Biomedical subjects
Publications and source records attributed to T Suda.
The various biological activities of side-chain mobility restricted analogs, four diastereomers at C(20) and C(22) of 22-methyl-1alpha,25-dihydroxyvitamin D3, were evaluated. The relationship between structure and the various activities of the analogs was discussed in terms of the active space region concept that we previously suggested.
Osteoclasts, the multinucleated giant cells that resorb bone, develop from monocyte-macrophage lineage cells. Osteoblasts or bone marrow stromal cells have been suggested to be involved in osteoclastic bone resorption. The recent discovery of new members of the tumor necrosis factor (TNF) receptor-ligand family has elucidated the precise mechanism by which osteoblasts/stromal cells regulate osteoclast differentiation and function. Osteoblasts/stromal cells express a new member of the TNF-ligand family "osteoclast differentiation factor(ODF)/osteoprotegerin ligand (OPGL)/TNF-related activation-induced cytokine (TRANCE)/receptor activator of NF-kB ligand (RANKL)" as a membrane associated factor. Osteoclast precursors which possess RANK, a TNF receptor family member, recognize ODF/OPGL/TRANCE/RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into osteoclasts in the presence of macrophage colony-stimulating factor. Mature osteoclasts also express RANK, and their bone-resorbingactivity is also induced by ODF/OPGL/TRANCE/RANKL which osteoblasts/stromal cells possess. Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF)/TNF receptor-like molecule 1 (TR1) is a soluble decoy receptor for ODF/OPGL/TRANCE/RANKL. Activation of NF-kB and c-Jun N-terminal kinase through the RANK-mediated signaling system appears to be involved in differentiation and activation of osteoclasts.
Recent studies have shown that long-term repopulating hematopoietic stem cells (HSCs) first appear in the aorta-gonad-mesonephros (AGM) region. Our immunohistochemistry study showed that TEK+ cells existed in the AGM region. Approximately 5% of AGM cells were TEK+, and most of these were CD34(+) and c-Kit+. We then established a coculture system of AGM cells using a stromal cell line, OP9, which is deficient in macrophage colony-stimulating factor (M-CSF). With this system, we showed that AGM cells at 10.5 days postcoitum (dpc) differentiated and proliferated into both hematopoietic and endothelial cells. Proliferating hematopoietic cells contained a significant number of colony-forming cells in culture (CFU-C) and in spleen (CFU-S). Among primary AGM cells at 10.5 dpc, sorted TEK+ AGM cells generated hematopoietic cells and platelet endothelial cell adhesion molecule (PECAM)-1(+) endothelial cells on the OP9 stromal layer, while TEK- cells did not. When a ligand for TEK, angiopoietin-1, was added to the single-cell culture of AGM, endothelial cell growth was detected in the wells where hematopoietic colonies grew. Although the incidence was still low (1/135), we showed that single TEK+ cells generated hematopoietic cells and endothelial cells simultaneously, using a single-cell deposition system. This in vitro coculture system shows that the TEK+ fraction of primary AGM cells is a candidate for hemangioblasts, which can differentiate into both hematopoietic cells and endothelial cells.
Although IL-10 has been implicated in the pathogenesis of several autoimmune diseases, the mechanisms by which this cytokine mediates inflammatory lesions remain to be elucidated. Exocrine gland destruction is an important early step in the development of Sjögren's syndrome. To better understand the role of IL-10 in Sjögren's syndrome, we made transgenic mice in which the mouse IL-10 gene was regulated by the human salivary amylase promoter. Transgenic expression of IL-10 induced apoptosis of glandular tissue destruction and lymphocyte infiltration consisting primarily of Fas-ligand (FasL)+ CD4+ T cells, as well as in vitro up-regulation of FasL expression on T cells. These data suggest that overexpression of IL-10 in the glands and their subsequent Fas/FasL-mediated bystander tissue destruction is a causal factor in the development of this disease.
