Generation of metastatic variants in populations of mutator and amplificator mutants.

Genetic instability has been hypothesized by P. C. Nowell and other investigators to be an important aspect of tumor progression that leads to the generation of metastatic variants. In this study we examined the rate of generation of metastatic variants in mutant cell lines having increased rates of spontaneous mutation and gene amplification. Parallel clonal populations of the spontaneous mutation rate mutant thy-49 and the gene amplification mutants YMP1 and YMP7 and their respective wild types were generated and grown to a critical population size. The number of metastatic variants in each clonal population was then determined following iv injection into nude mice. Lung tumors were scored 3-4 weeks after injection of cells, and the mean number per clonal population was determined. Analysis of the means with the Luria-Delbruck fluctuation test showed no significant differences in the rate of generation of metastatic variants produced in the genetically unstable lines compared to their normal counterparts. This study suggests that increased spontaneous mutation and gene amplification rates in mammalian cells are not sufficient on their own to increase the rate of generation of metastatic variants.

Etiology of hepatocellular carcinoma in Italian patients with and without cirrhosis.

We performed a case-control study to assess the role of hepatitis B virus (HBV), hepatitis C virus (HCV), GB virus C/hepatitis G virus (HGV), TT virus, alcohol intake, and tobacco smoking as risk factors for hepatocellular carcinoma (HCC) in the presence or absence of cirrhosis. We prospectively recruited 174 patients with a first diagnosis of HCC admitted to the main hospitals in Brescia, North Italy. On the basis of histological, clinical, and radiological criteria, the presence of cirrhosis was established in 142 cases, excluded in 21 cases, and remained undefined in 11 cases. Among the HCC cases without cirrhosis, a histological picture of normal liver was found in a single patient, chronic viral hepatitis was found in 11 patients, alcoholic hepatitis was found in 5 patients, nonspecific reactive hepatitis was found in 3 patients, and hemochromatosis was found in 1 patient. As controls, we also included 610 subjects unaffected by hepatic diseases and admitted to the same hospitals as cases. The odds ratios for having HCC according to positivity for HCV RNA, HBsAg and/or HBV DNA, and alcohol intake > 80 g/day (95% confidence interval) were as follows, in the presence and absence of cirrhosis, respectively: (a) 33.5 (17.7-63.4) and 19.7 (6-64.8) for HCV RNA; (b) 17.6 (9.0-34.4) and 20.3 (5.7-72.6) for HBsAg; and (c) 5.5 (3.1-9.7) and 4.6 (1.5-13.8) for alcohol intake. No association was found with HGV or TT virus infections or tobacco. This study has shown that most HCC cases arising in the area are due to HBV, HCV, or alcohol intake, in both the presence and absence of cirrhosis.

Hepatitis C virus infection in patients undergoing allogeneic bone marrow transplantation.

Antibody to the recently identified hepatitis C virus was investigated in sera of 128 patients treated with allogeneic bone marrow transplantation, to determine the prevalence of HCV infection and its role in post-transplant liver complications. The overall prevalence of anti-HCV positivity was 28.6% (38/128 patients). The presence of pretransplant anti-HCV positivity (in 10/35 tested patients) did not seem to predict a more severe liver disease. In fact 8/10 anti-HCV+ and 15/25 anti-HCV- patients had elevated transaminases at BMT, and posttransplant liver failure (due to VOD or subacute hepatitis), and post-BMT rises in transaminases occurred regardless of anti-HCV serology (P = 0.6 and 0.2, respectively). In patients tested for anti-HCV after BMT (n = 128), only two (one anti-HCV+ and one anti-HCV-) experienced VOD; the number of patients in whom liver failure contributed to death was comparable in anti-HCV-positive and anti-HCV- negative patients (P = 0.4). Among 17 patients with documented posttransplant seroconversion (from anti-HCV- to anti-HCV+) the appearance of anti-HCV was concomitant with hepatitis exacerbation in 9 (53%). Histologic changes demonstrated a more severe liver damage in anti-HCV+ patients: a chronic hepatitis was diagnosed in 9/11 anti-HCV+ versus 1/7 anti-HCV- cases. Based on these observations, we conclude that hepatitis C virus has a role in liver disease in such patients, although its evaluation by the anti-HCV test is still of limited accuracy, due to low sensitivity and incomplete specificity.

