A decline of LAMP- 2 predicts ursodeoxycholic acid response in primary biliary cirrhosis.

Biochemical response to ursodeoxycholic acid (UDCA) in patients with primary biliary cirrhosis (PBC) is variable. We have previously reported that augmented expression of lysosome-associated membrane protein 2 (LAMP-2) was correlated with the severity of PBC. This study aimed to determine whether serum LAMP-2 could serve as a predictor of biochemical response to UDCA. The efficiency of serum LAMP-2 to predict biochemical response was assessed after 1 year of UDCA treatment in PBC patients by a retrospective analysis. We found that the basal serum LAMP-2 level was increased in PBC, especially in patients with stage III-IV (p = 0.010) or TBIL > 1 mg/dL (p = 0.014). Baseline serum LAMP-2 was higher in non-responders than that in responders, but the difference was statistically insignificant. However, after UDCA treatment, serum LAMP-2 level decreased prominently in the first 3 months, which was more obvious in responders. Further studies showed that the 35% decline of LAMP-2 after treatment for 3 months could be stated as an indicator of UDCA response with the sensitivity of 62.9% and specificity of 75.0% by Paris criteria. Meanwhile the specificity and sensitivity were identified as 63.5% and 64.1% by Barcelona criteria. Together, a decline in LAMP-2 might help to predict the response to UDCA.

The association between bile salt export pump single-nucleotide polymorphisms and primary biliary cirrhosis susceptibility and ursodeoxycholic acid response.

BACKGROUND: Primary biliary cirrhosis (PBC) is a chronic and progressive cholestasis liver disease. Bile salt export pump (BSEP) is the predominant bile salt efflux system of hepatocytes. BSEP gene has been attached great importance in the susceptibility of PBC and the response rate of ursodeoxycholic acid (UDCA) treatment of PBC patients. METHODS: In this study, TaqMan assay was used to genotype four variants of BSEP, and the Barcelona criteria were used for evaluating the response rate of UDCA treatment. RESULTS: Variant A allele of BSEP rs473351 (dominant model, OR = 2.063; 95% CI, 1.254-3.393; P = 0.004) was highly associated with PBC susceptibility. On the contrary, variant A allele of BSEP rs2287618 (dominant model, OR = 0.617; 95% CI, 0.411-0.928; P = 0.020) provided a protective role and Barcelona evaluation criterion indicated that the frequency of variant allele at BSEP rs2287618 was significantly decreased in UDCA-responsive PBC patients (P = 0.021). CONCLUSION: These results suggested that BSEP rs473351 was closely associated with the susceptibility of PBC and if people with BSEP rs2287618 were diagnosed as PBC, the UDCA treatment was not satisfactory. Larger studies with mixed ethnicity subjects and stratified by clinical and subclinical characteristics are needed to validate our findings.

Meta-analysis of efficacy and safety of fast-track surgery in gastrectomy for gastric cancer.

BACKGROUND: Fast-track surgery (FTS) is a promising program for surgical patients and has been applied to several surgical diseases. FTS is much superior to conventional perioperative care. Our aim was to evaluate and compare the safety and efficacy of FTS and conventional perioperative care for patients undergoing gastrectomy using a systematic review. METHODS: We searched the literature in PubMed, SCOPUS, and EMBASE up to November 2013. No language restriction was applied. Weighted mean differences (WMDs) and odds ratios (ORs) with their 95 % confidence intervals (CIs) were used for analysis by a fixed or a random effects model according to the heterogeneity assumption. RESULTS: In the present meta-analysis, we included five randomized controlled trials and one controlled clinical trial from five studies. Compared with conventional care, FTS shortened the duration of flatus (WMD -21.08; 95 % CI -27.46 to -14.71, z = 6.48, p < 0.00001 in the open surgery group; WMD -8.20; 95 % CI -12.87 to -3.53, z = 3.44, p = 0.0006 in the laparoscopic surgery group), accelerated the decrease in C-reactive protein (WMD -15.56; 95 % CI 21.28 to 9.83, z = 5.33, p < 0.00001), shortened the postoperative stay (WMD -2.00; 95 % CI -2.69 to -1.30, z = 5.64, p < 0.00001), and reduced hospitalization costs (WMD -447.72; 95 % CI -615.92 to -279.51, z = 5.22, p < 0.00001). FTS made no significant difference in operation times (p = 0.93), intraoperative blood loss (p = 0.79), or postoperative complications (p = 0.07). CONCLUSIONS: Based on current evidence, the FTS protocol was feasible for gastric cancer patients who underwent gastrectomy (distal subtotal gastrectomy, proximal subtotal gastrectomy, or radical total gastrectomy) via open or laparoscopic surgery. Larger studies are needed to validate our findings.

