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Biomedical subjects

G Peltre

Publications and source records attributed to G Peltre.

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

Protein and allergen analysis of latex mattresses.

BACKGROUND: Latex allergy is a well-recognized health problem. The wide use of latex in daily life raised the question whether latex articles could be a source of allergens. The present study was carried out to analyse various latex mattresses for protein and allergen characterization. METHODS: Five latex foam mattresses were reduced to powder and proteins were extracted using either water or urea. The protein content of the extracts was analyzed by SDS-PAGE, IEF, 2-D gel electrophoresis and amino acid sequence analysis. The presence of specific IgE, directed toward mattress proteins found in human sera of latex-allergic patients, was tested using ELISA and immunoprints. RESULTS: The results showed that no protein or allergen could be extracted from synthetic foam. However, depending on the type of mattress and extraction procedure, various quantities of proteins could be extracted from mattresses containing natural latex. The ELISA levels of various latex-allergic patients were always more significant against urea extracts than against water extracts, except for one mattress. The protein and allergen patterns were qualitatively similar for all mattresses containing natural latex. Hev b 1 was identified by N-terminal amino acid sequence analysis. CONCLUSION: Because the natural rubber of the mattresses contains latex allergens, these allergens are a potential source of sensitization and could constitute a risk, at least to allergic individuals.

Allergens↗
PubMed1998

Mapping of Dermatophagoides farinae mite allergens by two-dimensional immunoblotting.

BACKGROUND: Allergens from the house dust mite Dermatophagoides farinae are responsible for frequent respiratory allergic disorders, but only 3 groups of these allergens are well characterized. OBJECTIVE: This study was performed to complete the repertoire of D farinae allergens using two-dimensional (2-D) electrophoresis. METHODS: D farinae mite allergens, extracted from whole cultures in the presence of a mild detergent, were separated by 2-D electrophoresis with subsequent immunoblotting. IgE-binding proteins were detected with individual mite-sensitive patient sera and the anti-D pteronyssinus human serum pool. Allergens were identified by an inhibition immunoblot test, by means of specific mAbs, or by biochemical characterization. The internal peptides of 2 allergens were microsequenced. RESULTS: 2-D immunoblotting with individual patient sera showed a marked heterogeneity in the isoelectric point of the allergens, as well as differences in the individual IgE-binding patterns. In addition to identification of allergens Der f 1, Der f 2, and Der f 3, new allergens have been characterized as Der f 4, Der f 5, and 2 high molecular mass allergens. Microsequencing of peptides from the latter allergens revealed significant homologies with allergen Mag 3 from D farinae and with a chitinase from prawn Penaeus japonicus. CONCLUSION: 2-D electrophoresis with subsequent immunoblotting and protein microsequencing allowed characterization of a more complete repertoire of D farinae allergens and their multiple isoforms, and identification of six new allergens.

Allergens↗
PubMed1998

Allergy to bovine beta-lactoglobulin: specificity of human IgE using cyanogen bromide-derived peptides.

BACKGROUND: Bovine beta-Lactoglobulin (Blg) is a major allergen involved in allergy to cow's milk proteins. Hydrolyzing Blg did not totally suppress its allergenicity; moreover its immunoreactivity may be increased. The aim of this work was to evaluate the specificity of serum IgE to different fragments of Blg in a group of 19 individuals allergic to cow's milk. METHODS: This study was performed using both direct and competitive inhibition ELISA involving immobilized native protein or peptides derived from Blg cyanogen bromide cleavage. RESULTS: Analyses of responses to each peptide revealed a large number of epitopes recognized by specific IgE of human allergic sera. However, there were differences in the specific determinants recognized, depending on the serum. Generally, peptides (25-107) and (108-145) retained substantial proportions of the immunoreactivity of the whole protein. Two other peptides, i.e. (8-24) and (146-162), were less recognized but were not inert. CONCLUSION: The main conclusion is that many epitopes were identified all along the Blg sequence by specific anti-Blg IgE from allergic humans.

Adolescent↗
PubMed1998

[Inter-relationship between allergenic pollens and air pollution].

Morbidity to pollens is increasing in the French population. Pollen is the vector of the male genome of the plant. It is the wind-borne pollens that are the most allergenic by release of allergen molecules that make contact with the mucosae. So every individual who is genetically allergic (atopic) may develop rhinitis and/or conjunctivitis and/or asthma.... More than 200 allergens have been identified, of which the three-dimensional structure is well-known. The urban pollution that is specific to a quarter under consideration (residential zone, pedestrianised, mid-town roadway and industrial zone) plays an important role in morbidity to pollen allergy. A significant coating of pollens is to be found on urban roads. Pollen sensitisation is increased by exposure to pollutants from 24 to 48 hours. The pollutants seem to make the surface of the exine more fragile, so triggering a mucosal reaction, making them more exposed to pollen allergens. The pollutant also plays the role of an adjuvant to the pollen allergen, and so is the origin of a greater production of IgE.