Lack of correlation between deoxyribonucleotide pool sizes, spontaneous mutation rates and malignant potential in Chinese hamster ovary cells.

To examine the relationship between altered spontaneous mutation rates and malignant characteristics of cells, two hydroxyurea-resistant Chinese hamster ovary cell lines, with alterations in ribonucleotide reductase, were examined for their rates of spontaneous mutation to 6-thioguanine and ouabain resistance, tumor growth rates and their ability to form experimental lung metastases. The most resistant cell line, HR-R2T, showed no changes in the rate of spontaneous mutation to 6-thioguanine or ouabain resistance compared to the parental wild-type cell line; however, the mutant line formed lung metastases in experimental metastasis assays with BALB/c nu/nu mice, and exhibited metastatic abilities significantly different from the wild-type population. Furthermore, the HR-R2T population did not show imbalances in any of the deoxyribonucleoside triphosphate pool sizes, which are frequently observed in cells altered in ribonucleotide reductase activity. The second hydroxyurea-resistant line, HNR-AT, had gross alterations in dCTP and dGTP pools and although the rate of spontaneous mutation to 6-thioguanione resistance was unaltered, it showed a moderate decrease in the rate of spontaneous mutation to ouabain resistance when compared to the parental wild-type population. Interestingly, the HNR-AT cell line did not form any lung metastases in the experimental metastasis assay. Both mutant cell lines, HR-R2T, and HNR-AT, had increased tumor growth rates in C57 BALB/c "beige" nude (nu/nu) mice as compared to the parental wild-type population. In total, the results obtained with the two mutant cell lines question the association of altered mutation rates with increased metastatic potential. Although several explanations are possible for the altered malignant properties exhibited by HR-R2T and HNR-AT cells, it is interesting to note that the results are consistent with earlier suggestions that changes in ribonucleotide reductase may accompany modifications in the malignant characteristics of cells.

Interferon therapy of cryptogenic chronic active liver disease and its relationship to anti-HCV.

In a randomized controlled trial of Interferon (IFN) in 60 patients (30 treated and 30 controls) with cryptogenic chronic active liver disease, 70% of treated patients showed complete response, but a high rate of biochemical relapse (62%) was noted. In these cases, a second response to higher doses of IFN has been more difficult and less frequent. A response to IFN was found in 88.5% of anti-HCV positive treated patients and only in 25% of anti-HCV negative. We suggest that serum anti-HCV is a suitable test to predict the response to IFN.

Significance of hepatitis C virus genotypes and viral load in patients with "essential" mixed cryoglobulinemia.

Several studies have established a strong association between hepatitis C virus (HCV) infection and essential mixed cryoglobulinemia (EMC). However, the mechanisms by which HCV infection may result in cryoglobulinemia in some patients but not in others remain unknown. In this paper we shall summarize some of the work done in our laboratories on certain aspects of HCV in patients with EMC.

A 1-year trial of lamivudine for chronic hepatitis B in children.

We carried out a 1-year trial to evaluate the efficacy and tolerability of lamivudine, an oral nucleoside analogue, in a small group of children with vertically acquired chronic hepatitis B. Patients were assessed for serum alanine aminotransferase (ALT) and serum hepatitis B virus (HBV) DNA at baseline and every 4 weeks thereafter, and for hepatitis B s antigen, hepatitis B e antigen and their antibodies every 12 weeks. Analysis of HBV mutation was undertaken at entry and on the occasion of the last positive control of HBV DNA. Lamivudine suppressed serum HBV DNA to undetectable levels in all treated patients within 24 weeks. Serum ALT levels returned to normal values within 36 weeks. Therapy was well tolerated, and although nausea and vomiting were reported in one child, it was not necessary to stop treatment. A new observation was that, contrary to previous data, seroconversion appeared to occur earlier in children with lower ALT levels at baseline.

[Treatment of cryptogenic chronic liver diseases with recombinant alpha-2a interferon. Preliminary results of a randomized controlled clinical trial]. / Trattamento delle epatopatie croniche criptogenetiche con interferone alfa-2a ricombinante. Risultati preliminari di uno studio clinico randomizzato controllato.

