or 30mg/kg, orally) or

vehicle (saline) 1h before stimulus with MSU (n=6). Mechanical hyperalgesia (electronic anaesthesiometer) and knee

joint edema (caliper) were evaluated 1-15h after stimulus. Leukocyte migration to the knee joint was evaluated 15h

after stimulus. HMC at 30mg/kg was chosen for subsequent experiments. Leukocyte recruitment was evaluated in the

histopathological analysis (HE staining) and in the joint wash of MSU-stimulated LysM-GFP+ mice by confocal

microscopy. Knee joint samples were also collected 15h after stimulus for determination of cytokine production and

NF-κB activation evaluated by ELISA. Inflammasome components, mRNA expression of gp91phox, nuclear factor

erythroid 2-related factor 2 (Nrf2), and heme-oxigenase-1 were evaluated by RT-qPCR. Oxidative stress by measuring

antioxidant glutathione levels, superoxide anion production (NBT assay), total antioxidant capacity (FRAP and ABTS

assays), In vitro analysis using LPS-primed bone marrow-derived macrophages was performed 5h after stimulation

with MSU to determine mature IL-1β secretion by ELISA. Data were analyzed by ANOVA followed by the Tukey’s test.

Results: HMC reduced in a dose-dependent manner MSU-induced mechanical hyperalgesia, edema, and leukocyte

recruitment to the knee joint (57%). HMC also inhibited MSU-induced Lysm-GFP+ cells infiltrate and synovitis score

(77%) as observed in the HE histopathological analysis. In terms of MSU-induced oxidative stress, treatment with

HMC restored GSH levels (88%) and total antioxidant capacity [FRAP (100%) and ABTS (100%) assays]. In addition,

HMC inhibited superoxide anion production (100%) and NO (100%). Moreover, HMC reduced gp91phox and Nrf2/HO-

1 mRNA expression. HMC also decreased pro-inflammatory cytokines production TNF-α, IL-1β, and IL-6 as well as

inhibited MSU-induced NFκB activation (83%) and the inflammasome components NLRP3 (87%), ASC (90%), Pro-

caspase-1 (89%). Moreover, HMC induced anti-inflammatory TGFβ production. However, HMC did not inhibit IL-1β

secretion by LPS-primed macrophages challenged with MSU crystals.

Conclusion: The present data shows that the anti-inflammatory and analgesic effect of HMC in MSU-induced arthritis

is related to the inhibition of NFκB activation, but not on direct inhibition of inflammasome. Moreover, the increase on

the mRNA expression of the antioxidant Nrf2/HO-1 signaling pathway may account to the beneficial effect herein

observed. Therefore, molecules that act in multiple pathways such as HMC are highly attractive and represent a

possible therapeutic approach for the treatment of GA.

Grants: This work was supported by grants from Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior

(CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação Araucária.


INFLUENCE OF MATERNAL TREATMENT WITH METFORMIN ON OFFSPRING’S METABOLIC AND

CARDIOVASCULAR PARAMETERS OF RATS

Moura, K.F1; Novi, D.R.B.S

1; Vidigal, C.B

1; Raquel, H.A.¹; Uchoa, ET

1; Martins-Pinge, M.C.

1; Gerardin, D.C.C.

1,

Ceravolo G.S. 1

1Universidade Estadual de Londrina, Department of Physiology, Londrina, PR, Brazil (e.g)

[email protected]

Keywords: Fetal exposure, maternal treatment, insulin, blood pressure

Introduction and objectives: Metformin (MET) is a biguanide used to treat type 2 diabetes, gestational diabetes,

insulin resistance and polycystic ovary syndrome. Although the MET crosses the placenta and has been detected in

the umbilical cord at the same concentration of the maternal blood, it is considered a safe drug throughout pregnancy.

Due to scarcity of studies evaluating the metabolic and cardiovascular consequences intrauterine exposure to MET,

the aim this study was to evaluate if maternal exposure to MET during pregnancy and lactation could cause metabolic

and cardiovascular alterations on adult offspring.

Material and methods: All experimental protocols were approved by the State University of Londrina Ethics

Committee for Animal Research (CEUA/UEL: 6996.2015.02). Wistar female rats were treated with MET

293mg/kg/day, by gavage from gestational day (GD) 0 to the GD 21 (METG) or GD 0 until post natal day (PND) 21

(METGL). Control dams received water by gavage at the same periods (CTRG and CTRGL). Dams’ weights were

obtained every three days during all pregnancy and lactation for dose adjusting, and weight gain variation was

calculated during gestation (D = PND1- GD0) and lactation (D= PND21-PND1). The food consumption of dams was

weekly evaluated. In male and female offspring (75 days) were evaluated the blood pressure mean (BPM) and heart

rate (HR); the insulin tolerance (kITT), obesity (Lee index and visceral fat pad) evaluation and biochemical

parameters: free fatty acids, total cholesterol and triglycerides. The results are shown as mean ± S.E.M. The variables

that presented normal distribution and homogeneity were analyzed by Student-t test or RM ANOVA (food

consumption). Values were considered statistically significant when P<0.05.

Results: The results demonstrated that BPM of adult male and female exposed to MET was similar to their controls.

The HR of female METGL was increased when compared to CTRGL, while female METG, and in male METG and

METGL the HR was similar to their respective controls. The constant rate for blood glucose decay (kITT), basal

glycemic levels, body weight, Lee index and biochemical parameters of male and female exposed to MET were similar

to control groups. In relation to body composition, female METG had reduction in perigonadal fat pad and male

METGL presented reduction in retroperitoneal fat pad deposition.

Conclusion: Altogether, it is possible to conclude that MET exposure during gestational and lactation periods was

safe for dams and offspring and this exposure probably does not program cardiovascular and metabolic alterations in

adult offspring.


INTRAUTERINE EXPOSURE TO METFORMIN DOES NOT LEAD TO CHANGES IN THE VASCULAR

SYSTEM OF ADULTHOOD OFFSPRING

Vidigal, C.B.1; Novi, D.R.B.S.

1; Moura, K.F.

1; Montagnini, B.G.¹; Gerardin, D.C.C.

1; Ceravolo, G.S.

1

¹Department of Physiological Sciences, Center of Biological Sciences, State University of Londrina, Londrina, PR,

Brazil.

[email protected]; [email protected]; [email protected];

[email protected]; [email protected]; [email protected]

Keywords: intrauterine exposure to metformin; perivascular adipose tissue; abdominal aorta; endothelial function;

vascular reactivity.

Introduction and objective: The biguanide metformin (MET) crosses the placenta and has been detected in the

umbilical cord at similar concentrations of maternal blood. Besides that, MET has been used for the treatment of

diabetes and polycystic ovary syndrome during gestation. In mice intrauterine exposure to MET causes increased

body weight and increased adipose tissue accumulation during adult age. However, there are no studies evaluating

the vascular consequences of MET exposure during initial phases of body development. The present study aimed to

investigate the effects of MET intrauterine exposure on abdominal aorta reactivity in adult offspring, analyzing the

endothelium and perivascular adipose tissue (PVAT) function.

