Biomedical subjects

A Muraguchi

Publications and source records attributed to A Muraguchi.

At least 37 records · Page 2Linked to original sources

PubMed1998

Immature thymocyte antigen-1: a novel thymocyte marker discriminating pre- and post-selected thymocytes.

Previously, we described a mAb (1-23) reacting with a novel cell surface antigen expressed on thymocytes at late CD4-CD8- [(double negative (DN)] to early CD4+CD8+ [(double positive (DP)] differentiation stage. Since the expression of this molecule was restricted to immature thymocytes, we designated it as immature thymocyte antigen-1 (IMT-1). In this study, we have investigated the relevance of IMT-1 expression to thymocyte selection using TCR transgenic mice, scid mice or RAG-2-/- mice. The IMT-1+ population in DP thymocytes was decreased in the thymuses of MHC class I-restricted or class II-restricted TCR transgenic mice with a positively selecting MHC background when compared with that of the mice with a non-selecting MHC background. IMT-1+ DP thymocytes were also decreased in TCR transgenic mice in which negative selection occurs. When DP thymocytes in H-Y TCR transgenic mice were stimulated with CD3epsilon mAb in vitro as well as in vivo, the expression of IMT-1 on DP thymocytes was decreased. Furthermore, the expression of IMT-1 on DN thymocytes from RAG-2-/- mice was drastically reduced when CD3epsilon mAb was challenged in vivo. These results suggest that the expression of IMT-1 on DP or DN thymocytes is down-regulated by stimulation through TCR as well as pre-TCR. Taken together, these results show that IMT-1 is a unique surface marker which exquisitely separates pre-selected thymocytes from post-selected thymocytes.

Animals↗

PubMed1998

Stromal cells and cytokines in the induction of recombination activating gene (RAG) expression in a human lymphoid progenitor cell.

The activation of recombination activating genes (RAGs) plays critical roles in the V(D)J gene recombination machinery and lymphocyte repertoire formation. However, the regulation of RAG gene expression in humans as well as animals is poorly understood. We show that RAG gene expression is activated in a human lymphoid progenitor cell line (FL8.2.4.4) by coculturing them on a bone marrow-derived stromal cell line (PA6) in the presence of cytokines. The RAG transcripts become detectable in 12 hours after initiation of culture, and the increased level is sustained at 24 hours. Among the cytokines, IL-3, IL-6, and IL-7, but not IL-2, IL-4, SCF, GM-CSF induces RAG activation. IL-3, IL-6, and IL-7 exert their effect synergistically on RAG activation. A cognate interaction between FL8.2.4.4 cells and PA6 stromal cells seems to be prerequisite for RAG activation. RAG transcripts are inducible in FL8.2.4.4 cells when cocultured on paraformaldehyde fixed-PA6 stromal cells in the presence of cytokines. These data indicate that two separate signals are both required for induction of RAG activation in lymphoid progenitors; one from the cell surface molecule(s) on stromal cells, and the other from recombinant cytokine(s). The expression of RAG mRNA in FL8.2.4.4 cells is concomitant with induction of recombinase activity. Thus, this system may provide a useful means for further understanding of the mechanisms controlling RAG activation and lymphocyte development in human system.

Cell Line↗

PubMed1996

Induction of recombination activating gene expression in a human lymphoid progenitor cell line: requirement of two separate signals from stromal cells and cytokines.

