Details of a Researcher - Kawakami, Yutaka

Papers - Kawakami, Yutaka

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Identification of a novel cancer-testis antigen CRT2 frequently expressed in various cancers using representational differential analysis

Hayashi Emiko, Matsuzaki Yuriko, Hasegawa Go, Yaguchi Tomonori, Kurihara Sachiko, Fujita Tomonobu, Kageshita Toshiro, Sano Makoto, Kawakami Yutaka

Clinical Cancer Research 13 ( 21 ) 6267 - 6274 2007.11

ISSN 1078-0432

Purpose: Cancer-testis antigens are promising targets for cancer immunotherapy. Identification of additional cancer-testis antigens with frequent expression in various cancers was attempted using representational differential analysis (RDA) and immunogenicity evaluation. Experimental Design: cDNAs preferentially expressed in testis were enriched using RDA by subtraction between testis and normal tissues. Thirty clones showing cancer-testis - like expression based on EST database analysis were evaluated by reverse transcription-PCR. A potential antigen, CRT2, was identified and its expression was analyzed with a newly generated anti-CRT2 antibody. The immunogenicity of CRT2 was examined based on reactivity with serum immunoglobulin G (IgG) from cancer patients, using Western blot and ELISA analysis, and on in vitro induction of tumor-reactive CTLs from HLA-A24 transgenic mice and human peripheral blood lymphocytes. Results: CRT2 was expressed in elongated spermatids of testis among normal tissues and in various cancer cell lines and tissues. The recombinant CRT2 protein was recognized by serum IgG from patients with various cancers in Western blot and ELISA analyses. ACRT2-derived peptide was identified as an HLA-A24-restricted T-cell epitope that induced tumor-reactive CTLs. Conclusion: CRT2 was identified as a new cancer-testis antigen expressed in elongated spermatids of testis and in cancer tissues (particularly melanoma) that is recognized by serum IgG from cancer patients. An HLA-A24-restricted T-cell epitope capable of inducing tumor-reactive CTLs was identified, suggesting that CRT2 may be useful for cancer diagnosis and immunotherapy.

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Suppression of choroidal neovascularization by dendritic cell vaccination targeting VEGFR2

Mochimaru Hiroshi, Nagai Norihiro, Hasegawa Go, Kudo-Saito Chie, Yaguchi Tomonori, Usui Yoshihiko, Kurihara Toshihide, Koto Takashi, Satojuka Shingo, Shinoda Hajime, Ozawa Yoko, Tsubota Kazuo, Kawakami Yutaka, Ishida Susumu

Investigative Ophthalmology and Visual Science 48 ( 10 ) 4795 - 4801 2007.10

ISSN 0146-0404

PURPOSE. To investigate whether the induction of cellular immunity against vascular endothelial growth factor receptor (VEGFR) 2 inhibits the development of choroidal neovascularization (CNV). METHODS. H-2Db-restricted peptide corresponding to amino acids 400 to 408 of VEGFR2 was used as an epitope peptide. Dendritic cells (DCs) were harvested from bone marrow progenitors of C57BL/6 mice. Six-week-old C57BL/6 mice received subcutaneous injections of the epitope peptide-pulsed mature DCs three times at 6-day intervals. After the third immunization, laser photocoagulation was performed to induce CNV. One week after photocoagulation, mice were killed to harvest the choroid and splenocytes. CNV volume was evaluated by volumetric measurements. To confirm the specific immunogenicity of the epitope peptides in C57BL/6 mice, CD8 T cells isolated from harvested splenocytes were restimulated to measure interferon (IFN)-γ and tumor necrosis factor (TNF)-α production through enzyme-linked immunospot assay and ELISA. To determine the T-cell subset responsible for the immunotherapy, mice were intraperitoneally injected with an anti-CD4 or anti-CD8 depletion antibody. RESULTS. CNV volume was significantly lower in mice immunized with the VEGFR2 epitope peptide than in those not immunized or immunized with a control peptide gp70. Cytokine assays showed the peptide-specific production of IFN-γ and TNF-α from the CD8 T cells in a dose-dependent manner. In vivo depletion of CD8, but not CD4, T cells significantly reversed the suppressive effect of the VEGFR2 peptide-pulsed DC vaccination on CNV to the level observed in nonimmunized or gp70-immunized animals. CONCLUSIONS. These results indicate that the VEGFR2 peptide-specific induction of cellular immunity inhibits CNV through the cytotoxicity of CD8 T cells. Results of the present study suggested the possibility of DC vaccination targeting VEGFR2 as a novel therapeutic strategy for CNV.

