Biomedical subjects

J Piette

Publications and source records attributed to J Piette.

At least 91 records · Page 5Linked to original sources

PubMed1995

1-Thiomethoxsalen and 1-thiopsoralen: synthesis, photobiological properties, and site specific reaction of thiopsoralens with DNA.

The sulfur analogues of psoralen and 8-methoxypsoralen (8-MOP) in the pyrone moiety were synthesized and compared to the parent compounds in terms of photoreactivity with viral M13mp19 RF DNA. The damaged viral DNA was transfected into Escherichia coli and scored for infectivity toward Ca-treated wild-type E. coli. This allowed a comparative study of the sulfur and oxygen analogues to be made in terms of photoreactivity. Furthermore, the DNA sequence specificity for the formation of monoadducts and cross-links of the four analogues was determined with 32P-labeled oligonucleotides containing thymidine in different sequences. The most site specific of the studied psoralens is 8-MOP, while 1-thiopsoralen is the most reactive analogue. This new thio analogue of psoralen leads to the efficient formation of monoadducts and cross-links in any pyrimidine-purine site.

Base Sequence↗

PubMed1995

NF-kappa B transcription factor and human immunodeficiency virus type 1 (HIV-1) activation by methylene blue photosensitization.

Reactive oxygen species like hydrogen peroxide (H2O2) have been shown to serve as messengers in the induction of NF-kappa B and then in the activation and replication of HIV-1 in human cells. Because singlet oxygen (1O2) is another very important reactive oxygen species whose action in transcription factor activation is totally undetermined, we started to investigate its role in both NF-kappa B and HIV-1 activation. For provoking unbalanced redox conditions, 1O2 was generated by photosensitization using methylene blue as photosensitizer. Lymphocytes or monocytes (ACH-2 or U1 respectively) latently infected with HIV-1 were treated by photosensitization mediated by methylene blue and the production of reactive oxygen species was monitored through their cytotoxic effect in infected cells. The generation of 1O2 by methylene blue turns out to be very efficient in inducing NF-kappa B as a heterodimer composed of the p50 and p65 subunits. This induction appears specific since other transcription factors like AP-1 are only weakly activated by this treatment. In comparison with other inducing treatments such as phorbol esters or tumor necrosis factor alpha (TNF-alpha), the methylene-blue-mediated activation of NF-kappa B is slow, becoming optimal 180 min after treatment. These kinetic data were obtained by following, on the same samples, both the emergence of NF-kappa B in the nucleus and the disappearance of I kappa B-alpha in the cytoplasmic extracts. Conjugated with the induction of this transcription factor, HIV-1 reactivation from these latently infected cells was also observed by the measurement of reverse transcriptase activity in the cell supernatants. These data allow us to postulate that 1O2 is a biologically important reactive oxygen species which could play a role in the establishment of oxidative stress conditions leading to HIV-1 activation via the presence of NF-kappa B in the nucleus of infected cells.

Base Sequence↗

PubMed1995

Distribution of varicella-zoster virus gpI and gpII and corresponding genome sequences in the skin.

In the course of varicella-zoster virus (VZV) infection, some viral capsid antigens are found in the epidermis and dermis. The aim of this study was to investigate the localisation of two major VZV glycoproteins (gpI and gpII) and of their respective genes in the skin. The distribution of VZV gpI and II in 27 formalin fixed paraffin embedded skin biopsies from herpes zoster eruptions were compared by immunohistochemistry. Double immunostaining was carried our to identify infected cells. The presence of viral nucleic acids coding for gpI and gpII was examined by in situ hybridisation. The distribution of gpI and gpII and their corresponding genome sequences was similar in the epidermis. gpI and gpII were also detected in dermal FXIIIa positive dendrocytes, in Mac 387 and CD68 positive macrophages, and in perineural and endothelial cells. However, the corresponding viral nucleic acids were rarely and barely detected in these cells of the dermis. It is concluded that VZV infection of epithelial cells follows a different course than in dermal cells.

Adult↗

PubMed1995

Immunohistochemical identification of varicella-zoster virus gene 63-encoded protein (IE63) and late (gE) protein on smears and cutaneous biopsies: implications for diagnostic use.

