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Background: Non-AT-III mediated heparin-resistance during CPB occurs by complex-forming with heparin-binding proteins. Currently, there are no specific recommendations for non-AT-III mediated heparin-resistance., Case Presentation: We present a fatal case of a 70-yr-old male-patient undergoing cardiac-surgery in which refractory heparin-resistance was observed. The massive AL amyloidosis found at autopsy is thought to be responsible and illustrates that awareness and knowledge of the etiology and perioperative strategies of non-AT-III mediated heparin-resistance is important., Conclusion: For anticoagulation during cardiopulmonary bypass surgery in case of a non-AT-III medicated heparin resistance, we refer to the decision tree added to this manuscript and if necessary to consider direct thrombin inhibitors, such as bivalirudin or argatroban, as it bypasses the complexing pathway., (© 2023. The Author(s).)

Glycosaminoglycans are important for cell signaling and therefore for proper embryonic development and adult homeostasis. Expressions of genes involved in proteoglycan/glycosaminoglycan (GAG) metabolism and of genes coding for growth factors known to bind GAGs were analyzed during skin development by microarray analysis and real time quantitative PCR. GAG related genes were organized in six categories based on their role in GAG homeostasis, viz. (1) production of precursor molecules, (2) production of core proteins, (3) synthesis of the linkage region, (4) polymerization, (5) modification, and (6) degradation of the GAG chain. In all categories highly dynamic up- and downregulations were observed during skin development, including differential expression of GAG modifying isoenzymes, core proteins, and growth factors. In two mice models, one overexpressing heparanase and one lacking C5 epimerase, differential expression of only few genes was observed. Data show that during skin development a highly dynamic and complex expression of GAG-associated genes occurs. This likely reflects quantitative and qualitative changes in GAGs/proteoglycans, including structural fine tuning, which may be correlated with growth factor handling.

Identifying collagen produced de novo by cells in a background of purified collagenous biomaterials poses a major problem in for example the evaluation of tissue-engineered constructs and cell biological studies to tumor dissemination. We have developed a universal strategy to detect and localize newly deposited collagen based on its inherent association with dermatan sulfate. The method is applicable irrespective of host species and collagen source.

Autologous skin grafts are the gold standard for the treatment of burn wounds. In a number of cases, treatment with autologous tissue is not possible and skin substitutes are used. The outcome, however, is not optimal and improvements are needed. Inspired by scarless healing in early embryonic development, we here set out a strategy for the design and construction of embryonic-like scaffolds for skin tissue engineering. This strategy may serve as a general approach in the construction of embryonic-like scaffolds for other tissues/organ. As a first step, key effector molecules upregulated during embryonic and neonatal skin formation were identified using a comparative gene expressing analysis. A set of 20 effector molecules was identified, from which insulin-like growth factor 2 (IGF2) and sonic hedgehog (SHH) were selected for incorporation into a type I collagen-heparin scaffold. Porous scaffolds were constructed using purified collagen fibrils and 6% covalently bound heparin (to bind and protect the growth factors), and IGF2 and SHH were incorporated either individually (~0.7 and 0.4 µg/mg scaffolds) or in combination (combined ~1.5 µg/mg scaffolds). In addition, scaffolds containing hyaluronan (up to 20 µg/mg scaffold) were prepared, based on the up- or downregulation of genes involved in hyaluronan synthesis/degradation and its suggested role in scarless healing. In conclusion, based on a comprehensive gene expression analysis, a set of effector molecules and matrix molecules was identified and incorporated into porous scaffolds. The scaffolds thus prepared may create an 'embryonic-like' environment for cells to recapitulate embryonic events and for new tissues/organs., (Copyright © 2013 John Wiley & Sons, Ltd.)

