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No known therapies can prevent anaphylaxis. Bruton's tyrosine kinase (BTK) is an enzyme thought to be essential for high-affinity IgE receptor (FcsRI) signaling in human cells. We tested the hypothesis that FDA-approved BTK inhibitors (BTKis) would prevent IgE-mediated responses including anaphylaxis. We showed that irreversible BTKis broadly prevented IgE-mediated degranulation and cytokine production in primary human mast cells and blocked allergen-induced contraction of isolated human bronchi. To address their efficacy in vivo, we created and used what we believe to be a novel humanized mouse model of anaphylaxis that does not require marrow ablation or human tissue implantation. After a single intravenous injection of human [CD34.sup.+] cells, NSG-SGM3 mice supported the population of mature human tissue-resident mast cells and basophils. These mice showed excellent responses during passive systemic anaphylaxis using human IgE to selectively evoke human mast cell and basophil activation, and response severity was controllable by alteration of the amount of allergen used for challenge. Remarkably, pretreatment with just 2 oral doses of the BTKi acalabrutinib completely prevented moderate IgE-mediated anaphylaxis in these mice and also significantly protected against death during severe anaphylaxis. Our data suggest that BTKis may be able to prevent anaphylaxis in humans by inhibiting Fc[epsilon]RI-mediated signaling., Introduction Anaphylaxis is an acute, potentially life-threatening systemic allergic reaction characterized by urticaria, angioedema, bronchospasm, nausea/vomiting, abdominal cramping, diarrhea, hypotension, and/or shock (1). IgE-mediated anaphylaxis requires antigen cross-linking of Fc[epsilon]RI-bound [...]

Food allergies are a major clinical problem and are driven by IgE antibodies (Abs) specific for food antigens (Ags). T follicular regulatory (Tfr) cells are a specialized subset of [FOXP3.sup.+] T cells that modulate Ab responses. Here, we analyzed the role of Tfr cells in regulating Ag-specific IgE using a peanut-based food allergy model in mice. Peanut-specific IgE titers and anaphylaxis responses were significantly blunted in Tfr cell-deficient Foxp3-Cre [Bcl6.sup.fl/fl] mice. Loss of Tfr cells led to greatly increased nonspecific IgE levels, showing that Tfr cells have both helper and suppressor functions in IgE production in the germinal center (GC) that work together to facilitate the production of Ag-specific IgE. Foxp3-Cre [Pten.sup.fl/fl] mice with augmented Tfr cell responses had markedly higher levels of peanut-specific IgE, revealing an active helper function by Tfr cells on Ag-specific IgE. The helper function of Tfr cells for IgE production involves IL-10, and the loss of IL-10 signaling by B cells led to a severely curtailed peanutspecific IgE response, decreased GCB cell survival, and loss of GC dark zone B cells after peanut sensitization. We thus reveal that Tfr cells have an unexpected helper role in promoting food allergy and may represent a target for drug development., Introduction Antibodies (Abs) that bind to antigen (Ag) with high affinity and help to eliminate pathogens and foreign substances are generated in the germinal center (GC) reaction (1-3). A specialized [...]

Allergic asthma is mediated by Th2 responses to inhaled allergens. Although previous experiments indicated that Notch signaling activates expression of the key Th2 transcription factor Gata3, it remains controversial how Notch promotes allergic airway inflammation. Here we show that T cell-specific Notch deficiency in mice prevented house dust mite-driven eosinophilic airway inflammation and significantly reduced Th2 cytokine production, serum IgE levels, and airway hyperreactivity. However, transgenic Gata3 overexpression in Notch-deficient T cells only partially rescued this phenotype. We found that Notch signaling was not required for T cell proliferation or Th2 polarization. Instead, Notch-deficient in vitro-polarized Th2 cells showed reduced accumulation in the lungs upon in vivo transfer and allergen challenge, as Notch-deficient Th2 cells were retained in the lung-draining lymph nodes. Transcriptome analyses and sequential adoptive transfer experiments revealed that while Notch-deficient lymph node Th2 cells established competence for lung migration, they failed to upregulate sphingosine-1-phosphate receptor 1 (S1PR1) and its critical upstream transcriptional activator Kruppel-like factor 2 (KLF2). As this KLF2/S1PR1 axis represents the essential cell-intrinsic regulator of T cell lymph node egress, we conclude that the druggable Notch signaling pathway licenses the Th2 response in allergic airway inflammation via promoting lymph node egress., Introduction Allergic asthma is a common but heterogeneous chronic inflammatory lung disease characterized by type 2 airway inflammation and bronchial hyperreactivity (1). Asthma patients experience symptoms such as wheezing, shortness [...]

