Enzyme subunit numbers in Trypanosoma cruzi zymodemes.
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Animals↗
Biomedical subjects
M D Chapman
Publications and source records attributed to M D Chapman.
In vitro investigations in asthmatic children undergoing hyposensitization with tyrosine-adsorbed Dermatophagoides pteronyssinus antigen.
Successful hyposensitization to Dermatophagoides pteronyssinus in perennial childhood asthma was associated with a slight mean rise of serum IgG antibody to the mite antigen in contrast to a placebo group in whom this antibody fell slightly. No relationship was detected between the effect on asthma and the magnitude of this change. Nor was there any consistent effect on IgE or IgA antibody. There was a wide range of total serum IgE and IgE, IgG and IgA antibody to D. pteronyssinus before treatment; this level did not predict the effect of treatment. Some patients lacked IgA antibody. IgE antibody to timothy grass pollen was raised in some but not others. These levels did not change systematically during the study and improvement occurred in those who had this antibody as well as IgE antibody to D. pteronyssinus. IgG and IgE antibodies to D. pteronyssinus were significantly correlated in the pre-treatment samples. Lymphocyte thymidine uptake was lower after 8 weeks of treatment than in the control group, not only after stimulus with D. pteronyssinus antigen, in vitro, but also with antigen from Candida albicans. This was not a serum effect. The late bronchial provocation response was lost only in those with serum IgA less than the log mean for age.
Adolescent↗
Trypanosoma cruzi from the Paraguayan Chaco: isoenzyme profiles of strains isolated at Makthlawaiya.
At Makthlawaiya, in the Paraguayan Chaco, the prevalence of Trypanosoma (Schizotrypanum) cruzi infection among both domestic Triatoma infestans and domestic dogs was 38%, and IgG anti-T. cruzi antibody was detected by the quantitative enzyme-linked immunosorbent assay (ELISA) in 80% (105/133) of human sera. Ninety percent (25/28) of T. cruzi strains isolated from both T. infestans and dogs showed heterozygous isoenzyme profiles for glucose phosphate isomerase, phosphoglucomutase and 6-phosphogluconate dehydrogenase. These strains appeared to be closely related to Bolivian zymodeme 2. Three Paraguayan T. cruzi strains showed homozygous isoenzyme profiles, similar to those of major Brazilian zymodemes. It was concluded that T. cruzi strains with heterozygous isoenzyme profiles predominate in domestic transmission cycles in this highly endemic area of the Paraguayan Chaco.
Animals↗
Recognition of two Dermatophagoides pteronyssinus-specific epitopes on antigen P1 by using monoclonal antibodies: binding to each epitope can be inhibited by serum from dust mite-allergic patients.
Monoclonal antibodies were raised against Antigen P1, the major allergen of the house dust mite (Dermatophagoides pteronyssinus). The majority were Antigen P1 specific, isotype IgG1, and did not react with a comparable D. farinae allergen. These antibodies bound 38 to 50% of 125I Antigen P1 in antigen-binding assays (titer greater than or equal to 1/1,000,000), and the quantities of IgG antibody in ascites were 2 to 4 logs greater than those in polyclonal mouse antiserum or in serum from a mite-allergic patient. Two IgM antibodies showed weak binding to Antigen P1 but reacted strongly with D. pteronyssinus in enzyme immunoassay (titer greater than or equal to 1/100,000). Assessments of the specificity of the IgG antibodies by using two inhibition radioimmunoassays suggested that they were directed against two different epitopes. Antibodies 10B9 F6 and 5H8 C12 were purified by preparative isoelectric focusing (isoelectric points of pI 6.25 and 7.4, respectively) and radiolabeled with 125I. Cross-inhibition experiments, using ascites dilutions to inhibit binding of each radiolabeled antibody to Antigen P1, confirmed that these antibodies recognized two distinct epitopes. Analysis of antibodies from 39 clones/hybrids showed that the majority were directed against the same epitopes as either 10B9 F6 or 5H8 C12 (3 out of 39 [8%] and 29 out of 39 [74%], respectively). None of the monoclonal antibodies significantly inhibited (greater than 10%) human IgE binding to Antigen P1 in the radioallergosorbent test. However, 12 of 14 sera from mite allergic patients inhibited binding by the monoclonal antibodies. One serum from a mite-allergic patient inhibited binding of both 10B9 F6 and 5H8 C12 by greater than 85% and showed parallel inhibition curves. The results suggest that these monoclonal antibodies could be used to assay Antigen P1 in both D. pteronyssinus and house dust extracts. It should also be possible to use monoclonal antibodies in inhibition assays to define the antigenic/allergenic determinants recognized by human IgG and IgE antibodies on this mite allergen.
