Objective To develop a novel padlock probe and rolling circle amplification(RCA) to detect 5 common cell culture contaminant Mollicute( Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycomplasma orale, and Acholeplasma laidlawii ). Methods "Universal" primers ( SPS1, SPA2 and SPS2, SPA1) were used to amplify the Mollicute 16S-23S rRNA intergenic spacer region.Amplicon was ligate Mollicute specific padlock probe. Probe amplified and monitored using a Corbett RotorGeneTM 6000 machine. Results Five reference Mollicute strains were correctly detected by RCA method.There was no cross-reaction. RCA method can sensitive detect 10 copies templates and show strong positive signal. Sixty-two cell culture specimens were detected. Thirty-seven samples were contained single specie Mollicute and 14 samples contained two Mollicutes. Eleven samples did not contained Mollicute. RCA detection results were concordant with previously species-specific PCR. Conclusion RCA can rapid, sensitive and specific detect contamination Mollicute.

Objective To examine the reliability,validity,and practicability of Cognitive Emotion Regulation Questionnaire (CERQ) in hypertensive patients in China.Methods Altogether 434 hypertensive patients and 462 healthy subjects were recruited. All the subjects were assessed with the CERQ-Chinese version (CERQ-C), Dysfunctional Attitude Scale (DAS), Mood and Anxiety Symptom Questionnaire-Short Form (MASQ-SF), and Center for Epidemiologic Studies Depression Scale (CES-D). We calculated the mean inter-item correlations for the total CERQ and for each of the subscales. Cronbach's alpha coefficient was used to analyze the inter-correlation and reliability, and confirmatory factor analysis was used to examine the 9-factor model. Results 1) Hypertension group reported significantly higher score than that of healthy ones on rumination (12.19±2.51 vs. 11.51±2.60, P<0.001), catastrophizing(8.82±2.19 vs.8.11±2.70,P<0.001),and blaming others(10.76±2.11 vs. 9.88±2.48,P<0.001), and had significantly lower score than that of healthy ones on positive reappraisal(13.80±3.55 vs.14.71±4.11,P<0.001).2)Reliability:In the hypertension group the Cronbach's alpha for the total CERQ was 0.80, and that for the 9 subscales ranged from 0.71 (self-blame) to 0.90 (rumination). In the healthy group the Cronbach's alpha for the total CERQ was 0.79, and that for the 9 subscales ranged from 0.71 (positive reappraisal) to 0.90 (rumination). The mean inter-item correlation coefficient for the 9 subscales was 0.21-0.42(the hypertension group)/0.19-0.32 (the healthy group). In the hypertension group,the test-retest reliability of the total scale was 0.82, the test-retest reliability of the 9 subscales ranged from 0.73 to 0.92. The confirmatory factor analysis showed that the 9 first-order factor data fitted both 2 samples well. Conclusion CERQ meets the psychometric standard and it is reliable and valid for cognitive emotion regulation strategies, which may be regarded as an appropriate assessment tool.

Objective To estimate the expression of ER and PR in the endometrium of both intrauterine adhesions (IUA) and non-IUA specimens. Methods The endometrium specimens from patients undergoing hysteroscopy for confirmed moderate IUA (n=20: 10 in proliferative phase, and 10 in secretory phase) were enrolled as the IUA group in Beijing Obstetrics and Gynecology Hospital from October 2014 to August 2015. The specimens scheduled for hysteroscopy due to infertility were recruited into the control group (n=26: 13 in proliferative phase, and 13 in secretory phase). Immunohistochemistry and quantificational real-time PCR (qRT-PCR) were used to detect the expression of ER-α, ER-β and PR in endometrium with different menstrual period in both groups. Results (1) Location: in both groups, the expression of ER-α, ER-β and PR appeared in the endometrial glandular epithelial cells and the stromal cells of the endometrium. The positive brown granules of ER-α, ER-β and PR appeared mainly in cell nucleus. (2) ER-α and ER-β in the endometrium:the protein expression of ER-α and ER-β in IUA group (proliferative phase: 0.657 ± 0.028, 0.493 ± 0.023; secretory phase: 0.537 ± 0.020, 0.365 ± 0.031) were significantly higher than those of control group (proliferative phase: 0.586 ± 0.025, 0.437 ± 0.022; secretory phase:0.459 ± 0.025, 0.323 ± 0.017;all P<0.01). And the ER-αand ER-βmRNA expressions in IUA group were 2.524 ± 0.296, 1.947 ± 0.339, higher than those of control group in the proliferative phase (all P<0.01), and in the secretory phase (1.977±0.333, 1.345±0.292) were also higher than those in the control group (all P<0.01). (3) PR in the endometrium: the protein expression of PR was not significantly different between IUA group (proliferative phase:0.248±0.025, secretory phase:0.194±0.024) and control group (proliferative phase: 0.234 ± 0.019, secretory phase: 0.186 ± 0.020; P=0.162, 0.359). Meanwhile, there were no statistical differences in the mRNA expression of PR in both groups with different menstrual period (proliferative phase: 1.144 ± 0.384 versus 0.981 ± 0.306, secretory phase: 0.763 ± 0.237 versus 0.631 ± 0.203; P=0.270, 0.166). (4) ER and PR expression in menstrual cycles: the expression of ER-α, ER-β and PR in the IUA group changed with the menstrual cycles, and their expression in the proliferative phase were higher than those in the secretory phase (all P<0.05). Conclusions The expression of ER-α and ER-β in the endometrium of IUA patients changes with menstrual cycle, and are higher compared with those in normal endometrium. No difference is found in the PR expression between the two groups.

