2.!South!Eastern!European!Immunology!School final.pdf · Sabina Rabatic: Innate immunity to...
Transcript of 2.!South!Eastern!European!Immunology!School final.pdf · Sabina Rabatic: Innate immunity to...
2. South Eastern European Immunology School
1 – 4 October 2010
Inter University Centre, Dubrovnik, Croatia
2
Programme Friday, 1 October 17.00 Registration at IUC 18.30 Keynote Lecture:
Seppo Meri: Microbial escape of innate immunity 20.00 Welcome reception at IUC Saturday, 2 October 09.15 Plenary Lecture:
Sabina Rabatic: Innate immunity to respiratory viruses 10.00 Plenary Lecture:
Hannes Stockinger: Yin/Yang of adaptive immunity between cure and destruction
10.45 Coffee break 11.15 Plenary Lecture:
Hans-Willi Mittruecker: Defence against intracellular bacteria 12:00 Plenary Lecture:
Bernhard Fleischer: Regulation of T cell activation: co-stimulation and co-inhibition
12.45 Lunch break (Restaurant MIMOZA) 15.00 Parallel Workshops
1. How to interpret FACS data 2. Defects of innate immunity and how to detect them
15:45 Coffee Break 16:30 Parallel Workshops 3. How to measure T cell immunity
4. Complement diagnostics, diagnostics of autoimmune diseases 5. Antibodies in research and therapeutics
17:15 Free evening for sight seeing
3
Sunday, 3 October 09.15 Plenary Lecture:
Moncef Zouali: B cells and autoimmunity 10:00 Plenary Lecture:
Annette Gospos: Autoantigens in diagnostics of human autoimmune diseases
10:45 Coffee break 11.15 Plenary Lecture:
Friedrich Haag: Mechanisms of tolerance 12:00 Plenary Lecture:
Seppo Meri: Complement activation and regulation 12.45 Lunch (Restaurant MIMOZA) 15:00 Parallel Workshops (repeated according to request by participants) 15:45 Coffee break 16.15 Poster session 20.00 Farewell party with dinner at Restaurant Kavana Poliksar, Old Harbour,
Ribarnica Monday, 4 October 09.15 Plenary Lecture:
Hannes Stockinger: Live-cell imaging to open new views for understanding of immune cell function
10.00 Plenary Lecture:
Hans-Joachim Seitz: Opportunities for young researchers from SE Europe in Germany
10:45 Final Discussion 11.00 Farewell, Certificates, Coffee, Sandwiches 12.00 Departure
4
Systemic low-grade inflammatory response following oral warfarin administration in rats Sandra Belij1, Djordje Miljkovic2, Marija Slavić3, Vesna Subota4, Aleksandra Popov1, Ivana Mirkov1, Dragan Kataranovski1, Milena Kataranovski1 1Department of Ecology, Institute for Biological Research “Sinisa Stankovic”, Bulevar Despota Stefana 142, Belgrade, Serbia, 2Department of Immunology, Institute for Biological Research “Sinisa Stankovic”, Bulevar Despota Stefana 142, Belgrade, Serbia, 3Department of Physiology, Institute for Biological Research “Sinisa Stankovic”, Bulevar Despota Stefana 142, Belgrade, Serbia, 4Institute for Medical Biochemistry, Military Medical Academy, Crnotravska 17, Belgrade, Serbia Objective: To investigate the existence of systemic inflammatory response to subchronic oral warfarin consummation in rats. Methods: Dark Agouti rats were treated with warfarin in drinking water (3.5 mg/l) for 30 days. Heparinized blood samples were collected (24 hours following 30-day period) and differential leukocyte counts were determined (May Grünwald-Giemsa protocol). Peripheral blood (PB) granulocyte (isolated on density gradient) viability was determined by MTT reduction assay. Oxidative activity (cytochemical NBT reduction), myeloperoxidase (MPO) intracellular content of PB granulocyte (oxidation of O-dianisidine dihydrochloride), levels of nitrites accumulated in medium conditioned by PB granulocytes (Griess assay), mRNA levels for inducible nitric oxide synthase (iNOS) (RT-PCR), plasma levels of IL-6 and TNF-α (ELISA) and superoxide dismutase (SOD) activity (red blood cell lysates) were analyzed as inflammatory parameters in rats following warfarin consumption. Changes in prothrombin time (PT) and partial thromboplastin time (PTT), as basic biological warfarin activity were determined as well. Statistical significance was defined by Mann-Whitney U test (functional granulocyte assays) and t-test (SOD activity, gene expression). Results: Significantly increased PT and PTT were noted following WF administration, documenting access of WF to general circulation. No numerical changes in differential leukocyte counts were noted, without direct cell toxicity on PB granulocyte, evaluated by MTT test. Increase of PMA-stimulated neutrophil NBT reduction capacity (neutrophil priming) and significantly increase in MPO intracellular content, levels of nitrites and mRNA levels for iNOS were noted in PB granulocytes following WF administration. Warfarin consummation resulted in no changes in plasma levels of IL-6 and TNF-α, but significant decrease in the SOD activity was detected, suggesting systemic oxidative activity. Conclusion: Presented data demonstrate a potent pro-inflammatory effect of anticoagulant warfarin on granulocytes, including potentiation of their effectors functions. Absence of the rise in inflammatory cytokines in circulation, suggest low-grade inflammation in these rats. These findings contribute to the list of biological activities of anticoagulants, other than those affecting hemostasis. This study was supported by the Ministry of Science and Technological Development of the Republic of Serbia, Grant # 143038.
