ACTGen Inc.

Komagane, Japan

ACTGen Inc.

Komagane, Japan
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Yamanishi Y.,Tokyo Medical University | Yamanishi Y.,University of Tokyo | Takahashi M.,Tokyo Medical University | Izawa K.,Tokyo Medical University | And 14 more authors.
Journal of Immunology | Year: 2012

Leukocyte mono-Ig-like receptor 5 (LMIR5, also called CD300b) is an activating receptor expressed in myeloid cells. We have previously demonstrated that T cell Ig mucin 1 works as a ligand for LMIR5 in mouse ischemia/reperfusion injury of the kidneys. In this article, we show that LMIR5 is implicated in LPS-induced sepsis in mice. Notably, neutrophils constitutively released a soluble form of LMIR5 (sLMIR5) through proteolytic cleavage of surface LMIR5. Stimulation with TLR agonists augmented the release of sLMIR5. LPS administration or peritonitis induction increased serum levels of sLMIR5 in mice, which was substantially inhibited by neutrophil depletion. Thus, neutrophils were the main source of LPS-induced sLMIR5 in vivo. On the other hand, i.p. administration of LMIR5-Fc, a surrogate of sLMIR5, bound to resident macrophages (Mφ) and stimulated transient inflammation in mice. Consistently, LMIR5-Fc induced in vitro cytokine production of peritoneal Mφ via its unknown ligand. Interestingly, LMIR5 deficiency profoundly reduced systemic cytokine production and septic mortality in LPS-administered mice, although it did not affect in vitro cytokine production of LPS-stimulated peritoneal Mφ. Importantly, the resistance of LMIR5-deficient mice to LPS- or peritonitis-induced septic death was decreased by LMIR5-Fc administration, implicating sLMIR5 in LPS responses in vivo. Collectively, neutrophil-derived sLMIR5 amplifies LPS-induced lethal inflammation. Copyright © 2012 by The American Association of Immunologists, Inc.


Koga Y.,National Cancer Center Hospital East | Yasunaga M.,National Cancer Center Hospital East | Kajikawa M.,ACTGen Inc. | Shimizu E.,ACTGen Inc. | And 12 more authors.
Cancer Science | Year: 2011

The current medical examinations for detecting endometrial cancer can sometimes be stressful and inconvenient for examinees and examiners. Therefore, we attempted to develop an autoscan-virtual cytology system for detecting endometrial cancer without relying on judgment by the human eye. Exfoliated cells from the uterus were retrieved using a tampon inserted for 3h. More than 100 monoclonal antibodies (mAb) developed by us were screened in three steps of immunohistochemistry to find mAb sets that would enable the cancer and normal endometrium to be perfectly distinguished. The exfoliated cells provided by 30 endometrial cancer patients and a total of 37 samples of 14 non-malignant volunteers including the menstrual cycle were analyzed using imaging cytometry. All samples contained epithelial cells and dysplasia cells, but the pathologist could not definitively diagnose all of them as endometrial cancer cells because most cells had degenerated. Twenty-two of 28 endometrial cancer tissues (79%) were positive with four mAb sets, CRELD1, GRK5, SLC25A27 and STC2, and 22 of 22 normal endometriums (100%) were negative. Our newly developed autoscan-virtual cytology for exfoliated endometrial cells showed overall sensitivity for endometrial cancer patients and overall specificity for volunteers of 50% (15/30) and 95% (35/37), respectively. Our autoscan-virtual cytology combined with cancer-specific mAb and imaging cytometry could be useful for endometrial cancer detection. Autoscan-virtual cytology for endometrial cancer deserves further evaluation for future endometrial cancer screening. © 2011 Japanese Cancer Association.


Enomoto Y.,Tokyo Medical University | Yamanishi Y.,Tokyo Medical University | Izawa K.,Tokyo Medical University | Kaitani A.,Tokyo Medical University | And 8 more authors.
Journal of Biological Chemistry | Year: 2010

Here we characterize leukocyte mono-Ig-like receptor 7 (LMIR7)/CLM-3 and compare it with an activating receptor, LMIR4/CLM-5, that is a counterpart of an inhibitory receptor LMIR3/CLM-1. LMIR7 shares high homology with LMIR4 in the amino acid sequences of its Ig-like and transmembrane domains. Flow cytometric analysis demonstrated that LMIR4 was predominantly expressed in neutrophils, whereas LMIR7 was highly expressed in mast cells and monocytes/macrophages. Importantly, LMIR7 engagement induced cytokine production in bone marrow-derived mast cells (BMMCs). Although FcRγ deficiency did not affect surface expression levels of LMIR7, it abolished LMIR7-mediated activation of BMMCs. Consistently we found significant interaction of LMIR7-FcRγ, albeit with lower affinity compared with that of LMIR4-FcRγ. Our results showed that LMIR7 transmits an activating signal through interaction with FcRγ. In addition, like LMIR4, LMIR7 synergizes with TLR4 in signaling. Analysis of several chimera receptors composed of LMIR4 and LMIR7 revealed these findings: 1) the transmembrane of LMIR7 with no charged residues maintained its surface expression at high levels in the absence of FcRγ; 2) the extracellular juxtamembrane region of LMIR7 had a negative effect on its surface expression levels; and 3) the strong interaction of LMIR4 with FcRγ depended on the extracellular juxtamembrane region as well as the transmembrane domain of LMIR4. Thus, LMIR7 shares similarities with LMIR4, although they are differentially regulated in their distribution, expression, and function. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.


