Biomedical subjects

R S Basch

Publications and source records attributed to R S Basch.

At least 37 records · Page 2Linked to original sources

PubMed1991

Thymic stromal cells in culture. 2. Binding of normal thymocytes to a cloned thymic stromal cell line.

Thymic stromal cell line TS-9 was found to selectively bind a subpopulation of normal murine thymocytes. Selective binding allowed the isolation and phenotypic characterization of the adherent and nonadherent subpopulations of thymocytes. Flow cytometric analysis of fluorescently labeled thymocytes revealed that the adherent and nonadherent populations differ in maturity, with the adherent population enriched in immature thymocytes of the PNAhi, Thy-1hi, CD3-/lo, and CD4+/CD8+ double positive surface phenotype. A quantitative microwell assay was developed to measure the binding of thymocytes to TS-9. Thymocytes labeled with vital DNA stain Hoechst 33342 were allowed to bind to TS-9 in microwells and the intense fluorescence of this label was readily detected with a scanning fluorometer. The binding was trypsin-sensitive and hyaluronidase and PI-PLC resistant. The binding was also temperature dependent and sensitive to cytochalasin B. A panel of monoclonal antibodies to cell surface antigens including CD2, LFA-I/ICAM-I, and Thy-1 was screened in a quantitative binding assay for their ability to inhibit the binding of thymocytes to TS-9. The binding was partially inhibited by the C3C12 monoclonal antibody which recognizes the recently identified and apparently unique gp23,gp45 complex expressed on murine stromal cells.

Animals↗

PubMed1991

Thymic stromal cells in culture. I. Establishment and characterization of a line which is cytotoxic for normal thymocytes and produces hematopoietic growth factor(s).

Lines of thymic stromal cells have been established. One of these, designated TS-9, has been cloned and studied extensively. This line expresses both acid and alkaline phosphatases. Despite repeated cloning, TS-9 cells remain morphologically heterogeneous. The origin of these cells is not clear. They express low levels of immunologically identifiable cytokeratins, produce laminin, a basement membrane protein, but express antigens typically found on bone marrow stromal cells. The TS-9 cells are MHC Class I+ but Class II-. They express the Thy-1, Pgp-1, and Mac-2 antigens but not other lineage markers of T cells or macrophages. Coculturing TSC with normal thymocytes or with the CTLL-1 cell line leads to a profound inhibition of lectin-induced and/or IL-2 induced T cell proliferation. This requires direct cell-cell contact and ultimately results in the death of the bound lymphocytes. It cannot be reproduced by culturing the thymocytes with TSC culture supernatants. These supernatants do contain hematopoietic growth factor(s) which augment the growth of some T lineage cells and support the growth of monocytic colonies in semi-solid culture medium. Both normal thymocytes and a variety of T cell tumors bind to TSC but only the normal cells are killed as a consequence of this interaction. Neither the binding nor the killing appear to be MHC restricted. We suggest that this killing may provide a model for the effector mechanism of the negative selection imposed by the thymus on developing T cells.

Animals↗

PubMed1990

Protection of mice against tumor growth by immunization with an oncogene-encoded growth factor.

The K-fgf/hst oncogene encodes a growth factor of the fibroblast growth factor (FGF) family that is secreted and transforms cells through a mechanism of autocrine cell proliferation. K-fgf-transformed cells are highly tumorigenic in immunocompetent allogeneic and syngeneic animals. BALB/c mice were immunized with a bacterial fusion protein consisting of a portion of the MS2 polymerase and of the human K-FGF precursor lacking only the first 4 amino acids or with a recombinant protein corresponding to the mature, secreted form of K-FGF (176 amino acids). They were then challenged with syngeneic K-fgf- or H-ras-transformed cells. Vaccinated animals exhibited a significant degree of protection against tumor induction, which was specific for K-fgf-transformed cells and correlated with the ability of the immunized mice to produce high titers of anti-K-FGF antibodies. Thus immunization with a single oncogene product can protect animals against tumor cells expressing this oncogene.

Animals↗

PubMed1990

Expression of CD4 by human megakaryocytes.

