Koichi Tsuneyama and Takeshi Oya : 腫瘍病理鑑別診断アトラス 肝癌 第2版 悪性リンパ腫および偽リンパ腫, 文光堂, Tokyo, Nov. 2020.
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Koichi Tsuneyama and Takeshi Oya :
文光堂, Nov. 2020.
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Minoru Matsumoto, Rodrigues M. Pedro, Sousa Laura, Koichi Tsuneyama, Mitsuru Matsumoto and Alves L. Nuno : The Ins and Outs of Thymic Epithelial Cell Differentiation and Function, Springer, Apr. 2019.
Masahiro Umemura, Akira Honda, Maho Yamashita, Takeshi Chida, Hidenao Noritake, Kenta Yamamoto, Takashi Honda, Mayuko Shimizu, Koichi Tsuneyama, Teruo Miyazaki, Nobuhito Kurono, C Patrick S Leung, Eric M Gershwin, Takafumi Suda and Kazuhito Kawata : High-fat diet modulates bile acid composition and gut microbiota, affecting severe cholangitis and cirrhotic change in murine primary biliary cholangitis., Journal of Autoimmunity, Vol.148, 2024.
(Summary)
Increasing evidence suggests that, in addition to a loss of tolerance, bile acid (BA) modulates the natural history of primary biliary cholangitis (PBC). We focused on the impacts of dietary changes on the immunopathology of PBC, along with alterations in BA composition and gut microbiota. In this study, we have taken advantage of our unique PBC model, a Cyp2c70/Cyp2a12 double knockout (DKO), which includes a human-like BA composition, and develops progressive cholangitis following immunization with the PDC-E2 mimic, 2-octynoic acid (2OA). We compared the effects of a ten-week high-fat diet (HFD) (60% kcal from fat) and a normal diet (ND) on 2OA-treated DKO mice. Importantly, we report that 2OA-treated DKO mice fed HFD had significantly exacerbated cholangitis, leading to cirrhosis, with increased hepatic expression of Th1 cytokines/chemokines and hepatic fibrotic markers. Serum lithocholic acid (LCA) levels and the ratio of chenodeoxycholic acid (CDCA)-derived BAs to cholic acid-derived BAs were significantly increased by HFD. This was also associated with downregulated expression of key regulators of BA synthesis, including Cyp8b1, Cyp3a11, and Sult2a1. In addition, there were increases in the relative abundances of Acetatifactor and Lactococcus and decreases in Desulfovibrio and Lachnospiraceae_NK4A136_group, which corresponded to the abundances of CDCA and LCA. In conclusion, HFD and HFD-induced alterations in the gut microbiota modulate BA composition and nuclear receptor activation, leading to cirrhotic change in this murine PBC model. These findings have significant implications for understanding the progression of human PBC.
Shun Takano, Koudai Kani, Kaichi Kasai, Naoya Igarashi, Miyuna Kato, Kana Goto, Yudai Matsuura, Mayuko Ichimura-Shimizu, Shiro Watanabe, Koichi Tsuneyama, Yukihiro Furusawa and Yoshinori Nagai : Antibiotic effects on gut microbiota modulate diet-induced metabolic dysfunction-associated steatohepatitis development in C57BL/6 mice., Genes to Cells, Vol.29, No.8, 635-649, 2024.
(Summary)
The potential involvement of the gut microbiota in metabolic dysfunction-associated steatohepatitis (MASH) pathogenesis has garnered increasing attention. In this study, we elucidated the link between high-fat/cholesterol/cholate-based (iHFC)#2 diet-induced MASH progression and gut microbiota in C57BL/6 mice using antibiotic treatments. Treatment with vancomycin (VCM), which targets gram-positive bacteria, exacerbated the progression of liver damage, steatosis, and fibrosis in iHFC#2-fed C57BL/6 mice. The expression levels of inflammation- and fibrosis-related genes in the liver significantly increased after VCM treatment for 8 weeks. F4/80 macrophage abundance increased in the livers of VCM-treated mice. These changes were rarely observed in the iHFC#2-fed C57BL/6 mice treated with metronidazole, which targets anaerobic bacteria. A16S rRNA sequence analysis revealed a significant decrease in α-diversity in VCM-treated mice compared with that in placebo-treated mice, with Bacteroidetes and Firmicutes significantly decreased, while Proteobacteria and Verrucomicrobia increased markedly. Finally, VCM treatment dramatically altered the level and balance of bile acid (BA) composition in iHFC#2-fed C57BL/6 mice. Thus, the VCM-mediated exacerbation of MASH progression depends on the interaction between the gut microbiota, BA metabolism, and inflammatory responses in the livers of iHFC#2-fed C57BL/6 mice.
(Keyword)
Animals / Gastrointestinal Microbiome / Mice, Inbred C57BL / Mice / Anti-Bacterial Agents / Male / Vancomycin / Diet, High-Fat / Liver / Fatty Liver / Bile Acids and Salts
Naoya Igarashi, Kaichi Kasai, Yuki Tada, Koudai Kani, Miyuna Kato, Shun Takano, Kana Goto, Yudai Matsuura, Mayuko Ichimura-Shimizu, Shiro Watanabe, Koichi Tsuneyama, Yukihiro Furusawa and Yoshinori Nagai : Impacts of liver macrophages, gut microbiota, and bile acid metabolism on the differences in iHFC diet-induced MASH progression between TSNO and TSOD mice., Inflammation Research, Vol.73, No.7, 1081-1098, 2024.
(Summary)
Tsumura-Suzuki non-obese (TSNO) mice exhibit a severe form of metabolic dysfunction-associated steatohepatitis (MASH) with advanced liver fibrosis upon feeding a high-fat/cholesterol/cholate-based (iHFC) diet. Another ddY strain, Tsumura-Suzuki diabetes obese (TSOD) mice, are impaired in the progression of iHFC diet-induced MASH. To elucidate the underlying mechanisms contributing to the differences in MASH progression between TSNO and TSOD mice. We analyzed differences in the immune system, gut microbiota, and bile acid metabolism in TSNO and TSOD mice fed with a normal diet (ND) or an iHFC diet. TSOD mice had more anti-inflammatory macrophages in the liver than TSNO mice under ND feeding, and were impaired in the iHFC diet-induced accumulation of fibrosis-associated macrophages and formation of histological hepatic crown-like structures in the liver. The gut microbiota of TSOD mice also exhibited a distinct community composition with lower diversity and higher abundance of Akkermansia muciniphila compared with that in TSNO mice. Finally, TSOD mice had lower levels of bile acids linked to intestinal barrier disruption under iHFC feeding. The dynamics of liver macrophage subsets, and the compositions of the gut microbiota and bile acids at steady state and post-onset of MASH, had major impacts on MASH development.
Kai Yan, Qing-Zhi Liu, Rong-Rong Huang, Yi-Hua Jiang, Zhen-Hua Bian, Si-Jin Li, Liang Li, Fei Shen, Koichi Tsuneyama, Qing-Ling Zhang, Zhe-Xiong Lian, Haixia Guan and Bo Xu : Spatial transcriptomics reveals prognosis-associated cellular heterogeneity in the papillary thyroid carcinoma microenvironment., Clinical and Translational Medicine, Vol.14, No.3, 2024.
(Summary)
The occurrence of tumour foci No. 2 and three enhanced ligand-receptor interactions in the AFC area/tumour foci reduced the relapse-free survival of PTC patients, potentially leading to improved prognostic strategies and targeted therapies for PTC patients.
Satoshi Sumida, Nobuo Satake and Koichi Tsuneyama : Two Ectopic Liver Lobes Discovered Incidentally at an Autopsy: A Case Report., Curēus, Vol.16, No.1, 2024.
(Summary)
The ectopic liver lobe is a rare anomaly and is most frequently reported as a solitary mass. Herein, we report a case of multiple (two) ectopic liver lobes detected at an autopsy. A Japanese man in his 70s died of an infectious disease associated with acquired immunodeficiency syndrome (AIDS). Autopsy revealed the incidental finding of two 1-cm masses, located anterior to the inferior vena cava. Both masses were composed of liver tissue and had internal microscopic structures resembling the porta hepatis, consisting of an outflow bile duct and blood vessels. The outflow bile duct appeared to be continuous with the common bile duct, but the connection point of the outflow vessel was unclear. The liver tissue showed fibrous thickening of the central veins and portal venopathy, including fibrosis in the portal area as well as narrowing and loss of the portal veins. There was no evidence of congestion, fibrosis, biliary stasis, or neoplasm. The incidence of hepatocellular carcinoma is higher in the ectopic liver lobe than in the proper liver, presumably due to the abnormal circulation and bile excretion pathways. The patient also presented with portal venopathy; this suggests the presence of abnormal circulatory dynamics.
Indigo naturalis (IN), derived from the leaves of the indigo plant, is a traditional Chinese medicine that has historically been used for its anti-inflammatory properties in the treatment of various diseases, including ulcerative colitis (UC). However, long-term use of IN in UC patients is incontrovertibly associated with the onset of pulmonary arterial hypertension (PAH). To investigate the mechanisms by which IN induces PAH, we focused on the raw material of IN, indigo leaves (IL). Only the condition of long-term chronic (6 months) and high-dose (containing 5% IL in the control diet) administration of IL induced medial thickening in the pulmonary arteries without right ventricular hypertrophy in our rat model. IL administration for a month did not induce pulmonary arterial remodeling but increased endothelin-1 (ET-1) expression levels within endothelial cell (EC) layers in the lungs. Gene Expression Omnibus analysis showed that ET-1 is a key regulator of PAH and that the IL component indican and its metabolite IS induced ET-1 mRNA expression via reactive oxygen species-dependent mechanism. We identified the roles of indican and IS in ET-1 expression in ECs, which were linked to pulmonary arterial remodeling in an animal model.
Saya Matsuzaki, Eiji Hase, Hiroki Takanari, Yuri Hayashi, Yusaku Hayashi, Haruto Oshikata, Takeo Minamikawa, Satoko Kimura, Mayuko Shimizu, Takeshi Yasui, Masafumi Harada and Koichi Tsuneyama : Quantification of collagen fiber properties in alcoholic liver fibrosis using polarization-resolved second harmonic generation microscopy., Scientific Reports, Vol.13, No.1, 22100, 2023.
(Summary)
Liver fibrosis is assessed mainly by conventional staining or second harmonic generation (SHG) microscopy, which can only provide collagen content in fibrotic area. We propose to use polarization-resolved SHG (PR-SHG) microscopy to quantify liver fibrosis in terms of collagen fiber orientation and crystallization. Liver samples obtained from autopsy cases with fibrosis stage of F0-F4 were evaluated with an SHG microscope, and 12 consecutive PR-SHG images were acquired while changing the polarization azimuth angle of the irradiated laser from 0° to 165° in 15° increments using polarizer. The fiber orientation angle (φ) and degree (ρ) of collagen were estimated from the images. The SHG-positive area increased as the fibrosis stage progressed, which was well consistent with Sirius Red staining. The value of φ was random regardless of fibrosis stage. The mean value of ρ (ρ-mean), which represents collagen fiber crystallinity, varied more as fibrosis progressed to stage F3, and converged to a significantly higher value in F4 than in other stages. Spatial dispersion of ρ (ρ-entropy) also showed increased variation in the stage F3 and decreased variation in the stage F4. It was shown that PR-SHG could provide new information on the properties of collagen fibers in human liver fibrosis.
Nana Makiuchi, Shun Takano, Yuki Tada, Kaichi Kasai, Naoya Igarashi, Koudai Kani, Miyuna Kato, Kana Goto, Yudai Matsuura, Mayuko Shimizu, Yukihiro Furusawa, Koichi Tsuneyama and Yoshinori Nagai : Dynamics of Liver Macrophage Subsets in a Novel Mouse Model of Non-Alcoholic Steatohepatitis Using C57BL/6 Mice., Biomedicines, Vol.11, No.10, 2659, 2023.
(Summary)
cells were not distinct populations in the iHFC-fed TSNO mice. Thus, differences in cholic acid content and mouse strain affect the macrophage subsets that accumulate in the liver.
Biopsy is a commonly used method for determining pathological diagnoses by directly using human tissues and cells. Biopsies are widely used to determine disease progression and treatment efficacy. Although organs and tissues are usually obtained by sacrifice during animal experiments, it is theoretically possible to use the same biopsy techniques in humans. In the present study, we examined the feasibility of performing four repeated liver biopsies in a spontaneous metabolic syndrome mouse model. Even though a small number of mice died accidently, most mice were able to undergo four liver biopsies without significant adverse events. We also performed three liver biopsies in mouse liver tumor carcinogen models at 4, 8, and 12 weeks of age. In addition to the sample collected at 16 weeks of age during sacrifice, we successfully collected four liver samples from the same mice at different stages of disease progression. The application of this liver biopsy technique might make it possible for direct evaluation of pathological conditions in the same individual over time, thereby reducing the number of experimental animals.
Takumi Kakimoto, Masato Hosokawa, Mayuko Shimizu, Hirohisa Ogawa, Yuko Miyakami, Satoshi Sumida and Koichi Tsuneyama : Accumulation of α-synuclein in hepatocytes in nonalcoholic steatohepatitis and its usefulness in pathological diagnosis., Pathology, Research and Practice, Vol.247, 154525, 2023.
(Summary)
Nonalcoholic steatohepatitis (NASH) is characterized by fat deposition, inflammation, and hepatocellular damage. The diagnosis of NASH is confirmed pathologically, and hepatocyte ballooning is an important finding for definite diagnosis. Recently, α-synuclein deposition in multiple organs was reported in Parkinson's disease. Since it was reported that α-synuclein is taken up by hepatocytes via connexin 32, the expression of α-synuclein in the liver in NASH is of interest. The accumulation of α-synuclein in the liver in NASH was investigated. Immunostaining for p62, ubiquitin, and α-synuclein was performed, and the usefulness of immunostaining in pathological diagnosis was examined. Liver biopsy tissue specimens from 20 patients were evaluated. Several antibodies against α-synuclein, as well as antibodies against connexin 32, p62, and ubiquitin were used for immunohistochemical analyses. Staining results were evaluated by several pathologists with varying experience, and the diagnostic accuracy of ballooning was compared. Polyclonal α-synuclein antibody, not the monoclonal antibody, reacted with eosinophilic aggregates in ballooning cells. Expression of connexin 32 in degenerating cells was also demonstrated. Antibodies against p62 and ubiquitin also reacted with some of the ballooning cells. In the pathologists' evaluations, the highest interobserver agreement was obtained with hematoxylin and eosin (H&E)-stained slides, followed by slides immunostained for p62 and α-synuclein, and there were cases with different results between H&E staining and immunostaining CONCLUSION: These results indicate the incorporation of degenerated α-synuclein into ballooning cells, suggesting the involvement of α-synuclein in the pathogenesis of NASH. The combination of immunostaining including polyclonal α-synuclein may contribute to improving the diagnosis of NASH.
Minoru Matsumoto, Hideyuki Yoshida, Koichi Tsuneyama, Takeshi Oya and Mitsuru Matsumoto : Revisiting Aire and tissue-restricted antigens at single-cell resolution, Frontiers in Immunology, Vol.14, 1176450, 2023.
(Summary)
The thymus is a highly specialized organ that plays an indispensable role in the establishment of self-tolerance, a process characterized by the "education" of developing T-cells. To provide competent T-cells tolerant to self-antigens, medullary thymic epithelial cells (mTECs) orchestrate negative selection by ectopically expressing a wide range of genes, including various tissue-restricted antigens (TRAs). Notably, recent advancements in the high-throughput single-cell analysis have revealed remarkable heterogeneity in mTECs, giving us important clues for dissecting the mechanisms underlying TRA expression. We overview how recent single-cell studies have furthered our understanding of mTECs, with a focus on the role of Aire in inducing mTEC heterogeneity to encompass TRAs.
Koudai Kani, Kaichi Kasai, Yuki Tada, Riko Ishibashi, Shun Takano, Naoya Igarashi, Mayuko Shimizu, Koichi Tsuneyama, Yukihiro Furusawa and Yoshinori Nagai : The innate immune receptor RP105 promotes metabolic syndrome by altering gut microbiota composition and intestinal barrier function., Biochemical and Biophysical Research Communications, Vol.664, 77-85, 2023.
(Summary)
mice. A 16S rRNA sequence analysis indicated that RP105 modified gut microbiota composition and was involved in the maintenance of its diversity. Thus, RP105 promotes metabolic syndrome by altering gut microbiota composition and intestinal barrier function.
Tetsuyuki Takahashi, Yuri Ando, Hirona Ichikawa, Koichi Tsuneyama and Takao Hijikata : Serum/glucose starvation strikingly reduces heterogeneous nuclear ribonucleoprotein A1 protein and its target, cyclin D1., The FEBS Journal, 2023.
(Summary)
Our investigation to explore cellular alterations related to undernutrition in cancer cells revealed that the protein level of heterogenous nuclear ribonucleoprotein A1 (hnRNP A1) is drastically decreased by serum/glucose starvation. Its loss was reversible, serum/glucose starvation-specific and universal throughout cell types and species. The hnRNP A1 mRNA level and hnRNP A1 mRNA/protein stability were not altered under this condition. CCND1 mRNA, which we newly identified as the binding target of hnRNP A1, was decreased by serum/glucose starvation. Under similar conditions, CCND1 protein was reduced in vitro and in vivo, whereas hnRNP A1 mRNA level and CCND1 mRNA level revealed no correlation in most clinical samples. Functional analyses revealed that CCND1 mRNA stability is certainly dependent on hnRNP A1 protein level and that RNA recognition motif-1 (RRM1) in hnRNP A1 plays a central role in maintaining CCND1 mRNA stability and subsequent protein expression. The injection of RRM1-deleted hnRNP A1-expressing cancer cells in the mouse xenograft model did not form any tumours, and that of hnRNP A1-expressing cancer cells retained CCND1 expression at the lesion adjacent to necrosis with a slight increase in tumour volume. Furthermore, RRM1 deletion caused growth suppression with the induction of apoptosis and autophagy, whereas CCND1 restoration completely recovered it. Our results indicate that serum/glucose starvation triggers entire hnRNP A1 protein loss, and its loss may play a role in CCND1 mRNA destabilization and CCND1-mediated cellular event inhibition, i.e. growth promotion, apoptosis induction and autophagosome formation.
Yuko Miyakami, Takeo Minamikawa, Hirohisa Ogawa, Mayuko Shimizu and Koichi Tsuneyama : Definitive Confirmation of Erythropoietic Protoporphyria via Re-biopsy Three Years After Initial Liver Biopsy at Age 15., Curēus, Vol.15, No.4, e38017, 2023.
(Summary)
Erythropoietic protoporphyria (EPP) is a rare inherited disorder of porphyrin metabolism that can cause liver damage and cholestatic hepatocellular failure. We report a case of EPP in a teenaged male who underwent liver biopsy for investigation of liver dysfunction of unknown cause. The diagnosis was not made until a re-biopsy approximately three years later, when the patient presented with recurrent skin lesions and elevated blood and urinary protoporphyrin levels. The liver biopsies contained brownish deposits that exhibited birefringence under polarized light and porphyrin fluorescence under fluorescence spectroscopy. EPP should be considered in young patients with unexplained liver dysfunction, skin symptoms, and seasonal changes in symptoms. Fluorescence spectroscopy of liver biopsy tissue can be a useful tool in the diagnosis of EPP.
Junko Morimoto, Minoru Matsumoto, Takeshi Oya, Koichi Tsuneyama and Mitsuru Matsumoto : Cooperative but Distinct Role of Medullary Thymic Epithelial Cells and Dendritic Cells in the Production of Regulatory T Cells in the Thymus., The Journal of Immunology, 2023.
(Summary)
Regulatory T cells (Tregs) are produced in the thymus to establish self-tolerance, and agonistic stimuli by self-Ags play a pivotal role in this process. Although two types of APCs, medullary thymic epithelial cells (mTECs) and dendritic cells (DCs), are responsible for presenting self-Ags together with costimulatory/cytokine signals, the distinct role of each APC in producing Tregs remains enigmatic. We have approached this issue by depleting the mTECs and DCs using mice expressing diphtheria toxin receptors driven by Aire and CD11c promoters, respectively. Depletion of mTECs showed an effect on Treg production quantitatively and qualitatively more profound than that of DCs followed by the development of distinct organ-specific autoimmune lesions in the hosts. Because self-Ags produced by mTECs are transferable to DCs through a process known as Ag transfer, we monitored the process of Ag transfer using mice expressing GFP from TECs. Although GFP expressed from total TECs was effectively transferred to DCs, GFP expressed from cortical TECs was not, suggesting that mTECs are the predominant source of self-Ags. We also found that GFP expressed not only from mature mTECs but also from immature mTECs was transferred to DCs, suggesting that a broad spectrum of molecules were subjected to Ag transfer during mTEC development. Interestingly, the numbers of recirculating non-Tregs producing IL-2, an important source for Treg expansion in the thymus, were reduced only in the mTEC-depleted mice. These results suggested the cooperative but distinct role of mTECs and DCs in the production of Tregs to avoid autoimmunity.
Maho Yamashita, Akira Honda, Shin Shimoyama, Masahiro Umemura, Kazuyoshi Ohta, Takeshi Chida, Hidenao Noritake, Nobuhito Kurono, Mayuko Shimizu, Koichi Tsuneyama, Teruo Miyazaki, Atsushi Tanaka, C Patrick S Leung, Eric M Gershwin, Takafumi Suda and Kazuhito Kawata : Breach of tolerance versus burden of bile acids: Resolving the conundrum in the immunopathogenesis and natural history of primary biliary cholangitis., Journal of Autoimmunity, Vol.136, No.103027, 2023.
(Summary)
Primary biliary cholangitis (PBC) is a classic autoimmune disease due to the loss of tolerance to self-antigens. Bile acids (BA) reportedly play a major role in biliary inflammation and/or in the modulation of dysregulated immune responses in PBC. Several murine models have indicated that molecular mimicry plays a role in autoimmune cholangitis; however, they have all been limited by the relative failure to develop hepatic fibrosis. We hypothesized that species-specific differences in the BA composition between mice and humans were the primary reason for this limited pathology. Here, we aimed to study the impact of human-like hydrophobic BA composition on the development of autoimmune cholangitis and hepatic fibrosis. We took advantage of a unique construct, Cyp2c70/Cyp2a12 double knockout (DKO) mice, which have human-like BA composition, and immunized them with a well-defined mimic of the major mitochondrial autoantigen of PBC, namely 2-octynoic acid (2OA). 2OA-treated DKO mice were significantly exacerbated portal inflammation and bile duct damage with increased Th1 cytokines/chemokines at 8 weeks post-initial immunization. Most importantly, there was clear progression of hepatic fibrosis and increased expression of hepatic fibrosis-related genes. Interestingly, these mice demonstrated increased serum BA concentrations and decreased biliary BA concentrations; hepatic BA levels did not increase because of the upregulation of transporters responsible for the basolateral efflux of BA. Furthermore, cholangitis and hepatic fibrosis were more advanced at 24 weeks post-initial immunization. These results indicate that both the loss of tolerance and the effect of hydrophobic BA are essential for the progression of PBC.
Minoru Matsumoto, Takuya Ohmura, Yuto Hanibuchi, Mayuko Shimizu, Yasuyo Saijo, Hirohisa Ogawa, Ryuichiro Miyazawa, Junko Morimoto, Koichi Tsuneyama, Mitsuru Matsumoto and Takeshi Oya : AIRE illuminates the feature of medullary thymic epithelial cells in thymic carcinoma., Cancer Medicine, 2023.
(Summary)
Despite the clear distinction between cortical (cTECs) and medullary thymic epithelial cells (mTECs) in physiology, the cell of origin of thymic carcinomas (TCs) and other thymic epithelial tumors remained enigmatic. We addressed this issue by focusing on AIRE, an mTEC-specific transcriptional regulator that is required for immunological self-tolerance. We found that a large proportion of TCs expressed AIRE with typical nuclear dot morphology by immunohistochemistry. AIRE expression in TCs was supported by the RNA-seq data in the TCGA-THYM database. Furthermore, our bioinformatics approach to the recent single-cell RNA-seq data on human thymi has revealed that TCs hold molecular characteristics of multiple mTEC subpopulations. In contrast, TCs lacked the gene signatures for cTECs. We propose that TCs are tumors derived from mTECs.
Shoko Yamashita, Chie Takasu, Yuji Morine, Hiroki Ishibashi, Tetsuya Ikemoto, Hiroki Mori, Shin-ichiro Yamada, Takeshi Oya, Koichi Tsuneyama and Mitsuo Shimada : Characteristic submucosal alteration in biliary carcinogenesis of pancreaticobiliary maljunction with a focus on inflammasome activation., Journal of Hepato-Biliary-Pancreatic Sciences, Vol.30, No.4, 462-472, 2023.
(Summary)
Activated fibroblasts and M2 macrophages in the GB lamina propria may be associated with biliary carcinogenesis of PBM, possibly through inflammasome activation.
Jie Long, Si-Yu Yang, Meng-Xing Huang, Pan-Yue Luo, Liang Li, Koichi Tsuneyama, A Aftab Ansari, Ling Lu, Eric M Gershwin, Zhe-Xiong Lian and Zhi-Bin Zhao : Spontaneous development of an autoimmune hepatitis - primary biliary cholangitis overlap syndrome in dnTGFβRII Aire-/- mice, The Journal of Pathology, 2023.
Although several angiogenesis-related factors are reportedly involved in the pathogenesis of ulcerative colitis (UC), the mechanisms by which they contribute to disease are unclear. We first examined the expression of angiogenesis-related factors in inflamed colorectal tissue of UC patients using antibody array, and identified the 5 factors with highest expression, which included matrix metalloproteinase-8, urokinase-type plasminogen activator (uPA), angiostatin/plasminogen, hepatocyte growth factor and endoglin. Subsequent real-time PCR experiments using additional colorectal tissues revealed that uPA mRNA levels were significantly higher in inflamed tissues than in non-inflamed tissues, and significantly correlated with the severity of UC. Mirror section immunohistochemistry revealed that uPA was expressed in the neutrophils of inflamed colorectal tissues. We administered dextran sulfate sodium (DSS) in drinking water to uPA knockout (uPA) mice, and found that the disease activity index in uPA mice was marginally lower and the histological score in uPA mice was significantly lower than those in wild-type mice, suggesting the importance of uPA in colitis. When an uPA-selective inhibitor, UK122, was administered to DSS-treated C57BL6J mice, the disease activity index and histological score in those mice were significantly lower compared with control mice. Multiple cytokine/chemokine assay using colorectal tissues from uPA and UK122-treated mice revealed significantly lowered level of RANTES. In conclusion, uPA was highly expressed in neutrophils of the inflamed mucosa of UC patients, and the expression level correlated with the severity of UC. Genetic uPA deletion or pharmacological uPA blockade significantly ameliorated colitis in mice, concomitant with downregulation of RANTES.
Kaichi Kasai, Naoya Igarashi, Yuki Tada, Koudai Kani, Shun Takano, Tsutomu Yanagibashi, Fumitake Usui-Kawanishi, Shiho Fujisaka, Shiro Watanabe, Mayuko Shimizu, Kiyoshi Takatsu, Kazuyuki Tobe, Koichi Tsuneyama, Yukihiro Furusawa and Yoshinori Nagai : Impact of Vancomycin Treatment and Gut Microbiota on Bile Acid Metabolism and the Development of Non-Alcoholic Steatohepatitis in Mice., International Journal of Molecular Sciences, Vol.24, No.4, 2023.
(Summary)
-recruited macrophages into the liver, forming hepatic crown-like structures, was enhanced by vancomycin treatment. The co-localization of this macrophage subset with collagen was greatly augmented in the liver of vancomycin-treated iHFC-fed mice. These changes were rarely seen with the administration of metronidazole, which targets anaerobic organisms, in iHFC-fed mice. Finally, the vancomycin treatment dramatically modulated the level and composition of bile acid in iHFC-fed mice. Thus, our data demonstrate that changes in inflammation and fibrosis in the liver by the iHFC diet can be modified by antibiotic-induced changes in gut microbiota and shed light on their roles in the pathogenesis of advanced liver fibrosis.
Shin-ichiro Yamada, Yuji Morine, Satoru Imura, Tetsuya Ikemoto, Yu Saitou, Mayuko Shimizu, Koichi Tsuneyama, Mitsue Nishiyama, Shiori Ishizawa and Mitsuo Shimada : Effect of daikenchuto (TU-100) on carcinogenesis in non-alcoholic steatohepatitis., The Journal of Medical Investigation : JMI, Vol.70, No.1.2, 66-73, 2023.
(Summary)
TU-100 regulates the intestinal microbiome and may suppress subsequent hepatocarcinogenesis in the NASH model. J. Med. Invest. 70 : 66-73, February, 2023.
Shin-ichiro Yamada, Yuji Morine, Tetsuya Ikemoto, Yu Saitou, Katsuki Miyazaki, Mayuko Shimizu, Koichi Tsuneyama and Mitsuo Shimada : Inhibitory effect of non-alcoholic steatohepatitis on colon cancer liver metastasis., EJSO - European Journal of Surgical Oncology, Vol.49, No.2, 410-415, 2023.
(Summary)
The WD-fed NASH mouse model showed an inhibitory effect on CLM. Suppressed interleukin-6/signal transducer and activator of transcription 3 signaling and serum amyloid A/matrix metalloproteinase 9 expression may affect this phenomenon.
Ryuichiro Miyazawa, Jun-Ichi Nagao, Ken-Ichi Arita-Morioka, Minoru Matsumoto, Junko Morimoto, Yoshida Masaki, Takeshi Oya, Koichi Tsuneyama, Hedeyuki Yoshida, Yoshihiko Tanaka and Mitsuru Matsumoto : Dispensable Role of Aire in CD11c+ Conventional Dendritic Cells for Antigen Presentation and Shaping the Transcriptome., ImmunoHorizons, Vol.7, No.1, 140-158, 2023.
(Summary)
Aire, the defect of which is responsible for the development of autoimmunity, is predominantly expressed in medullary thymic epithelial cells, and it controls a wide variety of genes, including those of tissue-restricted Ags, for establishing thymic tolerance. Aire is also expressed from APCs in the periphery, called extrathymic Aire-expressing cells (eTACs), and their complementing role to thymic tolerance has been suggested. eTACs are composed of two distinct classes of APCs, conventional dendritic cell (cDC)-type and group 3 innate lymphoid cell (ILC3)-like-type expressing retinoic acid receptor-related orphan receptor γt (RORγt). Although the essential role of Aire in the latter in the Th17-mediated immune response against Candida albicans has been reported, the role of Aire in the cDC-type eTACs for this action has not been examined. Furthermore, the significance of Aire in the production of the transcriptome of the cDC-type eTACs remains unknown. We have approached these issues using a high-fidelity Aire-reporter mouse strain. We found that although the cDC-type eTACs dominated ILC3-like-type eTACs in number and they served as efficient APCs for the immune response against an exogenous Ag as well as for the C. albicans-specific Th17 immune response, loss of Aire in cDC-type eTACs showed no clear effect on these functions. Furthermore, loss of Aire showed no major impact on the transcriptome from cDC-type eTACs. These results suggested that Aire in cDC-type eTACs may not have a cell-intrinsic role in the immune response in contrast to the role of Aire in ILC3-like-type eTACs.
Katsuhisa Omagari, Ayumi Maruta, Natsuki Yayama, Yuki Yoshida, Kyoko Okamoto, Bungo Shirouchi, Shouhei Takeuchi, Kazuhito Suruga, Kazunori Koba, Mayuko Shimizu and Koichi Tsuneyama : The Effects of Overnight Fasting Duration on Glucose and Lipid Metabolism in a Sprague-Dawley Rat Model of Nonalcoholic Steatohepatitis with Advanced Fibrosis., Journal of Nutritional Science and Vitaminology, Vol.69, No.5, 357-369, 2023.
(Summary)
Nonalcoholic steatohepatitis (NASH) can progress to hepatic fibrosis, and is associated with cardiovascular and liver-related mortality. To understand the pathogenesis of NASH, reliable animal models of the disease are useful. In animal studies, the animals are usually fasted overnight before biospecimens are taken, but little is known about the effects of fasting. Here, we investigated the impact of overnight fasting for approximately 9 to 17 h on glucose and lipid metabolism in a Sprague-Dawley (SD) rat model of diet-induced moderate and advanced NASH in comparison to normal SD rats. Our results revealed that in the moderate NASH model rats, the fasting duration did not affect glucose and lipid metabolism, the histopathological findings, or the hepatic mRNA expression levels of genes related to lipid metabolism, cholesterol metabolism, inflammation, fibrosis, and oxidative stress. In contrast, in the normal rats, significant fasting time-dependent reductions were observed in the epididymal fat pad weight and the hepatic mRNA expression levels of adipose differentiation-related protein and heme oxygenase-1. Moreover, in the advanced NASH model rats, a significant fasting time-dependent reduction and increase were observed in the serum insulin level and mRNA expression level of alpha-smooth muscle actin, respectively. Our present results suggest that the influence of the overnight fasting duration differs among the healthy condition, moderate NASH, and advanced NASH statuses. Further studies are needed in humans to determine the appropriate overnight fasting duration for the accurate evaluation of glucose and lipid metabolism in NASH patients.
Satoshi Sumida, Mayuko Shimizu, Yuko Miyakami, Takumi Kakimoto, Tomoko Kobayashi, Yasuyo Saijo, Minoru Matsumoto, Hirohisa Ogawa, Takeshi Oya, Yoshimi Bando, Hisanori Uehara, Shu Taira, Mitsuo Shimada and Koichi Tsuneyama : Histological and immunohistochemical analysis of epithelial cells in epidermoid cysts in intrapancreatic accessory spleen., The Journal of Medical Investigation : JMI, Vol.70, No.1.2, 251-259, 2023.
(Summary)
Novel histological features of epithelial cells of ECIPAS were indicated. Although more cases need to be evaluated, we propose that the cause of ECIPAS may be different from that of pancreatic ductal origin. J. Med. Invest. 70 : 251-259, February, 2023.
Tomio Uragami, Yugo Ando, Mamiko Aoi, Toshiro Fukui, Yasushi Matsumoto, Shunsuke Horitani, Takashi Tomiyama, Kazuichi Okazaki, Koichi Tsuneyama, Hajime Tanaka and Makoto Naganuma : Establishment of a Novel Colitis-Associated Cancer Mouse Model Showing Flat Invasive Neoplasia., Digestive Diseases and Sciences, Vol.68, No.5, 1885-1893, 2022.
(Summary)
Although feasibility and reproducibility of azoxymethane/CD4-dbTGFβRII appear to be disadvantages compared to the azoxymethane/dextran sodium sulfate model, this is the first report to demonstrate that the chronic inflammatory colitis model, CD4-dnTGFβRII also develops colitis-related colorectal cancer.
Yuki Tada, Kaichi Kasai, Nana Makiuchi, Naoya Igarashi, Koudai Kani, Shun Takano, Hiroe Honda, Tsutomu Yanagibashi, Yasuharu Watanabe, Fumitake Usui-Kawanishi, Yukihiro Furusawa, Mayuko Ichimura-Shimizu, Yoshiaki Tabuchi, Kiyoshi Takatsu, Koichi Tsuneyama and Yoshinori Nagai : Roles of Macrophages in Advanced Liver Fibrosis, Identified Using a Newly Established Mouse Model of Diet-Induced Non-Alcoholic Steatohepatitis., International Journal of Molecular Sciences, Vol.23, No.21, 2022.
(Summary)
cells are a macrophage subset that play an anti-inflammatory role and promote tissue repair in NASH. Taken together, our data revealed changes in liver macrophage subsets during the development of NASH and shed light on the roles of the recruited macrophages in the pathogenesis of advanced fibrosis in NASH.
Wenhua Shao, Orgil Jargalsaikhan, Mayuko Shimizu, Qinyi Cai, Hirohisa Ogawa, Yuko Miyakami, Kengo Atsumi, Mitsuru Tomita, Mitsuko Sutoh, Shunji Toyohara, Ryoji Hokao, Yasusei Kudo, Takeshi Oya and Koichi Tsuneyama : Spontaneous Occurrence of Various Types of Hepatocellular Adenoma in the Livers of Metabolic Syndrome-Associated Steatohepatitis Model TSOD Mice., International Journal of Molecular Sciences, Vol.23, No.19, 2022.
(Summary)
Male Tsumura-Suzuki Obese Diabetes (TSOD) mice, a spontaneous metabolic syndrome model, develop non-alcoholic steatohepatitis and liver tumors by feeding on a standard mouse diet. Nearly 70% of liver tumors express glutamine synthetase (GS), a marker of hepatocellular carcinoma. In contrast, approximately 30% are GS-negative without prominent nuclear or structural atypia. In this study, we examined the characteristics of the GS-negative tumors of TSOD mice. Twenty male TSOD mice were sacrificed at 40 weeks and a total of 21 tumors were analyzed by HE staining and immunostaining of GS, liver fatty acid-binding protein (L-FABP), serum amyloid A (SAA), and beta-catenin. With immunostaining for GS, six (29%) tumors were negative. Based on the histological and immunohistological characteristics, six GS-negative tumors were classified into several subtypes of human hepatocellular adenoma (HCA). One large tumor showed generally similar findings to inflammatory HCA, but contained small atypical foci with GS staining and partial nuclear beta-catenin expression suggesting malignant transformation. GS-negative tumors of TSOD mice contained features similar to various subtypes of HCA. Different HCA subtypes occurring in the same liver have been reported in humans; however, the diversity of patient backgrounds limits the ability to conduct a detailed, multifaceted analysis. TSOD mice may share similar mechanisms of HCA development as in humans. It is timely to review the pathogenesis of HCA from both genetic and environmental perspectives, and it is expected that TSOD mice will make further contributions in this regard.
Tihong Shao, C Patrick S Leung, Weici Zhang, Koichi Tsuneyama, M William Ridgway, A Howard Young, Zongwen Shuai, A Aftab Ansari and Eric M Gershwin : Treatment with a JAK1/2 inhibitor ameliorates murine autoimmune cholangitis induced by IFN overexpression., Cellular & Molecular Immunology, Vol.19, No.10, 1130-1140, 2022.
(Summary)
T cells. Additionally, ruxolitinib treatment also decreased the frequencies of germinal center B (GC B) cells and T follicular helper (Tfh) cells and led to lower serological AMA levels. Of note, liver and peritoneal macrophages were sharply decreased and polarized from M1 to M2 with a higher level of IRF4 expression after ruxolitinib treatment. Mechanistically, ruxolitinib inhibited the secretion of IL-6, TNF and MCP1 and the expression of STAT1 but promoted the expression of STAT6 in macrophages in vitro, indicating that M1 macrophage polarization to M2 occurred through activation of the STAT6-IRF4 pathway. Our data highlight the significance, both immunologically and clinically, of the JAK/STAT signaling pathway in autoimmune cholangitis.
Ryosuke Nagatomo, Haruki Kaneko, Shihori Kamatsuki, Mayuko Shimizu, Naozumi Ishimaru, Koichi Tsuneyama and Koichi Inoue : Short-chain fatty acid profiling in biological samples from a mouse model of Sjögrens syndrome based on derivatized LC-MS/MS assay., Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, Vol.1210, 123432, 2022.
(Summary)
An analytical platform is required to characterize the short-chain fatty acids (SCFAs) in a mouse model of pathological immune conditions. Therefore, liquid chromatography tandem mass spectrometry combined with 2-picolylamine derivatization and a comprehensive study of SCFAs distribution based on serum, saliva, feces, liver, and brain from a mouse model of Sjögren's syndrome (SS) is performed. The design of experiments is used to achieve efficient 2-picolylamine derivatization, and optimize the reaction conditions. Twelve SCFAs are derivatized, and separated on a reversed-phase C column. All SCFAs show high linearity (r > 0.995) and intra/inter-day accuracy values from 71.6% to 115.6% (precision < 13.7%). This method was used to determine SCFAs concentrations in the serum, saliva, feces, liver, and brain of an SS model mice, and isobutyric acid, valeric acid, isovaleric acid, and 2-methylbutyric acid in liver from SS were significantly different compared with control group. Moreover, the preliminary evaluation of propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid in saliva is conducted based on the respective SS stages and are correlated with these histological scores. This analytical platform for the widely SCFAs profiling in several tissues can be a clue for studying unclear immune pathophysiology.
Weici Zhang, Tihong Shao, C Patrick S Leung, Koichi Tsuneyama, Luke Heuer, A Howard Young, M William Ridgway and Eric M Gershwin : Dual B-cell targeting therapy ameliorates autoimmune cholangitis., Journal of Autoimmunity, Vol.132, 2022.
(Summary)
B cell subset which was sensitive to dual B-cell targeting therapy and depletion of this unique population was associated with reduced portal infiltration and bile duct damage. Taken together, our data indicate that dual B cell targeting therapy with anti-BAFF and anti-CD20 not only led to the efficient depletion of B cells both in the peripheral blood and tissues, but also led to significant clinical improvement. These findings highlight the potential application of combination of anti-BAFF and anti-CD20 in treating patients with PBC. However, additional studies in other animal models of PBC should be undertaken before considering human trials in those PBC patients who have incomplete responses to conventional therapy.
Mayuko Shimizu, Shiro Watanabe, Yuka Kashirajima, Ami Nagatomo, Hitomi Wada, Koichi Tsuneyama and Katsuhisa Omagari : Dietary Cholic Acid Exacerbates Liver Fibrosis in NASH Model of Sprague-Dawley Rats Fed a High-Fat and High-Cholesterol Diet., International Journal of Molecular Sciences, Vol.23, No.16, 2022.
(Summary)
Adding CA to the HFC diet altered bile acid metabolism and inflammatory response and triggered the development of fibrosis in the rat liver.
Consultation by subspecialty experts is the most common mode of rendering diagnosis in challenging cases in pathological practice. Our study aimed to highlight the diagnostic benefits of whole-slide image (WSI)-based remote consultation. We obtained diagnostically challenging cases from two institutions from the years 2010 and 2013, with histological diagnoses that contained keywords "probable," "suggestive," "suspicious," "inconclusive," and "uncertain." A total of 270 cases were selected for remote consultation using WSIs scanned at 40 × . The consultation process consisted of three rounds: the first and second rounds each with 12 subspecialty experts and the third round with six multi-expertise senior pathologists. The first consultation yielded 44% concordance, and a change in diagnosis occurred in 56% of cases. The most frequent change was from inconclusive to definite diagnosis (30%), followed by minor discordance (14%), and major discordance (12%). Out of the 70 cases which reached the second round, 31 cases showed discrepancy between the two consultants. For these 31 cases, a consensus diagnosis was provided by six multi-expertise senior pathologists. Combining all WSI-based consultation rounds, the original inconclusive diagnosis was changed in 140 (52%) out of 266 cases. Among these cases, 80 cases (30%) upgraded the inconclusive diagnosis to a definite diagnosis, and 60 cases (22%) changed the diagnosis with major or minor discordance, accounting for 28 cases (10%) and 32 cases (12%), respectively. We observed significant improvement in the pathological diagnosis of difficult cases by remote consultation using WSIs, which can further assist in patient healthcare. A post-study survey highlighted various benefits of WSI-based consults.
Takashi Yamaguchi, Katsunori Yoshida, Miki Murata, Kanehiko Suwa, Koichi Tsuneyama, Koichi Matsuzaki and Makoto Naganuma : Smad3 Phospho-Isoform Signaling in Nonalcoholic Steatohepatitis., International Journal of Molecular Sciences, Vol.23, No.11, 2022.
(Summary)
Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis with insulin resistance, oxidative stress, lipotoxicity, adipokine secretion by fat cells, endotoxins (lipopolysaccharides) released by gut microbiota, and endoplasmic reticulum stress. Together, these factors promote NAFLD progression from steatosis to nonalcoholic steatohepatitis (NASH), fibrosis, and eventually end-stage liver diseases in a proportion of cases. Hepatic fibrosis and carcinogenesis often progress together, sharing inflammatory pathways. However, NASH can lead to hepatocarcinogenesis with minimal inflammation or fibrosis. In such instances, insulin resistance, oxidative stress, and lipotoxicity can directly lead to liver carcinogenesis through genetic and epigenetic alterations. Transforming growth factor (TGF)-β signaling is implicated in hepatic fibrogenesis and carcinogenesis. TGF-β type I receptor (TβRI) and activated-Ras/c-Jun-N-terminal kinase (JNK) differentially phosphorylate the mediator Smad3 to create two phospho-isoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). TβRI/pSmad3C signaling terminates cell proliferation, while constitutive Ras activation and JNK-mediated pSmad3L promote hepatocyte proliferation and carcinogenesis. The pSmad3L signaling pathway also antagonizes cytostatic pSmad3C signaling. This review addresses TGF-β/Smad signaling in hepatic carcinogenesis complicating NASH. We also discuss Smad phospho-isoforms as biomarkers predicting HCC in NASH patients with or without cirrhosis.
Cheng-Bo Wang, Yan Wang, Yuan Yao, Jin-Jun Wang, Koichi Tsuneyama, Qiong Yang, Bin Liu, Carlo Selmi, Eric M Gershwin, Shu-Han Yang and Zhe-Xiong Lian : The gut microbiome contributes to splenomegaly and tissue inflammation in a murine model of primary biliary cholangitis., Annals of Translational Medicine, Vol.10, No.9, 507, 2022.
(Summary)
Splenomegaly was associated with more severe liver inflammation in our PBC murine model, and this effect was reversed by quadruple antibiotic treatment.
Hitoshi Nishijima, Mizuki Sugita, Natsuka Umezawa, Naoki Kimura, Hirokazu Sasaki, Hiroshi Kawano, Yasuhiko Nishioka, Minoru Matsumoto, Takeshi Oya, Koichi Tsuneyama, Junko Morimoto and Mitsuru Matsumoto : Development of organ-specific autoimmunity by dysregulated Aire expression., Immunology and Cell Biology, Vol.100, No.5, 371-377, 2022.
(Summary)
cells expressing AIRE in the peripheral blood before the treatment but not during the remission phase treated with immunosuppressive drugs. Thus, not only loss of function of AIRE/Aire but also augmented and/or dysregulated expression of AIRE/Aire should be considered for the pathogenesis of organ-specific autoimmunity. We suggest that further analyses should be pursued to establish a novel link between organ-specific autoimmune disease and dysregulated AIRE expression in clinical settings.
Shoko Yamashita, Masaaki Nishi, Kozo Yoshikawa, Toshihiro Nakao, Takuya Tokunaga, Chie Takasu, Hideya Kashihara, Yuma Wada, Toshiaki Yoshimoto, Yosuke Iwakawa, Takeshi Oya, Koichi Tsuneyama and Mitsuo Shimada : Gastric carcinoma with lymphoid stroma derived from hamartomatous inverted polyp with osteoclast-like giant cells: a case report., International Cancer Conference Journal, Vol.11, No.3, 196-200, 2022.
(Summary)
Gastric carcinomas with lymphoid stroma (GCLS) are characterized by prominent stromal infiltration of lymphocyte and account for 1-4% of gastric cancers. Although, osteoclast-like giant cells (OGC) have been reported in some GCLS, OGCs in gastric tumors is exceedingly rare. A 60-year-old woman presented to our hospital after the finding of a positive fecal blood test during a routine medical check. Esophagogastroduodenoscopy revealed a Type 0-III + IIc tumor in the middle part of the gastric body. Biopsy revealed a poorly differentiated tumor and she was referred to our department. Early phase computed tomography showed thickening of the wall in the middle of the gastric body and enlargement of nearby lymph nodes. Laparoscopic total gastrectomy was performed. Pathological examination revealed a hamartomatous inverted polyp (HIP) in the submucosal layer with tub2-por1 tumor in the HIP. Prominent lymphocytic infiltration and OGCs were found around the tumor. Immunohistochemical analysis showed that the tumor cells were negative for EBER, MLH-1, and MSH2, 6. These findings suggest that this tumor was a non-microsatellite instability (MSI)-high GCLS without Epstein-Barr virus (EBV) infection. The patient's postoperative course was uneventful and she was discharged 11 days after surgery. She remains well 3 years after surgery.
Fumika Nakamura, Yasushi Sato, Koichi Okamoto, Yasuteru Fujino, Yasuhiro Mitsui, kaizo kagemoto, Tomoyuki Kawaguchi, Hiroshi Miyamoto, Naoki Muguruma, Tomoko Sonoda, Koichi Tsuneyama and Tetsuji Takayama : Colorectal carcinoma occurring via the adenoma-carcinoma pathway in patients with serrated polyposis syndrome., Journal of Gastroenterology, Vol.57, No.4, 286-299, 2022.
(Summary)
Although serrated polyposis syndrome (SPS) is associated with an increased colorectal cancer (CRC) risk, the carcinogenic mechanisms remain unknown. We investigated clinicopathological characteristics and genetic abnormalities in colorectal polyps and CRC to elucidate carcinogenic mechanisms in SPS. We retrospectively analyzed clinicopathological features of colorectal polyps in 44 SPS patients, and examined mutations of genes including APC, RAS, BRAF, and TP53, and microsatellite instability (MSI) in CRC tissues. Of the 44 patients, 25 (56%) fulfilled WHO criterion 1, 11 (25%) fulfilled criterion 2, and 8 (18%) fulfilled both. A total of 956 polyps were observed; 642 (67%) hyperplastic polyps (HP), 204 (21%) sessile serrated lesions (SSL), 10 (1%) traditional serrated adenoma (TSA), and 100 (11%) adenomas. The median numbers of polyps (/patient) were 10.5 (IQR 2.75-23) HPs, 4.0 (2.0-6.0) SSLs, 0 (0-0) TSA, and 1 (0-3.3) adenoma. SSL and HP located preferentially in the proximal and distal colon, respectively. Twenty-two CRCs were found in 18 patients. Based on the histological coexistence of SSL/TSA, BRAF mutation and MSI, 5 CRCs (26%) were classified as serrated-neoplasia pathway. Conversely, based on the coexistence of adenoma, APC/RAS and TP53 mutations, 11 CRCs (58%) were classified as adenoma-carcinoma pathway. The remaining three were unclassifiable. Most CRCs through adenoma-carcinoma pathway were located in the left-side colorectum and patients bearing those met criterion 2, characterized by many HP and advanced adenomas. Adenoma was a significant risk factor for CRC. Our results suggest that more than half of the CRCs, particularly those in the left-side colorectum, developed through the adenoma-carcinoma pathway in SPS patients. Adenoma was a risk factor for CRCs, suggesting its importance in colorectal carcinogenesis.
Hirohisa Ogawa, Masahiko Azuma, Aya Umeno, Mayuko Shimizu, Kazutoshi Murotomi, Yasukazu Yoshida, Yasuhiko Nishioka and Koichi Tsuneyama : Singlet oxygen -derived nerve growth factor exacerbates airway hyperresponsiveness in a mouse model of asthma with mixed inflammation., Allergology International, Vol.71, No.3, 395-404, 2022.
(Summary)
Our findings suggest that neutrophil MPO-derived singlet oxygen induces increased NGF production, leading to AHR and 10- and 12-(Z,E)-HODEs production. These findings may help to develop new therapies targeting this mechanism and to establish a new biomarker for non-type 2 and refractory asthma.
Noriaki Murayama, Koichi Okamoto, Tadahiko Nakagawa, Jinsei Miyoshi, Kensei Nishida, Tomoyuki Kawaguchi, kaizo kagemoto, Shinji Kitamura, Beibei Ma, Hiroshi Miyamoto, Naoki Muguruma, Mitsuyasu Yano, Koichi Tsuneyama, Takahiro Fujimori, Yasushi Sato and Tetsuji Takayama : miR-144-3p/miR-451a promotes lymphovascular invasion through repression of PTEN/p19 in rectal neuroendocrine tumors., Journal of Gastroenterology and Hepatology, Vol.37, No.5, 919-927, 2022.
(Summary)
Although rectal neuroendocrine tumor (NET-G1) have potential metastatic capability, even among small tumors, no predictive biomarker for invasion and metastasis has been reported. We analyzed microRNA (miRNA) expression profiles in rectal NET-G1 tissues with and without lymphovascular invasion (LVI). Moreover, we then investigated their target genes to clarify the mechanism of invasion/metastasis in NET-G1. miRNA array analysis was performed using seven rectal NET-G1 tissues with LVI and seven without LVI. miRNA expression was confirmed by quantitative real-time PCR. A NET cell line H727 was transfected with miRNA mimic or target gene small interfering RNA, and migration and invasion assays were performed. The expression levels of miR-144-3p and miR-451a were significantly higher in NET-G1 with LVI versus without LVI, as determined by miRNA array analysis and RT-qPCR. A significant correlation was observed between miR-144-3p and miR-451a expression levels, strongly suggesting miR144/451 cluster overexpression in NET-G1 with LVI. Bioinformatic analysis of target genes revealed that miR-144-3p and miR-451a directly interact with PTEN and p19 mRNA, respectively. Immunohistochemistry revealed significantly lower expression of PTEN and p19 in NET-G1 tissues with LVI than in those without LVI. The miR-144-3p and miR-451a mimic significantly increased cell migration/invasion capability, respectively. Knockdown of PTEN and p19 induced significant augmentation of cell invasion and migration capability, respectively. Our data suggest that overexpression of miR-144/miR-451 cluster promotes LVI via repression of PTEN and p19 in rectal NET-G1 cells. miR-144/451 cluster may be a novel biomarker for predicting invasion/metastasis in rectal NET-G1.
Takeo Minamikawa, Eiji Hase, Mayuko Shimizu, Yuki Morimoto, Akihiro Suzuki, Takeshi Yasui, Satoko Nakamura, Akemi Tsutsui, Koichi Takaguchi and Koichi Tsuneyama : Assessment of Ultra-Early-Stage Liver Fibrosis in Human Non-Alcoholic Fatty Liver Disease by Second-Harmonic Generation Microscopy., International Journal of Molecular Sciences, Vol.23, No.6, 3357, 2022.
(Summary)
Non-alcoholic fatty liver disease (NAFLD) is associated with the chronic progression of fibrosis. In general, the progression of liver fibrosis is determined by a histopathological assessment with a collagen-stained section; however, the ultra-early stage of liver fibrosis is challenging to identify because of the low sensitivity in the collagen-selective staining method. In the present study, we demonstrate the feasibility of second-harmonic generation (SHG) microscopy in the histopathological diagnosis of the liver of NAFLD patients for the quantitative assessment of the ultra-early stage of fibrosis. We investigated four representative NAFLD patients with early stages of fibrosis. SHG microscopy visualised well-matured fibrotic structures and early fibrosis diffusely involving liver tissues, whereas early fibrosis is challenging to be identified by conventional histopathological methods. Furthermore, the SHG emission directionality analysis revealed the maturation of each collagen fibre of each patient. As a result, SHG microscopy is feasible for assessing liver fibrosis on NAFLD patients, including the ultra-early stage of liver fibrosis that is difficult to diagnose by the conventional histopathological method. The assessment method of the ultra-early fibrosis by using SHG microscopy may serve as a crucial means for pathological, clinical, and prognostic diagnosis of NAFLD patients.
Ya-Fei Xu, Yuan Yao, Min Ma, Shu-Han Yang, Peng Jiang, Jinjun Wang, Koichi Tsuneyama, Chan Wang, Xiangdong Liu, Liang Li and Zhe-Xiong Lian : The Proinflammatory Cytokines IL-18, IL-21, and IFN- γ Differentially Regulate Liver Inflammation and Anti-Mitochondrial Antibody Level in a Murine Model of Primary Biliary Cholangitis, Journal of Immunology Research, Vol.2022, 2022.
(Summary)
is responsible for both AMA production and liver inflammation in our model. Our results demonstrate that different proinflammatory cytokines can regulate different effector functions in PBC pathogenesis and need to be considered in PBC treatment.
Junko Morimoto, Minoru Matsumoto, Ryuichiro Miyazawa, Takeshi Oya, Koichi Tsuneyama and Mitsuru Matsumoto : No Major Impact of Two Homologous Proteins Ly6C1 and Ly6C2 on Immune Homeostasis., ImmunoHorizons, Vol.6, No.3, 202-210, 2022.
(Summary)
Ly6C comprises two homologous components of Ly6C1 and Ly6C2, and the expression of either of the Ly6C molecules defines unique functional subsets of monocytes. Ly6C is also expressed by other immune cell types, including Aire-expressing medullary thymic epithelial cells. Because the role of Ly6C expression in determining the functional subsets remains unclear, we generated mice deficient for both Ly6C1 and Ly6C2 with CRISPR-Cas9mediated deletion. Mice deficient for Ly6C1/Ly6C2 showed no major alterations in the subsets and function of monocyte and other immune cells, including the cells involved in the dextran sulfate sodium saltinduced colitis model. By generating the mice deficient for Ly6C1 alone, we have also investigated the expression pattern of Ly6C1 and Ly6C2 in immune cells. Except for medullary thymic epithelial cells and CD4 single-positive T cells, immune cells predominantly expressed Ly6C2. Thus, despite the importance as a marker with a unique differential expression pattern, the Ly6C molecules have no major impact on determining the functional subsets and maintaining immune homeostasis.
Junko Morimoto, Minoru Matsumoto, Ryuichiro Miyazawa, Hideyuki Yoshida, Koichi Tsuneyama and Mitsuru Matsumoto : Aire suppresses CTLA-4 expression from the thymic stroma to control autoimmunity., Cell Reports, Vol.38, No.7, 110384, 2022.
(Summary)
Impaired production of thymic regulatory T cells (Tregs) is implicated in the development of Aire-dependent autoimmunity. Because Tregs require agonistic T cell receptor stimuli by self-antigens to develop, reduced expression of self-antigens from medullary thymic epithelial cells (mTECs) has been considered to play a major role in the reduced Treg production in Aire deficiency. Here, we show that mTECs abnormally express co-inhibitory receptor CTLA-4 if Aire is non-functional. Upon binding with CD80/CD86 ligands expressed on thymic dendritic cells (DCs), the ectopically expressed CTLA-4 from Aire-deficient mTECs removes the CD80/CD86 ligands from the DCs. This attenuates the ability of DCs to provide co-stimulatory signals and to present self-antigens transferred from mTECs, both of which are required for Treg production. Accordingly, impaired production of Tregs and organ-specific autoimmunity in Aire-deficient mice are rescued by the depletion of CTLA-4 expression from mTECs. Our studies illuminate the significance of mTEC-DC interaction coordinated by Aire for the establishment of thymic tolerance.
Hitoshi Nishijima, Minoru Matsumoto, Junko Morimoto, Kazuyoshi Hosomichi, Nobuko Akiyama, Taishin Akiyama, Takeshi Oya, Koichi Tsuneyama, Hideyuki Yoshida and Mitsuru Matsumoto : Aire Controls Heterogeneity of Medullary Thymic Epithelial Cells for the Expression of Self-Antigens., The Journal of Immunology, Vol.208, No.2, 303-320, 2022.
(Summary)
The deficiency of Aire, a transcriptional regulator whose defect results in the development of autoimmunity, is associated with reduced expression of tissue-restricted self-Ags (TRAs) in medullary thymic epithelial cells (mTECs). Although the mechanisms underlying Aire-dependent expression of TRAs need to be explored, the physical identification of the target(s) of Aire has been hampered by the low and promiscuous expression of TRAs. We have tackled this issue by engineering mice with augmented Aire expression. Integration of the transcriptomic data from Aire-augmented and Aire-deficient mTECs revealed that a large proportion of so-called Aire-dependent genes, including those of TRAs, may not be direct transcriptional targets downstream of Aire. Rather, Aire induces TRA expression indirectly through controlling the heterogeneity of mTECs, as revealed by single-cell analyses. In contrast, Ccl25 emerged as a canonical target of Aire, and we verified this both in vitro and in vivo. Our approach has illuminated the Aire's primary targets while distinguishing them from the secondary targets.
Hitomi Fujishiro, Miharu Sumino, Daigo Sumi, Hitomi Umemoto, Koichi Tsuneyama, Takehisa Matsukawa, Kazuhito Yokoyama and Seiichiro Himeno : Spatial localization of cadmium and metallothionein in the kidneys of mice at the early phase of cadmium accumulation., The Journal of Toxicological Sciences, Vol.47, No.12, 507-517, 2022.
(Summary)
Chronic exposure to cadmium (Cd) leads to an accumulation of Cd in the kidneys. Metallothionein (MT) is a low-molecular-weight protein having a high affinity for Cd. Cd bound to MT in serum is filtered through the glomeruli of kidney nephrons and reabsorbed by endocytosis into the proximal tubules from the luminal side. Accumulation of Cd in renal cells induces MT synthesis, leading to long-term deposition of Cd and the suppression of Cd toxicity. Recently, many studies have investigated the tissue distribution of metals using laser ablation ICP-MS (LA-ICP-MS). However, little information has been available regarding renal Cd distribution. Hence, we dually investigated the renal distribution of Cd by LA-ICP-MS and that of MT by immunohistochemistry to clarify the dose- and time-dependent changes in the distributions of Cd and MT in mice exposed to Cd from drinking water for 1, 2, and 4 months. Both Cd and MT exhibited characteristic heterogeneous distribution patterns in the renal cortex. The accumulation of Cd and MT near the surface of the cortex suggests a preferential accumulation of Cd in the surface nephrons. MT distribution was more pronounced in the proximal tubules than in the distal tubules, and there were clear differences in MT immunostaining even among the proximal tubules. The heterogeneous localization of MT may reflect the nephron-specific accumulation of Cd. Combining elemental imaging of Cd with immunostaining of MT proved a successful strategy to reveal the characteristic renal Cd distribution, especially in the early stages of Cd accumulation.
Katsuhisa Omagari, Miku Uchida, Yumeno Tagawa, Mizuki Yogo, Kae Inagaki, Ryoko Hongo, Shouhei Takeuchi, Kazuhito Suruga, Kazunori Koba, Mayuko Shimizu and Koichi Tsuneyama : Influence of Fasting Time on Serum and Hepatic Lipid Profiles in a Sprague-Dawley Rat Model of Nonalcoholic Steatohepatitis., Journal of Nutritional Science and Vitaminology, Vol.68, No.5, 409-419, 2022.
(Summary)
Nonalcoholic steatohepatitis (NASH) is associated with several cardiovascular risk factors, including atherogenic dyslipidemia. Recently, fasting prior to lipid profile evaluation has been thought to be unnecessary for most individuals. We investigated the impact of fasting for up to 9 h on the serum and hepatic lipid profiles in Sprague-Dawley (SD) rats of dietary-induced NASH model in comparison to SD rats fed a normal diet. In both groups, fasting affected the serum and hepatic triglyceride (TG), serum free fatty acid (FFA) and leptin levels, histopathologically assessed hepatocyte ballooning, and hepatic mRNA expression levels of several genes related to lipid metabolism. In contrast, the serum adiponectin and aminotransferase levels, serum and hepatic total cholesterol contents, and liver histopathological findings of hepatic steatosis, lobular inflammation and fibrosis were not influenced by fasting. A significant fasting time-dependent reduction was seen in the serum TG level only in the normal SD rats group. Regarding the hepatic TG level, a significant fasting time-dependent increase was seen only in the NASH model rat group. A significant fasting time-dependent reduction was also seen in the serum FFA level only in the NASH model rat group. Our present results indicate that excessive fasting can be avoided before blood or hepatic tissue sampling for the evaluation of several parameters in non-NASH and/or NASH model rats. Further investigations are needed in humans to determine whether excessive fasting before blood or hepatic tissue sampling can be avoided in both healthy individuals and NASH patients.
Mayuko Shimizu, Yosuke Tsuchiyama, Yuki Morimoto, Minoru Matsumoto, Tomoko Kobayashi, Satoshi Sumida, Takumi Kakimoto, Takeshi Oya, Hirohisa Ogawa, Michiko Yamashita, Satoru Matsuda, Katsuhisa Omagari, Shu Taira and Koichi Tsuneyama : A novel mouse model of non-alcoholic steatohepatitis suggests that liver fibrosis initiates around lipid-laden macrophages, The American Journal of Pathology, Vol.192, No.1, 31-42, 2022.
(Summary)
Various cells, such as macrophages and hepatic stellate cells, interact in the generation of fibrosis in nonalcoholic steatohepatitis (NASH), but the mechanism remains unclear. We employed a high-fat/cholesterol/cholate (HFCC) diet to generate a model of NASH-related fibrosis and investigate the pathogenesis of fibrosis. Two mouse strains differing in susceptibility to obesity, the susceptible strain C57BL/6J and the relatively resistant strain A/J, developed hepatic histologic features of NASH, including fat deposition, intralobular inflammation, hepatocyte ballooning, and fibrosis, after 9 weeks of HFCC diet feeding. The severity of hepatic inflammation and fibrosis was greater in A/J mice than in C57BL/6J mice. A/J mice fed the HFCC diet exhibited characteristic CD204-positive lipid-laden macrophage aggregation in hepatic parenchyma. Polarized light visualized the Maltese cross (namely, cholesterol crystals within the aggregated macrophages). Moreover, fibrosis developed in a ring shape from the periphery of the aggregated macrophages (ie, the starting point of fibrosis could be visualized histologically). Furthermore, matrix-assisted laser desorption/ionization mass spectrometry imaging analysis detected a molecule at m/z 772.462, which corresponds to the protonated ion of phosphatidylcholine [P-18:1 (11Z)/18:0] and phosphatidylethanolamine [18:0/20:2 (11Z, 14Z)], in aggregated macrophages adjacent to the fibrotic lesions. In conclusion, the present HFCC diet-fed A/J model provides an ideal tool to study fibrogenesis and enables novel insights into the pathophysiology of NASH-related fibrosis.
Naohiro Nakamura, Katsunori Yoshida, Rinako Tsuda, Miki Murata, Takashi Yamaguchi, Kanehiko Suwa, Mayuko Ichimura, Koichi Tsuneyama, Koichi Matsuzaki, Toshiaki Nakano, Junko Hirohara, Toshihito Seki, Kazuichi Okazaki, Eric M Gershwin and Makoto Naganuma : Phospho-Smad3 signaling is predictive biomarker for hepatocellular carcinoma risk assessment in primary biliary cholangitis patients., Frontiers in Bioscience (Landmark edition), Vol.26, No.12, 1480-1492, 2021.
(Summary)
In this study, Smad phospho-isoform status showed promise as a biomarker predicting likelihood of HCC occurrence in PBC. Eventually, therapies to shift favorably Smad phospho-isoforms might decrease likelihood of PBC-related HCC.
Yasu-Taka Azuma, Takashi Fujita, Takeshi Izawa, Kana Hirota, Kazuhiro Nishiyama, Airi Ikegami, Tomoko Aoyama, Mikihito Ike, Yumi Ushikai, Mitsuru Kuwamura, Hideki Fujii and Koichi Tsuneyama : IL-19 Contributes to the Development of Nonalcoholic Steatohepatitis by Altering Lipid Metabolism., Cells, Vol.10, No.12, 2021.
(Summary)
Interleukin (IL)-19, a member of the IL-10 family, is an anti-inflammatory cytokine produced primarily by macrophages. Nonalcoholic steatohepatitis (NASH) is a disease that has progressed from nonalcoholic fatty liver disease (NAFLD) and is characterized by inflammation and fibrosis. We evaluated the functions of IL-19 in a NAFLD/NASH mouse model using a 60% high fat diet with 0.1% methionine, without choline, and with 2% cholesterol (CDAHFD). Wild-type (WT) and IL-19 gene-deficient (KO) mice were fed a CDAHFD or standard diet for 9 weeks. Liver injury, inflammation, and fibrosis induced by CDAHFD were significantly worse in IL-19 KO mice than in WT mice. IL-6, TNF-α, and TGF-β were significantly higher in IL-19 KO mice than in WT mice. As a mechanism using an in vitro experiment, palmitate-induced triglyceride and cholesterol contents were decreased by the addition of IL-19 in HepG2 cells. Furthermore, addition of IL-19 decreased the expression of fatty acid synthesis-related enzymes and increased ATP content in HepG2 cells. The action of IL-19 in vitro suppressed lipid metabolism. In conclusion, IL-19 may play an important role in the development of steatosis and fibrosis by directly regulating liver metabolism and may be a potential target for the treatment of liver diseases.
Mayuko Shimizu, Takeshi Kageyama, Takeshi Oya, Hirohisa Ogawa, Minoru Matsumoto, Satoshi Sumida, Takumi Kakimoto, Yuko Miyakami, Ryosuke Nagatomo, Koichi Inoue, Chunmei Cheng and Koichi Tsuneyama : Verification of the Impact of Blood Glucose Level on Liver Carcinogenesis and the Efficacy of a Dietary Intervention in a Spontaneous Metabolic Syndrome Model, International Journal of Molecular Sciences, Vol.22, No.23, 12844, 2021.
(Summary)
Metabolic syndrome (MS) is a risk factor for type 2 diabetes mellitus, vascular inflammation, atherosclerosis, and renal, liver, and heart diseases. Non-alcoholic steatohepatitis (NASH) is a progressive representative liver disease and may lead to the irreversible calamities of cirrhosis and hepatocellular carcinoma. Metabolic disorders such as hyperglycemia have been broadly reported to be related to hepatocarcinogenesis in NASH; however, direct evidence of a link between hyperglycemia and carcinogenesis is still lacking. Tsumura Suzuki Obese Diabetic (TSOD) mice spontaneously develop metabolic syndrome, including obesity, insulin resistance, and NASH-like liver phenotype, and eventually develop hepatocellular carcinomas. TSOD mice provide a spontaneous human MS-like model, even with significant individual variations. In this study, we monitored mice in terms of their changes in blood glucose levels, body weights, and pancreatic and liver lesions over time. As a result, liver carcinogenesis was delayed in non-hyperglycemic TSOD mice compared to hyperglycemic mice. Moreover, at the termination point of 40 weeks, liver tumors appeared in 18 of 24 (75%) hyperglycemic TSOD mice; in contrast, they only appeared in 5 of 24 (20.8%) non-hyperglycemic mice. Next, we investigated three kinds of oligosaccharide that could lower blood glucose levels in hyperglycemic TSOD mice. We monitored the levels of blood and urinary glucose and assessed pancreatic lesions among the experimental groups. As expected, significantly lower levels of blood and urinary glucose and smaller deletions of Langerhans cells were found in TSOD mice fed with milk-derived oligosaccharides (galactooligosaccharides and lactosucrose). At the age of 24 weeks, mild steatohepatitis was found in the liver but there was no evidence of liver carcinogenesis. Steatosis in the liver was alleviated in the milk-derived oligosaccharide-administered group. Taken together, suppressing the increase in blood glucose level from a young age prevented susceptible individuals from diabetes and the onset of NAFLD/NASH, as well as carcinogenesis. Milk-derived oligosaccharides showed a lowering effect on blood glucose levels, which may be expected to prevent liver carcinogenesis.
C Julio Valencia, A Rebecca Erwin-Cohen, E Paul Clavijo, Clint Allen, E Michael Sanford, Chi-Ping Day, M Megan Hess, Morgan Johnson, Jie Yin, M John Fenimore, A Ian Bettencourt, Koichi Tsuneyama, E Maria Romero, D Kimberly Klarmann, Peng Jiang, R Heekyong Bae, W Daniel McVicar, Glenn Merlino, F Elijah Edmondson, Niroshana Anandasabapathy and A Howard Young : Myeloid-Derived Suppressive Cell Expansion Promotes Melanoma Growth and Autoimmunity by Inhibiting CD40/IL27 Regulation in Macrophages., Cancer Research, Vol.81, No.23, 5977-5990, 2021.
(Summary)
autoinflammation and exacerbates underlying AID, even without evident antitumor responses. Recently, systemic lupus erythematosus (SLE) activity was found to drive myeloid-derived suppressor cell (MDSC) formation in patients, a known barrier to healthy immune surveillance and successful cancer immunotherapy. Cross-talk between MDSCs and macrophages generally drives immune suppressive activity in the tumor microenvironment. However, it remains unclear how peripheral pregenerated MDSC under chronic inflammatory conditions modulates global macrophage immune functions and the impact it could have on existing tumors and underlying lupus nephritis. Here we show that pathogenic expansion of SLE-generated MDSCs by melanoma drives global macrophage polarization and simultaneously impacts the severity of lupus nephritis and tumor progression in SLE-prone mice. Molecular and functional data showed that MDSCs interact with autoimmune macrophages and inhibit cell surface expression of CD40 and the production of IL27. Moreover, low CD40/IL27 signaling in tumors correlated with high tumor-associated macrophage infiltration and ICB therapy resistance both in murine and human melanoma exhibiting active IFNγ signatures. These results suggest that preventing global macrophage reprogramming induced by MDSC-mediated inhibition of CD40/IL27 signaling provides a precision melanoma immunotherapy strategy, supporting an original and advantageous approach to treat solid tumors within established autoimmune landscapes. SIGNIFICANCE: Myeloid-derived suppressor cells induce macrophage reprogramming by suppressing CD40/IL27 signaling to drive melanoma progression, simultaneously affecting underlying autoimmune disease and facilitating resistance to immunotherapy within preexisting autoimmune landscapes.
Katsuki Miyazaki, Yuji Morine, Shin-ichiro Yamada, Yu Saitou, Kazunori Tokuda, Shohei Okikawa, Shoko Yamashita, Takeshi Oya, Tetsuya Ikemoto, Satoru Imura, Haun Hu, Hisayoshi Morioka, Koichi Tsuneyama and Mitsuo Shimada : Stromal tumor-infiltrating lymphocytes level as a prognostic factor for resected intrahepatic cholangiocarcinoma and its prediction by apparent diffusion coefficient., International Journal of Clinical Oncology, Vol.26, No.12, 2265-2274, 2021.
(Summary)
Tumor-infiltrating lymphocytes (TILs) are a prognostic factor or an indicator of chemotherapy response for various malignancies. The aim of this study was to investigate the prognostic impact of TILs in resected intrahepatic cholangiocarcinoma (IHCC). We also investigated the usefulness of the apparent diffusion coefficient (ADC) in diffusion-weighted magnetic resonance imaging (DW-MRI) to predict TILs. We enrolled 23 patients with IHCC who underwent initial hepatic resection in Tokushima University Hospital from 2006 to 2017. We evaluated stromal TILs in the tumor marginal area and central area in surgical specimens. Patients were divided into low vs high stromal TILs groups. We analyzed the patients' clinicopathological factors, including prognosis, according to the degree of stromal TILs. We also analyzed the correlation between stromal TILs and the minimum ADC value. Stromal TILs in the marginal area reflected overall survival more accurately than that in the central area. Additionally, marginal low TILs was significantly associated with lymph node metastasis and portal vein invasion. Both overall- and disease-free survival rates in the marginal low TILs group were significantly worse than those in the marginal high TILs group (P < 0.05). In the multivariate analysis, marginal low TILs were an independent prognostic factor for both overall- and disease-free survival (P < 0.05), and marginal low TILs were significantly associated with lower minimum ADC values (P < 0.02). Stromal TILs, especially in the marginal area, might demonstrate prognostic impact in patients with IHCC. Moreover, the ADC values from MRI may predict TILs in IHCC tumor tissue.
(Keyword)
Bile Duct Neoplasms / Bile Ducts, Intrahepatic / Cholangiocarcinoma / Diffusion Magnetic Resonance Imaging / Humans / Lymphocytes, Tumor-Infiltrating / Prognosis
Shun-ichi Toki, Tetsuro Yoshimaru, Yosuke Matsushita, Hitoshi Aibara, Masaya Ono, Koichi Tsuneyama, Koichi Sairyo and Toyomasa Katagiri : The survival and proliferation of osteosarcoma cells are dependent on the mitochondrial BIG3-PHB2 complex formation., Cancer Science, Vol.112, No.10, 4208-4219, 2021.
(Summary)
Previous studies reported the critical role of the brefeldin A-inhibited guanine nucleotide exchange protein 3-prohibitin 2 (BIG3-PHB2) complex in modulating estrogen signaling activation in breast cancer cells, yet its pathophysiological roles in osteosarcoma (OS) cells remain elusive. Here, we report a novel function of BIG3-PHB2 in OS malignancy. BIG3-PHB2 complexes were localized mainly in mitochondria in OS cells, unlike in estrogen-dependent breast cancer cells. Depletion of endogenous BIG3 expression by small interfering RNA (siRNA) treatment led to significant inhibition of OS cell growth. Disruption of BIG3-PHB2 complex formation by treatment with specific peptide inhibitor also resulted in significant dose-dependent suppression of OS cell growth, migration, and invasion resulting from G2/M-phase arrest and in PARP cleavage, ultimately leading to PARP-1/apoptosis-inducing factor (AIF) pathway activation-dependent apoptosis in OS cells. Subsequent proteomic and bioinformatic pathway analyses revealed that disruption of the BIG3-PHB2 complex might lead to downregulation of inner mitochondrial membrane protein complex activity. Our findings indicate that the mitochondrial BIG3-PHB2 complex might regulate PARP-1/AIF pathway-dependent apoptosis during OS cell proliferation and progression and that disruption of this complex may be a promising therapeutic strategy for OS.
Takeshi Nishida, Koichi Tsuneyama, Yasuhiko Tago, Koji Nomura, Makoto Fujimoto, Takahiko Nakajima, Akira Noguchi, Takashi Minamisaka, Hideki Hatta and Johji Imura : Effect of Continuous Feeding of Ayu-Narezushi on Lipid Metabolism in a Mouse Model of Metabolic Syndrome, The Scientific World Journal, Vol.2021, 2021.
(Summary)
improved obesity and dyslipidemia in the TSOD mice and that the activation of fatty acid oxidation in the liver might contribute to these improvements.
Hideki Hatta, Takeshi Nishida, Takashi Minamisaka, Koichi Tsuneyama and Johji Imura : Utility of Ethylene-Diamine-Tetraacetic Acid Buffer Solution With Boric Acid for Immunostaining of Specimens Stored for an Extended Period., Curēus, Vol.13, No.8, 2021.
(Summary)
Antigen modification and denaturation are recognized causes of false negatives in immunostaining. Specimens that have been stored for an extended period at room temperature show decreased immunoreactivity and may mislead the diagnosis. Studies of the molecular targeting of drugs often involve immunostaining of previous samples and, in some situations, only unstained specimens can be used. The present study aimed to develop an effective staining method to recover antigen activation in unstained specimens stored for an extended period by using ethylene-diamine-tetraacetic acid (EDTA) buffer solution with boric acid. We compared several commonly used antigen retrieval solutions and found that Tris-borate-EDTA (TBE) buffer solution with a pH 8.3 provided sufficient antigen retrieval. However, pH values higher than 8.3 (9.0, 10.0, and 11.0) frequently caused severe tissue damage. Thus, TBE with pH 8.3 was the most suitable antigen retrieval solution for recovering the antigenicity of specimens stored for an extended period. This procedure may allow useful immunohistochemical information, even from sections that have been stored for an extended period.
Tomoko Kobayashi, Mayuko Shimizu, Takeshi Oya, Hirohisa Ogawa, Minoru Matsumoto, Yuki Morimoto, Satoshi Sumida, Takumi Kakimoto, Michiko Yamashita, Mitsuko Sutoh, Shunji Toyohara, Ryoji Hokao, Chunmei Cheng and Koichi Tsuneyama : Neonatal streptozotocin treatment rapidly causes different subtype of hepatocellular carcinoma without persistent hyperglycemia in 4CS mice fed on a normal diet., Pathology, Research and Practice, Vol.225, No.153559, 2021.
(Summary)
Although diabetes mellitus (DM) is a well-known risk factor for hepatocellular carcinoma (HCC), the underlying mechanisms have not yet to be defined. We previously reported that DIAR mice fed with standard murine diet developed type 1 diabetes and HCC at age of 16 weeks old with a neonatal streptozotocin treatment (n-STZ). Because DIAR mice did not manifest obesity nor develop steatohepatitis, hyperglycemia with streptozotocin trigger or streptozotocin alone might turn on the hepato-carcinogenesis. An insulin-recruitment to DIAR-nSTZ mice showed an increased frequency of HCC during the first 12 weeks of age, although the diabetic indications notably improved. To elucidate the role of hyperglycemia in hepato-carcinogenesis, we performed a head-to-head comparative study by using 4CS mice and DIAR mice with n-STZ treatment. Newborn 4CS mice and DIAR mice were divided into STZ treated group and control group. The blood glucose levels of DIAR-nSTZ mice increased at age of eight weeks, while that of 4CS-nSTZ mice were maintained in the normal range. At eight weeks old, three out of five DIAR-nSTZ mice (60%) and one out of ten 4CS-nSTZ mice (10%) developed multiple liver tumors. At age of 12 weeks old, all eight of DIAR-nSTZ mice (100%) and two of 10 4CS-nSTZ mice (20%) developed multiple liver tumors. At 16 weeks old, all animals of DIAR-nSTZ and 4CS-nSTZ mice occurred liver tumors. DIAR-nSTZ showed hyperglycemia and HCC, and 4CS-nSTZ developed HCC without hyperglycemia. These results were interpreted that the onset of HCC maybe not related to the presence or absence of hyperglycemia but nSTZ treatment. On the other hand, since the carcinogenesis of 4CS-nSTZ is delayed compared to DIAR-nSTZ, hyperglycemia may play a role in the progression of carcinogenesis. Histologically, the liver tumor appeared irregularly trabecular arrangements of hepatocytes with various degrees of nuclear atypia. By immunohistochemical analyses, all liver tumors showed positive staining of glutamine synthetase (GS), an established human HCC marker. The expression pattern of GS was divided into a strong diffuse pattern and weak patchy pattern, respectively. The liver tumor showing the weak GS-patchy pattern expressed biliary/stem markers, EpCAM, and SALL4, partially. Because 4CS-nSTZ mice did not show any metabolic complications such as gaining body weight or high blood glucose level, it is a unique animal model with a simple condition to investigate hepatic carcinogenesis by excluding other factors.
Shao-Yuan Chen, Koichi Tsuneyama, Mao-Hsiung Yen, Jiunn-Tay Lee, Jiun-Liang Chen and Shih-Ming Huang : Hyperbaric oxygen suppressed tumor progression through the improvement of tumor hypoxia and induction of tumor apoptosis in A549-cell-transferred lung cancer., Scientific Reports, Vol.11, No.1, 2021.
