Objective To investigate the effects of stretch load on the ultramicrostructure and c-kit expression of the bladder interstitial cells of cajal (ICCs) in rats. Methods Rat model with detrusor overactivity due to partial outflow obstruction with bladder neck ligation were established. The effects of stretch load on the changes of ultramicrostructure of the bladder ICCs were observed by scanning electron microscopy. The changes in c-kit gene expression of ICCs in stable, overactive, and normal detrusors were analyzed with RT-PCR. Results The ultramicrostructure of the ICCs changed significantly. The expression of c-kit was significantly higher in the overactive group than that in stable and normal groups (P

Objective To investigate the effects of stretch load on the quantity of rat bladder interstitial cells of Cajal (ICCs) and c-kit expression in the cells. Methods Rat models of detrusor instability with partial outflow obstruction were established. The quantity of ICCs and c-kit expression in ICCs from stable, unstable and normal detrusor were studied with light microscopy and Western blotting. Results The quantity of ICCs and expression of c-kit in the stable and unstable detrusors were higher than that in normal control group (P

Objective To investigate the effects of leptin on Th17 cells and the possible mechanism. Methods The leptin-deficient ( ob/ob) mice and their homologous wild-type mice were used in the study.The percentages of Th17 cells in peripheral blood samples, spleen tissues and lymph nodes were measured by flow cytometry ( FCM) analysis.The splenic CD4+T cells, separated from the ob/ob mice and the wild-type mice by using magnetic beads,were respectively cultured with leptin at various concentrations and with anti-leptin neu-tralization antibody to evaluate the effects of leptin on Th17 cells.The quantitative real-time PCR was performed to analyze Th17 cell-related cytokines at transcriptional levels.The levels of IL-6 and IL-17A in the supernatants of CD4+T cell culture were measured with Luminex technology.Results Compared with the wild-type mice, the ob/ob mice showed lower percentages of Th17 cells in both peripheral blood samples and spleen tissues (0.49%±0.03%vs 1.29%±0.1%, 1.56%±0.22%vs 2.47%±0.11%).There was a decrease in the percentages of Th17 cells upon the in vitro treatment of CD4+T cells from wild-type mice with anti-leptin antibody.The per-centages of Th17 cells were increased in a dose-dependent manner upon the in vitro treatment of CD4+T cells from ob/ob mice with leptin.Moreover, the levels of IL-17A and IL-6 and the transcriptional levels of RORγt, IL-17A and IL-6 in leptin deficiency group were lower than those of wild-type group, but were increased upon the treatment with leptin.No significant difference with the transcriptional levels of TGF-βand IL-23 was ob-served between the two groups with and without intervention.Conclusion Leptin deficiency seriously hampered the generation of Th17 cells in mice and resulted in a decreased expression of RORγt, IL-17A and IL-6 at mRNA level.The treatment of CD4 T cells with leptin might promote the generation of Th17 cells through up-regulating the transcription of RORγt and IL-6.

Objective To explore the expression of connexin 43 in the interstitial cells of Cajal (ICCs) from guinea pig bladder in vitro. Methods Bladder ICCs were primarily cultured from guinea pigs by collagenase digestion method. Primarily cultured bladder smooth muscle cells (SMCs) from guinea pig were taken as control. The expressions of c-kit and smooth muscle actin (SMA) were detected by immunofluorescent method. Immumofluorescent method and Western blotting were used to detect the expression of Connexin 43 in 2 groups of cells. Results The expression of c-kit was positive in ICCs but negative in the control group. On the contrary, the expression of SMA was negative in ICCs but positive in the control group. Connexin 43 expression was significantly higher in ICCs than that in the control group (P

Objective To explore the expression of neuron specific enolase(NSE)and neuronal nitric oxide synthase(nNOS)in the interstitial cells of Cajal(ICCs)in vitro from guinea pigs.Methods The ICCs of bladder were primary cultured from guinea pigs by collagenase digestion.The control group was smooth muscle cell(SMC)of bladder primary cultured from guinea pigs.The expression of c-kit and smooth muscle actin(SMA)were detected by immumofluorescence.RT-PCR and Western blotting were used to detect the mRNA level and protein content of NSE and nNOS at 1,3 and 5 d after primary culture.Results The expression of c-kit was positive in the ICCs of bladder but negative in the control group,while the expression of SMA was negative in the ICCs of bladder but positive in the control group.Both NSE and nNOS of the ICCs of bladder were high in the mRNA and protein content,but were negative in the control group.Conclusion NSE and nNOS were expressed stably in the ICCs of bladder in vitro,indicating that the ICCs of bladder may be involved in the regulation of bladder function.

OBJECTIVETo detect the expression levels of co-inhibitory molecules, including CTLA-4, LAG-3, PD-1 and CD39, on CD4⁺ T cells in peripheral blood or tumor tissues from NSCLC patients and to investigate their potential internal relationships with the progression of NSCLC.

