Omega-3 fatty acids impair miR-1-3p-dependent Notch3 down-regulation and alleviate sepsis-induced intestinal injury.

BACKGROUND: Sepsis is a troublesome syndrome that can cause intestinal injury and even high mortality rates. Omega-3 fatty acids (FAs) are known to protect against intestinal damage. Accordingly, the current study set out to explore if omega-3 FAs could affect sepsis-induced intestinal injury with the involvement of the microRNA (miR)-1-3p/Notch3-Smad axis. METHODS: First, cecal ligation and perforation (CLP) was performed to establish septic mouse models in C57BL/6J mice, and mouse intestinal epithelial MODE-K cells were induced by lipopolysaccharide (LPS) to establish sepsis cell models. The CLP-induced septic mice or LPS-exposed cells were subjected to treatment with Omega-3 FAs and activin (Smad signaling activator), miR-1-3p inhibitor and over-expressed/short hairpin RNA (oe-/sh)-Notch3 to explore their roles in inflammation, intestinal oxidative stress and cell apoptosis. A dual-luciferase reporter gene assay was further performed to verify the regulatory relationship between miR-1-3p and Notch3. RESULTS: Omega-3 FAs inhibited CLP-induced intestinal injury and ameliorated LPS-induced intestinal epithelial cell injury by down-regulating miR-1-3p, as evidenced by decreased levels of tumor necrosis factor-α, interleukin-1ß (IL-1ß) and IL-6, in addition to diminished levels of reactive oxygen species, malondialdehyde levels and superoxide dismutase activity. Furthermore, miR-1-3p could down-regulate Notch3, which inactivated the Smad pathway. CONCLUSION: Collectively, our findings indicated that omega-3 FAs elevate the expression of Notch3 by down-regulating miR-1-3p, and then blocking the Smad pathway to alleviate intestinal epithelial inflammation and oxidative stress injury caused by sepsis.

Abscisic-acid-induced cellular apoptosis and differentiation in glioma via the retinoid acid signaling pathway.

Retinoid acid (RA) plays critical roles in regulating differentiation and apoptosis in a variety of cancer cells. Abscisic acid (ABA) and RA are direct derivatives of carotenoids and share structural similarities. Here we proposed that ABA may also play a role in cellular differentiation and apoptosis by sharing a similar signaling pathway with RA that may be involved in glioma pathogenesis. We reported for the first time that the ABA levels were twofold higher in low-grade gliomas compared with high-grade gliomas. In glioma tissues, there was a positive correlation between the ABA levels and the transcription of cellular retinoic acid-binding protein 2 (CRABP2) and a negative correlation between the ABA levels and transcription of fatty acid-binding protein 5 (FABP5). ABA treatment induced a significant increase in the expression of CRABP2 and a decrease in the expression of peroxisome proliferator-activated receptor (PPAR) in glioblastoma cells. Remarkably, both cellular apoptosis and differentiation were increased in the glioblastoma cells after ABA treatment. ABA-induced cellular apoptosis and differentiation were significantly reduced by selectively silencing RAR-α, while RAR-α overexpression exaggerated the ABA-induced effects. These results suggest that ABA may play a role in the pathogenesis of glioma by promoting cellular apoptosis and differentiation through the RA signaling pathway.

Chloroquine, an autophagy inhibitor, potentiates the radiosensitivity of glioma initiating cells by inhibiting autophagy and activating apoptosis.

