Shared epitope classification methods according to Gregersen and de Vries allow adequate characterization of patients with rheumatoid arthritis in a Colombian population

Introduction: The most important genetic association in rheumatoid arthritis (RA) is presented with some alleles from the HLA-DRB1 gene that encode the shared epitope (SE). Objectives: To apply the SE classification methods of Gregersen, de Vries, Raychaudhuri, Mattey, and Tezenas du Montcel in a group of Colombian patients with RA and determine the most common HLA-DRB1 alleles in the population. Methods: RA diagnosis, genetic study of the HLA-DRB1 region using Luminex technology in 50 RA and 50 healthy subjects. For the classification analysis, Fisher's exact test and chi-squared test were applied. Tables were created to count the RA-related alleles. We used odds ratio to determine the risk between the presence of the shared epitope (SE) and anti-cyclic citrullinated peptides (Anti-CCP). Results: Gregersen and de Vries methods were suitable for the characterization of RA in this population (p = .006). The most prevalent HLA-DRB1 alleles in the RA group were 14:02,04:04, 08:02,04:05, and 10:01. High frequencies of the 07:01, 03:01,13:02,01:02, and 12:01 HLA-DRB1 alleles were found in the healthy population. HLA-DRB1 alleles with similar distribution in both populations were 04:07, 15:01, 11:01, 16:02, and 01:01. A high frequency of SE + was observed in Anti-CCP + individuals (63.15%); however, this was not statistically significant [OR2.4 (.63-9.01); p = .19]. Conclusion: The SE classification methods of Gregersen and de Vries were adequate in characterizing RA in a Colombian population group. An equivalence of 100% was verified between the susceptibility alleles defined by de Vries and the alleles assigned as SE according to Gregersen.

Introducción: La asociación genética más importante en artritis reumatoide (AR) se presenta con algunos alelos del gen HLA DRB1 que codifican el epítope compartido (EC). Objetivos: Aplicar los métodos de clasificación de EC de Gregersen et al., de Vries et al., Raychaudhuri et al., Mattey et al., y Tezenas du Montcel et al., en un grupo de pacientes colombianos con AR, y determinar los alelos HLA DRB1 más frecuentes en esta población. Métodos: Diagnóstico para AR, estudio genético de la región HLA DRB1 por tecnología Luminex® de 50 sujetos AR y 50 sanos. Para análisis comparativos de clasificaciones EC, se aplicaron las pruebas test exacto de Fisher y Chi-cuadrado y se realizaron tablas de conteos para los alelos relacionados con AR. Se estimó la razón de odds para determinar el riesgo entre la presencia de EC y los anticuerpos antipéptidos cíclicos citrulinados (anti-PCC). Resultados: Los métodos de Gregersen et al. y de Vries et al. fueron adecuados para la caracterización de AR en esta población (p = 0,006). Los alelos HLA DRB1 más prevalentes en el grupo AR fueron 14:02, 04:04, 08:02, 04:05 y 10:01. Se encontraron altas frecuencias de los alelos HLA DRB1 07:01, 03:01,13:02, 01:02 y 12:01 en población sana. Alelos HLA DRB1 con distribución similar en ambas poblaciones fueron: 04:07, 15:01, 11:01, 16:02 y 01:01. Se observó alta frecuencia de individuos EC+ en el grupo AR anti-PCC+ (63,15%); no obstante, sin asociación estadística (OR: 2,4 [0,63-9,01]; p = 0,19). Conclusión: Los métodos de clasificación para EC de Gregersen et al. y de Vries et al. fueron adecuados caracterizando AR en un grupo de población colombiana. Se corroboró equivalencia del 100% entre los alelos de susceptibilidad definidos por de Vries y los alelos asignados como EC según Gregersen et al.

Identificação, caracterização e determinação de padrões da resposta imune de epítopos imunogênicos de Plasmodium vivax / Identification, characterization and determination of immune response patterns of immunogenic epitopes of Plasmodium vivax

