Child, Preschool ; Female ; Humans ; Infant ; Male ; Rotavirus Infections ; diagnosis ; therapy ; Vomiting ; etiology

Objective To construct an eukaryotic expression plasmid PeDNA3.1 (+)/Lpp20 and to detect its expression in HeLa cells, and to observe the humoral and cellular immune responses in C57BL/6 mice induced by the Helicobacter pylori Lpp20 DNA vaccine injected intramuscularly. Methods The Lpp20 gene was amplified by PCR. PCR product was subcloned into the eukaryotic expression vector pcDNA3.1 (+)/ Lpp20, and the recombinant plasmid was transfected into HeLa cells using Liposome. After verifying that the Lpp20 antigen gene could be expressed in HeLa cells. Six weeks old C57BL/6 mice were immunized with pcDNA3.1 (+)/Lpp20 or pcDNA3.1 (+) or PBS buffer intramuscularly at 2-week interval for four times. ELISA was used for the quantitative detection of the specific IgG antibody in the sera of C57BL/6 mice and the cytokine IFN-γ in mice spleen lymphocyte culture medium after stimulating by Lpp20. The proliferation response of spleen cells was detected by MTT assay. The Lpp20 gene in muscle was identified by PCR. Results The significant specific antibody titers were detected by ELISA in DNA vaccine groups and the highest titer was 1:1024 after 6 weeks. The cytnkine IFN-γ in mice inoculated with pcDNA3.1 (+)/Lpp20 was increased and reached (410.36±56.23) pg/ml. A significant difference was tested between the experiment group and the control group[(25.26±10.85)pg/ml] ,P <0.01. The proliferation response of spleen cells of DNA vaccine group(SI: 2.37±0.22) was significantly higher than those of mice injected with pcDNA3.1 (+) (SI:1.53+0.47) ,P<0.01. Lpp20 gene could exist constantly in musculature cells of mice. Conclusion The eukaryotic expression recombinant pcDNA3.1 (+)/Lpp20 was successfully constructed. Strong humoral and cellular im-munity can be induced by DNA vaccine of pcDNA3.1(+)/Lpp20 in C57BL/6 mice, which might be helpful for further investigation concerning the immunoprotection of DNA vaccine.

Objective To evaluate the value of evoked potentials (EP) in diagnoses of minimal hepatic encephalopathy (MHE) for liver cirrhotic patients without overt hepatic encephalopathy (OHE). Methods A blind and self control study was conducted in 114 liver cirrhotic patients without OHE. All patients were tested for MHE by the number connection test-A(NCT-A), digit symbol test (DST), visual evoked potentials (VEP), brain-stem auditoru evoked potentials (BAEP), short latency somatosensory evoked potentials (SSEP), P300 auditory event-related potentials (P300ERP). MHE was identified when the NCT-A or/and DST was abnormal. The positive rate was compared among VEP, BAEP, SSEP and P300ERP for their reliability and validity in diagnosis of MHE. Results Of 114 patients, 60 patients were found with MHE (52. 6%), which was positively correlated with Child-Pugh classifications (r=0. 278, P = 0. 003). The positive rate was found 17.5% in VEP, 29.8% in BAEP, 38. 6% in SSEP and 57. 0% in P300ERP. There was no significant difference in diagnosis of MHE between P300ERP and NCT-A+DST (X2 =0. 432,P = 0. 511). The sensitivity of VEP, BAEP, SSEP or P300ERP for diagnosis of MHE was 13. 3%, 41. 7%, 46. 7% or 73. 3%, respectively, whereas the specificity was 77. 8%, 83. 3%, 70. 4% or 61. 1 %, respectively. The receiver operating characteristic curve revealed that the best sensitivity and specificity for the diagnosis of MHE was P300EERP (area under the curve was 0. 672, 95%CI 0. 572 * 0. 773). The agreement of NCT-A+DST with VEP, BAEP, SSEP or P300ERP was 43. 9%, 61. 4%, 57. 9% or 67. 5%. Conclusions P300ERP is a sensitive and specific method for the diagnosis of MHE. which can serve as a supplement but not instead of NCT-A+DST.

