Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Atsunobu Takeda Last modified date:2022.07.04

Associate Professor / Department of Ophthalmology, Graduate School of Medical Sciences / Department of Clinical Medicine / Faculty of Medical Sciences

1. Fukuda Y, Nakao S, Kaizu Y, Arima M, Shimokawa S, Wada I, Yamaguchi M, Takeda A, Sonoda KH, Morphology and fluorescence leakage in diabetic retinal microaneurysms: A study using multiple En face OCT angiography image averaging., Graefe’s Arch Clin Exp Ophthalmol., 2022.05.
2. Fukuda Y, Nakao S, Kohno R, Ishikawa K, Shimokawa S, Shiose S, Takeda A, Morizane Y, Sonoda KH., Postoperative Follow-up of Submacular Hemorrhage Displacement Treated with Vitrectomy and Subretinal Injection of Tissue Plasminogen Activator., Jpn J Ophthalmol., 10.1007/s10384-022-00910-7., 66, 3, 264-270, 2022.05.
3. Takeda A, Hasegawa E, Yawata N, Notomi S, Ishikawa K, Murakami Y, Hisatomi T, Kimura K, Sonoda KH, Increased vitreous levels of B cell activation factor (BAFF) and soluble interleukin-6 receptor in patients with macular edema due to uveitis related to Behçet’s disease and sarcoidosis, Graefe’s Arch Clin Exp Opthalmol., 10.1007/s00417-022-05600-1., 2022.03.
4. Funatsu J, Murakami Y, Shimokawa S, Nakatake S, Fujiwara K, Okita A, Fukushima M, Shibata S, Yoshida N, Koyanagi Y, Akiyama M, Notomi S, Nakao S, Hisatomi T, Takeda A, Paschalis E, Vavvas D, Ikeda Y, Sonoda KH. , Circulating inflammatory monocytes oppose microglia and contribute to cone cell death in retinitis pigmentosa., PNAS nexus, 10.1093/pnasnexus/pgac003., 2022.03.
5. Ishikawa K, Akiyama M, Mori K, Nakama T, Notomi S, Nakao S, Kohno RI, Takeda A, Sonoda KH, Drainage retinotomy confers risk of epiretinal membrane formation following vitrectomy for rhegmatogenous retinal detachment repair, American Journal of Ophthalmology, 10.1016/j.ajo.2021.07.028, 234, 20-27, 2022.02.
6. Yamana S, Shibata K, Hasegawa E, Arima M, Shimokawa S, Yawata N, Takeda A, Yamasaki S, Sonoda KH., Mucosal-associated invariant T cells have therapeutic potential against ocular autoimmunity., Mucosal Immunol., doi: 10.1038/s41385-021-00469-5., 15, 2, 351-361, 2022.02.
7. Shimokawa S, Murakami Y, Fujiwara K, Funatsu J, Nakatake S, Koyanagi Y, Akiyama M, Yoshida N, Takeda A, Ikeda Y, Sonoda KH, Recurrence Rate of Cystoid Macular Edema with Topical Dorzolamide Treatment and Its Risk Factors in Retinitis Pigmentosa, Retina, 10.1097/IAE.0000000000003286., 42, 1, 168-173, 2022.01.
8. Takeda A, Hasegawa E, Notomi S, Ishikawa K, Arima M, Murakami Y, Nakao S, Hisatomi T, Sonoda KH, Surgical Outcomes of Contrast Sensitivity and Visual Acuity in Uveitis-Associated Cataract, Clincal Ophthalmology, 10.2147/OPTH.S314173, 15, 2665-2673, 2021.06.
9. Takeda A, Hasegawa E, Nakao S, Ishikawa K, Murakami Y, Hisatomi T, Arima M, Yawata N, Oda Y, Kimura K, Yoshikawa H, Sonoda KH., Vitreous levels of interleukin-35 as a prognostic factor in B-cell vitreoretinal lymphoma, Scientific Reports, 10.1038/s41598-020-72962-z , 10, 1, 15715, 2020.09.
10. Okita A, Murakami Y, Shimokawa S, Funatsu J, Fujiwara K, Nakatake S, Koyanagi Y, Akiyama M, Takeda A, Hisatomi T, Ikeda Y, Sonoda KH., Changes of Serum inflammatory Molecules and Their Relationships with Visual Function in Retinitis Pigmentosa, Invest Ophthalmol Vis Sci., 10.1167/iovs.61.11.30., 2020.09.
11. Takuto Sakono, Akira Meguro, Masaki Takeuchi, Takahiro Yamane, Takeshi Teshigawara, Nobuyoshi Kitaichi, Yukihiro Horie, Kenichi Namba, Shigeaki Ohno, Kumiko Nakao, Taiji Sakamoto, Tsutomu Sakai, Tadashi Nakano, Hiroshi Keino, Annabelle A. Okada, Atsunobu Takeda, Takako Ito, Hisashi Mashimo, Nobuyuki Ohguro, Shinichirou Oono, Hiroshi Enaida, Satoshi Okinami, Nobuyuki Horita, Masao Ota, Nobuhisa Mizuki, Variants in IL23R-C1orf141 and ADO-ZNF365-EGR2 are associated with susceptibility to Vogt-Koyanagi-Harada disease in Japanese population, PloS one, 10.1371/journal.pone.0233464, 15, 5, 2020.05, Vogt-Koyanagi-Harada (VKH) disease is a systemic inflammatory disorder that affects pigment cell-containing organs such as the eye (e.g., chronic and/or recurrent granulomatous panuveitis). While the exact etiology and pathogenic mechanism of VKH disease are unclear, HLA-DR4 alleles have been documented to be strongly associated with VKH disease in various ethnic groups. Recently, a genome-wide association study (GWAS) found two new genetic risk factors (IL23R-C1orf141 and ADO-ZNF365-EGR2) in a non-HLA region from a Han Chinese population. In this study, we replicated these GWAS findings in a Japanese population. A total of 1,643 Japanese samples (380 cases with VKH disease and 1,263 healthy controls) were recruited. We assessed four single nucleotide polymorphisms (SNPs) shown in previous GWAS: rs78377598 and rs117633859 in IL23R-C1orf141, and rs442309 and rs224058 in ADO-ZNF365-EGR2. A significant allelic association with VKH disease was observed for all of the four SNPs (rs78377598: pc = 0.0057; rs117633859: pc = 0.0017; rs442309: pc = 0.021; rs224058: pc = 0.035). In genotypic association analysis, the minor alleles of IL23R-C1orf141 rs78377598 and rs117633859 had the strongest association with disease susceptibility under the additive model (pc = 0.0075 and pc = 0.0026, respectively). The minor alleles of ADO-ZNF365-EGR2 rs442309 and rs224058 were most strongly associated with disease susceptibility under the dominant model (pc = 0.00099 and pc = 0.0023, respectively). The meta-analysis of the current and previous studies found that all of the four SNPs exhibited a significantly strong association with VKH disease (meta-p < 0.00001: rs78377598, meta-odds ratio (OR) = 1.69; rs1176338, meta-OR = 1.82; rs442309, meta-OR = 1.34; rs224058, meta-OR = 1.33). In summary, our study replicated significant associations with VKH disease susceptibility reported in a previous GWAS. Thus, the IL23R-C1orf141 and ADO-ZNF365-EGR2 loci may play important roles in the development of VKH disease through genetic polymorphisms..
