Antigens, CD34 ; metabolism ; Cytokines ; metabolism ; Diagnosis, Differential ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Mucin-1 ; metabolism ; Perineum ; pathology ; Phosphopyruvate Hydratase ; metabolism ; Sarcoma ; metabolism ; pathology ; surgery ; Vimentin ; metabolism

Objective To evaluate the clinical and histological outcomes in a cohort of chronic hepatitis B (CHB) patients who had histologically confirmed severe liver fibrosis and received lamivudine (LAM) therapy for up to 10 years. Methods Thirty-nine CHB patients with severe liver fibrosis (Ishak fibrosis score≥4) were treated with LAM for up to 10 years. Disease progression liver histological improvement, virological and biochemical responses were evaluated during follow-up. Data were analyzed using paired t test, Fisher exact test and Willcoxon test. Results Twenty-eight patients completed the 10-year follow-up. There were 5 (17.9% ) patients with disease progression.At the end of follow up, 16 patients received a second liver biopsy, which showed significant improvement of histological activity index (1.1 ± 1.4 vs 7. 1 ± 3.2, t =- 0.82, P＜0.01 ) and Ishak fibrosis score (3.6±2.2 vs 5.3±0.7, t= -2.89, P＜0.05) compared to baseline. There were 3 cases with Ishak fibrosis score improved from F5 to F0. Among 27 patients, 3(11% ) cases achieved hepatitis B surface antigen (HBsAg) loss and 2 (7 % ) achieved HBsAg seroconversion. At the end of follow-up, 19 out of 23 (83% ) hepatitis B e antigen (HBeAg) positive patients obtained HBeAg loss and 9 (39 % ) obtained HBeAg seroconversion. During LAM treatment, 11 patients experienced virological breakthrough or detected documented LAM-related resistance mutation. The viral loads of all patients were below 1 ×103 copy/mL at the end of follow-up after rescued by add-on or switch to another nucleotide analog.Conclusions Long-term LAM therapy can delay the disease progression in CHB patients with severe liver fibrosis, increase HBsAg and HBeAg loss rates, sustain suppression of HBV replication at a low level and even totally reverse the liver fibrosis in some patients. The effect of LAM resistance mutation on disease outcomes would be reduced by rescue therapy.

ObjectiveTo investigate the histological features as well as the factors influencing liver disease progression in Chinese patients with chronic hepatitis C (CHC). MethodsA total of 102 CHC patients who underwent percutaneous liver biopsy between August 2007 and May 2010 were recruited. Age, gender, body mass index (BMI) and transmission route of recruited patients were recorded. Serum levels of alanine transaminase (ALT) and aspartate transaminase (AST), HCV genotypes, HCV viral load and liver histological changes were detected. Statistical analysis was done by t test and Logistic regression. ResultsThe serum levels of ALT and AST in CHC patients with histological activity index (HAI) ≥4 were much higher, while platelet (PLT) counts were lower than those with HAI ＜4(t=2.209, 2. 298 and 2. 565, respectively; all P＜0.05). Likewise, in patients with F≥3, the serum levels of ALT and AST as well as the mean age and the duration of infection were significantly elevated compared with F ＜ 3 group ( t = 3.497, 2. 758, 2. 340 and 2. 570,respectively; all P＜0. 05), while PLT counts were much lower (t = 2. 761, P=0. 007). The unvariate predictors for HAI≥4 were female, ALT＞1 × upper limits of normal (ULN), AST level,F≥3, HCV RNA≥6 lgIU/mL and PLT counts. By mutivariate analysis, the Ishak stage score was the only independent predictor for HAI≥4 (OR 3.098, 95％CI 1.884-5. 092; P＜0.01). Finally,the univariate predictors for F≥3 were age, BMI≥24 kg/m2 , ALT＞1 × ULN, AST level, HAI≥4,PLT counts and duration of infection≥ 15 years. Multivariate analysis revealed that age (OR 1. 074,95％CI 1.006-1. 146; P=0.033), ALT level (OR 1. 035, 95％CI 1.015－1.055; P＜0.01), ASTlevel (OR 0. 969, 95％CI 0. 948－0. 990; P=0. 005), the duration of infection ≥15 years (OR 37. 215, 95％CI 5. 816－238. 127; P＜0.01) and HAI≥4 (OR 1. 939, 95％CI 1. 426－2. 636; P＜0.01) were independent predictors for F≥ 3. ConclusionAge, ALT level, AST level, duration of infection≥15 years, HAI≥4 are independent predictors for liver fibrosis.

