STAT4 expression and activation is increased during mitosis in vitro and in vivo in skin- and mucosa-derived cell types: implications in neoplastic and inflammatory skin diseases.

BACKGROUND: The signal transducer and activator of transcription-4 (STAT4/Stat4) is a transcription factor known to convey signals from interleukin-12, interleukin-23, and interferon-alpha/beta to the nucleus, resulting in activation of dendritic cells, T-helper cell differentiation and production of interferon-gamma. OBJECTIVE: To demonstrate a novel role for STAT4 in cell mitosis. RESULTS: Phosphoserine STAT4 (pSerSTAT4) is increased in cells undergoing mitosis and is distributed throughout the cytoplasm during this stage of the cell cycle, whilst phosphotyrosine STAT4 (pTyrSTAT4) is confined to the chromosomal compartment. This distinct pattern of pSerSTAT4 during mitosis is seen in vitro in human keratinocytes and in other cell types. This is also present in vivo in cells undergoing mitosis in normal skin, psoriasis and squamous cell carcinoma. Inhibition of STAT4 phosphorylation by lisofylline and depletion of STAT4 by RNA interference results in a delay in progression of mitosis and leads to a reduction in cells completing cytokinesis. CONCLUSION: Our data demonstrate that STAT4 plays a role in enabling the normal and timely division of cells undergoing mitosis.

In vitro diagnostic assays are effective during the acute phase of delayed-type drug hypersensitivity reactions.

BACKGROUND: Previous reports have suggested that drug-specific lymphocyte proliferation assays (LPA) can be used retrospectively to confirm the culprit drug following delayed-type drug hypersensitivity reactions (DHR). However, only limited evidence supports their use in aiding acute clinical management. The aim of this study was to compare the LPA against combination cytokine assays for potential use in the acute setting. METHODS: A total of 43 patients with DHR (19 during the acute reaction, 20 after recovery, four during acute and after recovery) and 14 control subjects without DHR were investigated using ex vivo analysis of drug-specific proliferation, and interferon (IFN)-γ and interleukin (IL)-4 production. RESULTS: Healthy controls showed negative drug-specific proliferation and cytokine release in contrast to individuals with a known sensitivity (P < 0·0001). The assays demonstrated a test specificity of 95% (LPA), 83% (IFN-γ) and 92% (IL-4). The sensitivity of combined measurement of drug-specific IFN-γ and IL-4 cytokines during acute DHR was better than LPA (82% vs. 50%), but all assays were less sensitive during the recovery phase. The correlation between LPA and IFN-γ assays was strong (r = 0·7, P < 0·0001), whereas the IL-4 assay did not correlate as well with either of these assays. In contrast to LPA, drug enzyme-linked immunosorbent spot assays showed positive responses in patients concurrently taking immunosuppressive medication. CONCLUSIONS: In vitro assays of drug-specific IFN-γ and IL-4 production offer potential for use as rapid diagnostic tests. Cytokine detection offers distinct advantages over the LPA, including a shorter assay time, a greater sensitivity and effectiveness in testing immunosuppressed patients.

Prostaglandin E2 and nitric oxide mediate the acute inflammatory (erythemal) response to topical 5-aminolaevulinic acid photodynamic therapy in human skin.

