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For improving epitope immunogenicity and achieving the co-immunization, late protein 1 (L1) of HPV type 16 (HPV16L1) was selected as the vector to carry the dominant epitope of Toxoplasma gondii because of the shared common population between Toxoplasma gondii and human papillomavirus (HPV). RSepitope-HPV16L1 (RSepitope fused at the "N-terminus" of HPV16L1) and HPV16L1-RSepitope (RSepitope fused at the "C-terminus" of HPV16L1) chimeras were constructed. After transfection of COS-7 cells with the recombinants, Western blot, RT-PCR, and immunofluorescence experiments confirmed that RSepitope-HPV16L1 could successfully express the corresponding mRNA and protein of RSepitope and HPV16L1, but the HPV16L1-RSepitope construct could not. A "prime-boost" immunization program was applied in mice to further evaluate the immune response elicited by the constructs, and the RSepitope-HPV16L1 immunization group produced the most significantly increased humoral and cellular immune responses (the highest RSepitope-specific IgG antibody level and the highest IFN-γ production, respectively), in which both elevated Th1 and Th2 immune responses were obtained. Moreover, the advantage of HPV16L1 as an epitope carrier was remarkable for RSepitope-HPV16L1, which induced a more prominent immunological response than RSepitope alone (without fusion with HPV16L1). Our research indicated that the N-terminus of HPV16L1 could be a better insertion site for enhancing target epitope immunogenicity, and our study offers a design for epitope vaccine of reasonable combination.
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Animals , Mice , Antibody Formation , Epitopes , Immunization , Mice, Inbred BALB C , Toxoplasma , Vaccination , Vaccines, DNAABSTRACT
We predict in this paper B-cell epitopes of Epstein-Barr virus nuclear antigen-1 (EBNA-1) and analyze the results matched with the related autoantigens sequence of human. We selected EBV-1 standard strain NA-1 amino acid sequence as the basis. We predicted B-cell dominant epitopes of EBNA-1 with the methods of SOPMA, GOR and HNN, combined with the multi-parameter analysis of transmembrane domain, hydrophilicity profile, surface probability, antigenicity index, polarity and average flexibility. The blastp method was adopted to analyze the matched results between the predicted B-cell epitopes of EBNA-1 and the related autoantigens sequence of human. The results have shown that the possible B-cell dominant epitopes of EBNA-1 were located in the N terminal regions of 16-23, 35-78, 332-337, 340-357, 398-404, 419-432 and 620-637, in which different regions gained higher scores when matched with small nuclear ribonucleoprotein SmB, SmD, ribonucleoprotein SSA, heterogeneous nuclear ribonucleoprotein hnRNP A1, hnRNP G, respectively. It was available to predict B-cell dominant epitopes of EBNA-1 with multiparameter methods and to analyze the same or similar autoantigens sequences of human, which laid a theory foundation for the study of pathogenesis, diagnosis and treatment of autoimmune diseases.
Subject(s)
Humans , Amino Acid Sequence , Autoantigens , Allergy and Immunology , Base Sequence , Epitopes, B-Lymphocyte , Allergy and Immunology , Epstein-Barr Virus Nuclear Antigens , Allergy and Immunology , Molecular Sequence Data , Sequence Homology, Amino AcidABSTRACT
Objective To analyze the immunogenicity of selected B-cell epitopes of Epstein-Barr virus (EBV) latent membrane protein-2 (LMP2). Methods Three potential dominant B-cell epitopes of LMP2199-209, LMP2318-322 and LMP2381-391 from EBV LMP2 had been predicted using bioinformaties methods. The gene fragments of three epitopes were cloned respectively into pET32a(+) vector and transformed into E. coli strain BL21 (DE3). After identification by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, the expression products were purified by Ni-NTA agarose affinity chromatography. BALB/c mice in immunized groups were immunized by multi-point intracutaneous injection with the three purified epitope proteins,respectively; and mice in control groups were injected with pET32a (+) protein or phosphate buffered saline(PBS), respectively. The sera from mice at week O, week 3 and week 6 of injection were collected for determination of epitope-specific antibody IgG by enzyme linked immunosorbent assay (ELISA) using epitope proteins as coating antigens. The ability of serum antibody recognizing nature EBV antigen was determined at week 6 of immunization. Results Three epitope proteins of LMP2199-209 ,LMP2318-322 and LMP2381-391 were successfully expressed in prokaryotic system. Epitopespecific antibodies IgG could be detected respectively in the sera of all immunized mice, and the levels of antibodies increased with immunized time increasing. The antibody levels in LMP2318-322 immunized group at week 3 and week 6 were significantly higher than that of pET32a (+) protein control group (F= 493.85 and 773.99, respectively; both P＜0. 05), and the antibody levels in LMP2381-391 immunized group at week 3 and week 6 were also significantly higher than that of pET32a (+) protein control group (F= 926.33 and 309.14, respectively; both P＜0.05). Antibody level in LMP2199-209 immunized group at week 6 was significantly higher than that of pET32a ( + ) protein control group (F=87.27, P＜0.05). The antibody IgG in serum from immunized mice with three epitope proteins could all recognize nature EBV antigens, especially LMP2199-209 and LMP2381-391 immunized groups.Conclusions Three possible dominant epitopes of LMP2199-209, LMP2318-322 and LMP2381-391 from EBV LMP2 are prepared by prokaryotic expression system and exhibit obvious immunogenicity, which could be used for further research of EBV infection and related tumor vaccine.
