Biomedical subjects
Publications and source records attributed to R E Esch.
Allergen immunotherapy plays an important role in the treatment of allergic diseases and asthma. This article is a brief review of the current approaches, including patient and allergen selection, routes of administration, and use of standardized allergen vaccines. New approaches offering potentially useful strategies based on recent studies of T-cell epitopes, cytokines, and anti-IgE and DNA vaccines also are considered.
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Allergen extracts are prepared from a wide variety of source materials including pollens, fungi, arthropods, animal danders, foods, and dusts. The composition of allergen extracts can vary depending on the allergen source, manufacturing process, and storage conditions. Allergen-specific immunoglobulin E (IgE) assays and skin tests employ a variety of allergen-containing reagents that confer specificity on the test. Given that the allergen source materials are heterogenous mixtures of proteins, glycoproteins, carbohydrates, and other substances that are not allergenic, it is not unexpected that variability exists between test results obtained with different allergen-containing reagents. Variability within a single manufacturer's allergen product can be controlled by using reproducible extraction and processing procedures, single large lots of allergen source materials, and solid-phase supports. These controls do not, however, ensure the consistency of products between manufacturers or laboratories because allergen source materials, manufacturing procedures, and acceptance criteria for allergen reagents may vary.
This report documents the mapping of the second major epitope, previously described as site D, of grass group I allergens to residues 23-35 of meadow fescue group I (STFYGKPTGAGPK). Mapping was accomplished by screening fractions from a meadow fescue group I tryptic digest for peptide(s) that inhibit the ability of monoclonal antibody 24.64 (specific for site D) to bind to immobilized group I allergen. One such peptide, representing residues 23-35 of meadow fescue group I, was identified. Additional studies with the use of synthetic analogs of this peptide demonstrate that it binds mAb 24.64 directly. Examination of extracts containing group I glycoproteins from seven other species of grass confirms antigenic cross-reactivity due to this peptide. We also report based on protein sequence analysis that the amino terminal segment (which includes the site D epitope) of GpI allergens from seven different grass species is highly conserved and contains two hydroxyproline residues and an N-linked carbohydrate moiety.
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OBJECTIVE: To compare cutaneous reactivity to insect and arachnid allergens in clinically normal (control) and allergic dogs in the southeastern United States. DESIGN: Prospective, controlled study. ANIMALS: 26 clinically normal dogs and 82 allergic dogs from the southeastern United States. PROCEDURE: Intradermal skin testing with various dilutions of 13 insect and arachnid allergens was performed on control dogs to establish skin threshold concentrations (ie, concentrations to which < 25% of the dogs had positive reactions). These established threshold concentrations were then used to test allergic dogs for reactivity. Prevalence of single and multiple insect and arachnid reactions were determined. RESULTS: Flea allergen was the only allergen that caused a significantly higher prevalence of positive reactions in allergic dogs than in control dogs. CLINICAL IMPLICATIONS: Flea hypersensitivity is the most important arthropod hypersensitivity in dogs. The importance of reactivity to insect and arachnid allergens other than flea allergen can be determined only when prevalence of positive reactivity has been determined in an appropriate regional control group of dogs.
OBJECTIVES: To determine whether flea extract could be determined (via ELISA) to share allergenic epitopes with other insects, and to determine whether sera with different reactivities to insect extracts have different cross-reactivity patterns. SAMPLE POPULATION: 69 canine serum samples that were selected from samples submitted for routing ELISA allergy testing and had previously been found to have high reactivities to flea. PROCEDURE: Each serum sample was assessed by means of a direct ELISA for IgE binding to 11 common insects. Samples that were reactive primarily to flea extract alone were designated pool 1, samples that were reactive to small numbers of insects were designated pool 2, and samples that were reactive to all or almost all insects were designated pool 3. Samples that did not have any apparent patterns of cross-reactivity were not included in the rest of the study. Inhibition ELISA techniques were used with the 3 serum pools to determine whether multiple insect extracts inhibited reactivity on flea-coated ELISA plates. Those extracts were used to coat ELISA plates, and reciprocal inhibition studies were then performed. RESULTS: Black fly, black ant, and cockroach extracts were capable of > 50% inhibition of flea solid-phase IgE binding with all 3 serum pools. In the ELISA inhibition studies, flea extract was able to inhibit IgE binding to each extract with all pools, confirming reciprocal inhibition. CONCLUSIONS: Inhibition of IgE binding to solid-phase flea antigen by black ant, black fly, and cockroach extracts suggested sharing of allergenic epitopes among these species. Reciprocal inhibition studies further confirmed these findings. These results indicated in vitro cross-reactivity between flea, black ant, black fly, and cockroach extracts. These results need to be further investigated in vivo. CLINICAL RELEVANCE: It is possible that dogs may become sensitized to fleas via exposure to other insects, and flea allergenic dogs may have signs of pruritus, in the absence of fleas, if exposed to cross-reactive insects.
