Recombinant human proteoglycan 4 lowers inflammation and atherosclerosis susceptibility in female low-density lipoprotein receptor knockout mice.

Recombinant human proteoglycan 4 (rhPRG4) is a macromolecular mucin-like glycoprotein that is classically studied as a lubricant within eyes and joints. Given that endogenously produced PRG4 is present within atherosclerotic lesions and genetic PRG4 deficiency increases atherosclerosis susceptibility in mice, in the current study we investigated the anti-atherogenic potential of chronic rhPRG4 treatment. Female low-density lipoprotein receptor knockout mice were fed an atherogenic Western-type diet for 6 weeks and injected three times per week intraperitoneally with 0.5 mg rhPRG4 or PBS as control. Treatment with rhPRG4 was associated with a small decrease in plasma-free cholesterol levels, without a change in cholesteryl ester levels. A marked increase in the number of peritoneal foam cells was detected in response to the peritoneal rhPRG4 administration, which could be attributed to elevated peritoneal leukocyte MSR1 expression levels. However, rhPRG4-treated mice exhibited significantly smaller aortic root lesions of 278 ± 21 × 103 µm2 compared with 339 ± 15 × 103 µm2 in the aortic root of control mice. The overall decreased atherosclerosis susceptibility coincided with a shift in the monocyte and macrophage polarization states towards the patrolling and anti-inflammatory M2-like phenotypes, respectively. Furthermore, rhPRG4 treatment significantly reduced macrophage gene expression levels as well as plasma protein levels of the pro-inflammatory/pro-atherogenic cytokine TNF-alpha. In conclusion, we have shown that peritoneal administration and subsequent systemic exposure to rhPRG4 beneficially impacts the inflammatory state and reduces atherosclerosis susceptibility in mice. Our findings highlight that PRG4 is not only a lubricant but also acts as an anti-inflammatory agent. KEY POINTS: Endogenously produced proteoglycan 4 is found in atherosclerotic lesions and its genetic deficiency in mice is associated with enhanced atherosclerosis susceptibility. In this study we investigated the anti-atherogenic potential of chronic treatment with recombinant human PRG4 in hypercholesterolaemic female low-density lipoprotein receptor knockout mice. We show that recombinant human PRG4 stimulates macrophage foam cell formation, but also dampens the pro-inflammatory state of monocyte/macrophages, eventually leading to a significant reduction in plasma TNF-alpha levels and a lowered atherosclerosis susceptibility. Our findings highlight that peritoneal recombinant human PRG4 treatment can execute effects both locally and systemically and suggest that it will be of interest to study whether rhPRG4 treatment is also able to inhibit the progression and/or induce regression of previously established atherosclerotic lesions.

Scale-Dependent Rheology of Synovial Fluid Lubricating Macromolecules.

Synovial fluid (SF) is the complex biofluid that facilitates the exceptional lubrication of articular cartilage in joints. Its primary lubricating macromolecules, the linear polysaccharide hyaluronic acid (HA) and the mucin-like glycoprotein proteoglycan 4 (PRG4 or lubricin), interact synergistically to reduce boundary friction. However, the precise manner in which these molecules influence the rheological properties of SF remains unclear. This study aimed to elucidate this by employing confocal microscopy and multiscale rheometry to examine the microstructure and rheology of solutions containing recombinant human PRG4 (rhPRG4) and HA. Contrary to previous assumptions of an extensive HA-rhPRG4 network, it is discovered that rhPRG4 primarily forms stiff, gel-like aggregates. The properties of these aggregates, including their size and stiffness, are found to be influenced by the viscoelastic characteristics of the surrounding HA matrix. Consequently, the rheology of this system is not governed by a single length scale, but instead responds as a disordered, hierarchical network with solid-like rhPRG4 aggregates distributed throughout the continuous HA phase. These findings provide new insights into the biomechanical function of PRG4 in cartilage lubrication and may have implications in the development of HA-based therapies for joint diseases like osteoarthritis.

A structural and functional comparison between two recombinant human lubricin proteins: Recombinant human proteoglycan-4 (rhPRG4) vs ECF843.

