The Thr to Met substitution of amino acid 118 in hepatitis B virus surface antigen escapes from immune-assay-based screening of blood donors.

Hepatitis B surface antigen (HBsAg) is the main diagnosis marker for hepatitis B virus (HBV) infection. In this study, a novel HBV mutant from an HBV-positive blood donor with false-negative results during HBsAg screening was identified. DNA sequencing discovered two mutations at nt 353 (A to T) and nt 349 (T to A), leading to Thr to Met and Ser to Thr substitutions at aa 118 and 117 of HBsAg, respectively. Further analysis showed that eight of ten HBsAg ELISA kits failed to detect this HBsAg mutant. A mutagenesis assay indicated that the Thr to Met substitution at aa 118 was the determinant for escape from HBsAg ELISA detection. A small-scale screening of blood donors identified two individuals infected by this unique HBV mutant, suggesting a certain level of prevalence among the general population. In conclusion, our study identified the aa 118 mutation in HBV surface antigen and provided information for improvement of HBV diagnosis products.

Prevalence and associated factors of knee osteoarthritis in a rural Chinese adult population: an epidemiological survey.

BACKGROUND: The exact pathogenic mechanism of knee osteoarthritis (OA) is still unknown. With the exception of clinical treatment to alleviate symptoms, or total knee replacement, there is currently no effective treatment method. Consequently, an in-depth etiological and epidemiological study of knee OA can provide clues for diagnosis, treatment and scientific research, and will ultimately have a beneficial effect on public health. METHODS: A cross-sectional community study in the rural village of Gaoyou was conducted in 3428 Chinese adults (aged ≥ 40 years). Subjects completed an interviewer-administered questionnaire, evaluating knee pain and associated disability, analgesia, use of health services, past medical history, walking, income, smoking, and use of oral contraceptives, and standardized weight-bearing knee radiographs were obtained. Patient demographic characteristics and biochemical parameters were recorded. RESULTS: Single-factor regression analysis indicated that age, overweight, central adiposity, high low-density lipoprotein cholesterol (LDLC), high total cholesterol (TC), high triglycerides (TG), dyslipidemia, hypertension and low income were the associated factors for knee OA in females; age, high LDLC, hypertension, low income and frequent walking were the associated factors for knee OA in males. Interestingly, male heavy smokers were less likely to develop severe knee OA compared with non-smokers. Stepwise logistic regression analysis indicated that age and overweight were the associated factors for knee OA for all individuals. Although central adiposity, high LDLC, high TC, high TG, dyslipidemia, hypertension and low income appeared to be related to knee OA in females according to univariate analysis, these factors were not identified in stepwise logistic regression analysis. In addition although age, high LDLC, hypertension and frequent walking were also the associated factors for knee OA in males by stepwise logistic regression analysis, smoking as a protective factor was not identified in this analysis. CONCLUSIONS: In this study, aging, obesity, frequent walking, low income and relevant multiple metabolic disorders were the associated factors for knee OA. Smoking might be associated with a lower prevalence of OA in male smokers according to univariate analysis. A retrospective association of smoking with OA may constitute an important etiologic clue, but further well-designed, large-scale prospective controlled trials are required to confirm these findings.

Activation of α7 nicotinic acetylcholine receptors protects astrocytes against oxidative stress-induced apoptosis: implications for Parkinson's disease.

Astrocytes have been implicated in the immune responses associated with Parkinson's disease (PD). Inhibition of astrocyte apoptosis is a novel strategy for the treatment of PD. Recent studies suggest that α7 nicotinic acetylcholine receptors (α7-nAChRs) expressed in glial cells are critical links between inflammation and neurodegeneration in PD. However, little is known about their contribution to astrocyte apoptosis during the development of this disorder. In the present study, we showed that nicotine exerts a protective effect on H2O2-induced astrocyte apoptosis and glial cell-derived neurotrophic factor (GDNF) downregulation, and this effect was abolished by an α7-nAChR-selective antagonist. The underlying mechanisms might involve alleviation of mitochondrial membrane potential loss, stabilization of the Bax/Bcl-2 balance, and inhibition of cleaved caspase-9 activity through α7-nAChR activation. Systemic administration of nicotine dramatically alleviated MPTP-induced symptoms, protected dopaminergic neurons against degeneration, inhibited astrocytes and microglia activation in the substantia nigra pars compacta (SNpc) and blocked the decrease of GDNF in the striatum by activating α7-nAChRs. Taken together these findings demonstrate, for the first time, that nicotine suppresses H2O2-induced astrocyte apoptosis via the mitochondrial pathway through the stimulation of α7-nAChRs. Targeting α7-nAChRs expressed in astrocytes may be a novel therapeutic strategy for the treatment of neurodegenerative disorders.