Polymorphisms in the vitamin D receptor gene and risk of primary biliary cirrhosis: a meta-analysis.

BACKGROUND AND AIM: Primary biliary cirrhosis (PBC) is a chronic and progressive cholestatic autoimmune liver disease. Although many studies have evaluated the association between many functional polymorphisms in the vitamin D receptor (VDR) gene and PBC risk, debates still exist. Our aim is to evaluate the association between VDR gene polymorphisms, including TaqI (rs731236), BsmI (rs1544410), and ApaI (rs7975232), and the risk of PBC by a systematic review. METHODS: We searched literatures in PubMed, SCOPUS, and EMBASE until July 2013. We calculated pooled odds ratios (OR) and 95% confidence intervals (CIs) using a fixed effects model or a random effects model for the risk to PBC associated with different VDR gene polymorphisms. And the heterogeneity assumption decided the effect model. RESULTS: A total of six relevant studies, with 1322 PBC cases and 2264 controls, were included in this meta-analysis. The results indicated that TaqI (rs731236) polymorphism was significantly associated with PBC risk (for T vs t OR = 0.75, 95% CI 0.63, 0.89, Pz = 0.001; TT + Tt vs tt OR = 0.62, 95% CI 0.44, 0.86, Pz = 0.005; OR = 0.74, 95% CI 0.58, 0.94, Pz = 0.016 for recessive model), while ApaI (rs7975232) or BsmI (rs1544410) polymorphism did not. CONCLUSION: Based on current evidences from published studies, the cumulative effect of TaqI polymorphism in VDR was significantly associated with PBC. Larger studies with mixed ethnicity subjects and stratified by clinical and sub clinical characteristics are needed to validate our findings.

Cytotoxic T-lymphocyte antigen 4 gene +49A/G polymorphism significantly associated with susceptibility to primary biliary cirrhosis: a meta-analysis.

OBJECTIVE: To evaluate comprehensively the association of cytotoxic T-lymphocyte antigen 4 (CTLA-4) +49A/G polymorphism with susceptibility to primary biliary cirrhosis (PBC). METHODS: PubMed was used to search for the relevant published articles. The risk of PBC association with the CTLA-4+49A/G polymorphism was estimated for each study in a random-effects model. Odds ratio (OR) and 95% confidence interval (CI) were estimated for each study. Risks to PBC were estimated by stratified analysis in patients with different ethnicity and antimitochondrial antibody (AMA) status, as well as histological stages. RESULTS: A total of 12 articles were included in the study. An association between PBC and CTLA-4 G allele was found, overall OR = 1.20, 95% CI 1.03-1.41 (P = 0.02). However, stratification by ethnicity indicated a significant association between the G allele and PBC in Asians (OR = 1.36, 95% CI 1.12-1.65, P = 0.002), but not in Caucasians (OR = 1.15, 95% CI 0.95-1.39, P = 0.15). Moreover, AMA positive patients carrying G allele were more susceptible to PBC compared with AMA negative patients (OR = 1.23, 95% CI 1.06-1.43, P = 0.007; OR = 0.98, 95% CI 0.71-1.34, P = 0.88, respectively). CONCLUSIONS: Polymorphism in exon 1 of CTLA-4 gene at position 49 may act as a candidate of susceptibility locus to PBC. However, larger studies with participants of varying ethnicity and stratified by clinical and laboratory characteristics are needed to validate our findings.

[Observation on therapeutic alliance with UDCA and glucocorticoids in AIH-PBC overlap syndrome].

