Gabriele Pozzato

Drinking habits as cofactors of risk for alcohol induced liver damage

Background—The Dionysos Study is a cohort study of the prevalence of chronic liver disease in the general population of two northern Italian communities. It included 6917 subjects, aged 12–65 (69% of the total population). Aims—The aim of this part of the study was to examine the relationship of daily alcohol intake, type of alcoholic beverage consumed, and drinking patterns to the presence of alcohol induced liver damage in an open population. Patients and methods—6534 subjects, free of virus related chronic liver disease and participating in the first cross-sectional part of the study, were fully examined. Each subject underwent: (a) medical history and physical examination, (b) evaluation of alcohol intake using an illustrated dietary questionnaire, and (c) routine blood tests. More invasive diagnostic procedures were performed when indicated. Results—Multivariate analysis showed that the risk threshold for developing either cirrhosis or non-cirrhotic liver damage (NCLD) was ingestion of more than 30 g alcohol per day in both sexes. Using this definition, 1349 individuals (21% of the population studied) were at risk. Of these, only 74 (5.5% of the individuals at risk) showed signs of liver damage. The prevalence of “pure” alcoholic cirrhosis was 0.43% (30 of 6917), representing 2.2% of the individuals at risk, with a ratio of men to women of 9:1, while 44 (3.3% of the individuals at risk) showed persistent signs of NCLD. After 50 years of age, the cumulative risk of developing both NCLD and cirrhosis was significantly higher (p<0.0001) for those individuals who regularly drank alcohol both with and without food than for those who drank only at mealtimes. Conclusions—Our data show that in an open population the risk threshold for developing cirrhosis and NCLD is 30 g ethanol/day, and this risk increases with increasing daily intake. Drinking alcohol outside mealtimes and drinking multiple different alcoholic beverages both increase the risk of developing alcohol induced liver damage.

Clinical course and risk factors of hepatitis C virus related liver disease in the general population: report from the Dionysos study

BACKGROUND The severity, clinical course, and risk of hepatitis C virus (HCV) related chronic liver disease are still rather poorly defined. AIMS To investigate the prevalence, risk factors, and severity of HCV related liver disease in the general population, and investigate whether infection with a specific genotype is associated with an increased risk of cirrhosis or hepatocellular carcinoma. METHODS HCV RNA determination by polymerase chain reaction (PCR) and HCV genotyping were performed in all anti-HCV positive subjects belonging to the Dionysos study (6917 subjects). Diagnosis of cirrhosis and hepatocellular carcinoma was established by liver biopsy in all cases. All the data were analysed by univariate and multivariate statistics in all the cohort. To investigate the natural history of HCV infection, anti-HCV positive subjects were followed up every six months for three years with liver function tests and ultrasonograms. RESULTS The overall prevalence of HCV RNA positivity was 2.3%. Positivity increased progressively with age, and was higher in women (ratio of men to women = 0.7). Genotypes 1b and 2a were the most frequent (42 and 24% of HCV RNA positive patients), with a prevalence of 1 and 0.6% respectively. Intravenous drug use, blood transfusions received before 1990, history of previous hepatitis among the cohabiting, and history of animal (mainly dogs) bites were significantly (p<0.05) associated with HCV infection, independently of age and sex. Multivariate analysis showed that, independently of age, sex, and alcohol intake, genotype 1b infection, with or without coinfection with other genotypes, is the major risk factor associated with the presence of cirrhosis and/or hepatocellular carcinoma. During the three years of follow up, 57 (35%) of the HCV RNA positive subjects had consistently normal alanine aminotransferase and γ-glutamyltransferase values. Two of the 22 HCV RNA positive cirrhotic patients, all drinking more than 90 g of alcohol a day, developed hepatocellular carcinoma (incidence rate = 3.0% per year). CONCLUSIONS In the general population of Northern Italy, HCV infection is widespread, but only less than 50% of the anti-HCV positive subjects, particularly those infected with genotype 1b, are associated with a more severe liver disease. Alcohol consumption greater that 30 g a day significantly aggravates the natural course of the disease.

