Allergen-specific IgG Research Articles

IgE-blocking antibodies following SLIT with recombinant Mal d 1 accord with improved apple allergy

We recently reported that 16 weeks of sublingual immunotherapy (SLIT) with recombinant (r) Mal d 1, but not rBet v 1, significantly improved birch pollen-related apple allergy. Allergen-specific IgE-blocking IgG antibodies have been associated with clinical efficacy. We compared the quantity, quality, and IgE-blocking bioactivity of SLIT-induced Mal d 1-specific IgG antibodies in both treatment groups. Pre- and post-SLIT sera were assessed for rMal d 1-specific IgG antibodies in ELISA and for their ability to inhibit apple allergen-induced upregulation of CD63 on basophils from nontreated individuals with birch pollen-related apple allergy. Post-SLIT sera depleted of IgG1 or IgG4 were compared for their IgE-blocking activity. IgG1 binding to rMal d 1 was competed with rMal d 1 and rBet v 1 in ELISA. SLIT with rMal d 1 and rBet v 1 induced comparable levels of rMal d 1-specific IgG1, IgG2, IgG3, and IgG4 antibodies. Only post-rMal d 1 SLIT sera displayed IgE-blocking activity, which was significantly reduced by depletion of IgG1 and less so by IgG4 depletion. In competition ELISA, IgG1 binding to Mal d 1 in post-rMal d 1 SLIT sera was fully inhibited with rMal d 1 but not with rBet v 1. Correspondingly, Bet v 1 was the more potent competitor for IgG1 binding to Mal d 1 in post-rBet v 1 SLIT sera. rMal d 1 SLIT for 16 weeks induced functional, primarily Mal d 1-specific IgE-blocking antibodies, whereas rBet v 1 SLIT induced Bet v 1-specific, Mal d 1-cross-reactive IgG antibodies with limited cross-blocking activity. These results provide a possible explanation for the limited effectiveness of birch pollen immunotherapy in birch pollen-related food allergy and indicate a dominant protective role of functional IgE-blocking IgG1 antibodies in the early phase of allergy treatment.

Ragweed SLIT-Tablets Induce Similar Patterns of Allergen-Specific IgE and IgG4 Responses in Children and Adults with Allergic Rhinoconjunctivitis

Ragweed SLIT-Tablets Induce Similar Patterns of Allergen-Specific IgE and IgG4 Responses in Children and Adults with Allergic Rhinoconjunctivitis

The Establishment of EXiLE test for House Dust Mite Allergy

Successful allergen immunotherapy (AIT) is associated with an increase in the production of allergen-specific IgG antibodies including IgG4 which may contribute to IgE-blocking factor (IgE-BF) activity. Recently, in vitro allergy test method was reported as EXiLE (IgE crosslinking-induced luciferase expression) test using a luciferase-reporting humanized rat mast cell line. However, to understand immunomodulation effect after AIT, this functional assay has not been fully validated. This study is aimed to investigate effect of IgE-BFs induced by house dust mite (HDM)-AIT on EXiLE test. HDM allergic rhinitis patients’ sera before- and after-AIT were used. EXiLE test was performed as previously reported (Allergol. Int, 2012). Briefly, RS-ATL8 cells were plated into the wells of a 96 well-plate and incubated with diluted serum overnight. The cells were stimulated with allergen (HDM, 0.1-1000 ng/mL) without washing. Then, luciferase substrate was added to the cells, and chemiluminescence was measured. The data was expressed as luciferase expression (LE) levels which were represented as the fold increase compared with the allergen untreated conditions (baseline), and EC50 value was calculated by fitting a dose response curve. The optimal incubation time was 3hr with HDM (0.1-1000 ng/mL). The LE levels showed significant correlation with the value of Der p specific-IgE (r2>0.95). EC50 of LE levels in sera after-AIT was shifted from that in sera before-AIT (p=0.01) EXiLE test was validated using the HDM allergic rhinitis sera. This test can be utilized for immunomodulation effect induced by AIT.