Material and methods: The study was approved by the CEUA/UEL (6996.2015.02). Twenty-three Wistar rats were

treated with MET 293 mg/kg/day or water (CTR) by gavage during gestational period. It was evaluated in male

offspring (23 rats, 75-80 days) the abdominal aorta reactivity to phenylephrine (Phenyl), acetylcholine (Ach) and

sodium nitroprussiate (SNP) in ring with PVAT (PVAT+) and without PVAT (PVAT-), both in the presence (Endo+) or

absence of endothelium (Endo-). The comparison between groups was performed using the maxima response (maxR)

to the drugs and using pD2. Statistical analyses were performed using SPSS and the variables analyzed by two way

ANOVA, being results presented as mean ± standard error of the mean (SEM). Differences were considered

statistically significant if *p<0.05.

Results: The dependent variable maxR (% of KCl contraction) to Phenyl in the presence and absence of endothelium

did not present significant differences in relation to independent variables, exposure or not to MET and presence or

absence of PVAT [Endo+ PVAT+: CTR 155.86±9.25 vs MET 154.55±8.01; Endo+ PVAT-: CTR 154.12±8.36 vs MET

142.26±8.36; Endo- PVAT+: CTR 187.67±9.21 vs MET 180.24±8.82; Endo- PVAT-: CTR 215.46±9.21 vs MET

206.77±9.66], and pD2 to Phenyl was also similar between the groups [Endo+ PVAT+: CTR 6.00±0.14 vs MET

5.97±0.12; Endo+ PVAT-: CTR 5.78±0.13 vs MET 5.90±0.13; Endo- PVAT+: CTR 6.71±0.12 vs MET 6.86±0.11;

Endo- PVAT-: CTR 6.78±0.12 vs MET 6.83±0.12]. ACh and SNP induced concentration-dependent relaxation in all

abdominal aortic rings evaluated. There were no differences in maxR [Endo+ PVAT+: CTR 92.25±2.41 vs MET

92.15±2.41; Endo+ PVAT-: CTR 91.48±2.29 vs MET 91.71±2.41] and pD2 [Endo+ PVAT+: CTR 7.17±0.08 vs MET

6.98±0.08; Endo+ PVAT-: CTR 7.47±0.08 vs MET 7.50±0.08] to ACh, as well as there were no differences in the

maxR [Endo- PVAT+: CTR 98.434±2.32 vs MET 100.52±2.32; Endo- PVAT-: CTR 98.42±2.45 vs MET 101.42±2.32]

and pD2 [Endo- PVAT+: CTR 8.16±0.08 vs MET 8.14±0.08; Endo- PVAT-: CTR 8.44±0.09 vs MET 8.40±0.08] to NPS

between CTR and MET groups.

Conclusion: The intrauterine exposure to metformin was safe and did not lead to changes in the abdominal aorta

reactivity of adulthood offspring.

Grants: CAPES






KAURENOIC ACID ATTENUATES ZYMOSAN-INUCED ARTHRITIS AND PERITONITIS IN MICE

Ambrosio, F1; Fattori, V.

1; Manchope, M.F.

1; Staurengo-Ferrari, L.

1; Bertozzi M.M.

1; Mizokami, S.S.

1; Casagrande, R

2;

Arakawa, N.S.2; Verri, W.A

1

1 Universidade Estadual de Londrina, Departmento de Ciências Patológicas, Londrina, PR, Brazil.

2 Universidade Estadual de Londrina, Hospital Universitário, Departmento de Ciências Farmacêuticas, Londrina, PR,

Brazil.


Keywords: zymosan, arthritis, inflammation, kaurenoic acid, synovitis

Introduction and objectives: Pain is a symptom present in most inflammatory diseases, and may even lead to the

development of depression or loss of function of affected tissues or organs. During inflammation, peripheral activation

of mechanisms responsible for the induction of edema, leukocyte migration, and hyperalgesia occurs. Rheumatoid

arthritis (RA) is a chronic autoimmune disease characterized by debilitating pain, cartilage destruction, and loss of joint

function. Management of RA includes drugs that target NF-κB and downstream cytokine production. Therefore,

molecules that act by inhibiting this signaling pathway without the severe side effects of, for instance, corticoids would

be suitable therapeutic strategies. Kaurenoic acid (KA, ent-Kaur-16-on-19-oic acid) is a diterpene presented in several

plants and the main constituent of Sphagneticola trilobata (L.) Pruski. Studies have shown that KA possesses anti-

inflammatory, antioxidant, and antinociceptive activity. Importantly, KA as other diterpenes, may act inhibiting NF-κB

activation and thereby reducing downstream production of pro-inflammatory mediators. Therefore, we aimed at

evaluating the efficacy of KA in two different models: zymosan-induced arthritis and peritonitis.

Material and methods: Experiments were conducted on male Swiss mice. Animal care and handling procedures

were conducted accordingly with International Association for the Study of Pain (IASP) guidelines and with Londrina

State University Ethics Committee on Animal Research and Welfare approval under process number 12105.2012.67.

For the arthritis model, mice were treated with KA (3, 10, or 30 mg/kg, per oral) 60 min before the administration of

zymosan (500 μg/knee joint, intra-articular). Mechanical hyperalgesia (evaluated using the electronic version of von

Frey filaments), knee joint edema (caliper), and motor performance (rota-rod apparatus) were evaluated 1-7 h after

the stimulus. The knee joint wash was collected for analysis of total leukocytes and polymorphonuclear cells. For the

peritonitis model, mice were treated with KA (30 mg/kg, per oral) 60 min before the administration of zymosan (1

mg/cavity, intraperitoneal). Peritoneal wash was collected 7h after stimulus for evaluation of leukocytes recruitment,

cytokine production (TNF-α, IL-1β, and IL-6) by ELISA, and oxidative stress (NBT assay, superoxide anion

production).

Results: Treatment with KA decreased zymosan-induced mechanical hyperalgesia, edema, and leukocytes

recruitment to the knee joint (total and polymorphonuclear cells) in a dose-dependent manner. Importantly, KA did not

influence on motor capacity demonstrating that did not possess myorelaxant effect. Regarding the peritonitis model,

KA reduced zymosan-induced leukocytes recruitment to the peritoneal cavity in 47.6% and superoxide anion

production in 57.6%. Moreover, KA reduced the production of TNF-α in 32.1%, IL-1β in 23.2%, and IL-6 in 9.4% in the

peritoneal wash.

Conclusion: This study demonstrates that KA improves zymosan-induced arthritis and peritonitis by inhibiting

cytokine production (TNF-α, IL-6, and IL-1β), the recruitment of polymorphonuclear cells, and oxidative stress.

Therefore, KA is a promising molecule for the treatment of inflammatory diseases and certainly deserves further

studies.






