研究者詳細 - 河上　裕

論文 - 河上　裕

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Identification of human tumor antigens and its implications for diagnosis and treatment of cancer

Kawakami Yutaka, Fujita Tomonobu, Matsuzaki Yuriko, Sakurai Toshiharu, Tsukamoto Makoto, Toda Masahiro, Sumimoto Hidetoshi

Cancer Science 95 （ 10 ） 784 - 791 2004年10月

ISSN 1347-9032

Human tumor antigens recognized by T cells have been identified by means of various molecular biological and immunological methods, including cDNA expression cloning with patients' T cells and antibodies, cDNA subtraction using RDA and PCR differential display, systematic gene analysis such as DNA sequencing, CGH, DNA chip/microarray and SAGE, in vitro T cell induction and immunization of HLA transgenic mice. The identification of human tumor antigens has led to a better understanding of the nature of tumor antigens, anti-tumor immune responses in patients before and after immunotherapy, and tumor escape mechanisms. The information obtained from these researches has enabled us to develop and improve immunotherapy by attempting to overcome the identified problems, including intrinsically low immunogenicity of tumor antigens and several escape mechanisms, such as regulatory T cell induction. The existence of immunogenic unique antigens derived from genetic alterations in tumor cells, and the varied immunogenicity of shared tumor antigens among patients due to differing expression in tumor cells and immunoreactivity of patients, indicates that individualized immunotherapy should ideally be performed. The identified antigens will also be useful for development of diagnostic methods and molecular targeting therapy for cancer.

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Defective vasculogenesis in systemic sclerosis

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

Lancet 364 （ 9434 ） 603 - 610 2004年08月

ISSN 0140-6736

Background Typical vascular features of systemic sclerosis include low capillary density and vascular obliteration. The formation and repair of blood vessels in adults involve vasculogenesis, which is mediated through the recruitment of bone-marrow-derived circulating endothelial precursors (CEP). We investigated whether vasculogenesis is impaired in patients with systemic sclerosis. Methods Peripheral blood was obtained from 11 patients with systemic sclerosis, 11 with rheumatoid arthritis, and 11 healthy controls. Factors potentially affecting the CEP number were matched among the three groups. CEP (identified as circulating cells positive for CD34, CD133, and the type 2 receptor for vascular endothelial growth factor) were quantified by cell sorting and three-colour flow cytometry. The circulating concentrations of angiogenic factors were measured by ELISA. The potential of CEP to differentiate into endothelial cells was assessed by the upregulation of von Willebrand factor after in-vitro maturation treatment. Findings The absolute number of CEP was much lower in patients with systemic sclerosis than in patients with rheumatoid arthritis or healthy controls (median 274 [IQR 178-395] vs 1154 [653-1524] and 1074 [713-1186] per 20 mL peripheral blood, respectively; p<0·0001 by Kruskal-Wallis test. Paradoxically, circulating concentrations of most angiogenic factors were significantly higher in patients with systemic sclerosis than in healthy controls. The proportion of CEP that differentiated into endothelial cells was significantly lower in patients with systemic sclerosis than in healthy controls (p<0·0001, Mann-Whitney test). Interpretation Insufficient vascular repair machinery due to defective vasculogenesis might contribute to vasculopathy in systemic sclerosis. Relevance to practice As well as providing an important insight into the pathogenesis of this disorder, these findings suggest that dysregulated vasculogenesis might be important in other disorders with abnormalities in vascular formation, including those with excessive formation of new vessels such as cancer and those with deficient vessel formation such as atherosclerosis. Circulating endothelial precursors could be a novel target for therapeutic strategies for ischaemic complications in patients with systemic sclerosis.

