OBJECTIVE To investigate the change in antibiotic resistance of Burkholderia cepacia in hospital for reference of clinical antimicrobial strategy.METHODS Data were collected of the 859 bacteria strains isolated from clinical specimens from Jan 2004 to Dec 2006.Drug sensitivity tests were made for 22 antimicrobial agents.RESULTS All of the drug-resistance rates were higher than 90.00% including ampicillin,ampicillin/sulbactam,amoxicillin/clavulanic acid,cefazolin,gentamicin,tobramycin,amikacin,imipenem and nitrofurantoin.but that of the others such as ceftazidime(23.57%),cefepime(26.95%),ciprofloxacin(31.73%),cefoperazone/sulbactam(33.33%),and piperacillin/tazobactam(21.45%) were lower.The resistance rate to sulfamethoxazole/trimethoprim was 5.73% which was the lowest.CONCLUSIONS The isolation rate as well as the resistance to most of the antibiotics are increasing in clinical specimens.Antimicrobial treatment should be guided by drug sensitivity test.

Objective To investigate the impact of suppressor of cytokine signaling 1 (SOCS1) overexpression on dendritic cells (DC) functions and its therapeutic effect on acute liver failure (ALF) in mice.Methods Bone marrow derived dendritic cells (BMDC) from C57BL/6 mice were transfected with lentivirus encoding SOCS1 and negative control lentivirus at a MOI=50, and labeled as DC-SOCS1and DC-VNG, respectively after 96 hours of successful transduction.Then DCs were stimulated with lipopolysaccharides(LPS)1 mg/L and collected for flow cytometry analysis of surface costimulatory molecules, allogeneic mixed lymphocyte reaction (MLR) and western blot test of Janus kinase (JAK)/signaling transducers and activators of transcription (STAT) pathway.Afterwards, 90 mice were randomly assigned into 4 groups including 12 in normal control group, 26 in ALF group, 26 in treatment groups with DC-SOCS1 and 26 with the treatment of DC-VNG.All were received tail vein injection with normal saline, modified DC-VNG and DC-SOCS1 suspended in normal saline, respectively.Twelve hours after injection, LPS (10 μg/kg)/D-GaIN (600 mg/kg) were injected intraperitoneally to induce ALF model.The mortality, serum levels of alanine aminotransferase (ALT) and aspartate transaminase (AST), liver pathology and proportion of splenic regulatory T cells of each group were observed.Means in different groups were compared with one-way ANOVA analysis.Categorical variables were analyzed with x2 test.Variables were examined with normality test and homogeneity of variance with LSD test.Results The results of mixed lymphocyte reaction (MLR) revealed that T cell proliferation ratio in DC-SOCS1 group with mixture ratio of 100∶1 were (25.87±0.38)%, which was lower than that of mixture ratio of 10∶1 in the mDC group ([84.29±3.25]%) with statistical significance (x2=49.821, P<0.01);interleukin (IL)-10 concentration was higher than that in mDC group with mixture ratio of 10∶1 with statistical significance (F=20.112, P<0.05);IL-6 concentration was also lower with statistical significance (F=47.718, P<0.05).Compared to imDC, expression of JAK2 (t=0.525,0.523 and 0.489, respectively, all P<0.01), signal transduction factors and activation of transcription factors-1 (STAT1) (t=0.442,0.400 and 0.402, respectively, all P<0.01) and SOCS1 (t=0.322,0.363 and 1.090, respectively, all P<0.01) of mDC, DC-VNG and DC-SOCS1 after LPS stimulation increased significantly.Furthermore, the expressions of phosphorylated STAT1 (p-STAT1) and phosphorylated JAK2 (p-JAK2) of DC-SOCS1 were much lower than those of the mDC, with statistically significant difference (t=-3.840 and 0.254, respectively, both P<0.01).Pathological analysis revealed that there existed moderate hepatic cells necrosis and less immune cell infiltration in DC-SOCS1 group accompanied with higher regulatory T lymphocytes proportion than those in ALF group and DC-VNG group.Survival rate of ALF with DC-SOCS1 treatment group was significantly higher than that of ALF group with statistical difference (x2=12.87, P<0.05).Conclusions DC-SOCS1could sustain an immature state and exhibit as regulatory DC through negative regulation of JAK2/STAT1 pathway with overexpression of SOCS1.Infusion of DC-SOCS1 could ameliorate ALF by inhibiting aggressive inflammation response with increased proportion of regulatory T cells in mice, which shows good therapeutic effect for ALF mice.