Azithromycin attenuates pulmonary inflammation and emphysema in smoking-induced COPD model in rats.

INTRODUCTION: The role of inflammation and immunity in COPD treatment is increasingly being recognized. The relationship between anti-inflammation/immunoregulation and emphysema in COPD lungs remains to be elucidated. The aim of this study was to investigate the effects of azithromycin (Azm) on the development of emphysema in smoking-induced COPD in rats. METHODS: Sprague-Dawley rats (n = 50) were randomly assigned to normal, COPD, saline-treated, Azm-treated, and levofloxacin-treated (Lev) groups. The effects of treatment were assessed by measuring the levels of vascular endothelial growth factor (VEGF) by enzyme-linked immunosorbent assay and measuring the numbers of neutrophil and macrophage in bronchoalveolar lavage fluid, vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR2) protein expression by western blotting. Lung function measurements and histopathological evaluations (mean linear intercept and destructive index) were performed. RESULTS: FEV0.3/FVC and peak expiratory flow were lower in the COPD group than in the normal group. Mean linear intercept and destructive index were lower in the Azm-treated group than in the COPD, saline-treated, and Lev-treated groups. The numbers of neutrophil and macrophage in bronchoalveolar lavage fluid were lower in the Azm-treated group than in the COPD, saline-treated, and Lev-treated groups. As confirmed by western blotting, the levels of VEGF in lung homogenates were higher in the Azm-treated group than in the COPD, saline-treated, and Lev-treated groups. VEGFR2 protein expression was higher in the Azm-treated group than in the COPD, saline-treated, and Lev-treated groups. CONCLUSIONS: Azm attenuates pulmonary emphysema by partly reversing the decrease in the numbers of inflammatory cells (neutrophil and macrophage) and VEGF secretion and VEGFR2 protein expression in smoking-induced COPD in rats.

Concurrent measurement of dynamic changes in viral load, serum enzymes, T cell subsets, and cytokines in patients with severe fever with thrombocytopenia syndrome.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infection caused by a novel Bunyavirus. Analysis on the dynamic changes of clinical, laboratory, and immunological abnormalities associated with SFTS in a concurrent study is lacking. Thirty-three SFTS patients were admitted to Jiangsu People's Hospital, Nanjing, China, and diagnosis was made based on the clinical symptoms and positive viral RNA detected by RT-PCR. Four patients deceased and twenty-nine survived. Blood samples were collected every other day between Day 5 and Day 15 from the onset of fever. Samples from healthy volunteers were used as normal controls. Peak viral RNA load, serum enzymes, IL-6, and IL-10 were significantly higher in deceased patients compared to survivors. Viral load, serum enzymes, and cytokines declined in survivors within 2 weeks from onset of fever. CD69+ T cells were elevated early after infection while HLA-DR+ and CTLA4+ T cells were elevated during the recovery phase of those who survived. High level SFTSV viral load was concurrently observed with reduced PLT, elevated serum enzymes, elevated pro-inflammatory and anti-inflammatory cytokines, and activation of CD69+ T cells. The degree and pattern of changes in these parameters may indicate the clinical outcome in SFTSV-infected patients.

[Hepatitis B e antigen from chronic hepatitis B patients induces Th1/Th2 cytokine imbalance in vitro].

