Risk factors for alcoholic liver disease in China

AIM: To examine the association of daily alcohol intake,types of alcoholic beverage consumed, drinking patterns and obesity with alcoholic liver disease in China.METHODS: By random cluster sampling and a 3-year follow-up study, 1 270 alcohol drinkers were recruited from different occupations in the urban and suburban areas of Xi'an City. They were examined by specialists and inquired for information on: Medical history and family medical history, alcohol intake, types of alcoholic beverage consumed, drinking patterns by detailed dietary questionnaires. Routine blood tests and ultrasonography were done.RESULTS: Multivariate analysis showed that: (1) The risk threshold for developing alcoholic liver disease was ingestion of more than 20 g alcohol per day, keeping on drinking for over 5 years in men. The highest OR was at the daily alcohol consumption ≥160 g, the occurrencerate of ALD amounted to 18.7% (P<0.01). No ALD occurred when ingestion of alcohol was less than 20 g per day. (2) 87.9% of all drank only at mealtimes. The cumulative risk of developing ALD was significantly higher in those individuals who regularly drank alcohol without food than in those who drank only at mealtimes, especially for those who regularly drank hard liquors only and multiple drinks (P<0.05). (3) The alcohol consumption in those with BMI ≥25 was lower than in those with BMI<25, but the risk increased to 11.5%, significantly higher than that of general population, 6.5% (P<0.01). (4) Abstinence and weight reduction could benefit the liver function recovery.CONCLUSION: In the Chinese population the ethanol risk threshold for developing ALD is 20 g per day, and this risk increases with increased daily intake. Drinking 20 g of ethanol per day and for less than 5 years are safe from ALD. Drinking alcohol outside mealtimes and drinking hard liquors only and multiple different alcohol beverages both increase the risk of developing ALD. Obesity also increases the risk, Abstinence and weight reduction will directly affect the prognosis of ALD, Doctor's strong advice might influence the prognosis indirectly.

Antiproliferative effect of octreotide on gastric cancer cells mediated by inhibition of Akt/PKB and telomerase

AIM: To investigate the antiproliferative effect of octreotide,a long-acting analogue of somatostatin, on gastric cancer cell line SGC7901 and its possible molecular mechanisms.METHODS: Gastric cancer cell line SGC7901 employed in the study was treated with 0.008, 0.04, 0.2, 1, 5 and 25μg@ml-1 of octreotide respectively for 24 h to evaluate the antiproliferative effect of somatostatin analog on the tumor cells by MTT assay method. To elucidate the underlying mechanism, the cells were exposed to 1 μg@ml-1 of octreotide for 0, 12, 24 and 48 h, when their Akt/PKB and telomerase activities were respectively determined using PCR-ELSIA and nonradioactive protein kinase assay protocols. The same experimental procedures were also performed in the control cells that were treated with corresponding vehicles instead of somatostatin analog.RESULTS: After exposed to octreotide for 24 h at the concentrations of more than 1 μg@ml-1 SGC7901 cells exhibited a dose-dependent inhibition of growth with the inhibiting rate to be as high as 34.66 % when 25 μg@ml-1 of octreotide was applied. The Akt/PKB and telomerase activity of SGC7901 cells was significantly inhibited when the cells were exposed to 1 μg@ml-1 of octreotide for 12, 24 and 48 h compared with that of their control counterparts (P＜0.01),both of which exhibited in a time-dependent manner.CONCLUSION: The antiproliferative effect of octreotide on SGC7901 cells might be mediated by the inhibition of Akt/PKB and telomerase.

