Lrp1 is a host entry factor for Rift Valley fever virus.

Rift Valley fever virus (RVFV) is a zoonotic pathogen with pandemic potential. RVFV entry is mediated by the viral glycoprotein (Gn), but host entry factors remain poorly defined. Our genome-wide CRISPR screen identified low-density lipoprotein receptor-related protein 1 (mouse Lrp1/human LRP1), heat shock protein (Grp94), and receptor-associated protein (RAP) as critical host factors for RVFV infection. RVFV Gn directly binds to specific Lrp1 clusters and is glycosylation independent. Exogenous addition of murine RAP domain 3 (mRAPD3) and anti-Lrp1 antibodies neutralizes RVFV infection in taxonomically diverse cell lines. Mice treated with mRAPD3 and infected with pathogenic RVFV are protected from disease and death. A mutant mRAPD3 that binds Lrp1 weakly failed to protect from RVFV infection. Together, these data support Lrp1 as a host entry factor for RVFV infection and define a new target to limit RVFV infections.

Golgi apparatus-synthesized sulfated glycosaminoglycans mediate polymerization and activation of the cGAMP sensor STING.

Activation of the cyclic guanosine monophosphate (GMP)-AMP (cGAMP) sensor STING requires its translocation from the endoplasmic reticulum to the Golgi apparatus and subsequent polymerization. Using a genome-wide CRISPR-Cas9 screen to define factors critical for STING activation in cells, we identified proteins critical for biosynthesis of sulfated glycosaminoglycans (sGAGs) in the Golgi apparatus. Binding of sGAGs promoted STING polymerization through luminal, positively charged, polar residues. These residues are evolutionarily conserved, and selective mutation of specific residues inhibited STING activation. Purified or chemically synthesized sGAGs induced STING polymerization and activation of the kinase TBK1. The chain length and O-linked sulfation of sGAGs directly affected the level of STING polymerization and, therefore, its activation. Reducing the expression of Slc35b2 to inhibit GAG sulfation in mice impaired responses to vaccinia virus infection. Thus, sGAGs in the Golgi apparatus are necessary and sufficient to drive STING polymerization, providing a mechanistic understanding of the requirement for endoplasmic reticulum (ER)-to-Golgi apparatus translocation for STING activation.

Molecular mechanisms of proteoglycan-mediated semaphorin signaling in axon guidance.

The precise assembly of a functional nervous system relies on axon guidance cues. Beyond engaging their cognate receptors and initiating signaling cascades that modulate cytoskeletal dynamics, guidance cues also bind components of the extracellular matrix, notably proteoglycans, yet the role and mechanisms of these interactions remain poorly understood. We found that Drosophila secreted semaphorins bind specifically to glycosaminoglycan (GAG) chains of proteoglycans, showing a preference based on the degree of sulfation. Structural analysis of Sema2b unveiled multiple GAG-binding sites positioned outside canonical plexin-binding site, with the highest affinity binding site located at the C-terminal tail, characterized by a lysine-rich helical arrangement that appears to be conserved across secreted semaphorins. In vivo studies revealed a crucial role of the Sema2b C-terminal tail in specifying the trajectory of olfactory receptor neurons. We propose that secreted semaphorins tether to the cell surface through interactions with GAG chains of proteoglycans, facilitating their presentation to cognate receptors on passing axons.

The accomplices: Heparan sulfates and N-glycans foster SARS-CoV-2 spike:ACE2 receptor binding and virus priming.

