Genetic analysis of human glioblastomas using a genomic microarray system

Genomic microarray systems can simultaneously provide substantial genetic and chromosomal information in a relatively short time. We have analyzed genomic DNA from frozen sections of 30 cases of primary glioblastomas by GenoSensor Array 300 in order to characterize gene amplifications, gene deletions, and chromosomal information in the whole genome. Genes that were frequently amplified includedPFC2/CYLN2 (63.3%),EGFR (53.3%),IL6 (53.3%),ABCB1 (MDR1) (36.7%), andPDGFRA (26.7%). Genes that were frequently deleted includedFGFR2 (66.7%),MTAP (60.0%),DMBT1 (56.7%),CDKN2A (p16)/MTAP (50.0%),PIK3CA (43.3%), andEGR2 (43.3%), but deletion ofRB1 orTP53 was rarely detected. Chromosomal gains were observed frequently for 7q (33.3%), 7p (20.0%), and 17q (13.3%). Loss of the 10q was frequently detected in 13 of 30 cases (46.7%). Loss of the entire chromosome 10 was seen in 9 of 30 cases (30.0%), and was often accompanied byEGFR amplification (7 cases, 77.8%). The GenoSensor Array 300 proved to be useful for identification of genome-wide molecular changes in glioblastomas. The obtained microarray profile can also yield valuable insight into the molecular events underlying carcinogenesis of brain tumors and may provide clues about clinical correlations, including response to treatment.     

Endothelin-1 regulates cardiac sympathetic innervation in the rodent heart by controlling nerve growth factor expression

The cardiac sympathetic nerve plays an important role in regulating cardiac function, and nerve growth factor (NGF) contributes to its development and maintenance. However, little is known about the molecular mechanisms that regulate NGF expression and sympathetic innervation of the heart. In an effort to identify regulators of NGF in cardiomyocytes, we found that endothelin-1 specifically upregulated NGF expression in primary cultured cardiomyocytes. Endothelin-1-induced NGF augmentation was mediated by the endothelin-A receptor, Gibetagamma, PKC, the Src family, EGFR, extracellular signal-regulated kinase, p38MAPK, activator protein-1, and the CCAAT/enhancer-binding protein delta element. Either conditioned medium or coculture with endothelin-1-stimulated cardiomyocytes caused NGF-mediated PC12 cell differentiation. NGF expression, cardiac sympathetic innervation, and norepinephrine concentration were specifically reduced in endothelin-1-deficient mouse hearts, but not in angiotensinogen-deficient mice. In endothelin-1-deficient mice the sympathetic stellate ganglia exhibited excess apoptosis and displayed loss of neurons at the late embryonic stage. Furthermore, cardiac-specific overexpression of NGF in endothelin-1-deficient mice overcame the reduced sympathetic innervation and loss of stellate ganglia neurons. These findings indicate that endothelin-1 regulates NGF expression in cardiomyocytes and plays a critical role in sympathetic innervation of the heart.     

Implantation of dendritic cells in injured adult spinal cord results in activation of endogenous neural stem/progenitor cells leading to de novo neurogenesis and functional recovery

We report a treatment for spinal cord injury involving implantation of dendritic cells (DCs), which act as antigen-presenting cells in the immune system. The novel mechanisms underlying this treatment produce functional recovery. Among the immune cells tested, DCs showed the strongest activity inducing proliferation and survival of neural stem/progenitor cells (NSPCs) in vitro. Furthermore, in DC-implanted adult mice, endogenous NSPCs in the injured spinal cord were activated for mitotic de novo neurogenesis. These DCs produced neurotrophin-3 and activated endogenous microglia in the injured spinal cord. Behavioral analysis revealed the locomotor functions of DC-implanted mice to have recovered significantly as compared to those of control mice. Our results suggest that DC-implantation exerts trophic effects, including activation of endogenous NSPCs, leading to repair of the injured adult spinal cord.     

Human neural stem/progenitor cells, expanded in long-term neurosphere culture, promote functional recovery after focal ischemia in Mongolian gerbils

Transplantation of human neural stem cells (NSCs) is a promising potential therapy for neurologic dysfunctions after the hyperacute stage of stroke in humans, but large amounts of human NSCs must be expanded in long-term culture for such therapy. To determine their possible therapeutic potential for human stroke, human fetal neural stem/progenitor cells (NSPCs) (i.e., neurosphere-forming cells) were isolated originally from forebrain tissues of one human fetus, and expanded in long-term neurosphere culture (exceeding 24 weeks), then xenografted into the lesioned areas in the brains of Mongolian gerbils 4 days after focal ischemia. Sensorimotor and cognitive functions were evaluated during the 4 weeks after transplantation. The total infarction volume in the NSPC-grafted animals was significantly lower than that in controls. Approximately 8% of the grafted NSPCs survived, mainly in areas of selective neuronal death, and were costained with antibodies against neuronal nuclei antibody (NeuN), microtubule associated protein (MAP-2), glial fibrillary acidic protein (GFAP), and anti-2'3' cyclic nucleotide 3'-phosphodiesterase (CNPase). Synaptic structures between NSPCs-derived neurons and host neurons were observed. Furthermore, gradual improvement of neurologic functions was observed clearly in the NSPC-grafted animals, compared to that in controls. Human NSPCs, even from long-term culture, remarkably improved neurologic functions after focal ischemia in the Mongolian gerbil, and maintained their abilities to migrate around the infarction, differentiate into mature neurons, and form synapses with host neuronal circuits. These results indicate that in vitro-expanded human neurosphere cells are a potential source for transplantable material for treatment of stroke.     

