Publications by authors named "Nicola J Mason"

25 Publications

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Veterinary Cooperative Oncology Group-Common Terminology Criteria for Adverse Events (VCOG-CTCAE v2) following investigational therapy in dogs and cats.

Vet Comp Oncol 2021 Jan 11. Epub 2021 Jan 11.

National Cancer Institute, National Institutes of Health, Comparative Oncology Program, Center for Cancer Research, Bethesda, Maryland, USA.

The updated VCOG-CTCAE v2 guidelines contain several important updates and additions since the last update (v1.1) was released in 2011 and published within Veterinary and Comparative Oncology in 2016. As the Veterinary Cooperative Oncology Group (VCOG) is no longer an active entity, the original authors and contributors to the VCOG-CTCAE v1.0 and v1.1 were consulted for input, and additional co-authors sought for expansion and refinement of the adverse event (AE) categories. VCOG-CTCAE v2 includes expanded neurology, cardiac and immunologic AE sections, and the addition of procedural-specific AEs. It is our intent that, through inclusion of additional authors from ACVIM subspecialties and the American College of Veterinary Surgery, that we can more comprehensively capture AEs that are observed during clinical studies conducted across a variety of disease states, clinical scenarios, and body systems. It is also our intent that these updated veterinary CTCAE guidelines will offer improved application and ease of use within veterinary practice in general, as well as within clinical trials that assess new therapeutic strategies for animals with a variety of diseases. Throughout the revision process, we strived to ensure the grading structure for each AE category was reflective of the decision-making process applied to determination of dose-limiting events. As phase I trial decisions are based on these criteria and ultimately determine the maximally tolerated dose, there is impact on standard dosing recommendations for any new drug registration or application. This document should be updated regularly to reflect ongoing application to clinical studies carried out in veterinary patients.
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http://dx.doi.org/10.1111/vco.12677DOI Listing
January 2021

Comparative Immunology and Immunotherapy of Canine Osteosarcoma.

Authors:
Nicola J Mason

Adv Exp Med Biol 2020 ;1258:199-221

Department of Clinical Sciences and Advanced Medicine, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Approximately 800 people are diagnosed with osteosarcoma (OSA) per year in the USA. Although 70% of patients with localized OSA are cured with multiagent chemotherapy and surgical resection, the prognosis for patients with metastatic or relapsed disease is guarded. The small number of patients diagnosed annually contributes to an incomplete understanding of disease pathogenesis, and challenges in performing appropriately powered clinical trials and detecting correlative biomarkers of response. While mouse models of OSA are becoming increasingly sophisticated, they generally fail to accurately recapitulate tumor heterogeneity, tumor microenvironment (TME), systemic immune dysfunction, and the clinical features of tumor recurrence, metastases, and chemoresistance, which influence outcome. Pet dogs spontaneously develop OSA with an incidence that is 30-50 times higher than humans. Canine OSA parallels the human disease in its clinical presentation, biological behavior, genetic complexity, and therapeutic management. However, despite therapy, most dogs die from metastatic disease within 1 year of diagnosis. Since OSA occurs in immune-competent dogs, immune factors that sculpt tumor immunogenicity and influence responses to immune modulation are in effect. In both species, immune modulation has shown beneficial effects on patient outcome and work is now underway to identify the most effective immunotherapies, combination of immunotherapies, and correlative biomarkers that will further improve clinical response. In this chapter, the immune landscape of canine OSA and the immunotherapeutic strategies used to modulate antitumor immunity in dogs with the disease will be reviewed. From this immunological viewpoint, the value of employing dogs with spontaneous OSA to accelerate and inform the translation of immunotherapies into the human clinic will be underscored.
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http://dx.doi.org/10.1007/978-3-030-43085-6_14DOI Listing
December 2020

Sarcomas-A barren immunological wasteland or field of opportunity for immunotherapy?

Vet Comp Oncol 2020 Dec 27;18(4):447-470. Epub 2020 Apr 27.

Department of Clinical Sciences and Advanced Medicine, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Key advances in our understanding of immunobiology and the immunosuppressive mechanisms of the tumour microenvironment have led to significant breakthroughs in manipulating the immune system to successfully treat cancer. Remarkable therapeutic responses have occurred with tumours that carry a high mutational burden. In these cases, pre-existing tumour-specific T cells can be rejuvenated via checkpoint inhibition to eliminate tumours. Furthermore, durable remissions have been achieved in haematological malignancies following adoptive transfer of T cells that specifically target cell surface proteins where expression is restricted to the malignancy's cell of origin. Soft tissue sarcomas and bone sarcomas have a paucity of non-synonymous somatic mutations and do not commonly express known, targetable, tumour-specific antigens. Historically, soft tissue sarcomas have been considered immunologically 'cold' and as such, unlikely candidates for immune therapy. Here, we review the immune landscape of canine and feline sarcomas and the immunotherapeutic strategies that have been employed in veterinary clinical trials to improve patient outcome. We also provide insight into immunotherapeutic approaches being used to treat human sarcomas. Together, current data indicates that, rather than a barren immunological wasteland, sarcomas represent a field of opportunities for immunotherapies. Furthermore, we and others would suggest that strategic combinations of immunotherapeutic approaches may hold promise for more effective treatments for high grade soft tissue sarcomas and bone sarcomas.
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http://dx.doi.org/10.1111/vco.12595DOI Listing
December 2020

Molecular subtypes in canine hemangiosarcoma reveal similarities with human angiosarcoma.

