Expressão, purificação e caracterização imunológica de um fragmento recombinante (resíduos 179-281) da proteína G do vírus rábico

Dissertação (mestrado) - Universidade Federal de Santa Catarina. Programa de Pós-Graduação em Biotecnologia.A proteína G do virus rábico contém 505 aminoácidos em sua forma nativa, sendo exposta na superfície da partícula viral. Esta proteína é importante para a infectividade viral e imunidade protetora, sendo o antígeno que induz anticorpos neutralizantes contra o vírus. Diversos epítopos lineares, identificados por anticorpos monoclonais, foram identificados na região central da proteína. Nesse estudo, uma região compreendendo esses epítopos (resíduos 179-281, cepa ERA), denominada rGERA179-281, foi clonada e expressa em Escherichia coli cepa Rosetta, ligada a uma cauda de histidina na região N-terminal. A expressão resultou na formação de agregados insolúveis da proteína em corpos de inclusão. Os corpos de inclusão foram solubilizados com cloreto de guanidina 6M e a proteína foi purificada por cromatrografia de afinidade por íons metálicos imobilizados, em condições desnaturantes, sendo sua identidade confirmada por espectrometria de massa. A proteína purificada (13,8 kDa) foi reconhecida por anticorpos presentes na preparação comercial de imunoglobulina antirábica humana (HRIG), por meio de immunoblotting. Além disso, por um método de inibição de neutralização, a rGERA179-281 levou a uma redução mensurável na atividade neutralizante da HRIG. Para analisar a imunogenicidade da proteína, camundongos foram imunizados com a rGERA179-281. Observou-se, pela técnica de imunofluorescência indireta, que amostras de soro destes animais foram capazes de reconhecer o vírus rábico na forma nativa em células infectadas, embora não tenha sido observada uma indução de títulos neutralizantes acima de 0,5 UI/ml. Esses resultados, juntamente com o bom rendimento obtido, geram perspectivas de estudos posteriores mais detalhados das propriedades imunológicas da rGERA179-281, além da sua possível aplicação no desenvolvimento de novos métodos de diagnóstico. The G protein, which coats the outer surface of the rabies virus, contains 505 amino acids in its native form and it is important for virus infectivity and induction of the protective immunity. In this study, the region comprising linear epitopes (residues 179 to 281, ERA strain), named rGERA179-281, was cloned in frame with a hexahistidine tag coding sequence at its N-terminal end and overexpressed in Escherichia coli Rosetta strain. The expression yielded insoluble protein aggregates in the form of inclusion bodies. The inclusion bodies were solubilized with 6M guanidine HCl and the protein was purified by immobilized metal affinity chromatography under denaturing conditions. Mass spectrometry data confirmed the identity of the protein. The purified protein (13.8 kDa) showed significant reactivity with antibodies present in a therapeutic human rabies immune globulin (HRIG), as demonstrated by immunoblotting analysis. In addition, by an in vitro inhibition neutralization assay, rGERA179-281 led to a measurable reduction in the ability of HRIG to neutralize rabies virus. For analyzing the immunogenic property of the protein, mice were immunized with rGERA179-281. The antibodies elicited in the mouse serum samples recognized the native form of rabies virus in the virus-infected cells by immunofluorescence assay. However, significant titers of virus neutralizing antibodies were not detected. These results, along with the good yield obtained, encourage further studies on the more detailed immunological properties of rGERA179-281 and the production of anti-G monoclonal antibodies, which together, might be useful for the development of new diagnostic methods

Adipose Tissue-Derived Mesenchymal Stem Cells Increase Skin Allograft Survival and Inhibit Th-17 Immune Response

Adipose tissue-derived mesenchymal stem cells (ADSC) exhibit immunosuppressive capabilities both in vitro and in vivo. Their use for therapy in the transplant field is attractive as they could render the use of immunosuppressive drugs unnecessary. The aim of this study was to investigate the effect of ADSC therapy on prolonging skin allograft survival. Animals that were treated with a single injection of donor allogeneic ADSC one day after transplantation showed an increase in donor skin graft survival by approximately one week. This improvement was associated with preserved histological morphology, an expansion of CD4+ regulatory T cells (Treg) in draining lymph nodes, as well as heightened IL-10 expression and down-regulated IL-17 expression. In vitro, ADSC inhibit naïve CD4+ T cell proliferation and constrain Th-1 and Th-17 polarization. In summary, infusion of ADSC one day post-transplantation dramatically increases skin allograft survival by inhibiting the Th-17 pathogenic immune response and enhancing the protective Treg immune response. Finally, these data suggest that ADSC therapy will open new opportunities for promoting drug-free allograft survival in clinical transplantation

Report of East-Central South African Chikungunya virus genotype during the 2016 outbreak in the Alagoas State, Brazil

