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Biomedical subjects

Philippe Halfon

Publications and source records attributed to Philippe Halfon.

At least 37 records · Page 2Linked to original sources
PubMed2004

A loss of viral replicative capacity correlates with altered DNA polymerization kinetics by the human immunodeficiency virus reverse transcriptase bearing the K65R and L74V dideoxynucleoside resistance substitutions.

Mechanisms governing viral replicative capacity are poorly understood at the biochemical level. Human immunodeficiency virus, type 1 reverse transcriptase (HIV-1 RT) K65R or L74V substitutions confer viral resistance to 2',3'-dideoxyinosine (ddI) in vivo. The two substitutions never occur together, and L74V is frequently found in patients receiving ddI, while K65R is not. Here we show that recombinant viruses carrying K65R and K65R/L74V display the same resistance level to ddI (about 9.5-fold) relative to wild type. Consistent with this result, purified HIV-1 RT carrying K65R RT or K65R/L74V substitutions exhibits an 8-fold resistance to ddATP as judged by pre-steady state kinetics of incorporation of a single nucleotide into DNA. Resistance is due to a selective decrease of the catalytic rate constant k(pol): 22-fold (from 7.2 to 0.33 s(-1)) for K65R RT and 84-fold (from 7.2 to 0.086 s(-1)) for K65R/L74V RT. However, the K65R/L74V virus replication capacity is severely impaired relative to that of wild-type virus. This loss of viral fitness is correlated to a poor ability of K65R/L74V RT to use natural nucleotides relative to wild-type RT: 15% that of wild-type RT for dATP, 36% for dGTP, 50% for dTTP, and 25% for dCTP. The order of incorporation efficiency is wild-type RT > L74V RT > K65R RT > K65R/L74V RT. Processivity of DNA synthesis remains unaffected. These results explain why the two mutations do not combine in the clinic and might give a mechanism for a decreased viral fitness at the molecular level.

Cells, Cultured↗
PubMed2003

Mechanistic basis for reduced viral and enzymatic fitness of HIV-1 reverse transcriptase containing both K65R and M184V mutations.

HIV-1 drug resistance mutations are often inversely correlated with viral fitness, which remains poorly described at the molecular level. Some resistance mutations can also suppress resistance caused by other resistance mutations. We report the molecular mechanisms by which a virus resistant to lamivudine with the M184V reverse transcriptase mutation shows increased susceptibility to tenofovir and can suppress the effects of the tenofovir resistance mutation K65R. Additionally, we report how the decreased viral replication capacity of resistant viruses is directly linked to their decreased ability to use natural nucleotide substrates and that combination of the K65R and M184V resistance mutations leads to greater decreases in viral replication capacity. All together, these results define at the molecular level how nucleoside-resistant viruses can be driven to reduced viral fitness.

Adenine↗
PubMed2003

Apoptosis and proliferation kinetics of T cells in patients having experienced antiretroviral treatment interruptions.

Multiple failures of antiretroviral treatments, as a result of multidrug-resistant virus, have led to a proposal for structured therapeutic interruptions (STI). However, a significant decrease in CD4+ T cells may occur. The aim of our study was to determine the kinetics of T cell subpopulation changes, T cell apoptosis and peripheral blood mononuclear cell proliferation after STI. The impact of resistance mutation disappearance on T cell apoptosis was also studied. Ten patients were enrolled prospectively, and blood sampling was performed at weeks 0, 2, 4, 6, 8 and 12. The mean increase in viral load was 1.3 log(10) copies/ml, ranging from 0.1 to 3.2. CD4+ T cell count decreased to a mean of 80 cells/mm(3) from baseline to week 12. In the same period, CD8+ T cells decreased to a mean of 139 cells/mm(3). A significant increase in both T cell apoptosis and proliferation of mononuclear cells was observed. However, proliferation was an early and brief event. The increase in CD4+ T cell apoptosis was obvious in patients exhibiting complete reversion of resistance mutations to antiviral drugs. Our results suggest that during STI, apoptosis is an overwhelming phenomenon compared with proliferation, and may explain the limited immunological impact of this therapeutic option.

