Biomedical subjects

G J Dougherty

Publications and source records attributed to G J Dougherty.

51 records · Page 3Linked to original sources

PubMed1993

A putative marker for human melanoma. A monoclonal antibody derived from the melanoma gene in the Xiphophorus melanoma model.

A MoAb was raised against a peptide corresponding to an exposed domain of the putative tyrosine kinase receptor protein encoded by Xmrk, a gene involved in melanoma formation and/or progression in the Xiphophorus fish melanoma model. The antibody reacts specifically with cells from human melanocytic lesions, ie, common acquired nevi, primary and metastatic melanoma biopsies. No reactivity with other cells, including normal melanocytes, was observed in the biopsies or with cells in biopsies from normal tissue (skin, liver, lung, spleen) and from other malignancies including those of neuroectodermal origin. The reactivity was very weak and variable in metastatic melanomas but very strong and characteristic of a receptor-type antigen in primary melanomas, a stage in melanoma progression in which cells have acquired metastasizing potential. It is suggested that the antigen recognized may be involved in growth promotion and represents the human equivalent of the fish melanoma gene product.

Animals↗

PubMed1992

Genetic modification of a murine fibrosarcoma to produce interleukin 7 stimulates host cell infiltration and tumor immunity.

Retroviral-mediated gene transfer was used to introduce and express the gene for murine interleukin 7 (IL-7) in a fibrosarcoma tumor (FSA). The tumorigenicity of these genetically modified FSA cells was greatly decreased in immunologically intact syngeneic mice but was unaltered in T-cell-deprived mice. IL-7-infected tumors that did grow in intact animals from large size inocula did so slowly and had a high incidence of spontaneous regression. Furthermore, mice that had rejected tumors became specifically immune to challenge with uninfected parental tumor cells. IL-7-infected FSA growing in intact mice were heavily infiltrated with host T-cells that were presumably responsible for slow growth and tumor regression, and tumor cells were in the minority. Fluorescence-activated cell sorter analysis showed that there was a 530% increase in T-cells in IL-7-infected FSA compared with control tumors. CD8+ T-cells were particularly elevated, but CD4+ lymphocytes were also increased in number, as were eosinophils and basophils. The CD4+:CD8+ ratio in IL-7-infected FSA was 1:1.7 in comparison to 1:0.6 in control tumors. Lymphocytes isolated from IL-7-producing tumors had greatly enhanced cytotoxicity towards uninfected, parental FSA cells. Killing of non-cross-reacting fibrosarcoma line was also increased but to a much lesser extent. Injection of recombinant human IL-7 directly into established FSA tumors slowed their growth and, in a significant number of instances, caused complete regression. Mice that had rejected tumor became specifically immune. The dose that was needed for this effect was, however, somewhat large: 20 micrograms twice daily for 10 days. This result contrasts with the efficacy of IL-7 gene infection in stimulating responses to the same tumor. These considerations make IL-7 a good candidate for tumor-directed cytokine gene therapy.

Animals↗

PubMed1991

Molecular cloning of CD44R1 and CD44R2, two novel isoforms of the human CD44 lymphocyte "homing" receptor expressed by hemopoietic cells.

