Evaluation of an asynchronous teleconsultation system for diagnosis of skin cancer and other skin diseases.

We wished to assess both diagnostic accuracy and concordance among dermatologists when evaluating digital images of skin disease presented using a low-cost asynchronous (store-and-forward) format. Each of eight board-certified dermatologists reviewed 50 clinical cases presented in digital format on a 15-inch computer monitor. For each case, the teleconsultants made a primary diagnosis and differential diagnosis and indicated whether or not a biopsy should be performed both before and after reviewing a brief history of each case. Diagnostic accuracy was calculated for each teleconsultant on the basis of biopsy, culture, or wet mount results. Concordance was determined by comparing primary and differential diagnoses made by the teleconsultants on all 50 cases with those made by the dermatologists who originally examined the patients in person. For eight skin cancers, the diagnostic accuracy for the inperson dermatologist was 88% versus 90% (range, 75-100%) for the teleconsultants. For the 25 cases (including the 8 skin cancers) confirmed by either biopsy (20), culture (1), or wet mount (4), the in-person accuracy was 84% compared to 73% (range, 65-88%) for the teleconsultants. The concordance between the in-person and teleconsultant diagnoses were in agreement 77% of the time (90% if differential diagnoses were included). After evaluating the accompanying history, teleconsultants changed their primary diagnosis in 11% of the cases (range, 2-22%). Biopsy rates were not significantly different between teleconsultants (45%) and in-person dermatologists (40%). An asynchronous software application can provide levels of diagnostic accuracy and concordance equivalent to those reported using live teleconsultation.

Cyclooxygenases in human and mouse skin and cultured human keratinocytes: association of COX-2 expression with human keratinocyte differentiation.

Epidermal expression of the two isoforms of the prostaglandin H-generating cyclooxygenase (COX-1 and COX-2) was evaluated both by immunohistochemistry performed on human and mouse skin biopsy sections and by Western blotting of protein extracts from cultured human neonatal foreskin keratinocytes. In normal human skin, COX-1 immunostaining is observed throughout the epidermis whereas COX-2 immunostaining increases in the more differentiated, suprabasilar keratinocytes. Basal cell carcinomas express little if any COX-1 or COX-2 immunostaining whereas both isozymes are strongly expressed in squamous cell carcinomas deriving from a more differentiated layer of the epidermis. In human keratinocyte cultures, raising the extracellular calcium concentration, a recognized stimulus for keratinocyte differentiation, leads to an increased expression of both COX-2 protein and mRNA; expression of COX-1 protein, however, shows no significant alteration in response to calcium. Because of a recent report that failed to show COX-2 in normal mouse epidermis, we also looked for COX-1 and COX-2 immunostaining in sections of normal and acetone-treated mouse skin. In agreement with a previous report, some COX-1, but no COX-2, immunostaining is seen in normal murine epidermis. However, following acetone treatment, there is a marked increase in COX-1 expression as well as the appearance of significant COX-2 immunostaining in the basal layer. These data suggest that in human epidermis as well as in human keratinocyte cultures, the expression of COX-2 occurs as a part of normal keratinocyte differentiation whereas in murine epidermis, its constitutive expression is absent, but inducible as previously published.

12-O-tetradecanoylphorbol-13-acetate and the induction of prostaglandin E2 generation by human keratinocytes: a re-evaluation.

12-O-Tetradecanoylphorbol-13-acetate (TPA) induces prostaglandin E2 (PGE2) synthesis in mouse keratinocytes and is associated with the induction of keratinocyte proliferation as well as accelerated differentiation. In human keratinocytes, TPA has been reported not to induce the release of either 3H-labeled arachidonic acid or 3H-labeled prostaglandins, even though cell differentiation is stimulated. Because PGE2 has been associated with the modulation of cell differentiation and because of technical problems inherent in evaluating arachidonic acid metabolism using only radiolabeled substrates, we evaluated the ability of TPA to induce endogenous PGE2 generation by cultured human keratinocytes using a specific and sensitive enzyme immunoassay. With this technique, TPA was found to induce a dose-dependent (1.6 x 10(-12)-1.6 x 10(-8) M) increase in PGE2 generation. These results are consistent with observations made not only in mouse keratinocytes but in other mammalian and human cell types. Documenting the ability of TPA to stimulate PGE2 production in human keratinocytes is very relevant to current theories regarding the role of PGE2 in keratinocyte differentiation as well as to establishing parallels between the murine and human skin models.

