Role and recruitment of Th9 cells in liver cirrhosis patients

Objective: To investigate the role of T helper 9 (Th9) cells in liver cirrhosis (LC) patients and whether chemokine receptor type 6 (CCR6)/chemokine ligand 20 (CCL20) axis is involving in the recruitment of Th9 cells into liver. Methods: Peripheral blood and liver tissue from 30 LC patients and 18 normal controls were recruited. The frequency of Th9 cells and CCR4, CCR6 in the peripheral blood was tested by flow cytometry. Serum interleukin (IL)-9 and CCL20 levels were tested by enzyme-linked immunosorbent assay. Immunohistochemical staining was used to detect α-smooth muscle actin, CCR6 and CCL20 expression in liver tissue. Results: The frequency of Th9 cells in LC patients was significantly increased compared with controls (P < 0.05). The serum IL-9 level and CCL20 level increased markedly in LC patients compared with controls (P < 0.05), and IL-9 was positively correlated to Th9 cells and CCL20. Furthermore, the frequency of Th9 cells was correlated to prothrombin time, total bilirubin level, hyaluronic acid and type IV collagen in LC patients. We also found that Th9 cells in LC patients expressed higher frequency of CCR4

Role of Th9 cells and Th17 cells in the pathogenesis of malignant ascites

Objective: To assess the role of Th9 and Th17 cells in malignant ascites (MA). Methods: MA from 30 hepatic carcinoma patients and benign ascites from 30 cirrhotic patients were collected. Corresponding peripheral blood samples from these hepatic carcinoma and cirrhotic patients as well as 30 healthy subjects were collected. The frequency of Th9 and Th17 cells was tested by flow cytometry. Serum levels of interleukin (IL)-9 and IL-17 were examined by ELISA. Results: The observed frequency of Th9 and Th17 cells, and the IL-9 and IL-17 serum levels were significantly higher in MA patients than those in cirrhotic patients and healthy control samples ( P<. 0.05). Moreover, the Th9 cells demonstrated positive correlation with Th17 cells as well as IL-9 in MA patients; however, this positive correlation was not observed in the cirrhotic patients or healthy control samples. The frequency of Th9 and Th17 cells was distinctly higher in MA patients presenting with stage III or IV malignancy and with lymph node or distant metastasis than those in patients in stage I or II and without distant metastasis ( P<. 0.05). Conclusions: The increased frequency of Th9 and Th17 cells in MA patients suggests that these two T cell subsets play a synergistic role in MA pathogenesis. This study also demonstrated that Th9 and Th17 cells may perform their biological functions in conjunction with IL-9 production.

Activation of hepatocyte growth factor promotes apoptosis of hepatic stellate cells via the Rho pathway / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the role of activated hepatocyte growth factor (HGF) in apoptosis of hepatic stellate cells (HSCs) and in modulating the Rho signaling pathway.</p><p><b>METHODS</b>HSCs were divided into the following groups: blank control, consisting of HSCs without treatment; two treatment controls, consisting of HSCs exposed to exogenous HGF at 50 ng/ml and HSCs exposed to exogenous HGF activator (HGFA) at 70 ng/ml; three experimental groups, consisting of HSCs exposed to both exogenous HGF and HGFA, HSCs pre-incubated with the HGF inhibitor c-met at 500 ng/ml for 6 hours and then exposed to exogenous HGF and HGFA, and HSCs pre-incubated with the Rho pathway inhibitor Y-27632 at 10 ng/ml and then exposed to exogenous HGF and HGFA. Activation status of the cultured HSCs was determined by change in expression of alpha-smooth muscle actin (SMA). The optimal intervention concentration of Y-27632 was determined by MTT assay. The apoptotic status of HSCs was determined by flow cytometry. Expression of the HGF-alpha chain was detected by immunofluorescence. The expression of RhoA was evaluated by PCR (for mRNA) and by immunohistochemical staining and Western blot analysis (for protein).</p><p><b>RESULTS</b>Exposure to 10 mumol/L Y-27632 led to obvious growth inhibition of HGF + HGFA-induced HSCs, compared with the other concentrations tested (P less than 0.05). HGF + HGFA induced the expression of the HGF-alpha chain in a time-dependent manner (P less than 0.01); however, the increases in expression of HGF-alpha chain induced by HGF alone and HGFA alone were not significantly different from the level in the blank controls (P more than 0.05). Exposure to HGF alone and HGFA alone led to a time-dependent increase in apoptosis (24 h, 48 h, 72 h) but exposure to HGF + HGFA led to the highest levels of apoptosis (P less than 0.05). Exposure to HGF + HGFA led to a time-dependent decrease in RhoA mRNA and protein expression (P less than 0.01).</p><p><b>CONCLUSION</b>Activation of hepatocyte growth factor promotes apoptosis of hepatic stellate cells by suppressing RhoA expression and down-regulating the Rho signaling pathway.</p>

