Yasuharu Akasaki

Induction of a CD4+ T Regulatory Type 1 Response by Cyclooxygenase-2-Overexpressing Glioma

PGE2, synthesized by cyclooxygenase-2 (COX-2)-overexpressing tumor, is known to contribute to cellular immune suppression in cancer patients, but the mechanism remains unclear. We report the mechanism of a CD4+ T regulatory type 1 (Tr1) induction by CD11c+ mature dendritic cells (DCs) that phagocytose allogeneic and autologous COX-2-overexpressing glioma. A human glioma cell line, U-87MG, and primary cultured glioblastoma cells (MG-377) overexpressed COX-2. We did not detect IL-10Rα expression in these gliomas, and rIL-10 did not suppress their COX-2 expression. Exposure to COX-2-overexpressing glioma induced mature DCs to overexpress IL-10 and decreased IL-12p70 production. These DCs induced a Tr1 response, which is characterized by robust secretion of IL-10 and TGF-β with negligible IL-4 secretion by CD4+ T cells, and an inhibitory effect on admixed lymphocytes. Peripheral CD4+ T cell populations isolated from an MG-377 patient also predominantly demonstrated a Tr1 response against MG-377 cells. Selective COX-2 inhibition in COX-2-overexpressing gliomas at the time of phagocytic uptake by DCs abrogated this regulatory response and instead elicited Th1 activity. COX-2 stable transfectants in LN-18 (LN-18-COX2) also induced a Tr1 response. The effect of a COX-2 inhibition in LN-18-COX2 is reversible after administration of PGE2. Taken together, robust levels of PGE2 from COX-2-overexpressing glioma, which is unresponsive to IL-10 within the local microenvironment, may cause DCs to secrete high levels of IL-10. These results indicate that COX-2-overexpressing tumors induce a Tr1 response, which is mediated by tumor-exposed, IL-10-enhanced DCs.

Glioma Tropic Neural Stem Cells Consist of Astrocytic Precursors and Their Migratory Capacity Is Mediated by CXCR4

Malignant gliomas spawn disseminated microsatellites, which are largely refractory to currently employed therapies, resulting in eventual tumor recurrence and death. The use of tumor-tropic neural stem cells (NSCs) as delivery vehicles for therapeutic gene products represents an attractive strategy specifically focused at treating these residual neoplastic foci. We wished to elucidate the biological cues governing NSC tropism for glioma. In this context, we describe that tumor-tropic NSCs comprise largely of astrocytic progenitors expressing chemokine receptor 4 (CXCR4). Blocking of CXCR4 significantly inhibits NSC migration toward the tumor. These findings define specific characteristics associated with the cell populations within transplanted NSCs that demonstrate glioma-tracking behavior.

Small interference RNA modulation of IL-10 in human monocyte-derived dendritic cells enhances the Th1 response

RNA interference technology has been used to modulate dendritic cell (DC) function by targeting the expression of genes such as IL‐12 and NF‐kB. In this paper, we demonstrate that transfectionof DC with IL‐10‐specific double strands of small interference RNA (siRNA) resulted in potent suppression of IL‐10 gene expression without inducing DC apoptosis or blocking DC maturation. Inhibition of IL‐10 by siRNA was accompanied by increased CD40 expression and IL‐12 production after maturation, which endowed DC with the ability to significantly enhance allogeneic T cell proliferation. IL‐10 siRNA transfection did not affect MHC class II, CD86, CD83, or CD54 expression in mature DC. To further test the ability of IL‐10 siRNA‐treated DC to induce a T cell response, naive CD4 T cells were stimulated by autologous DC pulsed with KLH. The results indicated that IL‐10 siRNA‐transfected DC enhanced Th1 responses by increasing IFN‐γ and decreasing IL‐4 production. These findings suggest the potential for a novel immunotherapeutic strategy of using IL‐10 siRNA‐transfected antigen‐presenting cells as vaccine delivery agents to boost the Th1 response against pathogens and tumors that are controlled by Th1 immunity.

