[Effect and Persistent Effect of Thiolated Montmorillonite on Safe Production in Cadmium-contaminated Cropland].

To explore the effect and persistent effect of thiolated montmorillonite (TM) on safe production in cadmium (Cd) contaminated cropland, a two-year field experiment was conducted with different application amounts of TM. By adding to highly contaminated soils containing 2.46-3.81 mg·kg-1 Cd with no replenishment, the impacts of TM on concentrations of Cd in different parts of rice and available Cd in soils were investigated. The results showed that TM could significantly reduce the contents of Cd in brown rice as well as the contents and proportions of available Cd in soils, and its persistent effects on the passivation of Cd were obvious. After applying 0.5% or 1% TM to soils, the contents of Cd in different parts of the rice decreased significantly in the first season compared with that in the control. The contents of Cd in brown rice in the first season decreased to 0.16 mg·kg-1 and 0.08 mg·kg-1, respectively, by 84.0% and 91.9% compared with that of the control (0.98 mg·kg-1). Contents of Cd in brown rice were significantly lower than the maximum allowable amount (0.2 mg·kg-1) set by China (GB 2762-2017). Under the 0.5% and 1% treatments, the contents of Cd in brown rice of the subsequent three seasons under successive planting decreased by 50.2%-67.8% and 56.0%-81.6%, respectively, which were within the allowable amount. The proportions of available Cd in soils in the first season decreased from 48.4% under the control to 27.9% and 18.4%, respectively, which decreased by 20.5% and 29.9% under the 0.5% and 1% treatments. Compared with that in the control, proportions of available Cd in soils of the following three seasons decreased by 10.0%-17.1% and 12.4%-20.8%. There was a significant positive correlation between available Cd contents in soils and Cd contents in various parts of the rice. TM mainly reduced available Cd contents in soils, then reduced the absorption and accumulation of Cd in rice. The results of the two-year field experiment showed significant and continuous effects of TM on inhibiting Cd uptake by rice, which could be applied to the safe production in heavily Cd contaminated cropland.

Evaluation of phenotypic tests for detection of metallo-beta-lactamase-producing Pseudomonas aeruginosa strains in China.

A total of 264 nonduplicate strains of imipenem (IPM)-nonsusceptible Pseudomonas aeruginosa were isolated from hospitals in 16 different regions throughout China. These 264 IPM-nonsusceptible clinical isolates of P. aeruginosa were examined by PCR, a metallo-beta-lactamase (MBL) Etest, a double-disk synergy test (DDST), and a test using combined IPM disks supplemented with various amounts of EDTA. A total of 24 strains positive for MBLs were confirmed by PCR and DNA sequence analysis: 10 strains positive for the bla(VIM-2) gene, 13 strains positive for the bla(IMP-9) gene, and 1 strain positive for the bla(IMP-1) gene. Real-time reverse transcriptase PCR (RT-PCR) was used to verify whether the isolates harboring MBL genes produced the enzyme and was considered the standard for evaluation of the methodology in this study. Of these 24 MBL-positive stains, 21 were confirmed as MBL-producing strains by real time RT-PCR for MBL expression and the other 3 had no expression of MBLs. The sensitivities, specificities, and positive and negative predictive values for the MBL Etest, the DDST, and the combined disk (CD) assay were evaluated. The best method for screening for MBL production in P. aeruginosa strains from China was the CD assay (IMP-EDTA) using 750 microg of EDTA/disk with a breakpoint of >or=6 mm. In the CD assay (IPM-EDTA) with 290 microg and 750 microg EDTA, the zone diameter increases for VIM-2-producing P. aeruginosa isolates were greater than those for IMP-9-producing P. aeruginosa isolates (P = 0.00).

Sepsis resulting from Enterobacter aerogenes resistant to carbapenems after liver transplantation.