Proteoglycan 4 (PRG4, lubricin) is a mucin-like glycoprotein present on the ocular surface that has both boundary lubricating and anti-inflammatory properties. Full-length recombinant human PRG4 (rhPRG4) has been shown to be clinically effective in improving signs and symptoms of dry eye disease (DED). In vitro, rhPRG4 has been shown to reduce inflammation-induced cytokine production and NFκB activity in corneal epithelial cells, as well as to bind to and inhibit MMP-9 activity. A different form of recombinant human lubricin (ECF843), produced from the same cell line as rhPRG4 but manufactured using a different process, was recently assessed in a DED clinical trial. However, ECF843 did not significantly improve signs or symptoms of DED compared to vehicle. Initial published characterization of ECF843 showed it had a smaller hydrodynamic diameter and was less negatively charged than native PRG4. Further examination of the structural and functional properties of ECF843 and rhPRG4 could contribute to the understanding of what led to their disparate clinical efficacy. Therefore, the objective of this study was to characterize and compare rhPRG4 and ECF843 in vitro, both biophysically and functionally. Hydrodynamic diameter and charge were measured by dynamic light scattering (DLS) and zeta potential, respectively. Size and molecular weight was determined for individual species by size exclusion chromatography (SEC) with in-line DLS and multi-angle light scattering (MALS). Bond structure was measured by Raman spectroscopy, and sedimentation properties were measured by analytical ultracentrifugation (AUC). Functionally, MMP-9 inhibition was measured using a commercial MMP-9 activity kit, coefficient of friction was measured using an established boundary lubrication test at a latex-glass interface, and collagen 1-binding ability was measured by quart crystal microbalance with dissipation (QCMD). Additionally, the ability of rhPRG4 and ECF843 to inhibit urate acid crystal formation and cell adhesion was assessed. ECF843 had a significantly smaller hydrodynamic diameter and was less negatively charged than rhPRG4, as assessed by DLS and zeta potential. Size was further explored with SEC-DLS-MALS, which indicated that while rhPRG4 had 3 main peaks, corresponding to monomer, dimer, and multimer as expected, ECF843 had 2 peaks that were similar in size and molecular weight compared to rhPRG4's monomer peak and a third peak that was significantly smaller in both size and molar mass than the corresponding peak of rhPRG4. Raman spectroscopy demonstrated that ECF843 had significantly more disulfide bonds, which are functionally determinant structures, relative to the carbon-carbon backbone compared to rhPRG4, and AUC indicated that ECF843 was more compact than rhPRG4. Functionally, ECF843 was significantly less effective at inhibiting MMP-9 activity and functioning as a boundary lubricant compared to rhPRG4, as well as being slower to bind to collagen 1. Additionally, ECF843 was significantly less effective at inhibiting urate acid crystal formation and at preventing cell adhesion. Collectively, these data demonstrate ECF843 and rhPRG4 are significantly different in both structure and function. Given that a protein's structure sets the foundation for its interactions with other molecules and tissues in vivo, which ultimately determine its function, these differences most likely contributed to the disparate DED clinical trial results.

Evaluation of microMend wound closure device in repairing skin lacerations.

BACKGROUND: microMend, a novel microstaple skin closure device, may be able to close simple lacerations. This study aimed to evaluate the feasibility and acceptability of using microMend to close these wounds in the ED. METHODS: This was an open-label, single-arm clinical study conducted at two EDs within a large urban academic medical centre. Wounds closed with microMend underwent assessments performed at days 0, 7, 30 and 90. Photographs of treated wounds were rated by two plastic surgeons using a 100 mm visual analogue scale (VAS) and a wound evaluation scale (WES), which has a best possible score of 6. Participants rated pain during application and both participants and providers rated their satisfaction with the device. RESULTS: Thirty-one participants were enrolled in the study: 48% were female and the mean age of participants was 45.6 (95% CI 39.1 to 52.1). The mean wound length was 2.35 cm (95% CI 1.77 to 2.92), with a range of 1-10 cm. Mean VAS and WES scores at day 90 as evaluated by two plastic surgeons were 84.1 mm (95% CI 80.2 to 87.9) and 4.91 (95% CI 4.54 to 5.29), respectively. The mean pain score with application of the devices was 7.28 mm (95% CI 2.88 to 11.68) on a scale of 0-100 mm using VAS. Local anaesthesia was used in 9 patients (29%, 95% CI 20.7 to 37.3) of participants (of whom 5 required deep sutures). Ninety per cent (90%) of participants rated their overall assessment of the device as excellent (74%) or good (16%) at day 90. There were no serious adverse events in any participants in the study. CONCLUSION: microMend appears to be an acceptable alternative for closing skin lacerations in the ED, providing good cosmetic results, with high levels of satisfaction by patients and providers. Randomised trials are needed to compare microMend with other wound closure products. TRIAL REGISTRATION NUMBER: NCT03830515.

Recombinant Human Proteoglycan 4 (rhPRG4) Downregulates TNFα-Stimulated NFκB Activity and FAT10 Expression in Human Corneal Epithelial Cells.

Dry Eye Disease (DED) is a complex pathology affecting millions of people with significant impact on quality of life. Corneal inflammation, including via the nuclear factor kappa B (NFκB) pathway, plays a key etiological role in DED. Recombinant human proteoglycan 4 (rhPRG4) has been shown to be a clinically effective treatment for DED that has anti-inflammatory effects in corneal epithelial cells, but the underlying mechanism is still not understood. Our goal was to understand if rhPRG4 affects tumor necrosis factor α (TNFα)-stimulated inflammatory activity in corneal epithelial cells. We treated hTERT-immortalized corneal epithelial (hTCEpi) cells ± TNFα ± rhPRG4 and performed Western blotting on cell lysate and RNA sequencing. Bioinformatics analysis revealed that rhPRG4 had a significant effect on TNFα-mediated inflammation with potential effects on matricellular homeostasis. rhPRG4 reduced activation of key inflammatory pathways and decreased expression of transcripts for key inflammatory cytokines, interferons, interleukins, and transcription factors. TNFα treatment significantly increased phosphorylation and nuclear translocation of p65, and rhPRG4 significantly reduced both these effects. RNA sequencing identified human leukocyte antigen (HLA)-F adjacent transcript 10 (FAT10), a ubiquitin-like modifier protein which has not been studied in the context of DED, as a key pro-inflammatory transcript increased by TNFα and decreased by rhPRG4. These results were confirmed at the protein level. In summary, rhPRG4 is able to downregulate NFκB activity in hTCEpi cells, suggesting a potential biological mechanism by which it may act as a therapeutic for DED.

