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Biomedical subjects

M D Chapman

Publications and source records attributed to M D Chapman.

At least 55 records · Page 3Linked to original sources
PubMed1999

Cockroach allergens and asthma in Brazil: identification of tropomyosin as a major allergen with potential cross-reactivity with mite and shrimp allergens.

BACKGROUND: Cockroaches produce several proteins that induce IgE antibody responses. Although cockroaches are abundant in warm and humid areas, sensitization to cockroach allergens has not been investigated in Brazil. OBJECTIVE: The aims of this study were to investigate the frequency of cockroach allergy among patients with asthma, rhinitis, or both in Brazil and to identify American cockroach allergens. METHODS: Skin tests using cockroach extracts were performed on children and young adults with asthma, rhinitis, or both. A Periplaneta americana complementary (c)DNA library was screened by using IgE antibodies from Brazilian patients allergic to cockroaches. Reactivity of an mAb directed to Dermatophagoides pteronyssinus tropomyosin against cockroach tissue was examined by immunofluorescence. RESULTS: Cockroach allergy was present in 55% and 79% of the patients, as determined by using skin prick tests alone or combined prick and intradermal tests, respectively. Five cDNA clones reacted with IgE antibody and contained the same sequence. A representative clone (1300 bp), pa 12, coded for a protein that reacted with 50% of the sera from patients allergic to cockroaches on plaque immunoassay and showed a high degree of homology to tropomyosins, particularly those from invertebrates. P americana tropomyosin showed 80%, 81%, and 82% sequence identity to tropomyosins from D pteronyssinus, D farinae, and shrimp, respectively, which have been previously defined as important allergens. An mAb directed against D pteronyssinus tropomyosin, which also recognizes shrimp tropomyosin, showed binding to cockroach striated muscle. CONCLUSION: Our results support the recommendation that cockroach extracts should be routinely used for the evaluation of patients with asthma, rhinitis, or both in Brazil. The identification of P americana tropomyosin as an important allergen will make it possible to investigate cross-reactivity among cockroaches, mites, and food derived from invertebrates.

Adolescent↗
PubMed1999

Molecular cloning of Per a 1 and definition of the cross-reactive Group 1 cockroach allergens.

BACKGROUND: Sensitization to allergens produced by German and American cockroaches is strongly associated with the cause of asthma. Most of the cockroach allergens identified to date have been species specific. OBJECTIVE: The aim of this study was to identify and sequence cross-reactive cockroach allergens. METHODS: A Periplaneta americana cDNA library was screened with IgE antibody from patients in the United States who were allergic to cockroach and who were sensitized to Blattella germanica. RESULTS: A cDNA clone was isolated that contained an 870-bp sequence with a 695-bp open reading frame, encoding a 231 amino acid protein, molecular weight 26.2 kd. Plaque immunoassays using anti-Bla g 1 and anti-Per a 1 mAbs and a panel of human IgE antibodies showed that the protein expressed by these clones was Per a 1. Sequence homology searches showed that Per a 1 was homologous to 5 previously reported, but unidentified, sequences from B germanica and P americana. These sequences encoded proteins with multiple molecular sizes containing approximately 100 amino acid repeats. The Per a 1 sequence also showed 31% identity to a mosquito precursor protein, ANG12, which may be involved in digestion. The Per a 1 cDNA was expressed in Pichia pastoris to produce purified recombinant allergen (yield, 14 mg/L). CONCLUSION: The results define the molecular structure and antigenic relationships between a new family of cross-reactive "Group 1" allergens produced by both P americana and B germanica. These recombinant allergens and specific mAbs will provide tools to improve the diagnosis and treatment of allergic diseases caused by cockroaches.

Allergens↗
PubMed1999

High prevalence of sensitization to cat allergen among Japanese children with asthma, living without cats.