Objective To observe the expression and significance of hypoxia inducible factor (HIF)-1α,matrix metalloproteinase(MMP)-9 and transforming growth factor(TGF)-β1 in liver tissues of rats with hepatic fibrosis and the effects of TGF-β1 vaccine on the expressions of HIF-1α,MMP-9 and TGF-β1.Methods The hepatic fibrosis rat model was set up by dimethylnitrosamine (DMN).The rat model was injected with TGF-β1 vaccine.The expressions of HIF-1α,MMP-9 and TGF-β1 were detected by immunohistochemistry in rat fibrosis tissues after 6 weeks.Results The expressions of HIF-1α,MMP-9 and TGF-β1 in fibrosis model group and TGF-β1 vaccine group were all significantly higher than control group(P＜0.01),which was positively correlated with the fibrosis degree(r=0.911,0.814 and 0.836,respectively,P＜0.01).However,the expressions of HIF-1α,MMP-9 and TGF-β1 in TGF-β1 vaccine group were all significantly decreased than fibrosis model group (t=2.62,4.03,3.43;P＜0.01).Conclusion During the development of liver fibrosis,HIF-1α promotes the transcriptions and expressions of MMP-9 and TGF-β1.

Objective To study the effect of endotoxin tolerance (ETT) on chemokine receptor 7 (CXCR7) in the liver tissue of rats with acute liver failure (ALF).Methods SD male rats were randomly divided into three groups:normal group,ALF group and ETT group.The rats in the ETT group and ALF group were injected with lipopolysacharide (LPS) 0.1 mg/kg or saline respectively,one time / day for 5 days.At 24 hours after the 5th-day injection,all rats were injected with D-GalN 800 mg/kg and LPS 8μg/rat.Blood sample and liver tissue were collected on 2,6,12,24 and 48 hours after injection.The gene expressions of CXCR7 in the liver were measured by RT-PCR,and the protein expressions of CXCR7 were determined by Western Blot.The data analysis was performed by LSD,Dunnett's t test.Results The histological damage in the liver tissue was significantly mider in ETT group compared to ALF group.The gene expressions of CXCR7 were significantly milder in ETT group compared to ALF group (2 h:F =29.222,6 h:F=166.892,12 h:F=38.975,24h:F=34.603,48 h:F=18.929,allP＜0.01),but still severer than that of normal group.The CXCR7 protein expression in ALF group and ETT group peaked at 24 hours,but the expression of CXCR7 in ETT group was lower compared with that in in ALF group (2h:F=11.155,6 h:F=42.553,12h:F=17.082,all P＜0.01; 24 h:F=7.242,P＜0.05).Conclusions During the process of endotoxin tolerance,LPS pretreatment and D-GalN can decrease the liver injury,down-regulate the expressions of CXCR7mRNA and CXCR7.This suggests that CXCR7 may play an important role in the ETT.

Objective To investigate the mechanism of endotoxin tolerance (ETT) through observing the expression of OX40 in liver tissues of ETT rats.Methods SD male rats were randomly divided into three groups:normal group (n=6),acute liver failure (ALF) group (n=24) and ETT group (n=24).Lipopolysacharide (LPS) 0.1 mg/kg (ETT groups) or 0.9 ％NaC1 (ALF groups) was administered by five consecutive intraperitoneal injections at 24 h intervals,and at the sixth day,all animals were treated with intraperitoneal injections of D-galactosamine (D-GalN) 800 mg/kg and LPS 8 μg/rat.Blood and liver tissue were collected at 6,12,24 and 48 hours after the injection of D-GalN/LPS.The gene expression of OX40 in the liver was measured by reverse transcription-polymerase chain reaction (RT-PCR).The protein expression of OX40 was estimated by Western blot.The tumor necrosis factor (TNF)-α and interleukin (IL)-6 levels were determined by enzyme-linked immunosorbent assay (ELISA).The data analysis was performed by one-way ANOVA,lease significant difference (LSD) and Dunnett's T3 test.Results The expressions of TNF α and IL-6 were both significantly lower in ETT group compared to ALF group,but still higher than that of control group.The gene expressions of OX40 peaked at 12 hours and decreased gradually in ALF group.The gene expressions of OX40 were significantly lower in ETT group compared to ALF group (6 h:F=5.027,24 h:F=5.539,48 h:F=5.011,all P＜0.05; 12 h:F=36.688; P＜0.01),but still higher than that of normal group.The tendency of OX40 protein expression in ALF group was peaked at 12 hours and decreased at 24 hours.In ETT group,the expression of OX40 was lower,and the difference between ETT group and ALF group had statistical significance (6 h:F=8.658,P＜0.05; 12 h:F=34.611,24 h:F=28.176,48 h:F=16.747; all P＜0.01).Conclusions The level of OX40 is increased in ALF group,while the expressions of OX40,TNF-α and IL-6 are lower in ETT group,which suggested that OX40 may play an important role in the process of ETT.