Quadruped Gait and Regulation of Apoptotic Factors in Tibiofemoral Joints following Intra-Articular rhPRG4 Injection in Prg4 Null Mice.

Camptodactyly-arthropathy-coxa vara-pericarditis (CACP) syndrome leads to diarthrodial joint arthropathy and is caused by the absence of lubricin (proteoglycan 4-PRG4), a surface-active mucinous glycoprotein responsible for lubricating articular cartilage. In this study, mice lacking the orthologous gene Prg4 served as a model that recapitulates the destructive arthrosis that involves biofouling of cartilage by serum proteins in lieu of Prg4. This study hypothesized that Prg4-deficient mice would demonstrate a quadruped gait change and decreased markers of mitochondrial dyscrasia, following intra-articular injection of both hindlimbs with recombinant human PRG4 (rhPRG4). Prg4-/- (N = 44) mice of both sexes were injected with rhPRG4 and gait alterations were studied at post-injection day 3 and 6, before joints were harvested for immunohistochemistry for caspase-3 activation. Increased stance and propulsion was shown at 3 days post-injection in male mice. There were significantly fewer caspase-3-positive chondrocytes in tibiofemoral cartilage from rhPRG4-injected mice. The mitochondrial gene Mt-tn, and myosin heavy (Myh7) and light chains (Myl2 and Myl3), known to play a cytoskeletal stabilizing role, were significantly upregulated in both sexes (RNA-Seq) following IA rhPRG4. Chondrocyte mitochondrial dyscrasias attributable to the arthrosis in CACP may be mitigated by IA rhPRG4. In a supporting in vitro crystal microbalance experiment, molecular fouling by albumin did not block the surface activity of rhPRG4.

Decrease of core 2 O-glycans on synovial lubricin in osteoarthritis reduces galectin-3 mediated crosslinking.

The synovial fluid glycoprotein lubricin (also known as proteoglycan 4) is a mucin-type O-linked glycosylated biological lubricant implicated to be involved in osteoarthritis (OA) development. Lubricin's ability to reduce friction is related to its glycosylation consisting of sialylated and unsialylated Tn-antigens and core 1 and core 2 structures. The glycans on lubricin have also been suggested to be involved in crosslinking and stabilization of the lubricating superficial layer of cartilage by mediating interaction between lubricin and galectin-3. However, with the spectrum of glycans being found on lubricin, the glycan candidates involved in this interaction were unknown. Here, we confirm that the core 2 O-linked glycans mediate this lubricin-galectin-3 interaction, shown by surface plasmon resonance data indicating that recombinant lubricin (rhPRG4) devoid of core 2 structures did not bind to recombinant galectin-3. Conversely, transfection of Chinese hamster ovary cells with the core 2 GlcNAc transferase acting on a mucin-type O-glycoprotein displayed increased galectin-3 binding. Both the level of galectin-3 and the galectin-3 interactions with synovial lubricin were found to be decreased in late-stage OA patients, coinciding with an increase in unsialylated core 1 O-glycans (T-antigens) and Tn-antigens. These data suggest a defect in crosslinking of surface-active molecules in OA and provide novel insights into OA molecular pathology.

Proteoglycan 4 (PRG4) expression and function in dry eye associated inflammation.

Dry eye disease (DED) affects hundreds of millions of people worldwide. It is characterized by the production of inflammatory cytokines and chemokines as well as damaging matrix metalloproteinases (MMPs) at the ocular surface. While proteoglycan 4 (PRG4), a mucin-like glycoprotein present at the ocular surface, is most well known as a boundary lubricant that contributes to ocular surface integrity, it has been shown to blunt inflammation in various cell types, suggesting a dual mechanism of action. Recently, full-length recombinant human PRG4 (rhPRG4) has been shown to improve signs and symptoms of DED in humans. However, there remains a significant need for basic science research on rhPRG4's biological properties and its potential therapeutic mechanisms of action in treating DED. Therefore, the objectives of this study were to characterize endogenous PRG4 expression by telomerase-immortalized human corneal epithelial (hTCEpi) cells, examine whether exogenous rhPRG4 modulates cytokine and chemokine secretion in response to dry eye associated inflammation (TNFα and IL-1ß), explore interactions between rhPRG4 and MMP-9, and understand how experimental dry eye (EDE) in mice affects PRG4 expression. PRG4 secretion from hTCEpi cells was quantified by Western blot and expression visualized by immunocytochemistry. Cytokine/chemokine production was measured by ELISA and Luminex, while rhPRG4's effect on MMP-9 activity, binding, and expression was quantified using an MMP-9 inhibitor kit, surface plasmon resonance, and reverse transcription polymerase chain reaction (RT-PCR), respectively. Finally, EDE was induced in mice, and PRG4 was visualized by immunohistochemistry in the cornea and by Western blot in lacrimal gland lysate. In vitro results demonstrate that hTCEpi cells synthesize and secrete PRG4, and PRG4 secretion is inhibited by TNFα and IL-1ß. In response to these pro-inflammatory stresses, exogenous rhPRG4 significantly reduced the stimulated production of IP-10, RANTES, ENA-78, GROα, MIP-3α, and MIG, and trended towards a reduction of MIP-1α and MIP-1ß. The hTCEpi cells were also able to internalize fluorescently-labelled rhPRG4, consistent with a mechanism of action that includes downstream biological signaling pathways. rhPRG4 was not digested by MMP-9, and it did not modulate MMP-9 gene expression in hTCEpi cells, but it was able to bind to MMP-9 and inhibited in vitro activity of exogenous MMP-9 in the presence of human tears. Finally, in vivo results demonstrate that EDE significantly decreased immunolocalization of PRG4 on the corneal epithelium and trended towards a reduction of PRG4 in lacrimal gland lysate. Collectively these results demonstrate rhPRG4 has anti-inflammatory properties on corneal epithelial cells, particularly as it relates to mitigating chemokine production, and is an inhibitor of MMP-9 activity, as well as that in vivo expression of PRG4 can be altered in preclinical models of DED. In conclusion, these findings contribute to our understanding of PRG4's immunomodulatory properties in the context of DED inflammation and provide the foundation and motivation for further mechanistic research of PRG4's properties on the ocular surface as well as expanding clinical evaluation of its ability as a multifunctional therapeutic agent to effectively provide relief to those who suffer from DED.

