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Biomedical subjects

Martin D Chapman

Publications and source records attributed to Martin D Chapman.

At least 19 recordsLinked to original sources
PubMed2006

High-throughput fluorescent multiplex array for indoor allergen exposure assessment.

BACKGROUND: Current enzyme immunoassay methods for detection of common indoor allergens in environmental dust samples are labor-intensive and time consuming. OBJECTIVE: To develop and validate a fluorescent multiplex array to measure 6 (Der p 1, Der f 1, Der p 2, Der f 2, Fel d 1, and Can f 1) indoor allergen levels simultaneously. METHODS: A multiplex array for 6 allergens, using mAbs covalently coupled to fluorescent microspheres, was developed using a single universal standard composed of purified natural allergens. The multiplex array was validated by comparing the measured dust mite, cat, and dog allergen levels in household dust samples to those obtained by standard ELISA methods. RESULTS: Linear regression analysis showed a highly significant quantitative correlation between the multiplex array and ELISA for dust mite, cat, and dog allergens: R(2) values ranging from 0.90 to 0.99 (P < .001). In addition, the sensitivity, limit of detection (<0.1 ng/mL), reproducibility, intra-assay coefficient of variance (<5%), and interassay coefficient of variance (<25%) of the fluorescent multiplex array were shown to be equal to or better than the ELISA method. CONCLUSION: A multiplex array has been developed to measure simultaneously 6 indoor allergens from a single sample. The array will facilitate epidemiologic studies and indoor air quality assessments and can, in principle, be expanded to include other allergens and biologics. CLINICAL IMPLICATIONS: The multiplex array lends itself to clinical studies, population-based environmental surveys, and allergen avoidance studies comparing allergen exposure in large populations over several time points.

Air Pollution, Indoor↗
PubMed2006

Nomenclature and structural biology of allergens.

Purified allergens are named using the systematic nomenclature of the Allergen Nomenclature Sub-Committee of the World Health Organization and International Union of Immunological Societies. The system uses abbreviated Linnean genus and species names and an Arabic number to indicate the chronology of allergen purification. Most major allergens from mites, animal dander, pollens, insects, and foods have been cloned, and more than 40 three-dimensional allergen structures are in the Protein Database. Allergens are derived from proteins with a variety of biologic functions, including proteases, ligand-binding proteins, structural proteins, pathogenesis-related proteins, lipid transfer proteins, profilins, and calcium-binding proteins. Biologic function, such as the proteolytic enzyme allergens of dust mites, might directly influence the development of IgE responses and might initiate inflammatory responses in the lung that are associated with asthma. Intrinsic structural or biologic properties might also influence the extent to which allergens persist in indoor and outdoor environments or retain their allergenicity in the digestive tract. Analyses of the protein family database suggest that the universe of allergens comprises more than 120 distinct protein families. Structural biology and proteomics define recombinant allergen targets for diagnostic and therapeutic purposes and identify motifs, patterns, and structures of immunologic significance.

Air Pollution, Indoor↗
PubMed2006

Peanut allergen exposure through saliva: assessment and interventions to reduce exposure.

