Detection of Cell Surface Ligands for Human Synovial γδ T Cells.

Lack of understanding of the nature and physiological regulation of γδ T cell ligands has considerably hampered full understanding of the function of these cells. We developed an unbiased approach to identify human γδ T cells ligands by the production of a soluble TCR-γδ (sTCR-γδ) tetramer from a synovial Vδ1 γδ T cell clone from a Lyme arthritis patient. The sTCR-γδ was used in flow cytometry to initially define the spectrum of ligand expression by both human tumor cell lines and certain human primary cells. Analysis of diverse tumor cell lines revealed high ligand expression on several of epithelial or fibroblast origin, whereas those of hematopoietic origin were largely devoid of ligand. This allowed a bioinformatics-based identification of candidate ligands using RNAseq data from each tumor line. We further observed that whereas fresh monocytes and T cells expressed low to negligible levels of TCR-γδ ligands, activation of these cells resulted in upregulation of surface ligand expression. Ligand upregulation on monocytes was partly dependent upon IL-1ß. The sTCR-γδ tetramer was then used to bind candidate ligands from lysates of activated monocytes and analyzed by mass spectrometry. Surface TCR-γδ ligand was eliminated by treatment with trypsin or removal of glycosaminoglycans, and also suppressed by inhibition of endoplasmic reticulum-Golgi transport. Of particular interest was that inhibition of glycolysis also blocked TCR-γδ ligand expression. These findings demonstrate the spectrum of ligand(s) expression for human synovial Vδ1 γδ T cells as well as the physiology that regulates their expression.

Percutaneous Treatment of Herniated Lumbar Discs with Ozone: Investigation of the Mechanisms of Action.

PURPOSE: To elucidate the mechanism of action of intradiscal oxygen-ozone therapy for herniated intervertebral disc therapy. METHODS: Ozone's mechanism of action was investigated using 3 approaches: mathematical models of intervertebral disc space to explore the relationship between disc pressure and volume; ozonolysis experiments using glycosaminoglycans (GAGs) from a Chinese hamster ovary cell line that were similar in composition to GAGs found in human nucleus pulposus; and experiments in which live Yucatan miniature pigs received various concentrations of percutaneous, image-guided intradiscal oxygen-ozone treatment and were examined (after sacrifice) with histology and semiquantitative analysis of disc cytokine concentrations. RESULTS: Engineering calculations support observations that a small (6%) disc volume reduction can result in considerable (9.84%) intradiscal pressure reduction. Porcine disc histology and Chinese hamster ovary GAG ozonolysis results showed that administered ozone reacted with and fragmented disc proteoglycans, reducing disc volume through disc dehydration. Cytokine analysis of porcine discs found that each of 4 cytokines measured (interleukin [IL]-1ß, IL-6, IL-8, and tumor necrosis factor α) increased in concentration after 2 wt% ozone treatment. CONCLUSIONS: Oxygen-ozone therapy breaks down proteoglycan GAGs that maintain disc osmotic pressure, dehydrating the nucleus pulposus and reducing intervertebral disc volume. This is likely a primary mechanism by which ozone relieves nerve root compression and alleviates herniated disc-related pain. Additionally, 2 wt% ozone appears to interact with intradiscal cytokines, generating an antiinflammatory response that may contribute to symptom improvement.

anexVis: A Transcriptome Tool to Visualize Organ/Tissue-Specific Glycosaminoglycan Biosynthetic and Catabolic Pathways in Human Health and Diseases.

Our lab has developed a new visualization tool, anexVis, for transcriptome analysis of glycosaminoglycan-related genes. This tool allows one to analyze a large number of genes that are related to biosynthetic and catabolic pathways of all glycosaminoglycans, such as heparan sulfate, chondroitin sulfate, keratan sulfate, and hyaluronic acid, in parallel across various human tissues/organs. Such visual analyses have not been accessible to the broad research community despite the accumulation of a large amount of RNA-seq data. We use publicly available data provided by the GTEx project with NIH permission to generate this new framework. Herein, we describe the use of anexVis in understanding the relationship among all biosynthetic and catabolic enzymes, core protein proteoglycans, and various transporters and understanding factors that control organ-specific GAG biosynthesis and catabolism in humans at the transcriptome level. This visualization tool may also assist us in understanding the impact of lysosomal diseases and rare glycan-related diseases on specific organs in humans.

Enzymatic Alteration of ECM to Explore Muscle Function and Motor Control of a Learned Behavior.

Nerves and muscle interact to perform learned motor behavior such as birdsong. Glycosaminoglycans play a major role in the function of muscle as well as the formation and function of the neuromuscular junction. The alteration of GAG chains provides a unique opportunity to alter muscle behavior and thus motor control of a behavior. This chapter provides a method for observing the effects on mature birdsong of removal of GAG chains within syringeal muscle.

Methods for Assessing the Effects of Xylosides on Angiogenesis.

