Distal 5q deletion syndrome: phenotypic correlations.

We describe the phenotypes of two male sibs with partial monosomy of chromosome 5 [46,XY,der(5)inv ins(1;5)(p32;q35.4q34)]; maternally derived from a balanced insertion of 1 and 5 [inv ins (1;5)(p.32;q35.4q34)]. One sib had microcephaly, cleft lip and palate, facial anomalies, atrial (ASD) and ventricular (VSD) septal defects, camptodactyly 4th and 5th fingers, and developmental delay. The other sib showed microcephaly, facial anomalies, ASD, hypotonia, primary optic nerve hypoplasia, and developmental delay. Only seven other patients with 5q deletions distal to 5q33 have been reported and none showed the putative breakpoints identified in our two patients. All nine showed developmental delay or malformations of the CNS and facial anomalies; six of nine had defects of cardiac septation. Our two patients and one other were shown to have only one copy of the cardiac specific hCSX gene that defines in part the etiology of their ASD and VSD. The other components of their phenotypes cannot be related at present to genes identified in the deleted segments.

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.

Delayed postanoxic demyelination and arylsulfatase-A pseudodeficiency.

We report a patient with delayed postanoxic demyelination who had pseudodeficiency of arylsulfatase A, reducing his enzyme activity to 10 to 30% of normal. This may have implications regarding the pathogenesis of postanoxic demyelination.

Delivery of growth factor to wounds using a genetically engineered biological bandage.

Increasing the rate of wound healing of acute wounds and promoting the closure of chronic ulcers is an important goal in wound therapy. Growth factors have been shown to facilitate this process; however, the systems described for growth factor delivery are not ideal. In the present report we demonstrate the feasibility of a new method of delivering growth factors to the wound site using a genetically engineered biological bandage. The bandage consists of keratinocytes (SCC-13 cells) that are engineered by gene transfer to produce high levels of bovine growth hormone (bGH). bGH was selected for these studies because it can be easily distinguished from rat and human growth hormone in wound fluids and culture medium. The bGH-producing cells are contained and maintained in serum-free medium inside an envelope composed of a low protein binding, 0.2 micron pore size, polysulfone membrane. The genetically engineered cells cannot escape from the bandage, but the bGH is freely released into the surrounding culture medium. When placed onto a full-thickness, surgically generated wound on rats, the cells within the bandage continue to produce and release bGH into the wound for at least 3 days. This system is a safe and reliable way of providing real-time delivery of any desired biomolecule into the wound site.

Human keratinocytes contain keratin filaments that are glycosylated with keratan sulfate.

We have reported strong intracytoplasmic immunoreactivity for anti-keratan sulfate monoclonal antibodies in human keratinocytes. Consequently, ultrastructural immunogold studies were undertaken to identify the cytoplasmic components responsible for this immunoreactivity. Immunogold labeling of cultured keratinocytes identified keratin filaments as a source of keratan sulfate epitopes. Immunolabeling also marked desmosomal cytoplasmic plaques and amorphous electron-dense bodies. These observations were confirmed for epidermal keratinocytes of human skin. Further evidence was obtained that some keratins have epitopes for anti-keratan sulfate antibodies by Western blot analyses following fractionation of proteins by sodium dodecyl sulfate gel electrophoresis. Four bands were detected with apparent molecular weights of 58, 54, 50, and 48 kDa that reacted with anti-keratin monoclonal antibody mixture AE1/AE3. Keratins of 58 and 56 kDa immunoreacted with anti-keratan sulfate antibody 8C2 while all four keratins immunoreacted with anti-keratan sulfate antibody 5D4. Endo-beta-D-galactosidase and keratanase, enzymes that degrade keratan sulfate, removed all, or a portion, of specific keratan sulfate epitopes from keratin extracts. These results demonstrate that a portion of the cytoplasmic anti-keratan sulfate immunoreactivity is due to keratins that are glycosylated with carbohydrates that contain keratan sulfate epitopes or that keratan sulfate-containing molecules bind or comigrate in SDS-polyacrylamide gels with cytokeratins.

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.

Orthotopic liver transplantation in two adults with Niemann-Pick and Gaucher's diseases: implications for the treatment of inherited metabolic disease.

