The cytoskeleton in hereditary ichthyoses

The hereditary forms of ichthyosis can be considered to be models of impaired terminal epidermal differentiation. Analysis of the cytokeratin polypeptide pattern represents a new attempt at elucidating the mechanisms of keratinization mechanisms which are still unclear. We therefore studied the cytokeratin expression of the following types of ichthyosis: autosomal dominant ichthyosis vulgaris (n = 4), X-linked recessive ichthyosis vulgaris (n = 4), recessive non-bullous congenital ichthyosiform erythroderma (n = 1), recessive classical lamellar ichthyosis (n = 2), autosomal dominant lamellar ichthyosis (n = 1), and Netherton syndrome (n = 1). After dissection of frozen sections of the interfollicular epidermis, two-dimensional gel electrophoresis was performed. For immunofluorescence microscopy a panel of monoclonal cytokeratin antibodies (KG8.13, KK8.60, KA5 and AE1) was used. Cytokeratin polypeptide expression was basically unchanged compared with normal epidermis. In contrast, however, the antibody AE1 did not stain the basal cell layer in most types of ichthyosis, regardless of their genetic type. The cytokeratin polypeptides nos. 6 and 16, which are generally considered markers of hyperproliferation, were not expressed in either type of ichthyosis vulgaris (XRI or ADI), but were detected in trace amounts in various types of congenital ichthyosis.     

Tob is a potential marker gene for the basal layer of the epidermis and is stably expressed in human primary keratinocytes

BACKGROUND: Epidermis consists of multiple layers, from the proliferating basal layer to terminal differentiated cornified layers, and these layers are defined by differentiation status. Tob gene product is known to be a member of the BTG antiproliferative protein family. We investigated the expression pattern of Tob gene product to understand the possible role in differentiation of keratinocytes and epidermis. OBJECTIVES: In this study, we examined the expression of Tob gene product in the primary cultured human keratinocytes and in the in vivo epidermis. METHODS: The expression of Tob gene product was assessed by Western blotting analysis. Cellular localization of Tob was detected using the green fluorescent protein-tagged Tob cDNA expression construct. In vivo expression of Tob gene product in the epidermis was determined by immunohistochemistry with paraffin sections. RESULTS: Tob family members are degraded by the ubiquitine-proteasome system triggered by the growth signal. Tob is stably and abundantly expressed in primary cultured human keratinocytes. Furthermore, the expression of Tob in the keratinocytes persists during the differentiation induced by calcium; however, it was not detected in primary cultured fibroblasts. Also, the subcellular localization of Tob is mainly in the cellular membrane in the primary human keratinocytes. We evaluated Tob expression in normal skin, oral mucosa and different diseases, such as psoriasis, X-linked ichthyosis and squamous cell carcinoma (SCC). Using immunohistochemical analysis, we observed that Tob was selectively expressed in the basal layer of X-linked ichythyosis and the hyperproliferative basal layer of psoriasis and oral mucosa as well as in normal epidermis. In SCC, the expression of Tob gene product was relatively decreased. CONCLUSIONS: Tob is stably expressed in primary human keratinocytes and it is specifically expressed in the basal layer of in vivo epidermis.     

The calcium-activated neutral protease calpain I is present in normal foetal skin and is decreased in neonatal harlequin ichthyosis

