O efeito do tratamento com rapamicina em órgãos linfoides de camundongos propensos à senescência acelerada / The effect of rapamycin treatment on lymphoid organs of senescence accelerated mice prone

Envelhecer compreende um fenômeno complexo, natural e irreversível, que submete o organismo a inúmeras alterações nos processos biológicos, fisiológicos, ambientais, psicológicos, comportamentais e sociais. Esse processo é caracterizado por um declínio gradual dos mecanismos homeostáticos do organismo, intimamente relacionados com o estado senescente. A senescência, quando diz respeito ao sistema imunológico, é denominada de imunossenescência, que pode ser definida como uma parada estável do ciclo celular associada a mudanças, com uma resposta que limita a proliferação de células envelhecidas ou danificadas. A autofagia está diretamente relacionada com a manutenção do fenótipo senescente, em que a atividade autofágica exerce um papel essencial e ativo na influência da biossíntese de proteínas e organelas. Essa via é regulada naturalmente pela proteína mTOR e quimicamente pelo fármaco rapamicina. Assim, pretendemos investigar: (1) as alterações no perfil corporal e hematimêtrico dos animais ao longo do tratamento com rapamicina; (2) avaliar o perfil de citocinas; (3) observar as modificações histológicas em órgãos linfoides primários e secundário; (4) analisar as populações de células linfoides e mieloides; e (5) avaliar a capacidade proliferativa de linfócitos in vitro. Camundongos SAMP-8 e SAMR-1 foram tratados com rapamicina durante dois meses. A mensuração da massa corporal e análises hematológicas foram realizadas antes e durante o tratamento. Amostras de soro, medula óssea, timo e baço foram analisados em ensaios de ELISA, histologia, população e subpopulações de células. Alterações na massa corporal, parâmetros hematológicos e celularidade de células foram nítidas entre os dois modelos utilizados. Diferenças também foram percebidas na detecção de citocinas IL-1ß. IL-6 e TNF-α, com resultados significantes nas amostras de baço, timo e medula óssea. As citocinas IL-7 e IL-15 apresentaram diferenças de secreção entre os grupos, sendo a primeira maior detectada em camundongos com senescência acelerada tratados com rapamicina. Em nossa análise histológica observamos que os camundongos SAM-P8 apresentaram involução tímica. E nas subpopulações de linfócitos T do baço, células TCD4+ e TCD8+ estavam, respectivamente, em maior e menor quantidade nos camundongos SAM-P8 tratados com rapamicina. Dessa forma, o camundongo da linhagem SAM-P8 é um excelente modelo para se estudar as alterações da senescência, em que o mesmo apresenta características fisiológicas distintas dos camundongos utilizados como controle (SAM-R1). Além disso, verificamos que a dose de rapamicina empregada não desencadeou alterações que pudessem comprometer a resposta imunológica desses camundongos, bem como na possibilidade de atuar na resposta contra os efeitos complexos do envelhecimento

Aging comprises a complex, natural, and irreversible phenomenon, which subjects the organism to countless alterations in biological, physiological, environmental, psychological, behavioral, and social processes. This process is characterized by a gradual decline in the organism's homeostatic mechanisms, closely related to senescence effects. Senescence, when it concerns the immune system, is called immunosenescence, which can be defined as a stable cell cycle arrest associated with changes and is a response that limits the proliferation of aged or damaged cells. Autophagy is a genetically regulated, conserved cellular process and a metabolic pathway essential for maintaining cellular homeostasis, which plays a constitutive and active role in controlling the biosynthesis of proteins and organelles. This pathway is regulated naturally by mTOR or chemically by the drug rapamycin, having a direct relationship with cellular homeostasis and maintenance of the senescent phenotype. Thus, we intend to investigate: (1) the changes in the body and hematimetic profile of the animals throughout the rapamycin treatment; (2) evaluate the cytokine profile; (3) observe histological changes in primary and secondary lymphoid organs; (4) analyze lymphoid and myeloid cell populations; and (5) evaluate the proliferative capacity of lymphocytes in vitro. SAMP-8 and SAMR-1 mice were treated with rapamycin for two months. Body mass measurement and hematological analyses were performed before and during treatment. Serum, bone marrow, thymus and spleen samples were analyzed in ELISA assays, histology, cell population and subpopulations. Changes in body mass, hematological parameters, and cellularity were clear between the two models used. Differences were also noticed in the detection of cytokines IL-1ß. IL-6 and TNF-α, with significant results in the spleen, thymus and bone marrow samples. The cytokines IL-7 and IL-15 showed differences in secretion between groups, the former being higher detected in mice with accelerated senescence treated with rapamycin. In our histological analysis we observed that SAM-P8 mice showed thymic involution. And in the spleen T-lymphocyte subpopulations, TCD4+ and TCD8+ cells were, respectively, in higher and lower quantities in SAM-P8 mice treated with rapamycin. Thus, the SAM-P8 mouse is an excellent model to study the changes of senescence, since it presents physiological characteristics different from the control mice (SAM-R1). Furthermore, we verified that the dose of rapamycin used did not trigger changes that could compromise the immune response of these mice, as well as the possibility of acting in the modulatory response against the complex effects of aging

Protective effects of Baicalin injection on severe acute pancreatitis through regulating follistatin-like-1 signaling pathway by down-regulating miR-429 expression in mice

