Library

Notes: Summary Keratinocyte differentiation in psoriasis was examined using a panel of monospecific monoclonal antibodies to keratins (K), including two recently dcveloped monoclonal antibodies raised to carboxy terminal peptides of K6 (LL020) and Kl6 (LL025). Keratinocytes from normal skin, untreated psoriatic plaques and non-lesional psoriatic skin, were cultured using multiple in vitro Systems. Timelapse cinephotography was used to measure the intermitotic time of normal and psoriatic keratinocytes in both low calcium-defined and serum-containing media. The intermitotic time did not differ significantly between psoriatic and normal keratinocytes. Keratin expression of psoriatic and normal keratinocytes in vitro was examined by both gel electrophoresis and immunocytochemistry. K6. K16 and Kl7 were detected suprabasally in all culture Systems in vitro, but only in interfollicular psoriatic epidermis in vivo. and not in normal skin. Small subpopulations of keratinocytes expressed simple epithelial keratins K7, K8, Kl8 and K19 in cultures on plastic Substrates, but these keratins were absent in skin equivalents of normal or psoriatic skin. No psoriasis-spesfic pattern of differentiation was found in vitro. As the K6 peptide antibody reacted with basal cells of normal skin. probably due to K5 cross-reactivity. K16 expression determined by LL025 was found to be the most sensitive indicator of the psoriatic state of differentiation. and this antibody is recommended for future work on psoriasis. Kl7 had a distinct pattern of tissue distribution in normal skin: Kl7. but not K16. was present in basal myoepithelial cells in sweat glands. and the dccp outer root shealh. but Kl 7 distribution parallelcd that ol'Klfi in suprabasal psoriatic epidermis. As keratins K6. Kid and Kl 7 are expressed in keratinocyte hyperproliferation. when high Ievels of certain cytokines are also expressed. the role of growth factors and regulalory nuclear transcription factors in the control of K6, K16 and Kl7 cxpression in psoriasis requires further study, in order to provide insight into the relationship between proliferation and differentiation.

Notes: Summary Monospecific antibodies to individual keratin polypeptides can be used to examine the tissue and cellular coexpression of members of keratin pairs. Monospecific monoclonal and polyclonal antibodies have been raised to keratins 1 and 10 using both crude cytoskeletal extracts and synthetic peptides. The tissue distribution of these keratins has been determined against a panel of freshly frozen normal tissues from humans, rodents and pigs, Epidermal expression has been examined in psoriatic plaques, and healing wounds, as examples of epidermal hyperproliferation. Cultured keratinocytes in monolayer(low calcium), stratified (high calcium), and complex cultures, transformed keratinocytes, and tumour cell lines, have been examined for the in vitro expression of these keratins. The sensitivity and precise localization of reactivity with these monospecific antibodies gives a highly accurate picture of individual cell expression. There is confirmation of coexpression of keratins 1 and 10 in epidermal and mucosal sites, and with keratin 16 in hyperproliferative states. These monospecific antibodies provide an important means of examining keratin expression in epidermal tumours and keratinizing disorders, and of seeking keratin mutations in cell lines and in skin diseases.

Notes: Background  Several hereditary human diseases are now known to be caused by distinct mutations in genes encoding various desmosome components. Although the effects of some of these mutant genes have been analysed by targeted disruption experiments in mouse models, little is known about the cell and tissue changes in affected human patients.Objectives  To investigate the effects of heterozygous nonsense mutations in desmoplakin (Dp) and desmoglein (Dsg) 1 which cause the autosomal dominant disorder striate palmoplantar keratoderma (SPPK), focusing on changes in desmosome structure and composition and the associated keratin intermediate filament (KIF) network in palm skin, and in cultured keratinocytes generated from the same site.Methods  We analysed palm and nonpalm skin sections from four SPPK patients with Dp mutations and one patient with a Dsg1 mutation with respect to tissue and subcellular morphologies, and correlated the in vivo and in vitro findings.Results  Using electron microscopy, we found abnormalities of desmosomes and cell–cell adhesion in the suprabasal layers in the epidermis from patients with both Dsg1- and Dp-associated SPPK. These changes were more advanced in skin from patients with Dp mutations. Both Dp and Dsg1 mutations were accompanied by significantly reduced numbers of desmosomes in the suprabasal layers, while decreased desmosome size was evident only in Dsg1-associated SPPK. Confocal microscopy analysis showed marked differences in the expression of keratins and of desmosome components, both between the two types of SPPK, and between SPPK and normal skin. The expression of keratins K5, K14 and K10 was reduced in Dsg1-associated SPPK skin, whereas perinuclear aggregation of keratin filaments was more evident in Dp-associated SPPK. In both types of SPPK upregulation of K16 was pronounced and involucrin labelling was abnormal.Conclusions  Mutations in Dp and Dsg1 genes causing SPPK may be associated with perturbations in epidermal differentiation accompanied by a marked disruption of several components of the epidermal scaffold including desmosomes and the KIF network.

