Biomedical subjects
Publications and source records attributed to Hitoshi Ikeda.
Epidermoid cyst of the testis, extremely rare in children, is a non-teratomatous benign tumor, and testis-sparing surgery should be the treatment of choice. To prevent unnecessary orchiectomy, recognition of this rare tumor in children is essential.
Several reports on immunotherapy using dendritic cells-based vaccine have been published. We investigated findings using fusion cells (FCs) generated from rat dendritic cells and a syngeneic hepatic cancer cell line with regard to inducing anti-tumor immunity. Vaccination of rats using FCs protected against growth of the subcutaneously implanted tumor in vivo and induced infiltration of CD8(+) T cells into the tumor. At the site of CD8(+) T cell infiltration, there were apoptotic tumor cells. T cells from spleen of FCs-vaccinated rats with protective ability against tumor growth included tumor specific cytotoxic CD8(+) T cells restricted to major histocompatibility complex Class I. In addition, adaptive transfer of in vitro re-stimulated splenic T cells with FCs was effective in preventing tumor growth and in vivo vaccinations of rats with FCs after resection of the subcutaneous implanted tumor inhibited local tumor recurrences. Immunotherapy using FCs appears to be an effective method if used in combination with surgical or other anti-cancer therapies.
Hepatic stellate cells (HSCs) play a central role in the development of hepatic fibrosis. Recent evidence has revealed that HSCs also play a role in its resolution, where HSC apoptosis was determined. Moreover, induction of HSC apoptosis caused a reduction of experimental hepatic fibrosis in rats. Thus knowing the mechanism of HSC apoptosis might be important to clarify the pathophysiology and establish the therapeutic strategy for hepatic fibrosis. In HSCs, Rho and Rho kinase are known to regulate contraction, migration, and proliferation with modulation of cell morphology. Controversy exists as to the participation of Rho and Rho kinase on cell survival, and little is known regarding this matter in HSCs. In this study, we directed our focus on the role of the Rho pathway in the regulation of HSC survival. C3, an inhibitor of Rho, increased histone-associated DNA fragmentation and caspase 3 activity with enhanced condensation of nuclear chromatin in rat cultured HSCs. Moreover, Y-27632, an inhibitor of Rho kinase, had the same effects, suggesting that inhibition of the Rho/Rho kinase pathway causes HSC apoptosis. On the other hand, lysophosphatidic acid, which stimulates the Rho/Rho kinase pathway, decreased histone-associated DNA fragmentation in HSCs. Inhibition of the Rho/Rho kinase pathway did not affect p53, Bcl-2, or Bax levels in HSCs. Thus we concluded that the Rho/Rho kinase pathway may play a role in the regulation of HSC survival.
Using a rat myofibroblast-like hepatic stellate cell line, we studied the actomyosin-based cytoskeletal actions mediated by Rho-kinase and/or myosin light chain kinase (MLCK). Calmodulin/MLCK inhibitors W-7 and ML-7 attenuated cell migration dose-relatedly at concentrations from 10(-6) to 10(-4)M and collagen gel-contraction by the cells at 10(-4)M, respectively. Rho-kinase inhibitors Y-27632 and HA1077 attenuated the gel-contraction at concentrations from 10(-6) to 10(-4) M, respectively. These Rho-kinase inhibitors attenuated cell migration at 10(-7)M but enhanced the migration at 10(-4)M, respectively. They altered cell morphology showing prominent peripheral actin bundles and sparse central stress fibers, in comparison with the calmodulin/MLCK inhibitors. Both ML-7 and Y-27632 attenuated phosphorylation of myosin regulatory light chain and cell attachment to extracellular substrate. ML-7 attenuated the activation of GTP-binding protein Rac, while Y-27632 did not. These findings suggest that the actomyosin-based cytoskeletal actions can be functionally diverse depending on the Rho-kinase-mediated pathway and the MLCK-mediated pathway.