Fifty-six consecutive patients with cryptogenic chronic active liver disease were enrolled in a randomized controlled clinical trial of alpha-2a interferon and randomly assigned to a control group (28 patients) and to a treated one (28 patients). All had a histologic diagnosis of chronic active hepatitis (with superimposed cirrhosis in 50% of them) and presented a persistent elevation in aminotransferases, during the last year. Ad-interim analysis shows that 19 out of 28 treated patient (68%) have normalized the aminotransferases during the eight months of therapy, with a statistically significant difference (p less than 0.01) between treated and control group; nevertheless, in 58% of them, we noted rising aminotransferases at low doses of interferon with subsequent normalization when the dose was increased to previous effective levels. Retrospectively, the antibody directed to virus C (anti-HCV) was found positive in 84% of our patients, and its presence was strongly associated with response to interferon treatment. Our preliminary results seem to demonstrate that interferon is truly effective, mainly at high doses, in cryptogenic chronic active liver disease; these data with the high prevalence of anti-HCV and the association between anti-HCV and response to therapy, may confirm a possible etiologic role of virus C in causing this subgroup of liver disease.

Molecular and cellular characterization of drug resistant hamster cell lines with alterations in ribonucleotide reductase.

Ribonucleotide reductase consists of 2 protein components frequently called M1 and M2. Hydroxyurea specifically inhibits DNA synthesis by interacting with the M2 protein and destroying a unique tyrosyl-free radical. We have carried out a molecular and cellular characterization of 2 Chinese hamster ovary cell lines exhibiting either low (HN(R)-AT) or relatively high (H(R)-R2T) resistance to the cytotoxic effects of hydroxyurea. Both drug-resistant lines have an increased level of ribonucleotide reductase activity. EPR measurements for tyrosyl-free radical content and studies with M1-specific antibodies indicated that the elevation in enzyme activity was entirely due to an increase in the M2 component. Studies with M1 cDNA showed that both drug-resistant cell lines contained a wild-type level of M1 mRNA and a wild-type M1 gene copy number. Studies with M2 cDNA indicated that the 2 drug-resistant lines possessed elevated levels of M2 message that could explain the observed increase in M2 component. The elevation of M2 mRNA in the most resistant line, H(R)-R2T, was due to an increase in M2 gene copy number. The low resistant cell line, HN(R)-AT, exhibited a wild-type M2 gene copy number, indicating that the increase in M2 gene message occurred through a process other than gene amplification. Enzyme kinetic studies with partially purified preparations from both drug resistant lines showed reduced sensitivity to hydroxyurea and to the negative allosteric effector, dATP. In addition to hydroxyurea, H(R)-R2T cells were also resistant to several other drugs whose site of action is the M2 component. Furthermore, H(R)-R2T cells were not cross-resistant to colchicine or puromycin, suggesting that hydroxyurea-resistant cells do not share the multi-drug resistance phenotype, which is frequently associated with cross-resistance to these drugs.

A comparison of ribonucleotide reductase activities in normal human fibroblast strains with their transformed counterparts.

The objective of this investigation was to examine the relationship between levels of ribonucleotide reductase activity and transformation of two human cell strains. Enzyme activity levels were elevated by 3.2- to 3.5-fold in transformed cells compared directly to the normal human fibroblast strains from which they were derived. There did not appear to be a general correlation between elevated ribonucleotide reductase and increased proliferation abilities as has been previously observed with some rodent tumor cell lines. In keeping with the rise in reductase activity, human transformed cells were relatively more resistant to the cytotoxic effects of hydroxyurea, an antitumor agent whose site of action is ribonucleotide reductase. This indicates that an important point to be considered during drug therapy aimed at the reductase, is the greater sensitivity of normal compared to transformed cells due to differences in enzyme activity. The results of this investigation support the view that an increased ability to reduce ribonucleotides is an important step towards the development of a neoplastic program in human cells.

HIV antibody screening and confirmatory testing of Italian blood donors. One-year experience of a reference center.