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Inhibition of growth and invasive ability of melanoma by inactivation of mutated BRAF with lentivirus-mediated RNA interference

Sumimoto Hidetoshi, Miyagishi Makoto, Miyoshi Hiroyuki, Yamagata Shizuko, Shimizu Ayako, Taira Kazunari, Kawakami Yutaka

Oncogene 23 （ 36 ） 6031 - 6039 2004年08月

ISSN 0950-9232

Oncogenic mutations of molecules involved in the mitogen-activated protein kinase (MAPK) pathways provide signals mediating both tumor growth and invasion in various cancers including melanomas. BRAF somatic mutations, found in 66% of melanomas, have NIH3T3 transforming ability with the elevated kinase activity in vitro. We attempted to mediate RNA interference (RNAi) with HIV lentiviral vectors specific for either wild type or the most frequently mutated form of BMAF (V599E) in 10 melanoma cell lines, and found that RNAi inhibited the growth of most melanoma cell lines in vitro as well as in vivo, which was accompanied by decrease of both BRAF protein and ERK phosphorylation. Interestingly, the mutated BRAF (V599E)-specific siRNA inhibited the growth and MAPK activity of only melanoma cell lines with this mutation. Furthermore, BRAF RNAi inhibited matrigel invasion of melanoma cells accompanied with a decrease of matrix metalloproteinase activity and β₁ integrin expression. These results clarify that the mutated BRAF (V599E) is essentially involved in malignant phenotype of melanoma cells through the MAPK activation and is an attractive molecular target for melanoma treatment. The lentivirus-mediated RNAi specific for oncogenic mutations may be a powerful technique for gene therapy of cancer.

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Optimization of an siRNA-expression system with an improved hairpin and its significant suppressive effects in mammalian cells

Miyagishi Makoto, Sumimoto Hidetoshi, Miyoshi Hiroyuki, Kawakami Yutaka, Taira Kazunari

Journal of Gene Medicine 6 （ 7 ） 715 - 723 2004年07月

ISSN 1099-498X

Background. RNA interference (RNAi) is a phenomenon in which expression of an individual gene can be specifically silenced by introducing a double-stranded RNA, one complementary to the gene, into cells. This phenomenon can be observed in mammalian cells when small interfering RNAs (siRNAs) are used, and is receiving attention as the most powerful tool for reverse genetics in the post genome era. Several groups have developed vector-based siRNA-expression systems that can induce RNAi in living cells. Methods. We describe here a comparative analysis of various siRNA-expression systems, in which we examined the effects of stem length, loop sequence and insertion of mutation(s) and/or bulges in the stem sequence on silencing effects and on the stability of the vectors. Results. As a result of the comparative analysis, we determined the following optimized siRNA-expression system: U6 promoter-driven hairpin-type dsRNA with 21-nt stem length, three to four mutations in the sense strand only, and the optimized 9-nt loop sequence, derived from microRNA. Moreover, we demonstrate that the siRNA-expression system with a tetracycline-regulated U6 promoter(s) could have the potential to control RNAi in cells, and that the HTV vector-mediated transfer of an siRNA-expression cassette into cells resulted in efficient silencing of a target gene at a multiplicity of infection as low as five. Conclusion. The mutated hairpin siRNAs and their genetically stable coding vectors could be very useful for gene knockdown experiments, and could further benefit gene therapy using RNAi.

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Implantation of Dendritic Cells in Injured Adult Spinal Cord Results in Activation of Endogenous Neural Stem/Progenitor Cells Leading to De Novo Neurogenesis and Functional Recovery

Mikami Yuji, Okano Hideyuki, Sakaguchi Masanori, Nakamura Masaya, Shimazaki Takuya, Okano Hirotaka James, Kawakami Yutaka, Toyama Yoshiaki, Toda Masahiro

Journal of Neuroscience Research 76 （ 4 ） 453 - 465 2004年05月

ISSN 0360-4012

We report a treatment for spinal cord injury involving implantation of dendritic cells (DCs), which act as antigen-presenting cells in the immune system. The novel mechanisms underlying this treatment produce functional recovery. Among the immune cells tested, DCs showed the strongest activity inducing proliferation and survival of neural stem/progenitor cells (NSPCs) in vitro. Furthermore, in DC-implanted adult mice, endogenous NSPCs in the injured spinal cord were activated for mitotic de novo neurogenesis. These DCs produced neurotrophin-3 and activated endogenous microglia in the injured spinal cord. Behavioral analysis revealed the locomotor functions of DC-implanted mice to have recovered significantly as compared to those of control mice. Our results suggest that DC-implantation exerts trophic effects, including activation of endogenous NSPCs, leading to repair of the injured adult spinal cord.