PLoS One 2020 25;15(3):e0229728. Epub 2020 Mar 25.

Department of Pathology and Laboratory Medicine, Raymond and Ruth Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America.

Angiosarcoma (AS) is a rare neoplasm with limited treatment options and a poor survival rate. Development of effective therapies is hindered by the rarity of this disease. Dogs spontaneously develop hemangiosarcoma (HSA), a common, histologically similar neoplasm. Metastatic disease occurs rapidly and despite chemotherapy, most dogs die several months after diagnosis. These features suggest that HSA might provide a tractable model to test experimental therapies in clinical trials. We previously reported whole exome sequencing of 20 HSA cases. Here we report development of a NGS targeted resequencing panel to detect driver mutations in HSA and other canine tumors. We validated the panel by resequencing the original 20 cases and sequenced 30 additional cases. Overall, we identified potential driver mutations in over 90% of the cases, including well-documented (in human cancers) oncogenic mutations in PIK3CA (46%), PTEN (6%), PLCG1(4%), and TP53 (66%), as well as previously undetected recurrent activating mutations in NRAS (24%). The driver role of these mutations is further demonstrated by augmented downstream signaling crucial to tumor growth. The recurrent, mutually exclusive mutation patterns suggest distinct molecular subtypes of HSA. Driver mutations in some subtypes closely resemble those seen in some AS cases, including NRAS, PLCG1, PIK3CA and TP53. Furthermore, activation of the MAPK and PI3K pathways appear to be key oncogenic mechanisms in both species. Together, these observations suggest that dogs with spontaneous HSA could serve as a useful model for testing the efficacy of targeted therapies, some of which could potentially be of therapeutic value in AS.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0229728PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7094861PMC
June 2020

Generation and Validation of an Antibody to Canine CD19 for Diagnostic and Future Therapeutic Purposes.

Vet Pathol 2020 03 21;57(2):241-252. Epub 2020 Feb 21.

Department of Clinical Sciences and Advanced Medicine, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA.

The B-cell coreceptor, CD19 is a transmembrane protein expressed throughout B-cell ontogeny from pro-B cell to plasmablast. It plays an important role in B-cell development and function and is an attractive target for antibody-directed immunotherapies against B-cell malignancies, including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin lymphoma (B-NHL) in humans. With the rapid development of next-generation immunotherapies aimed at improving therapeutic efficacy, there is a pressing need for a clinically relevant, immune-competent, spontaneous animal model to derisk these new approaches and inform human immunotherapy clinical trials. Pet dogs develop spontaneous B-cell malignancies, including B-NHL and leukemias that share comparable oncogenic pathways and similar immunosuppressive features to human B-cell malignancies. Despite treatment with multiagent chemotherapy, durable remissions in canine B-NHL are rare and most dogs succumb to their disease within 1 year of diagnosis. Here we report the development and validation of an anti-canine CD19-targeting monoclonal antibody and its single-chain derivatives, which enable next-generation CD19-targeted immunotherapies to be developed and evaluated in client-owned dogs with spontaneous B-NHL. These future in vivo studies aim to provide important information regarding the safety and therapeutic efficacy of CD19-targeted mono- and combination therapies and identify correlative biomarkers of response that will help to inform human clinical trial design. In addition, development of canine CD19-targeted immunotherapies aims to provide better therapeutic options for pet dogs diagnosed with B-cell malignancies.
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http://dx.doi.org/10.1177/0300985819900352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7462180PMC
March 2020

Establishing a model system for evaluating CAR T cell therapy using dogs with spontaneous diffuse large B cell lymphoma.

Oncoimmunology 2020 23;9(1):1676615. Epub 2019 Oct 23.

Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Multiple rodent and primate preclinical studies have advanced CAR T cells into the clinic. However, no single model accurately reflects the challenges of effective CAR T therapy in human cancer patients. To evaluate the effectiveness of next-generation CAR T cells that aim to overcome barriers to durable tumor elimination, we developed a system to evaluate CAR T cells in pet dogs with spontaneous cancer. Here we report on this system and the results of a pilot trial using CAR T cells to treat canine diffuse large B cell lymphoma (DLBCL). We designed and manufactured CD20-targeting, second-generation canine CAR T cells for functional evaluation and using lentivectors to parallel human CAR T cell manufacturing. A first-in-species trial of five dogs with DLBCL treated with CAR T was undertaken. Canine CAR T cells functioned in an antigen-specific manner and killed CD20+ targets. Circulating CAR T cells were detectable post-infusion, however, induction of canine anti-mouse antibodies (CAMA) was associated with CAR T cell loss. Specific selection pressure on CD20+ tumors was observed following CAR T cell therapy, culminating in antigen escape and emergence of CD20-disease. Patient survival times correlated with product expansion. Altering product manufacturing improved transduction efficiency and skewed toward a memory-like phenotype of canine CAR T cells. Manufacturing of functional canine CAR T cells using a lentivector is feasible. Comparable challenges to effective CAR T cell therapy exist, indicating their relevance in informing future human clinical trial design.
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http://dx.doi.org/10.1080/2162402X.2019.1676615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6959441PMC
October 2019

Keeping the Engine Running: The Relevance and Predictive Value of Preclinical Models for CAR-T Cell Development.

ILAR J 2018 12;59(3):276-285

Department of Pathology and Laboratory Medicine, and Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania; and Parker Institute for Cancer Immunotherapy; and Corporal Michael J. Crescenz VA Medical Center, Philadelphia, Pennsylvania.

The cellular immunotherapy field has achieved important milestones in the last 30 years towards the treatment of a variety of cancers due to improvements in ex-vivo T cell manufacturing processes, the invention of synthetic T cell receptors, and advances in cellular engineering. Here, we discuss major preclinical models that have been useful for the validation of chimeric antigen receptor (CAR)-T cell therapies and also promising new models that will fuel future investigations towards success. However, multiple unanswered questions in the CAR-T cell field remain to be addressed that will require innovative preclinical models. Key challenges facing the field include premature immune rejection of universal CAR-T cells and the immune suppressive tumor microenvironment. Immune competent models that accurately recapitulate tumor heterogeneity, the hostile tumor microenvironment, and barriers to CAR-T cell homing, toxicity, and persistence are needed for further advancement of the field.
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http://dx.doi.org/10.1093/ilar/ilz009DOI Listing
December 2018

Checkpoint Blockade Reverses Anergy in IL-13Rα2 Humanized scFv-Based CAR T Cells to Treat Murine and Canine Gliomas.

Mol Ther Oncolytics 2018 Dec 28;11:20-38. Epub 2018 Aug 28.

Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

We generated two humanized interleukin-13 receptor α2 (IL-13Rα2) chimeric antigen receptors (CARs), Hu07BBz and Hu08BBz, that recognized human IL-13Rα2, but not IL-13Rα1. Hu08BBz also recognized canine IL-13Rα2. Both of these CAR T cell constructs demonstrated superior tumor inhibitory effects in a subcutaneous xenograft model of human glioma compared with a humanized EGFRvIII CAR T construct used in a recent phase 1 clinical trial (ClinicalTrials.gov: NCT02209376). The Hu08BBz demonstrated a 75% reduction in orthotopic tumor growth using low-dose CAR T cell infusion. Using combination therapy with immune checkpoint blockade, humanized IL-13Rα2 CAR T cells performed significantly better when combined with CTLA-4 blockade, and humanized EGFRvIII CAR T cells' efficacy was improved by PD-1 and TIM-3 blockade in the same mouse model, which was correlated with the levels of checkpoint molecule expression in co-cultures with the same tumor . Humanized IL-13Rα2 CAR T cells also demonstrated benefit from a self-secreted anti-CTLA-4 minibody in the same mouse model. In addition to a canine glioma cell line (J3T), canine osteosarcoma lung cancer and leukemia cell lines also express IL-13Rα2 and were recognized by Hu08BBz. Canine IL-13Rα2 CAR T cell was also generated and tested by co-culture with canine tumor cells and in an orthotopic model of canine glioma. Based on these results, we are designing a pre-clinical trial to evaluate the safety of canine IL-13Rα2 CAR T cells in dog with spontaneous IL-13Rα2-positive glioma, which will help to inform a human clinical trial design for glioblastoma using humanized scFv-based IL-13Rα2 targeting CAR T cells.
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http://dx.doi.org/10.1016/j.omto.2018.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6174845PMC
December 2018

Actionable mutations in canine hemangiosarcoma.

PLoS One 2017 30;12(11):e0188667. Epub 2017 Nov 30.

Department of Pathology and Laboratory Medicine, Raymond and Ruth Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States of America.

