Objective:To optimize the techniques for isolation and cryopreservation of primary rat hepatocytes.Methods:By improving traditional isolation methods and establishing an in situ recirculating collagenase perfusion through the portal vein,we want to improve the yield,viability and purity of primary rat hepatocytes and to reduce the cost of collagenase.Primary rat hepatocytes were cryopreserved at 1?10 7/ml in a suspension buffer containing 2% DMSO or 10% DMSO and were stored for 7 days,14 days or 2 months at -70℃ or in liquid nitrogen.After rapidly thawed hepatocytes were cultured for 7 days,their viabilities were measured by trypan blue exclusion (TBE) and MTT method,and the albumin (ALB) and lactodehydrogenase (LDH) in the supernatant of hepatocytes were analyzed.Results:By using the new technique,the hepatocyte yields have been improved to 1.584?0.525?10 8/100g rat body weight (BW),the viability of the hepatocytes is 95.2?2.9%,and purities better than 95%.At the same time,the cost of the collagenase has been reduced to 4.5mg/100g rat BW.The efficiency of cryopreservation is:(1)The viabilities of hepatocytes preserved in buffer containing 2% DMSO or 10% DMSO were ≥90% and ≥85% respectively.(2)The viabilities of hepatocytes cryopreserved in buffer containing 2% DMSO at -70℃ or in liquid nitrogen were all 90%-95%.(3)ALB synthesis was unaffected by different cryopreservation methods.(4)LDH in the supernatants of freshly cultured hepatocytes was equivalent to that of cryopreserved hepatocytes.Conclusion:Isolation technique of primary rat hepatocytes has been successfully improved.Freshly isolated hepatocytes suspended in buffer containing 2% MDSO can be cryopreserved for 2 moths at -70℃.These results have showed so far that the methods of isolation and cryopreservation of primary rat hepatocytes are optimized.

0.05),respectively.Sensitivity rate of 37 bacterial strains to gatifloxacin,ciprofloxacin,levofloxacin,sipafloxacin,cefataxime and penicillin(for gonococcus) were 100.0%,86.5%,94.6%,76.5%,91.9%,100.0%,respectively.Adverse events were generally mild,and adverse events rates of the two groups were 8.3% and 5.0%,respectively.Conclusion:Gatifloxacin is an highly effective and well tolerated antibacterial agent for the treatment of urinary tract and genital bacterial infections.

Objective:To evaluate the clinical efficacy and safety of gatifloxacin in the treatment of mild and moderate bacterial infections of respiratory tract,urinary tract,genital tract,skin and soft tissues.Methods:115 patients in study group were treated with gatifloxacin 200mg taken orally q12h,and 108 patients in control group were treated with ciprofloxacin 250mg taken orally q8h for 7days to 14 days.Results:The total cure rates of study group and control group were 90.4% and 77.8%( P 0.05),respectively.The bacterial eradication rates of the two groups were 97.1% and 89.2%( P 0.05),respectively.Conclusion:Gatifloxacin is a highly effective and well tolerated antibacterial agent for treatment of acute bacterial infections of respiratory tract,urinary tract,genital tract,skin and soft tissues.

Objective To investigate the diagnostic value of the T-SPOT.TB test for diagnosing tuberculous meningitis(TBM) by meta-analysis.Methods A systematic retrieval from the databases of PubMed,EMBASE,etc.was performed.The literature on the T-SPOT.TB test for diagnosing TBM was collected.Two reviewers independently screened the literature,extracted the data and judged the quality.The meta analysis was conducted by the Meta-Disc 1.4 software.Results 8 articles were included,involving 425 patients including 232 cases of TBM.In the peripheral blood group,the combined sensitivity was 80%(95%CI:0.74-0.85),the combined specificity was 74%(95%CI:0.67-0.80),the area under the curve(AUC)of summary receiver operating characteristic (SROC)was 0.858 7;the diagnostic odds ratio(DOR)was 15.50.In the CSF group,the combined sensitivity was 76%(95%CI:0.70-0.82),the combined specificity was 83%(95%CI:0.77-0.88),AUC was 0.892 7;DOR was 22.62.Conclusion Adopting the T-SPOT.TB test conduces to increase the diagnostic rate of TBM.The diagnostic accuracy of the T-SPOT.TB test for CSF may be higher than that for peripheral blood.

