Biomedical subjects

C Pasquier

Publications and source records attributed to C Pasquier.

At least 37 records · Page 2Linked to original sources

PubMed2000

Shift in HIV resistance genotype after treatment interruption and short-term antiviral effect following a new salvage regimen.

OBJECTIVE: To investigate the changes in genotypic drug-resistance pattern, plasma HIV RNA and CD4 cell count after treatment interruption and assess the short-term antiviral effect of a new salvage regimen. DESIGN: Prospective study of 38 patients with multiple failing regimens who had completely stopped all medication for 3 months before a three to five-drug regimen was reintroduced according to clinical guidelines. METHODS: Patients were tested for HIV resistance before and after treatment interruption by population-based sequencing and clonal analysis of selected patients. RESULTS: Discontinuation of therapy for 3 months was associated with a median increase in HIV RNA of 0.4 log10 and a median decrease in CD4 cell count of 43 x 10(6)/l. Sixty-one per cent of patients had a shift from the drug-resistant genotype to a predominantly wild-type genotype. The patients significantly likely to show genotype reversion were those in Centers for Disease Control groups A or B, who had been exposed to few drugs, had a low plasma HIV RNA, or a high CD4 cell count. The only independent factor predicting genotype reversion was the clinical stage. The median change in plasma HIV RNA at month 3 after treatment reintroduction was -2.3 log10 copies/ml in patients who had genotype reversion compared with -0.6 log10 copies/ml in patients without genotype reversion (P = 0.004). CONCLUSION: Suspending treatment for 3 months after multiple failures could be a suitable strategy for optimizing salvage therapy provided it is instituted early, before the HIV disease becomes too advanced.

Adult↗

PubMed2000

Sperm washing and virus nucleic acid detection to reduce HIV and hepatitis C virus transmission in serodiscordant couples wishing to have children.

BACKGROUND: Use of a motile spermatozoa isolation process was assessed for reducing the transmission of HIV and hepatitis C virus (HCV) during artificial insemination in HIV-serodiscordant couples in which the man is infected. PATIENTS: Thirty-two HIV-1-infected clinically asymptomatic men, having a median CD4 cell count of 396 x 10(6)/l and a median blood plasma HIV-1 RNA content of 414 copies/ml. Of these, 16 were infected with both HIV and HCV. METHODS: Motile spermatozoa were isolated from 51 semen samples by density gradient and 'swim-up'. HIV-1 and HCV genomes were detected and quantified in the blood plasma and seminal plasma, and detected in seminal cell fractions obtained during spermatozoa isolation. RESULTS: HIV-1 RNA was detected in 30% of seminal plasma samples. HIV-1 genomes were found in 18% of seminal cell samples, but in none of the motile spermatozoa fractions after 'swim-up'. There was no correlation between the HIV-1 RNA concentrations in the blood and seminal plasma. HIV-1 genome was detected intermittently in patients who gave more than one sample. HCV RNA was detected in 20% of seminal plasma samples from HCV viraemic patients, but in no seminal cells or motile spermatozoa fractions. CONCLUSIONS: Purification of motile spermatozoa by density gradient plus 'swim-up' reduced the HIV-1 and HCV genomes in the semen of infected individuals to undetectable levels. This method, associated with a standardized virus assay, could be useful for serodiscordant couples (males infected) who wish to have children.

Adult↗

PubMed2000

Prevalence of drug resistant mutants and virological response to combination therapy in patients with primary HIV-1 infection.

Baseline genotype resistance analysis was carried out in 48 adults with primary HIV-1 infection between 1995 and 1998 before starting early combination therapy. Seventeen percent (8/48) of the isolates displayed key mutations conferring resistance to reverse transcriptase (RT) inhibitors such as amino acid substitutions 215Y/F (5/48,10%), 70R (3/48, 6%), 184V (2%). Two percent (1/48) had a major mutation associated with resistance to protease inhibitors (D30N). Other mutations at positions 10, 15, 20, 33, 36, 46, 63, 71, 77, 82, 93 of the protease gene were frequent (73%). Among the 46 patients who were given antiretroviral combination therapy and who responded durably to treatment after 6 and 12 months, there was no significant difference between those harboring RT mutant strains (Group I) and those with wild-type isolates (Group II). No significant difference was found at months 6 and 12 between the two groups in terms of CD4+ cell counts. These findings suggest that the presence of drug-resistant strains at the time of primary HIV-1 infection does not necessarily predict drug failure. Other factors, such as adherence to treatment, tolerance and pharmacokinetics parameters are probably major determinants of virological response in patients with early therapeutic intervention.