Abstract In China, Scutellaria is used for treating inflammatory-related diseases. Baicalin is the main active component of Scutellaria and has protective effects on acute pancreatitis. However, the mechanism of Baicalin is still unclear. In this study, the protective effects of baicalin on acute pancreatitis induced by taurocholate and its mechanism are investigated. In this study, mice were randomly divided into three groups: sham operation, model, and treatment groups. Acute pancreatitis in mice was induced by intraperitoneal injection of taurocholate (35 mg/kg). The treatment group was given baicalin (100 mg/kg) 2 h before acute pancreatitis induction. The mRNA expression levels of miR-429, nuclear factor kappa B65(NF-kB65), toll-like receptor 4(TLR4), TNF receptor associated factor6 (TRAF6), NF-kappa-B inhibitor(IkB), Follistatin-like 1 (FSTL1), and interleukin-1 receptor-associated kinase (IRAK) in the liver tissues 24 h after intraperitoneal injection were detected by RT-PCR. Then, the expression levels of NF-kB65, p-NF-κB65, TLR4, TRAF6, IkB, FSTL1, IRAK, p- IRAK, and p- IkB-а proteins were detected by Western blot. IL-6, TNF-α and IL-1 ß in plasma were measured by ELISA, and histopathological changes in the pancreases of the mice were observed. The results showed that after baicalin treatment, miR-429 expression in the pancreatic tissues and the expression levels of NF-kB65, TLR4, TRAF6, p-IkB-а, FSTL1, and p-IRAK decreased. Similarly, pancreatic myeloperoxidase (MPO) activity and the plasma levels of IL-6, TNF-а, IL-12, IL-1ß1, endotoxin, serum amylase, and lipase were reduced. Thus, the pancreatic injury induced by taurocholate was alleviated. The present study indicates that pretreatment with Baicalin can alleviate acute pancreatic injury induced by taurocholate in mice. The mechanism may be associated with the decreased miR-429 expression, reduced FSTL1 signaling pathway activity, TLR4 and TLR4/MyD88 signaling pathway inhibition, and reduced pancreatic inflammation. FSTL1 is the regulatory target for miR-429

Evaluation of miR-15a, miR-16-1, ZAP-70, Ang-2, and Bcl-2 as potential prognostic biomarkers in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a blood cancer characterized by the accumulation of clonal B-lymphocytes. This study evaluated the mRNA gene expression of miR-15a, miR-16- 1, ZAP-70, and Ang-2 by qPCR, as well as the plasma levels of Bcl-2 by Elisa immunoassay, in CLL patients and healthy controls. Significant differences were observed when comparing patients and controls regarding miR-15a (p < 0.001), miR-16-1 (p < 0.001) mRNA, Ang-2 gene expression, and Bcl-2 plasma levels (p < 0.001). When stratified by risk, differences were maintained with a significantly reduced expression in high-risk patients. A positive correlation was observed between miR-15a and platelets (R2 = 0.340; p = 0.009) as well as between Bcl-2 and leukocytes (R2 = 0.310; p = 0.019). Conversely, negative correlations were observed between ZAP-70 and platelets (R2 = - 0.334; p = 0.011), between miR-15a and lymphocytes (R2 = - 0.376; p = 0.004), as well as between miR-16-and lymphocytes (R2 = - 0.515; p = 0.00004). The data suggest that a reduction in miR-15a and miR-16-1 expressions, in addition to an overexpression of Bcl-2, are associated with the reduction in apoptosis and, consequently, to a longer survival of lymphocytes, thus contributing to lymphocyte accumulation and aggravation of the disease. By contrast, Ang-2 expression was significantly higher in A than in B + C Binet groups. This context leads to the speculation that this biomarker should be investigated in more robust studies within populations with a still relevantly indolent form of the disease in an attempt to identify those patients with a greater potential for an aggravation of the disease

Avaliação da resposta de anticorpos IgG contra diferentes variantes alélicas do Antígeno 1 de Membrana Apical (AMA-1) de Plasmodium vivax em indivíduos de áreas endêmicas de malária / Evaluation of IgG antibody response against different variants of Plasmodium vivax apical membrane antigen 1 (AMA-1) in individuals from malaria endemic areas

O Plasmodium vivax é a espécie com maior distribuição geográfica no mundo e a que predomina nas Américas, incluindo o Brasil. Comparado ao Plasmodium falciparum, poucas vacinas contra o P. vivax encontram-se em fase de testes clínicos. Um dos antígenos de formas sanguíneas de P. vivax candidato a vacina é o Antígeno 1 de Membrana Apical (PvAMA-1). Entretanto, a diversidade antigênica do mesmo na natureza representa um grande desafio para seu uso no desenvolvimento de uma vacina de ampla cobertura. No presente estudo, avaliamos se os polimorfismos de sequências já descritos são capazes de influenciar na eficácia de uma vacina baseada em PvAMA-1. Para isso, geramos 9 proteínas recombinantes a partir da levedura Pichia pastoris, as quais são representativas de diferentes variantes alélicas do antígeno PvAMA-1, a saber: Belem, Chesson I, Sal-1, Indonesia XIX, SK0814, TC103, PNG_05_ESP, PNG_62_MU e PNG_68_MAS. Após expressão e purificação das proteínas selecionadas, avaliamos comparativamente por ELISA a resposta de anticorpos IgG naturalmente adquiridos em indivíduos expostos a malária, procedentes da Região Amazônica. Todas as proteínas foram obtidas com rendimento e pureza apropriados para os estudos propostos. A prevalência total de indivíduos expostos a malária com anticorpos contra PvAMA-1 Belem foi de 53,68%, em 611 amostras de soro testadas. Entre 100 das amostras sorologicamente positivas para PvAMA-1 Belem, os maiores valores de DO492 foram obtidos para as variantes Chesson I, SK0814 e Sal-1, sugerindo que epítopos comuns ou de reatividade cruzada estão sendo reconhecidos nessas variantes. Por outro lado, níveis mais baixos de DO492 foram obtidos para as variantes Indonesia XIX, TC103, PNG_05_ESP, PNG_62_MU e PNG_68_MAS, o que pode significar que essas variantes são menos prevalentes ou não circulam no Brasil. Soros policlonais de camundongos C57BL/6 previamente imunizados com PvAMA-1 Belem foram testados quanto ao reconhecimento das diferentes variantes por ELISA. Nossos resultados demonstraram que as variantes Chesson I, Indonesia XIX, SK0814, Sal-1 e a proteína homóloga foram predominantemente reconhecidas. Por fim, ensaios de competição baseados em ELISA revelaram que as proteínas Chesson I, Indonesia XIX, SK0814 e Sal-1, na fase solúvel, foram capazes de inibir a ligação de anticorpos à variante Belem aderida a placa, sugerindo a presença de epítopos comuns ou de reatividade cruzada entre as mesmas. Nossos dados sugerem que uma vacina baseada na variante PvAMA-1 Belem gera anticorpos variante-transcendentes. Entretanto, para gerar uma vacina universal baseada em PvAMA-1, uma formulação multi-alélica, incluindo variantes da Tailândia e Papua Nova Guiné, deverão ser testadas

