Thalidomide-induced angiopoietin 2, Notch1 and Dll4 downregulation under hypoxic condition in tissues with gastrointestinal vascular malformation and human umbilical vein endothelial cells.

OBJECTIVE: To determine the pathogenesis of gastrointestinal vascular malformation (GIVM) and the mechanism of thalidomide in treating GIVM by evaluating the expression of angiopoietin 2 (Ang2), Notch1, delta-like ligand 4 (Dll4) and hypoxia inducible factor 1α (Hif-1α). METHODS: Data of 10 patients with histology-confirmed GIVM were reviewed. Immunohistochemistry of surgically resected GIVM tissues and the adjacent mucosa of the patients and normal tissues from those who had undergone colonoscopy for health examination was performed to examine the expressions of Ang2, Notch1, Dll4 and Hif-1α. In addition, in vitro effect of thalidomide on Ang2, Notch1 and Dll4 in human umbilical vein endothelial cells (HUVEC) and on HUVEC proliferation was also investigated during normoxic and hypoxic conditions. RESULTS: GIVM lesions presented as tortuous, dilated arterioles, venules and capillaries. Ang2, Notch1 and Dll4 showed strong immunoreactivity in the cytoplasm and nuclei of GIVM lesions but negative or weak positivity in the intestinal mucosa of the adjacent tissues and normal mucosa. Under hypoxic condition the expressions of Hif-1α, Ang2, Notch1 and Dll4 were upregulated and the tube formation was more abundant with a greater diameter of tubes. Moreover, thalidomide downregulated their expression in HUVEC and HUVEC proliferation decreased in a concentration-dependent manner under both hypoxic and normoxic conditions. CONCLUSION: Ang2, Notch1, Dll4 and Hif-1α may play an important role in the pathogenesis of GIVM and may be potential targets of thalidomide in the treatment of the disease.

New ideas for future studies of Helicobacter pylori.

Gastric cancer (GC) is one of the inflammation-associated cancers. Helicobacter pylori is now thought to be responsible for more than 95% of all GCs, and its development is associated with at least four mechanisms that lead to genetic instability of the gastric mucosa. The risk of developing GC can be predicted by assessing the extent and severity of corpus atrophy and the degree of risk can be estimated by using non-invasive methods such as the pepsinogen test, or endoscopic or histological cancer risk scoring systems such as the operative link for gastritis assessment. The eradication of H. pylori will stop the progression of gastritis, prevent atrophy and thus decrease the risk of cancer. H. pylori eradication should follow the dictum "use what works best locally". There are several new developments in the diagnosis and treatment of H. pylori infection including serological antibody, fluorescent in situ hybridization and antibiotic resistance tests. It is still necessary to develop a preventive or therapeutic vaccine to prevent GC.

Differentiation of nonhuman primate embryonic stem cells into hepatocyte-like cells.

OBJECTIVE: To investigate whether cells derived from rhesus monkey embryonic stem cells (ESC) had hepatocyte characteristics after the differentiation. METHODS: Rhesus monkey ESC were induced towards hepatocyte-like cells via a four-step differentiation process: the formation of embryoid bodies (EB), EB in activin A and insulin-transferrin-selenium medium for 4 days, in fibroblast growth factor (FGF)-4 and bone morphogenetic protein-2 (BMP2) medium for 8 days, in hepatocyte culture medium containing hepatocyte growth factor for 3 days and then with oncostatin M and dexamethasone for another 5 days. Expression of albumin (ALB), glucose-6-phosphatase, α-fetoprotein (AFP) and α-1 antitrypsin (α1-AT) at the mRNA level in differentiated cells were detected by reverse transcription-polymerase chain reaction. The expression of hepatocyte markers AFP, ALB, hepatocyte nuclear factor 4 (HNF4), cytokeratin 8 (CK8), CK19 and cell proliferation marker, Ki67, in the differentiated cells were determined by immunocytochemistry. The ultrastructure of the differentiated cells was examined by electron microscopy. Indocyanine green (ICG) uptake was also explored. RESULTS: After induction, some differentiated cells were binucleate, which is typical of hepatocytes. Hepatocyte-specific genes ALB, glucose-6-phosphatase, AFP and α1-AT were expressed in the differentiated cells. The differentiated cells expressed hepatocyte markers AFP, ALB, HNF4, CK8 and CK19 at the protein level. The cells also expressed cell proliferation marker Ki67. Under electron microscopy, the ultrastructures of hepatocyte-like cells, such as mitochondrion and catalase-containing peroxisomes, were observed in the differentiated cells. ICG uptake test was positive in differentiated cells. CONCLUSIONS: With cytokine induction, rhesus monkey ESC differentiated into cells displaying morphological features, gene expression patterns and metabolic activities characteristic of hepatocytes.

