Inhibitory effect of 2­mercaptoethane sulfonate on the formation of Escherichia coli biofilms in vitro.

The biofilms (BF) formed by Escherichia coli (E. coli) is an important cause of chronic and recurrent infections due to its capacity to persist on medical surfaces and indwelling devices, demonstrating the importance of inhibiting the formation of E. coli BF and reducing BF infection. Although 2­mercaptoethane sulfonate (MESNA) exhibits a marked mucolytic effect clinically, the effect of MESNA on the inhibition of E. coli BF formation remains to be elucidated. The present study investigated whether MESNA inhibits the formation of E. coli BF in vitro. The minimum inhibitory concentration of MESNA on E. coli was determined to be 10 mg/ml. Subsequently, the effect of MESNA on BF early adhesion, extracellular polysaccharide (EPS) and extracellular protein were detected. The effect of a subinhibitory concentration of MESNA on BF formation was evaluated, and the inhibitory potency of MESNA against matured BF was assayed. The results revealed that MESNA inhibited early stage adhesion and formation of the E. coli BF, destroyed the mature BF membrane and reduced the EPS and extracellular proteins levels of the BF. In addition, the present study investigated the effects of MESNA on the expression of EPS­ and adhesion protein­associated genes using quantitative polymerase chain reaction analysis, which demonstrated that MESNA effectively inhibited the expression of these genes. These results suggested that MESNA possesses anti­BF formation capability on E. coli in vitro and may be used as a potential reagent for the clinical treatment of E. coli BF­associated infections.

Spread of carbapenemase-producing enterobacteria in a southwest hospital in China.

BACKGROUND: The rapid emergence and dissemination of carbapenem resistance in Enterobacteriaceae complicates the treatment of infections caused by these organisms. METHODS: We collected clinical isolates with meropenem inhibition zones of ≤ 22 mm from January 1, 2009, through December 31, 2010. We attempted to amplify the NDM-1 gene from these isolates and conducted the modified Hodge test (MHT). The minimal inhibitory concentration (MIC) of the MHT-positive strains was determined by the agar disk dilution method. The carbapenemase-encoding resistance genes of these strains were examined using polymerase chain reaction (PCR) analysis and a sequencing strategy to characterize these enzymes. The clonal relationship among isolates was analyzed by pulsed-field gel electrophoresis (PFGE). RESULTS: Among the 158 Enterobacteriaceae isolates that were collected, there were no NDM-1-positive strains and 26 MHT-positive strains. Among the latter, 18 strains were IMP-4-positive, and 1 was KPC-2-positive. In addition, 15 of the IMP-4-positive Klebsiella pneumoniae strains belonged to 4 PFGE genotypes, with 8 strains having the same genotype. CONCLUSION: These results suggest that nosocomial infections are one of the main reasons for the spread of these resistant strains.

Post-transcriptional regulation by miR-137 underlies the low abundance of CAR and low rate of bilirubin clearance in neonatal mice.

AIM: Jaundice, potentially fatal encephalopathy, is common in approximately two-thirds of all well term infants. It is largely due to low expression of constitutive androstane receptor (CAR) in newborns; however, the mechanisms for this low expression were poorly understood. MATERIALS AND METHODS: Expression of miR-137 and CAR was compared between neonatal and adult mice and between healthy and a mouse model of obstructive jaundice (OJ) using real-time RT-PCR and Western blot methods. Rate of bilirubin clearance was measured. DNA methylation of miR-137 was analyzed. KEY FINDINGS: Inverse expressions of miR-137 and CAR were consistently observed between newborn and adult mice, with a significantly higher miR-137 level and lower CAR protein and mRNA levels in neonatal liver than in adult liver. Similar reciprocal relationship was found existing between adult OJ mice and healthy control animals with a higher miR-137 level and lower CAR protein and mRNA levels in OJ than in healthy mice. Forced expression of miR-137 in primary hepatocytes repressed CAR protein levels, which was prevented by the inhibitor of miR-137. Knockdown of endogenous miR-137 by its inhibitor increased the rate of bilirubin clearance in OJ mice. Finally, we found that miR-137 was epigenetically over-activated due to hypomethylation in neonatal mice and in adult OJ mice, relative to adult healthy animals. SIGNIFICANCE: Our findings indicate that miR-137 is a repressor of CAR and thus a critical determinant of bilirubin clearance and may be considered a molecular target for the treatment of neonatal hyperbilirubinemia.