Dermatan sulfate (DS), also known as chondroitin sulfate (CS)-B, is a member of the linear polysaccharides called glycosaminoglycans (GAGs). The expression of CS/DS and DS proteoglycans is increased in several fibrotic renal diseases, including interstitial fibrosis, diabetic nephropathy, mesangial sclerosis and nephrosclerosis. Little, however, is known about structural alterations in DS in renal diseases. The aim of this study was to evaluate the renal expression of two different DS domains in renal transplant rejection and glomerular pathologies. DS expression was evaluated in normal renal tissue and in kidney biopsies obtained from patients with acute interstitial or vascular renal allograft rejection, patients with interstitial fibrosis and tubular atrophy (IF/TA), and from patients with focal segmental glomerulosclerosis (FSGS), membranous glomerulopathy (MGP) or systemic lupus erythematosus (SLE), using our unique specific anti-DS antibodies LKN1 and GD3A12. Expression of the 4/2,4-di-O-sulfated DS domain recognized by antibody LKN1 was decreased in the interstitium of transplant kidneys with IF/TA, which was accompanied by an increased expression of type I collagen, decorin and transforming growth factor beta (TGF-β), while its expression was increased in the interstitium in FSGS, MGP and SLE. Importantly, all patients showed glomerular LKN1 staining in contrast to the controls. Expression of the IdoA-Gal-NAc4SDS domain recognized by GD3A12 was similar in controls and patients. Our data suggest a role for the DS domain recognized by antibody LKN1 in renal diseases with early fibrosis. Further research is required to delineate the exact role of different DS domains in renal fibrosis.

Lyophilisomes are a novel class of proteinaceous biodegradable nano/microparticle capsules developed for tumor drug delivery. The in vivo characteristics of lyophilisomes are unknown and, therefore, the time course of biodistribution of sized albumin-based lyophilisomes in CD1 mice after intravenous administration was studied. Lyophilisomes, prepared from Dylight680-labeled albumin, were sized using a sucrose gradient centrifugation methodology and four fractions with a mean size of approximately 200nm, 400nm, 550nm, and 650nm were pooled for in/ex vivo localization, (immuno)histochemistry and biochemical analysis. Lyophilisomes were rapidly taken out of the circulation by the liver and spleen. Immunohistochemistry revealed that lyophilisomes were taken up in the liver by F4/80 positive macrophages, and in the spleen by Sign-R1 positive macrophages specifically located in the marginal zones. Lyophilisomes were most likely degraded by the liver and spleen and subsequently excreted via the urine, as high levels of degraded Dylight680-labeled albumin were detected in the urine. This was corroborated by electron microscopy of the spleen, which showed intact lyophilisomes in the marginal zone 5 and 30min after injection, but not after 2h. In conclusion, IV injected lyophilisomes are rapidly entrapped by liver and splenic macrophages, biodegraded, and excreted in the urine., (Copyright © 2015 Elsevier B.V. All rights reserved.)

HS (heparan sulfate) is a long linear polysaccharide, variably modified by epimerization and sulfation reactions, and is organized into different domains defined by the extent of modification. To further elucidate HS structural organization, the relative position of different HS structures, identified by a set of phage-display-derived anti-HS antibodies, was established. Two strategies were employed: inhibition of HS biosynthesis using 4-deoxy-GlcNAc, followed by resynthesis, and limited degradation of HS using heparinases. Using both approaches, information about the position of antibody-defined HS structures was identified. The HS structure recognized by the antibody NS4F5, rigorously identified as (GlcN6S-IdoA2S)₃, was found towards the non-reducing end of the HS chain.

Glycosaminoglycans (GAGs) are long, anionic polysaccharides involved in many basic aspects of mammalian physiology and pathology. Here we describe a method to extract GAGs from formalin-fixed, paraffin-embedded tissues and found that they are structurally comparable with GAGs extracted from frozen tissues. We employed this method to structurally characterize GAGs in tissues, including laser-dissected layers of skin and pathological specimens. This method enables the use of the archival paraffin-embedded material for detailed (structural) analysis of GAGs.

