Extracellular aaRSs drive autoimmune and inflammatory responses in rheumatoid arthritis via the release of cytokines and PAD4.

OBJECTIVES: Recent studies demonstrate that extracellular-released aminoacyl-tRNA synthetases (aaRSs) play unique roles in immune responses and diseases. This study aimed to understand the role of extracellular aaRSs in the pathogenesis of rheumatoid arthritis (RA). METHODS: Primary macrophages and fibroblast-like synoviocytes were cultured with aaRSs. aaRS-induced cytokine production including IL-6 and TNF-α was detected by ELISA. Transcriptomic features of aaRS-stimulated macrophages were examined using RNA-sequencing. Serum and synovial fluid (SF) aaRS levels in patients with RA were assessed using ELISA. Peptidyl arginine deiminase (PAD) 4 release from macrophages stimulated with aaRSs was detected by ELISA. Citrullination of aaRSs by themselves was examined by immunoprecipitation and western blotting. Furthermore, aaRS inhibitory peptides were used for inhibition of arthritis in two mouse RA models, collagen-induced arthritis and collagen antibody-induced arthritis. RESULTS: All 20 aaRSs functioned as alarmin; they induced pro-inflammatory cytokines through the CD14-MD2-TLR4 axis. Stimulation of macrophages with aaRSs displayed persistent innate inflammatory responses. Serum and SF levels of many aaRSs increased in patients with RA compared with control subjects. Furthermore, aaRSs released PAD4 from living macrophages, leading to their citrullination. We demonstrate that aaRS inhibitory peptides suppress cytokine production and PAD4 release by aaRSs and alleviate arthritic symptoms in a mouse RA model. CONCLUSIONS: Our findings uncovered the significant role of aaRSs as a novel alarmin in RA pathogenesis, indicating that their blocking agents are potent antirheumatic drugs.

Risk factors for excessive postoperative sliding of femoral trochanteric fracture in elderly patients: A retrospective multicenter study.

INTRODUCTION: The application of a load on the internal fixation of a trochanteric fracture exerts a moment along the lag screw, causing the proximal bone fragment to slide along the lag screw, allowing contact between the proximal and distal bone fragments, which promotes healing. However, excessive sliding is related to poor postoperative outcomes. We aimed to identify the risk factors for excessive sliding. MATERIALS AND METHODS: We conducted a multicenter retrospective study including 115 trochanteric fractures sustained through low-energy trauma in 19 male and 96 female patients aged 60 years or older (mean age: 82.9 years) between September 2013 and December 2014. We measured the postoperative sliding distance after osteosynthesis using a sliding hip screw or intramedullary nailing, and classified participants with ≥8 mm of sliding into the excessive sliding group (ESG) and with <8 mm into non-ESG. Finally, we investigated the risk factors of excessive postoperative sliding. RESULTS: Fifty participants were classified into the ESG and 65 participants into the non-ESG. Female sex (p = 0.0264), an A3 fracture type (p = 0.0003), greater tip-apex distance (p = 0.0250), and poor reduction in either the anteroposterior or lateral radiographic views (p = 0.0156) were identified as risk factors for excessive sliding by multivariate regression analysis. CONCLUSIONS: Female sex, an unstable fracture type, a greater tip-apex distance, and a poor reduction, in either the anteroposterior or lateral views, are associated with excessive postoperative sliding. Therefore, surgery should aim to achieve good reduction and stabilization from both radiographic views.

The fibroblast: An emerging key player in thymic T cell selection.

Fibroblasts have recently attracted attention as a key stromal component that controls the immune responses in lymphoid tissues. The thymus has a unique microenvironment comprised of a variety of stromal cells, including fibroblasts and thymic epithelial cells (TECs), the latter of which is known to be important for T cell development because of their ability to express self-antigens. Thymic fibroblasts contribute to thymus organogenesis during embryogenesis and form the capsule and medullary reticular network in the adult thymus. However, the immunological significance of thymic fibroblasts has thus far only been poorly elucidated. In this review, we will summarize the current views on the development and functions of thymic fibroblasts as revealed by new technologies such as multicolor flow cytometry and single cell-based transcriptome profiling. Furthermore, the recently discovered role of medullary fibroblasts in the establishment of T cell tolerance by producing a unique set of self-antigens will be highlighted.

Efficient ssODN-Mediated Targeting by Avoiding Cellular Inhibitory RNAs through Precomplexed CRISPR-Cas9/sgRNA Ribonucleoprotein.

