Nrdp1-mediated Macrophage Phenotypic Regulation Promotes Functional Recovery in Mice with Mild Neurological Impairment after Intracerebral Hemorrhage.

Neuregulin receptor degradation protein 1 (Nrdp1) is a ring finger E3 ubiquitin ligase involved in some inflammation through ubiquitination, including macrophage polarization following cerebral hemorrhage. However, there is limited understanding regarding the mechanisms through which Nrdp1 modulates macrophage polarization and the potential impact of this modulation on neurological function. Using stereotactic injection and adenoviral transfection techniques, the corresponding animal models were constructed through injecting adenovirus, saline, or blood into the mouse striatum at different periods of time in this research. The alteration in the ratio of various M1/M2 phenotype-associated markers (e.g., CD86, CD206, IL-6, IL-10, etc.) was evaluated through immunohistochemistry, immunofluorescence, western blotting, and elisa assays. Additionally, neurological function scores and behavioral tests were utilized to evaluate changes in neurological function in mice after cerebral hemorrhage. Our results show that overexpression of Nrdp1 promotes the expression of a variety of M2 macrophage-associated markers and enhance transcriptional activity of arginase-1 (Arg1) protein through ubiquitination for early regulation M2 macrophage polarization. Additionally, Nrdp1 promotes hematoma absorption, increases IL-10 expression, inhibits inducible nitric oxide synthase (iNOS), IL-6, and TNF-α production, alleviates neurological impairment and brain edema, and accelerates functional recovery. These findings suggest that modulating macrophage polarization through Nrdp1 could be a therapeutic strategy for neurofunctional impairment in cerebral hemorrhage.

Programmed death (PD)-1 attenuates macrophage activation and brain inflammation via regulation of fibrinogen-like protein 2 (Fgl-2) after intracerebral hemorrhage in mice.

Neuroinflammation plays an important role in the recovery of brain injury in ICH. Macrophage is the major executor in the neuroinflammation and initiates neurological defects. Programmed death 1 (PD-1) delivers inhibitory signals that regulate the balance between T cell activation, tolerance, and immunopathology. PD-1 expression by macrophages plays a pathologic role in the innate inflammatory response. However, the exact role of PD-1 on inflammatory responses following ICH has not been well identified. In this experiment, PD-1 KO (PD-1 -/-) ICH mice and Wild-type (WT) ICH mice were caused by intracranial injection of type IV collagenase. The level of macrophage activation, inflammatory cytokines and fibrinogen-like protein 2 (Fgl-2) were detected using immunofluorescence staining and ELISA assays. In addition, brain edema and neurological scores of ICH mice were also measured. Our data demonstrated that ICH promoted PD-1 expression of macrophage and enhanced inflammatory cytokines and Fgl-2 concentrations. PD-1 -/- mice exhibited significantly higher expression of the inflammatory cytokines which initiate Fgl-2, than did their wild-type (WT) littermates. As a result, macrophage activation, cerebral edema and neurological deficit scores of PD-1 -/- mice were higher. In conclusion, our data demonstrate that PD-1 plays a vital role in brain inflammation via regulation of Fgl-2 after ICH, and that manipulation of PD-1 might be a promising therapeutical target in ICH.

Recombinant adenovirus encoding NLRP3 RNAi attenuate inflammation and brain injury after intracerebral hemorrhage.

Numerous evidence have shown that microglia mediated inflammation plays a pivotal role in the development of brain injury after intracerebral hemorrhage (ICH). Therefore anti-inflammation therapy represents a potentially promising approach to ICH. Recently, NLRP3 inflammasome was discovered to facilitate the inflammatory response. However, the effect of NLRP3 inflammasome after ICH has not been fully studied. To explore the potential of NLRP3 inflammasome, we detected NLRP3 expression, inflammation, brain edema and neurological functions in vitro and in vivo. We found that ICH activated the NLRP3 inflammasome and inflammation. However, NLRP3 RNAi could attenuate inflammation and brain injury after ICH. Therefore, the findings suggested that recombinant adenovirus encoding NLRP3 RNAi might be valuable as a potential strategy for anti-inflammation therapy in ICH.

A novel nanoparticle containing neuritin peptide with grp170 induces a CTL response to inhibit tumor growth.

