Serological response to infection with different isolates of hepatitis C virus.

Different isolates of hepatitis C virus (HCV) show nucleotide sequence variability throughout the genome. Detection of antibodies to recombinant proteins derived from hepatitis C virus genotype 1, the prototype HCV clone HCV-PT, constitutes the main method for screening HCV infection. The influence of the genomic variability on the serological diagnosis of HCV by enzyme immunoassay remains poorly defined. The aim of this study was to assess the serological reactivity of a panel of well characterized French HCV isolates typed by sequence analysis from patients with chronic hepatitis. The 73 sera samples were tested in three third generation EIA tests and three confirmatory assays. HCV isolates were determined by RT-PCR and sequencing in NS5B region of the genome. The 73 sera were positive in the three EIA tests. The three confirmatory tests showed a weaker reactivity with NS5 protein whatever the genotype, and a lower reactivity in NS4 antigens of non-type 1 sequences, particularly for genotype 3. Even though the reactivity of the antigens differed among the HCV isolates, the 73 isolates from genotype 1-6 were reactive with the three commercial screening assays. These results demonstrate that using a single test is adequate in the routine diagnosis of HCV infection in clinical laboratory, as recommended by the last French and European consensus conference.

The expression of genes induced in melanocytes by exposure to 365-nm UVA: study by cDNA arrays and real-time quantitative RT-PCR.

Ultraviolet A radiation (UVA; 320-400 nm) constitutes more than 90% of the terrestrial UV solar energy. This type of radiation generates reactive oxygen species and consequently induces DNA damage. UVA irradiation is now considered to be an important carcinogen agent especially in the development of melanoma. UVA radiation is known to activate several pathways in mammalian cells. We have used cDNA arrays to analyze differential gene expression in primary cultures of human melanocytes in response to 365-nm UVA. Among 588 genes studied, 11 were overexpressed. These genes included genes involved in cell cycle regulation (GADD45, CIP1/WAF1), in stress response (HSP70, HSP40, HSP86), in apoptosis (GADD153, tristetraproline) and genes encoding transcription factors (EGR-1, ETR-101, c-JUN, ATF4). This coordinate gene regulation was confirmed by real-time quantitative RT-PCR.

Association of APOE promoter but not A2M polymorphisms with risk of developing Alzheimer's disease.

The APOE4 allele is widely accepted as a major risk factor for late-onset Alzheimer's disease (AD). Recently, it has been reported that polymorphisms in the APOE promoter and in the alpha2-macroglobulin gene (A2M) are associated with AD. We have analyzed the distribution of APOE alleles, -219T/G APOE promoter polymorphism, and A2M/A2Mdel polymorphism in a large case-control study. Our results showed that APOE genotype was the only informative marker of AD risk contrary to -219T/G and A2M/A2Mdel polymorphism. In AD patients however, a strong linkage disequilibrium was observed between the T allele of -219T/G polymorphism and APOE4 allele. This result indicates that -219T/G APOE promoter polymorphism is a risk factor for AD by increasing the APOE4-associated risk.

Integrity of the NS5A (amino acid 2209 to 2248) region in hepatitis C virus 1b patients non-responsive to interferon therapy.

BACKGROUND/AIMS: In hepatitis C virus-1b, it has been suggested that an amino acid stretch (aa 2209-2248) of the carboxy terminal half of the non-structural 5A (NS5A) region participates in the response to interferon treatment. We tested the hypothesis that absence of mutations in the NS5A (aa 2209-2248) sequence is required for interferon resistance. We also investigated the importance of different HCV-1b isolates in interferon response in France. METHODS: We determined the NS5A sequences of 70 patients with chronic hepatitis C before IFN therapy and then compared them with HCV-J prototype sequence. The isolates were determined by NS5B sequencing, the "gold standard" method for genotyping and subtyping. Pre-therapeutic viral load was also measured. RESULTS: No sustained virological response was observed in the patients without amino acid substitutions in the NS5A (aa 2209-2248) sequence, and in the patients with HCV-J isolates. Viral load was significantly higher in the patients with no amino acid substitutions in the NS5A (aa 2209-2248) sequence. CONCLUSIONS: In HCV-lb infected patients, an HCV-J strain with no amino acid substitution in the NS5A (aa 2209 2248) region indicates a poor prognosis for response to IFN therapy. The low interferon response rate in HCV-lb infection in Europe is probably not due to a difference between isolates.

Modulation of adenosine concentration by opioid receptor agonists in rat striatum.