OBJECTIVE: To study the immunoregulatory effect of hepatitis B virus (HBV) e antigen (HBeAg) on peripheral blood monocytes (PBMCs). METHODS: PBMCs were isolated from patients with chronic hepatitis B (CHB; both HBeAg- and HBeAg+) and healthy controls, and cultured with recombinant HBeAg. The HBeAg-induced changes in expression of PD-1/PD-L1 were measured by flow cytometry of the cells and in secreted cytokines were measured by enzyme-linked immunosorbent assay of the supernatants. Comparisons between two groups were made by the independent-samples t-test; the relationship between PD-1/B7-H1 level and HBV DNA copy number was evaluated by Spearman's correlation analysis. RESULTS: Exposure to HBeAg led to a significant decrease in CD3+CD4+ T lymphocyte-specific expression of IFNa for both the CHB patients' and healthy controls' samples (t = 2.382 and -4.190 respectively, P less than 0.01). For the HBeAg- CHB patients' and healthy controls' samples, the HBeAg exposure led to increased levels of secreted cytokines IL-6, IL-10 and TNFa (t = 2.504, 3.583 and 4.324, P less than 0.01 and t = 3.542, 6.246 and 5.273, P less than 0.01 respectively) and of CD14+ PBMC-specific expression of PD-L1 (t = 4.815 and 3.454, P less than 0.05 respectively). Compared to the HBeAg-negative CHB patients' and healthy controls' samples, the HBeAg+ CHB patients' samples had significantly lower CD3+CD4+ T cell-specific expression of IFNa (t = -3.177 and -4.541, P less than 0.01 respectively), but significantly higher levels of secreted IL-4 (t = 3.382 and 4.393, P less than 0.01 respectively), of CD3+ T cells-specific expression of PD-1/PD-L1 (t = 4.755, 2.942 and 4.518, 4.595, P less than 0.01 respectively), and of CD14+ T cells-specific expression of PD-L1 (t = 5.092 and 5.473, P less than 0.01 respectively). The CD3+ T cells-specific expression of PD-L1 was significantly higher in the samples from HBeAg- CHB patients than from the healthy controls (t = 3.214, P less than 0.01). CONCLUSION: HBeAg was able to down-regulate the production of Th1-type cytokines (IFNgamma), and up-regulate the secretion of Th2-type cytokines (IL-6, IL-10) and the expression of PD-1/PD-L1on monocytes. These changes are conducive to the formation of immune tolerance to HBV. Therefore, HBeAg may play an important role in immune tolerance to chronic HBV infection.

α7 nicotinic acetylcholine receptor-mediated neuroprotection against dopaminergic neuron loss in an MPTP mouse model via inhibition of astrocyte activation.

BACKGROUND: Although evidence suggests that the prevalence of Parkinson's disease (PD) is lower in smokers than in non-smokers, the mechanisms of nicotine-induced neuroprotection remain unclear. Stimulation of the α7 nicotinic acetylcholine receptor (α7-nAChR) seems to be a crucial mechanism underlying the anti-inflammatory potential of cholinergic agonists in immune cells, including astrocytes, and inhibition of astrocyte activation has been proposed as a novel strategy for the treatment of neurodegenerative disorders such as PD. The objective of the present study was to determine whether nicotine-induced neuroprotection in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model occurs via α7-nAChR-mediated inhibition of astrocytes. METHODS: Both in vivo (MPTP) and in vitro (1-methyl-4-phenylpyridinium ion (MPP+) and lipopolysaccharide (LPS)) models of PD were used to investigate the role(s) of and possible mechanism(s) by which α7-nAChRs protect against dopaminergic neuron loss. Multiple experimental approaches, including behavioral tests, immunochemistry, and stereology experiments, astrocyte cell cultures, reverse transcriptase PCR, laser scanning confocal microscopy, tumor necrosis factor (TNF)-α assays, and western blotting, were used to elucidate the mechanisms of the α7-nAChR-mediated neuroprotection. RESULTS: Systemic administration of nicotine alleviated MPTP-induced behavioral symptoms, improved motor coordination, and protected against dopaminergic neuron loss and the activation of astrocytes and microglia in the substantia nigra. The protective effects of nicotine were abolished by administration of the α7-nAChR-selective antagonist methyllycaconitine (MLA). In primary cultured mouse astrocytes, pretreatment with nicotine suppressed MPP(+)-induced or LPS-induced astrocyte activation, as evidenced by both decreased production of TNF-α and inhibition of extracellular regulated kinase1/2 (Erk1/2) and p38 activation in astrocytes, and these effects were also reversed by MLA. CONCLUSION: Taken together, our results suggest that α7-nAChR-mediated inhibition of astrocyte activation is an important mechanism underlying the protective effects of nicotine.

