Objective To study the changes of somatostatin(SOM) in plasma and cerebrospinal fluid (CSF) of children with convulsive diseases.Methods Sixty-seven children with convulsive diseases were studied as following:obtaining the samples of plasma in the 1st and 7th day after being in hospital,and the samples of CSF in the 1st after being in hospital.We investigated the changes of SOM in plasma and CSF with radioimmunoassay(RIA).Results 1.Convulsive group:the concentration of SOM in plasma in the 7th day(29.47?9.40 ng/L) was significant lower than that in the 1st day(39.23?11.00 ng/L)(t=21.530 P0.05).The concentration of SOM in plasma in the 1st day in control group was(19.58?6.04) ng/L.There were significant differences in convulsive group and encephalitis group without convulsion, control group(t= 6.847,7.921 P

OBJECTIVETo investigate the role of CD40 ligand (CD40L) in dendritic cells (DC) of CEA transgenic mice and to evaluate the specific cellular immunity induced by activated DC.

METHODSBone marrow cells of the CEA transgenic mice were used to generate immature dendritic cells under the condition of GM-CSF and IL-4. CD40L was added to activate dendritic cells into mature phenotype. Dendritic cells cancer vaccine was pulsed with CEA526-533 peptide which made the vaccine specific for cancer immunity. The immunophenotype molecules were identified by flow cytometry. The cytokines produced by cells were determined by ELISA. T cells proliferation was measured by (3)H-thymidine essays.

RESULTSImmunophenotype molecules expressions of CD40L-activated dendritic cells were significantly higher than those in control group. IL-12 secretion by CD40L-activated dendritic cells was (937.81+/-51.99) pg/10(6) DC, significantly higher than that in control group [(83.06+/-8.58) pg/10(6) DC, P<0.01]. CD8(+) T cells proliferation induced by CD40 L-activated dendritic cells was stronger as compared to control group (P<0.05), and the secretion of IFN-gamma was(33.900+/-4.550) ng/L, significantly higher than that in control group [(5.226+/-0.460) ng/L, P<0.01]. Splenocytes proliferation induced by CD40 L-activated dendritic cells was stronger as compared to control group (P<0.01), and the secretion of IFN-gamma was (69.802+/-11.407) ng/L, significantly higher than that in control group [(2.912+/-0.562) ng/L, P<0.01].

CONCLUSIONThe method of using CD40L to stimulate bone marrow-delivered dendritic cells promotes the maturation and activation of dendritic cells, which enhances the cellular immunity in CEA transgenic mice.

Animals ; CD40 Ligand ; immunology ; physiology ; Dendritic Cells ; cytology ; immunology ; metabolism ; Immunity, Cellular ; immunology ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic

Actins ; immunology ; metabolism ; Antibodies, Monoclonal ; metabolism ; Child ; Diagnosis, Differential ; Fibroma ; metabolism ; pathology ; surgery ; Follow-Up Studies ; Gastrointestinal Neoplasms ; metabolism ; pathology ; Gastrointestinal Stromal Tumors ; metabolism ; pathology ; Humans ; Leiomyoma ; metabolism ; pathology ; Male ; Pyloric Antrum ; pathology ; Stomach Neoplasms ; metabolism ; pathology ; surgery ; Vimentin ; metabolism

OBJECTIVETo evaluate the clinical performance of a probe melting analysis (PMA)-based real-time PCR detection kit in rapid detection of rifampin-resistant mutations in Mycobacterium tuberculosis (MTB).

METHODSThe specificity of the assay was evaluated by detecting 37 non-tuberculous mycobacteria (NTM), and the detection limit of the method was evaluated by genomic DNA of a standard strain H37Rv. Finally, 962 clinical isolates were analyzed with the PMA assay by detecting mutations in rifampin resistance-determining region (RRDR) of rpoB gene, and results were verified with DNA sequencing.

RESULTSAmong 37 NTM strains, three strains showed drug resistant mutation signals. The PMA method could detect down to 30 bacteria per reaction. Sample analysis showed that 186 of 962 isolates were mutants, 751 isolates were wild type and 25 isolates failed to give amplification signals. Among the mutant samples detected, 112 samples from November 2009 to April 2010 were further analyzed by sequencing, as well as 200 wild-type samples. The results showed a complete agreement with the PMA assay except for 5 samples failed in sequence analysis.

CONCLUSIONThe PMA assay is rapid, accurate and easy-to-use, and thus can be used for detection of rifampin-resistant in clinical isolate samples.

