Karen A. Holbrook

Calcium regulation of growth and differentiation of mouse epidermal cells in culture

Modification of the ionic calcium concentration in the culture medium markedly alters the pattern of proliferation and differentiation in cultured mouse epidermal cells. When medium calcium is lowered to 0.05--0.1 mM, keratinocytes proliferate rapidly with a high growth fraction and do not stratify, but continue to synthesize keratin. The cells grow as a monolayer for several months and can be subcultured and cloned in low Ca++ medium. Ultrastructural examination of cells cultured under low Ca++ conditions reveals widened intercellular spaces, abundant microvilli and perinuclear organization of tonofilaments and cellular organelles. Desmosomes are absent. Epidermal cells growing as a monolayer in low Ca++ can be induced to terminally differentiate by adding calcium to the level normally found in the culture medium (1.2 mM). Cell-to-cell contact occurs rapidly and desmosomes form within 2 hr. The cells stratify by 1--2 days and terminally differentiate with cell sloughing by 3--4 days. After Ca++ addition, DNA synthesis decreases with a lag of 5--10 hr and is totally inhibited within 34 hr. In contrast, RNA and protein synthesis continue at 40--50% of the low Ca++ level at day 3, a time when many cells are detaching from the culture dish. Keratin synthesis is unaffected by the Ca++ switch.

Revised clinical and laboratory criteria for subtypes of inherited epidermolysis bullosa: A consensus report by the Subcommittee on Diagnosis and Classification of the National Epidermolysis Bullosa Registry

Inherited epidermolysis bullosa encompasses a number of diseases, with the common finding of blister formation after minor mechanical trauma to the skin. In some forms significant, if not eventually fatal, extracutaneous disease activity may occur. In recent years application of newer technologies has contributed substantially to an overall understanding of this collection of inherited diseases. Concurrently, many new phenotypes have been recognized, in part the result of ongoing prospective patient registries in the United States and abroad. Unfortunately, this has resulted in a massive literature that may appear to be confounded by seemingly excessive or arbitrary subdivision of epidermolysis bullosa variants. With these concerns in mind a subcommittee was established by the National Epidermolysis Bullosa Registry to summarize the current literature and to make recommendations as to the best clinical and laboratory criteria for the practical diagnosis and subclassification of patients with inherited epidermolysis bullosa.

Calcium regulation of cell-cell contact and differentiation of epidermal cells in culture. An ultrastructural study.

Calcium modulation of keratinocyte growth in culture was studied by both transmission (TEM) and scanning electron microscopy (SEM). Under standard culture conditions (1.2-1.8 mM calcium), cells were connected by desmosomes and stratified to 4-6 cell layers. Many aspects of in vitro epidermal maturation were analogous to the in vivo process, with formation of keratohyalin granules, loss of nuclei, formation of cornified envelopes and shedding of cornified cells containing keratin filaments. When the medium calcium concentration was lowered to 0.02-0.1 mM, the pattern of keratinocyte growth was strikingly changed. Cells grew as a monolayer with no desmosomal connections and proliferated rapidly, shedding largely non-cornified cells into the medium. Large bundles of keratin filaments were concentrated in the perinuclear cytoplasm. The elevation of extracellular calcium to 1.2 mM induced low calcium keratinocytes to stratify, keratinize and cornify in a manner analogous to that seen when plated in standard calcium medium. The earliest calcium-induced ultrastructural change was the asymmetric formation of desmosomes between adjacent cells. Desmosomal plaques with associated tonofilaments were observed 5 min after calcium addition; symmetric desmosomes were formed within 1-2 h. This system is presented as a useful model for the study of the regulation of desmosome assembly and disassembly.

X-Linked Cutis Laxa: Defective Cross-Link Formation in Collagen Due to Decreased Lysyl Oxidase Activity

We studied several members of a family with an X-linked form of cutis laxa; the affected males have mild skin laxity, a characteristic facies, skeletal abnormalities, structural abnormalities of the genitourinary tract, and low serum copper levels. The activity of lysyl oxidase, a copper-dependent enzyme involved in cross-link formation in collagen, was decreased in skin-biopsy specimens (13 to 26 per cent of normal) and in culture medium from cells to two affected males (15 to 20 per cent of normal). Immunoreactive lysyl oxidase from skin of both patients was virtually undetectable by immunodiffusion assay. The amounts of lysyl-derived aldehydes (the product formed in collagen and elastin by lysyl oxidase) and of cross-links formed from these products were decreased in dermal fibroblasts in culture. Collagen extractability from these cells was increased in culture. These findings suggest that lysyl oxidase deficiency provides the biochemical basis of the X-linked form of cutis laxa.

Clinical and ultrastructural heterogeneity of type IV Ehlers-Danlos syndrome

SummaryEhlers-Danlos syndrome (EDS) type IV is a clinically and genetically heterogeneous disorder characterized by thin skin, prominent venous vascular markings, markedly increased bruising, and an increased likelihood of large bowel and large artery rupture. We studied two type IV EDS patients. Both have decreased amounts of type III collagen in skin, but ultrastructural examination of dermis showed massive dilation of rough endoplasmic reticulum in dermal fibroblasts in one, but not the other. Both had a major population of collagen fibrils of small diameter. Although previous studies suggested absent synthesis of type III collagen as the hallmark of one type of EDS IV, several abnormalities in metabolism of that type of collagen may be responsible for the phenotype in these disorders. Such disorders are likely to provide better understanding of the function of specific collagens in tissues.

