Experimental observations in the rat on the influence of cadmium on skin wound repair.
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Wound healing in the skin depends upon the availability of appropriate trace metals as enzyme cofactors and structural components in tissue repair. The present study forms part of a series of experimental investigations to examine the influence of xenobiotic elements with no known nutritional function and which are known to compete with essential trace metals. It was designed to investigate further the importance of trace metals in wound healing as an aid to wound management and to identify mechanisms of nonhealing which constitute a major problem in human medicine. Surgically induced skin wounds in young adult male Wistar rats were exposed topically to 0.2 ml of 0.01, 0.10 or 1.0% cadmium chloride (aq.) daily for up to 10 days. Control wounds received de-ionized water only. Wounds exposed to cadmium chloride at 0.01 or 0.10% healed in a similar fashion to controls and exhibited a comparable histological profile with metallothionein distribution. Wounds receiving 1.0% cadmium chloride failed to heal or fully re-epithelialize within 7 days and animals were humanely killed. They showed a persistent mass of inflammatory cell infiltration, oedema, wound debris and aberrant epidermal cell growth. Metallothionein concentrations in the epidermis and fibroblasts of the papillary dermis increased greatly by 5 days postwounding and remained high through the observation period. Cadmium was identified in the liver, kidney and wound sites. In the wound, 1.0% cadmium chloride induced statistically significant (P > 0.001) changes in local concentrations of zinc and calcium at key stages in the healing process, and as a consequence disturbed the trace metal balance necessary for normal wound repair. Zinc levels were increased twofold after 7 days, but calcium was markedly reduced. Local changes in the distribution of metallothionein indicate interaction of cadmium and trace metal carrier proteins as a probable mechanism for impaired wound healing. The cytotoxicity of cadmium is considered to be largely responsible. (+info)
Modification of fibroblast gamma-interferon responses by extracellular matrix.
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Fibroblasts from scaffold-based three-dimensional human cultures have been demonstrated to colonize ulcer wound beds and persist for at least 6 mo without rejection. This study examines the expression in these cultures of molecules associated with activation of the immune system in acute rejection. Studies in monolayer cultures showed that fibroblasts expressed CD40 at about 10% of the surface density seen in umbilical vein endothelial cells, whereas HLA-DR was undetectable. In these cultures, both molecules were induced by gamma-interferon. In scaffold-based three-dimensional cultures, however, a majority of the fibroblasts showed little induction of CD40 and HLA-DR in response to gamma-interferon, although HLA class I expression was increased. Fibroblasts re- isolated from the three-dimensional cultures and cultured in monolayers recovered HLA-DR induction in response to gamma-interferon. Fibroblasts cultured in an alternative three-dimensional system using collagen gels showed CD40 and HLA-DR induction by gamma-interferon in the same manner as monolayer cultures. Comparison of phosphorylation of signal transducer and activator of transcription 1 on tyrosine-701 showed it to be similar in monolayer and three-dimensional culture, and phospho-signal transducer and activator of transcription 1 moved into the nucleus. Induction of the class II transcription activator was greatly reduced, however. We propose that interaction of fibroblasts with the fibroblast-derived extracellular matrix is an important modulator of gamma-interferon responsiveness and that this interaction may play a role in the low immunogenicity of allogeneic fibroblasts grown on scaffolds. (+info)
Decreased susceptibility to Fas-induced apoptosis of systemic sclerosis dermal fibroblasts.
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OBJECTIVE: To determine whether dysregulated apoptosis of systemic sclerosis (SSc) fibroblasts contributes to progressive fibrosis by promoting fibroblast longevity. METHODS: We examined the pattern of fibroblast proliferation and apoptosis in SSc skin lesions and the susceptibility of cultured SSc dermal fibroblasts to apoptosis. Skin biopsy samples from SSc patients and control subjects were used to establish fibroblast cultures and were examined histologically. In skin sections, apoptosis was examined by TUNEL, and proliferation by immunostaining for proliferating cell nuclear antigen. Susceptibility of fibroblasts to apoptosis induced in vitro by different stimuli was studied by TUNEL. Expression of Bcl-2, Bcl-x, and Bax proteins in cultured fibroblasts was studied by Western blotting. RESULTS: Proliferation of dermal fibroblasts was not observed in normal skin but was present in skin from patients with SSc and other inflammatory skin diseases. Apoptosis of fibroblasts in SSc fibrotic skin lesions was not observed. In vitro, SSc fibroblasts were specifically resistant to apoptosis induced by Fas receptor stimulation but had normal susceptibility to apoptosis induced by nonspecific stimuli (protein kinase inhibition or serum withdrawal). Decreased susceptibility to Fas stimulation was not caused by decreased levels of surface Fas receptor. In SSc fibroblasts, quiescence induced by confluence and serum starvation was followed by an abnormal down-regulation of proapoptotic Bax protein. Up-regulation of the Bax:Bcl-2 ratio in SSc fibroblasts by Bcl-2 antisense oligonucleotides restored their susceptibility to Fas-mediated apoptosis. CONCLUSION: Our findings suggest that abnormal apoptotic regulation in fibroblasts can contribute to the pathogenesis of progressive fibrosis in SSc. Modulation of Bcl-2-related proteins appears to be a potential target for the development of apoptosis-based antifibrotic strategies. (+info)
Flow cytometric assessment of the reactivity of a panel of monoclonal antibodies (mAb) against two populations of human dendritic cells (DC).
