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L B Epstein

Publications and source records attributed to L B Epstein.

At least 19 recordsLinked to original sources
PubMed1996

Antisense manganese superoxide dismutase mRNA inhibits the antiviral action of interferon-gamma and interferon-alpha.

Manganese superoxide dismutase (MnSOD) is induced by interferon-gamma (IFN-gamma) in various cell lines. To determine whether MnSOD plays a role in the antiviral action of IFN-gamma, we employed an antisense strategy to inhibit the expression of MnSOD in the human melanoma cell line, A375. Three antisense-containing clones that exhibited reduced induction of MnSOD were investigated with respect to their response to the antiviral protective effects of IFN-gamma and IFN-alpha. We observed a striking decrease in the ability of IFN-gamma to protect antisense clones from vesicular stomatitis virus infection (VSV). The IFN-alpha induced antiviral state was also impaired, but to a lesser degree than was observed with IFN-gamma. We excluded the possibility that these effects were caused by a higher sensitivity of the antisense cells to VSV itself and found that the antisense clones actually were less sensitive to VSV. Therefore, we conclude that MnSOD is involved in the establishment of the IFN-gamma-induced antiviral state and to a lesser degree in the antiviral actions of IFN-gamma.

Antiviral Agents↗
PubMed1995

Superoxide radical and iron modulate aconitase activity in mammalian cells.

Aconitase is a member of a family of iron-sulfur-containing (de)hydratases whose activities are modulated in bacteria by superoxide radical (O2-.)-mediated inactivation and iron-dependent reactivation. The inactivation-reactivation of aconitase(s) in cultured mammalian cells was explored since these reactions may impact important and diverse aconitase functions in the cytoplasm and mitochondria. Conditions which increase O2-. production including exposure to the redox-cycling agent phenazine methosulfate (PMS), inhibitors of mitochondrial ubiquinol-cytochrome c oxidoreductase, or hyperoxia inactivated aconitase in mammalian cells. Overproduction of mitochondrial Mn-superoxide dismutase protected aconitase from inactivation by PMS or inhibitors of ubiquinol-cytochrome c oxidoreductase, but not from normobaric hyperoxia. Aconitase activity was reactivated (t1/2 of 12 +/- 3 min) upon removal of PMS. The iron chelator deferoxamine impaired reactivation and increased net inactivation of aconitase by O2-.. The ability of ubiquinol-cytochrome c oxidoreductase-generated O2-. to inactivate aconitase in several cell types correlated with the fraction of the aconitase activity localized in mitochondria. Extracellular O2-. generated with xanthine oxidase did not affect aconitase activity nor did exogenous superoxide dismutase decrease aconitase inactivation by PMS. The results demonstrate a dynamic and cyclical O2-.-mediated inactivation and iron-dependent reactivation of the mammalian [4Fe-4S] aconitases under normal and stress conditions and provide further evidence for the membrane compartmentalization of O2-..

Aconitate Hydratase↗
PubMed1995

Rapid mass spectrometric peptide sequencing and mass matching for characterization of human melanoma proteins isolated by two-dimensional PAGE.

We report a general mass spectrometric approach for the rapid identification and characterization of proteins isolated by preparative two-dimensional polyacrylamide gel electrophoresis. This method possesses the inherent power to detect and structurally characterize covalent modifications. Absolute sensitivities of matrix-assisted laser desorption ionization and high-energy collision-induced dissociation tandem mass spectrometry are exploited to determine the mass and sequence of subpicomole sample quantities of tryptic peptides. These data permit mass matching and sequence homology searching of computerized peptide mass and protein sequence data bases for known proteins and design of oligonucleotide probes for cloning unknown proteins. We have identified 11 proteins in lysates of human A375 melanoma cells, including: alpha-enolase, cytokeratin, stathmin, protein disulfide isomerase, tropomyosin, Cu/Zn superoxide dismutase, nucleoside diphosphate kinase A, galaptin, and triosephosphate isomerase. We have characterized several posttranslational modifications and chemical modifications that may result from electrophoresis or subsequent sample processing steps. Detection of comigrating and covalently modified proteins illustrates the necessity of peptide sequencing and the advantages of tandem mass spectrometry to reliably and unambiguously establish the identity of each protein. This technology paves the way for studies of cell-type dependent gene expression and studies of large suites of cellular proteins with unprecedented speed and rigor to provide information complementary to the ongoing Human Genome Project.