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The possibility and limitations of cell therapy. 2) The current status and overview of tumor-immune cell therapy--T cell-dendritic cell therapy

Kawakami Yutaka

Nihon Naika Gakkai zasshi. The Journal of the Japanese Society of Internal Medicine 96 ( 9 ) 1923 - 1928 2007.09

ISSN 0021-5384

Clinical significance of serum p53 antibodies in patients with ulcerative colitis and its carcinogenesis

Yoshizawa Shigeo, Matsuoka Katsuyoshi, Inoue Nagamu, Takaishi Hiromasa, Ogata Haruhiko, Iwao Yasushi, Mukai Makio, Fujita Tomonobu, Kawakami Yutaka, Hibi Toshifumi

Inflammatory Bowel Diseases 13 ( 7 ) 865 - 873 2007.07

ISSN 1078-0998

Background: For early detection of ulcerative colitis (UC)-associated colorectal cancer (CRC), surveillance colonoscopy is recommended in UC patients at high risk. However, poor acceptability deteriorates its effectiveness and a suitable marker for selecting patients at high risk is needed. Here we evaluated clinical usefulness of the measurement of anti-p53 antibodies (Abs) by enzyme-linked immunosorbent assay (ELISA) using sera samples from UC patients. Methods: Sera from 286 patients with UC, 82 patients with sporadic CRC, and 63 healthy controls (HC) were obtained. Serum anti-p53 antibodies were detected with ELISA. Immunohistochemical detection was also performed in patients who developed dysplasia or CRC. Results: Serum p53 Ab was positive in 15.0% of UC, while it was positive only in 1.6% of HCs. In sporadic CRCs, 52.4% of 82 patients were positive. In UC patients with disease duration equal to or longer than 8 years, positivity of serum p53 Ab was significantly higher than those in patients with shorter duration. Eight of 13 (61.5%) UC patients with CRC or dysplasia were positive for serum p53 Abs, which was significantly higher than that in patients without neoplasia. All UC patients with CRC were positive for p53 staining, while 2 were negative for serum p53 Ab. Finally, levels of serum p53 Ab had fallen in 4 patients with CRC we could monitor after surgery. Conclusions: This study revealed that p53 Ab developed in the progression of UC-associated CRC but not in all patients with neoplasia, suggesting that serological detection of p53 Abs by ELISA is not suitable in primarily selecting patients at high risk; however, it is helpful in salvaging patients who drop from a surveillance program.

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Preferential expression and frequent IgG responses of a tumor antigen, SOX5, in glioma patients

Ueda Ryo, Yoshida Kazunari, Kawase Takeshi, Kawakami Yutaka, Toda Masahiro

International Journal of Cancer 120 ( 8 ) 1704 - 1711 2007.04

ISSN 0020-7136

We previously reported to identify SOX5 as a glioma antigen by serological screening using a testis cDNA library. The present study was designed to analyze SOXS expression, its immunoreactivity, and the correlation between SOX5 IgG responses and clinical features in glioma patients to evaluate the possibility of its use as a diagnostic marker. Quantitative RT-PCR and Western blot analysis revealed that SOX5 was expressed in glioma tissues, but not in normal adult tissues, except in the testis. An immunohistochemical analysis showed that SOX5 was expressed in glioma cells, but only a few SOX5-positive cells were detected in non-neoplastic tissues from the cerebral cortex. IgG antibodies against SOX5 were detected in sera from 8 of the 27 glioma patients (27.6%), 0 of the 14 patients with other brain diseases (0%), 1 of the 54 other cancer patients (1.9%) and 1 of the 37 healthy individuals (2.7%). Patients with glioblastotna (GBM) who showed IgG responses against SOX5 exhibited significantly better survival periods than GBM patients without SOX5 antibodies. In summary, SOX5 is aberrantly expressed in glioma and can be recognized as a glioma antigen using IgGs from the sera of glioma patients. Furthermore, there is a statistically significant correlation between the presence of SOX5 IgGs and survival in GBM patients, suggesting that the glioma antigen SOX5 may be useful not only as a diagnostic marker, but also as a prognostic marker in glioma patients.