(Summary)
Tumor cells have long been recognized as a relative contraindication to hyperbaric oxygen treatment (HBOT) since HBOT might enhance progressive cancer growth. However, in an oxygen deficit condition, tumor cells are more progressive and can be metastatic. HBOT increasing in oxygen partial pressure may benefit tumor suppression. In this study, we investigated the effects of HBOT on solid tumors, such as lung cancer. Non-small cell human lung carcinoma A549-cell-transferred severe combined immunodeficiency mice (SCID) mice were selected as an in vivo model to detect the potential mechanism of HBOT in lung tumors. HBOT not only improved tumor hypoxia but also suppressed tumor growth in murine xenograft tumor models. Platelet endothelial cell adhesion molecule (PECAM-1/CD31) was significantly increased after HBOT. Immunostaining of cleaved caspase-3 was demonstrated and apoptotic tumor cells with nuclear debris were aggregated starting on the 14th-day after HBOT. In vitro, HBOT suppressed the growth of A549 cells in a time-dependent manner and immediately downregulated the expression of p53 protein after HBOT in A549 cells. Furthermore, HBOT-reduced p53 protein could be rescued by a proteasome degradation inhibitor, but not an autophagy inhibitor in A549 cells. Our results demonstrated that HBOT improved tissue angiogenesis, tumor hypoxia and increased tumor apoptosis to lung cancer cells in murine xenograft tumor models, through modifying the tumor hypoxic microenvironment. HBOT will merit further cancer therapy as an adjuvant treatment for solid tumors, such as lung cancer.
Shoko Yamashita, Yuji Morine, Satoru Imura, Tetsuya Ikemoto, Yu Saitou, Chie Takasu, Shinichiro Yamada, Kazunori Tokuda, Shohei Okikawa, Katsuki Miyazaki, Takeshi Oya, Koichi Tsuneyama and Mitsuo Shimada : A new pathological classification of intrahepatic cholangiocarcinoma according to protein expression of SSTR2 and Bcl2., World Journal of Surgical Oncology, Vol.19, No.1, 142, 2021.
(Summary)
This method could be used to classify IHCC into peripheral and perihilar type by embryological expression patterns of SSTR2 and Bcl2.
(Keyword)
Bile Duct Neoplasms / Bile Ducts, Intrahepatic / Cholangiocarcinoma / Humans / Prognosis / Proto-Oncogene Proteins c-bcl-2 / Receptors, Somatostatin
Shin Shimoyama, Kazuhito Kawata, Kazuyoshi Ohta, Takeshi Chida, Tetsuro Suzuki, Koichi Tsuneyama, Shinji Shimoda, Nobuhito Kurono, C Patrick S Leung, Eric M Gershwin, Takafumi Suda and Yoshimasa Kobayashi : Ursodeoxycholic acid impairs liver-infiltrating T-cell chemotaxis through IFN-γ and CX3CL1 production in primary biliary cholangitis., European Journal of Immunology, Vol.51, No.6, 1519-1530, 2021.
(Summary)
Ursodeoxycholic acid (UDCA) is the primary treatment for primary biliary cholangitis (PBC), but its mechanism of action remains unclear. Studies suggest that UDCA enhances NF erythroid 2-related factor 2 (NFE2L2) expression and that the interaction between IFN-γ and C-X3-C motif chemokine ligand 1 (CX3CL1) facilitates biliary inflammation in PBC. Therefore, we examined the effects of UDCA on the expression of IFN-γ and CX3CL1 in in vitro and in vivo PBC models such as human liver tissue, a murine model, cell lines, and isolated human intrahepatic biliary epithelial cells (IHBECs). We observed a significant decrease in IFN-γ mRNA levels and positive correlations between IFN-γ and CX3CL1 mRNA levels post-UDCA treatment in PBC livers. NFE2L2-mediated transcriptional activation was significantly enhanced in UDCA-treated Jurkat cells. In 2-octynoic acid-immunized mice, IFN-γ production by liver-infiltrating T cells was dependent on NFE2L2 activation. IFN-γ significantly and dose-dependentlyinduced CX3CL1 expression, which was significantly decreased in HuCC-T1 cells and IHBECs upon UDCA treatment. These results suggest that UDCA-induced suppression of IFN-γ and CX3CL1 production attenuates the chemotactic and adhesive abilities of liver-infiltrating T cells in PBC.
Mayuko Shimizu, Katsuhisa Omagari, Michiko Yamashita and Koichi Tsuneyama : Development of a novel mouse model of diet-induced nonalcoholic steatohepatitis-related progressive bridging fibrosis., Bioscience, Biotechnology, and Biochemistry, Vol.85, No.4, 941-947, 2021.
(Summary)
Nonalcoholic steatohepatitis (NASH) progresses to liver fibrosis and cirrhosis. Existing mouse models of NASH rarely develop diet-induced severe fibrosis. We aimed to establish a dietary model of NASH with rapid progression to fibrosis. Six-week-old male Tsumura-Suzuki obese diabetes (TSOD) mice (a model of spontaneous metabolic syndrome) and corresponding control Tsumura-Suzuki nonobese (TSNO) mice were fed a novel diet high in fat, cholesterol, and cholate (iHFC). Histologic steatohepatitis, including steatosis, inflammation, and fibrosis, were observed in both TSNO and TSOD iHFC diet-fed mice at 20 weeks of age. As compared with TSOD mice, TSNO mice developed much more severe fibrosis and reached stage 3 of bridging fibrosis within 14 weeks under the iHFC diet feeding. Perivenular/perisinusoidal pattern of fibrosis in TSNO mice resembled human NASH. Our model of NASH with advanced fibrosis by simple diet offers many advantages useful in studying the mechanism of liver fibrosis and preclinical drug testing.
Kotaro Matsumoto, Kentaro Kikuchi, Ayako Hara, Hiromichi Tsunashima, Koichi Tsuneyama and Shinpei Doi : Immunohistochemical detection of procalcitonin in fibrolamellar hepatocellular carcinoma., Clinical Journal of Gastroenterology, Vol.14, No.3, 827-830, 2021.
(Summary)
A 25-year-old woman with fever and epigastric pain was referred to our hospital. Blood examination showed significant liver dysfunction, markedly high C-reactive protein (CRP 19.1 mg/dL) and procalcitonin (48.3 ng/mL) levels. Dynamic computed tomography showed a tumor approximately 120 mm in size in the right lobe of the liver, but with no abscess formation. The patient was hospitalized and started on antibiotics; her CRP level improved, but the procalcitonin level did not decrease. Histopathological examination of the liver tumor biopsy revealed fibrolamellar hepatocellular carcinoma (FLC). Positive staining of the FLC with an anti-procalcitonin antibody suggested the production of procalcitonin.
Hironori Tanaka, Yoshihito Saijyo, Tetsu Tomonari, Takahiro Tanaka, Tatsuya Taniguchi, Shusuke Yagi, Koichi Okamoto, Hiroshi Miyamoto, Masahiro Sogabe, Yasushi Sato, Naoki Muguruma, Koichi Tsuneyama, Masataka Sata and Tetsuji Takayama : An Adult Case of Congenital Extrahepatic Portosystemic Shunt Successfully Treated with Balloon-occluded Retrograde Transvenous Obliteration, Internal Medicine, Vol.60, No.12, 1839-1845, 2021.
(Summary)
A 42-year-old woman visited our hospital due to syncope. Contrast-enhanced CT revealed portosystemic shunt, portal vein hypoplasia, and multiple liver nodules. The histological examination of a liver biopsy specimen exhibited portal vein hypoplasia and revealed that the liver tumor was positive for glutamine synthetase. The patient was therefore diagnosed with congenital extrahepatic portosystemic shunt type II, and with focal nodular hyperplasia (FNH)-like nodules. She had the complication of severe portopulmonary hypertension and underwent complete shunt closure by balloon-occluded retrograde transvenous obliteration (B-RTO). The intrahepatic portal vein was well developed at 1 year after B-RTO, and multiple liver nodules completely regressed. Her pulmonary hypertension also improved.
Katsuhisa Omagari, Mayu Yamasaki, Ngo Chi Thi Linh, Chiaki Koba, Asuka Nagata, Ayumi Fukuda, Kazuhito Suruga, Mayuko Ichimura and Koichi Tsuneyama : Influence of age in weeks on the development and progression of nonalcoholic steatohepatitis in a diet-induced Sprague-Dawley rat model, Acta Medica Nagasakiensia, Vol.65, No.1, 7-15, 2021.
(Summary)
<b>Background:</b> Understanding the pathogenesis of nonalcoholic steatohepatitis (NASH) in humans has been hampered by the lack of a comprehensive and physiological small animal model of NASH. We previously reported a dietary (high-fat and high-cholesterol; HFC diet) -induced NASH model that developed advanced fibrosis within a relatively short period (9 weeks) using Sprague-Dawley (SD) rats (age, 9 weeks). <b>Methods:</b> In this study, we evaluated the age-related alterations of NASH in 9-, 18-, and 27-week-old male SD rats that were fed an HFC diet (30% fat, 1.25% cholesterol, and 0.5% sodium cholate, w/w) for 9 weeks (six rats/group). <b>Results:</b> Age-dependent increases in serum transaminases, insulin, and insulin resistance index were observed with or without a significant difference after the 9-week rearing period. Histopathological findings such as hepatic steatosis, lobular inflammation, and hepatocyte ballooning were similar regardless of age, but hepatic fibrosis was more evident in the older groups. Rats in all three groups developed NASH at a high rate (83.3% or higher in each group). The mRNA levels of fibrosis-related genes encoding transforming growth factor-β (TGF-β) and α-smooth muscle actin (α-SMA) in the liver were low in the youngest group and high in the older groups, although this difference was not statistically significant. <b>Conclusion:</b> These results and those from our previous study indicate that a 9-week HFC diet-induced NASH model using SD rats can be applied a relatively wide range of ages (5-27 weeks of old), and that the risk of NASH-related fibrosis increases with age.
Tomohiro Umezu, Koichi Tsuneyama, Kohsuke Kanekura, Michiyo Hayakawa, Toshihito Tanahashi, Mitsuoki Kawano, Y-H Taguchi, Hidenori Toyoda, Akihiro Tamori, Masahiko Kuroda and Yoshiki Murakami : Comprehensive analysis of liver and blood miRNA in precancerous conditions., Scientific Reports, Vol.10, No.1, 2020.
(Summary)
Streptozotocin administration to mice (STZ-mice) induces type I diabetes and hepatocellular carcinoma (HCC). We attempted to elucidate the carcinogenic mechanism and the miRNA expression status in the liver and blood during the precancerous state. Serum and liver tissues were collected from STZ-mice and non-treated mice (CTL-mice) at 6, 10, and 12 W. The exosome enriched fraction extracted from serum was used. Hepatic histological examination and hepatic and exosomal miRNA expression analysis were serially performed using next-generation sequencing (NGS). Human miRNA expression analysis of chronic hepatitis liver tissue and exosomes, which were collected before starting the antiviral treatment, were also performed. No inflammation or fibrosis was found in the liver of CTL-mice during the observation period. In STZ-mice, regeneration and inflammation of hepatocytes was found at 6 W and nodules of atypical hepatocytes were found at 10 and 12 W. In the liver tissue, during 6-12 W, the expression levels of let-7f-5p, miR-143-3p, 148a-3p, 191-5p, 192-5p, 21a-5p, 22-3p, 26a-5p, and 92a-3p was significantly increased in STZ-mice, and anti-oncogenes of their target gene candidates were down-regulated. miR-122-5p was also significantly down-regulated in STZ-mice. Fifteen exosomal miRNAs were upregulated in STZ-mice. Six miRNAs (let-7f-5p, miR-10b-5p, 143-3p, 191-5p, 21a-5p, and 26a-5p) were upregulated, similarly to human HCC cases. From the precancerous state, aberrant expression of hepatic miRNAs has already occurred, and then, it can promote carcinogenesis. In exosomes, the expression pattern of common miRNAs between mice and humans before carcinogenesis was observed and can be expected to be developed as a cancer predictive marker.
Takeo Minamikawa, Mayuko Shimizu, Hiroki Takanari, Yuki Morimoto, Ryosuke Shiomi, Tanioka Hiroki, Eiji Hase, Takeshi Yasui and Koichi Tsuneyama : Molecular imaging analysis of microvesicular and macrovesicular lipid droplets in non-alcoholic fatty liver disease by Raman microscopy., Scientific Reports, Vol.10, No.1, 18548, 2020.
(Summary)
Predominant evidence of non-alcoholic fatty liver disease (NAFLD) is the accumulation of excess lipids in the liver. A small group with NAFLD may have a more serious condition named non-alcoholic steatohepatitis (NASH). However, there is a lack of investigation of the accumulated lipids with spatial and molecular information. Raman microscopy has the potential to characterise molecular species and structures of lipids based on molecular vibration and can achieve high spatial resolution at the organelle level. In this study, we aim to demonstrate the feasibility of Raman microscopy for the investigation of NAFLD based on the molecular features of accumulated lipids. By applying the Raman microscopy to the liver of the NASH model mice, we succeeded in visualising the distribution of lipid droplets (LDs) in hepatocytes. The detailed analysis of Raman spectra revealed the difference of molecular structural features of the LDs, such as the degree of saturation of lipids in the LDs. We also found that the inhomogeneous distribution of cholesterol in the LDs depending on the histology of lipid accumulation. We visualised and characterised the lipids of NASH model mice by Raman microscopy at organelle level. Our findings demonstrated that the Raman imaging analysis was feasible to characterise the NAFLD in terms of the molecular species and structures of lipids.
Takaaki Tsunematsu, Rieko Arakaki, Hidehiko Kawai, Jan Ruppert, Koichi Tsuneyama, Naozumi Ishimaru, C William Earnshaw, Michele Pagano and Yasusei Kudo : is required for the termination of chromosomal passenger complex activity upon mitotic exit., Journal of Cell Science, Vol.133, No.18, 2020.
(Summary)
terminates CPC activity upon mitotic exit and thereby contributes to proper control of DNA replication.
Makoto Kurano, Kazuhisa Tsukamoto, Masumi Hara, Koichi Tsuneyama, Takako Nishikawa, Hitoshi Ikeda and Yutaka Yatomi : Modulation of sphingosine 1-phosphate by hepatobiliary cholesterol handling., The FASEB journal, Vol.34, No.11, 14655-14670, 2020.
(Summary)
S1P, an exogenous S1P analog. Upregulation of apolipoprotein M (apoM) was involved in these modulations, although apoM was not necessary for these modulations. Moreover, the increase in the plasma S1P levels also observed in ABCG5/8-overexpressing mice was dependent on the elevation of the plasma apoM levels. In regard to other sphingolipids and lysophospholipids, ceramides were similarly modulated by NPC1L1 to S1P, while other lipids were differently influenced by NPC1L1 or ABCG5/8 from S1P. Hepatobiliary cholesterol handling might also regulate the functional lipids, such as S1P.
高松 直子, Yusuke Osaki, Hiroki Yamazaki, Kazutaka Kuroda, Hirohisa Ogawa, Hisanori Uehara, Koichi Tsuneyama, Hiroyuki Nodera and Yuishin Izumi : A case of led to diagnosis of malignancy by brachial plexus ultrasonography, Neurosonology, Vol.33, No.2, 36-40, 2020.
(Summary)
A 72-year-old man admitted to our hospital due to severe pain and drop hand in the left arm. MRI and CT of the head and neck at an outside hospital showed no abnormality. Neurological findings revealed distal weakness in the left upper extremity, vague pain between the radial aspect of the left hand and the middle of the digits 1-3, as well as brisk reflexes in the left extremities. Small masses were palpable in the posterior neck, the lower jaw, and the left neck. Clinically, left radial neuropathy and cervical radiculopathy were suspected. We performed ultrasound of the nerve roots, brachial plexus, and the radial nerve. There was a mass compressing the left brachial plexus from the caudolateral direction. Additionally, nerve swelling in the left arm was identified. Skin biopsy over the mass suggested metastatic adenocarcinoma. Chest CT scan showed a mass in the upper right lobe suggestive of a lung cancer. We concluded that the pain was due to radial neuropathy and upper and middle trunk disturbance of the left brachial plexopathy, of which neuromuscular ultrasound was useful in diagnosis.
Chronic intravenous administration of iron was associated with iron deposition in the liver, even when given the minimum recommended dosage. In long-term TPN patients, the iron dose should therefore be carefully considered.
(Keyword)
Aged / Aged, 80 and over / Autopsy / Female / Humans / Infusions, Intravenous / Iron / Liver / Male / Parenteral Nutrition
Mengling Li, Chengai Wu, Sualeh Jibran Muhammad, Dan Yan, Koichi Tsuneyama, Hideki Hatta, Zheng-Guo Cui and Hidekuni Inadera : Melatonin sensitises shikonin-induced cancer cell death mediated by oxidative stress via inhibition of the SIRT3/SOD2-AKT pathway., Redox Biology, Vol.36, 2020.
(Summary)
Recent research suggests that melatonin (Mel), an endogenous hormone and natural supplement, possesses anti-proliferative effects and can sensitise cells to anti-cancer therapies. Although shikonin (SHK) also possesses potential anti-cancer properties, the poor solubility and severe systemic toxicity of this compound hinders its clinical usage. In this study, we combined Mel and SHK, a potentially promising chemotherapeutic drug combination, with the aim of reducing the toxicity of SHK and enhancing the overall anti-cancer effects. We demonstrate for the first time that Mel potentiates the cytotoxic effects of SHK on cancer cells by inducing oxidative stress via inhibition of the SIRT3/SOD2-AKT pathway. Particularly, Mel-SHK treatment induced oxidative stress, increased mitochondrial calcium accumulation and reduced the mitochondrial membrane potential in various cancer cells, leading to apoptosis. This drug combination also promoted endoplasmic reticulum (ER) stress, leading to AKT dephosphorylation. In HeLa cells, Mel-SHK treatment reduced SIRT3/SOD2 expression and SOD2 activity, while SIRT3 overexpression dramatically reduced Mel-SHK-induced oxidative stress, ER stress, mitochondrial dysfunction and apoptosis. Hence, we propose the combination of Mel and SHK as a novel candidate chemotherapeutic regimen that targets the SIRT3/SOD2-AKT pathway in cancer.
Combinations of histological features were found to be helpful for differentiating AP-AIH from DILI, but we were not able to statistically identify an individual feature as definitive.
The roles of cancer-associated fibroblasts (CAFs) have been studied in the tumor progression, and CAFs are expected to become the new targets for cancer pharmacotherapies. CAFs contribute to tumor cell survival and proliferation, tumor angiogenesis, immune suppression, tumor inflammation, tumor cell invasion and metastasis, and extracellular matrix remodeling. However, detailed mechanisms of how CAFs function in the living system remain unclear. CAFs include α-smooth muscle actin, expressing activated fibroblasts similar to myofibroblasts, and are highly capable of producing collagen. Several reports have demonstrated the contributions of extracellular-signal-regulated kinase 5 (ERK5) in fibroblasts to the fibrotic processes; however, the roles of CAF-derived ERK5 remain unclear. To investigate the roles of CAF-derived ERK5 in the tumor progression, we created mice lacking the ERK5 gene specifically in fibroblasts. Colon-26 mouse colon cancer cells were implanted into the mice subcutaneously, and the histological analyses of the tumor tissue were performed after 2 weeks. Immunofluorescence analyses showed that recipient-derived fibroblasts existed within the tumor tissue. The present study demonstrated that fibroblast-specific ERK5 deficiency exacerbated tumor progression and it was accompanied with thicker tumor vessel formation and the increase in the number of activated fibroblasts. We combined the results of The Cancer Genome Atlas (TCGA) database analysis with our animal studies, and indicated that regulating ERK5 activity in CAFs or CAF invasion into the tumor tissue can be important strategies for the development of new targets in cancer pharmacotherapies.
Minoru Matsumoto, Koichi Tsuneyama, Junko Morimoto, Kazuyoshi Hosomichi, Mitsuru Matsumoto and Hitoshi Nishijima : Tissue-specific autoimmunity controlled by Aire in thymic and peripheral tolerance mechanisms., International Immunology, Vol.32, No.2, 117-131, 2020.
(Summary)
Tissue-specific autoimmune diseases are assumed to arise through malfunction of two checkpoints for immune tolerance: defective elimination of autoreactive T cells in the thymus and activation of these T cells by corresponding autoantigens in the periphery. However, evidence for this model and the outcome of such alterations in each or both of the tolerance mechanisms have not been sufficiently investigated. We studied these issues by expressing human AIRE (huAIRE) as a modifier of tolerance function in NOD mice wherein the defects of thymic and peripheral tolerance together cause type I diabetes (T1D). Additive huAIRE expression in the thymic stroma had no major impact on the production of diabetogenic T cells in the thymus. In contrast, huAIRE expression in peripheral antigen-presenting cells (APCs) rendered the mice resistant to T1D, while maintaining other tissue-specific autoimmune responses and antibody production against an exogenous protein antigen, because of the loss of Xcr1+ dendritic cells, an essential component for activating diabetogenic T cells in the periphery. These results contrast with our recent demonstration that huAIRE expression in both the thymic stroma and peripheral APCs resulted in the paradoxical development of muscle-specific autoimmunity. Our results reveal that tissue-specific autoimmunity is differentially controlled by a combination of thymic function and peripheral tolerance, which can be manipulated by expression of huAIRE/Aire in each or both of the tolerance mechanisms.
Kanehiko Suwa, Takashi Yamaguchi, Katsunori Yoshida, Miki Murata, Mayuko Shimizu, Koichi Tsuneyama, Toshihito Seki and Kazuichi Okazaki : Smad Phospho-Isoforms for Hepatocellular Carcinoma Risk Assessment in Patients with Nonalcoholic Steatohepatitis., Cancers, Vol.12, No.2, 286, 2020.
(Summary)
Nonalcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC) sometimes occurs in mildly fibrotic livers, while HCC incidence in NASH-related cirrhosis is lower than and less predictable than in hepatitis C virus (HCV)-related cirrhosis. Transforming growth factor (TGF)-β signaling in hepatocytic nuclei is implicated in fibrosis and carcinogenesis. TGF-βtype I receptor (TβRI) and c-Jun N-terminal kinase (JNK) differentially phosphorylate the mediator Smad3, resulting in 2 distinct phospho-isoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). In mature hepatocytes, oncogenic signaling via the JNK/pSmad3L pathway antagonizes signaling via the tumor-suppressive TβRI/pSmad3C pathway. We immunohistochemically examined domain-specific Smad3 phosphorylation in liver biopsy specimens from 30 NASH patients representing different fibrotic stages and 20 chronically infected hepatitis C patients as controls, correlating Smad3 phosphorylation with clinical course. HCC occurred during follow-up in 11 of 12 NASH patients with abundant pSmad3L and limited pSmad3C but in only 2 of 18 with limited pSmad3L. In contrast, HCC developed in 12 of 15 NASH patients with limited pSmad3C but only 1 of 15 with abundant pSmad3C. Two of fourteen NASH patients with mild fibrosis developed HCC, their hepatocytic nuclei showed abundant pSmad3L and limited pSmad3C. Five of sixteen patients with severe fibrosis did not develop HCC, their hepatocytic nuclei showed limited pSmad3L and abundant pSmad3C. Smad phospho-isoforms may represent important biomarkers predicting HCC in NASH and potential therapeutic targets for preventing NASH-related HCC.
Shingo Oda, Nanaka Miyazaki, Koichi Tsuneyama and Tsuyoshi Yokoi : Exacerbation of gefitinib-induced liver injury by glutathione reduction in mice., The Journal of Toxicological Sciences, Vol.45, No.8, 493-502, 2020.
(Summary)
Gefitinib (GEF) is the first selective tyrosine kinase inhibitor of epidermal growth factor receptor. It is associated with the occurrence of clinical drug-induced liver injury. Although GEF is metabolized to chemically reactive metabolites by cytochrome P450 3A and 1A enzymes and then conjugated to glutathione (GSH), whether these reactive metabolites contribute to GEF-induced toxicity remains unknown. In this study, we investigated whether GSH depletion can sensitize mice to liver injury caused by GEF. Male C57BL/6J mice were intraperitoneally pretreated with L-buthionine (S,R)-sulfoximine (BSO) at 700 mg/kg to inhibit GSH synthesis and then orally administered GEF at 500 mg/kg every 24 hr for 4 consecutive days. The coadministration of BSO and GEF increased plasma alanine aminotransferase (ALT) levels to approximately 700 U/L and 1600 U/L at 72 and 96 hr after the first administration, respectively, whereas the increase in plasma ALT levels in mice receiving GEF at 500 mg/kg alone was limited, suggesting that GSH plays a protective role in GEF-induced liver injury. Histological examination showed nuclear karyorrhexis and sporadic single hepatocyte death in the livers of BSO+GEF coadministered mice. In these mice, the hepatic expression levels of heme oxygenase 1 (Hmox1) and metallothionein 2 (Mt2) mRNA, caspase 3/7 enzymatic activity, and the amounts of 2-thiobarbiuric acid reactive substances were significantly increased, suggesting the presence of oxidative stress, which may be associated with hepatocellular death. Together, these results show that oxidative stress as well as the reactive metabolites of GEF are involved in GEF-induced liver injury in GSH-depleted mice.
(Keyword)
Animals / Buthionine Sulfoximine / Chemical and Drug Induced Liver Injury / Cytochrome P-450 CYP3A / Disease Progression / Gefitinib / Glutathione / Liver / Mice, Inbred C57BL / Oxidative Stress / Protein Kinase Inhibitors
Tsugunobu Andoh, Kazunari Suzuki, Mitsuhiro Konno, Koichi Tsuneyama and Yasushi Kuraishi : Pharmacological Characterization of a Novel Mouse Model of Cholestatic Pruritus., Biological & Pharmaceutical Bulletin, Vol.43, No.7, 1111-1117, 2020.
(Summary)
receptor antagonist) did not affect scratching. These results suggested that partial obstruction of bile secretion in mice induced anti-histamine-resistant itching and that central opioid system is involved in cholestatic itching.
Ayumi Fukuda, Marin Sasao, Eri Asakawa, Sumire Narita, Mei Hisano, Kazuhito Suruga, Mayuko Shimizu, Koichi Tsuneyama, Kazunari Tanaka and Katsuhisa Omagari : Dietary fat, cholesterol, and cholic acid affect the histopathologic severity of nonalcoholic steatohepatitis in Sprague-Dawley rats, Pathology, Research and Practice, Vol.215, No.11, 152599, 2019.
(Summary)
Understanding of the pathogenesis of nonalcoholic steatohepatitis (NASH)-associated fibrosis has been hampered by the lack of a comprehensive and physiological small animal model of NASH with fibrosis. Feeding a high-fat and high-cholesterol (HFC) diet supplemented with cholic acid to rats is known to replicate human NASH pathology, and it induces fibrosis earlier than with an HFC diet alone. In the present study, physiological and histopathological observations from 65 Sprague-Dawley (SD) rats fed an HFC diet with or without cholic acid for 9 or 18 weeks in our laboratory between January 2013 and February 2018 were retrospectively reviewed. The liver weight/body weight ratio at the end of the rearing period was higher in rats fed an HFC diet than in rats fed a normal diet in a cholesterol dose-, cholic acid dose-, or rearing period dependent manner. Dietary fat, cholesterol and/or cholic acid and rearing period affected the histopathologic severity of NASH. Overall, 56 (86.2%) of 65 SD rats fed an HFC diet for 9 or 18 weeks developed histopathologically proven NASH. It is noted that the SD rats fed an HFC diet supplemented with 2% (w/w) cholic acid for 18 weeks frequently developed advanced fibrosis, including cirrhosis. Thus, this diet-induced NASH rat model is likely to be a highly reproducible.
Chihiro Kageyama, Takuro Igawa, Yuka Gion, Noriko Iwaki, Tetsuya Tabata, Takehiro Tanaka, Eisei Kondo, Hajime Sakai, Koichi Tsuneyama, Kazuhiro Nomoto, Hiroko Noguchi, Tadashi Yoshino, Kenji Yokota and Yasuharu Sato : Hepatic Campylobacter jejuni infection in patients with Castleman-Kojima disease (idiopathic multicentric Castleman disease with thrombocytopenia, anasarca, fever, reticulin fibrosis, and organomegaly (TAFRO) syndrome)., Pathology International, Vol.69, No.10, 572-579, 2019.
(Summary)
Castleman-Kojima disease, also known as idiopathic multicentric Castleman disease with TAFRO syndrome (iMCD-TAFRO), is a recently recognized systemic inflammatory disorder with a characteristic series of clinical symptoms, including thrombocytopenia (T), anasarca (A), fever (F), reticulin fibrosis (R), and organomegaly (O). Patients with iMCD-TAFRO often develop severe abdominal pain, elevated alkaline phosphatase levels, and systemic inflammation, but the etiological factors are unknown. To investigate the potential role of bacterial infection in the pathogenesis of iMCD-TAFRO, we performed polymerase chain reaction (PCR) for the bacterial 16S rRNA gene with DNA extracted from liver specimens of three patients with iMCD-TAFRO, four patients with amyotrophic lateral sclerosis, and seven patients with inflammatory conditions. Sequencing of the PCR product showed 99% DNA sequence identity with Campylobacter jejuni in all three patients with iMCD-TAFRO and in two patients with inflammatory conditions. Immunohistochemical and electron microscopy analyses could not identify C. jejuni in patients with iMCD-TAFRO. The findings indicated that C. jejuni infection is not the pathological cause of iMCD-TAFRO; however, this ubiquitous bacterium may play a role in uncontrolled systemic hypercytokinemia, possibly through the development of cross-reactive autoantibodies.
Benedetta Beretta-Piccoli Terziroli, Giorgina Mieli-Vergani, Diego Vergani, M John Vierling, David Adams, Gianfranco Alpini, M Jesus Banales, Ulrich Beuers, Einar Björnsson, Christopher Bowlus, Marco Carbone, Olivier Chazouillères, George Dalekos, Andrea Gottardi De, Kenichi Harada, Gideon Hirschfield, Pietro Invernizzi, David Jones, Edward Krawitt, Antonio Lanzavecchia, Zhe-Xiong Lian, Xiong Ma, Michael Manns, Domenico Mavilio, Mm Eamon Quigley, Federica Sallusto, Shinji Shimoda, Mario Strazzabosco, Mark Swain, Atsushi Tanaka, Michael Trauner, Koichi Tsuneyama, Ehud Zigmond and Eric M Gershwin : The challenges of primary biliary cholangitis: What is new and what needs to be done., Journal of Autoimmunity, Vol.105, 2019.
(Summary)
Primary Biliary Cholangitis (PBC) is an uncommon, chronic, cholangiopathy of autoimmune origin and unknown etiology characterized by positive anti-mitochondrial autoantibodies (AMA), female preponderance and progression to cirrhosis if left untreated. The diagnosis is based on AMA- or PBC-specific anti-nuclear antibody (ANA)-positivity in the presence of a cholestatic biochemical profile, histologic confirmation being mandatory only in seronegative cases. First-line treatment is ursodeoxycholic acid (UDCA), which is effective in preventing disease progression in about two thirds of the patients. The only approved second-line treatment is obeticholic acid. This article summarizes the most relevant conclusions of a meeting held in Lugano, Switzerland, from September 23rd-25th 2018, gathering basic and clinical scientists with various background from around the world to discuss the latest advances in PBC research. The meeting was dedicated to Ian Mackay, pioneer in the field of autoimmune liver diseases. The role of liver histology needs to be reconsidered: liver pathology consistent with PBC in AMA-positive individuals without biochemical cholestasis is increasingly reported, raising the question as to whether biochemical cholestasis is a reliable disease marker for both clinical practice and trials. The urgent need for new biomarkers, including more accurate markers of cholestasis, was also widely discussed during the meeting. Moreover, new insights in interactions of bile acids with biliary epithelia in PBC provide solid evidence of a role for impaired epithelial protection against potentially toxic hydrophobic bile acids, raising the fundamental question as to whether this bile acid-induced epithelial damage is the cause or the consequence of the autoimmune attack to the biliary epithelium. Strategies are needed to identify difficult-to-treat patients at an early disease stage, when new therapeutic approaches targeting immunologic pathways, in addition to bile acid-based therapies, may be effective. In conclusion, using interdisciplinary approaches, groundbreaking advances can be expected before long in respect to our understanding of the etiopathogenesis of PBC, with the ultimate aim of improving its treatment.
Katsuhisa Omagari, Eri Asakawa, Marin Sasao, Sumire Narita, Mei Hisano, Ayumi Fukuda, Kazuhiro Suruga, Mayuko Shimizu and Koichi Tsuneyama : Age-related alterations of nonalcoholic steatohepatitis in Sprague-Dawley rats fed a high-fat and high-cholesterol diet., Journal of Nutritional Science and Vitaminology, Vol.65, No.4, 349-356, 2019.
(Summary)
Nonalcoholic steatohepatitis (NASH), a subtype of nonalcoholic fatty liver disease (NAFLD), has a potentially progressive course that can lead to liver cirrhosis. Age is strongly associated with the development and progression of NAFLD/NASH, but the natural history of pediatric NAFLD is still not fully understood. Here, we evaluated the age-related alterations of NASH in 5-, 9- and 13-wk-old male Sprague-Dawley rats that were fed a high-fat and high-cholesterol diet (30% fat, 1.25% cholesterol and 0.5% sodium cholate, w/w) for 9 wk (6 rats/group). Our results showed that the cumulative energy intake, body weight gain and food efficacy during the 9-wk rearing period were highest in the youngest group and lowest in the oldest group. Serologically, almost all parameters including the serum triglyceride and total cholesterol were similar regardless of age. Histopathological findings, such as hepatic steatosis, lobular inflammation and hepatocyte ballooning, were also similar regardless of age, but hepatic fibrosis was more evident in the oldest group. Also, the mRNA expression levels of some fibrogenic, inflammatory, oxidative stress and cholesterol or lipid metabolism-related genes in the liver were highest in the oldest group and lowest in the youngest group, although the difference was not statistically significant. These results indicated that aging is likely associated with the development of NASH. Because the cumulative energy intake and daily food intake/body weight were not similar among groups in the present study, further studies designed with an equivalent daily food intake/body weight among groups are needed in order to interpret the exact nutritional effect.
Ru Jia, Shingo Oda, Koichi Tsuneyama, Yuya Urano and Tsuyoshi Yokoi : Establishment of a mouse model of troglitazone-induced liver injury and analysis of its hepatotoxic mechanism., Journal of Applied Toxicology : JAT, 2019.
(Summary)
Drug-induced liver injury is a major problem in drug development and clinical drug therapy. Troglitazone (TGZ), a thiazolidinedione antidiabetic drug for the treatment of type II diabetes mellitus, was found to induce rare idiosyncratic severe liver injury in patients, which led to its withdrawal in 2000. However, in normal experimental animals in vivo TGZ has never induced liver injury. To explore TGZ hepatotoxic mechanism, we established a novel mouse model of TGZ-induced liver injury. Administration of BALB/c female mice with a single intraperitoneal TGZ dose (300 mg/kg) significantly elevated alanine aminotransferase and aspartate aminotransferase levels 6 hours after the treatment. The ratio of oxidative stress marker glutathione/disulfide glutathione was significantly decreased. The increased hepatic mRNA levels of inflammation- and oxidative stress-related factors were observed in TGZ-treated mice. Subsequently, hepatic transcriptome profiles of TGZ-exposed liver were compared with those of non-hepatotoxic rosiglitazone. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway was activated in TGZ-induced liver injury. The activation of the JAK/STAT pathway promoted phosphorylation of STAT3 in TGZ-treated mice. Consequently, upregulation of STAT3 activation increased mRNA levels of its downstream genes. In conclusion, a single intraperitoneal dose of TGZ exposure could induce liver injury in BALB/c female mice and, by a hepatic transcriptomic analysis, we found that the activation of JAK/STAT pathway might be related to TGZ-induced hepatotoxicity.
Fuhua Yang, Taiki Takeuchi, Koichi Tsuneyama, Tsuyoshi Yokoi and Shingo Oda : Experimental Evidence of Liver Injury by BSEP-Inhibiting Drugs With a Bile Salt Supplementation in Rats., Toxicological Sciences, Vol.170, No.1, 95-108, 2019.
(Summary)
The bile salt export pump (BSEP, ABCB11) mediates bile acid efflux from hepatocytes into bile. Although the inhibition of BSEP has been implicated as an important mechanism of drug-induced liver injury (DILI), liver injury caused by BSEP-inhibiting drugs is rarely reproduced in experimental animals, probably due to species differences in bile acid composition between humans and rodents. In this study, we tested whether supplementation with chenodeoxycholic acid (CDCA) sodium, a hydrophobic bile salt, could sensitize rats to liver injury caused by a BSEP-inhibiting drug. A potent BSEP inhibitor, ketoconazole (KTZ), which is associated with clinical DILI, was intragastrically administered simultaneously with CDCA at a nontoxic dose once a day for 3 days. Plasma transaminase levels significantly increased in rats receiving CDCA+KTZ, whereas neither treatment with CDCA alone, KTZ alone nor a combination of CDCA and miconazole, a safe analog to KTZ, induced liver injury. In CDCA+KTZ-treated rats, most bile acid species in the liver significantly increased compared with treatment with vehicle or CDCA alone, suggesting that KTZ administration inhibited bile acid excretion. Furthermore, hepatic mRNA expression levels of a bile acid synthesis enzyme, Cyp7a1, and a basolateral bile salt influx transporter, Ntcp, decreased, whereas a canalicular phosphatidylcholine flippase, Mdr2, increased in the CDCA+KTZ group to compensate for hepatic bile acid accumulation. In conclusion, we found that oral CDCA supplementation predisposed rats to KTZ-induced liver injury due to the hepatic accumulation of bile acids. This method may be useful for assessing the potential of BSEP-inhibiting drugs inducing liver injury in vivo.
Ayaka Iida, Sachi Kuranuki, Ryoko Yamamoto, Masaya Uchida, Masanori Ohta, Mayuko Shimizu, Koichi Tsuneyama, Takayuki Masaki, Masataka Seike and Tsuyoshi Nakamura : Analysis of amino acid profiles of blood over time and biomarkers associated with non-alcoholic steatohepatitis in STAM mice., Experimental Animals, Vol.68, No.4, 417-428, 2019.
(Summary)
The changes in free amino acid (AA) levels in blood during the progression from non-alcoholic steatohepatitis (NASH) to hepatocellular carcinoma (HCC) are unclear. We investigated serum AA levels, along with biochemical and histological events, in a mouse model of NASH. We induced NASH in male C57BL/6J mice with a streptozotocin injection and high-fat diet after 4 weeks of age (STAM group). We chronologically (6, 8, 10, 12, and 16 weeks, n=4-12 mice/group) evaluated the progression from steatohepatitis to HCC by biochemical and histological analyses. The serum AA levels were determined using an AA analyzer. Serum aspartate aminotransferase and alanine aminotransferase levels were higher in the STAM group than in the normal group (non-NASH-induced mice). Histological analysis revealed that STAM mice had fatty liver, NASH, and fibrosis at 6, 8, and 10 weeks, respectively. Moreover, the mice exhibited fibrosis and HCC at 16 weeks. The serum branched-chain AA levels were higher in the STAM group than in the normal group, especially at 8 and 10 weeks. The Fischer ratio decreased at 16 weeks in the STAM group, with increasing aromatic AA levels. These results suggested that this model sequentially depicts the development of fatty liver, NASH, cirrhosis, HCC, and AA metabolism disorders within a short experimental period. Additionally, serum amyloid A was suggested to be a useful inflammation biomarker associated with NASH. We believe that the STAM model will be useful for studying AA metabolism and/or pharmacological effects in NASH.
Makoto Kurano, Koichi Tsuneyama, Yuki Morimoto, Masako Nishikawa and Yutaka Yatomi : Apolipoprotein M suppresses the phenotypes of IgA nephropathy in hyper-IgA mice., The FASEB journal, Vol.33, No.4, 5181-5195, 2019.
(Summary)
Because the association between sphingosine 1-phosphate (S1P)/apolipoprotein M (ApoM) and chronic kidney diseases has not been established, we investigated the involvement of S1P/ApoM in the phenotypes of IgA nephropathy in hyper-IgA (HIGA) mice. The overexpression of ApoM in adenoviral gene transfer ameliorated the phenotypes of IgA nephropathy in HIGA mice, whereas the knockdown of ApoM with siRNA caused deterioration. When ApoM-overexpressing HIGA mice were treated with VPC23019, an antagonist against S1P receptor 1 (S1P1) and 3 (S1P3), we observed that the protective effects of ApoM were reversed, whereas JTE013, an antagonist against S1P2, did not inhibit the effects. We also found that S1P bound to albumin accelerated the proliferation of MES13 cells and the fibrotic changes of HK2 cells, which were inhibited by JTE013, whereas S1P bound to ApoM suppressed these changes, which were inhibited by VPC23019. These results suggest that S1P bound to ApoM possesses properties protective against the phenotypes of IgA nephropathy through S1P1 and S1P3, whereas S1P bound to albumin exerts deteriorating effects through S1P2. ApoM may be useful as a therapeutic target to treat or retard the progression of IgA nephropathy.-Kurano, M., Tsuneyama, K., Morimoto, Y., Nishikawa, M., Yatomi, Y. Apolipoprotein M suppresses the phenotypes of IgA nephropathy in hyper-IgA mice.
Yuki Izawa-Ishizawa, Masaki Imanishi, Yoshito Zamami, Toya Hiroki, Nagao Tomoko, Marin Morishita, Koichi Tsuneyama, Yuya Horinouchi, Yoshitaka Kihira, Kenshi Takechi, Yasumasa Ikeda, Koichiro Tsuchiya, Masanori Yoshizumi, Toshiaki Tamaki and Keisuke Ishizawa : Development of a novel aortic dissection mouse model and evaluation of drug efficacy using in-vivo assays and database analyses., Journal of Hypertension, Vol.37, No.1, 73-83, 2019.
(Summary)
Aortic dissection is a life-threatening disease. At present, the only therapeutic strategies available are surgery and antihypertensive drugs. Moreover, the molecular mechanisms underlying the onset of aortic dissection are still unclear. We established a novel aortic dissection model in mice using pharmacologically induced endothelial dysfunction. We then used the Japanese Adverse Drug Event Report database to investigate the role of pitavastatin in preventing the onset of aortic dissection. To induce endothelial dysfunction, Nω-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, was administered to C57BL/6 mice. Three weeks later, angiotensin II (Ang II) and β-aminopropionitrile (BAPN), a lysyl oxidase inhibitor, were administered with osmotic mini-pumps. False lumen formation was used as the pathological determinant of aortic dissection. The incidences of aortic dissection and death from aneurysmal rupture were significantly higher in the Nω-nitro-L-arginine methyl ester, Ang II, and BAPN (LAB) group than they were in the Ang II and BAPN (AB) group.Pitavastatin was administered orally to LAB mice. It significantly lowered the incidences of dissection and rupture. It also decreased inflammation and medial degradation, both of which were exacerbated in the LAB group. The Japanese Adverse Drug Event Report database analysis indicated that there were 113 cases of aortic dissection out of 95 090 patients (0.12%) not receiving statins but only six cases out of 16 668 patients receiving statins (0.04%) (odds ratio: 0.30; P = 0.0043). Our results suggest that endothelial dysfunction is associated with the onset of aortic dissection and pitavastatin can help prevent this condition.
Junko Morimoto, Yumiko Nishikawa, Takumi Kakimoto, Kohei Furutani, Naoki Kihara, Minoru Matsumoto, Koichi Tsuneyama, Yuko Kozono, Haruo Kozono, Katsuto Hozumi, Kazuyoshi Hosomichi, Hitoshi Nishijima and Mitsuru Matsumoto : Aire Controls in Trans the Production of Medullary Thymic Epithelial Cells Expressing Ly-6C/Ly-6G., The Journal of Immunology, Vol.201, No.11, 3244-3257, 2018.
(Summary)
Medullary thymic epithelial cells (mTECs), which express a wide range of tissue-restricted Ags (TRAs), contribute to the establishment of self-tolerance by eliminating autoreactive T cells and/or inducing regulatory T cells. Aire controls a diverse set of TRAs within Aire-expressing cells by employing various transcriptional pathways. As Aire has a profound effect on transcriptomes of mTECs, including TRAs not only at the single-cell but also the population level, we suspected that Aire (Aire+ mTECs) might control the cellular composition of the thymic microenvironment. In this study, we confirmed that this is indeed the case by identifying a novel mTEC subset expressing Ly-6 family protein whose production was defective in Aire-deficient thymi. Reaggregated thymic organ culture experiments demonstrated that Aire did not induce the expression of Ly-6C/Ly-6G molecules from mTECs as Aire-dependent TRAs in a cell-intrinsic manner. Instead, Aire+ mTECs functioned in trans to maintain Ly-6C/Ly-6G+ mTECs. Thus, Aire not only controls TRA expression transcriptionally within the cell but also controls the overall composition of mTECs in a cell-extrinsic manner, thereby regulating the transcriptome from mTECs on a global scale.
Yuki Moritoki, Koichi Tsuneyama, Yuka Nakamura, Kentaro Kikuchi, Akira Shiota, Yoshiyuki Ohsugi, Zhe-Xiong Lian, Weici Zhang, Guo-Xiang Yang, Shigeharu Ueki, Masahide Takeda, Ayumi Omokawa, Tomoo Saga, Akiko Saga, Daisuke Watanabe, Masahito Miura, Yoshiyuki Ueno, C Patrick S Leung, Atsushi Tanaka, Eric M Gershwin and Makoto Hirokawa : Anti-drug Antibodies Against a Novel Humanized Anti-CD20 Antibody Impair Its Therapeutic Effect on Primary Biliary Cholangitis in Human CD20- and FcγR-Expressing Mice., Frontiers in Immunology, Vol.9, 2018.
(Summary)
= 0.0054) were apparent. Accompanying these changes was a dramatic reduction in anti-mitochondrial antibodies (AMAs), interleukin (IL)-12p40 and IL-5, and elevated levels of the anti-inflammatory chemokine CXCL1/KC. In mice that developed ADAs, clinical improvements were less pronounced. Sustained treatment with B cell-targeted therapies may broadly inhibit effector pathways in PBC, but may need to be administered early in the natural history of PBC.
Kazuchika Nishitsuji, Syunsuke Watanabe, Jinzhong Xiao, Ryosuke Nagatomo, Hirohisa Ogawa, Takaaki Tsunematsu, Hitomi Umemoto, Yuki Morimoto, Hiroyasu Akatsu, Koichi Inoue and Koichi Tsuneyama : Effect of coffee or coffee components on gut microbiome and short-chain fatty acids in a mouse model of metabolic syndrome., Scientific Reports, Vol.8, No.1, 2018.
(Summary)
We previously showed that male Tsumura Suzuki obese diabetes (TSOD) mice, a spontaneous mouse model of metabolic syndrome, manifested gut dysbiosis and subsequent disruption of the type and quantity of plasma short-chain fatty acids (SCFAs), and daily coffee intake prevented nonalcoholic steatohepatitis in this mouse model. Here, we present a preliminary study on whether coffee and its major components, caffeine and chlorogenic acid, would affect the gut dysbiosis and the disrupted plasma SCFA profile of TSOD mice, which could lead to improvement in the liver pathology of these mice. Three mice per group were used. Daily intake of coffee or its components for 16 wk prevented liver lobular inflammation without improving obesity in TSOD mice. Coffee and its components did not repair the altered levels of Gram-positive and Gram-negative bacteria and an increased abundance of Firmicutes in TSOD mice but rather caused additional changes in bacteria in six genera. However, caffeine and chlorogenic acid partially improved the disrupted plasma SCFA profile in TSOD mice, although coffee had no effects. Whether these alterations in the gut microbiome and the plasma SCFA profile might affect the liver pathology of TSOD mice may deserve further investigation.
Hong-Di Ma, Zhi-Bin Zhao, Wen-Tao Ma, Qing-Zhi Liu, Cai-Yue Gao, Liang Li, Jinjun Wang, Koichi Tsuneyama, Bin Liu, Weici Zhang, Yongjian Zhou, Eric M Gershwin and Zhe-Xiong Lian : Gut microbiota translocation promotes autoimmune cholangitis., Journal of Autoimmunity, Vol.95, 47-57, 2018.
(Summary)
mice to investigate the influences of gut microbiota on autoimmune cholangitis. Firstly, we report that dnTGFβRII mice manifest altered composition of gut microbiota and that alteration of this gut microbiota by administration of antibiotics significantly alleviates T-cell-mediated infiltration and bile duct damage. Second, toll-like receptor 2 (TLR2)-deficient dnTGFβRII mice demonstrate significant exacerbation of autoimmune cholangitis when their epithelial barrier integrity was disrupted. Further, TLR2-deficiency mediates downregulated expression of tight junction-associated protein ZO-1 leading to increased gut permeability and bacterial translocation from gut to liver; use of antibiotics reduces microbiota translocation to liver and also decreases biliary pathology. In conclusion, our data demonstrates the important role of gut microbiota and bacterial translocation in the pathogenesis of murine autoimmune cholangitis.
Hirohisa Ogawa, Masahiko Azuma, Takaaki Tsunematsu, Yuuki Morimoto, Mayo Kondo, Toshifumi Tezuka, Yasuhiko Nishioka and Koichi Tsuneyama : Neutrophils induce smooth muscle hyperplasia via neutrophil elastase-induced FGF-2 in a mouse model of asthma with mixed inflammation., Clinical and Experimental Allergy, Vol.48, No.12, 1715-1725, 2018.
(Summary)
The IL-17/neutrophil axis may play an important role in airway remodelling by contributing to smooth muscle hypertrophy/hyperplasia in mixed allergic inflammation and accordingly represents an attractive therapeutic target for severe asthma.
Yuya Urano, Shingo Oda, Koichi Tsuneyama and Tsuyoshi Yokoi : Comparative hepatic transcriptome analyses revealed possible pathogenic mechanisms of fasiglifam (TAK-875)-induced acute liver injury in mice., Chemico-Biological Interactions, Vol.296, 185-197, 2018.
(Summary)
Fasiglifam (TAK-875), a G protein-coupled receptor 40 (GPR40) agonist, was a drug candidate for type 2 diabetes. However, its development was terminated in phase 3 trials due to liver safety concerns. Although TAK-875 was reported to inhibit hepatobiliary transporters and disturb bile acid disposition, pathogenic mechanisms of TAK-875-induced liver injury are not fully understood. In this study, we sought to identify the mechanisms with a hepatic genome-wide transcriptomic analysis in a murine model. We demonstrated that, among the three GPR40 agonists, TAK-875, AMG-837, and TUG-770, only TAK-875 induced acute liver injury in mice. Transcriptome profiles of TAK-875-exposed liver was compared with those of non-hepatotoxic analogues AMG-837 and TUG-770 as negative controls and those of classical hepatotoxicants concanavalin A and carbon tetrachloride as positive controls. The comparative hepatic transcriptome analyses revealed the enrichment of genes involved in inflammation, endoplasmic reticulum (ER) stress, apoptosis, and hepatic lipid accumulation, suggesting that these events play pathophysiologic roles in the development of TAK-875-induced liver injury. These results were validated by quantitative PCR with significant changes in chemokines, danger signals, ER stress mediators, proapoptotic factors, and hepatic steatosis markers only in TAK-875-exposed liver. Pretreatment of TAK-875-administered mice with an ER stress inhibitor 4-phenylbutyric acid (4-PBA) alleviated the liver injury. Consistent with the in vivo study, pretreatment of HepG2 cells with 4-PBA significantly improved the decrease of cell viability induced by TAK-875. In conclusion, by a comprehensive transcriptomic analysis, we found multiple possible processes that contribute to TAK-875-induced acute liver injury in mice.
(Keyword)
Animals / Benzofurans / Carbon Tetrachloride / Chemical and Drug Induced Liver Injury / Concanavalin A / Female / Liver / Liver Failure, Acute / Mice / Mice, Inbred ICR / Molecular Structure / Sulfones / Transcriptome
Mayo Kondo, Hirokazu Ogino, Hirohisa Ogawa, Tania Afroj, Yuko Toyoda, Satoshi Sakaguchi, Miki Tsuboi, Yoshimi Bando, Hisatsugu Goto, Koichi Tsuneyama and Yasuhiko Nishioka : A case of pulmonary pleomorphic carcinoma with malignant phenotypes induced by ZEB1-associated epithelial-mesenchymal transition., Respiratory Medicine Case Reports, Vol.25, 119-121, 2018.
(Summary)
A 60-year-old man was admitted to our hospital with non-small cell lung cancer (NSCLC). Imaging and pathological studies revealed NSCLC, not otherwise specified (NOS), at clinical stage T3N1M0 stage IIIA. We started radiotherapy alone because of obstructive pneumonia and end-stage renal disease, but the tumors progressed rapidly and resulted in death due to air obstruction by pharyngeal metastasis. The cancer was diagnosed as pleomorphic carcinoma in an autopsy. Viable lung tumor cells, which were resistant to radiotherapy, and the pharyngeal metastasis had mesenchymal phenotypes and expressed ZEB1 but not SNAI1. These observations indicated that ZEB1-associated epithelial-mesenchymal transition has malignant features including resistance to radiotherapy and aggressive metastatic potential. ZEB1-associated EMT may be an important mechanism to understand the pathophysiology of pleomorphic carcinoma.
Kaizo Kagemoto, Koichi Okamoto, Tohshi Takaoka, Yasushi Sato, Shinji Kitamura, Tetsuo Kimura, Masahiro Sogabe, Hiroshi Miyamoto, Naoki Muguruma, Koichi Tsuneyama and Tetsuji Takayama : Detection of aberrant crypt foci with image-enhanced endoscopy., Endoscopy International Open, Vol.6, No.8, E924-E933, 2018.
(Summary)
Conventional detection of aberrant crypt foci (ACF) with dye-spraying and magnifying observation is labor- and skill-intensive. We performed a prospective non-inferiority study to investigate the utility of image-enhanced endoscopy (IEE) for detection of ACF. Patients with a history of colorectal neoplasm were eligible. The number of ACF in the lower rectum was counted first using IEE magnification with narrow-band imaging (NBI) or blue-laser imaging (BLI), and subsequently using the methylene blue method. The primary endpoint was the ACF detection rate with IEE, i. e., the number of ACF detected with IEE relative to the number of ACF detected with methylene blue. The secondary endpoints were bowel preparation time, ACF detection time, and the detection rate with NBI or BLI. A total of 40 patients were enrolled (NBI 20 and BLI 20). The overall detection rate for ACF with IEE was 81.7 % (503/616; 95 %CI 78.8 - 84.6 %), meeting the primary endpoint. The detection rate for ACF with BLI (84.9 %, 258/304) was significantly higher than with NBI (78.5 %, 245/312; < 0.05). Both bowel preparation time and ACF detection time were significantly shorter with IEE versus the methylene blue method ( < 0.01, respectively). The detection rates for dysplastic and non-dysplastic ACF with IEE were 84.4 % (27/32) and 80.3 % (469/584), respectively. IEE is able to detect ACF during colonoscopy with sensitivity non-inferior to that of the conventional methylene blue method. IEE is simpler than the methylene blue method and is therefore a potentially useful new tool for ACF detection.
Cai-Yue Gao, Yuan Yao, Liang Li, Shu-Han Yang, Hui Chu, Koichi Tsuneyama, Xiao-Mei Li, M Gershwin Eric and Zhe-Xiong Lian : Tissue resident memory CD8 T cells mediate salivary gland damage in a murine model of Sjögren's syndrome., Arthritis & Rheumatology, 2018.
(Summary)
Although a role for CD4 T cells in the pathology of Sjögren's syndrome (SS) has been documented, the pathogenic significance of CD8 T cells is unclear. The aim of this study is to investigate the role of CD8 T cells in the development of SS. Flow cytometry and immunofluorescence were utilized to detect T cell infiltration within the labial glands of patients with primary SS. In parallel p40 CD25 mice were used as a murine model of SS. In addition CD4, CD8a and IFN-γ KO mice were crossed with p40 CD25 mice respectively to study the pathogenic significance of specific lineage subpopulations including functional salivary, histopathological and serological data. CD8 T cell specific depletion antibody was used to evaluate this potential therapeutic strategy. CD8 T cells with tissue resident memory phenotype outnumbered CD4 T cells in the labial gland of SS patients and were primarily co-localized with saliva duct epithelial cells and acinar cells. Furthermore, infiltrated CD8 T cells with resident phenotype CD69 CD103 with a significant elevation of IFN-γ production were dominant in the submandibular gland of this SS murine model. CD8a knockout abrogated the development of SS in these mice. Knockout of IFN-γ decreased CD8 T cell infiltration and gland destruction. More importantly, depletion of CD8 T cells fully protected mice from SS even after the onset of disease. Our data reveal pathogenic significance of CD8 T cells in the salivary gland pathology in SS. Treatment directed against CD8 T cells may be a rational therapy for human SS. This article is protected by copyright. All rights reserved.
Masaki Takeuchi, Shingo Oda, Koichi Tsuneyama and Tsuyoshi Yokoi : Comprehensive analysis of serum microRNAs in hepatic sinusoidal obstruction syndrome (SOS) in rats: implication as early phase biomarkers for SOS., Archives of Toxicology, 2018.
(Summary)
Sinusoidal obstruction syndrome (SOS) is a liver injury caused by clinical chemotherapy, of which pathogenesis is associated with the damage in liver sinusoidal endothelial cells (LSEC). The unavailability of appropriate specific biomarkers for the early diagnosis of SOS may potentially overlook SOS patients. In this study, we sought to find serum microRNAs (miRNAs) as non-invasive biomarkers for investigating SOS in rats. Male Sprague-Dawley rats were orally administered monocrotaline, and then, their livers and sera were collected after 0.25, 0.5, 1, 2, 4, and 7 days. The rats showed a typical SOS phenotype including LSEC damage as early as day 0.25, followed by severe hepatocyte damage on day 2, and developed hepatic fibrosis from days 4 to 7. The miRNA microarray showed that 65 serum miRNAs were increased in their levels on day 0.25, when LSEC damage was observed, while hepatocyte damage was absent. Among the increased serum miRNAs on days 0.25-1, miR-511-3p was enriched in normal LSECs and miR-21-5p was in both LSECs and hepatocytes, suggesting that they were released into blood from the damaged LSECs. The miR-122-5p, miR-192-5p, and miR-101b-3p, which were enriched in hepatocytes, reached the highest levels in serum on day 2, suggesting their utility as indicators for hepatocyte damage. No miRNA showing an increasing trend from days 4 to 7 was found as a biomarker for fibrosis. In conclusion, we found that LSEC-derived miR-21-5p and especially miR-511-3p in serum would serve as early phase biomarkers for SOS in response to LSEC damage.
Makoto Kurano, Koichi Tsuneyama, Yuki Morimoto, Tomo Shimizu, Masahiro Jona, Hidetoshi Kassai, Kazuki Nakao, Atsu Aiba and Yutaka Yatomi : Apolipoprotein M Protects Lipopolysaccharide-Treated Mice from Death and Organ Injury., Thrombosis and Haemostasis, 2018.
(Summary)
These results suggest that apoM possesses protective properties against LPS-induced organ injuries and could potentially be introduced as a novel therapy for the severe conditions that are consequent to sepsis.
Akiko Matsubara, Shingo Oda, Sho Akai, Koichi Tsuneyama and Tsuyoshi Yokoi : Establishment of a drug-induced rhabdomyolysis mouse model by co-administration of ciprofloxacin and atorvastatin., Toxicology Letters, Vol.291, 184-193, 2018.
(Summary)
Rhabdomyolysis is one of the serious side effects of ciprofloxacin (CPFX), a widely used antibacterial drug; and occasionally, acute kidney injury (AKI) occurs. Often, rhabdomyolysis has occurred in patients taking CPFX co-administered with statins. The purpose of this study is to establish a mouse model of drug-induced rhabdomyolysis by co-administration of CPFX and atorvastatin (ATV) and to clarify the mechanisms of its pathogenesis. C57BL/6J mice treated with L-buthionine-(S,R)-sulfoximine (BSO), a glutathione synthesis inhibitor, were orally administered with CPFX and ATV for 4 days. Plasma levels of creatinine phosphokinase (CPK) and aspartate aminotransferase (AST) were significantly increased in the CPFX and ATV-co-administered group. Histopathological examination of skeletal muscle observed degeneration in gastrocnemius muscle and an increased number of the satellite cells. Expressions of skeletal muscle-specific microRNA and mRNA in plasma and skeletal muscle, respectively, were significantly increased. The area under the curve (AUC) of plasma CPFX was significantly increased in the CPFX and ATV-co-administered group. Furthermore, cytoplasmic vacuolization and a positively myoglobin-stained region in kidney tissue and high content of myoglobin in urine were observed. These results indicated that AKI was induced by myoglobin that leaked from skeletal muscle. The established mouse model in the present study would be useful for predicting potential rhabdomyolysis risks in preclinical drug development.
Shota Fujimoto, Naoki Muguruma, Koichi Okamoto, Takeshi Kurihara, Yasushi Sato, Yoshihiko Miyamoto, Shinji Kitamura, Hiroshi Miyamoto, Takahiro Taguchi, Koichi Tsuneyama and Tetsuji Takayama : A Novel Theranostic Combination of Near-infrared Fluorescence Imaging and Laser Irradiation Targeting c-KIT for Gastrointestinal Stromal Tumors., Theranostics, Vol.8, No.9, 2313-2328, 2018.
(Summary)
It is difficult to distinguish gastrointestinal stromal tumors (GISTs) from other types of submucosal tumors under conventional gastrointestinal endoscopy. We aimed to detect GISTs by molecular fluorescence imaging using a near-infrared (NIR) photosensitizer (IR700)-conjugated anti-c-KIT antibody and to treat GISTs by photoimmunotherapy with NIR irradiation as a non-invasive theranostic procedure. We also investigated the therapeutic mechanisms. Human GIST cell lines GIST-T1 and GIST-882M were incubated with IR700-conjugated anti-c-KIT antibody, IR700-12A8, and observed by confocal laser microscopy. Mice with GIST-T1 xenografts or rats with orthotopic xenografts were injected with IR700-12A8 or AF488-conjugated antibody, and observed under IVIS or autofluorescence imaging (AFI) endoscopy. GIST cells were treated with IR700-12A8 and NIR light and vivo, and cell viability, histology and apoptosis were evaluated. Strong red fluorescence of IR700-12A8 was observed on the cell membrane of GIST cells and was gradually internalized into the cytoplasm. Tumor-specific accumulation of IR700-12A8 was observed in GIST-T1 xenografts in mice. Under AFI endoscopy, a strong fluorescence signal was observed in orthotopic GIST xenografts in rats through the normal mucosa covering the tumor. The percentage of dead cells significantly increased in a light-dose-dependent manner and both acute necrotic and late apoptotic cell death was observed with annexin/PI staining. Cleaved PARP expression was significantly increased after IR700-12A8-mediated NIR irradiation, which was almost completely reversed by NaN. All xenograft tumors (7/7) immediately regressed and 4/7 tumors completely disappeared after IR700-12A8-mediated NIR irradiation. Histologic assessment and TUNEL staining revealed apoptosis in the tumors. NIR fluorescence imaging using IR700-12A8 and subsequent NIR irradiation could be a very effective theranostic technology for GIST, the underlying mechanism of which appears to involve acute necrosis and supposedly late apoptosis induced by singlet oxygen.
Momoko Akashi, Kazuto Tajiri, Akinori Wada, Koichi Tsuneyama, Kengo Kawai, Satoshi Yasumura, Masami Minemura, Terumi Takahara and Toshiro Sugiyama : A Patient with Non-alcoholic Steatohepatitis Complicated by Multiple Myeloma., Internal Medicine, 2018.
(Summary)
A 68-year-old woman with liver dysfunction was diagnosed with nonalcoholic steatohepatitis (NASH) stage 1. Three years later, she showed massive ascites and jaundice. A trans-jugular liver biopsy confirmed advanced cirrhosis, suggesting that her liver fibrosis had progressed rapidly. At the same time, she was diagnosed with multiple myeloma (MM). In this case, the plasma levels of osteopontin (OPN), a proinflammatory cytokine that promotes liver fibrosis progression through the hedgehog pathway and is increased in patients with MM, were increased. This increased OPN expression was accompanied by the upregulation of the hedgehog pathway in this patient, suggesting that the MM-associated increase in OPN had promoted the progression of liver fibrosis through the hedgehog pathway. The progression of liver fibrosis should be monitored in patients with NASH if other diseases, such as MM, are present.
Satoru Joshita, Kaname Yoshizawa, Takeji Umemura, Hiromasa Ohira, Atsushi Takahashi, Kenichi Harada, Canh Nguyen Hiep, Koichi Tsuneyama, Masayoshi Kage, Masayuki Nakano, Jong-Hon Kang, Kazuhiko Koike, Mikio Zeniya, Tetsuya Yasunaka, Akinobu Takaki, Takuji Torimura, Masanori Abe, Osamu Yokosuka, Atsushi Tanaka and Hajime Takikawa : Clinical features of autoimmune hepatitis with acute presentation: a Japanese nationwide survey., Journal of Gastroenterology, 2018.
(Summary)
AIH with acute presentation is a newly recognized disease entity for which diagnostic hallmarks, such as ALT, fibrosis, and ANA, are needed. Further investigation is also required on the mechanisms of this disorder. Clinicians should be mindful of disease relapse during patient care.
Shingo Oda, Masaki Takeuchi, Sho Akai, Yuji Shirai, Koichi Tsuneyama and Tsuyoshi Yokoi : miRNA in Rat Liver Sinusoidal Endothelial Cells and Hepatocytes and Application to Circulating Biomarkers that Discern Pathogenesis of Liver Injuries., The American Journal of Pathology, Vol.188, No.4, 916-928, 2018.
(Summary)
Sinusoidal obstruction syndrome is a serious liver injury caused by toxic injury to liver sinusoidal endothelial cells (LSECs) during clinical chemotherapy. Although circulating miRNAs, such as hepatocyte-specific miR-122-5p and miR-192-5p, have been proposed as potential noninvasive biomarkers of hepatocellular liver injury, these miRNAs may not be specific to damage to other hepatic cell types, including LSECs. We characterized miRNA expression in LSECs and hepatocytes and investigated whether cell type-specific miRNAs in plasma can discern pathogenesis of liver injuries in rats. Comprehensive miRNA expression analyses found that 66 and 12 miRNAs were highly expressed in LSECs and hepatocytes isolated from nontreated rats, respectively. An LSEC-enriched miR-511-3p was relatively liver specific according to public data. For establishing LSEC and hepatocyte injury models, rats were orally treated with monocrotaline and thioacetamide, respectively. In monocrotaline-treated rats, a sinusoidal obstruction syndrome model, LSEC damage was observed 6 hours after dosing, whereas hepatocellular damage was observed after 48 hours. Interestingly, the level of miR-511-3p in plasma was increased as early as 6 hours after monocrotaline dosing, followed by an increase of miR-122-5p after 24 hours. In the thioacetamide-induced hepatocellular injury model, the level of miR-511-3p was not altered in plasma, whereas miR-122-5p levels were increased after 6 hours. In conclusion, we identified miR-511-3p in plasma as a possible biomarker for LSEC damage.
Hitoshi Nishijima, Tatsuya Kajimoto, Yoshiki Matsuoka, Yasuhiro Mouri, Junko Morimoto, Minoru Matsumoto, Hiroshi Kawano, Yasuhiko Nishioka, Hisanori Uehara, Keisuke Izumi, Koichi Tsuneyama, Il-mi Okazaki, Taku Okazaki, Kazuyoshi Hosomichi, Ayako Shiraki, Makoto Shibutani, Kunitoshi Mitsumori and Mitsuru Matsumoto : Paradoxical development of polymyositis-like autoimmunity through augmented expression of autoimmune regulator (AIRE)., Journal of Autoimmunity, Vol.86, 75-92, 2018.
(Summary)
Autoimmunity is prevented by the function of the autoimmune regulator [AIRE (Aire in mice)], which promotes the expression of a wide variety of tissue-restricted antigens (TRAs) from medullary thymic epithelial cells (mTECs) and from a subset of peripheral antigen-presenting cells (APCs). We examined the effect of additive expression of human AIRE (huAIRE) in a model of autoimmune diabetes in NOD mice. Unexpectedly, we observed that mice expressing augmented AIRE/Aire developed muscle-specific autoimmunity associated with incomplete maturation of mTECs together with impaired expression of Aire-dependent TRAs. This led to failure of deletion of autoreactive T cells together with dramatically reduced production of regulatory T cells in the thymus. In peripheral APCs, expression of costimulatory molecules was augmented. We suggest that levels of Aire expression need to be tightly controlled for maintenance of immunological tolerance. Our results also highlight the importance of coordinated action between central tolerance and peripheral tolerance under the common control of Aire.
Kazuchika Nishitsuji, Jinzhong Xiao, Ryosuke Nagatomo, Hitomi Umemoto, Yuki Morimoto, Hiroyasu Akatsu, Koichi Inoue and Koichi Tsuneyama : Analysis of the gut microbiome and plasma short-chain fatty acid profiles in a spontaneous mouse model of metabolic syndrome., Scientific Reports, Vol.7, No.1, 15876, 2017.
(Summary)
Male Tsumura Suzuki obese diabetes (TSOD) mice spontaneously develop obesity and obesity-related metabolic syndrome. Gut dysbiosis, an imbalance of gut microbiota, has been implicated in the pathogenesis of metabolic syndrome, but its mechanisms are unknown. Short-chain fatty acids (SCFAs) are the main fermentation products of gut microbiota and a link between the gut microbiota and the host's physiology. Here, we investigated a correlation among gut dysbiosis, SCFAs, and metabolic syndrome in TSOD mice. We detected enriched levels of Gram-positive bacteria and corresponding decreases in Gram-negative bacteria in 24-wk-old metabolic syndrome-affected TSOD mice compared with age-matched controls. The abundance of Bacteroidetes species decreased, the abundance of Firmicutes species increased, and nine genera of bacteria were altered in 24-wk-old TSOD mice. The total plasma SCFA level was significantly lower in the TSOD mice than in controls. The major plasma SCFA-acetate-decreased in TSOD mice, whereas propionate and butyrate increased. TSOD mice had no minor SCFAs (valerate and hexanoate) but normal mice did. We thus concluded that gut dysbiosis and consequent disruptions in plasma SCFA profiles occurred in metabolic syndrome-affected TSOD mice. We also propose that the TSOD mouse is a useful model to study gut dysbiosis, SCFAs, and metabolic syndrome.
Shohei Yamashita, Shingo Oda, Hideko Endo, Koichi Tsuneyama and Tsuyoshi Yokoi : Neutrophil depletion protects against zomepirac-induced acute kidney injury in mice., Chemico-Biological Interactions, Vol.279, 102-110, 2017.
(Summary)
Acyl glucuronide (AG) metabolites of carboxylic acid-containing drugs have been implicated in drug toxicity. Zomepirac (ZP) is a non-steroidal anti-inflammatory drug that was withdrawn from the market because of anaphylactic reactions and renal injury. We recently established a novel mouse model of ZP-induced kidney injury by increasing zomepirac acyl-glucuronide (ZP-AG) concentration via pretreatment with tri-O-tolyl phosphate, a nonselective esterase inhibitor, and l-buthionine-(S,R)-sulfoximine, a glutathione synthesis inhibitor. Although we have shown that ZP-AG is responsible for ZP-induced kidney injury in mice, the exact pathogenic mechanisms of ZP-induced kidney injury have not been investigated yet. In this study, we aimed to investigate the role of immune cells in the pathogenesis of ZP-induced kidney injury, as a representative of AG toxicity. We found that the counts of neutrophils and inflammatory monocytes increased in the blood of mice with ZP-induced kidney injury. However, clodronate liposome- or GdCl3-induced monocyte and/or macrophage depletion did not affect blood urea nitrogen and plasma creatinine levels in mice with ZP-induced kidney injury. Neutrophil infiltration into the kidneys was observed in mice with ZP-induced kidney injury, whereas anti-lymphocyte antigen 6 complex, locus G (Ly6G) antibody pretreatment prevented the renal neutrophil infiltration and partially protected against ZP-induced kidney injury. The mRNA expression of neutrophil-infiltrating cytokines and chemokines, interleukin-1 and macrophage inflammatory protein-2, increased in mice with ZP-induced kidney injury, whereas pretreatment with anti-Ly6G antibody resulted in a marked reduction of their expression. These results suggest that ZP-AG might be involved in kidney injury, partly via induction of neutrophil infiltration. Therefore, this study may provide an important understanding on toxicological role of ZP-AG in vivo that helps to understand toxicity of AG metabolites.
Yasuhiro Mouri, Yoshihiro Ueda, Tomoyoshi Yamano, Minoru Matsumoto, Koichi Tsuneyama, Tatsuo Kinashi and Mitsuru Matsumoto : Mode of tolerance induction and requirement for Aire are governed by the cell types that express self-antigen and those that present antigen, The Journal of Immunology, Vol.199, No.12, 3959-3971, 2017.
(Summary)
Aire controls the fate of autoreactive thymocytes (i.e., clonal deletion or development into regulatory T cells [Tregs]) through transcriptional control of the expression of tissue-restricted self-antigens (TRAs) from medullary thymic epithelial cells (mTECs) and bone marrow (BM)-derived cells. Although TRAs expressed by mTECs and BM-derived cells are suggested to complement each other to generate a full spectrum of TRAs, little is known about the relative contribution of TRAs from each component for establishment of self-tolerance. Furthermore, the precise role of Aire in specific types of Aire-expressing APCs remains elusive. We have approached these issues by generating two different types of transgenic mouse (Tg) model, which express a prefixed model self-antigen driven by the insulin promoter or the Aire promoter. In the insulin-promoter Tg model, mTECs alone were insufficient for clonal deletion, and BM-derived APCs were required for this action by utilizing Ag transferred from mTECs. In contrast, mTECs alone were able to induce Tregs, although at a much lower efficiency in the absence of BM-derived APCs. Importantly, lack of Aire in mTECs, but not in BM-derived APCs, impaired both clonal deletion and production of Tregs. In the Aire-promoter Tg model, both mTECs and BM-derived APCs could independently induce clonal deletion without Aire, and production of Tregs was impaired by the lack of Aire in mTECs, but not in BM-derived APCs. These results suggest that the fate of autoreactive thymocytes together with the requirement for Aire depend on the cell types that express self-antigens and the types of APCs involved in tolerance induction.
Akemi Tsutsui, Kenichi Harada, Koichi Tsuneyama, Tomonori Senoh, Takuya Nagano, Koichi Takaguchi, Midori Ando, Satoko Nakamura, Koichi Mizobuchi and Masatoshi Kudo : Clinicopathological Study of Autoimmune Hepatitis Cases That Were Difficult to Differentiate from Drug-Induced Liver Injury., Digestive Diseases, Vol.35, No.6, 506-514, 2017.
(Summary)
This study showed that DDW-J scale was useful for differentiating AIH from DILI in cases with a DDW-J scale score of 5. The histologic features of AIH were characterized by cobblestone hepatocellular change, interface hepatitis, and plasma cell infiltration of the portal region.
Metabolic syndrome (MS) is a worldwide healthcare issue and a dominant risk factor for the development of incurable diseases affecting the entire body. The hepatic manifestations of MS include nonalcoholic fatty liver disease (NAFLD) and its progressive variant, nonalcoholic steatohepatitis (NASH). NASH is known to progress to liver cirrhosis and hepatocellular carcinoma (HCC). Excellent animal models for determining the mechanism of pathogenesis and establishing therapeutic treatment of NASH/HCC are strongly required worldwide. We recently reported that two previously established mouse models of obesity and diabetes mellitus, namely, Tsumura-Suzuki Obese Diabetes (TSOD) mice and MSG mice, developed MS-associated NASH and that their clinical course and pathological characteristics closely mimicked those of human MS-NASH patients. Interestingly, most of the mice developed HCC with advancing age, and the pathological and functional characteristics of this condition were identical to those of human HCC. We further established a novel mouse model of HCC based on type 1 diabetes (DIAR-nSTZ mice) and reported its histopathological features. By comparing various aspects of these mouse models, specific and useful characteristics in a suitable model of MS-associated liver diseases, including hepato-carcinogenesis, can be highlighted.
R Heekyong Bae, L Deborah Hodge, Guo-Xiang Yang, C Patrick S Leung, Babu Sathi Chodisetti, C Julio Valencia, Michael Sanford, M John Fenimore, M Ziaur S Rahman, Koichi Tsuneyama, L Gary Norman, Eric M Gershwin and A Howard Young : The interplay of type I and type II interferons in murine autoimmune cholangitis as a basis for sex-biased autoimmunity., Hepatology, Vol.67, No.4, 1408-1419, 2017.
(Summary)
We have reported a murine model of autoimmune cholangitis, generated by altering the AU rich element by deletion of the IFN- 3'UTR region (coined ARE-Del(-/-) ), that has striking similarities to human primary biliary cholangitis (PBC) with female predominance. Previously, we suggested that the gender bias of autoimmune cholangitis was secondary to intense and sustained type I and II IFN signaling. Based on this thesis, and to define the mechanisms that lead to portal inflammation, we specifically addressed the hypothesis that type I IFNs are the driver of this disease. To accomplish these goals, we crossed ARE-Del(-/-) mice with IFN type I receptor alpha chain (Ifnar1) knockout mice. We report herein that loss of type I IFN receptor signaling in the double construct of ARE-Del(-/-) Ifnar1(-/-) mice dramatically reduces liver pathology and abrogated gender bias. More importantly, female ARE-Del(-/-) mice have an increased number of germinal center (GC) B cells as well as abnormal follicular formation, sites which have been implicated in loss of tolerance. Deletion of type I IFN signaling in ARE-Del(-/-) Ifnar1(-/-) mice corrects these GC abnormalities, including abnormal follicular structure. In conclusion, our data implicate type I IFN signaling as a necessary component of the gender bias of this murine model of autoimmune cholangitis. Importantly these data suggest that drugs that target the type I IFN signaling pathway would have potential benefit in the earlier stages of PBC. This article is protected by copyright. All rights reserved.
(Keyword)
Animals / Autoimmune Diseases / B-Lymphocytes / Cholangitis / Disease Models, Animal / Female / Flow Cytometry / Interferon Type I / Liver / Male / Mice / knockout mice / Sex Factors / signal transduction
Hayato Baba, Makoto Kurano, Takeshi Nishida, Hideki Hatta, Ryoji Hokao and Koichi Tsuneyama : Facilitatory effect of insulin treatment on hepatocellular carcinoma development in diabetes., BMC Research Notes, Vol.10, No.1, 478, 2017.
(Summary)
Insulin treatment promoted, rather than inhibited, the progression of liver carcinogenesis in DIAR-nSTZ mice. Hyperinsulinemia rather than hyperglycemia can accelerate the progression of HCC via insulin signaling.
Adipose tissue resident macrophages have important roles in the maintenance of tissue homeostasis and regulate insulin sensitivity for example by secreting pro-inflammatory or anti-inflammatory cytokines. Here, we show that M2-like macrophages in adipose tissue regulate systemic glucose homeostasis by inhibiting adipocyte progenitor proliferation via the CD206/TGF signaling pathway. We show that adipose tissue CD206(+) cells are primarily M2-like macrophages, and ablation of CD206(+) M2-like macrophages improves systemic insulin sensitivity, which was associated with an increased number of smaller adipocytes. Mice genetically engineered to have reduced numbers of CD206(+) M2-like macrophages show a down-regulation of TGF signaling in adipose tissue, together with up-regulated proliferation and differentiation of adipocyte progenitors. Our findings indicate that CD206(+) M2-like macrophages in adipose tissues create a microenvironment that inhibits growth and differentiation of adipocyte progenitors and, thereby, control adiposity and systemic insulin sensitivity.Adipose tissue contains macrophages that can influence both local and systemic metabolism via the secretion of cytokines. Here, Nawaz et al. report that M2-like macrophages, present in adipose tissue, create a microenvironment that inhibits proliferation of adipocyte progenitors due to the secretion of TGF-1.
Katsuhisa Omagari, Kazuhito Suruga, Akira Kyogoku, Satomi Nakamura, Ai Sakamoto, Shinta Nishioka, Mayuko Shimizu, Yuji Miyata, Koichi Tajima, Koichi Tsuneyama and Kazunari Tanaka : A fermented mixed tea made with camellia (Camellia japonica) and third-crop green tea leaves prevents nonalcoholic steatohepatitis in Sprague-Dawley rats fed a high-fat and high-cholesterol diet., Hepatobiliary Surgery and Nutrition, Vol.7, No.3, 175-184, 2017.
(Summary)
Established treatments for non-alcoholic steatohepatitis (NASH) are few, thus it is imperative to develop novel dietary strategies that can prevent NASH. A fermented mixed tea (FMT) made with Camellia japonica (Japanese camellia) and third- crop green tea leaves by tea-rolling processing was reported to reduce body weight and adipose tissue weight in Sprague-Dawley (SD) rats. Because visceral fat is one of the most important factors for the development of hepatic steatosis, this FMT supplementation can be a candidate dietary strategy for the prevention of NASH. Nine-week-old male SD rats were fed a high-fat and high-cholesterol (HFC) diets with or without FMT (camellia and third-crop green tea leaves at ratios of 1:5, 1:2 and 1:1) for 9 weeks (n=6-7/group). Histopathology, serology and expressions of fibrogenetic, proinflammatory, oxidative stress and lipid metabolism-related genes in the liver were evaluated. Histologically, HFC diet with FMT at a ratio of 1:5 dramatically reduced NASH progression (14%) compared to the HFC diet without FMT (100%). FMT at a ratio of 1:5 reduced hepatic steatosis due to the activation of microsomal triglyceride transfer protein, and FMT at a ratio of 1:2 reduced mRNA levels of some proinflammatory, lipid metabolism-related, fibrogenic and oxidative stress marker genes. Our data suggest that FMT at a ratio of 1:5 or 1:2 likely possesses a preventive effect on NASH progression.
Kotaro Matsumoto, Mayuko Shimizu, Koichi Tsuneyama, Yuki Moritoki, Hiromichi Tsunashima, Katsuhisa Omagari, Masumi Hara, Ichiro Yasuda, Hiroshi Miyakawa and Kentaro Kikuchi : Fructo-oligosaccharides and intestinal barrier function in a methionine-choline-deficient mouse model of nonalcoholic steatohepatitis., PLoS ONE, Vol.12, No.6, e0175406, 2017.