Amino Acid Sequence↗
PubMed1994

Enhanced synthesis of tumor necrosis factor-inducible proteins, plasminogen activator inhibitor-2, manganese superoxide dismutase, and protein 28/5.6, is selectively triggered by the 55-kDa tumor necrosis factor receptor in human melanoma cells.

We have demonstrated that A375 melanoma cells express mRNA for both types of tumor necrosis factor (TNF) receptors and receptor proteins on their plasma membranes. Specific agonist and blocking antibodies to either 55-kDa (TNF-R1) or 75-kDa (TNF-R2) TNF receptors combined with two-dimensional gel analysis were employed to determine which receptor type is responsible for mediating the induction of individual melanoma proteins. Our results indicate that the enhanced synthesis of proteins 21/>7 (M(r)/pI), 28/5.6, and 41/5.7 is selectively induced through TNF-R1. TNF induces these proteins; antagonist antibody to TNF-R1 prevents their induction by TNF, and TNF-R1 agonist induces them in the absence of TNF. Identification of these proteins by immunoblot analysis proved that 21/>7 is manganese superoxide dismutase, protein 28/5.6 is unrelated to 27/28-kDa heat shock protein, and protein 41/5.7 is plasminogen activator inhibitor-2. Furthermore, TNF cytotoxicity for A375 cells is also mediated by TNF-R1. These studies indicate that TNF-R1 is a critical signaling receptor for TNF action on A375 cells and demonstrate the potential use of TNF-R1 antibodies to selectively block or enhance specific effects of TNF on melanoma cells.

Base Sequence↗
PubMed1993

Overexpression of LFA-1 and ICAM-1 in Down syndrome thymus. Implications for abnormal thymocyte maturation.

The interaction between lymphocyte function-associated antigen 1 (LFA-1) on thymocytes and intercellular adhesion molecule 1 (ICAM-1) on the thymic stroma plays an important role in thymocyte maturation. Consequently, we examined the constitutive expression of LFA-1 and ICAM-1 in postnatal human thymus from children with Down syndrome (trisomy 21, DS) and age-matched control children. We studied DS thymuses because this aneuploid condition is associated with abnormal thymic anatomy and patterns of thymocyte maturation and the affected individuals have a greatly increased incidence of infection. In addition, the beta-chain for LFA-1 is encoded on human chromosome 21, suggesting that trisomy 21 thymocytes may overexpress this adhesion molecule. Using immunofluorescence and flow cytometry, LFA-1 beta expression was evaluated in eleven pairs of DS and age-matched control thymocytes. The mean channel of fluorescence for LFA-1 beta expression was significantly higher in DS thymocytes than in the controls (p < or = 0.01). Six of the 11 pairs were examined for LFA-1 alpha chain expression. DS thymocytes also showed significantly higher levels of LFA-1 alpha expression (p < or = 0.05), which is consistent with findings that surface expression of the LFA-1 alpha chain is dependent on beta-chain expression. Using immunohistochemical analysis and quantitative video imaging, we examined the level of ICAM-1 expression on frozen sections from four pairs of DS and control thymuses, and found nearly twofold higher levels of ICAM-1 expression in the DS thymuses (p < or = 0.05). DS thymuses also showed a diffuse pattern of ICAM-1 expression with elevated staining in both cortex and medulla and poor demarcation of staining at the cortico-medullary junctions. Given our recent observation that DS thymuses overexpress mRNA for IFN-gamma and TNF, and the fact that both of these cytokines induce ICAM-1 expression on cultured human thymic epithelial cells, we propose that increased levels of IFN-gamma and TNF contribute to the enhanced expression of ICAM-1 in DS thymuses. Our findings support a role for cytokines in the regulation of adhesion molecule expression in the thymus and suggest that the increased expression and abnormal distribution of adhesion molecules in DS thymuses alters the interaction between developing thymocytes and the thymic stroma and results in the abnormal thymocyte maturation observed in DS.

Cell Adhesion Molecules↗
PubMed1993

Mass spectrometric and Edman sequencing of lipocortin I isolated by two-dimensional SDS/PAGE of human melanoma lysates.