Can the incidence of gastric cancer be reduced in the new century?

Gastric cancer (GC) is one of the most common cancers in the world. The incidence and mortality rate of GC vary among different countries. It is suggested that GC is the result of the interaction between Helicobacter pylori (H. pylori) infection and the genetic and environmental factors in the host. H. pylori infection is the trigger of intestinal gastric adenocarcinoma. The incidence of GC is highest in East Asia and East Europe, but much lower in Africa; however, H. pylori infection is commonly seen in Africa, which is known as the African enigma. The 5-year survival of early GC is far better than that of advanced GC. A high detection rate of early GC could help us to conquer GC. A decreasing trend of GC incidence has been witnessed worldwide. With the improvement of living conditions and the achievements of scientific research, it seems possible that there will be a further reduction in the incidence of GC in the new century.

Predictive value of neutrophil infiltration as a marker of Helicobacter pylori infection.

AIM: To evaluate the predictive value of neutrophil infiltration as a marker of Helicobacter pylori (H. pylori) infection. METHODS: A total of 315 patients with dyspepsia symptoms who underwent upper gastrointestinal endoscopy were enrolled in this study. Biopsies were evaluated using the updated Sydney system. The medication history of all patients in the preceding 4 wk was recorded. The diagnosis of H. pylori infection was based on (13)C-urea breath test at least 4 wk after withdrawal of antisecretory drugs, antibiotics and related drugs. For the patients with subtotal gastrectomy, the diagnosis of H. pylori infection was based on anti-H. pylori immunoglobulin G (IgG) antibody. Serum anti-H. pylori IgG antibody was measured by enzyme-linked immunosorbent assays (Biohit, Finland). RESULTS: The sensitivity, specificity, positive predictive value and negative predictive value of neutrophil infiltration in the diagnosis of H. pylori infection were 92.3%, 83.5%, 77.4% and 94.7%, respectively. Neutrophil infiltration of gastric mucosa in the histological analysis was strongly associated with H. pylori infection (77.4% vs 5.3% in the neutrophil infiltration negative group, P = 0.000). Moderate neutrophil infiltration was more frequent in H. pylori infection when compared to mild infiltration (81.8% and 75%, respectively), but did not reach statistical significance. For those patients with negative rapid urease test, H. pylori was detected in 73.2% of patients with positive neutrophil infiltration on histology. In patients with subtotal gastrectomy, the diagnostic accuracy of neutrophil infiltration in H. pylori infection was 50%. CONCLUSION: Neutrophil infiltration is closely associated with H. pylori and may be recognized as a sign of this infection.

[The potential pathogenesis of gastrointestinal vascular malformation and the potential mechanism of thalidomide in the treatment of gastrointestinal vascular malformation].

OBJECTIVE: To study the pathogenesis of gastrointestinal vascular malformation (GIVM) and the potential mechanism of thalidomide in the treatment of gastrointestinal bleeding due to GIVM. METHODS: We collected the surgical intestinal specimens from 10 patients who suffered from massive hemorrhage of gastrointestinal tract owning to GIVM and the normal intestinal mucosa around the lesions, as well as normal intestinal mucosa from healthy subjects. Immunohistochemical (IHC) staining was carried out to investigate the differences of angiopoietin 2 (Ang2), Notch1 and delta like ligand 4 (Dll4) in the above three intestinal mucosa to find the relationship with the pathogenesis of GIVM. Human umbilical vein endothelial cells (HUVECs) were cultured with 0, 25, 50, 100 and 200 mg/L thalidomide for 24 or 48 hours to observe their mRNA and protein expressions of Ang2, Notch1, Dll4 by real-time PCR and Western blot. RESULTS: By IHC staining, more expressions of Ang2, Notch1 and Dll4 in the lesions were detected than those in the normal intestinal mucosa around the lesions and the normal intestinal mucosa in healthy people. The expressions of Ang2, Notch1 and Dll4 were significantly correlated (P = 0.016, r = 0.732), and the expressions of Notch1 and Dll4 were absolutely correlated (P = 0.000, r = 1.000). Real-time PCR and Western blot showed that thalidomide could down-regulate the expressions of them, which were in a concentration-dependent manner. CONCLUSION: Ang2, Notch1 and Dll4 may correlate with the pathogenesis of GIVM, while thalidomide can concentration-dependently down-regulate the expression of Ang2, Notch1 and Dll4, which may be one of the mechanism that thalidomide play a therapeutic role in GIVM.

