RESUMO
Hepatitis E Virus (HEV) is a zoonotic pathogen responsible for causing acute hepatitis in human, especially in developing countries. Diagnosis of HEV usually relies on the detection of antibodies mostly by enzyme-linked immunosorbent assay (ELISA). In the present study, we designed a new indirect ELISA (iELISA) based on a short recombinant peptide derived from the capsid protein (ORF2p) and demonstrated its potential for detecting human IgG against HEV genotype 3. The best polystyrene plate (Maxisorp®), optimal ORF2p coating antigen concentration (0,67µg/well) and primary antibody dilution (1:100) were determined. This iELISA showed a sensitivity of 91.4% and specificity of 95.9%. The comparison of our in house iELISA with a commercial assay (RecomWell, Mikrogen®) showed 94.25% of agreement and a kappa index of 0.88. The ORF2 recombinant ELISA was used to screen 780 blood donors for anti-HEV IgG and we found that 314 (40,25%) of these donors were IgG positive. This high prevalence of antibodies suggests, for the first time, that the Southern Brazil region might be endemic to Hepatitis E Virus genotype 3.
Assuntos
Doadores de Sangue , Endonucleases/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Vírus da Hepatite E/isolamento & purificação , Imunoglobulina G/imunologia , DNA Polimerase Dirigida por RNA/metabolismo , Brasil/epidemiologia , Vírus da Hepatite E/imunologia , Humanos , Proteínas Recombinantes/metabolismo , Estudos SoroepidemiológicosRESUMO
Hepatitis E, caused by hepatitis E virus (HEV), is a viral infectious pathology of great importance in the public health. Hepatitis E outbreaks were registered in developing countries with poor or no sanitation, where drinking water was contaminated with fecal material, but also in many industrialized countries probably due to consumption of HEV-positive swine meat. In this study, we present the development and characterization of a recombinant antigen from ORF2 HEV genotype 3. Viral RNA was extracted from swine feces infected with the native virus. A total of 267 residues from the C-terminal ORF2((394-661)) coding sequence were cloned into the pET20a vector and expressed in Escherichia coli ER2566. Recombinant protein was purified by liquid chromatography and the fragment obtained a 98% homology against other human or swine HEV genotype 3 ORF2 sequences. Wistar rats were inoculated with ORF2p, developing antibodies able to recognize both the homologous antigen and the native HEV genotype 3 ORF2 present in infected stool. In parallel, HEV-negative swine were experimentally challenged with HEV genotype 3. ORF2 was detected by PCR 14 days post-inoculation in three-fourth piglets' feces and one week later by dot blot. In conclusion, this study proved the immunogenic and antigenic properties of the recombinant protein ORF2p.
Assuntos
Antígenos Virais/imunologia , Fezes/virologia , Vírus da Hepatite E/imunologia , Hepatite E/diagnóstico , Fases de Leitura Aberta/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Brasil , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Vírus da Hepatite E/classificação , Vírus da Hepatite E/genética , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Viral/genética , RNA Viral/isolamento & purificação , Ratos , Ratos Wistar , Alinhamento de Sequência , Análise de Sequência de RNA , Suínos , Doenças dos SuínosRESUMO
Desde sua caracterização em 1983, o vírus da hepatite E (HEV) vem sendo associado à ocorrência de surtos ictéricos de hepatite aguda em regiões endêmicas como a India. Recentemente casos esporádicos de hepatite E comecaram a ser descritos e associados à transmissão zoonótica deste vírus a partir de reservatórios animais, principalmente suínos, cujos vírus foram caracterizados no final dos anos 90. Desta forma hepatite E é a única hepatite viral em que foi evidenciada a transmissão zoonótica Desde então, a descoberta de potenciais reservatórios animais vem cada vez mais contribuindo para mudança do padrão epidemiológico da hepatite E.
RESUMO
Desde sua caracterização em 1983, o vírus da hepatite E (HEV) vem sendo associado à ocorrência de surtos ictéricos de hepatite aguda em regiões endêmicas como a India. Recentemente casos esporádicos de hepatite E comecaram a ser descritos e associados à transmissão zoonótica deste vírus a partir de reservatórios animais, principalmente suínos, cujos vírus foram caracterizados no final dos anos 90. Desta forma hepatite E é a única hepatite viral em que foi evidenciada a transmissão zoonótica Desde então, a descoberta de potenciais reservatórios animais vem cada vez mais contribuindo para mudança do padrão epidemiológico da hepatite E.
