Anticoagulants ; pharmacology ; therapeutic use ; Cardiovascular Diseases ; drug therapy ; Humans

Objective To evaluate the expression of Toll-like receptor(TLR)on the monocyte- derived dendritic cells(DC)from chronic hepatitis B(CHB)patients and to analyze the expression pro- file and significance of the TLR such as TLR3,TLR4,TLR?,TLR8 and TLRg,which are associat- ed with immune response to viral infection.Methods Peripheral blood mononuclear cell(PBMC) centrifugated by the hydroxyethyl starch(HES)centrifugation were cultured and induced into DC by granulocyte-maerophage colony stimulating factor(GM-CSF)and interleukin-4(IL-4),and their mor- phology and phenotype were detected by the inverted microscope and flow cytometry respectively. Monocyte-derived DC were obtained from 10 chronically hepatitis B virus(HBV)-infected patients and 15 healthy volunteers.TLR3,TLR4,TLR7,TLRS,TLR9 expression on immature and mature DC were analyzed by FACS Calibur.DC was pulsed with HBcAg on day 3 and 5,then DC maturation and ability to process HBcAg and to stimulate autogeneic T cells were evaluated.Results Monocyte- derived DC developed different TLR expression patterns as they went through different maturation stages.TLR7,TLR8 expressions on immature DC and TLR3,TLR7 expressions on mature DC were lower in CHB than in control(for TLR7,TLR8 expression on immature DC:75.9%,1.0%vs 98.4%,15.4%,P

This study aims to establish an HPLC method for simultaneous determination of gastrodin and eight nucleosides and nucleobases components in Gastrodia elata. The separation was carried out on an Agilent Zorbax Bonus-RP (4.6 mm x 250 mm, 5 μm) column with a methanol-(0.04% acetic acid) water solution gradient elution program at a flow rate of 1.0 mL x min(-1). The column temperature was 36 degrees C, and the detection wavelength was 254 nm. The volume of injection was 20 μL. The nine components including gastrodin, cytosine, uracil, cytosine, adenine, thymine, uridine, guanosine and adenosine were well separated. The calibration curve was well linear in the range of 2.04-262.00 mg x L(-1), 0.20-24.67 mg x L(-1), 0.18-23.75 mg x L(-1), 0.20-25.83 mg x L(-1), 0.20-26.67 mg x L(-1), 0.16-20.00 mg x L(-1), 0.22-27.71 mg x L(-1), 0.20-24.29 mg x L(-1), 0.24-30.58 mg x L(-1), respectively, and the correlation coefficient was between 0.998 9-0.999 9. The average recovery of gastrodin and eight nucleosides and nucleobases were 96.4%-99.6%, RSD less than 2.7% (n = 6). The contents of gastrodin in all the seven Tibet cultured Gastrodia elata samples were over 2 mg x g(-1). Further, all samples contain higher contents of adenosine, guanosine, uridine and cytidine compared to low contents of cytosine, uracil, adenine and thymine. The established method is accurate, reproducible and suitable for the determination of gastrodin and eight nucleosides and nucleobases comppnents in Gastrodia elata.
Benzyl Alcohols ; analysis ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; analysis ; Gastrodia ; chemistry ; Glucosides ; analysis ; Nucleosides ; analysis ; Nucleotides ; analysis

OBJECTIVETo investigate the relationship between the thiopurine methytransferase (TPMT) gene polymorphisms and its enzymatic activity, and to clarify the significance of TPMT activity and gene polymorphisms on individualized therapy with thiopurines.

METHODSThe TPMT activity and gene polymorphisms were determined in an unrelated population of 250 Chinese healthy blood donors, 100 cords blood and 280 patients with acute leukemia. The TPMT genotyping assay was based on polymerase chain reaction (PCR), restriction digestion of PCR products, denaturing high-performance liquid chromatography (DHPLC) and SNaPshot sequencing and direct DNA sequencing in the TPMT exon 5 (G238C), TPMT exon7 (G460A) and TPMTexon10 (A719G). Erythrocyte TPMT activity was measured by high-performance liquid chromatography (HPLC).