We have integrated preparative two-dimensional polyacrylamide gel electrophoresis with high-performance tandem mass spectrometry and Edman degradation. By using this approach, we have isolated and identified, by partial sequencing, a human melanoma protein (34 kDa, pI 6.4) as lipocortin I. To our knowledge, this protein was not previously known to be associated with melanoma cells. The identity of the protein was confirmed by two-dimensional immunoblot analysis. High-energy collision-induced dissociation analysis revealed the sequence and acetylation of the N-terminal tryptic peptide and an acrylamide-modified cysteine in another tryptic peptide. Thus, knowledge concerning both the primary structure and covalent modifications of proteins isolated from two-dimensional gels can be obtained directly by this approach, which is applicable to a broad range of biological problems.

Amino Acid Sequence↗
PubMed1992

Tumor necrosis factor-alpha and IFN-gamma expression in human thymus. Localization and overexpression in Down syndrome (trisomy 21).

In vitro studies suggest that TNF-alpha and IFN-gamma regulate thymocyte proliferation, but little evidence exists for the constitutive production of these cytokines in normal human thymus. In paired experiments, we examined frozen sections of postnatal human thymus from four control children and four age-matched children with Down syndrome (DS) (trisomy 21) for TNF-alpha and IFN-gamma mRNA expression using in situ hybridization. We studied thymuses from children with DS because this aneuploid condition is associated with a greatly increased incidence of infection and has abnormal thymic anatomy and patterns of thymocyte maturation. We found cells expressing constitutive levels of TNF-alpha mRNA in the trabeculae, corticomedullary junctions, and medulla of both control and DS thymuses and the number of these cells was an average of 3.9-fold higher in DS thymuses than in age-matched control thymuses. DS thymuses also contained an average of 3 fold higher numbers of cells with mast cell morphology, identified by toluidine blue histologic staining and electron microscopy. In both DS and control thymuses the mast cells colocalized with TNF-alpha mRNA-expressing cells. In addition, TNF-alpha protein- expressing cells, identified by immunohistochemistry, displayed a granular pattern of staining that is characteristic of mast cells. These results suggest that mast cells may be one source of TNF-alpha in human postnatal thymus. Discrete cells expressing IFN-gamma mRNA were distinctly localized to the cortical region of both DS and control thymuses and were 2.4-fold more abundant in DS thymuses than in the controls. Our results demonstrate, for the first time, the constitutive production and location of TNF-alpha and IFN-gamma in postnatal human thymus. The overexpression of both of these cytokines in DS thymuses suggests a dysregulation in cytokine production in DS and may provide an explanation for the abnormal thymic anatomy and thymocyte maturation associated with this syndrome.

Down Syndrome↗
PubMed1992

Induction of leucine aminopeptidase by interferon-gamma. Identification by protein microsequencing after purification by preparative two-dimensional gel electrophoresis.

The protein previously called "Mr approximately 50/pI approximately 6.9," which we observed to be induced by the immunoregulatory cytokine interferon (IFN)-gamma in human fibroblasts, was purified from a total cell lysate by preparative two-dimensional gel electrophoresis and identified by partial amino-terminal sequencing as leucine aminopeptidase (LAP), a 53-kDa cytosolic exopeptidase. Induction of LAP protein by IFN-gamma, confirmed by immunoblotting with an antiserum raised against bovine lens LAP, is a consequence of induction of LAP mRNA and occurs in all four human cell lines examined: HS153 fibroblasts, ACHN renal carcinoma, A549 lung carcinoma, and A375 melanoma. Induction of LAP mRNA is a secondary response to IFN-gamma, blocked by inhibition of protein synthesis with cycloheximide.

Amino Acid Sequence↗
PubMed1992

Down syndrome (DS) peripheral blood contains phenotypically mature CD3+TCR alpha, beta+ cells but abnormal proportions of TCR alpha, beta+, TCR gamma, delta+, and CD4+ CD45RA+ cells: evidence for an inefficient release of mature T cells by the DS thymus.

