Topographical distribution of warmth, burning and itch sensations in healthy humans.

To gain information on the topographical distribution of warmth, burning and itch sensations in healthy humans, we delivered laser stimuli to elicit sensations of warmth, applied capsaicin cream for burning, and pricked histamine for itch on the skin of the face, shoulder, hand, thigh and foot in 12 healthy subjects. We found that whereas warm and burning sensations progressively increased from foot to face, itch sensation increased from face to foot (P<0.0001). Hence our findings indicate that unlike thermal and pain receptors, itch receptors are denser at distal than at proximal body sites. Our psychophysical study provides new information supporting the idea that specific unmyelinated neuronal pathways mediate sensations of warmth, burning and itch.

Serum-induced basophil CD63 expression by means of a tricolour flow cytometric method for the in vitro diagnosis of chronic urticaria.

BACKGROUND: Functional autoantibodies against the alpha-chain of the high-affinity IgE receptor (FcepsilonRIalpha) identify a subset of patients with chronic urticaria (CU) due to autoreactivity, as assessed by an in vivo positive response to autologous serum skin test (ASST). We performed a study to standardize the serum-induced basophil activation assay by flow cytometry (FCM) using a new tricolour method, assessing the diagnostic performance of this test in discriminating between ASST+ and ASST- CU patients. METHODS: Sera of 64 CU patients (22 ASST+ CU and 42 ASST- CU) and 10 healthy subjects were tested for their ability to induce basophil CD63 expression when incubated with whole blood of both atopic (DA) and non-atopic donors (DNA). Using a triple-labelled strategy with anti-CD123, anti-HLA-DR and anti-CD63 antibodies, CD63+ basophils were identified on a selected population of CD123+ HLA-DR- cells. In 3 ASST+ CU patients who underwent cyclosporine therapy, the assay was performed before and after treatment. RESULTS: The ASST+ CU sera resulted in a significant higher induction of basophil CD63 expression compared with ASST- CU and healthy donors sera; when whole blood from DA was used, sensitivity and specificity of the assay were 95.5% and 90.5% respectively. ASST+ CU serum activity was significantly decreased during cyclosporine A treatment, in parallel with clinical remission. CONCLUSIONS: Chronic urticaria serum-induced CD63 expression assay performed on DA whole blood by means of our tricolour FCM method could be the most useful tool for identification of a subset of patients with autoimmune CU and may become a promising tool also for monitoring treatment efficacy.

Increased IL-18 in patients with systemic lupus erythematosus: relations with Th-1, Th-2, pro-inflammatory cytokines and disease activity. IL-18 is a marker of disease activity but does not correlate with pro-inflammatory cytokines.

OBJECTIVE: The aim of this study was to investigate the imbalance between Th-1 and Th-2 cytokines in systemic lupus erythematosus patients (SLE) and to asses if any of these cytokines could be related to disease activity. METHODS: Twenty SLE patients and 20 healthy individuals were investigated. Blood samples were collected to evaluate, using ELISA method, serum levels of a wide array of cytokines including: Th-1 type cytokines (Interleukin (IL)-12, Interferon (IFN)-gamma), Th-2 cytokines (IL-4, IL-10), pro-inflammatory cytokines (tumor necrosis factor (TNF)-alpha, IL-1beta and IL-18). Disease activity was assessed using the SLE Disease Activity Index (SLEDAI). Data were evaluated using the Mann-Whitney and Spearman's rank tests. RESULTS: The SLE patients group had a higher IL-4, IL-10, IL-12 and IL-18 serum concentration compared to the normal control group. IL-18 was negatively correlated with IL-4 and positively correlated with IFN-gamma. No serum cytokine level was correlated with disease activity except for IL-18, which was found strongly correlated with "active disease", defined as SLEDAI > 8 points. IL-18 showed no correlation with pro-inflammatory cytokines. CONCLUSIONS: Our results show that Th-1 as well Th-2 cytokines can be elevated in SLE patients suggesting that lupus is a complex disease that may be supported by different cytokine patterns in different time-points. Only IL-18 has been found to be disease-activity related. The role of IL-18 in the pathogenesis of SLE might be important through apoptosis-mediating properties.