There is evidence that adenosine and morphine interact in the striatum. However, little is known about the precise role of the opioid receptor subtypes implicated in the modulation of adenosine tissue concentration and in adenosine receptor expression and function. We sought to evaluate, in the absence of withdrawal symptoms, the effects of the short-term administration of selective mu-, delta- or kappa-opioid receptor agonists on adenosine concentration and on adenosine A(2A) receptor function in rat striatum. Adenosine A(2A) receptor was chosen because the neuronal sub-population expressing this receptor coexpresses enkephalin, suggesting that adenosine A(2A) receptor may be regulated by opioid receptor agonists. Oxymorphone hydrochloride mu-opioid receptor agonist, 6 mg/kg/day), +[-(5 alpha,7 alpha, 8 beta)-(-)-N-methyl-N(7-(1-pyrrolidinyl)1-oxaspiro (4.5)dec-8-yl) benzenacetamide] (U69593) (kappa-opioid receptor agonist, 0.75 mg/kg/day), and (+)-4[(alpha R)-alpha-((2S,5R)-4-allyl-2, 5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide) (SNC80) (delta-opioid receptor agonist, 9 mm/kg/day), or vehicle, were administered i.p 3 x daily during 5 days to groups of rats (n=6). We also investigated the effects of opioid receptor agonists on adenosine uptake by striatal cell extracts. We found that administration of mu- or delta-opioid receptor agonists significantly decreased adenosine uptake in striatal cell extracts and increased adenosine concentration (mean+24% and +45% for mu- and delta-opioid receptor agonist, respectively, relative to controls). None of the receptor agonists tested induced obvious modifications of adenosine A(2A) receptor function. However, the delta-opioid receptor agonist induced an increase in adenosine A(2A) mRNA expression (mean 44%). We conclude that mu and delta receptor agonists inhibit adenosine uptake by striatal cell extracts and increase adenosine concentrations in rat striatum.

Mother-to-infant transmission of hepatitis C virus: molecular evidence of superinfection by homologous virus in children.

BACKGROUND/AIM: Vertical transmission of hepatitis C virus (HCV) is well established but its incidence is low. To assess the molecular evidence of mother-to-infant transmission or intrafamilial transmission of HCV, the NS5 B region and the hypervariable region 1 (HVR1) of the E2/NS1 region of the HCV genome from each member of a family were investigated. METHODS: A 35-year-old mother with chronic hepatitis C virus infection and her four infected boys were studied. The same HCV 1a genotype was found in all five. Phylogenetic analysis was done by the neighbor-joining, the maximum likelihood, and the maximum parsimony methods. RESULTS: Comparison of the phylogenetic trees in the NS5B and HVR1 regions showed that the sequences in the children were more closely related to the population of variants of their own mother than to any genotype la sequence available in the databases. However, four HVR1 clones from two brothers (E2 and E3) had a strong homology, but were significantly divergent from the variants of the mother. CONCLUSIONS: These results suggest that a cluster of HCV strains exists in the family and that E3 could have been superinfected by E2 HCV strains and reciprocally. In conclusion, phylogenetic analysis through variable regions of the genome suggests that at least two modes of transmission are involved in this family: perinatal and horizontal.

Cyclosporine A and purinergic receptors in rat kidney.

Previous reports have demonstrated that Cyclosporine A (CyA) chronically administered induces an increase in adenosine plasma concentration by inhibiting adenosine uptake by red blood cells (RBC). We hypothesized that this effect may modulate, by a down regulation, the mRNA expression of adenosine receptors in rat kidney. Since high blood pressure (HBP) is a classical side effect of CyA treatment, nicardipine, a dihydropyridine calcium channel blocker, is often associated with CyA in treatment. To distinguish between the effects of CyA-induced HBP and the effects of CyA by itself, we have evaluated the effects of CyA and/or nicardipine on the mRNA expression of A1 and A2a adenosine receptors. The study was performed on five groups of rats (n= 8) receiving during 21 days either serum saline (0.5 ml i.p), CyA (12 mg/kg/day, i.p), nicardipine (1.2 mg/kg i.p) or nicardipine + CyA. The last (or fifth) group was injected with vehicle (0.5 ml i.p). Blood samples for adenosine assay were collected in the renal artery at day 21, just before the rat kidneys were removed for quantitation of adenosine A1 and A2a mRNA concentration by RT-PCR. We make two conclusions :i) Nicardipine induces a decrease in mRNA expression of A1 but not of A2a adenosine receptors. However, because nicardipine lowered both blood pressure and A1 mRNA expression, it is not possible to conclude if A1 mRNA decrease is implicated in the nicardipine effects on blood pressure.ii) CyA induces an increase in renal artery adenosine concentration and a decrease in mRNA expression of A1 and A2a adenosine receptors.

