Pattern Of Glycosaminoglycans Research Articles

Protein glycosylation in lung cancer from a mass spectrometry perspective.

Lung cancer is a severe disease for which better diagnostic and therapeutic approaches are urgently needed. Increasing evidence implies that aberrant protein glycosylation plays a crucial role in the pathogenesis and progression of lung cancer. Differences in glycosylation patterns have been previously observed between healthy and cancerous samples as well as between different lung cancer subtypes, which suggests untapped diagnostic potential. In addition, understanding the changes mediated by glycosylation may shed light on possible novel therapeutic targets and personalized treatment strategies for lung cancer patients. Mass spectrometry based glycomics and glycoproteomics have emerged as powerful tools for in-depth characterization of changes in protein glycosylation, providing valuable insights into the molecular basis of lung cancer. This paper reviews the literature on the analysis of protein glycosylation in lung cancer using mass spectrometry, which is dominated by manuscripts published over the past 5 years. Studies analyzing N-glycosylation, O-glycosylation, and glycosaminoglycan patterns in tissue, serum, plasma, and rare biological samples of lung cancer patients are highlighted. The current knowledge on the potential utility of glycan and glycoprotein biomarkers is also discussed.

In silico and in vitro mapping of specificity patterns of glycosaminoglycans towards cysteine cathepsins B, L, K, S and V

Human cysteine cathepsins are lysosomal proteases, which are involved in different biological processes. Their enzymatic activity can be regulated by glycosaminoglycans (GAGs): long linear periodic negatively charged polysaccharides, which dimeric building blocks consist of uronic acid and hexosamine monosaccharide units. In this study, molecular docking simulations of chondroitin 4-sulfate, chondroitin 6-sulfate, heparin, heparan sulfate, dermatan sulfate and hyaluronic acid of various chain lengths were performed with cathepsins B, L, K, S and V and followed by molecular dynamics-based refinement and binding free energy analysis. We concluded that electrostatics might be a driving force for cathepsin-GAG interactions; indeed as in most of characterised systems, the increase of GAG chain length consequently leads to a more pronounced effect on the strength of cathepsin-GAG interactions. Results also suggest that binding of GAGs at different regions on cathepsins surface affect differently their enzymatic activity and could is dependent on cathepsin and GAG type. Present data contribute to systematic description of cathepsin-GAG interactions, which is helpful in understanding the subtle molecular mechanisms of protease regulation behind their biological functions.

Disease-specific glycosaminoglycan patterns in the extracellular matrix of human lung and brain

A wide variety of diseases throughout the mammalian organism is characterized by abnormal deposition of various components of the extracellular matrix (ECM), including the heterogeneous family of glycosaminoglycans (GAGs), which contribute considerably to the ECM architecture as part of the so-called proteoglycans. The GAG's unique sulfation pattern, derived from highly dynamic and specific modification processes, has a massive impact on critical mediators such as cytokines and growth factors. Due to the strong connection between the specific sulfation pattern and GAG function, slight alterations of this pattern are often associated with enormous changes at the cell as well as at the organ level. This review aims to investigate the connection between modifications of GAG sulfation patterns and the wide range of pathological conditions, mainly focusing on a range of chronic diseases of the central nervous system (CNS) as well as the respiratory tract.

The stoic tooth root: how the mineral and extracellular matrix counterbalance to keep aged dentin stable