(Summary)
Impairments in intestinal barrier function, epithelial mucins, and tight junction proteins have been reported to be associated with nonalcoholic steatohepatitis. Prebiotic fructo-oligosaccharides restore balance in the gastrointestinal microbiome. This study was conducted to determine the effects of dietary fructo-oligosaccharides on intestinal barrier function and steatohepatitis in methionine-choline-deficient mice. Three groups of 12-week-old male C57BL/6J mice were studied for 3 weeks; specifically, mice were fed a methionine-choline-deficient diet, a methionine-choline-deficient diet plus 5% fructo-oligosaccharides in water, or a normal control diet. Fecal bacteria, short-chain fatty acids, and immunoglobulin A (IgA) levels were investigated. Histological and immunohistochemical examinations were performed using mice livers for CD14 and Toll-like receptor-4 (TLR4) expression and intestinal tissue samples for IgA and zonula occludens-1 expression in epithelial tight junctions. The methionine-choline-deficient mice administered 5% fructo-oligosaccharides maintained a normal gastrointestinal microbiome, whereas methionine-choline-deficient mice without prebiotic supplementation displayed increases in Clostridium cluster XI and subcluster XIVa populations and a reduction in Lactobacillales spp. counts. Methionine-choline-deficient mice given 5% fructo-oligosaccharides exhibited significantly decreased hepatic steatosis (p = 0.003), decreased liver inflammation (p = 0.005), a decreased proportion of CD14-positive Kupffer cells (p = 0.01), decreased expression of TLR4 (p = 0.04), and increases in fecal short-chain fatty acid and IgA concentrations (p < 0.04) compared with the findings in methionine-choline-deficient mice that were not administered this prebiotic. This study illustrated that in the methionine-choline-deficient mouse model, dietary fructo-oligosaccharides can restore normal gastrointestinal microflora and normal intestinal epithelial barrier function, and decrease steatohepatitis. The findings support the role of prebiotics, such as fructo-oligosaccharides, in maintaining a normal gastrointestinal microbiome; they also support the need for further studies on preventing or treating nonalcoholic steatohepatitis using dietary fructo-oligosaccharides.
Mayumi Takeuchi, Kenji Matsuzaki, Koichi Tsuneyama, Masato Nishimura, Eri Takiguchi and Masafumi Harada : Ovarian Large Cell Neuroendocrine Carcinoma Associated with Serous Carcinoma: Correlation of Pathology with MR Imaging, Magnetic Resonance in Medical Sciences, Vol.16, No.4, 273-274, 2017.
Seitarou Shimada, Kazuto Tajiri, Hayato Baba, Masami Minemura, Koichi Tsuneyama, Masayuki Nakano and Toshiro Sugiyama : Peritumoral Hyperplasia in Hepatic Sclerosed Hemangioma., ACG Case Reports Journal, Vol.4, e61, 2017.
(Summary)
Peritumoral hyperplasia (PTH) is a hyperplastic lesion located around hypervascular tumors. Hepatic sclerosed hemangioma is a very rare form of hemangioma with sclerotic changes and is distinct from sclerosing hemangioma. We present a patient with non-alcoholic steatohepatitis-induced cirrhosis who presented with a hypervascular tumor. The tumor showed atypical findings of hemangioma and was treated with surgical resection because hepatic malignancy could not be ruled out. Histopathologic examination revealed the tumor was a sclerosed hemangioma with PTH. Lesions with carcinogenic potential were found in the PTH lesion. Sclerosed hemangioma should be observed and managed carefully.
Hiep Canh Nguyen, Kenichi Harada, Hirofumi Ouchi, Yasunori Sato, Koichi Tsuneyama, Masayoshi Kage, Masayuki Nakano, Kaname Yoshizawa, Atsushi Takahashi, Masanori Abe, Jong-Hon Kang, Kazuhiko Koike, Ayano Inui, Tomoo Fujisawa, Akinobu Takaki, Teruko Arinaga-Hino, Takuji Torimura, Yoshiyuki Suzuki, Keiichi Fujiwara, Mikio Zeniya, Hiromasa Ohira, Atsushi Tanaka and Hajime Takikawa : Acute presentation of autoimmune hepatitis: a multicentre study with detailed histological evaluation in a large cohort of patients., Journal of Clinical Pathology, 2017.
(Summary)
The acute presentation of AIH represents the entire histological spectrum of acute hepatitis and chronic hepatitis with various activity grades and fibrosis stages that clinically correspond to acute-onset AIH and acute exacerbation of classic AIH, respectively. Although there are no pathognomonic features for the pathological diagnosis, the prominent presence of lobular and perivenular necroinflammatory activity, pigmented macrophages and cobblestone appearance of hepatocytes in addition to the classic AIH features, such as plasma cell infiltration and emperipolesis, are useful for the pathological diagnosis of the acute presentation of AIH.
IL-33 is one of the critical cytokines that activates group 2 innate lymphoid cells (ILC2s) and mediates allergic reactions. Accumulating evidence suggests that IL-33 is also involved in the pathogenesis of several chronic inflammatory diseases. Previously, we generated an IL-5 reporter mouse and revealed that lung IL-5-producing ILC2s played essential roles in regulating eosinophil biology. In this study, we evaluated the consequences of IL-33 administration over a long period, and we observed significant expansion of ILC2s and eosinophils surrounding pulmonary arteries. Unexpectedly, pulmonary arteries showed severe occlusive hypertrophy that was ameliorated in IL-5- or eosinophil-deficient mice, but not in Rag2-deficient mice. This indicates that IL-5-producing ILC2s and eosinophils play pivotal roles in pulmonary arterial hypertrophy. Administration of a clinically used vasodilator was effective in reducing IL-33-induced hypertrophy and repressed the expansion of ILC2s and eosinophils. Taken together, these observations demonstrate a previously unrecognized mechanism in the development of pulmonary arterial hypertrophy and the causative roles of ILC2 in the process.
Cindy Chang, Ana Lleo, Anchasa Kananurak, Fabio Grizzi, Koichi Tsuneyama, Pietro Invernizzi, L Charles Bevins and L Christopher Bowlus : Human -Defensin 2 in Primary Sclerosing Cholangitis., Clinical and Translational Gastroenterology, Vol.8, No.3, e80, 2017.
(Summary)
Our data show that HBD2 serum levels and tissue expression are increased in PSC subjects, suggesting that this arm of innate immunity may be important in the etiopathogenesis of PSC.
Yuji Shirai, Shingo Oda, Sayaka Makino, Koichi Tsuneyama and Tsuyoshi Yokoi : Establishment of a mouse model of enalapril-induced liver injury and investigation of the pathogenesis., Laboratory Investigation; a Journal of Technical Methods and Pathology, Vol.97, No.7, 833-842, 2017.
(Summary)
Drug-induced liver injury (DILI) is a major concern in drug development and clinical drug therapy. Since the underlying mechanisms of DILI have not been fully understood in most cases, elucidation of the hepatotoxic mechanisms of drugs is expected. Although enalapril (ELP), an angiotensin-converting enzyme inhibitor, has been reported to cause liver injuries with a low incidence in humans, the precise mechanisms by which ELP causes liver injury remains unknown. In this study, we established a mouse model of ELP-induced liver injury and analyzed the mechanisms of its hepatotoxicity. Mice that were administered ELP alone did not develop liver injury, and mice that were pretreated with a synthetic glucocorticoid dexamethasone (DEX) and a glutathione synthesis inhibitor l-buthionine-(S,R)-sulfoximine (BSO) exhibited liver steatosis without significant increase in plasma alanine aminotransferase (ALT). In mice pretreated with DEX and BSO, ALT levels were significantly increased after ELP administration, suggesting that hepatic steatosis sensitized the liver to ELP hepatotoxicity. An immunohistochemical analysis showed that the numbers of myeloperoxidase-positive cells that infiltrated the liver were significantly increased in the mice administered DEX/BSO/ELP. The levels of oxidative stress-related factors, including hepatic heme oxygenase-1, serum hydrogen peroxide and hepatic malondialdehyde, were elevated in the mice administered DEX/BSO/ELP. The involvement of oxidative stress in ELP-induced liver injury was further supported by the observation that tempol, an antioxidant agent, ameliorated ELP-induced liver injury. In conclusion, we successfully established a model of ELP-induced liver injury in DEX-treated steatotic mice and demonstrated that oxidative stress and neutrophil infiltration are involved in the pathogenesis of ELP-induced liver injury.
Hong-Di Ma, Wen-Tao Ma, Qing-Zhi Liu, Zhi-Bin Zhao, Mu-Zi-Ying Liu, Koichi Tsuneyama, Jin-Ming Gao, M William Ridgway, A Aftab Ansari, Eric M Gershwin, Yun-Yun Fei and Zhe-Xiong Lian : Chemokine receptor CXCR3 deficiency exacerbates murine autoimmune cholangitis by promoting pathogenic CD8(+) T cell activation., Journal of Autoimmunity, Vol.78, 19-28, 2017.
(Summary)
CXC Chemokine Receptor 3 (CXCR3) is functionally pleiotropic and not only plays an important role in chemotaxis, but also participates in T cell differentiation and may play a critical role in inducing and maintaining immune tolerance. These observations are particularly critical for autoimmune cholangitis in which CXCR3 positive T cells are found around the portal areas of both humans and mouse models of primary biliary cholangitis (PBC). Herein, we investigated the role of CXCR3 in the pathogenesis of autoimmune cholangitis. We have taken advantage of a unique CXCR3 knockout dnTGFRII mouse to focus on the role of CXCR3, both by direct observation of its influence on the natural course of disease, as well as through adoptive transfer studies into Rag-/- mice. We report herein that not only do CXCR3 deficient mice develop an exacerbation of autoimmune cholangitis associated with an expanded effector memory T cell number, but also selective adoptive transfer of CXCR3 deficient CD8(+) T cells induces autoimmune cholangitis. In addition, gene microarray analysis of CXCR3 deficient CD8(+) T cells reveal an intense pro-inflammatory profile. Our data suggests that the altered gene profiles induced by CXCR3 deficiency promotes autoimmune cholangitis through pathogenic CD8(+) T cells. These data have significance for human PBC and other autoimmune liver diseases in which therapeutic intervention might be directed to chemokines and/or their receptors.
Mayuko Shimizu, Miki Masuzumi, Miku Kawase, Mika Sakaki, Shizuka Tamaru, Yasuo Nagata, Kazunari Tanaka, Kazuhito Suruga, Koichi Tsuneyama, Satoru Matsuda and Katsuhisa Omagari : A diet-induced Sprague-Dawley rat model of nonalcoholic steatohepatitis-related cirrhosis., The Journal of Nutritional Biochemistry, Vol.40, 62-69, 2017.
(Summary)
Certain modified diets containing saturated fatty acids, cholesterol or fructose lead to the development of nonalcoholic steatohepatitis (NASH)-related fibrosis in rodents; however, progression to cirrhosis is rare. Experimental liver cirrhosis models have relied on genetic manipulation or administration of hepatotoxins. This study aimed to establish a reliable dietary model of NASH-related cirrhosis in a relatively short period. Male Sprague-Dawley rats (9 weeks of age) were randomly assigned to normal, high-fat (HF), or two types (1.25% or 2.5% cholesterol) of high-fat and high-cholesterol (HFC) diets for 18 weeks. All HFC diets contained 2% cholic acid by weight. Histopathological analysis revealed that the HFC diets induced obvious hepatic steatosis, inflammation with hepatocyte ballooning and advanced fibrosis (stage 3-4) in all 12 rats at 27 weeks of age. In contrast, all five rats given the HF diet developed mild steatosis and inflammation without fibrosis. The amount of cholesterol in the liver and hepatocellular mitochondrial and microsomal fractions was significantly higher in rats fed the HFC diets than the normal or HF diets. The HFC diets significantly suppressed mRNA levels of microsomal triglyceride transfer protein, adenosine triphosphate binding cassette transporter G5, bile acid CoA: amino acid N-acyltransferase and bile salt export pump, as well as the enzymatic activity of carnitine palmitoyltransferase in the liver. In conclusion, the HFC diets induced liver cirrhosis in conjunction with hepatic features of NASH in Sprague-Dawley rats within 18 weeks, and altered gene expression and enzyme activity to accumulate lipid and bile acid in the liver.
Koichi Tsuneyama, Hayato Baba, Yuki Morimoto, Takaaki Tsunematsu and Hirohisa Ogawa : Primary Biliary Cholangitis: Its Pathological Characteristics and Immunopathological Mechanisms., The Journal of Medical Investigation : JMI, Vol.64, No.1.2, 7-13, 2017.
(Summary)
Primary biliary cholangitis (PBC), formerly known as primary biliary cirrhosis, is an organ-specific autoimmune disease that predominantly affects middle-aged women and is characterized by the chronic progressive destruction of small intrahepatic bile ducts with portal inflammation and, ultimately, fibrosis. The serological hallmark of PBC is the presence of anti-mitochondrial autoantibodies (AMA). Several mechanisms have been proposed for immune-mediated bile duct damage in PBC, including the roles of T cells, B cells, other cell phenotypes, and AMA. A sign of fragility of biliary epithelial cells caused by apoptosis, senescence, and autophagy has also been noted. Several complex steps and mechanisms appear to be involved in the induction and progression of cholangitis and biliary degeneration in patients with PBC. J. Med. Invest. 64: 7-13, February, 2017.
Shota Morimoto, Tomoya Ishikawa, Kumoinori Hyodo, Takahiro Yamazaki, Shu Taira, Koichi Tsuneyama and Yuko Iciyanagi : Preparation and characterization of newly developed matrix using functional γ-Fe2O3 nanoparticles for mass spectrometry in small molecules., Surface and Interface Analysis, Vol.48, No.11, 1127-1131, 2016.
Pathological stage and grade have limited ability to predict the outcomes of superficial urothelial bladder carcinoma at initial transurethral resection (TUR). AT-motif binding factor 1 (ATBF1) is a tumor suppressive transcription factor that is normally localized to the nucleus but has been detected in the cytoplasm in several cancers. Here, we examined the diagnostic value of the intracellular localization of ATBF1 as a marker for the identification of high risk urothelial bladder carcinoma. Seven anti-ATBF1 antibodies were generated to cover the entire ATBF1 sequence. Four human influenza hemagglutinin-derived amino acid sequence-tagged expression vectors with truncated ATBF1 cDNA were constructed to map the functional domains of nuclear localization signals (NLSs) with the consensus sequence KR[X10-12]K. A total of 117 samples from initial TUR of human bladder carcinomas were analyzed. None of the patients had received chemotherapy or radiotherapy before pathological evaluation. ATBF1 nuclear localization was regulated synergistically by three NLSs on ATBF1. The cytoplasmic fragments of ATBF1 lacked NLSs. Patients were divided into two groups according to positive nuclear staining of ATBF1, and significant differences in overall survival (P .021) and intravesical recurrence-free survival (P .013) were detected between ATBF1+ (n 10) and ATBF1- (n ) cases. Multivariate analysis revealed that ATBF1 staining was an independent prognostic factor for intravesical recurrence-free survival after adjusting for cellular grading and pathological staging (P .008). Cleavage of ATBF1 leads to the cytoplasmic localization of ATBF1 fragments and downregulates nuclear ATBF1. Alterations in the subcellular localization of ATBF1 due to fragmentation of the protein are related to the malignant character of urothelial carcinoma. Pathological evaluation using anti-ATBF1 antibodies enabled the identification of highly malignant cases that had been overlooked at initial TUR. Nuclear localization of ATBF1 indicates better prognosis of urothelial carcinoma.
Takeshi Nishida, Koichi Tsuneyama, Makoto Fujimoto, Kazuhiro Nomoto, Shinichi Hayashi, Shigeharu Miwa, Takahiko Nakajima, Yuko Nakanishi, Hideki Hatta and Johji Imura : Aberrant iron metabolism might have an impact on progression of diseases in Tsumura Suzuki obese diabetes mice, a model of spontaneous metabolic syndrome., Pathology International, Vol.66, No.11, 622-628, 2016.
(Summary)
Tsumura Suzuki obese diabetes (TSOD) mice spontaneously develop obesity and type 2 diabetes with aberrant accumulation of excessive iron in the spleen. Aberrantly accumulated iron may cause oxidative stress and result in various symptoms of metabolic syndrome in the mice. We investigated iron metabolism and oxidative stress in TSOD mice. Male TSOD and control mice were killed at 2, 3, 6, and 8 months of age, and blood and tissue samples were collected. The serum levels of ferritin and oxidized low-density lipoprotein (OxLDL) were measured. Total glutathione concentrations of liver and spleen were also measured. Serum ferritin and OxLDL were higher in TSOD mice than in control mice at 2 and 6 months. In addition, the glutathione concentrations in TSOD mice were lower in the liver and higher in the spleen at 3 and 6 months than those in control mice. These results suggest that abnormal iron metabolism and imbalanced oxidative stress occurs in young and old TSOD mice. We propose herein that TSOD mice might be a unique and valuable model for investigating the role of iron metabolism in pathogenesis of metabolic syndrome.
Adipose tissue hypoxia is an important feature of pathological adipose tissue expansion. Hypoxia-inducible factor-1 (HIF-1) in adipocytes reportedly induces oxidative stress and fibrosis, rather than neoangiogenesis via vascular endothelial growth factor (VEGF)-A. We previously reported that macrophages in crown-like structures (CLSs) are both hypoxic and inflammatory. In the current study, we examined how macrophage HIF-1 is involved in high-fat diet (HFD)-induced inflammation, neovascularization, hypoxia, and insulin resistance using mice with myeloid cell-specific HIF-1 deletion that were fed an HFD. Myeloid cell-specific HIF-1 gene deletion protected against HFD-induced inflammation, CLS formation, poor vasculature development in the adipose tissue, and systemic insulin resistance. Despite a reduced expression of Vegfa in epididymal white adipose tissue (eWAT), the preadipocytes and endothelial cells of HIF-1-deficient mice expressed higher levels of angiogenic factors, including Vegfa, Angpt1, Fgf1, and Fgf10 in accordance with preferable eWAT remodeling. Our in vitro study revealed that lipopolysaccharide-treated bone marrow-derived macrophages directly inhibited the expression of angiogenic factors in 3T3-L1 preadipocytes. Thus, macrophage HIF-1 is involved not only in the formation of CLSs, further enhancing the inflammatory responses, but also in the inhibition of neoangiogenesis in preadipocytes. We concluded that these two pathways contribute to the obesity-related physiology of pathological adipose tissue expansion, thus causing systemic insulin resistance.
Tetsuyuki Takahashi, Takeshi Nishida, Hayato Baba, Hideki Hatta, Johji Imura, Mitsuko Sutoh, Syunji Toyohara, Ryoji Hokao, Syunsuke Watanabe, Hirohisa Ogawa, Hisanori Uehara and Koichi Tsuneyama : Histopathological characteristics of glutamine synthetase-positive hepatic tumor lesions in a mouse model of spontaneous metabolic syndrome (TSOD mouse)., Molecular and Clinical Oncology, Vol.5, No.2, 267-270, 2016.
(Summary)
We previously reported that Tsumura-Suzuki obese diabetic (TSOD) mice, a polygenic model of spontaneous type 2 diabetes, is a valuable model of hepatic carcinogenesis via non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). One of the characteristics of tumors in these mice is the diffuse expression of glutamine synthetase (GS), which is a diagnostic marker for hepatocellular carcinoma (HCC). In this study, we performed detailed histopathological examinations and found that GS expression was diffusely positive in >70% of the hepatic tumors from 15-month-old male TSOD mice. Translocation of -catenin into nuclei with enhanced membranous expression also occurred in GS-positive tumors. Small lesions (<1 mm) in GS-positive cases exhibited dysplastic nodules, with severe nuclear atypia, whereas large lesions (>3 mm) bore the characteristics of human HCC, exhibiting nuclear and structural atypia with invasive growth. By contrast, the majority of GS-negative tumors were hepatocellular adenomas with advanced fatty change and low nuclear grade. In GS-negative tumors, loss of liver fatty acid-binding protein expression was observed. These results suggest that the histological characteristics of GS-positive hepatic tumors in TSOD mice resemble human HCC; thus, this model may be a useful tool in translational research targeting the NAFLD/NASH-HCC sequence.
G-X Yang, Y Sun, Koichi Tsuneyama, W Zhang, P S. C. Leung, X-S He, A A. Ansari, C Bowlus, W M. Ridgway and M E. Gershwin : Endogenous interleukin-22 protects against inflammatory bowel disease but not autoimmune cholangitis in dominant negative form of transforming growth factor beta receptor type II mice., Clinical and Experimental Immunology, Vol.185, No.2, 154-164, 2016.
(Summary)
During chronic inflammation, interleukin (IL)-22 expression is up-regulated in both CD4 and CD8 T cells, exerting a protective role in infections. However, in autoimmunity, IL-22 appears to have either a protective or a pathogenic role in a variety of murine models of autoimmunity and, by extrapolation, in humans. It is not clear whether IL-22 itself mediates inflammation or is a by-product of inflammation. We have taken advantage of the dominant negative form of transforming growth factor beta receptor type II (dnTGF-RII) mice that develop both inflammatory bowel disease and autoimmune cholangitis and studied the role and the biological function of IL-22 by generating IL-22(-/-) dnTGF-RII mice. Our data suggest that the influence of IL-22 on autoimmunity is determined in part by the local microenvironment. In particular, IL-22 deficiency exacerbates tissue injury in inflammatory bowel disease, but has no influence on either the hepatocytes or cholangiocytes in the same model. These data take on particular significance in the previously defined effects of IL-17A, IL-12p40 and IL-23p19 deficiency and emphasize that, in colitis, there is a dominant role of IL-23/T helper type 17 (Th17) signalling. Furthermore, the levels of IL-22 are IL-23-dependent. The use of cytokine therapy in patients with autoimmune disease has significant potential, but must take into account the overlapping and often promiscuous effects that can theoretically exacerbate inflammation.
Heekyong R. Bae, Patrick S. C. Leung, Koichi Tsuneyama, Julio C. Valencia, Deborah L. Hodge, Seohyun Kim, Tim Back, Megan Karwan, Anand S. Merchant, Nobuyuki Baba, Dechun Feng, Ogyi Park, Bin Gao, Guo-Xiang Yang, M Gershwin Eric and Howard A. Young : Chronic Expression of Interferon Gamma Leads to Murine Autoimmune Cholangitis with a Female Predominance., Hepatology, Vol.64, No.4, 1189-1201, 2016.
(Summary)
In most autoimmune diseases the serologic hallmarks of disease precede clinical pathology by years. Therefore the use of animal models in defining early disease events becomes critical. Herein we have taken advantage of a "designer" mouse with dysregulation of interferon gamma (IFN) characterized by prolonged and chronic expression of IFN through deletion of the IFN 3' UTR AU-rich element. These mice develop primary biliary cholangitis (PBC) with a female predominance that mimics human disease and is characterized by upregulation of total bile acids, spontaneous production of AMA, and portal duct inflammation. Transfer of CD4 T cells from ARE-Del-/- to B6/Rag1(-/-) mice induced moderate portal inflammation, and parenchymal inflammation, RNA-sequencing of liver gene expression revealed that upregulated genes potentially define early stages of cholangitis. Interestingly, upregulated genes specifically overlap with the gene expression signature of biliary epithelial cells in PBC, implying that IFN may play a pathogenic role in biliary epithelial cells (BEC) in the initiation stage of PBC. Moreover, differentially expressed genes in female mice have stronger Type I and II interferon signaling and lymphocyte-mediated immune responses and thus may drive the female bias of the disease. In conclusion, changes in IFN expression are critical for the pathogenesis of PBC. This article is protected by copyright. All rights reserved.
Atsushi Iwamura, Katsuhito Watanabe, Sho Akai, Tsubasa Nishinosono, Koichi Tsuneyama, Shingo Oda, Toshiyuki Kume and Tsuyoshi Yokoi : Zomepirac Acyl Glucuronide Is Responsible for Zomepirac-Induced Acute Kidney Injury in Mice., Drug Metabolism and Disposition, Vol.44, No.7, 888-896, 2016.
(Summary)
Glucuronidation, an important phase II metabolic route, is generally considered to be a detoxification pathway. However, acyl glucuronides (AGs) have been implicated in the toxicity of carboxylic acid drugs due to their electrophilic reactivity. Zomepirac (ZP) was withdrawn from the market because of adverse effects such as renal toxicity. Although ZP is mainly metabolized to acyl glucuronide (ZP-AG) by UDP-glucuronosyltransferase, the role of ZP-AG in renal toxicity is unknown. In this study, we established a ZP-induced kidney injury mouse model by pretreatment with tri-o-tolyl phosphate (TOTP), a nonselective esterase inhibitor, and l-buthionine-(S,R)-sulfoximine (BSO), a glutathione synthesis inhibitor. The role of ZP-AG in renal toxicity was investigated using this model. The model showed significant increases in blood urea nitrogen (BUN) and creatinine (CRE), but not alanine aminotransferase. The ZP-AG concentrations were elevated by cotreatment with TOTP in the plasma and liver and especially in the kidney. The ZP-AG concentrations in the kidney correlated with values for BUN and CRE. Upon histopathological examination, vacuoles and infiltration of mononuclear cells were observed in the model mouse. In addition to immune-related responses, oxidative stress markers, such as the glutathione/disulfide glutathione ratio and malondialdehyde levels, were different in the mouse model. The suppression of ZP-induced kidney injury by tempol, an antioxidant agent, suggested the involvement of oxidative stress in ZP-induced kidney injury. This is the first study to demonstrate that AG accumulation in the kidney by TOTP and BSO treatment could explain renal toxicity and to show the in vivo toxicological potential of AGs.
Takahiko Nakajima, Haruo Yagi, Hayato Baba, Takashi Minamisaka, Shigeharu Miwa, Shinichi Hayashi, Takeshi Nishida, Hideki Hatta, Koichi Tsuneyama and Johji Imura : Complete Resolution of Pseudomalignant Erosion in a Reflux Gastroesophageal Polyp with Proton Pump Inhibitor., Case Reports in Pathology, Vol.2015, 2015.
(Summary)
Pseudomalignant erosion is a diagnostic pitfall for pathologists in the differential diagnosis of malignant neoplasms. Here, we present a challenging case of a biopsy specimen from the eroded head of a polyp at the esophagogastric junction. A malignant neoplasm could not be ruled out due to the presence of bizarre stromal cells. A second biopsy performed after the administration of a proton pump inhibitor (PPI) for 4 weeks revealed endoscopic resolution of the polyp along with the complete histological resolution of the bizarre stromal cells and led to the diagnosis of pseudomalignant erosion in a reflux gastroesophageal polyp. In conclusion, histological and endoscopic response to PPI therapy is an important clue for the correct diagnosis of reflux gastroesophageal polyps with pseudomalignant erosion.
Haruka Wakae, Tomomi Higashide, Koichi Tsuneyama, Takahiro Nakamura, Kanji Takahashi and Kazuhisa Sugiyama : Immunohistochemical Characterization of the Ectopic Epithelium Devoid of Goblet Cells From a Posttraumatic Iris Cyst Causing Mucogenic Glaucoma., Journal of Glaucoma, 2015.
(Summary)
The ectopic epithelium of an iris cyst causing mucogenic glaucoma was most likely to originate from limbal basal cells, which showed dual direction of differentiation toward both the conjunctival and corneal epithelia. The membrane-bound mucin may have caused mucogenic glaucoma in the absence of goblet cells.
Jing-Bo Yang, Yin-Hu Wang, Wei Yang, Fang-Ting Lu, Hong-Di Ma, Zhi-Bin Zhao, Yan-Jie Jia, Wei Tang, Koichi Tsuneyama, M William Ridgway, Eric M Gershwin and Zhe-Xiong Lian : Successful treatment of murine autoimmune cholangitis by parabiosis: Implications for hematopoietic therapy., Journal of Autoimmunity, Vol.66, 108-117, 2015.
(Summary)
There is a significant unmet need in the treatment of primary biliary cirrhosis (PBC) despite significant data on the effector pathways that lead to biliary duct damage. We focused attention on a murine model of PBC, the dominant negative transforming growth factor receptor II (Tg) mice. To further define the pathways that lead to biliary pathology in these mice, we developed Tg mice deleted of CD4 cells (CD4(-/-)Tg). Interestingly, these mice developed more severe cholangitis than control Tg mice. These mice, which lack CD4 cells, manifested increased levels of IFN- produced by effector CD8 cells. It appears that increased cholangitis is due to the absence of CD4 Treg cells. Based on these data, we parabiosed CD4(-/-)Tg mice with established disease at 8-9 weeks of age with C57BL/6 control mice. Such parabiotic "twins" had a significant reduction in autoimmune cholangitis, even though they had established pathology at the time of surgery. We prepared mixed bone marrow chimera mice constructed from CD4(-/-)Tg and CD8(-/-) mice and not only was cholangitis improved, but a decrease in terminally differentiated CD8(+) T effector cells in the presence of wild type CD4 cells was noted. In conclusion, "correcting" the CD4 T cell subset, even in the presence of pathogenic CD8 T cells, is effective in treating autoimmune cholangitis.
Hiromichi Tsunashima, Koichi Tsuneyama, Yuki Moritoki, Masumi Hara and Kentaro Kikuchi : Accumulated myeloid-derived suppressor cells demonstrate distinct phenotypes and functions in two non-alcoholic steatohepatitis mouse models., Hepatobiliary Surgery and Nutrition, Vol.4, No.5, 313-319, 2015.
(Summary)
These results suggest that hepatic fatty changes promote MDSC accumulation, and inflammatory changes induce phenotypic and functional alteration in hepatic MDSCs in NASH mouse models.
Eita Sasaki, Azumi Iida, Shingo Oda, Koichi Tsuneyama, Tatsuki Fukami, Miki Nakajima and Tsuyoshi Yokoi : Pathogenetic analyses of carbamazepine-induced liver injury in F344 rats focused on immune- and inflammation-related factors., Experimental and Toxicologic Pathology, 2015.
(Summary)
Drug-induced liver injury is one of the major reasons for a drug to be withdrawn postmarketing. Carbamazepine (CBZ), an anticonvulsant agent, has been reported rarely to cause liver failure in humans. We recently generated a rat model of CBZ-induced liver injury using F344 rats for five consecutive days of CBZ administration combined with a glutathione (GSH) depletor, l-buthionine S,R-sulfoximine, treatment. The involvement of metabolic activation was demonstrated in developing CBZ-induced liver injury, and a difference in metabolic activation reactions between mice and rats was indicated. In this study, we analyzed the pathogenetic mechanism of CBZ-induced liver injury, primarily focusing on immune- and inflammation-related factors using the rat model for CBZ-induced liver injury. After the last CBZ administration, plasma alanine aminotransfearase (ALT) levels were drastically increased. In the histopathological evaluation, time-dependent hepatocellular degeneration and necrosis were observed in the centrilobular region. Different from mice, although hepatic mRNA expression levels of inflammation-related genes were increased, T-helper cell-related genes were not predominantly changed in rats. The number of ED1- and ED2-positive macrophages was increased in injured centrilobular areas in the liver with CBZ-induced liver injury. Treatment with a Kupffer cell depletor, gadolinium chloride, prevented the elevation of plasma ALT levels and an increase in the hepatic mRNA expression levels of inflammation-related genes. Hepatic adenosine triphosphate (ATP) contents were significantly decreased 24h after CBZ administration. Therefore, the Kupffer cells-mediated inflammation was predominant in the development of the CBZ-induced liver injury in rats.
Zheng-Guo Cui, Ryohei Ogawa, Koichi Tsuneyama, Gen Yan, Lingling Tao, Akiko Shimomura and Hidekuni Inadera : Insight into the molecular mechanism of heme oxygenase-1 induction by docosahexaenoic acid in U937 cells., Chemico-Biological Interactions, Vol.238, 180-188, 2015.
(Summary)
Heme oxygenase-1 (HO-1) has anti-inflammatory effects on myeloid cells in response to various stimuli. To date, little is known about whether fatty acids can affect HO-1 induction. Here, we report the induction of HO-1 by docosahexaenoic acid (DHA) and the associated molecular mechanisms in human myelomonocytic lymphoma U937 cells. When U937 cells were treated with DHA, eicosapentaenoic acid, palmitic acid or oleic acid, DHA was the most effective inducer of HO-1. The activation of AKT and glycogen synthase kinase-3 did not significantly change after DHA treatment. However, DHA increased the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), but not of other mitogen-activated protein kinases such as p38 and JNK. The increase in HO-1 expression was significantly inhibited by U0126, an ERK1/2 inhibitor. Nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf-2) and its binding to the HO-1 promoter significantly increased upon DHA treatment. An increase in intracellular reactive oxygen species was detected by dichlorofluorescein diacetate, but not by hydroethidium or 2-[6-(4-hydroxy)phenoxy-3H-xanthen-3-on-9-yl] benzoic acid after DHA treatment. Pretreatment with NAC dramatically inhibited the ERK1/2 activation, binding of Nrf-2 to antioxidant response elements (AREs) located in the HO-1 promoter and the induction of HO-1 by DHA. In conclusion, DHA increased HO-1 expression in U937 cells via activation of ERK1/2 and increased Nrf-2 binding to ARE in the HO-1 promoter. These findings will help develop better strategies for treating inflammatory disorders with DHA.
Koichiro Shinoda, Hirofumi Taki, Konen Obayashi, Yukio Ando, Akihiko Watanabe, Akira Komiya, Hideki Fuse, Koichi Tsuneyama and Kazuyuki Tobe : Wild-type ATTR amyloidosis of the ureter in a 56-year-old woman with rheumatoid arthritis and Sjögren's syndrome., International Journal of Clinical and Experimental Pathology, Vol.8, No.7, 8624-8627, 2015.
(Summary)
We present a case of acute pyelonephritis with right hydronephrosis in a middle-aged woman, who had suffered from rheumatoid arthritis and Sjögren's syndrome. She had successfully treated with antibiotics, however, ureteral stenosis sustained. She underwent ureteroscopy and stenting of right ureter. Biopsy specimen revealed submucosal amyloid deposition in the interstitium overlying a benign urothelium. Amyloid protein was positive for transthyretin (TTR) by immunohistochemistry and amyloid deposition was not demonstrated in other organs. The patient's TTR genes were wild type and she was diagnosed with wild-type ATTR (ATTR wt) amyloidosis. This is the first report about symptomatic ATTR wt amyloidosis, which was also called 'systemic senile amyloidosis (SSA)' in the ureter. We should aware that SSA can occur at younger age and cause symptomatic ureteral stenosis. Further investigation is needed to clarify the association of autoimmune diseases to develop ATTR wt amyloidosis.
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 26339445
Yin Fan Lam, Ka Kui Tong, Kin Ming Kwan, Koichi Tsuneyama, Shang-An Shu, Patrick S. C. Leung and Ka Hou Chu : Gastrointestinal Immune Response to the Shrimp Allergen Tropomyosin: Histological and Immunological Analysis in an Animal Model of Shrimp Tropomyosin Hypersensitivity., International Archives of Allergy and Immunology, Vol.167, No.1, 29-40, 2015.
(Summary)
Shellfish hypersensitivity is among the most common food allergies. A murine model of IgE-mediated shrimp allergy has been established in our laboratory. The aim of this study is to determine the intestinal histological changes and cytokine expression profile of this model sensitized with the major shellfish allergen tropomyosin. Female Balb/c mice orally sensitized and challenged with recombinant tropomyosin were sacrificed. Continuous sections of duodenum, jejunum and ileum were prepared using the Swiss roll technique for histological and immunological analysis. Duodenal epithelial cell apoptosis and migration were examined. mRNA expression of IL-4, IL-6, IL-10, IL-13, IL-18 and IFN- in intestinal tissue was measured via RT-PCR. In tropomyosin-sensitized and challenged mice, an increased number of eosinophils, mast cells and goblet cells was found 24 h after challenge. There were also increased mast cell and goblet cell numbers at 72 h after challenge, but the level of eosinophils decreased. Differences compared with control mice are most prominent at the duodenum compared to the distal regions. In addition, TUNEL assay indicates a significantly higher apoptosis rate in sensitized mice sacrificed 72 h after challenge, and mRNA expression showed a biased Th2/Th1 cytokine profile and a higher level of murine mast cell protease 1. This study documented a multitude of histological and immunological changes in the gut in a murine model of shrimp allergy. Even without repetitive intragastric challenge, shrimp tropomyosin induces an increase in the number of inflammatory cells to varying degrees within the small intestine. This model provides an important tool for testing new therapeutic interventions.
H Tanaka, G-X Yang, T Tomiyama, Koichi Tsuneyama, W Zhang, P S. C. Leung, R L. Coppel, T Joh, S G. Nadler, A A. Ansari, C Bowlus and M E. Gershwin : Immunological potential of cytotoxic T lymphocyte antigen 4 immunoglobulin in murine autoimmune cholangitis., Clinical and Experimental Immunology, Vol.180, No.3, 371-382, 2015.
(Summary)
Cytotoxic T lymphocyte antigen 4 (CTLA-4) immunoglobulin (Ig) is an important regulator of T cell activation and a fusion protein directed at CD80 and CD86; it blocks co-stimulatory signalling and T cell activation. We have taken advantage of a murine model of human primary biliary cirrhosis (PBC), mice expressing a transforming growth factor (TGF)- receptor II dominant negative (dnTGF-RII) transgene to address the potential therapeutic efficacy of CTLA-4 Ig. To mimic patients with PBC at different stages or duration of disease, we treated mice with either CTLA-4 Ig or control IgG three times weekly from 3 to 12 or 24 weeks of age, or from 12 to 24 weeks of age. CTLA-4 Ig treatment from 3 weeks of age significantly reduced liver inflammation to 12 weeks of age. Treatment initiated at 12 weeks of age also ameliorated the autoimmune cholangitis at 24 weeks of age. However, in mice treated at 3 weeks of age, suppression of liver inflammation was not sustained and colitis was aggravated when treatment was extended to 24 weeks of age. Our data indicate that, in dnTGF-RII mice, CTLA-4 Ig treatment has short-term beneficial effects on autoimmune cholangitis, but the effect varies according to duration of treatment and the time in which therapy was initiated. Further dissection of the events that lead to the reduction in therapeutic effectiveness of CTLA-4 Ig will be critical to determining whether such efforts can be applied to human PBC.
Kei Takahashi, Nao Nagai, Keisuke Ogura, Koichi Tsuneyama, Ikuo Saiki, Tatsuro Irimura and Yoshihiro Hayakawa : Mammary tissue microenvironment determines T cell-dependent breast cancer-associated inflammation., Cancer Science, Vol.106, No.7, 867-874, 2015.
(Summary)
Although the importance of the host tissue microenvironment in cancer progression and metastasis has been established, the spatiotemporal process establishing a cancer metastasis-prone tissue microenvironment remains unknown. In this study, we aim to understand the immunological character of a metastasis-prone microenvironment in a murine 4T1 breast tumor model, by using the activation of nuclear factor-b (NF-B) in cancer cells as a sensor of inflammatory status and by monitoring its activity by bioluminescence imaging. By using a 4T1 breast cancer cell line stably expressing an NF-B/Luc2 reporter gene (4T1 NF-B cells), we observed significantly increased bioluminescence approximately 7 days after metastasis-prone orthotopic mammary fat-pad inoculation but not ectopic s.c. inoculation of 4T1 NF-B cells. Such in vivo NF-B activation within the fat-pad 4T1 tumor was diminished in immune-deficient SCID or nude mice, or T cell-depleted mice, suggesting the requirement of host T cell-mediated immune responses. Given the fat-pad 4T1 tumor expressed higher inflammatory mediators in a T cell-dependent mechanism compared to the s.c. tumor, our results imply the importance of the surrounding tissue microenvironment for inflaming tumors by collaborating with T cells to instigate metastatic spread of 4T1 breast cancer cells.
Takahiko Nakajima, Toshiro Sugiyama, Hayato Baba, Hideki Hatta, Takeshi Nishida, Shigeharu Miwa, Shinichi Hayashi, Koichi Tsuneyama and Johji Imura : Bone metastasis in gastrointestinal stromal tumors preferentially occurs in patients with original tumors in sites other than the stomach., International Journal of Clinical and Experimental Pathology, Vol.8, No.5, 5955-5959, 2015.
(Summary)
Bone metastases are rare in gastrointestinal stromal tumors (GISTs) and data on the clinicopathological profiles are lacking. The purpose of this report was to identify the clinicopathological profiles of this rare clinical setting by evaluating 23 cases, four of which were our own and the additional 19 were from the relevant English literature. In 18 cases, the primary GISTs occurred in sites other than the stomach, although a high proportion of these tumors do arise in the stomach. All tumors at the disease presentation had more than a low risk of recurrence, with most tumors either at a high risk or initially malignant with liver metastasis. In four cases, bone metastasis was the primary metastatic manifestation. Although rare in GISTs, bone metastasis should be considered in patients with primary tumors at a high risk for recurrence or in initially malignant tumors with liver metastasis, especially with primary tumors in sites other than the stomach.
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 26191327
Akinori Wada, Aya Ito, Hirofumi Iitsuka, Koichi Tsuneyama, Takayoshi Miyazono, Jun Murakami, Naotoshi Shibahara, Hiroaki Sakurai, Ikuo Saiki, Takashi Nakayama, Osamu Yoshie, Keiichi Koizumi and Toshiro Sugiyama : Role of chemokine CX3CL1 in progression of multiple myeloma via CX3CR1 in bone microenvironments., Oncology Reports, Vol.33, No.6, 2935-2939, 2015.
(Summary)
Several chemokines/chemokine receptors such as CXCL12, CCL3, CXCR4 and CCR1 attract multiple myelomas to specific microenvironments. In the present study, we investigated whether the CX3CL1/CX3CR1 axis is involved in the interaction of the multiple myeloma cells with their microenvironment. The expression of CX3CR1 (also known as fractalkine) was detected in three of the seven human myeloma cell lines. CX3CL1-induced phosphorylation of Akt and ERK1/2 was detected in the CX3CR1-positive cell lines, but not in the CX3CR1-negative cell lines. In addition, CX3CL1-induced cell adhesion to fibronectin and vascular cell adhesion molecule-1 (VCAM-1) in the human myeloma RPMI-8226 cell line. We also investigated whether a relationship existed between myeloma cells and osteoclasts that may function via the CX3CL1/CX3CR1 axis. Conditioned medium from CX3CL1-stimulated RPMI-8226 cells drastically increased the osteoclast differentiation. Collectively, the results from the present study support the concept of the CX3CL1-mediated activation of the progression of the multiple myeloma via CX3CR1. Thus, CX3CR1 may represent a potential therapeutic target for the treatment of multiple myeloma in a bone microenvironment.
Shohei Takai, Shingo Oda, Koichi Tsuneyama, Tatsuki Fukami, Miki Nakajima and Tsuyoshi Yokoi : Establishment of a mouse model for amiodarone-induced liver injury and analyses of its hepatotoxic mechanism., Journal of Applied Toxicology : JAT, 2015.
Azumi Iida, Eita Sasaki, Azusa Yano, Koichi Tsuneyama, Tatsuki Fukami, Miki Nakajima and Tsuyoshi Yokoi : Carbamazepine-Induced Liver Injury Requires CYP3A-Mediated Metabolism and Glutathione Depletion in Rats., Drug Metabolism and Disposition, Vol.43, No.7, 958-968, 2015.
(Summary)
Carbamazepine (CBZ) is widely used as an antiepileptic agent and causes rare but severe liver injury in humans. It has been generally recognized that reactive metabolites formed via the metabolic activation reaction contribute to the onset of liver injuries by several drugs. However, the role of CBZ metabolism in the development of liver injury is not fully understood. In this study, we developed a novel rat model of CBZ-induced liver injury and attempted to elucidate the associated mechanisms by focusing on the metabolism of CBZ. The repeated administration of CBZ for 5 days in combination with l-buthionine sulfoximine (BSO), a glutathione (GSH) synthesis inhibitor, resulted in increases in the plasma alanine aminotransferase (ALT) levels and centrilobular necrosis in the liver that were observed in various degrees. The CBZ and 2-hydroxy-CBZ concentrations in the plasma after the last CBZ administration were lower in the rats with high plasma ALT levels compared with those with normal plasma ALT levels, showing the possibility that the further metabolism of CBZ and/or 2-hydroxy-CBZ is associated with the liver injury. Although a single administration of CBZ did not affect the plasma ALT levels, even when cotreated with BSO, pretreatment with dexamethasone, a CYP3A inducer, increased the plasma ALT levels. In addition, the rats cotreated with troleandomycin or ketoconazole, CYP3A inhibitors, suppressed the increased plasma ALT levels. In conclusion, reactive metabolite(s) of CBZ produced by CYP3A under the GSH-depleted condition might be involved in the development of liver injury in rats.
Shinichi Hayashi, Yoshiro Abe, Shigeki Tomita, Yuko Nakanishi, Shigeharu Miwa, Takahiko Nakajima, Kazuhiro Nomoto, Koichi Tsuneyama, Takahiro Fujimori and Johji Imura : Primary non-gestational pure choriocarcinoma arising in the ovary: A case report and literature review., Oncology Letters, Vol.9, No.5, 2109-2111, 2015.
(Summary)
Pure non-gestational choriocarcinoma is a primary germ cell neoplasm that has been defined as a tumor without other germ cell elements. The current study presents an extremely rare case of non-gestational pure choriocarcinoma in a postmenarcheal young female and describes details of the tumor, including the clinicopathological findings. The patient was a 10-year-old female who underwent salpingo-oophorectomy. Histologically, the extensive hemorrhagic tumor was composed of choriocarcinoma without additional germ cell tumor components. The tumor cells were immunohistochemically positive for epithelial markers, including cytokeratins and epithelial membrane antigens, and there was a positive cytoplasmic reaction for -human chorionic gonadotropin in the syncytiotrophoblasts. Furthermore, numerous tumor cells were immunohistochemically positive for the 2-microglobulin antibody. The patient received adjuvant cisplatin, etoposide and bleomycin chemotherapy, and is currently disease-free without evidence of recurrence or metastasis subsequent to 62 months of follow-up.
Fengying Gao, Satoru Yokoyama, Makoto Fujimoto, Koichi Tsuneyama, Ikuo Saiki, Yutaka Shimada and Yoshihiro Hayakawa : Effect of keishibukuryogan on genetic and dietary obesity models., Evidence-Based Complementary and Alternative Medicine : eCAM, Vol.2015, 801291, 2015.
(Summary)
Obesity has been recognized as one of the most important risk factors for a variety of chronic diseases, such as diabetes, hypertension/cardiovascular diseases, steatosis/hepatitis, and cancer. Keishibukuryogan (KBG, Gui Zhi Fu Ling Wan in Chinese) is a traditional Chinese/Japanese (Kampo) medicine that has been known to improve blood circulation and is also known for its anti-inflammatory or scavenging effect. In this study, we evaluated the effect of KBG in two distinct rodent models of obesity driven by either a genetic (SHR/NDmcr-cp rat model) or dietary (high-fat diet-induced mouse obesity model) mechanism. Although there was no significant effect on the body composition in either the SHR rat or the DIO mouse models, KBG treatment significantly decreased the serum level of leptin and liver TG level in the DIO mouse, but not in the SHR rat model. Furthermore, a lower fat deposition in liver and a smaller size of adipocytes in white adipose tissue were observed in the DIO mice treated with KBG. Importantly, we further found downregulation of genes involved in lipid metabolism in the KBG-treated liver, along with decreased liver TG and cholesterol level. Our present data experimentally support in fact that KBG can be an attractive Kampo medicine to improve obese status through a regulation of systemic leptin level and/or lipid metabolism.
Masashi Ikutani, Koichi Tsuneyama, Susumu Nakae and Kiyoshi Takatsu : Emerging roles of IL-33 in inflammation and immune regulation, Inflammation and Regeneration, Vol.35, No.2, 69-77, 2015.
(Summary)
Interleukin-33 (IL-33) belongs to the IL-1 family of cytokines and has been reported to play multiple roles in host defense, allergies and chronic inflammation. Constitutive expression of IL-33 in epithelial cells ensures rapid immune responses against invading pathogens such as parasites and viruses. Tissue damage caused by pathogens results in the release of extracellular IL-33 that in turn alerts a variety of immune cells such as group 2 innate lymphoid cells (ILC2s), eosinophils, basophils and mast cells. These cells mediate T helper type 2 (Th2) immune responses to destroy pathogens. IL-33 also plays central roles in mediating allergic diseases including asthma and atopic rhinitis. Although the functions of IL-33 in host defense and allergies initially received most attention, focus is turning to its roles in chronic inflammatory diseases. Recent advances, however, have led to problematic results. In addition to Th2 responses, IL-33 also promotes Th1 responses and there have been positive and negative roles reported for IL-33 in inflammatory diseases. This mini-review will summarize IL-33 biology and discuss issues regarding previously unrecognized roles of IL-33.
Ji-Ye Kee, Aya Ito, Shozo Hojo, Isaya Hashimoto, Yoshiko Igarashi, Koichi Tsuneyama, Kazuhiro Tsukada, Tatsuro Irimura, Naotoshi Shibahara, Ichiro Takasaki, Akiko Inujima, Takashi Nakayama, Osamu Yoshie, Hiroaki Sakurai, Ikuo Saiki and Keiichi Koizumi : CXCL16 suppresses liver metastasis of colorectal cancer by promoting TNF--induced apoptosis by tumor-associated macrophages., BMC Cancer, Vol.14, 949, 2014.
(Summary)
Inhibition of metastasis through upregulation of immune surveillance is a major purpose of chemokine gene therapy. In this study, we focused on a membrane-bound chemokine CXCL16, which has shown a correlation with a good prognosis for colorectal cancer (CRC) patients. We generated a CXCL16-expressing metastatic CRC cell line and identified changes in TNF and apoptosis-related factors. To investigate the effect of CXCL16 on colorectal liver metastasis, we injected SL4-Cont and SL4-CXCL16 cells into intraportal vein in C57BL/6 mice and evaluated the metastasis. Moreover, we analyzed metastatic liver tissues using flow cytometry whether CXCL16 expression regulates the infiltration of M1 macrophages. CXCL16 expression enhanced TNF--induced apoptosis through activation of PARP and the caspase-3-mediated apoptotic pathway and through inactivation of the NF-B-mediated survival pathway. Several genes were changed by CXCL16 expression, but we focused on IRF8, which is a regulator of apoptosis and the metastatic phenotype. We confirmed CXCL16 expression in SL4-CXCL16 cells and the correlation between CXCL16 and IRF8. Silencing of IRF8 significantly decreased TNF--induced apoptosis. Liver metastasis of SL4-CXCL16 cells was also inhibited by TNF--induced apoptosis through the induction of M1 macrophages, which released TNF-. Our findings suggest that the accumulation of M1 macrophages and the enhancement of apoptosis by CXCL16 might be an effective dual approach against CRC liver metastasis. Collectively, this study revealed that CXCL16 regulates immune surveillance and cell signaling. Therefore, we provide the first evidence of CXCL16 serving as an intracellular signaling molecule.
H Tanaka, W Zhang, G-X Yang, Y Ando, T Tomiyama, Koichi Tsuneyama, P Leung, R L. Coppel, A A. Ansari, Z X. Lian, W M. Ridgway, T Joh and M E. Gershwin : Successful immunotherapy of autoimmune cholangitis by adoptive transfer of forkhead box protein 3(+) regulatory T cells., Clinical and Experimental Immunology, Vol.178, No.2, 253-261, 2014.
(Summary)
Treatment of primary biliary cirrhosis (PBC) has lagged behind that of other autoimmune diseases. In this study we have addressed the potential utility of immunotherapy using regulatory T cells (Treg ) to treat murine autoimmune cholangitis. In particular, we have taken advantage of our ability to produce portal inflammation and bile duct cell loss by transfer of CD8(+) T cells from the dominant negative form of transforming growth factor beta receptor type II (dnTGF-RII) mice to recombination-activating gene (Rag)1(-/-) recipients. We then used this robust established adoptive transfer system and co-transferred CD8(+) T cells from dnTGF-RII mice with either C57BL/6 or dnTGF-RII forkhead box protein 3 (FoxP3(+) ) T cells. Recipient mice were monitored for histology, including portal inflammation and intralobular biliary cell damage, and also included a study of the phenotypical changes in recipient lymphoid populations and local and systemic cytokine production. Importantly, we report herein that adoptive transfer of Treg from C57BL/6 but not dnTGF-RII mice significantly reduced the pathology of autoimmune cholangitis, including decreased portal inflammation and bile duct damage as well as down-regulation of the secondary inflammatory response. Further, to define the mechanism of action that explains the differential ability of C57BL/6 Treg versus dnTGF-RII Treg on the ability to down-regulate autoimmune cholangitis, we noted significant differential expression of glycoprotein A repetitions predominant (GARP), CD73, CD101 and CD103 and a functionally significant increase in interleukin (IL)-10 in Treg from C57BL/6 compared to dnTGF-RII mice. Our data reflect the therapeutic potential of wild-type CD4(+) FoxP3(+) Treg in reducing the excessive T cell responses of autoimmune cholangitis, which has significance for the potential immunotherapy of PBC.
Hiroe Honda, Yoshinori Nagai, Takayuki Matsunaga, Naoki Okamoto, Yasuharu Watanabe, Koichi Tsuneyama, Hiroaki Hayashi, Isao Fujii, Masashi Ikutani, Yoshikatsu Hirai, Atsushi Muraguchi and Kiyoshi Takatsu : Isoliquiritigenin is a potent inhibitor of NLRP3 inflammasome activation and diet-induced adipose tissue inflammation., Journal of Leukocyte Biology, Vol.96, No.6, 1087-1100, 2014.
(Summary)
Inflammasome activation initiates the development of many inflammatory diseases, including obesity and type 2 diabetes. Therefore, agents that target discrete activation steps could represent very important drugs. We reported previously that ILG, a chalcone from Glycyrrhiza uralensis, inhibits LPS-induced NF-B activation. Here, we show that ILG potently inhibits the activation of NLRP3 inflammasome, and the effect is independent of its inhibitory potency on TLR4. The inhibitory effect of ILG was stronger than that of parthenolide, a known inhibitor of the NLRP3 inflammasome. GL, a triterpenoid from G. uralensis, had similar inhibitory effects on NLRP3 activity, but high concentrations of GL were required. In contrast, activation of the AIM2 inflammasome was inhibited by GL but not by ILG. Moreover, GL inhibited NLRP3- and AIM2-activated ASC oligomerization, whereas ILG inhibited NLRP3-activated ASC oligomerization. Low concentrations of ILG were highly effective in IAPP-induced IL-1 production compared with the sulfonylurea drug glyburide. In vivo analyses revealed that ILG potently attenuated HFD-induced obesity, hypercholesterolemia, and insulin resistance. Furthermore, ILG treatment improved HFD-induced macrovesicular steatosis in the liver. Finally, ILG markedly inhibited diet-induced adipose tissue inflammation and IL-1 and caspase-1 production in white adipose tissue in ex vivo culture. These results suggest that ILG is a potential drug target for treatment of NLRP3 inflammasome-associated inflammatory diseases.
Jun-Ling An, Qiao-Hong Ji, Ji-Jiang An, Shinji Masuda and Koichi Tsuneyama : Clinicopathological analysis of CD8-positive lymphocytes in the tumor parenchyma and stroma of hepatocellular carcinoma., Oncology Letters, Vol.8, No.5, 2284-2290, 2014.
(Summary)
Tumor-infiltrating lymphocytes may be a manifestation of antitumor immunity. In the present study, hepatocellular carcinoma (HCC) and pericancerous non-tumor liver tissues samples were obtained from 86 surgical patients who had not received preoperative treatment. The cellular expression levels of CD4 and CD8 were immunohistochemically examined in the two tissue groups using tissue microarrays, to evaluate their clinicopathological relevance. Immunohistochemically, CD4 and CD8 T cells were observed in the tumor parenchyma and tumor stroma, and the intensity of CD4 and CD8 immunoreactivity was homogeneous in all HCC samples examined. Morphometrically, the average numbers of CD4- and CD8-positive T cells were significantly increased in the tumor stroma, compared with those in the tumor parenchyma (tumor stroma versus tumor parenchyma: 22±3.6 versus 7.4±0.9 in CD4, 32.8±4.2 versus 16±2.5 in CD8; both P<0.01). Furthermore, the average numbers of CD8-positive T cells in the tumor parenchyma and stroma were significantly increased, compared with the average numbers of CD4-positive cells (P<0.05). In addition, in the tumor parenchyma and stroma, the average numbers of CD8 T cells were significantly higher in patients with tumor diameters 5 cm compared with those in patients with tumor diameters >5 cm (diameter 5 cm versus diameter >5 cm: 18.1±3.3 versus 12.2±3.8 in tumor parenchyma, 36.5±4.8 versus 21.9±8.9 in tumor stroma; both P<0.05). In addition, CD8 expression was significantly enhanced in patients with chronic hepatitis and cirrhosis, compared with paired tumor parenchymal tissues (P<0.01). Furthermore, a significant positive correlation was observed between CD4 and CD8 expression in the tumor parenchyma and stroma (both P<0.001). These observations suggest that tumor parenchyma- or stroma-infiltrating CD8 T cells may be involved in HCC tumor diameter control.
Koichi Tsuneyama, Takeshi Nishida, Hayato Baba, Shu Taira, Makoto Fujimoto, Kazuhiro Nomoto, Shinichi Hayashi, Shigeharu Miwa, Takahiko Nakajima, Mitsuko Sutoh, Emu Oda, Ryoji Hokao and Johji Imura : Neonatal monosodium glutamate treatment causes obesity, diabetes, and macrovesicular steatohepatitis with liver nodules in DIAR mice., Journal of Gastroenterology and Hepatology, Vol.29, No.9, 1736-1743, 2014.
(Summary)
Non-alcoholic steatohepatitis (NASH) is the hepatic manifestation of metabolic syndrome (MS). Monosodium glutamate (MSG)-treated ICR mice is a useful model of MS and NASH, but it shows the different patterns of steatosis from human NASH. Because inbred aged DIAR (ddY, Institute for Animal Reproduction) mice spontaneously show the similar pattern of steatosis as NASH, we analyzed their liver pathology after administering MSG. MSG-treated DIAR mice (DIAR-MSG) and untreated DIAR mice (DIAR-controls) were sacrificed and assessed histopathologically at 29, 32, 40, 48, and 54 weeks of age. The NASH activity score, body mass index, blood glucose level, and oral glucose tolerance test were also assessed. The body mass index and blood glucose levels of DIAR-MSG were significantly higher than controls. The oral glucose tolerance test revealed a type 2 diabetes pattern in DIAR-MSG. The livers of DIAR-MSG mice showed macrovesicular steatosis, lobular inflammation with neutrophils, and ballooning degeneration after 29 weeks. At 54 weeks, mild fibrosis was observed in 5/6 DIAR-MSG and 2/5 DIAR-control mice. In imaging mass spectrometry analysis, cholesterol as well as triglyceride accumulated in the liver of DIAR-MSG mice. Atypical liver nodules were also observed after 32 weeks in DIAR-MSG, some with cellular and structural atypia mimicking human hepatocellular carcinoma. The NASH activity score of DIAR-MSG after 29 weeks was higher than that of control mice, suggesting the development of NASH. DIAR-MSG had NASH-like liver pathology and liver nodules typically associated with MS symptoms. DIAR-MSG provides a valuable animal model to analyze NASH pathogenesis and carcinogenesis.
(Keyword)
Animals / Blood Glucose / Body Mass Index / Cholesterol / Diabetes Mellitus / Flavoring Agents / Humans / Liver / Male / Metabolic Syndrome X / Mice, Inbred Strains / Non-alcoholic Fatty Liver Disease / Obesity / Sodium Glutamate / Triglycerides
Wei Yang, Yuan Yao, Yan-Qing Yang, Fang-Ting Lu, Liang Li, Yin-Hu Wang, Takahiko Nakajima, Koichi Tsuneyama, William M. Ridgway, M Eric Gershwin and Zhe-Xiong Lian : Differential modulation by IL-17A of Cholangitis versus Colitis in IL-2R deleted mice., PLoS ONE, Vol.9, No.8, e105351, 2014.
(Summary)
IFN- is a signature Th1 cell associated cytokine critical for the inflammatory response in autoimmunity with both pro-inflammatory and potentially protective functions. IL-17A is the hallmark of T helper 17 (Th17) cell subsets, produced by T, CD8+ T, NK and NKT cells. We have taken advantage of our colony of IL-2R-/- mice that spontaneously develop both autoimmune cholangitis and inflammatory bowel disease. In this model CD8+ T cells mediate biliary ductular damage, whereas CD4+ T cells mediate induction of colon-specific autoimmunity. Importantly, IL-2R-/- mice have high levels of interferon (IFN-), and interleukin-17A (IL-17A). We produced unique double deletions of mice that were either IL-17A-/-IL-2R-/- or IFN--/-IL-2R-/- to specifically address the precise role of these two cytokines in the natural history of autoimmune cholangitis and colitis. Of note, deletion of IL-17A in IL-2R-/- mice led to more severe liver inflammation, but ameliorated colitis. In contrast, there were no significant changes in the immunopathology of double knock-out IFN--/- IL-2R-/- mice, compared to single knock-out IL-2R-/- mice with respect to cholangitis or colitis. Furthermore, there was a significant increase in pathogenetic CD8+ T cells in the liver of IL-17A-/-IL-2R-/- mice. Our data suggest that while IL-17A plays a protective role in autoimmune cholangitis, it has a pro-inflammatory role in inflammatory bowel disease. These data take on particular significance in the potential use of anti-IL-17A therapy in humans with primary biliary cirrhosis.
Jinjun Wang, Guo-Xiang Yang, Koichi Tsuneyama, M Eric Gershwin, William M. Ridgway and Patrick S. C. Leung : Animal models of primary biliary cirrhosis., Seminars in Liver Disease, Vol.34, No.3, 285-296, 2014.
(Summary)
Within the last decade, several mouse models that manifest characteristic features of primary biliary cirrhosis (PBC) with antimitochondrial antibodies (AMAs) and immune-mediated biliary duct pathology have been reported. Here, the authors discuss the current findings on two spontaneous (nonobese diabetic autoimmune biliary disease [NOD.ABD] and dominant negative transforming growth factor- receptor II [dnTGFRII]) and two induced (chemical xenobiotics and microbial immunization) models of PBC. These models exhibit the serological, immunological, and histopathological features of human PBC. From these animal models, it is evident that the etiology of PBC is multifactorial and requires both specific genetic predispositions and environmental insults (either xenobiotic chemicals or microbial), which lead to the breaking of tolerance and eventually liver pathology. Human PBC is likely orchestrated by multiple factors and hence no single model can fully mimic the immunopathophysiology of human PBC. Nevertheless, knowledge gained from these models has greatly advanced our understanding of the major immunological pathways as well as the etiology of PBC.
Hayato Baba, Koichi Tsuneyama, Takeshi Nishida, Hideki Hatta, Takahiko Nakajima, Kazuhiro Nomoto, Shinichi Hayashi, Shigeharu Miwa, Yuko Nakanishi, Ryoji Hokao and Johji Imura : Neonatal streptozotocin treatment causes type 1 diabetes and subsequent hepatocellular carcinoma in DIAR mice fed a normal diet., Hepatology International, Vol.8, No.3, 415-424, 2014.
(Summary)
Hepatocellular carcinoma (HCC) represents a major health challenge because of its increasing morbidity and mortality. The establishment of useful models of HCC can significantly contribute to unveiling its pathophysiology. We developed a novel mouse model of HCC based on type 1 diabetes and reported its histopathological features. Newborn male ddY, Institute for Animal Reproduction (DIAR) mice were divided into two groups on the basis of streptozotocin treatment, which induces type 1 diabetes. Streptozotocin was subcutaneously injected (60 mg/g) into the treated group (DIAR-nSTZ mice), whereas physiologic solution was injected into the control group (DIAR-control mice) at 1.5 days after birth. All mice were fed a normal diet and histopathologically assessed at 6, 8, 10, 12, 19, and 27 weeks of age. At 8 weeks, small hepatocytic nodules with mild to moderate cellular atypia were observed in the livers of DIAR-nSTZ mice, which progressed to large hepatocytic nodules with cellular atypia and infiltrating growth at 12 weeks, identical to those in well-differentiated human HCC. At 19 and 27 weeks, moderately differentiated HCC was observed in all DIAR-nSTZ mice. Conversely, no neoplastic findings were evident in DIAR-control mice. No steatosis or fibrosis was evident in either group. Immunohistochemical analysis revealed that all nodules observed in DIAR-nSTZ mice were positive for glutamine synthetase. In DIAR-nSTZ mice, the development of HCC with similarity to human HCC and high reproducibility can be achieved using a short and simple protocol. We believe that this model will be useful for studying liver carcinogenesis.
Mayuko Shimizu, Miku Kawase, Miki Masuzumi, Mika Sakaki, Yasuo Nagata, Kazunari Tanaka, Kazuhito Suruga, Shizuka Tamaru, Shigeko Kato, Koichi Tsuneyama and Katsuhisa Omagari : High-fat and high-cholesterol diet rapidly induces non-alcoholic steatohepatitis with advanced fibrosis in Sprague-Dawley rats., Hepatology Research, Vol.45, No.4, 458-469, 2014.
(Summary)
The development of fibrosis is considered an important phase in the progress of non-alcoholic steatohepatitis (NASH) towards the end stage of liver disease, including cirrhosis. However, few small animal models can display NASH-associated fibrosis. We aimed to establish a dietary model of NASH with rapid progression to fibrosis using genetically normal rats. Nine-week-old male Sprague-Dawley rats were fed with normal, high-fat (HF), or two types of high-fat and high-cholesterol (HFC) diets for 9 weeks (n = 5 each). All HFC diets contained 1.25% or 2.5% cholesterol. The rats fed with the HF diet developed mild steatosis and inflammation without fibrosis at 18 weeks of age, whereas all rats given the HFC diet developed obvious steatosis and inflammation with hepatocyte ballooning and fibrosis. Two of five (40%) rats given the HFC diet containing 2.5% cholesterol progressed to liver cirrhosis. Hepatic total cholesterol levels were significantly higher in rats given the HFC, than the normal or HF diets. The HFC diet significantly and dose-dependently decreased microsomal triglyceride transfer protein expression. Cholesterol tended to suppress carnitine palmitoyltransferase activity and adenosine triphosphate-binding cassette transporter G5 expression. Adding cholesterol to the HF diet modified hepatic lipid metabolism at the molecular level. The HFC diet induced hepatic features of NASH and eventually progressed cirrhosis in Sprague-Dawley rats within 9 weeks.
Yu Yamaura, Miki Nakajima, Naoyuki Tatsumi, Shingo Takagi, Tatsuki Fukami, Koichi Tsuneyama and Tsuyoshi Yokoi : Changes in the expression of miRNAs at the pericentral and periportal regions of the rat liver in response to hepatocellular injury: comparison with the changes in the expression of plasma miRNAs., Toxicology, Vol.322, 89-98, 2014.
(Summary)
MicroRNAs (miRNAs) in body fluids have received attention as potential biomarkers of organ damage because miRNAs that are highly or specifically expressed in a given organ are likely released into body fluids as a result of damage to that organ. We previously determined that the plasma miRNA profile in rats was dramatically changed due to acetaminophen (APAP)-induced pericentral necrosis and methapyrilene (MP)-induced periportal necrosis in the liver. The purpose of this study was to examine whether the expression of hepatic miRNAs is differentially modulated at different zones due to injury and to examine the relationship of the hepatic miRNA profile with the changes in the plasma miRNA expression profile. Through the laser microdissection of the periportal and periportal regions of the liver and TaqMan microRNA Array analysis, we found that 49 miRNAs are differentially expressed between the pericentral and periportal regions of control rats. In both APAP- and MP-treated rats, the miRNAs that presented decreased expression dominated in both the injured and non-injured areas compared with the miRNAs that exhibited increased expression. The changes in miRNA expression in each region of the liver were compared with those observed in the plasma. Of the 301 plasma miRNAs with expression that was changed as a result of APAP administration, only 21% were changed in the injured area of the liver. Of the 263 plasma miRNAs with expression that was changed due to MP administration, only 24% were changed in the injured area of the liver. Thus, the miRNA expression profiles in the plasma do not merely reflect the release of miRNAs from the damaged cells in the liver. This report provides the first demonstration of zonal miRNA expression in the liver and of the relationship of the miRNA expression profile in a tissue with the plasma miRNA profile.
Yuki Oda, Miki Nakajima, Koichi Tsuneyama, Masataka Takamiya, Yasuhiro Aoki, Tatsuki Fukami and Tsuyoshi Yokoi : Retinoid X receptor in human liver is regulated by miR-34a., Biochemical Pharmacology, Vol.90, No.2, 179-187, 2014.
(Summary)
Retinoid X receptor (RXR) forms a heterodimer with numerous nuclear receptors to regulate drug- or lipid-metabolizing enzymes. In this study, we investigated whether human RXR is regulated by microRNAs. Two potential recognition elements of miR-34a were identified in the RXR mRNA: one in the coding region and the other in the 3'-untranslated region (3'-UTR). Luciferase assays revealed that miR-34a recognizes the element in the coding region. The overexpression of miR-34a in HepG2 cells significantly decreased the endogenous RXR protein and mRNA levels. The stability of RXR mRNA was decreased by the overexpression of miR-34a, indicating that miR-34a negatively regulates RXR expression by facilitating mRNA degradation. We found that the miR-34a-dependent down-regulation of RXR decreases the induction of CYP26 and the transactivity of CYP3A4. miR-34a has been reported to be up-regulated by p53, which has an ability to promote liver fibrosis. The p53 activation resulted in an increase of the miR-34a level and a decrease of the RXR protein level. In addition, the miR-34a levels in eight fibrotic livers were higher than those in six normal livers, and the reverse trend was found for the RXR protein levels. An inverse correlation was observed between the miR-34a and the RXR protein levels in the 14 samples. Taken together, the data show that miR-34a negatively regulates RXR expression in human liver, and affects the expression of its downstream genes. This miR-34a-dependent regulation might be the underlying mechanism responsible for the decreased expression of the RXR protein in fibrotic livers.
(Keyword)
3' Untranslated Regions / Aged / Aged, 80 and over / Cytochrome P-450 CYP3A / Cytochrome P-450 Enzyme System / Female / Gene Expression Regulation / Genes, Reporter / HEK293 Cells / Hep G2 Cells / Humans / Liver Cirrhosis / Luciferases / Male / MicroRNAs / Middle Aged / Open Reading Frames / RNA Cleavage / RNA Stability / Retinoid X Receptor alpha / Signal Transduction / Transcription, Genetic / Tumor Suppressor Protein p53
Makoto Kurano, Masumi Hara, Koichi Tsuneyama, Hideyuki Sakoda, Tomo Shimizu, Kazuhisa Tsukamoto, Hitoshi Ikeda and Yutaka Yatomi : Induction of insulin secretion by apolipoprotein M, a carrier for sphingosine 1-phosphate., Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, Vol.1841, No.9, 1217-1226, 2014.
(Summary)
High-density lipoprotein (HDL) has been proposed to enhance -cell functions. Clinical studies have suggested that apolipoprotein M (apoM), which rides mainly on HDL, is involved in diabetes; however, the underlying mechanism has not yet been elucidated. Recently, apoM was shown to be a carrier for sphingosine 1-phosphate (S1P), a bioactive lipid mediator. In the present study, we investigated the modulation of insulin secretion by apoM through the action of S1P. We overexpressed apoM in the livers of C57BL6 mice using adenovirus gene transfer and found that the blood glucose levels under ad libitum feeding conditions were lower in the apoM-overexpressing mice. While an insulin tolerance test revealed that insulin sensitivity was not significantly affected, a glucose tolerance test revealed that apoM-overexpressing mice had a better glucose tolerance because of enhanced insulin secretion, a phenomenon that was reversed by treatment with VPC 23019, an antagonist against S1P1 and S1P3 receptor. In vitro experiments with MIN6 cells also revealed that apoM-containing lipoproteins enhanced insulin secretion, which was again inhibited by VPC 23019. ApoM retarded the degradation of S1P, and an increase in Pdx1 expression, the attenuation of endoreticulum stress, and the phosphorylation of Akt, AmpK, and Erk were observed as possible underlying mechanisms for the effect of S1P, maintained at a high concentration by apoM, on the increase in insulin secretion. ApoM augmented insulin secretion by maintaining the S1P concentration under both in vivo and in vitro conditions.
Kazuto Tajiri, Koichi Tsuneyama, Kengo Kawai, Yoshinari Atarashi, Masami Minemura, Shigeaki Sawada, Kazuhiro Tsukada, Johji Imura and Toshiro Sugiyama : A case of progressing focal nodular hyperplasia and its molecular expression pattern., Clinical Journal of Gastroenterology, Vol.7, No.3, 271-277, 2014.
(Summary)
We report the case of an adult male with progressing focal nodular hyperplasia (FNH). Although imaging studies suggested that the tumor was a classical FNH, the tumor biopsy showed glutamine synthetase expression and heat shock protein 70 in part of the tumor. As we could not definitely distinguish this case of FNH from early hepatocellular carcinoma (HCC), we performed laparoscopic partial hepatectomy. The surgical resected specimen showed that the tumor had a central scar with vascular and cholangiolar proliferation, which is compatible with FNH. Immunohistochemical analysis showed that the molecular expression pattern was compatible with FNH in the center of the tumor, whereas it partly resembled early HCC in the periphery of the tumor. FNH progression is occasionally found, and the molecular pattern of the progressing area in FNH might resemble that of early HCC due to morphologic and phenotypic changes induced by the regenerative mechanism and the alteration of blood flow. We should carefully observe progressing FNH.
Chen-Yen Yang, Xiong Ma, Koichi Tsuneyama, Shanshan Huang, Toru Takahashi, Naga P. Chalasani, Christopher L. Bowlus, Guo-Xiang Yang, Patrick S. C. Leung, Aftab A. Ansari, Linda Wu, Ross L. Coppel and M Eric Gershwin : IL-12/Th1 and IL-23/Th17 biliary microenvironment in primary biliary cirrhosis: implications for therapy., Hepatology, Vol.59, No.5, 1944-1953, 2014.
(Summary)
The interleukin (IL)-12/IL-23-mediated Th1/Th17 signaling pathway has been associated with the etiopathogenesis of primary biliary cirrhosis (PBC). To address the cytokine microenvironment specifically in the liver, we examined the localized expression of cytokine subunits and their corresponding receptors using previously optimized immunohistochemistry with an extensive panel of antibodies directed at IL-12p70, IL-12p35, interferon-gamma (IFN-), IL-12RB2, IL-23p40, IL-23p19, IL-17, and IL-23R using liver from PBC (n = 51) and non-PBC (n = 80) control liver disease patients. Multiple portal tracts in each patient were blindly evaluated and individually scored. We report herein that although IL-12/Th1 and IL-23/Th17 staining was detected in all of the liver sections, they were primarily localized around the damaged interlobular bile ducts in PBC. Most important, Th17 skewing was prominent in advanced PBC patients with intensive secretion of IL-23p19 by inflamed hepatocytes around IL-23R, IL-12RB2, and IFN- expressing degenerated cholangiocytes. Our novel finding on the direct association of Th17 skewing and disease severity illustrates the significance of the IL-23/Th17 pathway in the perpetuation of IL-12/Th1-mediated immunopathology in PBC. Furthermore, localized IL-23p19 production by hepatocytes may enhance profibrotic Th17 signaling and proinflammatory IFN- production that contribute to PBC pathology. Our data emphasize the pathogenic relevance of IL-12/Th1 and IL-23/Th17 in the evolution of PBC. Of significance, however, the shift from a Th1 to a Th17 imbalance at advanced stages of the disease suggests the necessity to consider modulation of the IL-23/Th17 pathway as a potential target for therapeutic intervention.
Y-R Deng, K Yoshida, Q L. Jin, M Murata, T Yamaguchi, Koichi Tsuneyama, Y Moritoki, J Q. Niu, K Matsuzaki and Z-X Lian : Reversible phospho-Smad3 signalling between tumour suppression and fibrocarcinogenesis in chronic hepatitis B infection., Clinical and Experimental Immunology, Vol.176, No.1, 102-111, 2014.
(Summary)
Transforming growth factor (TGF)-, type I receptor (TRI) and c-Jun N-terminal kinases (JNK) phosphorylate Smad3 differentially to create 2 isoforms phosphorylated (p) at the COOH-terminus (C) or at the linker region (L) and regulate hepatocytic fibrocarcinogenesis. This study aimed to compare the differences between how hepatitis B virus (HBV) infection affected hepatocytic Smad3 phosphorylated isoforms before and after anti-viral therapy. To clarify the relationship between Smad3 phosphorylation and liver disease progression, we studied 10 random patients in each stage of HBV-related fibrotic liver disease (F1-4) and also 10 patients with HBV-associated HCC. To examine changes in phosphorylated Smad3 signalling before and after anti-HBV therapies, we chose 27 patients with chronic hepatitis B who underwent baseline and follow-up biopsies at 52 weeks from the start of nucleoside analogue treatments (Lamivudine 100 mg daily or Telbivudine 600 mg daily). Fibrosis stage, inflammatory activity and phosphorylated Smad3 positivity in the paired biopsy samples were compared. Hepatocytic pSmad3C signalling shifted to fibrocarcinogenic pSmad3L signalling as the livers progressed from chronic hepatitis B infection to HCC. After nucleoside analogue treatment, serum alanine aminotransferase (ALT) and HBV-DNA levels in 27 patients with HBV-related chronic liver diseases were decreased dramatically. Decrease in HBV-DNA restored pSmad3C signalling in hepatocytes, while eliminating prior fibrocarcinogenic pSmad3L signalling. Oral nucleoside analogue therapies can suppress fibrosis and reduce HCC incidence by successfully reversing phosphorylated Smad3 signalling; even liver disease progressed to cirrhosis in chronic hepatitis B patients.
Zheng-Guo Cui, Jin-Lan Piao, Takashi Kondo, Ryohei Ogawa, Koichi Tsuneyama, Qing-Li Zhao, Loreto B. Feril and Hidekuni Inadera : Molecular mechanisms of hyperthermia-induced apoptosis enhanced by docosahexaenoic acid: implication for cancer therapy., Chemico-Biological Interactions, Vol.215, 46-53, 2014.
(Summary)
To develop a non-toxic enhancer for hyperthermia-induced cell death as a potential cancer treatment, we studied the effect and mechanism of docosahexaenoic acid (DHA) on hyperthermia-induced apoptosis. Treatment with 20M DHA and 44°C for 10min induced significant apoptosis, increased intracellular reactive oxygen species (ROS), and caspase-3 activation in U937 cells, but heat or DHA alone did not induce notable apoptosis. Decreased mitochondrial transmembrane potentials were dramatically increased by the combined treatment, accompanied by increased pro-apoptotic Bcl-2 family protein tBid, and decreased anti-apoptotic Bcl-2 and Bcl-xL. Combined hyperthermia-DHA treatment induced significant phosphorylation of protein kinase C (PKC)- (p-PKC-), and apoptosis in a DHA dose-dependent manner. Using both 20M DHA and 44°C for 10min induced significant PKC- cleavage and its translocation to mitochondria. These results were also seen in HeLa cells. However, MAPKs and Akt were not affected by the treatment. In conclusion, DHA enhances hyperthermia-induced apoptosis significantly via a mitochondria-caspase-dependent pathway; its underlying mechanism involves elevated intracellular ROS, mitochondria dysfunction, and PKC- activation.
Ryo Okada, Kinzo Matsumoto, Ryohei Tsushima, Hironori Fujiwara and Koichi Tsuneyama : Social isolation stress-induced fear memory deficit is mediated by down-regulated neuro-signaling system and Egr-1 expression in the brain., Neurochemical Research, Vol.39, No.5, 875-882, 2014.
(Summary)
We previously reported that social isolation (SI) rearing of rodents not only elicits a variety of behavioral abnormalities including attention deficit hyperactivity disorder-like behaviors, but also impairs fear memory in mice. This study aimed to clarify a putative mechanism underlying SI-induced conditioned fear memory deficit. Mice were group-housed (GH) or socially isolated for 2 weeks or more before the experiments. SI animals acquired contextual and auditory fear memory elucidated at 90 min and 4 h after training, respectively; however, they showed significantly impaired contextual and auditory memory performance at 24 h and 4 days after the training, respectively, indicating SI-induced deficit of the consolidation process of fear memory. Neurochemical studies conducted after behavioral tests revealed that SI mice had a significantly down-regulated level of Egr-1 but not Egr-2 in the hippocampal and cortical cytosolic fractions compared with those levels in the GH control animals. Moreover, in the SI group, phosphorylated levels of synaptic plasticity-related signaling proteins in the hippocampus, NR1 subunit of N-methyl-D-aspartate receptor, glutamate receptor 1, and calmodulin-dependent kinase II but not cyclic AMP-responsive element binding protein were significantly down-regulated compared with those levels in GH animals, whereas non-phosphorylated levels of these proteins were not affected by SI. These findings suggest that dysfunctions of Egr-1 and neuro-signaling systems are involved in SI-induced deficits of fear memory consolidation in mice.
(Keyword)
Animals / Behavior, Animal / Cerebral Cortex / Conditioning, Classical / Down-Regulation / Early Growth Response Protein 1 / Early Growth Response Protein 2 / Fear / Hippocampus / Male / Memory / Memory Disorders / Mice, Inbred ICR / Neuronal Plasticity / Signal Transduction / Social Isolation / Stress, Psychological
Yuan Yao, Wei Yang, Yan-Qing Yang, Hong-Di Ma, Fang-Ting Lu, Liang Li, Yan-Yan Tao, Koichi Tsuneyama, Weici Zhang, Scott Friedman, M Eric Gershwin and Zhe-Xiong Lian : Distinct from its canonical effects, deletion of IL-12p40 induces cholangitis and fibrosis in interleukin-2R-/- mice., Journal of Autoimmunity, Vol.51, 99-108, 2014.
(Summary)
The IL-12 family modulates T cell mediated autoimmune diseases and GWAS in PBC have suggested a critical role of IL-12 and its subunits in modulating portal inflammation. We have taken advantage of an aggressive model of portal inflammation and colitis in IL-2R(-/-) mice to study the specific role of IL-12 and, in particular, the immunobiology of p40(-/-)IL-2R(-/-) mice. Colonies of IL-2R(+/-), IL-2R(-/-) and p40(-/-)IL-2R(-/-) mice were studied for the natural history of immunopathology in liver and colon using histology and immunohistochemistry. Further, to focus on mechanisms, liver, spleen and mesenteric lymph node flow cytometry was employed to identify specific phenotypes; cytokine analysis on inflammatory cell populations was compared between groups. Finally, Real-Time PCR was used to focus on the genes involved in hepatic fibrosis. Surprisingly, p40(-/-)IL-2R(-/-) mice manifest more severe portal inflammation and bile duct damage, including signs of portal hypertension and liver fibrosis, but a significant reduction in colitis. Indeed, p40(-/-)IL-2R(-/-) mice reveal a profound hepatic CD8(+) T cell infiltrate, whose major component are effector memory cells as well as enhanced hepatic Th1 but reduced Th17 responses. These observations were confirmed by Real-Time PCR analysis of fibrosis-related genes in the liver. Distinct from its canonical effects, IL-12p40 plays a critical role in autoimmune cholangitis, including hepatic fibrosis. These data take on striking significance for any proposed human trials that modulate the IL-12p40 pathway in human PBC.
