Allergic Subjects Research Articles

Interleukin-13, Interleukin-10, Interferon-gamma and IDO Production in Response to Home Dust Mites in Allergic Asthma

BACKGROUND: Allergic asthma is a degenerative atopic disease caused by allergic or hypersensitivity type-1. More than 50% of people with allergic asthma are caused by the presence of house dust mites (HDMs) allergens.METHODS: The cellular immunity response was evaluated through a peripheral blood mononuclear cell (PBMC) culture isolated from blood, using the ficoll gradient technique. Subjects were atopic asthma groups and non-atopic asthma groups. PBMC from each subject cultured was stimulated with HDMs allergen, then incubated in a CO2 5% incubator, 37o C for 72 hours. With the multiplex assay method, interferon (IFN)-γ, interleukin (IL)-13 and IL-10 were measured, meanwhile indoleamine 2,3-dioxygenase level (IDO) was measured by the enzyme-linked immunosorbent assay (ELISA) sandwich methods.RESULTS: The IFN-γ production in the supernatant of PBMC cultures was stimulated by phytohemagglutinin (PHA), Roswell Park Memorial Institute (RPMI) medium and allergens. The IFN-γ production in allergen-stimulated supernatants showed higher level of IFN-γ in the nonatopic group (4,681,455±3,434,851) than atopic group (4,363,300±2,067,941) even though it was not statistically significant (p=0.078). There were no differences between the mean of IL-13 production in atopic asthma group and non-atopic group. The IL-10 production in allergenstimulated supernatants was shown to be higher in nonatopic group and were statistically significantly different (p=0.015). The IDO production in allergen-stimulated supernatants was shown to be higher in the non-atopic group (272,231±269,564) than in the actopic group (13,273±400), and it was significantly different (p=0.007).CONCLUSION: Cellular immune profile of subjects with allergic asthma to Dermatophagoides pterronyssinus (Der p) is characterized by a type-2 inflammatory response that is dominant compared to type-1 inflammation (higher IL-13 ratio compared to IFN-γ) and to the role of anti-inflammation (higher IL-13 ratio compared to IL-10). The decline in IDO production in allergic asthma subjects to Der p is thought to be related to the low cellular immune response in expressing IFN-γ compared to IL-13.KEYWORDS: interleukin-13, interleukin-10, IDO, PBMC, asthma

IgE, IgG1, and IgG4 Reactivity to Dermatophagoides pteronyssinus Glycosylated Extract in Allergic Patients.

Background House dust mites are important allergen sources and some of these allergenic proteins may contain carbohydrate moieties, which are able to be isolated using lectins, as Concanavalin A (ConA). This study aimed to investigate allergenicity (IgE) and antigenicity (IgG1 and IgG4) of ConA-unbound and ConA-bound Dermatophagoides pteronyssinus (Dpt) crude extracts using sera of mite-allergic patients as well as inhibition capacity of antibody binding. Material and Methods We obtained mannose-enriched and mannose-depleted fractions from Dpt by ConA affinity chromatography. Both ConA-bound and ConA-unbound fractions were evaluated by ELISA and Western Blotting for specific IgE, IgG1, and IgG4 reactivity with sera obtained from 95 mite-allergic patients (DP+) and 92 nonallergic (NA) subjects. Inhibition ELISA was used to assess cross-reactivity between Dpt extract and its fractions. Results Among the DP+ patients, no difference was found between ConA-unbound and ConA-bound fractions regarding the levels of specific IgE, IgG1, and IgG4. Nonallergic subjects had the same levels of specific IgG1 to both ConA-unbound and ConA-bound fractions, although for specific IgG4, values were higher for ConA-bound. A positive correlation was found among specific IgE, IgG1, and IgG4 levels when Dpt was compared to ConA-unbound and ConA-bound fractions. Recognition of crude Dpt by IgE, IgG1, and IgG4 was highly inhibited by ConA-unbound and ConA-bound fractions. Western Blotting revealed a broad spectrum of bands ranging from 14 to 116 kDa recognized by specific IgE and IgG4. However, IgG1 reached higher frequency values on high molecular weight polypeptides. Conclusion ConA-unbound and ConA-bound fractions derived from D. pteronyssinus crude extract revealed important components involved in the IgE recognition in allergic patients as well as IgG1 and/or IgG4 in allergic and healthy subjects.

