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190 virus infected monocyes

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190 virus infected monocyes

  1. 1. Procoagulant And Inflammatory Response of Virus-Infected Monocyes Provided by: Enrique Gurfinkel , MD, PhD Fundación Favaloro, Capital Federal, Buenos Aires, Argentina Editorial Slides VP Watch – November 13, 2002 - Volume 2, Issue 45
  2. 2. Introduction • Atherosclerosis is thought to be an inflammatory disease, but its origin still remains unanswered. • Chronic low-grade inflammation and combination of classical risk factors, plus novel risk factors such as the infectious burden seem to contribute to promote atherosclerosis.
  3. 3. Introduction • Monocytes play a prominent role in inflammation, coagulation and the ability to produce tissue factor and cytokines. • All of these factors, particularly the production of cytokines may contribute to acute coronary syndromes by eliciting plaque instability.
  4. 4. Background • During viral infections monocytes predominantly produce inflammatory cytokines. • In addition, continuing viral infection may sustain excess tissue factor production and exhaust the inhibitory effects of tissue factor pathway inhibitor.
  5. 5. As highlighted in VP Watch of this week, • Bouwman and co-workers tried to establish whether virus-infected monocytes initiate coagulation. And • To detect if the production of cytokines by monocytes may contribute to the chronic process of atherosclerosis in an in-vitro model.
  6. 6. Methods • Isolation of monocytes: monocytes obtained from fresh blood of healthy volunteers.
  7. 7. Methods • Preparation of virus stocks: Human embryonic lung, human epidermoid larynx carcinoma, and LLC-MK2 cells were cultured in Eagles´s minimum essential medium with Earle´s salts. • A clinical isolete of CMV was propagated in HEL cells • Chlamydia pneumoniae strain AR 39 was propagated in HEP-2 cells • Influenza A HIN1 86 Singapore was propagated in LLC-MK2 cells
  8. 8. Methods • Coagulation assay: Normal pooled plasma was prepared from the blood of nine healthy donors. • Cytokine assays: IL 6, 8, and 10 were measured in the supernatants of virus- infected monocytes.
  9. 9. It appeared that all strains could infect monocytes, but in all cases the infection was below 5% when undiluted virus stocks were used.  Fluorescence microscopy images of virus- infected monocytes. Magnification 200x after overnight incubation; Red background: uninfected monocytes. Green areas: virus-infected monocytes stained with FITC-labeled anti-virus antibodies. (a) CMV, specific staining of CMV in the nuclei of the infected cells. (b) Influenza A, smooth staining pattern of influenza A in monocytes. (c) Cp, more dense staining pattern of Cp in the cytoplasm of monocytes.
  10. 10. 80% of the monocytes were covered with activated CD41+ CD42+ Ficoll + VPT+ VPT+ Depletion Depletion Positive Selection CD41+ CD42+ 46% 78% 3%
  11. 11. CMV and Cp, like influenza, reduced the clotting time. The inititation of coagulation by virus-infected monocyes was a result of the expression of TF. Clottingtime(seconds) Uninfected monocytes Infected monocytes 140+-22.1 s 333+-22.1s
  12. 12. Infection with CMV and Cp induced the production of modest levels of IL6 and IL8, whereas infection with influenza A strongly stimulated the production of IL6 and 8. IL-6IL-8(pgml) CMV & Cp IL 6 1000 IL-8 H1N1 2000
  13. 13. Discussion • The results of the present study indicate that only small quantities of an infectious virus are needed to stimulate monocytes to exert considerable immunological effects.
  14. 14. Conclussions • The procoagulant activity of virus- infected monocytes is TF-dependent. • Influenza infection induced a pronounced expression of IL-6 and IL- 8, which could be associated with plaque rupture.
  15. 15. Questions • Is there any differences between the bacterial and viral infectious burden in promoting procoagulant activity? • Is it plausible to think that this could explain a systemic pro-thrombotic process after infection?
  16. 16. 1. Buja LM. Does atherosclerosis have an infectious etiology? Circulation 1996;94:872. 2. Burch GE, Tsui CY, Harb JM. Pathologic changes of aorta and coronary arteries of mice infected with Coxsackie B virus. Proc Soc Exp Biol Medl 1971;137:657–61. 3. Mosorin et al. Detection of Chlamydia pneumoniae-Reactive T lymphocytes in human atherosclerotic plaques of the carotid artery. Arterioscl Thromb Vasc Biol 2000;20:1061–7. 4. Blankenberg S, Rupprecht HJ, Bickel C, Espinola-Klein C, Rippin G et al. Cytomegalovirus infection with interleukine 6 response predicts cardiac mortality in patients with coronary artery disease. Circulation 2001;103 (24):2915–21. 5. Visseren FLJ, Bouwman JJM, Bouter KP, Diepersloot RJA, de Groot Ph, Erkelens DW. Procoagulant activity of endothelial cells after infection with respiratory viruses. Thromb Haemost 2000;84 (2):319–24. 6. Frostegard J, Ulfgren A, Nyberg P, Hedin U, Swedenborg J et al. Cytokine expression in advanced human atherosclerotic plaques: dominance of pro-inflammatory (Th1) and macrophage stimulating cytokines. Atherosclerosis 1999;145:33–43. 7. Neumann FJ, Ott I, Marx N, Luther T, Kenngott S et al. Effect of human recombinant Interleukin-6 and Interleukin-8 on Monocyte Procoagulant activity. Arteriosclerosis Thromb Vasc Biol 1997;17:3399–405. 8. Bouwman J. J. M. et al,. Eur J Clin Invest 2002; 32 (10): 759-766 References

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