Johji Imura, Shinichi Hayashi, Kazuhito Ichikawa, Shigeharu Miwa, Takahiko Nakajima, Kazuhiro Nomoto, Koichi Tsuneyama, Tatsuya Nogami, Hitoaki Saitoh and Takahiro Fujimori : Malignant transformation of hyperplastic gastric polyps: An immunohistochemical and pathological study of the changes of neoplastic phenotype., Oncology Letters, Vol.7, No.5, 1459-1463, 2014.
(Summary)
In spite of the evidence that the malignant transformation of gastric hyperplastic polyps (HPs) is a rare event, it must always be taken into account during diagnosis. The aim of the current study was to clarify the mechanism of the malignant transformation of gastric hyperplasia polyps, with focus on phenotypic expression, cell proliferation and p53 overexpression. Immunohistochemistry for mucin phenotypic markers, including MUC1, MUC2, MUC5AC, MUC6, tight junction factors (claudin-3, -4 and -18), an intestinal phenotypic marker [caudal type homeobox 2 (Cdx2)], Ki-67 proliferative index and p53 overexpression, was performed on archival specimens of gastric polyps excised from six patients. Histologically, the intermingled components of several lesions were present in these polyps. Furthermore, the cancer components were predominantly differentiated adenocarcinoma. Immunohistochemically, all hyperplastic components expressed MUC5AC, but did not exhibit positivity for MUC2. Additionally, the majority of hyperplastic components were immunonegative for claudin-3, while claudin-3 positivity was observed in the majority of areas of dysplasia and carcinoma. Expression of claudin-4 was also observed in the majority of cases and claudin-18 was preserved in the hyperplastic, dysplastic and adenocarcinomatous lesions of all cases. Nuclear accumulation of Cdx2 was detected in almost all the samples with dysplasia and carcinoma, while nuclear p53 was detected in 24-80% of the dysplastic areas and >85% of the cancer components. The Ki-67 labeling index appeared to correlate with neoplastic progression. The observations provided evidence that the mechanism underlying malignant transformation of gastric HPs may occur by multistep carcinogenesis, such as the hyperplasia-adenoma (dysplasia)-adenocarcinoma sequence, and these neoplastic cells may acquire various phenotypes during this process.
Makoto Fujimoto, Koichi Tsuneyama, Yuko Nakanishi, Thucydides L. Salunga, Kazuhiro Nomoto, Yoshiyuki Sasaki, Seiichi Iizuka, Mitsunobu Nagata, Wataru Suzuki, Tsutomu Shimada, Masaki Aburada, Yutaka Shimada, M Eric Gershwin and Carlo Selmi : A dietary restriction influences the progression but not the initiation of MSG-Induced nonalcoholic steatohepatitis., Journal of Medicinal Food, Vol.17, No.3, 374-383, 2014.
(Summary)
The metabolic syndrome is a major worldwide health care issue and a dominant risk factor for cardiovascular disease. The liver manifestations of this syndrome include nonalcoholic fatty liver disease (NAFLD) and its progressive variant nonalcoholic steatohepatitis (NASH). Although significant research has been performed, the basic pathogenesis of NAFLD/NASH remains controversial and effective treatments are still unavailable. We have previously reported on a murine model of NASH induced by the neonatal injection of monosodium glutamate (MSG), which includes the clinical manifestations of central obesity, diabetes, hyperlipidemia, and ultimately liver inflammation, fibrosis, and cancer. Although MSG is considered a safe food additive, its administration to pregnant rats increases the voracity and growth hormone levels in the offspring. To further understand the biology of this model, we have investigated the influence of the calorie intake on these clinical manifestations by feeding animals a restrictive diet. MSG-treated animals fed a restrictive diet continue to manifest obesity and early stage NASH but have improvements in serum lipid profiles. At 12 months of age, mice had manifestations of obesity, whether animals were fed a restricted or control diet, but animals fed a restrictive diet had a reduction in the progression of NASH. In conclusion, MSG appears to be a critical factor in the initiation of obesity, whereas calorie intake may modulate the progression of disease.
Hideki Hatta, Koichi Tsuneyama, Kazuhiro Nomoto, Shinichi Hayashi, Shigeharu Miwa, Takahiko Nakajima, Takeshi Nishida, Yuko Nakanishi and Johji Imura : A simple and rapid decalcification procedure of skeletal tissues for pathology using an ultrasonic cleaner with D-mannitol and formic acid., Acta Histochemica, Vol.116, No.5, 753-757, 2014.
(Summary)
Decalcification procedures are required in order to prepare histopathological preparations of hard tissues such as bone and teeth. Decalcification is usually performed by immersing the hard tissue in different decalcification fluids with various properties. These decalcification fluids typically include inorganic and organic acids, a neutral fluid containing a chelating agent, or a mixture of solutions. Unfortunately, there is no universal decalcification fluid that satisfies all the requirements of pathologists such as rapid decalcification, easy handling, and minimal tissue damage. Techniques involving use of microwaves (MW) or ultrasonic apparatus (US) have been shown to be useful for shortening the time for decalcification procedures. In the present study, we investigated a unique decalcification procedure that uses a common commercial ultrasonic cleaner and a decalcification fluid (formic acid) containing a free-radical scavenger (D-mannitol). The time required to complete the procedure is approximately half of that required to complete a standard decalcification procedure. In addition, tissue morphology and antigenicity is fairly well preserved after decalcification. The procedure is quick, easy to perform, and achieves decalcification of hard tissue with minimal tissue damage.
(Keyword)
Bone and Bones / Decalcification Technique / Formates / Humans / Mannitol / Pathology / Time Factors / Ultrasonics
J J. Wang, G-X Yang, W C. Zhang, L Lu, Koichi Tsuneyama, M Kronenberg, J L. Véla, M Lopez-Hoyos, X-S He, W M. Ridgway, P S. C. Leung and M E. Gershwin : Escherichia coli infection induces autoimmune cholangitis and anti-mitochondrial antibodies in non-obese diabetic (NOD).B6 (Idd10/Idd18) mice., Clinical and Experimental Immunology, Vol.175, No.2, 192-201, 2014.
(Summary)
Several epidemiological studies have demonstrated that patients with primary biliary cirrhosis (PBC) have a higher incidence of urinary tract infections (UTI) and there is significant homology of the immunodominant mitochondrial autoantigen, the E2 component of the pyruvate dehydrogenase complex (PDC-E2), between mammals and bacteria. Previous work has demonstrated that non-obese diabetic (NOD).B6 Idd10/Idd18 infected with Novosphingobium aromaticivorans developed liver lesions similar to human PBC. It was postulated that the biliary disease was dependent upon the presence of the unique N. aro glycosphingolipids in activating natural killer T (NK T) cells. To address this issue, we infected NOD.B6 Idd10/Idd18 mice with either Escherichia coli, N. aro or use of a phosphate-buffered saline (PBS) vehicle control and serially followed animals for the appearance of liver pathology and anti-mitochondrial autoantibodies (AMA). Of striking importance, the biliary disease of E. coli-infected mice was more severe than N. Aro-infected mice and the titre of AMA was higher in E. coli-infected mice. Furthermore, the immunopathology did not correlate with the ability of bacterial extracts to produce antigen-dependent activation of NK T cells. Our data suggest that the unique glycosphingolipids of N. aro are not required for the development of autoimmune cholangitis. Importantly, the data highlight the clinical significance of E. coli infection in a genetically susceptible host, and we suggest that the appearance of autoimmune cholangitis is dependent upon molecular mimicry. These data highlight that breach of tolerance to PDC-E2 is probably the first event in the natural history of PBC in genetically susceptible hosts.
Hayato Baba, Koichi Tsuneyama, Tokimasa Kumada, Keiko Aoshima and Johji Imura : Histopathological analysis for osteomalacia and tubulopathy in itai-itai disease., The Journal of Toxicological Sciences, Vol.39, No.1, 91-96, 2014.
(Summary)
Cadmium (Cd) is a widespread environmental contaminant that causes both renal tubulopathy and osteomalacia. Osteomalacia is thought to be a result of renal tubulopathy, but there are few studies about the histopathological relationship between the two pathoses. Therefore, in the present study, we examined specimens from cases of itai-itai disease (IID), the most severe form of chronic cadmium poisoning, to evaluate the relationship between them. We analyzed kidney and bone specimens of 61 IID cases and the data regarding Cd concentration in kidney and bone. Tubulopathy was graded on the basis of a three-step scale (mild, moderate, and severe) using the following three items: the degree of proximal tubular defluxion, thickness of renal cortex, and weight of the kidney. Osteomalacia was evaluated using the relative osteoid volume (ROV). There were 15 cases of mild, 19 cases of moderate, and 27 cases of severe tubulopathy. The average ROV was 24.9 ± 2.0%. ROV tended to increase as tubulopathy advanced in severity, and ROV was significantly higher in cases with severe tubulopathy than those with mild or moderate tubulopathy. ROV had a negative correlation with Cd concentration in the kidney but no correlation with that in the bone. Our results suggest that the development of osteomalacia was related to the development of tubulopathy.
(Keyword)
Aged / Aged, 80 and over / Bone and Bones / Cadmium Compounds / Cadmium Poisoning / Environmental Pollutants / Female / Humans / Kidney / Kidney Diseases / Kidney Tubules / Male / Middle Aged / Osteomalacia
To reveal the site of immunoglobulin (Ig)M production in primary biliary cirrhosis (PBC) we performed immunohistochemical analysis on spleens collected from patients with PBC. Splenic tissue samples were collected at the time of the autopsy from patients with hepatic failure. Immunostaining for IgM, CD21 and CXCL13 were performed using the splenic tissue samples. The samples from five out of eight cases with PBC but not in eight cases of chronic hepatitis C virus infection showed accumulation of IgM positive cells in CD21 positive lymph follicles. The CXCL13 positive cells also accumulated in the center of the lymph follicles where the IgM positive cells accumulated. The present results suggest that excess IgM is produced from the spleen of PBC. Furthermore, it was suggested that CXCL13 positive follicular dendritic cells possibly contribute to this process.
Katsuhisa Omagari, Chisato Yoshikawa, Shin-ichi Inoue, Yuna Tanaka, Toshie Murayama, Mayuko Shimizu, Ayako Miyata, Sawako Mori, Mai Kamogawa, Eri Hirao, Shigeko Kato, Kazuhito Suruga and Koichi Tsuneyama : Abdominal subcutaneous adipose tissue accumulation is positively correlated with hepatic steatosis in Sprague-Dawley rats, Acta Medica Nagasakiensia, Vol.59, No.2, 47-56, 2014.
(Summary)
The precise roles of visceral (VAT) or subcutaneous adipose tissue (SAT) on hepatic fat accumulation have not been fullyelucidated. In this report, we examined the correlation between VAT or SAT volume and severity of hepatic fat accumulation. Inthe present study, Sprague-Dawley (SD) rats were fed a standard diet containing 10% fat or a high-fat diet containing 45% or 60%fat for 16 weeks. Abdominal VAT and SAT volume, as well as fat percentage of the liver were measured by computed tomography(CT). Hepatic triglyceride (TG) content and histopathological findings of hepatic steatosis were also examined. Abdominal SATweight/body weight ratio was positively and strongly correlated with abdominal VAT weight/body weight ratio. Fat percentage ofthe liver by CT evaluation, hepatic TG content, and hepatic steatosis score by histopathological evaluation showed positive correlationswith one another. Fat percentage of the liver by CT evaluation and hepatic TG content was positively correlated withboth the abdominal VAT weight/body weight ratio and SAT weight/body weight ratio, respectively. Furthermore, hepatic TG contentwas negatively correlated with the abdominal VAT weight/SAT weight ratio. Our data suggest that abdominal SAT accumulationis positively correlated with hepatic steatosis in SD rats, rather than abdominal VAT accumulation. Further investigations areneeded in order to clarify the precise mechanisms of SAT and VAT effects on the development of hepatic fat accumulation.
Johji Imura, Kaori Abe, Yoshiaki Uchida, Masaharu Shibata, Kazue Tsunematsu, Motohiro Sathoh, Shigeharu Miwa, Takahiko Nakajima, Kazuhiro Nomoto, Shinichi Hayashi and Koichi Tsuneyama : Introduction and utility of liquid-based cytology on aspiration biopsy of peripheral nodular lesions of the lung., Oncology Letters, Vol.7, No.3, 669-673, 2013.
(Summary)
In the present study, aspiration biopsy cytology (ABC) was used for the diagnosis of peripheral nodular lesions in the lung (PNLL), and liquid-based cytology (LBC) was carried out on the material collected to evaluate it in comparison with the conventional method (CM). The subjects comprised 130 cases that underwent computed tomography (CT)-guided ABC for PNLL. A total of 73 cases received a tumor resection, with a diagnosis based on the pathology, while 57 cases were followed up, as the tumor showed no change on the radiological examinations. Biopsy samples from these patients and lavage fluid from the aspiration needles were used for analysis. Cellular material was obtained by centrifugation of the lavage fluid, and samples were prepared by two methods, direct smearing and LBC according to the ThinPrep method. The samples were categorized into three diagnoses: i) Benign, ii) suspicion of malignancy and iii) malignant. Appropriate samples were collected in 72% of cases by LBC, but only in 36% of cases by the CM. There was no marked difference in cellular images between the two methods, with the exception of a few specific cases. LBC on its own provided sensitivity at 68%, specificity at 61% and accuracy at 65%, while a combination of LBC and biopsy markedly improved these figures to 94, 81 and 84%, respectively. The introduction of LBC is considered useful for the cytopathological diagnosis of PNLL by CT-guided ABC. LBC enables the examination of appropriate samples rich in cellular components and supports a biopsy-based diagnosis. A combination of these two methods provides even higher diagnostic accuracy, and LBC is considered an excellent method to evaluate these pathological samples.
H Tanaka, G-X Yang, N Iwakoshi, S J. Knechtle, K Kawata, Koichi Tsuneyama, P Leung, R L. Coppel, A A. Ansari, T Joh, C Bowlus and M E. Gershwin : Anti-CD40 ligand monoclonal antibody delays the progression of murine autoimmune cholangitis., Clinical and Experimental Immunology, Vol.174, No.3, 364-371, 2013.
(Summary)
While there have been significant advances in our understanding of the autoimmune responses and the molecular nature of the target autoantigens in primary biliary cirrhosis (PBC), unfortunately these data have yet to be translated into new therapeutic agents. We have taken advantage of a unique murine model of autoimmune cholangitis in which mice expressing a dominant negative form of transforming growth factor receptor II (dnTGFRII), under the control of the CD4 promoter, develop an intense autoimmune cholangitis associated with serological features similar to human PBC. CD40-CD40 ligand (CD40L) is a major receptor-ligand pair that provides key signals between cells of the adaptive immune system, prompting us to determine the therapeutic potential of treating autoimmune cholangitis with anti-CD40L antibody (anti-CD40L; MR-1). Four-week-old dnTGFRII mice were injected intraperitoneally with either anti-CD40L or control immunoglobulin (Ig)G at days 0, 2, 4 and 7 and then weekly until 12 or 24 weeks of age and monitored for the progress of serological and histological features of PBC, including rigorous definition of liver cellular infiltrates and cytokine production. Administration of anti-CD40L reduced liver inflammation significantly to 12 weeks of age. In addition, anti-CD40L initially lowered the levels of anti-mitochondrial autoantibodies (AMA), but these reductions were not sustained. These data indicate that anti-CD40L delays autoimmune cholangitis, but the effect wanes over time. Further dissection of the mechanisms involved, and defining the events that lead to the reduction in therapeutic effectiveness will be critical to determining whether such efforts can be applied to PBC.
Kentaro Matsuo, Eita Sasaki, Satonori Higuchi, Shohei Takai, Koichi Tsuneyama, Tatsuki Fukami, Miki Nakajima and Tsuyoshi Yokoi : Involvement of oxidative stress and immune- and inflammation-related factors in azathioprine-induced liver injury., Toxicology Letters, Vol.224, No.2, 215-224, 2013.
(Summary)
Drug-induced liver injury (DILI) is a growing concern in the fields of drug development and clinical drug therapy because numerous drugs have been linked to hepatotoxicity. However, it is difficult to predict DILI in humans due to the lack of experimental animal models. Although azathioprine (AZA), which is a widely used immunosuppressive drug, is generally well tolerated, a small number of patients prescribed AZA develop severe hepatitis. However, the mechanism underlying this process has not yet been elucidated. In this study, we developed a mouse model of AZA-induced liver injury and investigated the mechanisms responsible for the hepatotoxicity of AZA. Female BALB/c mice were orally administered AZA. After AZA administration, the plasma levels of alanine aminotransferase and aspartate aminotransferase were increased, and liver damage was confirmed through a histological evaluation. In addition, the hepatic glutathione levels and superoxide dismutase activity were significantly decreased. The plasma levels of reactive oxygen species were significantly increased during the early phase of AZA-induced liver injury, and the hepatic mRNA levels of immune- and inflammation-related factors were also significantly changed. In conclusion, oxidative stress and the subsequently activated immune- and inflammation-related factors are involved in AZA-induced liver injury.
Xiao-Yu Luo, Terumi Takahara, Kengo Kawai, Masayuki Fujino, Toshiro Sugiyama, Koichi Tsuneyama, Kazuhiro Tsukada, Susumu Nakae, Liang Zhong and Xiao-Kang Li : IFN- deficiency attenuates hepatic inflammation and fibrosis in a steatohepatitis model induced by a methionine- and choline-deficient high-fat diet., American Journal of Physiology, Gastrointestinal and Liver Physiology, Vol.305, No.12, G891-9, 2013.
(Summary)
Cytokines play important roles in all stages of steatohepatitis, including hepatocyte injury, the inflammatory response, and the altered function of sinusoidal cells. This study examined the involvement of a major inflammatory cytokine, interferon- (IFN-), in the progression of steatohepatitis. In a steatohepatitis model by feeding a methionine- and choline-deficient high-fat (MCDHF) diet to both wild-type and IFN--deficient mice, the liver histology, expression of genes encoding inflammatory cytokines, and fibrosis-related markers were examined. To analyze the effects of IFN- on Kupffer cells in vitro, we examined the tumor necrosis factor- (TNF-) production by a mouse macrophage cell line. Forty two days of MCDHF diet resulted in weight loss, elevated aminotransferases, liver steatosis, and inflammation in wild-type mice. However, the IFN--deficient mice exhibited less extensive changes. RT-PCR revealed that the expression of tumor necrosis factor- (TNF-), transforming growth factor-, inducible nitric oxide synthase, interleukin-4 and osteopontin were increased in wild-type mice, although they were suppressed in IFN--deficient mice. Seventy days of MCDHF diet induced much more liver fibrosis in wild-type mice than in IFN--deficient mice. The expression levels of fibrosis-related genes, -smooth muscle actin, type I collagen, tissue inhibitor of matrix metalloproteinase-1, and matrix metalloproteinase-2, were dramatically increased in wild-type mice, whereas they were significantly suppressed in IFN--deficient mice. Moreover, in vitro experiments showed that, when RAW 264.7 macrophages were treated with IFN-, they produced TNF- in a dose-dependent manner. The present study showed that IFN- deficiency might inhibit the inflammatory response of macrophages cells and subsequently suppress stellate cell activation and liver fibrosis. These findings highlight the critical role of IFN- in the progression of steatohepatitis.
Kazuhito Kawata, Masanobu Tsuda, Guo-Xiang Yang, Weici Zhang, Hajime Tanaka, Koichi Tsuneyama, Patrick Leung, Xiao-Song He, Stuart Knechtle, Aftab A. Ansari, Ross L. Coppel and M Eric Gershwin : Identification of potential cytokine pathways for therapeutic intervention in murine primary biliary cirrhosis., PLoS ONE, Vol.8, No.9, e74225, 2013.
(Summary)
Primary biliary cirrhosis (PBC) is considered a model autoimmune disease, with the most highly directed and specific autoantibody in both murine and human autoimmunity, the anti-mitochondrial autoantibody (AMA). However, therapeutic advances in this disease have lagged behind. Herein we have taken advantage of our unique model of murine PBC in which mice immunized with 2-octynoic acid coupled to BSA (2OA-BSA), a compound identified by quantitative structure activity relationships (QSAR) of human AMA binding, develop an intense inflammatory cholangitis with striking similarities to humans with PBC. In particular, we have constructed several unique gene-deleted mice, including mice deleted of IL-12p40, IL-12p35, IFN-, IL-23p19, IL-17A, IL-17F and IL-22, immunized these animals with 2OA-BSA and followed the natural history of immunopathology to identify key pathways that might provide clues for successful therapy. Our data indicate that whereas both IL-12/Th1 and IL-23/Th17 are involved in cholangitis, it is the IL-12/Th1 signaling pathway that elicits pathology. In fact, deletion of IFN- prevents disease and suppresses autoantibodies. Importantly, deletion of the Th17 cytokines IL-17A and IL-22, but not IL-17F, reduces biliary damage; IL-17A-knockout mice have reduced levels of anti-mitochondrial antibody. We further demonstrate that the production of IFN- is significantly decreased in the liver of IL-23p19(-/-), IL-17A(-/-) and IL-22(-/-) mice compared with controls. However, the ability of T cells to produce IFN- was not affected in Th17 cytokine-deficient mice. Our data indicate that a deficient Th17 pathway suppresses the accumulation of IFN- producing cells in liver during the early phase of cholangitis. In conclusion, whereas IFN- has a pivotal role in the early events involved in the pathogenesis of autoimmune cholangitis induced by 2OA-BSA, the IL-23/Th17 pathway potentiates the effects of IL-12/IFN--mediated immunopathology.
Eita Sasaki, Kentaro Matsuo, Azumi Iida, Koichi Tsuneyama, Tatsuki Fukami, Miki Nakajima and Tsuyoshi Yokoi : A novel mouse model for phenytoin-induced liver injury: involvement of immune-related factors and P450-mediated metabolism., Toxicological Sciences, Vol.136, No.1, 250-263, 2013.
(Summary)
Drug-induced liver injury is an important issue for drug development and clinical drug therapy; however, in most cases, it is difficult to predict or prevent these reactions due to a lack of suitable animal models and the unknown mechanisms of action. Phenytoin (DPH) is an anticonvulsant drug that is widely used for the treatment of epilepsy. Some patients who are administered DPH will suffer symptoms of drug-induced liver injury characterized by hepatic necrosis. DPH-induced liver injury occurs in 1 in 1000 or 1 in 10 000 patients. Clinically, 75% of patients who develop liver injury develop a fever and 63% develop a rash. In this study, we established a mouse model for DPH-induced liver injury and analyzed the mechanisms for hepatotoxicity in the presence of immune-related or inflammation-related factors and metabolic activation. Female C57BL/6 mice were administered DPH for 5 days in combination with L-buthionine-S,R-sulfoximine. Then, the plasma alanine aminotransferase (ALT) levels were increased, hepatic lesions were observed during the histological evaluations, the hepatic glutathione levels were significantly reduced, and the oxidative stress marker levels were significantly increased. The inhibition of cytochrome P450-dependent oxidative metabolism significantly suppressed the elevated plasma ALT levels and depleted hepatic glutathione. Among the innate immune factors, the hepatic mRNA levels of NACHT, LRR, pyrin domain-containing protein 3, interleukin-1, and damage-associated molecular patterns were significantly increased. Prostaglandin E1 treatment ameliorated the hepatic injury caused by DPH. In conclusion, cytochrome P450-dependent metabolic activation followed by the stimulation of the innate immune responses is involved in DPH-induced liver injury.
Yan-Ru Deng, Hong-Di Ma, Koichi Tsuneyama, Wei Yang, Yin-Hu Wang, Fang-Ting Lu, Cheng-Hai Liu, Ping Liu, Xiao-Song He, Anna Mae Diehl, M Eric Gershwin and Zhe-Xiong Lian : STAT3-mediated attenuation of CCl4-induced mouse liver fibrosis by the protein kinase inhibitor sorafenib., Journal of Autoimmunity, Vol.46, 25-34, 2013.
(Summary)
There have been major advances in defining the immunological events associated with fibrosis in various chronic liver diseases. We have taken advantage of this data to focus on the mechanisms of action of a unique multi-kinase inhibitor, coined sorafenib, on CCl4-induced murine liver fibrosis, including the effects of this agent in models of both acute and chronic CCl4-mediated pathology. Importantly, sorafenib significantly attenuated chronic liver injury and fibrosis, including reduction in liver inflammation and histopathology as well as decreased expression of liver fibrosis-related genes, including -smooth muscle actin, collagen, matrix metalloproteinases and the tissue inhibitor of metalloproteinase-1. Furthermore, sorafenib treatment resulted in translocation of cytoplasmic STAT3 to the nucleus in its active form. Based on this observation, we used hepatocyte-specific STAT3 knockout (STAT3(Hep-/-)) mice to demonstrate that hepatic STAT3 was critical for sorafenib-mediated protection against liver fibrosis, and that the upregulation of STAT3 phosphorylation was dependent on Kupffer cell-derived IL-6. In conclusion, these data reflect the clinical potential of the multi-kinase inhibitor sorafenib for the prevention of fibrosis as well as the treatment of established liver fibrosis and illustrate the immunological mechanisms that underlie the protective effects of sorafenib.
Cheng-Pu Sun, Tzu-Hui Wu, Chun-Chi Chen, Ping-Yi Wu, Yao-Ming Shih, Koichi Tsuneyama and Mi-Hua Tao : Studies of efficacy and liver toxicity related to adeno-associated virus-mediated RNA interference., Human Gene Therapy, Vol.24, No.8, 739-750, 2013.
(Summary)
Adeno-associated virus (AAV)-mediated RNA interference shows promise as a therapy for chronic hepatitis B virus (HBV) infection, but its low efficacy and hepatotoxicity pose major challenges. We have generated AAV vectors containing different promoters and a panel of HBV-specific short hairpin RNAs (shRNAs) to investigate factors that contribute to the efficacy and pathogenesis of AAV-mediated RNA interference. HBV transgenic mice injected with high doses of AAV vectors containing the U6 promoter produced abundant shRNAs, transiently inhibited HBV, but induced severe hepatotoxicity. Sustained HBV suppression without liver toxicity can be achieved by lowering the dose of AAV-U6 vectors. AAVs containing the weaker H1 promoter did not cause liver injury, but their therapeutic efficacy was highly dependent on the sequence of the shRNA. Mice treated with the toxic U6-promoter-driven shRNA showed little change in hepatic microRNA levels, but a dramatic increase in hepatic leukocytes and inflammatory cytokines and chemokines. Hepatotoxicity was completely absent in immunodeficient mice and significantly alleviated in wild-type mice depleted of macrophages and granulocytes, suggesting that host inflammatory responses are the major cause of liver injury induced by the overexpressed shRNAs from AAV-U6 vectors. Our results demonstrate that selection of a highly potent shRNA and control its expression level is critical to achieve sustained HBV suppression without inducing inflammatory side effects.
Koichi Tsuneyama, Hayato Baba, Kentaro Kikuchi, Takeshi Nishida, Kazuhiro Nomoto, Shinichi Hayashi, Shigeharu Miwa, Takahiko Nakajima, Yuko Nakanishi, Shinji Masuda, Mitsuhiro Terada, Johji Imura and Carlo Selmi : Autoimmune features in metabolic liver disease: a single-center experience and review of the literature., Clinical Reviews in Allergy & Immunology, Vol.45, No.1, 143-148, 2013.
(Summary)
Non-alcoholic steatohepatitis (NASH) is the progressive phenotype of non-alcoholic fatty liver disease associated with the metabolic syndrome. The existence of autoimmune features in NASH has been reported, but its significance remains unclear. We herein report the autoantibody profile of 54 patients with histologically proven NASH and further determined the development of autoimmunity in three different murine NASH models (monosodium glutamate, CDAA (choline-deficient L-amino acid-defined), and TSOD (Tsumura Suzuki, Obese Diabetes)) at 48 weeks of age. Forty-eight percent (26/54) of NASH cases were positive for antinuclear (ANA) or antimitochondrial antibody and manifested histological signs of overlap with autoimmune hepatitis and primary biliary cirrhosis, respectively. These patients were significantly older (60 ± 10 versus 50 ± 16 years), more frequently women (81 % versus 43 %), and with more severe portal inflammatory infiltrate compared with patients without autoimmunity. In one third of mice, regardless of the model, we observed a marked lymphoid infiltrate with non-suppurative cholangitis, and several cases were ANA-positive, but none AMA-positive. Our data suggest that autoimmunity may share some pathogenetic traits with the chronic inflammation of NASH, possibly related to advanced age.
Kazuhito Kawata, Guo-Xiang Yang, Yugo Ando, Hajime Tanaka, Weici Zhang, Yoshimasa Kobayashi, Koichi Tsuneyama, Patrick S. C. Leung, Zhe-Xiong Lian, William M. Ridgway, Aftab A. Ansari, Xiao-Song He and M Eric Gershwin : Clonality, activated antigen-specific CD8(+) T cells, and development of autoimmune cholangitis in dnTGFRII mice., Hepatology, Vol.58, No.3, 1094-1104, 2013.
(Summary)
There are several murine models described with features similar to human primary biliary cirrhosis (PBC). Among these models, the one which has the closest serologic features to PBC is a mouse with a T-cell-restricted expression of the dominant negative transforming growth factor receptor type II (dnTGFRII). Our work has demonstrated that CD8(+) T cells from dnTGFRII mice transfer autoimmune cholangitis to Rag1(-/-) recipients. However, it remained unclear whether the autoimmune cholangitis was secondary to an intrinsic function within CD8(+) T cells or due to the abnormal TGFR environment within which CD8(+) T cells were generated. To address this mechanistic issue, we used our dnTGFRII, OT-I/Rag1(-/-) , OT-II/Rag1(-/-) mice and in addition generated OT-I/dnTGFRII/Rag1(-/-) , and OT-II/dnTGFRII/Rag1(-/-) mice in which the entire T-cell repertoire was replaced with ovalbumin (OVA)-specific CD8(+) or CD4(+) T cells, respectively. Importantly, neither the parental OT-I/dnTGFRII/Rag1(-/-) mice and/or OT-II/dnTGFRII/Rag1(-/-) mice developed cholangitis. However, adoptive transfer demonstrated that only transfer of CD8(+) T cells from dnTGFRII mice but not CD8(+) T cells from OT-I/Rag1(-/-) mice or from OT-I/dnTGFRII/Rag1(-/-) mice transferred disease. These data were not secondary to an absence of CD4(+) T cell help since a combination of CD8(+) T cells from OT-I/dnTGFRII/Rag1(-/-) and CD4(+) T cells from OT II/dnTGFRII/Rag1(-/-) or CD8(+) T cells from OT-I/dnTGFRII/Rag1(-/-) with CD4(+) T cells from OT-II/Rag1(-/-) mice failed to transfer disease. Defective TGFRII signaling, in addition to clonal CD8(+) T cells that target biliary cells, are required for induction of autoimmune cholangitis.
Kazuto Tajiri, Koichi Tsuneyama, Takahiro Miyazono, Kengo Kawai, Masami Minemura and Toshiro Sugiyama : A case of primary biliary cirrhosis that progressed rapidly after treatment involving rituximab., Case Reports in Gastroenterology, Vol.7, No.1, 195-201, 2013.
(Summary)
Primary biliary cirrhosis (PBC) is a progressive liver disease for which limited therapies are recommended. Rituximab, an anti-CD20 monoclonal antibody, is expected to be a useful therapeutic regimen for PBC. Previous studies indicated biochemical and immunological improvement in PBC after rituximab treatment. Although rituximab shows therapeutic potential for PBC, few cases have been reported and histological improvement and long-term outcome remain uncertain. Here, we report a case of PBC in a 66-year-old Japanese female patient who presented with a gastric lymphoma and who had been treated with a regimen containing rituximab for incidental malignant lymphoma. She showed biochemical and immunological improvements, and liver histology before and after rituximab treatment confirmed a decrease in liver inflammation. However, she developed liver cirrhosis a short time after rituximab treatment without biochemical or immunological worsening. Rituximab treatment for PBC might be considered and careful observation is required after treatment.
Mayuko Shimizu, Shigeko Kato, Koichi Tsuneyama, Sachiko Matsutake, Mai Kamogawa, Eri Hirao, Ayako Miyata, Sawako Mori, Noriaki Yamaguchi, Kazuhito Suruga and Katsuhisa Omagari : Phycocyanin prevents hypertension and low serum adiponectin level in a rat model of metabolic syndrome., Nutrition Research, Vol.33, No.5, 397-405, 2013.
(Summary)
Endothelial dysfunction is associated with hypertension, atherosclerosis, and metabolic syndrome. Phycocyanin is a pigment found in the blue-green algae, Spirulina, which possesses antihypertensive effect. In this study, we hypothesized that phycocyanin derived from Spirulina exerts antihypertensive actions by improving endothelial dysfunction in metabolic syndrome. Spontaneously hypertensive/NIH-corpulent (SHR/NDmcr-cp) rats were divided into 4 groups then fed a normal diet with or without phycocyanin (2500-, 5000-, or 10,000-mg/kg diet) for 25 weeks. At 34 weeks of age, although systolic blood pressure was not significantly different among groups, phycocyanin-fed groups exhibited a dose-dependent decrease in blood pressure. Serum levels of adiponectin and messenger RNA levels of adiponectin and CCAAT/enhancer-binding protein in the adipose tissue of rats fed diets containing phycocyanin tended to be higher than those of rats fed a normal diet, but the differences were not statistically significant. Immunohistochemistry analysis showed a significant and positive correlation between aortic endothelial nitric oxide synthase (eNOS) expression levels, a downstream target of the adiponectin receptor, and serum adiponectin levels, although there were no significant differences in eNOS expression among groups. There was also no significant correlation between eNOS expression levels and systolic blood pressure. These results suggest that long-term administration of phycocyanin may ameliorate systemic blood pressure by enhancing eNOS expression in aorta that is stimulated by adiponectin. Phycocyanin may be beneficial for preventing endothelial dysfunction-related diseases in metabolic syndrome.
(Keyword)
Adiponectin / Animals / Antihypertensive Agents / Aorta / Blood Pressure / Body Weight / CCAAT-Enhancer-Binding Protein-alpha / Cyanobacteria / Disease Models, Animal / Endothelium, Vascular / Energy Intake / Hypertension / Liver / Male / Metabolic Syndrome X / Nitric Oxide Synthase Type III / Organ Size / Phycocyanin / Rats / Rats, Inbred SHR / Receptors, Adiponectin
Hayato Baba, Koichi Tsuneyama, Megumi Yazaki, Kohei Nagata, Takashi Minamisaka, Tatsuhiro Tsuda, Kazuhiro Nomoto, Shinichi Hayashi, Shigeharu Miwa, Takahiko Nakajima, Yuko Nakanishi, Keiko Aoshima and Johji Imura : The liver in itai-itai disease (chronic cadmium poisoning): pathological features and metallothionein expression., Modern Pathology, Vol.26, No.9, 1228-1234, 2013.
(Summary)
Cadmium (Cd) is a highly hepatotoxic heavy metal, which is widely dispersed in the environment. Acute Cd hepatotoxicity has been well studied in experimental animals; however, effects of prolonged exposure to Cd doses on the liver remain unclear. In the present study, to evaluate chronic Cd hepatotoxicity, we examined specimens from cases of itai-itai disease, the most severe form of chronic Cd poisoning. We compared 89 cases of itai-itai disease with 27 control cases to assess Cd concentration in organs. We also examined 80 cases of itai-itai disease and 70 control cases for histopathological evaluation. In addition, we performed immunohistochemistry for metallothionein, which binds and detoxifies Cd. Hepatic Cd concentration was higher than Cd concentration in all other organs measured in the itai-itai disease group, whereas it was second highest following renal concentration in the control group. In the liver in the itai-itai disease group, fibrosis was observed at a significantly higher rate than that in the control group. Metallothionein expression was significantly higher in the itai-itai disease group than that in the control group. Prolonged exposure to low doses of Cd leads to high hepatic accumulation, which can then cause fibrosis; however, it also causes high expression of metallothionein, which is thought to reduce Cd hepatotoxicity.
(Keyword)
Aged / Aged, 80 and over / Autopsy / Cadmium / Cadmium Poisoning / Case-Control Studies / Chi-Square Distribution / Drug-Induced Liver Injury, Chronic / Environmental Pollutants / Female / Humans / Immunohistochemistry / Liver / Liver Cirrhosis / Metallothionein / Middle Aged / Predictive Value of Tests / Up-Regulation
S Fujisaka, I Usui, M Ikutani, A Aminuddin, A Takikawa, Koichi Tsuneyama, A Mahmood, N Goda, Y Nagai, K Takatsu and K Tobe : Adipose tissue hypoxia induces inflammatory M1 polarity of macrophages in an HIF-1-dependent and HIF-1-independent manner in obese mice., Diabetologia, Vol.56, No.6, 1403-1412, 2013.
(Summary)
As obesity progresses, adipose tissue exhibits a hypoxic and inflammatory phenotype characterised by the infiltration of adipose tissue macrophages (ATMs). In this study, we examined how adipose tissue hypoxia is involved in the induction of the inflammatory M1 and anti-inflammatory M2 polarities of ATMs. The hypoxic characteristics of ATMs were evaluated using flow cytometry after the injection of pimonidazole, a hypoxia probe, in normal-chow-fed or high-fat-fed mice. The expression of hypoxia-related and inflammation-related genes was then examined in M1/M2 ATMs and cultured macrophages. Pimonidazole uptake was greater in M1 ATMs than in M2 ATMs. This uptake was paralleled by the levels of inflammatory cytokines, such as TNF-, IL-6 and IL-1. The expression level of hypoxia-related genes, as well as inflammation-related genes, was also higher in M1 ATMs than in M2 ATMs. The expression of Il6, Il1 and Nos2 in cultured macrophages was increased by exposure to hypoxia in vitro but was markedly decreased by the gene deletion of Hif1a. In contrast, the expression of Tnf, another inflammatory cytokine gene, was neither increased by exposure to hypoxia nor affected by Hif1a deficiency. These results suggest that hypoxia induces the inflammatory phenotypes of macrophages via Hif1a-dependent and -independent mechanisms. On the other hand, the expression of inflammatory genes in cultured M2 macrophages treated with IL-4 responded poorly to hypoxia. Adipose tissue hypoxia induces an inflammatory phenotype via Hif1a-dependent and Hif1a-independent mechanisms in M1 ATMs but not in M2 ATMs.
Takashi Yamaguchi, Koichi Matsuzaki, Ryosuke Inokuchi, Rinako Kawamura, Katsunori Yoshida, Miki Murata, Junichi Fujisawa, Nobuyoshi Fukushima, Michio Sata, Masayoshi Kage, Osamu Nakashima, Akihiro Tamori, Norifumi Kawada, Koichi Tsuneyama, Steven Dooley, Toshihito Seki and Kazuichi Okazaki : Phosphorylated Smad2 and Smad3 signaling: Shifting between tumor suppression and fibro-carcinogenesis in chronic hepatitis C., Hepatology Research, Vol.43, No.12, 1327-1342, 2013.
(Summary)
Insight into hepatic fibrogenesis and carcinogenesis (fibro-carcinogenesis) caused by hepatitis C virus (HCV) infection has come from recent analyses of transforming growth factor (TGF)- signaling. TGF- type I receptor and pro-inflammatory cytokine-activated kinases differentially phosphorylate Smad2 and Smad3 to create C-terminally (C), linker (L) or dually (L/C) phosphorylated (p) isoforms. This study aimed to elucidate how HCV infection affected hepatic fibro-carcinogenesis, particularly via phospho-Smad signaling. We first studied phospho-Smad2/3 positivity of 100 patients in different stages of HCV-related chronic liver disease. To examine changes in phospho-Smad2/3 after HCV clearance, we analyzed 32 paired liver biopsy samples obtained before and after sustained virological response (SVR), dividing patients into two groups: 20 patients not developing hepatocellular carcinoma (HCC) after attaining SVR (non-HCC group), and 12 patients who developed HCC despite SVR (HCC group). Hepatocytic tumor-suppressive pSmad3C signaling shifted to carcinogenic pSmad3L and fibrogenic pSmad2L/C signaling as liver diseases progressed. In the non-HCC group, 13 patients (65%) displayed fibrotic regression and inflammation reduction after SVR. Interestingly, SVR restored cytostatic pSmad3C signaling in hepatocytes, while eliminating prior carcinogenic pSmad3L and fibrogenic pSmad2L/C signaling. In the HCC group, seven patients (58%) displayed unchanged or even progressed fibrosis despite smoothened inflammatory activity, reflecting persistently high numbers of hepatocytes with pSmad3L- and pSmad2L/C-signaling and low pSmad3C-signaling. HCV clearance limits fibrosis and reduces HCC incidence by switching inflammation-dependent phospho-Smad signaling from fibro-carcinogenesis to tumor suppression. However, progression to HCC would occur in severely fibrotic livers if an inflammation-independent fibro-carcinogenic process has already begun before HCV clearance.
Dong Han, Yasuhiko Yamamoto, Seiichi Munesue, So Motoyoshi, Hidehito Saito, Myat Thu Thu Win, Takuo Watanabe, Koichi Tsuneyama and Hiroshi Yamamoto : Induction of receptor for advanced glycation end products by insufficient leptin action triggers pancreatic -cell failure in type 2 diabetes., Genes to Cells, Vol.18, No.4, 302-314, 2013.
(Summary)
Glucolipotoxicity, which is exerted by free fatty acids (FFA) and prolonged hyperglycemia, is implicated in pancreatic -cell failure in diabetes. Pattern recognition receptors such as receptor for advanced glycation end products (RAGE) and toll-like receptors 2 and 4 could mediate danger signals in -cells. We examined whether RAGE contributes to -cell failure in a type 2 diabetes mouse model. Pancreatic islets were isolated from ob/ob, db/db, diet-induced obesity (DIO), RAGE-null (RAGE(-/-) ), and RAGE(+/+) wild-type (WT) control mice and dispersed into single cells for flow cytometry. RAGE expression was detected in insulin-positive -cells of ob/ob and db/db mice, but not of WT, DIO, or RAGE(-/-) mice: thus, inadequate leptin receptor signaling and RAGE expression may be linked. Compared with RAGE(+/+) db/db mice, RAGE(-/-) db/db mice showed higher -cell number and mass with less apoptosis as well as glucose tolerance with higher insulin secretion without any differences in serum levels of FFA and adiponectin. Palmitate or oleate pretreatment combined with a leptin antagonist induced RAGE expression, AGE-elicited apoptosis, and impaired glucose-stimulated insulin secretion by advanced glycation end products (AGE) in MIN6 cells. FFA elevation with concomitant AGE formation during prolonged hyperglycemia could cause -cell damage through insufficient leptin action and subsequent RAGE induction in type 2 diabetes.
Masanobu Tsuda, Weici Zhang, Guo-Xiang Yang, Koichi Tsuneyama, Yugo Ando, Kazuhito Kawata, Ogyi Park, C Patrick S Leung, L Ross Coppel, A Aftab Ansari, M William Ridgway, Bin Gao, Zhe-Xiong Lian, Richard Flavell, Xiao-Song He and Eric M Gershwin : Deletion of interleukin (IL)-12p35 induces liver fibrosis in dominant-negative TGFβ receptor type II mice., Hepatology, Vol.57, No.2, 806-816, 2013.
(Summary)
Mice with a dominant-negative transforming growth factor β receptor restricted to T cells (dnTGFβRII mice) develop an inflammatory biliary ductular disease that strongly resembles human primary biliary cirrhosis (PBC). Furthermore, deletion of the gene encoding interleukin (IL)-12p40 resulted in a strain (IL-12p40(-/-) dnTGFβRII) with dramatically reduced autoimmune cholangitis. To further investigate the role of the IL-12 cytokine family in dnTGFβRII autoimmune biliary disease, we deleted the gene encoding the IL-12p35 subunit from dnTGFβRII mice, resulting in an IL-12p35(-/-) dnTGFβRII strain which is deficient in two members of the IL-12 family, IL-12 and IL-35. In contrast to IL-12p40(-/-) mice, the IL-12p35(-/-) mice developed liver inflammation and bile duct damage with similar severity but delayed onset as the parental dnTGFβRII mice. The p35(-/-) mice also demonstrated a distinct cytokine profile characterized by a shift from a T-helper 1 (Th1) to a Th17 response. Strikingly, liver fibrosis was frequently observed in IL-12p35(-/-) mice. In conclusion, IL-12p35(-/-) dnTGFβRII mice, histologically and immunologically, reflect key features of PBC, providing a useful generic model to understand the immunopathology of human PBC.
Amy Dhirapong, Guo-Xiang Yang, Steven Nadler, Weici Zhang, Koichi Tsuneyama, Patrick Leung, Stuart Knechtle, Aftab A. Ansari, Ross L. Coppel, Fu-Tong Liu, Xiao-Song He and M Eric Gershwin : Therapeutic effect of cytotoxic T lymphocyte antigen 4/immunoglobulin on a murine model of primary biliary cirrhosis., Hepatology, Vol.57, No.2, 708-715, 2013.
(Summary)
Collectively, the data in both humans and murine models of human primary biliary cirrhosis (PBC) suggest that activated T cells, particularly CD8 T cells, play a critical role in biliary cell destruction. Under physiological conditions, T-cell activation involves two critical signals that involve the major histocompatibility complex and a set of costimulatory molecules, which include a receptor on T cells termed cytotoxic T lymphocyte antigen 4 (CTLA-4). Germane to the studies reported herein, signaling by CTLA-4 has the potential to modulate costimulation and induce inhibitory signals. In this study, we have taken advantage of our well-defined murine model of PBC, in which mice are immunized with 2-octynoic acid coupled to bovine serum albumin (2OA-BSA), leading to the production of high-titer antimitochondrial autoantibodies (AMAs) and portal cellular infiltrates. To investigate the potential of CTLA-4-Ig (immunoglobulin) as an immunotherapeutic agent, we treated mice both before and after induction of autoimmune cholangitis. First, we demonstrate that CTLA-4-Ig treatment, begun 1 day before 2OA-BSA immunization, completely inhibits the manifestations of cholangitis, including AMA production, intrahepatic T-cell infiltrates, and bile duct damage. However, and more critically, treatment with CTLA-4-Ig, initiated after the development of autoimmune cholangitis in previously immunized mice, also resulted in significant therapeutic benefit, including reduced intrahepatic T-cell infiltrates and biliary cell damage, although AMA levels were not altered. These data suggest that an optimized regimen with CTLA-4-Ig has the potential to serve as an investigative therapeutic tool in patients with PBC.
Shiro Watanabe and Koichi Tsuneyama : Eicosapentaenoic acid attenuates hepatic accumulation of cholesterol esters but aggravates liver injury and inflammation in mice fed a cholate-supplemented high-fat diet., The Journal of Toxicological Sciences, Vol.38, No.3, 379-390, 2013.
(Summary)
The administration of a sodium cholate-supplemented high-fat (CAHF) diet in mice induced the predominant accumulation of cholesterol esters (CE) in the liver and biochemical and histological features of liver injury. Cholesteryl oleate was the most abundant CE found in the liver of the mice fed the CAHF diet. We examined the effect of ethyl eicosapentaenoate (EPA) on hepatic CE accumulation and liver injury in the mice fed the CAHF diet. The EPA supplementation suppressed the elevation in the level of cholesteryl oleate in the liver. The expression levels of sterol O-acyltransferase-2 and stearoyl-CoA desaturase-1 mRNA in the liver were elevated in the mice fed the CAHF diet, but they were normalized by the EPA supplementation. However, the elevation in serum transaminase activity, the sign of inflammatory cell exudation and inflammatory gene responses in the liver of the mice fed the EPA-supplemented diet were enhanced compared with those of the mice fed the CAHF diet. We demonstrated that EPA supplementation attenuated CE accumulation but aggravated liver injury and liver inflammation in the mice fed the CAHF diet.
Masahisa Arahata, Kazuto Tajiri, Kazuhiro Nomoto, Koichi Tsuneyama, Shinji Minami and Yukihiro Shimizu : A novel type of selective immunoglobulin m deficiency in a patient with autoimmune liver cirrhosis with recurrent hepatocellular carcinoma: a case report and review of the literature., International Archives of Allergy and Immunology, Vol.161, No.1, 91-96, 2012.
(Summary)
A 64-year-old female with advanced liver cirrhosis who had never experienced severe infections presented in 2004 with general malaise. At the time, her serum showed low levels of immunoglobulin (Ig) M (11 mg/dl) with high levels of both IgG (2,942 mg/dl) and IgA (808 mg/dl). Because serum levels of IgG and IgA in previous cases of selective IgM deficiency were normal, this case could have a novel immunological mechanism. By 2006, serum IgM was undetectable (<5 mg/dl). Liver biopsy showed liver cirrhosis from autoimmune hepatitis. She had no other autoimmune diseases or hematological malignancies. She developed hepatocellular carcinoma (HCC) several times and died of liver failure. Immunological analyses performed before the first diagnosis of HCC showed polyclonal -globulin elevation, normal chromosome and normal gene rearrangement of immunoglobulin heavy chain Cµ. Peripheral blood showed low count B cells with few surface IgM-positive B lymphocytes, but the percentages of T cell subsets were normal. Expression of activation-induced cytidine deaminase (AID), which plays a critical role in immunoglobin class switching, was found to be overexpressed in the HCC tissue and B cells in bone marrow. This phenomenon could account for the clinical and immunological features of this case. In conclusion, we propose a novel type of selective IgM deficiency in association with the overexpression of AID.
Chikako Inada, Xoan Le Thi, Koichi Tsuneyama, Hironori Fujiwara, Takeshi Miyata and Kinzo Matsumoto : Endogenous acetylcholine rescues NMDA-induced long-lasting hippocampal cell damage via stimulation of muscarinic M(1) receptors: elucidation using organic hippocampal slice cultures., European Journal of Pharmacology, Vol.699, No.1-3, 150-159, 2012.
(Summary)
This study aimed to investigate a recuing role of cholinergic systems in the excitotoxicity-induced hippocampal cell damage. Organotypic hippocampal slice cultures (OHSCs) were prepared from 7-day-old mice and exposed to N-methyl-d-aspartate (NMDA) for 24h. After washing out the NMDA, OHSCs were incubated in medium containing test drugs for 0-6 days. Hippocampal cell damage was evaluated by propidium iodide staining, immunofluorescence, and Western blotting. NMDA (1-10 M) dose-dependently damaged hippocampal cells. The toxic effect of 3 M NMDA was also observed at 3-6 days, even after washing out NMDA, and was blocked by MK-801 from day 3 to day 6. Post-treatments with tacrine, donepezil, and galantamine reduced the NMDA-induced long-lasting hippocampal cell damage. The effect of tacrine was induced in a manner dependent on the incubation period after NMDA treatment and was confirmed by Nissl staining and immunostaining with NeuN, a marker of mature neurons. The effect of tacrine was attenuated by scopolamine and a muscarinic M(1) receptor antagonist, pirenzepine, but not by a muscarinic M(3) receptor antagonist, darifenacin, or a nicotinic receptor antagonist, mecamylamine. The protein kinase C inhibitor Ro-31-8220 abolished the effect of tacrine. The pretreatment with 3 M NMDA had no effect on the expression level of presynaptic cholinergic markers, choline acetyltransferase and vesicular acetylcholine transporter, in OHSCs. These results suggest that a low concentration of NMDA causes long-lasting hippocampal cell damage and that endogenous acetylcholine plays, via muscarinic M(1) receptor, a rescuing role in the excitotoxicity-induced long-lasting hippocampal cell damage.
Takeshi Nishida, Koichi Tsuneyama, Makoto Fujimoto, Kazuhiro Nomoto, Shinichi Hayashi, Shigeharu Miwa, Takahiko Nakajima, Yuko Nakanishi, Yoshiyuki Sasaki, Wataru Suzuki, Seiichi Iizuka, Mitsunobu Nagata, Tsutomu Shimada, Masaki Aburada, Yutaka Shimada and Johji Imura : Spontaneous onset of nonalcoholic steatohepatitis and hepatocellular carcinoma in a mouse model of metabolic syndrome., Laboratory Investigation; a Journal of Technical Methods and Pathology, Vol.93, No.2, 230-241, 2012.
(Summary)
Metabolic syndrome is a worldwide healthcare issue and a dominant risk factor for the development of incurable diseases that affect the entire body. The hepatic manifestations of this syndrome include nonalcoholic fatty liver disease (NAFLD) and its progressive variant nonalcoholic steatohepatitis (NASH). The basic pathogenesis of NAFLD/NASH remains controversial because it is difficult to clarify the disease process of NASH on the basis of metabolic syndrome alone. To determine the pathogenesis and effective treatment, an excellent animal model of NASH is required. Tsumura Suzuki obese diabetes (TSOD) male mice spontaneously develop diabetes mellitus, obesity, glucosuria, hyperglycemia, and hyperinsulinemia without any special treatments such as gene manipulation. In this study, we examined the histopathological characteristics of visceral fat and liver of 56 male TSOD mice aged 4-17 months and 9 male Tsumura Suzuki non-obesity (control) mice aged 6-12 months. In the visceral fat, enlargement of adipocytes and perivascular and pericapsular CD8-positive lymphoid aggregation were observed in 4-month-old mice. Abnormal expression of tumor necrosis factor-, interleukin-6, and lipid peroxidation endo products was observed in macrophages. In the liver, microvesicular steatosis, hepatocellular ballooning, and Mallory bodies were observed in 4-month-old mice, with severity worsening with increasing time. These pathological findings in the liver mimic those seen in patients with NASH. Interestingly, small liver nodules with high cellularity and absence of portal tracts were frequently observed after 12 months. Most of them showed nuclear and structural atypia, and mimicked human hepatocellular carcinoma. The degree of steatosis in the non-tumor portions of the liver improved when the liver nodules developed. These findings were not observed in control mice. Here, we report that TSOD male mice spontaneously developed NAFLD without any special treatment, and that these mice are a valuable model for assessing NASH and NASH carcinogenesis owing to metabolic syndrome.
Motoko Sasaki, Takashi Matsubara, Norihide Yoneda, Kazuhiro Nomoto, Koichi Tsuneyama, Yasunori Sato and Yasuni Nakanuma : Overexpression of enhancer of zeste homolog 2 and MUC1 may be related to malignant behaviour in intraductal papillary neoplasm of the bile duct., Histopathology, Vol.62, No.3, 446-457, 2012.
(Summary)
Intraductal papillary neoplasm of the bile duct (IPNB) usually has a favourable prognosis, but occasionally is associated with invasive carcinoma. Overexpression of the polycomb group protein enhancer of zeste homolog 2 (EZH2) is involved in the progression of malignant tumours. In this study, we examined the significance of EZH2 expression in IPNB and its association with clinicopathological features and the expression of p16(INK4a) , p53 and mucin core proteins. We examined immunohistochemically the expression of EZH2, p16(INK4a) , MUC mucin core proteins and p53 in 15 patients with IPNB without invasion, including the cystic variant [male/female ratio (M/F) = 9/6], and in 19 with IPNB associated with invasive carcinoma (M/F = 13/6). The expression levels of EZH2, p53 and MUC1 were significantly lower (P < 0.01), and of MUC6 were significantly higher (P < 0.05), in IPNB without invasion than in IPNB with invasion. Expression of EZH2 was significantly correlated with expression of MUC1 (P < 0.01) and inversely correlated with expression of MUC6 (P < 0.05). In cholangiocarcinoma cells (HuCTT-1 and TFK-1), knockdown of EZH2 and MUC1 by small interfering RNA decreased invasion and proliferation, whereas knockdown of MUC6 increased invasion. Overexpression of EZH2 may be associated with malignant behaviour in IPNB in parallel with up-regulated MUC1 expression and down-regulated MUC6 expression.
(Keyword)
Aged / Aged, 80 and over / Bile Duct Neoplasms / Bile Ducts, Intrahepatic / Disease Progression / Female / Humans / Immunohistochemistry / Male / Middle Aged / Mucin-1 / Mucin-6 / Neoplasm Invasiveness / Polycomb Repressive Complex 2 / RNA, Small Interfering / Real-Time Polymerase Chain Reaction / Reverse Transcriptase Polymerase Chain Reaction / Tumor Markers, Biological
Weici Zhang, Masanobu Tsuda, Guo-Xiang Yang, Koichi Tsuneyama, Xiao-Song He, A Aftab Ansari, M William Ridgway, L Ross Coppel, Zhe-Xiong Lian, C Patrick S Leung and Eric M Gershwin : Lymphoma-like T cell infiltration in liver is associated with increased copy number of dominant negative form of TGFβ receptor II., PLoS ONE, Vol.7, No.11, 2012.
(Summary)
Hepatosplenic T cell lymphoma (HSTCL) is a distinct and lethal subtype of peripheral T cell lymphoma with an aggressive course and poor outcome despite multiagent chemotherapy. Contradictory literature, an unknown etiology, and poor response to treatment highlight the need to define the malignant process and identify molecular targets with potential for successful therapeutic interventions. Herein, we report that mice homozygously expressing a dominant negative TGFβRII (dnTGFβRII) under the control of the CD4 promoter spontaneously develop lymphoma-like T cell infiltration involving both spleen and liver. Splenomegaly, hepatomegaly and liver dysfunction were observed in homozygous dnTGFβRII mice between 10 weeks and 10 months of age associated with a predominant infiltration of CD4(-)CD8(-)TCRβ(+)NK1.1(+) or CD8(+)TCRβ(+)NK1.1(-) T cell subsets. Notch 1 and c-Myc expression at the mRNA levels were significantly increased and positively correlated with the cell number of lymphoid infiltrates in the liver of dnTGFβRII homozygous compared to hemizygous mice. Further, 2×10(4) isolated lymphoma-like cells transplant disease by adoptive cell transfers. Collectively, our data demonstrate that increased copy number of dnTGFβRII is critical for development of lymphoma-like T cell infiltration.
Qi Zhao, Yimin Niu, Kinzo Matsumoto, Koichi Tsuneyama, Ken Tanaka, Takeshi Miyata and Takako Yokozawa : Chotosan ameliorates cognitive and emotional deficits in an animal model of type 2 diabetes: possible involvement of cholinergic and VEGF/PDGF mechanisms in the brain., BMC Complementary and Alternative Medicine, Vol.12, 2012.
(Summary)
These results suggest that CTS ameliorates diabetes-induced cognitive deficits by protecting central cholinergic and VEGF/PDGF systems via Akt signaling pathway and that CTS exhibits the anxiolytic effect via neuronal mechanism(s) independent of cholinergic or VEGF/PDGF systems in db/db mice.
(Keyword)
Animals / Brain / Cognition / Diabetes Mellitus, Type 2 / Disease Models, Animal / Drugs, Chinese Herbal / Emotions / Humans / Male / Mice / Mice, Inbred C57BL / Platelet-Derived Growth Factor / Vascular Endothelial Growth Factor A
Yugo Ando, Guo-Xiang Yang, Masanobu Tsuda, Kazuhito Kawata, Weici Zhang, Takahiko Nakajima, Koichi Tsuneyama, Patrick Leung, Zhe-Xiong Lian, Kazuichi Okazaki, M William Ridgway, L Gary Norman, A Aftab Ansari, Xiao-Song He, L Ross Coppel and Eric M Gershwin : The immunobiology of colitis and cholangitis in interleukin-23p19 and interleukin-17A deleted dominant negative form of transforming growth factor beta receptor type II mice., Hepatology, Vol.56, No.4, 1418-1426, 2012.
(Summary)
Dominant negative form of transforming growth factor beta receptor type II (dnTGFβRII) mice, expressing a dominant negative form of TGFβ receptor II under control of the CD4 promoter, develop autoimmune colitis and cholangitis. Deficiency in interleukin (IL)-12p40 lead to a marked diminution of inflammation in both the colon and the liver. To distinguish whether IL-12p40 mediates protection by the IL-12 or IL-23 pathways, we generated an IL-23p19(-/-) dnTGFβRII strain deficient in IL-23, but not in IL-12; mice were longitudinally followed for changes in the natural history of disease and immune responses. Interestingly, IL-23p19(-/-) mice demonstrate dramatic improvement in their colitis, but no changes in biliary pathology; mice also manifest reduced T-helper (Th)17 cell populations and unchanged IFN-γ levels. We submit that the IL-12/Th1 pathway is essential for biliary disease pathogenesis, whereas the IL-23/Th17 pathway mediates colitis. To further assess the mechanism of the IL-23-mediated protection from colitis, we generated an IL-17A(-/-) dnTGFβRII strain deficient in IL-17, a major effector cytokine produced by IL-23-dependent Th17 cells. Deletion of the IL-17A gene did not affect the severity of either cholangitis or colitis, suggesting that the IL-23/Th17 pathway contributes to colon disease in an IL-17-independent manner. These results affirm that the IL-12/Th1 pathway is critical to biliary pathology in dnTGFβRII mice, whereas colitis is caused by a direct effect of IL-23.
Kazuto Tajiri, Yukihiro Shimizu, Yoshiharu Tokimitsu, Koichi Tsuneyama and Toshiro Sugiyama : An elderly man with syncytial giant cell hepatitis successfully treated by immunosuppressants., Internal Medicine, Vol.51, No.16, 2141-2144, 2012.
(Summary)
Here, we report an elderly man with acute-on-chronic hepatitis accompanied by massive ascites. He showed elevated serum transaminase and anti-nuclear antibody (ANA) levels. Liver biopsy showed diffuse multinucleated giant hepatocytes with interface hepatitis, and he recovered with administration of azathioprine in addition to corticosteroids. Follow-up liver biopsy after recovery showed improvement of hepatic inflammation and reduction of giant hepatocyte formation. The patient is receiving low-dose corticosteroid maintenance therapy and he has remained healthy for 8 years to date. Active immunosuppressive treatment may be beneficial in patients with adult syncitial giant cell hepatitis (AGCH).
(Keyword)
Aged / Alanine Transaminase / Ascites / Azathioprine / Drug Therapy, Combination / Hepatitis, Viral, Human / Hepatocytes / Humans / Immunosuppressive Agents / Male / Prednisolone
Makoto Kurano, Masumi Hara, Koichi Tsuneyama, Koji Okamoto, Naoyuki Iso-O, Teruhiko Matsushima, Kazuhiko Koike and Kazuhisa Tsukamoto : Modulation of lipid metabolism with the overexpression of NPC1L1 in mouse liver., Journal of Lipid Research, Vol.53, No.11, 2275-2285, 2012.
(Summary)
Niemann-Pick C1-like 1 protein (NPC1L1), a transporter crucial in intestinal cholesterol absorption, is expressed in human liver but not in murine liver. To elucidate the role of hepatic NPC1L1 on lipid metabolism, we overexpressed NPC1L1 in murine liver utilizing adenovirus-mediated gene transfer. C57BL/6 mice, fed on normal chow with or without ezetimibe, were injected with NPC1L1 adenovirus (L1-mice) or control virus (Null-mice), and lipid analyses were performed five days after the injection. The plasma cholesterol levels increased in L1-mice, and FPLC analyses revealed increased cholesterol contents in large HDL lipoprotein fractions. These fractions, which showed α-mobility on agarose electrophoresis, were rich in apoE and free cholesterol. These lipoprotein changes were partially inhibited by ezetimibe treatment and were not observed in apoE-deficient mice. In addition, plasma and VLDL triglyceride (TG) levels decreased in L1-mice. The expression of microsomal triglyceride transfer protein (MTP) was markedly decreased in L1-mice, accompanied by the reduced protein levels of forkhead box protein O1 (FoxO1). These changes were not observed in mice with increased hepatic de novo cholesterol synthesis. These data demonstrate that cholesterol absorbed through NPC1L1 plays a distinct role in cellular and plasma lipid metabolism, such as the appearance of apoE-rich lipoproteins and the diminished VLDL-TG secretion.
Takahiko Nakajima, Kuniko Iihara, Junichi Fukushima, Koichi Tsuneyama, Seiko Saito, Toshirou Sugiyama and Hajime Horiuchi : [A case of gastrointestinal stromal tumor of the stomach mimicking a primary tumor of the omentum minus due to the extramural growth]., The Japanese Journal of Gastro-enterology, Vol.109, No.8, 1367-1371, 2012.
(Summary)
We report a case of gastrointestinal stromal tumor (GIST) of the stomach mimicking a primary tumor of the omentum minus. The tumor presented as an isolated mass in the omentum minus without any adhesion to the stomach. Microscopic examination revealed that the tumor pseudocapsule on the gastric side included a small smooth muscle tissue component. The patient was given a diagnosis of a gastric GIST that showed extensive extramural growth. GISTs should not be defined by the localization of the tumor.
Yukitaka Yoshikawa, Taishi Miyashita, Satonori Higuchi, Koichi Tsuneyama, Shinya Endo, Tohru Tsukui, Yasuyuki Toyoda, Tatsuki Fukami, Miki Nakajima and Tsuyoshi Yokoi : Mechanisms of the hepatoprotective effects of tamoxifen against drug-induced and chemical-induced acute liver injuries., Toxicology and Applied Pharmacology, Vol.264, No.1, 42-50, 2012.
(Summary)
Although estrogen receptor (ER)α agonists, such as estradiol and ethinylestradiol (EE2), cause cholestasis in mice, they also reduce the degree of liver injury caused by hepatotoxicants as well as ischemia-reperfusion. The functional mechanisms of ERα have yet to be elucidated in drug-induced or chemical-induced liver injury. The present study investigated the effects of an ERα agonist, selective ER modulators (SERMs) and an ER antagonist on drug-induced and chemical-induced liver injuries caused by acetaminophen, bromobenzene, diclofenac, and thioacetamide (TA). We observed hepatoprotective effects of EE2, tamoxifen (TAM) and raloxifene pretreatment in female mice that were exposed to a variety of hepatotoxic compounds. In contrast, the ER antagonist did not show any hepatoprotective effects. DNA microarray analyses suggested that monocyte to macrophage differentiation-associated 2 (Mmd2) protein, which has an unknown function, is commonly increased by TAM and RAL pretreatment, but not by pretreatment with the ER antagonist. In ERα-knockout mice, the hepatoprotective effects of TAM and the increased expression of Mmd2 mRNA were not observed in TA-induced liver injury. To investigate the function of Mmd2, the expression level of Mmd2 mRNA was significantly knocked down to approximately 30% in mice by injection of siRNA for Mmd2 (siMmd2). Mmd2 knockdown resulted in a reduction of the protective effects of TAM on TA-induced liver injury in mice. This is the first report of the involvement of ERα in drug-induced or chemical-induced liver injury. Upregulation of Mmd2 protein in the liver was suggested as the mechanism of the hepatoprotective effects of EE2 and SERMs.
Drug-induced liver injury is a major safety concern in drug development and clinical pharmacotherapy; however, advances in the understanding of the mechanisms of drug-induced liver injury are hampered by the lack of animal models. Carbamazepine (CBZ) is a widely used antiepileptic agent. Although the drug is generally well tolerated, only a small number of patients prescribed CBZ develop severe hepatitis. In the present study, we developed a mouse model of CBZ-induced liver injury and elucidated the mechanisms accounting for the hepatotoxicity of CBZ. Male BALB/c mice were orally administered CBZ for 5 days. The plasma levels of alanine aminotransferase and aspartate aminotransferase were prominently increased, and severe liver damage was observed via histological evaluation. The analysis of the plasma concentration of CBZ and its metabolites demonstrated that 3-hydroxy CBZ may be relevant in CBZ-induced liver injury. The hepatic glutathione levels were significantly decreased, and oxidative stress markers were significantly altered. Mechanistic investigations found that hepatic mRNA levels of toll-like receptor 4, receptor for advanced glycation end products, and their ligands were significantly increased. Moreover, the plasma concentrations of proinflammatory cytokines were also increased. Prostaglandin E(1) administration ameliorated the hepatic injury caused by CBZ. In conclusion, metabolic activation followed by the stimulation of immune responses was demonstrated to be involved in CBZ-induced liver injury in mice.
Takashi Matsubara, Yasunori Sato, Motoko Sasaki, Kenichi Harada, Kazuhiro Nomoto, Koichi Tsuneyama, Koichi Nakamura, Toshifumi Gabata, Osamu Matsui and Yasuni Nakanuma : Immunohistochemical characteristics and malignant progression of hepatic cystic neoplasms in comparison with pancreatic counterparts., Human Pathology, Vol.43, No.12, 2177-2186, 2012.
(Summary)
The recent World Health Organization classification for tumors of the digestive system defined grossly and histologically hepatic mucinous cystic neoplasms and intraductal papillary neoplasms of the bile duct separately. In this study, the immunohistochemical features of intraductal papillary neoplasm of the bile duct (19 cases) and hepatic mucinous cystic neoplasm (5 cases) were characterized and compared with those of similar pancreatic lesions, intraductal papillary mucinous neoplasm of the pancreas (12 cases), and pancreatic mucinous cystic neoplasm (6 cases) and with those of other biliary cystic lesions, peribiliary cysts (10 cases). Intraductal papillary neoplasm of the bile duct and intraductal papillary mucinous neoplasm of the pancreas frequently expressed cytokeratin 7; mucin core proteins 1, 2, 5AC, and 6; trypsin; and amylase. Hepatic and pancreatic mucinous cystic neoplasms frequently expressed cytokeratin 7, mucin core proteins 1 and 5AC, estrogen receptor, progesterone receptor, trypsin, and amylase. Estrogen and progesterone receptors were expressed in the subepithelial stromal cells. The groups with intraductal papillary neoplasm of the bile duct and intraductal papillary mucinous neoplasm of the pancreas were different from the groups with hepatic and pancreatic mucinous cystic neoplasm with respect to several phenotypes reflecting gastric and intestinal metaplasia and also the lack of expression of estrogen and progesterone receptors. The Ki-67 and p53 labeling indexes increased significantly with the malignant progression of intraductal papillary neoplasm of the bile duct and intraductal papillary mucinous neoplasm of the pancreas. The p16 labeling index decreased and EZH2 labeling index increased significantly with the malignant progression of intraductal papillary neoplasm of the bile duct and intraductal papillary mucinous neoplasm of the pancreas. In conclusion, intraductal papillary neoplasm of the bile duct and hepatic mucinous cystic neoplasm might be regarded as biliary counterparts of intraductal papillary mucinous neoplasm of the pancreas and pancreatic mucinous cystic neoplasm, respectively, and the mucinous cystic neoplasm and intraductal papillary neoplasm groups differed from each other. Labeling indexes of Ki-67, p53, p16, and EZH2 were comparable in intraductal papillary neoplasm of the bile duct and intraductal papillary mucinous neoplasm of the pancreas along with their malignant progression, suggesting a common carcinogenic process of the tumors.
S Shimoda, Koichi Tsuneyama, K Kikuchi, K Harada, Y Nakanuma, M Nakamura, H Ishibashi, S Hisamoto, H Niiro, C P S Leung, A A Ansari, E M Gershwin and K Akashi : The role of natural killer (NK) and NK T cells in the loss of tolerance in murine primary biliary cirrhosis., Clinical and Experimental Immunology, Vol.168, No.3, 279-284, 2012.
(Summary)
One of the major obstacles in dissecting the mechanism of pathology in human primary biliary cirrhosis (PBC) has been the absence of animal models. Our laboratory has focused on a model in which mice, following immunization with a xenobiotic chemical mimic of the immunodominant autoepitope of the E2 component of pyruvate dehydrogenase complex (PDC-E2), develop autoimmune cholangitis. In particular, following immunization with 2-octynoic acid (a synthetic chemical mimic of lipoic acid-lysine located within the inner domain of PDC-E2) coupled to bovine serum albumin (BSA), several strains of mice develop typical anti-mitochondrial autoantibodies and portal inflammation. The role of innate immune effector cells, such as natural killer (NK) cells and that NK T cells, was studied in this model based on the hypothesis that early events during immunization play an important role in the breakdown of tolerance. We report herein that, following in-vivo depletion of NK and NK T cells, there is a marked suppression of anti-mitochondrial autoantibodies and cytokine production from autoreactive T cells. However, there was no change in the clinical pathology of portal inflammation compared to controls. These data support the hypothesis that there are probably multiple steps in the natural history of PBC, including a role of NK and NK T cells in initiating the breakdown of tolerance. However, the data suggest that adaptive autoimmune effector mechanisms are required for the progression of clinical disease.
Recent accumulating evidence suggests that innate immunity is associated with obesity-induced chronic inflammation and metabolic disorders. Here, we show that a Toll-like receptor (TLR) protein, radioprotective 105 (RP105)/myeloid differentiation protein (MD)-1 complex, contributes to high-fat diet (HFD)-induced obesity, adipose tissue inflammation, and insulin resistance. An HFD dramatically increased RP105 mRNA and protein expression in stromal vascular fraction of epididymal white adipose tissue (eWAT) in wild-type (WT) mice. RP105 mRNA expression also was significantly increased in the visceral adipose tissue of obese human subjects relative to nonobese subjects. The RP105/MD-1 complex was expressed by most adipose tissue macrophages (ATMs). An HFD increased RP105/MD-1 expression on the M1 subset of ATMs that accumulate in eWAT. Macrophages also acquired this characteristic in coculture with 3T3-L1 adipocytes. RP105 knockout (KO) and MD-1 KO mice had less HFD-induced adipose tissue inflammation, hepatic steatosis, and insulin resistance compared with wild-type (WT) and TLR4 KO mice. Finally, the saturated fatty acids, palmitic and stearic acids, are endogenous ligands for TLR4, but they did not activate RP105/MD-1. Thus, the RP105/MD-1 complex is a major mediator of adipose tissue inflammation independent of TLR4 signaling and may represent a novel therapeutic target for obesity-associated metabolic disorders.
Kazuhiro Nomoto, Takeshi Nishida, Yuko Nakanishi, Makoto Fujimoto, Ichiro Takasaki, Yoshiaki Tabuchi and Koichi Tsuneyama : Deficiency in galectin-3 promotes hepatic injury in CDAA diet-induced nonalcoholic fatty liver disease., The Scientific World Journal, Vol.2012, 2012.
(Summary)
Nonalcoholic fatty liver disease (NAFLD) is increasingly recognized as a condition in which excess fat accumulates in hepatocytes. Nonalcoholic steatohepatitis (NASH), a severe form of NAFLD in which inflammation and fibrosis in the liver are noted, may eventually progress to end-stage liver disease. Galectin-3, a β-galactoside-binding animal lectin, is a multifunctional protein. This protein is involved in inflammatory responses and carcinogenesis. We investigated whether galectin-3 is involved in the development of NASH by comparing galectin-3 knockout (gal3(-/-)) mice and wild-type (gal3(+/+)) mice with choline-deficient L-amino-acid-defined (CDAA) diet-induced NAFLD/NASH. Hepatic injury was significantly more severe in the gal3(-/-) male mice, as compared to the gal3(+/+) mice. Data generated by microarray analysis of gene expression suggested that galectin-3 deficiency causes alterations in the expression of various genes associated with carcinogenesis and lipid metabolism. Through canonical pathway analysis, involvement of PDGF and IL-6 signaling pathways was suggested in galectin-3 deficiency. Significant increase of CD14, Fos, and Jun, those that were related to lipopolysaccharide-mediated signaling, was candidate to promote hepatocellular damages in galectin-3 deficiency. In conclusion, galectin-3 deficiency in CDAA diet promotes NAFLD features. It may be caused by alterations in the expression profiles of various hepatic genes including lipopolysaccharide-mediated inflammation.
Shu Taira, Shuichi Shimma, Issey Osaka, Daisaku Kaneko, Yuko Ichiyanagi, Ryuzo Ikeda, Yasuko Konishi-Kawamura, Shu Zhu, Koichi Tsuneyama and Katsuko Komatsu : Mass spectrometry imaging of the capsaicin localization in the capsicum fruits, International Journal of Biotechnology for Wellness Industries, Vol.1, No.1, 61-65, 2012.
Qinglong Jin, Yuki Moritoki, Ana Lleo, Koichi Tsuneyama, Pietro Invernizzi, Hitoshi Moritoki, Kentaro Kikuchi, Zhe-Xiong Lian, M Gideon Hirschfield, A Aftab Ansari, L Ross Coppel, Eric M Gershwin and Junqi Niu : Comparative analysis of portal cell infiltrates in antimitochondrial autoantibody-positive versus antimitochondrial autoantibody-negative primary biliary cirrhosis., Hepatology, Vol.55, No.5, 1495-1506, 2012.
(Summary)
Our data suggest a putative role of B-cell autoimmunity in regulating the portal destruction characteristic of PBC.
Hidehiro Tajima, Tetsuo Ohta, Hirohisa Kitagawa, Koichi Okamoto, Seisho Sakai, Jun Kinoshita, Isamu Makino, Hiroyuki Furukawa, Hironori Hayashi, Keishi Nakamura, Katsunobu Oyama, Masafumi Inokuchi, Hisatoshi Nakagawara, Hideto Fujita, Hiroyuki Takamura, Itasu Ninomiya, Sachio Fushida, Takashi Tani, Takashi Fujimura, Seiko Kitamura, Hiroko Ikeda and Koichi Tsuneyama : Neoadjuvant chemotherapy with gemcitabine for pancreatic cancer increases in situ expression of the apoptosis marker M30 and stem cell marker CD44., Oncology Letters, Vol.3, No.6, 1186-1190, 2012.
(Summary)
We examined the pathological effects of preoperative neoadjuvant chemotherapy (NAC) and the expression of markers of apoptosis, epithelial-to-mesenchymal transition (EMT) and cancer stem cells in resected pancreatic cancer specimens from patients treated with gemcitabine as NAC. Immunohistochemical expression of the apoptosis marker M30, EMT marker Snail and stem cell marker CD44 in surgically resected pancreatic cancer specimens were compared between patients treated (NAC group n=13) and not treated (control group n=21) with gemcitabine. In the NAC group, the tumor specimens showed tumor cell injury; however, there was no significant reduction of serosal, retroperitoneal, perineural or vascular invasion, lymph node metastasis or tumor size. The expression frequencies of M30 and CD44 were significantly higher in the NAC group (61.5 and 53.8%) compared to the control group (9.5 and 14.3%); however, no significant difference in Snail expression was noted between the two groups (53.8 versus 42.9%). Gemcitabine induced apoptosis of pancreatic cancer cells in vivo; however, it did not reduce the tumor burden. Moreover, the residual cancer tissues were rich in chemoresistant cancer stem cells. By contrast, marked EMT of cancer cells was observed in the specimens from the groups treated and not treated with gemcitabine.
Makoto Fujimoto, Koichi Tsuneyama, Takako Fujimoto, Carlo Selmi, Eric M Gershwin and Yutaka Shimada : Spirulina improves non-alcoholic steatohepatitis, visceral fat macrophage aggregation, and serum leptin in a mouse model of metabolic syndrome., Digestive and Liver Disease, Vol.44, No.9, 767-774, 2012.
(Summary)
Spirulina reduces dyslipidaemia in our metabolic syndrome model while ameliorating visceral adipose tissue macrophages. Human studies are needed to determine whether this safe supplement could prove beneficial in patients with metabolic syndrome.
Toru Takahashi, Tomofumi Miura, Junichiro Nakamura, Satoshi Yamada, Tsutomu Miura, Masahiko Yanagi, Yasunobu Matsuda, Hiroyuki Usuda, Iwao Emura, Koichi Tsuneyama, Xiao-Song He and Eric M Gershwin : Plasma cells and the chronic nonsuppurative destructive cholangitis of primary biliary cirrhosis., Hepatology, Vol.55, No.3, 846-855, 2012.
(Summary)
These data collectively suggest a role for plasma cells in the specific destruction of intrahepatic bile ducts in PBC and confirm the increasing interest in plasma cells and autoimmunity.
Yu Yamaura, Miki Nakajima, Shingo Takagi, Tatsuki Fukami, Koichi Tsuneyama and Tsuyoshi Yokoi : Plasma microRNA profiles in rat models of hepatocellular injury, cholestasis, and steatosis., PLoS ONE, Vol.7, No.2, 2012.
(Summary)
MicroRNAs (miRNAs) are small RNA molecules that function to modulate the expression of target genes, playing important roles in a wide range of physiological and pathological processes. The miRNAs in body fluids have received considerable attention as potential biomarkers of various diseases. In this study, we compared the changes of the plasma miRNA expressions by acute liver injury (hepatocellular injury or cholestasis) and chronic liver injury (steatosis, steatohepatitis and fibrosis) using rat models made by the administration of chemicals or special diets. Using miRNA array analysis, we found that the levels of a large number of miRNAs (121-317 miRNAs) were increased over 2-fold and the levels of a small number of miRNAs (6-35 miRNAs) were decreased below 0.5-fold in all models except in a model of cholestasis caused by bile duct ligation. Interestingly, the expression profiles were different between the models, and the hierarchical clustering analysis discriminated between the acute and chronic liver injuries. In addition, miRNAs whose expressions were typically changed in each type of liver injury could be specified. It is notable that, in acute liver injury models, the plasma level of miR-122, the most abundant miRNA in the liver, was more quickly and dramatically increased than the plasma aminotransferase level, reflecting the extent of hepatocellular injury. This study demonstrated that the plasma miRNA profiles could reflect the types of liver injury (e.g. acute/chronic liver injury or hepatocellular injury/cholestasis/steatosis/steatohepatitis/fibrosis) and identified the miRNAs that could be specific and sensitive biomarkers of liver injury.
K Nomoto, S Hayashi, Koichi Tsuneyama, T Hori and S Ishizawa : Cytopathology of cervical mesonephric adenocarcinoma: a report of two cases., Cytopathology, Vol.24, No.2, 129-131, 2012.
Shiro Watanabe and Koichi Tsuneyama : Cattle bile but not bear bile or pig bile induces lipid profile changes and fatty liver injury in mice: mediation by cholic acid., The Journal of Toxicological Sciences, Vol.37, No.1, 105-121, 2012.
(Summary)
Three types of animal bile preparation, bear bile (BB), cattle bile (CB) and pig bile (PB) differ in bile acid composition and are supposed to exert different pharmacotoxicological actions. Dietary supplementation with CB at 1% (w/w) for 4 weeks decreased triacylglycerol (TAG) level but increased total cholesterol (CHO) level in serum, which were associated with fatty liver injury in mice. The increased levels of cholesterol esters (CE) and monounsaturated fatty acids (MUFA) in the serum and liver were observed in the mice fed the CB-supplemented diet. Lipid abnormalities and fatty liver injury observed in the mice fed the CB diet were not induced by the supplementation with BB and PB. The supplementation with cholic acid (CA), the most abundant bile acid in CB, could induce lipid abnormalities and fatty liver injury, which were indistinguishable from those induced by CB supplementation. CB and CA supplementation induced similar changes in the expression levels of mRNAs in the liver. Thus, CB induced lipid abnormalities and fatty liver injury, which can be attributed to the actions of CA contained in CB. The inabilities of BB and PB to induce lipid abnormalities and fatty liver injury are supposed to be due to their limited contents of CA.
