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Biomedical subjects

A Jestin

Publications and source records attributed to A Jestin.

At least 37 records · Page 2Linked to original sources
PubMed1999

Flow cytometric and optical microscopic evaluation of poly(D, L-lactide-co-glycolide) microspheres phagocytosis by pig alveolar macrophages.

The phagocytosis of fluorescent poly(D,L-lactide-co-glycolide) microspheres by fresh and frozen pig alveolar macrophages was investigated by optical microscopy on adherent cell culture and by flow cytometry with cell suspension. The kinetic of phagocytosis was studied on a 360 min period as a function of the ratio between microspheres and macrophages (MS:AM ratio from 1:1 to 10:1). No difference of phagocytosis between fresh and frozen macrophages was observed whatever the MS:AM ratio following flow cytometric evaluation while a significant phagocytosis pattern was noticed following optical microscopic evaluation for the highest ratio. The intensity of phagocytosis was dependent on the duration of incubation and dependent, but not proportionally, to the MS:AM ratio showing that the highest efficiency was obtained with the MS:AM ratio of 1:1. Flow cytometry analysis has shown a correlation between cell population and fluorescent events suggesting that phagocytosis of nonfluorescent antigen-loaded particles with different characteristics could be investigated.

Animals↗
PubMed1999

Molecular characterization and expression of the S3 gene of muscovy duck reovirus strain 89026.

Although reovirus infection is one of the major virus diseases of muscovy ducks in France, no vaccine is available and nothing is known about the structure and function of the genes and proteins of the reovirus involved. The complete S3 genome segment of the muscovy duck reovirus strain 89026 has been cloned and the nucleotide and deduced amino acid sequences are reported here. The S3 genome segment is 1201 bp long and possesses the same terminal motifs (5' GCTTTTT and TATTCATC 3') as the S3 genome segment of known chicken reovirus strains. It contains one open reading frame that encodes a protein of 367 amino acids with a molecular mass of 40.8 kDa. The gene, encoding the sigmaB major outer-capsid protein, was cloned into two different baculovirus transfer vectors and expressed in insect cells as a glutathione S-transferase fusion protein or a non-fused protein. The antigenicity of the two recombinant proteins was demonstrated by immunoblot assay. The potential immunogenic role of sigmaB protein was studied in a protection assay against reovirus infection of specific-pathogen-free muscovy ducks. No antibodies could be detected by ELISA or immunoblot in ducks immunized with the recombinant proteins and no significant protection was noted after the challenge. However, whereas the weights of wild-type baculovirus-infected and challenge-control ducks were significantly lower than those of unchallenged ducks, the weights of male ducks previously immunized with the sigmaB recombinant proteins did not differ significantly from males of either group. This work is the first to provide molecular data for a duck reovirus.

Amino Acid Sequence↗
PubMed1998

Measurement of digestive disorders in the piglet at weaning and related risk factors.

A cohort study was carried out in France about postweaning digestive disorders in the piglet. One hundred and six farrow-to-finish farms were involved. In each of them, a batch of contemporary piglets was considered. A total of 12,034 piglets were ear-notched, evaluated during the suckling phase and weighed at weaning, at 14 and 28 days postweaning. Postweaning diarrhoea and mortality were recorded daily. Data were collected about diet composition and feed intake, housing and husbandry throughout the period. Weaning weight was 8.1 kg and weaning age was 27.2 days on average. Diarrhoea occurred in the pens after a 3-4-day latency period. Prevalence was maximum around 7 to 9 days after weaning and remained high until 21 days after weaning. Mortality was moderate (1.9%). Average daily gains were 283 and 489 g for the two subsequent 14-day periods postweaning. Descriptive multivariate methods indicated a strong pattern between diarrhoea, mortality and growth. The main risk factors associated with the digestive disorders were determined. The hygiene level at the reception of the piglets (cleanliness, level of temperature), management and husbandry level (air quality, group size and stocking procedure) were found to be important factors leading to risky or secure profiles. In addition, the feed intake of the piglet during the first week postweaning was strongly associated with the severity of the digestive disorders over the whole 28-day postweaning period of observation. It is concluded that prevention of postweaning digestive disorders could be based on the control of zootechnical conditions.

