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Biomedical subjects

G Rowden

Publications and source records attributed to G Rowden.

At least 37 records · Page 2Linked to original sources

Melanocytes freshly isolated from normal human skin express the cell membrane receptor for the adhesive glycoprotein thrombospondin.

Thrombospondin (TSP) is an adhesive protein with multiple binding sites, which is able to mediate several cell-to-cell and cell-to-matrix interactions, particularly through its cell membrane receptor (TSP-R). Because human keratinocytes are able to synthesize and express TSP, and as TSP is also localized at the dermal-epidermal junction in normal human skin, we questioned whether epidermal cells are able to bind available TSP, that is, to express TSP-R. To investigate this, we employed gold immunoelectron microscopy on epidermal cells freshly isolated from normal human skin; the TSP-R was detected by OKM5 monoclonal antibody. Epidermal cells showing ultrastructural characteristics of melanocytes were gold-stained on their plasma membrane, whereas keratinocytes, Langerhans cells and lymphocytes were unstained. Although functional studies are clearly necessary to clarify the role(s) played by the TSP-R on the cell surface of melanocytes, it is tempting to speculate that the TSP-R may be important for melanocyte adhesion to the dermal-epidermal junction and to keratinocytes. Such adhesion may not only subserve the steric localization of melanocytes, but also have important implications for those functional activities of melanocytes which have been shown to require close contact between these cells and adjacent keratinocytes and/or basement membrane components.

Antibodies, Monoclonal↗

The distribution of Merkel cells in human fetal and adult skin.

Merkel cells (MCs) have been a subject of investigation in human and animal studies for over a century, but their origin, function(s), and exact distribution in human skin remain largely unknown. The objectives of the present study were to quantify these cutaneous neuroendocrine cells in fetal and adult human skin, using an immunohistochemical marker (neuron-specific enolase) and morphometric methods. Our results indicate that, in postnatal life, MC are sparsely distributed along the undersurface of epidermal and occasionally adnexal epithelium in an approximate range of 0.1-1.0 MCs per centimeter of basal zone. They are most numerous on volar skin and least on genital skin, and on the face they have a primarily perifollicular orientation. Fetal samples have shown that MCs appear between the 15th and 18th week of gestation and are most abundant on volar skin. They appear to arise within the epidermis, and are initially numerous and later diminish with increasing gestational age. Their predominance in intrauterine life suggests a functional role in growth and development.

Adult↗

The relationship of mast cells and their secreted products to the volume of fibrosis in posttransplant hearts.

A series of 96 posttransplant endomyocardial biopsies taken from 11 patients was subjected to quantitative analysis of mast cells and fibrosis. Ultrastructural analysis showed that mast cell numbers were increased and there was obvious degranulation in some posttransplant hearts. Activated mast cells and their secreted products, which contain heparin and histamine, are toxic to the hearts and may contribute to interstitial and perimyocytic fibrosis. The numbers of mast cells and granules were correlated with volume of fibrosis (r = 0.63, P less than 0.025; r = 0.73, P less than 0.01). There were differences between the release of mast cell granule contents seen in the posttransplant hearts and the rapid and massive reaction of anaphylactic degranulation of mast cells. Some mast cells progressively lost their dense granule contents, displaying a variety of piecemeal degranulation that indicates a slow degranulation process. These events occurred from the first week; they lasted from weeks to months. The fibrosis developed quickly in the cases with more mast cells and degranulation. The cases with fewer mast cells and granules showed only mild increases in the volume of fibrosis. Mast cells appeared as early as the first posttransplantation week. Patients with greater numbers of mast cells underwent more severe rejection episodes. This study demonstrated that mast cells play an early and important role in the perimyocytic and interstitial fibrosis of posttransplant hearts. Mast cells may also be important in the inflammatory process of rejection reaction. The severity of fibrosis appears related to the density of mast cells and their granules.

Fibrosis↗

Comparative epidermal Langerhans cell migration studies in epidermal and epidermal/dermal equivalent grafts.

Immigration of Langerhans cell precursors from the peripheral blood to the skin was studied in human grafts placed on severe combined immunodeficient (SCID) mice. Monocyte fractions of human blood were injected intraperitoneally to SCID bearing either reconstituted (Langerhans cell free) epidermal sheets (E) or living skin equivalents (E/D) consisting of both epidermis and dermis. A range of immunocytochemical and ultrastructural markers was employed to monitor the colonization of the grafts, i.e., CD1a/c, Birbeck granules. In situ hybridization with probes against Alu sequences of human DNA were employed together with immunostaining for MHC class I mouse and human antigens to document graft survival. Although unequivocal LC were detected within E grafts, including both human (CD1a positive) and murine (NLDC-145 positive), no migration was achieved in the E/D situations.

