IgG4 antibodies in patients with house-dust-mite-sensitive bronchial asthma: relationship with antigen-specific immunotherapy

Sera from 40 patients with house-dust-mite (Dermatophagoides farinae)-sensitive bronchial asthma were evaluated by solid-phase radioimmunoassay for their mite-specific IgG4 antibody levels. Asthmatic patients undergoing specific immunotherapy possessed a significantly higher mean value of IgG4 antibodies than normal controls and asthmatics without immunotherapy (p less than 0.01 and p less than 0.05, respectively). Moreover, evaluation of 11 patients before and after immunotherapy showed that IgG4 antibodies tend to increase during immunotherapy. The clinical significance of the IgG4 subclass as blocking antibodies in immediate allergic reactions is also briefly discussed.     

Solid-phase enzyme immunoassay for anti-mite IgE and IgG antibodies in allergic patients

Solid-phase enzyme immunoassays for the measurement of anti-mite IgE and IgG antibodies in the sera of mite-allergic patients were developed. In both assays, microtiter plates coated with the crude extract of Dermatophagoides farinae were used. The solid-phase enzyme immunoassay detected mite-specific IgE and IgG antibodies in sera of 18 and 15 out of 20 allergic patients, respectively. The correlation coefficient between the two antibody titers in patients' sera was 0.74, which was statistically significant (P less than .01). Both of the assays require no special facilities for radioactive materials and are considered to be useful in practice for the diagnosis of allergy to house dust mites by combining the results of the two assays.     

Immunologic analysis of steroid-dependent asthma

In order to analyze steroid-dependent asthma immunologically, IgE antibodies to mite (Dermatophagoides farinae), Candida albicans, and Aspergillus fumigatus were measured in 112 asthmatic patients. IgG and IgG subclass antibodies to mite were also measured. The rate of patients who were positive to candida IgE RAST was higher in atopic steroid-dependent patients than in atopic steroid-independent patients (P less than .01). The rate of mite-sensitive patients who had not received immunotherapy with mite or house dust was higher than in the atopic steroid-dependent patients than in atopic steroid-independent patients (P less than .05). IgG1 and IgG4 antibodies to mite were higher in mite-sensitive steroid-independent patients than in mite-sensitive steroid-dependent patients. IgE antibodies to A. fumigatus were detected only in patients with allergic bronchopulmonary aspergillosis (ABPA). Based on these results, we were encouraged to try immunotherapy with house dust mite or C. albicans if patients were steroid-dependent and sensitive to these allergens except when the patients had ABPA.     

The change of allergen-specific IgG subclass antibodies during immunotherapy in mite-sensitive asthmatic children.

Fifty-six Dermatophagoid farinae (D.f)-sensitive asthmatic children were hyposensitized by D.f-crude extract for two years. Serum total IgG subclass antibodies and D.f-specific IgE and IgG subclass antibodies were measured by ELISA before and after 2 years of treatment. The results showed that 1) After two years of treatment, there were significantly higher levels of total serum IgG1 in both responder and non-responder groups than those before treatment (p less than 0.01). The responder group also had significantly higher values of total IgG2 and IgG4 after immunotherapy (IT) (p less than 0.05), but not in the non-responder group. 2) The serum levels of D.f-specific IgG3 and IgG4 antibodies in responder group increased significantly after IT (p less than 0.05). On the contrary, the D.f-specific IgE and IgG1 IgG1 in the responder group were significantly lower than those before IT. No signi- in the responder group were significantly lower than those before IT. No signi-body titres before and after IT was found in non-responder group. 3) There was a significant correlation between the total IgG4 and D.f-specific IgG4 antibody (r = 0.634, p less than 0.01). The correlation coefficient was 0.634. No correlation was found between the other IgG subclass antibodies and D.f-specific IgG subclass antibodies. 4) Correlations between the levels of D.f-specific IgE and IgG subclass antibodies were highly significant both in IT-responder and non-responder groups. There was a significant correlation between the levels of D.f-specific IgG1 and IgG4 in non-responders, while no relationship was observed in the responder group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dogs hypersensitive to house dust mite