12. Tomoko Ohno, Akira Meguro, Masaki Takeuchi, Takahiro Yamane, Takeshi Teshigawara, Nobuyoshi Kitaichi, Yukihiro Horie, Kenichi Namba, Shigeaki Ohno, Kumiko Nakao, Taiji Sakamoto, Tsutomu Sakai, Tadashi Nakano, Hiroshi Keino, Annabelle A. Okada, Atsunobu Takeda, Takako Fukuhara, Hisashi Mashimo, Nobuyuki Ohguro, Shinichirou Oono, Hiroshi Enaida, Satoshi Okinami, Nobuhisa Mizuki, Association Study of ARMC9 Gene Variants with Vogt-Koyanagi-Harada Disease in Japanese Patients, Ocular Immunology and Inflammation, 10.1080/09273948.2018.1523438, 27, 5, 699-705, 2019.07, Purpose: To investigate whether variants in the ARMC9 gene encoding KU-MEL-1 are associated with Vogt-Koyanagi-Harada (VKH) disease in a Japanese population. Methods: We recruited 380 Japanese patients with VKH disease and 744 Japanese healthy controls to genotype seven single-nucleotide polymorphisms (SNPs) in ARMC9. We also performed imputation analysis of the ARMC9 region and 195 imputed SNPs were included in the statistical analysis. Results: We observed an increased frequency of the A allele of rs28690417 in patients compared with controls (P = 0.0097, odds ratio (OR) = 1.46). The A allele had a dominant effect on VKH disease risk (P = 0.011, OR = 1.51). However, these significant differences disappeared after Bonferroni correction (corrected P > 0.05). The remaining 201 SNPs did not show any significant association with disease risk. Conclusions: Our study suggests that ARMC9 variants do not play a critical role in the development of VKH disease..
13. Ito T, Takeda A, Fujiwara K, Hasegawa E, Nakao S, Ohishi Y, Oda Y, Yoshikawa H, Sonoda KH., Risk factors for failure of vitrectomy cell block technique in cytological diagnosis of vitreoretinal lymphoma., Graefe’s Arch Clin Exp Opthalmol., 257, 5, 1029-1036, 2019.05.
14. Takeda A, Yamada H, Hasegawa E, Arima M, Notomi N, Myojin S, Yoshimura T, Hisatomi T, Enaida H, Yanai R, Kimura K, Ishibashi T, Sonoda KH., Crucial role of P2X7 receptor for effector T cell activation in experimental autoimmune uveitis., Jpn J Ophthalmol., 62, 3, 398-406, 2018.05.
15. Toshio Hisatomi, Takashi Tachibana, Shoji Notomi, Yoshito Koyanagi, Yusuke Murakami, Atsunobu Takeda, Yasuhiro Ikeda, Shigeo Yoshida, Hiroshi Enaida, Toshinori Murata, Taiji Sakamoto, Koh Hei Sonoda, Tatsuro Ishibashi, Internal limiting membrane peeling–Dependent retinal structural changes after vitrectomy in rhegmatogenous retinal detachment, Retina, 10.1097/IAE.0000000000001558, 38, 3, 471-479, 2018.03, Purpose: To examine retinal changes after vitrectomy with internal limiting membrane (ILM) peeling, we used 3-dimensional optical coherence tomography (3D-OCT) in rhegmatogenous retinal detachment cases. Methods: The 68 eyes from 67 patients with rhegmatogenous retinal detachment were studied, including 35 detached macula cases (51%) and 33 attached macula cases. Internal limiting membrane peeling was performed with fine forceps after brilliant blue G staining. The 3D-OCT images were obtained with volume-rendering technologies from cross-sectional OCT images. Results: The 3D-OCT detected 45 eyes (66%) with ILM peeling-dependent retinal changes, including dissociated optic nerve fiber layer appearance, dimple sign, temporal macular thinning, ILM peeling area thinning, or forceps-related retinal thinning. The ILM peeled area was detectable in only 9 eyes with 3D-OCT, whereas it was undetectable in other 59 eyes. The dissociated optic nerve fiber layer appearance was detected in 8 of the total cases (12%), and dimple signs were observed in 14 cases (21%). Forceps-related thinning was also noted in eight cases (24%) of attached macula cases and in four cases (11%) of detached macula cases. No postoperative macular pucker was noted in the observational period. Conclusion: The 3D-OCT clearly revealed spatial and time-dependent retinal changes after ILM peeling. The changes occurred in 2 months and remained thereafter..
16. Ueda S, Akahoshi M, Takeda A, Inoue Y, Omoto A, Ayano M, Kimoto Y, Mitoma H, Arinobu Y, Niiro H, Tsukamoto H, Horiuchi T, Hikita SI, Fukuhara T, Ishibashi T, Sonoda KH, Akashi K., Long-term efficacy of infliximab treatment and the predictors of treatment outcomes in patients with refractory uveitis associated with Behçet’s disease. , Eur J Rheumatol., 5, 1, 9-15, 2018.01.
17. Sato K, Takeda A, Hasegawa E, Jo YJ, Arima M, Oshima Y, Yanai R, Nakazawa T, Yuzawa M, Nakashizuka H, Shimada H, Kimura K, Ishibashi T, Sonoda KH., Interleukin-6 plays a crucial role in the development of subretinal fibrosis in a mouse model., Immunological Medicine, 41, 1, 23-29, 2018.01.