BACKGROUND: Sepsis due to Enterobacter aerogenes (E. aerogenes) is rare after liver transplantation but is also a serious infection that may cause liver abscess. The purpose of this case report is to relate an unusual presentation of liver transplantation to show how successive treatment can be an appropriate option in septic patients after liver transplantation. METHOD: We report on a patient with liver transplantation who developed sepsis due to extended spectrum beta-lactamases and AmpC-producing E. aerogenes. RESULTS: A 39-year-old man had a biliary fistula and then was found to have multiple liver abscesses through abdominal ultrasound and an abdominal computed tomography scan, and carbapenem-sensitive E. aerogenes infection was confirmed. The patient was not successfully treated with conservative treatment consisting of intravenous carbapenems, percutaneous transhepatic cholangial drainage, and biliary stent placement by endoscopic retrograde cholangiopancreatography, so a second liver transplantation followed. Carbapenem-resistant E. aerogenes was detected in bile and blood after a five-week course of carbapenem therapy. The patient developed septic shock and multiple organ dysfunction syndrome. CONCLUSIONS: We first report an unusual case of sepsis caused by E. aerogenes after liver transplantation in China. Carbapenem-resistant E. aerogenes finally leads to uncontrolled sepsis with current antibiotics. We hypothesize that the infection developed as a result of biliary fistula and predisposing immunosuppressive agent therapy. Further research is progressing on the aspect of immunomodulation therapy.

[Magnesium isoglycyrrhizinate in the treatment of chronic liver diseases: a randomized, double-blind, multi-doses, active drug controlled, multi-center study].

OBJECTIVE: To evaluate the efficacy and safety of Magnesium isoglycyrrhizinate in treatment of chronic liver diseases. METHODS: It is a randomized, double-blind, multi-doses, active drug controlled, multi-center study. 480 proper patients were randomly divided into group A (180 patients), group B (180 patients) or group C (120 patients). Patients in group A received magnesium isoglycyrrhizinate 100 mg once daily. Patients in group B received magnesium isoglycyrrhizinate 150 mg once daily. Patients in group C received compound glycyrrhizin 120 mg once daily. The treatment course was 4 weeks. Patients were followed up 2 weeks after the treatment. Patients visited once every 2 weeks. Clinical symptoms, ALT, AST were evaluated in all the patients before treatment, at week 2, at week 4 and at 2 weeks later after treatment. The other liver function test was done before treatment and at week 4. RESULTS: 412 patients completed the study according to the protocol,152 in group A, 160 in group B and 100 in group C. ALT and AST level were significantly decreased in all groups at week 2 and week 4 (P < 0.05). The degree of ALT decrease is greater in group B than in group C at week 2 (P < 0.01). The degree of ALT decrease was not significant different among three groups at week 4 (P > 0.05). The rates of ALT improvement at week 4 in group A, B, C were 92.59%, 91.76%, 88.29%, respectively (P > 0.05). The rates of symptoms improvement at week 4 in group A, B, C were 90.41%, 89.86%, 86.46% and 72.22%, 73.53%, 68.47%, respectively (P > 0.05). No relapse were found in all three groups after treatment. The rate of adverse event in three groups was similar (P > 0.05). CONCLUSION: Magnesium isoglycyrrhizinate is an effective and safe treatment for chronic liver diseases.

In vivo development of carbapenem resistance in clinical isolates of Enterobacter aerogenes producing multiple beta-lactamases.

Four clinical strains of extended-spectrum beta-lactamase- and AmpC-producing Enterobacter aerogenes were isolated successively from a liver transplantation patient. Isolates C(1) and C(2) were isolated prior to carbapenem therapy, whilst isolates C(3) and C(4) were recovered after 40 days of carbapenem therapy. The homology of these strains was analysed by pulsed-field gel electrophoresis (PFGE). beta-Lactamases were analysed by isoelectric focusing, polymerase chain reaction (PCR) and sequencing. Outer membrane proteins were analysed by PCR, sequencing, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot. Disruption of OmpE36 in C(1) in vitro was also performed by homologous gene recombination. The isolates demonstrated an indistinguishable PFGE pattern. Molecular characterisation revealed that, in addition to the pre-existing multiple beta-lactamases (DHA-1, TEM-1, SHV-5, CTX-M-3 and CTX-M-14) found in C(1) and C(2), isolates C(3) and C(4) failed to express OmpE36 owing to insertional inactivation by an IS903-like insertion sequence. Other resistance mechanisms, such as production of carbapenem-hydrolysing enzymes or expression of chromosomal efflux, were apparently not involved. Completely replacing OmpE36 by the kanamycin resistance gene (kan) resulted in a significant increase in carbapenem minimum inhibitory concentrations of an ompE36 mutant. Thus, C(3) and C(4) were apparently derived from the previously imipenem-susceptible isolates C(1) and C(2). Following carbapenem exposure, depletion of OmpE36 expression resulted in the collateral effect of carbapenem resistance.

