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The rising importance of the progress and application of the molecular and cellular medicine in clinical medicine, Research Institute of Interventional.

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Presentation on theme: "The rising importance of the progress and application of the molecular and cellular medicine in clinical medicine, Research Institute of Interventional."— Presentation transcript:

1 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

2 History Rudolf 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.

3 The development of modern cellular and molecular biology will lead in next years to paradigm change in diagnostic and therapy as well as in prevention of diseases. The use of molecular approach gives us the opportunity to explain the function of each cell und of particular cellular components, and to recognize the mechanisms and rules of their interactions in tissues and organs. By understanding of these biological processes the possibility arises for rational and specific therapy as opposed to up to now mostly symptomatic approach. Introduction

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

5 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

6 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

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9 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

10  Leuc µl  PMN 92%  Hb 9,2  Hct 29%  Thromb 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

11  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 µg/l) Lab. at referring

12  Granulocyte function test by use of Dehydrorodamin-Test (DHR) 123:  Phagocytosis 58%  Respiratory burst low 0,2% 3-17 (fMLP)  Respi. burst high 2% (PMA)  Respi. burst E.Coli 2% 91-97%  Respi. burst Staph. aur. 1% 91-97%  Monocyte function test:  Phagocytosis 52%  Respiratory burst 4 %  Respi. burst Staph. aur. 1% 48-80

13  Granulocyte function after Stimulation with IFN-gamma: before after  Phagocytosis 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 %

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17  IFN-gamma receptor (CD 119): normal  IL-12 receptor (IL-12Rbeta1): normal  Lymphocyte subpopulation by flow cytometry: normal

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21 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

22 Brain MRI 2 weeks after therapy before therapy after therapy

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24 Granulocyte function 2 wks. after therapy: before after  Phagocytosis 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 %

25 Granulocyte function after stimulation with IFN-gamma 2 wks. after therapy: before after  Phagocytosis 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%

26 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

27 Before therapy Brain MRT 6 wks. after therapy After therapy

28  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

29  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

30 Clinical application of molecular medicine in endocrinology Case

31 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

32 Findings at diagnosing by us: - Physical and neurological exam: normal findings - no fever, no weight loss, no night sweats, weight 50 P

33 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

34 Suspicion diagnosis: MODY 2 (Maturity Onset Diabetes of the Young type 2)

35 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)

36 MODY The following characteristics suggest the possibility of a diagnosis of MODY in hyperglycemic and diabetic patients: - Mild to moderate hyperglycemia (typically 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.

37 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.

38 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

39 Findings at diagnosing by us: Molecular genetic exam: Mutation of Glucokinase-Gene K420E

40 Therapy: Insulin discharged Dietary measures and sport The degree of hyperglycemia does not usually worsen with age and long-term diabetic complications are rare

41 Clinical application of molecular medicine in diabetology Case

42 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

43 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

44 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

45 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

46 Therapy: - No therapy, no dietary measures - No relationship with diabetes mellitus

47 Clinical application of molecular medicine in infection induced hepatitis Case

48 Oxidative Stress and Infectious Diseases

49 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)

50 Cell damages by ROS Aus: “Free Radicals Randox Ltd.

51 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.

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53 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.

54 Case report 10-yr-old male, unrelated healthy parents PH: gestation, birth, somatic and mental development normal normal  CC: chronic active hepatitis, interferon therapy in the department of paediatric hepatology of the department of paediatric hepatology of university children hospital in Germany university children hospital in Germany because of negative 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 The therapy was not tolerated, did not lead to improvement and was discharged after 4 wks. improvement and was discharged after 4 wks.

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

56 Case report Abdomen ultrasound: Mild increased echogenicity of liver paranchyme, otherwise normal findings

57 Normal range  CBC normal  Thromb 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.

58 Normal range Normal range  Selenium 58 µg/l  Beta-Carotin 0,38 mg/l 0,47-4,1  Ery.-Malondialdehyde (MDA) 432 nmol/g Hb  Gluthation-Peroxidase 26,2 U/g Hb Lab

59  Genotype of Hepatitis B-Virus: Point mutation rtA194T Point mutation rtA194T Lab.

60 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

61 Therapy results  significant improvement of physical and mental standards 2 wks. after therapy standards 2 wks. after therapy

62 before Therapy after 4 W 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 rtA 194t detectable Lab. 4 wks. after therapy

63 before therapy after 4 W 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

64 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

65 before after 6 W 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

66 Thank you for your attention

67 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

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69 Requirements for an effective antioxidant therapy Quality of antioxidants (antioxidative capacity) Presence of suitable carriers

70 Requirements for an effective antioxidant therapy Quality of antioxidants (antioxidative capacity)

71 Synergismus of nature 100 gr fresh apple contins approx. 5,7 mg Vit C The antioxidative capacity is equal to 1500 mg synthetic Vit C Nature 2000, 430: 903-4

72 The antioxidative capacity of 100g capacity of 100g apple (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 apples MARIAN V. EBERHARDT 1, CHANG YONG LEE 1 & RUI HAI LIU 1 Department of Food Science, 108 Stocking Hall, Cornell University, Ithaca, New York , USA Nature 405, (June 22 th, 2000)

73 Requirements for an effective antioxidant therapy Presence of suitable carriers

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78 Free radicals and oxidative 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


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