David I. Wilkinson

Peptide T improves psoriasis when infused into lesions in nanogram amounts

In each of 14 patients, two small but comparable psoriatic lesions were infused for 2 weeks with either saline or a saline solution of peptide T (as its analog D-ala1-peptide T amide) at 10(-7) mol/L with Alzet osmotic pumps worn extracorporeally. During infusion, lesions were photographed and scored for clinical features of psoriasis on a 9-point scale. After another 7 days, biopsy specimens were taken from the infused sites, and sections were scored for features of psoriasis on a 19-point scale. The differences between means for data from saline- and peptide T-infused lesions were evaluated statistically. Peptide T-infused lesions improved clinically; scores decreased from a mean of 4.35 initially to 1.57 at biopsy, whereas control lesions changed from 4.43 to 3.57 (p less than 0.01 for 1.57 vs 3.57). Histologic scores were also significantly different (5.28 for peptide T vs 10.00 for controls, 0.05 greater than p greater than 0.02). This study provides evidence that intralesionally infused peptide T demonstrates some clearing effect in psoriasis.

Nerve growth factor increases the mitogenicity of certain growth factors for cultured human keratinocytes: A comparison with epidermal growth factor

Abstract Newborn foreskin and adult skin keratinocytes (KTs) were cultured in 24‐well plates using keratinocyte basal medium (KBM) either alone or supplemented with epidermal growth factor (EGF) or nerve growth factor (NGF), plus one of the following: insulin (INS), insulin‐like growth factors (IGF)‐l or ‐2, transforming growth factor alpha (TGFα), basic fibroblast growth factor (bFGF). Culture was maintained until one group of cells reached about 30,000 cells/well, when cells were stained with crystal violet and the extracted dye used to quantify cell numbers. In some cases, cells were subjected to the hexosaminidase assay for enumeration. In KBM alone, EGF, IGF‐1, IGF‐2 and TGFα were milogenic to newborn KTs. In addition, NGF increased the growth of adult KTs, possibly by mechanisms involving synergy with autocrine growth factors. EGF augmented the growth of newborn cells in the presence of each of the growth factors except TGFα, but adult cells exhibited only additive effects. In the presence of IGF‐1 or IGF‐2, NGF stimulated the growth of both newborn and adult cells by as much as 150% above purely additive increases in cell numbers. NGF amplifies the effects of most neurotrophic factors that are also KT mitogens and may therefore be significant in psoriatic lesions, where many of these factors are overex‐pressed, and in wound healing, in promoting KT growth.

Effect of prostaglandins on cyclic nucleotide levels in cultured keratinocytes

Ginea pig ear epidermal cells (keratinocytes) were established in primary cultures using trypsin, and treated in their proliferative phase of growth with prostaglandins E1, D1, F1 alpha, E2, D2, or F2 alpha. This phase is induced by the addition of retinoic acid during cell plating. Intracellular content of cAMP and cGMP was measured by radioimmunoassay at various times after treatment. Maximum stimulation of cAMP levels was observed with PGD2, smaller increases with PGE2 and relatively transient rises with PGF2 alpha which were of low significance, but confirm earlier data. Similar results were observed with PGD1, PGE1, and PGF1 alpha with smaller increases. The effects of D and E PGs were biphasic. Significant increases in cGMP were immediately observed with PGD2 and PGE2. With PGF2 alpha, maximum cGMP levels were noted after some delay. All PGs tested showed some effect in elevating cyclic nucleotides in keratinocytes. The most striking result was the increase in cAMP on PGD2 treatment.

Effect of vitamin A acid on the growth of keratinocytes in culture.

SummaryThe effect of vitamin A acid on the culture of guinea pig ear keratinocytes was investigated. Use of this agent and treatment of the plastic dishes with collagen solution facilitated attachment and growth. Vitamin A acid elicited a wave of DNA synthesis which was studied by incoporation of tritiated thymidine and quantitation of cellular DNA, particularly during the period 24–45 h after plating. During this period, thymidine incorporation into DNA was increased ten-fold by vitamin A acid and showed a complex pattern of peaks and shoulders which may represent the S phase of synchronous keratinocytes.ZusammenfassungDer Einfluß von Vitamin A-Säure auf Gewebekulturen von Keratinocyten gewonnen von Meerschweinchen-Ohren wurde untersucht. Vitamin A-Säure sowie die Vorbehandlung der Plastik-Petri-Schalen mit einer Kollagen-Suspension erleichterte die Zelladhäsion und das Wachstum. Vitamin A-Säure versursachte eine DNS-Synthese-Welle, die durch den Einbau von 3H-Thymidin und die Bestimmung der cellulären DNS nachgewiesen werden konnte. Diese DNS-Synthese erfolgte 24–45 h nachdem die Keratinocyten angesetzt wurden. Während dieser Zeitspanne resultierte die Vorbehandlung mit Vitamin A-Säure in einer zehnfachen Erhöhung des Einbaus von 3H-Thymidin und zeigte ein komplexes Muster von Gipfeln und Schultern, welche die synchrone S-Phase der Keratinocyten darstellen könnte.

Polyunsaturated fatty acids of skin; Identification and14C-Acetate incorporation

Following exposure of the tissue to14C-acetate, polyunsaturated fatty acids were separated from the total fatty acids of newborn human preputial and mouse skin using argentative thin layer and gas liquid chromatographic procedures. They were characterized by conventional methods including oxidative cleavage and gas liquid chromatography of fission fragments. Members of the linoleic and linolenic acid classes were present in labeled form including the parent acids. Linoleic and arachidonic acids predominated, all others occurring in relatively small amounts. Patterns for the two types of skin were similar in distribution of both mass and radioactivity of individual acids.