BACKGROUND: Exposure to food allergens through saliva (kissing, utensils) can cause local and systemic allergic reactions. OBJECTIVE: To determine the time course of peanut allergen (Ara h 1) persistence in saliva after ingestion of peanut butter and to evaluate mouth cleansing interventions to reduce salivary peanut allergen. METHODS: Thirty-eight individuals ingested 2 tablespoons of peanut butter, and saliva was collected at various time points. At another time, samples were collected after 5 interventions (brushing teeth, brushing and rinsing, rinsing, waiting then brushing, waiting then chewing gum). Detection of Ara h 1 was performed by a monoclonal-based ELISA (detection limit, 15-20 ng/mL). RESULTS: Salivary Ara h 1 varied considerably immediately after ingestion, but included levels expected to invoke reactions (as much as 40 microg/mL). Most (87%) subjects with detectable peanut after a meal had undetectable levels by 1 hour with no interventions. None had detectable levels several hours later after a peanut-free lunch. This result indicates (95% confidence) that 90% would have undetectable Ara h 1 in saliva under these circumstances. All of the interventions reduced salivary Ara h 1, in some cases by >95%, but Ara h 1 remained detectable in approximately 40% of samples (though typically below thresholds reported to induce reactions). CONCLUSION: Patients with peanut allergy require counseling regarding risks of kissing or sharing utensils, even if partners have brushed teeth or chewed gum. Advice to reduce risks, though not as ideal as total avoidance, includes waiting a few hours plus eating a peanut-free meal. CLINICAL IMPLICATIONS: Waiting several hours and ingesting a peanut-free meal were more effective at reducing salivary peanut protein concentration than simple, immediate interventions.

Adolescent↗
PubMed2006

Bla g 6: a troponin C allergen from Blattella germanica with IgE binding calcium dependence.

BACKGROUND: The known cockroach allergens do not appear to account for the full repertoire of IgE responses. OBJECTIVE: To identify and investigate the importance of other Blattella germanica allergens contributing to cockroach allergy. METHODS: A B germanica cDNA library was screened with pooled sera from patients with cockroach allergy. Three isoallergens of troponin C (Bla g 6) were cloned and expressed in Pichia pastoris. Homology modeling was performed by using Swiss-Model. IgE responses to purified allergens were simultaneously measured in 104 sera by using a fluorescent multiplex array system. The effect of calcium on IgE binding was investigated by ELISA. RESULTS: Three isoallergens, Bla g 6.0101, Bla g 6.0201, and Bla g 6.0301, were identified which share homology with insect troponin Cs and vertebrate calmodulins (61% to 78% and 42% to 44% amino acid identity, respectively) and have 2 EF-hand calcium binding domains. Molecular models of Bla g 6 showed 2 structurally homologous lobes connected by a linker that confers flexibility to the allergen. The prevalence of IgE binding to recombinant Bla g 6 was 14%. Calcium depletion by 10 mmol/L ethyleneglycol-bis-(beta-aminoethylether)-N,N,N',N'-tetraacetic acid did not significantly affect IgE binding in most cases. Interestingly, addition of 10 mmol/L CaCl2 after calcium depletion increased IgE binding by approximately 2-fold, a finding not previously reported for calcium binding allergens. CONCLUSION: Bla g 6 is a troponin allergen with a calcium dependent IgE reactivity that may be involved in muscle contraction. CLINICAL IMPLICATIONS: Bla g 6 homologous allergens may occur among other insects and cause cosensitization or allergenic cross-reactivity.

Allergens↗
PubMed2005

Crystal structure of cockroach allergen Bla g 2, an unusual zinc binding aspartic protease with a novel mode of self-inhibition.

The crystal structure of Bla g 2 was solved in order to investigate the structural basis for the allergenic properties of this unusual protein. This is the first structure of an aspartic protease in which conserved glycine residues, in two canonical DTG triads, are substituted by different amino acid residues. Another unprecedented feature revealed by the structure is the single phenylalanine residue insertion on the tip of the flap, with the side-chain occupying the S1 binding pocket. This and other important amino acid substitutions in the active site region of Bla g 2 modify the interactions in the vicinity of the catalytic aspartate residues, increasing the distance between them to approximately 4A and establishing unique direct contacts between the flap and the catalytic residues. We attribute the absence of substantial catalytic activity in Bla g 2 to these unusual features of the active site. Five disulfide bridges and a Zn-binding site confer stability to the protein, which may contribute to sensitization at lower levels of exposure than other allergens.

Amino Acid Sequence↗
PubMed2005

Specific IgE and IgG antibody-binding patterns to recombinant cockroach allergens.

