Vitiligo: the evolution of cultured epidermal autografts and other surgical treatment modalities.
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Cell Transplantation↗
Biomedical subjects
M Yaar
Publications and source records attributed to M Yaar.
Thymidine dinucleotides inhibit contact hypersensitivity and activate the gene for tumor necrosis factor alpha1.
DNA is a target for ultraviolet-B-induced inhibition of contact hypersensitivity, and small DNA fragments such as thymidine dinucleotides (pTpT) can simulate several ultraviolet-induced effects. To determine whether pTpT mimics the suppressive influence of ultraviolet-B on contact hypersensitivity, we compared the effects of topical application of pTpT with those of ultraviolet-B irradiation on C57BL/6 mice sensitized to dinitrofluorobenzene. Mice pretreated with pTpT or ultraviolet-B irradiation showed markedly suppressed ear swelling responses to dinitrofluorobenzene challenge. Because tumor necrosis factor alpha mediates ultraviolet-B-induced suppression of contact hypersensitivity, and because pTpT exerts many ultraviolet-mimetic effects by augmenting mRNA and protein levels of effector molecules, we asked if pTpT mimics ultraviolet-B's upregulatory influence on tumor necrosis factor alpha expression. Using transgenic mice carrying a chloramphenicol acetyl transferase reporter linked to the tumor necrosis factor alpha promoter, we examined effects of ultraviolet-B irradiation versus intradermal injection of pTpT on tumor necrosis factor alpha gene transcription. Both treatments induced cutaneous chloramphenicol acetyl transferase activity. Ultra- violet-B or pTpT treatment of cultured dermal fibroblasts from these mice also stimulated chloramphenicol acetyl transferase activity. To determine whether human cells responded similarly, a well- differentiated ultraviolet-responsive human squamous cell carcinoma line was treated with pTpT. pTpT increased tumor necrosis factor alpha mRNA expression and protein secretion in a dose-dependent manner. Our findings expand the spectrum of ultraviolet effects mimicked by pTpT to include inhibition of contact hypersensitivity and activation of the tumor necrosis factor alpha gene. These results support the hypothesis that DNA photoproducts and/or their repair intermediates trigger many of the biologic consequences of ultraviolet irradiation.
Animals↗
Kinesin participates in melanosomal movement along melanocyte dendrites.
Movement of melanosomes along melanocyte dendrites is necessary for the transfer of melanin pigment from melanocytes to basal and suprabasal keratinocytes, an event critical to epidermal photoprotection and maintenance of normal skin color. Recent murine data suggest that in melanocyte dendrites the microtubule-associated melanosome movement is bidirectional and that actin-associated myosin V secures the peripheral melanosomes, preparing them to be transferred to surrounding keratinocytes. We now report that human melanocytes express high levels of kinesin, a molecule that participates in microtubule-associated transport of organelles in other cell types, and that ultrastructurally kinesin molecules are closely associated with melanosomes. To determine whether kinesin participates in melanosomal transport, cultured melanocytes were treated with sense or antisense oligonucleotides complementary to kinesin heavy chain sequences. Antisense oligonucleotides decreased kinesin protein levels and inhibited the bidirectional movement of the melanosomes, promoting their backward movement. Furthermore, guinea pigs were exposed to ultraviolet B irradiation, known to enhance transport of melanosomes from melanocytes to epidermal keratinocytes, and then were treated with kinesin sense or antisense oligonucleotides. The areas that were treated with kinesin antisense oligonucleotides showed significantly less pigmentation clinically and histologically than control (sense) oligonucleotide-treated areas. As observed ultrastructurally, in antisense-treated areas melanosomes remained in melanocyte dendrites but over several days were not transferred to the surrounding keratinocytes. Our study supports a major role for kinesin in microtubule-associated anterograde melanosomal transport in human melanocyte dendrites.
Animals↗
Role of cytoplasmic dynein in melanosome transport in human melanocytes.
