Nonatopic Subjects Research Articles

An Immunodiagnostic Assay for Quantitation of Specific IgE to the Major Pollen Allergen Component, Pas n 1, of the Subtropical Bahia Grass

Background: Pollens of the Panicoideae subfamily of grasses including Bahia (Paspalum notatum) are important allergen sources in subtropical regions of the world. An assay for specific IgE to the major molecular allergenic component, Pas n 1, of Bahia grass pollen (BaGP) would have immunodiagnostic utility for patients with pollen allergy in these regions. Methods: Biotinylated Pas n 1 purified from BaGP was coated onto streptavidin ImmunoCAPs. Subjects were assessed by clinical history of allergic rhinitis and skin prick test (SPT) to aeroallergens. Serum total, BaGP-specific and Pas n 1-specific IgE were measured. Results: Pas n 1 IgE concentrations were highly correlated with BaGP SPT (r = 0.795, p < 0.0001) and BaGP IgE (r = 0.915, p < 0.0001). At 0.23 kU/l Pas n 1 IgE, the diagnostic sensitivity (92.4%) and specificity (93.1%) for the detection of BaGP allergy was high (area under receiver operator curve 0.960, p < 0.0001). The median concentrations of Pas n 1 IgE in non-atopic subjects (0.01 kU/l, n = 67) and those with other allergies (0.02 kU/l, n = 59) showed no inter-group difference, whilst grass pollen-allergic patients with allergic rhinitis showed elevated Pas n 1 IgE (6.71 kU/l, n = 182, p < 0.0001). The inter-assay coefficient of variation for the BaGP-allergic serum pool was 6.92%. Conclusions: Pas n 1 IgE appears to account for most of the BaGP-specific IgE. This molecular component immunoassay for Pas n 1 IgE has potential utility to improve the sensitivity and accuracy of diagnosis of BaGP allergy for patients in subtropical regions.

Novel CLC3 transcript variants in blood eosinophils and increased CLC3 expression in nasal lavage and blood eosinophils of asthmatics

Eosinophilia is a characteristic feature of allergic airway inflammation and remodeling. Chloride channel-3 (CLC3) in eosinophils has been associated with superoxide generation and respiratory burst. The CLC3 gene may produce multiple transcript variants through alternative splicing. However, the presence of CLC3 variants in human eosinophils is unknown. We examined the expression of CLC3 transcript variants in peripheral blood eosinophils of allergic asthmatics and healthy individuals. Potential of these obligatory dimers to form homo- or hetero-dimers was examined in HEK293 cells co-transfected with CLC3b-GFP and CLC3e-RFP. Eosinophils were isolated from peripheral blood by negative selection. Expression of CLC3 and CLC3 transcript variants was examined by qPCR, Western blot, and immunofluorescence. Confocal micrographs were analyzed with Image J software. Higher levels of novel transcript variants of CLC3 (CLC3b and CLC3e) were found in peripheral blood eosinophils of asthmatics compared to healthy non-atopic subjects. We also found higher CLC3 protein expression in the blood and nasal lavage eosinophils of asthmatics than healthy subjects. Both membranous and intracellular CLC3 expression were observed. Also, we found the presence of both homodimers and heterodimers of CLC3b-GFP and CLC3e-RFP in HEK293 cells. Higher and differential expression of novel CLC3 transcript variants in mild-to-moderate and moderate-to-severe asthmatic eosinophils suggest their critical role in allergic asthma. Membranous and intracellular (granular) expression of CLC3 in nasal lavage and peripheral blood eosinophils suggest their involvement in the activation and migration of eosinophils in allergic asthma. Moreover, homo- and hetero-dimerization of these transcript variants may change the channel properties to exhibit these states. Presence of CLC3 variants may serve as a biomarker in allergic asthma and additional knowledge of interaction between CLC3 transcript variants and their specific role in the activation and migration of eosinophils will allow to explore novel therapeutic approach in allergic asthma.