Combined analysis of AFP and HCCR-1 as an useful serological marker for small hepatocellular carcinoma: a prospective cohort study.

Hepatocellular carcinoma (HCC) is one of the most frequent malignant tumors in the world. The only serological marker widely used for the diagnosis of HCC is alpha-fetoprotein (AFP). Despite that AFP is widely used for the diagnosis of HCC, it has a limit as a serological marker due to its low sensitivity and specificity. The human cervical cancer proto-oncogene 1 (HCCR-1) was previously reported as a new biomarker for HCC. To further evaluate the HCCR-1 as a biomarker for HCC, we conducted the prospective cohort study. We evaluated the significance of simultaneous measurement of 2 tumor markers in the diagnosis of HCC in China, Japan and Korea. Two markers for HCC, AFP and HCCR-1, were measured in the sera obtained from 1,338 patients at the time of initial diagnosis of HCC. Of the 1338 HCC patients, 616 (46%) and 686 (51.3%) were sero-positive for AFP and HCCR-1, respectively. The positive rate for HCC was increased up to 74.1% in combined use of AFP and HCCR-1. Many cases (54%) for AFP-negative HCC were positive for HCCR-1 and vice versa. More importantly, the diagnostic rate for small HCC (< 2 cm) was significantly improved in the combined analysis of AFP and HCCR-1 to 56.9% although it was only 40.1% and 23.4% in the single analysis of HCCR-1 and AFP, respectively. Our result suggests that the HCCR-1 could be an useful biomarker for HCC while the diagnostic rate could be significantly improved in the combined use of HCCR-1 and AFP.

Osteopontin enhances the expression and activity of MMP-2 via the SDF-1/CXCR4 axis in hepatocellular carcinoma cell lines.

BACKGROUND AND AIMS: Osteopontin, SDF-1α, and MMP-2 are important secreted molecules involved in the pathophysiology of human hepatocellular carcinoma (HCC). This study investigates the effect of the SDF-1α/CXCR4 axis on expression and activity of MMP-2 induced by osteopontin. METHODS: The expression of CXCR4, SDF-1α, MMP-2 and their associated cellular signaling cascades, involving Akt and MAP Kinases, were determined by Western blotting. The activities of MMP-2 and MMP-9 were assayed by gel zymography. The role of the osteopontin receptors integrin α(v)ß3 and CD44v6 was evaluated using neutralizing antibodies. We also established CXCR4-deficient SMMC7721 cell lines by transfection with miRNA-CXCR4 plasmids and determined cell invasion activity in a transwell assay. RESULTS: In comparison with untreated cells, recombinant human osteopontin (rhOPN) up-regulated CXCR4, SDF-1α, and MMP-2 expression about 5-, 4-, and 6-fold on the protein levels through binding to integrin α(v)ß3 and CD44v6 in hepatocellular carcinoma cells (SMMC7721 and HepG2). Inhibition of the SDF-1α/CXCR4 axis down-regulated the rhOPN-induced MMP-2 expression and activity. rhOPN also activated Akt, p38 and JNK. Down-regulation of CXCR4 decreased the rhOPN-induced invasion in SMMC7721 cells. CONCLUSION: These results indicate that rhOPN up-regulates MMP-2 through the SDF-1α/CXCR4 axis, mediated by binding to integrin α(v)ß3 and CD44v6 and activating the PI-3K/Akt and JNK pathways in HepG2 and SMMC7721 cells. Therefore, the osteopontin-SDF-1α/CXCR4-MMP-2 system may be a new therapeutic target for treating HCC progression.

Clinical utility of alpha fetoprotein and HCCR-1, alone or in combination, in patients with chronic hepatitis, liver cirrhosis and hepatocellular carcinoma.