Active intradermal skin reaction, respiratory resistance and respiratory patterns were examined in five dogs after intradermal injection or inhalation of house dust mite (Dermatophagoides farinae) extract and the results obtained were as follows: 1. Three out of the five dogs exhibited positive skin reactions against house dust mite extract solution at concentrations of 0.1 micrograms/ml to 10 micrograms/ml. 2. Inhalation of mite extract at a concentration of 1 mg/ml by the dogs that had positive skin reactions produced a marked increase in respiratory resistance and changes in spirogram. These results suggest that the dogs that exhibited positive skin reactions were spontaneously sensitized to D. farinae.     

Measurement of IgG antibodies to house dust mite and grass pollen by a solid-phase radioimmunoassay

Polystyrene microtubes, coated with extracts of either Dermatophagoides pteronyssinus (DPT) or grass pollens, were used as the solid-phase in a radioimmunoassay for the measurement of specific IgG antibodies to the corresponding allergen. Radiolabelled protein A from Staphylococcus aureus (SpA) was used to determine the IgG antibodies attached to the tubes. The binding of IgG from either normal or allergic sera to DPT-coated tubes was antigen specific and mediated by the Fab fragment of the immunoglobulin. IgG antibodies purified by affinity chromatography from non-allergic serum competed with IgE antibodies to DPT. IgE antibodies do not significantly interfere with the assay. Indeed, heating a reaginic serum resulted in a striking reduction of the (¹²⁵I) anti-IgE binding to allergen-coated tubes without modifying the (¹²⁵I)-SpA binding. Furthermore, filtration of a reaginic serum through Sephacryl S-200 separated a peak of IgE antibodies, characterized by a high binding of labelled a-IgE and a low binding of (¹²⁵I)-SpA from the peak of IgG antibodies defined by low a-IgE and high SpA binding. The solid phase method is more sensitive than a double-antibody technique employing the same DPT extract as labelled antigen. Non-allergic subjects had less IgG antibodies to DPT or grass pollens than allergic patients. In untreated patients, there was a good correlation between levels of IgG and IgE antibodies to grass pollens but not to DPT. Patients hyposensitized to house dust mite had on the average three times more specific IgG antibodies than untreated cases.     

The allergen specific B-cell response during immunotherapy

The paper examines the allergen specific B-cell response in peripheral blood from patients undergoing immunotherapy with house dust mite extract. The 12 patients were part of a double blind placebo controlled study, and they were treated with either Dermatophagoides pteronyssinus (n = 4), Dermatophagoides farinae extract (n = 3) (Alutard SQ, ALK, Denmark) or placebo (n = 5). Blood was taken every fortnight on day seven after hyposensitization and tested for IgM, IgG, IgA and IgE antibody secreting cells (AbSC) to D. pteronyssinus and D. farinae allergens and for the total number of immunoglobulin secreting cells (IgSC). The data showed a maximum of approximately 120 Der f I+II specific AbSC/10(6) mononuclear cells (MNC). A comparison of specific AbSC to the major allergens of the two house dust mites demonstrated that there was no measurable species specificity in the B-cell response that could be correlated to immunotherapy with either of the two extracts. The specific IgM, IgG, and IgA response to Der f I+II was examined in the placebo (39 measurements) and the actively treated (56 measurements) groups, and the results demonstrated a significant rise in specific IgM and IgA AbSC following immunotherapy. The number of specific IgG AbSC did not change. There was a mean of less than one specific IgE AbSC/10(6) MNC, and no detectable change following the treatment. It is speculated that immunotherapy to inhalant allergens causes the induction of specific IgA AbSC. It would then be these partly differentiated plasma cells that are detected on their way to the bronchial or gut mucosa to exert their protective function mediated by allergen specific secretory IgA.     