AIM: To examine the effect of cholecystokinin-octapeptide (CCK-8) on focal cerebral ischemia/reperfusion injury and its underlying mechanisms. METHODS: By using the suture model of focal cerebral ischemia and reperfusion, the effects of intracerebroventricular (icv) injection of CCK-8 and proglumide, nonselective CCK receptors antagonist, on the infarct size, regional cerebral blood flow (rCBF), and the levels of nitric oxide (NO), malondialdehyde (MDA) were observed in different brain regions of rats subjected to 1 h focal cerebral ischemia followed by 24 h reperfusion. RESULTS: (1) pretreatment with different doses of CCK-8 (0.3 ?g,1.0 ?g,2.0 ?g or 4.0 ?g) could attenuate the infarct size, but the statistically significant effects of CCK-8 were obtained only at the doses of 1.0 ?g and 2.0 ?g(P

To investigate whether LL-37 and human beta defensin-2 (hBD-2) is related to the patients with psoriasis seldom having skin infections and explore the role of the two peptides and CCR6 (the receptor of hBD-2) in the pathogenesis of psoriasis, the expression levels of mRNA of LL-37, hBD-2, and CCR6 in skin lesions of patients with psoriasis vulgaris were detected by using RT-PCR. The results showed that the mRNA expression levels of the two peptides and CCR6 in psoriatic lesions all increased compared with the normal skin (P<0.001). It was suggested that up-regulated expression of LL-37 and hBD-2 might be the main reason that result in the the skin of patients with psoriasis being seldom infected, and the two peptides and CCR6 might play crucial roles in the pathogenesis of psoriasis.
Adult ; Antimicrobial Cationic Peptides ; biosynthesis ; genetics ; Female ; Humans ; Male ; Middle Aged ; Psoriasis ; genetics ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, CCR6 ; Receptors, Chemokine ; biosynthesis ; genetics ; Up-Regulation ; beta-Defensins ; biosynthesis ; genetics

The histological and immunohistological features of two cases of cutaneous lymphadenoma was studied. A single, erythematous nodule with smooth surface developed on the face of both patients. The lesion slowly progressed. Histology revealed irregular epithelial lobules in the dermis which showed a peripheral palisaded border of basaioid-like cells as well as a center composed of clear cells. Some epithelial lobules and surrounding stroma were infiltrated by numerous small lymphocytes. Immunohistological study showed that the lymphocytic infiltration was predominantly composed of T cells (CD3 positive) along with a small number of B cells (CD20 positive). Within epithelial lobules and surrounded stroma, there were numerous dendritic cells that were positive for S-100 and CDia but negative for cytokeratin 7, cytokeratin 20 or carcino-embryonic antigen. In the center of epithelial lobules in one case, a few cells positive for epithe-lial membrane antigen and CD30 was observed. The diagnosis of cutaneous lymphadenoma was made according to the pathological and immunohistochemical findings, and the infiltration was predominated by CD3-positive lymphocytes in this uncommon epithelial neoplasm.

