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J Miyazaki

Publications and source records attributed to J Miyazaki.

At least 19 recordsLinked to original sources
PubMed2006

In vivo IL-10 gene delivery attenuates bleomycin induced pulmonary fibrosis by inhibiting the production and activation of TGF-beta in the lung.

BACKGROUND: Idiopathic pulmonary fibrosis is a devastating disorder for which there is no effective treatment. Transforming growth factor (TGF)-beta plays a critical role in provoking fibrosis. Interleukin (IL)-10 is a potent immunosuppressive cytokine but its effect on the fibrosing process is unclear. A study was undertaken to examine whether IL-10 affects the production and activation of TGF-beta and thus can attenuate the fibrosis. METHODS: Mice were given an intratracheal injection of bleomycin. On day 1 or 14, IL-10 gene was delivered by rapid intravenous injection of Ringer's solution containing plasmid. Two weeks after the plasmid injection the mice were examined for fibrosis. The effect of IL-10 on TGF-beta production by alveolar macrophages was assessed. RESULTS: Even when delivered during the fibrosing phase, IL-10 gene significantly suppressed the pathological findings, hydroxyproline content, and production of both active and total forms of TGF-beta1 in the lung. Immunohistochemical analyses showed that alveolar macrophages were one of the major sources of TGF-beta1 and IL-10 diminished the intensity of the staining. IL-10 also suppressed the expression of alphaV beta6 integrin, a molecule that plays an important role in TGF-beta activation, on lung epithelial cells. Alveolar macrophages from bleomycin injected mice produced TGF-beta1 spontaneously ex vivo, which was significantly suppressed by treatment of the mice in vivo or by treatment of the explanted macrophages ex vivo with IL-10. CONCLUSION: IL-10 suppresses the production and activation of TGF-beta in the lung and thus attenuates pulmonary fibrosis, even when delivered in the chronic phase.

Animals↗
PubMed2005

Propionibacterium freudenreichii component 1.4-dihydroxy-2-naphthoic acid (DHNA) attenuates dextran sodium sulphate induced colitis by modulation of bacterial flora and lymphocyte homing.

BACKGROUND AND AIMS: 1.4-Dihydroxy-2-naphthoic acid (DHNA), a bifidogenic growth stimulator from Propionibacterium freudenreichii, is thought to have a beneficial effect as a prebiotic; however, its in vivo effect on intestinal inflammation remains unknown. The aim of this study was to determine whether oral administration of DHNA can ameliorate dextran sodium sulphate (DSS) induced colitis and to determine the possible underlying mechanisms. METHOD: Colitis was induced in mice by treatment with 2.0% DSS for seven days. DHNA (0.6 or 2.0 mg/kg) was given in drinking water prior to (preventive study) or after (therapeutic study) DSS administration. Colonic damage was histologically scored, and mucosal addressin cell adhesion molecule 1 (MAdCAM-1) expression and beta7 positive cell infiltration were determined by immunohistochemistry. mRNA levels of proinflammatory cytokines (interleukin (IL)-1beta, IL-6 and tumour necrosis factor alpha (TNF-alpha)) were determined by quantitative real time polymerase chain reaction. In addition, bacterial flora in the caecum, concentrations of short chain acids, and luminal pH were examined. RESULTS: DHNA improved survival rate and histological damage score in mice administered DSS in both the preventive and therapeutic studies. DHNA significantly attenuated the enhanced expression of MAdCAM-1, the increased beta7 positive cell number, and the increased mRNA levels of IL-1beta, IL-6, and TNF-alpha in DSS treated colon. In addition, the decreased number of Lactobacillus and Enterobacteriaceae induced by DSS was recovered by DHNA. Preventive effects on decrease in butyrate concentration and decrease in pH level in mice administered DSS were also observed in the DHNA preventive study. CONCLUSION: DHNA, a novel type of prebiotic, attenuates colonic inflammation not only by balancing intestinal bacterial flora but also by suppressing lymphocyte infiltration through reduction of MAdCAM-1.

Animals↗
PubMed2005

Free fatty acid receptor 1 (FFA(1)R/GPR40) and its involvement in fatty-acid-stimulated insulin secretion.