Natural antibodies in man to Streptococcus mutans: specificity and quantification

Antibodies to whole cells of Streptococcus mutans were examined in 108 subjects by a solid-phase radioimmunoassay and quantified by reference to isotype-specific affinity-purified antibodies. Serum antibodies of each isotype were present in all subjects examined. The mean concentration of serum antibodies to S. mutans was calculated as about 84 micrograms/ml of IgG (range 33-140 micrograms/ml), 26 micrograms/ml of IgA (range 12-43 micrograms/ml) and 9 micrograms/ml of IgM (range 4-15 micrograms/ml). The mean antibody values accounted for about 0.7, 1 and 0.8% of the total IgG, IgA, and IgM, respectively. Overall the antibody binding to whole cells of S. mutans accounted for about 0.8% of the total immunoglobulin. Inhibition experiments using a variety of purified cell wall antigens revealed that the binding of antibodies to whole cells could be inhibited by about 30% with a purified protein antigen (SA I/II) and with glucosyltransferase (GTF), by 25% with c polysaccharide and by 16% with lipoteichoic acid. The protein antigens GTF and SA I/II appear to be major immunogenic cell wall antigens, but natural antibodies in man that bind to S. mutans whole cells have been induced by several antigens, some of which are specific to S. mutans and some of which are shared with other Gram-positive bacteria.     

Measurement of IgE, IgG1 and IgG4 antibodies against mite Sephadex fractions and purified allergens by means of enzyme-linked immunosorbent assay

Mite antigens (Dermatophagoides farinae) were fractionated by a Sephadex G-200 column and their reactivities with IgE, IgG1 and IgG4 antibodies were investigated with enzyme-linked immunosorbent assay (ELISA). High IgE antibody values were observed in fractions with low molecular weight (allergenic part), while high IgG1 and IgG4 antibody values were observed in fractions with high molecular weight. High IgG4 antibody values to crude mite extract and fractions with high molecular weight were detected in individuals who had received immunotherapy. However, IgG4 antibodies directed to allergenic part were found in only one out of 12 sera tested. IgG4-ELISA using DF1 (major allergen of Dermatophagoides farinae) as antigen was also performed. In the group treated with mite, significant IgG4 antibody levels were detected in only one out of 13 sera tested. In the group treated with house dust, significant IgG4 antibodies were detected in only one out of 12 sera tested. Patients who showed high IgG4 antibody responses to crude mite extract and to high molecular weight did not show responses to allergenic part and DF1. The only case who showed positive IgG4 responses to allergenic part also reacted with DF1. Those results suggest that IgG1 and IgG4 antibody values in ELISA using crude mite extract as antigen do not reflect major allergen-specific antibody values. The importance of the use of partially purified antigens in measuring major allergen-specific IgG4 antibodies was also suggested.     

The significance of specific IgE and IgG to dermatophagoides farinae according to the types of asthmatic reaction in house dust asthmatics

To investigate the role of specific IgE and IgG in the various types of asthmatic reaction, we measured specific IgE and IgG levels to Dermatophagoides farinae (D.farinae) using the D. farinae-radioallergosorbent test (RAST) and Phadebas IgG-RAST in 39 house dust asthmatics (11 early responders, 21 dual responders and 7 isolated late responders) and 12 negative responders on house dust bronchoprovocation. There were significant differences in the D. farinae-specific IgE level and skin reactivity to D. farinae and house dust among the 4 groups (p less than 0.05) and the specific IgE level of dual asthmatic responders was the highest and was significantly higher than that of early responders (p less than 0.05). The specific IgG level showed no differences among the 4 groups. These results suggested that the types of asthmatic reaction in house dust asthmatics were closely related to specific IgE level to D. farinae and the specific IgG level seemed not to be related to an isolated late response.     