Endogenous prostaglandin E2 modulates calcium-induced differentiation in human skin keratinocytes.

The concentration of extracellular calcium appears critical to the initiation of keratinocyte differentiation. Prostaglandins (PGs) have also been implicated in cell differentiation. Consequently, the participation of endogenous eicosanoids in calcium-induced differentiation of human keratinocytes was evaluated in vitro. Our results demonstrate that: (1) exogenously introduced PGE2, the major keratinocyte-derived eicosanoid, but not prostaglandin I2 (PGI2) or its stable metabolite 6-keto-PGF1 alpha, enhances calcium-induced cornified envelope formation, an established marker of keratinocyte differentiation; (2) increasing extracellular calcium increased endogenous PGE2 synthesis by cultured keratinocytes; (3) blocking endogenous PGE2 synthesis with indomethacin significantly suppresses calcium-induced formation of the cornified envelope; and (4) adding back PGE2 to indomethacin-treated keratinocytes is able to re-establish the control level of cornified envelope formation following stimulation by calcium. These data document the participation of endogenously generated PGE2 in the modulation of calcium-induced differentiation by human keratinocytes.

Peroxisomal abnormality in fibroblasts from involved skin of CHILD syndrome. Case study and review of peroxisomal disorders in relation to skin disease.

BACKGROUND AND DESIGN: Peroxisomal deficiency has been described in a number of syndromes characterized by chondrodysplasia punctata, including the Conradi-Hünermann (C-H) syndrome. Because of overlapping clinical features of X-chromosome inheritance, ichthyosis, and limb-reduction defects in C-H and CHILD (congenital hemidysplasia with ichthyosiform erythroderma and limb defects) syndromes, we examined peroxisomal content using diaminobenzidine cytochemistry and peroxisomal functions in fibroblasts from involved vs uninvolved skin of CHILD syndrome. RESULTS: Fibroblasts from involved skin of a patient with CHILD syndrome accumulated cytoplasmic lipid, visualized with the fluorescent probe, nile-red. Ultrastructurally, fibroblasts of involved skin of CHILD syndrome accumulated lamellated membrane and vacuolar structures. By diaminobenzidine ultracytochemistry, fewer peroxisomes were present. Moreover, the activities of two peroxisomal enzymes, catalase and dihydroxyacetone phosphate acyltransferase, were decreased (approximately 30% of normal). However, peroxisomal oxidation of very-long-chain and branched-chain fatty acids was preserved. Moreover, plasma very-long-chain fatty acids, plasma phytanic acid, and erythrocyte plasmalogen content were normal. CONCLUSIONS: The CHILD, C-H, and rhizomelic chondrodysplasia punctata syndromes are all characterized by ichthyosis, chondrodysplasia punctata, and limb defects, as well as peroxisomal deficiency. Thus, these syndromes may be related pathogenically. Because peroxisomes are involved in prostaglandin metabolism, peroxisomal deficiency may directly contribute to the previously reported alterations in prostaglandin metabolism in fibroblasts of involved skin of fibroblasts.

Norepinephrine-induced increase in sympathetic neuron-derived prostaglandins is independent of neuronal release mechanisms.

