Measurement of murine IgE antibody responses to dust mite allergens by in vitro assay.

In an analysis of murine immune responses to the dust mite allergen Der p 1, treatment with purified allergen induced a significant increase in the level of circulating IgE immunoglobulin (from less than 100 ng/ml in normal mice to 1,350 ng/ml in mice receiving the allergen). Even so, specific IgE antibodies binding to purified Der p 1 were not detected in a conventional ELISA, and the major response appeared to be the induction of high titre IgG antibodies. Specific circulating murine IgE antibodies were however detected using the following assay format: murine IgE was captured to anti-murine IgE antibody coated wells; Der p 1 was added and bound by immobilized anti-Der p 1 IgE antibodies; the captured Der p 1 was then detected by the addition of monoclonal IgG antibodies against Der p 1 and these antibodies were measured by the addition of anti-murine IgG antibody-enzyme conjugate with which colour development is produced after substrate addition. This assay establishes a procedure to measure circulating anti-Der p 1 IgE antibodies which are present together with competing high titre IgG anti-Der p 1 antibodies.     

Evidence that negative feedback between antibody concentration and affinity regulates humoral response consolidation to a non-infectious antigen in infants

The dynamics of human antigen-specific immunoglobulin (Ig) responses in early life are not well characterized. We have previously observed an inverse relationship between allergen-specific Ig concentration and allergen-Ig-binding affinity in allergen-sensitive atopic adults, suggesting a possible feedback relationship between these variables. We prospectively studied children (6 months to 6 years) with and without atopic sensitization to the Der p 1 major allergen. Experimental results showed the following trends. (1) In both study groups, there was little change with age in average Der p 1-specific Ig (IgG1 or IgE) concentrations or allergen-Ig-binding affinities, and concentrations and affinities were independent. (2) Among individuals, however, there was a negative correlation between Ig concentration changes and affinity changes with age. (3) The rate of increase with age of the non-atopic Der p 1-IgG1 total binding capacity (Ig concentration x Ig affinity) paralleled that for the atopic Der p 1-IgE total binding capacity, and there was a comparable 'consolidation' of responses with age reflected by a narrowing of the variance of total binding capacity values. Except for the Ig classes involved, development of a humoral response to a non-infectious allergen is similarly regulated in atopic and non-atopic children, with Ig total binding capacity as the key regulatory variable. These results also suggest that there is a time-dependent feedback relationship between Ig concentrations and affinities that establishes an optimal Ig total binding capacity for a given environmental 'antigen load'. A theoretical model is proposed to account for this relationship.     

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.     

Increased airway responsiveness, allergy-type-I skin responses and systemic anaphylaxis in a humanized-severe combined immuno-deficiency mouse model

BACKGROUND: In patients with allergic bronchial asthma, a strong relationship between elevated serum IgE antibody titres and the development of increased airway responsiveness (AR) has been demonstrated. To further elucidate the relationship between human (hu) IgE and development of increased AR, we developed an in vivo model utilizing immuno-compromised severe combined immuno-deficiency (SCID) mice. METHODS: SCID mice were either reconstituted with peripheral blood mononuclear cells (PBMC) from non-atopic, healthy or atopic individuals sensitized against house dust mite allergen (Der p), or passively sensitized with plasma from non-atopic, healthy or atopic individuals. RESULTS: In both systems, atopic hu-SCID mice developed increased AR. The following results suggest that these responses were mediated via IgE antibodies: increased AR did not occur after transfer of either PBMC or IgE-negative plasma from non-atopic individuals; increased AR occurred simultaneous with increased serotonin release detected 15 min after allergen-aerosol challenge in bronchoalveolar lavage fluid; and increased AR required at least two allergen-aerosol challenges. SCID mice reconstituted with serum containing anti-Der p IgE antibodies developed positive immediate-type skin test responses to intradermal injection of Der p as well as anti-hu-IgE antibody. In addition, IgE binding to skin mast cells was demonstrated by immunohistochemistry. Furthermore, intravenous challenge of hu anti-Der p positive SCID mice with Der p resulted in systemic anaphylaxis. CONCLUSION: These data provide evidence that passive immunization of SCID mice with hu IgE alters AR and that T cells and eosinophils were not a requirement for the development of increased AR in this model.     

