Regional variations in cytokeratin expression in palmo-plantar epidermis

The cytokeratin composition of palmo-plantar epidermis from sites with different degrees of mechanically induced thickening of the stratum corneum was analysed. The urea-soluble proteins of the stratum corneum were analysed by two-dimensional electrophoresis. Viable epidermal layers were analysed by immunofluorescence microscopy with polyclonal and monoclonal antibodies. A mouse monoclonal antibody specific for cytokeratin no. 9 was prepared for the study. Significant amounts of low molecular weight cytokeratins were found in suprabasal layers at sites with the most pronounced thickening of the stratum corneum. This was taken as evidence that palmo-plantar epidermis responds to mechanical stress with hyperproliferation. At sites where stratum corneum thickness is most increased this hyperproliferation appears to involve two different populations of cells--one capable of expressing high molecular weight, differentiation-related cytokeratins in the suprabasal epidermal layers, and one population that does not express these cytokeratins. At sites with intermediate epidermal hyperplasticity the high molecular weight cytokeratins were predominant in all suprabasal cells.     

Low-molecular-weight contact allergens in p-tert-butylphenol-formaldehyde resin

BACKGROUND: p-tert-Butylphenol-formaldehyde resin (PTBPFR) is a contact allergen that is included in most standard patch test series. This resin consists of a large number of substances most of which are unknown. OBJECTIVE: The aim of this study was to investigate if allergens in PTBPFR are molecules mainly of low (MW < 250), medium, or high molecular weight (MW > 1,000); to isolate and identify some of the low molecular weight components of the resin; and for new substances to determine if these were allergens. METHODS: Gel permeation chromatography, patch testing, high-pressure liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectrometry were used. RESULTS: Patch test reactions indicated allergens in low-, medium-, and high-molecular-weights fractions. The strongest patch test reactivity was seen to a medium molecular weight fraction constituting only 6% wt/wt of the resin for 4 of the patients. Two patients reacted positively to low molecular weight fractions, and one of these patients reacted only to these fractions. The following substances were isolated: 4-tert-butyl-[1,2]benzoquinone, 4-tert-butyl-2,6-bis-hydroxymethyl-phenol, 4-tert-butylbenzene-1,2-diol, 4-tert-butyl-2-hydroxymethyl-phenol, 5-tert-butyl-2-hydroxy-3-hydroxymethyl-benzaldehyde, 4-tert-butyl-2-hydroxymethyl-6-methoxymethyl-phenol, and p-tert-butylphenol. Patients reacted positively to 5-tert-butyl-2-hydroxy-3-hydroxymethyl-benzaldehyde and 4-tert-butyl-2-hydroxymethyl-6-methoxymethyl-phenol but not to 4-tert-butyl-[1,2]benzoquinone. CONCLUSION: Two new allergens in PTBPFR were found: 5-tert-butyl-2-hydroxy-3-hydroxymethyl-benzaldehyde and 4-tert-butyl-2-hydroxymethyl-6-methoxymethyl-phenol.     

III. THE RIBONUCLEASES OF HUMAN EPIDERMIS

Sodium acetate and sulphuric acid extracts of human epidermis can each be separated by chromatographic techniques into three or more fractions with ribonuclease activity. Eight of these fractions were compared with respect to molecular weight, pH activity profile, polyribonucleotide hydrolysis, and activity in the presence of low levels of spermidine. Sodium acetate and sulphuric acid extracts were also prepared from callus and from psoriatic lesions and compared with extracts from normal epidermis for their response to exogenous spermidine. All eight human epidermal ribonuclease fractions studied had an apparent molecular weight of 15,000 daltons. Seven of the ribonuclease fractions were optimally active at alkaline pHs (pH 7.3-7.6 in sodium phosphate and pH 8.I in Tris-HCl) while the eighth ribonuclease was most active at pH 5.6 in a citrate-phosphate buffer. All enzymes hydrolyzed polycytidylic acid and five also hydrolyzed polyuridylic acid. None hydrolyzed polyadenylic acid. Seven of the eight ribonucleases studied exhibited greater activity in the presence of added spermidine. The extracts from psoriatic scales showed markedly elevated ribonuclease levels which could not be raised further by the addition of spermidine.     