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BACKGROUND: The identification of antigens on human DC has been a very difficult and elusive task because of the lack of appropriate reagents. Therefore, we evaluated by flow cytometry a panel of mAb that recognize antigens on human DC, aiming to determine the kinetics of DC antigen expression at 7, 14, 21 and 28 days in (i) Dermal DC like cells (Mo-DC) and (ii) Langerhans cell like DC (Mo-LC). In addition we aimed to identify markers for DC subpopulations. RESULTS: It was found at day 7, that mAb BG6, HP-F1, BU10, RFD-1, CMRF-44 recognized <20% of Mo-DC. In contrast, 7H5, ZM3.8, CDlb/c, 55K-2, MMR1.16, MMR190.BB3 and L25 reacted with >50% of Mo-DC. Moreover, 7H5, ZM3.8, CMRF-56, CDlb/c, 55K-2, MMR1.16, MMR190.BB3 and L25 showed increased MFI reactivity against Mo-DC. mAb BG6, BU10 and CMRF-44 recognized <20% Mo-LC while RFD-1 reacted with 21% of Mo-LC. In contrast, HP-F1 showed 87% of Mo-LC positive. Also, 7H5, ZM3.8, RFD-7, MR15-2, CDlb/c, 55K-2, MMR1.16, MMR190.BB3 and L25 reacted with >50% of Mo-LC. The increase in % of positive cells was paralleled by MFI increases. At day 14, fourteen mAb recognized >50% of the Mo-DC, while five recognized 20-50% of Mo-DC. BG6 reacted with 7% of the Mo-DC. Nineteen mAb recognized >48% of Mo-LC while BG6 had negative reactivity. At day 21 and 28, all mAb reacted with >20% of Mo-DC and yielded a significant MFI with Mo-DC. Also nineteen mAb yielded significant MFI with Mo-LC while RFD-7 did not. CONCLUSIONS: The immunophenotyping assays demonstrated differences between the two DC populations as well as variations in the reactivity of the mAb at diverse time points, suggesting the existence of subpopulations within the Mo-DC and Mo-LC. (+info)
Abnormalities in fibrillin 1-containing microfibrils in dermal fibroblast cultures from patients with systemic sclerosis (scleroderma).
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OBJECTIVE: To determine if there are abnormalities in fibrillin 1-containing microfibrils in the extracellular matrix (ECM) of primary dermal fibroblasts explanted from patients with systemic sclerosis (SSc). METHODS: Explanted fibroblasts from unaffected skin of 12 SSc patients were used to examine fibrillin 1-containing microfibrils by immunofluorescence (IF) using a monoclonal antibody (mAb) to fibrillin 1. Metabolic labeling of the fibroblast cultures was used to study the synthesis, secretion, and processing of fibrillin 1, as well as to observe microfibril formation and stability. Microfibrils elaborated by the SSc cells were analyzed by electron microscopy for ultrastructural abnormalities, and the results were confirmed by immunoblotting. RESULTS: Control and SSc fibroblasts displayed a prominent meshwork of fibrillin 1-containing microfibrils when visualized by IF using a fibrillin 1 mAb. Paradoxically, metabolic studies indicated a paucity of fibrillin 1 in the ECM in the majority of the SSc fibroblast strains. Subsequent rotary-shadowed electron microscopy revealed reduced amounts of and ultrastructural abnormalities in the microfibrils elaborated by all strains of SSc cells. Immunoblots confirmed the lack of the high molecular weight form of fibrillin 1 in the SSc fibroblasts of Choctaw American Indians. Finally, in vitro studies indicated that the amount of fibrillin 1 in the ECM of SSc cells diminished at a faster rate than the amount of fibrillin 1 in the ECM of control cells with time. CONCLUSION: Although SSc fibroblasts assemble microfibrils, these microfibrils are unstable, suggesting that an inherent defect of fibrillin 1-containing microfibrils may play a role in the pathogenesis of SSc. (+info)
Human dermal fibroblasts express prohormone convertases 1 and 2 and produce proopiomelanocortin-derived peptides.