MALFORMATION OF THE DENTAL GERM IN MICE EXPOSET TO METHYLPHENIDATE DURING

PREGNANCY

Sestário,C.S1;Lima,K.S

2;Ramos,S.P

2;Martins,C.C.N

1;Salles,M.J.S

1

1Universidade Estadual de Londrina, Departamento de Biologia, Londrina,PR,Brasil

2Universidade Estadual de Londrina, Departamento de Histologia, Londrina, PR, Brasil

[email protected]

Key words: pregnancy, methylphenidate, odontogenesis

Methylphenidate hydrochloride (MFD) is a psychostimulant that has been getting space in the treatment of

Attention Deficit Disorder and Hyperactivity, however, the use of this drug during pregnancy is not quite safely

determined with regard to the risks offered to the embryo.In this way, the objective of this work was to evaluate the

effects of exposure to MFD on the embryonic development of the dental germ in mice. It has been used 32 pregnant

adult Swiss mice (Mus musculus), weighing out 35g each one. The drug used was Ritalin (Novartis). For the prenatal

study, females were divided into 2 groups, 1 control group and 1 treated group. Each group consists of 16 animals.

The treated group received 5 mg / kg of drug via gavage. The control group received the same volume of sterile

saline. For the histological evaluation of odontogenesis the first fetus of the right uterine horn of each female was

separated and had its head removed. The pieces were placed in previously identified containers containing 10%

formaldehyde for 48 hours. Then, they were processed for histological procedure and stained by hematoxylin and

eosin, and then 25 sagittal cuts of each piece, 7 micrometers thick, were performed. The cut plane chosen was

parallel to the longitudinal axis of the molar, demonstrating the organ region of the enamel and dental papilla. Three

cuts of each animal were selected, so the analysis of the beginning, middle and end of the dental germs could be

performed. The analysis was performed on 3 slides of each piece, and on each slide, 5 fields were analyzed. The

slides were photographed in a digital camera coupled to an optical microscope (Moticam, Motic Co., Xiamen, China)

and analyzed in Motic Image Plus 2.0 software (Motic Co, Xiamen, China) at 10 and 20X magnification. For the

analysis of the association between the dependent variables and the groups (control or treated) the chi-square and

exact tests were used. The parameter that presented statistical relevance was the occurrence of changes in 43.8% in

the form of the enamel organ (p = 0.035) of the treated group, while no alteration was observed in the control group.

In conclusion , we can see that the exposure of metylphenidate during the pragnancy alterns the development

the dental germ in mice. Administration of drugs during the first trimester of pregnancy, may affect the development,

differentiation and growth of dental germs. Is the dentist´s responsible for advise pregnant women about medications

that can affect fetal development.



MATERNAL EXPOSURE TO FLUOXETINE DURING GESTATION AND LACTATION UP-REGULATES

NEURONAL NITRIC OXIDE SYNTHASE EXPRESSION LEADING TO DECREASED AORTIC

CONTRACTION IN ADULT FEMALE PROGENY

Higashi, C. M.

1; Sartoretto, S.M.

2; Echem, C.

2; Carvalho, M. H. C.

2; Pelosi, G. G.

1; Pinge-Filho P.

3; Gerardin, D. C. C.

1;

Moreira, E. G.1; Akamine, E. H.

2; Ceravolo, G. S.

1.

1 Department of Physiological Sciences, Biological Sciences Center, State University of Londrina, Parana, Brazil.

2 Department of Pharmacology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil.

3 Department of Pathology, Biological Sciences Center, State University of Londrina, Londrina, Parana, Brazil.

Introduction: To pharmacological treatment of diseases as anxiety disorders and depression during pregnancy and

lactation, the serotonin reuptake inhibitor,fluoxetine (FLX),is worldwide prescribed due to its relative safety. However,

the maternal treatment with this drug may cause programming of disease in the progeny. In fact, itwas demonstrated

that maternal treatment with FLX during gestation and lactation causes aortic hypo-contraction and increases

plasmatic nitric oxide (NO) levels in adult female rat offspring, suggesting that early exposure to FLX programs

vascular alterations in rats’ female progeny. Therefore, the present study aimed to elucidate the mechanism involved

in the aortic hypo-reactivity in female rats maternally exposed to FLX.

Methods: Research approval by the Animal Research Ethical Committee: CEUA nº16166-2012.12. Wistar dams were

gavaged daily with FLX (5mg/kg/day, n=10) or water (CTL, n=10) during pregnancy and lactation. The thoracic aorta

of female offspring 75 days old was removed and cut in two rings, with (E+) and without (E-) endothelium. Cumulative

concentration-effect curves to phenylephrine (Phe: 1nM-30µM) were constructed in the absence or presence

of: nonspecific NO synthases (NOS) inhibitor (L-NAME 1μM), or inducible NOS inhibitor (L-NIL 1μM), or catalase

(100U/mL), or cyclooxygenases (COX) inhibitor (indomethacin, INDO 10μM), or COX2 inhibitor (NS-398 1μM),

or prostacyclin synthase inhibitor (tranylcypromine, tranyl 10μM). Also, in aortic tissue NO was measured by Griess

method and endothelial (eNOS) and neuronal (nNOS) NOS, COX1 and PGI2 expression were assayed by western

blot. Results expressed as mean±SEM. Statistical analysis: one or two-way ANOVA and Tukey's test, differences with

p<0.05.

Results: The maximal response (Rmax, g) to phenylephrine was reduced in aortic rings from FLX rats (1.52±0.11)

when compared to CTL (2.47±0.19), p=0.0001; and L-NAME increased the Rmax in both groups (CTL=3.20±0.20;

FLX=2.97±0.33), p=0.001. Neither L-NIL, catalase nor NS-398 interfered with aortic contraction from both groups.

INDO and tranyl increased contraction in FLX aorta (INDO: CTL=2.14±0.08 vs FLX=2.27±0.12; tranyl: CTL=2.22±0.23

vs FLX=2.23±0.28), matching the Rmax with CTL. NO aortic levels were increased in FLX group (5.7±0.40) compared

to CTL (3.8±0.22), p=0.0001. There was no difference in eNOS, COX1 and PGI2 expression in aorta between groups

and nNOS expression was increased in FLX aorta.

Conclusions: The aortic hypo-contraction, observed in aorta from adult female rats exposed to FLX during

intrauterine and lactation periods, involves NO increased production probably by up-regulation of nNOS expression.

Grants: CAPES/CNPq


MATERNAL TOXICITY OF TRICLOSAN IN A TWO-GENERATION STUDY IN WISTAR RATS

Montagnini, B.G1; Monteiro, M. C

1; Goés, M. L

1; Pernoncine, K. V

1; Kiss, A. C. I

2; Gerardin, D. C. C

1


2 Universidade Estadual Paulista ―Júlio de Mesquita Filho‖, Department of Physiology, Botucatu, SP, Brazil

[email protected]

Keywords: endocrine disruptor, triclosan, maternal toxicity, behavior.