Aging is a physiological process with profound impact on the biology and function of biosystems, including the human dentition. While resilient, human teeth undergo wear and disease, affecting overall physical, psychological, and social human health. However, the underlying mechanisms of tooth aging remain largely unknown. Root dentin is integral to tooth function in that it anchors and dissipates mechanical load stresses of the tooth-bone system. Here, we assess the viscoelastic behavior, composition, and ultrastructure of young and old root dentin using nano-dynamic mechanical analysis, micro-Raman spectroscopy, small angle X-ray scattering, atomic force and transmission electron microscopies. We find that the root dentin overall stiffness increases with age. Unlike other mineralized tissues and even coronal dentin, however, the ability of root dentin to dissipate energy during deformation does not decay with age. Using a deconstruction method to dissect the contribution of mineral and organic matrix, we find that the damping factor of the organic matrix does deteriorate. Compositional and ultrastructural analyses revealed higher mineral-to-matrix ratio, altered enzymatic and non-enzymatic collagen cross-linking, increased collagen d-spacing and fibril diameter, and decreased abundance of proteoglycans and sulfation pattern of glycosaminoglycans . Therefore, even in the absence of remodeling, the extracellular matrix of root dentin shares traits of aging with other tissues. To explain this discrepancy, we propose that altered matrix-mineral interactions, possibly mediated by carbonate ions sequestered at the mineral interface and/or altered glycosaminoglycans counteract the deleterious effects of aging on the structural components of the extracellular matrix. Statement of significanceGlobally, a quarter of the population will be over 65 years old by 2050. Because many will retain their dentition, it will become increasingly important to understand and manage how aging affects teeth. Dentin is integral to the protective, biomechanical, and regenerative features of teeth. Here, we demonstrate that older root dentin not only has altered mechanical properties, but shows characteristic shifts in mineralization, composition, and post-translational modifications of the matrix. This strongly suggests that there is a mechanistic link between mineral and matrix components to the biomechanical performance of aging dentin with implications for efforts to slow or even reverse the aging process.

The Influence of Polysaccharides-Based Material on Macrophage Phenotypes.

Macrophage polarization is a key factor in determining the success of implanted tissue engineering scaffolds. Polysaccharides (derived from plants, animals, and microorganisms) are known to modulate macrophage phenotypes by recognizing cell membrane receptors. Numerous studies have developed polysaccharide-based materials into functional biomaterial substrates for tissue regeneration and pharmaceutical application due to their immunostimulatory activities and anti-inflammatory response. They are used as hydrogel substrates, surface coatings, and drug delivery carriers. In addition to their innate immunological functions, the newly endowed physical and chemical properties, including substrate modulus, pore size/porosity, surface binding chemistry, and the mole ratio of polysaccharides in hybrid materials may regulate macrophage phenotypes more precisely. Growing evidence indicates that the sulfation pattern of glycosaminoglycans and proteoglycans expressed on polarized macrophages leads to the changes in protein binding, which may alter macrophage phenotype and influence the immune response. A comprehensive understanding of how different types of polysaccharide-based materials alter macrophage phenotypic changes can be beneficial to predict transplantation/implantation outcomes. This review focuses on recent advances in promoting wound healing and balancing macrophage phenotypes using polysaccharide-based substrates/coatings and new directions to address the limitations in the current understanding of macrophage responses to polysaccharides.

Biochemical, machine learning and molecular approaches for the differential diagnosis of Mucopolysaccharidoses.

This study was aimed to construct classification and regression tree (CART) model of glycosaminoglycans (GAGs) for the differential diagnosis of Mucopolysaccharidoses (MPS). Two-dimensional electrophoresis and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used for the qualitative and quantitative analysis of GAGs. Specific enzyme assays and targeted gene sequencing were performed to confirm the diagnosis. Machine learning tools were used to develop CART model based on GAG profile. Qualitative and quantitative CART models showed 96.3% and 98.3% accuracy, respectively, in the differential diagnosis of MPS. The thresholds of different GAGs diagnostic of specific MPS types were established. In 60 MPS positive cases, 46 different mutations were identified in six specific genes. Among 31 different mutations identified in IDUA, nine were nonsense mutations and two were gross deletions while the remaining were missense mutations. In IDS gene, four missense, two frameshift, and one deletion were identified. In NAGLU gene, c.1693C > T and c.1914_1914insT were the most common mutations. Two ARSB, one case each of SGSH and GALNS mutations were observed. LC-MS/MS-based GAG pattern showed higher accuracy in the differential diagnosis of MPS. The mutation spectrum of MPS, specifically in IDUA and IDS genes, is highly heterogeneous among the cases studied.

Fast fabrication of stable cartilage-like tissue using collagen hydrogel microsphere culture.

