Oronzo Schiraldi

Clinical role of MMP-2/TIMP-2 imbalance in hepatocellular carcinoma

An imbalance between the proteolytic activity of matrix metalloproteinase‐2 (MMP‐2) and the tissue inhibitor of MMP‐2 (TIMP‐2) is responsible for degradation of extracellular matrix (ECM) components and plays a critical role in tumor invasion and in metastasis formation. The occurrence of intra‐hepatic metastasis, which severely affects prognosis and long‐term survival, is commonly observed in the course of hepatocellular carcinoma (HCC). We investigated the expression of MT1‐MMP in tissues, whereas both MMP‐2 and TIMP‐2 were evaluated in the sera and tissues (primary and metastatic nodules) of HCC patients with and without metastasis, whose clinical outcome was followed over a 2‐year period. MT1‐MMP expression was similar among primary nodule tissues of patients with and without metastasis. Serum and tissue levels of MMP‐2 were not statistically different between patients with and without metastasis, but MMP‐2 was concentrated at the invasive edge of the metastatic tissue. On the contrary, serum and tissue levels of TIMP‐2 were significantly higher in HCC patients without metastasis than in those with. This situation was not only observed in the primary HCC tissues, but also in the metastatic nodules. These results correlate with the clinical outcome, because more than 90% of the patients with high levels of TIMP‐2 were still alive after 2 years, whereas less than 30% with low levels of TIMP‐2 had survived. Furthermore, we found a strict correlation between tissue and serum levels of TIMP‐2, this suggesting that a MMP‐2/TIMP‐2 imbalance and in particular TIMP‐2 levels, could represent an important prognostic factor in patients with HCC.

Transforming Growth Factor-β1 Triggers Hepatocellular Carcinoma Invasiveness via α3β1 Integrin

Metastasis occurrence in the course of hepatocellular carcinoma (HCC) severely affects prognosis and survival. We have shown that HCC invasive cells express α3β1-integrin whereas noninvasive cells do not. Here we show that transforming growth factor (TGF)-β1 stimulates α3-integrin expression at a transcriptional level in noninvasive HCC cells, causing transformation into a motile and invasive phenotype. Such activities are inhibited by neutralizing anti-α3- but not anti-α6-integrin monoclonal antibodies. HCC invasive cells secrete abundant levels of active TGF-β1 in comparison with noninvasive cells, but in the latter, addition of active matrix metalloproteinases-2 increases the concentration of active TGF-β1. In this way, the cells express α3-integrin at a transcriptional level and acquire motility on Ln-5. By contrast, an anti-TGF-β1-neutralizing antibody reduces α3-integrin expression and the invasive ability of HCC invading cells. In HCC patients, TGF-β1 serum concentrations and α3-integrin expression are strongly correlated. The integrin, absent in normal and peritumoral liver parenchyma, is abundantly expressed in HCC primary and metastatic tissue. In particular, patients with metastasis show higher levels of TGF-β1 serum concentrations and stronger expression of TGF-β1 and α3-integrin in HCC tissues. In conclusion, TGF-β1 may play an important role in HCC invasiveness by stimulating α3-integrin expression, and could therefore be an important target for new therapies.

Relationship Between Interferon-γ, Interleukin-10, and Interleukin-12 Production in Chronic Hepatitis C and In Vitro Effects of Interferon-α

In the current study, increased interferon (IFN)-γ, interleukin (IL)-10, and IL-12 p40 serum levels were observed in patients with chronic hepatitis C (CHC) compared to controls. Patients also displayed an increased spontaneous IFN-γ release but a deficient peripheral blood mononuclear cells (PBMC) IFN-γ production following stimulation with Staphylococcus aureus Cowan I strain (SAC). No difference was found with reference to spontaneous or phytohaemagglutinin (PHA)-induced IL-10 release between patients and controls, whereas a higher IL-12 p70 and IL-12 p40 secretion triggered by SAC was observed in patients. Moreover, IL-12 p40/p70 ratio following SAC stimulation was higher in patients compared to controls and a negative correlation was found between this ratio and IFN-γ amounts. Recombinant IL-12 (rIL-12) as well as neutralizing anti-IL-10 monoclonal antibodies (mAbs) were able to restore the compromised IFN-γ production. Of note, anti-IL-10 supplementation induced a lower IL-12 p40/p70 ratio in HCV subjects as compared to controls. Finally, IFN-α upregulated in vitro IFN-γ, IL-10, and IL-12 p70 release but not IL-12 p40 secretion, this giving rise to a normalization of IL-12 p40/p70 ratio. The data suggest the occurrence of an enhanced responsiveness to IL-10 modulating effects, likely mediated by an imbalance of IL-12 p40/p70 ratio, in chronic HCV infection. Cytokine balance restoration might thus contribute to achieve therapeutical results in chronic hepatitis C.