5
Hyper IgE in a HIV positive patient Hristomanova S, Grunevska V1, Balabanova M2, Trajkov D, Petlichkovski A, Kirijas M, Djulejic E, Senev A, Spiroski M Institut of Immunobiology and Human Genetics; 1Clinic for Infective Diseases; 2Clinic for Dermatovenerology; Faculty of Medicine, University “St Kiril and Metodij”, Skopje, Republic of Macedonia Introduction Since1999 on the Institute of Immunology and Human Genetics in Skopje, Republic of Macedonia operates laboratory for allergology. It encompasses three UniCap 100 systems and they are for different assignment. Starting from the early beginning from 1999 persistent increase of both the number of total analysis and the number of analysis per patient is seen. Thus we have started with only 41 analyses in 1999 and we have reached total of 8491 analysis last year. Case report: A patient, 63 years old, with loss of weight and frequent stool previously diagnosed and treated as enterocollitis. Also previously treated at the Dermatological Clinic for herpes zoster infection and undiagnosed allergy. Microbiology tests showed presence of Candida and Klebsiella pneumoniae in the patient's sputum which led to testing and confirmation of HIV/AIDS. After that Lues was diagnosed and because the therapy is with penicillin blood sample was sent to our laboratory, for penicillin allergy testing. Results The results showed increased level of total IgE for almost 12 times above normal ranges and also allergy to ampicillin was revealed. Additional laboratory tests showed increased IgA levels with normal IgM and IgG levels. From acute phase proteins the only significant deviation was extreme raise of CRP followed with decreased levels of transferrin, α2 macroglobulin, ceruloplasmin and albumin.It is important to mention that no skin changes were visible in the patient. Disscusion Highly increased levels of total IgE indicated the possibility for HIE Sy in this patient. HIE is a rare immunodeficiency disorder with an autosomal dominant inheritance pattern, associated with multiple abnormalities.Clinical manifestations of atopic allergy, drug reactions, and increased serum levels of IgE have already been described during the course of human immunodeficiency virus (HIV) infection.However the relationship of such findings to the immunologic derangements found in patients with HIV is not entirely clear.There are similarities between HIV-positive patients and those with primary HIE Sy, but there are also some important differences. Conclusion In conclusion, this is the first case of HIV positive patient with hyper IgE-like Sy in the Republic of Macedonia. We addressed the important laboratory findings and actual theories explaining the association between HIES and HIV/AIDS.
6
Cytokine expression levels in high-risk human papillomavirus (hrHPV) positive women Irina Huica, Coralia Bleotu, Anca Botezatu, Iulia V Iancu, Gabriela Anton Institute of Virology “Stefan S. Nicolau”, 285 Mihai Bravu Ave 030304 Bucharest – Romania Tel/Fax: +40-21-324147
High-risk types of human papillomaviruses (hrHPV) are known to causing cervical cancer. As the host immune response plays an important role in determining the regression of a cervical abnormality or persistence and progression to a malignancy, the purpose of our study was to investigate cytokine levels in persistent lesions of the uterine cervix. Materials and Methods: 184 hrHPV DNA positive patients (Roche), age 17-48 years, were monitored between years 2008-2010. HPV positive case patients with cytological interpretation of ASCUS or LGSIL, presenting HPV persistent infection were investigated for cytokine levels detection. The persistent infections were confirmed by the presence of the same HPV DNA type in consecutive testing. The control group consisted of 10 hrHPV-negative women. Using exfoliated cells collected by cervical cytology brush, we assessed pro- and anti-inflammatory cytokine expression [interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor a (TNF-a), and interferon γ (IFN-γ)] in RT-real-time PCR (Taqman). Results. Persistence was significantly greater by oncogenic HPV types 16, 18 and 51 alone or in association with low/medium risk types. By compare with control group, we found that IFN-γ levels was increased in ASCUS and seems to be associated with low viral oncogene expression. The pro-inflammatory cytokines expression levels (TNF-a, IL-6, and IL-10) were higher in LGSIL lesions. While IL-4 levels were higher in LSIL lesions, especially in HPV 16 and 18 positive cases, IL-2 levels were slightly increased in patients presenting ASCUS and HPV 18 positive infection. No correlation with other oncogenic types (31, 33 and 45) was observed. Conclusions. Among HPV-persistent infected women, pro-inflammatory cytokine expression levels are increased and that may be considered as a prognostic marker for oncogenic potential of high-risk HPV.
