RESUMO
BACKGROUND: Despite the genotype 4 has become the dominant cause of hepatitis E disease in China, none antigen derived from genotype 4 of hepatitis E virus (HEV) was used in current commercial anti-HEV immunoassay, and the serological reactivity of antigen derive from genotype 4 is not well-charactered. METHODS: We expressed and purified the 4 main immuno-dominant epitopes derived from genotype 1 and 4 including ORF2 (410-621aa) of genotype 4, ORF3 (47-114aa) of genotype 4, ORF2 (396-606aa) of genotype 1 and ORF3 (56-123aa) of genotype 4. RESULTS: The ORF2 of genotype 4 displayed good diagnostics performance according to ROC analysis using in-house panel, and the immunoassays based the ORF2 of genotype 4 was then developed to detect the anti-HEV IgG antibodies and evaluated further in 530 anti-HEV IgG positive specimens and 380 negative specimens. The sensitivity and the specificity is 98.1% (520/530) and 94.7% (360/380) for immunoassay based on ORF2 of genotype 4, 96.6% (512/530) and 92.6% (352/380) for commercial immunoassay based on genotype 1. It is noted that all of the positive samples will be detected by combing two assays together. The anti-HEV immunoassays based on genotype 4 are in accordance with Chinese anti-HEV national standard,and show an good agreement of 95.8% with commercial assay (kappa=0.913, P=0.014). CONCLUSIONS: The immunoassay based on ORF2G4 displays good performance, and combining assay based on genotype 1 together with genotype 4 will benefit the HEV diagnosis in large scale samples.
Assuntos
Antígenos Virais , Testes Diagnósticos de Rotina/métodos , Anticorpos Anti-Hepatite/sangue , Vírus da Hepatite E/imunologia , Hepatite E/diagnóstico , Epitopos Imunodominantes , Virologia/métodos , Antígenos Virais/genética , China , Humanos , Imunoensaio/métodos , Epitopos Imunodominantes/genética , Proteínas Recombinantes/genética , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: To study the pulmonary pathology in patients died of fatal human influenza A(H1N1) infection. METHODS: Eight cases of fatal human influenza A (H1N1) infection, including 2 autopsy cases and 6 paramortem needle puncture biopsies, were enrolled into the study. Histologic examination, immunohistochemitry, flow cytometry and Western blotting were carried out. RESULTS: The major pathologic changes included necrotizing bronchiolitis with surrounding inflammation, diffuse alveolar damage and pulmonary hemorrhage. Influenza viral antigen expression was detected in the lung tissue by Western blotting. Immunohistochemical study demonstrated the presence of nuclear protein and hemagglutinin virus antigens in parts of trachea, bronchial epithelium and glands, alveolar epithelium, macrophages and endothelium. Flow cytometry showed that the apoptotic rate of type II pneumocytes (32.15%, 78.15%) was significantly higher than that of the controls (1.93%, 3.77%). CONCLUSION: Necrotizing bronchiolitis, diffuse alveolar damage and pulmonary hemorrhage followed by pulmonary fibrosis in late stage are the major pathologic changes in fatal human influenza A (H1N1) infection.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Influenza Humana/patologia , Pulmão/patologia , Adolescente , Adulto , Idoso , Células Epiteliais Alveolares/patologia , Antígenos Virais/metabolismo , Apoptose , Autopsia , Biópsia por Agulha , Bronquiolite Viral/patologia , Criança , Pré-Escolar , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/metabolismo , Influenza Humana/mortalidade , Influenza Humana/virologia , Pulmão/imunologia , Pulmão/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/metabolismo , Alvéolos Pulmonares/patologia , Fibrose Pulmonar/patologia , Adulto JovemRESUMO
AIM: To evaluate the efficacy of a new hepatitis C virus (HCV) core antigen assay developed in China. METHODS: After the determination of HCV infection, 49 serial samples were selected from 11 regular plasma donors in 5 different plasma stations. To compare the performance of HCV core antigen detection and HCV PCR, these samples were genotyped, and each specimen was analyzed by ELISA for the detection of HCV core antigen and by qualitative HCV PCR. RESULTS: Among all of the sequential samples, the original 13 specimens were HCV RNA-negative, and 36 samples were HCV RNA-positive. Twenty-seven samples (75%) were HCV core antigen-positive from these HCV RNA-positive specimens. Conversely, 27 samples (93.1%) were found HCV RNA-positive in HCV core antigen-positive samples. Intervals between HCV RNA and HCV core antigen-positive, as well as between HCV core antigen-positive and HCV antibody-positive were 36.0 and 32.8 d, respectively. CONCLUSION: This HCV core antigen assay, developed in China, is able to detect much of anti-HCV-negative, HCV RNA-positive preseroconversion window period (PWP) plasma donations.
