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Biomedical subjects

E Eveno

Publications and source records attributed to E Eveno.

17 recordsLinked to original sources
PubMed2004

Risk factors for Salmonella enterica subsp. enterica shedding by market-age pigs in French farrow-to-finish herds.

Fattening-pigs carriers of Salmonella enterica are believed to be a main source of carcass and pork contamination at the later steps of the meat process. We did a prospective study in 2000-2001 in 105 French farrow-to-finish pig farms. In each farm, a batch of contemporary fattening pigs housed in the same room was followed throughout the fattening period. Salmonella shedding was assessed on environmental samples of faecal material (taken by means of pairs of gauze socks) analysed by classical bacteriological methods. 36.2% of the batches studied had at least one contaminated environmental sample and therefore were classified as Salmonella-shedding batches. Logistic regression was used to assess the association between managerial and hygiene practices and health status and the shedding risk at the end of the finishing period. Emptying the pit below the slatted floor after the previous batch of sows was removed and frequent removal of sow dung during the lactation period were protective. Presence of residual Salmonella contamination of the floor and pen partitions in the fattening rooms before loading the growing pigs also was a risk factor. The risk for Salmonella shedding at the end of the fattening period was increased when dry feed (versus wet feed) was provided during the fattening period. Lastly, Lawsonia intracellularis seroconversion and PRRSV seropositivity during the fattening period also was a risk factor for Salmonella shedding.

Animal Feed↗
PubMed2003

Risk factors for porcine post-weaning multisystemic wasting syndrome (PMWS) in 149 French farrow-to-finish herds.

A cross-sectional study involving 149 farms was carried out in France in 2000 and 2001 to assess the risk factors for post-weaning multisystemic wasting syndrome (PMWS). The farms were divided into three groups according to their current or past PMWS status: CASES (current and typical PMWS), CONTROLS#1 (PMWS-free farms), and CONTROLS#2 (farms which have recovered from PMWS). Two different comparisons were tested: CASES versus CONTROLS#1 and CASES versus CONTROLS#2. In the first comparison, the odds of PMWS were increased when fattening pigs tested positive for parvovirus (PPv) and porcine reproductive and respiratory syndrome (PRRS) virus (OR=4.4 and 6.5, respectively), when separate vaccines for parvovirus and Erysipela for the gilts versus associated vaccines were used (OR=2.5), and when on-farm semen collection was used versus all the semen purchased from an insemination centre (OR=4.6). Large pens in weaning facilities increased the odds of PMWS (OR=4.1); whereas long empty periods in weaning and farrowing facilities versus shorter (OR=0.2), regular treatment against external parasites (OR=0.1), and housing the sows in collective pens during pregnancy versus individual pens (OR=0.3) all decreased the odds of PMWS. The same kinds of risk factors were found with the second comparison with, in addition, a common pit for several adjacent fattening rooms versus separate pits (OR=6.7) and a high level of cross-fostering (OR=5.1). On the other hand, when farms had a self-replacement scheme for the gilts (OR=0.1), and when vaccination of the sows against E. coli was in place (OR=0.2), the odds of PMWS were decreased.

Animals↗
PubMed2003

Longitudinal serological responses to Salmonella enterica of growing pigs in a subclinically infected herd.