Mesenchymal stem cells (MSCs) had been increasingly regarded as a potent cell source for cartilage repair. However, due to the instability of MSC-derived chondrocyte phenotype and ossification of the synthesised cartilage matrix, regenerating a stable cartilage tissue by MSCs is still challenging. The fate of chondrogenesis from MSCs is regulated by their local microenvironment, which is of vital importance to the cell behaviours, chondrogenic phenotype and matrix synthesis. In this study, we fabricated cartilage-like tissues by the chondrogenesis of MSC in three different microenvironments, including cell pellets, collagen hydrogel bulk (CHB) and collagen hydrogel microspheres (CHMs) in vitro. After 15 days in culture, the cell number was increased to 472.6% in CHMs, compared to a 58.6% decrease in CHB and a 46.6% decrease in pellets; resulting in a 230% increase in CHM size, but a 36.8% decrease in CHB and only a 20.1% increase in pellets. Histological staining demonstrated a more intensive but less homogeneous glycosaminoglycan (GAG) pattern in pellets than in CHMs. The outer area of CHB showed a stronger GAG staining than its inner area from day 5 to day 15, but the staining was weaker than that in both pellets and CHMs. The PCR results showed that CHMs achieved a significantly higher chondrogenic gene (AGG, COL2A1, SOX9) expression and a lower hypertrophic gene (COL10A1) expression than pellets and CHB, suggesting a better chondrogenic differentiation potential with a more stable phenotype in CHMs. In summary, this study highlights the advantages of CHM microenvironments over those of CHB and pellets by a better mimicking of the natural MSC proliferation process and enhancing mass exchange in vitro. The CHM culture demonstrated potential to fabricate stable cartilage-like tissue in MSC based cartilage tissue regeneration.

Biochemical validity of imaging techniques (X-ray, MRI, and dGEMRIC) in degenerative disc disease of the human cervical spine—an in vivo study

Background ContextOn a molecular level, maturation or degeneration of human intervertebral disc is among others expressed by the content of glycosaminoglycans (GAGs). According to the degenerative status, the disc content can differ in nucleus pulposus (NP) and annulus fibrosus (AF), respectively. Research in this area was conducted mostly on postmortem samples. Although several radiological classification systems exist, none includes biochemical features. Therefore, we focused our in vivo study on a widely spread and less expensive imaging technique for the cervical spine and the correlation of radiological patterns to biochemical equivalents in the intervertebral discs. PurposeThe aim of this pilot study was to (1) measure the GAG content in human cervical discs, (2) to investigate whether a topographic biochemical GAG pattern can be found, and (3) whether there is a correlation between imaging data (X-ray and magnetic resonance imaging [MRI] including delayed gadolinium-enhanced MRI of cartilage [dGEMRIC] as a special imaging technique of cartilage) and the biochemical data. Study Design/SampleWe conducted a prospective experimental pilot study. Patient SampleOnly non-responders to conservative therapy were included. All subjects were physically and neurologically examined, and they completed their questionnaires. Outcome MeasuresVisual analogue scale neck and arm, Neck Disability Index score, radiological parameters (X-rays, MRI, dGEMRIC), and the content of GAG in the cervical disc were assessed. MethodsAfter surgical removal of 12 discs, 96 fractions of AF and NP were biochemically analyzed for the GAG content using dimethylmethylene blue assay. ResultsA quantitative pattern of GAGs in the human cervical disc was identified. There were (1) significantly (p<.001) higher values of GAGs (µg GAG/mg tissue) in the NP (169.9 SD 37.3) compared with the AF (132.4 SD 42.2), and (2) significantly (p<.005) higher values of GAGs in the posterior (right/left: 149.9/160.2) compared with the anterior (right/left: 112.0/120.2) part of the AF. Third, we found in dGEMRIC imaging a significantly (p<.008) different distribution of GAGs in the cervical disc (NP 1083.3 ms [SD 248.6], AF 925.9 ms [SD 137.6]). Finally, we found that grading of disc degeneration in X-ray and MRI was significantly correlated with neither AF- nor NP-GAG content. ConclusionsThe GAG content in human cervical discs can be detected in vivo and is subject to a significantly (p<.05) region-specific pattern (AF vs. NP; anterior vs. posterior in the AF). Up to the levels of AF and NP, this is reproducible in MRI in dGEMRIC technique, but not in X-ray or standard MRI sequences. Potentially, the MRI in dGEMRIC technique can be used as a non-invasive in vivo indicator for disc degeneration in the cervical spine.

