Development of diagnostic methods and study of the immunoreactivity of a mixture of recombinant core and E2 proteins fused to GST with control serum positive for hepatitis C

AbstractThe hepatitis C virus (HCV) is an enveloped virus that is about 50–70nm in diameter, has positive-strand RNA, and belongs to the genus Hepacivirus and the family Flaviridae. The detection and quantification of the core antigen, HCV nucleocapsid protein, has been successful in many trials and is considered a marker of viral replication since it presents a sequence of highly conserved amino acids, giving it high sensitivity and specificity. The E2 protein is an envelope glycoprotein of HCV with 11 glycosylation sites; most of these are well-conserved, making it a target antigen. The aim of this study is to develop high-sensitivity, low-cost diagnostic methods for HCV, which could be used for serological screening. The genomic regions encoding the core (part 136 aa) and E2 proteins of HCV were expressed in Escherichia coli Rosetta strain, cloned in expression vector pET-42a, and induced with 0.4mmolL−1 IPTG, producing recombinant proteins that were fused to glutathione S-transferase (GST) protein, which was then purified by affinity chromatography. The immunoreactivity was assessed by Western blot, Slot Blot, and developed and improved diagnostic methods (capture, indirect, and immunoblotting enzyme-linked immunosorbent assay (ELISA)). After applying the results to the formulas for determining the quality parameters, obtained for immunoblotting method 100% sensitivity and specificity and for ELISA 100% sensitivity and 87.5% specificity. The methods developed were more sensitive and specific using the mixture of the recombinant proteins fused to GST (core+E2)

Interação da proteina e2 do vírus da hepatite c com o receptor de ldl e cd81 presentes em células endoteliais e ativação da resposta inflamatória

Approximately 170 million people worldwide are chronically infected with hepatitis C virus (HCV). In Brazil, the numbers have already reached 3 million. The persistence of HCV infection leads to chronic infection in liver disease, cirrhosis and hepatocellular carcinoma. The envelope protein 2 (E2) of HCV is responsible for its coupling to the host cell through interaction with cell surface receptors such as the R-LDL and CD81, among others. Because changes in micro and macrovasculature liver, have been identified as key elements in the histogenesis and prognosis of the disease, the aim of this study was to evaluate the interaction of the protein E2 recombinant with the R-LDL present on the surface of endothelial cells (HUVEC and ECV304) under the influence of LDL and protein glycosylation. And also evaluate the inflammatory response of endothelial cells to interact with these proteins. The E2 protein was expressed in two heterologous systems, E. coli and P. pastoris for evaluation of glycosylation and interaction with LDL binding to the R-LDL cells by flow cytometry. The proteins were also evaluated in biological tests for induction of cellular production of reactive oxygen species (EROs - QL and H2O2 - flow cytometry), NO (QL in doing gas and Griess Reagent), arginase (spectrophotometry), IL-8 (ELISA), VEGF (ELISA) and cell death (MTT – spectrophotometry and Annexin V and PI - flow cytometry). The results obtained showed that the recombinant E2 protein can interact with R-LDL endothelial cell after binding to LDL and that this binding can be enhanced by glycosylation (E2L) (p <0.01 in relation to E2B). The NO test indicated that glycosylated proteins exhibit greater potential for the stimulation of the HUVEC cells (p <0.01 compared with negative control). Protein concentrations tested were not sufficient to stimulate an increase or decrease in the activity of arginase remarkable compared to the negative control. The recombinant protein ...Aproximadamente 170 milhões de pessoas no mundo estão cronicamente infectadas pelo vírus da Hepatite C (HCV). No Brasil, os números já chegam a 3 milhões. A persistência da infecção pelo HCV no fígado leva à cronificação da doença, cirrose e hepatocarcinoma. A proteína de envelope 2 (E2) do HCV é a responsável pelo seu acoplamento à célula hospedeira através da interação com receptores de superfície celular, como o R-LDL e o CD81, entre outros. Como as alterações na micro e macrovasculatura hepática, têm sido apontadas como elementos fundamentais na histogênese e prognóstico da doença, o objetivo deste estudo foi avaliar a interação da proteína E2 recombinante com os R-LDL presentes na superfície de células endoteliais (ECV304 e HUVEC) sob influência de LDL e da glicosilação protéica. Também avaliar a resposta inflamatória das células endoteliais à interação com estas proteínas. A proteína E2 foi expressa em dois sistemas heterólogos, E. coli e P. pastoris, para avaliação da glicosilação e da interação com LDL na ligação aos R-LDL das células através de citometria de fluxo. As proteínas também foram avaliadas em testes biológicos quanto à indução celular de produção de espécies reativas de oxigênio (EROs totais – QL e H2O2 - citometria de fluxo), NO (QL em fase gasosa e por método de Griess), arginase (espectrofotometria), IL-8 (ELISA), VEGF (ELISA) e morte celular (MTT - espectrofotometria e por anexina V e PI - citometria de fluxo). Os resultados obtidos revelaram que as proteínas E2 recombinantes podem interagir com os R-LDL das células endoteliais após a ligação ao LDL e que esta ligação pode ser melhorada pela glicosilação (E2L) (p<0,01 em relação à E2B). O teste de NO indicou que as proteínas glicosiladas exibem maior potencial para o estímulo deste nas células HUVEC (p<0,01 em relação ao controle negativo). As concentrações de proteínas testadas não ...Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Burst oxidativo dos neutrófilos humanos: estudo da influência do polimorfismo do receptor para IgG FeyRIIIb na cooperação com os receptores para complemento

