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AIM To uncover novel genetic markers that could contribute to predicting hepatocellular carcinoma (HCC) susceptibility in Caucasians. METHODS The present retrospective case-control study compared genotype frequencies between a cohort of HCC cases and two, independent, HCC-free, age/sex-matched control groups. The HCC cohort comprised 192 homogeneous patients that had undergone orthotopic liver transplantation. The first control group comprised 167 patients that were matched to the HCC cohort for the percentage of hepatitis B (HBV) and/or hepatitis C (HCV) infections. A second control group included 192 virus-free, healthy individuals that were used to evaluate the generalizability of the identified predictive markers. All cases and controls were Caucasian. The three study populations were characterized with a panel of 31 markers derived from 21 genes that encoded key proteins involved in hepatocarcinogenesis-related pathways. The study end-point was to assess the association between genetic variants and HCC onset. RESULTS Five genetic markers were identified as risk factors for HCC in high-risk patients infected with HBV/HCV. According to a dominant model, reduced HCC risk was associated with three polymorphisms: ERCC1 rs3212986 (OR = 0.46, 95%CI: 0.30-0.71, P = 0.0005), GST-P1 rs1138272 (OR = 0.41, 95%CI: 0.21-0.81, P = 0.0097), and CYP17A1 rs743572 (OR = 0.50, 95%CI: 0.31-0.79, P = 0.0032). Conversely, according to a recessive model, increased HCC risk was associated with two polymorphisms: XRCC3 rs1799794 (OR = 3.70, 95%CI: 1.02-13.39, P = 0.0461) and ABCB1 rs1128503 (OR = 2.06, 95%CI: 1.18-3.61, P = 0.0111). These associations remained significant in a subgroup analysis, where patients were stratified according to viral status (HBV- or HCV-positive serology). Two variants exhibited a serology-specific effect: ABCB1 rs1128503 (OR = 4.18, 95%CI: 1.55-11.29, P = 0.0048) showed an effect in the HBV-positive subgroup; and ERCC1 rs3212986 (OR = 0.33, 95%CI: 0.18-0.60, P = 0.0003) showed an effect in the HCV-positive subgroup. Among the five markers identified, ERCC1 rs3212986 (OR = 0.43, P < 0.0001) and CYP17A1 rs743572 (OR = 0.73, P = 0.0310) had a different distribution in patients with HCC compared to healthy individuals. With a recursive partitioning approach, we also demonstrated that significant gene-gene interactions between ERCC1 rs3212986, CYP17A1 rs743572, GST-P1 rs1138272, and the previously described UGT1A7*3 predictive marker, played a role in the complex trait of HCC susceptibility. CONCLUSION We identified five polymorphisms and interactions that contributed crucially to predicting HCC risk. These findings represented an important step towards improving HCC diagnosis and management.

Background & Aims The definition of new biomarkers of hepatocellular carcinoma (HCC) risk, especially in high-risk HBV/HCV-positive population, is urgently needed to improve HCC clinical management. This study focused on variants of UDP-glucuronosyltransferase 1A (UGT1A) enzymes that catalyse the reaction of glucuronidation, one of the most important chemical defence pathway of the body. The aim of this study was to elucidate the contribution of UGT1A polymorphisms in predicting HCC susceptibility in Caucasians. Methods In this retrospective case-control analysis, 192 HCC liver transplanted patients represent the study group. Two age/sex-matched groups were used as control, one composed of 167 HBV- and/or HCV-infected individuals, and the other of 192 healthy subjects. All the cases were characterized for a panel of UGT1A1, UGT1A7 and UGT1A9 variants. The study end-point was the association between UGT1A markers and HCC onset. Results UGT1A7*3 allele emerged as a protective marker for HCC development among both high-risk HBV/HCV-positive patients (OR=0.64, P=.0026), and healthy subjects (OR=0.47, P=.0051). UGT1A1*28 (OR=0.61, P=.0013) and UGT1A9*22 (OR=2.18, P=.0003) alleles were also associated to HCC occurrence, especially among healthy subjects. UGT1A haplotype, summarizing the UGT1A genetic alterations, confirmed the protective role against HCC development emerged for low-activity alleles. The observed associations could probably be linked to an increase of serum levels of health-beneficial molecules including free bilirubin. Conclusion A predictive effect of UGT1A polymorphisms on HCC risk was identified. If confirmed, these findings could contribute to improve the HCC surveillance, treatment tailoring and patients care.

This investigation was focused on the contribution of individual human leukocyte antigen (HLA)-DR and -DQ alleles to the human hepatitis C virus (HCV)(+) non-Hodgkin's lymphoma (NHL), with and without mixed cryoglobulinemia (MC), to study whether individual HLA class II alleles are expressed preferentially or equally in human HCV-specific NHL. For this purpose, peripheral blood mononuclear cells were obtained from two groups of patients with HCV(+) NHL and with or without MC (70 and 71 cases, respectively), and from 4575 blood donors. Eighty-three subjects with HCV infection only, and 118 patients with MC, only without lymphoma, were added as additional control groups. Individual HLA-DR and -DQ alleles were determined using high-resolution sequence-based typing and then data were collected by considering the HLA-DRB1 and DQB1 supertypes on the basis of common structural and functional features, proposed by in silico Bioinformatic studies. From the data, it is evidenced that the DR5-DQ3 HLA combination was strongly associated with the HCV (+) MC (+) NHL group of patients compared with bone marrow donor population (P

Although hepatitis C and B viruses and alcohol consumption are the major risk factors for hepatocellular carcinoma (HCC), dietary habits may also be relevant. A hospital-based case-control study was conducted in Italy in 1999-2002, including 185 HCC cases and 412 cancer-free controls. Dietary habits were assessed using a validated food-frequency questionnaire to compute nutrient intakes. Odds ratios (OR) and corresponding confidence intervals (CI) were calculated using the energy-adjusted residual models. Inverse association emerged for linoleic acid (OR=0.35 for highest versus lowest tertile; 95% CI: 0.18-0.69) and, possibly, beta-carotene (OR=0.48; 95% CI: 0.24-0.93). Among minerals, iron intake was associated with increased HCC risk (OR=3.00; 95% CI: 1.25-7.23), but the association was considerably reduced when iron from wine was excluded (OR=1.61; 95% CI: 0.78-3.30). In conclusion, a diet rich in linoleic acid containing foods (e.g. white meats and fish) and beta-carotene was inversely related to HCC risk.

