Michael J. Schumacher

Allergy to murine antigens in a biological research insitute

Symptomatic and immunologic responses to allergens from laboratory mice were studied in a research institute. Subjects who had been exposed to mice and 50 unexposed subjects were studied by questionnaire and by prick tests with seven prevalent aeroallergens and allergens from mouse urine and pelts. Of the 121 exposed subjects, 39 (32.2%) had respiratory, ocular, or cutaneous symptoms after exposure to mice; occurrence of these symptoms correlated with positive skin tests to purified mouse urinary proteins (MUP) and pelt allergens from CBA/H mice. Serum levels of IgG antibodies correlated with the frequency of mouse exposure. In subjects with seasonal allergic rhinitis, nasal symptoms from exposure to mice, positive prick tests to MUP, and IgE antibodies to MUP were significantly more prevalent. The possibility of genetic influences on susceptibility to mouse allergy were also suggested by a negative association between the incidence of HLA-DRW6 and positive prick-test responses to urinary proteins from C57BL and BALB/c mice among the 54 subjects who were exposed to mice and tested for DR locus antigens (p = 0.05). However, no significant differences in any of the loci studied could be shown in subjects with and without nasal symptoms from exposure to mice.

Pulmonary response to nasal-challenge testing of atopic subjects with stable asthma

Nasal deposition of allergen or histamine could cause bronchoconstriction in subjects with asthma by a reflex mechanism. To investigate this possibility, six atopic subjects with stable asthma and five normal control subjects were studied by nasal-challenge testing with saline, Bermuda grass-pollen allergen, and histamine on paper disks. Challenges were done on 3 separate days by use of a double-blind, randomized trial design. Fivefold serial dilutions were used to determine threshold doses for provocation of at least a twofold increase in the postsaline nasal airway resistance. No patient developed cough or wheezing or required treatment at the end of the nasal-challenge tests, even when persistent sneezing was provoked or in the subject who had a dual nasal response to nasal challenge. In spite of changes in nasal airway resistance that generally conformed to expectations, there were no significant effects of nasal challenge on lung function, including forced expiratory flow rates, vital capacity, residual volume, and partial expiratory flow-volume curves.

Rate and quantity of delivery of venom from honeybee stings

To determine the rate and completeness of delivery of venom from honeybee stings, European bees were collected at the entrance of a hive and studied with the use of two laboratory models. In one model bees were induced to sting the shaved skin of anesthetized rabbits. The stings were removed from the skin at various time intervals after autotomization, and residual venom was assayed with a hemolytic method. In the other model the bees were induced to sting preweighed filter paper disks, which were weighed again after removal of the sting at various intervals. Results of both experiments were in agreement, showing that at least 90% of the venom sac contents were delivered within 20 seconds and that venom delivery was complete within 1 minute. The data suggest that a bee sting must be removed within a few seconds after autotomization to prevent anaphylaxis in an allergic person. The extensive variation found in the amount of venom delivered at each time point may explain inconsistencies in relationships among reactions to field stings, sting challenge testing, venom skin tests and RAST.

Characterization of the major allergen of Cynodon dactylon (Bermuda grass) pollen, Cyn d I

An allergen from Cynodon dactylon (Bermuda grass) pollen, Cyn d I, has been purified by a combination of concanavalin A-Sepharose affinity chromatography, and carboxymethyl-Sepharose chromatography. The allergen constitutes the major allergenic component of the pollen extract as observed by immunoelectrophoretic techniques, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an IgE-inhibition experiment, and skin testing. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Cyn d I is presented as a dominant 32 kd band and a minor 29 kd band, both binding IgE. Both bands are identified by monospecific rabbit antibodies (Abs) raised against Cyn d I. These Abs only weakly precipitate allergens from other grass species, indicating that Cyn d I possesses some unique immunochemical properties. Two of four purified murine monoclonal Abs raised against Cyn d I also bind to both bands of Cyn d I, indicating that the bands represent isoallergens with slightly different immunochemical properties. All four monoclonal Abs cross-react with pollen components from other grass species, especially Poa pratensis and Dactylis glomerata. The NH2-terminal sequence corresponding to approximately 10% of the complete sequence was determined, and it revealed high homology to the corresponding sequence of the major allergen of Lolium perenne, Lol p I. From the amino acid composition determination and immunoelectrophoretic comparison, the amount of Cyn d I in the source whole-pollen extract was estimated to be 15% wt/wt.

Recognition of pollen and other particulate aeroantigens by immunoblot microscopy

Most grass-pollen types appear identical by normal light microscopy. Restricted antigenic cross-reactivity of Cynodon dactylon (Bermuda grass) pollen allowed development of a new method to identify antigens associated with grass-pollen grains. Pollen applied to the surface of an adhesive tape was blotted onto nitrocellulose, and the blots were identified by anti-Bermuda-grass antibodies, second antibody, and fluorescence microscopy. Of the 44 species of grass pollen studied for specificity of the method, the only species to demonstrate uniformly bright staining were Cynodon dactylon, Elymus triticoides, Elymus cinereus, and Koeleria cristata. Thirty-one species were negative, and nine other species demonstrated occasional brightly fluorescent spots, suggesting contamination. The immunoblotting method was used to study Tucson air collected continuously by a Burkard pollen and spore trap throughout April 1986. Each 2-hour transect of the adhesive tape from the trap was examined by immunoblotting and by normal light microscopy to compare antigen particle counts with grass-pollen counts. The mean antigen-particle concentration, 52.8/m3 of air, was higher than the mean grass-pollen concentration, 21.9/m3 of air, suggesting presence of amorphous Bermuda-grass antigens in air samples. Antigen-particle concentration, not grass-pollen concentration, correlated significantly with wind velocity, temperature, and time of day.

