Work in progress: Prostaglandin activity in human eccrine sweat

Thermal sweat collected from 14 healthy volunteers contained prostaglandin activity ranging from 0.2 to 1.5 ng of prostaglandin E₂ equivalents per milliliter (mean ± SE; 0.62 ± 0.43) as measured by a prostaglandin-specific radioimmunoassay. There was no statistically significant difference in the mean activity between males and females. The activity in sweat from two of five patients with atopic dermatitis, in one of four patients with psoriasis, and in one of two patients with hyperhidrosis was significantly ( ) above the mean activity in the sweat of the healthy volunteers. The importance of this prostaglandin activity is uncertain, but prostaglandins may participate in the regulation of sweat electrolytes, as suggested by their influence on electrolyte fluxes in the kidney and gut.     

Study of elastase-type activity in blister fluids of recessive dystrophic epidermolysis bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is characterized clinically by blister formation due to minor trauma and ultrastructurally by a progressive disappearance of anchoring fibrils at the dermoepidermal junction and of the oxytalan-type fibers which belong to the elastic fiber system. In this study, we determined the elastase-type activity in blister fluid obtained from 8 patients suffering from RDEB as compared to the suction fluid of experimental blisters in a healthy person and to the blister fluid of a patient suffering from epidermolysis bullosa simplex. One patient with dominant dystrophic epidermolysis of the albopapuloid type was also studied. Seven of the eight children with RDEB showed highly elevated values. The eighth child, treated with etretinate, as well as the patient suffering from dominant epidermolysis bullosa had moderately increased values. The determination of elastase-type activity in the blister fluid could therefore be useful to establish the differential diagnosis of recessive dystrophic epidermolysis bullosa.     

Identification of histamine releasing factor(s) in the late phase of cutaneous IgE-mediated reactions

We have shown that fluids collected from antigen-challenged skin blisters during the late phase reaction cause the release of substantial amounts of histamine (means = 42%, n = 14) from human basophils in vitro. Control fluids collected either during the immediate phase or from an unchallenged blister released less than or equal to 10% histamine from both basophils and lung mast cells. Late phase blister fluids induced low levels of histamine release from human lung cells (means = 11%, n = 4) that were slightly but not significantly greater than levels induced by control blister fluids. The characteristics of basophil release were similar to IgE-mediated stimuli in dose dependence, calcium and temperature requirements, and kinetics. The IgE dependence of the late phase blister fluid was demonstrated by desensitization of the basophils to anti-IgE, which obviated the response to anti-IgE and blister fluid but did not affect a non-IgE-mediated stimulus. Removal of the cell surface IgE with lactic acid also abolished the response to both anti-IgE and late phase blister fluid. Incubation of the "stripped" cells with serum containing IgE myeloma restored the response to anti-IgE but failed to affect response to late phase blister fluid. The characteristics of release obtained with this factor closely resemble those of an IgE-dependent histamine releasing factor from cultured macrophages previously described by our group.     

Hemidesmosomal Molecular Changes in Dermatitis Herpetiformis; Decreased Expression of BP230 and Plectin/HD1 in Uninvolved Skin

Recent BP230-knockout experiments with subsequent blistering and recently identified plectin/HD1 mutations in epidermolysis bullosa simplex patients suggest that defective expression of BP230 and plectin/HD1 may predispose to blister formation in human skin. We have studied the expression of the epithelial adhesion complex as well as the basement membrane and anchoring fibril antigens in uninvolved dermatitis herpetiformis skin to find out if alterations can be detected in these structures predisposing to the blister formation typical of the disease. Ten uninvolved dermatitis herpetiformis skin specimens, which all showed clear granular deposits of IgA under the basement membrane in direct immunofluorescence and five normal skin specimens, were studied by indirect immunofluorescence technique. Six uninvolved dermatitis herpetiformis skin specimens showed distinctly decreased immunoreaction for BP230 and four uninvolved dermatitis herpetiformis skin specimens showed distinctly decreased immunoreaction for plectin/HD1. All five skin controls showed strong immunoreactions for BP230 and plectin/HD1. Other hemidesmosomal proteins including BP180 and integrin 64, as well as basement membrane proteins laminin-5, laminin-1, nidogen and type IV collagen, and the anchoring fibril protein type VII collagen showed a normal strong expression. Our results suggest that alterations in BP230 and plectin/HD1 may contribute or predispose to blister formation in dermatitis herpetiformis skin.     