Although it is well established that the SARS-CoV-2 spike glycoprotein binds to the host cell ACE2 receptor to initiate infection, far less is known about the tissue tropism and host cell susceptibility to the virus. Differential expression across different cell types of heparan sulfate (HS) proteoglycans, with variably sulfated glycosaminoglycans (GAGs), and their synergistic interactions with host and viral N-glycans may contribute to tissue tropism and host cell susceptibility. Nevertheless, their contribution remains unclear since HS and N-glycans evade experimental characterization. We, therefore, carried out microsecond-long all-atom molecular dynamics simulations, followed by random acceleration molecular dynamics simulations, of the fully glycosylated spike:ACE2 complex with and without highly sulfated GAG chains bound. By considering the model GAGs as surrogates for the highly sulfated HS expressed in lung cells, we identified key cell entry mechanisms of spike SARS-CoV-2. We find that HS promotes structural and energetic stabilization of the active conformation of the spike receptor-binding domain (RBD) and reorientation of ACE2 toward the N-terminal domain in the same spike subunit as the RBD. Spike and ACE2 N-glycans exert synergistic effects, promoting better packing, strengthening the protein:protein interaction, and prolonging the residence time of the complex. ACE2 and HS binding trigger rearrangement of the S2' functional protease cleavage site through allosteric interdomain communication. These results thus show that HS has a multifaceted role in facilitating SARS-CoV-2 infection, and they provide a mechanistic basis for the development of GAG derivatives with anti-SARS-CoV-2 potential.

Discovery of a class of glycosaminoglycan lyases with ultrabroad substrate spectrum and their substrate structure preferences.

Glycosaminoglycan (GAG) lyases are often strictly substrate specific, and it is especially difficult to simultaneously degrade GAGs with different types of glycosidic bonds. Herein, we found a new class of GAG lyases (GAGases) from different bacteria. These GAGases belong to polysaccharide lyase 35 family and share quite low homology with the identified GAG lyases. The most surprising thing is that GAGases can not only degrade three types of GAGs: hyaluronan, chondroitin sulfate, and heparan sulfate but also even one of them can also degrade alginate. Further investigation of structural preferences revealed that GAGases selectively act on GAG domains composed of non/6-O-/N-sulfated hexosamines and d-glucoronic acids as well as on alginate domains composed of d-mannuronic acids. In addition, GAG lyases were once speculated to have evolved from alginate lyases, but no transitional enzymes have been found. The discovery of GAGases not only broadens the category of GAG lyases, provides new enzymatic tools for the structural and functional studies of GAGs with specific structures, but also provides candidates for the evolution of GAG lyases.

Glycosaminoglycans modulate long-range mechanical communication between cells in collagen networks.

Cells can sense and respond to mechanical forces in fibrous extracellular matrices (ECMs) over distances much greater than their size. This phenomenon, termed long-range force transmission, is enabled by the realignment (buckling) of collagen fibers along directions where the forces are tensile (compressive). However, whether other key structural components of the ECM, in particular glycosaminoglycans (GAGs), can affect the efficiency of cellular force transmission remains unclear. Here we developed a theoretical model of force transmission in collagen networks with interpenetrating GAGs, capturing the competition between tension-driven collagen fiber alignment and the swelling pressure induced by GAGs. Using this model, we show that the swelling pressure provided by GAGs increases the stiffness of the collagen network by stretching the fibers in an isotropic manner. We found that the GAG-induced swelling pressure can help collagen fibers resist buckling as the cells exert contractile forces. This mechanism impedes the alignment of collagen fibers and decreases long-range cellular mechanical communication. We experimentally validated the theoretical predictions by comparing the intensity of collagen fiber alignment between cellular spheroids cultured on collagen gels versus collagen­GAG cogels. We found significantly lower intensities of aligned collagen in collagen­GAG cogels, consistent with the prediction that GAGs can prevent collagen fiber alignment. The role of GAGs in modulating force transmission uncovered in this work can be extended to understand pathological processes such as the formation of fibrotic scars and cancer metastasis, where cells communicate in the presence of abnormally high concentrations of GAGs.

Impaired instructive and protective barrier functions of the endothelial cell glycocalyx pericellular matrix is impacted in COVID-19 disease.

The aim of this study was to review the roles of endothelial cells in normal tissue function and to show how COVID-19 disease impacts on endothelial cell properties that lead to much of its associated symptomatology. This places the endothelial cell as a prominent cell type to target therapeutically in the treatment of this disorder. Advances in glycosaminoglycan analytical techniques and functional glycomics have improved glycosaminoglycan mimetics development, providing agents that can more appropriately target various aspects of the behaviour of the endothelial cell in-situ and have also provided polymers with potential to prevent viral infection. Thus, promising approaches are being developed to combat COVID-19 disease and the plethora of symptoms this disease produces. Glycosaminoglycan mimetics that improve endothelial glycocalyx boundary functions have promising properties in the prevention of viral infection, improve endothelial cell function and have disease-modifying potential. Endothelial cell integrity, forming tight junctions in cerebral cell populations in the blood-brain barrier, prevents the exposure of the central nervous system to circulating toxins and harmful chemicals, which may contribute to the troublesome brain fogging phenomena reported in cognitive processing in long COVID disease.