7
Characterization of peripheral regulatory T cell subsets in patients with posttraumatic stress disorder Mladen Jergović (1), Krešo Bendelja (1), Anđelko Vidović (2), Katja Gotovac (1), Sabina Rabatić (1), Neda Aberle (3), Zlatko Sudac(3), Ante Sabioncello (1), 1Institute of Immunology, Department for Cellular Immunology, Zagreb, Croatia; 2University Hospital Dubrava, Department of Psychiatry, Zagreb, Croatia; 3Children's Department, General Hospital Dr Josip Bencević, Slavonski Brod, Croatia Posttraumatic stress disorder (PTSD) is associated with elevated risk for inflammatory and autoimmune diseases. Regulatory T cells play a major role in maintaining homeostasis of the immune system by controlling the activity of effector cells including auto-‐reactive T lymphocytes. Regarding the role of regulatory T cells in inflammatory and autoimmune diseases, we hypothesized that PTSD patients might exhibit changes in composition of regulatory T cells in peripheral blood. For this purpose we have analyzed absolute numbers of T cells as well CD4 andCD8 subpopulations in peripheral blood from PTSD patients (N=21) and age matched healthy controls (N=22). Frozen PBMC samples were later analyzed regarding regulatory T cell composition. Natural regulatory T cell (nTreg) population is characterized as CD4+CD25+FOXP3+ . Additionally, nTreg cells express other markers which define different subpopulations. Mature nTreg express low levels of CD127 and HLA-‐DR while terminally differentiated nTreg cells lose CD127 and display higher expression of FOXP3. Induced regulatory T cells (iTreg) are characterized by low expression of CD127 and lack of HLA-‐DR marker. Their precursors pTreg cells do not express CD127 or HLA-‐DR. In addition to these markers we have analyzed the expression of CD39, membrane ectoenzyme which hydrolyzes extracellular ATP and ADP into AMP. Although PTSD patients have a lower absolute number of T cells andT helper cells we found no differences in Tregs percentage and composition. These results indicate that PTSD patients might exhibit lower suppression of the immune response due to lower number of T helper cells but not changes in T regs frequency and composition.
8
Quercetin increases sensitivity of bladder cancer cells to cisplatin and hyperthermia Pavle Josipović*, Ivo Karač*, Domagoj Karković*, Branimir Lodeta+ and Nada Orsolić*. Faculty of Science (Roosvelt square 6, Zagreb, Croatia)[*], and General Hospital (Ivana Meštrovića bb, Varaždin, Croatia)[+]. Hyperthermia provokes regression of human cancer. Great differences in heat
sensitivity have been observed between different cell types and tissues. Considering that
hyperthermia has produced only limited results, attention has been focused on searching
for substances able to sensitize tumour cells to the effects of hyperthermia. Flavonoid
quercetine has been recognized as hyperthermic sensitizer in ovarian and uterine
cervical tumours, leukaemia and in prostate carcinoma cells. Quercetine and cisplatin
inhibit Ehrlich ascites tumor growth and mammary carcinoma induced peritoneal
carcinomatosis growth.
We have observed that both quercetine and cisplatin at doses of 1 and 50 µM synergize
with hyperthermia (43ºC), with the effect of reducing the clonogenic activity of T-
24/83 and UMUC-3 and inducing the inhibition cell growth in both cell lines. As
revealed by clonogenic assay and MTT analyses, quercetine and cisplatine have
reduced a number of colonies and increased the cell cytotoxicity in hypertermal
condition, in concentration- depedent manner. In addition, T-24/83 cell line are more
sensitive to quercetine, cisplatine and hypertermal treatment than UMUC-3 cells.
Higher reduction of heat shock proteins (Hsps) expression in the T-24, in relation to
UMUC-3 cell line, promotes the higher induction of apoptosis by quercetine, which
may be a reason for better antiproliferative mechanism in T-24 cell line.
Exact antitumor mechanisms of hyperthermic chemoimmunotheraphy effectiveness still
need to be elucidated. Quercetine and cisplatin can be usefully combined with
hyperthermia in therapy of bladder cancer cells.
9
Comparison of two strategies for resolution of ambiguous HLA typing results Meri Kirijas, Stefan Saltirovski, Aleksandar Senev, Mirko Trpevski, Slavica Hristomanova, Olivija Efinska-‐Mladenovska, Aleksandar Petlichkovski, Dejan Trajkov, Mirko Spiroski Institute of Immunobiology and Human Genetics, Faculty of Medicine, St Cyril and Methodius Universisty, Skopje, Republic of Macedonia Introduction: The precise identification of HLA Class I and Class II alleles is critical for successful transplantation of hematopoietic cells, development of peptide based viral and cancer vaccines, investigation of immune response and in anthropological studies. The DNA based techniques for HLA typing have been proved to be powerful in resolving many serological ambiguities and detecting of single nucleotide polymorphisms. However, there is still not a single technique that offers ambiguity-‐free MHC typing. Defining strategies to resolve ambiguities created by HLA DNA typing remains in focus of interest for HLA DNA typing professionals.
Aim: The aim of this study is to compare two strategies for resolution of ambiguities arising from the SSOP typing method. The first is based on using of a high resolution SSP typing method, and the second is based on statistical analysis using the HLA completion software.
Material and methods: Two hundred and fourteen unrelated random healthy Macedonian volunteers of Macedonian origin and nationality, Christian Orthodox religion and residents of different regions of the Republic of Macedonia were included in this study. Peripheral blood was drawn after signing of the informed consent and genomic DNA was extracted from the peripheral blood leukocytes using the standard phenol/chloroform procedure and stored in the anthropology project field of the Macedonian human DNA Bank (hDNAMKD) until processing. Representative ambiguous results were selected from the database and probabilistically resolved using the software tool available at: http://microsoft.com/science. In addition, 83 typings using the high resolution SSP method were performed. From the results compared, only 2 were not concordant, and overall concordance of 97,59% was achieved.
Conclusion: In conclusion, the statistical method for resolution of ambiguous HLA typings using HLA Completion tool is satisfactory add in anthropological research. However, additional method should be used when mistyping is not an option, such as setup of transplantation.