Adult↗

PubMed2000

Longitudinal analysis of hepatitis C virus replication and liver fibrosis progression in renal transplant recipients.

The pathogenesis of hepatitis C virus (HCV) infection was investigated by analysis of changes in viral and histologic parameters in 36 renal transplant recipients who were infected with HCV before transplantation. Each patient was classified according to development of liver fibrosis as assessed by 2 liver biopsies done 45 and 81 months after transplantation: 13 had progressing liver fibrosis (fibrosers) and 23 did not (nonfibrosers). All developed high-titer posttransplant viremia with a significant increase of 1.2 log RNA copies/mL. There were no significant differences in the increases in serum HCV RNA or genotype distributions in fibrosers and nonfibrosers. The hypervariable region (HVR)-1 of the HCV genome was analyzed by cloning and sequencing 20 clones per sample from 5 fibrosers and 5 nonfibrosers. Comparison of samples revealed that liver fibrosis progression was significantly associated with slower HVR-1 quasispecies diversification, suggesting the selection of more aggressive variants in fibrosers.

Adult↗

PubMed2000

Anethole dithiolethione regulates oxidant-induced tyrosine kinase activation in endothelial cells.

Interaction between neutrophils and endothelial cells is one of the first steps in the functional response of polymorphonuclear neutrophils (PMN), and is necessary for their migration toward damaged tissues. PMN activation, leading to their adhesion to and migration between endothelial cells, is part of a complex phenomenon that can be altered in pathological situations such as the ischemia-reperfusion syndrome, in which large numbers of PMN are recruited to the tissue and release reactive oxygen species (ROS) near the vessel wall. ROS have been implicated in the pathogenesis of various inflammatory diseases. The increased adhesion of PMN to ROS-stimulated endothelial cells involves an increase in tyrosine phosphorylation of a tyrosine kinase focal adhesion kinase (p125FAK) and several cytoskeleton proteins, including paxillin and p130 cas. We examined the role of glutathione (GSH) in the regulation of this adhesion phenomenon and in the increased tyrosine phosphorylation induced by ROS. For this purpose we used anethole dithiolthione (ADT), which increases the glutathione synthesis by activating gamma-glutamyl-cysteine synthetase. We found that ADT reduced both PMN adhesion to ROS-stimulated human umbilical vein endothelial cells (HUVEC) and tyrosine phosphorylation of p125FAK and paxillin. ADT increased redox status by increasing intracellular GSH content in oxidized cells. These results show that GSH can reverse the effect of oxidation on tyrosine kinase activation and phosphorylation, and thus plays an important role in cell signaling. They also confirm the antioxidant activity of ADT.

Anethole Trithione↗

PubMed2000

Genetic heterogeneity of hypervariable region 1 of the hepatitis C virus (HCV) genome and sensitivity of HCV to alpha interferon therapy.

