Objective To investigate the effects of dengue type 2 virus(DENV-2)on the apopto-sis and autophagy of primary human hepatic sinusoidal endothelial cells(HHSECs)and the expression of ICAM-1 and Beclin-1 at mRNA level and to analyze the possible pathogenic mechanism of DENV-2. Meth-ods Immunohistochemistry(IHC)and flow cytometry analysis(FCM)were performed to identify HHSECs by detecting factor Ⅷ and CD31. The DENV-2 strain was identified by using PCR and HindⅢ. The 50%tissue culture infective dose(TCID50 )of DENV-2 was calculated after infecting C6 / 36 cells with DENV-2. Dynamic changes of DENV-2 NS1 were measured by real-time PCR after infecting HHSECs with DENV-2. CCK-8 was used to dynamically detect the cytotoxicity of DENV-2 to HHSECs. The transcriptional levers of Beclin-1 and ICAM-1 in DENV-2-infected HHSECs were detected by real-time PCR. FCM was performed to analyze the apoptosis of HHSECs and the expression of LC3B and ICAM-1. Results The cells in the exper-imental group were stained brown by DAB and the positive expression rate of CD31 reached 87. 1% . The TICD50 of DENV-2 to C6 / 36 cells was 10-6. 845 / 0. 1 ml. Compared with the uninfected cells,partial se-quences of NS1 gene were expressed in DENV-2-infected HHSECs. DENV-2 suppressed the cell activities of HHSECs. The suppression rates of DENV-2 to HHSECs at 12 h,24 h,36 h and 48 h were respectively (10. 90±1. 24)% ,(16. 40±0. 42)% ,(17. 00±0. 46)% and(29. 60±0. 26)%(P﹤0. 05). The tran-scriptional levels of Beclin-1 and ICAM-1 in HHSECs were significantly increased at the time point of 24 h after DENV-2 infection,the 2-△△Ct values of which were 46. 77±2. 55 and 40. 97±4. 91,respectively. The expression of LC3B and ICAM-1 in DENV-2-infected HHSECs were increased,the peaks of which were reached at 24 h(14. 7% )and 36 h(35. 5% ),respectively. The apoptosis of DENV-2-infected HHSECs was remarkably enhanced at 12 h with an apoptosis rate of 13. 17% . Conclusion HHSECs was susceptible to DENV-2. DENV-2 induced the upregulation of ICAM-1 and the activation of HHSECs. Moreover,autoph-agy and apoptosis of HHSECs could also be induced by DENV-2.

Objective:To reveal the primary mechanism of changing permeability in DENV-2 infected pHDMECs. Methods:pHDMECs was incubated by DENV-2 on the concentration of 103 TCID50 ,and the penetrability of the cell was detected by Transwell at 4,8,12,24,48 h,respectively. Then,the partial sequence of DENV-2 NS1 was analyzed by Real time-PCR,and NS1 protein was detected by immunofluorescence and flow cytometer (FCM). The apoptosis rate of pHDMECs was assayed by FCM. Finally,IL-6 and IL-8 secreted by pHDMECs were analyzed by Real time-PCR and double antibody sandwich ELISA. Results:The relative expression of NS1 gene was elevated but NS1 protein was not detected;the permeability of DENV-2 infected pHDMECs had dramatically increased both at 24,48 h,but the apoptosis rate has little changed even been influenced by DENV-2 at 72 h. However,the relative expression of IL-6/IL-8 mRNA was boosted at 8,24 h[(2. 49±0. 50) and (6. 82±1. 69) fold,respectively,P<0. 05]. In protein level,compared with control(869. 6±50. 70)pg/ml,IL-6 secreted by DENV-2 infected pHDMECs could reach by(1 248. 8±86. 9)pg/ml(P<0. 05),and IL-8 was(1 331. 0±86. 3)pg/ml(P<0. 05) while the control was (967. 6±156. 6)pg/ml. Conclusion:Indeed,pHDMECs can be infected by DENV-2;the increasing permeability of DENV-2 infected pHDMECs may not be caused by the pHDMECs′ apoptosis but the enhancing of pro-inflammatory cytokine IL-6 /IL-8.