Calcium concentration is a critical factor for epidermal differentiation and is implicated in the expression and post-translational modification of numerous proteins in suprabasal cells of the epidermis. Calpains (calcium-activated neutral proteases) are believed to participate in signal transduction via highly regulated cytoplasmic protease activity. Here we investigate the expression of calpain I in normal human skin development and in neonatal harlequin ichthyosis (HI), a disorder of altered epidermal differentiation, especially the transition from the granular to the fully differentiated cornified layer. Calpain I was detected in developing foetal epidermis at 54 days estimated gestational age in the basal layer and the periderm of the developing foetal epidermis. By 125 days, calpain I was also detected in the granular layer. This pattern was maintained in newborn skin, but expression was significantly weaker in HI biopsies (n = 7). Reduced expression of calpain was specific to HI and was not observed in other skin diseases. Calpain was also normally expressed in the outer root sheath of hair follicles, in sebaceous glands and in sweat ducts and glands. Immunoblots of epidermal and keratinocyte extracts showed that the 78-kDa and 76-kDa active forms were generated via limited proteolysis of the 80-kDa inactive subunit; however, all forms were diminished in HI, consistent with findings in tissue sections. Our results show that calpain is present throughout the epidermis and is expressed from the early stages of development. These findings implicate calcium-mediated signalling events in the alteration of differentiation that occurs in HI.     

Movement of beta-irradiated epidermal basal cells to the spinous-granular layers in the absence of cell division.

Guinea-pig epidermis was irradiated with 3000 rad of beta rays 1 hr after two injections of [3-H]thymidine 5 hr apart (labeled cells in S phase and G2 phase) or 18 hr after injection (labeled early G1 cells). In nonirradiated epidermis labeled basal cells divided within 24 hr with daughter cells remaining in the basal layer, and approximately 50% of the labeled cells moved into the spinal layer by the 3rd day. Cell division in nonirradiated epidermis diluted the number of silver grains/nucleus, and lightly labeled cells were found in the granular layer by day 7. Beta irradiation inhibited cell division but it did not slow the rate of transit (ca 8 days) of irradiated labeled cells from basal to granular layer, some of these remaining heavily labeled. Although cell division may play some role in upward movement of basal cells in normal epidermis detachment of a basal cell from the basement membrane and its transit to the granular layer is unimpaired in the absence of cell division. These findings suggest that some radioresistant metabolic function(s), not cell division, is responsible for upward movement of basal cells.     

Cytochemical expression of epidermal peroxidase and cytochrome oxidase activities in pathological skin conditions of man

Summary The cytochemical expression of epidermal peroxidase and cytochrome oxidase activity was recently well documented in normal human skin.We report here its expression in basal and squamous cell carcinomas, actinic keratoses, psoriasis, allergic contact dermatitis, seborrheic keratoses, and autosomal dominant ichthyosis vulgaris. The two enzyme activities were evaluated using the diaminobenzidine method. If present, the two enzymes were always localized in the same organelles as in normal epidermis endogenous peroxidase in the nuclear envelope and endoplasmic reticulum, and cytochrome oxidase in mitochondria. In basal and squamous carcinomas, actinic keratoses and psoriasis, the keratinocytes lost their peroxidase activity, but maintained their cytochrome oxidase activity. In seborrheic keratoses, allergic contact dermatitis and ichthyosis vulgaris, the cytochrome oxidase activity was greatly reduced or abolished in keratinocytes, Langerhans' cells, and melanocytes, whereas the peroxidase activity was present as in normal epidermis. These results indicate that the two peroxidatic enzymes studied are not interrelated and alternatively suppressed by different cellular dysfunctions.A part of this work was presented at the combined 12th SCUR Annual Meeting and 6th International Dermatopathology Colloqium, April 1985, Florence, Italy     

丝聚蛋白的研究进展

丝聚蛋白在表皮含量丰富,主要存在于表皮的最外层,对表皮的屏障功能起着重要的作用.近年的研究发现,寻常性鱼鳞病及特应性皮炎的发病与丝聚蛋白的编码基因Flg有明显的相关性.Flg失功能突变使表皮丝聚蛋白表达减少或缺如,导致表皮的屏障功能受损,使环境中的变应原、刺激物等易于进入表皮,继而引起各种病理生理变化.     