Hepatitis C virus (HCV) populations persist in vivo as a mixture of heterogeneous viruses called quasispecies. The relationship between the genetic heterogeneity of these variants and their responses to antiviral treatment remains to be elucidated. We have studied 26 virus strains to determine the influence of hypervariable region 1 (HVR-1) of the HCV genome on the effectiveness of alpha interferon (IFN-alpha) therapy. Following PCR amplification, we cloned and sequenced HVR-1. Pretreatment serum samples from 13 individuals with chronic hepatitis C whose virus was subsequently eradicated by treatment were compared with samples from 13 nonresponders matched according to the major factors known to influence the response, i.e., sex, genotype, and pretreatment serum HCV RNA concentration. The degree of virus variation was assessed by analyzing 20 clones per sample and by calculating nucleotide sequence entropy (complexity) and genetic distances (diversity). Types of mutational changes were also determined by calculating nonsynonymous substitutions per nonsynonymous site (K(a)) and synonymous substitutions per synonymous site (K(s)). The paired-comparison analysis of the nucleotide sequence entropy and genetic distance showed no statistical differences between responders and nonresponders. By contrast, nonsynonymous substitutions were more frequent than synonymous substitutions (P </= 0.05) in responders, but there was no significant difference in nonresponders. Nonsynonymous substitutions tended to be more frequent than synonymous substitutions in women (P = 0.06) but not in men. Nucleotide entropy and genetic distances were significantly related to serum RNA concentration (P </= 0.01). Our findings suggest that after controlling for the major determinants of interferon response, neither complexity nor diversity of the HVR-1 region is associated per se with virus eradication. Because a higher proportion of nonsynonymous substitutions than synonymous substitutions was found only in responders, host anti-HCV-specific immune response rather than viral factors may be playing an important role in the interferon response.

Adult↗

PubMed1999

Molecular evidence for nosocomial transmission of hepatitis C virus in a French hemodialysis unit.

A systematic virological follow-up of hemodialysis patients identified 11 cases of de novo hepatitis C virus (HCV) infection in the same unit that were not due to blood transfusion. There were three groups of infection, each occurring within a period of 3 months: four infections with genotype 1b, two infections with genotype 1b, and five infections, four with genotype 1a and one with genotype 5a. The possibility of patient-to-patient transmission was addressed by sequencing the first hypervariable region of the HCV genome in sera taken shortly after infection. Phylogenetic analysis indicated clustering of most of the cases of de novo infections. Sequence homologies identified potential contaminators among already infected patients. All patients who were infected with closely related HCV isolates were found to have been treated in the same area and during the same shift or on the previous one. These infections could have been due to occasional breaches of the usual hygiene measures. Strict adhesion to hygiene standards and routines, continuously supervised, remains the key rule in the management of dialysis patients. Nevertheless, the isolation of patients with HCV could reduce the risk of infection because occasional lapses of preventive hygiene measures or unpredictable accidents can always take place in a hemodialysis unit. This policy needs to be evaluated by large-scale prospective studies.

Adult↗

PubMed1999

Using RT-PCR and bDNA assays to measure non-clade B HIV-1 subtype RNA.

The performance of the new version of RT-PCR assay (Amplicor HIV-1 Monitor v1.5) was assessed. The quantification of non-B subtype HIV-1 plasma RNA (30A, 1C, 1D, 3E, 2F, 3G) obtained using Monitor v1.5 was compared to the former version of this assay (Monitor v1.0) and to the Quantiplex v2.0 bDNA assay. The new primers used in Monitor v1.5 were similar to the former version in both specificity and sensitivity. The new primers corrected the detection and quantification defect observed previously for HIV-1 non-B subtypes and gave slightly higher RNA concentrations than those measured using the bDNA assay (+0.39 log copies/ml).

Female↗

PubMed1999

Anti-proteinase-3 (PR3) antibodies (C-ANCA) recognize various targets on the human umbilical vein endothelial cell (HUVEC) membrane.

Numerous studies suggest that C-ANCA are directly pathogenic in vasculitis by activating leucocytes (oxidative burst, enzyme release, endothelial cytotoxicity, etc.). We and others have shown that C-ANCA can also directly activate HUVEC, but the precise target on HUVEC is unknown. We show in this study that C-ANCA recognize various targets on the HUVEC membrane (different from PR3 in our model), leading to secondary cell activation. Polyclonal affinity-purified C-ANCA recognized targets on the unfixed endothelial membrane in fluorescent ELISA, flow cytometry, and immunoprecipitation studies. C-ANCA did not react with Fcgamma receptors. Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments showed that HUVEC did not express PR3. The targets of polyclonal and monoclonal anti-PR3 antibodies on the endothelial membrane were not the same. Some epitopes were lost after trypsin-EDTA digestion and formaldehyde fixation of cells, whereas anti-PR3 targeted unfixed HUVEC. This suggests that anti-PR3 react with the endothelial membrane and recognize conformational epitopes shared with PR3. Endothelial cells may thus participate in the inflammation associated with Wegener's granulomatosis and contribute to the emergence of clinical manifestations.

