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Background: Rubella is an infection caused by rubella virus (RV) and is generally regarded as a mild childhood disease. The disease continues to be of public health importance mainly because when the infection is acquired during early pregnancy, it often results in fetal abnormalities, which are classified as congenital rubella syndrome (CRS). An accurate diagnosis of rubella is thus of pivotal importance for proper treatment. Objective: The aim of the study was to produce a recombinant multiepitope protein (rMERUB) for the diagnosis of rubella, based on conserved immunodominant epitopes of glycoprotein E1 and E2. Methods: A synthetic gene was designed and cloned into vector pET21a with a 6xHis tag at the C-terminal for affinity purification and overexpressed in Escherichia coli cells. Biophysical analysis of rMERUB was performed by circular dichroism. Biological activity was assessed using an in-house ELISA assay. Results: Expression in Escherichia coli showed a ~22 kDa protein that was purified and used to per-form structural assays and an IgG ELISA. Structural analyses reveal that rMERUB has a β leaf pattern that promotes the exposure of epitopes, thus allowing antibody recognition. Evaluation of 33 samples (22=positive; 11=negative) was performed using in-house ELISA and this was compared with a com-mercial kit. The sensitivity was 100% (95% CI: 85-100) and specificity 90.91% (95% CI: 62-99). Excellent agreement (Kappa index = 0.9) was obtained between ELISA assays. Conclusions: The careful choice of epitopes and the high epitope density, coupled with simple-step purification, pinpoints rMERUB as a promising alternative for rubella diagnosis, with potential for the development of a diagnostic kit.

Background:The Human Cytomegalovirus (HCMV) has infected more than 90% of the world population and its prevalence can be related to the individuals geographical and socialeconomic status. Serological tests based on ELISA are pivotal for HCMV diagnosis. Due to the lack of standardization in the production/purification of antigens from viral preparations, ELISA tests are based on several recombinant proteins or peptides. As an alternative, multiepitope proteins may be employed.Objective:In this work, we developed a recombinant multiepitope protein (rMEHCMV) for HCMV diagnosis based on conserved and immunodominant epitopes derived from tegument (pp150, pp65 and pp28), glycoprotein gB (pp38) and DNA polymerase subunit (pp52) of HCMV.Methods:The rMEHCMV gene was synthesized de novo and overexpressed in Escherichia coli cells. The recombinant protein was purified to homogeneity using a Ni-NTA column. Biophysical analysis of recombinant protein was performed by circular dichroism. A preliminary biological activity test was performed using 12 positive human sera samples by using an in-house IgG ELISA. The following patents database were consulted: Espacenet, Google Patents and the National Institute of Intellectual Property (INPI, Brazil).Results:The recombinant multiepitope protein was successfully expressed in E. coli. The structural data obtained by circular dichroism spectroscopy showed that rMEHCMV is structurally disordered. An in-house IgG ELISA test with rMEHCMV was successfully used to recognized IgG from human serum samples.Conclusion:Together, our results show that rMEHCMV should be considered as a potential antigenic target for HCMV diagnosis.

Tese (doutorado)—Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Ciências Médicas, 2018. Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). A inflamação é um processo biológico complexo, altamente regulado definido como uma resposta de defesa do organismo frente a um agente agressor. Tem por objetivo recompor a homeostase do tecido lesado. Assim, a resposta inflamatória aguda é benéfica ao hospedeiro, reestabelecendo em pouco tempo a estrutura e função do tecido e/ou órgão. Entretanto, a resposta inflamatória crônica pode promover perda da função do órgão ou tecido levando ao desenvolvimento de doenças. Na indústria de processamento da castanha de caju, são obtidos a amêndoa e o óleo, tradicionalmente conhecido como Líquido da Castanha de Caju (LCC). Estudos mostram que os derivados do LCC possuem atividade antioxidante e anti-inflamatória. No entanto, o elevado desperdício do LCC no Brasil faz com que este subproduto da indústria alimentícia seja pouco explorado. O perfil farmacológico multi-alvo de ácido anacárdico encontrado no LCC o torna um alvo potencial para o desenvolvimento de novos agentes anti-inflamatórios. O presente trabalho propõe avaliar o efeito imunoprotetor e o efeito anti-inflamatório de dois derivados semisintéticos, exrtraídos a partir de lipídeos fenólicos de Anacardium occidentale L., denominados LDT11 e LDT13. A atividade biológica das moléculas foi avaliada em modelo celular in vitro. Foi avaliada a citotoxicidade dos compostos propostos, LDT11 e LDT13, na linhagem celular de macrófagos murinos, RAW264.7. Para avaliação do efeito protetor à inflamação, as células foram previamente tratadas com os derivados fenólicos e, em seguida, a células foram estimuladas com lipopolissacarídeo (LPS), nos tempos de 6, 24 e 48 horas. Para a avaliação do efeito anti-inflamatório, as células foram primeiramente estimuladas com LPS e, em seguida, tratadas com as moléculas estudas. Para comparação da atividade biológica, foram utilizados os fármacos comerciais, ácido acetilssalicílico (AAS) e dexametasona (DEXA). Foi realizada a análise da expressão gênica de marcadores inflamatórios TNFα, iNOS, COX-2, NF-κB, IL-1β e IL-6, dosagem de óxido nítrico (NO) e citocina IL-6. Os resultados mostraram que o LDT11 e o LDT13 influenciaram na modulação gênica dos mediadores inflamatórios. A quantificação relativa dos transcritos gênicos mostrou que os derivados fenólicos propostos apresentaram potencial efeito anti-inflamatório pela diminuição da expressão gênica em comparação ao grupo controle, células estimuladas com LPS. As dosagens de NO e IL-6 confirmam os resultados encontrados na expressão gênica. Os derivados fenólicos mostraram ser potenciais agentes anti-inflamatórios, tendo um efeito rápido e mais efeicaz que os fármacos comerciais. Inflammation is a complex, highly regulated biological process defined as a defense response of the organism to an aggressive agent. It aims to restore the homeostasis of the damaged tissue. Thus, the acute inflammatory response is beneficial to the host, reestablishing in a short time the structure and function of the tissue and / or organ. However, the chronic inflammatory response may promote loss of organ or tissue function leading to disease development. In the cashew processing industry, almonds and oil are traditionally known as Cashew Nut Shell Liquid (CNSL). Studies show that the derivatives of CNSL have antioxidant and anti-inflammatory activity. However, the high waste of the CNSL in Brazil makes this by-product of the food industry little explored. The multi-target pharmacological profile of anacardic acid found in CNSL makes it a potential target for the development of novel anti-inflammatory agents. This study proposes to evaluate the immunoprotective effect and the anti-inflammatory effect of two semisynthetic derivatives, extracted from phenolic lipids of Anacardium occidentale L., called LDT11 and LDT13. The biological activity of the molecules were evaluated in cellular model in vitro. The cytotoxicity of the proposed compounds, LDT11 and LDT13, in the murine macrophages cell line, RAW264.7, was evaluated. To evaluate the protective effect on inflammation, the cells were previously treated with the phenolic derivatives and then the cells were stimulated with lipopolysaccharide (LPS), at the times of 6, 24 and 48 hours. For the evaluation of the anti-inflammatory effect the cells were first stimulated with LPS, and then treated with the molecules studied. To compare the biological activity, commercial drugs, acetylsalicylic acid (ASA) and dexamethasone (DEXA) were used. The gene expression of inflammatory markers TNFα, iNOS, COX-2, NF-κB, IL-1β and IL-6, nitric oxide (NO) and IL-6 cytokine were analyzed. The results showed that LDT11 and LDT13 influenced the gene modulation of inflammatory mediators. The relative quantification of the gene transcripts showed that the proposed phenolic derivatives showed a potential anti-inflammatory effect due to the decrease of the gene expression in comparison to the control group, cells stimulated with LPS. The dosages of NO and IL-6 confirm the results found in the gene expression. Phenolic derivatives have been shown to be potential anti-inflammatory agents, having a rapid and more effective effect than commercial drugs.

