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R B Levy

Publications and source records attributed to R B Levy.

At least 37 records · Page 2Linked to original sources
PubMed1995

Perforin- and Fas-mediated cytotoxic pathways are not required for allogeneic resistance to bone marrow grafts in mice.

Failure to engraft is a major complication in allogeneic bone marrow transplantation (BMT) using T cell-depleted donor marrow. Previous work has demonstrated that radioresistant host natural killer (NK) cells, CD8+ T cells, and T cells with NK markers participate in the active rejection of donor hematopoietic stem cells in murine models of allogeneic BMT. However, the precise role of cell-mediated cytotoxic mechanisms in marrow allograft rejection remains controversial. To determine the role of perforin- and Fas-mediated cytotoxicity in allogeneic resistance, we transplanted T cell-depleted allogeneic bone marrow into perforin-deficient (perforin 0/0), Fas-ligand-defective (gld/gld), and normal mice. Short-term resistance was measured using a sensitive in vitro assay for colony formation by spleen cells from BMT recipients. The findings we report here demonstrate that strong allogeneic resistance remains largely intact in perforin-deficient and Fas-ligand-defective recipient mice. Thus, perforin- and Fas-mediated cytotoxic pathways are not required for resistance to bone marrow allografts in mice. We conclude that alternative pathways of cytotoxicity and/or soluble factors can mediate resistance to allogeneic BM.

Animals↗
PubMed1994

Endogenous superantigens in allogeneic bone marrow transplant recipients rapidly and selectively expand donor T cells which can produce IFN-gamma.

Despite the existence of many non-MHC disparities between MHC matched but non-MHC mismatched donors and recipients, graft-versus-host disease (GVHD) is not clinically apparent following a significant number of allogeneic bone marrow transplants (BMT) in experimental animals. The present studies examined V beta TcR expression and IFN-gamma production by donor T cells in a BMT model involving an MHC matched, allogeneic donor-recipient combination which included a unidirectional superantigen disparity (Mls). B10.D2-->BALB/c, but not BALB/c-->B10.D2 recipients develop GVHD and mortality ensues 8-12 weeks post-transplant. During the first 2 weeks post-transplant of B10.D2-->BALB/c, approximately 50% of all Thy1.2+ spleen and lymph node cells were found to express T cell receptors utilizing V beta 3. A similar rapid and selective expansion of V beta 3+ TcR bearing donor T cells was detected in two other H-2 matched superantigen disparate donor-recipient BMT combinations. An increased percentage of V beta 3+ T cells was noted among both the CD4+ and CD8+ populations. Thus, in these donor/recipient combinations, all TcR families were not equally expanded early following transplant. At 4-10 days post-transplant, IFN-gamma specific mRNA was readily detected in the spleens of B10.D2-->BALB/cBMT recipients containing large numbers of V beta 3+ T cells. Moreover, V beta 3+ donor T cells from these recipients contained IFN-gamma mRNA. Specific stimulation in vitro with immobilized anti-TcR moAbs demonstrated that V beta 3+ T cells secreted a large amount of the total IFN-gamma levels detected. The ability of endogenous superantigens to activate large numbers of T cells which can produce cytokines after BMT indicates that when present, such antigenic differences may contribute to events occurring during initial graft-versus-host reactions. Such antigens could therefore participate in the events influencing whether GVHD develops following BMT between certain donors and recipients.

Animals↗
PubMed1993

CD8+ and CD4+ T cells contribute to the exacerbation of class I MHC disparate graft-vs-host reaction by concurrent murine cytomegalovirus infection.

Concurrent infection with MCMV has been observed to result in the marked exacerbation of a P-->F1 GVHR across a class I MHC only donor-recipient disparity. We have previously determined that MCMV induces several alterations characteristic of severe GvHR/D which are not observed during GvHR or MCMV infection alone. Most notable of these alterations is the enhanced development of donor anti-host cytotoxic T cell activity. The present studies were performed to examine the requirement of specific donor and host cell populations. Only depletion of donor CD8+ T cells (not NK1.1+ or CD4+) prevented development of severe GVHR/D. By in vivo antibody treatments, depletion of host CD4+ but not CD8+ T cells also abrogated the development of severe GVHR/D. These findings therefore support the hypothesis that MCMV-activated CD4+ T cells enhance the production of a donor anti-host class I CD8+ response. Thus, the present findings support hypotheses attributing the association of viral infection and GVHD development with pathogen-induced immune responses.