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Identification of human cancer antigens by SEREX

Okada Tsutomu, Kawakami Yutaka

Biotherapy 18 （ 3 ） 205 - 211 2004年05月

ISSN 0914-2223

SEREX (serological identification of tumor antigens by recombinant cDNA expression cloning) is a powerful cDNA expression cloning technique for isolation of tumor antigens recognized by T cells using serum antibodies from cancer patients from whom it is difficult to raise tumor-reactive T cells. To efficiently identify tumor antigens, various modifications have been attempted for SEREX because of frequent isolation of antigens not associated with immune responses to tumor cells. These include use of a cDNA library made from testis, use of sera from patients who are immunized with autologous tumor-derived products who demonstrate tumor regression after treatment, and use of human IgG containing mice sera from immunodeficient mice implanted with human tumor tissues.

Development of Immunotherapy for Pancreatic Cancer

Kawakami Yutaka, Okada Takaho, Akada Masanori

Pancreas 28 （ 3 ） 320 - 325 2004年04月

ISSN 0885-3177

Human tumor antigens recognized by T cells have been recently identified in various cancers, including pancreatic cancer. With the identified antigens, new immunotherapies can be developed using more efficient immunologic intervention (due to sufficient amounts of antigens in a more immunogenic form), as well as more quantitative and qualitative immunomonitoring. Various immunotherapies for patients with various cancers, including pancreatic cancer, are currently under evaluation in clinical trials. These include adoptive transfer of tumor reactive T cells and LAK cells; nonmyeloablative stem cell transplantation; active immunization with the identified tumor antigens, various tumor-derived products, dendritic cells pulsed with tumor antigens, and gene-modified tumor cells. Although these efforts in the realm of pancreatic cancer are still limited, various groups in Japan continue to be actively involved in this field of research.

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Single nucleotide polymorphisms of the inflammatory cytokine genes in adults with chronic immune thrombocytopenic purpura

Satoh Takashi, Pandey Janardan P., Okazaki Yuka, Yasuoka Hidekata, Kawakami Yutaka, Ikeda Yasuo, Kuwana Masataka

British Journal of Haematology 124 （ 6 ） 796 - 801 2004年03月

ISSN 0007-1048

Single nucleotide polymorphisms (SNPs) of inflammatory cytokine genes were examined in 84 adult Japanese patients with chronic immune thrombocytopenic purpura (ITP) and 56 race-matched healthy controls. The SNPs examined were within the genes encoding tumour necrosis factor (TNF)-α (-238 G/A and -308 G/A), TNF-β (+252 G/A), and interleukin (IL)-1β (-511 C/T and +3953 T/C). Of these SNPs, the frequency of the TNF-β (+252) G/G phenotype was significantly higher in ITP patients than in healthy controls (21% vs. 7%, P = 0.04, odds ratio = 3.6, 95% confidence interval 1.1-11.1), while no significant association was detected for the other SNPs. The distribution of the TNF-β (+252) phenotype was not associated with human leucocyte antigen class II alleles or the therapeutic response in ITP patients. The frequency of circulating anti-glycoprotein IIb/IIIa antibody-producing B cells was significantly higher in ITP patients with the TNF-β (+252) G/G phenotype than in those with the G/A or A/A phenotype (11.9 ± 4.9 vs. 6.8 ± 4.9 and 3.7 ± 2.8 per 10⁵ peripheral blood mononuclear cells; P = 0.02 and P < 0.001, respectively). These findings suggest that the SNP located at TNF-β (+252) contributes to susceptibility to chronic ITP by controlling the autoreactive B-cell responses to platelet membrane glycoproteins.

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Augmentation of Antitumor Immune Responses by Multiple Intratumoral Inoculations of Replication-Conditional HSV and Interleukin-12