Objective To study the effect for albendazole therapy for alveolar echinococcus infection in gerbils and mice.Methods Mice and gerbils were infected of metacestode tissues by intraperitoneal (IP) transplantation and treated with albendazole-medicated feeds. The effects were evaluated by comparison of the treated and control groups in terms of host mortality, larval metastases to lungs and liver, final larval weight, histopathological and ultrastructural examination of metacestode tissues. Viability of metacestode tissues at necropsy of treated animals was tested by intraperitoneal transplantation into uninfected animals.Results Albendazole-medicated feeds significantly inhibited larval growth of Echinococcus multilocularis (E. multilocularis) both in mice and gerbils with markedly reduced host mortality and pulmonary and liver metastsaes. Viability test showed that albendazole therapy was parasiticidal in early stage of experimental infection. Light microscopic and ultrastructural examination of metacestode tissues of the treated animals revealed severe destruction and massive necrosis with marked calcification of protoscoleces and residual tissues.Conclusion Continuous long term albendazole therapy in animal models is parasiticidal against larval E. multilocularis especially in early stage of infection.

Hepatitis B ; virology ; Hepatitis B virus ; physiology ; Humans ; Virus Replication

OBJECTIVEBy studying the possibility of obtaining expression of human hepatitis B virus (HBV) genes and production in normal liver cells from heterologous species like normal primary duck hepatocytes (PDH), to investigate the species-specificity of HBV infection and replication.

METHODSTwo days after transfecting the complete HBV genome into PDH by electroporation (transfected group), HBsAg and HBeAg in the supernatants and lysates of PDH were measured by the IMX system. Meanwhile, replication of HBV in PDH was analyzed by Southern blotting and dot blotting procedures. PDH was electroporated as control.

RESULTSHBsAg in the lysate of transfected group was 9.10 (P/N values, positive?2.1), HBeAg was 1.0 (negative?2.1), both were negative in the supernatants of transfected group. dot blotting revealed that transfected group was strongly positive, whereas the control group was negative. Southern blot analysis of intracellular total DNA indicated that there were relaxed circular (RC), covalently closed circular (CCC) and single-stranded (SS) HBV DNA replicative intermediates in the transfected group, and there was no integrated HBV DNA in the cellular genome. Control groups were negative.

CONCLUSIONSReplication of HBV can occur in hepatocytes from nonmammalian species, which strongly supports the idea that replication of HBV has no critical species-specificity, and yet it depends on the endoenvironment of hepatocyte.

Animals ; Cells, Cultured ; DNA Replication ; physiology ; DNA, Viral ; biosynthesis ; Disease Models, Animal ; Ducks ; Electroporation ; Hepatitis B Surface Antigens ; analysis ; Hepatitis B e Antigens ; analysis ; Hepatitis B virus ; genetics ; physiology ; Hepatocytes ; metabolism ; virology ; Humans ; Species Specificity ; Transfection ; Virus Replication

OBJECTIVETo evaluate the short-term effect and safety of entecavir for the treatment of chronic hepatitis B (CHB) virus carriers.

METHODSNinety-three cases of CHB virus infection (hepatitis B surface antigen (HBsAg)-positive, hepatitis B e antigen (HBeAg)-positive, hepatitis B core antibody (HBcAb)-positive, HBV DNA≥1x10(5) copies/mL) were divided into two groups: CHB virus carrier (47 cases) and CHB (46 cases). All of the 93 cases were given 0.5 mg entecavir orally once a day for 48 weeks. Virology, serology and biochemistry tests were perrmed at treatment weeks 0, 4, 12, 24 and 48. Side effects of entecavir and the incidence of liver cirrhosis and hepatocellular carcinoma were recorded.