The activation and expression of recombination activating genes (RAGs) plays critical roles in V(D)J gene recombination machinery and lymphocyte development. We showed that RAG gene expression was induced in freshly isolated human bone marrow cells and a human lymphoid progenitor cell line, FL8.2.4.4, by coculture on a monolayer of a murine bone marrow-derived stromal cell line, PA6, in the presence of a mixture of recombinant cytokines. The RAG transcripts were detected 12 hours after initiation of culture, and the increased level was sustained at 24 hours. Among recombinant cytokines, interleukin-3 (IL-3), IL-6, and IL-7, but not IL-2, IL-4, stem cell factor (SCF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) could induce RAG-1 activation in FL8.2.4.4 cells, and a significant synergistic effect between IL-3, IL-6, and IL-7 was observed. Using a double chamber culture technique, it was shown that a cognate interaction between FL8.2.4.4 cells and PA6 stromal cells was a prerequisite for RAG-1 activation. Furthermore, RAG-1 transcripts were induced in FL8.2.4.4 cells when they were cocultured on paraformaldehyde-fixed PA6 stromal cells in the presence of cytokines. These results suggest that two separate signals are both required for induction of RAG-1 activation in lymphoid progenitors; one from the cell surface molecule(s) on stromal cells, and the other from the recombinant cytokine(s). Finally, we showed that expression of RAG mRNA in FL8.2.4.4 cells was concomitant with induction of recombinase activity. This system may provide useful means for further understanding the mechanisms controlling RAG activation and lymphocyte development in the human system.

Bone Marrow Cells↗

PubMed1996

Chromatin structure and transcriptional regulation of human RAG-1 gene.

The recombination activating genes (RAGs) play a critical role in V(D)J recombination machinery and lymphocyte development. Their expression is strictly regulated during lymphocyte ontogeny, with expression being rapidly lost as the lymphoid precursors differentiate into their progeny. To elucidate molecular mechanisms of regulation of human RAG-1 gene expression, we examined a chromatin structure of a approximately 24-kb DNA segment adjacent to a human RAG-1 promoter region in various cell lines by analyzing DNase I hypersensitive (DNase I HS) sites. In a RAG-1-expressing human pre-B-cell line, at least four DNase I HS sites (HS1, HS2, HS3, and HS4) were identified. Among these HS sites, one HS site (HS1) was ubiquitously detected in all cell lines examined, but the other three HS sites (HS2, HS3, and HS4) were associated only with RAG-1-expressing lymphoid cell lines. Using transient expression assays, we showed that the 5' upstream region of the major transcription start site showed low but significant promoter activity and that a DNA segment within HS3 located in the promoter region was indispensable to its basal promoter activity. Importantly, this promoter region was shown to be active in both RAG-1-expressing and RAG-1-nonexpressing cell lines. These results suggest that alteration of chromatin structure in the promoter region, in addition to other control elements outside of the promoter region, is one of the mechanisms regulating tissue- and stage-specific expression of human RAG-1 gene.

B-Lymphocytes↗

PubMed1996

Molecular cloning and characterization of a novel stromal cell-derived cDNA encoding a protein that facilitates gene activation of recombination activating gene (RAG)-1 in human lymphoid progenitors.

The activation and expression of recombination activation genes (RAGs) in lymphoid progenitors are regulated by signals from surface molecules of stromal cells and/or cytokines. Using a mRNA differential display method, we isolated a novel stromal cell-derived cDNA clone, C2.3, whose transcripts were intensively expressed in RAG-1-inducible stromal cell line, but rarely expressed in RAG-1-non inducible mutant cell line (PA6). The cDNA sequence had no homology to the known genes. The sequence revealed an open reading frame that encodes a 221 amino acid protein with 4 potential transmembrane domains, suggesting a possible role of C2.3 product as a membrane receptor. Introduction of C2.3 cDNA into PA6 mutant line restored the ability to activate RAG-1 gene in lymphoid progenitors, indicating that a C2.3 product may be involved in the induction of RAG-1 gene activation.

Amino Acid Sequence↗

PubMed1996

Isolation and characterization of a TATA-less promoter for the human RAG-1 gene.

