Antigen Der f I from the dust mite Dermatophagoides farinae: structural comparison with Der p I from Dermatophagoides pteronyssinus and epitope specificity of murine IgG and human IgE antibodies

The physicochemical and antigenic properties of an allergen purified from Dermatophagoides farinae, Der f I, were compared with Der p I from Dermatophagoides pteronyssinus. On SDS-PAGE, Der f I migrated as a single polypeptide chain with the same m.w. as Der p I (24,000). Two isoallergenic peaks of Der f I were identified on preparative isoelectric focusing (pI 5.7 to 6.3 and pI 6.6 to 6.95). Fractions from each peak were shown to have an identical amino acid composition (which was similar but not identical to Der p I) and the same N-terminal amino acid sequence. There was a good correlation between quantitative intradermal skin tests to both purified allergens and to D. farinae extract in mite-allergic patients, with positive results when using as little as 10(-5) micrograms/ml of Der f I. The majority of sera with detectable IgE antibody to D. farinae also had IgE antibody to Der f I both among children (29/42 = 69%) and adults (55/63 = 87%). By RAST, there was an excellent correlation between IgE antibody to Der f I and Der p I in sera from 42 mite-allergic children (n = 0.94, p less than 0.001). Polyclonal IgG antibodies from six mice immunized with Der f I showed preferential binding to that allergen, and most monoclonal antibodies (16 of 18) raised against Der f I did not bind Der p I. However, two monoclonal antibodies from this fusion showed cross-reactive binding to both allergens. Immunoabsorption experiments, using D. pteronyssinus and D. farinae extracts coupled to Sepharose, showed that a large proportion of murine antibodies (74% to Der p I and 60 to 93% to Der f I) could not be absorbed by the heterologous extract on the immunosorbent. In contrast, in sera from seven mite-allergic patients, most of the specific IgE and IgG antibody (i.e., greater than or equal to 82%) was removed by either immunosorbent. Thus, Der f I and Der p I represent a homologous pair of major allergens which possess both cross-reacting and species-specific epitopes. The antibody response in mice immunized with either allergen in complete Freund's adjuvant was largely directed against species-specific epitopes, whereas in allergic humans, IgE- and IgG-specific antibodies bound predominately to cross-reacting epitopes.     

The prevalence of increased serum IgE and Aspergillus sensitization in patients with COPD and their association with symptoms and lung function

Background

Allergy and Aspergillus hypersensitivity (AH) were shown to be associated with severe symptoms or worse lung function in COPD patients. The prevalence of elevated total IgE (T-IgE) and its association with clinical symptoms and lung function in COPD have not been studied. The prevalence of AH and its correlation with clinical characteristics in a COPD cohort of larger sample size is also lacking.

Methods

273 patients with COPD were evaluated by respiratory symptoms, blood test, chest HRCT, lung function, serum detection of T-IgE and Aspergillus specific IgE. Patients with T-IgE ≥ 1000 KU/L were further investigated for allergic bronchopulmonary aspergillosis (ABPA).

Results

The prevalence of elevated T-IgE and AH in patients with COPD was 47.3% and 15.0%, respectively. Eight patients (2.9%) met the diagnostic criteria for ABPA. Compared with the normal T-IgE group, patients with elevated T-IgE had a longer history of dyspnea (p < 0.01), an earlier onset of dyspnea after chronic cough/expectoration (p < 0.01), and were more likely to wheeze (p < 0.01). They also showed worse lung functions and more severe GOLD staging (p < 0.01). Analysis of the clinical data in male patients with smoking as the risk factor showed the same results. To evaluate the clinical characteristics of COPD with AH, patients with elevated T-IgE were further divided into subgroups with and without AH. When compared with the normal T-IgE group, both the two subgroups showed longer history of dyspnea (p < 0.01), an earlier onset of dyspnea (p < 0.01) and a worse status of lung function (p < 0.05). Correlation analysis demonstrated that T-IgE was correlated positively with the time length of dyspnea (r = 0.401, p < 0.001), and the ratio of duration of dyspnea to that of chronic cough/expectoration (r = 0.59, p < 0.001), but negatively with FEV1/FVC% (r = −0.194, p = 0.001), and FEV1%predicted (r = −0.219, p < 0.001).