“Default” versus “pre-atopic” IgG responses to foodborne and airborne pathogenesis-related group 10 protein molecules in birch-sensitized and nonatopic children

The route and dose of exposure are believed to be relevant factors in the sensitization process. Pathogenesis-related group 10 protein (PR-10) molecules are a family of allergenic proteins shared by many pollens (eg, birch and alder) and foods (eg, apple, peach, and soy). Children are exposed to both pollen-derived (inhaled) and food-derived (ingested) PR-10 molecules. We sought to investigate the role of route and dose of exposure in the evolution of IgG and IgE responses to recombinant PR-10 molecules. The German Multicentre Allergy Study examined a birth cohort born in 1990. Blood samples were collected at the ages of 1, 2, 3, 5, 6, 7, 10, and 13 years. Participants were included in the present analysis if they had (1) at least 1 serum sample at each of the 4 age periods or time points (1-3 years, 5-7 years, 10 years, and 13 years) and (2) IgE responses to birch (children with birch atopy) or no IgE response at all to 9 common aeroallergens and food allergens (nonatopic children). Therefore serum IgE antibodies to a panel of 4 airborne and 5 foodborne extracts, as well as to Bet v 1, were measured in singleplex assays, whereas IgG and IgE antibodies to a panel of 3 airborne PR-10 molecules (rBet v 1, rAln g 1, and rCor a 1.0101) and 7 foodborne PR-10 molecules (rCor a 1.0401, rMal d 1, rPru p 1, rGly m 4, rAra h 8, rApi g 1, and rDau c 1) were tested by using a multiplex microarray. In the present analyses we included 28 children with birch atopy and randomly selected 28 nonatopic children from the 190 children fulfilling the inclusion criteria. Two different patterns of IgG responses to PR-10 molecules were identified. Among nonatopic subjects, a "default" IgG response was directed mostly against foodborne PR-10, started often before age 2 years, stayed weak, and was mostly transient. Among all atopic subjects, the default IgG response at age 1 year was overwhelmed after age 2 years by an "pre-atopic" IgG response, which started with or shortly before the IgE response and was intense and persistent. This atopic IgG response, as well as the IgE response, involved progressively more foodborne PR-10 proteins with frequencies and levels related to their homology with Bet v 1. The results suggest that children have a default antibody response to PR-10 molecules, which is early, weak, and transient; does not involve IgE; and is initiated by foodborne PR-10. By contrast, an atopic antibody response to PR-10 molecules is delayed, strong, and persistent; involves both IgG and IgE; and is initiated by airborne PR-10.

Basophil expression of diamine oxidase: A novel biomarker of allergen immunotherapy response

Immunotherapy inhibits basophil histamine release, but the assay is cumbersome, and no one has studied the effects of immunotherapy withdrawal. Intracellular fluorochrome-labeled diamine oxidase (DAO) was used as a novel functional readout of basophil histamine release after immunotherapy. Results were compared with conventional basophil surface expression of activation markers. Subcutaneous immunotherapy (SCIT)-treated patients (n= 14), sublingual immunotherapy (SLIT)-treated patients (n= 12), participants who completed 3 years of treatment with grass pollen sublingual immunotherapy (the SLIT-TOL group; n= 6), patients with untreated seasonal allergic rhinitis (SAR; n= 24), and nonatopic control subjects (n= 12) were studied. Intracellularly labeled DAO(+) and surface expression of CD203c(bright), CD63(+), and CD107a(+) on chemoattractant receptor-homologous molecule expressed on TH2 lymphocytes (CRTh2)-positive basophils were measured by means of flow cytometry. Serum IgG4 levels and serum inhibitory activity for IgE-allergen complex binding to B cells (IgE-FAB) and basophil histamine release were also determined. Proportions of allergen-stimulated DAO(+)CRTh2(+) basophils were higher in participants in the SCIT, SLIT, and SLIT-TOL groups (all P< .0001) compared with those in patients in the SAR group. Similarly, there were lower proportions of CRTh2(+) basophils expressing surface CD203c(bright) (all P< .001), CD63 (all P< .001), and CD107a (allP< .01). Rhinitis symptoms were lower in the SCIT, SLIT, and SLIT-TOL groups (P< .001) compared with those in the SAR group. Serum inhibitory activity for IgE-FAB and basophil histamine release were also significantly greater in all immunotherapy groups (P< .05) compared with the SAR group. These results support long-term clinical and immunologic tolerance during and after grass pollen immunotherapy. Intracellularly labeled DAO expression by basophils merits further investigation as a surrogate biomarker for monitoring efficacy and tolerance after immunotherapy.