O Plasmodium vivax representa um grande desafio no controle da malária devido a sua vasta distribuição ao redor do globo, grande frequência de infecções sub microscópicas e habilidade de induzir recaídas em consequência das formas evolutivas que podem ficar latentes no fígado por longos períodos (hipnozoítos). O recente aumento de cepas de P. vivax resistentes aos fármacos disponíveis, a evolução de formas mais virulentas do parasito e a produção precoce de gametócitos, característica desta espécie, contribuem para classificar a malária vivax como um problema de saúde pública que merece atenção. Ainda que reconhecido por suas características biológicas peculiares e pelo agravamento recente de sua virulência, poucos investimentos têm sido feitos no desenvolvimento de ferramentas de controle para vivax. Portanto, o presente estudo teve como objetivo identificar e caracterizar novos alvos potenciais utilizando amostras de diferentes áreas endêmicas ao redor do mundo (Brasil, Mali, Camboja e Estados Unidos da América). Para tanto, investigamos e caracterizamos uma proteína recém descoberta na urina de pacientes naturalmente infectados (PvVir14); e descrevemos o potencial imunogênico de epítopos de células B de uma das proteínas mais bem caracterizadas de P. vivax, a PvAMA-1. Anti-IgG circulantes contra PvVir14 apareceram em 61% e 34.5% dos indivíduos do Brasil e Camboja, respectivamente, enquanto que indivíduos de Mali (infectados com falciparum e não expostos a vivax), tiveram 0% de reconhecimento. Ainda, os níveis de anti-PvVir14 correlacionaram-se com aqueles contra outros antígenos de vivax já bem caracterizados, como a PvCSP e a PvDBP, que foram reconhecidos por 7.6% e 42% dos indivíduos respectivamente. Com relação ao perfil celular, indivíduos sororreativos para PvVir14 apresentaram níveis significativamente maiores de células B atípicas circulantes (CD 21- CD 27-), sugerindo que tal tipo celular possa estar ligado à resposta anti-PvVir14. Entre as células T, os níveis de CD4+ e CD8+ diferiram entre indivíduos com e sem anticorpos contra PvVir14 (menor e maior expressão, respectivamente), enquanto que os níveis de células NKT foram mais expressivos em indivíduos sem anti-PvVir14. Se tratando da PvAMA-1, a antigenicidade dos peptídeos com epítopos para células B previamente selecionados foi avaliada através de múltiplos ensaios sorológicos utilizando amostras de indivíduos com infecção aguda por P.vivax do norte do Brasil. Os peptídeos sintéticos foram reconhecidos por 45.5%, 48.7% e 31.2% (PI, PII e PII, respectivamente) dos indivíduos selecionados para o estudo. Quando sintetizados em conjunto (tripeptídeo), a reatividade aumentou para 62%, porcentagem comparável àquela obtida pela proteína em sua forma e tamanho originais (57%). Além disso, a reatividade anti-IgG conta o tripeptídeo foi reduzida em 42% pós-depleção, indicando que tais epítopos podem ser responsáveis por parte considerável da imunogenicidade da proteína. Esses resultados representam uma excelente perspectiva na identificação de novos alvos com potencial imunogênico para compor uma vacina, ou auxiliar no desenvolvimento de outras medidas de controle, como testes diagnósticos, já que contemplar diversos alvos do ciclo de vida do parasito parece ser a chave para alcançar a resposta robusta e protetora que uma vacina contra a malária vivax precisa para ter sucesso.

Plasmodium vivax is a major challenge for malaria control due to its wide geographic distribution, high frequency of submicroscopic infections, and ability to induce relapses due to the latent forms present in the liver (hypnozoites). The recent increase in drug-resistant P. vivax strains, the evolution toward more virulent forms and the early production of gametocytes adds up to make P. vivax malaria a public health issue of increasing importance. Besides its tricky biological features and new awareness of its virulence, minimal investments have been made in vaccine discovery for P. vivax. Given that, this study aimed to discover and characterize potential new targets for future vaccine development using samples from different endemic areas around the world (Brazil, Mali, Cambodia and United States of America). For this purpose, we investigated and characterized a novel protein recently discovered in the urine of naturally infected subjects (PvVir14) and described the immunogenic potential of peptides from a well-known vivax protein (PvAMA-1), which has been proved to have important B cell epitopes that can induce specific immune response. Circulating antibodies against PvVir14 appeared in 61% and 34.5% of subjects from Brazil and Cambodia, respectively, versus none (0%) of the P. falciparum-infected subjects from Mali who have no exposure to P. vivax. PvVir14 antibody levels correlated with those against other well-characterized sporozoite/liver (PvCSP) and blood stage (PvDBP-RII) antigens, which were recognized by 7.6% and 42% of Brazilians, respectively. Concerning the cellular immune profiling of Brazilian subjects, PvVir14 seroreactive individuals displayed significantly higher levels of circulating atypical (CD21− CD27−) B cells, raising the possibility that atypical B cells may be contribute to the PvVir14 antibody response. Among T cells, CD4+ and CD8+ levels differed (lower and higher, respectively) between subjects with versus without antibodies to PvVir14, while NKT cell levels were higher in those without antibodies. As for PvAMA-1, the antigenicity of the selected B-cell peptides was assessed by multiple serological assays using sera from acute P.vivax infected subjects. The synthetic peptides were recognized by 45.5%, 48.7% and 32.2% of infected subjects for peptides I, II and III respectively. Moreover, when synthetized together (tripeptide), the reactivity increases up to 62%, which is comparable to the reactivity found against the whole protein PvAMA-1 (57%). Furthermore, IgG reactivity against the tripeptide after depletion was reduced by 42%, indicating that these epitopes may be responsible for a considerable part of the protein immunogenicity. These results represent an excellent perspective on discovering new targets with immunogenic potential to compose a vaccine, or even to assist the development of other control measures, such as diagnostic tools, since contemplating several targets seems to be the key to achieving a robust and protective response that a malaria vaccine needs to be successful.

Soy protein: a food allergen frequently used in the preparation of meat products / Proteína de soya: un alergeno alimentario altamente utilizado en la preparación de productos cárnicos

The addition of soy proteins, currently classified as a food allergen, into meat products is a commonly used practice due to its functional properties and low cost. Its addition to meat products can cause health problems for individuals allergic to these proteins. Allergic individuals can be affected by the ingestion of low amounts of the allergen. In Brazil, limits are set for the addition of soy proteins in meat products in order to avoide fraud. Starting in 2015 reporting the name of the added component became mandatory for all food labelling. Some studies have reported that food processing can reduce the allergenicity, either by irreversible removal of allergens or by modifying the allergen structure. However, the technological approach to decrease allergenicity has largely been empirical. This review describes the use of soy protein in meat products and the health risk for allergic individuals and consumers of these products. Finally, appropriate methodologies for the detection and quantification of these proteins must be further explored and established to avoid fraud and to preserve consumer health.

