Objective To establish the rational antibiotic treatment by accurate screening of highlevel aminoglycoside (HLA) resistant enterococci in clinical laboratory. Methods A single HLA gentamicin 500 μg/ml and streptomycin 200 μg/ml for agar dilution method, and gentamicin 120 μg/disk and streptomycin 300 μg/disk for disk diffusion method were used as parallel screening tests to detect high-level aminoglycoside resistance in 172 enterococci strains. Antibiotic time-kill test was performed to verify the accuracy and reliability of both methods. Results The resistant rates of the two methods for HLA gentamicin were 60.0％ and 59.3％, respectively. While for HLA streptomycin, both were 61.1%. In disk diffusion tests of 172 enterococci strains, the resistant rates for penicillin, ampicillin and vancomycin were 16.9 %,14.0%, and 1.7%, respectively. β-lactamase from 80 strains enterococci were all negative. Conclusion High-concentration aminoglycoside disk diffusion method is a simple, reliable method for screening the HLA resistant enterococci in clinical laboratory. The results can provide a rational base for physicians to treat enterococcal infections.

Objective To express major outer membrane protein (MOMP) of Chlamydia trachomatis E and to prepare rabbit polyclonal antibody. Methods The recombinant plasmid MOMP/ pGEX6p-l prepared by our lab was introduced into E. coli. The protein was expressed and purified by gel recycling, then was injected into New Zealand rabbits to produce polyclonal antibodies. Enzyme linked immunosorbent assay (ELISA) was used to detect the titer of antibody. The antibody specificity was identified by Western blot and immunofluorescence. Results The fusion recombinant protein glutathione S-transferase (GST)-MOMP was successfully expressed in E. coli. The titer of antibody recombinant protein detected by Western blot and to the endogenous MOMP of Chlamydia trachomatis in vitro detected by immunofluorescence. Conclusions The recombinant MOMP is successfully expressed and the MOMP antibody with high titer and high specificity is obtained. which will be helpful for Chlamydia trachomatis detection and related clinical research.

Objective To study the therapeutic effects of human umbilical cord mesenchymal stem cells (hUCMSCs) on acute liver failure ( ALF) induced by D-galactosamine (D-gal) and lipopolysaccharide (LPS) in rats. Methods The ALF model was obtained through intraperitoneal injection of D-gal(300 mg/kg)and LPS (20μg/kg)in Wister rats. The hUCMSCs were transplanted after intoxication. All rats were divided into four groups, and each group received either hUCMSCs or 0.9% NaCl solution through intraperitoneal or tail-intravenous injection. To evaluate the liver function of each group, the levels of alanine aminotransferase (ALT), total bilirubin (TBil) and serum albumin (Alb) were measured on the day of hUCMSCs transplantation and the following 1, 2, 3, 5 and 7 days. All rats were then sacrificed to examine the liver histology at day 7. Analyses were done by using Fisher's exact test, unpaired t test and Mann-Whitney U test. Results There were no significant differences of survival rates among four groups (Fisher's exact test, both P = 1. 00). The levels of ALT, TBil and Alb in group receiving hUCMSCs intraperitoneally were (804. 9 ± 88. 0) U/L,(17. 4±2. 7) μmol/L and (20. 9±0. 8) g/L, respectively after 2 days of injection, whereas in the corresponding control group, those were (1294. 3± 171. 4) U/L, (32. 3±5. 5) μmol/L and (16. 1±0. 9) g/L, respectively, which indicated that hUCMSCs transplantation significantly improved the liver function (t = 2. 640, P =0.020;t=2.529, P = 0. 025;t= - 3. 833, P = 0. 002). Both of hUCMSCs-transplanted groups showed no significant differences. Liver histological data showed that transplantation of hUSMSCs through either intraperitoneal or tail-intravenous injection alleviated liver damage (U=4. 500, P = 0. 005;U=4. 500, P = 0. 008) and the mitotic index also increased in hUCMSCs-transplanted groups (U=4. 000, P = 0. 005; U=5. 500, P = 0. 013). Conclusions The levels of ALT, TBil and Alb can rapidly normalize in ALF rats after injected with hUCMSCs either intraperitoneally or tail-intravenously. hUCMSCs application raises the mitotic index, enhances hepatocellular regeneration and improves histological status.