A papillary-structured collagen fibril membrane is created, mimicking the 3D-architecture of the human papillary dermis. Primary human keratinocytes cultured to confluency on papillar-structured films are compared to keratinocytes cultured on flat membranes. Microscopical evaluation reveals the presence of morphologically distinct cells at the base of the papillar structures that are not observed on flat membranes. Gene expression microarrays and RT-qPCR indicate that these cells are in a more proliferative/migrational state, whereas cells on flat membranes have a more differentiated expression profile. Immunohistochemical stainings confirm these results. In conclusion, specific collagen architecture can direct keratinocyte behavior, and this may be used to further improve skin regeneration., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Glycosaminoglycans, like heparin, are frequently incorporated in biomaterials because of their capacity to bind and store growth factors and because of their hydrating properties. Heparin is also often used in biomaterials for its anticoagulant activity. Analysis of biomaterial-bound heparin is challenging because most assays are based on heparin in solution. In this study, seven different methods were probed to analyze heparin covalently attached to collagen scaffolds. For each method, the basic mechanism and the advantages and disadvantages are given. An analysis by the factor Xa assay and the Farndale assay clearly indicated that the amount of immobilized heparin cannot be determined correctly when the scaffold is intact. Scaffolds had to be proteolytically digested or acid treated to obtain reliable measurements. Methods used to quantify the amount of bound heparin included a hexosamine assay, an uronic acid assay, a Farndale assay, agarose gel electrophoresis, and immuno-dot blot analysis. Location and semiquantification of heparin were accomplished by immunofluorescence. Although all assays had their advantages and disadvantages, the hexosamine assay turned out to be the most robust and is recommended as the preferred assay to quantify the amount of heparin bound to scaffolds. It is applicable to all scaffolds that are acid hydrolyzable. This study may allow researchers in the field to select the most appropriate method to analyze glycosaminoglycans in biomaterials.

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.

Parenchymal destruction, airspace enlargement, and loss of elasticity are hallmarks of pulmonary emphysema. Although the basic mechanism is unknown, there is a consensus that malfunctioning of the extracellular matrix is a major contributor to the pathogenesis of emphysema. In this study, we analyzed the expression of the elastic fiber protein fibrillin-1 in a large number (n=69) of human lung specimens with early-onset emphysema. Specimens were morphologically characterized by the Destructive Index, the Mean Linear Intercept, and the Panel Grading. We observed a strong correlation (P<0.001) of aberrant fibrillin-1 staining with the degree of destruction of lung parenchyma (r=0.71), airspace enlargement (r=0.47), and emphysema-related morphological abnormalities (r=0.69). There were no obvious correlations with age and smoking behavior. Staining for three other extracellular matrix components (type I collagen, type IV collagen, and laminin) was not affected. The aberrant fibrillin-1 staining observed in this study is similar to that observed in Marfan syndrome, a syndrome caused by mutations in the gene encoding fibrillin-1. Strikingly, emphysema is noticed in a number of Marfan patients. This, together with the notion that disruption of the fibrillin-1 gene in mice results in emphysematous lesions, makes fibrillin-1 a strong candidate to be involved in the etiology and pathogenesis of emphysema.

Dermatan sulfate (DS) is a member of the glycosaminoglycan (GAG) family and is primarily located in the extracellular matrix. Using a modified phage display procedure, we selected 2 different antibodies against DS of which one antibody, LKN1, was specific for DS. LKN1 was especially reactive with 4/2,4-di-O-sulfated DS, and did not react with other classes of GAGs including chondroitin sulfate and heparan sulfate. Immunohistochemical analysis of kidney, skin and tendon showed a typical fibrillar staining pattern, co-localizing with type I collagen. Staining was abolished by specific enzymatic digestion of DS. Immunoelectron microscopy confirmed the association of the DS epitope with collagen fibrils. The location of DS did not follow the main banding period of collagen, which is in line with the current concept that the core protein rather than the DS moiety of DS-proteoglycans specifically binds to collagen fibrils. This unique anti-DS antibody and the availability of its coding DNA may be instrumental in studies of the structure and function of DS.

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 anti-thrombin binding pentasaccharide sequence.

