ObjectiveTo explore the relation of VEGF-C and CD44V6 expression with cervical lymph metastases in papillary thyroid carcinoma.MethodsThe expression of VEGF-C and CD44V6 in 80 specimens of papillary thyroid carcinoma were evaluated with SP immunohistochemical methods. Lymphangiogenesis was examined by immunohistochemical staining with the specific D2-40 antibody and evaluated by lymphatic vessel density(LVD).In all the 80 cases,there were 38 cases with cervical lymph node metastases and 42 cases without cervical lymph node metastasis. ResultsThe positive expression rates of VEGF-C and CD44V6 in the cases with cervical metastases were 86.84 ％ (33/38) and 81.58 ％ (31/38) respectively,and in the cases without cervical lymph metastases were 61.90 ％ (26/42) and 52.38 ％ (22/42) respectively.There were significant differences in the positive expression rates of VEGF-C and CD44V6between two groups(P＜0.05). In the cases with lymph node metastases,there were positive correlations between VEGF-C and CD44V6 expression(r=0.525,P＜0.0l).Conclusion VEGF-C and CD44V6 play important roles in lymph metastasis and invasion of papillary thyroid carcinoma.VEGF-C and CD44V6 can be considered as a marker of pathological and biological behavior of papillary thyroid carcinoma.

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

Aim To investigate the effect of indirubin derivative PHⅡ-7 and TRAIL on proliferation in breast cancer cell MCF-7 and its MDR counterpart MCF-7/ADR and the mechanism.Methods Growth inhibition rate was examined respectively by MTT assay under treatment with TRAIL or PHⅡ-7 or in combination. Cell apoptosis and ROS production were examined by flow cytometry.The change of TRAIL receptors(DR4/DR5 )in mRNA was analysed by realtime PCR.Re-sults IC50 of PHⅡ-7 on MCF-7 and MCF-7/ADR was (4.49 ±1.55 ),(3.44 ±0.90 )μmol · L-1 respec-tively;MDA-MB-231 was TRAIL sensitive cell line, and apparently TRAIL induced apoptosis in MDA-MB-23 1 .Low concentration of PHⅡ-7 in combination with TRAIL could augment TRAIL-induced cytotoxic effect including apoptosis while TRAIL or PHⅡ-7 treatment alone had limited cytotoxity to those cells.Besides, PHⅡ-7 at this concentration had little toxicity to hu-man peripheral blood mononuclear cells even if in com-bination with TRAIL.PHⅡ-7 generated ROS produc-tion inside MCF-7 and MCF-7/ADR cells and up-regu-lated DR4/DR5 expression concentration dependently. Once upon ROS scavenger NAC involved,the effect of TRAIL receptors up-regualtion by expression was abro-gated.Conclusions PHⅡ-7 at low concentration could improve the sensitivities of breast cancer cell MCF-7 and MCF-7/ADR to TRAIL,the mechanism of which may be the ability of ROS production by PHⅡ-7 help up-regulated TRAIL receptor DR4,DR5 .Our re-search set a solid foundation for PHⅡ-7 in combination with TRAIL in future clinical application.

Objective To investigate the anticancer activities of resveratrol on malignant melanoma cells in vitro and involved mechanisms. Methods A375 human malignant melanoma cells and B16-F1mouse malignant melanoma cells were cultured and treated with various concentrations of resveratrol for different durations. The cell proliferation, apoptosis and cycle of both B16-F1 and A375 cells were detected with MTT assay, Annexin V-F1TC/propidium iodide (PI) double staining flow cytometry and propidium iodide flow cytometry, respectively. Western blot analysis was performed to measure the expression of Bcl-2 and Bax protein in both cells. Results Resveratroi inhibited the proliferation of A375 and B16-F1 cells in a time- and dose- dependent manner. The apoptosis rate of A375 cells was (16.7±2.1 )%, (17.2±1.7)% and (52.3±4.1 )% after treatment with resveratrol of 25 μmol/L for 24 hours, resveratrol of 100 μmol/L for 12 and 72 hours, respectively;, and resveratrol of 100 μmol/L induced the apoptosis of B16-F1 at a rate of ( 18.4±1.6)%, (39.6±3.3 )% and (56.7±4.5 )% at 12, 24 and 72 hours, respectively. Flow cytometry showed that A375 and B16-F1 cells treated with resveratrol were arrested in the G1 phase of cell cycle, and the blocking effect increased in a dose-dependent manner. The percentage of A375 and B16-F1 cells in G1 phase was (40.51±3.97 )% and (41.34±3.12 )%, respectively, after 24-hour treatment with resveratrol of 25 μmol/L,(55.64±4.95)% and (53.93±5.12)%, respectively with resveratrol of 100μmol/L for the same duration.The expression of Bcl-2 protein was decreased in malignant melanoma cells treated with resveratrol,while that of Bax protein increased. Conclusions Resveratrol can effectively inhibit the proliferation of malignant melanoma cells by regulating the cell cycle and inducing cell apoptosis, which seems to be associated with the regulation of Bcl-2 and Bax expressions.