Inhibition of allergen-IgE binding to B cells by IgG antibodies after grass pollen immunotherapy

BACKGROUND: Among atopic individuals, levels of allergen-specific IgG antibodies have been inversely associated with the degree of allergen sensitization. Additionally, allergen-specific IgG antibodies are markedly increased by allergen injection immunotherapy. These observations have led to proposals that allergen-specific IgG antibodies might have protective properties in atopic individuals. OBJECTIVE: We hypothesized that after grass pollen immunotherapy, these antibodies disrupt IgE-dependent allergen processing by antigen-presenting cells. METHODS: We have developed a novel flow cytometric assay based on detection of allergen-IgE binding to the low-affinity IgE receptor on B cells to examine the blocking effects of sera collected from 18 patients who participated in a double-blind, controlled trial of grass pollen immunotherapy for 1 year. RESULTS: In all 10 patients who received active therapy, there was induction of activity that inhibited allergen-IgE binding to B cells (P =.02, vs placebo subjects), as well as subsequent allergen presentation to T cells. This activity copurified with IgG and was allergen specific, because sera taken from patients treated with grass pollen immunotherapy but who were also birch pollen sensitive did not inhibit IgE-birch pollen allergen binding to B cells. CONCLUSION: We conclude that allergen-specific IgG antibodies induced by immunotherapy can disrupt formation of allergen-IgE complexes that bind to antigen-presenting cells and facilitate allergen presentation.     

Role of IgE in atopic dermatitis

Atopic dermatitis is a chronic inflammatory skin disease associated with elevated serum IgE levels and sensitization to a variety of inhalant, food and microbial allergens. Controlled challenges have provided substantial evidence that allergens can trigger acute IgE-mediated mast-cell dependent exacerbations of eczema in these patients. However, the sustained chronic skin inflammation that characterizes atopic dermatitis is likely to result from a local expansion of allergen-specific T helper type 2 cells that produce interleukin-4 and interleukin-5 and the concomitant infiltration of eosinophils. An important role for IgE in allergen presentation to T helper type 2 cells by Langerhans cells has been proposed. These observations may have important implications for the development of new approaches for the treatment of this increasingly common allergic disorder.     

Effect of mattress encasings on atopic dermatitis outcome measures in a double-blind,placebo-controlled study: the Dutch mite avoidance study

BACKGROUND: House dust mite (HDM) allergen might induce and maintain atopic dermatitis (AD). Reduction of allergen load by applying encasings might improve the clinical symptoms of AD. OBJECTIVE: We sought to investigate, in a randomized, double-blind, placebo-controlled study, whether reducing HDM allergen levels by using mattress, duvet, and pillow encasings for 12 months will result in improvement in AD symptoms. METHODS: Patients with AD (8-50 years old and allergic to HDM), having a Leicester sign score (a dermatitis score) of at least 1% extent and a severity score of 6 points or greater, were randomly allocated to an active (n = 45) or a placebo allergen-avoidance group (n = 41). Avoidance measures consisted of applying HDM-impermeable encasings for mattresses, pillows, and duvets for the active treatment group and cotton encasings for the placebo group. Effect on allergen concentrations (Der p 1 and Der p 1 plus Der f 1), Leicester sign score extent and severity, visual analogue scale scores for itching and sleeplessness, intradermal test results, atopy patch test results, total serum IgE levels, anti-Der p 1-specific IgE levels, and total blood eosinophil counts were studied. RESULTS: The active encasings reduced the Der p 1 allergen concentration in the mattress after 12 months with a factor 2.1 (P =.007) and the Der p 1 plus Der f 1 allergen concentration with a factor of 2.5 (P =.005); no significant change in allergen concentrations in mattresses was seen in the placebo group. Although the decrease in allergen load was significant, no differences in treatment-induced changes were seen between the placebo and active groups. CONCLUSIONS: Use of HDM-impermeable encasings resulted in a significant decrease in Der p 1 and Der p 1 plus Der f 1 allergen concentrations. However, this reduction in allergen load did not result in significant changes in clinical parameters between the groups. Reduction of allergens in other environments (work, school, and outdoors) might be equally important in improving symptoms of AD.     