Allergens↗
Quantitative differences in the expression of a 72,000 molecular weight cell surface glycoprotein (GP72) in Trypanosoma cruzi zymodemes.
A high affinity monoclonal antibody, 8G2 B9, was used to assess the expression of a 72,000 m.w. glycoprotein ( GP72 ) in isoenzyme-typed T. cruzi strains ( zymodemes ). Western blotting analysis of T. cruzi clones showed that 8G2 B9 bound strongly to GP72 and also suggested that this antigen was absent or weakly detectable in T. cruzi zymodeme 1 (Z1) strains. Purified 8G2 B9 was radiolabeled with 125I and used in an inhibition radioimmune binding assay to compare the quantities of GP72 in different zymodemes . Ninety-six T. cruzi strains were assayed, of which 36 were Z1, 36 were Z2, five were Z3 , and 19 were Z2 (heterozygous). Most (64%) Z1 strains lacked detectable GP72 , whereas this antigen was always detected Z2 and Z2 (heterozygous) strains. There was an 18-fold difference between geometric mean values for the quantities of GP72 (expressed as nanograms per milligram total cell protein) in Z1 and Z2 strains (Z1, 36 ng/mg; Z2, 639 ng/mg; p less than 0.001). There were also significant differences between the geometric mean values for Z2 and Z2 (heterozygous) strains, i.e., 639 ng/mg and 1648 ng/mg, respectively (p less than 0.001). GP72 was detected in four of the Z3 strains in quantities ranging from 740 to 3640 ng/mg. The absolute amounts of antigen in GP72 -positive strains were low, comprising less than 1% of the total cell protein. The specificities of two other anti- GP72 monoclonal antibodies, 7C6 D7 and WIC 29.26, were compared with 8G2 B9. Both antibodies completely inhibited the binding of 8G2 B9 to GP72 in solid phase immunoassays, suggesting that they reacted with the same antigenic determinants. The results show that monoclonal antibody-based assessments of the expression of GP72 correlate with zymodeme classification, and they also suggest that the monoclonal antibodies recognize major antigenic determinants on GP72 . It should be possible to use 8G2 B9 as an immunologic marker to additionally investigate the clinical significance of T. cruzi zymodemes and the biologic significance of GP72 .
Animals↗
Quantitative assessments of IgG and IgE antibodies to inhalant allergens in patients with atopic dermatitis.
Using antigen-binding radioimmunoassays, we have measured class specific antibodies against two major inhalant allergens, antigen P1 from D. pteronyssinus and Rye I from grass pollen, in sera from 69 patients with atopic dermatitis. The results show that many of the patients have IgE ab to these allergens in keeping with their skin tests. In all cases, the IgE ab was paralleled by IgG ab to the same allergen. In many sera, IgE ab to these inhalant allergens made a significant contribution to the total serum IgE. With two other allergens to which these patients had not been exposed, specific IgE ab was detected in only one serum, whereas the 42 sera tested did not contain IgE ab to diphtheria toxin. Eleven of the adult patients with atopic dermatitis had no history of asthma and had strongly positive skin tests. This group of patients had levels of total IgE and specific ab to antigen P1 that were very similar to those found in a comparable group of patients who had both atopic dermatitis and asthma. Our recent finding that allergens applied to the skin can induce delayed eczematous lesions provides a mechanism by which allergens could contribute to skin lesions. Our present results support the view that specific sensitivity to common allergens should be taken into account in considering the causes of these patients' skin lesions.
Adolescent↗
The role of dust mite allergens in atopic dermatitis.
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Adolescent↗
A monoclonal antibody with specificity for Trypanosoma cruzi, central and peripheral neurones and glia.
Of 26 hybridomas secreting anti-Trypanosoma cruzi antibodies, two reacted with murine brain and spinal cord extracts but not other tissues. Both antibodies (5H7 and 3H3) had identical isotypes (IgM) and specificities as judged by western blotting. Antigens of molecular weight 58,000 and 37,000 in mouse brain and spinal cord, and 58,000 and 35,000 in T. cruzi were detected. Different tissue antigens were recognized by a previously reported T. cruzi mammalian neural tissue cross-reacting monoclonal antibody CE5; on immunofluorescence 5H7 stained some glia, and some central and peripheral neurones in rat tissue sections. Pooled sera from mice chronically infected with T. cruzi competed with binding of 5H7 to T. cruzi antigens.