Two adults were seen with cirrhosis caused by different lipid storage diseases. A 42-yr-old woman with Niemann-Pick disease type B had marked hepatomegaly, ascites and recent variceal bleeding. Her evaluation showed chronic bilateral pulmonary infiltrates, multiple stigmata of chronic liver disease including the recent cessation of menses, diuretic-resistant sterile ascites, hepatic encephalopathy and variceal bleeding. Five percent of normal sphingomyelinase activity was measured in peripheral leukocytes. A 42-yr-old man with Gaucher's disease and a history of bilateral hip replacements presented with hepatomegaly, jaundice, refractory ascites and renal insufficiency. His evaluation showed 20% to 23% of normal glucocerebrosidase activity in peripheral leukocytes. Both patients underwent orthotopic liver transplantation with resolution of all aspects of decompensated liver function. Assessment of the underlying metabolic defect before and 6 to 14 mo after transplantation showed that after transplantation the patient with Niemann-Pick disease had above normal hepatic sphingomyelinase activity, a less-marked increase in peripheral leukocyte enzyme activity and lower than normal hepatic sphingomyelin and cholesterol content. In contrast, the patient with Gaucher's disease had only a 61% increase in hepatic glucocerebrosidase activity but had an elevated hepatic glucocerebroside content that was only 15% of the pretransplant level and decreased peripheral leukocyte enzyme levels. These findings suggest that variable relationships may exist between posttransplant hepatic and peripheral leukocyte enzyme activities in the different lipidoses, which may have implications for recurrence of glycolipid-induced liver damage.

Human keratinocytes cultured on collagen gels form an epidermis which synthesizes bullous pemphigoid antigens and alpha 2 beta 1 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.

Human keratinocytes contain carbohydrates that are recognized by keratan sulfate-specific monoclonal antibodies.

Monoclonal antibodies that recognize carbohydrate epitopes found in keratan sulfate glycosaminoglycan chains identified both intracytoplasmic and cell-surface carbohydrates of human keratinocytes. These carbohydrates were detected, using indirect immunoperoxidase methods, both in sections of paraffin-embedded tissues and in intact cultured keratinocytes. Of the seven anti-keratan sulfate monoclonal antibodies used in this study, five detected significant amounts of epitopes associated with keratinocytes. This indicates that only certain, specific types of keratan sulfate-like carbohydrates were expressed by these cells. The extent and localization of keratan sulfate-like carbohydrates appeared to be closely related to the differentiation status of cultured keratinocytes. These epitopes were very weakly expressed on surfaces of all monolayer keratinocytes, but flattened, suprabasal cells in high Ca++ cultures strongly expressed keratan sulfate-like carbohydrates on their surfaces. A much larger population of cultured keratinocytes expressed intracellular keratan sulfate-like carbohydrates identified by the same five antibodies that detected surface epitopes. In monolayer cells, keratan sulfate-like carbohydrates were predominantly found in a broad perinuclear zone. In addition, three of the five immunoreactive antibodies detected epitopes that appeared at cell boundaries, specifically at sites of close cell-to-cell contact. Thus, molecules bearing carbohydrates recognized by anti-keratan sulfate antibodies appear at developmentally important stages of keratinocyte differentiation, indicating that these carbohydrates may serve as markers for molecules important in the differentiation of human keratinocytes.

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.

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.

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.

Motor neuron disease and adult hexosaminidase A deficiency in two families: evidence for multisystem degeneration.

We studied three patients from two unrelated families with adult hexosaminidase A deficiency. A 30-year-old, non-Jewish proband in the first family had juvenile amyotrophic lateral sclerosis that evolved to mild dementia, ataxia, and axonal (neuronal) motor-sensory peripheral neuropathy. A 36-year-old Jewish proband in the second family had "pure" spinal muscular atrophy. One supposedly healthy brother of the first proband was found to have borderline IQ, mild spasticity, and ataxia but no evidence of motor neuron disease. Marked cerebellar atrophy was detected by head scans in all three patients. In both probands electromyograms were characterized by prominent, complex repetitive discharges in many muscles. Hexosaminidase A activities against the artificial substrate were similar to those reported in infantile Tay-Sachs disease; however, the hexosaminidase A level against GM2 substrates was higher than that found in infantile Tay-Sachs disease. The hexosaminidase A levels of the parents were in the heterozygous range. Motor neuron disease in our patients and in those previously described appears to be part of a multisystem degeneration of the nervous system.