Sulphated glycosaminoglycans and proteoglycans in the developing vertebral column of juvenile Atlantic salmon (Salmo salar).

In the present study, the distribution of sulphated glycosaminoglycans (GAGs) in the developing vertebral column of Atlantic salmon (Salmo salar) at 700, 900, 1100 and 1400 d° was examined by light microscopy. The mineralization pattern was outlined by Alizarin red S and soft structures by Alcian blue. The temporal and spatial distribution patterns of different types of GAGs: chondroitin-4-sulphate/dermatan sulphate, chondroitin-6-sulphate, chondroitin-0-sulphate and keratan sulphate were addressed by immunohistochemistry using monoclonal antibodies against the different GAGs. The specific pattern obtained with the different antibodies suggests a unique role of the different GAG types in pattern formation and mineralization. In addition, the distribution of the different GAG types in normal and malformed vertebral columns from 15 g salmon was compared. A changed expression pattern of GAGs was found in the malformed vertebrae, indicating the involvement of these molecules during the pathogenesis. The molecular size of proteoglycans (PGs) in the vertebrae carrying GAGs was analysed with western blotting, and mRNA transcription of the PGs aggrecan, decorin, biglycan, fibromodulin and lumican by real-time qPCR. Our study reveals the importance of GAGs in development of vertebral column also in Atlantic salmon and indicates that a more comprehensive approach is necessary to completely understand the processes involved.

Adhesion of adipose-derived mesenchymal stem cells to glycosaminoglycan surfaces with different protein patterns.

Proteins and glycosaminoglycans (GAGs) are the main constituents of the extracellular matrix (ECM). They act in synergism and are equally critical for the development, growth, function, or survival of an organism. In this work, we developed surfaces that display these two classes of biomacromolecules, namely, GAGs and proteins, in a spatially controlled fashion. The generated surfaces can be used as a minimalistic but straightforward model aiding the elucidation of cell-ECM interactions. GAGs (hyaluronic acid and heparin) were covalently bound to amino functionalized surfaces, and albumin or fibronectin was patterned by microcontact printing on top of them. We demonstrate that adipose-derived stem cells (ASCs) can adhere either on the protein or on the GAG pattern as a function of the patterned molecules. ASCs found on the GAG pattern had different morphology and expressed different surface markers than the cells adhered on the protein pattern. ASCs morphology and spreading were also dependent on the size of the pattern. These results show that the developed supports can also be used for ASCs differentiation into different lineages.

Influence of the scaffold geometry on the spatial and temporal evolution of the mechanical properties of tissue-engineered cartilage: insights from a mathematical model.

The production of tissue-engineered cartilage in vitro with inhomogeneous mechanical properties is a problem yet to be solved. Different geometries have been studied to overcome this caveat; however, the reported measurements are limited to average values of some properties and qualitative measures of spatial distributions. We will apply a coupled model to extend knowledge about the introduction of a macrochannel in a scaffold by calculating spatiotemporal patterns for several interest variables related to the remodeling of the mechanical properties. Model parameters were estimated based on experimental data on the temporal patterns of glycosaminoglycans, collagen and compressive Young's modulus for channel-free constructs. The model reproduced the experimental data trends in both geometries, with experimental-numerical correlations between 0.84 and 0.97. The channel had a higher impact on the reduction in spatial heterogeneities and delay of saturation of core properties than in the improvement of average properties. Despite the possible improvement of cell densities for longer periods than 56 days, it is estimated that it will not cause further significant improvements of the mechanical properties. The degrees of spatial heterogeneity of the Young's modulus and permeability in the channeled geometry are 23 and 27% of the channel-free values. While the average Young's modulus values are in the range of native cartilage, the permeabilities are one to three degrees of magnitude higher than the native cartilage, suggesting that limiting factors such as scaffold porosity and initial permeability are more relevant than scaffold geometry to effectively decrease the tissue permeability.