Hepatitis C virus and non-Hodgkin's lymphomas

Hepatitis C virus (HCV) seems to be the aetiologic agent of mixed cryoglobulinaemia, and as this ‘benign’ lymphoproliferative disorder can frequently develop into more aggressive haematological disorders, this study was undertaken to determine the prevalence of HCV infection in non‐Hodgkins lymphomas. 199 unselected subjects treated by three haematological centres in Northeast Italy were investigated for the presence of HCV infection. As controls, the prevalence of HCV infection was determined in a group of patients affected by other haematological malignancies (153 subjects) and in the general population of the same geographical area in the cohort study called the Dyonisos project (6917 subjects).

Clonal B-cell expansions in peripheral blood of HCV-infected patients

Summary. Clonal expansions of IgM‐producing B cells were investigated in 38 patients with a chronic hepatitis C virus infection. Eight patients were affected with type II mixed cryoglobulinaemia (two of whom also had non‐Hodgkins lymphoma and one had Waldenstroms disease), one with type III mixed cryoglobulinaemia, one with Waldenstroms disease, and 28 with chronic liver disease. To detect the clonal B‐cell expansions we used a RT/PCR procedure in which the CDR3/FW4 regions of the IgM heavy chain mRNAs were amplified and resolved in sequencing poly‐acrylamide gels. Clonal Ig gene rearrangements were detected in all patients with type II mixed cryoglobulinaemia and also at a high frequency (24%) in the HCV‐infected patients without cryoglobulinaemia. A polyclonal pattern was present in the patient with type III mixed cryoglobulinaemia and in the 15 normal individuals and 16 age‐related patients with HCV‐negative alcoholic liver disease which were investigated as controls. No association was found between the presence of a clonal B‐cell expansion and age, sex, liver histology, or levels of serum aminotransferase. The serum levels of rheumatoid factor were increased in all patients with a clonal expansion, suggesting that the expanded B‐cell clones belong to the rheumatoid factor producing B‐cell subset.

Association between molecular lesions and specific B-cell receptor subsets in chronic lymphocytic leukemia.

Genetic lesions and B-cell receptor (BCR) signaling are both oncogenic drivers in chronic lymphocytic leukemia (CLL). However, scant data are available on preferential associations between specific genetic alterations and stereotyped BCR subsets. By analyzing 1419 cases, 2 CLL subsets (2 and 8) harboring stereotyped BCR are enriched in specific molecular alterations influencing disease course. SF3B1 mutations are the genetic hallmark of IGHV3-21-CLL belonging to subset 2 (52%) but are evenly represented in nonstereotyped IGHV3-21-CLL. Trisomy 12 (87%) and NOTCH1 mutations (62%) characterize IGHV4-39-CLL belonging to subset 8 but occur with the expected frequency in IGHV4-39-CLL with heterogeneous BCR. Clinically, co-occurrence of SF3B1 mutations and subset 2 BCR configuration prompts disease progression in IGHV3-21-CLL, whereas cooperation between NOTCH1 mutations, +12, and subset 8 BCR configuration invariably primes CLL transformation into Richter syndrome. These findings provide a proof of concept that specific stereotyped BCR may promote or select molecular lesions influencing outcome.

Treatment with interferon(s) of community-acquired chronic hepatitis and cirrhosis type C

Two hundred and thirty-four patients with chronic non-A, non-B hepatitis, 86% positive for anti-HCV by ELISA, were treated with recombinant interferon-alpha 2a or with natural (human-leukocytes-derived) interferon-alpha using different dosage and periods of administration. Interim analysis of follow-up data indicate that 65-70% of patients treated initially with 6 MU, thrice weekly, of recombinant interferon-alpha 2a achieved a complete biochemical response (normalization of alanine aminotransferase: ALT) during therapy compared to 56-58% of those treated with 3 MU, thrice weekly, of recombinant or natural interferon-alpha. A 12-month schedule of interferon administration appeared superior to a 6-month schedule in reducing the probability of reactivation of liver disease after therapy withdrawal, although further data are needed to confirm such a conclusion. The probability of response to interferon in terms of maintaining normal ALT after withdrawal did not appear to be influenced by sex, while it was significantly higher in patients aged below 45 years and in those without cirrhosis.

