To obtain fusion protein of Mycobacterium bovis with high purity, the recombinant prokaryotic expression vector for Mb2277 gene was constructed and the immunogenicity of its products was initially investigated in the present study.A pair of primer was designed according the gene sequence Mb2277 from the genomic DNA of M.Bovis in GenBank. and was amplified by PCR using DNA of M.Bovis 93006 strain as template. The PCR product and pET-28a(+) was then digested by BamHⅠ and EcoR Ⅰdouble enzyme. To constructed a prokaryotic expression plasmid, the purified Mb2277 was cloned to pET28a(+). Then the recombinant plasmid was transformed into competent cell of E.coli BL21(DE3).The bacteria were induced by IPTG and its lysates were analyzed by SDS-PAGE and Western-blotting. In this way, the prokaryotic expression plasmid for M. bovis Mb 2277 protin was obtained, and a expression band with molecular of 25 ku could be found in SDS-PAGE analysis. As demonstrated by Western blotting this expression product showed excellent reactivity with rabbit immune sera against M. bovis.

Objective To explore the diagnostic value of MRI special use of breast and uhrasonography in axillary lymph node metastasis of early breast cancer.Methods Clinical data of 136 Ⅰ-Ⅲ A breast cancer patients accepted MRI examination before surgery had been retrospectively studied,analysing diagnostic value of MRI and ultrasonography in axillary lymph node metastasis of early breast cancer.Results The sensitivity,specificity,and accuracy obtained by MRI were 83.3％,88.6％ and 86.3％.And these data of ultrasonography were 73.1％,76.7％ and 75.0％.The sensitivity,specificity,and accuracy obtained MRI were better than that of ultrasonography.The sensitivity,specificity,and accuracy of ≥ 50 years old patients were 70.0％,77.8％ and 75.0％.And ＜ 50 years old patients were 85.7％,92.3％ and 88.9％.The sensitivity,specificity,and accuracy of ＜ 50 years old patients were better than ≥ 50 years old patients.Conclusions The MRI special use of breast have an important value in axillary lymph node metastasis of early breast cancer,especially to gounger than 50 years old patients.It can provide a scientific basis of the clinical accurate treatment for early breast cancer patients.

Objective To investigate the effects of proanthocyanidins on depressant-like behaviors and the structure of adrenal gland in chronic unpredictable mild stress (CUMS) rats. Methods Rats were randomly divided into 6 groups:control group, stressed group (CUMS + vehicle), three treatment groups (CUMS + proanthocyanidins 25,50,100 mg·kg-1,respectively) ,and imipramine group (CUMS + imipramine 10 mg·kg-1). Used the CUMS model in rats to investigate the effects of chronic oral administration (21 days) of proanthocyanidins and imipramine (ip) on the open-field;and forced swimming and sucrose consumption tests and the ratio of adrenal gland/body weight,and its thickness were examined by HE stain. Results Compared with control group, rats subjected to CUMS exhibited increased ratio of adrenal gland /body weight ( P < 0. 01), less sucrose consumption( P<0.01) and inhibited in the open-field test( P<0.01) as well as more despair time in the forced swimming test( P<0.01). While compared with stressed group,treatments with proanthocyanidins (25,50,100 mg·kg-1, po ,21 days) could significantly improve the activities in open-field test ((39.6±3.4) vs (49±4.5), (52.6±3.7),(54.1±1.8) ;all P<0.01) and sucrose consumption( (5.8±2.5)ml vs (8.1±3.3)ml,(8.5±4.1) ml, (9.2±2.6) ml; P < 0.05, P < 0.05, P < 0.01 respectively); Meanwhile, it could reduce the duration time in forced swimming test significantly( (103.5±10.2)s vs (83.7±8.8)s,(75.8±5.9)s,(67.2±6.5)s; all P<0.01) as well as thickness of the adrenal gland(P<0.05, P<0.01).Conclusions This study suggests that the proanthocyanidins (25,50,100 mg·kg-1) has an antidepressant-like effects in CUMS rats. The antidepressant actions of proanthocyanidins, in some degrees, may be related with the regulation of the adrenal gland's structure.