Aberrant expression of regulatory cytokine IL-35 and pattern recognition receptor NOD2 in patients with allergic asthma.

We investigated the plasma concentration of the novel regulatory cytokine IL-35 and intracytosolic pattern recognition receptors nucleotide-binding oligomerization domain (NOD)-like receptors in granulocytes and explored their potential implication in disease severity monitoring of allergic asthma. The expression of circulating IL-35 and other pro-inflammatory mediators in asthmatic patients or control subjects were evaluated using enzyme-linked immunosorbent assay (ELISA). The intracellular expressions of NOD1 and NOD2 in CCR3+ granulocytes were assessed using flow cytometry. Plasma concentrations of IL-35, IL-17A, basophil activation marker basogranulin, and eosinophilic airway inflammation biomarker periostin were significantly elevated in allergic asthmatic patients compared to non-atopic control subjects (all probability (p) <0.05). Both granulocyte markers exhibited significant and positive correlation with plasma IL-35 concentration in asthmatic patients (all p < 0.05). Significant positive correlation was also identified between plasma concentrations of IL-35 and periostin with disease severity score in asthmatic patients (both p < 0.05). The basophil activation allergenicity test was positive in allergic asthmatic patients but not in control subjects. Despite significantly elevated eosinophil count in allergic asthmatic patients, downregulation of NOD2 in CCR3+ granulocytes was observed in these patients (both p < 0.05). A negative correlation between plasma concentrations of tumor necrosis factor family member LIGHT and soluble herpesvirus entry mediator was observed in patients with elevated plasma concentration of IL-35 (p < 0.05). Aberrant expression of NOD2 in granulocytes may be contributed to the impaired innate immunity predisposing allergic asthma. IL-35 may serve as a potential surrogate biomarker for disease severity of allergic asthma.

Helicobacter pylori infection and allergic diseases in pediatric age. A retrospective study

s / Digestive and Liver Disease 46 (2014) e85–e127 e119 HELICOBACTER PYLORI INFECTION AND ALLERGIC DISEASES IN PEDIATRIC AGE. A RETROSPECTIVE STUDY Angela Lanzafame ∗, Stefania Tomarchio, Martina Filippelli, Salvatore Leonardi, Elena Lionetti Unit of Pediatric Pneumo-Allergology and Cystic Fibrosis, University of Catania, Catania, Italy Objective: The recent spread of allergic diseases in industrialized countries has been associated with a reduced prevalence of infectious diseases. To evaluate the hypothesis that Helicobacter pylori (Hp) infection can protect from allergic diseases, we determined the prevalence of Hp infection and atopic diseases in a pediatric population. Methods: 95 patients [male: 49 (51.6%), median age: 8 years, range: 1–18 years] living in the province of Catania were enrolled. The status of atopy was evaluated by performing skin prick test and radioallergosorbent test for principal allergens; Hp status was assayed through the detection of bacterial antigens in diluted fecal samples. Results: Overall, 17 out of 95 children enrolled (17.9%) were positive forHp infection; 4.8% (2/42) of atopic patientswereHppositive, as compared to28.3% (15/53) of non-atopic (P<0.01,OR=0.1). Conclusions: As in other geographic areas with a high standard of living, even in the province of Catania the prevalence ofHp infection in the 1–18 aged population is low. Our study shows that the prevalence ofHp infection is significantly higher in non-atopic subjects, confirming a protective role of Hp infection versus atopic diseases. http://dx.doi.org/10.1016/j.dld.2014.07.143 EVALUATION OF MAGNETIC RESONANCE ENTEROGRAPHY IN PEDIATRIC CROHN DISEASE: A RETROSPECTIVE PILOT-STUDY Martina Vallorani1,∗, Alessandra Palpacelli 1, Matilde Rossi1, Simona Gatti 1, Giovanni Pieroni2, Veronica Albano1, Carlo Catassi1 1 Dipartimento di Pediatria, Ospedale Salesi/Universita Politecnica delle Marche, Ancona, Italy 2 Dipartimento di Radiologia, Ospedale Salesi/Universita Politecnica delle Marche, Ancona,