Iizuka Yukihiko, Suzuki Ayuko, Kawakami Yutaka, Toda Masahiro

Journal of Immunotherapy 27 （ 2 ） 92 - 98 2004年03月

ISSN 1524-9557

Intratumoral inoculation with a herpes simplex virus (HSV) mutant, G207, as an in situ cancer vaccine has been shown to inhibit tumor growth by inducing tumor-specific immune responses. Here, as a step toward the clinical application of this therapeutic approach, we evaluated different protocols for enhancing the antitumor effect. First, in a bilaterally established tumor model with CT26 colon carcinoma, we demonstrated that multiple intratumoral inoculations with G207 induced a greater antitumor effect on both the inoculated and distant tumors than did 1 or 2 inoculations. Second, to boost this antitumor effect, we developed 2 strategies: multiple in situ cancer vaccines with G207 in combination with systemic administration of recombinant interleukin-12 (rIL-12) (G207/systemic rIL-12) or local administration of rIL-12 (G207/local rIL-12). The antitumor effects in both the inoculated and distant tumors by the combined treatments were significantly greater than by either G207 or rIL- 12 treatment alone. G207/systemic rIL-12 and G207/local rIL-12 mediated the complete regression of both the inoculated and distant tumors in 67% and 79% of the animals, respectively. These results indicate that multiple intratumoral inoculations of G207 and systemic or local rIL- 12 administration work synergistically to facilitate tumor regression and that this combination of treatments may have potential for treating cancer metastasis.

Identification of bladder cancer antigens recognized by IgG antibodies of a patient with metastatic bladder cancer

Ito Keiichi, Fujita Tomonobu, Akada Masanori, Kiniwa Yukiko, Tsukamoto Makoto, Yamamoto Aiko, Matsuzaki Yuriko, Matsushita Maiko, Asano Takako, Nakashima Jun, Tachibana Masaaki, Hayakawa Masamichi, Ikeda Hideyuki, Murai Masaru, Kawakami Yutaka

International Journal of Cancer 108 （ 5 ） 712 - 724 2004年02月

ISSN 0020-7136

To identify tumor antigens useful for the diagnosis and treatment of patients with bladder cancer, a lambda phage cDNA library constructed from a high-grade bladder cancer cell line was screened with autologous serum from a patient with metastatic bladder cancer. Forty-eight distinct antigens were isolated. By evaluating the immunogenicity and the tissue-specific expression, KU-BL-1 and KU-BL-2 were identified as immunogenic antigens with restricted tissue expression. KU-BL-1 was found to be a putative human lipoic acid synthetase with a metal-binding site, CXXXCXXC, that was expressed in bladder cancer cell lines and most bladder cancer tissues, as well as normal bladder mucosa and testis tissues. Immunoglobulin (Ig)G antibody to KU-BL-1 was detected in 2 of 28 patients with bladder cancer, but not in 30 healthy individuals. KU-BL-2 was found to be a putative human kelch-like protein that was homologous to Drosophila kelch, with a BTB/POZ domain and kelch repeats. KU-BL-2 was expressed in bladder cancer cell lines, most bladder cancer tissues, testis and heart, but not in normal bladder mucosa. IgG antibody to KU-BL-2 was detected in 8 of 28 patients with bladder cancer, but not in 16 healthy individuals. Tumor reactive T cells were induced from peripheral blood mononuclear cells (PBMC) by stimulation with one of the HLA-A24 binding KU-BL-2 peptides. Therefore, KU-BL-1 and KU-BL-2, which showed preferential expression in bladder cancer with restricted expression in normal tissues, as well as immunogenicity in multiple patients with bladder cancer, may be useful for the development of diagnostic and therapeutic methods for patients with bladder cancer.

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Identification of a human glioma antigen, SOX6, recognized by patients' sera

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

Oncogene 23 （ 7 ） 1420 - 1427 2004年02月

ISSN 0950-9232

To identify tumor antigens for glioma, a human testis cDNA library was screened by serological identification of antigens by recombinant expression cloning with sera from glioma patients. In this screening, the most frequently isolated antigen was SOX6, an Sry-related high-mobility group (HMG) box-containing gene. SOX6 is a transcriptional factor that is specifically expressed in the developing central nervous system and in the early stages of chondrogenesis in mouse embryos. IgG antibodies against SOX6 were detected in sera from 12 of 36 glioma patients (33.3%), 0 of 14 patients with other brain disease (0%), and one of 54 other cancer patients (1.9%). In sera from 37 healthy individuals, no IgG responses against SOX6 were detected, except in an elderly female. Furthermore, Western blot and ELISA analyses with sera from glioma patients revealed that the DNA-binding domain, the HMG box of SOX6, might be a dominant epitope of IgGs against SOX6. RT-PCR and Northern blot analysis revealed that the SOX6 gene was more highly expressed in glioma tissues than in normal adult tissues, except testis. Western blot analysis with an anti-SOX6 antibody demonstrated that the SOX6 protein was expressed in glioma tissues, but not in normal adult brain tissue. Immunohistochemical analysis with the anti-SOX6 antibody showed that all the glioma tissues analysed expressed SOX6 in tumor cells, but only a few SOX6-positive cells were detected in non-neoplastic tissues from the cerebral cortex. In summary, these results indicate that the developmentally regulated transcription factor SOX6 is aberrantly expressed in glioma and specifically recognized by IgGs from glioma patients' sera.