Background Anacardium occidentale L phenolic lipid (LDT11) is used in traditional medicine as anti-inflammatory, astringent, antidiarrheal, anti-asthmatic and depurative. Phenolic derivatives, such as anacardic acid, extracted from cashew nut shell liquid (CNSL) have demonstrated biological and pharmacological properties, and its profile makes it a candidate for the development of new anti-inflammatory agents. The objective of the present study was to evaluate the anti-inflammatory profile of a derivative, synthesized from LDT11, on an in vitro cellular model. Methods Organic synthesis of the phenolic derivative of CNSL that results in the hemi-synthetic compound LDT11. The cytotoxicity of the planned compound, LDT11, was analyzed in murine macrophages cell line, RAW264.7. The cells were previously treated with LDT11, and then, the inflammation was stimulated with lipopolysaccharide (LPS), in intervals of 6 h and 24 h. The analysis of the gene expression of inflammatory markers (TNFα, iNOS, COX-2, NF-κB, IL-1β and IL-6), nitric oxide (NO) dosage, and cytokine IL-6 were realized. Results The results showed that the phenolic derivative, LDT11, influenced the modulatory gene expression. The relative gene transcripts quantification demonstrated that the LDT11 disclosed an immunoprotective effect against inflammation by decreasing genes expression when compared with cells stimulated with LPS in the control group. The NO and IL-6 dosages confirmed the results found in gene expression. Discussion The present study evaluated the immunoprotective effect of LDT11. In addition to a significant reduction in the expression of inflammatory genes, LDT11 also had a faster and superior anti-inflammatory action than the commercial products, and its response was already evident in the test carried out six hours after the treatment of the cells. Conclusion This study demonstrated LDT11 is potentially valuable as a rapid immunoprotective anti-inflammatory agent. Treatment with LDT11 decreased the gene expression of inflammatory markers, and the NO, and IL-6 production. When compared to commercial drugs, LDT11 showed a superior anti-inflammatory action.

Hepatitis B is a liver inflammation caused by hepatitis B virus (HBV) and can be diagnosed in clinical stage by hepatitis B core antibody from IgM class (anti-HBcIgM). Hepatitis B core antibody from IgG class (Anti-HBcIgG) appears quickly after IgM, reaching high titers in chronic hepatitis, and remains even after cure. Since hepatitis B core antibody (anti-HBc) is the first antibody identified and sometimes the only marker detected during the course of infection, it can be used both to indicate HBV acute infection (anti-HBc-IgM) and to identify individuals who have come into contact with the virus (anti-HBc-IgG). In this work we propose a recombinant hepatitis B core multiepitope antigen (rMEHB) to be used for diagnosis of hepatitis B. For this purpose, a synthetic gene coding for rMEHB was designed and cloned into vector pET21a with a 6xHis tag at the C-terminal. Time course induction inE. colishowed an induced protein with an apparent molecular mass of ~21 kDa. Protein purification was performed by a single step with affinity chromatography Ni-NTA. Circular dichroism spectroscopy indicated rMEHB as a thermal stable protein at pH 7.0 and 8.0. In these conditions rMEHB was successfully used to perform an enzyme linked immuno sorbent assay (ELISA) with positive and negative sera.