Interleukin-1 (IL-1) is one of the most potent bone-resorbing factors involved in bone loss associated with inflammation. We previously reported that IL-1 prolonged the survival of multinucleated osteoclast-like cells (OCLs) formed in cocultures of murine osteoblasts/stromal cells and bone marrow cells via the prevention of spontaneously occurring apoptosis. It was reported that macrophage colony-stimulating factor (M-CSF/CSF-1) prolongs the survival of OCLs without the help of osteoblasts/stromal cells. The present study was conducted to determine whether IL-1 also directly induces the multinucleation and activation of OCLs. Mononuclear osteoclast-like cells (prefusion osteoclasts; pOCs) were purified using the "disintegrin" echistatin from cocultures of murine osteoblastic cells (MB 1.8 cells) and bone marrow cells. Both IL-1 and M-CSF prolonged the survival and induced the multinucleation of pOCs through their respective receptors. However, actin ring formation (a functional marker of osteoclasts) by multinucleated cells was observed in the pOC cultures treated with IL-1, but not those treated with M-CSF. We previously reported that enriched multinucleated OCLs as well as pOCs placed on bone/dentine slices formed few resorption pits, but their pit-forming activity was greatly increased by the addition of osteoblasts/stromal cells. Here, pit-forming activity of both pOCs and enriched OCLs placed on dentine slices was induced by adding IL-1, even in the absence of osteoblasts/stromal cells. M-CSF failed to induce pit-forming activity in pOC and enriched OCL cultures. These results indicate that IL-1 induces the multinucleation and bone-resorbing activity of osteoclasts even in the absence of osteoblasts/stromal cells.
The effect of L-dihydroxyphenylalanine (L-DOPA) on extracellular dopamine (DA) in the striatum was determined by microdialysis in 6-hydroxydopamine (6-OHDA)-lesioned rats treated with and without the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT). At the same time the intensity of L-DOPA-induced rotational behavior was assessed. In 6-OHDA-lesioned rats treated with 5,7-DHT, L-DOPA (50 mg/kg, i.p.) increased extracellular DA only to 20% of that measured in animals not treated with 5,7-DHT. Likewise, 6-OHDA-lesioned rats treated with 5,7-DHT exhibited a significantly lower number of L-DOPA-induced rotations. These results suggest that serotonergic terminals in the striatum can convert exogenously administered L-DOPA into DA that can be released into the extracellular space.
To identify genes expressed in myeloid differentiation, we isolated a cDNA fragment by differential display using RNA prepared from HT93A cells, a human cell line capable of differentiating into neutrophil and eosinophil lineages in response to retinoic acid (RA). Evaluation of the full-length clone isolated from an HT93A cDNA library showed that it encoded a 24 kDa protein comprised of several domains conserved in the Ras superfamily. Comparison of the deduced amino acid sequence of this clone with Rab proteins revealed that it had highest homology to a small GTP-binding protein, murine Rab3D. The mRNA expression of human Rab3D was upregulated in the course of myeloid differentiation, and it was preferentially expressed in granulocytes. These results suggest that human Rab3D may play a specific role in granulocytes, for example in exocytosis of neutrophil-specific granules or in degranulation of both eosinophils and basophils.
To determine whether dopamine release derived from exogenous l-DOPA is under inhibitory control of presynaptic dopamine D2 receptors in the dopaminergic denervated striatum, extracellular dopamine levels were measured in the striatum of 6-hydroxydopamine-lesioned rats using in vivo brain microdialysis. Quinpirole, a D2 agonist, dose-dependently (0.01-3 mg/kg s.c.) inhibited endogenous dopamine release both in the intact and dopaminergic denervated striatum. The dose-response curve obtained from the denervated striatum showed a shift to the right. Administration of l-DOPA (30 mg/kg i.p.) with carbidopa (25 mg/kg i.p.) increased dopamine release to 130% of basal levels in the intact striatum and 770% of basal levels in the denervated striatum. In the intact striatum, dopamine release was continuously inhibited by quinpirole pretreatment (1 mg/kg s.c.) even after l-DOPA administration. In the denervated striatum, l-DOPA-derived dopamine release was not affected by quinpirole pretreatment. These results suggest that, in the striatum with dopaminergic denervation, regulation by presynaptic D2 receptors is still operative on endogenous dopamine release but it does not work on dopamine release derived from exogenously administered l-DOPA.
Recent evidence suggests that leptin, the product of obese (ob) gene, may play an important role in the regulation of reproductive function. However, a possible role of leptin in the preovulatory surges of luteinizing hormone (LH) and prolactin (PRL) in rodents has yet to be explored, and thus examined in this study. Experiments were performed on both normally fed and 3-day starved rats, which were ovariectomized and primed with estradiol and progesterone. At 11:00 h on the day of the experiments, normally fed rats received an intracerebroventricular injection of artificial cerebrospinal fluid, anti-leptin serum, or normal rabbit serum. Three-day starved rats were given artificial cerebrospinal fluid or recombinant human leptin (2.5 microgram) via the same route. From 11:00 to 18:00 h, blood was collected every 30 min to measure LH and PRL. The 3-day starvation completely abolished both LH and PRL surges, but leptin resumed these hormonal surges to the levels of normally fed rats. In addition, anti-leptin serum given to normally fed rats significantly depressed LH surge and delayed the onset of PRL surge. This study is the first to demonstrate that leptin plays a physiologically important role in the generation of steroid-induced LH and PRL surges in female rats.