Antibodies, Antineutrophil Cytoplasmic↗

PubMed1999

A novel method for predicting transmembrane segments in proteins based on a statistical analysis of the SwissProt database: the PRED-TMR algorithm.

We present a novel method that predicts transmembrane domains in proteins using solely information contained in the sequence itself. The PRED-TMR algorithm described, refines a standard hydrophobicity analysis with a detection of potential termini ('edges', starts and ends) of transmembrane regions. This allows one both to discard highly hydrophobic regions not delimited by clear start and end configurations and to confirm putative transmembrane segments not distinguishable by their hydrophobic composition. The accuracy obtained on a test set of 101 non-homologous transmembrane proteins with reliable topologies compares well with that of other popular existing methods. Only a slight decrease in prediction accuracy was observed when the algorithm was applied to all transmembrane proteins of the SwissProt database (release 35). A WWW server running the PRED-TMR algorithm is available at http://o2.db.uoa. gr/PRED-TMR/

Algorithms↗

PubMed1999

An hierarchical artificial neural network system for the classification of transmembrane proteins.

This work presents a simple artificial neural network which classifies proteins into two classes from their sequences alone: the membrane protein class and the non-membrane protein class. This may be important in the functional assignment and analysis of open reading frames (ORF's) identified in complete genomes and, especially, those ORF's that correspond to proteins with unknown function. The network described here has a simple hierarchical feed-forward topology and a limited number of neurons which makes it very fast. By using only information contained in 11 protein sequences, the method was able to identify, with 100% accuracy, all membrane proteins with reliable topologies collected from several papers in the literature. Applied to a test set of 995 globular, water-soluble proteins, the neural network classified falsely 23 of them in the membrane protein class (97.7% of correct assignment). The method was also applied to the complete SWISS-PROT database with considerable success and on ORF's of several complete genomes. The neural network developed was associated with the PRED-TMR algorithm (Pasquier,C., Promponas,V.J., Palaios,G.A., Hamodrakas,J.S. and Hamodrakas,S.J., 1999) in a new application package called PRED-TMR2. A WWW server running the PRED-TMR2 software is available at http://o2.db.uoa.gr/PRED-TMR2

Animals↗

PubMed1999

Mutations conferring resistance to zidovudine diminish the antiviral effect of stavudine plus didanosine.

This study evaluated the influence of zidovudine (ZDV) resistance mutations on the antiviral effect of the combination of stavudine (D4T) plus didanosine (ddI) in patients treated previously with ZDV plus zalcitabine (ddC). Twenty patients who had been treated with ZDV plus ddC for a median duration of 11 months (range, 7-42 months) were switched to D4T (40 mg twice a day [BID]) + ddI (200 mg BID) in an open pilot study lasting 6 months. The CDC classes were A (n = 10) and B (n = 10). The median baseline CD4 count was 285/mm(3) and the median baseline plasma virus RNA (Amplicor HIV Monitor RT-PCR assay) was 4.6 log copies/ml. Population-based sequence analysis detected mutations associated with resistance to reverse transcriptase (RT) inhibitors in the RT domain of virus RNA from baseline plasma samples in 13/20 (65%) patients. Twelve patients had mutations associated with zidovudine resistance (3 T215Y - M41L - L210W; 3 T215Y - M41L; 2 T215Y - L210W; 3 T215Y; 1 K70R) and 1 patient had a multi-dideoxynucleoside resistance mutation (QI5IM). Patients with a resistance mutation had a significantly lower RNA suppression after 3 and 6 months (median RNA reduction -0.5 log and -0.1 log) than the remaining patients (-1.6 log and -2 log). Fifty percent of patients with wild-type viruses had undetectable plasma RNA after 24 weeks of D4T plus ddI therapy, whereas all those with mutated viruses had HIV RNA concentration > 3 log copies/ml at week 24 (P <.05). Our finding may have implications when deciding on a second line therapy with three or four drugs that includes two new nucleoside analogues. Cross-resistance between nucleoside analogues deserves maximal attention to ensure optimal antiretroviral therapy and design algorithms for antiretroviral management based on genotypic antiretroviral resistance testing.