Antiretroviral Therapy, Highly Active↗
PubMed2003

Kinetics of disappearance of resistance mutations and reappearance of wild-type during structured treatment interruptions.

OBJECTIVE: To monitor the disappearance of resistance-associated mutations and reappearance of wild-type (WT) virus during structured treatment interruptions (STI) using DNA sequencing and line probe assay. METHODS: Eleven HIV-1-infected patients participating in the MUTAVIR study undergoing a 3-month STI after multi-HAART failure were monitored biweekly. Genotypes were assessed by sequencing and VERSANT HIV-1 Resistance Assays (LiPA). RESULTS: At treatment interruption, 54 mutations in total were identified with both methods among the patients. LiPA provided a result for 93.3% of the codons at baseline. For 37 mutations, a complete reversion of mutant to WT was observed with one of the two methods. Among these, LiPA detected mutations in 23 codons for 7 to 52 days longer, in 10 codons for the same period, and in four codons for a shorter time than sequencing. Similarly, LiPA detected 35 WT codons 8 to 86 days earlier, and 15 at the same time point as sequencing. A sharp reduction in the number of mutations was observed at the time of viral load increase in five of the 11 patients. Taking only the codons detected by LiPA into consideration, two patients showed a complete reversion to WT according to both genotyping assays at the end of the STI period. CONCLUSIONS: The determination of the timepoint at which a virus population of an HIV-1 patient undergoing STI reverts to WT is dependent on the assay used. The viral load increase in most patients is compatible with the outgrowth of virus with fewer or no mutations.

Antiretroviral Therapy, Highly Active↗
PubMed2003

Lymph node enlargement within the hepatoduodenal ligament in patients with chronic hepatitis C reflects the immunological cellular response of the host.

BACKGROUND/AIMS: Lymph nodes in the hepatoduodenal ligament seem to be a common ultrasonographic finding in patients with chronic hepatitis C. Lymphadenopathic enlargement is associated with the histological hepatic features reflecting the immunological response of the host, but the correlation between lymphadenopathy, liver histology and the cellular immunoreactivity of the host has never been studied. AIM: (1) To specify the prevalence of lymph nodes within the hepatoduodenal ligament; and (2) to investigate whether lymphadenopathies might reflect the immunological response of the host. METHODS: One hundred and eleven patients were enrolled in this study. Eleven chronic hepatitis B patients and 34 healthy volunteers served as controls. RESULTS: Lymph nodes were detectable in 90 out of the 104 chronic hepatitis C patients studied. After logistic regression, a high CD8 level and the absence of post hepatitis C cirrhosis were associated with lymph node enlargement. The total lymph node volume was correlated with transaminase levels, inflammatory activity, and stage of fibrosis. CONCLUSIONS: (1) The prevalence of lymph nodes within the hepatoduodenal ligament is high; (2) lymph node enlargement is correlated with the immunological cellular response of the host; and (3) the total lymph node volume is correlated with hepatic necroinflammatory markers and the stage of fibrosis.

Adult↗
PubMed2003

Hepatitis C virus RNA load in relapsed patients: week two of treatment is the best time to predict the complete response.

OBJECTIVE: An early virological response has been shown to be predictive of a sustained virological response to antiviral treatment in chronic hepatitis C infection. The aim of the study was to analyse viral kinetics during the first 6 weeks of treatment (interferon plus ribavirin) in 18 relapsed hepatitis C patients after a first course of interferon monotherapy. METHODS: We studied 18 relapsed patients treated with interferon and ribavirin. A sustained virological response (negative HCV RNA measured by polymerase chain reaction 6 months after the end of therapy) was obtained in 12 patients. Samples were obtained before therapy and each week for 6 weeks during therapy; HCV RNA levels were determined using quantitative bDNA. RESULTS: At the end of week two, a viral-load drop of more than 2.20 log was observed in all the 12 patients with a sustained virological response and in none of the six other patients. When we considered the median of the viral load reduction from baseline for each week of treatment, week two appeared to be the time point most predictive of a sustained viral response (positive predictive value 83%; negative predictive value 92%). CONCLUSION: During treatment with interferon plus ribavirin in relapsed hepatitis C patients, viral kinetics showed that the second week of treatment appeared to be the time point most predictive of a sustained viral response.