Increased JNK1 signaling pathway is responsible for ABCG2-mediated multidrug resistance in human colon cancer.

Multidrug resistance remains a major obstacle to effective chemotherapy of colon cancer. ABCG2, as a half-transporter of the G subfamily of ATP-binding cassette transporter genes (ABC transporters), is known to play a crucial role in multidrug resistance. However, the molecular mechanism of controlling ABCG2 expression in drug resistance of colon cancer is unclear and scarcely reported. In the present study, we systematically investigate the potential role of the c-Jun NH2-terminal kinase (JNK) signal pathway in ABCG2-induced multidrug resistance in colon cancer. In the hydroxycamptothecin (HCPT) resistant cell line SW1116/HCPT from human colon cancer cell line SW1116, ABCG2 is the major factor for multidrug resistance, other than well-studied ABCB1 or ABCC1. Our findings indicate that blocking the JNK pathway by pathway inhibitor SP600125 reduces the expression level and transport function of ABCG2 in drug-resistant cells SW116/HCPT. Notably, the experiments of small interfering RNA directed against JNK1 and JNK2 show that only silence of JNK1 gene has the equal effect as SP600125 on dephosphorylation of transcription factor c-Jun and the expression of ABCG2 protein, while the corresponding phenomena were not observed after silence of JNK2 gene. Meanwhile, SP600125 induces the apoptosis of SW116/HCPT cells by promoting the cleavage of PARP and suppressing the anti-apoptotic protein survivin and bcl-2, and increases the sensitivity of SW1116/HCPT to HCPT. Taken together, our work demonstrated that JNK1/c-jun signaling pathway was involved in ABCG2-mediated multidrug resistance in colon cancer cells. Definitely, inhibition of the JNK1/c-jun pathway is useful for reversing ABCG2-mediated drug resistance in HCPT-resistant colon cancer cells.

Effect of the parvovirus H-1 non-structural protein NS1 on the tumorigenicity of human gastric cancer cells.

OBJECTIVE: To investigate the in vivo oncosuppressive effect of the non-structural protein NS1 of parvovirus H-1 on human gastric cancer cell lines. METHODS: Recombinant plasmid pcDNA3.1-NS1 containing the complete NS1 gene of parvovirus H-1 was constructed and characterized by restriction enzyme digestion and sequence analysis. The human gastric cancer cell lines MKN28, SGC7901 and MKN45 were stably transfected with empty or recombinant plasmids. NS1 gene transcription and protein expression in the latter transfectants were verified by reverse transcriptase polymerase chain reaction and Western blot, respectively. The oncosuppressive effect of the parvoviral protein NS1 on the gastric cancer cell lines was tested by comparing the tumorigenicity of empty and recombinant vector-transfected cells in nude mice. RESULTS: Well differentiated gastric cancer cells (MKN28) transfected with either empty plasmid or pcDNA3.1-NS1 were tumorigenic in nude mice. Moderately (SGC7901) and poorly (MKN45) differentiated gastric cancer cells transfected with empty plasmid were also tumorigenic, but no tumor resulted from the injection when they were transfected with pcDNA3.1-NS1. This NS1-associated suppression of SGC7901 and MKN45 tumors correlated with the decreased percentage of CD44 positive cells. CONCLUSIONS: NS1 expression in poorly differentiated gastric cancer cells prevents them from forming tumors, perhaps by impairing the stem-like phenotype. The parvoviral NS1 protein warrants further investigation for its therapeutic potential against cancer.

Chronic atrophic gastritis is a progressive disease: analysis of medical reports from Shanghai (1985-2009).