RESULTSThe CHB virus carrier and CHB group had complete virological response rates of 14.9% and 17.4% at week 4, 51.1% and 63.0% at week 12, 76.6% and 89.1% at week 24, and 97.9% and 100% at week 48, respectively; there was no significant difference between the two groups (P>0.05). The CHB virus carrier and CHB group had partial virological response rates of 42.6% and 47.8% at week 4, 57.44% and 65.2% at week 12, 85.0% and 89.1% at week 24, and 100% and 100% at week 48, respectively; there was no significant difference between the two groups (P>0.05). No cases in either group experienced virologic breakthrough during the treatment course. The CHB virus carrier and CHB group had serological response (HBeAg-negative) rates of 0 and 4.3% at week 4, 2.1% and 8.7% at week 12, 4.3% and 13.0% at week 24, and 8.5% and 21.7% at week 48, respectively; there was no significant difference between the two groups (P>0.05). The CHB virus carrier and CHB group had HBeAg seroconversion rates of 0 and 0 at week 4, 0 and 4.4% at week 12, 2.1% and 10.9% at week 24, and 6.4% and 17.4% at week 48, respectively; there was no significant difference between the two groups (P>0.05). No case in either group showed HBsAg-negativity and seroconversion during the treatment course. The CHB group had a biochemical response (alanine aminotransferase normalization) rate of 26.1% at week 4, 65.2% at week 12, 91.3% at week 24, and 97.8% at week 48.No case in either group showed biochemical breakthrough during the treatment course. There were no cases of liver cirrhosis or hepatocellular carcinoma in either group. There were no side effects of the entecavir treatment experienced in either group.

CONCLUSIONAntiviral therapy with entecavir is effective, safe and well tolerated in CHB virus carriers.

Alanine Transaminase ; Antiviral Agents ; Carcinoma, Hepatocellular ; Guanine ; analogs & derivatives ; Hepatitis B e Antigens ; Hepatitis B virus ; Hepatitis B, Chronic ; Humans ; Liver Neoplasms

Objective To explore the value of RT-PCRin diagnosis of patients with HIV-negative hypoimmunity and pneumocystis pneumonia (PCP).Methods Totally 71 phlegm samples of pulmonary infection patients with suspected PCP and HIV-negative hypoimmunity were selected.ITS-nested PCRand ITS-real-time fluorescence quantitative PCRwere used to detect pneumocystis jiroveci in samples.Results Among the 71 patients,18 cases were diagnosed as PCP.There were 25 cases with positive results by n-PCR,but the false-positive rate was 36.00%,and sensitivity,specificity and positive predictive values of n-PCRwere 88.89%,83.01% and 64.00%,respectively.If the threshold was 103 copies/mL,the specificity and positive predictive value of RT-PCRwere significantly higheRthan n-PCR.In patients with PCP,the the gene load of pneumocystis by RT-PCRwas significantly higheRthan non-PCP patients.Conclusion In patients with HIV-negative hypoimmunity,RT-PCRis more accurate and rapid than nested PCRin the diagnosis of PCP,and it can distinguish the infection and colonization of pneumocystis.

Objective To explore the value of RT-PCRin diagnosis of patients with HIV-negative hypoimmunity and pneumocystis pneumonia (PCP).Methods Totally 71 phlegm samples of pulmonary infection patients with suspected PCP and HIV-negative hypoimmunity were selected.ITS-nested PCRand ITS-real-time fluorescence quantitative PCRwere used to detect pneumocystis jiroveci in samples.Results Among the 71 patients,18 cases were diagnosed as PCP.There were 25 cases with positive results by n-PCR,but the false-positive rate was 36.00%,and sensitivity,specificity and positive predictive values of n-PCRwere 88.89%,83.01% and 64.00%,respectively.If the threshold was 103 copies/mL,the specificity and positive predictive value of RT-PCRwere significantly higheRthan n-PCR.In patients with PCP,the the gene load of pneumocystis by RT-PCRwas significantly higheRthan non-PCP patients.Conclusion In patients with HIV-negative hypoimmunity,RT-PCRis more accurate and rapid than nested PCRin the diagnosis of PCP,and it can distinguish the infection and colonization of pneumocystis.