Inhibition of Mast Cell Function and Proliferation by mTOR Activator MHY1485

Mast cells integrate innate and adaptive immunity and are implicated in pathophysiological conditions, including allergy, asthma, and anaphylaxis. Cross-linking of the high-affinity IgE receptor (FcεRI) initiates diverse signal transduction pathways and induces release of proinflammatory mediators by mast cells. In this study, we demonstrated that hyperactivation of mechanistic target of rapamycin (mTOR) signaling using the mTOR activator MHY1485 suppresses FcεRI-mediated mast cell degranulation and cytokine secretion. MHY1485 treatment increased ribosomal protein S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation, which are downstream targets of mTOR complex 1 (mTORC1), but decreased phosphorylation of Akt on mTOR complex 2 (mTORC2) target site serine 473. In addition, this activator decreased β-hexosaminidase, IL-6, and tumor necrosis factor α (TNF-α) release in murine bone marrow-derived mast cells (BMMCs) after FcεRI stimulation. Furthermore, MHY1485-treated BMMCs showed significantly decreased proliferation when cultured with IL-3. These findings suggested hyperactivation of mTORC1 as a therapeutic strategy for mast cell-related diseases.

Pruebas dermatológicas con autosuero en pacientes alérgicos y en pacientes autistas y sus madres / Autoserum skin tests in allergic patients, and in autistic patients and their mothers

Introducción: las alergias se encuentran con frecuencia en pacientes autistas y asimismo el autismo muestra una gran presencia entre los pacientes alérgicos. Objetivo: demostrar que las alergias y el autismo comparten algunos patrones inmunológicos similares. Métodos: la prueba dermatológica con autosuero se utilizó para demostrar la presencia de anti-IgE y/o de anticuerpos de receptores de Ig/E (FcεRIα). Resultados: la prueba ASST confirmó la frecuencia similar de positivos/positivos y de negativos/negativos en pacientes alérgicos y en pacientes autistas. Estas similitudes no existieron cuando se realizó la comparación con el grupo control. Se había hallado una correlación positiva con los resultados obtenidos en pacientes autistas y sus madres. Conclusiones: los pacientes autistas y los pacientes alérgicos comparten ciertas similitudes inmunológicas. Ambos se diferencian del grupo de controles sin estas condiciones. Resulta frecuente encontrar pacientes autistas con síntomas alérgicos y pacientes alérgicos con signos de autismo. Es motivo de análisis si los hallazgos inmunológicos representan un puente clínico entre ambos procesos. Asimismo se mostró una posible correlación genética entre los pacientes con autismo y sus madres(AU)

Introduction: allergies are frequently found among patients with autism and autism shows an increased frequency among the allergic patients. Objective: to demonstrate that allergies and autism share some similar immunological patterns. Methods: the autoserum skin test (ASST) was used to demonstrate the presence of anti-IgE and/or anti-IgE receptor antibodies (FcεRIα). Results: the ASST demonstrated similar frequency, positives/positives and negatives/negatives, considering allergic and autistic patients. These similarities didn't exist when comparing with the control group. A positive correlation had been found with the results of autistic patients and their mothers. Conclusions: autistic and allergic patients share some immunological similarities. Both differ from normal controls. It is not uncommon autistics with allergic symptoms and allergic patients with autism. If the immunological findings represent a clinical bridge between both processes, it is under discussion. Also it was demonstrated a possible genetic correlation between the patients with autism and their mothers(AU)

Antibody to FcεRIα Suppresses Immunoglobulin E Binding to High-Affinity Receptor I in Allergic Inflammation