INTRODUCTION: We aimed to examine the turnover of chronic atrophic gastritis (CAG) pathologically and endoscopically and explore its potential causes. METHODS: A retrospective analysis was conducted of prospective data collected from 1,592 patients who underwent gastroscopy three times or more during the period 1985-2009 at Renji Hospital, Shanghai, China. Pathological and endoscopic findings were analysed. Data collected included gender, age, length of follow-up period, family history, past medical history, history of Helicobacter (H.) pylori infection, drug history for the use of proton pump inhibitors (PPIs), antacids and non-steroidal anti-inflammatory drugs [NSAIDs], and lifestyle history, including the patients' eating habits. RESULTS: 23 (1.44%) patients presented with gastric cancers resulting from CAG and 349 (21.92%) patients had dysplasia. Pathological and endoscopic findings suggested that the proportion of patients with worsening gastric mucosa during the atrophic and intestinal metaplasia (IM) phases was over 35% with increasing age. Gastric mucosa was found to be pathologically aggravated by carbonated drinks and fast food, and pathologically degenerated by H. pylori infection. Smoking deteriorated the gastric mucosa. Side dishes of vegetables may benefit the gastric mucosa even in the atrophic and IM phases. CONCLUSION: Our findings support the consensus that CAG is a progressive disease. Potential factors that were found to affect the state of the gastric mucosa in our patient group were gender, H. pylori infection, use of PPIs or NSAIDs, and intake of vegetable side dishes, spicy food, carbonated drinks and fast food.

MicroRNA 506 regulates expression of PPAR alpha in hydroxycamptothecin-resistant human colon cancer cells.

Chemotherapeutic drug resistance remains a major obstacle to the successful treatment of colon cancer. Here, we show that 77 differentially expressed miRNAs were identified in SW1116/HCPT versus SW1116, and over-expressed miR-506 in SW1116/HCPT cells was validated. Then it was indicated that PPARα is a common target of miR-506 by using a luciferase reporter assay. Our results also demonstrated that cytotoxic ability of HCPT requires the concomitant presence of PPARα, and that loss of PPARα expression imparts resistance to HCPTs anti-tumor effects. All together, our studies indicate that miR-506 over-expression in established HCPT-resistant colon cancer cell line confers resistance to HCPT by inhibiting PPARα expression, then providing a rationale for the development of miRNA-based strategies for reversing resistance in HCPT-resistant colon cancer cells.

The role of HIF-1, angiopoietin-2, Dll4 and Notch1 in bleeding gastrointestinal vascular malformations and thalidomide-associated actions: a pilot in vivo study.

OBJECTIVE: To investigate plasma levels of hypoxia inducible factor-1 (HIF-1), angiopoietin-2 (Ang-2), Delta-like ligand 4 (Dll4) and Notch1 in patients with recurrent gastrointestinal bleeding due to gastrointestinal vascular malformation (GIVM) with or without thalidomide treatment. METHODS: Ten eligible patients with recurrent gastrointestinal bleeding due to GIVM, who received thalidomide 100 mg/d for 4 months, were followed up for 1 year. The effective response was the proportions of patients with yearly bleeding episodes reduced by ≥50% at 1 year after treatment. Plasma levels of HIF-1, Ang-2, Dll4 and Notch1 were measured using enzyme-linked immunosorbent assay in the GIVM thalidomide treatment group before and after treatment (10 patients), the GIVM non-thalidomide treatment group (25 patients) and the control group (18 participants). RESULTS: In the GIVM thalidomide treatment group, eight patients (8/10) achieved effective response and five (5/10) displayed complete cessation of bleeding. Mean plasma levels of HIF-1, Ang-2, Dll4 and Notch1 were all higher in the GIVM thalidomide and non-thalidomide treatment groups than in the control group (all P < 0.001). However, Ang-2 decreased more significantly in the effective subgroups (P = 0.003) and no-bleeding patients (P = 0.008). CONCLUSIONS: HIF-1, Ang-2, Dll4 and Notch1 might participate in the formation of GIVM. Thalidomide is an effective and safe treatment agent for GIVM and its associated bleeding. The reduction degree of Ang-2 after a 4-month treatment of thalidomide may offer values for evaluating its prognosis and efficacy.

