Biomedical subjects

O Rollman

Publications and source records attributed to O Rollman.

At least 19 recordsLinked to original sources

PubMed2006

Epithelial regeneration from bioengineered skin explants in culture.

BACKGROUND: Artificial skin substitutes are beneficial in the treatment of chronic wounds although their performance relative to authentic human skin is unclear. OBJECTIVES: We compared the rate of outgrowth and morphology of neoepidermis from a bioengineered skin construct (Apligraf) with normal adult human skin explants on de-epidermized human dermal growth substrate with or without intact epidermal basement membrane zone. METHODS: Epithelial outgrowth of air-exposed cultures in serum-supplemented keratinocyte medium was quantified by fluorescence imaging, morphology by light microscopy, biomarkers of keratinocyte activation, proliferation and migration by immunohistochemical analysis, and gelatinases by zymography. RESULTS: Resurfacing from bioengineereed skin explants started earlier than from normal skin but subsequently, from day 3 to day 9, the rate of epidermalization from bioengineered skin was only 40% (206 +/- 23 microm day(-1), mean +/- SEM) of that of authentic skin (521 +/- 17 microm day(-1), P < 0.001). At culture termination at day 11, normal human skin had formed a multilayered and well-structured neoepidermis covering 41.0 +/- 1.2 mm2 of the dermal substrate while bioengineered skin produced a thinner, less organized epithelium covering 20.4 +/- 3.0 mm2. At this later stage, a higher expression of beta-defensin-2, keratin 16, Ki67 and matrix metalloproteinase (MMP)-9 was found in neoepidermis formed from authentic skin than from bioengineered skin. Activated MMP-2 was elevated in bioengineered skin-derived neoepidermis. Minor epithelial outgrowth was noted with either skin type on the dermal substrate devoid of basement membrane zone. CONCLUSIONS: Cultured normal skin explants produced a more uniform and expansive in vivo-like neoepidermis than bioengineered skin explants.

Adult↗

PubMed2003

Antibiotic-resistant acne: lessons from Europe.

BACKGROUND: Propionibacterium acnes and P. granulosum are widely regarded as the aetiological agents of inflammatory acne. Their proliferation and metabolism are controlled using lengthy courses of oral and/or topical antibiotics. Despite numerous reports of skin colonization by antibiotic-resistant propionibacteria among acne patients, accurate prevalence data are available only for the U.K. OBJECTIVES: To determine the prevalence of skin colonization by antibiotic-resistant propionibacteria among acne patients and their contacts from six European centres. METHODS: Skin swabs were collected from 664 acne patients attending centres in the U.K., Spain, Italy, Greece, Sweden and Hungary. Phenotypes of antibiotic-resistant propionibacteria were determined by measuring the minimum inhibitory concentrations (MIC) of a panel of tetracycline and macrolide, lincosamide and streptogramin B (MLS) antibiotics. Resistance determinants were characterized by polymerase chain reaction (PCR) using primers specific for rRNA genes and erm(X), followed by nucleotide sequencing of the amplified DNA. RESULTS: Viable propionibacteria were recovered from 622 patients. A total of 515 representative antibiotic-resistant isolates and 71 susceptible isolates to act as control strains were characterized phenotypically. The prevalence of carriage of isolates resistant to at least one antibiotic was lowest in Hungary (51%) and highest in Spain (94%). Combined resistance to clindamycin and erythromycin was much more common (highest prevalence 91% in Spain) than resistance to the tetracyclines (highest prevalence 26.4% in the U.K.). No isolates resistant to tetracycline were detected in Italy, or in Hungary. Overall, there were strong correlations with prescribing patterns. Prevalence of resistant propionibacteria on the skin of untreated contacts of the patients varied from 41% in Hungary to 86% in Spain. Of the dermatologists, 25 of 39 were colonized with resistant propionibacteria, including all those who specialized in treating acne. None of 27 physicians working in other outpatient departments harboured resistant propionibacteria. CONCLUSIONS: The widespread use of topical formulations of erythromycin and clindamycin to treat acne has resulted in significant dissemination of cross-resistant strains of propionibacteria. Resistance rates to the orally administered tetracycline group of antibiotics were low, except in Sweden and the U.K. Resistant genotypes originally identified in the U.K. are distributed widely throughout Europe. Antibiotic-resistant propionibacteria should be considered transmissible between acne-prone individuals, and dermatologists should use stricter cross-infection control measures when assessing acne in the clinic.