Serum alpha fetoprotein (AFP) is the most widely used tumor marker in detecting patients with hepatocellular carcinoma (HCC). However, it has been indicated that HCCR-1 (human cervical cancer oncogene 1) might be supplementary to AFP in the detection. We conducted a prospective study in 120 normal and 524 liver disease patients to evaluate the significance of simultaneous measurement of 2 tumor markers (AFP and HCCR-1) in the diagnosis of HCC through the cohort study in Korea and China. We also performed immunohistochemical studies using 25 normal subjects (N), 32 liver cirrhosis (LC) and 116 HCC tissues. The sensitivities of AFP (20 ng/mL) and HCCR-1 (10 ng/mL) in HCC were 55.8% (164/294) and 44.2% (130/294), respectively. When AFP was combined with HCCR-1, sensitivities increased to 4.2% (N), 12.7% (chronic hepatitis; CH), 50.0% (LC), and 77.2% (HCC), respectively. Although there was no significant difference in the diagnostic rate for HCC between AFP and HCCR-1, many cases for AFP-negative HCC were positive for HCCR-1 and vice versa. Moreover, the combined use of AFP and HCCR-1 improved the diagnostic rate to 70.8% in small HCC (< 2 cm) and 81.6% in large HCC (⩾ 2 cm), respectively. AFP and HCCR-1 are independent markers. Our result suggests that the HCCR-1 could be an useful biomarker for HCC while the diagnostic rate could be significantly improved in the combined use of HCCR-1 and AFP.

[The study of expansion and function HBV special interferon-γ secreting human T lymphocytes in vitro].

AIM: To expand HBV special interferon-γ secreting human T lymphocytes in vitro and sustain the ability to recognize special T cell epitodes of HBcAg. METHODS: After stimulated by peptide 22 hours, feeded with autologous peripheral blood mononuclear cells ( PBMCs) deproliferated by mitomycin C and low concentration of IL-2 to expand in vitro for other 4 weeks. Then cells were collected and tested by flow cells assay every week. The ability of these cells to recognize special epitodes was tested by enzyme linked immunospot assay in which autologous lymphoblastoid cell lines (LCLs) produced from PBMC was transfected by epstein-barr virus pulsed with peptides and used as target cell. RESULTS: HBV special interferon-secreting human T lymphocytes were selected and expanded more than 1000 times in vitro. The ability of the T cells to recognize special epitodes is as same as unexpanded cells and the proportion of CD4 and CD8 positive cells likely remained native state. CONCLUSION: HBV special interferon-γ secreting T lymphocytes are effectively expanded in vitro and keep the ability to recognize stringently special T cell epotides.

Requirement of Osteopontin in the migration and protection against Taxol-induced apoptosis via the ATX-LPA axis in SGC7901 cells.

BACKGROUND: Autotaxin (ATX) possesses lysophospholipase D (lyso PLD) activity, which converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA). The ATX-LPA signaling axis has been implicated in angiogenesis, chronic inflammation and tumor progression. Osteopontin (OPN) is an important chemokine involved in the survival, proliferation, migration, invasion and metastasis of gastric cancer cells. The focus of the present study was to investigate the relationship between the ATX-LPA axis and OPN. RESULTS: In comparison with non-treated cells, we found that the ATX-LPA axis up-regulated OPN expression by 1.92-fold in protein levels and 1.3-fold in mRNA levels. The ATX-LPA axis activates LPA2, Akt, ERK and ELK-1 and also protects SGC7901 cells from apoptosis induced by Taxol treatment. CONCLUSIONS: This study provides the first evidence that expression of OPN induced by ATX-LPA axis is mediated by the activation of Akt and MAPK/ERK pathways through the LPA2 receptor. In addition, OPN is required for the protective effects of ATX-LPA against Taxol-induced apoptosis and ATX-LPA-induced migration of SGC7901 cells.

ATX-LPA axis induces expression of OPN in hepatic cancer cell SMMC7721.

