Objective To investigate the effects of different doses of estradiol on apoptosis of T lymphocytes from spleens in ovariectomized mice and explore the possible mechanisms.Methods The mice splenic T lymphocytes were isolated and divided into ten groups: young group, sham- ovariectomized group, ovariectomized group, ovariectomized plus estradiol(10-11, 10-10, 10-8 and 106groups, ovariectomized plus estradiol (10-10mol/L) plus 1CI182 780 (10-7tool/L) group, ovariectomized plus estradiol(10-10mol/L) plus 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPP, 10-6mol/L) group, ovariectomized plus estradiol(10-10mol/L) plus pyrroline dithiocarbamate(PDTC, 10-6mol/L) group. The apoptosis rates were determined by flow eytometry using Annexin V-FITC/ PI and the protein levels of ERa, ERβ, Bax, Bcl-2 and P65 were detected by Western blot. Results The apoptosis rate of ovariectomized group was(19. 4±2.5)%, which was higher than that of young group [(14.6±2.4%) 3 and sham-ovariectomized group [p (14.5±2.3)%], and the levels of Bcl-2 and nuclear P65 were lower than the young group [(0. 25±0. 05, 0. 09±0. 01) vs. (0. 40± 0.07,0. 15±0. 02), P<0.01]. The ovariectomized plus estradiol (10-10tool/L) group had lower apoptosis rate and higher Bcl-2 and P65 levels compared to the ovariectornized group[(16.6±1.8)% vs.(19.4±2.5)%,P<0.05;0.36±0.03 vs. 0.25～0.05, 0.14±0.01 vs. 0.09±0.01, P< 0. 01)], while the ovariectomized plus estradiol(10-8mol/L, 10-6tool/L) groups had higher apoptosis rates than the ovariectomized group[(22. 55±2. 5)% vs. (19. 4±2. 5)% ,P<0. 05;(27.8±3.1)% vs. (19.4 4±2. 5)%,P<0. 01, respectively]. The 2protein levels of ERa and ERβ of ovariectomized group were 0. 23±k0.01 and 0. 22±0. 03, respectively, which were lower than those of young((0. 27±0. 02) and (0. 29±0.04)] and sham-ovariectomized group [(0. 28±0. 03) and (0. 29±0.02)]. The ovariectomized plus estradiol(10-1110-1010-8tool/L) groups had higher while ovariectomized plus estradiol(10-6mol/L) group had lower ERα and ERβ protein levels (0. 09±0. 01,0. 14±0.02) than the ovariectomized group(P<0. 01). There was no significant difference between ovariectomized plus estradiol(10-10mol/L) plus ICI182 780 group or ovariectomized plus estradiol(10-10tool/L) plus PDTC group and ovariectomized group [(19.4±1.6)% vs. (19.4±2. 5)%, (21.0±2. 9)% vs. (19.4d±2. 5)%, P>0. 05). There were also no significant difference between ovariectomized plus estradiol(10-10mol/L) plus MPP group and ovariectomized plus estradiol (10-10mol/L) grou p[(16.9±2.2)% vs. (16.6±1.8)%, P>0.05]. Conclusions The ovariectomy of mice leads to increased apoptosis rates of splenic T lymphocytes. The effects of estradiol on the apoptosis of T lymphocytes in ovariectomized mice are dependent on doses: physiological dose of estradiol inhibits while higher dose of estradiol exacerbats the apoptosis of T lymphocytes in ovariectomized mice.Physiological dose of estradiol may act on Rice T lymphocytes via ERβ and NFkB signaling.