In addition to the 85-95 kD CD44 species found on most hemopoietic cell types, the human myelomonocytic cell line KG1a expresses proteins of approximately 115 kD and 130 kD that react with monoclonal antibodies belonging to CD44. The possibility that these higher molecular weight species may represent novel CD44 isoforms containing additional protein sequence was investigated. CD44 cDNA clones were isolated from a plasmid-based expression library prepared from KG1a mRNA. One of the three clones obtained (clone 2.3) was found to encode a CD44 molecule of approximately 130 kD in transfected COS cells. Sequences analysis indicated that the molecule encoded by this cDNA clone, designated CD44R1, was essentially identical to CD44 except for the presence of an additional 132 amino acids inserted into the extracellular domain. This inserted region is rich in serine and threonine residues that may serve as sites of O-linked glycosylation, and contains a potential site of N-linked glycosylation and a potential site of chondroitin sulphate attachment. PCR analysis using primers that flank the inserted region present within CD44R1 identified an additional CD44 isoform, designated CD44R2, that contains only the last 69 amino acids present within the unique region of CD44R1. Peripheral blood mononuclear cells and granulocytes from normal individuals and patients with chronic myelogenous leukemia, polycythemia vera, or acute myelomonocytic leukemia, express both CD44R1 and CD44R2. In contrast, CD44R1 and CD44R2 appear to be differentially expressed in various CD44-positive cell lines. Thus KG1a, and the Epstein-Barr Virus-transformed B cell lines WalkDR4 and Way-1 express both CD44 and the CD44 isoforms CD44R1 and CD44R2, while the myeloid cell lines HL60 and U937 express high levels of CD44, but only very low levels of CD44R1 and CD44R2. The CD44-negative cell lines DHL-4, DHL-10, Jurkat, and K562 are also negative for CD44R1 and CD44R2.

Amino Acid Sequence↗

PubMed1991

Mechanisms that regulate the cell cycle status of very primitive hematopoietic cells in long-term human marrow cultures. II. Analysis of positive and negative regulators produced by stromal cells within the adherent layer.

Numerous factors that can influence the proliferation and differentiation in vitro of cells at various stages of hematopoiesis have been identified, but the mechanisms used by stromal cells to regulate the cycling status of the most primitive human hematopoietic cells are still poorly understood. Previous studies of long-term cultures (LTC) of human marrow have suggested that cytokine-induced variations in stromal cell production of one or more stimulators and inhibitors of hematopoiesis may be important. To identify the specific regulators involved, we performed Northern analyses on RNA extracted from human marrow LTC adherent layers, or stromal cell types derived from or related to those present in the adherent layer. These analyses showed marked increases in interleukin-1 beta (IL-1 beta), IL-6, and granulocyte colony-stimulating factor (G-CSF) mRNA levels within 8 hours after treatments that lead to the activation within 2 days of primitive hematopoietic progenitors in such cultures. Increases in granulocyte-macrophage (GM)-CSF and M-CSF mRNA were also sometimes seen. Bioassays using cell lines responsive to G-CSF, GM-CSF, and IL-6 showed significant elevation in growth factor levels 24 hours after IL-1 beta stimulation. Neither IL-3 nor IL-4 mRNA was detectable at any time. In contrast, transforming growth factor-beta (TGF-beta) mRNA and nanogram levels of TGF-beta bioactivity in the medium were detected at all times in established LTC, and these levels were not consistently altered by any of the manipulations that stimulated hematopoietic growth factor production and primitive progenitor cycling. We also found that addition of anti-TGF-beta antibody could prolong or reactivate primitive progenitor proliferation when added to previously stimulated or quiescent cultures, respectively. Together, these results indicate a dominant negative regulatory role of endogenously produced TGF-beta in unperturbed LTC, with activation of primitive hematopoietic cells being achieved by mechanisms that stimulate stromal cells to produce G-CSF, GM-CSF, and IL-6. Given the similarities between the LTC system and the marrow microenvironment, it seems likely that the control of human stem cell activation in vivo may involve similar variations in the production of these factors by stromal cells.

Blotting, Northern↗

PubMed1990

Heterogeneous modifications of the 114/A10 protein of interleukin-3-dependent cells are concentrated in a highly repetitive amino-terminal domain.

