Irwin A. Schafer

Identification of monoclonal antibodies that recognize novel epitopes in native chondroitin/dermatan sulfate glycosaminoglycan chains: their use in mapping functionally distinct domains of human skin.

Five monoclonal antibodies (MAb), 7D4, 4C3, 6C3, 4D3, and 3C5, were produced in mice immunized with high buoyant density embryonic chick bone marrow proteoglycans (PGs) as antigen. All of these MAb recognized epitopes in native chick bone marrow and cartilage PGs which could be selectively removed by chondroitinase ABC and chondroitinase AC II, indicating that their epitopes were present in chondroitin sulfate glycosaminoglycans (GAGs). These MAb recognized epitopes present in purified cartilage PGs obtained from a wide variety of different vertebrate species. However, none of the new MAb detected epitopes in Swarm rat chondrosarcoma PG. On the basis of these results, we propose that these MAb recognize novel epitopes located in chondroitin sulfate/dermatan sulfate glycosaminoglycan (CS/DS GAG) chains, representing at least four and possibly five different structures. Immunocytochemical studies have shown that the epitopes identified by these new MAb are differentially distributed in tissues. All of these MAb immunocytochemically detected epitopes in embryonic chick cartilage and bone marrow. Three of them (4C3, 7D4, and 6C3) recognized epitopes in adult human skin. All three detected epitopes in the epidermis, one (6C3) strongly detected epitopes in the papillary dermis, and two (4C3, 7D4) detected epitopes in the reticular dermis. Immunostaining patterns in skin using the new MAb directed against native CS/DS structures were distinctly different from those obtained using MAb against the common CS isomers. The distribution of these CS epitopes in functionally distinct domains of different tissues implies that these structures have functional and biological significance.

Comparative observation of fibroblasts derived from the papillary and reticular dermis of infants and adults: growth kinetics, packing density at confluence and surface morphology

We have confirmed the reports of Harper and Grove (Science, 204 (1979) 526-527), and Azzarone and Macierira-Coehlo (J. Cell Sci., 57 (1982) 177-187) that fibroblasts derived from the papillary dermis have greater in vitro growth potential and longer replicative lifespans than genomically identical fibroblasts derived from the reticular dermis. In addition we demonstrate that the kinetics of cell replication differ for papillary and reticular fibroblasts derived from infant and adult donors. Infant papillary fibroblasts replicate at faster rates than reticular fibroblasts throughout the growth cycle. Adult papillary and reticular fibroblasts replicate at similar rates at low cell densities, but exponential growth of reticular fibroblasts slows at lower cell densities than papillary fibroblasts suggesting that they are more sensitive to density-dependent inhibition of replication. The surface morphologies of reticular fibroblasts and papillary fibroblasts at confluence correlate with their growth kinetics. The decreased cell yields of reticular fibroblasts appears related to the spreading behaviors of individual cells which stretch and occupy more area of the growth surface than do papillary fibroblasts. These data and the reports cited clearly show that one must account for the presence of at least two distinct populations of dermal fibroblasts when examining their biological properties in vitro.

The interaction of human papillary and reticular fibroblasts and human keratinocytes in the contraction of three-dimensional floating collagen lattices☆

Fibroblasts derived from the papillary and reticular dermis of human skin and human keratinocytes show differences in their abilities to contract floating three-dimensional gels constructed from type I collagen. Reticular fibroblasts produce greater gel contraction than papillary fibroblasts. When equal numbers of papillary and reticular fibroblasts are mixed in the gels, papillary fibroblasts consistently inhibit gel contraction by reticular fibroblasts indicating interaction between these cell types in the contraction process. Surprisingly, keratinocytes alone produce greater gel contraction than that produced by either fibroblast type. Cooperativity in the gel contraction process is observed when fibroblasts are incorporated into the collagen matrix and keratinocytes are seeded onto the gel surface. Keratinocytes and dermal fibroblasts adhere to the collagen fibril to induce gel contraction by different mechanisms. Fibroblast contraction of collagen gels does not require fibronectin but is a serum-dependent reaction. In contrast, keratinocyte contraction of collagen gels occurs in a serum-free environment. Polyclonal, affinity-purified antibodies to human plasma fibronectin at high concentrations do not inhibit gel contraction by keratinocytes, making unlikely the possibility that fibronectin synthesized by the keratinocyte is a significant factor in the gel contraction process. We are currently examining the possibilities either that keratinocytes are synthesizing other adhesion proteins or that receptors on the cell surface can interact directly with the collagen fiber.

Keratinocyte allografts accelerate healing of split-thickness donor sites: applications for improved treatment of burns.