POMC and fibroblast biology.

We evidenced in vitro proopiomelanocortin (POMC) mRNA-transcription in human dermal fibroblasts using Northern blot hybridization. Modulation of POMC gene expression by cytokines (transforming growth factor-beta, TGF-beta, and tumor necrosis factor-alpha, TNF-alpha) was investigated by incubating human normal fibroblasts with 1 and 10 ng/ml cytokines, either alone or in combination, for 24 hours. Our results show that dermal fibroblasts express POMC at significant levels under unstimulated conditions. POMC steady-state levels were significantly reduced by addition of TGF-beta. On the other hand, TNF-alpha exerted a stimulatory effect on POMC mRNA transcription, partially counteracting the effect of TGF-beta. These data provide the first demonstration of POMC gene expression in cultured skin fibroblasts. The opposite regulatory effect of TGF-beta and TNF-alpha, two cytokines primarily involved in extracellular matrix regulation, suggests a possible role for POMC-derived peptides in fibroblast activity. We also investigated POMC mRNA expression in keloid-derived fibroblasts in culture, and its regulation by TGF-beta added at the highest concentration documented for inhibition. Keloid-derived fibroblasts showed clearly detectable levels of POMC mRNA in basal conditions, and no alteration of POMC gene expression was observed when TGF-beta was added in culture. The altered TGF-beta regulation of POMC mRNA levels suggest that POMC-derived peptides may play a role in the pathogenesis of keloid formation through an autocrine/paracrine network, resulting in modulation of extracellular matrix synthesis.

The role of proopiomelanocortin-derived peptides in skin fibroblast and mast cell functions.

We have previously described proopiomelanocortin (POMC) gene-expression in human normal cultured dermal fibroblasts, and its dose- and time-dependent modulation by transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha). The aim of the work described here was to investigate POMC-derived peptide release in vitro by cultured fibroblasts following incubation with different concentrations of both TNF-alpha and TGF-beta for 24 hours (1, 5, and 10 ng/ml). The effect of simultaneous addition of both TNF-alpha and TGF-beta (10 ng/ml) was also evaluated. Culture supernatants of human skin fibroblasts were collected to detect adrenocorticotropin hormone (ACTH), alpha-melanotropin (alpha-MSH), and beta-endorphin (beta-EP) levels by specific immunoenzymatic assay. We investigated the in vitro histamine-releasing activity of the POMC-derived peptides, alpha-MSH and beta-EP, on human foreskin mast cells. Detection of cleavage products in supernatants from cultured normal human dermal fibroblasts indicated intracellular processing by POMC protein. We were able to measure detectable levels of all peptides in basal conditions. TNF-alpha addition resulted in an increase in beta-EP and ACTH levels. TGF-beta-stimulated fibroblasts showed no alteration in beta-EP and alpha-MSH levels, whereas ACTH release was significantly enhanced. Both alpha-MSH and beta-EP induced histamine release from human foreskin mast cells in vitro with beta-EP-induced histamine levels as high as those observed with the calcium ionophore, ionomycin. Our data document fibroblast POMC-derived peptide release and modulation by cytokines, suggesting that they have a possible role in extracellular matrix deposit regulation and skin inflammation.

Expression of Bcl-2, p53, c-jun and c-fos protooncogenes in keloids and hypertrophic scars.