In-situ immunophenotyping study of hepatic-infiltrating cytotoxic cells in chronic active hepatitis C

Objective: Beside the hypothesis of a direct viral cytopathy, several lines of evidence argue in favour of hepatic damage triggered by immune‐mediated mechanisms in hepatitis C virus (HCV) infection. The intrahepatic localization of HCV antigen‐specific cytotoxic T‐lymphocytes (CTLs) to disease sites has been described; however, very few data are available about the degree and the role of hepatic‐infiltrating natural killer (NK) cells in chronically HCV‐infected subjects. Design: In a series of percutaneous needle liver biopsies obtained from 35 consecutive untreated patients with chronic active hepatitis C, we performed an in‐situ immunophenotyping study to evaluate the degree of cytotoxic NK cell infiltration as compared to CTLs, the hepatocyte expression of human major histocompatibility complex antigens class I and class II (HLA‐I and HLA‐II), and cell adhesion molecules (CAM) in the context of liver inflammatory infiltrates. The data were correlated with the histological activity index (HAI) of disease. Results: In‐situ immunophenotyping analysis of CAM provided evidence for the intrahepatic expression of leucocyte adhesion molecules (CD11a and CD2) and their corresponding ligands on hepatocytes (CD54 and CD58) in all cases. A significant parallel expression of CD11a and CD54 as well as CD2 and CD58 structures, restricted to hepatic lobules within the disease sites, was also observed, even if their induction exhibited different degrees of correlation with biological and/or histological activities. A membranous pattern of HLA‐I and HLA‐II antigen expression on hepatocyte clusters was found in all tissue samples, although HLA‐I expression was significantly higher than HLA‐II. Moreover, lymphocyte subset analysis displayed a CD8+ T‐cell lobular infiltration within inflammatory and/or spotty necrosis areas in all cases, while CD4+ T‐cells were confined to the portal and periportal levels. A few scattered CD56+ and CD16+ NK cells, mainly distributed at periportal areas within inflammatory and/or necrotic foci, were detected in 7/35 (20%) and in 5/35 (14.2%) cases, respectively. On the other hand, CD8+ T‐cell lobular expression exhibited a linear correlation with HAI (r: 0.698, P<0.01). Finally, cytotoxic cell infiltration degree did not correlate with HCV serotypes. Conclusion: Our findings suggest a limited role for NK cells in the immune mechanism of liver injury in chronic active hepatitis C, while providing further support for the involvement of CD8+ T‐cells at disease sites.

Occurrence of portal vein tumor thrombus in hepatocellular carcinoma affects prognosis and survival. A retrospective clinical study of 150 cases

Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world. Despite improvements in diagnostic and therapeutic interventions, prognosis and survival are still poor. To identify factors influencing survival, we retrospectively examined 150 consecutive patients with HCC from the time of first diagnosis of cirrhosis to death. In a multivariate analysis, we found that patients with larger HCC lesions had shorter survival, while other pathologic features had no predictive value. The most important and reliable prognostic factor was the occurrence of tumor thrombus of the portal vein (P<0.01). Childs stage of underlying liver disease was relevant only in the univariate, but not in the multivariate analysis. The survival of patients with HCC is mainly affected by the biological ability of cancer cells to invade surrounding tissue and vessels. More studies are needed to elucidate the mechanisms that modulate tumor cell motility, in order to design more effective therapies.

Prevalence of antibodies to hepatitis C virus among family members of patients with chronic hepatitis C

In this study, 108 family members of 40 chronically HCV-infected patients (19 post-transfusion and 21 sporadic), and 45 families of 16 anti-HCV-negative index cases (control group) were tested for anti-HCV antibodies. Anti-HCV antibodies were found in 16 (14.8%) families of anti-HCV positive index cases (15% males and 14.6% females; p = NS), with no difference between families of index cases with post-transfusion and those with sporadic HCV infection. Out of the 16 anti-HCV positive family members, 12 (75%) had clinical and/or serological evidence of chronic liver damage. None of the control group subjects were anti-HCV-positive (p < 0.01). The rate of anti-HCV positivity was 34.4% among spouses, 14.3% among siblings, 16.7% among cohabitants and 2.3% among children; anti-HCV antibodies were not detected among parents. We found a positive correlation between the prevalence of anti-HCV antibodies among families and the severity of the HCV-related chronic liver damage of the index cases (p < 0.00005). In addition, to confirm that HCV infection and HCV-related chronic hepatitis may be transmitted intrafamiliarly, our findings also indicate that horizontal, especially sexual contact, is a more important route of HCV infection than vertical/perinatal transmission. Finally, the risk of acquiring HCV infection among families appears to be the highest when index cases are suffering from severe HCV-related chronic hepatitis.

A bioptic study of gastrointestinal mucosa in cholera patients during an epidemic in southern Italy.