Dissemination of imipenem-resistant Acinetobacter baumannii strains carrying the ISAba1 blaOXA-23 genes in a Chinese hospital.

An outbreak of 95 clinical infections with imipenem-resistant Acinetobacter baumannii in a Chinese hospital was investigated and the carbapenemase-encoding genes and their relationship with ISAba1 of these and a further 16 isolates recovered from the intensive care unit (ICU) environment were analysed. Almost all isolates were resistant to a wide range of antimicrobials; the lowest resistance rates were found for polymyxin E (17.1 %), cefoperazone/sulbactam (30.6 %) and ampicillin/sulbactam (67.6 %). Six pattern types defined by DNA macrorestriction patterns were distinguished among the clinical isolates with dissemination of pattern A (50 isolates) to patients in seven hospital units and pattern B (35 isolates) to eight units; the environmental isolates from ICUs were also of pattern A. All isolates were positive for the bla(OXA-66) and bla(OXA-23) genes. The OXA-23-encoding gene was located 34 bp downstream of ISAba1. No plasmids were detected and conjugal transfer of resistance was not demonstrated. The bla(OXA-23) probe hybridized with 200 and 220 kb ApaI chromosomal fragments for type patterns A and B, respectively.

[The degree of HBV suppression with 24 week telbivudine- or lamivudine-treatment in hepatitis B patients predicts the efficacy of the treatment at week 52].

OBJECTIVES: To investigate the possibilities of an association between the degrees of HBV suppression with nucleoside treatments at week 24 and week 52 in hepatitis B patients and to find a useful predictor for treatment efficacy. METHODS: In this phase III, double-blind, multicenter trial, we compared the efficacy of telbivudine treatment with lamivudine treatment in 332 Chinese compensated chronic hepatitis B patients. The patients were randomly assigned to a daily 600 mg telbivudine treatment group or daily 100 mg lamivudine group for 24 weeks. They were then categorized into 4 groups according to their serum HBV DNA levels (copies/ml) at week 24: a PCR-undetectable group (< 300 copies/ml); a QL- < 10(3) copies/ml group; a 10(3)-<10(4) copies/ml group; and a > or = 10(4) copies/ml group. The treatments were continued as they previously had been for another 28 weeks and the patients serum HBV DNA levels were examined again. RESULTS: At week 52, mean reductions of serum HBV DNA were significantly greater in the telbivudine-treated patients than in the lamivudine-treated group (6.2 log10 vs 5.4 log10, t = 3.6, P < 0.01). Viral resistance was twice as common in lamivudine-treated patients compared to those receiving telbivudine. Telbivudine was well-tolerated with an adverse event profile similar to that of lamivudine. The lower the HBV DNA level achieved at week 24, the higher HBV DNA non-detectable by PCR. ALT normalization and HBeAg seroconversion achieved at week 52, and viral resistance at week 48 decreased parallel to the degree of HBV DNA inhibition. CONCLUSION: HBV DNA PCR-undetectable at week 24 in nucleoside-treated hepatitis B patients suggests a better efficacy at week 52 and lower viral resistance at week 48. The degree of suppression of HBV at week 24 may be used as a predictor of 1-year outcome.

Insertion sequence ISEcp1-like element connected with a novel aph(2'') allele [aph(2'')-Ie] conferring high-level gentamicin resistance and a novel streptomycin adenylyltransferase gene in Enterococcus.

