AM3, a natural glycoconjugate, induces the functional maturation of human dendritic cells.

BACKGROUND AND PURPOSE: Dendritic cells (DCs) are dedicated antigen-presenting cells able to initiate specific immune responses and their maturation is critical for the induction of antigen-specific T-lymphocyte responses. Here, we have investigated the effects of Inmunoferon-active principle (AM3), the active agent of a commercial immunomodulatory drug, on human monocyte-derived DCs (MDDCs). EXPERIMENTAL APPROACH: MDDCs derived from healthy and hepatitis C virus (HCV)-infected patients were stimulated with AM3. We analysed the expression of cell surface proteins by flow cytometry, that of cytokine production by ELISA, and the expression of chemokines and chemokine receptors by RNase protection assays. T-lymphocyte proliferation was assessed in mixed lymphocyte reactions, protein expression by western blot and luciferase-based reporter methods, and Toll-like receptor (TLR)-blocking antibodies were employed to analyse TLR activity. KEY RESULTS: In MDDCs, AM3 induced or enhanced expression of CD54, CD83, CD86, HLA-DR, chemokines and chemokine receptors, interleukin (IL)-12p70 and IL-10. Furthermore, AM3 stimulated MDDCs to increase proliferation of allogenic T cells. AM3 triggered nuclear translocation of NF-kappaB and phosphorylation of p38 mitogen-activated protein kinase. AM3 promoted NF-kappaB activation in a TLR-4-dependent manner, and blocking TLR-4 activity attenuated the enhanced expression of CD80, CD83 and CD86 induced by AM3. AM3 enhanced the expression of maturation-associated markers in MDDCs from HCV-infected patients and increased the proliferation of T lymphocytes induced by these MDDCs. CONCLUSIONS AND IMPLICATIONS: These results underline the effects of AM3 in promoting maturation of MDDCs and suggest that AM3 might be useful in regulating immune responses in pathophysiological situations requiring DC maturation.

Polypodium leucotomos extract: antioxidant activity and disposition.

The extract of the fern Polypodium leucotomos (PL, Fernblock) is an oral photoprotectant with strong antioxidative properties. Recent studies to determine its chemical composition have shown 4-hydroxycinnamic acid (p-coumaric), 3 methoxy-4-hydroxycinnamic acid (ferulic), 3,4-dihydroxycinnamic acid (caffeic), 3-methoxy-4-hydroxybenzoic acid (vanillic) and 3-caffeoilquinic acid (chlorogenic) to be among its major phenolic components. No conclusive data are available, however, on the H2O2-scavenging capacity of these compounds, or on their absorption and metabolism following their oral intake. In the present work, their antioxidative capacity was assessed by the luminol/H2O2 assay, their absorption studied using Caco-2 cells to resemble the intestinal barrier, and their metabolism investigated using cultured primary rat hepatocytes. The antioxidant capacity of PL components increased in a concentration-dependent manner, with ferulic and caffeic acids the most powerful antioxidants. The apparent permeability results correspond to a human post-oral administration absorption of 70-100% for all tested substances. Coumaric, ferulic and vanillic acids were metabolized by CYP450-dependent mono-oxygenases and partially conjugated to glucuronic acid and sulfate. These phenolic compounds may contribute to the health benefits afforded by this oral photoprotectant.

Gene expression profile of T-cell-specific chemokines in human hepatocyte-derived cells: evidence for a synergistic inducer effect of cytokines and hepatitis C virus proteins.

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.

Increased intrahepatic cyclooxygenase 2, matrix metalloproteinase 2, and matrix metalloproteinase 9 expression is associated with progressive liver disease in chronic hepatitis C virus infection: role of viral core and NS5A proteins.

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-HCV- 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 HCV- 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.

Increased circulating and intrahepatic T-cell-specific chemokines in chronic hepatitis C: relationship with the type of virological response to peginterferon plus ribavirin combination therapy.

AIMS: 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-gamma-inducible protein-10 levels at baseline in genotype 1-infected patients were significantly associated with greater degrees of intrahepatic inflammation and fibrosis (P = 0.0046 and P = 0.02, respectively) and with virological non-response (P = 0.01). In patients with genotype 1, basal serum interferon-gamma-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-gamma-inducible protein-10 levels in genotype 1-infected patients are associated with virological non-response to peginterferon plus ribavirin combination therapy.

Hepatitis B virus X protein transactivates inducible nitric oxide synthase gene promoter through the proximal nuclear factor kappaB-binding site: evidence that cytoplasmic location of X protein is essential for gene transactivation.