Molecular insights into the promiscuous interaction of human pregnane X receptor (hPXR) with diverse environmental chemicals and drug compounds.

The nuclear receptor member human pregnane X receptor (hPXR) regulates enzymes and transporters involved in xenobiotic detoxification as well as maintains homeostatic balance of bile acids, thyroid and steroid hormones. hPXR can be recognized and activated by a structurally diverse array of environmental chemicals and drug compounds to initiate adverse biological effects, such as perturbing normal physiological functions and causing dangerous drug-drug interactions and exhibiting a high promiscuity in its ligand spectrum. Understanding of the molecular mechanism and biological implication underlying the promiscuous interaction of hPXR with its diverse ligands is fundamentally important for toxicological and pharmaceutical researches. In the current study, molecular docking and hybrid quantum mechanics/molecular mechanics (QM/MM) were employed to investigate the binding mode, structural basis and energetic property of hPXR interactions with various activators and non-activators. It was found that, as compared to non-activators, the activators adopt few dominant modes to tightly interact with hPXR, which are specified by few polar spots located on the hydrophobic surface of hPXR active pocket. Based on the findings, a novel method called multiple binding mode-based quantitative structure-activity relationship (MBMB-QSAR) that characterizes the nonbonded interaction profile of hPXR with its ligand in multiple binding modes was proposed to model and predict the activating potency of small-molecule compounds on hPXR. Several partial least square (PLS) predictors derived from the MBMB-QSAR modeling were demonstrated to be effective for quantitative characterization of the biological behavior of experimentally confirmed activators, and for qualitatively differentiating the activators from a large number of non-activators. From the predictor models it is suggested that the hydrophobic force and electrostatic interaction play an important role in hPXR-ligand binding, while steric factor contributes moderately to the binding.

Association of Hepatitis A exposure and TIM-1 with childhood allergic asthma.

BACKGROUND: Hepatitis A virus (HAV) receptor (TIM-1) polymorphism plays an important role in asthma and autoimmune diseases. Objective. To analyze the association of TIM-1 polymorphism and HAV infection with childhood allergic asthma in Southwest China. METHODS: TIM-1 exon 4 (157insMTTTVP) and two polymorphism loci, -416G>C and -1454G>A, in the HAV receptor promoter region were studied. Polymerase chain reaction (PCR) was used to test the genotypes of three polymorphism loci among 579 cases of asthma and 524 controls. The HAV infection status was determined in a case-control study with stratified analysis. RESULTS: HAV exposure associated with childhood allergic asthma in the study population was compared with controls (odds ratio (OR) = 0.181, 95% confidence interval (CI) 0.126-0.260, p < .001). The -416G>C polymorphism was associated with asthma (OR = 1.384, 95% CI 1.148-1.669, p < .001), but the insertion variant 157delMTTTVP of exon 4 and the -1454G>A polymorphism were not. CONCLUSION: Our results indicated that the -416G>C polymorphism of the TIM-1 gene is associated with childhood allergic asthma, providing a better understanding of the pathogenesis of the allergic asthma among children aged below 15 years in Southwest China.

[Relationship between TIM-4 polymorphism and childhood asthma].