Human recombination activating gene-1 (RAG-1) genomic DNA clones containing the first exon coding for the 5' untranslated region and the second exon coding for the remaining 5' untranslated region, coding region, and 3' untranslated region were cloned. Primer extension analysis and RNase protection analysis demonstrated the multiple RAG-1 transcription start sites, clustered in a 31 nucleotide (nt) region. Sequence analysis showed that the RAG-1 promoter lacked a TATA box as well as an initiator sequence. Transient expression assays using a luciferase reporter gene with truncated promoter fragments and substitution mutants, showed that the 5' promoter region containing the CCAAT box between -110 and -86, is indispensable for its basal promoter activity in RAG-1 expressing Nalm 6 cell line. Comparative transient expression assays in various cell lines revealed that the 854 nt upstream promoter region was active, not only in RAG-1 expressing cell lines but also in RAG-1 non-expressing cell lines. These data indicate that the 854 nt upstream region of RAG-1 gene confer basal promoter activity, and that the tissue- and stage-specific expression of RAG-1 is controlled by elements present outside of the promoter region and/or differential chromatin structure(s) of the individual cells.

Base Sequence↗

PubMed1996

Purification and characterization of an allergenic monomeric hemoglobin from a chironomid distributed worldwide, Polypedium nubifer.

The Pol n component MV, a potent experimental allergen for mice, was purified to homogeneity from extracts of a chironomid distributed worldwide, Polypedium nubifer (PN). The Pol n I component MV was shown to have cross-reactivity to hemoglobins (Hb) derived from all species of chironomids tested. Determination of the amino acid sequence of the first 37 N-terminal residues revealed that it had 30-59% homology to Hb of an European chironomid, Chironomus thummi thummi, which had been known as an important allergen for humans. By Western blot analysis, we showed that sera from asthmatic patients, which had positively reacted to the extract of the adult PN midge, bound to the purified Pol n I component MV. Furthermore, using rabbit polyclonal antibodies raised against synthetic polypeptides corresponding to the N-terminal residues, it was demonstrated that the N-terminal amino acid sequence between position 15 and 35 contained antigenic epitope(s) for human IgE. The results indicate that the Pol n I component MV is an allergen for human beings as well as for mice, and useful as a diagnostic tool for chironomid allergy.

Adolescent↗

PubMed1995

A novel cell surface antigen, immature thymocyte antigen-1, is involved in the differentiation of murine thymocytes.

A mAb, 1-23, which recognizes a novel cell surface Ag on immature murine thymocytes (designated as IMT-1 for immature thymocyte antigen-1) was prepared. IMT-1 was found to be expressed on 40 to 50% of CD4-8- double negative (DN), 5 to 10% of CD4-8+, and 5 to 20% of CD4+8+ double positive (DP) thymocytes in adult mice, but not expressed on CD4+8- thymocytes or peripheral T lymphocytes. It was not expressed on either nonlymphoid cell lines or thymic stromal cells. In subsets of DN thymocytes, IMT-1 was detected on 40 to 50% of the heat-stable Ag+, 15% of CD44+25+, 70% of CD44-25+, and 70% of CD44-25- populations, whereas it was not detected on heat-stable Ag- and CD44+25- populations. IMT-1+ thymocytes expressed low levels of or no CD3 molecules. In fetal thymuses, IMT-1 was expressed on a minor population of thymocytes at day 14.5 and 15.5 of gestation. However, at day 16.5 of gestation, a majority of DN as well as DP thymocytes became IMT-1-positive. Addition of the 1-23 Ab to the fetal thymus organ culture relatively increased CD8+ SP thymocytes. These results show that IMT-1 is expressed during the late DN stage as well as the early DP stage of thymocyte maturation and suggest the possible involvement of IMT-1 in thymocyte differentiation.

Animals↗

PubMed1995

Reciprocal IL-1 beta gene expression in medial and lateral hypothalamic areas in SART-stressed mice.

Specific alteration of rhythm of temperature (SART) stress has been found to induce thymic atrophy via activation of the hypothalamus-pituitary-adrenal (HPA) axis. We demonstrate here that SART stress induces increment of IL-1 beta mRNA levels in the medial hypothalamic area (MHA) and decrement of IL-1 beta mRNA levels in the lateral hypothalamic area (LHA). The altered levels of IL-1 beta expression in these loci return to those of non-stressed mice upon cessation of the stress. These data imply that the reciprocal wave of SART stress-induced IL-1 beta gene expression in MHA and LHA may contribute to activation of the HPA axis and the resulting immunological dysfunction.

