Manuel Lopez

A two-site monoclonal antibody ELISA for the quantification of the major Dermatophagoides spp. allergens, Der p I and Der f I

A two-site monoclonal antibody (Mab) ELISA was developed to measure the Group I allergens from Dermatophagoides spp., Der p I from D. pteronyssinus and Der f I from D. farinae. Species-specific Mabs were used to coat microtiter plates which were then incubated with allergen or house dust extracts. Bound allergen was detected using a biotinylated Mab which recognized a common epitope on both Der p I and Der f I, followed by the addition of streptavidin-peroxidase and ABTS/H2O2 substrate. The assay had low non-specific binding (approximately 0.08 absorbance units) and had a sensitivity of 5 ng/nl for aqueous allergen extracts (equivalent to 0.1 microgram allergen/g dust). 53 dust samples were assayed using the Mab ELISA and an RIA previously described using 125I-labelled Mab. The results showed a very good quantitative correlation between the assays (r = 0.96, p less than 0.001 for Der p I; r = 0.92, P less than 0.001 for Der f I). A further 132 dust samples from a different geographical areas were also assayed by both methods and gave correlation coefficients of 0.90 (P less than 0.001) and 0.86 (P less than 0.001) for Der p I and Der f I, respectively. The Mab ELISA will be useful in epidemiological studies of allergic asthma, both in the assessment of levels of dust mite allergen present in houses and the efficacy of allergen avoidance regimes.

Prevalence of dust mites in the homes of people with asthma living in eight different geographic areas of the United States.

The density and species prevalence of dust mites were determined at various times over a 5-year-period in 252 homes of dust mite sensitive people with asthma who lived in eight geographic areas of the United States (Cincinnati, Ohio; New Orleans, La.; Memphis, Tenn.; Galveston, Texas; Greenville, N.C.; Delray Beach, Fla.; San Diego and Los Angeles, Calif.). The most common dust mites found in the homes were Dermatophagoides farinae (DF), D. pteronyssinus (DP), Euroglyphus maynei (EM), and Blomia tropicalis. All homes in all locations contained Dermatophagoides spp. mites, but few homes were populated exclusively by either DF or DP alone. Most homes (81.7%) were coinhabitated by both DF and DP. In coinhabited homes one species was predominant and usually made up at least 75% of the total mite population. Prevalence of the dominant or only species present varied between homes within a geographic area. EM occurred in significant numbers in 35.7% of homes in New Orleans, Memphis, Galveston, Delray Beach, and San Diego. Blomia tropicalis occurred in these same cities but in low densities. For all dust samples, only 13 homes of the 252 sampled had 100 or fewer mites/gm dust, which is considered to be the threshold for sensitivity. Most homes had average mite densities of 500 or more mites/gm dust. The results of the present study suggest a significant and widespread occurrence of both DF and DP. Therefore extracts of both mite species should be considered for diagnostic tests and immunotherapy. Significant levels of EM were present in some areas. Thus sensitivity to EM should be considered in these areas.

Basidiomycete mycelia and spore-allergen extracts: Skin test reactivity in adults with symptoms of respiratory allergy

One hundred fifty adults, with respiratory-allergic disease, and 14 control subjects, without symptoms of respiratory allergy, were skin prick tested with 16 common inhalant allergens, 12 extracts of mycelia from Basidiomycetes grown in vitro, and/or 10 to 15 basidiospore extracts. Eighty-three subjects (58%) had positive skin tests to two or more of the common inhalant allergens. Twenty-seven percent of the study subjects had positive skin reactions to one or more of the Basidiomycete mycelia extracts, and 32% demonstrated positive skin reactions to one or more basidiospore extracts. None of the 14 control subjects had positive skin reactivity to basidiospore extracts. Skin prick reactivity of study subjects to 15 different basidiospore extracts ranged from 5% for Cantharellus cibarius to 17% for Scleroderma sp. The prevalence of skin test reactivity to basidiospores did not differ significantly from the reactivity to commercial mold extracts of several common species of the Fungi Imperfecti (6% for Cladosporium herbarum or Penicillium notatum to 13% for Alternaria tenuis). These results demonstrate that a significant number of individuals reporting symptoms of respiratory allergy have skin test reactivity to basidiospores and suggest that these spores are important fungal aeroallergens in the New Orleans environment.

