[Pasteurella multocida infection after a cat bite].
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Animals↗
Biomedical subjects
J Carreira
Publications and source records attributed to J Carreira.
Isolation by mAb based affinity chromatography of two Par j I isoallergens. Comparison of their physicochemical, immunochemical and allergenic properties.
We report the identification and separation of two isoallergen components of Par j I, the major allergen from Parietaria judaica pollen. First, electrophoretic conditions for consistently separating both isoforms in an SDS-PAGE system were established, and mol. wt values of 13,000 (isoallergen IA) and 10,500 (isoallergen IB) were estimated. Immunoblot, after SDS-PAGE experiments, with individual P. judaica-sensitive human sera revealed a slightly different IgE-binding pattern for each isoallergen. Four anti-Par j I mAbs were obtained from BALB/c mice immunized with a purified Par j I preparation comprising IA and IB isoallergens. Three mAbs were directed to an epitope shared by both isoallergens, and the fourth one recognized specifically one epitope on Par j IB. Dot-blot experiments with the deglycosylated allergen showed that the mAbs did not recognize the carbohydrate prosthetic group of the molecules. Affinity chromatography using the mAbs allowed the separation of the isoallergens that retained their IgE-binding ability after the purification process. Amino acid composition analyses and partial N-terminal sequencing demonstrated an extensive homology and also the existence of some structural differences between Par j I isoallergens, which is in agreement with the high, but not complete, cross-reactivity observed in competition ELISA experiments. Finally, skin prick tests performed on 28 P. judaica-sensitive patients showed that all of them recognized both isoforms and that allergenic epitopes present in Par j IA and IB are responsible for most of the allergenic activity of the whole extract.
Allergens↗
The main allergen of Olea europaea (Ole e I) is also present in other species of the Oleaceae family.
Three major pollen allergens from Fraxinus excelsior, Ligustrum vulgare and Syringa vulgaris belonging to the Oleaceae family were purified. Monoclonal antibodies previously raised against the main allergen of Olea europaea (Ole e I) were used for their purification by affinity chromatography. The three new purified allergens were able to bind human IgE from serum of olive-allergic patients in a way analogous to Ole e I. Crossed radioimmunoelectrophoresis of the four allergens, using anti-olive extract rabbit serum, showed a unique immunoprecipitation arc with the same characteristics. The four purified proteins had similar molecular weights on SDS-PAGE and the N-terminal sequences for the first 20 amino acids were identical. Furthermore, the concentration of the allergens could be determined using a two-side solid phase assay previously developed for the allergen Ole e I. Our results indicate that the four purified proteins share, to a great extent, antigenic and allergenic epitopes leading to cross-reactivities which could cause common clinical manifestations. We propose for the newly purified allergens the nomenclature of Fra e I, Lig v I and Syr v I.
Allergens↗
[The Reynolds syndrome. Its diagnostic accessibility and therapeutic intervention in primary care].
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Aged↗
[New trends in medical education. Evaluation of placements in health centers].
Whether to use just hospitals or a wider range of health resources is one of the debating-points in the updating of undergraduate medical training programmes. The Faculty of Medicine in the Autonomous University of Madrid integrated obligatory Primary Care placements for students in the last year of their course, into its syllabus as from 1988/89. This programme and the students' own assessment of it was studied by means of a systematic, voluntary and anonymous survey, which covered various aspects of their assignments to Primary Care teams. 93% (473) replied. Every student took part in the clinical activities, both on demand and as programmed. 76% went on home visits along with their tutors. 88% considered that their placement was active. The overall evaluation of the placement by the students is 8 out of 10. Half believe that a month is too short a period and suggest that more time is offered as an optional placement. 98% consider that the Centre in which they were placed was sufficiently or very capable of teaching at the undergraduate level. Placing medical students in Primary Care teams is an essential part of today's medical teaching and is feasible within the present health system. The relationship between the University and the general physicians who act as tutors is seen as the main obstacle that needs to be overcome.
Community Health Centers↗
Monoclonal antibodies against Olea europaea major allergen: allergenic activity of affinity-purified allergen and depleted extract and development of a radioimmunoassay for the quantitation of the allergen.