Acne Vulgaris↗

PubMed2002

Topical retinoic acid alters the expression of cellular retinoic acid-binding protein-I and cellular retinoic acid-binding protein-II in non-lesional but not lesional psoriatic skin.

Therapeutic retinoids have profound effects on psoriatic skin pathology but their interactions with various retinoid-binding proteins in lesional vs non-lesional skin have not been investigated. Using quantitative real-time PCR the mRNA expression of cellular retinol-binding protein I (CRBPI) and retinoic acid-binding protein I/II (CRABPI/CRABPII) was studied in psoriatic and healthy control (=normal) skin after 4 days of occlusive RA/vehicle treatment (n=6). Untreated psoriatic lesions showed a markedly elevated CRABPII/CRABPI ratio, while the CRBPI level was reduced in lesional and non-lesional skin as compared to normal skin. In RA-treated normal and non-lesional skin, the mRNA expression of CRBPI was unaltered while that of CRABPI and CRABPII was reduced by approximately 80% and increased approximately 5-fold, respectively, as compared to vehicle-treated skin. In contrast, lesional skin exposed to RA showed an almost 90% increase in CRBPI transcripts but unaltered expression of CRABPI and CRABPII, yet, the mRNA expression of several inflammatory mediators, e.g. inducible nitric oxide synthase, interferon-gamma and interleukin-1beta, was clearly reduced. Immunohistochemistry localized CRABPII to suprabasal keratinocytes in normal skin and revealed markedly elevated levels in lesional skin. RA treatment induced CRABPII protein expression in normal and non-lesional skin, to similar levels as in untreated lesions. The results indicate that the effects of RA differ in normal/non-lesional psoriatic skin and lesional skin. Whether the high expression of CRABPII in psoriatic skin lesions is due to increased amounts of endogenous retinoids in lesional skin or reflects an abnormal regulation of the CRABPII gene in psoriasis remains to be studied.

Administration, Topical↗

PubMed2002

Cutaneous gene transfer for skin and systemic diseases.

Recent progress in molecular genetics has illuminated the basis for a wide variety of inherited and acquired diseases. Gene therapy offers an attractive therapeutic approach capitalizing upon these new mechanistic insights. The skin is a uniquely attractive tissue site for development of new genetic therapeutic approaches both for its accessibility as well as for the large number of diseases that are amenable in principle to cutaneous gene transfer. Amongst these opportunities are primary monogenic skin diseases, chronic wounds and systemic disorders characterized by low or absent levels of circulating polypeptides. For cutaneous gene therapy to be effective, however, significant progress is required in a number of domains. Recent advances in vector design, administration, immune modulation, and regulation of gene expression have brought the field much nearer to clinical utility.

Epidermolysis Bullosa↗

PubMed1999

The vitamin A metabolism and expression of retinoid-binding proteins differ in HaCaT cells and normal human keratinocytes.