Skin irritancy from fish is related to its postmortem age

Scratch tests with different fish products (fish juice from fillets, meat (fillet), skin, slime, juice from fish boxes and hold in the fishing boats, and entrails) were performed in 145 volunteers. All fish products were able to cause irritant skin reactions. Itching and erythema were the predominant symptoms and severe itch reactions occurred more often than severe erythema. The symptoms, in general, were mild to moderate compared to histamine. We found that the postmortem age of the fish was of great importance to the frequency and severity of the symptoms. Only the protein fraction of fish products caused symptoms. Our results are in accordance with the subjective complaints and clinical findings among workers in the fish processing industry.     

Eczematous infiltrated plaques to subcutaneous heparin: a type IV allergic reaction

Two patients are described who had delayed reactions with eczematous plaques to subcutaneous injections of heparin. Subcutaneous challenge with high and low molecular weight heparin compounds were positive in both patients. Patch and intradermal tests with heparin were positive in one subject. Histological and immunohistochemical studies indicated that it was a type IV allergic reaction.     

A study of mosquito salivary gland components and their effects on man

To evaluate the mechanism of mosquito bite reaction in man, salivary gland extracts from female Aedes albopictus were prepared. When the extract of the gland was analysed by ion-exchange high performance liquid chromatography the amount of histamine in a pair of salivary glands of one mosquito was found to be below the limit of detection, an amount which is insufficient to produce an immediate reaction. Salivary gland extracts were fractionated to higher and lower molecular weight components. Intradermal injection tests with salivary gland extracts, which contained less than 100 ng of protein showed that the higher molecular weight fraction (molecular weight greater than 10,000) elicited an immediate and a delayed reaction similar to a bite reaction.     

Low-molecular-weight leukocyte chemotactic factors in psoriatic scales: contribution of lipid-soluble factors to the chemotactic activity

Psoriatic scales contain polymorphonuclear leukocyte (PMN) chemotactic factors which presumably are responsible for transepidermal PMN chemotaxis in psoriatic lesions. On molecular sieve chromatography, psoriatic scale extracts revealed PMN chemotactic activity in the peptide fractions eluted near 12 kDa in addition to low molecular weight (MW) chemotactic fractions. PMN chemotactic peptides with MW around 12 kDa that contain a C5-cleavage product have been characterized as a unique major chemotactic factor in the aqueous components. On the other hand, it is uncertain whether or not lipid-soluble low MW chemotactic factors, i.e., leukotriene B₄ (LTB₄) and its related compound 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), play major roles as PMN chemoattractants. In the present study, we found that the chemotactic activity in the aqueous psoriatic extracts was much higher than that in the organic extracts. Chemotactic activity in the low MW chemotactic fractions of psoriatic scale extracts, about half of which was recovered after extraction with ether at acidic pH, was heat-stable (100°C) and withstood protease digestion, but was partially inactivated by treatment with trypsin. Immunoreactive LTB₄ was demonstrated in these fractions. The previous findings and the present results suggest that the lipid-soluble chemotactic factors, LTB₄ and 12-HETE, which constitute the low MW fraction of the psoriatic scale extracts, do not play as major a role as a PMN chemoattractants as do the chemotactic peptide fractions.     

The determination of lipids and proteins in suction blister fluid.

The concentration of 5 different proteins in suction blister fluid and serum was determined by immunotechniques. These proteins, varying in size and molecular weight (6,600-2,300,000) were insulin, albumin, high density lipoprotein determined as apoprotein A-I, alpha 2-macroglobulin and low density lipoprotein measured as apoprotein B. The difference in the blister fluid/serum concentration ratio of the proteins was dependent on the molecular weight and followed mainly the law of diffusion. Moreover, the amounts of insulin, albumin and apoproteins A-I and B in suction blister fluid were the same as those reported in peripheral lymph. The results indicate that the sieve function of the capillary basement membrane remains intact during the formation of the suction blisters. Suction blister fluid might therefore be regarded as representative of interstitial fluid. The concentrations of 4 different lipids (cholesterol, cholesterolesters, triglycerides and phospholipids) were also determined and their blister fluid/serum concentration ratio proved to have a fairly constant value of 0.25.     