RESULTSThe frequency of TPMT polymorphism in 250 Chinese healthy blood donors, 100 cords blood and 280 patients with acute leukemia was low (3.5%), and all the varied alleles were TPMT* 3C (exon 10A719G). All of them were TPMT* 1/TPMT* 3C heterozygote. The TPMT activity was between 6 and 12 U. The activity in 95.1% was more than 12 U (13 - 32 U), while the activity in others (4.9%) was 6 - 12 U. TPMT activity and genotype were concordant. Of 630 subjects evaluated, TPMT activity of heterozygous individuals in Chinese healthy blood donors, cords blood and acute leukemia patients were 9.1 U, 9.3 U and 9.07 U, respectively, significantly lower than that in general population (17.6 U, 17.67 U and 18.6 U, respectively). In the samples analyzed, ten subjects with heterozygous phenotypes (6/15 acute leukemia children and 4/16 healthy blood donors and cords blood) did not have TPMT* 2, TPMT* 3A or TPMT* 3C. Therefore, other factors may affect on TPMT activity.

CONCLUSIONTPMT gene polymorphisms and its activity were concordant. The heterozygotes had low TPMT activity. Therefore, detection of TPMT genotype and its activity is useful. These findings hold a promise of improving the safety and efficacy of thiopurines therapy.

Acute Disease ; Child ; Chromatography, High Pressure Liquid ; Erythrocytes ; enzymology ; Exons ; Female ; Fetal Blood ; enzymology ; Genotype ; Humans ; Leukemia ; blood ; enzymology ; genetics ; Male ; Methyltransferases ; blood ; genetics ; Polymorphism, Single Nucleotide

BACKGROUNDThe technique of intersphincteric resection of tumors combined with coloanal anastomosis has been used to avoid permanent colostomy for patients with a rectal cancer located < 5 cm from the anal verge. This study aimed at assessing the preservation of continence function of the residual rectum and the clinical prognosis of patients with lower rectal cancer after intersphincteric resection using a prolapsing technique.

METHODSThis study included patients with the following inclusion criteria: (1) pathological evidence of rectal cancer and the tumors within distal margins located 5 cm or less from the anus by preoperative endoscopic examination; (2) no evidence by MRI of infiltration of either the external sphincter, puborectalis or the levator muscle; (3) the patients are eligible for intersphincteric resection and lower coloanal anastomosis with a preoperative biopsy showing the tumors with well-to-moderate differentiation. From January 2000 to June 2004, 23 patients with low rectal cancer were included in this study. We used the standard abdominoperineal approach to perform radical resection of tumors with excision of the mesorectum and total or part of the internal sphincters. The patients were followed for assessment of the function of the residual rectum and of cancer recurrence after the operations.

RESULTSThe median tumor distance from the anal margin was 4.5 (range 3.5 - 5.0) cm and the mean distal surgical margin 1.6 (range 1.0 - 2.0) cm. Cancer was classified into Stage I (30.4%), Stage II (47.8%), and Stage III (21.7%) according to the TNM classification. Two patients developed anastomotic fistula after the surgical resection and 2 patients (8.7%) developed later stages of anastomotic stricture at the site of coloanal anastomosis. The median follow-up period was 31.5 months (range 12 - 54) and 2 patients (8.7%) developed local recurrence. Three deaths were associated with distal organ metastasis. Twenty patients (87.0%) have maintained competence to control solid or liquid stool and the capacity of flatus continence after the surgery. Among these patients, 2 patients were able to control solid stool and occasionally lose continence of liquid stool. And only 1 patient (4.4%) has retained partial rectum function with good continence of solid stool but not liquid after the operations. Average times of defecation per day of 3, 6, 12, 24 and 36 months after the surgery were 13.1, 4.7, 3.1, 2.9, and 3.2 times/day. Anal manometer measurements showed a decrease of pressure during the resting time after intersphincteric resection and this change remained during the period of follow-up. The maximum squeeze pressure was improved after an initial decrease after the surgery.

CONCLUSIONSMore residual rectum function after the surgery may be preserved by intersphincteric resection of low rectum cancer. At the same time this technique is safe with few postoperative complication and low tumor recurrence after the surgery.