Plasmodium vivax has the largest geographical distribution Plasmodium species in the world, and is predominant in the Americas, including Brazil. Fewer P. vivax vaccines than P. falciparum vaccines have successfully reached clinical trials. One of the candidate antigens for a blood-stage P. vivax vaccine is the apical membrane antigen 1 (PvAMA-1). However, the high natural variability found in this antigen presents a major challenge for its development into a wide-range vaccine. In the present study, we evaluated whether sequence polymorphisms would influence a vaccine based on PvAMA-1. To achieve this, we generated 9 recombinant proteins from the yeast Pichia pastoris, representative of different allelic variants of the PvAMA-1 antigen: Belem, Chesson I, Sal-1, Indonesia XIX, SK0814, TC103, PNG_05_ESP, PNG_62_MU, and PNG_68_MAS. After expression and purification of these proteins, we compared, by ELISA and IgG blocking, the natural acquired response from malaria-exposed individuals in the Amazon Region. All proteins selected had the appropriate yield and purity for the proposed studies. The total prevalence of malaria-exposed individuals with reactivity to PvAMA-1 Belem was 53,68%, from 611 serum samples tested. One hundred of these serologically positive samples were further tested against recombinant proteins representing the other allelic variants. The highest OD values resulted from Sal-1, Chesson I and SK0814 variants, suggesting that common epitopes or cross-reactivity exist across the variants. On the other hand, the lowest OD values resulted from the variants Indonesia XIX, TC103, PNG_05_ESP, PNG_62_MU, and PNG_68_MAS, which may mean these variants are less prevalent or do not circulate in Brazil. Polyclonal sera from C57BL/6 mice immunized with PvAMA-1 Belem were tested for recognition of different variants by ELISA. Our results showed that the variants Chesson I, Sal-1, Indonesia XIX, SK0814 and the homologous protein were predominantly recognized. Lastly, ELISA-based competition assays revealed that Chesson I, Sal-1, Indonesia XIX and SK0814 proteins were able to inhibit antibody binding to the Belem variant, suggesting the presence of common epitopes or cross-reactivity between these variants. Our data suggest that a vaccine based on the PvAMA-1 Belem variant displays strain-transcendent antibodies. However, to generate a universal vaccine based on PvAMA-1, a multiallelic formulation including variants from Thailand and Papua New Guinea must be tested

Buchholzia coriacea (wonderful kola) seeds induce male reproductive toxicity by suppressing the pituitary-gonadal axis in Wistar rats

The methanolic extract of Buchholzia coriacea seeds (MEBC) has been reported to induce male reproductive toxicity by decreasing sperm parameters and fertility index. To elucidate the possible mechanism(s), the effects of graded doses of MEBC on sex hormones and sperm profile were investigated in this study. The MEBC (e.g., 50, 200, 400, and 600 mg/kg) was administered daily (p.o.) to male Wistar rats for 6 weeks, while a concurrent control group received distilled water (vehicle). Then, the animals were sacrificed under sodium pentobarbital anaesthesia. Weights of organs were recorded, and the sperm profile was determined microscopically. Testosterone, luteinizing hormone (LH), and follicle stimulating hormone (FSH) were assayed from the obtained serum using the ELISA technique. Sperm motility was significantly reduced by MEBC (i.e., 50 and 200 mg/kg), and sperm count reduced in all treated groups in a dose-dependent manner compared with that of the control. Serum testosterone, LH, and FSH decreased in treated rats. A histopathological examination of testes showed a considerable depletion and necrosis of the epithelium of seminiferous tubules. The result suggests that Buchholzia coriacea seeds induce male reproductive toxicity by suppressing the pituitary-gonadal axis.

A Review on COVID-19 Diagnosis Tests Approved for Use in Brazil and the Impact on Pandemic Control

Abstract With the COVID-19 pandemic, many diagnostic tests (molecular or immunological) were rapidly standardised, given the urgency of the situation, many are still in the process of being validated. The main objective of this study was to review the aspects of the diagnostic kits approved in Brazil and their application in the different federative units to gather epidemiological information. In order to achieve these objectives, a survey was carried out on the data available at the regulatory agency (ANVISA) and in the literature. The main countries that have registered products in Brazil are China (51.4%), Brazil (16.6%), South Korea (9.2%), USA (8.8%) and Germany (3.6%). The methodologies of these products are based on the detection of nucleic-acid (15.8%), antigen (13%) and antibody (71.2%). In the immunological tests, it was verified that the sensitivity ranged from 55 to 100% and the specificity from 80 to 100%. The percentage of cases in the samples tested in Brazil is elevated in almost all federative units since eight states showed 40% of positive cases in tested samples, while 18 states displayed between 20 and 40%. In conclusion, this review showed that Brazil is dependent on external technology to respond to pandemics, epidemics and endemics disease and needs to improve its biotechnological scheme to solve further diseases outbreaks.

Acurácia dos testes diagnósticos registrados na ANVISA para a COVID-19 / Acurácia dos testes diagnósticos registrados na ANVISA para a COVID-19