Assuntos
Antígenos Virais/sangue , Doadores de Sangue , Hepacivirus/imunologia , Hepatite C/diagnóstico , Testes Sorológicos/métodos , Proteínas do Core Viral/sangue , China , Análise Custo-Benefício , Ensaio de Imunoadsorção Enzimática/economia , Hepatite C/sangue , Hepatite C/transmissão , Humanos , Técnicas de Amplificação de Ácido Nucleico/economia , RNA Viral/sangueRESUMO
A gold nanoparticle probe-based assay (GNPA) was developed for ultrasensitive detection of Hepatitis C virus (HCV) core antigen. In the GNPA, after anti-HCV core antigen polyclonal antibodies and single-stranded barcode signal DNA were labeled on gold nanoparticle probe (NP), DNA enzyme was used to degrade the unbound barcode DNAs. The anti-HCV core antigen monoclonal antibodies were coated on magnetic microparticles probe (MMP). Then the NP-HCV core antigen-MMP sandwich immuno-complex was formed when the target antigen protein was added and captured. Magnetically separated, the immuno-complex containing the single-stranded barcode signal DNA was characterized by TaqMan probe based real-time fluorescence PCR. A detection limit of 1 fg/ml was determined for the HCV core antigen which is magnitude greater than that of ELISA (2ng/ml). The coefficients of variation (CV) of intra-assay and inter-assay respectively ranged from 0.22-2.62% and 1.92-3.01%. The improved GNPA decreased the interference of unbound barcode DNAs and may be an new way for HCV core antigen detection.
Assuntos
Antígenos Virais/análise , Ouro , Hepacivirus/isolamento & purificação , Hepatite C/diagnóstico , Imunoensaio/métodos , Nanopartículas , Proteínas do Core Viral/análise , Humanos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Human cytomegalovirus (hCMV), a ubiquitous beta-herpesvirus, has been associated with several autoimmune diseases. However, the direct role of hCMV in inducing autoimmune disorders remains unclear. Here we report the identification of an autoantibody that recognizes a group of peptides with a conserved motif matching the Pp150 protein of hCMV (anti-Pp150) and is shared among patients with various autoimmune diseases. Anti-Pp150 also recognizes the single-pass membrane protein CIP2A and induces the death of CD56(bright) NK cells, a natural killer cell subset whose expansion is correlated with autoimmune disease. Consistent with this finding, the percentage of circulating CD56(bright) NK cells is reduced in patients with several autoimmune diseases and negatively correlates with anti-Pp150 concentration. CD56(bright) NK cell death occurs via both antibody- and complement-dependent cytotoxicity. Our findings reveal that a shared hCMV-induced autoantibody is involved in the decrease of CD56(bright) NK cells and may thus contribute to the onset of autoimmune disorders.
Assuntos
Anticorpos Antivirais/metabolismo , Autoanticorpos/metabolismo , Doenças Autoimunes/patologia , Citomegalovirus/imunologia , Homeostase/efeitos dos fármacos , Células Matadoras Naturais/efeitos dos fármacos , Proteínas Virais/imunologia , Antígenos Virais/imunologia , Autoantígenos/imunologia , Sobrevivência Celular/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Células Matadoras Naturais/fisiologia , Proteínas de Membrana/imunologiaRESUMO
OBJECTIVE: To explore the mechanism of Bushen Qiangji Granule (, BSQJ) in restraining the osteogenic differentiation of ankylosing spondylitis (AS) fifibroblasts. METHODS: Hip joint capsules were obtained from AS patients (n=10) receiving total hip replacement and healthy hip joint capsules from patients with hip fracture (n=10) receiving surgery as a control. Finite fifibroblast lines were established from these tissue samples to observe the effect of BSQJ on suppressing osteogenic differentiation of fifibroblasts. The expression of osteogenic marker gene corebinding factor a1 (Cbfa1) and Smad family proteins were examined by Western blot and real-time quantitative polymerase chain reaction (qPCR). RESULTS: The mRNA expression level of Cbfa1 was significantly higher in AS fibroblasts than that in normal fibroblasts and the expression of pSmad1, pSmad5, Smad4 and Cbfa1 in AS fibroblasts was also higher, demonstrating the activation of the BMP/Smads signal pathway in AS fifibroblasts. BSQJ-medicated serum not only restrained the mRNA and protein expression levels of Cbfa1 and inhibited protein expression level of Smad4 but also decreased the expression quantities of pSmad1 and pSmad5. CONCLUSIONS: BSQJ can inhibit osteogenic differentiation of AS fifibroblasts in vitro by suppressing the activation of the BMP/Smads signal pathway. This may be the important molecular mechanism of BSQJ in regulating AS ossifification.