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Biomedical subjects

P Wattre

Publications and source records attributed to P Wattre.

At least 19 recordsLinked to original sources
PubMed2000

Simultaneous detection of 6 human herpesviruses in cerebrospinal fluid and aqueous fluid by a single PCR using stair primers.

A Herpes Consensus allows the simultaneous detection of 6 human herpesviruses: herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), human cytomegalovirus (HCMV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), and human herpes virus 6 (HHV-6). This technique was used first to examine retrospectively 100 DNA extracts from 95 CSF and 5 aqueous fluids, prepared by treatment by saturated NaCl followed by ethanol precipitation (n = 63) or by simple boiling (n = 37) and stored at -80 degrees C, and secondly to test prospectively 38 CSF samples for which two DNA extracts were prepared with commercially available DNA extraction kits. In all cases, the results were compared with those of an "in-house" PCR. Concordant results between both PCR and the Herpes Consensus techniques were obtained in 61 of 63 DNA extracts prepared by treatment by saturated NaCl (97%) and in only 31 of 37 boiled samples (84%). Both commercially available methods of DNA extraction examined appear to be suitable for Herpes Consensus PCR, although they cannot remove completely PCR inhibitors that must be sought in case of negative results. This preliminary study shows that the Herpes Consensus method should be of value for rapid diagnosis of herpesvirus infections on condition that it is performed on purified DNA extracts.

Aqueous Humor↗
PubMed1999

Role of interferon alpha (IFN-alpha) and interferon gamma (IFN-gamma) in the control of the infection of monocyte-like cells with human cytomegalovirus (HCMV).

The role of cytokines in the control of HCMV infection has been studied in THP-1 cells, a macrophage-like cell model and in MRC-5 cells. HCMV replication was studied by immune detection of viral immediate-early antigens (IEA) and virus yield was evaluated in MRC-5 cells by immunoperoxidase staining. Pretreatment of MRC-5 and phorbol 12-myristate 13-acetate (PMA)-treated THP-1 cells with IFN-alpha or IFN-gamma for 24 hr prior to the infection reduced the number of infected cells and virus yield. A synergistic anti-CMV activity in synthesis of early proteins was obtained with these cytokines in combination with TNF-alpha in differentiated THP-1 cells only. Treatment of HCMV-infected differentiated THP-1 cells or MRC-5 cells with IFN-alpha or IFN-gamma alone had no inhibitory effect on virus replication, however the virus yield was reduced with ganciclovir. A synergistic anti-CMV activity in virus yield was obtained only when infected differentiated THP-1 cells were treated with ganciclovir in combination with IFN-gamma. The current study shows that IFN-alpha and IFN-gamma can play a role in the reduction of HCMV replication in macrophage-like cells and in the efficiency of therapies with ganciclovir in this cell type and that the anti-CMV effect of cytokines may be different in fibroblasts and in macrophage-like cells.

Antiviral Agents↗
PubMed1999

MxA protein in capillary blood of children with viral infections.

Capillary blood of febrile children was lysed by using a lysis buffer containing ascorbic acid. MxA quantitation was performed by an immunochemiluminescent assay. The MxA values were significantly higher in capillary blood of infants with viral infections due to adenovirus (n = 5), rotavirus (n = 15), or respiratory syncytial virus (n = 28), than in capillary whole blood from infants with bacterial infections (n = 6) and healthy control patients (n = 20). A strong correlation was found between the MxA values in capillary whole blood and peripheral whole blood (r' = 0.86, P < 0.0001, n = 48). The MxA values found at these two sites were compared with the levels of IFN-alpha obtained by a dissociation enhanced lanthanide fluoroimmunoassay. A correlation between these two values was found. The results show that the combination of collection of blood by finger prick and specific immunochemiluminescent assay for MxA protein measurement may be of value for the diagnosis of viral infections in children.

Bacterial Infections↗
PubMed1998

A clinical and epidemiological study of human parvovirus B19 infection in fetal hydrops using PCR Southern blot hybridization and chemiluminescence detection.

