Jonathan G. Schoenecker

Protease-Activated Receptor-2 Signaling Triggers Dendritic Cell Development

Dendritic cells (DC) are potent antigen-presenting cells that govern the effector cell responses of the immune system. DC are thought to continuously develop from circulating progenitors in a process that is accelerated by inflammatory stimuli. However, the physiological signals that regulate the development of DC from precursor cells have not been well defined. Here we show that a serine protease acting via protease-activated receptor-2 (PAR-2) stimulates the development of DC from bone marrow progenitor cells cultured in granulocyte-macrophage colony-stimulating factor and IL-4. DC fail to develop in bone marrow cultures treated with soy bean trypsin inhibitor, a serine protease inhibitor, but this inhibition is overcome by a PAR-2 agonist peptide. DC do not spontaneously develop from the bone marrow of PAR-2-deficient mice, but can be stimulated to do so by inflammatory mediators. These results suggest that endogenous serine proteases stimulate DC development in vitro. Thus, serine proteases may help trigger adaptive immune responses in vivo.

Exposure to Topical Bovine Thrombin During Surgery Elicits a Response Against the Xenogeneic Carbohydrate Galactose α1-3Galactose

Exposure of humans to topical bovine thrombin has been associated with development of antibodies against bovine and human coagulation factors and blood coagulation abnormalities. However, the nature of this humoral response is unknown. In this study, numerous glycoproteins in the topical bovine thrombin were found to contain the Galα1-3Gal epitope, which is known to be highly immunogenic. More importantly, Galα1-3Gal is recognized by natural antibodies that are found in all normal individuals and are known to effectively mediate complement activation and subsequent destruction of xenogeneic tissues. Thus, primary exposure of normal individuals to topical bovine thrombin is expected to result in an immediate immune reaction against that reagent. Further, following exposure to topical bovine thrombin, the levels of anti-Galα1-3Gal IgG rose to levels tenfold greater than the average level of natural anti-Galα1-3Gal IgG in naive individuals. Thus, Galα1-3Gal in topical bovine thrombin accounts for, at least in part, the highly immunogenic nature of this reagent.

Micro-computed tomography assessment of the progression of fracture healing in mice

The mouse fracture model is ideal for research into the pathways of healing because of the availability of genetic and transgenic mice and the ability to create cell-specific genetic mutations. While biomechanical tests and histology are available to assess callus integrity and tissue differentiation, respectively, micro-computed tomography (μCT) analysis has increasingly been utilized in fracture studies because it is non-destructive and provides descriptions of the structural and compositional properties of the callus. However, the dynamic changes of μCT properties that occur during healing are not well defined. Thus, the purpose of this study was to determine which μCT properties change with the progression of fracture repair and converge to values similar to unfractured bone in the mouse femur fracture model. A unilateral femur fracture was performed in C57BL/6 mice and intramedullary fixation performed. Fractured and un-fractured contralateral specimens were harvested from groups of mice between 2 and 12 weeks post-fracture. Parameters describing callus based on μCT were obtained, including polar moment of inertia (J), bending moment of inertia (I), total volume (TV), tissue mineral density (TMD), total bone volume fraction (BV/TV), and volumetric bone mineral density (vBMD). For comparison, plain radiographs were used to measure the callus diameter (D) and area (A); and biomechanical properties were evaluated using either three-point bending or torsion. The μCT parameters J, I, TV, and TMD converged toward their respective values of the un-fractured femurs over time, although significant differences existed between the two sides at every time point evaluated (p<0.05). Radiograph measurement D changed with repair progression in similar manner to TV. In contrast, BV/TV and BMD increased and decreased over time with statistical differences between callus and un-fractured bone occurring sporadically. Similarly, none of the biomechanical properties were found to distinguish consistently between the fractured and un-fractured femur. Micro-CT parameters assessing callus structure and size (J, I, and TV) were more sensitive to changes in callus over time post-fracture than those assessing callus substance (TMD, BV/TV, and BMD). Sample size estimates based on these results indicate that utilization of μCT requires fewer animals than biomechanics and thus is more practical for evaluating the healing femur in the mouse fracture model.

