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Biomedical subjects

N Sakaguchi

Publications and source records attributed to N Sakaguchi.

At least 19 recordsLinked to original sources
PubMed2001

PU.1 is involved in the regulation of B lineage-associated and developmental stage-dependent expression of the germinal center-associated DNA primase GANP.

Germinal center-associated DNA primase (GANP) associated with MCM3 of the DNA replication complex is up-regulated selectively in germinal center B cells. We studied promoter activity of the 5' region involved in the developmental stage-dependent expression in B lineage cells by luciferase reporter assay. Selective regulation of ganp expression was observed in the -737-bp promoter region in B and plasma cell lines but was significantly low in pre-B and T cell lines. The deletion constructs displayed a gap decrease after shortening the region from -134 to -108 bp. Further narrowing suggested the involvement of the PU.1 consensus sequence at -126 bp by electrophoretic mobility shift assay. The protein component PU.1 complex is not inhibited with mutated probes at the consensus site but is inhibited with the known PU.1 probe of CD72 and with anti-PU.1 antibody. Moreover, introduction of PU.1 cDNA enhanced the reporter gene activity in a dose-dependent manner in B cells, whereas the reporter construct with the mutated PU.1 site did not respond. Anti-CD40 stimulation induced the reporter activity with a 100% increase, which is not observed with the PU.1-mutated reporter construct. These results demonstrate that the germinal center-associated DNA primase expression is partly regulated by the transcription factor PU.1 expressed in B lineage cells.

Acetyltransferases↗
PubMed2001

Germinal center-associated nuclear protein (GANP) has a phosphorylation-dependent DNA-primase activity that is up-regulated in germinal center regions.

Antigen stimulation induces a rapid proliferation of B cells for expansion of specific B cell clones and their further differentiation into antibody-producing cells in germinal centers of T-dependent antigen-immunized mice. Previously, we identified a 210-kDa germinal center-associated nuclear protein (GANP) that is up-regulated selectively in germinal centers and carries an MCM-binding domain in the carboxyl-terminal side. In addition, here, we found a region (from 414 to 550 aa) in GANP molecule that is slightly similar to the known DNA-primase component p49. The recombinant GANP fragment covering this region synthesizes RNA primers for extension by DNA polymerase I with single-stranded DNA templates in vitro. GANP DNA-primase activity is controlled by phosphorylation at Ser(502) that is induced by CD40-mediated signaling in vitro and in the germinal center B cells stimulated with antigen in vivo. Overexpression of ganp cDNA in Daudi B cells caused the increased DNA synthesis more than the levels of the mock-transfectants. These evidences suggested that the novel DNA-primase GANP is involved in regulation of cell proliferation of antigen-driven B cells in germinal centers.

Acetyltransferases↗
PubMed2001

IL-1 enhances T cell-dependent antibody production through induction of CD40 ligand and OX40 on T cells.

IL-1 is a proinflammatory cytokine that plays pleiotropic roles in host defense mechanisms. We investigated the role of IL-1 in the humoral immune response using gene-targeted mice. Ab production against SRBC was significantly reduced in IL-1alpha/beta-deficient (IL-1(-/-)) mice and enhanced in IL-1R antagonist(-/-) mice. The intrinsic functions of T, B, and APCs were normal in IL-1(-/-) mice. However, we showed that IL-1(-/-) APCs did not fully activate DO11.10 T cells, while IL-1R antagonist (-/-) APCs enhanced the reaction, indicating that IL-1 promotes T cell priming through T-APC interaction. The function of IL-1 was CD28-CD80/CD86 independent. We found that CD40 ligand and OX40 expression on T cells was affected by the mutation, and the reduced Ag-specific B cell response in IL-1(-/-) mice was recovered by the treatment with agonistic anti-CD40 mAb both in vitro and in vivo. These observations indicate that IL-1 enhances T cell-dependent Ab production by augmenting CD40 ligand and OX40 expression on T cells.

Adjuvants, Immunologic↗
PubMed2001

Localization of recombination activating gene 1/green fluorescent protein (RAG1/GFP) expression in secondary lymphoid organs after immunization with T-dependent antigens in rag1/gfp knockin mice.

