Analysis of differing patterns of cross-reactivity of honeybee and yellow jacket venom-specific IgE: use of purified venom fractions.

Prior studies of sera from insect sting-allergic patients have analyzed the relationship of coexisting honeybee venom- and yellow jacket venom-specific IgE. Radioallergosorbent (RAST)-inhibition tests with these venoms revealed four different patterns of activity. In this present study, purified fractions prepared from these venoms were used to analyze these varying patterns. The hyaluronidases of yellow jacket venom and honeybee venom showed extensive cross-reaction. The phospholipases from these venoms showed minimal cross-reactivity; antigen 5 was restricted to yellow jacket venom. There was a high molecular weight component in yellow jacket venom with immunologic properties similar to honeybee venom acid phosphatase. Sera from individual patients showed quantitative and qualitative differences in the reactions to the major components of both venoms. The differences in the RAST-inhibition patterns in patients with elevated levels of both honeybee venom- and yellow jacket venom-specific IgE are accounted for by these differences as well as by differences in the cross-reactivity between the individual components.

Further studies of patients with both honeybee- and yellow-jacket-venom-specific IgE.

Twenty-five sera from patients with high titers of both honeybee- and yellow-jacket-venom-specific IgE were analyzed in RAST inhibition experiments, using each venom as the coupling and inhibiting antigen. Eight sera had unique antibody activity with no cross-reactivity between yellow-jacket- and honeybee-venom-specific IgE. In 5 sera, the IgE antibody activity was directed at a major allergen in yellow jacket venom cross-reacting with a minor allergen in honeybee venom. Honeybee venom inhibited only the honeybee venom RAST; yellow jacket venom inhibited both the honeybee and the yellow jacket venom RAST. Five sera showed the opposite pattern with IgE antibody directed at a major allergen in honeybee venom cross-reacting with a minor allergen in yellow jacket venom. The fourth newly observed pattern was that of extensive IgE antibody cross-reaction found with 7 sera. Both honeybee venom and yellow jacket venom inhibited both the honeybee and yellow jacket venom RASTs. There were no clinical features such as age, sex, atopy or type of anaphylactic symptoms which could distinguish patients in each group. All but one patient had a history of multiple sting exposures. These data suggest multiple allergens in honeybee and yellow jacket venom with differing patterns of cross-reactivity and have important implications for proper venom immunotherapy.

Local intranasal immunotherapy with high-dose polymerized ragweed extract.

Thirty-one ragweed-allergic patients received preseasonal local intranasal immunotherapy (LNIT) with high doses of gluteraldehyde-polymerized ragweed extract (average total dose 544 micrograms antigen E). Minimal side effects were reported during treatment and did not interfere with the dosing schedule. During the ragweed pollen season, LNIT-treated patients had lower symptom scores for sneezing, rhinorrhea and nasal congestion than a comparable group of untreated ragweed-allergic patients. There was no difference in ragweed-induced eye symptoms between the two groups. Secretory ragweed-specific IgA and IgG rose following LNIT treatment. Absolute antibody titers and changes in titers did not correlate with clinical improvement. LNIT with the polymerized ragweed did not block the seasonal rise in serum ragweed-specific IgE. These results suggest that LNIT with high-dose polymerized ragweed extract is a safe, simple and effective form of immunotherapy.

Local nasal immunotherapy: efficacy of low-dose aqueous ragweed extract.

In previous studies preseasonal local nasal immunotherapy (LNIT) with moderate doses of aqueous ragweed extract (mean total dose 59 micrograms of AgE and 139 micrograms of AgE) was an effective treatment for ragweed hay fever; however, local adverse reactions during therapy were common. This study evaluated the clinical and immunologic responses to LNIT by use of lower doses of aqueous ragweed extract in order to minimize these adverse reactions. Patients were administered preseasonal LNIT for 7 wk and received a mean total dose of 4.7 micrograms of AgE. During the ragweed season, symptom/medication scores (SMS) of the treated patients were equivalent to SMS of untreated patients. Serum ragweed-specific IgE and nasal secretory ragweed-specific IgA rose slightly in the treated patients but not to the extent observed in previous studies. After the ragweed season treated and untreated patients had a substantial increase in serum ragweed IgE antibody titers. No correlation could be found between antibody responses and SMS. This study indicates that LNIT with lower doses of aqueous ragweed extract is clinically ineffective.

Comparison of measurements of serum venom-specific IgG by radioimmunoassay and enzyme immunoassay.