Down syndrome (DS) thymocytes have a markedly diminished proportion of cells expressing high levels of the alpha, beta T cell receptor (TCR alpha, beta) and the associated CD3 molecule. Thus, we examined the surface expression of TCR alpha, beta and CD3 as well as TCR gamma, delta, CD4, CD8, CD16, and CD45RA on peripheral blood lymphocytes (PBL) from 13 noninstitutionalized subjects with DS and 13 closely age-matched sibling controls using immunofluorescence and flow cytometry. DS PBL expressed high surface levels of TCR alpha, beta and CD3, but, as compared to controls, they had a lower proportion of cells expressing TCR alpha, beta (61% vs. 68%, respectively; P less than or equal to 0.05). Moreover, the absolute number of TCR alpha, beta+ cells was considerably lower for DS subjects than for controls (1634 +/- 229 vs. 2763 +/- 530, respectively; P less than or equal to 0.05). DS subjects had a markedly higher proportion of cells expressing TCR gamma, delta than did the controls (12% vs. 7%, respectively; P less than or equal to 0.02). In addition, DS subjects had a lower proportion of CD4+CD45RA+ cells than controls (22% vs. 35%, respectively; P less than or equal to 0.02), representing naive T cells which have recently emigrated from the thymus. The imbalance in the proportions of T cell subpopulations we have observed in DS PBL may contribute to the increased susceptibility to infection associated with DS and may represent a diminished efficiency in the production of newly differentiated T cells by the DS thymus.

Adolescent↗
PubMed1992

A role for tumor necrosis factor-alpha and interferon-gamma in the regulation of interleukin-4-induced human thymocyte proliferation in vitro. Heightened sensitivity in the Down syndrome (trisomy 21) thymus.

The influence of recombinant interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF) on IL-4-induced proliferation of postnatal human thymocytes from eight children with Down syndrome (DS, trisomy 21) and 18 control children was evaluated. DS thymuses were studied because they are characterized by cortical depletion and abnormal thymocyte differentiation. IL-4, without mitogen, induced a dose-dependent proliferation of both DS and control thymocytes. The proliferation was comparable to that induced by IL-2 and far greater than the proliferation mediated by IL-1 beta in the absence of mitogen. The level of IL-4 responsiveness correlated with the proportion of cells expressing the gamma, delta chains of the T cell receptor. Furthermore, thymocyte preparations greatly enriched for T cell receptor gamma, delta-bearing cells were found to vigorously proliferate when treated with IL-4. Both IFN-gamma and TNF inhibited IL-4-driven proliferation in a dose-dependent manner, but DS thymocytes were found to be significantly more sensitive to inhibition by both cytokines. Our studies suggest an important role for IL-4 in the proliferation of T cell receptor gamma, delta+ thymocytes and demonstrate regulatory functions for IFN-gamma and TNF in human thymocyte proliferation. The increased sensitivity of DS thymocytes to IFN-gamma and TNF may explain anatomical abnormalities in DS thymuses and suggests the involvement of genes encoded on human chromosome 21 in the responses to both IFN-gamma and TNF.

Cell Division↗
PubMed1992

The role of superoxide anions in the establishment of an interferon-alpha-mediated antiviral state.

It has been suggested that CuZn-superoxide dismutase (CuZnSOD) is required for the establishment of an interferon (IFN)-mediated antiviral state. To investigate this possibility further, a panel of 6 stably transfected HeLa clones, expressing CuZnSOD activity from 1.6 to 7.3 times the normal level, were treated with different concentrations of recombinant human interferon alpha A (rHuIFN-alpha A) followed by challenge with vesicular stomatitis virus (VSV). A biphasic response curve was generated (r = 0.87, p less than 0.025). Clones with up to 3-fold basal level CuZnSOD activity exhibited an inverse relationship between their ability to generate an IFN-alpha-mediated antiviral state and CuZnSOD activity: the higher the CuZnSOD activity, the lower the sensitivity to IFN-alpha and the more IFN-alpha required for antiviral defense. Clones with between 4 to 7.3 times higher CuZnSOD activity than the non-transfected HeLa control showed a direct relationship between the CuZnSOD activity and the sensitivity to IFN-alpha. Furthermore, in agreement with the results obtained with the SOD1-transfected HeLa cells with up to 3 times the basal SOD activity, fetal fibroblasts derived from SOD1-transgenic mouse strains, TgHS-229 and TgHS-218, which also express 3 times the basal CuZnSOD activity, required higher IFN-alpha to achieve 50% protection. These results suggest a possible role for superoxide anion in the establishment of IFN-mediated antiviral effect, especially in the dose-response region in which the inverse relationship between the generation of the IFN-alpha-mediated antiviral state and CuZnSOD activity was observed. To assess this possibility, allopurinol was used as a xanthine oxidase inhibitor and hydroxyl radical scavenger in the IFN-alpha-mediated antiviral assay. Addition of 3 mM allopurinol diminished the IFN-mediated antiviral effect by between 40 and 50% (p less than 0.01), and there was a reduction in superoxide generation (p less than 0.05). The degree of reduction caused by allopurinol treatment was higher at an IFN-alpha concentration of 10 U/ml than at 100 U/ml, and there was no correlation between CuZnSOD activity and the degree of reduction. To establish further the role of superoxide as an antiviral agent, paraquat was used as a superoxide generator in the absence of IFN-alpha in the antiviral assay. Although paraquat at high concentrations is toxic to the cells, it actually showed a protective effect against VSV infection, and an inverse relationship (r = 0.79, r less than 0.025) between cell survival and CuZnSOD activity was observed with 150 mM paraquat treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Allopurinol↗
PubMed1991