10
Proteomic approach for indirect quantification of breast cancer antigens in human serum Daria Ler1,2 1 Department of Genetics and Bioengineering, Faculty of Engineering and Natural Sciences, International
University of Sarajevo, 2 Institute of Genetic Engineering and Biotechnology, Sarajevo, Bosnia and
Herzegovina
Objectives: Immune system of patients with cancer gives response to tumor antigens
with the production of antibodies. The observed accumulation of such “auto-antibodies”
in cancer patients suggests them to be of high diagnostic/prognostic value. In this term,
specific ELISA-based approach was optimized to identify and validate new serum
tumor marker ecPKA (extracellular Protein Kinase A), potential candidate for
noninvasive detection of early human breast neoplasm. This auto-antibody ELISA is
thought to identify the cancer antigens by detecting the presence of auto-antibodies
against the tumor protein in human serum.
Method and Materials: The anti-ecPKA auto-antibodies levels were measured by solid
phase enzyme linked immunosorbent assay (ELISA). For this purpose a heterogeneous
group of breast cancer patients (BCP), respectively 120 serum samples, was tested as a
pilot project.
Results:
- Reproducible and robust assay, functional in quantifying specific anti-PKA auto-
antibodies.
- Two relatively high significant changes were obtained, namely between the two
BCP subgroups (adjuvants with HR+ and patients with metastasis), and between
one BCP subgroup (adjuvants with HR+) and healthy donors.
- No significant difference was obtained between the BCP group and the healthy
donors.
Conclusion: Anti-ecPKA auto-antibody ELISA represents a good alternative to the
current antigen determination method, it is functional, easy to perform, cost- and time
saving method but further studies are necessary to indicate the probable utility of this
new assay as a cancer screening tool and to define its promising role in early
diagnosis/prognosis.
11
The involvement of IL-17 in the development of zymosan-induced arthritis
in SCID mice
Viktoriya Milanova, Nina Ivanovska and Petya Dimitrova
Department of Immunology, Institute of Microbiology, Bulgarian Academy of Sciences, Sofia,
Bulgaria
IL-17 is a key cytokine involved in the regulation of immune response and is
abnormally produced in chronic inflammatory conditions. Recently, it is suggested that
IL-17 can participate in innate immune reactions including activation of dendritic cells,
NK cells and recruitment of neutrophils at inflammatory sites. In the present study we
have investigated the involvement of IL-17 in the development of zymosan-induced
arthritis (ZIA) in SCID and BALB/c mice. ZIA developed earlier and the inflammation
was more severe in BALB/c than in SCID mice. CD69 positive neutrophils appeared to
the synovium of both strains on day 3 and day 7 of ZIA. However, at late stage of
disease the percentage of activated neutrophils was significantly higher in SCID
compared to BALB/c mice. At the same time point SCID mice showed elevated
synovial level of IL-17. We further investigated the ability of IL-17 to sustain the in
vitro differentiation of osteoclasts isolated from SCID and BALB/c mice. Our data
suggest a complex role of IL-17 in zymosan-induced inflammation.
12
The role of target tissue in modulating CNS autoimmunity
Momčilović M1, Miljković Ž2, Miljković Đ1, Mostarica Stojković M2 1Institute of Biological Research “Siniša Stanković”, University of Belgrade, Belgrade, Serbia 2Institute of Immunology and Microbiology, School of Medicine, University of Belgrade,
Belgrade, Serbia
Objectives: Experimental autoimmune encephalomyelitis (EAE) is an animal model of
multiple sclerosis (MS), inflammatory, demyelinating disease of the CNS. In every
species tested so far some strains are resistant to the induction of EAE. The analysis of
the mechanism underlying EAE resistance can contribute to the understanding of the
EAE and MS pathogenesis. In order to find out the role of the target tissue in CNS
autoimmunity we examined potential differences between cells isolated from spinal
cords (SC) of AO and DA rat strains, EAE resistant and susceptible, respectively.
Methods: EAE was induced in AO and DA rats by immunization with rat spinal cord
homogenate mixed with complete Freund's adjuvant. Inflammatory cells were obtained
from the SC of rats perfused with sterile PBS and separated on the 30%/70% Percoll
gradient. Flow cytometry was used for phenotypization of CNS-infiltrating cells,
detection of apoptotic cells and IFN-gamma and IL-17-producing cells. IFN-gamma and
IL-17 gene expression in CNS infiltrating cells were measured using real time PCR.
Results: Although AO rats did not exhibit any neurologic signs of disease after the
immunization in contrast to severely diseased DA rats, infiltration of immune cells into
SC was evident in both strains. However, the infiltration was less extensive in AO rats,
and the higher percentage of CD4+ T cells was found among cells infiltrating SC of DA
rats. Further, CD4+ T cells were also more activated in DA rats, as judged from the
higher percentage of OX40+ and CD25+ cells within T cells isolated from SC of
immunized animals, and less prone to apoptosis induction. Also, cells infiltrating CNS
of AO rats are less capable to produce IFN-gamma and IL-17, major marker cytokines
of Th1 and Th17 effector cells, respectively.
Conclusion: Our results presented indicate that besides regulatory mechanisms working
in the lymphoid tissues outside the CNS, additional modulating effects exerted within
the CNS parenchyma contribute to the resistance of AO rats to the induction of EAE.
