G. Elizabeth Pluhar

The effect of recombinant human bone morphogenetic protein-2 on femoral reconstruction with an intercalary allograft in a dog model

This study compared the effect of augmentation of allograft–host bone junctions with recombinant human bone morphogenetic protein‐2 (rhBMP‐2) on an absorbable collagen sponge (ACS), autogenous cancellous bone graft (CBG), and a collagen sponge alone in a canine intercalary femoral defect model repaired with a frozen allograft. Outcome assessment included serial radiographs, dual energy X‐ray absorptiometry scans, and gait analyses, and mechanical testing and histology of post‐mortem specimens. The distal junction healed more quickly and completely with rhBMP‐2 than ACS alone based on qualitative radiography and histologic evaluations. The primary tissue in the unhealed gaps in the ACS group was fibrous connective tissue. The proximal allograft–host bone junction had complete bone union in the three treatment groups. There was significantly greater new bone callus formation at both junctions with rhBMP‐2 than with CBG or ACS alone that resulted in increased bone density around the allograft–host bone junctions. All dogs shifted their weight from the treated leg to the contralateral pelvic limb immediately after surgery. Weight bearing forces were redistributed equally between the pelvic limbs at 12 weeks after surgery with rhBMP‐2, at 16 weeks after surgery with CBG, and at 24 weeks after surgery with ACS alone. Bending and compressive stiffnesses of the whole treated femora were equal to the contralateral control femora in all treatment groups, whereas torsional rigidities of the whole treated femora for the CBG and ACS groups were significantly less than the control. Both the proximal and distal junctions the treated with rhBMP‐2 had torsional stiffnesses and strengths equal to intact control bones. Ultimate failure torques of the proximal junctions of the CBG group and of both junctions of the ACS group were significantly less than the BMP‐treated bones. Augmentation of the allograft–host bone junctions with rhBMP‐2 on an ACS gave results for all parameters measured that equaled or exceeded autogenous graft in this canine intercalary femoral defect model.

Optimization of adenoviral vector-mediated transgene expression in the canine brain in vivo, and in canine glioma cells in vitro

Expression of the immune-stimulatory molecule Fms-like tyrosine kinase 3 ligand (Flt3L) and the conditional cytotoxic enzyme herpes simplex virus type 1 thymidine kinase (HSV1-TK) provides long-term immune-mediated survival of large glioblastoma multiforme (GBM) models in rodents. A limitation for predictive testing of novel antiglioma therapies has been the lack of a glioma model in a large animal. Dogs bearing spontaneous GBM may constitute an attractive large-animal model for GBM, which so far has remained underappreciated. In preparation for a clinical trial in dogs bearing spontaneous GBMs, we tested and optimized adenovirus-mediated transgene expression with negligible toxicity in the dog brain in vivo and in canine J3T glioma cells. Expression of the marker gene beta-galactosidase (beta-Gal) was higher when driven by the murine (m) than the human (h) cytomegalovirus (CMV) promoter in the dog brain in vivo, without enhanced inflammation. In the canine brain, beta-Gal was expressed mostly in astrocytes. beta-Gal activity in J3T cells was also higher with the mCMV than the hCMV promoter driving tetracycline-dependent (TetON) transgene expression within high-capacity adenovirus vectors (HC-Ads). Dog glioma cells were efficiently transduced by HC-Ads expressing mCMV-driven HSV1-TK, which induced 90% reduction in cell viability in the presence of ganciclovir. J3T cells were also effectively transduced with HC-Ads expressing Flt3L under the control of the regulatable TetON promoter system, and as predicted, Flt3L release was stringently inducer dependent. HC-Ads encoding therapeutic transgenes under the control of regulatory sequences driven by the mCMV promoter are excellent vectors for the treatment of spontaneous GBM in dogs, which constitute an ideal preclinical animal model.

