Search PubMed⌕ Search

Biomedical subjects

D A Norris

Publications and source records attributed to D A Norris.

At least 73 records · Page 4Linked to original sources
PubMed1990

Cytokine modulation of adhesion molecules in the regulation of immunologic cytotoxicity of epidermal targets.

Immunologic cytotoxicity is an important endpoint of the immune response to tumors, viral infected cells, grafted tissues, and exogenous microorganisms, and is also an important mechanism of disease, especially in autoimmunity. There are multiple mechanisms of immunologic cytotoxicity, but each has three major stages: leukocyte/target attachment, specific recognition, and target lysis following effector activation. Adhesion molecules present on leukocytes and potential targets appear to be involved in all three stages of cytotoxicity. A major factor in all types of cellular cytotoxicity is the interaction of LFA-1 on leukocytes and CAM-1 on targets. Modulation of ICAM-1 levels on target by the cytokines TFN-g, IL-1, and TNF-a is a major point of control of the susceptibility of targets to cytotoxicity by many different cytotoxic mechanisms. It also appears that modulation of the avidity of LFA/ICAM-1 binding is another important control point in modulating immunologic cytotoxicity. Cytokines also have important effects on immunologic cytotoxicity in ways other than adhesion molecule induction: effector priming to better respond to specific recognition signals, effector mobilization into tissue, and expansion of cytotoxic effector populations. ICAM-1 on the surface of epidermal keratinocytes and melanocytes is likely to greatly influence cytotoxic damage of these cells in diseases as photosensitive lupus erythematosus, lichen planus, erythema multiforme, and vitiligo. It has been found that the epidermal staining pattern for ICAM-1 in each of these diseases in distinctive and different in each disease. It is proposed that disease-specific induction of ICAM-1 by factors such as UVR and herpes-virus is an important determinant in triggering these skin diseases and in determining the pattern of disease.

Cell Adhesion Molecules↗
PubMed1990

Metabolism of exogenous leukotrienes by cultured human keratinocytes.

Leukotrienes are involved in diseases associated with a neutrophilic infiltrate. The role of human keratinocytes in the metabolism and inactivation of leukotrienes has not been thoroughly examined. We added exogenous radioactive leukotrienes to cultured human keratinocytes and evaluated the metabolic products using high-performance liquid chromatography. Over a 24-h period, unstimulated cultured keratinocytes convert leukotriene B4 to unidentified polar molecules. Leukotriene C4 is converted to a leukotriene D4/leukotriene E4-like product. Cultured human keratinocytes have the ability to metabolize leukotrienes and thus the keratinocyte may play a major role in the in vivo metabolism of leukotrienes produced during inflammatory dermatoses.

Binding Sites↗
PubMed1989

Susceptible cytotoxicity to ultraviolet B light in fibroblasts and keratinocytes cultured from autoimmune-prone MRL/Mp-lpr/lpr mice.

The MRL/Mp-lpr/lpr (MRL/l) mouse is an autoimmune model of spontaneous lupus erythematosus (LE), in addition to lupus nephritis. In order to better understand the mechanisms of photosensitivity in LE, in vitro photocytotoxicity was examined by using fibroblasts and keratinocytes cultured from MRL/l mice, control MRL/Mp- +/+ (MRL/n) mice, and normal BALB/c mice. A colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and the acridine orange/ethidium bromide assay were used for determination of cytotoxicity. Fibroblasts cultured from newborn MRL/l mice showed higher susceptibility to single ultraviolet light B (UVB) light irradiation at a dose of 100-500 mJ than those from MRL/n, F1 hybrid of (MRL/l x MRL/n mice), and BALB/c mice. However, the susceptibility to UVB was not observed in young (1-month-old) and adult (4-month-old) MRL/l mice. UVA light irradiation was not cytotoxic. Keratinocytes cultured from MRL mice showed lower cytotoxicity to UVB irradiation than fibroblasts cultured. However, keratinocytes from newborn MRL/l mice showed higher cytotoxicity to 50 mJ UVB irradiation than cells from MRL/n mice. Syngeneic or allogeneic sera augmented UVB-induced cytotoxicity of fibroblasts cultured. UVB irradiation of spleen cells induced no significant difference of cytotoxicity between MRL/l and MRL/n mice. Based on the results of F1 hybrid of (MRL/l x MRL/n) mice, the susceptibility seemed to be associated with autoimmune traits and to be regulated by genetical background.

Animals↗
PubMed1989

Leukotrienes C4 and D4 as potent mitogens for cultured human neonatal melanocytes.