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Role of heat shock protein 47, a collagen-binding chaperone, in lacrimal gland pathology in patients with cGVHD

Ogawa Yoko, Razzaque Mohammed S., Kameyama Kaori, Hasegawa Go, Shimmura Shigeto, Kawai Masataka, Okamoto Shinichiro, Ikeda Yasuo, Tsubota Kazuo, Kawakami Yutaka, Kuwana Masataka

Investigative Ophthalmology and Visual Science 48 ( 3 ) 1079 - 1086 2007.03

ISSN 0146-0404

PURPOSE. Uncontrolled fibrosis due to excessive accumulation of extracellular matrix proteins in the lacrimal glands of patients with chronic graft-versus-host disease (cGVHD) is well documented. Heat-shock protein 47 (HSP47) is involved in the molecular maturation of collagen and has been shown to have a fibrogenic role in various fibrotic diseases. In this study, the role of HSP47 in the pathogenesis of lacrimal gland of patients with cGVHD was investigated. METHODS. The expression of HSP47, Ki67 (a proliferation marker), types I and III collagen, and α-smooth muscle actin (α-SMA) was examined in tissue sections and in primary cultures of fibroblasts obtained from the lacrimal glands of patients with cGVHD (n = 8) and Sjögren's syndrome (SS; n = 7). RESULTS. Tissue sections of the lacrimal glands of patients with cGVHD showed markedly increased expression of HSP47 in fibroblasts around the medium-sized ducts than did those from patients with SS. The elevated expression of HSP47 in patients with cGVHD was mostly detected in Ki67-positive fibroblasts and was associated with increased accumulation of types I and III collagen in and around the fibrotic areas. Primary fibroblast cultures generated from cGVHD lacrimal gland showed higher HSP47 mRNA expression than did fibroblasts isolated from SS biopsy tissue, as determined by RT-PCR (P < 0.05). In contrast, α-SMA was higher in the SS than cGVHD fibroblasts at both mRNA and protein levels, and more lacrimal gland fibroblasts in the SS were positive for α-SMA than cGVHD (P < 0.01). CONCLUSIONS. In cGVHD, increased expression of HSP47 may promote excessive collagen assembly in and around the periductal areas where fibroblasts are mostly in an active state. The less α-SMA in the cGVHD lacrimal gland fibroblasts suggests a relative lack of myofibroblastic transformation. It is likely that fibroblasts incapable of myofibroblastic transformation are the main source of HSP47 and collagen production, and the resultant effect is the periductal fibrotic changes seen in lacrimal glands of patients with cGVHD.

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T helper type 2-biased natural killer cell phenotype in patients with pemphigus vulgaris

Takahashi Hayato, Amagai Masayuki, Tanikawa Akiko, Suzuki Shigeaki, Ikeda Yasuo, Nishikawa Takeji, Kawakami Yutaka, Kuwana Masataka

Journal of Investigative Dermatology 127 ( 2 ) 324 - 330 2007.02

ISSN 0022-202X

Pemphigus vulgaris (PV) is an autoantibody-mediated bullous disease, but the role of natural killer (NK) cells in its pathogenic process has never been examined in detail. Circulating CD56⁺CD3^- NK cells as well as CD69⁺-activated NK cells were increased in PV patients compared with healthy controls and patients with other autoantibody-mediated autoimmune diseases, including immune thrombocytopenic purpura and myasthenia gravis. Gene expression analysis of highly purified NK cells demonstrated an increased expression of IL-10 and decreased expression of IL-12Rβ2, perforin, and granzyme B ex vivo in PV patients versus healthy controls. The NK cells from PV patients also showed impaired signal transducer and activator of transduction4 phosphorylation upon in vitro IL-12 stimulation. Moreover, NK cells from PV patients exhibited reduced IL-10 production in response to in vitro stimulation with IL-2/IL-12. Finally, IL-5 expression in NK cells was exclusively detected ex vivo in PV patients with active disease, and was lost in subsequent analyses performed during disease remission. Together these findings suggest that NK cells contribute to a T helper type 2-biased immune response in PV patients through impaired IL-12 signaling and an upregulation of IL-10 and IL-5.