OBJECTIVE: To explore the factors related to the length of hospital stay for cerebrovascular accident and to provide the basis for health administrative department to formulate measures, and for clinical department to develop treatment guidelines. METHODS: We collected the medical record of the hospitalized cerebrovascular accident patients from 2008 to 2013 in the Third Xiangya Hospital, Central South University. The collected data included demographic information, disease characteristics, treatment information and health economics information. Then we analyzed the factors related to the length of hospital stay for cerebrovascular accident. SPSS 13.0 was used for statistical analysis and logistic regression and nonparametric rank sum test was performed. RESULTS: The length of stay in hospital was from one day to 513 days, with a median of 10 days. The length of stay for women was shorter than that of men (OR=0.869). The length of stay for the older patients was longer than that of younger patients (OR=1.158). The length of stay for patients who implemented a surgery was 1.686 times longer than those who were not implemented a surgery (OR=1.686). The length of stay for ischemic cerebrovascular patients was shorter than that of the hemorrhagic cerebrovascular patients (OR=0.275). The patients with new rural cooperative medical insurance or without health insurance usually stayed a short time in hospital. CONCLUSION Sex, age, surgery, type of cerebrovascular accident and medicare type are the factors that affect the length of hospital stay for cerebrovascular accident.
Female ; Humans ; Length of Stay ; Logistic Models ; Male ; Stroke

Objective To explore the predictive value of neutrophil-to-lymphocyte ratio on the prognosis of H7N9 avian influenza.Methods A retrospective analysis was conducted on 28 H7N9 avian influenza patients (treatment group) at the First Affiliated Hospital of Soochow University from April 2013 to January 2016.Thirty healthy physical examiners in the same period were enrolled as the healthy control group.The 28 patients were followed up for half a year and divided into the improvement group (18 cases) and the death group (10 cases) according to the clinical prognosis.Inflammatory indicators including white blood cells (WBC),neutrophil (N),lymphocyte (L),monocytes (M),platelet (PLT),creatine kinase (CK),lactate dehydrogenase (LDH),high sensitive C reactive protein were collected at day 1,day 3 and week 1 of admission.Calculation of neutrophil-to-lymphocyte ratio (NLR),platelet-to-lymphocyte ratio (PLR),lymphocyte-to-monocyte ratio (LMR),△NLR3 (day 3 of admission NLR-on day 1 of admission NLR),△NLR7 (week 1 of admission NLR-day 3 of admission NLR) and so on calculating △PLR3,△PLR7,△LMR3,△LMR7.Differences of the above indicators between the improvement group and death group were compared.The measurement data with normal distribution were tested by t-test of two independent samples,and the count data with non-normal distribution were tested by Mann-Whitney U-test.Univariate and multivariate logistic regression analysis to explore the prognostic factors and the working characteristic curve of subjects was used to evaluate the predictive value of inflammatory response indexes for H7N9 avian influenza death.Results In the treatment group,the baseline WBC,L,N,PLT,the proportion of lymphocytes,neutrophils,monocytes,and NLR,PLR,and LMR were all statistically different compared with the healthy control group (all P ＜0.01).After treatment,day 3 NLR,△NLR3 in improvement group were both significantly decreased to 10.93 (15.71)and0.87 (-15.63),respectively when compared with death group (17.62[23.63] and 7.42[22.68],respectively) (Z =-2.16 and-2.014,respectively,both P＜0.05).Day 7 NLR,△NLR7 in improved group were 6.51 (13.23) and-0.37 (-12.38),respectively,which were both lower than those of death group (27.90 [25.64] and 11.54 [-26.22]) with statistically significant differences (Z =-2.444 and -2.111,respectively,both P ＜ 0.05).Multivariate logistic regression analysis indicates that △NLR3 is the main factor that affects the prognosis of the H7N9 infection (odds ratio [OR] =1.153,95％ confidence interval [CI]:1.052-1.263,P =0.002).Reciver operating characteristic curve analysis showed that the area under the curve was 0.733 (95 ％ CI:0.532-0.935,P =0.044).Based on the principle of Youden index,the cutoff value of △NLR3 to predict the death risk of H7N9 avian influenza was 5.453 with sensitivity of 0.700 and the specificity of 0.722.The mortality was higher when △NLR3 was higher than 5.453.Conclusions Dynamic monitoring NLR,especially △NLR3 may reflect the condition and prognosis of H7N9 infection,which is an independent predictor of death.

