Ultrasound-Guided Injections of HYADD4 for Knee Osteoarthritis Improves Pain and Functional Outcomes at 3, 6, and 12 Months without Changes in Measured Synovial Fluid, Serum Collagen Biomarkers, or Most Synovial Fluid Biomarker Proteins at 3 Months.

BACKGROUND: Prior studies have demonstrated improved efficacy when intra-articular (IA) therapeutics are injected using ultrasound (US) guidance. The aim of this study was to determine if clinical improvement in pain and function after IA hyaluronic acid injections using US is associated with changes in SF volumes and biomarker proteins at 3 months. METHODS: 49 subjects with symptomatic knee OA, BMI < 40, and KL radiographic grade II or III participated. Subjects with adequate aspirated synovial fluid (SF) volumes received two US-guided IA-HA injections of HYADD4 (24 mg/3 mL) 7 days apart. Clinical evaluations at 3, 6, and 12 months included WOMAC, VAS, PCS scores, 6 MWD, and US-measured SF depth. SF and blood were collected at 3 months and analyzed for four serum OA biomarkers and fifteen SF proteins. RESULTS: Statistical differences were observed at 3, 6, and 12 months compared to baseline values, with improvements at 12 months for WOMAC scores (50%), VAS (54%), and PCS scores (24%). MMP10 levels were lower at 3 months without changes in SF volumes, serum levels of C2C, COMP, HA, CPII, or SF levels of IL-1 ra, IL-4, 6, 7, 8, 15, 18, ILGFBP-1, 3, and MMP 1, 2, 3, 8, 9. Baseline clinical features or SF biomarker protein levels did not predict responsiveness at 3 months. CONCLUSIONS: Clinical improvements were observed at 12 months using US needle guidance for IA HA, whereas only one SF protein biomarker protein was different at 3 months. Larger studies are needed to identify which SF biomarkers will predict which individual OA patients will receive the greatest benefit from IA therapeutics.

Synovial Fluid Cytokines, Chemokines and MMP Levels in Osteoarthritis Patients with Knee Pain Display a Profile Similar to Many Rheumatoid Arthritis Patients.

BACKGROUND: There are currently no effective disease-modifying drugs to prevent cartilage loss in osteoarthritis and synovial fluid is a potentially valuable source of biomarkers to understand the pathogenesis of different types of arthritis and identify drug responsiveness. The aim of this study was to compare the differences between SF cytokines and other proteins in patients with OA (n = 21) to those with RA (n = 27) and normal knees (n = 3). METHODS: SF was obtained using ultrasound (US) guidance and an external pneumatic compression device. RA patients were categorized as active (n = 20) or controlled (n = 7) based upon SF white blood cell counts (> or <300 cells/mm3). Samples were cryopreserved and analyzed by multiplex fluorescent bead assays (Luminex). Between-group differences of 16 separate biomarker proteins were identified using ANOVA on log10-transformed concentrations with p values adjusted for multiple testing. RESULTS: Only six biomarkers were significantly higher in SF from active RA compared to OA-TNF-α, IL-1-ß IL-7, MMP-1, MMP-2, and MMP-3. Only MMP-8 levels in RA patients correlated with SF WBC counts (p < 0.0001). Among OA patients, simultaneous SF IL-4, IL-6, IL-8, and IL-15 levels were higher than serum levels, whereas MMP-8, MMP-9, and IL-18 levels were higher in serum (p < 0.05). CONCLUSION: These results support the growing evidence that OA patients have a pro-inflammatory/catabolic SF environment. SF biomarker analysis using multiplex testing and US guidance may distinguish OA phenotypes and identify treatment options based upon targeted inflammatory pathways similar to patients with RA.

Precision Medicine for Rheumatoid Arthritis: The Right Drug for the Right Patient-Companion Diagnostics.