The protein 114/A10 is expressed in structurally heterogeneous forms on the surfaces of all murine hemopoietic cells that are responsive to the growth factor interleukin-3, including multipotent progenitors. Despite their structural diversity, all forms of 114/A10 appear to be expressed from a single gene that encodes a peptide with a potential transmembrane segment, three sequences with homology to epidermal growth factor, and an N-terminal domain consisting of eight perfect or near perfect tandem repeats of a 27-amino acid peptide that has a very high content of serine and threonine. Constructed cDNAs that encode deleted or hybrid forms of the 114/A10 protein have been expressed on COS cells in order to localize sites of post-translational modification. The results demonstrate that the structural diversity of 114/A10 is confined to its N-terminal repeat domain and probably arises through extensive O-linked glycosylation within each of the repeats in that domain. The highly modified N terminus of 114/A10 projects from the outer surface of the cell and could serve as a ligand for lectin-like proteins or could modulate the activities of the adjacent epidermal growth factor-like domains of 114/A10.

Amino Acid Sequence↗

PubMed1989

Molecular cloning of 114/A10, a cell surface antigen containing highly conserved repeated elements, which is expressed by murine hemopoietic progenitor cells and interleukin-3-dependent cell lines.

Monoclonal antibody 114/A10, raised against the murine multipotential hemopoietic progenitor cell line B6SUtA, identifies an antigen highly expressed by primary myeloid progenitor cells, the myelomonocytic leukemia cell line WEHI-3, and various interleukin-3 (IL-3)-dependent cell lines. Western blotting studies indicate that the 114/A10 antigen has a mean relative molecular mass of 150,000 but varies greatly in size range between different cell types. cDNA clones encoding this protein were isolated from a plasmid-based expression vector library prepared from B6SUtA RNA. Three clones corresponding in size to the two major mRNA species detected in IL-3-dependent cell lines (3.0 and 2.2 kilobase pairs) and differing in their utilization of alternative polyadenylation signals were obtained. These clones contain a single long open reading frame of 573 amino acids possessing the typical characteristics of an integral membrane protein. A particularly striking feature of this sequence is the presence at the extracellular amino terminus of a series of eight highly conserved 27-amino acid, serine/threonine-rich (55%) tandem repeats that may serve as sites of extensive glycosylation. The extracellular domain also contains three epidermal growth factor-like cysteine-rich repeats. The distribution and structural characteristics of the 114/A10 antigen suggest a possible regulatory role in the cellular response to IL-3.

Amino Acid Sequence↗

PubMed1989

Identification and characterization of 114/A10, an antigen highly expressed on the surface of murine myeloid and erythroid progenitor cells and IL-3-dependent cell lines.

Monoclonal antibody (mAb) 114/A10, raised against the murine bone marrow-derived multipotential hemopoietic progenitor cell line B6SUtA, identifies an antigen highly expressed by various interleukin-3 (IL-3)-dependent cell lines, the myelomonocytic cell line WEHI-3, and a large proportion of primary myeloid and erythroid colony-forming cells. Spleen- and bone marrow-derived 114/A10-positive cells were shown to selectively proliferate in vitro in response to pokeweed mitogen-stimulated spleen cell-conditioned medium or recombinant IL-3. Western blot analysis indicated that the antigen recognized by mAb 114/A10 has a mean relative molecular mass of approximately 150,000, although it is extremely heterogeneous in nature, and differs greatly in size range among different cell lines.

Animals↗

PubMed1988

The function of human intercellular adhesion molecule-1 (ICAM-1) in the generation of an immune response.

Monoclonal antibody RR 1/1 directed against the putative LFA-1 ligand molecule intracellular adhesion molecule-1 (ICAM-1) was found to inhibit the T cell proliferative response to the antigen PPD. Interestingly, the percentage of unstimulated monocytes which expressed ICAM-1 on their surface appeared to vary greatly from person to person although the majority of monocytes did express high levels of ICAM-1 within their cytoplasm and surface expression could be rapidly induced on most cells by adherence to fibronectin. Resting T cells showed no evidence of surface or cytoplasmic ICAM-1 although expression was induced both within the cell and on the membrane as a result of activation with phytohemagglutinin or a combination of OKT3 and phorbol 12,13-dibutyrate. The significance of these findings with respect to the function of monocyte and T cell in the generation of an immune response is discussed.

