Targeted Analysis of the Size Distribution of Heavy Chain-Modified Hyaluronan with Solid-State Nanopores.

The glycosaminoglycan hyaluronan (HA) plays important roles in diverse physiological functions where the distribution of its molecular weight (MW) can influence its behavior and is known to change in response to disease conditions. During inflammation, HA undergoes a covalent modification in which heavy chain subunits of the inter-alpha-inhibitor family of proteins are transferred to its structure, forming heavy chain-HA (HC•HA) complexes. While limited assessments of HC•HA have been performed previously, determining the size distribution of its HA component remains a challenge. Here, we describe a selective method for extracting HC•HA from mixtures that yields material amenable to MW analysis with a solid-state nanopore sensor. After demonstrating the approach in vitro, we validate extraction of HC•HA from osteoarthritic human synovial fluid as a model complex biological matrix. Finally, we apply our technique to pathophysiology by measuring the size distributions of HC•HA and total HA in an equine model of synovitis.

Links between three chronic and age-related diseases, osteoarthritis, periodontitis, and osteoporosis, in a wild mammal (moose) population.

OBJECTIVE: Osteoarthritis, periodontitis and osteoporosis are chronic, age-related diseases which adversely impact millions of people worldwide. Because these diseases pose a major global public health challenge, there is an urgent need to better understand how these diseases are interrelated. Our objective was to document the age and sex-specific prevalence of each disease and assess interrelationships among the three diseases in a wild mammal (moose, Alces alces) population. METHODS: We examined the bones of moose dying from natural causes and recorded the severity of osteoarthritis (typically observed on the hip and lowest vertebrae), osteoporosis (osteoporotic lesions observed on the skull) and periodontitis (observed on maxilla and mandibles). RESULTS: Periodontitis was associated with a greater prevalence of both severe osteoarthritis and osteoporotic lesions in moose. We found no evidence to suggest that moose with osteoporotic lesions were more or less likely to exhibit signs of osteoarthritis or severe osteoarthritis. The prevalence of osteoarthritis, periodontitis and osteoporotic lesions was greater among males than for females. CONCLUSIONS: Our results were consistent with the hypothesis that bacterial pathogens causing periodontitis are a risk factor for osteoarthritis and osteoporosis. They are also consistent with the hypothesis that the inverse association between osteoarthritis and osteoporosis sometimes observed in humans may be influenced by shared risk factors, such as obesity, smoking or alcohol consumption, which are absent in moose. Together these results provide insights about three diseases which are expected to become more prevalent in the future and that cause substantial socio-economic burdens.

RELATIONSHIP BETWEEN REPRODUCTIVE AND BONE BIOMARKERS AND OSTEOARTHRITIS IN ZOO ASIAN (ELEPHAS MAXIMUS) AND AFRICAN (LOXODONTA AFRICANA) ELEPHANTS.

Osteoarthritis (OA) is common in zoo Asian (Elephas maximus) and African (Loxodonta africana) elephants. This study investigated the relationship between confirmed or suspected OA with ovarian cyclicity, gonadotropins, progestagens, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and collagen type I (CTX-I) in zoo elephants. In Asian elephants, odds of having confirmed or suspected OA decreased with cycling (OR = 0.22, P = 0.016; OR = 0.29, P = 0.020, respectively), however, not when adjusted for age (odds ratio [OR] = 0.31, P = 0.112; OR = 0.58, P = 0.369, respectively). In African elephants, none of the models between confirmed OA and cycling status were significant (P > 0.060), while the odds of having suspected OA decreased with cycling (OR = 0.12, P = 0.001), even after adjusting for age (OR = 0.15, P = 0.005). Progestagens (Asian elephants P > 0.096; African elephants P > 0.415), LH (Asian P > 0.129; African P > 0.359), and FSH (Asian P > 0.738; African P > 0.231) did not differ with confirmed or suspected OA status, unadjusted. CTX-I concentrations were not related to OA status (P > 0.655). This study concluded hormonal changes may not have a strong impact on OA, so additional investigation into other serologic biomarkers is warranted.

A Multidimensional Bioinformatic Platform for the Study of Human Response to Surgery.