Bacterial Proteins ; genetics ; Base Sequence ; DNA Mutational Analysis ; DNA, Bacterial ; genetics ; DNA-Directed RNA Polymerases ; Genotype ; Limit of Detection ; Microbial Sensitivity Tests ; Molecular Sequence Data ; Mutation ; Mycobacterium tuberculosis ; genetics ; isolation & purification ; Polymerase Chain Reaction ; methods ; Reagent Kits, Diagnostic ; Sensitivity and Specificity

OBJECTIVETo investigate the apoptosis induced by manumycin in U937 and HL-60 cell lines, and to explore the role of mitochondria apoptotic pathway in manumycin-inducing apoptosis.

METHODSLeukemic cells line U937 and HL-60 were treated by manumycin at 2 micromol/L for different time. Apoptosis of leukemia cells was detected by flow cytometry. The cytosolic proteins were extracted using a digitonin buffer. The protein expression of cytochrome C, caspase-9, caspase-8, and caspase-3 were determined by western blot. Mitochondrial membrane potential was detected by JC-1.

RESULTSIn U937 and HL-60 cells, manumycin induced mitochondrial depolarization after 6 h treatment. The average red/green fluorescence ratios at 6 h were significantly (P < 0.01) lower than those at time 0, being 0.51 +/- 0.07 and 0.41 +/- 0.06 for control group respectively. Manumycin induced cytochrome C release from the mitochondria into the cytosol after 6 h treatment, and activated caspase-9, caspase-8, and caspase-3 after a 16h treatment. The broad-spectrum caspase-inhibitor Z-VAD-fmk at 50 micromol/L was able to inhibit caspase cleavage completely, but only reduced the manumycin-induced apoptosis rates by 51.69% and 56.47% in U937 and HL-60, respectively.

CONCLUSIONManumycin induced apoptosis in U937 and HL-60 cell lines via mitochondria apoptotic pathway.

Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Caspase 9 ; metabolism ; Cytochromes c ; metabolism ; HL-60 Cells ; Humans ; Mitochondria ; drug effects ; metabolism ; physiology ; Polyenes ; pharmacology ; Polyunsaturated Alkamides ; pharmacology

OBJECTIVETo construct plasmid expressing pacemaker gene pIRES2-EGFP-HCN2 and study its effects in transfected atrial myocytes in vitro and in canine model of sick sinus syndrome (SSS).

METHODSmHCN2 gene was isolated from PTR plasmids and cloned into eukaryotic expression plasmid pIRES2-EGFP. Recombinant plasmids pIRES2-EGFP-HCN2 was transfected with by electroporation into neonatal atrial cardiomyocytes or injected to the sinoatrial (SA) region of canines with SSS induced by catheter and chemical ablation. pIRES2-EGFP-HCN2 expression was detected under fluorescence microscope and confirmed by reverse transcription-polymerase chain reaction (RT-PCR). Spontaneous beating rate in atrial cardiomyocytes was detected with light microscope.

RESULTSEGFP expression was seen in transfected atrial cardiomyocytes 24 to 48 hours after transfection and the spontaneous beating rate was significantly increased than that in non-transfected atrial cardiomyocytes [(180 +/- 11) bpm vs (140 +/- 14) bpm, P < 0.05]. Heart rate was significantly increased 24 hours post recombinant plasmids pIRES2-EGFP-HCN2 injection compared to saline injection in canines with SSS [(150 +/- 13) bpm vs (105 +/- 17) bpm, P < 0.05]. Green fluorescence was also detected in frozen SA tissue sections of canines injected with recombinant plasmids pIRES2-EGFP-HCN2 and the production amplified by RT-PCR was about 300 bp which is consistent with mHCN2 gene fragment.

CONCLUSIONThe recombinant eukaryotic expression plasmid pIRES2-EGFP-HCN2 can improve pacing function in atrial myocytes and in canine model of SSS.

Animals ; Disease Models, Animal ; Dogs ; Gene Expression ; Gene Transfer Techniques ; Genetic Therapy ; Genetic Vectors ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels ; In Vitro Techniques ; Ion Channels ; genetics ; Myocytes, Cardiac ; metabolism ; Plasmids ; Sick Sinus Syndrome ; therapy

OBJECTIVETo investigate the detection limit of multicolor combinational probe coding real-time PCR (MCPC-PCR) in detection of Salmonella and Staphylococcus aureus suspended in the food samples, and to apply MCPC-PCR to detect the samples of food poisoning.

METHODSSeries concentration of bacterium suspension (10(1) - 10(9) CFU/ml) was prepared by using 22 simulated samples including fresh meat and cakes and then MCPC-PCR was applied to detect Salmonella and Staphylococcus aureus in 22 samples. Enrichment broth of 101 frozen samples and 5 early patients' anal swabs in food poisoning cases were detected after the DNA samples were extracted.