INTRODUÇÃO: O coronavírus da Síndrome Respiratória Aguda Grave 2 (abreviado para SARSCoV-2, do inglês Severe Acute Respiratory Syndrome Coronavirus 2), anteriormente conhecido como novo coronavírus (2019-nCoV), é um agente zoonótico recémemergente que surgiu em dezembro de 2019, em Wuhan, China, causando manifestações respiratórias, digestivas e sistêmicas, que se articulam no quadro clínico da doença denominada COVID-19 (do inglês Coronavirus Disease 2019). Ainda não há informações robustas sobre a história natural da doença, tampouco sobre as medidas de efetividade para o manejo clínico dos casos de infecção pelo COVID19, restando ainda muitos detalhes a serem esclarecidos. No entanto, sabe-se que o vírus tem alta transmissibilidade e provoca uma síndrome respiratória aguda que varia de casos leves ­ cerca de 80% ­ a casos muito graves com insuficiência respiratória - entre 5% e 10% dos casos ­, os quais requerem tratamento especializado em unidades de terapia intensiva (UTI). Sua letalidade varia, principalmente, conforme a faixa etária. TECNOLOGIA: Os testes de diagnóstico para a COVID-19 se destacaram na pandemia de coronavírus em andamento como uma ferramenta essencial para rastrear a propagação da doença. Uma ampla gama de testes diagnósticos para o SARS-CoV-2 está disponível comercialmente, alguns dos quais receberam autorizações para uso por várias agências reguladoras nacionais. Com as informações da sequência genética devidamente identificadas, os testes de diagnóstico baseados na detecção da sequência viral por reação em cadeia da polimerase com transcriptase reversa (RT-PCR) ou plataformas de sequenciamento logo se tornaram disponíveis. Isso permitiu a confirmação do diagnóstico e melhores estimativas da atividade da infecção, que vêm aumentando em velocidades alarmantes. Para a detecção mais sensível de SARS-CoV, MERS-CoV e SARS-CoV-2, recomendavam-se a coleta e o teste de amostras respiratórias superiores e inferiores. O diagnóstico de casos suspeitos era confirmado por testes de RNA com RT-PCR em tempo real ou sequenciamento de próxima geração. Foi demonstrado que o RNA viral poderia ser detectado a partir do swab nasal e faríngeo, lavagem broncoalveolar e plasma sanguíneo usando RT-PCR direcionado ao gene do vírus (5). O padrão-ouro para diagnóstico laboratorial da COVID-19 é a reação da transcriptase reversa, seguida de reação em cadeia da polimerase (RT-PCR) para amostras coletadas no trato respiratório superior ou inferior. OBJETIVO: O objetivo deste relatório é analisar a acurácia dos testes diagnósticos registrados na Agência Nacional de Vigilância Sanitária (ANVISA) até a presente data. METODOLOGIA: foi realizada uma busca por diagnósticos para COVID-19 com registros vigentes na ANVISA. Para tal, foram utilizados os termos "COVID 19", SARS e coronavírus no campo de consulta de registro de produtos para saúde no site da Agência (https://consultas.anvisa.gov.br/#/saude/). Os passos para acesso ao banco de dados de produtos diagnósticos na ANVISA são: 1) consulta produtos; 2) consulta a banco de dados; 3) produtos para a saúde e 4) pesquisa de produtos para a saúde registrados. CONCLUSÕES: A ANVISA já avaliou mais de 120 pedidos de registro de produtos para testagens relacionadas à COVID-19 desde o dia 18 de março. A maior parte das petições aguarda complementação de informações por parte das empresas e outras estão sendo analisadas com prioridade. O tempo médio para avaliação dos registros na ANVISA gira em torno de 15 dias. Atualmente, mais da metade dos registros concedidos diz respeito a testes rápidos para anticorpos. Até a presente data, foram registrados 64 testes para diagnóstico da COVID-19, sendo 15 deles moleculares. O teste de polymerase chain reaction em tempo real (RT-PCR) para identificação de SARS-CoV-2 é um teste de elevada sensibilidade e especificidade, ainda que os doentes com maior carga viral possam ter maior probabilidade de um teste positivo. Os testes moleculares baseados em RNA exigem instalações laboratoriais específicas com níveis restritos de biossegurança e técnica. A sensibilidade e especificidade dos testes sorológicos variaram entre os fabricantes. É importante destacar que uma baixa sensibilidade do teste diagnóstico pode resultar em uma maior probabilidade de detectar falsos-negativos, o que poderia interferir principalmente em casos de indivíduos assintomáticos. Em geral, a sensibilidade dos testes foi superior a 85% e a especificidade, superior a 94%. Os testes sorológicos medem a quantidade de dois anticorpos (IgG e IgM) que o organismo produz quando entra em contato com um invasor. Contudo, o desenvolvimento da resposta de um anticorpo à infecção pode ser dependente do hospedeiro e levar tempo. No caso de SARS-CoV-2, estudos iniciais sugerem que a maioria dos pacientes se converte entre 7 e 11 dias após a exposição ao vírus, embora alguns pacientes possam desenvolver anticorpos mais cedo. Devido a esse atraso natural, o teste de anticorpos pode não ser útil no cenário de uma doença aguda (11). Os testes de anticorpos para SARS-CoV-2 podem facilitar (i) o rastreamento de contatos (os testes baseados em RNA também podem ajudar); (ii) a vigilância sorológica nos níveis local, regional, estadual e nacional; e (iii) a identificação de quem já teve contato com o vírus e, portanto, pode (se houver imunidade protetora) ser imune (11,12). Alguns conjuntos de reagentes para testes sorológicos foram autorizados pela ANVISA em caráter emergencial devido à gravidade da situação e à necessidade de ampliar a testagem da população, mas a validação desses reagentes pelos laboratórios é fundamental, uma vez que poucos trabalhos conseguiram ser publicados até o momento. As aprovações estão de acordo com a Resolução da Diretoria Colegiada (RDC) 348/2020, que define os critérios e os procedimentos extraordinários e temporários para tratamento de petições de registro de medicamentos, produtos biológicos e produtos para diagnóstico in vitro, e mudança pós-registro de medicamentos e produtos biológicos em virtude da emergência de saúde pública internacional decorrente do novo coronavírus. Na RDC, para registro de testes diagnósticos, a ausência de qualquer estudo de desempenho ou restrição de dados deve ser justificada por motivações técnicas que permitam a avalição da confiabilidade dos resultados e da efetividade diagnóstica do produto. Os registros concedidos nas condições dessa Resolução terão a validade de um ano, exceto para situações em que a avaliação da estabilidade seja apresentada por comparação com produtos similares e os demais critérios descritos no Regulamento sejam atendidos. Nesse caso, poderão ter a concessão regular de validade de registro de produtos para saúde por um período de 10 anos. Em resumo, as duas categorias de testes para SARS-CoV-2 podem ser úteis nesse surto, pois, eventualmente, a coleta de múltiplas amostras, regiões e em tempos diferentes durante a evolução da doença pode ser necessária para o diagnóstico da COVID-19.