Objective To explore the relationship among cytokine levels and Toll-like receptor (TLR) 2,4 in hepatitis B virus (HBV) related cirrhosis. Methods Heparin anticoagulated venous blood of 35 randomly selected HBV related cirrhosis and 35 healthy volunteers were collected aseptically. Plasma tumor necrosis factor (TNF)-α concentration was determined using enzyme-linked immunosorbent assay (ELISA). Peripheral blood mononuclear cells (PBMC) were separated and stained with anti-TLR2,4 monoclonal antibodies, then analysed by flow cytometry. Total RNA was extracted from PBMC and TLR2,4 mRNA expression levels were evaluated by real-time quantitative polymerase chain reaction (PCR) using SYBR Green detection. Means of normal distribution were compared by t test and one factor analysis of variance. The data of abnormal distribution were analyzed using Mann-Whithey U test, Kruskal-Wallis H test and Spearman correlation analysis. Results The plasma concentration of TNF-α in the cirrhotic patients was significantly higher than that in the healthy controls (33.52 ng/L vs 6.07 ng/L, Z=-6.584, P<0. 01), which was parellel with Child-Pugh score. TLR2 positive rate in PBMC from the cirrhotic patients was significantly higher than that from the healthy controls (20.65% vs 12.04%, Z=-4.458, P<0.01), which was positively correlated with plasma TNF-α level (r= 0.448 3, P<0.05), and parellel with Child-Pugh score. Between the cirrhotic and healthy groups, there was no significant difference of TLR4 positive rate in PBMC. The mRNA expression level of TLR2/GAPDH in PBMC from the cirrhotic patients was significantly higher than the controls (0.234 2 vs 0.043 1, Z=-6.83, P<0.01), which was positively correlated with plasma TNF-α level (r=0.411 1, P<0.05), and parellel with Child-Pugh score. Between the two groups, there was no significant difference of TLR4 mRNA expression level in PBMC. Conclusions The expression of TLR2 in PBMC from cirrhotic patients is significantly elevated, which is positively correlated with plasma TNF-α level and the severity of liver disease. The expression of TLR4 in PBMC from cirrhotic patients is unchanged. It suggests that TLR2 but not TLR4, plays an important role in the progression of liver cirrhosis.

The fermentation process of recombinant human Endostatin expression in Escherichia coli BL21 (DE3) was studied. The effects of factors such as concentration of IPTG, induction time, cultivation temperature and feeding strategies were investigated. Beside that, by changing the temperature to 40 degrees C after induction, the high-density cultivation finished in a much shorter period. After 9 hours cultivation, the optical density (OD) at 600 nm reached 140 and the yield of inclusion body was 3 g/L. While E. coli system was used, protein with better activity and stability was obtained. The cost was much lower and the producing process was much steadier. It will meet the demands of the industrial production.
Endostatins ; biosynthesis ; genetics ; Escherichia coli ; genetics ; growth & development ; metabolism ; Fermentation ; Humans ; Protein Engineering ; Recombinant Proteins ; biosynthesis ; genetics ; Thiogalactosides ; chemistry

The method of monoclonal antibody-based immunoassay has a great importance in the study of quality control of traditional Chinese medicine (TCM) and detection of trace components in vivo animals. Synthesis of small molecule artificial antigen is the prerequisite for the establishment of this method. In present study, catalpol-BSA was synthesized by sodium periodate oxidation method. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry ( MALDI-TOF-MS) and molecular exclusion chromatography showed that catalpol was successfully conjugated with BSA. The mice could specifically produce anti-catalpol antibodies with titer up to 1:8000. The artificial antigen of catalpol was successfully synthesized.
Animals ; Antibodies ; immunology ; Antigens ; chemistry ; immunology ; Immunoassay ; Iridoid Glucosides ; chemistry ; immunology ; Male ; Medicine, Chinese Traditional ; Mice ; Mice, Inbred BALB C ; Serum Albumin, Bovine ; chemistry ; immunology

AIMTo investigate the action and mechanism of Syn-1A in reversing the activation of K(ATP) channel induced by weak acidic pH.

METHODSThe patches excised from Kir6.2/SUR2A expressing HEK-293 cells were used to establish inside-out configuration. To examine the actions of weak acidic pH in activation of the channel and the reverse action of Syn-1A on it, the inside-out patches were continuously perfused with the solution of pH from 7.4, 7.0, 6.8, 6.5 to 6.0 with or without Syn-1A. In vitro binding was employed to study the influence of different pH to the binding of Syn-1A to SUR2A subunit.

