Capacity of purified antigens and whole pollen extracts to release histamine from leukocytes of hay fever patients

Eighty-seven consecutive patients appearing with complaints suggestive of spring and fall hay fever were subjected to basophil histamine release study with 4 antigenic preparations: whole ragweed extract, antigen E of ragweed, mixed grass pollen extract, and Group I antigen of rye grass. Among the 87 patients, 12 failed to release histamine to ragweed extract or antigen E and 16 failed to release histamine to grass extract or Group I. Among the remaining patients 5 reacted to whole ragweed extract but not antigen E and 2 reacted to mixed grass extract but not Group I. The patients with this pattern were all at the lower end of the spectrum of sensitivity for the crude extracts. These data tend to confirm that antigen E and Group I are the antigens of prime importance in the majority of hay fever patients with ragweed and grass pollen sensitivity respectively.     

Allergy to insect sting : III. Allergenic cross-reactivity among the vespid venoms

Recent reports have indicated that venoms may be more beneficial than whole body extracts for the diagnosis and treatment of Hymenoptera sensitive patients. These studies were undertaken to determine the cross-reactivity among the vespid venoms. Eighteen patients who were anaphylactically sensitive to vespid venoms were studied using in vitro leukocyte histamine release. The results (venom concentration for 50% histamine release) were analyzed by linear regression analysis; there was no allergenic cross-reactivity between any of the venoms, except for a modest association between yellow hornet and white hornet venom. In spite of this result 13 of the 18 patients studied were sensitive to three or four of the venoms tested. There is no clear explanation for this observation, but it suggests the existence of multiple major allergens in the vespid venoms, some of which are cross-reactive. Since immunotherapy with inappropriate proteins may lead to the development of IgE and the possibility of clinical sensitivity and since the majority of patients were not sensitive to all venom preparations, we suggest that appropriate diagnostic studies be carried out before the institution of therapy.     

Studies on the immunological relationship of ragweed pollen antigens E and Ra.3

This study compares the allergenic and antigenic activities of ragweed pollen antigens Ra.3 and E using the human leukocyte histamine release assay. Leukocytes from 23 antigen E-sensitive patients were challenged separately with varying concentrations of Ra.3 and E. Six were unresponsive to Ra.3, although the same cells released 60 to 100 per cent histamine on challenge with antigen E. The leukocytes of 16 patients gave maximal release of 50 to 100 per cent with Ra.3. Leukocyte sensitivity to Ra.3 relative to E ranged from 0.1 to 10⁴ and demonstrated little or no allergenic cross-reaction between the 2 antigens. Skin tests on an additional 19 patients confirmed this pattern. Leukocytes were challenged separately with mixtures of each allergen and dilutions of human antisera to antigen E (α-AgE) and water-soluble ragweed (α-AgWSR). Whereas α-AgWSR and α-AgE were equally inhibitory to E-induced histamine release, only AgWSR effectively inhibited Ra.3-induced release. Rabbit antiserum to Ra.3 and AgE in most patients inhibited the homologous allergen and demonstrated little or no cross-reactivity. In 2 patients, however, anti-AgE inhibited Ra.3-mediated histamine release while the homologous antiserum was inactive. We conclude that antigens Ra.3 and E are antigenically and allergenically distinct.     

Cyclic AMP metabolism in asthma: studies with leukocytes and lymphocytes

The effect of isoproterenol on cyclic AMP (cAMP) levels in lymphocytes and leukocytes from asthmatic and normal individuals has been studied. Lymphocyte cAMP levels rose in response to 10^?8 to 10^?2 M isoproterenol; the dose-response curve was biphasic with a maximum response at concentrations of 10^?6 to 10^?4 M. At all drug concentrations the response of cells from asthmatic individuals was less than the response of cells from normal control subjects. The difference between the two groups was not statistically significant, however. Similarly, basal cAMP levels were lower in the cells of asthmatic as compared with normal individuals, but again the difference was not significant. When these two parameters were combined and the results expressed as absolute cAMP levels after isoproterenol treatment, a significant difference was observed. Both the unstimulated. cAMP levels and the response to isoproterenol of leukocytes from normal and asthmatic subjects were influenced by the incubation medium. Use of a medium buffered with tris as compared with bicarbonate-phosphate resulted in lower basal cAMP levels and increased responsiveness to isoproterenol. Treatment of normal leukocytes with serum from asthmatic individuals did not alter their response to isoproterenol.     