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Evaluation of platelet kinetics in patients with liver cirrhosis

Kajihara Mikio, Okazaki Yuka, Kato Shinzo, Ishii Hiromasa, Kawakami Yutaka, Ikeda Yasuo, Kuwana Masataka

Journal of Gastroenterology and Hepatology (Australia) 22 ( 1 ) 112 - 118 2007

ISSN 0815-9319

Background: Thrombocytopenia is a common manifestation of liver cirrhosis (LC), but its underlying mechanism is not fully understood. The purpose of the present paper was to evaluate the platelet kinetics in LC patients by examining several non-invasive convenient markers. Methods: Fifty-seven LC patients, 32 patients with idiopathic thrombocytopenic purpura (ITP), 12 with aplastic anemia (AA), and 29 healthy individuals were studied. Plasma thrombopoietin was measured by enzyme-linked immunosorbent assay. Absolute reticulated platelet (RP) count and plasma glycocalicin were used as indices for thrombopoiesis, and the indices for platelet turnover were the RP proportion and the plasma glycocalicin normalized to the individual platelet count (GCI). Results: There was no difference in thrombopoietin levels between LC patients and healthy controls. The RP proportion and GCI were significantly higher and the absolute RP count and glycocalicin significantly lower in LC patients than in healthy controls. These markers in ITP and LC patients were comparable, but significantly different from those in AA patients. The bone marrow megakaryocyte density in LC and ITP patients was similar, and significantly higher than in AA patients. Conclusions: Cirrhotic thrombocytopenia is a multifactorial condition involving accelerated platelet turnover and moderately impaired thrombopoiesis. Thrombopoietin deficiency is unlikely to be the primary contributor to cirrhotic thrombocytopenia.

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Enhancement of immunologic tumor regression by intratumoral administration of dendritic cells in combination with cryoablative tumor pretreatment and Bacillus Calmette-Guerin cell wall skeleton stimulation

Udagawa Masaru, Kudo-Saito Chie, Hasegawa Go, Yano Kazuhito, Yamamoto Aiko, Yaguchi Masae, Toda Masahiro, Azuma Ichiro, Iwai Takehisa, Kawakami Yutaka

Clinical Cancer Research 12 ( 24 ) 7465 - 7475 2006.12

ISSN 1078-0432

Purpose: We developed an effective immunotherapy, which could induce antitumor immune responses against shared and unique tumor antigens expressed in autologous tumors. Experimental Design: Intratumoral administration of dendritic cells is one of the individualized immunotherapies; however, the antitumor activity is relatively weak. In this study, we attempted to enhance the antitumor efficacy of the i.t. dendritic cell administration by combining dendritic cells stimulated with Bacillus Calmette-Guerin cell wall skeleton (BCG-CWS) additionally with cryoablative pretreatment of tumors and analyzed the therapeutic mechanisms. Results: These two modifications (cryoablation of tumors and BCG-CWS stimulation of dendritic cells) significantly increases the antitumor effect on both the treated tumor and the untreated tumor, which was distant at the opposite side, in a bilateral s.c. murine CT26 colon cancer model. Further analysis of the augmented antitumor effects revealed that the cryoablative pretreatment enhances the uptake of tumor antigens by the introduced dendritic cells, resulting in the induction of tumor-specific CD8⁺ T cells responsible for the in vivo tumor regression of both treated and remote untreated tumors. This novel combination i.t. dendritic cell immunotherapy was effective against well-established large tumors. The antitumor efficacy was further enhanced by depletion of CD4⁺CD25 ⁺FoxP3⁺ regulatory T cells. Conclusions: This novel dendritic cell immunotherapy with i.t. administration of BCG-CWS-treated dendritic cells following tumor cryoablation could be used for the therapy of cancer patients with multiple metastases.

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Endothelial differentiation potential of human monocyte-derived multipotential cells

Kuwana Masataka, Okazaki Yuka, Kodama Hiroaki, Satoh Takashi, Kawakami Yutaka, Ikeda Yasuo