Conclusions

There was a high prevalence of elevated serum T-IgE and AH in patients with COPD. Serum T-IgE level was correlated with symptoms such as dyspnea and impairment of lung function. Allergens other than Aspergillus may have similar effects on disease expression or progression of COPD.     

Epitope mapping of two major inhalant allergens, Der p I and Der f I, from mites of the genus Dermatophagoides

The repertoire of antigenic sites on two major dust mite allergens, Der p I of Dermatophagoides pteronyssinus and Der f I of D. farinae, was studied using murine (BALB/c) monoclonal antibodies (Mab), polyclonal rabbit IgG antibodies, and human IgE antibodies. Fifty-three IgG Mab were analyzed from six different fusions (five vs Der p I, one vs Der f I). By antigen binding radioimmunoassay (RIA), most Mab were either Der p I or Der f I specific, and only 2/53 bound to both allergens. Epitope mapping studies using cold Mab to inhibit the binding of six 125I labeled Mab to solid phase allergen defined four nonrepeated, nonoverlapping epitopes on Der p I, a single species-specific epitope on Der f I and a cross-reacting epitope present on each allergen. All but one of the 53 Mab bound to one of these six epitopes. Seventy percent (25/35) of anti-Der p I Mab were directed to the same epitope, suggesting that this epitope is immunodominant for BALB/c mice. Similarly, 88% (16/18) of anti-Der f I Mab bound to the same epitope on Der f I. Parallel cross-inhibition curves were obtained using the species-specific Mab, 10B9, and the cross-reacting Mab, 4C1, to compete for binding to Der p I, suggesting that the epitopes defined by these two Mab on Der p I are adjacent to one another. Both murine Mab and polyclonal rabbit IgG antibodies to cross-reacting sites on both allergens were used to inhibit binding of human IgE antibodies to Der p I by using 19 sera from mite allergic patients. Cross-reacting rabbit IgG antibodies strongly inhibited all sera tested (mean 79.5% +/- 7.7) and two Mab, 10B9 and 4C1, partially inhibited (38% +/- 12). However, the four Mab directed against separate species-specific epitopes (including murine immunodominant sites) showed little or no inhibition (less than or equal to 20%). Our results suggest that most of the epitopes defined by Mab are not the same as, or close to, those defined by human IgE antibody. The striking differences in the repertoires of murine IgG and human IgE antibody responses to Der p I and Der f I could be explained by genetic differences or by altered antigen processing and presentation occurring as a result of different modes of immunization in mice and in mite allergic humans.     

Another smoking hazard: raised serum IgE concentration and increased risk of occupational allergy.

Individual smoking histories of a general population sample and of two groups of workers exposed to occupational allergens were related to serum IgE concentrations and results of radioallergosorbent and prick tests in the workers. The geometric mean IgE concentration was higher in smokers than in non-smokers. The distribution of serum IgE values in the two groups showed an apparent difference, with a bimodal appearance in the smokers. Evidence of sensitisation against occupational allergens was more common in workers who smoked. The adjuvant effect of smoking on IgE antibody production might be due to damage to airways mucosa and supports the mucosal theory of atopy.     