Structure analysis and expressions of a novel tetratransmembrane protein,lysosoma-associated protein transmembrane 4β associated with hepatocellular carcinoma

AIM: To analyze the structure and expressions of the protein encoded by an HCC-associated novel gene, lysosomeassociated protein transmembrane 4 β (LAPTM4B). METHODS: Primary structure and fundamental characteristics of LAPTM4B protein were analysed with bioinformatics. Expressions of LAPTM4B in HCC tissues and various cell lines were detected using polyclonal antibodies and Western blot. RESULTS: LAPTM4B encoded two isoforms of proteins with molecular masses 35-ku and 24-ku, respectively. The expression level of LAPTM4B-35 protein in HCC tissues was dramatically upregulated and related to the differentiation status of HCC tissues, and it was also high in some cancer cell lines. Computer analysis showed LAPTM4B was an integral membrane protein with four transmembrane domains. LAPTM4B showed relatively high homology to LAPTM4A and LAPTM5 in various species. CONCLUSION: LAPTM4B gene encoded two isoforms of tetratansmembrane proteins, LAPTM4B-35 and LAPTM4B-24. The expression of LAPTM4B-35 protein is upregulated and associated with poor differentiation in human HCC tissues, and also at high levels in some cancer cell lines. LAPTM4B is an original and conserved protein.

The Application of Complex PSA and Its Relative Indexes in the Detection of Prostate Cancer

Objective:To study the diagnostic value of complex PSA(cPSA),the calculated free/total PSA(f/t PSA) raio and total PSA(tPSA)in the differentiation of prostate cancer from benign prostate hyperplasia.Methods:The tPSA,cPSA and fPSA were measured using the Bayer ACS-180 chemiluminescence immuno-assay.152 patients(21 with prostate cancer and 131 with benign prostate hyperplasia proven by tissue pathology)whose serum total PSA ranged from 0.2-20.0ng/ml were accessed from July 2001 to May 2002 consecutively.The correlation between tPSA and cPSA was analyzed.The re-ceiver operator characteristic curves(ROC curve)were generated by plotting the sensitivity versus specificity.Areas under the curve were calculated for each assay.Logistic regression analysis was used to evaluate the ability of the indices as independent varia-bles to predict prostate cancer.Results:In the experimental group,the areas under the ROC curve of cPSA ,tPSA and fPSA/tPSA ratio were 0.811,0.799 and 0.376 respectively.The specificity for tPSA,fPSA/tPSA ratio and cPSA were 62%,57% and 4.7%,respectively,at cotoff yield-ing 95% sensitivity.Serum cPSA concentration was determined to be the best index among the three through logistic regression analy-sis.Conclusion:The serum levels of cPSA and tPSA are better indices than f/tPSA in the differentiation of prostate cancer from benign prostate hyperplasia.At the same level of sensitivity,cPSA has a higher specificity than tPSA.Serum cPSA may be a better indicator in the prediction of prostate cancer of early stage.

Two-dimensional polyacrylamide gel electrophoresis analysis of indomethacin-treated human colon cancer cells

AIM: To establish the two-dimensional gel electrophoresis (2-DE) profiles of indomethacin (IN)-treated human colon cancer cell line HCT116, and to provide a new way to study its anti-tumor molecular mechanism through analyzing a variety of protein maps.METHODS: Two-DE profiles of HCT116 were established in IN-treated and untreated groups. Total proteins were separated by immobilized pH gradient-based 2-DE. The gels were stained by silver, scanned by ImageScanner,and analyzed with Image Master software.RESULTS: Clear background, well-resolved and reproducible 2-DE patterns of HCT116 cells were acquired in IN-treated and untreated group. The average deviation of spot position was 0.896±0.177 mm in IEF direction and 1.106±0.289 mm in SDS-PAGE direction respectively. In IN-treated group,1 169±36 spots were detected and 1 061±32 spots were matched, the average matching rate was 90.6% in three gels. In untreated group, 1 256±50 spots were detected and 1 168±46 spots were matched, the average matching rate was 93.0% in three gels. Forty-five differential protein spots were displayed between IN-treated and untreated groups. Of which, 34 protein spots decreased and 9showed higher expression in IN-treated group, and only two protein spots showed an expression in untreated cells.CONCLUSION: Two-DE profiles of IN-treated and untreated HCT116 cells were established. Apparent 45 different protein spots were detected in IN-treated and untreated HCT116 cells. The analysis on differential protein spots may serve as a new way to study the molecule mechanism of IN-treated colon cancer.