Animals↗

Immunoalkaline phosphatase with cerium-based cytochemistry for double staining at the transmission electron microscopic level.

Immunoelectron microscopic localization of surface antigens on lymphocytes is possible using alkaline phosphatase combined with cerium-based cytochemical methods. Distinctive electron-dense deposits are easily identified at sites of antibody binding. Mouse splenocytes showing surface immunoglobulin localization and human peripheral blood lymphocytes stained for the MHC-Class II antigen HLA-DR illustrate the results. Double staining for murine Ia antigen by the alkaline phosphatase procedure, combined with immunogold labeling of antigens identified on dendritic cells, i.e., NLDC-145, demonstrates the utility of the cerium cytochemical method.

Alkaline Phosphatase↗

Keratin and carcinoembryonic antigen (CEA) in human melanoma cells.

Human melanomas are known to contain vimentin intermediate filaments but there has been some dispute about their expression of cytokeratins. The cytoplasm of human M21 melanoma cells maintained in culture reacted with a rabbit anti-keratin antibody and two monoclonal anti-keratin antibodies AE1 and AE2. Cells derived directly from subcutaneous xenografts of M21 melanoma in nude mice, however, failed to express cytokeratins. The presence of keratin filaments in cultured M21 cells was confirmed by electronmicroscopic and immuno-electronmicroscopic examinations of cell extracts. Polyacrylamide gel electrophoresis (PAGE), revealed 46 KD keratin proteins in cultured M21 cells. Small amounts of these low molecular weight keratins were detected by PAGE in M21 melanoma xenografts even though immunofluorescence and immunoperoxidase assays failed to demonstrate keratin at the light microscopic level. Immunofluorescence revealed keratin and carcinoembryonic antigen (hitherto undetected in human melanomas) first on the 9th day of culture of xenograft-derived M21 cells. The appearance of keratin and CEA in M21 melanoma cells in vitro was not affected by inhibition of cellular proliferation or as a result of exposure to methotrexate or adriamycin. However, adriamycin altered the cytoplasmic distribution of keratin.

Antibodies, Monoclonal↗

Neurohumoral regulation of excitation-contraction coupling in ventricular myocytes from cardiomyopathic hamsters.

STUDY OBJECTIVE: The aim was to evaluate the regulation of contractility by autonomic stimulation in the necrotic stage of cardiomyopathy in male Syrian hamsters. DESIGN: The electrical and contractile activity of isolated intracellularly stimulated ventricular myocytes has been recorded and dose-response curves to [Ca2+]o, and to beta adrenergic, alpha adrenergic, and muscarinic agonists and antagonists were examined. EXPERIMENTAL MATERIAL: Ventricular cardiomyocytes were isolated by enzymatic dissociation of hearts from 90 to 120 day old cardiomyopathic hamsters CHF 147 and from age matched non-myopathic controls: CHF 148 or golden hamsters. MEASUREMENTS AND MAIN RESULTS: The membrane potential was recorded by suction (patch) electrodes. The contractile activity was recorded by a video system as the shortenings of the myocytes. The contractile response (EC50) to beta adrenergic stimulation (isoprenaline) showed a bimodal distribution: 60% of the myopathic myocytes responded like the controls, while in the remaining 40% the sensitivity was significantly decreased. The electrical activity and beta adrenergic receptor density were not different from the controls. The alpha adrenergic stimulation (by phenylephrine) was enhanced, while response to the muscarinic agonist carbachol (in the presence of isoprenaline) was attenuated in the myopathic cells. Sensitivity to [Ca2+]o was unchanged. CONCLUSIONS: Profound changes occur in the contractile response of myocytes from cardio-myopathic hamster to stimulation by mediators of the autonomic nervous system, which at this necrotic stage are not related to any significant changes in basal contractile response to [Ca2+]o, to the electrical activity, or to the number of beta adrenergic receptors.

Animals↗

The histogenesis of inflammatory fibroid polyps of the gastrointestinal tract.