Gene expression profiling of laser microdissected airway smooth muscle tissue in asthma and atopy

Asthma and atopy share common characteristics including type 2 helper-T-cell-mediated inflammation. However, only asthma is associated with variable airways obstruction. The complex cellular and molecular pathways distinguishing asthma and atopy can now be captured by transcriptomic analysis (RNA-Seq). We hypothesized that the transcriptomic profile of airway smooth muscle (ASM) distinguishes atopic asthma from atopic healthy controls. First, we compared the ASM transcriptomic profiles of endobronchial biopsies between glucocorticoid-free, atopic asthma patients, and atopic and nonatopic healthy controls. Second, we investigated the association between ASM transcriptomic profiles and airway function. Twelve asthma patients and 12 control subjects (six atopic, six nonatopic) underwent bronchoscopy. RNA of laser-dissected ASM from 96 bronchial biopsy specimens was sequenced with Roche GS FLX. Gene networks were identified using Ingenuity Pathway Analysis. RNA-Seq reads were assumed to follow a negative binomial distribution. With the current sample size, the estimated false discovery rate was approximately 1%. One hundred and seventy four ASM genes were differentially expressed between asthma patients and atopic controls, 108 between asthma patients and nonatopic controls, and 135 between atopic and nonatopic controls. A set of eight genes discriminated asthma patients from nonasthmatic controls, irrespective of atopy. Four of these genes (RPTOR, VANGL1, FAM129A, LEPREL1) were associated with airway hyper-responsiveness (P<0.05). Airway smooth muscle from asthma patients can be distinguished from that of atopic and nonatopic control subjects by a specific gene expression profile, which is associated with airway hyper-responsiveness.

Is atopy in early childhood a risk factor for ADHD and ASD? A longitudinal study

ObjectivePrevious studies have found a temporal concordance in the increased prevalence of atopic diathesis/atopic diseases, attention-deficit hyperactivity disorder (ADHD), and autistic spectrum disorder (ASD) worldwide. But, the temporal association among these 3 distinct diseases is unknown. Method14,812 atopic subjects diagnosed with any atopic disease (asthma, atopic dermatitis, allergic rhinitis, or allergic conjunctivitis) before the age of 3 (atopic cohort) and 6944 non-atopic subjects with no lifetime atopic disease (non-atopic cohort), born between 1997 and 2000, were enrolled and followed to December 31, 2010 to identify the development of ADHD and ASD. ResultsThe presence of any atopic disease in early childhood increased the risk of developing ADHD (hazard ratio [HR]: 1.97) and ASD (HR: 3.40) in later life. Greater numbers of atopic comorbidities (4 comorbidities: ADHD: HR: 2.53; ASD: HR: 4.29) were significantly related to a greater risk of developing ADHD and ASD. DiscussionAtopic diathesis in early childhood elevated the risk of developing ADHD and ASD in later life, with the dose-dependent relationship of more atopic comorbidities with a greater likelihood of ADHD and ASD.

Skin prick test reactivity to lupin in comparison to peanut, pea, and soybean in atopic and non-atopic German subjects: A preliminary cross-sectional study.

