Biomedical subjects
Publications and source records attributed to J Carreira.
A case of acute ischaemic colitis, confirmed by colonoscopy and colon biopsy, caused by IgE mediated allergy to amoxicillin is presented. The damage to the gut seemed to occur as a result of the hypotension suffered during the anaphylactic episode.
BACKGROUND: The demonstration that some asthma epidemics have been caused by allergens of soybean-hull dust prompted us to develop a two-site ELISA, suitable for the quantification of the major allergen (Gly m 1), to be used for the prevention of new episodes. METHODS: BALB/c mice were injected with Gly m 1 purified from soybean hulls. After fusion and screening, 10 monoclonal antibodies (mAbs) were obtained that were shown to be specific for Gly m 1 Two of them (6G1 as the capture antibody; 1G10 as the tracer) were selected to develop a quantitative two-site ELISA for the indoor and outdoor determination of Gly m 1. RESULTS: The two-site ELISA developed is very sensitive, with a detection limit of less than 0.2 ng/ml and a practical working range of 0.4-10 ng/ml. The assay is also highly reproducible with an intra-assay coefficient of variation of 3.5% and an interassay coefficient of variation of 12.5%. The method was applied to measure the concentration of Gly m 1 in air-sampler filters and in house-dust samples. Our results illustrate that there is a good correlation between the content of Gly m 1 in a number of samples and the allergenic activity as measured by ELISA inhibition. CONCLUSIONS: A specific and sensitive method is presented that can be used for the quantification of Gly m 1. The application of this method may allow the establishment of risk limits for soybean dust, and thus may contribute to the control of environmental contamination and to the prevention of new asthma epidemics.
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A solid-phase, monoclonal antibody-based ELISA was set up to quantitate group 5 allergens in pollen extracts of wild and cultivated Pooideae grasses. The method was able to evaluate group 5 concentration in mass units with a sensitivity in the ng/ml range and a practical working range of 1-100 ng/ml. The group 5 ELISA was compared with rocket immunoelectrophoresis for determination of allergen levels in several Phleum pratense extracts, and a very good quantitative correlation was found (r = 0.98; P < 0.0001). A highly significant correlation (r > 0.8) was also obtained in comparing allergenic potency determined by RAST inhibition to group 5 content in several wild and cultivated grass species. The results proved the usefulness of the method in the standardization of Pooideae pollen extracts employed in diagnosis and treatment.
BACKGROUND: Fagales pollens are considered among the main agents responsible for allergic diseases in many countries of the northern hemisphere and single major allergens have been shown to be responsible for these responses. OBJECTIVE: To develop a solid phase immunoassay for the quantification of Bet v 1, the main allergen from Betula verrucosa (birch), and to assess its suitability for quantitating the equivalent major allergen in other Fagales species as well. METHODS: The assay is based on the use of two different anti-Bet v 1 monoclonal antibodies which were immobilized on the solid phase and, as a primary standard, affinity purified Bet v 1, the protein content of which was determined by amino acid analysis. RESULTS: The ELISA proved to measure less than 0.2 ng/mL of Bet v 1 with a practical range of 0.4-40 ng/mL and could be suitable to quantify the equivalent major allergen in other Fagales species such as Corylus avellana (hazel), Carpinus betulus (horbeam) and Alnus glutinosa (alder). The method was compared with quantitative electrophoresis and rocket immuno-electrophoresis for the determination of the allergen content in several Betula verrucosa extracts, and a very good quantitative correlation was found. Likewise, the Bet v 1 content exhibited a good correlation (r = 0.87; P < 0.005) with the allergenic potency values obtained by RAST inhibition. CONCLUSIONS: The results indicate that the Bet v 1-assay could be useful for standardization purposes in Fagales pollen extracts intended for clinical use.
OBJECTIVE: Evaluate relationships between the next variables in an adult natural population: total cholesterol, LDL-cholesterol, HDL-cholesterol, triglicerides, total cholesterol/HDL-cholesterol and glucose blood levels with blood pressure (systolic and diastolic). DESIGN: A crossover observation study with descriptive and analytic components. PATIENTS: Random sample of 251 individuals taken from census and representative of the adult general population from Mora de Toledo (with 10,000 inhabitants approximately). SETTING: A medical station in a health centre. MEASUREMENTS AND MAIN RESULTS: Blood pressure measurements and lipids and glucose blood levels were got in the selected patients. Some positive and significant relationships were got in the adult population from Mora de Toledo between systolic and diastolic blood pressure with the next variables: total cholesterol, LDL-cholesterol, total cholesterol/HDL-cholesterol, triglicerides and glucose blood levels. These associations are stronger in young people from 21-40 years-old. When we consider the relationship with HDL-cholesterol, we can see it is mildly positive but not significant. CONCLUSION: These results agree with others encountered in different countries and support the hypothesis that there is an association between blood pressure and the cardiovascular metabolic risk factors.
