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D Mochizuki

Publications and source records attributed to D Mochizuki.

30 records · Page 2Linked to original sources
PubMed1986

Regulation of antibody production in vitro by granulocyte-macrophage colony stimulating factor.

The precise molecular characteristics and the mode of action of the T cell derived lymphokines which augment antibody production in vitro remain uncertain. The use of ill-defined culture supernatants to dissect the cellular interactions in vitro involved in antibody production can lead to ambiguous results as the factors may act either on a contaminating non-B-lymphoid population or directly on the B lymphocyte. We report herein the development of a system for measuring in vitro primary antibody responses by murine spleen cells in which endogenous lymphokine production has been minimized by the in vivo administration of cytotoxic antibodies to deplete T lymphocytes and the addition of the glucocorticosteroid, dexamethasone, throughout the culture period. Using such an assay, a lymphokine activity was detected which was capable of augmenting the plaque forming cell response. This lymphokine was present in culture supernatant derived from the lectin activation of the T cell lymphoma, LBRM-33 and was distinct from other known B cell activators, notably IL-2 and IFN gamma. Biochemical purification of this activity indicated that it might be identical to granulocyte-macrophage colony stimulating factor (GM-CSF). The use of recombinant-derived GM-CSF protein unambiguously showed the role of this lymphokine in antibody production. These experiments demonstrated for the first time, the involvement of a hematopoietic factor in antigen-specific immune responses. Moreover, these results demonstrated an important regulatory circuit in the generation of antibody producing B cells in which GM-CSF, derived from activated T cells, stimulates macrophage function.

Animals↗
PubMed1984

Lymphokine purification by reversed-phase high-performance liquid chromatography.

Reversed-phase high-performance liquid chromatography has been used to purify to homogeneity two different lymphokines. Human IL-2 was purified on a C8 reversed-phase column in pyridine-acetate-propanol followed by chromatography on a C18 reversed-phase column in trifluoroacetic acid-acetonitrile. Protein sequence analysis of in situ-generated cyanogen bromide peptides obtained from this preparation established the homogeneity of this material and confirmed the amino acid sequence predicted from the published DNA sequence. Murine CSF-2 alpha was purified on a C18 reversed-phase column in trifluoroacetic acid-acetonitrile followed by chromatography on the same column in pyridine-acetate-propanol. The final preparation yielded a single band on a sodium dodecyl sulfate-polyacrylamide gel with a molecular weight of 24,500.

Amino Acid Sequence↗
PubMed1984

Biochemical comparison of murine colony-stimulating factors secreted by a T cell lymphoma and a myelomonocytic leukemia.

Factors that stimulate the proliferation and differentiation of murine bone marrow cells have been purified from a cloned T cell lymphoma, LBRM-33, and a cloned myelomonocytic leukemia cell line, WEHI-3. These colony-stimulating factors (CSF) have been purified by sequential fractionation by using salt precipitation, gel filtration, anion and cation exchange chromatography, and high pressure liquid chromatography. Both LBRM-33 and WEHI-3 cells secrete a CSF species with similar chemical and biologic properties. This CSF species appears to exist in two forms, termed CSF-2 alpha and CSF-2 beta, both of which stimulate the growth of bone marrow cells in the granulocyte, macrophage, megakaryocyte, mast cell, and erythrocyte lineages, as well as the growth of a CSF-dependent cell line, FDC-P2. These properties of CSF-2 alpha and -2 beta are similar to those reported for interleukin 3, hematopoietic cell growth factor, mast cell growth factor, and persisting cell growth factor. However, LBRM-33 cells secrete another CSF species, not produced by WEHI-3 cells. This CSF species, unique to LBRM cells, is termed here CSF-2 gamma and it stimulates the proliferation of granulocytes and macrophages from bone marrow but does not support the growth of FDC-P2 cells.

Animals↗
PubMed1981

Comparative mitogenic effects of herpes simplex virus and mycoplasma on murine lymphocytes.