Introduction and objectives: Triclosan (TCS) is a phenolic compound with antimicrobial action widely used in

cosmetics and other personal care products. Its widespread use over the decades has made TCS one of the most

commonly detected compounds in wastewater and effluent worldwide already being found in human urine, plasma

and milk. Studies have shown that TCS has the ability to interfere with the endocrine system, including the

reproductive system, with both estrogenic and anti-estrogenic effects in rodents. The present study aims to evaluate

the TCS effects on the maternal toxicity (maternal behavior, body weight and food consumption) in a two-generation

study.

Material and methods: Female Wistar rats (n = 17 group) were daily treated by gavage with TCS at the doses of 0.8,

2.4 and 8.0 mg/kg/day or corn oil (control group) over 10 weeks (F0) and over 14 weeks (F1) prior to mating and then

throughout mating, gestation and lactation until weaning of F1 and F2 generation respectively. Body weight and food

consumption were recorded during gestation and lactation. The maternal behavior was analyzed during the first 10

postpartum days and the target maternal behaviors were: pup grooming, nest building, off the nest, retrieving and

nursing pups. Data ± SEM were compared by ANOVA (*p < 0.05) (CEUA: 283.2015.27).

Results: Females from TCS 2.4 group of F0 generation presented decreased food consumption during lactation

period (F(3,59) = 3.408, *p = 0.023) and showed an increased frequency of maternal licking pup behavior, compared

to control animals (F(3,36) = 5.444, *p = 0.003). Mean body weight and food consumption during gestational and

lactational periods, as well as maternal behavior parameters were similar between F1 treated groups and their control

(p > 0.05).

Conclusion: The oral treatment with TCS compromised the maternal food intake and maternal behavior in F0 dams.

Results could be related to direct effects of TCS in hypothalamic pathways of appetite control and sexual and

maternal motivations.

Grants: CAPES and CNPq (454499/2014-0)


NITRIC OXIDE INVOLVEMENT IN ACUTE LUNG INJURY CAUSED BY Tityus bahiensis VENOM

Miyamoto, J.G.¹; Andrade, F.B.¹; Victorino, V.J.¹; Chaves, P.A.¹; Cândido, D.M.2; Pinge-Filho, P.¹; Ferraz, C. R.¹; Verri

Jr, W.A.¹; Kwasniewski, F.H.1


2Instituto Butantan,

Laboratório de Artrópodes, São Paulo, SP, Brazil.

[email protected]

Keywords: Tityus bahiensis, acute lung injury, nitric oxide.

Introduction and objectives: Acute lung injury with participation of inflammatory mediators can be triggered by

venoms from scorpions of medical importance. In Brazil, T. serrulatus and T. bahiensis are included in this group. It

was shown that nitric oxide (NO) is produced in experimental envenomation by T. serrulatus. The occurrence of lung

injury induced by other animals from our scorpiofauna (as T. bahiensis) and the participation of inflammatory

mediators, including NO, in such situation is unknown. We studied parameters of airways injury in rats receiving T.

bahiensis venom (Tbv) and the participation of NO on these parameters by pharmacological modulation.

Material and methods: Male rats received Tbv (200 µg/kg, intravenous), following the indicated times the animals

were killed by CO2 inhalation and airways edema (in 30 minutes, n=6 to 14) and hemorrhage (in 60 minutes, n=11 to

15) were investigated by Evans blue (EB) dye extravasation and cyanometahemoglobin concentration using Drabkin's

solution, respectively. After 4 hours of envenomation the cellularity in brochoalveolar lavage (BAL, n=5 to 10), activity

of myeloperoxidase (MPO, n=5 to 7), protein content (by Bradford method, n=5 to 7) and nitrite as estimative of NO

levels (by an adapted Griess method, n=5 to 8) in lung homogenates were analyzed. To evaluate the impact of NO,

the animals were pretreated 30 minutes before the envenomation with L-NAME (nonselective NO synthesis inhibitor,

10 mg/kg intravenous) or L-arginine (substrate for NO synthesis, 300 mg/kg intraperitoneal). Venom and drugs were

diluted in apirogenic NaCl 0,9%. Control data were obtained with rats injected with apirogenic NaCl 0,9%. The

procedures were approved by the institutional ethics committee (CEUA nº 16583.2013.29). Data were compared by

one-way ANOVA and Tuckey’s post-hoc, or Kruskal-Wallis and Dunn’s post-hoc. All analysis were performed in the

GraphPad Prism (5.0) software (San Diego, CA, USA) considering a significance level of α = 0.05. Results were

expressed as mean ± standard error of means (SEM).

Results: In the experimental scorpionism induced by Tbv L-NAME did not modify the EB extravasation, but induced

hemorrhage in trachea and upper bronchi whereas L-arginine increased EB extravasation in the trachea and upper

bronchi and inhibited hemorrhage in inner bronchi and lungs. In non-envenomed rats neither L-NAME nor L-arginine

had any effect on EB extravasation or hemoglobin content. The protein extravasation in BAL induced by Tbv was

increased by L-NAME and inhibited by L-arginine. Tbv induced neutrophil influx into lungs that was enhanced by L-

NAME and not modified by L-arginine; the same pattern of response was found in MPO activity. Nitrite content in

lungs was increased in envenomed rats and it was inhibited by L-NAME but not modified by L-arginine.

Conclusions: NO seems to be involved as an inhibitory mediator in the acute lung injury induced by Tbv.

Grants: Financial support from Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPQ (Process

457512/2014-8). JGM received scholarship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

(CAPES).


PERIPUBERAL EXPOSURE OF MALE RATS TO LOW DOSES OF MALATHION LEADS TO

HISTOPATHOLOGICAL AND INFLAMMATORY PROFILE ALTERATIONS IN TESTIS AND EPIDIDYMIS

Erthal, R. P.1,2

; Punhagui, A. P. F. 1,2

; Staurengo-Ferrari, L. 2; Fattori, V.

2; Verri Jr, W. A.


1



[email protected]

Keywords: male, toxicology, development, cytokines, histopathology

Introduction and objectives: Diseases currently considered as a public health problem, such as dengue,

chikungunya and zica, are caused by the Aedes aegypt mosquito. Prevention of such diseases depends on the

control of vector mosquitoes through the use of chemical insecticides being the organophosphates used in large

scale. From this insecticides class, an insecticide used on a large scale is malathion, which has been shown to cause

damage to reproductive parameters. Knowing the importance of peripuberty as a critical period for postnatal

development of organs of the male genital system, the present study aims to evaluate whether exposure to low doses

of malathion during the peripuberal period can cause damage to the testicular and epididymal development.