Several monoclonal antibodies (MAbs) were raised against Olea europaea pollen-extract components. Two of these antibodies, named OL 2 and OL 7, recognize two nonoverlapping, nonrepeating epitopes on the olive-allergen Ole e I, as demonstrated by different techniques. The allergen was purified in a single step by MAb-based affinity chromatography, and the allergen revealed a band at molecular weight 20 kd as well as a minor band at 18 kd on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The contribution of allergen Ole e I to the allergenic activity of O. europaea pollen extracts was determined from the effect of allergen depletion by affinity chromatography on skin reactivity and a histamine-release test. The removal of allergen caused a large reduction in the activity of the preparation in 25 monospecific olive-allergic patients. In agreement, the affinity-purified allergen demonstrated a similar response when it was compared with the whole extract in these assays. The results indicated that Ole e I is by far the most important olive-pollen allergen. A two-site solid-phase radioimmunoassay was developed for the quantitation of the allergen Ole e I in mass units. The assay was based on the MAbs, OL 2 and OL 7, and had a detection limit in the nanogram range. A good correlation was found between allergenic activity, as determined by RAST inhibition, and allergen content in 18 olive-pollen extracts. This result indicates that the assay can be a good alternative to RAST inhibition for the standardization of O. europaea extracts.
Allergens↗
Purification and characterization of Lep d I, a major allergen from the mite Lepidoglyphus destructor.
A major allergen of the storage mite Lepidoglyphus destructor (Lep d I) has been purified by affinity chromatography using an anti-Lep d I monoclonal antibody. The purity of the protein obtained by this procedure was assessed by reverse-phase HPLC. Lep d I displayed a molecular weight of 14 kD on SDS-PAGE under non-reducing conditions, and 16 kD in the presence of a reducing agent. Analytical IEF revealed a little charge microheterogeneity, showing three bands with pIs 7.6-7.8. Purified Lep d I retained IgE-binding ability, as proved by immunoblotting experiments after SDS-PAGE and RAST with individual sera from L. destructor-sensitive patients. Results from the latter technique demonstrated that 87% of L. destructor-allergic patients had specific IgE to Lep d I, and a good correlation between IgE reactivity with L. destructor extract and Lep d I was found. In addition, RAST inhibition experiments showed that IgE-binding sites on Lep d I are major L. destructor-allergenic determinants, since Lep d I could inhibit up to 75% the binding of specific IgE to L. destructor extract; on the other hand, Lep d I did not cross-react with D. pteronyssinus allergens.
Allergens↗
Purification and characterization of major inhalant allergens from soybean hulls.
Proteins responsible for respiratory allergy to soybean have been purified from an extract of soybean hulls. The purification procedure combined size exclusion and reverse-phase HPLC. Two pure glycoproteins (S1 and S2) exhibiting IgE-binding ability, as demonstrated by immunoblotting and ELISA techniques, were obtained. Both proteins displayed low molecular weight values on SDS-PAGE (S1, 7.0 kD; S2 7.5 kD). Protein S1 showed charge microheterogeneity, rendering two bands at pH 6.1-6.2 on IEF, whereas S2 showed a single band at pH 6.8. Amino acid composition analyses revealed a strong homology between S1 and S2 and, as a characteristic feature, a high percentage of hydrophobic residues, mainly leucine and isoleucine. Concerning the allergenic activity, both proteins were recognized by the specific IgE from 95% of patients who suffered asthma attacks during the asthma outbreaks of 1987 and 1988 in Cartagena (Spain), caused by soybean dust. Besides, proteins S1 and S2 were able to, separately, inhibit up to 75% the binding of specific IgE to the whole extract. Moreover, purified proteins totally crossreacted, even though protein S2 seemed to be slightly more active in all the immunochemical techniques employed. Results presented allow us to conclude that both proteins are isoallergens and to name them as Gly m IA (protein S2) and Gly m IB (protein S1), according to the IUIS-allergen nomenclature system.
Allergens↗
Presence of IgG4 on the membrane of human basophils. Histamine release is induced by monoclonal antibodies directed against the Fab but not the Fc region of the IgG4 molecule.
