Angiocidin promotes pro-inflammatory cytokine production and antigen presentation in multiple sclerosis.

Angiocidin was originally identified as a potent inhibitor of angiogenesis and tumor growth in vivo. In addition to its involvement in the regulation of carcinogenesis, recent studies indicate that angiocidin may also play a significant role in immune system modulation. This report describes the expression and potential function of angiocidin in multiple sclerosis (MS), a severe demyelinating, inflammatory and autoimmune disease of the central nervous system (CNS). We demonstrated that angiocidin and interleukin-7 (IL-7) are over-expressed in brain lesions of MS patients. Angiocidin-treated monocytes, peripheral blood T cells and primary astrocytes secreted various cytokines and chemokines including, IL-6, IL-7, GM-CSF, and MCP-1. Addition of recombinant angiocidin to cell cultures was able to promote differentiation of monocytes into a macrophage-like cell, induce MHC class I and class II gene expression and activate CD4(+) and CD8(+) T lymphocytes. Consistent with these findings, angiocidin induced mononuclear phagocyte migration and adhesion as well as increased the IL-2 response by antigen-specific T cells to myelin basic protein peptide presented to them by autologous mononuclear phagocytes. Furthermore, we examined STAT3 expression in angiocidin stimulated mononuclear phagocytes, T cells, and primary astrocytes. We found that angiocidin markedly stimulates STAT3 expression in these cell populations. Angiocidin, therefore appears to play a previously unappreciated and potentially important role in the regulation of immune response during the clinical course of MS.

Immunotherapy with a HER2-Targeting Listeria Induces HER2-Specific Immunity and Demonstrates Potential Therapeutic Effects in a Phase I Trial in Canine Osteosarcoma.

PURPOSE: Recombinant Listeria vaccines induce tumor-specific T-cell responses that eliminate established tumors and prevent metastatic disease in murine cancer models. We used dogs with HER2/neu(+) appendicular osteosarcoma, a well-recognized spontaneous model for pediatric osteosarcoma, to determine whether a highly attenuated, recombinant Listeria monocytogenes expressing a chimeric human HER2/neu fusion protein (ADXS31-164) could safely induce HER2/neu-specific immunity and prevent metastatic disease. EXPERIMENTAL DESIGN: Eighteen dogs that underwent limb amputation or salvage surgery and adjuvant chemotherapy were enrolled in a phase I dose escalation clinical trial and received either 2 × 10(8), 5 × 10(8), 1 × 10(9), or 3.3 × 10(9) CFU of ADXS31-164 intravenously every 3 weeks for 3 administrations. RESULTS: Only low-grade, transient toxicities were observed. ADXS31-164 broke peripheral tolerance and induced antigen-specific IFNγ responses against the intracellular domain of HER2/neu in 15 of 18 dogs within 6 months of treatment. Furthermore, ADXS31-164 reduced the incidence of metastatic disease and significantly increased duration of survival time and 1-, 2-, and 3-year survival rates when compared with a historical control group with HER2/neu(+) appendicular osteosarcoma treated with amputation and chemotherapy alone. CONCLUSIONS: These findings demonstrate that ADXS31-164 administered in the setting of minimal residual disease can induce HER2/neu-specific immunity and may reduce the incidence of metastatic disease and prolong overall survival in a clinically relevant, spontaneous, large animal model of cancer. These findings, therefore, have important translational relevance for children with osteosarcoma and adults with other HER2/neu(+) cancers. Clin Cancer Res; 22(17); 4380-90. ©2016 AACR.

A One Health overview, facilitating advances in comparative medicine and translational research.

