Heparan sulfates (HS) are long, linear polysaccharides with a high degree of variability. They bind to a vast number of proteins such as growth factors and cytokines, and these interactions are likely to be mediated by specific HS domains. To investigate the structural diversity and topological distribution of HS domains in tissues, we selected a panel of 10 unique anti-HS antibodies using phage display technology. All 10 antibodies recognize a specific HS epitope as demonstrated by enzyme-linked immunosorbent assay using defined synthetic HS oligosaccharides, modified HS/ heparin molecules, and HS isolated from a variety of organs. The chemical groups involved in the epitopes could be indicated and the position of sulfate groups is of major importance. All HS epitopes have a defined tissue distribution as shown by immunohistochemistry using rat organs. Taken together, the data show that in vivo, a large number of defined HS epitopes exist that do not occur randomly but are tightly, topologically regulated.Heparan sulfates (HS), 1 a class of glycosaminoglycans, are long linear complex polysaccharides covalently bound to a protein core. They have a ubiquitous distribution in the extracellular matrix and on cell surfaces and have been implicated in many basic biological phenomena such as cell migration, adhesion, and differentiation. They play a role in such diverse processes as growth factor/cytokine handling, enzyme regulation, lipid metabolism, blood coagulation, and viral entry (1-4). This involvement is mediated by the interactions of HS with a vast number of proteins such as growth factors/cytokines, enzymes, protease inhibitors, extracellular matrix molecules, and viral coat proteins (5, 6). The large number of interactions suggests an extensive structural variation within HS. Chemical analysis of HS-derived disaccharides indeed indicates a large structural diversity (7-9), which is brought about by specific chain modifications during HS biosynthesis. The importance of defined monosaccharide sequences for specific interactions with proteins has been demonstrated for the binding and activation of antithrombin III by HS/heparin (10, 11). In addition, specific structural requirements for binding to basic fibroblast growth factor and hepatocyte growth factor have been defined (12, 13). These observations indicate that HS modifications do not occur randomly but that a controlled expression of specific domains/sequences in HS exists. To investigate whether a large number of different HS domains indeed occur in tissues, we selected a panel of epitope-specific anti-HS antibodies using phage display technology. Using these antibodies, we chemically and topologically characterized the HS epitopes involved. We chose antibody phage display because it allows for the generation of antibodies against poorly immunogenic molecules such as HS. EXPERIMENTAL PROCEDURES MaterialsA human semisynthetic antibody phage display library (14, now officially named synthetic scFv Library No. 1) was generously provided by Dr G. Winter, C...

(4T3538).Autofluorescence can be a very disturbing factor in immunofluorescence microscopy. We present here a method to eliminate autofluotescence. The method is based on the fact that most autofluorescent compounds have broad-banded excitation and emission spectra, whereas specific fluorescent probes have M~O W spectra. Two images are tecorded and digitized, one at a wavelength exciting both the fluorescent probe and the autofluotescent molecules, and one at a wavelength exciting only the latter. Subtraction of the autofluorescence signal from the total fluorescence signal, using a selfdeveloped computer program, results in an autofluorescence-

Small human lung specimens are frequently used for cell biological studies of the pathogenesis of emphysema. In general, lung function and other clinical parameters are used to establish the presence and severity of emphysema/chronic obstructive pulmonary disease without morphological analysis of the specimens under investigation. In this study we compared three morphological methods to analyze emphysema, and evaluated whether clinical data correlate with the morphological data of individual lung samples. A total of 306 lung specimens from resected lung(lobes) from 221 patients were inflated and characterized using three morphological parameters: the Destructive Index, the Mean Linear Intercept, and Section Assessment. Morphological data were related to each other, to lung function data, and to smoking behavior. Significant correlations (P < .001) were observed between Section Assessment and Destructive Index (r ‫؍‬ 0.92), Mean Linear Intercept with Destructive Index (r ‫؍‬ 0.69) and Mean Linear Intercept with Section Assessment (r ‫؍‬ 0.65). Section Assessment, being much less time consuming than Mean Linear Intercept and Destructive Index, is the parameter of choice for initial analysis. Destructive Index is the most sensitive parameter. There was a significant (P < .001), but weak correlation for all three parameters with the diffusion capacity for CO (K CO ) (Destructive Index: r ‫؍‬ ؊0.28; Mean Linear Intercept: r ‫؍‬ ؊0.34; Section Assessment: r ‫؍‬ ؊0.32), and with FEV 1 /IVC (Destructive Index: r ‫؍‬ ؊0.29; Mean Linear Intercept: r ‫؍‬ ؊0.33; Section Assessment: r ‫؍‬ ؊0.28), but not with other lung function parameters. A significant difference (P < .05) between (ex-) smokers and never-smokers was observed for Destructive Index and Section Assessment. It is concluded that the application of the three morphological parameters represents a useful method to characterize emphysematous lesions in a (semi-)quantitative manner in small human lung specimens, and that Section Assessment is a suitable and fast method for initial screening. The extent of emphysema of individual lung specimens should be established by means of morphometry, rather than lung function data.