Background: The burden of proteinases from inflammatory cells in the lung of subjects with type Pi ZZ of alpha-1-antitrypsin deficiency is higher than in those without the deficiency. Cross-sectional studies have shown increased levels of biomarkers of extracellular matrix degradation in vivo. Longitudinal variability of these biomarkers is unknown but desirable for clinical studies with proteinase inhibitors., Methods: We measured three different types of biomarkers, including desmosines, elastase-formed fibrinogen fragments and heparan sulfate epitope JM403, in plasma and urine for a period of 7 weeks in a group of 12 patients who participated in a placebo-controlled study to assess the safety of a single inhalation of hyaluronic acid., Results: Effect of study medication on any of the biomarkers was not seen. Baseline desmosines in plasma and urine correlated with baseline CO diffusion capacity (R = 0.81, p = 0.01 and R = 0.65, p = 0.05). Mean coefficient of variation within patients (CVi) for plasma and urine desmosines was 18.7 to 13.5%, respectively. Change in urinary desmosine levels correlated significantly with change in plasma desmosine levels (R = 0.84, p < 0.01). Mean CVi for fibrinogen fragments in plasma was 20.5% and for JM403 in urine was 27.8%. No correlations were found between fibrinogen fragments or JM403 epitope and desmosines., Conclusion: We found acceptable variability in our study parameters, indicating the feasibility of their use in an evaluation of biochemical efficacy of alpha-1-antitrypsin augmentation therapy in Pi Z subjects.

Oxidants may play a role in hypoxia-induced respiratory muscle dysfunction. In the present study we hypothesized that hypoxia-induced impairment in diaphragm contractility is associated with elevated peroxynitrite generation. In addition, we hypothesized that strenuous contractility of the diaphragm increases peroxynitrite formation. In vitro force-frequency relationship, isotonic fatigability, and nitrotyrosine levels were assessed under hypoxic (Po(2) approximately 6.5 kPa) and hyperoxic (Po(2) approximately 88.2 kPa) control conditions and also in the presence of authentic peroxynitrite (60 min), ebselen (60 min), and the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine acetate (L-NMMA) (90 min). A hypoxia-induced downward shift of the force-frequency relationship was associated with elevated nitrotyrosine level in the diaphragm. During hypoxia, both ebselen and L-NMMA decreased nitrotyrosine levels but did not affect force generation. Strenuous contractions impaired force generation but did not affect nitrotyrosine levels in the diaphragm during hypoxia. But under hyperoxic conditions, fatiguing contractions were associated with elevated diaphragm nitrotyrosine levels. Under hyperoxic conditions exogenous peroxynitrite impaired force generation and increased nitrotyrosine level. These studies show that hypoxia-induced impairment in diaphragm contractility is associated with increased diaphragm protein nitration, but no causal relationship was found between diaphragm nitrotyrosine formation and in vitro force generation.

Heparan sulfates (HS), a class of glycosaminoglycans, are long linear complex polysaccharides covalently attached to a protein core. The HS molecules are made up of repeating disaccharides onto which modification patterns are superimposed. This results in a large structural heterogeneity and forms the basis of specific interactions of HS toward a vast array of proteins, including growth factors and proteases. To study HS heterogeneity in the lung, we used phage display technology to select seven antibodies against human lung HS. Antibodies reacted with HS/heparin, but not with other glycosaminoglycans or polyanions. Sulfate groups were essential for antibody binding. The amino acid sequence of the antibodies was established, the complementarity determining region 3 of the heavy chain containing basic amino acids. The antibodies defined HS epitopes with a characteristic tissue distribution. Antibody EV3A1 primarily stained macrophages. Other antibodies primarily stained basement membranes, but with different preference toward type of basement membrane. Antibody EV3C3 was the only antibody which clearly reacted with bronchiolar epithelial cells. In human lung parenchyma, basic fibroblast growth factor and vascular endothelial growth factor were largely bound by HS. Some antibodies blocked a basic fibroblast growth factor-binding site of HS, and one antibody blocked a vascular endothelial growth factor-binding site of heparin. Taken together, these data suggest a specific role for HS epitopes in human lung. The antibodies obtained may be valuable tools to study HS in pulmonary diseases.