Proteoglycan-4 and hyaluronan composition in synovial fluid and serum from clinical equine subjects: relationship to cartilage boundary lubrication and viscosity of synovial fluid.

Purpose: In experimental models of equine joint-injury and osteoarthritis synovial fluid (SF) composition (proteoglycan-4, hyaluronan) can vary, along with changes to SF mechanical function (lubrication, viscosity). The study hypotheses were a) clinical equine joint-injury and disease results in altered SF composition and diminished mechanical function, and b) serum composition (proteoglycan-4 or hyaluronan) changes concurrently. The objectives were to characterize composition (proteoglycan-4, hyaluronan), and function of SF and serum from normal horses compared to clinical groups: osteoarthritis, acute-joint-injury, and osteochondrosis.Materials and Methods: Equine samples of SF (from various joints) and blood were collected at the point-of-care. Proteoglycan-4 concentrations were measured by amplified-luminescence-proximity-assay and enzyme-linked-immunosorbent-assay in SF and serum, respectively. Molecular-weight of hyaluronan was characterized by agarose-gel-electrophoresis, and concentrations were measured by enzyme-linked-immunosorbent-assay kit. Biomechanical function of SF was characterized by an in vitro cartilage-on-cartilage friction test, and viscosity test.Results: SF proteoglycan-4 concentration increased in acute-joint-injury (1185 ± 276 versus normal 205 ± 106 µg/mL, µ± SEM, p < 0.01), with increased percentage of lower molecular-weight hyaluronan in acute-joint-injury and osteochondrosis. SF and serum proteoglycan-4 concentrations were correlated in normal horses (r2 = 0.85, p < 0.05), but not in clinical groups. Cartilage-lubricating ability was unchanged, although steady-shear viscosity of acute-joint-injury SF decreased from normal.Conclusion: Composition of SF from cases of equine acute-joint-injury changed; both proteoglycan-4 concentration and hyaluronan molecular-weight were altered, with decreased SF viscosity, but no associated changes to serum. Serum proteoglycan-4 and hyaluronan concentrations alone may not be useful biomarkers for equine joint-injury or disease.

Role of CD44 in Regulating TLR2 Activation of Human Macrophages and Downstream Expression of Proinflammatory Cytokines.

Osteoarthritis (OA) is a low-grade chronic inflammatory joint disease. Innate immunity contributes to OA progression, mediated by TLR2 and TLR4. We evaluated the role of cluster determinant 44 (CD44), a transmembrane glycoprotein, in regulating TLR2-linked macrophage activation and resultant proinflammatory responses. TLR2 stimulation was performed on differentiated THP-1 macrophages in the presence or absence of a CD44-specific Ab or hyaluronan (HA). NF-κB nuclear translocation, IL-1 ß and TNF-α gene expression, and protein concentrations were determined. Anti-CD44 Ab and HA treatments reduced NF-κB translocation, IL-1ß and TNF-α expression, and production (p < 0.001). Inhibition of proinflammatory response in macrophages by HA was mediated by CD44. Protein phosphatase 2A mediated the reduction in NF-κB translocation by HA. CD44 knockdown reduced NF-κB nuclear translocation and downstream IL-1ß and TNF-α protein production following TLR2 receptor stimulation (p < 0.001). CD44+/+ murine bone marrow-derived macrophages produced higher TNF-α compared with CD44-/- macrophages following TLR2 stimulation (p < 0.01). HA dose-dependently inhibited TLR2-induced TNF-α production by murine bone marrow-derived macrophages (p < 0.001). OA synovial fluids (SF) stimulated TLR2 and TLR4 receptors and induced NF-κB translocation in THP-1 macrophages. Anti-CD44 Ab treatment significantly reduced macrophage activation by OA SF (p < 0.01). CD44 regulated TLR2 responses in human macrophages, whereby a reduction in CD44 levels or engagement of CD44 by its ligand (HA) or a CD44-specific Ab reduced NF-κB translocation and downstream proinflammatory cytokine production. A CD44-specific Ab reduced macrophage activation by OA SF, and CD44 is a potentially novel target in OA treatment.