Cytoplasmic dynein is a microtubule-associated retrograde-directed motor molecule for transport of membrane-bound organelles. To determine whether cytoplasmic dynein is expressed in melanocytes, we performed reverse transcriptase polymerase chain reaction using melanocyte cDNA and primers complementary to human brain cytoplasmic dynein heavy chain. A polymerase chain reaction product of the expected molecular size was generated and the identity was confirmed by sequence analysis. Western blotting of total melanocyte proteins reacted with an anti-intermediate chain cytoplasmic dynein antibody identified the appropriate 74 kDa band. To determine whether cytoplasmic dynein plays a role in melanosome transport, duplicate cultures were treated with cytoplasmic dynein antisense or sense (control) oligodeoxynucleotides and the cells were observed by high-resolution time-lapse microscopy, which allows visualization of melanosomal aggregates and individual melanosomes. Antisense-treated melanocytes demonstrated a strong anterograde transport of melanosomes from the cell body into the dendrites, whereas melanosome distribution was not affected in sense-treated melanocytes. To determine whether ultraviolet irradiation modifies cytoplasmic dynein expression, melanocyte cultures were exposed to increasing doses of solar-simulated irradiation, equivalent to a mild to moderate sunburn exposure for intact skin. Within 24 h, doses of 5 and 10 mJ per cm2 induced cytoplasmic dynein protein, whereas doses of 30 mJ per cm2 or more were associated with decreased levels of cytoplasmic dynein compared with sham-irradiated controls. Our data show that cytoplasmic dynein participates in retrograde melanosomal transport in human melanocytes and suggest that the altered melanosomal distribution in skin after sun exposure is due, at least in part, to decreased cytoplasmic dynein levels resulting in augmented anterograde transport.
Base Sequence↗
Effect of daily versus intermittent sunscreen application on solar simulated UV radiation-induced skin response in humans.
BACKGROUND: Acute and chronic skin damage occurs as a consequence of solar UV radiation exposure. To diminish such skin damage, the dermatologic community advocates the daily use of sunscreens as part of a sun avoidance strategy. OBJECTIVE: We determined the effectiveness of a sunscreen product with a sunscreen protection factor (SPF) of 15 applied daily in preventing UV-induced histologic damage in human skin compared with the protection afforded by sunscreens with equal or higher SPF applied intermittently. METHODS: Twenty-four subjects were exposed to 2 minimal erythema doses of solar-simulated UV on 4 consecutive days. Three sunscreen products were applied to the buttock of each subject. One SPF 15 product was applied daily before exposure to UV and, to simulate intermittent product use, an SPF 15 or SPF 29 product was applied on 3 of 4 days, with one missed application on days 2, 3, or 4. Skin biopsy specimens were taken and processed for routine and immunohistochemical staining. Changes in number of sunburn cells and Langerhans cells as well as degree of inflammatory infiltrate and lysozyme immunostaining were determined. RESULTS: There was a statistically significant increase in the number of sunburn cells, degree of inflammation, and intensity of lysozyme staining, and there was a decrease in the number of Langerhans cells at sites where sunscreen application was missed as compared with unirradiated control and daily SPF 15 sunscreen-treated sites. CONCLUSION: Our data suggest that daily use of a sunscreen reduces the skin damage produced by UV exposure compared with intermittent use of equal or higher SPF products. The daily application of sunscreens in appropriate quantities reduces the harmful effects of solar UV radiation on skin. Compliance is essential for maximal benefit of sunscreens.
Adult↗
Mechanisms and implications of the age-associated decrease in DNA repair capacity.