Air Pollutants↗
PubMed1997

Expression, purification, and characterization of rat aromatic L-amino acid decarboxylase in Escherichia coli.

A cDNA encoding rat aromatic L-amino acid decarboxylase (AADC) was successfully expressed in Escherichia coli using a T7 RNA polymerase expression system. Two types of expression vectors were tested and revealed to be equivalent to produce AADC. The enzyme was purified in both cases. The ratio of recovery of the pure active recombinant protein was better when the purification of the protein was made easier by addition of a short His-Tag at the C-terminal moiety of AADC, as achieved in the case of pET-20b+ vector expression. Spectral characteristics of the bound pyridoxal-5'-phosphate were essentially identical to the spectral properties of rat AADC. Kinetic constants Km and Vmax of recombinant AADC for the natural substrates L-dihydroxyphenylalanine and 5-hydroxytryptamine were 0.14 mM and 8444 U/mg, and 0.066 mM and 1813 U/mg, respectively. These values were in good agreement with previously reported values for AADC of the rat and other mammalian species.

5-Hydroxytryptophan↗
PubMed1996

Quantitative analysis of multiple V-region interactions among normal human IgG.

There is to date no quantitative method for scoring putative V-region interactions among serum antibodies, and the available qualitative techniques are not amenable to routine utilization. This deficit may explain the paucity of observations on the characteristics of the immune network, in contrast with the multiplicity of phenomenological descriptions on idiotype regulation. We describe here a novel methodology that uses isoelectric focusing (IEF) to resolve human F(ab')2 preparations from large pools of normal serum IgG into multiple bands, and computer-aided data processing to analyze interactions between the resulting blotted proteins and normal serum IgG from individual donors. Our results show that in all normal human sera tested, there are IgG-mediated interactions with a large number of IEF fractions of human F(ab')2. These interactions are V region specific, as assessed by inhibition experiments and by lack of binding of IgG monoclonal antibodies, and are characterized by average affinities that are in the micromolar range, as measured by surface plasmon resonance.

Antibody Affinity↗
PubMed1996

Protein separation by electrophoresis in a nonsieving amphoteric medium.

A numerical model has been used to study the separation of protein mixtures by zone electrophoresis in a nonsieving amphoteric medium. An amphoteric buffer fixes the pH of the solution close to its isoelectric point, where the buffer molecules are unchanged: they thus contribute very little to the conductivity of the solution. This means that high field strengths can be used for rapid separation without sacrificing resolution. The numerical study shows that in this process the band spreading that can reduce resolution is essentially due to differences in migration rate between protein-rich and low-protein zones: both differences in solution conductivity and in protein mobility are involved. Rules for judging the buffer capacity of amphoteric molecules are presented and it is shown how, for a given protein, the effectiveness of this technique varies with the range of pH in which it is applied.

Animals↗
PubMed1996

Cloning, sequencing and immunological characterization of Dac g 3, a major allergen from Dactylis glomerata pollen.

Preliminary work showed that a 14-kDa allergen with a pI of 9 was recognized by more than 60% of sera from Dactylis glomerata (Dac g) pollen-allergic individuals. The N-terminal amino acid sequence of this Dac g allergen was determined by Edman degradation and compared with that of Lol p 3, a major allergen of Lolium perenne. A sequence identity of 65% was found, suggesting that the Dac g allergen could be the homologue of Lol p 3 and therefore named Dac g 3. We report the cloning and sequence analysis of a cDNA encoding the Dac g 3 pollen allergen. The recombinant allergen (rDac g 3) expressed in plasmid vector pGEX-2T contained IgE-reactive epitopes found in its natural counterpart, and induced histamine release from basophils of Dac g-allergic individuals, confirming that the recombinant protein has biological properties similar to the pollen extracted allergen. Computer analyses showed that, in spite of a high degree of sequence homology, even closely related allergens such as Dac g 3 and Lol p 3 have dissimilar predictive secondary structures and potential different antigenicity. Because it possesses the properties of the native counterpart, rDac g 3 could be a relevant tool for molecular studies in allergy.

Allergens↗
PubMed1996

Characterization of Dac g 4, a major basic allergen from Dactylis glomerata pollen.