Free fatty acids (FFA) have generally been proposed to regulate pancreatic insulin release by an intracellular mechanism involving inhibition of CPT-1. The recently de-orphanized G-protein coupled receptor, FFA(1)R/GPR40, has been shown to be essential for fatty-acid-stimulated insulin release in MIN6 mouse insulinoma cells. The CPT-1 inhibitor, 2-bromo palmitate (2BrP), was investigated for its ability to interact with mouse FFA(1)R/GPR40. It was found to inhibit phosphatidyl inositol hydrolysis induced by linoleic acid (LA) (100 muM in all experiments) in HEK293 cells transfected with FFA(1)R/GPR40 and in the MIN6 subclone, MIN6c4. 2BrP also inhibited LA-stimulated insulin release from mouse pancreatic islets. Mouse islets were subjected to antisense intervention by treatment with a FFA(1)R/GPR40-specific morpholino oligonucleotide for 48 h. Antisense treatment of islets suppressed LA-stimulated insulin release by 50% and by almost 100% when islets were pretreated with LA for 30 min before applying the antisense. Antisense treatment had no effect on tolbutamide-stimulated insulin release. Confocal microscopy using an FFA(1)R/GPR40-specific antibody revealed receptor expression largely localized to the plasma membrane of insulin-producing cells. Pretreating the islets with LA for 30 min followed by antisense oligonucleotide treatment for 48 h reduced the FFA(1)R/GPR40 immunoreactivity to background levels. The results demonstrate that FFA(1)R/GPR40 is inhibited by the CPT-1 inhibitor, 2BrP, and confirm that FFA(1)R/GPR40 is indeed necessary, at least in part, for fatty-acid-stimulated insulin release.

Animals↗
PubMed2005

Protective role for cytosolic phospholipase A2alpha in autoimmune diabetes of mice.

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) plays an important role in arachidonate pathway. To investigate the contribution of cPLA(2)alpha to autoimmune diabetes, we established non-obese diabetic (NOD) mouse, an excellent model for human type 1 diabetes, deficient in cPLA(2)alpha. These mice showed severe insulitis and a higher incidence of diabetes. In their macrophages, decreased prostaglandin E(2) (PGE(2)) induced by cPLA(2)alpha deficiency, and the increase in production of tumor necrosis factor (TNF)-alpha were observed. These results suggested that cPLA(2)alpha plays a protective role in progression of insulitis and development of autoimmune diabetes by suppression of TNF-alpha production from macrophages.

Animals↗
PubMed2005

In vivo demonstration of T lymphocyte migration and amelioration of ileitis in intestinal mucosa of SAMP1/Yit mice by the inhibition of MAdCAM-1.

The aetiology of Crohn's disease (CD) remains unknown. Since SAMP1/Yit mice have been reported to develop CD-like spontaneous enteric inflammation, such mice have been studied as an animal model of CD. In this study, using this model we examined T lymphocyte migration in microvessels of intestinal mucosa in vivo and the expression of adhesion molecules by immunohistochemistry. Fluorescence-labelled T lymphocytes isolated from AKR/J (control) mice were injected into the tail veins of recipient mice, and T lymphocyte migration in the postcapillary venules of Peyer's patches, submucosal microvessels, and villus capillaries of the terminal ileum was monitored using an intravital microscope. Adhesion of T lymphocytes was significantly increased in 35 week old SAMP1/Yit mice compared with that in AKR/J or 15 week old SAMP1/Yit mice. Immunohistochemical study showed increased infiltration of CD4, CD8 and beta7-integrin-positive cells and increased expression of MAdCAM-1 and VCAM-1 in the terminal ileum of SAMP1/Yit mice. Antibodies against MAdCAM-1 and VCAM-1 significantly inhibited adhesion of T lymphocytes to microvessels of the terminal ileum, and anti-MAdCAM-1 antibody showed stronger suppressive effect than the anti-VCAM-1 antibody. Periodical administration of anti-MAdCAM-1 antibody twice a week for 7 weeks significantly ameliorated ileitis of SAMP1/Yit mice, but submucosal hypertrophy was not significantly suppressed. Anti-VCAM-1 antibody treatment failed to show significant resolution of ileitis. In addition, anti-MAdCAM-1 antibody treatment also attenuated established ileitis. The results demonstrate that, although MAdCAM-1 and VCAM-1 play an important role in T lymphocyte-endothelial cell interactions in SAMP1/Yit mice, MAdCAM-1 may be a more appropriate target for therapeutic modulation of chronic ileitis.

Animals↗
PubMed2004

Kidney-targeted naked DNA transfer by retrograde injection into the renal vein in mice.

