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Purpose To identify molecular features that distinguish individuals with shared clinical features of granulomatous uveitis. Design Cross-sectional observational study. Participants Four eyes from patients with active granulomatous uveitis. Methods We performed single-cell RNA sequencing with antigen-receptor sequence analysis to obtain an unbiased gene expression survey of ocular immune cells and to identify clonally expanded lymphocytes. Main Outcomes Measures For each inflamed eye, we measured the proportion of distinct immune cell types, the amount of B- or T-cell clonal expansion, and the transcriptional profile of T and B cells. Results Each individual showed robust clonal expansion arising from a single T- or B-cell lineage, suggesting distinct, antigen-driven pathogenic processes in each patient. This variability in clonal expansion was mirrored by individual variability in CD4 T-cell populations, whereas ocular CD8 T cells and B cells were more similar transcriptionally among patients. Finally, ocular B cells displayed evidence of class switching and plasmablast differentiation within the ocular microenvironment, providing additional support for antigen-driven immune responses in granulomatous uveitis. Conclusions Collectively, our study identified both conserved and individualized features of granulomatous uveitis, illuminating parallel pathophysiologic mechanisms and suggesting that future personalized therapeutic approaches may be warranted.

Background\ud IgE to α-Gal is a cause of mammalian meat allergy and has been linked to tick bites in North America, Australia, and Eurasia. Reports from the developing world indicate that α-Gal sensitization is prevalent but has been little investigated.\ud \ud Objective\ud We sought evidence for the cause(s) of α-Gal sensitization and lack of reported meat allergy among children in less developed settings in Ecuador and Kenya.\ud \ud Methods\ud IgE to α-Gal and total IgE were assessed in children from Ecuador (n = 599) and Kenya (n = 254) and compared with children with (n = 42) and without known (n = 63) mammalian meat allergy from the southeastern United States. Information on diet, potential risk factors, and helminth infections was available for children from Ecuador. IgG4 to α-Gal and antibodies to regionally representative parasites were assessed in a subset of children.\ud \ud Results\ud In Ecuador (32%) and Kenya (54%), α-Gal specific IgE was prevalent, but levels were lower than in children with meat allergy from the United States. Sensitization was associated with rural living, antibody markers of Ascaris exposure, and total IgE, but not active infections with Ascaris or Trichuris species. In Ecuador, 87.5% reported consuming beef at least once per week, including 83.9% of those who had α-Gal specific IgE. Levels of α-Gal specific IgG4 were not high in Ecuador, but were greater than in children from the United States.\ud \ud Conclusions\ud These results suggest that in areas of the developing world with endemic parasitism, α-Gal sensitization is (1) common, (2) associated with Ascaris exposure, and (3) distinguished by a low percentage of specific/total IgE compared with individuals with meat allergy in the United States.

Females of child-bearing age are more resistant to infectious disease and have an increased risk of systemic lupus erythematosus (SLE). We hypothesized that estrogen-induced gene expression could establish an immunoactivated state which would render enhanced defense against infection, but may be deleterious in autoimmune development. Using peripheral blood mononuclear cells (PBMCs), we demonstrate enhanced responses with immunogen stimulation in the presence of 17β-estradiol (E2) and gene array analyses reveal toll-like receptor 8 (TLR8) as an E2-responsive candidate gene. TLR8 expression levels are up-regulated in SLE and PBMCs stimulated with TLR8 agonist display a female sex-biased, E2-sensitive response. Moreover, we identify a putative ERα-binding region near the TLR8 locus and blocking ERα expression significantly decreases E2-mediated TLR8 induction. Our findings characterize TLR8 as a novel estrogen target gene that can lower the inflammatory threshold and implicate an IFNα-independent inflammatory mechanism that could contribute to higher SLE incidence in women.