OBJECTIVE: To design and establish a prospective biospecimen repository that integrates multi-omics assays with clinical data to study mechanisms of controlled injury and healing. BACKGROUND: Elective surgery is an opportunity to understand both the systemic and focal responses accompanying controlled and well-characterized injury to the human body. The overarching goal of this ongoing project is to define stereotypical responses to surgical injury, with the translational purpose of identifying targetable pathways involved in healing and resilience, and variations indicative of aberrant peri-operative outcomes. METHODS: Clinical data from the electronic medical record combined with large-scale biological data sets derived from blood, urine, fecal matter, and tissue samples are collected prospectively through the peri-operative period on patients undergoing 14 surgeries chosen to represent a range of injury locations and intensities. Specimens are subjected to genomic, transcriptomic, proteomic, and metabolomic assays to describe their genetic, metabolic, immunologic, and microbiome profiles, providing a multidimensional landscape of the human response to injury. RESULTS: The highly multiplexed data generated includes changes in over 28,000 mRNA transcripts, 100 plasma metabolites, 200 urine metabolites, and 400 proteins over the longitudinal course of surgery and recovery. In our initial pilot dataset, we demonstrate the feasibility of collecting high quality multi-omic data at pre- and postoperative time points and are already seeing evidence of physiologic perturbation between timepoints. CONCLUSIONS: This repository allows for longitudinal, state-of-the-art geno-mic, transcriptomic, proteomic, metabolomic, immunologic, and clinical data collection and provides a rich and stable infrastructure on which to fuel further biomedical discovery.

Extracellular Vesicles as Biological Indicators and Potential Sources of Autologous Therapeutics in Osteoarthritis.

Along with cytokines, extracellular vesicles (EVs) released by immune cells in the joint contribute to osteoarthritis (OA) pathogenesis. By high-resolution flow cytometry, we characterized 18 surface markers and 4 proinflammatory cytokines carried by EVs of various sizes in plasma and synovial fluid (SF) from individuals with knee OA, with a primary focus on immune cells that play a major role in OA pathogenesis. By multiplex immunoassay, we also measured concentrations of cytokines within (endo) and outside (exo) EVs. EVs carrying HLA-DR, -DP and -DQ were the most enriched subpopulations in SF relative to plasma (25-50-fold higher depending on size), suggesting a major contribution to the SF EV pool from infiltrating immune cells in OA joints. In contrast, the CD34+ medium and small EVs, reflecting hematopoietic stem cells, progenitor cells, and endothelial cells, were the most significantly enriched subpopulations in plasma relative to SF (7.3- and 7.7-fold higher). Ratios of EVs derived from neutrophils and lymphocytes were highly correlated between SF and plasma, indicating that plasma EVs could reflect OA severity and serve as systemic biomarkers of OA joint pathogenesis. Select subsets of plasma EVs might also provide next generation autologous biological products for intra-articular therapy of OA joints.

Cartilage biomarkers in the osteoarthropathy of alkaptonuria reveal low turnover and accelerated ageing.

OBJECTIVE: Alkaptonuria (AKU) is a rare autosomal recessive disease resulting from a single enzyme deficiency in tyrosine metabolism. As a result, homogentisic acid cannot be metabolized, causing systemic increases. Over time, homogentisic acid polymerizes and deposits in collagenous tissues, leading to ochronosis. Typically, this occurs in joint cartilages, leading to an early onset, rapidly progressing osteoarthropathy. The aim of this study was to examine tissue turnover in cartilage affected by ochronosis and its role in disease initiation and progression. METHODS: With informed patient consent, hip and knee cartilages were obtained at surgery for arthropathy due to AKU (n = 6; 2 knees/4 hips) and OA (n = 12; 5 knees/7 hips); healthy non-arthritic (non-OA n = 6; 1 knee/5 hips) cartilages were obtained as waste from trauma surgery. We measured cartilage concentrations (normalized to dry weight) of racemized aspartate, GAG, COMP and deamidated COMP (D-COMP). Unpaired AKU, OA and non-OA samples were compared by non-parametric Mann-Whitney U test. RESULTS: Despite more extractable total protein being obtained from AKU cartilage than from OA or non-OA cartilage, there was significantly less extractable GAG, COMP and D-COMP in AKU samples compared with OA and non-OA comparators. Racemized Asx (aspartate and asparagine) was significantly enriched in AKU cartilage compared with in OA cartilage. CONCLUSIONS: These novel data represent the first examination of cartilage matrix components in a sample of patients with AKU, representing almost 10% of the known UK alkaptonuric population. Compared with OA and non-OA, AKU cartilage demonstrates a very low turnover state and has low levels of extractable matrix proteins.

Matrix metalloproteinase activity and prostaglandin E2 are elevated in the synovial fluid of meniscus tear patients.