We have studied the possible role of human IgG4 as an anaphylactic antibody. For that purpose, we have determined the induction of histamine release (HR) from human basophils by anti-IgE and anti-IgG4 monoclonal antibodies (MoAbs) recognizing different epitopes located at the Fc and Fab regions of the IgG4 molecule. The results show that anti-IgG4 (Fab) MoAb was able to induce HR in 93% of donors tested, with no differences between atopics and non-atopics. That HR is calcium dependent and is accompanied by the synthesis and release of leukotriene C4. In contrast, no HR could be induced by anti-IgG4(Fc) MoAbs in any individual, even in the presence of D2O or after a second challenge with a polyclonal goat anti-mouse IgG antibody. The results obtained suggest the presence of IgG4 on the basophil membrane and that the epitope recognized by the anti-IgG4 (Fc) MoAbs is probably hidden in cell-bound IgG4. This was demonstrated by immunofluorescence techniques: IgG4 bound to the basophil membrane could be detected with anti-IgG4(Fab) but not with anti-IgG4(Fc) MoAbs. In addition, we found that nine donors were unresponsive to an anti-IgE stimulus, while they released histamine efficiently after challenge with anti-IgG4(Fab), suggesting the existence of different receptors for both immunoglobulins.
Antibodies, Monoclonal↗
Food allergy: identification of the major IgE-binding component of peach (Prunus persica).
Since peaches are a relatively common cause of food allergy, we set out to identify the allergens involved. With the use of a panel of 48 sera from patients allergic to peach, we demonstrate that most of the allergenicity of that fruit is confined to the skin, rather than to the flesh of peaches, and corresponds to a protein doublet with an estimated molecular weight of 8-10 kD.
Adolescent↗
Sensitization to the storage mite Lepidoglyphus destructor in wheat flour respiratory allergy.
Occupational allergy due to hypersensitivity to cereal flours is relatively common among bakers and grain-store workers. Storage mites can contaminate wheat flour and could be an important cause of allergic symptoms due to inhalation. Forty-three patients with criteria for allergic sensitization to wheat flour (skin tests, specific IgE to wheat flour and positive challenge tests) were included in a study to investigate the prevalence of cosensitization to Lepidoglyphus destructor (Ld). This mite was the predominant species in the wheat flour samples supplied by our patients. We found that 30% of the patients had IgE-mediated hypersensitivity of Ld. Of these, 23% did not have a relationship with any bakery or agriculture. We conclude that the prevalence of sensitization to Ld in patients sensitized to wheat flour is important.
Adolescent↗
Monoclonal antibodies against human IgE. Identification of an epitope sharing properties with the high-affinity receptor binding site.
Three monoclonal antibodies to human IgE are described. One of them recognizes an epitope located within a region of 76 amino acids that has been shown to contain the Fc epsilon RI binding site. That epitope is shown to be susceptible to heating and to alkylation of cysteines involved in inter heavy chain bonds, but not to their reduction alone. In addition, this monoclonal antibody, although having a high affinity for free IgE, is unable to bind Fc epsilon RI-linked IgE. Based on these results, we discuss the possibility that the antibody recognizes the Fc epsilon RI binding site of the IgE molecule.
Alkylation↗
Identification of IgE-binding proteins from Lepidoglyphus destructor and production of monoclonal antibodies to a major allergen.
The allergen composition of one of the most important storage mites, Lepidoglyphus destructor, has been studied by immunodetection after SDS-PAGE with individual patient sera. An allergenic polypeptide of 14 kDa was identified with 95% of the sera. This major allergen was isolated in the supernatant of 60% ammonium sulfate salt precipitation of the whole extract, which was subsequently used to immunize BALB/c mice so as to produce monoclonal antibodies. Four mAbs recognizing molecules with IgE-binding ability were obtained. The specificity of the mAbs was assayed against different allergenic extracts, and the molecules recognized by them were characterized by immunoblotting. Two mAbs (Le5B5 and Le9E4) were directed to the 14-kDa allergen; the other two to several proteins of lesser allergenic significance.
Allergens↗
Liposomes as carriers for allergy immunotherapy.