Human herpesvirus 8 (HHV-8, Kaposi's sarcoma-associated herpesvirus, KSHV) is a new herpes virus isolated from patients with AIDS-associated Kaposi's sarcoma (AIDS-KS). The ORF59 protein of HHV-8 has recently been shown to encode a processivity factor (PF-8) for HHV-8-encoded DNA polymerase. By immunoscreening a cDNA library derived from the HHV-8-infected cell line TY-1, ORF59 antigen was identified in AIDS-KS patients. Immunoblotting revealed that recombinant ORF59 protein reacted with sera from patients with AIDS-KS. Enzyme-linked immunosorbent assay (ELISA) using ORF59-recombinant protein as the antigen revealed that 7 of 22 (31. 8%) AIDS-KS patients and 6 of 263 (2.2%) Japanese HIV-negative patients or healthy blood donors were positive for anti-ORF59 antibodies. Immunohistochemistry using anti-ORF59 rabbit antibodies revealed that this protein was expressed in some of the tumor cells found in KS tissues and that ORF59 protein was detected in 11 of 22 (50%) AIDS-KS tissues. In situ hybridization indicated that some of KS tumor cells were positive for HHV-8 T1.1 mRNA in the same specimen. These data suggest that ORF59 is one of the HHV-8 encoded antigens in patients with AIDS-KS and also indicated that viral replication occurred in some of KS tumor cells.
BACKGROUND AND OBJECTIVE: Precise evaluation of the prognostic factors for hematogenic recurrence after resection for colorectal cancer is important not only for the prediction of patient outcome but also for the determination of adjuvant therapy. The purpose of the current study was to elucidate the clinical significance of using clinicopathological variables in combination with p53 expression as a prognosticator for hematogenic recurrence. METHODS: One hundred forty-two patients with colorectal cancer were examined. The expression of p53 was determined by immunohistochemical staining. RESULTS: Eighteen (60%) of the 30 patients who were positive for both p53 overexpression and lymph node metastasis, 13 (41%) of the 32 patients who were positive for p53 and venous invasion, and 13 (39%) of the 33 patients who were positive for p53 and carcinoembryonic antigen (CEA) developed hematogenic recurrence. CONCLUSIONS: The combination of p53 overexpression and lymph node metastasis was an excellent prognostic indicator for hematogenic recurrence in colorectal cancer.
Hepatocellular carcinoma (HCC) mainly arises from the liver with chronic inflammation. Because telomere reduction reflects replicative history in somatic cells, we analyzed the possibility that liver tissues surrounding HCC consist of the cells carrying substantial reduction of telomere. We studied 20 HCC and surrounding noncancerous liver tissues (SL) obtained by surgical resection, and 10 laparoscopically obtained needle biopsy specimens of the liver with chronic inflammation including no overt HCC (CI). Five liver tissues without chronic liver diseases (ND) were also examined. Extracted genomic DNAs were blotted on a nylon membrane, and probed at first with radio-labeled d(TTAGGG)(3) and reprobed with radio-labeled d(CCT)(7). The intensity caused by d(TTAGGG)(3) was divided by that of d(CCT)(7). The ratio was defined as telomeric repeats content (TC). Dilution experiments reproducibly revealed almost the same TC. The reduction rate of telomere length through aging estimated by regression analysis of TC was 0.62% per year. Concomitant analyses of TC and average telomere length revealed that both values were significantly correlated (r =.45; P =.009). To compare TC in the liver with respect to chronic inflammation, the value was divided by TC in peripheral blood leukocytes (PBL) from the same donor. The ratio was defined as relative TC (RTC). There was a statistically significant decrease of RTC in CI compared with that in ND (P =.03). Furthermore, RTC in SL was significantly lower than that in CI (P =.0001). These observations suggest that RTC value in liver tissues may digitally indicate a replicative history of hepatocytes under chronic inflammation, and a risk of HCC development.