Function and contribution of two putative Enterococcus faecalis glycosaminoglycan degrading enzymes to bacteremia and catheter-associated urinary tract infection.

Enterococcus faecalis is a common cause of healthcare-acquired bloodstream infections and catheter-associated urinary tract infections (CAUTIs) in both adults and children. Treatment of E. faecalis infection is frequently complicated by multi-drug resistance. Based on protein homology, E. faecalis encodes two putative hyaluronidases, EF3023 (HylA) and EF0818 (HylB). In other Gram-positive pathogens, hyaluronidases have been shown to contribute to tissue damage and immune evasion, but the function in E. faecalis has yet to be explored. Here, we show that both hylA and hylB contribute to E. faecalis pathogenesis. In a CAUTI model, ΔhylA exhibited defects in bladder colonization and dissemination to the bloodstream, and ΔhylB exhibited a defect in kidney colonization. Furthermore, a ΔhylAΔhylB double mutant exhibited a severe colonization defect in a model of bacteremia while the single mutants colonized to a similar level as the wild-type strain, suggesting potential functional redundancy within the bloodstream. We next examined enzymatic activity, and demonstrate that HylB is capable of digesting both hyaluronic acid (HA) and chondroitin sulfate in vitro, while HylA exhibits only a very modest activity against heparin. Importantly, HA degradation by HylB provided a modest increase in cell density during the stationary phase and also contributed to dampening of lipopolysaccharide-mediated NF-κB activation. Overall, these data demonstrate that glycosaminoglycan degradation is important for E. faecalis pathogenesis in the urinary tract and during bloodstream infection.

Chemo-enzymatic synthesis of tetrasaccharide linker peptides to study the divergent step in glycosaminoglycan biosynthesis.

Glycosaminoglycans are extended linear polysaccharides present on cell surfaces and within the extracellular matrix that play crucial roles in various biological processes. Two prominent glycosaminoglycans, heparan sulfate and chondroitin sulfate, are covalently linked to proteoglycan core proteins through a common tetrasaccharide linker comprising glucuronic acid, galactose, galactose, and xylose moities. This tetrasaccharide linker is meticulously assembled step by step by four Golgi-localized glycosyltransferases. The addition of the fifth sugar moiety, either N-acetylglucosamine or N-acetylgalactosamine, initiates further chain elongation, resulting in the formation of heparan sulfate or chondroitin sulfate, respectively. Despite the fundamental significance of this step in glycosaminoglycan biosynthesis, its regulatory mechanisms have remained elusive. In this study, we detail the expression and purification of the four linker-synthesizing glycosyltransferases and their utilization in the production of fluorescent peptides carrying the native tetrasaccharide linker. We generated five tetrasaccharide peptides, mimicking the core proteins of either heparan sulfate or chondroitin sulfate proteoglycans. These peptides were readily accepted as substrates by the EXTL3 enzyme, which adds an N-acetylglucosamine moiety, thereby initiating heparan sulfate biosynthesis. Importantly, EXTL3 showed a preference towards peptides mimicking the core proteins of heparan sulfate proteoglycans over the ones from chondroitin sulfate proteoglycans. This suggests that EXTL3 could play a role in the decision-making step during glycosaminoglycan biosynthesis. The innovative strategy for chemo-enzymatic synthesis of fluorescent-labeled linker-peptides promises to be instrumental in advancing future investigations into the initial steps and the divergent step of glycosaminoglycan biosynthesis.

The significant role of glycosaminoglycans in tooth development.