Objective To investigate the expression of microRNA-548ah (miR-548ah) and microRNA-4804 (miR-4804) in peripheral blood mononuclear cells (PBMCs) of patients with chronic hepatitis B virus infection and their clinical significance.Methods PBMCs were collected from 29 patients with chronic hepatitis B (CHB),30 hepatitis B virus carriers (HBVC),26 inactive HBsAg carriers (IASC) and 28 healthy controls in Taizhou People's Hospital during September 2012 and August 2013.Expressions of miR-548ah and miR-4804 in PBMCs were detected by fluorescence real-time quantitative RT-PCR (qRT-PCR).Receiver operating characteristic (ROC) curve was used to evaluate the expression of miR-548ah and miR-4804 in distinguishing immune tolerance phase and clearance phase of HBV infection.Pearson correlation analysis was performed to assess the correlations of miRNAs expression with clinical markers alanine aminotrans ferase (ALT) and HBV DNA loads.Results There were significant differences in expressions of miR-548ah and miR-4804 in PBMCs between CHB,HBVC,IASC groups and control group (F =28.16 and 83.17,P ＜ 0.01).Compared with control group,miR-548ah was up-regulated in CHB group(P ＜ 0.01),and down-regulated in HBVC and IASC groups (P ＜ 0.01) ; miR-4804 was up-regulated in CHB group (P ＜ 0.01),down-regulated in HBVC group (P ＜ 0.01),while there was no significant difference between IASC group and control group in miR-4804 expressions(P ＞ 0.05).The areas under ROC curve (AUCs) of miR-548ah and miR4804 in differentiation of immune tolerance and immune clearance were 0.966 and 0.997,and the sensitivities and specificities were 89.7％,96.6％ and 99.6％,100.0％,respectively.No significant correlation was found between the expression of miR-548ah,miR-4804 and ALT,HBV DNA loads (r=0.14,0.18,-0.20 and-0.19,P＞0.05).Conclusion miR-548ah and miR-4804 may be involved in the immunopathogenesis of CHB,and their expression levels in PBMCs are helpful in differentiation of immune tolerance and immune clearance in HBV infection.

Objective To analyze the differences of immune responses against Mycobacterium tu-berculosis antigens induced in two different nonhuman primates and to provide rationales for the selection of suitable animal models for vaccine efficacy evaluation.Methods Expression of functional surface markers including CD69 and HLA-DR, the activation markers on CD4+and CD8+T cells from in rhesus macaques and cynomolgus monkeys were measured by flow cytometry analysis.PBMCs were isolated from rhesus ma-caques and cynomolgus monkeys with Mycobacterium tuberculosis infection and stimulated with PPD and pep-tide pools ( ESAT-6/CFP-10) .Enzyme-linked imunospot ( ELISPOT) assay was performed to detect IFN-γproducing lymphocytes.Results The CD4+and CD8+T cells isolated from rhesus macaques without Myco-bacterium tuberculosis infection expressed higher levels of CD69 and HLA-DR than those from healthy cyno-molgus monkeys (P<0.01).The numbers of IFN-γspot forming cells/106 PBMCs in rhesus macaques with Mycobacterium tuberculosis infection for 10 and 11 months were respectively 3 and 3.5 times higher than that of cynomolgus monkeys upon after the stimulation of PBMCs with PPD.The levels of IFN-γproduction by the cells from rhesus macaque group were also higher than those from cynomolgus monkey group upon after the stimulation of PBMCs with ESAT-6 or CFP-10 peptide pools.Conclusion More IFN-γproducing cells were induced in rhesus macaques than that in cynimolgus monkeys after stimulation with Mycobacterium tu-berculosis antigens.Therefore, the rhesus macaques might be a better animal model for evaluating immune responses induced by Mycobacterium tuberculosis vaccines.

Humans ; Male ; Middle Aged ; Neurofibroma ; pathology ; Pharyngeal Neoplasms ; pathology

Objective To examine the expression of ATP-binding cassette transporter A1(ABCA1)in eukaryotie cells and the effect of arsenic resistance after the transfection of eukaryotic expression vector containing ABCA1 gene.Methods HeLa cells were transfected with the recombinant plasmid by lipofectaonmine 2000 (recombinant plasmid group),empty plasmid and untransfected HeLa cell as the control group.The level of the mRNA was examined by real-time PCR,and the expression of ABCA1 protein wag examined by Western blot,the change of cell survival rate was examined by methyl thiazolyl tetrazolium(MTT)after exposure in a series of arsenic [0(contro1),4,8,16,32,64,128 μmol/L]for 48 hours.Results Expression level of ABCA1 mRNA in recombinant plasmid,empty plasmid and untransfeeted groups was(2.09±0.08)×10-4,(0.09±0.02)×10-4,(0.08±0.02)×10-4,there was a significant difference between the groups(F=1499.23,P<0.01).The level of ABCA1 mRNA in recombinant plasmid group was higher than empty plasmid and untransfected group(all P<0.01).Western blot showed that specific protein straps existed at 254×103 in all the three groups,with a similar size to the ABCA1 protein.The amount of the recombinant plasmid group was higher than the other two groups.MTT shows that arsenic concentration at 4,8,16,32,64,128 μmol/L,the survival rates of recombinant plasmid group was(94.8±0.9)%,(86.5 ± 2.6)%, (77.8 ± 2.0)%, (56.0 ± 2.0)%, (23.8 ± 1.7)%, (18.6 ± 0.6)%, higher than that of empty plasmid group[ (85.3 ± 1.1)%, (78.7 ± 0.6)%, (67.8 ± 2.4)%, (43.2 ± 1.5)%, (14.5 ± 1.3)%, (8.0 ± 0.4)%], the difference of survival rate had a statistical signifieance(t = 18.985,6.689,5.922,9.504,9.481,32.634, all P < 0.01). Conclusions ABCA1 protein is over expressed in HeLa cells after transfect ABCA1 gene. ABCA1 protein increases resistance of arsenic in HeLa cells.