The increasing use of lupin in food processing poses a problem of potential (cross-)allergic reactions. To evaluate the prevalence of sensitization to lupin in comparison to that of other legumes skin prick tests were performed with lupin, pea, peanut, and soybean in atopic (n = 81) and non-atopic (n = 102) German adults. Of these 183 subjects, 20 subjects had to be excluded due to invalid skin prick tests (reaction to histamine <3 mm or to sodium chloride >2 mm). Thus, skin prick tests of 163 subjects were included in final analyses. Of 163 subjects, 18 had a positive reaction to at least one legume tested. Overall skin prick test reactivity was different among non-atopic and atopic subjects (P = 0.005). Altogether, six subjects (4%) were sensitized to lupin, 12 (7%) to pea, 5 (3%) to peanut, and 8 (5%) to soybean. Two (2%) of the 92 non-atopic subjects and 4 (6%) of the 71 atopic subjects had a positive skin prick test to lupin. Of the 6 subjects sensitized to lupin, 3 (50%) were also sensitized to pea, 3 (50%) to peanut, and 5 (83%) to soybean. In conclusion, the prevalence rates of lupin sensitization were comparable to or even lower than those of pea, peanut, and soybean. To date, lupin allergy is suspected to be relatively uncommon in the overall German population since lupin sensitization occurred in only 2% of non-atopic subjects. However, there is a clear risk of a lupin allergy in predisposed subjects, since the frequency of lupin sensitization was 6% in atopic subjects. In particular, subjects with existing sensitization or allergy to other legumes are at higher risk for a sensitization or allergy to lupin due to cross-reactivity.

Filaggrin-stratified transcriptomic analysis of pediatric skin identifies mechanistic pathways in patients with atopic dermatitis

BackgroundAtopic dermatitis (AD; eczema) is characterized by a widespread abnormality in cutaneous barrier function and propensity to inflammation. Filaggrin is a multifunctional protein and plays a key role in skin barrier formation. Loss-of-function mutations in the gene encoding filaggrin (FLG) are a highly significant risk factor for atopic disease, but the molecular mechanisms leading to dermatitis remain unclear.ObjectiveWe sought to interrogate tissue-specific variations in the expressed genome in the skin of children with AD and to investigate underlying pathomechanisms in atopic skin.MethodsWe applied single-molecule direct RNA sequencing to analyze the whole transcriptome using minimal tissue samples. Uninvolved skin biopsy specimens from 26 pediatric patients with AD were compared with site-matched samples from 10 nonatopic teenage control subjects. Cases and control subjects were screened for FLG genotype to stratify the data set.ResultsTwo thousand four hundred thirty differentially expressed genes (false discovery rate, P < .05) were identified, of which 211 were significantly upregulated and 490 downregulated by greater than 2-fold. Gene ontology terms for “extracellular space” and “defense response” were enriched, whereas “lipid metabolic processes” were downregulated. The subset of FLG wild-type cases showed dysregulation of genes involved with lipid metabolism, whereas filaggrin haploinsufficiency affected global gene expression and was characterized by a type 1 interferon–mediated stress response.ConclusionThese analyses demonstrate the importance of extracellular space and lipid metabolism in atopic skin pathology independent of FLG genotype, whereas an aberrant defense response is seen in subjects with FLG mutations. Genotype stratification of the large data set has facilitated functional interpretation and might guide future therapy development.

The expression of cannabinoid receptor 1 is significantly increased in atopic patients

The expression of cannabinoid receptor 1 is significantly increased in atopic patients

Multiple House Dust Mite Allergen- Sensitization Profiles in Children with Allergic Asthma

Background: Allergic asthma is a common chronic inflammatory disorder which affects populations, with increasing prevalence among children. The risk of developing asthma is uncertain but depends on the interaction of environmental and genetic factors. The role of house dust mite (HDM) allergen exposure in the development of sensitization and asthma remains unclear. Objective: This study determined the sensitization profiles of common HDM species in a population of asthmatic children, which is essential in the development of highly specific and accurate diagnostic and therapeutic strategies for asthma in children. Methods: The immunoglobulin E-binding activity of allergens from the HDM species Blomia tropicalis (Bt), Dermatophagoides farina (Df), and Dermatopahagoides pteronyssinus (Dp) were determined in 250 age- and sexmatched paediatric allergic asthma and non-atopic Filipino subjects using Enzyme-linked immunosorbent assay and western blot analysis. Results: Majority of the allergic asthma patients tested were sensitized with multiple allergens from different HDM species where 33% exhibited sensitizations to any two HDM species and 26% were sensitized with allergens from three HDMs. HDM allergens of different molecular weights bind to IgE in allergic asthma patients tested. In addition a significant correlation was observed between total IgE and HDM-specific IgE level among allergic asthma patients (Bt p-value=0.038; Df p-value=0.045; Dp p-value=0.003), suggesting a significant contribution of dust mite allergens in the up-regulation of total serum IgE levels of allergic asthma patients. Conclusion: The results obtained in this study suggest that the HDM species Bt, Dp, and Df are important sources of allergens that trigger multiple sensitization in children with allergic asthma in the Filipino population. The incorporation of Bt, Dp, and Df allergens in the panel of diagnostic allergens for HDM allergy and allergic asthma is highly recommended.