With the aim of obtaining an efficient but safer vaccine for allergy immunotherapy, the possibility of using liposomes as adjuvants has been considered given their proven low toxicity, adjuvant properties and ability to maintain the encapsulated substance in their interior for some time in vivo. Different methods of encapsulating allergenic extracts into neutral, positively, and negatively charged DPPC: cholesterol liposomes have been investigated and the immune response provoked by these in mice was studied and compared to the immune response to free allergen or other adjuvants such as aluminium hydroxide. The results obtained show that allergen encapsulated in all three types of liposomes elicit an increase in specific IgG levels higher than that provoked by free allergen, however, both encapsulation efficiency and specific IgG titre were higher when positively charged (DPPC: cholesterol stearylamine) liposomes were used. Specific IgE levels to allergen in positive and neutral liposomes was lower than to allergen in negative liposomes or adsorbed to Al(OH)3. No difference were found in the behaviour of liposomes prepared by different methods (the results were obtained from pooled sera from different groups of mice so there is no statistical data). These results confirm the immunoadjuvant effect of liposomes for allergy immunotherapy. Further studies to determine their lack of toxicity, pharmacokinetic studies and human clinical studies are necessary to confirm their adequacy for human use and advantage over current immunotherapy methods.
Allergens↗
Identification of soybean proteins responsible for respiratory allergies.
Serum samples from 32 patients who suffered attacks during the asthma outbreaks of 1987 and 1988 in Cartagena, Spain, supposedly caused by soybean dust, were studied. At least 90% had specific IgE to shell components and only 13% showed specific IgE to shell-depleted soybean grains. A control group of 32 patients who also suffered asthma attacks but on different days from those of the outbreaks were negative. The shell's most important allergen with an apparent molecular weight of 8 kDa was not present in shell-depleted grains. This allergen as well as other less important shell allergens may be different from the allergens already identified by using serum from patients suffering food allergy to soya.
Allergens↗
Purification of Art v I, a relevant allergen of Artemisia vulgaris pollen.
An allergenic protein from Artemisia vulgaris pollen has been purified to homogeneity. Its molecular weight in native conditions is 47,000. The purified allergen, hereafter denominated Art v I, is a monomeric protein. It is a clinically relevant allergen since, at least, 70% of the individuals allergic to Artemisia vulgaris pollen have specific IgE in serum and in mast cells, demonstrated by ELISA and skin prick tests, respectively.
Allergens↗
HPLC purification of the main allergen of Parietaria judaica pollen.
A two-step purification procedure of Par j I from the whole Parietaria judaica pollen extract is described. The first step consisted of gel filtration HPLC using a TSKG 3000 SW column, and 0.1% trifluoroacetic acid as the eluant. By this method, proteins were separated from the highly colored material present in the extract. Then, Par j I-containing fractions were chromatographed on a reversed-phase HPLC column (Vydac C4) using an acetonitrile gradient. This second step yielded pure Par j I as assessed by SDS-PAGE and CIE. Previously reported microheterogeneity was still observed, but amino acid analysis of various RP-HPLC fractions suggested that the heterogeneity of Par j I might not be due to changes in its primary structure. Allergenic activity of Par j I was shown to be retained after the purification procedure by several immunochemical techniques.
Allergens↗
Obtention of monoclonal antibodies against human IgG4 using two different immunization strategies: development of a biotin-based ELISA for IgG4 quantitation.
Several monoclonal antibodies (MAbs) against human IgG4 have been produced in two fusion experiments. In fusion I, Balb/c mice were conventionally immunized and most of the antibodies obtained recognized IgG class-specific epitope(s). Therefore, a second fusion experiment was performed using IgG4-immunized mice that had been made tolerant to epitopes shared by the four IgG subclasses, and most of the hybridomas derived from this second fusion secreted antibodies specific for IgG4. Five MAbs which were found to be specific for both IgG4 isoallotypes (4a and 4b) were selected from the two fusion experiments. These antibodies define at least two non-overlapping epitopes located on the Fab and Fc regions of IgG4 molecule, respectively. Two MAbs were used to develop a two-site ELISA for IgG4 quantitation, in which one MAb (anti-Fab) was immobilized in the solid phase and the other one (anti-Fc) was biotin-labeled. The assay had a detection limit below 1 ng/ml and a practical working range between 4 and 200 ng/ml. The use of these MAbs should improve our understanding of the role of IgG4 in different clinical conditions.
Antibodies, Monoclonal↗