We recently developed a novel kidney-targeted gene transfer technique in rats, using the retrograde renal vein injection of naked plasmid DNA. Many animal disease models are created in mice by transgenic or knockout technologies. However, it is much harder to perform renal vein injection in mice than in rats because they have a thin and short vein. Here we transferred the mouse interleukin (IL)-10 gene into mice by retrograde renal vein injection, using an IL-10 and immunoglobulin fusion protein (IL-10/Fc) (96-kDa) expression plasmid, pCAGGS-IL10/Fc. We observed a dose-response relationship between serum IL-10 levels and the amount of injected DNA. The serum IL-10 levels peaked at day 1 and then were sustained for at least 2 weeks. These results demonstrate that the kidney-targeted naked plasmid DNA transfer of mice by retrograde renal vein injection can be achieved, and the kidney serves as a depot organ for the production of large proteins.

Animals↗
PubMed2004

Post-secretion neutralization of transgene-derived effect: soluble erythropoietin receptor/IgG1Fc expressed in liver neutralizes erythropoietin produced in muscle.

BACKGROUND: The regulation of transgene expression is a key issue for the development of safe gene therapy. Various strategies have been used to regulate protein production at the levels of transgene expression, transcription, translation, and secretion. Neutralization following secretion is another important backup system to prevent super-therapeutic levels of a protein from being expressed by gene transfer. METHODS: We tested whether the soluble human erythropoietin receptor (EpoR)/IgG(1)Fc could neutralize the rat Epo at the post-secretory level and suppress erythrocytosis. RESULTS: To assess whether soluble human EpoR could bind rat Epo in vitro, we used the Epo-dependent human leukemic cell line, AS-E2. EpoR/IgG(1)Fc significantly inhibited the growth of AS-E2 cells in Epo-containing medium. To test this neutralization effect of EpoR/IgG(1)Fc in vivo, we first transferred pCAGGS-Epo into rat muscle by in vivo electroporation, confirmed erythropoiesis for 3 weeks, and then delivered EpoR/IgG(1)Fc by liver-targeted gene transfer via tail-vein injection with hydrodynamics-based transfection. Reticulocyte counts and hematocrit levels in rats that received pCAGGS-EpoR/IgG(1)Fc injections were significantly lower than in rats that received pCAGGS-EpoR, pCAGGS-IgG(1)Fc, or no injection. CONCLUSIONS: These results demonstrate that liver-targeted pCAGGS-EpoR/IgG(1)Fc transfer by tail-vein injection with hydrodynamics-based transfection is useful for neutralizing Epo delivered by in vivo electroporation. This backup strategy at the level of post-secretion could facilitate the clinical application of gene therapy in the future.

Animals↗
PubMed2004

Endotoxin stimulates monocyte-endothelial cell interactions in mouse intestinal Peyer's patches and villus mucosa.

Although monocyte-endothelial cell interactions represent an initial step in controlling the recruitment of monocytes in inflamed tissues, their dynamic processes in microvessels of lymphoid (Peyer's patches) and non-lymphoid (villus) regions in gut-associated lymphoid tissue remain poorly understood. We monitored the migration of fluorescence-labelled monocytes derived from the spleen in intestinal microvessels with or without lipopolysaccharide (LPS) treatment and investigated the role of adhesion molecules, P-selectin, vascular cell adhesion molecule (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). In control mice, there were few interactions between infused monocytes and the endothelium of intestinal microvessels. The monocyte-endothelial interactions (both rolling and adhesion) were significantly increased in intestinal microvessels of LPS-treated mice compared with those in controls. Anti-P-selectin monoclonal antibody (MoAb) significantly suppressed the LPS-induced increase in monocyte rolling in postcapillary venules of Peyer's patches and submucosal venules. Anti-VCAM-1 MoAbs significantly suppressed the LPS-induced increase in monocyte adhesion to postcapillary venules (PCVs) of Peyer's patches, submucosal venules, and villus capillaries. In contrast, anti-ICAM-1 MoAb significantly suppressed the number of adherent monocytes in PCV of Peyer's patches but not in submucosal venules or villus capillaries. These observations demonstrated that LPS treatment resulted in a significant increase in recruitment of monocytes both in microvessels of lymphoid and non-lymphoid regions and that P-selectin, VCAM-1 and ICAM-1 appeared to play important roles in LPS-induced interactions.

Animals↗
PubMed2003

Ectopically expressed PDX-1 in liver initiates endocrine and exocrine pancreas differentiation but causes dysmorphogenesis.