Specific immunotherapy modifies allergen-specific CD4+ T-cell responses in an epitope-dependent manner

Understanding the mechanisms by which the immune system induces and controls allergic inflammation at the T-cell epitope level is critical for the design of new allergy vaccine strategies. We sought to characterize allergen-specific T-cell responses linked with allergy or peripheral tolerance and to determine how CD4(+) T-cell responses to individual allergen-derived epitopes change over allergen-specific immunotherapy. Timothy grass pollen (TGP) allergy was used as a model for studying grass pollen allergies. The breadth, magnitude, epitope hierarchy, and phenotype of the DR04:01-restricted TGP-specific T-cell responses in 10 subjects with grass pollen allergy, 5 nonatopic subjects, and 6 allergy vaccine-treated subjects was determined by using an ex vivo peptide-MHC class II tetramer approach. CD4(+) T cells in allergic subjects are directed to a broad range of TGP epitopes characterized by defined immunodominance hierarchy patterns and with distinct functional profiles that depend on the epitope recognized. Epitopes that are restricted specifically to either TH2 or TH1/TR1 responses were identified. Allergen-specific immunotherapy was associated with preferential deletion of allergen-specific TH2 cells and without a significant change in the frequency of TH1/TR1 cells. Preferential allergen-specific TH2 cell deletion after repeated high-dose antigen stimulation can be another independent mechanism to restore tolerance to allergen during immunotherapy.

Transcutaneous yellow fever vaccination of subjects with or without atopic dermatitis

Atopic dermatitis (AD) is a common inflammatory skin disease with a global prevalence ranging from 3% to 20%. Patients with AD have an increased risk for complications after viral infection (eg, herpes simplex virus), and vaccination of patients with AD with live vaccinia virus is contraindicated because of a heightened risk of eczema vaccinatum, a rare but potentially lethal complication associated with smallpox vaccination. We sought to develop a better understanding of immunity to cutaneous viral infection in patients with AD. In a double-blind randomized study we investigated the safety and immunogenicity of live attenuated yellow fever virus (YFV) vaccination of nonatopic subjects and patients with AD after standard subcutaneous inoculation or transcutaneous vaccination administered with a bifurcated needle. Viremia, neutralizing antibody, and antiviral T-cell responses were analyzed for up to 30 days after vaccination. YFV vaccination administered through either route was well tolerated. Subcutaneous vaccination resulted in higher seroconversion rates than transcutaneous vaccination but elicited similar antiviral antibody levels and T-cell responses in both the nonatopic and AD groups. After transcutaneous vaccination, both groups mounted similar neutralizing antibody responses, but patients with AD demonstrated lower antiviral T-cell responses by 30 days after vaccination. Among transcutaneously vaccinated subjects, a significant inverse correlation between baseline IgE levels and the magnitude of antiviral antibody and CD4(+) T-cell responses was observed. YFV vaccination of patients with AD through the transcutaneous route revealed that high baseline IgE levels provide a potential biomarker for predicting reduced virus-specific immune memory after transcutaneous infection with a live virus.

Prevalence of atopy, eosinophilia, and IgE elevation in IgG4-related disease

IgG4-related disease (IgG4-RD) is a fibroinflammatory disorder that can affect virtually every organ system. T-helper type 2 responses have been presumed to be pathogenic in this disease, and a high proportion of patients with IgG4-RD are reported to have longstanding allergies, peripheral blood eosinophilia, and serum IgE elevation. It has therefore been proposed that allergic mechanisms drive IgG4-RD. However, no epidemiological assessment of atopy, peripheral blood eosinophilia, and serum IgE concentrations has ever been undertaken in patients with IgG4-RD. In this study, we evaluated these parameters in a large cohort of patients with IgG4-RD in whom a wide range of organs were affected by disease. Our results demonstrate that the majority of patients with IgG4-RD are nonatopic. Nevertheless, a subset of nonatopic subjects exhibit peripheral blood eosinophilia and elevated IgE, suggesting that processes inherent to IgG4-RD itself rather than atopy per se contribute to the eosinophilia and IgE elevation observed in the absence of atopy.