Objective: To investigate the clinicopathologic characteristics, immunophenotype, differential and diagnostic features of atypical spindle cell lipomatous tumor (ASLT). Methods: Three cases of ASLT were collected from January 2010 to March 2017 at Zhejiang Provincial People′s Hospital. The clinical and imaging features, histomorphology, immunophenotype and prognosis were analyzed. Fluorescence in situ hybridization (FISH) was used to detect MDM2 gene amplification, and relevant literature was reviewed. Results: All three patients were adult males, aged 38, 43 and 54 years, respectively. One tumor originated in the subcutaneous soft tissue in the head and neck, one was located in the left primary bronchus and one in the latissimus dorsi muscle. Grossly, all three tumors were circumscribed and ranged from 4.0 to 5.8 cm in size. Microscopically, all showed a focally infiltrative front. These tumors were composed of variable proportions of spindle-shaped and adipocytic cells in a background of variable fibrous and edematous matrix. Scattered lipoblasts were easily seen. One tumor was composed predominately of spindle tumor cells, one of adipocytic cells, and one of equally mixed cell populations. The spindle tumor cells were generally bland-appearing with focal nuclear enlargement and hyperchromasia noted in one case. Mitosis was not seen in neither the spindle cells nor the adipocytic cells. By immunohistochemistry, diffuse and strong reactivity to CD34 of the spindle cells was noted in all cases, definite loss of Rb expression was noted in one of three cases, and S-100 protein was expressed only in the adipocytic cells. INI-1 was intact and Ki-67 index was 1% to 3%. All other markers including CDK4, MDM2, STAT6, SOX10, CD99, bcl-2, β-catenin, CD117, GFAP, CK, EMA, SMA and desmin were negative. FISH of MDM2 was done in two cases, and both showed no amplification. The ASLT in the head and neck had two recurrences during 17 months of follow-up, whereas the tumor in the latissimus dorsi was free of disease during 33 months of follow-up. Conclusions ASLT is a rare subtype of low-grade adipocytic neoplasm and is distinctive from atypical lipomatous tumor/well-differentiated liposarcoma. The histomorpholgy of ASLT has significant heterogeneity and forms a continuous spectrum. ASLT needs to be distinguished from a series of benign and malignant soft tissue tumors.

Objective: To investigate the mechanism of vascular endothelial growth factor(VEGF) inducing tolerogenic dendritic cells(DCs) in oral squamous cell carcinoma (OSCC). Methods: The DCs were divided into four groups: Control group (DC), VEGF group (VEGF added into DC), Co-culture group (DC co-cultured with SCC7) and Anti-VEGF group (anti-VEGF antibody added into DC co-cultured with SCC7). Flow cytometry (FCM) was used to detect DC surface markers. To detect the effect of DC on proliferation activity of T lymphocyte, the experiment included five groups: Nc group (T lymphocyte), Control group (T lymphocyte added into DC), VEGF group (T lymphocyte + DC + VEGF), Co-culture group (T lymphocyte + DC + supernatant of SCC7) and Anti-VEGF group (T lymphocyte + DC + supernatant of SCC7 + anti-VEGF antibody). Subsequently, the mixed lymphocyte reaction(MLR) was conducted. The expression levels of indole-2, 3-doxygenase(IDO)and programmed cell death 1 ligand 1(PD-L1)in DC were detected by western blot, real time PCR and FCM respectively. For the cytotoxic lymphocyte (CTL) assay, SCC7 cells and CTLs were mixed and CTL-mediated SCC7 cells cytotoxicity was tested. The experiment included four groups: Control group (T lymphocyte + DC), IDO inhibition group (T lymphocyte + DC + IDO inhibitor), Anti-PD-L1 antibody group (T lymphocyte + DC + anti-PD-L1 antibody) and Combination group (T lymphocyte + DC + IDO inhibitor + anti-PD-L1 antibody). The SCC7 tumor-bearing mice treated with IDO inhibitor and the anti-PD-L1 antibody were sacrificed and the tumor inhibition rate and the spleen index were determined. Results: Compared with Control group, exogenous VEGF or SCC7 co-culture inhibited the relative number of DC expressing CD11C, CD80, CD86, CD40 and MHC Ⅱ. The positive DCs were increased in the Anti-VEGF group compared with VEGF or Co-culture group. In VEGF or Co-culture group, the number of T cells stimulated by SCC7-pulsed DCs was decreased compared with Control group. However, the ability of Anti-VEGF group to induce T cell proliferation was significantly increased compared with VEGF or Co-culture group. Significantly increased expression of IDO and PD-L1 were observed in VEGF and Co-culture group. However, this was partially reversed by addition of anti-VEGF antibody into the co-culture system. Compared with Control group, the expressions of CD11C and CD86 in DC in both the IDO inhibition group and Anti-PD-L1 antibody group were increased, and were significantly higher in the Combination group compared with the single drug groups. The similar results were exhibited in MLR and CTL assay. In vivo, the results revealed that the tumors obtained from the mice in three experimental groups were smaller than those in the control group. Furthermore, the tumor volume of the Combination group was the smallest. The spleen index of each group was calculated and the results showed the spleen index of the three experimental groups was significantly higher than that of Control group. Conclusion VEGF in OSCC micro-environment inhibits the maturation and function of DC that are transformed into tolerogenic DC by high expression of IDO and PD-L1.