Positional isomers of monoene and diene fatty acids of human skin epidermal cells

Abstract Trypsin-released epidermal cells from human skin were incubated in a tissue culture medium containing acetate-1- 14 C, and their lipids subsequently extracted and converted to methyl esters. The patterns of positional isomers present in the principal even-carbon monoenoic acids, and in octadecadienoic acid were examined after oxidative cleavage of individual esters, and GLC of the fragments. In the case of the C 18 species, the radioactivity of the fragments was assayed. It was found that Δ9 acids predominated, and that cleavage products of 18: Δ9,12 contained radioactivity. Thus epidermal cells apparently have the ability to synthesize linoleic acid.

Camptothecin induces differentiation, tissue transglutaminase and apoptosis in cultured keratinocytes

Abstract: Cultured normal human adult keratinocytes were exposed to (S)‐(+)‐ camptothecin over the concentration range 10‐5 to 10‐10 M. The dose‐dependent inhibition of growth was recorded using cell counting. The induction of terminal differentiation was demonstrated by the relative increase in squamous and cornified cells, and the concomitant decrease in small, proliferative cells, with an overall decrease in total cell numbers on going from 10‐10 to 10‐6 M concentration of the drug. The induction of apoptosis was studied by assay of two types of transglutaminase, “tissue” and “keratinocyte”, and by assay of histonelinked mono‐ and oligonucleosomes. Induction of apoptosis was accompanied with increase in “tissue” transglutaminase and in the amount of nucleosomes, the latter being indicative of endonuclease activity. This activity was sharply increased at a camptothecin concentration of 10‐5 M, and may have been faciliated by “tissue” transglutaminase at lower concentrations. The data suggest that camptothecin restricts keratinocyte growth by several mechanisms including apoptosis and emphasize its possible use in topical therapy for psoriasis.

Effect of methylglyoxal bisguanylhydrazone on polyamine biosynthesis, growth, and differentiation of cultured keratinocytes

SummaryThe putrescine, spermidine, and spermine content of subcultured human newborn foreskin keratinocytes was determined during growth and early plateau phase and found to be highest during growth. Exposure of the cells to methylglyoxal bis(guanylhydrazone) during growth phase caused a dose-dependent fall in intracellular spermidine and spermine levels and an increase in putrescine levels at higher concentrations. These effects reflect inhibition of S-adenosyl methionine decarboxylase by the drug. At 8×10-6 M the drug reduced incorporation of leucine into protein, lowered or stopped the accumulation of DNA per dish, inhibited mitotic activity, and increased the histidine/leucine incorporation into protein. The last effect is regarded as induction of keratinization. All these effects were reversible if the use of the drug was discontinued after 3 days. Inhibition of the enzymes of polyamine biosynthesis may have value in psoriasis therapy.ZusammenfassungDer Putrescin-, Spermidin- und Spermingehalt in Subkulturen von menschlichen Vorhaut-Keratinocyten wurde während der Phase des schnellen Wachstums und der frühen Plateau-Phase bestimmt. Der höchste Wert wurde in der Wachstums-Phase gefunden. Zugabe von Methylglyoxal bis(guanylhydrazone) während der Wachstums-Phase resultierte in einer konzentrationsabhängigen Abnahme des intracellulären Spermidin- und Spermingehaltes und bei hoher Konzentration Zunahme des Putrescingehaltes. Diese Wirkungen reflektieren die Hemmung der S-adenosyl methionine Decarboxylase durch Methylglyoxal bis(guanylhydrazone). Eine Konzentration dieser Substanz von 8×10-6 M reduzierte den Einbau von radioaktivem Leucin in Eiweiß, reduzierte oder verhinderte die Ansammlung von DNA per Petrischale, inhibierte die mitotische Aktivität und erhöhte den Histidin/Leucin Einbau in Eiweiß. Letztere Wirkung wird als Induzierung der Epithelisierung angesehen. Alle diese Wirkungen waren reversibel, wenn die Inhibitor-Substanz nach 3 Tagen abgewaschen wurde. Hemmung der Enzyme der Polyamin Biosynthese können möglicherweise in der Psoriasistherapie wertvoll sein.

Effects of Nordihydroguaiaretic Acid, Phloretin, and Phloridzin on the Activity of Adenylate Cyclase, Lipoxygenase and Hexose Transport, and Growth of Cultured Keratinocytes

Compounds related to the flavonoid group of natural products may have potential as antipsoriatic drugs. The dihydrochalcone phloretin, its glycoside derivative phloridzin. and the structurally related compound nordihydroguaiaretic acid (NDCA) were selected for study, Phloretin and NDGA strongly inhibited keratinocyte growth hut had no effect on isoproterenol‐stimulated adenylate cyclase; phloridzin had no effect on growth hut potentiated the response of the enzyme. None had any effect on phosphodiesterase. Neither phloretin or phoridzin inhibited lipoxygenase or, surprisingly, decreased deoxyglucose transport. Phloretin and NDCA should he considered antipsoriatic agents.

Effect of β-adrenergic receptor blockade or refractoriness induced by isoproterenol on growth of keratinocytes in virto

Almost complete suppression of β‐adrenergic sensitivity of adenylate cyclase was produced in keratinocytes growing on collagen gels either by induction of refractoriness by isoproterenol or by receptor blockade by propranolol, these drugs being introduced 3 days post‐plating and continuously thereafter. Neither of these two manoeuvres affected growth of the keratinocytes. Despite this suppression, basal levels of cyclic AMP never fall below the levels observed in untreated cultures. On the other hand, growth inhibition is observed if isoproterenol or propranolol is added on plating.