Inflammatory fibroid polyps are lesions occurring in the submucosa of the gastrointestinal tract. These lesions have been identified by a variety of names, indicative of their uncertain histogenesis. Three cases were studied by light microscopy and cytochemistry and, in one case, by electronmicroscopy. The most characteristic feature consisted of concentric fibrovascular structures. Peroxidase reaction for muscle actin and Ulex europeus agglutinin was positive in the two main cellular components. Electronmicroscopy revealed that the two principal cell constituents were endothelial and myocytic cells. These observations support the conclusion that inflammatory fibroid polyps are lesions of vascular origin.

Actins↗

Subsets of keratinocytes and Langerhans' cells express epitopes associated with suppressor-inducer capabilities in resting normal human epidermis.

In recent years two cell populations with down-regulatory immune capabilities have been identified in murine epidermis. The present report demonstrates that even in human epidermis at least two populations of cells expressing suppressor-inducer phenotypes (i.e. CD45R-positive) exist, namely small subsets of keratinocytes and Langerhans' cells, respectively. Highly specific and sensitive 5-nm colloidal gold-immunoelectronmicroscopic techniques were carried out using anti-CD45R monoclonal antibodies, on freshly isolated crude epidermal cell suspensions, and 4000 cells were scrutinized in the electron microscope. Over 2% of the total epidermal cell population was CD45R+. Subpopulation analysis revealed that approximately 2% of keratinocytes and about 5% of the total Langerhans' cell population showed strong gold-plasma membrane staining, whilst the remaining epidermal cells were absolutely negative. Heterogeneity of staining together with this somehow surprising distribution of CD45R positivity on non-lymphoid epidermal cells was confirmed by the negative controls. These CD45R+ Langerhans' cells and keratinocytes are clearly candidates for the cells which have been functionally demonstrated as being capable of inducing down-regulation responsiveness in the human epidermis. However, functional investigations are needed to clarify the roles of the CD45R+ keratinocyte and Langerhans' cell subsets in the modulation of cutaneous immune responses.

Antigens, CD↗

The S-100 beta protein in normal human peripheral blood is uniquely present within a discrete suppressor-T-cell compartment.

The S-100-positive T lymphocytes, and, particularly, the S-100 beta subunit, are restricted, as demonstrated by quantitative subset analysis and double-labeling (gold-peroxidase) immunoelectron microscopy of T-cell subpopulations, to an unique T8-positive cell subset which interestingly was 9.3-negative and CD11b-positive. Since both the T8-positive, 9.3-negative and the T8-positive, CD11b-positive subpopulations have been demonstrated to show suppressive activities, the S-100-positive T cells seem to be closely restricted to a small T-suppressor-cell compartment. Although functional studies on viable isolated S-100 beta-positive cells are impossible to achieve, due to the lack of this protein on the cell membrane, its presence in a discrete T-suppressor compartment might suggest a possible role for the S-100 beta-positive T cells in the regulation of the immune system.

Antigens, Differentiation, T-Lymphocyte↗

Immunoelectronmicroscopic demonstration of S-100 protein in hairy cell leukemia cells.

Hairy cell leukemia (HCL) is generally considered a neoplastic proliferation of B-cell lineage. Among immunocompetent cells, however, S-100 protein is also demonstrated in "dendritic" cells (i.e., interdigitating reticulum cells, Langerhans cells, follicular dendritic cells) and in a small percentage of T8-cells. This study reports the surprising detection of S-100 protein in peripheral blood HCL cells of B-cell type, using immunofluorescence and immunoelectronmicroscopy. However, negative results for S-100 protein staining were obtained in paraffin splenic sections of HCL cases. This report stresses the need for caution in the interpretation of immunocytochemical methods and results, and emphasizes the diagnostic ambiguity of the S-100 protein.

Blood Cells↗

Identification of CD4+, 2H4+ (T8 gamma +) suppressor-inducer cells in normal human epidermis and superficial dermis.

Immunohistological staining of frozen sections of normal human skin demonstrated the presence of significant numbers of mononuclear cells expressing novel epitopes associated with CD4-positive suppressor-inducer functions. The cells were located around superficial vessels and within the basal layers of the epidermis and hair follicles. The antigen identified by the various antibodies has been shown to be functionally important in the induction of various suppressor cells capable of abrogating B cell responses to pokeweed mitogen. The presence in the skin of cells with possible down-regulatory functions in the immune response may be significant with respect to surveillance against neoplasms and control of appropriate responses to infectious agents.

Antibodies, Monoclonal↗

Preparation of "histocomposites" for direct immunohistochemical screening of monoclonal antibodies.