This review delves into the roles of glycosaminoglycans (GAGs), integral components of proteoglycans, in tooth development. Proteoglycans consist of a core protein linked to GAG chains, comprised of repeating disaccharide units. GAGs are classified into several types, such as hyaluronic acid, heparan sulfate, chondroitin sulfate, dermatan sulfate, and keratan sulfate. Functioning as critical macromolecular components within the dental basement membrane, these GAGs facilitate cell adhesion and aggregation, and play key roles in regulating cell proliferation and differentiation, thereby significantly influencing tooth morphogenesis. Notably, our recent research has identified the hyaluronan-degrading enzyme Transmembrane protein 2 (Tmem2) and we have conducted functional analyses using mouse models. These studies have unveiled the essential role of Tmem2-mediated hyaluronan degradation and its involvement in hyaluronan-mediated cell adhesion during tooth formation. This review provides a comprehensive summary of the current understanding of GAG functions in tooth development, integrating insights from recent research, and discusses future directions in this field.

Inhibitors of dermatan sulfate epimerase 1 decreased accumulation of glycosaminoglycans in mucopolysaccharidosis type I fibroblasts.

Genetic deficiency of alpha-L-iduronidase causes mucopolysaccharidosis type I (MPS-I) disease, due to accumulation of glycosaminoglycans (GAGs) including chondroitin/dermatan sulfate (CS/DS) and heparan sulfate (HS) in cells. Currently, patients are treated by infusion of recombinant iduronidase or by hematopoietic stem cell transplantation. An alternative approach is to reduce the L-iduronidase substrate, through limiting the biosynthesis of iduronic acid. Our earlier study demonstrated that ebselen attenuated GAGs accumulation in MPS-I cells, through inhibiting iduronic acid producing enzymes. However, ebselen has multiple pharmacological effects, which prevents its application for MPS-I. Thus, we continued the study by looking for novel inhibitors of dermatan sulfate epimerase 1 (DS-epi1), the main responsible enzyme for production of iduronic acid in CS/DS chains. Based on virtual screening of chemicals towards chondroitinase AC, we constructed a library with 1,064 compounds that were tested for DS-epi1 inhibition. Seventeen compounds were identified to be able to inhibit 27%-86% of DS-epi1 activity at 10 µM. Two compounds were selected for further investigation based on the structure properties. The results show that both inhibitors had a comparable level in inhibition of DS-epi1while they had negligible effect on HS epimerase. The two inhibitors were able to reduce iduronic acid biosynthesis in CS/DS and GAG accumulation in WT and MPS-I fibroblasts. Docking of the inhibitors into DS-epi1 structure shows high affinity binding of both compounds to the active site. The collected data indicate that these hit compounds may be further elaborated to a potential lead drug used for attenuation of GAGs accumulation in MPS-I patients.

Unveiling Charge-Pair Salt-Bridge Interaction Between GAGs and Collagen Protein in Cartilage: Atomic Evidence from DNP-Enhanced ssNMR at Natural Isotopic Abundance.

The interactions between glycosaminoglycans (GAGs) and proteins are essential in numerous biochemical processes that involve ion-pair interactions. However, there is no evidence of direct and specific interactions between GAGs and collagen proteins in native cartilage. The resolution of solid-state NMR (ssNMR) can offer such information but the detection of GAG interactions in cartilage is limited by the sensitivity of the experiments when 13C and 15N isotopes are at natural abundance. In this communication, this limitation is overcome by taking advantage of dynamic nuclear polarization (DNP)-enhanced magic-angle spinning (MAS) experiments to obtain two-dimensional (2D) 15N-13C and 13C-13C correlations on native samples at natural abundance. These experiments unveiled inter-residue correlations in the aliphatic regions of the collagen protein previously unobserved. Additionally, our findings provide direct evidence of charge-pair salt-bridge interactions between negatively charged GAGs and positively charged arginine (Arg) residues of collagen protein. We also identified potential hydrogen bonding interactions between hydroxyproline (Hyp) and GAGs, offering atomic insights into the biochemical interactions within the extracellular matrix of native cartilage. Our approach may provide a new avenue for the structural characterization of other native systems.

Multivalent, calcium-independent binding of surfactant protein A and D to sulfated glycosaminoglycans of the alveolar epithelial glycocalyx.