Monoclonal antibodies were produced against Dac g 4, a purified major basic allergen from Dactylis glomerata pollen. Their ability to be used for immunopurification of Dac g 4 was studied on a BIAcore apparatus (Pharmacia). The allergen was purified by affinity chromatography with one monoclonal antibody. Its precise molecular mass, 59,185 +/- 30 d, was determined by mass spectrometry. Its isoelectric point is 10.4. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting showed that Dac g 4-related proteins of similar molecular mass were detected in the majority of allergenic grass pollen species. By double-site ELISAs, we have estimated that Dac g 4 represents about 6% of the total proteins from a water-soluble extract. One monoclonal antibody (mAb H) recognized a 60 kd cross-reactive protein in other grass pollens, though none in any of the tree or weed pollens tested. Inhibition studies of IgE antibody binding to Dac g 4 with pollen extracts confirmed the presence of cross-reactive allergens in Secale cereale, Lolium perenne, Festuca elatior, Holcus lanatus, Bromus arvensis, Poa pratense, Hordeum sativum, and Phleum pratense.

Allergens↗
PubMed1996

Isotypic analysis of grass-pollen-specific antibodies in human plasma. III. Relationship to autoantibodies to IgE.

Since it has been shown that autoanti-IgE may be mistaken for antiallergen antibodies, thus appearing as pseudo-allergen-specific antibodies, it is crucial to separate true-from pseudo-allergen-specific antibodies and to determine to what extent autoanti-IgE appeared as pseudo-allergen-specific antibodies. For this purpose, human Ig pools were affinity-purified successively on a grass-pollen column and then on an antihuman-IgE column. IgG1-4, IgA, and IgM antibodies that were eluted from the grass-pollen column separated into pseudo- (approximately 30-40%) and true-allergen-specific antibodies that were coretained and not coretained, respectively, with the IgE on the anti-IgE column. Levels of autoanti-IgE were determined in individual plasma samples by surface plasmon resonance and statistically compared to the concentrations of allergen-specific antibodies obtained previously in the same plasma samples. A positive correlation between IgM autoanti-IgE levels and grass-pollen- "specific" IgM concentrations (P < 0.0002), and negative correlations between IgA autoanti-IgE and both IgE anti-grass pollen and IgG2 autoanti-IgE levels (P < 0.03, in both cases) were observed for the first time. This supports the contentions that: (1) autoanti-IgE antibodies appeared as pseudo-grass-pollen-specific antibodies, (2) they hid IgE antibodies when the latter were measured, and (3) they compete with one another in binding IgE. Lastly, a model of large Ig complexes is discussed.

Antibodies, Anti-Idiotypic↗
PubMed1996

Biochemical features of grain legume allergens in humans and animals.

Peanuts and soybeans are the major legumes involved in human food allergy, although some data exist on adverse reactions to temperate legumes including pea, green bean, sweet lupin, and lentil. An increasing number of legume proteins or glycoproteins have been characterized as food allergens. Limited data tend to indicate that they are usually different from legume inhalent allergens. Cross-recognition among legume allergens is immunochemically frequent but clinically less common. A common feature to most legume allergens is their natural resistance to thermal, chemical, and in some way, proteolytic denaturation. Finally, other mammals including preruminant calves, and piglets at the time of weaning, are prone to gut immune-mediated reactions to soybean and pea proteins.

Allergens↗
PubMed1996

Coexistence of two structurally similar but functionally different PII proteins in Azospirillum brasilense.

The coexistence of two different PII, proteins in Azospirillum brasilense was established by comparing proteins synthesized by the wild-type strain and two null mutants of the characterized glnB gene (encoding PII) adjacent to glnA. Strains were grown under conditions of nitrogen limitation or nitrogen excess. The proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or isoelectric focusing gel electrophoresis and revealed either by [32P]phosphate or [3H]uracil labeling or by cross-reaction with an anti-A. brasilense PII-antiserum. After SDS-PAGE, a single band of 12.5 kDa revealed by the antiserum in all conditions tested was resolved by isoelectric focusing electrophoresis into two bands in the wild-type strain, one of which was absent in the glnB null mutant strains. The second PII protein, named Pz, was uridylylated under conditions of nitrogen limitation. The amino acid sequence deduced from the nucleotide sequence of the corresponding structural gene, called glnZ, is very similar to that of PII. Null mutants in glnB were impaired in regulation of nitrogen fixation and in their swarming properties but not in glutamine synthetase adenylylation. No glnZ mutant is yet available, but it is clear that PII and Pz are not functionally equivalent, since glnB null mutant strains exhibit phenotypic characters. The two proteins are probably involved in different regulatory steps of the nitrogen metabolism in A. brasilense.

Amino Acid Sequence↗
PubMed1996

Comparison of the IgE titers to bovine colostral G immunoglobulins and their F(ab')2 fragments in sera of patients allergic to milk.

