Development and validation of a UPLC-UV method for the quantification of thiopurine methyltransferase enzyme activity in human erythrocytes.

Thiopurines are drugs widely used for the treatment of autoimmune conditions, inflammatory bowel disease or acute lymphoblastic leukemia. Determination of thiopurine methyltransferase activity (TPMT), a major determinant of thiopurines toxicity, has been suggested before implementing thiopurine treatment. An ultraperformance liquid chromatography (UPLC) method was developed and validated for the quantification of TPMT enzyme activity based on the conversion of 6-mercaptopurine (6-MP) to 6-methylmercaptopurine (6-MMP) using S-adenosyl-L-methionine (SAM) as methyl donor in red blood cell lysates (RBC). This method was improved from a previous laborious high performance liquid chromatography (HPLC) method, using a lower volume of injection and with a shorter runtime. After incubation and protein precipitation 6-MMP was separated on a HSS-T3 (2.1 × 50 mm, 1.8 µm) column and monitored by UV detection (290 nm). A change on the organic solvent used to dissolve 6-MP resulted in a reduction of interference by endogenous or non-enzymatic methylated 6-MMP. A full validation of the 6-MMP assay was performed according to the FDA and EMA guidelines. The method was linear from 0.125 to 2 nmol/mL, with acceptable values of accuracy and precision. The method was applied in 106 patients treated with thiopurines whose TPMT activity was previously quantified by HPLC. Evaluation through Bland-Altman plot showed that TPMT activities were in agreement between both methods.

Long-Term Clinical Impact of Adaptation of Initial Tacrolimus Dosing to CYP3A5 Genotype.

Pretransplantation adaptation of the daily dose of tacrolimus to CYP3A5 genotype is associated with improved achievement of target trough concentration (C0 ), but whether this improvement affects clinical outcomes is unknown. In the present study, we have evaluated the long-term clinical impact of the adaptation of initial tacrolimus dosing according to CYP3A5 genotype: The transplantation outcomes of the 236 kidney transplant recipients included in the Tactique study were retrospectively investigated over a period of more than 5 years. In the Tactique study, patients were randomly assigned to receive tacrolimus at either a fixed dosage or a dosage determined by their genotype, and the primary efficacy end point was the proportion of patients for whom tacrolimus C0 was within target range (10-15 ng/mL) at day 10. Our results indicate that the incidence of biopsy-proven acute rejection and graft survival were similar between the control and the adapted tacrolimus dose groups, as well as between the patients who achieve the tacrolimus C0 target ranges earlier. Patients' death, cancer, cardiovascular events, and infections were also similar, and renal function did not change. We conclude that optimization of initial tacrolimus dose using pharmacogenetic testing does not improve clinical outcomes.

Role of the lean body mass and of pharmacogenetic variants on the pharmacokinetics and pharmacodynamics of sunitinib in cancer patients.

INTRODUCTION: Sunitinib is a multikinase inhibitor active in various cancers types including renal cancers and endocrine tumors. The study analyzed the influence of the lean body mass (LBM) and of pharmacogenetic variants on the exposure to sunitinib and its active metabolite, SU12662, and on sunitinib toxicity and clinical activity. MATERIALS AND METHODS: Exposure to sunitinib and SU12662 was assessed on days 10 and 21 during the first treatment cycle. Acute toxicity was graded using the NCI 4.0 CTCAE ver. 4.0. The LBM and 14 common single nucleotide polymorphisms in the CYP3A4/3A5, NR1I2, NR1I3, ABCB1, and ABCG2 genes were analyzed according to the drug exposure at day 10. Determinants (including sunitinib exposure and pharmacogenetic variants) for toxicities were assessed, as well as the relationship between drug exposure and survival in renal cancer patients. RESULTS: Ninety-two patients (60 % with renal cancer) were assessable for pharmacokinetics, toxicity and survival, and 66 for genetic analysis. The LBM (p < 0.0001) and a polymorphism in the ABCG2 transporter (421C>A) (p = 0.014) were two independent parameters accounting for the variability of composite (sunitinib + SU12662) exposure. Advanced age (OR = 1.47 [1.01-2.15], p = 0.048) and high sunitinib exposure (OR = 1.16 [1.05-1.28], p = 0.005) were independently associated with any grade ≥ 3 acute toxicity, and high SU12662 exposure was associated with grade ≥ 2 thrombocytopenia (OR = 1.27 [1.03-1.57], p = 0.028). A high composite area under the curve (AUC) >1,973 ng/mL∙h at day 21 was associated with a doubled survival (35.2 vs 16.7 months; log-rank p = 0.0051) in renal cancer patients. CONCLUSIONS: This study indicates that LBM and drug monitoring may be helpful in the management of sunitinib-treated patients.

