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Biomedical subjects

M R Loken

Publications and source records attributed to M R Loken.

At least 91 records · Page 5Linked to original sources
PubMed1982

Changes in susceptibility to cytotoxic antibody among tumor cells surviving exposure to chemotherapeutic agents.

We determined that myeloma cells which survived drug treatment had an altered sensitivity to cytotoxic antibody. The effects of several chemotherapeutic agents differing in drug action were compared. The antiserum was directed against viral determinants on the surface of S107 myeloma cells. This antiserum was then used to inhibit the colony formation of drug-treated myeloma cells. Tumor cells were treated with melphalan (200 ng/ml), methotrexate (40 ng/ml), actinomycin D (5 ng/ml), or 0.5 mM thymidine for 24 hr and then washed and resuspended in fresh medium. On various days after drug treatment, aliquots of these cells were exposed to complement and dilutions of antiserum and then cloned in soft agar in order to quantify the degree of antibody-mediated inhibition. Melphalan, methotrexate, and thymidine caused a severalfold increased resistance of the tumor cells to the antiserum during the first 1 or 2 days after drug treatment. During the following days, however, myeloma cells showed markedly increased susceptibility to the antiserum. The biphasic effect of methotrexate or thymidine treatment was similar to that previously observed after melphalan treatment and differed from the effect of actinomycin D. Actinomycin D caused only an increased susceptibility of the tumor cells to the antibody for a period of 4 to 5 days immediately following drug treatment. Our studies indicate that several chemotherapeutic drugs at concentrations comparable to those used in humans have long-lasting antagonistic as well as synergistic effects on the sensitivity of tumor cells to antibody and complement, depending on the particular drug used and on the time interval between drug exposure and subsequent treatment with antibody. These results suggest a model for evaluating the use of antibody in the elimination of malignant cells which, despite exposure to chemotherapy, remain clonogenic and proliferative.

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PubMed1981

Lyt-2-/Lyt 3- variants of a cloned cytolytic T cell line lack an antigen receptor functional in cytolysis.

To investigate the role of Lyt-2 and Thy-1 in cytolysis, we have generated, by ethyl methanesulfonate mutagenesis and selection, variants of the cloned cytolytic T lymphocyte line L3 that specifically lack either Lyt-2 or Thy-1. An analysis of these variants indicates that neither Lyt-2 nor Lyt-3 is responsible for the lethal hit, but suggests that Lyt-2 and/or Lyt-3 are required for an antigen receptor functional in cytolysis. The data also suggest that the expression of Lyt-3 on the cell surface is not independent of the expression of Lyt-2. Finally the data indicate the Thy-1 plays no role in cytolysis.

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PubMed1981

Alteration of clonal profile. II. Studies on the capacity of BALB/c splenic B cells to perpetuate responsiveness to phosphorylcholine and T 15 idiotypic dominance.

(CBA/N x BALB/c)F1 hybrid male mice are unable to mount anti-phosphorylcholine (PC) plaque-forming cell (PFC) responses because they carry the CBA/N X-linked immune defect of B lymphocyte differentiation. Transplantation of splenic B cells from BALB/c mice restores responsiveness to thymus-dependent and thymus-independent PC antigens up to 8 months after cell transfer. Cytotoxicity studies demonstrate the donor origin of PFC generated in reconstituted (CBA/N x BALB/c)F1 mice. Although responsiveness to PC is restored permanently, a shift in idiotype expression that leads to the loss of T 15 idiotypic dominance 3 months after cell transfer can be detected. This shift originates from Ig- cells because Ig+ splenic cells purified in a fluorescence-activated cell sorter maintain T 15 dominance. Therefore, the Ig+ cells have a remarkable capacity to maintain responsiveness to antigens and can perpetuate idiotypic dominance if the stem cell pool is removed.

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PubMed1981

DNL 1.9: a monoclonal antibody which specifically detects all murine B lineage cells.