Objective This study aimed to investigate the effect of splenic CD11clow CD45RBhigh dendritic cell (DC)derived from endotoxin tolerance (ET)mice on the expression of zinc finger protein A20 in acute liver failure (ALF)and to clarify the possible mechanism.Methods ET mice were modeled. CD11clow CD45RBhigh DC were isolated from spleen by magnetic activated cell sorting (MACS).One hundred and twenty-six healthy male BALB/c mice were randomly divided into four groups:control group (group A,n=6),ALF group (group B,n =40),normal CD11clow CD45RBhigh DC-treated group (group C,n=40),ET-CD11clow CD45RBhigh DC-treated group (group D,n=40).Mice in group B,C and D were injected with D-galactosamine (D-GalN)600 mg/kg and lipopolysaccharides (LPS)10 μg/mouse.Mice in group A were given the same volume of normal saline (NS).Half an hour after the D-GalN/LPS injection,mice in group C were treated with splenic CD11clow CD45RBhigh DC derived from normal mice (1 ×10 6/mouse,0.2 mL/mouse).Mice in group D were treated with splenic CD11clow CD45RBhigh DC derived from ET mice (1 × 10 6/mouse,0.2 mL/mouse).Mice in group A and B were given the same volume of 0.9% NaCl solution (0.2 mL/mouse).Alanine aminotransferase (ALT)and aspartate aminotransferase (AST)levels were measured at each time point.Liver histopathological changes were confirmed by hematoxglin and eosin methods.Expressions of tumor necrosis factor-α (TNF-α),nuclear factor-kappa B (NF-κB),and zinc finger protein A20 were measured by reverse transcriptase polymerase chain reaction(RT-PCR)and Western blot.One-way analysis of variance was used to compare means between groups.Normal distribution and homogeneity of variance were tested.LSD test was conducted in patients accorded with homogeneity of variance.Results ALT and AST levels increased 2 h after modeling in group B and peaked at 24 h,which were significantly higher than groups A (t = 31 .00, 11 .52,both P <0.05).ALT and AST levels also increased after 2 h after modeling and peaked at 24 h in group C and group D,which were both significantly higher than group B (t =14.60,26.43,both P <0.05).The mRNA levels and protein expressions of TNF-αand NF-κB in group B increased gradually and peaked at 12 h after D-GalN/LPS injection.Compared to that of group A,the differences were both statistically significant (t = 427.58,122.42,179.35 ,165 .98,all P < 0.05 ).The mRNA level and protein expression of zinc finger protein A20 in group B decreased gradually and reached the minimum at 12 h after D-GalN/LPS injection,which was statistically different compared to group A (t = 90.80, 160.43,both P <0.05).On the contrary,the levels of zinc finger protein A20 in group C and D increased gradually and peaked at 12 h after D-GalN/LPS injection.The expression level of zinc finger protein A20 in group D was significantly higher than group C (t = 11 .21 ,24.80,both P < 0.05 ).Conclusion Treatment of splenic CD11clow CD45RBhigh DC derived from ET mice contributes to liver protection against D-GalN/LPS-induced ALF.