Adult↗
PubMed2003

Discrepancies between protease inhibitor concentrations and viral load in reservoirs and sanctuary sites in human immunodeficiency virus-infected patients.

The variable penetration of antiretroviral drugs into sanctuary sites may contribute to the differential evolution of human immunodeficiency virus (HIV) and the emergence of drug resistance. We evaluated the penetration of indinavir, nelfinavir, and lopinavir-ritonavir (lopinavir/r) in the central nervous system, genital tract, and lymphoid tissue and assessed the correlation with residual viral replication. Plasma, cerebrospinal fluid (CSF), semen, and lymph node biopsy samples were collected from 41 HIV-infected patients on stable highly active antiretroviral therapy regimens to determine drug concentrations and HIV RNA levels. When HIV RNA was detectable, sequencing of the reverse transcriptase and protease genes was performed. Ratios of the concentration in semen/concentration in plasma were 1.9 for indinavir, 0.08 for nelfinavir, and 0.07 for lopinavir. Only indinavir was detectable in CSF, with a concentration in CSF/concentration in plasma ratio of 0.17. Differential penetration into lymphoid tissue was observed, with concentration in lymph node tissue/concentration in plasma ratios of 2.07, 0.58, and 0.21 for indinavir, nelfinavir, and lopinavir, respectively. HIV RNA levels were <50 copies/ml in all CSF samples of patients in whom HIV RNA was not detectable in plasma. HIV RNA was detectable in the semen of three patients (two patients receiving nelfinavir and one patient receiving lopinavir/r), and its detection was associated with multiple resistance mutations, while the viral load in plasma was undetectable. HIV RNA was detectable in all lymph node tissue samples. Differential drug penetration was observed among the three protease inhibitors in the sanctuary sites, but there was no correlation between drug levels and HIV RNA levels, suggesting that multiple factors are involved in the persistence of viral reservoirs. Further studies are required to clarify the role and clinical relevance of drug penetration in sanctuaries in terms of long-term efficacy and drug resistance.

Adult↗
PubMed2003

Porphyria cutanea tarda, C282Y, H63D and S65C HFE gene mutations and hepatitis C infection: a study from southern France.

BACKGROUND: To evaluate the role of genetic factors in the pathogenesis of porphyria cutanea tarda (PCT) and their association with chronic hepatitis C. OBJECTIVE: To investigate the relations between hemochromatosis gene (HFE) mutations and PCT in the south of France and their links with chronic hepatitis C virus (HCV) infection. METHODS: The genotype for the C282Y, H63D and S65C mutations of HFE was determined in 33 patients with PCT, 46 patients with HCV infection but without PCT and 58 controls. Iron status and HCV, HBV and HIV serologies were studied in all patients. RESULTS: A statistically significant increase in the C282Y mutation was found in PCT patients. No difference was found for H63D or S65C mutations. The prevalence of HCV infection was higher in PCT patients than controls. CONCLUSIONS: C282Y mutations and HCV infection but not H63D or S65C mutations are PCT-triggering or associated factors in the south of France.

Female↗
PubMed2002

A prospective assessment of the inter-laboratory variability of biochemical markers of fibrosis (FibroTest) and activity (ActiTest) in patients with chronic liver disease.

