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Biomedical subjects

D A Norris

Publications and source records attributed to D A Norris.

At least 37 records · Page 2Linked to original sources
PubMed1996

Candida albicans induces selective expansion of human T lymphocytes expressing the T-cell receptor variable region V beta 5.1.

Candida albicans is a common pathogen which can present major problems as an opportunistic skin pathogen in patients with immunodeficiency. The exact nature of the T cell responses to C. albicans is poorly understood. The purpose of this study was to determine whether C. albicans could stimulate the selective expansion of T lymphocytes expressing particular V beta gene segments. Human T lymphocytes stimulated in vitro with an extract of C. albicans were analyzed for T cell receptor V beta gene expression by using a quantitative PCR technique. We found that stimulation of peripheral blood mononuclear cells (PBMC) produced a selective increase in the expression of V beta 5.1 and 5.2 gene transcripts. Using cytofluorographic analysis with available anti-V beta monoclonal antibodies, we verified that there was a significant selective expansion (P = 0.035) of V beta 5.1 positive T lymphocytes in PBMC from six subjects stimulated in vitro with C. albicans. PCR analysis of V beta 5.1 expansion in 10 subjects showed increases in V beta 5.1 gene transcripts in 7/10 subjects. More importantly, analysis of the T cell infiltrate 48 h after intradermal injections with C. albicans also showed significant expression of V beta 5.1 in the infiltrates, along with the infiltration of V beta 8.1 + T cells. The selective expansion of V beta 5.1 bearing T lymphocytes in PBMC stimulated with C. albicans and in skin test reactions to C. albicans suggests that a restricted population of T cells react to C. albicans. Furthermore, our present data raise the provocative possibility that one or more antigens in C. albicans can act as a superantigen, producing selective expansion of a population of T lymphocytes bearing a particular V beta specificity.

Antibodies, Monoclonal↗
PubMed1996

DOPA-negative melanocytes in the outer root sheath of human hair follicles express premelanosomal antigens but not a melanosomal antigen or the melanosome-associated glycoproteins tyrosinase, TRP-1, and TRP-2.

It is believed that DOPA-negative melanocytes in the outer root sheath of the human hair follicle are activated, become identifiable by DOPA staining, and migrate into the epidermis during the repigmenting phase of vitiligo. These cells are difficult to identify, however, and otherwise have not been characterized. These cells are readily identified by immunofluorescence, immunohistochemistry, and immunoelectronmicroscopy using the antibodies NKI/beteb and A4F11, which recognize premelanosome-related antigens. The majority of the outer root sheath melanocytes were found in the mid to the upper portion of the hair follicle. Double staining revealed that these cells were distinct from HLA-DR-bearing dendritic cells. Further immunohistochemical investigation using alpha-PEP-7, alpha-PEP-1, or TMH-1 and alpha-PEP-8 antibodies revealed that outer root sheath melanocytes cannot be identified by antibodies to tyrosinase, TRP-1, or TRP-2, respectively. These cells also did not react with HMB45 antibody, which recognizes a melanosome-associated cytoplasmic antigen. We believe that the inactive outer root sheath melanocytes contain some of the early structural proteins but not any of the enzymatic proteins necessary for melanogenesis. Therefore, activation is the process whereby outer root sheath melanocytes acquire all of the structural and enzymatic proteins necessary for melanogenesis.

Adult↗
PubMed1996

Expression of E and P-cadherin by melanoma cells decreases in progressive melanomas and following ultraviolet radiation.

The purpose of our study was to determine whether the degree of E- and P-cadherin expression in melanomas correlates with the invasive behavior of the clinical lesions from which the cell lines were derived. Cadherins comprise a family of calcium-dependent cellular adhesion molecules expressed on most cell types that form solid tissues. In the human epidermis, melanocyte cadherin expression may function to maintain the integrity of the epidermal-melanin unit. Employing both immunofluorescence microscopy and fluorescence-activated cell sorter analysis, we localized and quantitated E- and P-cadherin expression on melanoma cell lines derived from primary or metastatic lesions using the monoclonal antibodies HECD-1 and NNC-CAD-299, respectively. Human epidermal melanocytes isolated from neonatal foreskin were evaluated by similar techniques and served as a biologic control. Melanoma cell lines were isolated from primary or metastatic lesions of patients described as having "early," "intermediate," or "advanced disease." Melanoma E- and P-cadherin immunofluorescence, as quantified by fluorescence-activated cell sorter, varied inversely with disease progression. Selected log mean ratios of E-cadherin fluorescence, as compared to human epidermal melanocytes (arbitrarily = 1), ranged from 1.04 in the WM 35 melanoma cell line (low invasive potential) to 0.1 and 0.02 in the WM 983A and 1361A melanoma cell lines (derived from primary lesions with metastases), respectively. Although values for P-cadherin fluorescence were less, the trend of decreasing cadherin amounts with more advanced disease was observed. Melanoma cells appear to express E- and P-cadherin levels inversely related to disease progression. Ultraviolet radiation significantly decreased E- and P-cadherin expression in the human epidermal melanocytes and P-cadherin expression in the WM 35 melanoma cell line (p < 0.05). Although not statistically significant, E-cadherin expression in the WM 35 melanoma cell line decreased substantially. Thus, ultraviolet radiation may have a direct effect on human epidermal melanocytes and melanoma cell attachment through cadherins within the epidermis or tumor nodules.

