Adult ; Female ; Humans ; Motor Neuron Disease ; complications ; Neck ; pathology ; Thyroid Neoplasms ; complications

OBJECTIVE: To establish a method for simultaneous determination of voriconazole and its main metabolite N-oxidized voriconazole in blood, and monitor the levels of voriconazole and N-oxidized voriconazole in leukemia children aged 2-12 years. METHODS: Agilent ZORBAX SB-C18 column (4.6 mm×250 mm,5 μm) was used, and the mobile phase was acetonitrile, methanol and ammonium acetate buffer (10 mmol•L-1, pH adjusted with acetic acid to 3.0)=27∶23∶50, the flow rate was 1 mL•min-1, and the wavelength was set at 262 nm. The sample was extracted with ethyl acetate.Carbamazepine was used as the internal standard. RESULTS: The calibration curves of voriconazole and N-oxidized voriconazole were linear in the range of 0.1-8.0 μg•mL-1, the lower limit of quantitation was 0.1 μg•mL-1, and the intra-assay and inter-assay precision RSDs were between 2% and 10%. The accuracy, method recovery and stability all met the requirements.Twenty-nine patients were monitored with a total of 100 blood samples.The medians (IQRs) of voriconazole concentration, N-oxidized voriconazole concentration, and ratio of N-oxidized voriconazole to voriconazole were 0.87 μg•mL-1 (0.32-1.65 μg•mL-1) and 1.15 μg•mL-1 (0.55-1.67 μg•mL-1) and 1.10(0.66-1.22), respectively. There was a correlation relationship between the concentrations of voriconazole and N-oxidized voriconazole (r=0.603 1, P<0.000 1). CONCLUSION: The method is simple, accurate, highly specific, and can simultaneously monitor the concentrations of voriconazole and N-oxidized voriconazole, thus can be used for the pharmacokinetic research of voriconazole and clinical drug monitoring.

OBJECTIVETo evaluate the inhibitory effect of recombinant adenovirus carrying human endostatin gene (Ad-endo) on the growth of human pancreatic carcinoma xenograft in nude mice.

METHODSThe expression of endostatin in human pancreatic carcinoma Capan-2 cells was examined by RT-PCR after infection with Ad-endo. The supernatants of Capan-2 cells were collected after 48 h of infection with Ad-endo as the conditioned medium for human umbilical vein endothelial cells (HUVECs), whose proliferation in vitro was assayed. Capan-2 cell xenografts were established to determine the antitumoral effects of Ad-endo in vivo. The intratumoral microvessel density (MVD) was evaluated using CD31 staining.

RESULTSThe expression of endostatin gene was detected by PT-PCR in infected Capan-2 cells. The conditioned medium from Ad-endo-infected cells significantly inhibited HUVEC proliferation (P<0.05). Ad-endo significantly suppressed the growth of Capan-2 tumor xenografts in nude mice (P<0.05), and the MVD decreased significantly in the treated tumor (P<0.05) as compared with that in the control group.

CONCLUSIONAdenovirus carrying human endostatin gene produces inhibitory effects on the growth of human pancreatic carcinoma tumors in nude mice.

Adenoviridae ; genetics ; metabolism ; Angiogenesis Inhibitors ; metabolism ; pharmacology ; Animals ; Endostatins ; biosynthesis ; genetics ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Neovascularization, Pathologic ; genetics ; Pancreatic Neoplasms ; blood supply ; pathology ; therapy ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology

Kringle 1-3 domain is a recently found angiogenesis inhibitor with anti-angiogenesis and anti-tumor activity. The kringle 1-3 gene was amplified by PCR technique using angiostatin gene as template. After DNA sequencing, the PCR product was cloned into pPIC9K resulting in recombinant plasmid pPIC9K13 which was then transformed into Pichia pastoris GS115. The high copy integration transformants screened by PCR and G418 methods were cultivated in flasks. The K1-3 was expressed and secreted to the medium and has immunogenic activity as shown by SDS-PAGE and Western blotting. High cell density culture was carried out in 30-liter and 80-liter bioreactor, the biomass reaches 300 OD after methanol induction, and the expressed product is 200 mg/L. The fermentation supernatant was purified by Streamline SP and Phenyl Sepharose Chromatography, the product appears as a single band on SDS-PAGE, with a purity of 95%-96%. The purified product has anti-angiogenesis and anti-tumor activity.
Bioreactors ; Cloning, Molecular ; Fermentation ; Humans ; Kringles ; genetics ; Pichia ; genetics ; Plasmids ; Plasminogen ; genetics ; isolation & purification ; pharmacology ; Recombinant Proteins ; biosynthesis ; isolation & purification ; pharmacology

BACKGROUNDIt is still unclear whether viral genetic variability influences response to interferon (IFN)-alpha treatment. Recent reports suggest that IFN-alpha effects may be associated with hepatitis B virus (HBV) post-transcriptional regulation. This study was designed to explore the heterogeneity of HBV post-transcriptional regulatory elements (HPRE) and the relationship between the diversity of HPRE and the response to IFN-alpha treatment.