Secondary rearrangements of immunoglobulin gene segments that generate a new antibody repertoire in peripheral B cells have been described as receptor revision and occur by as yet unknown mechanisms. To determine the importance of recombination activating gene (RAG) expression in receptor revision, heterozygous rag1/green fluorescent protein (gfp) knockin mice were used to examine the location of RAG1 expression in the germinal centers (GCs) of lymphoid follicles after immunization with a variety of T-cell-dependent antigens. Immunization of rag1/gfp heterozygous mice or rag1 homozygous knockout mice reconstituted with rag1/gfp heterozygous spleen cells caused the down-regulation of RAG1/GFP signal in GCs. Although some RAG1/GFP(+) cells appeared in regions surrounding the peanut agglutinin (PNA)(+)GL-7(+) GC area, RAG1/GFP(+) cells did not accumulate in the central region. In addition, the stimulation of spleen B cells with anti-mu antibody plus interleukin-4 (IL-4) or with anti-CD40 monoclonal antibody plus IL-7 did not induce GFP signals at detectable levels in vitro. These results clearly demonstrate that RAG1 re-expression either does not occur or is at extremely low levels in antigen-driven B cells in GCs of secondary lymphoid follicles, suggesting that other mechanisms may mediate the gene rearrangements observed in receptor revision.

Animals↗
PubMed2001

CD40 expression is induced by the introduction of IgM receptor on the surface of pro-B cell line NFS70.

To examine the molecular mechanism of B cell differentiation, we introduced rearranged immunoglobulin (Ig) mu- and kappa-chain genes into the NFS70 pro-B cell line and observed their maturation. The IgR(+)-transfectants had characteristics of mature surface IgM (sIgM)+ B220high CD40+ CD38+ CD25+ B cells. CD40 expression levels were regulated by stimulation via the IgR. In comparison to wild type NFS70 cells, NF-kappaB activity was up-regulated in the IgR transfectants. Anti-IgR crosslinking of IgR+ cells induced down-regulation of CD40 expression that correlated with down-regulation of NF-kappaB activity in the IgR(+)-transfectants. Immature CD19+ sIgD- B cells from bone marrow also showed an alteration of CD40 expression in response to anti-IgR crosslinking. The results suggest that expression of IgR on the surface is one of the factors responsible for further maturation of B cells.

Animals↗
PubMed2001

Increased T cell autoreactivity in the absence of CD40-CD40 ligand interactions: a role of CD40 in regulatory T cell development.

Mutations in the CD40 ligand (CD40L) gene lead to X-linked immunodeficiency with hyper-IgM, which is often associated with autoimmune diseases. To determine the contribution of defective CD40-CD40L interactions to T cell autoreactivity, we reconstituted CD40-CD40L interactions by transferring T cells from CD40-deficient mice to syngenic athymic nude mice and assessed autoimmunity. T cells from CD40-deficient mice triggered autoimmune diseases accompanied with elevations of various autoantibodies, while those from wild-type mice did not. In CD40-deficient mice, the CD25(+) CD45RB(low) CD4(+) subpopulation which regulates T cell autoreactivity was markedly reduced. CD40-deficient APCs failed to induce T regulatory cells 1 producing high levels of an inhibitory cytokine, IL-10 in vitro. Furthermore, autoimmune development was inhibited when T cells from CD40-deficient mice were cotransferred with CD45RB(low) CD4(+) T cells from wild-type mice or with T regulatory cells 1 induced on CD40-expressing APCs. Collectively, our results indicate that CD40-CD40L interactions contribute to negative regulation of T cell autoreactivity and that defective interactions can lead to autoimmunity.

Animals↗
PubMed2001

Immunologic self tolerance maintained by T-cell-mediated control of self-reactive T cells: implications for autoimmunity and tumor immunity.

T-cell-mediated dominant control of self-reactive T cells is one mechanism for maintaining immunologic self tolerance. It also hampers the generation of immunity to autologous tumor cells. Abrogation of the control can evoke potent tumor immunity as well as autoimmunity in normal animals. This common regulatory mechanism for autoimmunity and tumor immunity can be exploited to devise a novel immunotherapy against cancer.

Animals↗
PubMed2001

Direct observation of a Ti/Cu interface with atomic displacement under electron irradiation.

UNLABELLED: A Ti film was deposited onto a Cu substrate by means of a radio frequency magnetron sputtering METHOD: Cross-sectional thin foils for TEM observation were prepared using a focused ion beam. Electron irradiation was carried out using a high-resolution high-voltage electron microscope operated at 1.25 MV. The Cu/Ti interface of the foils was irradiated at 623 K. In-situ observation images during electron irradiation were recorded by a CCD camera with a digital video cassette. The (020)Cu plane on the Cu/Ti interface preferentially moved towards the Ti film with irradiation. Composition analysis of the diffused region showed that its composition corresponded to Ti3Cu2.

Journal Article↗
PubMed2001

A rat model of hypereosinophilic syndrome.