Skin cancer incidence is clearly linked to UV irradiation and increases exponentially with age. We studied the rate of removal of thymine dimers and (6-4) photoproducts in UV-irradiated human dermal fibroblasts derived from donors of different ages. There was a significant decrease with aging in the repair rates of both thymine dimers and (6-4) photoproducts (P<0.001). In addition, there was an age-associated decrease in the protein levels of ERCC3, PCNA, RPA, XPA, and p53 that participate in nucleotide excision repair. Moreover, the mRNA levels of XPA, ERCC3, and PCNA were significantly reduced with aging, suggesting that these decreases are often regulated at the mRNA level. Furthermore, with age induction of p53 after UV irradiation was significantly reduced. Taken together, our data suggest that the age-associated decrease in the repair of UV-induced DNA damage results at least in part from decreased levels of proteins that participate in the repair process.
Adult↗
The pathogenesis of melanoma induced by ultraviolet radiation.
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DNA Repair↗
Thymidine dinucleotide mimics the effect of solar simulated irradiation on p53 and p53-regulated proteins.
The tumor suppressor protein p53 participates in DNA repair and cell cycle regulation in response to injuries like ultraviolet (UV) irradiation. We have previously reported that the thymidine dinucleotide (pTpT), a common target for DNA photoproduct formation by UV light, mimics many effects of UV irradiation in cultured skin-derived cells, at least in part through the activation of p53. In this report we compare the effects of solar-simulated irradiation and pTpT on p53 and p53-regulated proteins involved in cellular growth arrest and DNA repair in cultured human dermal fibroblasts. We find that, like UV irradiation, pTpT increases the levels of p53, p21, and proliferating-cell nuclear antigen. The magnitude and time course of the inductions are UV dose dependent and consistent with known regulatory interactions among these nuclear proteins. These data confirm and expand previous studies of UV effects on nuclear proteins involved in cell cycle regulation and DNA repair. Our observations suggest that such protective effects can also be induced by pTpT in the absence of initial DNA damage, rendering cells more capable of responding to subsequent DNA damage.
Cells, Cultured↗
Post-transcriptional regulation of UV induced TNF-alpha expression.
Ultraviolet (UV) irradiation exerts multiple effects on skin cells, including the induction of several cytokines involved in immunomodulation. Specifically, UV irradiation has been shown to upregulate the level of tumor necrosis factor-alpha (TNF-alpha) mRNA in keratinocytes. To determine whether the induction of TNF-alpha mRNA is regulated by transcriptional or post-transcriptional mechanisms, we examined cells of keratinocytic lineage (SCC12F) for steady state level, transcription rate, and stability of TNF-alpha mRNA after UV irradiation. Within 4 h there was a 20-40-fold induction of TNF-alpha mRNA that persisted at lower levels through 48 h. Consistently, TNF-alpha protein secretion increased at 24 and 48 h after UV irradiation. UV irradiation increased the half-life of TNF-alpha mRNA from approximately 35 min to approximately 10 h. Conversely, the transcription rate of the TNF-alpha gene increased < 2-fold at the time of peak mRNA steady state levels. Thus, post-transcriptional mechanisms play a major role in UV induced TNF-alpha transcript level.
Cell Line↗
Aging versus photoaging: postulated mechanisms and effectors.
The differences between intrinsic aging and photoaging are reviewed. The various model systems currently employed for the studies of aging and photoaging are discussed. Findings on age associated decrements in receptor/ligand mediated signaling as well as changes during cellular senescence in the expression of nuclear transcription factors are described. The role of telomere shortening and oxidative damage in the aging process is explained. At the cellular level, genetic and behavioral differences between aging and photoaging are illustrated with particular emphasis on changes in the structure and function of the tumor suppressor gene p53.
Aging↗
Binding of beta-amyloid to the p75 neurotrophin receptor induces apoptosis. A possible mechanism for Alzheimer's disease.