Adult↗

PubMed1988

Identification of a novel monocyte cell surface molecule involved in the generation of antigen-induced proliferative responses.

A monocyte molecule, identified by a monoclonal antibody (mAb) named 24, is involved in the antigen-specific proliferation of T cells. Several types of antigen-induced responses are blocked by mAb24, but mitogen responses, even at low doses, are not. The 24 molecule is found on circulating monocytes and no other cell type. Normally only a proportion of monocytes express the 24 molecule but it can be induced rapidly on monocytes in culture, possibly from intracellular sources. The epitope is present on a heterodimer of 175 and 95 kDa which is similar to the leukocyte cell adhesion molecule (Leu-CAM) family. These findings suggest that although the 24 molecule shows some functional and biochemical similarities with the Leu-CAM family of molecules, it represents a novel structure primarily associated with mononuclear phagocytes.

Antibodies, Monoclonal↗

PubMed1987

The role of monocyte lymphocyte function-associated antigen 1 (LFA-1) in accessory cell function.

Monoclonal antibodies directed against the common beta subunit shared by lymphocyte function-associated antigen-1 (LFA-1), CR3 and p150,95 or the alpha subunit of LFA-1, inhibited the T lymphocyte proliferative response to tetanus toxoid, pokeweed mitogen-1 and OKT3 but had little or no effect on the response to the leukoagglutinating lectins pokeweed mitogen-2 and phytohemagglutinin. The LFA-1 molecule was shown to participate in the adhesive interaction between monocytes and T lymphocytes that was required for the initiation of an immune response. Both monocyte and T lymphocyte LFA-1 were found to contribute to this interaction. These results further define the part played by the LFA-1 molecule in the activation of T lymphocytes.

Adult↗

PubMed1987

The human mononuclear phagocyte high-affinity Fc receptor, FcRI, defined by a monoclonal antibody, 10.1.

The properties of the mononuclear phagocyte (Mph) high-affinity Fc receptor, FcRI, were investigated using a novel monoclonal antibody (mAb) designated 10.1. This receptor was shown to be a protein of 71 kDa, presented chiefly on monocytes and the myeloid cell lines U937 and HL60. mAb 10.1 inhibited the binding to Mph of erythrocytes opsonized with rabbit IgG or human IgG3. It also blocked T cell proliferation induced by murine CD3 mAb of the IgG2a but not the IgG1 subclass. These results suggest that rabbit IgG, human IgG3 and murine IgG2a all bind to FcRI in a similar manner and that mAb 10.1 reacts with an epitope on FcRI near to the binding site for the Fc region of IgG. In addition, although it is well known that FcRI has a high affinity for both monomeric human IgG1 and IgG3, we show in this study that while erythrocytes opsonized with human IgG3 bind to Mph, equivalent cells opsonized with IgG1 surprisingly do not. These results define further the nature of the constraints on the interaction between Mph FcRI and particular IgGs.

Animals↗

PubMed1986

Immunoregulating activity of tumor-associated macrophages.

In this report, we examine the antigen nonspecific immunoregulating activity of macrophages isolated from the murine methylcholanthrene-induced fibrosarcoma FSA. These cells were shown to enhance the primary anti-CRBC PFC response of whole normal spleen cells in a dose-dependent fashion. This function was associated with a subpopulation of large Ia-negative macrophages and was mediated by a soluble macrophage-derived factor that appeared to act by stimulating the proliferation and/or differentiation of antigen-reactive T cells. The relationship of this factor to previously described monokines is discussed.

Animals↗

PubMed1986

Accessory cell activity of murine tumor-associated macrophages.