OBJECTIVES: Sphingosine 1-phosphate (Sph-1-P), a bioactive lipid derived from activated platelets, may play an important role in coronary artery spasm and hence the pathogenesis of ischemic heart diseases, since we reported that a decrease in coronary blood flow was induced by this lysophospholipid in an in vivo canine heart model [Cardiovasc. Res. 46 (2000) 119]. In this study, metabolism related to and cellular responses elicited by Sph-1-P were examined in human coronary artery smooth muscle cells (CASMCs). METHODS AND RESULTS: [3H]Sphingosine (Sph), incorporated into CASMCs, was converted to [3H]Sph-1-P intracellularly, but its stimulation-dependent formation and extracellular release were not observed. Furthermore, the cell surface Sph-1-P receptors of S1P family (previously called EDG) were found to be expressed in CASMCs. Accordingly, Sph-1-P seems to act as an extracellular mediator in CASMCs. Consistent with Sph-1-P-elicited coronary vasoconstriction in vivo, Sph-1-P strongly induced CASMC contraction, which was inhibited by JTE-013, a newly-developed specific antagonist of S1P(2) (EDG-5). Furthermore, C3 exoenzyme or Y-27632 inhibited the CASMC contraction induced by Sph-1-P, indicating Rho involvement. Finally, exogenously-added [3H]Sph-1-P underwent a rapid degradation. Since lipid phosphate phosphatases, ectoenzymes capable of dephosphorylating Sph-1-P, were expressed in CASMCs, Sph-1-P may be dephosphorylated by the ectophosphatases. CONCLUSIONS: Sph-1-P, derived from platelets and dephosphorylated on the cell surface, may induce the contraction of coronary artery smooth muscle cells through the S1P(2)/Rho signaling.
Transgenic rats expressing human CD21 gene (hCD21) driven by the mouse immunoglobulin enhancer were generated. hCD21 was expressed in lymphoid tissues, especially in the spleen and in the brain. Flow cytometric analysis indicated that about 20% of spleen cells, most having a B-lymphocyte marker, expressed hCD21. After Epstein-Barr virus (EBV) infection of spleen cells, EBV-determined nuclear antigen (EBNA) was first detected on Day 4 and reached a maximum of 0.3% on Day 5, but the infection was abortive and was not followed by blastogenesis, cellular DNA synthesis or proliferation. Reverse transcription-polymerase chain reaction (RT-PCR) analyses demonstrated that EBV-infected spleen cells expressed EBNA1 and EBV-encoded small RNA (EBER), but not other latent EBV products. EBNA promoter analysis by RT-PCR indicated that the Q promoter was active, whereas C and W promoters were not active. The present findings indicate that human and rat lymphocytes respond to EBV infection differently in vitro.
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OBJECTIVE: The International Neuroblastoma Staging System (INSS) and Pathology Classification (INPC) were applied to analyze the results of treatment of 644 patients with neuroblastoma treated in Japan during the years from 1995 to 1999, and it was found that the pathology classification (INPC) showed the strongest relevance to prognosis compared to other factors such as stage, MYNC amplification, DNA ploidy and 1p-deletion. Current results of treatment for advanced neuroblastoma are still not satisfactory, so innovative therapeutic methods have been sought during the past 10 years. METHODS: Prospects for irinotecan and recombinant human endostatin (rhEndostatin) were studied expertimentally and clinically. RESULTS: Irinotecan is a water-soluble derivative of camptothecin, which is isolated from a Chinese tree, Camptotheca acuminata; Its effectiveness against neuroblastoma was confirmed by in vivo preclinical studies, and phase I clinical trials in Japan concluded the maximum tolerated dose of this agent is 160-180 mg/m2/day for 3 consecutive days, repeated after 25 days off. Phase II trials with this dose began, and we could obtain some encouraging results with the clinical use of irinotecan. rhEndostatin has been studied in in vivo experimental models. The action of rhEndostatin was quite different from those of other cytotoxic chemotherapeutic agents, and continuous administration of this substance showed a more marked anti-effect than its intermittent use. CONCLUSION: Irinotecan appears to be promising when it is given to the patients neuroblastoma, whereas rhEndostatin needs to have more preclinical studies before it is used in patients.