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Objective To explore the HLA-A2 restriction and immunogenicity of 5 previously identified HCV-speeific CTL epitopes. Methods Based on T2 cell, to explore the HLA-A2 restriction of previously identified HCV-specific CTL epitopes by MHC-peptide complex stabilization assay;To detect pep-tide-specific CTL in HLA-A2~+ PBMC stimulated by HLA-A2-restricted peptides by intracellular cytokine staining(ICS) and ELISPOT; To explore the cytotoxicity of peptide-specific CTL to same peptide-loaded T2 cells (target cells) by CTL cytotoxicity test. Results Among 5 previously identified CTL epitopes NS4b_78 (SMMAFSAAL) and NS5a_367 (TVSSALAEL) have high-affinity for HLA-A2 molecules(FI 1) ;ELISPOT results shown that NS4b_78(SMMAFSAAL) and NSSa_367(TVSSALAEL) induced high levels of IFN-γ-se-creting cells [(60±6) SFC/10~4 PBMC vs (4±1 ) SFC/10~4 PBMC, P < 0.01 ; (10 ± 3 ) SFC/10~4 PBMC vs (2±1 ) SFC/10~4 PBMC, P <0.01, respectively] ;ICS results indicated that there were high percentages of CD8~+ IFN-γ~+ T cells in total CD8~+T cells stimulated by these peptides [(2.33 ±0.22 ) % vs (0.05±0.01)%, P <0.001 ; (0.36±0.06)% vs (0.03±0.01)%, P <0.001, respectively]. Furthermore,peptide-specific CTL could effectively kill same peptide-loadcd T2 cells. Conclusion NS4b_78 (SMMAF-SAAL) and NSSa_367 (TVSSALAEL) were identified as HLA-A2-restricted CTL epitopes which could in-duce immune response in vitro.
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To predict the B cell epitopes for major outer membrane protein (MOMP) of Chlamydia trachomatis (CT), the secondary structure of CT MOMP was predicted by the methods of GOR based on the sequence of amino acids of E serotype CT MOMP. By combining the comprehensive analysis of transmembrane domain, hydrophilicity profile, surface probability, antigenic index and average flexibility, the B cell predominant epitopes of CT MOMP were further predicted. The N-terminal No. 73-81, 217-225, 377-386, 261-270 and 161-175 were the predominant B cell epitopes. Prediction of the B cell epitopes for the CT MOMP by the multi-parameters is helpful for the identification of B cell epitopes.
Subject(s)
Amino Acid Sequence , Antigens, Bacterial , Allergy and Immunology , Chlamydia trachomatis , Classification , Allergy and Immunology , Epitopes, B-Lymphocyte , Allergy and Immunology , Molecular Sequence Data , Porins , Allergy and Immunology , Protein Conformation , Protein Structure, Secondary , SerotypingABSTRACT
OBJECTIVE: To evaluate the therapeutic effects of Chinese materia medica in treating patients with different syndromes by tongue image analysis software 1.0 based on tongue colors, and to discuss the feasibility of applying this computer science-based techniques into drug evaluation. METHODS: The tongue colors and the areas of tongue fur were examined and analyzed by the tongue image analysis software 1.0 in healthy persons and the patients with different syndromes before and after treatment. The parameters of tongue colors consisted of the followings: the hue (H), the lightness (L), the saturation (S), and the values of red (R), green (G) and blue (B). RESULTS: Obvious differences could be revealed in tongue color index between the healthy persons and the patients in five groups of different syndromes. There also existed some significant differences in those index between patients before and after treatment. CONCLUSION: The tongue image analysis software 1.0 based on tongue colors is helpful to evaluate the therapeutic effects of Chinese materia medica.