OBJECTIVE: To determine the effect of mold extracts with high protease activity on the biological activity of allergenic tree, grass, and weed extracts, using in vivo and in vitro methods, in atopic dogs. ANIMALS: 15 dogs with history and clinical signs of atopy. All dogs had strong positive reactions (3+ or 4+) to 1 or more preselected allergens and negative reactions (O) to molds. PROCEDURE: Mold extracts and saline solution were coincubated separately with tree, grass, and weed pollen extracts at 4 C for 30 and 80 days. Skin end-point titration (30-day incubation) and ELISA inhibition (30- and 180-day incubations) tests were performed on all samples. The biological activity of pollen extracts coincubated with mold extracts was compared with that of pollen extracts coincubated with saline solution. RESULTS: In the skin end-point titration test, weed pollen extracts coincubated with a mixed mold extract lost a statistically significant amount of biological activity, compared with saline coincubated controls. In the ELISA inhibition test, grass and weed pollen extracts incubated with a mixed mold extract lost a significant amount of biological activity, compared with saline coincubated controls. A significant correlation in the measurement of biological activity was found between a loss of end-point dilution in the skin end-point titration test and a decrease in relative potency, as measured by the ELISA inhibition test for allergenic grass and weed extracts. CONCLUSION AND CLINICAL RELEVANCE: Mold proteases can decrease the biological activity of certain grass and weed pollen extracts when coincubated in the same vial for 30 days. Separation of mold and pollen extracts, when preparing immunotherapy vaccines, may help prevent loss of pollen extract potency and increase the vaccine's stability and efficacy.
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Epitopes recognized by five mAb which block the binding of human IgE antibodies to grass group I (GpI) Ag were characterized and partially mapped. Site specificity studies defined four apparently non-overlapping blocking antibody binding sites on the meadow fescue GpI molecule, Fes e I. One of these sites (site A) was localized to a 14,000 m.w. fragment designated P3 generated by CNBr cleavage of purified Fes e I. The P3 peptide possessed human IgE binding sites as well as other epitopes (non-site A) defined by 19 other anti-GpI mAb. All of the P3 reactive antibodies recognized cross-reactive determinants found on GpI Ag isolated from five different grasses suggesting that P3 is a conserved portion of grass GpI molecules. The P3 fragment from Fes e I was used to immunize mice and induced antibodies which reacted with intact GpI Ag from all 5 different grasses currently being studied in this laboratory.
A series of peptides has been isolated from the Group I (GpI) allergen from five species of grasses currently under study in this laboratory. These peptides were generated by performing a limited tryptic digestion on each extensively reduced and alkylated GpI allergen and fractionating the digest by molecular sieve, ion-exchange and HPLC reverse-phase chromatography. Material in the elution profiles was assayed with a monoclonal antibody known to cross-react with all GpI allergens in this study. In addition, this particular monoclonal antibody has been previously shown to inhibit the ability of human IgE to bind to these GpI allergens. The peptides identified and isolated in this way were found to be highly homologous to one another.
The kinetics, quantitative yield, and sequence of solute release during the extraction of allergenic substances from short ragweed (Ambrosia artemisiifolia) pollen were compared with a conventional batch-type method and the novel technique of pollen grain column chromatography. With the batch method, 14.6 +/- 1.7 mg of pollen solutes were eluted per 100 mg of dried defatted pollen in 1 minute; the 24-hour solute yield was 27.4 +/- 2.7 mg. With the column method, 3.7 +/- 1.3 mg of pollen solutes were eluted in 1 minute; the 24-hour solute yield was 29.3 +/- 2.1 mg. The kinetics of solute release with the column method were modeled as the simultaneous first-order elution of ragweed-pollen solutes into three hypothetical compartments. The theoretical initial solute concentration was 50 gm/L. The isoelectric focusing patterns, optical properties, distributions of enzymes, Ra5, and antigen E activities were consistent with the sequential separation of ragweed-pollen solutes and the three compartment model. Enzyme activities were eluted either maximally in the first minute (phosphatases and N-acetyl-beta-glucaminidase) or delayed until 10 minutes (leucine aminopeptidase). Ra5 was eluted rapidly, whereas antigen E was eluted during a more prolonged period. Pollen grain chromatography provides a simple, reproducible method for studying pollen solute release.