BACKGROUND: Topical 5-aminolaevulinic acid photodynamic therapy (5-ALA-PDT) causes a clinical inflammatory response in human skin. While histamine mediates the immediate reaction, the mediators of the prolonged erythema are unknown. OBJECTIVES: To look for involvement of the proinflammatory mediators prostaglandin (PG)E2 and nitric oxide (NO) in topical PDT-induced erythema in human skin. METHODS: A series of studies was performed in healthy volunteers (n = 35). Following definition of the erythemal time course and dose response to 5-ALA-PDT, duplicate 5-ALA dose series were iontophoresed into the skin of each ventral forearm and exposed to 100 J cm(-2) broadband red light. Within subject, arms were randomized to control, or treatment with the cyclooxygenase and NO synthase inhibitors indometacin and Nω -nitro-l-arginine methyl ester (l-NAME), respectively, and the impact on 5-ALA-PDT-induced erythema was quantified. Additionally, release of PGE2 and NO was directly assessed by sampling dermal microdialysate at intervals following 5-ALA-PDT administration. RESULTS: A 5-ALA dose-related delayed erythema occurred by 3 h (r = 0·97, P < 0·01), with erythema persisting to 48 h post-PDT. Topical indometacin applied immediately post-PDT reduced the slope of erythemal response at 3 h and 24 h (P < 0·05). Intradermal injection of l-NAME into 5-ALA-PDT-treated sites reduced the slope of response at 24 h post-PDT (P < 0·001), while significantly inhibiting erythema from 3 h to 48 h post-PDT (P < 0·01). Analysis of dermal microdialysate showed release of NO and PGE2 following treatment. CONCLUSIONS: Topical 5-ALA-PDT upregulates PGE2 and NO in human skin, where they play a significant role in the clinical inflammatory response. The potential relevance of these mediators to PDT in human cutaneous pathology warrants study.

Repeated low-dose skin exposure is an effective sensitizing stimulus, a factor to be taken into account in predicting sensitization risk.

BACKGROUND: Contact sensitization by ingredients in personal products is an important clinical problem. It is not clear how sensitization is induced by the generally low concentrations at which they occur but it might be the result of repeated exposure. OBJECTIVES: To compare the strength of contact sensitization induced by a single exposure to 2,4-dinitrochlorobenzene (DNCB) (60 microg cm(-2)) or three repeated exposures to a subsensitizing dose (10 microg cm(-2)). METHODS: Two groups (n = 10) of healthy adult volunteers were randomized to receive either a single patch of DNCB 60 microg cm(-2) or three once-weekly applications to the same site of 10 microg cm(-2) DCNB. Four weeks after the last application, sensitization was quantified by measurement of responses (skinfold thickness) to a graded series of four challenge doses. RESULTS: All the volunteers were sensitized and the strength of the responses was virtually identical between the groups. CONCLUSIONS: The same degree of sensitization was induced by three exposures to DNCB 10 microg cm(-2) as by one exposure to 60 microg cm(-2) of DNCB. Thus repeated exposure to low doses of contact sensitizers may increase the sensitizing potency. This must be taken into account in future risk assessments.

BSACI guidelines for the management of drug allergy.

These guidelines have been prepared by the Standards of Care Committee (SOCC) of the British Society for Allergy and Clinical Immunology (BSACI) and are intended for allergists and others with a special interest in allergy. As routine or validated tests are not available for the majority of drugs, considerable experience is required for the investigation of allergic drug reactions and to undertake specific drug challenge. A missed or incorrect diagnosis of drug allergy can have serious consequences. Therefore, investigation and management of drug allergy is best carried out in specialist centres with large patient numbers and adequate competence and resources to manage complex cases. The recommendations are evidence-based but where evidence was lacking consensus was reached by the panel of specialists on the committee. The document encompasses epidemiology, risk factors, clinical patterns of drug allergy, diagnosis and treatment procedures. In order to achieve a correct diagnosis we have placed particular emphasis on obtaining an accurate clinical history and on the physical examination, as these are critical to the choice of skin tests and subsequent drug provocation. After the diagnosis of drug allergy has been established, communication of results and patient education are vital components of overall patient management.

Inflammatory disease processes and interactions with nutrition.