Ninety-eight samples from 80 cases of spontaneous abortions after fetal death or hydrops fetalis from 12,000 pregnant women were examined using PCR. DNA was extracted from amniotic fluid, fetal blood, ascitic fluid and fetal biopsies or placenta specimens using QIA amp kits (QIAGEN). A 270-bp length fragment located within the B19 gene NS1 was amplified using PCR followed by electrophoresis and southern-blot hybridization assay using a horseradish peroxidase-labelled probe and chemiluminescence detection. This assay was able to detect 1 to 10 DNA copies in a 10 microliters sample. Parvovirus B19 was identified in 11 cases (14% of fetal hydrops; 1 case for 1,100 pregnancies). Amniotic fluid was the most common and reliable sample to assess the diagnosis. Gestational age ranged from 17 to 28 weeks (mean 23 weeks). IgM antibodies were detected in 3 maternal sera, 2 patients of which reported an exposure to B19 infection during pregnancy. In 2 cases, intrauterine blood transfusions led to the cessation of symptoms and to birth of normal babies.

Adolescent↗
PubMed1998

Coxsackie B virus infection and beta cell autoantibodies in newly diagnosed IDDM adult patients.

BACKGROUND: Environmental agents such as viruses have been identified as potentially important determinants of insulin-dependent diabetes mellitus (IDDM). Enterovirus infections, Coxsackievirus B especially, could be linked to the beta cell damaging process and to the onset of clinical IDDM. OBJECTIVES: Enteroviral (EV) infection and beta cell autoimmunity were studied in adult patients at the onset of IDDM. STUDY DESIGN: A total of 14 newly diagnosed-IDDM patients with ketosis or ketoacidosis were compared to, anteriorly diagnosed IDDM patients with metabolic decompensation, non-IDDM patients with metabolic decompensation and healthy adults. EV infection was studied by genomic RNA detection in whole blood using a RT-PCR assay. In order to assess the level of beta cell autoantibodies at the time of the initial metabolic decompensation, serum specimens from IDDM patients were tested for GAD65 antibodies and islet cell antibodies (ICAs). RESULTS: Coxsackie B3 or B4 virus genome was detected and genotyped in five of 14 (35.7) newly diagnosed IDDM patients and in one of 12 (8%) patients in the course of IDDM. By contrast, none of the 12 non-IDDM patients and none of the 15 healthy adults was positive for enterovirus RNA detection in whole blood. Positive GAD65 antibodies and ICAs assays were not significantly correlated to a positive EV-RNA detection. CONCLUSION: The present study demonstrates that Coxsackie B virus RNA sequences can be detected in the peripheral blood from adult patients at the onset or in the course of IDDM and suggests that a Coxsackie B virus infection could initiate or accelerate beta cell autoimmune damaging process.

Adult↗
PubMed1998

Production of interleukin-4, interferon (IFN)-gamma and IFN-alpha in human immunodeficiency virus-1 infection: an imbalance of type 1 and type 2 cytokines may reduce the synthesis of IFN-alpha.

Interferon-alpha (IFN-alpha) is an important molecule in the antiviral response, but cells from HIV-1-infected individuals show a reduced ability to secrete IFN-alpha. We investigated an association between an imbalance of type 1/type2 cytokines and the production of IFN-alpha in HIV-1 infection. We used whole blood culture to study the cytokine production profile, interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), in response to HIV-1 antigens and to study the Sendai Virus and HSV-1-induced-production of IFN-alpha in seven HIV-1-infected patients. An impaired synthesis of IFN-alpha was obtained in patients with a predominant IL-4 production (IL-4 > IFN-gamma), and we found a positive correlation between the ex vivo production of IFN-alpha and the IFN-gamma/IL-4 ratio but not with the HIV RNA copy number in plasma. We investigated the role of T-cell-derived cytokines in the in vitro production of IFN-alpha by PBMC from eight healthy donors, activated with Sendai Virus or HSV-1. Whereas type 2 cytokines (IL-4, IL-13) inhibited virus-induced IFN-alpha synthesis, on the contrary, type 1 cytokines (IL-2, IFN-gamma) enhanced it. A disarray in the T-cell-derived cytokine response may play a role in the defect of IFN-alpha production in HIV-1-infected individuals. Further investigations are needed to explore this hypothesis.

Adult↗
PubMed1997

Procalcitonin as a marker of bacterial sepsis in patients infected with HIV-1.