La adición de proteínas de soya, actualmente clasificadas como alergeno alimentario, en los productos cárnicos es una práctica comúnmente utilizada debido a sus propiedades funcionales y bajo costo. Su adición en productos cárnicos puede causar problemas de salud en personas alérgicas a estas proteínas. Las personas alérgicas pueden verse afectadas por la ingestión de cantidades diminutas de alérgeno. En Brasil, se establecen límites para la adición de proteínas de soya en los productos cárnicos con el objetivo de evitar el fraude. Solo en 2015 se hizo obligatoria la declaración en la etiqueta de todos los alimentos que indicaban la presencia de sustancias alérgicas, así como el nombre del componente. Algunos estudios se refieren al procesamiento de alimentos para reducir la alergenicidad, ya sea mediante la eliminación irreversible de alergenos o modificando la estructura del alergeno; sin embargo, el enfoque tecnológico hasta ahora para disminuir la alergenicidad ha sido en gran medida empírico. Esta revisión describe el uso de proteína de soya en los productos cárnicos y el riesgo que puede causar para la salud de las personas alérgicas y a los consumidores de estos productos. Finalmente, las metodologías apropiadas para la detección y cuantificación de estas proteínas deben explorarse en profundidad y establecerse para evitar el fraude y preservar la salud de los consumidores.

Epitope of dengue virus E protein detect human antibodies associated with mild disease: a potential peptide for vaccine development

ABSTRACT The antigenic potential of seven immunogenic peptides of the dengue virus was evaluated in the sera of patients with dengue confirmed by IgM/IgG serology. Antibodies IgM and IgG against dengue virus peptides were analyzed by ELISA in 31 dengue sero-positive and 20 sero-negative patients. The P5 peptide showed significant IgG immunoreactivity mostly in the sera of patients with dengue without warning signs in comparison with patients with dengue with warning signs, correlating with mild disease. This finding suggests that the low antibody response against P5 epitope could be a risk factor for higher susceptibility to dengue virus infection with warning signs, and that P5 could be a potential antigen for vaccine development.

Diseño y evaluación de una proteína multiepitópica como candidata para vacuna contra la enfermedad de carrión / Design and evaluation of a multiepitopic protein as a candidate for a carrion disease vaccine

RESUMEN Objetivos. Diseñar y evaluar una proteína multiepítope como candidato a vacuna contra la enfermedad de Carrión. Materiales y métodos. Mediante herramientas bioinformáticas se seleccionó epítopes de proteínas de membrana externa y se diseñó una proteína multiepítope. El gen de la proteína multiepítope fue subclonado en el plásmido de expresión pET28b y transformado en E. coli BL21 pLys. La proteína multiepítope fue expresada usando isopropil-β-D-1-tiogalactopiranósido y purificada usando resina. Esta proteína purificada fue utilizada para inmunizar ratones BALB/c y se obtuvo anticuerpos policlonales. Se realizaron ensayos de invasión in vitro usando una cepa de Bartonella bacilliformis (B. bacilliformis) a eritrocitos humanos. Resultados. La proteína multiepítope M1 presenta epítopes conservados entre aislamientos de B. bacilliformis, no tóxicos, no homólogos a proteínas humanas y superficiales. Los ratones inmunizados presentaron niveles de anticuerpos IgG capaces de reducir in vitro la tasa de invasión de B. bacilliformis a eritrocitos humanos. Conclusiones. La proteína multiepítope M1 podría servir como candidato a vacuna contra la enfermedad de Carrión; sin embargo, se requiere de más estudios para caracterizar el uso de este antígeno como vacuna.

ABSTRACT Objectives. To design and assess a multiepitopic protein as a candidate for a vaccine against Carrion disease. Materials and Methods. Using bioinformatics tools, epitopes of external membrane proteins were selected and a multiepitopic protein was designed. The multiepitopic protein gene was subcloned into the expression plasmid pET28b and transformed into E. coli BL21 pLys. The multiepitopic protein was expressed using isopropyl-β-D-1-thiogalactopyranoside and purified using resin. This purified protein was used to immunize BALB/c mice obtaining polyclonal antibodies. In vitro invasion assays were conducted using a strain of Bartonella bacilliformis (B. bacilliformis) in human red blood cells. Results. The multiepitopic protein M1 presents preserved epitopes between isolates of B. bacilliformis with are non-toxic, and not homologous to human and surface proteins. Immunized mice presented IgG antibody levels capable of reducing in vitro the rate of invasion of B. bacilliformis into human red blood cells. Conclusions. Multiepitopic protein M1 may serve as a candidate for a Carrion disease vaccine; however, more studies are needed to characterize the use of this antigen as a vaccine.

Expression and purification of the transcription factor StMsn2 from Setosphaeria turcica in Escherichia coli

Background: In Saccharomyces cerevisiae, Msn2, which acts as a key transcription factor downstream the MAPKHOG cascade pathway, also regulates the expression of genes related to stress responses. However, little is known about the regulation mechanisms of the transcription factor in Setosphaeria turcica. Results: In this study, a zinc finger DNA-binding protein, designated as StMSN2, was cloned from S. turcica. Sequencing results showed that StMSN2 had a 1752 bp open reading frame (ORF), which was interrupted by an intron (135 bp) and encoded a putative 538-amino acid protein. Phylogenetic analysis further revealed that StMsn2 was more closely related to Msn2 of Aspergillus parasiticus. StMSN2 was cloned into the pET-28a vector with His (Histidine) tags and induced with 1 mM IPTG (isopropyl-ß-D-thiogalactoside) at 37°C. The recombinant His-tagged StMsn2 was purified, and a band of size approximately 58.8 kDa was obtained. The high specificity of the polyclonal antibody Msn2-2 was detected with the StMsn2 protein from S. turcica and prokaryotic expression system, respectively. Conclusions: A new gene, named StMSN2, with 1617 bp ORF was cloned from S. turcica and characterized using bioinformatics methods. StMsn2 was expressed and purified in a prokaryotic system. A polyclonal antibody, named Msn2-2, against StMsn2 with high specificity was identified.