Necrotizing vasculitis: a circulating immune complex producing inflammatory skin lesions

A patient with acute necrotizing vasculitis is described in whom tests for circulating immune complexes were negative. The patient's serum injected intradermally produced lesions which closely resembled those occurring spontaneously. When the serum was fractionated, the skin-reactive fraction was found to be associated with IgG, but was of a higher molecular weight than normal IgG. Immunofluorescent studies showed that lesions induced by this active fraction contained IgG and complement in the epidermal basement membrane zone and within the small dermal vessels. We conclude that an immune complex of relatively low molecular weight is present in the active fractions, and capable of initiating the lesions of acute necrotizing vasculitis.     

Dialysis for psoriasis —Preliminary remarks concerning mode of action

Summary In one patient treated by peritoneal dialysis for psoriasis the skin lesions cleared completely. In two persons hemodialysis gave unsatisfactory results.In treated and another three untreated patients cellular immunity was evidently suppressed. This phenomenon is similar to the immunologic changes in persons with uremia.Solutes in middle molecular weight range (SMMWR) suppress cellular immunity. These compounds were therefore investigated in psoriatics. The level of SMMWR was lower before than after each dialysis, although their concentration increased slightly during the dialytic treatment.This suggests that solutes in middle molecular weight are fixed in the epidermis and/or in the walls of the skin capillaries and that dialysis liberates them from these places.Peritoneal dialysis seems to be a more effective method of treating psoriasis than hemodialysis. Solutes in middle molecular weight range might play an important role in the pathomechanisms of this multifactorial disease.     

Mucopolysacchariduria in genetic dermatoses: hereditary epidermolysis bullosa, congenital ichthyosis and ectodermal dysplasia

4 patients suffering from epidermolysis bullosa, 11 persons with genetically determined ichthyosis, as well as 3 cases of x-linked recessive ectodermal dysplasia were investigated with regard to glycosaminoglycane(GAG)uria; the GAG fractions were analysed by means of GAG thin-layer chromatography. All three groups of patients showed increased GAG-uria. The GAG fractions proved to be chondroitin-6-sulphate, chondroitin-4-sulphate, and heparan-sulphate. The pathomechanism of the increased GAG-uria is supposed to be an increased GAG degradation process.     

Isolation of a low molecular weight glycoprotein in the human urine: comparison with urinary pemphigoid antigen

A glycoprotein was isolated from the urine of normal individuals and patients with bullous pemphigoid by means of ultrafiltration, ion exchange chromatography, and gel filtration. The yield of the purified glycoprotein from each liter of pooled urine was 0.35–0.40 mg. Analysis of the glycoprotein on SDS-polyacrylamide gel electrophoresis showed a single band of approximate molecular weight 23 kd. The glycoprotein contained approximately 2.5% (w/w) carbohydrate. The glycoprotein differed from the urinary pemphigoid antigen described by Diaz et al (1), though both were nondialyzable, cathodally migrating, low molecular weight glycoproteins in the human urine. The significance of the glycoprotein is unclear at present, but it might be useful for studies of the metabolic degradation process of serum proteins.     

Die Wirkung kleinmolekularer Substanzen auf die menschliche Haut

Interactions between low molecular weight compounds with cells of the skin result in reactions with different proteins which enable the uptake, metabolism and efflux of these compounds. It is unlikely, that small molecular weight compounds can achieve pharmacological concentrations within cells by diffusion alone. The pattern of influx proteins of keratinocytes is different from that of hepatocytes. If the balance between these systems is disturbed, the skin may become unable to function as a protective organ which can result in diseases including cancer or-more frequently-allergic contact dermatitis. Recent investigations of the sensitization to fragrances and p-phenylenediamine are discussed. An improved understanding of the metabolism of low molecular weight compounds can lead to new therapeutic strategies. One example is the introduction of photodynamic therapy with topical applied porphyrin precursors.     