Collapsing glomerulopathy associated lupus in a black female with homozygous APOL1 mutation.

Collapsing glomerulopathy (CG), characterized by collapse of the glomerular capillary loops onto the mesangial stalks is rarely associated to systemic lupus erythematosus (SLE). Recently a genetic predisposition to HIV associated nephropathy (HIVAN) has been shown in Afro-Americans: MYH9 polymorhism in 2008 and then APOL1 variants (G1 and G2 alleles) in 2010 were shown to be strongly associated with HIVAN. We describe here for the first time the association of CG in a young Afro-American female with SLE having a homozygous mutation of APOL1. The clinical history, laboratory findings and immunofluorescence all confirmed a diagnosis of SLE. However, studies for factors associated with collapsing glomerulopathy in other situations were consistently negative. As this Afro-American patient developed a CG, we performed genotyping of APOL1. It was found that she is homozygotic for the G2 allele of APOL1. Despite.

Genetic factors (VKORC1, CYP2C9, EPHX1, and CYP4F2) are predictor variables for warfarin response in very elderly, frail inpatients.

Determining the optimal dose of warfarin for frail elderly patients is a challenging task because of the low dose requirements in such patients, the wide interindividual variability of response, and the associated risk of bleeding. The objective of this study was to address the influence of 13 common variations in eight genes on the maintenance dose of warfarin in a cohort of frail elderly inpatients. For our study, we enrolled 300 Caucasian subjects who were hospital inpatients, with a mean age of 86.7 +/- 6 years. In addition to age, genetic variants of VKORC1, CYP2C9, CYP4F2, and EPHX1 were found to be significant predictor variables for the maintenance dose of warfarin, explaining 26.6% of dose variability. Among 132 patients in whom warfarin therapy was initiated with the same low-dose regimen, we studied the relative influences of genetic and nongenetic factors. The time to first international normalized ratio (INR) > or =2 was influenced by VKORC1 and CYP2C9 genotypes (P = 0.0003 and P = 0.0016, respectively); individuals with multiple variant alleles were at highest risk for overanticoagulation (INR >4) (odds ratio, 12.8; 95% confidence interval, 2.73-60.0). In this special population of frail elderly patients with multiple comorbidities and polypharmacy, we demonstrated the main impact of genetic factors on warfarin response.

Voriconazole pharmacokinetic variability in cystic fibrosis lung transplant patients.