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Quantitative analysis of PRAME for detection of minimal residual disease in leukemia.

Matsushita Maiko, Yamazaki Rie, Kawakami Yutaka

Methods in molecular medicine 97 267 - 275 2004年

ISSN 1543-1894

Immunohistochemical analysis of SOX6 expression in human brain tumors

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

Brain Tumor Pathology 21 （ 3 ） 117 - 120 2004年

ISSN 1433-7398

We previously demonstrated that the developmentally regulated gene, SOX6, is strongly expressed in glioma cells and in the fetal brain, but only faintly in the normal adult brain. Recent studies have indicated that brain tumor cells may share antigens, signaling systems, and behavior with neural stem/progenitor cells. To test the validity of this proposition, we analyzed the expression of SOX6 in various human central nervous system (CNS) tumors. Immunohistochemical analysis revealed that astrocytic and oligodendroglial tumors expressed SOX6; neuronal-glial cell tumors (central neurocytoma) and embryonal tumors (medulloblastoma), which arise from multipotential stem cell precursors, also showed a high intensity of SOX6 staining. In contrast, ependymal tumors (ependymoma and subependymoma), meningioma, and schwannoma, which are all well differentiated tumors, showed either no staining or only faint staining for SOX6. These results suggest that SOX6 may be expressed in bipotential or multipotential cells capable of neuronal and glial differentiation, but not in fully differentiated cells. SOX6 may be a useful marker for the diagnosis of tumors arising from immature bipotential cells that may differentiate into neuronal and glial cells.

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Expression of a transcriptional factor, SOX6, in human gliomas

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

Brain Tumor Pathology 21 （ 1 ） 35 - 38 2004年

ISSN 1433-7398

By screening a human testis cDNA library with glioma patients' sera, we isolated a transcriptional factor, SOX6. Here, we analyzed SOX6 expression in gliomas having a range of malignancy grades using immunostaining. Murine Sox6 is a transcriptional factor that is specifically expressed in the developing central nervous system and in the early stages of chondrogenesis in mouse embryos. The reverse transcription-polymerase chain reaction (RT-PCR) revealed that the SOX6 gene was more highly expressed in glioma tissues and fetal brain than in normal adult brain and other cancer cells, except melanoma cells. Immunohistochemical analysis with the anti-SOX6 antibody showed that all the glioma tissues analyzed (14 glioblastomas, 14 anaplastic astrocytomas, 3 anaplastic oligoastrocytomas, 5 diffuse astrocytomas, 1 oligodendroglioma, and 1 pilocytic astrocytoma) expressed SOX6 in tumor cells, but only a few SOX6-positive cells were detected in nonneoplastic tissues from the cerebral cortex. These results indicate that the developmentally regulated transcription factor SOX6 may be a potential diagnostic marker for gliomas.

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Development of immunotherapy for cancer

Kawakami Yutaka

Nihon Rinshō Men'eki Gakkai kaishi = Japanese journal of clinical immunology 27 （ 2 ） 87 - 98 2004年

ISSN 0911-4300

Identification of human melanoma antigens by various molecular biological and immunological techniques and evaluation of tumor reactive T cells in patients with the identified tumor antigen and HLA tetramer technology, not only provided us more profound understanding of anti-tumor immune responses in human, but also led to reveal basic problems in each step towards immunological tumor rejection, including systemic suppressive mechanisms such as regulatory T cell induction and local inhibitory environment in tumors. Based on these results obtained from the basic and clinical researches, various improvements have been applied for immunotherapy, including active immunization with modified antigenic peptides and recombinant virus, T cell adoptive transfer with lymphodepletive pretreatment, and administration of anti-CTLA-4 Ab, although further improvement is necessary. The translational research performed on melanoma, would facilitate development of immunotherapy for other cancers.