RESULTSThe limits of MCPC-PCR assay in detecting Salmonella and Staphylococcus aureus were about 10(2) copies/test; 101 frozen enrichment broth of samples in food poisoning cases were detected by MCPC-PCR assay, of 23 positive samples, 18 were confirmed by bacteriology techniques; 96 samples detected by MCPC-PCR and bacteriology techniques had the same results, and the coincidence rate was 95.05%. Anal swabs, collected from 5 of early patients in a food poisoning case gave a clue to be Vibrio parahaemolyticus by MCPC-PCR assay and then were perfectly consistent with bacteriology assay.

CONCLUSIONAs a method of high sensitivity and good specificity, MCPC-PCR assay can quickly and conveniently detect multiple pathogens existing in food samples, therefore we recommend it to be used in rapidly screening or simultaneous detection of food-borne diseases.

Bacteriological Techniques ; methods ; Food Contamination ; analysis ; Food Microbiology ; Molecular Probe Techniques ; Molecular Sequence Data ; Polymerase Chain Reaction ; methods ; Salmonella ; genetics ; isolation & purification ; Sensitivity and Specificity ; Staphylococcus aureus ; genetics ; isolation & purification

OBJECTIVETo investigate clinical results of percutaneous reduction and hollow screw internal fixation for the treatment of calcaneal fractures, and to compare therapeutic effects between close reduction hollow screw internal fixation and open reduction plate internal fixation.

METHODSFrom August 2007 to May 2010, 53 patients with calcaneal fractures were retrospectively analyzed. All the patients were divided into two groups, 25 patients in group A (PR group) treated with percutaneous reduction and hollow screw internal fixation, including 17 males and 8 females, with an average age of (39.4 +/- 9.9) years. While 28 patients in group B (OR group) treated with open reduction and plate internal fixation, including 18 males and 10 females, with an average age of (38.6 +/- 10.2) years. According to Sanders classification, there were 18 patients with type II fractures, 29 patients with type III and 6 type IV. In both groups, operative time, blood loss, postoperative complications and radiology were recorded. Functional recovery was evaluated by Maryland score.

RESULTSAll the patients were followed up, and the duration ranged from nine months to thirty-five months (averaged 20.4 months). There were no significant differences in sex, age, fracture type, fracture classification, initial Böhler angle, or late complications between the two groups. But significant difference can be seen between operative time, blood loss, and skin complications (in group A no nonunion and skin complications occurred, but subtalar posttraumatic arthritis occurred in 1 case; in group B, 3 patients had complications of skin necrosis, 1 patient suffered from a delayed union due to large defect filled with artificial bone, and 1 patient got subtalar posttraumatic arthritis). No difference were found in the latest X-ray film. According to Maryland score, in group A, 8 got an excellent result and 12 good. In group B, 10 got an excellent and 14 good. There were no significant differences between the two groups in Margland score.

CONCLUSIONThe results of this study suggest that in comparison with open reduction, percutaneous reduction and hollow screw internal fixation minimizes complications and achieves good results. Further study of this technique is needed.

Adolescent ; Adult ; Aged ; Bone Screws ; Calcaneus ; surgery ; Case-Control Studies ; Female ; Follow-Up Studies ; Fracture Fixation, Internal ; instrumentation ; methods ; Fractures, Bone ; surgery ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Treatment Outcome ; Young Adult

The study was aimed to investigate the expression of HLA class I molecules and MHC class I chain-related molecules A/B (MICA/MICB) in K562 and adriamycin (ADM)-resistant K562 cell lines (K562/AO2) and their effect on cytotoxicity of NK cells. Expression of HLA class I molecules and MICA/MICB on the surface of K562 and K562/AO2 cell lines were analyzed by flow cytometry. Cytotoxicity of NK cells (isolated from 3 healthy persons) against K562 and K562/AO2 cells were detected by LDH releasing assay at different effect-to-target cell ratios (E:T). In blocking experiments, anti-MHC class I monoclonal antibody (McAb) (W6/32, a pan anti-HLA class I antibody) and anti-MHC class I chain-related molecules McAb (BAMO-1, specifically against MICA and MICB) were added to the target cells at E:T of 10:1. The results showed that the expression of MHC class I chain-related molecules on K562 was higher than that on K562/AO2 (P=0.000), and HLA class I molecules were not detectable on both cells. Cytotoxicities of NK cells against K562 and K562/AO2 cells were (29.32 +/- 0.12)%, (45.33 +/- 0.78)%, (58.37 +/- 0.87)%, (72.37 +/- 0.96)% and (12.47 +/- 0.91)%, (24.36 +/- 1.11)%, (33.29 +/- 1.03)%, (53.87 +/- 1.27)% at E:T ratios of 5:1, 10:1, 20:1 and 30:1 respectively (P=0.000), the cytotoxicity of NK cells on K562 cells was significantly higher than that on K562/A02 cells at different E:T ratios. Blocking experiments confirmed that at E:T of 10:1 killing of NK cells against K562 and K562/AO2 cells was efficiently inhibited by BAMO-1, whereas W6/32 had no effect on K562 and K562/AO2 cells. It is concluded that the expression of MHC class I chain-related molecules on K562 and K562/AO2 cells is correlated with NK cell-mediated lysis. NK cells display higher cytotoxicity against parental K562 cells than multi-drug resistant K562/AO2 cells. Down-regulation of MICA/B in multi-drug resistant tumor cell lines leads to reduction of susceptibility to NK lysis.
Cytotoxicity, Immunologic ; immunology ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; immunology ; Genes, MHC Class I ; genetics ; Histocompatibility Antigens Class I ; immunology ; Humans ; K562 Cells ; Killer Cells, Natural ; immunology