Early and specific recognition of varicella zoster virus (VZV) infection is of vital concern in immunocompromised patients. The aim of this study was to compare the diagnostic accuracy of histochemical and immunohistochemical identification of the VZV ORF63 encoded protein (IE63) and of the VZV late protein gE on smears and formalin-fixed paraffin-embedded skin sections taken from lesions clinically diagnosed as varicella (n = 15) and herpes zoster (n = 51). Microscopic examinations of Tzanck smears and skin sections yielded a diagnostic accuracy of Herpesviridae infections in 66.7% (10/15) and 92.3% (12/13) of varicella, and 74.4% (29/39) and 87.8% (43/49) of herpes zoster, respectively. Immunohistochemistry applied to varicella provided a type-specific virus diagnostic accuracy of 86.7% (13/15; IE63) and 100% (15/15; gE) on smears, and of 92.3% for both VZV proteins on skin sections. In herpes zoster, the diagnostic accuracy of immunohistochemistry reached 92.3% (36/39; IE63) and 94.9% (37/39; gE) on smears, and 91.7% (44/48; IE63) and 91.8% (45/49; gE) on skin sections. These findings indicate that the immunohistochemical detection of IE63 and gE on both smears and skin sections yields a higher specificity and sensitivity than standard microscopic assessments.

Adolescent↗

PubMed1995

DNA probe hybridisation in microwells using a new bioluminescent system for the detection of PCR-amplified HIV-1 proviral DNA.

A new bioluminescent detection system combined with a sandwich DNA hybridisation reaction in microwells has been developed for the detection of human immunodeficiency virus type 1 (HIV-1) provirus DNA amplified by the polymerase chain reaction (PCR). First, a fragment of the HIV-1 gag gene was amplified. The amplified DNA fragments were denatured and hybridised to a capture probe immobilised in microwells and to a biotinylated detection probe. A streptavidin-pyruvate kinase conjugate could then react on the biotinylated probe and the kinase activity detected by means of the luciferin-luciferase system, with production of a bioluminescent signal. This sandwich assay followed by a bioluminescent reaction detected as little as 7 amol of target DNA. The bioluminescent assay detected 5 HIV copies generated after one round of PCR, even if no band was seen on an agarose gel. The assay was applied to the detection of HIV-proviral DNA in peripheral blood mononuclear cells after one round of PCR and allowed to clearly identify a positive sample as compared to nested PCR.

Base Sequence↗

PubMed1995

Iron chelation decreases NF-kappa B and HIV type 1 activation due to oxidative stress.

An important aspect of human immunodeficiency virus (HIV-1) infection is the regulation of its expression by nuclear factor kappa B (NF-kappa B) through redox-controlled signal transduction pathways. In this study, we demonstrate that iron chelation by deferoxamine (DFO) protects against the cytotoxic and reactivating effects of hydrogen peroxide (H2O2). These protective effects were observed both in lymphocytic (ACH-2) and promonocytic (U1) cells latently infected by HIV-1. Concomitantly, NF-kappa B activation by H2O2, when followed by gel retardation assay, was decreased in the DFO-treated U1 and ACH-2 cells. This latter DFO-mediated effect was specific, as DFO did not clearly affect AP-1 DNA-binding activity when studied after H2O2-induced stress. More importantly, DFO protected against the H2O2-induced activation of HIV-1 as evidenced by reverse transcriptase activity in the supernatant. DFO also protected against PMA-induced NF-kappa B activation as well as TNF-alpha-induced HIV-1 activation. Furthermore, DFO attenuated the p24 response in PBMC infected with HIV-1 and stimulated with IL-2. These different effects of DFO were obtained at DFO concentrations lower than 5 microM. Other chemically unrelated iron chelators also provided protection against cytotoxicity, NF-kappa B activation, and HIV-1 activation in U1 cells challenged with H2O2.

Base Sequence↗

PubMed1995

Varicella-zoster virus induces apoptosis in cell culture.

Apoptosis is an active mechanism of cell death which can be initiated in response to various stimuli including virus infections. In this work, we demonstrate that lytic infection by varicella-zoster virus (VZV), a human herpesvirus, is characterized by nuclear fragmentation of DNA into oligonucleosomal fragments and by chromatin condensation. In vitro, VZV-induced cell death is actually mediated by apoptosis. The mechanisms developed by cells to protect themselves against apoptosis could be one of the parameters allowing the establishment of virus latency. In the case of VZV, which can remain latent in sensory ganglia, we have not yet identified a cellular or viral protein which could play this protective role, since the observed apoptosis mechanism seems to be independent from Bcl-2, the most frequently described inhibitor of apoptosis.