Objective To isolate and culture splenic CD11clow CD45RBhigh dendritic cells (DC) derived from endotoxin tolerance (ET) mice and investigate its biological characterization.Methods Mice weighed 20 to 25 gram were completely randomized into two groups including ET group and control group with 6 each.ET mice were modeled by intraperitoneal injection of low-dose lipopolysaccharide (LPS) for several days (pretreated with LPS 0.1 μg/mouse for 5 d).Mice in control group were given the same volume of normal saline (NS).CD11clowCD45RBhighDC were isolated from spleen by magnetic activated cell sorting (MACS).The immunological phenotypes were detected by flow cytometry.The suppressive capacity of CD11clow CD45RBhigh DC was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay in allogenic mixed T cells reaction.The expressions of interleukin (IL)-10 and IL-12 produced by CD11clow CD45RBhigh DC were measured by enzyme-linked immunosorbent assay (ELISA).Statistical significance was analyzed through one-way analysis of variance (ANOVA).The homogeneity of variances was detected by Levene test.If variances were homogeneous,the least significant difference (LSD) test was used.If not,Dunnett T3 test was applied.Results The consistence of CD1 1 clow CD45RBhigh DC in control group was 30 ％,reaching the amount of (5.30±0.12) × 105/mouse ;In ET group,the percentage of CD11clow CD45RBhighDC achieved 80 ％ and the production was (1.20 ± 0.13) × 106/mouse the difference was statistically significant (t=3.23,P＜0.01).The cellar morphology in two groups showed no obvious difference.Compared to expression levels of all cell phenotypes (histocompatibility complex-Ⅱ,CD40 and CD80) in normal mice,the cell surface expression levels of CD11clowCD45RBhigh DC in ET mice were much lower.The difference in two groups was statistically significant.Splenic CD11clowCD45RBhighDC derived from ET mice with cell concentration of 1∶ 10,1∶50and 1∶100 had more obvious prohibitory effects on allogenic T cells (t1∶0 =1.36,P1∶10 ＜0.01,t1∶50 =2.49,P1∶50 ＜0.01,1∶100 =1.88,Pm00 ＜0.01).Secretion of IL-10 produced by CD11clowCD45RBhighDC of ET mice was significantly increased (t1∶0=13.63,P1∶10 ＜0.01,t1∶50 =13.45,P1∶50 ＜0.01,t1∶00 =9.31,P1∶00 ＜0.01),but the expression of IL-12 was lower (t1∶0 =2.62,P1∶0 =0.02,1∶∶50 =2.74,P1∶0=0.02,t1∶100 =2.99,P1∶100 =0.01).Conclusion Splenic CD11clow CD45RBhigh DC from ET mice have weaker ability of antigen presenting and allogeneic lymphocytes proliferation stimulating than those from normal mice.

Objective To investigate the effect of endotoxin tolerance(ETT) on the rats with acute liver failure (ALF) and Janus kinase/signal transducer and activator of transcription (JAK/ STAT) signal transduction pathway. Methods S-D male rats were divided randomly into three groups: control group, ALF model group anti ETT group, lipopolysacharide (LPS) 0.1 mg/kg(ETT groups) or saline(ALF groups)was administered by five consecutive intraperitoneal injections at 24 h intervals. On the sixth day all animals were treated with intraperitoneal injection of D-galactosamine (D-GaIN) 800 mg/kg and LPS 8 μg/rat. The blood was gathered from portal vein and livers were take out before and 2, 6,12, 24 and 48 h after the injection of D-GalN/LPS. Liver function and liver histopathology of each group were observed. The gene expressions of STAT3 and SOCS3 in the livers were measured by semi-quantitative reverse transcriptionpolymerase chain reaction(RT-PCR). The tumor necrosing factor(TNF)-α and interleukin(IL)-6 level were determined by enzyme-linked immunosorbent assay(ELISA). The data analysis was performed by using t test. Results The histological damage in the liver tissue was significantly milder in ETT group compared to ALF group, but still severer than that of control group (TNF-α: 6 h: t=2. 670,P<0.05,12 h: t=3. 604,24 h: t=6. 426, 48 h: t=3. 274,all P<0.01;IL-6:6 h: t=2. 333,P<0. 05,12 h: t=4. 266, 24 h: t=8. 063,48 h: t=4. 177, all P<0. 01). The gene expressions of STAT3 and SOCS3 in the liver were increased significantly in ALF group, however, in ETT group the expression of STAT3 was inhibited while the expression of SOCS3 was increased and much higher than those in ALF groups. Conclusions LPS pretreatment can induce ETT in rats, which will reduce the expression of TNF-α and IL-6. In ETT groups, the gene expression of STAT3 is lower while the gene expression of SOCS3 is higher compared to those in ALF groups. It suggests that JAK/STAT pathway may involve in mechanisms of ETT.