Background The sensitization and exposure to fungal allergens have been reported to be associated with asthma. The aim of this study was to clarify the impact of sensitization to Schizophyllum commune (S. commune) on the severity and exacerbations of asthma. Methods Ninety-two patients with asthma of various levels of severity [mild (n = 18), moderate (28), and severe (46)] and exacerbation severity [moderate (n = 43) and severe (6)] were retrospectively examined with regard to fungal sensitization such as specific IgE or intradermal skin reactions against S. commune and other common allergens. We also classified the patients into three groups: (1) three or more asthma attacks during the past year (F-BA) (n = 29),(2) one or two asthma attacks (NF-BA) (n = 20), and (3) no asthma attack (C-BA) (n = 43). Results The positive rate of late cutaneous reactions to S. commune was higher in patients with severe asthma (41.2%) than with moderate (26.1%) or mild asthma (6.7%), and was significantly different among the three groups (P < 0.05). Although the ratio did not show a significant difference between the patients with severe (83.3%) or moderate (36.1%) exacerbation, it was higher in F-BA (60.9%) than in NF-BA (21.1%) and C-BA patients (10.0%), and it was significantly different among the three groups (P = 0.0002). Multivariate analysis identified positive results for late-phase skin reactions to S. commune and the age of the patients as an independent determinant of asthma severity, and the skin results and %FVC an independent determinant of exacerbation frequency. Conclusion This study demonstrated that S. commune is an environmental fungus that appears to enhance both the severity of asthma and the exacerbation frequency. Keywords Asthma severity * Severity of exacerbation * Exacerbation frequency * Basidiomycetous fungus * Fungal sensitization * Schizophyllum commune, Introduction Throughout the Global Initiative for Asthma (GINA) Science Committee report in 2009 [1], emphasis is placed on the concept that the goal of asthma treatment is to achieve and [...]

Recently we described a family of peptides, unrelated in sequence to IgE, that form stable [beta]-hairpins in solution and inhibit IgE activity in the [micro]M range [Nakamura, G. R., Starovasnik, M. A., Reynolds, M. E. & Lowman, H. B. (2001) Biochemistry 40, 9828-9835]. Using an expanded set of peptide-phage libraries, we found a simpler motif, [X.sub.2]CP[X.sub.2]CYX, for binding to the high-affinity IgE receptor. In solution, one of these peptides spontaneously formed a covalent antiparallel dimer. We subsequently linked these monomers in a single-chain construct on phage and optimized receptor binding. Ultimately, peptides with 30 nM affinity were produced. NMR studies showed that the peptide adopts a stable fold consisting of two 'zeta' ([zeta])-shaped moieties. Structure-activity analyses reveal a single binding site created by the zeta-dimer, with two tyrosine residues important for structural stability and two proline residues important for Fc[epsilon]RI binding. The peptides inhibit histamine release from cultured cells and are extremely stable in biological fluids. The zeta peptides appear to act as competitive IgE inhibitors and suggest possibilities for design of novel IgE antagonists.

The interaction between immunoglobulin E (IgE) and its cellular receptor FcepsilonRI is a key occurence in the development of allergic response. Thus, the use of therapeutic compounds which would inhibit an interaction between IgE and FcepsilonRI is one approach to treating allergic disorders. These compounds take action as allergen-bound IgE mediating the allergic response by binding through its cellular receptor mast cells and basophils, which causes the release of chemical mediators. The structure of two retroenantiomeric compounds which mimic the high-affinity receptor for IgE is analyzed to understand this approach.

Using defined oligomers of IgE, our group previously studied the quantitative relationship between the aggregation of the high affinity receptors for IgE (Fc[Epsilon]RI) and the earliest signals initiated by such aggregation: the phosphorylation of tyrosines on the receptor. Notably, at certain doses of the oligomers such phosphorylation reached a plateau level well before the aggregation of the receptors had reached a maximum. These findings and others led us to propose that aggregates of the receptor were competing for a limited amount of the critical kinase - thought to be Lyn in this system. This paper describes a test of this proposal. We incubated cells with two distinguishable IgEs and examined the effect of aggregating one or the other or both types on the phosphorylation. When receptors binding antigen-specific IgE were aggregated with polyvalent antigen, they became rapidly phosphorylated as expected. Remarkably, however, Fc[Epsilon]RI that had already been phosphorylated by the binding of dimers of IgE, became dephosphorylated simultaneously. Furthermore, when the antigen-driven aggregates were dissociated with hapten, the phosphorylation pattern reverted to that seen prior to the addition of antigen: as the antigen-driven aggregates became dephosphorylated, the receptors stably aggregated by the bound oligomers became rapidly rephosphorylated. Dephosphorylation of oligomer-driven aggregates was also partially reversed during the 'spontaneous' dephosphorylation of the antigen-driven receptors seen at longer times after addition of antigen. Thus signal transduction in this system is in part regulated by the shuttling of limited amounts of the kinase that initiates the cascade of phosphorylations.