BACKGROUND: Aspergillosis is a high-risk complication in cystic fibrosis (CF) lung transplant patients. Azole antifungal drugs inhibit CYP3A4, resulting in significant metabolic drug-drug interactions. Voriconazole (VRZ) was marketed without therapeutic drug monitoring (TDM) recommendations, consistent with favorable pharmacokinetics, but regular determinations of plasma VRZ concentration were introduced in our center to manage interactions with calcineurin inhibitors and to document the achievement of therapeutic levels. METHODS: VRZ TDM data analysis for trough concentration (C0) and peak concentration (C2) was carried out, using validated liquid chromatography assay with ultraviolet detection, for 35 CF lung transplant patients (mean age 25 years, mean weight 47 kg, balanced sex ratio) since 2003. Therapeutic range (C0: 1.5 +/- 0.5 - C2 : 4.0 +/- 1.0 mg/L) was expressed relative to pivotal pharmacokinetic trial data. RESULTS: The duration of VRZ treatment ranged from 9 days to 22 months. The recommended standard dose of VRZ (200 mg twice a day, following the loading dose) resulted in significant plasma concentrations (>0.5 mg/L) in 20% of CF lung transplant patients. Therapeutic concentrations were obtained using higher doses (average 570 +/- 160 mg/day, +43%, P<0.01). Despite adaptation, C0 remained <0.5 mg/L (11%), even when the drug was administered intravenously, highlighting the variability of VRZ pharmacokinetics, possibly enhanced by CYP2C19 polymorphism. The risk of inefficacy during periods of underdosage was overcome by treatment with antifungal drug combinations (caspofungin, n=10). The therapeutic index was limited by neurologic effects (14%) and hepatic abnormalities (30%). VRZ concentrations correlated significantly (P<0.01) with aspartate aminotransferase levels but not with bilirubin levels. VRZ acted as a metabolic inhibitor of tacrolimus (C0 to dose ratio 5.8 +/- 2.6, n=31/VRZ versus 1.7 +/- 0.9 alone, P<0.001). Large changes in azole concentration affected the magnitude of the drug-drug interactions and adjustment requirements. CONCLUSIONS: TDM is required because VRZ levels are often undetectable in treated CF lung transplant patients, supporting the use of antifungal drug combinations until achievement of VRZ C0 at a steady state between 1 and 2 mg/L. Plasma VRZ concentrations should be determined for the quantitative, individualized management of drug-drug interactions in lung transplant patients, in particular immunosuppressant such as tacrolimus, considering VRZ to be both a target and an inhibitor of CYP3A4.

Characterisation of novel defective thiopurine S-methyltransferase allelic variants.

Human thiopurine S-methyltransferase (TPMT, EC 2.1.1.67) is a key enzyme in the detoxification of thiopurine drugs widely used in the treatment of various diseases, such as inflammatory bowel diseases, acute lymphoblastic leukaemia and rheumatic diseases. The TPMT gene is genetically polymorphic and the inverse relationship between TPMT activity and the risk of developing severe hematopoietic toxicity is well known. In this study, the entire coding sequence of TPMT, together with its 5'-flanking promoter region, was analysed in patients with an intermediate phenotype for thiopurine drug methylation. Four polymorphisms were identified, two previously described, c.356A>C (p.Lys(119)Thr, TPMT*9) and c.205C>G (p.Leu(69)Val, TPMT*21), and two novel missense mutations, c.537G>T (p.Gln(179)His, TPMT*24) and c.634T>C (p.Cys(212)Arg, TPMT*25). Structural investigations, using molecular modeling, were undertaken in an attempt to explain the potential impact of the amino acid substitutions on the structure and activity of the variant proteins. Additionally, in order to determine kinetic parameters (K(m) and V(max)) of 6-thioguanine (6-TG) methylation, the four variants were expressed in a recombinant yeast expression system. Assays were performed by HPLC and the results were compared with those of wild-type TPMT. The p.Leu(69)Val and the p.Cys(212)Arg substitutions encode recombinant enzymes with a significantly decreased intrinsic clearance compared to that of the wild-type protein, and, consequently, characterise non-functional alleles of TPMT. The p.Lys(119)Thr and the p.Gln(179)His substitutions do not affect significantly the catalytic activity of the corresponding variant proteins, which prevents to unambiguously describe these latter alleles as defective TPMT variants.

UDP-glucuronosyltransferase UGT1A7 genetic polymorphisms in hepatocellular carcinoma: a differential impact according to seropositivity of HBV or HCV markers?