A longitudinal survey was conducted in France in a subclinically Salmonella-infected farrow-to-finish pig farm to describe the time-course of the serological response to Salmonella enterica in growing pigs. We used three batches of sows and their corresponding litters (n = 31 litters). Among these, 256 pigs randomly selected and individually identified were followed from the first week of age until slaughter. Serial individual blood samples were submitted to indirect Salmonella-ELISA testing. Salmonella shedding was investigated by bacteriological testing of faecal material regularly collected from the sows and pigs and by environment swabs taken from the pens. Caecal contamination was checked at the slaughterhouse. Information about litter composition (filiation), location of the pigs in post-weaning and fattening pens, sanitary events, sex and body weights was recorded. 11.6% of the pigs shed S. enterica; 52% of pigs seroconverted before slaughter. The age-related variation of the natural logarithm of calibrated optical densities (COD) of pigs was described with two linear mixed models. From 8 to 61 days of age, the decrease in COD with age was fitted with a model including random effects of the animal and the dam on the intercept and slope, a batch random effect on the intercept and an individual birth-weight fixed effect on the intercept. The dam random effect was explained by the parity of the sow, Salmonella shedding by the sow during the farrowing phase and the value of the optical density of colostrum collected at parturition. A second model fitting the increase in COD from 61 days of age until slaughter included the random effect on intercept of the batch and the random effects on slope and intercept of the animal, the dam and the pen in which the followed animals were located during the fattening phase and the environmental contamination as fixed effect. In this second model, no relation was found between individual slaughter-bacteriological results and increasing COD values. Considering seroconversion time between 61 days of age and slaughter, survival function were constructed using the Cox proportional-hazards model. Both methods suggested that seroconversions generally occurred during the last third of the fattening phase (from 140 days of age to slaughter), while shedding was observed during the first half of the fattening period. The fitted models suggest the existence of clusters (such as pen and litter of origin).

Animal Husbandry↗
PubMed2003

An ORF2 protein-based ELISA for porcine circovirus type 2 antibodies in post-weaning multisystemic wasting syndrome.

Porcine circovirus type 2 (PCV2) plays a crucial role in the pathogenesis of post-weaning multisystemic wasting syndrome (PMWS) in swine. As PCV2 displays significant homology with PCV1 (a non-pathogenic virus) at the nucleotide and amino-acid level, a discriminative antigen is needed for specific serological diagnosis. The ORF2-encoded capsid protein from PCV2 was used to develop an indirect enzyme-linked immunosorbent assay (ELISA). GST-fused capsid protein from PCV2 and GST alone (both expressed in recombinant baculovirus-infected cells) were used as antigens for serodiagnosis. The specificity of the ELISA for detection of PCV2 antibodies was demonstrated in sera from pigs experimentally infected with PCV1, PCV2 and other swine viruses. The semi-quantitative nature of the test was evaluated versus an immunoperoxidase monolayer assay (IPMA). The ELISA was performed on 322 sera from pigs in eight Brittany herds and compared with IPMA. The sensitivity (98.2%) and specificity (94.5%) of this test were considered suitable for individual serological detection. High PCV2 seroprevalence was found in sows and pigs at the end of the growth phase (18-19 weeks) in all eight herds. The seroprevalence in piglets (11-17 weeks) was statistically correlated with clinical symptoms of PMWS (93% in affected versus 54%, in non-affected farms). A cohort study performed in PMWS-free farms showed that 57% of piglets exhibited active seroconversion after 13 weeks, indicating that PCV2 infection occurred earlier in PMWS-affected piglets.

Animals↗
PubMed1999

The Genexpress IMAGE knowledge base of the human brain transcriptome: a prototype integrated resource for functional and computational genomics.

Expression profiles of 5058 human gene transcripts represented by an array of 7451 clones from the first IMAGE Consortium cDNA library from infant brain have been collected by semiquantitative hybridization of the array with complex probes derived by reverse transcription of mRNA from brain and five other human tissues. Twenty-one percent of the clones corresponded to transcripts that could be classified in general categories of low, moderate, or high abundance. These expression profiles were integrated with cDNA clone and sequence clustering and gene mapping information from an upgraded version of the Genexpress Index. For seven gene transcripts found to be transcribed preferentially or specifically in brain, the expression profiles were confirmed by Northern blot analyses of mRNA from eight adult and four fetal tissues, and 15 distinct regions of brain. In four instances, further documentation of the sites of expression was obtained by in situ hybridization of rat-brain tissue sections. A systematic effort was undertaken to further integrate available cytogenetic, genetic, physical, and genic map informations through radiation-hybrid mapping to provide a unique validated map location for each of these genes in relation to the disease map. The resulting Genexpress IMAGE Knowledge Base is illustrated by five examples presented in the printed article with additional data available on a dedicated Web site at the address http://idefix.upr420.vjf.cnrs.fr/EXPR++ +/ welcome.html.