Azusa Yano, Satonori Higuchi, Koichi Tsuneyama, Tatsuki Fukami, Miki Nakajima and Tsuyoshi Yokoi : Involvement of immune-related factors in diclofenac-induced acute liver injury in mice., Toxicology, Vol.293, No.1-3, 107-114, 2012.
(Summary)
Drug-induced liver injury (DILI) is a major safety concern in drug development and clinical drug therapy. However, the underlying mechanism of DILI is little known. It is difficult to predict DILI in humans due to the lack of experimental animal models. Diclofenac, a non-steroidal anti-inflammatory drug rarely causes severe liver injury in human, but there is some evidence for immunoallergic idiosyncratic reactions. In this study, the mechanism of diclofenac-induced liver injury in mice was investigated. First, we established the dosing condition for liver injury in normal mice. Plasma ALT and AST levels were significantly increased in diclofenac-administered (80 mg/kg, i.p.) mice in a dose- and time-dependent manner. Among several interleukins (ILs) and chemokines, mRNA expression of helper T (Th) 17 cell-mediated factors, such as retinoid orphan receptor (ROR)-γt, and signal transducers and activators of transcription factor (STAT) 3 in the liver, and the plasma IL-17 level were significantly increased. Neutralization of IL-17 tended to suppress the hepatotoxicity of diclofenac, suggesting that IL-17 was partly involved. Gadolinium chloride (GdCl₃) administration demonstrated that Kupffer cells are not likely to be involved in diclofenac hepatotoxicity. Hepatic expressions of IL-1β mRNA and plasma IL-1β were significantly increased soon after the diclofenac administration. Then, the results of an in vivo neutralization study of IL-1β suggested that IL-1β was involved early in the time of pathogenesis of the diclofenac-induced liver injury. In conclusion, we firstly developed a diclofenac-induced acute liver injury model in normal mice, and the involvement of IL-17 and IL-1β was clarified.
(Keyword)
Animals / Anti-Inflammatory Agents, Non-Steroidal / Apoptosis / Chemical and Drug Induced Liver Injury / Diclofenac / Disease Models, Animal / Dose-Response Relationship, Drug / Female / Gene Expression Regulation / Injections, Intraperitoneal / Interleukin-17 / Interleukin-1beta / Kupffer Cells / Liver / Mice / Mice, Inbred BALB C / Necrosis / RNA, Messenger / Th17 Cells / Time Factors
C Patrick S Leung, Xiang Guo Yang, Amy Dhirapong, Koichi Tsuneyama, M William Ridgway and Eric M Gershwin : Animal models of primary biliary cirrhosis: materials and methods., Methods in Molecular Biology, Vol.900, 291-316, 2012.
(Summary)
Primary biliary cirrhosis (PBC) is a female-predominant autoimmune disease of the liver characterized by immune-mediated destruction of the intrahepatic bile ducts and the presence of antimitochondrial antibodies (AMAs). There have been limited advances in understanding the molecular pathogenesis of the disease because of the difficulty in accessing human tissues and the absence of appropriate animal models. Recently, several unique murine models that manifest the serological, biochemical, and histological features similar to human PBC have been described. In this article, we discuss the current data on three spontaneous and two induced murine models of PBC. The spontaneous models are: (a) NOD.c3c4, (b) dominant negative TGF-β receptor II (dnTGFβRII), and (c) IL-2Rα(-/-) mouse line models. The two induced models are: (a) xenobiotic and (b) Novosphingobium aromaticivorans immunized mice. These animal models provide various important platforms to further investigate the etiology and mechanisms of pathogenesis in PBC. Laboratory methodologies and the protocols that are used in evaluating these animal models are described. Finally, we stress the importance of realizing the strengths and limitations of the animal models are essential in data analysis and their application in therapeutic studies.
Taishi Miyashita, Yasuyuki Toyoda, Koichi Tsuneyama, Tatsuki Fukami, Miki Nakajima and Tsuyoshi Yokoi : Hepatoprotective effect of tamoxifen on steatosis and non-alcoholic steatohepatitis in mouse models., The Journal of Toxicological Sciences, Vol.37, No.5, 931-942, 2012.
(Summary)
Non-alcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid accumulation that starts with steatosis and progresses to non-alcoholic steatohepatitis (NASH). Recently, the number of patients with such liver diseases has increased, but the understanding of the fundamental mechanisms and appropriate therapies are lacking. Tamoxifen (TAM) is a selective estrogen receptor modulator. We previously reported that TAM plays a protective role against drug-induced and chemical-induced acute liver injuries. However, the effects of TAM on chronic liver injury, including steatosis and NASH, remain to be addressed. We first found that the administration of TAM to mouse models of steatosis and NASH significantly decreased the plasma ALT and AST levels. The administration of TAM decreased the accumulated fat and inflammation in the livers in both mouse models. In addition, we observed decreased hepatic mRNA levels of triglyceride synthesis, acyl-CoA: diacylglycerol acyltransferase 2 (DGAT2), proinflammatory cytokines, tumor necrosis factor (TNF) α, and chemokines, monocyte chemoattractant protein (MCP) -1. TAM increased the extracellular signal-regulated kinase (ERK) phosphorylation, which is related to the proliferation and regeneration of liver and to decreased DGAT2 gene expression. Furthermore, a decrease in eukaryotic translational initiation factor (eIF2α), which is involved in apoptosis, was observed in both models. These findings suggest that TAM treatment exerts a hepatoprotective effect against steatosis and NASH, presumably via up-regulation of the ERK pathways and attenuation of eIF2α activation. These pathways represent a potential therapeutic target for steatosis and NASH in drug development.
Masashi Ikutani, Tsutomu Yanagibashi, Masaru Ogasawara, Koichi Tsuneyama, Seiji Yamamoto, Yuichi Hattori, Taku Kouro, Atsuko Itakura, Yoshinori Nagai, Satoshi Takaki and Kiyoshi Takatsu : Identification of innate IL-5-producing cells and their role in lung eosinophil regulation and antitumor immunity., The Journal of Immunology, Vol.188, No.2, 703-713, 2011.
(Summary)
IL-5 is involved in a number of immune responses such as helminth infection and allergy. IL-5 also plays roles in innate immunity by maintaining B-1 B cells and mucosal IgA production. However, the identity of IL-5-producing cells has not been unambiguously characterized. In this report, we describe the generation of an IL-5 reporter mouse and identify IL-5-producing non-T lymphoid cells that reside in the intestine, peritoneal cavity, and lungs in naive mice. They share many characteristics with natural helper cells, nuocytes, and Ih2 cells, including surface Ags and responsiveness to cytokines. However, these phenotypes do not completely overlap with any particular one of these cell types. Innate non-T IL-5-producing cells localized most abundantly in the lung and proliferated and upregulated IL-5 production in response to IL-25 and IL-33. IL-33 was more effective than IL-25. These cells contribute to maintaining sufficient numbers of lung eosinophils and are important for eosinophil recruitment mediated by IL-25 and IL-33. Given that eosinophils are shown to possess antitumor activity, we studied lung tumor metastasis and showed that innate IL-5-producing cells were increased in response to tumor invasion, and their regulation of eosinophils is critical to suppress tumor metastasis. Genetic blockade or neutralization of IL-5 impaired eosinophil recruitment into the lung and resulted in increased tumor metastasis. Conversely, exogenous IL-5 treatment resulted in suppressed tumor metastasis and augmented eosinophil infiltration. These newly identified innate IL-5-producing cells thus play a role in tumor surveillance through lung eosinophils and may contribute to development of novel immunotherapies for cancer.
Yasuyuki Toyoda, Shinya Endo, Koichi Tsuneyama, Taishi Miyashita, Azusa Yano, Tatsuki Fukami, Miki Nakajima and Tsuyoshi Yokoi : Mechanism of exacerbative effect of progesterone on drug-induced liver injury., Toxicological Sciences, Vol.126, No.1, 16-27, 2011.
(Summary)
Drug-induced liver injury (DILI) is a major safety concern in drug development and clinical drug therapy. However, the underlying mechanism of DILI is little known. It is generally believed that women exhibit worse outcomes from DILI than men. Recently, we found that pretreatment of mice with estradiol attenuated halothane (HAL)-induced liver injury, whereas pretreatment with progesterone exacerbated it in female mice. To investigate the mechanism of sex difference of DILI, we focused on progesterone in this study. We found the exacerbating effect of progesterone in thioacetamide (TA), α-naphthylisothiocyanate, and dicloxacillin-induced liver injury only in female mice. Higher number of myeloperoxidase-positive mononuclear cells infiltrated into the liver and increased levels of Chemokine (C-X-C motif) ligand 1 and 2 (CXCL1 and CXCL2) and intercellular adhesion molecule-1 in the liver were observed. Interestingly, CXCL1 was slightly increased by progesterone pretreatment alone. Progesterone pretreatment increased the extracellular signal-regulated kinase (ERK) phosphorylation in HAL-induced liver injury. Pretreatment with U0126 (ERK inhibitor) significantly suppressed the exacerbating effect of progesterone and the expression of inflammatory mediators. In addition, pretreatment with gadolinium chloride (GdCl(3): inhibitor of Kupffer cells) significantly suppressed the exacerbating effect of progesterone pretreatment and the expression of inflammatory mediators. Moreover, posttreatment of RU486 (progesterone receptor antagonist) 1 h after the HAL or TA administration ameliorated the HAL- or TA-induced liver injury, respectively, in female mice. In conclusion, progesterone exacerbated the immune-mediated hepatotoxic responses in DILI via Kupffer cells and ERK pathway. The inhibition of progesterone receptor and decrease of the immune response may have important therapeutic implications in DILI.
Sumiyo Sasaki, Yoshinori Nagai, Tsutomu Yanagibashi, Yasuharu Watanabe, Masashi Ikutani, Ai Kariyone, Koichi Tsuneyama, Yoshikatsu Hirai and Kiyoshi Takatsu : Serum soluble MD-1 levels increase with disease progression in autoimmune prone MRL(lpr/lpr) mice., Molecular Immunology, Vol.49, No.4, 611-620, 2011.
(Summary)
MD-1 is a secreted protein that forms a complex with radioprotective 105 (RP105) and this complex plays a crucial role in lipopolysaccharide (LPS) recognition by B cells. Disease progression is known to improve in RP105-deficient lupus-prone MRL(lpr/lpr) mice. Furthermore, a soluble form of the homologous MD-2 protein is present in the plasma of septic patients and can opsonize gram-negative bacteria in cooperation with Toll-like receptor (TLR) 4. We have now established a flow cytometry-based assay to detect the soluble form of murine MD-1 (sMD-1) and explored potential roles in autoimmunity. The assay was quantitative and validated with sera from MD-1-deficient mice. Interestingly, heat-inactivated murine serum diminished the ability of sMD-1 to bind RP105. The sMD-1 was secreted by bone marrow-derived macrophages from C57BL/6 mice. Autoimmune prone MRL(lpr/lpr) mice had higher levels of sMD-1 than control MRL(+/+) mice, and levels markedly increased with disease progression. Expression of MD-1 but not MD-2 mRNA increased with age in the liver and kidney of MRL(lpr/lpr) mice. Finally, immunohistochemical analyses revealed that MD-1 was present in infiltrated macrophages within perivascular lesions of the MRL(lpr/lpr) kidney. This correlation suggests that sMD-1 may contribute to pathogenesis in this autoimmune disease model.
Qi Zhao, Kinzo Matsumoto, Koichi Tsuneyama, Ken Tanaka, Feng Li, Naotoshi Shibahara, Takeshi Miyata and Takako Yokozawa : Diabetes-induced central cholinergic neuronal loss and cognitive deficit are attenuated by tacrine and a Chinese herbal prescription, kangen-karyu: elucidation in type 2 diabetes db/db mice., Journal of Pharmacological Sciences, Vol.117, No.4, 230-242, 2011.
(Summary)
We investigated the effect of kangen-karyu (KK), a Chinese herbal prescription, on cognitive deficits and central cholinergic systems of type 2 diabetic db/db mice. Seven-week-old db/db (Y-db/db) mice received daily administration of test drugs during an experimental period of 12 weeks. At 18 weeks of age (O-db/db), the animals underwent the water maze test. Compared with age-matched control strain mice (O-m/m), vehicle-treated O-db/db mice showed impaired learning and memory performance. KK (100 - 200 mg/kg per day) and the reference drug tacrine (THA: 2.5 mg/kg per day) ameliorated the performance of O-db/db mice without affecting their serum glucose level. O-db/db mice had lower levels of brain-derived neurotrophic factor (BDNF) mRNA and its protein in the brain than O-m/m mice. Expression levels of central cholinergic marker proteins in the hippocampus and the number of cholinergic cells in the medial septum and basal forebrain were also significantly lower in O-db/db than in O-m/m mice, whereas no significant differences in the expression levels of these factors and the cell number were found between Y-m/m and Y-db/db mice. KK and THA treatment significantly reversed the down-regulated levels of cholinergic markers, choline acetyltransferase-positive cell number, and BDNF expression in db/db mice. These findings suggest that KK as well as THA prevents diabetes-induced cognitive deficits by attenuating dysfunction of central cholinergic systems.
Hiroyuki Hounoki, Koichiro Shinoda, Reina Ogawa, Hirofumi Taki, Koichi Tsuneyama and Kazuyuki Tobe : Simultaneously developed polymyositis and autoimmune hepatitis., BMJ Case Reports, Vol.2011, 2011.
(Summary)
The inflammatory myopaties such as polymyositis (PM) and dermatomyositis (DM) are autoimmune inflammatory muscle disorders characterised by the development of proximal and often symmetrical muscle weakness. Levels of serum muscle enzymes such as creatine kinase (CK), lactate dehydrogenase (LDH), asparate aminotransferase (AST) and alanine aminotransferase (ALT) are usually elevated. However, high levels of AST, ALT and LDH, without a determination of CK, are often misdiagnosed with hepatic diseases. Conversely, concomitant elevations of AST, ALT and LDH along with CK in patients with PM and DM may be considered to be due to myopathy itself even in a case of coexistence of liver injury. Oral administration of prednisolone was begun at a dose of 60 mg/day, resulting in a good outcome.
Koichi Tsuneyama, Yen-Chen Chen, Makoto Fujimoto, Yoshiyuki Sasaki, Wataru Suzuki, Tsutomu Shimada, Seiichi Iizuka, Mitsunobu Nagata, Masaki Aburada and Shao-Yuan Chen : Advantages and disadvantages of hyperbaric oxygen treatment in mice with obesity hyperlipidemia and steatohepatitis., The Scientific World Journal, Vol.11, 2124-2135, 2011.
(Summary)
The effect of hyperbaric oxygen treatment (HBOT) was examined using MSG mice, which are an animal model of obesity, hyperlipidemia, diabetes, and nonalcoholic fatty liver disease. Nineteen MSG male mice were divided into HBOT treated and control groups at 12 weeks of ages. The HBOT group was treated with hyperbaric oxygen from 12 to 14 weeks (first phase) and then from 16 to 18 weeks (second phase). Interestingly, the body weight of the HBOT group was significantly lower (P < 0.01) than that of the control group. In contrast, the serum lipid level did not show significant changes between the two groups. As for the effects of increasing oxidative stress, the liver histology of the HBOT group showed severer cellular damage and aberrant TNF-α expression. HBOT has the advantage of improving obesity in patients with metabolic syndrome, but the fault of causing organ damage by increasing oxidative stress.
Masanobu Tsuda, M Yoko Ambrosini, Weici Zhang, Guo-Xiang Yang, Yugo Ando, Guanghua Rong, Koichi Tsuneyama, Kosuke Sumida, Shinji Shimoda, L Christopher Bowlus, C Patrick S Leung, Xiao-Song He, L Ross Coppel, A Aftab Ansari, Zhe-Xiong Lian and Eric M Gershwin : Fine phenotypic and functional characterization of effector cluster of differentiation 8 positive T cells in human patients with primary biliary cirrhosis., Hepatology, Vol.54, No.4, 1293-1302, 2011.
(Summary)
In conclusion, our data demonstrate that CD45RO(high) CD57(+) CD8(high) T cells are a subset of terminally differentiated cytotoxic T(EM) cells, which could play a critical role in the progressive destruction of biliary epithelial cells.
M Y Ambrosini, G-X Yang, W Zhang, M Tsuda, S Shu, Koichi Tsuneyama, C P S Leung, A A Ansari, L R Coppel and E M Gershwin : The multi-hit hypothesis of primary biliary cirrhosis: polyinosinic-polycytidylic acid (poly I:C) and murine autoimmune cholangitis., Clinical and Experimental Immunology, Vol.166, No.1, 110-120, 2011.
(Summary)
A void in understanding primary biliary cirrhosis (PBC) is the absence of appropriate animal models. Our laboratory has studied a murine model of autoimmune cholangitis induced following immunization with 2-octynoic acid (2OA), an antigen identified following extensive quantitative structural activity relationship (QSAR) analysis, using human autoantibodies and three-dimensional analysis of the mitochondrial autoantigen, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2). Mice immunized with 2OA coupled to bovine serum albumin (BSA) develop anti-mitochondrial antibodies (AMAs) of the identical specificity as humans with PBC, and in addition develop inflammatory portal cell infiltrates in liver. However, the natural history of disease is less severe than in humans and does not include fibrosis. Data from human and autoimmune murine models suggest that environmental and/or infectious agents can exacerbate autoimmune reactions, and a model of PBC has been described in which polyinosinic-polycytidylic acid (poly I:C), a viral RNA mimetic and Toll-like receptor 3 (TLR-3) agonist induces low-titre AMAs and in mild portal infiltrates. We took advantage of our established model to determine whether immunization with 2OA-BSA coupled with poly I:C alters the disease process. Indeed, the addition of poly I:C produces a profound exacerbation of autoimmune cholangitis, including a significant increase in CD8(+) infiltrating T cells, as well as a marked increase of proinflammatory cytokines. In addition, mice have evidence of fibrosis. These findings lend support to the concept that besides breakdown of self-tolerance, there is a requirement of a second 'hit' during the breakdown process that leads to disease which more faithfully mimics human PBC.
Qi Zhao, Takako Yokozawa, Koichi Tsuneyama, Ken Tanaka, Takeshi Miyata, Notoshi Shibahara and Kinzo Matsumoto : Chotosan (Diaoteng San)-induced improvement of cognitive deficits in senescence-accelerated mouse (SAMP8) involves the amelioration of angiogenic/neurotrophic factors and neuroplasticity systems in the brain., Chinese Medicine, Vol.6, 2011.
(Summary)
Recovery of impaired neuroplasticity system and VEGF/PDGF systems may play a role in the ameliorative effects of CTS on cognitive dysfunction caused by aging and ischemic insult.
Ya-Hui Huang, Cheng-Chieh Fang, Koichi Tsuneyama, Ho-Yuan Chou, Wen-Yu Pan, Yao-Ming Shih, Ping-Yi Wu, Yin Chen, C Patrick S Leung, Eric M Gershwin and Mi-Hua Tao : A murine model of hepatitis B-associated hepatocellular carcinoma generated by adeno-associated virus-mediated gene delivery., International Journal of Oncology, Vol.39, No.6, 1511-1519, 2011.
(Summary)
A relevant animal model is critical for investigating the pathogenic mechanisms underlying hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC). Mice are not naturally infected by HBV, presumably due to the lack of HBV receptors on mouse hepatocytes. To bypass this entry step of HBV infection, we report generation of a novel HBV model in immunocompetent mice by hepatic delivery of the HBV genome using trans-splicing adeno-associated viral vectors (AAV/HBV). We confirmed production of HBV virions and proteins in the liver and circulation in all AAV/HBV-transduced mice in all four immunocompetent mouse strains tested. These mice produced antigen and antibody profiles similar to that observed in chronic HBV patients. Importantly, 12-16 months later, all 12 AAV/HBV-transduced mice developed macroscopically visible liver-tumor nodules. Ten of the twelve tumors were characterized with typical HCC features. This AAV/HBV-transduced murine HCC model provides a useful instrument for studying the pathogenesis of HBV-associated HCC and the development of HCC therapeutic interventions.
(Keyword)
Animals / Base Sequence / Carcinoma, Hepatocellular / Dependovirus / Disease Models, Animal / Gene Transfer Techniques / Genetic Vectors / Hepatitis B virus / Hepatitis B, Chronic / Liver Neoplasms / Male / Mice / Mice, Inbred BALB C / Mice, Inbred C57BL / Trans-Splicing / Viral Load
Satonori Higuchi, Masanori Kobayashi, Azusa Yano, Koichi Tsuneyama, Tatsuki Fukami, Miki Nakajima and Tsuyoshi Yokoi : Involvement of Th2 cytokines in the mouse model of flutamide-induced acute liver injury., Journal of Applied Toxicology : JAT, Vol.32, No.10, 815-822, 2011.
(Summary)
Drug-induced liver injury is a growing concern for pharmaceutical companies and patients because numerous drugs have been linked to hepatotoxicity and it is the most common reason for a drug to be withdrawn. Flutamide rarely causes liver dysfunction in humans, and immune allergic reactions have been suggested in some cases. In this study, we investigated the mechanisms of flutamide-induced liver injury in BALB/c mice. Plasma alanine aminotransferase and aspartate aminotransferase levels were significantly increased 3, 6 and 9 h after flutamide (1500 mg kg⁻¹ , p.o.) administration. The biomarker for oxidative stress was not changed, but Th2-dominant immune-related factors, such as interleukin (IL)-4, IL-5, STAT6 and GATA-binding protein (GATA)-3, were induced in flutamide-administered mice. The pre-administration of monoclonal-IL-4 antibody suppressed the hepatotoxicity of flutamide. In addition, we investigated the effect of 13,14-dihydro-15-keto-PGD₂ (DK-PGD₂; 10 µg per mouse, i.p.) administration on flutamide-induced acute liver injury. Coadministration of DK-PGD₂ and flutamide resulted in a significant increase in alanine aminotransferase and a remarkable increase of macrophage inflammatory protein-2. In conclusion, we demonstrated that flutamide-induced acute liver injury is mediated by Th2-dominant immune responses in mice.
Koichi Tsuneyama, Yuki Moritoki, Kentaro Kikuchi and Yasuni Nakanuma : Pathological features of new animal models for primary biliary cirrhosis., International Journal of Hepatology, Vol.2012, 2011.
(Summary)
Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by immune mediated biliary damage and frequent appearance of autoantibodies against mitochondrial enzymes. There is almost no useful animal model that is globally recognized and routinely used, however, several unique animal models manifested the characteristic clinical and pathological features of human PBC within the last 5 years. Herein, we compare the pathological features of previously reported and newly introduced novel animal models of PBC. Knowledge and understanding of the strengths and the limitations of each animal model have led to the development of promising therapies and novel tools to characterize these clinical conditions. Moreover, suitability of the model for the intended purpose should be confirmed by further research and analysis.
N Hashimoto, S Shimoda, H Kawanaka, Koichi Tsuneyama, H Uehara, T Akahoshi, N Kinjo, A Taketomi, K Shirabe, K Akashi, A Lleo, A A Ansari, E M Gershwin and Y Maehara : Modulation of CD4+ T cell responses following splenectomy in hepatitis C virus-related liver cirrhosis., Clinical and Experimental Immunology, Vol.165, No.2, 243-250, 2011.
(Summary)
Dysfunction of T cells is a common feature in chronic persistent viral infections, including hepatitis C virus (HCV), and although hepatic and peripheral T cells have been studied extensively in chronic HCV hepatitis, the role of splenic T cell responses in such patients is poorly defined. This is an important issue, as thrombocytopenia is a complication of HCV-related liver cirrhosis (LC), due to splenic platelet sequestration and bone marrow suppression; splenectomy has been proposed to treat such patients. Herein, we studied peripheral blood mononuclear cells (PBMC) and splenic lymphoid subpopulations from a total of 22 patients, including 15 with HCV-related LC with marked thrombocytopenia treated with splenectomy, and seven controls. CD4(+) T cells from peripheral blood and spleen were isolated and phenotype and function evaluated. Splenic CD4(+) T cells in patients with LC expressed molecules associated with inhibitory signalling, including increased frequency of negative markers such as cytotoxic T lymphocyte associated antigen-4 (CTLA-4) and programmed death 1 (PD-1) and decreased production of cytokines. Patients with LC manifest higher levels of splenic CD4(+) regulatory T cells and PD-L1- and PD-L2-expressing cells than controls. Blocking of PD-1/PD-1 ligand interaction reconstituted proliferative and cytokine responses of splenic mononuclear cells (SMC) from patients with LC. Splenectomy was followed by an increase in the ratio of interferon (IFN)-γ to interleukin (IL)-10 and a reduction of PD-1-expressing CD4(+) T cells in peripheral blood. Our data suggest that peripheral tolerance is promoted by the spleen in LC via the up-regulated expression of PD-1 ligands.
Yoshiyuki Sasaki, Tsutomu Shimada, Seiichi Iizuka, Wataru Suzuki, Hiroko Makihara, Ryutaro Teraoka, Koichi Tsuneyama, Ryoji Hokao and Masaki Aburada : Effects of bezafibrate in nonalcoholic steatohepatitis model mice with monosodium glutamate-induced metabolic syndrome., European Journal of Pharmacology, Vol.662, No.1-3, 1-8, 2011.
(Summary)
Recently, we reported that monosodium glutamate-treated mice (MSG mice) developed severe hyperlipidemia and diabetes mellitus and several complications of obesity. MSG mice acquired fatty livers and subsequently underwent changes that are characteristic of nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH). In the present study, the effects of bezafibrate on obesity, diabetes mellitus, and NAFLD/NASH were examined in MSG mice. A single dose of MSG (4 mg/g) was administered subcutaneously to neonatal male mice within 24h of birth. Bezafibrate was mixed into the normal feed for 8 weeks. The weight and body mass index of MSG mice increased significantly despite the unchanged intake of food. Triglyceride and total cholesterol levels in blood, visceral adipose tissue, and interscapular adipose tissue rose significantly. In the livers of MSG mice, moderate centrilobular microvesicular steatosis, ballooning degeneration with Mallory bodies, and scattered infiltration of neutrophils and lymphocytes were observed. Centrilobular hepatocytes were 4-hydroxynonenal-positive in MSG mice. Bezafibrate ameliorated the severity of diabetes mellitus, hyperinsulinemia, and hyperlipidemia. Adiponectin and leptin concentrations in blood improved, and the accumulation of visceral fat was inhibited. The expression of acyl-CoA oxidase, a beta-oxidation gene, and carnitine palmitoyl transferase, which regulates lipid metabolism, increased markedly on administration of bezafibrate. The liver pathology in MSG mice also improved with bezafibrate; specifically, macro- and microvesicles in hepatocytes nearly disappeared, and NAFLD activity score improved. It is concluded that bezafibrate inhibits the accumulation of visceral fat, following amelioration of hyperlipidemia, in MSG-induced obese mice, due to improvements in diabetes mellitus, fatty liver, and NAFLD.
Drug-induced liver injury (DILI) is one of the major problems in drug development and clinical drug therapy. In general, it is believed that women exhibit worse outcomes from DILI than men. It is known that halothane (HAL), an inhaled anesthetic, rarely induces severe liver injury. The risk factors for severe HAL-induced liver injury (HILI) are female sex, genetics and adult age. To investigate the underlying mechanism by which women are more susceptible to HILI, we focused on two major female sex hormones, estradiol (E2) and progesterone (Prog). In this study, we first found that pretreatment of mice with E2 attenuated HILI, whereas pretreatment with Prog exacerbated HILI. E2 and Prog had no effects on the degree of metabolic activation, the ratio of GSH/GSSG or oxidative stress in the liver. We observed higher numbers of neutrophils infiltrated into the liver and increased hepatic mRNA levels of proinflammatory cytokines, tumor necrosis factor (TNF) α, interleukin (IL)-1β and IL-6 and chemokines, CXCL1 and CXCL2 by pretreatment with Prog, whereas E2 pretreatment resulted in the opposite effects. These results suggest that E2 and Prog play a critical role in HILI via immune-related responses and female sex hormone balance might represent a risk factor for HILI.
Takao Hidaka, Akitoshi Nakashima, Toru Hasegawa, Kazuhiro Nomoto, Shin Ishizawa, Koichi Tsuneyama, Yasuo Takano and Shigeru Saito : Ovarian squamous cell carcinoma which metastasized 8 years after cervical conization for early microinvasive cervical cancer: a case report., Japanese Journal of Clinical Oncology, Vol.41, No.6, 807-810, 2011.
(Summary)
Squamous cell cervical carcinoma that metastasized to the ovary is common in patients with bulky tumors or locally advanced disease; however, ovarian squamous cell carcinoma that metastasized after cervical conization surgery for early microinvasive uterine cervical carcinoma is very rare. We present a case of ovarian squamous cell carcinoma that metastasized 8 years after cervical conization surgery for early microinvasive cervical carcinoma. She had no sign of recurrence in the uterine cervix. We detected human papillomavirus type 16 DNA in both cervical tissue and ovarian tissue, suggesting that ovarian squamous cell carcinoma is derived from microinvasive cervical cancer. Although there are very few cases of early microinvasive squamous cell carcinoma that metastasized to the ovary with delayed recurrence, we should pay attention strictly not only to the cervical condition but also to the ovarian condition on regular post-operative follow-up.
Diet-induced obesity is reported to induce a phenotypic switch in adipose tissue macrophages from an antiinflammatory M2 state to a proinflammatory M1 state. Telmisartan, an angiotensin II type 1 receptor blocker and a peroxisome proliferator-activated receptor-γ agonist, reportedly has more beneficial effects on insulin sensitivity than other angiotensin II type 1 receptor blockers. In this study, we studied the effects of telmisartan on the adipose tissue macrophage phenotype in high-fat-fed mice. Telmisartan was administered for 5 wk to high-fat-fed C57BL/6 mice. Insulin sensitivity, macrophage infiltration, and the gene expressions of M1 and M2 markers in visceral adipose tissues were then examined. An insulin- or a glucose-tolerance test showed that telmisartan treatment improved insulin resistance, decreasing the body weight gain, visceral fat weight, and adipocyte size without affecting the amount of energy intake. Telmisartan reduced the mRNA expression of CD11c and TNF-α, M1 macrophage markers, and significantly increased the expressions of M2 markers, such as CD163, CD209, and macrophage galactose N-acetyl-galactosamine specific lectin (Mgl2), in a quantitative RT-PCR analysis. A flow cytometry analysis showed that telmisartan decreased the number of M1 macrophages in visceral adipose tissues. In conclusion, telmisartan improves insulin sensitivity and modulates adipose tissue macrophage polarization to an antiinflammatory M2 state in high-fat-fed mice.
Shinji Shimoda, Kenichi Harada, Hiroaki Niiro, Ken Shirabe, Akinobu Taketomi, Yoshihiko Maehara, Koichi Tsuneyama, Yasuni Nakanuma, Patrick Leung, A Aftab Ansari, Eric M Gershwin and Koichi Akashi : Interaction between Toll-like receptors and natural killer cells in the destruction of bile ducts in primary biliary cirrhosis., Hepatology, Vol.53, No.4, 1270-1281, 2011.
(Summary)
These data highlight critical differences in the varied roles of Mo and NK cells following TLR3-L and TLR4-L stimulation.
Si-Jie Wu, Yao-Hsu Yang, Koichi Tsuneyama, C Patrick S Leung, Petr Illarionov, Eric M Gershwin and Ya-Hui Chuang : Innate immunity and primary biliary cirrhosis: activated invariant natural killer T cells exacerbate murine autoimmune cholangitis and fibrosis., Hepatology, Vol.53, No.3, 915-925, 2011.
(Summary)
Our data suggests a primary role of innate immunity in the exacerbation of autoimmune cholangitis and also become a logical explanation for the recurrence of PBC following liver transplantation in the absence of major histocompatability complex compatibility. We submit that PBC begins with loss of tolerance to PDC-E2 and a multilineage antimitochondrial response in which autoreactive CD8(+) T cells are critical. However, the perpetuation of disease and its exacerbation will also be modulated by innate immune mechanisms.
Hiroshi Oka, Hirozo Goto, Keiichi Koizumi, Shin Nakamura, Koichi Tsuneyama, Yue Zhou, Michiko Jo, Takako Fujimoto, Hiroaki Sakurai, Naotoshi Shibahara, Ikuo Saiki and Yutaka Shimada : Effect of Hachimijiogan against Renal Dysfunction and Involvement of Hypoxia-Inducible Factor-1α in the Remnant Kidney Model., Evidence-Based Complementary and Alternative Medicine : eCAM, Vol.2011, 2011.
(Summary)
In chronic renal failure, hypoxia of renal tissue is thought to be the common final pathway leading to end-stage renal failure. In this study the effects of hachimijiogan, a Kampo formula, were studied with respect to hypoxia-inducible factor (HIF). Using remnant kidney rats, we studied the effects of hachimijiogan on renal function in comparison with angiotensin II receptor blocker. The result showed that oral administration of hachimijiogan for seven days suppressed urinary protein excretion and urinary 8-OHdG, a marker of antioxidant activity, equally as well as oral administration of candesartan cilexetil. In contrast, the protein volume of HIF-1α in the renal cortex was not increased in the candesartan cilexetil group, but that in the hachimijiogan group was increased. In immunohistochemical studies as well, the expression of HIF-1α of the high-dose hachimijiogan group increased compared to that of the control group. Vascular endothelial growth factor and glucose transporter 1, target genes of HIF-1α, were also increased in the hachimijiogan group. These results suggest that hachimijiogan produces a protective effect by a mechanism different from that of candesartan cilexetil.
Yuki Moritoki, Masanobu Tsuda, Koichi Tsuneyama, Weici Zhang, Katsunori Yoshida, Zhe-Xiong Lian, Guo-Xiang Yang, M William Ridgway, S Linda Wicker, A Aftab Ansari and Eric M Gershwin : B cells promote hepatic inflammation, biliary cyst formation, and salivary gland inflammation in the NOD.c3c4 model of autoimmune cholangitis., Cellular Immunology, Vol.268, No.1, 16-23, 2011.
(Summary)
There are now several murine models of autoimmune cholangitis that have features both similar and distinct from human PBC. One such model, the NOD.c3c4 mouse, manifests portal cell infiltrates, anti-mitochondrial antibodies but also biliary cysts. The biliary cysts are not a component of PBC and not found in the other murine models. To address the immunopathology in these mice, we generated genetically B cell deficient Igμ(-/-) NOD.c3c4 mice and compared the immunopathology of these animals to control B cell sufficient NOD.c3c4 mice. B cell deficient mice demonstrated decreased number of non-B cells in the liver accompanied by reduced numbers of activated natural killer cells. The degree of granuloma formation and bile duct damage were comparable to NOD.c3c4 mice. In contrast, liver inflammation, biliary cyst formation and salivary gland inflammation was significantly attenuated in these B cell deficient mice. In conclusion, B cells play a critical role in promoting liver inflammation and also contribute to cyst formation as well as salivary gland pathology in autoimmune NOD.c3c4 mice, illustrating a critical role of B cells in modulating specific organ pathology and, in particular, in exacerbating both the biliary disease and the sialadenitis.
Yasuhiko Yamamoto, Ai Harashima, Hidehito Saito, Koichi Tsuneyama, Seiichi Munesue, So Motoyoshi, Dong Han, Takuo Watanabe, Masahide Asano, Shin Takasawa, Hiroshi Okamoto, Satoshi Shimura, Tadahiro Karasawa, Hideto Yonekura and Hiroshi Yamamoto : Septic shock is associated with receptor for advanced glycation end products ligation of LPS., The Journal of Immunology, Vol.186, No.5, 3248-3257, 2011.
(Summary)
Septic shock is a severe systemic response to bacterial infection. Receptor for advanced glycation end products (RAGE) plays a role in immune reactions to recognize specific molecular patterns as pathogen recognition receptors. However, the interaction between LPS, the bioactive component of bacterial cell walls, and RAGE is unclear. In this study, we found direct LPS binding to RAGE by a surface plasmon resonance assay, a plate competition assay, and flow cytometry. LPS increased TNF-α secretion from peritoneal macrophages and an NF-κB promoter-driven luciferase activity through RAGE. Blood neutrophils and monocytes expressed RAGE, and TLR2 was counterregulated in RAGE(-/-) mice. After LPS injection, RAGE(+/+) mice showed a higher mortality, higher serum levels of IL-6, TNF-α, high mobility group box 1, and endothelin-1, and severe lung and liver pathologies compared with RAGE(-/-) mice without significant differences in plasma LPS level. Administration of soluble RAGE significantly reduced the LPS-induced cytokine release and tissue damage and improved the LPS-induced lethality even in RAGE(-/-) as well as RAGE(+/+) mice. The results thus suggest that RAGE can associate with LPS and that RAGE system can regulate inflammatory responses. Soluble RAGE would be a therapeutic tool for LPS-induced septic shock.
(Keyword)
Amino Acid Sequence / Animals / Binding, Competitive / Cell Line, Tumor / Glycation End Products, Advanced / Humans / Inflammation Mediators / Ligands / Lipopolysaccharides / Male / Mice / Mice, Knockout / Molecular Sequence Data / NF-kappa B / Protein Binding / Rats / Receptor for Advanced Glycation End Products / Receptors, Immunologic / Shock, Septic / Signal Transduction / Survival Analysis / Tumor Necrosis Factor-alpha
Amy Dhirapong, Ana Lleo, Guo-Xiang Yang, Koichi Tsuneyama, Robert Dunn, Marilyn Kehry, A Thomas Packard, C John Cambier, Fu-Tong Liu, Keith Lindor, L Ross Coppel, A Aftab Ansari and Eric M Gershwin : B cell depletion therapy exacerbates murine primary biliary cirrhosis., Hepatology, Vol.53, No.2, 527-535, 2010.
(Summary)
Our results reflect a novel disease-protective role of B cells in PBC and suggest that B cell depletion therapy in humans with PBC should be approached with caution.
Drug-induced liver injury (DILI) is a major problem in drug development and clinical drug therapy. In most cases, the mechanisms are still unknown. It is difficult to predict DILI in humans due to the lack of experimental animal models. Dicloxacillin, penicillinase-sensitive penicillin, rarely causes cholestatic or mixed liver injury, and there is some evidence for immunoallergic idiosyncratic reaction in human. In this study, we investigated the mechanisms of dicloxacillin-induced liver injury. Plasma ALT and total-bilirubin (T-Bil) levels were significantly increased in dicloxacillin-administered (600 mg/kg, i.p.) mice. Dicloxacillin administration induced Th2 (helper T cells)-mediated factors and increased the plasma interleukin (IL)-4 level. Neutralization of IL-4 suppressed the hepatotoxicity of dicloxacillin, and recombinant mouse IL-4 administration (0.5 or 2.0 μg/mouse, i.p.) exacerbated it. Chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTh2) is a cognate receptor for prostaglandin (PG) D(2), and is suggested to be involved in Th2-dependent allergic inflammation. We investigated the effect of 13,14-Dihydro-15-keto-PGD(2) (DK-PGD(2); 10 μg/mouse, i.p.) administration on dicloxacillin-induced liver injury. DK-PGD(2)/dicloxacillin coadministration resulted in a significant increase of alanine aminotransferases and a remarkable increase of macrophage inflammatory protein 2 expression. In conclusion, to the best of our knowledge, this is the first report to demonstrate that dicloxacillin-induced liver injury is mediated by a Th2-type immune reaction and exacerbated by DK-PGD(2).
Kazuhiro Nomoto, Takashi Hori, Chieko Kiya, Junya Fukuoka, Akitoshi Nakashima, Takao Hidaka, Shigeru Saito, Yoshiki Mikami, Koichi Tsuneyama and Yasuo Takano : Endometrioid adenocarcinoma of the vagina with a microglandular pattern arising from endometriosis after hysterectomy., Pathology International, Vol.60, No.9, 636-641, 2010.
(Summary)
Primary endometrioid adenocarcinoma rarely occurs in the vagina. Occasionally, endometrioid adenocarcinoma has a microglandular pattern. Herein, a case of primary endometrioid adenocarcinoma of the vagina with a microglandular pattern arising from pre-existing endometriosis long after a hysterectomy, is described. A 57-year-old postmenopausal woman developed a vaginal discharge over one decade after undergoing a hysterectomy. Microscopic examination of the vaginal smear and a biopsy specimen demonstrated an atypical glandular proliferation composed of columnar cells with occasional intracytoplasmic mucin and bland nuclear morphology, showing microcysts and numerous neutrophils within and around cysts. Immunohistochemically, the neoplastic cells were diffusely positive for CK7, MUC1, ER, and PR, and focally positive for vimentin, CEA, CK5/6, p63, p16(INK4a), and p53. A portion of residual endometrioid adenocarcinoma was identified adjacent to foci of endometriosis in the vaginectomy specimen. The patient has done well without evidence of recurrent disease for 1 year after surgery. Pathologists are encouraged to be aware of the occurrence of endometrioid adenocarcinoma associated with endometriosis in the vaginal stump after hysterectomy, and microglandular morphology which might be a source of misinterpretation.
Qi Zhao, Takako Yokozawa, Noriko Yamabe, Koichi Tsuneyama, Xiaohan Li and Kinzo Matsumoto : Kangen-karyu improves memory deficit caused by aging through normalization of neuro-plasticity-related signaling system and VEGF system in the brain., Journal of Ethnopharmacology, Vol.131, No.2, 377-385, 2010.
(Summary)
These findings suggest that normalization of neuro-plasticity-related neuronal signaling and VEGF systems in the brain may be of the mechanisms underlying the ameliorative effects of KK on the cognitive deficits in older SAMP8.
Weici Zhang, Masanobu Tsuda, Guo-Xiang Yang, Koichi Tsuneyama, Guanghua Rong, M William Ridgway, A Aftab Ansari, A Richard Flavell, L Ross Coppel, Zhe-Xiong Lian and Eric M Gershwin : Deletion of interleukin-6 in mice with the dominant negative form of transforming growth factor beta receptor II improves colitis but exacerbates autoimmune cholangitis., Hepatology, Vol.52, No.1, 215-222, 2010.
(Summary)
The data from these observations emphasize that there are distinct mechanisms involved in inducing pathology in inflammatory bowel disease compared to autoimmune cholangitis. These data also suggest that patients with inflammatory bowel disease may not be the best candidates for treatment with anti-IL-6R if they have accompanying autoimmune liver disease and emphasize caution for therapeutic use of anti-IL-6R antibody.
Masanori Kobayashi, Satonori Higuchi, Katsuhiko Mizuno, Koichi Tsuneyama, Tatsuki Fukami, Miki Nakajima and Tsuyoshi Yokoi : Interleukin-17 is involved in alpha-naphthylisothiocyanate-induced liver injury in mice., Toxicology, Vol.275, No.1-3, 50-57, 2010.
(Summary)
Drug-induced liver injury (DILI) is a major safety concern in drug development and clinical drug therapy. The pathogenesis of DILI usually involves the participation of the parent drug or metabolites that either directly affect the cell biochemistry or elicit an immune response. However, in most cases the mechanisms are unknown. Alpha-naphthylisothiocyanate (ANIT) is known as a hepatotoxicant that causes biliary cell and hepatocyte damage and induces intense neutrophil infiltration in the liver. To investigate whether an immune-mediated mechanism is involved in ANIT-induced liver injury, we examined the plasma AST, ALT and T-Bil levels, hepatic expression of transcriptional factors, cytokines and CXC chemokine genes, plasma IL-17 level and histopathological changes in liver after ANIT administration in mice. Hepatic mRNA expression of retinoid related orphan receptor gamma t (ROR gamma t) and macrophage inflammatory protein (MIP-2) and plasma IL-17 level was significantly increased in ANIT-administered mice as well as the plasma AST, ALT and T-Bil. Neutralization of IL-17 using anti-IL-17 antibody (100 microg/mouse, single i.p.) suppressed the hepatotoxic effect of ANIT. Co-administration of recombinant IL-17 (1 microg/mouse, single i.p.) to ANIT-administered mice resulted in a remarkable increase of the plasma AST, ALT and T-Bil levels. In conclusion, it was firstly demonstrated that IL-17 is involved in the ANIT-induced liver injury in mice.
(Keyword)
1-Naphthylisothiocyanate / Animals / Chemical and Drug Induced Liver Injury / Female / Interleukin-17 / Mice / Mice, Inbred BALB C / Recombinant Proteins
Chia-Ming Chang, Chia-Hui Lo, Yao-Ming Shih, Yin Chen, Ping-Yi Wu, Koichi Tsuneyama, R Steve Roffler and Mi-Hua Tao : Treatment of hepatocellular carcinoma with adeno-associated virus encoding interleukin-15 superagonist., Human Gene Therapy, Vol.21, No.5, 611-621, 2010.
(Summary)
Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, but effective therapies are still needed. The liver has been identified as an important immune organ and is heavily populated with various lymphocyte subsets known to play important roles in cancer immunosurveillance. We hypothesized that activation of hepatic lymphocytes by interleukin (IL)-15, a cytokine known for its ability to trigger proliferation and activation of natural killer (NK) cells, natural killer T cells, and memory CD8(+) T cells, might offer an alternative therapy for HCC. We employed hepatotropic adeno-associated virus serotype 8 (AAV8) to deliver an IL-15 superagonist (IL-15-IL-15RalphaS), consisting of IL-15 covalently linked to the N-terminal sushi domain of the IL-15 receptor alpha chain, to achieve local sustained cytokine expression in the liver environment. We observed that a single injection of AAV8 expressing IL-15-IL-15RalphaS, but not IL-15 alone, greatly expanded the number of hepatic mononuclear cells, mainly NK cells, for at least 21 days. AAV8/IL-15-IL-15RalphaS treatment generated potent antitumor activity in a liver metastatic murine HCC model (BNL cells), and significantly prolonged the survival time of treated animals. The antitumor effect depended mainly on NK cells, not on CD8(+) and CD4(+) T cells, because AAV8/IL-15-IL-15RalphaS treatment greatly enhanced the cytolytic activity of hepatic NK cells and depletion of NK cells abrogated the therapeutic effect. Importantly, no apparent liver toxicity was observed during AAV8/IL-15-IL-15RalphaS treatment. Together, our data demonstrate that AAV8-delivered IL-15-IL-15RalphaS provides an effective and safe therapy against metastatic HCC.
Makoto Fujimoto, Koichi Tsuneyama, Hideki Kinoshita, Hirozo Goto, Yasuo Takano, Carlo Selmi, L Carl Keen, Eric M Gershwin and Yutaka Shimada : The traditional Japanese formula keishibukuryogan reduces liver injury and inflammation in patients with nonalcoholic fatty liver disease., Annals of the New York Academy of Sciences, Vol.1190, 151-158, 2010.
(Summary)
The Kampo formula keishibukuryogan (KBG, Guizhifulingwan) is frequently used in traditional Japanese and Chinese medicine to treat several symptoms and manifests anti-inflammatory and scavenging effects. Nonalcoholic fatty liver disease (NAFLD) is a common manifestation of the metabolic syndrome and has the potential to evolve to liver cirrhosis through chronic inflammation and steatohepatisis (NASH). We have recently reported the KBG significant effectiveness on liver injury in a NASH animal model that prompted us to prescribe to KBG (TJ-25). We performed a retrospective study and reviewed the charts of outpatients who were prescribed KBG for 8-12 weeks due to non-liver-related symptoms (n= 11) over the past year to evaluate the clinical outcome. In six of these cases, biochemical and ultrasound signs of NAFLD were observed. KBG led to a significant reduction in liver injury tests and blood cholesterol but had no effects on body weight in all NAFLD cases. Further, liver tests and lipid profiles returned to baseline values when KBG treatment was stopped. On the basis of data on a small number of subjects, we suggest that the use of KBG is a safe complementary treatment in patients with NAFLD. While it is unlikely that Kampo formulas may substitute the current nutritional approaches to the metabolic syndrome, future studies should address the possibility of an additive effect, possibly through anti-inflammatory mechanisms.
Yasuni Nakanuma, Yoh Zen, Kenichi Harada, Motoko Sasaki, Akitaka Nonomura, Takeshi Uehara, Kenji Sano, Fukuo Kondo, Toshio Fukusato, Koichi Tsuneyama, Masahiro Ito, Kenichi Wakasa, Minoru Nomoto, Hiroshi Minato, Hironori Haga, Masayoshi Kage, Hirohisa Yano, Joji Haratake, Shinichi Aishima, Tomoyuki Masuda, Hajime Aoyama, Aya Miyakawa-Hayashino, Toshiharu Matsumoto, Hayato Sanefuji, Hidenori Ojima, Tse-Ching Chen, Eunsil Yu, Ji-Hun Kim, Nyun Young Park and Wilson Tsui : Application of a new histological staging and grading system for primary biliary cirrhosis to liver biopsy specimens: Interobserver agreement., Pathology International, Vol.60, No.3, 167-174, 2010.
(Summary)
Recently the authors proposed a new staging and grading system for primary biliary cirrhosis (PBC) that takes into account necroinflammatory activity and histological heterogeneity. Herein is proposed a convenient version of this system. Scores for fibrosis, bile duct loss, and chronic cholestasis were combined for staging: stage 1, total score of 0; stage 2, score 1-3; stage 3, score 4-6; and stage 4, score 7-9. Cholangitis activity (CA) and hepatitis activity (HA) were graded as CA0-3, and HA0-3, respectively. Analysis of interobserver agreement was then conducted. Digital images of 62 needle liver biopsy specimens of PBC were recorded as virtual slides on DVDs that were sent to 28 pathologists, including five located overseas. All participants were able to apply this version in all 62 cases. For staging, kappa was 0.385 (fair agreement) and the concordance rate was 63.9%. For necroinflammatory activity, the kappa and concordance rate were 0.110 (slight agreement) and 36.9% for CA, and 0.197 (slight agreement) and 47% for HA, respectively. In conclusion, this new staging and grading system for PBC seems to be more convenient and practical than those used at present, but more instruction and guidance are recommended for the grading of necroinflammatory activity in practice.
Hideki Hatta, Koichi Tsuneyama, Takashi Kondo and Yasuo Takano : Development of an ultrasound-emitting device for performing rapid immunostaining procedures., The Journal of Histochemistry and Cytochemistry, Vol.58, No.5, 421-428, 2010.
(Summary)
Although intraoperative rapid diagnosis is conventionally performed using hematoxylin-eosin (HE)-stained specimens, the use of additional special staining, together with immunostaining techniques, has been examined in recent years to improve diagnostic accuracy. In intraoperative rapid diagnosis, immunostaining should be completed within 7-10 min, because the pathologist is typically presented with an HE-stained specimen within the same time period. We hypothesized that ultrasound may enhance antigen-antibody reactions and reduce the number of immunostaining steps. To clarify the ability of ultrasound to support immunostaining, we first created an ultrasonic generator specifically for immunostaining. Next, we explored the optimal conditions for immunostaining of formalin-fixed specimens to examine the utility of the ultrasonic generator. Finally, we tried immunostaining with the ultrasonic generator using frozen specimens to simulate intraoperative rapid diagnosis. We report herein that ultrasound enables immunostaining of frozen specimens in approximately 10 min.
Shinji Shimoda, Kenichi Harada, Hiroaki Niiro, Akinobu Taketomi, Yoshihiko Maehara, Koichi Tsuneyama, Kentaro Kikuchi, Yasuni Nakanuma, R Ian Mackay, Eric M Gershwin and Koichi Akashi : CX3CL1 (fractalkine): a signpost for biliary inflammation in primary biliary cirrhosis., Hepatology, Vol.51, No.2, 567-575, 2010.
(Summary)
There are common properties of ECs, LSECs, and BECs, whether derived from PBC or viral hepatitis, but there are also significant differences, particularly in the potential in PBC for LMCs to adhere to ECs and BECs and to produce TNF-alpha; such properties were associated with augmented CX3CL1 production by BEC from PBC liver. The processes defined herein suggest potential novel biotherapies for biliary specific inflammation.
Shinji Nakada, Koichi Tsuneyama, Ichiro Kato, Yoshiaki Tabuchi, Ichiro Takasaki, Yukihiro Furusawa, Hiroshi Kawaguchi, Makoto Fujimoto, Hirozo Goto, Hiroaki Hikiami, Takashi Kondo, Yasuo Takano and Yutaka Shimada : Identification of candidate genes involved in endogenous protection mechanisms against acute pancreatitis in mice., Biochemical and Biophysical Research Communications, Vol.391, No.3, 1342-1347, 2009.
(Summary)
We surveyed changes of the gene expression profile in caerulein-exposed pancreas using Affymetrix GeneChip system (39,000 genes). Up-regulation of genes coding for claudin 4, claudin 7, F11 receptor, cadherin 1, integrin beta 4, syndecan 1, heat shock proteins b1/90aa1, Serpinb6a, Serpinb6b, Serpinb9, Bax, Bak1, calpain 2, calpain 5, microtubule-associated protein 1 light chain 3 alpha, S100 calcium-binding proteins A4/A10 were found in mouse pancreas exposed to caerulein for 12h. In contrast, the anti-apoptotic gene Bcl2 was down-regulated. The functions of these genes concern tight junction formation, cell-cell/cell-matrix adhesions, stress response, protease inhibition, apoptosis, autophagy, and regulation of cytoskeletal dynamics. Caerulein-exposed pancreatic acinar cells were immunohistochemically stained for claudin 4, cadherin 1, integrin beta 4, heat shock protein b1, and Serpinb6a. In conclusion, we have newly identified a set of genes that are likely to be involved in endogenous self-protection mechanisms against acute pancreatitis.
Keiichi Koizumi, Yurika Saitoh, Takayuki Minami, Nobuhiro Takeno, Koichi Tsuneyama, Tatsuro Miyahara, Takashi Nakayama, Hiroaki Sakurai, Yasuo Takano, Miyuki Nishimura, Toshio Imai, Osamu Yoshie and Ikuo Saiki : Role of CX3CL1/fractalkine in osteoclast differentiation and bone resorption., The Journal of Immunology, Vol.183, No.12, 7825-7831, 2009.
(Summary)
The recruitment of osteoclast precursors toward osteoblasts and subsequent cell-cell interactions are critical for osteoclast differentiation. Chemokines are known to regulate cell migration and adhesion. CX3CL1 (also called fractalkine) is a unique membrane-bound chemokine that has dual functions for cells expressing its receptor CX3CR1: a potent chemotactic factor in its soluble form and a type of efficient cell adhesion molecule in its membrane-bound form. In this paper, we demonstrate a novel role of CX3CL1 in osteoblast-induced osteoclast differentiation. We found that osteoclast precursors selectively expressed CX3CR1, whereas CX3CL1 is expressed by osteoblasts. We confirmed that soluble CX3CL1 induced migration of bone marrow cells containing osteoclast precursors, whereas immobilized CX3CL1 mediated firm adhesion of osteoclast precursors. Furthermore, a blocking mAb against CX3CL1 efficiently inhibited osteoclast differentiation in mouse bone marrow cells cocultured with osteoblasts. Anti-CX3CL1 also significantly suppressed bone resorption in neonatal mice by reducing the number of bone-resorbing mature osteoclasts. Collectively, CX3CL1 expressed by osteoblasts plays an important role in osteoclast differentiation, possibly through its dual functions as a chemotactic factor and adhesion molecule for osteoclast precursors expressing CX3CR1. The CX3CL1-CX3CR1 axis may be a novel target for the therapeutic intervention of bone resorbing diseases such as rheumatoid arthritis, osteoporosis, and cancer bone metastasis.
Mika Ito, Akitoshi Nakashima, Takao Hidaka, Motonori Okabe, Duy Nguyen Bac, Shihomi Ina, Satoshi Yoneda, Arihiro Shiozaki, Shigeki Sumi, Koichi Tsuneyama, Toshio Nikaido and Shigeru Saito : A role for IL-17 in induction of an inflammation at the fetomaternal interface in preterm labour., Journal of Reproductive Immunology, Vol.84, No.1, 75-85, 2009.
(Summary)
Chorioamnionitis (CAM) is a major cause of preterm delivery. Inflammatory cytokines and chemokines play important roles in the pathogenesis of preterm delivery. Interleukin (IL)-17 is a key cytokine which induces inflammation and is critical to host defense. In this study, we examined the role of IL-17 in the pathogenesis of preterm delivery. The levels of cytokines including IL-17, IL-8 and tumor necrosis factor (TNF) alpha were measured by ELISA in amniotic fluid from 154 cases of preterm labor. Flow cytometry and immunohistochemical staining were performed to determine the distribution of IL-17-producing cells. IL-8 secretion was evaluated in primary cultured human amniotic mesenchymal (HAM) cells and human amniotic epithelial (HAE) cells stimulated with IL-17, TNFalpha or IL-1beta. We also studied the signaling pathway of IL-17 and TNFalpha in HAM cells. Levels of inflammatory cytokines in amniotic fluid were higher in preterm delivery cases than in term delivery cases. Furthermore, IL-8, IL-17 and TNFalpha levels were significantly higher in the preterm cases with CAM stage II or III than those without CAM. Flow cytometry and immunohistochemical staining revealed that CD3(+)CD4(+) T cells were the main source of IL-17 in the chorioamniotic membrane. Interestingly, TNFalpha-induced IL-8 secretion was enhanced by IL-17 in a dose-dependent manner in HAM cells. The IKK inhibitor BMS-345541 and mitogen-activated protein kinase (MAPK) inhibitors p38, JNK and p42/44 (ERK1/2 pathway) reduced IL-8 secretion by IL-17-stimulated and TNFalpha-stimulated HAM cells. These results indicate that IL-17, produced by T cells, promotes inflammation at the fetomaternal interface in preterm delivery.
Carlo Selmi and Koichi Tsuneyama : Nutrition, geoepidemiology, and autoimmunity., Autoimmunity Reviews, Vol.9, No.5, A267-70, 2009.
(Summary)
As well represented by the impaired immune function of malnourished individuals encountered in developing countries and the incidence of specific diseases following local nutrient deficiencies, nutrition and immunity have been linked to each other for centuries while the specific connection between dietary factors and autoimmunity onset or modulation is a more recent acquisition. Autoimmune diseases manifest limited prevalence rates in developing countries while numerous immunity-related claims have been proposed in the field of functional foods. Nevertheless, over the past years multiple lines of evidence have supported a major role for specific dietary factors (including vitamin D, vitamin A, selenium, zinc, omega-3 fatty acids, probiotics, and flavanols) in determining the immune responses involved in infections, allergies, and autoimmune diseases. Interestingly, the link between nutrition and autoimmunity may well contribute to the geoepidemiology observed for numerous conditions. In general terms, most data that will be discussed herein were obtained in experimental or animal models while human data from real-life clinical settings or randomized clinical trials remain largely unsatisfactory. Our current knowledge on the beneficial impact of nutrition on autoimmunity prompts us to encourage the search for evidence-based nutrition to support the everyday diet choices of patients.
Yuki Moritoki, Zhe-Xiong Lian, Keith Lindor, Joseph Tuscano, Koichi Tsuneyama, Weici Zhang, Yoshiyuki Ueno, Robert Dunn, Marilyn Kehry, L Ross Coppel, R Ian Mackay and Eric M Gershwin : B-cell depletion with anti-CD20 ameliorates autoimmune cholangitis but exacerbates colitis in transforming growth factor-beta receptor II dominant negative mice., Hepatology, Vol.50, No.6, 1893-1903, 2009.
(Summary)
These data suggest potential usage of anti-CD20 in early PBC resistant to other modalities, but raise a cautionary note regarding the use of anti-CD20 in inflammatory bowel disease.
Katsunori Yoshida, Guo-Xiang Yang, Weici Zhang, Masanobu Tsuda, Koichi Tsuneyama, Yuki Moritoki, A Aftab Ansari, Kazuichi Okazaki, Zhe-Xiong Lian, L Ross Coppel, R Ian Mackay and Eric M Gershwin : Deletion of interleukin-12p40 suppresses autoimmune cholangitis in dominant negative transforming growth factor beta receptor type II mice., Hepatology, Vol.50, No.5, 1494-1500, 2009.
(Summary)
These data indicate that in this mouse model of PBC, signaling by way of IL-12p40 is an essential requirement for the development of autoimmune cholangitis. The results of these studies will play an important role in identifying pathways and reagents that will selectively inhibit IL-12 signaling for the outlining of future therapeutic strategies for human PBC.
Yu Yamazaki, Isao Usui, Yukiko Kanatani, Yuji Matsuya, Koichi Tsuneyama, Shiho Fujisaka, Agussalim Bukhari, Hikari Suzuki, Satoko Senda, Shingo Imanishi, Kazuya Hirata, Manabu Ishiki, Ryuji Hayashi, Masaharu Urakaze, Hideo Nemoto, Masashi Kobayashi and Kazuyuki Tobe : Treatment with SRT1720, a SIRT1 activator, ameliorates fatty liver with reduced expression of lipogenic enzymes in MSG mice., American Journal of Physiology, Endocrinology and Metabolism, Vol.297, No.5, E1179-86, 2009.
(Summary)
Nonalcoholic fatty liver disease (NAFLD) is an abnormal liver metabolism often observed with insulin resistance and metabolic syndrome. Calorie restriction is a useful treatment for NAFLD and reportedly prolongs the life spans of several species in which sirtuin plays an important role. In this study, we examined whether the activation of SIRT1, a mammalian ortholog of sirtuin, may ameliorate the development of NAFLD. Monosodium glutamate (MSG) mice, which exhibited obesity and insulin resistance, were treated with SRT1720, a specific SIRT1 activator from the age of 6-16 wk. Sixteen-week-old MSG mice exhibited increased liver triglyceride content and elevated levels of aminotransferase. SRT1720 treatment significantly reduced these levels without affecting body weight or food intake. These results suggested that the administration of SRT1720 ameliorated the development of NAFLD in MSG mice. The expressions of lipogenic genes, such as sterol regulatory element-binding protein-1c, acetyl-CoA carboxylase, and fatty acid synthase, and the serum lipid profiles, including free fatty acids, were elevated in MSG mice and were reduced by SRT1720 treatment. SRT1720 treatment also reduced the expressions of lipogenic genes in cultured HepG2 cells. Furthermore, SRT1720 treatment decreased the expressions of marker genes for oxidative stress and inflammatory cytokines in the liver of MSG mice. Taken together, SRT1720 treatment may reduce liver lipid accumulation, at least in part, by directly reducing the expressions of lipogenic genes. The reduction of oxidative stress and inflammation may also be involved in the amelioration of NAFLD.
W Zhang, Y Moritoki, Koichi Tsuneyama, G-X Yang, Y Ilan, Z-X Lian and E M Gershwin : Beta-glucosylceramide ameliorates liver inflammation in murine autoimmune cholangitis., Clinical and Experimental Immunology, Vol.157, No.3, 359-364, 2009.
(Summary)
We have demonstrated spontaneous development of autoimmune cholangitis, similar to human primary biliary cirrhosis, in mice expressing a dominant negative form of the transforming growth factor-beta receptor (dnTGF-betaRII) restricted to T cells. The autoimmune cholangitis appears to be mediated by autoreactive CD8(+) T lymphocytes that home to the portal tracts and biliary system. Because the liver pathology is primarily secondary to CD8(+) T cells, we have determined herein whether administration of beta-glucosylceramide (GC), a naturally occurring plant glycosphingolipid, alters the natural history of disease in this model. We chose GC because previous work has demonstrated its ability to alter CD8(+) T cell responses and to down-regulate tissue inflammation. Accordingly, dnTGF-betaRII mice were treated with either GC or control for a period of 18 weeks beginning at 6 weeks of age. Importantly, in mice that received GC, there was a significant decrease in the frequency and absolute number of autoreactive liver-infiltrating CD8(+) T cells, accompanied by a significant decrease in activated CD44(high) CD8(+) T cell populations. Further, there was a significant reduction in portal inflammation in GC-treated mice. Interestingly, there were no changes in anti-mitochondrial antibodies, CD4(+) T cells, CD19(+) B cells or natural killer (NK) T cell populations, indicating further that the beneficial effects of GC on liver inflammation were targeted specifically to liver-infiltrating CD8(+) T cells. These data suggest that further work on GC in models of CD8(+) T-mediated inflammation are needed and point to a new therapeutic venue for potentially treating and/or modulating autoimmune disease.
Hiroyuki Takahashi, Shigeharu Miwa, Kazuhiro Nomoto, Shinichi Hayashi, Koichi Tsuneyama and Yasuo Takano : Peribiliary cyst of common bile duct: possible cause of refractory jaundice., Pathology International, Vol.59, No.9, 692-693, 2009.
(Keyword)
Aged / Autopsy / Cholestasis, Extrahepatic / Common Bile Duct / Common Bile Duct Diseases / Cysts / Fatal Outcome / Humans / Incidental Findings / Jaundice, Obstructive / Lung Diseases, Fungal / Male / Pneumonia / Zygomycosis
M1 and M2 ATMs constitute different subsets of macrophages. Insulin resistance is associated with both the number of M1 macrophages and the M1-to-M2 ratio. The increased expression of IL-10 after an HFD might be involved in the increased recruitment of M2 macrophages.
Yoshiyuki Sasaki, Wataru Suzuki, Tsutomu Shimada, Seiichi Iizuka, Satoko Nakamura, Mitsunobu Nagata, Makoto Fujimoto, Koichi Tsuneyama, Ryoji Hokao, Ken-Ichi Miyamoto and Masaki Aburada : Dose dependent development of diabetes mellitus and non-alcoholic steatohepatitis in monosodium glutamate-induced obese mice., Life Sciences, Vol.85, No.13-14, 490-498, 2009.
(Summary)
A single 4 mg/g dose of MSG is the most suitable as the obese model and induces not only severe obesity and diabetes mellitus, but also liver changes resembling human NAFLD/NASH. A small amount of MSG in the newborn develops obesity and the other complications without hyperphagia after a long term.
(Keyword)
Animals / Animals, Newborn / Body Mass Index / Body Weight / Diabetes Mellitus / Dose-Response Relationship, Drug / Female / Food Additives / Glucose / Liver / Liver Cirrhosis / Male / Mice / Mice, Inbred ICR / Mice, Obese / Sodium Glutamate
Yukitaka Yoshikawa, Mayu Morita, Hiroko Hosomi, Koichi Tsuneyama, Tatsuki Fukami, Miki Nakajima and Tsuyoshi Yokoi : Knockdown of superoxide dismutase 2 enhances acetaminophen-induced hepatotoxicity in rat., Toxicology, Vol.264, No.1-2, 89-95, 2009.
(Summary)
Drug-induced hepatotoxicity is a major problem in drug development, and oxidative stress is known as one of the causes. Superoxide dismutases (SODs) are important antioxidant enzymes against reactive oxygen species (ROS). Mitochondria are the major source of superoxide production, and SOD2 is mainly localized in mitochondria and, with other SODs, plays an important role in scavenging superoxide. Previously, we reported the establishment of an adenovirus vector with short hairpin RNA against rat SOD2 (AdSOD2-shRNA), and applied this to evaluate drug-induced cytotoxicity. In this study, infection of AdSOD2-shRNA to Fisher 344 rats resulted in a significant decrease of SOD2 mRNA, protein expression, and SOD2 enzyme activity to 28%, 35%, and 39%, respectively, 7 days after infection. Serum AST and ALT were significantly increased by single oral administration of acetaminophen (1000 mg/kg) to these SOD2-knockdown rats without fasting compared with the control adenovirus infected groups. Heme oxygenase-1 protein, known to be induced by oxidative stress, was detected in SOD2-knockdown rats administered acetaminophen. In addition, protein carbonyl and lipid peroxidation, also known to be induced by oxidative stress, were significantly increased in SOD2 knockdown rats. This is the first report of a SOD2-knockdown rat model that could be useful to evaluate the drug-induced hepatotoxicity with high sensitivity.
(Keyword)
Acetaminophen / Adenoviridae / Analgesics, Non-Narcotic / Animals / Animals, Genetically Modified / Biotransformation / Blotting, Western / Chemical and Drug Induced Liver Injury / Genetic Vectors / Heme Oxygenase-1 / Lipid Peroxidation / Liver Function Tests / Male / Mitochondria, Liver / Protein Carbonylation / RNA / Rats / Rats, Inbred F344 / Reverse Transcriptase Polymerase Chain Reaction / Superoxide Dismutase
Eisuke Kobayashi, Masanori Kobayashi, Koichi Tsuneyama, Tatsuki Fukami, Miki Nakajima and Tsuyoshi Yokoi : Halothane-induced liver injury is mediated by interleukin-17 in mice., Toxicological Sciences, Vol.111, No.2, 302-310, 2009.
(Summary)
Drug-induced liver injury is a major problem in drug development and clinical drug therapy. In most cases the mechanisms are still unknown, thus, it is difficult to predict or prevent these reactions. It has been known that halothane, an inhaled anesthetic, induces liver injury. To investigate the mechanisms of halothane-induced liver injury, we used a recently established mouse model of liver injury. The expression of transcription factors and cytokines specific for Th1 and Th2 (helper T cells), respectively, were compared between BALB/c and C57BL/6 mice. The mRNA expression ratios of mouse T-bet(a Th1-specific transcription factor)/GATA-binding protein (GATA-3, a Th2-specific transcription factor) and interferon gamma/interleukin (IL)-10 were lower in BALB/c mice compared with C57BL/6 mice, suggesting that a typical Th1 or Th2-dominant response could not be distinguished in halothane-induced liver injury. We observed increases of the plasma IL-17 level and hepatic macrophage inflammatory protein 2 expression in halothane-administrated BALB/c mice, as well as neutrophil infiltration. Neutralization of IL-17 suppressed the hepatotoxic effect of halothane. Administration of recombinant IL-17 (1 microg per mouse, single ip) to the halothane-treated mice resulted in a remarkable increase of alanine and aspartate aminotransferases. In conclusion, we demonstrated that IL-17 is involved in the halothane-induced liver injury.
(Keyword)
Anesthetics, Inhalation / Animals / Base Sequence / Chemical and Drug Induced Liver Injury / Cytokines / DNA Primers / Dinoprostone / Female / Gene Expression Regulation / Halothane / Interleukin-17 / Liver / Mice / Mice, Inbred BALB C / Mice, Inbred C57BL / Neutrophils / Recombinant Proteins / Reverse Transcriptase Polymerase Chain Reaction / Transcription Factors
Kazuto Tajiri, Yukihiro Shimizu, Koichi Tsuneyama and Toshiro Sugiyama : Role of liver-infiltrating CD3+CD56+ natural killer T cells in the pathogenesis of nonalcoholic fatty liver disease., European Journal of Gastroenterology & Hepatology, Vol.21, No.6, 673-680, 2009.
(Summary)
Intrahepatic CD3+CD56+ NKT cells are increased in NAFLD as NAS increased. These cells may enhance disease activity through cytokine production after the recognition of lipid antigens presented with CD1d in livers of NAFLD.
Mayu Morita, Sho Akai, Hiroko Hosomi, Koichi Tsuneyama, Miki Nakajima and Tsuyoshi Yokoi : Drug-induced hepatotoxicity test using gamma-glutamylcysteine synthetase knockdown rat., Toxicology Letters, Vol.189, No.2, 159-165, 2009.
(Summary)
Idiosyncratic drug-induced liver injury (DILI) is a major clinical problem for drug development. It is generally known that DILI is mainly caused by hepatic glutathione (GSH) depletion. The glutathione S-transferase activity of rodent is higher than that of human, which could make the prediction of DILI more difficult. Recently, we reported that an experimental rat model of GSH-depletion displayed high susceptibility to acetaminophen-induced hepatotoxicity. To deplete GSH, we used an adenovirus vector with short hairpin RNA against gamma-glutamylcysteine synthetase heavy chain subunit (AdGCSh-shRNA). In this study, we further investigated the usefulness of this rat model for determining drug-induced sensitive acute and subacute toxicity. Rats were administered diclofenac and flutamide which have been reported as idiosyncratic hepatotoxic drugs. In the acute (6 or 24h) or subacute (7 days) toxicity tests, rats were administered the drugs once or once a day for a week, respectively. Plasma biochemical markers for hepatotoxicity were measured. The 6 and 24h toxicity test of diclofenac, and the 24h and 7 days toxicity tests of flutamide showed significant ALT elevations. Additionally, the 24h toxicity test of flutamide showed a slight bilirubin elevation, and histological hepatotoxicity. The 7 days toxicity test of flutamide also demonstrated histological hepatotoxicity. In conclusion, this rat model would contribute to evaluating acute and subacute DILI in preclinical drug development.
(Keyword)
Androgen Antagonists / Animals / Anti-Inflammatory Agents, Non-Steroidal / Chemical and Drug Induced Liver Injury / Diclofenac / Dose-Response Relationship, Drug / Flutamide / Gene Silencing / Glutamate-Cysteine Ligase / Male / Rats / Toxicity Tests
Weici Zhang, Rahul Sharma, Shyr-Te Ju, Xiao-Song He, Yanyan Tao, Koichi Tsuneyama, Zhigang Tian, Zhe-Xiong Lian, Man Shu Fu and Eric M Gershwin : Deficiency in regulatory T cells results in development of antimitochondrial antibodies and autoimmune cholangitis., Hepatology, Vol.49, No.2, 545-552, 2009.
(Summary)
The lack of functional Foxp3 is a major predisposing feature for loss of tolerance that leads to autoimmune cholangitis. These findings reflect on the importance of regulatory T cells in other murine models as well as in patients with primary biliary cirrhosis.
Kengo Kawai, Yoshinari Atarashi, Terumi Takahara, Hiroshi Kudo, Kazuto Tajiri, Yoshiharu Tokimitsu, Yasuhiro Nakayama, Katsuharu Hirano, Yutaka Yata, Masami Minemura, Satoshi Yasumura, Yasuharu Onishi, Kazuhiro Tsukada, Koichi Tsuneyama, Yasuo Takano and Toshiro Sugiyama : Dietary supplement implicated in fulminant hepatic failure in a well-controlled Wilson disease patient., Clinical Journal of Gastroenterology, Vol.2, No.2, 119-124, 2009.
(Summary)
We encountered a patient with previously well-controlled Wilson disease who experienced fulminant hepatic failure with hemolytic anemia, possibly caused by the dietary supplement Health Proportion(®) (Jubilant Co., Ltd., Ehime, Japan). A 21-year-old woman was admitted to our hospital with marked liver dysfunction and severe hemolytic anemia. Free serum copper level was elevated at 101 μg/dl, and urinary copper excretion was extremely increased (25,600 μg/day). Plasma exchange and continuous hemodiafiltration were performed to remove serum copper and to treat the hemolytic anemia. However, liver function did not improve, and she underwent liver transplantation on 28th day after admission. Copper and iron contents in the resected liver were high at 851.9 μg and 551.7 μg/dry liver weight (g), respectively, despite the patient having regularly taken D-penicillamine since diagnosis and having a well-controlled copper level 1 year before her admission. Two months before admission, the patient had taken a dietary supplement made from soybeans for 1 month. This supplement was labeled as containing large amounts of copper and iron, and we assume that this caused fulminant hepatic failure with hemolytic crisis in this patient. It is important to be mindful of the micronutrient content of dietary supplements, especially for metabolic disorder patients.
Willy Hsu, Weici Zhang, Koichi Tsuneyama, Yuki Moritoki, M William Ridgway, A Aftab Ansari, L Ross Coppel, Zhe-Xiong Lian, Ian Mackay and Eric M Gershwin : Differential mechanisms in the pathogenesis of autoimmune cholangitis versus inflammatory bowel disease in interleukin-2Ralpha(-/-) mice., Hepatology, Vol.49, No.1, 133-140, 2009.
(Summary)
Interleukin-2 (IL-2) receptor alpha knockout (IL-2Ralpha(-/-)) mice have a deficiency of CD25 and a corresponding functional defect in T regulatory cells (Tregs). These mice spontaneously develop portal inflammation with biliary ductular damage and colitis with features similar to human inflammatory bowel disease with T cell infiltrates in both the liver and colon. In humans, inflammatory bowel disease may be accompanied by primary sclerosing cholangitis (PSC), but seldom primary biliary cirrhosis (PBC). We hypothesized that the effector mechanism responsible for T cell infiltrates would differ for colon versus liver. To address this thesis, we developed three colonies of double-knockout mice including IL-2Ralpha(-/-) CD4(-/-), IL-2Ralpha(-/-) CD8(-/-), and IL-2Ralpha(-/-) T cell receptor (TCR)-beta(-/-). Tissue immunopathology, body weight, and serum levels of cytokines, immunoglobulins, and anti-mitochondrial antibodies (AMA) were assayed at 3 months of age. Relative to IL-2Ralpha(-/-) mice, IL-2Ralpha(-/-) CD4(-/-) mice had increased biliary ductular damage but reduced inflammation in the colon. In contrast, IL-2Ralpha(-/-) CD8(-/-) mice had increased colon inflammation but markedly attenuated biliary ductular damage. Both IL-2Ralpha(-/-) CD4(-/-) and IL-2Ralpha(-/-) CD8(-/-) mice demonstrated elevated serum levels of tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), interleukin-12p40 (IL-12p40), and interleukin-2 (IL-2) compared with C57BL/6J controls, but only IL-2Ralpha(-/-) CD8(-/-) mice had increased serum levels of immunoglobulin A (IgA), AMA and interleukin-17 (IL-17). Finally, and of importance, IL-2Ralpha(-/-) TCR-beta(-/-) mice had abrogation of liver and colon pathological conditions and lacked AMA. In conclusion, on loss of Treg function in mice, CD8 T cells mediate biliary ductular damage whereas CD4 T cells mediate induction of colon-specific autoimmunity.
Z Ruth Y Lan, L Thucydides Salunga, Koichi Tsuneyama, Zhe-Xiong Lian, Guo-Xiang Yang, Willy Hsu, Yuki Moritoki, A Aftab Ansari, Claudia Kemper, Jeff Price, P John Atkinson, L Ross Coppel and Eric M Gershwin : Hepatic IL-17 responses in human and murine primary biliary cirrhosis., Journal of Autoimmunity, Vol.32, No.1, 43-51, 2008.
(Summary)
The emergence of new regulatory and pro-inflammatory immune cell subsets and cytokines dictates the need to re-examine the role of these subsets in various diseases involving the immune system. IL-17 has been recently identified as a key cytokine involved in numerous autoimmune processes. However, its role in liver autoimmune diseases remains unclear. Primary biliary cirrhosis (PBC) is characterized histologically by autoreactive CD4 and CD8 T cells surrounding damaged bile ducts. CD4(+) T cells are a major source of IL-17, which compose a distinct T helper subset (Th17). Thus we set out to determine the role of IL-17 in both human and a murine model of PBC in a liver-targeted manner. Our data demonstrate an increase in the frequency of IL-17(+) lymphocytic infiltration in liver tissues from PBC patients and those with other liver dysfunctions as compared to healthy livers. IL-2 receptor alpha knockout mice, a recently identified murine model of human PBC, also demonstrate marked aggregations of IL-17-positive cells within portal tracts and increased frequencies of Th17 cells in the liver compared to the periphery. Interestingly, CD4(+) T cells from livers of normal C57BL/6J mice also secreted higher levels of IL-17 relative to those from spleens, indicating a preferential induction of Th17 cells in liver tissues. Importantly, C57BL/6J cocultures of splenic CD4(+) T cells and liver non-parenchymal cells increased IL-17 production approximately 10-fold compared to T cells alone, suggesting a role of the liver microenvironment in Th17 induction in cases of liver autoimmunity and other liver inflammatory diseases.
Ying-Yi Li, Yu Wu, Koichi Tsuneyama, Tomohisa Baba and Naofumi Mukaida : Essential contribution of Ets-1 to constitutive Pim-3 expression in human pancreatic cancer cells., Cancer Science, Vol.100, No.3, 396-404, 2008.
(Summary)
We previously demonstrated that the proto-oncogene Pim-3 with serine/threonine kinase activity was aberrantly expressed in cancer cells but not in the normal cells of the pancreas. In order to elucidate the molecular mechanism underlying aberrant Pim-3 expression in pancreatic cancer cells, we constructed luciferase expression vectors linked to 5'-flanking deletion mutants of the human Pim-3 gene and transfected human pancreatic cancer cells with the resultant vectors. The region up to -264 bp was essential for constitutive Pim-3 gene expression, and the mutation in the Ets-1 binding site (between -216 and -211 bp) reduced luciferase activities. Moreover, Ets-1 mRNA and protein were constitutively expressed together with Pim-3 in human pancreatic cancer cell lines. Chromatin immunoprecipitation assay demonstrated constitutive binding of Ets-1 to the 5'-flanking region of human Pim-3 gene between -249 and -183 bp. Pim-3 promoter activity and its protein expression were induced by transfection with wild type-Ets-1 and were reduced by transfection with dominant negative-Ets-1 or Ets-1 small-interfering RNA (siRNA). Furthermore, dominant negative-Ets-1 and Ets-1 siRNA reduced the amount of Bad phosphorylated at its Ser(112) and induced apoptosis, when they were transfected into human pancreatic cancer cells. Finally, Pim-3 cDNA transfection reversed Ets-1 siRNA-induced increase in apoptosis and decrease in Bad phosphorylation at its Ser(112). These observations would indicate that the transcription factor Ets-1 can induce aberrant Pim-3 expression and subsequently prevent apoptosis in human pancreatic cancer cells.
K Wakabayashi, K Yoshida, C P S Leung, Y Moritoki, G-X Yang, Koichi Tsuneyama, Z-X Lian, T Hibi, A A Ansari, S L Wicker, M W Ridgway, L R Coppel, R I Mackay and E M Gershwin : Induction of autoimmune cholangitis in non-obese diabetic (NOD).1101 mice following a chemical xenobiotic immunization., Clinical and Experimental Immunology, Vol.155, No.3, 577-586, 2008.