Cadherins↗
PubMed1996

Staphylococcal toxins and protein A differentially induce cytotoxicity and release of tumor necrosis factor-alpha from human keratinocytes.

It has been proposed that toxins and other bacterial protein products of Staphylococcus aureus can act as triggers or persistence factors in several inflammatory skin diseases. In this study, we examined the S. aureus isolates from the skin of patients with atopic dermatitis and psoriasis. We found that the bacterial isolates from these patients exhibited either characteristic superantigenic toxins or thermolabile toxins believed to be staphylococcal alpha-toxin. All of these staphylococcal strains also secreted extracellular staphylococcal protein A. We found significant differences in the action of these toxins on human keratinocytes and keratinocyte cell lines. The superantigenic toxins toxic shock syndrome toxin-1, staphylococcal enterotoxins A and B, and exfoliative toxin-A, as well as staphylococcal protein A, did not induce significant cytotoxic damage in the keratinocyte cell line HaCaT, whereas the staphylococcal alpha-toxin produced profound cytotoxicity. Keratinocyte cytotoxicity induced by staphylococcal alpha-toxin was time and concentration dependent and demonstrated the morphologic and functional characteristics of necrosis, not apoptosis. Addition of alpha-toxin to keratinocytes simultaneously induced cell lysis and tumor necrosis factor-alpha release into the medium within 30 min; apparently, it was constitutive tumor necrosis factor-alpha. On the other hand, superantigenic toxins and, in particular, protein A showed stimulation of tumor necrosis factor-alpha secretion in keratinocytes and release of this cytokine after 6-12 h of incubation. Thus, staphylococcal protein A, alpha-toxin, and superantigenic toxins found in S. aureus isolates from patients with psoriasis and atopic dermatitis can produce direct pro-inflammatory effects on keratinocytes through the release of tumor necrosis factor-alpha. We propose that these effects may be relevant to the induction and persistence of lesions in these two diseases.

Bacterial Toxins↗
PubMed1996

Effective elimination of fibroblasts in cultures of melanocytes by lowering calcium concentration in TPA depleted medium following geneticin treatment.

Melanocytes grow well in culture media supplemented with phorbol ester. However, phorbol ester is metabolically stable and has prolonged effects on multiple cellular responses. Different groups have reported that they could cultivate melanocytes without phorbol ester. However, in medium without phorbol esters contaminating fibroblasts are often difficult to eliminate. We have developed an improved method of eliminating fibroblasts using geneticin in serum free medium with bFGF, endothelin-1 and no phorbol ester. Treatment with a low concentration of geneticin effectively eliminated contaminating fibroblasts and was not harmful to melanocytes if geneticin was added initially in a high calcium media (2 mM) followed by a change to a low (0.09 mM) calcium media immediately after removal of the geneticin. With this technique we could easily establish pure melanocyte culture. This culture method will provide a useful tool for studies on melanocyte cellular response without the influence of phorbol ester.

Anti-Bacterial Agents↗
PubMed1995

The role of superantigens in skin disease.

Staphylococcus aureus and streptococci secrete a large family of exotoxins involved in the pathogenesis of toxic-shock-like syndromes and have been implicated in several autoimmune disorders. These toxins act as prototypic superantigens capable of binding to major histocompatibility complex proteins on antigen-presenting cells outside the antigen peptide-binding groove and can thereby stimulate cytokine release from macrophages. The superantigen-major histocompatibility complex unit is recognized primarily by the variable region of the T-cell receptor beta chain, and by engaging this region, can activate a large portion of the T-cell repertoire. It is thought that the capacity of these toxins to cause the massive stimulation of T cells and accessory cells such as macrophages, Langerhans cells, and activated keratinocytes accounts for most of their pathologic effects. The current review examines the evidence that implicates a role for these superantigens in the pathogenesis of certain skin diseases.