Arachidonic acid and its metabolites (eicosanoids) are membrane-derived inflammatory mediators with a diverse set of biologic properties affecting numerous cells and organ systems, including the skin. They have been implicated in the pathogenesis of inflammatory skin disease and post-inflammatory hyperpigmentation. We have studied the ability of arachidonic acid, prostaglandin D2, prostaglandin E2, leukotriene B4, leukotriene C4, leukotriene D4, and leukotriene E4 to enhance the growth of cultured human melanocytes. Of these compounds, only leukotriene C4 and leukotriene D4 were capable of stimulating melanocyte proliferation. In addition, cultured melanocytes metabolized leukotriene C4 to leukotriene E4 with greater than 60% conversion in less than three hours. Melanocytes grown on suboptimal media (doubling time 12-20 days) respond in a dose-dependent fashion to leukotriene C4, with a significant difference from control noted at 28 days with a concentration of LTC4 of 30 nM and a doubling time of 5-8 days. We feel that leukotriene C4 and D4 could play an important role in post-inflammatory melanocyte hyperplasia.

Cells, Cultured↗
PubMed1989

Pattern of cutaneous immunoglobulin G deposition in subacute cutaneous lupus erythematosus is reproduced by infusing purified anti-Ro (SSA) autoantibodies into human skin-grafted mice.

Subacute cutaneous lupus and neonatal lupus are closely associated with the presence of anti-Ro (SSA) autoantibodies, but there is no direct evidence establishing a role for anti-Ro (SSA) in these diseases. After parental injection into mice, IgG from sera containing anti-Ro (SSA) will bind human skin grafted onto the mice. To determine whether the antibody binding is due to anti-Ro (SSA), affinity-purified anti-Ro (SSA) and serum depleted of anti-Ro (SSA) were prepared. After injection into human skin-grafted mice, purified anti-Ro (SSA) antibodies bound an antigen in the human skin graft, while preabsorbing anti-Ro (SSA) serum with Ro (SSA) virtually abolished binding to the human skin graft. Moreover, the pattern of IgG deposition was primarily epidermal and was identical in the human skin-grafted mice injected with purified anti-Ro (SSA) when compared with that found in five patients with subacute lupus (four adults, one neonate). These data directly show that anti-Ro (SSA) antibodies bind to the skin, and support the hypothesis that anti-Ro (SSA) autoantibodies are involved in the disease process that produces subacute cutaneous lupus and neonatal lupus.

Adult↗
PubMed1989

Mechanisms of cutaneous tissue damage in lupus erythematosus.

Cutaneous disease in lupus is clinically heterogeneous, and it is likely that mechanisms of disease are also heterogeneous. Nevertheless, in many cutaneous forms of lupus and in the systemic disease itself, there is compelling evidence that autoantibodies are involved. It is our belief that autoantibodies are of primary importance in the etiology of the cutaneous lupus lesions discussed in this chapter and that antibody specificities are critical in most cases in determining the clinical expression of disease. As discussed earlier, antibodies can effect tissue injury by several mechanisms. There is evidence that ADCC may be an important mechanism of keratinocyte cytotoxicity in cutaneous lesions characterized by antibody deposition and a mononuclear cell infiltrate. Vasculitic lesions that have an infiltrate of polymorphonuclear cells may be related to antibody- and complement-dependent neutrophil-mediated endothelial cytotoxicity. Ultraviolet light, sex steroids, and certain genes such as those of the major histocompatibility complex appear to be important modulating factors in lupus.

Adult↗
PubMed1988

Estradiol enhances binding to cultured human keratinocytes of antibodies specific for SS-A/Ro and SS-B/La. Another possible mechanism for estradiol influence of lupus erythematosus.