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-mediated up-regulation as well as the effect of the intracellular distribution of the HBx on its transacting function. Cotransfection of human hepatocyte-derived cells with wild-type or mutated iNOS promoter and with an HBx expression vector showed that functional inactivation of the proximal nuclear factor kappaB (NF-kappaB)-binding site of the iNOS promoter markedly reduced the HBx-mediated transcriptional activity. Mobility shift assays showed increased DNA-protein complexes comprising mainly the p50 and p65 members of NF-kappaB family in the nuclear extracts from HBx-transfected cells. Transient transfection experiments with HBx-expressing plasmids containing distinct cellular localization sequences showed that cytoplasmic location of this viral protein activated the iNOS promoter but when HBx was targeted to the nucleus, the HBx-induced luciferase activity was almost completely abrogated. In conclusion, cytoplasmic location of HBx protein is essential for the transcriptional activation of the iNOS gene through the nuclear translocation of p50-p65 heterodimers.

S-Adenosylmethionine modulates inducible nitric oxide synthase gene expression in rat liver and isolated hepatocytes.

BACKGROUND/AIMS: Hepatocellular availability of S-adenosylmethionine, the principal biological methyl donor, is compromised in situations of liver damage. S-Adenosylmethionine administration alleviates experimental liver injury and increases survival in cirrhotic patients. The mechanisms behind these beneficial effects of S-adenosylmethionine are not completely known. An inflammatory component is common to many of the pathological conditions in which S-adenosylmethionine grants protection to the liver. This notion led us to study the effect of S-adenosylmethionine administration on hepatic nitric oxide synthase-2 induction in response to bacterial lipopolysaccharide and proinflammatory cytokines. METHODS: The effect of S-adenosylmethionine on nitric oxide synthase-2 expression was assessed in rats challenged with bacterial lipopolysaccharide and in isolated rat hepatocytes treated with proinflammatory cytokines. Interactions between S-adenosylmethionine and cytokines on nuclear factor kappa B activation and nitric oxide synthase-2 promoter transactivation were studied in isolated rat hepatocytes and HepG2 cells, respectively. RESULTS: S-Adenosylmethionine attenuated the induction of nitric oxide synthase-2 in the liver of lipopolysaccharide-treated rats and in cytokine-treated hepatocytes. S-Adenosylmethionine accelerated the resynthesis of inhibitor kappa B alpha, blunted the activation of nuclear factor kappa B and reduced the transactivation of nitric oxide synthase-2 promoter. CONCLUSIONS: Our findings indicate that the hepatoprotective actions of S-adenosylmethionine may be mediated in part through the modulation of nitric oxide production.

Nuclear factor-kappa B in the liver of patients with chronic hepatitis C: decreased RelA expression is associated with enhanced fibrosis progression.

The mechanisms of liver damage in chronic hepatitis C virus (HCV) infection are poorly understood. The transcription factor, nuclear factor-kappa B (NF-kappa B), regulates the expression of genes involved in apoptosis, inflammation, and antiviral response. It plays a protective role in several forms of liver damage. In this study, we analyzed NF-kappa B by gel mobility shift assay and immunohistochemistry in liver biopsies from HCV-infected patients, and we have determined the hepatic levels of the components of the NF-kappa B system by semiquantitative polymerase chain reaction (PCR). We found that NF-kappa B was activated in the liver of patients with chronic hepatitis C. Neither NF-kappa B activity nor the RNA levels of NF-kappa B subunits showed correlation with liver inflammatory activity, viral load, or HCV genotype. By contrast, hepatic mRNA values of RelA, the main element of active NF-kappa B, correlated inversely with apoptosis (r = -.68; P <.05) and with the rate of fibrosis progression (r = -.51; P <.04). In intermediate/rapid fibrosers, RelA mRNA levels were significantly decreased as compared with slow fibrosers (P <.003) and with normal livers (P <.03). In conclusion, we found that NF-kappa B is activated in chronic HCV-infected livers, and that the expression of RelA is inversely correlated with liver cell apoptosis and with the rate of fibrosis progression. Our data thus suggest that RelA expression may protect against liver fibrosis and hepatocellular damage.

Effect of the hepatitis B virus HBx protein on integrin-mediated adhesion to and migration on extracellular matrix.

BACKGROUND/AIMS: The hepatitis B virus HBx protein is associated with the development of hepatocellular carcinoma (HCC). However, its possible contribution to tumor spreading has not been explored. The migration of tumor cells through the extracellular matrix (ECM) represents a crucial step in tumor metastasis. Our aim was to study the effect of HBx on the integrin-mediated cell-ECM interaction, and its possible consequences for cell migration. METHODS: Cell-ECM interaction was evaluated by static adhesion experiments, using blocking and stimulating anti-beta1 integrin mAbs. ECM receptor expression was analyzed by flow cytometry. The cellular distribution of the activated beta1 integrin subunit was determined by immunofluorescence analysis, and cell motility was determined by wound-healing assays. RESULTS: HBx-bearing cells showed decreased adhesion to fibronectin, which correlated with a decreased expression of the alpha5 integrin subunit. The activated beta1 subunit was redistributed to the tips of pseudopodial protrusions of HBx-bearing cells, whereas it was evenly localized in the control cells. HBx-induced cell migration was abrogated by irreversible stimulation of beta1 integrins. CONCLUSIONS: These results suggest that HBx might play a role in tumor spreading by modulating the adhesion-deadhesion balance of the cells in the primary tumor site and favoring integrin-mediated cell migration.