Enterococcus casseliflavus HZ95 is an enterococcus with high-level resistance to aminoglycosides. Nine genes responsible for high-level aminoglycoside resistance, including aac(6')-Ie-aph(2'')-Ia, aph(2'')-Ib, aph(2'')-Ic, aph(2'')-Id, aph(3')-IIIa, aac(6')-Ii, ant(3')-Ia, ant(4')-Ia and ant(6')-Ia, were not detected in HZ95. An 8 kb fragment from unconjugative plasmids of HZ95 was cloned, and expressed gentamicin resistance in Escherichia coli DH5alpha. The genetic structures ( approximately 8 kb DNA fragment) containing these aminoglycoside-modifying enzyme genes in Ent. casseliflavus HZ95 were determined. The deduced amino acid sequence of the novel aph(2'') allele, aph(2'')-Ie, had 93.7 % amino acid identity with APH(2'')-Id. The aph(2'')-Ie gene was bracketed upstream by an insertion sequence (IS)Ecp1-like element and downstream by a streptomycin adenylyltransferase gene (str). The streptomycin adenylyltransferase encoded by the str gene had 80.3 % amino acid identity with the protein encoded by aadE. The plasmid of approximately 16 kb could hybridize with a PCR-generated aph(2'')-Ie intragenic probe. The ISEcp1-like element had 91 % identity with ISEcp1. ISEcp1, which commonly acts as a key factor in the dissemination of CTX-M-type beta-lactamase genes in Gram-negative bacteria, has not been reported in Enterococcus.

Current therapy with nucleoside/nucleotide analogs for patients with chronic hepatitis B.

BACKGROUND: Currently, more and more nucleos(t)ide analogs are appearing as therapeutic options in the treatment of chronic hepatitis B (CHB). Their efficacy and safety profile in hepatitis B virus (HBV) infection have already been studied in detail worldwide. This review summarizes the efficacy of lamivudine, adefovir, entecavir and newer antiviral agents such as emtricitabine, telbivudine and clevudine in the treatment of hepatitis B in different clinical situations. DATA SOURCES: An English-language literature search using OVID and MEDLINE was performed and a total of 40 articles on the treatment of chronic hepatitis with nucleos(t)ide analogues were selected. RESULTS: Nucleos(t)ide analogs such as lamivudine, adefovir and entecavir are well tolerated and induce a decrease in serum HBV-DNA levels associated with normalization of serum alanine aminotransferase (ALT) levels. But their sustained response with HBeAg to anti-HBe seroconversion is rarely obtained and HBsAg loss is exceptional. The response is maintained during therapy which needs to be continued indefinitely in the majority of patients since withdrawal of treatment is generally followed by a rapid reactivation of hepatitis B. However, drug resistant mutations can be induced in long-term treatment. Other newer antiviral agents such as emtricitabine, telbivudine and clevudine in the treatment of hepatitis B are still under phase II or III clinical trials. CONCLUSIONS: Nucleos(t)ide analogs play an important role in the therapy of hepatitis B now and in the future. Lamivudine is limited by the frequent emergence of drug-resistant (HBV) mutants (YMDD). Adefovir and entecavir appear to be effective against both YMDD mutation and wild type. Therapeutic options against hepatitis B virus remain a major clinical challenge.

[A novel plasmid-mediated aminoglycosides-modifying enzyme gene, aph (2'')-Ie, in a strain of high-level gentamicin resistant Enterococcus casseliflavus].

OBJECTIVE: To identify the aminoglycoside resistance gene in the high-level gentamicin resistant (HLGR) enterococcus and its transmission mechanism. METHODS: A HLGR strain, HZ95, of Enterococcus casseliflavus was screened by agar dilution method. The aminoglycoside activation enzyme genes were screened by PCR. The PCR products underwent purification, cloned, and sequenced. Plasmids were extracted and underwent Southern hybridization. Plasmid-mediated aminoglycoside modifying enzymes were cloned. The resistance of the HZ05 strain to different antimicrobial agents was determined by K-B method or agar dilution method. RESULTS: A new aminoglycosides-modifying enzyme, APH (2'')-Ie, leading to HLGR, was found in the plasmid of the HZ95 bacteria. The aph (2'')-Ie gene and partial sequence of a streptomycin adenylyltransferase gene (str) were contained in the 8.7-kb cloned fragment, and the transposase gene (tnpA) was on the upper stream. The aph (2'')-Ie gene was located on the 16-kb plasmid with the amino acid sequence 96.1% homologous with chromosome-mediated APH (2'')-Id aminoglycoside-modifying enzyme. CONCLUSION: HLGR can be caused by a new plasmid-mediated aminoglycosides-modifying enzyme, APH (2'')-Ie, which can be transmitted among plasmids or chromosome with other resistant genes through transposase.