Mortality rates of hepatocellular carcinoma (HCC) are high in Italy compared with other Western countries. To elucidate further the role of hepatitis B virus (HBV), hepatitis C virus (HCV), alcohol drinking, and tobacco smoking in the etiology of HCC, we carried out a hospital-based case-control study in two areas of Italy: the province of Pordenone in the Northeast and the town of Naples in the South. A total of 229 HCC cases (median age, 66 years) and 431 controls (median age, 65 years) answered a questionnaire and provided blood samples between 1999 and 2002. Odds ratios (OR), percent attributable risks, and corresponding 95% confidence intervals were computed using unconditional multiple logistic regression. ORs for hepatitis B surface antigen (HBsAg) positive versus HBsAg negative and for anti-HCV antibody positive versus anti-HCV antibody negative were 20.2 and 15.6, respectively. Positivity for both markers was associated with an OR of 51.6. Sensitive molecular techniques applied to sera in a subset of HCC cases disclosed a very small number of occult hepatites. Maximal lifetime alcohol intake of ≥35 versus

The role of diet, except for alcohol drinking and aflatoxin contamination, in the etiology of hepatocellular carcinoma (HCC) is unclear. A hospital-based case-control study was conducted in Italy in 1999-2002, including 185 incident, histologically-confirmed cases of HCC. Controls were 412 subjects admitted to hospitals for acute, nonneoplastic diseases unrelated to diet. Dietary habits were assessed using a validated food-frequency questionnaire. Odds ratios (ORs) and the corresponding 95% confidence intervals (CI) were computed using unconditional multiple logistic regression, adjusting for hepatitis B (HBV) and hepatitis C (HCV) virus infection and alcohol drinking. Energy adjustment was carried out by means of the residual model. A significant inverse relation was found between intakes of milk and yoghurt (OR = 0.28; 95% CI: 0.13-0.61), white meats (OR = 0.44; 95% CI: 0.20-0.95), eggs (OR = 0.31; 95% CI: 0.14-0.69), and fruits (OR = 0.48; 95% CI: 0.22-1.05) and HCC risk. The favourable effect of high intakes of milk and yoghurt, white meats, eggs and fruits was consistent across strata of HBV and HCV infections. The present study supports the hypothesis of a role of diet in HCC aetiology. Dietary modifications may be indicated in subjects at high-risk for HCC.

Tobacco smoking is a well-documented risk factor for several cancers, but the role of cigarette smoking in the etiology of non-Hodgkin lymphoma (NHL) is inadequately understood. Hepatitis C virus (HCV) has been associated with NHL, but the interaction between HCV and smoking habits has not yet been studied. Between 1999 and 2002, we conducted a case-control study on the association of HCV, smoking habits and NHL in 2 areas of northern and southern Italy. Cases were 225 consecutive patients (median age, 59 years) with a new diagnosis of NHL that were admitted to reference and general hospitals. Controls were 504 patients (median age, 63 years) admitted to the same hospitals as cases, for a wide spectrum of acute, nonneoplastic, nonimmune-, nor tobacco-related conditions. Current, heavy smokers (≥20 cigarettes/day) had an odds ratio (OR) of NHL of 2.10 (95% confidence interval, CI: 1.07–4.12) compared to never smokers. The association between smoking and NHL was consistent across strata of sex and age. Compared to never smokers, current smokers of ≥20 cigarettes/day had ORs of 1.14 (95% CI: 0.37–3.56) for B-cell-low-grade, 2.10 (95% CI: 0.94–4.67) for B-cell-intermediate and high-grade, and 25.84 (95% CI: 1.95–342.17) for T-cell NHL. The effect of tobacco smoking and HCV were independent on the relative risk, leading a 4-fold elevated risk in current smokers HCV positive subjects. Tobacco smoking and hepatitis C virus (HCV) have been associated to non-Hodgkin lymphoma (NHL), but the interaction between HCV and smoking habits has not yet been studied. Our study confirms that tobacco is related to NHL, and reports on the combined effect of tobacco smoking and HCV. Infection acted together according to a multiplicative model, leading to a 4-fold elevated risk in current smokers HCV positive subjects. © 2005 Wiley-Liss, Inc.

HCV has been associated with NHL, but the evidence from case series and case-control studies is not totally consistent. Between 1999 and 2002, we conducted a hospital case-control study on the association between HCV, HBV and NHL in 2 areas of Italy where HCV infection is relatively frequent. Cases (n = 225, median age 59 years) were consecutive patients with a new diagnosis of NHL admitted to local specialized and general hospitals. Controls (n = 504, median age 63 years) were patients with a wide spectrum of acute conditions admitted to the same hospitals as cases. HCV prevalence was 19.6% among NHL cases and 8.9% among controls (adjusted OR = 2.6, 95% CI 1.6-4.3). The ORs for HCV were similar for low-grade and intermediate-/high-grade B-cell NHL (3.2 and 2.4, respectively) as well as for nodal and extranodal NHL (2.7 and 2.6, respectively). Positivity for HBsAg was found in 3.8% of cases and 0.9% of controls (OR = 4.1, 95% CI 1.2-14.4). An elevated OR was also found for history of hepatitis C (OR = 4.7, 95% CI 2.3-9.5). History of blood transfusion before 1990 was associated with HCV positivity among controls but not with NHL risk. In conclusion, HCV infection was associated with an increase in NHL risk, and the fraction of NHL cases attributable to HCV was 12.4% (range 6.3-18.5%). (C) 2004 Wiley-Liss, Inc.