Glycosaminoglycans (GAGs) are anionic polysaccharides present on cells and in the extracellular matrix (ECM). They likely play a role in tumor formation because of their capacity to bind and modulate a variety of proteins including growth factors, cytokines, and proteases. Using a panel of (human) phage display-derived anti-GAG antibodies, the location and expression of GAG epitopes in human cutaneous melanocytic lesions was studied. Antibodies EW4E1 and EW4G2 identified a melanoma-associated chondroitin sulfate/heparan sulfate epitope, whereas antibody EW4B7 recognized a melanoma-associated heparan sulfate epitope. These antibodies showed a high reactivity with blood vessels and ECM in cutaneous melanoma tumors, whereas their reactivity with nevi was very low. Using a set of defined oligosaccharides it was established that sulfate groups are of main importance in the binding to the antibodies and that glycomimetics can mimic natural oligosaccharides. In xenografts of melanoma cell line MeL57, a strong association of GAG epitopes with an injected fluorescent fluid flow tracer was observed. In uveal melanoma antibody, EW4E1 proved to be a sensitive probe for the detection of the geometry of ECM structures, known to have prognostic value. Taken together, data indicate that in melanoma a defined set and location of GAG epitopes are present with possible functional significance.

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.

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.

Resection of lung specimens results in deflated and distorted lung structures. If no major airway is present (as in the case of small lung specimens from biopsies), lung dimensions cannot be restored by inflation under 25 cmH20. This impedes morphological analysis of the tissue. This report describes a simple and easy procedure to restore alveolar dimensions in deflated small lung specimens. Small human lung samples were inflated using moderate vacuum conditions which are provided by a common water stream-driven vacuum device. Restoration of alveolar dimensions and morphology was evaluated for paraffin-embedded as well as frozen tissue, using morphometric and immunohistological analysis. Vacuum inflation results in restoration of original lung dimensions as judged by light and scanning electron microscopy, and by analysis of the mean linear intercept, and the average length, width, perimeter and surface area. It also results in markedly improved cutting characteristics, allowing reliable sectioning of 2 microm cryosections and achieving high resolution images in immunofluorescence. Vacuum inflation is a simple and easy procedure to restore lung architecture of small human lung specimens/biopsies with a concomitant improvement of cutting characteristics. It allows for correct histological analysis of small specimens which cannot be inflated otherwise.

The possible involvement of proteoglycans in the pathogenesis of emphysema was studied in rats by a single intratracheal instillation of p-nitrophenyl-beta-D-xylopyranoside (beta-D-xyloside), an inhibitor of proteoglycan synthesis. The first 3 days after instillation are characterized by mild hemorrhages, some infiltration of inflammatory cells, and edema. After 1 wk, lung morphology is normal again. Forty days after instillation, considerable parenchymal destruction has occurred as determined by the mean linear intercept (81 +/- 12 microns versus 57 +/- 5 microns for control [P < 0.001]). Pulmonary fibrosis is not observed. Instillation with p-nitrophenyl-alpha-D-xylopyranoside and p-nitrophenol do not induce parenchymal destruction, indicating the specificity of beta-D-xyloside action. Urinary glycosaminoglycan (GAG) content of the beta-D-xyloside-treated rats is increased 15-fold during the first day after instillation, mainly due to elevated levels of chondroitin sulfate and dermatan sulfate. The increase is correlated to the extent of parenchymal destruction after 40 days (r = 0.68; P < 0.002). At day 2 and thereafter, levels are normal again. A short-term increase in dermatan and chondroitin sulfate content is also observed in serum, bronchoalveolar lavage (BAL) fluid, and lung tissue. Heparan sulfate content is decreased in BAL fluid and lung tissue. Instillation with p-nitrophenyl-alpha-D-xylopyranoside and p-nitrophenol do not induce elevated GAG concentration in urine. We suggest that a disturbance in proteoglycan synthesis accompanied by an increase of (beta-D-xyloside-primed) free GAGs results in loss of stability and integrity of the alveolar wall, leading to parenchymal destruction and emphysematous lesions. beta-D-xyloside treatment may be an alternative experimental method for inducing emphysema.