TABLE OF CONTENTS: A1 One health advances and successes in comparative medicine and translational researchCheryl StroudA2 Dendritic cell-targeted gorilla adenoviral vector for cancer vaccination for canine melanomaIgor Dmitriev, Elena Kashentseva, Jeffrey N. Bryan, David T. CurielA3 Viroimmunotherapy for malignant melanoma in the companion dog modelJeffrey N. Bryan, David Curiel, Igor Dmitriev, Elena Kashentseva, Hans Rindt, Carol Reinero, Carolyn J. HenryA4 Of mice and men (and dogs!): development of a commercially licensed xenogeneic DNA vaccine for companion animals with malignant melanomaPhilip J. BergmanA5 Successful immunotherapy with a recombinant HER2-expressing Listeria monocytogenes in dogs with spontaneous osteosarcoma paves the way for advances in pediatric osteosarcomaNicola J. Mason, Josephine S. Gnanandarajah, Julie B. Engiles, Falon Gray, Danielle Laughlin, Anita Gaurnier-Hausser, Anu Wallecha, Margie Huebner, Yvonne PatersonA6 Human clinical development of ADXS-HER2Daniel O'ConnorA7 Leveraging use of data for both human and veterinary benefitLaura S. TremlA8 Biologic replacement of the knee: innovations and early clinical resultsJames P. StannardA9 Mizzou BioJoint Center: a translational success storyJames L. CookA10 University and industry translational partnership: from the lab to commercializationMarc JacobsA11 Beyond docking: an evolutionarily guided OneHealth approach to drug discoveryGerald J. Wyckoff, Lee Likins, Ubadah Sabbagh, Andrew SkaffA12 Challenges and opportunities for data applications in animal health: from precision medicine to precision husbandryAmado S. GuloyA13 A cloud-based programmable platform for healthHarlen D. HaysA14 Comparative oncology: One Health in actionAmy K. LeBlancA15 Companion animal diseases bridge the translational gap for human neurodegenerative diseaseJoan R. Coates, Martin L. Katz, Leslie A. Lyons, Gayle C. Johnson, Gary S. Johnson, Dennis P. O'BrienA16 Duchenne muscular dystrophy gene therapyDongsheng DuanA17 Polycystic kidney disease: cellular mechanisms to emerging therapiesJames P. CalvetA18 The domestic cat as a large animal model for polycystic kidney diseaseLeslie A. Lyons, Barbara GandolfiA19 The support of basic and clinical research by the Polycystic Kidney Disease FoundationDavid A. BaronA20 Using naturally occurring large animal models of human disease to enable clinical translation: treatment of arthritis using autologous stromal vascular fraction in dogsMark L. WeissA21 Regulatory requirements regarding clinical use of human cells, tissues, and tissue-based productsDebra A. WebsterA22 Regenerative medicine approaches to Type 1 diabetes treatmentFrancis N. KaranuA23 The zoobiquity of canine diabetes mellitus, man's best friend is a friend indeed-islet transplantationEdward J. RobbA24 One Medicine: a development model for cellular therapy of diabetesRobert J. Harman.

Assessment of canonical NF-κB activity in canine diffuse large B-cell lymphoma.

Companion dogs with spontaneous malignancies are clinically relevant models in which to study the corresponding human diseases and potential therapies. In both dogs and people, non-Hodgkin's lymphoma (NHL) is the most common hematopoietic malignancy. Diffuse large B-cell lymphoma (DLBCL) is the most common NHL subtype in dogs and people, sharing similar biologic, behavioral, genetic, and molecular characteristics in both species. One such molecular characteristic is the constitutive activation of the canonical NF-κB pathway, which in health regulates the expression of target genes that control cellular proliferation, survival, and immune and inflammatory responses as well as multidrug resistance. We found that canine and human DLBCL patients share similar NF-κB activity profiles. Using the cell-permeable NBD peptide, which blocks NF-κB signaling, we inhibited constitutive NF-κB activity and induced apoptosis of primary canine malignant B cells in vitro. In addition, we found that NBD peptide administration to dogs with relapsed B-cell lymphoma inhibited the expression of NF-κB target genes and reduced tumor burden. In this chapter, we describe our methods for processing canine malignant lymphoid tissue. We also describe our methods for treating the lymphocytes isolated from this tissue with NBD peptide and evaluating constitutive canonical NF-κB activity in these cells via immunoblot and electrophoretic mobility shift assay (EMSA). We highlight the nuances of working with canine primary cells.

A phase I clinical trial of systemically delivered NEMO binding domain peptide in dogs with spontaneous activated B-cell like diffuse large B-cell lymphoma.

Activated B-Cell (ABC) Diffuse Large B-Cell Lymphoma (DLBCL) is a common, aggressive and poorly chemoresponsive subtype of DLBCL, characterized by constitutive canonical NF-κB signaling. Inhibition of NF-κB signaling leads to apoptosis of ABC-DLBCL cell lines, suggesting targeted disruption of this pathway may have therapeutic relevance. The selective IKK inhibitor, NEMO Binding Domain (NBD) peptide effectively blocks constitutive NF-κB activity and induces apoptosis in ABC-DLBCL cells in vitro. Here we used a comparative approach to determine the safety and efficacy of systemic NBD peptide to inhibit constitutive NF-κB signaling in privately owned dogs with spontaneous newly diagnosed or relapsed ABC-like DLBCL. Malignant lymph nodes biopsies were taken before and twenty-four hours after peptide administration to determine biological effects. Intravenous administration of <2 mg/kg NBD peptide was safe and inhibited constitutive canonical NF-κB activity in 6/10 dogs. Reductions in mitotic index and Cyclin D expression also occurred in a subset of dogs 24 hours post peptide and in 3 dogs marked, therapeutically beneficial histopathological changes were identified. Mild, grade 1 toxicities were noted in 3 dogs at the time of peptide administration and one dog developed transient subclinical hepatopathy. Long term toxicities were not identified. Pharmacokinetic data suggested rapid uptake of peptide into tissues. No significant hematological or biochemical toxicities were identified. Overall the results from this phase I study indicate that systemic administration of NBD peptide is safe and effectively blocks constitutive NF-κB signaling and reduces malignant B cell proliferation in a subset of dogs with ABC-like DLBCL. These results have potential translational relevance for human ABC-DLBCL.