Background/Aims. The association between mixed cryoglobulinaemic, cryoglobulinaemic glomerulonephritis, and chronic hepatitis C virus infection has recently been described. The renal disease had usually been treated with immunosuppressive therapy, but, given the presence of viral infection, this therapy is no longer recommended. In this study, we compare steroid vs interferon therapy in a group of patients affected by hepatitis C virus positive cryoglobulinaemic glomerulonephritis in the stationary phase. Patients/Methods. The diagnosis of cryoglobulinaemic glomerulonephritis was made bearing in mind standard criteria. Patients were randomly assigned to 2 groups receiving oral prednisone 0.2 mg/kg/die for 6 months (6 patients, group A) or lymphoblastoid interferon 3 MU, three times a week for 6 months (7 patients, group B). Hepatitis C virus-RNA was determined by reverse transcription-polymerase chain reaction and hepatitis C virus genotype according to Okamoto. Hepatitis C virus-RNA quantitation was performed by competitive polymerase chain reaction. Results. The 2 groups were comparable in terms of age and severity of kidney failure. All genotypes of hepatitis C virus were found with a prevalence of Type 1 b. In group A, 4 patients showed a partial response; in group B, 1 patient achieved complete remission, 4 a partial response, 2 patients in both groups showed no response. At the end of the treatment, all patients in both groups relapsed. Only 1 patient in group B became hepatitis C virus-RNA negative, and recovered from cryoglobulinaemic glomerulonephritis. Conclusions. Interferon seems to be an effective drug in the treatment of cryoglobulinaemic glomerulonephritis, but dosage and length of treatment still need to be addressed by large multicentre studies.

Recently, a new, suspected hepatotropic virus has been identified. Named GBV-C/HGV, this virus shares with the hepatitis C virus (HCV) routes of transmission and molecular organization. Indeed, a proportion of HCV-infected patients (10-25%) are also carriers of GBV-C/HGV. Since mixed cryoglobulinaemia (MC) is closely associated with HCV infection, the aim of this study was to determine the prevalence of GBV-C/HGV infection in MC patients, and to investigate whether the double infection influenced the clinical and/or laboratory aspects of the disease. 52 patients affected by MC were studied. 100 patients affected by HCV-positive chronic liver disease (CLD) without MC were used as control group. To determine the prevalence of GBV-C/HGV infection in general population, 150 blood donors were studied, as well as 80 patients affected by non-A-E CLD. Among the MC patients, only five (9.6%) were positive for both HCV and GBV-C/HGV infection. No difference was found between patients with and without double infection as regards main clinical and laboratory aspects. Among HCV-positive CLD cases, 27 were positive for double infection. Among blood donors, the prevalence of GBV-C/HGV infection was 8.0%, whereas in cases with cryptogenetic CLD the prevalence was 5.0%. In conclusion, these data show that GBV-C/HGV infection does not play any role in the pathogenesis of MC.

A clonal expansion of peripheral blood mononuclear cells committed to IgM cryoprecipitating rheumatoid factor production has been demonstrated in hepatitis C virus (HCV)-associated mixed cryoglobulinemia (MC). To determine the role of HCV in B cell gene rearrangements we studied a series of 57 HCV-infected patients with and without MC. Clonal Ig gene rearrangements of both RNA and DNA were detected in 10 of the 13 patients with type II MC, 1 patient had gene rearrangement of the DNA only, and 2 had polyclonal patterns. 2 of the 17 patients with type III MC showed clonal rearrangement of both RNA and DNA, in 6 only the DNA was rearranged clonally and in 9 the patterns were completly normal. 14 of 27 patients with cryocrit

With the aim of investigating whether interleukin 28B gene (IL28B) rs1297860 polymorphism is associated with different hepatitis C (HCV) infection statuses, we compared IL28B allelic distribution in an Italian case series of 1050 patients with chronic infection and different outcomes, 47 individuals who spontaneously cleared HCV, and 178 blood donors. Furthermore, we compared IL28B variants among 3882 Caucasian patients with chronic infection, 397 with spontaneous clearance, and 1366 blood donors reported in PubMed. Overall data confirmed a relation between IL28B C allele and HCV spontaneous clearance. Furthermore, we found that IL28B T allele had a weak relation with chronic HCV progression to hepatocellular carcinoma. Study findings are in accordance with the hepatocellular carcinogenic model where IL28B TT genotype, by promoting a persistent chronic hepatitis which leads to both hepatocyte injury and chronic inflammation, could facilitate HCC development. Conversely, patients with lymphoproliferative disorders had not any significantly different IL28B rs1297860 allelic distribution than those with chronic HCV, but, like all chronic HCV-related diseases, they showed a lower CC frequency than patients who spontaneously cleared HCV. Study results confirmed the model of persistent HCV infection as a risk factor for the pathogenesis of both liver and lymphoproliferative disorders.

We found a rare hepatitis C virus (HCV) genotype in 11 Italian and three French patients. The HCV core gene sequence for these patients showed nucleotide similarities of 93–98% to each other, 88–92% to genotypes III (2a) and IV (2b) isolates, and only 82–85% to genotypes I (1a), II (1b), 1c, and V (3a) isolates. The NS5b sequence also indicated the distinctness of this group of HCV, its relatively close relationship to genotype III (2a) and IV (2b), and a high degree of similarity to several isolates provisionally classified as ‘2c’. The El region sequence determined in one French patient also had a high similarity to another ‘2c’ isolate reported previously. Although the prevalence of this ‘2c’ HCV in Italian patients was low ( 11 173 : 6%), it was of clinical interest. This genotype was associated with poor responsiveness to interferon therapy and also with poor antibody response to C100-3 antigen, and two of the 11 Italian patients had mixed cryoglobulinemia. Interestingly, all the three French patients with the ‘2c’ HCV were from Italy, and virtually Italians.