Animal Husbandry↗
PubMed1998

Development of a PCR-based method coupled with a microplate colorimetric assay for the detection of Porcine Parvovirus and application to diagnosis in piglet tissues and human plasma.

A new method for Porcine Parvovirus (PPV) diagnosis was developed. The method is based on polymerase chain reaction (PCR) amplification followed by hybridization and colorimetric detection of PCR products in microwell plates. A highly specific and sensitive amplification step was ensured by primers carefully selected in the VP2 structural gene and optimized PCR conditions. Uracyl-DNA-Glycosylase (UDG) in combination with dUTP was used to avoid false-positive results, and 100 copies of internal control (IC) were added to each PCR reaction to reveal any false-negative samples. Biotinylated amplified fragments were hybridized on specific capture probes covalently linked to microwell plates. Finally, the detection of hybridized PCR products was performed by means of a colorimetric reaction, which was automated. The method permitted the detection of 10(3) copies (6 fg) of replicative form DNA (RF-DNA) in 20 mg of lung sample, and 500 copies (3 fg) in 100 microl of plasma. It was used to analyse 24 field piglet tissue samples, and 35 human plasma or serum specimens collected from patients treated with porcine Factor VIII concentrates.

Animals↗
PubMed1998

Molecular variation in the nucleoprotein gene (ORF7) of the porcine reproductive and respiratory syndrome virus (PRRSV).

The nucleoprotein gene (ORF7) of 15 European isolates of porcine reproductive and respiratory syndrome virus (PRRSV) was sequenced and compared with corresponding sequences of other PRRSV isolates (2 European and 13 American) and one isolate each of other arteriviruses (the lactate dehydrogenase elevating virus (LDV), the simian haemorrhagic fever virus (SHFV) and the equine arteritis virus (EAV)). Their phylogenetic relationships were established using neighbour-joining and parsimony methods. Four lineages (PRRSV, LDV, SHFV and EAV) were discriminated. Two genotypes of PRRSV, European and American, could be further identified. The European genotype of PRRSV was highly conserved. Analysis of the nucleotide and amino acid substitutions in PRRSV ORF7 revealed four stable regions, probably conserved because of their requirement for nucleocapsid function and/or structure. No constant mutations accumulation in the ORF7 could be determined precisely when either synonymous or non-synonymous mutations were studied. Passage of the European PRRSV in vivo had little influence on the ORF7 sequence: only a small number of synonymous substitutions in ORF7 was detectable, confirming its low variability.

Amino Acid Sequence↗
PubMed1998

Transcriptional organization of the avian adenovirus CELO.

A detailed map of the transcriptional organization of the CELO virus genome was produced. Recent computer analysis of CELO virus has indicated the presence of 38 putative open reading frames (ORFs). This study, based on analysis of the transcriptional products of CELO in vitro, confirmed the presence of RNAs for 26 of these 38 ORFs. All of the results were obtained by cDNA isolation or specific reverse transcriptase PCR. Observation of ORF transcription kinetics postinfection revealed the existence of early and late expression, with the earliest starting at 2 h postinfection. The 5' untranslated regions of some RNAs were also studied, and this revealed the existence of a bipartite leader sequence, comparable in structure to the tripartite leader of mastadenovirus. The leader most probably involved in transcriptional activity was observed in most of the structural protein genes of the CELO virus genome. This suggests some homology in transcriptional organization between the avian adenovirus CELO and known mastadenoviruses such as human adenovirus.

Adenoviridae Infections↗
PubMed1998

Perifusion analysis of insulin secretion from specific pathogen-free large-white pig islets shows satisfactory functional characteristics for xenografts in humans.