BACKGROUND: We conducted a case-control study to evaluate the role of UDP-glucuronosyltransferase 1A7 (UGT1A7) polymorphisms in the onset of hepatocellular carcinoma (HCC). METHODS: The study included 165 patients with HCC, 134 with cirrhosis and 142 controls without liver disease, matched for age and hospital. All were men younger than 75 years. HCC and cirrhosis patients were stratified according to time since cirrhosis diagnosis. RESULTS: We found a positive association between the UGT1A7*3/*3 genotype and HCC when the comparison was restricted to patients whose disease was of viral origin [OR = 3.4 (0.3-45)] but a negative association when it included only alcoholic patients [OR = 0.1 (0.02-0.6), p = 0.01]. CONCLUSION: Our study shows that UGT1A7 may play a role in hepatocellular carcinogenesis and that this role may differ according to the primary cause of the cirrhosis.

[Pharmacogenetics of anticancer drugs]. / Pharmacogénétique des médicaments anticancéreux.

Much progress has been made in treating human malignancies and there are now multiple treatment options with similar efficacy for nearly every type of cancer. However, the narrow therapeutic index of most chemotherapeutic agents and the severe consequences of undertreatment or overdosing have led to research molecular predictive factors of the toxicity and efficacy of cancer treatments. Genetic factors affecting drug metabolism and transport partly explain interindividual variability in drug response. Pharmacogenetic focuses on the molecular mechanisms involved in drug response, and its ultimate goal is the optimisation of the treatments, that combines the optimal efficacy and the minimal risk of severe side effects. Polymorphisms in genes encoding specific drug-metabolising enzymes can result in individuals in the general population being characterised as low, rapid or even ultra-rapid metabolisers. Phenotyping and genotyping tests are now available that determine or predict the metabolic status of an individual and, thus, enable the evaluation of risk of drug failure or toxicity. Some clinical applications of pharmacogenetics (5-FU, irinotecan, thiopurines) have already been developed in routine medicine resulting in significant improvement in patient treatment. The clinical validation of an increasing number of pharmacogenetic tests, as well as the development of new highly efficient technologies for genotyping (real-time PCR, DNA chips...) should further promote pharmacogenetics in clinical practice and lead to the development of a patient-tailored drug therapy.

TEGDMA modulates glutathione transferase P1 activity in gingival fibroblasts.

Dental resinous materials can contain large amounts (from 30 to 50%) of triethylene-glycol-dimethacrylate (TEGDMA). This compound leaches into aqueous media and is toxic to dental pulp, as well as to gingival fibroblasts in vitro. To elucidate the mechanism of TEGDMA toxicity, we investigated the effects on glutathione (GSH) level and glutathione transferase P1 (GSTP1) activity in cultured human gingival fibroblasts. TEGDMA cytotoxic concentrations (from 0.5 to 2 mM) induced a depletion of GSH without formation of oxidized GSH (GSSG). In fibroblasts expressing the wild-type GSTP1, TEGDMA both inhibited and potentiated GSTP1 activity at high (IC50 = 1.1 mM) and low concentrations, respectively. In contrast, cells expressing the GSTP1 *A/*B variant showed a weak inhibition of GST activity only, associated with greater sensitivity to drug toxicity. Biochemical analysis of GSTP1 inhibition revealed that TEGDMA is a non-competitive antagonist with respect to GSH and substrate. Thus, TEGDMA interference with GSH and GSTP1 activity may contribute to dental-resin-induced adverse effects.

[Pharmacogenetics or the promise of a personalized medicine: variability in drug metabolism and transport]. / La pharmacogénétique ou la promesse d'une médecine personnalisée : variations du métabolisme et du transport des médicaments.

There is much variability in the manner individuals respond to drugs, such that the management of some drugs is problematic. In France, the incidence of hospital admissions related to adverse drug reactions is estimated to be 3.2 %, at an annual cost of over 300 millions euros. Genetic factors affecting the pharmacokinetics and pharmacodynamics of drugs partly explain interindividual variability in drug response. Pharmacogenetic focuses on the molecular mechanisms involved in drug response, and its ultimate goal is the optimisation of drug treatments, both in terms of efficacy and safety. Numerous polymorphisms in genes encoding drug-metabolising enzymes, transporters and receptors have been described and their consequences on disposition and effect of a substantial number of medications have been elucidated. This review focuses on variability of drug metabolism and transport to define the objectives of pharmacogenetics, the molecular bases of interindividual variation in drug response and the methods used for the evaluation of the individual risk of drug failure or toxicity. Some clinical applications of pharmacogenetics have already been developed in routine medicine resulting in significant improvement in patient treatment. The clinical validation of an increasing number of pharmacogenetic tests, as well as the development of new highly efficient technologies for genotyping (real-time PCR, DNA chips) should further promote pharmacogenetics in clinical practice and lead to the development of a patient-tailored drug therapy.