18. Taki R, Takeda A, Yoshikawa H, Fukuhara T, Arita R, Suehiro Y, Che I, Kumano Y, Nakamura T, Ishibashi T, Clinical features of systemic metastatic retinal lymphoma in Japanese patients., Ocul Immunol Inflamm., 25, 5, 654-662, 2017.10.
19. Kaori Higashi, Akira Meguro, Masaki Takeuchi, Takahiro Yamane, Nobuyoshi Kitaichi, Yukihiro Horie, Kenichi Namba, Shigeaki Ohno, Kumiko Nakao, Taiji Sakamoto, Tsutomu Sakai, Hiroshi Tsuneoka, Hiroshi Keino, Annabelle A. Okada, Atsunobu Takeda, Takako Fukuhara, Hisashi Mashimo, Nobuyuki Ohguro, Shinichirou Oono, Hiroshi Enaida, Satoshi Okinami, Nobuhisa Mizuki, Investigation of the association between IL10 gene polymorphisms and Vogt-Koyanagi-Harada disease in a Japanese population, Ophthalmic Genetics, 10.3109/13816810.2016.1145698, 38, 2, 187-189, 2017.03.
20. Hiroshi Enaida, Shinji Nagata, Atsunobu Takeda, Shintaro Nakao, Yasuhiro Ikeda, Tatsuro Ishibashi, Changes in chorioretinal blood flow velocity and cerebral blood flow after carotid endarterectomy, Japanese Journal of Ophthalmology, 10.1007/s10384-016-0472-y, 60, 6, 459-465, 2016.11, Purpose: To investigate the changes in chorioretinal blood flow velocity and cerebral blood after carotid endarterectomy (CEA). Methods: Nine patients with moderate to severe internal carotid artery stenosis underwent CEA. Chorioretinal blood flow velocity was measured by laser speckle flowgraphy (LSFG), while cerebral blood flow (CBF) was measured by single-photon emission computed tomography (SPECT), on the affected side both before and after CEA. LSFG was evaluated in five areas to determine mean blur rate, while CBF was calculated from regional CBF and cerebrovascular reactivity (CVR), at the middle cerebral artery (MCA) region of each patient. Results: Five cases showed an increase (mean 3.49 %, range −29.82 to 35.59 %) of average chorioretinal blood flow velocity using LSFG after CEA. A particularly averaged increase in chorioretinal blood flow was observed in the macular area compared with other areas. Similarly, there was an increase in CBF at rest (mean 11.46 %, range −14.51 to 74.14 %) observed using SPECT after surgery. Improvement of CVR was confirmed in four cases. All general and visual symptoms disappeared after CEA. Severe adverse effects, including hyperperfusion syndrome, were not observed in any cases. Conclusions: LSFG may be useful for the analysis of chorioretinal blood flow changes after CEA..
21. Sayaka Myojin, Takeru Yoshimura, Shigeo Yoshida, Atsunobu Takeda, Yusuke Murakami, Yoichi Kawano, Yuji Oshima, Tatsuro Ishibashi, Koh Hei Sonoda, Gene expression analysis of the irrigation solution samples collected during vitrectomy for idiopathic epiretinal membrane, PloS one, 10.1371/journal.pone.0164355, 11, 10, 2016.10, Purpose: The analysis of gene expression in idiopathic epiretinal membranes (iERMs) may help elucidate ERM formation and its pathology. Here, we conducted a case-control study, in order to determine the expression levels of cytokines and other genes in eyes with macular hole (MH) or iERM. Methods: Twenty eyes, obtained from seven male and 13 female patients, were included in the study. The average age of the study subjects was 69.1 ± 7.67 years, and 15 eyes had iERM, while five eyes had MH. Irrigation solution samples were collected during vitrectomy, centrifuged, and the levels of cytokine and other mRNAs in the sediment were assessed using real-time PCR. The expression level of 11 cytokine genes, four transcription factor genes, two cytoskeletal genes, and genes encoding two extracellular matrix proteins in eyes with MH or iERM were determined and compared. Results: The expression levels of interleukin 6 (IL6), tumor growth factor B2 (TGFB2), vascular endothelial growth factor A (VEGFA), chemokine C-X-C motif ligand 1 (CXCL1), v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA), glial fibrillary acidic protein (GFAP), and tenascin C (TNC) were significantly higher in eyes with iERM than in eyes with MH. The expression of these genes was not associated with the preoperative visual acuity of the investigated patients. Conclusions: The obtained results indicate that real-time PCR analysis of irrigation solution samples collected during vitrectomy can help assess the expression levels of several genes, and that iERM is associated with the expression of pro-inflammatory genes and the genes expressed during angiogenesis and wound healing process (IL6, TGFB2, VEGFA, CXCL1, RELA, GFAP, and TNC)..
22. Muneo Yamaguchi, Shintaro Nakao, Yoshihiro Kaizu, Yoshiyuki Kobayashi, Takahito Nakama, Mitsuru Arima, Shigeo Yoshida, Yuji Oshima, Atsunobu Takeda, Yasuhiro Ikeda, Shizuo Mukai, Tatsuro Ishibashi, Koh Hei Sonoda, High-Resolution Imaging by Adaptive Optics Scanning Laser Ophthalmoscopy Reveals Two Morphologically Distinct Types of Retinal Hard Exudates, Scientific reports, 10.1038/srep33574, 6, 2016.09, Histological studies from autopsy specimens have characterized hard exudates as a composition of lipid-laden macrophages or noncellular materials including lipid and proteinaceous substances (hyaline substances). However, the characteristics of hard exudates in living patients have not been examined due to insufficient resolution of existing equipment. In this study, we used adaptive optics scanning laser ophthalmoscopy (AO-SLO) to examine the characteristics of hard exudates in patients with retinal vascular diseases. High resolution imaging using AO-SLO enables morphological classification of retinal hard exudates into two types, which could not be distinguished either on fundus examination or by spectral domain optical coherence tomography (SD-OCT). One, termed a round type, consisted of an accumulation of spherical particles (average diameter of particles: 26.9 ± 4.4 μm). The other, termed an irregular type, comprised an irregularly shaped hyper-reflective deposition. The retinal thickness in regions with round hard exudates was significantly greater than the thickness in regions with irregular hard exudates (P = 0.01 â †'0.02). This differentiation of retinal hard exudates in patients by AO-SLO may help in understanding the pathogenesis and clinical prognosis of retinal vascular diseases..