Epidermal protein synthesis in newborn rat skin

Protein synthesis in newborn rat epidermis was assessed by in vivo labeling of epidermal proteins with radioactive glycine and the isolation of neutral buffer, sodium-dodecylsulfates (SDS) dithiothreitol soluble proteins, and 0.1 M NaOH soluble protein fractions of the epidermis. There was an increase in the labeling of proteins in the buffer and SDS fractions for 5 h after injection and then no further increase at 19 h after injection. In the 0.1 M NaOH soluble proteins and the final pellet after these extractions there was a progressive increase in glycine incorporation up to 19 h after injection. SDS gel electrophoresis of the labeled proteins showed early synthesis and continued synthesis of proteins of 56,000 and 65,000 daltons corresponding to the main fibrous proteins (alpha-keratins) of the epidermis. At 19 h a new protein band of 45,000 daltons first appeared and was associated with a decrease in the labeling of very high molecular weight (or highly aggregated) proteins. This later protein is presumptively identified as keratohyalin. Free amino acid pools were associated with both the neutral buffer and SDS extractable fractions.     

Skin temperature and skin symptoms among workers in the fish processing industry

196 workers employed in the fish processing industry participated in a survey of skin disorders. 156 (80%) had experienced skin problems during their work with fish on some occasions. The symptoms were itching, redness and stinging. Although the fingers are in direct contact with fish meat and juice, skin symptoms only seldom occur here, but instead almost exclusively on the forearms (70%) and the backs of the hands (26%). The skin temperature of the fingers and palms of the hands ranged from 17 degrees C to 20 degrees C, while the temperature on the backs of the hands and forearms ranged from 25 degrees C to 30 degrees C. Skin temperatures less than 20 degrees C abolish itch and reduce vasodilation by half. We suggest that the low temperature on the fingers affords protection against the development of some irritant skin reactions and that differences in skin temperature may be an important reason for the location of skin symptoms.     

Molecular size of secreted and cell-associated plasminogen activators from cultured epidermal cells

The molecular sizes of secreted and cell-associated plasminogen activators from four cultured cell types were determined using an SDS-PAGE technique in which plasminogen and casein were included during polymerization of the polyacrylamide gel. The major bands of plasminogen activators secreted by human neonatal epidermal cells, human adult epidermal cells and transformed human squamous cells migrated the same distance as the high molecular weight band of authentic urokinase, indicating that the apparent molecular weight of these plasminogen activators was approximately 55,000 daltons. Plasminogen activator extracted from normal adult human epidertnis also migrated with this major band of plasminogen activator, and a minor higher molecular weight band was also detected. In contrast, plasminogen activators secreted by transformed mouse squamous cells migrated between the high molecular weight band (～ 55K) and the low molecular weight band of urokinase (～ 32K), indicating that plasminogen activators of mouse epidermal cells differ from those of human epidermal cells. The mobility of the major bands of plasminogen activators detected in cell lysates of the four cell types was identical to that of secreted plasminogen activators.     

Skin irritants of the sun spurge (Euphorbia helioscopia L)

Euphorbia helioscopia is a common herbaceous weed found in the British Isles and parts of Europe. It has been responsible for poisoning of livestock resulting in severe inflammation particularly of mucous membranes and the eyes. Four esters of 12-deoxyphorbol were isolated from the fresh aerial parts of the plant and their irritant doses 50% (I.D.50) ascertained using female L.A.C.A. mice. 12-Deoxyphorbol-13-phenylacetale-20-acetate was found to be the major component of the toxic fraction and occurred in yields of 0.0012 % w/w. This compound was also the most irritant substance isolated and exhibited an I.D.₅₀ of 0.038μg, and is accordingly considered to be responsible for the major part of the toxicity of E. helioscopia. A high molecular weight aliphatic ester, 12-deoxyphorbol-13-dodec-dienoate-20-acetate, was also obtained in low yields. This compound exhibited an T.D-no of 0.12μg. The final two esters obtained from the toxic fraction of the plant were the low molecular weight homologues, 12-deoxyphorbol-13-[2-methyl-cis-2-butenoate].-20-acetate and 12-deoxyphorbol-13-[2-methyl-cis-2-butenoate]. These compounds exhibited I.D.₅0 of 3.09 and 0.72 μg, respectively and were the least irritant of the series of compounds isolated.     