Measurement of IgG blocking antibodies by interference in the radioallergosorbent test

We previously found that sera of patients immunized with ragweed pollen extract contained a factor that interfered with the binding of IgE antibodies to solid-phase allergens in the radioallergosorbent test (RAST). We now describe an assay, RAST interference, to measure this factor, and we present evidence that the factor is IgG blocking antibody. Sera from immunized allergic patients were heated at 56 degrees C for 4 hr to destroy heat-labile Fc determinants on IgE and were tested for their ability to prevent binding of additional IgE antibody to solid-phase allergens in the RAST. Eight of 10 sera from allergic immunized patients gave RAST interference dose-response curves that did not differ from the arbitrary standard. The factor causing interference showed specificity for the immunizing antigen, was heat-stable, eluted from Sephadex G-200 in the 7S peak, was present only in sera of immunized patients, and rose after initiation of immunization. These results indicated that RAST interference can be used to measure IgG blocking antibodies with the same reagents employed for the measurement of IgE antibodies, provided the antiserum to IgE is specific for the heat-labile FC determinants on IgE.     

B cell epitopes of the major timothy grass pollen allergen, phl p 1, revealed by gene fragmentation as candidates for immunotherapy.

Group 1 grass pollen allergens are recognized by IgE antibodies of almost 40% of allergic individuals and therefore belong to the most important elicitors of Type I allergy worldwide. We have previously isolated the cDNA coding for the group 1 allergen from timothy grass, Phl p 1, and demonstrated that recombinant Phl p 1 contains most of the B cell as well as T cell epitopes of group 1 allergens from a variety of grass and corn species. Here we determine continuous B cell epitopes of Phl p 1 by gene fragmentation. IgE antibodies of grass pollen allergic patients identified five continuous epitope-containing areas that on an average bound 40% of Phl p 1-specific IgE antibodies and were stably recognized in the course of disease. In contrast to untreated patients, patients undergoing grass pollen immunotherapy started to mount IgG(4) antibodies to the recombinant IgE-defined fragments in the course of immunotherapy. The protective role of these IgG(4) antibodies is demonstrated by observations that 1) increases in rPhl p 1 fragment-specific IgG(4) were in parallel with decreases in Phl p 1-specific IgE, and 2) preincubation of rPhl p 1 with patients sera containing rPhl p 1 fragment-specific IgG(4) blocked histamine release from basophils of an untreated grass pollen allergic patient. We propose to use recombinant Phl p 1 fragments for active immunotherapy in order to induce protective IgG responses against IgE epitopes in grass pollen allergic patients. This concept may be applied for the development of allergy vaccines whenever the primary sequence or structure of an allergen is available.     

Carriage of DRB1*13 is associated with increased posttreatment IgE levels against Schistosoma mansoni antigens and lower long-term reinfection levels

Praziquantel treatment for Schistosoma mansoni infection enhances Th2 responsiveness against parasite Ags, but also increases the variance in Ab isotype levels. This effect may arise partly from genetic heterogeneity. In this study, associations between HLA polymorphisms at three loci (HLA-DQB1, HLA-DQA1, and HLA-DRB1) and posttreatment Ig responses to S. mansoni Ags were assessed in 199 individuals aged 7-50 years from Uganda. Blood samples were assayed for IgG1, IgG4, and IgE levels against soluble worm Ag (SWA), soluble egg Ag, tegument Ag, and a recombinant tegumental Ag (rSm 22.6) 7 wk after treatment. Multivariate ANOVA analysis initially revealed associations between carriage of DRB1*13 and increased levels of IgG1, IgG4, and IgE against SWA, tegument Ag, and rSM22.6. Subsequent analysis of covariance, which controlled for correlations between isotype levels and also included pretreatment IL-4, IL-5, and IL-13 responsiveness against SWA as covariates, revealed an independent association only between DRB1*13 and a factor score summarizing IgE levels to worm-derived Ags, which was strongest in adults. A post hoc age- and sex-stratified analysis revealed lower reinfection intensities at 1 year, 22 mo, and 6 years after the first round of treatment among carriers of DRB1*13. These results indicate that genetic background has a prominent influence on the posttreatment Th2 immune response to S. mansoni Ags, as well as a downstream association with long-term reinfection levels.     