Background Red meat allergy has historically been understood as a rare disease of atopic children, but the discovery of the “α-Gal syndrome,” which relates to IgE to the oligosaccharide galactose-α-1,3-galactose (α-Gal), has challenged that notion. Objective To describe the clinical and immunologic characteristics of a large group of subjects with self-reported allergy to mammalian meat. Methods This was an observational study of 261 children and adults (range, 5-82 years) who presented for evaluation for allergic reactions to mammalian meat. Results were based on serum assays and a detailed questionnaire. Results α-Gal specific IgE ≥ 0.35 IU/mL was detected in 245 subjects and symptom onset occurred ≥2 hours after eating mammalian meat in 211 (81%). Component testing supported a diagnosis of α-Gal syndrome in 95%, pork-cat syndrome in 1.9%, and primary beef allergy in 1.1%. Urticaria was reported by 93%, anaphylaxis by 60%, and gastrointestinal symptoms by 64%. Levels of IgE and IgG specific to α-Gal were similar in subjects who reported early- or delayed-onset symptoms, and in those with and without anaphylaxis. Levels of α-Gal specific IgE and severity of reactions were similar among those with and without traditional atopy, and among children (n = 35) and adults (n = 226). Blood group B trended toward being under-represented among α-Gal-sensitized subjects; however, α-Gal specific IgE titers were high in symptomatic cases with B-antigen. Conclusions The α-Gal syndrome is a regionally common form of food allergy that has a characteristic but not universal delay in symptom onset, includes gastrointestinal symptoms, can develop at any time in life, and is equally common in otherwise nonatopic individuals.

Determination of metabolic changes that occur following the development of and after red meat challenge in alpha-gal allergic individuals will aid in biomarkers for patient identification and targeted therapeutic development.

BACKGROUND: Although sensitization to indoor allergens is strongly associated with asthma, there are questions as to how this relates to asthma symptoms.OBJECTIVE: We sought to study the relevance ...

Rationale: IgE antibodies to the mammalian oligosaccharide galactose-α-1,3-galactose (α-gal) are common in the southeastern United States. These antibodies, which are induced by ectoparasitic ticks, can give rise to positive skin tests or serum assays with cat extract. Objectives: To evaluate the relationship between IgE antibodies to α-gal and asthma, and compare this with the relationship between asthma and IgE antibodies to Fel d 1 and other protein allergens. Methods: Patients being investigated for recurrent anaphylaxis, angioedema, or acute urticaria underwent spirometry, exhaled nitric oxide, questionnaires, and serum IgE antibody assays. The results were compared with control subjects and cohorts from the emergency department in Virginia (n = 130), northern Sweden (n = 963), and rural Kenya (n = 131). Measurements and Main Results: Patients in Virginia with high-titer IgE antibodies to α-gal had normal lung function, low levels of exhaled nitric oxide, and low prevalence of asthma symptoms. Among patients in the emergency department and children in Kenya, there was no association between IgE antibodies to α-gal and asthma (odds ratios, 1.04 and 0.75, respectively). In Sweden, IgE antibodies to cat were closely correlated with IgE antibodies to Fel d 1 (r = 0.83) and to asthma (P < 0.001). Conclusions: These results provide a model of an ectoparasite-induced specific IgE response that can increase total serum IgE without creating a risk for asthma, and further evidence that the main allergens that are causally related to asthma are those that are inhaled.

Background Although associated allergies are common and the mechanism may include long-term exposure to allergens, measurement of serum specific IgE levels has not been studied in pediatric eosinophilic esophagitis (EE). Objective To compare the results of serum IgE testing, patch testing, and epicutaneous skin testing to measure allergic sensitization in pediatric patients with EE. Methods In a cross-sectional study of 53 pediatric patients with EE, relevant history was obtained by questionnaire, and patch testing to foods was performed. Food and inhalant sensitivities were also assessed using skin prick testing and serum specific IgE measurement. Streptavidin CAP was used to measure specific IgE to cross-reactive carbohydrate determinants and Helicobacter pylori . Results The overall prevalence of food and inhalant sensitization was 80%, with higher total IgE levels in sensitized vs nonsensitized patients (median, 150 vs 13 IU/mL; P P = .001). Patch test results were interpreted as positive in 39% of patients (rye, wheat, and soy were the most common). Conclusions Most, but not all, patients with EE are highly atopic individuals with frequent allergic sensitivities. Thus, serum IgE measurement of low-titer IgE antibody may be useful in identifying relevant food sensitivities and in distinguishing subgroups of patients with EE, making a more directed approach to food avoidance possible.