Characterisation of CD154+ T cells following ex vivo allergen stimulation illustrates distinct T cell responses to seasonal and perennial allergens in allergic and non-allergic individuals

BackgroundAllergic sensitisation has been ascribed to a dysregulated relationship between allergen-specific Th1, Th2 and regulatory T cells. We sought to utilise our short-term CD154 detection method to further analyse the relationship between these T cell subsets and investigate differences between seasonal and perennial allergens. Using peripheral blood samples from grass-allergic, cat-allergic and healthy non-atopic subjects, we compared the frequencies and phenotype of CD154-positive T helper cells following stimulation with seasonal (grass) and perennial (cat dander) allergens.ResultsWe identified a higher frequency of CD154+ T cells in grass-allergic individuals compared to healthy controls; this difference was not evident following stimulation with cat allergen. Activated Th1, Th2 and Tr1-like cells, that co-express IFNγ, IL4 and IL10, respectively, were identified in varying proportions in grass-allergic, cat-allergic and non-allergic individuals. We confirmed a close correlation between Th1, Th2 and Tr1-like cell frequency in non-allergic volunteers, such that the three parameters increased together to maintain a low Th2: Th1 ratio. This relationship was dysregulated in grass-allergic individuals with no correlation between the T cell subsets and a higher Th2: Th1 ratio. We confirmed previous reports of a late-differentiated T cell phenotype in response to seasonal allergens compared to early-differentiated T cell responses to perennial allergens.ConclusionsThe findings confirm our existing work illustrating an important balance between Th1, Th2 and Tr1-like responses to allergens in health, where Th2 responses are frequently observed, but balanced by Th1 and regulatory responses. We confirm previous tetramer-based reports of phenotypic differences in T cells responding to seasonal and perennial allergens.

Cultured Mast Cells from Patients with Asthma and Controls Respond with Similar Sensitivity to Recombinant Der P2‐Induced, IgE‐Mediated Activation

The function of cultured mast cells may depend on genetic or environmental influence on the stem cell donor. This study investigates whether asthma or atopy in the donor influenced the growth and sensitivity of mast cells cultured from patients with asthma and healthy controls under identical conditions. Mast cells were cultured from peripheral blood from twelve patients with an objectively confirmed asthma diagnosis and eight healthy subjects. During the last 2 weeks of culture, mast cells were incubated with IL-4 and 80 kU/l recombinant human IgE containing two clones (7% + 7%) specific for mite allergen Der p2. The sensitivity of IgE-mediated activation of mast cells was investigated as FcεRI-mediated upregulation of CD63. Ten subjects were atopic, defined as a positive skin prick test (>3 mm) to at least one of ten common allergens. After activation with recombinant Der p2, the maximum CD63 median fluorescence intensity was 20 456 ± 1640 (SE) for patients with asthma and 22,275 ± 1971 (SE) for controls (ns). The fraction of CD63 positive cells was 54.4% in patients with asthma and 48.4% in controls (ns). The allergen concentration inducing 50% of the maximal CD63 response was similar in patients with asthma [-0.4795 log ng/ml ± 0.092 (SE)] and controls (-0.6351 log ng/ml ± 0.083, ns) and in atopic and non-atopic subjects. When cultured, sensitized and activated under identical conditions, mast cells from allergic asthmatics and healthy controls respond similar. Activation of cultured mast cells appears to depend on culture conditions (IL-4, IgE) rather than on donor status as atopy and asthma.