Three-dimensional structure of the cross-reactive pollen allergen Che a 3: visualizing cross-reactivity on the molecular surfaces of weed, grass, and tree pollen allergens

Two EF-hand calcium-binding allergens (polcalcins) occur in the pollen of a wide variety of unrelated plants as highly cross-reactive allergenic molecules. We report the expression, purification, immunological characterization, and the 1.75-A crystal structure of recombinant Che a 3 (rChe a 3), the polcalcin from the weed Chenopodium album. The three-dimensional structure of rChe a 3 resembles an alpha-helical fold that is essentially identical with that of the two EF-hand allergens from birch pollen, Bet v 4, and timothy grass pollen, Phl p 7. The extensive cross-reactivity between Che a 3 and Phl p 7 is demonstrated by competition experiments with IgE Abs from allergic patients as well as specific Ab probes. Amino acid residues that are conserved for the two EF-hand allergen family were identified in multiple sequence alignments of polcalcins from 15 different plants. Next, the three-dimensional structures of rChe a 3, rPhl p 7, and rBet v 4 were used to identify conserved amino acids with high surface exposition to visualize surface patches as potential targets for the polyclonal IgE Ab response of allergic patients. The essentially identical three-dimensional structures of rChe a 3, rPhl p 7, and rBet v 4 explain the extensive cross-reactivity of allergic patients IgE Abs with two EF-hand allergens from unrelated plants. In addition, analyzing the three-dimensional structures of cross-reactive Ags for conserved and surface exposed amino acids may be a first approach to mapping the conformational epitopes on disease-related Ags that are recognized by polyclonal patient Abs.     

Eosinophil cationic protein, specific IgE and IgG4 in human toxocariasis

Among 67 French patients presenting a toxocaral infection, various demographic, environmental, clinical and laboratory parameters (blood eosinophil count, eosinophil cationic protein (ECP), serum total IgE, specific IgE against common inhalant allergens, specific IgE and IgG4 against Toxocara excretory-secretory antigens) were investigated. Correlation studies and logistic regression analyses were conducted, testing elevated levels of ECP, specific anti-Toxocara IgE or IgG4 as outcome variables An elevated ECP level was significantly associated with both cough and rhinitis, a high level of specific anti-Toxocara IgE with itchy rashes and possible atopic status, and an increase of specific anti-Toxocara IgG4 with rural residence.     

Interaction of smoking and atopy in producing specific IgE antibody against a hapten protein conjugate.

A survey was carried out of a population of workers exposed to tetrachlorophthalic anhydride, an acid anhydride epoxy resin curing agent known to cause asthma. Using a radioallergosorbent test with a tetrachlorophthalic anhydride human serum albumin conjugate, specific IgE antibody was detected in serum from 24 out of 300 factory floor workers exposed to tetrachlorophthalic anhydride. Of these 24, 20 (83.3%) were current smokers compared with 133 (48.2%) of 276 without antibody (p less than 0.01), and there was a weaker association with atopy, defined by skin tests with common allergens. Smoking and atopy interacted, the prevalence of antibody being 16.1% in atopic smokers, 11.7% in non-atopic smokers, 8.3% in atopic non-smokers, and nil in non-atopic non-smokers (p less than 0.025). Smoking may predispose to, and interact with atopy in, the production of specific IgE antibody to this hapten protein conjugate.     

Antigens of Ambrosia elatior (short ragweed) pollen. III. Crossed radioimmunoelectrophoresis of ragweed-allergic patients' sera with special attention to quantification of IgE responses

