A newly identified secreted larval antigen elicits basophil-dependent protective immunity against N. brasiliensis infection.

Hookworms infect more that 400 million people and cause significant socio-economic burden on endemic countries. The lack of efficient vaccines and the emergence of anthelminthic drug resistance are of major concern. Free-living hookworm larvae infect their hosts via the skin and live as adult worms in the small intestine where they feed on host tissue and blood. Excretory/secretory (E/S) products, released by helminths as they migrate through their host, are thought to play a key role in facilitating infection and successful establishment of parasitism. However, E/S products can also elicit protective immune responses that might be harnessed for vaccine development. By performing Western blots with serum of Nippostrongylus brasiliensis (Nb) infected mice as a model for human hookworm infection, we identified a largely overlapping set of IgG1- and IgE-reactive antigens in E/S from infective L3 stage larvae. Mass spectrometry analysis led to the identification of a new protein family with 6 paralogues in the Nb genome which we termed Nb-LSA1 for "Nippostrongylus brasiliensis larval secreted protein 1". The recombinantly expressed 17 kDa family member Nb-LSA1a was recognized by antibodies in the serum of Nb immune mice. Immunization of mice with Nb-LSA1a in alum elicited a strong IgG1 response but no detectable antigen-specific IgE. Most importantly, immunized mice were largely protected against a challenge Nb infection. This effect was dependent on the presence of basophils and occurred before the parasites reached the intestine. Therefore, basophils appear to play a critical role for rapid control of infection with L3 stage larvae in mice immunized with a single secreted larval protein. A better understanding of basophil-mediated protective immunity and identification of potent larval antigens of human hookworms could help to develop promising vaccination strategies.

Histological Identification and Quantification of Eosinophils and Ascites in Leghorn Chickens Treated with High Oral Concentrations of NaCl-Pilot Study.

The purpose of this pilot study was to determine the role played by eosinophils in NaCl poisoning and right cardiac hypertrophy (ascitic syndrome) in Leghorn chickens, as well as the histological findings in the central nervous system (CNS), liver, and kidney. Moreover, the hypertrophy of the right ventricle index (HRVI) as an indicator of ascites was evaluated. Male SPF Leghorn birds at 28 days of age were submitted to two experiments. Food and water (FW) experiment: birds were treated with food plus 3.3% NaCl for the next 27 days and 1% NaCl in their drinking water from days 22 to 27. Water experiment (W): birds were treated with 1% NaCl in their drinking water for 5 days. In both experiments, the chickens exhibited loss of appetite, diuresis, and watery, green diarrhea during treatment days; at 24−27 td-FW and experiment W, the birds showed nervous signology (prostration, running movements, tremors, and comatose state). In the leukogram at 28 td-FW, an increase (p < 0.05) in heterophiles and basophils was observed. CNS eosinophilia was not observed in birds intoxicated with NaCl, though they did present demyelination in the brain and spinal cord, hepatic degeneration, mesangial proliferative glomerulopathy, and acute proximal renotubular necrosis.

Structural and allergenic properties of the fatty acid binding protein from shrimp Litopenaeus vannamei.

BACKGROUND: The shrimp Litopenaeus vannamei is an important source of food allergens but its allergenic repertoire is poorly characterized. Cross-reactivity between crustacean and mites has been reported, with tropomyosin, the most relevant allergen involved. The aim of this study was to investigate the structural and immunological properties of a recombinant Fatty Acid Binding Protein (FABP) family from L. vannamei (LvFABP). METHODS: ELISA, skin prick test (SPT) and basophil activation assays were performed to determine IgE reactivity and allergenic activity of LvFABP. LC-MS/MS and Circular Dichroism experiments were done for structural analysis. B-cell epitope mapping with overlapping peptides, and cross-inhibition studies using human sera were done to identify antigenic regions and cross-reactivity. RESULTS: The recombinant LvFABP bound serum IgE from 27% of 36 shrimp allergic patients and showed allergenic activity when tested for basophil activation and SPT in a selected number of them. CD-spectroscopy of LvFABP revealed that the protein is folded with a secondary structure composed of mainly ß-strands and a smaller fraction of α helices. This is consistent with molecular modelling results, which exhibit a typical ß barrel fold with two α-helices and ten ß-strands. Epitope mapping identified two IgE-binding antigenic regions and inhibition assays found high cross-reactivity between LvFABP and Blo t 13, mediated by the antigenic region involving amino acids 54 to 72. CONCLUSIONS: Our results show that LvFABP is a shrimp allergen that cross reacts with the house dust mite allergen Blo t 13 and has allergenic activity, which suggest that it could be clinically relevant in case of shellfish allergy. This new allergen, named Lit v 13, will also help to understand basic mechanisms of sensitization to shrimp.