Brain Chemistry↗
PubMed1999

The genexpress IMAGE knowledge base of the human muscle transcriptome: a resource of structural, functional, and positional candidate genes for muscle physiology and pathologies.

Sequence, gene mapping, and expression data corresponding to 910 genes transcribed in human skeletal muscle have been integrated to form the muscle module of the Genexpress IMAGE Knowledge Base. Based on cDNA array hybridization, a set of 14 transcripts preferentially or specifically expressed in muscle have been selected and characterized in more detail: Their pattern of expression was confirmed by Northern blot analysis; their structure was further characterized by full-insert cDNA sequencing and cDNA extension; the map location of the corresponding genes was refined by radiation hybrid mapping. Five of the 14 selected genes appear as interesting positional and functional candidate genes to study in relation with muscle physiology and/or specific orphan muscular pathologies. One example is discussed in more detail. The expression profiling data and the associated Genexpress Index2 entries for the 910 genes and the detailed characterization of the 14 selected transcripts are available from a dedicated Web server at. The database has been organized to provide the users with a working space where they can find curated, annotated, integrated data for their genes of interest. Different navigation routes to exploit the resource are discussed.

Base Sequence↗
PubMed1997

Retrovirus-mediated gene transfer corrects DNA repair defect of xeroderma pigmentosum cells of complementation groups A, B and C.

With the aim to devise a long-term gene therapy protocol for skin cancers in individuals affected by the inherited autosomal recessive xeroderma pigmentosum we transferred the human DNA repair XPA, XPB/ERCC3 and XPC cDNAs, by using the recombinant retroviral vector LXSN, into primary and immortalized fibroblasts obtained from two XP-A, one XP-B (associated with Cockayne's syndrome) and two XP-C patients. After transduction, the complete correction of DNA repair deficiency and functional expression of the transgenes were monitored by UV survival, unscheduled DNA synthesis and recovery of RNA synthesis, and Western blots. The results show that the recombinant retroviruses are highly efficient vectors to transfer and stably express the human DNA repair genes in XP cells and correct the defect of DNA repair of group A, B and C. With our previous results with XPD/ERCC2, the present work extends further promising issues for the gene therapy strategy for most patients suffering from this cancer-prone syndrome.

Blotting, Western↗
PubMed1997

A mutation in the XPB/ERCC3 DNA repair transcription gene, associated with trichothiodystrophy.

Trichothiodystrophy (TTD) is a rare, autosomal recessive disorder characterized by sulfur-deficient brittle hair and nails, mental retardation, impaired sexual development, and ichthyosis. Photosensitivity has been reported in approximately 50% of the cases, but no skin cancer is associated with TTD. Virtually all photosensitive TTD patients have a deficiency in the nucleotide excision repair (NER) of UV-induced DNA damage that is indistinguishable from that of xeroderma pigmentosum (XP) complementation group D (XP-D) patients. DNA repair defects in XP-D are associated with two additional, quite different diseases; XP, a sun-sensitive and cancer-prone repair disorder, and Cockayne syndrome (CS), a photosensitive condition characterized by physical and mental retardation and wizened facial appearance. One photosensitive TTD case constitutes a new repair-deficient complementation group, TTD-A. Remarkably, both TTD-A and XP-D defects are associated with subunits of TFIIH, a basal transcription factor with a second function in DNA repair. Thus, mutations in TFIIH components may, on top of a repair defect, also cause transcriptional insufficiency, which may explain part of the non-XP clinical features of TTD. Besides XPD and TTDA, the XPB gene product is also part of TFIIH. To date, three patients with the remarkable conjunction of XP and CS but not TTD have been assigned to XP complementation group B (XP-B). Here we present the characterization of the NER defect in two mild TTD patients (TTD6VI and TTD4VI) and confirm the assignment to X-PB. The causative mutation was found to be a single base substitution resulting in a missense mutation (T119P) in a region of the XPB protein completely conserved in yeast, Drosophila, mouse, and man. These findings define a third TTD complementation group, extend the clinical heterogeneity associated with XP-B, stress the exclusive relationship between TTD and mutations in subunits of repair/transcription factor TFIIH, and strongly support the concept of "transcription syndromes."