丝聚蛋白的研究进展

丝聚蛋门在表皮含量丰富,主要存在于表皮的最外层,对表皮的屏障功能起着重要的作用。近年的研究发现,寻常性鱼鳞病及特应性皮炎的发病与丝聚蛋白的编码基因Flg有明显的相关性。Flg失功能突变使表皮丝聚蛋白表达减少或缺如,导致表皮的屏障功能受损,使环境中的变应原、刺激物等易于进入表皮,继而引起各种病理生理变化。     

Profile of epidermal metabolic activity in autosomal dominant ichthyosis and small bowel disorders

The in vitro incorporation of 14C acetate by the epidermis has been studied in patients with autosomal dominant ichthyosis and in patients with a dry, itchy, slightly scaly skin associated with a disorder of the small bowel. Analysis of 14C acetate containing lipid moieties by thin layer chromatography has indicated that there are both quantitative and qualitative differences in the uptake of 14C acetate between autosomal dominant ichthyosis and normal. In particular an increased incorporation into the triglyceride and phosphatidyl choline fractions was noted. No such differences were apparent in those patients with disorders of the small bowel. In addition the in vitro incorporation of radioactively labelled thymidine, proline and histidine has been studied in these patients. In both groups of patients the rate of incorporation of tritiated thymidine and histidine into epidermal macromolecules was found not to differ significantly from normal. On the other hand the rate of incorporation of tritiated proline was increased in both groups of patients.     

Retinol-binding protein in serum and epidermis of patients with ichthyosis vulgaris

Fifteen patients with ichthyosis vulgaris of the dominant type were studied by immunological techniques for the detection of retinol-binding protein (RBP) in serum and epidermis. The serum RBP levels were all within the normal range thus indicating a normal vitamin A status in the blood. The epidermal RBP pattern was abnormal as judged by an indirect immuno-fluorescence technique. It is suggested that the decreased binding of RBP to ichthyosis epidermis is secondary to a reduced number of granular cells and not due to a disturbed vitamin A uptake per se.     

Monoclonal antibody 4F2 reactive with basal layer keratinocytes: studies in the normal and a hyperproliferative state

To establish a method for separating different keratinocyte subpopulations in the epidermis, we studied the specificity of monoclonal antibody 4F2 for keratinocytes. Preliminary screening experiments had previously demonstrated 4F2 reactivity with the epidermis. 4F2 reacted with a subpopulation (19.29 +/- 5.23%) of human epidermal cells in suspension. The membrane antigen identified by 4F2 continues to be expressed by cultured keratinocytes. In frozen tissue section using an indirect immunofluorescence technique, the 4F2-positive cells in the basal layer are sharply demarcated from the negative suprabasilar layers. Even in the hyperproliferative state of psoriasis, the 4F2 reactivity is confined to the basal layer. Cell suspensions of psoriatic epidermis demonstrated a greater percentage of reactivity with 4F2 (49.51% +/- 6.50%), probably reflecting the expanded population of basal layer cells. Monoclonal 4F2, therefore, reacts with a membrane antigen present on basal keratinocytes, and provides a probe for use in the isolation of the basal keratinocyte subpopulation. Thus, this antibody should be useful in studies of normal and aberrant differentiation of the epidermis.     

四种遗传性鱼鳞病临床表型和组织病理的比较研究

目的：比较四种鱼鳞病（寻常型鱼鳞病；X-连锁鱼鳞病；板层状鱼鳞病；大疱性鱼鳞病）临床表型和组织病理的差异。方法：对鱼鳞病患者进行临床表型分析、皮损组织病理和电镜检测。结果：1．寻常型鱼鳞病：浅褐色多角形鳞屑，组织病理角化过度伴局灶性角化不全，颗粒层变薄；电镜示透明角质颗粒数量减少且结构异常。2．X-连锁鱼鳞病：粗大深褐色鳞屑，组织病理中度角化过度，颗粒层正常或稍增厚。3．板层状鱼鳞病：粗大黑色板样鳞屑，组织病理致密板层状角化过度伴灶性角化不全，局灶颗粒层增厚，棘层不规则增生肥厚；电镜示角质包膜异常。4．大疱性鱼鳞病：棕色疣状鳞屑，组织病理致密角化过度，颗粒层显著增厚，颗粒层及棘层中上部细胞核周空泡改变。电镜可见异常角蛋白分布于基底层以上全部表皮，棘细胞松解，颗粒层细胞核周围有异常聚集的角蛋白丝。结论：四种鱼鳞病患者临床表型、组织病理和严重程度有明显差异。     