Beyond eosinophilia: inflammatory patterns in patients with asthma.

Background: Recently, inflammatory cell ratios have gained importance as useful indicators in the categorization of asthma.Objective: We compared the concentration of white blood cells in peripheral blood, as well as their respective inflammatory cell ratios, between patients with asthma and a healthy control group.Methods: We performed cross-sectional analyses of the data obtained from 53 adult patients with asthma and 109 adult controls. In our study, we estimated and compared the following inflammatory cell ratios: Neutrophil-Lymphocyte Ratio (NLR), Eosinophil-Lymphocyte Ratio (ELR), Eosinophil-Neutrophil Ratio (ENR), Eosinophil-Monocyte Ratio (EMR), and Platelet-Lymphocyte Ratio (PLR). The magnitude of association was quantified with the odds ratio.Results: In both groups, the average age was 33 years. In asthmatic patients, we obtained the following results: eosinophils ≥ 400 cells/µl, accounted for 37.7%; basophils ≥ 110 cells/µl, comprised 37.7%; and monocytes < 320 cells/µl, reached 11.3%. In the control group, the results were as follows: 4.6%, 9.2% and 0.9%, respectively. When compared to the control group, asthmatic patients had higher odds of eosinophils ≥ 400 cells/µl (OR = 12.61, p < 0.0001); higher odds of basophils ≥ 110 cells/µl (OR = 6.00, p < 0.0001); and increased odds of monocytes < 320 cells/µl (OR = 13.79, p = 0.017). NLR did not differ between our two groups; however, ELR, ENR, EMR and PLR were significantly higher in the asthma group.Conclusions: Overall, patients with asthma have a higher concentration of eosinophils and basophils, fewer monocytes in their blood, and higher ratios of increased chronic inflammation.

Causal Relationship Between Anti-TPO IgE and Chronic Urticaria by In Vitro and In Vivo Tests.

PURPOSE: Immunoglobulin (Ig) E autoantibodies against thyroid antigens such as thyroid peroxidase (TPO) have been demonstrated in chronic spontaneous urticaria (CSU) patients in higher frequency than healthy subjects. However, if these IgE autoantibodies can trigger urticaria is still a matter of study. The aim of this study was to investigate the relationship between concomitant IgE autoantibodies against thyroid antigens in CSU. METHODS: Patients with CSU, healthy subjects and patients with autoimmune thyroid disease (ATD) were recruited. Total IgE and specific anti-TPO IgE and IgG were assessed in all subjects. The basophil activation test and skin tests with TPO were performed to demonstrate whether this antigen could selectively induce urticaria reaction in subjects with positive anti-TPO IgE. RESULTS: Anti-TPO IgE was present in all 3 groups (CSU: 34.0%, ATD: 16.6%, healthy subjects: 8.1%). Anti-TPO IgE levels were higher in CSU patients, whereas anti-TPO IgG were higher in ATD patients. After exposure to TPO, CD203c expression from patients with CSU and anti-TPO IgE significantly increased in comparison to the other groups; 33.0% vs. 14.0% in ATD patients and 9.0% in control subjects (P < 0.05). Skin reactions with TPO were higher in patients with CSU according to the intradermal (CSU: 18.0%, ATD: 3.3%, control: 8.0%) and skin prick tests (12.0%, 0%, 0%, respectively). Passive transfer of anti-TPO IgE from a CSU patient to the skin of control subjects without anti-TPO IgE induced a positive skin reaction. CONCLUSIONS: Anti-TPO IgE is not a specific biomarker for CSU. However, IgE against TPO plays a pathogenic role in inducing effector cell activation and skin exacerbation in some patients with CSU.