The Redox State of Glutathione,Cysteine and Homocysteine in the Extracellular Fluid in the Skin

Glutathione, the most abundant low-molecular weight thiol in the skin, has been shown to protect the skin from both photobiological and chemical injury. The thiols, glutathione in particular, have also been shown to be crucially involved in defence against contact allergens. Since the levels of extracellular thiol concentrations are important determinants of intracellular thiol status, we have compared the normal concentrations and the redox status of the main low-molecular weight thiol components in the extracellular fluid at the dermo-epidermal junction with the corresponding plasma levels. In their sulfhydryl form, all three thiols, i.e. glutathione, cysteine and homocysteine, were more abundant in experimental skin blister fluid than in plasma, as were the free disulfides of glutathione and homocysteine, whereas the free disulfides of cysteine were about the same in blister fluid and in plasma. Protein mixed disulfide levels were higher in plasma than in blister fluid. The present results provide information concerning the extracellular defence in the skin.     

The redox state of glutathione, cysteine and homocysteine in the extracellular fluid in the skin

Glutathione, the most abundant low-molecular weight thiol in the skin, has been shown to protect the skin from both photobiological and chemical injury. The thiols, glutathione in particular, have also been shown to be crucially involved in defence against contact allergens. Since the levels of extracellular thiol concentrations are important determinants of intracellular thiol status, we have compared the normal concentrations and the redox status of the main low-molecular weight thiol components in the extracellular fluid at the dermo-epidermal junction with the corresponding plasma levels. In their sulfhydryl form, all three thiols, i.e. glutathione, cysteine and homocysteine, were more abundant in experimental skin blister fluid than in plasma, as were the free disulfides of glutathione and homocysteine, whereas the free disulfides of cysteine were about the same in blister fluid and in plasma. Protein mixed disulfide levels were higher in plasma than in blister fluid. The present results provide information concerning the extracellular defence in the skin.     

Enrichment and function of urushiol (poison ivy)-specific T lymphocytes in lesions of allergic contact dermatitis to urushiol

The frequencies of urushiol (poison ivy)-specific T cells were determined in the lesional skin and peripheral blood of patients with allergic contact dermatitis to urushiol. Lesions of urushiol dermatitis were biopsied and the T cells retrieved. Frequencies of Ag-specific cells were determined by limiting dilution assay. Enrichment of urushiol specific T cells was detected in lesional skin or blister fluid of five of five of these donors as well as seven of seven donors with urushiol patch tests. There was also enrichment relative to tetanus toxoid specific T cells in blood and skin of six of seven donors. The frequency of lesional T cells specific for urushiol was less than 1/100 and generally less than 1/1000. Furthermore, the frequency of urushiol specific T cells was often less than 1/10,000 in the peripheral blood during an acute eruption. Autoreactive T cells that proliferated to autologous irradiated PBMC were also enriched in skin lesions relative to blood in 5/5 urushiol patch tests. These autoreactive T cells were expanded and found to be CD4+. It has been proposed that such autoreactive T cells recognize autologous DR and may have a role in immunoregulation. Urushiol-specific T cells were expanded from limiting dilution wells of two donors and characterized. The majority of the urushiol-specific cells were CD8+, and 13/43 lines were suppressive for PWM-induced IgG synthesis.     

Prostacyclin,thromboxane and prostaglandin F2αin suction blister fluid of human skin: Effect of systemic aspirin and local glucocorticosteroid treatment

Changes in prostaglandin (PG) metabolism are known to be involved in various skin diseases. To elucidate the behavior of hree vasoactive PGs in human skin, namely prostacyclin (PG1₂), thromboxane A₂ (TxA₂) and PGF_2α, their stable metabolites, 6-keto-PGF_1α. TxB₂, and 13, 14 dihydro-15-keto PGF_2α (MPGF_2α), respectively, were measured by radioimmunoassays in suction blister fluids of 29 healthy male subjects. Nine of them were treated with acetylsalicylic acid (0.5 g × 4/day for one day beforehand), eight with local glucocorticoid (clobetasol-17-propionate, Dermovat^R Cream, twice a day for seven days) and 12 served as controls. All three PGs were detected in blister fluid. In controls the mean (±SD) concentration of 6-keto-PGF_1α was 1160 ± 470 pg/ml (n=12) that of TxB₂ 1590 ± 610 pg/ml (n=12) and that of MPGF_2α 1800 ± 710 pg/ml (n=12), levels which are higher than the respective concentrations in human plasma. The preceding aspirin treatment decreased the 6-keto-PGF_1α levels by 40 % (P<0.005), the TxB₂ levels by 80 % (P<0.001) and MPGF_2α levels by 35 % (P<0.05), whereas the preceding local glucocorticoid caused no changes in these PG levels. The results show that 1) PG1₂, TxA₂, and PGF_2α are locally released in the suction blister fluid of healthy human skin, 2) systemic treatment with a PG synthesis inhibiting drug, acetysalicylic acid, reduces this release, and 3) locally applied clobetasol-17-propionate does not affect the levels of prostaglandins and thromboxane as measured by our methods.     