Objective To evaluate the therapeutic effects of recombinant expression plasmid containing hepatocyte growth factor (HGF) and augmenter of liver regeneration (ALR) on rats with hepatic fibrosis. Methods Ninety Sprague-Dawley rats, which had been established into hepatic fibrosis models, were equally divided into 6 groups: blank group, pcDNA3.1 therapy group,pcDNA3.1-HGF therapy group, pcDNA3. 1-ALR therapy group, pcDNA3.1-HGF and pcDNA3. 1-ALR combined therapy group, and pcDNA3. 1-HGF-ALR therapy group. Zero point one μmol of blank or plasmid was injected into model rats in each group by tail vein once a day for 3 days. Model rats in blank group didn't receive any treatment. Additional 10 rats were chosen as control group, which were not given any interference during the experiment. All rats were sacrificed 4 days after end of treatment. Liver tissues were reserved for observing pathologic changes after HE staining and detecting proliferating cell nuclear antigen (PCNA) and c-jun by immunohistochemistry. Measurement data were compared by single-factor analysis of variance. Comparison between groups was done by SNK test. Enumeration data were analyzed by Fisher's exact test. Results In blank group and pcDNA3.1 therapy group, hyperplasia of fibrous connective tissue was very obvious, false lobules were formed. There was no significant difference between these two groups (x2 =0. 317,P= 1. 000).In the 4 remaining groups, hepatic fibrosis all achieved different degree of amelioration, and the therapeutic effect of pcDNA3.1-HGF-ALR was optimal. In control group, the expressions of PCNA and c-jun in liver tissues were low, with absorbance value of 8.6±1.9 and 3.2 ± 1.2, respectively. In blank group and pcDNA3. 1 therapy group, the expressions of PCNA and c-jun were obviously increased, with absorbance value of 24. 1±3.0, 24.5±4.3 and 23.8±3.1, 24.9±4.2, respectively,which were significant different from control group (all P＜0.01). In the 4 remaining groups, the expressions of PCNA were all obviously increased, and expressions of c-jun were all obviously decreased. The maximum change scope was observed in pcDNA3. 1-HGF-ALR therapy group.Conclusions The recombinant expression plasmid pcDNA3. 1-HGF-ALR can effectively ameliorate experimental hepatic fibrosis of rats. The anti-fibrosis effects are achieved probably by up-regulating PCNA expression and down-regulating c-jun expression.

Objective To investigate the effect of rapamycin(RPM) on the gene expression of Toll-like receptor (TLR)-4 by inhibiting Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway in rats with acute liver failure (ALF).Methods The ALF model of rats was induced by D-galactosamine (D-GalN) 800 mg/kg and lipopolysaccharide (LPS) 8 μg.The blood and liver tissue samples were collected at 2,6,12,24 and 48 h after D-GalN/LPS injection.SD rats were randomly divided into three groups:normal control group (n=6),ALF group (n=30) and RPM treatment group (n=30).The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) at each time points were tested.The levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 were tested by enzyme-linked immunosorbent assay (ELISA),and the mRNA expressions of TLR-4 in liver tissue were determined by reverse transcription-polymerase chain reaction (RT-PCR).The data analysis was performed using t test.Results The levels of TNF-α and IL-6 were increased markedly 2 h after GalN/LPS iniection and peaked at 6 h.Treatment with RPM could significantly inhibit the levels of TNF-α and IL-6.The TLR-4 mRNA expression levels in liver tissues at 6,12,24,48 h in ALF group were 0.745±0.135,1.092±0.175,1.115±0.152 and 0.812±0.13,respectively;those in RPM group were 0.545±0.118,0.798±0.124,0.857±0.109 and 0.595±0.152,respectively.The differences between two groups at each time points were all significant (t=2.726,3.349,3.382 and 2.567,respectively,all P＜0.05).In addition,TLR-4 mRNA expression was positively correlated with ALT and AST levels(r=0.722 and 0.712,respectively,both P＜0.01).Conclusions Inhibition of JAK/STAT pathway can markedly down regulate TLR-4 gene expression in liver tissue of rats with ALF,which indicates that JAK/STAT pathway may be involved in the regulation of TLR-4 mRNA expression during ALF.