Stem Cells 24 ( 12 ) 2733 - 2743 2006.12

ISSN 1066-5099

We previously reported a unique CD14⁺CD45⁺CD34 ⁺ type I collagen⁺ cell fraction derived from human circulating CD14⁺ monocytes, named monocyte-derived multipotential cells (MOMCs). This primitive cell population contains progenitors capable of differentiating along the mesenchymal and neuronal lineages. Here, we investigated whether MOMCs can also differentiate along the endothelial lineage. MOMCs treated with angiogenic growth factors for 7 days changed morphologically and adopted a caudate appearance with rod-shaped microtubulated structures resembling Weibel-Palade bodies. Almost every cell expressed CD31, CD144, vascular endothelial growth factor (VEGF) type 1 and 2 receptors, Tie-2, von Willebrand factor (vWF), endothelial nitric-oxide synthase, and CD146, but CD14/CD45 expression was markedly downregulated. Under these culture conditions, the MOMCs continued to proliferate for up to 7 days. Functional characteristics, including vWF release upon histamine stimulation and upregulated expression of VEGF and VEGF type 1 receptor in response to hypoxia, were indistinguishable between the MOMC-derived endothelial-like cells and cultured mature endothelial cells. The MOMCs responded to angiogenic stimuli and promoted the formation of mature endothelial cell tubules in Matrigel cultures. Finally, in xenogenic transplantation studies using a severe combined immunodeficient mouse model, syngeneic colon carcinoma cells were injected subcutaneously with or without human MOMCs. Cotransplantation of the MOMCs promoted the formation of blood vessels, and more than 40% of the tumor vessel sections incorporated human endothelial cells derived from MOMCs. These findings indicate that human MOMCs can proliferate and differentiate along the endothelial lineage in a specific permissive environment and thus represent an autologous transplantable cell source for therapeutic neovasculogenesis.

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Effects of a Helicobacter pylori eradication regimen on anti-platelet autoantibody response in infected and uninfected patients with idiopathic thrombocytopenic purpura

Asahi Atsuko, Kuwana Masataka, Suzuki Hidekazu, Hibi Toshifumi, Kawakami Yutaka, Ikeda Yasuo

Haematologica 91 ( 10 ) 1436 - 1437 2006.10

ISSN 0390-6078

Prolonged thrombocytopenia after allogeneic hematopoietic stem cell transplantation

Yamazaki R., Kuwana M., Mori T., Okazaki Y., Kawakami Y., Ikeda Y., Okamoto S.

Bone Marrow Transplantation 38 ( 5 ) 377 - 384 2006.09

ISSN 0268-3369

To evaluate the mechanisms underlying prolonged thrombocytopenia after allogeneic hematopoietic stem cell transplantation (SCT), an index for plasma glycocalicin normalized for the individual platelet count (GCI), plasma thrombopoietin (TPO), and circulating B cells producing anti-GPIIb-IIIa antibodies were measured in 50 SCT recipients with or without prolonged thrombocytopenia, 42 patients with idiopathic thrombocytopenic purpura, nine patients with aplastic anemia, and 22 healthy individuals. All three indices were significantly higher in the SCT recipients with thrombocytopenia than in those without (P<0.01 for all comparisons), and were significantly correlated with the platelet count in SCT recipients. Stepwise multiple regression analysis of the samples from the SCT recipients revealed that GCI and TPO independently pointed to specific mechanisms of thrombocytopenia. The GCI and TPO status in SCT recipients with thrombocytopenia had a pattern similar to that seen in aplastic anemia, suggesting a major role for impaired thrombopoiesis. An antiplatelet antibody response was frequently detected in SCT recipients, but the development of thrombocytopenia is likely to depend on additional factors, such as reticuloendothelial function. In summary, post transplant prolonged thrombocytopenia is associated with complex mechanisms, including impaired thrombopoiesis and increased platelet turnover.

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The BRAF-MAPK signaling pathway is essential for cancer-immune evasion in human melanoma cells

Sumimoto Hidetoshi, Imabayashi Fumie, Iwata Tomoko, Kawakami Yutaka

Journal of Experimental Medicine 203 ( 7 ) 1651 - 1656 2006.07

ISSN 0022-1007

The mitogen-activated protein kinase (MAPK) pathway is frequently activated in human cancers, leading to malignant phenotypes such as autonomous cellular proliferation. Here, we demonstrate a novel role of the activated MAPK pathway in immune evasion by melanoma cells with the mutation of BRAF, which encodes a MAPKKs, (BRAF^V600E). MEK inhibitor U0126 or RNA interference (RNAi) for BRAF V600E decreased production of the immunosuppressive soluble factors interleukin (IL)-10, VEGF, or IL-6 from melanoma cells to levels comparable to those after signal transducer and activator of transcription (STAT)3 inactivation. The suppressive activity of the culture supernatants from the melanoma cells on the production of inflammatory cytokines IL-12 and tumor necrosis factor α by dendritic cells upon lipopolysaccharide stimulation was markedly reduced after transduction with BRAF^V600E RNAi, comparable to the effects observed with STAT3 RNAi transduction. No additive or synergistic effects were observed by the simultaneous transduction of RNAi for both BRAF^V600E and STAT3. Furthermore, specific DNA binding and transcriptional activity of STAT3 were not affected by down-regulation of the MAPK signaling with the BRAF RNAi. These results indicate that the MAPK signal, along with the STAT3 signal, is essential for immune evasion by human melanomas that have constitutively active MAPK signaling and is a potential molecular target for overcoming melanoma cell evasion of the immune system. JEM

