Prognosis of endotherapy versus splenectomy and devascularization for variceal bleeding in patients with hepatitis B-related cirrhosis.

OBJECTIVE: This study aimed to compare the long-term outcome of endotherapy versus a combination of splenectomy and devascularization for variceal bleeding in patients with hepatitis B-related cirrhosis (HBRC). MATERIALS AND METHODS: A total of 1074 patients with HBRC and acute variceal bleeding (AVB) treated with endotherapy and 248 patients with HBRC treated with a combination of splenectomy and devascularization surgery were included in the analysis. After one-to-one propensity score matching, 151 paired patients were selected. The primary end-point was death. The secondary outcomes were 3-year survival, 5-year survival, and rebleeding. Complications were recorded. RESULTS: The median follow-up time was 1165 days in the endoscopic group and 1709 days in the surgical group. Before matching, the 1-year, 3-year, and 5-year survival rates were significantly lower in the endoscopic group than in the surgical group (91.1 vs 96.3%, P = 0.017; 79.6 vs 91.6%, P = 0.001; 65.2 vs 81.3%, P = 0.001). After matching, no significant differences were found between groups (94.5 vs 95.2%, P = 0.767; 87.0 vs 88.9%, P = 0.635; 77.9 vs 77.9%, P = 0.905). The rebleeding rate was lower in the surgical group than in the endoscopic group; the rebleeding-free survival rate was similar in the two groups. No patient died of complications. No statistically significant difference was observed in complications between groups. CONCLUSIONS: Both endotherapy and a combination of splenectomy and devascularization are good choices for patients with AVB. The rebleeding rate was lower after the surgical procedure, but the long-term prognosis was similar.

Clinical Features and Outcomes of Repeated Endoscopic Therapy for Esophagogastric Variceal Hemorrhage in Cirrhotic Patients: Ten-Year Real-World Analysis.

OBJECTIVE: This study is aimed at evaluating the survival of cirrhotic patients with different etiologies after endoscopic therapy for acute variceal bleeding and the effect of repeated endotherapy on patients' prognosis. METHODS: We retrospectively evaluated the clinical features and outcomes between cirrhotic patients with chronic HBV or HCV infections and other etiologies. The 3-year and 5-year survival rates and rehemorrhage rate in one year between the viral and nonviral cirrhosis patients were compared by Kaplan-Meier curves and log-rank test. Cox analysis was used to identify the impact factors that affect the long-term survival of patients with cirrhosis and variceal bleeding after endotherapy. RESULTS: Out of 2665 patients with liver cirrhosis and variceal hemorrhage selected from our medical center between September 2008 and December 2017, a total of 1342 patients were included for analysis. The median follow-up duration was 32.9 months (range 0.16-111.4 months), the 3- and 5-year cumulative survival rates were 75.3% and 52.8%, respectively. The median survival time was significantly longer in viral cirrhosis patients (47.1 months [95% CI: 24.9-69.1]) compared with nonviral cirrhosis patients (37.0 months [95% CI: 25.0-56.0], p = 0.001). The 3-year and 5-year survival rates of the viral group were higher than the nonviral group. The rehemorrhage rate at one year was higher in nonviral patients than in viral patients (p < 0.001). CONCLUSION: Repeated endotherapy combined with effective antiviral therapy is helpful for long-term survival of cirrhotic population with variceal hemorrhage and HBV or HCV infection.

New model predicting gastroesophageal varices and variceal hemorrhage in patients with chronic liver disease.

INTRODUCTION AND OBJECTIVES: The predictors for gastroesophageal varices (GOV) and hemorrhage development have not been well studied in different liver diseases or different population. This study aimed to evaluate whether a new algorithm focusing on chronic hepatitis B (CHB) patients is also applicable to other chronic liver diseases (CLDs) in Chinese population. PATIENTS OR MATERIALS AND METHODS: We retrospectively analyzed 659 CHB patients and 386 patients with other CLDs. A total of 439 CHB patients were included in training set, the other 220 CHB patients and other patients with CLDs were included in validation set. A new algorithm for diagnosing GOV was established and its sensitivity and specificity for predicting the varices was verified. RESULTS: Multivariable logistic regression revealed that the rough surface of the liver (p<0.001), splenic thickness (p<0.001), and liver stiffness (p=0.006) were independent predictors of GOV. The new algorithm was considered to be a reliable diagnostic model to evaluate the presence of varices. The AUROC was 0.94 (p<0.001) in CHB validation set and 0.90 (<0.001) in non-CHB validation set. When the cut-off value was chosen as -1.048, the sensitivity and specificity in diagnosing GOV in CHB population were 89.1% and 82.5%, respectively. Importantly, the new algorithm accurately predicted the variceal hemorrhage not only in CHB patients, but also in patients with other CLDs. CONCLUSION: The new algorithm is regarded as a reliable model to prognosticate varices and variceal hemorrhage, and stratified not only the high-risk CHB patients, but also in patients with other CLDs for developing GOV and variceal bleeding.

