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Biomedical subjects

M R Duncan

Publications and source records attributed to M R Duncan.

At least 37 records · Page 2Linked to original sources
PubMed1989

Differential regulation of collagen, glycosaminoglycan, fibronectin, and collagenase activity production in cultured human adult dermal fibroblasts by interleukin 1-alpha and beta and tumor necrosis factor-alpha and beta.

In order to clarify the role played by immunologically derived cytokines in dermal connective tissue synthesis and degradation, we investigated the effect of human recombinant (hu-r) interleukin (IL) 1-alpha and beta, hu-r tumor necrosis factor (TNF)-alpha and beta, hu-r IL 2, and hu-r granulocyte-macrophage colony-stimulating factor (GM-CSF) on the production of collagen, glycosaminoglycan, fibronectin, and collagenase activity by three lines of cultured human adult dermal fibroblasts. Our results show that 24-72 h treatment of confluent fibroblast cultures with IL 1-alpha or beta or TNF-alpha or beta causes concentration (1 to 1 X 10(4) U/ml) dependent increases in collagen, glycosaminoglycan, and collagenase activity production, but decreases in fibronectin production. In contrast, treatment with IL 2 and GM-CSF had no effect on fibroblast functions. The data show that IL 1-alpha and beta and TNF-alpha and beta differentially regulate fibroblast functions, and that increases in catabolic functions like collagenase activity production are more than tenfold greater than increases in anabolic functions like collagen production. When these results are considered along with other reports, they suggest that IL 1 and TNF may play predominately a catabolic role in situ during dermal fibrotic responses by directly inhibiting fibronectin production and indirectly causing the degradation of collagen and glycosaminoglycan by significantly increasing dermal fibroblast elaboration of collagenase and proteoglycanase activities.

Adult↗
PubMed1989

Pentoxifylline inhibits normal human dermal fibroblast in vitro proliferation, collagen, glycosaminoglycan, and fibronectin production, and increases collagenase activity.

Fibroblasts from normal human adult skin were cultured in vitro in the presence and absence of different concentrations of pentoxifylline or a pentoxifylline analog, A81-3138 (10(-1)-10(3) micrograms/ml). Similar concentration dependent reductions in normal proliferation of fibroblasts in fetal calf serum-driven subconfluent cultures were detected following treatment with pentoxifylline or A81-3138. Fibroblasts assayed as confluent cultures produced sub-normal amounts of collagen, glycosaminoglycans (GAGs), and fibronectin in a fashion dependent upon the concentration of pentoxifylline. In contrast, fibroblasts exposed to pentoxifylline elaborated double the collagenase activity produced by normal, untreated fibroblasts. The reduced proliferation and reduced synthetic activities were not due to a lethal toxic effect on fibroblasts by pentoxifylline and A81-3138, nor was the reduction in collagen synthesis simply due to an inability to secrete newly synthesized intracellular collagen. Unlike pentoxifylline-induced inhibition of collagen and fibronectin production, which was detected only in cultures supplemented with serum, pentoxifylline inhibits, to a similar degree, both constitutive and serum-driven production of GAGs. The addition of IL1 beta (2.5 and 10.0 U/ml) to serum-driven fibroblast cultures resulted in greater proliferation, which was inhibitable by the presence of pentoxifylline and A81-3138 as anti-fibrotic agents in certain disorders of fibrosis.

Cell Division↗
PubMed1988

Stimulation of mono (ADP-ribosyl)ation by reduced extracellular calcium levels in human fibroblasts.

Lowering extracellular calcium in cultures of human diploid fibroblast-like cells caused a rapid depletion of NAD pools. This loss of NAD was reversed by restoring extracellular Ca2+ and was inhibited by 3-aminobenzamide, an inhibitor of ADP-ribosyl transfer reactions. The concentrations of 3-aminobenzamide needed to inhibit the loss of NAD were consistent with those required to inhibit cellular mono(ADP-ribosyl) rather than poly(ADP-ribosyl) reactions. Calcium depletion did not inhibit the biosynthesis of NAD. These results suggest that mono(ADP-ribosyl)ation is involved in the regulation of cellular Ca2+ levels.

Adenosine Diphosphate Ribose↗
PubMed1987

Persistence of a reduced-collagen-producing phenotype in cultured scleroderma fibroblasts after short-term exposure to interferons.