Urinary Glycosaminoglycan Electrophoresis With Optimized Keratan Sulfate Separation Using Peltier System for the Screening of Mucopolysaccharidoses

The purpose of this communication is to indicate a simple and rapid method with a small volume of urine sample to detect urine glycosaminoglycan (GAG) and serve as a screening procedure for mucopolysaccharidoses (MPSs). Total GAG measurement for patients with MPS disorders is considered to be the first step in diagnosis of those heterogeneous group of lysosomal storage disorders presenting clinical phenotype. In this study, modified 9-dimethylmethylene blue method is used for total GAG measurement. Following GAG quantitation, the procedure described here allows GAG isolation from a very a small volume of urine sample and subjected to high-resolution GAG electrophoresis, which can be easily performed in routine clinical diagnostic laboratories. Glycosaminoglycan precipitation is a modified method based on total GAG concentration in the urine. For optimized isolation of total GAG for electrophoresis, instead of considering the urine creatinine concentration, 300 μg/mL GAG containing urine is considered to be the target concentration for the best precipitation with 1000 μL cetylpyridinium chloride (CPC)/citrate buffer. Glycosaminoglycan concentration-based precipitation of urine with CP Ca llows the laboratory to be able to work with as mall volume of urine sample by keeping the precipitating ratio with CPC constant for samples that contain GAG less than 300 μg/mL. Based on the effect of cold buffer using low voltage, GAGs high-resolution electrophoresis banding patterns described here enable a clear separation of keratan sulfate from chondroitin sulfate as well as dermatan sulfate (DS1 and DS2) and heparan sulfate. By this procedure, GAG patterns are more clear, easily identified, and provide a guide for the enzyme analysis deficient in the MPS disorders.

Biohybrid networks of selectively desulfated glycosaminoglycans for tunable growth factor delivery.

Sulfation patterns of glycosaminoglycans (GAG) govern the electrostatic complexation of biomolecules and thus allow for modulating the release profiles of growth factors from GAG-based hydrogels. To explore options related to this, selectively desulfated heparin derivatives were prepared, thoroughly characterized, and covalently converted with star-shaped poly(ethylene glycol) into binary polymer networks. The impact of the GAG sulfation pattern on the network characteristics of the obtained hydrogels was theoretically evaluated by mean field methods and experimentally analyzed by rheometry and swelling measurements. Sulfation-dependent differences of reactivity and miscibility of the heparin derivatives were shown to determine network formation. A theory-based design concept for customizing growth factor affinity and physical characteristics was introduced and validated by quantifying the release of fibroblast growth factor 2 from a set of biohybrid gels. The resulting new class of cell-instructive polymer matrices with tunable GAG sulfation will be instrumental for multiple applications in biotechnology and medicine.

Interference of an algal nutritional supplement with a urinary metabolic screening test for glycosaminoglycans in a dog suspected to have a storage disease

The finding of excess urinary glycosaminoglycans (GAG) is the first step in the laboratory diagnosis of mucopolysaccharidosis (MPS). Urinary screening tests are based upon the binding of GAG to dimethylmethylene blue. Alternatively, paper spot tests using toluidine blue are used in human and veterinary laboratory medicine. Positive samples undergo GAG isolation for subsequent characterization. Here, we describe a 3-year-old English Cocker Spaniel with a positive urinary GAG test, but without other clinical signs of MPS. Urine samples were strongly positive with the dimethylmethylene blue test, and isolated GAG subjected to electrophoresis on cellulose acetate revealed a band co-migrating with dermatan sulfate. However, the isolated GAG were resistant to digestion with chondroitinase ABC, suggesting that the band did not represent dermatan sulfate. This was confirmed by mobility of the isolated GAG different from dermatan sulfate on agarose gel electrophoresis. MPS types VI and VII were excluded by enzyme assay. To test the hypothesis of a nutritional source, a healthy control dog was fed the same dog food as the index case. His urine showed a comparable abnormal GAG screening test and electrophoretic pattern. In addition, the analysis of an algal supplement present in the administered dog food showed a similar electrophoretic GAG pattern. The Cocker Spaniel was not available for further testing after withdrawal of the supplement. Algae contain highly sulfated fucans and galactans, and it appears that commercial dog food containing such algal, and possibly other, supplements can give rise to false-positive urinary MPS screening tests.