PURPOSE: Meniscus tears are a common knee injury and are associated with the development of post-traumatic osteoarthritis (OA). The purpose of this study is to evaluate potential OA mediators in the synovial fluid and serum of meniscus tear subjects compared to those in the synovial fluid of radiographic non-OA control knees. MATERIALS AND METHODS: Sixteen subjects with an isolated unilateral meniscus injury and six subjects who served as reference controls (knee Kellgren-Lawrence grade 0-1) were recruited. Twenty-one biomarkers were measured in serum from meniscus tear subjects and in synovial fluid from both groups. Meniscus tear subjects were further stratified by tear type to assess differences in biomarker levels. RESULTS: Synovial fluid total matrix metalloproteinase (MMP) activity and prostaglandin E2 (PGE2) were increased 25-fold and 290-fold, respectively, in meniscus tear subjects as compared to reference controls (p < 0.05). Synovial fluid MMP activity and PGE2 concentrations were positively correlated in meniscus tear subjects (R = 0.83, p < 0.0001). In meniscus tear subjects, synovial fluid levels of MMP activity, MMP-2, MMP-3, sGAG, COMP, IL-6, and PGE2 were higher than serum levels (p < 0.05). Subjects with complex meniscus tears had higher synovial fluid MMP-10 (p < 0.05) and reduced serum TNFα and IL-8 (p < 0.05) compared to other tear types. CONCLUSIONS: Given the degradative and pro-inflammatory roles of MMP activity and PGE2, these molecules may alter the biochemical environment of the joint. Our findings suggest that modulation of PGE2 signaling, MMP activity, or both following a meniscus injury may be targets to promote meniscus repair and prevent OA development.

Does a lack of physical activity explain the rheumatoid arthritis lipid profile?

BACKGROUND: In rheumatoid arthritis (RA), cardiovascular risk is associated with paradoxical reductions in total cholesterol, low density lipoprotein-cholesterol (LDL-C), and high density lipoprotein-cholesterol (HDL-C). Concentrations of small LDL (LDL-P) and HDL (HDL-P) particles are also reduced with increased inflammation and disease activity in RA patients. Here we sought to identify which measure(s) of inflammation, disease activity and cardiometabolic risk contribute most to the RA-associated lipoprotein profile. METHODS: NMR lipoprotein measurements were obtained for individuals with RA (n = 50) and age-, gender-, and body mass index (BMI)-matched controls (n = 39). Groups were compared using 39 matched pairs with 11 additional subjects used in RA only analyses. Among RA patients, relationships were determined for lipoprotein parameters with measures of disease activity, disability, pain, inflammation, body composition, insulin sensitivity and exercise. Percentage of time spent in basal activity (<1 metabolic equivalent) and exercise (≥3 metabolic equivalents) were objectively-determined. RESULTS: Subjects with RA had fewer total and small LDL-P as well as larger LDL and HDL size (P < 0.05). Among RA patients, pain and disability were associated with fewer small HDL-P (P < 0.05), while interleukin (IL)-6, IL-18, and TNF-α were associated with LDL size (P < 0.05). BMI, waist circumference, abdominal visceral adiposity and insulin resistance were associated with more total and small LDL-P, fewer large HDL-P, and a reduction in HDL size (P < 0.05). Most similar to the RA lipoprotein profile, more basal activity (minimal physical activity) and less exercise time were associated with fewer small LDL-P and total and small HDL-P (P < 0.05). CONCLUSIONS: The RA-associated lipoprotein profile is associated with a lack of physical activity. As this was a cross-sectional investigation and not an intervention and was performed from 2008-13, this study was not registered in clinicaltrials.gov.

Dietary fatty acid content regulates wound repair and the pathogenesis of osteoarthritis following joint injury.

OBJECTIVE: The mechanisms linking obesity and osteoarthritis (OA) are not fully understood and have been generally attributed to increased weight, rather than metabolic or inflammatory factors. Here, we examined the influence of fatty acids, adipokines, and body weight on OA following joint injury in an obese mouse model. METHODS: Mice were fed high-fat diets rich in various fatty acids (FA) including saturated FAs (SFAs), ω-6 polyunsaturated FAs (PUFAs), and ω-3 PUFAs. OA was induced by destabilising the medial meniscus. Wound healing was evaluated using an ear punch. OA, synovitis and wound healing were determined histologically, while bone changes were measured using microCT. Activity levels and serum cytokines were measured at various time-points. Multivariate models were performed to elucidate the associations of dietary, metabolic and mechanical factors with OA and wound healing. RESULTS: Using weight-matched mice and multivariate models, we found that OA was significantly associated with dietary fatty acid content and serum adipokine levels, but not with body weight. Furthermore, spontaneous activity of the mice was independent of OA development. Small amounts of ω-3 PUFAs (8% by kcal) in a high-fat diet were sufficient to mitigate injury-induced OA, decreasing leptin and resistin levels. ω-3 PUFAs significantly enhanced wound repair, SFAs or ω-6 PUFAs independently increased OA severity, heterotopic ossification and scar tissue formation. CONCLUSIONS: Our results indicate that with obesity, dietary FA content regulates wound healing and OA severity following joint injury, independent of body weight, supporting the need for further studies of dietary FA supplements as a potential therapeutic approach for OA.

