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Dr.Amro El-karef Assistant Professor of Pathology Department of Pathology Mansoura University

Expression of Large Tenascin -C Splice Variants by Hepatic Stellate Cells in Chronic Hepatitis C. Dr.Amro El-karef Assistant Professor of Pathology Department of Pathology Mansoura University. Introduction. * Chronic Hepatitis C is a worldwide liver

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Dr.Amro El-karef Assistant Professor of Pathology Department of Pathology Mansoura University

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  1. Expression of Large Tenascin-C Splice Variants by Hepatic Stellate Cells in Chronic Hepatitis C Dr.Amro El-karef Assistant Professor of Pathology Department of Pathology Mansoura University

  2. Introduction * Chronic Hepatitis C is a worldwide liver disease that causes liver fibrosis, cirrhosis & hepatocellular carcinoma. (Lauer 2001) * Tenascin-C (TN-C), a hexameric ECM glyco- protein, is a part of provisional matrix that modulates cellular functions during tissue remodleing & cancer invasion. (Chiquet-Ehrismann 1993) * TN-C has different isoforms due to alternative splicing of its mRNA. (Jones 2001) * Serum levels of large TN-C was found to correlate well with the grades of piecmeal necrosis. (Tanaka 2005 – in press)

  3. The structure of human TN-C molecule and the recognition sites of different anti-TN-C antibodies

  4. The structure of human TN-C molecule and the recognition sites of different anti-TN-C antibodies

  5. The structure of human TN-C molecule and the recognition sites of different anti-TN-C antibodies FNIII Domains TAEGFL RepeatsAlternatively Spliced Domains Fbg 6C4TT 4C8MS 4F10TT

  6. Materials and methods

  7. Materials and methods A) Monoclonal antibodies preparation (4F10TT, 6C4TT & 4C8MS)

  8. Materials and methods A) Monoclonal antibodies preparation (4F10TT, 6C4TT & 4C8MS) B) In vivo study: liver biopsies of chronic HCV patients - H&E & Sirus red To diagnose & grade the activity (Ishak’s HAI) - Immunohistochemistry * TN-C * α-SMA - Combined IHC & In situ hybridization α-SMA & human TN-C mRNA probes

  9. Materials and methods A) Monoclonal antibodies preparation (4F10TT, 6C4TT & 4C8MS) C) In vitro study: - LI90 human HSC line culturestimulated with PDGF-BB and TGF-B. - Immunoblottingof culture medium & cell lysate to detect TN-C & its splice variants. - RNA isolation, RT-PCR & Real time PCRto quantify TN-C mRNA. - Sequencing of transcripts of TN-C to conform the different splice variants B) In vivo study: liver biopsies of chronic HCV patients - H&E & Sirus red To diagnose & grade the activity (Ishak’s HAI) - Immunohistochemistry * TN-C * α-SMA - Combined IHC & In situ hybridization α-SMA & human TN-C mRNA probes

  10. Immunostaining of liver biopsies of Chronic HCV Patients with different Anti-TN-C Antibodies Immunostaining of liver biopsies of Chronic HCV Patients with different Anti-TN-C Antibodies 4F10TT 6C4TT 4C8MS

  11. Immunostaining of liver biopsies of Chronic HCV Patients with different Anti-TN-C Antibodies Immunostaining of liver biopsies of Chronic HCV Patients with different Anti-TN-C Antibodies 4F10TT 6C4TT 4C8MS Normal

  12. Immunostaining of liver biopsies of Chronic HCV Patients with different Anti-TN-C Antibodies Immunostaining of liver biopsies of Chronic HCV Patients with different Anti-TN-C Antibodies 4F10TT 6C4TT 4C8MS Normal Piecemeal necrosis

  13. Immunostaining of liver biopsies of Chronic HCV Patients with different Anti-TN-C Antibodies Immunostaining of liver biopsies of Chronic HCV Patients with different Anti-TN-C Antibodies 4F10TT 6C4TT 4C8MS Normal Piecemeal necrosis Confluent necrosis

  14. Immunostaining of liver biopsies of Chronic HCV Patients with different Anti-TN-C Antibodies Immunostaining of liver biopsies of Chronic HCV Patients with different Anti-TN-C Antibodies 4F10TT 6C4TT 4C8MS Normal Piecemeal necrosis Confluent necrosis

  15. Immunostaining of liver biopsies of Chronic HCV Patients with different Anti-TN-C Antibodies 4F10TT 6C4TT 4C8MS Normal Piecemeal necrosis Confluent necrosis

  16. Immunostaining of liver biopsies of Chronic HCV Patients with different Anti-TN-C Antibodies 4F10TT 6C4TT 4C8MS Normal Piecemeal necrosis Confluent necrosis

  17. TN-C expression Vs Activity Grades and Fibrosis Stage

  18. HSCs are major sources of TN-C in the liver TN-C α-SMA α-SMA + TN-C α-SMA + TN-C mRNA

  19. TN-C expression Vs HSC Count

  20. Control Control Control PDGF-BB PDGF-BB PDGF-BB Immunoblotting of TN-C Protein and its Large Variants from LI90 HSC after Induction with PDGF-BB Medium Lysate Lysate 4F10TT 4F10TT 6C4TT 4C8MS KDa KDa 250 - 250 - TGF-b TGF-b Control PDGF-BB PDGF-BB stimulated LI90 HSC to produce small and large TN-C variants more than TGF-β compared to control Most of large TN-C variants expressed by PDGF-BB contained A1/A4 and B domains as detected by 6C4 and 4C8 respectively

  21. mRNA levels of Total TN-C and its large variants in LI90 HSC Line

  22. mRNA levels of Total TN-C and its large variants in LI90 HSC Line Conventional PCR Real time PCR Total TN-C mRNA increased with PDGF-BB stimulation more than TGF-β or control cells TN-C - 344 - 540 b-actin TGF-b Control PDGF-BB TGF-b Control PDGF-BB bp - 2000 - 1600 * * 1500 - * * Most of TN-C expressed contains alternatively spliced domains * * - 1000 1000 - * * * * - 500 500 - * * TGF-b BamHI-HindIII Control PDGF-BB TGF-b Control PDGF-BB

  23. TN-C variants produced by LI90 HSC

  24. Conclusion 1- The previously characterized TN-C is only the small variants. 2- TN-C is markedly upregulated in active liver lesions especially the large variants. 3- Large TN-C variants is strongly well correlated with piecemeal and confluent necrosis, the most reliable prognostic lesions of chronic hepatitis. 4- Hepatic stellate cells are major sources of large TN-C variants after their activation by growth factors.

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