Despite the growing number of biologic and JAK inhibitor therapeutic agents available to treat various systemic autoimmune illnesses, the lack of a validated companion diagnostic (CDx) to accurately predict drug responsiveness for an individual results in many patients being treated for years with expensive, ineffective, or toxic drugs. This review will focus primarily on rheumatoid arthritis (RA) therapeutics where the need is greatest due to poor patient outcomes if the optimum drug is delayed. We will review current FDA-approved biologic and small molecule drugs and why RA patients switch these medications. We will discuss the sampling of various tissues for potential CDx and review early results from studies investigating drug responsiveness utilizing advanced technologies including; multiplex testing of cytokines and proteins, autoantibody profiling, genomic analysis, proteomics, miRNA analysis, and metabolomics. By using these new technologies for CDx the goal is to improve RA patient outcomes and achieve similar successes like those seen in oncology using precision medicine guided therapeutics.

A human pluripotent stem cell model for the analysis of metabolic dysfunction in hepatic steatosis.

Nonalcoholic fatty liver disease (NAFLD) is currently the most prevalent form of liver disease worldwide. This term encompasses a spectrum of pathologies, from benign hepatic steatosis to non-alcoholic steatohepatitis, which have, to date, been challenging to model in the laboratory setting. Here, we present a human pluripotent stem cell (hPSC)-derived model of hepatic steatosis, which overcomes inherent challenges of current models and provides insights into the metabolic rewiring associated with steatosis. Following induction of macrovesicular steatosis in hepatocyte-like cells using lactate, pyruvate, and octanoate (LPO), respirometry and transcriptomic analyses revealed compromised electron transport chain activity. 13C isotopic tracing studies revealed enhanced TCA cycle anaplerosis, with concomitant development of a compensatory purine nucleotide cycle shunt leading to excess generation of fumarate. This model of hepatic steatosis is reproducible, scalable, and overcomes the challenges of studying mitochondrial metabolism in currently available models.

Multiple breath washout: A noninvasive tool for identifying lung disease in symptomatic military deployers.

RATIONALE: Military deployments to austere environments since November 9, 2001 may put "deployers" at risk for respiratory disease. Sensitive, noninvasive tools for detecting large and small airways injury are needed to identify early disease and help inform management for this at-risk population. OBJECTIVES: We examined multiple breath washout (MBW) as a tool for identifying deployment-related airways disease and assessed host and exposure risk factors compared to healthy controls. METHODS: Between March 2015 and March 2020, 103 healthy controls and 71 symptomatic deployers with asthma and/or distal lung disease completed a questionnaire, spirometry and MBW testing. SAS v. 9.4 was used to compare MBW parameters between deployers and controls via univariate analyses and adjusted for demographic factors using multiple linear regression. MEASUREMENTS AND MAIN RESULTS: Deployers were significantly more likely than controls to have an abnormal lung clearance index (LCI) score indicating global ventilation inhomogeneity. Adjusting for sex, smoking status, smoking pack-years and body mass index, LCI scores were significantly more abnormal among those with deployment-related asthma and distal lung disease compared to controls. The unadjusted variable Sacin (a marker of ventilation inhomogeneity in the acinar airways) was higher and thus more abnormal in those with both proximal and distal airways disease. Deployers who reported more frequent exposure to explosive blasts had significantly higher LCI scores. CONCLUSIONS: This study demonstrates the utility of MBW in evaluating exposure-related airways disease in symptomatic military personnel following deployment to austere environments, and is the first to link exposure to explosive blasts to measurable small airways injury.

Multiple breath washout test data for healthy controls.

This article includes pulmonary function data collected via multiple breath nitrogen washout for 103 healthy U.S. adults recruited at National Jewish Health in Denver, Colorado. Testing was performed by certified technicians and reviewed by expert pulmonologists for quality and consistency. Data were collected from a diverse population that included 52 males and 51 females with an average age of 39 years (range 20-77 years). Participants were of non-Hispanic White (85%), African-American/Black (6%), Hispanic (4%), more than one race (4%) or American Indian/Alaskan Native (1%) race/ethnicity. The majority were never smokers (85%), but 12% were former smokers and 3% were current smokers. Height, weight, and body mass index (BMI) were collected in addition to multiple breath washout (MBW) test parameters such as the lung clearance index (LCI) score.