HaCaT keratinocytes differ from normal human epidermal keratinocytes (HEK) by constitutive expression of differentiation markers which are normally suppressed by vitamin A. In search of an explanation for this discrepancy we compared the vitamin A content, the expression of retinoid-binding proteins, and the vitamin A metabolism in the two cell types. The concentrations of retinol and 3,4-didehydroretinol in cultured HaCaT cells were less than one-fifth those in HEK, and the content of fatty acyl esters was even lower. Similarly, the concentrations of cellular retinol-binding protein and cellular retinoic acid-binding protein (CRBPI and CRABPII, respectively) were 10-30 times lower in HaCaT cells than in HEK corresponding to a reduced mRNA expression of these proteins. Unexpectedly, HaCaT cells expressed RARbeta in addition to RARalpha, RARgamma and RXRalpha, which are nuclear receptors normally found in HEK. Radioactive retinol added to the culture medium appeared only transiently in HaCaT cells, and pulse labeling confirmed a defective cellular retention of retinyl esters. After 24 h of incubation with [3H]retinol, cell-associated radioactivity corresponding to retinol, 3,4-didehydroretinol, all-trans-retinoic acid and 3,4-didehydroretinoic acid was found in both HaCaT cells and HEK. [3H]Retinoic acid showed a more rapid metabolism to 4-hydroxy/4-keto-retinoic acid in HaCaT cells than in HEK, which could be explained by a higher expression of cytochrome p450RAI in the former cells. In conclusion, the abnormal uptake of vitamin A and low levels of retinoid binding proteins in HaCaT cells, linked with an aberrant metabolism of retinol, may help to explain why these cells differentiate also in the presence of retinoids.

Biomarkers↗

PubMed1998

Clones of normal keratinocytes and a variety of simultaneously present epidermal neoplastic lesions contain a multitude of p53 gene mutations in a xeroderma pigmentosum patient.

A patient with xeroderma pigmentosum group C was extensively examined for mutations in the p53 gene in normal skin exposed to varying degrees of sunlight and in excisional biopsies of basal cell cancer, squamous cell cancer, and squamous cell dysplasia. Seventy-three samples were analyzed by microdissection of small cell clusters, followed by PCR and direct DNA sequencing. In skin taken from areas that most likely had never been exposed to the sun, no mutations were found. However, in skin exposed to the sun, we observed a multitude of mutations in the p53 gene. UV light-induced mutations were found in all types of lesions, as well as in clusters of morphologically normal epidermal cells. Twenty-nine distinct mutations were found in exons 5-8, all missense or nonsense, of which 27 (93%) were UV-specific C --> T or CC --> TT transitions at dipyrimidine sites of the nontranscribed strand. Two types of normal skin areas containing p53 mutations were observed: areas that stain strongly with p53 antibody (p53 patches) and those that do not stain. Because no silent or intron mutations were found in these cell clusters, the alterations in the p53 gene of morphologically normal cells are likely to have resulted in a selective growth advantage. The poor correlation between mutations and morphological phenotypes demonstrates that p53 mutations alone do not determine the phenotypes observed.

Adolescent↗

PubMed1998

Oral isotretinoin therapy in severe acne induces transient suppression of biochemical markers of bone turnover and calcium homeostasis.

Although dietary vitamin A is required for normal growth and development, long-term or high-dose administration of vitamin A derivatives (retinoids) may produce a variety of skeletal side-effects in man. In this study we investigated the early effects of oral isotretinoin therapy on bone turnover and calcium homeostasis in eleven consecutive patients with nodulocystic acne. The effects on bone metabolism were correlated to radiological and bone mineral density measurements following drug therapy for six months. Markers of bone turnover, i.e. serum osteocalcin, the carboxyterminal propeptide of type I collagen, bone specific alkaline phosphatase, the carboxyterminal telopeptide of type I collagen, and urine levels of calcium and hydroxyproline decreased significantly within five days of treatment (p < 0.05). There was also a statistically significant decrease in serum calcium, with a minimum on day five, and a marked increase in serum parathyroid hormone (p < 0.05). With continued treatment, however, the abnormal levels of these markers returned to baseline values within 14 days. No significant roentgenological changes or effects on bone mineral density were found in response to the drug. The observed inhibitory effects of isotretinoin on bone turnover, despite elevated parathyroid hormone levels, indicates that the drug exerts a direct effect on bone tissue.

Acne Vulgaris↗

PubMed1998

Retinyl palmitate injections reduce serum levels and effects of endotoxin on systemic haemodynamics and oxygen transport in the pig.