Osteopontin (OPN) and autotaxin (ATX) are important chemokines involved in the survival, proliferation, migration, invasion, and metastasis of many cancer cells. The focus of the study was to investigate the relationship between OPN and ATX-lysophosphatidic acid (LPA) axis. The expression of OPN and its cellular cascades were determined by western blot and real-time quantitative transcription polymerase chain reaction (real-time PCR) analyses. Cell migration activity was determined by a Transwell-migration assay. In comparison with nontreated cells, we found that the ATX-LPA axis upregulated OPN expression by 2.91-fold in protein levels and 2.52-fold in mRNA levels. The ATX-LPA axis activates Akt and ATX/LPC-induced OPN expression in SMMC7721 cells was largely reduced by the inhibitors of phosphatidylinositol 3-kinase (PI3K)/Akt or LPA receptor. This study provides the first evidence that the induction of the OPN expression by ATX-LPA axis was mediated by the activation of Akt through LPA receptors and OPN was required for migration of SMMC7721 cells induced by ATX-LPA axis.

Gastric atrophy and intestinal metaplasia before and after Helicobacter pylori eradication: a meta-analysis.

OBJECTIVE: Whether gastric atrophy (GA) and intestinal metaplasia (IM) are reversible after the eradication of Helicobacter pylori remains controversial. The purpose of this meta-analysis was to systematically review histological alterations in GA and IM by comparing histological scores before and after H. pylori eradication. METHODS: English-language articles in the medical literature containing information about the association between infection with H. pylori and gastric premalignant lesions (i.e. GA and IM) were identified by searching the Medline, PubMed, and EMBASE databases with suitable key words up to December 2009. Review Manager 4.2.8 was used for the meta-analysis. RESULTS: Twelve studies containing a total of 2,658 patients were included in the first meta-analysis. Before treatment, 2,648 patients had antrum GA, 2,401 patients had corpus GA, 2,582 patients had antrum IM, and 2,460 patients had corpus IM. Comparing the histological alterations before and after H. pylori eradication, the pooled weighted mean difference (WMD) with 95% CI for antral GA was 0.12 (0.00-0.23), p = 0.06. For corpus GA, the pooled WMD was 0.32 (0.09-0.54), p = 0.006. For antral IM, the pooled WMD was 0.02 (-0.12-0.16), p = 0.76, and for corpus IM, the pooled WMD was -0.02 (-0.05-0.02), p = 0.42. CONCLUSION: Our study shows that eradication of H. pylori results in significant improvement in GA in the corpus but not in the antrum; it also does not improve gastric mucous IM. Consequently, all patients with GA in the corpus should be tested for H. pylori infection, and eradication therapy should be prescribed for H. pylori-positive patients in those with GA in corpus.

Soluble CD40 ligand-activated human peripheral B cells as surrogated antigen presenting cells: A preliminary approach for anti-HBV immunotherapy.

BACKGROUND: We aimed to clarify whether soluble CD40 ligand (sCD40L) activated B cells may be loaded with HBcAg18-27 peptide and served as antigen-producing cells (APCs) to induce HBV-specific cytolytic T lymphocytes (CTLs). RESULTS: Human B cells could be cultured in the presence of sCD40L up to 54 days, and the proportion of B cells in the S phase increased from 0% to 8.34% in the culture. The expression of CD80, CD86, major histocompatibility complex (MHC) classes I and II molecules on the sCD40L-activated B cell was significantly increased after long-time culture. Cytometry and fluorescence microscopy showed that more than 98% sCD40L-activated B cells were loaded by the HBcAg peptide. Furthermore, the peptide-pulsed activated B cells could induce HBcAg18-27 specific CTLs. CONCLUSIONS: Our results demonstrate that sCD40L-activated B cells may function as APCs and induce HBV-specific CTLs.

Epidermal growth factor induces HCCR expression via PI3K/Akt/mTOR signaling in PANC-1 pancreatic cancer cells.