Random outcomes of allergen-specific responses in atopic families

BACKGROUND: Allergens are common non-infectious antigens to which people will mount T cell dependent humoral responses. Among genetically susceptible individuals, an antigen-specific response results involving the production of allergen-specific IgE (atopy). OBJECTIVE: Determine if this susceptibility is manifested as an inherited, allergen-specific trait or a random response to allergens among susceptible people. METHODS: We evaluated allergen-specific outcomes in 1099 members of families with positive atopic history (26 multi-generation and 112 nuclear families). Each was tested for sensitivity to 14 common allergens by standardized skin prick test (SPT), a marker of specific IgE production. Over 15,000 individual SPT's were evaluated. Among five randomly selected multi-generation families (N=163), semi-quantitative determinations of Amb a 1-specific IgA1,2 and IgG1-4 were determined in three groups: (A) Amb a SPT(+)/Amb a 1-IgE(+), (B) Amb a SPT(-)/Amb a 1-IgE(+), (C) Amb a SPT(-)/Amb a 1-IgE(-). RESULTS :By rank correlation statistics, there were no discernible 'patterns' of specific SPT outcomes among any of the multi-generation families, suggesting that environmental exposure rather than allergen-specific inheritance determined the responses. This was confirmed among the nuclear families since the conditional SPT outcomes among children were independent of the SPT responses of their parents. Among five randomly selected multi-generation families, the relative proportionate concentrations of the Amb a 1-specific IgA and IgG subclasses were comparable, regardless of atopic sensitization to the ragweed allergen Amb a. CONCLUSION: While the general propensity for atopy may be inherited, an individual's specific atopic outcome is a random variable independent of familial sensitization patterns.     

C3-containing IgE immune complexes in asthmatic patients.

Higher levels of IgE-containing immune complexes (IC) have been reported in sera from patients with allergic diseases than in sera from controls. To evaluate the possibility of an IC-mediated mechanism in the pathogenesis of bronchial asthma, we measured circulating C3-containing IgE IC (C3-IgE IC) using anti-C3 ELISA from 20 house dust mite (HDM)-sensitive asthmatics, 20 non-atopic asthmatics, and 14 non-atopic controls. C3-IgE IC levels were significantly higher in HDM-sensitive asthmatics (mean +/- S.D.: 12.2 +/- 7.8 AU/ml) than in non-atopic asthmatics (6.5 +/- 7.5 AU/ml) or controls (5.8 +/- 4.4 AU/ml). C3-IgE IC levels were significantly correlated with HDM-specific IgE levels (r = 0.50, p < 0.05), but not with total IgE levels (r = 0.36, p > 0.05) in HDM-sensitive atopic asthmatics. C3-IgE IC levels in sera did not significantly change during HDM-bronchoprovocation test in six HDM-sensitive asthmatics who showed positive reaction. Part of C3-IgE IC could be precipitated by protein G coupled beads. In conclusion, C3-IgE IC levels were elevated in sera from HDM-sensitive asthmatics; moreover IgG antibodies might be a component of C3-IgE IC. Our results suggest that an IgE IC-mediated mechanism could be involved in the pathogenesis of atopic asthma.     

Association between severity of atopic eczema and degree of sensitization to aeroallergens in schoolchildren