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Frequent immune response to a melanocyte specific protein KU-MEL-1 in patients with Vogt-Koyanagi-Harada disease

Otani S., Sakurai T., Yamamoto K., Fujita T., Matsuzaki Y., Goto Y., Ando Y., Suzuki S., Usui M., Takeuchi M., Kawakami Yutaka

British Journal of Ophthalmology 90 ( 6 ) 773 - 777 2006.06

ISSN 0007-1161

Aim: To isolate autoantigens possibly involved in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease. Methods: Autoantigens recognised by immunoglobulin G antibodies (IgG Ab) in sera from VKH patients were isolated by screening the lambda phage cDNA libraries made from melanocytes and a highly pigmented melanoma cell line with the patients' sera. Presence of IgG specific for the autoantigens in sera from patients with various panuveitis and healthy individuals was evaluated. Relation between the specific IgG and various clinicopathological features was examined. Results: KU-MEL-1 was found to be one of the 81 isolated positive clones representing 35 distinct genes, which is a previously isolated melanoma antigen preferentially expressed in melanocytes. The IgG Ab specific for KU-MEL-1 was detected in sera from patients with VKH in significantly higher amounts than in sera from patients with Behçet's disease, sarcoidosis, and from healthy individuals. Positive serum KU-MEL-1 Ab was significantly associated with HLA-DRB1*0405 and male VKH patients. Conclusion: KU-MEL-1 was identified as a new autoantigen for VKH. The highly frequent induction of IgG Ab for KU-MEL-1 in HLA-DRB1*0405 positive VKH patients may suggest the possible involvement of KU-MEL-1 specific CD4 ⁺ T cells in the pathogenesis of VKH, suggesting the possible use in the development of diagnostic and therapeutic treatments for VKH patients.

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Increase in circulating endothelial precursors by atorvastatin in patients with systemic sclerosis

Kuwana Masataka, Kaburaki Junichi, Okazaki Yuka, Yasuoka Hidekata, Kawakami Yutaka, Ikeda Yasuo

Arthritis and Rheumatism 54 ( 6 ) 1946 - 1951 2006.06

ISSN 0004-3591

Objective. To evaluate whether atorvastatin can increase bone marrow-derived circulating endothelial precursors (CEPs) and improve the vascular symptoms in patients with systemic sclerosis (SSc; scleroderma). Methods. The study was designed as an open-label, prospective study involving 14 patients with SSc who received 10 mg/day of atorvastatin for 12 weeks and were followed up for the subsequent 4 weeks. CEPs were quantified at weeks 0 (pretreatment), 4, 8, 12 (during treatment), and 16 (posttreatment) by cell sorting followed by 3-color flow cytometry. Raynaud's phenomenon variables, global measures, and psychological scales as well as circulating angiogenic factors and endothelial activation/injury markers were serially assessed. The potential of CEPs to differentiate into mature endothelial cells was examined in cultures with angiogenic stimuli. Results. None of the patients experienced an adverse event, but 1 dropped out because of an excessive decrease in serum total cholesterol. Atorvastatin treatment resulted in a 1.7- to 8.0-fold increase in CEPs from baseline levels (P < 0.0001), but the numbers returned to within baseline levels at posttreatment. However, 8 patients (62%) experienced a gradual decrease in the number of CEPs, even while taking atorvastatin. Variables indicating the extent of Raynaud's phenomenon improved significantly, and up-regulated levels of angiogenic factors and vascular endothelial activation/injury markers decreased significantly during atorvastatin treatment. These variables returned to within baseline levels after discontinuation of the drug. In contrast, atorvastatin failed to improve the in vitro maturation potential of CEPs. Conclusion. The results of this pilot study suggest that atorvastatin treatment can increase CEPs and may be effective in improving Raynaud's phenomenon, even in SSc patients who have CEP dysfunction intrinsically.