OBJECTIVE: To elucidate whether the polymorphism of asthma immune regulator gene TIM-4 is associated with the risk of childhood allergic asthma in the southwest region of China. METHODS: TIM-4 gene promoter region RS6882076 and intron RS4704727 were studied. PCR-RFLP was used to test the genotypes of two polymorphism loci among 579 cases (average 7.2 years old) of asthma and 524 controls (average 7.6 years old) in a case-control study. RESULTS: There were significant differences in the frequency of gene types at RS4704727 site between the asthma and the control groups (P<0.01). The results of PCR-RFLP showed that the polyporphisms of RS6882076 and RS4704727 in TIM-4 gene were present in this study population. The frequency of T allele at the RS4704727 site in the asthma group was significantly lower than that in the control group (OR=1.603; 95%CI 1.304-1.971; P<0.01). There were no significant differences in the frequencies of gene types and allele at RS6882076 site between the two groups (P>0.05). CONCLUSIONS: RS4704727 polymorphism of TIM-4 gene may be associated with childhood asthma, providing a better understanding of the pathogenesis of childhood asthma in the Southwest region of China.

[Human fetal liver nonparenchymal mesenchymal stem cells differentiate into functional hepatocyte-like cells in vitro].

OBJECTIVE: To induce nonparenchymal mesenchymal stem cells (NPMSCs) differentiating into functional hepatocyte-like cells in vitro, and to identify the molecular biology and functional characteristics of those hepatocyte-like cells. METHODS: Human NPMSCs were isolated and cultured with cell culture technique. NPMSCs were induced (on 1% Matrigel as a matrix and then submitted to 2.5 mmol/L AZA pretreatment for 10-12 h), by adding HGF 10 microg/L + FGF4 10microg/L + HGM into the culture medium. The characteristics of proliferation and growth of human NPMSCs were studied with methyl thiazolyl tetrazolium (MTT). The phenotypes of NPMSCs were identified by flow cytometry, immunohistochemistry and RT-PCR. Albumin (Alb) levels in culture supernatants were determined with ELISA. Staining for glycogen of undifferentiated NPMSCs and NPMSCs derivated hepatocyte-like cells was conducted with periodic acid-Schiff (PAS) test. RESULTS: Growth and division of adherent cells obtained from fetal livers were good and the amount of NPMSCs resourced from each fetus could be amplified to 109 cells after 10 serial subcultivations. The phenotype of NPMSCs was CD166 positive and CD34 negative. The shape of NPMSCs plated on Matrigel with FGF4 and HGF changed from long fusiform to polygonal or round on days 21-28. The rate of cell rounding was 40% and the ratio of dikaryocytes was 5%. Immunohistochemical and RT-PCR detection showed that undifferentiated NPMSCs expressed few alpha fetoprotein (AFP) and AFP mRNA, and did not express any of the liver-specific transcription factors or cytoplasmic markers. Many cells in early induction expressed GATA4, AFP and CK18 proteins and their mRNAs, and their expressions were reduced at the late induction, but the expressions of Alb, CK18, GST-and hepatocyte transcription factor HNF1increased gradually. The ratio of Alb and CK18 positive cells was 60%. Undifferentiated NPMSCs did not produce Alb. Alb production by induced NPMSCs increased in a time-dependent manner. Glycogen storage was first seen on day 14, and maximum levels were seen after day 28. CONCLUSIONS: There are MSCs among nonparenchymal cells of fetal livers. A high ratio of hepatocyte-like cells was obtained under our induction condition. NPMSCs differentiate firstly into hepatocyte precursors, and then differentiate into mature hepatocytes and hepatocyte-like cells with positive hepatocyte markers. The induced NPMSCs have hepatocyte specific functional features.

[Isolation, cultivation and biological identification of human fetal marrow mesenchymal stem cells].

