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M I Greene

Publications and source records attributed to M I Greene.

At least 127 records · Page 7Linked to original sources

Identification of tissue specific nuclear proteins: DNA sequence and protein binding regions in the T cell receptor beta J-C intron.

We have determined the DNA sequences in the J2-C2 intron of the T cell receptor (TCR) beta gene and analyzed nuclear proteins binding to this region. Previously, we identified two tissue-specific DNase I hypersensitive regions, potential regulatory regions, in the J-C intron. The DNA sequence of the J2-C2 intron revealed that both DNase I hypersensitive regions have similar DNA sequences, suggesting that these regions are evolutionarily conserved. We have also identified tissue-specific nuclear-protein binding regions downstream of the DNase hypersensitive regions. Although transcriptional enhancer activity was not observed in the hypersensitive regions or the adjacent protein binding regions in the J-C intron, our findings suggest that the TCR-beta J-C intron may contain some other type of regulatory element.

Animals↗

Expression of the neu gene-encoded protein (P185neu) in human non-small cell carcinomas of the lung.

The neu protooncogene is a recently described transforming gene originally isolated from ethylnitrosourea-induced rat neuroblastomas. We have examined the expression of the neu gene in human non-small cell lung carcinomas using immunoprecipitation and immunohistochemistry. The neu protein product (p185neu) was present in eight of 22 non-small cell carcinoma cell lines derived from human lung tumors. Expression of p185neu was found in all histological subtypes of non-small cell carcinomas including large cell carcinomas, squamous cell carcinomas, and adenocarcinomas. Extension of these data to biopsy specimens of human lung tumors demonstrated that normal ciliated bronchial epithelium of the peripheral airways expressed p185neu at low levels. Neoplastic cells in four of 12 adenocarcinomas and three of five squamous cell carcinomas also expressed p185neu at levels higher than the normal ciliated bronchial epithelium. Together these studies indicate that p185neu expression is a common feature of human lung tumors.

Biopsy↗

Analysis of c-erbB-2 protein expression in conjunction with DNA content using multiparameter flow cytometry.

Breast cancer is a leading cause of cancer death among women. Factors useful for determining the prognosis of breast cancer include axillary lymph node involvement, tumor size, hormonal receptor status, nuclear grade, and relative DNA content. The c-erbB-2 protooncogene is amplified in 10-40% of primary breast tumors, as well as in breast cancer cell lines; where it is amplified there is increased expression of its product. We have investigated the DNA content and c-erbB-1 protein expression in tumor cell lines and in breast cancer patient specimens by multiparameter flow cytometry. The study was enabled by the discovery that both cellular integrity and c-erbB-2 antigen reactivity were preserved in cells and tissues following fixation in 70% ethanol. We demonstrate that flow cytometric analysis of c-erbB-2 expression in populations of ethanol-fixed tumor cells is a reliable and sensitive quantitative method that correlates well with previously documented semiquantitative techniques. This is a feasible method for analyzing archived clinical samples, and further allows correlations between c-erbB-2 levels and other cellular parameters. Additionally, this method detects abnormal populations not identified by DNA content analysis alone. Further studies utilizing this approach are necessary to evaluate the prognostic value of this oncoprotein in human breast cancer.

Adenocarcinoma↗

Origin and selection of peripheral CD4-CD8- T cells bearing alpha/beta T cell antigen receptors in autoimmune gld mice.

We have analyzed the origin and development of unusual CD4-CD8- alpha/beta T cell receptor-positive peripheral T cells produced in large numbers by mice homozygous for the gld mutation (C3H-gld/gld). These mice may be an important model for investigating processes controlling T cell development. Bone marrow transfers demonstrated that the gld defect was intrinsic to bone marrow-derived cells. Clonal deletion of potentially autoreactive cells was observed in peripheral gld CD4-CD8-, CD4+CD8-, and CD4-CD8+ T cells, as well as mature thymocytes. This suggests that gld CD4-CD8- T cells have passed through the thymus in ontogeny and that gld autoimmunity does not result from a general defect in elimination of self-reactive thymocytes. These observations, combined with demethylation of the CD8 gene in the CD4-CD8- population, support prior expression of CD4 and/or CD8 in gld CD4-CD8- T cell ontogeny, perhaps at a CD4+CD8+ stage. Steroid sensitivity of gld thymocytes and CD4-CD8- T cells was normal. Therefore, we found no gross abnormalities in two major mechanisms of inducible cell death in the gld thymus, the clonal deletion process associated with tolerance and the steroid-inducible endogenous endonuclease thought to be involved in apoptosis of unselected thymocytes. The data suggest that if gld CD4-CD8- T cells arise via escape from normal elimination in the thymus, they must do so by a novel defect in thymic selection (perhaps related to aberrant positive signals) and/or are expanded by an extrathymic process which allows clonal deletion to occur.