[Genotypes of aminoglycoside-modifying enzyme and clinical study of high-level gentamycin resistant enterococcus].

OBJECTIVE: To determine the antibiotics resistance, aminoglycoside-modifying enzymes and homology of high-level gentamycin resistant enterococcus in clinical specimens. METHODS: The high-level gentamicin resistant (HLGR) isolates were screened by the agar method and the resistance of 14 antimicrobial agents was determined by K-B method. The aminoglycoside-modifying enzyme genes were detected by polymerase chain reaction (PCR). Pulsed-field gel electrophoresis (PFGE) was used to analyze the homology of HLGR isolates. RESULTS: The ratio of HLGR was 64.2% (68/106). Among the HLGR,there were no isolates resistant to linezolid, vancomycin and tecoplanin, and Enterococcus faecium was more resistant to beta-lactam antibiotics and quinolone than Enterococcus faecalis. The positive rate of aac(6')-Ie-aph(2')-Ia was 92.6% and 3 isolates had the resistance gene mostly similar to aph(2')-Id. And among 51 HLGR isolates from the hospitalized patients, PFGE grouped 17 E. faecalis isolates into 4 clusters (A-D), and 33 E. faecium isolates into 8 clusters (A-H) with A cluster as predominant. CONCLUSION: HLGR has become the important antibiotic resistance bacteria which results in nosocomial infection; and aac(6')-Ie-aph(2')-Ia is the main aminoglycoside-modifying enzyme gene which causes HLGR.

Nosocomial spread of multi-resistant Klebsiella pneumoniae containing a plasmid encoding multiple beta-lactamases.

Six Klebsiella pneumoniae isolates that exhibited resistance to a wide spectrum of antibiotics were recovered from the intensive care units in the First Affiliated Hospital, Zhejiang University, Hangzhou, China. All isolates contained two plasmids of approximately 95 kb and 200 kb. The 95 kb plasmid was shown to be transferable by conjugation experiments. Isoelectric focusing patterns of the beta-lactamases extracted from the six transconjugants were identical, displaying five pI bands: 5.4, 7.75, 8.0, 8.2 and 8.4. The band corresponding to a pI of 7.75 could be inhibited by cloxacillin but not clavulanic acid, while the other bands could be inhibited by clavulanic acid but not cloxacillin. The 95 kb plasmid was digested with HindIII and a recombinant plasmid pT948 was obtained. The insert was found to contain blaDHA-1, regulatory gene ampR and an insertion element (IS26), which was downstream of blaDHA-1. PCR and DNA sequencing results confirmed that the 95 kb plasmid encoded at least four beta-lactamase genes: blaTEM-1, blaSHV-12), blaCTX-M-3 and blaDHA-1. Epidemiological typing by PFGE of the six clinical isolates of K. pneumoniae demonstrated identical genotypic patterns. In conclusion, all results indicated that the six multi-drug resistant clinical isolates of K. pneumoniae most probably originated from one clone and caused a localized epidemic in the intensive care units.

Intestinal microflora in rats with ischemia/reperfusion liver injury.