Procalcitonin (ProCT) is a recently described marker of severe sepsis. It was decided to assess the value of proCT as a marker of secondary infection in patients infected with HIV-1. ProCT plasma levels were measured by immunoluminometric assay in a prospective study in 155 HIV-infected individuals: 102 asymptomatic and 53 with lever or suspected secondary infections. The baseline plasma level of ProCT was low (0.5 ng/ml +/- 0.37), even in the latest stages of the disease, and did not differ from the values of healthy subjects (0.54 ng/ml +/- 0.08). EDTA-treated whole blood was collected from patients before starting specific antimicrobial therapy. No elevation of ProCT level was detected in HIV-infected patients with evolving secondary infections including PCP (n = 4), cerebral toxoplasmosis (n = 4), viral infections (n = 9), mycobacterial infections (n = 5), localized bacterial (n = 12) and fungal infections (n = 4), malignancies (n = 3), and in various associated infectious and non-infectious febrile events (n = 13). All these plasma values were lower than 2.1 ng/ml. In contrast, high ProCT plasma levels were detected in one HIV-infected patient with a septicaemic Haemophilus influenzae infection (16.5 ng/ml) and another one with a septicaemic Pseudomonas aeruginosa infection (44.1 ng/ ml), ProCT values decreased rapidly under appropriate therapy. ProCT seems to be a specific marker of bacterial sepsis in HIV-infected patients, as no increase in other secondary infections could be detected in those patients. A rapid determination of ProCT level could be useful to confirm or refute bacterial sepsis for a better management of febrile HIV-infected patients.

AIDS-Related Opportunistic Infections↗
PubMed1997

A macrophage-like cell model for testing anti-CMV drugs.

Monocyte/macrophage cells can be infected with human cytomegalovirus (HCMV) and could be a reservoir and a vehicle for virus spread in infected patients. The monocytic-like cell line THP-1 differentiated along the monocyte pathway with a phorbol ester (phorbol 12-myristate 13-acetate, PMA) for 24h was infected with cell free-supernatant of Towne CMV-strain infected fibroblasts MRC-5. We designed experiments to study the replication of HCMV in PMA treated THP-1 cells, exposed to DHPG (Cymevan, ganciclovir, Syntex). We incubated PMA treated THP-1 cells with DHPG for one to five days at 37 degrees C before or after the infection. The cells were cultivated for 5 days at 37 degrees C then supernatants of culture were collected and they were added to fibroblast cultures. The infection of the fibroblasts was studied on the basis of immune detection of viral immediate early antigens (IEA) in nucleus 24h after culture. Incubation of infected THP-1 cells with 11.1 microM DHPG for 5 days at 37 degrees C resulted in 95 +/- 2% inhibition of the replication whereas incubation of the infected cells with the same concentration of DHPG for two days resulted in 50 +/- 5% inhibition. Pretreatment of cells with DHPG for 5 days induced a 50 +/- 4% decrease of the level of infectious particles produced by HCMV infected cells. This is the first report of the antiviral effect of DHPG in macrophagic-like cells.

Antiviral Agents↗
PubMed1997

An interferon-gamma (IFN-gamma) based whole blood assay to detect T cell response to antigens in HIV-1 infected patients.

Recently it has been reported that cytokine production by T cells in response to antigens may be more sensitive test than lymphoproliferation. T cell reactivities to antigens is usually performed on isolated PBMCs, however whole blood is being used frequently for cytokine production studies. A whole blood assay is described to measure T cell mediated immune responses to HIV-1 and recall antigens. The cultures were performed in 96-well plates in which only 25 microliters of whole blood was required. We studied the production of IFN gamma in short term culture (24 hours) of 1/10 diluted heparinized whole blood (HWB) from 22 HIV-1 (+) patients grouped according to the 1993 classification of the CDC. IFN gamma was measured with an immunoassay in supernatants of HWB cultured in parallel experiments in the presence of supernatant of HIV-1LAI infected CD4+ T cells, p24 HIV antigen, PPD, tetanus toxoid (TET) and PHA. We found no production of IFN gamma in response to HIV-1 antigens in 15 HIV-1 (-) subjects; whereas a specific IFN gamma production in the presence of HIV-1 antigen was obtained in all of the 9 group A patients, in 7 of 8 group B patients and in 2 of 5 group C patients. In response to recall antigens (TET, PPD), we obtained IFN gamma production in 6 of 9 group A patients, 5 of 8 group B patients and in 1 of 5 group C patients, the response to PHA decreased but remained preserved until late in the disease. The HWB assay is a quick and simple potentially valuable tool for assessing cellular immune function in HIV-1+ patients.