Glutathione-associated enzymes in head and neck squamous cell carcinoma and response to cisplatin-based neoadjuvant chemotherapy.

Glutathione S-transferases (GSTs) are metabolic phase II enzymes that promote reactive metabolite elimination by conjugating them to glutathione (GSH). Because of their important role in xenobiotic metabolism and detoxification, they have been implicated in carcinogenesis processes, especially epithelium transformation. Moreover, their influence on response to chemotherapy in cancer patients has been demonstrated. Genetic polymorphisms for GSTM1, GSTT1 and GSTP1 have been found in human populations and have been shown to have phenotypic consequences. To investigate the role of GST enzymes in carcinogenesis and in response to chemotherapy in patients with head and neck squamous cell carcinoma (HNSCC), GSTP1, GSTM1 and GSTT1 were studied prospectively in a large series of HNSCC patients. Correlations between GST alterations, p53 mutation status and clinical response to chemotherapy were investigated. We showed that the risk of developing laryngeal cancer was increased by 2.6-fold [95% CI 1.6--6.1] in patients with the GSTM1 null genotype and by 2.8-fold [95% CI 0.9--8.1] in patients with the homozygous GSTP1 val105 genotype. Furthermore, individuals with this latter genotype were over-represented in the p53 mutation group (p = 0.05). After storage duration and hemolysis adjustment, a significantly lower plasmatic GSTP1 level was observed in complete responders compared with partial and non-responders (mean: 4.4 +/- 0.06 microg/l, 4.7 +/- 0.06 microg/l and 4.7 +/- 0.07 microg/l; p = 0.05), respectively. The prevalence of p53-mutated tumors was significantly higher in the group of non-responders (81%) compared with partial (60%) and complete responders (64%) (p = 0.05). Two types of multivariate analysis were performed including parameters that have been shown to influence response to chemotherapy significantly in univariate analysis. p53 mutations and high tumor stage are independent factors of non-response to chemotherapy, whereas plasmatic GSTP1 levels and low tumor stage are independent factors of complete response. Our data suggest that GST enzymes are associated with larynx cancer and that their use as predictive factors and treatment targets should be further explored.

Genetic polymorphisms of cytochrome P450 2A6 in a case-control study on lung cancer in a French population.

Cytochrome P450 2A6 (CYP2A6) is involved in the C-oxidation of nicotine and in the metabolic activation of tobacco nitrosamines. Recent data have suggested that CYP2A6 genetic polymorphisms might play a role in tobacco dependence and consumption as well as in lung cancer risk. However, the previously published studies were based on a genotyping method that overestimated the frequencies of deficient alleles, leading to misclassification for the CYP2A6 genotype. In this study, we genotyped DNA from 244 lung cancer patients and from 250 control subjects for CYP2A6 (wild-type allele CYP2A6*1, and two deficient alleles: CYP2A6*2, and CYP2A6*4, the latter corresponding to a deletion of the gene) using a more specific procedure. In this Caucasian population, we found neither a relation between genetically impaired nicotine metabolism and cigarette consumption, nor any modification of lung cancer risk related to the presence of defective CYP2A6 alleles (odds ratio = 1.1, 95% confidence interval = 0.7-1.9).

Structural characterization of a new variant of the CYP2A6 gene (CYP2A6*1B) apparently diagnosed as heterozygotes of CYP2A6*1A and CYP2A6*4C.