(Summary)
Our laboratory has suggested that loss of tolerance to pyruvate dehydrogenase (PDC-E2) leads to an anti-mitochondrial antibody response and autoimmune cholangitis, similar to human primary biliary cirrhosis (PBC). We have suggested that this loss of tolerance can be induced either via chemical xenobiotic immunization or exposure to select bacteria. Our work has also highlighted the importance of genetic susceptibility. Using the non-obese diabetic (NOD) congenic strain 1101 (hereafter referred to as NOD.1101 mice), which has chromosome 3 regions from B6 introgressed onto a NOD background, we exposed animals to 2-octynoic acid (2OA) coupled to bovine serum albumin (BSA). 2OA has been demonstrated previously by a quantitative structural activity relationship to react as well as or better than lipoic acid to anti-mitochondrial antibodies. We demonstrate herein that NOD.1101 mice immunized with 2OA-BSA, but not with BSA alone, develop high titre anti-mitochondrial antibodies and histological features, including portal infiltrates enriched in CD8(+) cells and liver granulomas, similar to human PBC. We believe this model will allow the rigorous dissection of early immunogenetic cause of biliary damage.
Hikari Suzuki, Ichiro Kato, Isao Usui, Ichiro Takasaki, Yoshiaki Tabuchi, Takeshi Oya, Koichi Tsuneyama, Hiroshi Kawaguchi, Koichi Hiraga, Shin Takasawa, Hiroshi Okamoto, Kazuyuki Tobe and Masakiyo Sasahara : Characterization of diabetic nephropathy in CaM kinase IIalpha (Thr286Asp) transgenic mice., Biochemical and Biophysical Research Communications, Vol.379, No.1, 38-42, 2008.
(Summary)
Detailed studies were performed on diabetic kidneys derived from transgenic mice overexpressing the mutant form (Thr286Asp) of Ca(2+)/calmodulin-dependent protein kinase IIalpha (CaM kinase IIalpha) in pancreatic beta-cells. Kidney weight/body weight ratio, urinary albumin/creatinine ratio, serum BUN level, and mesangial/glomerular area ratio were all significantly higher in transgenic mice than in wild-type mice. cDNA microarray analysis revealed 17 up-regulated genes and 12 down-regulated genes in transgenic kidney. Among up-regulated genes, cyclin D2 (6.70-fold) and osteopontin (2.35-fold) were thought to play important roles in the progression of diabetic nephropathy. Transgenic glomeruli and tubular epithelial cells were strongly stained for osteopontin, a molecule which induces immune response. In quantitative real-time RT-PCR analyses, expressions of not only M1 macrophage marker genes but also M2 macrophage marker genes were elevated in renal cortex of transgenic mice. Overall results indicate that CaM kinase IIalpha (Thr286Asp) transgenic mice serve as an excellent model for diabetic nephropathy.
Yuki Moritoki, Weici Zhang, Koichi Tsuneyama, Katsunori Yoshida, Kanji Wakabayashi, Guo-Xiang Yang, Christopher Bowlus, M William Ridgway, Yoshiyuki Ueno, A Aftab Ansari, L Ross Coppel, R Ian Mackay, A Richard Flavell, Eric M Gershwin and Zhe-Xiong Lian : B cells suppress the inflammatory response in a mouse model of primary biliary cirrhosis., Gastroenterology, Vol.136, No.3, 1037-1047, 2008.
(Summary)
B cells have a suppressive effect on the inflammatory response in the dnTGF-betaRII model of primary biliary cirrhosis.
Takuya Ishibe, Akihiko Kimura, Yuko Ishida, Tatsunori Takayasu, Takahito Hayashi, Koichi Tsuneyama, Kouji Matsushima, Ikuhiro Sakata, Naofumi Mukaida and Toshikazu Kondo : Reduced acetaminophen-induced liver injury in mice by genetic disruption of IL-1 receptor antagonist., Laboratory Investigation; a Journal of Technical Methods and Pathology, Vol.89, No.1, 68-79, 2008.
(Summary)
Acetaminophen (APAP) induced increases in intrahepatic expression of interleukin (IL)-1 alpha, IL-1 beta, and IL-1 receptor antagonist (IL-1ra), when administered intraperitoneally. These observations prompted us to define the pathophysiological roles of IL-1ra in APAP-induced liver injury. Compared with wild-type (WT) mouse-derived hepatocytes, IL-1ra-deficient (IL-1ra KO)-derived hepatocytes exhibited more resistance against APAP but not APAP-derived major toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Moreover, the amounts of a major APAP adduct (selenium-binding protein), an indicator of NAPQI generation from APAP, was significantly lower in IL-1ra KO mice than WT mice with depressed intrahepatic expression of CYP1A2, CYP2E1, and CYP3A11, the enzymes crucially involved in NAPQI generation from APAP. These observations would indicate that IL-1ra deficiency impaired APAP metabolism. IL-1 alpha and IL-1 beta were expressed to similar extents in livers of untreated IL-1ra KO and WT mice. By contrast, the intranuclear amount of p65 of NF-kappaB, which can suppress the gene expression of CYP1A2, CYP2E1, and CYP3A11, was higher in untreated IL-1ra KO than WT mice. Moreover, when mice were intraperitoneally administered APAP (200 mg/kg), IL-1ra KO mice exhibited attenuated APAP-induced liver injury as evidenced by reductions in serum alanine transferase levels and histopathological changes such as centrilobular necrosis, hemorrhages, and leukocyte infiltration. Finally, when given 12 h before APAP challenge, IL-1 alpha repressed the intrahepatic expression of CYP1A2, CYP2E1, and CYP3A11, eventually reducing APAP-induced liver injury, along with reduction in APAP adducts. Collectively, NF-kappaB was activated without any stimuli by the genetic disruption of IL-1ra, and suppressed cytochrome P450 enzyme expression, thereby reducing APAP-induced liver injury.
(Keyword)
Acetaminophen / Analgesics, Non-Narcotic / Animals / Cells, Cultured / Chemical and Drug Induced Liver Injury / Cytochrome P-450 CYP1A2 / Cytochrome P-450 CYP2E1 / Drug Resistance / Gene Expression / Hepatocytes / Interleukin 1 Receptor Antagonist Protein / Interleukin-1alpha / Liver / Liver Diseases / Male / Mice / Mice, Inbred BALB C / Mice, Knockout / NF-kappa B / Transcription Factor RelA
Kanji Wakabayashi, Zhe-Xiong Lian, C Patrick S Leung, Yuki Moritoki, Koichi Tsuneyama, J Mark Kurth, S Kit Lam, Katsunori Yoshida, Guo-Xiang Yang, Toshifumi Hibi, A Aftab Ansari, M William Ridgway, L Ross Coppel, R Ian Mackay and Eric M Gershwin : Loss of tolerance in C57BL/6 mice to the autoantigen E2 subunit of pyruvate dehydrogenase by a xenobiotic with ensuing biliary ductular disease., Hepatology, Vol.48, No.2, 531-540, 2008.
(Summary)
these data provide a persuasive argument in favor of an environmental origin for human PBC.
Yuko Nakanishi, Koichi Tsuneyama, Kazuhiro Nomoto, Makoto Fujimoto, L Thucydides Salunga, Takahiko Nakajima, Shigeharu Miwa, Yoshihiro Murai, Shinichi Hayashi, Ichiro Kato, Koichi Hiraga, K Daniel Hsu, Fu-Tong Liu and Yasuo Takano : Nonalcoholic steatohepatitis and hepatocellular carcinoma in galectin-3 knockout mice., Hepatology Research, Vol.38, No.12, 1241-1251, 2008.
(Summary)
We believe that it would be interesting to use this murine model to investigate liver carcinogenesis based on a natural history of NAFLD. Furthermore, CT scanning might be a useful tool for longitudinal evaluation of morphological changes in vivo.
Guo-Xiang Yang, Zhe-Xiong Lian, Ya-Hui Chuang, Yuki Moritoki, Y Ruth Lan, Kanji Wakabayashi, A Aftab Ansari, A Richard Flavell, M William Ridgway, L Ross Coppel, Koichi Tsuneyama, R Ian Mackay and Eric M Gershwin : Adoptive transfer of CD8(+) T cells from transforming growth factor beta receptor type II (dominant negative form) induces autoimmune cholangitis in mice., Hepatology, Vol.47, No.6, 1974-1982, 2008.
(Summary)
These data suggest that in this model of PBC, autoreactive CD8(+) cells destroy bile ducts.
Katsuhisa Omagari, Shigeko Kato, Koichi Tsuneyama, Chisato Inohara, Yu Kuroda, Hiroe Tsukuda, Eri Fukazawa, Keiko Shiraishi and Masatoshi Mune : Effects of a long-term high-fat diet and switching from a high-fat to low-fat, standard diet on hepatic fat accumulation in Sprague-Dawley rats., Digestive Diseases and Sciences, Vol.53, No.12, 3206-3212, 2008.
(Summary)
To investigate the effects of a long-term high-fat diet and switching from high-fat to a low-fat diet on hepatic fat accumulation in Sprague-Dawley (SD) rats, 3-week-old male SD rats were fed a high-fat diet (HFD) containing 45% fat (kilocalories) for 43 weeks (HDHD group), an HFD for 23 weeks followed by a low-fat, standard diet (LFD) containing 10% fat for 20 weeks (HDLD group), and an LFD for 43 weeks (LDLD group). Histopathologically, steatosis and lobular inflammation was obvious in the HDLD and HDHD groups at 46 weeks of age, and ballooning hepatocytes and Mallory hyalines were seen in the HDHD group. Mild fibrosis was observed in 5 of 13 (38%) rats in the HDHD or HDLD groups. Our results demonstrate that a long-term high-fat diet can induce nonalcoholic steatohepatitis (NASH) in SD rats. Switching to a low-fat, standard diet prevented the progression of NASH, although steatosis was not improved.
Shigeko Kato, Katsuhisa Omagari, Koichi Tsuneyama, Eri Fukazawa, Hiroe Tsukuda, Chisato Inohara, Yu Kuroda, Keiko Shiraishi and Masatoshi Mune : A possible rat model for non-alcoholic steatohepatitis: Histological findings in SHR/NDmcr-cp rats., Hepatology Research, Vol.38, No.7, 743-744, 2008.
Jiro Ogawa, Hirokazu Kanegane, Koichi Tsuneyama, Rika Kanezaki, Takeshi Futatani, Keiko Nomura, Shin Ishizawa, Masakiyo Sasahara, Etsuro Ito and Toshio Miyawaki : Platelet-derived growth factor may be associated with fibrosis in a Down syndrome patient with transient myeloproliferative disorder., European Journal of Haematology, Vol.81, No.1, 58-64, 2008.
(Summary)
Transient myeloproliferative disorder (TMD) is experienced by approximately 10% of neonates with Down syndrome (DS). Most TMD is asymptomatic and the patients undergo spontaneous remission within a few months. However, some cases are fatal because of systemic organ dysfunctions including hepatic fibrosis. Some cytokines such as platelet-derived growth factor (PDGF) may be involved in the development of hepatic fibrosis in TMD. The report describes a fatal case of TMD accompanying DS. The patient presented with pulmonary hypertension and hepatic failure. An autopsy disclosed severe fibrosis in the lung, liver, kidney and pancreas. Immunohistochemical analysis revealed high expression of PDGF receptor beta in the severe fibrotic areas of the fibrotic tissues. A real-time polymerase chain reaction (PCR) analysis demonstrated the expression of PDGFalpha and PDGFbeta in the peripheral blood samples of the patient. The finding indicates that the PDGF pathway may play an important role in the fibrosis of several organs in patients with TMD.
Shinji Shimoda, Kenichi Harada, Hiroaki Niiro, Tomoharu Yoshizumi, Yuji Soejima, Akinobu Taketomi, Yoshihiko Maehara, Koichi Tsuneyama, Minoru Nakamura, Atsumasa Komori, Kiyoshi Migita, Yasuni Nakanuma, Hiromi Ishibashi, Carlo Selmi and Eric M Gershwin : Biliary epithelial cells and primary biliary cirrhosis: the role of liver-infiltrating mononuclear cells., Hepatology, Vol.47, No.3, 958-965, 2008.
(Summary)
We submit that the proinflammatory activity of BECs in PBC is secondary to the intervention of LMNCs and is not determined per se. These data support the hypothesis that BECs are in fact "innocent victims" of autoimmune injury and that the adaptive immune response is critical in PBC.
Makoto Fujimoto, Koichi Tsuneyama, Mosaburo Kainuma, Nobuyasu Sekiya, Hirozo Goto, Yasuo Takano, Katsutoshi Terasawa, Carlo Selmi, Eric M Gershwin and Yutaka Shimada : Evidence-based efficacy of Kampo formulas in a model of non alcoholic fatty liver., Experimental Biology and Medicine, Vol.233, No.3, 328-337, 2008.
(Summary)
Data on the efficacy of herbal compounds are often burdened by the lack of appropriate controls or a limited statistical power. Treatments to prevent the progression of non alcoholic fatty liver disease (NAFLD) to steatohepatitis (NASH) remain unsatisfactory. A total of 56 rabbits were arrayed into 7 groups fed with standard rabbit chow (SRC), SRC with 1% cholesterol, or each of the five experimental treatments (Kampo formulas 1% keishibukuryogan [KBG], 1% orengedokuto [OGT], and 1% shosaikoto [SST]; vitamin E [VE]; or pioglitazone [PG]) in a 1% cholesterol SRC. We analyzed changes after 12 weeks in plasma and liver lipid profiles, glucose metabolism, adipocytokines, oxidative stress, and liver fibrosis. Data demonstrated that all five treatments were associated with significant amelioration of lipid profiles, oxidative stress, and liver fibrosis compared to no supplementation. KBG was superior to VE and PG in the reduction of liver total cholesterol (P < 0.01) and lipid peroxidase levels (P < 0.05), urinary 8-hydroxy-2'-deoxyguanosine (P < 0.05), hepatic alpha-smooth muscle actin positive areas (P < 0.01) and activated stellate cells (P < 0.01). In conclusion, there was a statistically significant benefit of Kampo formulas (KBG in particular) on a dietary model of NAFLD/NASH. Future studies need to be directed at the mechanisms in the treatment of NASH.
Tomohiko Kutsuna, Huachuan Zheng, Osman Hekmat Abdel-Aziz, Yoshihiro Murai, Koichi Tsuneyama, Isao Furuta and Yasuo Takano : High JC virus load in tongue carcinomas may be a risk factor for tongue tumorigenesis., Virchows Archiv, Vol.452, No.4, 405-410, 2008.
(Summary)
The John Cunningham virus (JCV) asymptomatically infects a large proportion (approximately 90%) of the population worldwide but may be activated in immunodeficient patients, resulting in progressive multifocal leukoencephalopathy. Recent reports demonstrated its oncogenic role in malignancies. In this paper, the presence of JCV-targeting T antigen was investigated in tongue carcinoma (TC, n = 39), dysplastic tongue epithelium (DTE, n = 15) and glossitis (n = 15) using real-time polymerase chain reaction (PCR) and in situ PCR and immunohistochemistry, and JCV copies were analyzed with the clinicopathological parameters of TCs. The results demonstrated that glossitis and DTEs had significantly lower copies of JCV (410.5 +/- 44.3 and 658.3 +/- 53.3 copies/mug DNA respectively) than TCs (981.5 +/- 14.0, p < 0.05). When they were divided into three groups with 0-200 copies/mug DNA (low), 201-1,000 (moderate) and more than 1001 (high), TCs showed 3 (7.6%) in the low group, 21 (53.8%) in the moderate group and 15 (38.4%) in the high group and glossitis showed 11 (73.3%) in the low group, 0 (0%) in the moderate group and 4 (26.6%) in the high group. The DTEs occupied an intermediate position between them (p < 0.001). In situ PCR demonstrated that the nuclei of TC and DTE cells are sporadically T-antigen positive but not in nasal turbinate epithelial cells. Immunohistochemistry for T-antigen protein revealed four positive cases only in TCs. The existence of JCV T-antigen DNA was not associated with the clinicopathological variables of TCs. In conclusion, the presence of JCV may be a risk factor of tongue carcinogenesis.
Sabine Oertelt-Prigione, K Tin Mao, Carlo Selmi, Koichi Tsuneyama, A Aftab Ansari, L Ross Coppel, Pietro Invernizzi, Mauro Podda and Eric M Gershwin : Impaired indoleamine 2,3-dioxygenase production contributes to the development of autoimmunity in primary biliary cirrhosis., Autoimmunity, Vol.41, No.1, 92-99, 2008.
(Summary)
The immunomodulatory effects of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) have been elucidated at a cellular level and implicated in the pathogenesis of several complex diseases. Defects within the regulatory T cell compartment are one of the characteristics of primary biliary cirrhosis (PBC), an autoimmune chronic cholestatic liver disease, a phenotype that has also been shown in disease-mimicking animal models of this disease. We hypothesized that IDO dysregulation could lead to altered frequency and/or function of T cells at the level of antigen processing/presentation and we thus investigated IDO in peripheral monocytes and bile duct cells from patients with PBC. Both expression and activation manifested an impaired IFN-gamma response in peripheral monocytes while a peculiar IDO expression profile in bile duct cells characterized early stage PBC. Further, we observed an increased frequency of a gain-of-function SNP within the TGF-beta promoter region, a molecule known to suppress IDO transcription. In conclusion, we submit that an impaired IDO induction characterizes PBC and might represent a contributing factor in disease pathogenesis in association with several specific defects in the target tissue.
Osman Hekmat Abdel-Aziz, Yoshihiro Murai, Ichiro Takasaki, Yoshiaki Tabuchi, Hua-chuan Zheng, Kazuhiro Nomoto, Hiroyuki Takahashi, Koichi Tsuneyama, Ichiro Kato, K Daniel Hsu, Fu-tong Liu, Koichi Hiraga and Yasuo Takano : Targeted disruption of the galectin-3 gene results in decreased susceptibility to NNK-induced lung tumorigenesis: an oligonucleotide microarray study., Journal of Cancer Research and Clinical Oncology, Vol.134, No.7, 777-788, 2008.
(Summary)
Disrupted galectin-3 may attenuate the lung carcinogenesis due to its regulatory role in the B-cell receptor, ERK/MAPK, and PPAR signal pathways.
Takuya Akashi, Keiichi Koizumi, Koichi Tsuneyama, Ikuo Saiki, Yasuo Takano and Hideki Fuse : Chemokine receptor CXCR4 expression and prognosis in patients with metastatic prostate cancer., Cancer Science, Vol.99, No.3, 539-542, 2008.
(Summary)
The chemokine receptor CXCR4 has been reported to be aberrantly expressed in human cancers and has also been shown to participate in the development of cancer metastasis. The present study was carried out to assess immunohistochemically the pattern of CXCR4 expression in patients with metastatic prostate cancer. We analyzed whether there may be an association between CXCR4 expression and prognosis. Fifty-two patients who received hormonal therapy were enrolled. Specimens were obtained from transperineal needle biopsy before treatment, and were stained with antihuman CXCR4 antibody. We also evaluated the pathological grade, extent of bony metastasis, clinical response to hormonal therapy, and patient prognosis. CXCR4 was detected in 94.2% patients. Its expression showed no association with pathological grade, extent of bony metastasis, or clinical response to hormonal therapy. Patients with a high expression of CXCR4 in tumors had poorer cancer-specific survival than those with low expression of CXCR4. CXCR4 expression is a useful prognostic factor for patients with metastatic prostate cancer treated with androgen-withdrawal therapy.
(Keyword)
Aged / Aged, 80 and over / Antineoplastic Agents, Hormonal / Biomarkers, Tumor / Bone Neoplasms / Humans / Immunohistochemistry / Male / Middle Aged / Multivariate Analysis / Prognosis / Prostatic Neoplasms / Receptors, CXCR4 / Survival Analysis
Kazuhiro Nomoto, Koichi Tsuneyama, Hiroyuki Takahashi, Yoshihiro Murai and Yasuo Takano : Cytoplasmic fine granular expression of 8-hydroxydeoxyguanosine reflects early mitochondrial oxidative DNA damage in nonalcoholic fatty liver disease., Applied Immunohistochemistry & Molecular Morphology : AIMM, Vol.16, No.1, 71-75, 2008.
(Summary)
To clarify the possible role of oxidative stress in hepatocytes in nonalcoholic fatty liver disease, the hepatic expression of 8-hydroxydeoxyguanosine (8-OHdG), a good marker of oxidative DNA damage, was immunohistochemically investigated in nonalcoholic steatohepatitis (NASH) and steatosis. In double immunostaining, the cytoplasmic fine granular 8-OHdG expression was considered to reflect 8-OHdG-positive mitochondrial DNA affecting oxidation stress. In steatosis, 4 of 8 cases showed cytoplasmic 8-OHdG, 1 case showed nuclear 8-OHdG and 1 case showed both cytoplasmic and nuclear 8-OHdG. In contrast, 8-OHdG expression was more frequently detected in NASH (12 of 13 cases, 92%). Immunoreactivity for 8-OHdG was observed only in the cytoplasm with a fine granular pattern (1 of 13 cases, 8%), only in the nucleus (6 of 13 cases, 46%), and in both the cytoplasm and the nucleus (5 of 13 cases, 38%). Megamitochondria also exhibited 8-OHdG intensely. We indicate that 8-OHdG expression in the cytoplasm with a fine granular pattern reflects oxidative damage to the mitochondrial DNA of hepatocytes in both NASH and steatosis. We propose herein that the evaluation of cytoplasmic 8-OHdG may be a sensitive diagnostic marker of early nonalcoholic fatty liver disease events.
Osman Hekmat Abdel-Aziz, Yoshihiro Murai, Mei Hong, Tomohiko Kutsuna, Hiroyuki Takahashi, Kazuhiro Nomoto, Shuichi Murata, Koichi Tsuneyama and Yasuo Takano : Detection of the JC virus genome in lung cancers: possible role of the T-antigen in lung oncogenesis., Applied Immunohistochemistry & Molecular Morphology : AIMM, Vol.15, No.4, 394-400, 2007.
(Summary)
The JC virus (JCV) infects a large proportion of the population worldwide and 80% to 90% of adults are seropositive and it may be activated in immunodeficient patients, resulting in progressive multifocal leukoencephalopathy. Recent reports described the possibility of its oncogenetic role in several malignancies. To clarify whether JCV might have a potential role in the genesis of lung cancers, we investigated the presence of its genome in 62 tumors, along with 23 samples of normal lung tissue, targeting the T-antigen, VP, and Agnoprotein by nested polymerase chain reaction/Southern blotting followed by direct DNA sequencing. Immunohistochemistry was performed to assess links between p53 and beta-catenin in lung cancers and the presence of T-antigen. The T-antigen was detected in 25 of 62 lung cancers but only 4 of 23 normal lung samples (P=0.048). In total, the JCV genome was present in 33 of the lung cancers and 10 of the normal samples. Furthermore, T-antigen was found in cancer cells in metastatic lymph nodes in 3 of 4 cases (P=0.042) and was more frequently detected in adenocarcinomas than in squamous cell carcinomas (P=0.038). Immunohistochemistry showed significant correlations between T-antigen and p53 (P=0.022) and also nuclear detection of beta-catenin (P=0.021). It is concluded that the JCV genome might be present in cancer cells in approximately half of all Japanese lung cancer cases, and that the T-antigen may play a role in oncogenesis of lung cancers through inactivation of p53 and dysregulation of the Wnt signaling pathway.
Huachuan Zheng, Hiroyuki Takahashi, Yoshihiro Murai, Zhengguo Cui, Kazuhiro Nomoto, Koichi Tsuneyama and Yasuo Takano : Low expression of FHIT and PTEN correlates with malignancy of gastric carcinomas: tissue-array findings., Applied Immunohistochemistry & Molecular Morphology : AIMM, Vol.15, No.4, 432-440, 2007.
(Summary)
To clarify the roles of FHIT (fragile histidine triad) and PTEN (phosphatase and tensin homology deleted from human chromosome 10) expression in the genesis and progression of gastric cancers, we examined expression of FHIT and PTEN on tissue microarray containing gastric normal mucosa (n=49), adenoma (n=49), noncancerous mucosa adjacent to carcinoma (n=84) and carcinoma (n=249) by immunohistochemistry. Their expression was compared with clinicopathologic parameters of tumors, including expression of p53 and cysteine protease protein 32 as well as survival time of patients with carcinoma. The results showed expression of FHIT and PTEN were lower in gastric carcinoma than those in normal mucosa, noncancerous mucosa adjacent to carcinoma and adenoma of the stomach (P<0.05). FHIT and PTEN expression showed a significantly negative association with depth of invasion, lymphatic invasion, and lymph node metastasis, liver metastasis, and Union Internationale Contre le Cancer staging of gastric carcinoma (P<0.05). Intestinal-type gastric carcinomas highly expressed FHIT and PTEN protein, compared with diffuse-type ones (P<0.05). Expression of FHIT and PTEN were positively related with expression of p53 and cysteine protease protein 32 in gastric carcinoma (P<0.05), as well as favorable prognosis of the patients with the tumors (P<0.05). There was positive relationship between FHIT and PTEN expression in gastric carcinoma (P<0.05). It was suggested that down-regulated expression of FHIT and PTEN contributed to gastric carcinogenesis possibly by involving in the imbalance between apoptosis and proliferation of cells. Their altered expression underlay the molecular basis of invasion, metastasis, differentiation of gastric carcinoma.
Ichiro Nozaki, Takuo Watanabe, Makoto Kawaguchi, Hiroyasu Akatsu, Koichi Tsuneyama, Yasuhiko Yamamoto, Kazuyo Ohe, Hideto Yonekura, Masahito Yamada and Hiroshi Yamamoto : Reduced expression of endogenous secretory receptor for advanced glycation endproducts in hippocampal neurons of Alzheimer's disease brains., Archives of Histology and Cytology, Vol.70, No.5, 279-290, 2007.
(Summary)
The receptor for advanced glycation endproducts (RAGE) is a cell-surface multiligand receptor, which interacts with amyloid beta (Abeta), a key protein in Alzheimer's disease (AD). RAGE-Abeta interaction is thought to be associated with pathological progression in AD. A splice variant of RAGE, endogenous secretory RAGE (esRAGE) can act as a decoy receptor for RAGE ligands that would prevent the progression of some pathologic conditions. In this study, the expression of esRAGE in the hippocampal tissues from AD brains compared with control (non-AD) was examined by immunohistochemistry and Western blot analysis. Semiquantitative immunohistochemical analysis of hippocampal tissues using esRAGE-specific antibody revealed significantly decreased immunoreactivities in pyramidal cells in CA1 and CA3 regions of AD compared with non-AD. On the other hand, immunoreactivities of astrocytes for esRAGE significantly increased in those regions. Dentate granule cells and astrocytes showed essentially invariant immunoreactivities between AD and non-AD. Changes in esRAGE immunoreactivity in CA3 neurons and astrocytes were observed from the early pathological stages. Moreover, the esRAGE-immunoreactive bands of AD samples were weaker than those of non-AD samples in Western blot analysis. The results indicate that low expression of esRAGE in the hippocampus would be associated with the development of AD.
(Keyword)
Aged, 80 and over / Alzheimer Disease / Female / Hippocampus / Humans / Immunohistochemistry / Male / Receptor for Advanced Glycation End Products / Receptors, Immunologic
Atsushi Tanaka, Koichi Tsuneyama, Masaki Mikami, Satoko Uegaki, Mitsuhiko Aiso and Hajime Takikawa : Gene expression profiling in whole liver of bile duct ligated rats: VEGF-A expression is up-regulated in hepatocytes adjacent to the portal tracts., Journal of Gastroenterology and Hepatology, Vol.22, No.11, 1993-2000, 2007.
(Summary)
Gene expression profiling in the whole liver of the BDL rats revealed 38 up-regulated and 17 down-regulated transcripts. In addition, the up-regulated expression of VEGF was mainly observed in hepatocytes surrounding to the portal tracts.
(Keyword)
Animals / Bile Ducts / Blotting, Western / Cell Proliferation / Cholestasis / Disease Models, Animal / Gene Expression Profiling / Hepatocytes / Immunohistochemistry / Ligation / Liver / Male / Oligonucleotide Array Sequence Analysis / Portal System / Rats / Rats, Sprague-Dawley / Time Factors / Up-Regulation / Vascular Endothelial Growth Factor A
Hua-Chuan Zheng, Koichi Tsuneyama, Hiroyuki Takahashi, Shigeharu Miwa, Toshiro Sugiyama, Konstantinova Boryana Popivanova, Chifumi Fujii, Kazuhiro Nomoto, Naofumi Mukaida and Yasuo Takano : Aberrant Pim-3 expression is involved in gastric adenoma-adenocarcinoma sequence and cancer progression., Journal of Cancer Research and Clinical Oncology, Vol.134, No.4, 481-488, 2007.
(Summary)
Aberrant Pim-3 expression was involved in gastric adenoma-adenocarcinoma sequence and subsequent invasion and metastasis process in gastric cancer. Moreover, Pim-3 may be employed to predict the prognosis of gastric cancer patients. Distinct Pim-3 expression underlies the molecular mechanisms for the differentiation of intestinal-type and diffuse-type carcinomas.
Hiroyuki Takahashi, Yoshihiro Murai, Koichi Tsuneyama, Kazuhiro Nomoto, Eikichi Okada, Hideharu Fujita and Yasuo Takano : High labeling indices of cdc25B is linked to progression of gastric cancers and associated with a poor prognosis., Applied Immunohistochemistry & Molecular Morphology : AIMM, Vol.15, No.3, 267-272, 2007.
(Summary)
To clarify the significance of cdc25B, which plays an important physiologic role in regulation of the G2/M check point, in progression of gastric cancer, 125 samples of paraffin-embedded gastric cancers were investigated by immunohistochemistry. In addition, 3 human gastric cancer cell lines were studied to determine the cellular localization of cdc25B by immunohistochemistry and cell fractionation followed by Western blotting. In the cell lines cdc25B was found to be present in both nuclei and cytoplasm, but predominantly in nuclei. High labeling indices of cdc25B in invasion front of gastric cancer was observed in 31 out of 125 cases (24.8%), linked to an advanced depth of cancer invasion (P=0.02), high rates of lymphatic invasion (P=0.03), and lymph node metastasis (P<0.01). Furthermore, the Kaplan-Meier method demonstrated a poor prognosis for cdc25B high labeling indices cases (P=0.02), although multivariate analysis revealed it not to be an independent factor. In conclusion, it seems likely that cdc25B is located predominantly in nuclei when overexpressed and this has some linkage with progression of gastric cancer.
L Thucydides Salunga, Yoshiaki Tabuchi, Ichiro Takasaki, B Loreto Feril, Qing-Li Zhao, Kenzo Ohtsuka, Koichi Tsuneyama and Takashi Kondo : Identification of genes responsive to paeoniflorin, a heat shock protein-inducing compound, in human leukemia U937 cells., International Journal of Hyperthermia, Vol.23, No.6, 529-537, 2007.
(Summary)
The present results indicate that PF affects the expression of many genes including Hsp70 and will provide a better understanding on the molecular mechanism of action of this compound in inducing HSPs in cells.
C Patrick S Leung, Ogyi Park, Koichi Tsuneyama, J Mark Kurth, S Kit Lam, A Aftab Ansari, L Ross Coppel and Eric M Gershwin : Induction of primary biliary cirrhosis in guinea pigs following chemical xenobiotic immunization., The Journal of Immunology, Vol.179, No.4, 2651-2657, 2007.
(Summary)
Although significant advances have been made in dissecting the effector mechanisms in autoimmunity, the major stumbling block remains defining the etiological events that precede disease. Primary biliary cirrhosis (PBC) illustrates this paradigm because of its high degree of heritability, its female predominance, and its extraordinarily specific and defined immune response and target destruction. In PBC, the major autoantigens belong to E2 components of the 2-oxo-acid dehydrogenase family of mitochondrially located enzymes that share a lipoylated peptide sequence that is the immunodominant target. Our previous work has demonstrated that synthetic mimics of the lipoate molecule such as 6-bromohexoanate demonstrate a high degree of reactivity with PBC sera prompted us to immunize groups of guinea pigs with 6-bromohexanoate conjugated to BSA. In this study, we provide serologic and immunohistochemical evidence that such immunized guinea pigs not only develop antimitochondrial autoantibody responses similar to human PBC, but also develop autoimmune cholangitis after 18 mo. Xenobiotic-immunized guinea pigs are the first induced model of PBC and suggest an etiology that has implications for the causation of other human autoimmune diseases. The data also reflect the likelihood that, in PBC, the multilineage antimitochondrial response is a pathogenic mechanism and that loss of tolerance and subsequent development of biliary lesions depends on either modification of the host mitochondrial Ag or a similar breakdown due to molecular mimicry.
H Zheng, O Aziz H Abdel, Y Nakanishi, S Masuda, H Saito, Koichi Tsuneyama and Y Takano : Oncogenic role of JC virus in lung cancer., The Journal of Pathology, Vol.212, No.3, 306-315, 2007.
(Summary)
The JC virus (JCV) infects a large proportion of the population world wide and can cause progressive multifocal leucoencephalopathy in the context of immunodeficiency. Recent reports provide evidence that it may also be oncogenic. Here, JCV was examined by targeting its T-antigen in lung carcinomas (n=103) and normal lung tissues (n=18) by nested-PCR followed by Southern blot, real-time PCR, immunohistochemistry, in situ hybridization and in situ PCR. Additionally, expression of Ki-67, caspase-3, beta-catenin, p53, and Rb was analysed by immunohistochemistry on tissue microarrays of lung carcinomas. Copy numbers of JCV were compared with clinicopathological features. Normal lung tissue was positive significantly less frequently, and contained a lower copy number of JCV than lung carcinomas (p<0.05), and copies were lower in lung adenocarcinomas than in squamous, small or large cell carcinomas (p<0.05). In situ PCR and immunolabelling revealed JCV positivity in the nuclei of lung carcinoma cells. The JCV copy number correlated closely with sex, and expression of Ki-67 and membrane beta-catenin (p<0.05), but not with age, tumour size, pleural invasion, lymph node metastasis, expression of caspase-3, cytoplasmic beta-catenin, p53 or Rb, prognosis, smoking or cancer family history (p>0.05). Age and UICC staging were independent prognostic factors for lung carcinoma patients. These data suggest that JCV may be involved in lung carcinogenesis, especially in tumour types other than adenocarcinoma. Lung carcinomas with higher JCV copy numbers display high proliferation and down-regulation of cell adhesion mediated by membrane beta-catenin.
Sho Akai, Hiroko Hosomi, Keiichi Minami, Koichi Tsuneyama, Miki Katoh, Miki Nakajima and Tsuyoshi Yokoi : Knock down of gamma-glutamylcysteine synthetase in rat causes acetaminophen-induced hepatotoxicity., The Journal of Biological Chemistry, Vol.282, No.33, 23996-24003, 2007.
(Summary)
Drug-induced hepatotoxicity is mainly caused by hepatic glutathione (GSH) depletion. In general, the activity of rodent glutathione S-transferase is 10 to 20 times higher than that of humans, which could make the prediction of drug-induced hepatotoxicity in human more difficult. Gamma-glutamylcysteine synthetase (gamma-GCS) mainly regulates de novo synthesis of GSH in mammalian cells and plays a central role in the antioxidant capacity of cells. In this study, we constructed a GSH-depletion experimental rat model for the prediction of human hepatotoxicity. An adenovirus vector with short hairpin RNA against rat gamma-GCS heavy chain subunit (GCSh) (AdGCSh-shRNA) was constructed and used to knock down the GCSh. In in vitro study in H4IIE cells, a rat hepatoma cell line, GCSh mRNA and protein were significantly decreased by 80% and GSH was significantly decreased by 50% 3 days after AdGCSh-shRNA infection. In the in vivo study in rat, the hepatic GSH level was decreased by 80% 14 days after a single dose of AdGCSh-shRNA (2 x 10(11) pfu/ml/body), and this depletion continued for at least 2 weeks. Using this GSH knockdown rat model, acetaminophen-induced hepatotoxicity was shown to be significantly potentiated compared with normal rats. This is the first report of a GSH knockdown rat model, which could be useful for highly sensitive tests of acute and subacute toxicity for drug candidates in preclinical drug development.
(Keyword)
Acetaminophen / Animals / Cell Line / Chemical and Drug Induced Liver Injury / Disease Models, Animal / Drug Synergism / Gene Silencing / Glutamate-Cysteine Ligase / Glutathione / RNA, Small Interfering / Rats
Takahito Hayashi, Naho Hirayama, Ayako Ro, Norimasa Kageyama, Yuko Ishida, Koichi Tsuneyama, Akihiko Kimura, Tatsushige Fukunaga and Toshikazu Kondo : Severe brainstem compression by an unruptured giant vertebral aneurysm--an autopsy case., Legal Medicine, Vol.9, No.6, 322-325, 2007.
(Summary)
We describe an autopsy case of sudden unexpected death due to severe brainstem compression by an unruptured giant vertebral aneurysm. A 71-year-old male was found dead in his bedroom. The forensic autopsy revealed no severe trauma leading to his death. On internal examination, a giant intracranial aneurysm (3.4 x 2.6 x 2.7 cm) was observed on the trunk of the right vertebral artery. The aneurysm compressed the right side of the lower one-third of the pons and adjacent medulla oblongata. On sectioning, almost all of the aneurysm lumen was filled with a firm, clearly laminated organized thrombus. There was no evidence of subarachnoid hemorrhage. Histopathological analyses revealed congestion and hypoxic tissue changes in all organs examined. In microscopic sections of the giant vertebral aneurysm, thick fibrotic walls, intimal hyperplasia and organized thrombi in the lumen were found. Lots of intrathrombotic clefts with fresh erythrocytes were also observed. Moreover, Elastica van Gieson staining revealed fragmentation and disruption of the intimal elastic lamina in the aneurysmal wall. Collectively, we considered that some triggers in his daily life, including head rotation, might have caused the rapid onset of respiratory disturbance due to severe brainstem compression by a giant vertebral aneurysm.
L Thucydides Salunga, Zheng-Guo Cui, Shinji Shimoda, Hua-Chuan Zheng, Kazuhiro Nomoto, Takashi Kondo, Yasuo Takano, Carlo Selmi, Gianfranco Alpini, Eric M Gershwin and Koichi Tsuneyama : Oxidative stress-induced apoptosis of bile duct cells in primary biliary cirrhosis., Journal of Autoimmunity, Vol.29, No.2-3, 78-86, 2007.
(Summary)
There has been a relative paucity of effort at defining effector mechanisms of biliary damage in PBC. We hypothesize that biliary cells are destroyed secondary to the immunologic relationships of inflammation and biliary epithelial apoptosis and, in particular, that biliary damage is a result of reduced levels of glutathione-S-transferase (GST), the production of hypochlorous acid (HOCl) and its association with eosinophil peroxidase (EPO). To address this issue, we examined the expression of EPO and GST in PBC and control livers and demonstrated an increase of EPO within the portal areas of PBC. We also demonstrated that macrophages have evidence of phagocytosed EPO. Furthermore, we studied the influence of HOCl on apoptosis in cultured human biliary epithelial cells (BEC) as well as the associated activity of Bcl-2, Bax, p-JNK, JNK, p53, Fas and caspase-3. HOC1-induced apoptosis in BEC in a dose-dependent fashion increased the activity of caspase-3 and the expression of p53 and p-JNK. Pretreatment with l-buthionine-(S,R)-sulfoximine, a glutathione (GSH) inhibitor, potentiated the sensitivity of BEC to HOCl-induced apoptosis. We conclude that intracellular GSH reduction leads directly to BEC apoptosis. Modulation of these events will be critical to reduce immune-mediated destruction.
Shozo Hojo, Keiichi Koizumi, Koichi Tsuneyama, Yoshihisa Arita, Zhengguo Cui, Kanna Shinohara, Takayuki Minami, Isaya Hashimoto, Takashi Nakayama, Hiroaki Sakurai, Yasuo Takano, Osamu Yoshie, Kazuhiro Tsukada and Ikuo Saiki : High-level expression of chemokine CXCL16 by tumor cells correlates with a good prognosis and increased tumor-infiltrating lymphocytes in colorectal cancer., Cancer Research, Vol.67, No.10, 4725-4731, 2007.
(Summary)
CXCL16 is a new member of the chemokine superfamily, which exists in a transmembrane as well as a soluble form. Its receptor CXCR6 is detected on CD4(+) T cells, CD8(+) T cells, and natural killer T cells. Here, we report a significant correlation of CXCL16 expression by tumor cells with the infiltration of T cells and prognosis in colorectal cancer (CRC). We first found that CXCL16 expression was consistently up-regulated more in tumor tissues than in normal mucosa derived from the same CRC patients. Four human CRC cell lines also expressed CXCL16 mRNA and secreted soluble CXCL16. We next examined the expression of CXCL16 and infiltration of lymphocytes in CRC specimens (n = 58) by immunohistochemistry. CRC patients with high levels of CXCL16 expression (n = 43) had higher levels of CD4(+) and CD8(+) tumor-infiltrating lymphocytes (TIL; P < 0.01) than those with low levels of CXCL16 expression (n = 15). Furthermore, the high CXCL16 expression group showed significantly better prognosis than the low CXCL16 expression group (P < 0.05). Collectively, our data suggest that the expression of CXCL16 by tumor cells enhances the recruitment of TILs, thereby bringing about a better prognosis in CRC. Thus, CXCL16 is a new prognostic biomarker and may be useful for the development of a more effective therapeutic strategy for CRC.
Miao Yu, Huachuan Zheng, Koichi Tsuneyama, Hiroyuki Takahashi, Kazuhiro Nomoto, Huimian Xu and Yasuo Takano : Paradoxical expression of maspin in gastric carcinomas: correlation with carcinogenesis and progression., Human Pathology, Vol.38, No.8, 1248-1255, 2007.
(Summary)
Maspin, a serine protease inhibitor related to the serpin family, can suppress invasion and metastasis of malignancies. To clarify the role of maspin in the genesis and progression of gastric carcinomas, its expression pattern and level were studied by immunohistochemistry on tissue microarrays containing gastric carcinoma (n = 237), normal gastric mucosa (n = 23), intestinal metaplasia (n = 38), and adenoma (n = 42); and the findings were compared with clinicopathological parameters. Furthermore, maspin expression in the gastric carcinoma cell lines (HCG-27, MKN28, and MKN45) was examined by immunohistochemistry and Western blotting. We found that cytoplasmic and nuclear maspin expression paralleled each other (P < .05) and decreased from intestinal metaplasia, adenoma, and carcinoma to normal gastric mucosa (P < .05). A significant positive association was noted with depth of invasion, lymphatic invasion, lymph node metastasis, and TNM stage (P < .05) but not with sex or Lauren's classification (P > .05). Univariate and multivariate analyses indicated that expression of maspin correlated negatively with cumulative patient survival in gastric carcinoma (P < .05) but was not an independent factor in the prognosis. The 2 independent factors, depth of invasion and lymphatic invasion, influenced the relation between nuclear maspin expression and survival, whereas only depth of invasion correlated with cytoplasmic maspin. Our study indicated that maspin expression experiences upregulation in gastric precancerous lesions and then slight downregulation with malignant transformation. High expression may paradoxically promote invasion and metastasis of gastric carcinomas and could be considered a good marker for the pathobiological behaviors of gastric carcinomas.
Tomoko Miyashita, Takahito Hayashi, Yuko Ishida, Koichi Tsuneyama, Akihiko Kimura and Toshikazu Kondo : A fatal case of pontine hemorrhage related to methamphetamine abuse., Journal of Forensic and Legal Medicine, Vol.14, No.7, 444-447, 2007.
(Summary)
In this report, we describe a fatal case of pontine hemorrhage related with methamphetamine abuse. A 54-year-old male was found dead in a prone position in his parents' house, and a medico-legal autopsy was carried out to determine the cause of his death. Externally, although an injection mark-like injury with subcutaneous hemorrhage was observed in the left cubital fossa, the autopsy revealed no severe trauma leading to death. Internally, every organ was moderately congested. The brain weighed 1330 g. Macroscopically, there was no vascular abnormality such as aneurysm or malformation. In the sections of the brain stem, a massive hematoma occupied the central area of the pons. Drug screening test using Triage was weakly positive for amphetamines. Moreover, in the blood and urine samples, methamphetamine was quantitatively detected at concentrations of 0.4 and 0.6 mg/l, respectively, by gas chromatography-mass spectrometry. Other drugs and poison were not detected in the blood and urine samples collected at autopsy. Histopathologically, necrotizing angiitis characterized by fibrinoid necrosis of the intima and media was observed with cell infiltration. Thus, the pontine hemorrhage seemingly resulted from methamphetamine-induced angiitis, with an acute elevation of blood pressure after methamphetamine abuse.
(Keyword)
Amphetamine-Related Disorders / Central Nervous System Stimulants / Forensic Pathology / Humans / Intracranial Hemorrhages / Male / Methamphetamine / Middle Aged / Pons
Konstantinova Boryana Popivanova, Ying-Yi Li, Huachuan Zheng, Kenji Omura, Chifumi Fujii, Koichi Tsuneyama and Naofumi Mukaida : Proto-oncogene, Pim-3 with serine/threonine kinase activity, is aberrantly expressed in human colon cancer cells and can prevent Bad-mediated apoptosis., Cancer Science, Vol.98, No.3, 321-328, 2007.
(Summary)
We previously observed that Pim-3 with serine/threonine kinase activity, was aberrantly expressed in malignant lesions of endoderm-derived organs, liver and pancreas. Because Pim-3 protein was not detected in normal colon mucosal tissues, we evaluated Pim-3 expression in malignant lesions of human colon, another endoderm-derived organ. Pim-3 was detected immunohistochemically in well-differentiated (43/68 cases) and moderately differentiated (23/41 cases) but not poorly differentiated colon adenocarcinomas (0/5 cases). Moreover, Pim-3 proteins were detected in adenoma (35/40 cases) and normal mucosa (26/111 cases), which are adjacent to adenocarcinoma. Pim-3 was constitutively expressed in SW480 cells and the transfection with Pim-3 short hairpin RNA promoted apoptosis. In the same cell line, a pro-apoptotic molecule, Bad, was phosphorylated at Ser(112) and Ser(136) sites of phosphorylation that are representative of its inactive form. Ser(112) but not Ser(136) phosphorylation in this cell line was abrogated by Pim-3 knockdown. Furthermore, in human colon cancer tissues, Pim-3 co-localized with Bad in all cases (9/9) and with phospho-Ser(112)Bad in most cases (6/9). These observations suggest that Pim-3 can inactivate Bad by phosphorylating its Ser(112) in human colon cancer cells and thus may prevent apoptosis and promote progression of human colon cancer.
Huachuan Zheng, Yoshihiro Murai, Mei Hong, Yuko Nakanishi, Kazuhiro Nomoto, Shinji Masuda, Koichi Tsuneyama and Yasuo Takano : JC [corrected] virus detection in human tissue specimens., Journal of Clinical Pathology, Vol.60, No.7, 787-793, 2007.
(Summary)
Nested PCR whose amplicons should be confirmed by Southern blot and sequencing was a comparatively sensitive approach to detect JCV genomic DNA in human non-neural tissues. Real-time PCR might be employed to quantify copy number of JCV. In-situ PCR was a good method to observe the JCV location in cells, given appropriate modulation of amplification cycles. Combinations of various approaches will be adopted to explore the oncogenic roles of JCV in malignancies.
Yoshihiro Murai, Hua-chuan Zheng, Osman Aziz Hekmat Abdel, Hong Mei, Tomohiko Kutsuna, Yuko Nakanishi, Koichi Tsuneyama and Yasuo Takano : High JC virus load in gastric cancer and adjacent non-cancerous mucosa., Cancer Science, Vol.98, No.1, 25-31, 2007.
(Summary)
The JC virus (JCV) infects a large proportion of the worldwide population and approximately 90% of adults are seropositive. Recent reports have described the possibility of its oncogenetic role in several malignancies. The aim of the present study was to assess the oncogenetic significance of JCV for gastric cancer. Twenty-two sample pairs of fresh tumor and adjacent non-cancerous tissue (ANCT) as well as 10 normal gastric mucosa specimens were investigated on the basis of nested polymerase chain reaction (PCR) followed by Southern blotting, DNA direct sequencing, real-time PCR, in situ PCR and immunohistochemistry. The T antigen sequence was detected in 86.4% of gastric cancers and ANCT, and in 100% of the normal mucosa samples, as for virus capsid protein, 54.1%, 68.1% and 70%, respectively. A generally low incidence was noted for agnoprotein. The JCV DNA load was approximately 10-fold higher in both gastric cancers and paired ANCT (4784 +/- 759 and 5394 +/- 1466 copies/microg DNA, respectively) than in normal gastric tissue (542.4 +/- 476.0 copies/microg DNA, P < 0.0001). In situ PCR revealed sporadic JCV genome-positive cancer cells and foveolar epithelial cells. T antigen protein expression assessed by immunohistochemistry was detected only in one case (1/22; 4.5%), probably because the half life of T antigen might be short. It was concluded that the gastric epithelium in most Japanese people is infected with JCV at a low rate but levels of infection are increased markedly in both cancer cells and ANCT, indicating that multiplication of JCV copies might be a risk factor and a background for gastric carcinogenesis.
Huachuan Zheng, Koichi Tsuneyama, Hiroyuki Takahashi, Shigeharu Miwa, Kazuhiro Nomoto, Hiroshi Saito, Shinji Masuda and Yasuo Takano : Expression of PTEN and FHIT is involved in regulating the balance between apoptosis and proliferation in lung carcinomas., Anticancer Research, Vol.27, No.1B, 575-581, 2007.
(Summary)
PTEN and FHIT may contribute to regulation of the balance between apoptosis and proliferation in physiological events in normal lung and during development of lung carcinoma. Down-regulated PTEN appears closely linked to frequent lymphoangiogenic invasion and low FHIT expression, and could provide a molecular basis for differences in genetic sensitivity between men and women to lung carcinogens. PTEN could be a good prognostic factor for lung carcinomas, regardless of the histological types.
(Keyword)
Acid Anhydride Hydrolases / Adenocarcinoma / Adult / Aged / Aged, 80 and over / Apoptosis / Carcinoma, Large Cell / Carcinoma, Small Cell / Carcinoma, Squamous Cell / Cell Proliferation / Chi-Square Distribution / Disease Progression / Female / Humans / Kaplan-Meier Estimate / Lung Neoplasms / Male / Membrane Proteins / Middle Aged / Neoplasm Proteins / PTEN Phosphohydrolase / Prognosis / Sex Factors / Tissue Array Analysis
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 17348444
Huachuan Zheng, Koichi Tsuneyama, Chunmei Cheng, Hiroyuki Takahashi, Zhengguo Cui, Yoshihiro Murai, Kazhiro Nomoto and Yasuo Takano : Maspin expression was involved in colorectal adenoma-adenocarcinoma sequence and liver metastasis of tumors., Anticancer Research, Vol.27, No.1A, 259-265, 2007.
(Summary)
Up-regulated maspin expression was involved in colorectal adenoma-adenocarcinoma sequence. Low maspin expression is closely linked to the liver metastasis of CRA possibly through degradation of the extracellular matrix-tenascin to enhance carcinoma cell mobility.
Hiroto Kita, Yuichi Hikichi, Kouki Hikami, Koichi Tsuneyama, Zheng-Guo Cui, Hiroyuki Osawa, Hirohide Ohnishi, Hiroyuki Mutoh, Hiroko Hoshino, L Christopher Bowlus, Hironori Yamamoto and Kentaro Sugano : Differential gene expression between flat adenoma and normal mucosa in the colon in a microarray analysis., Journal of Gastroenterology, Vol.41, No.11, 1053-1063, 2006.
(Summary)
This is the first report characterizing the genes differentially expressed in flat adenomas using a microarray analysis. Considerable differences in the gene expression profiles of flat adenomas also exist between the right and left colon. These data should lead to new insights into the pathogenesis of flat adenomas in the colon as well as to new therapeutic strategies.
K Nomoto, Koichi Tsuneyama, O Aziz H Abdel, H Takahashi, Y Murai, Z-G Cui, M Fujimoto, I Kato, K Hiraga, K D Hsu, F-T Liu and Y Takano : Disrupted galectin-3 causes non-alcoholic fatty liver disease in male mice., The Journal of Pathology, Vol.210, No.4, 469-477, 2006.
(Summary)
Galectin-3, a beta-galactoside-binding animal lectin, is a multifunctional protein. Previous studies have suggested that galectin-3 may play an important role in inflammatory responses. Non-alcoholic fatty liver disease (NAFLD) is increasingly recognized as a liver condition that may progress to end-stage liver disease and based on the known functions of galectin-3, it was hypothesized that galectin-3 might play a role in the development of NAFLD. Thus, this study investigated the role of galectin-3 in NAFLD by comparing galectin-3 knockout (gal3(-/-)) mice and wild-type (gal3(+/+)) mice. The livers of gal3(-/-) male mice at 6 months of age histologically displayed mild to severe fatty change. The liver weight per body weight ratio, serum alanine aminotransferase levels, liver triglyceride levels, and liver lipid peroxide in gal3(-/-) mice were significantly increased compared with those in gal3(+/+) mice. Furthermore, the hepatic protein levels of advanced glycation end-products (AGE), receptor for AGE (RAGE), and peroxisome proliferator-activated receptor gamma (PPARgamma) were increased in gal3(-/-) mice relative to gal3(+/+) mice. In conclusion, this study suggests that the absence of gal3 can cause clinico-pathological features in male mice similar to those of NAFLD.
(Keyword)
Alanine Transaminase / Animals / Body Weight / Fatty Liver / Galectin 3 / Glycation End Products, Advanced / Immunohistochemistry / Liver / Male / Membrane Proteins / Mice / Mice, Knockout / Organ Size / PPAR gamma / Perilipin-2 / RNA, Messenger / Receptor for Advanced Glycation End Products / Receptors, Immunologic / Sterol Regulatory Element Binding Protein 1
Mosaburo Kainuma, Makoto Fujimoto, Nobuyasu Sekiya, Koichi Tsuneyama, Chunmei Cheng, Yasuo Takano, Katsutoshi Terasawa and Yutaka Shimada : Cholesterol-fed rabbit as a unique model of nonalcoholic, nonobese, non-insulin-resistant fatty liver disease with characteristic fibrosis., Journal of Gastroenterology, Vol.41, No.10, 971-980, 2006.
(Summary)
Cholesterol-fed rabbits share several physiopathological features of NAFLD. Because this model did not show insulin resistance or obesity, it may be useful for elucidating the mechanism of NAFLD related mainly to hyperlipidemia.
Kazuto Tajiri, Yukihiro Shimizu, Koichi Tsuneyama and Toshiro Sugiyama : A case report of oxidative stress in a patient with anorexia nervosa., The International Journal of Eating Disorders, Vol.39, No.7, 616-618, 2006.
(Summary)
Our present case demonstrates that oxidative stresses in hepatocytes, which might be associated with iron deposition, could be involved in the pathogenesis of liver injury in patients with AN.
Kanji Wakabayashi, Zhe-Xiong Lian, Yuki Moritoki, Y Ruth Lan, Koichi Tsuneyama, Ya-Hui Chuang, Guo-Xiang Yang, William Ridgway, Yoshiyuki Ueno, A Aftab Ansari, L Ross Coppel, R Ian Mackay and Eric M Gershwin : IL-2 receptor alpha(-/-) mice and the development of primary biliary cirrhosis., Hepatology, Vol.44, No.5, 1240-1249, 2006.
(Summary)
Recently, we identified a child born with a genetic deficiency of IL-2 receptor alpha (IL-2Ralpha, CD25) expression who had several clinical manifestations of primary biliary cirrhosis (PBC). In addition, there has been suggestive evidence in both patients with PBC and their first-degree relatives that a deficiency of regulatory T cells (Tregs) is an integral component for susceptibility to PBC. Based on these observations, we generated IL-2Ralpha/CD25 deficient (IL-2Ralpha(-/-)) mice and wild-type littermate controls and followed them longitudinally for the natural history of liver immunopathology and appearance of antimitochondrial antibodies (AMAs). The analyses included immunohistochemical staining of liver and portal tract infiltrates as well as FACS profiles of lymphoid subpopulations in liver and spleen. In addition, serum cytokine profiles were quantitated. Importantly, IL-2Ralpha(-/-), but not littermate controls, develop portal inflammation and biliary ductular damage similar to human patients with PBC. CD4(+) and CD8(+) T cells predominate among portal cell infiltrates and sera reflect a Th1 cytokine bias with increased levels of IFN-gamma, TNF-alpha, IL-2 and IL-12p40. Of importance is the finding that the IL-2Ralpha(-/-) mice not only develop significantly increased serum levels of IgG and IgA, but they also develop AMAs with specificity for PDC-E2, which maps to the inner lipoyl domain of the autoantigen, all characteristics which are hallmarks of human PBC. In conclusion, the IL-2Ralpha(-/-) mice should facilitate studies of the early events in PBC and especially the tantalizing connection between Treg deficiency and autoimmunity specifically directed to mitochondrially located PDC-E2 and subsequent biliary ductular cell damage.
H-C Zheng, H Takahashi, Y Murai, Z-G Cui, K Nomoto, S Miwa, Koichi Tsuneyama and Y Takano : Upregulated EMMPRIN/CD147 might contribute to growth and angiogenesis of gastric carcinoma: a good marker for local invasion and prognosis., British Journal of Cancer, Vol.95, No.10, 1371-1378, 2006.
(Summary)
Tumour growth depends on angiogenesis, which is closely associated with vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). Extracellular MMP inducer (EMMPRIN) was reported to involve in the progression of malignancies by regulating expression of VEGF and MMPs in stromal cells. To clarify the role of EMMPRIN in progression and angiogenesis of gastric carcinoma, expression of EMMPRIN, ki-67, MMP-2, MMP-9 and VEGF was examined on tissue microarray containing gastric carcinomas (n=234) and non-cancerous mucosa adjacent to carcinoma (n=85) by immunohistochemistry. Additionally, microvessel density (MVD) was assessed after labelling with anti-CD34 antibody. Extracellular MMP inducer expression was compared with clinicopathological parameters of tumours, including levels of ki-67, MMP-2, MMP-9 and vascular endothelial growth factor (VEGF), MVD as well as survival time of carcinoma patients. Gastric carcinoma cell lines (HGC-27, MKN28 and MKN45) were studied for EMMPRIN expression by immunohistochemistry and Western blot. Extracellular MMP inducer expression was gradually increased from normal mucosa to carcinomas through hyperplastic or metaplastic mucosa of the stomach (P<0.05). There was strong EMMPRIN expression in all gastric carcinoma cell lines despite different levels of glycosylation. Extracellular MMP inducer expression was positively correlated with tumour size, depth of invasion, lymphatic invasion, expression of ki-67, MMP-2, MMP-9 and VEGF of tumours (P<0.05), but not with lymph node metastasis, UICC staging or differentiation (P>0.05). Interestingly, there was a significantly positive relationship between EMMPRIN expression and MVD in gastric carcinomas (P<0.05). Survival analysis indicated EMMPRIN expression to be negatively linked to favourable prognosis (P<0.05), but not be independent factor for prognosis (P>0.05). Further analysis showed three independent prognostic factors, depth of invasion, lymphatic and venous invasion, to influence the relationship between EMMPRIN expression and prognosis. Upregulated expression of EMMPRIN possibly contributes to genesis, growth and local invasion of gastric carcinomas. Altered EMMPRIN expression might enhance growth, invasion and angiogenesis of gastric carcinoma via upregulating MMP expression of both stromal fibroblasts and gastric cancer cells and could be considered as an objective and effective marker to predict invasion and prognosis.
Osman Hekmat Abdel-Aziz, Ichiro Takasaki, Yoshiaki Tabuchi, Kazuhiro Nomoto, Yoshihiro Murai, Koichi Tsuneyama and Yasuo Takano : High-density oligonucleotide microarrays and functional network analysis reveal extended lung carcinogenesis pathway maps and multiple interacting genes in NNK [4-(methylnitrosamino)-1-(3-pyridyle)-1-butanone] induced CD1 mouse lung tumor., Journal of Cancer Research and Clinical Oncology, Vol.133, No.2, 107-115, 2006.
(Summary)
These results suggest that application of gene expression profiling may provide an improved strategy for therapeutic targeting of tobacco smoking-induced lung cancer.
Hiroyuki Takahashi, Yoshihiro Murai, Koichi Tsuneyama, Kazuhiro Nomoto, Eikichi Okada, Hideharu Fujita and Yasuo Takano : Overexpression of phosphorylated histone H3 is an indicator of poor prognosis in gastric adenocarcinoma patients., Applied Immunohistochemistry & Molecular Morphology : AIMM, Vol.14, No.3, 296-302, 2006.
(Summary)
Ki-67 immunostaining is commonly used for assessing cell proliferation, but studies of its use as a prognostic indicator have revealed discordant results in gastric cancer patients. Recently, antibodies for phosphorylated histone H3 have been used to identify dividing cells because of its precise overexpression in mitosis. The authors tested the hypothesis that phosphorylated histone H3 overexpression might be a good prognostic indicator for gastric cancer patients by conducting an immunohistochemical comparison with Ki-67 in gastric cancer samples. One hundred twenty-two surgically resected primary cases were selected and histologically categorized in accordance with Lauren's classification. No correlation was found between phosphorylated histone H3 and Ki-67 regarding overexpression. However, correlations between phosphorylated histone H3 overexpression and clinicopathologic variables were noted for histologic type (intestinal type predominant in high labeling indices [LIs], defined as over the value of the 75th percentile; P<0.01), vessel invasion (positive in high LIs; P=0.05), and lymph node metastasis (positive in high LIs; P=0.04). With regard to Ki-67 overexpression, no correlation was evident with the clinicopathologic variables except histologic type (intestinal type predominant; P=0.05). By the Kaplan-Meier method with the log-rank test, cases overexpressing phosphorylated histone H3 showed a poorer prognosis than cases with low expression (P<0.01). In contrast, Ki-67 expression did not influence prognosis. Multivariate analyses indicated phosphorylated histone H3 overexpression to be an independent prognostic factor, together with lymphatic invasion and venous invasion (P<0.01). In conclusion, it seems likely that phosphorylated histone H3 plays an important role in the prognosis of gastric cancer, and its immunohistochemical investigation is useful for the prediction of prognosis in gastric cancer.
Khin-Mar Myint, Yasuhiko Yamamoto, Toshio Doi, Ichiro Kato, Ai Harashima, Hideto Yonekura, Takuo Watanabe, Harumichi Shinohara, Masayoshi Takeuchi, Koichi Tsuneyama, Noriyoshi Hashimoto, Masahide Asano, Shin Takasawa, Hiroshi Okamoto and Hiroshi Yamamoto : RAGE control of diabetic nephropathy in a mouse model: effects of RAGE gene disruption and administration of low-molecular weight heparin., Diabetes, Vol.55, No.9, 2510-2522, 2006.
(Summary)
Diabetic nephropathy is a major microvascular complication in long-standing diabetic patients who eventually undergo renal dialysis or transplantation. To prevent development of this disease and to improve advanced kidney injury, effective therapies directed toward the key molecular target are required. In this study, we examined whether inhibition of the receptor for advanced glycation end products (RAGE) could attenuate changes in the diabetic kidney. Here, we show that inactivation of the RAGE gene in a mouse model of diabetic nephropathy results in significant suppression of kidney changes, including kidney enlargement, increased glomerular cell number, mesangial expansion, advanced glomerulosclerosis, increased albuminuria, and increased serum creatinine compared with wild-type diabetic mice. The degree of kidney injury was proportional to RAGE gene dosage. Furthermore, we show that low-molecular weight heparin (LMWH) can bind RAGE at a mean equilibrium dissociation constant (K(d)) value of approximately 17 nmol/l and act as an antagonist to RAGE. LMWH treatment of mice significantly prevented albuminuria and increased glomerular cell number, mesangial expansion, and glomerulosclerosis in a dose-dependent manner; it also significantly improved the indexes of advanced-stage diabetic nephropathy. This study provides insight into the pathological role of RAGE in both early- and advanced-phase diabetic nephropathy and suggests that RAGE antagonists will be a useful remedy in the treatment of diabetic nephropathy.
(Keyword)
Animals / Diabetic Nephropathies / Disease Models, Animal / Female / Glycation End Products, Advanced / Heparin, Low-Molecular-Weight / Male / Mice / Receptor for Advanced Glycation End Products / Receptors, Immunologic
Huachuan Zheng, Hiroyuki Takahashi, Yoshihiro Murai, Zhengguo Cui, Kazuhiro Nomoto, Hideki Niwa, Koichi Tsuneyama and Yasuo Takano : Expressions of MMP-2, MMP-9 and VEGF are closely linked to growth, invasion, metastasis and angiogenesis of gastric carcinoma., Anticancer Research, Vol.26, No.5A, 3579-3583, 2006.
(Summary)
MMP-2, MMP-9 and VEGF largely contribute to the angiogenesis and progression of gastric carcinomas. PTEN might inhibit the processes by down-regulating VEGF expression. These parameters should be regarded as good markers to indicate pathobiological behaviours of gastric carcinomas.
Anchalee Sistayanarain, Koichi Tsuneyama, Huachuan Zheng, Hiroyuki Takahashi, Kazuhiro Nomoto, Chunmei Cheng, Yoshihiro Murai, Atsushi Tanaka and Yasuo Takano : Expression of Aurora-B kinase and phosphorylated histone H3 in hepatocellular carcinoma., Anticancer Research, Vol.26, No.5A, 3585-3593, 2006.
(Summary)
Aberrant expression of Aurora-B and H3-P plays a role in hepatocarcinogenesis. Alterative splicing of Aurora-B produces different sizes of proteins in HCC. Temporally altered phosphorylation of histone-H3 in the entire cell cycle may up-regulate the entry of HCC into the cell cycle to enhance their proliferation.
Chunmei Cheng, Koichi Tsuneyama, Huachuan Zheng, Takeshi Oya, Zhengguo Cui, B Loreto Feril and Yasuo Takano : Enhanced scavenging of lipid substances is a possible effect of corticosteroids in the treatment of cholesterol crystal embolism., Pathology, Research and Practice, Vol.202, No.8, 591-598, 2006.
(Summary)
Cholesterol crystal embolism (CCE) is a systemic refractory disease especially prevalent amongst elderly patients suffering from atherosclerosis. Treatment of this condition remains controversial due to difficulties in diagnosis. Corticosteroid therapy may be an important treatment option despite its elusive mechanisms. To clarify the role of corticosteroid in CCE therapy, we collected the samples from six autopsied subjects with CCE, three of whom were clinically given various doses of corticosteroid to investigate stable atherosclerosis-related substances, advanced glycation end-products (AGE), and several AGE receptors such as scavenger receptor class B type 1 (SR-B1), receptor for AGE (RAGE), and galectin-3 in the liver tissues and atherosclerotic areas by immunostaining using a tissue macro-array technique. An intense expression of AGE and its receptors was identified in the enlarged Kupffer cells of CCE cases, which were given relatively high doses of corticosteroid. In addition, numerous mononuclear cells in the intimal atheromatous plaque presented strong expressions of AGE and SR-B1. In conclusion, we speculated that corticosteroid treatment for CCE may upregulate the activations, including phagocytic capacity of Kupffer cells mediated by overexpression of RAGE and scavenger receptors, resulting in efficient clearance of the lipid substances from the blood circulation released from atherosclerotic areas.
(Keyword)
Aged / Aged, 80 and over / cholesterol / Embolism, Cholesterol / Female / Fluorescent Antibody Technique, Indirect / Glucocorticoids / Glycation End Products, Advanced / Humans / Immunoenzyme Techniques / Liver / Male / Scavenger Receptors, Class B
Huachuan Zheng, Hiroyuki Takahashi, Takahiko Nakajima, Yoshihiro Murai, Zhengguo Cui, Kazuhiro Nomoto, Koichi Tsuneyama and Yasuo Takano : MUC6 down-regulation correlates with gastric carcinoma progression and a poor prognosis: an immunohistochemical study with tissue microarrays., Journal of Cancer Research and Clinical Oncology, Vol.132, No.12, 817-823, 2006.
(Summary)
Down-regulation of MUC6 may contribute to malignant transformation of gastric epithelial cells and underlie the molecular bases of growth, invasion, metastasis and differentiation of gastric carcinoma. Altered expression might therefore be employed as an indicator of pathobiological behaviors and prognosis of gastric carcinoma.
Ya-Hui Chuang, Zhe-Xiong Lian, Koichi Tsuneyama, Bor-Luen Chiang, A Aftab Ansari, L Ross Coppel and Eric M Gershwin : Increased killing activity and decreased cytokine production in NK cells in patients with primary biliary cirrhosis., Journal of Autoimmunity, Vol.26, No.4, 232-240, 2006.
(Summary)
Although the pathogenesis of primary biliary cirrhosis (PBC) remains enigmatic, the immune system plays a key role in the initiation and subsequent development of pathology. Previous studies have indicated a critical role of the innate immune system. Importantly, natural killer (NK) cells are abundant in liver where they serve as sentinels of the immune system. In addition, NK cells have significant biologic activity based on their production of immunoregulatory cytokines. To address this issue, we have investigated several qualitative and quantitative activities of NK cells in patients with PBC as well as normal and liver diseased controls. We report herein a marked increase in the frequency and absolute number of blood and liver NK cells in PBC patients. Moreover, the cytotoxic activity and perforin expression by isolated NK cells were significantly increased in PBC patients associated with increased levels of plasma IL-8 and the expression of CD128a (IL-8 receptor) on NK cells. In contrast, the levels of IFN-gamma, IL-6 and IL-8 synthesized by NK cells were significantly decreased in PBC patients as compared to controls. In conclusion, data from this study provide compelling evidence supporting a biologic role of NK cells in the immunopathogenesis of PBC.
Huachuan Zheng, Hiroyuki Takahashi, Yoshihiro Murai, Zhengguo Cui, Kazuhiro Nomoto, Shigeharu Miwa, Koichi Tsuneyama and Yasuo Takano : Pathobiological characteristics of intestinal and diffuse-type gastric carcinoma in Japan: an immunostaining study on the tissue microarray., Journal of Clinical Pathology, Vol.60, No.3, 273-277, 2006.
(Summary)
Intestinal-type gastric carcinomas with a more favourable prognosis frequently show high levels of proliferation and apoptosis, and always accompany strong expression of FHIT, PTEN and mutant p53 and EMMPRIN. EMMPRIN expression might underlie the molecular basis of liver metastasis and higher proliferation of intestinal-type gastric carcinomas in Japan. Lauren's classification thus proved pathologically relevant for the clinical treatment of gastric carcinomas.
Ai Harashima, Yasuhiko Yamamoto, Chunmei Cheng, Koichi Tsuneyama, Mar Khin Myint, Akihiko Takeuchi, Kazunobu Yoshimura, Hui Li, Takuo Watanabe, Shin Takasawa, Hiroshi Okamoto, Hideto Yonekura and Hiroshi Yamamoto : Identification of mouse orthologue of endogenous secretory receptor for advanced glycation end-products: structure, function and expression., The Biochemical Journal, Vol.396, No.1, 109-115, 2006.
(Summary)
The cell-surface RAGE [receptor for AGE (advanced glycation end-products)] is associated with the development of diabetic vascular complications, neurodegenerative disorders and inflammation. Recently, we isolated a human RAGE splice variant, which can work as a decoy receptor for RAGE ligands, and named it esRAGE (endogenous secretory RAGE). In the present study, we have isolated the murine equivalent of esRAGE from brain polysomal poly(A)+ (polyadenylated) RNA by RT (reverse transcription)-PCR cloning. The mRNA was generated by alternative splicing, and it encoded a 334-amino-acid protein with a signal sequence, but lacking the transmembrane domain. A transfection experiment revealed that the mRNA was actually translated as deduced to yield the secretory protein working as a decoy in AGE-induced NF-kappaB (nuclear factor kappaB) activation. RT-PCR and immunoblotting detected esRAGE mRNA and protein in the brain, lung, kidney and small intestine of wild-type mice, but not of RAGE-null mice. The esRAGE expression was increased in the kidney of diabetic wild-type mice. The present study has thus provided an animal orthologue of esRAGE for clarification of its roles in health and disease.
(Keyword)
Alternative Splicing / Amino Acid Sequence / Animals / Blotting, Western / COS Cells / Cell Line, Tumor / Chlorocebus aethiops / Diabetes Mellitus, Experimental / Glioma / Glycation End Products, Advanced / Humans / Intestine, Small / Kidney / Ligands / Lung / Male / Mice / Mice, Inbred C57BL / Molecular Sequence Data / NF-kappa B / Nerve Tissue Proteins / Organ Specificity / RNA, Messenger / Rats / Receptor for Advanced Glycation End Products / Receptors, Immunologic / Reverse Transcriptase Polymerase Chain Reaction / Sequence Alignment / Sequence Homology, Amino Acid / Solubility / Species Specificity / Structure-Activity Relationship / Surface Plasmon Resonance / Transfection
Huachuan Zheng, Koichi Tsuneyama, Chunmei Cheng, Hiroyuki Takahashi, Zhengguo Cui, Kazuhiro Nomoto, Yoshihiro Murai and Yasuo Takano : Expression of KAI1 and tenascin, and microvessel density are closely correlated with liver metastasis of gastrointestinal adenocarcinoma., Journal of Clinical Pathology, Vol.60, No.1, 50-56, 2006.
(Summary)
Up-regulated KAI1 expression may play an important part in malignant transformation of gastrointestinal epithelial cells. Reduced expression of KAI1 and tenascin might underlie the molecular basis of liver metastasis of GIA. Angiogenesis is a key event in the invasion and metastasis of GIA. These markers might be used to indicate liver metastasis of GIA in clinicopathological practice.
Huachuan Zheng, Koichi Tsuneyama, Chunmei Cheng, Hiroyuki Takahashi, Zhengguo Cui, Yoshihiro Murai, Kazuhiro Nomoto and Yasuo Takano : An immunohistochemical study of P53 and Ki-67 in gastrointestinal adenoma and adenocarcinoma using tissue microarray., Anticancer Research, Vol.26, No.3B, 2353-2360, 2006.
(Summary)
The up-regulated expressions of mutant p53 and ki-67 are involved in the carcinogenesis and progression of GIA. They appear to be objective and effective markers to reflect the development of GIA.
Hideki Hatta, Koichi Tsuneyama, Tokimasa Kumada, Huachuan Zheng, Chunmei Cheng, Zhengguo Cui, Hiroyuki Takahashi, Kazuhiro Nomoto, Yoshihiro Murai and Yasuo Takano : Freshly prepared immune complexes with intermittent microwave irradiation result in rapid and high-quality immunostaining., Pathology, Research and Practice, Vol.202, No.6, 439-445, 2006.
(Summary)
We have previously reported that intermittent microwave irradiation shortened the primary or secondary antibody incubation time to 10 min in a special moist chamber. To achieve precise immunostaining within 1h, we attempted to generate a novel procedure, "freshly prepared immune complex with intermittent microwave irradiation (f-IC-M)". The advantage of this immunostaining procedure lies in a one-step incubation instead of primary and then secondary antibody application. In this study, we employed five primary antibodies to examine the efficiency and quality of this procedure. As expected, every primary antibody examined brought about precise immunostaining within 45 min for formalin-fixed, paraffin-embedded sections, and within 15 min for frozen sections. In addition, this procedure is able to generate double-immunoenzymatic staining with different enzyme-labeled primary antibodies if desired. As any combination of primary and secondary antibodies is possible by this one-step application, f-IC-M increases the efficiency of immunostaining without losing quality. Therefore, this procedure is able to rapidly provide diagnostic information to the pathologists.
Yuko Ishida, Toshikazu Kondo, Akihiko Kimura, Koichi Tsuneyama, Tatsunori Takayasu and Naofumi Mukaida : Opposite roles of neutrophils and macrophages in the pathogenesis of acetaminophen-induced acute liver injury., European Journal of Immunology, Vol.36, No.4, 1028-1038, 2006.
(Summary)
Neutrophils and macrophages infiltrate after acetaminophen (APAP)-induced liver injury starts to develop. However, their precise roles still remain elusive. In untreated and control IgG-treated wild-type (WT) mice, intraperitoneal APAP administration (750 mg/kg) caused liver injury including centrilobular hepatic necrosis and infiltration of neutrophils and macrophages, with about 50% mortality within 48 h after the injection. APAP injection markedly augmented intrahepatic gene expression of inducible nitric oxide synthase (iNOS) and heme oxygenase (HO)-1. Moreover, neutrophils expressed iNOS, which is presumed to be an aggravating molecule for APAP-induced liver injury, while HO-1 was mainly expressed by macrophages. All anti-granulocyte antibody-treated neutropenic WT and most CXC chemokine receptor 2 (CXCR2)-deficient mice survived the same dose of APAP, with reduced neutrophil infiltration and iNOS expression, indicating the pathogenic roles of neutrophils in APAP-induced liver injury. However, APAP caused more exaggerated liver injury in CXCR2-deficient mice with reduced macrophage infiltration and HO-1 gene expression, compared with neutropenic WT mice. An HO-1 inhibitor, tin-protoporphyrin-IX, significantly increased APAP-induced mortality, implicating HO-1 as a protective molecule for APAP-induced liver injury. Thus, CXCR2 may regulate the infiltration of both iNOS-expressing neutrophils and HO-1-expressing macrophages, and the balance between these two molecules may determine the outcome of APAP-induced liver injury.
(Keyword)
Acetaminophen / Analgesics, Non-Narcotic / Animals / Chemical and Drug Induced Liver Injury / Fluorescent Antibody Technique / Gene Expression / Heme Oxygenase-1 / Immunohistochemistry / Liver Diseases / Macrophages / Male / Mice / Mice, Inbred BALB C / Mice, Knockout / Neutrophils / Nitric Oxide Synthase Type II / Receptors, Interleukin-8B / Reverse Transcriptase Polymerase Chain Reaction
Y Ruth Lan, Chunmei Cheng, Zhe-Xiong Lian, Koichi Tsuneyama, Guo-Xiang Yang, Yuki Moritoki, Ya-Hui Chuang, Takafumi Nakamura, Shigeru Saito, Shinji Shimoda, Atsushi Tanaka, L Christopher Bowlus, Yasuo Takano, A Aftab Ansari, L Ross Coppel and Eric M Gershwin : Liver-targeted and peripheral blood alterations of regulatory T cells in primary biliary cirrhosis., Hepatology, Vol.43, No.4, 729-737, 2006.
(Summary)
CD4+CD25high regulatory T cells (Tregs) play a critical role in self-tolerance, as seen in murine autoimmunity. Studies on Tregs in human autoimmunity have focused primarily on peripheral blood samples. A study targeting diseased tissue should identify direct relationships between Tregs and autoimmunity. Peripheral blood samples were collected from 91 patients with primary biliary cirrhosis (PBC), 28 immediate relatives, and 41 healthy controls, and Treg frequencies were determined as a percentage of CD4+CD25high T cells in CD4+TCR-alphabeta+ T cells. A tissue-targeted determination of frequency and distribution of FoxP3+ Tregs was also performed on 90 different liver tissue specimens exhibiting PBC (n = 52), chronic hepatitis C (CHC) (n = 30), and autoimmune hepatitis (AIH) (n = 8). Treg suppression studies were performed on 50 PBC patients and 27 controls. Patients with PBC demonstrated a relative reduction of Tregs compared with controls (P < .0002). Interestingly, a deficiency in CD4+CD25+ Tregs was also found in the daughters and sisters of PBC patients compared with controls (P < .0007). However, functional studies did not reveal a global PBC Treg defect. The level of FoxP3-expressing Tregs was markedly lower in affected PBC portal tracts compared with CHC and AIH (P < .001). In addition, the CD8+T cell/FoxP3+ Treg ratio was significantly higher in livers of late-stage PBC compared with those of CHC (P < .001) and early-stage AIH (P < .001). In conclusion, these data provide support for a genetic modulation of Treg frequency and illustrate the role Tregs play in the loss of tolerance in PBC.
Hideki Abe, Koichi Tsuneyama, Kazuhiro Tsukada and Masatoshi Makuuchi : Five-year survival following a medial pancreatectomy for an invasive ductal carcinoma from the body of the pancreas., World Journal of Gastroenterology : WJG, Vol.12, No.5, 822-824, 2006.
(Summary)
We report a rare case of a patient who survived for 5 years after undergoing a medial pancreatectomy for invasive ductal carcinoma originating from the body of the pancreas. A 63-year-old woman was diagnosed as a small cancer of the pancreatic body, and surgery was performed. Even though the tumor was a carcinoma, its small size prompted us to perform a medial pancreatectomy with regional lymph nodes dissection. Additional chemoradiation was performed and, five years after surgery, the patient is well with no signs of recurrence. Medial pancreatectomy for invasive ductal carcinoma has not ever been reported. Furthermore, long-term survival after a lumpectomy for invasive ductal carcinoma has never been reported in the literatures. The current case suggests that long-term survival in patients with invasive ductal carcinoma of the pancreas may be associated with the pathological or biological features of pancreatic carcinoma.
Masakuni Noguchi, Mitsuharu Earashi, Hisatake Fujii, Koichi Yokoyama, Ken-ichi Harada and Koichi Tsuneyama : Radiofrequency ablation of small breast cancer followed by surgical resection., Journal of Surgical Oncology, Vol.93, No.2, 120-128, 2006.
(Summary)
RF ablation is promising as a minimally invasive ablation technique in the local treatment of invasive or non-invasive breast cancer. However, further study is necessary before RF ablation can replace conventional breast conservation therapy for patients with small breast cancer.
Kazuhiro Nomoto, Koichi Tsuneyama, Chunmei Cheng, Hiroyuki Takahashi, Ryota Hori, Yoshihiro Murai and Yasuo Takano : Intrahepatic cholangiocarcinoma arising in cirrhotic liver frequently expressed p63-positive basal/stem-cell phenotype., Pathology, Research and Practice, Vol.202, No.2, 71-76, 2005.
(Summary)
In general, intrahepatic cholangiocarcinoma (ICC) is not related to liver cirrhosis. However, a few cases have been reported in which ICC was accompanied by severe liver fibrosis. Some researchers have proposed that hepatocellular and cholangiocellular (HC-CC) carcinoma, an intermediate mixed phenotype possibly arising in cirrhotic liver, might originate from hepatic precursor cells. In the liver, hepatocytes and cholangiocytes form the epithelial element, but stromal and mesenchymal elements may be produced by hepatic stem cells. Based on these aspects, not only HC-CC, but also other combinations of cellular phenotypes, would cover all the cancers with stem cell features. In this study, which aimed at determining the characteristics of the ICC phenotype, we used immunohistochemistry to examine the expression of basal/stem-cell markers, i.e., p63 in ICC with and without liver cirrhosis, as well as the expressions of cytokeratin (CK) 34 beta E12, specific for the basal-cell marker, and c-kit, specific for the stem-cell marker. Aberrant p63 was frequently expressed in ICC arising in cirrhotic liver. This result suggests that ICC cancer cells originate from hepatic precursor cells with a hidden multi-differentiation potential.
