Regulatory T cells inhibit FoxP3 to increase the population of tumor initiating cells in hepatocellular carcinoma.

PURPOSE: Tumor initiating cells (TICs) or cancer stem cells (CSCs) are considered to be the main culprit of hepatocellular carcinoma (HCC) initiation and progression, nevertheless the mechanism by which tumor microenvironment maintains the HCC 'stemness' is not fully understood. This study aims to investigate the effect of regulatory T cells (Tregs) on the TICs characteristics of HCC. METHODS: Immunocytochemistry, flow cytometry, real-time PCR, western blot, in vitro sphere-formation, and in vivo tumorigenesis assay were used to detect HCC 'stemness'. Additionally, after forced expression or inhibition of FoxP3, ß-catenin expression and HCC 'stemness' were investigated. RESULTS: Tregs enhanced the 'stemness' of HCC cells by upregulating TIC-related markers CD133, Oct3/4, Sox2, c-Myc, Klf4, Nanog, CD13, EpCAM, and inducting epithelial to mesenchymal transition (EMT), increasing TICs ratio, as well as promoting tumorigenic ability. Moreover, ß-catenin and c-Myc were upregulated in HCC cells after co-cultured with Tregs. HCC 'stemness' was inhibited after treatment with Wnt/ß-catenin pathway inhibitor. Furthermore, forced expression of FoxP3 resulted in increased GSK3ß, decreased ß-catenin and TIC ratio in HCC. In contrast, FoxP3 interference reduced GSK3ß, enhanced ß-catenin and TIC ratio of HCC. CONCLUSION: This study, for the first time, demonstrated that Tregs increased the population of TICs in HCC by inhibiting FoxP3 as well as promoting ß-catenin expression.

Topographic distribution features of the choroidal and retinal nerve fiber layer thickness in Chinese school-aged children.

AIM: To explore the topographic distribution features of choroidal thickness (CT) and retinal nerve fiber layer thickness (RNFLT), and determine the relationship between CT and ocular parameters in school-aged children. METHODS: The healthy school-aged children with low ametropia or emmetropia in Wenzhou were recruited for this cross-sectional study. With high-density optical coherence tomography (HD-OCT) combined with MATLAB software, the CT and RNFLT values in the macular area were measured at different locations and compared. Statistical analyses were performed to evaluate the correlation between CT and ophthalmic parameters, such as spherical equivalent (SE) and the axial length (AL). RESULTS: A total of 279 school-aged children with 8.00±1.35 years of mean age (range, 6-10y) were included. The mean AL was 23.66±0.86 mm. The mean CT in CT-C (264.31±48.93 µm) was thicker than that in CT-N1 (249.54±50.52 µm), and the average CT in the parafoveal region was also thicker than that in CT-N2 (235.65±50.63 µm). The subfoveal CT also varied substantially across refractive errors (P<0.001), and those with myopia (250.59±47.01 µm) exhibited a thinner choroid compared with those with emmetropia (278.74±48.06 µm). CT negatively correlated with AL (y=-21.72x+779.17; R 2=0.1458), and positively correlated with SE (y=15.76x+271.9; R 2=0.0727, OD; y=18.31x+269.8; R 2=0.1007, OS). The average RNFLT was thickest in the peripapillary region (236.35±19.03 µm), the mean RNFLT-S (131.10±15.16 µm) was thicker than the RNFLT-I (128.20±16.59 µm), and the mean RNFLT-T (76.54±11.99 µm) was thicker than the RNFLT-N (64.28±8.55 µm). The variations in the RNFLT between quadrants did differ between those with myopia and emmetropia (P<0.05). CONCLUSION: We establish demographic information for the choroid and RNFLT. These findings provide information that should be considered in future analyses of the CT and RNFLT in OCT studies in school-aged children.

Topographic distribution features of the choroidal and retinal nerve fiber layer thickness in Wenzhou school-aged children.

AIM: To explore the topographic distribution features of choroidal thickness (CT) and retinal nerve fiber layer thickness (RNFLT), and determine the relationship between CT and ocular parameters in school-aged children. METHODS: The healthy school-aged children with low myopia or emmetropia in Wenzhou were recruited for this cross-sectional study. With high-density optical coherence tomography (HD-OCT) combined with MATLAB software, the CT and RNFLT values in the macular area were measured at different locations and compared. Statistical analyses were performed to evaluate the correlation between CT and ophthalmic parameters, such as spherical equivalent (SE) and the axial length (AL). RESULTS: A total of 279 school-aged children with 8.00±1.35 years of mean age (range, 6-10y) were included. The mean AL was 23.66±0.86 mm. The mean CT in CT-C (264.31±48.93 µm) was thicker than that in CT-N1 (249.54±50.52 µm), and the average CT in the parafoveal region was also thicker than that in CT-N2 (235.65±50.63 µm). The subfoveal CT also varied substantially across refractive errors (P<0.001), and those with myopia (250.59±47.01 µm) exhibited a thinner choroid compared with those with emmetropia (278.74±48.06 µm). CT positively correlated with AL (y=11.12x-4.15; R 2=0.18), and positively correlated with SE (y=90.07x+17.916; R 2=14.2). The average RNFLT was thickest in the peripapillary region (236.35±19.03 µm), the mean RNFLT-S (131.10±15.16 µm) was thicker than the RNFLT-I (128.20±16.59 µm), and the mean RNFLT-T (76.54±11.99 µm) was thicker than the RNFLT-N (64.28±8.55 µm). The variations in the RNFLT between quadrants did differ between those with myopia and emmetropia (P<0.05). CONCLUSION: We establish demographic information for the choroid and RNFLT. These findings provide information that should be considered in future analyses of the CT and RNFLT in OCT studies in school-aged children.