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Objective To explore the mechanism of fibrosis in autoimmune hepatitis(AIH). Methods By using synaptophysin (SYN) as a new marker for hepatic stellate cells (HSCs),HSCs,collagen I,collagen IV,and MT-MMP-1 were detected by immunohistochemistry,and the expressions of MT-MMP-1 and TIMP-1 mRNA were assessed by in situ hybridization in liver tissues obtained by needle biopsy from 36 AIH patients. Results The HSCs were observed in the portal tracts,fibrotic septa and lobules of AIH liver tissues where inflammation was active,especially in the interface of inflammatory and non-inflammatory areas. The number of HSCs increased in proportion to the increase in histoligical active index (HAI,Knodell),while the deposition of Col I and Col IV were increased with increase in hepatic fibrosis stages (Knodell). MT-MMP-1 and its mRNA were mainly expressed in mesenchymal cells which were distributed in the areas of interface of inflammation and borders of fibrotic septa. It was also observed in a few hepatocytes. The expression of MT-MMP-1 was parallel to collagen IV distribution,and increased with advancement of HAI and fibrosis stages,reaching the peak at S4-5 stage. In addition,the expression of TIMP-1 mRNA was similar to that of MT-MMP-1 mRNA. Conclusions The results of immunohistochemistry and in situ hybridization suggested persistant active inflammation,triggering the activation and proliferation of HSC,and the resultant deposition of extracellular matrix such as collagen IV and I might be one of pathogenetic mechnisms of hepatic fibrosis in AIH. The increased expression of MT-MMP-1 in liver tissues of AIH in parallel with the advancement fibrotic stages also suggested that the relative lower level of ECM degeneration due to metalloproteinase suppression might be another reason for fibrogenesis and development of fibrosis in AIH. In addition,it was shown that synaptophysin was another good marker for HSC.
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Objective To study the effect of the traditional Chinese medicinal herbs, Fufangbiejiaruanganpian (FFBJRGP), in an experimental model of hepatic fibrosis and its pharmacodynamics. Methods An experimental model of hepatic fibrosis induced by carbon tetrachloride in rat was reproduced, and FFBJRGP was given in high, moderate, and low dosage for 0, 1, 3 and 6 months respectively. Six months after the treatment, matrix metalloproteinase MMP-2, MMP-13, MT-MMP-1, MT-MMP-2 and their inhibitor TIMP-1 and TIMP-2, total extracellular matrix and collagen I, Ⅲ and Ⅳ, and active hepatic stellate cells in the fibrotic livers were qualitatively and quantitatively examined at the protein and/or mRNA expression levels by using immunohistochemistry, in situ hybridization, image analysis and Chevallier's scoring system. Meanwhile, enzyme-degrading activities of MMP-2 and MMP-13 were assessed with gelatin or collagen substrate zymography respectively. Results Compared with the control group, in which rats with hepatic fibrosis were not treated with FFBJRGP, the histological examination of rat livers in the treatment groups showed that the total scores of hepatic fibrosis in treatment groups with varions dosage were significantly decreased 3,6 months after the treatment and 3,6 months after the termination of treatment (P
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Objective To explore the mechanism of Fufangbiejiaruanganpian(FFBJRGP) treating liver fibrosis. Methods Needle biopsies before and after treatment with FFBJRGP were done in 65 patients with chronic viral hepatitis B, and the liver tissues were studied with Ishak scoring system to evaluate the effects of treatment. The activation, proliferation and apoptosis of hepatic stellate cells (HSCs) in the liver specimens were determined by using the double immunohistochemical staining of in situ terminal deoxynucleotidyl transferase mediated dUTP nick end labelling method (TUNEL) and smooth muscle actin (SMA). Results Compared with the specimens before treatment, the stages of liver fibrosis and histological activity grades in liver tissues were significantly improved after treatment with FFBJRGP for 6 months (mean value: P
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Objective To assess the efficacy of extensive esophagogastric devascularization with splenectomy for surgical treatment of portal hypertension complicating cirrhosis of liver, and to explore the pathogenesis of chronic congestive splenomegaly. Methods A retrospective analysis of clinical data of 232 patients of portal hypertension complicating cirrhosis of liver having undergone extensive esophagogastric devascularization with splenectomy was made. Pathological alterations and extracellular matrix productive cells of the congestive splenomegaly were studied both immunohistochemically and histologically. Results The functional markers including the numbers of PLT, WBC, and PTA in the peripheral blood and serum Alb were significantly improved after the operation compared with that of before the operation. Careful pre-operation preparation, replenishment of blood loss during the operation, postoperative drainage of the splenic bed, and prevention of complications were efficiently carried out. The mean volume of CCS spleens was 1 423.67?738.69cm 3. There was an obvious increase in the numbers of vimentin-, ?-SMA-positive cells in the CCS tissues, as well as CD68-positive macrophages. Conclusions The results indicated that extensive esophagogastric devascularization with splenectomy was a reasonably effective alternative therapy for patients with portal hypertension complicated by esophageal varices and congestive splenomegaly. Adequate replacement of blood lost in prevention of operation and postoperative complications were essential for satisfactory recovery of the petient. The activation of macrophagic system, with proliferation of fibroblasts and myofibroblasts, might participate in the pathogenesis of congestive splenomegaly.