METHODSThe HPRE sequences from 31 Chinese patients infected with HBV were determined by directly sequencing of polymerase chain reaction (PCR) product, and comparing them to those from Caucasian patients. Subsequently, eukaryotic expression vectors containing HPRE at various points were constructed and transfected into HepG2 cells, which were then exposed to recombinant human cytokines.

RESULTSThe T to C point mutation at nt 1504 and the C to T (G) at nt 1508 in HPRE were found in 21 and 19 patients with chronic hepatitis B, respectively; the C to T point mutation at nt 1509 was found in 17 patients. These point mutations did not exist in the HPRE of the Caucasian patients. The activity of the CAT gene obviously increased in the case of T to C point mutation at nt 1504, but did not change in the case of the C to T (G) mutations at nt 1508 and 1509. The activity of the CAT gene at these point mutations of HPRE could be inhibited by IFN-alpha/gamma and tumor necrosis factor (TNF)-alpha except for the point mutations at nt 1508 of HPRE which may escape the suppression role of IFN-alpha on HPRE.

CONCLUSIONSThere are point mutations between the HPRE of Chinese and Caucasian HBV patients, which might be correlated with response to IFN-alpha. The variation of HPRE might affect the function of HPRE and influence the regulative function of IFN-alpha other than that of IFN-gamma or TNF-alpha on HPRE.

Chloramphenicol O-Acetyltransferase ; metabolism ; Genes, Regulator ; Hepatitis B virus ; drug effects ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Interferon-alpha ; pharmacology ; Interferon-gamma ; pharmacology ; Plasmids ; Point Mutation ; Tumor Necrosis Factor-alpha ; pharmacology

Objective To evaluate the effect of PTH gene polymorphisms on bone mineral density (BMD) at multiple skeletal sites in normal females.Methods PTH gene phenotype was determined by PCR-RFLP of restriction enzyme Bst BⅠin 596 females aged (46.3?13.7) years (20-80 years),and PCR products with or without enzymolytic site were considered as genotype B or genotype b respectively.BMDs of the anteropesterior spine (AP) and supine lateral spine (Lat) of lumbar vertebrae (L_1-L_4),femoral neck (FN),total hip (T-hip), Ward's triangle (Ward),Trochanter (Troch),forearm [radius+ulna ultradistal (RUUD) and total area of radius + ulna (RUT) ] were measured by DEXA (QDR4500A).Results (1) Hardy-Weinberg equilibrium was evident for PTH polymorphisms.The frequencies of genotype were BB 0.784,Bb 0.208,bb 0.008 and frequencies of alleles B,b were 0.888 and 0.112 respectively in 596 normal females.Frequencies of BB,Bb,bb genotypes were 0.781,0.210,and 0.009 respectively in 347 postmenopausal women and their frequencies of alleles B,b were 0.886,0.114.No significant difference was found between post- and premenopausal women in genotype frequen- cy.(2) Comparing their BMDs of AP,Lat,FN,T-hip,Ward,Troch,RUUD and RUT,there was no significant difference between BB and Bb genotypes of pre- and postmenopansal women groups.(3) Logistic regression analysis failed to show any statistical difference between normal and osteoporosis women with regard to PTH phenotype.Conclusion PTH gene polymorphism has little effect on BMD in normal females.

