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 Department of Pathology Mansoura University

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

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

5. 12345A1A2A3A4BAD2AD1CD678 FNIII Domains TA EGFL Repeats Alternatively Spliced Domains Fbg 4F10TT 6C4TT4C8MS The structure of human TN-C molecule and the recognition sites of different anti-TN-C antibodies

6. Materials and methods

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

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

9. 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 Materials and methods A) Monoclonal antibodies preparation (4F10TT, 6C4TT & 4C8MS) C) In vitro study: - LI90 human HSC line culture stimulated with PDGF-BB and TGF-B. - Immunoblotting of culture medium & cell lysate to detect TN-C & its splice variants. - RNA isolation, RT-PCR & Real time PCR to quantify TN-C mRNA. - Sequencing of transcripts of TN-C to conform the different splice variants

10. 4F10TT6C4TT4C8MS Immunostaining of liver biopsies of Chronic HCV Patients with different Anti-TN-C Antibodies

11. Normal 4F10TT6C4TT4C8MS Immunostaining of liver biopsies of Chronic HCV Patients with different Anti-TN-C Antibodies

12. Normal Piecemeal necrosis 4F10TT6C4TT4C8MS Immunostaining of liver biopsies of Chronic HCV Patients with different Anti-TN-C Antibodies

13. Normal Piecemeal necrosis Confluent necrosis 4F10TT6C4TT4C8MS Immunostaining of liver biopsies of Chronic HCV Patients with different Anti-TN-C Antibodies

14. Normal Piecemeal necrosis Confluent necrosis 4F10TT6C4TT4C8MS Immunostaining of liver biopsies of Chronic HCV Patients with different Anti-TN-C Antibodies

15. Normal Piecemeal necrosis Confluent necrosis 4F10TT6C4TT4C8MS

16. Normal Piecemeal necrosis Confluent necrosis 4F10TT6C4TT4C8MS

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

20. Medium 250 - KDa Lysate 4F10TT Control PDGF-BB TGF-  Control PDGF-BB TGF-  KDa 6C4TT4C8MS 250 - Lysate Control PDGF-BB 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

22.  - actin TN - C - 344 - 540 Control PDGF-BB TGF-  500 - 1000 - 1500 - - 500 - 1000 - 1600 - 2000 Control PDGF-BB TGF-  * * * * * * bp * * * * * * BamHI-HindIII Total TN-C mRNA increased with PDGF-BB stimulation more than TGF-β or control cells Most of TN-C expressed contains alternatively spliced domains Conventional PCR Real time PCR Control PDGF-BB TGF-  Control PDGF-BB TGF- 

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.