Animals↗
PubMed1992

Neu differentiation factor: a transmembrane glycoprotein containing an EGF domain and an immunoglobulin homology unit.

We recently reported that a 44 kd glycoprotein secreted by transformed fibroblasts stimulates tyrosine phosphorylation of the product of the neu proto-oncogene and induces differentiation of mammary tumor cells to milk-producing, growth-arrested cells. A partial amino acid sequence of the protein, termed Neu differentiation factor (NDF), enabled cloning of the corresponding complementary DNA. The deduced structure of the precursor of NDF indicated that it is a transmembrane protein whose extracellular portion contains an EGF-like domain that probably functions as a receptor recognition site. In addition, the ectodomain contains one immunoglobulin homology unit. Despite the lack of a recognizable hydrophobic signal peptide at the N-terminus, a recombinant NDF, like the natural molecule, is released into the medium of transfected COS-7 cells in a biologically active form. Northern blot analysis indicated the existence of several NDF transcripts, the major ones being 1.8, 2.6, and 6.7 kb in size. Transformation by the ras oncogene dramatically elevated the expression of NDF in fibroblasts.

Amino Acid Sequence↗
PubMed1992

Isolation of the neu/HER-2 stimulatory ligand: a 44 kd glycoprotein that induces differentiation of mammary tumor cells.

The neu/HER-2 proto-oncogene (also called erbB-2) encodes a transmembrane glycoprotein related to the epidermal growth factor receptor. We have purified to homogeneity a 44 kd glycoprotein from the medium of ras-transformed cells that stimulates phosphorylation of the Neu protein and retains activity after elution from the polyacrylamide gel. The protein is active at picomolar concentrations and displays a novel N-terminal sequence. Cross-linking experiments with radiolabeled p44 result in specific labeling of Neu, indicating that p44 is a ligand for Neu or a related receptor. The purified protein induces phenotypic differentiation of cultured human breast cancer cells, including altered morphology and synthesis of milk components. This is accompanied by an increase in nuclear area, inhibition of cell growth (probably by cell cycle arrest at the late S or the G2/M phases), and induction of DNA polyploidy. We propose the name Neu differentiation factor (NDF) for p44.

Amino Acid Sequence↗
PubMed1992

Phagocytosis by the murine testicular TM4 Sertoli cell line in culture.

To investigate phagocytosis, an assay enabling flow cytometric analysis of single cells having internalized fluorescent carboxyl microspheres was employed. Greater than 80% of murine testicular Sertoli line (TM4) cells were found to phagocytose one or more microspheres within six hours and electron microscopy confirmed carboxyl microsphere internalization. This level was equivalent to that of a macrophage-like cell line and much greater than the levels of testicular Leydig (TM3) cells. Reducing extracellular calcium or using a calcium channel blocker profoundly inhibited phagocytosis suggesting that phagocytosis by Sertoli cells requires extracellular Ca++. Although follicular stimulating hormone, luteinizing hormone, and testosterone had no significant effects on Sertoli cell phagocytosis, insulin, epidermal growth factor, and hydrocortisone enhanced activity. In contrast, beta-endorphin and 8-bromoadenosine-cyclic monophosphate had an inhibitory effect. In contrast to augmenting macrophage phagocytosis, 1,25-(OH)2D3, interferon-gamma, prostaglandin E2, and lipopolysaccharides, had no apparent effect on that by Sertoli cells. Additionally, neither C3bi receptors (Mac-1 antigen) nor FcRII could be detected on Sertoli cells. In total, the findings demonstrated that the murine Sertoli line exhibits potent phagocytic function and suggest the regulation of this activity may differ from that in "professional" phagocytic cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

1,25-Dihydroxyvitamin D3 down-regulation of HLA-DR on human peripheral blood monocytes.

The regulatory activity of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on human leucocyte antigen (HLA)-DR (MHC class II) antigen expression in monocytes from normal human peripheral blood was examined. Using forward light and side scatter by flow cytometry most cells within the discrete monocyte area expressed high levels of HLA-DR antigens following 4-day culture in medium alone (culture-enhanced HLA-DR) and expression was further up-regulated in the presence of interferon-gamma (IFN-gamma) (IFN-gamma-enhanced HLA-DR). Treatment with 1,25-(OH)2D3 markedly inhibited both culture and IFN-gamma-enhanced HLA-DR but not HLA-ABC (MHC class I). This 1,25-(OH)2D3 inhibition was as effective as PGE2 and hydrocortisone. To ascertain if HLA-DR was specifically down-regulated on monocytes, the effect of vitamin D3 analogues in CD33+ cells was examined. Incubation of the CD33+ cells with 1,25-(OH)2D3, 24-25-(OH)2D3 and 25-OHD3 resulted in dose-dependent inhibition of culture-enhanced HLA-DR paralleling the vitamin D3-receptor affinities of these compounds. Northern analysis also demonstrated that 1,25-(OH)2D3 treatment markedly decreased both expression of culture-enhanced and IFN-gamma-enhanced HLA-DR beta chain messenger RNA (mRNA) in monocyte-enriched populations. In total, our findings are consistent with the proposal that vitamin D3 analogues can contribute to down-regulating immune responses as a consequence of inhibiting class II expression.