Objective To investigate the expressions of human telomerase reverse transcriptase(hTERT) mRNA and protein in pheochromocytoma and paraganglioma and their significance as diagnostic markers in predicting the biological behaviour of these tumours.Methods Expression of hTERT mRNA was determined by in situ hybridization in 45 pheochromocytomas/paragangliomas(31 benign,7 suspected malignant and 7 malignant) and 9 normal adrenal medulla samples,hTERT protein was determined by immunohistoebemistry.Results hTERT mRNA was expressed in 5/7 malignant turnouts and 5/7 suspected malignant tumours as compared with 3/31 benign tumours(P

The quantitative identification and enrichment of viable regulatory T cells (Treg) requires reliable surface markers that are selectively expressed on Treg. Foxp3 is the accepted marker of natural Treg, but it cannot be used to isolate cells for functional studies. CD127 is a new surface marker expressed in Treg cells. In this study, two populations of Treg, including CD4(+)CD25(+)CD127(low/-) and CD4(+)CD25(+)Foxp3(+)T cells, and profiles of the Foxp3 expression in CD4(+)CD25(+)CD127(low/-) cells were compared to evaluate which population is better. The peripheral blood cells were collected and spleen suspension of BALB/C mice were prepared, and using triple staining CD4, CD25, CD127 and CD4, CD25, Foxp3. The profiles of Treg, including CD4(+)CD25(+)CD127(low/-) and CD4(+)CD25(+)Foxp3(+) were detected by flow cytometry. The quadruple staining CD4, CD25, Foxp3 and CD127 were used to determine the CD127 expression in CD4(+)CD25(+)Foxp3(+) cells. The results showed that on T cell subset the median expression levels of CD4(+), CD4(+)CD25(+) were 39.02%, 5.35% in peripheral blood and 23.49%, 3.86% in spleen. On CD4(+) T cell subset, the median expression level of CD4(+)CD25(+)CD127(low/-) and CD4(+)CD25(+)Foxp3(+)T cells were 7.13%, 3.97% in peripheral blood and 12.8%, 8.23% in spleen. The ratio of CD4(+)CD25(+)CD127(low/-) T cells was higher than that of CD4(+)CD25(+)Foxp3(+) cells in both peripheral blood and spleen cells (P < 0.01). The CD4(+)CD25(+)CD127(low/-) cells highly expressed Foxp3, while the CD4(+)CD25(+)Foxp3(+)T cells lowly expressed CD127. It is concluded that compared with the CD4(+)CD25(+)Foxp3(+) populations, CD4(+)CD25(+)CD127(low/-) T cells better fit the definition of naturally occurring regulatory T cells in peripheral blood cells and spleen of BALB/C mice. CD127(low/-) is a characteristic marker on surface of CD4(+)CD25(+) Treg cells, and has been confirmed to be more specific marker for quantitatively sorting Treg cells.
Animals ; Biomarkers ; blood ; Female ; Interleukin-7 Receptor alpha Subunit ; analysis ; Mice ; Mice, Inbred BALB C ; Spleen ; cytology ; T-Lymphocytes, Regulatory ; metabolism

BACKGROUNDMany researchers suggest that adult mammalian central nervous system (CNS) is incapable of completing self-repair or regeneration. And there are accumulating lines of evidence which suggest that endogenous neural stem cells (NSCs) are activated in many pathological conditions, including stroke in the past decades, which might partly account for rehabilitation afterwards. In this study, we investigated whether there was endogenous neural stem cell activation in intracerebral hemorrhagic (ICH) rat brains.

METHODSAfter ICH induction by stereotactical injection of collagenase type VII into globus pallidus, 5-Bromo-2 Deoxyuridine (BrdU) was administered intraperitoneally to label newborn cells. Immunohistochemical method was used to detect Nestin, a marker for neural stem cells, and BrdU.

RESULTSNestin-positive or BrdU-Labeled cells were predominantly located at 2 sites: basal ganglion around hemotoma, ependyma and nearby subventricular zone (SVZ). No positive cells for the 2 markers were found in the 2 sites of normal control group and sham group, as well as in non-leisioned parenchyma, both hippocampi and olfactory bulbs in the 4 groups. Nestin+ cells presented 4 types of morphology, and BrdU+ nucleus were polymorphologic. Positive cell counting around hemotoma showed that at day 2, Nestin+ cells were seen around hemotoma in model group, the number of which increased at day 4, day 7 (P <0.01), peaked at day 14 (P <0.05), and reduced significantly by day 28 (P <0.01).

CONCLUSIONEndogenous neural stem cells were activated in experimental intracerebral hemorrhagic rat brains.

Animals ; Brain ; pathology ; Bromodeoxyuridine ; metabolism ; Cerebral Hemorrhage ; pathology ; Intermediate Filament Proteins ; analysis ; Male ; Nerve Tissue Proteins ; analysis ; Nestin ; Neurons ; pathology ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; pathology