This work was supported by the Serbian Ministry of Science (grants 143029 and
145066)
13
KIR gene frequencies in women with infertility problems Aleksandar Petlichkovski, Dejan Trajkov, Slavica Hristomanova, Eli Djulejic, Mirko Trpevski, Olivija Efinska-‐Mladenovska, Meri Kirijas, Aleksandar Senev, Mirko Spiroski Institute of Immunobiology and Human Genetics, Faculty of Medicine, St Cyril and Methodius University, Skopje, Republic of Macedonia Introduction: Natural killer (NK) cells are the predominant lymphocyte population in the decidua. Being the most abundant leucocytes, the activity of NK cells is important in different immuno-‐pathological conditions, such as recurrent spontaneous abortions, infertility and problems in implantation. The NK cells recognize HLA class I molecules on trophoblasts trough killer immunoglobulin-‐like receptors (KIRs) found on their surface. The KIRs are classified as either activating or inhibitory, regarding the effect they produce on NK cells upon interaction with corresponding ligand. Since KIR genes exhibit extensive polymorphism and individuals differ in both the number and kind (activating vs. inhibitory) of KIR genes, it is hypothesized that the KIR gene content might influence the pregnancy outcome. The Aim: The aim of this pilot study is to analyze the frequency of different KIR genes in women with infertility problems, and compare them to healthy women. Material and Methods: Total of 122 healthy women and 29 women with reproductive problems participated in this study. After signing of written consent DNA was isolated from peripheral blood using phenol/chloroform method. The genotyping of 16 KIR genes was performed using commercially available kit from Dynal Biotech, (Pel-‐Freez Clinical Systems, Brown Deer, WI, USA), based on SSP method. Results: All the framework genes (KIR3DL3, KIR3DP1, KIR2DL4 and KIR3DL2) were present in all studied individuals. In the patient group, the most prevalent activating gene was KIR2DS4, present in all individuals (100%), while the most frequent inhibitory KIR genes were: KIR2DL1, KIR2DL3 and KIR3DL1, all with estimated frequency of 80%. These findings were comparable with the group of healthy control individuals. Conclusion: In conclusion, simple KIR gene frequencies do not significantly differ among two studied groups. Further analysis of frequencies of corresponding genotypes or in the ratio of activating/inhibiting genes content in two groups are needed.
14
Toll-Like Receptors 2, 4, 9 and MyD88 Expression Correlates with Disease Activity in Experimental Autoimmune Encephalomyelitis Savić E1, Popadić D1, Marković M1, Mostarica-Stojković M1 1Institute of Microbiology and Immunology, University of Belgrade, School of Medicine, Belgrade, Republic of Serbia In multiple sclerosis (MS) and its model, experimental autoimmune encephalomyelitis (EAE), helper T cells (Th), primed in periphery and differentiated into Th1 and Th17 subsets, orchestrate the process of neuronal damage of the central nervous system (CNS). Recently, innate immunity and foremost Toll-like receptors (TLRs) were found crucial for shaping the adaptive response. TLR expression is increased in MS brain lesions. However, the results demonstrating the role of several TLRs (2, 3, 4 and 9) in EAE are controversial. Nevertheless, there is a consensus that mice lacking adaptor protein MyD88 do not develop EAE. Dark Agouti (DA) and Albino Oxford (AO) rats, susceptible and resistant to EAE, respectively, were immunized with rat spinal cord homogenate in complete Freund’s adjuvant. Draining lymph nodes (DLNs) and caudal portion of spinal cord (SC) were taken in total for Real Time PCR analysis of mRNA for TLR2, TLR3, TLR4, TLR9 and MyD88 before onset (3 dpi), with first neurological signs (7 dpi), in the peak of disease (14 dpi) and in recovery phase (21 dpi). Susceptible DA strain had significantly higher TLR2, 4, 9 and MyD88 mRNA expressions in SC in peak of clinical illness compared to EAE-resistant AO rats. Additionally, AO strain exhibited significantly higher TLR3 expression in SC in the induction period. Our data supported the studies in which TLR2, 4, 9 and MyD88 expressions correlated with more severe EAE and TLR3 signaling contributed to milder disease. (Supported by grant No 145066, Ministry of Science and Technological Development, Republic of Serbia)
15
Allele frequency of HLA-DQB1 locus in Macedonian population Aleksandar Senev, Meri Kirijas, Mirko Trpevski, Slavica Hristomanova, Olivija Efinska-‐Mladenovska, Aleksandar Petlichkovski, Dejan Trajkov, Mirko Spiroski Institute of Immunobiology and Human Genetics, Faculty of Medicine, St Cyril and Methodius Universisty, Skopje, Republic of Macedonia Aim. The aim of this paper was to genotype HLA-‐DQB1 locus in healthy unrelated
Macedonian population.
Material and Methods. Reverse Line Strip typing for HLA-‐DQB1 locus was
performed on a population of 217 samples from healthy individuals. The results
were obtained as alleles, and as NMDP Codes. Alleles were genotyped and
expressed as high resolution with more than four digits with slash between
ambiguous alleles. NMDP Codes were expressed as four digits for unambiguous
results, and with combination of numbers and characters for the certain
ambiguous combination. We did not found any genotypic ambiguities in HLA-‐
DQB1.
Results. We have found 33 different alleles of HLA-‐DQB1 in Macedonian
population, 12 of which were unambiguous with the frequency of 40.55%, and 21
were ambiguous with the frequency of 58.53%. The biggest frequency (33.64%)
was found for 05 unambiguous groups, and for 03 ambiguous groups (36.40%).
The biggest allele frequency in Macedonian population for HLA-‐DQB1 was found
for 03NX (030101/0102/09/13) with 27.88%, followed with 0502 with 13.82%,
and 02MN (0201/02/03) with 10.37%.