Meta-analysis: circulating adiponectin levels and risk of colorectal cancer and adenoma.

OBJECTIVE: To provide a systematic review with a meta-analysis for addressing the association between circulating adiponectin levels and the risk of colorectal cancer and adenoma. METHODS: Multiple electronic sources including MEDLINE, EMBASE and the Science Citation Index Expanded databases were searched to identify relevant studies for this systematic review. All existing observational studies that examined the relationship between circulating adiponectin and colorectal cancer or adenoma were included. Weighted mean difference and 95% confidence intervals (CI) were estimated and pooled using meta-analysis methods. RESULTS: Overall 13 case control or nested case control studies met the inclusion criteria. A total of 6175 participants and 3015 cases of colorectal cancer and adenoma were included in this meta-analysis. The weighted mean difference (95% CI) were -1.084 µg/mL (-1.836, -0.331), P = 0.005 in colorectal cancer and -1.43 µg/mL (-2.231, -0.628), P = 0.000 in adenoma. In men, a 2% decreased risk of colorectal neoplasm for a 1 µg/mL increment in adiponectin levels was observed (OR = 0.98, 95% CI 0.96-0.99) whereas among women there is no evidence of such a trend (OR = 0.99, 95% CI 0.97-1.01). CONCLUSIONS: Patients with colorectal cancer and adenoma demonstrated markedly lower adiponectin values than controls, yet there was significant heterogeneity among studies. A negative dose response relationship between levels of adiponectin and the risk of colorectal neoplasm was observed in men.

Efficacy of thalidomide for refractory gastrointestinal bleeding from vascular malformation.

BACKGROUND & AIMS: Patients with recurrent bleeding from gastrointestinal vascular malformations are a challenge to treat. We investigated the long-term efficacy and safety of thalidomide for refractory bleeding from gastrointestinal vascular malformations in an open-label, randomized study. METHODS: Eligible patients were randomly assigned to groups that were given either 100 mg thalidomide (n = 28) or 400 mg iron (n = 27, controls), daily for 4 months; patients were followed for at least 1 year (mean, 39 months). Bleeding was defined by a positive result from an immunoassay fecal occult blood test. The primary end point was the effective response rate, defined as the proportion of patients in whom bleeding episodes had decreased by ≥ 50% in the first year of the follow-up period. The secondary end points included the rates of cessation of bleeding, blood transfusion, overall hospitalization, and hospitalization for bleeding. We also quantified yearly bleeding episodes, bleeding duration, levels of hemoglobin, and yearly requirements for transfusions of red cells, numbers of hospitalizations for bleeding, and hospital stays. Plasma levels of vascular endothelial growth factor were measured in the group given thalidomide. RESULTS: Rates of response in the thalidomide and control groups were 71.4% and 3.7%, respectively (P < .001). All secondary end points differed significantly different between groups; thalidomide was more effective. No severe adverse effects were observed, although minor side effects were common among patients in the thalidomide group. Levels of vascular endothelial growth factor were significantly reduced by thalidomide (P < .001). CONCLUSIONS: Thalidomide is an effective and relatively safe treatment for patients with refractory bleeding from gastrointestinal vascular malformations. Mechanisms of thalidomide activity might involve vascular endothelial growth factor.

Efficacy and safety of adalimumab in Crohn's disease: meta-analysis of placebo-controlled trials.

OBJECTIVE: To evaluate the efficacy and safety of adalimumab in inducing and maintaining remission of Crohn's disease. METHODS: Electronic databases were searched. Placebo-controlled trials of adalimumab used in patients with Crohn's disease were included. Data were analyzed with Review Manager 4.2. RESULTS: Four studies enrolling 1402 patients were confirmed as meeting our criteria. Remission rates of inducing and maintaining remission in patients with Crohn's disease were higher for adalimumab than placebo (P<0.05). Adalimumab significantly improves the quality of life in patients with Crohn's disease. No significant difference in total adverse events was found in maintaining remission. Moreover, there were significantly less serious adverse events from taking adalimumab than from taking the placebo in long-term treatment. Patients with increased baseline C-reactive protein may benefit more from adalimumab therapy than those without. CONCLUSIONS: Adalimumab is effective and safe in Crohn's disease. However, studies of a larger number of patients are still required for better assessing the safety profile of adalimumab.

