Yasushi Naganuma

Isocitrate dehydrogenase 2 mutation is a frequent event in osteosarcoma detected by a multi-specific monoclonal antibody MsMab-1.

Somatic mutations of isocitrate dehydrogenase (IDH) 1 and IDH2 occur in gliomas, acute myeloid leukemia, and cartilaginous tumors. Somatic mosaic IDH1/2 mutations are also reported in Ollier disease and Maffucci syndrome, which are characterized by multiple central cartilaginous tumors. Although IDH1/2 mutation analysis against osteosarcoma has been performed in several studies, no IDH1/2 mutation has been reported. Herein, we newly report the IDH2‐R172S mutation in three of 12 (25%) osteosarcoma patients, which was detected by direct DNA sequencing. No monoclonal antibody (mAb) has been reported against IDH2‐R172S mutation. However, we demonstrate that the IDH2‐R172S peptide was recognized by our established multi‐specific anti‐mutated IDH1/2 mAb, MsMab‐1, in enzyme‐linked immunosorbent assay. Western blot analysis revealed that MsMab‐1 reacts with PA tag combined recombinant proteins of IDH2‐R172S. Furthermore, MsMab‐1 stained IDH2‐R172S‐expressing osteosarcoma tissues in immunohistochemistry. The MsMab‐1 stained nine of 32 (28.1%) osteosarcomas in a tissue microarray. This report is the first describing IDH2 mutations in osteosarcoma, which can be detected by MsMab‐1 mAb. Taken together, these results show that MsMab‐1 can be anticipated for use in immunohistochemical determination of IDH1/2 mutation‐bearing osteosarcoma.

Lipoteichoic acid modulates inflammatory response in macrophages after phagocytosis of titanium particles through Toll-like receptor 2 cascade and inflammasomes

Toll-like receptor 2 (TLR2) and nucleotide-binding and oligomerization domain-like receptors with a pyrin domain 3 (NLRP3) inflammasomes have been presumed to participate in the pathogenesis of aseptic implant loosening. The aim of this study is to analyze the cellular localization of TLR2 and NLRP3 inflammasomes in the periprosthetic tissue from aseptically loose hip implants as well as the expression of these molecules in macrophages stimulated in vitro with titanium particles (Ti) coated with lipoteichoic acid (LTA). Using immunohistochemistry, immunoreactivity of TLR2 and NLRP3 inflammasomes was found in macrophages within the foreign body granulomatosis. Using RAW264.7 cells, stimulation with Ti increased the messenger RNA (mRNA) levels of TLR2 and TNF-α. Stimulation with LTA-coated Ti enhanced mRNA levels of NLRP3 and IL-1β, whereas reinforced secretion of IL-1β was not detected in spite of marked release of TNF-α. Finally, the same cells with silenced Irak2, an adaptor protein in the TLR2 cascade, suppressed this NLRP3 upregulation. This study suggests that TLR2 and NLRP3 inflammasomes are factors involved in cross-talk mediating the foreign body type response to wear particles. In addition, discrepant behavior in the release between TNF-α and IL-1β release may explain the variable pathomechanisms of aseptic implant loosening without acute inflammatory reactions.

Complications of Distal Radius Fractures Treated by Volar Locking Plate Fixation

The current study investigated the incidence of complications after surgery for distal radial fractures. This multicenter retrospective study was conducted at 11 institutions. A total of 824 patients who had distal radius fractures that were treated surgically between January 2010 and August 2012 were identified. The study patients were older than 18 years and were observed for at least 12 weeks after surgery for distal radius fractures with a volar locking plate. Sex, age, fracture type according to AO classification, implants, wrist range of motion, grip strength, fracture consolidation rate, and complications were studied. Analysis included 694 patients, including 529 women and 165 men, with a mean age of 64 years. The mean follow-up period was 27 weeks. The fracture consolidation rate was 100%. There were 52 complications (7.5%), including 18 cases of carpal tunnel syndrome, 12 cases of peripheral nerve palsy, 8 cases of trigger digit, 4 cases of tendon rupture (none of the flexor pollicis longus), and 10 others. There was no rupture of the flexor pollicis longus tendon because careful attention was paid to the relationship between the implant and the tendon. Peripheral nerve palsy may have been caused by intraoperative traction in 7 cases, temporary fixation by percutaneous Kirschner wires in 3 cases, and axillary nerve block in 1 case; 1 case appeared to be idiopathic. Tendon ruptures were mainly caused by mechanical stress. [Orthopedics.2016; 39(5):e893-e896.].