Malignant glioma is among the most challenging of all cancers to treat successfully. Despite recent advances in surgery, radiotherapy and chemotherapy, current treatment regimens have only a marginal impact on patient survival. In this study, we constructed a novel nanoparticle containing neuritin peptide with grp170. The nanoparticle could elicit a neuritin-specific cytotoxic T lymphocyte response to lyse glioma cells in vitro. In addition, the nanoparticle could inhibit tumor growth and improve the lifespan of tumor-bearing mice in vivo. Taken together, the results demonstrated that the nanoparticle can inhibit tumor growth and represents a promising therapy for glioma.

Adenovirus encoding Smad4 suppresses glioma cell proliferation and increases apoptosis through cell cycle arrest at G1 phase.

Mothers against decapentaplegic homologue 4 (Smad4) is associated with several human cancers. However, the exact mechanism of Smad4 in human glioma is still unknown. In this study, we constructed a recombinant adenovirus encoding Smad4 and transduced it into glioma cells. The results demonstrated that the overexpression of Smad4 not only suppressed glioma cell proliferation but also increased cell apoptosis by promoting cell cycle arrest at G1 phase. Furthermore, an adenovirus encoding Smad4 suppressed tumor formation in nude mice. These findings clearly demonstrate that Smad4 plays an important role in human glioma development by regulating cell proliferation. Moreover, Smad4 may represent a potential therapeutic target in glioma.

Hypoxia induces microglia autophagy and neural inflammation injury in focal cerebral ischemia model.

Much evidence demonstrated that autophagy played an important role in neural inflammation response after ischemia stroke. However, the specific effect of microglia autophagy in cerebral ischemia is still unknown. In the current study, we constructed focal cerebral ischemia model by permanent middle cerebral artery occlusion (pMCAO) in mice. We detected microglia autophagy and inflammation response in vivo, and observed infarct brain areas, edema formation, and neurological deficits of mice. We found that pMCAO induced microglia autophagy and inflammatory response. The suppression of autophagy using either pharmacologic inhibitor (3-MA) not only decreased the microglia autophagy and inflammatory response, but also significantly decreased infarct size, edema formation and neurological deficits in vivo. Taken together, these results suggested that cerebral ischemia induced microglia autophagy contributed to ischemic neural inflammation and injury. In addition, our findings also provided novel therapeutic strategy for ischemic stroke.

Regulatory T cells inhibit microglia activation and protect against inflammatory injury in intracerebral hemorrhage.

Numerous evidence demonstrate that microglia mediated inflammatory injury plays a critical role in intracerebral hemorrhage (ICH). Therefore, the way to inhibit the inflammatory response is greatly needed. Treg cells have been shown to play a critical role in immunologic self-tolerance as well as anti-tumor immune responses and transplantation. In the current study, we transfered Treg cells in the ICH model, and investigated the effect. The cytokines of microglia were measured by ELISA, JNK/ERK and NF-κB were measured by Western blot and EMSA (Electrophoretic Mobility Shift Assay), animal behavior was evaluated by animal behavioristics. We found that Treg cells could inhibit microglia mediated inflammatory response through NF-κB activation via the JNK/ERK pathway in vitro, and improve neurological function in vivo. Our findings suggest that Treg cells could suppress inflammatory injury and represent a novel cell-based therapeutical strategy in ICH.

Dendritic cells transduced with CPEB4 induced antitumor immune response.

Much evidence leads to the exploration of immunologic approaches for eliminating tumor cells. Cytoplasmic polyadenylation element binding protein 4 (CPEB4) is considered to be a novel therapeutical target for glioblastoma. In this study, we transduced DCs with CPEB4 to explore the immune response in vivo. We found that DCs transduced with recombinant adenovirus encoding CPEB4 could induce specific cytotoxic T lymphocytes (CTLs) to lyse glioma cells and augment the number of IFN-γ secreting T-cells in mice. In addition, the modified DCs could effectively protect mice from lethal challenges against glioma cells, reduce tumor growth and increase the mice life span. These results suggest that the DC transduced with CPEB4 may induce anti-tumor immunity against glioma cells and might be used as an efficient tumor vaccine in clinical applications.