Probing the Molecular Interactions and Lubrication Mechanisms of Purified Full-Length Recombinant Human Proteoglycan 4 (rhPRG4) and Hyaluronic Acid (HA).

Probing the adsorption and lubrication behavior of lubricin, also known as proteoglycan 4 (PRG4), is important for understanding the ultralow friction of cartilage lubrication. Most previous research has focused on native lubricin either purified from synovial fluid or articular cartilage explant culture media. In this work, the adsorption behavior and lubrication mechanism of full-length recombinant human PRG4 (rhPRG4) on mica as well as the effect of adding hyaluronic acid (HA, a polysaccharide) were systematically investigated using a surface forces apparatus (SFA) technique. A low friction coefficient (µ ∼ 0.04) was measured when multilayer rhPRG4 (∼31 nm) was confined in between mica surfaces, even when the load increased to ∼1.2 MPa. Intriguingly, a previously unreported ultralow friction coefficient (µ < 0.005) was observed at a low sliding velocity ( v = 0.14 µm/s) with the applied load P reaching ∼3.6 MPa when a diluted rhPRG4 solution (∼90 µg/mL) was used. The distinct friction behavior is likely due to the smooth and more close-packed lubricin coating, as made evident by the atomic force microscope imaging. Adding HA onto multilayer rhPRG4-coated mica increased the friction coefficient µ to ∼0.1; however, the load bearing property increased, indicating potential synergistic effect between rhPRG4 and HA, which was further demonstrated by the weak adhesion observed when separating rhPRG4-coated mica and HA-coated aminopropyltriethoxysilane-mica (APTES-mica). Alternatively, adding premixed rhPRG4-HA on mica had a friction coefficient (µ ∼ 0.1) close to that of injecting concentrated rhPRG4 (∼450 µg/mL) with lower load sustainability. Our results provide fundamental insights into the adsorption and lubrication behavior of lubricin and its interaction with HA, with useful implications for the underlying mechanism of ultralow friction provided by synovial fluid.

cAMP attenuates TGF-ß's profibrotic responses in osteoarthritic synoviocytes: involvement of hyaluronan and PRG4.

Osteoarthritis (OA) is characterized by synovitis and synovial fibrosis. Synoviocytes are fibroblast-like resident cells of the synovium that are activated by transforming growth factor (TGF)-ß to proliferate, migrate, and produce extracellular matrix. Synoviocytes secrete hyaluronan (HA) and proteoglycan-4 (PRG4). HA reduces synovial fibrosis in vivo, and the Prg4-/- mouse exhibits synovial hyperplasia. We investigated the antifibrotic effects of increased intracellular cAMP in TGF-ß-stimulated human OA synoviocytes. TGF-ß1 stimulated collagen I (COL1A1), α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase (TIMP)-1, and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) expression, and procollagen I, α-SMA, HA, and PRG4 production, migration, and proliferation of OA synoviocytes were measured. Treatment of OA synoviocytes with forskolin (10 µM) increased intracellular cAMP levels and reduced TGF-ß1-stimulated COL1A1, α-SMA, and TIMP-1 expression, with no change in PLOD2 expression. Forskolin also reduced TGF-ß1-stimulated procollagen I and α-SMA content as well as synoviocyte migration and proliferation. Forskolin (10 µM) increased HA secretion and PRG4 expression and production. A cell-permeant cAMP analog reduced COL1A1 and α-SMA expression and enhanced HA and PRG4 secretion by OA synoviocytes. HA and PRG4 reduced α-SMA expression and content, and PRG4 reduced COL1A1 expression and procollagen I content in OA synoviocytes. Prg4-/- synovium exhibited increased α-SMA, COL1A1, and TIMP-1 expression compared with Prg4+/+ synovium. Prg4-/- synoviocytes demonstrated strong α-SMA and collagen type I staining, whereas these were undetected in Prg4+/+ synoviocytes and were reduced with PRG4 treatment. We conclude that increasing intracellular cAMP levels in synoviocytes mitigates synovial fibrosis through enhanced production of HA and PRG4, possibly representing a novel approach for treatment of OA synovial fibrosis.

Ultrasonography versus computed tomography for suspected nephrolithiasis.