IgE antibodies from schistosomiasis patients to recognize epitopes in potato apyrase

Abstract INTRODUCTION: High percentages of structural identity and cross-immunoreactivity have been reported between potato apyrase and Schistosoma mansoni ATP diphosphohydrolase (SmATPDases) isoforms, showing the existence of particular epitopes shared between these proteins. METHODS: Potato apyrase was employed using ELISA, western blot, and mouse immunization methods to verify IgE reactivity. RESULTS: Most of the schistosomiasis patient's (75%) serum was seropositive for potato apyrase and this protein was recognized using western blotting, suggesting that parasite and plant proteins share IgE-binding epitopes. C57BL/6 mice immunized with potato apyrase showed increased IgE antibody production. CONCLUSIONS: Potato apyrase and SmATPDases have IgE-binding epitopes.

The situation of lymphatic filariasis in the municipality of Paulista, Pernambuco, Brazil

Cases of Lymphatic Filariasis (LF) have been notified since 1959 in the municipality of Paulista, yet it is still considered an LF-free area. The purpose of this study was to describe the situation of Paulista Health Department. The data were gathered via antigenic surveys carried out in the town, using POC-ICT-AD12 tests. A total of 1,000 individuals, aged 10 and over, were examined in the neighborhoods of Mirueira, Engenho Maranguape, Janga and Maranguape II (250 individuals in each district). Among the individuals evaluated, seven (0,7%) tested positive for antigens using CFA POC-ICT-AD12, 5 out of 250 (2.0%) in the Engenho Maranguape neighborhood and 2 out of 250 (0.8%) in Janga. In this group, one particular individual presented microfilaremia, quantified at 5 Mf/mL. These results suggest that the municipality of Paulista might be a "silent" source of LF continuous transmission, fact that could impact negatively on the goals of the GPELF program meant to provide certification of parasitic disease control and elimination by the year 2022

Analysis of Bm86 conserved epitopes: is a global vaccine against Cattle Tick Rhipicephalus microplus possible? / Análise de epítopos conservados da Bm86: é possível uma vacina global contra Carrapato-do-Boi Rhipicephalus microplus?

Abstract The cattle tick Rhipicephalus microplus causes significant economic losses in agribusiness. Control of this tick is achieved mainly through the application of chemical acaricides, often resulting in contamination of animal food products and of the environment. Another major concern associated with acaricide use is the increasing reports of resistance of this tick vector against the active ingredients of many commercial products. An alternative control method is vaccination. However, the commercially available vaccine based on a protein homologous to Bm86 exhibits variations in efficacy relative to the different geographical locations. This study aimed to identify antigenic determinants of the sequences of proteins homologous to Bm86. Phylogenetic analyses were performed to determine the extent of divergence between different populations of R. microplus to identify the sequence that could be used as a universal vaccine against the multiple geographically distinct populations of R. microplus and related tick species. Considering the extensive sequence and functional polymorphism observed among strains of R. microplus from different geographical regions, we can conclude that it may be possible to achieve effective vaccination against these cattle ticks using a single universal Bm86-based antigen.

Resumo O carrapato Rhipicephalus microplus é responsável por perdas significativas no agronegócio. O controle deste carrapato é feito principalmente por meio da aplicação de acaricidas químicos, geralmente resultando na contaminação de produtos de origem animal e do meio ambiente. Outra preocupação importante associada ao uso de acaricidas é o crescente aumento de relatos sobre a resistência deste carrapato a princípios ativos de vários produtos comerciais. Uma alternativa de controle é por meio de vacinação. Porém, a vacina comercializada contendo proteína homóloga à Bm86, apresenta variações de eficácia em relação às diferentes localizações geográficas. Este estudo buscou identificar determinantes antigênicos das sequencias de proteínas homólogas a Bm86. As análises filogenéticas foram feitas para determinar a extensão da divergência entre diferentes populações de R. microplus com o objetivo de identificar a sequência que poderia ser usada como vacina universal contra as múltiplas populações geograficamente distintas de R. microplus e espécies de carrapatos relacionados. Considerando-se a extensa sequência e o polimorfismo observados entre linhagens de R. microplus de diferentes regiões geográficas, podemos concluir que pode ser possível obter uma vacinação efetiva contra esses carrapatos bovinos utilizando um único antígeno universal baseado em Bm86.

Mapping of the continuous epitopes displayed on the Clostridium perfringens type D epsilon-toxin

Abstract The epsilon toxin, produced by Clostridium perfringens, is responsible for enterotoxemia in ruminants and is a potential bioterrorism agent. In the present study, 15 regions of the toxin were recognized by antibodies present in the serum, with different immunodominance scales, and may be antigen determinants that can be used to formulate subunit vaccines.