BACKGROUND: A subgroup of patients with atopic eczema exhibits aggravation through contact with aeroallergens. Little is known from population-based studies, however, about the association between the severity of eczematous skin disease and the degree of aeroallergen sensitization. OBJECTIVE: We sought to investigate the relationship between IgE-mediated allergic sensitization to aeroallergens and severity of atopic eczema in schoolchildren. METHODS: A nested case-control analysis on atopic eczema was performed on the basis of a cross-sectional study of 2201 East German schoolchildren aged 5 to 14 years. Atopic eczema and its severity was identified by dermatologic examination. Total and allergen-specific IgE antibodies to grass and birch pollen, Cladosporium herbarum, Dermatophagoides pteronyssinus, and cat epithelium in serum were determined, and additional information was obtained by means of standardized questionnaire. RESULTS: The overall prevalence of actual atopic eczema was 2.5%. Thirty-seven percent of the children were sensitized to at least one allergen. Children with atopic eczema were significantly more often sensitized than those without skin disease (75.0% vs 36.3%; odds ratio, 5.27; 95% confidence interval, 2.54-11.15). This was observed for each single allergen. The prevalence of atopic eczema increased significantly with increasing RAST class (chi(2) trend test for each allergen, P <.0001). Also, the prevalence of sensitization increased with the severity of the disease (chi(2) trend test for each allergen, P <.0001). This association was pronounced for house dust mite and cat allergen. Multiple linear regression analyses showed significant associations between the severity score of atopic eczema and concentrations of allergen-specific IgE to dust mite (P =.032) and cat (P =.014) allergens after adjustment for sex, age, location, and parental predisposition. CONCLUSIONS: The degree of sensitization is directly associated with the severity of atopic eczema. We speculate that early epicutaneous sensitization to aeroallergens may be enhanced by damage of the skin barrier function. The specific IgE response seems to contribute to the severity of the disease in a dose-dependent fashion.     

Variable binding affinities for allergen suggest a 'selective competition' among immunoglobulins in atopic and non-atopic humans

Atopy is a persistent, aberrant humoral response to certain classes of proteins (allergens) characterized by the presence of allergen-specific IgE. Yet, in both atopic and non-atopic individuals, allergen-specific responses involving the IgA and IgG subclasses have been observed, which evidence does not support models suggesting inherited differences in sensitivity to certain protein classes. Using the major ragweed component Amb a 1 as a model allergen, we assessed the humoral responses in three groups of unrelated donors: (A) atopic, ragweed sensitive; (B) atopic, but not ragweed sensitive; (C) non-atopic. As expected, Amb a 1-specific IgE was present in group A only. However, there were essentially no differences in the relative proportions of Amb a 1-specific IgA(1,2) and IgG(1-4) among the groups. We also determined the Amb a 1 binding affinities for IgG(1) and IgG(4) in the three groups, and compared these to Amb a 1-specific IgE binding affinities in group A. Group A donors' Amb a 1-IgE had extremely high affinities (10(8) to 10(11)M(-1)), but their Amb a 1-IgG(1) and Amb a 1-IgG(4) affinities were significantly lower (10(7) to 10(10)M(-1)). The average IgG(4) binding affinities in groups B and C were slightly higher than that of IgG(4) in group A, although not statistically significant. However, the IgG(1) affinity for Amb a 1 among group C, non-atopic donors was significantly elevated and comparable to the IgE affinity observed in group A, ragweed atopics. Inhibition studies with allergen-specific IgE-free serum showed that all isotypes recognized the major epitopes seen by IgE. These results suggest that there may be a "selective competition" among isotypes for allergens that is driven by the ability to produce high affinity, allergen-specific immunoglobulins.     

Allergen-specific IgG1 provides parsimonious heritability estimates for atopy-associated immune responses to allergens

Although serum total immunoglobulin E (IgE) is generally elevated in atopic conditions, it is an unreliable trait for dissecting the genetic and environmental components contributing to atopic immune responses, because it can be significantly confounded by demographic factors (age, gender, and race) and clinical status (atopic vs nonatopic). Allergen-specific IgE is a discontinuous trait present only in those with sensitivity to allergens. However, all people will produce allergen-specific immunoglobulin G1 (IgG1), which is elevated among those atopically sensitized to specific allergens. We screened 91 Caucasian nuclear families (N = 367) with medical histories of atopic diseases and used variance components analysis to compare heritability estimates for total IgE and IgG1 produced against the common major allergen from house dust mite Dermatophagoides pteronyssinus (Der p 1). An estimate of total IgE heritability was about 48%, although this was significantly confounded by age, gender, and clinical atopic status. In contrast, Der p 1-IgG1 demonstrated a significant inherited component of about 62% that was not influenced by age, gender, or clinical status. For genetic studies of atopic humoral responses, allergen-specific IgG1 may be a more reliable quantitative trait than serum IgE. Moreover, atopy is an inherited deregulation of immune responses to noninfectious antigens, involving antibody isotypes other than IgE.     