Chemopreventive effect of epigallocatechin-3-gallate (EGCG) and folic acid on the N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastrointestinal cancer in rat model.

OBJECTIVE: To investigate the chemopreventive effect and mechanisms of epigallocatechin-3-gallate (EGCG) and folic acid on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastrointestinal cancer in rats, and to investigate and compare the combinatorial effects of EGCG and folic acid on the chemoprevention of gastrointestinal carcinogenesis. METHODS: A total of 159 healthy male Wistar rats were randomly divided into seven groups to have the MNNG in drink (group M), MNNG in drink and EGCG in the feed (group ME), MNNG in drink and folic acid in the feed (group MF), MNNG in drink and EGCG+folic acid in the feed (group MEF), EGCG in the feed (group E), folic acid in the feed (group F) or normal feed (group C), respectively. At 44 weeks, all the rats were killed and assessed for the presence of gastrointestinal tumor. The occurrence of cancer was evaluated by histology. Ki-67 in cancerous tissues and in situ apoptosis were determined by immunohistochemical staining or terminal deoxyribonucleotide transferase-mediated nick-end labeling (TUNEL) assay, respectively. RESULTS: The experiment was completed in 157 rats (98.74%). As compared with group M, the tumor incidence of group MEF decreased significantly (P=0.011). Ki-67 expression in cancerous tissues of group ME and MEF also decreased significantly (P=0.038, P=0.009), while apoptosis of group ME, MF and MEF increased significantly (P=0.000, P=0.003, P=0.000). CONCLUSION: EGCG combined with folic acid has an obvious chemopreventive effect on gastrointestinal carcinogenesis induced by MNNG in rats.

Risk factors that predict the requirement of aggressive therapy among Chinese patients with Crohn's disease.

OBJECTIVES: Crohn's disease is now increasingly considered as a disabling disease. Conventionally, the disease is managed by step-up therapy. In recent years, the top-down strategy has been proposed and is thought to benefit the patients in whom the condition is likely to rapidly deteriorate toward disabling. However, this strategy has severe adverse effects which have to be weighed against its benefits. The aim of this study is to identify the risk factors that can predict the requirement of top-down therapy among Chinese patients. METHODS: We included 207 Chinese patients who had histories of Crohn's disease for ≥ 5 years, or those who had Crohn's disease for <5 years and at least one criterion of disabling disease. The risk factors related to the 5-year disabling course and the 2-year disabling course of Crohn's disease were separately analyzed in the same cohort by logistic regression. RESULTS: Among the 207 patients, the rate of disabling disease was 80.19% for 5-year, and 71.01% for 2-year. The risk factors of age <40 years at diagnosis, steroids requirement for treating acute exacerbation, and presence of perianal disease at diagnosis were significantly associated with a 5-year disabling course. In the same cohort, the risk factors related to 2-year disabling course were likewise steroids requirement for treating acute exacerbation and presence of perianal disease at diagnosis. CONCLUSION: The risk factors associated with disabling Crohn's disease, which entails the requirement of top-down therapy in Chinese patients, are requirement of steroids for treating acute exacerbation and the presence of perianal disease at diagnosis.

RNA interference (RNAi) of Ufd1 protein can sensitize a hydroxycamptothecin-resistant colon cancer cell line SW1116/HCPT to hydroxycamptothecin.

OBJECTIVE: To investigate whether RNA interference (RNAi) of the ubiquitin fusion-degradation 1-like protein (Ufd1) could sensitize hydroxycamptothecin (HCPT)-resistant colon cancer cell line SW1116/HCPT to the cytotoxic effect of HCPT. METHODS: SW1116/HCPT cells were transfected with plasmids containing Ufd1-specific small interfering RNA (siRNA) (Ufd1 knockdown cells) and non-specific siRNA (control cells). A drug sensitivity analysis, 3-(4,5)-dimethylthiahiazol (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) assay was performed on Ufd1 knockdown cells and control cells. After treating the cells with HCPT, a caspase-3 and caspase-4 activity assay, flow cytometric analysis and Western blot for detecting phosphorylated c-Jun N-terminal kinase (p-JNK), phosphorylated protein kinases B (p-Akt), P53, ubiquitin, GADD 153 and Grp78/Bip were performed. RESULTS: According to the MTT assay, the survival rate of knockdown cells was significantly lower than that of the control cells (P < 0.01). Both caspase-3 and caspase-4 activity assay showed higher activation level in Ufd1 knockdown cells than that in the control cells (P < 0.01). A flow cytometric analysis revealed more severe S-phase arrest in the Ufd1 knockdown cells than that in the control cells (P < 0.05). The Western blot showed that increasing the concentration of HCPT resulted in a higher expression level of p-JNK, P53, ubiquitin, GADD 153 and Grp78/Bip in the Ufd1 knockdown cells than that in the control cells. CONCLUSION: Ufd1 plays a key role in HCPT resistance of SW1116/HCPT and RNAi of Ufd1 can sensitize SW1116/HCPT to the cytotoxic effect of HCPT via strengthening the activation of caspase-3 pathway and disturbing endoplasmic reticulum functions.

