Correlation between lymphocyte responses and immediate hypersensitivity to purified allergens

Seventy-nine unrelated subjects were selected for high allergic sensitivity to ragweed and/or grass pollens. Sensitivities to ragweed antigens E and Ra5 and rye grass group I were measured by intradermal skin testing and by histamine release in vitro. Lymphocyte responses were determined by incorporation of ³H-thymidine in vitro in the presence of antigen. Skin-test and histamine-release sensitivities were significantly correlated with lymphocyte response in the case of antigen E, but not for group I and Ra5. In addition, lymphocyte response to antigen E was significantly correlated with total serum IgE level. Six antigen E-sensitive individuals had marked delayed, but no immediate, reactions to Ra5 and almost all showed lymphocyte responses to Ra5. We conclude that immediate sensitivity and lymphocyte responses of allergic individuals to antigen E are significantly correlated, and that lymphocyte responses and delayed reaction to an antigen without immediate sensitivity or IgG antibody are possible even in allergic subjects.     

LACK OF ASSOCIATION OF THE IMMUNE RESPONSE TO RAGWEED ANTIGEN E,Ra 3 AND Ra5 WITH THE HLA SYSTEM

One hundred and sixty-five individuals with ragweed rhinitis and asthma were studied. The associations between the HLA system and: Ra3 skin-test response, Ra3 RAST, Ra3/antigen E skin-test reactivity ratio, Ra5 skin-test response, Ra5 RAST, Ra5/antigen E skin-test reactivity ratio, serum IgE levels, antigen E skin-test response, and antigen E RAST were studied. In addition, the influence of the serum IgE levels of these immune parameters were evaluated. No highly significant associations were noted.     

Human immunoglobulin E and immunoglobulin G antibody responses to the “minor” ragweed allergen Ra3: Correlation with skin tests and comparison with other allergens

We report here the use of an antigen-binding assay to measure serum IgE and IgG binding activity (BA) for the “minor” ragweed antigen, Ra3. These studies were carried out in ragweed-allergic individuals, many of whom had been skin tested with Ra3 as well as with the “major” ragweed allergen, antigen E (AgE). IgE BA for Ra3 showed a weak quantitative correlation with skin-test sensitivity to Ra3. More strikingly, there was a threshold of skin sensitivity (<10^?3 μg/ml) above which serum IgE BA was likely to be detectable. Serum IgG BA for Ra3 measured in parallel with IgE BA was found in all sera that showed detectable IgE BA for Ra3. By contrast, $\text{11}\text{13}$ ragweed-allergic individuals who were skin-test negative for Ra3 showed no detectable IgG BA for Ra3. These results support the view that the known genetic controls over Ra3 sensitivity control IgG antibody as well as IgE antibody responses. Individuals receiving injections of ragweed extract showed increases in IgG BA for Ra3 following therapy. Among nine persons who had no IgG BA for Ra3 before treatment, four out of nine developed a low degree of IgG BA for Ra3. However, the patients who developed IgG BA did not develop detectable IgE BA for Ra3.     

Immunological studies on Alternaria sensitivity use of crossed radioimmunoelectrophoresis, precipitins and enzyme-linked immunosorbent assay

Characterization of the immunological response to Alternaria in sensitive subjects is not complete. We used crossed radioimmunoelectrophoresis (CRIE) to identify antigens in Alternaria extracts reacting with IgE antibody in five patients with Alternaria-sensitive asthma, four with Alternaria-induced rhinitis, three non-allergic asthmatics, and three normal controls. All five Alternaria-asthma patients and three of four Alternaria-rhinitis patients showed IgE binding to a single antigen peak in the CRIE. One patient exhibited specific radio-staining to a second peak, and two other patients had IgE binding to a third antigen. These results suggest an analogy of Alternaria antigens with that found in ragweed pollen extracts, i.e. that IgE antibody is directed against more than one antigen. Using the enzyme-linked immunosorbent assay (ELISA), we found a significant difference (P < 0·05, unpaired Student's t-test) in igG binding between Alternaria-sensitive asthmatics and normal controls. There was no apparent difference in IgG binding between untreated Alternaria-sensitive asthmatics and those receiving high-dose immunotherapy.     