Anti-tumor immune response correlates with neurological symptoms in a dog with spontaneous astrocytoma treated by gene and vaccine therapy

Gene therapy and vaccination have been tested in malignant glioma patients with modest, albeit encouraging results. The combination of these therapies has demonstrated synergistic efficacy in murine models but has not been reported in large animals. Gemistocytic astrocytoma (GemA) is a low-grade glioma that typically progresses to lethal malignancy despite conventional therapies. Until now there has been no useful animal model of GemA. Here we report the treatment of a dog with spontaneous GemA using the combination of surgery, intracavitary adenoviral interferon gamma (IFNgamma) gene transfer, and vaccination with glioma cell lysates mixed with CpG oligodeoxynucleotides. Surgical tumor debulking and delivery of Ad-IFNgamma into the resection cavity were performed. Autologous tumor cells grew slowly in culture, necessitating vaccination with allogeneic tumor lysate in four of the five vaccinations. Transient left-sided blindness and hemiparesis occurred following the fourth and fifth vaccinations. These neurological symptoms correlated with a peak in the levels of tumor-reactive IgG and CD8(+) T cells measured in the blood. All symptoms resolved and this dog remains tumor-free over 450 days following surgery. This case report preliminarily demonstrates the feasibility of treating dogs with spontaneous glioma using immune-based therapy and warrants further study using this therapeutic approach.

Efficacy of nonviral gene transfer in the canine brain

OBJECT The purpose of this study was to evaluate the gene transfer capability and tolerability of plasmid DNA/polyethylenimine (PEI) complexes in comparison with adenovirus and naked plasmid DNA in the canine brain. METHODS Plasmid or adenoviral vectors encoding firefly luciferase were injected directly into the cerebral parenchyma of five adult dogs at varying doses and volumes. Serial physical and neurological examinations, as well as blood and cerebrospinal fluid (CSF) analyses, were conducted before and after the surgery for 3 days. Three days after gene delivery, a luciferase activity assay and immunofluorescence analysis were used to test the brain tissue for gene expression. RESULTS Injection into the brain parenchyma resulted in gene transfer throughout the cerebrum with every vector tested. Luciferase expression was highest when adenovirus vectors were used. Injection of plasmid DNA/PEI complexes and naked DNA resulted in similar levels of luciferase expression, which were on average 0.5 to 1.5% of the expression achieved with adenovirus vectors. Immunofluorescent microscopy analysis revealed that plasmid DNA/PEI complexes transduced mainly neurons, whereas adenovirus transduced mainly astrocytes. No significant acute side effects or neurological complications were observed in any of the dogs. Mononuclear cell counts significantly increased in the CSF after adenovirus injection and modestly increased after injection of plasmid DNA/PEI complexes, suggesting that a mild, acute inflammatory response occurred in the central nervous system (CNS). CONCLUSIONS Compared with rodent models that are limited by very small brains, the dog is an excellent preclinical model in which to assess the distribution and safety of emerging gene transfer technologies. In this study, short-term gene transfer was evaluated as a prelude to long-term expression and safety studies. The authors conclude that the viral and nonviral vectors tested were well tolerated and effective at mediating gene transfer throughout a large portion of the canine brain. The nonviral plasmid vectors were less effective than adenovirus, yet they still achieved appreciable gene expression levels. Due to reduced gene transfer efficiency relative to viral vectors, nonviral vectors may be most useful when the expressed protein is secreted or exerts a bystander effect. Nonviral vectors offer an alternative means to genetically modify cells within the CNS of large mammals.

Vaccination for Invasive Canine Meningioma Induces in Situ Production of Antibodies Capable of Antibody-Dependent Cell-Mediated Cytotoxicity

Malignant and atypical meningiomas are resistant to standard therapies and associated with poor prognosis. Despite progress in the treatment of other tumors with therapeutic vaccines, this approach has not been tested preclinically or clinically in these tumors. Spontaneous canine meningioma is a clinically meaningful but underutilized model for preclinical testing of novel strategies for aggressive human meningioma. We treated 11 meningioma-bearing dogs with surgery and vaccine immunotherapy consisting of autologous tumor cell lysate combined with toll-like receptor ligands. Therapy was well tolerated, and only one dog had tumor growth that required intervention, with a mean follow up of 585 days. IFN-γ-elaborating T cells were detected in the peripheral blood of 2 cases, but vaccine-induced tumor-reactive antibody responses developed in all dogs. Antibody responses were polyclonal, recognizing both intracellular and cell surface antigens, and HSP60 was identified as one common antigen. Tumor-reactive antibodies bound allogeneic canine and human meningiomas, showing common antigens across breed and species. Histologic analysis revealed robust infiltration of antibody-secreting plasma cells into the brain around the tumor in posttreatment compared with pretreatment samples. Tumor-reactive antibodies were capable of inducing antibody-dependent cell-mediated cytotoxicity to autologous and allogeneic tumor cells. These data show the feasibility and immunologic efficacy of vaccine immunotherapy for a large animal model of human meningioma and warrant further development toward human trials.