Manganese superoxide dismutase is induced by IFN-gamma in multiple cell types. Synergistic induction by IFN-gamma and tumor necrosis factor or IL-1.

We have previously characterized more than 20 proteins induced by the immunoregulatory lymphokine IFN-gamma in human fibroblasts by their m.w. and isoelectric points determined in two-dimensional gels. Some of these proteins are induced uniquely by IFN-gamma, whereas others are also induced by IFN-alpha, TNF, or IL-1. Recent technologic advances have allowed us to begin to rapidly identify proteins induced by IFN-gamma and other cytokines by sequencing the induced proteins from blots of preparative two-dimensional gels of total cell lysates. In this study, we show that the approximately 21 kDa, isoelectric point greater than 7 protein induced by IFN-gamma is manganese superoxide dismutase (Mn-SOD), a mitochondrial protective enzyme encoded by a nuclear gene. Mn-SOD is induced by IFN-gamma and also by TNF in all four human cell lines examined: HS153 fibroblasts, ACHN renal carcinoma, A549 lung carcinoma, and A375 melanoma. Induction of Mn-SOD mRNA is a primary, rapid, and dose-dependent response to IFN-gamma. In ACHN renal carcinoma cells, Mn-SOD mRNA and protein are induced synergistically by IFN-gamma in combination with either TNF or IL-1, and the induced protein is enzymatically active. IFN-gamma and TNF together induce Mn-SOD mRNA by more than 100-fold relative to its level in untreated ACHN cells. The induction of Mn-SOD by IFN-gamma and its synergistic induction by IFN-gamma in combination with TNF and IL-1 should protect healthy cells from the toxicity of O2- during an immune response, and may provide a mechanism for selective killing of infected cells.

Amino Acid Sequence↗
PubMed1990

Coordinated induction of autocrine tumor necrosis factor and interleukin 1 in normal human monocytes and the implications for monocyte-mediated cytotoxicity.

Cytokine production and cytotoxicity for tumor cells are two important aspects of monocyte function and the inflammatory response against tumors and infectious agents. In the present studies we provide direct evidence at the mRNA and protein levels for the existence of autocrine tumor necrosis factor (TNF) and its importance as a mediator of human monocyte cytotoxicity for WEHI-164 tumor cells. The induction of TNF and interleukin 1 beta (IL-1 beta) mRNA by exogenous TNF or IL-1 beta, as determined by Northern blot analysis, is time dependent in normal human monocytes isolated by countercurrent elutriation. With either TNF or IL-1 beta as the stimulus, TNF mRNA is induced first, peaks within 1-3 h, and declines to nearly undetectable levels by 9 h. TNF mRNA accumulation is enhanced in the presence of cycloheximide indicating that de novo protein synthesis is not required for maximal TNF mRNA induction. In contrast, IL-1 beta mRNA is induced later, peaks at 3-9 h, and remains considerably elevated at 18 h. IL-1 beta mRNA accumulation is partially suppressed in the presence of cycloheximide. TNF and IL-1 beta protein production as assayed by specific enzyme-linked immunosorbent assays correlates well with respective mRNA induction. Both TNF and IL-1 beta enhance monocyte cytotoxicity as single agents; however, their combined effect is less than additive. When both agents are combined, TNF mRNA levels, as assessed by densitometric analysis of slot blots, are approximately equal to those induced by TNF alone. In contrast, IL-1 beta mRNA levels are additive. Our studies provide evidence for highly coordinated and interrelated pathways of autocrine TNF and IL-1 induction in human monocytes and demonstrate the role of TNF and IL-1 in regulating monocyte-mediated cytotoxicity for tumor cells.