IL-4 induces the differentiation of monocytes toward dendritic cells (DCs). The activity of many cytokines is modulated by glycosaminoglycans (GAGs). In this study, we explored the effect of GAGs on the IL-4-induced differentiation of monocytes toward DCs. IL-4 dose-dependently up-regulated the expression of DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), CD80, CD206, and CD1a. Monocytes stained positive with Abs against heparan sulfate (HS) and chondroitin sulfate (CS) B (CSB; dermatan sulfate), but not with Abs that recognize CSA, CSC, and CSE. Inhibition of sulfation of monocyte/DC cell surface GAGs by sodium chlorate reduced the reactivity of sulfate-recognizing single-chain Abs. This correlated with hampered IL-4-induced DC differentiation as evidenced by lower expression of DC-SIGN and CD1a and a decreased DC-induced PBL proliferation, suggesting that sulfated monocyte cell surface GAGs support IL-4 activity. Furthermore, removal of cell surface chondroitin sulfates by chondroitinase ABC strongly impaired IL-4-induced STAT6 phosphorylation, whereas removal of HS by heparinase III had only a weak inhibitory effect. IL-4 bound to heparin and CSB, but not to HS, CSA, CSC, CSD, and CSE. Binding of IL-4 required iduronic acid, an N-sulfate group (heparin) and specific O sulfates (CSB and heparin). Together, these data demonstrate that monocyte cell surface chondroitin sulfates play an important role in the IL-4-driven differentiation of monocytes into DCs.

Antibodies against heparan sulfate (HS) are useful tools to study the structural diversity of HS. They demonstrate the large sequential variation within HS and show the distribution of HS oligosaccharide sequences within their natural environment. We analyzed the distribution and the structural characteristics of the oligosaccharide epitope recognized by anti-HS antibody HS4C3. Biosynthetic and synthetic heparin-related oligosaccharide libraries were used in affinity chromatography, immunoprecipitation, and enzyme-linked immunosorbent assay to identify this epitope as a 3-O-sulfated motif with antithrombin binding capacity. The antibody binds weakly to any N-sulfated, 2-O-and 6-O-sulfated hexa-to octasaccharide fragment but strongly to the corresponding oligosaccharide when there is a 3-O-sulfated glucosamine residue present in the sequence. This difference was highlighted by affinity interaction and immunohistochemistry at salt concentrations from 500 mM. At physiological salt conditions the antibody strongly recognized basal lamina of epithelia and endothelia. At 500 mM salt conditions, when 3-O sulfation is required for binding, antibody recognition was more restricted and selective. Antibody HS4C3 bound similar tissue structures as antithrombin in rat kidney. Furthermore, antithrombin and antibody HS4C3 could compete with one another for binding to heparin. Antibody HS4C3 was also able to inhibit the anti-coagulant activities of heparin and Arixtra as demonstrated using the activated partial thromboplastin time clotting and the anti-factor Xa assays. In summary, antibody HS4C3 selectively detects 3-O-sulfated HS structures and interferes with the coagulation activities of heparin by association with the antithrombin binding pentasaccharide sequence. Heparan sulfate (HS)3 proteoglycans consist of a core protein with covalently linked HS side chains, and occur on cell surfaces and in the extracellular matrix. HS polysaccharides consist of up to ϳ200 repeating disaccharide units (glucosamine ␣1-4-glucuronic acid ␤1-4 and glucosamine ␣1-4-iduronic acid ␣1-4), which are variably modified by N-acetyl/N-sulfate and O-sulfate groups (1, 2). The HS chains have fundamental roles in embryonic development, homeostasis, and disease, by interaction with regulatory proteins (morphogens, growth factors, enzymes etc.), mediated by specific HS domains (3). HS-protein interactions are believed to be dictated not only by the overall charge of the HS chain but also by the distribution and positioning of the negatively charged carboxyl and sulfate groups within the HS chain (4). The structural diversity within the HS chain arises through the ordered action of sulfotransferases and an epimerase (1, 5, 6) during HS biosynthesis within the Golgi apparatus and may be further affected by the extracellular action of endosulfatases after biosynthesis (7). The biosynthetic HS modification reactions include N-deacetylation/N-sulfation of the glucosamine (GlcN) residues by N-deacetylase/N-sulfotransferases, C 5 epimerization of glucuronic ...