Lung surfactant collectins, surfactant protein A (SP-A) and D (SP-D), are oligomeric C-type lectins involved in lung immunity. Through their carbohydrate recognition domain, they recognize carbohydrates at pathogen surfaces and initiate lung innate immune response. Here, we propose that they may also be able to bind to other carbohydrates present in typical cell surfaces, such as the alveolar epithelial glycocalyx. To test this hypothesis, we analyzed and quantified the binding affinity of SP-A and SP-D to different sugars and glycosaminoglycans (GAGs) by microscale thermophoresis (MST). In addition, by changing the calcium concentration, we aimed to characterize any consequences on the binding behavior. Our results show that both oligomeric proteins bind with high affinity (in nanomolar range) to GAGs, such as hyaluronan (HA), heparan sulfate (HS) and chondroitin sulfate (CS). Binding to HS and CS was calcium-independent, as it was not affected by changing calcium concentration in the buffer. Quantification of GAGs in bronchoalveolar lavage (BAL) fluid from animals deficient in either SP-A or SP-D showed changes in GAG composition, and electron micrographs showed differences in alveolar glycocalyx ultrastructure in vivo. Taken together, SP-A and SP-D bind to model sulfated glycosaminoglycans of the alveolar epithelial glycocalyx in a multivalent and calcium-independent way. These findings provide a potential mechanism for SP-A and SP-D as an integral part of the alveolar epithelial glycocalyx binding and interconnecting free GAGs, proteoglycans, and other glycans in glycoproteins, which may influence glycocalyx composition and structure.NEW & NOTEWORTHY SP-A and SP-D function has been related to innate immunity of the lung based on their binding to sugar residues at pathogen surfaces. However, their function in the healthy alveolus was considered as limited to interaction with surfactant lipids. Here, we demonstrated that these proteins bind to glycosaminoglycans present at typical cell surfaces like the alveolar epithelial glycocalyx. We propose a model where these proteins play an important role in interconnecting alveolar epithelial glycocalyx components.

A close-up view of the Hunter syndrome.

The Lysosomal Storage disease known as Mucopolysaccharidosis type II, is caused by mutations affecting the iduronate-2-sulfatase required for heparan and dermatan sulfate catabolism. The central nervous system (CNS) is mostly and severely affected by the accumulation of both substrates. The complexity of the CNS damage observed in MPS II patients has been limitedly explored. The use of mass spectrometry (MS)-based proteomics tools to identify protein profiles may yield valuable information about the pathological mechanisms of Hunter syndrome. In this further study, we provide a new comparative proteomic analysis of MPS II models by using a pipeline consisting of the identification of native protein complexes positioned selectively by using a specific antibody, coupled with mass spectrometry analysis, allowing us to identify changes involving in a significant number of new biological functions, including a specific brain antioxidant response, a down-regulated autophagic, the suppression of sulfur catabolic process, a prominent liver immune response and the stimulation of phagocytosis among others.

Comprehensive evaluation of pathogenic protein accumulation in fibroblasts from all subtypes of Sanfilippo disease patients.

Sanfilippo disease is a lysosomal storage disorder from the group of mucopolysaccharidoses (MPS), characterized by storage of glycosaminoglycans (GAGs); thus, it is also called MPS type III. The syndrome is divided into 4 subtypes (MPS III A, B, C and D). Despite the storage of the same GAG, heparan sulfate (HS), the course of these subtypes can vary considerably. Here, we comprehensively evaluated the levels of protein aggregates (APP, ß-amyloid, p-tau, α-synuclein, TDP43) in fibroblasts derived from patients with all MPS III subtypes, and tested whether lowering GAG levels results in a decrease in the levels of the investigated proteins and the number of aggregates they form. Elevated levels of APP, ß-amyloid, tau, and TDP43 proteins were evident in all MPS III subtypes, and elevated levels of p-tau and α-synuclein were demonstrated in all subtypes except MPS IIIC. These findings were confirmed in the neural tissue of MPS IIIB mice. Fluorescence microscopy studies also indicated a high number of protein aggregates formed by ß-amyloid and tau in all cell lines tested, and a high number of aggregates of p-tau, TDP43, and α-synuclein in all lines except MPS IIIC. Reduction of GAG levels by genistein led to the decrease of levels of all tested proteins and their aggregates except α-synuclein, indicating a relationship between GAG levels and those of some protein aggregates. This work describes for the first time the problem of deposited protein aggregates in all subtypes of Sanfilippo disease and suggests that GAGs are partly responsible for the formation of protein aggregates.