Alzheimer's disease is a neurodegenerative disorder characterized by the extracellular deposition in the brain of aggregated beta-amyloid peptide, presumed to play a pathogenic role, and by preferential loss of neurons that express the 75-kD neurotrophin receptor (p75NTR). Using rat cortical neurons and NIH-3T3 cell line engineered to stably express p75NTR, we find that the beta-amyloid peptide specifically binds the p75NTR. Furthermore, 3T3 cells expressing p75NTR, but not wild-type control cells lacking the receptor, undergo apoptosis in the presence of aggregated beta-amyloid. Normal neural crest-derived melanocytes that express physiologic levels of p75NTR undergo apoptosis in the presence of aggregated beta-amyloid, but not in the presence of control peptide synthesized in reverse. These data imply that neuronal death in Alzheimer's disease is mediated, at least in part, by the interaction of beta-amyloid with p75NTR, and suggest new targets for therapeutic intervention.
3T3 Cells↗
Fas/Fas ligand interaction contributes to UV-induced apoptosis in human keratinocytes.
Keratinocytes in human skin undergo apoptosis during various inflammatory processes and after ultraviolet (UV) irradiation. To determine if keratinocyte apoptosis may be mediated by the Fas/APO-1 receptor (CD95), a signal transduction pathway known to initiate programmed cell death of lymphocytes, we investigated Fas expression, modulation, and function in keratinocytes. Keratinocytes constitutively expressed the 2.5- and 1.9-kb Fas transcripts, as well as the 43-kDa Fas protein. Treatment of interferon-gamma-stimulated keratinocytes with Fas agonistic antibody significantly promoted their cell death, indicating that Fas in keratinocytes is functional. UV irradiation induced Fas mRNA expression within 16 to 24 h and Fas protein within 24 h and through 48 h after irradiation. Furthermore, keratinocytes constitutively expressed Fas ligand (FasL) mRNA and protein. UV irradiation induced FasL mRNA as early as 4 h after irradiation and elevated FasL mRNA levels were maintained for at least 24 h postirradiation. Moreover, a FasL neutralizing antibody significantly reduced UV-induced apoptosis of IFN-gamma-treated keratinocytes. Our data strongly suggest that the Fas system contributes to keratinocyte apoptosis in UV-irradiated human skin.
Antibodies, Monoclonal↗
Purpuric drug eruption secondary to itraconazole.
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Aged↗
Nerve growth factor: its significance in cutaneous biology.
Work in the past 10 y has greatly expanded our knowledge regarding the role of neurotrophins and, in particular, nerve growth factor, in skin. During fetal development, neurotrophins are expressed both in the mesenchyme and the epithelium of developing skin, and they play a trophic role supporting the survival of innervating neurons. Nerve growth factor appears to have an important role also in the adult organism. It serves as a regulatory molecule during skin inflammation and repair, affecting both the neuronal and the immune systems. Recent studies also suggest that nerve growth factor is an important autrocrine growth factor and survival factor for keratinocytes, cells that express both high- and low-affinity receptors for nerve growth factor. Neural crest-derived melanocytes also express receptors for nerve growth factor. Nerve growth factor appears to have a major role during skin injury, e.g., preventing melanocyte apoptotic cell death following UV irradiation. Thus, epidermal events interact to maintain the number and function of both melanocytes and keratinocytes in skin. These recent insights into the mechanisms underlying nerve growth factor effects in skin may eventually result in the development of novel therapeutic modalities.
Animals↗
Human melanocytes as a model system for studies of Alzheimer disease.
The aging process leads to increased vulnerability to injury and disease, resulting in a decline in 1 or more organ systems that is incompatible with life. One of the most devastating age-associated neurodegenerative disorders, Alzheimer disease, is characterized by neuronal loss and the extracellular deposition in the brain of beta-amyloid peptide, which is presumed to be causally related. Using cultured neural crest-derived cutaneous melanocytes, we find that in the presence of beta-amyloid, melanocytes, like neurons, undergo programmed cell death (apoptosis). Nerve growth factor, which has been reported to attenuate the loss of cholinergic neurons in Alzheimer disease, protects melanocytes from apoptosis induced by beta-amyloid. Moreover, beta-amyloid is a ligand for the 75-kD transmembrane neurotrophin receptor that belongs to the family of apoptotic receptors that generates a cell-death signal on activation. Our data suggest that neuronal death in Alzheimer disease is mediated by the interaction of beta-amyloid with the 75-kD neurotrophin receptor. Human melanocytes provide a valuable in vitro model for studies of Alzheimer disease and for development of potential therapies.