Sequence similarity between thyroid self-protein and hepatitis c virus polyprotein: possible triggering mechanism of autoimmune thyroiditis / Similaridade de sequências entre auto-proteínas da tireoide e poliproteínas do vírus da hepatite C: possível mecanismo de desencadeamento da tireoidite auto-imune

ABSTRACT Background - Exposure to viral antigens that share amino acid sequence similar with self- antigens might trigger autoimmune diseases in genetically predisposed individuals, and the molecular mimicry theory suggests that epitope mimicry between the virus and human proteins can activate autoimmune disease. Objective - The purpose of this study is to explore the possible sequence similarity between the amino acid sequences of thyroid self-protein and hepatitis C virus proteins, using databanks of proteins and immunogenic peptides, to explain autoimmune thyroid disease. Methods - Were performed the comparisons between the amino acid sequence of the hepatitis C virus polyprotein and thyroid self-protein human, available in the database of National Center for Biotechnology Information on Basic Local Alignment Search Tool. Results - The sequence similarity was related each hepatitis C virus genotype to each thyroid antigen. The similarities between the thyroid and the viral peptides ranged from 21.0 % (31 identical residues out of 147 amino acid in the sequence) to 71.0% (5 identical residues out of 7 amino acid in the sequence). Conclusion - Bioinformatics data, suggest a possible pathogenic link between hepatitis C virus and autoimmune thyroid disease. Through of molecular mimicry is observed that sequences similarities between viral polyproteins and self-proteins thyroid could be a mechanism of induction of crossover immune response to self-antigens, with a breakdown of self-tolerance, resulting in autoimmune thyroid disease.

RESUMO Contexto - A exposição a antígenos virais que compartilham sequência de aminoácidos semelhantes a auto-antígenos pode provocar doenças auto-imunes em indivíduos predispostos geneticamente, e a teoria do mimetismo molecular sugere que o mimetismo entre epítopos de vírus e proteínas humanas pode ativar doenças auto-imunes. Objetivo - O objetivo deste estudo foi explorar a possível semelhança entre as sequências de aminoácidos de auto-proteinas da tireóide e proteínas do vírus da hepatite C, utilizando bancos de dados de proteínas e peptídeos imunogênicos, para explicar a doença auto-imune da tireóide. Métodos - Foram realizadas comparações entre as sequências de aminoácidos de poliproteínas do vírus da hepatite C e auto-proteinas da tireóide humana, disponível na base de dados do National Center for Biotechnology Information no Basic Local Alignment Search Tool. Resultados - A semelhança de sequências foi relacionada para cada genótipo de vírus da hepatite C e proteínas da tireóide. As semelhanças entre proteínas da tireóide e os peptídeos virais variaram de 21,0% (31 resíduos idênticos da sequência de 147 aminoácidos) a 71,0% (cinco resíduos idênticos da sequência de 7 aminoácidos). Conclusão - Dados de bioinformática sugerem uma possível ligação entre vírus da hepatite C e doença auto-imune da tireóide. Através de mimetismo molecular observa-se que as semelhanças entre as sequências de poliproteínas virais e auto-proteínas da tireóide pode ser um mecanismo de indução de resposta imune resultando em doença auto-imune da tireóide.

Expression of Hemagglutinin-Neuraminidase and fusion epitopes of Newcastle Disease Virus in transgenic tobacco

Background: Newcastle disease is an important avian infectious disease that brings about vast economic damage for poultry industry. Transgenic plants represent a cost-effective system for the production of therapeutic proteins and are widely used for the production of poultry vaccines. In an attempt to develop a recombinant vaccine, a plant expression binary vector pBI121, containing the genes encoding Hemagglutinin-Neuraminidase (HN) and Fusion (F) epitopes of Newcastle Disease Virus (NDV) under the control of CaMV35S promoter and NOS terminator was constructed and introduced into the tobacco ( Nicotiana tabacum) plant by Agrobacterium-mediated transformation. Results: Putative transgenic plants were screened in a selection medium containing 50 mg/L kanamycin and 30 mg/L meropenem. Integration of the foreign gene in plant genome was confirmed by PCR. Expression of foreign gene was analyzed at transcription level by RT-PCR and at translation level by means of dot blotting and ELISA. All analyses confirmed the expression of recombinant protein. Conclusion: Developments in genetic engineering have led to plant-based systems for recombinant vaccine production. In this research, tobacco plant was used to express F and HN epitopes of NDV. Our results indicate that for the production of recombinant vaccine, it is a novel strategy to use concatenated epitopes without their genetic fusion onto larger scaffold structure such as viral coat protein.

Fenômeno de epitope spreading: caracterização clínico imunológica em pacientes portadores de dermatoses bolhosas autoimunes / Epitope spreading" phenomena: clínical and immunopathological characterization in patients with bullous dermatosis