Objective To describe the distribution of mannose binding lectin (MBL) genetic polymorphisms in non-acquired immunodeficiency syndrome (AIDS) patients with cryptococcosis in China and to verify the association of MBL polymorphisms with susceptibility to cryptococcosis.Methods The case-controlled genetic association study was conducted and 167 non-AIDS patients with cryptococcosis and 208 healthy controls were recruited. Genome DNA was extracted from the peripheral blood and MBL gene was amplified by polymerase chain reaction (PCR). Six singlenucleotide polymorphisms ( SNP) of MBL gene were sequenced. The association of MBL polymorphisms with susceptibility to cryptococcosis were analyzed. The comparison between patients and controls was performed by chi square test or Fisher's exact test. The differences of MBL plasma concentrations between groups with different MBL genotypes were compared by single factor variance analysis. Results There were no differences between patients and controls in terms of MBL genotype frequencies, haplotypes and genotypes (all P>0. 05). Compared with healthy control, the deficient MBL-producing genotypes were strongly associated with cryptococcal meningitis (16. 5% vs 8. 7%,χ2=4.25, P=0.0392, OR = 2.09), particularly in patients without underlying immunocompromised conditions (21. 4% vs 8. 7%, χ2 =7. 15, P = 0. 0075, OR = 2. 88). Individuals with MBL deficiency genotypes showed significantly higher rates of central nervous system (CNS) cryptococcal infection rather than non-CNS cryptococcosis (16. 5% vs 3. 1%, Fisher's exact test, P = 0. 010, OR = 6. 13).The difference was even more significant in the immunocompetent patients (21. 4% vs 4. 0%, P =0.009, OR= 6. 55). Conclusion MBL deficiency is associated with cryptococcal meningitis and may play a role in CNS Cryptococcus infection.

Objective To explore the influence factors on hepatitis B virus (HBV) relapse after nucleos(t)ide analogues (NA) withdrawal in the chronic hepatitis B (CHB) patients who met NA cessation criteria. Methods Eighty-one consecutive CHB patients were treated with NA, 38 with lamivudine (LAM), 25 with adefovir dipivoxil (ADV), 12 with entecavir (ETV), 6 with LAM +ADV. Among recruited patients, 40 were hepatitis B virus e antigen (HBeAg) positive, 41 were HBeAg negative, 67 of them were initial treatment, 14 were retreatment due to resistance to NA at baseline. The treatment was discontinued after meeting China therapeutic end-point criteria. HBV DNA, HBV serological markers, alanine aminotransferase (ALT) were measured respectively at baseline, every month before virological response, every 3 months after virological response, every month within first 6 months and every 2 months over 6 months after drugs withdrawal. Twelve probable influence factors on relapse which were sex, age, HBV family history, baseline HBV DNA,baseline HBeAg status, baseline ALT, virological response time, total duration of treatment, duration of additional treatment, the level of hepatitis B virus surface antigen (HBsAg) at cessation therapy,initial treatment or retreatment, drug category were analyzed with univariate, multivariate Cox regression modle and stratified analysis. The cumulative relapse was calculated by the Kaplan-Meier method. Results A total of 36 patients (44. 4%) relapsed within 1 year. Initial treatment or retreatment, HBV family history, virological response time, the level of HBsAg at cessation therapy were independent risk factors. The relapse rate of retreatment was higher than that of initial treatment (78.6% vs 37. 3% , χ2 = 7. 983, P = 0. 005) , those of patients with HBV family history higher than without family history (64. 5% vs 15.0%, χ2 =12. 096,P = 0.002), those of patients obtained virological response within 3 months lower than after 3 months(34. 0% vs 64. 3% , χ2 =6. 823,P=0. 009) , those of patients with HBsAg≤150 μg/L at cessation therapy lower than >150 μg/L(27. 6% vs 53. 8%, χ2=5. 199,P=0. 023). Conclusions Retreatment, HBV family history, later virological response and higher HBsAg level at cessation therapy are risk factors of relapse after NA withdrawal. Such patients should be treated with prolonged duration after meeting end-point criteria to strengthen the efficacy.

Objective To investigate expressions of programmed cell death ligand 1 (PD-L1) in hepatic tissues at the different stages of hepatitis B virus ( HBV) infection, and clarify its role in the mechanism of chronic hepatitis B virus infection. Methods The expressions of PD-L1 were detected by immunohistochemistry and computer image quantitative analysis in the hepatic tissues of 65 chronic HBV infected patients and 5 healthy controls. The correlations between PD-L1 expression and inflammatory grading in the hepatic tissues, total bilirubin (TBil), alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum HBV DNA level were analyzed. Results The PD-L1 expressions in hepatic tissues of HBV infection with G0 - G4 inflammatory grades were 3. 07 % ±0.93%, 8.01%±1.49%, 11.60%±2.60%, 18.41%±2.21% and 26. 04% ±3. 41%, respectively,which were all significantly stronger than that in controls (0. 64%±0. 28%). PD-L1 expression was a positively correlated with inflammation grading of hepatitis tissues, TBil, ALT and AST level in serum (r=0. 917, 0. 787, 0. 483, 0. 628; all P<0. 05), and negatively correlated with serum HBV DNA load (r=-0. 620, P<0. 05). Conclusion The upregulated PD-L1 expression may be probably involved in the chronicity of HBV infection.