A rat anti-mouse monoclonal antibody, DNL 1.9, has been produced and shown to bind to all cells of the B lineage in mice. By quantitative immunofluorescence, DNL 1.9 binds to approximately 50% of spleen cells, 20-30% of lymph node cells, but no to thymocytes. In bone marrow and neonatal liver, 70-80% of the small cells and 10-20% of the large cells were identified by this antibody. Two-color immunofluorescence studies showed that all immunoglobulin-positive cells were also DNL 1.9-positive, while Thy-1.2-positive cells did not bind the monoclonal antibody. All pre-B cells in bone marrow , as identified by cytoplasmic IgM staining, were labeled by the DNL 1.9 antibody. A significant proportion of lymphocytes in the bone marrow which did not express cell surface immunoglobulin did bind DNL 1.9. Both direct and indirect plaque-forming cells were also shown to bind DNL 1.9 antibody. The antigen recognized by this monoclonal antibody was not immunoglobulin nor was the antigen present in normal mouse serum. All seven mouse strains tested showed similar patterns of antigen expression. These results show that all of the cells indentifiable as being of the B lineage were identified by this monoclonal antibody. It is possible that the cells which bind DNL 1.9 but do not express cell surface immunoglobulin may be very early precursors of B lymphocytes.

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PubMed1981

Structural differences in cell surface T25 polypeptides from thymocytes and cloned T cells.

The molecule bearing the Thy-1.2 antigen, T25, has been isolated from fractionated thymocytes as well as from cloned cytolytic and helper T cells by immune precipitation with monoclonal antibodies. Using NaDodSO4/polyacrylamide gels cross-linked with N,N'-diallytartardiamide, electrophoretic analyses of the precipitates revealed a heterogeneous pattern of polypeptide bands, ranging in apparent molecular weight from 28,000-36,000 daltons. Thymocytes separated on the basis of Thy-1.2 surface antigen density were found to differ not only in amount of Thy-1.2 expressed, but also in the structure of the surface T25 molecules isolated from each subpopulation. This structural variation was evidenced in changes detected in the relative density and electrophoretic mobility of T25 band patterns obtained on SDS gels. Differences in T25 band patterns were also observed in polypeptides isolated from cloned cytolytic or helper T cells. These data suggest that although surface T25 is structurally heterogeneous, the pattern of heterogeneity can differ from cell to cell, or from population to population. Changes in T25 structure appear to correlated with differences in either T lymphocyte maturation or T cell function.

Animals↗
PubMed1980

Simultaneous quantitation of Hoechst 33342 and immunofluorescence on viable cells using a fluorescence activated cell sorter.

The cytochemical stain Hoechst 33342 has been used to quantify DNA in viable cells and has been used under nonsaturating conditions to discriminate between lymphoid cell types. In order to correlate the quantitative emission from Hoechst 33342 with cell surface antigens, a fluorescence activated cell sorter was modified to simultaneously detect emission from the UV excited Hoechst dye and fluorescein attached to the cell surface by immunofluorescence techniques. A special set of laser mirrors was installed in an argon ion laser so that all the lines from 351-488 nm could be used to illuminate the cells. Appropriate emission filters were used to separate the light emitted by Hoechst 33342 from the fluorescein. An electronic cross-over circuit was used to compensate for special overlap between the two dyes. Analysis of murine lymph node cells stained both with Hoechst 33342 under nonsaturating conditions and anti-Thy 1.2 indicated that the cells that stained dimly with the Hoechst dye expressed the Thy 1.2 marker while the cells that were brightly stained with Hoechst 33342 lacked this differentiation antigen. The correlation of cell surface myeloma protein with cell cycle on an in vitro cell line indicated that the heterogeneity of cell surface antigen expression could not be accounted for solely by variations occurring during the cell cycle.

Animals↗
PubMed1980

Interaction between I region loci influences the expression of a cell surface Ia antigen.

Loci clustered in the I region of the murine H-2 gene complex control the capacity to generate an immune response against foreign antigens (Ir loci) and control differentiation antigens which appear to serve as structures used by cells to interact with and regulate one another (Ia loci). Both genetic and functional studies suggest that Ia antigens may be products of Ir loci. Recent studies have shown that interaction between closely linked Ir loci is required for generating immune responses to certain foreign antigens, and that interaction between H-2-linked loci determines the appearance of an Ia glycoprotein (the Ae chain) on lymphocyte cell surfaces. In this report, we show that one Ia locus regulates the quantitative expression of the product (the E alpha chain) of a second Ia locus. This regulatory locus is dominantly expressed and exerts its effects in either the cis or trans chromosomal position. Thus, the quantitative as well as the qualitative expression of some Ia products is dependent on interaction between tightly linked loci. Our results suggest a possible molecular basis for this regulation: the synthesis and intracellular association of Ae and E alpha chains may be an absolute requirement for the expression of normal levels of either polypeptide chain on the lymphocyte cell surface. The implications these findings have for I region control of immune responses and study of human HLA-D antigens are discussed.

