Akira Tominaga

Galectin-9 suppresses tumor metastasis by blocking adhesion to endothelium and extracellular matrices

We previously described an inverse correlation between galectin-9 (Gal-9) expression and metastasis in patients with malignant melanoma and breast cancer. This study verified the ability of Gal-9 to inhibit lung metastasis in experimental mouse models using highly metastatic B16F10 melanoma and Colon26 colon cancer cells. B16F10 cells transfected with a secreted form of Gal-9 lost their metastatic potential. Intravenous Gal-9 administration reduced the number of metastases of both B16F10 and Colon26 cells in the lung, indicating that secreted Gal-9 suppresses metastasis. Analysis of adhesive molecule expression revealed that B16F10 cells highly express CD44, integrin alpha1, alpha 4, alpha V, and beta1, and that Colon26 cells express CD44, integrin alpha2, alpha 5, alpha V, and beta1, suggesting that Gal-9 may inhibit the adhesion of tumor cells to vascular endothelium and the extracellular matrix (ECM) by binding to such adhesive molecules. Indeed, Gal-9 suppressed the binding of hyaluronic acid to CD44 on both B16F10 and Colon26 cells, and also suppressed the binding of vascular cell adhesion molecule-1 to very late antigen-4 on B16F10 cells. Furthermore, Gal-9 inhibited the binding of tumor cells to ECM components, resulting in the suppression of tumor cell migration. The present results suggest that Gal-9 suppresses both attachment and invasion of tumor cells by inhibiting the binding of adhesive molecules on tumor cells to ligands on vascular endothelium and ECM.

A role for CD44 in an antigen-induced murine model of pulmonary eosinophilia

Previous studies established that IL-5-producing CD4(+) T cells play a pivotal role in allergic respiratory inflammation. It was also reported that CD4(+) T cells express higher levels of CD44 in the airway than in peripheral blood of patients with allergic respiratory diseases. We have used experimental pulmonary eosinophilia induced in mice by Ascaris suum (Asc) extract to investigate the role of CD44 in the development of allergic respiratory inflammation. Intraperitoneal administration of anti-CD44 mAb prevented both lymphocyte and eosinophil accumulation in the lung. Anti-CD44 mAb also blocked antigen-induced elevation of Th2 cytokines as well as chemokines (CCL11, CCL17) in bronchoalveolar lavage fluid (BALF). Treatment with anti-CD44 mAb inhibited the increased levels of hyaluronic acid (HA) and leukotriene concentrations in BALF that typically result from antigen challenge. Anti-CD44 mAb also blocked antigen-induced airway hyperresponsiveness. An anti-CD44 mAb (IM7) inhibited the HA-binding ability of splenocytes associated with decreased levels of CD44. Soluble CD44 levels in serum were increased in Asc-challenged IM7-treated mice, but not in KM201-treated mice, compared with Asc-challenged rat IgG-treated mice. Abs that block CD44-HA binding reduced allergic respiratory inflammation by preventing lymphocyte and eosinophil accumulation in the lung. Thus, CD44 may be critical for development of allergic respiratory inflammation.

Characterization of microglia induced from mouse embryonic stem cells and their migration into the brain parenchyma.

We derived microglia from mouse embryonic stem cells (ES cells) at very high density. Using the markers Mac1(+)/CD45(low) and Mac1(+)/CD45(high) to define microglia and macrophages, respectively, we show that Mac1(+) cells are induced by GM-CSF stimulation following neuronal differentiation of mouse ES cells using a five-step method. CD45(low) expression was high and CD45(high) expression was low on induced cells. We used a density gradient method to obtain a large amount of microglia-like cells, approximately 90% of Mac1(+) cells. Microglia-like cells expressed MHC class I, class II, CD40, CD80, CD86, and IFN-gammaR. The expression level of these molecules on microglia-like cells was barely enhanced by IFN-gamma. Intravenously transferred GFP(+) microglia derived from GFP(+) ES cells selectively accumulated in brain but not in peripheral tissues such as spleen and lymph node. GFP(+) cells were detected mainly in corpus callosum and hippocampus but were rarely seen in cerebral cortex, where Iba1, another marker of microglia, is primarily expressed. Furthermore, both GFP(+) and Iba1(+) cells exhibited a ramified morphology characteristic of mature microglia. These studies suggest that ES cell-derived microglia-like cells obtained using our protocol are functional and migrate selectively into the brain but not into peripheral tissues after intravenous transplantation.