Combined with CRISPR-Cas9 technology and single-stranded oligodeoxynucleotides (ssODNs), specific single-nucleotide alterations can be introduced into a targeted genomic locus in induced pluripotent stem cells (iPSCs); however, ssODN knockin frequency is low compared with deletion induction. Although several Cas9 transduction methods have been reported, the biochemical behavior of CRISPR-Cas9 nuclease in mammalian cells is yet to be explored. Here, we investigated intrinsic cellular factors that affect Cas9 cleavage activity in vitro. We found that intracellular RNA, but not DNA or protein fractions, inhibits Cas9 from binding to single guide RNA (sgRNA) and reduces the enzymatic activity. To prevent this, precomplexing Cas9 and sgRNA before delivery into cells can lead to higher genome editing activity compared with Cas9 overexpression approaches. By optimizing electroporation parameters of precomplexed ribonucleoprotein and ssODN, we achieved efficiencies of single-nucleotide correction as high as 70% and loxP insertion up to 40%. Finally, we could replace the HLA-C1 allele with the C2 allele to generate histocompatibility leukocyte antigen custom-edited iPSCs.

Refracture of pediatric both-bone diaphyseal forearm fracture following intramedullary fixation with Kirschner wires is likely to occur in the presence of immature radiographic healing.

PURPOSE: Refracture of pediatric both-bone diaphyseal forearm fractures (PBDFFs) may occur, even if the fractures are treated with intramedullary nailing. The purpose of this study was to investigate the risk of refracture of PBDFFs treated with intramedullary Kirschner wires (K-wires), which are commonly used in our clinic. METHODS: The present multicenter retrospective study included 60 consecutive patients with 60 PBDFFs who were treated with intramedullary K-wires at 5 hospitals between 2007 and 2016. The age of the patients at the time of the primary fracture ranged from 2 to 15 years. The characteristics of the primary fractures and treatment course were evaluated. RESULTS: Refracture occurred in 6 patients (10.0%). Three of the patients were young girls; the other 3 were adolescent boys. Refractures were caused by falling or during sports activity. The duration from primary fracture to refracture ranged from 46 to 277 days, and in 5 of the 6 patients refractures occurred within 6 months. Although we were unable to identify factors significantly contributing to refracture (e.g. fracture type or treatment procedures), radiographs at the latest visit before refracture demonstrated findings of immature healing in five of six patients. Both K-wires and external immobilization had been removed before complete fracture healing in a large proportion of patients with refracture (80.0%). CONCLUSIONS: Refracture of PBDFF may occur several months after treatment with intramedullary K-wires if the primary fracture shows immature healing. Physicians should pay special attention when judging radiographic fracture healing, even when the fracture is deemed to have clinically healed.

Assessment of baseline bone turnover marker levels and response to risedronate treatment: Data from a Japanese phase III trial.

BACKGROUND: Risedronate increases bone mineral density (BMD) and reduces fracture risk, but treatment response may depend on the baseline state of bone turnover. Data regarding the selection of therapeutic drugs or the prediction of therapeutic effects with baseline levels of bone turnover markers (BTMs) as a reference are insufficient. We hypothesized that when the baseline levels of BTMs are higher, baseline BMD might be lower, changes in BMD at 12 months after risedronate treatment might be higher, and the reduction of fracture incidence might be greater. This study aimed to analyze the data of a phase III clinical trial of risedronate from Japan to investigate the relationships between baseline BTM levels and (1) baseline BMD, (2) changes in BMD at 12 months after the start of treatment, and (3) the incidence of new vertebral fractures. METHODS: This post-hoc analysis included 788 postmenopausal women with osteoporosis whose baseline BTM levels as well as baseline and endpoint BMDs were measured. Relationships between baseline BTM levels and BMD at baseline and 12 months after risedronate treatment and new vertebral fractures were examined. One-way analysis of variance, two-tailed Student's t-test, and Fisher's exact test were used to analyze the data. RESULTS: Baseline BMD showed a significant upward trend when baseline BTM levels were lower in the analysis by tertiles. New vertebral fractures tended to occur in patients with prevalent vertebral fractures, but the relationship between new fractures and BTM levels was not statistically significant. Regardless of BTM types, BMD percentage increments (%) and increments (g/cm2) with the 12-month treatment were high when pretreatment BTM levels were high (P < 0.0001), and a >5.0% increase in BMD was observed even if baseline BTM levels were within the normal range. A new vertebral fracture occurred in only six patients (0.77%), and there was not enough statistical power to clarify the relationship between baseline BTM levels and fracture risk reduction. CONCLUSIONS: When pretreatment BTM levels increased, baseline BMD tended to be lower and the increase in BMD with 12-month risedronate treatment was higher. However, BMD could still be increased even if the baseline BTM levels are within the normal range. Combined with available evidence, baseline BTMs may not have an important role in deciding the optimal therapy. To elucidate the relationship between baseline BTM levels and long-term fracture risk, it will be necessary to conduct more large-scale studies with a longer follow-up period in severe osteoporotic patients with a high fracture risk. MINI ABSTRACT: We evaluated the significance of baseline bone turnover markers in the response to risedronate treatment. The increase in the bone mineral density (BMD) with the 12-month treatment may be higher when the state of bone turnover at baseline is higher, and BMD could still be increased even if the baseline bone turnover is within the normal range.

Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping.

Prolonged expression of the CRISPR-Cas9 nuclease and gRNA from viral vectors may cause off-target mutagenesis and immunogenicity. Thus, a transient delivery system is needed for therapeutic genome editing applications. Here, we develop an extracellular nanovesicle-based ribonucleoprotein delivery system named NanoMEDIC by utilizing two distinct homing mechanisms. Chemical induced dimerization recruits Cas9 protein into extracellular nanovesicles, and then a viral RNA packaging signal and two self-cleaving riboswitches tether and release sgRNA into nanovesicles. We demonstrate efficient genome editing in various hard-to-transfect cell types, including human induced pluripotent stem (iPS) cells, neurons, and myoblasts. NanoMEDIC also achieves over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy (DMD) patient iPS cells. Finally, single intramuscular injection of NanoMEDIC induces permanent genomic exon skipping in a luciferase reporter mouse and in mdx mice, indicating its utility for in vivo genome editing therapy of DMD and beyond.

Spontaneous bilateral femoral neck fractures in a young male adult: a case report and literature review.

BACKGROUND: Simultaneous bilateral femoral neck fracture is a very rare condition, even in osteoporotic elderly individuals. We report an atypical case of a young male adult who developed simultaneous bilateral femoral neck fractures without previous trauma or overuse. CASE PRESENTATION: A 33-year-old man presented with discomfort in the bilateral groin, which had started 2 weeks previously. Bilateral femoral neck fractures were observed on a radiograph, and in addition, a fracture line was seen at the right subchondral region of the acetabulum using magnetic resonance imaging (MRI). Although the patient had no obvious risk factors associated with bone fragility, his bone mineral density measured using dual X-ray absorptiometry indicated severe osteoporosis (lumber spine: T score - 3.4 standard deviation [SD]; femoral neck: T score - 2.8 SD). Serum 25-hydroxyvitamin D level was deficient (19 ng/mL), which was considered to be partly due to non-sunlight exposure for 3 years owing to social withdrawal. Bilateral osteosynthesis was performed, considering his young age, although more than 2 weeks had passed since the onset of the fracture. Bone union and non-occurrence of osteonecrosis of the femoral head were confirmed via radiography and MRI 8 months after the surgery. CONCLUSIONS: Our case suggests that simultaneous non-traumatic bilateral femoral neck fractures can occur in healthy young men.

The depth of the low-intensity band on the T1-weighted MR image is useful for distinguishing subchondral insufficiency fracture from osteonecrosis of the collapsed femoral head.

PURPOSE: To verify the hypothesis that depth of the low-intensity band on T1-weighted MR image is useful for distinguishing subchondral insufficiency fracture (SIF) from osteonecrosis of the collapsed femoral head (ON). PATIENTS AND METHODS: We reviewed 35 consecutive hips in 35 patients with radiological evidence of subchondral collapse of the femoral head and available MR images and histology between May 2013 and January 2016. Both clinical and radiological appearances were investigated. The ratios of distance from articular surface of the femoral head to the T1 low-intensity band to femoral head diameter (band depth ratio: BDR) on (1) mid-coronal slice of MR images and that on (2) coronal slice of MR images in which the highest BDR was observed, were calculated. RESULTS: The mean age in SIF group was significantly higher than that in ON group (SIF: 68 years, ON: 49 years, P = 0.0017). The rates of history of steroid intake or alcohol consumption in SIF group were significantly lower than those in ON group (P = 0.0022 and P = 0.0408, respectively). The mean BDRs in SIF group were (1) 0.16 and (2) 0.23, which were significantly lower than those in ON group [(1) 0.42 and (2) 0.59] (P < 0.0001 for both). The cut-off BDR values to differentiate SIF from ON were (1) 0.22 and (2) 0.38, respectively. CONCLUSION: The results of the study suggest that depth of the low-intensity band on T1-weighted MR image is useful for distinguishing SIF from ON in cases with collapsed femoral heads.

