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Aim: To investigate the prevalence of autoantibodies and their associations with clinical features in Chinese patients with chronic hepatitis B (CHB)., Methods: A total of 325 Chinese patients with CHB were enrolled in this retrospective, hospital-based study. Patients with chronic hepatitis C (CHC), autoimmune hepatitis (AIH), or primary biliary cirrhosis (PBC) were included, with healthy donors acting as controls. A panel of autoantibodies that serologically define AIH and PBC was tested by indirect immunofluorescence assay and line immunoassay. The AIH-related autoantibody profile included homogeneous anti-nuclear antibodies (ANA-H), smooth-muscle antibodies, anti-liver kidney microsome type 1, anti-liver cytosolic antigen type 1, and anti-soluble liver antigen/liver pancreas; the PBC-related antibodies were characterized by ANA-nuclear dots/membranous rim-like, anti-mitochondrial antibodies-M2 (AMA-M2), anti-BPO (recombinant antigen targeted by AMA-M2), anti-Sp100, anti-promyelocytic leukemia protein (anti-PML), and anti-gp210. The dichotomization of clustering was used to unequivocally designate the AIH or PBC profiles for each case. Anti-Ro52 antibodies were also tested., Results: The prevalence of any autoantibody in CHB amounted to 58.2%, which was similar to the 66.2% prevalence in CHC, significantly higher than the 6.7% in the healthy controls (P < 0.001), and lower than the 100% found in AIH and PBC (P = 0.004 and P < 0.001, respectively). There were more anti-PML and anti-gp210 antibodies among the CHB patients than the CHC patients (11.1% vs 0%, P = 0.003; 12.6% vs 0%, P < 0.001, respectively). The prevalence and titer of AMA, anti-BPO, anti-PML, and anti-gp210 were higher in PBC than in those with CHB. Among the CHB patients, the prevalence of ANA, especially ANA-H, was significantly lower in patients with compensated and decompensated cirrhosis compared with patients without cirrhosis. Thirty-eight cases of hepatocellular carcinoma (HCC) in CHB showed a significant difference compared with non-HCC patients in the prevalence of anti-PML (0% vs 12.5%, P = 0.013). Dichotomization of the autoantibodies revealed that the PBC profile was more prevalent in patients with CHB than in those with CHC, and that it was strongly correlated with both compensated and decompensated cirrhosis. In contrast, the prevalence of the AIH profile was significantly higher in non-cirrhosis patients with CHB than in those with compensated cirrhosis (18.5% vs 8.2%, P = 0.039). Moreover, the AIH profile was also closely associated with hepatitis B e-antigen positivity., Conclusion: ANA-H could be an indicator of early-stage CHB. Dichotomizing the autoantibody profiles revealed that the PBC profile is strongly associated with cirrhosis in CHB.

Chibby (Cby) inhibits Wnt/β-catenin-mediated transcriptional activation by competing with Lef-1 (the transcription factor and target of β-catenin) to bind to β-catenin. This suggests that Cby could be a tumor suppressor protein. In the present study, we examined Cby expression in laryngeal squamous cell carcinoma (LSCC) and its function and mechanism in laryngeal carcinoma cell lines. Cby expression levels were investigated by immunohistochemistry in a panel of 36 LSCC patient cases. The expression of β-catenin, c-myc and cyclin D1 in Hep-2 were determined through RT-PCR and western blot analysis. Activity of Wnt/β-catenin signaling pathway after overexpression of Cby was measured by TCF/LEF luciferase reporter gene assay. Proliferation, clone forming ability, cell cycle distribution and cell apoptosis of Hep-2 cells were detected by MTT assay, plate colony forming assay, flow cytometry and TUNEL assay, respectively. This study showed that expression of Cby protein was strongly downregulated in LSCC tumor tissues in comparison to normal laryngeal mucosa samples. No significant correlation was found between the expression of Cby in tumor tissue and gender, age, clinical stage and tumor differentiation of laryngeal cancer patients. When Cby was overexpressed in Hep-2 cells, the expression of cyclin D1 was reduced and β-catenin activity was inhibited. Proliferation and plate colony forming assays revealed a significant inhibitory effect of Cby on growth and colony formation ability of Hep-2 cells after Cby overexpression in comparison to control and mock-infected cells. In addition, we also found that upregulated expression of Cby resulted in accumulation of numbers of cells in G0/G1 phase with concomitant decrease in S phase by cell cycle assay. TUNEL staining demonstrated that, compared with the control group, the rate of apoptosis in the plv-cs2.0-Cby group was significantly increased. Taken together, downregulation of Cby was observed in LSCC, but with no significant correlation to the clinicopathological features of LSCC patients. Overexpression of Cby effectively suppressed laryngeal carcinoma cell growth and promoted its apoptosis. A better understanding of the mechanisms of Cby gene activation in LSCC could provide potential novel therapeutic targets for human laryngeal carcinoma.

Wnt-β-catenin signaling participates in the epithelial-mesenchymal transition (EMT) in a variety of cancers; however, its involvement in hepatocellular carcinoma (HCC) and downstream molecular events is largely undefined. HNF4α is the most prominent and specific factor maintaining the differentiation of hepatic lineage cells and a potential EMT regulator in HCC cells. However, the molecular mechanisms by which HNF4α maintains the differentiated liver epithelium and inhibits EMT have not been completely defined. In this study, we systematically explored the relationship between Wnt-β-catenin signaling and HNF4α in the EMT process of HCC cells. Our results indicated that HNF4α expression was negatively regulated during Wnt-β-catenin signaling-induced EMT through Snail and Slug in HCC cells. In contrast, HNF4α was found to directly associate with TCF4 to compete with β-catenin but facilitate transcription co-repressor activities, thus inhibiting expression of EMT-related Wnt-β-catenin targets. Moreover, HNF4α may control the switch between the transcriptional and adhesion functions of β-catenin. Overexpression of HNF4α was found to completely compromise the Wnt-β-catenin-signaling-induced EMT phenotype. Finally, we determined the regulation pattern between Wnt-β-catenin signaling and HNF4α in rat tumor models. Our studies have identified a double-negative feedback mechanism controlling Wnt-β-catenin signaling and HNF4α expression in vitro and in vivo, which sheds new light on the regulation of EMT in HCC. The modulation of these molecular processes may be a method of inhibiting HCC invasion by blocking Wnt-β-catenin signaling or restoring HNF4α expression to prevent EMT.