Distribution of podoplanin in synovial tissues in rheumatoid arthritis patients using biologic or conventional disease-modifying anti-rheumatic drugs.

OBJECTIVE Podoplanin (PDPN) mediates tumor cell migration and invasion, which phenomena might also play a role in severe rheumatoid arthritis (RA). Therefore, the precise cellular distribution of PDPN and its relationships with inflammation was studied in RA treated with biologic disease-modifying anti-rheumatic drugs (DMARD) or conventional DMARDs (cDMARD). METHODS PDPN+ cells were immunostained by NZ-1 mAb, and scored (3+; >50%/ area, 2+; 20%- 50%, 1+; 5%-20%, 0: <5%) in synovial tissues from RA treated with biologic DMARDs (BIO, n=20) or cDMARD (n=20) for comparison with osteoarthritis (OA, n=5), followed by cell grading of inflammation and cell-typing. RESULTS Inflammatory synovitis score was 1.4 in both BIO and cDMARD, compared to only 0.2 in OA. PDPN+ cells were found in the lining layer (BIO 1.6, cDMARD 1.3, OA 0.2) and lymphoid aggregates (BIO 0.6, cDMRD 0.7, OA 0.2), and correlated with RA-inflammation in BIO- and cDMARD-groups in both area (r=0.7/0.9, r=0.6/0.7, respectively p<0.05). PDPN was expressed in CD68+ type A macrophage-like and 5B5+ type B fibroblast-like cells in the lining layer, and in IL- 17+ cells in lymphoid aggregates in RA. CONCLUSION PDPN was markedly increased in the immunologically inflamed RA synovitis, which was surgically treated due to BIO- and cDMARD-resistant RA. PDPN may have potential of a new marker of residual arthritis in local joints for inflammation-associated severe RA.

Comparison of the Effects of Osteochondral Autograft Transplantation With Platelet-Rich Plasma or Platelet-Rich Fibrin on Osteochondral Defects in a Rabbit Model

Background: Although osteochondral autograft transplantation (OAT) provides satisfactory outcomes for osteochondral defects, for large defects OAT is often inadequate because of graft availability. Osteochondral allograft transplantation is an alternative treatment for large defects, but this approach is limited by graft storage constraints and carries disease transmission risks. Platelet-rich fibrin (PRF) is a second-generation platelet concentrate, and its positive effect on articular cartilage has been reported. However, the effect of PRF with OAT of osteochondral defects is unknown. Purpose: To compare the effects of OAT with platelet-rich plasma (PRP) and PRF on osteochondral defects in a rabbit model. Study Design: Controlled laboratory study. Methods: Forty-two juvenile rabbits were divided into control, PRP, and PRF groups. In the control and PRP groups, a cylindrical osteochondral defect (5 mm in diameter and 2 mm in depth) was created on the patellar groove, and an osteochondral graft (3.5 mm in diameter and 5 mm in length) harvested from the contralateral side was inserted into the distal portion of the defect. After wound closure, either normal saline or PRP was injected in the knee. In the PRF group, a PRF clot was placed in the defect before grafting. The surgical site was macroscopically and histologically assessed after 3 and 12 weeks. Results: At 3 weeks, the PRF group (n = 8) was macroscopically healed compared with the other 2 groups (control, n = 7; PRP, n = 6) (P < .005). Histologically, osteochondral graft cartilage of the PRF group had normal cellularity and higher amounts of safranin O staining relative to the other 2 groups (P < .005). At 12 weeks, all 3 groups (n = 8 per group) were macroscopically healed with normal or nearly normal cartilage, and osteochondral graft cartilage was histologically hyaline cartilage. In contrast, the PRF group healed with hyaline-like cartilage at nongrafted defects, whereas the other 2 groups healed with fibrocartilage (P < .001). Conclusion: OAT with PRF maintained hyaline cartilage, and the nongrafted defect healed with hyaline-like cartilage. Clinical Relevance: PRF has the potential to improve clinical outcomes of OAT used to treat osteochondral lesions.