Adolescent↗
PubMed1996

Long-term complementation of DNA repair deficient human primary fibroblasts by retroviral transduction of the XPD gene.

Due to their limited life time in culture and their relative resistance to DNA transfection, primary fibroblasts derived from UV-hypersensitive patients could not be used for cloning DNA repair gene and studying stable complementation with wild-type DNA repair genes. Primary cells were only used for complementation analysis after transient expression through cell fusion. DNA microinjection and transfection. We report the retroviral-mediated highly efficient transfer and stable expression of XPD/ERCC2 gene in fibroblast strains from eight different patients using the LXPDSN retroviral vector. Cells derived from skin biopsies of xeroderma pigmentosum and trichothiodystrophy patients were incubated with vector-containing suspension and selected with the neomycin-analog G418. LXPDSN vector specifically complemented cells belonging to the XP-D group. Long-term reversion of repair-deficient phenotype, monitored by UV survival and UDS analysis, has been achieved in these diploid fibroblasts. We demonstrate this methodology is a powerful tool to study phenotypic reversion of nucleotide excision repair-deficient cells such as cellular DNA repair properties and we suggest that it may be used to study other cellular parameters (cell cycle regulation, p53 stability or immunosurveillance-controlling factors) involved in UV-induced skin cancers and which reliability requires the use of untransformed cells.

Cell Line↗
PubMed1995

Different removal of ultraviolet photoproducts in genetically related xeroderma pigmentosum and trichothiodystrophy diseases.

To understand the heterogeneity in genetic predisposition to skin cancer in different nucleotide excision repair-deficient human syndromes, we studied repair of cyclobutane pyrimidine dimers (CPDs) and of pyrimidine(6-4)pyrimidone (6-4PP) photoproducts in cells from trichothiodystrophy (TTD) patients. TTD is not associated with increased incidence of skin cancer, although 50% of the patients are photosensitive and carry a defect in the nucleotide excision repair pathway, similar to Xeroderma pigmentosum patients. However, in striking contrast to TTD, Xeroderma pigmentosum is highly prone to cancer. To address this apparent paradox, two types of studies were conducted: (a) reactivation of UV-irradiated plasmids harboring actively transcribed reporter genes, with or without photolyase treatment before transfection of SV40-transformed fibroblasts; and (b) the kinetics of removal of UV-induced CPDs and 6-4PPs in genomic DNA by immunoblot analysis using lesion-specific mAbs in SV40-transformed and untransformed fibroblasts representative of all genetic TTD complementation groups. Results showed that all cell lines from photosensitive TTD patients efficiently express Cat or luciferase genes in transfected plasmids carrying non-CPD lesions, including 6-4PP, and display wild-type or near-wild-type (50-70% in 3 cell lines) 6-4PP repair in the overall genome after immunoblot analysis. However, CPD lesions (the repair of which is defective in the overall genome) also block the expression of the reporter gene in transfected plasmids. Two cell lines from nonphotosensitive TTD patients showed wild-type levels of repair for both photoproducts in overall genome. A model on the lesion-specific repair in the context of the molecular defect in TTD is proposed. The implication of the defective CPD repair and efficient 6-4PP repair subpathways in cancer prevention in TTD patients is discussed.

Cell Line↗
PubMed1995

Stable SV40-transformation and characterisation of some DNA repair properties of fibroblasts from a trichothiodystrophy patient.

