The rising importance of the progress and application of the molecular and cellular medicine in clinical medicine

,

Research Institute of Interventional

Allergology and Immunology Cologne/Bonn, Germany

Prof. Prof.h.c. Alireza Ranjbar, M.D.,Ph.D

The 5th international Congress of the International Prof. Dr. Alireza Yalda Academic Forundation

in Medical Sciences

6-10th 2014, Tehran University of Medical Sciences

HistoryRudolf Virchow published a concept that the origin of diseases should be found in error of cellular function.

Robert Koch showed that some illnesses are caused by microorganisms. It was the base of today´s understanding of pathogenesis of infectious diseases.

Paul Ehrlich expanded the approach of cellular and molecular levels. His perception of substances which react specifically with microorganisms or body´s own cells through of specific receptors, introduced the beginning of molecular orientated therapy.

The Max Delbrück´s work about the nature of gene mutation and gene structure was the base of modern molecular Genetics and molecular biology.

The development of modern cellular and molecular biology will lead in next years to paradigm change in diagnostic and therapy as well as inprevention of diseases.

The use of molecular approach gives us the opportunity to explain thefunction of each cell und of particular cellular components, and torecognize the mechanisms and rules of their interactions intissues and organs.

By understanding of these biological processes the possibility arises for rational and specific therapy as opposed to up to now mostlysymptomatic approach .

Introduction

Clinical application of molecular and ceullar medicine in infectious diseases and immunology

Case

Chief complaints

• A 17-years-old male patient, related parents

• Persistent cerebral TB and chronic meningitis since the past 12 months with severe headache

• Skin abscess

• Resistance to anti TB and antibiotic therapy

• Episodes of recurrent otitis, sinusitis, pneumonia and skin abscesses

• Recurrent hospitalization and surgical cleaning of skin abscesses

• At 2 years of age CGD was diagnosed by use of NBT-Test(Nitroblue Tetrazolim Test)

Past medical history

Therapy and course of disease:

appropriate antibiotic therapy repeated surgical cleaning of skin abscess appropriate anti-tuberculosis treatment with INH, Rifampicine,

Pyrazinamide, Ethambutol, Streptomycine Subcutanous interferron gamma with a dosage of 50 µg/ m2

body surface 3 times per week

All therapy interventions were without success

Leuc 20000 µl PMN 92% Hb 9,2 Hct 29% Thromb. 520000 µl ESR 82 mm CRP 78 mg/l RF negativ ANA negativ Anti-ss-DNS negativ IgG 2400 mg/dl IgM 180 mg/dl IgA 80 mg/dl

Lab. at referring

Aspiration of skin abscess: Staph. aureus

CSF : Mycobacterium tuberculosis by use of PCR und cultur, sensitive to INH und Rifampicine

HIV-Ab : negative

HIV-Ag mittels PCR: negative

Zinc, copper within normal range, iron deminished, Ferritin increased

Selenium: 32 µg/l (normal range 100-150 µg/l)

Lab. at referring

Granulocyte function test by use of Dehydrorodamin-Test (DHR) 123: Phagocytosis 58% 84-96Respiratory burst low 0,2% 3-17 (fMLP)Respi. burst high 2% 91-97 (PMA)Respi. burst E.Coli 2% 91-97%Respi. burst Staph. aur. 1% 91-97%

Monocyte function test:

Phagocytosis 52% 60-76Respiratory burst 4 % 48-80Respi. burst Staph. aur. 1% 48-80

Granulocyte function after Stimulation with IFN-gamma: before afterPhagocytosis 58% 62 %Respiratory burst low 0,2% 0,2% (fMLP)Respi. burst high 2% 4% (PMA)Respi. burst E.Coli 2% 3%Respi. burst Staph. aur. 1% 1%

Monocyte function test:

Phagocytosis 52% 64%Respiratory burst 4 % 7%Respi. burst Staph. aur. 1% 4 %

IFN-gamma receptor (CD 119): normal

IL-12 receptor (IL-12Rbeta1): normal

Lymphocyte subpopulation by flow cytometry: normal

142,4

37,2

1000IL-

4 lev

els (p

g/ml)

IL-4 and IFN-gamma und IL-4 levels in PBMC

Controlgroup

28,2

124

150

IFN-ga

mma l

evels

(U

/ml)

Patient

Patient Controlgroup

Therapy and follow up

Prednisolone pulse therapy weekly for 2 cycles Selenium in term of sodium selenite with a dosage of 10µg/kg

BW per day Unchanged continuation of antituberculous therapy.