Goat Milk with Different Alpha-s1 Casein Genotype (CSN1S1) Fermented by Selected Lactobacillus paracasei as Potential Functional Food

The characteristics of fermented milk are affected by the type of milk used and the microorganisms involved in the fermentation process. Goat milk has been widely suggested as a possible alternative to cow milk in allergic subjects, because of the high genetic variability in alpha-s1 casein (CSN1S1) content, which is associated with different technological and nutritional properties of milk. The aim of the study was to evaluate the suitability of goat milk with low and high CSN1S1 to produce fermented milk. In addition, the performance as starter of selected Lactobacillus paracasei FS109 strain compared to no-selected L. paracasei strains was investigated. Initially, the selected L. paracasei FS109 strain was tested for adhesion ability to HT-29 and Caco-2 cells and immunomodulation effect. Then, the strain was used to produce fermented milk from goat milk with a low and high casein CSN1S1 genotype. The results indicated that greater acidifying activity was obtained for L. paracasei FS109 after 24 h of fermentation than the other two strains tested independently by the CSN1S1 genotype. L. paracasei FS109 grew well during fermentation, reaching a higher value (>8.5 log CFU/mL). Interestingly, the same strain maintained a high viable population (about 9 log CFU/mL) during the 30-day cold storage of the product. The present study shows for the first time the suitability of the goat milk with low CSN1S1 genotypes to produce fermented milk and highlight the importance of strain selection in determination of technological and beneficial traits. Combining goat milk with low CSN1S1 and selected strains could be a strategy of improving traditional and functional fermented milk market.

Characterization of IgE-binding proteins in the salivary glands of Simulium nigrogilvum (Diptera: Simuliidae).

Simulium dermatitis is an IgE-mediated skin reaction in animals and humans caused by the bites of black flies. Although Simulium nigrogilvum has been incriminated as the main human-biting black fly species in Thailand, information on its salivary allergens is lacking. Salivary gland extract of S. nigrogilvum females was subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis, and the separated components were applied onto nitrocellulose membranes for immunoblotting, which was performed by probing the protein blots with sera from 17 individuals who were allergic to the bites of S. nigrogilvum. IgE-reactive protein bands were characterized further by liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Nine protein bands (79, 42, 32, 25, 24, 22, 15, 13, and 11kDa) were recognized in the serum of the subjects. Four of the nine protein bands (32, 24, 15, and 11kDa) showed IgE reactivity in all (100%) of the tested sera, and they were identified as salivary secreted antigen 5-related protein, salivary serine protease, erythema protein, and hypothetical secreted protein, respectively. Three other proteins, salivary serine protease (25kDa), salivary D7 secreted protein (22kDa), and hypothetical protein (13kDa), reacted with > 50% of the sera. The relevance of the identified protein bands as allergens needs to be confirmed by using pure recombinant proteins, either in the in vivo skin prick test or in vitro detection of the specific IgE in the serum samples of allergic subjects. This will be useful for the rational design of component-resolved diagnosis and allergen immunotherapy for the allergy mediated by the bites of black flies.

Discovery of Alpha-Gal-Containing Antigens in North American Tick Species Believed to Induce Red Meat Allergy.