Animals↗

PubMed1995

Expression of 18.6/CD23 antigen on human lymphoid progenitor cell lines and phorbol 12-myristate 13-acetate (PMA)-induced microglia-shaped cells.

The nature of lymphoid progenitors and factor(s) determining commitment to either the T- or B-lymphocyte pathway are poorly understood in the human system. In this study, we generated a monoclonal antibody (MoAb), 18.6, that recognizes a cell surface antigen on a human lymphoid progenitor cell line (FL4.4). MoAb 18.6 reacted with lymphoid progenitor lines, B lymphoid cell lines, and myelomonocytic cell lines. It did not react with any T cell or erythroid leukemic cell lines. Two color FACS analyses of normal lymphoid tissues showed that MoAb 18.6 reacted with a majority of CD20+ mature B cells and a minority of CD64+ monocytes. Molecules of 3 different sizes with MW of 34, 45, and 68 Kd were precipitated with MoAb 18.6 from the lymphoid progenitor cell line. The 18.6 antigen was not expressed on a fetal liver-derived lymphoid progenitor-like cell line, FL1.4, which has the capacity to differentiate into microglia-shaped cells upon PMA-stimulation. Stimulation of FL1.4 cells with PMA induced expression of the 18.6 antigen within 24 hr and the microglia-shaped cells stained positively with MoAb 18.6. Finally, cloning of a cDNA that encoded the 18.6 antigen revealed that the 18.6 antigen is identical to the CD23 antigen. Taken together, these data suggest that the 18.6/CD23 antigen is expressed on lymphoid precursors at a very early stage of differentiation.

Adult↗

PubMed1994

Signal transmission through MHC class II molecules in a human B lymphoid progenitor cell line: different signaling pathways depending on the maturational stages of B cells.

The function of MHC class II HLA-DR molecules expressed on a human B lymphoid progenitor cell line FL8.2.4.4 (abbreviated as FL4.4) was examined. FL4.4 cells expressed HLA-DR molecules and stimulation of the DR molecules by anti-DR mAb or by superantigen TSST-1 induced strong augmentation of homocytic aggregation and protein tyrosine phosphorylation in FL4.4 cells. Induced homocytic aggregation in FL4.4 consists both of LFA-1/ICAM-1-dependent and -independent pathways as revealed by mAb blocking experiments. Metabolic inhibitors, NaN3 and cytochalasin B, blocked the induced homocytic aggregation of FL4.4. Early mature Daudi B cell lines also showed a similar type of homocytic aggregation by stimulation with anti-DR mAb. Daudi cells are more sensitive to protein kinase inhibitors herbimycin A and H7 than FL4.4 cells in their blocking of induced homocytic aggregation, while W7 showed stronger inhibitory effects on FL4.4 cells than on Daudi cells. Western blotting analysis revealed that the stimulation of DR molecules induced protein tyrosine phosphorylation of 100-kDa, 90-kDa, 60-kDa and 55-kDa proteins in FL4.4 cells, while, in Daudi cells 110-kDa, 100-kDa and 80-kDa proteins were phosphorylated. These results suggest that different signaling pathways through class II molecules are employed depending on the maturational stage of B-cell differentiation.

Antibodies, Monoclonal↗

PubMed1994

An in vivo study of hepatic and splenic interleukin-1 beta mRNA expression following oral PSK or LEM administration.