Objective To study the antigen-specific immune response induced by the graphene oxide (GO) in mice.Methods OVA-loaded GO nano-immunocomplexes (GO-OVA) were prepared by co-incubation of nano GO with model antigen ovalbumin (OVA).Nano GO was characterized by atomic force microscopy and laser particle sizeanalyzer.The cytotoxicity of GO to mouse bone marrow dendritic cells (BMDCs) was detected by cell counting kit (CCK-8).The GO-OVA uptake of BMDCs were observed by fluorescent staining.C57BL/6 mice were divided into OVA group,aluminum adjuvant OVA (Al-OVA) group and GO-OVA group (6 mice in each group) by body weight for in vivo immunization.The levels of OVA-specific antibody IgG (total IgG,IgG1,and IgG2a) in serum of mice were detected by enzyme-linked immunosorbent assay (ELISA).The T lymphocyte subsets in spleen and inguinal lymph nodes of mice were detected by flow cytometry.Results The average particle size of the prepared nano GO was (294.34±4.68) nm,and the polydispersity coefficient was 0.208.Nano GO has less toxicity to mouse BMDCs.The results of in vitro experiments indicated that GO-OVA nanovaccine can be efficiently internalized by mouse BMDCs.The antigen-specific IgG antibodies induced by the GO-OVA was similar to that of aluminum adjuvant and the difference was not statistically significant (P＞0.05),and the Th1-type response was predominant.The proportions of CD4+ and CD8+ T lymphocytes in the spleen and inguinal lymph nodes in GO-OVA group were significantly higher than those in OVA and Al-OVA groups,and the differences were statistically significant (all P＜0.05).Conclusions GO-OVA nano-immunocomplexes can induce both humoral and cellular immune responses in mice,which provides basis for the development of novel vaccine vectors and adjuvants.

Objective To study the maturation and activation effects of hyaluronic acid (HA) modified polymer nanoparticles co-delivering adjuvants and antigens on mouse bone marrow dendritic cells (BMDCs). Methods HA-modified polylactic acid-glycolic acid copolymer (PLGA) and cationic lipid DOTAP were used as nanocarriers (DOTAP-PLGA) to co-deliver adjuvant CpG with model antigen ovalbumin (OVA). In the drug-loaded nanocarriers, CpG was covalently bound to the surface of HA, and OVA was physically blended into DOTAP-PLGA nanocarriers. The nanoparticles were characterized by transmission electron microscopy and dynamic light scattering. The in vitro release of CpG and OVA in the nanoparticles was investigated. The uptake and distribution of nanoparticles in mouse BMDCs were studied by flow cytometry and laser scanning confocal microscopy. The maturation and cytokine expression of mouse BMDCs were evaluated by flow cytometry and enzyme-linked immunosorbent assay, respectively. Results The CpG-HA-OVA-PLGA nanoparticles loading CpG and OVA were prepared. The average particle size was (305.1±2.2) nm and the polydispersity index was 0.203. A core-shell structure of the nanoparticles modified by HA was clearly observed by transmission electron microscopy. Cellular experiment results showed that CpG-HA-OVA-PLGA nanoparticles could be efficiently uptaken by mouse BMDCs, and promote lysosomal release of CpG and cytoplasmic delivery of antigen OVA. Compared with free OVA group and free OVA+CpG group, the CpG-HA-OVA-PLGA nanoparticles significantly up-regulated the expression of co-stimulatory molecules CD86 and CD40 (all P<0.01), major histocompatibility complex I (MHC-I) (P<0.01), and cytokine tumor necrosis factor-α (TNF-α) (P<0.01). Conclusions HA-modified CpG and OVA nanoparticle co-delivery vectors can effectively promote the maturation and activation of dendritic cells, which provides a basis for the development of novel vaccine vectors for the co-delivery of antigens and adjuvants.

Objective To estimate the dose-response relationship between sedentary behavior with mortality in patients with type 2 diabetes. Methods A total of 17786 type 2 diabetic patients were recruited as participants, who were included in National Basic Public Health Service in Changshu County of Suzhou City, Qinghe District and Huai'an District in Huai'an City of Jiangsu Province. Cox proportional hazards regression model and restricted cubic spline model were employed to estimate the dose-response relationship between sedentary behavior with all-cause and cause specific mortality in patients with type 2 diabetes. Results Among 78114.34 person-years of the fo1low-up, the median of follow-up time was 4 years, and 1285 deaths occurred during that period. Compared to patients with sedentary behavior≤2 h/d, the multivariate adjusted hazard ratios of all-cause death associated with sedentary behavior levels of 3-4 h/d, 5-6 h/d, and≥7 h/d were 1.05(95％CI 0.92-1.20), 1.20(95％CI 1.03-1.42), and 1.39 (95％CI 1.16-1.65), respectively. Eevry increase of 1 h/d in sedentary behavior was associated with an increased hazard of death from cardiovascular disease(CVD) of 4％(HR=1.04, 95％CI 1.01-1.07) and from other causes of 6％( HR=1.06, 95％CI 1.03-1.09) . However, no significant association between sedentary behavior and malignant tumor death was found. The multivariable restrictive cubic spline regression indicated that the linear dose-response relationships were found between sedentary time with the all-cause, CVD cause, and other cause of mortality ( Non-linear test, P>0.05) . Conclusion Longer sedentary behavior could increase the risk of mortality in patients with type 2 diabetes.