Objective To study the SLC26A4 mutations in children with non -syndromic hearing loss by ge-netic testing method ,for the purpose of investigating etiology and mutation regularity of hearing loss ,and to provide basic information for the molecular diagnosis of hearing loss .Methods Blood samples and clinical data of 137 spo-radic cases with non -syndromic hearing loss and 126 normal controls were collected .The SLC26A4 gene of the pa-tients and normal controls were amplified by polymerase chain reaction (PCR) ,then subjected to automatic DNA se-quencing .Results Pathologic SLC26A4 mutations were identified in 23 out of 137 patients ,and in 23 out of 119 bi-lateral deafness ,mutate rate were 16 .79% and 19 .33% ,respectively .SLC26A4 mutations were identified in 19 out of 20(95% ) patients with bilateral LVA .A total of 11 mutations were identified in the present study ,including 4 novel mutations (E29K(c .85G>A) ,R79X(c .235C> T) ,C282G(c .844T>G) ,V285I(c .853G>A) )and 7 repor-ted mutations .In the present study ,IVS7-2A>G was the most common mutation ,and was detected in 19 out of 23(82 .61% ) patients with SLC26A4 mutations .Conclusion SLC26A4 mutations ,the common reason for non -syndromic hearing loss ,were closely related with LVA .IVS7-2A>G was the most common mutation in SLC26A4 mutant .

OBJECTIVETo identify the relationship between coagulation factor V (FV) gene single nucleotide polymorphisms (SNPs) and venous thromboembolism (VTE).

METHODSThe FV clotting activity (FV: C) and FV antigen (FV: Ag) in plasma of VTE group (111 patients) and normal control (110 patients) were detected using one-stage clotting assay and ELISA, respectively. Five pairs of primers of the F V polymorphisms including Asp79His, Arg306The, Arg306Gly, Arg506Gln and Ile359The/His1299 Arg were synthesized and amplified by PCR. The PCR products were digested by restriction enzyme using PCR-RFLP. The detected polymorphisms were confirmed by direct sequencing. The samples containing the polymorphisms were screened for coding regions of all F V exons with direct sequencing.

RESULTSThe plasma levels of F V: C and F V: Ag of VTE group and normal control were (106.9 +/- 28.0)%, (110.4 +/- 33.3)% and (102.4 +/- 30.9)%, (102.1 +/- 24.1)%, respectively. The plasma level of FV: Ag was significantly different between VTE group and normal control. However, there was no difference in F V: C levels. Polymorphisms for the fore mentioned 5 primer pairs were not found in either patients or normal controls. Polymorphism of His1299Arg was identified in 5 patients with VTE and 3 normal controls. And these 5 cases also combined Met1736Val polymorphism, 3 of them combined another Asp2194Gly polymorphism.

CONCLUSIONThe higher plasma level of F V: Ag contribute to venous thromboembolism. There is no relationship between polymorphisms of Asp79His, Arg306The, Arg306Gly, Arg506Gln, Ile359The and venous thromboembolism in Chinese studied. Polymorphism His1299Arg is higher in VTE group than in normal control, but has no statistical difference. Polymorphisms of His1299Arg, Met1736Val and Asp2194Gly are linked disequilibrium in Chinese Han population.

Factor V ; genetics ; Female ; Gene Frequency ; Humans ; Linkage Disequilibrium ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; Venous Thromboembolism ; genetics

AIMTo explore effects of intrathecal injection of U0126 on morphine withdrawal response and the spinal Phospho-CREB expression in morphine-induced withdrawal rats.

METHODSAll the rats were divided into 5 groups: control group, dependence group, withdrawal group, U0126 group (5 microg, it) and DMSO group. Morphine withdrawal score, touch evoked agitation scores(TEA score), immunohistochemical and Western-blotting technique were used to evaluate morphine withdrawal response and the expression of Phospho-CREB in the spinal cord.

RESULTSIntrathecal injection of MEK inhibitor U0126 significantly alleviated morphine withdrawal symptoms. Morphine withdrawal scores in U0126 group (22.5 +/- 4.09) were significantly lower than that of withdrawal group (28.6 +/- 4.89, P < 0.05). TEA score of withdrawal group was 13.5 +/- 2.55, which was significantly higher than that of U0126 group (10.0 +/- 2.76, P < 0.05). Phospho-CREB positive neurons in the spinal dorsal horn of withdrawal group were 380 +/- 71, which is higher than that of U0126 group (293 +/- 47, P < 0.05). Compared with withdrawal group, level of Phospho-CREB protein detected by Western blot in spinal cord of U0126 group was significantly lower.