Objective: To establish chemiluminescence enzyme immunoassay (CLEIA) for quantitative detection of procollagen III N-terminal peptide (P III NP) in serum., Methods: A sandwich reaction was preformed with horseradish peroxidase labeled monoclonal antibody of P III NP as the catalytic enzyme and the luminol as the luminescence reagent. Several reactions liquid's concentration and reaction conditions were optimized. The method was evaluated in all aspects such as linear range, sensitivity, specificity, stability and so on. The CLEIA was compared with imported ELISA kits, by detecting clinical serum., Results: The linear range was 0.8-85 ng/ml. The detection limit was 0.5 ng/ml. Inter-assay and intra-assay RSD were both less than 10%. The recoveries of three different spiked concentration samples were 96.2%, 91.2% and 101.1%. After stored at 4 degrees C and 37 degrees C for 3, 5, 7 days, the analysis showed correlation coefficient higher than 0.99 and RSD lower than 6%. The detected results of clinical sera with CLEIA closely corresponded to those with imported ELISA., Conclusion: Established CLEIA for quantity determination of serum P III NP has high accuracy, sensitivity and repeatability.

Objective: To establish enzyme-linked immunosorbent assay (ELISA) for quantitative detection of Golgi protein73 (GP73) in serum., Methods: A sandwich reaction was preformed with horseradish peroxidase labeled monoclonal antibody of GP73 as the catalytic enzyme. Several reactions liquid's concentration and reaction conditions were optimized. The method was evaluated in all aspects such as linear range, sensitivity, specificity, stability and so on., Results: The linear range was 25-500 ng/ml. The detection limit was 18.5 ng/ml. Inter-assay and intra-assay RSD were both less than 10%. The recoveries of three different spiked concentration samples were 95.3%, 92.6% and 103.7%. After stored at 4 degrees C and 37 degrees C for 3, 5, 7 days, the analysis showed correlation coefficient higher than 0.98 and RSD lower than 10%., Conclusion: Established ELISA for quantity determination of serum GP73 has high accuracy, sensitivity and repeatability.

Objective: To establish enzyme-linked immunosorbent assay (ELISA) for detection of hepatitis B virus large surface protein(HBV-LP) in serum., Methods: A sandwich reaction was preformed with horseradish peroxidase labeled monoclonal antibody of HBV-LP as the catalytic enzyme. Several reactions liquid's concentration and reaction conditions were optimized. The method was evaluated in all aspects such as sensitivity, specificity, stability and so on., Results: The detection limit was 5 ng/ml. Interassay and intra-assay RSD were both less than 10%. After stored at 4 degrees C and 37 degrees C for 3, 5, 7 days, the analysis showed correlation coefficient higher than 0.98 and RSD lower than 10%., Conclusion: Established ELISA for determination of serum HBV-LP has high sensitivity and repeatability. Enzyme-linked immunosorbent assay;

Objective: To explore the levels of serum GP73 in patients with fatty liver disease., Methods: The sera GP73 were determined by ELISA in 178 patients with fatty liver disease and 100 healthy controls., Results: Serum GP73 levels were significantly increased in patients with various fatty liver diseases(70.62 +/- 60.60 ng/ml), compared with those of control population (35.61 +/- 12.22 ng/ml). In patients with alcoholic fatty liver disease, acute liver injury, chronic hepatitis B, and non-alcoholic fatty liver disease, their serum GP73 concentration were 81.86 +/- 47.82 ng/ml, 82.77 +/- 77.73 ng/ml, 63.84 +/- 50.62 ng/ml, and 65.75 +/- 62.20 ng/ml, respectively. But no significant difference was found between these groups (P > 0.05). In 68 patients with F > or = 1.0 (71.46 +/- 66.48 ng/ml), 75 patients with F> or = 2.0 (69.58 +/- 62.31 ng/ml), and 34 patients with F3-F4 (71.65 +/- 43.89 ng/ml), there were also no marked differences was observed between these fatty groups (F = 0.02, P = 0.98)., Conclusion: Serum GP73 levels were increased in patients with different liver diseases, but its concentrations were seems not related with degree of fatty injury.

Objective: To clone and express human Golgi glycoprotein73 protein, and prepare the monoclonal antibody (mAb) against the protein., Methods: GP73 gene was amplified from HepG2 cells by RT-PCR, then ligated with pQE31 to form recombinant plasmid pQE-GP73 and transformed into E. coli BL21. The protein induced by IPTG was purified by 6 x His-tag and used to immunize the BALB/c mice. The specific monoclonal antibodies (mAbs) were prepared by the cell fusion technique. Western Blot was used to detect specificity of mAbs., Results: The prokaryotic plasmid expressing the recombinant protein was constructed, and the GP73 recombinant protein was expressed and purified. Five hybridoma cell lines that secreted anti-GP73 mAbs were obtained. 2 of 5 mAbs were the IgG1 subtype. Western Blot indicated the mAbs showed specific combination with GP73 protein., Conclusion: The GP73 recombinant protein is highly purified and has strong antigenicity. The anti-GP73 mAbs were prepared successfully.

Objective: To establish microplate chemiluminescence enzyme immunoassay (CLEIA) for quantitative analysis of tissue inhibitor of metalloproteinases I (TIMP I) in human serum., Methods: A sandwich reaction was preformed with horseradish peroxidase(HRP) labeled monoclonal antibody of TIMP I as the catalytic enzyme and the H2O2-luminol as the luminescence reagent. Several physical and chemical parameters were studied and optimized such as immunoreaction conditions, the dilution ratio of TIMP I-HRP, luminescence reaction time and so on. In order to evaluate the method, recovery test, heat stabilization test and comparison test were carried out., Results: The linear range was 0. 2-12 ng/ml with r = 0.996. The detection limit was 0.12 ng/ml. Inter-assay and intra-assay RSD were both less than 10%. The recoveries of three different spiked concentration samples were 100.6%, 96.5% and 106.5%. After stored at 4 degrees C and 37 degrees C for 3, 5, 7 days, the analysis showed correlation coefficient higher than 0. 998 and RSD lower than 6%. The detected results with CLEIA closely corresponded to those with imported ELISA in 60 patients sera with liver fibrosis., Conclusion: Established CLEIA for quantity determination of serum TIMP I has high accuracy, sensitivity and repeatability.