Neutrophils Fuel Effective Immune Responses through Gluconeogenesis and Glycogenesis.

Neutrophils can function and survive in injured and infected tissues, where oxygen and metabolic substrates are limited. Using radioactive flux assays and LC-MS tracing with U-13C glucose, glutamine, and pyruvate, we observe that neutrophils require the generation of intracellular glycogen stores by gluconeogenesis and glycogenesis for effective survival and bacterial killing. These metabolic adaptations are dynamic, with net increases in glycogen stores observed following LPS challenge or altitude-induced hypoxia. Neutrophils from patients with chronic obstructive pulmonary disease have reduced glycogen cycling, resulting in impaired function. Metabolic specialization of neutrophils may therefore underpin disease pathology and allow selective therapeutic targeting.

Consensus Guidelines for Evaluation and Management of Pulmonary Disease in Sjögren's.

BACKGROUND: Pulmonary disease is a potentially serious yet underdiagnosed complication of Sjögren's syndrome, the second most common autoimmune rheumatic disease. Approximately 16% of patients with Sjögren's demonstrate pulmonary involvement with higher mortality and lower quality of life. RESEARCH QUESTION: Clinical practice guidelines for pulmonary manifestations of Sjögren's were developed by the Sjögren's Foundation after identifying a critical need for early diagnosis and improved quality and consistency of care. STUDY DESIGN AND METHODS: A rigorous and transparent methodology was followed according to American College of Rheumatology guidelines. The Pulmonary Topic Review Group (TRG) developed clinical questions in the PICO (Patient, Intervention, Comparison, Outcome) format and selected literature search parameters. Each article was reviewed by a minimum of two TRG members for eligibility and assessment of quality of evidence and strength of recommendation. Guidelines were then drafted based on available evidence, expert opinion, and clinical importance. Draft recommendations with a clinical rationale and data extraction tables were submitted to a Consensus Expert Panel for consideration and approval, with at least 75% agreement required for individual recommendations to be included in the final version. RESULTS: The literature search revealed 1,192 articles, of which 150 qualified for consideration in guideline development. Of the original 85 PICO questions posed by the TRG, 52 recommendations were generated. These were then reviewed by the Consensus Expert Panel and 52 recommendations were finalized, with a mean agreement of 97.71% (range, 79%-100%). The recommendations span topics of evaluating Sjögren's patients for pulmonary manifestations and assessing, managing, and treating upper and lower airway disease, interstitial lung disease, and lymphoproliferative disease. INTERPRETATION: Clinical practice guidelines for pulmonary manifestations in Sjögren's will improve early identification, evaluation, and uniformity of care by primary care physicians, rheumatologists, and pulmonologists. Additionally, opportunities for future research are identified.

Activation of transcription factor circuity in 2i-induced ground state pluripotency is independent of repressive global epigenetic landscapes.

Mouse embryonic stem cells (mESCs) cultured with MEK/ERK and GSK3ß (2i) inhibitors transition to ground state pluripotency. Gene expression changes, redistribution of histone H3K27me3 profiles and global DNA hypomethylation are hallmarks of 2i exposure, but it is unclear whether epigenetic alterations are required to achieve and maintain ground state or occur as an outcome of 2i signal induced changes. Here we show that ESCs with three epitypes, WT, constitutively methylated, or hypomethylated, all undergo comparable morphological, protein expression and transcriptome changes independently of global alterations of DNA methylation levels or changes in H3K27me3 profiles. Dazl and Fkbp6 expression are induced by 2i in all three epitypes, despite exhibiting hypermethylated promoters in constitutively methylated ESCs. We identify a number of activated gene promoters that undergo 2i dependent loss of H3K27me3 in all three epitypes, however genetic and pharmaceutical inhibition experiments show that H3K27me3 is not required for their silencing in non-2i conditions. By separating and defining their contributions, our data suggest that repressive epigenetic systems play minor roles in mESC self-renewal and naïve ground state establishment by core sets of dominant pluripotency associated transcription factor networks, which operate independently from these epigenetic processes.