OBJECTIVE: To observe the efficacy of ursodeoxycholic acid (UDCA) combined with glucocorticoids in the treatment of autoimmune hepatitis-primary biliary cirrhosis (AIH-PBC) overlap syndrome. METHODS: 19 patients with AIH-PBC overlap syndrome were divided randomly into two groups: initiate combined group and initiate UDCA-monotherapy group. Biochemical responses and pathological features before and after treatment were analyzed retrospectively with student's t test, Wilcoxon rank sum test and Fisher's exact method. RESULTS: In the initiate combination group, biochemical responses in terms of AIH features (ALT decline to normal, IgG is less than or equal to 16 g/L) and PBC features (ALP decline ≥ 40% or to normal) were achieved. In UDCA-monotherapy group, no statistical difference existed in biochemical responses before adding glucocorticoids, whereas the levels of ALT, AST, GLB and IgG decreased significantly when combined with glucocorticoids. No statistical difference of rates of biochemical responses eixted between the two groups, whereas variance could be seen in different pathological stages. Alleviation of inflammatory infiltration after therapy appeared in 3 patients. CONCLUSION: Combination therapy of UDCA with glucocorticoids could be suitable for AIH-PBC overlap syndrome. Early treatment is of benefit for achieving better biochemical response and pathological improvement.

[Comparison between two diagnostic criteria for PBC-AIH overlap syndrome].

Not Abstract.

[A two-year follow-up study on the efficacy of ursodeoxycholic acid on primary biliary cirrhosis in different stages].

OBJECTIVE: To assess the therapeutic effect of primary biliary cirrhosis(PBC) in different stages with ursodeoxycholic acid (UDCA). METHODS: 91 patients with PBC were divided into 4 periods based on levels of liver test and symptoms. Clinical manifestations, biochemical changes and pathological changes were observed for 2 years on UDCA therapy. RESULTS: The levels of alkaline phosphatase (ALP) and glutamyltranspetidase (GGT) at the second PBC period were declined by 51.9% and 67.3% respectively after a 6-month UDCA therapy. The biochemical responses were 81.25% (Paris criteria) and 93.75% (Barcelona criteria). The levels of ALP and GGT at the third PBC period were declined by 48.8% and 46.6% after 6 months of UDCA therapy, and the biochemical responses were 36.84% (Paris criteria) and 57.89% (Barcelona criteria). Symptoms like fatigue, pruritus and jaundice after UDCA therapy were better than before. Same results also appeared at the fourth period. 11 patients in different periods underwent pathological examinations before and after UDCA therapy and no progression found in the first and the second periods, however difference found in the third and the fourth periods with the lymphocyte infiltration was less than before UDCA treatment. CONCLUSION: Good biochemical responds appear in patients at the second, third and forth periods after UDCA therapy, in which the second period is best. Symptoms could be improved after UDCA treatment. Early UDCA therapy is benefit for slowing down the progression of liver pathology.

[Construction of shRNA expression vector pWH1 and its function in silencing the HIF1 gene].

AIM: To construct the expression vector of small hairpin RNA(shRNA) and to test its efficacy in silencing the Hypoxia-inducible Factor-1 (HIF1) gene. METHODS: The H1 gene promoter was amplified from the genome of the human blood cells by PCR. Then the promoter was cloned into the pEGFP-C1 vector digested with the restriction enzyme. The constructed vector was named pWH1. The primer was designed to target the human HIF1 cDNA gene. The annealed primer fragment was cloned into pWH1. The new constructed plasmid was transfected into the SGC7901 cell line. Then the expression level of HIF1 gene was assayed by RT-PCR and Western blot. RESULTS: The newly constructed plasmid expressed shRNA to target the HIF1 gene.The results of RT-PCR and Western blot showed the expression of HIF1 gene was reduced dramatically in mRNA and at protein level. CONCLUSION: The successful construction of shRNA expression vector (pWH1) provides a tool for further research into the function of a novel gene.

[Osteopontin expression and its relation to invasion and metastases in gastric cancer].