Short ragweed allergenic extract has been studied by means of crossed radioimmunoelectrophoresis (CRIE) with the use of sera from 37 allergic patients and the relevant control sera. In this study 22 of 52 antigens, detectable in crossed immunoelectrophoresis (CIE) against polyspecific rabbit anti-ragweed IgG, were able to bind specific human IgE to their corresponding immunoprecipitates. This binding was semiquantified by comparison with the binding of a standard serum pool. Nine antigens were identified as important allergens, including the previously isolated components, AgE, AgK, and Ra6. Certain allergens (e.g., AgE, AgK, and Ag 31) bound IgE in almost all patients' sera, whereas others showed a bimodal distribution for sera of responder and nonresponder patients. The total CRIE score was found to correlate significantly both with ragweed-specific serum IgE antibody determined by RAST (rs = 0.88; p less than 0.001) and with total IgE level (rs = 0.55; p less than 0.01). Patient's CRIE scores to AgE also correlated significantly with their specific IgE antibody to AgE measured by RIA (r = 0.47; p less than 0.01) and with skin-test sensitivity to AgE (r = 0.44; p less than 0.05). It was concluded that CRIE is well suited for identification of important ragweed allergens without the previous need for laborious isolation procedures.     

Allergen-specific IgE and IgG4 are markers of resistance and susceptibility in a human intestinal nematode infection

IgG4 has been proposed to act as a 'blocking antibody' due to its ability to compete for the same epitopes as IgE thus preventing IgE-dependent allergic responses. IgG4 and IgE are both elevated in helminth infections and strong anti-parasite IgE responses are associated with resistance to infection. We wished to determine the relationship between anti-parasite IgG4 and IgE and Ascaris lumbricoides infection status. We examined anti-parasite responses, including antibody levels to recombinant Ascaris allergen-1A (rABA-1A), a target of serum IgE in endemic populations. Worm burden was indirectly estimated by measuring parasite egg output in a cross-sectional human population (N = 105). Levels of anti-parasite IgG4 and IgE in patients' plasma were quantified by immunoassay. Global anti-parasite antibody responses did not bear any significant relationships with intensity of Ascaris infection. Individuals who had detectable levels of IgE but not IgG4 to rABA-1A (11%) had lower average levels of infection compared with individuals who produced anti-rABA-1A IgG4 (40%) and sero-negative individuals (49%) (P = 0.008). The ratio of IgG4/IgE in rABA-1A responders positively correlated with intensity of infection (P < 0.025). IgG4 levels positively correlated with infection level in younger children (age 4-11) where average levels of infection were increasing (P = 0.038), whereas allergen specific IgE emerged as a correlate of immunity in older children and adults (age 12-36) where infection levels were decreasing (P = 0.048). Therefore, in a gastrointestinal helminth infection, differential regulation of anti-allergen antibody isotypes relate to infection level. Our results are consistent with the concept that IgG4 antibody can block IgE-mediated immunity and therefore allergic processes in humans.     

The determination of allergen-specific IgE and IgG antibodies in patients sensitized to the house dust mite Dermatophagoides pteronyssinus

45 patients, hypersensitive to house-dust mites, were examined by the method of skin tests to D. pteronyssinus allergen. Besides, in their blood sera the levels of allergen-specific IgE antibodies were determined in the radioallergosorbent test and allergen-specific IgG antibodies, in the enzyme immunoassay. These tests revealed that in 91% of the patients the results of skin tests were positive, in 68% an elevated level of specific IgE antibodies and in 93% of the patients an elevated level of specific IgG antibodies were detected. All patients showed the positive result in one of the above-mentioned tests. The largest group of the patients (55%) included persons showing the positive result of the skin test and having elevated levels of allergen-specific IgG and IgE antibodies. Thus, in cases of hypersensitivity to house-dust mites the levels of allergen-specific IgG and IgE antibodies in the patients' blood sera should be determined.     

