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R S Basch

Publications and source records attributed to R S Basch.

At least 19 recordsLinked to original sources
PubMed1995

Flow cytometric determination of apoptosis in heterogeneous cell populations.

We have shown that apoptosis and surface antigen expression can be detected simultaneously using multicolor flow cytometry. Apoptosis was measured with an in situ assay that makes use of the ability of the enzyme terminal deoxyribonucleotidyl transferase to catalyze the addition of biotinylated nucleotides to the free 3'-OH groups produced during endonucleolytic cleavage of DNA. The incorporation of the biotinylated nucleotides was quantified using fluorochrome-coupled streptavidin. Immunofluorescence was used to identify the phenotype of apoptotic cells using three color flow cytometry. In model systems, apoptosis was detected in human PBMNC treated with the DNA topoisomerase I inhibitor CAM and in murine thymocytes treated with DEX. The simultaneous application of this method for detecting apoptosis and immunofluorescence offers several advantages. (1) Apoptotic and necrotic cells can be distinguished. (2) The phenotype of the cells undergoing apoptosis can be determined in heterogeneous systems. Thus, we could show that T cells are relatively resistant to CAM-induced apoptosis compared to other peripheral blood mononuclear cells and that both double negative and single positive thymocytes are resistant to steroid-induced apoptosis. (3) The flow cytometric TdT assay to detect apoptosis-associated DNA degradation method is extremely sensitive compared with gel electrophoresis. As few as 2-5 x 10(3) cells give an adequate signal and it is possible to detect DNA strand breaks even when only a small proportion of the cells are undergoing apoptosis. Neither the sensitivity nor specificity of this method can be matched by any electrophoretic method of detecting apoptosis.

Animals↗
PubMed1992

B-cell subsets and platelet counts in HIV-1 seropositive subjects.

A subset of B lymphocytes positive for the CD5 antigen have been implicated in several autoimmune disorders. To investigate their role in human immunodeficiency virus type 1 (HIV-1) infection, we studied peripheral-blood B and T lymphocytes from HIV-1-positive patients with (n = 13) and without (n = 18) thrombocytopenia, 8 patients with classic autoimmune thrombocytopenia, and 16 healthy controls. The proportion of CD5-positive B cells was significantly higher in the HIV-1-positive thrombocytopenic patients than in the healthy controls, as a result of both higher numbers of CD5-positive B cells and lower numbers of CD5-negative B cells. Platelet count was positively correlated with CD5-negative B-cell count (r = 0.6, p less than 0.001) and negatively correlated with proportion of B cells that were CD5 positive (r = -0.5, p less than 0.01) among the HIV-1-positive patients. The high concentrations of IgM-containing immune complexes in HIV-1-positive patients with autoimmune disorders may be due to changes in the CD5-positive B-cell subset.

Adult↗
PubMed1992

T560: an (H-2b x H-2a) F1 hybrid, phosphorylcholine (PC)-binding, murine B cell lymphoma that bears receptors for IgA and IgG, presents antigen and secretes IL-4.

We describe T560, a tissue culture-adapted B lymphoma derived from the gut-associated lymphoid tissue (GALT) of a (B10 x B10.H-2a H-4b)F1 hybrid mouse. This lymphoma is interesting and useful not only because it bears an unusual IgA receptor, fully described elsewhere, but also because it is potentially capable of presenting antigen to T cells restricted by the MHC of either parent. Here we document that T560 cells are IgG2a kappa +, Ia+, B220+, J11d.2+, CD3-, CD4-, CD5-, Mac 1-, Mac 2-, non-specific esterase-. They bind bromelain-treated mouse RBC (BrMRBC) in a PC chloride-inhibitable manner but do not bind SRBC, ox RBC (ORBC) or TNP-ORBC. Two lines, T560.1 and T560.2, and several clones are available. T560.1 and its clones contain low numbers of IgA rosette-forming cells (RFC), intermediate numbers of IgG2a RFC and moderately high numbers of IgG2b RFC; T560.2 and its clones contain moderately high numbers of IgA RFC and low numbers of both IgG2a and IgG2b RFC. Both lines stimulate both B10 and B10.A cells in mixed lymphocyte reactions (MLR) and present keyhole limpet hemocyanin (KLH) to KLH-reactive T cells. T560.2 populations are, however, more efficient possibly because they have somewhat higher proportions of brightly fluorescent Ia+ cells and secrete larger quantities of lymphokine than T560.1 cells. They present PC-conjugated KLH (PC-KLH) approximately 20 times more efficiently than unconjugated KLH, suggesting that their PC binding receptors function in antigen uptake. They constitutively produce IL-1, IL-4 and IL-6, but not IL-2, IL-5 or TGF beta. Neither their IgA nor their IgG receptor expression is affected by IL-4 or by IFNs-alpha, -beta, or -gamma. In their ability to bind BrMRBC and secrete IL-4, they resemble the CH12 lymphoma but differ from it in that they are of F1 hybrid origin, are CD5-, bear IgG2a rather than IgM, do not bind sheep erythrocytes and have a receptor for IgA not present on CH12.

Animals↗
PubMed1992

Separation of self-renewing hematopoietic progenitors on the basis of CD45 (T-200) antigen expression.

Using monoclonal antibodies recognizing the shared determinants of the CD45 (T-200) antigen we have been able to distinguish spleen colony-forming units (CFU-s) whose progeny are self-renewing from other colony formers on the basis of their quantitative expression of this antigen(s). We have also been able to identify a population of cells that is capable of producing 12-day colonies but has only a limited capacity to produce 8-day colonies. CFU-s8 are found primarily in the dim T-200 population, whereas CFU-s12 were found in both the bright and dim population, but the cells within the colonies produced by these two populations differ in their capacity for self-renewal as CFU-s. Only the colonies dissected after 12 days from the spleens of mice receiving T-200-bright bone marrow cells contained significant numbers of cells that were capable of forming colonies after retransplantation. We calculate the frequency of these cells in total bone marrow to be approximately 1 in 5000.

Animals↗
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↗