A new concept of triggering mechanisms of IgE-mediated histamine release.

It is generally accepted that an initial step of reaginic hypersensitivity reactions is a bridging of mast cell--bound IgE antibody molecules by antigen. Since IgE molecules are firmly bound to receptors on mast cells, bridging of cell-bound IgE molecules probably brings receptor molecules into close proximity. A hypothesis was therefore presented that such a local change in membrane structure and/or possible interaction between adjacent receptor molecules may be triggering mechanisms of IgE-mediated histamine release. The hypothesis was tested by use of antibodies against "exposed portion" of receptor molecules on rat basophilic leukemia cells. It was found that antireceptor antibodies and its F(ab')2fragments induced noncytotoxic histamine release from normal rat mast cells without participation of IgE, while the monovalent Fab' fragments of the antibody failed to do so. However, sensitization of normal rat skin with the Fab' fragments followed by an intravenous injection of antirabbit IgG induced skin reactions. These findings support the concept that bridging of receptors rather than polymerization of IgE molecules is responsible for the activation of membrane-associated enzymes which in turn leads to histamine release.     

The role of ragweed pollen in autumnal asthma

Thirty-nine ragweed-allergic seasonal asthmatics were studied from 1972 to 1974. After quantitative skin tests, antigen E-induced leukocyte histamine release, quantitative inhalation bronchial challenge with ragweed extract to determine PD35 (provocation dose of allergen causing 35% decrease in specific airways conductance), and radioallergosorbent test (RAST) determinations were done, patients were paired based on PD35 values and randomly assigned to treatment or placebo groups, receiving either aqueous ragweed extract or placebo prior to the 1973 ragweed season. Treated patients received a mean cumulative dose of extract equivalent to 11.7 microng antigen E (4,180 protein nitrogen units [PNU]). Twenty-nine patients were followed through the ragweed season with daily symptom diaries and biweekly physician examinations. Severity of disease was not predictable by PD35 data, skin tests, leukocyte histamine release, or radioallergosorbent test (RAST) values. Although all patients were ragweed-allergic by objective tests, only 13/29 had asthma symptoms correlating with ragweed counts. Mold spore counts were related significantly to symptoms in some patients. Asthma and hay fever symptoms correlated significantly in 24/29 patients. This dose of immunotherapy caused no significant difference to be found in asthma or hay fever symptoms in treated versus placebo patients for the 1973 reporting period as determined by physician evaluations or daily symptom diaries. No patients showed significant improvement in PD35 values after treatment in 1973. Similar findings were obtained for a smaller group of patients followed through the 1974 ragweed season who received a mean dose of 31.2 microng antigen E (11,140 PNU). The failure of these patients to show a response to immunotherapy could be due to a combination of the relatively low dose of ragweed extract and their sensitivity to other allergens.     

Quantitative inhalation bronchial challenge in ragweed hay fevery patients: a comparison with ragweed-allergic asthmatics.

Fourteen ragweed hay fever nonasthmatic patients comparably sensitive to a group of ragweed-allergic asthmatics by skin test and leukocyte histamine release were tested by quantitative inhalation bronchial challenge with ragweed extract. The provocation dose of ragweed extract producing 35% decrease in airway conductance was determined and designated PD35. PD35 values in the hay fever patients were not significantly different from PD35 values in the asthmatic group. These data suggest that carefully performed skin tests may be as diagnostically useful as bronchial challenge in routinely confirming the allergic etiology of seasonal asthma.     