Bivalent promoter hypermethylation in cancer is linked to the H327me3/H3K4me3 ratio in embryonic stem cells.

BACKGROUND: Thousands of mammalian promoters are defined by co-enrichment of the histone tail modifications H3K27me3 (repressive) and H3K4me3 (activating) and are thus termed bivalent. It was previously observed that bivalent genes in human ES cells (hESC) are frequent targets for hypermethylation in human cancers, and depletion of DNA methylation in mouse embryonic stem cells has a marked impact on H3K27me3 distribution at bivalent promoters. However, only a fraction of bivalent genes in stem cells are targets of hypermethylation in cancer, and it is currently unclear whether all bivalent promoters are equally sensitive to DNA hypomethylation and whether H3K4me3 levels play a role in the interplay between DNA methylation and H3K27me3. RESULTS: We report the sub-classification of bivalent promoters into two groups-promoters with a high H3K27me3:H3K4me3 (hiBiv) ratio or promoters with a low H3K27me3:H3K4me3 ratio (loBiv). HiBiv are enriched in canonical Polycomb components, show a higher degree of local intrachromosomal contacts and are highly sensitive to DNA hypomethylation in terms of H3K27me3 depletion from broad Polycomb domains. In contrast, loBiv promoters are enriched in non-canonical Polycomb components, show lower intrachromosomal contacts and are less sensitive to DNA hypomethylation at the same genomic resolution. Multiple systems reveal that hiBiv promoters are more depleted of Polycomb complexes than loBiv promoters following a reduction in DNA methylation, and we demonstrate that H3K27me3 re-accumulates at promoters when DNA methylation is restored. In human cancer, we show that hiBiv promoters lose H3K27me3 and are more susceptible to DNA hypermethylation than loBiv promoters. CONCLUSION: We conclude that bivalency as a general term to describe mammalian promoters is an over-simplification and our sub-classification has revealed novel insights into the interplay between the largely antagonistic presence of DNA methylation and Polycomb systems at bivalent promoters. This approach redefines molecular pathologies underlying disease in which global DNA methylation is aberrant or where Polycomb mutations are present.

Respiratory Diseases in Post-9/11 Military Personnel Following Southwest Asia Deployment.

OBJECTIVE: Persistent respiratory symptoms following post-9/11 military deployment to Iraq and Afghanistan are well-recognized, but the spectrum of respiratory diseases remains poorly characterized. This study describes deployment-related respiratory diseases and the diagnostic utility of resting and exercise pulmonary function testing. METHODS: Between 2009 and 2017, 127 consecutive military workers ("deployers") with new-onset respiratory symptoms underwent clinical evaluation. Deployment-related respiratory diseases were classified as proximal and/or distal. Using descriptive statistics and logistic regression, we analyzed lung function parameters associated with deployment-related distal lung disease (DDLD). RESULTS: Common deployment-related respiratory diseases included asthma (31.5%), intermittent laryngeal obstruction (14.2%), rhinosinusitis (15%), and DDLD (68.5%). Decreased diffusion capacity (odds ratio [OR] = 4.6, 95% confidence interval [CI]: 1.4 to 15.1, P = 0.01) was significantly associated with DDLD. CONCLUSIONS: A comprehensive diagnostic approach may identify a spectrum of proximal and distal respiratory diseases that can occur in symptomatic post-9/11 deployers, requiring a personalized approach to care.

Non-alcoholic fatty liver disease (NAFLD) is associated with dynamic changes in DNA hydroxymethylation.

