Kenji Kameda

NG2 proteoglycan-expressing microglia as multipotent neural progenitors in normal and pathologic brains

Rat primary microglia (MG) acquired a multipotent property to give rise to neuroectodermal cells through two‐step culture in 10 and 70% serum‐supplemented media for 5 days. Such multipotent MG, called promicroglioblasts (ProMGBs), formed cell aggregates, which generated cells with neuroectodermal phenotypes shortly after their transfer into serum‐free medium. As revealed by immunohistochemistry, there were a few MG expressing NG2 chondroitin sulfate proteoglycan (NG2) in the neonatal rat brain. Primary culture from the neonatal brain contained NG2+ MG, which appeared to be the source of NG2+ ProMGB aggregates. The aggregates were MG marker+/NG2+/GFAP+/NCAM+/S‐100β− and had alkaline phosphatase activity. The marked accumulation of NG2+ MG was observed close to stab wounds made in the mature rat brain. The accumulated NG2+ MG in the wound gradually decreased in number, but the cells persisted up to 150 days postlesioning. In addition, GFAP immunoreactivity increased markedly around the wound. The NG2+ MG in the wounds separated with trypsin‐EDTA formed NG2+ aggregates in 70% serum‐supplemented medium and then transformed into cells with neuroectodermal phenotypes in serum‐free medium. Although it is difficult to separate viable neurons from mature brains, cells from stab wounds generated process‐bearing β‐tubulin III+ cells in vitro easily. These data suggest that NG2+ MG in normal developing or pathologic brains are involved in the genesis or regeneration of the brain.

Circulating Endothelial Progenitor Cells During Normal Pregnancy and Pre-Eclampsia

Endothelial progenitor cell (EPC), which mediates neovascularization of uterine endometrium may be involved in the neovascularization in the utero‐placental circulation. We evaluated whether EPC proliferation in pre‐eclampsia (PE) differed from that in normal pregnancy.

Nodal Lymphangiogenesis and Metastasis Role of Tumor-Induced Lymphatic Vessel Activation in Extramammary Paget's Disease

Nodal lymphangiogenesis promotes distant lymph node (LN) metastasis in experimental cancer models. However, the role of nodal lymphangiogenesis in distant metastasis and in the overall survival of cancer patients remains unknown. Therefore, we investigated mechanisms that might facilitate regional and distant LN metastasis in extramammary Pagets disease (EMPD). We retrospectively analyzed the impact of tumor-induced lymphatic vessel activation on the survival of 116 patients, the largest cohort with EMPD studied to date. Nodal lymphangiogenesis was significantly increased in metastatic, compared with tumor-free, LNs (P = 0.022). Increased lymphatic invasion within regional LNs was significantly associated with distant metastasis in LN (P = 0.047) and organs (P = 0.003). Thus, invasion within regional LNs is a powerful indicator of systemic tumor spread and reduced patient survival in EMPD (P = 0.0004). Lymphatic vessels associated with tumors expressed stromal cell-derived factor-1 (SDF-1), whereas CXCR4 was expressed on invasive Paget cells undergoing epithelial-mesenchymal transition (EMT)-like process. A431 cells overexpressing Snail expressed increased levels of CXCR4 in the presence of transforming growth factor-beta1. Haptotactic migration assays confirmed that Snail-induced EMT-like process promotes tumor cell motility via the CXCR4-SDF-1 axis. Sinusoidal lymphatic endothelial cells and macrophages expressed SDF-1 in subcapsular sinuses of lymph nodes before Paget cell arrival. Our findings reveal that EMT-related features likely promote lymphatic metastasis of EMPD by activating the CXCR4-SDF-1 axis.

Myasthenia gravis experimentally induced with muscle-specific kinase.

Here we present the first evidence that muscle‐specific kinase (MuSK) antigen can cause myasthenia in animals. MuSK is expressed at the postsynaptic membranes of neuromuscular junctions (NMJ) and forms complexes with acetylcholine receptors (AChR) and rapsyn. MuSK is activated by agrin, which is released from motoneurons, and induces AChR clustering and subsequent formation of NMJ in embryos. Notably, autoantibodies against MuSK were found in a proportion of patients with generalized myasthenia gravis (MG) but without the characteristic AChR autoantibodies. However, MuSK autoantibodies had no known pathogenic potential, and animals immunized with purified MuSK proteins did not develop MG in former studies. In contrast, we have now injected rabbits with MuSK ectodomain protein in vivo and evoked a MG‐like muscle weakness with a reduction of AChR clustering at the NMJ. Our results showed that MuSK is required for maintenance of synapses and that interference with that function by MuSK antibodies causes myasthenic weakness. In vitro, AChR clustering in myotubes is induced by agrin and agrin‐independent inducers, which do not activate MuSK. Neither the receptor nor the activation mechanisms of AChR clustering induced by agrin‐independent inducers has been identified with certainty, but MuSK autoantibodies in myasthenic animals inhibited both agrin and agrin‐independent AChR clustering. MuSK plays multiple roles in pre‐patterning of the postsynaptic membrane before innervation and formation of NMJ in embryos. Some of these mechanisms may also participate in the maintenance of mature NMJ. This model system would provide new knowledge about the molecular pathogenesis of MG and MuSK functions in mature NMJ.