A Novel Protein Highly Expressed in Testis Is Overexpressed in Systemic Sclerosis Fibroblasts and Targeted by Autoantibodies

Yasuoka Hidekata, Ihn Hironobu, Medsger Thomas A., Hirakata Michito, Kawakami Yutaka, Ikeda Yasuo, Kuwana Masataka

Journal of Immunology 171 （ 12 ） 6883 - 6890 2003年12月

ISSN 0022-1767

Nearly all autoantibody specificities in sera from patients with systemic sclerosis (SSc) target proteins distributed ubiquitously, and Abs against proteins whose expression is restricted to the affected sites have not been identified. In this study we describe SSc-specific autoantibody to a novel testicular Ag, termed protein highly expressed in testis (PHET), which is ectopically over-expressed in SSc dermal fibroblasts. A partial cDNA encoding PHET was isolated by immunoscreening of a HepG2 cDNA library with an SSc serum. PHET appeared to be a member of the UniGene cluster Hs.129872, but had a unique exon composition and a characteristic mRNA expression profile restricted to the testis. Serum Abs to a recombinant PHET fragment were detected in nine (8.4%) of 107 SSc patients, but in none of 50 systemic lupus erythematosus patients or 77 healthy controls. In SSc patients, the presence of anti-PHET Abs was associated with diffuse cutaneous SSc and lung involvement (p = 0.02 and 0.01, respectively). PCR-based quantitative analysis of PHET mRNA expression in cultured dermal fibroblasts showed increased expression of PHET mRNA in SSc fibroblasts compared with control fibroblasts. PHET-reactive Abs purified from SSc sera stained the cytoplasm of SSc dermal fibroblasts, and the staining intensity tended to be more prominent on SSc compared with control fibroblasts. These findings suggest that the autoantibody response to PHET can be induced by ectopic overexpression of PHET in dermal fibroblasts in SSc patients.

HLA Class II Alleles in Systemic Sclerosis Patients with Anti-RNA Polymerase I/III Antibody

Kuwana Masataka, Pandey Janardan P., Silver Richard M., Kawakami Yutaka, Kaburaki Junichi

Journal of Rheumatology 30 （ 11 ） 2392 - 2397 2003年11月

ISSN 0315-162X

Objective. To examine HLA class II gene associations with anti-RNA polymerase (RNAP) I/III antibody responses in patients with systemic sclerosis (SSc). Methods. HLA-DRB1, DRB3, DRB4, and DQB1 alleles were determined using polymerase chain reaction-based methods in 257 SSc patients (129 Japanese and 128 Caucasians) and 271 race-matched regional controls (138 Japanese and 133 Caucasians). Anti-RNAP I/III antibodies were identified by immunoprecipitation assay, and reactivities to individual RNAP subunits were determined by immunoblots using affinity-purified RNAP I, II, and III. Results. Serum anti-RNAP I/III antibody was detected in 10 (8%) Japanese and 24 (19%) Caucasian patients with SSc. The presence of anti-RNAP I/III antibodies was associated with DRB1*0405, DRB4*01, and DQB1*0401 in Japanese, and with DRB3*02 in Caucasians, but these associations were weak and inconsistent between these 2 ethnic groups. When anti-RNAP I/III-positive SSc patients were divided into 2 groups based on the presence or absence of reactivities to individual RNAP subunit proteins, significant associations of anti-IIa/IIo reactivity with DRB3*02, anti-Ia reactivity with DRB1*04, anti-43-kDa subunit reactivity with DRB4*01, and anti-34-kDa subunit reactivity with DRB1*15 were detected. These HLA associations with subunit reactivities were generally shared by Japanese and Caucasian patients with SSc. Conclusion. Our results suggest that in patients with SSc, anti-RNAP I/III antibodies are composed of subsets defined by combinations of reactivities to individual RNAP subunits having specific HLA class II correlations.

HLA class II alleles in systemic sclerosis patients with anti-RNA polymerase I/III antibody: associations with subunit reactivities.

Kuwana M, Pandey JP, Silver RM, Kawakami Y, and Kaburaki J.