Tax (p40Tax) encoded by the env-pX gene of human T-cell leukemia virus type I (HTLV-I) interacts with cyclic adenosine-3',5'-monophosphate response element binding protein/activation transcription factor 1 (CREB/ATF-1) transcription factors of host cells and activates the viral long terminal repeat (LTR) promoter. This molecular interaction induces augmentation of viral gene expression and may result in development of HTLV-I-associated diseases, including adult T-cell leukemia, HTLV-I associated myelopathy/tropical spastic paraparesis, and HTLV-I uveitis. To inhibit this pathway, a dominant negative molecule of ATF-1, ATF-1DN, was used. We transduced ATF-1DN into joint fibroblastic cells derived from transgenic rats carrying the LTR-env-pX-LTR gene of HTLV-I, using the Sindbis virus-based vectors. Expression of the pX gene in cells transduced with ATF-1DN was lower than that in cells with control transfection. A possible application of ATF-1DN to suppress viral gene expression in HTLV-I infected cells can be considered.
PURPOSE: The authors examined whether recombinant human endostatin (rhEndostatin), an antiangiogenic agent, is effective against a human neuroblastoma cell line (designated TNB9) using a human neuroblastoma xenograft model and investigated whether continuous infusion is more effective than intermittent administration. METHODS: In the first experiment, when tumors on the back of nude mice reached a weight of 90 to 95 mg, rhEndostatin, 10 mg/kg/d mouse weight, was administered subcutaneously to the mice (n = 5) every day for 10 consecutive days. In the second experiment, the same daily dose of rhEndostatin was administered continuously to the TNB9-bearing mice (n = 6) via subcutaneous infusion pumps for 3 consecutive days with total dose being 30% of that in the first experiment. Nestin and factor VIII expression levels were studied immunohistochemically to elucidate whether histologic evidence of the effects of rhEndostatin was present on day 4 in the second experiment. RESULTS: In the first experiment, relative tumor weight in treated mice (n = 5) was significantly less than that in controls (n = 12) on day 2 only after treatment initiation (P <.05). The maximum inhibition rate (MIR) of TNB9 xenograft growth by rhEndostatin was 46.4%, indicating lack of efficacy. In the second experiment, the effects of rhEndostatin were much more marked than those in the first experiment, with an MIR of 60.7%. The mean relative tumor weight in the treated group (n = 6) in the second experiment was significantly less than that in controls (n = 10) on days 2, 4, and 6 (P <.01) as well as on days 8 and 10 (P <.05). Nestin staining in the endothelium of control tumors (n = 2) was marked, whereas it showed a loss of fibrillar structure in rhEndostatin-treated tumors (n = 2). The number of vessels immunostained with antifactor VIII antibody was markedly reduced in tumors (n = 2) from rhEndostatin-treated mice compared with that in tumors from control animals (n = 2). CONCLUSIONS: Continuous administration of rhEndostatin resulted in more significant tumor regression than intermittent administration of the agent in the same model. This indicates that rhEndostatin, if administered in continuous fashion, could become an effective agent for treating patients with neuroblastoma in the future.