23. Yoshiyuki Kobayashi, Shigeo Yoshida, Yedi Zhou, Takahito Nakama, Keijiro Ishikawa, Mitsuru Arima, Shintaro Nakao, Yukio Sassa, Atsunobu Takeda, Toshio Hisatomi, Yasuhiro Ikeda, Akira Matsuda, Koh Hei Sonoda, Tatsuro Ishibashi, Tenascin-C promotes angiogenesis in fibrovascular membranes in eyes with proliferative diabetic retinopathy, Molecular vision, 22, 436-445, 2016.04, Purpose: We previously demonstrated that tenascin-C was highly expressed in the fibrovascular membranes (FVMs) of patients with proliferative diabetic retinopathy (PDR). However, its role in the pathogenesis of FVMs has not been determined. The purpose of this study was to investigate what role tenascin-C plays in the formation and angiogenesis of FVMs. Methods: The level of tenascin-C was determined by sandwich enzyme-linked immunosorbent assay in the vitreous samples collected from patients with PDR and with a macular hole as control. The locations of tenascin-C, α- smooth muscle actin (SMA), CD34, glial fibrillary acidic protein (GFAP), and integrin αV in the FVMs from PDR patients were determined by immunohistochemistry. We also measured the in vitro expression of the mRNA and protein of tenascin-C in vascular smooth muscle cells (VSMCs) stimulated by interleukin (IL)-13. The effects of tenascin-C on cell proliferation, migration, and tube formation were determined in human retinal endothelial cells (HRECs) in culture. Results: The mean vitreous levels of tenascin-C were significantly higher in patients with PDR than in patients with a macular hole (p<0.001). Double immunofluorescence analyses of FVMs from PDR patients showed that tenascin-C co-stained FVMs with α-SMA, CD34, and integrin αV but not with GFAP. In addition, IL-13 treatment increased both the expression and secretion of tenascin-C by VSMCs in a dose-dependent manner. Tenascin-C exposure promoted proliferation, migration, and tube formation in HRECs. Tenascin-C neutralizing antibody significantly blocked the tube formation by HRECs exposed to VSMC-IL-13-conditioned medium. Conclusions: Our findings suggest that tenascin-C is secreted from VSMCs and promotes angiogenesis in the FVMs associated with PDR..
24. Takeda A, Yoshikawa H, Fukuhara T, Hikita S, Otomo T, Arita R, Oshima Y, Hisatomi T, Kimura K, Yoshida S, Kawano Y, Sonoda KH, Ishibashi T, Distinct profiles of soluble cytokine receptors between B-cell vitreoretinal lymphoma and uveitis, Invest Ophthalmol Vis Sci., 56, 12, 7516-7523, 2015.12.
25. Kimura K, Yang Y, Takeda A, Ishibashi T, Sonoda KH, Inhibition of the TGF-β–induced epithelial-mesenchymal transition in mouse retinal pigment epithelial cells as well as attenuation of subretinal fibrosis in vivo by an RAR-γ agonist., Journal of Molecular Medicine, 10.1007/s00109-015-1289-8, 93, 2015.06.
26. Takeda A, Hasegawa E, Fukuhara T, Hirakawa S, Yamada H, Yang Y, Yoshimura T, Hisatomi T, Oshima Y, Yoshida H, Sonoda KH, Ishibashi T, EBI3 is pivotal for the initiation of experimental autoimmune uveitis., Experimental Eye Research, doi: 10.1016/j.exer.2014.06.004., 125, 107-113, 2014.08.
27. Koh Hei Sonoda, Takako Fukuhara, Horoshi Yoshikawa, Atsunobu Takeda, Takeru Yoshimura, Mitsuteru Akahoshi, Hirotake Kusumoto, Kentaro Kohno, Koji Kato, Koichi Akashi, Kenichi Kohashi, Shinichi Aijima, Kenichi Namba, Tatsuro Ishibashi, [A case report of malignant lymphoma receiving infliximab therapy with Behçet's disease]., Nippon Ganka Gakkai zasshi, 118, 5, 440-445, 2014.05, To report a case of malignant lymphoma occurring in Behçet's disease (BD) with infliximab therapy. A 62-year-old man was diagnosed with BD in 1997. Despite treatment with colchicine, cyclosporine and prednisolone, he had frequent bilateral posterior ocular attacks. He was started on infliximab in August 2007 and for 6 months had no ocular attacks. Cyclosporine was therefore reduced. After 4 years of infliximab administration, he had neither ocular attacks nor general symptoms. However, he had general malaise and weight loss from the end of March 2012. Peripheral blood examination showed abnormal cells, so we terminated the infliximab. Bone marrow aspiration showed diffuse proliferation of medium to large lymphoid cells, and the histological diagnosis was diffuse large B-cell lymphoma. He was treated with 8 cycles of chemotherapy and 4 times intrathecal chemotherapy, and is now in remission. After termination of infliximab, he had no further ocular attacks. Although malignant lymphoma associated with BD is rare, attending ophthalmologists need to keep it in mind..
28. Ryoji Yanai, Atsunobu Takeda, Takeru Yoshimura, Koh Hei Sonoda, Pathophysiology and new treatment of uveitis, Japanese Journal of Clinical Immunology, 10.2177/jsci.37.74, 37, 2, 74-82, 2014, Uveitis is narrow-defined inflammation of the uvea, also clinically include all inflammatory conditions in the eye. Uveitis may occur as a consequence of various causes and background, such as autoimmune diseases, infections, and hematopoietic malignancy. We have to treat uveitis not only controlling the inflammation but also maintaining up the visual function of the eye because the most uveitis is chronic and relapsing inflammatory disorder. Behçét's disease is a systemic disease and results in loss of vision without adequate treatment. Behçét's disease was a representative of vision loss uveitis because Behçét's patient usually had treatment resistance of conventional treatment, such as colchicine and cyclosporine. However, biological therapy with TNF-α, which started from 2007, has revolutionized the treatment strategy of Behçét's disease. It is not too much to say that Behçét's patient is free from fear of vision loss by the dramatic decrease of ocular attach. Biological therapy is not approved as a treatment of uveitis except Behçét's disease. Some protracted cases of Sarcoidosis and Vogt-Koyanagi-Harada disease are resistant to corticosteroid therapy and require new treatment. In this review, we discuss the pathophysiology of uveitis and report new treatment of Behçét's disease by biological therapy..