To investigate the epidemic and evolutionary trends of enterovirus (EV) in the external environment of Yunnan Province, China, molecular typing was performed on 4 EV strains that were isolated from environmental sewage in Yunnan. The VP1 region of isolates was amplified by RT-PCR using universal enterovirus primers, and the amplified VP1 region was sequenced for GenBank BLAST search and genotype analysis. The 4 EV strains were identified as ECHO7. Their nucleotide and amino acid homologies with the VP1 sequences of 68 ECHO7 strains retrieved from GenBank were measured by Mega software analysis. Our findings showed that ECHO7 strains from environmental sewage and population samples were in different evolutionary branches. These strains showed typical geographical and temporal differences; In addition, there were different transmission chains at the same time and in the same area. ECHO7 strains isolated from sewage water and patients with acute flaccid paralysis during the same period in Yunnan belonged to different clusters and evolved at different speeds. Special concerns are needed for this problem. Continuous molecular biological surveillance of human EV in the external environment of Yunnan will provide strong support for early warning of EV diseases.
China ; Databases, Genetic ; Enterovirus ; genetics ; isolation & purification ; Evolution, Molecular ; Humans ; Molecular Epidemiology ; Sequence Analysis ; Sewage ; virology

Permeability is a key factor in the bioavailability of oral drugs. Therefore, in the early stage of drug discovery, accurate and efficient evaluation of drug permeability is essential. The parallel artificial membrane permeability assay (PAMPA) with Caco-2 cells model was used by the industry as early evaluation methods. At present, the Ussing chamber rat model is also widely used. This review summarizes the human data for the in vivo single-pass perfusion technique (Loc-I-Gut) – the gold standard, and then focuses on the basic principles, experimental operation, and efficiency of the three in vitro methods, with correlation to the effective permeability coefficient (P_eff) and fractional absorbed (Fa) in man. We provide recommendations for the use of proper permeability methods at different stages in drug discovery and development.

Objective:To explore the expression of TNF a and IL-10 in different layers of laryngeal tissues after laryngeal transplantation and its relationship with acute rejection.Methods:Laryngeal heterotopic transplantation was performed in Wistar and SD rats(Wistar→SD rats).The SD rats were divided into 4 groups:GroupⅠ:Sham control(receive no transplantation): GroupⅡ(receive transplantation,without cyelosporine A treatment);GroupⅢ(receive transplantation.with 5 mg?kg~(-1)?d cyelosporine A treatment):and GroupⅣ(receive transplantation.with 10 mg?kg~(-1)?d~(-1)cyelosporine A treatment).The transplanted larynx was harvested on 3,7 and 11 days after transplantation for pathological examination.The expression of TNF-?and IL-10 in different layers of grafts was detected immunohistochemically.Results:Pathological observation showed mild,moderate and severe acute rejection in GroupⅡandⅢon 3.7 and 11 days after transplantation,respectively:there was no obvious rejection in GroupⅣ.Immunohistochemical staining showed expression of TNF-?and IL-10 in GroupⅡ.Ⅲ.andⅣ,not in GroupⅠ.The ratios of the positive areas of TNF-?and IL-10 in the mucosal and submucosal layers were significantly higher than those in the muscle and cartilage layers of laryngeal tissues(P