Antigens, CD↗
PubMed1991

Oncogenic forms of the neu/HER2 tyrosine kinase are permanently coupled to phospholipase C gamma.

The neu/HER2 proto-oncogene encodes a transmembrane tyrosine kinase homologous to receptors for polypeptide growth factors. The oncogenic potential for the presumed receptor is released through multiple genetic mechanisms including a specific point mutation, truncation at the extracellular domain and overexpression of the protooncogene. Here we show that all these modes of oncogenic activation result in a constitutively phosphorylated neu protein and an increase in tyrosine phosphorylation of a phosphatidylinositol-specific phospholipase (PLC gamma). The examined transforming neu/HER2 proteins, unlike the normal gene product, also co-immunoprecipitated with PLC gamma molecules. A kinase-defective mutant of a transforming neu failed to mediate both tyrosine phosphorylation and association with PLC gamma, suggesting direct interaction of the neu kinase with PLC gamma. This possibility was examined by employing a chimeric protein composed of the extracellular ligand-binding domain of the epidermal growth factor receptor and the neu cytoplasmic portion. The chimeric receptor mediated rapid ligand-dependent modification of PLC gamma on tyrosine residues. It also physically associated, in a ligand-dependent manner, with the phosphoinositidase. Based on the presented results we suggest that the mechanism of cellular transformation by the neu/HER2 receptor involves tyrosine phosphorylation and activation of PLC gamma.

Base Sequence↗
PubMed1991

Effect of concurrent graft-versus-host reaction on tissue distribution and infectious titer of murine cytomegalovirus.

Infection with cytomegalovirus is a major concern following bone marrow transplantation. Previously, we have described a model whereby infection by MCMV concurrent with the injection of parental strain lymphoid cells into F1 recipient mice results in a severe graft-versus-host reaction (GvHR) when the donor and host differ at the class I MHC locus (i.e., class I MHC disparate GvHR). The present studies were performed to determine whether one consequence of this severe GvHR is the alteration of the tissue distribution and/or levels of infectious virus as compared to mice not undergoing GvHR. Using PCR to detect MCMV DNA, it was observed that the tissue distribution of virus 3 days after injection was identical in recipients of virus alone and of GvHR plus virus inocula. At two weeks post injection, virus was recovered from the salivary gland and pancreas in both groups. In contrast to recipients of MCMV alone, lung tissue from GvHR plus virus mice also contained viral DNA. Notably, these results were corroborated by the plaque assay. Moreover, salivary gland and pancreas from recipients of GvHR plus virus were found to contain higher titers of infectious virus. In total, the data demonstrate that, with the notable exception of the lung, the distribution of virus was not changed in the animals despite the presence of a concurrent severe GvHR. However, viral replication in infected tissues appeared less restricted in these recipients.

Animals↗
PubMed1990

Heterodimerization of the erbB-1 and erbB-2 receptors in human breast carcinoma cells: a mechanism for receptor transregulation.

The erbB-1 and erbB-2 protooncogenes encode homologous membrane receptors that respectively bind epidermal growth factor (EGF) and a still incompletely characterized ligand. Binding of EGF to its receptor is known to increase tyrosine phosphorylation of the erbB-2/neu receptor in tumor cells. To investigate the mechanism of this transregulatory pathway, we analyzed the interactions between the two receptors in SKBR-3 human breast carcinoma cells. Chemical cross-linking of 125I-labeled EGF revealed that the radiolabeled EGF receptor coimmunoprecipitates with the erbB-2/neu receptor. In addition a cross-linked species of 360-kdalton molecular mass is also coimmunoprecipitated. The formation of the latter species is absolutely dependent on the presence of EGF receptor and thus appears to represent a heterodimer of the erbB-1 and erbB-2 receptors. In vitro kinase reaction assays revealed that receptor heterodimerization is induced by EGF binding and leads to a dramatic increase in the self-phosphorylation capacity of the dimerized receptors. Moreover, analysis of living SKBR-3 cells suggested that most of the EGF-induced transregulation of the erbB-2/neu receptor is due to receptor heterodimerization. In conclusion, heterodimers of erbB-1 and erbB-2 receptors may provide a mechanism for dual transductory functions of growth factors of breast tumor cells.