Objective To estimate the value of BIOMED-2 primers for the detection of T cell receptor γ (TCR-γ) gene rearrangements in different types of specimens from patients with mycosis fungoides (MF).Methods Totally,15 paraffin-embedded tissue specimens,14 fresh tissue specimens and 18 whole blood specimens were obtained from 28 patients with MF,and subjected to DNA extraction.BIOMED-2 multiplex PCR tubes TCRγ (A+B) were used for the analysis of TCRγgene rearrangements.Data were processed by SPSS 13.0 software,and statistical analysis was done by chi-square test and Fisher's exact probability test.Results TCR-γ gene rearrangements were detected in 3 paraffin-embedded tissue specimens,11 fresh tissue specimens and 12 blood specimens,with significant differences in the detection rate between the three samples (x2 =13.047,P ＜ 0.01).The fresh tissue samples showed a significantly higher detection rate than the paraffin-embedded tissue samples (X2 =12.523,P ＜ 0.01).The detection rate of TCRγgene rearrangements was 3/6 in paraffin-embedded tissue samples collected in 2011,significantly higher than that in the other 9 paraffin-embedded tissue samples collected before 2011 (Fisher's exact probability test,P =0.044),but similar to that in 14 fresh tissue specimens (12/14,Fisher's exact probability test,P =0.044).Decreased detection rate of TCRγ gene rearrangements was observed in blood samples compared with fresh tissue specimens,but no statistical difference was observed between the two types of specimens (x2 =2.358,P ＞ 0.05).Conclusions BIOMED-2 multiplex PCR tubes TCRγ(A+B) are suitable for the detection of clonal rearrangements of TCRγgene in different types of specimens,especially in fresh tissue specimens,from patients with MF.

Objective To investigate the mechanism of soluble β-1, 3-D-glucan in G-test positive serum in inhibiting ROS-dependent killing of Candida albicans ( C.albicans ) mediated by neutrophil Dectin-1.Methods The expression and distribution of internalized Dectin-1 and triggered ROS in human neutrophils were detected by using confocal/two-photon laser scanning microscopy upon stimulation with C.albicans (MOI=10) which was pretreated with β-1, 3-D-glucanase (10 U/ml) or not.Abrogation test was used to analyze whether intracellular Dectin-1 was involved in C.albicans-triggered ROS production in human neutrophils.Furthermore, flow cytometry analysis was performed to detect the expression of intracel-lular Dectin-1 and ROS in neutrophils which were pretreated respectively with G-test positive serum at differ-ent dilutions for 60 min and then stimulated with C.albicans for another 60 min at 37℃.Results After stimulated with C.albicans (MOI=10) for 60 min, the expression of Dectin-1 in neutrophils was recruited to the spores of opsonophagocytized C.albicans, and partly co-localized with the triggered ROS production . However, the expression of intracellular Dectin-1 was not observed in neutrophils when stimulated with β-1, 3-D-glucanase pretreated C.albicans for 60 min at 37℃.Abrogation test further showed that C.albicans-trig-gered ROS production in neutrophils was partly and irreversibly inhibited by adding Dectin -1 blocking mAb of 5 μg/ml.In addition , both the triggered expression of intracellular Dectin-1 and ROS production in neu-trophils stimulated with C.albicans ( MOI=10 ) in the presence of G-test positive serum were significantly lower than those of neutrophils stimulated only with C.albicans (LSD-t test, P<0.01).Linear regression a-nalysis suggested that the triggered intracellular Dectin-1 and ROS production in neutrophils upon stimulation with C.albicans were both inhibited by soluble β-1, 3-D-glucan in a dose-dependent manner (Dectin-1,R2=0.702,P<0.01;ROS,R2=0.588,P<0.01 ).Conclusion Taken together, these results demonstrated that the soluble β-1, 3-D-glucan in G-test positive serum may play a role in inhibiting the ROS-dependent killing of C.albicans by interfering with internalized expression of neutrophil Dectin-1.