Though the pig appears to be the islet donor of choice for grafts in diabetic patients, there may be a risk of transmission of infectious agents. In this context, we adopted a strategy of islet isolation from pigs raised and killed in specific pathogen-free (SPF) conditions as a minimum with regard to the concept of quality assurance. Accordingly, the present study investigated the function of SPF pig islets to determine whether they react qualitatively and quantitatively to nutriments, hormones and neuromediators with which they would be confronted in man and could therefore provide effective regulation during physiologic or physiopathologic situations. beta cells from 18 Large-White SPF pigs were functionally intact after 7 days in culture. Insulin stimulation indexes (SI) of 3.1 +/- 0.2, 2.2 +/- 0.1, and 4.4 +/- 0.3 were found respectively for 30 mmol/l K+, 100 mumol/l tolbutamide and 10 mmol/l theophylline. Basal insulin secretion (72.2 +/- 7.6 muU/min) had already increased significantly (p < 0.001) with 5.5 mmol/l glucose (184.2 +/- 25.5 muU/min, SI: 2.5 +/- 0.6), indicating that the threshold stimulatory concentration was comparable to that of human islets. Insulin secretion increased in a glucose dose-dependent manner (p < 0.001): SI: 3.1 +/- 0.3 and 3.6 +/- 0.2 with 11.0 mmol/l and 22.0 mmol/l glucose, which showed a satisfactory magnitude with reference to human islets. Even the subtle phenomenon of "glucose memory" was apparent in these pig islets. Arginine stimulated (p < 0.001) insulin secretion dose-dependently (SI: 2.2 +/- 0.3 with 5 mmol/l and 2.9 +/- 0.2 with 10 mmol/l). The ketone body beta-hydroxybutyrate (10 mmol/l) also induced insulin secretion (SI: 4.3 +/- 0.3). Insulin release was stimulated by 4 mumol/l gastric inhibitory peptide, revealing sensitivity to the hormonal enteroinsular axis, and by 2 mumol/l glucagon. Parasympathetic cholinergic influence was studied using 500 mumol/l carbamylcholine, which increased insulin secretion. The influence of orthosympathetic control and of stress situations was also studied. As in human islet response, epinephrine and the alpha 2-agonist clonidine (50 mumol/l) inhibited insulin secretion. Finally pre-culture of islets may be beneficial for graft outcome, provided that no deterioration in islet function occurs. A prolonged 21-day culture of SPF pig islets showed no decrease in insulin response to glucose, arginine and potassium, even with an unaltered threshold stimulatory glucose concentration. Thus, Large-White SPF pigs and the application of our isolation procedure provided islets with functional characteristics reproducibly compatible with potential utilisation for effective regulation of glycaemia under physiologic and physiopathologic situations in humans.

Animals↗
PubMed1998

Detection of porcine endogenous retrovirus: possible involvement in pig islet xenotransplantation.

One requirement prior to xenotransplantation of porcine islets during Type 1 diabetes is to eliminate the risk of transmitting infectious agents, particularly retroviruses, even when specific pathogen-free (SPF) pigs are used. We developed two sensitive complementary PCR-derived detection tests to assess this risk. This first is intended to detect a novel endogenous retrovirus pol sequence related to a recently described human endogenous retrovirus (HERV-L) and to foamy retroviruses. Primers for species-specific detection of this porcine endogenous sequence were designed and tested. The second test is a product-enhanced reverse transcriptase (PERT) assay optimised for the detection of porcine reverse transcriptase activity. These tests, which were used to detect HERV-L-related sequences and reverse transcriptase activity in certain SPF pig cells and porcine cell lines, may be useful in studying the risk of transmitting retroviruses by pig islet xenotransplantation in the immunogenetic context of Type 1 diabetes.

Animals↗
PubMed1997

Development of a semiquantitative PCR assay using internal standard and colorimetric detection on microwell plate for pseudorabies virus.

We have developed a semiquantitative PCR assay on microtitre plates for quantitation of pseudorabies virus (PRV). The test is based on co-amplification with an internal control (IC) of the target viral DNA, followed by hybridization of the biotin-amplified products on a capture probe covalently immobilized to a Covalink-NH MicroWells plate and then visualization with colorimetric enzymatic reactions. PCR was performed in the presence of uracil-N-glycolsylase (UNG) with dUTP instead of dTTP to prevent false positive results due to carry-over contamination. Our colorimetric test had a 3.5 log dynamic range with a detection level of 30 DNA copies per PCR reaction. A standard curve for quantitation of pseudorabies virus was established from co-amplification of 10 to 10(5) PRV molecules with 1000 IC molecules. Ratios of viral optical density/IC optical density were plotted against the number of PRV DNA target molecules in the PCR amplification. Integration of 96-well formats and automation using robots at different steps of the test ensured a good repeatability. Calibration of the quantitative test using samples from experimentally-infected pigs is in progress.