The diagnostic benefit of antibodies against ribosomal proteins in systemic lupus erythematosus

Abstract Background Anti-ribosomal P (anti-Rib-P) antibody is a specific serological marker for systemic lupus erythematosus (SLE) and routinely tested by targeting the common epitope of three ribosomal proteins of P0, P1 and P2. This study aimed to investigate if testing antibodies against individual ribosomal protein, but not the common epitope, is required to achieve the best diagnostic benefit in SLE. Methods The study included 82 patients with SLE and 22 healthy donors. Serum antibodies were determined by ELISA and immunoblot. Results The prevalence of each antibody determined by ELISA was 35.4% (anti-Rib-P), 45.1% (anti-Rib-P0), 32.9% (anti-Rib-P1) and 40.2% (anti-Rib-P2) at 99% specificity, respectively. Of 53 patients with negative anti-Rib-P antibody, 21 (39.6%) were positive for anti-Rib-P0, 9 (17.0%) for anti-Rib-P1 and 12 (22.6%) for anti-Rib-P2 antibody. The positive rate of anti-Rib-P antibody detected by ELISA was close to the results by immunoblot (33.4%). Patients with any of these antibodies were featured by higher disease activity and prevalence of skin rashes than those with negative antibodies. Moreover, each antibody was particularly related to some clinical and laboratory disorders. The distribution of subclasses of IgG1-4 was varied with each antibody. Anti-Rib-P0 IgG1 and IgG3 were strongly correlated with disease activity and lower serum complement components 3 and 4. Conclusions Anti-Rib-P antibody is not adequate to predict the existence of antibodies against ribosomal P0, P1 and P2 protein. The examination of antibodies against each ribosomal protein is required to achieve additional diagnostic benefit and to evaluate the association with clinical and serological disorders as well.(AU)

Preparation and in vitro characterization of monoclonal antibody ranibizumab conjugated magnetic nanoparticles for ocular drug delivery

Gold coated magnetite nanoparticles were prepared and coated with ranibizumab as an ocular drug delivery system. The surface morphologies of the nanoparticles were determined by Scanning Electron Microscopy (SEM). The size and surface charge were determined by using the dynamic light scattering (DLS) technique. Crystallographic properties of the gold coated Fe3O4 nanoparticles were recorded on X-ray diffractometer (XRD) the XRD pattern of nanoparticlees were shown to have uniqe Fe3O4 and gold peaks. Conjugation of ranibizumab onto nanoparticles was achieved using the physical adsorption method. The amount of ranibizumab on the surface of the nanoparticles was determined by thermogravimetric analysis (TGA). In the in vitro release studies performed using UV spectroscopy; it was found that almost 60% of antibodies were released within the first 30 minutes. Antibody activity after release studies was also proved with ELISA. Non-toxicity of gold coated Fe3O4 particles were proved with MTT. Results of the studies, showed that the antibody conjugated magnetic nanoparticle system could be a potential treatment system for ocular diseases.

Hesperidin inhibits the epithelial to mesenchymal transition induced by transforming growth factor-ß1 in A549 cells through Smad signaling in the cytoplasm

Hesperidin, a natural compound, suppresses the epithelial-to-mesenchymal transition through the TGF-ß1/Smad signaling pathway. However, studies on the detailed effects and mechanisms of hesperidin are rare. The present study showed that, for A549 alveolar epithelial cells, the anti-proliferative effects of hesperidin occurred in a dose-dependent manner, with an IC50= 216.8 µM at 48 h. TGF-ß1 was used to activate the Smad signaling pathway and induce the epithelial to mesenchymal transition in cells. Treatment with hesperidin or SB431542 was used for antagonism of Smad pathway activation. Hesperidin inhibited the increase in ɑ-SMA and Col1ɑ-1 and the decrease in E-cadherin in a dose-dependent manner from concentration of 20 µM to 60 µM, as assessed by both ELISA and Western blotting assays; however, there was no significant effect on cellular morphological alterations. Moreover, the Western blotting assay showed that, in the cytoplasm, hesperidin and SB431542 had no significant effect on the protein expression of Smad 2, 3, 4, or 7 as well as 2/3. However, 60 µM hesperidin and SB431542 significantly decreased p-Smad2/3 protein expression. From the above results, it is concluded that hesperidin can partly inhibit the epithelial to mesenchymal transition in human alveolar epithelial cells; the effect accounts for the blockage of the phosphorylation of Smad2/3 in the cytoplasm rather than a change in Smad protein production in the cytoplasm

Methotrexate use, not interleukin 33, is associated with lower carotid intima-media thickness in patients with rheumatoid arthritis

Abstract Background: Rheumatoid arthritis is a risk factor for early mortality due to cardiovascular disease. Interleukin-33 appears to protect against the development of atherosclerosis. The purpose of this study was to investigate the relationship between serum levels of interleukin-33 and its soluble receptor with the presence of subclinical carotid atherosclerosis in rheumatoid arthritis patients. Methods: Rheumatoid arthritis patients without atherosclerotic disease were subjected to clinical and laboratory assessments, including carotid ultrasound. Interleukin-33 and its soluble receptor serum levels were measured by ELISA. Results: 102 patients were included. The prevalence of carotid plaques was 23.5% and the median intima-media thickness was 0.7 mm. The median interleukin-33 and its soluble receptor concentration was 69.1 and 469.8 pg/ml. No association was found between serum interleukin-33 or its soluble receptor and intima-media thickness or plaque occurrence. Each 0.1 mm increase of intima-media thickness raised the odds of plaque occurrence by 5.3-fold, and each additional year of rheumatoid arthritis duration increased the odds of plaque occurrence by 6%. Each additional year in patients age and each one-point increase in the Framingham Risk Score were associated with a 0.004 mm and 0.012 mm increase in intima-media thickness. Methotrexate use was associated with a 0.07 mm reduction in intima-media thickness. Conclusions: Interleukin-33 and its soluble receptor were not associated with subclinical atherosclerosis. Traditional risk factors for atherosclerosis and rheumatoid arthritis duration were associated with intima-media thickness and plaque occurrence; methotrexate use was associated with a lower intima-media thickness.

Anti-collagen type v: a marker of early systemic sclerosis?