Objective To investigate the relationship between single nucleotide polymorphism (SNP) of Fas ligand (FasL) and fulminant hepatitis B in Han Chinese. Methods HBV infected subjects were enrolled in this case-control study, including 233 cases of inactive HBsAg carrier, 68 patients with fulminant hepatitis B,100 cases of spontaneous hepatitis B clearance, 102 patients with hepatitis B virus (HBV) related cirrhosis and 112 patients with HBV related primary hepatocellular carcinoma. The blood samples and clinical data were collected. FasL-844T/C polymorphisms of enrolled subjects were examined by TaqMan real time fluorescent genotyping polymerase chain reaction (RT-PCR). A adjusted odds ratios (OR)and 95% confidence intervals (CI)were calculated using the Logistic regression model. Results After adjusting the factors of gender and age, binary Logistic regression analyses indicated that the genotype frequencies of FasL-844 CC,CT,TT in inactive HBsAg carriers were 50. 64% ,39. 91% and 9. 44% respectively, and those in cases of fulminant hepatitis B were 79. 41%, 17. 65% and 2. 94%, respectively. The analysis also revealed that FasL-844CC genotype in inactive HBsAg carriers was high risk factor of developing fulminant hepatitis B (OR =4. 729,95%CI:0. 510 - 21. 282,P = 0. 043), while there were no statistic significances in other cases (P>0. 05). Conclusion The inactive HBsAg carriers harboring FasL-844CC may have greater susceptibility to fulminant hepatitis B, which need arouse high attention.

Objective To investigate the role of natural killer cells (NK cells) in patients with chronic hepatitis B (CHB). Methods Blood samples and liver biopsies from 108 CHB patients including 43 mild, 28 moderate and 37 severe CHB patients were collected. The levels of interferon (IFN) gamma in liver tissues and peripheral blood were detected by intracellular cytokine staining.The data were analyzed by t test. Results The levels of IFN gamma produced by NK cells in liver tissues and peripheral blood of patients with severe CHB were (8. 12±4. 26) % and (4. 59±2. 95) % ,respectively, which were significantly higher than those in patients with mild CHB [(3. 53±1. 56)%and (2. 26±0.61)%, respectively; t=3. 80 and t = 4. 30, respectively; both P<0. 05] and those in patients with moderate CHB [(4. 59±1. 98)% and (2. 48±1. 05)%, respectively;t=2. 62 and t =3. 28, respectively; both P<0. 05]. While there were no differences of IFN gamma produced by NK cells both in liver tissues and peripheral blood between mild and moderate CHB patients (t=0. 99 and t=1. 27, respectively; both P>0. 05). The levels of IFN gamma produced by NK cells in liver tissues and blood samples in severe phase of severe CHB were (10. 02 ± 4. 15)% and (6. 78 + 2. 91)%, respectively, those in recovery phase were (6. 13±2. 01)% and (3. 13± 1. 52)%, respectively (t = 1. 89 and t=1. 74, respectively; both P<0. 05]. Conclusion NK cells may play a pivotal role in the pathogenesis of severe CHB.

Objective To investigate the association between single nucleotide polymorphism (SNP) of interleukin-12 (IL-12) p40 3'untranslated region rs3212227 site and hepatitis C virus (HCV) infection. Methods Patients with hepatitis C (n=127) were genotyped and analyzed for the SNP of IL-12 p40 rs3212227 using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing. The serum HCV RNA levels of patients with hepatitis C were detected using real time fluorescence quantitative-polymerase chain reaction (FQPCR). Inter-group comparisons of genotype and allele frequency were analyzed using chi-square test.Results In patients with hepatitis C, the frequencies of AA, AC and CC genotypes of IL-12 p40 rs3212227 site were 34. 6% ,40. 9% and 24. 4% , respectively,and the frequencies of allele A, C of IL12 p40 rs3212227 site were 55.1% and 44. 9%, respectively. The frequency of rs3212227 C allele in patients with HCV RNA ≥2. 0× 106 copy/mL was higher than that in patients with HCV RNA <2. 0 ×106 copy/mL (χ2 =7. 367, P = 0. 007). The frequency of rs3212227 C allele in responders to interferon (IFN) therapy was lower than that in patients with nonresponse to IFN therapy (χ2 =4. 942,P=0. 026). Conclusions The SNP of rs3212227 is correlated with HCV infection. The carriers with C allele may be susceptible to HCV infection, while resistant to IFN therapy.

Objective To investigate the changes of CD4+ CD25+ regulatory T lymphocyte (Treg) and expressions of folkhead helix transcription factor 3 (FoxP3) in intestinal mucosa in human immunodeficiency virus (HIV) infected patients. Methods Twenty-one HIV infected patients and 17 control subjects without HIV infection were included in this study. The expression of FoxP3, which was considered as a specific marker of CD4+ CD25 + Treg, was detected in intestinal mucosa specimens from HIV infected patients by immunohistochemistry. Meanwhile, the in situ expression of CD4+ T lymphocyte was also determined by immunohistochemistry. The data were analyzed by t test. Results The positive labeling index of CD4+ T lymphocyte in intestinal mucosa was significantly lower in HIV infected patients compared to the controls (11. 56%±4. 44% vs 43. 49% ±8. 90% ,t=-11. 86,P<0. 01). The positive labeling index of FoxP3 in intestinal mucosa was also significantly lower in HIV infected patients compared to the controls (0.46% ± 0.20% vs 1. 18% ± 0. 44% ,t= - 5. 98,P<0.01). Conclusion The depletion of CD4+ CD25+ Treg is accompanied with the depletion of CD4 + T lymphocyte and the reduction of FoxP3 expression in intestinal mucosa of HIV infected patients.