Anti-galactose-α-1,3-galactose IgE from allergic patients does not bind α-galactosylated glycans on intact therapeutic antibody Fc domains

Background Reports from several African countries have noted an increasing prevalence of asthma in areas of extensive urbanization. Objective To investigate the relevance of allergen-specific sensitization and body mass index (BMI) to asthma/wheezing and exercise-induced bronchospasm (EIB) among children from affluent and poorer communities within a large town in Ghana. Methods Children with physician-diagnosed asthma and/or current wheezing aged 9–16 years (n=99; cases) from three schools with differing socio-economic backgrounds [urban affluent (UA), urban poor (UP) or suburban/rural (SR)] were recruited from a cross-sectional study (n=1848) in Kumasi, Ghana, and matched according to age, sex and area of residence with non-asthmatic/non-wheezy controls. We assayed sera for IgE antibodies to mite, cat, dog, cockroach, Ascaris and galactose-α-1,3-galactose. Results Children from the UA school had the lowest total serum IgE. However, cases from the UA school had a higher prevalence and mean titre of sIgE to mite (71.4%, 21.2 IU/mL) when compared with controls (14.3%, 0.8 IU/mL) or cases from UP (30%, 0.8 IU/mL) and SR community (47.8%, 1.6 IU/mL). While similar findings were observed with EIB in the whole population, among cases there was no difference in IgE antibody prevalence or titre between children with or without EIB. BMI was higher among UA children with and without asthma; in UP and SR communities, children with EIB (n=14) had a significantly higher BMI compared with children with asthma/wheezing without EIB (n=38) (18.2 vs. 16.4, respectively, P

IgE to mammalian allergens can contribute significantly to asthma risk. Studying the details of the relationship between animal sensitization and asthma is simpler in an environment where mite, fungal, and cockroach allergens make little or no contribution to asthma risk. Methods: Quantitative assays for IgE to eight allergens were carried out on 963 sera from 19-year-olds in a population-based cohort in northern Sweden, and associations with questionnaire data from ages 7, 12, and 19 on asthma symptoms, diagnosis, and treatment were tested. Results: Overall, 79 (53%) of the students with a physician diagnosis of asthma were positive to one or more of the mammalian allergens (cat, dog, or horse danders) tested. Of the allergens assessed, only mammalian allergens, birch, and timothy grass pollen showed a significant relationship with asthma diagnosis. Multivariate analysis showed that high titer (>17.5 IU/ml) IgE to any mammalian allergen had the strongest relationship with asthma at age 19 (odds ratio 5.1 [3.0-8.6]). Furthermore, IgE to mammalian allergens gave an odds ratio of 8.5 [4.9-15] for asthma that started before age 12 and was still present at age 19. Sensitization to Fel d 1 and Fel d 4 was strongly associated with asthma and significantly reduced in cat owners. Conclusions: Sensitization to cat and dog related allergens, and specifically to the components Fel d 1 and Fel d 4, is a major risk factor for the persistence and severity of asthma in an area where these are the only significant perennial allergens.

In 2009, we reported a novel form of delayed anaphylaxis to red meat related to serum IgE antibodies to the oligosaccharide galactose-alpha-1,3-galactose (alpha-gal). Although patients were remarkably consistent in their description of a 3- to 6-hour delay between eating mammalian meat and the appearance of symptoms, this delay has not been demonstrated under observed studies.We sought to formally document the time course of clinical symptoms after the ingestion of mammalian meat in subjects with IgE to alpha-gal and to monitor ex vivo for the appearance of markers of an allergic reaction.Open food challenges were performed with mammalian meat in 12 subjects with a history of severe urticarial reactions 3 to 6 hours after eating beef, pork, or lamb, as well as in 13 control subjects. Blood samples were taken hourly during each challenge.Ten of 12 subjects with IgE to alpha-gal had clinical evidence of a reaction during the food challenge (vs none of the control subjects, P.001). The reactions occurred 3 to 7 hours after the initial ingestion of mammalian meat and ranged from urticaria to anaphylaxis. Tryptase levels were positive in 3 challenges. Basophil activation, as measured by increased expression of CD63, correlated with the appearance of clinical symptoms.The results presented provide clear evidence of an IgE-mediated food allergy that occurs several hours after ingestion of the inciting allergen. Moreover, here we report that in vivo basophil activation during a food challenge occurs in the same time frame as clinical symptoms and likely reflects the appearance of the antigen in the bloodstream.