A Peroxisome Proliferator-activated Receptor-γ Agonist, Troglitazone, Facilitates Caspase-8 and -9 Activities by Increasing the Enzymatic Activity of Protein-tyrosine Phosphatase-1B on Human Glioma Cells

Despite dramatic advances in adjuvant therapies, patients with malignant glioma face a bleak prognosis. Because many adjuvant therapies seek to induce glioma apoptosis, strategies that lower thresholds for the induction of apoptosis may improve patient outcomes. Therefore, elucidation of the biological mechanisms that underlie resistance to current therapies is needed to develop new therapeutic strategies. Here we proposed a novel mechanism of proapoptotic effect induced by a pharmacological peroxisome proliferator-activated receptor-γ (PPARγ) agonist, troglitazone, that facilitates caspase signaling in human glioma cells. Troglitazone activates protein-tyrosine phosphatase (PTP)-1B, which subsequently reduces phosphotyrosine 705 STAT3 (pY705-STAT3) via a PPARγ-independent pathway. Reduction of pY705-STAT3 in glioma cells caused down-regulation of FLIP (FADD-like IL-1β-converting enzyme-inhibitory protein) and Bcl-2. Furthermore, troglitazone induced Ser-392 phosphorylation of p53 via a PPARγ-dependent pathway and up-regulation of Bax in a p53 wild-type glioma. When given with tumor necrosis factor-related apoptosis-inducing ligand or caspase-dependent chemotherapeutic agents, such as etoposide and paclitaxel, troglitazone exhibited a synergistic effect by facilitating caspase-8/9 activities. A PPARγ antagonist, GW9662, did not block this effect, although a PTP inhibitor abrogated it. Knockdown of STAT3 by STAT3-small interfering RNA negated the inhibitory effect of PTP inhibitor on troglitazone, indicating that troglitazone uses a STAT3 inactivation mechanism that makes caspase-8/9 activities susceptible to cytotoxic agents in glioma cells and that PTP1B plays a critical role in the down-regulation of activated STAT3, as well as FLIP and Bcl-2. When taken with caspase-dependent anti-neoplastic agents, troglitazone may be a promising drug for use against malignant gliomas because it facilitates the caspase cascade, thereby lowering thresholds for the apoptosis induction of glioma cells.

Dendritic cell-based immunotherapy for malignant gliomas

Despite dramatic advances in surgical techniques, imaging and adjuvant radiotherapy or chemotherapy, the prognosis for patients with malignant glial tumors remains dismal. Based on the current knowledge regarding immune responses in the healthy CNS and glioma-bearing hosts, this review discusses dendritic cell-based immunotherapeutic approaches for malignant gliomas and the relevance of recent clinical trials and their outcomes. It is now recognized that the CNS is not an immunologically tolerated site and clearance of arising glioma cells is a routine physiologic function of the normal, noncompromised immune system. To escape from immune surveillance, however, clinically apparent gliomas develop complex mechanisms that suppress tumoricidal immune responses. Although the use of dendritic cells for the treatment of glioma patients may be the most appropriate approach, an effective treatment paradigm for these tumors may eventually require the use of several types of treatment. Additionally, given the heterogeneity of this disease process and an immune-refractory tumor cell population, the series use of rational multiple modalities that target disparate tumor characteristics may be the most effective therapeutic strategy to treat malignant gliomas.

Manipulation of proliferation and differentiation of human bone marrow-derived neural stem cells in vitro and in vivo

Recent evidence has demonstrated that neural stem cells (NSC) can be expanded from a variety of sources, including embryos, fetuses, and adult bone marrow and brain tissue. We have previously reported the generation of adult rat bone marrow‐derived cellular spheres that are morphologically and phenotypically similar to neurospheres derived from brain NSC. Here we show that adult human bone marrow‐derived neural stem cells (HBM‐NSC) are capable of generating spheres that are similar to brain neural‐derived neurospheres. Additionally, we sought to promote proliferation and differentiation of HBM‐NSC through transduction with nonreplicative recombinant adenovirus encoding the cDNA sequence for Gli, rADV‐Gli‐1; sonic hedgehog, rADV‐Shh; or Nurr1, rADV‐Nurr1. Immunocytochemistry and RT‐PCR analysis showed that HBM‐NSC could be efficiently expanded and differentiated in vitro and that HBM‐NSC transduced with rADV‐Gli‐1 or rADV‐Shh dramatically increased NSC time‐related proliferation; however, Nurr1 had no effect on proliferation. We also transplanted HBM‐NSC into chicken embryos to examine their potential function in vivo. We found that transduction of HBM‐NSC with rADV‐Gli‐1 or rADV‐Shh and subsequent transplantation into chicken embryos increased HBM‐NSC proliferation, whereas rADV‐Nurr1 promoted migration and differentiation in vivo. Our findings suggest that HBM‐NSC can be efficiently expanded and differentiated in vitro and in vivo by overexpressing Gli‐1, Shh or Nurr1.