As dermatoses bolhosas autoimunes são um grupo heterogêneo de afecções da pele e/ou mucosas associadas à produção de autoanticorpos dirigidos às moléculas de adesão epitelial. Podem ser classificadas em dermatoses bolhosas intraepidérmicas (pênfigos) ou subepidérmicas (penfigóides, epidermólise bolhosa adquirida). Nos últimos anos, a transição entre dermatoses bolhosas autoimunes ou coexistência de autoanticorpos de diferentes dermatoses têm sido relatadas em alguns pacientes e atribuída ao fenômeno de epitope spreading (ES): a diversificação de epítopos reconhecidos pelo sistema imune evocaria uma reação secundária a antígenos distintos e não relacionados aos da doença primária. Neste trabalho avaliamos a ocorrência de fenômenos de ES em pacientes portadores de pênfigo. CASUÍSTICA E MÉTODOS: Inicialmente, foi realizada análise de dados clínicos e laboratoriais (exame histopatológico, de imunofluorescência direta-IFD, indireta IFI e ELISA) de 351 pacientes portadores de pênfigos acompanhados no Ambulatório de dermatoses bolhosas autoimunes do Departamento de Dermatologia da Faculdade de Medicina da Universidade de São Paulo no período de dezembro de 2002 a dezembro de 2012. Foram selecionados pacientes com quadro sugestivo de conversão à dermatose bolhosa distinta da doença primária. RESULTADOS: Nove pacientes apresentaram sinais sugestivos de fenômeno de ES e foram incluídos no estudo: 8 com a conversão de Pênfigo vulgar (PV) a foliáceo (PF) 2,3% (grupo1) e um de PF a Epidermólise bolhosa adquirida (EBA) 0,3% (grupo 2). No grupo 1 o intervalo mediano para a conversão foi de 3,5 anos. Cinco pacientes apresentaram modificação histopatológica de clivagem intraepidérmica na camada suprabasal para clivagem na camada subcórnea durante a suspeita de ES; 2 apresentaram clivagem na camada epidérmica média durante a transição e um manteve clivagem suprabasal, apesar de quadro clínico sugestivo de PF. Todos os pacientes apresentavam...

Autoimmune bullous skin diseases represent a heterogeneous group of disorders of skin and mucosa associated with autoantibodies against distinct adhesion molecules. They can be classified, based on the level of loss of adhesion in intraepidermal and sub epidermal dermatosis. The shift from an autoimmune blistering disease to another has been recently described and attributed to the "epitope spreading" (ES) phenomena. It occurs when a primary inflammatory/autoimmune process releases "hidden" epitopes which are recognized by the lymphocytes and evoke a secondary reaction to antigens distinct from, and non-cross-reactive, with the disease causing-epitope. This study attempted to characterize the occurrence of ES in pemphigus patients. METHODS: We analyzed data from 351 pemphigus patients treated ambulatorially at the Department of Dermatology, Faculty of Medicine, University of São Paulo, from December 2002 to December 2012. A careful search for clinical and laboratorial (histopathology, direct-DIF and indirect-IIF immunofluorescence, ELISA) changes suggestive of shift to a secondary bullous disease was performed. RESULTS: Nine out of 351 patients presented clínical shift and were included in the study: eight from pemphigus vulgaris (PV) to foliaceus (PF) 2.3% (group 1) and one from PF to epidermolysis bullosa acquisita (EBA) 0.3% (group 2). In group 1, median interval of disease shift was 3.5 years. Of 8 patients with clinical PF, five showed change of histopathology pattern from suprabasilar cleavage to subcorneal acantholysis, two had cleavage within the middle epidermal layer, and one sustained the suprabasilar acantholysis. One shifted back to PV after clinical and histopatological changes of PF. All patients showed intercellular IgG and/or C3 deposits during PV and PF diagnosis by DIF. IIF titers varied from 1:160 to 1:5120. ELISA index for Dsg1 varied from 22 to 319; and for Dsg3 from 0.4 to 224 (positive if > 20)....

Pesticide exposure and health conditions of terrestrial pesticide applicators in Córdoba Province, Argentina / Exposición a plaguicidas y condiciones de salud de agroaplicadores terrestres de la Provincia de Córdoba, Argentina / Exposição aos agrotóxicos e condições de saúde dos aplicadores de pesticidas da Província de Córdoba, Argentina

Agricultural workers represent a population that is highly vulnerable to the toxic effects of pesticide exposure. This cross sectional study aimed to describe the health conditions of terrestrial pesticide applicators in Córdoba Province, Argentina, their work practices and socio-demographic characteristics, by means of a standardized self-administered questionnaire (n = 880). A descriptive analysis reported a high prevalence of occasional or frequent symptoms: 47.4% had symptoms of irritation, 35.5% fatigue, 40.4% headache and 27.6% nervousness or depression. Using logistic regression models, risk and protective factors were found for symptoms of irritation, medical consultation and hospitalization. Among the occupational exposure variables, marital status, length of time in the job, low level of protection with regard to the use of personal protective equipment, combined use of different pesticides and the application of the insecticide endosulfan, were associated with a higher frequency of reported symptoms and higher consultation rates and hospitalization.

Los trabajadores agrícolas son una población altamente vulnerable a los efectos tóxicos de la exposición a plaguicidas. Con el objetivo de describir las condiciones de salud de agroaplicadores terrestres de plaguicidas de la Provincia de Córdoba, Argentina, sus prácticas laborales y características sociodemográficas, se realizó un estudio transversal, mediante cuestionario (n = 880). Un análisis descriptivo reportó alta prevalencia de sintomatología ocasional o frecuente: 47,4% síntomas irritativos, 35,5% cansancio, 40,4% cefalea y 27,6% ansiedad o depresión. Mediante modelos logísticos se detectaron factores protectores y de riesgo que explican la presencia de síntomas irritativos, la consulta médica y la hospitalización. El estado civil, la antigüedad en la tarea, el nivel de protección considerando uso de equipo de protección personal, la exposición múltiple a plaguicidas y la aplicación del insecticida endosulfán, se asociaron a mayor frecuencia de reporte de síntomas, consultas médicas y hospitalizaciones por causas relacionadas con la exposición a plaguicidas.