Identification of Myeloid Derived Suppressor Cells in Dogs with Naturally Occurring Cancer

Dogs with naturally occurring cancer represent an important large animal model for drug development and testing novel immunotherapies. However, poorly defined immunophenotypes of canine leukocytes have limited the study of tumor immunology in dogs. The accumulation of myeloid derived suppressor cells (MDSCs) is known to be a key mechanism of immune suppression in tumor-bearing mice and in human patients. We sought to identify MDSCs in the blood of dogs with cancer. Peripheral blood mononuclear cells (PBMCs) from dogs with advanced or early stage cancer and from age-matched healthy controls were analyzed by flow cytometry and microscopy. Suppressive function was tested in T cell proliferation and cytokine elaboration assays. Semi-quantitative RT-PCR was used to identify potential mechanisms responsible for immunosuppression. PBMCs from dogs with advanced or metastatic cancer exhibited a significantly higher percentage of CD11b+CD14−MHCII− cells compared to dogs diagnosed with early stage non-metastatic tumors and healthy dogs. These CD11b+ CD14−MHCII− cells constitute a subpopulation of activated granulocytes that co-purify with PBMCs, display polymorphonuclear granulocyte morphology, and demonstrate a potent ability to suppress proliferation and IFN-γ production in T cells from normal and tumor-bearing donors. Furthermore, these cells expressed hallmark suppressive factors of human MDSC including ARG1, iNOS2, TGF-β and IL-10. In summary our data demonstrate that MDSCs accumulate in the blood of dogs with advanced cancer and can be measured using this three-marker immunophenotype, thereby enabling prospective studies that can monitor MDSC burden.

Adenoviral-mediated gene transferinto the canine brain in vivo

OBJECTIVEGlioblastoma multiforme (GBM) is a devastating brain tumor for which there is no cure. Adenoviral-mediated transfer of conditional cytotoxic (herpes simplex virus [HSV] 1-derived thymidine kinase [TK]) and immunostimulatory (Fms-like tyrosine kinase 3 ligand [Flt3L]) transgenes elicited immune-mediated long-term survival in a syngeneic intracranial GBM model in rodents. However, the lack of a large GBM animal model makes it difficult to predict the outcome of therapies in humans. Dogs develop spontaneous GBM that closely resemble the human disease; therefore, they constitute an excellent large animal model. We assayed the transduction efficiency of adenoviral vectors (Ads) encoding β-galactosidase (βGal), TK, and Flt3L in J3T dog GBM cells in vitro and in the dog brain in vivo. METHODSJ3T cells were infected with Ads (30 plaque-forming units/cell; 72 h) encoding βGal (Ad-βGal), TK (Ad-TK), or Flt3L (Ad-Flt3L). We determined transgene expression by immunocytochemistry, βGal activity, Flt3L enzyme-linked immunosorbent assay, and TK-induced cell death. Ads were also injected intracranially into the parietal cortex of healthy dogs. We determined cell-type specific transgene expression and immune cell infiltration. RESULTSAdenoviral-mediated gene transfer of HSV1-TK, Flt3L, and βGal was detected in dog glioma cells in vitro (45% transduction efficiency) and in the dog brain in vivo (10-mm2 area transduced surrounding each injection site). T cells and macrophages/activated microglia infiltrated the injection sites. Importantly, no adverse clinical or neuropathological side effects were observed. CONCLUSIONWe demonstrate effective adenoviral-mediated gene transfer into the brain of dogs in vivo and support the use of these vectors to develop an efficacy trial for canine GBM as a prelude to human trials.