Development of specific IgE antibodies to the oligosaccharide galactose-α-1, 3-galactose (α-gal) following tick bites has been shown to be the source of red meat allergy. In this study, we investigated the presence of α-gal in four tick species: the lone-star tick (Amblyomma americanum), the Gulf-Coast tick (Amblyomma maculatum), the American dog tick (Dermacentor variabilis), and the black-legged tick (Ixodes scapularis) by using a combination of immunoproteomic approach and, carbohydrate analysis. Anti-α-gal antibodies identified α-gal in the salivary glands of both Am. americanum and Ix. scapularis, while Am. maculatum and De. variabilis appeared to lack the carbohydrate. PNGase F treatment confirmed the deglycosylation of N-linked α-gal-containing proteins in tick salivary glands. Immunolocalization of α-gal moieties to the salivary secretory vesicles of the salivary acini also confirmed the secretory nature of α-gal-containing antigens in ticks. Am. americanum ticks were fed on human blood (lacks α-gal) using a silicone membrane system to determine the source of the α-gal. N-linked glycan analysis revealed that Am. americanum and Ix. scapularis have α-gal in their saliva and salivary glands, but Am. maculatum contains no detectable quantity. Consistent with the glycan analysis, salivary samples from Am. americanum and Ix. scapularis stimulated activation of basophils primed with plasma from α-gal allergic subjects. Together, these data support the idea that bites from certain tick species may specifically create a risk for the development of α-gal-specific IgE and hypersensitivity reactions in humans. Alpha-Gal syndrome challenges the current food allergy paradigm and broadens opportunities for future research.

Discriminatory Molecular Biomarkers of Allergic and Nonallergic Asthma and Its Severity.

Asthma is a complex disease comprising various phenotypes and endotypes, all of which still need solid biomarkers for accurate classification. In a previous study, we defined specific genes related to asthma and respiratory allergy by studying the expression of 94 genes in a population composed of 4 groups of subjects: healthy control, nonallergic asthmatic, asthmatic allergic, and nonasthmatic allergic patients. An analysis of differential gene expression between controls and patients revealed a set of statistically relevant genes mainly associated with disease severity, i.e., CHI3L1, IL-8, IL-10, MSR1, PHLDA1, PI3, and SERPINB2. Here, we analyzed whether these genes and their proteins could be potential asthma biomarkers to distinguish between nonallergic asthmatic and asthmatic allergic subjects. Protein quantification was determined by ELISA (in serum) or Western blot (in protein extracted from peripheral blood mononuclear cells or PBMCs). Statistical analyses were performed by unpaired t-test using the Graph-Pad program. The sensitivity and specificity of the gene and protein expression of several candidate biomarkers in differentiating the two groups (and the severity subgroups) was performed by receiver operating characteristic (ROC) curve analysis using the R program. The ROC curve analysis determined single genes with good sensitivity and specificity for discriminating some of the phenotypes. However, interesting combinations of two or three protein biomarkers were found to distinguish the asthma disease and disease severity between the different phenotypes of this pathology using reproducible techniques in easy-to-obtain samples. Gene and protein panels formed by single biomarkers and biomarker combinations have been defined in easily obtainable samples and by standardized techniques. These panels could be useful for characterizing phenotypes of asthma, specifically when differentiating asthma severity.

Human BCR analysis of single-sorted, putative IgE+ memory B cells in food allergy

R. Jimenez-Saiz, Y. Ellenbogen and K. Bruton et al. developed a method to identify bona fide IgE+ MBCs in humans, demonstrated their extreme rarity in circulation and cautioned against the clinical utility of their assessment.

Validation of Strasbourg environmental exposure chamber (EEC) ALYATEC® in mite allergic subjects with asthma

Objective: Environmental Exposure Chamber (EEC) should have standardized and controlled allergenic and non-allergenic exposures to perform reproducible clinical studies. The aim was to demonstrate that mite exposure in the Alyatec® EEC could induce early (EAR) and/or late asthmatic reactions (LAR) in at least 60% of subjects allergic to mite.Methods: The EEC has a volume of 147-m3 with 20 seats. The nebulized particle number, airborne Der p1, endotoxins, and volatile organic compound (VOC) concentrations were measured. Twenty-four asthmatics allergic to mite were randomly exposed to 15, 25, and 46 ng/m3 Der p1. Specificity was assessed in not mite-sensitized asthmatics.Results: No significant endotoxin or VOC contamination was measured. The mean inter-assay CVs were 12.5% for the airborne particle number and 28.7% for airborne Der p1 concentrations. For the three Der p1 concentrations, at least 88% of the subjects developed EAR and/or LAR, and at least 46% developed a dual response. No reaction occurred with placebo or in the control group. No severe bronchial reaction occurred.Conclusions: The Alyatec® EEC demonstrated a tight control of allergenic and non-allergenic exposures. The EEC was clinically validated, with airborne Der p1 levels close to levels found in natural settings.