Honeybee venom (BV)-, yellow jacket venom (YJV)- and honeybee venom phospholipase A (PLA)-specific IgG were measured by radioimmunoassay (RIA) and enzyme immunoassay (RIA) in 54 sera of insect-allergic patients. In the majority of sera, results of the two tests were similar. Discordant results were found in 1 of 21 sera in tests with PLA, 4 of 23 sera in tests with BV, and 1 of 16 sera in tests with YJV. The RIA measured higher antibody titers in 5 of these 'outlier' sera. The results suggest that with a few exceptions, the two assays are similar for detection of venom- and PLA-specific IgG.

Allergenic components of bald-faced hornet (V. maculata) venom.

Bald-faced hornet (V. maculata) venom collected by electrical stimulation was fractionated using molecular exclusion gel-filtration and ion-exchange chromatography. Four fractions were selected for in-depth analysis. These were analyzed for phospholipase, hyaluronidase and protease enzyme activities, antigenicity as measured by reaction with anti-hornet venom rabbit serum, and allergenic activity, as determined by RAST reaction with sera from hornet-sensitive patients. The results of these studies suggest that the fractions containing phospholipase, hyaluronidase and protease possess allergenic activity. In addition, there appear to be other allergenic components in hornet venom. Allergic patients differ in their reactivity to the various allergenic components in hornet venom and may have IgE antibodies to one or more of these components.

Further evaluation of local intranasal immunotherapy with aqueous and allergoid grass extracts.

In a double-blind study, 45 grass-allergic patients received local nasal immunotherapy (LNIT) with either aqueous mixed-grass extract, formaldehyde-treated, mixed-grass extract (allergoid), or histamine placebo. Twenty-four patients received LNIT for a second successive year, and 21 patients received LNIT for the first year. The aqueous extract-treated patients had significantly lower symptom-medication scores than either allergoid- or placebo-treated subjects. There was no difference in symptom-medication scores in patients receiving allergoid and placebo treatment or in patients receiving 1 and 2 yr of LNIT. The aqueous extract stimulated a rise in serum grass-specific IgE. There was no serum or local antibody response after allergoid-extract treatment. Postseasonal rises in serum-IgE titers were observed in all three groups. These data suggest that LNIT with aqueous mixed-grass extract significantly reduces the symptoms of allergic rhinitis. The allergoid grass extract was ineffective in the second year of treatment. No cumulative effect of LNIT could be demonstrated in successive years of therapy.

Studies of coexisting honeybee and vespid-venom sensitivity.

Honeybee and vespid venom-specific IgE were measured by RAST in randomly selected sera of 87 patients who had had anaphylactic reactions after insect stings. Overall there was a poor correlation between the titers of honeybee venom and yellow jacket or hornet venom-specific IgE. Sera from nine patients with high titers of both honeybee venom and yellow jacket venom-specific IgE were selected for RAST-inhibition studies, with these two venoms as coupling and inhibiting antigens. Three patterns of IgE-antibody specificity were detected. Four patients had unique antibody activity with no cross-reactivity between the yellow jacket and honeybee venom-specific IgE. This is probably the most common pattern in patients with dual sensitivity. Three patients reacted to a major allergen in yellow jacket venom cross-reacting with a minor allergen in honeybee venom. Their RAST-inhibition patterns demonstrated that the yellow jacket-venom RAST was inhibited by yellow jacket venom only and the honeybee-venom RAST was inhibited by both yellow jacket venom and honeybee venom. Two patients had the opposite pattern with honeybee-venom RAST inhibited by honeybee venom only and the yellow jacket RAST inhibited by both honeybee venom and yellow jacket venom. These latter patients reacted to a major allergen in honeybee venom that was cross-reacting with a minor allergen in yellow jacket venom. Studies with rabbit antisera raised to vespid and honeybee venoms demonstrated major antigens that were unique to each family that did not cross-react and several minor cross-reacting antigens.

Comparison of the biochemical, immunologic and allergenic properties of vespid venoms collected in early and late summer.

The biochemical, immunologic and allergenic properties of yellow hornet (Vespula arenaria) and bald-faced hornet (Vespula maculata) venoms collected in early and late summer were compared. The phospholipase A content of both hornet venoms decreased in late summer while protease, hyaluronidase and acid phosphatase contents were unchanged. The antigenic and allergenic properties of the two venoms, as measured by their reaction with rabbit antisera and sera from insect-allergic patients, respectively, were unchanged. These results suggest no changes in venom properties during the summer which influence the allergic response to insect stings.

Use of dialyzed venoms in the radioallergosorbent test.

Optimal concentrations of honeybee, hornet and yellow jacket venoms were used in the radioallergosorbent test (RAST) before and after dialysis. RAST titers in the majority of sera from insect-allergic patients increased significantly with the dialyzed yellow jacket and hornet venom-coupled discs. Of 26 sera which had negative RASTs with crude venom, 20 became positive with dialyzed venom. No change in RAST results was found with crude and dialyzed honeybee venom. The low molecular weight component in the vespid venoms removed by dialysis had no specific activity in RAST inhibition experiments, suggesting nonspecific blocking of binding sites. These results indicate the importance of the appropriate antigen preparation coupled to discs in the RAST.