Transient exposure to inflammation-associated, fibroblast-stimulatory factors appears to initiate fibrosis by inducing the persistently activated phenotypes displayed by fibroblast cultures derived from scleroderma skin and other fibrotic tissues. To determine whether one class of fibroblast-inhibitory factors, the interferons (IFNs), plays a role in terminating fibrosis by acting as persistent fibroblast deactivators, we inhibited (40-60%) the growth and collagen production of normal dermal fibroblasts and hypercollagen-producing scleroderma fibroblasts by short-term exposure to IFN-alpha, beta, or gamma. During subsequent subculture in the absence of IFNs, the growth and collagen production of normal fibroblasts and the growth of scleroderma fibroblasts increased to untreated control levels after two to three passages. In contrast, collagen production by scleroderma fibroblasts remained inhibited for at least five passages (18 cell doublings) and was not further suppressed by subsequent IFN exposure. These data suggest that IFNs may help terminate fibrosis by suppressing persistently activated fibroblast functions.

Cell Division↗
PubMed1985

Gamma interferon is the lymphokine and beta interferon the monokine responsible for inhibition of fibroblast collagen production and late but not early fibroblast proliferation.

Human peripheral blood mononuclear cells activated with concanavalin A (Con A) or lipopolysaccharide (LPS) produce, respectively, lymphokines (LK) of 50,000 Mr or monokines (MK) of 20,000 Mr that inhibit the growth and collagen production of cultured human dermal fibroblasts. Because antigenic typing of the antiviral activity of these LK and MK preparations revealed that LK contained mainly gamma interferon (IFN-gamma), and MK, primarily IFN-beta, we investigated if any of the fibroblast-inhibiting activities could be attributed to human IFN. Unlike LK and MK, which act within 24 h to inhibit the growth of subconfluent foreskin and adult dermal fibroblasts, samples of purified, natural derived IFN-alpha, -beta, and -gamma and recombinant DNA-derived IFN-alpha 2 and -gamma were ineffective inhibitors at 24 h and required 48-72 h to significantly inhibit growth. However, all IFN did mimic LK/MK action in causing concentration-dependent inhibition of collagen production by confluent fibroblast microcultures. Furthermore, the collagen production-inhibiting activity of Con A-induced LK supernatant and its 50,000 Mr fraction was completely suppressed by 10(3) neutralizing U/ml of either polyclonal or monoclonal antibody to IFN-gamma, while polyclonal antibodies to IFN-alpha and -beta had no effect. Similarly, the collagen production-inhibiting activity of LPS-induced MK supernatant and its 20,000 Mr fraction was suppressed by polyclonal anti-IFN-beta but not by anti-IFN-alpha or -gamma. Anti-IFN failed to reverse early-acting LK or MK growth-inhibiting activities. These data suggest collagen production-inhibiting LK and MK are IFN-gamma and IFN-beta, respectively, and that early acting, growth-inhibiting LK and MK are not IFN.

Cell Division↗
PubMed1985

Interferon enhancement of HLA-DR antigen expression on epidermal Langerhans cells.

Langerhans cells (LCs) are dendritic epidermal cells whose ability to function as accessory/stimulatory cells in initiating the immune response is, like that of macrophages, dependent on the expression of class II major histocompatibility antigens. In normal human skin approximately 50% of LCs identified by cell surface T6 antigenicity also express HLA-DR histocompability determinants. We report here that recombinant DNA-derived human interferon (IFN)-gamma, but not IFN-alpha 2, induces the expression of HLA-DR antigens by the population of human epidermal LCs on which such antigens normally are not detected. IFN-gamma effectively induced HLA-DR on both neonatal and adult epidermal LCs and such induction was blocked by neutralization with a murine monoclonal antibody to IFN-gamma. IFN-gamma induction of LC HLA-DR expression is inhibited by prostaglandin E2 (PGE2) and is mimicked by the presence of fatty acid cyclooxygenase inhibitors, known to reduce PGE2 production. These results suggest that IFN-gamma may play a role in regulating skin-associated immune responses through enhanced expression of HLA-DR antigens on LCs and that such enhancement may be mediated by alterations in arachidonic acid metabolism.

Cyclooxygenase Inhibitors↗
PubMed1984

Histone acetylation and deacetylation in senescent human diploid fibroblasts.