Efficient differentiation of newly derived human embryonic stem cells from discarded blastocysts into hepatocyte-like cells.

OBJECTIVE: To establish an optimal method to isolate and culture human embryonic stem (hES) cells from discarded blastocysts and to differentiate these cells into hepatocyte-like cells. METHODS: Discarded human blastocysts of days 5-6 were cultured on mouse embryonic fibroblast cell feeder layers. Cells from the inner cell mass were isolated and subsequently cultured in vitro. To induce the formation of embryoid bodies (EB), dissociated ES cells were cultured in hanging drops for 5 days. The resulting EB were plated onto 100 mm plastic gelatin-coated dishes and allowed to attach for the outgrowth culture in the presence of hepatocyte growth factor for an additional 15 days. RESULTS: The hES cells had typical morphological characteristics: round or elliptic nest-like colonies with distinct cell borders and a smooth surface, significant swelling growth, compact cell aggregation, and relatively big nucleus compared to a small cytoplasm. The hES cells were identified by positive alkaline phosphatase staining. All had normal karyotypes and expressed octamer-binding transcription factor and cell surface markers, including stage-specific embryonic antigens SSEA-3, SSEA-4, and tumor rejection antigens TRA-1-60 and TRA-1-81. The cells could be differentiated to form teratomas in vivo. The hES cells could be induced to differentiate into hepatocyte-like cells and 70-80% of the cells expressed liver-associated proteins. CONCLUSION: Three hES cell lines have been successfully isolated and cultured from discarded human blastocysts. The hES cells can be efficiently differentiated into hepatocyte-like cells using the EB system. These cells possess the potential for treatment of liver diseases.

Comparison of the efficacy of triple versus quadruple therapy on the eradication of Helicobacter pylori and antibiotic resistance.

OBJECTIVE: Our aim was to compare the efficacy and safety of Helicobacter pylori (H. pylori) eradication with pantoprazole-based 7-day standard triple therapy (PAC) and 10-day quadruple therapy (PBMT), and to study the primary resistance of H. pylori to amoxicillin, clarithromycin, metronidazole and tetracycline. METHODS: A total of 170 patients with non-ulcer dyspepsia (NUD) and H. pylori infection were allocated to two study groups. The PAC group received pantoprazole 40 mg b.i.d., amoxicillin 1.0 g b.i.d. and clarithromycin 500 mg b.i.d. for 7 days; the PBMT group received pantoprazole 40 mg b.i.d., colloidal bismuth subcitrate 220 mg b.i.d, metronidazole 400 mg t.i.d. and tetracycline 750 mg b.i.d. for 10 days. A total of 80 H. pylori strains were isolated and antibiotic resistance was measured by the agar dilution method. RESULTS: A total of 166 patients completed the therapy. The intention-to-treat eradication rates in the PAC and PBMT groups were 63.5% and 89.4%, respectively (P < 0.05). By per protocol analysis, the eradication rates of the two groups were 65.1% and 91.6%, respectively (P < 0.05). Overall 77 clinical isolated H. pylori strains were cultured successfully. The H. pylori primary resistance rates to metronidazole and clarithromycin were 41.6% and 20.8%, respectively, whereas all the H. pylori isolates were sensitive to amoxicillin and tetracycline. CONCLUSION: The efficacy of PAC declines in many regions in China because of high antibiotic resistance rates. The PBMT regimen achieved a high eradication rate of H. pylori and might be used as a first-line therapy.