Chitosan: antimicrobial action upon staphylococci after impregnation onto cotton fabric

Background: High levels of viable Staphylococcus aureus, which are often found on inflamed skin surfaces, are usually associated with atopic dermatitis. Textiles, owing to their high specific surface area and intrinsic hydrophilicity, retain moisture while also providing excellent environmental conditions for microbial growth and proliferation. Recently, a number of chemicals have been added to textiles, so as to confer antimicrobial activity. Aims: To evaluate the antimicrobial action of chitosan upon selected skin staphylococci. Methods and Results: We isolated staphylococci from normal skin of 24 volunteers and studied their survival upon contact with chitosan‐impregnated cotton fabric. Low and high molecular weight chitosans were used at two concentrations; all four did effectively reduce the growth of some staphylococci (namely Staph. aureus), by up to 5 log cycles, thus unfolding a potential towards control and even prevention of related skin disorders. Conclusion: Our data suggest an effective, but selective antibacterial action of chitosans towards skin bacteria. Significance and Impact of the Study: The possibility to use a natural biopolymer incorporated in a textile to alleviate and even treat some of the symptoms associated with this skin condition may raise an alternative to existing medical treatments. The selectivity observed prevents full elimination of bacteria from the skin surface, which is an advantage.     

Suction blister fluid as potential body fluid for biomarker proteins

Early diagnosis is important for effective disease management. Measurement of biomarkers present at the local level of the skin could be advantageous in facilitating the diagnostic process. The analysis of the proteome of suction blister fluid, representative for the interstitial fluid of the skin, is therefore a desirable first step in the search for potential biomarkers involved in biological pathways of particular diseases. Here, we describe a global analysis of the suction blister fluid proteome as potential body fluid for biomarker proteins. The suction blister fluid proteome was compared with a serum proteome analyzed using identical protocols. By using stringent criteria allowing less than 1% false positive identifications, we were able to detect, using identical experimental conditions and amount of starting material, 401 proteins in suction blister fluid and 240 proteins in serum. As a major result of our analysis we construct a prejudiced list of 34 proteins, relatively highly and uniquely detected in suction blister fluid as compared to serum, with established and putative characteristics as biomarkers. We conclude that suction blister fluid might potentially serve as a good alternative biomarker body fluid for diseases that involve the skin.     

Radioautography of Noradrenaline-14C in Atopic Dermatitis

Since storage-time of administered noradrenaline in the skin of patients with atopic dermatitis may be prolonged, it would be of interest to demonstrate the site of uptake of noradrenaline-¹⁴C in atopic dermatitis as compared with other eczematous and normal skins. Two adult patients with longstanding atopic dermatitis, a patient with contact dermatitis to nickel and one with normal skin, were studied. Identical sites in the four patients were injected intradermally with 0.02 μg. DL-noradrenaline-7-¹⁴C acetate. An 8-mm. punch biopsy of the injected site was performed 24 hours later. Radioautographs were developed between three and 199 days, according to the technique of Kopriwa and Leblond.² At 199 days, the number of grains in atopic dermatitic skin was greater than in contact dermatitis or normal skin. There was a concentration of grains over arrectores pilorum muscles and the upper one-third of the epidermis of atopic skin. Grains were also visible in proximity to arteriolar walls. There were few grains visible in the control sections. The results confirm earlier studies suggesting that atopic dermatitic skin retains noradrenaline longer than other dermatoses. Noradrenaline concentrates in the arrectores pilorum muscles and the upper epidermis. These findings may explain the cutis anserina (goose-flesh) appearance in atopic dermatitis.     

Epidermal necrolysis: mechanisms of keratinocyte apoptosis

Toxic epidermal necrolysis and Stevens-Johnson syndrome are acute and severe adverse reaction to drugs, characterized by the widespread destruction of the epithelium of the skin and mucous membranes. This destruction by massive apoptosis leads to a clinical pattern of epidermal necrolysis resembling second degree burns, with sheets of necrotic epidermis detached from the underlying dermis. The mechanisms of acute and extensive destruction of the skin are not yet fully understood. At the onset of the reaction blisters develop from the fluid that accumulates between the dead epidermis and the dermis. High concentrations of mononuclear cells are present in this blister fluid, principally CD8 T-lymphocytes that may exhibit a drug specific MHC class I restricted cytotoxicity against autologous cells. The intervention of soluble mediators such as TNFalpha, perforin/granzyme, or Fas-Ligand may be necessary for amplifying the apoptosis of keratinocytes. A strong association between epidermal necrolysis to certain drugs and rare HLA-B genotypes suggests that direct interaction between these drugs and HLA-B molecules may initiate a reaction resembling the acute rejection of allogeneic epidermis.     