Journal of Rheumatology. 30 （ 11 ） 2392-2397 2003年11月

研究論文（学術雑誌）, 共著

Human circulating CD14⁺ monocytes as a source of progenitors that exhibit mesenchymal cell differentiation

Kuwana Masataka, Okazaki Yuka, Kodama Hiroaki, Izumi Keisuke, Yasuoka Hidekata, Ogawa Yoko, Kawakami Yutaka, Ikeda Yasuo

Journal of Leukocyte Biology 74 （ 5 ） 833 - 845 2003年11月

ISSN 0741-5400

Circulating CD14⁺ monocytes are precursors of phagocytes, such as macrophages and dendritic cells. Here we report primitive cells with a fibroblast-like morphology derived from human peripheral blood CD14⁺ monocytes that can differentiate into several distinct mesenchymal cell lineages. We named this cell population monocyte-derived mesenchymal progenitor (MOMP). MOMPs were obtained in vitro from human peripheral blood mononuclear cells cultured on fibronectin in the presence of fetal bovine serum alone as a source of growth factors. MOMPs had a unique molecular phenotype-CD14 ⁺CD45⁺CD34⁺type I collagen⁺-and showed mixed morphologic and molecular features of monocytes and endothelial and mesenchymal cells. MOMPs were found to be derived from a subset of circulating CD14⁺ monocytes, and their differentiation required that they bind fibronectin and be exposed to one or more soluble factors derived from peripheral blood CD14^- cells. MOMPs could be expanded in culture without losing their original phenotype for up to five passages. The induction of MOMPs to differentiate along multiple limb-bud mesodermal lineages resulted in the expression of genes and proteins specific for osteoblasts, skeletal myoblasts, chondrocytes, and adipocytes. Our findings represent the first evidence that human circulating CD14⁺ monocytes are a source of progenitors that exhibit mesenchymal cell differentiation.

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Results of a phase I clinical study using autologous tumour lysate-pulsed monocyte-derived mature dendritic cell vaccinations for stage IV malignant melanoma patients combined with low dose interleukin-2

Nagayama Hitomi, Sato Katsuaki, Morishita Mariko, Uchimaru Kaoru, Oyaizu Naoki, Inazawa Takeshi, Yamasaki Tomoko, Enomoto Makoto, Nakaoka Takashi, Nakamura Tetsuya, Maekawa Taira, Yamamoto Akifumi, Shimada Shinji, Saida Toshiaki, Kawakami Yutaka, Asano Shigetaka, Tani Kenzaburo, Takahashi Tsuneo A., Yamashita Naohide

Melanoma Research 13 （ 5 ） 521 - 530 2003年10月

ISSN 0960-8931

We conducted a pilot study to assess the feasibility and efficacy of immunotherapy for stage IV malignant melanoma patients resistant to conventional therapies involving vaccination with mature dendritic cells (mDCs) combined with administration of low dose interleukin-2, Autologous monocytes were harvested from a single apheresis and cultured for 7 days with granulocyte-macrophage colony-stimulating factor and interleukin-4, yielding immature dendritic cells (iDCs), which were then cryopreserved until use. For 4 days prior to vaccination, iDCs were exposed to autologous tumour lysate combined with tumour necrosis factor-α to induce terminal differentiation into mDCs. Patients were then vaccinated weekly with 10⁷ mDCs for 10 weeks and given 350-700 kIU of interleukin-2 three times per week. Of the 10 patients in the study, one showed stable disease, seven showed progressive disease, and two showed mixed responses, including partial tumour regression, and were therefore given 20 additional injections. Only minimal adverse events were noted, including localized skin reactions and mild fever (NIH-CTC grade 0-1). Median survival from the first vaccination was 240 days (range 31-735 days). In vitro, melanoma patient-derived dendritic cells (DCs) showed reduced cell surface expression of CD1a antigen on iDCs and reduced CD86 and HLA-DR expression on mDCs. In addition, antigen uptake, chemotaxis and antigen presentation were all attenuated in DCs from the patients. In summary, although improvement of clinical efficacy will require further research, autologous tumour lysate-pulsed monocytederived mDCs could be safely harvested, cryopreserved and administrated to patients without obvious complications.

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