Association of Helicobacter pylori with Elevated Blood Ammonia Levels in Cirrhotic Patients: A Meta-Analysis

PURPOSE: The association between Helicobacter pylori (H. pylori) and blood ammonia levels in cirrhotic patients is controversial. We aimed to clarify this controvercy by performing a meta-analysis of published studies. MATERIALS AND METHODS: We searched PubMed, EMBASE and Cochrane library for studies which explored the association between H. pylori and blood ammonia levels in cirrhotic patients before May 2012. Six cohort studies involved in 632 H. pylori positive and 396 H. pylori negative cirrhotic patients were eligible for our analysis. The summary estimates were presented as standard means differences (SMD) and 95% confidence intervals (CI) from individual studies. RESULTS: Overall, there was significant association between H. pylori infection and the elevated blood ammonia levels in cirrhotic patients (SMD=0.34, 95% CI=0.21-0.47, I2=42.1%). Sensitivity analysis further confirmed this association. Subgroup analysis showed that the association was found only in Asian ethnicity, but not in Caucasian ethnicity. CONCLUSION: H. pylori infection is associated with elevated blood ammonia levels in cirrhotic patients, and more large scale studies and stratify analysis are warranted in order to further evaluate this association.

Activation of hepatocyte growth factor-induced apoptosis in hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To determine whether apoptosis is induced in rat hepatic stellate cells (HSCs) in response to activation of the hepatocyte growth factor (HGF) by hepatocyte growth factor activator (HGFA) by using a co-culture system of bone marrow mesenchymal stem cells (BMSCs) and HSCs.</p><p><b>METHODS</b>In this study, cells were divided into the following five groups: HSC control group: HSCs co-cultured with fibroblast cells; HSCs blank group: HSCs cultured alone; BMSCs blank group: BMSCs cultured alone; Experimental group: BMSCs + HSCs; HGFA intervention group: HSCs treated with 70 ng/mL of HGFA. The culture systems were established in culture plates with transwell inserts, and cells were assessed at 24, 48, and 72 h of growth. Dynamic changes in cell morphology were observed under an inverted phase contrast microscope. The surface markers of BMSCs and the apoptosis rate of HSCs were detected by Annexin-V-FITC/propidium iodide (PI). Expression of a-smooth muscle actin (SMA) in HSCs was evaluated by immunohistochemistry. The presence of activated HGF (HGF-a chain) was determined by immunofluorescent staining. HSC proliferation was measured by MTT assay, and the concentrations of HGF and HGFA were quantified by enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>MTT results indicated that treatment with HGF alone had no effect on HSC proliferation rate (vs. HSC blank group, P more than 0.05), but that 24 h treatment with HGFA significantly inhibited the proliferation rate (0.26 ± 0.00 vs. blank group: 0.13 ± 0.04, P = 0.02); moreover, this effect was concentration-dependent. Expression of HGF-a was lower in the experimental group than in the HGFA intervention group at 72 h (37.24 ± 1.03 vs. 40.44 ± 0.77, P = 0.04), and both of these groups had higher expression than the control group at all time points examined (P less than 0.05). The apoptosis rate was consistently higher in the experimental group than in the HGFA intervention group, but most robustly at 72 h (40.77 ± 1.16% vs. 33.35 ± 2.04%, P = 0.00); moreover, the apoptosis rate was significantly higher than that in the control group at all time points examined (P less than 0.01). The concentration of HGF in the experimental group and the HGFA intervention group showed a time-dependent reduction, and was consistently lower than that in the HSCs control group (P less than 0.05). Finally, the concentration of HGFA was higher in the experimental group than in the blank group at all time points examined (P less than 0.05).</p><p><b>CONCLUSION</b>The BMSC-HSC co-culture system can promote secretion of HGFA from HSCs and HGF activation, thereby inducing apoptosis of HSCs.</p>