BACKGROUND: The specificity of serum antibody responses to different cockroach allergens has not been studied. OBJECTIVE: We sought to quantitate serum IgE and IgG antibodies to a panel of purified cockroach allergens among cockroach-sensitized subjects. METHODS: IgE antibodies to recombinant cockroach allergens (rBla g 1, rBla g 2, rBla g 4, rBla g 5, and rPer a 7) were measured in sera containing IgE antibodies to Blattella germanica extract (n = 118) by using a streptavidin CAP assay and a multiplex flow cytometric assay. Specific IgG antibodies were determined by using radioimmunoprecipitation techniques. RESULTS: Specific IgE antibodies measured by means of CAP assay and multiplex assay were strongly correlated ( r = 0.8, P < .001). The sum of IgE antibodies (in international units per milliliter) against all 5 allergens equated to IgE antibodies to cockroach extract. Although the prevalence of IgE antibodies was highest for rBla g 2 (54.4%) and rBla g 5 (37.4%), patterns of IgE antibody binding were unique to each subject. Surprisingly, only 16% of cockroach-sensitized subjects with IgE antibodies to house dust mite exhibited IgE antibody binding to cockroach tropomyosin (rPer a 7). Specific IgE antibodies were associated with increased IgG antibody levels, although detection of IgG in the absence of IgE was not uncommon. CONCLUSION: The techniques described offer a new approach for defining the hierarchy of purified allergens. IgE antibodies directed against 5 allergens constitute the majority of the IgE antibody repertoire for cockroach. Such distinct patterns of IgE-IgG responsiveness to different cockroach allergens highlight the complexity of B-cell responses to environmental allergens.

Adolescent↗
PubMed2005

Cockroach allergen Bla g 2: an unusual aspartic proteinase.

BACKGROUND: Enzymatic activity of mite, fungal, and bee venom allergens is thought to potentiate their allergenicity. Bla g 2 is a potent cockroach allergen, but despite sharing sequence homology with aspartic proteinases, it contains critical amino acid substitutions that impair proteolytic activity. The biologic function of Bla g 2 remains unclear. OBJECTIVE: We sought to investigate the effects of specific amino acid substitutions on enzymatic activity, and the peptide-binding capability of Bla g 2. METHODS: Site-directed mutagenesis was used to produce a recombinant Bla g 2 mutant (Mut) with corrected canonical triads and a flap region. Another mutant (MutF - ) was expressed after additional mutations in the flap region of Mut. Bla g 2 wild-type (Wt), Mut, and MutF - were assayed for aspartic proteinase activity, and Bla g 2 Wt was tested for pepstatin binding. RESULTS: Recombinant Bla g 2 Wt and Mut did not show enzymatic activity in a milk-clotting and hemoglobin assay. By using a modified hemoglobin assay, residual activity inhibited by pepstatin was detected for MutF - and Wt at 20 microg/mL, whereas pepsin was active at a 1000-fold lower concentration. Most of Bla g 2 binding to pepstatin-agarose was nonspecific. CONCLUSION: Residual proteolytic activity was found for Bla g 2 at concentrations of approximately 4 mM. This weak activity suggests that proteolysis is not the primary function of this allergen and that it is unlikely to contribute to the allergenicity of Bla g 2. Bla g 2 has a cleft that might specifically bind ligands other than pepstatin.

Amino Acid Sequence↗
PubMed2005

A prospective study of wheezing in young children: the independent effects of cockroach exposure, breast-feeding and allergic sensitization.