Significant improvement of subjective symptoms and headache 2 weeks after therapy

Brain MRI 2 weeks after therapy

before therapy after therapy

142,4

37,2

1000IL-

4 lev

els (p

g/ml)

IL-4 and IFN-gamma und IL-4 levels in PBMC2 wks. after therapy

Nach Th

28,2124

150

IFN-ga

mma l

evels

(U

/ml)

Vor Th

Vor Th Nach Th

Granulocyte function 2 wks. after therapy: before afterPhagocytosis 58% 66 %Respiratory burst low 0,2% 0,4% (fMLP)Respi. burst high 2% 12% (PMA)Respi. burst E.Coli 2% 22%Respi. burst Staph. aur. 1% 18%

Monocyte function:

Phagocytosis 52% 58%Respiratory burst 4 % 17 %Respi. burst Staph. aur. 1% 12 %

Granulocyte function after stimulation with IFN-gamma 2 wks. after therapy: before afterPhagocytosis 66 % 66%Respiratory burst low 0,4% 12% (fMLP)Respi. burst high 12% 80% (PMA)Respi. burst E.Coli 22% 82%Respi. burst Staph. aur. 18% 78%

Monocyte function:

Phagocytosis 58% 60%Respiratory burst 17 % 62%Respi. burst Staph. aur. 12 % 84%

Therapy and follow up

Continuation of selenium therapy Continuation of antituberculous treatment Interferron gamma 50 µg/ m2 body surface, subcutanous 3

times per week

Symptom-free 4 wks. after therapy,no headache, no skin abscess

Before therapy

Brain MRT 6 wks. after therapyAfter therapy

CSF : Cultur: steril, M. tuberculosis was not grown, PCR : still positive

Selenium : 82 µg/l

CBC: mild anemia and leucocytosis, otherwise normal findings

CRP : 12 µg/l (Norm <5)

ESR: 42 mm

Lab. 3 months after therapy

CSF : Cultur: steril, M. tuberculosis was not grown, PCR for M. tuberculosis: negative

Selenium : 140 µg/l

CBC: normal

CRP : 3 µg/l (Norm <5)

ESR: 32 mm

Lab. 6 months after therapy

Clinical application of molecular medicine in endocrinology

Case

9-yr-old male, unrelated parents

PH: gestation, birth, somatic and mental development

normal

FH: Diabetes mellitus in 4 persons in 2 generations

CC: since 6 months tiredness, listlessness,low performance

after diagnosing of Diabetes mellitus type I and insulin

therapy in an external pediatric clinic.

The patient showed a significant variability of BS levels

and hypoglycemic shocks despite low dose insulin

therapy

Findings at diagnosing by us:

- Physical and neurological exam: normal findings

- no fever, no weight loss, no night sweats, weight 50 P

Findings at diagnosing by us:

- FBS: 220 mg/dl

-Urin: Glucose++, Keton +, otherwise normal findings

- HbA1c: 6,5 (normal range: 4,7-6,4) - FT3,FT4,TSH, free corstisol in serum and all rotine lab: normal

- TPO-Ab,TG-Ab, TSH-Receptor-Ab: negative

- Islet cells-Ab, GADII-Ab, Tyrosinphosphatase IA 2-Ab and Insulin-Ab: negative

Suspicion diagnosis:

MODY 2 (Maturity Onset Diabetes of the Young type 2)

MODY

Maturity Onset Diabetes of the Young (MODY) is a group of diabetes disorders that affects about 2-5% of people with diabetes. MODY is often not recognised and people may be treated as Type 1 or Type 2 diabetes.