Os receptores para a porção Fc das IgG (FcgR) estão amplamente expressos nas células do sistema imune e podem estimular uma variedade de respostas efetoras. Três classes de FcgR são descritas nos humanos: FcgRI, FcgRII e FcgRIII. O neutrófilo expressa constitutivamente as isoformas FcgRIIa (CD32) e FcgRIIIb (CD16), as quais apresentam um polimorfismo funcional decorrente do polimorfismo alélico. No FcgRIIa, a substituição da histidina (H) pela arginina (R) na posição 131 resulta nas variantes polimórficas FcgRIIa- R131 e FcgRIIa-H131, que diferem quanto à capacidade de ligação à IgG2 humana. Quanto ao FcgRIIIb (CD16), o polimorfismo genético é responsável pela expressão dos antígenos de neutrófilo (NA)1, NA2 e SH e resulta em um maior número de sítios de glicosilação em NA2. O FcgRIIIb é responsável pela aproximação dos ligantes na membrana para o FcgRIIa e também interage com o receptor para complemento tipo 3 (CR3) na superfície celular. A cooperação entre os FcgRIIa/IIIb com o CR3 promove a estimulação máxima das respostas do neutrófilo, dentre elas o burst oxidativo. Uma vez que a interação FcgRIIIb/CR3 ocorre via sítio de ligação lectina-sacarídeo, o objetivo deste estudo foi avaliar se o polimorfismo alélico do FcgRIIIb poderia afetar a cooperação entre os FcgR e os CR em mediar o burst oxidativo dos neutrófilos. As freqüências dos alelos H/R-131 e NA1/NA2/SH foram analisadas por genotipagem com oligonucleotídeos alelo-específicos e reação em cadeia da polimerase e a densidade de expressão dos receptores (FcgRIIa, FcgRIIIb, CR1 e CR3) foi determinada por citometria de fluxo. A distribuição dos genótipos para o FcgRIIIb (n=169) foi NA1/NA2 (52,1%), NA2 (27,8%) e NA1 (20,1%). Dentre 174 indivíduos, apenas 6,3% apresentaram o gene SH. Quanto aos genótipos para o FcgRIIa (n=143), a distribuição...Receptors for immunoglobulin G (FcgR) are known to be expressed on many cells of the immune system and they can trigger a variety of biological responses. Human FcgR belong to the Ig superfamily and three classes of these receptors have been recognized: FcgRI, FcgRII and FcgRIIl, with different affinities and specificities for IgG subclasses. Neutrophils express the FcgRIIa (CD32) and FcgRIIlb (CD16) isoforms, which display a functional polymorphism due to biallelic polymorphisms. In the case of FcgRIIa, an arginine (FcgRIIa-R131) or histidine (FcgRIIa-H131) at amino acid position 131 determines receptor affinity for IgG2, FcgRIIa-H131 being the isoform with highest affinity. On neutrophils, the FcgRIIIb bears the neutrophil antigen (NA) polymorphism, NA1, NA2 and SH, NA2 being the more glycosylated isoform. Cooperation of FcgRIIIb with FcgRIIa and CR3 (complement receptor type 3) is necessary for efficient neutrophil responses, including the oxidative burst. Since the FcgRIIIb/CR3 cooperation occurs via lectin-sacharide-like interaction, the aim of this study was to evaluate whether the allelic polymorphism, NA1 and NA2, could influence the cooperation of FcgRIIIb with CR3 in mediating the oxidative burst of neutrophils. FcgRIIa and FcgRIIIb genotyping analysis were performed by polymerase chain reaction with allelespecific primers and surface expressions of FcgRIIa, FcgRIIIb, CR1 and CR3 on neutrophils were determined by flow cytometry. The FcgRIIIb genotype distribution (n=169) was NA1/NA2 (52.1%), NA2 (27.8%) and NA1 (20.1%). SH gene was found in 11 (6.3%) volunteers from 174 subjects. With respect to the FcgRIIa genotype (n=143), the distribution observed was R131 (41.2%), H/R131 (38.5%) and H (20.3%). Neutrophils were stimulated with immune complexes (IC)-IgG opsonized or not with complement and the oxidative burst... (Complete abstract click electronic access below)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Tratamiento de la hiperhidrosis palmar primaria por acupuntura: a propósito de un caso