Psoriasis occurring in lamellar ichthyosis: response to Epilyt

Psoriasis occurring in a patient with lamellar ichthyosis is reported. Similar self-limited episodes had occurred earlier. On histopathologic examination of a biopsy specimen, an eruptive plaque showed parakeratosis and a reduction in the granular layer. Electrophoretic analysis of the keratins isolated from the epidermis of a plaque showed a reduction in the amount of the 67 kd polypeptide compared to the keratins of ichthyosis epidermis. Both of these findings support the diagnosis of psoriasis. Epilyt, applied daily, was effective in removing scales.     

Dermatoglyphic characteristics in hereditary ichthyosis

Examination of 530 dermatoglyphic patterns of the palms and fingers in 265 patients with 5 nosologic forms of hereditary ichthyoses (autosomal dominant ichthyosis vulgaris, X-linked, congenital, lamellar, epidermolytic ichthyoses) have revealed significant differences in the pattern intensities and in the incidence rate of certain types of these patterns, associated with this or that form of ichthyosis; abnormalities in the flexor wrinkles of the ridge skin have been observed in all the studied forms of the disease, except the X-linked condition. The studies have revealed an abnormal roughness of the papillae on the epidermal ridges in epidermolytic ichthyosis and an obliterated dermatoglyphic pattern in lamellar ichthyosis. The detected changes in the ridge skin and the dermatoglyphic phenotypes may be useful for the differential diagnosis of these conditions.     

Ichthyosis vulgaris: identification of a defect in synthesis of filaggrin correlated with an absence of keratohyaline granules

Ichthyosis vulgaris is an autosomal dominant disorder of keratinization characterized histologically by absent or reduced keratohyaline granules in the epidermis and mild hyperkeratosis. The basic defect in ichthyosis vulgaris is unknown. We have tested for the presence of filaggrin and its precursor, profilaggrin, in the epidermis of affected and unaffected individuals from 2 families with ichthyosis vulgaris and correlated its presence and relative quantity with ultrastructure findings in the same individuals. Filaggrin was present on stained sodium dodecyl sulfate gels and immunoblots of epidermal proteins from controls and unaffected family members. It was absent from the more severely affected individuals in each family and reduced in intensity in the less severely affected family members. Immunohistology in controls showed localization of filaggrin-related protein in the stratum corneum and within the granular layer. In contrast, tissue from affected individuals showed little or no reaction. Electron microscopic studies showed that keratohyaline granules were absent in 3 severely affected individuals, and reduced in number in the others. The relative amount of keratohyalin by electron microscopy correlated with the amount of filaggrin detectable on immunoblots. The stratum corneum was thicker than in normals but showed the typical "keratin pattern" staining suggesting that filaggrin is not essential for keratin filament aggregation and may have another function in vivo. We have demonstrated that the structural proteins, profilaggrin and filaggrin, are reduced or absent in 5 patients from 2 pedigrees with ichthyosis vulgaris. This biochemical abnormality correlates with the morphologic reduction in the amount of keratohyalin, and with the clinical severity of the disorder.     