Konakahara S.,Tokyo University of Science | Saitou M.,Tokyo University of Science | Hori S.,Tokyo University of Science | Hori S.,Ono Pharmaceutical Co. | And 11 more authors.
FEBS Letters | Year: 2011

Leucine-rich repeat and fibronectin type III domain-containing (LRFN) family proteins are thought to be neuronal-specific proteins that play essential roles in neurite outgrowth and synapse formation. Here, we focused on expression and function of LRFN4, the fourth member of the LRFN family, in non-neural tissues. We found that LRFN4 was expressed in a wide variety of cancer and leukemia cell lines. We also found that expression of LRFN4 in the monocytic cell line THP-1 and in primary monocytes was upregulated following macrophage differentiation. Furthermore, we demonstrated that LRFN4 signaling regulated both the transendothelial migration of THP-1 cells and the elongation of THP-1 cells via actin cytoskeleton reorganization. Our data indicate that LRFN4 signaling plays an important role in the migration of monocytes/ macrophages. © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.


Konakahara S.,Tokyo University of Science | Suzuki Y.,Tokyo University of Science | Kawakami T.,Tokyo Medical University | Kawakami T.,Medical ProteoScope Co. | And 4 more authors.
FEBS Letters | Year: 2012

We previously reported that leucine-rich repeat and fibronectin type III domain-containing 4 (LRFN4) functioned in migration and morphological change (i.e. cell elongation) of monocytic cells. Here, we examined a molecular mechanism regulating LRFN4-mediated cell elongation. We found that 14-3-3 and NCK proteins complexed with LRFN4, and they were involved in LRFN4-mediated cell elongation. We also identified the regions of LRFN4 interacting with NCK1 and 14-3-3s. Finally, we demonstrated that a Rac1 small GTPase was involved in LRFN4-mediated cell elongation. These results indicated that LRFN4 complexed with 14-3-3s and NCK1 to mediate elongation in monocytic cells via Rac-1-mediated actin cytoskeleton reorganization. Crown Copyright © 2012 Published by Elsevier B.V. on behalf of Federation of European Biochemical society. All rights reserved.


Takahashi M.,Tokyo Medical University | Izawa K.,Tokyo Medical University | Kashiwakura J.-I.,Nihon University | Kashiwakura J.-I.,RIKEN | And 16 more authors.
Journal of Biological Chemistry | Year: 2013

CD300C is highly homologous with an inhibitory receptor CD300A in an immunoglobulin-like domain among the human CD300 family of paired immune receptors. To clarify the precise expression and function of CD300C, we generated antibodies discriminating between CD300A and CD300C, which recognized a unique epitope involving amino acid residues CD300A(F56-L57) and CD300C(L63-R64). Notably, CD300C was highly expressed in human monocytes and mast cells. Cross-linking of CD300C by its specific antibody caused cytokine/chemokine production of human monocytes and mast cells. Fc receptor γ was indispensable for both efficient surface expression and activating functions of CD300C. To identify a ligand for CD300A or CD300C, we used reporter cell lines expressing a chimera receptor harboring extracellular CD300A or CD300C and intracellular CD3ζ, in which its unknown ligand induced GFP expression. Our results indicated that phosphatidylethanolamine (PE) among the lipids tested and apoptotic cells were possible ligands for both CD300C and CD300A. PE and apoptotic cells more strongly inducedGFPexpression in the reporter cells through binding to extracellular CD300A as compared with CD300C. Differential recognition of PE by extracellular CD300A and CD300C depended on different amino acid residues CD300A(F56-L57) and CD300C(L63-R64). Interestingly, GFP expression induced by extracellular CD300C-PE binding in the reporter cells was dampened by co-expression of full-length CD300A, indicating the predominance of CD300A over CD300C in PE recognition/signaling. PE consistently failed to stimulate cytokine production in monocytes expressing CD300C with CD300A. In conclusion, specific engagement of CD300C led to Fc receptor γ-dependent activation of mast cells and monocytes. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.


Kobayashi E.,Tokyo University of Science | Motoi S.,Tokyo University of Science | Sugiura M.,ACTGen Inc. | Kajikawa M.,ACTGen Inc. | And 3 more authors.
Immunology Letters | Year: 2014

Antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells is a major mechanism of tumor therapy with antibodies. NK cells not only manifest cytotoxicity but also secrete a variety of cytokines/chemokines that regulate immune responses. Using a retroviral vector, in this study we established a KHYG-1 cell line that stably expresses FcγRIIIA (CD16A). The KHYG-1/FcγRIIIA cells exerted potent antibody concentration-dependent ADCC, whereas parental KHYG-1 cells did not. In contrast, without antibody, the natural killer activity of KHYG-1/FcγRIIIA cells was less potent than that of parental KHYG-1 cells. During the course of ADCC, KHYG-1/FcγRIIIA cells secreted IFN-γ and MIP-1α dependent upon antibody concentration, but parental KHYG-1 cells did not. These results suggest that KHYG-1/FcγRIIIA cells would be useful in studies to elucidate the function of NK cells and the mechanism of ADCC. © 2014 Elsevier B.V.

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