RESUMO
BACKGROUND: The natural outcome of infection with hepatitis C virus (HCV) varies substantially among individuals. However, little is known about host and viral factors associated with a self-limiting or chronic evolution of HCV infection. METHODS: From 1 January 2001 through 31 December 2008, a consecutive series of 65 patients from Rio de Janeiro, Brazil, with a well-documented diagnosis of acute HCV infection, acquired via various routes, were enrolled in this study. Patients were prospectively followed up for a median of 40 months after the estimated date of HCV infection with serial measurements of serum alanine aminotransferase, HCV RNA, and anti-HCV antibodies. Spontaneous viral clearance (SVC) was defined as undetectable levels of HCV RNA in serum, in the absence of treatment, for 3 consecutive HCV polymerase chain reaction tests within the first 6 months of follow-up. Cox proportional hazards regression was used to identify host and viral predictors of SVC. RESULTS: The cumulative rate of SVC was 44.6% (95% confidence interval, 32.3%-57.5%). Compared with chronic HCV evolution, patients with self-limiting disease had significantly lower peak levels of anti-HCV antibodies (median, 109.0 vs 86.7 optical density-to-cutoff ratio [od/co]; P<.02), experienced disease symptoms more frequently (69.4% vs 100%; P<.001), and had lower viral load at first clinical presentation (median, 4.3 vs 0.0 log copies; P=.01). In multivariate analyses, low peak anti-HCV level (<93.5 od/co) was the only independent predictor for SVC; the hazard ratio compared with high anti-HCV levels (> or =93.5 od/co) was 2.62 (95% confidence interval, 1.11-6.19; P=.03). CONCLUSION: Our data suggest that low levels of anti-HCV antibodies during the acute phase of HCV infection are independently related to spontaneous viral clearance.
Assuntos
Hepatite C/patologia , Hepatite C/fisiopatologia , Adulto , Idoso , Alanina Transaminase/sangue , Brasil , Feminino , Seguimentos , Hepatite C/imunologia , Anticorpos Anti-Hepatite C/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , RNA Viral/sangue , Adulto JovemRESUMO
BACKGROUND/AIMS: Although eosinophils are considered to play an important role in the pathogenesis of various parasitic, allergic and autoimmune digestive diseases, their role in fulminant hepatic failure (FHF) is unknown. Our contribution was to identify and quantify eosinophils and cytokine levels [interleukin (IL)-6, IL-5 and macrophage inflammatory protein (MIP)-1alpha] in liver parenchyma and peripheral blood from FHF patients at pre- and post-transplantation steps. METHODS: Histochemical methods were used to identify/quantify eosinophils in liver samples. Liver and plasma cytokine levels were quantified using immunofluorescence methods. RESULTS: Fulminant hepatic failure patients showed a high number of intrahepatic eosinophils concomitant with an increased expression of IL-6, besides the IL-6-positive eosinophils associated with the lack of IL-5. Also, an increased number of eosinophils and soluble IL-6 and MIP-1alpha with a low expression of IL-5 in peripheral blood at the pretransplantation step was observed. CONCLUSIONS: The increased number of intrahepatic eosinophils, besides the high production of IL-6, may be involved in liver dysfunction. In addition, the low presence of IL-5 in liver and peripheral blood may represent a particular pattern of eosinophil behaviour in human liver failure, which may also involve MIP-1alpha. Further ex vivo studies are necessary to evaluate the specific role of eosinophils in FHF.
Assuntos
Eosinofilia/sangue , Eosinófilos/imunologia , Interleucina-5/sangue , Interleucina-6/sangue , Falência Hepática Aguda/sangue , Adolescente , Adulto , Idoso , Biomarcadores/sangue , Movimento Celular , Quimiocina CCL3/sangue , Pré-Escolar , Eosinofilia/fisiopatologia , Feminino , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Falência Hepática Aguda/fisiopatologia , Falência Hepática Aguda/cirurgia , Transplante de Fígado , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
In the study of extensive canine tracheal replacement, heterotopic homologous bioprosthesis were implanted in eighteen healty mongrel dogs, of both sexes, weighing between 15 - 22kg. These animals were divided into two groups: group I - nine dogs with bioprosthesis envelopment in a muscular flap (platisma); group II - nine dogs with bioprosthesis envelopment in an omental flap (large omentum). The gross and microscopic evaluation showed the incorporation of the flap in both groups. However after the 45th day, the inflamatory process in the second group was the most organised and without excessive proliferative granulation tissue. In the comparision control qualitaty of the bioprosthesis in vitro (biophysical and microbiological) was comproved that tracheal swine segment was the most indicated for small animal tracheal reconstruction.
