At the conclusion of this learning activity, participants should be familiar with the history, pathogenesis, clinical features, and treatment of scurvy in adults.

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The presence of certain types of human papillomavirus (HPV) is a known risk factor for the development of anogenital squamous cell carcinomas (SCCs). A similar association has been hypothesized for cutaneous SCCs, although, to our knowledge, no studies to date have combined sensitive HPV DNA detection techniques with epidemiologic data controlling for known risk factors to explore the association. We designed a case-control study examining HPV prevalence using highly sensitive PCR-detection assays in tissue samples from 85 immunocompetent patients with histologically confirmed SCCs and 95 age-matched individuals without a prior history of skin cancer. A standardized interview was administered to all study subjects to collect information pertaining to potential confounding variables. The overall detection rate of HPV DNA was high in case lesions (54%) and perilesions (50%) and in both sun-exposed normal tissue (59%) and non-sun-exposed normal tissue (49%) from controls. In comparing case tissue to control tissue, there was no differential detection of HPV DNA across various HPV species. However, HPV DNA from beta-papillomavirus species 2 was more likely to be identified in tumors than in adjacent healthy tissue among cases (paired analysis, odds ratio=4.0, confidence interval=1.3-12.0). The high prevalence of HPV DNA detected among controls suggests that HPV DNA is widely distributed among the general population. However, the differential detection of HPV beta-papillomavirus species in tumors among cases suggests that certain HPV types may be involved in the progression of cutaneous SCCs.

Thrombospondin, a high molecular weight glycoprotein secreted by platelets in response to activation by thrombin, has been identified by immunofluorescence in bovine aortic endothelial cells, human foreskin fibroblasts, and human aortic smooth muscle cells. Immunofluorescence patterns were found to be similar using antisera raised to thrombospondins purified either from bovine aortic endothelial cells or from human platelets. Radioimmune precipitation of pulse-labeled cellular proteins confirmed the presence of thrombospondin in positively stained cells. A sensitive quantitative enzyme-linked immunosorbent assay (ELISA) was developed and used to determine that the accumulation of secreted thrombospondin was similar for endothelial cells and fibroblasts but was higher for smooth muscle cells. The presence of thrombospondin in a variety of cells suggests that its function may not be limited to an involvement in platelet interactions.Thrombospondin (TS), a high molecular weight glycoprotein, is released from a-granules after activation of platelets by thrombin (1, 2). After release, the protein binds to the activated platelet surface in a calcium-dependent fashion (3) and may participate in platelet-platelet interactions (4). Earlier work in this laboratory (5) identified a high molecular weight glycoprotein secreted by endothelial cells in culture that represented a substantial portion of the noncollagenous protein synthesized and secreted by these cells. This glycoprotein was subsequently shown to be indistinguishable from TS by a variety of criteria, including co-purification, molecular weight, amino acid composition, immunological cross-reactivity, and peptide maps (6).Although TS has been shown to have lectinlike activity in platelet-platelet interactions (4), its function in endothelial cells is not known. This led us to examine other cells in culture for the synthesis and secretion of TS. We have now identified TS in a variety of mesenchymal cells. Since TS is secreted and deposited in the cell layer of these cells in culture, we postulate that it may function as a matrix protein in vivo. MATERIALS AND METHODS Cell CultureBovine aortic endothelial (BAE) ceils were provided by Dr. S. Schwartz (University of Washington). They were maintained in Waymouth's medium supplemented with 10% fetal calf serum (FCS) (Reheis Chemical Company, Phoenix, AZ), penicillin (100 U/nil), and streptomycin (100 gg/ml). Human dermal fibroblasts were isolated from foreskin explants and maintained in Dulbecco's modified Eagle's medium (DME) supplemented with 10% FCS and antibiotics. Smooth muscle ceils were obtained from human aortae and were provided by Dr. Russell Ross (University of Washington). They were maintained in DME supplemented with 20% FCS and antibiotics. Ceils were grown at 37°C in an atmosphere of 5% CO2. Purification of Thrombospondins BAE cell TS was isolated according to the method of McPherson el al. (6).Human platelet TS was isolated from l-d-old blood bank platelets according to the procedure of Lawler a...