Clinical outcomes in relation to locations of bone marrow edema lesions in patients with a subchondral insufficiency fracture of the hip: a review of fifteen cases.

OBJECTIVE: The prognosis of patients with a subchondral insufficiency fracture remains unclear. The purpose of this study was to investigate the correlation between locations of bone marrow edema (BME) lesions and clinical outcome in patients with a subchondral insufficiency fracture of the hip. METHODS: We retrospectively reviewed 15 consecutive hips in 14 patients who were diagnosed with subchondral insufficiency fracture of the hip at our institution between April 2013 and September 2014. This study included five males (six hips) and nine females (nine hips), ranging from 36 to 83 years of age (mean age: 66 years). The mean duration from the onset of hip pain to MRI examination was 1.8 months (range 0.5-5 months). Both clinical and imaging findings were investigated. RESULTS: Based on the findings of MR images, BME lesion in the femoral head alone was observed in six patients (six hips), BME lesion in the acetabulum alone was observed in one patient (two hips) and BME lesions in both the femoral head and acetabulum were observed in seven patients (seven hips). 3 of 15 hips resulted in rapidly destructive arthrosis and their BME lesions were observed in both the femoral head and acetabulum. 8 of 15 hips successfully healed by conservative treatment and BME lesions in 7 of these 8 hips were observed in only the femoral head or acetabulum. CONCLUSION: The results of this study indicate that the locations of BME lesions (femoral side alone, acetabular side alone or both) may be related to the clinical outcome in patients with a subchondral insufficiency fracture of the hip. ADVANCES IN KNOWLEDGE: Patients with subchondral insufficiency fracture of the hip in whom BME lesions were observed in both the femoral head and acetabulum may have a higher risk to need to undergo total hip arthroplasty.

Combined adjuvants of poly(I:C) plus LAG-3-Ig improve antitumor effects of tumor-specific T cells, preventing their exhaustion.

Therapeutic cancer vaccines are designed to treat cancer by boosting the endogenous immune system to fight against the cancer. In the development of clinically effective cancer vaccines, one of the most practical objectives is to identify adjuvants that are capable of optimizing the vaccine effects. In this study, we explored the potential of polyinosinic-polycytidylic acid (poly(I:C)) and LAG-3-Ig (soluble recombinant protein of lymphocyte activation gene-3 [LAG-3] extracellular domain fused with human IgG Fc region) as adjuvants for P1A tumor antigen peptide vaccine in a pre-established P815 mouse tumor model with a transfer of tumor-specific T cells. Whereas the use of poly(I:C) or LAG-3-Ig as a signal adjuvant induced a slight enhancement of P1A vaccine effects compared to incomplete Freund's adjuvant, combined treatment with poly(I:C) plus LAG-3-Ig remarkably potentiated antitumor effects, leading to complete rejection of pre-established tumor and long-term survival of mice. The potent adjuvant effects of poly(I:C) plus LAG-3-Ig were associated with an enhanced infiltration of T cells in the tumor tissues, and an increased proliferation and Th1-type cytokine production of tumor-reactive T cells. Importantly, the combined adjuvant of poly(I:C) plus LAG-3-Ig downregulated expressions of PD-1, LAG-3, and TIGIT on P1A-specific T cells, indicating prevention of T cell exhaustion. Taken together, the results of the current study show that the combined adjuvants of poly(I:C) plus LAG-3-Ig with tumor peptide vaccine induce profound antitumor effects by activating tumor-specific T cells.

Brown tumor of the patella caused by primary hyperparathyroidism: a case report.

It has been reported that the common sites of brown tumors are the jaw, pelvis, ribs, femurs and clavicles. We report our experience in a case of brown tumor of the patella caused by primary hyperparathyroidism. An initial radiograph and CT showed an osteolytic lesion and MR images showed a mixed solid and multiloculated cystic tumor in the right patella. One month after the parathyroidectomy, rapid bone formation was observed on both radiographs and CT images.