Abstract Objective: To evaluate the frequency of anti-collagen type V in humans with early systemic sclerosis (SSc) compared to defined SSc patients and healthy controls, since collagen type V was shown to be overexpressed in early SSc patients' skin and there is no data concerning the presence of this antibody in early stages of human SSc. Experimental studies showed that animal models immunized with collagen type V developed a disease similar to human systemic sclerosis (SSc), with antibodies production, mainly in early stages post-immunization. Methods: Eighty-one female SSc patients were included and divided into two groups: early-SSc (18 patients-EULAR Preliminary Criteria) and defined-SSc (63 patients-ACR Criteria 1980). The control group consisted of 19 healthy women age-matched to Early-SSc group. Anti-collagen type V was performed by ELISA. Data was analyzed by appropriate tests. Results: The prevalence of anti-collagen type V in early-SSc, defined-SSc and control groups was respectively 33, 17 and 5% (p = 0.07). SSc patients with anti-collagen type V had shorter disease duration compared to those without this antibody (8.8 ± 5.1 vs. 14.7 ± 8.9, p = 0.006). Likewise, early-SSc patients with anti-collagen V also had a shorter disease duration than patients negative for this antibody (4.6 ± 2.2 vs. 9.7 ± 5.2, p = 0.04). No association with clinical subsets or scleroderma antibodies specificities was observed (p > 0.05). Conclusion: The production of anti-collagen type V in SSc seems to be an early event independent of other antibodies specificities. Further studies are necessary to determine if the underlying mechanism for this chronology involves a primary immune response to abnormal expression of collagen type V.(AU)

Epidemiological, clinical and immune factors that influence the persistence of antiphospholipid antibodies in leprosy

Abstract Introduction: Antiphospholipid antibodies (aPL) are described in individuals with leprosy without the clinical features of antiphospholipid antibody syndrome (APS), a condition involving thromboembolic phenomena. We have described the persistence of these antibodies for over 5 years in patients with leprosy after specific treatment. Objectives: To determine whether epidemiological, clinical and immunological factors played a role in the longterm persistence of aPL antibodies in leprosy patients after multidrug therapy (MDT) had finished. Methods: The study sample consisted of 38 patients with a diagnosis of leprosy being followed up at the Dermatology and Venereology Outpatient Department at the Alfredo da Matta Foundation (FUAM) in Manaus, AM. ELISA was used to detect anticardiolipin (aCL) and anti-β2 glycoprotein I (anti-β2GPI) antibodies. Patients were reassessed on average of 5 years after specific treatment for the disease (MDT) had been completed. Results: Persistence of aPL antibodies among the 38 leprosy patients was 84% (32/38), and all had the IgM isotype. Mean age was 48.1 ± 15.9 years, and 23 (72.0%) were male. The lepromatous form (LL) of leprosy was the most common (n = 16, 50%). Reactional episodes were observed in three patients (9.4%). Eighteen (47.37%) were still taking medication (prednisone and/or thalidomide). Mean IgM levels were 64 U/mL for aCL and 62 U/mL for anti-β2GPI. In the multivariate binary logistic regression the following variables showed a significant association: age (p = 0.045, OR = 0.91 and CI 95% 0.82-0.98), LL clinical presention (p = 0.034; OR = 0.02 and CI 95% = 0.0-0.76) and bacterial index (p = 0.044; OR = 2.74 and CI 95% = 1.03-7.33). We did not find association between prednisone or thalidomide doses and positivity for aPL (p = 0.504 and p = 0.670, respectively). No differences in the variables vascular thrombosis, pregnancy morbidity, diabetes, smoking and alcoholism were found between aPL-positive and aPL-negative patients. Conclusion: Persistence of positivity for aPL antibodies was influenced by age, clinical presentation and bacterial index. However, further studies are needed to elucidate the reason for this persistence, the role played by aPL antibodies in the disease and the B cell lineages responsible for generation of these antibodies.

Development of an indirect ELISA based on recombinant capsid protein to detect antibodies to bovine leukemia virus

Abstract Serological testing and culling infected animals are key management practices aiming eradication of bovine leukemia virus infection. Here, we report the development of an indirect ELISA based on BLV recombinant capsid protein (BLVp24r) to detect anti-BLV antibodies in cattle serum. The BLVp24r was expressed in Escherichia coli and purified by affinity chromatography, and then used to set up the ELISA parameters. The Polysorp ® plate coated with 50 ng of antigen/well and bovine serum diluted 1:100 gave the best results during standardization. Using sera from infected and non-infected cattle we set up the cutoff point at 0.320 (OD450 nm) with a sensitivity of 98.5% and specificity of 100.0%. Then, we tested 1.187 serum samples from dairy (736 samples) and beef cattle (451 samples) with unknown status to BLV. We found that 31.1% (229/736) and 9.5% (43/451) of samples amongst dairy and beef cattle, respectively, had IgGs to BLV. The rate of agreement with a commercial competitive ELISA was 84.3% with a κ value of 0.68. Thus, our BLVp24r iELISA is suitable to detect BLV infected animals and should be a useful tool to control BLV infection in cattle.

Produção da proteína recombinante humana TGF-ß1 (fator do crescimento transformante beta 1) em células de mamífero / Production of recombinant human protein TGF-ß1 (Transforming Growth Factor Beta 1) in mammalian cells

O fator de crescimento transformante beta tipo 1, TGF-ß1, é uma proteína extracelular homodimérica secretada por vários tipos celulares, que pode ter ação parácrina ou endócrina. Essa proteína está envolvida em processos celulares de diferenciação, proliferação, mobilidade e formação de matriz extracelular. Além disso, é parte importante dos processos de regeneração tecidual, atuando, de maneira decisiva, no reparo, atraindo macrófagos e fibroblastos para o local da injúria e estimulando a angiogênese. Assim, considerando o papel desse peptídeo no processo regenerativo, o uso de TGF-ß1 como proteína terapêutica na área de Bioengenharia Tecidual é bastante promissor. Apesar disso, a venda dessa proteína, para fins terapêuticos, é inexistente no mercado e a proteína recombinante vendida, que só pode ser utilizada em pesquisas científicas, não é produzida nacionalmente e chega a custar R$200.000,00/mg. Nesse contexto, o objetivo do presente trabalho é desenvolver uma metodologia de produção do fator recombinante TGF-ß1 em células de ovário de hamster chinês (CHO), visando à obtenção de níveis altos de rendimento, e, futuramente, a transferência da tecnologia de produção para a iniciativa privada, tornando possível seu uso na Medicina Regenerativa, sozinho ou em combinação com outros fatores de crescimento. O cDNA de TGF-ß1 foi amplificado a partir de um banco de cDNA humano e clonado no vetor proprietário pNU1 de expressão de mamífero. A construção pNU1/TGF-ß1 foi utilizada para transfectar estavelmente células CHO DG44 e uma estratégia de co-amplificação foi utilizada para selecionar células transfectantes com maior número de cópias da sequência correspondente a TGF-ß1. Estas culturas foram submetidas ao processo de amplificação gênica com concentrações crescentes de metotrexato. Ensaios de Western Blot e ELISA foram realizados utilizando-se o meio condicionado pelas populações selecionadas e por clones superprodutores. Entre os 41clones obtidos, cinco apresentaram maiores níveis de produção de TGF-ß1, entre 1.000 e 2.000 ng/mL. Estes clones foram selecionados para a realização de testes de atividade in vitro utilizando-se células A549, que permitem avaliar a transição epitélio-mesênquima. Um ensaio de cicatrização de feridas em peles do dorso de camundongos foi padronizado e utilizado para avaliar a atividade in vivo do clone que apresentou melhor resultado in vitro. A proteína TGF-ß1 foi parcialmente purificada por HPLC em uma coluna de afinidade. Portanto, a proteína TGF-ß1 humana recombinante foi produzida, apresentando atividade biológica in vitro e in vivo, sendo capaz de reparar eficientemente feridas cutâneas. Essa iniciativa pode oferecer aos pacientes uma alternativa para o tratamento de lesões teciduais, acelerando a cicatrização de feridas e o reparo de tecidos