To date, the potency of pancreatic and duodenal homeobox gene 1 (PDX-1) in inducing differentiation into insulin-producing cells has been demonstrated in some cells and tissues. In order to carry out efficient screening of somatic tissues and cells that can transdifferentiate into beta-cell-like cells in response to PDX-1, we generated CAG-CAT-PDX1 transgenic mice carrying a transgene cassette composed of the chicken beta-actin gene (CAG) promoter and a floxed stuffer DNA sequence (CAT) linked to PDX-1 cDNA. When the mice were crossed with Alb-Cre mice, which express the Cre recombinase driven by the rat albumin gene promoter, PDX-1 was expressed in more than 50% of hepatocytes and cholangiocytes. The PDX-1 (+) livers expressed a variety of endocrine hormone genes such as insulin, glucagon, somatostatin, and pancreatic polypeptide. In addition, they expressed exocrine genes such as elastase-1 and chymotrypsinogen 1B. However, the mice exhibited marked jaundice due to conjugated hyperbilirubinemia, and the liver tissue displayed abnormal lobe structures and multiple cystic lesions. Thus, the in vivo ectopic expression of PDX-1 in albumin-producing cells was able to initiate but not complete the differentiation of liver cells into pancreatic cells. The conditional PDX-1 transgenic mouse system developed in this study appeared to be useful for efficient screening of PDX-1 responsive somatic tissues and cells.

Animals↗
PubMed2003

Hydrodynamics-based transfer of PCR-amplified DNA fragments into rat liver.

A high level of plasmid DNA expression in rat liver can be achieved by the rapid injection of a large volume of a naked DNA solution into the tail vein, called the 'hydrodynamics-based procedure.' The preparation of PCR-amplified DNA fragments is easier than that of naked DNA. In this paper we evaluated the effects of expressing the erythropoietin (Epo) gene in the rat liver by injecting fCAGGS-Epo, an Epo-expressing PCR-amplified DNA fragment, via the tail vein. After injection of 5 pmol fCAGGS-Epo (10 microg) or pCAGGS-Epo (18.4 microg), plasmid DNA, the serum Epo levels peaked at week 1, then persisted for at least 12 weeks. Transgene-derived Epo secretion resulted in significant erythropoiesis. These results demonstrated that transfer of PCR-amplified DNA fragments into the rat liver via rapid tail vein injection can be achieved. This method may provide a useful means for studying the physiologic function of a putative gene.

Animals↗
PubMed2003

beta-cell neogenesis induced by adenovirus-mediated gene delivery of transcription factor pdx-1 into mouse pancreas.

beta-cell neogenesis is expected to provide a new therapy for diabetes. Numerous studies have demonstrated that transcriptional regulation involving pdx-1 is essential for endocrine neogenesis in vivo and in vitro. Therefore, it is possible that ectopic expression of pdx-1 in the pancreas could induce endocrine neogenesis. To test this possibility, we performed safe and efficient gene delivery of the pdx-1 gene into the mouse pancreas through the common bile duct using adenoviral vectors, and examined the effects of the ectopic expression of pdx-1. Here we show that adenovirus-mediated expression of pdx-1 can activate the endogenous pdx-1 gene, leading to beta-cell neogenesis and ductal proliferation. This technique is similar to the endoscopic retrograde cholangiopancreatography, which has been already established as a safe procedure for humans. Thus, beta-cell neogenesis induced by adenovirus-mediated expression of pdx-1 provides a novel strategy for gene therapy for a cure for diabetes mellitus.

Adenoviridae↗
PubMed2003

Hydrodynamics-based delivery of the viral interleukin-10 gene suppresses experimental crescentic glomerulonephritis in Wistar-Kyoto rats.

Gene therapy is expected to revolutionize the treatment of kidney diseases. Viral interleukin (vIL)-10 has a variety of immunomodulatory properties. We examined the applicability of vIL-10 gene transfer to the treatment of rats with crescentic glomerulonephritis, a T helper 1 (Th 1) predominant disease. To produce the disease, Wistar-Kyoto rats were injected with a rabbit polyclonal anti-rat glomerular basement membrane antibody. After 3 h, a large volume of plasmid DNA expressing vIL-10 (pCAGGS-vIL-10) solution was rapidly injected into the tail vein. pCAGGS solution was similarly injected into control rats (pCAGGS rats). We confirmed the presence of vector-derived vIL-10 mainly in the liver and observed high serum vIL-10 levels in pCAGGS-vIL-10-injected rats. Compared with the pCAGGS rats, the pCAGGS-vIL-10 rats showed significant therapeutic effects: reduced frequency of crescent formation, decrease in the number of total cells, macrophages, and CD4+ T cells in the glomeruli, decrease in urine protein, and attenuation of kidney dysfunction. Using quantitative real-time polymerase chain reaction, we also observed that this model was Th1-predominant in the glomeruli and that the ratio of the transcripts of CD4, interferon-gamma, tumor necrosis factor-alpha, and monocyte chemotactic protein-1 to the transcripts of glucose-6-phosphate dehydrogenase in the glomeruli were all significantly lower in the pCAGGS-vIL-10 rats than in the pCAGGS rats. These results demonstrate that pCAGGS-vIL-10 gene transfer by hydrodynamics-based transfection suppresses crescentic glomerulonephritis.