The objective of this study was to evaluate risk factors for persistent wheezing in a group of 2-4-year-old children after an index-wheezing episode in infancy. Eighty infants who had been seen at the Emergency Department for an episode of acute wheezing were followed for 2 yr in this prospective study. Caregivers completed a questionnaire, and children underwent clinical evaluation and skin prick testing 2 yr following the index-wheezing episode. Detection of respiratory viruses and analysis of exposure to major indoor allergens were carried out at enrollment. Immunoglobin E antibodies were measured at the beginning of the study and at the end of follow-up, using the CAP system. Logistic regression analysis was performed to identify factors associated with persistent wheezing. Seventy-three children (44 boys) completed the study. After 2 yr, 38 (52%) reported three or more wheezing episodes in the past 12 months (persistent wheezers). Independent risk factors for persistence of wheezing were allergic sensitization and exposure to cockroach allergen in the kitchen. Breast-feeding for at least 1 month was a protective factor. A strong association between allergic sensitization and persistence of wheezing was found in a group of very young children living in a subtropical area.

Allergens↗
PubMed2004

Exposure to indoor allergens in homes of patients with asthma and/or rhinitis in southeast Brazil: effect of mattress and pillow covers on mite allergen levels.

BACKGROUND: Exposure and sensitization to indoor allergens have been associated with the development of asthma and other allergic diseases in many parts of the world. It is important to establish the degree of exposure, and to evaluate whether allergen control measures are effective in a particular area. METHODS: Concentrations of major allergens of mites, cockroach, cat and dog were measured in dust samples from homes of 24 mite-allergic patients living in Ribeirão Preto, Brazil. Allergen concentrations were quantitated by monoclonal antibody-based ELISA. Mite-impermeable mattress and pillow covers were applied to beds in the homes of 19 of the 24 patients, and group 1 mite allergen levels were measured 1 and 6 months following intervention. RESULTS: Patients were exposed to high concentrations of mite allergens in their homes. 87.5% of the homes presented concentrations of group 1 allergens >10 microg/g of dust in at least one site. Cockroach allergen concentrations were low in most samples. Dog allergen concentrations were significantly higher in homes with dogs as compared to those without dogs. Mean concentrations of cat allergen were 0.1 microg/g. Mite allergen concentrations in bedding samples dropped from 24 microg/g at baseline to 0.9 and 1.0 microg/g, respectively, 1 and 6 months following encasement of mattresses and pillows. CONCLUSIONS: Significant mite allergen reduction can be achieved in an area of high degree of exposure to mite allergens. Further studies evaluating the effect of this reduction on symptoms and airway inflammation will be necessary to establish the effectiveness of mite avoidance measures in our area.

Adolescent↗
PubMed2004

Risk factors for wheezing in a subtropical environment: role of respiratory viruses and allergen sensitization.

BACKGROUND: Risk factors for acute wheezing among children in subtropical areas are largely unknown. OBJECTIVE: To investigate the role of viral infections, allergen sensitization, and exposure to indoor allergens as risk factors for acute wheezing in children 0 to 12 years old. METHODS: One hundred thirty-two children 0 to 12 years of age who sought emergency department care for wheezing and 65 children with no history of wheezing were enrolled in this case-control study. Detection of respiratory syncytial virus antigen, rhinovirus and coronavirus RNA, adenovirus, influenza, and parainfluenza antigens was performed in nasal washes. Total IgE and specific IgE to mites, cockroach, cat, and dog were measured with the CAP system. Major allergens from mites, cockroach, cat, and dog were quantified in dust samples by ELISA. Univariate and multivariate analyses were performed by logistic regression. RESULTS: In children under 2 years of age, infection with respiratory viruses and family history of allergy were independently associated with wheezing (odds ratio, 15.5 and 4.2; P = .0001 and P = .008, respectively). Among children 2 to 12 years old, sensitization to inhalant allergens was the major risk factor for wheezing (odds ratio, 2.7; P = .03). High-level allergen exposure, exposure to tobacco smoke, and lack of breast-feeding showed no association with wheezing. CONCLUSIONS: Some risk factors for wheezing previously identified in temperate climates were present in a subtropical area, including respiratory syncytial virus infection in infants and allergy in children older than 2 years. Rhinovirus was not associated with wheezing and did not appear to be a trigger for asthma exacerbations.

Allergens↗
PubMed2004

Distribution of peanut allergen in the environment.