Previous work indicated that herpes simplex virus type 1 (HSV-1) is a mitogen for mouse spleen cultures, as monitored by uptake of 3H-thymidine. We observed variable responses of mouse spleen cultures to different viral preparations. The variable responses, which did not follow normal dose-response relationships, were not due to HSV-1 strain differences, altered response kinetics or the presence or absence of defective viral particles, but to mycoplasma contamination of viral stocks. Mycoplasma-free (MF) HSV-1 stocks were prepared by transfection of MF NHF cells with HSV-1 DNA. MF HSV-1 infection of spleen cultures resulted in a five- to sixfold stimulation of DNA synthesis and stimulation of a polyclonal antibody response. Heat treatment (56 degrees C for 1 h) and antibiotics were used to distinguish mycoplasma and HSV-1-induced spleen culture mitogenic responses. The mycoplasma-induced mitogenic activity was found to be heat labile and sensitive to gentamicin and chloramphenicol. In contrast, the HSV-1 induced response was not affected by gentamicin or chloramphenicol and heat treatment resulted in only a 50% loss of activity.

Animals↗
PubMed1981

Biochemical and biologic characterization of lymphocyte regulatory molecules. VIII. Purification of interleukin 2 from a human T cell leukemia.

The ability of interleukin 2 (IL 2) to stimulate and maintain growth in IL 2-dependent cell lines distinguishes this substance from interleukin 1 and other T cell lymphokines. Jurkat-FHCRC, a human T cell leukemia cell line, has been shown to produce relatively high quantities of IL 2 after stimulation with the T cell mitogens phytohemagglutinin or concanavalin A. In this paper we describe techniques used to isolate human IL 2 from Jurkat supernatants and the biochemical properties of the resulting material. IL 2 produced by this cell line is eluted from a DEAE-Sephacel ion-exchange column with 0.05 M NaCl and has a m.w. determined by AcA54 gel chromatography in the range of 9000 to 18,000. Isoelectric focusing yields active material with a pI value of 7.75. Elution of IL 2 from a SDS-polyacrylamide gel shows it to have a m.w. of approximately 13,500. This material is capable of stimulating mouse as well as human IL 2-dependent cell lines.

Animals↗
PubMed1981

Purification of murine helper T cell-replacing factors.

Humoral factors secreted by T lymphocytes and macrophages appear to play a role in cell communication leading to the triggering of immune responses. A factor has been purified from the culture supernatants of concanavalin A(Con A)-activated murine spleen cells with lymphokine activity in four assay systems. 1) stimulation of antibody responses to erythrocyte antigens in BALB/c.nu spleen cultures 2) amplification of production of cytotoxic T cells in thymocyte cultures, 3) stimulation of mitogenic responses to Con A in thymocyte cultures where the cell density is too low to support responses to Con A alone, and 4) maintenance of continuous T cell growth. The biologic activity has been sequentially purified by salt precipitation, gel filtration, chromatography, ion-exchange chromatography, and isoelectric focusing (IEF). These four lymphokine activities appear to be due to one class of molecules, termed Interleukin 2 (IL-2), with an apparent molecular weight of 30,000 daltons. A T cell lymphoma, LBRM-33 has been found to produce high titers of IL-2 activity upon mitogen stimulation. LBRM-derived IL-2 possesses similar chemical and biological properties to splenic-derived IL-2. The mode of action of IL-2 appears to be the clonal expansion of antigen- or mitogen-activated T cells. While mitogen-activated LBRM cells secrete IL-2, which appears to exert helper T cell-replacing activity via the expansion of pre-T cells in nude spleens, mitogen-activated spleen cells appear to secrete both IL-2 and another class of T cell-replacing factor (TRF). These findings raise the question of whether there exist multiple classes of factors with TRF activity. The identification of these molecules may lead to an understanding of the mechanism that regulates the induction of antigen-sensitive cells.

Animals↗
PubMed1980

Biochemical and biologic characterization of lymphocyte regulatory molecules. IV. Purification of Interleukin 2 from a murine T cell lymphoma.