Automation↗
PubMed1997

Development of a RT-PCR test coupled with a microplate colorimetric assay for the detection of a swine Arterivirus (PRRSV) in boar semen.

Transmission of porcine reproductive and respiratory syndrome virus (PRRSV) through boar semen has been demonstrated, stressing the need for a reliable semen PRRSV detection test. A diagnostic assay was developed based on amplification of the PRRSV RNA by reverse transcription and polymerase chain reaction (RT-PCR) followed by detection of the amplification products by hybridization and colorimetric assay in microwell plates. A highly reproducible and efficient method of viral RNA isolation from semen samples was set up. A combined RT-PCR procedure was performed, incorporating the use of uracil-N-glycosylase (UNG) in combination with dUTP instead of dTTP to prevent false positive results due to carry-over contamination. An RNA internal control was added during the RNA isolation procedure to detect false negative results. The colorimetric detection in microwell plates of amplification products from either PRRSV or IC RNA gave specific and objective results and was automated. A cut-off value of 1000 RNA copies or 10 TCID50 of PRRSV per ml of semen samples could be detected with this assay. Semen samples collected from experimentally-infected boars were tested with this assay and showed PRRSV excretion early after infection and for an extended period.

Animals↗
PubMed1997

Minimisation of microbial contamination for potential islet xenografts using specific pathogen-free pigs and a protected environment during tissue preparation.

One major risk of islet xenotransplantation is transmission of infections. We thus compared microbial contamination during preparation of islets from 4 pigs conventionally breeded and slaughtered or 8 specific pathogen free (SPF) pigs, and different environmental conditions during pancreas excision. Pancreas harvested in a slaughterhouse (for conventional pigs) or in a protected autopsy room (for SPF pigs) were soaked in betadine solution and submitted to enzymatic digestion with collagenase. Islets were purified on histopaque gradient with a COBE 2991 processor. For each step of the process, a 10 ml aliquot was harvested and microbial contamination was analysed. For all animals, contamination of livers, which were not soaked in betadine solution, was also examined. Analysis of livers from the 4 conventional pigs showed polymicrobial contaminations (1,122 +/- 841 CFU/mg) with several species of Staphylococcus, Streptococcus, Bacillus and Enterobacteriaceae. For these conventional pigs, soaking of pancreas in betadine solution and presence of antibiotics in all media decreased the pancreatic contamination compared to hepatic contamination, but were unable to suppress it, as transport solution and crude suspension obtained after the digestion step with collagenase showed persistent contamination (9.7 +/- 2.4 and 10.5 +/- 4 CFU/ml, respectively). After islet purification by histopaque gradient, no medium remained contaminated. During analysis of the 8 SPF pigs, no liver exhibited contamination. Analysis of medium from each preparation step showed complete absence of contamination for 7 pancreases. Only one contamination with Staphylococcus simulans was observed for one pancreas in transport solution (6 CFU/ml), and persisted in digestion medium (16 CFU/ml). Finally, all purified suspensions were completely sterile. In conclusion, breeding conditions of pig islet donors, and controlled environment for pancreas excision, considerably influence the risk of microbial contamination. In order to limit the risk, SPF pigs are a suitable and compulsory source of islets.

Animals↗
PubMed1996

A fluorescence-based quantitative PCR method for investigation of pseudorabies virus latency.

A quantitative PCR method was developed in order to quantitate the number of copies of Pseudorabies virus (PRV) genome present in tissues from infected pigs. The method is based on the use of an internal standard that differs from the target DNA by a deletion of ten base pairs, and that is co-amplified with the target DNA. The resulting PCR products are labelled with a fluorescent primer and are then separated and detected by means of an automated sequencer. The assay was found to be specific and sensitive, allowing the detection of five copies of viral DNA among 10(6) host cells. The method was used successfully to quantitate the number of PRV DNA copies in trigeminal ganglia samples from infected pigs during the acute and the latent stages of the infection. Between 12 and 3.10(5) copies of viral genome per 10(6) neuronal cells were detected in these tissues which is consistent with data published previously.