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Objective To clone and express the fused gene fragment coding rhoptry protein ROP2 and major surface protein P30 from Toxoplasma gondii as a preparation for the construction of the complex ROP2,P30 antigen by gene engineering.Methods The gene fragment encoding P30 was amplified by PCR from T.gondii RH strain and subcloned into the recombinant plasmid pUC119/ROP2 already constructed.The recombinant plasmid pUC119/ROP2,P30 was digested by SacⅠ/HindⅢ and inserted into the same site of expression vector pET28b.The recombinant plasmid of pET28b/ROP2,P30 was transformed to E.coli and expressed under the induction of IPTG.Results The gene fragment 700 bp encoding P30 was obtained from the total DNA of T.gondii by PCR.The recombinant plasmid pET28b/ROP2,P30 was successfully constructed,which was highly expressed in E.coli,a fusion protein with molecular weight of 69 000.Conclusion The fusion gene encoding the rhoptry protein ROP2 and the major surface protein P30 of T.gondii has been successfully cloned and expressed to be an expected recombinant fusion protein ROP2,P30 with molecular weight 69 000.
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Objective To explore the clinical pathological features of Wilson's disease and the pathogenesis of liver fibrosis. Methods The clinical data and liver biopsy specimens obtained from 48 children with Wilson's disease were analysed. The pathological changes were studied with light microscopy, electron microscopy, combined with rhodanine and rubeanic acid for copper staining, Gordon-Sweet's staining for reticular fibers and Masson's staining for collagen fibers. Meanwhile, immunohistochemistry was used to investigate the expression of tissue inhibitors of metalloproteinase (TIMP) -1 and TIMP-2. The apoptosis of activated hepatic stellate cells (HSC) in liver tissues was illustrated with in situ end labeling (ISEL)(TUNEL POD method) and ?-SMA double staining. Results In all the cases, the mean onset age was 10.0?3.8 years, and the positive rates of family history, Kayser-Fleischer’s ring and decreased serum ceruloplasmin level were 29.2%, 68.8% and 93.0%, respectively. The levels of serum ALT, AST, ALP and ?-Glo were 3.6, 3.0, 2.7 and 2.0 fold of normal cutoff values. The major pathological changes in childhood patients with Wilson’s disease presented various chronic inflammatory changes in hepatic acini and portal tracts, interface hepatitis, focal or diffuse vesicular/ microvesicular steatosis, with large and irregular apoptotic bodies, Mallory's bodies, glycogenated nuclei, and eosinophilc granular hepatocytes. Among all the cases, 77.0% of liver specimens were positive for rhodanine and rubeanic acid staining for copper in hepatocyts, especially in the zone I of acinus. Ultrastructural observation showed swollen and unusual giant mitochondria, increased lysosomes and vesicular inclusions in hepatocytes. The incidence of hepatic fibrosis was 100%, presenting expanded portal tracts in the early, fibrotic septa in the moderate and cirrhosis in the late stage. The extent of TIMP-1 and TIMP-2 expression and number of activated HSC were increased in various degrees in all the liver specimens, while apoptotic HSCs were obviously decreased in the majority of cases. Conclusions The clinical and pathological changes of children with Wilson’s disease are varied and relatively obscure, and liver fibrosis appears early and progressive. Excessive activation and proliferation of HSC stimulated by liver injury and inflammation due to copper deposition and the decrease in activity of matrix degradation enzymes might be the important mechanisms underlying the occurrence and progression of hepatic fibrosis in Wilson's disease.
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Objective Liver function in perioperative periods, postoperative complications, and pathological changes in the liver were studied and compared between patients undergoing emergent and elective surgical intervention (extensive esophagogastric devascularization, EED) for portal hypertension (PTH), with the purpose to elucidate the pathogenesis of PTH. Methods The clinical data and liver biopsies from 150 cases of inpatients with hepatis cirrhosis and PTH who underwent either emergent (28 cases) or elective (100 cases) surgical intervention including extensive esophagogastric devascularization (EED) in 302 th Hospital of PLA were analysed. Liver biopsy was done in 128 patients, and the expression of ?-smooth muscle actin (?-SMA), tubulin?and ?in hepatic stellate cells (HSCs), and endothelin-1 (ET-1) was histochemically studied in the liver tissue. Results It was found that the mean internal diameter of portal veins before surgery was larger and the incidence of pre-operative acute variceal haemorrhage was significantly higher in the emergent EED group than those in the elective EED group (P