Material and methods: Juvenile male Wistar rats were divided into 3 experimental groups: control (saline), malathion

10 (saline + malathion 10 mg/kg) (M10) and malathion 50 (saline + malathion 50 mg/kg) (M50). The rats were

exposed to malathion or vehicle from postnatal day (PND) 25 until PND 65 by gavage (oral route). At PND 65, the rats

were anesthetized and euthanized. The protocol was approved by the Ethics Committee on Animal Use of State

University of Londrina (protocol number: 12305.2016.65 - CEUA/UEL). The right testes and epididymis were collected

and submitted to histopathological analysis (n=5). The left testes and epididymis were used to determine the

inflammatory profile (myeloperoxidase activity and N-Acetyl-b-D-glucosaminidase activity) and cytokines levels (n=5).

The data were compared using ANOVA followed by Dunnet’s post-hoc test. Differences were considered significant

for p<0.05. Statistical analyses were performed using GraphPad InStat (version 3.02).

Results: Histopathological analysis in testes and epididymis revealed that M50 group showed significantly increase of

abnormal seminiferous tubules when compared to the control group. The main alterations observed were the increase

in vacuoles and immature germ cells in the lumen of tubules, both in testes and epididymis. On the other hand, no

alterations were observed at M10 group. In relation to inflammatory profile, the exposure to M50 lead to an increase in

the recruitment of neutrophils and macrophages in caput epididymis in relation to control group. There were no

alterations in the testes and cauda epididymis in neutrophil or macrophages recruitment after exposure to M10 or

M50. In caput epididymis, the levels of IL-1 decreased after exposure to M10 and M50, and of IL-6 also decreased

after exposure to M10 in relation to control group. IL-10 levels increased in testes after exposure do M10 in relation to

control group. The levels of TNF-α did not show any alteration in both organs.

Conclusion: These results appoints to an alteration in epididymal and testicular postnatal development for altering

inflammatory profile and/or histopathological parameters, which can lead to reproductive damage.


PROTECTIVE EFFECT OF TRANS-CHALCONE IN A MODEL OF ACETIC ACID-INDUCED COLITIS IN

MICE: REDUCTION OF NEUTROPHIL RECRUITMENT, PRO-INFLAMMATORY CYTOKINES

PRODUCTION, AND NF-κB ACTIVATION

Andrade, K.C.

1; Guazelli, C.F.S.

1; Fattori, V.

1; Colombo, B.B.

1; Casagrande, R.

2; Baracat, M.M.

2; Verri, W.A.

1


2Universidade Estadual

de Londrina, Department of Pharmaceutical Sciences, Londrina, PR, Brazil


Keywords: Inflammatory bowel diseases, trans-chalcone, ulcerative colitis, flavonoids

Introduction and objectives: The idiopathic inflammatory bowel diseases (IBD) comprise two types of chronic

intestinal disorders: Crohn's disease and ulcerative colitis. This condition represents a serious health problem.

Intestinal inflammation is associated with increased cytokines and chemokines production, neutrophil recruitment, and

oxidative stress. Naturally occurring molecules, such as the flavonoids, usually present low toxicity, which allows their

use in high dose and therefore are highly attractive therapies for inflammatory diseases. Flavonoids possess anti-

inflammatory and antioxidant activity. In particular, trans-chalcone (TC), which lacks broad investigation on its

biological activity in inflammatory diseases, such as IBD. TC possesses antioxidant and anti-inflammatory that is

partially related to the inhibition of NF-κB activation and downstream targets. We aim to investigate the efficacy of TC

in a model of acetic acid-induced ulcerative colitis in mice.

Material and methods: The experiments were conducted in male Swiss mice with Londrina State University Ethics

Committee on Animal Research and Welfare approval under process number 1494.2015.56. Colon inflammation was

induced by a single intracolonic administration of 7.5% acetic acid (200 μL). The colitis groups received per oral

treatment with TC (0.3, 1, or 3 mg/kg; per oral; 20% tween 80 in saline) or vehicle. For assessment of inflammatory

parameters, mice were treated 2 h before and 10 h after colitis induction. Mice were euthanized 18th hour after

induction of colitis. One cm of the distal colon was collected for determination of neutrophil recruitment by evaluating

the myeloperoxidase activity, colon edema evaluated as the percentage of weight increase in one cm of colon,

macroscopic score damage, whole colon length (in cm), microscopic score damage (HE staining), cytokines

production and NF-κB activation measurements by ELISA. Data were analyzed using one-way ANOVA followed by

the Newman Keuls’s test.

Results: Treatment with TC, significantly reduced neutrophil recruitment as observed by reduction in

myeloperoxidase activity in 50%, edema in 67%, and macroscopic in 67% and microscopic in 75% lesions induced by

intracolonic administration of 7.5% acetic acid solution. These effects were accompanied by the reduction of acetic

acid-induced pro-inflammatory cytokines production TNF-α in 44%, IL-1β in 44%, IL-6 in 50%, and IL-33 in 60%.

Moreover, TC reduced NF-κB in 50% activation in the colon.

Conclusion: This study demonstrates that TC ameliorates acetic acid-induced ulcerative colitis by reducing macro

and microscopic lesion score in the colon. Moreover, TC inhibited acetic acid-induced neutrophils recruitment and pro-

inflammatory cytokines such as TNF-α, IL-1β, IL-6, and IL-33 in a NF-κB-dependent manner. Therefore, TC is a

promising molecule for the treatment of inflammatory diseases and certainly deserves further studies on its pre-clinical

and clinical applicability.






QUERCETIN LOADED MICROCAPSULES REESTABLISH LIVER GLYCOGEN LEVELS IN A MODEL

OF HYPERGLYCEMIA INDUCED BY 66% FRUCTOSE IN RATS

Semeão, L. O.1; Higachi, L.

1; Farias, C. C.

1; Matsumoto, A. K.

1; Michelin, A. P.

1; Martins, R. F. D. B.

1; Bonifácio, K. L.

1;

Brinholi, F. F.1; Casagrande, R.

2; Baracat, M. M.

2; Barbosa, D. S.

3


2 State University of Londrina, Department of pharmaceutical sciences



Keywords: quercetin loaded microcapsules, glycogen, fructose

Introduction and objectives: Increased fructose consumption contributes to the development of obesity

accompanied by glucose intolerance, fatty liver, dyslipidemia, and hyperuricemia. Fructose is absorbed in the intestine

and metabolized in the liver to glucose, lactate, glycogen, and lipids. Plasma fructose levels are much lower than

plasma glucose levels. However, plasma fructose levels are positively correlated with glycemic control. Fructose has

more potent cytotoxicity because of increased advanced glycation end product production. As a result, the aim of the

study was to evaluate whether quercetin or quercetin-loaded microcapsules improve glycogen levels.

Material and methods: All the experimental procedures were conducted using male Wistar Albino rats 150g (±25g).