Genetic and cellular evidence of decreased inflammation associated with reduced incidence of posttraumatic arthritis in MRL/MpJ mice.

OBJECTIVE: To examine the relationship between inflammation and posttraumatic arthritis (PTA) in a murine intraarticular fracture model. METHODS: Male C57BL/6 and MRL/MpJ "superhealer" mice received tibial plateau fractures using a previously established method. Mice were killed on day 0 (within 4 hours of fracture) and days 1, 3, 5, 7, 28, and 56 after fracture. Synovial tissue samples, obtained prior to fracture and on days 0, 1, 3, 5, and 7 after fracture, were examined by reverse transcription-polymerase chain reaction for gene expression of proinflammatory cytokines and chemokines. Synovial fluid and serum samples were collected to measure cytokine concentrations, using enzyme-linked immunosorbent assay. Whole joints were examined histologically for the extent of synovitis and cartilage degradation, and joint tissue samples from all time points were analyzed immunohistochemically to evaluate the distribution of interleukin-1 (IL-1). RESULTS: Compared to C57BL/6 mice, MRL/MpJ mice had less severe intraarticular and systemic inflammation following joint injury, as evidenced by lower gene expression of tumor necrosis factor α and IL-1ß in the synovial tissue and lower protein levels of IL-1α and IL-1ß in the synovial fluid, serum, and joint tissues. Furthermore, after joint injury, MRL/MpJ mice had lower gene expression of macrophage inflammatory proteins and macrophage-derived chemokine (CCL22) in the synovial tissue, and also had reduced acute and late-stage infiltration of synovial macrophages. CONCLUSION: C57BL/6 mice exhibited higher levels of inflammation than MRL/MpJ mice, indicating that MRL/MpJ mice are protected from PTA in this model. These data thus suggest an association between joint tissue inflammation and the development and progression of PTA in mice.

Immune system-related plasma extracellular vesicles in healthy aging.

Objectives: To identify age-related plasma extracellular vehicle (EVs) phenotypes in healthy adults. Methods: EV proteomics by high-resolution mass spectrometry to evaluate EV protein stability and discover age-associated EV proteins (n=4 with 4 serial freeze-thaws each); validation by high-resolution flow cytometry and EV cytokine quantification by multiplex ELISA (n=28 healthy donors, aged 18-83 years); quantification of WI-38 fibroblast cell proliferation response to co-culture with PKH67-labeled young and old plasma EVs. The EV samples from these plasma specimens were previously characterized for bilayer structure, intra-vesicle mitochondria and cytokines, and hematopoietic cell-related surface markers. Results: Compared with matched exo-EVs (EV-depleted supernatants), endo-EVs (EV-associated) had higher mean TNF-α and IL-27, lower mean IL-6, IL-11, IFN-γ, and IL-17A/F, and similar mean IL-1ß, IL-21, and IL-22 concentrations. Some endo-EV and exo-EV cytokine concentrations were correlated, including TNF-α, IL-27, IL-6, IL-1ß, and IFN-γ, but not IL-11, IL-17A/F, IL-21 or IL-22. Endo-EV IFN-γ and exo-EV IL-17A/F and IL-21 declined with age. By proteomics and confirmed by flow cytometry, we identified age-associated decline of fibrinogen (FGA, FGB and FGG) in EVs. Age-related EV proteins indicated predominant origins in the liver and innate immune system. WI-38 cells (>95%) internalized similar amounts of young and old plasma EVs, but cells that internalized PKH67-EVs, particularly young EVs, underwent significantly greater cell proliferation. Conclusion: Endo-EV and exo-EV cytokines function as different biomarkers. The observed healthy aging EV phenotype reflected a downregulation of EV fibrinogen subpopulations consistent with the absence of a pro-coagulant and pro-inflammatory condition common with age-related disease.

Anti-CMV IgG Seropositivity is Associated with Plasma Biomarker Evidence of Amyloid-ß Accumulation.