BACKGROUND: There is a lack of consensus about whether the initial imaging method for patients with suspected nephrolithiasis should be computed tomography (CT) or ultrasonography. METHODS: In this multicenter, pragmatic, comparative effectiveness trial, we randomly assigned patients 18 to 76 years of age who presented to the emergency department with suspected nephrolithiasis to undergo initial diagnostic ultrasonography performed by an emergency physician (point-of-care ultrasonography), ultrasonography performed by a radiologist (radiology ultrasonography), or abdominal CT. Subsequent management, including additional imaging, was at the discretion of the physician. We compared the three groups with respect to the 30-day incidence of high-risk diagnoses with complications that could be related to missed or delayed diagnosis and the 6-month cumulative radiation exposure. Secondary outcomes were serious adverse events, related serious adverse events (deemed attributable to study participation), pain (assessed on an 11-point visual-analogue scale, with higher scores indicating more severe pain), return emergency department visits, hospitalizations, and diagnostic accuracy. RESULTS: A total of 2759 patients underwent randomization: 908 to point-of-care ultrasonography, 893 to radiology ultrasonography, and 958 to CT. The incidence of high-risk diagnoses with complications in the first 30 days was low (0.4%) and did not vary according to imaging method. The mean 6-month cumulative radiation exposure was significantly lower in the ultrasonography groups than in the CT group (P<0.001). Serious adverse events occurred in 12.4% of the patients assigned to point-of-care ultrasonography, 10.8% of those assigned to radiology ultrasonography, and 11.2% of those assigned to CT (P=0.50). Related adverse events were infrequent (incidence, 0.4%) and similar across groups. By 7 days, the average pain score was 2.0 in each group (P=0.84). Return emergency department visits, hospitalizations, and diagnostic accuracy did not differ significantly among the groups. CONCLUSIONS: Initial ultrasonography was associated with lower cumulative radiation exposure than initial CT, without significant differences in high-risk diagnoses with complications, serious adverse events, pain scores, return emergency department visits, or hospitalizations. (Funded by the Agency for Healthcare Research and Quality.).

Degradation of proteoglycan 4/lubricin by cathepsin S: Potential mechanism for diminished ocular surface lubrication in Sjögren's syndrome.

Sjögren's syndrome (SS) is an autoimmune disease affecting the lacrimal and salivary glands with hallmark clinical symptoms of dry eye and dry mouth. Recently, markedly increased cathepsin S (CTSS) activity has been observed in the tears of SS patients. Proteoglycan 4 (PRG4), also known as lubricin, is an effective boundary lubricant that is naturally present on the ocular surface. While PRG4 is susceptible to proteolytic digestion, the potential effect of CTSS on PRG4 remains unknown. The objective of this study was to assess the ability of CTSS to enzymatically degrade purified PRG4, and PRG4 naturally present in human tears, and alter ocular surface boundary lubricating properties. To assess the potential time course and dose-dependency of PRG4 digestion by CTSS, full-length recombinant human PRG4 (rhPRG4) was incubated at 37 °C with or without CTSS in an enzymatic digestion buffer. Digestion of PRG4 by CTSS was also examined within normal human tear samples, both with and without supplementation by rhPRG4. Finally, digestion of endogenous PRG4 by CTSS, and the effect of a CTSS inhibitor, was examined in SS tears on Schirmer strips. Digestion products were separated on 3-8% SDS-PAGE and visualized by protein staining and western blotting. The boundary lubricating ability of rhPRG4 samples was assessed using an in vitro human eyelid-cornea friction test. Finally, SDS-PAGE protein stain bands resulting from rhPRG4 digestion were submitted for tandem mass spectrometry analysis to confirm their identity as PRG4 and identify non-tryptic cleavage sites. CTSS digested rhPRG4 in a time and dose dependent manner. CTSS digestion of rhPRG4 at 1% (where % is the mass ratio of CTSS to rhPRG4) resulted in a time dependent decrease in the full-length, ∼460 kDa, monomeric rhPRG4 band, and an appearance of lower MW fragments. After 20 h, no full-length rhPRG4 was observed. Furthermore, with an increased relative enzyme concentration of 3%, no protein bands were observed after 2 h, indicating complete digestion of rhPRG4. Western blotting demonstrated PRG4 is present in normal human tears, and that rhPRG4, tears, and tears supplemented with rhPRG4 incubated with 3-9% CTSS demonstrated decreased intensity of high MW PRG4 bands, indicative of partial degradation by CTSS. Similarly, western blotting of PRG4 in SS tears incubated with CTSS demonstrated decreased intensity of high MW PRG4 bands, which was reversed in the presence of the CTSS inhibitor. CTSS treatment of rhPRG4 resulted in an increased friction coefficient, compared to untreated controls. Lastly, the lower MW bands were confirmed to be PRG4 fragments by tandem mass spectrometry, and 6 non-tryptic cleavage sites were identified. rhPRG4 is susceptible to proteolytic digestion by CTSS, both alone and in human tears, which results in diminished ocular surface boundary lubricating ability. Moreover, endogenous PRG4 is susceptible to proteolytic digestion by CTSS, both in normal and SS tears. Given the elevated activity of CTSS in SS tears, and the role intact PRG4 plays in ocular surface health and lubrication, degradation of PRG4 by CTSS is a potential mechanism for diminished ocular surface lubrication in SS. Collectively these results suggest that tear supplementation of PRG4 may be beneficial for SS patients.

Interdisciplinary Development of an Improved Emergency Department Procedural Work Surface Through Iterative Design and Use Testing in Simulated and Clinical Environments.