Ryouta Hori, Yoshihiro Murai, Koichi Tsuneyama, Osman Hekmat Abdel-Aziz, Kazuhiro Nomoto, Hiroyuki Takahashi, Chun-mei Cheng, Tomohiko Kuchina, V Brian Harman and Yasuo Takano : Detection of JC virus DNA sequences in colorectal cancers in Japan., Virchows Archiv, Vol.447, No.4, 723-730, 2005.
(Summary)
JC virus (JCV), a ubiquitous polyoma virus that commonly infects humans, was first identified as the etiologic agent for the fetal demyelinating disease, progressive multifocal leukoencephalopathy. Recently, a number of reports have documented detection of JCV in samples derived from several types of neural as well as non-neural human tumors. It has been suggested that oncogenicity of JCV depends on a T antigen having a strict structural homology to the T antigen of simian virus 40. To clarify whether JCV might have a potential role with regard to colorectal cancers, we investigated the presence of its genome in a series of cases along with colorectal adenomas and normal colonic mucosa, targeting T antigen, VP and agnoprotein by nested polymerase chain reaction and Southern blotting and T antigen by immunohistochemistry. While VP and agnoprotein were not found in any of the samples examined, T antigen was detected in 6 of 23 colorectal cancers (26.1%) and 1 of 21 adenomas (4.8%), but none of 20 samples of normal colonic mucosa. No clear and diffuse staining with anti-T-antigen antibodies (1:100) could be detected, and there was no correlation with CD20-positive cells, which might have indicated JCV latent infection of B lymphocytes. Presence of T antigen did not influence clinicopathological variables, including survival. In one colonic cancer case positive for T antigen together with lymph node metastasis, DNA extracted from cancer cells in the lymph node revealed no detection of T antigen. Our results are in the intermediate position between the high T antigen rate (81%) in one report and the lack of it (0%) in another focused on colon cancers. It was concluded that T antigen might be integrated in cancer cells in approximately one fourth of Japanese colon cancer cases without clear and diffuse expression of the protein, suggesting a possible role in oncogenesis which might involve a hit-and-run mechanism.
Chunmei Cheng, Koichi Tsuneyama, Rieko Kominami, Harumichi Shinohara, Shigeru Sakurai, Hideto Yonekura, Takuo Watanabe, Yasuo Takano, Hiroshi Yamamoto and Yasuhiko Yamamoto : Expression profiling of endogenous secretory receptor for advanced glycation end products in human organs., Modern Pathology, Vol.18, No.10, 1385-1396, 2005.
(Summary)
The receptor for advanced glycation end products (RAGE) is a cell surface multiligand receptor of the immunoglobulin superfamily, which participates in physiological and pathological processes such as neuronal development, diabetes, inflammation, neurodegenerative disorders, and cancer. A novel splice variant of RAGE-endogenous secretory decoy form (esRAGE) was recently identified and is thought to be a prospective candidate to modify these RAGE-associated conditions. Here, we investigated the expression and distribution of esRAGE and RAGE proteins with domain-specific antibodies. We studied a wide variety of adult normal human preparations obtained from surgical and autopsy specimens using a tissue microarray technique. The results revealed that esRAGE was widely distributed and we classified its expression into four patterns. In pattern A, the cytoplasm is stained diffusely in neurons, vascular endothelium, pneumocytes, mesothelium, pancreatic beta cells, and macrophages/monocytes. In pattern B, dot-like granules are stained in the supranuclear regions facing the luminal surface of the bile ducts, salivary glands, digestive tracts, renal tubules, prostate, skin, thyroid, and bronchioles. Pattern C is represented by diffuse staining in the stromal area of the arterial walls. Pattern D shows diffuse and strong staining of secreted materials such as thyroidal colloid, crystals in renal tubular lumen, and glandular lumen in prostate. This study provides, for the first time, a histopathological basis for understanding the physiological roles of esRAGE in humans, and will contribute to elucidating the participation of esRAGE in pathological processes and to exploring novel diagnostic and therapeutic concepts.
(Keyword)
Glycation End Products, Advanced / Humans / Immunohistochemistry / Membrane Proteins / Organ Specificity / Protein Isoforms / Receptor for Advanced Glycation End Products / Receptors, Immunologic
Mei Hong, Yoshihiro Murai, Tomohiko Kutsuna, Hiroyuki Takahashi, Kazuhiro Nomoto, Chun-Mei Cheng, Shin Ishizawa, Qing-Li Zhao, Ryohei Ogawa, V Brian Harmon, Koichi Tsuneyama and Yasuo Takano : Suppression of Epstein-Barr nuclear antigen 1 (EBNA1) by RNA interference inhibits proliferation of EBV-positive Burkitt's lymphoma cells., Journal of Cancer Research and Clinical Oncology, Vol.132, No.1, 1-8, 2005.
(Summary)
These findings point to potential therapeutic applications for vector-mediated siRNA delivery to control EBV-associated malignant disorders.
Chronic sclerosing sialadenitis (CSS) is a cryptogenic tumor-like condition of the salivary gland(s). While immune-mediated processes are suspected in its pathogenesis, and CSS is occasionally reported to be associated with sclerosing pancreatitis, an IgG4-related disease, the exact immunopathologic processes of CSS remain speculative. In this study, we examined the clinicopathologic findings of CSS (12 cases) in comparison with sialolithiasis (8 cases) and Sjogren's syndrome (13 cases), and tried to clarify whether CSS is an IgG4-related disease or not. Submandibular gland(s) were affected in all cases of CSS. CSS cases could be divided into two types: 5 cases were associated with sclerosing lesions in extrasalivary glandular tissue (systemic type), while only salivary gland(s) were affected in the remaining 7 cases (localized type). In the former type, which showed male predominance, bilateral salivary glands were frequently affected, and eosinophilia and elevations of gamma-globulin and IgG in serum were frequently found. Histologically, all cases of CSS showed marked lymphoplasmacytic infiltration admixed with fibrosis and the destruction of glandular lobules. Obliterative phlebitis was found in the affected salivary glands in all cases of CSS. Immunohistochemically, the proportion of IgG4/IgG-positive plasma cells was more than 45% in CSS, while it was less than 5% in controls. The resemblance of the clinicopathologic features of CSS with those of sclerosing pancreatitis suggests the participation of a similar immunopathologic process with IgG4 disturbance in CSS. The abundance of IgG4-positive plasma cells in the lesions would be useful for distinguishing CSS from other forms of sialadenitis.
Chun-Mei Cheng, Koichi Tsuneyama, Kazuhiro Matsui, Hiroyuki Takahashi, Shin Ishizawa and Yasuo Takano : Cytoplasmic expression of c-erbB2 in non-small cell lung cancers., Virchows Archiv, Vol.446, No.6, 596-603, 2005.
(Summary)
The aim of this study was to investigate the expression of c-erbB2 in non-small cell lung cancers (NSCLCs) with attention both to membranous and cytoplasmic reaction, and to try to elucidate the meaning of cytoplasmic expression of c-erbB2 in NSCLCs. Immunohistochemical c-erbB2 expression and related clinico-pathological features were examined in 312 surgically resected patient tissues of NSCLCs, including 175 cases of adenocarcinoma and 137 cases of squamous cell carcinoma. Immunostaining of inner- and ecto-domain of c-erbB2, mRNA expression and the quantitation of soluble c-erbB2 in cultured media were performed in five NSCLC cell lines. Cytoplasmic expression of c-erbB2 was observed more frequently than membranous, both in patient tissues and cell lines. Neither membranous nor cytoplasmic expression of c-erbB2 was significantly correlated with short outcome in NSCLCs. Membranous c-erbB2 was expressed by both inner and ecto-domain, while cytoplasmic c-erbB2 was expressed by either or both inner and ecto-domain. c-erbB2 mRNA was produced in most cell lines; however, the soluble form was only detectable in a cell line that only presented a membranous c-erbB2. In conclusion, cytoplasmic c-erbB2 of NSCLCs was not a full-length protein only expressed in cellular membrane, but reflected degenerated c-erbB2 fragments with less functional ability.
Chifumi Fujii, Yasunari Nakamoto, Peirong Lu, Koichi Tsuneyama, K Boryana Popivanova, Shuichi Kaneko and Naofumi Mukaida : Aberrant expression of serine/threonine kinase Pim-3 in hepatocellular carcinoma development and its role in the proliferation of human hepatoma cell lines., International Journal of Cancer, Vol.114, No.2, 209-218, 2005.
(Summary)
Most cases of human hepatocellular carcinoma develop after persistent chronic infection with human hepatitis B virus or hepatitis C virus, and host responses are presumed to have major roles in this process. To recapitulate this process, we have developed the mouse model of hepatocellular carcinoma using hepatitis B virus surface antigen transgenic mice. To identify the genes associated with hepatocarcinogenesis in this model, we compared the gene expression patterns between pre-malignant lesions surrounded by hepatocellular carcinoma tissues and control liver tissues by using a fluorescent differential display analysis. Among the genes that were expressed differentially in the pre-malignant lesions, we focused on Pim-3, a member of a proto-oncogene Pim family, because its contribution to hepatocarcinogenesis remains unknown. Moreover, the unavailability of the nucleotide sequence of full-length human Pim-3 cDNA prompted us to clone it from the cDNA library constructed from a human hepatoma cell line, HepG2. The obtained 2,392 bp human Pim-3 cDNA encodes a predicted open reading frame consisting of 326 amino acids. Pim-3 mRNA was selectively expressed in human hepatoma cell lines, but not in normal liver tissues. Moreover, Pim-3 protein was detected in human hepatocellular carcinoma tissues and cell lines but not in normal hepatocytes. Furthermore, cell proliferation was attenuated and apoptosis was enhanced in human hepatoma cell lines by the ablation of Pim-3 gene with RNA interference. These observations suggest that aberrantly expressed Pim-3 can cause autonomous cell proliferation or prevent apoptosis in hepatoma cell lines.
Yoh Zen, Kenichi Harada, Motoko Sasaki, Koichi Tsuneyama, Kazuhiro Matsui, Joji Haratake, Shotaro Sakisaka, Shiro Maeyama, Kazuhide Yamamoto, Masayuki Nakano, Kazuhide Shimamatsu, Masayoshi Kage, Nozomu Kurose, Akio Uchiyama, Yasuharu Kaizaki, Gotaro Toda and Yasuni Nakanuma : Are bile duct lesions of primary biliary cirrhosis distinguishable from those of autoimmune hepatitis and chronic viral hepatitis? Interobserver histological agreement on trimmed bile ducts., Journal of Gastroenterology, Vol.40, No.2, 164-170, 2005.
(Summary)
This study revealed that 66.9% of PBC cases could be diagnosed based on trimmed bile ducts alone. Bile duct injury similar to that in PBC could be encountered in AIH.
(Keyword)
Bile Ducts / Female / Hepatitis, Autoimmune / Hepatitis, Chronic / Hepatitis, Viral, Human / Humans / Liver Cirrhosis, Biliary / Male / Middle Aged / Observer Variation
Yoshihiro Murai, Shinichi Hayashi, Hiroyuki Takahashi, Koichi Tsuneyama and Yasuo Takano : Correlation between DNA alterations and p53 and p16 protein expression in cancer cell lines., Pathology, Research and Practice, Vol.201, No.2, 109-115, 2005.
(Summary)
To investigate the interaction between DNA abnormalities, p53 and p16 gene mutations, and methylation and protein expression, 20 cancer cell lines were examined by Western blotting. A clear relation was found to exist between p53 accumulation and mutation status. Of 20 cell lines examined, 14 demonstrated p53 homozygous mutations in exons 3-10, including 12 missense mutations, one nonsense mutation, and one frameshift mutation. Overexpression of p53 was always linked to missense mutations in exons 6-8. Intermediate expression of p53 was noted in cells with missense mutations or polymorphism to proline at codon 72 in exons 4-5, whereas there was slight or no visible expression in wild type cells and in cells with nonsense and frameshift mutations. DNA aberration in the p16 promoter gene correlated significantly with protein expression of the p16 suppressor gene. Overexpression was noted in six cell lines, intermediate expression in two, and slight or no visible expression in 12. Methylation-caused disappearance of p16 protein was noted in 40% (8/20) of the cell lines. Of six cell lines overexpressing p16 protein, two could be amplified with primers for both unmethylated and methylated forms in a methylation-specific RCP analysis. One cell line with no visible expression could also be amplified with both primers. Overexpression or disappearance of p16 protein may readily occur when one of two alleles has been methylated.
(Keyword)
Biomarkers, Tumor / Blotting, Western / Cell Line, Tumor / Chromosome Aberrations / Cyclin-Dependent Kinase Inhibitor p16 / DNA / DNA Methylation / DNA Mutational Analysis / DNA Primers / Humans / Neoplasms / Reverse Transcriptase Polymerase Chain Reaction / Tubulin / Tumor Suppressor Protein p53
Tokimasa Kumada, Koichi Tsuneyama, Hideki Hatta, Shin Ishizawa and Yasuo Takano : Improved 1-h rapid immunostaining method using intermittent microwave irradiation: practicability based on 5 years application in Toyama Medical and Pharmaceutical University Hospital., Modern Pathology, Vol.17, No.9, 1141-1149, 2004.
(Summary)
Immunostaining depending on antigen-antibody specificity is the commonest approach for determining the localization of specific antigens in tissue sections. This procedure is applicable not only with frozen or specially fixed samples, but also has proved reliable with formalin-fixed paraffin-embedded tissue sections through improvement of antigen-retrieval. Immunostaining is thus firmly established as a tool for diagnostic pathology and in our institute multiple antibodies are applied for 13-15% of the cases examined, as well as H and E staining. With the standard approach, approximately 3 h is necessary from the beginning of deparaffinization till covering sections with the Envision system. We utilized intermittent microwave irradiation for 10 min during hybridization with primary and secondary antibodies in a special moist-chamber, to achieve all immunostaining steps within 1 h in 178 primary antibodies frequently used for diagnostic pathology. According to our 5 years experience, such microwave irradiation not only obtained significant specific staining for enhancing the specificity of antigen-antibody reactions, but also inhibited nonspecific binding. We present herein the details of the methodology and recommendations for its application with particular primary antibodies. This method can contribute to savings in time and energy, allowing pathologists to rapidly obtain diagnostic information.
(Keyword)
Antibodies, Monoclonal / Carcinoma, Squamous Cell / Gastrointestinal Stromal Tumors / Hospitals, University / Humans / Immunohistochemistry / Japan / Keratin-5 / Keratins / Microwaves / Proto-Oncogene Proteins c-kit / Reproducibility of Results / Schools, Medical / Staining and Labeling / Time Factors
Yoh Zen, Kenichi Harada, Motoko Sasaki, Yasunori Sato, Koichi Tsuneyama, Joji Haratake, Hiroshi Kurumaya, Kazuyoshi Katayanagi, Shinji Masuda, Hideki Niwa, Hideo Morimoto, Atsuo Miwa, Akio Uchiyama, C Bernard Portmann and Yasuni Nakanuma : IgG4-related sclerosing cholangitis with and without hepatic inflammatory pseudotumor, and sclerosing pancreatitis-associated sclerosing cholangitis: do they belong to a spectrum of sclerosing pancreatitis?, The American Journal of Surgical Pathology, Vol.28, No.9, 1193-1203, 2004.
(Summary)
Sclerosing cholangitis (SC) is a heterogeneous disease entity. Different etiologies such as choledocholithiasis, biliary tumor, or pericholangitis can manifest as SC. Hepatic inflammatory pseudotumor (IP) is rarely associated with SC (sclerosing cholangitis associated with hepatic inflammatory pseudotumor; SC-hepatic IP), but sclerosing pancreatitis (SP) is not infrequently associated with bile duct lesions (sclerosing pancreatitis-associated sclerosing cholangitis; SP-SC). In this study, we compared the histologic changes of hepatic hilar and extrahepatic bile duct lesions of SC (7 cases), SC-hepatic IP (5 cases), SP-SC (5 cases), and typical primary sclerosing cholangitis (PSC) (5 cases). Histologically, all SP-SC cases showed extensive and dense fibrosis with marked lymphoplasmacytic infiltration, many eosinophils, and obliterative phlebitis. Four cases of SC showed bile duct lesions similar to those of SP-SC, whereas other three cases of SC showed milder lymphoplasmacytic infiltration, scant eosinophilic cell infiltration, and no obliterative phlebitis. All SC-hepatic IP cases showed bile duct lesions identical to those of SP-SC. Immunohistochemically, many IgG4-positive plasma cells were found in the bile duct lesions of all SP-SC cases, 4 SC cases with marked lymphoplasmacytic infiltration, and all SC-hepatic IP cases. By contrast, IgG4-positive plasma cells were scarce or hardly found in the remaining 3 SC cases and all PSC cases. In conclusion, 4 SC cases and all SC-hepatic IP cases showed bile duct lesions identical to those of SP-SC, suggesting that these three conditions may be a single disease entity. Their pathogenesis may be similar or closely related to that of SP, and in that respect they may represent an IgG4-related biliary disease. They may respond to steroid therapy as SP does.
Koichi Tsuneyama, Motoko Sasaki, Tomonori Shimonishi and Yasuni Nakanuma : Expression of MAGE-A3 in intrahepatic cholangiocarcinoma and its precursor lesions., Pathology International, Vol.54, No.3, 181-186, 2004.
(Summary)
MAGE-A3 antigen is known to be neo-expressed in a large proportion of tumors but not detectable in normal tissues, and could be a target antigen recognized by autologous cytotoxic T lymphocytes. In the present study, the expression of MAGE-A3 at protein and mRNA levels was examined in intrahepatic cholangiocarcinoma (ICC) and its precursor lesions. Carcinomatous and dysplastic biliary cells expressed MAGE-A3 in their cytoplasm diffusely, although there was no MAGE-A3 expression in normal and hyperplastic biliary cells. MAGE-A3 was expressed in one of 10 cases (10%) of low-grade dysplasia, four of 13 (31%) cases of high-grade dysplasia/in situ carcinoma, and 32 of 68 invasive ICC cases (47%), respectively. The MAGE-A3 mRNA expression pattern was similar to that of MAGE-A3 protein. The incidence and intensity of MAGE-A3 expression increased along the progression of biliary neoplasia (P < 0.05). There was no correlation between MAGE-3 expression and histological differentiation or anatomical locations of invasive ICC. MAGE-A3 is a promising target molecule for the specific immunotherapy of ICC.
(Keyword)
Adult / Aged / Aged, 80 and over / Antigens, Neoplasm / Bile Duct Neoplasms / Bile Ducts, Intrahepatic / Biomarkers, Tumor / Cholangiocarcinoma / Female / Humans / Immunohistochemistry / In Situ Hybridization / Male / Middle Aged / Neoplasm Proteins / Precancerous Conditions / RNA, Messenger
Motoko Sasaki, Koichi Tsuneyama, Takahito Saito, Hiroaki Kataoka, Jan Mollenhauer, Annemarie Poustka and Yasuni Nakanuma : Site-characteristic expression and induction of trefoil factor family 1, 2 and 3 and malignant brain tumor-1 in normal and diseased intrahepatic bile ducts relates to biliary pathophysiology., Liver International, Vol.24, No.1, 29-37, 2004.
(Summary)
The intrahepatic biliary tree shows a site-characteristic expression and induction of TFF1,2,3 and DMBT1. In large bile ducts, TFF1,3 were constitutively expressed and increased in pathologic bile ducts. In small bile ducts, TFF2/DMBT1 is induced in damaged ducts irrespective of etiologies. However, the cytoprotective/repair property of TFF2/DMBT1 may not be enough to prevent the following bile duct loss in PBC.
Motoko Sasaki, Koichi Tsuneyama, Akira Ishikawa and Yasuni Nakanuma : Intrahepatic cholangiocarcinoma in cirrhosis presents granulocyte and granulocyte-macrophage colony-stimulating factor., Human Pathology, Vol.34, No.12, 1337-1344, 2003.
(Summary)
Intrahepatic cholangiocarcinoma (ICC) and combined hepatocellular and cholangiocarcinoma (HC-CC) are known to arise occasionally in hepatitis-related cirrhosis, although their clinicopathological features remain unclarified. In this study, we characterized the ICC (9 cases) and ICC elements of HC-CC (11 cases) arising in nonbiliary cirrhosis. Thirty-three hepatocellular carcinomas (HCC) associated with nonbiliary cirrhosis and 24 ICC without cirrhosis were used as controls. Prominent neutrophilic infiltration was frequent in ICC with cirrhosis (78%) and ICC elements of combined HC-CC (72%). Neutrophilic infiltration-related cytokines (interleukin 8, granulocyte colony-stimulating factor [G-CSF], and granulocyte macrophage colony-stimulating factor [GM-CSF]) were expressed frequently and intensely in carcinoma cells of ICC with cirrhosis (40%, 80%, and 60%, respectively) and in ICC elements of the combined one (13%, 38%, and 63%, respectively). Interleukin 8 was expressed in 18% of ICC without cirrhosis, irrespective of neutrophilic infiltration. Neutrophilic infiltration and expression of G-CSF and GM-CSF were in parallel (P < 0.05). G-CSF and GM-CSF mRNA were detected by RT-PCR in tissue specimens expressing G-CSF and GM-CSF at the protein level. Such neutrophilic infiltration and expression of G-CSF and GM-CSF were not evident in controls. The expressions of c-kit and c-Met, as a hematopoietic and hepatic stem cell marker, were seen frequently in ICC with cirrhosis (80% and 80%, respectively) and ICC elements of the combined one (63% and 50%, respectively). The present study revealed that the frequent expression of G-CSF and GM-CSF is a characteristic of ICC with cirrhosis and ICC in combined carcinoma, probably representing a phenotype of fetal hepatic parenchymal cell. The expression of these cytokines may be causally related to prominent neutrophilic infiltration.
Takashi Kamihira, Shinji Shimoda, Kenichi Harada, Akira Kawano, Mizuki Handa, Eishi Baba, Koichi Tsuneyama, Minoru Nakamura, Hiromi Ishibashi, Yasuni Nakanuma, Eric M Gershwin and Mine Harada : Distinct costimulation dependent and independent autoreactive T-cell clones in primary biliary cirrhosis., Gastroenterology, Vol.125, No.5, 1379-1387, 2003.
(Summary)
In PBC, costimulation-independent autoreactive T cells, which do not become anergic, increase and maintain the autoimmune response. In controls, although autoantigens are expressed on BEC and autoantigen-reactive T cells exist around BEC, autoantigen-reactive T cells are costimulation dependent and will become anergic and maintain peripheral tolerance.
Yuko Ishida, Toshikazu Kondo, Koichi Tsuneyama, Peirong Lu, Tatsunori Takayasu and Naofumi Mukaida : The pathogenic roles of tumor necrosis factor receptor p55 in acetaminophen-induced liver injury in mice., Journal of Leukocyte Biology, Vol.75, No.1, 59-67, 2003.
(Summary)
Acetaminophen (APAP) causes a massive production of intrahepatic tumor necrosis factor alpha (TNF-alpha). However, it still remains elusive regarding the roles of TNF-alpha in APAP-induced liver injury. Hence, we examined pathogenic roles of the TNF-alpha-TNF receptor with a molecular weight of 55 kDa (TNF-Rp55) axis in APAP-induced hepatotoxicity using TNF-Rp55-deficient [TNF-Rp55-knockout (KO)] mice. When wild-type (WT) BALB/c and TNF-Rp55-KO mice were intraperitoneally injected with a lethal dose of APAP (750 mg/kg), the mortality of TNF-Rp55-KO mice was marginally but significantly reduced compared with WT mice. Upon treatment with a nonlethal dose (600 mg/kg), WT mice exhibited an increase in serum transaminase levels. Histopathologically, centrilobular hepatic necrosis with leukocyte infiltration was evident at 10 and 24 h after APAP challenge. Moreover, mRNA expression of adhesion molecules, several chemokines, interferon-gamma (IFN-gamma), and inducible nitric oxide synthase (iNOS) was enhanced in the liver. On the contrary, serum transaminase elevation and histopathological changes were attenuated in TNF-Rp55-KO mice injected with APAP (600 mg/kg). The gene expression of all molecules except for IFN-gamma and iNOS was significantly attenuated in TNF-Rp55-KO mice. Moreover, anti-TNF-alpha neutralizing antibodies alleviated liver injury when administered at 2 or 8 h after but not at 1 h before APAP challenge to WT mice. Collectively, the TNF-alpha-TNF-Rp55 axis has pathogenic roles in APAP-induced liver failure.
(Keyword)
Acetaminophen / Alanine Transaminase / Animals / Antigens, CD / Cell Adhesion Molecules / DNA Primers / Gene Expression Regulation / Interleukins / Leukocytes / Liver / Male / Mice / Mice, Inbred BALB C / Receptors, Tumor Necrosis Factor / Receptors, Tumor Necrosis Factor, Type I / Reverse Transcriptase Polymerase Chain Reaction / Transcription, Genetic
M Sasaki, S-F Huang, M-F Chen, Y-Y Jan, T-S Yeh, A Ishikawa, J Mollenhauer, A Poustka, Koichi Tsuneyama, Y Nimura, K Oda and Y Nakanuma : Decrease of deleted in malignant brain tumour-1 (DMBT-1) expression is a crucial late event in intrahepatic cholangiocarcinoma., Histopathology, Vol.43, No.4, 340-346, 2003.
(Summary)
DMBT-1 expression is increased in IPN-L and non-invasive cholangiocarcinoma as well as in biliary epithelia in hepatolithiasis. Decreased expression of DMBT-1 and homozygous deletion of the DMBT-1 gene in invasive cholangiocarcinoma suggest that they occur in the late stage of cholangiocarcinogenesis.
Motoko Sasaki, Koichi Tsuneyama and Yasuni Nakanuma : Aberrant expression of trefoil factor family 1 in biliary epithelium in hepatolithiasis and cholangiocarcinoma., Laboratory Investigation; a Journal of Technical Methods and Pathology, Vol.83, No.10, 1403-1413, 2003.
(Summary)
Stepwise progression of intrahepatic cholangiocarcinoma (ICC) has been proposed in hepatolithiasis. We examined the participation of trefoil factor family 1 (TFF1), which is critical for mucosal protection and tumor suppression in the stomach, in the development and progression of ICC. We used 16 livers of ICC with hepatolithiasis, 11 of biliary epithelial dysplasia with hepatolithiasis, 16 of hepatolithiasis without dysplasia or carcinoma, 18 of ICC without hepatolithiasis, and 39 control livers. TFF1 expression in the biliary epithelium was increased in hepatolithiasis compared with control livers (p < 0.01). In biliary epithelial dysplasia and noninvasive ICC with hepatolithiasis, TFF1 was extensively expressed and MUC5AC gastric mucin was usually colocalized with TFF1. However, TFF1 expression was significantly decreased in invasive ICC despite preserved expression of MUC5AC. A total of four missense mutations were detected: three in two noninvasive ICC with hepatolithiasis (28.6%) and one in invasive ICC (11%). Loss of heterozygosity of the TFF1 gene was not detectable. The decreased expression of TFF1 in invasive ICC may be explained by the methylation of the TFF1 promoter region. Up-regulation of TFF1 coupled with MUC5AC in biliary epithelium in hepatolithiasis, biliary epithelial dysplasia, and noninvasive ICC may reflect the gastric metaplasia and early neoplastic lesion. Under such conditions, decreased TFF1 expression may lead to increased cell proliferation and then to the invasive character of ICC.
Koichi Tsuneyama, K Ohba, Y Zen, Y Sato, H Niwa, H Minato and Y Nakanuma : A comparative histological and morphometric study of vascular changes in idiopathic portal hypertension and alcoholic fibrosis/cirrhosis., Histopathology, Vol.43, No.1, 55-61, 2003.
(Summary)
In AF/C, a reduction in portal venous lumen was associated with an increase of hepatic arterial lumen and of angiogenesis-related cells in portal tracts. However, such compensatory arterial changes were not evident in IPH, and this compensatory failure may be a feature of IPH.
Motoko Sasaki, Shiu-Feng Huang, Miin-Fu Chen, Yi-Yin Jan, Ta-Sen Yeh, Akira Ishikawa, Jan Mollenhauer, Annemarie Poustka, Koichi Tsuneyama, Yuji Nimura, Koji Oda and Yasuni Nakanuma : Expression of deleted in malignant brain tumor-1 (DMBT1) molecule in biliary epithelium is augmented in hepatolithiasis: possible participation in lithogenesis., Digestive Diseases and Sciences, Vol.48, No.7, 1234-1240, 2003.
(Summary)
Deleted in malignant brain tumor-1 (DMBT1) is a mucin-like molecule participating in mucosal immune defense. Given that bovine gallbladder mucin, which accelerates cholesterol crystallization, is a DMBT1 homolog, DMBT1 expression was examined immunohistochemically in biliary epithelial cells in livers with hepatolithiasis (N = 25), primary sclerosing cholangitis (N = 7), large bile duct obstruction (N = 12), and control normal livers (N = 10). DMBT1 protein was determined in the hepatic bile samples of hepatolithiasis (N = 12) and other hepatobiliary diseases (N = 8) by immunoblot. While DMBT1 was faintly expressed in normal livers (20%), it was significantly augmented in hepatolithiasis (76%) (P < 0.05). DMBT1 was mildly expressed in primary sclerosing cholangitis and large bile duct obstruction. DMBT1 protein was detected frequently in hepatic bile samples of hepatolithiasis (50%) (P < 0.05), but in the other bile samples. The percentage of cholesterol in intrahepatic calculi was significantly higher in the patients with DMBT1-positive bile. Augmented expression and secretion of DMBT1 in intrahepatic large bile ducts in hepatolithiasis suggests its role in lithogenesis.
Kenichi Harada, Kumiko Isse, Koichi Tsuneyama, Hajime Ohta and Yasuni Nakanuma : Accumulating CD57 + CD3 + natural killer T cells are related to intrahepatic bile duct lesions in primary biliary cirrhosis., Liver International, Vol.23, No.2, 94-100, 2003.
(Summary)
This indicates that an immune disturbance induced by a selective reduction of V alpha 24-J alpha Q + NKT cells is unlikely to occur in PBC. Inversely, it can be postulated that auto aggressive CD57 + CD3 + NKT cells are recruited to regulate the altered immunity of the periductal microenvironment in PBC.
Koichi Tsuneyama, Yasuharu Kaizaki, Kenji Doden, Eiichi Kidani, Kenichi Harada, Motoko Sasaki and Yasuni Nakanuma : Combined hepatocellular and cholangiocarcinoma with marked squamous cell carcinoma components arising in non-cirrhotic liver., Pathology International, Vol.53, No.2, 90-97, 2003.
(Summary)
We report a surgical case of liver tumor, 40 x 35 mm in size, with squamous cell carcinoma (SCC) and hepatocellular carcinoma (HCC) components in a 60-year-old Japanese man with steatohepatitis. Most of the SCC component showed typical intercellular bridge and keratinization, while most of the HCC components showed a thick trabecular pattern with mild to moderate nuclear atypia. Both components transit each other without undifferentiated foci; however, a small foci showing glandular structure was intermediated. No cyst formation was found in the liver. The primary site of the squamous cell carcinoma was not detected in general clinical and radiological examination. Immunohistochemical analysis revealed that part of the HCC components neighboring the SCC showed patchy and weak expression of cytokeratin 7. There are several possibilities for the origin of squamous cell carcinoma in this case: marked squamous metaplastic change of cholangiocarcinoma and/or HCC, and carcinoma originating from pleuripotential stem cells. Irregular fatty changes, scattered giant mitochondria and acellular fibrosis with bridging were seen in the liver; however, this patient had no episode of hepatitis-associated viral infection. This is an interesting case of combined hepatocellular and cholangiocarcinoma with marked SCC components arising in a non-cirrhotic fibrotic liver.
Kenichi Harada, Satoru Ozaki, Yoshiko Sudo, Koichi Tsuneyama, Hajime Ohta and Yasuni Nakanuma : Osteopontin is involved in the formation of epithelioid granuloma and bile duct injury in primary biliary cirrhosis., Pathology International, Vol.53, No.1, 8-17, 2003.
(Summary)
Recently, it was shown that osteopontin (OPN) is involved as a chemoattractant cytokine in the recruitment of macrophages and T lymphocytes in the granulomas of diverse etiologies and also plays an important role in the production of autoantibodies and development of autoimmune diseases. Primary biliary cirrhosis (PBC) is characterized by immune-mediated bile duct damage with frequent epithelioid granulomas. In this study, the expression of OPN was immunohistochemically examined in 25 PBC and 52 control livers. Epithelioid cells within granuloma in PBC expressed OPN variably. These cells were also positive for CD68, suggesting their histiocyte/macrophage lineage. In addition, strong expression of OPN was seen in the cytoplasm of mononuclear cells infiltrating around granulomas and also damaged bile ducts in PBC. The number of such positive mononuclear cells and the ratio of OPN-positive cells/total infiltrating cells in portal tracts were higher in PBC than in controls. The majority of these OPN-positive cells were found to be IgG- or IgM-producing plasma cells. These suggest that in PBC, OPN is an important immune molecule in portal tracts, and contributes to the recruitment of mononuclear cells into epithelioid granuloma and also participates in bile duct injury via B-cell differentiation and plasma cell expansion.
Kenichi Harada, Koichi Tsuneyama, Mitsue Yasoshima, Yoshiko Kanemori, Hajime Ohta, Shinji Masuda, Nobuyuki Onai, Kouji Matsushima and Yasuni Nakanuma : Type1 and type2 memory T cells imbalance shown by expression of intrahepatic chemokine receptors relates to pathogenesis of primary biliary cirrhosis., Hepatology Research, Vol.24, No.3, 2002.
(Summary)
Primary biliary cirrhosis (PBC) is characterized by the immunopathologic destruction of interlobular bile ducts. Generally, immunereaction induced by the antigenetic stimulation is divided to cellular and humoral reactions, which are regulated by type1 and type2 memory T cells, respectively. The relative strength of type1 and type2 responses is an important factor in the pathophysiology of immune-mediated diseases. Chemokine receptors are disproportionally expressed in type1 and type2 T cells, providing a basis for tissue-specific recruitment of helper and cytotoxic T cell subsets. In this study, the expression of CXC chemokine receptor 3 (CXCR3) and CC chemokine receptor 4 (CCR4), which are preferentially expressed on type1 and type2, respectively, were examined to clarify their imbalance in the PBC liver tissue (23 cases). As a control, 16 livers of chronic viral hepatitis (CVH) were used. Reverse transcription-polymerase chain reaction and semiquantitative analysis revealed that the relative ratio of CXCR3/CCR4 mRNAs was higher in early PBC (0.76+/-0.56) rather than in advanced PBC (0.25+/-0.11), and was comparable to that in active CVH (0.67+/-0.43). By immunohistochemistry, the ratio of CXCR3-/CCR4-positive mononuclear cells in portal tracts of early PBC (4.77+/-1.70) is significantly higher than that of advanced PBC (3.11+/-1.26), and also than that of mild and of active CVH. In early PBC, mononuclear cells positive for CXCR3 were dense around the damaged bile ducts. These data suggest that the enhanced shift toward type1 occurs in portal tracts of early PBC and that the imbalance of type1/type2 changes during the course of PBC. This study provides further support for the state of enhanced type1 response in early PBC and potentially offers a possibility to suppress the bile duct lesions and to prevent the progression of PBC from early to advanced stages by reversing the type1/type2 imbalance.
Yoh Zen, Kenichi Harada, Motoko Sasaki, Koichi Tsuneyama, Kazuyoshi Katayanagi, Yui Yamamoto and Yasuni Nakanuma : Lipopolysaccharide induces overexpression of MUC2 and MUC5AC in cultured biliary epithelial cells: possible key phenomenon of hepatolithiasis., The American Journal of Pathology, Vol.161, No.4, 1475-1484, 2002.
(Summary)
Bacterial infection, bile stasis, mucin hypersecretion, and an alteration of the mucin profile such as an aberrant expression of gel-forming apomucin (MUC2 and MUC5AC) in the intrahepatic biliary tree are thought to be important in the lithogenesis of hepatolithiasis. So far, there have been no detailed studies linking bacterial infection to altered mucus secretion of biliary epithelium. In this study, the influence of lipopolysaccharide (LPS), a bacterial component, on apomucin expression in cultured murine biliary epithelial cells was examined with emphasis on the participation of tumor necrosis factor (TNF)-alpha. It was found that LPS up-regulated the expression of MUC2 and MUC5AC in cultured murine biliary epithelial cells. LPS also induced the expression of TNF-alpha in biliary epithelial cells and its secretion into the culture medium. The up-regulation of these apomucins was inhibited by pretreatment with TNF-alpha antibody. TNF-alpha alone also induced the overexpression of MUC2 and MUC5AC in cultured biliary epithelial cells. This overexpression was inhibited by pretreatment with calphostin C, an inhibitor of protein kinase C. These findings suggest that LPS can induce overexpression of MUC2 and MUC5AC in biliary epithelial cells via synthesis of TNF-alpha and activation of protein kinase C. This mechanism might be involved in the lithogenesis of hepatolithiasis.
Yoh Zen, Hajime Sunagozaka, Koichi Tsuneyama, Kekichi Masutomi, Shuichi Terasaki, Shuichi Kaneko, Kenichi Kobayashi and Yasuni Nakanuma : Incomplete septal cirrhosis associated with Wegener's granulomatosis., Liver, Vol.22, No.5, 388-393, 2002.
(Summary)
Incomplete septal cirrhosis, which is included in the spectrum of hepatoportal sclerosis, is characterized by parenchymal nodularity, incomplete fibrous septa, clustered or dispersed portal tract remnants, and abnormal spacing of portal tracts and hepatic veins. Hepatoportal sclerosis is known to be associated with collagen vascular diseases. Here, we describe a 73 year-old-female with incomplete septal cirrhosis. At 57 years, she presented with respiratory symptoms, and lung biopsy disclosed active arteritis with granuloma. Perinuclear antineutrophilic cytoplasmic antibody was also positive. Immunosuppressive therapy was done under the diagnosis of Wegener's granulomatosis. At 63 years, liver dysfunction was noted, and laparoscopy revealed uneven surface of the liver and dilatation of the umbilical vein. Liver dysfunction progressed, and she developed encephalopathy and massive ascites. She died of sepsis at 73 years. At autopsy the liver (700 g) was macronodular with several deep depressions. The parenchyma showed fine and diffuse nodularity. Grossly visible portal and hepatic veins were patent. The above-mentioned histologic features characterizing incomplete septal cirrhosis were found. This is the first report of incomplete septal cirrhosis associated with Wegener's granulomatosis implying that vascular and extravascular lesions of Wegener's granulomatosis might have been related to the pathogenesis of incomplete septal cirrhosis.
Koichi Tsuneyama, Kenichi Harada, Naoko Kono, Motoko Sasaki, Takahito Saito, Eric M Gershwin, Mamoru Ikemoto, Hiroyuki Arai and Yasuni Nakanuma : Damaged interlobular bile ducts in primary biliary cirrhosis show reduced expression of glutathione-S-transferase-pi and aberrant expression of 4-hydroxynonenal., Journal of Hepatology, Vol.37, No.2, 176-183, 2002.
(Summary)
The data suggest that lipid peroxidation in the bile ducts with reduced expression of glutathione-S-transferase-pi, may be an important pathologic process leading to the bile duct damage of primary biliary cirrhosis.
Koichi Tsuneyama, Kenichi Harada, Kazuyoshi Katayanagi, Kishichiro Watanabe, Hiroshi Kurumaya, Hiroshi Minato and Yasuni Nakanuma : Overlap of idiopathic portal hypertension and scleroderma: report of two autopsy cases and a review of literature., Journal of Gastroenterology and Hepatology, Vol.17, No.2, 217-223, 2002.
(Summary)
Idiopathic portal hypertension (IPH) is characterized by dense fibrosis of portal tracts and portal venous obliteration. Little is known about the etiopathogenesis of IPH. Association of various autoimmune diseases such as systemic lupus erythematosus in IPH suggests that IPH may share immunological disturbances with such autoimmune diseases. We recently experienced two autopsy cases presenting with both diffuse scleroderma and IPH. Dense fibrosis was found in both the dermis and intrahepatic portal tract of these cases. In addition, small vascular damages were commonly observed to various degrees in these fibrotic areas of both organs. The activation of fibroblasts and vascular damages mediated by various growth factors and cytokines reportedly involved in the dermis in scleroderma might have also been operative in portal tracts in these two cases of IPH. A review of literature disclosed eight overlapping cases of IPH and scleroderma (middle- to old-aged females), and scleroderma was diagnosed earlier than IPH. These findings suggest that similar pathogenetic processes are operative in the dermis as well as in the portal tracts of the liver in these cases.
Takahito Saito, Kenichi Harada, Koichi Tsuneyama, Masaaki Hirano, Susumu Amaya, Motoko Sasaki, Shuichi Kaneko, Kenichi Kobayashi and Yasuni Nakanuma : Primary squamous cell carcinoma of the liver producing parathyroid hormone-related protein., Journal of Gastroenterology, Vol.37, No.2, 138-142, 2002.
(Summary)
We report here an autopsy case of primary squamous cell carcinoma of the liver in a 63-year-old man who had hypercalcemia and an elevated serum level of parathyroid hormone-related protein. At autopsy, primary squamous cell carcinoma of the liver was found, without distinct preceding or associated hepatic or biliary diseases; no extrahepatic primary focus of squamous cell carcinoma was found. Bone involvement was not demonstrated, either radiologically or pathologically. Immunohistochemically, parathyroid hormone-related protein was detectable in the squamous cell carcinoma cells and it may have been responsible for the hypercalcemia. Such a case has not been reported so far in the English-language or the Japanese literature.
(Keyword)
Carcinoma, Squamous Cell / Fatal Outcome / Humans / Hypercalcemia / Immunohistochemistry / Liver Neoplasms / Magnetic Resonance Imaging / Male / Middle Aged / Parathyroid Hormone-Related Protein / Proteins
Koichi Tsuneyama, Wataru Kouda and Yasuni Nakanuma : Portal and parenchymal alterations of the liver in idiopathic portal hypertension: a histological and immunochemical study., Pathology, Research and Practice, Vol.198, No.9, 597-603, 2002.
(Summary)
Idiopathic portal hypertension (IPH) is characterized by presinusoidal portal hypertension owing to the intrahepatic, presinusoidal portal venous block, whereas the primary cause and initial vascular lesions(s) remain only speculative. In this study, a total of 97 IPH livers were histopathologically and immunohistochemically examined, placing emphasis on hepatic parenchymal fibrosis and atrophy as well as on portal tract fibrosis. Alcoholic cirrhosis and normal livers were used as controls. When compared with normal livers, the expression of connective tissue growth factor (CTGF) in periductal mononuclear cells was significant. Matrix metalloproteinase (MMP)9-positive mononuclear cells were fewer in number in the portal tract of IPH liver, when compared with alcoholic cirrhosis. These findings suggest a possible pathogenesis of collagen and elastin deposition because of increased CTGF expression and decreased MMP-9 expression in portal tracts of IPH. Sinusoidal dilatation associated with hepatocellular atrophy and apoptosis occurred frequently, but focally in 20% of the IPH cases. These changes were most often found in hyperplastic hepatocellular areas and in the perivenular areas of hepatic lobules. In these areas, pericellular fibrosis and thin fibrous septa were also frequently seen. In these fibrotic areas, there were deposited not only collagen fibers, but also elastic fibers, in which alpha-smooth muscle actin-positive sinusoidal cells, reflecting activated hepatic stellate cells, were frequently detected. It is possible that in IPH cases, continuing portal venous blood insufficiency may be responsible for hepatic parenchymal damage, which may be followed by hepatocellular apoptotic dropout and then by hepatic parenchymal atrophy and fibrosis.
Y Sudo, K Harada, Koichi Tsuneyama, K Katayanagi, Y Zen and Y Nakanuma : Oncocytic biliary cystadenocarcinoma is a form of intraductal oncocytic papillary neoplasm of the liver., Modern Pathology, Vol.14, No.12, 1304-1309, 2001.
(Summary)
Biliary cystadenocarcinoma with oncocytic differentiation was first reported in 1992. This is a report of a second case. The patient (a 71-year-old man) was admitted to our hospital complaining of abdominal fullness. Multicystic lesions were identified in the left hepatic lobe radiologically. The patient died of peritoneal dissemination of carcinoma 20 months later. At autopsy, the tumor of the left hepatic lobe was found to be composed of adjoining multiple cystic lesions and a solid lesion with infiltration of the hepatic hilus and peritoneal dissemination. Histologically, the multicystic lesions were covered by papillary neoplastic epithelial cells with an eosinophilic granular cytoplasm resembling that of oncocytes and a fine fibrovascular core. The cyst wall was fibrous, but there was no mesenchymal stroma. In the solid lesion and infiltrated areas, acidophilic and granular carcinoma cells formed small glandular or solid cord patterns with much mucin secretion (mucinous carcinoma). Immunohistochemically, carcinoma cells of both components were found to contain many mitochondria and showed the phenotypes of hepatocytes and cholangiocytes. Interestingly, the intrahepatic biliary tree also was invaded by carcinoma cells. This may be a case of intraductal oncocytic papillary neoplasm of the left hepatic lobe followed by secondary cystic dilatation of the affected bile duct.
Koichi Tsuneyama, K Harada, N Kono, K Hiramatsu, Y Zen, Y Sudo, E M Gershwin, M Ikemoto, H Arai and Y Nakanuma : Scavenger cells with gram-positive bacterial lipoteichoic acid infiltrate around the damaged interlobular bile ducts of primary biliary cirrhosis., Journal of Hepatology, Vol.35, No.2, 156-163, 2001.
(Summary)
LTA derived from bacterial fragments may reach the bile, not only in the diseased state but also under normal conditions. Such LTA may be involved in the development and progression of portal tract lesions, particularly bile duct lesions, in PBC.
S Amaya, M Sasaki, Y Watanabe, M W Tsui, Koichi Tsuneyama, K Harada and Y Nakanuma : Expression of MUC1 and MUC2 and carbohydrate antigen Tn change during malignant transformation of biliary papillomatosis., Histopathology, Vol.38, No.6, 550-560, 2001.
(Summary)
Biliary papillomatosis could undergo overt malignant transformation along with altered phenotypic expression of MUC proteins and mucin carbohydrate antigens.
Koichi Tsuneyama, M Nose, M Nisihara, K Katayanagi, K Harada and Y Nakanuma : Spontaneous occurrence of chronic non-suppurative destructive cholangitis and antimitochondrial autoantibodies in MRL/lpr mice: possible animal model for primary biliary cirrhosis., Pathology International, Vol.51, No.6, 418-424, 2001.
(Summary)
MRL/MP mice bearing the lymphoproliferative gene lpr (known as MRL/MP-lpr/lpr or MRL/Ipr mice) are known to spontaneously develop severe autoimmune diseases such as glomerulonephritis, arteritis and arthritis at an early stage of their life. They have also been reported to develop severe sialadenitis, suggesting that this mouse could be a model for Sjögren's syndrome. Primary biliary cirrhosis, an autoimmune disease characterized by chronic non-suppurative destructive cholangitis and the occurrence of antimitochondrial antibodies, is frequently associated with Sjögren's syndrome. In this study, we examined whether cholangitis and/or antimitochondrial antibodies occur in this mouse model, using more than 100 young and old MRL/Ipr mice. We frequently found portal inflammation associated with cholangitis of small intrahepatic bile ducts, especially in older mice. There was also infiltration of inflammatory cells (monocytes) as well as CD4-positive T cells. Epithelioid granuloma and bile-duct loss were also occasionally found. These histological features resemble primary biliary cirrhosis. In addition, antimitochondrial antibodies were shown by immunocytochemistry to be present in the sera of MRL/Ipr mice. There is currently no established animal model for primary biliary cirrhosis. Therefore, further studies on MRL/Ipr mice, with respect to pathogenesis of primary biliary cirrhosis, are warranted.
H Sabit, Koichi Tsuneyama, T Shimonishi, K Harada, J Cheng, H Ida, T Saku, K Saito and Y Nakanuma : Enhanced expression of basement-membrane-type heparan sulfate proteoglycan in tumor fibro-myxoid stroma of intrahepatic cholangiocarcinoma., Pathology International, Vol.51, No.4, 248-256, 2001.
(Summary)
To investigate the molecular mechanism for enhanced fibrous stroma formation in intrahepatic cholangiocarcinoma (ICC), we surveyed the expression pattern of basement-membrane-type heparan sulfate proteoglycan (HSPG; also known as perlecan) at the core protein and the mRNA level in ICC as well as in other liver neoplasms and reactive hepatic diseases. Immunohistochemistry of paraffin-embedded liver sections with hyaluronidase pretreatment showed that HSPG was present in small amounts in normal liver around the bile ducts and the blood vessels within the portal area. There was no evident expression within the hepatic lobules. Intense immunoexpression of HSPG was seen in the tumor-specific fibro-myxoid stroma of ICC and metastatic liver cancer originating from the colon. However, tumor-specific stroma of hepatocellular carcinomas showed little or no expression of HSPG. At the mRNA level, signals for HSPG were found in tumor cells of cholangiocarcinoma and metastatic colonic carcinomas, and in myofibroblasts in the tumor fibro-myxoid-specific stroma. From immunoprecipitation and reverse transcription-polymerase chain reaction (RT-PCR) analyses, a cultured human intrahepatic cholangiocarcinoma cell line (CCKS1), was found to express high levels of HSPG core protein and mRNA. These findings suggest that biliary and metastatic colon carcinoma cells as well as stromal myofibroblasts have a potential for HSPG production. In order to investigate the growth, invasion and metastatic ability of ICC, further study of the 'self-made' stromal component of ICC may provide a new approach.
K Harada, Koichi Tsuneyama, Y Sudo, S Masuda and Y Nakanuma : Molecular identification of bacterial 16S ribosomal RNA gene in liver tissue of primary biliary cirrhosis: is Propionibacterium acnes involved in granuloma formation?, Hepatology, Vol.33, No.3, 530-536, 2001.
(Summary)
The etiopathogenesis of primary biliary cirrhosis (PBC) remains speculative. Epithelioid granulomas are often found in the vicinity of damaged interlobular bile ducts in PBC, raising the possibility of a reaction to microbial materials. In this study, we tried to detect and identify bacterial DNA within granulomatous lesions in PBC. Using liver sections from 9 patients with PBC and 13 control livers, granuloma in portal tracts, portal tracts without granuloma, and adjacent hepatic parenchyma were selectively microdissected from sections, and then DNA was extracted from them. First, part of the bacterial 16S ribosomal RNA (rRNA) gene was amplified from DNA samples extracted from 5 PBC and 6 control livers, and their amplicons were sequenced for the identification of bacterial species. Several indigenous bacteria were identified. Among them, Propionibacterium acnes (P. acnes) was detected as a major clone in 20% to 50% of sequenced clones from granuloma of PBC, but the detection rate of P. acnes was 0% to 20% in those cloned from adjacent hepatic parenchyma of PBC. Then, a P. acnes-specific PCR was performed using all microdissected samples. Distinct PCR products were identified in epithelioid granuloma in all 9 PBC cases. The result that P. acnes DNA is present as a major clone in granulomas of PBC, suggest that P. acnes is involved in the pathogenesis of granuloma in PBC.
Y Zen, K Katayanagi, Koichi Tsuneyama, K Harada, I Araki and Y Nakanuma : Hepatocellular carcinoma arising in non-alcoholic steatohepatitis., Pathology International, Vol.51, No.2, 127-131, 2001.
(Summary)
The incidence and significance of hepatocellular carcinoma (HCC) in non-alcoholic steatohepatitis (NASH) has not been previously evaluated in detail. We recently experienced a case of NASH with multicentric HCC in a female patient. At the age of 58 years, the patient was diagnosed with non-insulin-dependent diabetes mellitus, treated by insulin therapy. The patient did not drink alcohol. She was negative for all serological markers of hepatitis B and C virus infection. Because of liver dysfunction, a needle biopsy was performed at the age of 62 years, and pathological findings, such as fatty change, Mallory's body, nuclear glycogen and pericellular fibrosis, suggested a diagnosis of NASH. Subsequently, four nodules were detected in the liver by imaging. Liver biopsies were performed from each nodule. One nodule was pathologically diagnosed as a pseudolymphoma, while three other nodules were moderately differentiated HCC (10 years after the diagnosis of non-alcoholic steatohepatitis), well-differentiated HCC (11 years later) and dysplastic nodule (11 years later), suggesting multicentric occurrence of HCC. This case suggests that HCC could be a late complication of NASH.
Koichi Tsuneyama, K Harada, M Yasoshima, K Hiramatsu, R C Mackay, R I Mackay, E M Gershwin and Y Nakanuma : Monocyte chemotactic protein-1, -2, and -3 are distinctively expressed in portal tracts and granulomata in primary biliary cirrhosis: implications for pathogenesis., The Journal of Pathology, Vol.193, No.1, 102-109, 2001.
(Summary)
The monocyte chemotactic proteins (MCPs) form a distinct structurally related subclass of C-C chemokines. MCPs select specific target cells due to binding to a distinct set of chemokine receptors and because of their effects on monocytes, and may participate in the process of granuloma formation during bacterial and/or mycobacterial infections. The aetiology of primary biliary cirrhosis (PBC) is still unclear, although bacterial infection and autoimmune processes have been implicated. In this study, the expression of three of the most potent monocyte chemoattractants, MCP-1, -2, and -3, was examined in patients with PBC and the data were compared with results for other liver diseases including primary sclerosing cholangitis (PSC), chronic viral hepatitis C, hepatic sarcoidosis, and normal liver. MCP-1 was expressed mainly in biliary epithelial cells of all liver specimens, irrespective of the cause of disease. Some mononuclear leukocytes in the portal tract expressed MCP-1 in all the disease groups examined and there were no significant differences in frequency between these groups. In contrast, more than 80% of PBC livers showed MCP-2- and MCP-3-positive mononuclear leukocyte infiltration in portal tracts, particularly around the bile ducts, whereas such cells were far less frequent in the other disease groups or in normal livers. Epithelioid granulomata of PBC patients contained MCP-2- and MCP-3-positive cells at their edge. In double staining experiments, more than 60% of the MCP-positive mononuclear cells co-expressed CD68, suggesting that a proportion of MCP-2- and MCP-3-positive cells are derived from monocytes. These monocytes expressing MCP-2 and MCP-3 may be responsible for the chemotactic activity of more monocytes. Such an expression pattern of MCP-1, -2 and -3 in portal tracts seems to be distinctive for PBC. This pattern underlines the importance of MCP-1, -2, and -3 in the recruitment of monocytes and possibly T lymphocytes into portal tracts, around the injured bile ducts, and into epithelioid granulomata in PBC. The data further implicate bacterial materials derived from bile in the overall pathogenesis of PBC.
Y Nakanuma, Koichi Tsuneyama, M Ohbu and K Katayanagi : Pathology and pathogenesis of idiopathic portal hypertension with an emphasis on the liver., Pathology, Research and Practice, Vol.197, No.2, 65-76, 2001.
(Summary)
Idiopathic portal hypertension (IPH) is characterized by a long-standing presinusoidal portal hypertension of unknown etiology in adults. Some unidentified agent(s) affect(s) the intrahepatic small portal veins or portal tracts. Immunological disturbance, thromboembolism, infectious etiology and/or increased fibrogenesis in portal tracts are suspected as being candidates for the primary agent(s). During the long clinical course of IPH, several pathological changes may occur, including subcapsular parenchymal atrophy, atrophy of the liver, portal and parenchymal fibrosis, and portal venous phlebosclerosis and thrombosis. The last-named of these lesions is mostly found in patients with a history of splenectomy. Subcapsular parenchymal and hepatic atrophy may result from a hepatocellular dropout via apoptosis or necrosis because of intrahepatic hemodynamic disturbances, particularly chronic portal venous blood insufficiency. Pericellular fibrosis and thin fibrous septa are also frequently found and associated with activated perisinusoidal cells positive for smooth muscle actin. At the same time, vague nodular hyperplasia of hepatocytes not surrounded by fibrous septa is not infrequently seen. It may resemble nodular regenerative hyperplasia, partial nodular transformation, or focal nodular hyperplasia. However, liver cirrhosis does not occur even at the terminal stage. Taking these findings into consideration, a new staging of IPH with a combination of hepatic parenchymal atrophy and portal venous thrombosis was proposed: non-atrophic liver without subcapsular parenchymal atrophy (stage I), non-atrophic liver with subcapsular parenchymal atrophy (stage II), atrophic liver with subcapsular parenchymal atrophy (stage III), and portal venous occlusive thrombosis (stage IV). IPH livers are likely to progress from stage I to stage III. Stage IV, which occurs relatively late, has a poor prognosis. This staging is applicable to clinical and autopsy cases without any histological data.
Y Nakanuma, Koichi Tsuneyama and K Harada : Pathology and pathogenesis of intrahepatic bile duct loss., Journal of Hepato-Biliary-Pancreatic Surgery, Vol.8, No.4, 303-315, 2001.
(Summary)
In recent years, the pathology and pathogenesis of bile duct loss have been extensively studied, and a number of hepatobiliary diseases have been added to the list of ductopenic diseases. In addition, the biology of biliary epithelial cells is now being studied with respect to bile duct loss, as well as biliary epithelial neoplasia. In this review, recent advances in pathogenetic and pathological studies of intrahepatic bile duct loss are described, with an emphasis on immune-mediated cholangiopathies. The bile duct loss, an acquired and pathologic process that occurs in the biliary tree, is recognizable as an absence of bile duct in an individual portad tract, and also as such absence in the vicinity of parallel running hepatic arterial branches that constitute the portal triad. Immunostaining with biliary cytokeratin and other carbohydrate materials is useful for the identification of biliary elements in the inflamed portal tracts or fibrous septa. The underlying processes responsible for bile duct loss include immunological, ischemic, infectious, metabolic, and toxic processes. Bile duct loss in primary biliary cirrhosis and primary sclerosing cholangitis is immune-mediated, that in interventional radiology using hepatic arterial branches is related to biliary ischemia, while that in hepatic allograft rejection is related to both immunological and ischemic insults. Bacterial and viral cholangitis with bile duct loss is an example of infectious cholangitis. The biliary tree maintains its homeostasis by renewal and dropout, and bile duct loss occurs mainly via biliary apoptosis. In some patients with bile duct loss, such as occurs in drug-induced injuries, the bile ducts regenerate and finally redistribute in the liver, while in other types of bile duct loss, the loss is progressive and is followed by vanishing bile duct syndrome, leading to biliary cirrhosis or liver transplantation. More analysis of the biology of biliary epithelial cells is mandatory for the evaluation of the pathobiology of bile duct loss, as well as for the effective restoration of biliary epithelial cells, in ductopenic liver diseases.
(Keyword)
Bile Duct Diseases / Bile Ducts, Intrahepatic / Epithelial Cells / Humans / Liver / Liver Diseases / Syndrome
T Shimonishi, K Isse, F Shibata, I Aburatani, Koichi Tsuneyama, H Sabit, K Harada, K Miyazaki and Y Nakanuma : Up-regulation of fas ligand at early stages and down-regulation of Fas at progressed stages of intrahepatic cholangiocarcinoma reflect evasion from immune surveillance., Hepatology, Vol.32, No.4 Pt 1, 761-769, 2000.
(Summary)
We examined immunohistochemically the possible participation of the Fas/Fas ligand (FasL) system in intrahepatic cholangiocarcinoma (ICC) during the escape from immune surveillance, using 68 cases of ICC, 29 cases of normal intrahepatic large bile ducts, and 18 cases of biliary dysplasia. Apoptosis of tumor-infiltrating lymphocytes (TIL) was examined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Fas was weakly expressed in normal intrahepatic bile ducts. Almost all biliary dysplasia and well-differentiated ICCs showed moderate to marked expression of Fas, while Fas expression was variable in moderately and poorly differentiated ICCs. Down-regulation of Fas expression was significantly correlated with histologic de-differentiation, vascular invasion, the size of ICCs, and short survival of ICC patients. By in situ hybridization, FasL mRNA were frequently and strongly expressed in biliary dysplasia compared with non-neoplastic intrahepatic bile duct. In well-differentiated ICCs, FasL mRNA expression was frequent and intense. But, the expression gradually decreased in moderately and poorly differentiated ICCs. Down-regulation of FasL mRNA expression in ICCs was correlated with perineural invasion and tumor size (over 4 cm) (P <.05). Apoptotic TIL were more frequent in ICC foci than in non-neoplastic foci remote from ICC foci. These findings suggest that a tumor evasion mechanism involving Fas/FasL exists in ICC; frequent and intense expression of FasL mRNA in well-differentiated ICCs enable them to escape immune surveillance by counterattacking Fas-bearing TIL. This counterattack becomes insensitive in poorly differentiated ICCs, in which the down-regulation of Fas gives them a resistance against the FasL-expressing TIL. These mechanisms may be involved in the tumor progression.
(Keyword)
Adult / Aged / Aged, 80 and over / Apoptosis / Bile Duct Neoplasms / Bile Ducts / Bile Ducts, Intrahepatic / Cholangiocarcinoma / Fas Ligand Protein / Female / Humans / In Situ Nick-End Labeling / Male / Membrane Glycoproteins / Middle Aged / RNA, Messenger / fas Receptor
Y Nakanuma, Koichi Tsuneyama, M Sasaki and K Harada : Destruction of bile ducts in primary biliary cirrhosis., Best Practice & Research. Clinical Gastroenterology, Vol.14, No.4, 549-570, 2000.
(Summary)
Primary biliary cirrhosis is characterized by the immune-mediated, progressive destruction of interlobular bile ducts. Lymphoid cells migrate into the biliary epithelial layer through integrin alpha(4)/fibronectin interaction and are responsible for chronic destructive cholangitis. The bile ducts express intercellular adhesion molecule-1 (ICAM-1) and vascular cellular adhesion molecule-1 (VCAM-1), and infiltrating lymphocytes express LFA1 and VLA4, facilitating their interaction. Epithelioid granulomas contain foamy cells ingesting biliary lipids, and CD1d was detectable in epithelioid granulomas, suggesting that the biliary substance(s) which are leaked is a trigger for chronic destructive cholangitis. Apoptotic biliary destruction is brought about by antigen-specific and non-specific reactions. Shrunken biliary epithelial cells with pyknotic nuclei positive for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) may reflect apoptotic processes. Increased expression of caspase-3 and -8 with DNA fragmentation factor on the bile ducts may reflect molecular events during apoptosis, and down-regulation of Bcl-2 of biliary epithelial cells seems to facilitate apoptosis. Multiple factors, particularly the Fas system, are stimuli of apoptosis. Anoikis with decreased biliary expression of integrin 6, a ligand for laminin, may also be involved in biliary epithelial apoptosis.
K Hiramatsu, K Harada, Koichi Tsuneyama, M Sasaki, S Fujita, T Hashimoto, S Kaneko, K Kobayashi and Y Nakanuma : Amplification and sequence analysis of partial bacterial 16S ribosomal RNA gene in gallbladder bile from patients with primary biliary cirrhosis., Journal of Hepatology, Vol.33, No.1, 9-18, 2000.
(Summary)
The present results raise several possible roles of gram-positive bacteria in bile in the etiopathogenesis of primary biliary cirrhosis. However, these results could also reflect an epiphenomenon due to decreased bile flow in the patients with primary biliary cirrhosis at an advanced stage.
W Koda, K Harada, Koichi Tsuneyama, N Kono, M Sasaki, O Matsui and Y Nakanuma : Evidence of the participation of peribiliary mast cells in regulation of the peribiliary vascular plexus along the intrahepatic biliary tree., Laboratory Investigation; a Journal of Technical Methods and Pathology, Vol.80, No.7, 1007-1017, 2000.
(Summary)
Our pilot study disclosed that tryptase-positive mast cells (MC) were densely distributed around the intrahepatic bile ducts (peribiliary MC). In this study, the pathophysiologic roles of these MC were examined with respect to the microcirculation around the bile duct in 71 cases of histologically normal liver, 24 cases of chronic hepatitis, and 45 cases of liver cirrhosis. The tryptase-positive MC were very close to the microvessels of the peribiliary vascular plexus (PVP), which supply the intrahepatic biliary tree. The tryptase-positive MC were frequently found adjacent to vascular smooth muscle cells, including pericytes. The location of the tryptase-positive MC was confirmed by ultrastructural analysis. In cirrhosis, the numbers of both microvessels of PVP and peribiliary MC increased in parallel. Peribiliary MC were immunoreactive for endothelin 1 (ET-1), and were variably immunoreactive for histamine, chymase, inducible nitric oxide synthase (iNOS), and endothelin A and B (ET(A) and ET(B)) receptors, particularly in cirrhotic livers. On vascular endothelial cells of PVP, endothelial nitric oxide synthase (eNOS) and ET-1 were consistently detectable, and ET(A) receptors, ET(B) receptors, and iNOS were variably detectable. Pericytes of PVP expressed ET(A) and ET(B) receptors in addition to ET-1 and iNOS. Biliary epithelial cells also focally expressed iNOS, ET-1, and ET(A) and ET(B) receptors. These vasoactive substances were strongly expressed on the cellular components in cirrhotic liver. By in situ hybridization, iNOS mRNA signals were observed on iNOS-immunoreactive cell components, including peribiliary MC. These morphologic and immunohistochemical findings suggest that the cellular components displaying vasoactive substances in the milieu of the intrahepatic biliary tree are very dynamic in the vasoregulation of PVP in normal livers, even more so in cirrhosis, and that peribiliary MC exert local effects on the microcirculation of PVP, directly and indirectly.
Koichi Tsuneyama, K Saito, H B Ruebner, I Konishi, Y Nakanuma and E M Gershwin : Immunological similarities between primary sclerosing cholangitis and chronic sclerosing sialadenitis: report of the overlapping of these two autoimmune diseases., Digestive Diseases and Sciences, Vol.45, No.2, 366-372, 2000.
(Summary)
Primary sclerosing cholangitis (PSC) is characterized by destructive inflammation and fibrosis affecting the bile ducts. The etiology of PSC is still unknown, although lymphocytic infiltration in the portal areas suggests an immune-mediated destruction of the bile ducts. Patients with one autoimmune disease often suffer from one or more other autoimmune diseases. It is well known that there is a close relationship between PSC and inflammatory bowel disease, particularly ulcerative colitis(UC). However, the pathological findings in UC and other overlap diseases do not resemble those of PSC. In the present study, we report a patient with chronic sclerosing sialadenitis (Kuttner's tumor) and PSC. It is compared the sclerosing changes in both salivary glands and bile ducts histologically. In addition, the expression pattern of mast cell tryptase, b-FGF, and HLA-DR were examined in both tissues immunohistochemically. Histological features of sclerosing change in both salivary and bile ducts were quite similar. Marked mast cell infiltration and b-FGF expression were seen in the sclerosing areas in both tissues. In active inflammatory areas of the salivary glands, HLA-DR expression was also seen. We hypothesized that similar immune reactions occur in both the salivary gland and bile ducts and are responsible for the fibrosis that follows.
M Yasoshima, Koichi Tsuneyama, K Harada, M Sasaki, E M Gershwin and Y Nakanuma : Immunohistochemical analysis of cell-matrix adhesion molecules and their ligands in the portal tracts of primary biliary cirrhosis., The Journal of Pathology, Vol.190, No.1, 93-99, 2000.
(Summary)
Intraepithelial migration of lymphoid cells (epitheliotropism) through the basement membrane of the bile duct is a key event in the development of chronic non-suppurative destructive cholangitis (CNSDC) and eventual immune-mediated bile duct loss in primary biliary cirrhosis (PBC). Cell-to-cell and cell-to-extracellular matrix proteins may play an important role in the development of CNSDC. To address the events of epitheliotropism in CNSDC, the expression of cell-matrix adhesion molecules such as integrins alpha1, alpha3, alpha4, alpha5, and alpha6 and the distribution of fibronectin, laminin, and collagen type IV were studied immunohistochemically, with emphasis on both infiltrating lymphocytes and the bile ducts, in frozen sections from 15 PBC cases and 34 controls (chronic viral hepatitis, extrahepatic biliary obstruction, and normal liver). In PBC and chronic viral hepatitis, most of the infiltrating lymphoid cells expressed integrin alpha4, while such expression was less common in extrahepatic biliary obstruction and normal liver. A biliary basement membrane-like structure was delineated by immunostaining of collagen type IV and laminin in PBC and controls. On the basement membrane of CNSDC, fibronectin, a ligand of integrin alpha4, was strongly and frequently expressed in PBC, while such expression on the biliary basement membrane was rare in other controls. These results suggest that increased fibronectin expression on the biliary basement membrane and integrin alpha4-fibronectin interaction facilitate adhesion and the penetration of infiltrating alpha4-expressing lymphocytes into the biliary epithelial layer in PBC.
Koichi Tsuneyama, N Kono, M Yamashiro, W Kouda, A Sabit, M Sasaki and Y Nakanuma : Aberrant expression of stem cell factor on biliary epithelial cells and peribiliary infiltration of c-kit-expressing mast cells in hepatolithiasis and primary sclerosing cholangitis: a possible contribution to bile duct fibrosis., The Journal of Pathology, Vol.189, No.4, 609-614, 1999.
(Summary)
Hepatolithiasis and primary sclerosing cholangitis (PSC) are intractable chronic biliary diseases. In hepatolithiasis, bilirubin-calcium stones are packed in multiple irregularly dilated intrahepatic bile ducts. In PSC, small bilirubin-calcium stones develop terminally. The progressive periductal fibrosis with dilated and stenotic bile ducts in these two diseases may play a role in their incurability. This immunohistochemical study has investigated the expression of some factors that might be involved in fibrogenesis in hepatolithiasis and PSC. Many mast cells positive for c-kit were found in the periductal and ductal fibrosis around the intrahepatic large bile ducts and also around the proliferative peribiliary glands. These mast cells also expressed basic fibroblast growth factor and/or tumour necrosis factor-alpha, which are known as fibrogenetic factors. It was of interest that the aberrant expression of stem cell factor (SCF), a ligand of c-kit, was demonstrated on biliary epithelia of the dilated and stenotic bile ducts showing periductal fibrosis and inflammation and also of the proliferated peribiliary glands in hepatolithiasis and PSC, while no such expression was seen in non-affected bile ducts in hepatolithiasis or in the bile ducts in normal livers. Some of the infiltrating mononuclear cells around the SCF-expressing bile ducts were also positive for SCF. It seems likely that aberrantly expressed SCF on biliary epithelial cells accumulates and stimulates mast cells via the c-kit receptor and that these up-regulated mast cells induce progressive periductal and portal fibrosis by displaying fibrogenetic factors in hepatolithiasis and PSC.
K Harada, Y Sudo, N Kono, S Ozaki, Koichi Tsuneyama, E M Gershwin and Y Nakanuma : In situ nucleic acid detection of PDC-E2, BCOADC-E2, OGDC-E2, PDC-E1alpha, BCOADC-E1alpha, OGDC-E1, and the E3 binding protein (protein X) in primary biliary cirrhosis., Hepatology, Vol.30, No.1, 36-45, 1999.
(Summary)
The characteristic serological feature of primary biliary cirrhosis (PBC) is the presence of antimitochondrial antibodies (AMAs), and the major proteins recognized by AMAs are subunits of the 2-oxo acid dehydrogenase complexes (2-OADC), including the E2 components of the pyruvate dehydrogenase complex (PDC), the 2-oxo-glutarate dehydrogenase complex (OGDC), the branched-chain 2-oxoacid dehydrogenase complex (BCOADC), the E3 binding protein (E3BP or protein X) and the E1a component of mammalian PDC. Previous work has postulated that either E3BP, or a molecule cross-reactive with the PDC-E2 molecule, is uniquely expressed on the surface of biliary epithelial cells in PBC. To address this issue, we performed in situ hybridization for all of the major 2-OADC components at the mRNA level, including PDC-E2, BCOADC-E2, OGDC-E2, PDC-E1a, BCOADC-E1a, OGDC-E1, and E3BP using 13 PBC and 9 control livers using 7 mitochondrial antisense probes. In both PBC and controls, the expression of all 2-OADC component mRNA studied herein were found in hepatocytes and infiltrating mononuclear cells, without significant differences. Interestingly, however, despite published data on immunohistochemical staining, interlobular bile ducts including the injured bile ducts in PBC were generally negative or only faintly positive, with the exception of 1 bile duct in 1 of 13 cases of PBC and 1 of 9 control liver specimens. Moreover, confocal microscopic examination and image analysis revealed that the mRNA signal intensity of each of the 2-OADC components in the bile ducts of PBC was relatively lower in comparison with control liver diseases. These data suggest that continuous enhanced synthesis of the 2-OADC components is not likely to be occurring in the biliary epithelial cells in PBC, and that an increase of PDC-E2 or E3BP immunoreactivity in PBC is caused by exogenous imported or cross-reactive molecules.
S Quaranta, H Shulman, A Ahmed, Y Shoenfeld, J Peter, B G McDonald, J de Water Van, R Coppel, C Ostlund, J H Worman, M Rizzetto, Koichi Tsuneyama, Y Nakanuma, A Ansari, F Locatelli, S Paganin, F Rosina, M Manns and E M Gershwin : Autoantibodies in human chronic graft-versus-host disease after hematopoietic cell transplantation., Clinical Immunology, Vol.91, No.1, 106-116, 1999.
(Summary)
Primary biliary cirrhosis (PBC) and graft-versus-host disease (GVHD) are thought to have common immunopathologic features and previous studies have reported that 5.2 to 81% of patients with chronic GVHD after allogeneic hematopoietic cell transplant have antimitochondrial antibodies (AMA). We studied a total of 89 patients with chronic GVHD and 60 controls for AMA reactivity by ELISA and immunoblotting using recombinant PDC-E2, BCOADC-E2, and OGDC-E2, immunoblotting of beef heart mitochondrial proteins, and reactivity to nuclei, smooth muscle (ASMA), ribonucleoprotein JO-1, extractable nuclear antigen, nuclear proteins SSA/ SSB, ribonucleic P proteinase III, cardiolipin (ACA), liver kidney microsomal, thyroid microsomal, myeloperoxidase, and the reactivity of rheumatoid factor. A subset of 60 chronic GVHD sera were tested for reactivity to gp210 and LBR. Finally, liver tissue from patients with chronic GVHD and PBC was studied by immunohistochemistry to determine whether there was comparable abnormal apical staining of biliary epithelial cells using PDC-E2-specific monoclonal antibodies. Surprisingly, there were no AMA found in the sera from the 89 patients with chronic GVHD. Review of published data on AMA in GVHD suggests that previous results were primarily false positives. In contrast, sera from the patients with GVHD did have a variety of other autoantibodies and, in particular, 20/89 (22.4%) positive ANA, 23/89 (25.8%) positive ASMA, and 9/89 (10.1%) positive ACA. The other autoantibodies assayed were not statistically different from controls. Finally, abnormal biliary epithelial luminal staining of bile ducts was found, as expected, in liver tissue of patients with PBC but was absent in chronic GVHD.
Koichi Tsuneyama, M Hoso, N Kono, M Kitagawa, S Masuda, N Matsuki and Y Nakanuma : An unusual case of epithelial-myoepithelial carcinoma of the liver., The American Journal of Surgical Pathology, Vol.23, No.3, 349-353, 1999.
(Summary)
The authors present an unusual case of an epithelial-myoepithelial carcinoma of the liver in a 67-year-old man who was admitted for resection of a gastric adenocarcinoma. At operation, a 3 x 3 cm mass in the right liver lobe was also removed. This mass consisted of duct-like structures with dual differentiation. The inner layer was composed of an epithelial lining, and the outer layer consisted of clear cells, all unrelated to the moderately well-differentiated gastric adenocarcinoma. The clear cells were positive for S-100 and alpha-smooth muscle actin, suggesting myoepithelial origin. The mass was considered to be low-grade epithelial-myoepithelial carcinoma. However, the patient had a history of an oral nodule present since childhood, resected 10 years previously. These slides were reviewed and revealed a mixture of clear cells and basal cells with squamous differentiation. In addition, there were duct-like structures with the two-layer pattern found in the liver tumor. This tumor had numerous mitotic figures and showed perineural invasion, suggesting a high grade of malignancy. These findings led to an interpretation of the oral tumor as also being epithelial-myoepithelial carcinoma, which had remained as "benign" for more than 50 years and subsequently underwent malignant transformation. During this long period, liver metastases may have occurred and remained low-grade. Alternatively, the liver and oral tumors may have arisen separately in the foregut during embryologic development, remaining low-grade until malignant transformation occurred.
Koichi Tsuneyama, M Sasaki, A Sabit, K Yokoi, Y Arano, T Imai and Y Nakanuma : A case report of gastric carcinosarcoma with rhabdomyosarcomatous and neuroendocrinal differentiation., Pathology, Research and Practice, Vol.195, No.2, 93-7;, 1999.
(Summary)
We report herein an unusual gastric carcinosarcoma with rhabdomyosarcomatous and neuroendocrinal differentiation in a 63-year-old Japanese male. The tumor was a pedunculated large polypoid tumor (7 x 6.5 x 3.5 cm) located in the pylorus. Histologically, it invaded to the subserosa and was composed of both adenocarcinomatous and sarcomatous components. Adenocarcinomatous foci generally showed tubular to solid patterns and occupied the parts facing the gastric lumen, while the sarcomatous components showed a generally irregular and solid arrangement. There were transitions between the sarcomatous and carcinoma elements. In addition, carcinoma cells with a cord-like or trabecular arrangement similar to that seen in endocrine carcinoma expressed chromogranin A, and were mainly observed in an intermediate area between the adenocarcinomatous and sarcomatous foci. The sarcomatous areas were mainly composed of spindle cells and occasionally contained a sarcomatous component showing rhabdomyosarcomatous differentiation. This is an interesting case to consider how the variety of cell type appeared in such a type of tumor in the stomach.
Y Nakanuma, K Harada, K Katayanagi, Koichi Tsuneyama and M Sasaki : Definition and pathology of primary sclerosing cholangitis., Journal of Hepato-Biliary-Pancreatic Surgery, Vol.6, No.4, 333-342, 1999.
(Summary)
Although primary sclerosing cholangitis (PSC) is not a common disease, it is important in the differential diagnosis of hepatobiliary tract diseases in clinical practice. A diagnosis of PSC should be made only after the exclusion of similar diseases with well known etiologies or pathogeneses. In this review, the pathology of classical PSC and its variants or related diseases is highlighted. PSC is histologically characterized by progressive periductal fibrosis with luminal stenosis or obliteration, along with the formation of a fibrous core, as well as dilatation (cholangiectasis). Its etiology is unknown. Bacterial ascending cholangitis is superimposed on its long clinical course. Such a heterogeneous distribution of biliary lesions with biliary obliteration and cholangiectasis is responsible for the radiological demonstration of biliary abnormalities, particularly the beaded appearance. Sampling variability is common in needle or wedge biopsied specimens. As a result of biliary damage, the liver shows progressive cholestatic change followed by biliary fibrosis and cirrhosis, and this hepatic progression is divisible into four stages. There are several variants of PSC or related diseases, such as localized biliary sclerosis and stenosis, sclerosing cholangitis associated with inflammatory pseudotumor, and PSC-autoimmune hepatitis overlapping syndrome. Cholelithiasis, including secondary hepatolithiasis and, to a lesser degree, biliary carcinoma and dysplasia, are also known to develop at the perihilar bile ducts as a late complication of PSC.
M Yamashiro, W Kouda, N Kono, Koichi Tsuneyama, O Matsui and Y Nakanuma : Distribution of intrahepatic mast cells in various hepatobiliary disorders. An immunohistochemical study., Virchows Archiv, Vol.433, No.5, 471-479, 1998.
(Summary)
There is evidence that mast cells are involved in a number of pathophysiological processes. The significance of mast cells in hepatic fibrosis was examined in 28 patients with histologically normal livers, 34 with acute liver diseases, 51 with chronic liver diseases, and 59 with cholestatic biliary diseases, using immunostaining of the mast cell-specific proteinase, tryptase. Mast cells that were positive for tryptase and for chymase were significantly increased in frequency in fibrotic portal tracts and fibrous septa, particularly in cholestatic/biliary diseases. Mast cells were also increased in frequency around the fibrotic septal and intrahepatic large bile ducts and peribiliary glands of biliary diseases. However, they were less common or even rare in the sclerotic bile ducts and in scarred portal or septal fibrosis. More than half of these more numerous mast cells were positive for histamine, and some were also positive for basic fibroblast growth factor. These two substances were detectable by immunoelectron microscopic in the cytoplasmic granules of mast cells. In contrast, mast cell numbers were not significantly increased in acute viral or drug-induced hepatitis, or in zones 2 and 3 of the hepatic acinus with respect to pericellular and perivenular fibrosis in chronic liver diseases. These findings suggest that mast cells increase in number in cholestatic/biliary diseases, and to a lesser degree in chronic liver diseases, and are involved in the active fibrous enlargement of portal tract and fibrous septa formation and also in the fibrosis of the intrahepatic bile ducts as they display fibrosis-promoting factors such as tryptase, fibroblast growth factor and histamine.
Koichi Tsuneyama, K Harada, M Yasoshima, K Kaji, E M Gershwin and Y Nakanuma : Expression of co-stimulatory factor B7-2 on the intrahepatic bile ducts in primary biliary cirrhosis and primary sclerosing cholangitis: an immunohistochemical study., The Journal of Pathology, Vol.186, No.2, 126-130, 1998.
(Summary)
Co-stimulatory factors B7-1 (CD80) and B7-2 (CD86) and their ligands, including CD28, are important for the efficient presentation and persistence of an antigen-specific immune reaction. Hitherto, there has been a paucity of data on the roles of such co-stimulatory factors in immune-mediated biliary diseases. In this investigation, the hepatic immunohistochemical expression of B7-1 and B7-2 has been studied, with emphasis on intrahepatic biliary epithelia, using wedge biopsies from 22 patients with primary biliary cirrhosis (PBC), seven with primary sclerosing cholagitis (PSC), and, as controls, eight cases of extrahepatic biliary obstruction, eight of chronic viral hepatitis C, and three histologically normal livers. In 10/22 (45 per cent) patients with PBC and 3/7 (43 per cent) patients with PSC, B7-2, but not B7-1, was expressed on the epithelial cells of small intrahepatic bile ducts and bile ductules. This expression was manifest as diffuse but variable cytoplasmic staining. Such B7-2-positive bile ducts were not seen in controls. Positive staining was found only in the early stage of PBC and PSC. In PBC and PSC, almost all lymphocytes in the portal tracts, including those around the damaged bile ducts, were positive for CD28, a ligand of B7-2. These results suggest that B7-2 expression on biliary epithelial cells is involved in antigen presentation and perhaps in bile duct destruction in PSC and PBC.