Background: Some human studies have identified infection with cytomegalovirus (CMV), a member of the alpha herpesvirus family, as a risk factor for Alzheimer's disease and related dementias (ADRD). To our knowledge, no studies have evaluated associations of CMV seropositivity with plasma biomarkers of ADRD risk in middle-aged adults. Objective: In participants recruited for an exercise study, we evaluated cross-sectional associations of CMV seropositivity with: Aß42/Aß40 ratio, a low ratio suggestive of central nervous system Aß accumulation; glial fibrillary acidic protein (GFAP), a measure of neuroinflammation; and neurofilament light (NfL), a measure of neurodegeneration. Methods: Anti-CMV IgG was quantified by ELISA. Plasma ADRD biomarkers were quantified using the ultrasensitive SIMOA assay. We used linear regression to evaluate associations of CMV seropositivity with the ADRD biomarkers, adjusting for age, sex, and race (n = 303; Age = 55.7±9.2 years). For ADRD biomarkers significantly associated with CMV seropositivity, we evaluated continuous associations of anti-CMV IgG levels with the ADRD biomarkers, excluding CMV seronegative participants. Results: 53% of participants were CMV seropositive. CMV seropositivity was associated with a lesser Aß42/Aß40 ratio (ß=-3.02e-03 95% CI [-5.97e-03, -7.18e-05]; p = 0.045). In CMV seropositive participants, greater anti-CMV IgG levels were associated with a lesser Aß42/Aß40 ratio (ß=-4.85e-05 95% CI[-8.45e-05, -1.25e-05]; p = 0.009). CMV seropositivity was not associated with plasma GFAP or NfL in adjusted analyses. Conclusions: CMV seropositivity was associated with a lesser plasma Aß42/Aß40 ratio. This association may be direct and causally related to CMV neuro-cytotoxicity or may be indirect and mediated by inflammatory factors resulting from CMV infection burden and/or the immune response.

Liver-derived plasminogen mediates muscle stem cell expansion during caloric restriction through the plasminogen receptor Plg-RKT.

An intriguing effect of short-term caloric restriction (CR) is the expansion of certain stem cell populations, including muscle stem cells (satellite cells), which facilitate an accelerated regenerative program after injury. Here, we utilized the MetRSL274G (MetRS) transgenic mouse to identify liver-secreted plasminogen as a candidate for regulating satellite cell expansion during short-term CR. Knockdown of circulating plasminogen prevents satellite cell expansion during short-term CR. Furthermore, loss of the plasminogen receptor KT (Plg-RKT) is also sufficient to prevent CR-related satellite cell expansion, consistent with direct signaling of plasminogen through the plasminogen receptor Plg-RKT/ERK kinase to promote proliferation of satellite cells. Importantly, we are able to replicate many of these findings in human participants from the CALERIE trial. Our results demonstrate that CR enhances liver protein secretion of plasminogen, which signals directly to the muscle satellite cell through Plg-RKT to promote proliferation and subsequent muscle resilience during CR.

Effect of Remotely Supervised Weight Loss and Exercise Training Versus Lifestyle Counseling on Cardiovascular Risk and Clinical Outcomes in Older Adults With Rheumatoid Arthritis: A Randomized Controlled Trial.

OBJECTIVE: To compare a remotely supervised weight loss and exercise intervention to lifestyle counseling for effects on cardiovascular disease risk, disease activity, and patient-reported outcomes in older patients with rheumatoid arthritis (RA) and overweight/obesity. METHODS: Twenty older (60-80 years), previously sedentary participants with seropositive RA and overweight/obesity were randomized to 16 weeks of either Supervised Weight loss and Exercise Training (SWET) or Counseling Health As Treatment (CHAT). The SWET group completed aerobic training (150 minutes/week moderate-to-vigorous intensity), resistance training (two days/week), and a hypocaloric diet (7% weight loss goal). The CHAT control group completed two lifestyle counseling sessions followed by monthly check-ins. The primary outcome was a composite metabolic syndrome z-score (MSSc) derived from fasting glucose, triglycerides, high density lipoprotein-cholesterol, minimal waist circumference, and mean arterial pressure. Secondary outcomes included RA disease activity and patient-reported outcomes. RESULTS: Both groups improved MSSc (absolute change -1.67 ± 0.64 in SWET; -1.34 ± 1.30 in CHAT; P < 0.01 for both groups) with no between-group difference. Compared with CHAT, SWET significantly improved body weight, fat mass, Disease Activity Score-28 C-reactive protein, and patient-reported physical health, physical function, mental health, and fatigue (P < 0.04 for all between-group comparisons). Based on canonical correlations for fat mass, cardiorespiratory fitness, and leg strength, component-specific effects were strongest for (1) weight loss improving MSSc, physical health, and mental health; (2) aerobic training improving physical function and fatigue; and (3) resistance training improving Disease Activity Score-28 C-reactive protein. CONCLUSION: In older patients with RA and overweight/obesity, 16 weeks of remotely supervised weight loss, aerobic training, and resistance training improve cardiometabolic health, patient-reported outcomes, and disease activity. Less intensive lifestyle counseling similarly improves cardiovascular disease risk profiles, suggesting an important role for integrative interventions in the routine clinical care of this at-risk RA population.