Capteurs de pollens et pollution : étude européenne AIS Life

The RNSA participated in a European study in 2015 and 2016 with the aim of assessing the amount of pollen and allergen content in several public gardens in Paris and in Lyon and, on the basis of the results obtained, making recommendations to protect allergic subjects. The conclusion is that these parks contain many allergenic species (birches, plane trees, cypresses, ash trees and so on).

IL-4 receptor engagement in human neutrophils impairs their migration and extracellular trap formation

Type 2 immunity serves to resist parasitic helminths, venoms, and toxins, but the role and regulation of neutrophils during type 2 immune responses are controversial. Helminth models suggested a contribution of neutrophils to type 2 immunity, whereas neutrophils are associated with increased disease severity during type 2 inflammatory disorders, such as asthma. We sought to evaluate the effect of the prototypic type 2 cytokines IL-4 and IL-13 on human neutrophils. Human neutrophils from peripheral blood were assessed without or with IL-4 or IL-13 for (1) expression of IL-4 receptor subunits, (2) neutrophil extracellular trap (NET) formation, (3) migration toward CXCL8 invitro and in humanized mice, and (4) CXCR1, CXCR2, and CXCR4 expression, as well as (5) in nonallergic versus allergic subjects. Human neutrophils expressed both types of IL-4 receptors, and their stimulation through IL-4 or IL-13 diminished their ability to form NETs and migrate toward CXCL8 invitro. Likewise, invivo chemotaxis in NOD-scid-Il2rg-/- mice was reduced in IL-4-stimulated human neutrophils compared with control values. These effects were accompanied by downregulation of the CXCL8-binding chemokine receptors CXCR1 and CXCR2 on human neutrophils on IL-4 or IL-13 stimulation invitro. Exvivo analysis of neutrophils from allergic patients or exposure of neutrophils from nonallergic subjects to allergic donor serum invitro impaired their NET formation and migration toward CXCL8, thereby mirroring IL-4/IL-13-stimulated neutrophils. IL-4 receptor signaling in human neutrophils affects several neutrophil effector functions, which bears important implications for immunity in type 2 inflammatory disorders.

Broad IgG repertoire in patients with chronic rhinosinusitis with nasal polyps regulates proinflammatory IgE responses

Chronic rhinosinusitis with nasal polyps (CRSwNP) is often characterized by local production of polyclonal IgE idiotypes. Although tissue IgE concentrations can be in the range of several thousand kilounits per liter, the regulatory mechanisms by which IgE-mediated inflammation is controlled in patients with nasal polyps are not well understood. We sought to determine whether locally induced IgG antibodies in patients with nasal polyps can inhibit an IgE-mediated proallergic response. Nasal polyp homogenates were collected from patients with grass pollen allergy with CRSwNP and nonallergic control subjects. IgE levels were measured using the Immuno Solid-phase Allergen Chip assay. IgE-containing nasal polyp homogenates with or without IgG depletion were evaluated for their capacity to promote IgE-facilitated allergen presentation, basophil activation, and histamine release. Local IgE and IgG repertoires were evaluated using Immunoglobulin 454 sequencing. We show that IgG plays a key role in controlling IgE-mediated inflammatory responses in patients with nasal polyps. Depletion of IgG from nasal homogenates resulted in an increase in CD23-mediated IgE-facilitated allergen binding to Bcells but also enhanced FcεRI-mediated allergen-driven basophil activation and histamine release. Asimilar response was observed in relation to specific IgE antibodies to Staphylococcus aureus enterotoxins. The capacity of IgG in nasal polyps to limit IgE-mediated inflammation is based on the fact that IgG repertoires widely share the antigen targets with the IgE repertoires in both allergic and nonallergic subjects. Polyclonal IgE idiotypes in patients with CRSwNP are functional, promote IgE-mediated proallergic inflammation, and are partially antagonized by corresponding IgG idiotypes. This is most likely due to the fact that IgE and IgG clonotypes are widely shared in patients with nasal polyps.