Digestive System Surgical Procedures ; methods ; Female ; Follow-Up Studies ; Humans ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Postoperative Complications ; etiology ; Prognosis ; Rectal Neoplasms ; mortality ; pathology ; physiopathology ; surgery ; Rectum ; pathology

The aim of the present study was to gain an insight into the thiopurine methytransferase (TPMT) genotyping assay, which was based on polymerase chain reaction (PCR), allele-specific PCR, restriction digestion of PCR products, denaturing high-performance liquid chromatography (DHPLC) and SNaPshot sequencing and in combination with direct DNA sequencing. Among the f our methods to test TPMT genetic SNPs based on PCR, allele specific PCR was not able to differentiate wild type from varied type. BsiYI, MwoI and AccI to digest PCR products were used so that SNP in TPMT exon 5, 7 and 10 tested. It showed that there were no differences between the results of digestion of PCR products and those of DNA sequence analysis. Therefore, this method was reliable. But some other methods were still needed to look for a compensation, because no restriction map changing resulted from the 2 SNPs in TPMT promotor was found. As to the results of DHPLC, those for the screening of TPMT exon-5 and -10 for SNPs were the same as restriction analysis of PCR products and direct DNA sequencing. But the variation of the heterozygotes in exon-7 was high, which was different from the results of direct DNA sequencing. After changing the Tm of DNA step by step, It was found that all the samples showed single peak when the temperature was 54 degrees C. But this result was unbelievable because a heterozygote in exon 7 as positive control could not be found. Therefore, it was necessary to test the sensitivity and accuracy of DHPLC, though DHPLC could be used as an effective method of SNPs screening. The results of the SNaPshot sequencing were also same as those of restriction analysis of PCR products and direct DNA sequencing. And the results showed that the bases of TPMT promoter -91 and -168 were G, instead of A and T. The results of the four methods to detect TPMT genetic SNPs based on PCR showed that SNPs analysis technique should be a combination of the techniques above-mentioned. One technique alone could not satisfy the need in clinics and research. The compensation of each other was very important.
Chromatography, High Pressure Liquid ; Exons ; Humans ; Methyltransferases ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by the accu-mulation of immature myeloid progenitor cells in the bone marrow,compromising of normal hematopoi-esis and ultimately resulting in bone marrow failure. Chemotherapy is the mainstay treatment for all AML patients,however,drug resistance and clinical relapse limits its efficacy.The 5-year survival rate of AML patients is only 26.6%.Survival rates are even lower among patients ages 65 to 74 years (5.3%)and 75 years or older(1.6%).Therefore,exploring novel therapeutic agents is urgent for improving the outcome of patients with AML. Saponins are amphipathic glycosides found in traditional Chinese medicines. In the present study, we isolated a panel of saponins from Paris forrestii (Takht.) H. Li, a unique plant found in Tibet and Yunnan provinces, China. By examining their activities in suppressing acute myeloid leukemia cell proliferation, total saponins from Paris forrestii (TSPf) displayed more potent activity than individual ones.TSPf induced more than 40% AML cell apoptosis within 24 h and decreased the viability of all leukemia cell lines. TSPf-induced apoptosis was confirmed by both Annexin V staining and caspase-3 activation.TSPf downregulated pro-survival proteins Mcl-1,Bcl-xL and Bcl-2,but upreg-ulated the expression of tumor suppressor proteins p53,p27,Bax and Beclin 1.The AKT/mTOR signaling pathway is frequently over activated in various AML cells,and TSPf was found to suppress the activa-tion of both AKT and mTOR,but had no effects on their total protein expression.This was further con-firmed by the inactivation of 4EBP-1 and p70S6K,two typical downstream signal molecules in the AKT/mTOR pathway. More specifically, TSPf-inactivated AKT/mTOR signaling was found to be associated with downregulated RNF6, a recently identified oncogene in AML. RNF6 activated AKT/mTOR, and consistently, knockdown of RNF6 led to inactivation of the AKT/mTOR pathway. Furthermore, TSPf suppressed the growth of AML xenografts in nude mice models. Oral administration of 100 mg·kg-1 body weight almost fully suppressed tumor growth within 14 d, without gross toxicity. This study thus demonstrated that TSPf displays potent anti-AML activity by suppressing the RNF6/AKT/mTOR pathway. Given its low toxicity,TSPf could be developed for the treatment of AML.

OBJECTIVETo establish a chromatography-based method for simultaneous analysis of the concentrations of amoxicillin and clavulanate potassium in human blood.

METHODSWith paracetamol as the internal control, human plasma samples, after treatment with methanol for protein sedimentation and centrifugation, were loaded for analysis with high-performance liquid chromatography (HPLC). HPLC analysis was carried out using a C18 column (5 µm, 4.6 mm×150 mm) with the mobile phase of acetonitrile-PBS (0.05 mol/L) of 10:90 (pH 2.3), UV detection wavelength of 220 nm, flow rate of 1.0 ml/min, and column temperature of 25 degrees celsius;.