No estudo para substitutos para segmentos extensos de traqueja canina, foram implantados heterotopicamente, biopróteses traqueais homólogas em 18 cães, sem raça definida, pesando entre 15 e 22kg, machos e fêmeas clinicamente sadios. Os animais foram divididos em dois grupos: Grupo I - nove cães com biopróteses implantadas em retalho muscular (Platisma). Grupo II -nove cães com bioprótese implantadas em retalho de grande omento. A avaliação macro e microscópica demonstrou a presença do processo de incorporação a partir do quadragésimo quinto dia nos dois grupos, acrescentando que o processo inflamatório apresentava-se mais organizado no segundo grupo sem a exuberância do tecido de granulação encontrado no primeiro grupo. Dentro do controle de qualidade das biopróteses, foi adicionado a este trabalho análises comparativas in vitro (biofísicas e microbiológicas) onde foi comprovado que a bioprótese traqueal suína apresentava-se estruturalmente como a mais indicada para a reconstrução traqueal em pequenos animais.
RESUMO
In the study of extensive canine tracheal replacement, heterotopic homologous bioprosthesis were implanted in eighteen healty mongrel dogs, of both sexes, weighing between 15 - 22kg. These animals were divided into two groups: group I - nine dogs with bioprosthesis envelopment in a muscular flap (platisma); group II - nine dogs with bioprosthesis envelopment in an omental flap (large omentum). The gross and microscopic evaluation showed the incorporation of the flap in both groups. However after the 45th day, the inflamatory process in the second group was the most organised and without excessive proliferative granulation tissue. In the comparision control qualitaty of the bioprosthesis in vitro (biophysical and microbiological) was comproved that tracheal swine segment was the most indicated for small animal tracheal reconstruction.
No estudo para substitutos para segmentos extensos de traqueja canina, foram implantados heterotopicamente, biopróteses traqueais homólogas em 18 cães, sem raça definida, pesando entre 15 e 22kg, machos e fêmeas clinicamente sadios. Os animais foram divididos em dois grupos: Grupo I - nove cães com biopróteses implantadas em retalho muscular (Platisma). Grupo II -nove cães com bioprótese implantadas em retalho de grande omento. A avaliação macro e microscópica demonstrou a presença do processo de incorporação a partir do quadragésimo quinto dia nos dois grupos, acrescentando que o processo inflamatório apresentava-se mais organizado no segundo grupo sem a exuberância do tecido de granulação encontrado no primeiro grupo. Dentro do controle de qualidade das biopróteses, foi adicionado a este trabalho análises comparativas in vitro (biofísicas e microbiológicas) onde foi comprovado que a bioprótese traqueal suína apresentava-se estruturalmente como a mais indicada para a reconstrução traqueal em pequenos animais.
RESUMO
The contents of the present research is the result of experiments in dogs, substituting the third part of the tracheal length for bioprosthesis of homograft tracheas preserved in buffered glutaraldehyde at 2.5%. Fourteen mongreal dogs weighing between 10 and 20kg, had ortotopic implants of bioprosthesis by means of end-to-end anastomosis enveloped in a muscular flap (sternothyroideus e sternohyoideus). Post-operative clinic attendance, anatomopathological examinations, radiology and endoscopy were performed at the 7, 15, 30 and 45th days after the implantation.
O conteúdo do presente estudo é resultado da substituição de um terço do comprimento da traqueja de cães, por bioprótese de tecido traqueal homólogo preservado em glutaraldeído tamponado a 2,5%. Quatorze cães sem raça definida, pesando entre 10 e 20kg, receberam os implantes ortotópicos através de anastomose término-terminal, que em seguida foram envelopados com retalho muscular (sternothyroideus e sternohyoideus). Os animais foram acompanhados clinicamente durante o pós-operatório e as biopróteses avaliadas através de exames anatomo-patológicos, radiológicos e endoscópicos, no sétimo, décimo quinto, trigésimo e quadragésimo dia após a implantação.
RESUMO
The contents of the present research is the result of experiments in dogs, substituting the third part of the tracheal length for bioprosthesis of homograft tracheas preserved in buffered glutaraldehyde at 2.5%. Fourteen mongreal dogs weighing between 10 and 20kg, had ortotopic implants of bioprosthesis by means of end-to-end anastomosis enveloped in a muscular flap (sternothyroideus e sternohyoideus). Post-operative clinic attendance, anatomopathological examinations, radiology and endoscopy were performed at the 7, 15, 30 and 45th days after the implantation.
O conteúdo do presente estudo é resultado da substituição de um terço do comprimento da traqueja de cães, por bioprótese de tecido traqueal homólogo preservado em glutaraldeído tamponado a 2,5%. Quatorze cães sem raça definida, pesando entre 10 e 20kg, receberam os implantes ortotópicos através de anastomose término-terminal, que em seguida foram envelopados com retalho muscular (sternothyroideus e sternohyoideus). Os animais foram acompanhados clinicamente durante o pós-operatório e as biopróteses avaliadas através de exames anatomo-patológicos, radiológicos e endoscópicos, no sétimo, décimo quinto, trigésimo e quadragésimo dia após a implantação.