The blister and the second degree burn in guinea pigs: the effect of exposure.

A reproducible partial-thickness burn was inflicted on 100 albino guinea pigs. With the blister intact, the evaporative water loss from the burn surface was essentially the same as from normal unburned skin. When the blister was removed, the rate of water loss was initially more than 100 times normal, and it remained 20 to 50 times the normal rate throughout the first week. This high rate of water loss was associated with an increasing depth of wound destruction (from dehydration) and a thickening of the overlying crust. Microscopic examination showed that previously viable dermis was incorporated in this crust. The depth of dermal loss was negligible when the blister was left intact, and improved healing resulted. We believe it is important to leave burn blisters intact, whenever possible.     

Therapeutic Efficacy and Immunological Response of CCL5 Antagonists in Models of Contact Skin Reaction

Skin-infiltrating T-cells play a predominant role in allergic and inflammatory skin diseases such as atopic dermatitis, psoriasis and allergic contact dermatitis. These T-cells are attracted by several chemotactic factors including the chemokine CCL5/RANTES, a CC chemokine inducing both the migration and activation of specific leukocyte subsets. CCL5 has been found to be associated with various cell-mediated hypersensitive disorders such as psoriasis, atopic dermatitis and irritant contact dermatitis. We have used two antagonists, the first, Met-CCL5, a dual CCR1/CCR5 antagonist and the second, a variant in which GAG binding is abrogated, ⁴⁴AANA⁴⁷-CCL5, which acts as a dominant negative inhibitor of CCL5. The antagonists were tested in two models of contact skin reaction. The first, irritant contact dermatitis (ICD) is a pathological non-specific inflammatory skin condition arising from the release of pro-inflammatory cytokines by keratinocytes in response to haptens, usually chemicals. The second, contact hypersensitivity (CHS) is a T-cell dependent model, mimicking in part the T-cell-mediated skin diseases such as psoriasis. In both models, the CCL5 antagonists showed therapeutic efficacy by reducing swelling by 50% as well as the reduction of soluble mediators in homogenates derived from challenged ears. These results demonstrate that blocking the receptor or the ligand are both effective strategies to inhibit skin inflammation.     

Blistering in keratinocyte cultures: a regular phenomenon associated with differentiation

Blisters have previously been observed in keratinocyte cultures depleted of vitamin A, and in cultures of keratinocytes from patients with epidermolysis bullosa. We have found that blistering may occur in keratinocyte cultures from normal human epidermis, grown under standard conditions, and our aim was to further characterize the mechanism of blister formation. Keratinocytes were seeded at 10(5) cells per 35 mm collagen-coated dish with a 3T3 feeder layer. Blisters were macroscopic, fluid-filled structures which formed irrespective of donor site, or donor age, and were noted on various alternative substrates (collagen, 3T3 + plastic, plastic alone). Blistering commenced around day 12, prior to confluency, and new blisters were formed for up to 5 weeks post-plating. Maximal numbers (up to 70 per dish) were present around days 12 to 20. Cleavage occurred at the cell/collagen interface to form a blister roof composed of 6 to 9 cell layers. The lowest layer appeared metabolically active, but, in contrast to peri-blister regions, lacked hemidesmosomes. The central 2 to 3 layers contained membrane-coating granules and keratohyalin granules while the superficial strata resembled rudimentary corneocytes. Cultures supplemented with 10(-5) M vitamin A formed no blisters, which correlated with suppressed differentiation. Ouabain (10(-7) M) caused blister collapse and a reversible inhibition of new blister formation. We conclude that blisters are a consistent finding in keratinocyte cultures grown under standard conditions. Their formation may be associated with active transport and triggered during differentiation. Further examination of this phenomenon might shed light on whether differentiation itself has an influence on keratinocyte attachment to substrate.     