Objectives To investigate the expression of B lymphocyte stimulator (BLyS) and its receptor BAFF-R(a receptor of B-cell activator factor, belonging to the TNF family) in peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE), and to explore their clinical significance. Methods The patients were separated into active group (n = 28) and inactive group (n = 24) according to the SLE disease activity index (SLEDAI). The mRNA and protein expression of BLyS and its receptor BAFF-R were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western-blot in PBMCs from the patients and 21 healthy volunteers. The relationship between the expression of BLyS and BAFF-R and SLEDAI was analyzed. Results In the case of the expression of mRNA and protein of BLyS and BAFF-R, the patients had a higher level than the healthy controls (P 0.05). Conclusions These findings indicate that BLyS and its receptor BAFF-R might be involved in the pathogenesis of SLE. Also, BLyS expression level might be a new parameter for the evaluation of SLE disease activity and therapeutic effect.

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 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 discrepancy of anorectal function in patients of Parkinson's disease (PD) with constipation and functional constipation (FC).Methods Fifteen consecutive male PD patients with constipation and 45 male FC patients were recruited for the study.All subjects underwent colonoscopy or barium enema in order to exclude organic colon diseases.Every patient underwent anorectal manometry and was categorized into subgroups of either dyssynergic defecation (F3a) or inadequate defecatory propulsion (F3b).Results The ages of PD with constipation and FC patients were (70 ± 11) and (68 ± 11) years old respectively.The rectal resting pressure in PD with constipation was higher than that in FC group without statistical significance [9.0 (4.0,15.0) mm Hg vs 6.0 (3.0,9.5) mm Hg,P=0.082,1 mm Hg =0.133 kPa].The anal resting pressure in PD group was not different from FC group [(51.2±17.2) mm Hg vs (59.7 ± 20.4) mm Hg,P =0.152].During anal squeezing,the maximal contraction pressure and area under the squeeze curve in PD with constipation group were both significantly lower than FC patients [maximal contraction pressure:(136.9 ± 43.8) mm Hg vs (183.0 ± 62.1) mm Hg,P=0.010; area under the squeeze curve:(823.5 ±635.7) mm Hg · s vs (1392.4± 939.9) mm Hg · s,P =0.033].During forced defecation,both of the defecation rectal pressure and defecation anal pressure in PD with constipation group were significantly lower than that of FC patients [22.0(15.0,30.0) vs42.0(31.0,55.0)mm Hg,P=0.000; and (46.3 ±23.3) vs (77.9 ±35.1) mm Hg,P =0.002].The proportions of F3a subtype were 10/15 and 46.7％ (21/45) in PD with constipation and FC patients respectively.There was no significant difference in the constituent ratio (P =0.120).Initial rectal sensory volumes were (91.3 ± 56.9) ml and (67.2 ± 38.9) ml in PD with constipation and FC patients respectively.Even both volumes were higher than the normal controls,there was no significant difference between the two groups (P =0.074).Conclusions Both PD with constipation and FC patients have abnormal anorectal motility and sensation comparing to the FC group,the parameters of anal contraction and defecation are significantly lower,F3b is dominant,and rectal sensory threshold is higher in PD with constipation patients.These parameters could possibly characterize the anorectal manometry for PD with constipation patients,which is helpful to understand the pathogenesis of PD and differentiate from other diseases.

Objective To compare the reactive oxygen species (ROS) expression in human neutrophils phagocytizing Sporothrix Schenckii and Candida albicans yeast cells,and to compare the fungicidal activity of human neutrophils against Sporothrix Schenckii and Candida albicans.Methods Human neutrophils were isolated from peripheral blood by using density gradient centrifugation method,and cultured with the presence of the yeast phase of a Sporothrix Schenckii clinical isolate and a standard strain of Candida albicans (ATCC 90028)at a multiplicity of infection of 10 or 1 for 60-210 minutes.Subsequently,flow cytometry with ROS probe (2',7'-dichlorofluorescein diacetate,DCFH-DA) was carried out to for the real time detection of intracellular ROS level,confocal laser scanning microscopy (CLSM) for the observation of ROS distribution.In addition,the fungicidal efficiency of neutrophils against Sporothrix Schenckii and Candida albicans was estimated by the number of colonies after additional culture of neutrophil lysates on brain-heart infusion agar (BHIA) medium.Statistical analysis was done by using univariate analysis of variance and LSD-t test.Results The intracellular ROS level peaked at 60 minutes in neutrophils incubated with Sporothrix Schenckii yeast cells,then decreased rapidly from 60 minutes to 210 minutes.Compared with the neutrophils incubated with Candida albicans yeast cells,those with Sporothrix Schenckii yeast cells showed a higher ROS level (expressed as mean fluorescence intensity) at 60minutes (159.67 ± 11.34 vs.112.22 ± 9.66,P＜ 0.01),but a lower ROS level at 120 minutes (89.01 ± 9.81 vs.110.25 ± 7.28,P＜ 0.05) and 180 minutes (57.63 ± 8.46 vs.109.98 ± 9.00,P＜ 0.01).CLSM revealed that ROS was mainly distributed in neutrophils with phagocytized fungal spores,and especially on the surface of phagocytized spores.Furthermore,the percentage of yeast cells killed by neutrophils was significantly lower for Sporothrix Schenckii than for Candida albicans at 180 minutes (19.21％ ± 3.68％ vs.26.63％ ± 4.97％,P ＜ 0.01).Conclusions Differential expression of intracellular ROS was observed in neutrophils after phagocytosis of Candida albicans and Sporothrix schenckii.Neutrophils exert a stronger fungicidal activity against Sporothrix Schenckii in comparison with Candida albicans,which may be associated with the rapid decrease of ROS level in neutrophils after phagocytosis.

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.