RESULTSSyn-1A blocked pH 6.5, 6.8 and 7.0 induced activation of the channel, and Syn-1A binding to SUR2A were increased by reducing pH from 7.4 to 6.0.

CONCLUSIONSyn-1A would assert some inhibition of the KATP channels, which might temper the fluctuation of acidic pH-induced K(ATP) channel opening that could induce fatal re-entrant arrhythmias.

HEK293 Cells ; Humans ; Hydrogen-Ion Concentration ; KATP Channels ; metabolism ; Patch-Clamp Techniques ; Potassium Channels ; metabolism ; Potassium Channels, Inwardly Rectifying ; metabolism ; Syntaxin 1 ; pharmacology

OBJECTIVETo create an externally-applied TCD plaster (Laosun Yutie plaster) for the lumbar muscle strain (LMS).

METHOD(1) The raw material such as angelica, safflower, frankincense, myrrh, clove and so on were turned into solid TCD plaster by a serial of modern treatment technology-distillation, extraction, condense, dehydration. Meanwhile, the naphtha from the distillation process and borneol were dissolved into ethanol solution and the dilution was thus made; (2) The solid TCD plaster and the dilution from the aboved-mentioned process were put together to form the TCD plaster and the corresponding safety experiments were applied to rabits' skin.

RESULTThe experimental results showed that it is neither of urgent toxicity, stimulary function, nor of allergy-inducing response.

CONCLUSIONThe laosun Yutie plaster was a newly externally-applied TCD plaster which could be used safely and handily.

Administration, Cutaneous ; Animals ; Drug Combinations ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; toxicity ; Exanthema ; chemically induced ; Female ; Guinea Pigs ; Hypersensitivity ; etiology ; Male ; Plants, Medicinal ; chemistry ; Rabbits ; Skin Irritancy Tests

OBJECTIVEThe purpose of this research was to study the genetic diversity of F-ATPase subunit gene uncEBF derived from Streptococcus mutans (S. mutans) clinical isolates, furthermore to investigate the relationship between the genetic diversity of F-ATPase and S. mutans aciduric ability.

METHODS38 S. mutans strains included 18 high acid tolerance strains and 20 low acid tolerance strains. Gene uncEBF of these isolates were amplified with specific primers from S. mutans genomic DNA, and the PCR products were analyzed by RFLP and sequenced. SPSS 11.0 statistic software assayed the results.

RESULTSIt was testified that two genotypes A and B of PCR-RFLP were revealed when digested with Alu I and Dde I digested fragments of uncEBF displayed two different patterns C and D. Fisher exact two-tail test showed that the distributions of A and B genotype strains with different acidurance were different (P < 0.05), and the proportion of A genotype strains from high acidurance group was higher than that from low acidurance one. Some of these amplified uncEBF genes from different genotype were sequenced and testified that there existed variation of Alu I and Dde I recognized sites.

CONCLUSIONThis study indicated that uncEBF gene of S. mutans F-ATPase obviously exhibited genetic diversity.

Adenosine Triphosphatases ; Dental Caries ; Genetic Variation ; Genotype ; Humans ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Streptococcus mutans

OBJECTIVETo study the genetic diversity and the gene expression of membrane-bound proton-translocating ATPase (F-ATPase) subunit gene uncG derived from Streptococcus mutans (S. mutans) clinical isolates.

METHODS38 S. mutans strains derived from caries-active and caries-free individuals including 18 strains displaying high acid tolerance and 20 strains displaying low acid tolerance. Gene uncG was amplified with specific primers from S. mutans genomic DNA, then the PCR product was analyzed by RFLP and sequenced. The relative expression quantity of uncG gene against the housekeeping gene recA was determined by using RT-PCR method. A gel documentation system and QUANTITY ONES software were used to analyze the data results.

RESULTSIt was testified that four genotypes A, B, C and D of PCR-RFLP were revealed when respectively digested with Alu I and Bsr I, but the distributions of the four genotype strains showed no difference (P > 0.05). The differences of uncG gene transcript quantities derived from different genotype or different aciduranc strains had no significance (P > 0.05).

CONCLUSIONThis study indicated that uncG gene of F-ATPase obviously displayed genetic diversity and existed polymorphism at mRNA expression level, while the Alu I-RFLP genotypes and the expression levels would not be responsive to different acid tolerance of S. mutans strains.

Adenosine Triphosphatases ; Dental Caries ; Genetic Variation ; Genotype ; Humans ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length ; RNA, Messenger ; Streptococcus mutans