OBJECTIVES: To investigate the intestinal microflora status related to ischemia/reperfusion (I/R) liver injury and explore the possible mechanism. METHODS: Specific pathogen free grade Sprague-Dawley rats were randomized into three groups: Control group (n=8), sham group (n=6) and I/R group (n=10). Rats in the control group did not receive any treatment, rats in the I/R group were subjected to 20 min of liver ischemia, and rats in the sham group were only subjected to sham operation. Twenty-two hours later, the rats were sacrificed and liver enzymes and malondialdehyde (MDA), superoxide dismutase (SOD), serum endotoxin, intestinal bacterial count, intestinal mucosal histology, bacterial translocation to mesenteric lymph nodes, liver, spleen, and kidney were studied. RESULTS: Ischemia/reperfusion increased liver enzymes, MDA, decreased SOD, and was associated with plasma endotoxin elevation in I/R group compared to those in the sham group. Intestinal Bifidobacterium and Lactobacillus decreased and intestinal Enterobacteria and Enterococci, bacterial translocation to kidney increased in the I/R group compared to the sham group. Intestinal microvilli were lost, disrupted and the interspace between cells became wider in the I/R group. CONCLUSION: I/R liver injury may lead to disturbance of intestinal microflora and impairment of intestinal mucosal barrier function, which contributes to endotoxemia and bacterial translocation to kidney.

[Study on the genetic structure and transmission mechanism of a plasmid-mediated AmpC beta-lactamase].

OBJECTIVE: To clone the gene of plasmid-mediated AmpC beta-lactamase from the plasmid of multiple-drug resistance Klebsiella pneumoniae producing plasmid-mediated AmpC beta-lactamase and demonstrate its mechanism of transmission. METHODS: Plasmids of the transconjugant were extracted and digested with restriction endonuclease HindIII. Taq DNA polymerase was applied to fill the recessed 3' termini, and a single deoxyadenosine was added to the 3' termimi of fragments. Then these fragments were ligated with pGEM-T Easy vector. E. coli DH5alpha containing recombinant plasmid was selected on MacConkey agar plates containing ampicillin and cefoxitin. Insert fragments were sequenced by primer walking. MIC determinations and isoelectric focusing electrophoresis (IFE) were utilized to analyze recombinant. RESULTS: The recombinant plasmid pT948 containing a 5.2-kb insert was obtained. The inserted fragment contained a bla(DHA-1) and a regulatory gene ampR. The insertion sequence (IS26), qacEDelta1 and sulI genes of the I type integron were obtained near the bla(DHA-1) gene. Recombinant expressed a beta-lactamase with pI of 7.7. MIC determinations showed that recombinant was resistant to cefoxitin and the resistance to ceftazidime could be induced by the cefoxitin. CONCLUSION: The plasmid-mediated ampC gene cloned was identified as bla(DHA-1). IS26 observed on the flanks of the bla(DHA-1) maybe relate to the translocation of bla(DHA-1) gene region from the chromosome to plasmid.

[Therapeutic effect of ceftazidime in a rabbit model of peritonitis caused by Escherichia coli producing CTX-M-14 extended-spectrum beta-lactamase].

OBJECTIVE: To study the therapeutic effect of ceftazidime (CAZ) in a rabbit model of peritonitis caused by Escherichia coli producing CTX-M-14 extended-spectrum beta-lactamase (ESBL). METHODS: The peritonitis model was produced by intra-abdominal injection of a mixture of 3 x 10(8) CFU/ml Escherichia coli producing CTX-M-14 ESBL and 10% BaSO(4). All rabbits were randomized into four groups. Four hours after the bacteria were injected, three groups were intramuscularly administered ceftazidime (100 mg/kg, twice a day), cefotaxime (100 mg/kg, twice a day) and piperacillin/tazobactam (225 mg/kg, every 8 h) respectively. The fourth group did not receive any therapy as a control. The temperature, leucocyte counts and percentage of neutrophils were closely observed. The dying time of rabbits was recorded and anatomized immediately, and pathology of the great omentum was examined. RESULTS: The temperature of the control group became normal on the 6th day, while the temperature of the other three groups returned normal 48 h after the injection of the bacteria. Compared with the mortality of the cefotaxime-treated group (50%) and the control group (52%), the mortality of the ceftazidime-treated group (22%) was significantly decreased (chi(2) = 5.64, 6.13, P < 0.05). The administration of ceftazidime also inhibited the incidence of abdominal abscess in the survived rabbits (16%) in comparison with the control group (50%, chi(2) = 3.93, P < 0.05). In addition, the leucocyte counts of the ceftazidime-treated group returned normal at the end of treatment, while those of the cefotaxime group and the control group were still high. The above measurements showed no difference between the ceftazidime-treated group and the piperacillin/tazobactam-treated group (P > 0.05). CONCLUSION: Ceftazidime showed good therapeutic effect in peritonitis caused by Escherichia coli producing CTX-M-14 ESBL.