Prueba de activación de basófilos: aspectos técnicos, metodológicos y su utilidad clínica / Basophil activation test: technical aspects, methodology and clinical utility

Resumen Introducción. La prueba de activación de basófilos (PAB) se considera una técnica confiable y segura para el diagnóstico de problemas alérgicos. Objetivo. Profundizar en el estado del arte de la PAB y su utilidad clínica. Materiales y métodos. Se realizó una revisión narrativa de la literatura mediante la búsqueda electrónica en las bases de datos y metabuscadores Ovid Medline, Google Scholar y PubMed, sin limitar la búsqueda por fecha, idioma o tipo de artículo. Se buscaron artículos sobre los detalles técnicos de la PAB y su utilidad clínica en el manejo de las enfermedades alérgicas. Resultados. De los marcadores de activación, CD63 ha sido el más estudiado y es el que mejor representa un evento de degranulación anafiláctica, mientras que CD203c es representativo de varias formas de degranulación. La superioridad de uno sobre otro como prueba diagnóstica depende del problema alérgico estudiado. En cuanto a los métodos de detección de basófilos, su selección con un único marcador, CCR3, se propone como una opción con buena relación de costo-efectividad. Conclusiones. La PAB es una herramienta prometedora para evaluar en clínica las reacciones alérgicas de forma segura. Es necesario una mayor estandarización de protocolos para obtener resultados más reproducibles.

Abstract Introduction: Basophil activation test (BAT) is considered a reliable and safe technique for diagnosing allergic diseases. Objective: To make a thorough analysis of the state of the art regarding BAT and its clinical utility. Materials and methods: A narrative literature review of BAT and its clinical utility in the management of allergic diseases was performed in the Ovid Medline, Google Scholar and PubMed databases and metasearch engines, without limiting the search by date, language or type of article. Results: Regarding basophil activation markers, CD63 is the most studied and the best representative of anaphylactic degranulation, while CD203c is representative of several types of degranulation. The superiority of one activation marker over the other as a diagnostic tool depends on the allergic disease under study. With respect to basophil detection methods, selection with a single marker, CCR3, is proposed as an option with a good cost-effectiveness ratio. Conclusions: BAT is a promising tool to evaluate clinically allergic reactions in a safe manner. Better standardization of protocols is necessary to achieve reproducible results.

[Determination of a new cow's milk-based formula allergenic capacity in 7 infants]. / Determinación de la capacidad alergénica de una nueva fórmula infantil a base de leche de vaca en 7 lactantes.

BACKGROUND: Cow's milk protein allergy is the main allergic problem during the first year of life, possibly owing to immune and gastrointestinal systems poor maturation. To prevent allergic reactions, the content and type of proteins in infant formulas resemble those of breast milk. We believe that reactions are due rather to the amount than to the type of protein. OBJECTIVE: To design a new formula with cow's milk that provides the infant with the main nutrients at an affordable cost and with lower risk for the development of allergies. METHODS: Three-phase project: product design, industrial production and ex vivo assay to assess for anemia and type I allergic reaction by CD63 expression in basophils. RESULTS: For every 100 calories, the content of protein was 2.0 g, carbohydrates 7.2 g and fat 0.5 g, which is higher than the indicated maximum value (4.5 g). Microbiologically, it was an innocuous food. CD63 expression was low in 57.1% of the babies and high in 42.9%. CONCLUSION: The new formula did not trigger any allergenic responses and can therefore be supplied to non-atopic infants.

Antecedentes: La alergia a las proteínas de la leche de vaca es el principal problema alérgico durante el primer año de vida, debido a la poca maduración de los sistemas inmunológico y gastrointestinal. Para evitar reacciones alérgicas, el contenido y tipo de proteínas de las fórmulas infantiles se asemejan a los de la leche materna. Conforme un principio de alergología, probablemente las reacciones se deben a la cantidad de proteínas más que al tipo. Objetivo: Diseñar una nueva fórmula con leche de vaca que aporte al lactante los principales nutrientes, a un bajo costo y con menor riesgo de padecer alergias. Métodos: Proyecto realizado en 3 fases: diseño del producto, producción industrial y ensayo ex vivo para evaluar mediante la expresión del CD63 en basófilos la presencia de anemia y reacción alérgica tipo I en lactantes. Resultados: Por cada 100 calorías, el contenido de proteínas fue de 2 g, de carbohidratos de 7.2 g y de grasa de 0.5 g, mayor al valor máximo indicado (4.5 g). Microbiológicamente, la fórmula láctea propuesta se trató de un alimento inocuo. La expresión del CD63 fue baja en 57.1 % de los lactantes y alta en 42.9 %. Conclusión: La nueva fórmula no desencadenó respuesta alergénica, por lo tanto, puede suministrarse a lactantes no atópicos.