The transforming growth factor beta 1, TGF-ß1, is a homodimeric extracellular protein secreted by several cell types, which may have paracrine or endocrine action. This protein is involved in cellular processes of differentiation, proliferation, mobility and formation of extracellular matrix. In addition, it is an important part of the tissue regeneration processes, acting decisively on repair, attracting macrophages and fibroblasts to the site of injury and stimulating angiogenesis. Therefore, considering the role of this peptide in the regenerative process and the use of TGF-ß1 as a therapeutic protein in the field of Tissue Bioengineering is very promising. Despite this, the sale of this protein for therapeutic purposes is nonexistent in the market and the recombinant protein available in the market, which can only be used in scientific research, is not produced nationally and the costs are in the order of R$ 200,000.00/mg. In this context, the objective of the present work is to develop a methodology for the production of the TGF-ß1 recombinant factor in Chinese hamster ovary (CHO) cells, aiming at obtaining high yields, and, in the future, transfering the production technology to the private initiative, allowing its use in Regenerative Medicine, alone or in combination with other growth factors. The TGF-ß1 cDNA was amplified from a human cDNA library and cloned into the proprietary pNU1 mammalian expression vector. The pNU1/TGF-ß1 construct was used to stably transfect CHO DG44 cells, and a co-amplification strategy was used to select transfectant cells with the largest number of gene copies. These cultures were subjected to the process of gene amplification with methotrexate. Western Blot and ELISA were used to assay the conditioned medium obtained from the selected cell populations and from overproducing cell clones. Among the 41 clones obtained, five presented higher levels of TGF-ß1 production, between 1,000 and 2,000 ng/mL. These clones were selected for in vitro activity testing using A549 cells to evaluate the epithelial-mesenchymal transition. Awound healing assay on mouse dorsal skin was standardized and used to evaluate the in vivo activity of the cell clone which displayed the highest result in vitro. The TGF-ß1 protein was partially purified by HPLC on an affinity column. Therefore, the recombinant human TGF-ß1 protein was produced and shown to display biological activity both in vitro and in vivo, being able to eficiently repair cutaneous wounds. This initiative may provide patients with an alternative treatment for tissue damage, accelerating wound healing and tissue repair

Clinical markers associated with acute laboratory-confirmed Dengue infection: results of a national epidemiological surveillance system / Marcadores clínicos asociados con la infección de Dengue confirmada por laboratorio: resultados del sistema nacional de vigilancia epidemiológica

ABSTRACT Objective To evaluate the association of several clinical markers with acute laboratory-positive Dengue Virus infection. Methods A hospital-based case-control study was conducted in the state of Colima, Mexico, by using information from the National System of Epidemiological Surveillance (Sistema Nacional de Vigilancia Epidemiológica [SINAVE]) for Dengue. Data from 2 732 cases and 2 775 frequency-matched controls were analyzed. Odds Ratio (OR) and the 95 % Confidence Interval (CI), estimated by means of logistic regression models, were used. Results The presence of skin rash (OR=1,7; 95 % CI 1,5-2,1), persisting vomiting (OR=1,8; 95 % CI 1,5-2,3) and increased capillary fragility (petechiae, ecchymosis, hematoma or positive tourniquet test; OR=1,8; 95 % CI 1,2-2,6) were associated with laboratory-positive infection. Conclusions Three clinical markers were significantly associated with an increased risk of acute laboratory-confirmed dengue infection. These findings would support accurate and timely diagnosis of dengue in laboratory-limited settings.(AU)

RESUMEN Objetivo Evaluar la asociación de distintos marcadores clínicos con la infección por virus de Dengue, confirmada por laboratorio. Métodos Se condujo un estudio hospitalario de casos y controles, en el estado de Colima, México, usando información del Sistema Nacional de Vigilancia Epidemiológica (SINAVE) para dengue. Se analizó la información de 2 732 casos y 2 775 controles de frecuencia compatible/pareada. Se utilizó la Razón de Momios (RM) y el Intervalo de Confianza de 95 % (IC), estimado con modelos de regresión logística. Resultados La presencia de exantema (OR=1,7; 95 % CI 1,5-2,1), vómito persistente (OR=1,8; 95 % CI 1,5-2,3) y fragilidad capilar aumentada (petequias, equimosis, hematomas o prueba del torniquete positiva; OR=1,8; 95 % CI 1,2-2,6) se asociaron con la infección por dengue confirmada por laboratorio. Conclusiones Tres marcadores clínicos se asociaron significante con un riesgo incrementado de la infección aguda por dengue confirmada por laboratorio. Estos hallazgos pueden apoyar al preciso y oportuno diagnóstico de la infección en sitios con acceso limitado a laboratorios.(AU)

Detección de proteínas transgénicas en harinas de maíz comercializadas en Bogotá, Colombia / Detection of transgenic proteins in maize flour marketed in Bogotá, Colombia