OBJECTIVE: To investigate the correlation between expression of the osteopontin (OPN) and invasion and metastases in gastric cancer. METHODS: The expression of OPN, NF-kappaB p65 and matrix metallo-proteinase 9 (MMP-9) was detected by immunohistochemistry in non-cancer gastric tissue (n = 12 cases) and gastric cancer tissue (n = 72 cases). RESULTS: (1) OPN, NF-kappaB p65 and MMP-9 were not expressed in 12 non-cancer gastric tissue samples(group A). Their expression rates were 43.3%, 40.0% and 46.7% respectively in 30 gastric cancer samples without lymph nodes metastasis (group B), but they increased to 76.9%, 73.1% and 80.8% in 26 gastric cancer samples with lymph nodes metastases (group C), and 87.5%, 81.3% and 93.8% respectively in 16 gastric cancer samples with lymph node and distant metastases (group D). (2) There were statistically significant differences in their expressions between group D and group B (P(a) = 0.004, P(c) = 0.007, P(e) = 0.002), and between group C and group B (P(b) = 0.011, P(d) = 0.013, P(f) = 0.009). (3) Despite some differences in positive expression rates, correlations existed between OPN and NF-kappaB p65, and between NF-kappaB p65 and MMP-9 (P(1) = 0.042, P(2) = 0.013; r(1)= 0.67, r(2)= 0.72). CONCLUSION: Osteopondin espression is closely related to the invasion and metastases of gastric cancer. It may upregulate the expression of metastasis-related molecule MMP-9 by activating NF-kappaB pathway.

Helicobacter pylori specific immune response induced by conservative flagellin linear B-cell epitope.

AIM: To testify the immunogenicity of a conservative B-cell linear epitope of Helicobacter pylori (H pylori) flagellin A. METHODS: Different programs were used to analyze the secondary structure, molecular hydropathy, and surface accessibility of H pylori flagellin A. Linear B-cell epitopes were estimated based on the structural and physiochemical information. Analysis of residue divergence was proposed to screen a conservative linear epitope. The 29-peptide (Pep29mer) synthesized by chemical method, including the predicted conservative B-cell epitope and a known K2d compatible T-cell epitope, was used to immunize mice, and then H pylori-specific antibodies were detected by ELISA. RESULTS: Based on the analyses of divergent amino acid residues, structural and physiochemical characteristics, it was strongly suggested that the short fragment NDSDGR was the core of a conservative linear epitope in flagellin A. Animals immunized by Pep29mer acquired efficient immune response. In detail, serum H pylori-specific IgA and IgG1 increased significantly in immunized group, while IgG2a only had an insignificant change. H pylori-specific IgA in gastrointestinal flushing fluid also increased significantly. CONCLUSION: The conservative short fragment NDSDGR is the core of a linear B-cell epitope of flagellin A.

[Expressions and activities of Rho GTPases in hypoxia and its relationship with tumor angiogenesis].

OBJECTIVE: To study the expressions and activities of Rho GTPases in hypoxia and its relationship with tumor angiogenesis. METHODS: Three tumor cell lines were used in this study: gastric cancer cell lines AGS, SGC7901 and hepatocellular carcinoma cell line HepG2. Expression level of Rac1 mRNA was detected by semi-quantitative RT-PCR. Activity of Rac1 was determined by pull-down assay and expression of HIF-1alpha, VEGF, p53 and PTEN protein was detected by Westernblot. RESULTS: The expression level of Rac1 mRNA was significantly increased in hypoxia compared to normoxia. Pull-down assay showed that hypoxia-induced activity of Rac1 was elevated in a time-dependent manner and climaxed at 3 hours. The expressions of HIF-1alpha and VEGF protein were up-regulated, while those of PTEN and p53 protein were down-regulated. CONCLUSION: These results indicate that hypoxia enhances Rac1 expression which might be involved in tumor angiogenesis by reacting with hypoxia-responsive genes.

[Expression of RhoC in gastric cancer cell lines and construction and identification of RhoC-specific siRNA expression vector].

AIM: To study the expression of RhoC in human gastric cancer cell lines and to construct and identify the RhoC-specific siRNA expressing vector. METHODS: The expression of RhoC in human gastric cancer cell lines was detected by Western blot. According to the computer aided design(CAD),RhoC-specific siRNA gene was synthesized and cloned into the expression vector mU6pro. The constructed RhoC-siRNA was transiently transfected into high-metastatic gastric cancer cell line AGS and the inhibition effect of RhoC-siRNA on expression of RhoC in AGS cells was detected using Western blot. RESULTS: The expression level of RhoC was up-regulated in high metastatic cells lines. Double enzyme digestion analysis and DNA sequencing confirmed that the RhoC-specific siRNA expression vector was constructed successfully. RhoC expression in AGS cells was specifically suppressed after transfection of RhoC-siRNA. CONCLUSION: The RhoC-specific siRNA expression vector has been successfully constructed, which may provide a novel applicable strategy for gene therapy of gastric cancer.