A strong association between anti-SS-A/Ro and anti-SS-B/La antibodies and skin lesions has been well documented in subacute cutaneous lupus erythematosus and neonatal lupus erythematosis in which 70 to 80% of patients are female. In order to better understand the mechanisms of the influence of sex hormones on cutaneous lupus, we designed immunopathological in vitro experiments to evaluate the effects of estradiol and other sex steroids on the binding of SS-A/Ro- and SS-B/La-specific antibodies to cultured human keratinocytes from neonates. Cultured human keratinocytes incubated with antisera specific for SS-A/Ro or SS-B/La Ag were fixed with either acetone or paraformaldehyde and then analyzed in indirect immunofluorescent assays or by FACS analysis to detect cell surface IgG binding as an indirect measure of SS-A/Ro and SS-B/La Ag expression on the cell surface of keratinocytes. Estradiol (10(-5) to 10(-7) M) augmented binding of antiserum probes on the surface of cultured keratinocytes, with 10(-7) M estradiol showing the highest induction of cell surface binding of antisera specific for SS-A/Ro plus SS-B/La Ag (24.5% of cells were positive). In contrast, dihydrotestosterone, testosterone, and progesterone showed no augmentation. The augmentation by estradiol was partially inhibited by the antiestrogen nafoxidine. Estradiol augmented the relative incidence and absolute number of small or cuboidal cells binding antibodies specific for SS-A/Ro and SS-B/La Ag, whereas the number and incidence of larger differentiated cells binding anti-SS-A/Ro and anti-SS-B/La decreased significantly in cell cultures stimulated with estradiol. Flow cytometric analysis utilizing monospecific anti-SS-A/Ro or anti-SS-B/La sera showed that estradiol induced binding of anti-SS-A/Ro in 13.1% of cultured keratinocytes, of anti-SS-A/La in 14.4%, and of sera specific for both Ag in 21.4%. This direct association between estradiol and the augmentation of binding to the cell surface of human keratinocytes of IgG from antisera specific for SS-A/Ro and SS-B/La Ag may be a trigger factor of immunologic damage in lupus and may be important in the different sex rates observed in skin manifestation of subacute cutaneous and neonatal lupus erythematosis.

Adjuvants, Immunologic↗
PubMed1988

Cryptic chemotactic activity of fibronectin for human monocytes resides in the 120-kDa fibroblastic cell-binding fragment.

Monocytes and lymphocytes form a second wave of infiltrating blood leukocytes in areas of tissue injury. The mechanisms for monocyte accumulation at these sites are not completely understood. Recently, however, fragments from extracellular matrix proteins including collagen, elastin, and fibronectin have been shown to induce monocyte chemotaxis. In this report we demonstrate that chemotactic activity for human monocytes is expressed when a 120-kDa fragment containing the RGDS cell-binding peptide is released from intact fibronectin or from larger fibronectin fragments. Monocytes, either from mononuclear cell Ficoll-Hypaque preparations (10-20% monocytes, 89-90% lymphocytes) or from elutriation preparations (95% monocytes, 5% lymphocytes), but not lymphocytes, migrated toward 120-kDa fragment preparations (10(-7) M) in blind-end chambers when the cells were separated from the chemoattractant by a 5-micron pore polycarbonate filter either alone or overlying a 0.45-micron pore nitrocellulose filter. Neutrophils migrated toward zymosan-activated serum but not toward 10(-5)-10(-8) M concentrations of the 120-kDa fragment. Intact fibronectin had no chemotactic activity for human monocytes. Fibronectin was isolated from citrated human plasma by sequential gelatin-Sepharose affinity and DEAE ion-exchange chromatography in the presence of buffers containing 1 mM phenylmethylsulfonyl fluoride to prevent fragmentation. Controlled enzymatic digestion with thermolysin cleaved fibronectin into 30 kDa fibrin, 45 kDa collagen, and 150/160-kDa cell and heparin domains. Upon prolonged digestion, purified 150/160-kDa fragments were cleaved into 120-kDa cell and 30/40-kDa heparin-binding fragments. Even though the intact fibronectin molecule, the 150/160-kDa fragments, and the 120-kDa fragment, have cell binding activity for Chinese hamster ovary fibroblasts, only the 120-kDa fragment expressed chemotactic activity for human monocytes. Thus, the 120-kDa fibroblastic cell-binding fragment contains a cryptic site for monocyte chemotaxis which is expressed upon enzymatic cleavage of fibronectin.

Chemotaxis, Leukocyte↗
PubMed1988

Evidence for immunologic mechanisms in human vitiligo: patients' sera induce damage to human melanocytes in vitro by complement-mediated damage and antibody-dependent cellular cytotoxicity.

Human vitiligo is a common depigmenting disorder that is often associated with polyendocrinopathies. The etiology of vitiligo is unknown although there is indirect evidence of a strong association between antimelanocyte antibodies in animal and human vitiligo. We report direct evidence that vitiligo patients' sera containing antimelanocyte antibodies can lyse cultured human melanocytes by both complement activation and antibody-dependent cellular cytotoxicity (ADCC). Melanocyte cytotoxicity was measured using an ethidium bromide/acridine orange viability assay, after 4 and 16 h incubation with sera from vitiligo patients and from normal controls. Significant melanocyte cytotoxicity was seen with vitiligo patients' sera as an antibody source with both complement-mediated cytotoxicity (p less than 0.01) and ADCC (p less than 0.05) as effector mechanisms. Nine of 11 vitiligo patients' sera produced cytotoxicity by complement-mediated lysis or ADCC. No cytotoxicity was seen using fibroblast targets and vitiligo patients' sera. The lysis of human melanocytes by vitiligo patients' sera by two different effector mechanisms provides direct support for the autoimmune hypothesis of human vitiligo.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1988

Development of an in vitro keratinocyte model for use in the study of HSV specific cytotoxicity.

