L. J. F. Youlten

Antibodies to purified bee venom proteins and peptides: I. Development of a highly specific RAST for bee venom antigens and its application to bee sting allergy

IgE antibodies to purified proteins and peptides from honeybee venom have been measured by the RAST. Trace amounts (less than 0.1%) of the major venom protein phospholipase A2 (PLA2) grossly distorted the measurement of IgE antibody to the other venom proteins, acid phosphatase (Acid P) and hyaluronidase (HYAL), and overemphasized their importance. Reduction of antigen coupled to the cellulose paper discs, which were used in the assay, diluted out the contaminating PLA2 without apparent loss in sensitivity. The reduction of disc-bound antigen increased the competition between IgE and IgG antibodies but did not affect measurement of IgE antibodies in sera taken from 35 untreated patients who had a history of general allergic reactions to bee stings. In 54% of sera from bee venom--allergic patients, the greatest IgE antibody response was to PLA2. In all, IgE antibodies to PLA2 were present in 91% of these sera. IgE antibodies to Acid P, HYAL, or melittin were present in 60%, 51%, and 31% of sera, respectively, and accounted for the highest level of binding in 17%, 17%, and 6% of these. Only 6% of sera were positive for whole venom but negative for the isolated antigens. A low level of IgE antibody was found to peptide 401 in 6% of sera. No IgE antibodies were found to apamin. While confirming the central role played by PLA2 in bee sting allergy, these results show that other venom components are also important in some patients.

The incidence and nature of adverse reactions to injection immunotherapy in bee and wasp venom allergy

The incidence, time course and nature of systemic reactions to injections of bee and wasp venom during immunotherapy have been estimated in an open, prospective, single centre study. One hundred and nine survivors of moderate to severe systemic reactions to stings from hymenoptera, received courses of bee or wasp venom by monthly subcutaneous injection for up to 3 years. Systemic reactions were recorded after 7.5% of 946 weekly venom injections during the initial phase of treatment, and after 2.1% of 1789 monthly maintenance injections. In both phases of treatment, reactions were more frequent after bee (17% of initial phase, 7.8% of maintenance treatment) than after wasp (3% of initial phase, 0.3% of maintenance treatment) venom injections. The percentage of patients experiencing at least one reaction was also higher for bee (46%) than for wasp (14%) sensitive patients. Over 80% of reactions began within 30 min of injection, over 90% within 1 h and only two (2%), between 1 and 2 h, the remaining six (5.5%) starting more than 2 h after injection. Only 0.47% of venom injections produced a systemic reaction which was severe enough to require adrenaline treatment. The female patients experienced more reactions (21% of the wasp, 60% of the bee, sensitive) than the males (5.5% wasp, 20% bee). Age and atopy did not appear to be significant risk factors for systemic reactions. We conclude that wasp and bee venom immunotherapy in a conventional dosage regimen was generally well tolerated. No safety advantage was apparent in keeping the patients under observation for more than 1 h after injections. Reactions are more likely to occur during the initial phase of treatment, in women and after bee, rather than wasp, venom injections.

Effect of aspirin in "aspirin sensitive" patients.

Eighteen patients with a history of urticaria or asthma, or both, induced by aspirin were studied before and after provocation of symptoms with aspirin. The plasma prostaglandin F2 alpha concentration, which was characteristically raised before challenge, fell significantly at the time of adverse reactions. Repeated administration of aspirin up to a dose of 650 mg daily induced tolerance in most of the patients, and several developed bronchodilator responses to aspirin. Although median total IgE concentrations may be raised in patients with aspirin sensitivity, it appears likely that pharmacological rather than immunological mechanisms are chiefly responsible for the phenomena of aspirin sensitivity and desensitisation.

The generation and cellular distribution of leukotriene C4 in human eosinophils stimulated by unopsonized zymosan and glucan particles

Human eosinophils (EOSs) stimulated under optimal conditions with 5 X 10(8) unopsonized zymosan particles at 37 degrees C for 30 minutes produced an average total immunoreactive leukotriene (LT) C4 of 1.6 ng per 10(6) EOSs, and 30% to 60% of the generated product remained cell associated. The dose-response characteristics of zymosan-induced LTC4 generation were different from those of phagocytosis, suggesting that the two events were independent. Pretreatment of EOSs with 10(-8) mol/L of formyl-methionyl-leucyl-phenylalanine for 30 minutes led to a twofold to fivefold augmentation of LTC4 generation by cells subsequently activated by unopsonized zymosan. Optimal EOS activation with 1 mumol/L of the calcium ionophore A23187 at 37 degrees C for 15 minutes produced more than 100 times greater quantities of LTC4 than with zymosan. The amount of immunoreactive LTC4 that remained cell associated after calcium ionophore A23187 stimulation reached a maximum after 5 minutes and then declined. Of the relatively small amount generated in the first minute, 71% was cell associated, but this figure declined to 9% after 15 minutes, by which time there had been a redistribution of the LTC4 to the supernatant. Inflammatory leukocytes may respond to zymosan because the cells recognize either one or both of its major polysaccharide components, glucan and mannan. Glucan, but not mannan, stimulated EOSs to generate LTC4 in a dose- and time-dependent manner. Under optimal conditions, there was no significant difference in the total quantities of LTC4 elaborated by EOSs stimulated by glucan and by unopsonized zymosan. This suggests that zymosan may induce leukotriene generation in the human EOS through a glucan recognition mechanism.