Induction of osteoarthritis and metabolic inflammation by a very high-fat diet in mice: effects of short-term exercise.

OBJECTIVE: To test the hypotheses that obesity due to a very high-fat diet induces knee osteoarthritis (OA), and that short-term wheel-running exercise protects against obesity-induced knee OA by reducing systemic inflammation and metabolic dysregulation. METHODS: Male C57BL/6J mice were fed either a control diet (13.5% kcal from fat) or a very high-fat diet (60% kcal from fat) from age 12 weeks to age 24 weeks. From 20 to 24 weeks of age, half of the mice were housed with running wheels. The severity of knee OA was determined by assessing histopathologic features, and serum cytokines were measured using a multiplex bead immunoassay and enzyme-linked immunosorbent assays. Body composition was quantified by dual-energy x-ray absorptiometry, and insulin resistance was assessed by glucose tolerance testing. RESULTS: Feeding mice with a very high-fat diet increased knee OA scores and levels of serum leptin, adiponectin, KC (mouse analog of interleukin-8 [IL-8]), monokine induced by interferon-γ (CXCL9), and IL-1 receptor antagonist to an extent in proportion to the gain in body fat (3-fold increase in percent body fat compared to controls). Wheel-running exercise reduced progression of OA in the medial femur of obese mice. In addition, exercise disrupted the clustering of cytokine expression and improved glucose tolerance, without reducing body fat or cytokine levels. CONCLUSION: Obesity induced by a very high-fat diet in mice causes OA and systemic inflammation in proportion to body fat. Increased joint loading is not sufficient to explain the increased incidence of knee OA with obesity, as wheel running is protective rather than damaging. Exercise improves glucose tolerance and disrupts the coexpression of proinflammatory cytokines, suggesting that increased aerobic exercise may act independently of weight loss in promoting joint health.

Diet-induced obesity significantly increases the severity of posttraumatic arthritis in mice.

OBJECTIVE: Obesity and joint injury are primary risk factors for osteoarthritis (OA) that involve potential alterations in the biomechanical and inflammatory environments of the joint. Posttraumatic arthritis is a frequent long-term complication of intraarticular fractures. Obesity has been linked to primary OA and may potentially contribute to the development of posttraumatic arthritis by a variety of mechanisms. The objectives of this study were to determine whether diet-induced obesity influences the severity of posttraumatic arthritis in mice and to examine the interrelationships between joint degeneration and serum levels of the inflammatory cytokines and adipokines that are involved in this response. METHODS: C57BL/6 mice were fed either normal chow (13% fat) or a high-fat diet (60% fat) starting at 4 weeks of age. At 16 weeks of age, half of the mice in each group were subjected to a closed intraarticular fracture of the left knee. At 8 weeks postfracture, knee OA was assessed by cartilage and synovium histology in addition to bone morphology. Serum cytokine concentrations were determined with multiplex assays. RESULTS: Fractured knee joints of mice receiving a high-fat diet showed significantly increased OA degeneration compared with nonfractured contralateral control knees, while fractured knee joints of mice receiving a low-fat diet did not demonstrate significant differences from nonfractured contralateral control knees. A high-fat diet increased serum concentrations of interleukin-12p70 (IL-12p70), IL-6, and keratinocyte-derived chemokine while decreasing adiponectin concentrations. Joint injury also increased IL-12p70 concentrations in mice receiving a high-fat diet. Systemic levels of adiponectin were inversely correlated with synovial inflammation in control limbs. CONCLUSION: Diet-induced obesity significantly increased the severity of OA following intraarticular fracture. Obesity and joint injury together can alter systemic levels of inflammatory cytokines such as IL-12p70.

Relationships amongst osteoarthritis biomarkers, dynamic knee joint load, and exercise: results from a randomized controlled pilot study.