Anesthetic management for non-cardiac operation with transesophageal echocardiography in a patient with left atrial myxoma

A 68-year-old female with left atrial myxoma underwent osteosynthesis of the night tibia. During the operation we observed the motion of the tumor continuously by transesophageal echocardiography (TEE). The operation was finished successfully without any cardiovascular complications. We think that TEE was useful for observation of left atrial myxoma during this non-cardiac surgery.     

IV-DSA is a new diagnostic tool for axillary lymph node metastasis in breast cancer patients

BACKGROUND AND OBJECTIVE: The aim of this study is to know whether intravenous digital subtraction angiography (IV-DSA) is useful to detect axillary lymph node metastasis of breast cancer and to evaluate the anigiogenesis of lymph nodes in the axilla. PATIENTS AND METHODS: Forty three primary breast cancer patients (N0: 26 cases, N1: 5 cases, N2: 2 cases) who underwent IV-DSA between January and November 2000 were included in the study. Infinix CB apparatus (Toshiba, Japan) was used to collect IV-DSA images and when a mass became stained in the axilla, it was considered to be metastatic. The angiogenesis was studied by examining microvessel density (MVD) after lymph node immunostaining for factor VIII. Primary tumor was detected by IV-DSA in all 43 cases. RESULTS: Axillary lymph node metastases were detected by IV-DSA in 34.9% of cases (15/43), and by pathology in 37.2% (16/43). The sensitivity, specificity, and accuracy of the diagnostic method were 75.0% (12/16), 88.9% (24/27), and 83.7% (36/43), respectively. MVD, calculated after immunostaining for factor VIII, was significantly lower in the in metastatic region of lymph nodes identified by DSA (88.5 +/- 35.0) than in metastasis-free lymph nodes (141.1 +/- 34.0, P < 0.0001). CONCLUSIONS: IV-DSA is useful in the diagnosis of axillary lymph node metastasis of breast cancer. Our results suggest that the primary factors involved in the mechanism of DSA display may be different from high/low MVC values.     

Huntingtin-interacting protein-1-related protein of rat (rHIP1R) is localized in the postsynaptic regions

We cloned a rHIP1R (GenBank Accession No., AB005052) encoding a Sla2/huntingtin-interacting protein (HIP1) family protein from a rat brain cDNA library. Localization of rHIP1R was investigated in the rat brain using an antibody specific to the HIP1R antibody. The rHIP1R protein was enriched in the synaptic plasma membrane fraction along with huntingtin, a synaptic protein and a causal protein for Huntington's disease. The electron microscopic examination revealed that HIP1R was localized at postsynaptic spines. Localization of HIP1R in the small vesicular structures in the spine, possible sites of vesicular transport of synaptic proteins, together with the structure-based analysis, suggested a role of HIP1R for vesicle trafficking through interaction with F-actin and working together with huntingtin and HIP1 at the synaptic sites.     

Implication of cyclooxygenase-2 on enhanced proliferation of neural progenitor cells in the adult mouse hippocampus after ischemia

Global ischemia promotes neurogenesis in the dentate gyrus of the adult mouse hippocampus. Cyclooxygenase (COX)-2, the principal isoenzyme in the brain, modulates inflammation, glutamate-mediated cytotoxicity, and synaptic plasticity. We demonstrated that delayed treatment with different classes of COX inhibitor significantly blunted enhancement of dentate gyrus proliferation of neural progenitor cells after ischemia. COX-2 immunoreactivity was observed in both neurons and astrocytes in the dentate gyrus, but not in neural progenitor cells in the subgranular zone. Moreover, in the postischemic dentate gyrus of heterozygous and homozygous COX-2 knockout mice, proliferating bromodeoxyuridine-positive cells were significantly fewer than in wild-type littermates. These results demonstrate that COX-2 is an important modulator in enhancement of proliferation of neural progenitor cells after ischemia.     

Proliferation of neural and neuronal progenitors after global brain ischemia in young adult macaque monkeys

To investigate the effect of global cerebral ischemia on brain cell proliferation in young adult macaques, we infused 5-bromo-2'-deoxyuridine (BrdU), a DNA replication indicator, into monkeys subjected to ischemia or sham-operated. Subsequent quantification by BrdU immunohistochemistry revealed a significant postischemic increase in the number of BrdU-labeled cells in the hippocampal dentate gyrus, subventricular zone of the temporal horn of the lateral ventricle, and temporal neocortex. In all animals, 20-40% of the newly generated cells in the dentate gyrus and subventricular zone expressed the neural progenitor cell markers Musashi1 or Nestin. A few BrdU-positive cells in postischemic monkeys were double-stained for markers of neuronal progenitors (class III beta-tubulin, TUC4, doublecortin, or Hu), neurons (NeuN), or glia (S100beta or GFAP). Our results suggest that ischemia activates endogenous neuronal and glial precursors residing in diverse locations of the adult primate central nervous system.