Las grandes directrices de la investigación en homeopatía (parte 1 de 2) / The broad guidelines of research in homeopathy (part 1 of 2)

La Homeopatía se fundó sobre un método que es, al mismo tiempo, experimental y empírico. La Homeopatía es parcialmente experimental debido a que su semiología nació con la experimentación sobre el hombre sano, efectuada según el método hahnemanniano; asimismo, es parcialmente empírica porque se basa en el trabajo de observación de los médicos homeópatas y en la adquisición de la experiencia práctica, la cual ha sido transmitida a alumnos deseosos de utilizar este método terapéutico. La investigación en Homeopatía se ha desarrollado progresivamente, tratando de integrar las aportaciones de estos dos métodos: experimental y empírico. De hecho, los esfuerzos realizados por situar a la Homeopatía dentro de una metodología rigurosa se remontan al inicio mismo de esta disciplina, inscrita desde su origen por Hahnemann dentro de la línea científica y médica original. (AU)

Homeopathy is in part experimental because the semiology was born with the experimentation on the healthy man, which was performed according to Hahnemann´s method; it is also partly empirical because much work is based on the observation of homeopaths and the acquisition of practical experience, which has been transmitted to students eager to use this therapeutic method. Research in Homeopathy has been gradually developed, trying to integrate the contributions of these two methods: experimental and empirical. In fact, efforts have been made to place homeopathy within a rigorous methodology dating back to the beginning of the discipline itself, from its origin registered by Hahnemann within the original scientific and medical field. (AU)

Las grandes directrices de la investigación en homeopatía (parte 1 de 2) / The broad guidelines of research in homeopathy (part 1 of 2)

La Homeopatía se fundó sobre un método que es, al mismo tiempo, experimental y empírico. La Homeopatía es parcialmente experimental debido a que su semiología nació con la experimentación sobre el hombre sano, efectuada según el método hahnemanniano; asimismo, es parcialmente empírica porque se basa en el trabajo de observación de los médicos homeópatas y en la adquisición de la experiencia práctica, la cual ha sido transmitida a alumnos deseosos de utilizar este método terapéutico. La investigación en Homeopatía se ha desarrollado progresivamente, tratando de integrar las aportaciones de estos dos métodos: experimental y empírico. De hecho, los esfuerzos realizados por situar a la Homeopatía dentro de una metodología rigurosa se remontan al inicio mismo de esta disciplina, inscrita desde su origen por Hahnemann dentro de la línea científica y médica original...

Homeopathy is in part experimental because the semiology was born with the experimentation on the healthy man, which was performed according to Hahnemann´s method; it is also partly empirical because much work is based on the observation of homeopaths and the acquisition of practical experience, which has been transmitted to students eager to use this therapeutic method. Research in Homeopathy has been gradually developed, trying to integrate the contributions of these two methods: experimental and empirical. In fact, efforts have been made to place homeopathy within a rigorous methodology dating back to the beginning of the discipline itself, from its origin registered by Hahnemann within the original scientific and medical field...

Peroxidase activity in Phrynops geoffroanus' (Testudines Chelidae) basophils / Atividade peroxidásica em basófilos de Phrynops geoffroanus (Testudines Chelidae)

As peroxidases, presentes nos peroxissomos e lisossomos, pertencem às oxidases e atuam como catalítico para o peróxido de hidrogênio (H2O2), posteriormente decomposto pela oxidação de cossubstratos, evitando danos celulares.(Õ) Foi aplicada a técnica da peroxidase(2) em esfregaços sanguíneos de Phrynops geoffroanus, comparando com sangue humano, para avaliação da atividade e controle da reação. O esfregaço sanguíneo humano apresentou marcações em neutrófilos, fagócitos com muitos lisossomos e peroxissomos (Figura 1). Nos esfregaços sanguíneos de Phrynops geoffroanus, as marcações apresentaram-se nos basófilos (Figura 2), que representam de 10 por cento a 25 por cento dos leucócitos de quelônios e possuem grande número de granulações citoplasmáticas,(3) sugerindo a presença de grande quantidade de enzimas e organelas como lisossomos e peroxissomos, possivelmente associadas a sua participação em reações imunes. A atividade peroxidásica representa resposta do organismo a ações ambientais danosas, servindo como marcador biológico.

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...