Opsonic Activity of Sera and Blister Fluid from Severely Burned Patients Evaluated by a Chemiluminescence Method

Burn wound sepsis is the most common and severe complication in the patients with severe burn. To know the systemic and local defect in immunity of burned patients, we measured the luminol-enhanced chemiluminescence (CL) response of normal polymorphonuclear leukocytes (PMNs) upon exposure to zymosan particles, bacteria or Candida albicans that were opsonized with any of patient's serum, blister fluid of burn wound or pooled normal serum (blood type AB). Sera from patients exhibited lower opsonic activities than those of pooled normal serum in the early postburn days. The levels of serum immunoglobulins, complement components and plasma fibronectin were found to correlate well with opsonin-index (OI), which was determined based on the CL response data obtained during the course of infusion therapy with fresh frozen plasma. Furthermore, patient's blister fluid showed much lower opsonic activity against bacteria such as Pseudomonas aeruginosa than patient's own serum. These results indicate that blister fluid is also not effective to opsonize bacteria because of the marked depression of the levels of immunoglobulins and complement components. Destruction of the skin barrier by thermal injury and impairment of systemic or local humoral immunity may predispose these patients to burn wound sepsis.     

A Novel Investigation of a Blister-Like Syndrome in Aquarium Echinopora lamellosa

This study investigates potential causes of a novel blister-like syndrome in the plating coral Echinopora lamellosa. Visual inspections of this novel coral syndrome showed no obvious signs of macroparasites and the blisters themselves manifested as fluid-filled sacs on the surface of the coral, which rose from the coenosarc between the coral polyps. Histological analysis of the blisters showed that there was no associated necrosis with the epidermal or gastrodermal tissues. The only difference between blistered areas and apparently healthy tissues was the presence of proliferated growth (possible mucosal cell hyperplasia) directly at the blister interface (area between where the edge of the blister joined apparently healthy tissue). No bacterial aggregates were identified in any histological samples, nor any sign of tissue necrosis identified. We conclude, that the blister formations are not apparently caused by a specific microbial infection, but instead may be the result of irritation following growth anomalies of the epidermis. However, future work should be conducted to search for other potential casual agents, including viruses.     

A PATTERN OF EPIDERMAL CELL MIGRATION DURING WOUND HEALING

Epidermal repair during wound healing is under investigation at both the light and electron microscopic levels. Suction-induced subepidermal blisters have been employed to produce two complementary model wound healing systems. These two model systems are: (a) intact subepidermal blisters, and (b) opened subepidermal blisters (the blister roof was removed immediately after induction, leaving an open wound). From these studies a pattern of movement for epidermal cells in wound healing is proposed. This pattern of movement is the same for both model systems. Epidermal cells appear to move by rolling or sliding over one another. Fine fibers oriented in the cortical cytoplasm may play an important role in the movement of these epidermal cells. Also instrumental in mediating this movement are intercellular junctions (desmosomes) and a firm attachment to a substrate through hemidesmosomes. In the intact subepidermal blisters hemidesmosomal attachment is made to a continuous and homogeneous substrate, the retained basal lamina. In the opened subepidermal blisters contact of epidermal cells is made to a discontinuous substrate composed of sporadic areas of fibrin and underlying mesenchymal cells.     

Prostaglandins and skin tumor promotion: inhibition of tumor promoter-induced ornithine decarboxylase activity in epidermis by inhibitors of prostaglandin synthesis.

Application of 12-O-tetradecanoylphorbol-13-acetate to mouse skin led to a dramatic induction of epidermal ornithine decarboxylase (EC 4.1.1.17; L-ornithine carboxy-lyase) activity. The degree of induction was remarkably depressed by prior treatment of skin with indomethacin, acetylsalicylic acid or flufenamic acid, inhibitors of prostaglandin synthesis. In contrast, dexamethasone, a steroidal anti-inflammatory drug, was ineffective. The inhibition of tumor promoter-induced ornithine decarboxylase activity by the non-steroidal anti-inflammatory drugs was completely counteracted by treatment with prostaglandin E₁ and E₂ but not with prostaglandin F_1α or F_2α.     

Hair follicle stem cell progeny heal blisters while pausing skin development

Injury in adult tissue generally reactivates developmental programs to foster regeneration, but it is not known whether this paradigm applies to growing tissue. Here, by employing blisters, we show that epidermal wounds heal at the expense of skin development. The regenerated epidermis suppresses the expression of tissue morphogenesis genes accompanied by delayed hair follicle (HF) growth. Lineage tracing experiments, cell proliferation dynamics, and mathematical modeling reveal that the progeny of HF junctional zone stem cells, which undergo a morphological transformation, repair the blisters while not promoting HF development. In contrast, the contribution of interfollicular stem cell progeny to blister healing is small. These findings demonstrate that HF development can be sacrificed for the sake of epidermal wound regeneration. Our study elucidates the key cellular mechanism of wound healing in skin blistering diseases.