Xylosides are small synthetic molecules consisting of a xylose molecule attached to an aglycone group and serve as primers in the assembly of core protein free glycosaminoglycans using cellular machinery. Synthetic xylosides hold great promise in many biomedical applications and as therapeutics. Recent advances in the study of xylosides have opened up the possibility of developing xylosides as therapeutics to achieve a desirable biological outcome through their selective priming and inhibitory activities toward glycosaminoglycan biosynthesis. The approach described, herein, will serve as a general strategy to comprehensively screen xylosides and evaluate their ability to promote or inhibit angiogenesis, a critical biological process that is dysregulated in over 70 human diseases.

Manipulation of Glycosaminoglycans Using Synthetic Xylosides to Study Their Roles in Lung Branching Morphogenesis in Ex Vivo Lung Bud Culture System.

The primary left and right bronchial buds grow and sprout secondary bronchi, which in turn develop tertiary bronchi, and so on. Branching continues for a total of 6-8 generations in the mouse and for about 23 generations in humans, forming the estimated 50 million branches of the human lung. Thus, patterns of branching are incalculably complex. However, these branches are rarely random, implying that they are under genetic control. Genomic information alone cannot specify the patterning information in terms of where the branching occurs and the direction it grows as well as their size and shape. There is a complex choreography among glycosaminoglycans and growth factors/morphogens that provide a highly complex instructive cues that control lung branching and development of the functional lung. Herein, we describe the use of xylosides in the manipulation of glycosaminoglycan (GAG) biosynthesis and study the effect of xyloside-primed GAGs in the regulation of lung branching events.

Xyloside Derivatives as Molecular Tools to Selectively Inhibit Heparan Sulfate and Chondroitin Sulfate Proteoglycan Biosynthesis.

Glycosaminoglycan (GAG) side chains of proteoglycans are involved in a wide variety of developmental and pathophysiological functions. Similar to a gene knockout, the ability to inhibit GAG biosynthesis would allow us to examine the function of endogenous GAG chains. However, ubiquitously and irreversibly knocking out all GAG biosynthesis would cause multiple effects, making it difficult to attribute a specific biological role to a specific GAG structure in spatiotemporal manner. Reversible and selective inhibition of GAG biosynthesis would allow us to examine the importance of endogenous GAGs to specific cellular, tissue, or organ systems. In this chapter, we describe the chemical synthesis and biological evaluation of xyloside derivatives as selective inhibitors of heparan sulfate and chondroitin/dermatan sulfate proteoglycan biosynthesis.

Investigating the Roles of Heparan Sulfate Structures in Alpha-Synuclein Aggregation in Cell Culture Models.

Glycosaminoglycans (GAGs), belonging to a family of negatively charged linear polysaccharides, have been found in the cores of amyloid inclusions such as Lewy bodies, which are the central pathological features in Parkinson's disease (PD), a neurodegenerative disease. Lewy bodies/neurites are mostly composed of α-synuclein protein (α-syn) aggregates. Recent studies have shown that α-syn aggregates can propagate via neurons in a prion-like fashion by seeding the endogenous cellular α-syn. Various GAGs, especially heparan sulfate (HS), have been shown to be very critical in the aggregation of α-syn. HS chains of heparan sulfate proteoglycans (HSPGs) mediate the uptake of α-syn aggregates and help seed intracellular accumulation and further neuronal spread. Methods that inhibit the binding of these aggregates to HSPG have been shown to decrease the aggregate uptake and propagation. Here, we describe a cell-based assay to screen inhibitors of HS and α-syn interactions.

Methods for the Production of Recombinant Heparosan, a Critical Heparin Precursor, from Nonpathogenic E. coli Strains.

Heparin is an essential anticoagulant drug discovered over a century ago. Heparin is the second most highly used natural drug and remains a mainstay of therapy with an expected global market share of more than $14 billion in the next 10 years. However, it is still naturally derived from unsustainable animal sources, such as bovine lungs and porcine intestines, as an unfractionated, heterogeneous complex mixture with unpredictable pharmacokinetic properties. Extensive research has been done in devising bioengineering and chemical approaches to produce structurally specific heparin and heparin-like polymers. Though several challenges remain, one of the main bottlenecks is the rapid, high-yield production of recombinant heparosan, a heparin precursor, which is originally isolated from a pathogenic E. coli K5 strain. Herein, we outline the methods for producing metabolically engineered size-specific heparosan, by transforming the essential heparosan biosynthetic genes into nonpathogenic E.coli strain BL21(DE3), in a highly controlled manner. The methods described herein are promising and can be easily scaled up for large-scale production of heparin-like structures.

Applications of Xylosides in the Manipulation of Stem Cell Niche to Regulate Human Neural Stem Cell Differentiation and Neurite Outgrowth.

The extracellular matrix (ECM) plays a pivotal role in the regulation of neural stem cell differentiation, axon guidance and growth, and neural plasticity. Glycosaminoglycans, such as heparan sulfate and chondroitin sulfate, are significant components of brain ECM that dictates neurogenesis and neural repair. Herein, we describe a simple method to assess the effect of xylsoides, which serve as primers and inhibitors of GAG biosynthesis, on human neural stem cell differentiation and neurite outgrowth in in vitro culture conditions.