Each male was housed individually in controlled environmental conditions, i.e., temperature at 21 ± 2 °C, 12 h

dark/light cycle (lights on at 07:00 AM) and free access to food and tap water. All experimental procedures foreseen in

this project were approved by the Ethics Committee in Animal Experimentation of the State University of Londrina

(CEUA: 31855.2012.76). For the induction of hyperglycemia, a fed prepared with 66% fructose was given to the

animals for 9 weeks. These animals were treated by gavage throughout the experiment. The rats were divided in 8

experimental groups with 10-13 rats each: control (treated with standard regular diet for rodents - NUVILAB®) +

saline; fructose-fed + saline; fructose-fed + tween 80 (20%) (quercetin vehicle); fructose-fed + quercetin

(100mg/kg/day); fructose-fed + inert microcapsules (100mg/kg/day); fructose-fed+ quercetin loaded microcapsules

(10mg/kg/day); fructose-fed + quercetin loaded microcapsules (30mg/kg/day); fructose-fed + quercetin loaded

microcapsules (100mg/kg/day). At the end of the experiment, the rats were anesthetized with thiopental 70mg / kg

(Thiopentax®). The animal was perfused with 140 mM phosphate buffer, pH 7.4, the liver was dissected and weighed.

The median lobe of the liver was packed in containers containing 10% formalin (formalin) at room temperature until

the material was processed.

The blades were stained with Schiff Periodic Acid for the analysis of glycogen. These were analyzed under optical

microscope by two trained students. Classification of the evaluated images: glycogen = 1+: discrete; 2+: moderate;

3+: accentuated; 4+: very accentuated. For the variables was used the Chi-square test followed by the z-test to

compare the percentages among the groups.

Results: The control group had presence of 52.5% liver glicogen caracterizing in standart frequency 3. The groups

presented frequency 2 were fructose (81%), tween 80 (68.5%), quercetin 100mg / kg (66.5%), inert microcapsule

(78.5%), quercetin loaded microcapsules 10mg (59%). Despite this, the quercetin loaded microcapsules 30mg / kg

(87%) and 100mg / kg (86%) showed frequency of 3, indicating that these concentrations reestablished glycogen

levels.

Conclusion: Fructose diet induces depletion of glycogen, in liver. The quercetin loaded microcapsules at

concentrations above 30mg / kg can be used as an alternative treatment in replenish the levels of glycogen in liver.


SUPPRESSIVE EFFECT OF INSULIN ON HEPATIC GLUCOSE PRODUCTION AND

GLYCOGENOLYSIS STIMULATED BY CAMP.

Galia, W. B. S.1; Diaz, B. F.

1; Ferraz, L. S.

1; Souza, H. M.

1; Bertolini, G. L

1.



Keywords: AMPc, glycogen catabolism, insulin, liver perfusion.

Introduction and objectives: Cyclic adenosine monophosphate (cAMP) is described as a second messenger

mediating the action of various hormones, such as glucagon and adrenaline. Studies from liver perfusion have directly

showed that cAMP stimulates glucose production and glycogenolysis in fed rats. In addition, it has been shown that

insulin inhibits hepatic glycogenolysis and the glycogen catabolism stimulated by AMPc by a mechanism that involves

lowering the intracellular concentration of cAMP. The aim of the present study was to reproduce the suppressive effect

of insulin on hepatic glucose production and glycogenolysis stimulated by cAMP.

Material and methods: For the surgical procedure, Wistar male rats (250 g, 8 weeks of age) were anesthetized by

intraperitoneal injection of sodium pentobarbital (40 mg/kg) and subjected to in situ liver perfusion with cAMP (3 µM),

in the presence or absence of physiological (20 mU/mL) concentrations of insulin, to evaluate the hepatic glucose

production and glycogenolysis. The experimental protocols were approved by Animal Experimentation Ethics

Committee of the State University of Londrina (protocol nº 2483.2015.51). Normal distribution and variance

homogeneity were tested and one-way ANOVA, followed by the Newman-Keuls test, was employed to analyse the

results. Statistical analysis was carried out with the Program GraphPad Prism 6.0 and significance was accepted

when p<0.05. Data were expressed as the mean±standard error of the mean of 6-7 animals.

Results: The infusion of cAMP (3 µM) in the liver stimulated (p<0.05) hepatic glucose production and glycogenolysis.

The infusion of 20 mU/mL insulin in the liver decreased (p<0.05) the hepatic glucose production and glycogenolysis

stimulated by cAMP.

Conclusion: Insulin suppressed the hepatic glucose production and glycogenolysis stimulated by cAMP. The

stimulation of hepatic glucose production and glycogenolysis by cAMP was probably mediated by activation of cAMP-

dependent protein kinase (PKA) and subsequent phosphorylation and activation of glycogen phosphorylase. The

suppressive effect of insulin on hepatic glycogenolysis can be attributed to a reduction in the intracellular

concentration of cAMP, due to the activation of phosphodiesterase-3B (PDE3B), an enzyme that promotes the

hydrolysis of cAMP. PDE3B is activated by Akt/PKB, via stimulation of PI3K associated with IRS-1.


THE FLAVONOID HESPERIDIN METHYL CHALCONE AMELIORATES ACETIC ACID-INDUCED

COLITIS BY REDUCING NEUTROPHIL RECRUITMENT, PRO-INFLAMMATORY CYTOKINES

PRODUCTION, AND NF-κB ACTIVATION IN MICE

Andrade, K.C. 1; Guazelli, C.F.S.

1; Fattori, V.

1; Colombo, B.B.

1; Casagrande, R.

2; Baracat, M.M.

2; Verri, W.A.

1


2Universidade Estadual

de Londrina, Department of Pharmaceutical Sciences, Londrina, PR, Brazil


Keywords: Inflammatory bowel diseases, hesperidin methyl chalcone, ulcerative colitis, flavonoids.

Introduction and objectives: Inflammatory bowel diseases (IBD) are chronic intermittent inflammatory conditions

that primarily affect young adults and represent a serious health problem. Intestinal inflammation is associated with

increased cytokines and chemokines production, neutrophil recruitment and oxidative stress. Neutrophil recruitment

towards intestinal tissues is an important event given that in the inflammatory foci, these cells produce ROS and NF-

κB-dependent pro-inflammatory cytokines, which contribute to tissue damage. One of the management choice for the

treatment of IBD is an anti-TNF-α monoclonal antibody. However, this therapy is often limited by a loss of efficacy due

to the development of adaptive response, underscoring the need for novel therapies. Flavonoids possess anti-

inflammatory and antioxidant activity and importantly, these molecules usually present low side effects. Hesperidin

methyl chalcone (HMC) is a biflavonoid with antioxidant and anti-inflammatory properties through inhibition of NF-κB

activation and therefore is a promising molecule for the treatment of IBD. We aim to investigate the efficacy of HMC in

a model of acetic acid-induced ulcerative colitis in mice.