BACKGROUND: Glioblastoma is refractory to conventional treatment, which is combined of surgery, chemotherapy and radiotherapy. Recent studies have shown that glioma initiating cells (GICs) contribute to tumorigenesis and radioresistance. Recently, other studies showed that the GICs use the autophagy as the major pathway to survive. Chloroquine, an anti-malarial chemical, is an autophagic inhibitor which blocks autophagosome fusion with lysosome and slows down lysosomal acidification. The aim of this study was to explore the mechanisms of chloroquine on the radiosensitivity of GICs. METHODS: Human glioblastoma cell lines U87 were investigated. MTT and clonogenic survival assay were used to evaluate the cell viability and survival from radiation. The formation of autophagosomes were evaluated by immunofluorescence. Annexin V-FITC/PI staining and flow cytometry were used to quantify the apoptotic cells. The expression levels of proteins were analyzed by Western blot. Cell cycle status was analyzed by checking DNA content after staining with PI. A comet assay was used to assess the DNA repair in the cells. Tumorsphere assay was used for evaluating GICs' renewal ability. RESULTS: Treatment of U87 GICs with chloroquine (10-80 nmol/L) alone inhibited the cell growth in a dose-dependent manner. A dose of chloroquine (20 nmol/L) obviously enhanced the radiation sensitivity of U87 GICs., we found more punctate patterns of microtubule-associated protein LC3 immunoreactivity in radiation-treated U87 GICs, and the level of membrane-bound LC3-II was obviously enhanced. A combination of radiation and chloroquine obviously enhanced the U87 GICs' apoptosis, as demonstrated by the enhanced levels of caspase-3, and reduced level of Bcl-2. In additon, combination of radiation and chloroquine cause G1/G0 cell cycle arrest. what's more, Chloroquine obviously weakened the repair of radiation-induced DNA damage as reflected by the tail length of the comet. Combination treatment of irradiation and chloroquine has synergistic effects on decreasing the GICs' tumorsphere number and diameter. CONCLUSION: Chloroquine enhances the radiosensitivity of GICs in vitro, suggesting the feasibility of joint treatment with chloroquine with radiation for GBM.

The prognostic value of Foxp3+ tumor-infiltrating lymphocytes in patients with glioblastoma.

Forkhead box protein 3 (Foxp3) is known as a specific marker for regulatory T cells which contribute to immunosuppression in tumor microenvironment. However, existing studies regarding clinical significance of Foxp3+ tumor-infiltrating lymphocytes (TILs) in glioblastoma (GBM) remained discrepant. In this study, we aimed to explore whether this subtype of TILs correlated with prognosis in patients with GBM. Foxp3+ TILs as well as CD8+ ones were detected by immunohistochemistry on paraffin-embedded tumor samples from 62 patients. Staining for p53, MGMT and Ki-67 were also performed. The correlation of TIL subtypes with clinicopathologic features were analyzed. Progression-free survival (PFS) and overall survival (OS) were estimated by Kaplan-Meier method and compared using log-rank test. Independent prognostic factors for PFS and OS were determined through univariate and multivariate analysis. Significant correlation was found between Foxp3 and CD8 expression (P = 0.003), but not between TIL subtypes and clinicopathologic characteristics. Patients with higher density of Foxp3+ TILs showed relatively shorter PFS (P < 0.001) and OS (P = 0.003) whereas patients with higher density of CD8+ TILs obtained no significant differences in survival. Survival analysis based on molecular classifications further clarified these predictive values. Univariate and multivariate analysis revealed that frequency of Foxp3+ TILs was probably associated with both PFS (P = 0.002) and OS (P = 0.003). In conclusion, the results suggest that Foxp3 positive infiltrates could provide an independent predictive factor in GBM.

B7-H4 expression is elevated in human U251 glioma stem-like cells and is inducible in monocytes cultured with U251 stem-like cell conditioned medium.

Previous studies indicated that B7-H4, the youngest B7 family, negatively regulates T cell-mediated immunity and is significantly overexpressed in many human tumors. Tumor stem cells are purported to play a role in tumor renewal and resistance to radiation and chemotherapy. However, the link between B7-H4 and tumor stem cells is unclear. In this study, we investigated B7-H4 expression in the medium of human glioma U251 cell cultures. Immunofluorescence results showed that U251 cells cultured in serum-free medium (supplemented with 2% B27, 20 ng/mL epidermal growth factor, 20 ng/mL basic fibroblast growth factor) maintained stem-like cell characteristics, including expression of stem cell marker CD133 and the neural progenitor cell markers nestin and SOX2. In contrast, U251 cells cultured in serum-containing medium highly expressed differentiation marker glial fibrillary acidic protein. Flow cytometry analysis showed serum-free medium-cultured U251 cells expressed higher intracellular B7-H4 than serum-containing medium-cultured U251 cells (24%-35% vs. 8%-11%, P < 0.001). Immunofluorescence in purified monocytes from normal human peripheral blood mononuclear cells revealed moderate expression of B7-H4 after stimulation with conditioned medium from U251 cells cultured in serum-containing medium. Moreover, conditioned medium from U251 stem-like cells had a significant stimulation effect on B7-H4 expression compared with serum-containing conditioned medium (P < 0.01). Negative costimulatory molecule B7-H4 was preferentially expressed in U251 stem-like cells, and conditioned medium from these cells more effectively induced monocytes to express B7-H4 than conditioned medium from U251 cells cultured in the presence of serum. Our results show that U251 stem-like cells may play a more crucial role in tumor immunoloregulation with high expression of B7-H4.