29. Yang Y, Takeda A, Yoshimura T, Oshima Y, Sonoda KH, Ishibashi T, IL-10 is significantly involved in HSP70-regulation of experimental subretinal fibrosis, Plos one, 8, 12, e80288, 2013.12.
30. Sunao Sugita, Manabu Ogawa, Norio Shimizu, Tomohiro Morio, Nobuyuki Ohguro, Kei Nakai, Kazuichi Maruyama, Kenji Nagata, Atsunobu Takeda, Yoshihiko Usui, Koh-hei Sonoda, Masaru Takeuchi, Manabu Mochizuki, Use of a comprehensive polymerase chain reaction system for diagnosis of ocular infectious diseases, Ophthalmology, 120, 9, 1761-1768, 2013.09.
31. Han Zhang, Yang Yang, Atsunobu Takeda, Takeru Yoshimura, Yuji Oshima, Koh-hei Sonoda, Tatsuro Ishibashi, A novel platelet-activating factor receptor antagonist inhibits choroidal neovascularization and subretinal fibrosis., Plos one, 8, 6, e68173, 2013.06.
32. Eiichi Hasegawa, Koh Hei Sonoda, Takashi Shichita, Rimpei Morita, Takashi Sekiya, Akihiro Kimura, Yuji Oshima, Atsunobu Takeda, Takeru Yoshimura, Shigeo Yoshida, Tatsuro Ishibashi, Akihiko Yoshimura, IL-23-independent induction of IL-17 from γδT cells and innate lymphoid cells promotes experimental intraocular neovascularization, Journal of Immunology, 10.4049/jimmunol.1202495, 190, 4, 1778-1787, 2013.02, Choroidal neovascularization (CNV) is a characteristic of age-related macular degeneration. Genome-wide association studies have provided evidence that the immune system is involved in the pathogenesis of age-related macular degeneration; however, the role of inflammatory cytokines in CNV has not been established. In this study, we demonstrated that IL-17 had a strong potential for promoting neovascularization in a vascular endothelial growth factor-independent manner in laser-induced experimental CNV in mice. Infiltrated γδT cells and Thy-1+ innate lymphoid cells, but not Th17 cells, were the main sources of IL-17 in injured eyes. IL-23 was dispensable for IL-17 induction in the eye. Instead, we found that IL-1β and high-mobility group box 1 strongly promoted IL-17 expression by γδT cells. Suppression of IL-1β and high-mobility group box 1, as well as depletion of γδT cells, reduced IL-17 levels and ameliorated experimental CNV. Our findings suggest the existence of a novel inflammatory cytokine network that promotes neovascularization in the eye..
33. Shoji Notomi, Toshio Hisatomi, Atsunobu Takeda, Yasuhiro Ikeda, Hiroshi Enaida, tatsuro ishibashi, Dynamic increase in extracellular ATP accelerates photoreceptor apoptosis via ligation of P2RX7 in subretinal hemorrhage., Plos one, 10.1371/journal.pone.0053338., 8, 1, 53338, 2013.01.
34. Hasegawa E, Oshima Y, Takeda A, Saeki K, Yoshida H, Sonoda KH, Ishibashi T., IL-27 inhibits pathological ocular neovascularization due to laser burn., J Leukoc Biol, 22045869, 91, 2, 267-273, 2012.02.
35. Notomi S, Hisatomi T, Ikeda Y, Enaida H, Takeda A, Kanemaru , Kroemer G, Ishibashi T, Critical involvement of extracellular ATP acting on P2RX7 purinergic receptors in photoreceptor cell death, Am J pathol, 179, 6, 2798-2809, 2011.12.
36. Jo YJ, Sonoda KH, Oshima Y, Takeda A, Kohno RI, Yamada Y, Hamuro J, Nodomi Hisatomi T, Ishibashi T., Establishment of a new animal model of focal subretinal fibrosis that resembles disciform lesion in advanced age-related macular degeneration, Invest Ophthalmol Vis Sci., 2010.11.
37. Takeshi Fujimoto, Koh Hei Sonoda, Kuniaki Hijioka, Kohta Sato, Atsunobu Takeda, Eiichi Hasegawa, Yuji Oshima, Tatsuro Ishibashi, Choroidal neovascularization enhanced by chlamydia pneumoniae via toll-like receptor 2 in the retinal pigment epithelium, Investigative Ophthalmology and Visual Science, 10.1167/iovs.09-4464, 51, 9, 4694-4702, 2010.09, PURPOSE. Choroidal neovascularization (CNV) is directly related to visual loss in persons with age-related macular degeneration (AMD) and other macular disorders. Chlamydia pneumoniae, a prokaryotic pathogen that causes chronic inflammation, is recognized as a risk factor for cardiovascular diseases. In this study, the authors investigated the association between C. pneumoniae infection and AMD using a laser-induced CNV model in mice. METHODS. C57BL/6 mice, myeloid differentiation factor (MyD) 88 knockout (KO) mice, Toll-like receptor (TLR) 2 KO mice, and TLR4 KO mice were used. Experimental CNV was induced by rupturing the Bruch's membrane by laser photocoagulation (PC). Seven days after PC, the eyes were enucleated and the areas of CNV were measured in choroidal flat mounts. Cytokine gene expression by quantitative real-time PCR in the primary cultured retinal pigment epithelium (RPE) cells was also examined. RESULTS. Vitreous injection of the C. pneumoniae antigen increased the size of CNV. Although lipopolysaccharide stimulation can induce multiple cytokines, cultured mouse RPE cells from C57BL/6 mice expressed IL-6 and VEGF, but not TNF-α mRNA, in response to C. pneumoniae antigen. RPE cells from either MyD88 KO mice or TLR2 KO mice did not respond to the C. pneumoniae antigen. TLR2 KO mice did not augment the size increase of experimental CNV by C. pneumoniae antigen in vivo. CONCLUSIONS. C. pneumoniae can trigger inflammatory responses in the eye and promote experimental CNV in a TLR2- dependent manner. These data provide experimental evidence to imply persistent C. pneumoniae infection is a risk factor for AMD..