To characterize nucleotide excision repair properties of cells from trichothiodystrophy (TTD) patients genetically-related to the xeroderma pigmentosum (XP) group D, TTD skin fibroblasts from two unrelated patients (TTD1VI and TTD2VI) belonging to the TTD/XPD group were transformed with a plasmid containing SV40 large T antigen-coding sequences and some DNA repair properties, such as unscheduled DNA synthesis (UDS), UV-survival, in vitro repair synthesis of cell extracts and reactivation of UV-irradiated reporter plasmid were studied. Results showed that: a) both untransformed and transformed TTD cells present a reduced UV-survival, compared to wild-type cells, but at significantly less reduced levels than XP-D cells; b) reduced repair activities were detected in both TTD and XP-D transformed cells by using in vitro cell free extract repair and reactivation of UV-irradiated plasmid procedures, and these relative reduced extents correlated with respective UV-survival; c) surprisingly, near wild-type UDS levels were detected in TTD2VILas transformed cells at different passages after the crisis, suggesting a phenotypic reversion of this transformed cell line; d) fluoro-cytometric analysis of TTD2VILas cells revealed a strong increase of a cell population containing a DNA amount more than twice as high than that of untransformed cells; finally, e) when UDS data were normalized to the DNA content in TTD2VILas cells, it appeared that the repair efficiency was only slightly higher than in untransformed cells. This implies that in transformed cells DNA repair properties should be evaluated, taking into account additional parameters. We obtained an immortalized TTD cell line which maintains DNA repair properties similar to those of parental untransformed cells and may be used to characterize the TTD defect at genetic, molecular and biochemical levels.

Cell Extracts↗
PubMed1995

Functional retroviral vector for gene therapy of xeroderma pigmentosum group D patients.

Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder characterized by an increased frequency of skin cancer following minimal sunlight exposure. Cells isolated from XP patients are also hypersensitive to UV rays and UV-like chemicals. This sensitivity is directly related to a defect in the early steps of nucleotide excision repair (NER) of damaged DNA. No efficient treatment is available for this disease and skin cancer prevention can only be achieved by strict avoidance of sunlight exposure. Thus, we are developing a model for gene therapy in XP, particularly for patients belonging to group D. We report here the construction of a retroviral vector (LXPDSN) containing the XPD (ERCC2) cDNA, which fully complements the DNA repair deficiency of primary skin fibroblasts. Efficient integration, mRNA synthesis, and protein expression of the XPD gene were obtained in all LXPDSN-transduced XP-D fibroblasts tested. Full correction of the DNA repair defect was observed with all DNA repair assays used, such as an increased survival after UV-radiation of the transduced cells, a normal level of DNA repair synthesis (UDS), and the reactivation of a UV-irradiated reporter vector. This retroviral vector will be used to modify keratinocytes genetically to produce repair proficient reconstituted skin for engraftment to XP patients.

Cell Survival↗
PubMed1995

Development of a new easy complementation assay for DNA repair deficient human syndromes using cloned repair genes.

Nucleotide excision repair (NER)-deficient human cells have been assigned so far to a genetic complementation group by a somatic cell fusion assay and, more recently, by microinjection of cloned DNA repair genes. We describe a new technique, based on the host cell reactivation assay, for the rapid determination of the complementation group of NER-deficient xeroderma pigmentosum (XP), Cockayne's syndrome (CS) and photosensitive trichothiodystrophy (TTD) human cells by cotransfection of a UV-irradiated reporter plasmid with a second vector containing a cloned repair gene. Expression of the reporter gene, either chloramphenicol acetyltransferase (CAT) or luciferase, reflects the DNA repair ability restored by the introduction of the appropriate repair gene. All genetically characterized XP, CS and TTD/XP-D cells tested failed to express the UV-irradiated reporter gene, this reflecting their NER deficiency whereas cotransfection with the repair plasmid expressing a gene specific for the given complementation group increased the enzyme activity to the level reached by normal cells. Selective recovery of both reporter enzyme activities was observed after cotransfection with the XPC gene for the XP17VI cells and with the XPA gene for both XP18VI and XP19VI cells. Using this method, we assigned three new NER-deficient human cells obtained from patients presenting clinical symptoms described as classical XP to either XP group A (XP18VI and XP19VI) and XP group C (XP17VI). Therefore, this technique increases the range of methods now available to determine the complementation group of new NER deficient patients with the advantage, unlike the somatic cell fusion assay or the microinjection procedure, of being simple, rapid, and inexpensive.