IgE antibody response to vertebrate meat proteins including tropomyosin

BACKGROUNDnAlthough meat is a main source of proteins in western diets, little information is available regarding allergy to vertebrate meats or the allergens implicated in these reactions.nnnOBJECTIVEnTo evaluate the in vitro IgE antibody response to different vertebrate meats in suspected meat-allergic subjects, as well as the possible role of tropomyosin in meat allergy and to analyze the cross-reactivity between vertebrate meats and the effect of heating on the IgE-binding to meat proteins.nnnMETHODSnFifty-seven sera from suspected meat-allergic subjects were tested by grid blot to extracts of beef, lamb, pork, venison, chicken, and turkey and to four mammalian tropomyosins of different origins.nnnRESULTSnMeat-allergic subjects have IgE antibodies to proteins in different mammalian meats (43/57 subjects); cross-reactivity with avian meat was limited: less than 50% (19/43) of meat positive sera reacted to chicken. In contrast, most of the poultry-positive sera also reacted to different mammalian meats. In general, there was stronger IgE reactivity to raw meats in comparison to cooked meats; an exception was six cases in which IgE reactivity to cooked poultry was stronger. Weak IgE reactivity to tropomyosin was detected in only 2/57 sera tested.nnnCONCLUSIONSnSuspected meat-allergic subjects have serum IgE directed to meat proteins. In vitro cross-reactivity among mammalian meats appears to be important, while cross-reactivity to poultry is limited indicating mammalian-specific proteins. Although cooking in general denatures meat proteins rendering them less allergenic, in some cases the process of cooking may result in the formation of new allergenic moieties. The muscle protein tropomyosin is not an important vertebrate meat allergen.

Identification of bovine IgG as a major cross-reactive vertebrate meat allergen.

Background: Although beef is a main source of protein in Western diets, very little has been published on allergic reactions to beef or the main allergens implicated in these reactions. The aim was to evaluate the IgE antibody response to beef in suspected meat‐allergic subjects and assess cross‐reactivity of beef with other vertebrate meats.

Allergenicity and immunogenicity of basidiomycetes

Species selected from six families of the class Basidiomycetes were evaluated for allergenicity in atopic and nonatopic individuals and for immunogenicity and antigenic cross-reactivity in experimental animals. Between 42% and 68% of atopic asthmatics demonstrated positive Type 1 wheal-and-flare skin reactivity to basidiomycete metabolic and somatic antigens. Sixty-four percent of skin test-positive atopic asthmatics exhibited positive RAST to as basidiomycete metabolic antigen and 50% were positive to somatic antigen. Negative RAST results were obtained in all nonatopic control sera. Only an occasional individual demonstrated positive serum antibasidiomycete precipitins by counterimmunoelectrophoresis. All basidiomycete species studied were highly immunogenic in the rabbit and most appeared to contain an electrophoretically heterogenous group of antigens with predominant anodal mobility. Ouchterlony double-diffusion analysis employing hyperimmune rabbit antisera indicated the presence of shared antigens among all basidiomycete species with the exception of Pleurotus. Rabbit antibasidiomycete sera did not cross-react with antigens of several common species of the Fungi imperfecti. Results indicate that many atopic asthmatics of the Gulf South area demonstrate IgE-medicated hypersensitivity to basidiomycete antigens as evidenced by positive wheal-and-flare skin reactivity and/or RAST. Basidiomycetes are also immunogenic in the rabbit and possess antigens that do not cross-react with those of certain Fungi imperfecti.