Animals↗
Basophils in allergen-induced patch test sites in atopic dermatitis.
Atopic dermatitis often occurs in patients who have high IgE levels and positive immediate skin tests to several common allergens. However, there is considerable doubt about the role played by allergens in this disease. Patch testing for 48 h at superficially abraded skin sites revealed that allergens could induce eczematous lesions in atopic dermatitis patients but only in those who also gave a positive immediate skin reaction to the same allergen. Lesions induced by the purified house dust mite antigen, antigen P1 contained mononuclear cells, basophils, eosinophils, and neutrophils. These patients also had raised specific serum IgE against antigen P1, and their leucocytes released histamine upon exposure to the same antigen. Thus an acute eczematous lesion can be induced by the application of inhalant allergens to the skin.
Adolescent↗
Mite faeces are a major source of house dust allergens.
The association between house dust allergy and asthma has long been recognized, and it has been demonstrated that a major allergen in house dust is related to the presence of mites of the genus Dermatophagoides. Using extracts of mite culture for skin testing, as many as 10% of the population and up to 90% of allergic asthmatics give positive immediate reactions. Although mites may occasionally become airborne during bed-making, it has also been demonstrated that they 'secrete or excrete' some allergen. Recently, we have shown that up to three-quarters of the serum IgE antibodies to mites are directed against a major allergen-antigen P1 (molecular weight 24,000). Using a radioimmunoassay it is possible to measure the concentration of this glycoprotein in both dust samples and mite cultures. These measurements, which are reported here, show that more than 95% of the allergen accumulating in mite cultures is associated with faecal particles.
Allergens↗
Human immunoglobulin E and immunoglobulin G antibody responses to the "minor" ragweed allergen Ra3: correlation with skin tests and comparison with other allergens.
We report here the use of antigen-binding assay to measure serum IgE and IgG binding activity (BA) for the "minor" ragweed antigen, Ra3. These studies were carried out in ragweed-allergic individuals, many of whom had been skin tested with Ra3 as well as with the "major" ragweed allergen, antigen E (AgE). IgE BA for Ra3 showed a weak quantitative correlation with skin-test sensitivity to Ra3. More strikingly, there was a threshold of skin sensitivity (< 10(-3) microgram/ml) above which serum IgE BA was likely to be detectable. Serum IgG BA for Ra3 measured in parallel with IgE BA was found in all sera that showed detectable IgE BA for Ra3. By contrast, 11/13 ragweed-allergic individuals who were skin-test negative for Ra3 showed no detectable IgG BA for Ra3. These results support the view that the known genetic controls over Ra3 sensitivity control IgG antibody as well as IgE antibody responses. Individuals receiving injections of ragweed extract showed increases in IgG BA for Ra3 following therapy. Among nine persons who had no IgG BA for Ra3 before treatment, four out of nine developed a low degree of IgG BA for Ra3. However, the patients who developed IgG BA did not develop detectable IgE BA for Ra3.
Adolescent↗
The distribution of dust mite allergen in the houses of patients with asthma.
Using an inhibition radioimmunoassay for the major allergen from Dermatophagoides pteronyssinus (antigen P1), we studied the distribution of this dust allergen in the houses of patients with asthma. Both bed and floor dust samples contained a wide range of antigen P1, 100 to 100,000 ng/g of fine dust, and this concentration correlated well with the number of mite bodies (r = 0.81, p less than 0.001). We were unable to detect antigen P1 in the air of undisturbed rooms. However, during domestic activity, between 1 and 30 ng were collected on a filter than sampled air for 45 min at 17 L/min. Using a cascade impactor it was shown that greater than 80% of the airborne antigen P1 was associated with particles greater than 10 mu in diameter. Some of the particles containing allergen could be identified because they formed precipitin rings when impacted onto agarose containing rabbit antimite antiserum. These particles had the physical appearance of mite feces, which are the major source of antigen P1 in mite cultures. The results suggested that natural exposure to this dust allergen allows occasional fecal particles to enter the lungs and that these particles contain very concentrated allergen.
Animals↗
Antibody response following prolonged hyposensitization with Dermatophagoides pteronyssinus extract.