Inflammation is a stereotypical physiological response to infections and tissue injury; it initiates pathogen killing as well as tissue repair processes and helps to restore homeostasis at infected or damaged sites. Acute inflammatory reactions are usually self-limiting and resolve rapidly, due to the involvement of negative feedback mechanisms. Thus, regulated inflammatory responses are essential to remain healthy and maintain homeostasis. However, inflammatory responses that fail to regulate themselves can become chronic and contribute to the perpetuation and progression of disease. Characteristics typical of chronic inflammatory responses underlying the pathophysiology of several disorders include loss of barrier function, responsiveness to a normally benign stimulus, infiltration of inflammatory cells into compartments where they are not normally found in such high numbers, and overproduction of oxidants, cytokines, chemokines, eicosanoids and matrix metalloproteinases. The levels of these mediators amplify the inflammatory response, are destructive and contribute to the clinical symptoms. Various dietary components including long chain omega-3 fatty acids, antioxidant vitamins, plant flavonoids, prebiotics and probiotics have the potential to modulate predisposition to chronic inflammatory conditions and may have a role in their therapy. These components act through a variety of mechanisms including decreasing inflammatory mediator production through effects on cell signaling and gene expression (omega-3 fatty acids, vitamin E, plant flavonoids), reducing the production of damaging oxidants (vitamin E and other antioxidants), and promoting gut barrier function and anti-inflammatory responses (prebiotics and probiotics). However, in general really strong evidence of benefit to human health through anti-inflammatory actions is lacking for most of these dietary components. Thus, further studies addressing efficacy in humans linked to studies providing greater understanding of the mechanisms of action involved are required.

Control of salicylate intolerance with fish oils.

We report three patients with disabling salicylate-induced intolerance who experienced abrogation of symptoms following dietary supplementation with omega-3 polyunsaturated fatty acids (PUFAs). All three patients experienced severe urticaria, asthma requiring systemic steroid therapy and anaphylactic reactions. After dietary supplementation with 10 g daily of fish oils rich in omega-3 PUFAs for 6-8 weeks all three experienced complete or virtually complete resolution of symptoms allowing discontinuation of systemic corticosteroid therapy. Symptoms relapsed after dose reduction. Fish oil appears a safe and effective treatment for this difficult and often serious condition.

What's new in the management of cutaneous T-cell lymphoma?

The aetiology and clinical management of primary cutaneous T-cell lymphoma (CTCL) and specifically of mycosis fungoides and Sezary syndrome are poorly defined. Interesting new insights into CTCL disease biology as well as a number of emerging of novel therapeutic interventions make this an increasingly interesting area for dermatologists and oncologists involved in the treatment of CTCL. This review article covers much of this new information including new drugs, such as denileukin diftitox (Ontak) a targeted cytotoxic biological agent, Bexarotene an RXR selective retinoid, anti-CD4 monoclonal antibodies (mAb), new cytotoxics agents and vaccines.

Photochemotherapy of psoriasis: DNA damage in blood lymphocytes.

DNA synthesis assessed by 3H thymidine incorporation was measured in lymphocytes from patients with psoriasis receiving photochemotherapy with 8-methoxypsoralen (8-MOP) and UV-A (PUVA). The emission from the UVA source contained sufficient short wavelengths (UV-B) to impair 3H-thymidine incorporation but could be screened out with clear plastic screens. When lymphocytes were irradiated in vitro in a 1/10 dilution of plasma containing 8-MOP, increasing doses of UV-A produced progressive inhibition of phytohaemagglutinin (PHA)-induced DNA synthesis. Cells from patients given several previous treatments were inhibited significantly more than those from patients receiving their first exposure. This was also true for unirradiated cells sugesting either an accumulation of 8-MOP within cells, persistence of DNA damage or alteration of the lymphocyte population. Lymphocytes removed immediately after patients were irradiated showed less 3H thymidine incorporation than cells taken just prior to irradiation, which confirms that PUVA treatment exerts in vivo effects on circulating lymphocytes.

TNF-alpha and IL-8 are upregulated in the epidermis of normal human skin after UVB exposure: correlation with neutrophil accumulation and E-selectin expression.