Differences in titres of IgE, IgG4 and other IgG subclass anti- Der p 2 antibodies in allergic and non-allergic patients measured with recombinant allergen

Background The mite allergens are recognized as major causes of allergic disease such as bronchial asthma, allergic rhinitis and atopic dermatitis. The functions of allergen-specific IgG subclass antibodies are not defined.
Objective In order to clarify the relationship between IgE and IgG subclasses, we examined scrum levels of the Dermatophagoides pteronyssisus group 2 (Der p 2)-specific antibodies of IgH. IgG total and IgG subclasses in children with mite allergy.
Methods We prepared a recombinant Der p 2 fusion protein and examined serum levels of Der p 2 antigen-specific antibodies by enzyme-linked immunosorbent assay (FLISA) systems developed in our laboratory using a recombinant Der p 2 as target antigen. Sera from 240 children with mite allergy and 25 controls were measured.
Results The serum levels of specific IgE and, to lesser degree, lgG4 were higher in allergic children than non-allergic controls, while in the levels of the other IgG subclasses there was no difference between the two groups. There was no correlation between levels of specific IgF and IgG4 or in those between specific IgG4 and other IgG subclasses.
Conclusion Results indicate that the induction of Der p 2-specific lgG4 in allergic diseases is independent to IgE as well as other IgG subclasses.     

Association between the occurrence of the anticardiolipin IgM and mite allergen-specific IgE antibodies in children with extrinsic type of atopic eczema/dermatitis syndrome

BACKGROUND: As the literature has only controversial data on the role of nonallergen-specific antibodies in atopic eczema dermatitis syndrome, the authors investigated the link between the occurrence of the antiphospholipid [anticardiolipin (ACL), anti-beta2-glycoprotein I] and allergen-specific immunoglobulin E (IgE) antibodies in 72 children with atopic eczema/dermatitis syndrome (AEDS). METHODS: The measurement of antiphospholipid antibodies was carried out by enzyme-linked immunosorbent assay (ELISA), serum total IgE by nephelometry, and allergen-specific IgE by immunoblotting assay. The statistical analysis was carried out by Fisher's exact test and odds ratio was calculated. RESULTS: Thirteen of 72 children with AEDS (mean age 8.3 years) had elevated serum levels of ACL, and eight anti-beta2-glycoprotein I antibodies. The presence of allergen-specific IgE against inhalant allergens and nutritive allergens was among eight of 13 and three of eight in the cases with elevated ACL. The ratio of patients with highly increased severity scoring of atopic dermatitis (SCORAD) index (>75) was significantly higher in the group with elevated (4/13) than in those with the normal ACL levels (2/59). There was a significant association between the appearance of mite (Dermatophagoides pteronyssinus, D. farinae)-specific IgE and ACL IgM antibodies (6/13). CONCLUSION: These findings show that there are significant linkage and association between the appearance of ACL IgM or the production of allergen-specific IgE against inhalant (mainly mite) allergens in children with atopic eczema/dermatitis syndrome.     

Epitope mapping and structural analysis of the anti-Der p 1 monoclonal antibody: insight into therapeutic potential

Group 1 allergen from Dermatophagoid pteronyssinus (Der p 1) belongs to the papain-like cysteine protease family and is a major cause of allergic rhinitis and asthma. An anti-Der p 1 monoclonal antibody, mAb W108, was selected and isolated from Der p-specific IgG2b-producing hybridoma clones. Two-dimensional electrophoresis and immunoblotting showed that mAb W108 reacted with four components of Der p extracts with a molecular mass of 35 kDa and pI values varying from 4 to 6; it also reacted with IgE antibodies in the sera of Der p-sensitive patients. In the competitive assay and using azocasein as a substrate, we found that mAb W108 inhibited not only the binding of Der p 1, but also its cysteine protease activity in a dose-dependent manner. The two peptide segments of Der p 1 identified by mAb W108 (aa 151–197 and 286–320) were parts of inter-connecting loops located in the substrate-binding cleft and on the surface of the domain comprising mainly β-sheets. From the predicted interaction between the amino acid sequence in the CDR3 of mAb W108 and Der p 1-binding epitopes, the possible binding sites for mAb W108 to Der p 1 may sterically hinder the IgE epitope and the active site of cysteine protease activity. Administration of mAb W108 in the Der p-sensitized murine model of asthma alleviated allergen-induced airway inflammation and the Th2 cytokine immune response, suggesting its therapeutic potential. These findings can provide new insights into understanding IgE-mediated disease and the design of modified allergen vaccines for future allergen-specific immunotherapy.     