Conclusion. Allele frequency of HLA-‐DQB1 in Macedonian population is similar
with that found in other European populations and can be used for anthropology
and disease association studies.
16
Isolation and characterization of monoclonal antibodies specific for murine
cytomegalovirus m74 gene product
Faruk Skenderi1,2, Tihana Lenac Rovis2, Barbara Adler4, Stipan Jonjic2,3* 1Department of Physiology and Biochemistry, University of Sarajevo Medical Faculty, Bosnia
and Herzegovina, 2Center for Proteomics, University of Rijeka Medical faculty, Croatia, 3Department of Histology and Embryology, University of Rijeka Medical faculty, Croatia, 4Max
von Pettenkofer-Institut, München, Germany
Human cytomegalovirus (CMV) is a frequent cause of morbidity of immuno-
compromised patients and major viral cause of congenital infections. Recent data
suggest possible role of CMV infection in autoimmune disease and some other diseases.
Many aspects of immunobiology and pathogenesis of CMV infection are still
insufficiently defined. Murine cytomegalovirus (MCMV) infection is generally
accepted as a suitable model for human cytomegalovirus (HCMV) infection. Many
studies focus on dissection of MCMV genome, transcriptome and proteome. HCMV
UL74 gene product, glycoprotein O (gO), is believed to play a role in virus entry into
the cell, assembly of viral particles and viral egress. MCMV gene m74 represents a
position homolog of the HCMV UL74. The product of MCMV m74 gene is MCMV
glycoprotein O (gO) whose role in infection is still to be clarified.
We used hybridoma technology to generate mouse monoclonal antibodies specific to
MCMV gO. BALB/c mice were immunized with UV inactivated MCMV virions. After
seroconversion, the mouse spleen was harvested and lymphocytes fused with mouse
myeloma SP2/0 cell. About 800 hybridomas were screened by ELISA using the lysates
of MCMV infected cells. Selected mother wells were additionally screened by FACS.
In total we obtained 105 hybridoma cell lines which were specific for viral proteins. To
select for a gO specific antibody we created BALB-3T3-m74HA transfectant cell line
expressing MCMV m74 gene product.
Our results show that we have generated several MCMV gO specific monoclonal
antibodies. Further characterization by using western blot, immunoprecipitation and
neutralization assay will eventually find suitability of different mAbs as tools to study
biology of this viral glycoprotein and its role in infection.
*Corresponding author. Mailing address: Medical Faculty Rijeka, Department of Histology and Embryology, B.Branchetta 20, 51000 Rijeka, Croatia. Phone: +38551651206. Email: [email protected]
17
Effects of cardiac resynchronization therapy on reverse left ventricle remodeling A. E. Stanciu, PhD1, Marcel Stanciu, PhD2, R. Vatasescu3, C. Iorgulescu3, M. Dorobantu, PhD3
1. ”Prof. Dr.Al.Trestioreanu” Institute of Oncology, Bucharest, Romania 2. University “Politehnica” of Bucharest, Bucharest, Romania 3. Clinic Emergency Hospital Bucharest, Bucharest, Romania
Background: Remodeling reflects the structural and functional deterioration that occurs in heart failure. Indices of remodeling constitute an important marker of the severity of heart failure, and reverse remodeling is an accepted goal in the treatment of heart failure. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are matrix-degrading enzymes that have been demonstrated to influence left ventricle (LV) properties and serve as targets of potential anti-remodeling agents. Objectives: The purpose of the study was to investigate the effects of CRT on serum levels of N-terminal pro-brain natriuretic peptide (NT-proBNP), MMP-2 and TIMP-2 in patients with CHF. Methods and patients: 27 patients (21M/8F, aged 64 ± 11 years) with CHF (II-IV NYHA functional class) were investigated for immune activation before (T0 – baseline) and 1 week (T1), 3 (T2), 6 (T3), 12 (T4) months after CRT treatment. In all patients, blood specimens were drawn from a peripheral vein. The serum levels of MMP-2, TIMP-2 and NT-proBNP were measured at the same time by an ELISA method. Cardiac function was assessed echocardiographically. Results: In 100% of the patients there was a rapid and significant clinical improvement. There were no HF hospitalizations or deaths in this cohort. There was an immediate and persistent reduction in LV asynchrony, which was associated with an important and progressive improvement in LV systolic function and extensive LV reverse remodeling. There was also a significant reduction of the serum levels of NT-proBNP, MMP-2 and MMP-2/TIMP-2. CRT positively influences extracellular matrix remodeling by decreasing serum levels of MMP-2 and increasing TIMP-2. The MMP-2/TIMP-2 ratio had decreased from 6.84 ± 7.55 before CRT to 1.95 ± 0.64 at 12 months after CRT treatment. There was a good correlation between changes in ejection fraction (EF) and MMP-2/TIMP-2 (r = 0.45, p < 0.05) and septal-to-posterior wall motion delay (SPWMDSax) and MMP-2/TIMP-2 (r = 0.61, p< 0.05) after CRT. Conclusions. At 12 months follow-up, CRT was associated with a discordant change in serum MMP-2 and TIMP-2 levels. These changes in MMP-2/TIMP-2 ratio lead to reverse LV remodeling in patients with CHF.