Animals↗

PubMed1995

Varicella-zoster virus gene 63 encodes an immediate-early protein that is abundantly expressed during latency.

Varicella-zoster virus (VZV) gene 63 encodes a protein with a predicted molecular mass of 30.5 kDa which has amino acid similarities with the immediate-early (IE) protein 22 (ICP-22) of herpes simplex virus type 1. In order to study the expression of this protein during lytic and latent infection, gene 63 was cloned in frame and downstream from the glutathione-S-transferase gene, expressed as a fusion protein, and purified. In VZV-infected Vero cells, antibodies directed against this protein detect two polypeptides of 45 and 38 kDa which are localized both in the cytoplasm and in the nucleus. Using a sequential combination of transcription and protein synthesis inhibitors (actinomycin D and cycloheximide, respectively), we demonstrated the immediate-early nature of this protein, which can thus be named IE63. Using a rat model of VZV latency, we showed that IE63 is heavily expressed, essentially in neurons, during latency. IE63 can also be detected in the skin of patients showing early herpes zoster symptoms.

Animals↗

PubMed1995

The impact of age on the quality of life in persons with HIV infection.

The authors administered the Medical Outcomes Study (MOS 20) Short Form Health Survey to 369 persons with HIV disease. The MOS survey measures six domains of health: physical function, role function, social function, mental health, health perception, and pain. Additional data included sociodemographics, HIV risk group, time since HIV diagnosis, symptoms (dyspnea, diarrhea, fever, chills, sweats, weight loss, weakness, numbness, memory trouble, seizures), and CD4 lymphocyte count within 3 months of the MOS survey. Bivariate analyses revealed worse MOS scores associated with older age in five health domains: physical function (p less than .01), health perception (p <.10), role function (n.s.), social function (n.s.), and mental health (n.s.). Older subjects reported less pain. When controlling for CD4 count and for sociodemographic and clinical variables, older age was significantly (p less than .05) associated with worse MOS scores in physical function, social function, and health perception, nonsignificantly associated with worse MOS scores in role function and mental health, and nonsignificantly associated with less reporting of pain.

Adolescent↗

PubMed1995

Varicella-zoster virus latency in the adult rat is a useful model for human latent infection.

A model of latent infection by varicella-zoster virus (VZV) was obtained in the adult rat. Inoculation of VZV-infected cells in the skin led to infection of the peripheral nervous system. Latency was characterized by a long-lasting presence of the viral genome, of selected viral gene transcripts, and of at least one viral protein in the dorsal root ganglia. Reactivation has not been obtained in vivo, but has occurred ex vivo after repeated stresses. Many similarities with VZV latency in humans were found, making this model useful for vaccine and antiviral studies.

Animals↗

PubMed1995

Varicella-zoster virus gene regulation.

The varicella-zoster virus genome contains 71 open reading frames (ORFs), five of which (ORF62, ORF4, ORF63, ORF61, and ORF10) encode regulatory proteins. ORF62 codes for the major immediate early protein of the virus exhibiting DNA-binding and regulatory functions. This protein, localized in the cell nucleus, is a functional homologue to ICP4 of herpes simplex virus type 1 (HSV-1). It trans-activates several varicella-zoster virus promoters of the various gene classes and autoregulates its own expression. ORF4 protein activates gene promoters provided they have basal activities, but it is not a functional homologue of HSV-1 ICP27. Gene regulation activity appears to be linked to its cysteine-rich C-terminal region. ORF63 codes for an immediate early protein mainly located in the cell nucleus. The regulatory functions it performs are still unclear. ORF61 protein is the functional homologue of HSV-1 ICP0. Its N-terminal region exhibits a RING domain responsible for trans-activating and trans-repressing activities. ORF10 protein exhibits similarities with HSV-1 VP16 and activates the ORF62 promoter.

Chromosome Mapping↗

PubMed1995

Localization of varicella-zoster virus nucleic acids and proteins in human skin.