A clinical evaluation of rush immunotherapy in adult patients with severe bronchial asthma

Rush immunotherapy (RIT) using house dust (HD) antigen were performed in ten adult patients with chronic severe bronchial asthma who had mite allergy. All cases have reached to the maintenance dose of 10(-1) X 0.50 ml within 10 days and been able to introduce maintenance therapy smoothly. After the RIT, remarkable improvement of asthmatic symptom was seen in 4 cases and one of them was considered to be completely cured of the asthma. Although side effects such as local skin reaction, rhinorrhea and asthmatic attack were seen, severe side effect was not observed at all. IgE-RAST scores for both HD and mite were unchanged before and after RIT. On the other hand, the values of specific IgG4 for both HD and mite antibody were significantly increased 2 or 4 weeks after RIT. These results indicated that RIT can be performed safely and securely, and rapid immunological response as well as clinical effectiveness are able to be expected for adult severe asthmatics.     

Circulating immune complexes in patients with house-dust-mite-sensitive bronchial asthma

Sera from forty patients with house-dust-mite-sensitive bronchial asthma were examined for the presence of circulating immune complexes (CIC) by the sensitive and quantitative CIq solid-phase radioimmunoassay (Clq-SP) and a monoclonal rheumatoid factor solid-phase radioimmunoassay (mRF-SP). Compared to fifteen normal individuals, the asthmatic patients showed significantly higher mean values of Clq-reactive materials; however, there was no difference between the results from the patients treated by immunotherapy using Dermatophagoides farinae extract and those not so treated. Moreover, immune complexes in eight patients before and after immunotherapy showed that the amount of the complexes tend to decrease during immunotherapy. Furthermore, the presence of complexes had no relationship with the amounts of mite-specific IgG antibody. Similar results were also obtained in tests using mRF-SP. These data suggest that complexes in the sera of the house-dust-mite-sensitive asthmatic patients are not necessarily associated either with immunotherapy or with the mite-specific IgG antibodies.     

Prospective observations on stopping prolonged venom immunotherapy

After a decade spent establishing the safety, efficacy, and optimal techniques for venom immunotherapy, we have begun a series of studies to determine how long venom immunotherapy must be continued. In retrospective surveys, patients who had stopped venom immunotherapy after 1 to 2 years had a substantial risk (25%) of systemic sting reactions, but this was less than 50% of the risk in untreated patients. In this first prospective study, 30 patients elected to stop venom immunotherapy after at least 5 years of therapy. Skin test sensitivity had decreased significantly during therapy in 18/30 patients but remained clearly positive in 23/30 (seven patients became equivocal or negative). Serum venom-specific IgE antibodies were at the lower limit of detection (1 ng/ml) in 11/30 patients. After stopping treatment, the mean serum venom-specific IgG antibody level declined from 5.5 +/- 0.6 micrograms/ml to 2.4 +/- 0.3 micrograms/ml by 9 months, which is the same as the mean venom IgG in untreated patients. After 12 months without therapy, live sting challenge caused no systemic reaction in 29 patients. The mean venom IgG level 1 month after the sting had risen significantly to 4.1 +/- 0.5 micrograms/ml, but there was no significant increase of venom IgE. These results suggest that prolonged venom immunotherapy leads to isotype-specific suppression of the venom IgE antibody response and may provide persistent clinical protection by mechanisms other than IgG blocking antibodies. The observations are to be interpreted very cautiously. Further investigations are needed to extend these observations in additional patients and for longer periods of time, and to examine possible mechanisms for this apparent loss of clinical reactivity.     