Non-alcoholic fatty liver disease (NAFLD) is now the commonest cause of liver disease in developed countries affecting 25-33% of the general population and up to 75% of those with obesity. Recent data suggest that alterations in DNA methylation may be related to NAFLD pathogenesis and progression and we have previously shown that dynamic changes in the cell lineage identifier 5-hydroxymethylcytosine (5hmC) may be important in the pathogenesis of liver disease. We used a model of diet-induced obesity, maintaining male mice on a high-fat diet (HFD) to generate hepatic steatosis. We profiled hepatic gene expression, global and locus-specific 5hmC and additionally investigated the effects of weight loss on the phenotype. HFD led to increased weight gain, fasting hyperglycaemia, glucose intolerance, insulin resistance and hepatic periportal macrovesicular steatosis. Diet-induced hepatic steatosis associated with reversible 5hmC changes at a discrete number of functionally important genes. We propose that 5hmC profiles are a useful signature of gene transcription and a marker of cell state in NAFLD and suggest that 5hmC profiles hold potential as a biomarker of abnormal liver physiology.

DNA Methylation Directs Polycomb-Dependent 3D Genome Re-organization in Naive Pluripotency.

The DNA hypomethylation that occurs when embryonic stem cells (ESCs) are directed to the ground state of naive pluripotency by culturing in two small molecule inhibitors (2i) results in redistribution of polycomb (H3K27me3) away from its target loci. Here, we demonstrate that 3D genome organization is also altered in 2i, with chromatin decompaction at polycomb target loci and a loss of long-range polycomb interactions. By preventing DNA hypomethylation during the transition to the ground state, we are able to restore to ESC in 2i the H3K27me3 distribution, as well as polycomb-mediated 3D genome organization that is characteristic of primed ESCs grown in serum. However, these cells retain the functional characteristics of 2i ground-state ESCs. Our findings demonstrate the central role of DNA methylation in shaping major aspects of 3D genome organization but caution against assuming causal roles for the epigenome and 3D genome in gene regulation and function in ESCs.

Military Occupational Specialty Codes: Utility in Predicting Inhalation Exposures in Post-9/11 Deployers.

OBJECTIVE: The aim of this study was to examine military occupational specialty (MOS) codes to identify those at risk from inhalation exposures during Southwest Asia deployment. METHODS: Exposure intensity to diesel exhaust, sandstorms, burn pit smoke, combat dust, and occupational vapors/dusts/gases/fumes (VDGF) were scored for all Army/Marine MOS codes by an expert panel. Based on MOS code, panel-rated exposure scores were compared with questionnaire data from military personnel with postdeployment respiratory illnesses. RESULTS: All exposures except VDGF were rated higher (range P < 0.0001 to P = 0.003) for combat versus noncombat MOS codes. Deployers with respiratory illnesses reported more intense exposure to diesel exhaust (P < 0.0001), burn pit smoke (P < 0.0001), and sandstorms (P = 0.005) compared with panel raters. These deployers clustered in MOS codes rated highest for inhalation hazard exposure intensity. CONCLUSIONS: MOS codes are useful in identifying high-risk military occupations where medical surveillance and exposure control should be focused.

Ultrasound measurement of knee synovial fluid during external pneumatic compression.

Synovial fluid based biomarker research has been limited by the small volumes of synovial fluid from the knees of some patients. We used ultrasound (US) to determine if synovial fluid could be displaced into an access port during pneumatic compression to 100 mmHg. Forty knees from 37 consecutive arthritis patients with rheumatoid arthritis -25, osteoarthritis -8, psoriatic arthritis -2, and 1 each with systemic lupus erythematosus and gout were evaluated. This group of 28 females and 9 males with a median age of 59 years and an average body mass index of 26.9 kg/m2 had previously undergone a diagnostic arthrocentesis and or a therapeutic knee injection using this pneumatic compression device. Blinded digital image analysis of the anechoic region on ultrasound demonstrated an increase in fluid within the 9 cm × 6 cm access port (anterolateral or anteromedial joint) during inflation in all patients with a 2.5-3.5 fold increase in fluid area and a 2-3 fold increase in fluid depth after inflation, p < 0.001. Statement of clinical significance: External pneumatic compression to the knee provides a larger volume of synovial fluid under positive pressure which should allow investigators to achieve greater success in obtaining synovial fluid during arthrocentesis for biomarker research or provide more precise therapeutic injections than traditional non image-guided anatomical landmark-based techniques. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

A Cell/Cilia Cycle Biosensor for Single-Cell Kinetics Reveals Persistence of Cilia after G1/S Transition Is a General Property in Cells and Mice.