Curcumin inhibits MCF-7 cells by modulating the NF-κB signaling pathway.

The present study investigated the inhibitory effect of curcumin on human breast cancer MCF-7 cells and investigated the potential underlying molecular mechanisms. MCF-7 cells were cultured with curcumin at different concentrations and time points. The effects of curcumin treatment on breast cancer cell proliferation were studied using a MTT assay. Reverse transcription-quantitative polymerase chain reaction and western blot analysis were used to assess the mRNA and protein expression levels of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax), nuclear factor-κ-light-chain-enhancer of activated B cells (NF-κB) and inhibitor of NF-κB-α (IκBα). The proliferation of MCF-7 cells in the group treated with curcumin was markedly decreased compared with the control, with the greatest inhibitory effect at a concentration of 20 µM. The expression of Bax mRNA was increased and Bcl-2 mRNA expression was decreased compared with the control. Additionally, protein expression of NF-κB and IκB was increased. The data indicate that curcumin is able to inhibit breast cancer cell proliferation, possibly by regulating the NF-κB signaling pathway.

Protective effects of BMSCs in combination with erythropoietin in bronchopulmonary dysplasia-induced lung injury.

Bronchopulmonary dysplasia (BPD) is the most common type of chronic lung disease in infancy, for which no effective therapy is currently available. The aim of the present study was to investigate the effect of treatment with bone marrow mesenchymal stem cells (BMSCs) in combination with recombinant human erythropoietin (rHuEPO) on BPD­induced mouse lung injury, and discuss the underlying mechanism. The BPD model was established by the exposure of neonatal mice to continuous high oxygen exposure for 14 days, following which 1x106 BMSCs and 5,000 U/kg rHuEPO were injected into the mice 1 h prior to and 7 days following exposure to hyperoxia. The animals received four treatments in total (n=10 in each group). After 14 days, the body weights, airway structure, and levels of matrix metalloproteinase­9 (MMP­9) and vascular endothelial growth factor (VEGF) were detected using histological and immunohistochemical analyses. The effect on cell differentiation was observed by examining the presence of platelet endothelial cell adhesion molecule (PECAM) and VEGF using immunofluorescence. Compared with the administration of BMSCs alone, the body weight, airway structure, and the levels of MMP­9 and VEGF were significantly improved in the BMSCs/rHuEPO group. The results of the present study demonstrated that the intravenous injection of BMSCs significantly improved lung damage in the hyperoxia­exposed neonatal mouse model. Furthermore, the injection of BMSCs in combination with intraperitoneal injection of rHuEPO had a more marked effect, compared with BMSCs alone, and the mechanism may be mediated by the promoting effects of BMSCs and EPO. The results of the present study provided information, which may assist in future clinical trials.

Suberoylanilide hydroxamic acid, an inhibitor of histone deacetylase, suppresses vasculogenic mimicry and proliferation of highly aggressive pancreatic cancer PaTu8988 cells.

BACKGROUND: Pancreatic cancer is one of the most aggressive human malignancies with a extremely low 5-year survival rate. Hence, the search for more effective anti-pancreatic cancer agents is urgent. METHODS: PaTu8988 pancreatic cancer cells were treated with different concentrations of suberoylanilide hydroxamic acid (SAHA), cell survival, proliferation, migration and vasculogenic mimicry (VM) were analyzed. Associated signaling changes were also analyzed by RT-PCR and Western blots. RESULTS: Here, we reported that SAHA, a histone deacetylase inhibitor (HDACi), exerted significant inhibitory efficiency against pancreatic cancer cell survival, proliferation, migration and VM. SAHA dose-dependently inhibited PaTu8988 pancreatic cancer cell growth with the IC-50 of 3.4 ± 0. 7 µM. Meanwhile, SAHA suppressed PaTu8988 cell cycle progression through inducing G2/M arrest, which was associated with cyclin-dependent kinase 1 (CDK-1)/cyclin-B1 degradation and p21/p27 upregulation. Further, SAHA induced both apoptotic and non-apoptotic death of PaTu8988 cells. Significantly, SAHA suppressed PaTu8988 cell in vitro migration and cell-dominant tube formation or VM, which was accompanied by semaphorin-4D (Sema-4D) and integrin-ß5 down-regulation. Our evidences showed that Akt activation might be important for Sema-4D expression in PaTu8988 cells, and SAHA-induced Sema-4D down-regulation might be associated with Akt inhibition. CONCLUSIONS: This study is among the first to report the VM formation in cultured human pancreatic cancer cells. And we provided strong evidence to suggest that SAHA executes significant anti-VM efficiency in the progressive pancreatic cancer cells. Thus, SAHA could be further investigated as a promising anti-pancreatic cancer agent.