Wnt signalling through β-catenin and the lymphoid-enhancing factor 1/T-cell factor (LEF1/TCF) family of transcription factors maintains stem cell properties in both normal and malignant tissues; however, the underlying molecular pathway involved in this process has not been completely defined. Using a microRNA microarray screening assay, we identified let-7 miRNAs as downstream targets of the Wnt-β-catenin pathway. Expression studies indicated that the Wnt-β-catenin pathway suppresses mature let-7 miRNAs but not the primary transcripts, which suggests a post-transcriptional regulation of repression. Furthermore, we identified Lin28, a negative let-7 biogenesis regulator, as a novel direct downstream target of the Wnt-β-catenin pathway. Loss of function of Lin28 impairs Wnt-β-catenin-pathway-mediated let-7 inhibition and breast cancer stem cell expansion; enforced expression of let-7 blocks the Wnt-β-catenin pathway-stimulated breast cancer stem cell phenotype. Finally, we demonstrated that the Wnt-β-catenin pathway induces Lin28 upregulation and let-7 downregulation in both cancer samples and mouse tumour models. Moreover, the delivery of a modified lin28 siRNA or a let-7a agomir into the premalignant mammary tissues of MMTV-wnt-1 mice resulted in a complete rescue of the stem cell phenotype driven by the Wnt-β-catenin pathway. These findings highlight a pivotal role for Lin28/let-7 in Wnt-β-catenin-pathway-mediated cellular phenotypes. Thus, the Wnt-β-catenin pathway, Lin28 and let-7 miRNAs, three of the most crucial stem cell regulators, connect in one signal cascade.

Objective: To prepare the monoclonal antibody (mAb) against tissue inhibitor of metalloproteinases I (TIMP-I) fusion protein., Methods: TIMP-I gene was amplified from fibrotic human liver tissue by RT-PCR, then ligated with pQE31 to form recombinant plasmid pQE-TIMP-I and transformed into E. coli BL21. The protein induced by IPTG was purified by 6 x His-tag and used to immunize the BALB/c mice. The specific monoclonal antibodies (mAbs) were prepared by the cell fusion technique. Western Blot were used to detect specificity of mAbs., Results: The prokaryotic plasmid expressing the recombinant protein was constructed, and the TIMP-I recombinant protein was expressed and purified. Four hybridoma cell lines that secreted anti-TIMP-I mAbs were obtained. 3 of 4 mAbs were the IgG1 subtype. Western Blot indicated the mAbs showed specific combination with TIMP-I protein., Conclusion: The TIMP-I recombinant protein is highly purified and has strong antigenicity. The anti- TIMP-I mAbs were prepared successfully.

Objective: To evaluate the clinical performance of careHPV16/18/45 DNA testing of cervical specimens as a triage testing for women with positive findings during the cervical cancer screening., Methods: Eligible women aged 25-65 years were enrolled from two high-risk communities in Yangcheng County,Shanxi Province.After providing written informed consent on a voluntary base,women underwent questionnaire-based interview,gynecological examination,and sample collection.Hybrid capture 2 technology(HC2),careHPV,Avantage HPV E6 test,and visual inspection with acetic acid(VIA)were conducted as the primary screening tests at the enrollment visit.Women with any positive finding were invited to receive a second VIA and colposcopy.careHPV16/18/45 was performed as a triage testing.Any visible lesion under colposcopy was directly biopsied.Women with pathology confirmed cervical intraepithelial neoplasia grade 2 and worse(CIN2+)were treated with standard procedures., Results: For the self-collected and doctor-collected samples,the application of careHPV16/18/45 as a triage testing decreased the colposcopy referral to 3.2% and 3.1%,respectively.Meanwhile,the sensitivity,specificity,and positive predictive value(PPV)for CIN2+were 50.0%,97.6%,and 26.7% for women with positive self-sampling careHPV results and 63.0%,97.9%,and 34.0% for women with positive doctor-sampling careHPV results., Conclusion: careHPV16/18/45 is promising as a triage testing among women with positive screening findings in low-resource settings.

Objective: To establish a purificatory method of alpha-fetoprotein variant (AFP-L3) based on microspincolumn with lens culinaris agglutinin (LCA)., Methods: LCA was isolated by ammonium sulfate precipitation method from lens culinaris. AFP-L3 affinity adsorption microspincolumns which were made from LCA coupled with activated Sepharose 4B were prepared. By adding into the centrifuge column, serum was absorbed and eluted to purify AFP-L3. The results of purified AFP-L3 detection of 10 cases AFP positive sera by electro-chemiluminescence immunoassay were compared with traditional crossed affinity immunoelectrophoresis., Results: 8 of 10 cases AFP-L3 concentration were greater than 5 ng/ml in purified sera. Six cases show positive reaction in affinity immune cross electrophoresis experiment., Conclusion: Successfully established purification method of AFP-L3 by affinity absorption based on microspincolumn. The method was more conducive to clinical laboratory applications due to its high sensitive and easy operation.

Objective: To estimate the consistency of two VITROS 3600 chemiluminescent analyzers according to the requirement of ISO15189., Methods: Verification tests were made for precision and accuracy of anti-HCV in two instruments. While 40 serum samples including Anti-HCV negative (10 cases) , positive (10 cases) , and weakly positive (20 cases). and the test results were statistical analised., Results: Two instruments negative and positive control samples intra-batch precision and coefficients of variation were 5% , 4% and 7. 14% , 7. 23% , inter-batch precision and coefficients of variation were 9. 47% , 7. 7% and 8.04%, 7. 6%, are less than requirement CV (15%) by ISO15189. The accuracy of two instrument were 100% , The test results of the control samples showed no significant difference (P < 0. 05). The correlation analysis of the test results of clinical samples R2 =0. 9984, with good consistency., Conclusion: Test results of two Vitros 3600 has good consistency and comparability.

Objective: Evaluated the chemiluminescence and enzyme-linked immunosorbent assay (ELISA) to detect HIV antibodies, and compared the results, to provide a reference for the selection and clinical application of HIV screening., Methods: 3000 cases of our hospital patients were measured by enzyme-linked immunosorbent assay and chemiluminescence immunoassay, using comfirmming experimental results as gold standards. Comparing sensitivity, specificity and other Indicators., Results: In the diagnosis of HIV infection, enzyme-linked immunosorbent assay and chemiluminescence immunoassay had no significant difference. The positive rate of enzyme-linked immunosorbent assay was 0.93%, while the sensitivity and specificity were 89.66%, 99.93%, the positive rate of chemiluminescence immunoassay was 1.03%, while the sensitivity and specificity were 100%, 99.93%, respectively., Conclusion: Both methods are suitable for screening of HIV, having high specificity, and chemiluminescence has greater sensitivity than ELISA.