Immune status markers dynamics during rotational diet therapy in obesity

BACKGROUND: Сhronic immune inflammation in obesity is pathogenetically associated with the development insulin resistance and the metabolic syndrome. However, there are a number of works devoted to the relationship of obesity and allergic diseases, in which data were obtained on models of bronchial asthma and allergic rhinitis. In obesity against a background of chronic immune inflammation, a more severe course of concomitant allergic disease is observed, accompanied by an increase in pro-inflammatory cytokine levels. In this regard, obesity and food allergies should be considered from the standpoint of comorbidity, and the study of immune status markers dynamics in obese patients with concomitant food allergies at the background of diet therapy will create prerequisites for determining the criteria of the effectiveness of the treatment.
 AIM: To study the of immune status markers dynamics on the background of rotational diet therapy for obesity.
 METHODS: A single-site, randomized controlled trial was conducted. The duration of observation of the patients was 14 days. There were 68 patients with obesity, aged 47.4 5.4 years, randomly divided into 2 homogeneous groups. Levels of allergen-specific IgG to 90 food allergens were measured to all patients of the main group by enzyme immunoassay method. The comparison group received a standard low-calorie diet. In both groups, the levels of pro-inflammatory markers (IL-1, TNF, IL-6) were determined using commercial Biosource kits (Belgium).
 RESULTS: In evaluating of the cytokine status and the level of non-specific markers of inflammation at the initial stage of the study, patients with a body mass index above 35 showed a significant increase in concentrations of C-reactive protein, IL-1, and IL-6. Analysis of allergen-specific IgG to 90 food allergens showed that food intolerance was found often to milk, dairy products, gluten. Analyzing the dynamics of the level of pro-inflammatory markers during treatment, a significant decrease in the level of CRP, IL-6, IL-1 was detected in the main group 15 % (p 0.05), 14 % (p 0.05), 10 % respectively, while in the comparison group the decrease in the level of IL-6 was 9 %, IL-1 by 6 %,CRP by 10 % (p 0.05).
 CONCLUSION: In patients with obesity with concomitant food intolerance there are changes in the immune status in the form of increased levels of pro-inflammatory markers. During the observation a more significant decrease was found in the studied parameters in the main group was detected at the background of rotational diet therapy in relation to the comparison group. The decrease in the concentrations of the studied markers in the serum during the study is an indicator of the effectiveness of rotational diet therapy.

Алгоритм диагностического поиска при IgE-независимом аллергическом рините

Allergic rhinitis is a widespread disease located at the junction of two specialties: otorhinolaryngology and allergology. Currently, it is characterized by an early onset, often continuously recurring course and refractory to traditional methods of treatment. Unstable remissions, a tendency to complications, and the lack of effectiveness of the therapy suggest that allergic rhinitis occurs against the background of secondary immunodeficiency, a favorable factor for the development of bacterial superinfection on the damaged mucous membrane of the nasal cavity. In conditions of impaired immune homeostasis, the microbial landscape of the nasal mucosa is characterized by polymorphism, bacterial agents can act as an infectious antigen. The article is devoted to a current problem – chronic IgE-independent allergic rhinitis due to allergen-specific lymphocytes or IgG antibodies with the formation of immune complexes. The article reveals the content of the concept of “chronic infectious-dependent allergic rhinitis”, its characteristic features are highlighted and described, the key stages of diagnosis are considered. The importance of knowledge about mediators of chronic eosinophilic inflammation for practitioners is reflected. The work is interdisciplinary in nature, written at the intersection of otorhinolaryngology, allergology and immunology and will be interesting to doctors of various specialties. Keywords: chronic rhinitis, allergic rhinitis, microbial landscape, immunity, diagnostic algorithm.