The pathogenic mechanisms involved in varicella-zoster virus (VZV) infections remain elusive. The pattern of cutaneous distribution of the IE63 protein and of the gpI (gE) and gpII glycoproteins with their corresponding genome sequences during VZV infections was studied by immunohistochemistry and in situ hybridization. Skin biopsy specimens were obtained from immunocompetent and immunocompromised patients with varicella, herpes zoster, or atypical VZV lesions. The first evidence for VZV infection consisted of the presence of IE63 in keratinocytes. In the vesicles and pustules, the viral transcripts gpI, gpII, and IE63 and the corresponding nucleic acids for gpI and gpII were identified in keratinocytes, sebocytes, Langerhans cells, dermal dendrocytes, monocytes/macrophages, and endothelial cells. The gpI and gpII glycorpoteins were essentially located on the cellular membranes while IE63 expression was generally restricted to the nuclei. In three biopsies of early herpes zoster, viral proteins were disclosed in dermal nerves and in perineurial type I dendrocytes. This was never encountered in varicella. Vasculitic changes and endothelial cell involvement were more prominent in varicella than in herpes zoster. It is concluded that the secondary viremia in varicella that affects the dermal endothelial cells is followed by a cell-to-cell spread to keratinocytes. In herpes zoster, the viral progression through cutaneous nerves primarily extends to the pilosebaceous units with a secondary involvement of epidermal keratinocytes, followed by a further spread to dermal cells.

DNA, Viral↗

PubMed1994

Epithelial-to-mesenchymal transition in HPV-33-transfected cervical keratinocytes is associated with increased invasiveness and expression of gelatinase A.

The invasive potential of a set of HPV-33- and HPV-33 + ras-transfected cervical keratinocytes was investigated. These cell lines were previously separated into 2 groups according to their behavior on collagen rafts. Cell lines from the first group reconstituted CINIII-like lesions, whereas cell lines from the second group reconstituted epithelia comparable to micro-invasive carcinomas. They were thus postulated to represent distinct stages of cervical carcinogenesis. The present results have shown that lines from group I, which have conserved an epithelial morphology in monolayer, (i) could not invade matrigel when tested in a modified Boyden chamber assay, (ii) produced solely gelatinase B and (iii) were unable to activate exogenous gelatinase A. On the other hand, lines from group II associated epithelial-to-mesenchymal transition (acquisition of elongated morphology, vimentin positivity) with high in vitro invasive potential and with the ability both to produce and to activate gelatinase A. These results strongly support the hypothesis that the epithelial-to-mesenchymal transition and the associated events might be implicated in the progression to the metastatic phenotype.

Blotting, Northern↗

PubMed1994

Differentiation ability and oncogenic potential of HPV-33- and HPV-33 + ras-transfected keratinocytes.

Five HPV-33-immortalized and 5 HPV-33 + ras-transfected cell lines were characterized in terms of growth in soft agar, tumorigenic potential in nude mice, p21 expression, morphology and expression of differentiation markers in organotypic cultures. No striking differences were observed between the HPV-33-immortalized cell lines and their corresponding ras-transfected counterparts as regards their tumorigenicity in nude mice (only one cell line was able to develop tumors in nude mice) or their behavior on lifted collagen gels. However, all the ras-transfected cell lines gave rise to colonies in soft agar while only 2 HPV-33-transfected lines (CK1 and CK4) displayed this property. The 10 cell lines could be divided into 2 groups with respect to their phenotype in monolayer and in organotypic cultures. Lines from group I (CK2, 3, 5 and their ras-transfected homologous lines) shared a typical epithelial phenotype in monolayer and the ability (a) to form an epithelium similar to a CIN-III lesion and (b) to strongly express keratins K1-K10 and involucrin in organotypic cultures. On the other hand, for the lines from group II (CK1, CK4, CK1EJ7 and CK4EJ5), there was a correlation between an elongated phenotype in monolayer and the property (a) to form a structure similar to a microinvasive carcinoma and (b) to express vimentin and keratins K8-K18. These cell lines, exhibiting various transformation-associated alterations, can be considered as an in vitro model representing various stages of HPV-33-associated cervical carcinogenesis.

Animals↗

PubMed1994

In vivo and in vitro analysis of electrical activity-dependent expression of muscle acetylcholine receptor genes using adenovirus.

