Comparison of CT with intra-operative navigation reported implant position utilising a robotic assisted technique in total knee arthroplasty.

BACKGROUND: Validation of navigated total knee arthroplasty (TKA) systems assists clinicians in making treatment decisions. The aim of this study was to independently review a navigation assisted robotic system for use in TKA. METHODS: We evaluated 87 patients (92 knees) undergoing robotic assisted TKA. Position estimated by the navigation software and postoperative CT scan were compared. Post-operative CT scans were interpreted by a senior radiologist blinded to intra-operative component position. Recorded were femoral varus/valgus, tibial varus/valgus and overall limb alignment in the coronal plane. In the sagittal plane tibial slope and femoral flexion/extension. Femoral component rotation was assessed in relation to the transepicondylar axis (TEA). RESULTS: Mean difference between software estimation and postoperative CT scan of the femoral component position in the coronal plane was 1.02° (0.86-1.18, 95%CI). Tibial coronal position was 1.19° (0.97-1.41). Sagittal plane component position for the femur was 1.64° (1.41-1.87). Tibial slope was 1.44° (1.21-1.68). Mean femoral component rotation was 1.27° (1.01-1.53). Overall 94.57% of intraoperative measures were within 3° of the component position measured on CT. CONCLUSION: Robotic assisted navigation used in combination with a novel balancing system can result in very accurate component positioning during total knee arthroplasty.

Mast cells enhance sterile inflammation in chronic nonbacterial osteomyelitis.

Chronic nonbacterial osteomyelitis (CNO) is an autoinflammatory bone disease, and patients with active or recurrent bone inflammation at multiple sites are diagnosed with chronic recurrent multifocal osteomyelitis (CRMO). The Chronic multifocal osteomyelitis (CMO) mouse model develops IL-1ß-driven sterile bone lesions reminiscent of severe CRMO. The goal of this study was to evaluate the potential involvement of mast cells in CMO/CRMO. Here, we show that mast cells accumulate in inflamed tissues from CMO mice and that mast cell protease Mcpt1 can be detected in the peripheral blood. A transgenic model of connective tissue mast cell depletion (Mcpt5-Cre:Rosa26-Stopfl/fl-DTa) was crossed with CMO mice and the resulting mice (referred to as CMO/MC-) showed a significant delay in disease onset compared with age-matched CMO mice. At 5-6 months of age, CMO/MC- mice had fewer bone lesions and immune infiltration in the popliteal lymph nodes that drain the affected tissues. In bone marrow-derived mast cell cultures from CMO mice, cytokine production in response to the alarmin IL-33 was elevated compared with wild-type cultures. To test the relevance of mast cells to human CRMO, we tested serum samples from a cohort of healthy controls and from CRMO patients at diagnosis. Interestingly, mast cell chymase was elevated in CRMO patients as well as in patients with oligoarticular juvenile arthritis. Tryptase-positive mast cells were also detected in bone lesions from CRMO patients and patients with bacterial osteomyelitis. Together, our results identify mast cells as cellular contributors to bone inflammation in CMO/CRMO and provide rationale for further study of mast cells as therapeutic targets.

SLAP Is a Negative Regulator of FcεRI Receptor-Mediated Signaling and Allergic Response.

Binding of antigen to IgE-high affinity FcεRI complexes on mast cells and basophils results in the release of preformed mediators such as histamine and de novo synthesis of cytokines causing allergic reactions. Src-like adapter protein (SLAP) functions co-operatively with c-Cbl to negatively regulate signaling downstream of the T cell receptor, B cell receptor, and receptor tyrosine kinases (RTK). Here, we investigated the role of SLAP in FcεRI-mediated mast cell signaling, using bone marrow derived mast cells (BMMCs) from SLAP knock out (SLAP KO) mice. Mature SLAP-KO BMMCs displayed significantly enhanced antigen induced degranulation and synthesis of IL-6, TNFα, and MCP-1 compared to wild type (WT) BMMCs. In addition, SLAP KO mice displayed an enhanced passive cutaneous anaphylaxis response. In agreement with a negative regulatory role, SLAP KO BMMCs showed enhanced FcεRI-mediated signaling to downstream effector kinases, Syk, Erk, and Akt. Recombinant GST-SLAP protein binds to the FcεRIß chain and to the Cbl-b in mast cell lysates, suggesting a role in FcεRI down regulation. In addition, the ubiquitination of FcεRIγ chain and antigen mediated down regulation of FcεRI is impaired in SLAP KO BMMCs compared to the wild type. In line with these findings, stimulation of peripheral blood human basophils with FcεRIα antibody, or a clinically relevant allergen, resulted in increased SLAP expression. Together, these results indicate that SLAP is a dynamic regulator of IgE-FcεRI signaling, limiting allergic responses.