CONCLUSIONMEK inhibitors U0126 could suppress expression of Phospho-CREB in the spinal cord.

Animals ; Butadienes ; pharmacology ; therapeutic use ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Injections, Spinal ; Male ; Morphine Dependence ; drug therapy ; metabolism ; Nitriles ; pharmacology ; therapeutic use ; Protein Kinase Inhibitors ; pharmacology ; therapeutic use ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; drug effects ; metabolism ; Substance Withdrawal Syndrome ; drug therapy ; metabolism

Coal Mining ; Humans ; Microbial Sensitivity Tests ; Pneumoconiosis ; microbiology ; Pseudomonas Infections ; microbiology ; Pseudomonas aeruginosa ; drug effects ; enzymology ; Respiratory Tract Infections ; microbiology ; Sputum ; microbiology ; beta-Lactam Resistance

OBJECTIVETo investigate the possible roles of adenosine and the cytokines TNF-alpha and IL-10 in the pathogenesis of acute bacterial prostatitis (ABP) in rats.

METHODSForty-eight male Wistar rats were randomly divided into groups A (ABP), B (ABP + theophylline intervention), C (sham) and D (blank control). ABP models were established by injecting Escherichia coli 0157 into the prostate, and those in group B were treated by intraperitoneal injection of theophylline immediately after modeling. At 4 and 14 days, the prostate tissues of the rats were collected for detection of the expressions of TNF-alpha and IL-10 by immunohistochemistry and the concentration of adenosine by high-performance liquid chromatography.

RESULTSAt 4 and 14 days, the concentrations of adenosine were significantly higher in group A ([48.38 +/- 17.27] and [26.54 +/- 11.22] microg/g) than in C ([0.45 +/- 0.25] and [0.46 +/- 0.29] microg/g) and D ([0.41 +/- 0.23] and [0.43 +/- 0.27] microg/g) (P < 0.05), and so were the expressions of TNF-alpha in A (0.23 +/- 0.08 and 0.21 +/- 0.03) than in C (0.07 +/- 0.03 and 0.07 +/- 0.01) and D (0.07 +/- 0.06 and 0.07 +/- 0.06) (P < 0.05), and those of IL-10 in A (0.13 +/- 0.03 and 0.25 +/- 0.01) than in C (0.07 +/- 0.03 and 0.07 +/- 0.03) and D (0.07 +/- 0.01 and 0.07 +/- 0.02) (P < 0.05). Compared with group A, the rats in group B showed significant increases at 4 and 14 days in the severity of inflammation, concentration of adenosine ([86.64 +/- 32.87] and [51.17 +/- 22.96] microg/g, P < 0.05) and expression of TNF-alpha (0.37 +/- 0.08 and 0.32 +/- 0.06, P < 0.05), but exhibited no remarkable difference in the expression of IL-10 (0.12 +/- 0.06 and 0.15 +/- 0.06, P > 0.05).

CONCLUSIONAdenosine may affect the progression of inflammation by regulating the expressions of the cytokines TNF-alpha and IL-10 in ABP rats through the adenosine receptor signaling pathway.

Adenosine ; physiology ; Animals ; Escherichia coli O157 ; Interleukin-10 ; metabolism ; Male ; Prostate ; drug effects ; metabolism ; Prostatitis ; metabolism ; microbiology ; Random Allocation ; Rats ; Rats, Wistar ; Theophylline ; pharmacology ; Tumor Necrosis Factor-alpha ; metabolism