Koichi Tsuneyama, M Yasoshima, K Harada, K Hiramatsu, E M Gershwin and Y Nakanuma : Increased CD1d expression on small bile duct epithelium and epithelioid granuloma in livers in primary biliary cirrhosis., Hepatology, Vol.28, No.3, 620-623, 1998.
(Summary)
Cluster of differentiation 1 (CD1) is a family of four distinct nonpolymorphic major histocompatibility complex class I-like molecules that can present microbial nonpeptide lipid antigens to T cells. Among the CD1 gene family, CD1d is found in a wide range of tissues including the intestine and liver, and has been proposed to play an important role in mucosal immunity. Primary biliary cirrhosis (PBC) is an immune-mediated liver disease involving the intrahepatic small bile ducts, which also belong to the mucosal immune system. In this study, we studied the expression of CD1d in patients with PBC and compared the data with those of patients with hepatic sarcoidosis, primary sclerosing cholangitis (PSC), chronic viral hepatitis (CVH), and normal liver as controls. CD1d was found to be expressed in hepatocytes in all cases examined, and in epithelioid granuloma cells in 19 of 22 PBC livers and in 4 of 4 livers with hepatic sarcoidosis. In addition, CD1d was focally expressed on epithelial cells of the small bile ducts in approximately 50% of the PBC patients but in no controls. Such bile duct epithelial staining of CD1d was seen in early-stage PBC and virtually absent in late-stage PBC. Moreover, there was no evidence of expression of CD1d in large bile duct epithelial cells of PBC. The CD1d on biliary epithelial cells in PBC may be involved in the antigen presentation of microbial lipid antigen(s) to surrounding T cells. Alternatively, modified endogenous lipidic compounds may share analogy with bacterial lipid antigens and explain CD1d expression, a possible epiphenomenon rather than a proof of bacterial involvement.
K Harada, N Kono, Koichi Tsuneyama and Y Nakanuma : Cell-kinetic study of proliferating bile ductules in various hepatobiliary diseases., Liver, Vol.18, No.4, 277-284, 1998.
(Summary)
These findings suggest that typical ductules reflect active proliferation of preexisting ductules. Atypical ductules might be classifiable into two categories: those in PBC and PSC primarily reflect ductular transformation (metaplasia) of periportal hepatocytes, while those in EBO and CH reflect active proliferation and transformation of hepatocytes. Apoptosis via perforin/granzyme B pathway may be involved in the maintenance of homeostasis in ductular proliferation as degrading fraction.
Koichi Tsuneyama, N Kono, M Hoso, H Sugahara, K Yoshida, K Katayanagi, E M Gershwin, K Saito and Y Nakanuma : aly/aly mice: a unique model of biliary disease., Hepatology, Vol.27, No.6, 1499-1507, 1998.
(Summary)
An autosomal recessive murine mutation, coined "aly/aly" or "alymphoplasia," was recently reported. Homozygotes for aly are defective in both humoral and cell-mediated immune function and have diffuse lymphoid cell infiltration of various tissues, particularly around the conduit ducts of the pancreas and salivary glands. In pilot studies in our laboratories, aly/aly mice were found to have peculiar biliary tract lesions, which were analyzed histologically and immunohistochemically in the present study. The livers of aly/aly mice older than 8 weeks consistently showed a variable lymphoid cell infiltration with lymph follicle formation in portal tracts; intrahepatic biliary epithelial cells showed various types of damage including pseudopyloric gland metaplasia and proliferative changes. In addition, the extrahepatic bile duct and intrahepatic large bile duct were found to contain an acidophilic substance in their epithelial cytoplasm. In the lumen and occasionally in the cytoplasm of these bile ducts, acidophilic crystals were also seen. Ultrastructurally, the intracytoplasmic acidophilic substances consisted of membrane-bound intracytoplasmic inclusions with homogeneous electron density, likely derived from rough endoplasmic reticulum (ER). Immunohistochemically, the cytoplasmic acidophilic substances were simultaneously positive for cystatin C, gastrin, serotonin, and somatostatin. In contrast, the acidophilic crystals did not react with any of these antibodies. These findings suggest that the intracytoplasmic acidophilic substances may contain a precursor of the peptide hormones, possibly because of defective secretion or intracellular transport. We believe that the aly/aly mouse is a useful model for the analysis of biliary metabolic events, and for studies of the interaction of the immune system and biliary destruction.
Y Nakanuma, K Harada, K Kaji, S Terasaki, Koichi Tsuneyama, S Moteki, J de Water Van, S P Leung and E M Gershwin : Clinicopathological study of primary biliary cirrhosis negative for antimitochondrial antibodies., Liver, Vol.17, No.6, 281-287, 1997.
(Summary)
Primary biliary cirrhosis (PBC) is characterized by the occurrence of antimitochondrial antibodies (AMA) and the progressive destruction of intrahepatic bile ducts, followed by biliary cirrhosis. However, there are about 5% of PBC patients who show clinicopathological features of PBC but are negative for AMA. In this study, clinicopathological features, as well as antibody reactivity against recombinant (r)-mitochondrial polypeptides, were examined in 30 AMA negative PBC patients and 38 AMA positive PBC patients, in whom the presence of AMA had been determined by indirect immunofluorescence (IF). There were few differences in the clinical and serological features between both groups. Histopathologic features, including staging, bile duct lesions and granuloma, were also similar in both groups. Among the 30 IF-tested AMA negative patients, 29 were also negative against beef heart mitochondrial proteins, but 24 reacted to one or more of the following r-polypeptides, as determined by immunoblotting: E1 alpha of pyruvate dehydrogenase complex, the E2 subunit of pyruvate dehydrogenase complex, and the branched-chain 2-oxo-acid dehydrogenase complex. The remaining six AMA-negative patients were asymptomatic, and histologically resembled having stage 1 of the disease, with relatively mild lymphocytic piecemeal necrosis. One case was positive for anti-smooth muscle antibody. The other clinicopathological features of these patients were similar to those of other AMA negative patients. The present study found that a majority of the AMA-negative patients fulfilling other clinicopathological criteria of PBC, had features similar to the AMA-positive PBC patients, and that a majority of IF AMA-negative patients were positive for r-polypeptides of the 2-oxo-acid dehydrogenase complex. It seems that nearly all the AMA negative patients possess a broad spectrum of antibody profile of AMA, in addition to clinicopathological and serological features.
K Kaji, Koichi Tsuneyama, Y Nakanuma, K Harada, M Sasaki, S Kaneko and K Kobayashi : B7-2 positive cells around interlobular bile ducts in primary biliary cirrhosis and chronic hepatitis C., Journal of Gastroenterology and Hepatology, Vol.12, No.7, 507-512, 1997.
(Summary)
Bile duct damage in patients with chronic hepatitis C (hepatitis-associated bile duct lesion) as well as that in patients with primary biliary cirrhosis (PBC; chronic non-suppurative destructive cholangitis), may be causally related to immunological assaults. Efficient antigen presentation is known to require the provision of a costimulatory signal which is dependent on the CD28 on T cell surfaces, and that at least two molecules, B7-1 and B7-2, work as costimulatory ligands for CD28. In this study, we examined immunohistochemically, the expression of B7-2 in portal tracts of liver biopsy specimens obtained from 75 patients with chronic hepatitis C who had hepatitis-associated bile duct lesions, and from 63 PBC patients with chronic non-suppurative destructive cholangitis. B7-2 positive cells were recognizable as large mononuclear cells scattered in portal tracts. Some of these cells showed a dendritic cell-like appearance. B7-2 positive cells were observed more frequently (41%) in PBC liver specimens than in chronic hepatitis C specimens (17%, P < 0.05). In PBC livers, such cells were preferentially observed around the damaged bile duct with a few located in the biliary epithelial layer. There was no such finding in chronic hepatitis C livers. The frequency and density of B7-2 positive cells in the liver specimens tended to decrease according to the stage of PBC (45% in stages 1 and 2, and 33% in stages 3 and 4; P = 0.10), whereas with chronic hepatitis C, no such tendency was observed. These findings suggest that B7-2 positive cells may play a role in the bile duct lesions that appear in the early histological stages of PBC and that the immunological mechanisms of bile duct damage, particularly of antigen presentation and B7-2 expression, differ between PBC and chronic hepatitis C.
(Keyword)
Antigens, CD / B7-2 Antigen / Bile Ducts / Chronic Disease / Hepatitis C / Humans / Immunohistochemistry / In Situ Hybridization / Liver / Liver Cirrhosis, Biliary / Membrane Glycoproteins
Y Nakanuma, M Yasoshima, Koichi Tsuneyama and K Harada : Histopathology of primary biliary cirrhosis with emphasis on expression of adhesion molecules., Seminars in Liver Disease, Vol.17, No.1, 35-47, 1997.
(Summary)
In the initiation and progression of immune-mediated destruction of interlobular bile ducts and hepatocytes in primary biliary cirrhosis, T-cell-mediated responses to target antigen(s) expressed on the bile ducts and hepatocytes, as well as cellular adhesions via various adhesion molecules are critical. Intercellular adhesion molecule 1 and, to a lesser degree, vascular adhesion molecule 1 are increasingly expressed on the damaged bile ducts in primary biliary cirrhosis. In addition, lymphocyte function-associated antigens, very late antigens, endothelial-leukocyte adhesion molecule 1, and other adhesion molecules on the vascular endothelial cells and/or inflammatory cells, particularly activated lymphocytes, are also expressed in the portal tracts and hepatic parenchyma. These adhesion molecules are involved in the extravasation as well as epitheliotropic processes of inflammatory cells. Dendritic cells, particularly interdigitating ones in the periductal tissue, are positive for these immune molecules and also for the B-7 family. They may also be important in antigen presentation to CD4+ helper T cells and their activation. However, there is still controversy about whether the B-7 family is expressed on the bile ducts and, then, whether biliary epithelial cells work as an antigen presenting cell. Expression of a very late antigen family on the basolateral surface of bile ducts may be involved in the cell-cell and cell-matrix interactions. Soluble adhesion molecules may be involved in the regulation of immune-mediated bile duct lesions.
Koichi Tsuneyama, J De Water Van, K Yamazaki, K Suzuki, S Sato, Y Takeda, B Ruebner, A B Yost, Y Nakanuma, L R Coppel and E M Gershwin : Primary biliary cirrhosis an epithelitis: evidence of abnormal salivary gland immunohistochemistry., Autoimmunity, Vol.26, No.1, 23-31, 1997.
(Summary)
Primary biliary cirrhosis (PBC) is an autoimmune liver disease of unknown etiology. Nearly 93% of patients with PBC exhibit evidence of focal sialoadenitis. In an earlier study, we reported evidence of aberrant expression of PDC-E2, or a mimeotope, in the salivary glands of patients with PBC that had Sjogren's syndrome. At the time of the previous study, data was not yet available regarding patients with PBC without sicca complaints. Therefore, to investigate the extent of salivary gland involvement in PBC, we collected lip biopsy sections from 9 PBC patients diagnosed as PBC by liver biopsy, without clinical or histologic features of Sjogren's syndrome and 9 PBC patients with established Sjogren's syndrome. Using immunohistochemical staining with both a murine monoclonal antibody. C355.1, and a human combinatorial antibody, SP4, we examined the ducts of these salivary glands for the presence of the characteristic aberrant staining pattern found in patients with PBC. We report that 6/9 PBC patients fulfilling established Sjogren's syndrome criteria and 6/9 PBC patients lacking features of Sjogren's syndrome showed intense staining of the ductal epithelial cells of the salivary gland. These data suggest that the PBC-specific antigen recognized by C355.1 and SP4 in bile duct epithelial cells is expressed aberrantly in the salivary gland in 66% of patients with PBC, independent of Sjogren's syndrome. This finding suggests a common disease process in these two tissues. Further, expression of this molecule may be an early marker of salivary gland involvement in patients with PBC.
Y Takeoka, Y S Chen, L R Boyd, Koichi Tsuneyama, N Taguchi, S Morita, H Yago, S Suehiro, A A Ansari, D L Shultz and E M Gershwin : A comparative analysis of the murine thymic microenvironment in normal, autoimmune, and immunodeficiency states., Developmental Immunology, Vol.5, No.2, 79-89, 1997.
(Summary)
It is widely accepted that the thymic microenvironment regulates normal thymopoiesis through a highly coordinated and complex series of cellular and cytokine interactions. A direct corollary of this is that abnormalities within the microenvironment could be of etiologic significance in T-cell-based diseases. Our laboratory has developed a large panel of monoclonal antibodies (mAbs) that react specifically with epithelial or nonepithelial markers in the thymus. We have taken advantage of these reagents to characterize the thymic microenvironment of several genetic strains of mice, including BALB/cJ, C57BL/6J, NZB/BlnJ, SM/J, NOD/Ltz, NOD/Ltz-scid/sz, C57BL/6J-Hcphme/Hcphme, and ALY/NscJcl-aly/aly mice, and littermate control animals. We report herein that control mice, including strains of several backgrounds, have a very consistent phenotypic profile with this panel of monoclonal antibodies, including reactivity with thymic epithelial cells in the cortex, the medulla and the corticomedullary junction, and the extracellular matrix. In contrast, the disease-prone strains studied have unique, abnormal staining of thymic cortex and medulla at both the structural and cellular levels. These phenotypic data suggest that abnormalities in interactions between developing thymocytes and stromal cells characterize disease-prone mice.
S Cha, S P Leung, J de Water Van, Koichi Tsuneyama, E R Joplin, A A Ansari, Y Nakanuma, J P Schatz, S Cwirla, E L Fabris, M J Neuberger, E M Gershwin and L R Coppel : Random phage mimotopes recognized by monoclonal antibodies against the pyruvate dehydrogenase complex-E2 (PDC-E2)., Proceedings of the National Academy of Sciences of the United States of America, Vol.93, No.20, 10949-10954, 1996.
(Summary)
Dihydrolipoamide acetyltransferase, the E2 component of the pyruvate dehydrogenase complex (PDC-E2), is the autoantigen most commonly recognized by autoantibodies in primary biliary cirrhosis (PBC). We identified a peptide mimotope(s) of PDC-E2 by screening a phage-epitope library expressing random dodecapeptides in the pIII coat protein of fd phage using C355.1, a murine monoclonal antibody (mAb) that recognizes a conformation-dependent epitope in the inner lipoyl domain of PDC-E2 and uniquely stains the apical region of bile duct epithelium (BDE) only in patients with PBC. Eight different sequences were identified in 36 phage clones. WMSYPDRTLRTS was present in 29 clones; WESYPFRVGTSL, APKTYVSVSGMV, LTYVSLQGRQGH, LDYVPLKHRHRH, AALWGVKVRHVS, KVLNRIMAGVRH and GNVALVSSRVNA were singly represented. Three common amino acid motifs (W-SYP, TYVS, and VRH) were shared among all peptide sequences. Competitive inhibition of the immunohistochemical staining of PBC BDE was performed by incubating the peptides WMSYPDRTLRTS, WESYPDRTLRTS, APKTYVSVSGMV, and AALWGVKVRHVS with either C355.1 or a second PDC-E2-specific mAb, C150.1. Both mAbs were originally generated to PDC-E2 but map to distinct regions of PDC-E2. Two of the peptides, although selected by reaction with C355.1, strongly inhibited the staining of BDE by C150.1, whereas the peptide APKTYVSVSGMV consistently inhibited the staining of C355.1 on biliary duct epithelium more strongly than the typical mitochondrial staining of hepatocytes. Rabbit sera raised against the peptide WMSYPDRTLRTS stained BDE of livers and isolated bile duct epithelial cells of PBC patients more intensively than controls. The rabbit sera stained all size ducts in normals, but only small/medium-sized ductules in PBC livers. These studies provide evidence that the antigen present in BDE is a molecular mimic of PDC-E2, and not PDC-E2 itself.
M Hoso, Y Nakanuma, M Kawano, K Oda, Koichi Tsuneyama, J de Water van and E M Gershwin : Granulomatous cholangitis in chronic hepatitis C: a new diagnostic problem in liver pathology., Pathology International, Vol.46, No.4, 301-305, 1996.
(Summary)
A case of chronic hepatitis C at the pre-cirrhotic stage complicated with hepatocellular carcinoma is reported. The patient, a 64 year old female, showed elevated levels of serum alkaline phosphatase and immunoglobulin M. Antimitochondrial antibodies were negative by indirect immunofluorescence. Western blotting using beef heart mitochondria and recombinant polypeptides coding for mitochondrial antigens revealed that the patient's serum was positive only for the E2-subunit of the branched chain ketoacid dehydrogenase complex. In the non-neoplastic liver, chronic non-suppurative cholangitis surrounded by epithelioid granuloma, resembling the granulomatous destructive cholangitis of primary biliary cirrhosis, was found. The damaged bile ducts were immunohistochemically minimally positive or ambiguous for HLA-DR, and their expression of the E2-subunit of the pyruvate dehydrogenase complex E2 (PDC-E2) was diffuse or granular, and not typical of primary biliary cirrhosis. There was no bile duct loss, and orcein-positive copper binding granules reflecting chronic cholestasis were negative in periportal hepatocytes. The overall features in this case were consistent with primary biliary cirrhosis presenting an infrequent profile of antimitochondrial antibodies and atypical expression of HLA-DR and PDC-E2 on biliary epithelial cells, with late superimposition on chronic hepatitis C. However, it is also possible that this is a case of chronic hepatitis C with hepatitis-associated bile duct damage accompanied by granulomatous reaction. Either way, this case raises new diagnostic issues in the differential diagnosis of chronic liver diseases presented with granulomatous cholangitis.
Koichi Tsuneyama, J De Water Van, D Thiel Van, R Coppel, B Ruebner, Y Nakanuma, R E Dickson and E M Gershwin : Abnormal expression of PDC-E2 on the apical surface of biliary epithelial cells in patients with antimitochondrial antibody-negative primary biliary cirrhosis., Hepatology, Vol.22, No.5, 1440-1446, 1995.
(Summary)
The presence of antimitochondrial antibodies (AMA) is a major criterion for the diagnosis of primary biliary cirrhosis (PBC). Although it is not clear that AMA are involved in the pathogenesis of the disease, the study of these autoantibodies has enabled much information to be accumulated about the specificity of this response. The autoantigens have been identified as components of a functionally related enzyme family, the 2-oxo-acid-dehydrogenase complex. Within this complex, pyruvate dehydrogenase E2 subunit (PDC-E2) has been determined to be the immunodominant autoantigen. Using a panel of mouse monoclonal antibodies and human combinatorial autoantibodies, it has been demonstrated that patients with PBC, but not controls, have an abnormal expression of either PDC-E2 or a cross-reacting molecule in the apical region of biliary epithelium. Others have shown a similar reaction using rabbit sera directed to PDC-E2. Our previous studies have concentrated on AMA-positive patients. In this study, the presence of PDC-E2, class II, immunoglobulin (Ig) A, and B7/BB1 in the bile duct epithelial cells of AMA-positive as well as AMA-negative patients is addressed. Most patients with AMA-negative PBC (seven of nine) react in a fashion similar to AMA-positive patients with intense staining of the apical region of the bile duct epithelial cells of "PDC-E2," increased IgA expression, and little major histocompatibility complex (MHC) class II staining in the early-stage patients. Interestingly, the two AMA-negative patients that did not express PDC-E2 on the apical side of their biliary epithelium had anticentromere antibodies and Sjögren's syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)
(Keyword)
Adult / Aged / Aged, 80 and over / Animals / Autoantibodies / Autoantigens / Bile Ducts, Intrahepatic / Dihydrolipoyllysine-Residue Acetyltransferase / Epithelium / Female / Histocompatibility Antigens Class II / Humans / Liver Cirrhosis, Biliary / Male / Mice / Microscopy, Confocal / Middle Aged / Mitochondria / Pyruvate Dehydrogenase Complex / Rabbits / Rheology
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 7590661
Y Nakanuma, Koichi Tsuneyama, E M Gershwin and M Yasoshima : Pathology and immunopathology of primary biliary cirrhosis with emphasis on bile duct lesions: recent progress., Seminars in Liver Disease, Vol.15, No.4, 313-328, 1995.
(Keyword)
Autoantigens / Bile Ducts / Humans / Liver Cirrhosis, Biliary
Koichi Tsuneyama, J de Water Van, S P Leung, S Cha, Y Nakanuma, M Kaplan, R Lellis De, R Coppel, A Ansari and E M Gershwin : Abnormal expression of the E2 component of the pyruvate dehydrogenase complex on the luminal surface of biliary epithelium occurs before major histocompatibility complex class II and BB1/B7 expression., Hepatology, Vol.21, No.4, 1031-1037, 1995.
(Summary)
Primary biliary cirrhosis (PBC) is a chronic autoimmune liver disease characterized histologically by nonsuppurative destructive cholangitis. Sera from patients with PBC react with a series of intramitochondrial enzymes with the immunodominant response directed against the E2 component of the pyruvate dehydrogenase complex (PDC-E2). Recently, using tissue sections of late-stage PBC, we showed that there is increased expression in biliary epithelial cells of patients with PDC-E2 or a molecule cross-reactive with PDC-E2. Previous work has shown that biliary epithelial cells of patients with PBC express an increased amount of class II. To address the sequence of events in the evolution of PBC, we have focused our attention in this study on early biliary epithelial lesions. In particular, we have studied the liver of 22 female patients with PBC that was diagnosed as either stage I or stage II using both a mouse monoclonal antibody that has reactivity similar to human autoantibodies as well as a human Fab combinatorial prepared from the lymph node of a PBC patient. Tissues were simultaneously stained using antibodies to PDC-E2, class II, and BB1/B7. As a positive control, tissues from late-stage PBC were studied concurrently. By determining the order of expression among the three molecules, PDC-E2, class II, and BB1/B7, we report that the expression of PDC-E2 or a PDC-E2-like molecule on biliary duct epithelium of patients with PBC precedes the expression of BB1/B7 and major histocompatibility complex (MHC) class II molecules. The alteration of an autoantigen in biliary duct epithelium may be the earliest lesion in PBC.
M Yasoshima, Y Nakanuma, Koichi Tsuneyama, J de Water Van and E M Gershwin : Immunohistochemical analysis of adhesion molecules in the micro-environment of portal tracts in relation to aberrant expression of PDC-E2 and HLA-DR on the bile ducts in primary biliary cirrhosis., The Journal of Pathology, Vol.175, No.3, 319-325, 1995.
(Summary)
We have examined immunohistochemically the expression of adhesion molecules in the micro-environment of portal tracts and their relationship to the expression of the pyruvate dehydrogenase E2 complex (PDC-E2) and HLA-DR in liver biopsy specimens. Ten cases of primary biliary cirrhosis (PBC) and 19 controls were examined, including four cases of extrahepatic biliary obstruction, six of chronic viral hepatitis, and nine normal livers. In PBC, the damaged small bile ducts demonstrated an increased expression of PDC-E2 and an aberrant expression of HLA-DR; about half of these damaged bile ducts also expressed intercellular adhesion molecules (ICAM)-1 and a few expressed vascular adhesion molecule (VCAM)-1. In addition, lymphocyte function-associated antigen (LFA)-1 and very late antigen (VLA)-4 were expressed on infiltrating lymphocytes around these bile ducts. In contrast, in control livers, these alterations in antigen expression on the bile ducts were either not observed or were only focal and weak, when present. These findings suggest that ICAM-1/LFA-1 and also VCAM-1/VLA-4 linkages between the damaged bile ducts and lymphocytes may facilitate antigen-specific reactions such as the presentation of antigens, possibly PDC-E2, to the periductal lymphocytes in PBC. ICAM-1, VCAM-1, and E-selectin were strongly expressed on the endothelial cells of some vessels in the portal tracts in PBC, suggesting the facilitation of the recruitment of lymphocytes around the bile ducts of PBC. VCAM-1, a member of the immunoglobulin superfamily, has not hitherto been reported on bile ducts.
Y Nakanuma, Koichi Tsuneyama, N Kono, M Hoso, J de Water Van and E M Gershwin : Biliary epithelial expression of pyruvate dehydrogenase complex in primary biliary cirrhosis: an immunohistochemical and immunoelectron microscopic study., Human Pathology, Vol.26, No.1, 92-98, 1995.
(Summary)
It has been reported recently that there is a unique distribution of the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2) on biliary epithelial cells in patients with primary biliary cirrhosis (PBC) but not primary sclerosing cholangitis. This distribution has been demonstrated using a mouse monoclonal antibody, coined C355.1. The epitope recognized by C355.1 is near the lipoic acid binding site of PDC-E2. C355.1 inhibits PDC-E2 activity in vitro and, unlike a panel of other monoclonal antibodies against different regions of PDC-E2, appears to bind not only to mitochondria but also to a unique antigen expressed predominantly on the luminal side of biliary epithelial cells in PBC. We have extended these observations by studying the subcellular reactivity of C355.1 using postembedding immunoelectron microscopy on the intrahepatic small bile ducts of PBC livers, extrahepatic biliary obstruction (EBO) livers, and normal livers. We report that the reactivity of C355.1 can be classified into two categories. The first category is characterized by small foci of reaction products that were randomly dispersed in cytoplasm, particularly in supranuclear areas; the ultrastructural characterization of these foci was impossible to define but was similar in PBC and EBO. However, of particular interest was the second category of reactivity, which was characterized by deposition of reaction products around the biliary lumen, including microvilli and adjacent subluminal ectoplasm and secretory substances in the biliary lumen. This staining pattern was frequent in PBC livers, only occasionally evident in EBO livers, and not found in normal livers. These data further define and highlight the unique subcellular distribution of PDC-E2 around the biliary lumen in PBC livers and suggest that this abnormality is related to the pathogenesis of bile duct lesions.
K Kaji, Y Nakanuma, M Sasaki, M Unoura, K Kobayashi, A Nonomura, Koichi Tsuneyama, J de Water Van and E M Gershwin : Hepatitic bile duct injuries in chronic hepatitis C: histopathologic and immunohistochemical studies., Modern Pathology, Vol.7, No.9, 937-945, 1994.
(Summary)
Interlobular bile ducts are reported to be damaged in viral hepatitis. Such damages are called hepatitic duct injuries and mimic chronic nonsuppurative destructive cholangitis of primary biliary cirrhosis. In this study, hepatitic bile duct injuries were evaluated histopathologically and immunohistochemically in 149 needle liver biopsy specimens from patients with chronic hepatitis C and compared immunohistochemically with primary biliary cirrhosis. Fifty-one of the needle biopsies from patients with chronic hepatitis C (34.2%) showed hepatitic bile duct injuries which were distributed focally in the liver and showed variable epithelial damages such as cytoplasmic swelling, vacuolation and acidophilia, nuclear pleomorphism, and loss of nuclear polarity. Some of the injured bile ducts were embedded within lymphoid aggregates, whereas others were surrounded by lymphocytes as well as other inflammatory cells to varied degrees. The majority of lymphoid cells around hepatitic bile duct injury were B- and T-cells mixed in various proportions, and activated T-cells were occasionally found within the biliary epithelial layer. Histopathologic and serial section studies disclosed that bile duct loss was rare in chronic hepatitis C. Statistical analysis revealed that advancement of chronic hepatitis and the degree of necroinflammatory processes of the liver, particularly in the portal tracts, were positively correlated with the occurrence of hepatitic bile duct injuries. Immunohistochemical studies demonstrated that, whereas strong ectopic expression of HLA-DR and enhanced expression of HLA-A,B,C, and pyruvate dehydrogenase E2-complex in biliary epithelial cells were frequently observed in chronic nonsuppurative destructive cholangitis of primary biliary cirrhosis, such unusual expressions were generally absent or mild, even if present, in bile duct injuries in chronic hepatitis C.(ABSTRACT TRUNCATED AT 250 WORDS)
Koichi Tsuneyama, J de Water Van, Y Nakanuma, S Cha, A Ansari, R Coppel and E M Gershwin : Human combinatorial autoantibodies and mouse monoclonal antibodies to PDC-E2 produce abnormal apical staining of salivary glands in patients with coexistent primary biliary cirrhosis and Sjögren's syndrome., Hepatology, Vol.20, No.4 Pt 1, 893-898, 1994.
(Summary)
An increase in the incidence of Sjögren's syndrome in patients with primary biliary cirrhosis has been noted. Indeed, primary biliary cirrhosis has been described as a ductal disease with involvement not only of the biliary tract but of epithelial ductal cells in other organs. We have previously reported the development of a panel of mouse monoclonal antibodies directed at PDC-E2, the major autoantigen of primary biliary cirrhosis. One such antibody, C355.1, but none of the other monoclonal antibodies, reacted not only with mitochondria but also with the apical region of biliary epithelium of patients with primary biliary cirrhosis but not in similar specimens from patients with other liver disease or normal human liver. In addition, we have reported the development of human combinatorial antibodies specific for PDC-E2; these reagents also reacted uniquely with the biliary epithelium of patients with primary biliary cirrhosis. In this paper, we have performed a similar study and have compared the staining of monoclonal antibody C355.1 and a human combinatorial antibody, SP4, with control monoclonal antibodies with respect to their reactivity of salivary glands in 9 patients with primary biliary cirrhosis associated with Sjögren's syndrome, 11 patients with Sjögren's syndrome alone and 7 control patients. Interestingly, the apical region of the salivary gland epithelial cells of approximately 50% of patients with coexisting primary biliary cirrhosis and Sjögren's syndrome had a staining pattern similar to that seen in primary biliary cirrhosis biliary epithelium. In contrast, we did not observe this reactivity in any patient with Sjögren's syndrome alone or in any control patient.(ABSTRACT TRUNCATED AT 250 WORDS)
Y Nakanuma, Koichi Tsuneyama, S Masuda and T Tomioka : Hepatic inflammatory pseudotumor associated with chronic cholangitis: report of three cases., Human Pathology, Vol.25, No.1, 86-91, 1994.
(Summary)
We present three cases of hepatic inflammatory pseudotumor associated with chronic cholangitis. In these three cases the inflammatory pseudotumor was solitary, was present in the hepatic hilar region, and had been misdiagnosed as cholangiocarcinoma by imaging modalities and/or gross pathologic examination. Hilar bile ducts were embedded within the tumor. Histologically, these "tumors" consisted of an 2ad-mixed proliferation of lymphocytes, plasma cells, and fibroblasts with variable hyaline fibrosis. The phenotypes of lymphocytes and plasma cells within the tumor were immunohistochemically heterogeneous. Bile ducts and peribiliary glands embedded within these tumors showed nonspecific fibrosis and inflammation, and inflammatory changes of the bile ducts were imperceptibly merged with those of the inflammatory tumor, raising the possibility that these tumors may have arisen in relation to cholangitis. The bile ducts adjacent to the tumor also showed similar but milder nonspecific inflammatory changes. The pathogenesis of cholangitis was unknown in two cases and was speculated to be due to fungal infection in the remaining case. These cases may indicate that biliary tract disease(s) should be explored as the etiology of hepatic inflammatory pseudotumor.
K Kizawa, Koichi Tsuneyama, T Terada, Y Nakanuma, U Miwa and H Kurumaya : [An autopsied case of primary hepatic malignant lymphoma associated with chronic active hepatitis C]., The Japanese Journal of Gastro-enterology, Vol.90, No.9, 2147-2151, 1993.
Katsuki Miyazaki, Yuji Morine, Satoru Imura, Tetsuya Ikemoto, Yu Saitou, Shin-ichiro Yamada, Kazunori Tokuda, Shohei Okikawa, Shoko Yamashita, Takeshi Oya, Koichi Tsuneyama and Mitsuo Shimada : Preoperative lymphocyte/C-reactive protein ratio and its correlation with CD8 tumor-infiltrating lymphocytes as a predictor of prognosis after resection of intrahepatic cholangiocarcinoma., Surgery Today, Vol.51, No.12, 1985-1995, 2021.
(Summary)
To clarify whether the preoperative lymphocyte/C-reactive protein (CRP) ratio (LCR) is a prognostic factor for patients with intrahepatic cholangiocarcinoma (IHCC), and investigate its mechanism via tumor-infiltrating lymphocytes. The subjects of this retrospective study were 42 patients who had undergone hepatectomy for IHCC. We divided the patients into low LCR and high LCR groups (cutoff value: 8780) and analyzed their overall survival (OS) and disease-free survival (DFS) with respect to LCR and other clinicopathological factors. We also investigated the levels of stromal tumor-infiltrating lymphocytes (TILs) and CD8 TILs in surgical specimens, and the relationship between LCR and TILs. A low LCR was identified in 21 patients and was significantly correlated with older age, a high CRP-albumin ratio, and advanced disease stage, and was a prognostic factor for OS and DFS. Multivariate analysis revealed that a low LCR was an independent prognostic factor for worse OS (HR 10.40, P = 0.0077). Although the LCR and levels of stromal TILs were not significantly related, LCR and levels of CD8 TILs were significantly related (P = 0.0297). The preoperative LCR may predict the postsurgical prognosis of patients with IHCC and reflect the CD8 TILs.
(Keyword)
Bile Duct Neoplasms / Biomarkers, Tumor / C-Reactive Protein / CD8-Positive T-Lymphocytes / Cholangiocarcinoma / Female / Humans / Lymphocyte Count / Lymphocytes, Tumor-Infiltrating / Male / Predictive Value of Tests / Preoperative Period / Prognosis
Shoko Iwahashi, Tetsuya Ikemoto, Yuji Morine, Satoru Imura, Shuichi Iwahashi, Yu Saitou, Shin-ichiro Yamada, Toshiaki Yoshimoto, Koichi Tsuneyama and Mitsuo Shimada : Two cases of non-mucinous cystadenomas of the pancreas with pancreatobiliary phenotype and ovarian-like stroma., Surgical Case Reports, Vol.5, No.1, 117, 2019.
(Summary)
Diagnosis of cystic tumor of the pancreas is based on the World Health Organization criteria that classify pancreatic cystadenomas into four types: intra-ductal papillary mucinous neoplasms, mucinous cystic neoplasms (MCNs), serous cystic neoplasms, and solid pseudo-papillary neoplasms depending on their secretion and presence of ovarian-like stroma. Recently, Albores-Saavedra identified non-mucinous cystadenomas of the pancreas with pancreato-biliary phenotype and ovarian-like stroma. This precipitated examination of the proportions of these rare tumors in patients treated at Tokushima University Hospital. Case 1 was a 40-year-old woman with a cystic tumor in the tail of the pancreas. Computed tomography (CT) revealed a diffuse and non-enhanced cystic tumor in the tail of the pancreas. This tumor was diagnosed as a simple cyst at this point. However, 2ears later, the tumor had increased in size by 3m. Thus, laparoscopic distal pancreatectomy was performed. The content of the cyst was serous. The epithelial cells were lined with a single layer of cuboidal cells and the tumor had ovarian-like stroma pathologically. The final pathological diagnosis was non-mucinous cystadenoma of the pancreas with ovarian-like stroma. In Case 2, a cystic tumor in the pancreas was found by medical examination in a woman in her sixties who presented without symptoms. CT showed a 1.5-cm cystic tumor in the tail and body of the pancreas and a septum in the cyst. Nine years later, the tumor had grown to 2.4m in diameter and had a clear septum in the cyst. This tumor was diagnosed preoperatively as MCN. Thus, laparoscopic distal pancreatectomy was performed. The cyst contained serous fluid. Microscopic examination showed no ovarian-like stroma and the epithelial cells were lined by a single layer of cuboidal cells. The final pathological diagnosis was non-mucinous cystadenoma of the pancreas with ovarian-like stroma. Accurate preoperative diagnosis of this type of pancreatic cystic tumor may be difficult, although it occurs more often than expected. Non-mucinous cystadenomas of the pancreas with ovarian-like stroma need to be considered as a differential diagnosis.
Azumi IIDA, azusa YANO, Koichi Tsuneyama, Tatsuki FUKAMI, Miki NAKAJIMA and Tsuyoshi YOKOI : Involvement of metabolic activation of carbamazepine in liver injury in rat, Annual Meeting of the Japanese Society of Toxicology, Vol.40, No.0, 1003038, 2013.
Mayuko Shimizu, Maiko Hatanaka, Koichi Tsuneyama, Shigeko Kato and Katsuhisa Omagari : An SHR/NDmcr-cp rat model of non-alcoholic steatohepatitis with advanced fibrosis induced by a high-fat, high-cholesterol diet., J Obes Wt Loss Ther, Vol.5, 1, 2015.
(Keyword)
SHR/NDmcr-cp rat / cholesterol
2.
Tamehito Onoe, Tadashi Konoshita, Koichi Tsuneyama, Ryoko Hamano, Ichiro Mizushima, Yasushi Kakuchi, Kazunori Yamada, Kenshi Hayashi, Masahiro Kuroda, Satoshi Kagitani, Hideki Nomura, Masakazu Yamagishi and Mitsuhiro Kawano : Situs inversus and cystic kidney disease: Two adult patients with this Heterogeneous syndrome., The American Journal of Case Reports, Vol.14, 20-25, 2013.
(Summary)
Situs inversus is a rare complication of cystic kidney diseases. Only three genes, INVS (NPHP2), NPHP3 and PKD2 have been proved to be responsible for some cases, while the responsible genes in many others are still unknown. Here we report two male patients with situs inversus combined with cystic kidney disease without any family history of polycystic kidney disease. Their renal function was normal in childhood but culminated in end stage renal disease in middle age. No pathogenic mutations were found in mutation analysis of INVS, IFT88, PKD2, UMOD or NPHP3 in them. Past reported cases of situs inversus and cystic kidney diseases were divided into three groups, i.e., gestational lethal renal dysplasia group, infantile or juvenile nephronophthisis group and polycystic kidney disease group. The present patients are different from each of these groups. Moreover, the renal lesions of the present two cases are quite different from each other, with one showing mildly atrophic kidneys with small numbers of cysts and the other an enlarged polycystic kidney disease, suggesting very heterogeneous entities.
Yuko Nakanishi, Koichi Tsuneyama, Makoto Fujimoto, L Thucydides Salunga, Kazuhiro Nomoto, Jun-Ling An, Yasuo Takano, Seiichi Iizuka, Mitsunobu Nagata, Wataru Suzuki, Tsutomu Shimada, Masaki Aburada, Masayuki Nakano, Carlo Selmi and Eric M Gershwin : Monosodium glutamate (MSG): a villain and promoter of liver inflammation and dysplasia., Journal of Autoimmunity, Vol.30, No.1-2, 42-50, 2008.
(Summary)
Chronic inflammation is a common theme in a variety of disease pathways, including autoimmune diseases. The pathways of chronic inflammation are well illustrated by nonalcoholic steatohepatitis (NASH), which is of a serious concern due to its increasing prevalence in the westernized world and its direct correlation with lifestyle factors, particularly diet. Importantly, NASH may ultimately lead to the development of hepatocellular carcinoma. We previously reported that injection of monosodium glutamate (MSG) in ICR mice leads to the development of significant inflammation, central obesity, and type 2 diabetes. To directly address the long-term consequences of MSG on inflammation, we have performed serial analysis of MSG-injected mice and focused in particular on liver pathology. By 6 and 12 months of age, all MSG-treated mice developed NAFLD and NASH-like histology, respectively. In particular, the murine steatohepatitis at 12 months was virtually undistinguishable from human NASH. Further, dysplastic nodular lesions were detected in some cases within the fibrotic liver parenchyma. We submit that MSG treatment of mice induces obesity and diabetes with steatosis and steatohepatitis resembling human NAFLD and NASH with pre-neoplastic lesions. These results take on considerable significance in light of the widespread usage of dietary MSG and we suggest that MSG should have its safety profile re-examined and be potentially withdrawn from the food chain.
(Keyword)
Animals / Chemical and Drug Induced Liver Injury / Diabetes Mellitus, Type 2 / Fatty Liver / Humans / Liver / Liver Cirrhosis / Mice / Obesity / Sodium Glutamate
Ya-Hui Chuang, Zhe-Xiong Lian, Chun-Mei Cheng, Y Ruth Lan, Guo-Xiang Yang, Yuki Moritoki, Bor-Luen Chiang, A Aftab Ansari, Koichi Tsuneyama, L Ross Coppel and Eric M Gershwin : Increased levels of chemokine receptor CXCR3 and chemokines IP-10 and MIG in patients with primary biliary cirrhosis and their first degree relatives., Journal of Autoimmunity, Vol.25, No.2, 126-132, 2005.
(Summary)
Infiltrating memory T cells play an important role in the destruction of the biliary tract in primary biliary cirrhosis (PBC) and inflammatory chemokines control lymphocyte traffic through their interactions with T cell chemokine receptors. In the present study, we measured plasma levels of chemokines interferon-gamma-inducible protein-10 (IP-10) and monokine induced by gamma interferon (MIG), and also studied the expression of CXCR3 chemokine receptors in 105 subjects, including 53 patients with PBC, 26 first degree relatives and 26 healthy controls. Interestingly, plasma IP-10 and MIG levels in PBC were increased significantly compared to controls and appeared to increase with disease progression. By immunohistochemistry, IP-10 and MIG expressions were evident in the portal areas in PBC. Further, the frequency of CXCR3-expressing cells in peripheral blood was also significantly higher in PBC, and CXCR3-positive cells were also found in the portal areas of diseased livers, primarily on CD4+ cells. Finally, the daughters and sisters of PBC patients also demonstrated increased plasma levels of IP-10 and MIG, but, in contrast, displayed normal frequency of CXCR3+ expressing peripheral blood lymphocytes. Our data imply a role for specific chemokine-chemokine receptor interactions in the pathogenesis of PBC and also highlight the familial risk factor.
K Harada, S Ozaki, N Kono, Koichi Tsuneyama, K Katayanagi, K Hiramatsu and Y Nakanuma : Frequent molecular identification of Campylobacter but not Helicobacter genus in bile and biliary epithelium in hepatolithiasis., The Journal of Pathology, Vol.193, No.2, 218-223, 2001.
(Summary)
Bacterial infection of the biliary tree and bile stasis may be causally related to hepatolithiasis, but which bacterial species are involved and their roles in the pathogenesis of hepatolithiasis have not been ascertained. Recently, the Helicobacter genus was detected in human bile and biliary mucosal samples by molecular techniques, and its association with several biliary diseases has been suggested. The Campylobacter genus, which is closely related to the Helicobacter genus, has also recently been identified as causative of human gastrointestinal diseases. This study attempted to elucidate whether Helicobacter and/or Campylobacter bacteria are present in bile samples and biliary mucosal specimens from hepatolithiasis patients and whether they are involved in the pathogenesis of hepatolithiasis. The 16S rRNA gene of the Helicobacter and of the Campylobacter genus was examined by polymerase chain reaction in DNA samples extracted from bile and/or microdissected biliary epithelium from 69 patients with hepatolithiasis and control patients with choledocholithiasis, cholecystolithiasis, and normal gall bladders. The Helicobacter genus was detected in 1 of 8 (13%) biliary epithelial samples in hepatolithiasis and 1 of 10 (10%) bile samples in choledocholithiasis. The Campylobacter genus was detected in 3 of 14 (21%) bile samples and 5 of 8 (63%) epithelial samples in hepatolithiasis, and in 2 of 15 (13%) bile samples and 1 of 8 (13%) epithelial samples in cholecystolithiasis. The detection rate for Campylobacter in biliary epithelium of hepatolithiasis was significantly higher than in the bile or biliary epithelium of control groups (p<0.05). By a phylogenetic analysis based on nucleotide sequences, the Campylobacter genuses detected in hepatolithiasis were clustered with C. rectus or C. showae. The frequent detection of the Campylobacter 16S rRNA gene in bile, and especially in biliary epithelium of hepatolithiasis, suggests a pathogenetic relationship with Campylobacter infection.
(Keyword)
Aged / Bile / Campylobacter / Case-Control Studies / Chi-Square Distribution / Cholelithiasis / Electrophoresis, Agar Gel / Female / Helicobacter / Humans / Male / Middle Aged / Polymerase Chain Reaction / RNA, Bacterial
M Iwata, K Harada, K Hiramatsu, Koichi Tsuneyama, S Kaneko, K Kobayashi and Y Nakanuma : Fas ligand expressing mononuclear cells around intrahepatic bile ducts co-express CD68 in primary biliary cirrhosis., Liver, Vol.20, No.2, 129-135, 2000.
(Summary)
The results suggest that in PBC, FasL expressing CD68+ monocytes are at least partly involved in apoptotic bile duct loss mediated by the Fas system, and a surface molecule, CD14, participates in this process.
Mayuko Shimizu, Khuleshwari Kurrey, Misaki Miyata, Takuya Dezawa, Koichi Tsuneyama and Masami Kojima : Emerging Insights into the Role of BDNF on Health and Disease in Periphery., Biomolecules, Vol.14, No.4, Apr. 2024.
(Summary)
Brain-derived neurotrophic factor (BDNF) is a growth factor that promotes the survival and growth of developing neurons. It also enhances circuit formation to synaptic transmission for mature neurons in the brain. However, reduced BDNF expression and single nucleotide polymorphisms (SNP) are reported to be associated with functional deficit and disease development in the brain, suggesting that BDNF is a crucial molecule for brain health. Interestingly, BDNF is also expressed in the hypothalamus in appetite and energy metabolism. Previous reports demonstrated that BDNF knockout mice exhibited overeating and obesity phenotypes remarkably. Therefore, we could raise a hypothesis that the loss of function of BDNF may be associated with metabolic syndrome and peripheral diseases. In this review, we describe our recent finding that BDNF knockout mice develop metabolic dysfunction-associated steatohepatitis and recent reports demonstrating the role of one of the BDNF receptors, TrkB-T1, in some peripheral organ functions and diseases, and would provide an insight into the role of BDNF beyond the brain.
Koichi Tsuneyama, Yuki Morimoto, Shoma Kaji and Takeshi Oya : Progress of Artificial Intelligence and Molecular Pathology, Shikoku Acta Medica, Vol.76, No.1,2, 23-28, Apr. 2020.
(Summary)
A pathological diagnosis that incorporates a molecular biology technique into a conventional pathological diagnosis based on morphology is called a molecular pathological diagnosis. Immunostaining, which detects proteins derived from oncogenes and tumor suppressor genes with specific antibodies, and FISH, which uses a fluorescently labeled nucleic acid probe to examine the amplification of a target gene, are already in clinical practice. In recent years, the application of genome analysis using a next-generation sequencer to pathological diagnosis has become available, and cancer genomic medicine has been rapidly expanding and evolving. In addition, new technologies such as liquid biopsy technology that detects proteins, DNA, microRNA, methylated DNA, etc. from samples that are less invasive at the time of collection, such as blood, are being incorporated into pathological diagnosis. The application of artificial intelligence(AI)is being studied around the world to reflect the diagnosis. This article introduces the application of rapid mass spectrometry(PESI-MS)to liquid biopsy and the development of diagnostic aids for urine cytology using AI.
Koichi Tsuneyama : New animal models for the translational study of metabolic syndrome-associated liver diseases, Shikoku Acta Medica, Vol.71, No.5,6, 113-120, Dec. 2015.
(Summary)
Metabolic syndrome (MS) is a worldwide healthcare issue and a dominant risk factor for the development of incurable diseases that affect the entire body. The hepatic manifestations of MS include nonalcoholic fatty liver disease (NAFLD) and its progressive variant nonalcoholic steatohepatitis (NASH). NASH is known to extend into liver cirrhosis and hepatocellular carcinoma (HCC). To determine the pathogenesis and effective treatment, an excellent animal model of NASH/HCC is required. We recently succeeded to develop two MS associated NASH mice model (TSOD mice and DIAR-MSG mice). Their clinical course and pathological characters were quite similar to those of human MS-NASH patients. Interestingly, most of them developed HCC in aged,which pathological and functional characters were identical to those of human HCC. In addition, we established a novel mouse model of HCC based on type 1 diabetes (DIAR-nSTZ mice) and reported its histopathological features. To compare these mice models from various aspects, we can highlight specific and useful characters of MS associated liver diseases including hepatocarcinogenesis.
Naoya Okubo, Eiji Hase, Kazuki Yasumaru, Yu Tokizane, M. Ichimura-Shimizu, Koichi Tsuneyama, Takeo Minamikawa and Takeshi Yasui : Quantitative evaluation of lipid mechanical properties in metabolic dysfunction-associated steatotic liver disease by use of Brillouin scattering microscopy, OPIC2024: BISC2024, BISCp-16, Apr. 2024.
2.
Noriaki Minakawa, Noriko Saito-Tarashima, Takaaki Koma, NAOTO Hinotani, YOSHIDA Keigo, OGASA Moka, AKIHO Murai, INOUE Shuya, Tomoyuki Kondo, Naoya Doi, Koichi Tsuneyama and Masako Nomaguchi : 3-Deazaguanosine exhibits anti-SARS-CoV-2 activity and blocks the development of COVID-19 pneumonis in hamsters., Supra FIBER International Summit for Nucleic Acids (S-FISNA) 2024, Mar. 2024.
3.
Koichi Tsuneyama, Takumi Kakimoto and Mayuko Shimizu : Connexin 32 expression and alpha-synuclein accumulation in ballooning cells of non-alcoholic steatohepatitis (NASH) and other inflammatory liver diseases, AASLD 2024, Nov. 2023.
4.
Koichi Tsuneyama : New imaging techniques useful for pathological diagnosis, 90 YEARS OF PATHOLOGY -ACHIEVEMENTS IN SCIENCE AND TECHNOLOGY INTERNATIONAL SCIENTIFIC WEBINAR CONFERENCE, Jun. 2023.
5.
Mayuko Shimizu, M Kojima, M Miyata, S Suzuki and Koichi Tsuneyama : Brain-derived neurotrophic factor knock-out mice develop nonalcoholic steatohepatitis, EASL, Jun. 2023.
Kazuki Yasumaru, Mayuko Shimizu, Eiji Hase, Takeshi Yasui, Koichi Tsuneyama and Takeo Minamikawa : Raman and SHG spectroscopic analyses of lipid droplets accumulated in the liver of a mouse model of nonalcoholic fatty liver disease, pLED International symposium 2023: Exploring Invisible Light Technology, P-11, Tokushima, Mar. 2023.
8.
Tomohiro Tamaki, Takeo Minamikawa, Eiji Hase, Yuki Morimoto, Akihiro Suzuki, Takeshi Yasui, Satoko Nakamura, Akemi Tsutsui, Koichi Takagushi and Koichi Tsuneyama : Second-harmonic generation polarization microscopy to analyze ultra-early-stage liver fibrosis in human non-alcoholic fatty liver disease, pLED International symposium 2023: Exploring Invisible Light Technology, P-5, Tokushima, Mar. 2023.
9.
Miyakami Yuko, Mayuko Shimizu, Yoshimura Kentaro, Tsutsui Akemi and Koichi Tsuneyama : ESTABLISHMENT of EARLY FIBROSIS MARKER in NASH USING SERUM PHOSPHOLIPIDS, APASL2023, Feb. 2023.
10.
Tetsuji Takayama, Yoshifumi Kida, Toshiya Okahisa, Yasushi Sato, Masahiro Bando, shota Fujimoto, Tomoyuki Kawaguchi, Fumika Nakamura, Koichi Okamoto, Hiroshi Miyamoto, Koichi Tsuneyama and Masahiro Sogabe : The relevance of urokinase-type plasminogen activator in pathogenesis of ulcerative colitis., WCOG2022 (World Congress of Gastroenterology), Dubai, Dec. 2022.
11.
Koichi Tsuneyama, Wenhua Shao and Mayuko Shimizu : Various type of hepatocellular adenomas occurred spontaneously in the liver of metabolic syndrome-associated steatohepatitis model, TSOD mice, AASLD The Liver meeting 2022, Nov. 2022.
12.
Takumi Kakimoto, Mayuko Shimizu, Koichi Tsuneyama, Minoru Matsumoto, Takeshi Oya, Hosokawa Masato and Koichi Tsuneyama : Accumulation of α-synuclein in hepatocytes in NASH liver and usefulness in pathological diagnosis, Digestive Disease Week 2022, May 2022.
13.
Kikuchi Kentaro, Matsumoto Kotaro, Takai Atsuko, Mayuko Shimizu, Moritoki Yuki, Koichi Tsuneyama and Hara Masumi : Fructo-oligosaccharides ameliorate steatohepatitis via increased production of short-chain fatty acids in mice, JSH International Liver Conference 2021, Oct. 2021.
14.
Mayuko Shimizu, 富永 俊弼 and Koichi Tsuneyama : A novel image analysis technology that can verify quantification and progress pattern of NASH fibrosis, The Liver Meeting 2020, Boston, Nov. 2020.
15.
Hirohisa Ogawa, Masahiko Azuma, Umeno Aya, Shimizu Mayuko, Takaaki Tsunematsu, Mayo Kondou, Murotomi Kazutoshi, Koichi Tsuneyama and Yasuhiko Nishioka : Nerve growth factor exacerbates airway hyperresponsiveness via epithelial damage by neutrophils-derived singlet oxygen in a mouse model of asthma with mixed inflammation, JSA/WAO Joint Congress 2020, WEB, Sep. 2020.
16.
Seidai Satou, Yamamoto Takushi, Waki Kana, Morimoto Yuki, Imakura Takeshi, Murakami Kojin, Takahashi Naoki, Nobuhito Naito, Kozo Kagawa, Kazuya Koyama, Hiroshi Kawano, Koichi Tsuneyama and Yasuhiko Nishioka : Analysis of localization of nintedanib and cyclophosphamide in pulmonary fibrosisusing the iMScope TRIOS, ERS International Congress 2020 Virtual, WEB, Sep. 2020.
Mayuko Shimizu and Koichi Tsuneyama : Phellodendron bark and its component berberine prevent nonalcoholic steatohepatitis-related fibrosis in mice, 29th Asian Pacific Association for the Study of the Liver (APASL) 2020, Bali, Mar. 2020.
18.
Eiji Hase, Hiroki Takanari, Mayuko Shimizu, Hayashi Yuri, Takeo Minamikawa, Takeshi Yasui and Koichi Tsuneyama : Characterization of fibrosis in non-alcoholic steatohepatitis by use of second-harmonic-generation microscopy, Asian Pacific Association for the Study of the Liver 2020 (APASL 2020), Bali, Indonesia, Mar. 2020.
19.
Takeo Minamikawa, Mayuko Shimizu, Hiroki Takanari, Shiomi Ryosuke, Kusaka Hiroki, Tanioka Hiroki, Eiji Hase, Takeshi Yasui and Koichi Tsuneyama : Molecular imaging analysis of accumulated fats in non-alcoholic steatohepatitis by Raman microscopy, Asian Pacific Association for the Study of the Liver 2020 (APASL 2020), Bali, Indonesia, Mar. 2020.
20.
Minoru Matsumoto, Hitoshi Nishijima, Ryuichiro Miyazawa, Junko Morimoto, Koichi Tsuneyama and Mitsuru Matsumoto : Characterization of Aire-expressing DCs with high-sensitivity and high-fidelity Aire-reporter strain, The 48th Annual Meeting of the Japanese Society for Immunology, Hamamatsu, Japan, Dec. 2019.
21.
Mayuko Shimizu and Koichi Tsuneyama : Fibrosis begins around lipid-phagocytic macrophages in high fat/cholesterol/cholate diet-induced nonalcoholic steatohepatitis model, The Liver Meeting 2019, Boston, Nov. 2019.
22.
Mayuko Shimizu, Hirohisa Ogawa, Minoru Matsumoto and Koichi Tsuneyama : Establishment of a novel dietary-induced mouse model showing steatohepatitis with severe fibrosis, UEG Week 2019, Barcelona, Oct. 2019.
23.
Mitsuru Matsumoto, Junko Morimoto, Minoru Matsumoto, Koichi Tsuneyama, Ryuichiro Miyazawa and Hitoshi Nishijima : Aire-dependent establishment of self-tolerance, The 17th International Congress of Immunology, Beijing, Oct. 2019.
24.
Mayuko Shimizu, Takeshi Oya and Koichi Tsuneyama : Fibrogenesis of nonalcoholic steatohepatitis begins around cholesterol-laden macrophage in the experimental mouse model, UEG Week 2019, Barcelona, Oct. 2019.
25.
Mitsuru Matsumoto, Junko Morimoto, Minoru Matsumoto, Koichi Tsuneyama, Ryuichiro Miyazawa and Hitoshi Nishijima : Aire-dependent establishment of self-tolerance studied by genetically modified mice, EMBO Workshop ThymE, Israel, May 2019.
26.
Koichi Tsuneyama : Pathological features of new animal models for primary biliary cholangitis, Eric Gershwin symposium, Tokyo Universitu, Tokyo, Feb. 2019.
27.
Koichi Tsuneyama : Novel animal models for analyzing metabolic syndrome-associated liver diseases, 2nd SCUT international symposium of liver imunology, Feb. 2019.
28.
Koichi Tsuneyama : Novel diet-induced NASH midel showing severe liver fibrosis, Monthly meetng, Novonordisk in Beijing, Jan. 2019.
29.
Koichi Tsuneyama : Elucidating the mechanism of fibrogenesis and carcinogenesis in NASH, Annual meeting of Pharmaceutical labs, the Seoul National University, Dec. 2018.
30.
Mitsuru Matsumoto, Minoru Matsumoto, Junko Morimoto, Koichi Tsuneyama and Hitoshi Nishijima : Tissue-specific autoimmunity manipulated by Aire in thymic and peripheral tolerance, Immunology of Diabetes Society Congress 2018, London, Oct. 2018.
31.
Koichi Tsuneyama : Comparative Pathology of Murine Model of PBC, Lugano meeting of PBC, Sep. 2018.
32.
Minoru Matsumoto, Hitoshi Nishijima, Junko Morimoto, Koichi Tsuneyama and Mitsuru Matsumoto : Manipulation of thymic and peripheral tolerance by AIRE defines distinct tissue-specific autoimmunity, 11th International Congress on Autoimmunity, Lisbon, Portugal, May 2018.
33.
Mitsuru Matsumoto, Junko Morimoto, Minoru Matsumoto, Koichi Tsuneyama and Hitoshi Nishijima : A novel Aire-dependent subset of mTECs with tolerogenic functions is defined by Ly6 family protein expression, ThymOz8, Heron Island, Australia, Mar. 2018.
34.
Minoru Matsumoto, Hitoshi Nishijima, Junko Morimoto, Koichi Tsuneyama and Mitsuru Matsumoto : Acquisition of the resistance to autoimmune diabetes by the expression of human AIRE in BM-derived APCs in NOD, ThymOz8, Heron Island, Australia, Mar. 2018.
35.
Koichi Tsuneyama : Pathological & Immunological characters of animal models for analyzing metabolic syndrome-associated liver disease, International conference of immunological liver diseases in SCTU, Mar. 2018.
36.
Ayumi Sugitani, Mayuko Shimizu, Shu Taira, Takaaki Tsunematsu and Koichi Tsuneyama : Analysis of fibrogenic roles of liver infiltrating macrophages in a novel Non-alcoholic steatohepatitis (NASH) rat model with liver cirrhosis, Hepatology, Vol.66, No.S1, 1099A-1100A, Oct. 2017.
37.
Kazuchika Nishitsuji, Koichi Inoue, Hiroyasu Akatsu and Koichi Tsuneyama : Gut dysbiosis and altered plasma short chain fatty acid profile underlie the pathogenesis of nonalcoholic steatohepatitis, Hepatology, Vol.66, No.S1, 450A, Oct. 2017.
Shota Fujimoto, Naoki Muguruma, Koichi Okamoto, Misaki Miwa, Yoshihiko Miyamoto, Yoshifumi Takaoka, Koichi Tsuneyama and Tetsuji Takayama : A New Theranostic Combination of Fluorescent Molecular Imaging and Laser Therapy Targeting C-Kit in Gastrointestinal Stromal Tumors., Digestive Disease Week2017, Chicago, May 2017.
40.
Koichi Tsuneyama, Ryosuke Bessho, Masahiro Miki, Hayato Baba, Tetsuyuki Takahashi, Hirohisa Ogawa and Hisanori Uehara : Hyperinsulinemia, not hyperglycemia accelerates the progression of hepatocellular carcinoma in neonatal streptozotocin induced mouse model, Annual meeting, American Association for the study of the Liver, Nov. 2015.
41.
Koichi Tsuneyama, Hayato Baba, Takeshi Nishida and Johji Imura : Daily moderate coffee intake inhibits pancreatic beta-cell damage and nonalcoholic steatohepatitis without improving obesity in a mouse model of spontaneous metabolic syndrome., 24th Conference of the Asian Pacific Associaton for the Study of the Liver (APASL ). 3/ 12 - 15, 2015, Istanbul, Turke, Mar. 2015.
42.
Mayuko Shimizu, Kawase Miku, Masuzumi Miki, Sakaki Mika, Nagata Yasuo, Tanaka Kazunari, Suruga Kazuhito, Koichi Tsuneyama and Omagari Katsuhisa : High-fat and high-cholesterol diet rapidly induces nonalcoholic steatohepatitis with advanced fibrosis in Sprague-Dawley rats, 23th Asian Pacific Association for the Study of the Liver, Mar. 2014.
Proceeding of Domestic Conference:
1.
Junji Chida, Eiji Hara, Mayuko Shimizu, Koichi Tsuneyama and Suehiro Sakaguchi : Anti-prion antibody treatment attenuates liver inflammation and fibrosis in experimental non-alcoholic steatohepatitis mouse modelAnti-prion antibody treatment attenuates liver inflammation and fibrosis in experimental non-alcoholic steatohepatitis mouse model,
Ryo Fujimaru, Takaaki Tsunematsu, Koichi Tsuneyama and Michiko Yamashita : A case of IGF-2-producing hepatocellular carcinoma with severe hypoglycemia, Proceedings of the Japanese Society of Pathology, Mar. 2024.
5.
Yasuyo Saijo, Koichi Tsuneyama, Minoru Matsumoto and Takeshi Oya : A case of biliary adenofibroma with malignant transformation, 第113回日本病理学会総会, Mar. 2024.
6.
立花 尚太郎, Hirohisa Ogawa, Koichi Tsuneyama and Mayuko Shimizu : 慢性気管支喘息においてリゾフォスファチジン酸(LPA)はLPA1受容体を介して杯細胞過形成を促進する, 第113回日本病理学会総会, Mar. 2024.
7.
前川 勇輔, Hirohisa Ogawa, Koichi Tsuneyama and Mayuko Shimizu : 喘息の気道周囲の線維化におけるM1,M2マクロファージの役割, 第113回日本病理学会総会, Mar. 2024.
8.
黒田 翔, Mayuko Shimizu, Hirohisa Ogawa and Koichi Tsuneyama : 代謝障害関連脂肪肝炎における マクロファージに対するBerberineの作用, 第112回日本病理学会総会, Mar. 2024.
Natsumi Ohara, Yumiko Yamashita, Ayumi Masumi, Shingen Nakamura, Hirohisa Ogawa, Koichi Tsuneyama and Michiko Yamashita : A case of ascitic plasmacytosis needed to distinguish to the fluid overload large B cell lymphoma FO-LBCL, 第267回徳島医学会学術集会, Aug. 2023.
21.
Ryo Fujimaru, Michiko Yamashita, Takaaki Tsunematsu, Izumi Fukuda, Katsuyoshi Tamaki and Koichi Tsuneyama : IGF-2producing hepatocellular carcinoma with non-islet cell tumor hypoglycemia as the paraneoplastic syndrome, 第267回徳島医学会学術集会, Aug. 2023.
Mayuko Shimizu, 西村 百夏, 中原 穂乃佳, Hirohisa Ogawa and Koichi Tsuneyama : Inhibitory effects of berberine on liver fibrosis in NASH model mice, 第77回日本栄養・食糧学会大会, May 2023.
Honoka Nakahara, Asuka Watanabe, Mayuko Shimizu, Hirohisa Ogawa and Koichi Tsuneyama : Inhibitory effects of berberine on liver fibrosis in NASH model mice, 第111回日本病理学会総会, Apr. 2022.
52.
Soichiroh Ishimaru, Mayuko Shimizu, Hirohisa Ogawa and Koichi Tsuneyama : An Application of Raman Microscopy to Analyzing Dermal Changes in Cutaneously Sensitized Food Allergy Mice, 第111回日本病理学会総会, Apr. 2022.
53.
Minoru Matsumoto, Yasuyo Saijo, Mayuko Shimizu, Hirohisa Ogawa, Koichi Tsuneyama, Mitsuru Matsumoto and Takeshi Oya : 胸腺癌におけるAIRE発現と胸腺髄質上皮細胞との類似性, Proceedings of the Japanese Society of Pathology, Vol.111, No.1, 243, Apr. 2022.
柿本 拓海, Mayuko Shimizu, Hirohisa Ogawa, Minoru Matsumoto, Takeshi Oya and Koichi Tsuneyama : Validation of α -synuclein immunohistochemical staining in the diagnosis of non-alcoholic steatohepatitis, The 67th Autumn Annual Meeting of Japanese Society of Pathology, Nov. 2021.
Takumi Kakimoto, Mayuko Shimizu, Hirohisa Ogawa and Koichi Tsuneyama : 非アルコール性脂肪性肝炎の診断におけるα-synuclein免疫組織化学染色の有用性, Proceedings of the Japanese Society of Pathology, Vol.110, No.2, 102, Oct. 2021.
(Keyword)
*免疫組織化学 *Alpha-Synuclein *肝疾患-非アルコール性脂肪性(病理学) ヒト
64.
Hirohisa Ogawa, 立花 尚太郎, Mayuko Shimizu and Koichi Tsuneyama : Lysophosphatidic acid is involved in the formation of airway remodeling in chronic bronchial asthma, Proceedings of the Japanese Society of Pathology, Vol.110, No.2, 98, Oct. 2021.
Takeo Minamikawa, Mayuko Shimizu and Koichi Tsuneyama : Molecular structural imaging analysis for non-alcoholic fatty liver disease by Raman spectroscopy, The 57th Annual Meeting of the Japan Society of Hepatology, Jun. 2021.
Katsuki Miyazaki, Yu Saitou, Mayuko Shimizu, Yuji Morine, Satoru Imura, Tetsuya Ikemoto, Shin-ichiro Yamada, Kazunori Tokuda, Koichi Tsuneyama and Mitsuo Shimada : Endoplasmic reticulum stress response deficiency is a major cause of poor liver regeneration after hepatectomy in nonalcoholic steatohepatitis, 第33回日本肝胆膵外科学会学術集会, Jun. 2021.
74.
Mayuko Shimizu, 平 修, Hirohisa Ogawa and Koichi Tsuneyama : Phospholipids Imaging of the Liver and Brain in a Mouse Model of Nonalcoholic Steatohepatitis, 第69回質量分析総合討論会2021, May 2021.
(Keyword)
pathogen lipids / Mass spectrometry imaging
75.
Yuri Hirokane, Minoru Matsumoto, Koichi Tsuneyama and Takeshi Oya : von Hippel-Lindau病に関連したendolymphatic sac tumorの一例, 第110回日本病理学会総会, Apr. 2021.
76.
Mayuko Shimizu, 遠藤 亜衣子, 石丸 漱一郎, 小島 拓海, Hirohisa Ogawa and Koichi Tsuneyama : Epicutaneous sensitization conditions for food allergens in animal models, 第110回日本病理学会総会, Apr. 2021.
77.
Michiko Yamashita, Koichi Tsuneyama and Takeshi Oya : Bone marrow microenvironment on Bone marrow gelatinous degeneration, Proceedings of the Japanese Society of Pathology, Vol.110, No.1, 355, Apr. 2021.
Hirohisa Ogawa, Kayu Kinouchi, Mayuko Shimizu and Koichi Tsuneyama : 喘息モデルにおける脳由来神経栄養因子(BDNF)の産生とその役割, Proceedings of the Japanese Society of Pathology, Vol.110, No.1, 291, Mar. 2021.
Mayuko Shimizu, Riku Yamaguchi, CHIYAMOTO Mana, OSAKI Yui and Koichi Tsuneyama : Antibacterial agent for external use promotes epicutaneous sensitization of food allergen in experimental mice, 第74回日本栄養食糧学会大会, May 2020.
Koichi Tsuneyama : 免疫チェックポイント阻害薬による肝障害をきたした4例の病理学的特徴, 日本肝臓学会総会, May 2019.
104.
Koichi Tsuneyama and Mayuko Shimizu : 免疫チェックポイント阻害薬による肝障害を来した4例の病理学的特徴, 第55回日本肝臓学会総会, May 2019.
(Keyword)
免疫チェックポイント阻害薬 / 肝障害
105.
飯田 綾香, 甲斐 瑠衣子, 倉貫 早智, Mayuko Shimizu, Koichi Tsuneyama and 中村 強 : Nonalcoholic fatty liver Disease in Western Diet-Loaded C57BL/6J mice, 第73回日本栄養食糧学会大会, May 2019.
(Keyword)
nonalcoholic steatohepatitis / 疾患モデル動物
106.
福田 歩美, 佐々尾 真鈴, 浅川 惠理, 成田 純令, 久野 萌偉, 駿河 和仁, Mayuko Shimizu, Koichi Tsuneyama, 田中 一成 and 大曲 勝久 : Incidence of non-alcoholic steatohepatitis in SD rats fed a high-fat and high-cholesterol diet., 第73回日本栄養食糧学会大会, May 2019.
(Keyword)
nonalcoholic steatohepatitis / mouse model / high fat and high cholesterol diet
107.
Mayuko Shimizu, 大曲 勝久 and Koichi Tsuneyama : Establishment of dietary model of nonalcoholic steatohepatitis-related fibrosis, 第73回日本栄養食糧学会大会, May 2019.
(Keyword)
Liver fibrosis / nonalcoholic steatohepatitis / mouse model / high fat and high cholesterol diet
108.
Mayuko Shimizu, Takeshi Oya, Hirohisa Ogawa, 大曲 勝久 and Koichi Tsuneyama : Cholesterol-loaded macrophage is associated with development of hepatic fibrosis in mice, 第108回日本病理学会総会, May 2019.
Mayuko Shimizu, 大曲 勝久 and Koichi Tsuneyama : The morphological changes of the liver of TSOD and TSNO mice fed the high-fat plus cholesterol diet, 第107回日本病理学会総会, Jun. 2018.
Minoru Matsumoto, Hitoshi Nishijima, Junko Morimoto, Koichi Tsuneyama and Mitsuru Matsumoto : Transgenic human AIRE expression in NOD acquired resistance to the diabetes due to the impaired presentation of self-antigens in the pancreas, 第46回日本免疫学会学術集会, Dec. 2017.
Ayumi Sugitani, Ryusei Takahashi, Kohki Kai, Mayuko Shimizu, 平 修, 川口 誠, Hirohisa Ogawa, Takaaki Tsunematsu, Koichi Tsuneyama, 野本 一博 and Koichi Tsuneyama : A case of lipid-rich carcinoma of the breast with numerous hyaline globules, Proceedings of the Japanese Society of Pathology, Vol.106, No.1, 492-493, Mar. 2017.
146.
Michiko Yamashita, Shingo Wakatsuki, 遠藤 早希子, 佐竹 宣法, 堀口 英久, 小川 博久 and Koichi Tsuneyama : 「病理夏の学校in徳島」における，検査値解析を用いた全学年参加CPC, Proceedings of the Japanese Society of Pathology, Vol.106, No.1, 327, Mar. 2017.
147.
Takaaki Tsunematsu, Yasusei Kudo, Akiko Yamada, Rieko Arakaki, Hirohisa Ogawa, Koichi Tsuneyama and Naozumi Ishimaru : 胎児性癌細胞におけるユビキチンプロテアソーム経路による未分化性維持機構, Proceedings of the Japanese Society of Pathology, Vol.106, No.1, 356, Mar. 2017.
148.
堀口 英久, 柿内 聡司, 米田 直樹, 板東 悠太郎, 清久 泰司 and Koichi Tsuneyama : 抗PD-1抗体治療中に致死性肺障害が生じた肺腺癌患者の剖検例, Proceedings of the Japanese Society of Pathology, Vol.106, No.1, 421, Mar. 2017.
149.
Koichi Tsuneyama, 渡邊 俊介, 高橋 徹行, 馬場 逸人, 森本 友樹, Takaaki Tsunematsu and Hirohisa Ogawa : 自然発症メタボリックシンドローム-NASHモデル動物を用いた少量のコーヒー摂取による病態抑制効果の検討, Proceedings of the Japanese Society of Pathology, Vol.106, No.1, 441, Mar. 2017.
150.
Hirohisa Ogawa, Takaaki Tsunematsu, 森本 友樹 and Koichi Tsuneyama : IL-17/好中球は気道粘膜上皮透過性を増加させFGF-2産生を介した気道平滑筋肥厚を誘導する, Proceedings of the Japanese Society of Pathology, Vol.106, No.1, 474, Mar. 2017.
151.
杉谷 鮎美, 高橋 立成, 甲斐 幸樹, Mayuko Shimizu, 平 修, 川口 誠, Hirohisa Ogawa, Takaaki Tsunematsu, Koichi Tsuneyama, 野本 一博 and 常山 幸一 : A case of lipid-rich carcinoma of the breast with numerous hyaline globules(和訳中), Proceedings of the Japanese Society of Pathology, Vol.106, No.1, 492-493, Mar. 2017.
152.
甲斐 幸樹, 常松 貴明, 杉谷 鮎美, 高橋 立成, Hirohisa Ogawa, 森本 友樹 and Koichi Tsuneyama : コーヒーに含まれるクロロゲン酸(5-カフェオイルキナ酸:5-CQA)の抗腫瘍効果の検討, Proceedings of the Japanese Society of Pathology, Vol.106, No.1, 513, Mar. 2017.
Mayumi Takeuchi, Kenji Matsuzaki, Masafumi Harada, Eri Takiguchi, Masato Nishimura, 渡邉 俊介 and Koichi Tsuneyama : 卵巣漿液性癌を背景に発生した大細胞性神経内分泌癌の1例, 第30回日本腹部放射線学会, Jun. 2016.
156.
信 蔵野, Koichi Tsuneyama, 政弘 常名 and 裕 矢冨 : アポ蛋白Mを用いた播種性血管内凝固症候群，多臓器不全の新規治療法の開発, Japanese Journal of Thrombosis and Hemostasis, Vol.27, No.2, 250, May 2016.
Koichi Tsuneyama, Hirohisa Ogawa, Takaaki Tsunematsu and Hisanori Uehara : 新生児期ストレプトゾトシン投与4CS系マウスは通常食飼育によって持続高血糖なしに肝腫瘍を発症する, Proceedings of the Japanese Society of Pathology, Vol.105, No.1, 384, Apr. 2016.
159.
Hirohisa Ogawa, Takaaki Tsunematsu, Hisanori Uehara and Koichi Tsuneyama : ダニ抗原経鼻感作はIL-17/好中球を増加させ平滑筋肥厚を伴う気道リモデリングを誘導する, Proceedings of the Japanese Society of Pathology, Vol.105, No.1, 386, Apr. 2016.
160.
Hisanori Uehara, Hirohisa Ogawa, Takaaki Tsunematsu and Koichi Tsuneyama : 前立腺癌細胞の骨内増殖におけるlipolysis stimulated lipoprotein receptorの役割, Proceedings of the Japanese Society of Pathology, Vol.105, No.1, 466, Apr. 2016.
161.
秀樹 八田, 隆彦 中嶋, Koichi Tsuneyama, 伸一 林, 重治 三輪, 尚 南坂, 健志 西田, ゆう子 中西 and 穣二 井村 : 免疫染色におけるホウ酸含有緩衝液の有効性 室温放置により抗原性が減弱した標本の賦活法, Proceedings of the Japanese Society of Pathology, Vol.105, No.1, 529, Apr. 2016.
162.
健志 西田, Koichi Tsuneyama, 伸一 林, 重治 三輪, 隆彦 中嶋, 尚 南坂, 秀樹 八田 and 穣二 井村 : 肥満・糖尿病マウスを用いた，メタボリックシンドロームに対する地域伝承発酵食品の有効性の検証, Proceedings of the Japanese Society of Pathology, Vol.105, No.1, 543, Apr. 2016.
163.
Takaaki Tsunematsu, Yasusei Kudo, Akiko Yamada, Rieko Arakaki, Hirohisa Ogawa, Hisanori Uehara, Naozumi Ishimaru and Koichi Tsuneyama : 染色体パッセンジャー複合体タンパク質Borealinのユビキチン分解の意義とその癌化への関与, Proceedings of the Japanese Society of Pathology, Vol.105, No.1, 548, Apr. 2016.
164.
良祐 別所, Takaaki Tsunematsu, Hirohisa Ogawa, Hisanori Uehara and Koichi Tsuneyama : 自然発症2型糖尿病モデルマウスにおける膵島の外分泌腺組織への変化がβ細胞の機能不全に果たす役割, Proceedings of the Japanese Society of Pathology, Vol.105, No.1, 595, Apr. 2016.
165.
徹行 高橋, Hisanori Uehara, Koichi Tsuneyama and 貴雄 土方 : プロスタグランジンE2受容体シグナリング阻害は膵臓がんにおけるIGF-1受容体シグナリングを介した増殖刺激を消失させる, 日本薬学会年会要旨集, No.4, 202, Mar. 2016.
Masaki TAKEUCHI, Shingo ODA, Koichi Tsuneyama and Tsuyoshi YOKOI : Identification of serum miRNAs as diagnostic biomarkers for sinusoidal obstruction syndrome in rats, Annual Meeting of the Japanese Society of Toxicology, Vol.43, No.0, P-80, 2016.
猪子 未希, Hirokazu Ogino, Mayuri Nakano, 岡崎 弘泰, 大塚 憲司, Hisatsugu Goto, Jun Kishi, Masaki Hanibuchi, Yoshimi Bando, Koichi Tsuneyama and Yasuhiko Nishioka : 脳，口蓋垂，喉頭および小腸への遠隔転移を形成する稀な臨床経過をたどった右上葉原発多形癌の一剖検例, 第54回日本呼吸器学会中国・四国地方会, Dec. 2015.
169.
Mayuko Shimizu, 畑中 麻衣子, Koichi Tsuneyama, 加藤 滋子 and 大曲 勝久 : An animal model of NASH with advanced fibrosis induced by high-fat and high-cholesterol diet, 第67回日本栄養食糧学会大会, May 2013.
(Keyword)
NASH / SHR/NDmcr-cp rat / high fat and high cholesterol diet / fibrosis
170.
藤坂 志帆, 薄井 勲, Takeshi Oya, 金谷 由紀子, 山崎 夕, Agussalim Bukhari, 鈴木 ひかり, 仙田 聡子, 高崎 一朗, Koichi Tsuneyama, 浦風 雅春, 小林 正 and 戸辺 一之 : テルミサルタンは脂肪組織のM1マクロファージを選択的に減少させることでインスリン抵抗性を改善する, Journal of the The Japan Diabetes Society, Vol.51, No.Suppl.1, S-348, Apr. 2008.
Kohta Hata, Ryoga Yuasa, Sarina Takeuchi, Michiko Yamashita, Takeshi Oya, Koichi Tsuneyama and Mayuko Shimizu : 骨髄膠様変性時，臨床検査値と造血幹細胞ニッチはどう変化しているか, THE TOKUSHIMA JOURNAL OF MEDICAL TECHNOLOGY, 28, Dec. 2021.
9.
Ryoga Yuasa, Kohta Hata, Sarina Takeuchi, Michiko Yamashita, Ryohei Sumitani, Hisanori Uehara, Yoshimi Bando and Koichi Tsuneyama : 同種骨髄移植後の造血微小環境についての病理学的研究, THE TOKUSHIMA JOURNAL OF MEDICAL TECHNOLOGY, 27, Dec. 2021.
Sarina Takeuchi, Yurie Kobayashi, Michiko Yamashita, Takeshi Oya and Koichi Tsuneyama : Immunohistochemical study of hematopoietic niche-forming cells in gelatinous degeneration of bone maro, THE TOKUSHIMA JOURNAL OF MEDICAL TECHNOLOGY, 18, Dec. 2020.
12.
Yurie Kobayashi, Sarina Takeuchi, Michiko Yamashita, Koichi Tsuneyama and Mayuko Shimizu : Statistical study of laboratory values correlated with gelatinous degeneration of bone marrow, THE TOKUSHIMA JOURNAL OF MEDICAL TECHNOLOGY, 19, Dec. 2020.
Elucidation of the pathogenesis and new therapeutic strategies for brain- and gut-related steatohepatitis based on brain-derived neurotrophic factor (Project/Area Number: 22K07024 )
Effects of metabolic syndrome on locomotor function and the mechanism of exercise therapy. (Project/Area Number: 20K09435 )
Functional Consequences of Neural Sodium Channel Nav1.8 in an Experimental Model of Electrical Storm Associated With QT Prolongation (Project/Area Number: 20K08450 )
Mechanism of suppressive effect for intrahepatic recurrence of hepatocellular carcinoma by hepatic stellate cell regulation after liver transplantation (Project/Area Number: 18K08573 )
Elucidation of the role of bile acids and short-chain fatty acids in the onset and progression of non-alcoholic steatohepatitis (Project/Area Number: 18K07069 )
Development of integrated treatment for impaired liver regeneration on non-alcoholic steatohepatitis (Project/Area Number: 18H02871 )
Establishment of new analytical method for visualizing chemical agents and xenobiotics on pathology speciments (Project/Area Number: 15K15098 )
Pathological analysis of cadmium and its microenvironment in patients with itai-itai disease (Project/Area Number: 25670175 )
Effect of obesity and diabetes on liver cancer and tumor-associated macrophage polarization (Project/Area Number: 25461333 )
Development of methods for reducing volume and reutilization of waste seaweeds with a novel species bacterium Myt-1 capable of degrading seaweeds. (Project/Area Number: 25340121 )
Kampo medicine pharmacology-based clarification and identification of etiologic factors and their function responsible for ADHD (Project/Area Number: 24659347 )
New analytical technology in the field of pathology clarified effectiveness of Kampo formula (Project/Area Number: 24390181 )
Detection of novel actions of kampo medicines for lipid metabolism by lipidomic analysis (Project/Area Number: 23590873 )
Sharing pathogenic mechanism between non-alcoholic steatohepatitis and primary biliary cirrhosis (Project/Area Number: 21590433 )
Studies on endogenous neuronal mediator with responsibility for anti-dementia effect of Kampo medicine (Project/Area Number: 20390197 )
Molecular pathological analysis of pathogenesis of non-alcoholic steatohepatitis: Propose of new treatment approach (Project/Area Number: 18590324 )
Role of p 53 codon 72 polymorphism in EBV-related gastric cancer (Project/Area Number: 17590297 )
Pathological and biological analysis of congenital hepatic fibrosis and Caroli's disease -A comparative study using an animal model for the disease (Project/Area Number: 12470044 )