Oncogenic KIT-induced aggressive systemic mastocytosis requires SHP2/PTPN11 phosphatase for disease progression in mice.

Acquired mutations in KIT are driver mutations in systemic mastocytosis (SM). Here, we tested the role of SHP2/PTPN11 phosphatase in oncogenic KIT signaling using an aggressive SM mouse model. Stable knock-down (KD) of SHP2 led to impaired growth, colony formation, and increased rates of apoptosis in P815 cells. This correlated with defects in signaling to ERK/Bim, Btk, Lyn, and Stat5 pathways in P815-KD cells compared to non-targeting (NT). Retro-orbital injections of P815 NT cells in syngeneic DBA/2 mice resulted in rapid development of aggressive SM within 13-16 days characterized by splenomegaly, extramedullary hematopoiesis, and multifocal liver tumors. In contrast, mice injected with P815 SHP2 KD cells showed less disease burden, including normal spleen weight and cellularity, and significant reductions in mastocytoma cells in spleen, bone marrow, peripheral blood and liver compared to NT controls. Treatment of human mast cell leukemia HMC-1 cells or P815 cells with SHP2 inhibitor II-B08, resulted in reduced colony formation and cell viability. Combining II-B08 with multi-kinase inhibitor Dasatinib showed enhanced efficacy than either inhibitor alone in blocking cell growth pathways and cell viability. Taken together, these results identify SHP2 as a key effector of oncogenic KIT and a therapeutic target in aggressive SM.

SHP2 phosphatase promotes mast cell chemotaxis toward stem cell factor via enhancing activation of the Lyn/Vav/Rac signaling axis.

SHP2 protein-tyrosine phosphatase (encoded by Ptpn11) positively regulates KIT (CD117) signaling in mast cells and is required for mast cell survival and homeostasis in mice. In this study, we uncover a role of SHP2 in promoting chemotaxis of mast cells toward stem cell factor (SCF), the ligand for KIT receptor. Using an inducible SHP2 knockout (KO) bone marrow-derived mast cell (BMMC) model, we observed defects in SCF-induced cell spreading, polarization, and chemotaxis. To address the mechanisms involved, we tested whether SHP2 promotes activation of Lyn kinase that was previously shown to promote mast cell chemotaxis. In SHP2 KO BMMCs, SCF-induced phosphorylation of the inhibitory C-terminal residue (pY507) was elevated compared with control cells, and phosphorylation of activation loop (pY396) was diminished. Because Lyn also was detected by substrate trapping assays, these results are consistent with SHP2 activating Lyn directly by dephosphorylation of pY507. Further analyses revealed a SHP2- and Lyn-dependent pathway leading to phosphorylation of Vav1, Rac activation, and F-actin polymerization in SCF-treated BMMCs. Treatment of BMMCs with a SHP2 inhibitor also led to impaired chemotaxis, consistent with SHP2 promoting SCF-induced chemotaxis of mast cells via a phosphatase-dependent mechanism. Thus, SHP2 inhibitors may be useful to limit SCF/KIT-induced mast cell recruitment to inflamed tissues or the tumor microenvironment.

Role of SHP2 phosphatase in KIT-induced transformation: identification of SHP2 as a druggable target in diseases involving oncogenic KIT.

Intracellular mechanism(s) that contribute to promiscuous signaling via oncogenic KIT in systemic mastocytosis and acute myelogenous leukemia are poorly understood. We show that SHP2 phosphatase is essential for oncogenic KIT-induced growth and survival in vitro and myeloproliferative disease (MPD) in vivo. Genetic disruption of SHP2 or treatment of oncogene-bearing cells with a novel SHP2 inhibitor alone or in combination with the PI3K inhibitor corrects MPD by disrupting a protein complex involving p85α, SHP2, and Gab2. Importantly, a single tyrosine at position 719 in oncogenic KIT is sufficient to develop MPD by recruiting p85α, SHP2, and Gab2 complex to oncogenic KIT. Our results demonstrate that SHP2 phosphatase is a druggable target that cooperates with lipid kinases in inducing MPD.

SH2 domain-containing phosphatase 2 is a critical regulator of connective tissue mast cell survival and homeostasis in mice.