The Incidence of Atypical Femoral Fractures in Patients with Rheumatic Disease: Yamagata Prefectural Committee of Atypical Femoral Fractures (YamaCAFe) Study

Atypical femoral fractures (AFFs) have been reported to occur with minimal or spontaneous subtrochanteric and femoral shaft fractures with a characteristic transverse pattern, compared with typical femoral fractures in young patients with high-energy trauma. AFFs are related to long-term use of bisphosphonates (BPs), glucocorticoids and rheumatic diseases. We have estimated a blind analysis of AFFs in rheumatic patients receiving BPs and glucocorticoids ordinary over a long time in all Yamagata prefectural area through radiographic examination. The 123 AFFs including suspected cases over six years were collected and reviewed by two independent orthopedic surgeons. We found 86 patients with a total of 99 AFFs between 2009 and 2014 (1.43 cases/100,000 person/year). Of these 99 AFFs, 11 were in 8 rheumatic patients including three patients with bilateral AFFs. The incidence of AFFs in rheumatic patients had trend to increase from 2012. The mean age of all 8 patients was 54.9 years. All 8 patients received BPs and 7/8 received prednisolone (PSL). The mean dose of PSL was 14 mg/day. Compared to patients with unilateral AFFs, those with bilateral AFFs in rheumatic patients were on a higher dose of PSL (20 mg/day vs. 7 mg/day) and had less femoral neck-shaft angle (129° vs. 136°, p < 0.05). In conclusion, the incidence of AFFs in rheumatic patients showed a trend to increase from 2012 to 2014 in Yamagata prefecture. Careful management of AFFs is of particular importance in rheumatic patients who have taken high doses of PSL and have small femoral neck-shaft angle.

Danger of frustrated sensors: Role of Toll-like receptors and NOD-like receptors in aseptic and septic inflammations around total hip replacements

Summary The innate immune sensors, Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), can recognize not only exogenous pathogen-associated molecular patterns (PAMPs), but also endogenous molecules created upon tissue injury, sterile inflammation, and degeneration. Endogenous ligands are called damage-associated molecular patterns (DAMPs), and include endogenous molecules released from activated and necrotic cells as well as damaged extracellular matrix. TLRs and NLRs can interact with various ligands derived from PAMPs and DAMPs, leading to activation and/or modulation of intracellular signalling pathways. Intensive research on the innate immune sensors, TLRs and NLRs, has brought new insights into the pathogenesis of not only various infectious and rheumatic diseases, but also aseptic foreign body granuloma and septic inflammation of failed total hip replacements (THRs). In this review, recent knowledge is summarized on the innate immune system, including TLRs and NLRs and their danger signals, with special reference to their possible role in the adverse local host response to THRs. Translational potential of this article: A clear understanding of the roles of Toll-like receptors and NOD-like receptors in aseptic and septic loosening of joint replacements will facilitate potential strategies to mitigate these events, thereby extending the longevity of implants in humans.