B cell activating factor (BAFF) and platelet activating factor (PAF) could both be markers of non-IgE-mediated reactions

Background B cell activating factor (BAFF) is a member of the tumor necrosis factor superfamily and an important regulator of peripheral B cell survival, maturation and immunoglobulin class-switch recombination. Many studies suggest that BAFF might be a new mediating mechanism in foodrelated inflammation. Higher levels in non-atopic compared with atopic patients, and no correlation between BAFF and IgE, suggest that BAFF might be particularly involved in non-IgE-mediated reactions [1]. According to Finkelman there are 2 pathways of systemic anaphylaxis: antigens can cause systemic anaphylaxis in mice through the classic pathway by cross-linking IgE bound to mast cell FceRI, stimulating histamine and PAF release, or the alternative pathway by forming complexes with IgG that cross-link macrophage FcgRIII, stimulating only PAF release [2]. The aim of this study is to evaluate the correlation between BAFF and PAF in non-atopic subjects.

TLR3, TLR4 and TLRs7-9 Induced Interferons Are Not Impaired in Airway and Blood Cells in Well Controlled Asthma.

Defective Rhinovirus induced interferon-β and interferon-λ production has been reported in bronchial epithelial cells from asthmatics but the mechanisms of defective interferon induction in asthma are unknown. Virus infection can induce interferon through Toll like Receptors (TLR)3, TLR7 and TLR8. The role of these TLRs in interferon induction in asthma is unclear. This objective of this study was to measure the type I and III interferon response to TLR in bronchial epithelial cells and peripheral blood cells from atopic asthmatics and non-atopic non-asthmatics. Bronchial epithelial cells and peripheral blood mononuclear cells from atopic asthmatic and non-atopic non-asthmatic subjects were stimulated with agonists to TLR3, TLR4 & TLRs7–9 and type I and III interferon and pro-inflammatory cytokine, interleukin(IL)-6 and IL-8, responses assessed. mRNA expression was analysed by qPCR. Interferon proteins were analysed by ELISA. Pro-inflammatory cytokines were induced by each TLR ligand in both cell types. Ligands to TLR3 and TLR7/8, but not other TLRs, induced interferon-β and interferon-λ in bronchial epithelial cells. The ligand to TLR7/8, but not those to other TLRs, induced only type I interferons in peripheral blood mononuclear cells. No difference was observed in TLR induced interferon or pro-inflammatory cytokine production between asthmatic and non-asthmatic subjects from either cell type. TLR3 and TLR7/8,, stimulation induced interferon in bronchial epithelial cells and peripheral blood mononuclear cells. Interferon induction to TLR agonists was not observed to be different in asthmatics and non-asthmatics.

Racial differences in the association of CD14 polymorphisms with serum total IgE levels and allergen skin test reactivity

BackgroundThe CD14 C-159T single nucleotide polymorphism (SNP) has been investigated widely as a candidate genetic locus in patients with allergic disease. There are conflicting results for the association of the CD14 C-159T SNP with total serum immunoglobulin E (IgE) levels and atopy. There are limited data regarding the association of the CD14 C-159T SNP in subjects of African ancestry. The aim of the study was to determine whether the C-159T SNP and other CD14 SNPs (C1188G, C1341T) were associated with total serum IgE levels and with allergy skin test results in nonatopic and atopic subjects; as well as in Caucasian and African American subjects.MethodsA total of 291 participants, 18–40 years old, were screened to determine whether they were atopic and/or asthmatic. Analyses were performed to determine the association between CD14 C-159T, C1188G, or C1341T genotypes with serum IgE levels and with the number of positive skin tests among Caucasian or African American subjects.ResultsWe found no significant association of serum total IgE level with CD14 C-159T, C1188G, or C1341T genotypes within nonatopic or atopic subjects. Subjects with CD14-159 T alleles had significantly more positive allergen skin tests than subjects without CD14-159 T alleles (P = 0.0388). There was a significant association between the CD14 1188 G allele, but not the CD14 1341 T allele, with the number of positive skin-test results in Caucasians, but not in African Americans.ConclusionThese results support a possible association between CD14 polymorphisms and atopy. CD14-159 T or CD14 1188 G alleles were associated with atopic disease. For subjects with CD14 1188 G alleles, the association with atopic disease was stronger in Caucasians compared to African Americans.