Hepatoblastoma is a common hepatic tumor in children. Although evidence regarding cytogenetic and molecular genetic alterations in hepatoblastomas has been reported, the molecular events affecting the biologic characteristics of this tumor, including alterations of the gene expression profile, are largely unknown. To identify genes differentially expressed between nondiseased liver (NDL) and hepatoblastoma tumor (HBT), we analyzed the gene expression profile in 14 NDL and 16 HBT samples using a high-density oligonucleotide DNA array. Using Mann-Whitney U test followed by the k-nearest neighbor algorithm, we identified 26 genes (predictor genes) that were able to assign unknown samples derived from NDL and HBT to either the NDL group or HBT group with 100% accuracy. Using a cross-validation approach, we confirmed that the k-nearest neighbor algorithm assigned the particular samples derived from NDL and HBT to either the NDL or HBT group with 93.3% (28/30 samples) accuracy. In the 26 predictor genes, we found alteration of the expression of genes regulating cell division (NAP1L1, STMN1, CCNG2, and CDC7L1) and tumor cell growth (IGF2 and IGFBP4) in HBT. Four predictor genes (ETV3, TPR, CD34, and NR1I3) were also found to be mapped to the chromosomal region 1q21 approximately q32, which has been reported to be frequently involved in the development of hepatoblastoma. The findings obtained in this study suggest that alteration of the expression of some genes regulating cell division and tumor cell growth may be characteristics of the gene expression profile in HBT, and that alteration of the expression of the four predictor genes mapped to chromosomal region 1q21 approximately q32 may also contribute to the differences in gene expression profile between NDL and HBT.
Transgenic rats carrying the env-pX gene of human T cell leukemia virus type-I (env-pX rats) develop various collagen vascular diseases. Since autoantibodies are present in their sera, env-pX rats are considered to be a prototype model for autoimmune diseases. Adoptive transfers of spleen cells from syngenic non-transgenic rats decreased the incidence of diseases in env-pX rats, thus suggesting that normal spleen contains cells, which suppress autoimmune diseases. Murine peripheral CD25(+)CD4(+)T cells play roles in maintaining immunological self-tolerance. To examine if alterations of immunoregulatory cells may be evident in env-pX rats, quantitative and qualitative analyses of splenic CD25(+)CD4(+)T cells were done before these rats developed autoimmune diseases. Env-pX and non-transgenic rats had equivalent number of CD25(+)CD4(+)T cells. However, CD25(+)CD4(+)T cells from env-pX rats did not suppress proliferation of T cells stimulated by anti-CD3 antibodies (Ab) in vitro, whereas those from non-transgenic rats did. Additionally, env-pX CD25(+)CD4(+)T cells showed autologous and anti-CD3 Ab-mediated proliferation, in contrast to the anergic features in non-transgenic rats. These findings appear to be the first evidence that CD25(+)CD4(+)immunoregulatory T cells are altered in animal models, which naturally develop autoimmune diseases.
Human endogenous retrovirus-R (HERV-R) is one of a full length HERV with a long open reading frame in the env region. The env transcripts are expressed in various human tissues. To investigate the biological role of HERV-R in vivo, we established two lines of transgenic rats carrying a full sequence of HERV-R under control of its own long terminal repeat (LTR) promoter. One line with tandem integration of multiple copies of the transgene expressed HERV-R mRNA in various organs with different expression levels and relatively higher in Harderian and submandibular salivary glands. In another line, the transgene was integrated as a single copy in a haploid and the expression was detected only in Harderian and submandibular salivary glands. In the placenta, one of the tissues with high levels of the HERV-R expression in humans, the transcription was evident starting the 12th day after gestation. A rabbit antiserum against synthetic peptides corresponding with the HERV-R env gene sequence led to detection of an 85 kDa product as a glycoprotein in the Harderian glands. While no pathological significance was observed in either line, the transgenic rat may prove to be a suitable model for analyzing the role of HERV-R function in vivo.