AUTORADIOGRAPHIC STUDY ON THE UPTAKE OF 3H-AMINO ACIDS BY NORMAL AND PATHOLOGICAL HUMAN EPIDERMIS

Incorporation of some tritiated amino acids in normal and pathological human epidermis was studied by autoradiography. In the case of methionine, glycine and histidine, silver particles were detected in the cytoplasm of nucleated cells. In a test after 1–2 hours incubation the density of these particles was greater in the upper squamous cell layer as compared with the lower layer. In the case of tyrosine, phenylalanine, valine and leucine, the particles were distributed more densely in the lower squamous layer as compared with other layers. After 4–6 hours incubation, all of these amino acids were observed more uniformly in the entire epidermal layers except the horny layer, the labelling being highest in the basal layer. It may be interpreted here that the incorporation into the upper layers after 1–2 hours is related to the enzyme activity or energy of the epidermal cell, and that into the lower layers, especially the basal layer after 4–6 hours, to protein synthesis accompanied by cellular proliferation. In parakeratotic epidermis associated with psoriasis, chronic dermatitis, verruca vulgaris, etc., the radioactive particles of ³H-tyrosine, phenylalanine, valine and leucine were found in both the upper and lower squamous layers after 1–2 hours incubation, while they were observed only in the lower squamous layer in the normal epidermis. A similar tendency was found in epidermis with hyper- and parakeratosis induced by radiation with ultraviolet lights or stripping of the horny layer. The more the parakeratotic changes, the greater is the tendency. It is therefore suggested that protein synthesis or metabolism occuring in each level of the epidermis is accelerated by a shortened life-span in the parakeratotic epidermis. In ichthyosis vulgaris, the so-called keratogenous zone is distinctly seen as a narrow layer immediately beneath the horny layer, showing fairly intensive labelling from ³H-glycine. In the dyskeratotic cells of skin cancer and molluscum, a few silver particles were distributed with some amino acids.     

Study of normal and pathological keratinization using anti-keratin polypeptide sera (author's transl)

Anti-keratin polypeptide sera were obtained against the different bands of polyacrylamide gel electrophoresis of fibrous proteins of stratum corneum derived from normal human epidermis. The sera were tested by indirect immunofluorescence of immunoperoxidase techniques. It was demonstrated that antibodies against P1 and P2 polypeptides, of MW 67,000 and 62,000 daltons respectively, were directed towards cytoplasmic antigens of keratinocytes of the upper Malpighian layers, while no labelling could be detected in the basal cell layer. Anti-P3 polypeptide sera and the anti-whole keratin serum labelled the whole epidermis, including the basal cell layer. Ultrastructural immuno-labelling performed on free epidermal cells obtained after trypsinization demonstrated that receptors for anti-P1 polypeptide sera were tonofilaments. These results showed that some keratin components (P1 and P2 polypeptides) might be absent in basal cell tonofilaments. This in in favour of various differentiation stages of the keratinizing cells. Keratin polypeptide 1 could be a useful marker of keratinization. According to preliminary studies, the expression of this keratin antigens was markedly disturbed in tumors such as basal and squamous cell carcinoma, in warts and in ichthyosis.     

Differentiation-associated localization of small prolne-rich rotein in normal and diseased human skin

Summary The expression of SPRR (small proline-rich protein) was investigated in normal human skin and in diseased skin from patients with psoriasis, squamous cell carcinoma, basal cell epithelioma. Naevus pigmentosus, ichthyosis vulgaris and several inflammatory skin diseases, by immunohistochemical staining. A polyclonal antibody was raised against a synthetic peptide for a C-terminal common region for SPRR l and SPRR 3. In immunoblot analysis, a positive band of 18kDa was detected, which showed the presence of SPRR l in human epidermal keratinocytes. In normal epidermis, positive staining for SPRK was observed in keratinocytes in the granular layer and the uppermost or two spinous cell layers, with no staining of the other spinous or basal layers. The staining was obvious at the cell periphery, weak at the cytoplasm, and absent in the nucleus. Staining was observed in several outer layers of the follicular infundibulum to the isthmus. No staining was detected in the inner root sheath of the hair follicles, hair matrix, sebaceous gland, eccrine gland, eccrine duct, melanocytes. Langerhans cells or fibroblasts. The arrectores pilorum, striated muscles, muscle layers of vessels, and myoepithelia of eccrine gland, were weakly stained. In psoriatic skin, stained keratinocytes were distributed in the spinous cell layers except for the basal layer, in ichthyosis vulgaris. SPRR was barely expressed in the uppermost living cell layers of the epidermis in epidermolytic hyperkeratosis. degenerated squamous cells widely expressed SPRR. In Darier's disease, dyskeratolic cells were clearly stained. In squamous cell carcinoma, staining was observed in keratotic cells around horny pearls. In basal cell epithelioma, naevus pigmentosus, and malignant melanoma, the tumour cells or naevus cells were not stained. The distribution of SPRR was similar to that of involucrin in normal and several diseased skin, except for ichthyosis vulgaris. We conclude that SPRR is expressed in close association with epidermal differentiation in normal skin and skin diseases. The alteration of the expression of the proteins correlated to terminal differentiation, and differs from disease to disease.     