The expression of malarial invasion-related molecules is affected by two different nitric oxide-based treatments.

The host immune response to parasitic infections plays an important role in controlling multiplication of the parasite and reducing clinical symptoms and life-threatening complications. Nitric oxide (NO), an important innate immune factor and classic Th1 immune effector, may play a role in inhibiting plasmodium infection. In this study, we used two different approaches (L-Arginine [precursor of NO] and NOC5 [short-time NO donor]) to prove the roles of NO in malaria infection. We used 6-8 week-old female BALB/c mice infected with the rodent malaria Plasmodium yoelii Landau, Michel et Adam, 1968 - strain 17XL (P.y17XL) as a model. For L-Arg treatment, mice were administered with an oral dose of 1.5 mg/g L-Arg daily for seven consecutive days prior to infection with Py17XL. L-Arg pretreatment resulted in the decrease of the mRNA level of the apical membrane antigen 1 (AMA1) gene, which encodes a protein involved in host invasion. For NOC5 treatment, NOC5 was injected intraperitoneally into the P.y17XL infected mice on day 5 post-infection or incubated in vitro with purified Py17XL schizonts. Both in vivo and in vitro treatments with NOC5 led to down-regulation of the transcript and protein levels of invasion-related molecules (AMA1, merozoites surface protein 1 and Py235). Our results confirmed the protective role of NO in the asexual blood stage of parasitic infection, which may be partially due to reduced expression of parasite invasion molecules.

Age-related CD4(+)CD25(+)Foxp3(+) regulatory T-cell responses during Plasmodium berghei ANKA infection in mice susceptible or resistant to cerebral malaria.

Different functions have been attributed to CD4(+)CD25(+)Foxp3(+) regulatory T-cells (Tregs) during malaria infection. Herein, we describe the disparity in Treg response and pro- and anti-inflammatory cytokines during infection with Plasmodium berghei ANKA between young (3-week-old) and middle-aged (8-month-old) C57BL/6 mice. Young mice were susceptible to cerebral malaria (CM), while the middle-aged mice were resistant to CM and succumbed to hyperparasitemia and severe anemia. The levels of pro-inflammatory cytokines, such as TNF-α, in young CM-susceptible mice were markedly higher than in middle-aged CM-resistant mice. An increased absolute number of Tregs 3-5 days post-inoculation, co-occurring with elevated IL-10 levels, was observed in middle-aged CM-resistant mice but not in young CM-susceptible mice. Our findings suggest that Treg proliferation might be associated with the suppression of excessive pro-inflammatory Th1 response during early malaria infection, leading to resistance to CM in the middle-aged mice, possibly in an IL-10-dependent manner.

[L-arginine enhances Th1 immune response against Plasmodium yoelii 17XL infection in DBA/2 mice via activation of dendritic cells].

OBJECTIVE: To investigate the effect of L-arginine (L-Arg) during blood-stage infection by P.y17XL in DBA/2 mice. METHODS: DBA/2 mice were divided into 2 groups, 20 mice in each group. Mice were respectively administered with L-Arg (1.5 g/kg, L-Arg group) and normal saline (control group) 7 days before they were infected intraperitoneally by 1 x 10(6) pRBC. Parasitemia were detected by Giemsa stained thin-smear microscopy and survival rate were monitored daily. Flow cytometry was introduced to detect the subsets of splenic CD4+CD69+ T cells, F4/80+CD36+ macrophages, myeloid dendritic cells (mDCs) (CD11c+CD11b+), plasmacytoid dendritic cells (pDCs) (CD11c+B220+) on day 3, 5 post infection (p.i.). The levels of IFN-gamma and NO in the supernatant of splenocytes culture were detected by ELISA and Griess reaction, respectively. RESULTS: Pre-treatment of mice with L-Arg significantly decreased the parasitemia from 45% to 20% and shortened self-cure time from 22d to 20d after infection. The level of F4/80+CD36+ macrophages [(29.61 +/- 0.47)%], IFN-gamma [(485.84 +/- 39.31) pg/ml], CD4+CD69+ T cells [(7.3 +/- 0.68)%], NO [(42.51 +/- 1.32) micromol/L], mDCs(CD11c+CD11b+) [(5.51 +/- 0.87)%] and pDCs(CD11c+B220+) [(5.60 +/- 0.85)%] in L-Arg group was higher than those in control group [(36.46 +/- 1.33)%, (767.86 +/- 20.56) pg/ml, (11.27 +/- 0.97)%, (78.66 +/- 2.89) micromol/L, (10.02 +/- 0.37)%] and (9.01 +/- 0.53)%, respectively]. CONCLUSION: L-Arg enhances Th1 immune responses during the early stage of P.y17XL infection in DBA/2 mice via the activation of DCs.