Novel mode of action of c-kit tyrosine kinase inhibitors leading to NK cell-dependent antitumor effects

Mutant isoforms of the KIT or PDGF receptors expressed by gastrointestinal stromal tumors (GISTs) are considered the therapeutic targets for STI571 (imatinib mesylate; Gleevec), a specific inhibitor of these tyrosine kinase receptors. Case reports of clinical efficacy of Gleevec in GISTs lacking the typical receptor mutations prompted a search for an alternate mode of action. Here we show that Gleevec can act on host DCs to promote NK cell activation. DC-mediated NK cell activation was triggered in vitro and in vivo by treatment of DCs with Gleevec as well as by a loss-of-function mutation of KIT. Therefore, tumors that are refractory to the antiproliferative effects of Gleevec in vitro responded to Gleevec in vivo in an NK cell-dependent manner. Longitudinal studies of Gleevec-treated GIST patients revealed a therapy-induced increase in IFN-gamma production by NK cells, correlating with an enhanced antitumor response. These data point to a novel mode of antitumor action for Gleevec.     

Local low‐dose lovastatin delivery improves the bone‐healing defect caused by Nf1 loss of function in osteoblasts

Postfracture tibial nonunion (pseudoarthrosis) leads to lifelong disability in patients with neurofibromatosis type I (NF1), a disorder caused by mutations in the NF1 gene. To determine the contribution of NF1 in bone healing, we assessed bone healing in the Nf1 conditional mouse model lacking Nf1 specifically in osteoblasts. A closed distal tibia fracture protocol and a longitudinal study design were used. During the 21‐ to 28‐day postfracture period, callus volume, as expected, decreased in wild‐type but not in Nf1 mice, suggesting delayed healing. At these two time points, bone volume (BV/TV) and volumetric bone mineral density (vBMD) measured by 3D micro–computed tomography were decreased in Nf1 callus‐bridging cortices and trabecular compartments compared with wild‐type controls. Histomorphometric analyses revealed the presence of cartilaginous remnants, a high amount of osteoid, and increased osteoclast surfaces in Nf1 calluses 21 days after fracture, which was accompanied by increased expression of osteopontin, Rankl, and Tgfβ. Callus strength measured by three‐point bending 28 days after fracture was reduced in Nf1 versus wild‐type calluses. Importantly, from a clinical point of view, this defect of callus maturation and strength could be ameliorated by local delivery of low‐dose lovastatin microparticles, which successfully decreased osteoid volume and cartilaginous remnant number and increased callus BV/TV and strength in mutant mice. These results thus indicate that the dysfunctions caused by loss of Nf1 in osteoblasts impair callus maturation and weaken callus mechanical properties and suggest that local delivery of low‐dose lovastatin may improve bone healing in NF1 patients. © 2010 American Society for Bone and Mineral Research     

Differential inhibitory effect of fondaparinux on the procoagulant potential of intact monocytes and monocyte-derived microparticles

Monocytes and monocyte-derived microparticles (MMPs) play a major role in acute coronary syndrome (ASC). Activated monocytes (ac-M) and MMPs support thrombin generation via tissue factor (TF). The aim of this study was to evaluate the inhibitory effect of fondaparinux, a selective Xa inhibitor, on thrombin generation supported by activated monocytes and MMPs. Monocytes were purified by elutriation. They were activated by LPS, allowing to obtain both ac-M and MMPs. Thrombin generation was performed using Fluoroscan^® in these two cell models, in comparison with a cell-free model (TF 5 pM final). Two concentrations of ac-M (0.2 × 10⁶ and 1 × 10⁶/well) and four concentrations of MMPs (40,000; 80,000; 120,000 and 160,000/well) were tested. TGT was evaluated for increasing fondaparinux concentrations (0, 0.1, 0.4, 0.7 and 1.2 μg/ml). Without fondaparinux, 0.2 × 10⁶ ac-M and 160,000 MMPs induced comparable results. Fondaparinux inhibited thrombin generation in the three models. Inhibition was fondaparinux concentration dependent. Rate index was the most sensitive parameter, compared to lag-time, peak and endogenous thrombin potential. The rate index IC₅₀ were 0.69 ± 0.03 μg/ml for ac-M, 0.20 ± 0.03 μg/ml for MMPs, and 0.22 ± 0.02 μg/ml for cell-free model. Fondaparinux exerted an inhibitory effect at all concentrations, including the lowest (0.1 μg/ml). The extend of inhibition was similar between MMPs and cell-free models, and stronger than ac-M model. We assume that the efficacy of fondaparinux 2.5 mg once daily in ACS patients may be in part attributed to its inhibitory effect on MMPs.     

Treatment of chronic disruption of the patellar tendon in Osteogenesis Imperfecta with allograft reconstruction

We present a case of chronic disruption of the patellar tendon in a patient with Osteogenesis Imperfecta. This patient was treated with a customized extensor mechanism allograft. Results were excellent at 5 years follow up. To our knowledge this treatment has not previously been published in this situation. We present this as a reliable treatment option.     

Cortical stimulation mapping using epidurally implanted thin-film microelectrode arrays

Stimulation mapping of motor cortex is an important tool for assessing motor cortex physiology. Existing techniques include intracortical microstimulation (ICMS) which has high spatial resolution but damages cortical integrity by needle penetrations, and transcranial stimulation which is non-invasive but lacks focality and spatial resolution. A minimally invasive epidural microstimulation (EMS) technique using chronically implanted polyimide-based thin-film microelectrode arrays (72 contacts) was tested in rat motor cortex and compared to ICMS within individual animals. Results demonstrate reliable mapping with high reproducibility and validity with respect to ICMS. No histological evidence of cortical damage and the absence of motor deficits as determined by performance of a motor skill reaching task, demonstrate the safety of the method. EMS is specifically suitable for experiments integrating electrophysiology with behavioral and molecular biology techniques.