We compared a home-made sequencing system to analyze plasma samples from patients with chronic HBV infection with the commercial TRUGENE(®) HBV Genotyping Assay. A PCR and sequencing protocol based on published primers was applied to detect the viral genotypes as well as the major patterns of point mutations leading to resistance to lamivudine, adefovir and entecavir. For the determination of HBV genotypes the obtained sequences were aligned with a database created within the RIDOM TraceEdit program and publicly available reference sequences. Our results showed perfect correlation with the commercial system, with types D (72%) and A (22%) being the most frequent genotypes. The resistance loci were also reliably detected with mostly combined L180M and M204V/I mutations as the local patterns. M204I mutations were more frequent in genotype D, M204V in genotype A isolates. G173L mutations were not found. The only genotype C isolate tested revealed a different pattern (E263D and I269L). These data speak for the usability of this rapid amplification and sequencing approach for routine genotyping of HBV isolates and simultaneous determination of the drug resistance profile of the dominant viral species.

The gamma delta T cell receptor (TcR) lymphocytes constitute 3-10% of human peripheral blood lymphocytes. Only a very small fraction of these cells is recognized by the delta TCS1 monoclonal antibody, directed against the V delta 1 chain of the receptor. We describe the immunological, virological and clinical data of a small group of seropositive subjects having high levels of gamma delta TcR T cells in the peripheral blood. Our flow cytometric studies show that most of these cells belong to the delta TCS1+ (V delta 1+), CD8 +/- (dim staining) subset. Patients with high gamma delta TcR T cell numbers were not characterized by the presence of an acute (IgM positive) or reactivated (as defined by high IgG titres against early antigen or IgA titres against viral capsidic antigen) Epstein-Barr virus infection. Cytomegalovirus infection was excluded by serological assays, and other herpes viral infections were not found after clinical examination. HIV p24 antigenaemia was present in two out of 11 subjects. AIDS patients had very high percentages of gamma delta TcR T cells. Altogether these data show that the selective expansion of delta TCS1+ cells in HIV1 seropositive subjects is not related to some exogenous antigen stimulation, but may be related to peculiar pathologic processes involving the immune system.

The role of coffee in the aetiology of hepatocellular carcinoma has raised great interest. In Italy, coffee consumption is high, thus allowing the investigation of the topic over a broad range of consumption. A hospital-based case-control study was conducted in Italy in 1999-2002, including 185 incidents, histologically confirmed cases of hepatocellular carcinoma aged 43-84 years. Controls were 412 subjects admitted to the same hospitals' networks for acute, non-neoplastic diseases unrelated to diet. Coffee and tea consumption were assessed using a validated food-frequency questionnaire. Odds ratios (ORs) and corresponding the 95% confidence intervals (CI) were computed using unconditional multiple logistic regression, adjusting for hepatitis viruses seropositivity, alcohol intake, smoking habits and other potential confounding factors. Compared to people who drunk14 cups/week of coffee, the risk of hepatocellular carcinoma decreased for increasing levels of consumption (OR=0.4, 95% CI: 0.2-1.1 foror=28 cups/week, p for trend = 0.02). In the present study, inverse relations were observed across strata of hepatitis C and, B virus infections and alcohol drinking. No significant association emerged with consumption of decaffeinated coffee (OR=0.7, 95% CI=0.2-2.5) or tea (OR=1.4, 95% CI=0.8-2.7). The present study supports the hypothesis of a favourable effect of coffee, though not decaffeinated coffee and tea, on the risk on hepatocellular carcinoma.

Background: Dietary habits have been suggested as a factor related to the increase of non-Hodgkin lymphoma (NHL) incidence in western populations, but the role of individual nutrients is still unclear. Patients and methods: A hospital-based case-control study was conducted in Italy, 1999–2002. Cases: 190 incident, histologically-confirmed NHL cases aged 18–84 years. Controls: 484 subjects admitted to hospital for acute, non-neoplastic diseases unrelated to diet. Dietary habits were assessed by a validated food-frequency questionnaire; nutrient intakes were computed using the Italian food composition database. Odds ratios (ORs) and corresponding 95% confidence intervals (CI) for tertiles of intake of nutrient were computed using the energy-adjusted residual models. Results: Inverse association emerged for polyunsaturated fatty acids (OR=0.6; 95% CI: 0.4–0.9), linoleic acid (OR=0.6; 95% CI: 0.4–0.9), and vitamin D (OR=0.6; 95% CI: 0.4–0.9). The protective effect for linoleic acid (OR=0.3; 95% CI: 0.2–0.7) and vitamin D (OR=0.4; 95% CI: 0.2–0.9) was stronger in women; no differences emerged according to age. Linoleic acid was inversely related to follicular and diffuse large B-cell lymphoma; the protective effect of vitamin D emerged most clearly for follicular subtypes. Conclusions: Our study suggests that a diet rich in polyunsaturated fatty acids and vitamin D is associated with a reduced risk of NHL.