STUDY OBJECTIVE: A stable and readily accessible work surface for bedside medical procedures represents a valuable tool for acute care providers. In emergency department (ED) settings, the design and implementation of traditional Mayo stands and related surface devices often limit their availability, portability, and usability, which can lead to suboptimal clinical practice conditions that may affect the safe and effective performance of medical procedures and delivery of patient care. We designed and built a novel, open-source, portable, bedside procedural surface through an iterative development process with use testing in simulated and live clinical environments. METHODS: The procedural surface development project was conducted between October 2014 and June 2016 at an academic referral hospital and its affiliated simulation facility. An interdisciplinary team of emergency physicians, mechanical engineers, medical students, and design students sought to construct a prototype bedside procedural surface out of off-the-shelf hardware during a collaborative university course on health care design. After determination of end-user needs and core design requirements, multiple prototypes were fabricated and iteratively modified, with early variants featuring undermattress stabilizing supports or ratcheting clamp mechanisms. Versions 1 through 4 underwent 2 hands-on usability-testing simulation sessions; version 5 was presented at a design critique held jointly by a panel of clinical and industrial design faculty for expert feedback. Responding to select feedback elements over several surface versions, investigators arrived at a near-final prototype design for fabrication and use testing in a live clinical setting. This experimental procedural surface (version 8) was constructed and then deployed for controlled usability testing against the standard Mayo stands in use at the study site ED. Clinical providers working in the ED who opted to participate in the study were provided with the prototype surface and just-in-time training on its use when performing bedside procedures. Subjects completed the validated 10-point System Usability Scale postshift for the surface that they had used. The study protocol was approved by the institutional review board. RESULTS: Multiple prototypes and recursive design revisions resulted in a fully functional, portable, and durable bedside procedural surface that featured a stainless steel tray and intuitive hook-and-lock mechanisms for attachment to ED stretcher bed rails. Forty-two control and 40 experimental group subjects participated and completed questionnaires. The median System Usability Scale score (out of 100; higher scores associated with better usability) was 72.5 (interquartile range [IQR] 51.3 to 86.3) for the Mayo stand; the experimental surface was scored at 93.8 (IQR 84.4 to 97.5 for a difference in medians of 17.5 (95% confidence interval 10 to 27.5). Subjects reported several usability challenges with the Mayo stand; the experimental surface was reviewed as easy to use, simple, and functional. In accordance with experimental live environment deployment, questionnaire responses, and end-user suggestions, the project team finalized the design specification for the experimental procedural surface for open dissemination. CONCLUSION: An iterative, interdisciplinary approach was used to generate, evaluate, revise, and finalize the design specification for a new procedural surface that met all core end-user requirements. The final surface design was evaluated favorably on a validated usability tool against Mayo stands when use tested in simulated and live clinical settings.

Friction-Induced Mitochondrial Dysregulation Contributes to Joint Deterioration in Prg4 Knockout Mice.

Deficiency of PRG4 (lubricin), the boundary lubricant in mammalian joints, contributes to increased joint friction accompanied by superficial and upper intermediate zone chondrocyte caspase-3 activation, as shown in lubricin-null (Prg4-/-) mice. Caspase-3 activity appears to be reversible upon the restitution of Prg4 either endogenously in vivo, in a gene trap mouse, or as an applied lubricant in vitro. In this study we show that intra-articular injection of human PRG4 in vivo in Prg4-/- mice prevented caspase-3 activation in superficial zone chondrocytes and was associated with a modest decrease in whole joint friction measured ex vivo using a joint pendulum method. Non-lubricated Prg4-/- mouse cartilage shows caspase cascade activation caused by mitochondrial dysregulation, and significantly higher levels of peroxynitrite (ONOO- and -OH) and superoxide (O-2) compared to Prg4+/+ and Prg4+/- cartilage. Enzymatic activity levels of caspase 8 across Prg4 mutant mice were not significantly different, indicating no extrinsic apoptosis pathway activation. Western blots showed caspase-3 and 9 activation in Prg4-/- tissue extracts, and the appearance of nitrosylated Cys163 in the active cleft of caspase-3 which inhibits its enzymatic activity. These findings are relevant to patients at risk for arthrosis, from camptodactyl-arthropathy-coxa vara-pericarditis (CACP) syndrome and transient lubricin insufficiency due to trauma and inflammation.

Role of lubricin and boundary lubrication in the prevention of chondrocyte apoptosis.

Osteoarthritis is a complex disease involving the mechanical breakdown of articular cartilage in the presence of altered joint mechanics and chondrocyte death, but the connection between these factors is not well established. Lubricin, a mucinous glycoprotein encoded by the PRG4 gene, provides boundary lubrication in articular joints. Joint friction is elevated and accompanied by accelerated cartilage damage in humans and mice that have genetic deficiency of lubricin. Here, we investigated the relationship between coefficient of friction and chondrocyte death using ex vivo and in vitro measurements of friction and apoptosis. We observed increases in whole-joint friction and cellular apoptosis in lubricin knockout mice compared with wild-type mice. When we used an in vitro bovine explant cartilage-on-cartilage bearing system, we observed a direct correlation between coefficient of friction and chondrocyte apoptosis in the superficial layers of cartilage. In the bovine explant system, the addition of lubricin as a test lubricant significantly lowered the static coefficient of friction and number of apoptotic chondrocytes. These results demonstrate a direct connection between lubricin, boundary lubrication, and cell survival and suggest that supplementation of synovial fluid with lubricin may be an effective treatment to prevent cartilage deterioration in patients with genetic or acquired deficiency of lubricin.