Animals↗

Inhibition of T cell development in thymic organ culture: implications for the mechanism of action of cyclosporin A.

We have examined the effects of the immunosuppressive drug cyclosporin A (CsA) on the phenotypic maturation of T cells in thymic organ cultures begun at day 16 of gestation. CsA specifically inhibited the generation of cells expressing high levels of alpha/beta TcR/CD3 complexes and a mature phenotype defined by CD4 and CD8 surface markers. Adding interleukin (IL) 1 beta, IL 2 or IL 4 failed to reverse the effects of CsA, and major histocompatibility complex class II expression in the thymic medulla was preserved. Possible mechanisms of CsA-mediated inhibition of T cell development are discussed.

Animals↗

T-cell receptor and autoimmune disease.

Since the genes encoding the TCR have been cloned, their structure, organization, pattern of rearrangement, diversification and expression in ontogeny have been classified. However, there are still many important questions to be addressed, such as the nature of thymic education, tolerance, the mechanism of MHC-restricted antigen recognition and the relation between TCR repertoire and autoimmunity. In the future, new approaches to study these issues, such as transgenic mice, X-ray crystallography, and severe combined immune deficiency mice reconstituted with human hematopoietic cells will lead to a more profound understanding of these questions. This will hopefully allow us to manipulate the immune response in different and more effective ways than are currently available.

Animals↗

Antibody geometry and form: three-dimensional relationships between anti-idiotypic antibodies and external antigens.

Anti-idiotypes have been developed to mimic a wide variety of antigens in studies of antibody-antigen interactions. Molecular, biochemical and structural analysis of antibody-anti-idiotype pairs presents an excellent model for study of complex protein-protein interactions. An understanding of the ways in which anti-idiotypes mimic antigens and the features determining binding characteristics will facilitate rational design of compounds with biological, enzymatic, pharmaceutical and chemical applications.

Amino Acid Sequence↗

Ligand binding to the cell surface receptor for reovirus type 3 stimulates galactocerebroside expression by developing oligodendrocytes.

Viruses utilize normal cell surface structures as attachment sites. Interaction of viral components with these structures may alter target cell growth. In the present study, the expression and function of the cell surface receptor for reovirus type 3 (Reo3R) was studied in neonatal rat optic nerve glial cultures. The Reo3R is expressed by mature oligodendrocytes and astrocytes but not by O-2A progenitor cells. It appears at an early stage of oligodendrocyte development, coincident with the O4 marker but prior to galactocerebroside or myelin basic protein. Anti-Reo3R antibodies stimulate the expression of galactocerebroside by developing oligodendrocytes. Divalent Reo3R-binding peptides are similarly active. Maximal stimulation of galactocerebroside expression occurs with treatment as short as 4 hr, consistent with a receptor-mediated process. Cell surface structures used as an attachment site by reovirus type 3 may also play a role in the regulation of oligodendrocyte differentiation.

Amino Acid Sequence↗

Self-reactive T cells can escape clonal deletion in T-cell receptor V beta 8.1 transgenic mice.

To study the mechanisms of tolerance in detail, we have constructed transgenic mice expressing a V beta 8.1-D beta 2-J beta 2.3-C beta 2 T-cell receptor (TCR) gene. Since expression of V beta 8.1 is known to correlate with reactivity of CD4+CD8- T cells to minor lymphocyte-stimulating locus 1a (Mls-1a), we expected to induce tolerance in most CD4+CD8- T cells in V beta 8.1 transgenic mice of the Mls-1a allele. In one line of Mls-1b V beta 8.1 transgenic mice, the V beta 8.1 TCR was expressed on greater than 98% of mature T cells and their response to Mls-1a was highly enriched. In Mls-1a V beta 8.1 transgenic mice, CD4+CD8- T cells in these mice were severely reduced among both peripheral T cells and thymocytes. However, the deletion of these cells was not complete, and most of the residual CD4+CD8- mature T cells still expressed normal densities of V beta 8.1 TCR. The residual CD4+CD8- T cells did not respond to Mls-1a but were still able to proliferate in response to other stimuli via the TCR. Interestingly, CD4+CD8- V beta 8.1+ T-cell clones isolated from Mls-1a V beta 8.1 transgenic mice could respond to Mls-1a. We suggest that these types of T cells escape clonal deletion in the thymus.

Animals↗

Mechanisms of autoimmunity in the context of T-cell tolerance: insights from natural and transgenic animal model systems.