[The overexpression and significance of MKP-1 in oxygen-deprived gastric cancer cell line SGC7901].

OBJECTIVE: To investigate the expression status of Mitogen-activated Protein Kinase Phosphatase-1 in oxygen-deprived gastric cancer cell line SGC7901 and its role in HIF-1 regulation. METHODS: The expression of MKP-1 in gastric cell line SGC7901 was detected with Western blot and semiquantity RT-PCR; Eukaryotic sense expression vector was constructed based on DNA recombination technology. Transfections of SGC7901 were performed using liposome; The luciferase activity was determined using Dual Luciferase Reporter System and the levels of VEGF in SGC7901cells under normoxia and hypoxia were measured by ELISA. RESULTS: (1) Semiquantity RT-PCR and Western blot suggested that the expression of MKP-1 was elevated in oxygen-deprived gastric cancer cell line SGC7901; (2) 48 hours after transfection, the phosphorylated form of HIF-1alpha in cell line transfected with recombinant plasmids was lower compared with that in cell line transfected with empty vectors after 12 hours of exposure to hypoxia; (3) There was very low luciferase activity under nomoxia while under hypoxia luciferase activity increased in a time-dependent manner and at all time points there was significant lower luciferase activity in SGC7901 cells than in cells transfected with empty plasmids. (4) At different points of time course, the expression of VEGF in SGC7901 was significantly higher under hypoxia than that in SGC7901 under nomorxia, while under hypoxia, the expression of VEGF in SGC7901 transfected with recombinant plasmids was significantly lower than that in SGC7901 transfected with empty vectors at all time points as indicated. CONCLUSION: The expression of MKP-1 in SGC7901 was elevated under hypoxia, which could downregulate the HIF-1 trans-activition activity thereby repressing the expression of downstream target gene VEGF.

Screening and identification of mimotope of gastric cancer associated antigen MGb1-Ag.

AIM: Using a monoclonal antibody against gastric cancer antigen named MGb1 to screen a phage-displayed random peptide library fused with coat protein pIII in order to get some information on mimotopes. METHODS: Through affinity enrichment and ELISA screening, positive clones of phages were amplified. 10 phage clones were selected after three rounds of biopanning and the ability of specific binding of the positive phage clones to MGb1-Ab were detected by ELISA assay (DNA sequencing was performed and the amino acid sequences were deduced) By blocking test, specificity of the mimic phage epitopes was identified. RESULTS: There were approximately 200 times of enrichment about the titer of bound phages after three rounds of biopanning procedures. DNA of 10 phage clones after the third biopanning was assayed and the result showed that the positive clones had a specific binding activity to MGb1-Ab and a weak ability of binding to control mAb or to mouse IgG. DNA sequencing of 10 phage clones was performed and the amino acid sequences were deduced. According to the homology of the amino acid sequences of the displayed peptides, most of the phage clones had motifs of H(x)Q or L(x)S. And these 10 phage clones could also partly inhibit the binding of MGb1-Ab to gastric cancer cell KATO-III. The percentage of blocking was from (21.0+/-1.6) % to (39.0+/-2.7) %. CONCLUSION: Motifs of H(x)Q and L(x)S selected and identified show a high homology in the mimic epitopes of gastric cancer associated antigen. There may be one or more clones which can act as candidates of tumor vaccines.

[In-vivo screening and characterization of peptides specific for vasculature of gastric cancer].