Objective: Establish a confirmatory test based on ELISA, and use to verify the authenticity of HBsAg weak positive samples, pick and get rid of the false result, and avoid the mistake diagnosis., Method: The particles (reagent A) coated by streptavidin and biotinylated HBsAb (reagent B) were mixed in different proportions, then neutralized with serum whose the COI of HBsAg > 20 by ELISA in order to identify the activity of HBsAb in confirmatory reagent. 30 pieces of HBsAg weak positive serum neutralized with the confirmatory reagent, the serum were considered to be positive if rate of decline of HBsAg COI > 50%. The results were compared to Roche confirmatory Kit., Result: Confirmatory reagent was able to neutralized with HBsAg. 24 of 30 pieces of HBsAg weak positive samples were judged to be positive, while 6 poeces were negative. The ELISA comfirm method is fully consistent with Roche confirmatory Kit., Conclusion: The ELISA confirmatory test for suspicious HBsAg positive samples is a simple, accurate and low cost initial validation method, After further clinical trials, should be widely applied.

Objective: To understand the hemagglutination inhibition antibody level in patients with influenza A H1N1., Methods: Sera from 28 patients with influenza A H1N1 at different time points after illness onset were collected and measured by hemagglutination inhibition assay., Results: The serum hemagglutination inhibition antibody titers at 1, 5, 15, 22, 37, 49 and 58 days after illness onset were 5.36, 9.39, 39.02, 57.99, 137.92, 55.19 and 57.99 respectively. The top geometric mean titer of hemagglutination inhibition antibody was 148.55. The antibody seroconversion rate and seroprotection rate were occurred in 96.4% (27/28) of patients., Conclusion: The patients with influenza A H1N1 have effective immune response.

Aims: Wnt signalling is involved in cellular homeostasis and development. Dysregulation of the Wnt signalling pathway has been linked to colorectal cancer. The orphan nuclear receptor TR3 plays important roles in proliferation and apoptosis. In this study, we investigated how TR3 suppresses intestinal tumorigenesis by regulating Wnt signalling., Methods: Intestinal polyps were quantified in Apc(min/+), Apc(min/+)/TR3(-/-) and Apc(min/+)/villin-TR3 mice. Wnt signalling activity was evaluated by assessing β-galactosidase activity in a BAT-Gal reporter strain. The TR3 agonist cytosporone B was used to evaluate the role of TR3 in intestinal tumorigenesis. Crosstalk between TR3 and β-catenin/TCF4 was analysed by molecular methods in colorectal cancer cells. The phosphorylation of TR3 by glycogen synthase kinase (GSK) 3β and the correlation between GSK3β activity and TR3 phosphorylation were evaluated in clinical samples and colorectal cancer cells., Results: TR3 was found to significantly suppress Wnt signalling activity and the proliferation of intestinal epithelial cells. Apc(min/+)/TR3(-/-) mice developed more intestinal polyps than Apc(min/+)/TR3(+/+) mice, whereas either transgenic overexpression of TR3 in the intestine or treatment with cytosporone B in Apc(min/+) mice significantly decreased intestinal tumour number. Mechanistically, TR3 disrupted the association of β-catenin and TCF4 on chromatin and facilitated the recruitment of transcriptional co-repressors to the promoters of Wnt signalling target genes. However, TR3 was phosphorylated by GSK3β in most clinical colorectal cancers, which attenuated the inhibitory activity of TR3 towards Wnt signalling., Conclusions: TR3 is a negative regulator of Wnt signalling and thus significantly suppresses intestinal tumorigenesis in Apc(min/+) mice. This inhibitory effect of TR3 may be paradoxically overcome through phosphorylation by GSK3β in clinical colorectal cancers.

Two new C(13)-norisoprenoids, named lyratols E and F (1-2), were isolated from the whole plant of Solanum lyratum Thunb, and their structures were elucidated by extensive spectroscopic analyses. In vitro, two new compounds were found to show significant cytotoxicity against selected cancer cells including P-388 and HT-29.

Objective: Establish a kind of confirmation method based on ELISA, and use to verify authenticity of HBsAb + in HBsAg + HBsAb + serum, pick and get rid of the false masculine gender the result, and avoid the mistake diagnosis., Method: Collect 60 pieces of serum whose thick degree of HBsAg at 1000 COI above tested by ECLIA as confirm serum, mixed the confirm serum of different dilution with HBsAb positive serum to screen and verify best thick degree of HBsAg. Collected 40 pieces of HBsAg + HBsAb + serum, ELISA tested the descend rate of HBsAb COI after neutralized with confirm serum in order to confirm authenticity of HBsAb + in pieces of HBsAg + HBsAb + serum., Result: When thick degree of HBsAg is 2000 COI, the performance of neutralization to HBsAb is best. The ELISA confirmatory test is fully consistent with the ECLIA method with true positive of 37 pieces of HBsAg + HBsAb + serum while false-positive of 3 pieces of serum., Conclusion: The ELISA confirm method is a simple, accurate and low cost initial validation method.

Objective: To evaluate the clinical value of an enzyme-linked immunosorbent assay by the Helicobacter pylori stool antigen (HpSA) test for the detection of H. pylori infection., Methods: 328 patients were measured upper gastrointestinal endoscopic examination which as gold standards and HpSA test in the meantime, comparing accuracy, sensitivity, specificity and other Indicators., Results: In the diagnosis of Hp infection, HpSA test had no significant difference comparing with gold standards and had a P value of over 0.5. The sensitivity of HpSA test was 94.6%, while the specificity, veracity, expected positive value, and expected negative value were 96.9%, 96.3%, 89.7% and 98.4%, respectively., Conclusion: The H. pylori stool antigen test is a simple, non-invasive method for accurate diagnosis of H. pylori infection.