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Fibroblastos/patologia , Osteogênese/efeitos dos fármacos , Soro/metabolismo , Proteínas Smad/metabolismo , Espondilite Anquilosante/patologia , Adulto , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Pessoa de Meia-Idade , Osteogênese/genética , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Espondilite Anquilosante/genética , Adulto JovemRESUMO
OBJECTIVE: To explore the effects of Elemene, the essential oil of Curcuma wenyujin, on Bone morphogenetic protein/drosophila mothers against decapentaplegic proteins (BMP/SMADs) signal pathway in ankylosing spondylitis (AS) fibroblasts. METHODS: Hip joint capsules were obtained from AS patients (n=10) receiving total hip replacement. Healthy hip joint capsules from patients with hip fracture (n=10) receiving surgery were included as a control. Primary fibroblast cell lines were established from these tissue samples. Fibroblasts were incubated with Elemene for 48 hours. The protein expression was detected by Western blot. The mRNA expression was detected by real-time fluorescent quantitative polymerase chain reaction (PCR). RESULTS: The results showed that the expression of proteins including SMAD1, pSMAD1, SMAD4 and Runt-related transcription factor 2 (RUNX2), and mRNA of RUNX2, which were over-expressed in AS fibroblasts were decreased in the AS fibroblasts cultured in medium with Elemene. CONCLUSIONS: Ele could have a hand in anti-osteogenic differentiation of AS fibroblasts by inhibiting the BMP/SMADs signal pathway and subsequently blocking expression of ossification marker genes RUNX2 that initiate the osteogenic differentiation.
Assuntos
Curcuma , Fibroblastos/metabolismo , Sesquiterpenos/farmacologia , Espondilite Anquilosante/metabolismo , Adulto , Artroplastia de Quadril , Proteínas Morfogenéticas Ósseas/biossíntese , Diferenciação Celular , Linhagem Celular , Subunidades alfa de Fatores de Ligação ao Core/biossíntese , Fibroblastos/efeitos dos fármacos , Articulação do Quadril/metabolismo , Humanos , Cápsula Articular/metabolismo , Masculino , Pessoa de Meia-Idade , Ossificação Heterotópica/metabolismo , Osteoblastos/metabolismo , Óleos de Plantas/farmacologia , Transdução de Sinais , Proteínas Smad/biossíntese , Espondilite Anquilosante/cirurgia , Adulto JovemRESUMO
AIM: To analyze the amino acid sequences of hypervariable region 1 (HVR1) of HCV isolates in China and to construct a combinatorial chimeric HVR1 protein having a very broad high cross-reactivity. METHODS: All of the published HVR1 sequences from China were collected and processed with a computer program. Several representative HVR1's sequences were formulated based on a consensus profile and homology within certain subdivision. A few reported HVR1 mimotope sequences were also included for a broader representation. All of them were cloned and expressed in E.coli. The cross-reactivity of the purified recombinant HVR1 antigens was tested by ELISA with a panel of sera from HCV infected patients in China. Some of them were further ligated together to form a combinatorial HVR1 chimera. RESULTS: Altogether 12 HVR1(s) were selected and expressed in E.coli and purified to homogeneity. All of these purified antigens showed some cross-reactivity with sera in a 27 HCV positive panel. Recombinant HVR1s of No. 1, 2, 4, and 8# showing broad cross-reactivities and complementarity with each other, were selected for the ligation elements. The chimera containing these 4 HVR1s was highly expressed in E.coli. The purified chimeric antigen could react not only with all the HCV antibody positive sera in the panel but also with 90/91 sera of HCV -infected patients. CONCLUSION: The chimeric antigen was shown to have a broad cross-reactivity. It may be helpful for solving the problem caused by high variability of HCV, and in the efforts for a novel vaccine against the virus.