The CD49d/CD29 complex is physically and functionally associated with CD38 in B-cell chronic lymphocytic leukemia cells

CD49d and CD38 are independent negative prognostic markers in chronic lymphocytic leukemia (CLL). Their associated expression marks a disease subset with a highly aggressive clinical course. Here, we demonstrate a constitutive physical association between the CD49d/CD29 integrin complex and CD38 in primary CLL cells and B-cell lines by (i) cocapping, (ii) coimmunoprecipitation and (iii) cell adhesion experiments using CD49d-specific substrates (vascular-cell adhesion molecule-1 or CS-1/H89 fibronectin fragments). The role of CD38 in CD49d-mediated cell adhesion was studied in CD49d+CD38+ and CD49d+CD38− primary CLL cells, and confirmed using CD38 transfectants of the originally CD49d+CD38− CLL-derived cell line Mec-1. Results indicate that CD49d+CD38+ cells adhered more efficiently onto CD49d-specific substrates than CD49d+CD38− cells (P<0.001). Upon adhesion, CD49d+CD38+ cells underwent distinctive changes in cell shape and morphology, with higher levels of phosphorylated Vav-1 than CD49d+CD38− cells (P=0.0006) and a more complex distribution of F-actin to the adhesion sites. Lastly, adherent CD49d+CD38+ cells were more resistant to serum-deprivation-induced (P<0.001) and spontaneous (P=0.03) apoptosis than the CD49d+CD38− counterpart. Altogether, our results point to a direct role for CD38 in enhancing CD49d-mediated adhesion processes in CLL, thus providing an explanation for the negative clinical impact exerted by these molecules when coexpressed in neoplastic cells.

Platelet production and destruction in liver cirrhosis

BACKGROUND & AIMS Thrombocytopenia is common in liver cirrhosis (LC) but the mechanisms are not fully understood. The purpose of our work was to evaluate platelet kinetics in LC with different etiologies by examining platelet production and destruction. METHODS Ninety-one consecutive LC patients (36 HCV, 49 alcoholics, 15 HBV) were enrolled. As controls, 25 subjects with idiopathic thrombocytopenic purpura, 10 subjects with aplastic anemia, and 40 healthy blood donors were studied. Plasma thrombopoietin (TPO) was measured by ELISA. Reticulated platelets (RP) were determined using the Thiazole Orange method. Plasma glycocalicin (GC) was measured using monoclonal antibodies. Platelet associated and serum antiplatelet antibodies were detected by flow cytometry. B-cell monoclonality in PBMC was assessed by immunoglobulin fingerprinting. RESULTS Serum TPO was significantly lower in LC (29.9±18.1 pg/ml) compared to controls (82.3±47.6 pg/ml). The GC levels were higher in LC (any etiology) than in healthy cases. Conversely, the absolute levels of RP were lower in LC (any etiology) than in healthy controls. The platelet-associated and serum anti-platelet antibodies were higher in HCV+ LC compared to healthy subjects (p<0.0064), alcoholic LC (p<0.018), and HBV+ LC (p<0.0001). B-cell monoclonality was found in 27% of the HCV+LC, while it was not found in HBV+ or alcoholic LC. CONCLUSIONS Patients with LC present decreased plasma TPO, accelerated platelet turnover, and reduced platelet production. This indicates that LC thrombocytopenia is a multifactorial condition involving both increased platelet clearance and impaired thrombopoiesis.

Pegylated-interferon plus ribavirin for HCV-positive indolent non-Hodgkin lymphomas.