[Lipopolysaccharide (LPS) increases tumor necrosis factor-alpha related apoptosis induced-ligand (TRAIL) in macrophages killing HepG2 cells].

OBJECTIVE: To investigate the influence of lipopolysaccharide (LPS) on macrophages expressing TNF-alpha related apoptosis induced-ligand (TRAIL) and its relation to apoptosis of HepG2 cell line. METHODS: Membrane-bound TRAIL (mTRAIL) was measured by flow cytometry; soluble TRAIL in supernatant was detected by enzyme-linked immunoabsorbent sandwich assay (ELISA); cytotoxicity of TRAIL to HepG2 cell line was measured by chromium release assay, and apoptosis of HepG2 cell was confirmed by Annexin V staining. RESULTS: LPS only slightly increased membrane-bound TRAIL expression of macrophages. On the other hand, soluble TRAIL in the supernatant was increased with LPS stimulation, and the optimal concentration of LPS was 100 ng/ml (sTRAIL value 67.40 ng/ml+/-5.08 ng/ml). The soluble TRAIL in the supernatant was cytotoxic to HepG2 cells, and this activity can be blocked by TRAIL neutralizing antibodies. CONCLUSION: LPS increases the expression of soluble TRAIL in macrophages, and soluble TRAIL is toxic to HepG2 cells. All of our results indicate that TRAIL may play an important role in the pathogenesis of viral hepatitis.

Mutations in precore and core promoter region of HBV in patients with hepatic failure.

OBJECTIVE: To clarify the association of hepatitis B virus mutants in precore and core promoter regions in patients with hepatic failure and HBeAg state. METHODS: Precore and core promoter regions of 25 HBV isolates from the patients with hepatic failure were analyzed by polymerase chain reaction (PCR) direct sequencing approach. RESULTS: Precore G-to-A(1896) mutants were identified in 16 (64%) of the 25 isolates. The "hot spot" mutations at A-to-T(1762) and G-to-A(1764) were present together in 19 (76%) of the 25 isolates, while C-to-T(1653) and T-to-C(1753) existed in a mutually exclusive manner and more frequently in hepatic failure with liver cirrhosis group than in hepatic failure with chronic hepatitis group (100% vs 50%). Both A(1896) and T(1762)-A(1764) could be found frequently in HBeAg-positive subjects (77.8% and 88.9%), whereas T(1653)/C(1753) was more prevalent in anti-HBe-positive subjects than in HBeAg-positive subjects (93.8% vs 33.3%). CONCLUSIONS: The whole frequency of mutations in precore and core promoter gene will become more frequent as HBV infection is to be persistent. Mutation to T(1653)/C(1753) may be useful as a marker for hepatic failure. It requires further study whether the mixed infection of mutants and wilds will develop and affect the condition of HBeAg in serum along the progression of liver disease.

Cloning and expression of cDNAs from hepatitis E virus structural gene.

OBJECTIVE: To obtain recombinant antigen for development of anti-HEV ELISA kit and vaccine against hepatitis E virus infection. METHODS: A 492 base cDNA was collected from 5-terminus of open reading frame 2 (ORF2) among epidemic hepatitis E virus (HEV) isolated from Xinjiang, Western China. The fragment was digested with BamH I and EcoR I, and inserted into vector pGEX-4T-3 which was also digested by the same enzyme. The recombinant plasmid was transformed into E.coli TG-1 and the fusion protein expressed was confirmed by Western blot analysis using serum of a patient with hepatitis E. RESULTS: The recombinant plasmid was identified and confirmed with enzyme digestion, polymerase chain reaction (PCR) and sequencing, respectively. A protein band of about 46 kDa was demonstrated by SDS-PAGE and designated GST-pORF2. The result of Western blot analysis suggested that the fusion protein reacted with anti-HEV positive sera at a dilution of 1:100. CONCLUSION: The recombinant protein GST-pORF2 may be useful in developing anti-HEV ELISA kit and vaccine against hepatitis E virus infection.