Uncovering Biphasic Catalytic Mode of C5-epimerase in Heparan Sulfate Biosynthesis

Heparan sulfate (HS), a highly sulfated polysaccharide, is biosynthesized through a pathway involving several enzymes. C(5)-epimerase (C(5)-epi) is a key enzyme in this pathway. C(5)-epi is known for being a two-way catalytic enzyme, displaying a "reversible" catalytic mode by converting a glucuronic acid to an iduronic acid residue, and vice versa. Here, we discovered that C(5)-epi can also serve as a one-way catalyst to convert a glucuronic acid to an iduronic acid residue, displaying an "irreversible" catalytic mode. Our data indicated that the reversible or irreversible catalytic mode strictly depends on the saccharide substrate structures. The biphasic mode of C(5)-epi offers a novel mechanism to regulate the biosynthesis of HS with the desired biological functions.

Wild blueberry (V. angustifolium)-enriched diets alter aortic glycosaminoglycan profile in the spontaneously hypertensive rat

Glycosaminoglycans (GAGs) are essential polysaccharide components of extracellular matrix and cell surface with key roles on numerous vascular wall functions. Previous studies have documented a role of wild blueberries on the GAG profile of the Sprague–Dawley rat with a functional endothelium as well as in the vascular tone of the spontaneously hypertensive rat (SHR) with endothelial dysfunction. In the present study, the effect of wild blueberries on the composition and structure of aortic GAGs was examined in 20-week-old SHRs after 8 weeks on a control (C) or a wild blueberry-enriched diet (WB). Aortic tissue GAGs were isolated following pronase digestion and anion-exchange chromatography. Treatment of the isolated populations with specific GAG-degrading lyases and subsequent electrophoretic profiling revealed the presence of three GAG species, i.e., hyaluronic acid (HA), heparan sulfate (HS) and galactosaminoglycans (GalAGs). A notable reduction of the total sulfated GAGs and a redistribution of the aortic GAG pattern were recorded in the WB as compared to the C group: a 25% and 10% increase in HA and HS, respectively, and an 11% decrease in GalAGs. Fine biochemical analysis of GalAGs at the level of constituent disaccharides with high-performance capillary electrophoresis revealed a notable increase of nonsulfated (18.0% vs. 10.7%) and a decrease of disulfated disaccharides (2.2% vs. 5.3%) in the WB aorta. This is the first study to report the redistribution of GAGs at the level of composition and their fine structural characteristics with implications for the endothelial dysfunction of the SHR.

Effect of the gonadal status and the gender on glycosaminoglycans profile in the lower urinary tract of dogs

Glycosaminoglycans (GAGs) form a functional component of connective tissues that affect the structural and functional integrity of the lower urinary tract (LUT). The specific GAGs of physiological relevance are both nonsulfated (hyaluronan) and sulfated GAGs (chondroitin sulphate [CS], dermatan sulphate [DS], keratan sulphate [KS], and heparan sulphate [HS]). As GAG composition in the LUT is hormonally regulated, we postulated that gonadectomy-induced endocrine imbalance alters the profile of GAGs in the canine LUT. Four regions of the LUT (body and neck of the bladder as well as the proximal and distal urethra) from 20 clinically healthy dogs (5 intact males, 5 intact anoestrus females, 4 castrated males, and 6 spayed females) were collected, wax-embedded and sectioned. Alcian blue staining at critical electrolyte concentrations was performed on the sections to determine total GAGs, hyaluronan, total sulfated GAGs, combined components of CS and DS, as well as KS and HS. The amount of staining was evaluated in 3 tissue layers, i.e., epithelium, subepithelial stroma and muscle within a region. Overall, hyaluronan (67.1%) was the predominant GAG in the LUT. Among sulfated GAGs, a combined component of KS and HS was found to be 61.8% and 38.2% for CS and DS. Gonadal status significantly affected GAG profiles in the LUT (P < 0.01). All GAG components were lower (P < 0.05) in body of the bladder of gonadectomized dogs. Total sulfated GAGs and a combined component of KS and HS were lower (P < 0.05) in all 4 regions of gonadectomized dogs. Except for a combined component of CS and DS, decreases in all GAGs were found more consistently in the muscle compared to other tissue layers. Differences between genders became obvious only when considered along with the effect of gonadal status. In gonadectomized dogs, changes in GAG components in the LUT were more consistent in females compared to males; this may partly explain different levels of risk in the development of urinary incontinence between genders. Quantitative differences in GAG profiles found between intact and gonadectomized dogs indicate a potential role of gonadectomy-induced endocrine imbalance in modifying GAG composition in the canine LUT. Profound alteration in the pattern of GAGs in gonadectomized dogs may compromise structural and functional integrity of the LUT and is possibly involved in the underlying mechanism of urinary incontinence post neutering.