BACKGROUND: Human cervical cancer oncoprotein 1 (HCCR-1), reported as a negative regulator of p53, is over-expressed in a variety of human cancers. However, it is yet unknown whether HCCR-1 plays any role in pancreatic cancer development. The aim of this study was to investigate the effect of epidermal growth factor on the expression of HCCR in pancreatic cancer cells, and to explore if PI3K/Akt/mTOR signaling pathway mediated this expression. METHODS: A polyclonal antibody against HCCR protein was raised by immunizing Balb/c mice with the purified recombinant protein pMBPc-HCCR. Tissue samples were constructed on a tissue chip, and the expression of HCCR was investigated by immunohistochemistry assay and Western blotting. Pancreatic cell line, PANC-1 cells were stably transfected with plasmids containing sense-HCCR-1 fragment and HCCR siRNA fragment. MTT and transwell assay were used to investigate the proliferation and invasion of stable tansfectants. The specific inhibitor of PI3K and mTOR was used to see if PI3K/mTOR signal transduction was involved in the induction of HCCR gene expression. A Luciferase assay was used to see if Akt can enhance the HCCR promoter activity. RESULTS: HCCR was up-regulated in pancreatic tumor tissues (mean Allred score 4.51+/-1.549 vs. 2.87+/-2.193, P<0.01), especially with high expression in poorly differentiated pancreatic cancer. The growth of cells decreased in HCCR-1 siRNA transfected cells compared with vector transfectants. The number of invasion cells was significantly lower in HCCR-1 siRNA transfected cells (24.4+/-9.9) than that in vector transfectants (49.1+/-15.4). Treatment of PANC-1 cells with epidermal growth factor increased HCCR protein level in a dose- and time-dependent manner. However, application of LY294002 and rapamycin caused a dramatic reduction of epidermal growth factor-induced HCCR expression. Over-expression of exogenous constitutively active Akt increased the HCCR promoter activity; in contrast, dominant negative Akt decreased the promoter activity. CONCLUSIONS: EGF-induced HCCR-1 over-expression is mediated by PI3K/AKT/mTOR signaling which plays a pivotal role in pancreatic tumor progression, suggesting that HCCR-1 could be a potential target for cancer therapeutics.

Involvement of mTOR and survivin inhibition in tamoxifen-induced apoptosis in human hepatoblastoma cell line HepG2.

Patients with advanced hepatocellular carcinoma (HCC) have shown to benefit from tamoxifen treatment. The mechanisms of tamoxifen effects in HCC, however, are not yet clearly understood. The PI3K/Akt/mTOR signal pathway is involved in cell proliferation, tumorigenesis, and apoptosis. Over-expression of survivin has played an important role in leading to antiapoptosis. The current study investigated changes in mTOR pathway and survivin expression in hepatocarcinoma cell line HepG2 treated with tamoxifen. We detected apoptosis of hepatocarcinoma cells by flow cytometry assay. Survivin transcription level and p70S6k was demonstrated by PCR, dual-luciferase reporter assay and western blot analysis respectively. Our results are showed as follows: tamoxifen leads to apoptosis of the cells, and reduction in survivin expression, as well as a dramatic reduction in the activated form of p70S6 kinase. Twenty micromoles tamoxifen treatment significantly depresses transcription of survivin mRNA. Treating HepG2 cells with rapamycin, a specific mTOR inhibitor, significantly reduce survivin protein level but did not affect survivin transcription, which indicated that tamoxifen and rapamycin were synergistic in regards to down-regulation of survivin expression in hepatocellular carcinoma cells. Our results suggest that tamoxifen down-regulate survivin expression in HepG2 cells is mediated by transcriptional and posttranscriptional level via PI3K/Akt/mTOR pathway to induce apoptosis.

[Down-regulation of mTOR activity and survivin expression during tamoxifen-induced apoptosis in hepatoblastoma cells].

OBJECTIVE: The aim of this study was to investigate the changes in mTOR activity and survivin expression in liver cancer cell line HepG2 cells treated with tamoxifen. METHODS: Survivin transcription level and p70S6K was demonstrated by PCR, dual-luciferase reporter assay and Western blot analysis, respectively, and the apoptosis in the HepG2 cells was detected by flow cytometry. RESULTS: Tamoxifen leads to apoptosis of the cells and reduction in survivin expression, as well as a dramatic reduction in the activated form of p70S6K. Treating HepG2 cells with rapamycin, a specific mTOR inhibitor, significantly reduced the survivin protein level but not affected the survivin transcription, indicating that tamoxifen and rapamycin were synergistic in regards to down-regulation of survivin expression in hepatocellular carcinoma cells. CONCLUSIONS: Our results suggest that tamoxifen down-regulates survivin expression in HepG2 cells and it is mediated by transcriptional and post-transcriptional level via PI3K/Akt/mTOR pathway to induce apoptosis.