Material and methods: The experiments were conducted in male Swiss mice with Londrina State University Ethics

Committee on Animal Research and Welfare approval under process number 1494.2015.56. Experimental colitis was

induced by a single intracolonic administration of 7.5% acetic acid (200 μL). Mice were treated with HMC (10, 30, or

100 mg/kg; per oral; in saline) or vehicle. For assessment of inflammatory parameters, mice were treated 2 h before

and 10 h after colitis induction. Mice were euthanized 18th hour after colitis induction. The distal colon (1 cm) was

collected for determination of neutrophil recruitment by measuring myeloperoxidase activity through a colorimetric

method, edema determination evaluated as percentage of weight increase in 1 cm of colon, macroscopic damage

score, whole colon length (in cm), microscopic damage evaluation (HE staining), determination of cytokine levels and

NF-κB activation by ELISA. Data were analyzed using one-way ANOVA followed by the Newman Keuls’s test.

Results: Treatment with HMC, significantly reduced neutrophil recruitment in approximately 43%, edema in 67%, and

macroscopic in 46% and microscopic in 64% lesions induced by intracolonic administration of 7.5% acetic acid

solution. Moreover, HMC prevented the acetic acid-induced decrease in colon length in 33%. These effects were

accompanied by the reduction of the acetic acid-induced TNF-α in 66%, IL-1β in 50%, IL-6 in 50%, and IL-33 in 67%.

Moreover, treatment with HMC inhibited the activation of NF-κB in 50% the colon.

Conclusion: This study demonstrates that HMC ameliorates acetic acid-induced ulcerative colitis through reduction of

neutrophil recruitment and inhibition of NF-κB activation and NF-κB-dependent pro-inflammatory cytokines such as

TNF-α, IL-1β, IL-6, and IL-33. Therefore, HMC is a prosperous molecule for the treatment of IBD.






VINPOCETINE AMELIORATES ACETIC ACID-INDUCED COLITIS BY INHIBITING OXIDATIVE

STRESS, PRO-INFLAMMATORY CYTOKINES PRODUCTION, NF-κB ACTIVATION AND REDUCING

VISCERAL MECHANICAL HYPERALGESIA IN MICE

Zaninelli, T. H.1; Colombo, B. B.

1; Guazelli, C. F. S.

1; Fattori, V.

1; Ruiz-Miyazawa, K. W.

1; Carvalho, T. T.

1; Ferraz, C.

R.1; Bussmann, A. J. C.

2; Casagrande, R.

3; Verri A. W. Jr.

1 1Universidade Estadual de Londrina, Department of Pathological Sciences, Londrina, PR, Brazil. 2Universidade Estadual de Londrina, Laboratory of Pathological Anatomy, Londrina, PR, Brazil.

3Universidade Estadual de Londrina, Department of Pharmaceutical Sciences, Londrina, PR, Brazil.

[email protected]

Keywords: vinpocetine, colitis, inflammation, oxidative stress, abdominal hyperalgesia

Introduction and objectives: The Inflammatory Bowel Disease (IBD) is characterized by chronic inflammation of the

gastrointestinal tract and comprises the ulcerative colitis and Chron’s Disease. Its incidence has been increasing in a

fashion that it can become a global disease that has a great impact on the individuals’ life quality. IBD occurs due to

an unregulated immune response triggered against intestinal microbiota. The main event is the recruitment of

neutrophils and macrophages towards intestinal tissue that produce reactive oxygen species and NF-κB-dependent

pro-inflammatory cytokines, which contribute to tissue damage. The current and most applied therapies in IBD

treatment consist of aminosalicylates, corticosteroids, antibiotics, and anti-TNF-α monoclonal antibodies. These

therapies are often limited by loss of efficiency, development of adaptive responses and side effects, highlighting the

need of novel therapies. Vinpocetine is a synthetic ethyl ester, derived from vincamine, an alkaloid isolated from the

leaves of Vinca minor. This nootropic drug is known to have antioxidant, analgesic, and anti-inflammatory properties,

partly by inhibition of NF-κB and its downstream cytokines. Therefore, in this study we aimed at investigating the

efficacy of vinpocetine in a model of acetic acid-induced colitis.

Material and methods: Experiments were conduced in male Swiss mice under Universidade Estadual de Londrina

Ethics Committee on Animal Research and Welfare approval (3307.2015.37). Colitis was induced through intracolonic

injection of acetic acid 7.5%. Vinpocetine treatment was performed orally and it was divided in two protocols. In the

first protocol, mice were treated with vinpocetine 10 and 4h before, and 2h after the colitis induction. In this protocol,

mice were euthanized 4h after colitis and a distal colon portion (1cm length) was collected for determination of tissue

edema and oxidative stress, through GSH and ABTS assays. Visceral mechanical hyperalgesia (electronic

anaethesiometer) was assessed 3h after colitis. In the second protocol, mice were treated with vinpocetine 2h before

and 4, 10, and 16h after colitis induction. In this protocol, mice were euthanized 18h after colitis, and a distal colon

portion (1cm length) was collected for determination of neutrophil recruitment (myeloperoxidase activity), cytokines

dosage (IL-1β, IL-10, IL-33, and TNF-α) by ELISA, and NF-κB activation by western blot. Visceral mechanical

hyperalgesia was assessed 3 and 17h after induction. Macroscopic score was determined by the intensity and

progression of acetic acid-induced colitis using the entire colon. The most effective dose of vinpocetine was

determined from a range of 1, 3, 10, or 30mg/kg after edema and MPO activity analysis.

Results: Vinpocetine at 30mg/kg inhibited acetic acid-induced myeloperoxidase activity in 41%, edema (72%), and

macroscopic score (42%). The treatment with vinpocetine also decreased acetic acid-induced visceral mechanical

hyperalgesia (52%), NF-κB activation (44%), and production of the pro-inflammatory cytokines IL-1β (60%), TNF-α

(54%), and IL-33 (56%) in the colon. In addition, vinpocetine normalized the levels of the anti-inflammatory cytokine

IL-10 and restored the total antioxidant capacity and GSH levels.

Conclusion: This study demonstrates for the first time that vinpocetine has anti-inflammatory, antioxidant, and

analgesic activity in experimental colitis in mice. Therefore, vinpocetine is a promising molecule for IBD treatment.

Grants: Coodenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação Araucária, Paraná State

Government and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).


VINPOCETINE ATTENUATES SUPEROXIDE ANION-INDUCED PAIN BY INHIBITING PRO-

INFLAMMATORY CYTOKINE PRODUCTION AND BY INCREASING NRF2 EXPRESSION IN MICE

Lourenco-Gonzalez, Y.1; Fattori, V.

1; Rossaneis, A.C.

2, Casagrande, R.

2; Verri, W.A.

1 1Universidade Estadual de Londrina, Departamento de Ciências Patológicas, Londrina, PR, Brasil

2Departamento de Ciências Farmacêuticas, Centro de Ciências da Saúde, Hospital Universitário, Universidade

Estadual de Londrina, Av. Robert Koch, 60, 86038-350 Londrina, PR, Brasil.