Sinomenine inhibits microglia activation and attenuates brain injury in intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) causes morbidity and mortality and commonly follows the reperfusion after an ischemic event. Microglial activation mediated cytokine and protease secretion contributes to brain injury in ICH. Previous studies have shown that sinomenine possesses potent immunoregulatory properties. However, little is known about its exact role in ICH. In the present study, to investigate the effect of sinomenine on microglial cells inflammation, we treated ICH-challenged BV2 microglial cells with sinomenine in vitro, and explored its neuroprotection role in intracerebral hemorrhage in vivo. Changes in inflammatory cytokines, such as TNF-α, IL-1ß and IL-6, reactive oxygen species (ROS) and NF-κB activation NF-κB were observed. In addition, the neurological deficit and cerebral water content of ICH mice were studied. The results demonstrated that sinomenine could inhibit the release of these cytokines and attenuate ROS production in a dose-dependent manner, and reduce NF-κB activation. Furthermore, sinomenine markedly inhibited cerebral water content and neurological deficit. In conclusion, our findings suggest that sinomenine played the protective effects through inhibition of microglial inflammation, and the findings also provided a novel therapy to treat ICH induced brain injury.

Isthmin inhibits glioma growth through antiangiogenesis in vivo.

Among glioma treatment strategies, antiangiogenesis emerges as a meaningful and feasible treatment approach for inducing long-term survival. Isthmin is a gene highly expressed in the isthmus of the midbrain-hindbrain organizer in Xenopus, and has recently been identified as a novel angiogenesis inhibitor. However, the potential of isthmin on the glioma angiogenesis has not been well studied. In the present study, we demonstrated that the recombinant adenovirus isthmin (Ad-isthmin) could inhibit VEGF-stimulated endothelial cell proliferation and induce apoptosis through a caspase-dependent pathway. In addition, Ad-isthmin significantly suppressed glioma growth through antiangiogenesis without apparent side effects. Taken together, our results demonstrated that isthmin could act as a novel angiogenesis inhibitor and might be utilized in the glioma antiangiogenesis therapy.

The effect of galectin-3 genetic variants on the susceptibility and prognosis of gliomas in a Chinese population.

The aim of this study is to explore the association between the polymorphisms of galectin-3 gene and clinico-pathological characteristics and prognosis of gliomas. We enrolled 190 histologically diagnosed gliomas and 210 healthy controls in this study. Two genetic variants at galectin-3 single nucleotide polymorphism (SNP) sites (galectin-3 +191 A>C and +292 A>C) were determined. We found that the A/A genotype at galectin-3 gene +292 A>C was significantly more prevalent in gliomas patient than in controls (42.1% vs. 29.0%, P=0.021); the A allele frequency was markedly higher in gliomas subjects than in controls (61.8% vs. 45.0%, P=0.008). There was a markedly higher prevalence of AA carriers in high-grade subgroup than in low-grade subgroup (50.5% vs. 31.8%, P=0.012). The Kaplan-Meier analyses showed that the gliomas patients carrying AA genotype of galectin-3 gene +292 A>C had marked shorter overall survival period than those did not (AA vs. AC+CC, 22.2±3.8 months vs. 38.3 months±7.9; P=0.04). The SNPs at +191 A>C of galectin-3 gene did not show positive association with clinico-pathological characteristics and prognosis of gliomas. The results of this study suggest the SNPs at +292 A>C, not SNPs at +191 A>C, of galectin-3 gene were associated with the tumor grade and prognosis of gliomas.

Identification of novel HLA-A(*)0201-restricted CTL epitopes from Pokemon.

Pokemon is a member of the POK family of transcriptional repressors and aberrant overexpressed in various human cancers. Therefore, the related peptide epitopes derived from Pokemon is essential for the development of specific immunotherapy of malignant tumors. In this study, we predicted and identified HLA-A(*)0201-restricted cytotoxic T lymphocyte (CTL) epitopes derived from Pokemon with computer-based epitope prediction, peptide-binding assay and testing of the induced CTLs toward different kinds of carcinoma cells. The results demonstrated that effectors induced by peptides of Pokemon containing residues 32-40, 61-69, 87-95, and 319-327 could specifically secrete IFN-γ and lyse tumor cell lines of Pokemon-positive and HLA-A2-matched. The results suggest that Pokemon32, Pokemon61, Pokemon87, and Pokemon319 peptides are novel HLA-A(*)0201-restricted restricted CTL epitopes, and could be utilized in the cancer immunotherapy against a broad spectrum of tumors.