BACKGROUND: Biochemical markers for liver fibrosis (FibroTest) and necroinflammatory features (ActiTest) are an alternative to liver biopsy in patients with chronic hepatitis C. Our aim was to assess the inter-laboratory variability of these tests, and their 6 components (gamma-glutamyl transpeptidase, alanine aminotransferase, alpha2-macroglobulin, haptoglobin, apolipoprotein A1, and total bilirubin) and to identify factors associated with this variability. RESULTS: Serum of 24 patients with chronic hepatitis C or severe alcoholic liver disease were prospectively recorded and analyzed in one reference center and in 8 additional laboratories. When gamma-glutamyl transpeptidase and alanine aminotransferase were expressed in international units, there was no significant difference between laboratories in the results of FibroTest or ActiTest; kappa statistics were greater than 0.50 with only 0.8% of cases (3/384) with a discordance of more than one stage. The main factor significantly associated with variability was the expression of gamma-glutamyl transpeptidase and alanine aminotransferase, as multiples of upper limit of reference values. The use of standardized method with pyridoxal phosphate reduced the variability of alanine aminotransferase expression, and standardized original Szasz method reduced the variability of gamma-glutamyl transpeptidase expression. CONCLUSIONS: The variability of FibroTest and ActiTest was acceptable without clinical consequences for the prediction of the stage of liver fibrosis and grade of activity. Standardized methods and assay calibration should be used and expression of alanine aminotransferase and gamma-glutamyl transpeptidase in multiples of the upper limit of reference values should not be employed.

Journal Article↗
PubMed2002

Human leukocyte antigen class II alleles may contribute to the severity of hepatitis C virus-related liver disease.

Whether the host's immune response genes influence the severity of hepatitis C virus (HCV) liver disease is controversial. Human leukocyte antigen (HLA) class II alleles were analyzed in 233 HCV RNA-positive patients with chronic active hepatitis (197 patients with Knodell index of fibrosis F0-F3 and 36 patients with index of F4). The 2 groups did not differ by sex, duration of infection, mode of contamination, alcohol consumption, or HCV genotype. Patients with cirrhosis were older than those without (56+/-12 vs. 46+/-14 years; P<10-4) and had a lower DRB1*11 allele frequency (5.6% vs. 14.5%; P=.037), whereas DRB1*03 and DQB1*0201 frequencies appeared to be higher (DRB1*03, 18.1% vs. 9.6%; DQB1*0201, 37.5% vs. 23.4%; P=.04, corrected P value is not significant). Mean index of fibrosis was higher in DR3-positive than in DR11-positive patients (2.14 vs. 1.58; P=.05). By multivariate analysis, cirrhosis was associated with male sex and age >50 years. HLA class II alleles may weakly contribute to the severity of HCV liver disease. Of persons infected with HCV, only 15%-20% spontaneously clear the virus, and the rest become chronically infected.

Adolescent↗
PubMed2002

Hydration free energy a fragmental model and drug design.

The aim of this work was to provide a simple model to determine hydration free energies of molecular compounds at ambient temperature in order to help drug design. Since the free energy of interaction between molecules and water can be approximated by an additive function of their constituent groups, a model based on a fragmental analysis was developed with all the hydration free energies determined from experimental values. The model gives accurate hydration free energies on small molecules by simply adding the hydration energies of the fragments constituting the molecule. The correlation factor r(2) is 0.990 on the 125 molecules used to establish the model parameters. As experimental values are determined at ambient temperature (25 degrees C), the values obtained from the model will correspond to this temperature. For mean size molecules or big molecules as proteins, it was not possible to calibrate the model owing to the absence of experimental values. However, the model makes it possible to estimate the hydration free energy of such molecules. The fragmental representation of any molecule with coded colours, depending on the fragment nature (hydrophobic, hydrophilic or neutral), can be useful to understand molecule behaviour and help drug design.

Drug Design↗
PubMed2002

The antibody response to hepatitis B virus vaccination is negatively influenced by the hepatitis C virus viral load in patients with chronic hepatitis C: a case-control study.