The CD4 antigen, which serves as the receptor for human immunodeficiency virus type 1 (HIV-1) on T cells, has been detected on human megakaryocytes. Recent evidence of impaired thrombopoiesis in HIV-1-related thrombocytopenia suggested that these cells could be directly infected by the virus and prompted a search for a receptor on megakaryocytes of normal subjects that could permit entry of HIV-1. Bone marrow specimens from uninfected normal control subjects were centrifuged over Ficoll-Hypaque (1.077 g/ml) and analyzed by three-color analysis with a flow cytometer utilizing monoclonal antibodies against CD4 and a glycoprotein present on the surface of megakaryocytes and platelets (GPIIb/IIIa; CD41), as well as 7-aminoactinomycin D, a stain for DNA. Cells presumed to be megakaryocytes were identified by having a DNA content greater than tetraploid and staining brightly with anti-CD41. Approximately 0.4% of the nucleated cells of the marrow met these criteria. Twenty-five percent of these megakaryocytes stained as brightly as CD4+ T cells. Several clones of antibody recognizing different epitopes of the CD4 molecule gave similar results. Platelets were CD4-. Staining of megakaryocytes with anti-CD4 was confirmed by direct microscopic examination of Percoll-gradient-enriched megakaryocytes employing two-color (CD4-phycoerythrin and CD41-fluorescein) immunofluorescence analysis and phase-contrast microscopy. The proportion of double-labeled cells among 112 phase-contrast-identifiable megakaryocytes from five bone marrow specimens varied between 20% and 26% with a mean and SD of 22% +/- 2.5%. Thus some human megakaryocytes express CD4 on their surface that should be capable of binding the HIV-1 gp120 envelope protein. This could serve as a portal of entry for HIV-1.

Antibodies, Monoclonal↗

PubMed1989

L3T4 antigen expression by hemopoietic precursor cells.

L3T4 (CD4) is expressed on immature hematopoietic cells. Sorting bone marrow cells on the basis of their expression of this antigen produces populations of cells that are markedly enriched for multipotential stem cells (CFU-s) and for myeloid precursors (CFU-c). We believe that L3T4 is transiently expressed by most, if not all, hematopoietic precursors early in their maturation. We suggest that the expression of CD4 molecules on the surface of immature precursors is required for their interaction with Ia bearing cells within the hemopoietic inductive microenvironment(s) of the marrow and thymus.

Animals↗

PubMed1989

A Thy-1 negative lymphoma cell variant defective in the formation of glycosyl-phosphatidylinositol membrane protein anchors.

Thy-1 is a glycoprotein present on the membrane of murine cells of the T-lineage. The mature Thy-1 is anchored to the membrane via a glycolipid, phosphatidylinositide. In order to study the regulation of the synthesis and membrane insertion of this protein, the biochemical properties of a Thy-1.2 negative variant T-lymphoma cell (RL male 1.4) were studied. It contains intracellular Thy-1 protein but fails to express it on the cell surface. While the wild type and the mutant show similar labelling of the intracellular Thy-1 glycoprotein with amino acids, no ethanolamine is incorporated into the Thy-1 molecule of RL male 1.4. A plasmid, pT1, containing the normal Thy-1.2 gene and bacterial gpt gene was transfected into RL male 1.4 and into the murine plasmacytoma cell, J558L. A transfected plasmacytoma, T1J2, synthesized a normal sized Thy-1 protein and displayed the antigen on the membrane. In contrast, the mycophenolic acid resistant RL male 1.4 transfectants did not display Thy-1.2 on the cell surface, despite the presence of substantial amounts of Thy-1 intracellularly. Two other antigens known to be anchored in the membrane by phospholipid, Ly-6e and Qa-2, were also examined in RL male 1.4. RL male 1.4 did not express Ly-6e after alpha interferon induction. In addition, the expression of Qa-2 antigen was greatly diminished in RL male 1.4 in comparison to RL male 1.3. Thus, the defect in RL male 1.4 is not restricted to Thy-1.2, but includes other similarly anchored glycoproteins as well. This implies that the addition of phospholipid to core proteins is similar, if not identical, for all these molecules and that the RL male 1.4 cell lacks the capacity to from the lipid glycoprotein linkage required for the expression of these proteins on the cell surface.

Animals↗

PubMed1985

N,N'-Bis(4-azidobenzoyl)cystine--a cleavable photoaffinity reagent.

A water-soluble, cleavable, heterobifunctional photoaffinity label has been synthesized in one step from N-hydroxysuccinimidyl 4-azidobenzoate and cystine. The resultant compound, N,N'-bis(4-azidobenzoyl) cystine [(ABC)2], reacts with protein sulfhydryl groups through disulfide exchange to generate photoactive derivatives. Since [35S]cystine of high specific activity is readily available, it is possible to produce highly radioactive (ABC)2. ABC-derivatized ovalbumin is antigenic in vivo, and monoclonal antibodies specific for ABC have been produced. The antigen binding site of these antibodies was covalently labeled with ABC.

Affinity Labels↗

PubMed1985

Thy-1 antigen expression by murine hematopoietic precursor cells.