Deletion of the C6orf120 gene with unknown function ameliorates autoimmune hepatitis induced by concanavalin A.

The present study was conducted to characterize the C6orf120 gene, by using C6orf120 gene-deleted rats (C6orf120-/-), to determine its role in the development and severity of autoimmune hepatitis induced by concanavalin A (Con A), as well as the underlying mechanisms. We found that following Con A injection, C6orf120-/- rats were less susceptible to developing autoimmune hepatitis with low levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) post challenge. Additionally, C6orf120 deficiency increased the frequency of cluster of differentiation (CD)4+ CD25+ Forkhead box P3+ regulatory T cells (Tregs) among intrahepatic lymphocytes, splenocytes, peripheral blood mononuclear cells, and CD4+ T in vitro. Moreover, C6orf120 deficiency downregulated interleukin (IL)-1ß, IL-6, tumor necrosis factor alpha-α, interferon-γ and IL-17a secretion in the plasma and liver tissues. Our results indicated that the C6orf120 gene-deleted rats were less susceptible to Con A-induced autoimmune hepatitis, which may be partly related to the increased frequency of Tregs and inhibited secretion of inflammatory cytokines.

Glycosyltransferase GLT8D2 positively regulates ApoB100 protein expression in hepatocytes.

Non-alcoholic fatty liver disease (NAFLD) is characterized by triglyceride (TG) accumulation in hepatocytes. Very low density lipoprotein (VLDL) is a major secretory product of the liver that transports endogenously synthesized TG. Disrupted VLDL secretion may contribute to the accumulation of TG in hepatocytes. ApoB100 (apolipoprotein B100) is a glycoprotein and an essential protein component of VLDL. Its glycosylation may affect VLDL assembly and secretion. However, which glycosyltransferase catalyzes apoB100 glycosylation is unknown. In this study, we cloned the GLT8D2 (glycosyltransferase 8 domain containing 2) gene from HepG2 cells and generated a series of plasmids for in vitro studies of its molecular functions. We discovered that GLT8D2 was localized in the ER, interacted with apoB100, and positively regulated the levels of apoB100 protein in HepG2 cells. Based on these results, we propose that GLT8D2 is a glycosyltransferase of apoB100 that regulates apoB100 levels in hepatocytes.

[Establishment of a quantitative detection method for Golgi protein73].

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.

[Quantitative detection of serum procollagen III N-terminal peptide chemiluminescence enzyme immunoassay].

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.

[Serum GP73 were increased in patients with fatty liver disease].

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.

[Establishment of a detection method for hepatitis B virus large surface protein (HBV-lP)].

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;

[Microplate chemiluminescence enzyme immunoassay for the quantitative analysis of tissue inhibitor of metalloproteinases I].

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.

[Cloning and expressing of golgi protein73 gene fragment and preparation of monoclonal antibodies against the recombinant protein].

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.

Role of C6ORF120, an N-glycosylated protein, is implicated in apoptosis of CD4⁺ T lymphocytes.