Community consensus for Heparan sulfate as a biomarker to support accelerated approval in Neuronopathic Mucopolysaccharidoses.

Mucopolysaccharidoses (MPS) disorders are a group of ultra-rare, inherited, lysosomal storage diseases caused by enzyme deficiencies that result in accumulation of glycosaminoglycans (GAGs) in cells throughout the body including the brain, typically leading to early death. Current treatments do not address the progressive cognitive impairment observed in patients with neuronopathic MPS disease. The rarity and clinical heterogeneity of these disorders as well as pre-existing brain disease in clinically diagnosed patients make the development of new therapeutics utilizing a traditional regulatory framework extremely challenging. Children with neuronopathic MPS disorders will likely sustain irreversible brain damage if randomized to a placebo or standard-of-care treatment arm that does not address brain disease. The United States Food and Drug Administration (FDA) recognized these challenges, and, in 2020, issued final guidance for industry on slowly progressive, low-prevalence, rare diseases with substrate deposition that result from single enzyme defects, outlining a path for generating evidence of effectiveness to support accelerated approval based on reduction of substrate accumulation [1]. Neuronopathic MPS disorders, which are characterized by the accumulation of the GAG heparan sulfate (HS) in the brain, fit the intended disease characteristics for which this guidance was written, but to date, this guidance has not yet been applied to any therapeutic candidate for MPS. In February 2024, the Reagan-Udall Foundation for the FDA convened a public workshop for representatives from the FDA, patient advocacy groups, clinical and basic science research, and industry to explore a case study of using cerebrospinal fluid (CSF) HS as a relevant biomarker to support accelerated approval of new therapeutics for neuronopathic MPS disorders. This review provides a summary of the MPS presentations at the workshop and perspective on the path forward for neuronopathic MPS disorders.

Causes of death in mucopolysaccharidoses.

Mucopolysaccharidoses are inherited metabolic diseases caused by mutations in genes encoding enzymes required for degradation of glycosaminoglycans. A lack or severe impairment of activity of these enzymes cause accumulation of GAGs which is the primary biochemical defect. Depending on the kind of the deficient enzyme, there are 12 types and subtypes of MPS distinguished. Despite the common primary metabolic deficit (inefficient GAG degradation), the course and symptoms of various MPS types can be different, though majority of the diseases from the group are characterized by severe symptoms and significantly shortened live span. Here, we analysed the frequency of specific, direct causes of death of patients with different MPS types, the subject which was not investigated comprehensively to date. We examined a total of 1317 cases of death among MPS patients, including 393 cases of MPS I, 418 cases of MPS II, 232 cases of MPS III, 45 cases of MPS IV, 208 cases of MPS VI, and 22 cases of MPS VII. Our analyses indicated that the most frequent causes of death differ significantly between MPS types, with cardiovascular and respiratory failures being predominant in MPS I, MPS II, and MPS VI, neurological deficits in MPS III, respiratory issues in MPS IV, and hydrops fetalis in MPS VII. Results of such studies suggest what specific clinical problems should be considered with the highest priority in specific MPS types, apart from attempts to correct the primary causes of the diseases, to improve the quality of life of patients and to prolong their lives.

Shedding light on the effects of blood on meniscus tissue: the role of mononuclear leukocytes in mediating meniscus catabolism.

OBJECTIVE: Traumatic meniscal injuries can cause acute pain, hemarthrosis (bleeding into the joint), joint immobility, and post-traumatic osteoarthritis (PTOA). However, the exact mechanism(s) by which PTOA develops following meniscal injuries is unknown. Since meniscus tears commonly coincide with hemarthrosis, investigating the direct effects of blood and its constituents on meniscus tissue is warranted. The goal of this study was to determine the direct effects of blood and blood components on meniscus tissue catabolism. METHODS: Porcine meniscus explants or primary meniscus cells were exposed to whole blood or various fractions of blood for 3 days to simulate blood exposure following injury. Explants were then washed and cultured for an additional 3 days prior to collection for biochemical analyses. RESULTS: Whole blood increased matrix metalloproteinase (MMP) activity. Fractionation experiments revealed blood-derived red blood cells did not affect meniscus catabolism. Conversely, viable mononuclear leukocytes induced MMP activity, nitric oxide (NO) production, and loss of tissue sulfated glycosaminoglycan (sGAG) content, suggesting that these cells are mediating meniscus catabolism. CONCLUSIONS: These findings highlight the potential challenges of meniscus healing in the presence of hemarthrosis and the need for further research to elucidate the in vivo effects of blood and blood-derived mononuclear leukocytes due to both hemarthrosis and blood-derived therapeutics.