Objective To investigate the relationship between blood pressure variability (BPV) and neurological deteri?oration (ND) during the acute phase in patients with hypertensive minor ischemic stroke. Methods A total of 200 hyperten?sive patients with acute minor ischemic stroke were recruited in this study. Patients were divided into two groups: stable group (n=182) and deterioration group (n=18) according to the neurological prognosis. Values of BPV in 24 h ambulatory blood pressure, 24 h systolic blood pressure variation coefficient (24 h CVSBP), 24 h diastolic blood pressure variation coeffi?cient (24 h CVDBP), day time systolic blood pressure variation coefficient (dCVSBP), day time diastolic blood pressure variation coefficient (dCVDBP), night time systolic blood pressure variability (nCVSBP) and night time diastolic blood pressure variability (nCVDBP) were compared between two groups. The related factors of BPV were analyzed by binary logistic method in the acute phase of patients with hypertensive minor ischemic stroke. Results There were significantly higher levels of 24 h CVSBP [17.75%(17.54%,19.26%) vs 12.78% (10.67%,14.39%)], 24 h CVDBP [25.48%(20.77%,27.87%) vs 17.95% (14.88%, 21.46%)], dCVSBP [18.61%(17.65%,20.65%) vs 12.30%(10.10%,14.75%)], dCVDBP [25.65%(21.25%,29.78%) vs 17.76%(14.89%,22.19%)] in deterioration group than those of stable group (P<0.01). Results of binary logistic regression analysis showed that values of 24 h CVSBP and dCVSBP were risk factors for neurological deterioration in the acute phase of patients with hypertensive minor ischemic stroke. Conclusion The increased 24 h BPV and day time BPV are correlated with neurologi?cal deterioration during the acute phase in hypertensive minor ischemic stroke patients. BPV should be concerned in the acute phase and secondary prevention in patients with ischemic stroke.

Objective To investigate the correlation of the expressions of advanced glycation end products(AGE) and the receptor for advanced glycation end products(RAGE) in serum and placenta with the pathogenesis of preeclampsia. Methods From December 2013 to June 2014, 32 women with severe preeclampsia who received cesarean section in the Affiliated Hospital of Qingdao University were recruited in the study, defined as the severe preeclampsia group. 30 healthy pregnant women who received cesarean section in the same hospital were recruited as the control group. ELISA was used to measure the maternal serum AGE, soluble receptor for advanced glycation end products (sRAGE) and tumor necrosis factor-α(TNF-α) in these women. Furthermore, ELISA was also used to measure AGE and TNF-α in the placenta. The localizations of AGE and RAGE protein in placentas were detected by immunohistochemical SP method. RAGE and TNF-α mRNA expression in placentas were measured by real-time quantitative PCR. AGE, RAGE and TNF-αprotein expression in placentas were measured by western blot, respectively. Results (1) The serum levels of AGE,sRAGE and TNF-αin the severe preeclampsia group were (538 ± 75),(367 ± 86) and (322 ± 40) ng/L,respectively. They were significantly higher than those in the control group[(454 ± 50), (286 ± 35) and (270 ± 35) ng/L, respectively](P<0.05). The levels of AGE showed positive correlation with the levels of TNF-α(r=0.588,P<0.05),while the levels of sRAGE showed no correlation with TNF-α(r=-0.041, P>0.05). (2) In the severe preeclampsia group, the levels of AGE and TNF-αin placentas were (500 ± 82) and (334 ± 57) ng/L, which were higher than those in the control group [(431 ± 74) and (263 ± 46) ng/L, respectively](P<0.05). The levels of AGE showed positive correlation with the levels of TNF-ɑ(r=0.406,P<0.05). (3)AGE and RAGE protein mainly located in the syncytiotrophoblasts, macrophages and vascular endothelial cells in the placentas of the two groups. AGE expressed mainly in the cytoplasm, and RAGE expressed in the cytoplasm and cell membranes.(4)RAGE and TNF-αmRNA expression in the placentas of the severe preeclampsia group were 12.6 ± 4.6 and 10.4 ± 2.4, which were significantly higher than those in the control group (0.9 ± 0.4 and 3.5 ± 0.9,P<0.01). (5) The expressions of AGE、RAGE and TNF-αprotein in placentas of the severe preeclampsia group were 0.68 ± 0.06, 0.82 ± 0.08 and 0.76 ± 0.08. All were significantly higher than those of the control group (0.46 ± 0.05,0.42 ± 0.09 and 0.52 ± 0.07;P<0.01). Conclusions The levels of AGE and RAGE in serum and placentas elevated in the severe preeclampsia group, and the expression of TNF-αalso elevated. These indicated that AGE and RAGE might be involved in the systemic inflammatory response and local inflammatory response in placentas, and then caused the preeclampsia.