Abstract # 3081 Impaired cognition and fatigue; the role of sleep and neuroinflammation for non-specific symptoms in allergy

Allergy is associated with non-specific symptoms such as fatigue and impaired cognition. Allergic patients also report worse sleep compared to healthy controls. As part of a study where we investigated seasonal changes in the glial cell marker translocator protein (TSPO), we studied fatigue, sleep and cognitive functions in allergic patients, in relation to seasonal changes in the glial cell marker translocator protein (TSPO) as measured using positron emission tomography (PET). We examined 18 patients with severe seasonal allergy and 13 healthy subjects in and out of pollen season, using TSPO PET, subjective ratings of fatigue and sleepiness, objective and subjective sleep measures and a test-battery of cognitive tests (CANTAB). Allergic subjects had shorter total sleep time, regardless of season, compared to healthy controls. Subjects with allergy also reported worse sleep quality during pollen season, but had a higher percentage of deep sleep. These effects were paralleled by increased fatigue and sleepiness, as well as increased levels of IL-5 and TNF-α during pollen season, but were unrelated to brain TSPO levels. Allergic patients showed lower scores on intra-extra dimensional set shift during pollen season, indicating lower capacity in set-shifting and flexibility of attention. We speculate that impaired sleep, together with neuroinflammatory mechanisms, contribute to a sickness-like state in allergy, characterized by fatigue, and dysregulated exteroceptive – and potentially increased interoceptive – attention.

Gender differences in dendritic cell population in nasal and oral cavity between allergic and non-allergic subjects.

Gender differences in dendritic cell population in nasal and oral cavity between allergic and non-allergic subjects.

Possible sources of Bermuda sensitization in Timothy grass allergic subjects

Possible sources of Bermuda sensitization in Timothy grass allergic subjects

A 5-year Summary of Real-life Dietary Egg Consumption after Completion of a 4-year Egg OIT Protocol

Long-term dietary implications of egg OIT (eOIT), including desensitization and sustained unresponsiveness (SU), are not well described. Previously described egg allergic subjects (5-11 yo) were randomized to eOIT (n=40, treated with eOIT up to 4 years) or placebo (n=15, 1 year). Subjects passing a 10g OFC on therapy were considered desensitized and underwent a 10g OFC and open feeding 4-6 weeks after discontinuing therapy to determine SU. Long-term follow-up questionnaires (LFQ) assessing egg consumption and symptoms were administered annually for 5 years following trial completion. At study completion, 20/40 (50%) eOIT subjects were classified as SU, 11/40 (28%) subjects desensitized, and 9/40 (22%) not desensitized. Annual LFQ completion ranged from 76-82% of all subjects (85-95% of the SU group). At year 5, 93% (30/32) of eOIT subjects were ingesting some egg compared to 64% (7/11) of placebo (p=0.029). 100% (19/19) of SU subjects were ingesting both concentrated and baked egg versus 43%, 17%, and 36% in the desensitized, not desensitized, and placebo groups, respectively. Decreased frequency and amount of egg ingestion and increased symptoms were observed in all non-SU groups versus the SU group. Epinephrine was required after concentrated egg ingestion in 3 non-SU subjects. Five years after study completion, eOIT treated subjects achieving SU were highly likely to consume and tolerate dietary egg, while other groups had more variable egg consumption and increased symptoms associated with consumption. Further study to develop biomarkers predictive of treatment response is necessary to mitigate risk and assess clinical outcomes with OIT.

Determining Best Tests of Baked Egg Tolerance in Egg Allergic Subjects

Determining Best Tests of Baked Egg Tolerance in Egg Allergic Subjects

Allergen-specific CD8+ T cells in peanut-allergic individuals

CD8+ T cells are seldom considered in IgE mediated food allergy; we show that peanut specific CD8+ T cells are increased in peanut allergic human subjects.