Current understanding of the effects of reducing exposure to cat allergens is limited. It has also become clear that there are different forms of immune response to cat allergens.To investigate changes in skin tests and cat specific IgG and IgE antibodies when students from a home with a cat move to a college dormitory.Ninety-seven college students participated in a prospective study that consisted of allergy skin prick testing and serum measurement of IgE and IgG antibodies to cat at the beginning and end of one academic year in college. A subgroup returned for follow-up at the end of 2 years.Among 97 students, 33% had IgG antibodies to Fel d 1 but no evidence of sensitization, 25% had positive skin test results and/or serum IgE antibodies, and 42% had negative skin test results and no detectable serum antibodies. Among the non-cat sensitized students with IgG antibodies, the titers decreased during 8 months (P = .002). Titers of IgG4 to Fel d 1 also decreased (P.001). Among the sensitized students, no change in IgE antibodies to cat occurred in 8 months (P = .20), whereas Fel d 1 specific IgG antibodies decreased (P.001). Thus, ratios of IgG to IgE decreased highly significantly (P = .007). Among the students with negative skin test results who returned for follow-up (n = 56), none developed positive skin test results or serum IgE antibodies.Under conditions of marked decrease in exposure, no participants developed new-onset sensitization. Among the individuals sensitized at study entry, there were major decreases in the ratio of IgG to IgE.

Background Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by significant gender bias. Previous studies have demonstrated the contributions of hormones, particularly estrogen (E2), to SLE pathogenesis. E2 has been shown to regulate the expression of multiple genes through ligand activation of estrogen receptors (ER) α and β. While many ER-target genes have been previously described, many others remain to be characterized. In this work, we examined ZAS3 and toll-like receptors (TLRs) as potential targets of E2-mediated regulation. ZAS3 is a member of the ZAS family of zinc-finger transcription factors and is thought to be involved in controlling cellular growth and inflammation (1). Furthermore, recent work with large patient cohorts has shown that TLRs appear to have a strong association as SLE susceptibility markers. TLRs have also been of interest as potential autoimmune response triggers (2). Objectives Since sex hormones have been shown to play a significant role in SLE pathogenesis, we examined the effects of estrogen on gene expression in peripheral blood mononuclear cells (PBMCs) from premenopausal healthy individuals and SLE patients. Methods RNA was isolated from the peripheral blood of SLE patients and healthy donors. In addition, PBMCs were isolated and treated with a physiological dose of testosterone, E2, and/or TLR agonists. Western blotting and real-time RT-PCR of protein and mRNA expression were then analyzed, respectively. Nuclear extracts were isolated for EMSA analysis from E2-treated lymphocyte cell lines. Results In whole blood, SLE patients expressed higher levels of ZAS3 and all endosome-associated TLRs: TLR3, TLR7, TLR8, and TLR9. Also, E2-induced expression of endosomal TLRs and ZAS3 was confirmed in healthy PBMCs in vitro , while no effects were observed with testosterone treatment. EMSA analysis with intronic estrogen receptor response element probes within the ZAS3 region revealed that ERα binds directly and that E2 stimulation enhanced complex formation. Using TLR agonists to treat these cells, we identified an autocrine loop with positive feedback over TLR expression. This mechanism was observed in all endosomal TLRs. Furthermore, we discovered that TLR8 agonist stimulation of PBMCs accentuates TLR8 expression in the presence of estrogen. Not only was an enhanced response with E2 and TLR8 agonist observed, but female PBMCs were more sensitive to this effect. Conclusions Thus, the E2/ERα-mediated stimulation of ZAS3 expression occurs through direct, genomic interactions with intragenic enhancing elements. Furthermore, primary human PBMCs display heightened expression of endosome-associated TLRs in response to E2. Taken together, we propose that estrogen-induced upregulation of ZAS3 and endosomal TLRs, along with the TLR autocrine loop, lowers the inflammatory threshold. These novel mechanisms could explain, in part, the gender bias in SLE. 1. Hong JW, Allen CE, Wu LC. Inhibition of NF-kappaB by ZAS3, a zinc-finger protein that also binds to the kappaB motif. Proc Natl Acad Sci U S A 2003;100(21):12301-6. 2. Li M, Zhou Y, Feng G, Su SB. The critical role of Toll-like receptor signaling pathways in the induction and progression of autoimmune diseases. Curr Mol Med 2009;9(3):365-74. Disclosure of Interest None Declared