A cloned murine T cell line, LBRM-33 5A4, growing in culture can be activated by phytohemagglutinin (PHA) to secrete Interleukin 2 (IL-2) activity. In the absence of PHA, LBRM cells do not produce IL-2. The chemical and biologic properties of LBRM-derived IL-2 are compared to those of murine spleen-derived IL-2. The fractionation of culture supernatants from PHA-activated LBRM cells by ion-exchange chromatography, gel filtration, and isoelectric focusing (IEF) reveals that IL-2 activity is contained in a class of molecules indistinguishable in size (30,000) and charge (pI 4.3-4.7 and 4.9-5.1) from murine spleen IL-2. LBRM-derived IL-2 also exhibits the entire spectrum of biologic activities exhibited by spleen IL-2, stimulating the growth of established T cell lines in culture, the generation of cytotoxic T lymphocytes in thymocyte cultures and the induction of antibody responses in nude spleen cultures. Both LBRM and splenic-derived IL-2 activities exhibit similar properties after digestion with proteases, and various chemical and temperature treatments. Since IL-2 is derived from a cloned cell line, it is likely these lymphocyte assays are detecting a single class of molecules that act directly on activated T cells. The use of LBRM cells as a source of IL-2 should greatly facilitate studies on the molecular characterization and biologic activity of this class of lymphocyte regulatory molecules.

Animals↗
PubMed1979

Biochemical and biological characterization of lymphocyte regulatory molecules. I. Purification of a class of murine lymphokines.

Murine spleen cells activated by concanavalin A (Con A) in culture produce a class of lymphokine molecules which possess biological activity in a number of lymphocyte response assays. Lymphokines with a mol wt of 30,000, as estimated from gel filtration studies, can be resolved into two components which differ by charge, with isoelectric point (pI) values of 4.3 and 4.9, respectively. Both components stimulate (a) the growth of established T-cell lines in culture, (b) the proliferation of thymocytes in the presence of Con A under culture conditions where Con A alone is nonmitogenic, (c) the induction of antibody responses to heterologous erythrocyte antigens in athymic (nude) spleen cultures, (d) the generation of cytotoxic T lymphocytes (CTL) in thymocyte cultures, and (e) the generation of CTL in nude spleen cultures. In each of these culture systems we suggest that the assays are detecting a single class of lymphokine which acts directly on activated T cells. Nonactivated T cells must be stimulated by either antigen or mitogen before becoming responsive to lymphokine, but do not require antigen or mitogen for continued growth with lymphokine. The two molecular species, separable by isoelectric focusing are referred to as the T-cell growth factor (TCGF). A lymphokine, similar in size (30,000 daltons) to TCGF but heterogeneous in charge (pI 3.0--4.0), stimulates immune responses to erythrocyte antigens in T-cell-depleted spleen cultures but has no stimulatory activity in the other lymphocyte assay systems described. The data have been interpreted as showing the two molecular forms of murine TCGF (pI 4.3 and 4.9) are responsible for many of the lymphokine activities described elsewhere as thymocyte mitogenic factor, nonspecific T-cell-replacing factor and killer helper factor or costimulator. The other lymphokine, separable from TCGF by charge, appears to have true T-cell-replacing activity.

Animals↗
PubMed1977

The interaction of Herpes Simplex Virus with murine lymphocytes. I. Mitogenic properties of herpes simplex virus.

Herpes simplex virus (HSV) stimulates DNA synthesis in mouse spleen cultures prepared from normal, macrophage-depleted, and T-cell-depleted spleen cells, but not from thymocytes. In addition, a polyclonal antibody response is observed in HSV-infected spleen cultures. These findings indicate that the cells stimulated to undergo DNA synthesis after HSV infection appear to be the bone marrow-derived lymphocytes. The newly synthesized DNA is host cell and not of viral origin. Heat treatment and ultraviolet irradiation of HSV before addition to spleen cultures prevents the induction of DNA synthesis. We consider the use of this system as assay for the study of cell transformation by HSV and also for the study of host cell control of the expression of the viral genome.

Animals↗
PubMed1975

Hospital radiopharmacy training program.

Training of radiopharmacists, including self-study and a hospital short-term course, is discussed. A systematic approach for self-study is suggested. The 30-day hospital training program described includes both didactic material and on-the-job experience.

Curriculum↗