Multipotential lymphohematopoietic stem cells detected by a 12-day spleen colony assay are uniformly Thy-1 positive. As these cells differentiate into stem cells with restricted developmental potential, the antigen is lost. The various restricted progenitor cells differ in their susceptibility to the cytolytic effects of anti-Thy-1 antiserum. Erythroid progenitors appear to lose the antigen more rapidly than any of the others tested, while macrophage precursors retain the antigen until relatively late in their developmental history. These differences in Thy-1 expression permit discrimination among the various progenitors.

Animals↗

PubMed1984

Isolation of highly malignant Thy-1-positive revertants from cultured cloned Thy-1-negative lymphoma cells of low tumorigenicity.

We have used a positive immunoselection method to isolate a Thy-1-positive revertant of a Thy-1-negative line. The technique makes use of the ability of surface bound antigen-antibody complexes containing the enzyme catalase to protect cells from the toxic effects of hydrogen peroxide. The revertant cells produce a Thy-1 molecule indistinguishable from that produced by the original parent line, and they resemble that line in being far more tumorigenic than the Thy-1-negative variant.

Animals↗

PubMed1984

Gene transfer in lymphoid cells: expression of the Thy-1.2 antigen by Thy-1.1 BW5147 lymphoma cells transfected with unfractionated cellular DNA.

We have transferred the gene coding for the Thy-1.2 alloantigen into a Thy-1.1-bearing T-cell lymphoma. BALB/c thymocyte DNA, precipitated with calcium phosphate, was used to effect the transfer. We report the stable transformation of lymphoid cells by total cellular DNA. To our knowledge, this has not been previously reported. Transient expression of the transfected gene could be detected by flow cytofluorometry, and 5% (range, 1.5%-11%) of the recipient cells had Thy-1.2 antigen detectable on their surface 48 hr after transfer. "Stable" transformants were isolated by repeatedly selecting for cells expressing the Thy-1.2 antigen, by use of fluorescence-activated cell sorting or "panning." No metabolic selection was required. The transferred gene, detected by Southern blotting, encoded a product that is indistinguishable from the normal antigen by immunoprecipitation and NaDod-SO4/PAGE.

Animals↗

PubMed1984

A monoclonal antibody (AE3.d3) with mitogenic properties for murine B cells.

A monoclonal antibody, AE3.d3, derived from the fusion of rat splenocytes immunized against mouse brain with the myeloma SP2, has been produced, which has the property of inducing the proliferation of mouse lymphocytes. The mitogenic effect is highest in spleen and lymph node cells, where up to a 10-fold stimulation of 3H-TdR incorporation is observed. B lymphocytes are the most susceptible to this proliferative stimulus, and they are induced to differentiate into plaque-forming cells. T lymphocytes and "null" cells (defined by the absence of Thy-1 or Ig on their surface) do proliferate, although to a smaller extent. The T cell subpopulation, isolated from either spleen or thymus, requires additional "helper factors" in order to proliferate. The mitogenic response is not genetically restricted by H-2 type, and strains such as C3H/HeJ and CBA/N, in which B cell function is defective, as well as T cell-deficient strains such as BALB/c nude mice, are capable of responding to the stimulus of AE3.d3. Using immunofluorescence, we also examined the distribution of AE3.d3-positive cells in various lymphoid organs. The highest percentage of stained cells is found in the spleen (approximately 28%) and lymph node (18%), whereas only 14% of the bone marrow and 5% of the cells of the thymus are brightly stained with AE3.d3.

Animals↗

PubMed1983

Identification of a new marker (Ly RL male 1) for cells of the T lineage by an auto-antithymocyte serum.

The sera of mice surviving challenge with a Thy-1-negative variant of the thymoma RL male 1 contain antibodies which identify a new cell surface antigen (Ly RL male 1) present on cells of the T lineage. This antigen appears early in the development of the lineage and it can be detected on most thymocyte precursors. Its presence on prothymocytes can serve to distinguish these cells from their multipotential precursors. The antigen is present on many thymocytes, and dividing thymocytes are more susceptible to its cytotoxic activity than is the total population. Ly RL male 1-antigen-positive cells can be detected in peripheral lymphoid tissues by both functional assays and absorption. Treatment of peripheral lymphoid cells with the antiserum leads to significant reduction in MLR and helper activity but does not alter mitogen reactivity or lymphocytotoxicity. Animals with significant serum levels of anti-RL male 1 are deficient in their ability to produce IgG antibody to sheep erythrocytes.

Animals↗

PubMed1983

Cell separation using positive immunoselective techniques.