Assuntos
Reações Cruzadas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologia , Sequência de Aminoácidos , Humanos , Dados de Sequência MolecularRESUMO
OBJECTIVE: To detect humoral immune response against different function regions of hepatitis C virus (HCV) in chronic patients, and further to investigate the correlativity between anti-HCV antibody titers and HCV RNA concentration. METHODS: Using recombinant dominate epitope antigens, e.g. HCV Core, NS3, NS4, NS5 and chimeric HVR1, a set of ELISA test reagents was formulated. Then, titers of antibodies against HCV different regions and the RNA concentration of HCV in chronic patient sera were detected by ELISA and quantitative RT-PCR technique, respectively. RESULTS: Great differences have been noted in antibody titers and positive rate of different HCV function regions in chronic patients. Antibodies against HCV Core and HVR1 have the highest positive rate, then NS3, NS4, and NS5 in sequence. CONCLUSION: The titer of antibodies against different regions of HCV in chronic patients has good correlation with HCV RNA concentration.
Assuntos
Anticorpos Anti-Hepatite C/sangue , Hepatite C Crônica/imunologia , RNA Viral/sangue , Hepatite C Crônica/virologia , HumanosRESUMO
AIM: To evaluate the presence and cross-reactive antibodies against hypervariable region 1 (HVR1) in hepatitis C virus (HCV) infected patients and its relationship with the progression of the disease. METHODS: Sixteen representative HVR1 proteins selected from a unique set of 1600 natural sequences were used to semiquantitate the cross-reactivity of HVR1 antibodies in the sera of HCV patients. Fifty-five chronic HCV patients including 23 with asymptomatic mild hepatitis, 18 with chronic hepatitis and 16 with liver cirrhosis patients were studied. RESULTS: The degree of the cross-reactivity of anti-HVR1 antibodies in 23 patients with mild asymptomatic hepatitis was 3.09 ± 2.68, which was significantly lower than in those with chronic hepatitis (5.44 ± 3.93, P < 0.05) and liver cirrhosis (7.44 ± 3.90, P < 0.01). No correlation was observed between the broadness of the cross-reactivity anti-HVR1 antibodies and patient's age, infection time, serum alanine aminotransferase activity, or serum HCV-RNA concentration. It was the breath of cross-reactivity rather than the presence of anti-HVR1 antibody in HCV sera that was associated with the progression of liver disease. CONCLUSION: The broadly cross-reactive HVR1 antibodies generated in natural HCV patients can not neutralize the virus, which results in persistent infection in patients with chronic hepatitis.
Assuntos
Formação de Anticorpos/imunologia , Reações Cruzadas/imunologia , Anticorpos Anti-Hepatite C/imunologia , Hepatite C Crônica/imunologia , Proteínas Virais/imunologia , Adulto , Sequência de Aminoácidos , Feminino , Hepatite C Crônica/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Análise Serial de Proteínas , Alinhamento de SequênciaRESUMO
Tetramer analysis is a novel technique in immunological research that has dramatically changed our knowledge of the immune response to pathogens, tumors and autoimmune disease. Through the formation of major histocompatibility complex (MHC)-peptide tetrameric complexes, it can provide accurate counts of antigen-specific T-cells and it allows their phenotypical and functional analysis. The tetramer is composed of the human leukocyte antigen (HLA) heavy chain, beta-2 microglobulin (beta-2m), the nominal peptide, and streptavidin. The HLA heavy chain and the beta-2m are expressed in Escherichia coli. But up to now, all laboratories have been expressing these two proteins by using isopropyl beta-d-thiogalactopyranoside IPTG. IPTG is very expensive, and it is tedious and laborious to induce expression protein. So it is difficult to scale up to express the objective protein. To address this problem, extracellular fractions of HLA-A0201 and beta-2m (absent signal peptide) genes were cloned from peripheral blood mononuclear cells (PBMCs) by RT-PCR. DNA coding for a Gly-Ser linker and a BSP (15-amino acid substrate peptide for BirA-dependent biotinylation) was added to the COOH-terminus of the extracellular fraction of HLA-A0201 by PCR, using an HLA-A0201 as the template. Then the HLA-A0201-BSP and beta-2m genes were cloned into pBV220 vector and expressed, respectively. The expressed proteins were purified and detected by ELISA and Western blot analyses. High-efficient expressions of HLA-A0201-BSP and beta-2m proteins lay a good foundation for further expression and purification in prokaryotic system and constructing MHC class I-peptide tetramer complexes to study the function of CTLs.