Autoradiography↗
PubMed1990

Decreased level of T cell receptor expression by Down syndrome (trisomy 21) thymocytes.

Individuals with Down syndrome (DS) have an enhanced susceptibility to viral and bacterial infections. Previous studies by our laboratory demonstrated alterations in the proportions of peripheral T cell subpopulations and decreased proliferative, interleukin-2, and antibody responses to viral and bacterial antigens in DS. These data suggested that DS lymphocytes have a diminished ability to recognize and respond to specific antigen. It has been proposed that the abnormalities in T cell function in DS may be a result of aberrant T cell maturation within the DS thymus. Therefore, we examined by immunofluorescence and flow cytometry the cell surface expression of the alpha,beta chains of the T cell receptor (TCR alpha,beta) and the associated CD3 molecule on thymocytes from 10 DS and 27 control children. A significantly smaller proportion of cells expressing high levels of TCR alpha,beta was observed in DS thymuses compared to controls (17.0% vs. 34.3%, respectively; P less than 0.01). A similar observation was made for CD3, a molecule responsible for signal transduction through the TCR, where a lower proportion of cells expressing high levels of CD3 was found in DS compared to controls (18.4% vs. 43.3%, respectively; P less than 0.001). These data are evidence for aberrant T cell maturation in DS. In addition, our findings of decreased acquisition of high levels of the molecules which are critical for antigen-specific recognition by T cells suggest a possible mechanism for the decreased T cell function found in individuals with DS.

Antigens, CD↗
PubMed1990

Synergistic induction of polypeptides by tumor necrosis factor and interferon-gamma in cells sensitive or resistant to tumor necrosis factor: assessment by computer based analysis of two-dimensional gels using the PDQUEST system.

Tumor necrosis factor (TNF) synergistically enhanced the antiproliferative activity of interferon-gamma (IFN-gamma) in both TNF-sensitive and TNF-resistant variants of the cervical carcinoma line, ME-180. TNF alone had no apparent effect on the levels of synthesis of individual proteins in either of these variant cell lines as assessed by computerized two-dimensional gel analysis of cell lysates using the PDQUEST system. However, IFN-gamma enhanced the levels of 18 polypeptides and suppressed the levels of 10 polypeptides in both cell lines. When used in combination in both cell lines, TNF and IFN-gamma induced the synthesis of 10 polypeptides that were not induced by either agent alone. These synergistically induced polypeptides may be crucial to the mechanism of the synergistic antiproliferative action of TNF and IFN-gamma in ME-180 cells.

Cell Division↗
PubMed1990

Down syndrome (trisomy 21) thymuses have a decreased proportion of cells expressing high levels of TCR alpha, beta and CD3. A possible mechanism for diminished T cell function in Down syndrome.

T cell differentiation antigen expression by thymocytes from nine individuals with Down syndrome (DS) and 27 controls was examined using immunofluorescence and flow cytometry. We found no significant differences between DS and controls in the proportion of CD1+ or CD2+ cells or in the percentages of CD4-8-, CD4+8+, CD4-8+, or CD4+8- cells. However, a significantly smaller proportion of cells expressing high levels of T cell receptor alpha, beta (TCR alpha, beta) was observed in DS thymuses compared to controls (28.0% vs 47.5%, respectively; P less than or equal to 0.01). A similar observation was made for CD3, a signal-transducing complex for the TCR, where the proportion of cells expressing high levels of CD3 in DS was 24.3% compared to 53.3% for controls (P less than or equal to 0.001). These data demonstrate aberrant T cell maturation in DS. Furthermore, our observation of diminished expression of critical molecules for antigen-specific recognition by T cells suggests a possible mechanism for decreased T cell function in DS.

Antigens, CD↗