Interaction of Helicobacter pylori with genetic variants in the MDM2 promoter, is associated with gastric cancer susceptibility in Chinese patients.

BACKGROUND AND AIMS: MDM2 is a major negative regulator of p53, and a single nucleotide polymorphism (SNP) in the MDM2 promoter region SNP309 (rs2279744) has been shown to increase the affinity of the transcriptional activator Sp1, resulting in elevated MDM2 transcription and expression in some cancers. The aim of this study was to determine whether SNP309 is associated with susceptibility to gastric cancer in Chinese patients. METHODS: Two hundred and sixty patients with gastric cancer and 260 healthy controls were genotyped for MDM2 SNP309 using the PCR-RFLP method. Helicobacter pylori's infection status was determined using a validated serology test. RESULTS: The healthy control group was found to be in Hard-Weinberg equilibrium at the polymorphic loci studied (chi(2) = 3.63, p = 0.06). Multiple logistic regression analyses revealed that the risk of gastric cancer for SNP309 (G/G) was significantly increased when compared with T carriers (adjusted odds ratio (OR): 2.05, 95% confidence interval (CI): 1.31-3.20). Further stratification analyses based on the recessive models reveal that a significantly increased risk of gastric cancer associated with the GG genotype was evident among subjects with H. pylori infection (adjusted OR: 2.52, 95% CI: 1.45-4.37), and noncardia gastric cancer patients (adjusted OR: 2.29, 95% CI: 1.41-3.71), compared with the T carriers. When stratified by histologic subtype of gastric cancer, the risk was also statistically significant. There was no significant difference in age at diagnosis among genotypes. CONCLUSIONS: The MDM2 promoter SNP309 is associated with the presence of gastric cancer in Chinese patients especially those with H. pylori infection.

[Establishment of long-term culture system for human B cells derived from peripheral blood mononuclear cells activated via soluble CD40 ligand].

OBJECTIVE: To develop a method for long-term culture of human B cells from peripheral blood mononuclear cells (PBMCs) by means of human soluble CD40 ligand (sCD40L), and to investigate the proliferation and antigen-presenting function of the CD40-activated B cells. METHOD: Peripheral blood sample of 30 ml was collected from a healthy person. PBMCs were isolated and cultured in the presence of sCD40L. Flow cytometry was used to examine the proliferation, DNA cycle, and cell surface markers: CD86, CD80, major histocompatibility complex (MHC) class II, and MHC Class I of the B cells. The cells cultured for 45 days were divided into 2 groups: Group 1 incubated with the fluorescein isothiocyanate (FITC)-conjugated hepatitis-B core antigen (HBcAg-FITC) and Group 2 used as control group. Eighteen hours later cytometry and fluorescence microscopy were used to detect the peptide pulsing. RESULTS: The B cells could be cultured up to 50 days in the sCD40L culture system. The ratio of B cells in the PBMCs was 8.21% at the beginning, and increased to 70.67% by day 47, and the B cell absolute count was 6.56 x 10(5) at the beginning and increased to 8.61 x 10(6) at day 50, about 10 times higher. sCD40L promoted a strong up-regulation of cell surface markers. The expression rates of CD80, CD86, MHC-II, and MHC-I of the sCD40L-activated B cells were 83.97%, 84.73%, 99.59%, and 98.70% respectively. Flow cytometry showed that 98.10% of the B cells co-incubated with HBcAg-FITC were loaded with HBcAg. Fluorescence microscopy showed that the HBcAg was in the cytoplasm of the B cells. CONCLUSION: A human sCD40L culture system has been developed with the ability to culture human peripheral blood B cells, thus realizing the long-term proliferation and activation of human peripheral blood B cells that function as antigen-presenting cells.