Author for correspondence: [email protected]

Keywords: vinpocetine, hyperalgesia, inflammatory pain, potassium superoxide

Introduction and objectives: Vinpocetine (3α, 16α-Eburnamenine-14- carboxylic acid ethyl ester) is a derivative of

vincamine molecule, an alkaloid extracted from the leaves of Vinca minor. It’s a drug currently used to treat the

symptoms of cognitive deterioration (deterioration of the learning process) and prevention of cardiovascular disorders.

Studies have shown that vinpocetine has anti-inflammatory and analgesic effects in other models of pain and

inflammation. Given the close relationship between oxidative stress and pain, this study aimed at evaluating the

analgesic and anti-inflammatory effects of vinpocetine in model of superoxide anion induced-pain.

Material and methods: The experiments were performed using male Swiss mice with Londrina State University

Ethics Committee on Animal Research and Welfare approval under process number 6166.2014.85. Potassium

superoxide (KO2) was used as superoxide anion donor for induce pain-like behavior and inflammation. Mice (n=6 per

group) were treated with vinpocetine at doses 3, 10, or 30mg/Kg or vehicle orally 1 h before intraplantar (30 μg) or

intraperitoneal (1 mg) injection of KO2 or saline (control group). Overt pain-like behaviors were determined by counting

the total number of abdominal writhings, number of paw flinches and time spent licking the paw. Mechanical and

thermal hyperalgesia were determined 1-7h after stimulus using an electronic anesthesiometer and hot plate,

respectively. Leukocytes migration to the inflamed paw was quantified through the measurement of the enzymes

myeloperoxidase (MPO, neutrophil marker) and N-acety-β-D-lglucosaminidase (NAG, macrophage marker) by

colorimetric assay. Cytokine concentration was determined in the paw skin by ELISA. Antioxidant capacity was

assessed by nitrobluetretrazolium assay (NBT, superoxide anion production), and by total antioxidant capacity [ferric

reducing antioxidant power (FRAP) and ABTS radical scavenging ability. The mRNA expression of Nrf2 transcription

factor, heme oxygenase-1 (HO-1), gp91phox, endothelin-1, and Cox-2 mRNA expression were determined by RT-

qPCR. Data were analyzed by ANOVA followed by Tukey’s post hoc and considered significant when p < 0.05.

Results: Treatment with vinpocetine was able to reduce overt pain-like behaviors such as the number of abdominal

writhes in a dose-dependent manner, as well as paw flinches and time spent licking the paw. Moreover, vinpocetine

reduced mechanical and thermal hyperalgesia in a dose-dependent manner. Vinpocetine at 30 mg/kg reduced the

activity of MPO and NAG in 69.8% and 65.1% respectively. Moreover, vinpocetine decreased the production of the

pro-inflammatory cytokines IL-1β in 59.9%, IL-33 in 56.6%, TNF-α in 54%. Vinpocetine also reduced oxidative stress

as observed by reduction in superoxide anion production in 67.3%, gp91phox mRNA expression, and restored in total

antioxidant capacity. Furthermore, vinpocetine increased the expression of the transcription factor Nrf2 and

downstream target HO-1. Finally, vinpocetine reduced the mRNA expression of endothelin-1 and Cox-2.

Conclusion: Vinpocetine possesses antinociceptive and anti-inflammatory properties. This effect was related to the

reduction neutrophil and macrophage recruitment to the inflammatory foci. Moreover, vinpocetine reduced oxidative

stress, as observed by reduction in superoxide anion production and increase in total antioxidant capacity, possibly by

modulating Nrf2 signaling pathway. Furthermore, vinpocetine decreased pro-inflammatory and pro-hyperalgesic

mediators such as IL-1β, IL-33, TNF-α, and endothelin-1. Therefore, vinpocetine present promising analgesic effect

and certainly deserves future clinical studies addressing this issue.



da Ciência, Tecnologia e Inovação (SETI), Fundação Araucária and Paraná State Government.


VITAMIN D DECREASES RADICAL COMPOUNDS PRODUCED BY NEUTROPHILS IN VITRO

Michelin, A. P.1; Bonifácio, K. L.

1; Camargo, S. M.

1; Matsumoto A. K.

1; Semeão, L. O.

1; Martins, R. F. D. B.

1; Barbosa,

D. S. 2; Venturini D.

2;


2 State University of Londrina, Department of Pathology, Clinical and Toxicological Analysis, Londrina, PR, Brazil


Keywords: vitamin D, respiratory burst, antioxidante activity;

Introduction and objectives: The term vitamin D encompasses a group of molecules derived from 7-

dehydrocholesterol. Vitamin D occurs in two major forms, ergocalciferol or vitamin D2, synthesized in the epidermis by

the action of ultraviolet radiation from sunlight and cholecalciferol or vitamin D3. The D2 and D3 forms differ only by

the presence of an additional double bond and a methyl group incorporated into the long side chain of the biological

form called D2. The literature shows that vitamin D has benefits in relation to oxidative stress, which is the imbalance

between compounds with oxidizing action and compounds with antioxidant action. Reactive oxygen species (ROS)

and nitrogen (RNS) can cause damage to cells and may damage the body's homeostasis. In view of the above, the

objective of this study was to analyse if vitamin D is able to decrease the production of radical compounds produced

by neutrophils in vitro.

Material and methods: To perform the test, first, it was necessary to convert vitamin D from international units (IU) to

μg. In the literature it is known that 1 IU of vitamin D biologically equates to 0.025 μg cholecalciferol/ergocalciferol.

Thus, a capsule containing 50.000 IU of vitamin D amounts to 1250 μg of vitamin D. A capsule containing 1250 μg of

vitamin D in 4 ml of dimethylsulfoxide (DMSO) was then diluted. The neutrophils ROS production was evaluated by

chemiluminescence in microplate reader (Victor X-3, PerkinElmer™, USA. Human neutrophils were isolated from

blood by Histopaque ficoll concentration gradient and centrifugation. Respiratory burst was induced by

phorbolmyristate acetate (PMA) in the presence of DMSO (control group) and 10,41 μg (416,4 UI) of vitamin D and

the results are expressed in counts per minute (CPM). The experiment for was composed of 7 replicates. Initially,

comparisons were performed according to the normal distribution of data in the Kolmogorov-Smirnov test, as well as

the variance analysis by Levene`s test. Difference among groups were analysed by the dependent T test (parametric

data). Antioxidant activity results are presented as means _ standard error mean (SEM) and the results were

considered statistically significant if p value < 0.05. This study was approved by the Ethics Research Committee at

UEL (number CAAE 41718014.9.0000.5231).

Results: Respiratory burst the results are expressed as CPM. The mean replicates of the DMSO control curve were

43527 ± 860.75 and the vitamin D at the concentration of 10,41 μg/mL (416,4 UI), the mean was 25028 ± 775.39.

Conclusion: With these results it is possible to conclude that vitamin D has antioxidant activity in vitro, by inhibiting

radical species produced by neutrophils induced by PMA at the concentration of 10,41 μg/mL (416,4 UI).

Grants: CNPq

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