BACKGROUND: Retinyl palmitate [(RP) 230 IU.kg(-1)] modulates the circulatory and respiratory responses of a subsequent infusion of endotoxin in the pig. The aims of this study were: I. To determine if RP (2300 IU.kg(-1)) affects the serum endotoxin levels in this model. II. To evaluate the effect of this dose of RP on circulatory and respiratory variables in our porcine model. III. To investigate the levels of RP and neutrophil count in porcine endotoxaemia. METHODS: Ten anaesthetized pigs were randomly given 2300 IU.kg(-1) of RP or the solvent i.m. prior to the continuous i.v. infusion of E. coli endotoxin (10 microg.kg(-1).h(-1)). Another 4 sham animals were given either i.m. RP (n=2) or i.m. solvent (n=2) followed by an infusion of saline. Haemodynamics and oxygen extraction were monitored and samples taken for analysis of endotoxin, RP and blood cells. RESULTS: I. Endotoxin levels in serum were lower (P<0.001) in the RP-pretreated pigs. II. These animals had higher cardiac index (P<0.05), mean arterial pressure and left ventricular stroke work index (both P<0.001), and lower oxygen extraction (P<0.01). III. RP-Pre=pretreatment caused a paradoxical decrease in serum retinyl (P<0.001) and a more rapid restitution of neutrophil count (P<0.05). CONCLUSION: Pretreatment with RP (2300 IU.kg(-1)) counteracts the progressive increase in serum endotoxin levels in porcine endotoxaemia.

Animals↗

PubMed1997

Cadexomer iodine in ulcers colonised by Pseudomonas aeruginosa.

The clinical and bacteriological efficacy of topical cadexomer iodine on venous leg ulcers colonised with Pseudomonas aeruginosa was evaluated in an open, uncontrolled, multicentre pilot study. Nineteen patients with venous leg ulcers (median ulcer area 15.5 cm2) were treated with cadexomer iodine paste and short-stretch bandaging for 12 weeks or until the ulcer was healed within that period. Bacteriological cultures for growth of Pseudomonas aeruginosa and ulcer area measurements were regularly performed during the study period. Seven patients were withdrawn during the study period for the following reasons: pain in the ulcer (n = 2), ulcer enlargement (n = 2), treatment of cellulitis in non-study ulcer with a systemic antibiotic that is effective against Pseudomonas aeruginosa (n = 1), increased thyroid-stimulating hormone level (n = 1) and poor compliance (n = 1). In addition, one patient was erroneously withdrawn. After one week's treatment, 11 out of 17 patients (65%) had a negative culture of Pseudomonas aeruginosa. At 12 weeks, six out of eight patients (75%) had a negative culture and complete healing was achieved in three patients (16%). The median ulcer area reduction obtained at 12 weeks was 32.9%. The results indicate that cadexomer iodine paste might be the treatment of choice for venous leg ulcers colonised with Pseudomonas aeruginosa. However, a larger controlled clinical study is needed for final evaluation.

Adult↗

PubMed1996

Biosynthesis and function of all-trans- and 9-cis-retinoic acid in parathyroid cells.

We demonstrate that cultured human and bovine parathyroid cells incubated with all-trans-[11,12-3H]-retinol convert this tracer into all-trans- and 9-cis-retinoic acid. By using RT-PCR, cellular retinol-binding protein type I (CRBP I), cellular retinoic acid binding protein I and II (CRABP I and II), retinoic acid receptors (RARs) alpha, beta and gamma, and 9-cis-retinoic acid receptor (RXR) alpha transcripts were detected in human parathyroid cDNA. CRBP I and CRABP I expression was confirmed by immunohistochemistry. Both 9-cis- and all-trans-RA were found to suppress parathyroid hormone (PTH) secretion from dispersed human adenomatous parathyroid cells, which was augmented by combined treatment with 1mM RA and 100 nM 1,25 (OH)2D3. The present data establish parathyroid gland as a target for retinoids and as a site of synthesis of the hormonal forms of vitamin A (retinol), all-trans- and 9-cis-retinoic acid.

Alitretinoin↗

PubMed1996

Differential uptake of chloroquine by human keratinocytes and melanocytes in culture.

