Foreign-infection associated Bacterial, Viral, Fungal origin

1. PLASMA NUCLEIC ACID CLEARANCE- A NOVEL LINK BETWEEN OPTIMAL INSULIN THERAPY, GOOD GLYCAEMIC CONTROL AND IMMUNOREGULATION G. Kocic, G. Bjelakovic, Lj. Saranac,R. Kocic, T. Jevtovic, D. Sokolovic, S. Stojanovic, D. Pavlovic Institute of Biochemistry and Clinic for Endocrinology Medical Faculty Nis, Serbia

3. Nucleic acids in blood Foreign-infection associated Bacterial, Viral, Fungal origin Foreign-food (meat) Endogenously generated (cell turnover, apoptosis, necrosis)

4. A key attribute to an effective immune system is the sensing of foreign nucleic acids DNA ssRNA dsRNA CpG

5. CpG APC stimulation through TLR produces pro-inflammatory cytokines and chemokines, induction of co-stimulatory molecules that are critical for adaptive immune responses and pro-inflammatory cytokine secretion

6. ssRNA

7. Toll-like receptor localisation • Cells of the non-specific immune response (macrophagal, dendritic) • Cells of specific immune response (T,B) • Non-immune cells (endothelial and epithelial origin)

9. Cell damage Normal cell turnover scavenger receptors

10. The presence of detectable levels of RNA in the blood of individuals who have active, chronic forms of some autoimmune diseases may be recognized as a potentially valuable marker for development of autoimmune disease.

11. A possible importance of circulating RNases in this way would be to protect against infectious agents by destroying unprotected or transiently revealed nucleic acids and oligonucleotides in the blood stream and cell entry. In this way a little if any self ssRNAs ever reach the antigen presenting cells

12. RNase -free -inhibitor bound RNases 1-5 RNases 1-4 preffer poly(C) RNase 5 preffers poly (U) “pancreatic type (pt)”, “non-pancreatic type (npt)” “pt/npt” ribonucleases. Peripheral tissues

13. AIM AIM • This study will be undertaken in order to examine: • i) plasma nuclease activity of diabetic patients in relation to the different substrates-TLR ligands; • ii) free and inhibitory-bound nuclease activity of diabetic patients in relation to the different types of diabetes or onset age; • iii) nuclease activity in newly-diagnosed insulin-dependent patients in relation to metabolic regulation; • v) in vitro time and dose dependent effect of hyperglycaemia on nucleases glycation; • vi) ribonucleic acids quantitative concentration and qualitative fashion in diabetic populations, compared to corresponding healthy population; • vii) in vitro effect of plasma oligonuclotides, purified from diabetic patients and healthy persons, on immune cell proliferation in culture, compared to commercial oligonucleotides and standard immune stimulators (Concavaline A)

14. Physical characteristics and fasting metabolic profile

15. U/l        *** *** *** *  *** * *** *** *** *** *** *** poly(C) rRNA poly(U) *** p<0.001 compared to the corresponding control * p<0.05 compared to the corresponding control Legend: Control children Juvenile diabetetic children Adult controls p<0.05 compared between controls IDDM patients (onset before yr 14 )  p<0.001 compared to the juvenile diabetics IDDM patients (onset after yr 14) NIDDM patients

16. U/l   U/l       *** ***    *** *** ***   *** * * *** ***  *** *** *** *** *** *** *** *** *** *** * *  * *** rRNA poly(C) poly(U) poly (I:C) poly (A:U) CpG Legend: *** p<0.001 compared to the corresponding control * p<0.05 compared to the corresponding control Control children Juvenile diabetetic children Adult controls p<0.05 compared between controls IDDM patients (onset before yr 14 )  p<0.001 compared to the juvenile diabetics IDDM patients (onset after yr 14) NIDDM patients

18. 1hour 24hours Fig.4a In vitro effect of glucose on plasma RNase activity after different time intervals (1h and 24h) of incubation

19. 1hour 24hours Fig.4b In vitro effect of glucose on commercial RNase A activity after different time intervals (1h and 24h) of incubation

20. The quantity of purified RNA oligonucleotides in plasma ***    * * * Control adults IDDM adults <14yr >14yr NIDDM Control Juvenile Children IDDM

21. Diabetic values Maximal control value 270 Figure 5a. Spectrophotometric scan analysis of purified RNA and oligonucleotides from plasma of control subjects and diabetic patients

22. 270 Figure 5b. Spectrophotometric scan analysis of different standards

23. 270 Figure 5c. Maximal absorbance of control and diabetic samples corresponds only to polyC homopolynucleotide maximal absorbance

24. D1 C1 C2 C3 C4 C5a D2 C5 C1a C2a C3a C4a D1a D3 RNA lader2 D2a D2aII D1aII D3a D6 D4 D8 D7 D9 D8 D5 RNA lader1 PolyC DNA

25. Flow cytometric analysis of lymphocyte proliferation by measuring the expression of proliferating cell nuclear antigen (PCNA), an auxiliary cyclin protein necessary for DNA polymerase, maximally expressed in mid S-phase . Proliferative activity of thymocytes was evaluated after 72 hours incubation, by using anti-PCNA monoclonal antibody, according to the manufacturer instructions Juvenile diabetic patients ConA Control children

26. NIDDM IDDM Control adult ConA

27. ssRNA dsRNA PolyI:C polyA:U polyA polyC polyU

28. The level of NOx and NTY P<0.001 P<0.05 NOx mol/l NTY mol/g prot Control NOx Control NTY Dabetes NTY Diabetes NOx

29. Altered Th1-Th2 balance • Promotion of Th1 response Loss of INF feed back regulation (growth factor deprivation Oxidative stress proinflammatory cytokines secretion IFN secretion) Virus Toxins Immunomodulatory actions MHC expression Cell apoptosis Acummulation of different-sized oligonucleotide fragments (acid-soluble nucleotides) Cell source Native or partially destroyed nucleic acids and olygonucleotides Blood RNase Deffective RNase activity Viral source Dietary source ?

30. Toll-like Receptors Toll-like Receptors RNase ssRNA dsRNA oligonucleotides

31. Activation of cells through TLRs by different oligonucleotides induces APCs to present antigens to T cells, MHC class I and class II expression, followed by the activation of CD8+ T cells, which can induce damage of the pancreatic islets. It leads to local inflammation, followed by the production of pro-inflammatory cytokines (IL-1ß and IFNs) and eventual islet invasion by mononuclear cells expressing FasL. The observed preliminary results may suggest a hypothesis that persisting increase of oligonucleotide fragments, most probably due to the insufficient RNase activity, may lead to T-cell hyperactivity in diabetes and higher susceptibility toward infections β-cells

32. At the same time most human diets are rich in nucleic acids. But the observed results may provide resonable explanations for the virtual starvation diets or strict meat diets which had been previously beleived as the oldest therapy for diabetes introduced many centuries ago, before insulin was widely available. It is in fact, nucleotide-free formula diet. The association was observed between the development of diabetes and meat consumption. If the trensfection of nucleic acids into cells can cause such marked results it is somewhat suprising that these results have not been observed previously, ant it is important that they be considered in the future ( Suzuki et al. 1999)

33. Thank you for your attention!