Animals↗
PubMed1995

In three types of interface dermatitis, different patterns of expression of intercellular adhesion molecule-1 (ICAM-1) indicate different triggers of disease.

We found distinct patterns of intercellular adhesion molecule-1 (ICAM-1) expression in three diseases characterized by interface dermatitis with mononuclear infiltrates and keratinocyte cytotoxicity: lichen planus (LP), subacute cutaneous lupus erythematosus (SCLE), and erythema multiforme (EM). In LP, basal keratinocytes show strong ICAM-1 expression associated with a dermal infiltrate, but ICAM-1 expression in the rest of the epidermis is minimal. In SCLE, there is diffuse epidermal ICAM-1 expression, sometimes with accentuation on the cell surface of basal cells. In EM, there is strong basal cell expression of ICAM-1 with evident cell surface accentuation, and also pockets of suprabasal expression with cell surface accentuation. These patterns are associated with different factors that trigger cytokine release in different locations. Both tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) produce greater relative ICAM-1 expression in basal keratinocytes than in more differentiated keratinocytes. In LP, the pure basal keratinocyte expression of ICAM-1 appears to be caused by cytokines, predominantly IFN-gamma, released by dermal lymphocytes. The pattern of ICAM-1 in SCLE corresponds to the pattern induced by ultraviolet radiation (UVR): diffuse epidermal ICAM-1 expression, sometimes with basal accentuation. Some individuals are "responders" to TNF-alpha or UVR, showing high levels of ICAM-1 expression following UVR or TNF-alpha stimulation in vitro or UVR stimulation in vivo. We propose that the pattern of ICAM-1 induction in SCLE is dependent on UVR-induced TNF-alpha release. EM is associated with apparent latent Herpes simplex virus, and Herpes simplex virus (HSV)-infected keratinocytes show enhanced ICAM-1 expression. We propose that in EM suprabasal ICAM-1 expression may be induced directly by HSV infection or indirectly through TNF-alpha release induced by HSV reactivation. Induction of ICAM-1 within the epidermis is stratified and individually variable. Basal keratinocytes show maximal induction of ICAM-1 expression due to innate sensitivity to TNF and IFN-gamma stimulation, and to location adjacent to dermal sources of cytokines. Suprabasal ICAM-1 can be induced by UVR and epidermal TNF-alpha release, and by factors such as viral infection. Different triggers of cytokine release and adhesion molecule induction may influence the different patterns of inflammation seen in diverse inflammatory skin diseases.

Erythema Multiforme↗
PubMed1995

Cytokine-induced ICAM-1 expression in human keratinocytes is highly variable in keratinocyte strains from different donors.

Induction of intercellular adhesion molecule 1 (ICAM-1) expression in the epidermis is felt to be an important initiator of leukocyte/keratinocyte interactions in many inflammatory skin diseases. The purpose of this project was to determine the individual variability of cytokine-induced ICAM-1 expression in human keratinocytes obtained from different donors. In 55 different keratinocyte strains, there was significant individual variability in ICAM-1 expression by either tumor necrosis factor alpha (TNF-alpha) or interferon-gamma. There was no correlation (r = 0.266, p = 0.06) in response of the same strain to either TNF-alpha or interferon-gamma. Multiple (n = 22) keratinocyte strains showed no significant induction of ICAM-1 expression to IL-1. The level of ICAM-1 expression in response to TNF-alpha and ultraviolet radiation (UVR) in individual strains was highly correlated in three different comparisons: level of stimulated response versus baseline (TNF-alpha and UVR both p < 0.0001); stimulation index TNF-alpha versus UVR (p = 0.00947); and variability of stimulated response versus variability of baseline (TNF p < 0.001; UVR p = 0.002). UVR-induced release of TNF from keratinocytes also showed variability among different keratinocyte strains. The UVR-induced ICAM-1 response in human keratinocytes and transformed epithelial cell was variably blocked with anti-TNF antibodies. The release of TNF from keratinocytes by UVR and the individually variable but linked characteristics of UVR and TNF-alpha stimulated ICAM-1 expression support the hypothesis that TNF-alpha is a major mediator of UVR-induced ICAM-1 expression.

Cells, Cultured↗
PubMed1995

Melanocyte mitogens induce both melanocyte chemokinesis and chemotaxis.