Bronchoprovocation studies in basidiospore-sensitive allergic subjects with asthma

Bronchoprovocation challenge with basidiospore extracts was performed in eight subjects with asthma and with positive wheal-and-flare skin reactivity and RAST to basidiospore allergens. Five of the eight patients demonstrated a significant decrease in FEV1 ranging from 20% to 47% after basidiospore-extract challenge. Both immediate and late-phase reactivity was observed. Six atopic control subjects with asthma and with negative skin reactivity to basidiospore extracts did not exhibit significant bronchospasm after basidiospore challenge. These results demonstrate that basidiospore extracts can induce bronchospasm in subjects with asthma who demonstrate IgE antibodies to basidiospore allergens.

Neutrophil chemotactic activity in toluene diisocyanate (TDI)-induced asthma

We quantitated serum neutrophil chemotactic activity (NCA), which is associated with mast cell or basophil activation, to determine if mast cell or basophil mediators are released during bronchoprovocation-inhalation challenge with subirritant levels of toluene diisocyanate (TDI). Four subjects with suspected TDI-induced asthma and four mite-sensitive subjects with asthma who served as a comparison group were studied. NCA was measured in a multiwell, microchemotaxis chamber. Blood samples were collected, and FEV1 measurements were performed before challenge and at regular intervals during the subsequent 24 hours. Three of four workers clinically sensitive to TDI reacted to a subirritant TDI exposure. There was no increase in NCA during placebo challenges. NCA increased in the three TDI-sensitive workers during early and late asthmatic reactions in quantities proportional to the FEV1 decline. No increase in NCA was found during TDI exposures in the TDI-negative worker. Gel filtration analysis demonstrated the main NCA fraction eluted with macromolecules of an estimated molecular weight greater than 440,000 daltons. This characteristic is compatible with neutrophil chemotactic factor of basophil or mast cell origin. The kinetics of NCA release were similar in mite- and TDI-induced asthmatic reactions. A high correlation (r = 0.97; p = 0.0006) was obtained between the percent decrease in FEV1 during early asthmatic reactions and percent increase in NCA. These observations support the hypothesis that activation of mast cells or basophils is associated with TDI-induced early and late asthmatic reaction.

Occupational asthma caused by high-molecular-weight substances.

More than 250 agents that are encountered in the workplace have been shown to induce asthma in susceptible individuals. It is estimated that 2% to 15% of cases of asthma may be occupational. High-molecular-weight substances, such as plant and animal proteins, enzymes, and large carbohydrate molecules, can induce IgE-mediated occupational asthma. The incidence of disease varies among industries and is dependent on the physiochemical properties of the agent, the level and duration of exposure, industrial hygiene, engineering practices, and host factors. Risk factors, common high-molecular-weight workplace antigens, diagnosis, treatment, and prognosis are discussed.

Lymphocyte proliferative response and tissue distribution of methylmercury sulfide and chloride in exposed rats

The immunotoxic effects and tissue distribution of different forms of methylmercury compounds were studied in rats. Methylmercury sulfide or methylmercury chloride was fed to rats at concentrations of 5 or 500 microg/L in drinking water for 8 wk. T-cell lymphocyte proliferative response to phytohemagglutinin (PHA) and determination of tissue distribution of mercury by gas chromatography using electron capture were assayed. Four different forms of mercury compounds were employed: MeHgS-, (MeHg)2S, (MeHg)3S+, and MeHgCl. Results indicated that exposure to methylmercury significantly enhanced lymphocyte responsiveness in most of the exposed groups at the low concentration of 5 microg/L, with the highest proliferative response (fourfold increase) in the MeHgCl group. At 500 microg/L, a significant decrease in the lymphocyte proliferative response was observed in the (MeHg)3S+ and MeHgCl groups; conversely, the MeHgS(-)- and (MeHg)2S-exposed animals had a modest increase of the lymphocyte proliferative response. The largest concentrations of all four mercury forms were detected in the kidney and spleen. The levels of mercury found in kidney, spleen, liver, brain, and testis were lower in the MeHgCl group than in those exposed to (MeHg)2S and (MeHg)3S+. These data indicate that the organ distribution of mercury and immune alteration may vary according to the chemical structure of the compound. This observation may have important implications in humans potentially exposed to low levels of methylmercury present in the environment, since the immune system plays an important regulatory role in the host-defense mechanisms.