Intrahepatic expression of inducible nitric oxide synthase in acute liver allograft rejection: evidence of modulation by corticosteroids.

Nitric oxide (NO) has been proposed to have an important role in the immune response. Plasma nitrate levels increase during acute rejection and decrease after treatment with corticosteroids, but little is known about its potential cellular source. We studied inducible NO synthase (iNOS) expression in liver biopsy specimens of 12 patients with acute rejection compared with biopsy specimens from the same patients after treatment with high doses of intravenous corticosteroids. We also compared iNOS expression during acute rejection with a control group (9 patients without histological rejection). iNOS expression was assessed by immunohistochemistry. Intrahepatic iNOS expression was only observed in the cytoplasm of hepatocytes, which were diffusely distributed throughout hepatic lobules. iNOS expression could not be shown in portal tracts, inflammatory cells, or endothelial and sinusoidal lining cells. In patients with acute rejection, iNOS expression was significantly stronger than in the control group (2 +/- 0.7 v 0.6 +/- 0.7; P <.05). After treatment with corticosteroids, iNOS expression decreased significantly (2 +/- 0.7 v 1.3 +/- 0.9; P <.05). In conclusion, the findings of the present study show that during acute liver rejection, hepatocytes are the main cellular source for NO production and treatment with corticosteroids induces significant downregulation of intrahepatic iNOS expression.

Characterization of pathogenic and prognostic factors of nonalcoholic steatohepatitis associated with obesity.

BACKGROUND/AIMS: Nonalcoholic steatohepatitis is an emerging clinical problem among the obese population. However, risk factors of progression to advanced forms of liver disease in this particular group of patients remain to be defined. METHODS: The demographics and clinical and histologic features of 46 obese patients were evaluated. The intrahepatic immunological phenotype was assessed in all liver biopsy samples by immunohistochemistry. RESULTS: Histologic findings of nonalcoholic steatohepatitis were observed in 69.5% of the obese population studied and significant fibrosis was evident in 41% of patients with nonalcoholic steatohepatitis. Age (p=0.003), degree of steatosis (p=0.000002), and grade of inflammation (p=0000) at liver biopsy were independent variables positively associated with fibrosis. Intrahepatic expression levels of several immunologic markers of inflammation as well as nitric oxide derivatives were significantly higher in the severe forms of nonalcoholic steatohepatitis than in the mildest forms. CONCLUSIONS: Obese persons with higher age, with greater degrees of hepatic steatosis, and specially those with increased grades of intrahepatic inflammation have the greatest risk for progression to fibrotic liver disease. An oxidative stress-triggered intrahepatic inflammatory response appears to be important in the pathogenesis of nonalcoholic steatohepatitis in obesity.

Intrahepatic accumulation of nitrotyrosine in chronic viral hepatitis is associated with histological severity of liver disease.

BACKGROUND/AIMS: The toxicity of nitric oxide is thought to be engendered, at least in part, by its reaction with superoxide yielding peroxynitrite, a potent oxidant that promotes the formation of nitrotyrosine within cells and tissue lesions. In this study we assessed the intrahepatic localization and distribution of the inducible nitric oxide synthase (iNOS) and nitrotyrosine (NTY) in patients with viral and non-viral liver disease. METHODS: We carried out single and double immunostaining experiments on cryostat liver biopsy sections using monoclonal antibodies against iNOS and NTY. We also performed a comparative analysis between the intrahepatic immunostaining score of NTY and the histological activity index of chronic viral hepatitis. RESULTS: We found a marked hepatocellular expression of iNOS with a diffuse lobular pattern in all liver samples from patients with viral liver disease, whereas NTY localization was mainly restricted to cellular foci consisting of hepatocytes and Kupffer cells. Interestingly, we demonstrated by means of double immunostaining experiments the existence of hepatocellular co-localization of iNOS and NTY in the majority of NTY-expressing liver cells. The amount of NTY was significantly higher in liver biopsies from viral liver disease than in non-viral liver disease. In addition, a statistically significant association between the intrahepatic amount of NTY and the severity of viral liver disease was found. CONCLUSIONS: Nitric oxide-mediated nitration of hepatocellular proteins is markedly induced in the inflamed liver tissue from patients with chronic viral hepatitis, and appears to be associated with the histological severity of viral chronic liver disease.

The hepatitis B virus X protein activates nuclear factor of activated T cells (NF-AT) by a cyclosporin A-sensitive pathway.