Herpes simplex virus (HSV) specific immune mediated cytotoxicity may be involved in control of HSV infections and in the tissue damage induced by HSV infection or HSV related skin disease such as herpes associated erythema multiforme. Developing an in vitro model to study this process has proved difficult due to the lack of an appropriate target cell that will express HSV antigens but is not simultaneously subject to viral induced cell death. The purpose of this study was to develop a model in which keratinocytes express cell surface HSV specific antigens but at the same time are protected from death due to viral infection. We found that keratinocytes infected with HSV in the presence of acyclovir (ACV) expressed such antigens yet remained viable for a period of time after the onset of antigen expression such that cytotoxicity studies could be successfully performed. Rabbit skin cells, a transformed keratinocyte line, or second passage human neonatal foreskin keratinocytes were grown in culture medium with or without 200 microM ACV and were infected with HSV. Examination by direct immunofluorescence with anti-HSV antibody revealed uniform HSV antigen expression by cells both with and without ACV by 8 h after infection. Cells infected without ACV exhibited marked structural abnormalities including formation of multinucleated giant cells, while cells with ACV showed fewer such changes throughout a 24-h period. An Ethidium Bromide-Acridine Orange cytotoxicity assay demonstrated significant increases in the cytotoxicity of infected cells not protected by ACV compared to that of cells with ACV (p less than .001). This in vitro model should prove useful in the investigation of HSV specific immune mediated cytotoxicity.

Acridine Orange↗
PubMed1988

Characterization and practical benefits of keratinocytes cultured in strontium-containing serum-free medium.

Strontium (Sr2+) can substitute for Ca2+ and stimulate a variety of functions of numerous types of cells. The purpose of this study was to investigate the details of the biologic effects of Sr2+ on human keratinocyte growth, cell cycle, viability, and differentiation and to compare these effects with Sr2+ effects on cultured skin melanocytes. Cultured keratinocytes stimulated with 1.0-3.0 mM Sr2+ showed higher viability and almost a twofold increase in cell number compared with those grown in a standard calcium concentration. Time course studies revealed that 2.0 mM Sr2+ had no effects on growth of cultured melanocytes or fibroblasts. Sr2+ increased the percentage of cultured keratinocytes in G2/M phase, with a decrease in cells in G0/G1 phase. This effect of Sr2+ on the cell cycle was not seen in cultured melanocytes or fibroblasts. A 2 mM concentration of Sr2+ produced an increase in low-density keratinocytes separated by a Percoll gradient. In addition, increased expression of human fibronectin was observed in the cytoplasm and on cell membranes of keratinocytes cultured in Sr2+. Sr2+ can be of practical benefit in the culture of human keratinocytes in serum-free medium, increasing the viability and proliferative rate and producing a more uniform population of basaloid cells with increased expression of cell surface fibronectin.

Cell Count↗
PubMed1988

Ro/SSA antigen in human epidermis.

It has been shown previously by an immunofluorescence technique, that whole serum from patients who have anti-Ro/SSA autoantibodies reacts with a component or components of the epidermis. We have now demonstrated by immunoblotting that the antigen identified in human epidermis by anti-Ro/SSA sera is Ro/SSA antigen, and that Ro/SSA antigen is present both in adult and in neonatal epidermis. The presence of this antigen in tissues which are injured in the anti-Ro/SSA-associated syndromes subacute cutaneous lupus erythematosus and neonatal lupus erythematosus supports the hypothesis that anti-Ro/SSA antibodies react with Ro/SSA antigen in the skin and are important in the initiation of tissue damage.

Adult↗
PubMed1987

Serum-free serial culture of adult human keratinocytes from suction-blister roof epidermis.

Coating cell culture flasks with natural extracellular matrix (ECM) enhanced the culture of adult human keratinocytes from suction-blister roof epidermis in an environment without fetal calf serum (FCS), bovine pituitary extracts or cellular feeder layers. A higher incidence of cell attachment on natural ECM was observed than on collagen and human fibronectins (HFN)-coated plastic dishes, and natural ECM was necessary for growth and proliferation of attached cells under the culture conditions used. Cells in primary culture grew to confluency on natural ECM-coated surfaces within about 14 days, and subsequent serial passage could be made up to fourth passage in collagen- and HFN-coated plastic flasks. Cultured keratinocytes in this serum-free environment formed colonies of small cuboidal, healthy cells with little keratinization or stratification and demonstrated antigenic characteristics of human basal cells.

Adolescent↗