Local nasal immunotherapy for ragweed-allergic rhinitis. III. A second year of treatment.

In 1979, pre-seasonal local nasal immunotherapy (LNIT) was found to be an effective treatment for ragweed hay fever. In 1980, this study was continued to evaluate the clinical and immunologic responses of a second year of LNIT. Patients received either pre-seasonal treatment with an unmodified ragweed extract (RW) or a polymerized ragweed extract (PRW), or no treatment. The results of the second year of treatment were the same as the first year. Adverse reactions were significantly higher in the RW-treated group than in the PRW-treated group (P less than 0.001). Symptom/medication scores (SMS) in the RW-treated group were significantly lower than in the control group (P less than 0.005). Although SMS in the PRW-treated group were lower than in the control group, this difference was not significant. The immunologic response was evaluated by measurements of serum (S) RW-specific IgE and IgG and nasal secretory (NS) RW-specific IgE, IgG, and IgA. After treatment, serum IgE titres and secretory IgA titres rose in the RW-treated patients. Nasal secretory-IgG and NS-IgA titres increased with PRW treatment. The only immunologic response observed in the control group was a rise in S-IgE titres after the ragweed season. There was no substantial difference in immunologic measurements observed in the 1979 and 1980 seasons, except that the pre-treatment NS-IgE level was higher in 1980 (P less than 0.02). No significant correlations were found between antibody response and SMS. This study supports the efficacy of LNIT but does not support the protective role for NS-ragweed-specific IgA or IgG.

Effect of prolonged venom immunotherapy on serum venom-specific IgE and IgG.

Serum venom-specific IgE and IgG were monitored in twenty-three patients receiving venom immunotherapy for more than 3 years. Two response patterns of IgE antibody were found. Following initiation of therapy, seven patients had a rise in serum venom-specific IgE, peaking at one year, then decreasing. Sixteen patients had a persistent fall in IgE antibody titres following initiation of therapy. At the end of 3 years, levels of serum venom-specific IgE in both groups were comparable. The presence of atopy may have influenced the rising IgE antibody response. Serum venom-specific IgG either rose or remained elevated if the pretreatment titres were high. After several years of therapy, there was generally a decrease in serum venom-specific IgG.

Local intranasal immunotherapy for grass-allergic rhinitis.

In a double-blind controlled study, local intranasal immunotherapy was evaluated for the treatment of grass pollenosis. On the basis of serum grass-specific IgE levels, 50 grass-allergic patients were randomly divided into three groups and treated with either an aqueous solution of mixed-grass extract, a formaldehyde-modified mixed-grass extract (allergoid), or a histamine solution (placebo). Intranasal solutions were administered in gradually increasing doses over a preseasonal 10 wk period, adverse local reactions from the aqueous grass extract were frequent during treatment. Few adverse reactions occurred from the allergoid or histamine solutions. During the pollen season, patients receiving both grass extracts had much lower symptom/medication scores than patients receiving placebo. The severity of eye symptoms was the same in all groups. After treatment, serum grass-specific IgE rose in patients receiving aqueous and allergoid extract; no change was noted in patients receiving placebo therapy. Grass nasal secretory-specific IgA titers in each group did not change during the study. The results of this study suggest that local intranasal immunotherapy with either aqueous or allergoid grass extracts is clinically effective for the treatment of grass pollenosis. Adverse reactions associated with the aqueous extract may limit its usefulness. No correlation was present between the secretory immune response and clinical benefit.

Allergy to honeybee body components: distinction from bee venom sensitivity.

Two patients with inhalant allergy to whole bee body components are described, documented by positive skin tests to whole bee body extract and the presence of serum bee body-specific IgE. Both had evidence of mild sensitivity to bee venom. RAST inhibition studies indicated that the IgE antibodies directed at whole bee body components and bee venom were distinct. These observations suggest that venom allergy from insect stings and bee body inhalant allergy are caused by different antigens.

Comparison of the allergenicity and antigenicity of Polistes venom and other vespid venoms.