During the course of investigating the frequency of a CYP2A6 whole deletion-type polymorphism (CYP2A6*4C) in Japanese, an unexpectedly large population of heterozygotes for CYP2A6*4C and the wild-type (CYP2A6*1A) was found. Cloning of a cDNA encoding CYP2A6 from the liver of individuals judged as heterozygotes for CYP2A6*4C and the CYP2A6*1A was carried out to identify the causal allele(s) responsible for a possible overestimation. A clone isolated from the liver cDNA library possessed 58 bp sequences in the 3'-untranslated region, which was replaced with the corresponding region of the CYP2A7 gene. The same gene conversion existed in the genomic DNA, indicating that the replacement was not a cloning artifact. Based on the gene structure of the allele (CYP2A6*1B), this variant was thought to be one of the causal alleles responsible for overestimation of heterozygotes for CYP2A6*4C and CYP2A6* A. To investigate this further, we developed a genotyping method which could distinguish the CYP2A6*A, CYP2A6*1B and CYP2A6*4C alleles from each other. The results clearly showed that CYP2A6*1B was the sole allele responsible for the overestimation. We conclude that the new genotyping method allows determination of six genotypes of the CYP2A6 gene, simultaneously and precisely, in both Oriental and Caucasian populations.

Permissiveness of human biliary epithelial cells to infection by hepatitis C virus.

The cellular tropism of hepatitis C virus (HCV) is an important but much debated issue. Permissivity to HCV of biliary cells has never been demonstrated. In this context, we used gallbladder epithelial cells (GBEC) as a model of the more proximal biliary epithelium. These cells were isolated from HCV-positive and -negative individuals and cultured for up to 40 days. Biliary cells from HCV-negative subjects were infected in vitro with various inocula. The retention of GBEC functional characteristics was assessed by the expression of cystic fibrosis transmembrane conductance regulator (CFTR). All 12 GBEC tested from HCV-negative patients were successfully infected by HCV. This was assessed by: 1) the detection of HCV-RNA positive and negative strands; 2) the detection of the viral capsid by immunofluorescence; and 3) the combination of single-strand conformation polymorphism (SSCP) and HVR1 sequence analysis demonstrating the distinct majoritary HCV genomes in serum and in GBEC. The level of HCV RNA in cell extracts and supernatants was low, but HCV infection was highly reproducible. Our results expand those showing the cellular tropism of HCV, and demonstrate the sensitivity of biliary cells to HCV infection. This might have an important impact in terms of pathogenesis and pathological features of HCV infection. In addition, given the easy access to these cells and the high reproducibility of in vitro infection, they should constitute an important tool for studies aimed at analyzing the issue of HCV penetration and neutralizing antibodies.

Influence of human immunodeficiency virus infection on chronic hepatitis B in homosexual men.

The aim of this study was to assess the influence of human immunodeficiency virus (HIV) infection on chronic hepatitis B. In a series of 132 (65 anti-HIV positive) homosexual non-drug addicted men with chronic hepatitis B, the liver function was assessed with biochemical tests; the degree of hepatitis B virus (HBV) replication was assessed with serum HBV DNA level and with immunoperoxidase staining of hepatitis B core (HBc) antigen on liver specimens; and the severity of liver lesions was assessed with an histology activity index. Anti-HIV-positive and anti-HIV-negative patients were not different for serum aspartate transaminase activity, bilirubin, prothrombin, and histology activity index. Anti-HIV-positive patients had lower serum alanine transaminase activity levels (P =.0001), lower serum albumin levels (P =.0009), and higher serum HBV DNA levels (P =.01). There was a higher prevalence of cirrhosis in anti-HIV-positive patients (P =.04). In homosexual men with chronic hepatitis B, HIV infection is associated with a higher level of HBV replication and a higher risk for cirrhosis without increased liver necrotico-inflammatory process.

[Characterization of liver regeneration in the albumin-urokinase transgenic mouse]. / Caractérisation de la régénération hépatique chez la souris transgénique albumine-urokinase.