PURPOSE: High-affinity receptor I (FcεRI) on mast cells and basophils plays a key role in the immunoglobulin E (IgE)-mediated type I hypersensitivity mediated by allergen cross-linking of the specific IgE-FcεRI complex. Thus, prevention of IgE binding to FcεRI on these cells is an effective therapy for allergic disease. We have developed a strategy to disrupt IgE binding to FcεRI using an antibody targeting FcεRIα. MATERIALS AND METHODS: Fab fragment antibodies, which lack the Fc domain, with high affinity and specificity for FcεRIα and effective inhibitory activity against IgE-FcεRI binding were screened. IgE-induced histamine, β-hexosaminidase and Ca2+ release in basophils were determined by ELISA. A B6.Cg-Fcer1a(tm1Knt) Tg(FCER1A)1Bhk/J mouse model of passive cutaneous anaphylaxis (PCA) was used to examine the inhibitory effect of NPB311 on allergic skin inflammation. RESULTS: NPB311 exhibited high affinity to human FcεRIα (KD=4 nM) and inhibited histamine, β-hexosaminidase and Ca2+ release in a concentration-dependent manner in hFcεRI-expressing cells. In hFcεRIα-expressing mice, dye leakage was higher in the PCA group than in controls, but decreased after NPB311 treatment. NPB311 could form a complex with FcεRIα and inhibit the release of inflammation mediators. CONCLUSION: Our approach for producing anti-FcεRIα Fab fragment antibody NPB311 may enable clinical application to a therapeutic pathway in IgE/FcεRI-mediated diseases.

Antibody to FcεRIα Suppresses Immunoglobulin E Binding to High-Affinity Receptor I in Allergic Inflammation

PURPOSE: High-affinity receptor I (FcεRI) on mast cells and basophils plays a key role in the immunoglobulin E (IgE)-mediated type I hypersensitivity mediated by allergen cross-linking of the specific IgE-FcεRI complex. Thus, prevention of IgE binding to FcεRI on these cells is an effective therapy for allergic disease. We have developed a strategy to disrupt IgE binding to FcεRI using an antibody targeting FcεRIα. MATERIALS AND METHODS: Fab fragment antibodies, which lack the Fc domain, with high affinity and specificity for FcεRIα and effective inhibitory activity against IgE-FcεRI binding were screened. IgE-induced histamine, β-hexosaminidase and Ca2+ release in basophils were determined by ELISA. A B6.Cg-Fcer1a(tm1Knt) Tg(FCER1A)1Bhk/J mouse model of passive cutaneous anaphylaxis (PCA) was used to examine the inhibitory effect of NPB311 on allergic skin inflammation. RESULTS: NPB311 exhibited high affinity to human FcεRIα (KD=4 nM) and inhibited histamine, β-hexosaminidase and Ca2+ release in a concentration-dependent manner in hFcεRI-expressing cells. In hFcεRIα-expressing mice, dye leakage was higher in the PCA group than in controls, but decreased after NPB311 treatment. NPB311 could form a complex with FcεRIα and inhibit the release of inflammation mediators. CONCLUSION: Our approach for producing anti-FcεRIα Fab fragment antibody NPB311 may enable clinical application to a therapeutic pathway in IgE/FcεRI-mediated diseases.

Treatment of Chronic Spontaneous Urticaria

Chronic spontaneous urticaria is defined as persistent symptoms of urticaria for 6 weeks or more. It is associated with autoimmunity in approximately 45 percent of patients. Therapy is often difficult however the initial approach should employ high-dose non-sedating antihistamines; 4-6 tablets/day may be necessary. It has been shown that the response to 4 tablets/day exceeds 3, and exceeds 2, which exceeds 1. However the dose that corresponds to the maximal dose of first generation antihistamines (hydroxyzine, diphenhydramine) used previously, is 6/day. Yet over half the patients are refractory to antihistamines and other agents should be tried next. Whereas current guidelines (published) often add leukotriene antagonists and/or H2 receptor antogonists next, these are of little utility. Likewise drugs effective for urticarial vasculitis (colchicine, dapsone, sulfasalazine, hydroxychloroquine) are effective in a small percentage of patients and no study suggests that the response rate of any of them exceeds the 30% placebo responses seen in most double-blind, placebo controlled studies. The drugs that are effective for antihistamine-resistant chronic spontaneous urticaria are corticosteroids, cyclosporine, and Omalizumab. Use of steroids is limited by toxicity. If used at all, a dose of no more than 10 mg/day should be employed with a weekly reduction of 1 mg. The response rates to cyclosporine and Omalizumab are each close to 75%. Cyclosporine can be used effectively if care is taken to monitor blood pressure, urine protein, blood urea nitrogen, and creatinine, every 6 weeks. Omalizumab has the best profile in terms of efficacy/toxicity and, once approved by federal agencies for use in chronic spontaneous urticaria, a dramatic change in the treatment paradigm, whether associated with autoimmunity or not, is predicted. A phase 3 trial is currently in place. Refractoriness to both Omalizumab and cyclosporine is expected to be less than 5 percent of patients. Other agents, can then be tried.