In vivo porphyrin production by P. acnes in untreated acne patients and its modulation by acne treatment

Propionibacterium acnes is often discussed as a contributing pathogenic factor in the aetiology of acne lesions. The aim of this study was to test which porphyrin patterns are synthesized by P. acnes in vivo in untreated acne patients and during standard acne regimens. These photosensitive compounds are potential targets for photo-dynamic therapy of acne and need to be better characterized in the skin. Using high-performance liquid chromatography coproporphyrin III was the main porphyrin identified in all patients. Coproporphyrin I and protoporphyrin were found at considerably lower concentrations. When the porphyrin concentration of individual patients receiving isotretinoin was analysed repeatedly over time, clinical improvement was associated with lowered levels of porphyrins. Statistical analysis demonstrated a significant reduction in the porphyrin fractions only in the isotretinoin group which was associated with clinical improvement 2 months after starting therapy.     

Isolation and characterization of proteinase from Candida albicans: substrate specificity

Candida albicans was able to produce a keratinolytic proteinase (KPase) when cultivated in a medium containing human stratum corneum as a nitrogen source. The KPase was purified to 108.5-fold by ion-exchange chromatography and gel filtration. The molecular weight of the enzyme was estimated to be 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration through Sephacryl S-200, while the isoelectric point was determined to be at pH 4.5. The enzyme had an optimum pH of 4.0 and was "inactive" below pH 2.5 and above pH 6.0. The activity of KPase after preincubation at various temperatures was stable up to 50 degrees C. The keratinolytic activity was not affected by the addition of nonionic detergents and divalent cations. The enzyme was a glycoprotein and contained a high content of aspartic acid residues (172/1000). Pepstatin and chymostatin inhibited the activity in a dose-dependent manner; however, neither the other group specific inhibitors tested nor the pepsin specific inhibitors, DAN or EPNP, showed any effect on the enzyme. From these inhibitory profiles, this enzyme was determined to be a carboxyl proteinase such as cathepsin D. Among the various substrates for proteolytic enzymes, KPase digested human stratum corneum as much as albumin and hemoglobin. In the three fractions (water soluble, keratin filamentous, and membranous) prepared from human stratum corneum, the keratin filamentous fraction was more susceptible to degradation by KPase than the other two fractions were. KPase also digested much less human fingernail (13%) than human stratum corneum, but did not show any signs of there being any digestion of human scalp hair. These studies suggest that KPase from C. albicans may play an important role in superficial infection by affecting the human stratum corneum of the skin and nail.     

A chymotrypsin-like proteinase that may be involved in desquamation in plantar stratum corneum

Summary We have recently reported that unipolar cell shedding from plantar stratum corneum incubated in vitro, and the associated degradation of the desmosomal protein desmoglein I, are dependent on the activity of a proteinase that can be inhibited by aprotinin, chymostatin and zinc ion. The aim of this work was to find a proteinase in plantar stratum corneum that fulfils the criteria for being the responsible enzyme. Dissociated plantar corneocytes were incubated with the chymotrypsin substrate 3-carbomethoxypropionyl-l-Arg-l-Pro-l-Tyr-p-nitroanilide hydrochloride (S-2586) and H-d-Ile-Pro-Arg-p-nitroanilide dihydrochloride (S-2288), a substrate for a wide range of serine proteinases with arginine specificity. There was a significant rate of hydrolysis of S-2586, but S-2288 was hydrolysed only very slowly. Extraction of dissociated corneocytes with buffers containing KCl or sodium dodecyl sulphate released one major proteinase that could be detected by electrophoresis in polyacrylamide gels with copolymerized casein and subsequent incubations of the gels. Both the caseinolytic activity and the S-2586-hydrolysing activities were inhibited by aprotinin, chymostatin and zinc ion, but not by leupeptin. The S-2586-hydrolysing activity was also inhibited by soybean trypsin inhibitor and phenylmethylsulphonyl fluoride. Both activities were optimal at pH 7–8 but were also significant at pH 5.5. On gel exclusion chromatography, the S-2586-hydrolysing and caseinolytic activities were eluted with an apparent molecular weight of around 18 kDa. When analysed by electrophoresis in the presence of sodium dodecyl sulphate under non-reducing conditions the caseinolytic enzyme had an apparent molecular weight of around 25 kDa. It is concluded that the enzyme studied may be responsible for the degradation of intercellular cohesive structures during in vitro cell shedding from plantar stratum corneum, and that it does have properties compatible with a role in stratum corneum turnover under physiological conditions.