Hepatitis C virus (HCV) causes not only chronic liver disease, but is also implicated in several lymphoproliferative disorders (Pozzato et al, 1994). The identification of CD81 as the main HCV receptor could explain this lymphotropism because this receptor is expressed in B lymphocytes (Pileri et al, 1998). The CD81 engagement determines cell activation with overexpression of CD69, CD71, CD86, and the chemokinereceptor CXCR3 (Rosa et al, 2005). In addition, an increased prevalence of t(14,18) translocation has been reported in HCV-patients, even in the absence of lymphoproliferative disorders (Zignego et al, 2002). This interaction of HCV with the immune system might explain the role of HCV in several extra-hepatic diseases, such as non-Hodgkin lymphoma (NHL). As HCV-positive lymphoproliferative disorders have been successfully treated with antiviral therapy (Gisbert et al, 2005), we compared the traditional treatment of interferon plus ribavirina (IFN + RIBA) with the newest antiviral therapy i.e. pegylated interferon alpha plus ribavirina (PEG-IFN +RIBA) in a small group of indolent NHL. Eighteen patients affected by HCV-related B-NHL were consecutively included in the study from February 1998 to January 2004. Criteria for patient’s selection were: (i) lowgrade B-NHL at first diagnosis or relapse, (ii) indolent course, (iii) HCV infection (HCV-RNA positivity in serum). Patients that carried the hepatitis B surface antigen (HBsAg) or were positive for anti-human immunodeficiency virus antibodies were excluded. All patients gave their informed consent before entry in the study. B cell monoclonality was analysed in the peripheral blood mononuclear cells and bone marrow samples of four cases by the previously described isotype-specific IGH VDJ Gene Scanning technique: briefly, a forward primer labelled with 6-carboxyfluorescein (FAM) fluorochrome in the second VDJ polymerase chain reaction (PCR) round was used. The VDJ PCR product and the internal size standard GeneScan 400 HD (Applied Biosystem, Forster City, CA, USA) were added to deionized formamide, and then subjected to capillary electrophoresis. The detection of IGHV gene segments was performed using VDJ FR1 PCR protocol, in which a second multiplexing PCR, IGHV3, IGHV4, IGHV5, IGHV6 primers and the IGHJ region were added. IGHV1-IGHV4, and IGHV3-IGHV6 primers were each labelled with a different fluorescent dye: FAM, HEX, NED or ROX. PCR products were added to the internal GeneScan 500 LIZ size standard. This mixture was subjected to capillary electrophoresis. When a monoclone was found, the PCR product was sequenced by standard methods. Eight patients (Group A) received 3 MU of IFN alpha-2b three times a week for 48 weeks. Ten patients (Group B) received PEG-IFN alpha-2b 1Æ5 lg/kg once a week for 48 weeks. Both groups received ribavirin 1000 mg or 1200 mg daily according to body weight (lower or greater than 75 kg) for 48 weeks (Manns et al, 2001). The patients were randomized on the basis of a computer-generated random list. The patient diagnoses were: one follicular lymphoma, one splenic lymphoma with villous lymphocytes, and 16 lymphoplasmacytoid lymphomas. The follicular lymphoma and the splenic lymphoma had node involvement (axillary and mediastinal/retroperitoneal, respectively). HCV genotyping showed genotype 1b in 11 cases (61%) while genotype 2a/2c was detected in the others. Eleven cases had chronic hepatitis at liver biopsy. Thirteen patients had dosable levels of cryoglobulins (from 2% to 35%) and high rheumatoid factor levels (from 30 to 21 700 U/l). Therapy outcome (Table I) was as follows:

IgM-producing chronic lymphocytic leukemia cells undergo immunoglobulin isotype-switching without acquiring somatic mutations.

The malignant B cells in chronic lymphocytic leukemia (CLL) typically express low-density membrane IgM or IgM/IgD. In vitro experiments have shown that the CLL cells can be induced to differentiate into cells that secrete immunoglobulin (Ig) and can occasionally undergo heavy (H) chain class switching. We now show that the CLL cells also undergo isotype-switching in vivo, since gamma and/or alpha H chain transcripts with identical FW3/CDR3/FW4 regions as the mu CLL transcripts were detected in all of the 13 investigated patients with IgM+ CLL. In most cases switching had occurred to alpha1 and gamma3, but CLL transcripts corresponding to the other gamma chain isotypes were also detected. In one case both the productively and nonproductively rearranged allele were found to undergo H chain class switching. CLL gamma transcripts were also present in surface IgG+ sorted B cells, demonstrating that a small subset of the CLL cells express membrane IgG. In addition, transcripts encoding secretary gamma2 and gamma3 H chains were detected in two cases, which suggests that some serum IgG could be produced by the leukemic clone. Analysis of sorted PBL showed that isotype-switching occurs in CLL cells that express the CD5 antigen. Finally, nucleotide sequence analysis showed that the mu, alpha, and gamma CLL transcripts are identical, demonstrating that the CLL cells do not accumulate somatic mutations in their variable region genes after the H chain class switching. These data provide in vivo evidence that isotype-switching is a frequent phenomenon in CLL, and indicate that a subset of the CLL lymphocytes progress to later stages of B cell differentiation.