Yeast phase Candida albicans (ATCC No. 10231) was grown in a nonantigenic medium, harvested and lyophilized. Ammonium sulfate fractions of an aqueous extract of the lyophilized cells were evaluated and the fraction yielding the highest specific delayed cutaneous reactivity in sensitized guinea-pigs was used to prepare a C. albicans skin test antigen (CASTA). The safety of the antigen was evaluated by measuring immediate and delayed (0.25, 6, 24, 48 and 72 h) cutaneous reactions in atopic and nonatopic human subjects. The outcome of three repetitive monthly Mantoux skin tests with 0.01-1 microgram antigen doses was used to test for booster effects in 14 subjects and to estimate a safe initial test antigen dose. The utility of a single skin test as a measure of cell-mediated immunity was evaluated in 40 healthy subjects. Reactor rates (greater than or equal to 2 mm, 48 h) of 40% and 85% were detected, respectively, with doses of 0.0316 and 1 microgram. Using a skin test reaction diameter greater than or equal to 5 mm at 48 h, the reactor rate was 50% for the 1-microgram dose. The only adverse reaction (45 mm, 0.25 h) was detected with the 1-microgram dose in an atopic subject who also exhibited exquisite scratch test reaginic hypersensitivity to C. albicans allergen. The prevalence of other adverse reactions to this antigen compared favorably with that to other antigens used for recall antigen testing. These studies suggest the 1-microgram CASTA dose can be used for effective, safe recall antigen skin tests.
As part of a study to probe the immunochemical basis for allergenic cross-reactivity among grass pollens, a series of useful reagents has been prepared. The major grass-pollen allergen, designated group I (GpI), was isolated from five grass pollens (meadow fescue, June grass, sweet vernal grass, redtop grass, and perennial ryegrass). The purified GpI antigens were used to immunize individual groups of BALB/c mice. A total of 123 hybridoma-derived anti-GpI monoclonal antibodies (mAbs) was produced. These mAbs were used to evaluate the antigenic relationship among the GpI antigens by means of two types of ELISA. The experiments revealed a high level of epitope diversity and demonstrated a wide range of antibody specificities. A cross-reactivity ELISA was used to identify and compare antigenic determinants on the GpI molecules, and it was possible to define mAbs with specificities unique for the immunizing allergen and other mAbs that cross-reacted with one or more other members of the test panel of allergens. These murine mAbs reflect the range of specificities present in sera from grass-allergic individuals.
It has been well documented, worldwide, that inhalation and/or contact with airborne particulate insect products has resulted in sensitivity to insect proteins and is manifested by such common entities as dermatitis, conjunctivitis, rhinitis, and asthma. However, the deliberate ingestion of a variety of insects (undertaken to prove their edibility and nutrient value) resulted in subsequent sensitization of some individuals. Such an outcome has not previously been reported in the literature. The objective was to document the anaphylactic reaction to the purposeful ingestion of mealworm in an individual known to be sensitized to the inhalation of beetle larvae. We used the occasion of the Centennial Celebration of The New York Entomological Society to expose members and guests of the Society to the ingestion of various insects. The subjects of the study consisted of: 1) Three members were adversely affected; 2) One individual with Baker's asthma; and 3) A number of controls with no known hypersensitivity to insect products. The investigation was undertaken by food challenges, inhalation challenges, skin testing to the individual insect allergens, a) Tenebrio molitor (TM), b) Zophobas morio (ZM), c) Blattella germanica (BG), skin testing to common indoors and outdoor allergens, and direct bind ELISA and ELISA inhibition. One individual manifesting hypersensitivity both by ingestion and inhalation to mealworm was identified. This sensitivity was documented clinically as well as by objective testing.