Keloids and hypertrophic scars represent a model of altered wound healing characterized by overproduction of extracellular matrix and dermal fibroblasts with high mitotic rate. Alteration of apoptosis and cell proliferation has been implicated in the etiology of keloids. The bcl-2 protooncogene encodes a protein that protects cells from programmed cell death while p53 protein functions as negative regulator of cell proliferation. Both protooncogenes have been shown to play a role in tissue homeostasis as apoptotic regulatory genes. The c-jun and c-fos protooncogenes are transactivating factors also involved in fibroblast proliferation. In our study we investigated, by immunohistochemistry, skin specimens from three clinically active hypertrophic scars and keloids, two resting keloids and two early phase morphea to detect both bcl-2 and p53 protein expression, in order to evaluate these apoptotic regulatory genes in different fibrotic conditions. The c-jun and c-fos, at protein and mRNA level, and Ki67 nuclear antigen expression were also investigated. In hypertrophic scars and active keloids we could detect intense Bcl-2 staining in basal keratinocytes and in scattered fibroblast-like and perivascular spindle-shaped cells, while no p53 expression could be demonstrated. The c-jun and c-fos mRNA and protein expression was mainly found in dermal fibroblast-like cells and elongated perivascular cells in all skin biopsies, and similar immunostaining pattern was observed for Ki67 antigen. No protooncogene expression in morphea patients and normal skin, unless Bcl-2 staining in the basal layer of normal epidermis, was documented. Our results suggest that Bcl-2, c-jun and c-fos protein expression and lack of p53 detection in fibroblast-like and perivascular spindle cells are related to increased fibroblast proliferation, confirmed by Ki67 positivity, probably due to alteration of these regulatory apoptotic genes resulting in pathological scarring.

Propiomelanocortin (POMC) gene expression by normal skin and keloid fibroblasts in culture: modulation by cytokines.

Originally described as a product of the pituitary gland, propiomelanocortin (POMC) has recently been identified in other tissues, such as in human skin, where it may accumulate in response to various stimuli. Thus far, epidermal keratinocytes, as well as melanocytes and macrophages, have been shown to express POMC. This study investigated the expression of POMC mRNA in cultured dermal fibroblasts derived from either normal skin or keloids. Using Northern blot hybridization with a POMC cDNA generated by RT-PCR of mRNA isolated from cardiac muscle, we demonstrated that dermal fibroblasts express POMC, as significant levels of mRNA were detected in unstimulated cells in culture. POMC transcript steady-state levels were strongly reduced by transforming growth factor-beta (TGF-beta), whereas tumor necrosis factor-alpha (TNF-alpha) counteracted the effect of TGF-beta and exerted a stimulatory activity on POMC mRNA levels. Reduction of POMC transcript levels by TGF-beta was also observed in cultured keratinocytes. Clearly detectable levels of POMC mRNA were detected in cultured keloid-derived fibroblasts; however, little, if any, regulation by TGF-beta was observed. These data represent the first demonstration of POMC expression by fibroblasts and down-regulation by TGF-beta. Furthermore, our results indicate altered TGF-beta regulation of POMC gene expression in keloid-derived fibroblasts, suggesting that POMC may play a role in the pathogenesis of keloid formation.

Cyclosporin G inhibits proliferation of A431 cells in a dose- and time-dependent manner comparable to cyclosporin A.

Cyclosporin A (CyA), a fungal metabolite with potent immunosuppressive activity and an antiproliferative effect on epithelial cells, i.e. normal and transformed keratinocytes, is currently proposed in the treatment of psoriasis, where its use is limited mainly by possible nephrotoxicity and/or hepatotoxicity. Numerous analogs of CyA have been produced and studied. The most promising of these is the immunosuppressive analog cyclosporin G (CyG), in which norvaline is substituted for alpha-aminobutyric acid at the 2 position. This would maintain strong immunological activity, with reduced to absent nephrotoxic and hepatotoxic effects. The authors compared the antiproliferative effect of CyG and CyA on the epidermoid carcinoma cell line A431 in vitro, performing the MTT-microculture tetrazolium colorimetric assay based on the ability of viable cells to reduce the MTT compound to a blue formazan product. Subconfluent A431 cells were incubated with CyA or CyG or solvent only, for 24, 48, 72 or 96 h at concentrations of in vivo relevance (0.3, 0.6, 1.25, 2.5, 5, 7.5, 10 micrograms/ml). CyA and CyG showed similar antiproliferative effects, in low-serum-containing media in a dose- and time-dependent manner. After 24 h of incubation, the inhibition of the growth rate was irrelevant. A striking inhibition of the growth rate at the higher concentrations of the drugs (7.5 and 10 micrograms/ml) at 72 and 96 h of incubation was evident. Therefore CyG has been demonstrated to exercise an antiproliferative effect on the A431 cell line. These data suggest possible use for CyG in the treatment of immune-mediated disease, particularly in the treatment of dermatologic diseases characterized by epidermal hyperplasia and/or keratinocyte hyperproliferation.