During the first year (1986) of blood donor screening for antibody to HIV, 201,750 subjects were tested in 40 blood banks of Lombardia (Italy). All sera repeatedly positive by ELISA were submitted to our reference center for confirmation by Western blot (WB). Only 40 (0.02%) of 286 repeatedly reactive donors were positive by WB, whereas another 45 (0.022%) gave atypical antibody reactivities on WB, mainly directed against HIV core proteins. Of the 16 donors with inconclusive WB results followed for 4-12 months, 3 developed a full-blown antibody response, 5 maintained the anti-core reactivity throughout the follow-up period, and 8 lost all reactivities. The use of recombinant env and core antigen ELISAs seems to decrease the proportion of sera with inconclusive WB reactions, and to identify as true positive all seroconverting donors in advance of the WB test. The large majority (35 out of 40) of WB-positive donors and all seroconverters for antibody to HIV admitted to belong to a group at risk for AIDS. Among the 19 first-time donors with HIV infection, we found 3 subjects with serological evidence of LAV-2 infection. We describe also the diagnostic and ethical issues when a donor notification policy is based on WB confirmatory procedures.

Hydroxy[14C]urea uptake by normal and transformed human cells: evidence for a mechanism of passive diffusion.

The antitumor agent hydroxyurea is a potent inhibitor of cell division and selectivity toxic for rapidly proliferating cells. This drug has been used in the treatment of human cancer and, since drug transport is an important aspect of drug action, we investigated the mechanism of hydroxy[14C]urea uptake by human diploid fibroblasts and their SV40-virus-transformed counterparts. Kinetic analysis of drug uptake, studies with metabolic inhibitors, and estimates of cell/medium distribution ratios and temperature coefficient (Q10) values indicated that hydroxyurea enters normal and SV40-virus-transformed human cells by a mechanism of diffusion.

[Epidemiology of tetanus]. / Epidemiologia del tetano.

The vaccinal preventive measures which were made compulsory since 1963 for the new working levies of some professional high risk categories, and since 1968 for the newborns, contributed to a large extent to the tetanus control in our country. In the last twenty years a progressive drop in the notified tetanus cases was observed. The epidemiology of this disease is conditioned, on the one hand by the correct application of the legal directions in force; on the other hand, by the level of health education, acquired by those who have not yet been involved, so far, by the compulsory antitetanus vaccination.

Altered expression of ribonucleotide reductase and role of M2 gene amplification in hydroxyurea-resistant hamster, mouse, rat, and human cell lines.

Five hamster, mouse, and rat cell lines resistant to the cytotoxic effects of hydroxyurea have been characterized. All cell lines contained increased ribonucleotide reductase activity, elevated levels of the M2 component of ribonucleotide reductase as judged by electron paramagnetic resonance spectroscopy, and increased copies of M2 mRNA as determined by Northern blot analysis. Two species of M2 mRNA were detected in rodent cell lines, a high-molecular-weight species of approximately 3.4 kb in hamster and rat cells and about 2.1 kb in mouse cells. The low molecular-weight M2 mRNA was about 1.6 kb in all rodent lines. Northern blot analysis showed that the mRNA for the other component of ribonucleotide reductase, M1, was not markedly elevated in the drug-resistant cells and existed as a single 3.1-kb species. Four of the five resistant lines contained an M2 gene amplification as determined by Southern blot analysis, providing direct evidence to support earlier suggestions that hydroxyurea resistance is often accompanied by amplification of a ribonucleotide reductase gene. An increase in gene dosage was detected even in cells exhibiting only modest drug-resistance properties. No evidence for amplification of the M1 gene of ribonucleotide reductase was found. In keeping with these observations with drug-resistant rodent lines, a human (HeLa) cell line resistant to hydroxyurea was also found to contain increased levels of two M2 mRNA species (about 3.4 and 1.6 kb) and exhibited M2 gene amplification. One hamster cell line resembled the other resistant rodent lines in cellular characteristics but did not show amplification of either the M1 or M2 gene, providing an example of a drug-resistant mechanism in which an elevation of M2 mRNA has occurred without a concomitant increase in M2 gene copy number.

Prevention of viral hepatitis in dialysis units: isolation and technical management of dialysis.