IGF-I gene transfer by electroporation promotes regeneration in a muscle injury model

The goal of this study was to determine whether insulin-like growth factor-I (IGF-I) gene delivery by electroporation promotes repair after muscle injury. An injury–repair model was created using mice in which a hamstring muscle was cut and sutured. A total of 50u2009μg of IGF-I DNA or green fluorescent protein (GFP) DNA (both in pCAGGS) was injected into the lesion and introduced into muscle cells by electrostimulation using an electric pulse generator. The number of regenerating muscle fibers in the IGF-I DNA group was significantly more than that in the GFP DNA group at 2 weeks after injection. The diameter of regenerating muscle fibers from the IGF-I DNA group was larger than that of the GFP DNA group at 4 weeks after injection. There was no significant difference in the serum IGF-I concentration between the IGF-I DNA group and the GFP DNA group at 1, 2, and 4 weeks after injection. However, muscle IGF-I concentration in the IGF-I DNA injection group was significantly greater than that in the GFP DNA injection group at 2 weeks after injection. These results demonstrated that the effects of enhanced IGF-I production were local and limited to the injected area. The ratio (injected/uninjected; intact) of the amplitude of compound muscle action potentials (CMAP) in the IGF-I DNA injection group was greater than that in the GFP DNA injection group at 4 weeks after injection and of the control group. In conclusion, IGF-I gene transfer by electroporation proved to be a simple, safe, inexpensive, and effective method to promote the regeneration of injured muscles in our injury model.

Marked eosinophilia in interleukin-5 transgenic mice fails to prevent Trichinella spiralis infection.

In order to study the role of eosinophils in the host defense against Trichinella spiralis infection, worm recovery after infection with T. spiralis was compared between interleukin-5 transgenic (IL-5 Tg) mice with a constant high level of peripheral eosinophils and nontransgenic C3H/HeN mice. No significant difference in the recovery of muscle larvae or adult worms in the small intestine, fecundity of female adult worms, or infectivity of newborn larvae was observed between nonimmunized C3H/HeN and IL-5 Tg mice or C3H/HeN and IL-5 Tg mice immunized with somatic antigen of T. spiralis. However, a significant difference was observed in the fecundity of female adult worms and recovery of muscle larvae between nonimmunized and immunized IL-5 Tg mice or C3H/HeN mice. These results demonstrate that having more eosinophils does not improve immunity against the various aspects of T. spiralis infection.

Development and functional analysis of eosinophils from murine embryonic stem cells

We have established a culture system for the development of eosinophils from murine embryonic stem (ES) cells. After transferring ES cells from embryonic fibroblast cells onto macrophage colony‐stimulating factor‐deficient stromal cells, OP9, ES cells were cultured in the presence of interleukin (IL)‐5 with either IL‐3 or granulocyte–macrophage colony stimulating factor (GM‐CSF) for 20u2003d to obtain approximately 50% eosinophils. Electron microscopy confirmed the presence of crystallized major basic protein (MBP) in the granules of some of these cells. Neither IL‐5, IL‐3, GM‐CSF nor eotaxin alone could induce eosinophils as efficiently as the conditions described above. Eotaxin induced eosinophil development in combination with either IL‐3 or IL‐5. Levels of GATA‐1, Friend of GATA (FOG)‐1, PU.1, CCAAT/enhancer binding protein (C/EBP)α, C/EBPβ, IL‐3 receptor α (IL‐3Rα), GM‐CSF receptor α (GM‐CSFRα), and MBP mRNAs were increased in ES cells 10u2003d after transfer onto OP9 cells. In contrast, C/EBPɛ, IL‐5Rα, and eosinophil peroxidase mRNAs were induced in response to IL‐3 and IL‐5 after transfer onto OP9 cells. Eosinophils that developed in this system expressed Gr‐1, F4/80, B220, CCR3, IL‐3Rα, IL‐5Rα, and DX5. Finally, eosinophils developed from ES cells produced reactive oxygen species in response to Leishmania as do peripheral blood eosinophils.

Eosinophilia, IL-5 level and recovery of larvae in IL-5 transgenic mice infected with Toxocara canis.

Eosinophil counts, interleukin 5 (IL-5) concentrations in peripheral blood and larval recovery after infection with Toxocara canis were compared between IL-5 transgenic (Tg) and nontransgenic counterparts, C3H/HeN mice. In Tg mice characterized by a constant high level of peripheral eosinophils, eosinophils in peripheral blood fell to the lowest level on day 4 of a T. canis infection and then returned to the preinfection level on day 14. Serum IL-5 level fell to the lowest level on day 4 of infection, then recovered rapidly and peaked on day 14 postinfection. In contrast, the eosinophil and IL-5 levels in the peripheral blood peaked on days 11 and 7 of infection, respectively in C3H/HeN mice. The degree of eosinophil infiltration into the lung 4 days after infection was far more pronounced in Tg than C3H/HeN mice. The highest number of larvae recovered from the lungs of infected Tg and C3H/HeN mice occurred on day 4 postinfection. Strains of Tg and C3H/ HeN mice vaccinated with excretory and secretory (ES) antigens of T. canis larvae were infected with T. canis and the recovery of larvae on day 21 analysed. There were no significant differences in the mean number of larvae recovered from nonvaccinated Tg and C3H/HeN mice or vaccinated Tg and C3H/HeN mice. However, significant differences were demonstrated in the mean total number of larvae recovered from vaccinated and nonvaccinated Tg or C3H/HeN mice. These results suggest that immunocompetent cells other than eosinophils may play a significant role in the expulsion and killing of T. canis larvae in infected mice.