Hematopoietic stem cells prevent hair cell death after transient cochlear ischemia through paracrine effects.

Transplantation of hematopoietic stem cells (HSCs) is regarded to be a potential approach for promoting repair of damaged organs. Here, we investigated the influence of hematopoietic stem cells on progressive hair cell degeneration after transient cochlear ischemia in gerbils. Transient cochlear ischemia was produced by extracranial occlusion of the bilateral vertebral arteries just before their entry into the transverse foramen of the cervical vertebra. Intrascalar injection of HSCs prevented ischemia-induced hair cell degeneration and ameliorated hearing impairment. We also showed that the protein level of glial cell line-derived neurotrophic factor (GDNF) in the organ of Corti was upregulated after cochlear ischemia and that treatment with HSCs augmented this ischemia-induced upregulation of GDNF. A tracking study revealed that HSCs injected into the cochlea were retained in the perilymphatic space of the cochlea, although they neither transdifferentiated into cochlear cell types nor fused with the injured hair cells after ischemia, suggesting that HSCs had therapeutic potential possibly through paracrine effects. Thus, we propose HSCs as a potential new therapeutic strategy for hearing loss.

Elevated Levels of Adhesion Molecules Derived from Leukocytes and Endothelial Cells in Patients with Pregnancy‐Induced Hypertension

Objective. Our purpose was to elucidate the role of adhesion molecules in the pathogenesis of pregnancy‐induced hypertension (PIH). Methods. Sera, peripheral lymphocytes, and polymorphonuclear cells (PMN) from PIH patients, normal pregnant women, and nonpregnant women were collected. Soluble intercellular adhesion molecule‐1 (sICAM‐1) in sera was measured by ELISA. ICAM‐1 expression on endothelial cells (EC) incubated with sera was analyzed by flow cytometry and RT‐PCR. CD11a, CD11b, and CD18 expression on lymphocytes and PMN were also measured by flow cytometory. Results. CD11a and CD18 expression levels on PMN and lymphocytes of PIH patients were significantly higher than those of normal pregnant women (p<0.05). The expression of CD11b was significantly increased in normal pregnancy compared with that in nonpregnant women (p<0.05). Serum sICAM‐1 in PIH patients was higher than that in normal pregnant women (p<0.05). ICAM‐1 expression level on EC incubated with PIH serum for 24 hr was significantly higher than that with normal pregnant serum (p<0.0005). ICAM‐1 mRNA expression after 12‐hr incubation with PIH serum was also significantly increased compared with serum from normal pregnant women (p<0.05). Conclusion. Adhesion molecules may play an important role in the pathogenesis of PIH.

Targeted Disruption of Organic Cation Transporter 3 (Oct3) Ameliorates Ischemic Brain Damage through Modulating Histamine and Regulatory T Cells

The organic cation transporters OCT1, 2, and 3 (SLC22A1-3) have been implicated in the elimination of biogenic amines such as histamine. Among them, OCT3 was identified as an uptake-2 transporter, responsible for clearance of histamine. Because increasing evidence suggests the involvement of histamine in cerebral ischemia, we investigated the effects of targeted disruption of organic cation transporter-3 (Oct3) on the severity of ischemic brain damage. Transient focal ischemia for 1 hour was induced by occlusion of the middle cerebral artery (MCA) of homozygous Oct3-deficient mice and their wild-type (Wt) littermates. Although targeted disruption of Oct3 did not affect physiological parameters after MCA occlusion, this disruption significantly increased histamine content in the ischemic cortex and significantly reduced the infarct volume after cerebral ischemia. Furthermore, targeted disruption of Oct3 prevented the reduction of regulatory T-cell proportion after cerebral ischemia while this disruption did not affect Th1 and Th2 cells proportions after ischemia. Since repeated administration of L-histidine (a precursor of histamine) to Wt mice also showed the same effects, our observations suggested that OCT3 is the molecule responsible for clearance of ischemia-induced histamine in the brain and targeted disruption of Oct3 ameliorated ischemic brain damage through an increase in regulatory T cells.