Endoplasmic reticulum chaperone glucose regulated protein 170-Pokemon complexes elicit a robust antitumor immune response in vivo.

Previous evidence suggested that the stress protein grp170 can function as a highly efficient molecular chaperone, binding to large protein substrates and acting as a potent vaccine against specific tumors when purified from the same tumor. In addition, Pokemon can be found in almost all malignant tumor cells and is regarded to be a promising candidate for the treatment of tumors. However, the potential of the grp170-Pokemon chaperone complex has not been well described. In the present study, the natural chaperone complex between grp170 and the Pokemon was formed by heat shock, and its immunogenicity was detected by ELISPOT and (51)Cr-release assays in vitro and by tumor bearing models in vivo. Our results demonstrated that the grp170-Pokemon chaperone complex could elicit T cell responses as determined by ELISPOT and (51)Cr-release assays. In addition, immunized C57BL/6 mice were challenged with subcutaneous (s.c.) injection of Lewis cancer cells to induce primary tumors. Treatment of mice with the grp170-Pokemon chaperone complex also significantly inhibited tumor growth and prolonged the life span of tumor-bearing mice. Our results indicated that the grp170-Pokemon chaperone complex might represent a powerful approach to tumor immunotherapy and have significant potential for clinical application.

siRNA directed against Livin inhibits tumor growth and induces apoptosis in human glioma cells.

Livin, a novel member of the human inhibitors of apoptosis protein family, plays an important role in tumor progression and occurrence by inhibiting cell apoptosis. It is selectively expressed in the most common human neoplasms and appears to be involved in tumor cell resistance to chemotherapeutic agents. The present study was designed to investigate the potential of using RNA interference (RNAi) technique to downregulate Livin expression, and the subsequent effect on human glioma cells. The results showed that knockdown of Livin expression by short interfering RNA (siRNA) significantly inhibited glioma cell proliferation and increased cell apoptosis through cell arrest in the G(1)/G(0) phase of cell cycle in vitro. Furthermore, Livin siRNA significantly suppressed tumor growth in nude mice. Together, these findings suggest that RNAi-mediated downregulation of Livin expression could lead to potent antitumor activity in glioma cells and might serve as a novel therapeutic strategy in clinic.

A novel vaccine containing EphA2 epitope and LIGHT plasmid induces robust cellular immunity against glioma U251 cells.

EphA2 is a receptor tyrosine kinase and can be acted as an attractive antigen for glioma vaccines. In addition, LIGHT plays an important role on enhancing T cell proliferation and cytokine production. To improve the CTL mediated immune response against glioma cells, we prepared the novel vaccine containing EphA2(883-891) peptide (TLADFDPRV) and LIGHT plasmid and utilized it to immunize the HLA-A2 transgenic HHD mice. In addition, trimera mice were immunized with the novel vaccine to elicit the antitumor immune response. The results demonstrated that the novel vaccine could induce robust cellular immunity against glioma U251 cells without lysing autologous lymphocytes. Moreover, the novel vaccine could significantly inhibit the tumor growth and prolong the life span of tumor bearing mice. These findings suggested that the novel vaccine containing EphA2 epitope and LIGHT plasmid could induce anti-tumor immunity against U251 cells expressing EphA2, and provided a promising strategy for glioma immunotherapy.

Lewis X oligosaccharides-heparanase complex targeting to DCs enhance antitumor response in mice.

Heparanase has been proved as an promising tumor antigen for the therapeutical target. However, the antigen alone cannot fully elicit the immune response in vivo. In this study, Lewis X oligosaccharides-heparanase complex was prepared, which can target to the dendritic cells (DCs) via dendritic cell-specific intercellular-adhesion-molecule-grabbing non-integrin (DC-SIGN). In addition, the DCs were loaded with the complex, and then were utilized to immunize mice to detect the immune response. Our data demonstrated that the modified DCs could enhance the specific IFN-γ production and cytotoxic T cell response. Furthermore, the modified DCs could also significantly suppress the established tumor growth and prolong the life span of tumor-bearing mice. Therefore, the Lewis X oligosaccharides-heparanase complex might be regarded as an ideal vaccine, and represent a novel way for the therapeutical strategy of tumor.

WITHDRAWN: Dual targeting of glioma U251 cells and neovasculature with a nanoparticle encoding vasohibin and RGD peptides prevents tumor angiogenesis and inhibits tumor growth.

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