The cilia and cell cycles are inextricably linked. Centrioles in the basal body of cilia nucleate the ciliary axoneme and sequester pericentriolar matrix (PCM) at the centrosome to organize the mitotic spindle. Cilia themselves respond to growth signals, prompting cilia resorption and cell cycle re-entry. We describe a fluorescent cilia and cell cycle biosensor allowing live imaging of cell cycle progression and cilia assembly and disassembly kinetics in cells and inducible mice. We define assembly and disassembly in relation to cell cycle stage with single-cell resolution and explore the intercellular heterogeneity in cilia kinetics. In all cells and tissues analyzed, we observed cilia that persist through the G1/S transition and into S/G2/M-phase. We conclude that persistence of cilia after the G1/S transition is a general property. This resource will shed light at an individual cell level on the interplay between the cilia and cell cycles in development, regeneration, and disease.

A reassessment of DNA-immunoprecipitation-based genomic profiling.

DNA immunoprecipitation followed by sequencing (DIP-seq) is a common enrichment method for profiling DNA modifications in mammalian genomes. However, the results of independent DIP-seq studies often show considerable variation between profiles of the same genome and between profiles obtained by alternative methods. Here we show that these differences are primarily due to the intrinsic affinity of IgG for short unmodified DNA repeats. This pervasive experimental error accounts for 50-99% of regions identified as 'enriched' for DNA modifications in DIP-seq data. Correction of this error profoundly altered DNA-modification profiles for numerous cell types, including mouse embryonic stem cells, and subsequently revealed novel associations among DNA modifications, chromatin modifications and biological processes. We conclude that both matched input and IgG controls are essential in order for the results of DIP-based assays to be interpreted correctly, and that complementary, non-antibody-based techniques should be used to validate DIP-based findings to avoid further misinterpretation of genome-wide profiling data.

Modelling non-alcoholic fatty liver disease in human hepatocyte-like cells.

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of liver disease in developed countries. An in vitro NAFLD model would permit mechanistic studies and enable high-throughput therapeutic screening. While hepatic cancer-derived cell lines are a convenient, renewable resource, their genomic, epigenomic and functional alterations mean their utility in NAFLD modelling is unclear. Additionally, the epigenetic mark 5-hydroxymethylcytosine (5hmC), a cell lineage identifier, is rapidly lost during cell culture, alongside expression of the Ten-eleven-translocation (TET) methylcytosine dioxygenase enzymes, restricting meaningful epigenetic analysis. Hepatocyte-like cells (HLCs) derived from human embryonic stem cells can provide a non-neoplastic, renewable model for liver research. Here, we have developed a model of NAFLD using HLCs exposed to lactate, pyruvate and octanoic acid (LPO) that bear all the hallmarks, including 5hmC profiles, of liver functionality. We exposed HLCs to LPO for 48 h to induce lipid accumulation. We characterized the transcriptome using RNA-seq, the metabolome using ultra-performance liquid chromatography-mass spectrometry and the epigenome using 5-hydroxymethylation DNA immunoprecipitation (hmeDIP) sequencing. LPO exposure induced an NAFLD phenotype in HLCs with transcriptional and metabolomic dysregulation consistent with those present in human NAFLD. HLCs maintain expression of the TET enzymes and have a liver-like epigenome. LPO exposure-induced 5hmC enrichment at lipid synthesis and transport genes. HLCs treated with LPO recapitulate the transcriptional and metabolic dysregulation seen in NAFLD and additionally retain TET expression and 5hmC. This in vitro model of NAFLD will be useful for future mechanistic and therapeutic studies.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.