The X gene product of the human hepatitis B virus (HBx) is a transcriptional activator of various viral and cellular genes. We recently have determined that the production of tumor necrosis factor-alpha (TNF-alpha) by HBV-infected hepatocytes is transcriptionally up-regulated by HBx, involving nuclear factor of activated T cells (NF-AT)-dependent activation of the TNF-alpha gene promoter. Here we show that HBx activates NF-AT by a cyclosporin A-sensitive mechanism involving dephosphorylation and nuclear translocation of the transcription factor. Luciferase gene expression assays demonstrated that HBx transactivates transcription through NF-AT-binding sites and activates a Gal4-NF-AT chimeric protein. DNA-protein interaction assays revealed that HBx induces the formation of NF-AT-containing DNA-binding complexes. Immunofluorescence analysis demonstrated that HBx induces the nuclear translocation of NF-AT, which can be blocked by the immunosuppressive drug cyclosporin A. Furthermore, immunoblot analysis showed that the HBx-induced activation and translocation of NF-AT are associated with its dephosphorylation. Thus, HBx may play a relevant role in the intrahepatic inflammatory processes by inducing locally the expression of cytokines that are regulated by NF-AT.

The hepatitis B virus X protein up-regulates tumor necrosis factor alpha gene expression in hepatocytes.

Human hepatocytes infected by hepatitis B virus (HBV) produce the proinflammatory cytokine, tumor necrosis factor (TNF-). In this study, we explored the mechanism of induction of TNF- synthesis by HBV. We found that the stable HBV-transfected hepatoma cell line, 2. 2.15, expressed high-molecular-weight (HMW) TNF- mRNAs, which were absent in the parent HepG2 cells. Treatment of 2.2.15 cells with interferon alfa (IFN-) and/or interleukin-1beta (IL-1beta) reduced both viral gene transcription and TNF- mRNA expression. Transient or stable transfection of hepatocyte-derived cell lines with HBV X protein (HBx) expression vectors induced the production of biologically active TNF-. In these cells, the HBx-induced TNF- was detected both as cell-associated and soluble forms. Luciferase gene-expression assays showed that the TNF- gene promoter contained target sequences for HBx trans-activation within the proximal region of the promoter. These results indicate that the hepatocyte TNF- synthesis induced by HBV is transcriptionally up-regulated by HBx. Thus, HBx may have a role in the induction of the intrahepatic inflammatory processes that take place during acute and chronic hepatitis B.

Intrahepatic enhanced expression of beta2-microglobulin conformational epitope in acute liver allograft rejection: evidence of modulation by glucocorticoids.

The mechanisms by which glucocorticoids are effective in acute liver rejection therapy are not entirely clear. The aims of this study were to characterize the intrahepatic immunological phenotype in acute liver rejection, as well as the effect of glucocorticoids on cytokine-stimulated hepatocyte cell lines. Biopsy sections from these patients were studied by immunohistochemistry. Cytokine-stimulated hepatocyte cell lines treated with glucocorticoids were evaluated by flow cytometry. The intrahepatic expression of both beta2-microglobulin conformational epitope and intercellular adhesion molecule-1 was higher in acute rejection than in resolving rejection. Interestingly, glucocorticoids were able to modulate in vitro the cytokine-induced expression of these molecules on hepatocyte cell lines. Beneficial effects of the glucocorticoid treatment appear to be associated with a modulation of a beta2-microglobulin conformational epitope and the intercellular adhesion molecule-1 on intrahepatic cellular targets in the acute rejection process.

Inducible nitric oxide synthase expression in chronic viral hepatitis. Evidence for a virus-induced gene upregulation.

Increased nitric oxide (NO) production may contribute to the pathological changes featuring in some inflammatory diseases, but the role of NO in chronic viral hepatitis is still unknown. We compared the inducible NO synthase (NOS2) expression in the liver of patients with chronic viral hepatitis with that of both nonviral liver disease and histologically normal liver. NOS2 expression was assessed by immunohistochemical and in situ hybridization studies of liver biopsy sections. An intense hepatocellular NOS2 reactivity was detected in chronic viral hepatitis, whereas it was weakly or not observed in nonviral liver disease or normal liver, respectively. In addition, we determined whether the hepatitis B virus (HBV) might regulate the synthesis of this enzyme. NOS2 mRNA and protein levels as well as enzyme activity were assessed in cytokine-stimulated HBV-transfected and untransfected hepatoma cells. Transfection with either HBV genome or HBV X gene resulted in induction of NOS2 mRNA expression, and the maximal induction of this transcript and NO production was observed in cytokine-stimulated HBV-transfected cells. These results indicate that hepatotropic viral infections are able to upregulate the NOS2 gene expression in human hepatocytes, suggesting that NO may mediate important pathogenic events in the course of chronic viral hepatitis.