Objective To investigate the mechanism hy which Dectin-1 mediates the killing of Candida albicans(C.albicans) by human neutrophils in a manner dependent on the production of reactive oxygen species (ROS).Methods After stimulation with FITC-C.albicans at a multiplicity of infection (MOI) of 10 for 30 or 60 min,PE-anti-human Dectin-1 monoclonal antibody (2.5 μg/106 cells) was used to detect the expression of Dectin-1 in human neutrophils by flow cytometry.For Dectin-1 inhibition test and ROS assay,human neutrophils (2×106/ml) were respectively pre-incubated with different concentrations of blocking antibody (0.5,1,2.5 and 5 μg/ml) for 60 min at 4℃,and then with 25 μmol/L 2′,7′-Dichlorofluorescein diacetate for another 20 min at room temperature.Afterwards,under stimulation with live C.albicans at a MOI of 10,the rate of intracellular ROS production over time in blocking and control groups was measured continuously at 10 min intervals for up to 120 min.In addition,localization of Dectin-1 and ROS in human neutrophils was observed by confocal/two-photon laser scanning microscopy after stimulation with live C.albicans.For the detection of candicidal activity,after pre-treatment with different concentrations of Dectin-1 blocking antibody as mentioned above,neutrophils were stimulated with live C.albicans (MOI=1) for 60 min,serial dilutions of cell lysate were plated onto yeast agar,and CFU were enumerated after incubation at 37℃ for 48 h.The candicidal activity was represented as [1-(CFUblocking group/CFUbuffer)] × 100％.Results Under stimulation with FITC-C.albicans at a MOI of 10 for 30 and 60 min,positive percentage of intracellular Dectin-1-expressing neutrophils increased significantly when compared with initial level (0 min,8.32％ ; 30 min,16.82％ ; 60 min,23.88％) (versus 0 min,P＜0.01).However,positive percentage of cell-surface Dectin-1-expressing neutrophils remained almost unchanged after stimulation for 30 and 60 min (versus 0 min,P＞0.05).Upon blockage of Dectin-1,the stimulated ROS generation (R2=0.306,P＜0.01) and candicidal activity (R2=0.251,P＜0.01) of neutrophils were partly and irreversibly inhibited in a dose-dependent manner when compared with control group.In addition,the intracellular Dectin-1 is recruited and co-distributed with ROS on the surface of phagocytized C.albicans as observed by confocal microscopy.Conclusion Taken together,these results demonstrated that an internalized expression pattern of human Dectin-1 might contribute to the ROS-dependent killing of serum-opsonzied C.albicans which was phagocytized by human neutrophils.

Objective Intrauterine adhesions cause serious damage to women′s reproductive health, to establish animal dis?ease mode, pure mechanical damage is the most similar cause to human pathogenesis, that is very necessary to conduct prospective studies. The study aimed to investigate the efficiency and significance of different mechanical damage methods to establish intrauterine adhesions model in rats. Methods 45 female SD rats were randomly( random number table) divided into three groups:pure scratch group (n=15), 2 mm diameter curet was used to scrape endometrial tissue; Incision?suture group (n=15), a longitudinal incision was made in the uterus, a blade was used to scrape endometrial tissue, the incision was then sutured with absorbable thread;Control group ( n=15) , sham operation, then the abdomen was closed after exposed to air for 20 minutes, the uterus were not injured. Endometri?al keratin immunohistochemical staining, Intrauterine AFS score, fi?brosis semi?quantitative score and pregnancy outcomes were observed to compare the injury of the two methods in establishing the model. Results ( 1) The degree of endometrial glandular epithelium keratin staining decreased significantly in both pure scratch and incision?su?ture groups compared to the control group. (2) AFS scores in pure scratch group and incision?suture group[3.0 (2.0-3.8), 5.0 (3.8-8.0)] were higher than control group[0 (0-0)], the difference was statistically significant (P<0.017); Incision?suture group was higher than pure scratch group, but the difference was not statistically significant(P>0.017). Endometrial fibrosis semi?quantitative scores in pure scratch group and incision?suture group[5.0 (4.0-5.3), 6.5 (5.8-8.0)] were higher than control group[0 (0-0)], the difference was statistically significant ( P<0.017);Incision?suture group was higher than pure scratch group, but the difference was not statistically significant (P>0.017). (3) Embryo number in pure scratch and incision?suture group side [0.5 (0-3.3), 0 (0-2.3)] was lower than the normal side [7.6 (6.8-8.0), 8.0 (7.8-9.0)],the difference was statistically significant (P<0.05) Conclusion Pure mechanical damage including pure scratch and incision?suture method to establish IUAs model were feasible. Both of the two methods could help to do research about pathogenesis and pathophysiological mechanisms of IUAs. Incision?suture method maybe can cause heavier fibrosis degree.