Amphirionin-4 with potent proliferation-promoting activity on bone marrow stromal cells from a marine dinoflagellate amphidinium species.

A linear polyketide, amphirionin-4 (1), has been isolated from cultivated algal cells of the marine dinoflagellate Amphidinium species. The structure was elucidated on the basis of detailed analyses of 1D and 2D NMR data, and the absolute configurations of C-4 and C-8 were determined using the modified Moshers method. Amphirionin-4 (1) exhibited extremely potent proliferation-promoting activity on murine bone marrow stromal ST-2 cells (950% promotion) at a concentration of 0.1 ng/mL.

Renal angiomyoadenomatous tumor: fluorescence in situ hybridization.

To the Editor: Renal angiomyoadenomatous tumor (RAT) is a very rare tumor and it was first described by Michal et al. 1 This tumor is histologically characterized by components including adenomatous cells endowed with bleb-like apical snouts (Fig. 1a) and surrounded by capillary network (Fig. 1b), and smooth muscle stroma focally forming abortive vessels (Fig. 1c). In this article we report on the chromosomal change of four RATS. Three of the four tumors have been previously described. Fluorescence in situ hybridization (FISH) was performed using the centromeric probe of chromosome 1 (D1Z5), chromosome 11 (D11Z1) and chromosome 16 (D16Z3; Vysis, Downers Grove, IL, USA). FISH was carried out by the Cytogenetic Testing Group, Molecular Genetic Testing Department, Clinical Testing Center, Mitsubishi Chemical Medience Corporation, Kyoto, Japan. For the results more than 400 neoplastic cells were evaluated and the percentages of one, two and three signals per cell were calculated. The cut-off for monosomy was >20%. The FISH results are summarized in Table 1. In case 1, tumorous cells had disomy for chromosome 1 and monosomy for chromosomes 11 and 16. In cases 2, 3 and 4, neoplastic cells demonstrated monosomy of all three chromosomes (Fig. 2). To date there have been no reports on chromosomal changes in RAT. We found monosomy of chromosomes 11 and 16 in all four RATS cases and monosomy of chromosome 1 in three of four RATS cases on FISH. We consider that RAT is characterized by monosomy of chromosomes 1, 11 and 16 on FISH. To the best of our knowledge, no renal tumors show the combination of abnormalities of chromosomes 1, 11 and 16 on FISH. Tumorous cells of clear cell renal cell carcinoma (RCC) often have loss of 3p including 3p13p14, 3p 21.3 and 3p24pter. In papillary RCC, neoplastic cells were generally known to show gain of chromosomes 7, 17, 12, 16 and 20, and loss of chromosome Y. Chromophobe RCC generally exhibits monosomy of chromosomes 1, 2, 6, 10, 13, 17 and 21. In collecting duct carcinoma, neoplastic cells had polysomy of chromosomes 3, 7 and 17, although the histological features were not described in that study. Neoplastic cells of mucinous tubular and spindle cell carcinoma demonstrate monosomy of chromosomes 15 and 22 and, furthermore, disomy or polysomy of chromosomes 7 and 17. Monosomy of chromosomes 1, 3, 4, 6 and 9 has also been reported. Renal oncocytoma shows loss of chromosomes Y and 1, normal chromosome or non-specific results. In metanephric adenoma, gain of chromosomes 7 and 17 and loss of sex chromosome may be seen, but some reports are inconsistent with this result. Therefore, we strongly believe that RAT is a genetically distinct entity of renal tumors. In contrast, the analysis of chromosomes 1, 11 and 16 on FISH may contribute to accurate diagnosis on histological suspicion of RAT.

Inhibitory effects of edible marine algae extracts on degranulation of RBL-2H3 cells and mouse eosinophils

Inhibitory effects on degranulation of rat basophilic leukemia (RBL-2H3) cells and mouse eosinophils by marine algae extracts were examined. More than 50% of the degranulation of RBL-2H3 cells was inhibited by water extracts of Ecklonia cava and Chrysymenia wrightii at a concentration of 100 μg/mL. More than 50% of the degranulation of RBL-2H3 cells was inhibited by methanol extracts of Petalonia binghamiae, Scytosiphon lomentaria, Undaria pinnatifida, Porphyra dentata, Codium fragile and Ulva japonica at a concentration of 200 μg/mL. Most inhibitory substances in the methanol extracts were partitioned into ethyl acetate and hexane layers. The ethyl acetate-partitioned layer of methanol extract of Petalonia binghamiae had higher inhibitory effects than the hexane-partitioned layer on the degranulation of RBL-2H3 cells. By contrast, the hexanepartitioned layer of the same extract had a higher inhibitory effect than the ethyl acetate-partitioned layer on the degranulation of mouse eosinophils. The ethyl acetate-partitioned layer of methanol extract of Petalonia binghamiae was further separated into eight fractions by silica gel column chromatography. Most inhibited the degranulation of RBL-2H3 cells, but not that of mouse eosinophils significantly. These results suggest methanol extract of Petalonia binghamiae contains materials that inhibit the degranulation of basophils and eosinophils differentially.