Preparation of Isotope-Enriched Heparan Sulfate Precursors for Structural Biology Studies.

Heparan sulfate (HS) plays numerous important roles in biological systems through their interactions with a wide array of proteins. Structural biology studies of heparan sulfate are often challenging due to the heterogeneity and complexity of the HS molecules. Radioisotope metabolic labeling of HS in cellular systems has enabled the elucidation of HS structures as well as the interactions between HS and proteins. However, radiolabeled structures are not amenable for advanced structural glycobiology studies using sophisticated instruments such as nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). The utilization of stable isotope-enriched HS precursors is an appealing approach to overcome these challenges. The application of stable isotope-enriched HS precursors has facilitated the HS structural analysis by NMR spectroscopy and mass spectrometry. Herein we describe two simple methods to prepare isotopically enriched HS precursors and HS.

Methods to Analyze the Effect of Diet-Derived Metabolites on Endothelial Inflammation and Cell Surface Glycosaminoglycans.

The glycocalyx is a biologically active barrier that covers the luminal side of the vascular endothelium and it is comprised of proteoglycans [core proteins with glycosaminoglycans (GAG) side chains], glycoproteins, and plasma proteins. Evidence shows that the disruption in the structure and function of the endothelial glycocalyx exacerbates vascular inflammation and atherosclerosis. The GAG components of the glycocalyx undergo remodeling in the setting of diabetes and these alterations in endothelial GAGs negatively impact the vascular function. Hence, the preservation and restoration of GAGs in altered vasculature may be a novel strategy to ameliorate vascular complications in diabetes and metabolic syndrome. Human studies support the beneficial vascular effects of flavonoids which are widely found in fruits and vegetables. Flavonoids are extensively metabolized by the intestinal microbiota and digestive enzymes in humans, suggesting that their biological activities may be mediated by their circulating metabolites. Studies indicate that counteracting the damage to GAGs using dietary compounds improve vascular complications. In this article, we describe the methods to analyze the effect of diet-derived metabolites such as metabolites of flavonoids on endothelial inflammation and cell surface glycosaminoglycans.

Procedures to Evaluate the Role of Heparan Sulfate on the Reactivity of Resistance and Conductance Arteries Ex Vivo.

Evidence is emerging that disruption of the endothelial glycocalyx might contribute importantly to arterial dysfunction in the context of diabetes. One approach to assess the integrity of the endothelium and the vascular smooth muscle cell layer, in the absence of neural, humoral, and mechanical influences, is by measuring arterial vasomotion ex vivo. Here we describe a procedure to assess non-receptor-mediated vasoconstriction, receptor-mediated vasoconstriction, and endothelium-dependent and -independent vasodilation, in resistance and conductance arteries pressurized to 60 mmHg. In addition to evaluating vasoreactivity using isobaric approaches, the same experimental set-up can be used to initiate a pressure gradient across the artery such that intraluminal, flow-mediated vasodilation can be measured. After recording endothelium-dependent vasodilation using isobaric or flow-mediated approaches, identical interventions can be completed in the presence of enzymes that cleave biologically active heparan sulfates into inactive disaccharide and oligosaccharide fragments to assess the contribution from: (a) endothelial-derived substances (e.g., nitric oxide via nitric oxide synthase inhibition); or (b) important components of the glycocalyx (e.g., removal of heparan sulfate via heparitinase III treatment). Here, we show that acute disruption of a predominant glycosaminoglycan i.e., heparan sulfate impairs intraluminal flow-mediated vasodilation in murine resistance arteries.

Preparation of Nanosensors for Detecting the Activity of Glycosaminoglycan Cleaving Enzymes.

Glycosaminoglycans (GAGs) play crucial roles in several biological processes including cell division, angiogenesis, anticoagulation, neurogenesis, axon guidance and growth, and viral and bacterial infections among others. The GAG cleaving hydrolases/lyases play a major role in the control of GAG structures, functions, and turn over. Dysregulation of GAG cleaving enzymes in vivo are linked to a number of human diseases including cancer, diabetes, atherosclerosis, arthritis, inflammation, and cardiovascular diseases. Several GAG cleaving enzymes are widely used for studying GAG glycobiology: heparitinases, chondroitinases, heparanases, hyaluronidases, and keratanases. Herein, we describe a method to synthesize four distinct nanometal surface energy transfer (NSET)-based gold-GAG-dye conjugates (nanosensors). Heparin, chondroitin sulfate, heparan sulfate, and hyaluronic acid are covalently linked with distinct fluorescent dyes and then immobilized on gold nanoparticles (AuNPs) to build nanosensors that serve as excellent substrates for GAG cleaving enzymes. Upon treatment of nanosensors with their respective GAG cleaving enzymes, dye-labeled oligosaccharides/disaccharides are released from AuNPs resulting in enhanced fluorescence recovery. These nanosensors have a great promise as diagnostic tools in various human pathophysiological conditions for detecting dysregulated expression of GAG cleaving enzymes and also as a sensitive analytical tool for assessing the quality control of pharmaceutical grade heparin polysaccharides that are produced in millions of small- and medium-sized animal slaughter houses worldwide.