We compared the antigen-specific antibody isotypes and lymphokine secretion by CD[4.sup.+] T cells in BALB/c mice immunized intradermally with either Escherichia coli [Beta]-galactosidase ([Beta]-gal) or plasmid DNA (pDNA) encoding [Beta]-gal in a cytomegalovirus-based expression vector (pCMV-LacZ). pCMV-LacZ induced mainly IgG2a, whereas [Beta]-gal in saline or alum induced IgG1 and IgE [Beta]-gal-specific antibodies. In addition, splenic CD[4.sup.+] T helper (Th) cells isolated from pDNA-immunized mice secreted interferon-[Gamma] but not inter-leukin (IL)-4 and IL-5, whereas Th cells from [Beta]-gal-injected mice secreted IL-4 and IL-5 but not interferon-[Gamma] after in vitro stimulation with antigen. Together these data demonstrate that pDNA immunization induced a T helper type 1 (T[h.sub.1]) response, whereas protein immunization induced a T helper type 2 (T[h.sub.2]) response to the same antigen. Interestingly, priming of mice with pCMV-LacZ prevented IgE antibody formation to a subsequent i.p. [Beta]-gal in alum injection. This effect was antigen-specific, because priming with pCMV-LacZ did not inhibit IgE anti-ovalbumin antibody formation. Most importantly, intradermal immunization with pCMV-LacZ (but not pCMV-OVA) of [Beta]-gal in alum-primed mice caused a 66-75% reduction of the IgE anti-[Beta]-gal titer in 6 weeks. Also, pCMV-LacZ induced specific IgG2a antibody titers and interferon-[Gamma] secretion by Th cells in the [Beta]-gal in alum-primed mice. The data demonstrate that gene immunization induces a T[h.sub.1] response that dominates over an ongoing protein-induced T[h.sub.2] response in an antigen-specific manner. This suggests that immunization with pDNA encoding for allergens may provide a novel type of immunotherapy for allergic diseases.

A study of the assembly and intracellular transport of the human high- affinity receptor for immunoglobulin E shows that the steric hindrance caused by the cytoplasmic domain of the gamma chain causes the masking of the endoplasmic reticulum (ER) retention motif. Dilysine ER retention signal allows the transport out of the ER of the fully assembled receptor due to the masking during gamma chain assembly.

Background Linear nevus sebaceous syndrome (LNSS) is a rare congenital neuroectodermal disorder characterized by involvement of the skeleton and central nervous system. Case We report the case of a 5-year-old girl who had LNSS with hypophosphatemic rickets and multiple fractures of her extremities. Biochemical tests revealed a high serum level of fibroblast growth factor-23 (FGF-23) but normal levels of immunoglobulin E (IgE) and parathormone (PTH). FGF-23 mRNA expression in the skin lesions of our patient's skin was found to be below the limit of detection in all samples tested by quantitative-PCR analysis. Conclusions It is possible that an as-yet unidentified substance increases FGF-23 expression LNS lesions., Introduction Linear nevus sebaceous syndrome (LNSS) is a rare congenital neuroectodermal disorder characterized by involvement of the skeleton and central nervous system [1]. Endocrinological complications in LNSS have rarely been [...]

Experimental studies using chemical crosslinking help measure the association between the aggregation of the receptors with high affinity for IgE and p53/56 (lyn) protein-tyrosine kinase. An in vitro assay reveals a that the phosphorylation of lyn associated with the aggregated receptors is much higher than that predicted on the basis of the amount of lyn associated with the receptors. A model suggests that additional molecules of lyn kinase increase the transphosphorylation of tyrosine on the receptors and associated proteins, leading to a cascade of biochemical changes.

Examination of the relationship between the aggregation of the high-affinity receptors for IgE on mast cells and phosphorylation of protein tyrosines reveals a linear correlation between phosphorylation of the exogenous substrates and time after aggregation. Both aggregated and disaggregated receptors increase the kinase activity toward exogenous substrates and the yield of enhanced kinetic activity exhibits a direct relationship with the state of phosphorylation of the receptors. A mechanistic study reveals transphosphorylation to be the mechanism for the phosphorylation of endogenous substrates by kinase.

Two isozymes of protein kinase C (PKC), PKC-beta and -epsilon, participate in c-fos and C-jun mRNA accumulation in the rat basophilic leukemia cell line, RBL-2H3. Aggregation of high-affinity IgE receptors has no influence on induction of c-fos and c-jun at high strengths of PKC-beta and -epsilon. Aggregation influences induction at low concentrations. PKC-beta and -epsilon connects the cell surface receptor and gene expression.

The expression of high levels of the gamma chain of the high-affinity receptor for IgE (Fc-epsilon-RI-gamma) as one invariant subunit of their T-cell antigen receptor (TCR)/CD3 complex results from the isolation of gamma delta T-cells from spleen and intestinal epithelial tissues. This is not evident in most alpha beta T-cells. Fc-epsilon-RI-gamma is present as a homodimer and a heterodimer, linked to the TCR gamma chain.

Anaphylaxis is caused by the activation of most cells and basophils. The basophils are activated by immunoglobin E (IgE) and allergen. Different types of IgE receptors are expressed, and the contribution of IgE receptors is determined by the generation Fc-epsilon-RI deficient mice. The deficient mice are normal in appearance and have normal amount of mast cells, but are resistant to cutaneous reactions. This indicates that the IgE dependent anaphyloctic reaction require IgE.

Byline: Tejas. Patel, Parvati. Patel, Manish. Barvaliya, C. Tripathi Background: Epidemiological data on drug-induced anaphylactic reactions are limited in India and are largely depending on studies from developed countries. Aim: [...]