The accessory cell activity of macrophages associated with the murine 3-methylcholanthrene-induced fibrosarcoma FSa was investigated. On the basis of Fc receptor expression and phagocytic activity, 20-25% of cells present within enzymatically disaggregated tumor cell suspensions could be classified as macrophages. These cells were approximately 50% I-Ak positive but did not express the Mac-1 antigen. T-cells played an important role in regulating I-Ak expression, and macrophages obtained from tumors grown in nu/nu mice were I-Ak negative. Tumor-associated macrophages were shown to possess potent accessory cell activity and were fully capable of reconstituting the primary anti-calf red blood cell plaque-forming cell (PFC) response of Sephadex G-10-passed spleen cells. This function required the presence of the I-Ak-positive subpopulation, and macrophages treated with anti-Ia serum and complement or obtained from tumors grown in nu/nu hosts lacked accessory cell activity. Tumor-associated macrophages were also able to provide the essential accessory cell function required for cooperation between tumor-specific TH cells and normal B-cells in the generation of an anti-trinitrophenyl (TNP) PFC response in the presence of TNP-coupled FSa antigen. These results suggest that progressive growth of the FSa tumor in vivo cannot be readily attributed to a defect in the accessory cell function of tumor-associated macrophages.

Animals↗

PubMed1984

Macrophage heterogeneity.

Macrophages perform a large number of diverse and on occasion mutually antagonistic functions. It seems unlikely given the magnitude of the task, that any one cell could carry out all these different demands. This raises the possibility that distinct subsets of macrophages exist each capable of performing only certain functions. It is indeed the case that although all macrophages have many features in common they are by no means uniform. Two major types of macrophage heterogeneity have been described. The first is termed "Interpopulation" heterogeneity and refers to differences between populations of macrophages obtained from different tissue sites. The second is termed "Intrapopulation" heterogeneity and refers to differences between subpopulations of macrophages obtained from within a particular site. We propose that such macrophage heterogeneity could be generated by 2 main mechanisms--"Differentiation and Modulation". It is assumed that as macrophages differentiate from bone marrow precursor cells they sequentially express a series of functionally distinct phenotypes. Such phenotypes are however relatively dynamic and may be readily altered by exposure to various microenvironmental and nonmicroenvironmental modulating signals. The responsiveness of cells to modulating signals is likely to depend both on their differentiation stage and previous exposure to other such signals. Assuming that the cells entering different tissue sites are essentially identical, then "Interpopulation" heterogeneity may be generated as a direct result of different microenvironmental modulating signals acting on the cells. "Intrapopulation" heterogeneity on the other hand may be generated by both differentiation and modulation. Macrophages at various differentiation stages are found within a tissue. Some cells will have recently arrived from the blood stream while others may have resided in the tissue for some time. In addition, the cells within a tissue may have been exposed to a different spectrum of modulating signals, or exposed to the same signals for various periods of time. The suggested existence of distinct macrophage sublineages adds an additional level of complexity to the subject of macrophage heterogeneity. It has been shown in a relatively small number of experiments that colonies of macrophages, clonogenically derived from individual bone marrow precursor cells in vitro, may express different phenotypes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMedDate not supplied

The effects of cytokine gene transfer into tumors on host cell infiltration and regression.

New strategies are becoming available that promise to revolutionize cancer immunotherapy. Although the task of generating what is in essence a pathogenic autoimmune anti-tumor response in the face of local and systemic immune suppression is likely to remain a formidable one, advances in molecular strategies for enhancing tumor immunity have been made that show considerable promise, in particular those based on gene transfer technology. For example, introduction of certain cytokine genes into murine tumor cells have been shown to enhance tumor immunogenicity and induce regression. Caution is needed in properly interpreting the relevance of observations derived from murine models for human cancer, but clinical trials are underway that will test the utility of cytokine gene therapy for cancer and that will generate data that will be useful for the design of future strategies. Because of the magnitude of the problem of inducing tumor regression, it is argued that, even if genetically engineering can be used to successfully enhance anti-tumor immunity, combination of such strategies with other existing conventional anti-cancer therapies, that increase the effectiveness of both, may be necessary to reliably achieve cure.

Animals↗