Adenosine and the nervous system: pharmacological data and therapeutic perspectives.

1. Adenosine acts on a family of G-protein-coupled receptors called purinoreceptors. 2. Four subtypes have been cloned and pharmacologically characterized. 3. The principal pharmacological data and structure-function relations for agonist interactions with P1 receptors are presented. 4. We conclude that the potent role of adenosine in the nervous system may be interesting for the development of drugs targeted at purines and their receptors.

Assessment of spontaneous fluctuations of viral load in untreated patients with chronic hepatitis C by two standardized quantitation methods: branched DNA and Amplicor Monitor.

Quantitation of hepatitis C virus (HCV) RNA in serum has been used to predict and monitor the efficacy of interferon therapy in chronic HCV infection. We prospectively studied the fluctuation of viremia by a longitudinal follow-up of HCV RNA levels for 2 months in six untreated patients. Spontaneous fluctuations of HCV RNA ranged from 2.8- to 5.7-fold with branched DNA assay and from 2.9- to 5.6-fold with Monitor. These large spontaneous fluctuations (up to 0.75 log), observed daily, weekly, and monthly, raise doubt about the clinical value of a single assessment of pretherapeutic viremia.

Prevalence of hepatitis G virus RNA in a monocentric population of French haemophiliacs.

Hepatitis G virus (HGV) and hepatitis GB virus (GBV-C) have been reported as possible causes of non-A-E transfusional hepatitis. To assess the prevalence of hepatitis G virus infection in haemophiliacs we retrospectively investigated the presence of viral RNA in 92 patients with and without HCV infection. HGV/GBV-C RNA was reverse transcribed and amplified with primers from the 5' non-coding region of the genome. RNA was detected in 16/92 patients (17.4%). Restriction enzyme analysis revealed that the 16 patients belonged to the HGV-like genotype. Serology with E2-specific antibodies demonstrated that HGV viraemia underestimates previous infection by HGV. 33 patients were positive for HGV; all but two have cleared HGV RNA. 47/92 patients had a marker of prior infection by HGV. No difference between HGV RNA positive and negative patients was observed concerning age, diagnosis, HIV and HCV status. Previous HBV infection correlated with the frequency of HGV infection. There was no difference in alanine aminotransferase levels between HGV positive and negative patients. All 18 patients exposed to only virally inactivated plasma-derived concentrates were negative for both HGV RNA and anti E2 antibodies. Prior exposure to untreated concentrates correlated with HGV viraemia (P=0.03), HGV seropositivity (P=0.0002), and markers of HGV infection (P<0.0001). In haemophiliacs with a past exposure to non-inactivated concentrates, persistence of HCV RNA (53/74 patients) was more frequent than HGV RNA persistence (16/74 patients) although HGV viraemia is more frequent than HCV viraemia in blood donors. This may be related to a greater ability of individuals to clear HGV infection and suggests that hepatitis G virus infection in multi-transfused patients has a better outcome than infection with other blood-borne viruses.

[Comparative efficacy of sustained release verapamil and captopril in mild to moderate arterial hypertension by ambulatory measurement and occasional measurement]. / Efficacité comparée du vérapamil à libération prolongée et du captopril dans l'hypertension artérielle légére à modérée par la mesure ambulatoire et mesure "occasionnelle".

The evaluation of mild to moderate hypertension must be carried out under the conditions in which treatments are usually prescribed, i.e., in general practice. After specific training of the physicians in the methods used, we evaluated the efficacy and safety of a new formulation of verapamil by comparing it with a reference drug: captopril. The main assessment criterion was the restoration of normal blood pressure in mildly to moderately hypertensive patients (blood pressure in excess of 160/95 mmHg). Blood pressure was evaluated by two methods: a mercury column sphygmomanometer, after the patient had rested in a half-sitting position for 10 minutes, and the ambulatory measurement of blood pressure (AMBP) using the SpaceLabs system. The results of this study involving 40 patients followed up for 3 months by 8 GPs in collaboration with our blood pressure unit were as follows: on verapamil, 47% of patients recovered normal values after 30 days of treatment and 71% after 60 days (with no change in dosage). On captopril, the normalization rates were 22 and 27% respectively. The highly significant reduction of blood pressure found by the "occasional" measurement for both treatments (p less than 0.001) was only faintly reflected by AMBP. Verapamil induced a reduction of nighttime blood pressure with no significant impact on heart rate. The clinical, paraclinical and electrocardiographic safety of both treatments was good.