The contribution of exocytosis to norepinephrine-stimulated prostaglandin release from sympathetic postganglionic neurons was evaluated in homogenates of adult rat superior cervical ganglia. Incubation of ganglion homogenates with norepinephrine (1 mM) for 30 min caused an increased release of prostaglandin E2 and prostaglandin I2 (measured as the stable metabolite, 6-keto-PGF1a). Neither tetrodotoxin (10 mM), K+ (120 mM), nor EDTA in Ca(2+)-free buffer affected prostaglandin generation under basal and norepinephrine-stimulated conditions. These results suggest that the increase in prostaglandin production by sympathetic neurons after norepinephrine administration is not through the release of previously synthesized intracellular stores. Instead, the increase in prostaglandins in response to norepinephrine appears to be explained by de novo synthesis.

Noradrenaline-induced prostaglandin production by sympathetic postganglionic neurons is mediated by alpha 2-adrenergic receptors.

In this study we have demonstrated that noradrenaline increases the levels of prostaglandin E2 and prostaglandin I2 (detected as the stable metabolite 6-keto-prostaglandin F1 alpha) synthesized by homogenates of superior cervical ganglia from the adult rat. This noradrenaline-induced prostaglandin production was further characterized: (a) Selective destruction of adrenergic sympathetic postganglionic neurons in the ganglia using 6-hydroxydopamine abolished both basal and stimulated prostaglandin production. (b) Elimination of preganglionic cholinergic sympathetic nerve terminals in the ganglia had no effect. (c) Mepacrine (a phospholipase inhibitor) and indomethacin (a cyclooxygenase inhibitor) attenuated both basal and stimulated prostaglandin production. (d) Yohimbine, but not prazosin, suppressed the noradrenaline dose-response curve for prostaglandin production. The results of these experiments show that, in vitro, noradrenaline stimulates de novo synthesis of prostaglandin E2 and prostaglandin I2 by sympathetic postganglionic neurons. This stimulation by noradrenaline appears to result from action at an alpha 2-adrenergic receptor.

Noxious stimulus-induced increase in spinal prostaglandin E2 is noradrenergic terminal-dependent.

Prostaglandins (PGs) were measured in perfusate from the lumbar intrathecal (IT) space of pentobarbital anaesthetized rats. The level of PGE2, but not of PGF2 alpha or 6-keto PGF1 alpha, was increased by immersion of a hindpaw in water at a noxious temperature (50 degrees C). No increase in PGE2 was produced by non-noxious thermal stimulation (35 degrees C water). The noxious stimulus-evoked increase in PGE2, and increases in PGE2 during norepinephrine infusion (10 micrograms/ml), were significantly decreased in rats pretreated with intrathecal 6-hydroxydopamine. These data suggest that noxious stimuli induce an increase in the production of spinal PGE2 and that this production derives from, or requires the presence of noradrenergic terminals in the spinal cord.

Production of hyperalgesic prostaglandins by sympathetic postganglionic neurons.

Prostaglandin E2 and prostacyclin (prostaglandin I2) produce hyperalgesia in animals and humans. Because there is evidence that prostaglandins contribute to pain maintained by sympathetic nervous system activity, we evaluated whether sympathetic postganglionic neurons synthesize these hyperalgesic prostaglandins, and whether production of prostaglandins by these neurons can contribute to sensitization of primary afferent nociceptors. Intradermal injection of arachidonic acid but not linoleic acid, in the rat hindpaw, produces a decrease in mechanical nociceptive threshold. This hyperalgesic effect is prevented by indomethacin, an inhibitor of prostaglandin synthesis or by prior surgical removal of the lumbar sympathetic chain. To test the hypothesis that sympathetic postganglionic neurons are the source of prostaglandins, we measured production of prostaglandin E2 and 6-keto-prostaglandin F1 alpha (the stable metabolite of prostacyclin) by homogenates of adult rat sympathetic postganglionic neurons from superior cervical ganglia. These homogenates produced significant amounts of prostaglandin E2 and 6-keto-prostaglandin F1 alpha, and most of this production is eliminated by neonatal administration of 6-hydroxydopamine which selectively destroys sympathetic postganglionic neurons. These results demonstrate that sympathetic postganglionic neurons produce prostaglandins, and supports further the hypothesis that the release of prostaglandins from sympathetic postganglionic neurons contributes to the hyperalgesia associated with sympathetically maintained pain.