BACKGROUND: Quantitation of Der 1 and Der 2 in dust samples by specific monoclonal antibodies is a method used increasingly to evaluate mite allergen exposure. The level of Der 1 has been proposed as a risk factor for sensitization. AIM: We report a drastic decrease in the Der 1/Der 2 ratio when dust samples are collected in bakeries. METHODS: Wheat flour and purified mites were extracted simultaneously; levels of Der p 1 and Der p 2 and cysteine protease activity were determined by ELISA and inhibition experiments. RESULTS: High titers of Der 2, but only trace amounts of Der p 1, were detected in dust collected from bakeries. Both the level and proteolytic activity of Der p 1 appeared greatly decreased when mites and wheat flour were coextracted. CONCLUSION: Group 1 protein was found to be masked by flour components, resulting in an underestimation of the mite content in bakery dust. This problem was not found for group 2 allergen.
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In order to evaluate the antigenic contribution of different regions of the penicillin molecule, monoclonal antibodies were raised against amoxicillin-protein conjugates and their specificities analysed in detail. A random sample of the clones produced was analysed by a quantitative inhibition-ELISA, using, as inhibitors, monomeric conjugates of the following antibiotics to butylamine (BA), amoxicillin (AX), ampicillin (AMP), benzylpenicillin (BP) and the nuclear part of these, 6-aminopenicillanic acid (6-APA); and different parts of the following molecules: N-(p-hydroxyphenyl)-glycine (PHPG), N-phenylglycine (NPG), phenylacetic acid (PA) and thiazolidine (TIAZ). The results showed that 92% of the antibodies recognized an epitope in which the side chain was a major constituent, although with variable contributions from other regions of the molecule. There was a high degree of crossreactivity with aminopenicillins, but low or absent crossreactivity with BP. None of the antibodies recognized the thiazolidine ring or the conjugated nuclear region of the penicillins. Finally, one antibody seemed to recognize, equally, all the different structures tested. The possible relevance of these results to penicillin allergy is discussed.
BACKGROUND: Plants of the genus Parietaria, Urticaceae family, represent a major cause of pollinosis in the Mediterranean area. Different Parietaria species crossreact to a great extent, but studies on the crossreactivity among the major allergens of these pollens have not been carried out so far. OBJECTIVE: To develop an immunochemical method to quantify the major Parietaria judaica allergen, Par j 1, as well as to verify the presence of Par j 1-like proteins in different Urticaceae pollens. These proteins would be purified in order to study the cross-reactivity among them. METHODS: Immunoaffinity chromatography with a monoclonal antibody, solid-phase enzyme-linked immunoassays and SDS-PAGE. RESULTS: A monoclonal antibody-based ELISA for the quantification of Par j 1 has been developed. The assay has a sensitivity of 0.2 ng/mL and shows a high correlation with the allergenic activity of P. judaica extracts determined by radioallergosorbent assay (RAST) inhibition. By means of this assay, proteins homologous to Par j 1 were detected in P. officinalis and P. mauritanica. These proteins (Par o 1 and Par m 1, respectively) were purified by affinity chromatography using the same monoclonal antibody employed in the ELISA. Crossed-inhibition experiments demonstrated that Par j 1, Par o 1, and Par m 1, competed for the binding of specific IgE from a P. judaica-sensitive patients serum pool. CONCLUSION: The results here described suggest that shared allergenic epitopes are present in the three main allergens investigated, which may simplify the diagnosis and therapy for Parietaria allergy.
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The most relevant allergen of the storage mite Lepidoglyphus destructor (Lep d I) has been characterized. Lep d I is a monomer protein of 13273 Da. The primary structure of Lep d I was determined by N-terminal Edman degradation and partially confirmed by cDNA sequencing. Sequence polymorphism was observed at six positions, with non-conservative substitutions in three of them. No potential N-glycosylation site was revealed by peptide sequencing. The 125-residue sequence of Lep d I shows approximately 40% identity (including the six cysteines) with the overlapping regions of group II allergens from the genus Dermatophagoides, which, however, do not share common allergenic epitopes with Lep d I.
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Olea europaea (Ole e) I-specific cDNA sequences were amplified by 3'-RACE-PCR, using specific primers based on the N-terminal sequence of the allergen, and cloned into appropriate vectors. The nucleotide sequence data obtained revealed the presence of isogenic variation in Ole e I gene(s). The molecular mass, pI, amino acid composition and sequence of the predicted polypeptides agree with data previously obtained by analysis of purified Ole e I from pollen. Furthermore, by treatment of purified Ole e I with specific glycopeptide hydrolases it has been demonstrated the presence of N-glycosylation in the allergen, and there is a unique concensus site for N-linked glycosylation at positions 111-113 of the deduced amino acid sequence. The Ole e I predicted sequence shows a significant homology with three putative proteins encoded respectively by the another-specific LAT52 gene from tomato and the pollen specific genes Zmc13 from maize and OSPSG from rice, suggesting that these proteins could have a role in one of the development processes unique to male gametophytes.
We have purified four allergens from Helianthus annuus (sunflower) pollen, hereafter named as allergens a, b, c, and d. Under native conditions, allergen a has a mol. mass of 32, allergen b has one of 24, and allergens c and d each have one of 55 kDa. At the least, allergens b, c, and d demonstrate charge heterogeneity, and the electrophoretic mobility of allergens c and d increases when these allergens are deglycosylated with trifluoromethanesulfonic acid. Cross-reactivity among the four allergens and with the whole extract is very high, and each allergen recognizes IgE in a high proportion of patients sensitized to sunflower pollen.
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