Exposure of Mice to Topical Bovine Thrombin Induces Systemic Autoimmunity

Bovine thrombin is used as an aid to hemostasis in medical and surgical procedures. At least 500,000 Americans are exposed to this therapeutic annually and reports suggest that exposure is associated with the development of autoreactive antibodies. To determine whether bovine thrombin can induce pathological autoimmunity we exposed nonautoimmune-prone galactose-alpha1-3-galactose-deficient mice to the two bovine thrombin preparations currently approved for use in the United States. We found that, like humans exposed to bovine thrombin, mice developed an immune response against the therapeutic and the xenogeneic carbohydrate galactose-alpha1-3-galactose, and some mice developed autoantibodies against clotting factors. Further, unexpectedly, a single exposure to this therapeutic also induced autoimmunity with features characteristic of systemic lupus erythematosus including antibodies against nuclear antigens, native DNA, double-stranded DNA, and cardiolipin. High levels of these autoantibodies correlated with glomerulonephritis in all mice evaluated. This autoimmune syndrome was detected in mice 15 weeks after a secondary exposure to bovine thrombin and female mice were found to develop the syndrome at a significantly greater frequency than males. Thus, these studies indicate that exposure to bovine thrombin preparations can induce a pathological systemic autoimmune syndrome with lupus-like serology.

Protease-activated Receptor (PAR) 1 and PAR4 Differentially Regulate Factor V Expression from Human Platelets

With the recent interest of protease-activated receptors (PAR) 1 and PAR4 as possible targets for the treatment of thrombotic disorders, we compared the efficacy of protease-activated receptor (PAR)1 and PAR4 in the generation of procoagulant phenotypes on platelet membranes. PAR4-activating peptide (AP)–stimulated platelets promoted thrombin generation in plasma up to 5 minutes earlier than PAR1-AP–stimulated platelets. PAR4-AP–mediated factor V (FV) association with the platelet surface was 1.6-fold greater than for PAR1-AP. Moreover, PAR4 stimulation resulted in a 3-fold greater release of microparticles, compared with PAR1 stimulation. More robust FV secretion and microparticle generation with PAR4-AP was attributable to stronger and more sustained phosphorylation of myosin light chain at serine 19 and threonine 18. Inhibition of Rho-kinase reduced PAR4-AP–mediated FV secretion and microparticle generation to PAR1-AP–mediated levels. Thrombin generation assays measuring prothrombinase complex activity demonstrated 1.5-fold higher peak thrombin levels on PAR4-AP–stimulated platelets, compared with PAR1-AP–stimulated platelets. Rho-kinase inhibition reduced PAR4-AP–mediated peak thrombin generation by 25% but had no significant effect on PAR1-AP–mediated thrombin generation. In conclusion, stimulation of PAR4 on platelets leads to faster and more robust thrombin generation, compared with PAR1 stimulation. The greater procoagulant potential is related to more efficient FV release from intracellular stores and microparticle production driven by stronger and more sustained myosin light chain phosphorylation. These data have implications about the role of PAR4 during hemostasis and are clinically relevant in light of recent efforts to develop PAR antagonists to treat thrombotic disorders.

Bisphosphonates Inhibit Osteosarcoma‐Mediated Osteolysis Via Attenuation of Tumor Expression of MCP‐1 and RANKL