18
Influence of hyperthermic environmental temperature on adenosine deaminase (ADA) activity in serum and lymphoid organs Dejan Trajkov1, Dinevska-‐Kjovkarova Suzana2, Aleksandar Petlichkovski1, Biljana Miova2, Olivija Efinska-‐Mladenovska1, Slavica Hristomanova1, Meri Kirijas1, Aleksandar Senev1, Mirko Trpevski1, Mitev Slavcho2, Mirko Spiroski1 1Institute of Immunobiology and Human Genetics, Faculty of Medicine, University "Ss. Kiril and Metodij", Skopje, Republic of Macedonia; 2Department of Physiology and Biochemistry, Faculty of Natural Sciences, University "Ss. Kiril and Metodij", Skopje, Republic of Macedonia Introduction: Adenosine deaminase (ADA) is a cytoplasmatic enzyme contained in different tissues. ADA is a catalyser of the irreversible hydrolytic deamination of adenosine into inosine. The influence of ADA on the cell mediated immunity is expressed by its participation in the T lymphocytes and monocytes differentiation and proliferation. Aim: The aim of this study was to analyze the influence of the hypertermic environmental temperature (35±10C) on the ADA activity in tissues rich with lymphoid cells in Wistar rats. Matherial and methods: The activity of ADA was examined in thymus, spleen, Peyer’s patches, mesenteric lymph nodes and serum. The experimental animals (n=11 male rates) were exposed (acclimated) on hyperthermic environment (35±10C) and relative air humidity of 20-‐30% within a period of 30 days. The control group of animals (n=10) were kept on room temperature (18-‐220C). The ADA activity was measured according to the modified method of Giusti. The results obtained were processed with the Student’s t-‐test. The values p<0.05 were considered to be statistically significant. Results: The results obtained have shown that there were different changes. The body weight statistically significant decrease from 175.35±21 g in the control group to 159.69±11.14 g in the experimental animals (p<0.015). The spleen mass was reduced from 375.94±176.56 mg/100 g to 237.59±74.06 mg/100 g (p<0.05), and the thymus mass from 245.52±51.55 mg/100g to 204.84±29.92 mg/100 g (p<0.05). Decreased of relative mass of both Peyer’s patches and mesenteric lymph nodes was not statistically significant. Activity of ADA in the thymus was significantly increased from 10.58±1.57 U/g in the control group of rats to 13.38±2.7 U/g in rats exposed on 35±10C (p<0.012), and in the spleen it raised from 5.17±3.29 U/g to 7.8±1.97 U/g (p<0.05). The ADA activity in the other lymphoid organs (Peyer’s patches and mesenteric lymph nodes) and serum showed no statistically significant changes between the two groups of examined rats. Conclusion: It could be concluded that the influence of hyperthermic environment (3535±10C, 30 days), provokes decrease in body weight, spleen and thymus relative mass, increase of the enzyme activity of ADA in thymus and spleen of rats, but there are no changes in the other lymphoid organs and tissues.
19
List of participants Belij, Sandra, Institute für Biological Research “Sinisa Stankovic”, Belgrad, Serbia mail: [email protected] Berisha, Dr. Naser, MD University Clinical Center, Obstetrics & Gynecology University of Prishtina, Prishtina, Kosovo mail: [email protected] Bijelic, Dr. Bibijana, MD Clinical Hospital Centre Osijek, Scientific Research Unit, Osijek, Croatia mail: [email protected] Bijelic, Dr. Nikola, MD University of J.J. Strossmayer, Faculty of Medicine, Osijek, Croatia mail: [email protected] Curaj, Teuta Universal Hospital Center, Tirana, Albania mail: [email protected] Dervisevic, Dr. Amela MD Medical Faculty, University of Sarajevo, Sarajevo, Bosnia and Herzegovina mail: [email protected] Drace, Dr. Zahida MD Clinical Center, University of Sarajevo Sarajevo, Bosnia and Herzegovina mail: [email protected] Dzafic, Dr. Fejzo MD University Clinical Center Tuzla, Tuzla, Bosnia and Herzegovina mail: [email protected] Gavranovic, Dr. Marija MD Clinical Center of Montenegro, Podgorica, Montenegro mail: [email protected] Godinjak, Dr. Amina MD Clinical Center University of Sarajevo Clinic for Endocrinology, Sarajevo, Bosnia and Herzogovina mail: [email protected] Hristomanova, Dr. Slavica MD Institut of Immunobiology and Human Genetics, Faculty of Medicine, Skopje, Macedonia
mail: [email protected] Huica, Irina Institute of Virology “Stefan S. Nicolau”, Bucharest, Romania mail: [email protected] Jergovic, Mladen Institute of Immunology, Zagreb, Croatia mail: [email protected] Josipovic, Pavle, Faculty of Sciences, Department of Animal Physiology, Zagreb, Croatia mail: [email protected] Kapovic, Dr. Agneza Marija MD Children’s Hospital, Zagreb, Croatia mail: [email protected] Karac, Ivo Faculty of Sciences, Department of Biology, Zagreb, Croatia mail: [email protected] Karkovic, Domagoj Faculty of Sciences, Department of Biology, Zagreb, Croatia mail: [email protected] Kirijas, Dr. Meri MD Institute of Immunobiology and Human Genetics, Skopje, Macedonia mail: [email protected] Kurti-Prifti, Dr. Margarita MD Laboratory of Immunology, University Hospital Center, Tirana, Albania mail: [email protected] Lacevic, Dr. Dzenana MD Institute of Medical Biochemistry, Clinical Centre of University, Sarajevo, Bosnia and Herzegovina mail: [email protected] Latifi-Pupovci, Prof.ass. Hatixhe, MD Chair for Physiology and Immunology, Medical Faculty, University of Prishtina, Prishtina, Kosovo mail: hpupovci@uni-‐pr.edu Ler, Daria Institute for Genetic Engineering and Biotechnology, University of Sarajevo, Sarajevo, Bosnia and Herzegovina mail: [email protected]