Evidence of an oncogenic gammaherpesvirus in domestic dogs.

In humans, chronic infection with the gammaherpesvirus Epstein-Barr virus is usually asymptomatic; however some infected individuals develop hematological and epithelial malignancies. The exact role of EBV in lymphomagenesis is poorly understood partly because of the lack of clinically relevant animal models. Here we report the detection of serological responses against EBV capsid antigens in healthy dogs and dogs with spontaneous lymphoma and that dogs with the highest antibody titers have B cell lymphoma. Moreover, we demonstrate the presence of EBV-like viral DNA and RNA sequences and Latent Membrane Protein-1 in malignant lymph nodes of dogs with lymphoma. Finally, electron microscopy of canine malignant B cells revealed the presence of classic herpesvirus particles. These findings suggest that dogs can be naturally infected with an EBV-like gammaherpesvirus that may contribute to lymphomagenesis and that dogs might represent a spontaneous model to investigate environmental and genetic factors that influence gammaherpesvirus-associated lymphomagenesis in humans.

NEMO-binding domain peptide inhibits constitutive NF-κB activity and reduces tumor burden in a canine model of relapsed, refractory diffuse large B-cell lymphoma.

PURPOSE: Activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) is an aggressive, poorly chemoresponsive lymphoid malignancy characterized by constitutive canonical NF-κB activity that promotes lymphomagenesis and chemotherapy resistance via overexpression of antiapoptotic NF-κB target genes. Inhibition of the canonical NF-κB pathway may therefore have therapeutic relevance in ABC-DLBCL. Here, we set out to determine whether dogs with spontaneous DLBCL have comparative aberrant constitutive NF-κB activity and to determine the therapeutic relevance of NF-κB inhibition in dogs with relapsed, resistant DLBCL. EXPERIMENTAL DESIGN: Canonical NF-κB activity was evaluated by electrophoretic mobility shift assays and immunoblot analyses, and NF-κB target gene expression was measured by quantitative real time PCR. Primary malignant canine B lymphocytes were treated with the selective IKK complex inhibitor NF-κB essential modulator-binding domain (NBD) peptide and evaluated for NF-κB activity and apoptosis. NBD peptide was administered intranodally to dogs with relapsed B-cell lymphoma and NF-κB target gene expression and tumor burden were evaluated pre- and post-treatment. RESULTS: Constitutive canonical NF-κB activity and increased NF-κB target gene expression were detected in primary DLBCL tissue. NBD peptide inhibited this activity and induced apoptosis of primary malignant B cells in vitro. Intratumoral injections of NBD peptide to dogs with relapsed DLBCL inhibited NF-κB target gene expression and reduced tumor burden. CONCLUSIONS: This work shows that dogs with spontaneous DLBCL represent a clinically relevant, spontaneous, large animal model for human ABC-DLBCL and shows the therapeutic relevance of NF-κB inhibition in the treatment of ABC-DLBCL. These results have important translational relevance for ABC-DLBCL treatment in human patients.

The novel angiogenic inhibitor, angiocidin, induces differentiation of monocytes to macrophages.

We previously showed that angiocidin, a tumor and vascular associated protein, is a potent inhibitor of angiogenesis and tumor growth. Angiocidin is a multidomain protein that exerts its antiangiogenic activity through multiple mechanisms, including effects on cell matrix interaction. Here, we describe another activity of angiocidin that may contribute to its antitumor activity. We show that angiocidin activates monocytes to secrete a mixture of proinflammatory cytokines and induces them to differentiate into macrophage-like cells. Using the monocytic cell line THP-1, we show that angiocidin induces the cells to become adherent and phagocytic, express macrophage markers, and secrete matrix metalloproteinase-9. Microarray analysis of control and angiocidin-treated THP-1 cells revealed that angiocidin up-regulated p105/p50, p100/p52, and rel B, components of the nuclear factor-kappaB (NF-kappaB) pathway. We confirmed the microarray data and showed that angiocidin induced phosphorylation of I kappa beta, p50, and p65 and translocation of p50 and p65 to the nucleus. We also showed that angiocidin activated up-stream mediators of NF-kappaB, such as the mitogen-activated protein kinase (MAPK) pathway and phosphoinositide-3 kinase (PI3K). Blockage of NF-kappaB and MAPK activation with small molecule inhibitors completely prevented angiocidin-mediated secretion of cytokines from THP-1 cells, but did not inhibit their adhesive phenotype. Blocking PI3K inhibited both secretion of cytokines, as well as the adhesive phenotype. These data suggest that angiocidin activates monocytes to secrete cytokines and differentiates them to a macrophage-like phenotype through at least two pathways mediated by MAPK and NF-kappaB, as well as PI3K.