BACKGROUND & AIMS: Sphingosine 1-phosphate (S1P), a ligand for G protein-coupled endothelial differentiation gene-1 (Edg-1), Edg-3, Edg-5, Edg-6, and Edg-8, elicits a variety of responses by cells. Prominent among these is cell proliferation. S1P is abundantly stored in platelets and released upon their activation, suggesting that S1P plays a pathophysiologic role in vivo. Because the major part of injected S1P was distributed into the liver in mice, we wondered whether the liver would be one of its targets. The effects of S1P on hepatocytes, the major constituent cells in the liver, were examined. METHODS & RESULTS: Northern blot analysis revealed the expression of Edg-1 and Edg-5 messenger RNA (mRNA) in cultured rat hepatocytes, in which S1P decreased DNA synthesis induced by hepatocyte growth factor (HGF) or epidermal growth factor (EGF) without affecting total protein synthesis. This inhibitory effect was attenuated by inactivation of small GTPase Rho with C3 exotoxin but not by inactivation of G(i) with pertussis toxin. Moreover, in the presence of JTE-013, a newly developed and specific binding antagonist for Edg-5, the inhibitory effect was also cancelled. Finally, the administration of S1P after 70% partial hepatectomy in rats reduced the peak of DNA synthesis in hepatocytes with increased Rho activity. Furthermore, Edg-5 but not Edg-1 mRNA expression was enhanced in hepatocytes 24-72 hours after partial hepatectomy, which coincides with decreasing hepatocyte proliferation. CONCLUSIONS: S1P has an antiproliferative property in rat hepatocytes by activating Rho via Edg-5. Our results raise the possibility that S1P is a negative regulator in liver regeneration.
Apoptosis of the spinal oligodendrocytes is the main factor linked to the pathogenesis of human T-lymphocyte virus type I (HTLV-I)-induced myeloneuropathy in rats (HAM rat). To clarify apoptosis-related mechanisms, expression of apoptosis-related genes in the spinal cord of these rats was chronologically examined by means of a semiquantitative reverse transcriptase-polymerase chain reaction. Provirus expansion and increment of HTLV-I pX mRNA were evident at 7 months after the induced infection. Tumor necrosis factor-alpha increased gradually soon after pX expression. The expression of a major apoptosis-resistant gene, bcl-2, was markedly suppressed at a period of the provirus expansion and bax was also down-regulated. p53 was consistently expressed at high levels. These findings were never observed in spinal cords of HAM-resistant strains with HTLV-I infection even throughout their entire life. Collective evidence suggests that the local provirus expansion and deregulation of apoptosis-related genes, especially down-regulation of bcl-2, may lead to apoptosis of oligodendrocytes, thus being a major pathogenetic pathway in the HTLV-I-induced myeloneuropathy.
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Sphingosine 1-phosphate (Sph-1-P), a bioactive lysophospholipid capable of inducing a wide spectrum of biological responses, acts as an intercellular mediator, through interaction with the endothelial differentiation gene (EDG)/S1P family of G protein-coupled receptors. In this study, the effects of JTE-013, a specific antagonist of the migration-inhibitory receptor EDG-5, on Sph-1-P-elicited responses were examined in human umbilical vein endothelial cells (HUVECs) and vascular smooth muscle cells (SMCs), which expressed EDG-5 protein weakly and abundantly, respectively. This pyrazolopyridine compound reversed the inhibitory effect of Sph-1-P on SMC migration and further enhanced Sph-1-P-stimulated HUVEC migration. In contrast, its effect on Sph-1-P-induced intracellular Ca(2+) mobilization was marginal. Our results indicate that specific regulation of Sph-1-P-modulated migration responses in vascular cells can be achieved by EDG-5 antagonists and that manipulation of Sph-1-P biological activities by each EDG antagonist may lead to a therapeutical application to control vascular diseases.
Branched-chain amino acids (BCAAs) modulate various cellular functions, in addition to providing substrates for the production of proteins. In this study, we examined the effect of BCAAs on the secretion of hepatocyte growth factor (HGF) by hepatic stellate cells. A hepatic stellate cell clone was cultured in medium supplemented with various concentrations of valine, leucine, or isoleucine. Of these BCAAs, leucine markedly induced an increase in the levels of HGF in the medium in a dose-dependent manner. The addition of valine or isoleucine had no significant effect on HGF levels in the medium. The difference in levels of HGF in the medium between leucine-treated and non-treated cells was enhanced by the incubation period. These results demonstrate that, among BCAAs, leucine stimulates the secretion of HGF by cultured hepatic stellate cells.