Perfil de ativação de basófilos e evidência de fatores liberadores de histamina na urticária crônica idiopática / Basophils activation profile and evidence of histamine release factor in chronic idiopathic urticaria

INTRODUÇÃO: A Urticária Crônica é caracterizada pelo aparecimento de pápulas eritematosas, pruriginosas recorrentes e transitórias que duram por mais de seis semanas. Na maioria dos pacientes a causa é indeterminada, definida como idiopática (UCI), entretanto, um sub-grupo apresentam autoanticorpos contra a cadeia alfa do receptor de alta afinidade para IgE (FceRI), que são expressos na superfície de mastócitos e basófilos, tornando-os células alvo nesta doença. OBJETIVOS: Avaliar em pacientes com UCI, submetidos ao teste intradérmico de soro autólogo (ASST), o perfil de ativação dos basófilos, pela intensidade de expressão de marcadores de ativação/desgranulação e pela capacidade dos basófilos em responder aos estímulo com a IL-3 e anticorpo anti-IgE. Além disto, a presença de fator liberador de histamina foi avaliado nos soros dos pacientes. METODOLOGIA: Pacientes com UCI (n= 37) foram selecionados no Ambulatório de Urticária do Departamento de Dermatologia do Hospital das Clínicas da Faculdade de Medicina da USP e submetidos ao ASST. O grupo controle foi constituído por indivíduos saudáveis (n=38). A análise da expressão de FceRI, CD63, CD123 e CD203c em basófilos de sangue periférico foi realizada por citometria de fluxo. No ensaio in vitro de estimulação dos basófilos com anti-IgE, as células foram previamente incubadas com IL-3. O ensaio de liberação de histamina mediada por soros de pacientes com UCI foi realizado com três diferentes doadores de leucócitos e a histamina liberada dosada por ELISA de competição. RESULTADOS: Há um baixo número de basófilos no sangue periférico nos pacientes com UCI, coincidente com o baixo nível sérico de histamina. Os escassos basófilos no sangue periférico mostram elevada expressão de FceRI e uma regulação positiva da expressão de CD203c e CD63, independentemente do ASST. A análise funcional dos basófilos, mostra que somente a incubação com IL-3 recombinante já induz aumento significante da expressão de CD203c...

INTRODUCTION: Chronic Urticaria is characterized by recurrent, transitory, pruritic and erythematous wheals present for at least six weeks. In most patients the cause is unknown, defined as idiopathic (CIU), however, a sub-group has autoantibodies against the alfa chain of the high affinity IgE receptor (FceRIa) expressed on mast cells and basophils surface making it the target cells in this disease. OBJECTIVES: To evaluate in CIU patients, undergone autologous serum skin test (ASST), the activation profile of the basophils assessed by the expression of activation/degranulation markers and by the ability to release histamine in response to IL-3 priming and cross-linking with anti-IgE antibodies. Furthermore, the presence of histamine releasing factor in sera of patients was evaluated. METHODS: CIU patients (n = 37) were selected from the Dermatological Outpatient Clinic of the Hospital das Clínicas de São Paulo (HC-FMUSP) and submitted to the ASST. The control group consisted of healthy subjects (n=38). The analysis of the expression of FceRI, CD63, CD123 and CD203c on basophils from peripheral blood was assessed by flow cytometry. For the in vitro stimulation with anti-IgE antibodies, the cells were previously primed with human recombinant IL-3. The histamine release assay mediated by sera from patients with CIU was performed with three different donors of leukocytes and released histamine measured by competition ELISA. RESULTS: There is a low number of basophils in peripheral blood of patients with CIU, reflecting a low serum levels of histamine. The scarce basophils in peripheral blood show high expression of FceRI and an up-regulation of CD203c and CD63 marker expression, independently of the ASST. The functional analysis of basophils, revealed that recombinant IL-3 per se induces a significant increase in CD203c expression and the histamine release from basophils of patients with CIU, which are enhanced followed for 15 and 40 minutes...