Incidence of non-Hodgkin lymphoma (NHL) has been rising worldwide, but the reasons are undefined. Dietary habits may play a role in the etiology of NHL by influencing the metabolic pathways of several cells of the immune system. This case-control study investigated the relation between food consumption and NHL risk. Between 1999 and 2002, we conducted a hospital-based case-control study on NHL in 2 areas of Italy. Cases were 190 patients (median age 58 years) with incident NHL admitted to specialized and general hospitals. Controls were 484 patients (median age 63 years) with acute non-neoplastic conditions admitted to the same hospitals network of cases. A validated food-frequency questionnaire was used to assess habitual diet 2 years before interview. Unconditional multiple logistic regression was used to estimate the odds ratios (OR) and the corresponding 95% confidence intervals (CI), with allowance for energy intake, according to the residual model. Consumption of highest versus lowest quartile of pasta/rice (OR = 1.87,95% CI: 1.04-3.36) and cheese (OR = 1.66, 95% CI: 0.98-2.83) were associated with a significantly increased NHL risk. Inverse association was found for vegetables (OR = 0.49, 95% CI: 0.28-0.87), fruits (OR = 0.51, 95% CI: 0.304.85), and egg consumption (OR = 0.59, 95% CI: 0.36-0.97). The association of pasta/rice was also supported by an increased risk of high glycemic load levels (OR = 1.86, 95% CI: 1.04-3.32). In conclusion, our results suggested that diet could affect NHL risk. (c) 2005 Wiley-Liss, Inc.

Background Conflicting results were obtained in the assay of anti-transglutaminase (anti-tTG) autoantibodies in patients with chronic liver disease. In order to establish whether this was attributable to methodological differences, anti-tTG antibodies were assayed in a large number of patients suffering from liver cirrhosis (LC). Methods 54 patients with LC and 29 patients suffering from celiac disease (CD), used as controls, were tested for IgA and IgG anti-tTG with 11 different commercial methods. Results In the patients with LC, positivity ranged from 0% to 33.3% for IgA anti-tTG and from 0% to 11.1% for anti-tTG of the IgG class. The largest number of false positives was found with methods that used tTG in association with gliadin peptides as antigen substrate. A significant association was found between IgA anti-tTG antibodies and serum immunoglobulin concentration. Conclusions The results of the various methods of assaying anti-tTG antibodies in patients with LC are highly variable, and the positives found are generally false positives, partly due to the high immunoglobulin concentration.

Comparison of human leukocyte antigen (HLA) frequencies in patients with hepatitis C virus (HCV)-associated hepatocellular carcinoma (HCC) and in patients with HCV-associated non-Hodgkin’s lymphoma (NHL) has not been addressed previously. To this aim, we investigated the distribution of HLA class II alleles in two selected groups of HCV-infected patients. Group 1 included 50 patients with HCV-associated NHL; group 2 included 29 patients with HCV-associated HCC. A control group included 144 hospitalized patients without NHL or HCC and who were negative for HCV, hepatitis B virus, and human immunodeficiency virus antibodies. Polymerase chain reaction sequence DRB1 and DQB1 specific-primer methods were used. DRB1*1101/DQB1*0301 haplotype, which mainly favors the spontaneous clearance of HCV infection, was lower in HCC subjects than in controls, whereas HLA-DRB1*1104/DQB1*0301, was higher in NHL patients. These findings suggest different pathogenic pathways in HCC and in NHL development. In patients with HCV-associated HCC, a major protective role of DQB1*0301 allele, rather than DRB1*11, was found, probably because of a better HLA class II-associated virus clearance. By contrast, the same allele as HLA-DRB1*04 showed an increase in HCV-associated NHL. These data suggest that NHL and HCC development may be associated to a different response with respect to chronic HLA class II-restricted antigen presentation (perhaps a switch toward CD4+Th2 response in NHL?) or, alternatively, that these alleles could be in linkage disequilibrium to unrelated gene(s), or are in synergy with other immunomodulatory genes that may confer increased risk for NHL.

The primary objective of AMCLI is to provide efficient, high quality continuing education.The CoTeLab has prepared a questionnaire including the main groups of interest of Laboratory Technologists, either of a specific or general nature, as well as other eventual arguments, suggestions and observations in order to understand the real necessity of training. The questionnaire was answered by 464 technicians of which 71% females and 29% males, most of interviewed were between 41 and 45 years of age with variable experience. Scolarity was not homogeneous due to Italian evolution in the past years. The necessity to up-date knowledge in the laboratory in which day-to-day work is performed and an elevated interest in molecular biology was evidenced. Quality control, biological hazard and hospital infections were the arguments most requested. The procedure of accreditation and/or certification already obtained in many laboratories, and the problems involved in new technologies are certainly factors that bring attention to Quality Control. The response in respect of biological hazards is due to the implementation of DL 626/94 and its successive modifications. Hospital infections have always been an important subject of microbiology and have gained much attention thanks to the new technologies offered (molecular epidemiology) Theoretical and practical training is mainly requested and evidences the technician’s need to understand routine procedures and not only to perform them. New technologies are the general arguments most requested and preferred. Surprisingly long distance education is not requested, probably since it has not yet been activated and the modality is not clear. In conclusion this study has hallowed us to have a reliable perspective of the educational needs of technologists and will permit us to gradually construct a high quality, efficient continuing education program that will certainly give positive results in the total quality of results referred to physicians.

HLA-A*02:374 differs from HLA-A*02:01:01 by one amino acid change at codon 112 where G is replaced by V.