Animals↗
PubMed1996

Expression of muscovy duck parvovirus capsid proteins (VP2 and VP3) in a baculovirus expression system and demonstration of immunity induced by the recombinant proteins.

The gene encoding the muscovy duck parvovirus (DPV)-strain 89384 capsid proteins VP2 and VP3 was cloned in a baculovirus expression system and expressed in insect cells. The recombinant proteins were found to react with specific anti-DPV serum by Western blotting and to be located in the nucleus of insect cells (Sf9) as shown by immunofluorescence. Empty virus-like particles (VLPs) identical in size and appearance to DPV virions were observed by electron microscopy. The antigenicity and immunogenicity of the recombinant proteins were evaluated by ELISA and seroneutralization. Immunization of 3-week-old muscovy ducklings induced anti-DPV antibodies; neutralizing antibody titres were consistent with those observed in ducklings inoculated with a commercial inactivated vaccine. The way to develop these promising results is discussed.

Animals↗
PubMed1995

Incorporation of pseudorabies virus gD into human immunodeficiency virus type 1 Gag particles produced in baculovirus-infected cells.

The human immunodeficiency virus type 1 (HIV-1) Pr55gag precursors were previously shown to assemble and bud efficiently as noninfectious virus-like particles (VLPs) when expressed in baculovirus-infected insect cells. In this study, we examined the abilities of foreign antigens to be incorporated on the outer surface of HIV-1 Gag particles. We have used a dual recombinant baculovirus, expressing the HIV-1 Gag gene and gD gene under the control of the P10 and polyhedrin promoters, respectively, to obtain hybrid VLPs. Transmission electron microscopy of insect cells infected with the dual recombinant revealed very large aggregates of particles budding from the cell membrane. The release of VLPs into the culture medium was clearly different for a recombinant baculovirus producing solely HIV-1 Gag, for which particles were uniformly distributed all around the cell surface. Biochemical analysis of hybrid particles indicated that glycoprotein gD was packaged into HIV-1 Gag VLPs. Moreover, the carboxy-terminal p6 region of Gag polyprotein and the glycoprotein gD intracytoplasmic domain were not required for gD incorporation. The experiments described here clearly demonstrate that glycoprotein gD can be packaged with HIV-1 Gag particles and released from insect cells.

Animals↗
PubMed1994

Pseudorabies virus latency: a quantitative approach by polymerase chain reaction.

Some data dealing with the establishment of a quantitative PCR assay are presented. The assay is based on the use of an internal standard (mimic) which differs from the target by a deletion of a few base pairs and which is co-amplified with the target DNA. The resulting PCR products are labelled with fluorescent primers and then separated and detected by an automated sequencer. A highly specific and sensitive PCR assay for the envelope glycoprotein gp50 gene has been developed. This assay is highly reproducible with a detection limit of one copy of PRV DNA. Several mimics were then constructed. As a result we can confirm that the strategy that we have chosen is adequate for the quantification of low amounts of virus DNA present in latently infected swine.

Animals↗
PubMed1994

Chemiluminescent detection of amplified pseudorabies virus gp50 DNA with immobilized probes on microtiter wells.

A highly specific and sensitive PCR assay for the envelope glycoprotein gp50 has been developed for the detection of PRV-DNA sequences. Primer pairs from PRV gp50 gene were used with the enzyme uracil N-glycosylase and dUTP instead dTTP to prevent contamination due to PCR product carry-over. Biotinylated PCR products were captured in microtiter wells by specific oligonucleotide covalently linked to the polystyrene wells. After recognition of the biotinylated PCR product with a streptavidin phosphatase conjugate, a chemiluminescent detection was realised. Different factors influencing the binding, the hybridization and the detection efficiency have been tested.

Base Sequence↗