An antigen-binding radioimmunoassay was used to measure IgG, IgA and IgE antibody (ab) to Dermatophagoides pteronyssinus in sera from patients who had been hyposensitized with either aqueous D. pteronyssinus extract or placebo. An allergen, F4P1, was obtained from D. pteronyssinus culture and 125I-labelled for use in the assay. The radioallergosorbent test was also used to measure IgE ab to D. pteronyssinus and IgE ab to Dermatophagoides farinae. The radioimmunoassay results showed a selective rise in IgG ab in sera from D. pteronyssinus-treated (DP) patients. An increase of greater than twofold in IgG ab in post-treatment sera was found in 27 of 29 DP patients compared to 1 of 29 placebo-treated ('placebo') patients (p less than 0.001). The mean rise in IgG ab in DP patients was sixfold, and the maximum rise was 50-fold. There was little change in the levels of IgA ab or IgE ab in either DP or 'placebo' patients during treatment. The DP patients showed significant clinical improvement compared with the 'placebo' patients (p less than 0.01). However, no correlation was found between post-treatment IgG ab and post-treatment symptom scores, or between the changes in IgG ab and the changes in symptom scores during treatment. The time course of the antigen-binding assay was investigated using very low concentrations of F4P1. The results showed that antigen-binding was slow, even in the presence of an excess of IgG ab. The role of IgG ab in hyposensitization is discussed.
Animals↗
Purification and characterization of the major allergen from Dermatophagoides pteronyssinus-antigen P1.
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Allergens↗
Cleavage by trypsin at N-epsilon-formyl-lysine residues [proceedings].
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Amino Acid Sequence↗
Measurement of IgG, IgA and IgE antibodies to Dermatophagoides pteronyssinus by antigen-binding assay, using a partially purified fraction of mite extract (F4P1).
An extract of Dermatophagoides pteronyssinus culture has been fractionated by chromatography on Sephadex G-100 and Pevikon block electrophoresis to obtain a partially purified allergen (F4P1). This preparation has a molecular weight of between 15--25,000 Dalton, migrates slowly on electrophoresis, and is colourless in solution. The skin-test reactivity of F1P1 was comparable to that of crude D. pteronyssinus extract. F4P1 was radio-labelled with 125I and used in an antigen-binding radioimmunoassay to measure IgG, IgA and IgE antibody (ab) to D. pteronyssinus. IgG, ab was detected in serum from 32/34 (94%) mite-allergic persons, and from 10/31 (30%) nonallergic persons. IgA ab and IgE ab were found in sera from 22/34 (65%) and 37/34 (79%) allergic persons respectively. Neither IgA nor IgE ab could be detected in sera from non-allergic persons. An excellent correlation was found between radioallergo-sorbent technique (RAST), using crude D. pteronyssinus extract and IgE-binding activity (BA) for F4P1, (r=0.94, P less than 0.001). The antigen-binding assay for IgE BA was as sensitive as RAST, but less sensitive than PK testing. There was a very good quantitative correlation between IgG BA and IgE BA (r = 0.84, P less than 0.001). IgG BA was shown to rise in the serum of three patients treated with injections of D. pteronyssinus extract.
Adolescent↗
Reduction in IgE binding to allergen variants generated by site-directed mutagenesis: contribution of disulfide bonds to the antigenic structure of the major house dust mite allergen Der p 2.
Site-directed mutagenesis was used to investigate the contribution of disulfide bonds to the antigenic structure of Der p 2. Single amino acid variants were generated at cysteine residues, preventing the formation of disulfide bonds at positions 21-27, 73-78, and 8-119. The variants were tested for binding to murine monoclonal antibodies (mAb) and human IgE antibodies (Ab) in an inhibition enzyme immunoassay. Removal of the disulfide linking the amino-carboxy termini (C8-C119) had no effect on mAb binding, however, IgE Ab binding was reduced by up to 10-fold. The other two disulfides form small loops and disruption of these bonds gave different binding patterns. The variant lacking the C21-C27 bond showed up to a 40-fold reduction in antibody binding, while the variant lacking the C73-C78 bond showed more than a 100-fold reduction in IgE Ab binding and failed to bind 3 of 4 mAb. Intradermal skin testing with the C73-C78 variant supported the in vitro findings; the variant was 10 to 100-fold less reactive than rDer p 2. These two bonds thus make markedly different contributions to stabilizing the antigenic determinants of Der p 2. The results suggest that the C73-C78 bond plays a critical role in stabilizing the antigenic structure of this major mite allergen.
Allergens↗