A new DNA probe assay (INNO LiPA Mycobacteria; Innogenetics, Ghent, Belgium) for the simultaneous identification, by means of reverse hybridization and line-probe technology, of Mycobacterium tuberculosis complex, Mycobacterium kansasii , Mycobacterium xenopi , Mycobacterium gordonae , the species of the Mycobacterium avium complex (MAC), Mycobacterium scrofulaceum , and Mycobacterium chelonae was evaluated on a panel of 238 strains including, besides representatives of all the taxa identifiable by the system, a number of other mycobacteria, some of which are known to be problematic with the only other commercial DNA probe system (AccuProbe; Gen-Probe, San Diego, Calif.), and two nocardiae. The new kit, which includes a control probe reacting with the whole genus Mycobacterium , correctly identified 99.6% of the strains tested; the one discrepancy, which remained unresolved, concerned an isolate identified as MAC intermediate by INNO LiPA Mycobacteria and as Mycobacterium intracellulare by AccuProbe. In five cases, because of an imperfect checking of hybridization temperature, a very slight, nonspecific, line was visible which was no longer evident when the test was repeated. Two strains whose DNA failed amplification at the first attempt were regularly identified when the test was repeated. Interestingly, the novel kit dodged all the pitfalls presented by the strains giving anomalous reactions with AccuProbe. A unique feature of INNO LiPA Mycobacteria is its ability to recognize different subgroups within the species M. kansasii and M. chelonae , while the declared overlapping reactivity of probe 4 with some M. kansasii and Mycobacterium gastri organisms and of probe 9 with MAC, Mycobacterium haemophilum , and Mycobacterium malmoense , may furnish a useful aid for their identification. The turnaround time of the method is approximately 6 h, including a preliminary PCR amplification.

Aims/background Current therapy for chronic hepatitis C virus (HCV) infection is based on the administration of interferon alpha (IFN) alone or in combination with other anti-viral agents. However, such therapy is effective in only a minority of selected patients. Long-term ursodeoxycholic acid (UDCA) treatment has been reported to improve liver function and structure especially in cholestatic disorders. We investigated the effect of long-term UDCA treatment on liver function in respect to the severity of chronic liver disease and HCV genotypes. Methods Forty-five patients with non-cholestatic laparoscopy-biopsy proven HCV-associated chronic hepatitis (n=16) or cirrhosis (n=29) who had not responded to, or were unsuitable for IFN, were randomly assigned to receive UDCA (600 mg/day; n=23) or no therapy (n=22) for 12 months. At entry, all patients were evaluated by means of conventional and quantitative liver function tests (LFTs), including galactose elimination capacity and antipyrine clearance, HCV antibodies, HCV-RNA and HCV genotypes. LFTs were measured at 6 and at 12 months, whereas HCV-RNA was determined again after treatment. Results Baseline characteristics were comparable in the two study groups. Long-term UDCA therapy was well tolerated. Based on the analysis of variance, there was a significant decrease in serum transaminase, LDH and GGT levels in UDCA treated patients. By contrast, the activities of these enzymes increased in untreated patients, with AST levels reaching statistical significance only. Statistical analysis also showed that the improvement in biochemical markers was more pronounced in UDCA treated patients with liver cirrhosis than in those with chronic hepatitis but was similar in patients with HCV genotype 1b and non-1b. However, HCV-RNA was positive in all patients after treatment. Quantitative LFTs remained, on average, stable over the 12 months of the trial in all groups. Conclusions Long-term UDCA treatment is well tolerated in patients with HCV-associated chronic liver disease. The effect appears to be greater in cirrhotics than in patients with chronic hepatitis but is independent of HCV genotypes. Thus, long-term UDCA treatment, despite the absence of an anti-viral effect, seems beneficial in reducing disease activity in patients with chronic hepatitis or cirrhosis who are unsuitable for IFN therapy.

A pathogenetic role of the hepatitis C virus (HCV) has been hypothesized for a subset of B-cell non-Hodgkin's lymphomas (NHLs). However, the preliminary characterization of B-cell NHLs in HCV-infected individuals has been poorly addressed. In the present study, we report detailed information on 35 consecutive patients with overt B-cell NHL of recent onset and HCV infection; all patients referred to a single oncological center in Northeast Italy. Histopathologic evaluation was performed by a single reference hemopathologist, and the link with the two relevant autoimmune diseases predisposing to B-cell NHL and in which HCV has been implied, ie, “essential” mixed cryoglobulinemia (EMC) and Sjögren's syndrome, was investigated. Control groups included 122 consecutive HCV-negative patients with B-cell NHL and 464 consecutive histopathologic cases of B-cell NHL referred to the same center, as well as 127 consecutive patients with HCV infection and without lymphoma referred to a different center in the same geographical area. B-cell NHLs in HCV-infected patients frequently presented at onset (1) an extranodal localization with peculiar target organs of HCV infection (ie, the liver and major salivary glands) being significantly overrepresented; (2) a diffuse large cell histotype without any prior history of low-grade B-cell malignancy or bone marrow involvement; and (3) a weak association with a full-blown predisposing autoimmune disease, although serum autoimmune features were common and cryoglobulins were always present. Therefore, the HCV-related B-cell NHLs in this oncological series presented distinctive features compared with B-cell NHLs in HCV-negative patients, and they differed from bone marrow low-grade NHLs frequently diagnosed in HCV-positive patients with EMC. Such novel information may be relevant for future research aimed at clarifying the possible link between HCV infection, autoimmunity, nonmalignant B-cell lymphoproliferation, and overt B-cell malignancy.

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-Hodgkin's 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). The presence of anti-HCV antibodies was determined by a commercial kit and, in positive cases, by PCR amplification of the 5' untranslated region of the virus. The HCV genotype was also obtained by PCR amplification of the Core region with type-specific primers. The presence of serum cryoglobulins was determined in each case of NHL. HCV infection was significantly (P < 0.00000001) higher in patients with non-Hodgkin's lymphomas (28.0%) when compared with that of the general population (2.9%), and with the group of patients affected by other malignancies (3.1%). The prevalence is particularly high in low-grade (38.4%), as compared with intermediate (11.4%), or high-grade (15.2%) lymphomas. The presence of the virus is significantly (P < 0.000001) associated with the presence of detectable levels of cryoglobulins. On the basis of these findings. HCV seems to play an important role in the development of low-grade non-Hodgkin's lymphomas.

