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Background: Cyclooxygenase 2 (COX-2) and matrix metalloproteinases (MMPs) have been implicated in tissue injury and fibrogenesis in animal models but little is known regarding their role in hepatitis C virus (HCV) related liver disease in humans. Aims: To characterise the intrahepatic expression pattern of COX-2 and MMPs in chronic HCV infection and determine whether HCV core and NS5A proteins could promote their expression in cultured hepatocyte derived cell lines. Patients: Thirty two anti-HCV+ and 10 anti-HCV2 patients were studied. Methods: Western blot, reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay, and immunohistochemistry were used to assess the expression pattern of COX-2 and MMPs in liver biopsy samples from all patients. COX-2 gene expression and MMP-9 protein levels were also determined by immunoblot, RT-PCR, and luciferase assays in core and NS5A transfected hepatocyte derived cells. Results: The intrahepatic expression level of COX-2, MMP-2, and MMP-9 was significantly higher in HCV+ than in HCV2 patients, increasing with the fibrotic stage of liver disease. We further demonstrated that COX-2 mRNA, protein, and activity were induced in resting and activated core and NS5A transfectants. Both viral proteins induced transcriptional activity of the COX-2 gene promoter whereas core, but not NS5A, exerted an inducer effect on MMP-9 protein levels in cultured hepatocyte derived cells. Conclusions: Intrahepatic COX-2, MMP-2, and MMP-9 overexpression is associated with progressive hepatic fibrosis in chronic HCV infection, suggesting their pathogenic role in fibrogenesis. HCV core and NS5A proteins were able to upregulate COX-2 and MMP-9 gene expression in hepatocyte derived cells, providing a potential mechanism for hepatic fibrosis during chronic HCV infection.

SUMMARYAims: To determine the serum and intrahepatic levels of T-helper-1-associated chemokines in patients with chronic hepatitis C before, during and after peginterferon plus ribavirin combination therapy and to search for correlations with baseline characteristics of hepatitis C virus-related chronic liver disease and type of therapeutic response. Methods: Serum chemokine levels were determined by enzyme-linked immunosorbent assays and intrahepatic chemokine messenger RNA and protein levels were tested by ribonuclease protection assay and immunohistochemistry. Results: Serum and intrahepatic chemokine levels were elevated in all patients with chronic hepatitis C and showed a marked decrease in patients who obtained a virological response vs. non-responders. Increased serum interferon-c-inducible protein-10 levels at baseline in genotype 1-infected patients were significantly associated with greater degrees of intrahepatic inflammation and fibrosis (P ¼ 0.0046 and P ¼ 0.02, respectively) and with virological non-response (P ¼ 0.01). In patients with genotype 1, basal serum interferon-c-inducible protein-10 levels greater than 299 pg/mL identified 80% of non-responders and lower than 299 pg/mL identified 63% of responders. Conclusions: Circulating and intrahepatic T-helper-1-associated chemokines are abnormally elevated in patients with chronic hepatitis C. Increased baseline serum interferon-c-inducible protein-10 levels in genotype 1-infected patients are associated with virological non-response to peginterferon plus ribavirin combination therapy.