Effect of sphingosine kinase 1 on the apoptosis, migration and invasion of colon cancer HT-29 cells and its molecular mechanisms / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the effect of sphingosine kinase 1 (SphK1) on the proliferation, apoptosis, migration and invasion of colon cancer TH-29 cells and to explore its molecular mechanisms.</p><p><b>METHODS</b>Phorbol 12-myristate 13-acetate (PMA) was used to induce the activity of SphK1 and N, N-dimethylsphingosine (DMS) was used to suppress the activity of SphK1. Cell prolieration and apoptosis were detected by MTT assay and flow cytometry, respectively. The migration and invasion capabilities of the cells were assessed in Transwell chambers. The activity of SphK1 was assayed by autoradiography. Western blot was used to evaluate the protein expression of SphK1, p38, phosphorylated p38 (p-p38) and SAPK/JNK.</p><p><b>RESULTS</b>PMA and DMS were able to induce and suppress the activity and protein expression of SphK1 in a time-dependent manner, respectively. PMA enhanced and DMS suppressed the cell viability in a time- and dose-dependent manner. Being treated with 100 nmol/L PMA or 50 µmol/L DMS for 0, 6, 12, 24 h, the cell apoptosis rates of PMA group were (9.35 ± 0.84)%, (7.61 ± 0.48)%, (5.53 ± 0.76)% and (0.56 ± 0.33)%, contrastly, that of DMS group were (9.18 ± 0.94)%, (12.06 ± 1.41)%, (19.80 ± 2.36)% and (31.85 ± 3.60)%, respectively. Compared with the control group, the cell migration and invasion capabilities of the PMA group were significantly enhanced, and that of the DMS group were significantly suppressed. The migration cell number of control, PMA and DMS groups were 68.75 ± 6.15, 109.33 ± 11.63 and 10.83 ± 2.48, the invasion cell number of control, PMA and DMS groups were 55.42 ± 4.50, 90.58 ± 7.06 and 9.58 ± 2.39, respectively. With the elevating activity and expression of SphK1, the protein expressions of p38, p-p38 and SAPK/JNK were strikingly suppressed. On the contrary, after treating with DMS the protein expressions of p38, p-p38 and SAPK/JNK were enhanced.</p><p><b>CONCLUSIONS</b>SphK1 potently enhances the prolieration, migration and invasion of colon cancer HT-29 cells, meanwhile suppresses the cell apoptosis. The suppressing of the p38 and SAPK/JNK signalling pathways may be one of its molecular mechanisms.</p>

Association of HLA-DQA1 loci with Helicobacter pylori infection in children of Guangxi Zhuang, Mulam nationalities, China and central Jing nationality, Vietnam / 中华儿科杂志

<p><b>OBJECTIVE</b>To compare the prevalence of Helicobacter pylori (Hp) infection in children of Zhuang and Mulan ethnic groups, Guangxi Luocheng county, China and in children of Jing ethnic group, Central Vietnam and to analyze the association of HLA-DQA1 alleles of these ethnic groups' children with Hp infection.</p><p><b>METHODS</b>Serodiagnosis by determining Hp antibody with ELISA and determination of serum CagA, VacA and urease antibodies by immunoblotting were performed for 54 Zhuang, 76 Mulan and 109 Jing children. Polymerase chain reaction-single strand polymorphism (PCR-SSP) technique was applied to determine the polymorphism of the HLA-DQA1 locus of these children and then the association of HLA-DQA1 alleles of these minority children with Hp infection was analyzed by SAS software.</p><p><b>RESULTS</b>The prevalence of Hp infection were 39% in Vietnamese Jing nationality, which was significantly lower than that in children of Guangxi Luocheng county (65% in Zhuang nationality and 58% in Mulan nationality) (P < 0.01). The distribution of HLA-DQA1 locus was not significantly different among the 3 groups. The frequency of HLA-DQA1 * 0104 allele was significantly higher in children with Hp infection than in children without Hp infection in each of the 3 groups (P < 0.01).</p><p><b>CONCLUSION</b>The results indicated that the prevalence of Hp infection in Zhuang and Mulan minority ethnic groups in Guangxi, China was higher than that in Vietnamese Jing ethnic group children. HLA-DQA1 * 0104 allele may be associated with susceptibility to Hp infection.</p>