Phage displaying peptides mimic schistosoma antigenic epitopes selected by rat natural antibodies and protective immunity induced by their immunization in mice

AIM: To obtain the short peptides mimic antigenic epitopes selected by rat natural antibodies to schistosomes, and to explore their immunoprotection against schistosomiasis in mice.METHODS: Adults worm antigens (AWA) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked transferred immunoblotting methods with normal SD rat sera (NRS). The killing effects on schistosomula with fresh and heat-inactivated sera from SD rats were observed. Then the purified IgG from sera of SD rats was used to biopan a phage random peptide library and 20 randomly selected positive clones were detected by ELISA and 2 of them were sequenced.Sixty female mice were immunized thrice with positive phage clones (0, 2nd, 4th wk). Each mouse was challenged with 40 cercariae, and all mice were killed 42 d after challenge. The worms and the liver eggs were counted. RESULTS: NRS could specifically react to the molecules of 75 000, 47 000, 34 500 and 23 000 of AWA. Sera from SD rats showed that the mortality rate of schistosomula was 76.2%, and when the sera were heat-inactivated in vitro, the mortality rate was decreased to 41.0% after being cultured for 48 h. The specific phages bound to IgG were enriched about 300-folds after three rounds of biopanning. Twenty clones were detected by ELISA, 19 of them bound to the specific IgG of rat sera. Immunization with these epitopes was carried out in mice. Compared with the control groups, the mixture of two mimic peptides could induce 34.9% (P = 0.000) worm reduction and 67.6% (P = 0.000) total liver egg reduction in mice. Two different mimic peptides could respectively induce 31.0% (P = 0.001), 14.5% (P = 0.074) worm reduction and 61.2% (P = 0.000), 35.7% (P = 0.000) total liver egg reduction. The specific antibody could be induced by immunization of the mimic peptides, and the antibody titer in immunized mice reached more than 1:6 400 as detected by ELISA.CONCLUSION: Specific peptides mimic antigenic molecules can be obtained by biopanning the phage random peptide library and a partially protective immunity against schistosome infection can be stimulated by these phage epitopes in mice.

Sequence evolution of putative cytotoxic T cell epitopes in NS3 region of hepatitis C virus

AIM:Quasispecies of hepatitis C virus (HCV) are the foundation for rapid sequence evolution of HCV to evade immune surveillance of hosts. The consensus sequence evolution of a segment of HCV NS3 region, which encompasses putative cytotoxic T cell epitopes,was evaluated.METHODS:Three male patients, infected with HCV through multiple transfusions,were identified from clinical symptoms and monitored by aminotransferase for 60 months.Blood samples taken at months 0, 32, and 60 were used for viral RNA extraction. A segment of HCV NS3 region was amplified from the RNA extraction by RT-PCR and subjected to subcloning and sequencing.HLA types of these three patients were determined using complement-dependent microlymphocytotoxic assay. CTL epitopes were predicted using MHC binding motifs.RESULTS:No patient had clinical symptoms or elevation of aspartate/alanine arninotransferase. Two patients showed positive HCV PCR results at all 3 time points. The other one showed a positive HCV PCR result only at month 0.A reported HLA-A2-restricted CTL epitope had no alteration in the HLA-A2-negative carrier over 60 months.In the HLA-A2-positive individuals, all the sequences from 0 month 0 showed an amber mutation on the initial codon of the epitope. Most changes of consensus sequences in the same patient occurred on predicted cytotoxic T cell epitopes.CONCLUSION:Amber mutation and changes of consensus sequence in HCV NS3 region may be related to viral immune escape.

Serum Treponema IgM Antibody Test for Syphilis Diagnosis

Objective: To evaluate the clinical utility of testing serum anti-treponema pallidum IgM antibody in the diagnosis of syphilis patients. Methods: Seventy-two cases of syphilis were tested for specific IgM antibody with ELISA, and the results were compared with RPR and TPPA.Results: The sensitivity of IgM antibody was 73.3 %(11/15) in primary syphilis, 88.9% (16/18) in sec-ondary syphilis, and there was no significant differ-ence between these values (x^2=1.6363, P>0.10). The sensitivity of IgM antibody in diagnosing latent syphi-lis was only 26.1% (6/23), much lower than the detec-tion rate in symptomatic earlv svDhilis (x^2=17.6189. P<0.005). RPR and TPPA were both 100% sensitive in latent and early symptomatic syphilis. Two were posi,five for IgM in the 16 cases who had received regular treatments 2 to 24 months before enrolled.Conclusions: Specific IgM antibody detection doees not appear superior to RPR and TPPA in diagnosing primary syphilis. The diagnosis of latent syphilis should mainly rely on RPR and TPPA, since there are low titers of IgM antibody at that stage. IgM antibody testing alone should not be recommended for monitor-ing syphilis development or treatment efficacy. Fur-ther studies should be concerned.