Human serum albumin and Tween 80 as stabilizers of allergen solutions

Intradermal skin testing may often give inaccurate results because of poor stability of allergens and loss of protein by adsorption to the walls of containers and syringes during the process of making the extreme dilutions required with potent extracts. To test the ability of stabilizers to prevent such losses of allergenic activity, three diluents for allergens were compared: standard phosphate-buffered saline (PBS), pH 7.4, containing 0.4% phenol and the same buffer containing either 0.03% human serum albumin (HSA) or 0.005% Tween 80. Tenfold dilution series of ragweed, grass, Altemaria, and dust allergens were tested by the intradermal threshold dilution technique in the same group of patients five times over six months, comparing stored dilutions with dilutions freshly made from the same batches of lyophilized extracts. Results with Tween 80 and HSA buffers were identical and highly reproducible; however, each new set of dilutions in standard buffers frequently showed within 24 to 48 hr after preparation a lower skin test potency which varied unpredictably between ten and one thousandfold. Furthermore, upon prolonged storage at 4 °C, dilutions in standard buffer lost further activity. Storage of radiolabeled antigen E (AgE) in ordinary glass tubes for 24 hr showed adsorption of about 5% of the labeled protein to glass in the absence of stabilizers but only 0.5% in the presence of stabilizers. We conclude that stabilizing agents should be added to diluting fluids in preparing allergens for skin testing or immunotherapy.     

Allergy to insect stings. II. Phospholipase A: the major allergen in honeybee venom.

In order to determine the proteins of major allergenic importance in honeybee venom (Apis mellifera) it was chromatographed on G-50 Sephadex. The four major protein peaks eluted were identified as hyaluronidase, phospholipase, melittin, and apamin. Testing these preparations on the leukocytes of 6 honeybee-sensitive patients, with the in vitro method of histamine release, revealed that all individuals were most sensitive to phospholipase A. IgE antibodies against phospholipase A (RAST) were found in the sera of honeybee-sensitive patients and IgG antibodies to this venom component were found in the sera from beekeepers and venom-treated patients. Melittin appeared to be allergenic in several patients, but the results were variable and were possibly due to contamination with phospholipase. All patients were insensitive to the hyaluronidase and apamin preparations. We conclude that phospholipase A is the major allergen of honeybee venom and, since this protein is readily available, it should be useful for diagnostic and therapeutic studies as well as for the standardization of materials used in the management of honeybee-sensitive patients.     

Comparisons of alum-precipitated and unprecipitated aqueous ragweed pollen extracts in the treatment of hay fever

In 1969 and 1970, groups of patients with ragweed hay fever never before treated were started on preseasonal courses of immunization with an alum precipitate of aqueous ragweed extract. A comparison between these two groups of patients and a similar group of patients treated with unprecipitated aqueous extract in 1968 shows that treatment with alum precipitate was safely initiated with fewer injections even though a higher dose was administered. The larger cumulative dose appeared to give better IgG antibody responses and greater relief of symptoms. A repeat preseasonal course the next year again required fewer injections of the alum-precipitated extract than a repeat course of aqueous extract.     

Allergy to insect stings. I. Diagnosis of IgE-mediated hymenoptera sensitivity by venom-induced histamine release