Aging↗
Innervation of melanocytes in human skin.
Communication between the nervous system and epidermal melanocytes has been suspected on the basis of their common embryologic origin and apparent parallel involvement in several disease processes, but never proven. In this study, confocal microscopic analysis of human skin sections stained with antibodies specific for melanocytes and nerve fibers showed intraepidermal nerve endings in contact with melanocytes. This intimate contact was confirmed by electron microscopy, which further demonstrated thickening of apposing plasma membranes between melanocytes and nerve fibers, similar to synaptic contacts seen in nervous tissue. Since many intraepidermal nerve fibers are afferent nerves that act in a "neurosecretory" fashion through their terminals, cultured human melanocytes were stimulated with calcitonin gene-related peptide (CGRP), substance P, or vasoactive intestinal peptide, neuropeptides known to be present in cutaneous nerves, to examine their possible functions in the epidermal melanin unit. CGRP increased DNA synthesis rate of melanocytes in a concentration- and time-dependent manner. Cell yields after 5 d were increased 25% compared with controls maintained in an otherwise optimized medium. Furthermore, stimulation by CGRP induced rapid and dose-dependent accumulation of intracellular cAMP, suggesting that the mitogenic effect is mediated by the cAMP pathway. These studies confirm and expand a single earlier report in an animal model of physical contact between melanocytes and cutaneous nerves and for the first time strongly suggest that the nervous system may exert a tonic effect on melanocytes in normal or diseased human skin.
Calcitonin Gene-Related Peptide↗
Aging affects epidermal growth factor receptor phosphorylation and traffic kinetics.
With increasing donor age, cultured human fibroblasts express fewer epidermal growth factor receptors and display decreased mitogenic responsiveness to epidermal growth factor. To determine age-associated differences in epidermal growth factor receptor phosphorylation and traffic kinetics, we studied in fibroblasts derived from donors of different ages autophosphorylation of the receptor after ligand binding and trafficking of the receptor-ligand complexes. We now report an age-associated delay in the rate of receptor phosphorylation after epidermal growth factor stimulation. Furthermore, receptor/ligand trafficking is affected by aging. There is an age-associated decrease and delay in the number of occupied receptors that are transported intracellularly and in their rate of clearance from the plasma membrane. Our data show that aging affects receptor/ligand activation and processing and suggest that the decreased cellular mitogenic response with aging may be, at least in part, the result of decrements in receptor activation and processing.
Biological Transport↗
Nerve growth factor rescues pigment cells from ultraviolet-induced apoptosis by upregulating BCL-2 levels.
Apoptosis plays an important role in eliminating dysfunctional damaged cells. For skin, the best characterized injurious environmental agent is ultraviolet (UV) irradiation. Most of the damaging UV irradiation is absorbed in the epidermis and leads to apoptosis of keratinocytes. However, epidermal melanocytes appear to be protected from UV-induced apoptosis. We now report that in pure cultures melanocytic cells undergo characteristic apoptosis after physiologic UV exposures. However, nerve growth factor (NGF) supplementation protects them from this programmed cell death. Furthermore, we show that NGF protects melanocytic cells from UV-induced apoptosis by upregulating BCL-2 protein in these cells and that prior downregulation of BCL-2 abrogates the NGF protective effect on melanocytes. Our data suggest that NGF, known to be constitutively produced by epidermal keratinocytes and induced in these cells after UV irradiation, may preserve the population of cutaneous melanocytes that would otherwise be depleted by casual sun exposure.
Apoptosis↗