UNLABELLED: Models of liver regeneration are essential to understand mechanisms of hepatic carcinogenesis, correct genetic diseases by gene transfer or hepatocyte transplantation. The expression in the liver of transgenic mice of a gene coding for a urokinase-type plasminogen activator (uPA mouse) induces hepatotoxicity and prolonged post-native liver regeneration from cellular clones which have inactivated the transgene. This model may have major applications but it remains necessary to characterize the liver regeneration pattern. METHODS: Histological and immunohistochemical studies of the liver of uPA and non-transgenic mice, 3, 7, 14, 21, 28, 42 and 56 days-old. Markers of cellular proliferation: 5-bromo-2'deoxyuridine (BrdU) and proliferating cell nuclear antigen (PCNA). RESULTS: Regenerative nodules were seen from day 14. These nodules then grew, became confluent and by 8 weeks constituted the entire liver mass. A semi-quantitative study of BrdU and PCNA showed a maximal labeling at day 7 (300 to 350 labeled cells/10 microscopic fields, mag 400). When the nodules appeared, 60 to 80% of the cells were labeled. The proportion of labeled cells decreased but was still greater than that observed in non transgenic mice up to day 56 (92 to 106 labeled cells vs 10 to 28, on day 28). CONCLUSIONS: In uPA mouse liver regeneration is significantly expanded, as compared to the regeneration following partial hepatectom. This study therefore has allowed to determine the best conditions for using this model.

Hepatitis C virus persistence in human hematopoietic cells injected into SCID mice.

The issue of infection of peripheral blood mononuclear cells (PBMC) by the hepatitis C virus (HCV) has potentially important implications, but is still debated. We have used the severe combined immunodeficiency (SCID) mouse model to test for the persistence of HCV in PBMC. Hematopoietic cells isolated from 14 subjects infected by HCV were inoculated intraperitoneally into SCID mice. Serum and blood cell samples from these mice were obtained with a mean follow-up of 8 weeks. As controls, human fibroblasts and sheep PBMC, preincubated with a human HCV-positive serum, were inoculated concomitantly into mice and analyzed. HCV-RNA positive strands were detected in 7 of 26 serum samples and 8 of 26 cell fractions from SCID mice inoculated with HCV-positive PBMC, after 8 weeks of follow-up. In contrast, no HCV RNA was detectable in the 10 control mice. HCV-RNA negative strands were detected in only 2 of 10 tested samples from 2 mice, and both positive mice had been inoculated with PBMC from HCV-positive subjects with malignant hematopoietic syndrome. Our study offers strong evidence for the persistence of HCV infection in mononuclear cells. Our results are also consistent with a low rate of HCV multiplication. This SCID mouse model might therefore be useful in analyzing the mechanisms of HCV persistence in mononuclear cells.

Prolonged interferon-alpha therapy of hepatitis B virus-related decompensated cirrhosis.

Interferon alpha therapy of hepatitis B virus-related decompensated cirrhosis with the dose and the duration generally used is frequently associated with severe side-effects and reactivations. Between 1989 and 1996, 15 patients with hepatitis B virus-related decompensated cirrhosis received prolonged (3-48 months) low-dose (3 million units) IFN-alpha therapy. Ten patients (66%) had a sustained loss of serum hepatitis B virus DNA and hepatitis Be antigen (if present initially) associated with a decrease of aminotransferase levels into the normal range. During follow-up of these 10 patients, seven had a marked clinical improvement and are alive and fully active. One has an hepatocellular carcinoma, and two died without reactivation. Among the five other patients, two had a transient loss of serum HBV DNA followed by reactivation and three did not respond to therapy. During follow-up, one of these five patients died and one underwent liver transplantation. Severe complications, possibly related to interferon were uncommon and included bacterial infection in one case and variceal bleeding in two cases. Eleven of the 15 patients treated are alive after 1.5-7 years of follow-up. Hence, in patients with hepatitis B-related cirrhosis, prolonged low-dose IFN-alpha therapy is relatively well tolerated and may induce a sustained inhibition of hepatitis B virus replication with marked clinical improvement.