The failure to distinguish sensitive from normal individuals by skin testing with whole body extracts of Hymenoptera led us to study whether “allergic” reactions to these insects were based on an immunologic mechanism and to attempt the development of a useful diagnostic test for this condition. Pure venoms of honeybee, yellow jacket, yellow and white-faced hornets, and bumblebees were used as antigens for leukocyte histamine release. The leukocytes from $\text{13}\text{16}$ patients judged clinically to have had systemic reactions to a stinging insect released > 50 per cent of their histamine with 0.001 to 1.0 μg per milliliter of venom. Whether the few patients who had a negative response were truly allergic or sensitive to venoms not used for testing remains to be seen. None of the leukocytes of the 12 control patients, however, released histamine at 1,000-fold greater concentrations of venom. We confirmed previous studies which showed that skin testing with Hymenoptera whole body extracts failed to separate these two groups. Passive sensitization of normal leukocytes for venom-induced histamine release was accomplished with sera from several of the Hymenoptera-sensitive patients. Heating these sera to 56 ° C. or passing them through an IgE immunoabsorbent column markedly reduced their sensitizing ability. We conclude that venom-induced histamine release from the leukocytes of Hymenoptera-sensitive patients is mediated by antibodies of the IgE class and suggest that the great majority of reactions to Hymenoptera stings are truly allergic. The clear-cut diagnostic utility of pure venoms in vitro warrants further exploration of their diagnostic utility by in vivo skin testing.     

Diagnostic tests in ragweed-allergic asthma. A comparison of direct skin tests, leukocyte histamine release, and quantitative bronchial challenge

Thirty-six patients with a history of seasonal ragweed asthma were tested. Direct intradermal skin tests, leukocyte histamine release, and quantitative inhalation bronchial challenge correlated significantly. Data reported suggest that quantitative skin tests are as diagnostically valuable as quantitative inhalation bronchial challenge or leukocyte histamine release. Data also suggest that lung and skin mast cells and circulating basophils respond as a single population of cells to ragweed antigens.     

The clinical and immunologic specificity of immunotherapy

In order to study the specificity of immunotherapy for respiratory allergy, a group of patients sensitive to both ragweed and grass pollens were selected. From 87 volunteers with a history of both spring and fall hay fever, 42 patients with evidence of strong sensitivity by basophil histamine release to both ragweed pollen and mixed grass pollen extracts were selected for study. On the basis of the histamine release data, the patients were divided into two groups matched for sensitivity to both grass and ragweed pollens. In 1970, May and June symptom diaries showed the two groups to suffer quite similar severity of symptoms during the grass pollination season. One group of patients was started on a preseasonal course of immunotherapy with alum-precipitated aqueous extract of ragweed pollen while the other group received placebos containing histamine. By fall there had been a considerable rise in IgG-blocking antibodies to ragweed in the treated group. Symptom diaries in August and September showed that the treated group showed significantly less severe symptoms than the placebo group. Both groups received booster injections at 2-wk intervals from the fall of 1970 to the fall of 1971. Doses in the treated group were raised to attempt to administer the largest possible dose. Again there was no difference in the symptoms reported by the two groups during the grass pollination season, but an even greater difference emerged between the two groups during the ragweed season. The following year 1972 the same results were obtained. These data demonstrate that treatment with ragweed pollen extracts has little or no effect on grass pollen symptoms and confirm that immunotherapy is clinically as well as immunologically specific. Antibody responses to the second year of high-dosage booster injections was not greater than responses to a comparatively short preseasonal course given the first year.     

Bronchoprovocation: effect on priming and desensitization phenomenon in the lung.

Priming, or increased sensitivity to antigen, has not been demonstrated in the lung and could play a role in asthmatic symptomatology during seasonal pollen exposure. It is also an important consideration in the design of any experimental protocol requiring serial bronchoprovocations with antigen. Thirteen patients with a history of asthma symptoms during the pollen season and a positive skin test to ragweed extract were selected. Patients were given bronchial challenge out of season on 4 successive days with stepwise inhalations of antigen, and airways conductance was monitored in the body plethysmograph. Antigen dose-response curves were drawn, and the cumulative dose required for a 35% reduction in specific airway conductance was calculated and designated Provocation Dose (PD35). No regular trend toward either priming or desensitization was noted. The daily changes in antigen sensitivity did not correlate with daily variation of baseline pulmonary function. To determine if there was any priming due to natural exposure to pollen, 9 patients were brought back and rechallenged during the pollen season with no significant increase in bronchial sensitivity to ragweed extract. The PD35 method provides figures useful for comparing dose-response curves and shows a one-log variation from day to day. Any evaluation by bronchial challenge of antigen sensitivity or drug efficacy must take into account such variation.     