AIM: To study effects of hyperbaric oxygen (HBO) and cyclosporin A (CsA) on the contents of active oxygens and nitric oxide (NO) in spleens of skin transplanted mice. METHODS: The donor mice BALB/C and receptor mice Cs7BL/6 were tested for skin transplantation. The HBO group mice were,treated with 99.2 % oxygen under 0.25 MPa for 1.5 hours, while CsA group mice were treated with CsA 0.5 rmg· kg- t· d- 1 by abdomen injection. After 14 days, the spleen were extracted the contents of malondialdehyde (MDA) and NO and the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH - PX), catalase (CAT) and NO synthases (NOS) were determined. RESULTS: (1) Compared with the control group, the transplantation group, HBO group and CsA group have markedly increased the content of MDA and the activities of GSH - PX and CAT; Compared with the transplantation group, the CsA group have markedly increased activity of SOD and reduced activities of GSH - PX and CAT; the HBO group have markedly reduced the activity of GSH - PX and increased the activities of CAT and SOD (P < 0.01 ). (2) Compared with the control group, the transplantation group have markedly increased the content of NO and the activity of NOS; Compared with the transplantation group, the HBO group have markedly increased the activity of NOS and reduced the content of NO ( P < 0.01 ); The content of NO and the activity of NOS in CsA group was not changed significantly. CONCLUSION: In the lymphocytes of the transplantation group, the peroxidation is intensified, and the content of NO and the activity of NOS increased. HBO and CsA may activate the systems of oxidation/antioxidation and NO/NOS in spleen, which may be related to their mechanism of inhibition rejection.

Mesenchymal chondrosarcoma originated in the primitive mesenchymal tissue .It usually devel-ops in the short bones such as hand ,foot and body bone ,while extremeIy rare in the orbit .We report a case of mesenchymal chondrosarcoma of the orbit which is confirmed by pathology .

Objective To detect the expression and function of ZNRF3 in different kinds of thyroid cancer tissues and cells.Methods Immunohistochemistry was used to detect the expressions of ZNRF3 protein in 35 cases of papillary thyroid carcinoma and 10 cases of poorly differentiated thyroid carcinoma.The expressions of ZNRF3 gene in TPC-1 and 8505C were detected by RT-PCR,and the cell lines were derived from papillary thyroid carcinoma and poorly differentiated thyroid carcinoma respectively.After silenced ZNRF3 gene expression with lentivirus,the proliferation ability of TPC-1 cells were detected with CCK-8,the invasion and metastasis ability of TPC-1 cells were detected with Transwell.Results According to results of immunohistochemistry and RT-PCR,the expressions of ZNRF3 in papillary thyroid carcinoma cells and tissues were higher than those in poorly differentiated thyroid carcinoma cells and tissues,the differences were statistically significant (4.83±0.44 vs.3.13 ±0.59,t =2.20,P ＜0.05;1.01±0.06 vs.0.21±0.04,t =11.80,P＜0.01).After ZNRF3 geng silencing,according to the results of CCK8,the proliferation ability of TPC-1 cells was significantly enhanced in 72 h,the difference was statistically significant (0.96 ± 0.10 vs.0.64 ± 0.05,t =3.19,P ＜ 0.05);and according to the results of Transwell,the TPC-1 cell's invasion (0.12 ± 0.01 vs.0.09 ±0.00,t =5.48,P＜0.01) and migration (0.22 ±-0.01 vs.0.17 ±0.01,t =4.58,P ＜0.05) also increased,the differences were statistically significant.Conclusion The expression of ZNRF3 in papillary thyroid carcinoma is higher than that in poorly differentiated thyroid cancer.ZNRF3 is tumor suppressor gene in the thyroid tumors.

T cell immunoglobulin and mucin domain 3 (Tim-3) is well known to negatively regulate T cells responses, but its role in burn-induced T cells immune suppression remains unclear. In the present study, in order to identify the relationship between Tim-3 expression and post-burn T cells immune suppression, C57BL/6 mice were subjected to burn injury or sham injury, and the liver and spleen were harvested at the day 1 after operation. The expression level of Tim-3 on hepatic or splenic T cells and the functional properties of Tim-3(+) T cells were evaluated. It was found burn injury induced dramatically elevated Tim-3 expression on both hepatic and splenic CD4(+) and CD8(+) T cells in contrast with the post-burn depletion of T cells. Furthermore, Tim-3 expression was correlated with the suppressive phenotype of T cells following burn injury, including increased expression of anti-inflammatory cytokine IL-10, decreased expression of pro-inflammatory cytokines IFN-γ and TNF-α, reduced T cell proliferation and elevated co-expression of Tim-3 and PD-1. Moreover, Tim-3(+) T cells subsets were more prone to spontaneous apoptosis than Tim-3(-) T cells subsets. Our findings reinforce the idea that the up-regulated expression of Tim-3 on T cells after burn injury plays an important role in the development and maintenance of burn-induced T cell immune suppression.