The appearance, density and distribution of melanocytes in human embryonic and fetal skin revealed by the anti-melanoma monoclonal antibody, HMB-45

SummaryThe presence, densities, and patterns of distribution of melanocytes in the epidermis of human embryos and fetuses, ranging in age from 40 d to 140 d estimated gestational age (EGA), were studied using the HMB-45 monoclonal antibody that recognizes an antigen in melanoma cells and fetal melanocytes. Immunostained sections of skin and epidermal sheets revealed dendritic melanocytes within the basal or intermediate layers of 50 d EGA and older skin. Melanocytes could not be identified by immunostaining or electron microscopy in younger (40–50 d EGA) epidermis or in cultured epidermal cells from these specimens. However, skin from a 45 d EGA embryo grown in organ culture for 11 d stained positively with HMB-45, suggesting that melanocytes are present at that age either in the epidermis or dermis of the explant. Double-labeling experiments using ATPase and HMB-45 confirmed the specificity of HMB-45 for melanocytes and demonstrated that melanocytes and Langerhans cells are nonoverlapping populations. Melanocytes were present in the embryonic epidermis in relatively high numbers (mean value of ∼1050 cells/mm2); they increased in density to ∼2300 cells/mm2 during the late first trimester and early second trimester, then declined during later stages of development to a density of ∼800 cells/mm2, within the range of values for the newborn child and young adult. Equivalent numbers of melanocytes were recognized by silver staining and with the HMB-45 antibody in an 87 d EGA test sample, indicating that HMB-45 reacted with the total melanocytic population. Melanocytes appeared to be distributed in epidermal sheets in a regular pattern. Statistical tests used to evaluate the randomness of a population revealed a tendency toward a non-random distribution in specimens younger than 80 d EGA, just prior to appendage formation and epidermal stratification into multiple layers, however there was variability in the degree of randomness for any given age. The results of this study have closed the gap in timing between the conclusion of neural crest formation and migration (around 6 weeks) and the appearance of melanocytes in the skin between 40–50 d EGA.

Fatal cardiovascular disease and cutis laxa following acute febrile neutrophilic dermatosis

Acute neutrophilic dermatosis (Sweet syndrome) is a benign self-limited disease in adults. A child with apparent evolution of acute neutrophilic dermatosis to postinflammatory cutis laxa and elastolysis then developed fatal vascular involvement. One other patient with postinflammatory cutis laxa with aortic regurgitation and sudden fatal unrecognized occlusive coronary arterial disease is discussed. If cardiovascular symptoms or signs develop during the course of Sweet syndrome or postinflammatory cutis laxa, a thorough investigation is warranted to rule out potentially fatal coronary arterial disease. Coronary bypass surgery may be the only effective treatment for the severely fibrosed proximal coronary arterial system.

Abnormal alpha 2-chain in type I collagen from a patient with a form of osteogenesis imperfecta.

Dermal fibroblasts in culture from a woman with a mild to moderate form of osteogenesis imperfecta synthesize two species of the pro alpha 2-chain of type I procollagen. One chain is normal. The abnormal chain has a slightly faster mobility than normal during electrophoresis in sodium dodecyl sulfate polyacrylamide gels. Analysis of cyanogen bromide peptides of the pro alpha-chain, the alpha-chain, and of the mammalian collagenase cleavage products of the pro alpha- and alpha-chains indicates that the abnormality is confined to the alpha 2(I)CB4 fragment and is consistent with loss of a short triple-helical segment. Type I collagen production was decreased, perhaps because the molecules that contained the abnormal chain were unstable, with a resultant alteration in the ratio of type III to type I collagen secreted into culture medium. Collagen fibrils in bone and skin had a normal periodicity but their diameters were 50% of control; the bone matrix was undermineralized. The structural abnormality in the alpha 2(I)-chain in this patient may affect molecular stability, intermolecular interactions, and collagen-mineral relationships that act to decrease the collagen content of tissues and affect the mineralization of bone.

Postinflammatory elastolysis and cutis laxa: A case report

One of the rarest forms of cutis laxa is postinflammatory elastolysis and cutis laxa, a disease previously reported only in children in Africa and South America. This disease is characterized by an urticarial or papular eruption followed by acute destruction of elastic tissue that results in atrophy and severe disfigurement. It is distinguished from anetoderma and acquired cutis laxa by its clinical features, its occurrence in young children, and its relatively benign course. This article describes the first case of postinflammatory elastolysis and cutis laxa reported in a white child from North America.

Growth and Differentiation of Mouse Epidermal Cells in Culture: Effects of Extracellular Calcium

The pattern of proliferation and differentiation in cultured mouse epidermal cells in markedly altered by modifying the ionic calcium concentration in the culture medium. When medium calcium is lowered from 1.44 mM to 0.05-0.1 mM, keratinocytes proliferate rapidly with a high growth fraction, do not stratify, but continue to synthesize keratin. The cells grow as a monolayer for several months and can be subcultured in low Ca++ medium. Ultrastructural examination of cells cultured under low Ca++ conditions reveals widened intercellular spaces with an absence of desmosomes. Microvilli are numerous, and tonofilaments and cellular organelles are organized perinuclearly. Epidermal cells growing as a monolayer in low Ca++ can be identified to terminally differentiate by adding calcium to the level normally found in the culture medium. Contact between cells occurs rapidly and desmosomes form within 2 hours. The cells stratify in 1-2 days and terminally differentiate in 3-4 days. After Ca++ addition, DNA synthesis decreases after a lag of 5-10 hours and is totally inhibited within 36 hours. In contrast, RNA and protein synthesis continue at 40-50% of the control level at Day 3, a time when many cells are detaching from the culture dish. Keratin synthesis is unaffected by the Ca++ switch. Manipulation of epidermal proliferation and differentiation by altering extracellular calcium levels should enhance the usefulness of epidermal cell cultures in the study of differentiation and carcinogenesis.