BACKGROUND: Cat allergy is common among children with asthma. Many cat-allergic patients in Japan and elsewhere do not keep cats, but nonetheless become sensitized through environmental exposure to cat allergen. OBJECTIVE: To assess the frequency of cat allergy and cat-specific immunoglobulin E (IgE) and immunoglobulin G (IgG) antibody responses in young Japanese patients with asthma in relation to self-reported cat exposure and Fel d 1 levels in dust samples. METHODS: Cat dander-specific IgE antibody was measured in sera from asthma patients using the CAP system. IgE and IgG antibody to Fel d 1 was measured by antigen binding radioimmunoassay and by chimeric enzyme immunoassay. Fel d 1 levels in dust samples from a subset of patients' homes were measured by monoclonal antibody-based enzyme immunoassay. RESULTS: Cat-specific IgE (CAP class>/=2) was found in sera from 70% of 44 patients who kept cats and 34% of 394 patients who had never kept cats. The prevalence of sensitization increased progressively to age 6 years (40%: positive), and then increased gradually to age 16 years (approximately 60%: positive) in patients who had never kept cats. There was an excellent correlation between cat CAP values and IgE levels to Fel d 1. The absolute amount of IgE antibody to Fel d 1 ranged from 0.01 to 15.6% of total IgE. Most patients who did not keep cats were exposed to Fel d 1 levels ranging from 0.07-8 microg/g dust. CONCLUSIONS: Sensitization to cat allergen is common among young asthmatic patients in Japan, even among patients who do not keep cats. Use of CAP and the chimeric enzyme-linked immunosorbent assay allows accurate diagnosis of cat allergy and quantification of specific IgE antibody levels.

Adolescent↗
PubMed1999

Allergenic differences between the domestic mites Blomia tropicalis and Dermatophagoides pteronyssinus.

BACKGROUND: House dust mite allergens are the most important indoor allergens associated with asthma and rhinitis in Singapore and the tropics. Recent data to suggest that besides the Dermatophagoides spp., the domestic mite Blomia tropicalis (Bt) is also an important source of allergens in these regions. OBJECTIVE: To evaluate the degree of allergenic cross-reactivity between Bt and D. pteronyssinus (Dp). METHODS: Cross-reactivity between extracts of Bt and Dp was evaluated by fluorescent allergosorbent (FAST) inhibition studies and cross enzyme immunoelectrophoresis. Additionally, the major Dp allergens - Der p 1, Der p 2 and Der p 5, were also compared with the Bt extract by dot blot inhibition. Skin prick and intradermal end-point titration were then carried out to compare the homologous allergens of the mite species, Blo t 5 and Der p 5. RESULTS: FAST inhibition studies showed low to moderate cross-reactivity between the two dust mite extracts (maximum cross-inhibition, 60%). Native allergens studied by cross enzyme immunoelectrophoresis using mite allergic sera also showed similar results but with at least four cross-reactive IgE binding antigens. Dot blot inhibition studies using allergens of Dp, Der p 1, Der p 2, and Der p 5, showed little cross-reactivity between these allergens with components of the crude Bt extracts. Further, evaluation of a recombinant allergen of Bt, Blo t 5, showed low levels of cross-reactivity even with its homologous Dp counterpart, Der p 5. CONCLUSION: These results provide evidence that Bt allergens are distinct and have relatively low to moderate cross-reactivity with Dermatophagoides spp. allergens. Bt allergens should therefore be included in the diagnostic panel for the evaluation of allergic disorders in the tropics, and the development of new diagnostic and therapeutic strategies should include allergens of Bt.

Allergens↗
PubMed1998

Novel allergen structures with tandem amino acid repeats derived from German and American cockroach.

Cockroaches produce potent allergens that are an important cause of asthma. The two principal domiciliary cockroach species, Blattella germanica and Periplaneta americana, secrete major allergens, Bla g 1 and Per a 1. Here, we report the molecular cloning of three Bla g 1 cDNA clones, which showed 70% amino acid sequence identity with Per a 1. Plaque immunoassays with human IgE antibodies or murine monoclonal antibodies showed that these allergens were antigenically cross-reactive. The Bla g 1 sequences also showed homology to five previously undefined cockroach allergen sequences. An unusual feature of all these sequences was that they contained multiple tandem amino acid repeats of approximately 100 amino acid residues. Between one and seven repeat units were identified by dot-plot matrix analysis. The sequences also showed homology to a mosquito protein involved in digestion (ANG12 precursor) and to mitochondrial energy transfer proteins. High levels of Bla g 1 were found in cockroach hindgut and proventriculus. Amino acid sequencing of natural Bla g 1 and Per a 1 suggested that these allergens are cleaved by trypsin-like enzymes following secretion into the digestive tract. The repeat sequences appear to have evolved by duplication of an ancestral amino acid domain, which may have arisen from the mitochondrial energy transfer proteins.