We measured the kinetics of histone H4 hyperacetylation and kinetics of deacetylation for all core histones in young and senescent human diploid fibroblast-like cells. Both cell populations contained a fraction of histone H4 characterized by very rapid acetylation and deacetylation. The kinetics of these reactions were similar in young and senescent cells. The distribution of acetylated species of core histones was also similar in young and senescent human diploid fibroblast-like cells. These results indicated that previously-reported alterations in chromatin template activity in senescent cells were due to a mechanism other than histone acetylation.

Acetylation↗
PubMed1984

Lymphokine/monokine inhibition of fibroblast proliferation and collagen production: role in progressive systemic sclerosis (PSS).

A normal fibrotic response to inflammatory stimuli appears to be dependent on the balanced production of a number of stimulatory and inhibitory fibroblast-regulatory mediators by activated mononuclear cells (MNL). To investigate whether altered mediator production contributes to the fibrosis observed in progressive systemic sclerosis (PSS), we stimulated human peripheral blood MNL with concanavalin A (Con A) and lipopolysaccharide (LPS) to produce macromolecular mediators that inhibit the proliferation and the collagen production of cultured normal human fibroblasts. The two Con A-induced mediators were lymphokines (LK) as they were exclusively produced by activated T cells and they coeluted from a Sephacryl S-200 column with a Mr of 50,000. In contrast, the two LPS-induced mediators were monokines (MK) as they were exclusively produced by activated monocytes, and they coeluted in the Mr 20,000 range. Each pair of inhibitory LK and MK may also be distinct as inhibition of collagen production still occurred in proliferatively quiescent cultures. A quantitative comparison of the levels of fibroblast-inhibitory LK/MK produced by normal volunteers and long-term PSS patients revealed that although PSS MNL produced normal levels of both collagen production inhibitory mediators, they were aberrant producers of both proliferation inhibitory mediators, being hypo-producers (-49%) of the LK and hyper-producers (+196%) of the MK. These results suggest that reduced production of proliferation inhibitory LK may allow stimulatory mediators to induce the unrestricted fibroblast proliferation observed in early active PSS, which then may be stabilized in long-term PSS by the increased production of proliferation inhibitory MK.

Adult↗
PubMed1983

Kinetics of histone hyperacetylation and deacetylation in human diploid fibroblasts.

We have utilized sodium butyrate-treated normal human diploid fibroblasts to study core histone hyperacetylation kinetics. We report a small, distinct population of core histone characterized by a very rapid rate of hyperacetylation (t1/2 approximately equal to 10-15 min for monoacetylated histone H4) compared to the slower rate (t1/2 approximately equal to 140-200 min for monoacetylated H4) observed for bulk histone. Two rates of core histone deacetylation were also detected and we demonstrated that the rapidly hypermodified histone H4 population was also rapidly deacetylated. The kinetics of histone H4 hyperacetylation and deacetylation in these cells were not significantly altered, regardless of whether cultures were exponentially growing, confluent or arrested in an essentially non-mitotic state.

Acetylation↗
PubMed1983

Circulating immune complexes in childhood malignancies: a Pediatric Oncology Group study.

Pretreatment serum samples obtained at diagnosis from 89 children with various pediatric malignancies were examined for circulating immune complexes (CIC) using the [125I]Clq binding assay. The study population consisted of 35 children with acute lymphocytic leukemia (ALL), 22 children with acute non-lymphocytic leukemia (ALL), 24 with neuroblastoma (NB), and eight with osteosarcoma (OS). Concomitant quantitation of immunoglobulins was performed in 55 patients, revealing normal values for age. Increased levels of CIC at diagnosis were found in 9%, 22%, 42%, and 50% of children with ALL, AML, NB, and OS, respectively. Except for a higher proportion of CIC-positive patients observed in stage IV NB (nine of 17) compared to stage I-III NB (one of seven), no correlation was observed between initial CIC level and presenting clinical features, response to treatment, prognosis, or presence of infection. Longitudinal sampling of six NB and two OS patients did not reveal a clear relationship between disease activity and quantity of CIC. For the pediatric malignancies studied, these data demonstrate minimal value in quantitating CIC as a means of assessing disease activity or predicting response to treatment and are in contrast to the apparently adverse effect of elevated pretreatment CIC on response to therapy and survival observed in adults with ALL, AML, and OS.

Adolescent↗
PubMed1982

Concanavalin A-induced human lymphocyte mitogenic factor: activity distinct from interleukin 1 and 2.