CHILD syndrome. Phenotypic dichotomy in eicosanoid metabolism and proliferative rates among cultured dermal fibroblasts.

Dermal fibroblasts from a patient with CHILD syndrome (an acronym for congenital hemidysplasia with ichthyosiform erythroderma and limb defects) were obtained and successfully maintained in culture. Fibroblasts from an area of chronically hyperkeratotic skin were compared with fibroblasts from the corresponding contralateral area of normal skin in regard to proliferative activity and to both unstimulated and stimulated generation of PGE2, an eicosanoid with documented effects on both epidermal cell and fibroblast function. Compared with the uninvolved skin fibroblasts, those from involved skin showed (a) a slower rate of proliferation, (b) a cyclical pattern of PGE2 synthesis, and (c) an approximately 20-fold greater synthesis of PGE2 in response to human purified IL-1, a cytokine known to be secreted by epidermal keratinocytes. Furthermore, we were able to demonstrate that the cyclical generation of PGE2 by the involved skin fibroblasts is responsible for their slower rate of growth when compared with the uninvolved skin fibroblasts. These data document a phenotypic dichotomy between the uninvolved and involved skin fibroblasts in CHILD syndrome that may be exploited to increase our understanding of the nature of dermal influences that may affect epidermal growth and differentiation.

Stimulated T cell and natural killer (NK) cell lines fail to synthesize leukotriene B4.

The ability of leukotriene B4 (LTB4) to influence T cell and natural killer (NK) cell functions makes the question of LTB4 generation by these cells important to address. Consequently, LTB4 generation was evaluated in a human (Jurkat), and in a murine (EL-4) T cell line as well as in a rat NK cell line (RNK-16). Incubation of each of the 3 cell lines with [1-14C]arachidonic acid alone or in the presence of phytohemagglutinin (PHA), of calcium ionophore A23187, or of concanavalin A (Con A) plus the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) failed to generate radiolabelled LTB4 or other eicosanoids as determined by thin layer radiochromatography. Using two different radioimmunoassays for LTB4 also failed to demonstrate the generation of LTB4 under basal or stimulated conditions. These results support earlier studies that demonstrate that T cells are not capable of de novo synthesis of prostaglandins, thromboxanes, or leukotrienes and also provide evidence that NK cells also do not have the capacity to generate LTB4 or other eicosanoids. Our findings are also critically discussed in relation to studies claiming eicosanoid synthesis by T cells.

A novel eicosanoid is the major arachidonic acid metabolite of cultured human monocytes.

Human peripheral blood monocyte-macrophages (M phi) generate a novel eicosanoid during in vitro culture. The metabolite is generated during incubation of the cells with 14C - arachidonic acid (AA). Lack of prior recognition of this metabolite probably results from the facts that: 1) on thin-layer chromatography (TLC) in two standard solvent systems, the novel metabolite co-chromatographed with either prostaglandin D2 or thromboxane B2, and 2) its generation, under the conditions studied, does not occur until between 90 and 180 minutes after culture initiation which is a time period beyond that used for most leukocyte studies. The generation of the metabolite is inhibited by nordihydroguaiaretic acid (NDGA) but not by indomethacin. Base hydrolysis did not alter its migration on TLC. On both reversed phase and straight phase high pressure liquid chromatography (HPLC), the novel peak isolated by TLC elutes as a single major peak of radioactivity with a retention time different from the known leukotrienes, hydroxy acids, or their metabolites. Furthermore, the peak isolated on HPLC has a single ultraviolet absorption maximum at 270 nm. M phi cultured for 1 week prior to a 24 hour incubation with 14C-AA generated proportionally less of the novel eicosanoid (roughly 68% of total radiolabeled product) than did M phi cultured for 3 weeks prior to a similar incubation with 14C-AA (roughly 86% of total radiolabeled product). Under the conditions studied, the novel eicosanoid is the major AA metabolite generated from exogenous AA by cultured M phi and it appears to be generated in increasing quantity as the M phi differentiate.