Effect of immunoglobulin preparations on the aggregation of human erythrocytes.

Abstract. The aggregation of human erythrocytes induced by four kinds of immunoglobulin preparations was examined by a low shear rheoscope. After removing anti‐A+ and anti‐B+ activities contaminated in all preparations by incubating with erythrocytes of different blood groups, the facilitating effect on the rouleau formation of erythrocytes was compared: (i) The effect of polyethyleneglycol‐treated preparation was the same in A+‐, B+‐, AB+‐ and O+‐erythrocytes. (ii) Sulfonation did not affect the velocity of rouleau formation. (iii) Some of pepsin‐treated preparations showed the strongest facilitation for A+‐, B+‐ and AB+‐erythrocytes, but the facilitation was much weaker for O+‐erythrocytes. The others showed the weak facilitation for all types of erythrocytes (especially O+‐erythrocytes). (iv) Plasmin treatment markedly decreased the velocity of rouleau formation of AB+‐ and O+‐erythrocytes, but was not of A+‐ and B+‐erythrocytes.

In vivo subcellular imaging of tumors in mouse models using a fluorophore‐conjugated anti‐carcinoembryonic antigen antibody in two‐photon excitation microscopy

Recently, there has been growing interest in applying fluorescence imaging techniques to the study of various disease processes and complex biological phenomena in vivo. To apply these methods to clinical settings, several groups have developed protocols for fluorescence imaging using antibodies against tumor markers conjugated to fluorescent substances. Although these probes have been useful in macroscopic imaging, the specificity and sensitivity of these methods must be improved to enable them to detect micro‐lesions in the early phases of cancer, resulting in better treatment outcomes. To establish a sensitive and highly specific imaging method, we used a fluorophore‐conjugated anti‐carcinoembryonic antigen (CEA) antibody to perform macroscopic and microscopic in vivo imaging of inoculated cancer cells expressing GFP with or without CEA. Macroscopic imaging by fluorescence zoom microscopy revealed that bio‐conjugation of Alexa Fluor 594 to the anti‐CEA antibody allowed visualization of tumor mass consisting of CEA‐expressing human cancer cells, but the background levels were unacceptably high. In contrast, microscopic imaging using a two‐photon excitation microscope and the same fluorescent antibody resulted in subcellular‐resolution imaging that was more specific and sensitive than conventional imaging using a fluorescence zoom microscope. These results suggest that two‐photon excitation microscopy in conjunction with fluorophore‐conjugated antibodies could be widely adapted to detection of cancer‐specific cell‐surface molecules, both in cancer research and in clinical applications.

Oct-3/4 modulates the drug-resistant phenotype of glioblastoma cells through expression of ATP binding cassette transporter G2

BACKGROUND Drug resistance is a major obstacle for the efficacy of chemotherapeutic treatment of tumors. Oct-3/4, a self-renewal regulator in stem cells, is expressed in various kinds of solid tumors including glioblastoma. Although Oct-3/4 expression has been implicated in the malignancy and prognosis of glioblastomas, little is known of its involvement in drug resistances of glioblastoma. METHODS The involvement of Oct-3/4 in drug resistance of glioblastoma cells was assessed by lactate dehydrogenase assay, efflux assay of an anticancer drug, poly ADP-ribose polymerase cleavage, and in vivo xenograft experiments. Involvement of a drug efflux pump ATP binding cassette transporter G2 in Oct-3/4-induced drug resistance was evaluated by quantitative PCR analysis and knockdown by shRNA. RESULTS Oct-3/4 decreased the susceptibility to chemotherapeutic drugs by enhancing excretion of drugs through a drug efflux pump gene, ATP binding cassette transporter G2. Moreover, the expression of Oct-3/4 was well correlated to ATP binding cassette transporter G2 expression in clinical GB tissues. CONCLUSION Oct-3/4 elevated the ATP binding cassette transporter G2 expression, leading to acquisition of a drug-resistant phenotype by glioblastoma cells. GENERAL SIGNIFICANCE If the drug-resistance of glioblastoma cells could be suppressed, it should be a highly ameliorative treatment for glioblastoma patients. Therefore, signaling pathways from Oct-3/4 to ATP binding cassette transporter G2 should be intensively elucidated to develop new therapeutic interventions for better efficacy of anti-cancer drugs.