The in vivo response to ultraviolet B (UVB) radiation in skin is characterized by the accumulation of both mononuclear and polymorphonuclear cells within the dermis and an induction of vascular endothelial adhesion molecules. Epidermal production of cytokines (IL-8 and TNF-alpha) has been strongly implicated in the development of UVB-induced inflammation. In the current study, we examined the time course of IL-8 and TNF-alpha mRNA and protein expression in the epidermis over a 24-h period after in vivo UVB irradiation. Also, the induction of adhesion molecule expression and the accumulation of neutrophils within the dermis were followed. We found constitutive expression of both cytokines (mRNA and protein) in the epidermis of unirradiated skin. IL-8 was rapidly upregulated after irradiation and mRNA and protein increased at 4 h, reaching a maximum between 8 and 24 h. TNF-alpha mRNA and protein was minimally increased by 8 h after UVB irradiation and reached a maximum by 24 h. No significant alteration in ICAM-1 or VCAM-1 expression was observed. E-selectin expression, which was absent from control samples, was increased from 4 h onward and also reached a maximum at 24 h, coinciding with peak neutrophil accumulation. A strong correlation (r = 0.96) was found between number of E-selectin-positive vessels and numbers of infiltrating neutrophils at this time. Moreover, because E-selectin expression was increased before any apparent increase in TNF-alpha protein (4 h), TNF-alpha does not appear to be involved in the early induction of the adhesion molecule, but cytokines such as TNF-alpha and IL-8 may act subsequently to augment the inflammatory response.

Dietary fish-oil supplementation in humans reduces UVB-erythemal sensitivity but increases epidermal lipid peroxidation.

Ultraviolet radiation (UVR)-induced erythema may be mediated in part by free radical-generated tissue damage, including lipid peroxidation. We have examined the effect of dietary fish oil rich in omega-3 fatty acids upon susceptibility to UVB-induced erythema and epidermal lipid peroxidation. Fifteen volunteers took 10 g fish oil, containing 18% eicosapentaenoic acid and 12% docosahexaenoic acid, daily for 3 or 6 months. Sensitivity to UVB was assessed at intervals on fish oil, and 2.5 months after stopping treatment. Paired skin shave biopsies were taken from six subjects, at baseline and 3 months, from both irradiated and control skin. Fatty acid composition was analyzed and thiobarbituric acid-reactive substances measured as an index of lipid peroxidation. With increasing time on fish oil the minimal erythema dose rose progressively, from 18.9 +/- 13.9 mJ/cm2 (mean +/- SD) at baseline to 41.1 +/- 16.6 mJ/cm2 at 6 months, p < 0.01. Ten weeks after stopping fish oil the minimal erythema dose fell to 23.1 +/- 4.9 mJ/cm2, p < 0.05. Epidermal total omega-3 fatty acids rose from 1.8 +/- 0.4% total fatty acids (mean +/- SEM) to 24.2 +/- 3.9% at 3 months, p < 0.01. This was accompanied by a rise in thiobarbituric acid-reactive substances in irradiated skin from 6 +/- 0.3 (mean +/- SEM) to 18.5 +/- 2.6 A532/g skin, p < 0.01. Hence dietary omega-3 fatty acids produce a pronounced reduction in UVB-erythemal sensitivity, although susceptibility of skin to lipid peroxidation is increased. Thus, omega-3 fatty acids may act as an oxidizable buffer, protecting more vital structures from free radical damage.

Subclass distribution of IgG autoantibodies in bullous pemphigoid.

The distribution of IgG subclasses in the antibasement membrane zone autoantibody of pemphigoid in skin and serum was analyzed by use of monoclonal antibodies to human IgG subclasses. The predominant subclass was IgG4 which was present in 23 of 24 skin biopsies, IgG1 was next and IgG3 was found only occasionally. In 3 of 24 biopsies IgG4 was the only IgG subclass detected, C3 was absent in 2 of these, the third contained IgM and C3. Serum autoantibodies were similarly analyzed by indirect immunofluorescence (IIF) when again IgG4 autoantibody was the dominant subclass. No IgE autoantibody was detected by IIF.

Dietary fish oil reduces basal and ultraviolet B-generated PGE2 levels in skin and increases the threshold to provocation of polymorphic light eruption.