SP1 Promotes HDAC4 Expression and Inhibits HMGB1 Expression to Reduce Intestinal Barrier Dysfunction, Oxidative Stress, and Inflammatory Response after Sepsis.

As a serious and elusive syndrome caused by infection, sepsis causes a high rate of mortality around the world. Our investigation aims at exploring the role and possible mechanism of specificity protein-1 (SP1) in the development of sepsis. A mouse model of sepsis was established by cecal ligation perforation, and a cellular model was stimulated by lipopolysaccharide (LPS), followed by determination of the SP1 expression. It was determined that SP1 was poorly expressed in the intestinal tissues of septic mice and LPS-treated cells. Next, we examined the interactions among SP1, histone deacetylase 4 (HDAC4), and high mobility group box 1 (HMGB1) and found that SP1 bound to the HDAC4 promoter to upregulate its expression, thereby promoting the deacetylation of HMGB1. Meanwhile, gain- or loss-of-function approaches were applied to evaluate the intestinal barrier dysfunction, oxidative stress, and inflammatory response. Overexpression of SP1 or underexpression of HMGB1 was observed to reduce intestinal barrier dysfunction, oxidative stress, and inflammatory injury. Collectively, these experimental data provide evidence reporting that SP1 could promote the HDAC4-mediated HMGB1 deacetylation to reduce intestinal barrier dysfunction, oxidative stress, and inflammatory response induced by sepsis, providing a novel therapeutic target for sepsis prevention and treatment.

Unilateral Subfrontal Approach for Giant Tuberculum Sellae Meningioma: Single Center Experience and Review of the Literature.

BACKGROUND: Microsurgical Transcranial approach (mTCA) is the primary choice for the resection of giant Tuberculum Sellae Meningiomas (TSM). The objective of this study is to explore surgical details of unilateral subfrontal approach. METHODS: Ten patients with giant TSM treated by unilateral subfrontal approach were included from January 2018 to June 2021. Demographic characteristics, surgical data, post-procedure complications and outcomes of patients have been descriptive analyzed, combined with systematic literature review to explore the surgical details and the prognosis of unilateral subfrontal approach. RESULTS: Ten patients include six male and four females, age range from 35 to 77 years, duration of visual impairment from 1 to 12 months, were all performed unilateral subfrontal approach. Nine patients achieved radical resection (Simpson grades I-II) through post-operative imaging confirmation, and Simpson IV resection was performed in the remaining one due to cavernous sinus invasion. The postoperative visual acuity was improved or maintained in 8 patients. Visual acuity decreased in 2 cases, including 1 case of optic nerve atrophy and the other case of optic canal not opening. Five cases with frontal sinus opened were repaired during the operation and there was no postoperative cerebrospinal fluid leakage or intracranial infection. One patient suffered from postoperative anosmia, one patient developed left limb weakness, but their symptoms have improved in the follow-up. CONCLUSION: Summarize the experience of our center and previous literature, unilateral forehead bottom craniotomy is a feasible surgical approach for giant tuberculum sellae meningioma. Intraoperative application of EC glue and pedicled fascia flap to repair the frontal sinus can prevent complications associated with frontal sinus opening. Optic canal unroofing has huge advantage in visual improvement.

Development and clinical application of nursing-sensitive indicators for midline catheterization care using evidence-based methods.