BACKGROUND: Little is known about the relationships of circulating levels of biomarkers of cartilage degradation with biomechanical outcomes relevant to knee osteoarthritis (OA) or biomarker changes following non-pharmacological interventions. The objectives of this exploratory, pilot study were to: 1) examine relationships between biomarkers of articular cartilage degradation and synthesis with measures of knee joint load during walking, and 2) examine changes in these biomarkers following 10 weeks of strengthening exercises. METHODS: Seventeen (8 male, 9 female; 66.1 +/- 11.3 years of age) individuals with radiographically-confirmed medial tibiofemoral OA participated. All participants underwent a baseline testing session where serum and urine samples were collected, followed by a three-dimensional motion analysis. Motion analysis was used to calculate the external knee adduction moment (KAM) peak value and impulse. Following baseline testing, participants were randomized to either 10 weeks of: 1) physiotherapist-supervised lower limb muscle strengthening exercises, or 2) no exercises (control). Identical follow-up testing was conducted 11 weeks after baseline. Biomarkers included: urinary C-telopeptide of type II collagen (uCTX-II) and type II collagen cleavage neoepitope (uC2C), serum cartilage oligomeric matrix protein (sCOMP), serum hyaluronic acid (sHA) and serum C-propeptide of type II procollagen (sCPII). Linear regression analysis was used to examine relationships between measures of the KAM and biomarker concentrations as baseline, as well as between-group differences following the intervention. RESULTS: KAM impulse predicted significant variation in uCTX-II levels at baseline (p = 0.04), though not when controlling for disease severity and walking speed (p = 0.33). KAM impulse explained significant variation in the ratio uCTX-II;sCPII even when controlling for additional variables (p = 0.04). Following the intervention, changes in sCOMP were significantly greater in the exercise group compared to controls (p = 0.04). On average those in the control group experienced a slight increase in sCOMP and uCTX-II, while those in the exercise group experienced a reduction. No other significant findings were observed. CONCLUSIONS: This research provides initial evidence of a potential relationship between uCTX-II and knee joint load measures in patients with medial tibiofemoral knee OA. However, this relationship became non-significant after controlling for disease severity and walking speed, suggesting further research is necessary. It also appears that sCOMP is amenable to change following a strengthening intervention, suggesting a potential beneficial role of exercise on cartilage structure. TRIAL REGISTRATION: Clinicaltrials.gov NCT01241812.

Effects of Leptin and Body Weight on Inflammation and Knee Osteoarthritis Phenotypes in Female Rats.

Leptin is a proinflammatory adipokine that contributes to obesity-associated osteoarthritis (OA), especially in women. However, the extent to which leptin causes knee OA separate from the effect of increased body weight is not clear. We hypothesized that leptin is necessary to induce knee OA in obese female rats but not sufficient to induce knee OA in lean rats lacking systemic metabolic inflammation. The effect of obesity without leptin signaling was modeled by comparing female lean Zucker rats to pair fed obese Zucker rats, which possess mutant fa alleles of the leptin receptor gene. The effect of leptin without obesity was modeled in female F344BN F1 hybrid rats by systemically administering recombinant rat leptin versus saline for 23 weeks via osmotic pumps. Primary OA outcomes included cartilage histopathology and subchondral bone micro-computed tomography. Secondary outcomes included targeted cartilage proteomics, serum inflammation, and synovial fluid inflammation following an acute intra-articular challenge with interleukin-1ß (IL-1ß). Compared to lean Zucker rats, obese Zucker rats developed more severe tibial osteophytes and focal cartilage lesions in the medial tibial plateau, with modest changes in proximal tibial epiphysis trabecular bone structure. In contrast, exogenous leptin treatment, which increased plasma leptin sixfold without altering body weight, caused mild generalized cartilage fibrillation and reduced Safranin O staining compared to vehicle-treated animals. Leptin also significantly increased subchondral and trabecular bone volume and bone mineral density in the proximal tibia. Cartilage metabolic and antioxidant enzyme protein levels were substantially elevated with leptin deficiency and minimally suppressed with leptin treatment. In contrast, leptin treatment induced greater changes in systemic and local inflammatory mediators compared to leptin receptor deficiency, including reduced serum IL-6 and increased synovial fluid IL-1ß. In conclusion, rat models that separately elevate leptin or body weight develop distinct OA-associated phenotypes, revealing how obesity increases OA pathology through both leptin-dependent and independent pathways. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Increased Effusion Synovitis for Those With a Dysregulated Inflammatory Response After an Anterior Cruciate Ligament Injury.