Osteosarcoma is the most common primary malignant tumor of bone and accounts for around 50% of all primary skeletal malignancies. In addition to novel chemotherapies, there is a need for adjuvant therapies designed to inhibit osteosarcoma proliferation and tumor‐induced osteolysis to attenuate tumor expansion and metastasis. As such, studies on the efficacy of bisphosphonates on human osteosarcoma are planned after feasibility studies determined that the bisphosphonate zoledronic acid (ZOL) can be safely combined with conventional chemotherapy. However, the molecular mechanisms responsible for, and means of inhibiting, osteosarcoma‐induced osteolysis are largely unknown. We establish that osteosarcoma growth directly correlates with tumor‐induced osteolysis and activation of osteoclasts in vivo. In vitro, tumor cells were determined to expresses surface, but not soluble, receptor activator of NF‐κB ligand (RANKL) and stimulated osteoclastogenesis in a manner directly proportional to their malignant potential. In addition, an aggressive osteosarcoma cell line was shown to secrete monocyte chemoattractant protein‐1 (MCP‐1), resulting in robust monocyte migration. Because MCP‐1 is a key cytokine for monocyte recruitment and surface‐bound RANKL strongly supports local osteoclastogenesis, we suggest that high levels of these signaling molecules are associated with the aggressive potential of osteosarcoma. Consistent with these findings, abundant expression of RANKL/MCP‐1 was observed in tumor in vivo, and MCP‐1 plasma levels strongly correlated with tumor progression and osteolysis. ZOL administration directly attenuates osteosarcoma production of RANKL/MCP‐1, reducing tumor‐induced bone destruction. In vivo, these findings also correlated with significant reduction in osteosarcoma growth. ZOL attenuates tumor‐induced osteolysis, not only through direct inhibition of osteoclasts, but also through direct actions on tumor expression of osteoclast activators. These data provide insight regarding the effect of ZOL on osteosarcoma essential for designing the planned upcoming prospective randomized trials to determine the efficacy of bisphosphonates on osteosarcoma in humans.

Complications and outcomes of diaphyseal forearm fracture intramedullary nailing: a comparison of pediatric and adolescent age groups.

Background: Flexible intramedullary nailing (IMN) has become a popular technique for the management of unstable or open forearm fractures. Recent publications have suggested an increased incidence of delayed union and poor outcomes in older children and adolescents. The objective of this study was to review forearm fractures treated with IMN, comparing the rate of complications and outcomes between the 2 age groups. Our hypothesis was that IMN is an effective technique with a similar rate of complications in both age groups. Methods: An Institutional Review Board-approved retrospective review was conducted of pediatric forearm fractures treated from 1998 to 2008 at a single institution. Over the study time period, 4161 pediatric forearm fractures were managed nonoperatively (92%) and 353 were treated operatively with plate, cross-pin, or intramedullary fixation (8%). Patients with inadequate follow-up, cross-pin, or plate fixation were excluded. Medical records were reviewed for indications and complications. Complications were graded with a modification of the Clavien-Dindo classification. Outcomes were judged by a new grading system. Results: A total of 205 forearm fractures treated with IMN in 203 patients were identified. The mean age was 9.7 years (range, 1.7 to 16.2 y) and mean follow-up was 42 weeks. Operative indications were failure of closed treatment in 165 (80%) and open fracture in 40 (20%). Mean time from injury to IMN was 5.9 days (range, 0 to 25 d). Single bone IMN was performed in 40 of 185 both bone fractures (26%); there were 20 single-bone forearm fractures treated with IMN. Open reduction was required in 61/165 (37%) of closed fractures. Asymptomatic delayed union (grade 1 complication) was observed in 9 fractures (4%). More severe complications were noted in 17% (grade 2 to 4 complications). Postoperative compartment syndrome occurred in 3 isolated forearm fractures with a significant younger mean age (6.0 vs. 10 y, P=0.031). Overall, complications were significantly more frequent in children older than 10 years of age (25/101) as compared with younger children (13/104, P=0.031). In particular, delayed union was more common in children over the age of 10 years (9/101 vs. 1/104, OR=9.99, P=0.009). Outcomes were good or excellent in 91% of fractures. There was no statistical association of patient age with a fair or poor outcome. Conclusions: IMN is an effective technique for pediatric forearm fractures with good to excellent outcomes in 91%. Complications are not infrequent with this technique, with complications of grade 2 to 4 severity in 17%. There was a 2-fold increase in the rate of complications in children over the age of 10 years. Compartment syndrome was more common in younger children. Patients and families should be counseled about the risks preoperatively. Level of Evidence: Level III—retrospective comparative study.