Chikungunya virus (CHIKV) causes a self-limiting disease characterized by the onset of fever, skin rash and persistent arthralgia. In the last decade, it has emerged as a serious public health problem causing several outbreaks around the world. Here, we report the CHIKV genotype characterization during the 2016 CHIKV outbreak in Alagoas State, Brazil. Partial E1 sequence from CHIKV-positive samples coming from different cities of Alagoas were submitted to DNA sequencing followed by phylogenetic analysis thus characterizing the virus genotype. The circulating CHIKV virus in Alagoas during 2016 outbreak belongs to the East-Central South African genotype. In this way, virus genotyping to monitoring the spread of CHIKV is needed to continued surveillance supporting the development of prevention strategies, mainly in endemic areas of mosquitoes and arboviruses co-circulation

In pulmonary paracoccidioidomycosis IL-10 deficiency leads to increased immunity and regressive infection without enhancing tissue pathology

BACKGROUND: \ud Cellular immunity is the main defense mechanism in paracoccidioidomycosis (PCM), the most important systemic mycosis in Latin America. Th1 immunity and IFN-γ activated macrophages are fundamental to immunoprotection that is antagonized by IL-10, an anti-inflammatory cytokine. Both in human and experimental PCM, several evidences indicate that the suppressive effect of IL-10 causes detrimental effects to infected hosts. Because direct studies have not been performed, this study was aimed to characterize the function of IL-10 in pulmonary PCM.\ud METHODOLOGY/PRINCIPAL FINDINGS: \ud Wild type (WT) and IL-10(-/-) C57BL/6 mice were used to characterize the role of IL-10 in the innate and adaptive immunity against Paracoccidioides brasiliensis (Pb) infection. We verified that Pb-infected peritoneal macrophages from IL-10(-/-) mice presented higher phagocytic and fungicidal activities than WT macrophages, and these activities were associated with elevated production of IFN-γ, TNF-α, nitric oxide (NO) and MCP-1. For in vivo studies, IL-10(-/-) and WT mice were i.t. infected with 1×10(6) Pb yeasts and studied at several post-infection periods. Compared to WT mice, IL-10(-/-) mice showed increased resistance to P. brasiliensis infection as determined by the progressive control of pulmonary fungal loads and total clearance of fungal cells from dissemination organs. This behavior was accompanied by enhanced delayed-type hypersensitivity reactions, precocious humoral immunity and controlled tissue pathology resulting in increased survival times. In addition, IL-10(-/-) mice developed precocious T cell immunity mediated by increased numbers of lung infiltrating effector/memory CD4(+) and CD8(+) T cells. The inflammatory reactions and the production of Th1/Th2/Th17 cytokines were reduced at late phases of infection, paralleling the regressive infection of IL-10(-/-) mice.\ud CONCLUSIONS/SIGNIFICANCE: \ud Our work demonstrates for the first time that IL-10 plays a detrimental effect to pulmonary PCM due to its suppressive effect on the innate and adaptive immunity resulting in progressive infection and precocious mortality of infected hosts.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 04/14518-2)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 2011/51258-2)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 2010/52275-5)CAPE

Retrospective clinical and epidemiological analysis of scorpionism at a referral hospital for the treatment of accidents by venomous animals in Alagoas State, Northeast Brazil, 2007-2017

Scorpionism has a high incidence rate in Brazil. It is considered a serious public health problem mainly in tropical and subtropical regions around the world. The number of scorpion accidents have increased over the years and the highest frequencies have been reported mainly in the Brazilian Northeast region. Therefore, in this study we report a retrospective clinical and epidemiological analysis of scorpion stings from 2007 to 2017 in Alagoas State, Northeast Brazil, at a referral hospital for assistance and treatment of accidents by venomous animals. During the analyzed period, the referral hospital treated 27,988 cases, and an increase in the number of cases has taken place over the years. The highest frequency of scorpion stings was observed in females, and the age range most affected was from 20 to 29 years old. The most stung body site was the foot, followed by finger, toe or hand. Regarding the severity, most severe cases were reported in children up to 4 years old (69.4%) and 50% of the total cases treated with serotherapy corresponded to patients in this age range. Interestingly, it was also found that the occurrence of systemic manifestations and the severity of the cases were significantly associated with pediatric patients. In this way, this study highlights the scorpionism as an environmental public health problem in Alagoas State, Northeast Brazil, as well as the need to intensify the epidemiological surveillance and educational campaigns to prevent and control scorpion accidents throughout the year

Immune regulatory properties of adipose-derived mesenchymal stem cells in the treatment of experimental autoimmune diabetes.