BACKGROUND: Midline catheters (MCs) have been widely applied in clinical settings as they can provide painless venous access, thus improving the quality of life and reducing medical costs. Nursing-sensitive indicators (NSIs) are real and effective measures of nursing quality. Using evidence-based methods, we established the NSIs of MC care, with an attempt to provide a basis for evaluating and monitoring nursing quality for MC use. METHODS: An electronic search was performed in 5 databases including China National Knowledge Infrastructure (CNKI), Chinese Scientific Journal Database (VIP), Wanfang Data, PubMed, and Web of Science to identify studies that evaluated nursing quality during MC use. Two evaluators independently selected literature, extracted data, and evaluated the risk of bias. According to the Donabedian's structure-process-outcome model, we divided the NSIs into 3 levels. RESULTS: The established NSIs for MC use included 3 indicators (i.e., structure indicators, process indicators, and outcome indicators), among which there were 3 level-2 indicators and 7 level-3 indicators at the structure level, 2 level-2 indicators and 9 level-3 indicators at the process level, and 5 level-2 indicators and 17 level-3 indicators at the outcome level. CONCLUSIONS: The established NSIs for MC use offer a set of objective criteria for evaluating nursing performance during MC use and will help to improve nursing quality control.

Exchange transfusion of least incompatible blood for severe hemolytic disease of the newborn due to anti-Rh17.

HDN attributed to the rare Rh variants has become more and more significant caused by anti-D, but the compatible blood is usually very difficult to obtain when exchange transfusion is required. We treated a 10-hour neonate of O, D + C + c - E - e+ blood group with severe HDN due to anti-Rh17 with least incompatible blood typed O, D + C - c + E + e-. The neonatal hemolysis was relieved obviously and bilirubin was reduced gradually after exchange transfusion. The infant was discharged in good health 13 days after birth with 135.0 g/L, 28.0 micromol/L and 10.7 micromol/L of Hb, total bilirubin and direct bilirubin, respectively. No sequelae were observed in a three-year follow-up. The result suggesting that the least incompatible blood is an alternative choice for exchange transfusion in severe HDN due to anti-Rh17 in case that Rh17 antigen-negative blood is unavailable.

[Neuroendoscopy for treatment of multilocular brain abscess in children].

OBJECTIVE: Multilocular brain abscess in children is a serious neurosurgical emergency and remains a serious, life-threatening disease. This study evaluated the role of neuroendoscopy in treating multilocular brain abscess in children. METHODS: Between January 2002 and June 2007, 16 children with multilocular brain abscess underwent an operation using a pure endoscopic procedure. RESULTS: Increased intracranial pressure was relieved after operation in the 16 patients. CT/MRI after operation showed the abscess cavities disappeared and only the residual abscess walls existed in the 16 patients. Fourteen patients were followed up for 6 months to 5 years after surgery. Abscess walls disappeared in 13 patients and abscess recurred only in 1 patient. CONCLUSIONS: Neuroendoscopy for treatment of multilocular brain abscess is safe and effective in children.

HSPB1 Enhances SIRT2-Mediated G6PD Activation and Promotes Glioma Cell Proliferation.

Heat shock proteins belong to a conserved protein family and are involved in multiple cellular processes. Heat shock protein 27 (Hsp27), also known as heat HSPB1, participates in cellular responses to not only heat shock, but also oxidative or chemical stresses. However, the contribution of HSPB1 to anti-oxidative response remains unclear. Here, we show that HSPB1 activates G6PD in response to oxidative stress or DNA damage. HSPB1 enhances the binding between G6PD and SIRT2, leading to deacetylation and activation of G6PD. Besides, HSPB1 activates G6PD to sustain cellular NADPH and pentose production in glioma cells. High expression of HSPB1 correlates with poor survivalrate of glioma patients. Together, our study uncovers the molecular mechanism by which HSPB1 activates G6PD to protect cells from oxidative and DNA damage stress.

B7-H4(B7x)-Mediated Cross-talk between Glioma-Initiating Cells and Macrophages via the IL6/JAK/STAT3 Pathway Lead to Poor Prognosis in Glioma Patients.