Fresh human mononuclear cells (MNL) cultured in serum-free medium and stimulated with concanavalin A (Con A) produce a lymphokine functionally similar to the previously described antigen-induced lymphokine human lymphocyte mitogenic factor (LMF). T lymphocytes appear to be the source of Con A-induced LMF and the presence of adherent monocytes is required for maximal production. The proliferative response to LMF, assayed either as supernatant fluids in the presence of alpha-methyl-D-mannoside or as partially purified 27,000 to 35,000 m.w. fractions from Bio-Gel P-100 columns, indicates LMF is distinct from Con A. LMF acts almost exclusively on B lymphocyte-enriched (E rosette-negative) MNL while exhibiting only minimal activity on T lymphocytes and no activity on monocytes. LMF preparations induce both B cell proliferation and the maturation and proliferation of E rosette-negative T cell progenitors into E rosette-positive cells. Assay of interleukin 1 (IL 1) and interleukin 2 (IL 2) preparations for LMF activity implicate IL 2 as the T cell progenitor stimulating factor. Assay of the proliferation of human IL 2-dependent alloreactive T lymphocyte line/ATLL) indicates that LMF preparations contain IL 2, IL 2 activity, however, is completely removed by absorption of LMF preparations by ATLL cells, whereas LMF activity (B cell proliferation) is unabsorbable, thus distinguishing LMF from IL 2. These absorbed LMF preparations contain no IL 1 activity as assayed by murine thymocyte proliferation, nor does IL 1 have LMF activity. These results indicate that Con A-induced LMF contains a functionally distinct lymphokine with the ability to act as a B cell growth factor.

Animals↗
PubMed1979

Superoxide dismutase specific activities in cultured human diploid cells of various donor ages.

It has been postulated that superoxide dismutase (SOD) protects cells from free radical-induced damage. In these experiments SOD specific activity was measured as established human diploid cell lines from various donor ages progressed through their in vitro lifespan. Significant elevations in activity occurred during the in vitro lifespans of cells from fetal and newborn donors, but no change in activity was detected during the lifespan of cells from an adult donor. In addition, a direct relationship between enzyme activity and donor age was detected with the following relative activities: adult greater than newborn greater than fetal. The possible relationship between these findings and the free radical theory of aging is discussed.

Aging↗
PubMed1978

Differential sensitivity of normal and transformed human cells to reovirus infection.

Normal and simian virus 40-transformed WI-38 cells exhibited a differential sensitivity to infection with type 3 reovirus. A progressive decrease in viability began 24 to 36 h after infection of transformed cells terminating in complete lysis of cultures by 96 h. Normal cells were productively infected and continued to produce and release virus for as long as 14 days after infection, but exhibited no detectable cytopathology. Inhibition of cellular DNA synthesis began 15 to 18 h after infection in transformed cells before development of cytopathology. No inhibition of DNA synthesis was detected in infected normal cells. No significant differences were noted in the adsorption or early replication characteristics of reovirus in normal and transformed cells. Virus replication and host cell DNA synthesis in normal and transformed human cells were compared to reovirus-infected L-929 mouse fibroblast cells.

Cell Division↗
PubMed1977

Relationship of heat labile glucose-6-phosphate dehydrogenase and multiple molecular forms of the enzyme in senescent human fibroblasts.

Human fibroblasts derived from newborn foreskin and designated CF-3 were assayed for heat labile glucose-6-phosphate dehydrogenase (G6PD) when they had grown to confluency, as well as when they were arrested in an essentially nonmitotic state. Under both culture conditions there was an increase in heat labile G6PD (up to 25% of the total activity) as cells progressed through their in vitro lifespan; however, arrested cells exhibited less heat labile G6PD than did comparable growth controls (5-10% decrease). Acrylamide gel electrophoresis of crude G6PD preparations revealed three distinct bands of enzymatic activity. One of the bands was tentatively identified as the dimeric form of the enzyme and another as the tetrameric form. The tetrameric form was more heat sensitive, and the percent of the total activity of this form increased as the cells became senescent. The percent of total activity of the tetrameric form was comparable to the percent heat labile enzyme assayed at a given population doubling level. These results indicated that the observed increase in heat lability of G6PD with age may be due to a shift in equilibrium to the tetrameric form of the enzyme rather than the synthesis of aberrant enzyme molecules.

Cell Division↗