Allergens↗
PubMed1998

High-level expression of cockroach allergen, Bla g 4, in Pichia pastoris.

Exposure to cockroach allergens is a risk factor for allergic disease and has been linked to an increase in asthma morbidity among cockroach-sensitive inner-city children. Bla g 4 is a ligand-binding protein (or calycin) that causes IgE antibody responses in 40% to 60% of patients allergic to cockroaches. Recombinant Bla g 4 was expressed in Escherichia coli as an 18 kd protein but provided poor yields (only 0.25 mg/L culture). To improve yields, Bla g 4 was expressed in the Pichia pastoris yeast system as a 23 kd secreted protein at concentrations of 50 mg allergen/L. By cross-inhibition radioimmunoassay, Bla g 4 expressed in E. coli or P. pastoris provided overlapping inhibition curves. Both allergen preparations bound comparable levels of serum IgE antibody and showed similar skin test reactivity in individuals allergic to cockroaches (10[-1] to 10[-3] microg/ml). Deglycosylation of Pichia-expressed Bla g 4 with endoglycosidase F resulted in an 18 to 20 kd doublet, and liquid chromatography-mass spectrometry results suggested that the 20 kd band contained residual sugar residues. Both glycosylated and deglycosylated Pichia Bla g 4 showed comparable inhibition of IgE antibody binding in radioimmunoassay. Pichia-produced Bla g 4 had the same antigenic reactivity as that produced in E. coli, and glycosylation had no effect on IgE antibody binding. The high yield of Bla g 4 obtained in the Pichia system will facilitate studies on the structure and function of calycin allergens and on the immune response of asthma patients to cockroach allergens.

Allergens↗
PubMed1998

Immune-reactivity of recombinant isoforms of the major house dust mite allergen Der p 2.

BACKGROUND: Recombinant Der p 2, expressed in yeast, lacked reactivity with 5 monoclonal antibodies against natural Der p 2. OBJECTIVE: The aim of this study was to investigate whether the lack of reactivity with recombinant Der p 2 can be explained by the existence of isoforms. METHODS: By site-directed mutagenesis three recombinant isoforms of Der p 2 were produced. Reactivity with monoclonal antibodies and human IgE was analysed by means of RAST and RAST-inhibition. RESULTS: All five monoclonals that lacked reactivity with the originally selected isoform, showed reactivity upon replacement of aspartic acid by asparagine at position 114. The other two substitutions (at position 26 and 47) had no effect. Binding of human IgE (n = 10) was not significantly influenced by the isogenetic variation at position 114. CONCLUSIONS: Monoclonal antibodies raised against natural Der p 2 can sometimes discriminate between different isoforms, allowing the study of the natural occurrence of isoforms. For application in allergen-measurement assays, non-discriminating monoclonal antibodies should be selected.

Animals↗
PubMed1998

The effect of dry heat on mite, cat, and dog allergens.

BACKGROUND: Various techniques have been tried in an attempt to reduce allergen levels in homes. This study investigated the effect of dry heat on mite, cat, and dog allergens. METHODS: Samples (50 mg) of Dermatophagoides pteronyssinus and D. farinae cultures, and of house dust rich in the major cat and dog allergens Fel d 1 and Can f 1 were heated for 5, 10, 15, 30, and 60 min at 60 degrees, 80 degrees, 100 degrees, 120 degrees, and 140 degrees C. Control samples remained at room temperature. Extracts were assayed with the appropriate two-site mono- or mono/polyclonal sandwich ELISA. RESULTS: For Der p 1, the breakdown was proportional to temperature and heating time; after 30 min at 120 degrees C, allergen levels were reduced to < 1% of control. Der p 2 was more heat stable, requiring 140 degrees C for 30-60 min to achieve > 99% reduction. D. farinae groups 1 and 2 allergens showed results similar to those obtained with D. pteronyssinus. In contrast, Can f 1 and Fel d 1 were considerably more thermostable, with 50% and 70%, respectively, of allergen remaining after 60 min at 140 degrees C. CONCLUSIONS: The effect of dry heat on allergens increased with increasing time and temperature, cat and dog allergens demonstrating greater heat resistance than mite allergens. Dry heating methods may represent an alternative technique for removal of mite allergens; however, the greater stability of Fel d 1 and Can f 1 suggests that this procedure may not be appropriate for pet allergens.

Allergens↗
PubMed1998

Distribution, aerodynamic characteristics, and removal of the major cat allergen Fel d 1 in British homes.