T cells and orbital connective tissue in endocrine orbitopathy.

In order to analyse the immunological changes in patients with endocrine orbitopathy (EO) the antigenic character of orbital connective tissue was studied. Counter-stimulation assays of patients' lymphocytes with autologous retrobulbar fibroblasts resulted in a markedly increased lymphocyte proliferation in comparison to incubation with retrobulbar control fibroblasts. Proliferation tests of retrobulbar T cell lines showed significant responses to autologous retro-orbital connective tissue proteins, with molecular weights of 6-10 kD and 19-26 kD. Phenotypic analysis of orbital T cell lines indicates that they consisted predominantly of CD4+ cells. Hyaluronic acid production of orbital fibro blasts following co-cultivation with lymphocytes of EO patients or controls revealed a threefold increased synthesis in patients with EO. Furthermore, distribution pattern of orbital extracellular matrix glycosaminoglycans (GAG) differs in EO patients in comparison to controls. The results suggest the presence of autoreactive T cells directed against antigens of orbital fibroblasts, whose stimulation results in an augmented GAG synthesis in patients with EO.

The Structure of Glycosaminoglycans and their Interactions with Proteins

Glycosaminoglycans (GAGs) are important complex carbohydrates that participate in many biological processes through the regulation of their various protein partners. Biochemical, structural biology and molecular modelling approaches have assisted in understanding the molecular basis of such interactions, creating an opportunity to capitalize on the large structural diversity of GAGs in the discovery of new drugs. The complexity of GAG-protein interactions is in part due to the conformational flexibility and underlying sulphation patterns of GAGs, the role of metal ions and the effect of pH on the affinity of binding. Current understanding of the structure of GAGs and their interactions with proteins is here reviewed: the basic structures and functions of GAGs and their proteoglycans, their clinical significance, the three-dimensional features of GAGs, their interactions with proteins and the molecular modelling of heparin binding sites and GAG-protein interactions. This review focuses on some key aspects of GAG structure-function relationships using classical examples that illustrate the specificity of GAG-protein interactions, such as growth factors, anti-thrombin, cytokines and cell adhesion molecules. New approaches to the development of GAG mimetics as possible new glycotherapeutics are also briefly covered.

Monocytes secrete factors regulating glycosaminoglycan synthesis in mesangial cellsin vitro

The present study was conducted to determine the manner in which monocytes increase mesangial matrices, particularly glycosaminoglycans (GAGs) which interact with various other matrix components such as collagens, laminin, fibronectin and lipoproteins. A supernatant of human peripheral blood monocyte cultures activated by lipopolysaccharide (LPS) contains stimulating factors for glycosaminoglycan synthesis in rat mesangial cells (MCs). The culture supernatant in this study was concentrated and fractionated by gel chromatography and the GAG-stimulatory factor was found to have a molecular weight of 10-17 kD. This factor was shown to be present in fractions different from that of IL-1. Gel and ion-exchange chromatography studies of GAGs synthesized by MCs indicated the elution patterns of GAGs in the presence and absence of the monocyte culture supernatant to be essentially the same. Local infiltration of monocytes into the glomerulus, often seen in various types of glomerular injury, may be an important factor in the accumulation of the mesangial matrix.