Purification,structural and immunological characterization of a timothy grass (Phleum pratense) pollen allergen,Phl p 4, with cross-reactive potential

Almost 500 million people worldwide suffer from Type I allergy, a genetically determined immunodisorder which is based on the production of IgE antibodies against per se harmless antigens (allergens). Due to their worldwide distribution and heavy pollen production, grasses represent a major allergen source for approximately 40% of allergic patients. We purified Phl p 4, a major timothy grass (Phleum pratense) pollen allergen with a molecular mass of 61.3 kDa and a pl of 9.6 to homogeneity. Circular dichroism spectroscopical analysis indicates that Phl p 4 contains a mixed alpha-helical/beta-pleated secondary structure and, unlike many other allergens, showed no reversible unfolding after thermal denaturation. We show that Phl p 4 is a major allergen which reacts with IgE antibodies of 75% of grass pollen allergic patients (n=150) and induces basophil histamine release as well as immediate type skin reactions in sensitized individuals. Phl p 4-specific IgE from three patients as well as two rabbit-anti Phl p 4 antisera cross-reacted with allergens present in pollen of trees, grasses, weeds as well as plant-derived food. Rabbit antibodies raised against Phl p 4 also inhibited the binding of allergic patients IgE to Phl p 4. Phl p 4 may thus be used for diagnosis and treatment of sensitized allergic patients.     

Recognition of two Dermatophagoides pteronyssinus-specific epitopes on antigen P1 by using monoclonal antibodies: binding to each epitope can be inhibited by serum from dust mite-allergic patients

Monoclonal antibodies were raised against Antigen P1, the major allergen of the house dust mite (Dermatophagoides pteronyssinus). The majority were Antigen P1 specific, isotype IgG1, and did not react with a comparable D. farinae allergen. These antibodies bound 38 to 50% of 125I Antigen P1 in antigen-binding assays (titer greater than or equal to 1/1,000,000), and the quantities of IgG antibody in ascites were 2 to 4 logs greater than those in polyclonal mouse antiserum or in serum from a mite-allergic patient. Two IgM antibodies showed weak binding to Antigen P1 but reacted strongly with D. pteronyssinus in enzyme immunoassay (titer greater than or equal to 1/100,000). Assessments of the specificity of the IgG antibodies by using two inhibition radioimmunoassays suggested that they were directed against two different epitopes. Antibodies 10B9 F6 and 5H8 C12 were purified by preparative isoelectric focusing (isoelectric points of pI 6.25 and 7.4, respectively) and radiolabeled with 125I. Cross-inhibition experiments, using ascites dilutions to inhibit binding of each radiolabeled antibody to Antigen P1, confirmed that these antibodies recognized two distinct epitopes. Analysis of antibodies from 39 clones/hybrids showed that the majority were directed against the same epitopes as either 10B9 F6 or 5H8 C12 (3 out of 39 [8%] and 29 out of 39 [74%], respectively). None of the monoclonal antibodies significantly inhibited (greater than 10%) human IgE binding to Antigen P1 in the radioallergosorbent test. However, 12 of 14 sera from mite allergic patients inhibited binding by the monoclonal antibodies. One serum from a mite-allergic patient inhibited binding of both 10B9 F6 and 5H8 C12 by greater than 85% and showed parallel inhibition curves. The results suggest that these monoclonal antibodies could be used to assay Antigen P1 in both D. pteronyssinus and house dust extracts. It should also be possible to use monoclonal antibodies in inhibition assays to define the antigenic/allergenic determinants recognized by human IgG and IgE antibodies on this mite allergen.     

Enhancing blood-stage malaria subunit vaccine immunogenicity in rhesus macaques by combining adenovirus, poxvirus, and protein-in-adjuvant vaccines