OBJECTIVETo investigate cytotoxicity and genotoxicity of benzo(a)pyrene (B(a)P) by 16HBE-CYP1A1 cells which are human bronchial epithelial cell with CYP1A1 transformed.

METHODSExpression of CYP1A1 and mEH of cell models were tested by real-time quantitative polymerase chain reaction. Cells were treated with 0, 1, 5, 10 and 20 micromol/L B(a)P for 24 h. Adverse effects of B(a)P were tested by cytokinesis-block micronucleus (CBMN) cytome assays. Cytotoxicity was assessed by the nuclear division index (NDI), frequency of necrotic and apoptotic cells. Genetic damages were assessed by frequencies of CBMN, nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs).

RESULTSHigh levels of CYP1A1 and mEH were found in 16HBE-CYP1A1 cells (relative mRNA content was 7.8 x 10(-4) and 0.030 respectively). In 16HBE-CYP1A1 cells, NDI were decreased in 1, 5, 10 and 20 micromol/L B(a)P treated groups, 1.92 +/- 0.04, 1.71 +/- 0.01, 1.61 +/- 0.04, and 1.41 +/- 0.01, respectively; and lower than control group (2.08 +/- 0.03). Compared with control group ((82.67 +/- 6.66)%), the binucleated cells ratios were decreased, (76.33 +/- 3.51)%, (66.33 +/- 0.58)%, (51.67 +/- 1.53)% and (39.0 +/- 1.0)% respectively.Necrotic cells ratios were (1.93 +/- 0.42)%, (2.20 +/- 0.53)%, (8.07 +/- 0.90)% and (15.27 +/- 2.80)%, respectively, higher than control group ((0.47 +/- 0.11)%). The differences were significant (F values were 899.94, 303.33, 240.87, P < 0.01). Apoptotic cells were increased at lower groups and decreased to normal at higher groups treated by B(a)P. They were (1.20 +/- 0.53)%, (2.00 +/- 0.20)%, (1.47 +/- 0.12)%, (1.20 +/- 0.00)% and (1.20 +/- 0.00)%, respectively. Analysis on biomarkers of genetic damage, the significant dose-effect relationship were observed in NPBs and NBUDs (F values were 50.23, 121.09, P < 0.01, respectively). Frequencies of NPBs were (4.67 +/- 2.89) per thousand, (7.33 +/- 1.53) per thousand, (10.67 +/- 2.08) per thousand and (11.00 +/- 1.00) per thousand respectively. Frequencies of NBUDs were (2.33 +/- 0.58) per thousand, (4.00 +/- 1.00) per thousand, (5.00 +/- 1.00) per thousand, and (7.67 +/- 1.16) per thousand respectively. However, the dose-relationship of CBMN last only to 10 micromol/L B(a)P treated groups in 16HBE-CYP1A1 cells, and frequencies of CBMN were (8.33 +/- 3.21) per thousand, (14.67 +/- 1.15) per thousand, respectively. Frequency of CBMN was (16.67 +/- 2.88) per thousand in 20 micromol/L B(a)P treated group, lower than 10 micromol/L B(a)P treated group ((17.67 +/- 2.08) per thousand). In 16HBEV control cells, the cytotoxicity was found only in higher B(a)P treated groups and frequencies of CBMN, NPBs and NBUDs were increased also. While no significant differences were observed between 5, 10, 20 micromol/L B(a)P treated groups (they were (6.37 +/- 2.08) per thousand, (9.33 +/- 1.52) per thousand, (9.33 +/- 3.21) per thousand; (4.33 +/- 1.53) per thousand, (6.00 +/- 2.65) per thousand, (5.33 +/- 1.53) per thousand and (2.33 +/- 0.58) per thousand, (3.33 +/- 1.16) per thousand, (3.67 +/- 1.16) per thousand, respectively).

CONCLUSIONSThe genetic damages were more severe after treated with activated B(a)P, which may be induced by decreased NDI, increased necrotic cells and inhibition of apoptosis.

Apoptosis ; drug effects ; Benzo(a)pyrene ; toxicity ; Cell Division ; drug effects ; Cell Line, Transformed ; DNA Damage ; Humans ; Micronuclei, Chromosome-Defective