An association between serum testosterone level and HLA phenotype

The concentration of testosterone was determined in sera of 122 HLA-typed women. Subsequently the women were classified in the category below or above the mean serum testosterone concentration. When the frequencies of HLA antigens were compared in these two categories of women, it was found that HLA-B5 and HLA-B12 antigens were significantly increased in the category of women with serum testosterone level above the mean concentration (P < 0.01 and P < 0.05, respectively). The frequency of HLA-B8 antigen was significantly decreased in the same category of women (P < 0.05). The comparisons of the mean testosterone values of each HLA antigen and the variance analysis have also shown significant differences between the mean of HLA-B8 antigen and the means of other HLA antigens — HLA-A2, A3, A9, B5, B12 and Bw35. These results gave further conclusive evidence that gene(s) inside HLA region influence either the androgen hormone metabolism itself or cellular sensitivity to hormonal action as it has been presented for congenital adrenal hyperplasia.     

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.     

An HLA-associated nonresponsiveness to mellitin: a component of bee venom

Previous work has demonstrated a close association between certain histocompatibility antigens and the gene that controls the IgE response to certain ragweed allergens. For example, there is a 90% association between IgE production to the short ragweed allergen, Amb a V, and an HLA class II allele. To assess whether these HLA linkages are specific for ragweed, we have investigated the association between HLA antigens and the capacity of individuals to mount a specific IgE response to melittin in patients with bee-venom allergy. Twenty-two subjects with bee-venom sensitivity, 22 healthy beekeepers without bee-venom allergy, and a normal population of 149 unselected individuals were studied. With serologic tissue typing and restriction fragment length polymorphism analysis, we have demonstrated a significant decrease in the HLA-DR4 and DQw3 alleles in subjects who are allergic to melittin compared to the control populations. There was also a negative association between the presence of HLA-DR4 and DQw3 alleles with the capacity of the individuals to mount an IgE response to phospholipase A2 (PLA2). The bee-venom sensitive subjects had a slightly lower titer of anti-PLA2 IgG when these subjects were compared to the bee-venom insensitive beekeepers. These results support the view that either HLA-DR or HLA-DQ has a protective role in controlling the IgE immune response. Lack of an IgE response to melittin or PLA2 is unlikely to be due to a failure to recognize allergen.     

HLA antigen frequencies and natural history in Alternaria-sensitive and perennial nonallergic asthmatics

The frequency of HLA-A, -B, and -C loci antigens in random populations of Alternaria-sensitive (N = 100) and perennial nonallergic asthmatics (N = 87) were compared with age- (±5 yr) and sex-matched controls from the same geographic region. There was no association between HLA antigens as measured by frequency analyses and Alternaria-sensitive or perennial nonallergic asthma. Moreover there was no association between HLA antigens and the age of onset of asthma, associated allergic disorders, various environmental factors provoking asthma, total serum IgE levels, and Alternaria-specific IgE antibody.     

HLA-A, B, C and HLA-DR antigens in extrinsic allergic alveolitis (budgerigar fancier's lung disease)

Twenty-three patients with extrinsic allergic alveolitis due to an allergy to inhaled budgerigar serum protein (budgerigar fancier's lung disease) were typed for HLA-A, B, C and HLA-DR antigens. Antigen frequencies were compared with those found in 154 healthy control subjects. No statistically significant variation in the frequency of any HLA antigen was detected. Exclusion of two patients who had concurrent coeliac disease, and subdivision of the population into those with acute and chronic disease, failed to reveal any significant association with an HLA specificity. A non-significant increase in B8-DR3 amongst the patients with acute disease was noted. Possible reasons for the apparent HLA associations previously reported by others for extrinsic allergic alveolitis are discussed.     

Reactivity of ragweed allergens with IgE antibodies. Analyses by leukocyte histamine release and the radioallergosorbent test and determination of cross-reactivity.