A sensitive reverse ELISA for the measurement of specific IgE to Der p 2, a major Dermatophagoides pteronyssinus allergen.

BACKGROUND: Epidemiologic studies have shown that the presence of IgE antibodies to house dust mite and other indoor allergens is an important risk factor for asthma. OBJECTIVE: The aim of this study was to develop a reverse ELISA (rELISA) for measuring specific IgE to Der p 2, a major Dermatophagoides pteronyssinus (Dpt) allergen, as a potential tool for followup of allergen immunotherapy. METHODS: Recombinant Der p 2 allergen or a monoclonal antibody to Der p 2 was used to coat plates in conventional ELISA (cELISA) and rELISA, respectively. Sera from 48 asthmatic patients with positive skin prick test (SPT+) to D. pteronyssinus extract were analyzed for total IgE and specific IgE to Der p 2, and the results were compared with a group of 41 SPT asthmatic and 30 SPT- control subjects. RESULTS: The sensitivity of the two assays for Der p 2-specific IgE was 3.9 EU/mL and their specificities were confirmed by inhibition tests, in a dose-dependent manner. There was a significant positive correlation between cELISA and rELISA (r = 0.74; P < 0.0001). However, rELISA was more sensitive than was cELISA, regarding both the positive sera percentage (70.8% vs 52.1%) and the Der p 2-specific IgE levels (28.4 vs 4.5 EU/mL) in SPT+ asthmatic patients. CONCLUSIONS: rELISA has shown to be a sensitive and alternative method for measuring Der p 2-specific IgE without using radioactive techniques. Detection of specific IgE to major allergens and relevant peptides, and identification of B cell epitopes in allergens will provide valuable information for the design of allergen analogs and peptides for immunotherapy.     

Peptide specificity and HLA restriction do not dictate lymphokine production by allergen-specific T-lymphocyte clones.

Human and murine CD4+ T lymphocytes can be subdivided into distinct subsets [T-helper type 0 (Th0), Th1 or Th2], based on their lymphokine production profiles. Not much is known about the factors that determine these restricted lymphokine secretion profiles. Peptide specificity and human leucocyte antigen (HLA) restriction may be such factors. As it is well established that allergen-specific T lymphocytes from atopic individuals and non-atopic controls differ in their lymphokine secretion profile, we studied two allergen-specific T-lymphocyte clones (TLC) with identical peptide specificity and HLA restriction that were generated from the peripheral blood of an atopic donor and a non-atopic control donor. The two CD4+ TLC recognize the same epitope (20-33) of the house dust mite Dermatophagoides pteronyssinus major allergen Der p II. Both TLC recognize the epitope in an HLA-DQB1*0602-restricted manner. However, the lymphokine production profiles of these TLC show clear differences after allergen-specific or polyclonal activation. As expected, TLC JBD4 from the atopic donor produced high levels of interleukin-4 (IL-4) without detectable interferon-gamma (IFN-gamma), whereas TLC PBA1 from the non-atopic donor produced both IFN-gamma and IL-4 upon allergen-specific or polyclonal activation. Inasmuch as both TLC recognized the same epitope of Der p II in association with the same HLA-DQ molecule, these data suggest that peptide specificity and HLA restriction of human allergen-specific TLC do not dictate their lymphokine secretion profile.     