It is believed that during repigmentation of vitiligo, inactive melanocytes in the outer root sheath of the hair follicle become activated, proliferate, and migrate into the depigmented skin. However, the mechanisms controlling melanocyte migration remain to be elucidated. In this study, we investigated the effects of well-described melanocyte growth factors on melanocyte migration. Using time-lapse photography, we demonstrated that melanocyte chemokinetic movement was induced by basic fibroblast growth factor, stem cell factor, and endothelin-1, with the greatest effect noted using 100 nM endothelin-1. Similar results were reported previously with leukotriene C4. When surrounded by these stimuli, melanocytes moved in a random, nonlinear fashion and showed no desensitization at the concentrations studied. In Boyden chamber checkerboard analysis, basic fibroblast growth factor, leukotriene C4 and endothelin-1 were chemotactic. They produced directional migration and showed desensitization at higher concentrations. The greatest effect again was seen with 100 nM endothelin-1. Stem cell factor showed no effect in this assay system at the concentrations tested. The four melanocyte mitogens--leukotriene C4, endothelin-1, basic fibroblast growth factor, and stem cell factor--stimulate melanocyte migration, and this migration may be either chemokinetic (activated random movement) or chemotactic (requiring a gradient, directional, and showing desensitization), depending on the conditions used. We believe that these factors may be effective in stimulating vitiligo repigmentation by inducing proliferation and migration of hair-follicle outer-root-sheath melanocytes into the depigmented epidermis.

Cell Adhesion Molecules↗
PubMed1995

Clinical, pathological and epidemiological aspects of flood plain staggers, a corynetoxicosis of livestock grazing Agrostis avenacea.

Flood plain staggers, a corynetoxicosis of grazing livestock, occurred on flood plains of the Darling river in northern New South Wales between spring 1990 and autumn 1991, associated with the grazing of Agrostis avenacea with diseased inflorescences. Over this period 1722 cattle, 2466 sheep and 11 horses died on 31 farms. Clinical signs were similar in sheep and cattle, being characterised by intermittent episodes of cerebral convulsion superimposed on varying degrees of cerebellar dysfunction. Pathological changes were variable and non-specific, principally reflecting trauma and the generalised nature of the intoxication.

Animal Feed↗
PubMed1995

Evidence for a streptococcal superantigen-driven process in acute guttate psoriasis.

Recent studies have suggested that T cells play a critical role in the pathogenesis of psoriasis. Guttate psoriasis is a well-defined form of psoriasis frequently associated with streptococcal throat infection. This study tested the hypothesis that T cells in acute guttate psoriasis skin lesions may be activated by streptococcal superantigens. Peripheral blood as well as lesional and perilesional skin biopsies were analyzed for T cell receptor V beta repertoire using monoclonal antibodies against 10 different V beta families. Skin biopsies from all patients with acute guttate psoriasis, but not skin biopsies from patients with acute atopic dermatitis or inflammatory skin lesions induced in normal subjects with sodium lauryl sulfate, demonstrated selective accumulation of V beta 2+ T cells (P < 0.05). The expansion of V beta 2+ T cells occurred in both the CD4+ and the CD8+ T cell subsets. Sequence analysis of T cell receptor beta chain genes of V beta 2-expressing T cells from skin biopsies of patients with guttate psoriasis showed extensive junctional region diversity that is more compatible with a superantigen rather than a conventional (nominal) antigen-driven T cell response. All streptococcal isolates from patients with guttate psoriasis secreted streptococcal pyrogenic exotoxin C, a superantigen known to stimulate marked V beta 2+ T cell expansion. These data support the concept that acute guttate psoriasis is associated with superantigenic stimulation of T cells triggered by streptococcal superantigen(s).

Acute Disease↗
PubMed1994

Ras mutations in human melanoma: a marker of malignant progression.

In this study we address whether there is an association between ras mutations and disease progression in malignant melanoma. DNA was extracted from 100 paraffin-embedded melanomas and sequences around the 12th, 13th and 61st codons of N-, H-, and K-ras were amplified using the polymerase chain reaction and probed for single base pair mutations using synthetic oligonucleotide probes. Thirty-six melanomas contained mutations, which in 25 cases (69%) occurred at the 61st codon of N-ras. The results from dot blot hybridizations were confirmed by subcloning and sequencing the polymerase chain reaction products from two tumors. No ras mutations were found in Clark's level I melanomas, whereas 19% of level II and 45% of the more advanced primary tumors contained ras mutations (Chi squared test: p < 0.05). The median Breslow thickness of primary melanomas with ras mutations was 0.72 mm, significantly thicker than the 0.42 mm of melanomas without mutations (Mann-Whitney U test, p = 0.042). Ras mutations were found more frequently in primary tumors from continuously exposed skin (56%) than tumors from intermittently or non-sun exposed sites (21%). Fifty percent of locally recurrent and 47% of metastatic melanomas had ras mutations. We conclude that ras mutations occur in a subset of melanomas from sun-exposed skin as a feature of tumor progression.