Five distinct proteins with allergenic activity have been isolated from short ragweed pollen. We initially tested three of these, AgE, AgK, and Ra3, for reactivity with IgE antibodies by leukocyte histamine release and by the radioallergosorbent test (RAST). We found highly significant correlations between the reactivities of these allergens by leukocyte histamine release and by the RAST, consistent with the view that both procedures detected comparable allergenic activity. We next tested the allergenic cross-reactivity of all five ragweed allergens. AgE, AgK, Ra3, Ra4, and Ra5, by RAST inhibition. With solid-phase AgE the only nonhomologous inhibitor was AgK, which cross-reacted weakly and required a 140-fold mass excess of AgK compared to AgE. With solid-phase AgK both AgK and AgE produced significant inhibition; AgE was slightly more potent than the homologous AgK, Ra3 and Ra5 were allergenically unique, because only the homologous allergen produced 50% inhibition. Ra4 was weakly inhibited by AgE, Ra3, and Ra5 when these allergens were added in 300- to 5---fold mass excesses; this weak inhibition may represent either cross-reaction or cross-contamination. We found that RAST inhibition could be used as an assay for the individual ragweed allergens and we demonstrated the presence of all of the allergens in a whole ragweed extract. The sensitivity of the RAST inhibition assay ranged from 10 ng to 100 ng for 50% inhibition. Finally, the solid-phase ragweed allergens were used to determine the frequency of elevated IgE antibody levels in 65 patients with ragweed hay fever. Virtually all of the patients reacted with AgE (97%), while 88% reacted with AgK, 51% reacted with Ra3, 28% reacted with Ra4, and 17% reacted with Ra5. These results highlight the usefulness of the RAST as a specific and sensitive tool for immunochemical studies of allergens.     

Assessment of the influence of irrelevant IgE on allergic sensitivity to two independent allergens.

Serum samples from 31 patients sensitive to both ragweed and rye grass were quantitated for IgE specific for ragweed antigen E (AgE) and rye grass group I (rye I) antigens employing the previously described radioallergosorbent test (RAST) elution technique. IgE anti-AgE ranged from 0.4 to 320 ng/ml and comprised 0.4% to 14.2% of total serum IgE. The same sera contained IgE anti-rye I ranging from 7.9 to 1,168 ng/ml, comprising 1.6% to 29.6% of total serum IgE. A new theoretical parameter (RF), representing the percent of total IgE-Fc receptors of target cells occupied by allergen-specific IgE, was calculated for each IgE specificity by using the determinations of allergen-specific IgE antibody, total serum IgE, and assuming an equilibrium constant for binding of IgE molecules to basophils of 10(9)M-1. This "% occupancy" parameter correlated with skin test titration, basophil cell sensitivity, and hay fever symptom scores as well as, but not better than, the absolute values of allergen-specific IgE. This finding suggests that in most atopic patients, the quantity of irrelevant IgE plays a relatively minor role in determining allergic sensitivity to a given allergen. The implications of this finding are discussed.     

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.     

The immune response of patients with ragweed hay fever treated with polymerized ragweed antigens.

This report describes the immune response of patients with ragweed hay fever treated with polymerized ragweed antigens (PRW). Their IgG antibody responses to crude ragweed extract, antigen E, antigen K, and antigen Ra3 were determined by a solid-phase radioimmunoassay. The results indicate that PRW contains an array of clinically important antigens that are available for immunologic processing and result in an immune response in patients treated with this new form of immunotherapy for ragweed hay fever.     

Immunological properties of the human Goodpasture target antigen.

IgE rheumatoid factor activity was found to be significantly elevated (P less than 0.01) using an ELISA assay when paired synovial fluid and sera from 13 patients with active RA were compared to ten control samples. Synovial fluid IgE RF activity was higher than predicted by diffusion alone in 7/11 (64%) of the RA synovial fluids studied when coefficients of diffusion were determined. The specificity of IgE RF activity as measured by the ELISA was confirmed using immunoaffinity chromatography. Mast cells, obtained by enzymatic dispersion of rheumatoid synovial tissue, were sensitized with sera containing either IgE antibodies directed against ragweed or IgE with RF activity. Histamine was released upon challenge with anti-IgE antibodies (33.2% +/- 11), ragweed antigen E (34.6% +/- 11), or aggregated gamma globulin (47.6% +/- 17.9). No histamine release was observed if antigen challenge occurred in the absence of appropriate sensitization, with C3 anaphylatoxin, or after immunoadsorption of IgE from sera containing IgE RF activity.     