PURPOSE: The objective of this study was to evaluate clinical significance and immunosuppressive mechanisms of B7-H4 (B7x/B7S1), a B7 family member, in glioma. EXPERIMENTAL DESIGN: B7-H4 levels in glioma tissue/cerebral spinal fluid (CSF) were compared between different grades of glioma patients. Survival data were analyzed with Kaplan-Meier to determine the prognostic value of B7-H4. Cytokines from CD133(+) cells to stimulate the expression of B7-H4 on human macrophages (Mφs) were investigated by FACS, neutralizing antibodies, and Transwell chemotaxis assay. shRNA, reporter vector, and chromatin immunoprecipitation were used to determine the binding of STAT3 to the B7-H4 promoter. The function of B7-H4(+) Mφs in vitro was evaluated through phagocytosis, T-cell proliferation/apoptosis, and cytokine production as well as in the xenografted model for in vivo analysis. RESULTS: We found that B7-H4 expression in tumors was associated with prognosis of human glioblastoma and correlated directly with malignant grades. Mechanistically, glioma initiating CD133(+) cells and Mφs/microglia cointeraction activated expression of B7-H4 via IL6 and IL10 in both tumor cells and microenvironment supporting cells. IL6-activated STAT3 bound to the promoter of B7-H4 gene and enhanced B7-H4 expression. Furthermore, CD133(+) cells mediated immunosuppression through B7-H4 expression on Mφs/microglia by silencing of B7-H4 expression on these cells, which led to increased microenvironment T-cell function and tumor regression in the xenograft glioma mouse model. CONCLUSIONS: We have identified B7-H4 activation on Mφs/microglia in the microenvironment of gliomas as an important immunosuppressive event blocking effective T-cell immune responses. Clin Cancer Res; 22(11); 2778-90. ©2016 AACR.

Evidence of brain tumor stem progenitor-like cells with low proliferative capacity in human benign pituitary adenoma.

Tumor stem cells have been implicated as cancer-initiating cells in malignant brain tumors. However, whether benign brain tumors also contain tumor stem cells are largely unexplored. Here, we investigated whether tumor stem-like cells were present in pituitary adenoma similar to malignant brain tumors. By immunocytochemistry, we found that pituitary adenoma tissues expressed neural stem cell marker. These cells could form neurospheres in vitro, expressed neural stem/progenitor cell markers and generated daughter cells with the capacity to differentiate into three neural lineages. Importantly, compared with non-invasive pituitary adenomas, we found that CD133 expression was significantly increased in invasive pituitary adenomas, suggesting that the proliferative capacity was correlated with the malignance of pituitary adenomas. Finally, invasive pituitary adenomas cells displayed lower proliferative ability than glioblastoma. Our data indicate that a subpopulation of stem/progenitor-like cells are present in pituitary adenomas, and these cells may be responsible for benign tumor initiation and maintenance.

Tumstatin transfected into human glioma cell line U251 represses tumor growth by inhibiting angiogenesis.

BACKGROUND: Angiogenesis is a prerequisite for tumor growth and plays an important role in rapidly growing tumors, such as malignant gliomas. A variety of factors controlling the angiogenic balance have been described, and among these, the endogenous inhibitor of angiogenesis, tumstatin, has drawn considerable attention. The current study investigated whether expression of tumstatin by glioma cells could alter this balance and prevent tumor formation. METHODS: We engineered stable transfectants from human glioma cell line U251 to constitutively secrete a human tumstatin protein with c-myc and polyhistidine tags. Production and secretion of the tumstatin-c-myc-His fusion protein by tumstatin-transfected cells were confirmed by Western blotting analysis. In the present study, we identify the anti-angiogenic capacity of tumstatin using several in vitro and in vivo assays. Student's t-test and one-way analysis of variance (ANOVA) test were used to determine the statistical significance in this study. RESULTS: The tumstatin transfectants and control transfectants (stably transfected with a control plasmid) had similar in vitro growth rates compared to their parental cell lines. However, the conditioned medium from the tumstatin transfected tumor cells significantly inhibits proliferation and causes apoptosis of endothelial cells. It also inhibits tube formation of endothelial cells on Matrigel. Examination of armpit tumors arising from cells overexpressing tumstatin repress the growth of tumor, accompanying the decreased density of CD31 positive vessels in tumors ((5.62 ± 1.32)/HP), compared to the control-transfectants group ((23.84 + 1.71)/HP) and wild type U251 glioma cells group ((29.33 + 4.45)/HP). CONCLUSION: Anti-angiogenic gene therapy using human tumstatin gene may be an effective strategy for the treatment of glioma.

U87MG glioma cells overexpressing IL-17 acclerate early-stage growth in vivo and cause a higher level of CD31 mRNA expression in tumor tissues.