Recombinant retroviruses are most commonly used in hematopoietic stem cell gene therapy trials, but gene transfer efficiency is still inadequate with the present vectors. One approach for overcoming this problem is to develop methods of selecting and enriching the successfully transduced cells. We investigated the feasibility of using the green fluorescent protein (GFP) gene as a selectable marker of hematopoietic cells. When M1 murine leukemia cells were electroporated with GFP expression vectors, a red-shifted mutant (S65T) GFP showed several-fold greater fluorescence than the wild-type GFP and generated readily detectable green light under control of SRalpha or CAG promoter. We then inserted an SRalpha-S65T GFP cassette into the MSCV retrovirus vector and established virus producer cells. Infection of primary murine bone marrow cells resulted in a distinct population with green fluorescence, which was separated by fluorescence-activated cell sorting. The fractionated bright cells gave rise to fluorescent spleen colonies in lethally irradiated mice, while the fluorescence-negative cells yielded only dark colonies. These results indicated that GFP is a faithful marker in gene transfer into hematopoietic progenitor/stem cells, facilitating selection of the transduced cells and tracking of their progeny in vivo.
Osteoclasts, the multinucleated giant cells that resorb bone, develop from hematopoietic cells of the monocyte/ macrophage lineage. Osteoblasts, as well as bone marrow stromal cells, support osteoclast development through a mechanism of cell-to-cell interaction with osteoclast progenitors. We recently purified and molecularly cloned osteoclastogenesis inhibitory factor (OCIF), which was identical to osteoprotegerin (OPG). OPG/OCIF, a secreted member of the tumor necrosis factor (TNF) receptor family, inhibited differentiation and activation of osteoclasts. A single class of high-affinity binding sites for OPG/OCIF appeared on a mouse bone marrow stromal cell line, ST2, in response to 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] and dexamethasone (Dex). When the binding sites were occupied by OPG/OCIF, ST2 cells failed to support the osteoclast formation from spleen cells. To identify an OPG/OCIF ligand, we screened a cDNA expression library of ST2 cells treated with 1,25(OH)2D3 and Dex using OPG/OCIF as a probe. The cloned molecule was found to be a member of the membrane-associated TNF ligand family, and it induced osteoclast formation from mouse and human osteoclast progenitors in the presence of macrophage colony-stimulating factor (M-CSF) in vitro. Expression of its gene in osteoblasts/stromal cells was up-regulated by osteotropic factors, such as 1,25(OH)2D3, prostaglandin E2 (P(GE2), parathyroid hormone (PTH), and interleukin (IL)-11. A polyclonal antibody against this protein, as well as OPG/OCIF, negated not only the osteoclastogenesis induced by the protein, but also bone resorption elicited by various osteotropic factors in a fetal mouse long bone culture system. These findings led us to conclude that the protein is osteoclast differentiation factor (ODF), a long sought-after ligand that mediates an essential signal to osteoclast progenitors for their differentiation into active osteoclasts. Recent analyses of ODF receptor demonstrated that RANK, a member of the TNF receptor family, is the signaling receptor for ODF in osteoclastogenesis, and that OPG/OCIF acts as a decoy receptor for ODF to compete against RANK. The discovery of ODF, OPG/OCIF, and RANK opens a new era in the investigation of the regulation of osteoclast differentiation and function.
We previously reported that osteoblasts/stromal cells are essentially involved in the activation as well as differentiation of osteoclasts through a mechanism involving cell-to-cell contact between osteoblasts/stromal cells and osteoclast precursors/osteoclasts. Osteoclast differentiation factor (ODF, also called RANKL/OPGL/TRANCE) and macrophage colony-stimulating factor (M-CSF, also called CSF-1) are two essential factors produced by osteoblasts/stromal cells for osteoclastogenesis. In other words, osteoblasts/stromal cells were not necessary to generate osteoclasts from spleen cells in the presence of both ODF/RANKL and M-CSF. In the present study, we examined the precise roles of ODF/RANKL and M-CSF in the activation of osteoclasts induced by calvarial osteoblasts. Osteoclasts were formed in mouse bone marrow cultures on collagen gel-coated dishes in response to a soluble form of ODF/RANKL (sODF/sRANKL) and M-CSF, and recovered by collagenase digestion. When recovered osteoclasts were further cultured on plastic dishes, most of the osteoclasts spontaneously died within 24 h. Osteoclasts cultured for 24 h on dentine slices could not form resorption pits. Addition of sODF/sRANKL to the recovered osteoclasts markedly enhanced their survival and pit-forming activity. M-CSF similarly stimulated the survival of osteoclasts, but did not induce their pit-forming activity. When primary mouse osteoblasts were added to the recovered osteoclasts, resorption pits were formed on dentine slices. Bone-resorbing factors such as 1alpha,25-dihydroxyvitamin D3, parathyroid hormone, or prostaglandin E2 enhanced pit-forming activity of osteoclasts only in the presence of osteoblasts. M-CSF-deficient osteoblasts prepared from op/op mice similarly enhanced pit-forming activity of osteoclasts. The pit-forming activity of osteoclasts induced by sODF/sRANKL or osteoblasts was completely inhibited by simultaneous addition of osteoprotegerin/osteoclastogenesis inhibitory factor, a decoy receptor of ODF/RANKL. Primary osteoblasts constitutively expressed ODF/RANKL mRNA, and its level was upregulated by treatment with 1alpha,25-dihydroxyvitamin D3, parathyroid hormone, and prostaglandin E2. These results, obtained by using an assay system that unequivocally assesses osteoclast activation, suggest that ODF/RANKL but not M-CSF mediates osteoblast-induced pit-forming activity of osteoclasts, and that bone-resorbing factors stimulate osteoclast activation through upregulation of ODF/RANKL by osteoblasts/stromal cells.