Grafting with split-thickness autograft skin remains the most effective method for treating burn wounds. When insufficient donor sites are present, decreasing the time required for healing of available donor sites permits more frequent reharvests to continue the grafting process. Cultured human keratinocytes speed wound healing by providing cover and by producing growth factors and extracellular matrix proteins. In this study we compare the rates of healing induced by allografts of cultured keratinocytes applied to split-thickness donor sites with healing by a standard treatment. Sheets of cultured human keratinocytes derived from neonatal foreskins are applied to a portion of a split-thickness donor site while the remainder is covered with a temporary skin substitute. The wound is inspected at 5, 7, 9, 11, 14, 17, 20, and 23 days. Biopsies are obtained at 7 days for light and electron microscopy. In 10 patients the average time to healing for sites covered with keratinocytes was 6.6 +/- 1.96 days compared with 12.6 +/- 4.32 days for control sites (p < 0.002). By day 7 most keratinocyte-covered sites showed reepithelization with the formation of a basement membrane and hemidesmosomes at the dermal-epidermal junction. Control areas were unhealed without epithelial coverage. The reepithelized donor sites from three patients treated with cultured keratinocytes were reharvested. In each case these grafts took, and they were equivalent to skin used from donor sites harvested for the first time. Keratinocyte allografts speed healing of split-thickness donor sites, thereby increasing the availability of autograft skin for burn wound coverage.

Changes in the composition and structure of glycosaminoglycans in the human placenta during development.

Extract: Glycosaminoglycans (acid mucopolysaccharides) are ubiquitous in their distribution in the body, yet information as to their biologic function is scanty. Studies of their structure and physical properties in solution suggest that they could function as gel nitration and exchange resins in vivo, thereby playing an important role in regulation of the passage of molecules through the ground substance of connective tissue. The glycosaminoglycan(s) (GAG) composition of the human placenta and the molecular structure of specific GAG has been studied by chemical, enzymatic, and physical methods at 12–18 weeks and at 40 weeks gestational age to explore this postulated structure-function relation.The young placenta contained more GAG (222 mg/100 mg dry defatted tissue) than did the term placenta (155 mg/100 g dry defatted tissue). Sulfated GAG comprised 56% of the total GAG in the young placenta versus 74% in the term placenta due to increased concentrations of dermatan sulfate (25% term versus 13% young placenta) and heparan sulfate (22% term versus 15% young placenta). Ghondroitin was a major component in the young placenta and comprised 22% of the total GAG, whereas the term placenta contained only 9%. Both young and term placenta showed similar quantities of hyaluronic acid and chondroitin 4− and 6-sulfates. Ghondroitin sulfate from the young placenta differed from the polymer in the term placenta in that it contained a higher proportion of unsulfated dissaccharides. Differences were also found in the molecular structure of dermatan sulfate. Hyaluronidase digestion of purified dermatan sulfate from young placenta produced a 50% reduction in average molecular weight compared with a 30% reduction in the molecular weight of dermatan sulfate isolated from term placenta. The smaller molecular weight fragments of dermatan sulfate from young placenta indicate differences in molecular structure due either to the number of glucuronic acid substitutions or to their position in the polymer chain, or to changes in the concentration of hybrid molecules.Speculation: The age-related changes in the composition and molecular structure of placental glycosaminoglycans will result in ground substance gels of differing physical properties. This could alter the transport of molecules through placental connective tissue and affect the rate of fetal growth.

Human keratinocytes cultured on collagen gels form an epidermis which synthesizes bullous pemphigoid antigens and α2β1 integrins and secretes laminin, type IV collagen, and heparan sulfate proteoglycan at the basal cell surface

Single cell suspensions of human keratinocytes when seeded onto floating three-dimensional gels constructed with type I collagen form a tissue resembling epidermis. These morphogenetic events occur in a serum-free environment in the absence of fibroblasts. Light and transmission electron microscopy show that cells form a basal layer plus suprabasilar cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum. The suprabasilar keratinocyte layers show morphologies which resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-fillagrin granules. The basal cell layer differs from skin in vivo in that there is no connection to a basement membrane via hemidesmosomes. Cells in the basal layers are polarized as evidenced by the secretion of type IV collagen, heparan sulfate proteoglycans, and laminin at the cell membrane interface with the collagen gel. These proteins are not organized into a cytological basement membrane. Bullous pemphigoid antigen, a protein component of hemidesmosomes, is synthesized by basal keratinocytes, but like the basement membrane proteins it is not incorporated into a definable cytological structure. Keratinocytes in the basal and suprabasilar layers also synthesize alpha 2 beta 1 integrins. The mechanisms of keratinocyte adhesion to the gel may be through the interactions of this cell surface receptor with laminin and type IV collagen synthesized by the cell and/or direct interactions between the receptor and type I collagen within the gel. This in vitro experimental system is a useful model for defining the molecular events which control the formation and turnover of basement membranes and the mechanisms by which keratinocytes adhere to type I collagen when sheets of keratinocytes are used clinically for wound coverage.