Protein-in-adjuvant formulations and viral-vectored vaccines encoding blood-stage malaria Ags have shown efficacy in rodent malaria models and in vitro assays against Plasmodium falciparum. Abs and CD4(+) T cell responses are associated with protective efficacy against blood-stage malaria, whereas CD8(+) T cells against some classical blood-stage Ags can also have a protective effect against liver-stage parasites. No subunit vaccine strategy alone has generated demonstrable high-level efficacy against blood-stage infection in clinical trials. The induction of high-level Ab responses, as well as potent T and B cell effector and memory populations, is likely to be essential to achieve immediate and sustained protective efficacy in humans. This study describes in detail the immunogenicity of vaccines against P. falciparum apical membrane Ag 1 in rhesus macaques (Macaca mulatta), including the chimpanzee adenovirus 63 (AdCh63), the poxvirus modified vaccinia virus Ankara (MVA), and protein vaccines formulated in Alhydrogel or CoVaccine HT adjuvants. AdCh63-MVA heterologous prime-boost immunization induces strong and long-lasting multifunctional CD8(+) and CD4(+) T cell responses that exhibit a central memory-like phenotype. Three-shot (AdCh63-MVA-protein) or two-shot (AdCh63-protein) regimens induce memory B cells and high-titer functional IgG responses that inhibit the growth of two divergent strains of P. falciparum in vitro. Prior immunization with adenoviral vectors of alternative human or simian serotype does not affect the immunogenicity of the AdCh63 apical membrane Ag 1 vaccine. These data encourage the further clinical development and coadministration of protein and viral vector vaccine platforms in an attempt to induce broad cellular and humoral immune responses against blood-stage malaria Ags in humans.     

B cell responses to paramyosin. Isotypic analysis and epitope mapping of filarial paramyosin in patients with onchocerciasis.

To examine the fine specificity of the human immune response to filarial paramyosin, the antigenicity of an expressed rcDNA (2.55 kb) of Dirofilaria immitis paramyosin was detailed by ELISA. Using sera from patients infected with Onchocerca volvulus, we analyzed both the entire paramyosin molecule and six subcloned fragments for their IgG, IgG subclasses, and IgE responses. Patients from both Guatemala (64% positive) and Ghana (100% positive) reacted to paramyosin with specific IgG levels above normal controls. Although there was no anti-paramyosin subclass restriction common to all patients, the IgG3 response in the Ghananians was significantly greater than that of Guatemalans (p less than 0.001). IgE anti-paramyosin responses showed positive correlations with IgG2 (p less than 0.001), IgG4 (p less than 0.002), and IgG1 (p less than 0.04) responses. Epitope mapping using the IgG response to the six subclones demonstrated preferential recognition of the amino terminal end of the molecule (nucleotides 1 to 360). IgG2 reactivity was clearly localized to the most amino-terminal 120 amino acids, and the IgG4 antibodies recognized amino acids immediately adjacent to this fragment. These studies examining the fine specificity of anti-filarial immune reactions should provide a method for understanding how parasites either evade or induce host immune responses.     

Beta(1,2)-xylose and alpha(1,3)-fucose residues have a strong contribution in IgE binding to plant glycoallergens

Primary structures of the N-glycans of two major pollen allergens (Lol p 11 and Ole e 1) and a major peanut allergen (Ara h 1) were determined. Ole e 1 and Ara h 1 carried high mannose and complex N-glycans, whereas Lol p 11 carried only the complex. The complex structures all had a beta(1,2)-xylose linked to the core mannose. Substitution of the proximal N-acetylglucosamine with an alpha(1, 3)-fucose was observed on Lol p 11 and a minor fraction of Ole e 1 but not on Ara h 1. To elucidate the structural basis for IgE recognition of plant N-glycans, radioallergosorbent test analysis with protease digests of the three allergens and a panel of glycoproteins with known N-glycan structures was performed. It was demonstrated that both alpha(1,3)-fucose and beta(1,2)-xylose are involved in IgE binding. Surprisingly, xylose-specific IgE antibodies that bound to Lol p 11 and bromelain did not recognize closely related xylose-containing structures on horseradish peroxidase, phytohemeagglutinin, Ole e 1, and Ara h 1. On Lol p 11 and bromelain, the core beta-mannose is substituted with just an alpha(1,6)-mannose. On the other xylose-containing N-glycans, an additional alpha(1,3)-mannose is present. These observations indicate that IgE binding to xylose is sterically hampered by the presence of an alpha(1,3)-antenna.     