BACKGROUND: Patients with peanut allergy can have serious reactions to very small quantities of peanut allergen and often go to extreme measures to avoid potential contact with this allergen. OBJECTIVE: The purpose of this study was to detect peanut allergen under various environmental conditions and examine the effectiveness of cleaning agents for allergen removal. METHODS: A monoclonal-based ELISA for Arachis hypogaea allergen 1 (Ara h 1; range of detection, 30-2000 ng/mL) was used to assess peanut contamination on cafeteria tables and other surfaces in schools, the presence of residual peanut protein after using various cleaning products on hands and tabletops, and airborne peanut allergen during the consumption of several forms of peanut. RESULTS: After hand washing with liquid soap, bar soap, or commercial wipes, Ara h 1 was undetectable. Plain water and antibacterial hand sanitizer left detectable Ara h 1 on 3 of 12 and 6 of 12 hands, respectively. Common household cleaning agents removed peanut allergen from tabletops, except dishwashing liquid, which left Ara h 1 on 4 of 12 tables. Of the 6 area preschools and schools evaluated, Ara h 1 was found on 1 of 13 water fountains, 0 of 22 desks, and 0 of 36 cafeteria tables. Airborne Ara h 1 was undetectable in simulated real-life situations when participants consumed peanut butter, shelled peanuts, and unshelled peanuts. CONCLUSION: The major peanut allergen, Ara h 1, is relatively easily cleaned from hands and tabletops with common cleaning agents and does not appear to be widely distributed in preschools and schools. We were not able to detect airborne allergen in many simulated environments.

Adult↗
PubMed2004

Environmental detection of mouse allergen by means of immunoassay for recombinant Mus m 1.

BACKGROUND: Mouse urinary allergens are an important cause of occupational asthma in animal facilities. Domestic exposure to mouse allergens is a risk factor for asthma among inner-city residents. OBJECTIVE: We sought to develop a sensitive and specific assay for assessing environmental mouse allergen exposure. METHODS: An ELISA for recombinant (r)Mus m 1 was developed by using rabbit polyclonal antibodies to rMus m 1 that were affinity purified against the natural allergen. Assay specificity was established by means of immunoblotting and ELISA. Mus m 1 levels in mouse, other mammalian allergenic products, and house dust samples from inner-city homes were compared. RESULTS: Polyclonal antibodies to Mus m 1 showed a single 20-kd band on immunoblots against rMus m 1 and male mouse urine. Parallel dose-response curves were obtained by using mouse urine extract and natural Mus m 1 or rMus m 1. Mus m 1 was detected in mouse allergenic products (0.10-10.0 microg/mL) and in gerbil allergenic products (0.1 microg/mL) but was less than the limit of detection in epithelial extracts from 10 other animal species. Environmental measurements showed an excellent correlation between Mus m 1 levels in house dust extracts from inner-city asthma studies by using 2 different Mus m 1 standards (n=22; r=0.99; P <.001). CONCLUSIONS: A highly sensitive ELISA has been developed with rMus m 1. This assay is suitable for monitoring domestic and environmental exposure to mouse urinary allergens.

Allergens↗
PubMed2004

Prediction of residential pet and cockroach allergen levels using questionnaire information.