38. Albuquerque RJC, Hayashi T, Cho W, Kleinman ME, Dridi S, Takeda A, Baffi JZ, Yamada K, Kaneko H, Chappell J, Peichl L, Hartmann MG, St.Leger J, Samuelson DA, Pesavento PA, Agnew DW, Ziang HY, Wang D, Yang Z, Zhang K, Yamagami S, Amano S, Alexander JS, Peterson ML, Brekken RA, Hirashima M, Capoor S, Usui T, Ambati BK, Ambati J., Alternatively spliced VEGF receptor-2 is an essential endogenous lymphangiogenesis inhibitor, Nature Medicine, 15, 9, 1023-1030, 2009.09.
39. Takeda A, Baffi JZ, Kleinman ME, Cho W, Nozaki M, Yamada K, Kaneko H, Albuquerque RJC, Dridi S, Saito K, Raisler BJ, Budd SJ, Geisen P, Munitz A, Ambati BK, Rich MG, Ishibashi T, Wright JD, Humbles AA, Gerard CJ, Ogura Y, Pan Y, Smith JR, Grisanti S, Hartnett ME, Rothenberg ME, Ambati J., CCR3 is a therapeutic and diagnostic non-inflammatory target for neovascular age-related macular degeneration, Nature, 460, 7252, 225-230, 2009.07.
40. Takashi Igawa, Hitoshi Nakashima, Atsushi Sadanaga, Kohsuke Masutani, Katsuhisa Miyake, Sakiko Shimizu, Atsunobu Takeda, Shinjiro Hamano, Hiroki Yoshida, Deficiency in EBV-induced gene 3 (EBI3) in MRL/lpr mice results in pathological alteration of autoimmune glomerulonephritis and sialadenitis, Modern Rheumatology, 10.1007/s10165-008-0117-1, 19, 1, 33-41, 2009, MRL/lpr mice develop a systemic autoimmune disease that is reminiscent of systemic lupus erythematosus (SLE) and Sjögren's syndrome in humans. To investigate the role of IL-27 in the development of autoimmune disorders in MRL/lpr mice, we disrupted the EBV-induced gene 3 (EBI3), which is a subunit of IL-27. Consequently, the pathophysiology of glomerulonephritis and sialadenitis, which develops in MRL/lpr mice, was drastically changed. EBI3-/- MRL/lpr mice developed disease that resembles human membranous glomerulonephritis (MGN), not diffuse proliferative glomerulonephritis (DPGN), with a predominance of IgG1 in glomerular deposits, and different type sialadenitis from Sjögren's syndrome, with IgG1 producing plasma cell infiltration in salivary glands, accompanied by increased IgG1 and IgE in the sera. T cells in these mice displayed significantly reduced IFN-γ production along with elevated IL-4 expression. Loss of EBI3 thus favors Th2-type autoimmune responses, suggesting that the Th1/Th2 balance may be a pivotal determinant of phenotypes of human autoimmune diseases..
41. Aya Takeuchi, Masaru Takeuchi, Kosuke Oikawa, Koh Hei Sonoda, Yoshihiko Usui, Yoko Okunuki, Atsunobu Takeda, Yuji Oshima, Keiichi Yoshida, Masahiko Usui, Hiroshi Goto, Masahiko Kuroda, Effects of dioxin on vascular endothelial growth factor (VEGF) production in the retina associated with choroidal neovascularization, Investigative Ophthalmology and Visual Science, 10.1167/iovs.08-2299, 50, 7, 3410-3416, 2009, PURPOSE. Cigarette smoking is the most consistent risk factor for age-related macular degeneration (AMD), especially the choroidal neovascularization (CNV)-mediated exudative type. Dioxins and dioxin-like compounds have various effects on living organisms and are also contained in cigarette smoke. However, the effects of dioxins on the eye remain elusive. In this study, the authors examined the association between dioxins and neovascularization in the eye. METHODS. C57BL/6 mice were injected intraperitoneally with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) every other day for 14 days. Messenger RNA expression of cytochrome P450 (CYP)1A1, CYP1B1, vascular endothelial growth factor (VEGF)-A and VEGF-B, and VEGF production were examined in the eyes of TCDD-treated mice and in human retinal pigment epithelial cell lines (ARPE-19) exposed to TCDD. In addition, CNV was induced by photocoagulation in mice injected with TCDD, and the volume of CNV was compared by fluorescencelabeled choroidal flat mount. RESULTS. TCDD injected intraperitoneally increased CYP1A1 mRNA expression in the iris/ciliary body and retina, indicating that TCDD acts directly on ocular tissues through the aryl hydrocarbon receptor (AhR) to promote the transcription of target genes. TCDD also promoted VEGF-A mRNA expression in the retina and the retinal pigment epithelium. TCDD-induced VEGF production at the molecular level was also observed in vivo by immunohistochemistry and in vitro using ARPE-19. Moreover, the injection of TCDD significantly exacerbated photocoagulation-induced CNV in mice. CONCLUSIONS. The authors demonstrate that dioxins are among the factors inducing abnormal vascularization in the eye through VEGF production mediated by AhR signaling..
42. Salvatore Ponticelli, Daniela Marasco, Valeria Tarallo, Romulo J.C. Albuquerque, Stefania Mitola, Atsunobu Takeda, Jean Marie Stassen, Marco Presta, Jayakrishna Ambati, Menotti Ruvo, Sandro De Falco, Modulation of angiogenesis by a tetrameric tripeptide that antagonizes vascular endothelial growth factor receptor 1, Journal of Biological Chemistry, 10.1074/jbc.M806607200, 283, 49, 34250-34259, 2008.12, Vascular endothelial growth factor receptor-1 (VEGFR-1, also known as Flt-1) is involved in complex biological processes often associated to severe pathological conditions like cancer, inflammation, and metastasis formation. Consequently, the search for antagonists of Flt-1 has recently gained a growing interest. Here we report the identification of a tetrameric tripeptide from a combinatorial peptide library built using non-natural amino acids, which binds Flt-1 and inhibits in vitro its interaction with placental growth factor (PlGF) and vascular endothelial growth factor (VEGF) A and B (IC50 ∼ 10 μM). The peptide is stable in serum for 7 days and prevents both Flt-1 phosphorylation and the capillary-like tube formation of human primary endothelial cells stimulated by PlGF or VEGF-A. Conversely, the identified peptide does not interfere in VEGF-induced VEGFR-2 activation. In vivo, this peptide inhibits VEGF-A- and PlGF-induced neoangiogenesis in the chicken embryo chorioallantoic membrane assay. In contrast, in the cornea, where avascularity is maintained by high levels of expression of the soluble form of Flt-1 receptor (sFlt-1) that prevents the VEGF-A activity, the peptide is able to stimulate corneal mouse neovascularization in physiological condition, as reported previously for others neutralizing anti-Flt-1 molecules. This tetrameric tripeptide represents a new, promising compound for therapeutic approaches in pathologies where Flt-1 activation plays a crucial role..