There are a number of mechanisms which cooperate to produce and maintain T-cell tolerance. First, and perhaps most important, is the clonal deletion in the thymus of T cells with high affinity for self antigens. However, to ensure that a wide repertoire of T cells is available in the periphery to combat foreign antigens, the threshold of clonal deletion may be set low enough so that T cells whose TCR's have sub-threshold affinity for self antigens mature and migrate to the periphery. T cells which recognize self antigen-derived peptides not expressed or presented in the thymus will also fail to be deleted. For those self-reactive T cells which are not deleted in the thymus, other mechanisms may produce tolerance, including an undefined alteration of signalling pathways which produces clonal anergy, and lowering the avidity of the TCR for its ligand by downregulating coreceptor and accessory molecules. Active suppression of T-cell responses in another well-described phenomenon whose mechanism is undefined. From our observations with the model systems discussed here, we have observed three distinct mechanisms by which T-cell tolerance can be circumvented, allowing autoimmune phenomena to occur. These mechanisms may have relevance for different types of autoimmune diseases seen in humans. In gld mice, the autoimmune disease seems to be related to a global defect in T-cell differentiation and function, which allows for the expansion of autoimmune B cells. While we showed that clonal deletion of V beta-bearing T cells is appropriate in certain cases, aberrant lymphokine secretion by the abnormal T cells or disruption of immune system regulation are most probably responsible for allowing autoantibody production. While human lupus erythematosis shares much of the pathology of lpr and gld mice, there is no expansion of T cells with a similar phenotype in human lupus. There are environmental factors which must play a role in the development of human lupus, since the incidence of the disease does not follow an absolute genetic pattern. The escape from clonal deletion and subsequent reactivation of autoimmune T cells which we observed in V beta 8.1 TCR-transgenic mice can be a model for human autoimmune diseases such as multiple sclerosis and type I diabetes, in which T cells are directed against a specific autoantigen. According to this model, susceptibility loci for autoimmune disease such as the MHC would function by producing different repertoires of T cells which in some cases could gain autoreactivity following activation.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

Reovirus transport--studies using lymphocytosis promoting factor.

To explore how bacteria and their products may modulate viral infection, we investigated the effect of a well-characterized and highly purified product of Bordetella pertussis, a pertussis toxin, also known as lymphocytosis promoting factor (LPF), on enteric reovirus infection. LPF is known to have a variety of effects, including modulation of circulation and homing of lymphoid cells. When adult mice are inoculated with reovirus type 1 perorally, reovirus first enters the Peyer's patches (PP) through M cells, and then spreads to mesenteric lymph nodes (MLN) and spleen with minimal dissemination to other peripheral tissues. In view of the profound effect of LPF on lymphoid tissues, we evaluated whether LPF might influence the early stages of type-1 reovirus infection following peroral inoculation. Pretreatment of adult BALB/c mice with LPF significantly inhibited the spread of reovirus in a manner dependent upon the route of inoculation; LPF inhibited the extra-intestinal spread of virus from PP to MLN after intragastric inoculation; in contrast there was enhancement of the spread of blood-borne viruses to MLN after intravenous inoculation. This result, together with the fact that the efferent lymph from PP reaches MLN, suggests that a proportion of reoviruses were conveyed from PP to MLN in association with lymphoid cells along the lymphatic channels and that LPF affects reovirus, in part, by blocking cell movement.

Animals↗

Neu (c-erbB-2), a tumor marker in carcinoma of the female breast.

The c-erbB-2 oncogene encodes a transmembrane phosphoglycoprotein. This molecule appears to be a growth factor receptor in the family of tyrosine kinase growth factor receptors; however, its ligand has not yet been identified. Amplification and/or overexpression of c-erbB-2 in breast adenocarcinomas occurs frequently and its occurrence implies a more advanced malignancy. This functional tumor marker is readily identified by appropriate DNA and antibody probes. The large external domain of the c-erbB-2 gene product is a promising target for immunodiagnostic and immunotherapeutic modalities.

Adenocarcinoma↗

Anti-receptor antibodies reverse the phenotype of cells transformed by two interacting proto-oncogene encoded receptor proteins.

The neu oncogene product, p185neu, is a tyrosine kinase receptor with structural similarity to the epidermal growth factor (EGF) receptor. We have recently described that coexpression of EGF receptors and high levels of normal p185c-neu lead to transformation of rodent fibroblasts. Anti-EGF receptor and anti-p185neu monoclonal antibodies inhibited tumorigenic growth of these transformants implanted into nude mice. These monoclonal antibodies also suppressed focus formation of the cells transformed by the synergistic action of these receptor proteins in vitro. However, EGF enhanced focus formation and stimulated cell growth when added to cells transfected just with the EGF receptor encoding cDNA. These data suggest that receptor specific effectors may have potentially useful applications in cancer therapy for neoplasms which demonstrate increased receptor densities. In addition the data suggest novel differences in the actions of tyrosine kinases when acting alone or in concert with other receptors.

Animals↗

Immunohistochemical evaluation of c-erbB-2 oncogene expression in ductal carcinoma in situ and atypical ductal hyperplasia of the breast.