The effects of orally administered biological response modifiers (BRMs) in preventing postoperative micro liver metastasis of primary colorectal cancer were examined in experimental animals. The two BRMs tested were Krestin (PSK) and Lentinus edodes mycelia (LEM). In previous experiments, we found that oral administration of PSK or LEM suppressed liver metastasis and prolonged the survival period. We also found that these agents elevated the liver natural killer (NK) and liver macrophage activities. In the present study in vivo, using reverse transcriptase-polymerase chain reaction (RT-PCR), we examined whether or not the liver and spleen have cytokines which would induce NK cells and macrophages, and whether or not the liver and spleen have cytokines induced by NK cells or macrophages. We placed emphasis on the examination of interleukin (IL)-1 beta expression in the liver and spleen in vivo. Two to six hours after oral administration of PSK or LEM (1 g/kg) to mice, IL-1 beta levels in the liver and spleen rose, and they returned to their baseline levels 24 h later. These findings suggest two possibilities: (1) hepatic IL-1 beta is potentiated by these agents soon after administration, resulting in activation of liver NK cells or macrophages, or (2) these agents stimulate IL-1 beta production by liver macrophages, and the produced IL-1 beta activates liver NK cells or liver macrophages (Kupffer cells). The results of this in vivo study suggest that the potentiation of hepatic and splenic IL-1 beta by PSK and LEM is involved in the early phases of suppression of micro liver metastases of colorectal cancer.

Administration, Oral↗

PubMed1993

Induction of tyrosine phosphorylation in human B lineage cells by crosslinking MB-1 molecule of B cell receptor-related heterodimer complex.

B cell antigen receptor is composed of immunoglobulin and associated MB-1 and B29. Here, we found that anti-human MB-1 stimulation induced tyrosine phosphorylation in immature B cells (FL4.4 and Nalm-6) but not in mature B cells (Daudi). Coprecipitated complex with the heterodimer component in Daudi and Nalm-6 contained the kinase molecule(s) which act on the heterodimer protein, while the complex in early lymphoid cell with germ line antigen receptor genes (FL4.4) did not. Candidate Fyn and Lyn are expressed in Nalm-6 and Daudi but are not expressed in FL4.4. These results suggested that src-type tyrosine kinases as Fyn and Lyn are responsible for the phosphorylation of MB-1 and B29 heterodimer, but anti-MB-1 stimulation can induce tyrosine phosphorylation reaction mediated by other kinase molecule(s) in the progenitor type cells.

Amino Acid Sequence↗

PubMed1993

Generation of cells with morphological and antigenic properties of microglia from cloned EBV-transformed lymphoid progenitor cells derived from human fetal liver.

Single-cell clones from the Epstein Barr virus transformed lymphoid progenitor-like cell line established from human fetal liver at 8-week gestation, have been derived and characterized. These clones retained immunoglobulin (Ig) and T cell receptor (TCR) genes in their germ line configuration. They expressed HLA-DR and some B lymphoid markers such as CD19, CD20, and in some, the T lymphoid marker, CD2. They did not express surface Igs, CD3, CD4, CD8 or TCRs (alpha/beta, gamma/delta). A sensitive RT-PCR assay revealed that they did not express mRNA for a recombination activating gene-1, which is expressed after commitment to lymphoid cells. These results suggest that the established cloned lines are very early lymphoid progenitors that have not yet been committed to lymphoid cell lineage. In one of the lines, FL8.2.1.4, a marked morphological change that resembled microglia was induced when the cells were cultured in the presence of phorbol myristate acetate (PMA). After 72 hr of culture, 5-10% of FL8.2.1.4 cells developed a microglial morphology when stimulated with 10 to 100 ng/ml PMA. The newly generated cells with microglial morphology expressed HLA-DR and stained with Recinus communis agglutinin-1, which has been reported to bind specifically to brain microglia. In contrast, expression of lymphoid markers on cells with microglia-shaped morphology was remarkably diminished by PMA stimulation. Thus, the early lymphoid progenitor cells have the capacity to differentiate into cells with the morphological and antigenic properties of microglia cells. This system might be useful for further understanding of the characteristics and functions of microglia cells distributed in the central nerve system.

Antigens, CD↗

PubMed1992

Expression of the CD2 molecule on human B lymphoid progenitors.

