Biochemical composition of suction blister fluid determined by high resolution multicomponent analysis (capillary gas chromatography--mass spectrometry and two-dimensional electrophoresis).

The biochemical composition of blister fluid was compared with serum and with blister fluid from erythematous lesions induced by ultraviolet irradiation. By using glass capillary gas chromatography--mass spectrometry over 100 metabolites were determined and with the aid of two-dimensional high resolution electrophoresis (the ISO-DALT system) several hundred protein spots were seen. The results show that the suction blister fluid qualitatively have a serum-like pattern but that the concentration of each compound was smaller than in serum. Also in suction blisters raised on erythematous reactions induced by ultraviolet light the same pattern was seen. The content of sodium, potassium and chloride was the same in suction blisters raised on erythematous and normal skin as that of serum.

Genotypic and phenotypic markers in the differentiation of Neisseria gonorrhoeae strains.

Restriction enzyme (RE) patterns, using Hind III enzyme, were analysed on 101 urogenital strains of Neisseria gonorrhoeae. The strains were also analysed serologically, using monoclonal antibodies, and tested for nutritional requirements by auxotyping. In addition, testing susceptibility to doxycycline was performed by an agar-dilution method. The strains were distributed into 23 RE patterns, of which the five most common accounted for 67.3% of all isolates. The same strains were distributed into 17 serovars, of which the four most common accounted for 77.3% of all isolates. Eleven auxotypes were demonstrated, of which the four most common accounted for 85.1% of all isolates. When the methods were combined, 38 combinations of RE patterns and serovars, 25 combinations of RE patterns and auxotypes, and 31 combinations of serovars and auxotypes were seen. Combining all three methods, 40 combinations of RE patterns, serovars and auxotypes were found. Correlations were particularly seen between RE patterns and auxotypes. The strains were moderately sensitive to doxycycline, those with serological markers for WII/WIII being less sensitive than those with markers for WI.

Acid hydrolases in experimental and spontaneous bullae.

The activities of eight acid hydrolases have been measured in blister fluid from various bullous disorders and from experimental suction blisters. The levels of all enzymes increased dramatically for at least 24 h after induction of the blister. No correlation was found between acid hydrolase activity and any individual disease. The levels of activity in spontaneous bullae were similar to those reached after 24 h in experimental suction blisters which had been raised on previously inflamed skin. We conclude that the release of acid hydrolases may be a consequence rather than the cause of blister formation. This conclusion is supported by the lack of inflammatory response to the i.c. injection of blister fluid into normal skin.

Flow cytometry (FCM) of human epidermal cells. A preparation method for epidermal cells and demonstration of circadian variations in the proportion of S-phase cells.

A suction blister technique on the abdominal skin to separate pure human epidermis from its basement membrane is described. After incubation with proteolytic enzymes (trypsin, pepsin) and mechanical shaking, isolated cells and nuclei were stained with ethidium bromide. DNA-specific fluorescence was measured in a flow cytometer using UV excitation light. Cell-cycle phase distributions were obtained from the resulting histograms using a planimetric method. The proportion of cells with an S-phase DNA content showed circadian variations with peak values shortly before noon, and lower values during the rest of the 24 h period.

DNA replication in short-time organ cultures of human epidermis. Inhibition by aphidicolin, and detection of DNA polymerases alpha, beta, and gamma.

Human epidermis uncontaminated by fibroblasts was isolated by a suction blister method. DNA synthesis in short-time organ cultures of isolated epidermis was strongly inhibited by aphidicolin, suggesting that DNA polymerase alpha is involved in DNA replication in human epidermis. On the basis of their responses to inhibitors, primer-template requirements, and chromatographic properties, DNA polymerases alpha, beta, and gamma were all identified in epidermal extracts.

A simple assay for determining keratin and collagen degradation in vitro.

A simple assay measuring degradation of human epidermal keratin and bovine tendon collagen is presented. Insoluble protein substrate (30 mg) was incubated with 1 ml buffer and enzyme sample for 1 h at 37 degrees C, following addition of 1 ml distilled water and removal of the remaining substrate by filtration/centrifugation. The protein content was determined in the filtrate/supernatant by the Lowry method. Keratin was prepared as follows: Freeze-drying, homogenization in a mortar or beetling mill, extraction in 0.9% (w/v) NaCl followed by water and 100% ethanol, drying at 37 degrees C. The assay was tested with pig pepsin, bovine trypsin, and crude extract of fish stomach, demonstrating that these preparations are effective in degrading human epidermal keratin.

Degradation of human epidermal keratin by fish pepsin.