MODY diabetes is based on genes mutations which are responsible for glucose transport and metabolism and insulin secretion.Till now 6 different genes have been recognised.

•hepatic nuclear factor (HNF)-1alpha (MODY 1)•Glucokinase (GCK) (MODY 2)•HNF-1beta (MODY 3)•HNF-4alpha (MODY 4)•insulin promoter factor-1 (MODY 5)•NeuroD/BETA2 (MODY 6)

MODY

The following characteristics suggest the possibility of a diagnosis of MODY in hyperglycemic and diabetic patients:

- Mild to moderate hyperglycemia (typically 130-250 mg/dl, or 7-14 mM) - Discovered before 25 years of age.- A first degree relative with a similar degree of diabetes.- Absence of positive antibodies or other autoimmunity (e.g.,thyroiditis) in patient and family.- Persistence of a low insulin requirement (e.g., less than 0.5 u/kg/day) past the usual "honeymoon" period.- Absence of obesity (though obese people can get MODY), or other problems associated with type 2 diabetes or metabolic syndrome (e.g. hypertension, hyperlipidemia, polycystic ovary syndrome)- Cystic kidney disease, hypoplasia or malformations of sexual organs (e.g. vaginal hypoplasia) in patient or close relatives (MODY 5)- Non-transient neonatal diabetes, or apparent type 1 diabetes with onset before 6 months of age.

MODY 2

- MODY 2 is due to any of several mutations in the GCK gene on chromosome 7 for glucokinase.

- Glucokinase serves as the glucose sensor for the beta cell and is an important enzyme for the glycogen synthesis in the lever.

Findings at diagnosing by us:

Because MODY 2 does not show any significant pathological rising of postprandial BS , we discharged insulin and meseured BS pre and postprandial within 5 days.

The results did not show pathological postprandial BS levels

Findings at diagnosing by us:

Molecular genetic exam:

Mutation of Glucokinase-Gene K420E

Therapy:

Insulin discharged

Dietary measures and sport

The degree of hyperglycemia does not usually worsen with age and long-term diabetic complications are rare

Clinical application of molecular medicine in diabetology

Case

4-yr-old female , related healthy parents (cousin 1. grade)

PH: gestation, birth, somatic and mental development normal CC: before 3 wks. detection of glucose in urin (glucose ++++) by a preventional exam The child was symptom free, no polydipsia or polyuria, no signs for infections

FH: Gestational diabetes by the mother, diabetes type 2 by the grandmother

Findings in an external pediatric clinic

- Urin exam: Glucose ++++, Keton negative, otherwise normal findings, amino acids: negative SDS-Electrophoresis in 24h-urin: normal findings

- FBS, HbA1c, FT3,FT4,TSH, free corstisol in Serum and all routine lab: normal - Oral Glucose Tolerance Test: normal

- Islet cells-Ab, GADII-AK, Tyrosinphosphatase IA 2-Ab: negative

- Ultrasound of abdomen and kidney: normal findings

- Ophtholmologic and ENT exam: normal findings

Findings at diagnosing by us:

- Physical and neurological exam: normal findings

-Urin: Glucose ++++, Keton negative, otherwise negative

- Urin exam by the parents: normal - Suspicion diagnosis: benigne renal glucosuria autosomal recessive form

Molecular genetic exam on mutation of Na+/Glucose-Cotransporter-Gene typ 2 (SGLT2=SLC5A2):

Child: Gene mutation E421K in homozygotic form

Both parents: Gene mutation E421K in heterozygotic form

Therapy:

- No therapy , no dietary measures

- No relationship with diabetes mellitus

Clinical application of molecular medicine in infection induced hepatitis

Case

Oxidative Stress and

Infectious Diseases

Free oxygen radicals (reactive oxygen species=ROS) are atoms, molecules or residues which carry single electrones in the outer membane. They possess a great potential to damage the vital cells because of reaction with proteins, lipids and DNA.

Die important ROS in the biological systems : Singulett-Oxygen (1O2) Superoxide anions (O2- ) H2O2 OH- Organic peroxide (ROOH)

Cell damages by ROS

Aus: “Free Radicals Randox Ltd.