Relative purity of thrombin-based hemostatic agents used in surgery

BACKGROUND Hemostatic agents used in surgery contain thrombin isolated from either a bovine or human source. The use of thrombin derived from a bovine source has been associated with the development of an abnormal immune response, but a study of the immunoreactivity of the various commercially available thrombin preparations has not been conducted. This study determined the relative purity of commercially available thrombin preparations, if humans have natural antibodies that recognize these preparations, and if elicited antibodies against bovine thrombin cross-react with other bovine or human hemostatic agents. STUDY DESIGN The purity of hemostatic agents was determined by protein and substrate assays, electrophoresis, and immunoblotting. The natural antigenicity and cross-reactivity of elicited antibodies were measured by ELISA using serum samples from 82 donors from the Red Cross and serum collected from patients exposed to bovine thrombin, respectively. RESULTS All of the bovine thrombin preparations were found to contain the xenogeneic carbohydrate galactosealpha1-3galactose. The natural antigenicity of the bovine thrombin preparations was greater than that of a human thrombin preparation and similar to that of porcine aortic endothelial cells. Antibodies elicited against bovine thrombin were found to cross-react with other bovine preparations and other xenoantigens but not with human hemostatic preparations. CONCLUSIONS All patients have antibovine thrombin antibodies, even before exposure to bovine thrombin-containing hemostatic agents. The cross-reactivity of elicited antibovine thrombin antibodies indicates that if a patient has been sensitized to a bovine product, it is likely safer to use a human-derived product in lieu of a bovine product.

Fibrinolysis is essential for fracture repair and prevention of heterotopic ossification.

Bone formation during fracture repair inevitably initiates within or around extravascular deposits of a fibrin-rich matrix. In addition to a central role in hemostasis, fibrin is thought to enhance bone repair by supporting inflammatory and mesenchymal progenitor egress into the zone of injury. However, given that a failure of efficient fibrin clearance can impede normal wound repair, the precise contribution of fibrin to bone fracture repair, whether supportive or detrimental, is unknown. Here, we employed mice with genetically and pharmacologically imposed deficits in the fibrin precursor fibrinogen and fibrin-degrading plasminogen to explore the hypothesis that fibrin is vital to the initiation of fracture repair, but impaired fibrin clearance results in derangements in bone fracture repair. In contrast to our hypothesis, fibrin was entirely dispensable for long-bone fracture repair, as healing fractures in fibrinogen-deficient mice were indistinguishable from those in control animals. However, failure to clear fibrin from the fracture site in plasminogen-deficient mice severely impaired fracture vascularization, precluded bone union, and resulted in robust heterotopic ossification. Pharmacological fibrinogen depletion in plasminogen-deficient animals restored a normal pattern of fracture repair and substantially limited heterotopic ossification. Fibrin is therefore not essential for fracture repair, but inefficient fibrinolysis decreases endochondral angiogenesis and ossification, thereby inhibiting fracture repair.

Autocrine VEGF/VEGFR1 Signaling in a Subpopulation of Cells Associates with Aggressive Osteosarcoma

Osteosarcoma is the most common primary bone malignancy and accounts for more than half of primary skeletal malignancies in children and young adults. Although vascular endothelial growth factor (VEGF) expression in osteosarcoma has been associated with poor outcome, its role in the pathogenesis of osteosarcoma remains controversial. Here, VEGF and VEGFR1 expression in both human and murine osteosarcoma cells associated with increasing malignant potential. Autocrine VEGF/VEGFR1 signaling resulted in constitutive activation of VEGFR1 in highly aggressive osteosarcoma cells. In addition, survival and proliferation of highly aggressive osteosarcoma cells was dependent on autocrine VEGF/R1 signaling in vitro. The effect of VEGFR1 expression on in vivo tumor growth and angiogenesis was evaluated by immunoselecting subpopulations of osteosarcoma cells that express high or low levels of VEGFR1. Cell enriched for high VEGFR1 expression showed increased VEGF production, tumor growth, tumor angiogenesis, and osteolysis in vivo. In addition, it was demonstrated that VEGF and VEGFR1 are coexpressed by a subset of tumor cells in human osteosarcoma, similar to what was observed in the murine osteosarcoma cells. These results suggest that autocrine VEGF/VEGFR1 signaling in a subpopulation of tumor cells plays a pivotal role in osteosarcoma progression. Implications: Aggressive osteosarcoma phenotypes are mediated by autocrine VEGF/VEGFR1 signaling and improved stratification measures and novel anti-angiogenic strategies may benefit this specific tumor type. Mol Cancer Res; 12(8); 1100–11. ©2014 AACR.