Influence of conventional house-dust-mite immunotherapy on histamine releasability from the basophil / 천식및알레르기

BACKGROUND: Allergen-specific immunotherapy has been shown to be clinically effective in the treatment of patients with atopic asthma, but the mechanisms are still unclear. Some of the immunologic changes include increase of an allergen-specific IgG antibody, decrease of allergen-specific IgE after transient increase, allergen-specific T-cell shift in cytokine expression from Th2 to Th1 cytokines, and decrease of basophil histamine releasability. OBJECTIVE: To investigate the influence of immunotherapy on basophil releasability, we examined the changes of IgE-mediated and non-IgE-mediated basophil histamine releasability during immunotherapy. METHODS: Fourteen Dermatophagoides farinae (D.f) sensitive asthmatic children with conventional immunotherapy were investigated. Basophil histamine releasability was measured prior to immunotherapy and 4 and 9 months after immunotherapy. Basophils were stimulated with D.f and goat antihuman IgE antibody as IgE-mediated stimuli which act on IgE-receptor, and formyl-Met-Leu-Phe (fMLP) as non-IgE-mediated stimuli which acts on non-IgE receptor, and calcium ionophore A23187 as non-IgE-mediated stimuli which does not act on cell surface receptors. Histamine was measured by automated fluorometric technique. RESULTS: Before immunotherapy, there were significant correlations between histamine release by D.f and histamine release by fMLP, and between histamine release by D.f and histamine release by anti-IgE antibody, but no correlation between histamine release by D.f and histamine release by calcium ionophore. Histamine release by D.f and by anti-IgE antibody decreased at and 9 months after immunotherapy compared to those before immunotherapy. Histamine release by fMLP and by calcium ionophore showed no significant changes after immunotherapy. There was no significant change of total histamine release after immunotherapy. CONCLUSION: Conventional immunotherapy has influenced only the IgE-mediated basophil releasability. IgE-mediated and non-IgE-receptor-mediated basophil releasability was correlated before immunotherapy, but only IgE-receptor-mediated basophil releasability decreased after immunotherapy. These findings suggest that a kind of physicochemical change may happen on the IgE receptors of basophil, which may induce decrease of IgE-mediated basophil histamine releasability after immunotherpy.

Studies on the prognostic marker in minimal change nephrotic syndrome

Childhood minimal change nephrotic syndrome (MCNS)is often associated with hyper-sensitivity reactions and considered to be caused by immune dysfunction. The elevated serum IgE levels and atopic symptoms have been frequently associated in these patients. The present therapy for MCNS mainly depends on corticosteroids, alkylating agents such as chlorambucil, cyclophosphamide and cyclosporin A (Cy, A). However, frequent relapses and severe side effects caused by such therapy necessitate development of a more specific and effective therapeutic regimen.Recently, a T cell derived cytokine, interleukin 4 (IL-4)is being recognized as a major cytokine up-regulating IgE production and response, while interferon- (IFN- )counteracts IL-4 actions to down-regulate the IL-4 induced IgE response. Hence, the present study is aimed to investigate the role of IL-4 in MCNS. Using freshly isolated peripheral blood from active MCNS patients and normal controls, it was found that not only serum IgE levels, but also membrane-bound IgE receptor expression and soluble IgE receptor production were also significantly higher in MCNS than in mormal controls, which in turn correlated well with the increased IL-4 activities in the patients' sera. Interestingly among MCNS patients receiving a steroid therapy, those who responded with a clinical remission demonstrated a noticeable decrease in IgE receptor levels on their B cells. From these results it is concluded that MCNS is highly associated with not only increased serum IgE, but also elevated IL-4 activities and IgE receptor expression. In addition, IgE receptor could be a good prognostic marker in MCNS.