The effect of osteoporosis treatments on fatigue properties of cortical bone tissue

Bisphosphonates are commonly prescribed for treatment of osteoporosis. Long-term use of bisphosphonates has been correlated to atypical femoral fractures (AFFs). AFFs arise from fatigue damage to bone tissue that cannot be repaired due to pharmacologic treatments. Despite fatigue being the primary damage mechanism of AFFs, the effects of osteoporosis treatments on fatigue properties of cortical bone are unknown. To examine if fatigue-life differences occur in bone tissue after different pharmacologic treatments for osteoporosis, we tested bone tissue from the femurs of sheep given a metabolic acidosis diet to induce osteoporosis, followed by treatment with a selective estrogen reception modulator (raloxifene), a bisphosphonate (alendronate or zoledronate), or parathyroid hormone (teriparatide, PTH). Beams of cortical bone tissue were created and tested in four-point bending fatigue to failure. Tissue treated with alendronate had reduced fatigue life and less modulus loss at failure compared with other treatments, while tissue treated with PTH had a prolonged fatigue life. No loss of fatigue life occurred with zoledronate treatment despite its greater binding affinity and potency compared with alendronate. Tissue mineralization measured by microCT did not explain the differences seen in fatigue behavior. Increased fatigue life with PTH suggests that current treatment methods for AFF could have beneficial effects for restoring fatigue life. These results indicate that fatigue life differs with each type of osteoporosis treatment.

CD8+ T Cell–Independent Tumor Regression Induced by Fc-OX40L and Therapeutic Vaccination in a Mouse Model of Glioma

Despite the growing number of preclinical and clinical trials focused on immunotherapy for the treatment of malignant gliomas, the prognosis for this disease remains grim. Although some promising advances have been made, the immune response stimulated as a result of immunotherapeutic protocols has been inefficient at complete tumor elimination, primarily due to our lack of understanding of the necessary effector functions of the immune system. We previously demonstrated that a tumor lysate vaccine/Fc-OX40L therapy is capable of inducing enhanced survival and tumor elimination in the GL261 mouse glioma model. The following experiments were performed to determine the mechanism(s) of action of this therapy that elicits a potent antitumor immune response. The evidence subsequently outlined indicates a CD8+ T cell–independent and CD4+ T cell–, NK cell–, and B cell–dependent means of prolonged survival. CD8+ T cell–independent tumor clearance is surprising considering the current focus of many cancer immunotherapy protocols. These results provide evidence for CD8+ T cell–independent means of antitumor response and should lead to additional examination of the potential manipulation of this mechanism for future treatment strategies.

Biomechanical Analysis of Stacked Plating Techniques to Stabilize Distal Radial Fractures in Small Dogs

OBJECTIVE To evaluate the fatigue life of partially stacked and fully stacked (1.5/2.0 and 2.0/2.7 mm) veterinary cuttable plates (VCP) in a fracture gap model of the distal aspect of the radius. STUDY DESIGN In vitro biomechanical study. METHODS Constructs (n=4/group) were assembled for each of 8 groups using 8-hole plates (1.5/2.0 and 2.0/2.7 mm VCP) in the following configurations: unstacked; 2-hole stacked centered over the gap (COG); 4-hole stacked COG; and fully stacked. Plate(s) were secured to 2 separate polyvinylchloride pipe lengths, mounted to a mechanical testing system with a custom jig, and were loaded in axial compression for 10(6) cycles at 10 Hz or until failure at 6-60 N for the 1.5/2.0 mm VCP and 10-100 N for the 2.0/2.7 mm VCP. Differences in number of cycles, stiffness, and failure mode were recorded. RESULTS All construct failures occurred through a screw hole adjacent to the gap. Fully stacked and 4-hole stacked 1.5/2.0 and 2.0/2.7 mm VCP withstood 10(6) cycles. Fatigue life and stiffness of the 1.5/2.0 or 2.0/2.7 mm unstacked constructs were significantly less than the other constructs. Differences were identified in stiffness among the 1.5/2.0 mm stacked constructs and in fatigue life among the 2.0/2.7 mm VCP stacked constructs. CONCLUSION Four-hole partially stacked VCP (either 1.5/2.0 or 2.0/2.7 mm) have comparable mechanical properties to fully stacked VCP. CLINICAL RELEVANCE Partial stacking of 2 holes of VCP on both sides of the fracture gap may provide sufficient strength for healing, but this premise must be tested in vivo.