Epidermolysis bullosa simplex Dowling-Meara due to an arginine to cysteine substitution in exon 1 of keratin 14

Epidermolysis bullosa simplex (EBS) is a blistering disorder affecting the basal layer of the epidermis usually inherited in an autosomal dominant fashion. Most cases are caused by mutations in the genes encoding keratin 5 (K5) and keratin 14 (K14) and are characterized by cytolysis within the basal layer of the epidermis. We report a patient manifesting the Dowling-Meara variant of EBS in whom we characterized a cytosine to thymine transition at codon 125 (R125C) in K14. This missense mutation is located at the amino terminus of the helical rod domain of the keratin 14 molecule, resulting in defective pairing with K5, thereby disrupting keratin tonofibril integrity.     

Histologic and ultrastructural features of the ichthyotic skin in X-linked dominant chondrodysplasia punctata

The ichthyotic skin in X-linked dominant chondrodysplasia punctata was investigated in a four-week-old baby and a fourteen-year-old girl. Histologically, the ichthyosiform erythroderma of the newborn and the ichthyosis of the older child presented as a retention hyperkeratosis with several distinctive features such as calcification of the keratotic follicular plugs, atrophy of the hair follicles and focal hyperpigmentation of the basal keratinocytes. On ultrastructural examination, small to medium sized vacuoles were regularly seen in the thinned granular layer. Some of these vacuoles contained needle-like calcium inclusions. The histologic and ultrastructural findings are therefore characteristic for this rare type of ichthyosis.     

Histologic distribution of staining by a monoclonal antibody (psi-3) in psoriasis and occurrence of psi-3 antigen in other cutaneous diseases

psi-3 is a monoclonal antibody that recognizes a 135,000 molecular weight structural component of maturing keratinocytes in psoriasis (the psi-3 antigen) but fails to bind to any constituent of keratinocytes in normal epidermis. This paper describes the occurrence of the psi-3 antigen in a variety of dermatopathologic conditions using immunoperoxidase (biotin-avidin-peroxidase) and immunofluorescence methods which show excellent concordance. In 35 of 36 specimens of psoriasis vulgaris, psi-3 antibody consistently immunolabels the cytoplasm of keratinocytes above the basal layer. At the edges of psoriatic plaques, psi-3 antibody staining extends for a variable distance into lesion-free epidermis. A similar pattern has been found in a certain number of other conditions described in the paper, including squamous cell carcinoma and condyloma acuminatum, but not Darier's disease, basal cell carcinoma, nor lamellar ichthyosis. In all but one condition, the outermost or basal layer of cells is never stained. The only disease in which the lowermost cell layer is stained is a lichen planus-like lesion. The occurrence of psi-3 antigen cannot be correlated with any histologic feature of psoriasis such as acanthosis, loss of the granular layer, or hyperproliferation. The antigen appears to be a unique keratinocyte constituent which is expressed in certain pathologic conditions and which is not detected by any other histologic or immunophenotyping method. It is a potentially valuable addition to the panel of antibodies available for characterizing epithelial cells.