The important origins of ROS are:

- Respiratory chain - NAD(P)H-Oxidase of neutrophils, macrophages and endothelial

cells of blood vessels

The human body has a complex protecting system to eleminate these toxic substances which is called as antioxidants.

The imbalance between anti- and prooxidants in favour of prooxidants is defined as oxidative stress, by which different illness can be initiated.

Macrophage

Neutrophils

Lysozyme

LTB4fMLPC5a

T cells

B cells

IL-1

IL-6

IL-8

TNF-alpha

GM-CSF

Myeloperoxidase

Elastase

Collagenase

Muraminidase

Oxygen free radicals

H2O2OHO2

-

--

Schematic outline of the immunologic process inthe inflammation (Ranjbar A.)

Elimination ofmicroorganism

Destruction ofconnective and

mesenchymal tissues

ROS and mutation of microorganism

• ROS may cause mutation of microorganisms, which lead to an increase of pathogenicity of microorgnisms, to resistance to antibiotics and to araise of cancerogenicity.

Case report10-yr-old male , unrelated healthy parents

PH: gestation, birth, somatic and mental development normal

CC: chronic active hepatitis , interferon therapy in the department of paediatric hepatology of university children hospital in Germany because of negative Anti-HBe , high Hep. B virus load in blood (measured by PCR) and increas of transaminase The therapy was not tolerated , did not lead to improvement and was discharged after 4 wks.

Case report

PE: normal physical and neurological findings, mild icterus , tiredness, listlessness , low performance, decline in physical and mental standards

Case report

Abdomen ultrasound:

Mild increased echogenicity of liver paranchyme, otherwise normal findings

Normal range

CBC normal Thromb. 251000 µl ESR 8 / 14 mm CRP < 5 mg/l < 5 Protein-Electr. normal Bilirubin total 1,73 mg/dl < 1 Bilirubin direct 1,2 mg/dl Gamma GT 47 U/l < 19 SGOT 170 U/l < 47 SGPT 224 U/l < 39 HBs-Ag qualitative positive HBc-IgM negative HBc-IgG positive HBs-Ab negative HBe-Ab negative HBV-DNA quantitaive (PCR) 9,2 exp7 Copy/ml Hepatitis A ,C,D,E serology negative All autoantibodies negative AFP and CEA negative

Lab.

Normal range

Selenium 58 µg/l 100-150

Beta-Carotin 0,38 mg/l 0,47-4,1

Ery.-Malondialdehyde (MDA) 432 nmol/g Hb 48-123

Gluthation-Peroxidase 26,2 U/g Hb 25-54

Lab

Genotype of Hepatitis B-Virus:

Point mutation rtA194T

Lab.

Therapy

- Sodium-Selenit oral 300µg/day

- Beta-Carotin 10 mg /day

- fruit and vegetables approx. 500 gr. /day

- Reduction of saturated fatty acids

- Reduction of red meats

Therapy results

significant improvement of physical and mental standards 2 wks. after therapy

before Therapy after 4 W Bilirubin total 1,73 mg/dl 1,32 Bilirubin direct 1,2 mg/dl 0,8 Gamma GT 47 U/l 22 SGOT 170 U/l 98 SGPT 224 U/l 128 HBs-Ag qualitative positive positive HBc-IgM negative negative HBc-IgG positive positive HBs-Ab negative negative HBe-Ab negative negative HBV-DNA quantitaive(PCR) 9,2 exp7 copy/ml 8,8 exp7