Adult↗

PubMed1999

Intermittent selection pressure with zidovudine plus zalcitabine treatment reduces the emergence in vivo of zidovudine resistance HIV mutations.

The development of mutations conferring drug resistance was investigated in 49 antiretroviral-naive asymptomatic HIV-1 subjects with CD4+ cell counts of 250-500/mm3 given intermittent (6-week courses, 6 weeks apart) or continuous treatment with zidovudine (AZT) plus zalcitabine (ddC) over 54 weeks. The concentration of human immunodeficiency virus type 1 RNA in the plasma and the CD4 cell counts were measured every 6 weeks. The rate of decrease of HIV-1 RNA concentration in plasma after a 6-week course of AZT + ddC was similar for each treatment cycle (approximately 1-log reduction). The plasma HIV-1 RNA concentration returned to its initial level at each treatment interruption. The mean CD4 cell counts after 54 weeks in the two treatment groups were similar. Genotype analysis by sequencing the reverse transcriptase coding region from plasma viral RNA on treatment showed a lower frequency of AZT resistance mutations after 54 weeks in patients given intermittent treatment (18%) than in those treated continuously (79 %, P < 0.001). No mutations conferring ddC resistance or multidideoxynucleoside resistance were observed in either group. These findings may have clinical implications for long-term treatment strategies.

Adult↗

PubMed1998

Decay of HIV-1 DNA in patients receiving suppressive antiretroviral therapy.

We have examined the effect of potent antiretroviral regimens on the latent reservoirs of HIV-1. The HIV-1 DNA in the peripheral blood mononuclear cells (PBMC) of 10 patients with undetectable plasma HIV-1 RNA (<20 copies/ml) who had received combination antiretroviral therapy was assayed every 12 weeks. No evidence of residual viral replication was found in the PBMC after 24 weeks of treatment. Although HIV-1 DNA remained detectable in all patients, it decreased significantly from 3.5 log copies/10(6) cells (range, 1.8-4.7 log copies/10(6) cells) to 2.3 log copies/10(6) cells (range, 0.6-3.1 log copies/10(6) cells) after 60 weeks of suppressive therapy. Analysis based on 6 patients who reached 60 weeks showed a slow decline with an estimated half-life of 40 weeks (range, 26-163 weeks). Genotypic analysis by sequencing the HIV-1 pol gene revealed no changes in the reverse transcriptase or protease coding regions after 48 to 60 weeks of therapy. The findings suggest that, in addition to potent antiretroviral regimens, new strategies must be developed to ensure eradication of the latent reservoir of provirus, and hence of the virus itself.

Adult↗

PubMed1998

Hydroxyl radical as a potential intracellular mediator of polymorphonuclear neutrophil apoptosis.

We investigated reactive oxygen species (ROS) involvement in polymorphonuclear neutrophilic leukocyte (neutrophil) apoptosis triggering. Neutrophils were incubated with xanthine oxidase (XO), which produces superoxide anion (O2.-) and hydrogen peroxide (H2O2) or glucose oxidase (GO), which produces only H2O2. Both XO and GO accelerated apoptosis when compared to spontaneously aged neutrophils. Catalase inhibited both spontaneous apoptosis and XO- or GO-accelerated apoptosis, but superoxide dismutase did not. Hydrogen peroxide can enter the cell, thus generating intracellular oxidation, which was observed by flow cytometry. Furthermore, the intracellular reduced glutathione content fell in the presence of XO or GO; however, apoptosis was not accelerated in the presence of buthionine sulfoximine (BSO), suggesting that the fall in glutathione in the presence of XO or GO is a consequence of oxidative stress but not a trigger of apoptosis. Hydrogen peroxide can react with iron to form hydroxyl radicals (HO.); we observed that two iron chelators, deferoxamine and hydroxybenzyl ethylenediamine (HBED), both inhibited spontaneous and accelerated apoptosis, suggesting that HO. may mediate neutrophil apoptosis.