Hypereosinophilia-occurring rats without chemical and antigen treatment have been maintained in our laboratory. The rat, Matsumoto Eosinophilia Shinshu (mes), showed hypereosinophilia at the age of 9 weeks or older and developed eosinophil-related inflammatory lesions in many organs. These lesions included: aortitis, granulomatous lesion in the mesenteric lymph node, inflammatory fibroid polyp of the stomach and pulmonary vasculitis with septal infiltration. These lesions were involved with cellular infiltration of eosinophils and macrophages, and deposition of eosinophilic crystals which immunohistologically showed major basic protein and eosinophilic peroxidase derived from eosinophilic lysosomal constituents. Although the distribution of lesions in mes is a little different from that of hypereosinophilic syndrome (HES) in humans, in that endomyocardial fibrosis appears in HES while aortitis appears in mes, mes is probably comparable with HES. The present paper describes the pathological aspects of the lesions in mes and discusses the pathogenesis of tissue injury related to eosinophilic infiltration.

Animals↗
PubMed2001

Histological diversity of vasculitic lesions in MPO-ANCA-positive autopsy cases.

To investigate the variety of histological features of vasculitic lesions in myeloperoxidase-specific antineutrophil cytoplasmic antibody (pANCA)-related vasculitis, retrospective pathological analysis was done on 13 autopsy cases, collected from 1990 to 1998 at five hospitals. These cases were classified into three groups: (i) pulmonary-renal syndrome characterized by capillaritis of lung and glomeruli with occasional small-vessel arteritis and/or phlebitis; (ii) glomerular capillaritis without pulmonary involvement associated with significant small-vessel arteritis; and (iii) extensive distribution of small-vessel arteritis with no capillary involvement. The results suggest that pANCA-related vasculitis encompasses a wide variety of vasculitic syndromes, including pulmonary-renal syndrome, microscopic polyarteritis nodosa, and classic polyarteritis nodosa. pANCA may contribute to pathogenesis in all of these cases.

Aged↗
PubMed2001

Reference values and limited serological survey for the Iriomote cat in Japan.

Clinical investigations and hematological, serum biochemical, and serological surveys were carried out on 11 male and 6 female Iriomote cats (Felis iriomotensis) in Japan. Examined Iriomote cats were considered clinically healthy by the inspection for the general physical conditions. However, urinalysis suggested the inflammation of the urinary tract in all the cats. Antibody for feline panleukopenia virus was positive in one of the examined Iriomote cats, which suggested a previous infection.

Animals↗
PubMed2000

Structure, expression, and chromosomal localization of the human gene encoding a germinal center-associated nuclear protein (GANP) that associates with MCM3 involved in the initiation of DNA replication.

A 210kDa protein named GANP is upregulated in germinal center (GC)-B cells in the spleen of antigen-immunized mouse. We studied a human ganp gene (hganp) encoding a putative polypeptide of 1980 amino acids. The carboxyl-terminal 721-amino-acid sequence of hGANP is identical to Map80, that is presumably generated by alternative splicing of hganp/Map80 gene. The genomic segment carrying hganp and Map80 genes was isolated, and the chromosomal location was determined on 21q22.3. Northern blot analysis with RNAs from various organs demonstrated a single band of 7kb hganp mRNA, which suggests a preferential transcription of hganp gene from the hganp/Map80 locus. The hGANP expression was upregulated in GCs of the tonsil, as demonstrated by in-situ RNA hybridization and immunohistochemical analyses. The hGANP, with the domain (Map-box) capable of binding to MCM3 in B cells, might be involved in regulation of cell-cycle progression and DNA replication of GC-B cells.

Acetyltransferases↗
PubMed2000

Immunologic self-tolerance maintained by CD25(+)CD4(+) regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4.

This report shows that cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) plays a key role in T cell-mediated dominant immunologic self-tolerance. In vivo blockade of CTLA-4 for a limited period in normal mice leads to spontaneous development of chronic organ-specific autoimmune diseases, which are immunopathologically similar to human counterparts. In normal naive mice, CTLA-4 is constitutively expressed on CD25(+)CD4(+) T cells, which constitute 5-10% of peripheral CD4(+) T cells. When the CD25(+)CD4(+) T cells are stimulated via the T cell receptor in vitro, they potently suppress antigen-specific and polyclonal activation and proliferation of other T cells, including CTLA-4-deficient T cells, and blockade of CTLA-4 abrogates the suppression. CD28-deficient CD25(+)CD4(+) T cells can also suppress normal T cells, indicating that CD28 is dispensable for activation of the regulatory T cells. Thus, the CD25(+)CD4(+) regulatory T cell population engaged in dominant self-tolerance may require CTLA-4 but not CD28 as a costimulatory molecule for its functional activation. Furthermore, interference with this role of CTLA-4 suffices to elicit autoimmune disease in otherwise normal animals, presumably through affecting CD25(+)CD4(+) T cell-mediated control of self-reactive T cells. This unique function of CTLA-4 could be exploited to potentiate T cell-mediated immunoregulation, and thereby to induce immunologic tolerance or to control autoimmunity.