The relevance of allergic sensitization, as judged by titers of serum IgE antibodies, to the risk of an asthma exacerbation caused by rhinovirus is unclear.We sought to examine the prevalence of rhinovirus infections in relation to the atopic status of children treated for wheezing in Costa Rica, a country with an increased asthma burden.The children enrolled (n= 287) were 7 through 12 years old. They included 96 with acute wheezing, 65 with stable asthma, and 126 nonasthmatic control subjects. PCR methods, including gene sequencing to identify rhinovirus strains, were used to identify viral pathogens in nasal washes. Results were examined in relation to wheezing, IgE, allergen-specific IgE antibody, and fraction of exhaled nitric oxide levels.Sixty-four percent of wheezing children compared with 13% of children with stable asthma and 13% of nonasthmatic control subjects had positive test results for rhinovirus (P.001 for both comparisons). Among wheezing subjects, 75% of the rhinoviruses detected were group C strains. High titers of IgE antibodies to dust mite allergen (especially Dermatophagoides species) were common and correlated significantly with total IgE and fraction of exhaled nitric oxide levels. The greatest risk for wheezing was observed among children with titers of IgE antibodies to dust mite of 17.5 IU/mL or greater who tested positive for rhinovirus (odds ratio for wheezing, 31.5; 95% CI, 8.3-108; P.001).High titers of IgE antibody to dust mite allergen were common and significantly increased the risk for acute wheezing provoked by rhinovirus among asthmatic children.

Rationale IgE antibodies to galactose-alpha-1,3-galactose (alpha-gal) have become increasingly prevalent in the southeastern United States. These antibodies have recently been linked to bites from the tick Amblyomma americanum, but the possibility of a role for other parasites remains. Methods Assays for IgE were performed on patients with delayed urticaria or anaphylaxis to meat and controls in Virginia (n=125), as well as a cohort from Esmeraldas Province, Ecuador (n=295) where Echinococcus is not endemic. Results The significant association between IgE antibodies to alpha-gal and IgE to Echinococcus was strongest in Virginia (r=0.74 vs r=0.62 for Ecuador, both p

In 2009, we reported a novel form of delayed anaphylaxis to red meat that is related to serum IgE antibodies to the oligosaccharide galactose-α-1,3-galactose (alpha-gal). Most of these patients had tolerated meat for many years previously. The implication is that some exposure in adult life had stimulated the production of these IgE antibodies.We sought to investigate possible causes of this IgE antibody response, focusing on evidence related to tick bites, which are common in the region where these reactions occur.Serum assays were carried out with biotinylated proteins and extracts bound to a streptavidin ImmunoCAP.Prospective studies on IgE antibodies in 3 subjects after tick bites showed an increase in levels of IgE to alpha-gal of 20-fold or greater. Other evidence included (1) a strong correlation between histories of tick bites and levels of IgE to alpha-gal (χ(2) = 26.8, P .001), (2) evidence that these IgE antibodies are common in areas where the tick Amblyomma americanum is common, and (3) a significant correlation between IgE antibodies to alpha-gal and IgE antibodies to proteins derived from A americanum (r(s) = 0.75, P .001).The results presented here provide evidence that tick bites are a cause, possibly the only cause, of IgE specific for alpha-gal in this area of the United States. Both the number of subjects becoming sensitized and the titer of IgE antibodies to alpha-gal are striking. Here we report the first example of a response to an ectoparasite giving rise to an important form of food allergy.