NEUROCHEMISTRY OF THE MUCOPOLYSACCHARIDOSES: BRAIN GLYCOSAMINOGLYCANS, LIPIDS AND LYSOSOMAL ENZYMES IN MUCOPOLYSACCHARIDOSIS TYPE III B (α-N-ACETYLGLUCOSAMINIDASE DEFICIENCY)

Glycosaminoglycans, lipids and lysosomal enzymes were measured in brain, liver and spleen of a patient with mucopolysaccharidosis Type III B (α‐N‐Acetylglucosaminidase deficiency).

Cell-associated pentosidine as a marker of aging in human diploid cells in vitro and in vivo.

Cellular aging is characterized by alterations at both the morphological and molecular levels, some of which are decreased mitotic rate, increased cytoplasmic vacuolization, and changes in intrinsic cellular constituents (Stanulis-Praeger, 1987. Mech. Ageing Dev. 38, 1-48). In the present investigation, glycoxidation is studied as a marker for cellular aging by measuring cell-associated pentosidine levels in human skin fibroblasts as a function of replicative life span and in human peripheral blood T lymphocytes as a function of chronological age. Fibroblasts were isolated from culture by detachment/centrifugation while lymphocytes were isolated from blood by a Ficoll-Paque/Lympho-Kwik T-Cell Prep technique. Pentosidine levels were measured in acid-hydrolyzed cell pellet suspensions by high-pressure liquid chromatography. Results show that pentosidine was detected in early and late cultured reticular and papillary fibroblasts. Pentosidine, expressed as either protein, DNA, or cell number, significantly (P < 0.0006) increased with in vitro passage and was significantly (P < 0.01) related to cell proliferation as measured by cell density and cell doublings per day during culture. Cell-associated pentosidine was measured in T lymphocytes isolated from healthy, diabetic, and uremic individuals. In healthy controls, levels significantly (P < 0.0003) increased with age. In uremic individuals, a large variation was observed with many values above the 95% confidence intervals determined for controls. Since a previous study showed that plasma pentosidine in healthy subjects does not increase with age, these results suggest that cellular turnover perhaps coupled to a deterioration in cellular anti-glycoxidation defensive mechanisms play a substantial role in explaining increased pentosidine concentrations during cellular aging.

Multiangle light scattering flow photometry of cultured human fibroblasts: comparison of normal cells with a mutant line containing cytoplasmic inclusions.

Multi-angle light scattering flow photometry was used to study the light scattering properties of normal cultured fibroblasts and a mutant fibroblast line containing cytoplasmic lysosomal inclusions. The effect of glutaraldehyde fixation on the light scattering properties of the cells was also examined and correlated with their ultrastructure. Normal fibroblasts showed uniform organelle distribution with few vacuoles or dense bodies in the cytoplasm while the mutant line showed abnormal cytoplasmic inclusions of varying morphology, density and lucency. As predicted by light scattering theory, the mutant cells containing the cytoplasmic inclusions scattered more light at large angles (greater than theta = 1.85 degrees) than did the normal cells. Glutaraldehyde fixation decreased light scattering at small angles (less than theta = 1.85 degrees), increased light scattering at larger angles (greater than theta = 1.85 degrees) in both normal and mutant cells and enhanced resolution of the light scattering signatures. The mutant line scattered 2-3 times more light at a wide angle (greater than theta = 12.74 degrees) than did the normal cells. These data suggest that abnormal lysosomal storage inclusion bodies in the cytoplasm of the cells can be detected by differential light scattering methods.

Glycosaminoglycan composition of human amniotic fluid.

The glycosaminoglycan composition of human amniotic fluid between 12–21 weeks gestation has been studied by Dowex column chromatography coupled with enzymatic analyses of the specific glycosaminoglycan in each column fraction. The total uronic acid recovered from the columns consisted of “glycopeptides” (7%), hyaluronic acid (34%), nonsulfated chondroitin (14%), chondroitin-4-sulfate (13%), chondroitin-6-sulfate (20%), dermatan sulfate (5%), and heparan sulfate (6%). Based on these studies a simple screening procedure was devised to detect increased quantities of heparan sulfate and dermatan sulfate in 5–10-ml samples of amniotic fluid and tested in the antenatal diagnosis of Hurler and Hunters syndrome. A false negative result was recorded in a Hunter fluid obtained early gestation and a false positive result recorded in a normal fluid obtained at 2212 weeks. These data suggest that the time in gestation when amniotic fluid is sampled for chemical analysis is an important variable affecting glycosaminoglycan composition in both normal and pathological pregnancies.