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Increased levels of chemokines (CK) in chronic hepatitis C virus (HCV) infection have been found. Given that NS5A and core can function as transcriptional transactivators, we aimed to determine whether these HCV proteins might induce CK gene expression in human hepatocyte-derived cells. We assessed (i) regulated upon activation, normal T cells expressed and activated (RANTES), interferon gamma-inducible protein-10 (IP-10), and monokine induced by interferon-gamma (MIG) mRNA levels in NS5A and core stably transfected Chang liver (CHL) cells, stimulated or not with a cytokine mixture (CM), by reverse transcriptase-polymerase chain reaction (RT-PCR) and (ii) quantitative enzyme-linked immunosorbent assay (ELISA) measurements of these CK in the supernatants of CHL cells. Induction of RANTES transcripts in resting HCV-transfected cells was clearly observed, being augmented fourfold in resting NS5A-transfected cells and threefold in resting core-transfected cells over that in resting mock-transfected (control) cells, as well as to a similar extent in CM-stimulated NS5A- and core-transfected cells. Increased RANTES secretion followed the same pattern observed for mRNA expression. Both IP-10 and MIG, such as mRNA and protein levels, were undetectable in resting HCV-transfected and -untransfected cells, whereas IP-10 and MIG mRNA expression was increased by seven- and fivefold in CM-stimulated NS5A-transfected cells and by 10- and 3.5-fold in CM-stimulated core-transfected cells, respectively, above that in CM-stimulated control cells. IP-10 and MIG secretion was enhanced by 2.6- and threefold in CM-stimulated NS5A-transfected cells and by 3.6-fold and 3.7-fold in CM-stimulated core-transfected cells, respectively over that in CM-stimulated control cells. These results demonstrate that NS5A and core proteins, alone or by the synergistic effect of cytokines, are capable of upregulating RANTES, IP-10 and MIG gene expression in cultured human hepatocyte-derived cells.

Intrahepatic chemokine signaling could play a key role regulating significant pathological events during chronic hepatitis C, opening new avenues for therapeutic interventions based on chemokine activities.

Nitric oxide appears to play a central role in the pathogenesis of many inflammatory disorders. We have previously shown that there is an enhanced intrahepatic expression of the inducible nitric oxide synthase (iNOS) gene during chronic hepatitis B virus infection, and that viral X protein (HBx) transcriptionally activates this cellular gene, but the molecular basis for this activation remains to be defined. We aimed to explore the involvement of different cis-acting elements of the human iNOS promoter in the HBx- Nitric oxide (NO) is a highly reactive gaseous molecule that plays an important role in the nonspecific immune response to infection, acting as antimicrobial. 1 However, a long-term production of large amounts of NO due to overexpression of the inducible nitric oxide synthase (iNOS) gene can have detrimental consequences, such as cytotoxicity and carcinogenesis. 2,3 Further supporting this assumption, we have recently shown that the expression of iNOS was markedly enhanced by hepatitis B virus (HBV) infection in hepatocytes from patients with chronic hepatitis B (CHB) 4 and, as expected, an intrahepatic accumulation of NO-derived reactive species closely associated with histologic severity of liver disease was also observed in patients with chronic HBV infection, 5 suggesting that NO could mediate important pathogenic events in the course of HBV infection.Cloning and functional analysis of the human iNOS gene promoter identified 2 copies of nuclear factor B (NF-B) response elements, one located Ϫ102 base pairs proximal to the transcription initiation site and the other as distal as Ϫ8,270 base pairs upstream. Furthermore, 2 copies of activator protein 1 (AP-1) binding sites have also been found. 6 Although it has been suggested that HBV X protein (HBx) transactivates the iNOS gene promoter through the induction of NF-B, 7 the mechanism of this activity remains only partially understood. It is conceivable, therefore, that understanding how HBx activates the iNOS gene transcription can provide insights into the pathogenic role of the enhanced intrahepatic iNOS expression that we observed in patients with CHB. 4,5 The HBV genome encodes a small protein known as HBx, 8 which is essential for viral infectivity 9 and is a potential cofactor in HBV-mediated carcinogenesis. 10,11 In addition, it has been shown that HBx behaves as a transcriptional transactivator of a number of viral and cellular gene promoters through direct interaction with transcriptional factors, such as the RPB5 subunit of RNA polymerase II, 12 TATA-binding protein 13 and ATF/CREB, 14 and activation of signal transduction pathways, such as the Ras/Raf/MAP kinase cascade. 15 In this regard, the role of the intracellular distribution of HBx still remains controversial, but data from Doria et al. 16 suggest that HBx could have a dual function on transcription machinery, acting on signal transduction pathways in the cytoplasm and transcription factors in the nucleus. The same investigators have shown that the cytoplasmic distribution of HBx wa...