T-helper 2 involvement in the pathogenesis of bullous pemphigoid: role of soluble CD30 (sCD30).

Bullous pemphigoid (BP) is an autoimmune blistering skin disease in which autoantibodies are directed against hemidesmosomal proteins of basal keratinocytes. The presence of activated T helper cells in lesions and peripheral blood of BP patients, the eosinophilia, the high levels of serum IgE, eosinophil cationic protein and soluble immune products such as IL-2, sIL-2R, IL-5, soluble CD23 (sCD23) strongly suggest the involvement of a cell-mediated immune reaction in which Th2 lymphocytes could play a pivotal role. To seek evidence to support this hypothesis we evaluated serum levels of IL-4 and sCD30, a specific activation marker of cells able to produce Th2-like cytokines, in 25 patients affected with BP. Serum from both healthy donors and pemphigus vulgaris (PV) patients were used as controls. Our results demonstrated significantly higher levels of IL-4 and sCD30 in patients with BP in relation to both normal individuals (16.6 +/- 7.9 vs 4.5 +/- 2.2 pg/ml, P < 0.0001; 30.3 +/- 10 vs 10.5 +/- 4 U/ml, P < 0.0001) and PV patients (6.2 +/- 4 pg/ml, P < 0.0001; 16 +/- 8.5 U/ml, P < 0.0001). Furthermore, a positive correlation between IL-4 and sCD30 was found (R = 0.85, P < 0.0001). In a subset of seven patients observed after systematic immunosuppressive therapy, we detected a significant reduction in sCD30 serum level (36.9 +/- 7.3 vs 16.3 +/- 6.8 U/ml, P = 0.002), with a parallel improvement in their clinical condition. These results seem to be consistent with the systematic involvement of Th2 lymphocytes in the pathogenesis of BP and suggest a role for sCD30 as a serological marker of disease activity.

Lack of sympathetic involvement in dermatitis confined to the median nerve territory. A case report.

Both decreased and increased sympathetic nerve activity has been suggested as a possible underlying mechanism in inflammatory skin lesions. Modulation of sympathetic function has been proposed in the treatment of dermatitis. This case report describes the investigation strategy and normal findings in a case of dermatitis strictly confined to the median nerve territory, illustrating the need for specific tests of sympathetic function when pharmacological as well as physical sympatho-modulatory therapies are considered.

Cyclosporine A inhibits tPA mRNA transcription in A431 cell line.

Cyclosporine A (CyA), a well established treatment of psoriasis, is a highly lipophilic cyclic undecapeptide mainly used for its immunosuppressive properties and exerting a wide spectrum of biological activities including fungicidal antiproliferative and anti-inflammatory effects. Plasminogen activators (PA), urokinase (UK, M(r) 55,000) and tissue type plasminogen activators (tPA, M(r) 74,000), physiologically catalyze the conversion of the plasminogen to the wide spectrum proteinase plasmin. UK and tPA are involved in cell growth, differentiation and migration. It has recently been shown that psoriatic epidermis is provided with abnormal tPA-dependent PA activity and that in lesional epidermis elevated tPA mRNA levels are present. It has been suggested that the tPA-dependent PA activity is a marker of disease activity and is reversible with different topical and systemic treatments. In this preliminary study we investigate the effect of CyA on the tPA mRNA transcription on A431 keratinocytes cell line. Subconfluent A431 cell cultures have been treated with CyA at in vivo relevant concentrations (10, 7.5, 5 micrograms/ml) for 48 h. Northern blot analysis of total RNA extracted from cultured A431 cell line has been performed for detecting tPA mRNA. mRNA for tPA has been detected in the control samples whereas an evident decrease of tPA mRNA expression has been detected in the CyA-treated samples. These data suggest that CyA could have an effect in clearing psoriatic lesions also modulating the abnormal plasminogen activation i.e. tPA-dependent serinoproteinase activity.