Allergen-specific IgG+ memory B cells are temporally linked to IgE memory responses

IgE is the least abundant immunoglobulin and tightly regulated, and IgE-producing B cells are rare. The cellular origin and evolution of IgE responses are poorly understood. The cellular and clonal origin of IgE memory responses following mucosal allergen exposure by sublingual immunotherapy (SLIT) were investigated. In a randomized double-blind, placebo-controlled, time course SLIT study, PBMCs and nasal biopsy samples were collected from 40 adults with seasonal allergic rhinitis at baseline and at 4, 8, 16, 28, and 52 weeks. RNA was extracted from PBMCs, sorted B cells, and nasal biopsy samples for heavy chain variable gene repertoire sequencing. Moreover, mAbs were derived from single B-cell transcriptomes. Combining heavy chain variable gene repertoire sequencing and single-cell transcriptomics yielded direct evidence of a parallel boost of 2 clonally and functionally related B-cell subsets of short-lived IgE+ plasmablasts and IgG+ memory B cells. Mucosal grass pollen allergen exposure by SLIT resulted in highly diverse IgE and IgGE repertoires. These were extensively mutated and appeared relatively stable as per heavy chain isotype, somatic hypermutations, and clonal composition. Single IgGE+ memory B-cell and IgE+ preplasmablast transcriptomes encoded antibodies that were specific for major grass pollen allergens and able to elicit basophil activation at very low allergen concentrations. For the first time, we have shown that on mucosal allergen exposure, human IgE memory resides in allergen-specific IgG+ memory B cells. These cells rapidly switch isotype, expand into short-lived IgE+ plasmablasts, and serve as a potential target for therapeutic intervention.

Biomarkers in Food Allergy Immunotherapy.

Investigational allergen immunotherapies (AITs) including oral immunotherapy (OIT), sublingual immunotherapy (SLIT), and epicutaneous immunotherapy (EPIT) have proven to increase allergen thresholds required to elicit an allergic reaction in a majority of subjects. However, these studies lack consistent biomarkers to predict therapy outcomes. Here, we will review biomarkers that are currently being investigated for AIT. The mechanisms underlying the therapeutic benefit of AIT involve various cell types, including mast cells, basophils, T cells, and B cells. Skin prick and basophil activation tests assess effector cell sensitivity to allergen and are decreased in subjects on AIT. Allergen-specific IgE increases initially and decreases with continued therapy, while allergen-specific IgG and IgA increase throughout therapy. Allergen-induced regulatory T cells (Tregs) increase throughout therapy and were found to be associated with sustained unresponsiveness after OIT. Subjects on OIT and SLIT have decreased Th2 cytokine production during therapy. Although trends have been reported, a common limitation of these biomarkers is that none are able to reproducibly predict prognosis during AIT. Further studies are needed to expand the currently available biomarker repertoire to provide personalized approaches to AIT.

Duration of allergen immunotherapy for inhalant allergy.

We evaluated the time-course of clinical and immunologic changes that occur during and after cessation of sublingual and subcutaneous allergen immunotherapy for inhalant allergies. Increases in production of inhibitory cytokines, such as IL-10 and allergen-specific IgE and IgG4 antibodies are induced within weeks of starting immunotherapy for both seasonal and perennial allergens. In general, 2-4 months' immunotherapy is needed for onset of efficacy whereas maximal clinical effect is achieved within 1-2 years of treatment. Therefore, assuming optimal patient selection, good compliance and at least moderate allergen exposure, if immunotherapy is ineffective at 2 years, it is reasonable to discontinue the treatment. For long-term clinical efficacy, at least 3 years of either subcutaneous or sublingual immunotherapy is required and this results in clinical and immunologic tolerance -- persistence of clinical benefits and suppression of type 2 immunity for years after discontinuation of treatment. Both sublingual and subcutaneous immunotherapy are effective and well tolerated for respiratory allergy. Clinical and immunological changes occur at early stages of treatment. Long-term evaluations support recommendations in international guidelines that both routes of administration should be continued for a minimum of 3 years to achieve disease modification and long-term tolerance.

Two years of treatment with the recombinant grass pollen allergy vaccine BM32 induces a continuously increasing allergen-specific IgG4 response.