Objetivo Detectar la presencia o ausencia de proteínas transgénicas derivadas de cultivos genéticamente modificados (GM) en harina de maíz comercializadas en Bogotá D.C., Colombia. Métodos Se evaluaron 11 protocolos de extracción de proteína total en 17 harinas pre-cocidas, dos no cocidas y tres controles positivos. Posteriormente, se determinó la presencia de 7 proteínas transgénicas (CP4EPSPS, Cry1Ab, Cry1Ac, Cry1F, Cry2A, Cry34Ab1 y Cry3Bb1) utilizando kits de ELISA comerciales. Resultados Se determinó que el mejor protocolo de extracción de proteína total fue el buffer con Tritón X-100, con el que se obtienen concentraciones de proteína mayores a 0,5 mg/g de harina y no genera interferencia con la técnica de ELISA. Se detectaron cuatro proteínas transgénicas: CP4EPSPS, Cry1F, Cry1Ab y Cry34Ab1 en harinas pre-cocidas y sin pre-cocción, con porcentajes que varían entre el 20 y 100 %. Conclusión Siete de las 19 harinas de maíz evaluadas contienen trazas de proteínas transgénicas (B2,B8,A3,O3,O1,C1 y C2), que confieren resistencia a lepidópteros o coleópteros y tolerancia al herbicida glifosato (CP4EPSPS, Cry1Ab, Cry1F, Cry34Ab1 y Cry3Bb1), todos los eventos detectados están aprobados para el consumo humano en Colombia según el Ministerio de Salud y Protección Social.(AU)

Objective To detect the presence or absence of transgenic proteins derived from GM crops in maize flour marketed in Bogota D.C., Colombia. Methods 11 extraction protocols for total protein were evaluated in 17 precooked flour, two uncooked and three positive controls. Subsequently, the presence of 7 transgenic proteins (CP4-EPSPS, Cry1Ab, Cry1Ac, Cry1F, Cry2A, Cry34Ab1 and Cry3Bb1) using commercial ELISA kits was determined. Results It was determined that the best protocol for total protein extraction was buffer with Triton X-100, which allowed obtaining protein concentrations greater than 0.5 mg per gram of flour and does not generate interference with the ELISA technique. Four transgenic proteins were detected: CP4EPSPS, Cry1F, Cry1Ab and Cry34Ab1 in precooked and uncooked flour with percentages varying between 20 and 100 %. Conclusion Seven of the 19 maize flours contain traces of transgenic protein (B2,B8,A3,O3,O1,C1 and C2) that provide resistance to lepidopterans and coleopterans, and tolerance to glyphosate herbicide, (CP4EPSPS- Cry1Ab, Cry1F, Cry34Ab1 and Cry3Bb1). All detected events are approved for human consumption in Colombia, according to the Ministry of Health and Social Protection.(AU)

Teste de ELISA indireto para diagnóstico sorológico de leishmaniose visceral em canídeos silvestres / Indirect ELISA for the serological diagnosis of visceral leishmaniasis in wild canids

Na América do Sul, alguns canídeos silvestres são considerados reservatórios naturais da Leishmania chagasi. A resposta imunológica desses animais à Leishmania é pouco conhecida, havendo a necessidade de métodos diagnósticos adequados para esse fim. No presente estudo, é descrita a padronização do ensaio imunoenzimático indireto (ELISA) para o diagnóstico sorológico de leishmaniose visceral em canídeos silvestres brasileiros. Foram estudadas amostras de soro e plasma de 12 canídeos cativos: sete lobos-guará (Chrysocyon brachyurus), três raposinhas (Lycalopex vetulus) e dois cachorros-do-mato (Cerdocyon thous). As amostras de um C. brachyurus e uma L. vetulus, cativos em área endêmica para LV, que apresentavam doença clínica e positividade em testes de Imunofluorescência Indireta e Reação em Cadeia de Polimerase, foram utilizadas como controles positivos. Foram comparados os conjugados anti-IgG de cão e proteína A, ambos ligados a peroxidase, cujos testes detectaram quatro (04/12) e três (03/12) C. brachyurus soropositivos para anticorpos anti-Leishmania sp., respectivamente. As médias das densidades ópticas (DOs) das amostras negativas foram nitidamente mais baixas do que as médias das DOs dos positivos tanto no ELISA com anti-IgG de cão (4,8 vezes) como com proteína A (15,5 vezes). Os soros de três C. brachyurus positivos no ELISA indireto foram avaliados por Western blotting e identificaram 22 bandas, sendo imunodominantes as de peso molecular de 19, 22, 24, 45 e 66 kDa. Os testes ELISA com a proteína A e o conjugado anti-IgG de cão apresentaram respectivamente concordância excelente (Kappa = 1; p<0,001) e moderada (Kappa = 0,8; p<0,0015), com o Western blotting. Ambos foram, portanto, considerados adequados a avaliações de triagem de animais cuja resposta humoral de anticorpos indica contato com o parasito, úteis para subsidiar estudos para adequação de metodologias específicas para os canídeos silvestres.

In South America, some wild canids are considered natural reservoirs of Leishmania chagasi. The immunological response of wild canids to Leishmania is not well understood, and the development of diagnostic methods is necessary for such purpose. In the present study, the standardization of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of visceral leishmaniasis (VL) in Brazilian species of wild canids is described. Serum and plasma samples from 12 captive wild canids were studied: seven from maned wolves (Chrysocyon brachyurus), three from hoary foxes (Lycalopex vetulus), and two from crab-eating foxes (Cerdocyon thous). Samples from C. brachyurus and L. vetulus, both captive in an endemic area for VL, presenting clinical disease and positivity in Indirect Immunofluorescence Reaction and Polymerase Chain Reaction tests were used as positive controls. The antibody anti-dog IgG and Protein A, both conjugated with horseradish peroxidase, were compared in indirect ELISA tests which detected four (04/12) and three (03/12) seropositive C. brachyurus for anti-Leishmania antibodies, respectively. The ELISA tests were able to clearly distinguish negative from positive samples, as the mean optical density (OD) of the negative samples was 4.8 and 15.5 times lower than those of the positive ones either using anti-dog IgG and Protein A, respectively. Samples from three ELISA - positive C. brachyurus were analyzed by Western blotting and identified immunodominant bands of 19, 22, 24, 45 and 66 kDa, among 22 protein bands detected. The ELISAs with protein A and anti-dog IgG showed respectively excellent (Kappa = 1.0; p<0.001) and moderate (Kappa = 0.8; p<0.0015) agreement with the Western blotting assay. The ELISA tests showed to be adequate for screening studies to identify antibody responses, thus indicating contact with Leishmania infection by wild canids.