Targeting of the Bmi-1 oncogene/stem cell renewal factor by microRNA-128 inhibits glioma proliferation and self-renewal.

MicroRNAs (miR) show characteristic expression signatures in various cancers and can profoundly affect cancer cell behavior. We carried out miR expression profiling of human glioblastoma specimens versus adjacent brain devoid of tumor. This revealed several significant alterations, including a pronounced reduction of miR-128 in tumor samples. miR-128 expression significantly reduced glioma cell proliferation in vitro and glioma xenograft growth in vivo. miR-128 caused a striking decrease in expression of the Bmi-1 oncogene, by direct regulation of the Bmi-1 mRNA 3'-untranslated region, through a single miR-128 binding site. In a panel of patient glioblastoma specimens, Bmi-1 expression was significantly up-regulated and miR-128 was down-regulated compared with normal brain. Bmi-1 functions in epigenetic silencing of certain genes through epigenetic chromatin modification. We found that miR-128 expression caused a decrease in histone methylation (H3K27me(3)) and Akt phosphorylation, and up-regulation of p21(CIP1) levels, consistent with Bmi-1 down-regulation. Bmi-1 has also been shown to promote stem cell self-renewal; therefore, we investigated the effects of miR-128 overexpression in human glioma neurosphere cultures, possessing features of glioma "stem-like" cells. This showed that miR-128 specifically blocked glioma self-renewal consistent with Bmi-1 down-regulation. This is the first example of specific regulation by a miR of a neural stem cell self-renewal factor, implicating miRs that may normally regulate brain development as important biological and therapeutic targets against the "stem cell-like" characteristics of glioma.

CD24 on the resident cells of the central nervous system enhances experimental autoimmune encephalomyelitis.

CD24 is a cell surface glycoprotein that is expressed on both immune cells and cells of the CNS. We have previously shown that CD24 is required for the induction of experimental autoimmune encephalomyelitis (EAE), an experimental model for the human disease multiple sclerosis (MS). The development of EAE requires CD24 expression on both T cells and non-T host cells in the CNS. To understand the role of CD24 on the resident cells in the CNS during EAE development, we created CD24 bone marrow chimeras and transgenic mice in which CD24 expression was under the control of a glial fibrillary acidic protein promotor (AstroCD24TG mice). We showed that mice lacking CD24 expression on the CNS resident cells developed a mild form of EAE; in contrast, mice with overexpression of CD24 in the CNS developed severe EAE. Compared with nontransgenic mice, the CNS of AstroCD24TG mice had higher expression of cytokine genes such as IL-17 and demyelination-associated marker P8; the CNS of AstroCD24TG mice accumulated higher numbers of Th17 and total CD4+ T cells, whereas CD4+ T cells underwent more proliferation during EAE development. Expression of CD24 in CD24-deficient astrocytes also enhanced their costimulatory activity to myelin oligodendrocyte glycoprotein-specific, TCR-transgenic 2D2 T cells. Thus, CD24 on the resident cells in the CNS enhances EAE development via costimulation of encephalitogenic T cells. Because CD24 is increased drastically on resident cells in the CNS during EAE, our data have important implications for CD24-targeted therapy of MS.

IFN-alpha-induced signal transduction, gene expression, and antitumor activity of immune effector cells are negatively regulated by suppressor of cytokine signaling proteins.

Proteins belonging to the suppressors of cytokine signaling (SOCS) family have been shown to regulate cytokine signal transduction in various cell types but their role in modulating the response of immune cells to IFN-alpha has not been fully explored. We hypothesized that SOCS proteins would inhibit the antitumor activity of IFN-alpha-stimulated immune cells. Transcripts for SOCS1, SOCS2, SOCS3, and cytokine-inducible Src homology 2-containing protein were identified in total human PBMC (PBMCs, NK cells, and T cells) within 1-2 h of stimulation with IFN-alpha (10(3)-10(5) U/ml). Immunoblot analysis confirmed the expression of these factors at the protein level. Transcripts for SOCS proteins were rapidly but variably induced in PBMCs from patients with metastatic melanoma following the i.v. administration of IFN-alpha-2b (20 million units/m(2)). Overexpression of SOCS1 and SOCS3, but not SOCS2, in the Jurkat T cell line inhibited IFN-alpha-induced phosphorylated STAT1 and the transcription of IFN-stimulated genes. Conversely, small inhibitory RNA-mediated down-regulation of SOCS1 and SOCS3 in Jurkat cells and normal T cells enhanced the transcriptional response to IFN-alpha. Loss of SOCS1 or SOCS3 in murine immune effectors was associated with enhanced IFN-induced phosphorylated STAT1, transcription of IFN-stimulated genes, and antitumor activity. Of note, IFN-alpha treatment eliminated melanoma tumors in 70% of SOCS1-deficient mice, whereas IFN-treated SOCS-competent mice all died. The antitumor effects of IFN-alpha in tumor-bearing SOCS1-deficient mice were markedly inhibited following depletion of CD8(+) T cells. These results indicate that the antitumor response of immune effector cells to exogenous IFN-alpha is regulated by SOCS proteins.

High-resolution 8 Tesla imaging of the formalin-fixed normal human hippocampus.

The purpose of this study was to evaluate the capacity of high-resolution magnetic resonance imaging (MRI) to visualize the normal anatomic features of the human hippocampus in vitro, using high field imaging equipment, parameters, and acquisition times appropriate for imaging human subjects in vivo. This research compared high field, high-resolution MRI of formalin-fixed normal human hippocampus specimens to histologic sectioning of the same hippocampus samples. Four specimens were evaluated using an 8 Tesla (T), 80 cm bore whole-body MRI scanner equipped with a 12.7 cm single strut transverse electromagnetic resonator (TEM) coil. Hahn spin echo images were acquired with a repetition time (TR) of 800 msec, echo times (TE) of 20, 50, 90, and 134 msec, and an acquisition time (TA) of 3.25 min. The image quality was superb with demonstration of most of the features of the hippocampus. High field, high-resolution MRI can be used to depict multiple layers of the formalin-fixed human hippocampus in vitro using an 8 T whole-body scanner, a TEM coil, and short acquisition times compatible with human imaging in vivo.

Limits of 8-Tesla magnetic resonance imaging spatial resolution of the deoxygenated cerebral microvasculature.

PURPOSE: To quantify the minimum magnetic resonance imaging (MRI) spatial resolution of the visible deoxygenated microscopic vessels of the human brain at 8 T. MATERIALS AND METHODS: This study compared 8-T gradient echo (GE) images of a human cadaver brain having an in-plane resolution of 195 x 195 microm to corresponding digital photographs of 205 cryomicrotome sections of the same cadaver brain, along with summed images of 25 contiguous cryomicrotome sections. One-millimeter-thick GE images of a 1-cm-thick unfixed whole coronal brain section were acquired using techniques similar to those commonly utilized for 8-T human imaging in vivo. RESULTS: There was excellent MR visualization of the deoxygenated microscopic vessels within the brain down to a resolution of approximately 100 microm. CONCLUSION: By taking advantage of magnetic susceptibility-based blood oxygenation level-dependent (BOLD) contrast, deoxygenated microscopic blood vessels smaller than the pixel dimensions used for imaging can be visualized using a whole-body 8-T MRI system.