OBJECTIVE: Noting the morphological and cytobiology characteristics and phenotypes of MMSCs, to establish an isolation and culture method for fetal MMSCs in order to provide a source of marrow mesenchymal stem cells (MMSCs). METHODS: Fetal MMSCs were isolated and cultured with in vitro cell culture technique; the characteristics of the proliferating and growing fetal MMSCs were studied with MTT and image analysis; the phenotypes of MMSCs were identified by flow cytometry and immunohistochemistry. RESULTS: Bone marrow of 12 fetuses was isolated within 0.5-2 h, and about 300+/-80 adherent cells were obtained at 24 h. Colonies with more than 5 cells were 15+/-6, growth detention period of culture cell was at 1-3 d after planting, log phase growth period was at day 4, and the amount of disintegration phase cells was reduced significantly. Original culture and serial subcultivations showed that cells divided prosperiously; unequal divisions special for stem cells were observed, and the amount of MMSCs harvested from each fetus was as much as 10(11)-10(12) cells after 10 serial subcultivations. The phenotype of MMSCs was CD166 positive and CD34 negative. Serial subcultivated MMSCs expressed a microamount of AFP and did not express albumine or CK18. CONCLUSION: Fetal MMSCs are easily isolated and proliferate prosperouly. Serial subcultivated MMSCs did not differentiate into hepatocyte-like cells under common culture condition and are feasibile as seed cells for tissue engineering reconstruction.

Effects of hemoperfusion adsorption and/or plasma exchange in treatment of severe viral hepatitis: a comparative study.

AIM: Non-bioartificial liver has been applied to clinic for quite a long time, but the reported efficacy has been very different. The aim of this study was to compare the efficacy and safety of hemoperfusion adsorption, plasma exchange and plasma exchange plus hemoperfusion adsorption in treatment of severe viral hepatitis. METHODS: Seventy-five patients with severe viral hepatitis were treated with hemoperfusion adsorption therapy (24 cases), plasma exchange therapy (17 cases) and plasma exchange plus hemoperfusion adsorption therapy (34 cases). The data of liver function, renal function, blood routine test, prothrombin time (PT) and prothrombin activity (PTa) pre- and post-therapy were analyzed. RESULTS: Clinical symptoms of patients improved after treatment. The levels of aminotransferase, total bilirubin, direct bilirubin decreased significantly after 3 therapies (P<0.05 or P<0.01). PT, the level of total serum protein decreased significantly and PTa increased significantly after plasma exchange therapy and plasma exchange plus hemoperfusion adsorption therapy (P<0.05 or P<0.01). The side effects were few and mild in all patients. CONCLUSION: Three therapies were effective in the treatment of severe viral hepatitis. Plasma exchange therapy and plasma exchange plus hemoperfusion adsorption therapy are better than hemoperfusion adsorption therapy.

[Effects evaluation of middle mode artificial liver in treatment of severe hepatitis].

OBJECTIVE: To evaluate the clinical efficacy and safety of middle mode artificial liver-plasma pheresis in treatment of severe hepatitis. METHODS: Seventeen patients with severe hepatitis were treated with plasma pheresis. The results of liver function, renal function, blood routine, prothrombin time (PT), prothrombin time activity (PTa) before and after the treatment were analyzed. All patients were observed closely. RESULTS: Symptoms of patients treated with plasma pheresis were improved, and the total effective rate reached 58.8 percent, but the survival rate was only 11.8 percent. Compared with those before the therapy, there were significant differences in aminotransferase, total bilirubin, direct bilirubin, PT, PTa and total protein level after treatment (P<0.05 or P<0.01). The side-effects were mild. CONCLUSION: Middle artificial liver is effective in the treatment of severe hepatitis.

[Preliminary evaluation on the effects of a hybrid bioartificial liver support system in the treatment of hepatic failure].

OBJECTIVES: To construct a novel hybrid artificial liver support system and evaluate its clinical efficacy in the treatment of hepatic failure. METHODS: A hybrid bioartificial liver support system consisting of plasma exchange device, charcoal perfusion column, and bioreactor cultured human or porcine hepatocytes was developed. 30 patients with hepatic failure were treated using this hybrid system. RESULTS: Both the excellent rate and effectual rate of the artificial liver support system were 43.3% (13/30). The total effectual rate was 86.7%. Finally, eleven out of 30 patients recovered completely. Six patients were bridged to liver transplantation. Six patients (20%) died of hepatic failure and seven patients (23.3%) discharged due to worsening of disease. CONCLUSIONS: The hybrid artificial liver support system has prominent liver support effects for hepatic failure, which can be regarded as an efficient measure for the treatment of severe hepatitis.