BACKGROUND: Although CD4(+) T cell apoptosis and CD8(+) T cell responses have been extensively studied during HIV infection, how apoptosis signals being initiated in CD4(+) T cells still need to be elucidated. The present study was designed to characterize the function-unknown gene, C6orf120, and elucidates its primary role in tunicamycin-induced CD4(+) T apoptosis. METHODS: The C6orf120 coding sequence was amplified from peripheral blood mononuclear cells (PBMCs) total RNA of AIDS patients. The DNA fragment was inserted into the pET-32a expression system, transformed into Escherichia coli, and preparation of C6ORF120 recombinant protein. The magnetic cell separation technology was used to prepare primary CD4(+) T cells and CD8(+) T cells. The primary T cells were cultured at 1 × 10(6) cells/ml, treated with 0, 0.1, 1, 10, 100, and 200 ng/ml of C6orf120 recombinant protein for 48 hours, then harvested for cell cycle and apoptosis analysis. Tunicamycin (0.5 µmol/L) was used to induce endoplasmic reticulum stress in Jurkat cells. The biomarker 78 KDa glucose-regulated protein (GRp78) and growth arrest and DNA damage (GADD) were used to evaluate endoplasmic reticulum stress of Jurkat cells. RESULTS: We prepared C6ORF120 recombinant protein and its polyclonal antibody. Immunohistochemical analysis showed that C6orf120 mainly expressed in hepatocytes and cells in germinal center of lymph node. At concentration of 0.1, 1, 10, 100, and 200 ng/ml, C6orf120 recombinant protein could induce apoptosis of Jurkat cells and primary CD4(+) T cells, and promoting G2 phase of its cell cycle. Western blotting analysis showed that C6ORF120 recombinant protein increased the expression of GRp78 and GADD in Jurkat cells in vitro. CONCLUSION: Our results suggested that C6ORF120 could induce apoptosis of CD4(+) T cells, at least in part, mediated with endoplasmic reticulum stress.

[Expression, purification and polyclonal antibody preparation for a novel gene BC097361].

OBJECTIVE: To express and purify of the BC097361 recombinant protein, and to prepare the BC097361 specific rabbit polyclonal antibody. METHODS: BC097361 cDNA was ligated into the prokaryotic expressive vector pET-32a (+), and the resulting plasmid was transformed into E.coli BL21 (DE3). The protein expression was induced with IPTG and the protein was analyzed with SDS-PAGE and western blotting. The expressed product was purified using Ni+ affinity column chromatography.Then the purified pET-32a (+) -BC097361 fusion protein was used to immunize New Zealand rabbits to gain polyclonal antibody. The specificity and potency of polyclonal antibody were evaluated by Western blot and ELISA. RESULTS: The BC097361 fusion protein was highly expressed.The protein was mainly in the inclusion body. ELISA indicated the titer of polyclonal antibody more than 1:320000. The high specificity was confirmed with Western blot. CONCLUSIONS: The recombinant BC097361 fusion protein and the BC097361 specific polyclonal antibody will be valuable tools for the investigation on the biological function of BC097361.

Different plasma levels of interleukins and chemokines: comparison between children and adults with AIDS in China.

BACKGROUND: The immunological differences between children and adults with AIDS in China are not well documented. Th1/Th2 cytokines and chemokines are two types of immune factors intimately involved in disease progression of HIV-1 infection. This study aimed to identify changes in plasma levels of Th1/Th2 cytokines inerleukin (IL)-18, IL-16, IL-10 and chemokines regulated on activation, normal T cell expressed and secreted (RANTES), stromal cell-derived factor-1 (SDF-1) and monocyte chemoattractant protein-1 (MCP-1) in HIV-1-infected children and adults in China. METHODS: Seventy-five children with AIDS and 35 adult AIDS patients were recruited and clinical data were collected. CD4(+) T lymphocyte counts were measured by flow cytometery and plasma HIV RNA levels were measured by quantitative RT-PCR. Plasma levels of IL-18, IL-10, IL-16, RANTES, MCP-1, SDF-1alpha and SDF-1beta were quantified by enzyme-linked immunosorbent assay. The levels of beta2-microglobulin (beta2-MG) and soluble Fas (sFas) were measured to validate the level of humoral and cellular immune activation. RESULTS: The mean levels of all cytokines in pediatric and adult AIDS patients were significantly higher than in their healthy controls (P < 0.01). The mean levels of these cytokines were higher in pediatric patients than in adult patients (P < 0.05, except for SDF-1alpha and beta2-MG). Some of the cytokine levels in patients younger than 6 years old was higher than in older children and adults with AIDS (IL-10, IL-18, SDF-1alpha, MCP, RANTES and sFas, P < 0.05). Levels of IL-18, IL-10, RANTES and beta2-MG of pediatric patients increased as the levels of viral load increased (P < 0.05). CONCLUSIONS: Abnormal immune activation can be measured in Chinese pediatric and adult patients with AIDS, and is higher in children than in adult patients. The cytokines levels coincide with disease progression of AIDS, but have no direct relationship with total CD4(+) T cell count.