The contamination of dialysis machines is an important risk factor for the spread of viral hepatitis in hemodialysis units. The use of reserved machines in separate rooms is a safe prophylactic measure.

Hepatitis C virus antibody response in acute and chronic non-A, non-B hepatitis.

Patients with post-transfusion, community-acquired or hemodialysis-acquired non-A, non-B hepatitis (NANBH) were tested for antibody to hepatitis C virus (HCV) during acute-phase and resolving or chronicized illness. HCV appears to be involved in most cases of post-transfusion and hemodialysis-acquired NANBH, but only in 40% of community-acquired NANBH. Second generation HCV antibody assays are more specific and sensitive, favoring early detection of HCV seroconversion and identification of HCV-antibody-positive individuals years after exposure to the virus.

Hepatitis C virus genotype in anti-HCV-positive hemodialysed patients.

We investigated the influence of hepatitis C virus (HCV) genotypes on the clinical course of HCV infection in a haemodialysis population. In June 1991, a 4 year prospective follow-up programme was implemented in 184 consecutive haemodialysis patients. Alanine aminotransferase (ALT) and gamma glutamine transferase (GGT) were performed every 2 months. When HCV antibody (Ab) (by second-generation ELISA) was positive, it was confirmed by RIBA 2 and HCV RNA amplification by PCR. The pattern of nucleotide sequence variability in the 5' non-coding region was categorized according to Simmonds' genotype classification. Risk factors including blood transfusions were evaluated. The levels of hepatic enzymes in HCV Ab-positive patients were retrospectively studied over a mean period of 11.8 years. ALT and GGT levels were assigned a score for every year of infection (0 = normal, 1 = fluctuating 2 = high levels). Fifty-two patients were HCV Ab reactive (30.4%), eight were RIBA undetermined and 44 were RIBA positive; 40 of these were HCV RNA positive (91%). Twelve patients were HCV RNA negative, suggesting that they had recovered from the infection. Four genotypes were identified: 1b [26 patients (65%)], 1a (one patient), 2 [12 patients (30%)] and 3 (one patient). The genotype distribution was not different from that found in patients with chronic hepatitis C and normal renal function of the same geographical area. Genotype 1b accounted for 75% of the cases before 1985 and an equal prevalence of the two major genotypes was observed after 1985. Patients infected with HCV subtype 1 had normal mean ALT levels, but higher levels in the follow-up period (28 +/- 15.6 IU/l) and higher ALT and GGT personal scores in the retrospective study. Genotype 1 patients had higher mean ALT levels after 6 months. HCV RNA-negative patients had lower ALT levels after 24 months. RIBA pattern could differentiate the patients. Patients with genotype 1 received a higher number of transfusions, while only 50% of HCV RNA-negative patients had been transfused. Our data suggest a worse course of HCV infection in haemodialysis patients infected with HCV subtype 1, but the severity of HCV infection can only be assessed by histology. Transaminases are only loosely correlated with severity.

Acute non-A, non-B hepatitis in Italy: a 16-year prospective epidemiological study. The possible role of hepatitis C virus.

During a survey of acute symptomatic viral hepatitis conducted in Padua over the last 16 years, 404 (20%) cases of non-A, non-B hepatitis were observed, including 55% with overt parenteral exposure (35% drug abusers) and 45% with unknown exposure. Between 1978 and 1982 the attack rate of the disease increased significantly (p < 0.01) in males, (from 3.8 to 17.3/10(5) inhabitants), in adolescents and in youths. The prevalence of drug abusers rose up to 58% in 1982 suggesting the occurrence of an outbreak in this risk group. In subsequent years the attack rate returned to initial levels in males, although drug abuse still remains the single most important route of infection, and declined in females, especially after the disappearance of post-transfusion hepatitis since 1991. Retrospective anti-HCV testing of patients seen up to 1990 and prospective investigation of patients hospitalized later have shown an antibody prevalence of 88% among parenterally transmitted cases, and of 29% in the other patients, without significant differences between the prospective and the retrospective study. These findings suggest that an outbreak of hepatitis C occurred in our area in the early eighties and that drug abuse is still the most important mode of transmission of acute hepatitis C.