IntroductionMicroRNAs (miRNAs), endogenous small noncoding RNAs regulating the activities of target mRNAs and cellular processes, are present in human plasma in a stable form. In this study, we investigated whether miRNAs are also stably present in synovial fluids and whether plasma and synovial fluid miRNAs could be biomarkers of rheumatoid arthritis (RA) and osteoarthritis (OA).MethodsWe measured concentrations of miR-16, miR-132, miR-146a, miR-155 and miR-223 in synovial fluid from patients with RA and OA, and those in plasma from RA, OA and healthy controls (HCs) by quantitative reverse transcription-polymerase chain reaction. Furthermore, miRNAs in the conditioned medium of synovial tissues, monolayer fibroblast-like synoviocytes, and mononuclear cells were examined. Correlations between miRNAs and biomarkers or disease activities of RA were statistically examined.ResultsSynovial fluid miRNAs were present and as stable as plasma miRNAs for storage at -20°C and freeze-thawing from -20°C to 4°C. In RA and OA, synovial fluid concentrations of miR-16, miR-132, miR-146a, and miR-223 were significantly lower than their plasma concentrations, and there were no correlation between plasma and synovial fluid miRNAs. Interestingly, synovial tissues, fibroblast-like synoviocytes, and mononuclear cells secreted miRNAs in distinct patterns. The expression patterns of miRNAs in synovial fluid of OA were similar to miRNAs secreted by synovial tissues. Synovial fluid miRNAs of RA were likely to originate from synovial tissues and infiltrating cells. Plasma miR-132 of HC was significantly higher than that of RA or OA with high diagnosability. Synovial fluid concentrations of miR-16, miR-146a miR-155 and miR-223 of RA were significantly higher than those of OA. Plasma miRNAs or ratio of synovial fluid miRNAs to plasma miRNAs, including miR-16 and miR-146a, significantly correlated with tender joint counts and 28-joint Disease Activity Score.ConclusionsPlasma miRNAs had distinct patterns from synovial fluid miRNAs, which appeared to originate from synovial tissue. Plasma miR-132 well differentiated HCs from patients with RA or OA, while synovial fluid miRNAs differentiated RA and OA. Furthermore, plasma miRNAs correlated with the disease activities of RA. Thus, synovial fluid and plasma miRNAs have potential as diagnostic biomarkers for RA and OA and as a tool for the analysis of their pathogenesis.

While it is well known that Staphylococcus aureus establishes chronic implant-associated osteomyelitis by generating and persisting in biofilm, research to elucidate pathogen and host specific factors controlling this process has been limited due to the absence of a quantitative in vivo model. To address this, we developed a murine tibia implant model with ex vivo region of interest (ROI) imaging analysis by scanning electron microscopy (SEM). Implants were coated with Staphylococcus aureus strains (SH1000, UAMS-1, USA300LAC) with distinct in vitro biofilm phenotypes, were used to infect C57BL/6 or Balb/c mice. In contrast to their in vitro biofilm phenotype, results from all bacteria strains in vivo were similar, and demonstrated that biofilm on the implant is established within the first day, followed by a robust proliferation phase peaking on Day 3 in Balb/c mice, and persisting until Day 7 in C57BL/6 mice, as detected by SEM and bioluminescent imaging. Biofilm formation peaked at Day 14, covering ~40% of the ROI coincident with massive agr-dependent bacterial emigration, as evidenced by large numbers of empty lacunae with few residual bacteria, which were largely culture negative (80%) and PCR positive (87.5%), supporting the clinical relevance of this implant model.

Osteomyelitis is a devastating disease caused by microbial infection of bone. While the frequency of infection following elective orthopedic surgery is low, rates of reinfection are disturbingly high. Staphylococcus aureus is responsible for the majority of chronic osteomyelitis cases and is often considered to be incurable due to bacterial persistence deep within bone. Unfortunately, there is no consensus on clinical classifications of osteomyelitis and the ensuing treatment algorithm. Given the high patient morbidity, mortality, and economic burden caused by osteomyelitis, it is important to elucidate mechanisms of bone infection to inform novel strategies for prevention and curative treatment. Recent discoveries in this field have identified three distinct reservoirs of bacterial biofilm including: Staphylococcal abscess communities in the local soft tissue and bone marrow, glycocalyx formation on implant hardware and necrotic tissue, and colonization of the osteocyte-lacuno canalicular network (OLCN) of cortical bone. In contrast, S. aureus intracellular persistence in bone cells has not been substantiated in vivo, which challenges this mode of chronic osteomyelitis. There have also been major advances in our understanding of the immune proteome against S. aureus, from clinical studies of serum antibodies and media enriched for newly synthesized antibodies (MENSA), which may provide new opportunities for osteomyelitis diagnosis, prognosis, and vaccine development. Finally, novel therapies such as antimicrobial implant coatings and antibiotic impregnated 3D-printed scaffolds represent promising strategies for preventing and managing this devastating disease. Here, we review these recent advances and highlight translational opportunities towards a cure.