Colostral G immunoglobulins (IgGs) are described in many recent studies as having a beneficial effect for the treatment of viral, bacterial and parasitic diarrhea in animals and humans. The specific IgE titers to bovine colostral IgG, to bovine serum IgG, and to F(ab')2 fragments of IgG were immunoenzymatically quantified in sera of patients allergic to milk, to statistically evaluate and compare their relative immunoreactivity towards these purified antigens. The results clearly indicated that 36% of the population tested was potentially allergic to colostral IgG, and serum IgG globally elicited significantly lower IgE titers. The F(ab')2 fragments lead to a significantly decreased immunoreactivity as compared to colostral IgG. This study shows the interesting use of peptic hydrolysis of IgG in producing fragments with preserved therapeutic immunoactivity and reduced potential allergenicity.

Animals↗
PubMed1996

Lactate dehydrogenase as a marker of Plasmodium infection in malaria vector Anopheles.

Lactate dehydrogenase (Ldh) electrophoresis showed the presence of Plasmodium yoelii yoelii in Anopheles stephensi and An. gambiae. The Ldh appeared as an additional band (pLdh) whose activity was more intense with 3-acetyl pyridine adenine dinucleotide as coenzyme than with beta nicotin-amide adenine dinucleotide. Several allelic forms occurred both in the vector and the host. The isoelectric point of Ldh, similar in the vector and host, differed from those of Ldh from mosquito and mouse. The presence of pLdh was detected from the 2nd to the 28th day of infection. The pLdh appeared to be proportional to the number of sporozoites present in infected salivary glands. However, pLdh was not found in salivary glands or midguts, but it was detected in the rest of the corresponding mosquito. The origin and use of pLdh as a marker of Plasmodium in its vector is discussed.

Animals↗
PubMed1996

Isotypic analysis of grass pollen-specific antibodies in human plasma. 4. Biological activity of allergen-specific and autoanti-IgE antibody fractions on basophil histamine release.

BACKGROUND: Blocking antibodies are defined as antibodies that compete with IgE for binding to allergens due to their specificity for those allergens. Thus, they may inhibit allergen-induced basophil and mast cell IgE-dependent mediator release both in vivo and in vitro. OBJECTIVE: The present study was designed to evaluate the ability of antibodies isolated from human plasma samples on a Dactylis glomerata (Cocksfoot) pollen affinity-column to inhibit the Dactylis pollen-induced histamine release from human basophils (BHR) in vitro. METHODS: Antibodies from Ig pools containing either high or low IgG4 anti-Dactylis pollen were purified on a Dactylis pollen affinity-column and then separated on an antihuman IgE column. Obtained Ig fractions were incubated for 30 min with Dactylis pollen allergens prior to incubation with basophils from Dactylis pollen-allergic donors. Cell supernatants were assessed for histamine content and the inhibition of BHR was calculated. RESULTS: Unlike control non-isolated Igs, the antibodies isolated on the Dactylis pollen column were able to inhibit efficiently and in a dose-dependent manner Dactylis pollen-induced BHR. The inhibitory activity was increased in isolated antibody samples that had high IgG4 levels. Antibodies isolated on the Dactylis pollen column, however, consisted not only of true allergen-specific (potentially blocking) antibodies but also of autoanti-IgE binding to allergen-specific IgE and mistaken for allergen-specific antibodies thus opening to question the involvement of the true allergen-specific antibodies in the BHR-inhibitory activity. Unlike the true allergen-specific antibodies, the autoanti-IgE were retained on and eluted from the anti-IgE column. Results showed that both the autoanti-IgE-depleted and the autoanti-IgE-containing fractions accounted for the inhibition observed with the related non-depleted sample that had been isolated on the Dactylis pollen column. CONCLUSION: For the first time, the true blocking activity of allergen-specific antibodies is demonstrated, that is, in the absence of the autoanti-IgE which can also inhibit BHR.

Allergens↗
PubMed1994

DMISA (dissociated membrane immunosorbent assay), a new ELISA technique performed with blotted samples.

This study describes the use of electrophoretically purified antigens blotted onto nitrocellulose, as solid phase antigens for enzyme-linked immunosorbent assays. This procedure is called DMISA, for dissociated membrane immunosorbent assay. The method is illustrated using immunoblotted antigens of Dactylis glomerata grass pollen extract. The band of interest was located on a print of nitrocellulose by light staining (India ink), then the corresponding strip of nitrocellulose was cut out. Immediately after its solubilization in ethyleneglycol monomethyl ether, the antigen coated nitrocellulose was precipitated by the addition of buffer. In this way the bulk of the antigen remained bound to the membrane. The resulting suspension was carefully washed, and used as a solid phase antigen in an enzyme-linked immunosorbent assay. Two different electrophoretic methods were used to separate the Dactylis glomerata antigens. We compared the results obtained with classical immunoblot and with DMISA, for IgG4 and IgE quantification using sera from patients allergic to D. glomerata and purified blotted antigens present at the nanogram level.

Allergens↗