As células-tronco isoladas a partir do tecido adiposo (ADMSCs) se tornaram promissoras para o tratamento de diversas doenças autoimunes devido a suas propriedades imunomoduladoras. O objetivo deste estudo foi avaliar o potencial terapêutico das ADMSCs em modular a resposta imune no diabetes autoimune experimental em camundongos NOD. ADMSC alogênicas foram administradas em camundongos NOD diabéticos (glicemia > 240 mg/dl) nos dias 0, 7 e 14 sendo então a glicemia monitorada por 12 semanas. A administração de ADMSCs resultou na reversão da hiperglicemia em 78% dos animais por 8 semanas após o tratamento. O tratamento com ADMSCs pôde melhorar de forma efetiva o diabetes autoimune em camundongos NOD pela atenuação da resposta autoimune envolvida concomitante a expansão de células T reguladoras, provendo o desenvolvimento futuro de novas perspectivas de estratégias terapêuticas de terapia celular para o DMT1.Adipose-derived mesenchymal stem cells (ADMSCs) display immunosuppressive properties representing a promising therapeutic approach for several autoimmune diseases. The aim of this study was to investigate the immune regulatory properties of allogeneic ADMSCs therapy in T cell-mediated experimental autoimmune diabetes in NOD mice. Diabetic NOD mice (blood glucose > 240 mg/dl) were treated or not with ADMSC at days 0, 7 and 14 and blood glucose was monitored once a week for 12 weeks after treatment. ADMSC reversed the hyperglycemia levels of early onset T1D in 78% of diabetic-treated mice for 8 weeks after treatment. ADMSC therapy efficiently ameliorates T1D pathogenesis in diabetic NOD mice by attenuating the Th1 immune response concomitantly with the expansion of Treg cells, thereby contributing to maintenance of functional -cells. This study may thus provide a new therapeutic perspective for the development of ADMSC-based cellular therapies for T1D

Immune regulatory properties of multipotent mesenchymal stromal cells: Where do we stand?

Multipotent mesenchymal stromal cells (MSC) can be isolated and efficiently expanded from almost every single body tissue and have the ability of self-renewal and differentiation into various mesodermal cell lineages. Moreover, these cells are considered immunologically privileged, related to a lack of surface expression of costimulatory molecules required for complete T cell activation. Recently, it has been observed that MSC are capable of suppressing the immune response by inhibiting the maturation of dendritic cells and suppressing the function of T lymphocytes, B lymphocytes and natural killer cells in autoimmune and inflammatory diseases as a new strategy for immunosuppression. The understanding of immune regulation mechanisms by MSC is necessary for their use as immunotherapy in clinical applications for several diseases

Vacina de DNA: uma alternativa para o tratamento do melanoma canino: revisão de literatura

O melanoma é considerado um dos tumores cutâneos de maior importância em cães devido ao seu caráter agressivo, à capacidade de produzir metástases em estágios precoces e à baixa resposta aos tratamentos não cirúrgicos. Recentemente, as vacinas de DNA apresentam-se promissoras na terapêutica do melanoma canino. O objetivo deste trabalho é apresentar uma revisão bibliográfica sobre o assunto. As vacinas de DNA são baseadas em plasmídeos que contêm o gene codificante para o antígeno alvo, expressando-o na célula do hospedeiro e apresentando, assim, vantagens em relação às vacinas tradicionais, como: facilidade de produção, estabilidade térmica, baixo custo e estimulação da resposta imune celular (linfócitos T CD8+). Devido ao sucesso limitado das terapias padrões, o estabelecimento de uma vacina de DNA efetiva aumenta a possibilidade de uma terapia promissora para o melanoma canino, podendo trazer novas expectativas aos animais portadores dessa neoplasia

Leptin as a link between the immune system and kidney-related diseases: leading actor or just a coadjuvant?

Food intake and nutritional status modify the physiological responses of the immune system to illness and infection and regulate the development of chronic inflammatory processes, such as kidney disease. Adipose tissue secretes immune-related proteins called adipokines that have pleiotropic effects on both the immune and neuroendocrine systems, linking metabolism and immune physiology. Leptin, an adipose tissue-derived adipokine, displays a variety of immune and physiological functions, and participates in several immune responses. Here, we review the current literature on the role of leptin in kidney diseases, linking adipose tissue and the immune system with kidney-related disorders. The modulation of this adipose hormone may have a major impact on the treatment of several immune- and metabolic-related kidney diseases.Brazilian foundation FAPESPBrazilian Foundation - FAPESP [09/50450-7, 2010/52180-4, 2012/02270-2, 07/07139-3]International Associated Laboratory in Renal Immunopathology (CNPq/Inserm)International Associated Laboratory in Renal Immunopathology (CNPq/Inserm)INCT Complex FluidsComplex Fluids INCTCNPqCNP

Leptin as a link between the immune system and kidney-related diseases: leading actor or just a coadjuvant?

Food intake and nutritional status modify the physiological responses of the immune system to illness and infection and regulate the development of chronic inflammatory processes, such as kidney disease. Adipose tissue secretes immune-related proteins called adipokines that have pleiotropic effects on both the immune and neuroendocrine systems, linking metabolism and immune physiology. Leptin, an adipose tissue-derived adipokine, displays a variety of immune and physiological functions, and participates in several immune responses. Here, we review the current literature on the role of leptin in kidney diseases, linking adipose tissue and the immune system with kidney-related disorders. The modulation of this adipose hormone may have a major impact on the treatment of several immune- and metabolic-related kidney diseases.Brazilian foundation FAPESPBrazilian Foundation - FAPESP [09/50450-7, 2010/52180-4, 2012/02270-2, 07/07139-3]International Associated Laboratory in Renal Immunopathology (CNPq/Inserm)International Associated Laboratory in Renal Immunopathology (CNPq/Inserm)INCT Complex FluidsComplex Fluids INCTCNPqCNP