OBJECTIVE: To study the effect of bioactive glass (BG) on the dentin bond strength and the microleakage of hybrid layer. METHODS: In the study, 30 dentin planes were prepared from the third molars with no caries and equally assigned to the control group, BG group, and sodium trimetaphosphate (STMP)-polyacrylic acid (PAA)-BG group (S-P-BG group), randomly. After etched with 35% phosphoric acid, the dentin planes of BG group were pretreated with 0.5 g/L BG, and the dentin planes of S-P-BG group were pretreated with 5% STMP, 5% PAA and 0.5 g/L BG. No additional pretreatment was done to the dentin planes of control group. Then the dentin planes were bonded using 3M Single Bond 2 adhesive to 3M Z350XT composite resin, and cut into 0.9 mm×0.9 mm column samples, which were stored at 37 ℃ artificial saliva (AS). After 24 hours, 1 month, and 3 months, the microtensile bond strength test was performed. The data were analyzed using one-way ANOVA and LSD method. The morphology of the bond fracture interface was observed with scanning electron microscope. Other 27 teeth were collected and the enamel layer and roots cut off, with the pulp chamber exposed. 0.1% rhodamine B was added to the 3M Single Bond 2 adhesive, and then the adhesive was applied to complete the bonding procedures as above. The teeth were stored in 37 ℃ AS for 24 hours, 1 month, 3 months, and then 0.1% sodium fluorescein solution was placed in the chambers and stained for 1 hour. Confocal laser scanning microscopy was used to observe the interface morphology and microleakage of the hybrid layer. RESULTS: At the end of 24 hours and 1 month, there was no significant difference in the microtensile bond strength among the three groups (P>0.05). After 3 months of soaking, the S-P-BG group [(36.91±7.07) MPa] had significantly higher microtensile bond strength than the control group [(32.73±8.06) MPa] (P=0.026); For the control group and the BG group, the microtensile bond strength significantly decreased at the end of 3 months compared with 24 hours (control group: P=0.017, BG group: P=0.01); The microtensile bond strength of S-P-BG group af the end of 3 months had no significant difference in compared with 24 hours [(37.99±7.98) MPa] (P>0.05). Observation of the fracture surface at the 24 hours showed no obvious mineralization in all the three groups. After 1 and 3 months, mineral formation was observed in BG group and S-P-BG group, and no obvious collagen exposure was observed in S-P-BG group. Confocal laser scanning microscopy revealed no obvious differences in the morphology and quantity of the resin tag in the control group, BG group and S-P-BG group. At the end of 24 hours, leakage was found in all the three groups. The microleakage of the control group increased at the end of 3 months, while the microleakage of the BG and S-P-BG groups decreased. CONCLUSION BG pretreatment of dentin bonding interface can induce mineralization at the bonding interface and reduce the microleakage of the hybrid layer; pretreating the dentin bonding interface with STMP, PAA and BG may enhance the maintaining of the dentin bonding durability.
Dentin ; Dentin-Bonding Agents ; Glass ; Resin Cements ; Tensile Strength

Induced pluripotent stem cells (iPSCs) can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for the extra-embryonic tissues. This iPSC technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large numbers of disease-specific cells for biomedical research. However, the low efficiency of reprogramming and genomic integration of oncogenes and viral vectors limit the potential application of iPSCs. Chemical-induced reprogramming offers a novel approach to generating iPSCs. In this study, a new combination of small-molecule compounds (SMs) (sodium butyrate, A-83-01, CHIR99021, Y-27632) under conditions of transient folate deprivation was used to generate iPSC. It was found that transient folate deprivation combined with SMs was sufficient to permit reprogramming from mouse embryonic fibroblasts (MEFs) in the presence of transcription factors, Oct4 and Klf4, within 25 days, replacing Sox2 and c-Myc, and accelerated the generation of mouse iPSCs. The resulting cell lines resembled mouse embryonic stem (ES) cells with respect to proliferation rate, morphology, pluripotency-associated markers and gene expressions. Deprivation of folic acid, combined with treating MEFs with SMs, can improve the inducing efficiency of iPSCs and reduce their carcinogenicity and the use of exogenous reprogramming factors.

The most of secreted proteins are exported by Sec translocase (secretion pathway). SecA ATPase is one of the most important subunit in the Sec translocase, which is preprotein translocase nanomotor that undergo membrane insertion and deinsertion to drive preprotein across the bacterial inner membrane, and SecA is indispensable to bacteria. It should be presumed that the compound which inhibits the activity of SecA ATPase probably can be used as the candidate of bactericide. A secA gene from Pseudomonas aerugi- nosa PAO1 was amplified and expressed in Escherichia coli BL21.19 (secA13). It has been shown that the wild-type SecA of Pseudomonas aeruginosa could fully complement the E. coli amber (secA13) mutant at the non-permissive temperature. So a cell level screening model targeting on SecA was established based on the above result. The inhibition of PaSecA ATPase activity was applied to validate the specificity of the cell-based method. Two positive samples based on both of cell and enzyme activities will be further studied.