Mast cells require KIT receptor tyrosine kinase signaling for development and survival. Here, we report that SH2 domain-containing phosphatase 2 (SHP2) signaling downstream of KIT is essential for mast cell survival and homeostasis in mice. Using a novel mouse model with shp2 deletion within mature mast cells (MC-shp2 knockout [KO]), we find that SHP2 is required for the homeostasis of connective tissue mast cells. Consistently with the loss of skin mast cells, MC-shp2 KO mice fail to mount a passive late-phase cutaneous anaphylaxis response. To better define the phenotype of shp2-deficient mast cells, we used an inducible shp2 knockout approach in bone marrow-derived mast cells (BMMCs) or cultured peritoneal mast cells and found that SHP2 promotes mast cell survival. We show that SHP2 promotes KIT signaling to extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase and downregulation of the proapoptotic protein Bim in BMMCs. Also, SHP2-deficient BMMCs failed to repopulate mast cells in mast cell-deficient mice. Silencing of Bim partially rescued survival defects in shp2-deficient BMMCs, consistent with the importance of a KIT → SHP2 → Ras/ERK pathway in suppressing Bim and promoting mast cell survival. Thus, SHP2 is a key node in a mast cell survival pathway and a new potential therapeutic target in diseases involving mast cells.

SNARE-mediated membrane traffic is required for focal adhesion kinase signaling and Src-regulated focal adhesion turnover.

Integrin signaling is central to cell growth and differentiation, and critical for the processes of apoptosis, cell migration and wound repair. Previous research has demonstrated a requirement for SNARE-dependent membrane traffic in integrin trafficking, as well as cell adhesion and migration. The goal of the present research was to ascertain whether SNARE-dependent membrane trafficking is required specifically for integrin-mediated signaling. Membrane traffic was inhibited in Chinese hamster ovary cells by expression of dominant-negative (E329Q) N-ethylmaleimide-sensitive fusion protein (NSF) or a truncated form of the SNARE SNAP23. Integrin signaling was monitored as cells were plated on fibronectin under serum-free conditions. E329Q-NSF expression inhibited phosphorylation of focal adhesion kinase (FAK) on Tyr397 at early time points of adhesion. Phosphorylation of FAK on Tyr576, Tyr861 and Tyr925 was also impaired by expression of E329Q-NSF or truncated SNAP23, as was trafficking, localization and activation of Src and its interaction with FAK. Decreased FAK-Src interaction coincided with reduced Rac activation, decreased focal adhesion turnover, reduced Akt phosphorylation and lower phosphatidylinositol 3,4,5-trisphosphate levels in the cell periphery. Over-expression of plasma membrane-targeted Src or phosphatidylinositol 3-kinase (PI3K) rescued cell spreading and focal adhesion turnover. The results suggest that SNARE-dependent trafficking is required for integrin signaling through a FAK/Src/PI3K-dependent pathway.

SH2 domain-containing phosphatase-2 protein-tyrosine phosphatase promotes Fc epsilon RI-induced activation of Fyn and Erk pathways leading to TNF alpha release from bone marrow-derived mast cells.

Clustering of the high affinity IgE receptor (Fc(epsilon)RI) in mast cells leads to degranulation and production of numerous cytokines and lipid mediators that promote allergic inflammation. Initiation of FFc(epsilon)RI signaling involves rapid tyrosine phosphorylation of Fc(epsilon)RI and membrane-localized adaptor proteins that recruit additional SH2 domain-containing proteins that dynamically regulate downstream signaling. SH2 domain-containing phosphatase-2 (SHP2) is a protein-tyrosine phosphatase implicated in Fc(epsilon)RI signaling, but whose function is not well defined. In this study, using a mouse model allowing temporal shp2 inactivation in bone marrow-derived mast cells (BMMCs), we provide insights into SHP2 functions in the Fc(epsilon)RI pathway. Although no overt defects in Fc(epsilon)RI-induced tyrosine phosphorylation were observed in SHP2 knock-out (KO) BMMCs, several proteins including Lyn and Syk kinases displayed extended phosphorylation kinetics compared with wild-type BMMCs. SHP2 was dispensable for Fc(epsilon)RI-induced degranulation of BMMCs, but was required for maximal activation of Erk and Jnk mitogen-activated protein kinases. SHP2 KO BMMCs displayed several phenotypes associated with reduced Fyn activity, including elevated phosphorylation of the inhibitory pY531 site in Fyn, impaired signaling to Grb2-associated binder 2, Akt/PKB, and IkappaB kinase, and decreased TNF-alpha release compared with control cells. This is likely due to elevated Lyn activity in SHP2 KO BMMCs, and the ability of Lyn to antagonize Fyn activity. Overall, our study identifies SHP2 as a positive effector of Fc(epsilon)RI-induced activation of Fyn/Grb2-associated binder 2/Akt and Ras/Erk pathways leading to TNF-alpha release from mast cells.