Positive immunoselection is the direct selection and recovery of cells which express a given specificity from among a heterogeneous group of contaminating cells. A variety of methods are available to effect such separations. The principles of affinity chromatography, using solid-phase matrices or cellular immunoadsorbents, are extensively used. Liquid-phase positive immunoselection can also be performed using either a fluorescence-activated cell sorter or by using 'cellular engineering' to protect a cell from an otherwise noxious environment. The enzyme catalase coupled to specific antibody has been used for this purpose and renders cells resistant to hydrogen peroxide. The various positive immunoselection techniques available are reviewed and evaluated in the following report.

Animals↗

PubMed1983

MG-1: a specificity identifying members of the macrophage and granulocyte lineages of mice.

An antigen (MG-1) which behaves as a lineage marker for immature granulocytes and cells of the monocytic series in mice is described. It is identified by rabbit anti-mouse brain antiserum, which has been exhaustively absorbed with thymocytes, red blood cells, and a Thy-1 negative variant of a T-cell lymphoma. MG-1 is present on immature granulocytes, declines in its surface expression as the cells differentiate and is absent from the most mature cells (segmented) of the series. Early monocytes are strongly positive for MG-1 and, with maturity, the amount of cell surface antigen increases. Adherent, phagocytic macrophages are brilliantly positive when stained with anti-MG-1 antiserum in an immunofluorescence assay. The antigen is present on most of the adherent cells of the lung, spleen and peritoneum. Many multipotential stem cells also express low levels of this antigen as do some bone marrow B cells.

Animals↗

PubMed1982

Thy-1 determinants are present on many murine hematopoietic cells other than T cells.

The development of a highly amplified immunofluorescence assay and the availability of monoclonal anti-Thy-1 antibodies have provided the methodology to reexamine the presence of Thy-1 antigen on murine lymphohematopoietic cells. The representation of this antigen is not, as previously believed, restricted to the T cell compartment of these cells. It is present on a significant number of mouse bone marrow cells (25-30%), including, as in the rat, multipotential stem cells, prothymocytes and some B cell precursors. Eosinophils and some immature myeloid cells are also antigen-positive.

Animals↗

PubMed1982

Serologic identification of early members of the T cell lineage.

The SC-1 antigen, identified by thymus-absorbed rabbit anti-mouse brain antiserum, is present on multipotential stem cells. Its presence on cells of the T lineage has been examined by immunofluorescence, and we have demonstrated that it is also a marker for thymic development. Although it is present on the majority of fetal and neonatal thymocytes, its expression declines rapidly and, as early as 1 mo after birth, adult levels are reached. In normal animals, these do not change during adult life, even in mice destined to develop a T cell leukemia. SC-1 does reappear transiently on cells in the regenerating thymus of sublethally irradiated mice. Although the antigen is not associated with preleukemic changes in the thymus, it is expressed on some cells of all spontaneous T lymphomas, and it is uniformly present on tissue culture lines of T cell lymphomas.

Animals↗

PubMed1981

Demonstration of a hematopoietic stem cell antigen (SC-1) on a murine lymphoma and isolation of variants lacking the antigen.

Murine multipotential hematopoietic stem cells (CFU-s) bear an antigen (SC-1) which is recognized by heterologous antisera to mouse brain. We have found that cloned Thy-1 negative variants of the T-cell lymphoma RL male 1 are sensitive to complement-mediated cytolysis by anti-brain serum and can absorb the anti-stem cell activity from the antiserum. We have isolated several subclones derived from a primary Thy-1 negative variant which are not susceptible to anti-brain serum. The surface of the resistant lines has little or no antigen capable of binding anti-mouse brain antibodies as measured by either immunofluorescence or a radioimmunoassay. These lines are also unable to absorb the antibodies responsible for the cytotoxic effect of rabbit anti-mouse brain serum against CFU-s. We conclude that the predominant antigen, serologically detectable on Thy-1 negative variants of RL male 1, is SC-1.

Animals↗

PubMed1981

Positive immunoselection using antibody-enzyme complexes.

We have developed an immunoselection technique using catalase-anti-catalase complexes coupled to specific antibodies to protect antigen positive target cells from the lethal effects of H2O2. The antibody-enzyme complexes are bound to the target cells through an antibody bridge with specificity for both the complexes and an anti-target cell antibody. In a model system, Thy-1 positive (RL male 1-3) cells were protected by incubation with rabbit anti-brain-associated theta antigen (BAT), sheep anti-rabbit IgG [F(ab)'2] and catalase-anti-catalase complexes. The amount and composition of the complexes adhering to the cells were measured by dual radiolabeling of the catalase and anti-catalase immunoglobulin. This technique provides a means of identifying and isolating large numbers of cells bearing any antigen for which specific antisera are available.

Animals↗