Os trabalhadores agrícolas são uma população altamente vulnerável aos efeitos tóxicos da exposição a pesticidas. Este estudo transversal teve o objetivo de descrever as condições de saúde de aplicadores terrestres de pesticidas da Província de Córdoba, Argentina, suas práticas de trabalho e características sociodemográficas, por meio de um questionário padronizado autoadministrado (n = 880). A análise descritiva relatou alta prevalência de sintomas ocasionais ou frequentes: 47,4% sintomas irritativos, 35,5% fadiga, 40,4% dor de cabeça e 27,6% ansiedade ou depressão. Mediante modelos logísticos foram detectados os fatores protetores e do risco que explicam a presença de sintomas irritativos, consulta médica e hospitalização. O estado civil, anos de trabalho, o nível de proteção considerando o uso de equipamentos de proteção individual, a exposição a vários pesticidas e aplicação do inseticida endosulfan, foram associados com maior frequência de sintomas, consultas médicas e hospitalização por causas relacionadas à exposição ao agrotóxico.

Análise da variabilidade imunológica e da expressão dos genes FREPs entre as espécies Biomphalaria glabrata e B. straminea com diferentes perfis de suscetibilidade ao parasito Schistosoma mansoni / Analysis of immune variability and expression of FREPS genes between the species Biomphalaria glabrata and B. straminea with different susceptibility profiles to infection to by Schistosoma mansoni

Algumas espécies do gênero Biomphalaria se apresentam como potenciais hospedeiras ao parasito Schistosoma mansoni, estando a suscetibilidade a este parasito, neste gênero, ligada ao sistema interno de defesa de cada espécie de Biomphalaria. Um dos componentes importantes no sistema imune de invertebrados é a enzima fenoloxidase, que ainda apresenta muitos aspectos desconhecidos no sistema de defesa do gênero Biomphalaria. Foi relatado também que os genes de proteínas relacionadas ao fibrinogênio (FREPs) possuem importância na resposta imune de Biomphalaria glabrata, entre esses, as subfamílias dos FREPs 3 e 4 são diferencialmente expressas em linhagens susceptíveis e resistentes frente a infecção com trematódeos. No entanto os trabalhos existentes em sua maioria estudam a espécie Biomphalaria glabrata, excluindo a espécie Biomphalaria straminea, amplamente distribuída no Brasil e principal responsável pela disseminação da esquistossomose. Tendo em vista a falta de conhecimento sobre a resposta imune destes moluscos hospedeiros, principalmente em relação à expressão de genes imune relevantes e ao tipo de resposta, o presente trabalho se propôs a estudar a variação do número de hemócitos, da produção de fenoloxidase e da expressão dos genes dos FREP 3 e FREP 4 envolvidos com a ligação a antígenos de trematódeos, nas espécies Biomphalaria glabrata, Biomphalaria straminea pós-infecção com S. mansoni, bem como em caramujos pré-expostos a antígenos de S. mansoni...

Nutritional repercussions in patients submitted to bariatric surgery / Repercussões nutricionais em pacientes submetidos à cirurgia bariátrica

BACKGROUND: Few studies evaluated the association between nutritional disorders, quality of life and weight loss in patients undergoing bariatric surgery. AIM: To identify nutritional changes in patients undergoing bariatric surgery and correlate them with weight loss, control of comorbidities and quality of life. METHOD: A prospective cohort, analytical and descriptive study involving 59 patients undergoing bariatric surgery was done. Data were collected preoperatively at three and six months postoperatively, evaluating nutritional aspects and outcomes using BAROS questionnaire. The data had a confidence interval of 95%. RESULTS: The majority of patients was composed of women, 47 (79.7%), with 55.9% of the series with BMI between 40 to 49.9 kg/m². In the sixth month after surgery scores of quality of life were significantly higher than preoperatively (p<0.05) and 27 (67.5 %) patients had comorbidities resolved, 48 (81.3 %) presented BAROS scores of very good or excellent. After three and six months of surgery 16 and 23 presented some nutritional disorder, respectively. There was no relationship between the loss of excess weight and quality of life among patients with or without nutritional disorders. CONCLUSION: Nutritional disorders are uncommon in the early postoperative period and, when present, have little or no influence on quality of life and loss of excess weight. .

RACIONAL: Poucos estudos avaliam a associação entre distúrbios nutricionais, qualidade de vida e perda de peso em pacientes submetidos à cirurgia bariátrica. OBJETIVO: Identificar alterações nutricionais em pacientes submetidos à cirurgia bariátrica e correlacioná-las com perda de peso, controle de comorbidades e qualidade de vida. MÉTODO: Estudo de coorte, prospectivo, analítico e descritivo envolvendo 59 pacientes submetidos à cirurgia bariátrica. Os dados foram coletados no pré-operatório e aos três e seis meses pós- operatórios, quantificando aspectos nutricionais e utilizando o Bariatric Analysis and Reporting Outcomes System (BAROS) como ferramenta de sucesso. Os dados usaram intervalo de confiança de 95%. RESULTADOS: O total de mulheres foi 47 (79,7%), sendo 55,9% com IMC entre 40-49,9 kg/m². No sexto mês depois da operação os escores de qualidade de vida foram significativamente maiores do que no pré-operatório (p<0,05) e 27 (67,5%) pacientes tinham todas comorbidades resolvidas, 48 (81,3%) apresentaram conceito BAROS muito bom ou excelente. Após três e seis meses 16 e 23 pacientes apresentaram algum distúrbio nutricional, respectivamente. Não houve relação entre a perda do excesso de peso e qualidade de vida entre pacientes com ou sem distúrbio nutricional. CONCLUSÃO: os distúrbios nutricionais são pouco frequentes no pós-operatório precoce e, quando presentes, têm pouca ou nenhuma influência na qualidade de vida e na perda do excesso de peso. .