METHODSEighty-eight patients including 53 NSCLC, 17 disease control cases and 18 healthy controls were studied. All the peripheral blood and 13 cases of tumor and tumor-adjacent tissues from surgically treated NSCLC patients were obtained. The expression levels of co-inhibitory molecules CTLA-4, LAG-3, PD-1 and CD39 were assayed by flow cytometry (FCM).

RESULTSThe ratios of CD4⁺ CTLA-4⁺ T cells, CD4⁺ LAG-3⁺ T cells, CD4⁺ PD-1⁺ T cells and CD4⁺ CD39⁺ T cells in the peripheral blood of NSCLC patients were (2.49 ± 2.43)%, (2.47 ± 3.50)%, (12.94 ± 5.96)% and (6.78 ± 5.21)%, respectively, the ratio of CD4⁺ CTLA-4⁺ T cells was significantly higher in the peripheral blood of NSCLC patients than that in the disease controls and healthy controls (P < 0.05) . The ratio of CD4(+)PD-1⁺ T cells was also highly raised in the peripheral blood of NSCLC patients than that in the healthy controls (P < 0.05). Further stratified analysis indicated that the ratio of CD4⁺ PD-1⁺ T cells was (13.21 ± 5.96)% in NSCLC patients entering stages III and IV, also significantly increased as compared with that of (11.06 ± 3.42)% in the patients undergoing stages I and II (P < 0.05). More CD4⁺ CTLA-4⁺ T cells, CD4⁺ LAG-3⁺ T cells and CD4⁺ PD-1⁺ T cells were verified in the cancer tissues (5.07 ± 2.11)%, (7.86 ± 3.24)% and (40.20 ± 18.84)%, respectively, than those in their matched peripheral blood (3.13 ± 1.01)%, (2.65 ± 1.48)% and (15.79 ± 5.69)%, (P < 0.05 for all), and especially, CD4⁺ CTLA-4⁺ T cells and CD4⁺ PD-1⁺ T cells were also highly increased than those in matched cancer-adjacent tissues (P < 0.05 for all).

CONCLUSIONSThe increased expression levels of co-inhibitory molecules CTLA-4, LAG-3 and PD-1 on CD4⁺ T cells in peripheral blood and tumor tissues may be one of the mechanisms related to immune escape of tumor cells, acceleration of disease progression and poor prognosis in NSCLC patients.

Antigens, CD ; metabolism ; Apyrase ; metabolism ; CD4-Positive T-Lymphocytes ; metabolism ; CTLA-4 Antigen ; metabolism ; Carcinoma, Non-Small-Cell Lung ; diagnosis ; metabolism ; Disease Progression ; Flow Cytometry ; Humans ; Lung Neoplasms ; diagnosis ; metabolism ; Programmed Cell Death 1 Receptor ; metabolism

Objective To investigate the effects of leptin on Treg cells and the possible mecha-nism. Methods Leptin-deficient ( ob/ob) mice and homologous wild-type mice were used in this study. The percentages of Treg cells in spleen tissues and peripheral blood samples were measured by flow cytometry ( FCM) . Differences in Treg cell functionality were compared between the two groups. Splenic CD4+T cells, separated from the ob/ob mice and the wild-type mice by magnetic beads, were respectively cultured with leptin and anti-leptin neutralization antibody to evaluate the effects of leptin on Treg cells. Quantitative real-time PCR was performed to analyze the expression of Treg cell-related cytokines at transcriptional level. The levels of IL-10 and TGF-β in the supernatants of CD4+T cell culture were measured with Luminex technolo-gy. Results Compared with the wild-type mice, the ob/ob mice showed higher percentages of Treg cells in both peripheral blood samples and spleen tissues [(11. 56 ± 0. 72)％ vs (5. 47 ± 0. 81)％, (10. 16 ± 0.93)％ vs (6.29±0. 69)％]. Treg cells isolated from the ob/ob mice had stronger immunosuppressive effects on the proliferation of effector T ( Teff) cells and the secretion of TNF-α and IFN-γ than those from the wild-type mice [TNF-α:(1. 6±0. 2)％ vs (2. 4±0. 5)％, IFN-γ:(4. 3±0. 3)％ vs (7. 2±1. 2)％]. The percentages of Treg cells were decreased from (12. 2±1. 8)％ to (7. 6±0. 9)％ upon the in vitro treat-ment of CD4+ T cells from the ob/ob mice with leptin and the immunosuppressive effects of Treg cells were also weakened. However, the percentages of Treg cells were increased from (7. 8±0. 85)％ to (13. 1± 1. 5)％ upon the in vitro treatment of CD4+T cells from the wild-type mice with anti-leptin antibody and the immunosuppressive effects of Treg cells were improved as well. Moreover, the expression of Foxp3, IL-10 and TGF-β at transcriptional level and the levels of IL-10 and TGF-β in the ob/ob group were higher than those in the wild-type group. Conclusions Leptin deficiency significantly promoted the generation of Treg cells in mice and resulted in an increased expression of Foxp3, IL-10 and TGF-βat mRNA level and elevat-ed levels of IL-10 and TGF-β. The treatment of CD4+T cells with leptin might inhibit the generation of Treg cells through down-regulating the transcription of Foxp3, IL-10 and TGF-β.