Primary hyperhidrosis is an excessive sweating disorder that occurs in axillae, palms, soles, and craniofacial region. According to Traditional Chinese Medicine (TCM) it is a water metabolism disorder. This study presents a case report treated with TCM, showing more satisfactory results than those by conventional treatments.La hiperhidrosis primaria es un trastorno de transpiración excesiva que ocurre en axilas, palmas de las manos, plantas de los pies y región craneofacial. De acuerdo con la Medicina Tradicional China (MTC) se trata de un trastorno del metabolismo del agua. En el presente estudio se presenta el informe de un caso tratado por la MTC, con resultados más satisfactorios que los presentados por los tratamientos convencionales

Rapid Fabrication of Microfluidic Devices for Biological Mimicking: A Survey of Materials and Biocompatibility

Microfluidics is an essential technique used in the development of in vitro models for mimicking complex biological systems. The microchip with microfluidic flows offers the precise control of the microenvironment where the cells can grow and structure inside channels to resemble in vivo conditions allowing a proper cellular response investigation. Hence, this study aimed to develop low-cost, simple microchips to simulate the shear stress effect on the human umbilical vein endothelial cells (HUVEC). Differentially from other biological microfluidic devices described in the literature, we used readily available tools like heat-lamination, toner printer, laser cutter and biocompatible double-sided adhesive tapes to bind different layers of materials together, forming a designed composite with a microchannel. In addition, we screened alternative substrates, including polyester-toner, polyester-vinyl, glass, Permanox® and polystyrene to compose the microchips for optimizing cell adhesion, then enabling these microdevices when coupled to a syringe pump, the cells can withstand the fluid shear stress range from 1 to 4 dyne cm2. The cell viability was monitored by acridine orange/ethidium bromide (AO/EB) staining to detect live and dead cells. As a result, our fabrication processes were cost-effective and straightforward. The materials investigated in the assembling of the microchips exhibited good cell viability and biocompatibility, providing a dynamic microenvironment for cell proliferation. Therefore, we suggest that these microchips could be available everywhere, allowing in vitro assays for daily laboratory experiments and further developing the organ-on-a-chip concept

Endothelial Cell Culture Under Perfusion On A Polyester-Toner Microfluidic Device

Abstract This study presents an inexpensive and easy way to produce a microfluidic device that mimics a blood vessel, serving as a start point for cell culture under perfusion, cardiovascular research, and toxicological studies. Endpoint assays (i.e., MTT reduction and NO assays) were used and revealed that the components making up the microchip, which is made of polyester and toner (PT), did not induce cell death or nitric oxide (NO) production. Applying oxygen plasma and fibronectin improved the adhesion and proliferation endothelial cell along the microchannel. As expected, these treatments showed an increase in vascular endothelial growth factor (VEGF-A) concentration profiles, which is correlated with adherence and cell proliferation, thus promoting endothelialization of the device for neovascularization. Regardless the simplicity of the device, our “vein-on-a-chip” mimetic has a potential to serve as a powerful tool for those that demand a rapid microfabrication method in cell biology or organ-on-a-chip research

Inflammatory response of endothelial cells to hepatitis C virus recombinant envelope glycoprotein 2 protein exposure

The hepatitis C virus (HCV) encodes approximately 10 different structural and non-structural proteins, including the envelope glycoprotein 2 (E2). HCV proteins, especially the envelope proteins, bind to cell receptors and can damage tissues. Endothelial inflammation is the most important determinant of fibrosis progression and, consequently, cirrhosis. The aim of this study was to evaluate and compare the inflammatory response of endothelial cells to two recombinant forms of the HCV E2 protein produced in different expression systems (Escherichia coli and Pichia pastoris). We observed the induction of cell death and the production of nitric oxide, hydrogen peroxide, interleukin-8 and vascular endothelial growth factor A in human umbilical vein endothelial cells (HUVECs) stimulated by the two recombinant E2 proteins. The E2-induced apoptosis of HUVECs was confirmed using the molecular marker PARP. The apoptosis rescue observed when the antioxidant N-acetylcysteine was used suggests that reactive oxygen species are involved in E2-induced apoptosis. We propose that these proteins are involved in the chronic inflammation caused by HCV