Enzyme-Linked Immunosorbent Assay for Determination of Major Excrement Allergens of House Dust Mite Species D. pteronyssinus, D. farinae and D. microceras

Species-specific enzyme-linked immunosorbent assays (ELISA) for major excrement allergens (Dp42, Df6 and Dm6) of D. pteronyssinus, D. farinae and D. microceras in house dust were established, using immunoabsorbed, monospecific rabbit antibodies, coupled to horse radish peroxidase. The limit of detection was 13, 4 and 38 ng/ml, respectively. The coefficient of variation for the entire procedure, including dust sieving (212 micron) and extraction was 5-16% for allergen levels above 1000 ng/g dust. Allergen concentration by ELISA correlated well with the number of mite bodies identified and counted by microscopy in 31 dusts (r = 0.88, 0.86 and 0.82 for combined Dermatophagoides sp., D. pteronyssinus and D. farinae group, resp.) Dermatophagoides allergen was recorded in 21/22 mattress dusts (median: 26,000 ng/g; maximum: 290,000 ng/g). D. pteronyssinus allergen occurred in largest amounts (median 7,500 ng/g) followed by D. microceras (median 650 ng/g) and D. farinae (median 240 ng/g).     

Enzyme-linked immunosorbent assay for quantifying antigens of Dermatophagoides farinae and D. pteronyssinus (Acari: Pyroglyphidae) in house dust samples

Double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody as capture and detector antibodies was developed for quantifying antigens of Dermatophagoides farinae Hughes and D. pteronyssinus (Trouessart) mites contained in house dust samples. This monoclonal antibody was directed against mite antigens that were also reactive to immunoglobulin E antibody in all of 10 serum samples obtained from patients allergic to mites. Histological study using fluorescent antibody revealed that the monoclonal antibody was bound to the major part of the D. farinae mite body section including fecal matter and cuticles. The detection limit of the assay system was 0.17 microgram of soluble antigens of both mite species and the antigen amount corresponding to 0.5 mites per microplate well, whether in live or dead mites. This system did not react to antigens of Tyrophagus putrescentiae (Schrank) and Cheyletus malaccensis Oudemans. Slight inhibition of less than or equal to 21.3% was observed with nonspecific substances contained in house dust, such as wool, cotton, human dander, human hair, soil, and biscuit, but no direct ELISA reactions were obtained with any of these materials. In 49 house dust samples, ELISA was significantly correlated with the conventional microscopic observation method.     

The role of IgG4 as blocking antibodies in asthmatics and in bee keepers

Sera from 40 asthmatic patients and from 77 bee keepers were evaluated by solid-phase radioimmunoassay for their allergen-specific IgG4 antibody levels. The results indicated that allergen-specific IgG4 antibodies become prominent upon repeated parenteral stimulation with antigen, i.e. immunotherapy and bee stings, and suggested the possible association of IgG4 with blocking antibodies in these allergic conditions.     

The effect of immunotherapy on the in vitro productions of histamine, prostaglandin E2 and leukotriene C4 in asthmatic children

In order to elucidate the working mechanisms of immunotherapy (IT), the in vitro productions of histamine, prostaglandin E2 (PGE2) and leukotriene C4 (LTC4) were studied in 18 newly diagnosed and 20 hyposensitized (greater than 2 yr) asthmatic children. All were sensitive to house dust and dust mites. (D. pteronyssinus). Ten age-matched normal children were included as control. Polymorphonuclear (PMNs) and mononuclear (MNCs) leukocytes were separated by density gradient centrifugation and dextran sedimentation. PMNs (2 x 10(7) cells/ml) and MNCs (2 x 10(7) cells/ml) were stimulated with mite allergen (10 micrograms/ml) and calcium ionophore A23187 (1 microgram/ml) for 15 minutes. The plasma and culture supernatant (sup) histamine levels and sup PGE2 and LTC4 were measured by RIA. The results showed; 1) When compared to new patients, the treated patients had much lower plasma and sup histamine (p less than 0.001), no matter whether PMNs and MNCs were stimulated with allergen or A23187 and the normals had the lowest histamine level among 3 groups; 2) LTC4 in A23187-stimulated sup was lower in treated patients (p less than 0.05); 3) The PGE2 in allergen-stimulated sup was markedly increased in treated patients as compared to new patients (p less than 0.01) and the PGE2 in sup of normals was also much higher than that of new patients. Thus, immunotherapy is able to reverse the abnormal secretory pattern of inflammatory mediators of allergic patients, and this change may account, partly, for its clinical effectiveness.     