Cytokines, fibrinolysis and vasculitis.

Endothelial cells are critical elements in the evolution of all types of cutaneous inflammation. They participate the pathological process through the synthesis and secretion of pro-inflammatory cytokines, including interleukin 1 (IL1), IL6, IL8, and the three colony stimulating factors G-CSF, M-CSF, and GM-CSF and the two chemotactic factors gro-alpha and MCP. They also express a series of cell-surface proteins and glycoproteins known as cell adhesion molecules that allow circulating leukocytes to selectively bind to endothelial cells. In this paper we discuss the role of endothelial cells in the evolution of cutaneous necrotizing vasculitis, an immunologically mediated clinical disorder associated with segmental inflammation and fibrinoid necrosis of the dermal venules, through the release of cytokines or their response to cytokines locally produced from leukocytes themselves primarily involved in the endothelial cells injury. This interaction seems to involve and modulate other biologically active systems including the fibrinolytic system that can act amplifying and self-perpetuating the tissue damage through a non-immunologic mechanism.

Ultrastructural study of hyaluronic acid before and after the use of a pulsed electromagnetic field, electrorydesis, in the treatment of wrinkles.

BACKGROUND: Treatment of wrinkles has become an increasing problem for dermatologists. Hyaluronic acid is a component of the family of glycosaminoglycans (GAGS, substances known for their property of retaining water), that significantly decreases with aging and in wrinkles. A new technique that uses a specific pulsed electromagnetic field, electrorydesis, has been introduced in the treatment of wrinkles associated with aging. The treatment is based on the reported in vitro effects of specific electromagnetic fields on fibroblast cultures (e.g., an increase in DNA synthesis and in the production of collagen and presumably also of GAGS). METHODS: The in vivo effects of the electromagnetic field on aged skin (3 subjects aged 50, 56 and 60 years), with particular focus on the ultrastructural modifications and GAGS amount before and after the treatment, were evaluated by electron microscope. RESULTS: The ultrastructural study (tissue stained with alcian blue) showed after treatment a significant increase (p < 0.005) of the electron-dense granules (corresponding to hyaluronic acid), located in collagen elastic fibers, and in the soluble matrix. This presumably leads to subsequent edema that was clinically evident after the treatment. CONCLUSIONS: These data suggest that the increased levels of GAGS and the subsequent edema of the dermis could explain at least in part the clinical changes observed after electrorydesis treatment (e.g., swelling and "disappearance" of the wrinkle).

Treatment of aquagenic pruritus with topical capsaicin cream.

BACKGROUND: Aquagenic pruritus is characterized by pruritus after contact with water; there are no objective cutaneous changes. Capsaicin, which induces the release of neuropeptides from A delta and C cutaneous nerve fibers, has been successfully used in the treatment of several dermatoses associated with pruritus. Among the many different neuropeptides present in human skin, the undecapeptide substance P has been shown to cause pruritus. OBJECTIVE: We evaluated the clinical effect and searched for alterations in cutaneous neuropeptidergic fibers before and after treatment with capsaicin cream. METHODS: Five patients with aquagenic pruritus were treated with capsaicin cream 0.025%, 0.5% or 1.0% three times daily for 4 weeks. Direct immunofluorescence (DIF) was performed before and after treatment to evaluate the storage of neuropeptides in the A delta and C type cutaneous nerve fibers. RESULTS: Before treatment (when by DIF the neuropeptidergic fibers appeared filled with neuropeptides), contact with water consistently provoked itching. After capsaicin treatment (when by DIF the neuropeptidergic fibers were depleted of neuropeptides), contact with water did not evoke pruritus. Areas of skin treated with the vehicle alone showed no clinical improvement or change in neuropeptide content. CONCLUSION: This study suggests that neuropeptides, including substance P, may contribute to mediating the itch in aquagenic pruritus.