BACKGROUND: There is little information available in the mechanism of radiation-induced salivary gland injury, and its treatment and prevention are still at the exploratory stage.OBJECTIVE: To establish a rat model of radiation-induced xerostomia with 18 Gy electron beam and to observe the pathological changes of the submandibular gland and changes in saliva ingredients.METHODS: Totally 115 Wistar rats were randomly divided into exposure and control groups: the rats in the exposure group were subjected to anesthesia, and the submandibular gland received 18 Gy electron beam radiation to establish the model of radiation-induced xerostomia. The rats in the control group were only anesthetized but not exposed to radiation. The water intake was recorded at 21 dys after modeling. The saliva was collected and the submandibular gland was removed at 1, 3, 7, 14, 21, 28, 35 and 42 days to detect the saliva volume and submandibular gland index,and the morphological changes of the submandibular gland were observed by hematoxylin-eosin staining.RESULTS AND CONCLUSION: At 1-21 days after modeling, the average daily water intake was (6.42±1.91) mL in the exposure group and (4.82±1.20) mL in the control group, respectively (P < 0.05). During 42 days after modeling, the saliva secretion volume in the exposure group was lower than that in the control group, which was the lowest on day 7,and the difference was significant at 7, 21, 28 and 42 days after modeling between two groups (P < 0.05). The submandibular gland index in the exposure group was significantly lower than that in the control group at 1 and 21-42 days after modeling (P < 0.05). Hematoxylin-eosin staining results showed that in the exposure group, the rat submandibular gland appeared with inflammatory infiltration, glandular atrophy and karyopyknosis that were aggravated with time until day 42. To conclude, the rat model of radiation-induced xerostomia is established successfully with 18 Gy beam, characterized as increased water intake, decreased saliva volume and progressive aggravation of pathological injury of the submandibular gland.

OBJECTIVETo explore the indication and optimum time for treating myelodysplastic syndrome (MDS) by allogeneic hematopoietic stem cell transplantation (allo-HSCT) with HLA identical sibling grafts.

METHODSFrom June 1997 to Sep. 2004, a total of 30 patients with MDS were treated with allo-HSCT from HLA-identical sibling donors in our institute. On HSCT, 4 patients had refractory anemia (RA) , 2 RA with ringed sideroblasts (RARS) , 7 RA with excess blasts(RAEB) , 14 RAEB in transformation (RAEB-t) , 3 already progressed to secondary AML. For IPSS system, 6 patients were in intermediate- I risk group, 11 in intermediate- li risk group, and 13 in high risk group. The modified BU/CY conditioning regimen was used. Four patients received bone marrow transplantation (BMT), 8 received peripheral blood stem cell transplantation (PBSCT) , and 18 received BMT + PBSCT.

RESULTSThe 3-year expected overall survival (OS) was 63.61%, 3-year expected disease-free survival ( DFS) 61.41%, and relapse rate 5.26%; OS for RA/ RAS, RAEB and RAEB-t/AML subgroup was 83.33%, 34.29% and 66.67% , respectively, and all had no statistic difference among them. OS for IPSS-intermediate and high risk subgroup was 64.7% , and 69.0% respectively, also had no statistic difference. 3-year expected OS in no aGVHD,grade I - II aGVHD and grade III - IV aGVHD group was 57.75% , 100% and 0% , respectively (P = 0.009). Pre-HSCT chemotherapy, disease subtype and cGVHD all had no correlation with LFS or OS (P > 0.05).

CONCLUSIONFor young MDS patients having HLA-identical sibling donors, HSCT should be the first line therapy and performed as soon as possible.

Adolescent ; Adult ; Contraindications ; Female ; Follow-Up Studies ; Hematopoietic Stem Cell Transplantation ; methods ; Histocompatibility Testing ; Humans ; Male ; Middle Aged ; Myelodysplastic Syndromes ; mortality ; surgery ; Prognosis ; Survival Rate ; Transplantation Conditioning ; Transplantation, Homologous

To explore the reasonable procedures and strategies of diagnosis and treatment of congenital neutropenia (CN), clinical data and laboratory examination results of a boy suspected of CN were collected; gene ELA2, GFI1, HAX1, and WASp of whom were sequenced, granulocyte colony-stimulating factor receptor (G-CSFR) expression on neutrophil was analyzed, and cytoplasmic domain of G-CSFR was sequenced. The results showed that the diagnosis of non-syndromic variants of CN (NSVCN) was made on this patient according to the criteria; sequencing results revealed no mutation occurred in ELA2, GFI1, HAX1 and WASp; a normal expression level of G-CSFR on neutrophil from this patient was detected and no truncated mutation was found in the intracellular domain of G-CSFR. It is concluded that reasonable procedure of diagnosis and treatment of CN is established, and a sporadic NSVCN with no recognized pathogenic mutation is confirmed in this patient.
Child ; DNA Mutational Analysis ; Humans ; Male ; Neutropenia ; congenital ; diagnosis ; genetics ; therapy ; Receptors, Granulocyte Colony-Stimulating Factor ; metabolism