The antimalarial drug chloroquine has a high affinity for melanin and accumulates in melanin-rich compartments such as those of the eye. Chloroquine is also deposited in cutaneous tissue, but whether the drug distribution is restricted to melanin-producing cells of the skin is not known. In the present study, the uptake of chloroquine by normal human epidermal keratinocytes was compared with that by melanocytes. Selectively cultivated cells were incubated at drug concentrations ranging between 0 and 10000 ng/ml for periods of up to 48 h. Chloroquine was quantified in cells and medium using high performance liquid chromatography and fluorometric detection. In both types of cells there was a rapid uptake of chloroquine within the first 2 h, followed by a slower uptake for 2-6 h until a steady-state condition was reached. Dose dependency was linear, with no sign of saturation, and approximately ten times higher drug concentrations were attained in melanocytes as compared with keratinocytes. No formation of desethylchloroquine, the major systemic metabolite, was detected in either cell type. The observed affinity of chloroquine for normal epidermal melanocytes in vitro suggests that the density and melanogenic activity of skin pigment cells may influence the cutaneous drug disposition of chloroquine.

Adult↗

PubMed1996

Increased concentrations of 3,4-didehydroretinol and retinoic acid-binding protein (CRABPII) in human squamous cell carcinoma and keratoacanthoma but not in basal cell carcinoma of the skin.

Retinoids are biologic response modifiers that are present in normal skin and may possibly be perturbed in carcinogenesis. To examine this possibility in human skin, we analyzed vitamin A and cytosolic retinoid binding proteins (cellular retinol binding protein and cellular retinoic acid binding protein [CRABP]) in a total of 38 non-melanoma skin tumors and 25 healthy skin samples using high performance liquid chromatography, radioligand electrophoresis, and reverse transcriptase-polymerase chain reaction. The mean +/- SEM retinol concentration was normal in basal cell carcinoma (0.60 +/- 0.10 microM) and seborrheic keratosis (0.47 +/- 0.07 microM), but increased in keratoacanthoma (1.60 +/- 0.41 microM) and squamous cell carcinoma (1.17 +/- 0.28 microM) (p < 0.05 for both). Also, the concentrations of 3,4-didehydroretinol, a major vitamin A metabolite produced in human skin, were markedly elevated (6-7 times normal) in keratoacanthoma and squamous cell cancer. All types of tumors showed moderately increased levels of cellular retinol binding protein. In addition, keratoacanthoma and squamous cell cancer showed markedly increased levels (6-7 times normal) of CRABPII protein. Transcriptional activity of the CRABPII gene was demonstrated in both normal and neoplastic epidermis, but clear CRABPI mRNA expression was found only in basal cell carcinoma. The data indicate that characteristic perturbations of the vitamin A and retinoid binding protein levels occur in squamous cell-derived skin tumors, but whether these reflect intrinsic errors in retinoid metabolism or are secondary to abnormal cellular differentiation is unknown.

Aged↗

PubMed1996

Increased expression of inducible nitric oxide synthase in psoriatic skin and cytokine-stimulated cultured keratinocytes.

Since nitric oxide (NO) has been implicated in the pathogenesis of various hyperproliferative and inflammatory diseases, the mRNA expression of constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) were investigated in psoriatic skin by reverse transcriptase coupled to the polymerase chain reaction (PCR). The study showed that the mRNA expression of brain nitric oxide synthase (bNOS), one of two isoforms of cNOS, was weak in both psoriatic plaques lesions and uninvolved skin, while mRNA transcripts for the second isoform, endothelial nitric oxide synthase (eNOS), were not detectable using the present method. In contrast, the mRNA expression of iNOS was markedly increased in lesional skin as compared to uninvolved skin. Cultured human keratinocytes exposed to a combination of interleukin-1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF-alpha) for 4 h, showed strong gene expression of iNOS, while in 24 h, the expression had returned to baseline expression. In summary, the study demonstrates that mRNA for the inducible form of NOS is over-expressed in psoriatic lesions. The cause of this may be the local presence of inflammatory cytokines. These findings imply that iNOS may play an important part in local regulation of NO synthesis in psoriasis and other inflammatory dermatoses.