Screening and selection of hybrids producing relevant antibodies in monoclonal technology usually rely on rapid and sensitive adsorption assays of the ELISA type. To identify clones producing antibodies with unexpected specificities direct immunohistological screening may be applied, but this is both tedious and expensive. Histocomposites made from a number of tissue types permit testing of supernatants at the required early stage after fusion. The multiple antigenic specificities displayed in such test specimens ensure detection of a broad range of antibodies. A simple method for production of the histocomposites is described.

Animals↗

One half of the CD11b+ human peripheral blood T lymphocytes coexpresses the S-100 protein.

The expression of the CD11b antigen and the presence of the S-100 (and, specifically, its beta subunit) protein within the T4- subpopulation of normal human peripheral blood lymphocytes were investigated by panning techniques, immunofluorescence analysis and immunoelectronmicroscopy. Both antigens are known to be absent in the T4+ lymphocytes. However, CD11b+ T lymphocytes represented about 30% of the T4- population; a part of them (over 1/3) belonged to and completely filled up the T4- T8- subpopulation, whereas the remaining part (almost 2/3) shared the T8 positivity. Interestingly, S-100+ T lymphocytes, which always were CD11b+ too, represented about one half of the CD11b+ T cells, but were excluded from the T4- T8- CD11b+ subpopulation, whereas they represented up to 80% of the T4- T8+ CD11b+ subset. Such findings demonstrate that the S-100+ T lymphocytes are exclusively restricted to a discrete T cell compartment which shows the T8+ CD11b+ immunophenotype. Since such T8+ CD11b+ cells had been shown to possess suppressive capabilities, we herein propose that S-100+ lymphocytes might to some extent modulate the immune responses. However, the exact functional significance of the S-100 protein still remains unknown.

Antigens, Differentiation, T-Lymphocyte↗

S-100-positive T cells are largely restricted to a CD8-positive, 9.3-negative subset.

The present report concerns the demonstration of the exclusive detection among peripheral blood T-lymphocytes of the S-100 protein within the CD8-positive subpopulation which lacks the antigen recognized by the 9.3 monoclonal antibody. Highly purified human peripheral blood T-cell subsets, obtained by means of panning techniques, were first stained, by an immunofluorescence method, with purified anti-S-100 protein antibodies. The vast majority of S-100 protein- (and, specifically, its beta subunit) positive cells were detected in the CD8-positive, 9.3-negative subset. This subset had previously been shown to comprise all the alloantigen-specific and histamine-inducible suppressor T-cells. Other T-subsets, even those showing either CD8-positivity (but 9.3-positivity) or 9.3-negativity (but CD8-negativity), were, as a rule, S-100 negative. Immunoelectronmicroscopy confirmed that the S-100-positive cells, showing peroxidase activity within the cytoplasm, were found exclusively within the CD8-positive, 9.3-negative subset. This finding of S-100 protein in cells of a specific T8 suppressor subset extends the range of the known distribution of this protein and may have important implications concerning its role in the modulation of immune responses.

Antibodies, Monoclonal↗

Secretion of a suppressor cell inducing factor by an interleukin-3 dependent cell line with natural cytotoxic activity.

This report describes the morphology, surface markers, growth requirements, and functional activity of the M1-A5 cell line, which was established by the limiting dilution of spleen cells from a mouse bearing a large methylcholanthrene-induced fibrosarcoma. The M1-A5 cells share many of the morphological features of large granular lymphocytes and, in addition, express asialo GM1 and Ly-5 surface markers which are commonly found on natural killer cells (NK) cells. There is no expression of T-cell differentiation antigens, surface immunoglobulin, or the granulocyte/macrophage marker, MAC-1. M1-A5 cells are dependent on exogenous growth factor(s) for survival and will proliferate if cultured in interleukin 3 (IL-3), but not in interleukin 1 (IL-1), interleukin 2 (IL-2), or granulocyte/macrophage colony stimulating factor (GM-CSF). In addition, the M1-A5 cells do not absorb IL-2. Despite their morphology and surface characteristics, the M1-A5 cells do not lyse NK targets such as YAC-1 and RLM1 in 4- or 18-hr cytotoxic assays but do lyse the natural cytotoxic (NC) susceptible target, WEHI-164, and to a very small extent, the M-1 fibrosarcoma cells, in an 18-hr assay. Thus they exhibit NC-like cytotoxic activity. In addition, the M1-A5 cells secrete a small molecular weight factor which activates suppressor cells capable of inhibiting antibody synthesis by cocultured syngeneic spleen cells.

Animals↗