While Staphylococcus aureus osteomyelitis is considered to be incurable, the major bacterial reservoir in live cortical bone has remained unknown. In addition to biofilm bacteria on necrotic tissue and implants, studies have implicated intracellular infection of osteoblasts and osteocytes as a mechanism of chronic osteomyelitis. Thus, we performed the first systematic transmission electron microscopy (TEM) studies to formally define major reservoirs of S. aureus in chronically infected mouse (Balb/c J) long bone tissue. Although rare, evidence of colonized osteoblasts was found. In contrast, we readily observed S. aureus within canaliculi of live cortical bone, which existed as chains of individual cocci and submicron rod-shaped bacteria leading to biofilm formation in osteocyte lacunae. As these observations do not conform to the expectations of S. aureus as non-motile cocci 1.0–1.5 µm in diameter, we also performed immunoelectron microscopy (IEM) following in vivo BrdU labeling to assess the role of bacterial proliferation in canalicular invasion. The results suggest that the deformed bacteria: 1) enter canaliculi via asymmetric binary fission; and 2) migrate toward osteocyte lacunae via proliferation at the leading edge. Additional in vitro studies confirmed S. aureus migration through a 0.5 µm porous membrane. Collectively, these findings define a novel mechanism of bone infection, and provide possible new insight as to why S. aureus implant related infections of bone tissue are so challenging to treat.

Towards development of a methicillin-resistant S. aureus (MRSA) vaccine we evaluated a neutralizing anti-glucosaminidase (Gmd) monoclonal antibody (1C11) in a murine model of implant-associated osteomyelitis, and compared its effects on LAC USA300 MRSA versus placebo (alpha-T2m) and a Gmd-deficient isogenic strain (delta-Gmd). 1C11 significantly reduced infection severity, as determined by bioluminescent imaging of bacteria, micro-CT assessment of osteolysis and histomorphometry of abscess numbers (p<0.05). Histology also revealed infiltrating macrophages, and the complete lack of staphylococcal abscess communities (SAC), in marrow abscesses of 1C11 treated mice. In vitro, 1C11 had no direct effects on proliferation, but electron microscopy demonstrated that 1C11 treatment phenocopies delta-Gmd defects in binary fission. Moreover, addition of 1C11 to MRSA cultures induced the formation of large bacterial aggregates (megaclusters) that sedimented out of solution, which was not observed in delta-Gmd cultures or 1C11 treated cultures of a protein A-deficient strain (delta-Spa), suggesting that the combined effects of Gmd inhibition and antibody-mediated agglutination are required. Finally, we demonstrated that macrophage opsonophagocytosis of MRSA and megaclusters is significantly increased by 1C11 (p<0.01). Collectively, these results suggest that the primary mechanism of anti-Gmd humoral immunity against MRSA osteomyelitis is macrophage invasion of SAC and opsonophagocytosis of megaclusters.

In human inflammatory sites, PD-1hiCXCR5−CD4+ T cells are involved in the formation of ectopic lymphoid-like structures (ELSs) by the secretion of chemokine CXCL13, but how the transcription of CXCL13 is regulated in CD4+ T cells is still unclear. Here we show that Sox4 is a key transcription factor for CXCL13 production in human CD4+ T cells under inflammatory conditions. In vitro TGF-β+, IL-2-neutralizing culture conditions give rise to PD-1hiCXCR5−CD4+ T cells that preferentially express CXCL13, and transcriptome analysis and lentiviral overexpression indicate Sox4 association with the CXCL13 transcription. In vivo, Sox4 is significantly upregulated in synovial CD4+ T cells, when compared with blood CD4+ T cells, from patients with rheumatoid arthritis (RA), and further correlates with ELS formation in RA synovium. Overall, our studies suggest that Sox4 contributes to CXCL13 production and ELS formation at inflammatory sites in humans.