Animals↗
PubMed1990

Isolated peripheral T cells from GvHR recipients exhibit defective IL-2R expression, IL-2 production, and proliferation in response to activation stimuli.

Graft-vs-host reactions (GvHR) following the injection of class I/II MHC disparate parental cells into unirradiated F1 recipient mice result in the development of marked immune dysfunction. Following negative selection using adherence and antibody and complement depletion, highly purified T cells were examined to determine their ability to undergo activation. Three weeks after GvHR initiation, unstimulated splenic T cells from GvHR mice displayed normal CD3 and IL-2R expression but elevated expression of class I MHC and Ly-6A/E antigens. Despite culture with normal F1 accessory cells, both CD4+ and CD8+ GvHR T cells exhibited low levels of proliferation to both Con A and anti-CD3 mAb. Although following exposure for 12 h to either of these stimuli, GvHR T cells expressed normal levels of IL-2R, expression was greatly decreased vs normal T cells between 24 and 48 h. In addition, at no timepoint was detectable IL-2 produced by GvHR T cells. Importantly, mixing experiments did not demonstrate detectable suppressive activity in the purified GvHR T cell subsets. GvHR T cells were also tested for their ability to respond to stimuli in the absence of any accessory cell population. These cells again did not proliferate to levels equivalent to normal T cells. Incubation with PMA and either cytokines (Con A supernatant, rIL-7) or anti-CD3 mAb resulted in only low levels of proliferation in GvHR T cells. Notably, at high ionomycin concentrations together with PMA, GvHR T cells did proliferate to equivalent levels as normal cells. However, with decreasing concentrations of ionophore, these cells failed to proliferate as well as normal cells. In total, these findings demonstrate that GvHR T cells are phenotypically and functionally distinct from normal T cells. The results suggest that GvHR T cells themselves may contribute to the well-characterized immune depression occurring in recipients undergoing GvHR.

Animals↗
PubMed1990

The use of primed cytotoxic T lymphocytes as a highly sensitive and accurate method for the detection of MHC disparate allogeneic cells.

The present studies have investigated the use of primed cytotoxic T cells (CTL) for the purpose of detecting the presence of an MHC disparate allogeneic cell population. The findings indicate that restimulation of primed CTL in the presence of activated T cell supernatants is an extremely sensitive method capable of detecting one allogeneic cell within a population containing 10,000 total cells (0.01%). In addition, this primed lymphocyte cytotoxicity assay (PLCA) was shown to be at least as accurate as flow cytometric analysis in determination of low numbers and percentages of allogeneic cells within the cell population being examined. Furthermore, the nature of the allogeneic mononuclear population did not influence the sensitivity or accuracy of this method. To examine this method's applicability for the detection of chimeric populations in vivo, spleen and thymic tissue from neonatally tolerized animals were examined by PLCA analysis. Allogeneic cells were readily detected in 100% of individual host spleen and thymuses tested. In total, the results showed that the PLCA is a highly reproducible and accurate method for the detection of extremely low numbers of allogeneic cells. This approach should be useful to monitor the presence of foreign cells in experimental and clinical situations in which individuals are exposed to allogeneic cell populations.

Animals↗
PubMed1990

T lymphocytes can recognize determinants unique to neuropeptides of guinea pig myelin basic protein containing a single D-isomer amino acid substitution.

The present studies were undertaken to examine how the substitution of racemized forms of selected amino acids in synthetic peptides of guinea pig myelin basic protein (GPMBP) would alter the host's immunological ability to recognize such molecules. Using peptides from the 69-84 sequence of GPMBP containing a D-serine at position 70 or 75 (69-84[D-ser70 or D-ser75]) or D-aspartate at position 82 (69-84[D-asp82]), the findings demonstrated that the position of the diastereomer substitution on these neuropeptides was critical with respect to the ability of the immune system to recognize the molecule. Thus substitution of D-asp at position 82 or D-ser at position 75 abrogated the ability of these peptides to induce experimental autoimmune encephalitis and proliferation of host T cells. In contrast, a peptide containing a D-ser70 residue was capable of inducing clinical disease in rats, as well as stimulating T lymphocytes from 69-84-(D-ser70)-injected animals. Moreover, although this D-peptide was shown to share at least some determinant(s) with the 69-84 peptide, the use of 69-84(D-ser70)-stimulated cell lines demonstrated that some epitope(s) unique to this molecule could stimulate CD4+ syngeneic T cells.