Introduction The progression to posttraumatic osteoarthritis (PTOA) after an anterior cruciate ligament (ACL) injury is likely multifactorial, involving biological, mechanical, and psychosocial factors. Following acute joint trauma, there appears to be a subset of patients that demonstrate a dysregulated inflammatory response. This pro-inflammatory phenotype, or "Inflamma-type," is characterized by an amplified pro-inflammatory response combined with a lack of attendant anti-inflammatory response and has been observed following both an ACL injury and an intra-articular fracture. The aims of this study were to: 1) compare magnetic resonance imaging (MRI)-measured effusion synovitis between those with vs. without a dysregulated inflammatory response, and 2) assess the correlations between effusion synovitis and synovial fluid concentrations of proinflammatory cytokines, degradative enzymes, and synovial fluid biomarkers of cartilage degradation. Methods A cluster analysis was previously performed with synovial fluid concentrations of biomarkers of inflammation and cartilage degradation from 35 patients with acute ACL injuries. Patients were then categorized into two groups: a pro-inflammatory phenotype ("Inflamma-type") and those with a more normal inflammatory response to injury (NORM). Effusion synovitis measured from each patient's preoperative clinical MRI scan was compared between the Inflamma-type and NORM groups using an independent, two-tailed t-test. In addition, Spearman's rho non-parametric correlations were calculated to evaluate the relationship between effusion synovitis and each of the synovial fluid concentrations of pro-inflammatory cytokines, degradative enzymes, and biomarkers of cartilage degradation and bony remodeling. Results Effusion synovitis was significantly greater for the Inflamma-type (10.9±3.8 mm) than the NORM group (7.4±4.4 mm, p=0.04, Cohen's d=0.82). Effusion synovitis significantly correlated with matrix metalloproteinase-3 (rho=0.63, p<0.001), matrix metalloproteinase-1 (rho=0.50, p=0.003), and sulfated glycosaminoglycan (rho=0.42, p=0.01). No other significant correlations were present. Conclusion Effusion synovitis was significantly greater for those that demonstrated a dysregulated inflammatory response after acute ACL injury than those with a more normal response to injury. Effusion synovitis was also found to significantly correlate with synovial fluid concentrations of degradative enzymes and a biomarker of early cartilage degradation. Future work is needed to determine if non-invasive methods, such as MRI or ultrasound, may accurately identify patients within this pro-inflammatory phenotype and whether this subset is more prone to more rapid PTOA changes after injury.

Tryptophan Metabolism and Neurodegeneration: Longitudinal Associations of Kynurenine Pathway Metabolites with Cognitive Performance and Plasma Alzheimer's Disease and Related Dementias Biomarkers in the Duke Physical Performance Across the LifeSpan Study.

BACKGROUND: The kynurenine pathway (KP) comprises a family of tryptophan-derived metabolites that some studies have reported are associated with poorer cognitive performance and an increased risk of Alzheimer's disease and related dementias (ADRD). OBJECTIVE: The objective of this study was to determine the associations of plasma KP metabolites (kynurenine [KYN], kynurenic acid [KA], and tryptophan [TRP]) with a panel of plasma ADRD biomarkers (Aß42/ ß40 ratio, pTau-181, glial fibrillary acidic protein [GFAP], and neurofilament light [NfL]) and cognitive performance in a subset of older adults drawn from the Duke Physical Performance Across the LifeSpan (PALS) study. METHODS: The Montreal Cognitive Assessment (MoCA) was used to assess cognitive performance. We used multivariate multiple regression to evaluate associations of the KYN/TRP and KA/KYN ratios with MoCA score and plasma ADRD biomarkers at baseline and over two years (n = 301; Age = 74.8±8.7). RESULTS: Over two years, an increasing KYN/TRP ratio was associated with increasing plasma concentrations of plasma p-Tau181 (ß= 6.151; 95% CI [0.29, 12.01]; p = 0.040), GFAP (ß= 11.12; 95% CI [1.73, 20.51]; p = 0.020), and NfL (ß= 11.13; 95% CI [2.745, 19.52]; p = 0.009), but not MoCA score or the Aß42/Aß40 ratio. There were no significant associations of KA/KYN with MoCA score or plasma ADRD biomarkers. CONCLUSION: Our findings provide evidence that greater concentrations of KP metabolites are associated longitudinally over two years with greater biomarker evidence of neurofibrillary tau pathology (pTau-181), neuroinflammation (GFAP), and neurodegeneration (NfL), suggesting that dysregulated KP metabolism may play a role in ADRD pathogenesis.