Mild exercise expedites joint clearance and slows joint degradation in a joint instability model of osteoarthritis in male rats.

OBJECTIVE: Exercise remains a hallmark treatment for post-traumatic osteoarthritis (PTOA) and may maintain joint homeostasis in part by clearing inflammatory cytokines, cells, and particles. It remains largely unknown whether exercise-induced joint clearance can provide therapeutic relief of PTOA. In this study, we hypothesized that exercise could slow the progression of preclinical PTOA in part by enhancing knee joint clearance. DESIGN: Surgical medial meniscal transection was used to induce PTOA in 3-month-old male Lewis rats. A sham surgery was used as a control. Mild treadmill walking was introduced 3 weeks post-surgery and maintained to 6 weeks post-surgery. Gait and isometric muscle torque were measured at the study endpoint. Near-infrared imaging tracked how exercise altered lymphatic and venous knee joint clearance during discrete time points of PTOA progression. RESULTS: Exercise mitigated joint degradation associated with PTOA by preserving glycosaminoglycan content and reducing osteophyte volume (effect size (95% Confidence Interval (CI)); 1.74 (0.71-2.26)). PTOA increased hind step widths (0.57 (0.18-0.95) cm), but exercise corrected this gait dysfunction (0.54 (0.16-0.93) cm), potentially indicating pain relief. Venous, but not lymphatic, clearance was quicker 1-, 3-, and 6-weeks post-surgery compared to baseline. The mild treadmill walking protocol expedited lymphatic clearance rate in moderate PTOA (3.39 (0.20-6.59) hrs), suggesting exercise may play a critical role in restoring joint homeostasis. CONCLUSIONS: We conclude that mild exercise has the potential to slow disease progression in part by expediting joint clearance in moderate PTOA.

Glycosaminoglycan-induced proinflammatory cytokine levels as disease marker in mucopolysaccharidosis.

Recently, it has been shown disturbances in oxidant/antioxidant system and increases in some inflammatory markers in animal studies and in some Mucopolysaccharidoses (MPSs) patients. In this study, we aimed to determine the oxidative stress/antioxidant parameters and pro-inflammatory cytokine levels in the serum of MPS patients, in order to evaluate the possible role of inflammation in these patient groups regarding to accumulated metabolites. MPS I (n = 3), MPS II (n = 8), MPS III (n = 4), MPS IVA (n = 3), MPS VI (n = 3), and VII (n = 1) patients and 20 age-matched healthy subjects were included into the study. There was no statistically significant change in activities of SOD, Catalase, GSH-Px and lipid peroxidation levels in erythrocytes between the MPS patients and healthy controls. While IL-1alpha (p = 0.054), IL-6 (p = 0.008) levels, and chitotriosidase activity (p = 0.003) elevated in MPS3 patients, IL1α (p = 0.006), IL-1ß (p = 0.006), IL-6 (p = 0.006), IFNγ (p = 0.006), and NFκB (p = 0.006) levels increased in MPS-6 patients. Elevated levels of IL-6, IL1α and chitotriosidase activity demonstrated macrophage activation in MPSIII untreated with enzyme replacement. Our study showed for the first time that high levels of IL1α, IL-6, IL1ß and NFκB were present in MPSVI patients, demonstrating the induction of inflammation by dermatan sulphate. The low level of paraoxonase in MPSVI patients may be a good marker for cardiac involvement. Overall, this study provides important insights into the relationship between lysosomal storage of glycosaminoglycan and inflammation in MPS patients. It highlights possible pathways for the increased release of inflammatory molecules and suggests new targets for the development of treatments.