The sunburn response is markedly reduced by dietary fish oil rich in omega-3 polyunsaturated fatty acids. Because prostaglandins mediate the vasodilatation, we examined the effect of fish oil on ultraviolet (UV) B-induced prostaglandin metabolism. In addition we assessed the potential photoprotective effect of fish oil in light-sensitive patients. Thirteen patients with polymorphic light eruption received dietary supplements of fish oil rich in omega-3 polyunsaturated fatty acids for 3 months. At baseline and 3 months, the minimal erythema dose of UVB irradiation was determined, and a graded UVA challenge given to a forearm to assess the threshold dose for papule provocation. Suction blisters were raised on the other forearm, on control skin, and on skin irradiated with four times the minimal erythema dose of UVB 24 h previously, and blister fluid prostaglandin E2 was measured by radioimmunoassay. Following 3 months of fish oil, the mean minimal erythema dose of UVB irradiation increased from 19.8 +/- 2.6 to 33.8 +/- 3.7 mJ/cm2 (mean +/- SEM), p < 0.01. The UVA provocation test was positive in 10 patients at baseline, and after 3 months nine of these showed reduced sensitivity to papule provocation, p < 0.001. Before fish oil, PGE2 increased from 8.6 (SEM 2.1) ng/ml in control skin to 27.2 (11) ng/ml after UVB, p < 0.01. Following 3 months of fish oil, PGE2 decreased to 4.1 (1) and 9.6 (2.4) ng/ml in control and irradiated skin, respectively, p < 0.05. Reduction of UV-induced inflammation by fish oil may be due, at least partially, to lowered prostaglandin E2 levels. The photoprotection against UVA-provocation of a papular response suggests a clinical application for fish oil in polymorphic light eruption.

Ultraviolet-B-induced erythema is mediated by nitric oxide and prostaglandin E2 in combination.

Ultraviolet-B-induced erythema (one, two, or four times the minimal erythema dose) was reduced but not abolished by application of 1% indomethacin gel immediately after irradiation of human skin. Continuous synthesis of prostaglandins is reflected by similar levels of indomethacin-mediated inhibition of erythema at any time within 48 h after irradiation. Repeated applications of indomethacin did not increase the inhibition. Twenty-four hours after irradiation with four minimal erythema doses, mean prostaglandin E2 levels in suction blisters were 27.2 ng per ml (SEM 11) compared with 8.6 ng per ml in unirradiated skin (n = 25; p < 0.01). Prosta glandin E2 levels in dermal tissues, sampled by microdialysis (depth 0.6 +/- 0.1 mm), were 310 pg per ml (SEM 123) and 237 pg per ml (SEM 88) in irradiated and unirradiated skin, respectively (n = 7, n.s.). Nitric oxide also made a significant contribution to ultraviolet-B-induced erythema. Ultraviolet erythema was inhibited by L-NAME in a dose-related fashion with 2 mM L-NAME causing total abolition of the response. L-NAME was effective at all time points up to 48 h suggesting that NO was produced continuously. NO was undetectable in suction blister fluid but in dermal microdialysate NO was present at 44.3 ng per ml (SEM 6.2) following ultraviolet B compared with 26.0 ng per ml (SEM 8.0) in unirradiated skin (p < 0.05), approximately 1000 times the molar concentration of prostaglandin E2. These findings confirm prostaglandin E2 and NO to be mediators of ultraviolet-induced erythema. They also show that there is prolonged synthesis of both mediators within the erythemal response and that synthesis of NO is induced by lower doses of ultraviolet B compared with that of prostaglandin E2.

UVR-induced oxidative stress in human skin in vivo: effects of oral vitamin C supplementation.

Previous studies of cultured skin cells and murine skin in vivo have indicated that UVR-induced damage involves the generation of reactive oxygen species and depletion of endogenous antioxidant systems. In order to explore the relevance of this to UVR-induced damage to human skin, we have undertaken a detailed examination of the time-course of changes in markers of oxidative stress in human skin following exposure to physiological amounts of UVR in vivo. In addition, we have examined the skin bioavailability of a common nutritional antioxidant, vitamin C, and have assessed the effects of supplementation on markers of oxidative stress. Our hypothesis was that acute exposure of human skin to UVR in vivo would lead to oxidation of cellular biomolecules that could be prevented by prior vitamin C treatment. A UVR-challenge of 120 mJ/cm2 of broadband UVB (peak 310 nm, range 270-400 nm) was applied to buttock skin of 8 healthy volunteers. This caused a rapid and significant rise in activity of skin catalase at 1 h and an increase in the oxidized/total glutathione ratio at 6 h post-UVR. AP-1 DNA binding also peaked at 1-6 h post-UVR, then declined rapidly to baseline levels. No significant changes were seen in skin malonaldehyde content. Oral vitamin C supplements (500 mg/day) were taken by 12 volunteers for 8 weeks resulting in significant rises in plasma and skin vitamin C content. Supplementation had no effect on the UVR-induced erythemal response. The skin malonaldehyde content was reduced by vitamin C supplementation, but surprisingly, reductions in the skin content of total glutathione and protein thiols were also seen. We speculate that this apparently paradoxical effect could be due to regulation of total reductant capacity by skin cells, such that vitamin C may have been replacing other reductants in these cells. No evidence was obtained for an effect of the supplementary vitamin C on the mild oxidative stress seen in human skin following UVR exposure.