43. N. Sugiyama, H. Nakashima, T. Yoshimura, A. Sadanaga, S. Shimizu, K. Masutani, T. Igawa, M. Akahoshi, K. Miyake, A. Takeda, A. Yoshimura, S. Hamano, H. Yoshida, Amelioration of human lupus-like phenotypes in MRL/lpr mice by overexpression of interleukin 27 receptor α(WSX-1), Annals of the Rheumatic Diseases, 10.1136/ard.2007.077537, 67, 10, 1461-1467, 2008.10, Objective: In the present work, we investigate the role of interleukin (IL)27/IL27 receptor α (Rα) (WSX-1) in the development of autoimmune disorders in the MRL/lpr mouse, which is considered as an experimental model of systemic lupus erythaematosus (SLE) in humans. Methods: We generated two strains of WSX-1 transgenic mice in the MRL/lpr background with different expression levels of WSX-1, and investigated the effect of WSX-1 overexpression on survival, glomerulonephritis and immunological properties. Results: In comparison with wild type (WT) MRL/lpr and transgenic (Tg) low (TgL) mice, Tg high (TgH) mice exhibited a prolonged lifespan and no apparent development of autoimmune nephritis. Production of anti-dsDNA antibody and total IgG and lgG2a were significantly lower in TgH mice than those of TgL and WT mice. The expressed amounts of interferon (IFN)y and IL4 mRNA by CD4+ T cells from Tg mice decreased in a dose-dependent fashion. CD4+ splenic lymphocytes in TgH mice were more subject to the IL27-mediated suppression of cytokine production. In vitro stimulation of CD4+ T cells by IL27 resulted in over phosphorylation of STAT3 in TgH cells than in WT cells. Conclusion: WSX-1 overexpression in the MRL/lpr background rendered the autoimmune prone mice protected from the development of autoimmune diseases. Our results suggest that IL27 signalling may be a therapeutic target against autoimmune diseases, including human SLE..
44. Kleinman ME*, Yamada K*, Takeda A*, Chandrasekaren V, Nozaki M, Baffi JZ, Albuquerque RJC, Yamasaki S, Itaya M, Pan Y, Appukutan B, Gibbs D, Yang Z, Kariko K, Ambati BK, Wilgus TA, DiPietro LA, Sakurai E, Zhang K, Smith JR, Taylor EW, Ambati J., Sequence- and target-independent suppression of angiogenesis by siRNA via TLR3, Nature, 452, 7187, 591-597, 2008.03.
45. Sonoda KH, Yoshimura T, Takeda A, Ishibashi T, Hamano S, Yoshida H, WSX-1 plays a critical role for an initiation of experimental autoimmune uveitis., Int Immunol, 19, 1, 93-98, 2007.01.
46. Ambati BK, Nozaki M*, Singh N*, Takeda A*, Jani PD*, Suthar T, Albuquerque RJC, Richter E, Sakurai E, Newcomb MT, Kleinman ME, Caldwell RB, Lin Q, Ogura Y, Orecchia A, Samuelson DA, Agnew DW, Leger JS, Green WR, Mahasreshti PJ, Curiel DT, Kwan D, Marsh H, Ikeda S, Leiper LJ, Collinson JM, Bogdanovich S, Khurana TS, Shibuya M, Baldwin ME, Ferrara N, Gerber HP, De Falco S, Witta J*, Baffi JZ*, Raisler BJ*, Ambati J., Corneal avascularity is due to soluble VEGF receptor-1, Nature, 443, 7114, 993-997, 2006.10.
47. Yoshimura T, Takeda A, Hamano S, Miyazaki Y, Kinjyo I, Ishibashi T, Yoshimura A, Yoshida H, Two-sided roles of IL-27; induction of Th1 differentiation on naïve CD4+ T cells vs. suppression of pro-inflammatory cytokine production including IL-23-induced IL-17 on activated CD4+ T cells partially through STAT3-dependent mechanism., J Immunol , 177, 8, 5377-5385, 2006.08.
48. Takeda A, Hamano S, Shiraishi H, Yoshimura T, Ogata H, Ishii K, Ishibashi T, Yoshimura A, Yoshida H., Hyperproliferation and hyperproduction of cytokines by WSX-1-overexpressing CD4+ T cells in response to TCR stimulation, Int Immunol, 17, 7, 889-897, 2005.07.
49. Satomi Shiose, Yasuaki Hata, Yoshihiro Noda, Yukio Sassa, Atsunobu Takeda, Hiroshi Yoshikawa, Kimihiko Fujisawa, Toshiaki Kubota, Tatsuro Ishibashi, Fibrinogen stimulates in vitro angiogenesis by choroidal endothelial cells via autocrine VEGF, Graefe's Archive for Clinical and Experimental Ophthalmology, 10.1007/s00417-004-0910-2, 242, 9, 777-783, 2004.09, Background: The purpose of this study is to investigate the effect of fibrinogen on angiogenesis in vitro formed by cultured bovine choroidal endothelial cells (BCECs) and the involvement of vascular endothelial growth factor (VEGF) in this mechanism. Methods: For in vitro tube formation assay, BCECs were seeded on collagen gel containing fibrinogen (0-1.5 mg/ml). After 3 days of cultivation, the total length of the tubular structure was measured using Macscope Analyzer. Total RNA and conditioned media were collected after fibrinogen treatment and subjected to Northern and Western blot analyses, respectively. Transcription factor HIF-1α was also analyzed by Western blot analysis using cytosolic and nuclear fraction of BCECs. Involvement of VEGF in fibrinogen-dependent in vitro tube formation was evaluated using anti-VEGF neutralizing antibody or VEGF receptor 2-selective inhibitor (SU5416). Results: Formation of the tubular structure was enhanced 20-50 times in fibrinogen-containing gel in a concentration-dependent manner. The treatment of BCECs with fibrinogen resulted in a significant increase in VEGF gene and protein expression. Accumulation of HIF-1α protein in the nuclear fraction was also detected after the treatment with fibrinogen. Finally, fibrinogen-induced tube formation was significantly inhibited in the presence of anti-VEGF-neutralizing antibody (52.0% inhibition at the concentration of 1 μg/ml, P<0.05) or SU5416 (54.8% inhibition at the concentration of 3 μM, P<0.05). Conclusions: Extravasated fibrinogen might play an important role in the development of choroidal neovascularization associated with age-related macular degeneration, at least in part, through the function of VEGF in an autocrine manner. Transcription factor HIF-1 appears to be involved in fibrinogen-induced VEGF expression..