We assessed the accuracy of questionnaire reports of cat and dog ownership and presence of cockroaches in predicting measured allergen concentrations in house dust. We collected dust samples in the homes of 932 newborns living in New England. Dust samples were taken from the main living area and the infant's bedding. Allergen content of house dust was measured by enzyme-linked immunosorbent assays (ELISA) and related to questionnaire information on past and current cat and dog ownership and presence of cockroaches. Allergen levels were dichotomized using the limit of detection and the following cut points: 1.0 microg/g and 8.0 microg/g for cat, 2.0 microg/g and 10.0 microg/g for dog, and 2 U/g and 8 U/g for cockroach allergen. For the upper cut point, both specificity and sensitivity of questionnaire-reported cat and dog ownership and presence of cockroaches were high. For the limit of detection and lower cut point, specificity was high (> 80%), whereas sensitivity was low, particularly for current cat and dog ownership (21-60%). Taking pet ownership during the preceding 2 years into account increased the sensitivity by 10%, but it remained relatively poor. In conclusion, questionnaire-reported pet ownership and presence of cockroaches predicts allergen levels above the upper cut point but is a relatively poor measure of allergen exposure above the limit of detection and the lower cut point. Knowledge of past pet ownership can improve pet allergen exposure assessment by means of questionnaire. However, for epidemiologic purposes, measured concentrations of allergens are necessary.

Adult↗
PubMed2003

Monitoring peanut allergen in food products by measuring Ara h 1.

BACKGROUND: Peanut allergy is an important health problem in the United States, affecting approximately 0.6% of children. Inadvertent exposure to peanut is a risk factor for life-threatening food-induced anaphylaxis. OBJECTIVE: The purpose of this investigation was to develop an immunoassay for a major peanut allergen, Ara h 1, to detect peanut allergen in foods so that the risk of inadvertent exposure can be reduced. METHODS: A specific 2-site monoclonal antibody-based ELISA was developed to measure Ara h 1 in foods. The sensitivity of the assay was 30 ng/mL. Ara h 1 was measured in foods (n = 83) with or without peanut and in experiments to optimize allergen yield and to determine peanut contamination in spiked foods. RESULTS: Ara h 1 levels in food products ranged from less than 0.1 microg/g to 500 microg/g. Ara h 1 measured in ng/mL was transformed to microg/g for food products. Peanut butter contained the highest amounts of Ara h 1. Peanut extracts contained from 0.5 to 15 mg Ara h 1/g of peanut depending on the extraction conditions. Optimal extraction of Ara h 1 was obtained by using phosphate buffer with 1 mol/L NaCl and Tween at 60 degrees C. Ara h 1 was not always detected in presence of chocolate under the extraction conditions tested. Spiking experiments showed that the assay could detect approximately 0.1% Ara h 1 contamination of food with ground peanut. There was an excellent correlation between Ara h 1 levels and peanut content measured by using a commercial polyclonal antibody-based ELISA (r = 93, n = 31, P <.001). CONCLUSION: A new sensitive and specific monoclonal antibody-based ELISA was used to monitor Ara h 1 content in food products. This assay should be useful for monitoring peanut contamination in the food manufacturing and processing industry and in developing thresholds for sensitization or allergic reaction in persons with peanut allergy.

Allergens↗
PubMed2002

Cockroach allergen Bla g 2: structure, function, and implications for allergic sensitization.

Exposure to German cockroach (Blattella germanica) allergens is associated with the development of chronic respiratory diseases, especially asthma. The mechanism by which allergic patients develop specific immunoglobulin E (IgE) responses to environmental allergens is unknown. However, recent reports provided evidence that enzyme activity, especially proteolytic activity, was a major contributor to allergenicity. Bla g 2 is one of the most potent cockroach allergens (prevalence of IgE responses of 60 to 80%) and shows homology to the aspartic proteinase family of enzymes. We investigated whether the allergenicity of Bla g 2 was linked to its putative enzymatic function. A molecular model of Bla g 2, based on the high resolution crystal structures of pepsin and chymosin, showed that the overall three-dimensional structure of Bla g 2 was similar to that of aspartic proteinases with a well-defined binding pocket. However, critical amino acid substitutions in the catalytic triads and in the "flap" region of the molecule suggested that Bla g 2 was inactive and homologous to mammalian pregnancy-associated glycoproteins. This was confirmed experimentally by enzyme assay. The results show dissociation between enzymatic activity and allergenicity for Bla g 2 and suggest that other genetic and environmental factors are important determinants of sensitization.

Amino Acid Sequence↗