Cases of ductal carcinoma in situ (DCIS) and atypical ductal hyperplasia (ADH) of the breast were examined for expression of the protein product of the c-erbB-2 (neu, HER-2) oncogene using two different polyclonal antibodies via an avidin-biotin immunoperoxidase method on formalin- or Bouin'-fixed, paraffin-embedded tissue. Fifty-five percent (18/33) of DCIS and 10% (2/21) of ADH were positive. Significant c-erbB-2 expression in DCIS was generally divided on histologic grounds: ten of ten comedocarcinomas showed strong membrane staining, while only one of 14 small cell DCIS cases (micropapillary or cribiform patterns) showed immunostaining (which was weak and basilar in this single case). DCIS cases of mixed histology were strongly positive in areas of comedocarcinoma. In two of three cases of associated Paget's disease strong membrane staining was seen. The two c-erbB-2-positive ADH cases showed weak basilar staining akin to the small cell DCIS cases. Five cases of lobular neoplasia (atypical lobular hyperplasia or lobular carcinoma in situ) associated with DCIS or ADH were negative for c-erbB-2 expression. We conclude that comedocarcinoma in situ and Paget's disease frequently express the c-erbB-2 protein and are both histologically and biochemically distinct from ADH and small cell patterns of DCIS. We advocate precise subclassification of DCIS on histopathologic reports, particularly in view of reports that overexpression of the c-erbB-2 oncogene in infiltrating breast carcinomas may be associated with a poor prognosis.

Breast↗

Species tropism of HIV-1 infectivity of interspecific cell hybridomas implies non-CD4 structures are required for cell entry.

Studies have demonstrated that CD4 may be necessary but not sufficient for human immunodeficiency virus (HIV) infection. Murine cell lines constructed to express the human CD4 glycoprotein demonstrate a restriction in their ability to support infection by the HIV-1 retrovirus. Such restrictions may indicate that molecules in addition to CD4 may be necessary for HIV-1 infection. HIV-1 infectivity has been examined in a panel of murine and human cell lines. It has been demonstrated that CD4 expression allows infectivity on a wide range of human cell lines. Some interspecific hybridomas constructed between human and murine cell lines can, when expressing the human CD4 glycoprotein, become infectable in a manner similar to human cell lines. These studies imply that surface molecules, in addition to CD4, are necessary for HIV-1 entry and infectivity. Such molecules will be important for understanding the pathogenesis of HIV-1.

Animals↗

Synergistic interaction of p185c-neu and the EGF receptor leads to transformation of rodent fibroblasts.

The protein product of the rodent neu oncogene, p185neu, is a tyrosine kinase with structural similarity to the epidermal growth factor receptor (EGFR). Transfection and subsequent overexpression of the human p185c-erbB-2 protein transforms NIH 3T3 cells in vitro. However, NIH 3T3 cells are not transformed by overexpressed rodent p185c-neu. NIH 3T3 transfectants overexpressing EGF receptors are not transformed unless incompletely transformed. Several groups have recently demonstrated EGF-induced, EGFR-mediated phosphorylation of p185c-neu. During efforts to characterize the interaction of p185c-neu with EGFR further, we created cell lines that simultaneously overexpress both p185c-neu and EGFR and observed that these cells become transformed. These observations demonstrate that two distinct, overexpressed tyrosine kinases can act synergistically to transform NIH 3T3 cells, thus identifying a novel mechanism that can lead to transformation.

Animals↗

Immune response to a molecularly defined internal image idiotope.

A monoclonal anti-idiotope termed 87.92.6 mimics the neutralization/cell-attachment site of the reovirus type 3 hemagglutinin (HA3). The second complementarity determining regions of the VH and VL of 87.92.6 share sequence similarity with a determinant on the HA3. We have used synthetic peptides (termed VH, VL, and Reo peptides, respectively) to probe the immunologic significance of this sequence similarity. Antibodies specific for Reo peptide or VL peptide neutralized reovirus type 3 infectivity. Although Reo peptide was an effective immunogen by itself, free VL peptide or VH peptide were unable to elicit antibodies unless they were linked to each other (VH-VL peptide). Immunization with reo peptide, 87.92.6, or the HA3 elicited a specific lymphocyte proliferative response to VH peptide, indicating that VH peptide may bear an important TH determinant. As found previously for 87.92.6, VL peptide elicited a delayed-type hypersensitivity response specific for reovirus type 3. Reovirus type 3 specific cytolytic lymphocytes specifically lysed targets coated with VH-VL peptide, but not VH or VL peptide alone. These results suggest that immune cross-reactivity between an external Ag and an internal image antibody can be understood at the primary structural level. These observations may have important implications for understanding the development of autoantibodies, network interactions, and the regulation of immune responses.

Amino Acid Sequence↗