Specific IgE antibody responses to ragweed allergens Ra5S and Ra5G associated with distinct HLA-DR beta genes

Previous studies have established that sensitivity (IgE antibody response) to Ra5S, a 5000 mol. wt protein of short ragweed pollen, is significantly associated with host possession of HLA-DR2. The same allele was implicated [Goodfriend et al. (1985) Molec. Immun. 22, 899-906] in sensitivity to Ra5G, a 4400 mol. wt homologue of Ra5S in giant ragweed pollen, based on frequency of co-sensitivity to both proteins. However, data reported here generated in HLA-DR assays of mono-sensitive individuals demonstrate that sensitivity to Ra5S and Ra5G is associated with separate alleles: DR2 and DRw52 respectively. Results consistent with the same sensitivity/DR associations were obtained in immunoabsorption studies with sera from co-sensitive individuals. As HLA-DR2 and DRw52 have identical alpha but different beta chain types (beta 1 and beta 3), it was considered that IgE antibody responses to Ra5S and Ra5G are associated with distinct DR-beta genes.     

Measurement of IgE antibodies by the radioallergosorbent test: II. Analyses of quantitative relationships in the test

IgE antibodies have been measured by the radioallergosorbent test (RAST) and the relative amounts present in serum determined by comparison with reference standards. In this study we analyzed the quantitative aspects of the binding of IgE antibody to solid-phase ragweed antigens. With the volumes of allergic serum usually tested, IgE antibodies are in excess in the first step of the RAST. The inability of solid-phase antigens to remove IgE antibody appeared to be due to an insufficient quantity of antigen on the particles rather than steric interference with binding of IgE antibody. Dose-response curves with serums from several ragweed-sensitive subjects were not parallel when plotted on a semilog scale. In contrast, log-log plots of dose-response curves with serums from ragweed-sensitive subjects were nearly parallel. Log-log plots of dose-response curves with serums from subjects with ragweed and grass sensitivity and tested with the appropriate solid-phase antigen also were nearly parallel. Because of the latter finding, RAST could be standardized using a reference serum in every assay and plotting the results on a log-log scale. Finally, because IgE antibody is in excess in the RAST as it is usually performed, the final result reflects both the quantity and the affinity of the IgE antibody.     

The role of human leucocyte antigens in children with hydatid disease: their association with clinical condition and prognosis

Hydatid disease (HD) is a parasitosis caused by Echinococcus granulosus, which is still an important health problem worldwide, and our country is an endemic region for HD. There is little information regarding the role of human leucocyte antigen (HLA) in genetic susceptibility or resistance to HD. In this study, we aimed to investigate the HLA profile of Turkish children with HD and to compare them with healthy individuals. We also planned to investigate whether HLAs have a potential role in the predisposition to or prevention of the occurrence of HD and to study the relationship between the clinical features of HD and the HLA profile of the patients. The study included 81 children (25 boys, 56 girls) with HD aged between 3 and 18 years. All the patients’ and control subjects’ HLA class I and II antigens were examined, antigen allele frequencies were calculated, and clinical characteristics were also evaluated. The frequency of HLA-B18, -DR1, and -DR15 alleles were significantly different between the patients and healthy groups; HLA-DR15 antigen might be associated with HD occurrence, and the presence of HLA-B18 and HLA-DR1 antigens might be associated with HD resistance. Compared with the healthy group, patients with lung HD had a significant increase in HLA-B44 frequency, and liver HD patients had a significant increase in HLA-DR15 antigen frequency. Furthermore, presence of HLA-DR11 was found to be a significant factor associated with cure of the disease. We concluded that HLA types have significant impact on the development of HD and clinical course of disease.     