Objective: To quantitatively detect intrahepatic HBV covalently closed circular DNA (cccDNA) and serum HBsAg; and to analyze the relationship between the two parameters and with serum HBV DNA level., Methods: Intrahepatic cccDNA (copies/cell) was quantitated by plasmid-safe ATP-dependent Danes (PSAD) digestion in combination with rolling circle amplification and gap-spanning selective real-time PCR assay using formalin fixed paraffin-embedded liver biopsy samples. HBsAg was measured by chemiluminescence's reagent manufactured by Abbott Company using sera sampled at time-point of liver biopsy., Results: Intrahepatic cccDNA level was positively correlated with serum HBsAg level (r = 0.459, P < 0.001), but not correlated with serum HBV DNA level. Serum HBsAg level was positively correlated with serum HBV DNA level (r = 0.328, P = 0.015), and reversely correlated with HBV replicative efficiency defined as the ratio of serum HBV DNA to cccDNA (r = -0.373, P = 0.005)., Conclusion: In patients with chronic hepatitis B, intrahepatic cccDNA level is correlated with serum HBsAg level. The two parameters combined with serum HBV DNA may comprehensively reflect HBV replication activity and help evaluation of antiviral therapeutic efficacy.

Aim: To develop a sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of serum hepatitis B virus large surface protein (HBV-LP), and study the clinical value of HBV-LP., Methods: Serum HBV-LP levels and a panel of other HBV markers were investigated in a large population of patients with chronic HBV. The clinical value of HBV-LP was evaluated by comparing the coincidence of detection of HBV markers and the change of serum HBV-LP level during antiviral therapy., Results: The ELISA was found to be sensitive and specific for the detection of HBV-LP. Serum HBV-LP level was positively correlated with HBV DNA (r=0.743) in HBV patients. Among the five HBV markers tested, HBV-LP displayed the highest coincidence rate (94.7%) with HBV DNA., Conclusions: Serum HBV-LP was strongly correlated with HBV DNA. This ELISA therefore offers a promising approach for the diagnosis and treatment monitoring of HBV patients., (Copyright © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Glioma is a common malignant tumor of the human neural system, and Wnt signaling activation is closely connected with glioma malignancy. Pygopus 2 (Pygo2) was recently discovered as a component of the Wnt signaling pathway regulating β-catenin/Tcf dependent transcription. However, the role of Pygo2 in glioma cells has not yet been defined. In the current study, we investigated the role of Pygo2 in rat glioma C6 cells for the first time. Our results showed that over-expression of Pygo2 promoted cell proliferation as well as enhanced cell cycle progression from G₁ to S phase associated with an increase in the expression of the Wnt target gene cyclin D1. In contrast, knockdown of Pygo2 suppressed cell proliferation with cell cycle block from G₁ to S phase and down-regulation of cyclin D1. In addition, the expression of Pygo2 and cyclin D1 in 67 glioma tissue samples was quantified by real-time reverse transcription polymerase chain reaction (RT-PCR) and immunochemistry. The data indicated that tumor grade was significantly associated with over-expression of Pygo2 and cyclin D1. We conclude that Pygo2 is highly expressed in and promotes the growth of glioma cells by an increase in the expression of cyclin D1 to improve G₁/S transition.

We analyzed changes in immunologic values over time for 28 hospitalized patients with pandemic (H1N1) 2009. Levels of interleukin-6, interferon-y, and interleukin-10 increased 1 day after illness onset and then decreased to baseline levels. Levels of virus-specific antibody were undetectable 1 day after illness onset and peaked 36 days later.

Objective: Assessment of detection of IgM antibodies for human enterovirus 71 (EV 71) in early diagnosis for the hand, foot and mouth disease (HFMD)., Method: The sera and throat swabs from 38 patients which were clinical diagnosis as HFMD, were continuous daily collected in our hospital in 2010. These specimens were detected by EV 71 IgM antibodies assay, real time RT-PCR methods for EV 71 and Enterovirus., Results: Among 38 HFMD patients, the cumulative positive rates of EV 71 IgM antibodies were: 60.5% on day 1, 71.1% on day 2, 81.5% in the first 3-4 days, 92.1% on day 5, 92.1% on day 6, and the positive rate of nucleic acid detected by the real time RT-PCR for EV 71 and Enterovirus were 60.5%, 73.6%., Conclusion: The positive rate of EV 71 IgM antibodies in the hand, foot and mouth disease just can occur on day 1, and reach to peak on day 5, which can be used as one of indicators of early diagnosis of hand, foot and mouth disease.

Objective: To establish the real-time fluorescent PCR method for detecting enterovirus, enterovirus 71 and Coxsackievirus A 16 nucleic acid., Methods: Primers and MGB probe were chosen for virus gene. The samples of 38 HFMD patients were analyzed by TaqMan-MGB PCR technique on a fluorescence real-time PCR instrument,and the results were compared with those by conventional RT-PCR., Results: The real-time fluorescent PCR positive rates of EV, EV71 and Cox A16 were 73.7%, 60.5%, 13.2%; the conventional RT-PCR were 71.1%, 55.3%, 13.2%. There were no significant differences between the two methods., Conclusion: The real-time fluorescent PCR detecting method of EV, EV71 and Cox A16 nucleic acid have been established successfully.

Objective: To verify a new kit of "universal and novel influenza A (H1N1) virus nucleic acid double-detection methods (PCR-fluorescence probe)"., Methods: 150 cases of throat swab specimens were collected consecutively. After RNA was extracted, the specimens were detected by the verified kit. At the same time, the same specimens were detected by Real-time PCR diagnostic kit from Beijing CDC as the control. The data were analysed by the Kappa in agreement and by McNemar chi2 in difference test., Results: The consistency rate of the verified kit and the Beijing CDC kit was universal primer M 97.33%, H1N1 98.67% respectively. The Kappa test and McNemar chi2 test showed that two methods had a higher consistency. Compared to the CDC kit, the "false negative rate" and "false-positive rate"of double-check kit were lower., Conclusion: The kit of "universal and novel influenza A (H1N1) virus nucleic acid double-detection methods (PCR-fluorescence probe)" from Shanghai Kehua Bio-Engineering Co., Ltd can be used to detect influenza A and novel influenza A (H1N1).