Affinity-purified antisera against thrombospondin were used to locate the presence of this glycoprotein in frozen sections of several human tissues by immunofluorescence techniques. Immunostaining was observed in the peritubular connective tissue and in basement membrane regions beneath glandular epithelium in skin and lung. Intense immunostaining was observed at the dermal-epidermal junction in skin and in small blood vessels throughout this tissue. Skeletal muscle exhibited positive staining with anti-thrombospondin antisera within interstitial areas.

Thrombospondin (TS), a protein first described in platelets, was recently shown to be synthesized and secreted by endothelial cells, fibroblasts, and smooth muscle cells. The presence of TS in the extracellular matrix of cultured cells has prompted us to examine the associations of this protein with matrix macromolecules . Interactions of TS with both matrix and serum proteins were tested using an enzyme-linked immunosorbent assay . With this assay we assessed the binding of TS in solution to proteins adsorbed to polystyrene microtiter plates . Among collagens, platelet TS bound to type V but not to types I, III, or IV. This selective interaction was confirmed in experiments using proteins linked to cyanogen bromide-activated Sepharose . TS released from platelets in response to thrombin activation, as well as that secreted by endothelial cells in culture, bound to type V but not to type I collagen-Seph arose. No binding was observed to denatured type V collagen-Sepharose . The binding region for type V collagen was located in a chymotrypsin-produced fragment of TS with chains of M, = 70,000, after reduction . Interactions of TS with a number of other proteins, including fibronectin, fibrinogen, and laminin, could be demonstrated using the enzyme-linked immunosorbent assay technique but the interpretation of these findings is difficult since comparable binding to protein-Sepharose was not always observed. Our findings suggest that both the extravascular distribution and function of TS in vivo may involve an interaction with type V Collagen.Thrombospondin (TS)' is a glycoprotein consisting of three, possibly identical, disulfide-bonded chains of -140,000 mol wt (25, 31). The protein was initially described in platelets and was shown to be released from storage in a-granules by the action of thrombin (2, 18). A fraction of the secreted TS binds to the platelet surface (15, 39) . Subsequently TS, or a protein very similar to it, was shown to be secreted by endothelial cells (9,34,36,42,45) and by various other cells in culture, including fibroblasts (21, 40), smooth muscle cells (40), and granular pneumocytes (43). The observation by immunofluorescence of TS in a fibrillar extracellular meshwork in cultured cells (21,40) suggested that TS may function as a normal component of the extracellular matrix in vivo.It has been proposed that TS acts as an endogenous lectin in platelet aggregation by binding to fibrinogen associated 'Abbreviations used in this paper: ELISA, enzyme-linked immunosorbent assay; TS, thrombospondin . 646with the activated platelet surface (20). Support for this proposal has come from the observation that fibrinogen in solution can form a complex with TS adsorbed to a plastic surface (26). In addition, Lahav et al. (22) have provided evidence for the interaction ofTS with fibronectin during platelet adhesion and aggregation. Using a radioactive cross-linking agent, an interaction was observed between TS released from activated platelets and fibronectin or collagen adsorbed to a glass surf...

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Thrombospondin (TS), a 450,000 molecular weight glycoprotein, is released from alpha-granules of thrombin-activated platelets and is secreted and incorporated into the extracellular matrix by several cell types in culture. We have examined the effects of cell density and transformation on the production of TS in cell culture. The levels of TS, per cell, in the culture medium of endothelial cells, smooth muscle cells, and fibroblasts were greater at lower cell densities; in fibroblasts the levels of two other extracellular matrix proteins, fibronectin and collagen, were unaffected by cell density. Our evidence indicates that the higher levels of TS in the culture medium, determined for lower-density cells, were achieved by an increased secretion of the protein rather than by a reduction in degradation or incorporation into the extracellular matrix. TS production by normal and transformed WI-38 fibroblasts was the same, although the fibronectin level in the culture medium of the transformed cells was substantially decreased. These findings suggest that the production of TS by cells in culture is regulated in a different fashion from that of fibronectin or collagen.