AB0381 Expression of Toll-like Receptors and Distribution of Immune-Cells in Rheumatoid Synovial Tissues of The Patients Treated by Abatacept

Background Rheumatoid arthritis (RA) is a progressive chronic autoimmune and autoinflammatory disease affecting multiple joints simultaneously1. Abatacept (ABT) which is cytotoxic T-lymphocyte-associated protein (CTLA) 4-Ig agent is one of biologics2. Objectives The aim of this study is to examine the effect of ABT for evaluating immune-reactive cells with toll-like receptors in the synovial tissue samples of RA patients with ABT therapy (ABT groups) compared to them with non-biological therapy (Non-BIO groups). Methods Synovial tissue samples of 20 RA (10 ABT and 10 Non-BIO samples) were immunohistochemically stained with TLR+ and immune-reactive cells including T cells (CD2), B cells (CD20), macrophage (CD68), conventional dendritic cells (cDC; S100, DC-LAMP), regulatory T cells (FoxP3) and indoleamine 2,3-dioxygenase+ cells (IDO). Positive cells were counted per 10 fields (x 200) by microscopy. TLR positive cells were analyzed by double Immunofluorescent methods with T cell, B cell, macrophage, cDC and plasmacytoid DC (pDC; CD123)-markers and IDO. Synovial inflammation was estimated by Krenn grading score. Results ABT and non-BIO group was shown the similar grading scores (2.3 vs 2.0), DAS28CRP4 (5.1 vs 4.3) and CRP (1.3 vs 0.9). IDO+ and FoxP3+ cells, not CD3+, CD20+ and CD68+ cells were correlated significant with DAS28CRP (4) in lymphoid aggregation in ABT groups (p<0.01). TLR immunoreactivity was also confirmed in pDCs and IDO+ cells by immunofluorescent staining (Fig. 1). Conclusions ABT was reported the similar remarkable effect with other biologics. We have already shown the residual inflammation of synovitis with TLR expressions in the RA patients who were received TNF-blockers3. TLR+ cells were observed with T cells and B cells related local inflammatory grading in ABT groups, however regulatory immune-cells might be increased and regulate inflammatory simultaneously. CTLA-4 and IDO was recently revealed the generation of osteoclast 4,5. IDO+ cells increased in synovial tissues of ABT group compared to no-BIO group. ABT may not only regulate the immunosuppression but also suppress the activation of osteoclast in RA. References Klareskog L, et al: Lancet 2004; 363: 675–681, Sokolove J, ARD, 2015, [Epub ahead of print] Takakubo Y, et al: Clin Rheumatol, 2013; 32: 853–61, Bozec A, et al: SciTranMed, 2014; 7; 235:235ra60 Schiff M, et al: ARD, 2014;73: 2174–7. Acknowledgement We sincerely thank a lot to Ms. Eiko Saito for her skillful technical support in immunohistochemistry. Disclosure of Interest None declared

THU0118 Trend of Atypical Femoral Fractures in Rheumatic Patients in The Highly Super Aging Area of North Japan