Acetylcholine receptor (AChR) genes are repressed in extrajunctional domains of adult muscle fiber by neurally evoked electrical activity. Denervation elicits upregulation of AChR gene transcription in extrasynaptic areas. We have used an adenovirus (Ad)-based strategy to analyze in vitro and in vivo the electrical activity-dependent transcription of the chicken AChR alpha 1 subunit gene. The luciferase gene placed under the control of wild-type and mutated fragments of the alpha 1 subunit promoter was inserted in a defective Ad vector designed for the study of transcriptional regulation. Animals were infected by intramuscular injection and in vivo luciferase levels were normalized by coinfection with an Ad vector containing the chloramphenicol acetyltransferase gene driven by an electrical activity-insensitive promoter. Our results demonstrate that although both proximal MyoD binding sites of the alpha 1 promoter are required for muscle-specific expression of the alpha 1 gene, only one is necessary, albeit insufficient, to enhance alpha 1 promoter activity after denervation. Parallel results were obtained with cultured muscle cells in vitro following tetrodotoxin blocking of spontaneous electrical activity. These results substantiate a direct contribution of MyoD factors in electrical activity-dependent regulation of AChR expression and further indicate that Ad-based vectors constitute a powerful tool in the field of transcriptional regulation.

Adenoviruses, Human↗

PubMed1994

Meningoradiculoneuritis due to acyclovir-resistant varicella zoster virus in an acquired immune deficiency syndrome patient.

Varicella zoster virus (VZV) is recognized as one of the major viral pathogens reactivated in patients with the acquired immune deficiency syndrome (AIDS). We report the case of meningoradiculoneuritis in an AIDS patient,associated with the isolation in the cerebrospinal fluid (CSF) of a thymidine kinase (TK)-deficient, acyclovir (ACV)-resistant strain of VZV. Although the virus was sensitive in vitro to phosphonoformate (PFA), the patient did not improve during PFA therapy and finally died. Several VZV strains isolated from this patient (including two isolates from the patient's CSF) were analyzed for their TK activity and subsequently the viral TK gene was sequenced showing a major deletion leading to a truncated protein. Their susceptibility to several antiviral agents including ACV, PFA, (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), 9-beta-D-arabinofuranosyladenine (vidarabine), (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine (HPMPC), and (S)-9-(3-hydroxy-2-phosphonyl-methoxypropyl)adenine (HPMPA) was evaluated. All the virus strains isolated from this patient remained sensitive to HPMPA and HPMPC, pointing to the potential usefulness of these acyclic nucleoside phosphonates for the treatment of ACV-resistant VZV infections in immunocompromised patients.

Acquired Immunodeficiency Syndrome↗

PubMed1994

HIV-1 reactivation after an oxidative stress mediated by different reactive oxygen species.

An important aspect of infection by the human immunodeficiency virus (HIV-1) type 1 is its long clinical latency period, suggesting that the provirus may remain latent for extended periods of time after primary infection. Numerous factors such as cytokines, tumor promoters, co-infection by several viruses and physical agents are able to reactivate latent virus. Since a common denominator, shared by several of these agents, is their ability to cause stress conditions, we have examined the effects of an oxidative stress mediated by reactive oxygen species on HIV-1 latently infected monocytes (U1) or lymphocytes (ACH-2). Exposure of these two cell lines to hydrogen peroxide causes a decrease of cell viability but among the cells surviving the treatment, a HIV-1 reactivation can be observed as measured by increased RT activities depicted in cell supernatants or by the appearance of HIV-1 antigens inside cells. Singlet oxygen (1O2) when generated either in the cytoplasm or in the cell nucleus can also promote an important HIV-1 reactivation from treated cells. However, extracellular generation of 1O2 cannot trigger the HIV-1 reactivation although this kind of treatment is highly cytotoxic. These experiments demonstrate that different reactive oxygen species are able to lead to an intracellular pro-oxidant state initiating one or several signalling pathways which lead in fine to the HIV-1 LTR transactivation by regulatory proteins.

Cell Line↗

PubMed1994

Photobiological activity of sulphur and selenium analogues of psoralen.

Eight psoralen analogues, in which sulphur or selenium replaces one or both intracyclic oxygen atoms, were synthesized. Photoreaction with M13mp19 RF DNA in the presence and absence of oxygen (wavelength, greater than 320 nm) was studied. The damaged viral DNA was transfected into Escherichia coli and scored for infectivity towards Ca-treated wild-type E. coli. This allowed a comparative evaluation to be made of the heteropsoralens in terms of the photoreaction with DNA and the photodynamic effect. Most of the seleno- and thio-psoralens show very high photoactivity towards DNA compared with psoralen and 8-methoxypsoralen (8-MOP). Their photoreactivity is due mainly to a [2 + 2] photoreaction, since only a minor influence of molecular oxygen could be detected. Some of the studied seleno- and thio-psoralens are very efficient DNA photoinactivating agents and show great promise in photochemotherapy.

Bacteriophage M13↗