BackgroundBM32, a grass pollen allergy vaccine containing four recombinant fusion proteins consisting of hepatitis B-derived PreS and hypoallergenic peptides from the major timothy grass pollen allergens adsorbed on aluminium hydroxide has been shown to be safe and to improve clinical symptoms of grass pollen allergy upon allergen-specific immunotherapy (AIT). We have investigated the immune responses in patients from a two years double-blind, placebo-controlled AIT field trial with BM32.MethodsBlood samples from patients treated with BM32 (n = 27) or placebo (Aluminium hydroxide) (n = 13) were obtained to study the effects of vaccination and natural allergen exposure on allergen-specific antibody, T cell and cytokine responses. Allergen-specific IgE, IgG, IgG1 and IgG4 levels were determined by ImmunoCAP and ELISA, respectively. Allergen-specific lymphocyte proliferation by 3H thymidine incorporation and multiple cytokine responses with a human 17-plex cytokine assay were studied in cultured peripheral blood mononuclear cells (PBMCs).FindingsTwo years AIT comprising two courses of 3 pre-seasonal injections of BM32 and a single booster after the first pollen season induced a continuously increasing (year 2 > year 1) allergen-specific IgG4 response without boosting allergen-specific IgE responses. Specific IgG4 responses were accompanied by low stimulation of allergen-specific PBMC responses. Increases of allergen-specific pro-inflammatory cytokine responses were absent. The rise of allergen-specific IgE induced by seasonal grass pollen exposure was partially blunted in BM32-treated patients.InterpretationAIT with BM32 is characterised by the induction of a non-inflammatory, continuously increasing allergen-specific IgG4 response (year 2 > year1) which may explain that clinical efficacy was higher in year 2 than in year 1. The good safety profile of BM32 may be explained by lack of IgE reactivity and low stimulation of allergen-specific T cell and cytokine responses.FundingsGrants F4605, F4613 and DK 1248-B13 of the Austrian Science Fund (FWF).

A hypoallergenic peptide mix containing T cell epitopes of the clinically relevant house dust mite allergens.

BackgroundIn the house dust mite (HDM) Dermatophagoides pteronyssinus, Der p 1, 2, 5, 7, 21, and 23 have been identified as the most important allergens. The aim of this study was to define hypoallergenic peptides derived from the sequences of the six allergens and to use the peptides and the complete allergens to study antibody, T cell, and cytokine responses in sensitized and nonsensitized subjects.MethodsIgE reactivity of HDM‐allergic and non‐HDM‐sensitized individuals to 15 HDM allergens was established using ImmunoCAP ISAC technology. Thirty‐three peptides covering the sequences of the six HDM allergens were synthesized. Allergens and peptides were tested for IgE and IgG reactivity by ELISA and ImmunoCAP, respectively. Allergenic activity was determined by basophil activation. CD4+ T cell and cytokine responses were determined in PBMC cultures by CFSE dilution and Luminex technology, respectively.ResultsHouse dust mite allergics showed IgE reactivity only to complete allergens, whereas 31 of the 33 peptides lacked relevant IgE reactivity and allergenic activity. IgG antibodies of HDM‐allergic and nonsensitized subjects were directed against peptide epitopes and higher allergen‐specific IgG levels were found in HDM allergics. PBMC from HDM‐allergics produced higher levels of IL‐5 whereas non‐HDM‐sensitized individuals mounted higher levels of IFN‐gamma, IL‐17, pro‐inflammatory cytokines, and IL‐10.ConclusionIgG antibodies in HDM‐allergic patients recognize peptide epitopes which are different from the epitopes recognized by IgE. This may explain why naturally occurring allergen‐specific IgG antibodies do not protect against IgE‐mediated allergic inflammation. A mix of hypoallergenic peptides containing T cell epitopes of the most important HDM allergens was identified.

Recombinant allergens for immunotherapy: state of the art.

Purpose of reviewMore than 30 years ago, the first molecular structures of allergens were elucidated and defined recombinant allergens became available. We review the state of the art regarding molecular AIT with the goal to understand why progress in this field has been slow, although there is huge potential for treatment and allergen-specific prevention.Recent findingsOn the basis of allergen structures, several AIT strategies have been developed and were advanced into clinical evaluation. In clinical AIT trials, promising results were obtained with recombinant and synthetic allergen derivatives inducing allergen-specific IgG antibodies, which interfered with allergen recognition by IgE whereas clinical efficacy could not yet be demonstrated for approaches targeting only allergen-specific T-cell responses. Available data suggest that molecular AIT strategies have many advantages over allergen extract-based AIT.SummaryClinical studies indicate that recombinant allergen-based AIT vaccines, which are superior to existing allergen extract-based AIT can be developed for respiratory, food and venom allergy. Allergen-specific preventive strategies based on recombinant allergen-based vaccine approaches and induction of T-cell tolerance are on the horizon and hold promise that allergy can be prevented. However, progress is limited by lack of resources needed for clinical studies, which are necessary for the development of these innovative strategies.