Abatacept↗
PubMed2000

LDL receptor-related protein as a component of the midkine receptor.

Midkine (MK) is a heparin-binding growth factor with migration-promoting and survival-promoting activities. To identify signaling receptor(s) of MK, membrane glycoproteins with MK-binding activity were isolated from day 13 mouse embryos by lectin- and MK-affinity chromatography. SDS-PAGE followed by protein sequence analysis revealed the presence of LDL receptor-related protein (LRP) and NCAM in the fraction. The dissociation constant of binding between LRP and MK was 3.5 nM. Receptor-associated protein (RAP), which interfered with the binding, inhibited MK-dependent survival of embryonic neurons. Brushin/megalin, which is also a high molecular weight protein belonging to the LDL receptor family, bound to MK less strongly than LRP. These findings suggest that LRP is a component of the receptor complex for MK.

Amino Acid Sequence↗
PubMed2000

A novel nuclear phosphoprotein, GANP, is up-regulated in centrocytes of the germinal center and associated with MCM3, a protein essential for DNA replication.

Antigen (Ag) immunization induces formation of the germinal center (GC), with large, rapidly proliferating centroblasts in the dark zone, and small, nondividing centrocytes in the light zone. We identified a novel nuclear protein, GANP, that is up-regulated in centrocytes. We found that GANP was up-regulated in GC B cells of Peyer's patches in normal mice and in spleens from Ag-immunized mice. GANP-positive cells appeared in the light zone of the GC, with coexpression of the peanut agglutinin (PNA) (PNA)-positive B220-positive phenotype. The expression of GANP was strikingly correlated with GC formation because Bcl6-deficient mice did not show the up-regulation of GANP. GANP-positive cells were mostly surrounded by follicular dendritic cells. Stimulation with anti-micro and anti-CD40 induced up-regulation of ganp messenger RNA as well as GANP protein in B220-positive B cells in vitro. GANP is a 210-kd protein localized in both the cytoplasm and nuclei, with a homologous region to Map80 that is associated with MCM3, a protein essential for DNA replication. Remarkably, GANP is associated with MCM3 in B cells and MCM3 is also up-regulated in the GC area. These results suggest that the up-regulation of GANP might participate in the development of Ag-driven B cells in GCs through its interaction with MCM3.

Acetyltransferases↗
PubMed2000

Recurrent idiopathic iridocyclitis after autologous peripheral blood stem-cell transplantation followed by G-CSF administration for acute lymphoblastic leukemia.

We describe a patient who experienced a recurrence of idiopathic iridocyclitis on day 12 after autologous peripheral blood stem-cell transplantation (auto-PBSCT) followed by G-CSF administration for acute lymphoblastic leukemia (ALL). Autologous SCT has been reported to be effective and safe in achieving dose intensification of chemotherapeutic drugs for the treatment of hematopoietic malignancies, but its therapeutic effect on autoimmune diseases is not definite. The findings from the present case suggest that auto-PBSCT followed by G-CSF administration for patients with a history of some kind of autoimmune disorders may induce exacerbation or recurrence of its symptoms after hematopoietic recovery.

Combined Modality Therapy↗
PubMed2000

Role of reactive oxygen species in gallic acid-induced apoptosis.

We earlier demonstrated that gallic acid (3,4,5-trihydroxybenzoic acid) induced apoptosis in promyelocytic leukemia HL-60RG cells, which was inhibited by catalase and intracellular Ca2+ chelator. In this study, we further studied the involvement of reactive oxygen species (ROS) and intracellular Ca2+ in gallic acid-induced apoptosis. The enhancement of intracellular ROS in HL-60RG cells was detected dose-dependently as early as 5 min after stimulation with gallic acid by using 5,6-carboxy-2',7'-dichlorofluorescin diacetate (DCFH-DA). Further studies that used various antioxidants and ROS scavengers showed that the intracellular peroxide level was well correlated with the potency to induce apoptosis and that the increased intracellular peroxides after gallic acid treatment seemed likely to result from the influx of H2O2 derived from superoxide which were generated extracellularly. In addition, gallic acid, HX/XO, and H2O2-induced apoptosis was completely inhibited by pretreatment with intracellular Ca2+ chelator 1,2-bis(2-aminophenoxyethane)-N,N,N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), but increase of intracellular peroxide levels by gallic acid were suppressed only slightly. It is suggested that intracellular ROS induced by gallic acid plays an important role in eliciting an early signal in apoptosis. Especially, H2O, which is derived from superoxide anion generated extracellularly may increase intracellular Ca2+ levels or cooperate with intracellular Ca2+, thus resulting in apoptosis induction.

Antioxidants↗