In patients with emphysema the integrity of the extracellular matrix (connective tissue skeleton) is compromised. In this study we analyzed glycosaminoglycans, which are main constituents of this matrix, in urines from patients with chronic obstructive pulmonary disease (COPD)/emphysema. Glycosaminoglycans (GAGs) were purified by anion exchange chromatography and quantified using the 1,9-dimethylmethylene blue assay. Heparan sulfate (HS) was assayed using three different chemical methods: digestion with heparitinase or with nitrous acid and by use of an adapted 1,9-dimethylmethylene blue assay. A specific epitope on the HS molecule, defined by the monoclonal antibody JM403, was determined using an inhibition enzyme immunoassay. In patients with COPD total urinary glycosaminoglycan and HS content were not altered. The JM403 epitope of HS, however, was greatly decreased in patients (0.6 versus 4.1 units/mg creatinine for control subjects, p < 0.0001). A similar pattern was observed when patients with bronchial carcinoma with and without emphysema were compared (0.4 versus 2.4 units/mg creatinine respectively, p < 0.0005). Patients with sarcoidosis did not show a decreased epitope content. These results indicate a structural change or an altered processing of the HS molecule in patients with emphysema. Taking into consideration the importance of HS for the stability of the alveolar extracellular matrix, this change may be associated with the pathogenesis of emphysema.

Autofluorescence can be a very disturbing factor in immunofluorescence microscopy. We present here a method to eliminate autofluorescence. The method is based on the fact that most autofluorescent compounds have broad-banded excitation and emission spectra, whereas specific fluorescent probes have narrow spectra. Two images are recorded and digitized, one at a wavelength exciting both the fluorescent probe and the autofluorescent molecules, and one at a wavelength exciting only the latter. Subtraction of the autofluorescence signal from the total fluorescence signal, using a self-developed computer program, results in an autofluorescence-free image. The procedure is demonstrated for elimination of elastin-derived autofluorescence in human lung alveoli and for elimination of lipofuscin-derived autofluorescence in human heart muscle. The autofluorescence signal is positively correlated with tissue section thickness (r = 0.93; p < 0.0001), and can be used to correct the specific fluorescence signals for section thickness.

A rapid, sensitive, and nonradioactive method has been developed for the quantification and characterization of glycosaminoglycans. The method is based on the separation of different types of glycosaminoglycans in agarose gel and subsequent fixation and staining with the cationic dye azure A, followed by silver enhancement. Densitometric analysis of the silver deposition gives a linear response between 1 and 20 ng for chondroitin and dermatan sulfate and between 2 and 40 ng for heparan sulfate. The detection limit is about 250 pg. The staining procedure was applied for the quantification and characterization of glycosaminoglycans in human serum, urine, and lung and in mouse kidney glomeruli. It requires only 10 microliters serum, 2 microliters urine, and only a single cryosection in case of tissue. The method is at least as sensitive as staining with radioactive markers and about 200 times more sensitive than conventional glycosaminoglycan-staining methods.

Basement membrane (BM) components were studied on human muscle and skeletal muscle cells cultured on different media by immunofluorescence and electron microscopy. Their topographical relation with acetylcholine receptors was investigated. Myotubes cultured on a combination of the serum substitute Ultroser G and brain extract show a continuous layer of heparan sulfate proteoglycans (HSPGs), laminin, and type IV collagen. In contrast, myotubes cultured on serum-containing media are associated with granular depositions of HSPG and laminin and only with wisps of type IV collagen. Omission of brain extract or substitution by chicken embryo extract results in an intermediate staining pattern. For all types of cultures, fibronectin is localized in and around mononuclear cells, but hardly associated with myotubes. A codistribution between clusters of acetylcholine receptors and HSPG and laminin and Vicia villosa B4 lectin-positive material exists only in Ultroser G/brain extract-based myotubes like in muscle in vivo. No clustering is observed in serum-based myotubes. Electron microscopy reveals that the former myotubes are surrounded by a continuous BM consisting of a lamina lucida, lamina densa, and lamina fibroreticularis. Proteoglycans are present on the external site of the lamina densa and associated in a regular fashion with collagen fibrils. In conclusion, BMs associated with myotubes cultured on Ultroser G/brain extract resemble in many ways the in vivo situation, including synaptic specializations. Cultured myotubes may serve as a model system for studies on the structure and function of human muscular (synaptic) BM under normal and pathological conditions.