A comparison of different methods of the allergen-specific immunotherapy in patients with pollinosis: the results of open randomized study

Background. Allergen-specific immunotherapy (ASIT) is viewed as the only treatment that influences all patho-genetically significant parts of the allergic process in the initial and late phases of the IgE-mediated allergic reaction and modifies the abnormal immune reactivity to a specific allergen. Currently, sublingual (SLIT) and subcutaneous (SCIT) immunotherapy are most commonly used in clinical practice. Despite long experience of sublingual and subcutaneous immunotherapy application, questions remain about the preferred ASIT method and comparative effectiveness of different ASIT methods. This article evaluates the efficacy, benefits of SCIT and SLIT and highlights new findings related mechanisms and potential biomarkers. The aim of the study. To evaluate the comparative efficacy of different methods of ASIT (subcutaneous and sublingual) based on clinical data and biomarkers in the blood serum and other biological fluids in adult patients with allergic rhinoconjunctivitis (with/without asthma). Materials and methods. 60 patients with allergic rhinoconjunctivitis (with/without asthma) aged 18 to 50 were randomly assigned to 3 groups treated by sublingual immunotherapy with extracts of allergens, subcutaneous immunotherapy with extracts of allergens and subcutaneous with modified allergens (allergoids) respectively. Results. The efficiency of the first course of preseason ASIT (SCIT and SLIT) with extracts of allergens and aller-goids in the control of symptoms of allergic rhinoconjunctivitis (with/without asthma) was demonstrated. After the end of the first year pre-season ASIT data analysis scales (Total Symptom Score -TSS, Medircation Score -MS) revealed the best performance in the group of patients receiving SCIT with allergoids compared with patients receiving the SLIT with extracts of allergens: the scales of the TSS (p=0.023), MS (p=0.002). In addition, at the end of the maintenance phase of ASIT in patients treated with SCIT with allergoids the level of eosinophilic cationic protein (ECP) in the nasal lavage decreased by 22% (p=0.012), secretory immunoglobulin A (sIgA) in the nasal lavage increased by 70% (p=0.001), interleukin-10 (IL-10) in serum increased by 126% (p=0.006), allergen-specific IgG4 increased by 42% (p=0.01) from the initial values, that correlates with a decrease in the severity of clinical manifestations. In pollen season ECP level in nasal lavage was significantly (p=0.007) lower in a group of patients who received SCIT with allergoids compared with patients who received the SLIT with extracts of allergens. The most significant changes of serum level of IL-10 in the pollen season occurred in a group of patients receiving SCIT with allergoids compared with patients who received SLIT (p=0.013) and SCIT (p=0.001) with extracts of allergens. Conclusion. The study results deepen the existing understanding of the mechanisms of SCIT and SLIT. They allow to develop a comprehensive assessment of the therapy efficacy scheme based on clinical parameters and on monitoring of local (ECP, sIgA) and systemic biomarkers (IL-10, allergen-specific IgG4) as well.

Maternal allergen-specific IgG might protect the child against allergic sensitization