Gliomas are common malignant tumors of the human neural system, and Wnt signaling activation is closely related to glioma malignancy. Human Pygopus 2 (Pygo2) was recently discovered to be a component of the Wnt signaling pathway, which is required for β-catenin/Tcf-dependent transcription. However, the role of Pygo2 in glioblastoma cell growth and survival remains uncertain. In the present study, Pygo2 expression was evaluated in 80 glioma tissue samples. Results demonstrated that tumor grade exhibited a positive correlation with overexpression of Pygo2. In addition, small hairpin RNA (shRNA) was used to specifically knockdown Pygo2 expression in human glioblastoma U251 cell lines. Results showed that inhibition of Pygo2 expression resulted in inhibited cell proliferation and invasiveness, as well as increased cell cycle arrest at the G(1) stage and decreased expression of the Wnt target gene cyclin D1. These results demonstrated that Pygo2 was highly expressed in glioma tissue and required for growth of glioblastoma cells.

Objective: To investigate the characters and changes of peripheral white blood cells and lymphocyte subsets of patients with pandemic influenza A virus (H1N1) infection and to provide evidences for diagnosis, treatment and prognosis of influenza A (H1N1) infection., Methods: Peripheral white blood cell parameters and the percentages of lymphocyte subsets in acute and recovery phases of 59 cases of influenza A virus (H1N1) infectious patients (42 mild cases and 17 severe cases) were investigated and analyzed, and compared respectively with those of 43 cases of healthy adults as control (HC) and 24 cases of general influenza A virus (no-H1N1) infectious using whole blood cell analysis and flow cytometry., Results: Peripheral white blood cell counts of mild cases decreased greatly but those of severe cases did not decrease significantly; the neutrophils of severe cases increased significantly in acute phase; similar to general influenza A virus (no-H1N1) infectious, the peripheral lymphocytes, CD3, CD4, CD8 and B cells of all patients with influenza A virus (H1N1) infection decreased greatly in acute phase and quickly recovered in recovery phase; NK and NKT cells absolute counts of severe cases decreased significantly in acute phase, and the decreasing extent of which were more than 20%., Conclusion: There were similar characteristics of change in peripheral white blood cells and lymphocyte subsets between patients with pandemic influenza A virus (H1N1) infection and general pandemic A virus (No-H1N1); the great decrease of NK and NKT cells absolute counts may suggest the severe tendency of diseases.

Objective: To determine the association between elevated levels of serum cancer antigen (CA) 125 and hepatitis cirrhosis in different stage, and also to explore the clinical application value of serum CA-125., Methods: During June to December in 2008, 200 cases with hepatitis cirrhosis were random selected in our hospital. CA-125 levels were measured by electrochemiluminescence immunization assay in sera collected from these cases which were termed with Child-Paugh classification and analyzed by SAS., Results: Serum CA-125 levels were correlated closely with ascites, primary peritonitis and liver function Child-Paugh classification, but no associated with primary carcinoma of liver and other liver function index,such as ALT, AST, ALB, TBIL and PT., Conclusion: The levels of serum CA-125 in hepatitis cirrhosis patients were osculating correlating with lesion of liver and ascite degree, could serve as a sensitive and conventional laboratory index for liver lesion degree and monitoring ascite generation. It was necessary to further study on the association with serum CA-125 level with primary hepatic carcinoma.

Objective: To study the prevalence rate of upper respiratory tract group A Streptococcus (GAS) carriage in school-age children from Xinjiang Province., Methods: A total of 478 children at age of 9-12 years from Tulufan City and Buerjin County of Xinjiang Province were enrolled by random cluster sampling. Throat swab cultures were performed once each season for the determination of presence of GAS., Results: In the 1 827 samples, 196 GAS strains were isolated, with a GAS carrier rate of 10.7%. The prevalence rate of GAS carrier in Tulufan City ranged from 3.7%-16.5% compared with 4.7%-21.4% in Buerjin County (P < 0.05). The prevalence rate of GAS carrier in winter is the highest, followed by in autumn, spring and summer in both regions. There were significant differences in the GAS carriage rate in autumn between the two regions. There were no significant differences in the GAS carriage rate between boys and girls. Of the 196 GAS strains, 133 from Han, 22 from Uygur and 41 from Hazakh children. There were significant differences in the prevalence rate of GAS carriage among children with different ethic groups., Conclusions: The prevalence rate of GAS carriage is high in school-age children from Tulufan and Buerjin of Xinjiang Province. The GAS carrier rate is associated with the season and ethic group. The children from Buerjin County present a higher GAS carrier rate than those from Tulufan City.

Objective: To analysis the clinical and laboratory characteristics of Patients infected with new influenza A (HIN1) virus., Methods: All cases with new influenza A (H1N1) confirmed on polymerase chain reaction assay on throat swabs. There were included in a prospective evaluation of clinical characteristics, laboratory results, treatment and overcome of new influenza A (H1N1)., Results: There were 35 patients in the epidemic. Clinical illness developed within a mean of 1.7 days. Fever occurred in 97.1%, sore throat 65.7% cough 51.4%, headache 28.6%, and myalgia 31.4%. All patients were treated with oseltamivir lasted 5 days. The mean duration of viral shedding was 4.5 days. All were cured and left hospital after day 7., Conclusion: It was infected by new influenza A (H1N1) typically in this epidemic.

Considering the non-Newtonian gravity proposed in grand unification theories, we show that the stability and observed global properties of neutron stars cannot rule out the supersoft nuclear symmetry energies at suprasaturation densities. The degree of possible violation of the inverse-square law of gravity in neutron stars is estimated using an equation of state of neutron-rich nuclear matter consistent with the available terrestrial laboratory data.

Within an isospin- and momentum-dependent hadronic transport model, it is shown that the recent FOPI data on the pi;{-}/pi;{+} ratio in central heavy-ion collisions at SIS/GSI energies [Willy Reisdorf, Nucl. Phys. A 781, 459 (2007)10.1016/j.nuclphysa.2006.10.085] provide circumstantial evidence suggesting a rather soft nuclear symmetry energy E&#95;{sym}(rho) at rho> or =2rho&#95;{0} compared to the Akmal-Pandharipande-Ravenhall prediction. Some astrophysical implications and the need for further experimental confirmations are discussed.