Animals↗
PubMed1990

HSV-1 enhances GvHR-associated parent anti-F1 alloreactivity in vivo and in vitro.

The present studies were performed to demonstrate whether concurrent HSV-1 infection could enhance the immune alterations and dysfunction associated with P----F1-induced graft-versus-host reactions. Examination of phenotypic and functional parameters revealed that Gv-HR-related immune abnormalities in the (C3H.SW X H-2bm1)F1 recipient were dependent on the parental donor inoculum. Together with HSV-1 infection, virus was found to exacerbate the phenotypic changes and functional abnormalities induced in this GvHR model. In addition, the presence of concurrent HSV-1 was shown to augment the level of specific in vivo donor anti-host reactivity present in F1 recipient spleen cells. Moreover, in vitro studies demonstrated that HSV-1 also enhanced the levels of parent anti-F1 allospecific cytotoxic activity. In total, these findings support the hypothesis that viral exacerbation of GvHR is mediated by its enhancement of donor anti-host alloreactive responses.

Animals↗
PubMed1990

Regulation and selective expression of Ly-6A/E, a lymphocyte activation molecule, in the central nervous system.

The Ly-6 locus encodes a group of cell surface molecules found predominantly on lymphoid cells in the mouse. These proteins share several structural and functional characteristics with Thy-1, a molecule expressed in both lymphoid and neuronal tissue. Utilizing anti-Ly-6A/E monoclonal antibodies, the present results demonstrate in situ expression of these molecules in brain tissue. The findings also indicated that these molecules are not expressed during embryonic or neonatal stages of development. Moreover, although Ly-6b haplotype mice exhibited staining primary associated with vascular elements throughout the brain, Ly-6a mice exhibited staining predominantly associated with white matter limited to the hippocampal and midbrain regions. Although cultured glial and neuronal cells expressed marginally detectable levels of Ly-6A/E, the majority of GFAP+ cells in these cultures expressed high levels of Ly-6A/E following incubation with cytokines including rIFN-gamma. In addition, northern blot analysis of RNA from enriched astrocytic cultures corroborated the induction of Ly-6A/E expression. These findings have therefore established that Ly-6 is amongst those groups of genes expressed in both brain and lymphoid tissues.

Aging↗
PubMed1990

Differential regulation and expression of major histocompatibility complex (MHC) and Ly-6 gene products on mouse testicular Leydig and Sertoli cell lines.

The expression and regulation of Class I and Class II major histocompatibility complex (MHC) and Ly-6 antigens were examined in BALB/c testicular cells. Studies were performed utilizing differentiated murine Leydig (TM3) and Sertoli (TM4) cell lines. Neither Class I (Dd) nor Class II (IA/Ed) MHC antigens were detectable on untreated TM3 cells. However, concanavalin-A activated spleen cell supernatant (Con-A sup) or interferon-gamma (IFN-gamma) treatment resulted in the marked induction of both Class I and Class II MHC antigens on virtually all of the Leydig cells. MHC Class II mRNA, which was not detected in resting cells, was clearly induced following IFN-gamma incubation. Sertoli cells were found to constitutively express low levels of Class I (Dd) but not Class II (IA/Ed) antigens. However, in contrast to the enhanced MHC expression in TM3 cells, Con-A sup or IFN-gamma treatment of TM4 cells resulted in marked augmentation of Class I, but not Class II, MHC antigens. Northern blot analysis failed to detect Class II mRNA in either the resting or IFN-gamma treated TM4 populations. Neither ethanol nor tumor necrosis factor (TNF) alone, or together with IFN-gamma head significant effects on MHC expression by TM3 and TM4 cells. Ly-6 antigens, predominantly expressed on hematopoietic cells, were found to be present on both TM3 and TM4 cells. Expression of this non-MHC encoded product was also shown to be markedly enhanced by IFN-gamma treatment on both testicular cell lines. In total, these findings demonstrated that cytokines can differentially affect discrete cell populations arising from a particular tissue with respect to the un-regulation of MHC and non-MHC gene products. These findings are discussed in the context of autoimmune responses directed against this tissue.

Animals↗