BackgroundAnalysis of allergen-specific IgE responses in birth cohorts with microarrayed allergens has provided detailed information regarding the evolution of specific IgE responses in children. High-resolution data regarding early development of allergen-specific IgG are needed.ObjectiveWe sought to analyze IgG reactivity to microarrayed allergens in mothers during pregnancy, in cord blood samples, in breast milk, and in infants in the first years of life with the aim to investigate whether maternal allergen-specific IgG can protect against IgE sensitization in the offspring.MethodsPlasma samples from mothers during the third trimester, cord blood, breast milk collected 2 months after delivery, and plasma samples from children at 6, 12, and 60 months of age were analyzed for IgG reactivity to 164 microarrayed allergens (ImmunoCAP ISAC technology) in 99 families of the Swedish birth cohort Assessment of Lifestyle and Allergic Disease During Infancy (ALADDIN). IgE sensitizations to microarrayed allergens were determined at 5 years of age in the children.ResultsAllergen-specific IgG reactivity profiles in mothers, cord blood, and breast milk were highly correlated. Maternal allergen-specific IgG persisted in some children at 6 months. Children’s allergen-specific IgG production occurred at 6 months and reflected allergen exposure. Children who were IgE sensitized against an allergen at 5 years of age had significantly higher allergen-specific IgG levels than nonsensitized children. For all 164 tested allergens, children from mothers with increased (>30 ISAC standardized units) specific plasma IgG levels against an allergen had no IgE sensitizations against that allergen at 5 years of age.ConclusionThis is the first detailed analysis of the molecular IgG recognition profile in mothers and their children in early life. High allergen-specific IgG reactivity in the mother’s plasma and breast milk and in cord blood seemed to protect against allergic sensitization at 5 years of age.

Allergen-Specific Antibodies Regulate Secondary Allergen-Specific Immune Responses.

Immunoglobulin E (IgE)-associated allergy is the most common immunologically-mediated hypersensensitivity disease. It is based on the production of IgE antibodies and T cell responses against per se innocuous antigens (i.e., allergens) and subsequent allergen-induced inflammation in genetically pre-disposed individuals. While allergen exposure in sensitized subjects mainly boosts IgE production and T cell activation, successful allergen-specific immunotherapy (AIT) induces the production of allergen-specific IgG antibodies and reduces T cell activity. Under both circumstances, the resulting allergen-antibody complexes play a major role in modulating secondary allergen-specific immune responses: Allergen-IgE complexes induce mast cell and basophil activation and perpetuate allergen-specific T cell responses via presentation of allergen by allergen presenting cells to T cells, a process called IgE-facilitated antigen presentation (FAP). In addition, they may induce activation of IgE memory B cells. Allergen-induced production of specific IgGs usually exerts ameliorating effects but under certain circumstances may also contribute to exacerbation. Allergen-specific IgG antibodies induced by AIT which compete with IgE for allergen binding (i.e., blocking IgG) inhibit formation of IgE-allergen complexes and reduce activation of effector cells, B cells and indirectly T cells as FAP is prevented. Experimental data provide evidence that by binding of allergen-specific IgG to epitopes different from those recognized by IgE, allergen-specific IgG may enhance IgE-mediated activation of mast cells, basophils and allergen-specific IgE+ B cells. In this review we provide an overview about the role of allergen-specific antibodies in regulating secondary allergen-specific immune responses.

Measurement of Specific IgG4 Anti-mouse Urine Antibodies.

IgG4 and its role in immune tolerance has been investigated widely. Symptom reduction and improved clinical outcomes in immunotherapy trials are associated with significant increases in allergen-specific IgG4 antibodies. Natural immune tolerance observed in beekeepers and cat owners has also been associated with elevated levels of bee venom and cat allergen-specific IgG4, respectively. Functionally, allergen-specific IgG4 has been shown to reduce the binding of IgE-allergen complexes to B cells, a key step in the initiation of the type 1 hypersensitivity allergic response. In laboratory animal allergy, IgG4 has been described as a "protective/blocking" antibody. However, a consensus on the exact relationship between exposure, IgG4, and tolerance has yet to be reached. In this chapter, we review the factors that require consideration when developing an ELISA for the quantification of allergen-specific IgG4.

Measurement of Specific IgG Anti-Fel d 1 Antibodies.

There is currently considerable interest in the role of specific IgG antibodies in allergy. Several studies suggest that specific IgG antibodies may play a protective role in allergy. Successful immunotherapy is associated with increases in allergen-specific IgG antibodies which correlate with clinical outcome. Other studies have identified an inverse relationship between exposure to cat and sensitization, which was associated with high-titer-specific IgG and IgG4. This immune response was described as a modified Th2 response, since both IgE and IgG4 require Th2 cytokine IL-4 for their production. A modified Th2 response was described with laboratory animal allergy, where there was almost a twofold reduction in the risk of developing work-related chest symptoms.In this chapter, we review the major factors to be considered in the development of an ELISA for the determination of specific IgG and IgG4 antibodies.