Skin and radioallergosorbent tests in patients with sensitivity to bee and wasp venom

Intradermal (ID) and prick tests with bee or wasp venom (Pharmalgen) have been performed on 102 subjects with a history of adverse reactions to stings and forty‐six control subjects giving no such history. Venom was diluted 100, 10 and 1 μg/ml for prick testing and 10‐2, 10‐2, 10‐3 and 10‐4%mUg/ml for ID injections.

Type I Allergy to Egg and Milk Proteins: Comparison of Skin Prick Tests with Nasal, Buccal and Gastric Provocation Tests

Provocation tests with egg or milk antigens were performed on symptomatic patients or those who were skin prick test positive to these antigens. Skin test positive patients responded immediately in 12/13 to nasal, in 7/15 to buccal and in 5/15 to gastric provocation tests. An immediate gastric response was within 1 h. The threshold dose for a positive result showed that tissue sensitivity from greatest to least was in the order: skin, nasal, buccal and gastric. None of the skin test negative group responded to any of the provocation tests. The results, particularly the nasal provocation, validate the skin prick test as a sensitive measure of type I allergy to defined foods. The relative insensitivity of buccal and gastric mucosae may explain positive skin test responses in asymptomatic subjects.

Histamine Release from Peripheral Blood Leukocytes with Purified Bee Venom Allergens: Effect of Hyperimmune Beekeeper Plasma

The response of 15 strongly bee-venom-allergic patients to highly purified venom allergens was compared using skin prick test titration, peripheral blood leukocyte (PBL) histamine release and radioallergosorbent test with three highly purified bee venom allergens: phospholipase (PLA2), hyaluronidase (HYAL) and acid phosphatase (ACID P). Sensitivity to the three allergens ranked in the same order for all three tests and in each case PLA2 was found to the most potent allergen. In the presence of hyperimmune beekeeper plasma, maximum histamine release was reduced significantly for all three allergens (p less than 0.001). Furthermore, hyperimmune beekeeper plasma increased the amount of allergen required for a comparable release of histamine (mean shift in dilution curve PLA2 917-fold; HYAL, 492-fold; ACID P, 61-fold). The release of histamine from whole blood was also compared with PBL + 10% normal human serum (NHS). For all three allergens maximum release was much lower from whole blood compared with washed cells + 10% NHS (p less than 0.001). These data confirm PLA2 as the major bee venom allergen by all three tests. Hyperimmune beekeeper plasma reduces maximum histamine release and increases its threshold. Histamine release in response to ACID P appears harder to block with hyperimmune beekeeper plasma than that provoked by PLA2 or HYAL (p less than 0.01). Whole blood releases less histamine and requires more allergen than washed cells, indicating that sensitivity of PBL in vivo is unlikely to be as great as washed PBL in vitro.

Specific desensitization in ‘aspirin-sensitive’ urticaria; plasma prostaglandin levels and clinical manifestations

Six out of eight patients with a history of aspirin‐provoked urticaria/angioedema responded with adverse reactions, including urticaria and bronchospasm, to provoking doses of oral aspirin from 30‐515 mg. The other two patients did not react to 1.2 g of aspirin on three occasions. Five of the six patients who had reacted became desensitized after their initial aspirin reaction, tolerating 650 mg on the second day. They then took 650 mg day−1 of aspirin for three weeks, during which time the ingestion of foods which had previously caused a variety of moderate or severe reactions caused no symptoms. The resting plasma PGF2α in ten ‘aspirin‐sensitive’ urticaria patients (24.89 + 2‐79 pg m−1) was significantly higher than the levels in ten normal subjects (6.75 + 1‐1 pg ml−1) (P < 0.01). In the patient group the lowest levels of PGF2x were found in the two patients who subsequently did not experience a positive reaction after aspirin provocation. The PGF2α/PGE2 ratio in ‘aspirin‐sensitive’ urticaria patients (1‐83 + 0.026) was significantly higher than that in normal subjects (0‐63 + 0.14) (P < 0.01).

Clinical and Biochemical Aspects of "Aspirin-Sensitivity"

Progressively increasing doses of aspirin (acetylsalicylic acid--ASA) were tolerated by 14 out of 15 patients with confirmed aspirin-sensitive urticaria and in 7 out of 9 patients with aspirin-sensitive asthma. Blood levels of histamine and prostaglandin (PG) F2 alpha were significantly raised in these patients before ASA administration. PGF2 alpha levels fell to within the normal range after challenge doses of ASA which were sufficient to cause symptoms. Skin prick testing with histamine and codeine phosphate did not show evidence of abnormal tissue reactivity or mast cell reactivity. A wider spectrum of mediators will need to be considered if the mechanism of symptom production is to be understood.