The purpose of this work was to evaluate in a case-control study the immunogenicity of a recombinant hepatitis B virus (HBV) vaccine in patients with chronic hepatitis C. Seventy-seven patients with histologically proven chronic hepatitis C without cirrhosis were included in a prospective trial and matched for sex and age to 231 healthy adult subjects. Recombinant HBV vaccine was administered at a dose of 20 microg at months 0, 1 and 2. The definition of 'responder to vaccination' was anti-HBs titre > 10 mIU/ml after the three injections. Forty-nine (63.6%) chronic hepatitis C patients were responders to vaccination, compared with 217 (93.9%) controls (P < 0.0001). After the three injections, anti-HBs titres were 156 +/- 260 and 615 +/- 435 mIU/ml (P < 0.0001), respectively. Chronic hepatitis C patients who were non-responders to vaccination had significantly higher viral load than responders to vaccination. Moreover, a negative correlation was observed between viral load and anti-HBs concentration (r = -0.36, P = 0.003). No significant side effects were observed. There was no effect of vaccination on alanine aminotransferase (ALT) levels and hepatitis C virus (HCV) viral load during or after vaccination. In multivariate analysis, the main predictive factors of response to HBV vaccine were absence of anti-HCV antibodies (OR = 7.65, P < 0.0001), weight < 75 kg (OR = 1.99, P < 0.035), and age < 50 years (OR = 1.58, P < 0.082). Our results suggest that viral load seems to negatively influence the response to HBV vaccine.

Adult↗
PubMed2002

Use of phylogenetic analysis of hepatitis C virus (HCV) hypervariable region 1 sequences to trace an outbreak of HCV in an autodialysis unit.

Hemodialysis patients are at high risk of infection by hepatitis C virus (HCV). The aim of this study was to investigate an HCV outbreak that occurred in an autodialysis unit by using epidemiological and molecular methods. Seroconversion to HCV antibody (anti-HCV) was observed in two patients over an 18-month period; two other patients had previously been recorded as anti-HCV positive. All four patients involved in the outbreak were tested for HCV RNA, and hepatitis C genotype determination was accomplished by a reverse hybridization assay. Furthermore, part of hypervariable region 1 (HVR1) of the hepatitis C genome was amplified and sequenced in samples from all HCV RNA-positive patients. Phylogenetic analysis of the nucleotide sequences obtained was carried out in order to investigate any possible epidemiological linkages among patients. The nucleotide sequences of the HVR1 regions of both newly infected patients were found to be identical to sequences of samples from previously recorded anti-HCV-positive original patients, suggesting that they were infected by the same isolate. Molecular and epidemiological analysis suggested that nosocomial patient-to-patient transmission was the most likely explanation for the virus spread in the autodialysis unit under study.

Disease Outbreaks↗
PubMed2002

Evaluation of the VERSANT HCV RNA 3.0 assay for quantification of hepatitis C virus RNA in serum.

We assessed the performance of a new assay (VERSANT HCV RNA 3.0 [bDNA 3.0] assay [Bayer Diagnostics]) to quantitate HCV RNA levels and compared the results of the bDNA 3.0 assay to results of the Quantiplex HCV RNA 2.0 (bDNA 2.0) assay. Samples used in this study included 211 serum specimens from hepatitis C virus (HCV)-infected persons from two sites (Bordeaux and Marseille, France) with different genotypes; 383 serum specimens from HCV antibody-negative, HCV RNA-negative persons; and serial dilutions of World Health Organization (WHO) HCV RNA standard at a titer of 100,000 IU/ml. The specificity of the bDNA 3.0 assay was 98.2%. A high correlation was observed between expected and observed values in all dilutions of WHO standard (r = 0.9982), in serial dilutions of pooled samples (r = 0.9996), and in diluted sera from different HCV genotypes (r = 0.9930 to 0.9995). The standard deviations (SD) for the within-run and between-run reproducibility of the bDNA 3.0 assay were <or=0.2 and <or=0.14, respectively. The intersite SD ranged from 0.03 to 0.14. The bDNA 3.0 assay results were positively correlated with the bDNA 2.0 assay results (r = 0.9533). Taking in account the overall performance, this assay could be used as a routine tool for the HCV RNA quantification.

Branched DNA Signal Amplification Assay↗