Pollen allergy and health behavior: patients trivializing their disease

Allergies are increasing in prevalence worldwide, with socioeconomic impacts and effects on quality of life. The aim of this study was to explore the health behavior and the utilization of different treatment options via questionnaires and to investigate for relationships of the above with socioeconomic factors. This cross-sectional survey was carried out among pollen allergic subjects in 2016, using questionnaires. A total of 679 allergics participated in the study (61.2% females). Their average age was 26.8 ± 8.8 years. Their symptom severity was 6.1 ± 1.9, measured on a 10-step scale and symptoms lasted for 9.0 ± 6.8 weeks during pollen season. Of all allergics, 9.1% were not aware of the causative agent of their allergy and 17.4% had never undergone allergy testing. Symptoms, especially in females, had strong impact on social life, everyday routines and sleep quality. Almost half of the participants treated their allergy without medical supervision, while only 32.3% sought medical support. Nevertheless, three quarters reported self-management of their allergies with oral antihistamines. Compared to males, females sought significantly more medical support, medications and allergen avoidance strategies. Knowledge about allergy increased the likelihood of treatment under supervision of a medical expert than no treatment, as well as symptom severity and interaction between female gender and symptom severity. The attitude of not considering allergy as a serious disease significantly reduced the likelihood of undergoing specific immunotherapy. This survey not only highlights the negative impact of pollen allergies on everyday life of allergics, but also that allergies are often neglected and untreated because of their trivialization by allergic subjects themselves.

An integrated framework using high-dimensional mass cytometry and fluorescent flow cytometry identifies discrete B cell subsets in patients with red meat allergy.

B cells play a critical role in the development and maintenance of food allergy by producing allergen-specific IgE. Despite the importance of B cells in IgE-mediated food allergy, the identity of sIgE-producing human B cells and how IgE is regulated are poorly understood. To identify the immunophenotypes of circulating B cells associated with the production of galactose-alpha-1,3-galactose-specific IgE production in patients with red meat allergy. B cells in PBMC samples obtained from 19 adults with physician-diagnosed red meat allergy and 20 non-meat allergic healthy controls were assessed by mass cytometry along with a bioinformatics analysis pipeline to identify discrete B cell phenotypes that associated with serum sIgE. Fluorescent flow cytometry was then applied to sort purify discrete B cell subsets, and B cells were functionally evaluated on an individual cell level for the production of sIgE by ELISPOT. Discrete B cell phenotypes abundant in meat allergic subjects compared to non-meat allergic controls were found in peripheral blood that do not share typical characteristics of classical isotype-switched memory B cells that express high levels of CD27. These B cell subsets shared higher IgD and lower IgM expression levels coupled with CXCR4, CCR6 and CD25 expression. In vitro polyclonal stimulation of purified B cell subsets from meat allergic subjects demonstrated that these subsets were enriched for cells induced to secrete sIgE. Circulating B cells display increased abundance of discrete B cell subsets in meat allergic subjects. This observation, coupled with the capacity of individual B cell subsets to produce sIgE following activation, implicates these novel B cell phenotypes in promoting IgE in meat allergy.

Immunological Profile of Iraqi Atopic Patients

The aims of the study are determine some immunological aspects namely total IgE, specific IgE, eosinophil count, IFN-γ, IL-10 and CCL-17 in serum of atopic patients and apparently healthy control to determine their relationship to atopic diseases. The age group of 21–40 years seems to be the most affect group with atopic diseases (P =0.0001) and asthmatic and allergic rhinitis patients are at this age group also (P =0.005, P =0.001respectively). There is a major differences (P < 0.001) in level of total IgE between allergic subjects and controls and among three atopic patients groups (P < 0.001) but Specific IgE is significantly higher in asthmatics than allergic rhinitis and atopic dermatitis patients (P < 0.05). Inhalation of outdoor allergens is significantly affected asthmatic patients and allergic rhinitis patients (P < 0.01). Controversially, indoor allergens are significantly affecting atopic dermatitis (P < 0.01). The mean of IFN-γ serum level in asthma patients was lower than IFN-γ serum level of the control group and atopic dermatitis patients (P < 0.05). The allergic rhinitis patients mean of serum level of IL-10 was significantly low as compared to the mean of the serum level of IL-10 control group (P= 0.020). All of three groups of atopic patients are significantly have high level of CCL-17 compare to mean of serum level of control group (P < 0.01).