CD2 expression on human B lymphoid progenitor cells was examined. By immunofluorescence analysis, a small fraction of bone marrow B cells was found to express CD2 on their surface. CD2 expression was not demonstrated on peripheral B cells. Epstein-Barr virus-transformed B cell lines derived from fetal liver at 8 weeks of gestation were analyzed to delineate the expression and function of CD2 at the early stage of human B cell development. Characterization of surface and genomic phenotypes of cell lines revealed that the established cell lines represent at least three different phenotypic characteristics of early B lineage cell: B progenitor, pre B, or early B cell. None of the 18 cell lines and 13 subclones with the phenotype of the early B lymphoid cells initially expressed CD2 antigen. However, CD2 expression was induced by the successive cultivation of some cloned B progenitor cell lines. In spite of the expression of CD2, these clones cell lines were unable to form rosettes with sheep red blood cells. By immunoprecipitation analysis, an identical 50 kDa protein was precipitated with anti-CD2 antibody from the lysates of the radioiodinated CD2+B progenitor cell line and peripheral blood T cells. Anti-CD2 antibody induced significant enhancement of proliferation of the CD2+B progenitor subline. These data indicate that human CD2 is expressed on a fraction of B lineage cells at a very early differentiation stage and may play a role in B lymphopoiesis.

Antigens, CD↗

PubMed1992

[Regulation of immune response by cytokine network].

Cytokines are a highly diverse group of intracellular messages. Many cDNAs that encode the cytokines have been cloned in 1980's and the structure of the molecules have been determined. The main function of the cytokines is to amplify the immune and inflammatory responses, keeping them under control at the same time. They orchestrate the response and maintain a proper balance among the various cell types. In this chapter, we summarized the immunoregulation by cytokine network, focussing on the control of antibody production and immunoglobulin class switch by various cytokines produced by helper T cell subsets.

Animals↗

PubMed1991

The prognostic significance of urinary interleukin 6 in IgA nephropathy.

Our previous study has shown that interleukin 6 (IL-6), a multifunctional cytokine, is closely associated with the pathogenesis of mesangial proliferative glomerulonephritis (mesPGN). To investigate whether urinary IL-6 can be used as an indicator in the prognosis of patients with IgA nephropathy, we monitored IL-6 activity in the urine of patients with IgA nephropathy for 10 months and compared IL-6 activity with clinical data as well as the histological changes of the kidneys obtained from the patients. It was found that among the patients who had continuously high urinary IL-6 activity, histological progression of IgA nephropathy was observed. On the other hand, among the patients whose urinary IL-6 became undetectable during the 10-month follow-up, histological improvement of IgA nephropathy was observed. These data suggest that the measurement of urinary IL-6 is a helpful tool for monitoring the progression of IgA nephropathy.

Biomarkers↗

PubMed1989

Involvement of IL-6 in mesangial proliferative glomerulonephritis.

In this study, we demonstrated that IL-6 was a possible autocrine growth factor for rat mesangial cells (MC). rIL-6 induced in vitro growth of rat MC at a concentration of 2 to 200 ng/ml and IL-6 activity was found in the supernatant of cultured rat MC. Northern blot analysis as well as in situ hybridization revealed that IL-6 mRNA was expressed in the cultured MC. Of urine samples from patients with mesangial proliferative glomerulonephritis (PGN) 50% were found to contain significant IL-6 activity (ranging from 30 to 126 pg/ml). Urine samples from other type of primary glomerular diseases such as minimal change nephrotic syndrome or healthy volunteers contained no detectable IL-6 activity. Only 2 of 27 urine samples from membranous nephropathy contained detectable amount of IL-6. Furthermore, there was some relationship between the levels of urine IL-6 and the progressive stage of PGN. Finally, by immunohistochemical staining using an anti-IL-6 mAb, it was shown that MC in the affected glomeruli of PGN patients produced IL-6, whereas MC obtained from the patients with membranous nephropathy, minimal change nephrotic syndrome or normal kidney were not found to produce IL-6. These data suggest that deregulated production of IL-6 is involved in PGN and the measurement of urine IL-6 is helpful for the differential diagnosis of PGN as well as for monitoring the progression of PGN.

Animals↗