CORR® ORS Richard A. Brand Award for Outstanding Orthopaedic Research: Engineering flexor tendon repair with lubricant, cells, and cytokines in a canine model.

BACKGROUND: Adhesions and poor healing are complications of flexor tendon repair. QUESTIONS/PURPOSES: The purpose of this study was to investigate a tissue engineering approach to improve functional outcomes after flexor tendon repair in a canine model. METHODS: Flexor digitorum profundus tendons were lacerated and repaired in 60 dogs that were followed for 10, 21, or 42 days. One randomly selected repair from either the second or fifth digit in one paw in each dog was treated with carbodiimide-derivatized hyaluronic acid, gelatin, and lubricin plus autologous bone marrow stromal cells stimulated with growth and differentiation factor 5; control repair tendons were not treated. Digits were analyzed by adhesion score, work of flexion, tendon-pulley friction, failure force, and histology. RESULTS: In the control group, 35 of 52 control tendons had adhesions, whereas 19 of 49 treated tendons had adhesions. The number of repaired tendons with adhesions in the control group was greater than the number in the treated group at all three times (p = 0.005). The normalized work of flexion in treated tendons was 0.28 (± 0.08), 0.29 (± 0.19), and 0.32 (± 0.22) N/mm/° at Day 10, Day 21, and Day 42 respectively, compared with the untreated tendons of 0.46 (± 0.19) at Day 10 (effect size, 1.5; p = 0.01), 0.77 (± 0.49) at Day 21 (effect size, 1.4; p < 0.001), and 1.17 (± 0.82) N/mm/° at Day 42 (effect size, 1.6; p < 0.001). The friction data were comparable to the work of flexion data at all times. The repaired tendon failure force in the untreated group at 42 days was 70.2 N (± 8.77), which was greater than the treated tendons 44.7 N (± 8.53) (effect size, 1.9; p < 0.001). Histologically, treated repairs had a smooth surface with intrinsic healing, whereas control repairs had surface adhesions and extrinsic healing. CONCLUSIONS: Our study provides evidence that tissue engineering coupled with restoration of tendon gliding can improve the quality of tendon healing in a large animal in vivo model. CLINICAL RELEVANCE: Tissue engineering may enhance intrinsic tendon healing and thus improve the functional outcomes of flexor tendon repair.

Activated chlorine dioxide solution can be used as a biocompatible antiseptic wound irrigant.

OBJECTIVE: The purpose of this study was to compare cosmesis at 3 to 4 months and infection in simple lacerations irrigated with normal saline (NS) versus activated chlorine dioxide (CD). DESIGN: This was a prospective, randomized trial of a convenience sample of patients. This study was approved by the institutional review board and Food and Drug Administration as a physician-sponsored trial (FDA investigational new drug no. 68762). SETTING: The study was conducted in a large urban, academic emergency department. PATIENTS: Patients aged 18 to 100 with simple, uncomplicated lacerations requiring repair that were less than 8 hours old were enrolled. INTERVENTIONS: Patients were randomized to receive either NS or CD wound irrigation. MAIN OUTCOME MEASURES: Demographics, infection, and cosmesis were analyzed and assessed. Cosmetic outcome was assessed at 3 to 4 months using a visual analog scale (VAS), wound evaluation score (WES), patient VAS (VASPt), and digital imaging VAS by 2 plastic surgeons (VASPlast). MAIN RESULTS: One hundred ninety-three patients were enrolled. Data analysis was available for 175 cases (86 NS and 89 CD). Wound infection follow-up was obtained in 74.9% of the patients. The 3- to 4-month cosmesis follow-up was 37.7% for VAS/WES, 40.0% for VASPt, and 37.7% for VASPlast. There were no significant differences in demographics, key wound characteristics, infection, adverse reactions, and cosmesis. CONCLUSION: The authors report the use of a novel antimicrobial irrigation solution. Chlorine dioxide appears to be a safe biologically acceptable antiseptic wound irrigant that does not appear to interfere with cosmetic outcomes.

Prediction of anemia in real-time using a smartphone camera processing conjunctival images.

Anemia is defined as a low hemoglobin (Hb) concentration and is highly prevalent worldwide. We report on the performance of a smartphone application (app) that records images in RAW format of the palpebral conjunctivae and estimates Hb concentration by relying upon computation of the tissue surface high hue ratio. Images of bilateral conjunctivae were obtained prospectively from a convenience sample of 435 Emergency Department patients using a dedicated smartphone. A previous computer-based and validated derivation data set associating estimated conjunctival Hb (HBc) and the actual laboratory-determined Hb (HBl) was used in deriving Hb estimations using a self-contained mobile app. Accuracy of HBc was 75.4% (95% CI 71.3, 79.4%) for all categories of anemia, and Bland-Altman plot analysis showed a bias of 0.10 and limits of agreement (LOA) of (-4.73, 4.93 g/dL). Analysis of HBc estimation accuracy around different anemia thresholds showed that AUC was maximized at transfusion thresholds of 7 and 9 g/dL which showed AUC values of 0.92 and 0.90 respectively. We found that the app is sufficiently accurate for detecting severe anemia and shows promise as a population-sourced screening platform or as a non-invasive point-of-care anemia classifier.