Adult↗

PubMed1996

Vitamin D analogs affect the uptake and metabolism of retinol by human epidermal keratinocytes in culture.

Human epidermis utilizes retinol as precursor for local production of a range of bioactive vitamin A metabolites including 3,4-didehydroretinol, retinoic acid, and 3,4-didehydroretinoic acid. These endogenously formed retinoids bind to nuclear retinoic acid receptors (RARs), thereby altering gene transcription. Because 9-cis-retinoic acid receptors (RXRs) form heterodimers both with RARs and the vitamin D3 receptor (VDR), it is plausible that vitamin D3 may affect retinol metabolism if altered transcription is involved in the regulation of vitamin A-metabolizing enzymes. To investigate the potential effect of vitamin D on retinol metabolism in human skin keratinocytes, HaCaT cells were preincubated with various vitamin D3-analogs at 10(-7)M for 24 h followed by the addition of [3H]retinol for another 24 h period. The uptake and metabolism of the radioactive tracer was monitored by HPLC-radiochromatography. It was found that all synthetic vitamin D-analogs tested (MC903, KH1060, EB1089, and EB1213) reduced the amount of cell-associated [3H]retinoid activity by 35-50% as compared to the vehicle. More specifically, the appearance of the parent substrate and two of its main metabolites, e.g., 3,4-didehydroretinol (ddROH) and 3,4-didehydroretinoic acid (ddRA), was inhibited by the synthetic vitamin D-analogs. The effects on retinol metabolism were not potentiated by coincubation of cells with vitamin D-analogs plus retinoic acid (RA) or 9-cis-RA. This study demonstrates that synthetic vitamin D3 interferes with both the uptake and the metabolism of retinol by human epidermal keratinocytes. Whether the effects are due to direct inhibition of cellular retinol uptake and metabolism or involve VDR-mediated transcriptional alteration of vitamin A metabolizing enzymes remains to be clarified.

Cells, Cultured↗

PubMed1995

Increased mRNA expression of manganese superoxide dismutase in psoriasis skin lesions and in cultured human keratinocytes exposed to IL-1 beta and TNF-alpha.

Because reactive oxygen species have been implicated in the pathogenesis of various hyperproliferative and inflammatory diseases, the mRNA expression of the antioxidant enzyme superoxide dismutase was studied in psoriatic skin tissue. By using reverse transcription-PCR we found similar expression of copper, zinc superoxide dismutase (CuZnSOD) in the involved vs. uninvolved psoriatic skin. In contrast, the level of the manganese superoxide dismutase (MnSOD) mRNA message was consistently higher in lesional psoriatic skin as compared to adjacent uninvolved skin and healthy control skin. Parallel investigation of those cytokines that are thought to be direct or indirect inducers of the MnSOD activity revealed an increased mRNA expression of IL-1 beta, TNF-alpha, and GM-CSF in lesional psoriatic skin. To study if these cytokines exert a direct effect on dismutase expression in epidermal cells, human keratinocytes in culture were challenged with IL-1 beta, TNF-alpha, and GM-CSF. It was found that IL-1 beta and TNF-alpha, but not GM-CSF, induced the mRNA expression of MnSOD, and an additive effect was demonstrated for the two former cytokines. Further, the expression of both CuZnSOD and MnSOD transcripts was similar in cultured keratinocytes maintained at low differentiation (low Ca2+ medium) and cells forced to terminal differentiation (by high Ca2+ medium). Our results indicate that the abnormal expression of MnSOD mRNA in lesional psoriatic skin is not directly linked to the pathologic state of keratinocyte differentiation in the skin. It seems more likely that the cutaneous overexpression of MnSOD in psoriatic epidermis represents a protective cellular response evoked by cytokines released from inflammatory cells invading the diseased skin.

Adult↗

PubMed1994

Expression of cytosolic retinoid-binding protein genes in human skin biopsies and cultured keratinocytes and fibroblasts.