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A new melanoma antigen fatty acid-binding protein 7, involved in proliferation and invasion, is a potential target for immunotherapy and molecular target therapy

Goto Yasufumi, Matsuzaki Yuriko, Kurihara Sachiko, Shimizu Ayako, Okada Tsutomu, Yamamoto Kazuhiko, Murata Hiroshi, Takata Minoru, Aburatani Hiroyuki, Hoon Dave S B, Saida Toshiaki, Kawakami Yutaka

Cancer Research 66 ( 8 ) 4443 - 4449 2006.04

ISSN 0008-5472

The identification of molecules that are preferentially expressed in melanoma cells and involved in their malignant phenotypes is important for understanding melanoma biology and the development of new diagnostic and therapeutic methods. By comparing the expression profile of a melanoma cell line with those of various normal tissues using GeneChip and by confirming the actual expression of the selected genes by reverse transcription-PCR and Northern and Western blot analyses, fatty acid-binding protein 7 (FABP7), which is frequently expressed in melanomas, was identified. Immunohistochemical examination revealed that FABP7 was expressed in 11 of 15 melanoma tissues. By down-regulating the FABP7 expression with FABP7-specific small interfering RNAs, in vitro cell proliferation and Matrigel invasion were suppressed in two of six melanoma cell lines. Overexpression of FABP7 in a FABP7-negative embryonic kidney cell line 293T by transfecting with the FABP7 cDNA resulted in enhanced cell proliferation and Matrigel invasion, indicating that FABP7 plays a role in the malignant phenotype of some melanoma cell lines. IgG antibodies specific for the phage or bacterial recombinant FABP7 protein were detected in 14 of 25 (56%) or in 8 of 31 (26%) sera from melanoma patients, respectively, but not in sera from healthy individuals, indicating that FABP7 is an immunogenic antigen in melanoma patients. These results showed that FABP7 is frequently expressed in melanoma, may be involved in cell proliferation and invasion, and may be a potential target for development of diagnostic and therapeutic methods.

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Neurogenic potential of progenitors derived from human circulating CD14⁺ monocytes

Kodama Hiroaki, Inoue Takafumi, Watanabe Ryuichi, Yasutomi Daisuke, Kawakami Yutaka, Ogawa Satoshi, Mikoshiba Katsuhiko, Ikeda Yasuo, Kuwana Masataka

Immunology and Cell Biology 84 ( 2 ) 209 - 217 2006.04

ISSN 0818-9641

We previously reported a primitive cell fraction derived from human circulating CD14⁺ monocytes, named monocyte-derived multipotential cells (MOMC), that can differentiate along mesenchymal lineages, including bone, cartilage, fat, skeletal muscle and cardiac muscle. In this study, we investigated whether MOMC can differentiate into the neuronal lineage. MOMC were fluorescently labelled and cocultivated with a primary culture of rat neurons for up to 4 weeks. The protein and gene expressions of neuron-specific markers in the human MOMC were evaluated over time using immunohistochemistry, in situ hybridization and reverse transcription followed by PCR. Shortly after cocultivation with rat neurons, nearly all the MOMC expressed early neuroectodermal markers, Mash1, Neurogenin2 and NeuroD, together with nestin, an intermediate filament expressed in neurogenesis. After 14 days of coculture, a subpopulation of MOMC displayed a multipolar morphology with elongated neurites and expressed mature neuron-specific markers, including neurofilament, microtubule-associated protein type 2, β3-tubulin, NeuN and Hu. Transdifferentiation of monocytes into the neuroectodermal lineage was shown by the simultaneous expression of proneural markers and CD45/CD14 early in the differentiation process. The cocultivated MOMC retained their proliferative capacity for at least 16 days. Finally, the neuronal differentiation of MOMC was observed when they were cultured with neurons without cell-to-cell contact. The capacity of MOMC to differentiate into both mesodermal and neuroectodermal lineages suggests that circulating CD14⁺ monocytes are more multipotential than previously thought.

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Identification of a glioma antigen, GARC-1, using cytotoxic T lymphocytes induced by HSV cancer vaccine

Iizuka Yukihiko, Kojima Hidefumi, Kobata Tetsuji, Kawase Takeshi, Kawakami Yutaka, Toda Masahiro