BACKGROUND: Sensitisation to cat allergen (Fel d 1) is an important risk factor for asthma in the UK. A study was undertaken to investigate the distribution of cat allergen in British homes, the aerodynamic characteristics and particle size distribution of airborne Fel d 1, and the method of removing it. METHODS: Dust was collected from 50 homes with a cat and from 50 homes without a cat, and airborne levels of Fel d 1 were measured in 50 homes with a cat and 75 homes without a cat. Particle size distribution was determined using an Andersen sampler (8 hours/day) in 10 homes with cats. This was repeated on five separate days in a house with four cats, and then one, two, four, seven, and 14 days after the cats were removed from the living room area. The effect of high efficiency particulate air (HEPA) cleaner on airborne levels of Fel d 1 was investigated in seven homes with cats. Samples were collected on two separate days from two rooms of each house concurrently, one of which contained the cat, one day with the HEPA cleaner on and the other day as a control. Three one hourly samples were collected over a nine hour period (baseline, 4-5 hours, 8-9 hours) using a high volume dust sampler (air flow rate 60 l/min) and the air sample was collected onto a microglass fibre filter (pore size 0.3 micron). RESULTS: Fel d 1 concentrations were much lower in houses without a cat than in those with a cat (260-fold difference (95% CI 167 to 590) in living room carpets: geometric mean (GM) 0.9 microgram/g (range 0.06-33.93) versus 237 micrograms/g (range 2.8-3000); 314-fold difference (95% CI 167 to 590) in upholstered furniture: 1.21 micrograms/g (range 0.06-61.9) versus 380 micrograms/g (range 7.1-6000); 228-fold difference (95% CI 109 to 478) in bedroom carpets: 0.24 microgram/g (range 0.06-2.24) versus 55 micrograms/g (range 0.06-2304); and 215-fold difference (95% CI 101 to 456) in mattresses: 0.2 microgram/g (range 0.06-2.3) versus 55 micrograms/g (range 0.06-3400). Airborne levels of Fel d 1 were detected in all houses with cats, and the levels varied greatly between the homes (range 0.7-38 ng/m3). Low concentrations of airborne Fel d 1 (range 0.24-1.78 ng/m3) were found in 22 of 75 homes without a cat. Although airborne Fel d 1 was mostly associated with large particles (> 9 microns, approximately 49% of the allergen recovered), small particles (< 4.7 microns) comprised approximately 23% of the total airborne allergen. Total airborne Fel d 1 was reduced by 61.7% two days after removal of the cat but this was due predominantly to the decrease in larger particles (> 4.8 microns) which fell to 13% of their baseline level. Fel d 1 levels associated with small particles (< 4.8 microns) remained largely unchanged on days 1, 2 and 4 and then slowly decreased to 33% of the baseline levels at day 14. With HEPA cleaner a significant reduction in airborne Fel d 1 was observed compared with the control sampling (GM 5.04-0.88 ng/m3 versus 3.79-1.56 ng/m3 at baseline and 8 hours, active versus control group; p = 0.008). CONCLUSIONS: Airborne Fel d 1 was detectable in undisturbed conditions in all homes with cats and in almost a third of homes without cats. In houses with cats a significant proportion (23%) of airborne Fel d 1 was associated with small particles (< 4.7 microns diameter). Removal of the cat from the living room and bedroom areas of the home and the use of HEPA air cleaner reduced airborne levels of cat allergen in homes with cats, but the reduction following cat removal was not evenly spread across the particle size range.

Air Pollution, Indoor↗
PubMed1997

Expression and secondary structure determination by NMR methods of the major house dust mite allergen Der p 2.

There exists a strong correlation between asthma and sensitization to indoor allergens. This study reports on the secondary structure of the major house dust mite allergen Der p 2, determined using heteronuclear NMR methods. The DNA was subcloned from the yeast expression vector pSAY1 into the high yield bacterial expression vector pET21a, resulting in yields of 50 mg/liter. The recombinant protein was shown to have immunoreactivity comparable with that of the natural mite protein using competitive inhibition enzyme-linked immunosorbent assay (ELISA) and a modified monoclonal radioallergosorbent test (RAST). The secondary structure was determined by examining chemical shifts, short and long range NOESYs, JHN-HA coupling constants, and amide exchange rates. From these data, it is clear that Der p 2 is composed of beta-sheets and random coil. Based on long range distance constraints, a number of beta-strands were aligned into two three-stranded, anti-parallel beta-sheets.