Stomach extract of Atlantic herring Clupea harengus, Atlantic salmon Salmo salar, cod Gadus morhua, redfish Sebastes marinus, and plaice Pleuronectes platessa, degraded human epidermal keratin effectively in vitro. The keratin-degrading activity of all extracts showed a pH optimum around 3.3-3.4, and sheets of plantar callus were degraded with about the same efficacy as keratin. Pepstatin sensitivity, heat lability, and the acidic pH optimum demonstrated that the keratin-degrading activity was pepsin. The keratin-degrading activity of cod stomach extract had a temperature optimum of around 42 degrees C at optimal pH, and showed a similar pH dependency with collagen as with keratin as substrate. The keratin-degrading activity of pepsin I and pepsin II purified from cod showed a pH optimum of 3.7 and 3.1, respectively, similar to that obtained with hemoglobin as substrate. Pig pepsin showed a pH optimum of about 2 with keratin, hemoglobin, and collagen as substrates. The present investigation demonstrates that fish pepsin is effective in degrading human epidermal keratin in vitro, and in a contemporary study the same was shown with fish trypsin. This may suggest a possible mechanism for the development of irritative hand eczema caused by exposure to fish and acidified fish material.

Degradation of human epidermal keratin by cod trypsin and extracts of fish intestines.

Cod Gadus morhua and bovine trypsin degraded human epidermal keratin with similar efficacies in vitro around optimal pH, which was at pH 8.4 for cod trypsin and at pH 9.5 for bovine trypsin. Extract of intestines of cod, Atlantic herring Clupea harengus, Atlantic salmon Salmo salar, and redfish Sebastes marinus degraded keratin with similar efficacies with pH optima between 8.5 and 9.5. Sheets of plantar callus were degraded with somewhat lower efficacy than keratin. The keratin-degrading activity of extract of cod intestines had a temperature optimum around 45 degrees C. Inhibition with benzamidine and 4-phenylbutylamine showed that trypsin amounted to more than 2/3 of the keratin-degrading activity in all extracts of fish intestines. Apart from cod intestines, which had the lowest chymotrypsin content, chymotrypsin made a smaller but significant contribution to the keratin-degrading activity. The present investigation demonstrates that fish trypsin and extract of fish intestines are effective in degrading human epidermal keratin in vitro, and in a recent investigation the same was shown with fish pepsin. This may suggest a possible mechanism for the development of irritative contact eczema caused by exposure to fish.

Reversible cellular damage by dimethyl sulfoxide reflected by release of marker enzymes for intracellular fractions.

Irritative human skin reactions were induced by dimethyl sulfoxide (DMSO). Suction blisters were raised on these areas within 1.5 h after their induction and simultaneously on normal skin. The activities of marker enzymes for subcellular fractions in the suction blisters were determined. In suction blisters raised on the DMSO induced wheals significantly higher values of the cytosol enzyme lactate dehydrogenase and also some higher values of the lysosomal marker alpha-mannosidase were found than in blisters produced on normal skin. Membrane-bound marker enzymes for subcellular fractions were not elevated. Since the skin is macroscopically completely normal 24 h after application of DMSO, our results indicate that the induction of a certain membrane damage with release of intracellular enzymes does not necessarily lead to cellular necrosis.

Release from intracellular enzymes from cutaneous cells after non-necrotizing damage by ultraviolet light.

Experimental blisters were produced with suction on normal human skin and simultaneously on skin inflamed after exposure to middle wave ultraviolet light. Total proteins and marker enzymes for the plasma membrane, cytosol, lysosomes, peroxisomes, mitochondria, and microsomes were assayed in the blister fluid. In blisters on erythematous skin, a large increase of lactate dehydrogenase from cytosol was noted. A small increase of the plasma membrane marker phosphodiesterase I and some increase of alpha-mannosidase from lysosomes was found. No significant increase in total proteins or in microsomal marker enzymes were not detectable. It is concluded that cutaneous cells to some extent may lose intracellular enzymes without visible signs of irreversible damage (necrosis), but that an UVB-induced injury/regeneration cycle probably explains the enzyme release.

Enzymatic repair of premutagenic DNA lesions in human epidermis. Quantitation of O6-methylguanine-DNA methyltransferase and uracil-DNA glycosylase activities.

Extracts of human epidermis prepared by the suction blister method were used to measure O6-methylguanine-DNA methyltransferase and uracil-DNA glycosylase activities. Although both activities were detected in all extracts examined, a 4-5-fold interindividual variation in activity was found. No obvious correlation of the two enzyme activities with the age of the patient was observed. Neither was there any correlation between the level of uracil-DNA glycosylase activity and O6-methylguanine-DNA methyltransferase activity.

Elevated expression of human nonpancreatic phospholipase A2 in psoriatic tissue.

In involved psoriatic tissue, which is characterized by chronic inflammation in both epidermis and dermis, elevated levels of arachidonic acid and eicosanoids have been measured. This implies that a phospholipase A2 (PLA2) may be involved in the pathogenesis of psoriasis. The PLA2's are a group of enzymes that release unsaturated fatty acids from the sn2-position of membrane phospholipids. Once released, the fatty acids are converted by various enzymes into biologically very important signaling molecules. Release of arachidonate initiates the arachidonate cascade, leading to the synthesis of eicosanoids such as prostaglandins, thromboxanes, leukotrienes, and lipoxines. Eicosanoids are important in a variety of physiological processes and play a central role in inflammatory mediators, such as lyso-PAF (a precursor for PAF) and other lysophospholipids, may also be formed through the action of a PLA2. We report for the first time the detection of transcripts of nonpancreatic phospholipase A2 (npPLA2, type II) and cytosolic (c) PLA2 in human skin, and overexpression of npPLA2 in involved skin from patients with psoriasis (plaque psoriasis and pustular psoriasis). Limited amounts of npPLA2 enzyme are detected immunologically in the uppermost layers of epidermis from healthy persons. Both involved and uninvolved psoriatic epidermis contain higher levels of npPLA2 than normal skin. Positive cells in dermis showed significantly higher levels of npPLA2 than epidermal cells. In dermis from healthy persons, only weak staining of a few cells could be detected. The two PLA2 enzymes detected in psoriatic skin (cytosolic and nonpancreatic) may both be involved in eicosanoid overproduction in psoriatic tissue, and the npPLA2 may also be involved in potentiating cell activation, especially T cells.