Maternal allergen-specific IgG may protect the child against allergic sensitization – literature review

Maternal allergen-specific IgG may protect the child against allergic sensitization – literature review

A light-initiated chemiluminescent assay for rapid quantitation of allergen-specific IgG4 in clinical samples

BackgroundAn increase in allergen-specific IgG4 (sIgG4), which serves as a blocking antibody, is associated with acquisition of immune tolerance after immunotherapy. In this study, we developed a rapid, sensitive, and homogeneous immunoassay based on the light-initiated chemiluminescent assay (LICA) technology for quantifying allergen sIgG4 in serum samples. MethodsAllergen sIgG4 was measured in vitro by incubating the sample with biotinylated allergens and chemiluminescent beads coated with anti-human IgG4 antibody, followed by the addition of streptavidin-coated sensitizer beads. Multiple tests were performed to optimize the working conditions of the LICA and evaluate its performance. ResultsWe established the optimal concentration of biotinylated allergens (250 ng/mL), the optimal dilution range (1:8 for Gal d 1, Gal d 2 sIgG4 and 1:4 for Gal d 3, Gal d 4 sIgG4), and the optimal incubation time (20 min for Gal d 1, Gal d 2 sIgG4 and 40 min for Gal d 3, Gal d 4 sIgG4). The lower limit of quantification (LLOQ) was 0.261 ng/mL. The coefficient variation (CV) of the LICA was <10%. The assay was unaffected by general interfering substances at physiological concentrations.It exhibited excellent accuracy to detect allergen-sIgG4 in human serum. Additionally, we demonstrated that the levels of Gal d 1, Gal d 2, and Gal d 3-sIgG4 were significantly higher in the egg allergy group (p < .05), but no differences were found between the groups for Gal d 4-sIgG4. ConclusionsThe LICA demonstrated satisfactory performance and can be used for quantifying allergen sIgG4 in clinical practice.

IL-10 Indirectly Downregulates IL-4-Induced IgE Production by Human B Cells.

Controlled allergic disease is associated with decreased allergen-specific IgE and increased allergen-specific IgG4. Although IL-10 has been shown to contribute to these changes, the underlying mechanisms are largely unknown. This study explored how IL-10 differentially regulates human IgE and IgG4 production. Highly purified B cells and B cell subsets from healthy individuals were cultured with combinations of anti-CD40, IL-4, and IL-10. In other experiments, PBMCs isolated from healthy donors or from autosomal dominant hyper-IgE syndrome (STAT3 loss-of-function) subjects were cultured with combinations of IL-4 and IL-10. In B cell cultures, IL-10 had no significant effect on IL-4–induced IgE production but increased IL-4–induced IgG4 production over 20-fold. IL-4-induced transcription of Cε and Cγ4 germline transcripts (GLTs) by isolated B cells was not affected by IL-10. In PBMC cultures, IL-4 induced production of both IgE and IgG4 and increased expression of Cε and Cγ4 GLTs above baseline. Unlike in purified B cells, IL-10 diminished IL-4–induced IgE production and expression of Cε GLTs without affecting IgG4 production or expression of Cγ4 GLTs. PBMCs from autosomal dominant hyper-IgE syndrome individuals failed to consistently modulate IgE production in response to IL-4 and IL-10. As measured by flow cytometry, the frequency of IL-10R+ cells was similar between IgE+ and IgG4+ B cells. These data suggest that IL-10 acts indirectly through accessory cells to modulate the production of IgE. For IgG4, IL-10 appears to act directly on B cells to drive IgG4 production, with its effects being downstream of germline transcription.

Vaccination of nonallergic individuals with recombinant hypoallergenic fragments of birch pollen allergen Bet v 1: Safety, effects, and mechanisms

Summary This is the first double-blind, placebo-controlled study showing that vaccination with recombinant hypoallergenic allergen-derivatives is safe and induces sustained allergen-specific IgG responses which block allergic patients IgE binding to the allergen.