50. Atsushi Yamanaka, Shinjiro Hamano, Yoshiyuki Miyazaki, Kazunari Ishii, Atsunobu Takeda, Tak W. Mak, Kunisuke Himeno, Akihiko Yoshimura, Hiroki Yoshida, Hyperproduction of Proinflammatory Cytokines by WSX-1-Deficient NKT Cells in Concanavalin A-Induced Hepatitis, Journal of Immunology, 10.4049/jimmunol.172.6.3590, 172, 6, 3590-3596, 2004.03, Administration of Con A induces liver injury that is considered to be an experimental model for human autoimmune or viral hepatitis, where immunopathology plays roles mediated by activated lymphocytes, especially NK1.1+ CD3+ NKT cells, and inflammatory cytokines, including IFN-γ and IL-4. In the present study we investigated the role of WSX-1, a component of IL-27R, in Con A-induced hepatitis by taking advantage of WSX-1 knockout mice. WSX-1-deficient mice were more susceptible to Con A treatment than wild-type mice, showing serum alanine aminotransferase elevation and massive necrosis in the liver. Although the development of NKT cells appeared normal in WSX-1 knockout mice, purified NKT cells from the knockout mice produced more IFN-γ and IL-4 than those from wild-type mice in response to stimulation with Con A both in vitro and in vivo. In addition, hyperproduction of proinflammatory cytokines, including IL-1, IL-6, and TNF-α, was observed in the knockout mice after Con A administration. These data revealed a novel role for WSX-1 as an inhibitory regulator of cytokine production and inflammation in Con A-induced hepatitis..
51. Shinjiro Hamano, Kunisuke Himeno, Yoshiyuki Miyazaki, Kazunari Ishii, Atsushi Yamanaka, Atsunobu Takeda, Manxin Zhang, Hajime Hisaeda, Tak W. Mak, Akihiko Yoshimura, Hiroki Yoshida, WSX-1 is required for resistance to Trypanosoma cruzi infection by regulation of proinflammatory cytokine production, Immunity, 10.1016/S1074-7613(03)00298-X, 19, 5, 657-667, 2003.11, WSX-1 is a class I cytokine receptor with homology to the IL-12 receptors and is essential for resistance to Leishmania major infection. In the present study, we demonstrated that WSX-1 was also required for resistance to Trypanosoma cruzi. WSX-1-/- mice exhibited prolonged parasitemia, severe liver injury, and increased mortality over wild-type mice. WSX-1 -/- splenocytes produced enhanced levels of Th2 cytokines, which were responsible for the prolonged parasitemia. Massive necroinflammatory lesions were observed in the liver of infected WSX-1-/- mice, and IFN-γ that was overproduced in WSX-1-/- mice compared with wild-type mice was responsible for the lesions. In addition, vast amounts of various proinflammatory cytokines, including IL-6 and TNF-α, were produced by liver mononuclear cells in WSX-1-/- mice. Thus, during T. cruzi infection, WSX-1 suppresses liver injury by regulating production of proinflammatory cytokines, while controlling parasitemia by suppression of Th2 responses, demonstrating its novel role as an inhibitory regulator of cytokine production..
52. Takeda A, Hata Y, Shiose S, Sassa Y, Honda M, Fujisawa K, Sakamoto T, Ishibashi T., Suppression of experimental choroidal neovascularization utilizing KDR selective receptor tyrosine kinase inhibitor, Graefe’s Arch Clin Exp Opthalmol, 10.1007/s00417-003-0688-7, 241, 9, 765-772, 2003.09.
53. Takeda A, Hamano S, Yamanaka A, Hanada T, Ishibashi T, Mak TW, Yoshimura A, Yoshida H., Role of IL-27/WSX-1 signaling for induction of T-bet through activation of STAT1 during initial Th1 commitment., J Immunol, 170, 10, 4886-4890, 2003.05.
54. Hiromitsu Hara, Atsunobu Takeda, Michiyo Takeuchi, Andrew C. Wakeham, Annick Itié, Masafumi Sasaki, Tak W. Mak, Akihiko Yoshimura, Kikuo Nomoto, Hiroki Yoshida, The apoptotic protease-activating factor 1-mediated pathway of apoptosis is dispensable for negative selection of thymocytes, Journal of Immunology, 10.4049/jimmunol.168.5.2288, 168, 5, 2288-2295, 2002.03, Negative selection is a process to delete potentially autoreactive clones in developing thymocytes. Programmed cell death or apoptosis is thought to play an important role in this selection process. In this study, we investigated the role of apoptotic protease-activating factor 1 (Apaf1), a mammalian homologue of CED-4, in programmed cell death during the negative selection in thymus. There was no developmental abnormality in thymocytes from newborn Apaf1-/- mice in terms of CD4 and CD8 expression pattern and thymocyte number. Clonal deletion by endogenous male H-Y Ag of Apaf1-deficient thymocytes with transgenic expression of H-Y Ag-specific TCRs (H-Y Tg/Apaf1-/- thymocytes) was normally observed in lethally irradiated wild-type mice reconstituted with fetal liver-derived hemopoietic stem cells. Clonal deletion induced in vitro by a bacterial superantigen was also normal in fetal thymic organ culture. Thus, Apaf1-mediated pathway of apoptosis is dispensable for the negative selection of thymocytes. However, H-Y Tg/Apaf1-/- thymocytes showed partial resistance to H-Y peptide-induced deletion in vitro as compared with H-Y Tg/Apaf1+/- thymocytes, implicating the Apaf1-mediated apoptotic pathway in the negative selection in a certain situation. In addition, the peptide-induced deletion was still observed in H-Y Tg/Apaf1-/- thymocytes in the presence of a broad spectrum caspase inhibitor, z-VAD-fmk, suggesting the presence of caspase-independent cell death pathway playing roles during the negative selection. We assume that mechanisms for the negative selection are composed of several cell death pathways to avoid failure of elimination of autoreactive clones..