Remission and relapse of chronic plaque psoriasis treated once a week with clobetasol propionate occluded with a hydrocolloid dressing versus twice daily treatment with clobetasol propionate alone.

BACKGROUND: Many studies have shown the clinical efficiency of occlusion therapy for psoriasis, particularly corticosteroids used under hydrocolloid dressings. However, there are no data from comparative clinical studies evaluating the remission and relapse characteristics of such occlusion therapy compared with orthodox topical steroid monotherapy. METHODS: In a randomised, open-label, parallel group study from three centres, the remission and relapse characteristics were investigated for the use of a hydrocolloid dressing (HCD) over clobetasol propionate 0.05% lotion once a week compared with the same steroid in ointment formulation used unoccluded twice a day in 61 patients with stable chronic plaque psoriasis. RESULTS: There was a pronounced treatment difference in favour of the HCD + clobetasol propionate lotion group with respect to time to clearance, but there was little evidence for a difference with respect to time to relapse. CONCLUSION: The combination of the HCD + clobetasol propionate lotion provides a fast and highly effective remission induction.

Successful treatment of therapy-resistant atopic dermatitis with clobetasol propionate and a hydrocolloid occlusive dressing.

During recent years, 48 patients with therapy-resistant chronic skin lesions of atopic dermatitis have been treated once a week with clobetasol propionate lotion left under Duoderm occlusive patches. They had previously failed to respond, or responded only sparsely, to topical corticosteroids. The lesions resolved completely in 44 patients, while partial remission was observed in the remaining 4. The mean time needed to obtain complete remission was, for lichenifications, 2 weeks; pruriginous lichenoid papules, 12 days; chronic hand eczema, 2.5 weeks; nummular eczema, 8 days; perioral eczema, 11 days, and breast eczema, 10 days. Adverse experiences were mild and infrequent. The amount of topical corticosteroid required was reduced to at most one-twentieth and to as little as one-hundredth of the amount of common topical steroid treatment needed. We conclude that clobetasol propionate and Duoderm once a week is the best treatment for resistant lesions of atopic dermatitis.

Short-contact treatment at home with Micanol.

The efficacy of home treatment of psoriasis with a new dithranol formulation, Micanol, was investigated in three studies. They were carried out according to a randomized, within-patient comparison design. In a 4-week pilot study Micanol was compared with placebo (10 patients) and in two 6-week studies Micanol was compared with dithranol 1% in petrolatum (33 patients) and with dithranol 1% in Amitase stick (16 patients). Micanol was found to be effective and well suited for treatment at home in all studies. The vehicle itself did not improve the psoriasis plaques. The rate of clinical improvement was faster for dithranol in petrolatum and Amitase stick than for Micanol. The prevalence and severity of erythema, burning and staining of skin and clothing were far less for Micanol. Approximately half the number of patients preferred dithranol in petrolatum or Amitase stick and the other half preferred Micanol.

Aspects of the treatment of mycosis fungoides. A report from the Scandinavian Mycosis Fungoides Study Group.

The Scandinavian Mycosis Fungoides Study Group includes dermatologic clinics in Denmark, Norway, and Sweden. The results of the first three years of activity are presented herein. In plaque stage, topical nitrogen mustard was highly effective. Preceding intravenous tolerance induction seems to be of no value. PUVA induced equally high remission rates. Both modalities were also highly effective in cutaneous tumor stage. In advanced tumor stage and in case of extracutaneous involvement systemic chemotherapy was given. Topical treatment alone was more effective on the cutaneous lesions including tumors than systemic chemotherapy alone. Therefore a combination of topical and systemic treatment is recommended in advanced stages of mycosis fungoides.

Normalized light reactions in mycosis fungoides patients after complete remission of skin lesions.

Eleven mycosis fungoides patients were phototested on apparently normal skin before treatment with mechlorethamine, topically in 8 patients and with methoxsypsoralen followed by longwave ultraviolet light (PUVA) in 3 patients. Abnormal photosensitivity to UVB was seen before treatment in 4, to UVA in 4, and to visible light in one patient. The abnormal photosensitivity to all wavelength regions was normalized after complete remission of the cutaneous lesions. Two of the PUVA-treated patients demonstrated that special care should be taken during the initial phase of this treatment because of the light sensitivity, especially on lesional skin.