Mycobacterium tuberculosis epitope-specific interferon-g production in healthy Brazilians reactive and non-reactive to tuberculin skin test

The interferon (IFN)-γ response to peptides can be a useful diagnostic marker of Mycobacterium tuberculosis (MTB) latent infection. We identified promiscuous and potentially protective CD4+ T-cell epitopes from the most conserved regions of MTB antigenic proteins by scanning the MTB antigenic proteins GroEL2, phosphate-binding protein 1 precursor and 19 kDa antigen with the TEPITOPE algorithm. Seven peptide sequences predicted to bind to multiple human leukocyte antigen (HLA)-DR molecules were synthesised and tested with IFN-γ enzyme-linked immunospot (ELISPOT) assays using peripheral blood mononuclear cells (PBMCs) from 16 Mantoux tuberculin skin test (TST)-positive and 16 TST-negative healthy donors. Eighty-eight percent of TST-positive donors responded to at least one of the peptides, compared to 25% of TST-negative donors. Each individual peptide induced IFN-γ production by PBMCs from at least 31% of the TST-positive donors. The magnitude of the response against all peptides was 182 ± 230 x 106 IFN-γ spot forming cells (SFC) among TST-positive donors and 36 ± 62 x 106 SFC among TST-negative donors (p = 0.007). The response to GroEL2 (463-477) was only observed in the TST-positive group. This combination of novel MTB CD4 T-cell epitopes should be tested in a larger cohort of individuals with latent tuberculosis (TB) to evaluate its potential to diagnose latent TB and it may be included in ELISPOT-based IFN-γ assays to identify individuals with this condition.

Production and evaluation of chicken antibodies against a synthetic peptide from glial growth factor / Producción y evaluación de anticuerpos de gallinas contra un péptido sintético del factor de crecimiento glial

Neuregulins (NRG) are proteins that belong to the family of epidermal growth factors. It is well established that these factors are essential for the development and maintenance of the nervous system. Due to the difficulty of purifying enough quantities of these factors and the lack of specificity from commercially available antibodies, the aim of this work was to produce antibodies against a synthetic peptide capable to detect and identify neuregulin GGFb isoforms. To accomplish this goal, polyclonal antibodies were raised in hens against a synthetic peptide designed from the GGFb1 extracellular sequence. The sequence analysis was made using different epitope-predicting programs. Our results showed that the peptide sequence selected was immunogenic because it was capable of inducing a specific type B immune response in the experimental animal model. These antibodies were also capable of recognizing a recombinant GGF protein and GGF isoforms present in different samples. Our results suggest that the development of immunoglobulin Y (IgY) using synthetic peptides represents, a valuable tool for neuroscience research.

Las Neuregulinas (NRG) son proteínas que pertenecen a la familia de los factores de crecimiento epidermal. Se ha demostrado que estos factores son esenciales para el desarrollo y mantenimiento de la funcionalidad del sistema nervioso. Debido a la dificultad para purificar estas proteínas y la falta de especificidad de los anticuerpos disponibles comercialmente, el objetivo de este trabajo fue producir anticuerpos contra un péptido sintético capaz de detectar e identificar una isoforma de la Neuregulina (GGFb). Para lograr este objetivo, se desarrollaron anticuerpos en gallinas (IgY) contra un péptido sintético diseñado a partir de la secuencia aminoacídica de la región extracelular de GGFb, utilizando programas de predicción de epítopes. Los resultados demuestran que el péptido seleccionado fue immunogénico debido a que estimuló una respuesta inmune específica tipo B en el modelo utilizado. Estos anticuerpos fueron también capaces de reconocer una proteína recombinante e isoformas de GGF presentes en diferentes muestras biológicas. Nuestros resultados demuestran el potencial valor de las inmunoglobulinas Y (IgY) contra péptidos sintéticos como una herramienta de aplicación para la investigación en neurociencia.

Mapeo computacional de epítopos de células B presentes en el virus del dengue / B-cell epitopes mapping of dengue virus

Dengue es una infección viral de importancia en salud pública. El desarrollo de una vacuna va a depender del conocimiento de aquello epítopos que neutralicen la infección y por ello, se generaron e identificaron computacionalmente aquellos epítopos lineales consenso de células B presentes en el virus del dengue, a partir de los datos depositados en la base de datos Immune Epitope Database (IEDB). Posteriormente, se agruparon en bloques de ocho aminoácidos por cada serotipo mediante el programa Nomad, y cada uno de ellos fue reevaluado con el programa BepiPred para determinar su antigenicidad. Se lograron postular 172 de los 1.239 obtenidos en la IEDB.

Dengue virus has emerged as a major public health problem. An epitope-based approach to the development of vaccines, and for this reason, based on Immune Epitope Database database (IEDB), linear consensus B-cell epitopes were computationally generated and identified from dengue virus. Then, amino acids were grouped into blocks of eight amino acids per serotype through Nomad program. Each block was evaluated using BepiPred for determining antigenic prediction. 172 out of 1239 were obtained from IEDB.