Using reverse transcription coupled to polymerase chain reaction we have studied the mRNA expression of serum retinol-binding protein and cytosolic receptors for retinol and retinoic acid in skin biopsies, and in cultured epidermal keratinocytes and dermal fibroblasts. Transcripts for cellular retinol-binding protein (CRBP) I and cellular retinoic-acid-binding protein (CRABP) I were found in normal skin, keratinocytes, and fibroblasts. CRABP II transcripts were detected in skin and keratinocytes. A decreased mRNA expression of CRABP I and an increased mRNA expression of CRABP II were found in lesional psoriatic skin compared with uninvolved skin. mRNA transcripts for serum retinol-binding protein (s-RBP) were detected in all tissues and cells. The biological importance of s-RBP expression in keratinocytes and fibroblasts is not known, but hypothetically this protein may be involved in the intracellular shuttling of retinol and retinoic acid, or in the retransportation of cellular retinoids into the extracellular space.

Base Sequence↗

PubMed1993

Biosynthesis of 3,4-didehydroretinol from retinol by human skin keratinocytes in culture.

The uptake and metabolism of radiolabelled retinol was studied in cultivated human skin cells. Normal epidermal keratinocytes in primary culture were able to incorporate unbound [11,12-3H]all-trans-retinol from the growth medium and transform it into 3,4-didehydroretinol (dehydroretinol) in a dose- and time-dependent manner. A total of 23% of the radioactive label became cell-associated during a 48-h incubation period when added at 7 nM to differentiated keratinocytes submerged in serum-containing, high-calcium (1.56 mM) culture medium. At that time point, 25-30% of cell-bound radioactive retinol had been converted into dehydroretinol, with no labelled retinal, dehydroretinal, retinoic acid or dehydroretinoic acid being detected in cells or medium. Thus dehydroretinol, which occurs physiologically in mammalian skin tissue in vivo, was identified as the predominant neutral retinol metabolite in cultured keratinocytes using h.p.l.c. and anhydro-derivatization procedures. At least 94% of the product, along with its precursor, was present in the cells in esterified form, with no traces of the compound being secreted into the cell environment. The rate of formation of dehydroretinol from its precursor was significantly lower in keratinocytes grown in serum-free, low-calcium (0.09 mM) culture medium, and in medium pre-incubated with excess unlabelled substrate. Furthermore, the application of 13-cis-retinoic acid (isotretinoin), a therapeutic retinoid drug known to markedly reduce dehydroretinol levels in human skin, blocked the biosynthesis of this metabolite in cultured keratinocytes. The 3,4-dehydrogenation pathway observed in this study could not be shown to operate to any significant extent in cultures of human epidermal melanocytes or dermal fibroblasts, supporting the hypothesis that keratinocytes represent the principal cell type involved in dehydroretinol formation from retinol in human skin.

Cells, Cultured↗

PubMed1993

Detection of mRNA transcripts for retinoic acid, vitamin D3, and thyroid hormone (c-erb-A) nuclear receptors in human skin using reverse transcription and polymerase chain reaction.

Differentiation of keratinocytes involves both non-genomic and genomic events. The genomic effects are regulated by ligand-dependent transcription factors, e.g. the steroid/thyroid super-family of nuclear receptors. In the present study we examined mRNA expression of receptors for retinoic acid, thyroid hormone, and vitamin D3 in normal human skin and cultured keratinocytes using reverse transcription coupled to the polymerase chain reaction. The vitamin D3 receptor and the retinoic acid receptor (RAR) gamma together with the more distantly related RXR alpha were amplified extensively in skin and cultured keratinocytes. RAR alpha was amplified at a lower level, and RAR beta was almost undetectable. The thyroid hormone receptors alpha 1 and beta 1 were weakly amplified, but to comparable levels. Because receptors for retinoic acid, thyroid hormones, and vitamin D3 are all expressed in human epidermis differentiation of keratinocytes is probably regulated at transcriptional level by these molecules. It remains to be seen whether alterations in the expression of the nuclear receptors occur in certain skin disorders.

Base Sequence↗