Inhibitory effects of reserpine and carbonyl cyanide m-chloro-phenylhydrazone on fluoroquinolone resistance of Acinetobacter baumannii

Mechanisms of bacterial resistance to fluoro-quinolones may be grouped intothree principal categories: gene mutations of DNA topoisomerase Ⅱ (GyrA or GyrB), DNA topoisomeraseⅣ ( ParC or ParE), decrease of outer membrane permeation and upregulation of multi-drug effluxpump (active efflux system). Efflux pumps are transport proteins removing toxic substrates(including virtually all classes of clinically relevant antibiotics) from cells to the externalenvironment. These proteins exist in both Gram positive bacteria and Gram negative bacteria as wellas in fungi and mammalian (tumour) cells. It has been reported that alkaloid reserpine and carbonylcyanide m-chlorophenylhydrazone (CCCP) can inhibit NorA multi-drug efflux. In order to explore theuniversality of drug efflux in microorganisms, 85 strains of Acinetobacter baumannii (A. baumannii)were tested using reserpine and CCCP. The quinolone-resistant-determining region (QRDR) of gyrA andparC genes in 35 isolates of A. baumannii were amplified by polymerase chain reaction (PCR) andsequenced by DNA sequencer. The correlation between resistant mutation regularity and bacterial drugefflux were analysed.

Relationship between antimicrobial resistance and aminoglycoside-modifying enzyme gene expressions in Acinetobacter baumannii

Background Acinetobacter baumannii is one of the main gramnegative bacilli in clinical practice Nosocomial infections caused by multidrug resistance Acinetobacter baumannii is very difficult to treat This study was designed to investigate the antimicrobial resistance characteristics and four resistant gene expressions of aminoglycosidemodifying enzymes including Nacetyltransferases and Ophosphotransferases in Acinetobacter baumannii Methods Bacterial identification and antimicrobial susceptibility test were performed by PhoenixTM system in 247 strains of Acinetobacter baumannii Minimal inhibitory concentrations (MICs) of seven aminoglycosides including gentamicin, amikacin, kanamycin, tobramycin, netilmicin, neomycin and streptomycin in 15 strains of multidrug resistant Acinetobacter baumannii were detected by agar dilution Four aminoglycosidemodifying enzyme genes were amplified by polymerase chain reaction (PCR) and verified by DNA sequencerResults The resistance rates of 247 strains of Acinetobacter baumannii against cefotaxime, levofloxacin, piperacillin, aztreonam, tetracycline, ciprofloxacin and chloramphenicol were more than 50% Imipenem and meropenem showed high antibacterial activities with resistance rates of 32% and 41% MIC50 and MIC90 of gentamicin, amikacin, streptomycin and kanamycin in 15 strains of multidrug resistant Acinetobacter baumanii were all more than 1024 mg/L, and the resistance rates were 100%, 100%, 100% and 933%, respectively But their resistance rates to tobramycin, netilmicin and neomycin were 867%, 933% and 467%, respectively Three modifying enzyme genes, including aacC1, aacC2 and aacA4 genes, were found in 15 strains, but aphA6 had not been detected Their positive rates were 933%, 200% and 200%, respectively These three genes existed simultaneously in No19 strain Nucleotide sequences of aacC1, aacC2 and aacA4 genes shared 100%, 979% and 997% identities with GenBank genes (AY307113, S68058 and AY307114)Conclusion Multidrug resistant Acinetobacter baumannii strains are rapidly spreading in our hospital, and their resistance to aminoglycosides may be associated with aminoglycosidemodifying enzyme gene expressions