Antibiotic‐loaded bone cement (ALBC) is broadly used to treat orthopaedic infections based on the rationale that high‐dose local delivery is essential to eradicate biofilm‐associated bacteria. However, ALBC formulations are empirically based on drug susceptibility from routine laboratory testing, which is known to have limited clinical relevance for biofilms. There are also dosing concerns with nonstandardized, surgeon‐directed, hand‐mixed formulations, which have unknown release kinetics. On the basis of our knowledge of in vivo biofilms, pathogen virulence, safety issues with nonstandardized ALBC formulations, and questions about the cost‐effectiveness of ALBC, there is a need to evaluate the evidence for this clinical practice. To this end, thought leaders in the field of musculoskeletal infection (MSKI) met on 1 August 2019 to review and debate published and anecdotal information, which highlighted four major concerns about current ALBC use: (a) substantial lack of level 1 evidence to demonstrate efficacy; (b) ALBC formulations become subtherapeutic following early release, which risks induction of antibiotic resistance, and exacerbated infection from microbial colonization of the carrier; (c) the absence of standardized formulation protocols, and Food and Drug Administration‐approved high‐dose ALBC products to use following resection in MSKI treatment; and (d) absence of a validated assay to determine the minimum biofilm eradication concentration to predict ALBC efficacy against patient specific micro‐organisms. Here, we describe these concerns in detail, and propose areas in need of research.

Background Because immunity against Staphylococcus aureus has not been fully elucidated, there is no diagnostic test to gauge how robust a patient's host response is likely to be. Therefore, we aimed to develop a test for specific antibodies in serum with diagnostic and prognostic potential. Questions/Purposes We describe the development and validation of a multiplex immunoassay for characterizing a patient's immune response against 14 known S aureus antigens, which we then used to answer four questions: (1) Do certain antigens predominate in the immune response against S aureus? (2) Is there a predominant pattern of antigens recognized by patients and mice with infections? (3) Is the immunoglobulin G (IgG) response to any single antigen a useful predictor of ongoing S aureus infection? (4) Does measurement of the combined response against all 14 antigens provide a better predictor of ongoing infection? Methods A case-control study was performed. Sera were collected from 35 consecutive patients with S aureus culture-confirmed (methicillin-sensitive S aureus or methicillin-resistant S aureus) musculoskeletal infections The institution of one or more of the authors (EMS, SLK, JLD, CAB) has received, during the study period, funding from the National Institutes of Health, NIAMS (Bethesda, MD, USA) and (EMS, SLK, JLD) the AOTrauma Clinical Priority Program (Davos, Switzerland). One of the authors certifies that he (JLD) or she or a member of his or her immediate family, has received or may receive payments or benefits, during the study period, an amount of USD 10,000 to 100,000 from Telephus Medical LLC (San Diego, CA, USA) and an amount of less than USD 10,000 from Calorics Pharmaceuticals (Waltham, MA, USA). One of the authors certifies that he (EMS) or she or a member of his or her immediate family, has received or may receive payments or benefits, during the study period, an amount of USD 10,000 to 100,000 from Telephus Medical LLC (San Diego, CA, USA). One of the authors certifies that he (SLK) or she or a member of his or her immediate family, has received or may receive payments or benefits, during the study period, an amount of less than USD 10,000 from Surgical Excellence Healthcare Quality Consulting, and an amount of USD 10,000 to 100,000 from Sage Publications (Thousand Oaks, CA, USA) One of the authors certifies that he (CAB) or she or a member of his or her immediate family, has received or may receive payments or benefits, during the study period, an amount of USD 10,000 to 100,000 from the FDA (Silver Spring, MD, USA), and an amount of USD 10,000 to 100,000 from Boston Scientific (Marlborough, MA, USA), an amount of USD 10,000 to 100,000 from Lundbeck Inc (Deerfield, IL, USA), an amount of USD 10,000 to 100,000 from Patient-Centered Outcomes Research Institute (PCORI, Washington, DC, USA), and an amount of less than USD 10,000 from Auspex Pharmaceuticals (La Jolla, CA, USA). All ICMJE Conflict of Interest Forms for authors and Clinical Orthopaedics and Related Research 1 editors and board mem...