Immunological alterations have been reported to be involved in glioma, the most common malignant disease of the adult brain. Our recent study identified higher levels of IL-17 in glioma specimens. The present study investigated the role and possible mechanisms of IL-17 in glioma tumorigenesis. Human IL-17 cDNA was cloned and inserted into the eukaryotic pEGFP-N1 expression vector, which was used to transfect the glioma U87MG cell line, resulting in a high level of IL-17 expression in these cells. The cells were then transfected with IL-17 (pEGFP-N1-IL-17-U87MG) or mock (pEGFP-N1-U87MG) vector or left untransfected (U87MG) and subcutaneously inoculated into the right flank of nude mice. The results revealed that the pEGFP-N1-IL-17-U87MG cells grew more rapidly in the early stages (P<0.05, determined on day 32 post-inoculation compared with the other two groups). Quantitative (q)PCR detected higher mouse (m)CD31 mRNA levels in the IL-17-transfected group (P<0.01) compared with the mock-transfected and untransfected groups. IL-17 transfection altered the mRNA expression of a panel of molecules that are associated with immunity and inflammation in U87MG cells in vitro. An effect of the vector was identified, whereby the mock transfection strongly inhibited cell growth in vivo and dramatically altered the mRNA levels of multiple molecules in the cell culture in vitro compared with the untransfected cells. The present study confirmed that IL-17 overexpression may enhance glioma cell growth in vivo, which may be associated with accelerated angiogenesis. IL-17 overexpression may also alter the cellular mRNA expression of immune-related molecules.

Th17-relevant cytokines vary with sera of different ANA staining patterns.

Antinuclear antibodies (ANA) react with components located in the cell nucleus and cytoplasm. Differing ANA staining patterns may reflect the specificity of autoantibodies in sera and indicate some autoimmune diseases specifically, to some extent. Th17-relevant cytokines have been shown to be involved in a variety of autoimmune diseases, but not consistently. In this study, we investigated whether differences in Th17-relevant cytokines exist between different ANA pattern sera. Sera of 64 ANA-positive patients (12 homogeneous, 13 speckled particle, 11 nucleolar, 15 centromere, 6 peripheral nuclear) and 16 healthy donors were analyzed for IL-17, IL-6, IL-21, IL-22, IL-23 (p19), and TGF-ß, and subsequently correlations between IL-17 and IL-6, IL-21, IL-22, IL-23, and TGF-ß were analyzed. Results showed that these Th17-relevant cytokines varied with different ANA-positive sera compared with healthy donors, except TGF-ß. Among them, IL-21 and IL-22 were higher with all ANA-positive sera and IL-17, IL-6, and IL-23 were higher with three or more ANA staining sera. No significant difference in these cytokines was seen between the different ANA staining sera except IL-17 levels in sera of peripheral nuclear staining positive subjects were higher than nucleolar. Additionally, in ANA-positive sera, IL-17 correlated with IL-6, IL-21, IL-22, and IL-23, but not with TGF-ß. Thus, we demonstrated that Th17-relevant cytokines varied with different ANA staining pattern sera, suggesting that Th17-relevant cytokines play differing roles in autoimmune diseases.

Co-delivery of pEGFP-hTRAIL and paclitaxel to brain glioma mediated by an angiopep-conjugated liposome.

In this study, we report an angiopep-2 modified cationic liposome (ANG-CLP) for the efficient co-delivery of a therapeutic gene encoding the human tumour necrosis factor-related apoptosis-inducing ligand (pEGFP-hTRAIL) and paclitaxel (PTX) to glioma. The dual targeting co-delivery system (ANG-CLP/PTX/pEGFP-hTRAIL) improved uptake and gene expression not only in U87 MG cells and BCECs, but also in the glioma bed and infiltrating margin of intracranial U87 MG glioma-bearing models. The system selectively induces apoptosis in U87 MG cells while reducing toxicity to BCECs. The results of the pharmacodynamics studies showed that the apoptosis of glioma cells in in vitro BBB models and in U87 MG glioma-bearing mice induced by ANG-CLP/PTX/pEGFP-hTRAIL was more apparent and widespread than that induced by single medication systems and unmodified co-delivery system. More importantly, the median survival time of brain tumour-bearing mice treated with ANG-CLP/PTX/pEGFP-hTRAIL was 69.5 days, significantly longer than that of other groups, even longer than the commercial temozolomide group (47 days). Therefore, the dual targeting co-delivery system is a promising drug delivery strategy against glioma.