Biopsy↗
PubMed1994

Correction by the ERCC2 gene of UV sensitivity and repair deficiency phenotype in a subset of trichothiodystrophy cells.

Trichothiodystrophy (TTD) is a rare genetic disease with heterogeneous clinical features associated with specific deficiencies in nucleotide excision repair. Patients have brittle hair due to a reduced content of cysteine-rich matrix proteins. About 50% of the cases reported in the literature are photosensitive. In these patients an altered cellular response to UV, due to a specific deficiency in nucleotide excision repair, has been observed. The majority of repair-defective TTD patients have been assigned by complementation analysis to group D of xeroderma pigmentosum (XP). Recently, the human excision repair gene ERCC2 has been shown to correct the UV sensitivity of XP-D fibroblasts. In this work we describe the effect of ERCC2 on the DNA repair deficient phenotype of XP-D and on two repair-defective TTD cell strains (TTD1VI and TTD2VI) assigned by complementation analysis to group D of XP. ERCC2 cDNA, cloned into a mammalian expression vector, was introduced into TTD and XP fibroblasts via DNA-mediated transfection or microneedle injection. UV sensitivity and cellular DNA repair properties, including unscheduled DNA synthesis and reactivation of a UV-irradiated plasmid containing the chloramphenicol acetyltransferase reporter gene (pRSVCat), were corrected to wild-type levels in both TTD and XP-D cells. These data show that a functional ERCC2 gene is sufficient to reestablish a wild-type DNA repair phenotype in TTD1VI and TTD2VI cells, confirming the genetic relationship between TTD and XP-D. Furthermore, our findings suggest that mutations at the ERCC2 locus are responsible for causing a similar phenotype in TTD and XP-D cells in response to UV irradiation, but produce quite different clinical symptoms.

Animals↗
PubMed1993

Expression of the dystrophin gene in cultured fibroblasts.

The dystrophin whose defect is responsible for Duchenne and Becker muscular dystrophies is present in muscle, brain and cerebellum. We describe here the detection of dystrophin in human cultured skin fibroblasts, L809 cells and murine 3T6 cell line. Dystrophin transcripts initiated at the muscle specific first exon can also be amplified by cDNA-PCR from various fibroblastic cells. The expression of the dystrophin gene in fibroblasts could account for some abnormalities observed in patient's fibroblast cultures.

Animals↗
PubMedDate not supplied

Development of a complete ELISA using Salmonella lipopolysaccharides of various serogroups allowing to detect all infected pigs.

Although poultry is recognized as the major source of food-poisoning caused by Salmonella, pork also contributes to human infections. This study was therefore undertaken in order to develop a reliable serological method for the evaluation of the Salmonella status of piglets. A complete ELISA was performed using lipopolysaccharides of Salmonella Typhimurium, Anatum, Hadar and Infantis because these serovars were representative of the serogroups isolated from 30 contaminated fattening farms. S. Enteritidis was also added because of its importance in human infection and to include the O:9 antigen. This method potentially detects 100% of infected pigs. A significant correlation was found between this serological method and the bacteriological data from mesenteric lymph nodes (p = 0.01). In addition, both sensitivity and specificity were high (97% and 94% respectively). The ELISA test was therefore used in a cross-sectional study on 4 farms to evaluate when pigs became contaminated: seropositive pigs were only found for the 20 week old finishing pigs. The antibody response to Salmonella in piglets was also investigated: maternal antibodies persisted until 7 weeks of age and post-Salmonella contamination seroconversion was detected from 8 weeks of age onwards.

Animals↗