International Journal of Cancer 118 ( 4 ) 942 - 949 2006.02

ISSN 0020-7136

Despite several ongoing clinical trials of immunotherapies against glioma, few glioma-specifk antigens recognized by cytotoxic T lymphocytes (CTLs) have been identified. We recently demonstrated that intratumoral inoculation with herpes simplex virus (HSV) as a cancer vaccine activates tumor-specific CTLs. To identify glioma antigens recognized by CTLs, we used the HSV cancer vaccine to vaccinate mice harboring a syngeneic mouse glioma cell line, GL261. From the splenocytes of the immunized mice, we generated an H-2D^b-restricted CTL line, GCL-1, that was specific for GL261. Then, a cDNA expression library generated from GL261 was screened with GCL-1, and a new gene encoding glioma antigen, GARC-1, was isolated. Sequence analysis revealed that the GARC-1 gene isolated from GL261 had a point mutation causing an amino acid change (Asp to Asn at position 81). T-cell epitope analysis revealed that the mutated peptide GARC-1_77-85 (AALLNKLYA) but not the wild-type peptide (AALLDKLYA), was recognized by GCL-1. These results suggest that HSV cancer vaccination may be a useful method for inducing tumor-specific CTLs and identifying tumor antigens. Furthermore, this GL261/GARC-1 murine glioma model may be useful for the development of immunotherapy for brain tumors.

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Autoantibody to CD40 ligand in systemic lupus erythematosus

Nakamura M., Tanaka Y., Satoh T., Kawai M., Hirakata M., Kaburaki J., Kawakami Y., Ikeda Y., Kuwana M.

Rheumatology 45 ( 2 ) 150 - 156 2006.02

ISSN 1462-0324

Objectives. To examine the prevalence, clinical associations and pathogenic roles of autoantibodies to CD40 ligand (CD40L) in patients with systemic lupus erythematosus (SLE). Methods. Plasma anti-CD40L antibodies from 125 patients with SLE, 24 with primary antiphospholipid syndrome (APS) and 90 with idiopathic thrombocytopenic purpura (ITP) and from 62 healthy individuals were measured with an enzyme-linked immunosorbent assay (ELISA). HeLa cells transfected with human CD40L cDNA (HeLa/CD40L) were used to confirm the presence of anti-CD40L autoantibodies. The effect of anti-CD40L antibodies on the CD40L-CD40 interaction was evaluated by observing CD40L-induced IκB activation in CD40-expressing fibroblasts. Results. Anti-CD40L autoantibody was detected in seven (6%) SLE, three (13%) primary APS and 11 (12%) ITP patients, but in no healthy controls. Antibody binding in an ELISA was competitively inhibited by membrane components of HeLa/CD40L. Anti-CD40L antibody-positive IgG specifically bound the surface of living HeLa/CD40L, as shown by flow cytometry. The frequency of thrombocytopenia was significantly higher in SLE patients with the anti-CD40L antibody than in those without (100 vs 14% P<0.00001), whereas there was no association between the anti-CD40L antibody and thrombosis. Binding of the anti-CD40L antibodies in patients' plasma to CD40L was competitively inhibited by a series of mouse anti-CD40L monoclonal antibodies. Anti-CD40L antibody-positive IgG failed to inhibit CD40L-induced IκB activation. Conclusions. Anti -CD40L autoantibody is associated with thrombocytopenia but not thromboembolism. Our findings are potentially useful in understanding the complex roles of CD40L in the pathophysiology of thrombosis and haemostasis as well as the thromboembolic complications that occur during treatment with anti-CD40L humanized antibody.

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Effective inhibition of cell growth and invasion of melanoma by combined suppression of BRAF (V599E) and Skp2 with lentiviral RNAi

Sumimoto Hidetoshi, Hirata Kenro, Yamagata Shizuko, Miyoshi Hiroyuki, Miyagishi Makoto, Taira Kazunari, Kawakami Yutaka

International Journal of Cancer 118 ( 2 ) 472 - 476 2006.01

ISSN 0020-7136

p27^Kip1 that regulates the G1/S transition of cell cycle and inhibits Rho A signaling is frequently lost in several cancers leading to the deregulation of cell growth and cell motility. Mitogen-activated protein kinases (MAPK) regulate the export of p27^Kip1 from nucleus to cytoplasm, followed by the degradation with proteases. Skp-2, a subunit of an SCF ubiquitin-protein ligase complex responsible for the ubiquitination of p27 ^Kip1, is upregulated frequently in several cancers, leading to the decrease of p27^Kip1. We applied human immunodeficiency virus (HIV) lentivirus-mediated RNA interference (RNAi) to melanoma cells with the BRAF mutation (V599E) and overexpressed Skp-2 and found that the simultaneous suppression of these activated oncogenes resulted in the effective inhibition of in vitro cell growth and invasive ability of melanoma cells accompanied by the additional increase of p27^Kip1. Our results suggest that gene therapy against melanoma with the enhanced MAPK and ubiquitin-proteasomal pathways could be a specific and effective therapeutic strategy for cancers.

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