Cell Culture Techniques↗
PubMed1996

Detection of enteroviral RNA by polymerase chain reaction in endomyocardial tissue of patients with chronic cardiac diseases.

Enteroviruses are suspected to be etiologic agents in myocarditis and cardiomyopathy. The prevalence of enteroviral (EV) heart infection in patients with chronic cardiomyopathy was determined through detection of specific EV genomic sequences using reverse transcription and polymerase chain reaction (RT-PCR) followed by slot blotting. Endomyocardial biopsies from the explanted hearts of 19 patients with dilated cardiomyopathy (DCM) and 14 patients with chronic coronary disease (CCD) were examined. EV genome was detected in 11 of 19 patients with DCM and in 8 of 14 patients with CCD. Ventricular biopsies from the control group, which included 35 healthy heart patients and 33 patients with myocardial infarction, were negative by EV RT-PCR. The percentage of patients showing presence of EV-RNA was almost similar in the DCM (57.9%) and CCD (57.1%) groups. The present study demonstrates that enterovirus RNA sequences persist in the myocardium in a significant proportion of patients suffering from end-stage ischaemic and dilated cardiac diseases and supports the hypothesis of a possible direct link between EV infection and the pathogenesis of chronic heart disease.

Adult↗
PubMed1996

Relationship between CMV and graft rejection after heart transplantation.

This study, which included 153 heart transplant patients, was designed to determine whether the cytomegalovirus (CMV) status of both donor and recipient may influence graft rejection. The follow-up was 1 year and they all received the same triple-drug immunosuppressive regimen with induction (antilymphocyte serum). There was no difference in the total rejection rate, but an increase in repeated rejection rate was shown in transplant recipients with hearts from CMV seropositive donors (P < 0.05). These data strongly suggest the impact of CMV in enhancement but not in induction of rejection. To prevent iterative rejection in the CMV seropositive donor group, antiviral therapy could be proposed during enhancement of antirejection therapy.

Cytomegalovirus Infections↗
PubMed1996

Multicenter evaluating of a commercially available PCR assay for diagnosing enterovirus infection in a panel of cerebrospinal fluid specimens.

Thirteen laboratories participated in blind tests of a panel of 20 coded cerebrospinal fluid specimens (7 uninfected samples, 3 samples infected with 1 50% tissue culture infective dose [TCID50]/0.1 ml [nonenterovirus strains], and 10 samples infected with 10, 1, or 0.1 TCID50/0.1 ml [three different enterovirus serotypes]) on the Amplicor enterovirus PCR assay (Roche Diagnostic Systems). The panel was also evaluated by in-house PCR (two nested-PCR and three one-step PCR assay) or tissue culture (eight laboratories). The viral load was shown to influence greatly the sensitivity of the assay. The average sensitivity of the Amplicor test ranged from 67 to 98% for viral titers of 1 to 10 TCID50/0.1 ml, respectively; titers of 0.1 TCID50/0.1 ml resulted in a sensitivity of only 16%. The overall specificity of the Amplicor test was 98%. The Amplicor assay compared favorably to the five in-house PCR tests (no significant difference in either sensitivity or specificity) and was much more sensitive than tissue culture (P < 0.001), even for high viral loads. It was easy to perform, rapid (about 6 h), well-standardized, and appeared to be suitable for the diagnosis of enterovirus meningitis on a routine basis in laboratories trained in molecular biology techniques.

Base Sequence↗
PubMed1996

[Apoptosis and human viral infections].