Objective: To evaluate the detection method of ELISA and Enhanced Chemiluminescence Immunoassay (ECLIA) in use to determine serum hyaluronate acid (HA), laminin (LN), type IV collagen (IV-C) and type III procollagen (PC III)., Methods: 253 patients with chronic hepatitis B were determined the four liver fibrosis serum markers with both the ECLIA and ELISA, and then compared with pathology results separately., Results: Both the detection results of ELISA and ECLIA can reflect that the patient's liver fibrosis from hepatitis to liver cirrhosis aggravated gradually. Compared with ELISA, the results of ECLIA and pathology have a better correlation., Conclusions: The detection of four liver fibrosis serum markers by ECLIA could indicate the better the response of the state of live fibrosis.

Objective: To explore the significance of Lens culinaris-reactive alpha-Fetoprotein (AFP-L3) detection in primary hepatocellular carcinoma., Methods: AFP-L3 was isolated by using microspin column coupled with lens culinaris agglutinin (LCA), AFP and AFP-L3 were detected with chemiluminescent immunoassay, the proportion of AFP L3 levels were calculated, and the relationship between the elevated AFP-L3 (%) levels and benign and malignant liver disease was analyzed., Results: There were significant differences in positive rate between the patients of HCC, suspected HCC and other liver disease (81.80%, 73.68%, 11.80%, respectively, P < 0.05). Among the undetermined HCC (suspected HCC, liver disease) patients, 12 out of 21 cases of AFP-13 positive were diagnosed to be HCC within 6 months, and 6 of them were diagnosed to be the single small HCC at the early stage through B-Ultrasonic Diagnosis or CT. Among 62 cases of AFP-L3 negative, 3 cases were diagnosed to be HCC within 6 months and the risk of occurrence of HCC for AFP-L3 positive increased 11.9 times., Conclusion: AFP-L3 has no correlation with AFP value, and it can be used as an independent HCC diagnosis factor. The detection of AFP-L3 has a significant implication for the identification of benign or malignant liver disease and the early stage predictive diagnosis of HCC while AFP increases.

In the present study, immunoproteomic analysis was utilized to systemically characterize global autoantibody profiles in autoimmune hepatitis (AIH). Sera from 21 patients with AIH and 15 healthy controls were analyzed for the antibody reactivity against the protein antigens of HepG2, a human hepatoma cell line. The lysates of HepG2 cells were separated by two-dimensional electrophoresis and then immunoblotted with each serum sample. Matrix-assisted laser desorption/ionization mass spectrometry or/and nanoelectrospray ionization MS/MS were then used to identify antigens, among which a bifunctional enzyme in mitochondrial, fumarate hydratase (FH), was further analyzed by ELISA using recombinant FH as a coating antigen. A total of 18 immunoreactive spots were identified as 13 proteins, 8 of which have not been reported in AIH. Immune reactivity to FH was detected in 66.67% of patients with AIH, 19.35% of patients with primary biliary cirrhosis (PBC), 12.31% of patients with chronic hepatitis B (CHB), 6.35% of patients with chronic hepatitis C (CHC), 11.32% of patients with systemic lupus erythematosus (SLE), and 3.57% of normal individuals. The differences of prevalence between AIH patients and healthy controls as well as other diseases were of statistical significance (P<0.001). These data demonstrate the serological heterogeneity in AIH and suggest the diversity of the mechanisms underlying AIH. FH, recognized mainly in AIH rather than in viral hepatitis and other autoimmune diseases, may have utility in improved diagnosis of AIH.

Background: Severe acute respiratory syndrome (SARS) is a disease with a mortality of 9.56%. Although SARS is etiologically linked to a new coronavirus (SARS-CoV) and functional cell receptor has been identified, the pathogenesis of the virus infection is largely unclear., Methods: The clinical specimens were processed and analyzed using an indirect enzyme-linked immunosorbent assay (ELISA) in-house. Further investigations of target antigen included reviews of phage display technique, rapid amplification of cDNA ends (RACE) technique, protein expression and purification, Western blotting validation, serological and immunohistochemical staining in postmortem tissue., Results: A type of medium or low titer anti-lung tissue antibodies were found in the sera of SARS patients at the early stage of the disease. Human long interspersed nuclear element 1 (LINE1) gene endonuclease (EN) domain protein was one of the target autoantigens and it was aberrantly expressed in the lung tissue of SARS patients. Anti-EN antibody was positive in the sera of 40.9% of SARS patients., Conclusions: Human LINE1 endonuclease domain was identified as a putative target of SARS-associated autoantibodies, which were presented in the serum of SARS patients and may be involved in the pathogenesis of SARS.

Aim: To construct a prokaryotic expression vector containing human interleukin-10 (IL-10) gene, express and identify the protein., Methods: Human IL-10 gene from HepG2 was isolated and identified by DNA sequencing, and then cloned into the expression vector PET-28 a (+) to construct prokaryotic expression vector. The recombinant protein was expressed in BL21(DE3) and identified by Western blot and ELISA., Results: The expressed protein was mainly located in the inclusion body. The relative molecular mass of the expressed product was identical to that of prediction. The expressed protein had binding activity with specific antibody., Conclusion: The human IL-10 gene was successfully cloned and expressed in E.coli. The expressed product had antigenicity which provides foundation for preparation of monoclonal antibodies against IL-10.

Background: To explore the significance of hepatitis B virus PreS1-Ag, PreS2-Ag, large protein (LP) detection and the prediction of viral replication., Methods: PreS1-Ag, PreS2-Ag, LP and HBV markers were measured by enzyme linked immunosorbent assay (ELISA) in 201 cases of infected serum. Serum HBV DNA level was quantitatively detected by real-time polymerase chain reaction (PCR)., Results: There were significant differences in positive rate between the PreS1-Ag, PreS2-Ag, LP, and HBsAg; the positive rate of PreS2-Ag and LP were higher than that of the HBeAg. No significant differences were found in the positive rates between LP and the levels of HBV DNA and there was a positive correlation between quantitations of HBV DNA and HBV-LP., Conclusion: Serum PreS1-Ag, PreS2-Ag and LP were laboratory markers that can accurately reflect HBV DNA reproduction, and were helpful complementarity to traditional HBV M. There is a close correlation between the number of copies of HBV DNA and the levels of HBV-LP.