Evidence of an affinity threshold for IgE‐allergen binding in the percutaneous skin test reaction

BACKGROUND: Atopy is an aberrant immune response involving allergen-specific IgE production, though serum IgE concentration is not an entirely reliable diagnostic tool, particularly for epidemiological and genetic studies. There is no clear correlation between IgE and other indicators of atopy such as skin prick tests (SPT)s, and physiological associations are difficult to justify in cases with detectable IgE but negative SPT results. OBJECTIVE: IgE reflects the number of molecules available to produce an atopic response, but the degree of the response is determined by the binding strength (affinity) between receptor-bound IgE and the allergen. We sought to determine if there was an association between binding affinity and SPT results in people with histories of atopy. METHODS: Standard SPTs (whole allergen extracts) were administered to people with histories of sensitivities to ragweed and house dust mite. The concentrations and affinities of serum allergen-specific IgEs were determined using the purified allergens Amb a 1 and Der p 1. RESULTS: There was a positive correlation between weal area and allergen-specific IgE among SPT-positive donors. However, for those individuals with detectable amounts of allergen-specific IgE, there was considerable overlap of IgE values between SPT-positive and -negative groups. Among sensitized donors, IgE-allergen interactions were characterized by two or three specific reactions of very high affinity (K(A) range 10(8) -10(11) M). Negative SPT reactions were associated with lowered IgE binding affinities to major allergens. This delimited two groups with atopic disorders: specific IgE(+)/ SPT(+) and specific IgE(+)/SPT(-). CONCLUSION: The product of antibody affinity and concentration, which we define as antibody capacity (CAP = K(A) x IgE), is more informative with regard to describing allergen sensitivity than antibody concentration alone. Antibody binding capacity provides physiological evidence of atopy in some subjects who do not test positively by common methods and suggests an affinity threshold to produce a positive SPT reaction.     

CD14 promoter polymorphisms in atopic families: implications for modulated allergen-specific immunoglobulin E and G1 responses

BACKGROUND: CD14 promoter DNA sequence polymorphisms for the endotoxin receptor gene have been implicated in modulating allergen-specific immunoglobulin (Ig)E responses in randomly selected individuals with atopy. We sought to determine if a single nucleotide polymorphism in the CD14 promoter region is associated with atopy in atopic families, and to assess its influence on serum levels of CD14 and allergen-specific IgE and IgG1 responses. METHODS: We screened 367 members of 91 Caucasian nuclear families with a history of asthma for pulmonary function by spirometry, including methacholine challenge to detect bronchial hyperreactivity, and atopy by serum total IgE and skin prick test to 14 allergens. The CD14 promoter single nucleotide polymorphism was analyzed in DNA isolated from peripheral blood mononuclear cells to identify C/C, C/T and T/T genotypes. Serum tests were done for soluble CD14 (sCD14) and dust mite-specific antibody (Der p 1-IgG1). RESULTS: Serum sCD14 levels were not associated with clinical phenotypes (asthma, bronchial hyperreactivity or atopy). However, sCD14 levels were inversely related to both allergen-specific IgE and Der p 1-IgG1 production, but only among those with evidence of atopic sensitization. Linear regression analysis, accounting for random family effects, demonstrated a higher production of allergen-specific IgE or Der p 1-IgG1 associated with the T/T genotype and a lower level of specific IgE and IgG1 production associated with sCD14 levels. CONCLUSIONS: An element of the innate immune system (CD14) has profound effects upon modulating the acquired allergen-specific immunoglobulin responses among those with an inherited atopic predisposition.     

Purification and IgE binding capacity of Der s 3, a major allergen from Dermatophagoides siboney

Background Sensitization to the house dust mite Dermatophagoides siboney has been demonstrated in asthmatic patients. Previously, Dermatophagoides siboney group 1 and group 2 allergens, named Der s 1 and Der s 2, respeetively, have been purified. Objectives The aim of this study was to purify and to study the IgE reactivity of a 30 kDa component, suspected to correspond to group 3 allergens. Methods The protein was purified by affinity chromatography using anti-Der f 3 monoclonal antibodies and semi-preparative SDS-PAGE. The IgE binding capacity of the purified fractions was tested with sera from 106 mite- sensitive asthmatic patients using a modified chemiluminiscent method. Results Affinity chromatography resulted in fractions containing the 30 kDa component which was further purified to homogeneity by SDS-PAGE. Seventythree per cent of the sera showed IgE reactivity to this protein, indicating that it is a major allergen. The protein also reacted with anti Der f 3 polyclonal antibodies and had tryptic activity. There were differences in the reaetivity to Der s 3 according to the age of the patients. Conclusion Based on the frequency of IgE reactions and the reactivity with antibodies directed to Der f 3, it is proposed to name this 30 kDa allergen from D. siboney, Der s 3.