Background A number of recent case reports and series have identified atypical fractures of the femoral shaft. Atypical femoral fractures (AFFs) with lack of trauma or less-energy have been reported to relate using of the bisphosphonates (BPs) and glucocorticoids (GCs) for a long time, affecting collagen diseases1,2. Objectives We have analyzed the AFFs in rheumatic patients in the highly super aging area of North Japan, in which the rate more than 65 year-old people in the population was 28%. Methods We investigated retrospectively all cases of AFF summarized by the American Society for Bone and Mineral Research (ASBMR) Task Force 20131 including affected rheumatic disease patients in all hospitals of our prefectural area from 2009 to 2014. Two independent orthpaedic surgeons have diagnosed blindly AFF and estimated proximal femoral geometry by using radiographic analyses. Observation end of this study was settled on Feb 2016. Results We have 86 cases 99 AFFs in our prefectural area from 2009 to 2014 (1.43 cases/100,000 person/year). The rheumatic patients with AFFs were eleven femurs in eight women in that periods and increased year by year (Fig. 1). Three cases have bilateral AFFs (six femurs, 55%). The mean age of them was 54.9 year-old (33–80). As comorbid conditions, five patients have systematic lupus erythematosus, two dermatomyositis and rheumatoid arthritis, one polyarteritis nodosa and systemic sclerosis. Fracture types were seven subtrochanteric and four diaphyseal femoral fractures. Mean femoral neck angle was 133 degrees (120–141) and femoral shaft angle was 6.1 degree (3–10). Six patients with AFF had prodromal pain (55%). All patients received BPs and all but one prednisolone (PSL). Mean duration of receiving those drugs was 52 months (24–120) and 109 months (36–120), respectively. Mean dosage of PSL was 14 mg/day (0–30). In addition, bilateral AFF group has more PSL dose and duration of PSL, more varus deformity of their femur compared to unilateral AFF group (p<0.05). After affecting the fractures, BPs were quitted in seven femurs except four. The surgery using intramedullary nail fixation were performed in all cases excluding one femur using a locking plate. Teriparatide and therapy of low-intensity pulsed ultrasound was induced for five cases after healing their operated wounds. Mean duration of post-operative observation was 28 months (12–70). At the latest follow-up, ten femurs were observed the sign of union at fracture site on X-ray or computed tomography of their femurs, but not one femur. Mean duration of union of the fracture site was 12.6 months (6–24) in ten femurs. Conclusions Eleven AFFs were observed in 2009–14 in rheumatic patients and the numbers were increased year by year in the highly super aging area of North Japan. The careful management and treatment for the AFFs in rheumatic patients are required even after the surgery, because our all cases have been observed the delayed union or non-union of fracture site at their latest follow-up3. In addition, AFF patients who show varus deformity of their femurs and receive much dose of PSL have highly risk of contralateral AFF. References Shane E, et al. JMBR, 2014, Blacks DM, et al. NEJM, 2010, Thompson RN, et al. JBJS, 2012. Acknowledgement We really appreciate all members of Yamagata Prefectural Committee of Atypical Femoral Fractures Study group. Disclosure of Interest None declared

AB0408 Are Atypical Femoral Fractures in Rheumatic Patients Increasing

Background Atypical femoral fractures with low-energy or lack of trauma have been reported to relate using of the bisphosphonates (BPs) and glucocorticoids for a long time, affecting collagen diseases1,2. Objectives The aim of this study was to analyze the atypical femoral fractures in rheumatic patients in our four institutes retrospectively. Methods We investigated the cases of atypical femoral fractures summarized by the American Society for Bone and Mineral Research (ASBMR) Task Force 20101 among our out-rheumatic patients from 2009 to 2014. Results We have 1,091 out-rheumatic patients/year in our four institutes from 2009 to 2014. The patients with atypical femoral fractures were 8 limbs in six women (0.12%) in six years. Three limbs were injured at 2013, and five at 2014, including two cases has both side atypical femoral fractures (Fig. 1). The mean age of them was 51 year-old (38-73). As comorbid conditions, two patients has dermatomyositis, systematic lupus erythematosus, one rheumatoid arthritis and one polyarteritis nodosa. Fracture types were seven subtrochanteric fractures and one diaphyseal femoral fracture. All patients received BPs and prednisolone (PSL). Mean duration of receiving the drugs was 75 months (36-120) and 114 months (60-180), respectively. Mean dosage of PSL was 15 mg/day (5-30). After affecting the fractures, BPs were quitted and the surgery using intramedullary nail fixation were performed in all cases. One case had the surgical-site infection. Teriparatide was induced excepted one case and therapy of low-intensity pulsed ultrasound was started for all cases after healing their operated wounds. Mean duration of post-operative observation was 12 months (5-23). At the latest follow-up, five femurs were observed the sign of union at fracture site on X-ray or computed tomography of their femurs, but not other three femurs. Mean duration of union of the fracture site was 10 months (6-13) in five femurs. Conclusions Eight atypical femoral fractures were observed in 2013-14, but not in 2009-2012. Atypical femoral fracture may increase year by year. The careful management and treatment for the atypical femoral fractures in rheumatic patients were required even after the surgery, because our all cases have been observed the delayed union or non-union of fracture site at their latest follow-up3. References Shane E, et al. JMBR, 2010. Blacks DM, et al. NEJM, 2010. Thompson RN, et al. JBJS 2012. Disclosure of Interest None declared