Prediction of late asthmatic responses to inhaled allergen

Relationships between cutaneous and bronchial responses to allergen were examined in nineteen atopic asthmatics. Allergen inhalation tests elicited an isolated early asthmatic response (EAR) in ten subjects and a dual asthmatic response (DAR) in nine subjects. Ragweed IgE RAST, performed with the sera of those patients tested with ragweed antigen, yielded higher values in all but one patient who experienced DAR than any of the patients with EAR. In one patient with annual symptoms in the ragweed season, positive skin tests with ragweed antigen and DAR to inhaled ragweed extracts, the IgE RAST was entirely negative and the serum IgE concentration was low. Dilutions of the allergen used in each individual for inhalation were also used in skin-prick tests. Early cutaneous allergic response (ECAR) mean wheal diameters were obtained at 10 min and late cutaneous allergic response (LCAR) mean diameters at 6-8 hr. Early asthmatic response (EAR) subjects differed modestly from DAR subjects in the relationships between ECAR and LCAR; in the EAR group, a significantly larger wheal diameter (P < 0.01) was required before an LCAR ensued, however there was some overlap. Once LCAR developed, there was no difference between EAR and DAR groups in the magnitude of the LCAR. There was a trend (not significant) towards a requirement for a higher antigen concentration in the EAR group to elicit an LCAR. In conclusion, correlates of an isolated EAR from inhaled antigen include: (i) a low positive IgE RAST result with the antigen, (ii) ECAR 6 mm or greater with the antigen which does not proceed to a LCAR, and (iii) a high concentration of antigen is needed in skin tests to elicit an LCAR. Correlates of a DAR include:(i) a high IgE RAST result with the antigen, (ii) an ECAR wheal diameter less than 5 mm with the antigen proceeds to a LCAR, and (iii) a low antigen concentration in skin tests elicits an LCAR. The observed correspondence between the tendency to late skin and late airway responses is evidence of a common immunologic basis.     

Lymphocyte proliferation to antigen E: Demonstration of the restriction of antigen E-specific T cells to ragweed-allergic donors

Allergic sensitivity to ragweed is common among atopic individuals in North America and can be associated with symptoms of seasonal hay fever and increased airway reactivity in asthma. This sensitivity is mediated by IgE antibody to ragweed antigens that in turn is presumed to be the product of B-lymphocytes regulated by various T cell subsets. Proliferation in vitro by lymphocytes obtained from individuals allergic to ragweed and cultured in the presence of ragweed antigen E (AgE) has been repeatedly described, but a comprehensive study of this proliferation has questioned the specificity of this response. We have examined this question and found that in the first week of culture, the specific lymphocyte proliferation to AgE may be obscured by high background and mitogen-like proliferation. However, by carrying the cells for a longer period of time in culture and providing a second in vitro boost with AgE, specific proliferation could be clearly documented. Lymphocytes from atopic ragweed-allergic donors proliferated at levels 20 to 50 times beyond background in the presence of AgE. Cells from nonragweed-allergic donors (either nonatopic or atopic) did not do so. The AgE-responsive cells could be expanded in culture and demonstrated to be T cells. Moreover, AgE-responsive T cells could only be cloned from AgE-allergic donors and, after expansion and subcloning, demonstrated to respond to AgE but not partially purified dust mite antigen. In contrast, a clone of T cells from a dust mite-sensitive individual proliferated in response to the dust mite antigen but not AgE.     

HLA-A, B and C and HLA-DR antigens in intrinsic and allergic asthma

Some 103 patients with asthma and 100 healthy volunteers have been typed for HLA-A, B and C and HLA-DR antigens. The 103 patients consisted of thirty-three with intrinsic asthma, thirty-four with extrinsic asthma, and thirty-six known to have precipitins to Aspergillus fumigatus. No increase in frequency of any of the A, B, C, or DR antigens was found to be significant after correction for the number of comparisons was made. However certain trends comparable to findings in other immunopathic disorders were noted. For example B12 was increased in the allergic asthmatics (46 vs 29% controls) and it is suggested that B12 is associated with the ability to produce the IgE antibodies. A3/B7/DRw2 (which are in linkage disequilibrium) all show a decreased frequency in intrinsic asthma (24, 12 and 9%vs 32, 26 and 24% respectively in controls). Finally B8 and DRw3, which showed a moderate increase in frequency in all three groups of asthmatics, were found in five of seven patients with low atopy but persisting antibodies to A. fumigatus. Further detailed studies of these asthmatic subgroups is warranted.