Objectives. As a close relationship has been established between mixed cryoglobulinaemia and hepatitis C virus (HCV) infection, the clinical, histological and virological findings of patients affected by mixed cryoglobulinaemia were determined. Design. Hepatitis C virus infection was investigated by the presence of anti-HCV antibodies and PCR amplification of the 5′ untranslated region (5′ UTR), and the genotype of HCV was also determined according to Okamoto. A bone marrow biopsy was performed in all patients and liver and kidney biopsies when indicated. Subjects. Eighty-two subjects affected by mixed cryoglobulinaemia were enrolled in this study. Results. The prevalence of anti-HCV antibodies was high (83%); PCR amplification of the 5′UTR region was performed in 52 subjects and in 44 of them (85%) the results were positive. In the same subjects, the Core region amplification was positive in 46 cases (88%). A high prevalence of genotype II was found (54%). Chronic liver disease was present in 55 patients (67%). Bone marrow biopsies showed the presence of low-grade non-Hodgkin's lymphomas in 11 cases (13%). Membrano-proliferative glomerulonephritis was found in seven subjects (8%). Conclusions. Mixed cryoglobulinaemia is associated with HCV infection in the nearly all cases. Several HCV genotypes are involved in the pathogenesis of this disease. Mixed cryoglobulinaemia is associated with a high prevalence of chronic liver disease, low-grade non-Hodgkin's lymphomas and membranoproliferative glomerulonephritis.

The aim of the study was to assess the role of different viral strains of hepatitis C virus (HCV) in determining the outcome of the alpha-interferon (IFN) therapy. Fifty-seven patients (34 from Italy and 23 from Japan) with HCV-positive liver disease were enrolled in the study. The NS4 region of HCV was amplified in sera by “nested” polymerase chain reaction (PCR) using a primer pair synthesized according to the sequence of JK-1. The NS4 region was positive in 14 (41%) Italian and in 13 (56%) Japanese patients. In positive patients the sequence of the NS4 region was also obtained. Subsequently, HCV genotype was determined in all patients by PCR amplification of the core region. All patients received recombinant alpha2a-interferon (IFN), 6 million units 3 times a week for 1 month followed by 3 million units 3 times a week for 5 months. The patients were followed for 1 year after the end of treatment. At the end of the follow-up, 17 (30%) had sustained normal levels of serum alanine aminotransferase (ALT). The outcome of treatment was not correlated with race, age, sex, histology, and pretreatment ALT level, but was significantly (P < 0.00001) associated with the presence of both the NS4-JK-1 region and HCV type II. Among the 27 NS4-positive patients, only 1 patient (3.7%) achieved a complete response, whereas the remaining 26 patients (96.3%) either were non-responders or relapsed after IFN was discontinued. In contrast, among the 30 NS4-JK-1-negative patients, 15 (53%) had a sustained remission. HCV genotyping showed type I in 3 (6%), type II in 40 (74%), type III in 4 (7%), and type IV in 3 (6%) cases. Coinfection was present in 4 (7%), while in 3 cases amplification was not obtained. Patients with type II were all non-responders or relapsers, while a response to the treatment was oberved in 17 of 17 (100%) of the remaining patients. These data indicate that the presence of JK-1 variant of HCV or HCV type II is almost always predictive of a poor response rate Of IFN therapy. © 1995 Wiley-Liss, Inc.

The presence of the “Japanese type” NS4 region was investigated in two series of patients (53 from Italy and 58 from Japan) with hepatitis C virus (HCV)-related chronic liver disease. The two populations were homogeneous as regard to age, male/female ratio, histological diagnosis, and serum aminotransferase activities. Genomic amplification was carried out by “nested” poly-merase chain reaction (PCR) with a pair of primers synthesized according to the sequence of JK-1 isolated in Japan. The presence of viral replication was confirmed further by PCR amplification of the 5′NC region. The NS4 region of the Japanese strain was detected in 24 sera (45%) from Italy and in 44 (71%) from Japan. NS4-posi-tive patients were significantly older and showed an ALT serum level significantly lower (P < 0.01) than NS4 negative cases in each group. Cirrhosis was significantly (P < 0.0007) more common in NS4-positive than in NS4-negative patients. The HCV genotype was subsequently obtained according to Okamoto. All the NS4-positive patients were infected by Type II, whereas in NS4-negative patients all four genotypes were present though Type II still constituted the majority. Cirrhosis was associated exclusively with Type II both in NS4-positive and -negative subjects. These data indicate that, although the pos-itivity for NS4 “Japanese” region seems to be associated with a more aggressive liver disease, the most prevalent Type II predicts more specifically those who are likely to develop cirrhosis. Since the same findings were obtained in two population from different areas of the world, the genotype of HCV appears to be one of the major determinants of the progression of chronic active hepatitis to cirrhosis. © 1994 Wiiey-Liss, Inc.

Objective To explain the lack of genotype-phenotype correlation observed in a patient double heterozygous for the 852del22 and F508del mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Design Case report. Setting Medical laboratory department. Patient(s) A 42-year-old asymptomatic patient underwent genetic screening for in vitro fertilization (IVF). Intervention(s) CFTR genetic screening (commercial kit aimed at detecting 57 mutations), segregation analysis, evaluation of the polymerase chain reaction (PCR) products using a denaturing high performance liquid chromatography (DHPLC), and sequence analysis. Main Outcome Measure(s) To avoid diagnostic errors and improve genetic counseling. Result(s) Segregation analysis allowed us to establish that the mutations were in trans . Analysis of the PCR products using a DHPLC apparatus showed a heteroduplex formation indicative of a heterozygous variant in exon 6A. Direct sequencing characterized the heterozygous variant as an A to T transversion at nucleotide position 875+11. Therefore, the change of one single nucleotide in a portion surrounding the 852del22 mutation facilitated the aspecific interaction between the commercial oligonucleotide probe and the amplified genomic DNA, which explains the 852del22 mutation false molecular positivity that was detected by the line probe assay. Conclusion(s) The individualization of 852del22 mutation by a standard genetic panel should be confirmed by more extensive analyses.