Animals↗
PubMed2002

Locally expressed CTLA4-Ig in a pancreatic beta-cell line suppresses accelerated graft rejection response induced by donor-specific transfusion.

AIMS/HYPOTHESIS: This study examined whether locally expressed CTLA4-Ig can suppress the accelerated islet allograft rejection that is induced by donor-specific transfusion. METHODS: CTLA4-Ig-transfected or parental MIN6 cells were transplanted subcutaneously into the right flank of streptozotocin-induced diabetic C3H/Hej mice with or without donor-specific transfusion. For donor-specific transfusion, spleen cells from C57BL/6 mice were injected i.v. at the time of transplantation. In other experiments, CTLA4-Ig-transfected and parental MIN6 cells were transplanted separately into each flank, together with donor-specific transfusion. Rejection was defined as a blood glucose concentration of more than 300 mg/dl in two consecutive measurements, and graft survival was confirmed by hyperglycaemia after the grafts were removed. The effect of an anti-CTLA4 antibody on the survival of CTLA4-Ig-transfected MIN6 cells was also examined. RESULTS: In 7 of 12 donor-specific transfusion sensitised mice, CTLA4-Ig-transfected MIN6 cells remained viable 20 days after grafting, whereas all parental MIN6 cells ( n = 10) were rejected promptly, within 14 days. The prolonged allograft survival was observed even in the absence of detectable levels of serum CTLA4-Ig, while the surviving allografts continued to produce CTLA4-Ig in situ. This protection was abrogated by an anti-CTLA4 antibody, but not by a control antibody. Furthermore, six animals that maintained normoglycaemia after the separate transplantation of parental and CTLA4-Ig-transfected MIN6 cells into each flank all showed abrupt hyperglycaemia after the CTLA4-Ig/MIN6 graft was removed, suggesting that this protection operated locally. CONCLUSION/INTERPRETATION: A beta-cell line genetically engineered to secrete CTLA4-Ig can protect a graft locally from the alloimmune response induced by donor-specific transfusion.

Abatacept↗
PubMed2002

Gene transfer of Fc-fusion cytokine by in vivo electroporation: application to gene therapy for viral myocarditis.

Among a number of techniques for gene transfer in vivo, the direct injection of plasmid DNA into muscle is simple, inexpensive and safe. Although combining direct DNA injection with in vivo electroporation increases the efficiency of gene transfer into muscle, applications of this method have remained limited because of the relatively low expression level. To overcome this problem, we developed a plasmid vector that expresses a secretory protein as a fusion protein with the noncytolytic immunoglobulin Fc portion and used it for electroporation-mediated viral interleukin 10 (vIL-10) expression in vivo. The fusion cytokine vIL-10/mutFc was successfully expressed and the peak serum concentration of vIL-10 was almost 100-fold (195 ng/ml) higher than with a non-fusion vIL-10 expression plasmid. The expressed fusion cytokine suppressed the phytohemagglutinin-induced IFN-gamma production by human peripheral blood mononuclear cells and decreased the mortality in a mouse viral myocarditis model as effectively as vIL-10 expression. These results demonstrate that the transfer of plasmid DNA expressing a noncytolytic Fc-fusion cytokine is useful to deliver enhanced levels of cytokine without altering general biological activities. This simple and efficient system should provide a new approach to gene therapy for human diseases and prove very useful for investigating the function of newly discovered secretory protein genes.

Animals↗
PubMed2001

Intravenous delivery of naked plasmid DNA for in vivo cytokine expression.

We previously demonstrated that electroporation-mediated cytokine gene delivery into muscle is an effective approach for long-term systemic delivery of cytokines. Here we show that hydrodynamics-based gene delivery into mice by intravenous administration of naked plasmid DNA is a more efficient procedure for expressing cytokines in vivo. A large volume of Ringer's solution containing an interleukin-10 (IL-10) expression plasmid pCAGGS-IL10 was rapidly injected into the tail vein of mice. Serum IL-10 levels increased in a dose-dependent manner with a saturation level (50.8 +/- 12.1 microg/ml) 10,000-fold higher than we obtained by the electroporation-mediated method. High levels of serum IL-10 were sustained for at least 2 weeks following a single injection. These results demonstrate that hydrodynamics-based gene delivery could induce sustained high-level expression of cytokines, which would be useful for further studies of cytokine function in vivo and the development of novel immunotherapeutic strategies for systemic cytokine gene therapy.

Animals↗