Genotype Point mutation no point mutation rtA 194t detectable

Lab. 4 wks. after therapy

before therapy after 4 W

Selenium 58 µg/l 82

Beta-Carotin 0,38 mg/l 4,2

Ery.-Malondialdehyde (MDA) 432 nmol/g Hb 82

Gluthation-Peroxidase 26,2 U/g Hb 38,6

Lab. 4 wks. after therapy

Therapy

- Interferon s.c. 5 Mio. E/m2 3 x per week

- Sodium-Selenit oral 200µg/day

- Fruit and vegetables approx. 500 gr. /day

- Reduction of saturated fatty acids

- Reduction of red meats

before after 6 W Bilirubin gesamt 1,32 mg/dl 0,8 Gamma GT 22 U/l 12 SGOT 98 U/l 46 SGPT 128 U/l 56 HBs-Ag qualitative positive positive HBc-IgM negativ negative HBc-IgG positive positive HBs-Ab negative negative HBe-Ab negative positive HBV-DNA quantitaive (PCR) 8,8 exp7 copy/ml 3,8 exp3

Lab. 6 wks. after Interferon therapy

Thank you for your attention

Antioxidants („Scavenger“)

Vitamins-A -C -E

etc ...

Carotinoids- - -Carotin- - -Carotin- Lycopin - Lutein- Zeaxanthin- ...

Trace elements- Selenium as cofactors- Iron - Copper- Zinc - Mangan

Phytoestrogens- Isoflavonoide- ...

Enzyme- Catalase- SOD Superoxiddismutase- Glutathion peroxidase- ...

Polyphenole- Phenolic acid- Flavonoids- ...

Sulfide- Allicin- ...

FreeRadicals

S ynergie!

Ubichinon

Vitamin C

Vitamin E

M n SO D-

Sperm inSperm id in

SAM

GST( , , , )

M DA

8-OHdG m t

DNS

X-R

XR-GSH

M ethioninCystein

NAC-L iponsre

HNEPGF2

8-OHdGnkLD L

ATP

Cu,Zn SOD-

CATALASE

RO S

GSH

GSSG

GPXGR

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Requirements for an effective antioxidant therapy

• Quality of antioxidants (antioxidative capacity)

• Presence of suitable carriers

Requirements for an effective antioxidant therapy

• Quality of antioxidants (antioxidative capacity)

Synergismus of nature

100 gr fresh apple contins approx.

5,7 mg Vit C

The antioxidative capacity

is equal to

1500 mg synthetic Vit CNature 2000, 430:

903-4

The antioxidative capacity of 100gapple (5,7mg Vit. C)Is equal to 1500 mg synthetic vitamin C

total oxyradical-scavenging capacity(TOSC; mol vitamin C equivalents per g)

Nutrition: Antioxidant activity of fresh applesMARIAN V. EBERHARDT1, CHANG YONG LEE1 & RUI HAI LIU1

Department of Food Science, 108 Stocking Hall, Cornell University, Ithaca, New York 14853-7201, USANature 405, 903 - 904 (June 22th, 2000)

Requirements for an effective antioxidant therapy

• Presence of suitable carriers

Selenium levels in serum of children with AD (n = 62) in comparisonwith control group (n = 78)

198

50

40

30

20

10

Control group

Selen

iu

m levels in

seru

m µ

g/l,

mean

+

/- S

D100

60

70

80

90

Text

p < 0.01

Children with AD

Selenium levels in whole blood of children with AD (n = 62) in comparisonwith control group (n = 78)

180

75

60

45

30

15

Control group

Selen

iu

m levels in

seru

m µ

g/l,

mean

+

/- S

D150

90

105

120

135

Text

p < 0.01

Children with AD

104

162

100

170

100

80

60

40

20

Weeks

0 40 4

µg

/l,

mean

+/-

S

DSelenium levels of children with moderate AD

before and after oral therapy with sodium seleniteresponder (n=52)vs. non-responder(n=10)

ns

p < 0.01

p < 0.01

132

198

115130

p < 0.01

20

20

Whole blood

Serum

Responder Non-Responder

170165

100

80

60

40

20

Weeks

0 4

µg

/l,

mean

+/-

S

DSelenium levels of non-responder children with

moderate AD before and after oral therapy with sodium selenite and

fruit /vegetables (n=8)

ns

130

210

p < 0.05

20

20

Whole blood

Serum

Free radicals andoxidative stress

„If you don‘t get enough antioxidants, it is the equivalent of irradiating yourself.“

„It‘s the same as stepping unprotected in front of an xray machine.“

B.N. Ames, JAMA 1995