AIM: To screen in vivo from a phage-displayed peptide library polypeptide fragments specific binding to vascular endothelial cells of gastric cancer xenografts, so as to provide for anti-angiogenesis therapy of tumor. METHODS: Immunosupressed mice models for human gastric cancer xeno-grafts were established by subrenal capsular assay (SR-CA). The 12-peptide library was panned through 4 rounds. Phages were recovered and titrated from tumor xenografts and control tissue (brain). The distribution of phage were detected in transplanted tumor tissues by immunohistochemical staining. RESULTS: Phage homing to gastric cancer xenografts were enriched through four rounds of panning,being 3.4-fold of that recovered from brain tissue. Peptide sequences were characterized for randomly picked-upclones and the peptide sequence YESIRIGVAPSQ appeared most frequently. Immunohistochemical staining for the homing phage revealed a specific vascular endothelial cell localization in gastric cancer xenografts 5 min after injection of the enriched phages. When the specific phage individually test-ed, the phage recovered from gastric cancer xenografts were as 4. 2 times as those from control tissue ( brain) , as 4.9 times as those from lung, as 5.4 times as those from heart. CONCLUSION: The tumor-specific homing peptides may provide a effective tool for targeting tumor vasculature in anti-angiogenesis therapy of cancer. The in vivo selection technique in this study was feasible and applicable to screening peptides homing to vascular endothelial cells.

Expression and function of classical protein kinase C isoenzymes in gastric cancer cell line and its drug-resistant sublines.

AIM: To investigate the expression and function of classical protein kinase C (PKC) isoenzymes in inducing MDR phenotype in gastric cancer cells. METHODS: Two cell lines were used in the study: gastric cancer cell SGC7901 and its drug-resistant cell SGC7901/VCR stepwise-selected by vincristine 0.3, 0.7 and 1.0 mg.L(-1), respectively. The expression of classical PKC (cPKC) isoenzymes in SGC7901 cells and SGC7901/VCR cells were detected using immunofluorescent cytochemistry, laser confocal scanning microscope and Western blot. The effects of anti-PKC isoenzymes antibody on adriamycin accumulation in SGC7901/VCR cells were determined using flow cytometric analysis. RESULTS: (1)SGC7901 cells exhibited positive staining of PKC-alpha. SGC7901/VCR cells exhibited stronger staining of PKC-alpha than SGC7901 cells. The higher dosage vincristine selected, the much stronger staining of PKC-alpha was observed on SGC7901/VCR cells. (2)Both SGC7901 and SGC7901/VCR cells exhibited positive staining of PKC-beta I and PKC-beta II with no significant difference. (3) Compared with SGC7901, SGC7901/VCR cells had decreased adriamycin accumulation and retention. Accumulation of adriamycin in SGC7901 was 5.21+/-2.56 mg.L(-1),in SGC7901/VCR 0.3 was 0.85+/-0.29 mg.L(-1), in SGC7901/VCR 0.7 was 0.81+/-0.32 mg.L(-1), and in SGC7901/VCR 1.0 was 0.80+/-0.33 mg.L(-1); Retention of adriamycin in SGC 7901 was 2.51+/-1.23 mg.L(-1), in SGC7901/VCR 0.3 was 0.47+/-0.14 mg.L(-1), in SGC7901/VCR 0.7 was 0.44+/-0.15 mg.L(-1), and in SGC 7901/VCR 1.0 was 0.41+/-0.11 mg.L(-1). (4) Fluorescence intensity presented adriamycin accumulation in SGC7901/VCR cells was increased from 1.14+/-0.36 to 2.71+/-0.94 when cells were co-incubated with anti-PKC-alpha but not with anti-PKC-beta I PKC-beta II and PKCgamma antibodies. CONCLUSION: PKC-alpha, but not PKC-beta I, PKC-beta II or PKCgamma, may play a role in multidrug resistance of gastric cancer cells SGC7901/VCR.

Cloning and identification of an angiostatic molecule IP10/crg -2.

AIM:To obtain human and murine cDNAs encoding IFN-gamma inducible protein 10 (IP-10) and cytokine responsive gene-2 (Crg-2).METHODS:The encoding genes of IP-10 and Crg-2 were amplified by RT-PCR from cultured human fibroblast cells and Balb/c mouse liver treated by IFN-gamma and TNF-alpha,respectively, and cloned into plasmids of pUC19 and pGEM3Zf(+).RESULTS:The nucleotide sequences of the amplified DNA were confirmed by endonucleases digestion and sequencing.CONCLUSION:Recombinant IP-10/crg-2 gene clones with 306bp and 314bp inserts were established for further research on biological activities and ligands of hIP-10/mCrg-2.