Prevalence and determinants of HIV infection were assessed in 313 parenteral drug addicts admitted to five Centers for Drug-Addict Assistance and two prisons located in the northeast of Italy (Friuli Venezia-Giulia), an area some distance from the major Italian cities first reached by the AIDS epidemic. The overall prevalence of HIV positivity was also rather high in this area, ie 30% with 24-36% confidence interval. The most important risk factors (besides syringe sharing applicable to all drug addicts), turned out to be of a geographical nature, ie living in Pordenone province (where a US military base is located) or coming from other endemic areas and having travelled long distances in the past three years. Prostitution also seemed to increase the risk of infection, but duration of drug addiction had little effect. In addition to persistent generalized lymphadenopathy (closely linked to HIV infection), reversal of helper:suppressor T-cell ratio was found in 67% of HIV positive individuals, but also in 31% of HIV negative ones. Clinical signs and symptoms strongly linked with HIV positivity were fatigue, weight loss, splenomegaly, fever and nocturnal sweats.

Seroepidemiological, clinical, immunological, and pathological features were studied in 315 intravenous drug abusers (IVDA) seen in five centers for drug addicts' assistance in the Friuli Venezia-Giulia region of Italy, close to the borders of Austria and Yugoslavia. No case of AIDS has been observed. Sixty-five (21%) were affected by persistent generalized lymphadenopathy (PGL). HTLV-III seropositivity was noted in 86 (27%) of the overall 315 IVDA, in 50 (77%) of 65 patients with PGL, and in 1 (0.5%) of 205 blood donors tested as a control group. Patients with PGL had a significantly lower OKT4/OKT8 ratio than the rest of the IVDA population and controls. Systemic symptoms were present in 52% of the patients with PGL, the most frequent symptoms being fatigue and night sweats. In 20 patients with PGL, DR typing revealed a significant increase in DR-5 frequency and a significant decrease of DR-2 frequency. The predominant histological features in the lymph nodes taken from 25 patients consisted of an exuberant follicular hyperplasia, capillary vessel proliferation, and plasmacytosis. Nineteen (22%) females reporting occasional prostitution were compared to 10 non-IVDA female prostitutes and concomitantly evaluated. HTLV-III seropositivity was noted in 11 (58%) of 19 IVDA female prostitutes and in none of the 10 non-IVDA prostitutes. Thirty-five couples composed of both IVDA were compared to 24 couples composed of an IVDA and a non-IVDA. Among the 24 couples of whom one or both partners were seropositive, concordance in HTLV-III seropositivity was present in 5/11 (45%) couples composed of both IVDA, and in only 1/13 (8%) couples composed of an IVDA and a non-IVDA. This suggests that the sharing of contaminated needles, universally practiced by our IVDA population, plays a more important role in the transmission of HTLV-III than sexual contact.

We have used 4B4 and 2H4 monoclonal antibodies in conjunction with OKT4 to quantify T cell subsets in lymph node suspensions from human immunodeficiency virus (HIV) positive subjects with lymphadenopathy syndrome. The data indicate that the reduced OKT 4:OKT 8 ratio was due to a depletion of the OKT 4 + 4B4 + subset. In contrast, there were no differences compared to reactive controls, considering the OKT 8 + subpopulation. These alterations may be related to the immunological deficiency associated with HIV infection.

The CD4 antigen has long been used as a surface marker for the indentification of a subset of T cells. CD4 acts also as the receptor for the Human Immunodeficiency Virus 1 HIV-1 and as a signal transducing molecule. Recently CD4 positive cells have been shown to release a soluble form of CD4 (sCD4). This molecule can interact with free gp 120 viral antigen and with serum immunoglobulins. Because of the importance of CD4 in the natural history of HIV-1 infection, we decided to monitor it's serum levels in HIV-1 seropositive subjects. sCD4 increases, on a per cell basis, in the late stages of infection, when CD4 cell counts dramatically fall and antigenemia is frequently found. Asymptomatic patients had a mean of 6 Units/ml of sCD4 every 100 CD4 T cells, while in AIDS patients we found a mean of 51 Units/ml every 100 CD4 T cells. These data suggest that sCD4 may be of value in monitoring the involvement of CD4 T cells during a pathological event.

Hepatitis C virus (HCV) has been implicated as the major etiologic factor sustaining B-cell clonal expansion in type II mixed cryoglobulinemia (MC). A putative pathogenetic role of HCV in the development of MC-associated B-cell malignancies has also been speculated. We report for the first time the localization of HCV within a parotid non-Hodgkin's lymphoma (NHL) lesion in the course of HCV- related type II essential MC, an important step to implicate any infectious agent in the lymphomagenesis. Plus and minus strand HCV RNA was first demonstrated by polymerase chain reaction on the whole RNA from the lesion. Further immunohistochemical studies localized HCV c22 proteins in the residual ductal or acinar parotid structures, which also abnormally expressed HLA-DR antigens. Weak c22 signals were inconstantly detected in cells strictly confined around the residual epithelium, while all the remaining infiltrating cells in the parotid lesion stained c-22-negative. Staining for c33 and c100 HCV antigens was negative. In situ hybridization (ISH) studies again identified the residual parotid epithelial cells as the site of HCV infection and replication in the NHL lesion. Sialotropic viruses previously involved in lymphoproliferation, ie, Epstein-Barr virus and human herpesvirus-6, were absent in the same tissue lesion. According to the current models of B-cell lymphomagenesis, a role of HCV as an exogenous antigenic stimulus should be considered for NHL development in the present case, whereas malignant B cells do not appear permissive of active HCV replication. Further efforts would be worthwhile to clarify a role of HCV infection in the development of some B-cell malignancies.