Nasal dyspnea: The place of rhinomanometry in its objective assessment

Background “Nasal dyspnea” describes the common symptom of nasal airway obstruction, which usually is assessed subjectively. Objective, quantitative methods are needed to assess the nasal airway adequately. Methods Rhinomanometry, in use for >30 years, was reviewed. Results No other symptom of rhinitis can be measured as objectively and accurately as that of nasal obstruction. Determinations by rhinomanometry are precise, usually better than 5%. Most rhinomanometers meet the published requirements of standardization by the International Rhinologic Society. An improved system for scoring symptoms of nasal dyspnea is suggested. Rhinomanometry, potentially a gold standard for the objective measurement of obstructed nasal airflow, has a proven place in the selection of patients for septal and nasal valve reconstruction. Conclusion Recommendations are given for extension of the standards to account for the near universal computerization of equipment. Miniaturization of equipment will increase its application to trials of drugs for rhinitis.

An immunoelectrophoretic analysis of the allergens of Cynodon dactylon (Bermuda grass) pollen

A crude extract of Cynodon dactylon (Bermuda grass) pollen was obtained by extraction, centrifugation, dialysis, and lyophilization. The corresponding polyspecific rabbit antibodies were obtained by immunization, bleeding, and purification and were used for a cross immunoelectrophoretic analysis of the crude extract. At least 52 antigens (Ags), of which 47 migrated toward the anode, and five, toward the cathode, were revealed. Crossed radioimmunoelectrophoretic analysis performed with sera from 32 patients allergic to Bermuda grass and a pool from 1000 normal individuals revealed specific IgE binding to 13 of these Ags. The approximate molecular weights (MWs) for 10 of these IgE-binding Ags were determined by a combination of gel filtration and immunochemical analysis. These Ags had apparent MWs greater than 14 kd. A combination of preparative sucrose gradient isoelectric focusing and immunochemical analysis was used to determine the approximate isoelectric point values of five of the IgE-binding Ags. Most precipitating Ags had isoelectric points between 4.5 and 5.5. Four of the Ags (Ag 24, MW, less than 65 kd; Ag 31, MW, 33 kd; Ag 33, MW, 20 kd; and Ag 34/35, MW, 32 kd) were classified as major allergens.

Stability of fresh and preserved fetal and adult lymphocyte cell surface markers

Delay between blood collection and immunophenotyping of peripheral blood (PB) and umbilical cord blood (UCB) lymphocytes occurs frequently. Holding media address this problem, but there are few reports of their limitations. We tested the ability of Cyto‐Chex (CC) to preserve the ability of lymphocyte subpopulations to be immunophenotyped after time delays. Ten UCB and 10 PB specimens were kept up to 48 hr and then placed in CC for 1 week, with removal of aliquots for staining with CD3, CD4, CD8, CD19, and CD45 at intervals. There were no significant changes in the number of nonpreserved PB or UCB cells stained with any of the reagents after 48 hr. Aside from a small decrease in UCB B‐cell numbers, there were no changes in UCB or PB lymphocyte numbers after placing them in CC for 1 week. However, PB and UCB CD19+ cells and CD8+ T cells lost capacity for bright staining after 1 week in CC. The results suggest that UCB and PB can be held for up to 48 hr before being placed in CC and then kept for up to 1 week in CC with no decrease in T‐cell numbers and only a minor reduction of B‐cell numbers, albeit with a marked reduction in staining in‐ tensity. J. Clin. Lab. Anal. 14:320–326, 2000.

Airborne contact dermatitis from Ambrosia deltoidea (triangle-leaf bursage)

3 cases of contact dermatitis due to exposure to airborne allergens from Ambrosia deltoidea (triangle‐leaf bursage) are presented. Although airborne plant dermatitis was suggested in each case by a history of aggravation of the rash by outdoor exposure and typical skin lesions, patch testing with an oleoresinous extract of A. deltoidea leaves identified the source of the sensitizer, not previously reported. As A. deltoidea is a ragweed that probably contains sesquiterpene lactones nearly identical to those of ragweeds widely prevalent in most of North America, the patients presented indicate the need for commercially available, standardized, plant extracts for patch testing in this country. The finding of positive patch tests with filters from an air sampler placed in 1 patients domestic environment suggests a new avenue of investigation in airborne contact dermatitis.

The preparation and testing of the proposed International Reference (IRP) Bermuda grass (Cynodon dactylon)-pollen extract.

A lyophilized candidate extract of Bermuda-grass (Cynodon dactylon) pollen, intended for use as an International Reference Standard, was prepared by pooling four individual candidate extracts. In preliminary investigations, the four candidate extracts encompassed a variety of extraction methods. The collaborative testing program simultaneously analyzed the proposed reference and the individual extracts and included 11 laboratories performing RAST inhibition, histamine release, crossed immunoelectrophoresis, crossed radioimmunoelectrophoresis, isoelectric focusing, sodium dodecyl sulfate gel electrophoresis, and protein determinations with a variety of reagents and methods. The four candidate extracts and the pooled reference were found to be equivalent. The stability of this extract has also been studied. This International Reference Preparation of Bermuda grass-pollen extract should be useful for research and industry.