Background: To investigate the biological functions of a novel hepatitis B virus e antigen (HbeAg) interacting protein AK026018, and to use cDNA microarray technique to screen genes regulated by the protein., Methods: The AK026018 coding DNA fragment was amplified by reverse transcription polymerase chain reaction (RT-PCR) technique from HepG2 cell. The expressive vector of pcDNA3.1-AK was constructed by routine molecular biological methods. The HepG2 cells were transfected with pcDNA3.1 and pcDNA3.1-AK, respectively by using lipofectamine. The total RNA was isolated and reverse transcribed. The cDNA of each sample was subjected to microarray screening with 8,464 cDNA probes and analyzed by bioinformatics., Results: The expressive vector was constructed and confirmed by DNA sequencing analysis and restriction enzyme digestion. High quality mRNA and cDNA of transfected HepG2 cells had been prepared and successful microarray screening conducted. From the scanning results, there were 122 differential expression genes, of which 36 genes were down-regulated, and 16 genes were up-regulated., Conclusion: Microarray technique was successfully used to screen the genes trans-regulated by AK026018. The expression of AK026018 protein affects the expression spectrum of HepG2 cells.

Objective: To screen and clone the genes in hepatocytes which encode protein that can interact with hepatitis B e antigen(HBeAg) by yeast-two hybridization., Methods: Recombined HBeAg bait plasmid (pGBKT7-eAg) was transformed into yeast AH l09, followed by mating with yeast Yl87 containing liver cDNA library plasmid in 2 x YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-Ade-His) which contains X-a-gal for selecting positive blue clones. Then positive clones were selected and plasmids were prepared and sequenced. Finally, bioinformatics analysis was performed., Results: Totally 245 positive colonies were selected and 101 colonies were sequenced. Through sequences alignment, 6 novel genes and 35 recorded genes were screened., Conclusion: Genes of HBeAg interacting proteins have been cloned successfully, which brings some new clues for further studies on the biological functions of HBeAg and the related proteins.

Objective: Test the cytokines secretion by peripheral blood mononuclear cells (PBMC) from hepatitis C patients after stimulation with highly variable region (HVR1) synthetic peptides., Methods: ELISA was used to test the cytokines secretion, flow cytometry to group the proliferated cells, and the antigenicity of synthetic peptide was predicted by the computer modeling., Results: (1)There were PBMC proliferation when stimulated by HVR1 antigen synthetic peptides. (2) There was a rise of IFN-gamma, IL-4 and IL-10. But no rise of IL-2 and TNF-gamma was found. (3) The proliferated cells were mainly CD4+ lymphocytes., Conclusion: PMBC from hepatitis C patients tend to secret Th2 cytokines after stimulation with HVR1 designed by the authors.

Objective: To explore whether autoimmune phenomena exist in SARS patients, and to seek for unusual autoimmune antibodies in SARS patients., Methods: Autoantibodies against cell nuclei (ANA), autoantibodies against smooth muscles (SMA), autoantibodies against parietal cells (PCA), autoantibodies against heart cells (HRA) were detected by using immunofluorescence, and autoantibodies against live-kidney microsomes (LKM) and anti-M2 antibodies were detected by ELISA in sera taken from 27 SARS patients and 18 healthy controls. Immunofluorescence was used to localize the targets antigens in slides with biochips of lung (monkey) of SARS associated antibodies., Results: ANA, AMA, LKM and SMA were found positive in 3, 1, 1, and 1 of the 27 SARS sera. In 18 healthy control sera, one ANA and one AMA were positive. Statistical analysis showed that there were no difference between two groups in every item detected. Twenty-six of 27 SARS patients and 5 of 18 healthy controls had strongly stained columnar epithelia of the bronchiole, especially the lumen border of the epithelia?the difference between two groups was significant., Conclusion: No antibodies against organs but lung were found in SARS patients. There are auto antibodies against lung tissues in sera of SARS patients.

With an isospin- and momentum-dependent transport model, we find that the degree of isospin diffusion in heavy-ion collisions at intermediate energies is affected by both the stiffness of the nuclear symmetry energy and the momentum dependence of the nucleon potential. Using a momentum dependence derived from the Gogny effective interaction, recent experimental data from NSCL-MSU on isospin diffusion are shown to be consistent with a nuclear symmetry energy given by E(sym)(rho) approximately 31.6(rho/rho(0))(1.05) at subnormal densities. This leads to a significantly constrained value of about -550 MeV for the isospin-dependent part of the isobaric incompressibility of isospin asymmetric nuclear matter.

Aim: To investigate the interaction between HBeAg and CD81., Methods: The CD81 gene was amplified by RT-PCR from HepG2 cells. The recombinant expression vector pGADT7-CD81 was constructed by routine molecular biological method. The auxotroph yeast cells were cotransfected with pGADT7-CD81 and pGBKT7-eAg and plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-alpha-gal. The binding of HBeAg to CD81 was also detected in vitro translation and coimmunoprecipitation test., Results: The recombinant expression vector was constructed and confirmed by restriction enzyme (EcoR I and BamH I) digestion and DNA sequencing analysis. The positive yeast clones could grow and from blue colonies on SD/-Trp-Leu-His-Ade/x-alpha-gal medium. The result of immunocoprecipitation showed that HBeAg could bind to CD81., Conclusion: HBeAg can bind to CD81, suggesting that CD81 plays an important role in the pathogenesis of HBV.

Signal regulatory protein (SIRP) alpha1 is a member of the SIRP family that undergoes tyrosine phosphorylation and binds SHP-2 tyrosine phosphatase in response to various mitogens. The expression levels of SIRPalpha1 were decreased in HCC tissues, compared with the matched normal tissues. Exogenous expression of wild type SIRPalpha1, but not of a mutant SIRPalpha1 lacking the tyrosine phosphorylation sites, in SIRPalpha1-negative Huh7 human HCC cells resulted in suppression of tumor cell growth both in vitro and in vivo. Treatment of Huh7 transfectants with EGF or HGF induced tyrosine phosphorylation of SIRPalpha1 and its association with SHP-2, which were accompanied by reduced ERK1 activation. Expression of SIRPalpha1 significantly suppressed activation of NF-kappaB and also sensitized Huh7 cells to TNFalpha or cisplatin-induced cell death. In addition, SIRPalpha1-transfected Huh7 cells displayed reduced cell migration and cell spreading in a fashion that was dependent on SIRPalpha1/SHP-2 complex formation. In conclusion, a negative regulatory effect of SIRPalpha1 on hepatocarcinogenesis is exerted, at least in part, through inhibition of ERK and NF-kappaB pathways., (Copyright 2004 American Association for the Study of Liver Diseases)