Allergens↗
PubMed1997

Induction of IgE antibody responses by glutathione S-transferase from the German cockroach (Blattella germanica).

We report that a major 23-kDa allergen from German cockroach (Blattella germanica) is a glutathione S-transferase (EC 2.5.1.18; GST). Natural B. germanica GST, purified from cockroach body extracts by glutathione affinity chromatography, and recombinant protein expressed in Escherichia coli using the pET21a vector, showed excellent IgE antibody binding activity. B. germanica GST caused positive immediate skin tests in cockroach-allergic patients using as little as 3 pg of recombinant protein. The NH2-terminal sequence of the natural protein and the deduced amino acid sequence from cDNA were identical except for one substitution (Phe9 --> Cys). Assignment of this protein to the GST superfamily was based on binding to glutathione and sequence identity (42-51%) to the GST-2 subfamily from insects, including Anopheles gambiae and Drosophila melanogaster. B. germanica GST contained 18 of the 26 invariable residues identified in mammalian GST by x-ray crystallography and exhibited enzymic activity against a GST substrate. Our results show that cockroach GST causes IgE antibody responses and is associated with asthma. The data strongly support the view that the immune response to GST plays an important role in allergic diseases.

Allergens↗
PubMed1997

The role of domestic allergens.

The documented increase in asthma has been almost entirely in perennial asthma and a large proportion of the cases are allergic to one of the common allergens found all year round in houses, i.e. house dust mites, cats, dogs or cockroaches. In population and case-control studies sensitization to one of these allergens is the strongest risk factor for asthma (adjusted odds ratios > or = 4). Using monoclonal antibody-based assays for the major indoor allergens it has been shown that sensitization to house dust mites is directly related to the concentration of Group 1 mite allergen in dust. This led to the hypothesis that increases in mite allergen secondary to changes in houses were responsible for increases in asthma. However, asthma has also increased in areas of the world where mites do not flourish. In these dry areas sensitization to one of the other indoor allergens is the major risk factor for asthma. Although sensitization of asthmatics reflects the concentration of allergens in their houses, these measurements of exposure do not accurately predict severity of symptoms. Other factors that can contribute to the symptoms of asthma may also have increased. In particular, diesel particulates, ozone, beta 2-agonists, endotoxin and rhinovirus infection have each been shown to enhance the inflammatory response to inhaled allergens. Increases in asthma must relate to some aspect of our predominantly sedentary indoor lifestyle; this could be either increased exposure to allergens or an increase in factors that enhance the response of the lungs to foreign proteins.

Allergens↗
PubMed1997

Production of a mouse/human chimeric IgE monoclonal antibody to the house dust mite allergen Der p 2 and its use for the absolute quantification of allergen-specific IgE.

A chimeric human IgE monoclonal antibody was developed against the house dust mite allergen Der p 2. This chimeric antibody (hIgE-Dp2A) was composed of the heavy-chain variable domains and light chains of the original murine monoclonal antibody retaining its binding characteristics, whereas the heavy-chain constant domains were exchanged with the human IgE heavy chain. The chimeric IgE expression level was IgE 600 IU/ml (1 IU = 2.4 ng/ml). The binding of the chimeric hIgE-Dp2A to mite extract was indistinguishable from that of the original mouse monoclonal antibody. Parallel dose-response curves were found when the binding of hIgE-Dp2A to mite extract and anti-IgE coupled to sepharose were compared. Binding levels were not identical; however, hIgE-Dp2A bound significantly better to the mite-extract sepharose. This result indicates that the commonly used anti-IgE on solid phase calibration systems may lead to an overestimation of the amount of allergen-specific IgE present in the serum sample. The less efficient binding of the detector anti-IgE in case of the anti-IgE sepharose is likely to be because of the occupation of epitopes of the IgE by the sepharose-bound anti-IgE. Dose-response curves of serial dilutions of patient samples were parallel with the hIgE-Dp2A dose-response curve, which indicates that hIgE-Dp2A behaves like natural IgE antibodies in binding to allergen coupled to solid phase. This antibody is well suited for use as a reference reagent in the RAST and enables the expression of the amount of allergen-specific IgE present in a patient sample in absolute amounts.

Allergens↗