[Relationship of CD4+ CD25hi regulatory T (Treg) cells to disease progression in HIV-infected patients].

OBJECTIVE: Analyzing the relationships between peripheral blood CD4+ CD25hi regulatory T (Treg) cells and peripheral blood immune status or plasma HIV-lviral load in HIV-infected individuals,so as to determine whether Treg were related to the progression of HIV-infected disease. METHODS: 116 HIV-infected patients in different stages and 21 healthy control individuals were included in this study. The CD4+ and CD8+ T cell counts were determined by a standard 4-color flow cytometry technique. The Treg cells were examined with 3-color immune staining flow cytometry. The plasma HIV-1 viral load was detected by real time PCR. RESULTS: The frequencies of Treg cells decreased in HIV-infected individuals with high CD4+ T cell counts( > 300/microl) compared with normal controls. With the progression of disease the frequencies of Treg cells were raised gradually, until were increased in HIV-infected individuals with low levels of CD4+ T cell counts ( < 100/microl). In addition, the frequencies of Treg cells were inversely related to CD4+ T cell counts and CD4+ /CD8+ ratio, data showed a statistically significant (respectively, r = -0.564, P < 0.001; r = -0.377, P < 0.001). Furthermore, the proportions of Treg cells were closely related to plasma HIV-1 RNA viral load (r = 0.514, P < 0.001). CONCLUSION: CD4 CD25hi Treg cells should be a kind of important cells participating the immunopathogenesis of AIDS. It may play different roles in different stages of HIV-infected disease. The exact mechanism of Treg cells in the progression of the HIV-infected disease needs to be investigate further.

[The association of TGF beta1 and AT1R gene polymorphisms with hereditary susceptibility and clinical phenotype of HBV-induced liver cirrhosis].

OBJECTIVE: To investigate the association of TGF beta1 and AT1R gene polymorphisms with hereditary susceptibility and clinical phenotype of HBV-induced liver cirrhosis. METHODS: Peripheral blood samples were collected from 102 patients with HBV-induced liver cirrhosis and 106 healthy blood donors. The polymorphisms of the promoter site -509C/T of TGF beta1 and 1166A/C of AT1R gene were determined by PCR-RFLP. RESULTS: The frequency of the homozygote CC of -509C/T of TGF beta1 gene in the group of liver cirrhosis was higher than that the control group (P<0.05); and the frequency rate of homozygote CC was higher in group C than in group A and group B of liver cirrhosis (P<0.05), but there was no significant difference in allele frequency among these group (P>0.05). There was no significant difference in genotypes and allele frequency of AT1R gene 1166A/C between the liver cirrhosis group and the control group (P>0.05). CONCLUSION: The polymorphism of the promoter site -509C/T of TGF beta1 gene is associated with hereditary susceptibility to liver cirrhosis and severity of HBV-induced liver cirrhosis; the polymorphism of AT1R gene 1166A/C is not associated with hereditary susceptibility to HBV-induced liver cirrhosis.

[Impact of highly active antiretroviral therapy on plasma MCP-1 and MSP in AIDS patients].

OBJECTIVE: To study the effect of highly active antiretroviral therapy (HAART) on plasma levels of MSP and MCP-1 in AIDS patients. METHODS: Forty Chinese AIDS patients were treated with HAART for 3 months and 84 German AIDS patients with HAART for 3 to 6 years. The pre-treatment and post-treatment plasma levels of MSP and MCP-1 were measured by enzyme-linked immunosorbent assay (ELISA), and their correlations with CD4+ cell counts and viral loads were analyzed. RESULT: The mean levels of MCP-1 were significantly higher and MSP were significantly lower in HIV-infected patients compared with the HIV-negative controls (P <0.01). After HAART for three months, there were no significant changes in the levels of these cytokines. But after long-term HAART (for 3 to 6 y), the level of MCP-1 was increased and that of MSP decreased significantly (P<0.01). There was a negative correlation between MSP and MCP-1 levels, and the same for MSP level and CD4+ cell counts; while there was a positive correlation between MCP-1 levels and CD4+ cell counts. CONCLUSION: The changed plasma levels of MSP and MCP-1 are associated with HIV-1 infection and HAART may reverse the levels of these two cytokines.