Enzyme-like immunosorbent assay (ELISA) for immunoglobulin G antibodies against insect venoms

IgG "blocking" antibodies were measured in patients receiving insect venom immunotherapy. The enzyme-linked immunosorbent assay (ELISA) described herein was found to be sensitive and reproducible. Results with ELISA correlated well with values obtained with a radioimmunoassay and with inhibition of the release of histamine from sensitive basophils. Also, specific antibody titers against phospholipase A and whole bee venom were correlated. Serial determinations of venom-specific IgG antibodies were made in 17 patients receiving Polistes wasp or bee venom immunotherapy. The majority of patients showed a rise in IgG antibodies, which peaked after administration of approximately 500 micrograms of venom. Only one out of 13 of these venom-treated patients had allergic symptoms after an insect sting while on maintenance therapy.     

Clinical relevance of the venom-specific immunoglobulin G antibody level during immunotherapy

Parameters associated with successful venom immunotherapy in insect allergy were sought by comparison of treatment failures with successes. Half-dose treatment was completely protective in 32 patients (successes) but was only partially effective in eight (failures). The outcome of treatment was not related to the severity of pretreatment sting reactions, to the degree of skin-test sensitivity, to an atopic personal history, or to age or gender. The mean yellow jacket venom-specific IgG antibody level (by the Staph-A solid-phase radioimmunoassay) was significantly less in the failures (3.9 ± 0.6 μg/ml) than in the successes (7.3 ± 1.1 μg/ml) (p < 0.01). When the failures were successfully treated, their mean IgG level (6.1 ± 1.3 μg/ml) was significantly greater than that associated with treatment failure (p < 0.025). Patients with an IgG antibody level above 5.0 μg/ml were significantly more likely to be fully protected (p < 0.02). Those whose IgG levels were less than 5 μg/ml had a risk of reaction similar to that in untreated patients. We conclude that early in the maintenance phase of low-dose venom immunotherapy, the risk of a reaction to a challenge sting is significantly greater for those patients with low levels of venom-specific IgG antibodies.     

Comparison of a multi-allergen dipstick IgE assay to skin-prick test and RAST

A multi-allergen dipstick enzyme immunoassay 'Quidel Allergy Screen' (QAS) has recently been developed commercially for measuring IgE antibodies against nine allergens (house dust 1, house dust 2, Dermatophagoides pteronyssinus, D. farinae, Japanese cedar, ragweed, cat dander, sweet vernal grass, and egg white) at one time. To assess whether this assay is useful in screening allergen-specific IgE antibody, we compared the titres of IgE antibodies against the nine allergens measured by QAS to those in the skin-prick test and by RAST in 93 atopic asthmatics and 22 normal subjects. We found a good overall agreement between the results of the skin-prick test and the results of QAS (sensitivity = 47.1-81.4%, specificity = 84.5-100%, and agreement = 78.9-88.9%). The sensitivities against house dust 1, D. pteronyssinus, and D. farinae ranged from 77.2 to 81.4%. However, the sensitivities against house dust 2, Japanese cedar, ragweed, and cat dander were low (47.1-68.8%). We also found a good overall agreement between the results of RAST and the results of QAS, except for egg white (sensitivity = 46.2-94.4%, specificity = 87.4-100%, and agreement = 77.4-96.5%). The sensitivities against house dust 1 and 2, D. pteronyssinus, D. farinae, and Japanese cedar ranged from 86.0 to 94.4%. The sensitivities against ragweed, cat dander, and sweet vernal grass were low (46.2-52.6%). There were strong correlations between the titres of RAST and the titres of QAS except cat dander and egg white (r = 0.701-0.924 for the seven allergens). Thus, we conclude that QAS is useful in screening IgE antibodies against multiple allergens at one time. However, because the sensitivities against some allergens tested were low, further improvement of some allergen preparations seems to be necessary in the assay.