Notes: The culture of keratinocytes from normal skin was attempted for many years but was largely hampered by a tendency for keratinocytes to differentiate in vitro rather than proliferate, and problems with fibroblast overgrowth. However, the report from Rheinwald and Green (1975), which utilizes a mouse 3 T 3 substrate and mitogens, led major advances in culture techniques permitting serial passaging of keratinocyte cultures without fibroblast contamination. Other systems using defined media (Boyce & Ham, 1983) and additives such as epidermal growth factor (Rheinwald & Green, 1977) and bovine pituitary extract (Peehl & Ham, 1980) provide expanded growth without a feeder system. This ability to expand, greatly, a given population of keratinocytes is of obvious clinical application in the repair of epidermal defects and already has been used in the treatment of young burns patients in the States (O'Connor et al., 1981). We report the use of cultured skin in the grafting of an elderly patient with stasis ulceration.

Notes: Monoclonal antibodies to keratin filament proteins can be used as markers of differentiation within the skin. Two major cell groups can be detected in the interfollicular skin—namely basal and suprabasal compartments. Within the hair follicle small subpopulations of cells stain with simple epithelial antibodies; in particular, the inner hair root sheath and a small zone of upper hair follicle cells. Previous biochemical studies suggested that basal cell carcinomas derive from the hair follicle. We have studied a series of basal cell carcinomas (n= 2o) using (1) SDS gel electrophoresis and (2) immunocytochemistry, using a large panel of monoclonal antibodies including peptide-specific monoclonal antibodies (LE61 to keratin 18; LE41 to keratin 8 and LP2K to keratin 19). Biochemically, tumour cells failed to express high molecular weight (MW) keratins but variable expression of low MW keratins. Immunocytochemistry showed (a) failure to express suprabasal differentiation (LHP1, LHP2, LHP3); (b) invariable expression of interfollicular basal antigens; and (c) occasional expression of simple epithelial antibodies only. There was no evidence to suggest that basal cell carcinoma cells originate from hair follicle cells.

Notes: Cultured keratinocytes were used as allografts to treat 51 patients with chronic venous ulccration or rheumatoid ulcers unresponsive to all previous conventional treatments including split skin grafts. Although early epithelialization could be seen in the centre of some ulcers, a major effect appeared to be healing from the previously indolent edge. This treatment appears to provide some clinical benefit in healing of chronic ulceration.

Notes: Monoclonal antibodies provide unique opportunities to detect subtle changes in the structure of the basal lamina particularly in the diagnosis of different subgroups of epidermolysis bullosa. The monoclonal antibody LH7:2 was produced following immunization of Balb C mice with 1% NP40 extract of trypsin separated epidermal cells (predominantly basal cells). By indirect immunofluorescence of skin sections, strong staining of the basal laminae of interfollicular and follicular epidermis, sweat glands and sebaceous glands was obtained. The basal laminae of other stratified squamous epithelia such as oral, oesophageal and cervical epithelia react with LH7:2 but not those of most simple epithelia including gastric and renal epithelia. Weak placental staining is obtained.The basal laminae of blood vessels do not react with LH7:2. Immuno-autoradiography, ELISA testing and immuno-blotting show no reactivity with laminin, entactin, fibronectin and types I-VI collagen. Immuno-electronmicroscopy shows deposition in the lamina densa and possibly anchoring fibrils. LH7:2 antigen has been shown to be absent in generalized, severe recessive dystrophic epidermolysis bullosa (RDEB) (n= 8) and present but reduced in localized recessive dystrophic epidermolysis bullosa (n= 2), but normal in dominant dystrophic epidermolysis bullosa (n= 6). Staining is normal in junctional epidermolysis bullosa (n= 4) and epidermolysis bullosa simplex (n= 2).This antibody may be useful in the rapid diagnosis of generalized severe RDEB, especially in antenatal diagnosis in affected families and in the distinction between localized RDEB and DDEB for genetic counselling.