The src-family protein tyrosine kinase, Lyn, has been reported to play a crucial role in the regulation of B-cell antigen receptor (BCR)-mediated signalling. To elucidate the role of Lyn in the maintenance of immunological tolerance and the prevention of B-1 cell activation and its autoantibody production, Lyn-deficient mice were crossed with transgenic mice carrying the immunoglobulin heavy and light chain genes encoding an autoantibody against mouse red blood cells. In the transgenic mice, most peripheral B cells expressed the B-1 cell phenotype. When the transgenic mice were bred in specific pathogen-free (SPF) conditions, B-1 cells were anergic and did not produce any autoantibody. In contrast, Lyn-deficient transgenic mice kept in the same SPF conditions revealed markedly increased numbers of activated B-1 cells and developed severe autoimmune haemolytic anaemia. Moreover, the mice had a huge splenomegaly containing a remarkable accumulation of erythroblasts, resulted from extramedullary erythropoiesis, in addition to the increased numbers of lymphoblast-like cells of the B-1 cell lineages. The present study demonstrates a crucial role of Lyn kinase in the regulation of B-1 cell activation and maintenance of tolerance.
Several anti-Fas ligand (FasL) inhibitory mAb (FLIM) were raised and characterized in this study. One, FLIM58, showed more potent neutralizing activity than Fas-Fc, the previously established artificial neutralizing agent for FasL. Several murine models of acute graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation have been used to show that both FasL and perforin, the major effector molecules of cytotoxic T lymphocytes, are involved in this disease. In our GVHD model, FasL rather than perforin was associated with lethality. Administration of FLIM58 or Fas-Fc reduced the weight loss and mortality caused by GVHD, although other signs of GVHD, such as skin lesions, lymphoid hypoplasia and mononuclear cell infiltration in the liver, did not improve significantly. FLIM58 was more effective than Fas-Fc in reducing mortality. Our results demonstrated that neutralizing agents for FasL are therapeutic for lethal GVHD.
Adrenomedullin (AM), a hypotensive peptide isolated from human pheochromocytoma, inhibits the proliferation of mesangial cells (MC) induced by mitogens such as platelet-derived growth factor. Quite recently, we have demonstrated that transmural pressure applied to cultured MC increased DNA synthesis and cell proliferation through protein kinase C and tyrosine kinase pathways. However, the modulatory effect of AM on pressure-induced cell proliferation is as yet unknown. In the present study, we examined the effect of AM on transmural pressure-induced DNA synthesis in cultured rat MC. Pressure was applied to cells placed in a sealed chamber using compressed helium. Application of pressure resulted in an increase in [(3)H]thymidine incorporation (approximately 2.0-fold). AM clearly inhibited pressure-induced DNA synthesis in a concentration-dependent manner. This inhibition was paralleled by an increase in cellular cAMP levels evoked by AM. Forskolin and dibutyryl cAMP mimicked the inhibitory effect of AM. The protein kinase A inhibitor H-89 significantly attenuated the effect of AM. Human AM(22-52)-NH(2), a putative AM receptor antagonist, reversed the inhibitory effects of AM more potently than did human CGRP(8-37), a calcitonin gene related peptide receptor antagonist. Our results suggest that AM, by acting mainly on AM-sensitive receptors, inhibits pressure-induced DNA synthesis in cultured rat MC through activation of protein kinase A. AM may play a protective role against MC proliferation in certain pathological conditions.