20
Lokaj-Berisha, Dr. Violeta MD University of Prishtina , Medical Faculty Institute of Physiology and Immunology Prishtina, Kosovo mail: [email protected] Lumezi, Dr. Besa MD Medical Faculty, University of Prishtina, Department of Physiology and Immunology Prishtina, Kosovo mail: [email protected] Marinovic Terzic, Dr. Ivana MD PhD Department of Immunology and Medical Genetics, Split University Medical School, Split, Croatia mail: [email protected] Masic, Dr. Admir MD Clinic for Neurology diseases,Clinical Center of University Sarajevo, Sarajevo, Bosnia and Herzogovina mail: [email protected] Milanova, Viktoriya Department of Immunology, Institute of Microbiology, Bulgarian Academy of Science, Sofia, Bulgaria mail: [email protected] Momcilovic, Dr. Miljana PhD Institute for Biological Research “Sinisa Stankovic”, Department of Immunology Belgrad, Serbia mail: [email protected] Okic, Dr. Anel MD Canton Hospital Zenica Zenica, Bosnia and Herzogovina mail: [email protected] Pecani, Dr. Arbi MD University Hospital Center, “Nene Tereza” Tirana, Albania mail: [email protected] Petlichkovski, Dr. Aleksandar MD Institute of Immunobiology and Human Genetics, Faculty of Medicine, Skopje, Macedonia mail: [email protected] Prljaca-Zecevic, Dr. Lamija MD Clinical Center, University of Sarajevo Sarajevo, Bosnia and Herzogovina mail: [email protected]
Savic, Dr. Emina, MD Institute of Microbiology and Immunology, School of Medicine, University of Belgrad Belgrad, Serbia mail: [email protected] Semanaj, Valentina Laboratory of Immunology University Hospital Center “Mother Teresa”, Tirana, Albania mail: [email protected] Senev, Dr. Aleksandar MD Institute of Immunobiology and Human Genetics, Faculty of Medicine, Skopje, Macedonia mail: [email protected] Serdarevic, Dr. Fadila MD Institute of Immunology University of Sarajevo, Bosnia and Herzegovina Mail: [email protected] Shrestha, Dilip Department of Biophysics and Cell Biology, University of Debrecen, Hungary mail: [email protected] Skenderi, Dr. Faruk MD Department of Physiology and Biochemistry, University of Sarajevo, Medicial Faculty Sarajevo, Bosnia and Herzogovina mail: [email protected] Skific, Marijana University Hospital Centre, Dept. of Clinical Transfusiology and Cellular Therapy, Zagreb, Croatia mail: mskific@kbc-‐zagreb.hr Stanciu, Dr. Adina Elena Prof. Al. Trestioreanu Institute of Oncology, Bucharest, Romania mail: [email protected] Trajkov, Dr. Dejan Institute of Immunobiology and Human Genetics, Faculty of Medicine, Skopje, Macedonia mail: [email protected] Zecevic-Masic, Dr. Mina, MD Clinic for Pulonary Diseases and TB, Clinical Centre of University of Sarajevo, Sarajevo, Bosnia and Herzegovina mail: [email protected]
21
Faculty Fleischer, Bernhard, Prof., Bernhard Nocht Institute for Tropical Medicine, D-‐20359 Hamburg, Germany, www.bnitm.de; and Institute of Immunology, University Medical Centre Hamburg-‐Eppendorf, D-‐20246 Hamburg, Germany, mail: [email protected] Gospos, Annette, Dr., Director, Division Medical Scientific Information, Euroimmun Inc., D-‐23560 Luebeck, Germany, www.euroimmun.de; mail: [email protected] Haag, Friedrich, Prof., Institute of Immunology, University Medical Centre Hamburg-‐Eppendorf, D-‐20246 Hamburg, Germany, www.uke.de/institute/immunologie; mail: [email protected] Meri, Seppo, Prof, Head, Haartman InstituteDepartment of Bacteriology and Immunology, PO Box 21, Haartman Institute, FIN-‐00014 University of Helsinki, Finland, mail: [email protected] Mittruecker, Hans-‐Willi, Prof. , Institute of Immunology, University Medical Centre Hamburg-‐Eppendorf, D-‐20246 Hamburg, Germany, www.uke.de/institute/immunologie; mail: [email protected] Rabatic, Sabina, President, Croatian Immunological Society, Institute of Immunology Rockefeller str.10, 100000 Zagreb, Croatia; mail: [email protected]
Seitz, Hans-‐Joachium, Prof., Southeast-‐Europe Cooperation, University Medical Centre Hamburg-‐Eppendorf, Martinistraße 52, D-‐20246 Hamburg, Germany; mail: [email protected] Stockinger, Hannes, Prof. , Chairman, Department of Molecular Immunology, Center of Physiology, Pathophysiology and Immunology, Medical University of Vienna; Internet: http://www.meduniwien.ac.at/immunology; mail: [email protected] Zouali, Moncef, Prof., Director of Research, Institut National de la Santé et de la Recherche Médicale (INSERM), University Denis Diderot, Paris, France; mail: [email protected]