The crossantigenicity of Polistes venom with other vespid venoms was examined with rabbit and human antisera. Venom preparations from various Polistes species were obtained by electrical stimulation of individual insects and venom sac dissection. Rabbit antibodies were raised to the venom (P. apachus) and venom sac extract (P. exclamans). Human antisera were obtained from patients allergic to Polistes and other vespid venoms. The venom appeared to be more potent than the venom sac preparations in reactions with rabbit IgG and human IgE antibodies. Among the Polistes species, P. exclamans, P. instablis, and P. apachus venoms showed several lines of precipitation with rabbit antisera, and P. annularis and P. fuscatus venoms only one line, suggesting quantitative or qualitative antigenic differences. In RAST analysis, most sera reacted equally to all Polistes species but occasional exceptions were noted, again suggesting differences in venom allergens. P. exclamans-coupled discs gave the most consistent results. In gel diffusion experiments, there was no crossreactivity between Polistes and yellow jacket venoms and only limited crossreactivity between Polistes and hornet venoms. Patients sensitive to Polistes venom showed varying degrees of reactivity to yellow jacket and hornet venoms in RAST analysis. Patients sensitive to other vespid venoms also showed varying degrees os sensitivity to Polistes venom. Polistes venom appears to contain a genus-unique antigen (allergen). In addition, there appear to be some crossreacting antigens in Polistes and other vespid venoms but to a much lesser degree than found previously in the analysis of the relationship of yellow jacket and hornet venoms.

Immunologic and biochemical evaluation of the potency of whole insect body extracts.

Recent studies have indicated that currently available whole body extracts have little potency and are ineffective for diagnosis and treatment of stinging insect allergy. Pure venom is a potent effective allergen but is difficult to obtain in sufficient quantities from all Hymenoptera species. In these studies, an attempt was made to prepare a potent whole body extract. Whole bee body extracts were prepared with different extraction periods and at cold and room temperatures. Potency was examined biochemically by measurements of phospholipase A (PLA) activity and immunologically by PLA and bee venom radioallergosorbent test (RAST) inhibition experiments and gel diffusion studies with the use of rabbit antisera. All extracts prepared in the laboratory had some potency, indicating that it is possible to make a whole body extract containing small quantities of PLA or bee venom. However, the potency of these extracts was minimal as compared with bee venom. Three commercial extracts were almost devoid of detectable immunologic activity. While further attempts may be made to prepare a potent whole body insect extract, these results suggest that it is necessary to obtain venom in relatively pure form for the diagnosis and treatment of stinging insect allergy.

Evaluation of RAST inhibition as a method for standardization of ragweed pollen extracts.

The RAST inhibition technique was evaluated as a method for standardization of ragweed pollen extracts. Seven different ragweed extracts were examined. There was no relationship between the RAST inhibition results and the commonly used parameters of extract potency including weight/volume, PNU and total protein measurements. Intradermal skin test end point titration was variable in different patients and could not correlated with RAST inhibition results. However, RAST inhibition potency did correlate with the antigen E content of the extract and this finding suggests that RAST inhibition might provide a method for better standardization of allergen extracts.

Detoxified hymenoptera venoms: preliminary studies of in vitro cytotoxicity and antigenicity of Apis mellifera 'venomoid'.

The preparation of detoxified venom (venomoid) of Apis mellifera is described. The venomoid obtained by formaldehyde detoxification retained significant antigenicity. Radioallergosorbent test (RAST) inhibition studies indicated a loss of the affinity of the venomoid for the venom-specific human IgE antibody. In rabbits the venomoid was capable of producing precipitating antibodies which cross-reacted with the original venom. Cytotoxic activity of venomoid as measured by nonimmunological histamine release and hemolytic activity with human cells was not detectable in vitro. Further studies seem warranted for the establishment of a venomoid as a diagnostic and/or therapeutic agent in the management of the hypersensitivity reactions to venom.

Allergenic potency of bee antigens measured by RAST inhibition.

The potency of various bee antigens including bee venom, several whole bee body extracts and fractions of bee venom was studied using the RAST inhibition method. As compared to whole bee body extract, bee venom was a much more potent inhibitor of both bee venom and whole body RAST, suggesting that venom has a greater capacity to bind specific bee IgE antibodies. Whole body extracts also varied substantially in their inhibiting activity. Phospholipase A and hyaluronidase were the most potent of the bee venom fractions suggesting their potential use as an assay for standardization of insect extracts.

Studies of the antigenicity and allergenicity of phospholipase A2 of bee venom.

The antigenic and allergenic properties of phospholipase A2 (PLA2) and whole bee venom were compared by measuring the IgG and IgE antibody responses in animals and man. Precipitating antibodies raised in rabbits and reaginic and other antibodies raised in mice reacted about equally with both bee venom and PLA. The majority of human sera containing bee venom-specific IgE also contained PLA-specific IgE, although in somewhat lower titers. Similarly, most human sera with significant amounts of total antibodies reacting with bee venom also had antibodies reacting with PLA. Histamine and SRS-a release from leukocytes of sensitive patients followed challenge with whole bee venom and PLA in the majority of instances. However, mediator release from several patients' cells was obtained with bee venom only. These studies suggest that although PLA is a major allergen and antigen in bee venom, significant exceptions in patients' reactivity may limit its potential diagnostic and therapeutic usefulness.