RESULTSThe retention time of acetaminophen for potassium clavulanate, amoxicillin sodium and the internal control was 5.3, 7.2, and 8.5 min, respectively, and no interference by the endogenous impurities in the plasma samples was found. Amoxicillin sodium showed a good linearity within the concentration range of 0.52-4.16 µg/ml (r(2)=0.9996), and potassium clavulanate had a good linearity within the range of 0.266-2.14 µg/ml (r(2)=0.9998). The minimum detectable concentrations of amoxicillin sodium and potassium clavulanate were 0.065 µg/ml and 0.066 µg/ml, respectively. The relative recoveries of amoxicillin sodium were 95.9%-96.5% (n=5), and those of clavulanate potassium were 92.5%-98.8% (n=5); the intra- and inter-day RSD of amoxicillin sodium was 1.84%-6.4% and 2.1%-7.8%, as compared to that of potassium clavulanate of 3.57%-8.6% and 1.8%-9.1%, respectively.

CONCLUSIONThis method is simple, accurate, sensitive, specific and reproducible for analyzing the concentrations of amoxicillin and clavulanate potassium simultaneously in human plasma.

Amoxicillin ; blood ; Chromatography, High Pressure Liquid ; methods ; Clavulanic Acid ; blood ; Drug Stability ; Humans

OBJECTIVETo describe the prevalence of metabolic syndrome (MS) in Jiangsu province, using IDF 2005 and ATP III 2005 definition.

METHODSMultistage cluster sampling method was used and the participants were interviewed by trained health workers under a structured questionnaire. The number of research subjects was 5888. Fasting plasma glucose (FPG), blood lipids, body mass index, blood pressure (BP) of all sample population (5888 subjects) were measured.

RESULTSThe prevalence of MS was 17.48% by IDF 2005 definition and 21.95% by ATP lII 2005 definition. Women had higher prevalence than men (P < 0.01). The prevalence of MS increased with age. Abrupt increase of the prevalence started at age of 50 in women. The prevalence varied by education, marital status, occupation and income. People from the urban and the south had a higher prevalence than from the rural and from the northern part of the country. The most common combination of MS individual components were 1) central obesity, dislipidemia and high BP; 2) central obesity, dislipidemia, high BP and high FPG.

CONCLUSIONThe prevalence of MS was high in Jiangsu province. Clustering of MS components was common. It is necessary to discuss the cut-off points of central obesity for the waist circumference diagnostic criteria of MS in Chinese population. The importance of prevention of MS should be strengthened both by health professionals and the government.

China ; Female ; Humans ; Male ; Metabolic Syndrome ; epidemiology ; Middle Aged ; Obesity, Abdominal ; epidemiology ; Prevalence ; Waist Circumference

OBJECTIVETo compare the significance of the application in Jiangsu population using the diagnostic criteria for metabolic syndrome(MS) proposed by the IDF in 2005, ATP III in 2005 and CDS in 2004.

METHODSBased on the populations in Jiangsu province from a project of Multiple Metabolic Disorders and MS, the study was conducted including 5888 cases, with data of plasma glucose, lipid profile, blood pressure, serum insulin etc. MS was diagnosed and compared according to these three definitions respectively.

RESULTSThe age-adjusted MS prevalence rates were 17.48%, 21.95% and 9.59% according to the IDF(2005), ATP III (2005) and CDS (2004) respectively. The agreement in the diagnosis of MS using ATP III (2005) and CDS(2004) definitions was 85.11%, and the agreement in the diagnosis of MS using IDF(2005) and CDS definitions was 87.35%. The agreement in the diagnosis of MS using IDF (2005) and ATP III (2005) definitions was 95.14%. The MS subjects diagnosed by the ATP III (2005) was 1.26 higher than subjects diagnosed by the IDF(2005) definition. The ratios of prevalence rates of high waist circumference(WC), MS_IDF (2005) and MS_ATP III (2005) was 2.17, 1.99 and 1.54 in sex ratio (woman to man).

CONCLUSIONThe agreement in the diagnosis of MS using the IDF(2005) and ATP III (2005) definition was higher than using CDS(2004) and other two definitions. For diagnosing MS, the cut off of WC in IDF(2005) and ATP III (2005) seemed not appropriate and the diagnostic criteria used for ATP III (2005) (waist circumference of man 85 cm, woman 80 cm) could identify more MS.

Blood Glucose ; Blood Pressure ; China ; Humans ; Insulin ; blood ; Lipids ; blood ; Metabolic Syndrome ; diagnosis ; Reference Values ; Waist Circumference