Quantifying anti-inflammatory agents' potency by measurement of response to dinitrochlorobenzene challenge.

Classical assays of topical corticosteroid potency based on the induction of vasoconstriction are unsatisfactory for a number of reasons. These include the doubtful relevance of vasoconstriction to immune inflammation, and more importantly, the inability to compare non-steroidal agents with corticosteroids. Here we describe a simple assay in which the inhibitory effect of agents upon delayed type hypersensitivity response to dinitrochlorobenzene can be quantified by measurements of reaction as skinfold thickness with Harpenden callipers. Using this system we have confirmed the greater potency of clobetasol propionate (Dermovate) compared with betamethasone valerate (Betnovate), but the evidence for an inhibitory effect of topical cyclosporin (10% cream) compared with base on this response is less convincing.

Oral cyclosporin inhibits the expression of contact hypersensitivity in man.

The expression of delayed contact hypersensitivity was studied in 6 patients with chronic contact dermatitis treated with cyclosporin A (CsA) 5 mg/Kg/day. Quantitative patch test challenge was used to establish individual dose-response curves and threshold concentration to certain allergens in the European Standard Battery. In all 6 patients, responses were reduced over the whole range of allergen concentrations, and in the 5 in whom the threshold for expression of contact hypersensitivity could be determined, the threshold was raised by CsA therapy. In addition, the clinical manifestations of allergic contact dermatitis underwent complete resolution within 2-3 weeks of CsA therapy. It was concluded that CsA inhibits expression of delayed contact hypersensitivity reactions in human skin.

Intracellular calcium modulates the responses of human melanocytes to melanogenic stimuli.

Ultraviolet radiation (UVR), the synthetic diacyglycerol (DAG), 1-oleoyl-2-acetylglycerol (OAG), and cyclic AMP (cAMP) stimulants, including cholera toxin (CT) have all been shown to increase melanogenesis in cultured human melanocytes. Indirect evidence suggests that an increase in intracellular free Ca2+ ([Ca2+]i) may be important in stimulated melanogenesis. Therefore, to determine whether melanogenic responses are modulated by [Ca2+]i, the Ca2+ in the culture medium of melanocytes ([Ca2+]o) was raised from 70 microM to 1 mM. This switch in [Ca2+]o was associated with a biphasic increase in [Ca2+]i, with an early transient rise, over minutes, and a delayed sustained rise in [Ca2+]i, over hours. The early increase was blocked by nickel chloride (NiCl2), but not affected by depletion of [Ca2+]i stores by thapsigargin, suggesting that this [Ca2+]i rise was due to Ca2+ entry across the plasma membrane. Melanocytes cultured in the absence of CT had a reduced basal melanin content following the switch to 1 mM [Ca2+]o, but in the presence of CT, which acts by stimulating cAMP synthesis, the basal level was increased. Raising [Ca2+]o resulted in enhanced melanogenic responses to UVR and OAG, in the presence or absence of CT, suggesting that Ca(2+)-dependent mechanisms are important. UVR also stimulated a delayed rise in [Ca2+]i, over 24 h, but OAG did not. These results indicate that while [Ca2+]i is not essential for melanogenesis, it plays an important role in modulating the responses of melanocytes to melanogenic stimuli.