Homeostasis of cell numbers in tissues is maintained by a critical balance between cell proliferation and programmed cell death or apoptosis. Many human viruses are able to develop suitable strategies for modifying apoptosis in virus-infected cells and in virus-primed T cells. Apoptosis is characterized by the fragmentation of nuclear DNA into 180-200 bp apoptotic bodies and can be analysed microscopically or by flow cytometry using staining with various dyes. Moreover DNA cleavage can be identified by electrophoresis and by specific labeling using in situ nucleotidyltransferase assay (ISNT), terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling technique (Tunel), or by Elisa. Adenovirus E1A induces expression of protooncogenes c-myc and c-fos which sensitize cells to apoptosis; EBV EBNA-5, and adenovirus E1A, HPV E7, and polyomavirus large T act in the same way by displacing pRB-bound E2F. EBV EBNA-5, HPV E6, Adenovirus E1B 55 kDa inactivate the tumor suppressor protein p53 and engage the cells in the transformation process. EBV LMP-1, HHV6, and HTLV1 tax induce the antiapoptotic bcl-2 protein. EBV BHRF1 encodes proteins with homology to bcl-2 and Adenovirus E1B 19 kDa encodes proteins that have protective functions similar to bcl-2. Activated lymphocytes responding to viral infections express high levels of fas and are susceptible to apoptosis. TNF alpha can down- or up-regulate fas and down-regulates TNF-R. Adenovirus E1B 19 kDa blocks the proapoptotic activity of TNF alpha. Inversly, Cytomegalovirus, hepatitis C virus and Myxoviruses up-regulate fas antigen prior to undergoing apoptosis. In HIV-infected patients, CD4+ T-cell apoptosis is mediated by the cytopathic effect of the virus and the cell surface expression of gp 120-env protein. Moreover, an accelerated T-cell apoptosis in HIV-infected individuals is characterized by (i) HIV gp120-CD4+ cross-linking and subsequent aberrant signaling of T-cells, (ii) involvement of TNF alpha-fas/Apo-1 (TNF-R) binding, (iii) involvement of accessory cells as an apoptosis inducer and as a result of defective antigen presentation, (iv) possible superantigen activity induced by HIV products and cofactors. Many viruses also encode proteins with protease activity which could induce apoptosis. The induction of apoptosis may result in virus clearance, in contrast the inhibition of apoptosis may result in virus cell transformation and viral persistence. Indirectly, the apoptosis of infected cells may be induced by CTLs, NK cells and cytokines. In addition, apoptosis-mediated physiological depletion of T lymphocytes in the course of viral infection can silence the immune response and can induce immunodeficiency.

Apoptosis↗
PubMed1995

Detection of enterovirus-specific RNA sequences in explanted myocardium biopsy specimens from patients with dilated or ischemic cardiomyopathy.

Enteroviral RNA (EV-RNA) was detected in endomyocardial tissue by means of retrotranscription and polymerase chain reaction (RT-PCR) followed by slot-blot hybridization. The myocardial biopsy specimens studied were taken at the time of heart transplantation from 15 patients with dilated cardiomyopathy (DCM) and from 10 patients with ischemic cardiomyopathy (ICM). Specimens from 18 (72%) of the 25 patients were positive for EV-RNA, whereas no control specimens (myocardial specimens from 29 healthy organ donors and atrial specimens from 15 patients with acute myocardial infarction treated by anatomic bypass) yielded evidence of EV-RNA. The rates of EV-RNA detection for the two groups requiring heart transplantation did not differ significantly (66.7% vs. 80.0%; chi 2 test). Our findings support a link between enteroviral infection in both DCM and ICM and suggest a pathogenic role for the enteroviruses.

Adult↗
PubMed1995

[Value of gene amplification of herpesviruses in the diagnosis and treatment of acute viral encephalitis].

Positive diagnosis of Herpes simplex virus (HSV) encephalitis was rarely obtained in the past, when brain biopsy had been performed. Other tests (HSV antigen and HSV antibodies detection and interferon alpha measurement, in cerebrospinal fluid) failed to prove HSV infection. Polymerase chain reaction has been proposed for accurate and rapid diagnosis of HSV encephalitis. With 35 cycles of a DNA polymerase sequence duplication, sensitivity reaches 95% and specificity 100%. HSV PCR is a useful tool for the diagnosis of acute encephalitis. This should be available in many neurologic clinics. Therapeutic consequences include rapid disruption of aciclovir when clinical features, MRI study and negative PCR suggest non herpetic encephalitis.

Acute Disease↗