Animals↗
PubMed1980

Separation of viable T and B lymphocytes using a cytochemical stain, Hoechst 33342.

Data are presented that show that a histochemical stain, Hoeschst 33342, can be used to discriminate between viable B and T lymphocytes in the mouse. Quantitative analysis of the staining of cells from various lymphoid tissues with Hoechst 33342 using a Fluorescence-Activated Cell Sorter (FACS) indicates that two populations of cells can be identified. In the spleen approximately 60% of the lymphocytes can be classified as brightly stained with 1 microgram/ml of Hoechst 33342, while in the lymph node only 40% of the cells stain brightly. Thymocytes exhibit only the dull staining profile. Separation of these two populations from the spleen using the FACS and reanalyzing them for cell surface antigenic markers shows that the lymphocytes stained brightly with Hoechst 33342 are predominantly immunoglobulin positive, while the cells that stain less brightly express Thy 1.2. This indicates that a histochemical stain correlates directly with classical immunological markers on cell surfaces.

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PubMed1980

Evaluating optical filter efficiency in a flow cytometer.

A simple yet effective method of testing optical filters under normal operating conditions on a Fluorescence Activated Cell Sorter is described. The spectral characteristics of three filters are compared to demonstrate the methods of determining which filter is the best in a particular situation. These tests include determining the efficiency of transmitting fluorescent light, determining the efficiency of removing scattered light, and detecting fluorescence originating from the filter itself.

Animals↗
PubMed1979

Clonal evolution of myeloma cells leads to quantitative changes in immunoglobulin secretion and surface antigen expression.

We report that a cloned population of tumor cells can rapidly produce variants that differ in their quantitative expression of surface proteins and in their rate of immunoglobulin secretion. A fresh clonal isolate of S107 myeloma cells possessing large amounts of surface IgA was continuously passaged in vitro for 2 years. During this period, fluorescence-activated cell sorter analysis indicated the development of subpopulations possessing decreased amounts of surface IgA. Cells from these variant subpopulations were isolated by first using the cell sorter to enrich for cells with decreased amounts of surface IgA and then cloning the selected population in soft agar. The 50 sublines that were isolated showed heritable differences in their levels of surface IgA and H-2 antigens and in their rates of myeloma protein secretion. Sublines having either large amounts, intermediate amounts, or absence of surface IgA also had corresponding large amounts, intermediate amounts, or absence of myeloma protein secretion. In contrast, a decrease or loss of surface Ig did not correlate with a decrease or loss of viral envelope glycoprotein gp71 and H-2 antigens. The variants did not resemble the phenotypes of less-differentiated normal lymphocyte populations of the B-cell lineage. The isolation and characterization of these variants allows us to explore the mechanisms and pathways of tumor cell differentiation as well as to study the regulation and function of cell surface proteins.

Animals↗
PubMed1978

Expression of IgD by murine lymphocytes. Loss of surface IgD indicates maturation of memory B cells.

B lymphocytes capable of generating primary IgM and IgG plaque-forming cells (PFC) responses to burro erythrocytes have surface IgD, as do primary IgM PFC. IgG memroy cells arising after one injection of antigen are divided into two groups, one of which expresses surface IgD while the other has no detectable membrane IgD. PFC generated from the IgG memory cells lacking surface IgD show a higher average avidity than those arising from IgD-positive IgG memory cells, indicating that mature IgG memory cells do not have surface IgD. After more than one injection of antigen, few, if any, IgG memory cells have surface IgD. IgG PFC arising in primary or secondary immune response lack membrane-bound IgD. These data provide the outlines for a B-cell maturation pathway in which IgD marks unprimed and early memory B cells and is lost in mature memory cells. Studies presented here were conducted by isolating IgD+ and IgD- cells with the fluorescence-activated cell sorter and functional testing of the isolated populations in adoptive transfer experiments.

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