Effect of ambient air PM2.5-bound heavy metals on blood metal(loid)s and children’s asthma and allergy pro-inflammatory (IgE,IL-4 and IL-13) biomarkers

BackgroundWe investigated the concentrations of metals in fine particulate matter PM_2.5 in the outdoor air around the home sites of 123 male children from Ahvaz, average age 7.56, along with their blood samples to measure pro-inflammatory responses (Immunoglobulin E and cytokines: IgE, IL-4 and IL-13).MethodsWe measured 6 metals (As, Cd, Cr, Hg, Ni and Pb) in three Ahvaz’s regions including industrial (Padad), vehicle traffic (Golestan) and control (Kianpars).ResultsThe higher concentrations of metals in the Padad as the industrial ambient air i.e., arsenic, cadmium, chromium, mercury and nickel coincided with the higher concentrations of those metals in exposed children (P < 0.05) versus the controls. Children in Golestan, the high traffic air pollution area had the highest lead concentrations (p < 0.05). Also a significant association was shown in Padad between blood arsenic and IgE (β = 26.59, P < 0.001), IL-4 (β = 172.1, P < 0.001) and IL-13 (β = 14.84, P < 0.001), blood chromium and IgE (β = 10.38, P < 0.001), IL-4 (β = 75.27, P < 0.001) and IL-13 (β = 5.27, P < 0.001) and blood mercury and IgE (β = 13.11, P < 0.001), IL-4 (β = 108.09, P < 0.001) and IL-13 (β = 7.96, P < 0.001) and blood lead and IgE(β = 0.92, P = 0.025), IL-4(β = 7.16, P < 0.001) and IL-13(β = 0.58, P = 0.003). However, no significant relation was found for Cadmium, Nickel in blood with IgE, IL-4 and IL-13 levels. Moreover, children from industrial areas showed significantly higher concentrations of IgE (mean = 146.44 pg/200landa, P < 0.001), IL-4 (mean = 548.23 pg/200landa, P < 0.001) and IL-13 (mean = 52.93 pg/200landa, P < 0.001) versus Golestan and Kianpars.ConclusionChildren residing in an industrial area with high concentrations of metals in PM_2.5 had high metals in blood and high production of IgE, IL-4 and IL-13, reflecting an immune dysregulation and brisk inflammatory responses.     

Analysis of epitope-specific immune responses induced by vaccination with structurally folded and unfolded recombinant Bet v 1 allergen derivatives in man

Previously, we have constructed recombinant derivatives of the major birch pollen allergen, Bet v 1, with a more than 100-fold reduced ability to induce IgE-mediated allergic reactions. These derivatives differed from each other because the two recombinant Bet v 1 fragments represented unfolded molecules whereas the recombinant trimer resembled most of the structural fold of the Bet v 1 allergen. In this study, we analyzed the Ab (IgE, IgG subclass, IgA, IgM) response to Bet v 1, recombinant and synthetic Bet v 1-derived peptides in birch pollen allergic patients who had been vaccinated with the derivatives or adjuvant alone. Furthermore, we studied the induction of IgE-mediated skin responses in these patients using Bet v 1 and Bet v 1 fragments. Both types of vaccines induced a comparable IgG1 and IgG4 response against new sequential epitopes which overlap with the conformational IgE epitopes of Bet v 1. This response was 4- to 5-fold higher than that induced by immunotherapy with birch pollen extract. Trimer more than fragments induced also IgE responses against new epitopes and a transient increase in skin sensitivity to the fragments at the beginning of therapy. However, skin reactions to Bet v 1 tended to decrease one year after treatment in both actively treated groups. We demonstrate that vaccination with folded and unfolded recombinant allergen derivatives induces IgG Abs against new epitopes. These data may be important for the development of therapeutic as well as prophylactic vaccines based on recombinant allergens.