Apoptosis↗

PubMed1998

Quantification of HIV-1 proviral DNA by a standardized colorimetric PCR-based assay.

A simple method was developed for measuring human immunodeficiency virus type 1 (HIV-1) proviral DNA in mononuclear cells based on the commercially available Amplicor(TM) HIV-1 polymerase chain reaction (PCR) assay and the limiting dilution method. The lowest limit of detection was four proviral genomes per 10(6) cells. The accuracy was demonstrated by using serial dilutions of LAV-8E5 cells, and the interassay variability was 0.2 log. The technique was used to measure HIV-1 proviral DNA in the peripheral blood mononuclear cells (PBMC) of 18 antiretroviral drug-naive HIV-1-positive individuals before and 4 weeks after initiating double nucleoside therapy. The DNA proviral titers at baseline (median = 3.45, range = 2.11-4.7 log copies/10(6) cells) were 2.08 log greater than the infectivity titers, but there was a correlation between these two parameters (r = 0.63, P = 0.009). The mean decrease in the proviral DNA titer after 4 weeks of therapy was 0.31 log, whereas the decrease in the infectivity titer was 0.81 log and the decrease in the plasma RNA concentration was 1.29 log. The technique was also used to measure HIV-1 proviral DNA in the PBMC of 11 patients who had undetectable plasma HIV-1 RNA after being placed on combination antiretroviral therapy. Although proviral DNA remained detectable in all patients after 36 weeks of treatment, a gradual decline with an estimated half-life of 21-58 weeks was observed. The reliability of this simple and convenient colorimetric PCR-based technique indicates its suitability for assessing the effect of current antiretroviral regimens on the latent reservoirs of provirus.

Adult↗

PubMed1998

Assessment of a hepatitis C virus antibody assay in saliva for epidemiological studies.

The performance of a commercially available assay for detection of hepatitis C virus (HCV) antibody in saliva samples was assessed. Samples of saliva were collected from 270 individuals whose HCV antibody status was determined by serum assay (161 HCV-positive, 109 HCV-negative). The saliva samples were tested for the presence of HCV antibodies using a modified protocol. The sensitivity was 94.4% (95% CI, 89.3-97.2%) and the specificity 99.1% (95% CI, 94.3-100%). Although the optical density in tests on HIV-positive individuals was lower than that among HIV-negative individuals, the HIV status had no significant influence on the results of the HCV assay in saliva. These findings suggest that tests on saliva can be useful in epidemiological studies for estimating the prevalence of HCV in populations that are difficult to reach.

Antibodies, Viral↗

PubMed1998

Congenital disorders sharing oxidative stress and cancer proneness as phenotypic hallmarks: prospects for joint research in pharmacology.

In spite of very distinct genotypic assets, a number of congenital conditions include oxidative stress as a phenotypic hallmark. These disorders include Fanconi's anaemia, ataxia telangiectasia, xeroderma pigmentosum and Bloom's syndrome, as well as two frequent congenital conditions: Down's syndrome and cystic fibrosis. Cancer proneness is a clinical feature shared by these disorders, while other manifestations include early ageing, neurological symptoms or congenital malformations. The onset of oxidative stress has been related to excess formation, or defective detoxification, of reactive oxygen species (ROS). This can arise from either the abnormal expression or inducibility of ROS-detoxifying enzymes, or by defective absorption of nutrient antioxidants. Resulting oxidative injury has been characterized through: (i) DNA, protein or lipid oxidative damage; (ii) excess ROS formation (in vitro and ex vivo); (iii) sensitivity to oxygen-related toxicity; (iv) improvement of cellular defects by either hypoxia or antioxidants; and (v) circumstantial evidence for in vivo oxidative stress (as e.g. clastogenic factors). Investigations conducted so far have been confined to individual disorders. Comparative studies of selected indicators for oxidative stress could provide further insights into the pathogenesis of each individual condition. Such a unified approach may have wide-ranging consequences for studies of ageing and cancer.

Aging↗