Genes, ras↗
PubMed1993

The herpes-specific immune response of individuals with herpes-associated erythema multiforme compared with that of individuals with recurrent herpes labialis.

Infection with herpes simplex virus (HSV) is the most common precipitating factor in the development of erythema multiforme (EM). It is not known why only a few of the many individuals who experience recurrent HSV infection also develop herpes-associated EM (HAEM), although a difference in the HSV-specific immune response has been postulated. The purpose of this study was to compare the HSV-specific immune response of individuals with HSV infection alone with that of individuals with HAEM. There were 21 patients in each of the two groups. Four parameters of the HSV-specific immune response were examined: (1) anti-HSV IgG titers were measured by ELISA; (2) antibody neutralization was assessed using a plaque assay; and (3) antibody-dependent complement-mediated cytotoxicity, and (4) antibody-dependent cellular cytotoxicity were investigated using a previously described in vitro HSV-specific cytotoxicity assay. No statistically significant differences were detected between the two patient groups. Thus, a difference in these HSV-specific immune mechanisms does not explain the development of HAEM in some individuals with recurrent HSV infection.

Antibody Formation↗
PubMed1993

Dissolved inorganic nitrogen budget for a forested catchment at Beddgelert, North Wales.

An input-output budget for dissolved inorganic-N in a small forested catchment in North Wales is presented. From 1982 to 1990, bulk precipitation inputs averaged 10.3 kg ha(-1) year(-1), whereas throughfall inputs in 1983-1984 were 20.3 kg ha(-1) year(-1). Streamwater outputs were consistently larger than bulk precipitation inputs, averaging 14.6 kg ha(-1) year(-1). Inorganic-N in the forest stream was predominantly nitrate and concentrations were substantially higher than in a nearby moorland stream. Both streams showed seasonal trends in nitrate concentration, with highest concentrations occurring in summer in the forest stream but in winter in the moorland stream. Nitrate concentration in the forest stream increased with increasing soil temperature up to approximately 7 degrees C and decreased at higher temperatures. Nitrification is thought to be responsible for nitrate production at temperatures both below and above 7 degrees C, but root uptake becomes significant only at the higher temperatures. In the forest, dry deposition and cloudwater inputs of inorganic-N are responsible for increased nitrogen fluxes in throughfall compared with wet deposition. Mineralization and nitrification in excess of plant needs causes the organic soil horizons to act as a net source of dissolved inorganic-N. Nitrogen transformations in the soil lead to soil acidification at a rate of 1.0 keq ha(-1) year(-1).

Journal Article↗
PubMed1993

Cultured human keratinocytes synthesize and secrete endothelin-1.

The human epidermal-melanin unit exists as a complex interplay of cell-cell interactions. Melanocytes synthesize melanin and transfer it to the surrounding keratinocytes, which, in turn, produce factors that affect melanocyte homeostasis, growth, and melanization. Endothelin-1 (ET-1), a vasoconstrictor peptide produced by endothelial cells, has recently been shown to stimulate human melanocyte proliferation and tyrosinase activity. To investigate the possibility that keratinocytes synthesize and secrete ET-1, we grew human keratinocytes in a defined serum-free medium and measured ET-1 levels in the keratinocytes and the keratinocyte-conditioned medium. Northern analysis of keratinocyte total RNA also was performed. We found that human keratinocytes express preproET-1 mRNA and translate the message to ET-1 protein, which is secreted into the keratinocyte medium. Human keratinocytes produced ET-1 in a time-dependent manner with total production of 20.1 +/- 1.1 pg ET-1/10(6) cells at 24 h (n = 7). Although total ET-1 production (secreted plus cell-associated ET-1) was similar, the proportion of secreted versus cell-associated ET-1 varied widely among the different donors. We have found that human keratinocytes synthesize and secrete ET-1 in vitro. From these data we believe that the keratinocyte could be an in vivo epidermal source of this melanocyte growth and pigmentation factor.

Blotting, Northern↗