Isolation and preliminary characterization of proteoglycan aggregates from cultured dermal fibroblasts.

A1 proteoglycan fractions were prepared by isopycnic cesium chloride density gradient centrifugation from early passage cultured fibroblasts derived from the skin of human infants. In five different fibroblast strains, A1 preparations from the medium and the cells, respectively, contained 36-63% and 59-79% of the sulfated proteoglycans applied to the gradients. A subpopulation of A1 proteoglycans was excluded from Sepharose CL-2B columns under associative conditions (0.5 M guanidium chloride (GdmCl]. The excluded proteoglycans comprised 45-55% of the medium and 28-40% of the cell-associated A1 preparations, showed reversible association-dissociation behaviors on Sepharose CL-2B columns, were composed primarily of chondroitin sulfate and dermatan sulfate, and behaved as monomers with a Kav of 0.33 following mild reduction and alkylation. These data indicate that the excluded peaks were supramolecular aggregates composed of proteoglycan subunits. The mechanism of aggregate formation was examined in A1D1 and D1 fractions. Hyaluronic acid induced aggregation of medium A1D1 proteoglycans in one cell strain, but not in two others. Aggregation was not observed with hyaluronic acid in any cell-associated A1D1 fraction. D1 medium proteoglycans chromatographed on Sepharose CL-2B columns under associative conditions showed two types of aggregates; one was disrupted with 4 M GdmCl indicating that a portion of the aggregate formed through noncovalent interactions that may not require interaction with hyaluronic acid. A second type of aggregate persisted in 4 M GdmCl and after mild reduction and alkylation of the core protein but was disrupted by 0.2% sodium dodecyl sulfate, suggesting that aggregation had occurred through proteoglycan interaction with lipids or hydrophobic proteins. These data indicate that human skin fibroblasts synthesize proteoglycans which share some of the properties of chondrocyte proteoglycans, but also have distinctive macromolecular characteristics.

Treatment of inborn errors of urea synthesis: activation of alternative pathways of waste nitrogen synthesis and excretion.

Children with inborn errors of urea synthesis accumulate ammonium and other nitrogenous precursors of urea, leading to episodic coma and a high mortality rate. We used alternative pathways for the excretion of waste nitrogen as substitutes for the defective ureagenic pathways in 26 infants. These pathways involve synthesis and excretion of hippurate after sodium benzoate administration, and of citrulline and argininosuccinate after arginine supplementation. The children were treated for seven to 62 months; 22 survived. The mean plasma level of ammonium ( +/- S.E.) was 36 +/- 2 mumol per liter, and that of benzoate was 1.5 +/- 1.0 mg per deciliter. Alternative pathways accounted for between 28 and 59 per cent of the total "effective" excretion of waste nitrogen. Nineteen infants had normal height, weight, and head circumference, and 13 had normal intellectual development. Activation of alternative pathways of waste nitrogen excretion can prolong survival and improve clinical outcome in children with inborn errors of urea synthesis.

Prenatal diagnosis of Maroteaux-Lamy syndrome.

Maroteaux-Lamy syndrome exhibits deficient activity of the enzyme arylsulfatase-B in cultured skin fibroblasts. Prenatal diagnosis was successfully attempted in two pregnancies of a consanguineous Chaldean couple whose first child is affected with Maroteaux-Lamy syndrome. In both instances, deficient arylsulfatase-B activity was observed in amniotic fluid cell cultures, and the diagnosis was confirmed by 35S-sulfate studies and postmortem enzymology and electron microscopy. The prenatal diagnosis of Maroteaux-Lamy syndrome remains problematic. Residual activity of arylsulfatase-B in the affected homozygote can make interpretation difficult, and the behavior of many lysosomal enzymes varies greatly in response to tissue culture conditions and enzyme extraction processes.