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In the last few years it has become apparent that the skin is a locoregional source for several proopiomelanocortin-derived peptides including alpha-melanocyte-stimulating hormone, adrenocorticotropin, and beta-endorphin. The enzymes that regulate expression of these neuropeptides are the prohormone convertases 1 and 2. In this study we demonstrate, by reverse transcriptase polymerase chain reaction and Western immunoblotting, that cultured human dermal fibroblasts express prohormone convertases 1 and 2 as well as 7B2, which is an essential cofactor for enzymatic activity of prohormone convertase 2. Immunofluorescence studies revealed prohormone convertase 1 to be mainly expressed in the perinuclear region in vesicular structures resembling the trans-Golgi network, whereas prohormone convertase 2 was found in the trans-Golgi network as well as in vesicular structures diffusely distributed in the peripheral cytoplasm. Expression of both enzymes was also confirmed in fibroblasts of normal adult human skin by immunohistochemistry using antibodies against prohormone convertases 1 and 2 and vimentin. To assess the relevance of prohormone convertase 1 and 2 expression in human dermal fibroblasts, we studied the expression of proopiomelanocortin and proopiomelanocortin-derived peptides. Proopiomelanocortin expression was detected by reverse transcriptase polymerase chain reaction and Western immunoblotting. Alpha-melanocyte-stimulating hormone, adrenocorticotropin, and beta-endorphin were mainly located in vesicular structures as demonstrated by immunofluorescence. Production of these peptides was confirmed by radioimmunoassay, immunoradiometric assay, or enzyme immunoassay. Among several stimuli tested, interleukin-1 was found to upregulate production of alpha-melanocyte-stimulating hormone in human dermal fibroblasts. In summary, we have shown that human dermal fibroblasts express the enzymatic machinery for proopiomelanocortin processing and make proopiomelanocortin, alpha-melanocyte-stimulating hormone, adrenocorticotropin, and beta-endorphin. Production of proopiomelanocortin peptides by human dermal fibroblasts may be relevant for fibroblast functions such as collagen degradation and/or regulation of dermal immune responses. (+info)
Spl phosphorylation induced by serum stimulates the human alpha2(I) collagen gene expression.
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Serum has been known to stimulate collagen production by dermal fibroblasts. As part of an ongoing study of the molecular mechanisms of collagen production, we have investigated transcriptional regulation of the human alpha2(I) collagen gene by serum in human dermal fibroblasts. Serum responsive elements were mapped by deletion analysis between bp -353 and -264, and between -148 and -108 in the alpha2(I) collagen promoter. Further functional analysis of the alpha2(I) collagen promoter containing various substitution mutations revealed that serum stimulation of this promoter is mediated equally by a GC-rich region located between bp -303 and -271 and by the TCCTCC motif located between bp -123 and -128, both of which constitute binding sites for transcription factor Spl and Sp3. No differences were observed in electrophoretic mobility shift assays between unstimulated and serum stimulated fibroblasts. The Spl inhibitor mithramycin blocked stimulation of the alpha2(I) collagen promoter activity by serum. Furthermore, immunoprecipitation analysis showed that serum stimulation increased Spl phosphorylation. In conclusion, this study characterized response elements that mediate serum stimulation of the human alpha2(I) collagen promoter and suggests that serum stimulation was mediated via Sp1/Sp3 binding sites in this promoter. (+info)
Plasma and skin concentration of 5-methoxypsoralen in psoriatic patients after oral administration.
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The aim of this work was to investigate the distribution of 5-methoxypsoralen in the skin after oral administration of the drug and to examine the correlation between skin and plasma concentrations. 5-Methoxypsoralen skin concentration was measured in both healthy and psoriatic sites of 10 psoriatic patients after single and multiple oral doses. The results obtained show that 5-methoxypsoralen accumulates at higher levels in the more external layers of the skin after oral administration. The high affinity of drug for the stratum corneum was confirmed by in vitro skin affinity measurements. The concentration of 5-methoxypsoralen in the skin was similar in both psoriatic and healthy sites, indicating that the pathology does not influence drug distribution in the skin. After single dose administration, a linear correlation was found between skin and plasma drug concentration. After multiple dose administration, drug concentration in the skin was fairly constant despite the variable plasma concentrations in different subjects. (+info)