Polistes wasp hypersensitivity: diagnosis by venom-induced release of histamine in vitro.

Polistes wasps cause a majority of Hymenoptera-induced anaphylactic reactions in Texas. Using the in vitro release of histamine from basophils of patients allergic to Polistes stings, we have studied the cross-reactivity of venoms from three species of Polistes wasps as well as the cross-reactivity among Polistes, honeybee, and Vespula maculifrons (yellow jacket) venoms. Venom collected by an extrusion technique from Pollistes exclamans, Pollistes apachus, and Pollistes carolina caused release of histamine in seven Polistes-sensitive individuals. The dose-response curves from all three Polistes species were quite similar, suggesting extensive cross-reactivity among these species. None of these patients showed significant release of histamine from leukocytes exposed to yellow jacket or honeybee venom. We conclude that a source of Polistes venom is available for further study and possibly for therapy. It appears that any of three local common species of Polistes wasps could be used. Our studies confirmed earlier reports that Hymenoptera sensitivity if often genus-specific.     

Diagnostic tests in ragweed hay fever. A comparison of direct skin tests, IgE antibody measurements, and basophil histamine release

In 87 patients with both spring and fall hay fever symptoms the radioallergosorbent test (RAST) technique for specific IgE antibodies to ragweed was compared with basophil histamine release and direct intradermal skin testing by the threshold dilution technique. The three techniques gave good agreement except with the leastsensitive patients, some of whom had a positive skin test but undetectable histamine release or IgE antibodies. Twenty-one patients who were highly sensitive to ragweed as measured by all three techniques were followed without specific immunotherapy. There was significant agreement between the level of positivity of all three tests and the symptom index obtained during the ragweed season. In 14 of the 21 patients there was a significant correlation between daily ragweed pollen counts and daily symptom indexes during the season. On the other hand, among the 16 least-sensitive patients (as judged by histamine release) the correlation between daily ragweed pollen counts and symptom indexes was significant in only 3 patients. Other significant allergens could not be identified in the latter group, and the cause of their symptoms is not clearly identified but appears not to be ragweed. The RAST is a quantitative technique that gives diagnostically useful information in ragweed hay fever, although not significantly different from basophil histamine release or carefully performed skin testing. The convenience to the patient may, however, offer a noticeable advantage.     

Studies on allergoids from naturally occurring allergens III. Preparation of ragweed pollen allergoids by aldehyde modification in two steps

We have devised a new process for modifying heat-labile allergens, which employs a sequential “two-step” incubation at temperatures of 10° C and 30° to 32° C. This process was found to produce effective ragweed “allergoids” with low allergenicity and good immunogenicity, which makes them useful for the therapy of allergic humans. Modification with formaldehyde produced derivatives (“formallergoids”) that were about 10-fold less allergenic in allergic humans (as measured by leukocyte histamine-release assay), and similarly or more immunogenic in guinea pigs, than glutaraldehyde-modified allergens (“glutarallergoids”). Further analysis by RAST inhibition showed that a ragweed formallergoid was sixfold less reactive than a glutarallergoid with a pool of human IgE antibodies. However, the formallergoid had retained the ability to induce a wide array of antibodies against native ragweed antigens, since rabbit anti-formallergoid serum was able to recognize at least 12 different ragweed antigens, including AgE. Gel-filtration experiments showed that both the formallergoid and glutarallergoid materials contained polymers having apparent molecular weights distributed around 260,000 and 230,000 daltons, respectively (approximate range 30,000 to 900,000 daltons). Our studies provide the immunochemical basis for the use of these allergoids in the therapy of allergic humans.