A histological biopsy study of gastric and jejunal mucosa of eight acute cholera patients during an epidemic in Southern Italy was carried out. The study demonstrated in all patients an intact epithelial lining of gastric and jejunal mucosa, a moderate degenerative process of enterocytes, presence of inflammatory lesions manifested by edema, vascular congestion, mononuclear cell infiltrate of lamina propria, and discharge of goblet-cells mucus. These changes reverted to normal in a few days. The authors emphasize that, contrary to cholera patients of Asiatic areas in whom an underlying chronic spruelike enteropathy is very common, the histological picture observed in Western patients may be considered more specific sinceVibrio cholerae acts upon a normal intestinal mucosa.

Costimulatory molecules and cytotoxic T cells in chronic hepatitis C: defence mechanisms devoted to host integrity or harmful events favouring liver injury progression? A review.

The recruitment of antigen-specific lymphocytes at liver site represents a prominent feature in patients chronically infected with hepatitis C virus (HCV). However, despite the strong and multispecific response, chronic infection leads in a significant number of cases to the development of cirrhosis and hepatocellular carcinoma. The finding that the expression of CD80 structure positively correlates with disease histological worsening points out a role for the costimulatory pathway in the progression of liver cell injury. On the other hand, the demonstration of CD95 and CD95-ligand positive cells in the context of periportal areas, a pattern which is not strictly associated to HCV tissue distribution, indicates the occurrence of either virus-infected or innocent bystander hepatocyte killing. Nonetheless, the persistence of HCV, in spite of cytotoxic T lymphocyte (CTL) liver recruitment, suggests a possible in-situ imbalance of cytotoxic activities, above all referred to perforin-granzyme-dependent necrosis. Altogether, these findings outline that several factors might be involved in HCV-driven immunopathogenesis. Therefore, the fully clarification of these mechanisms may offer a suitable therapeutical approach for the improvement of clinical outcome in chronic hepatitis C.

sICAM-1, sCD95 and sCD95L levels in chronic liver diseases of different etiology.

Abstract The release of soluble circulating molecules represents a prominent feature during the course of immune-mediated clinical conditions. To further assess the relationship between serum concentrations of adhesion or apoptotic-related soluble structures and liver diseases, we evaluated the levels of intercellular adhesion molecule-1 (sICAM-1), Fas receptor (CD95) and Fas ligand (sCD95L) in a group of patients affected by Hepatitis C Virus (HCV)induced chronic hepatitis (CH-C), HCV-positive liver cirrhosis with superimposed hepatocellular carcinoma (HCC), autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC) and alcoholic liver cirrhosis (ALC). Results show that sICAM-1 values were in all instances significantly elevated when compared to those seen in healthy donors. Similar findings were noted in subjects with liver diseases in terms of sCD95 concentrations, even if to a different degree of statistical significance. Finally, sCD95L amounts were augmented in AIH, PBC, ALC and CH-C in comparison to controls, while in the HCC counterpart sCD95L levels fell within normal range. All together, these findings emphasize the occurrence of circulating soluble molecules in patients with various chronic liver diseases, likely reflecting the involvement of several pathogenetic mechanisms.

Immunoresponsiveness in chronic hepatitis C patients : Correlation between tissue and serum findings

In the present study, intrahepatic CD8+ lymphocyte infiltrates as well as HLA class I and CD54 (ICAM-1) antigen expression at both tissue and serum levels were evaluated in 54 untreated patients with chronic hepatitis C stratified on the basis of histological diagnosis (Chronic Persistent Hepatitis/Chronic Lobular Hepatitis -CPH/CLH- and Chronic Active Hepatitis -CAH-: 22 and 32 subjects, respectively). The relationships between soluble HLA-I (sHLA-I) and ICAM-1 (sICAM-1) serum levels and their membrane-bound counterparts, CD8+ liver infiltration and serum alanine aminotransferase (ALT) were also studied. A strong HLA-I and CD54 tissue expression, associated to the presence of CD8+ cell infiltrates in necro-inflammatory areas, and elevated sHLA-I and sICAM-1 serum amounts were observed in all patients. At the same time, no difference was found at tissue level between the two groups of patients with respect to the mean scores of HLA-I and CD54 expression, while CAH subjects displayed a significantly higher CD8 periportal and lobular reactivity in comparison to the other subset. Serological assays outlined higher values of circulating HLA-I molecules in CPH/CLH patients and higher sICAM-1 levels in the CAH group. Finally, a negative correlation was found between sHLA-I and ALT in CAH subjects while, in all patients, sICAM-1 positively correlated with both CD8 tissue infiltration and ALT. Our findings confirm the occurrence of an immune activation status during chronic hepatitis C and suggest that sHLA-I molecules might play a down-modulating role on immunoresponsiveness of these patients.