Background: Angiosarcomas (AS) are rare in humans, but they are a deadly subtype of soft tissue sarcoma. Discovery sequencing in AS, especially the visceral form, is hampered by the rarity of cases. Most diagnostic material exists as archival formalin fixed, paraffin embedded tissue which serves as a poor source of high quality DNA for genome-wide sequencing. We approached this problem through comparative genomics. We hypothesized that exome sequencing a histologically similar tumor, hemangiosarcoma (HSA), that occurs in approximately 50,000 dogs per year, may lead to the identification of potential oncogenic drivers and druggable targets that could also occur in angiosarcoma.

Methods: Splenic hemangiosarcomas are common in dogs, which allowed us to collect a cohort of archived matched tumor and normal tissue samples suitable for whole exome sequencing. Mapping of the reads to the latest canine reference genome (Canfam3) demonstrated that >99% of the targeted exomal regions were covered, with >80% at 20X coverage and >90% at 10X coverage.

Results And Conclusions: Sequence analysis of 20 samples identified somatic mutations in PIK3CA, TP53, PTEN, and PLCG1, all of which correspond to well-known tumor drivers in human cancer, in more than half of the cases. In one case, we identified a mutation in PLCG1 identical to a mutation observed previously in this gene in human visceral AS. Activating PIK3CA mutations present novel therapeutic targets, and clinical trials of targeted inhibitors are underway in human cancers. Our results lay a foundation for similar clinical trials in canine HSA, enabling a precision medicine approach to this disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0188667PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5708669PMC
December 2017

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

Clin Transl Med 2016 Aug;5(Suppl 1):26

VetStem Biopharma, Inc., Poway, CA, 92064, USA.

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.
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http://dx.doi.org/10.1186/s40169-016-0107-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4996801PMC
August 2016

Feasibility and Safety of RNA-transfected CD20-specific Chimeric Antigen Receptor T Cells in Dogs with Spontaneous B Cell Lymphoma.

Mol Ther 2016 09 12;24(9):1602-14. Epub 2016 Jul 12.

Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Preclinical murine models of chimeric antigen receptor (CAR) T cell therapy are widely applied, but are greatly limited by their inability to model the complex human tumor microenvironment and adequately predict safety and efficacy in patients. We therefore sought to develop a system that would enable us to evaluate CAR T cell therapies in dogs with spontaneous cancers. We developed an expansion methodology that yields large numbers of canine T cells from normal or lymphoma-diseased dogs. mRNA electroporation was utilized to express a first-generation canine CD20-specific CAR in expanded T cells. The canine CD20 (cCD20) CAR expression was efficient and transient, and electroporated T cells exhibited antigen-specific interferon-gamma (IFN-γ) secretion and lysed cCD20+ targets. In a first-in-canine study, autologous cCD20-ζ CAR T cells were administered to a dog with relapsed B cell lymphoma. Treatment was well tolerated and led to a modest, but transient, antitumor activity, suggesting that stable CAR expression will be necessary for durable clinical remissions. Our study establishes the methodologies necessary to evaluate CAR T cell therapy in dogs with spontaneous malignancies and lays the foundation for use of outbred canine cancer patients to evaluate the safety and efficacy of next-generation CAR therapies and their optimization prior to translation into humans.
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http://dx.doi.org/10.1038/mt.2016.146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5113111PMC
September 2016

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

Clin Cancer Res 2016 Sep 18;22(17):4380-90. Epub 2016 Mar 18.

Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania.

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.
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http://dx.doi.org/10.1158/1078-0432.CCR-16-0088DOI Listing
September 2016

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

Methods Mol Biol 2015 ;1280:469-504

Department of Professional Studies in the Health Sciences, Drexel University, Philadelphia, PA, 19102, USA.

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.
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http://dx.doi.org/10.1007/978-1-4939-2422-6_29DOI Listing
November 2015

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.

PLoS One 2014 5;9(5):e95404. Epub 2014 May 5.

Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America; Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0095404PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4010398PMC
June 2015

Polymorphisms in canine platelet glycoproteins identify potential platelet antigens.

Comp Med 2013 Aug;63(4):348-54

Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Human alloimmune thrombocytopenic conditions caused by exposure to a platelet-specific alloantigen include neonatal alloimmune thrombocytopenia, posttransfusion purpura, and platelet transfusion refractoriness. More than 30 platelet-specific alloantigens have been defined in the human platelet antigen (HPA) system; however, there is no previous information on canine platelet-specific alloantigens. Using the HPA system as a model, we evaluated the canine ITGB3, ITGA2B, and GP1BB genes encoding GPIIIa (β3), GPIIb (αIIb), and GPIbβ, respectively, which account for 21 of 27 HPA, to determine whether amino acid polymorphisms are present in the orthologous canine genes. A secondary objective was to perform a pilot study to assess possible association between specific alleles of these proteins and a diagnosis of idiopathic thrombocytopenic purpura (ITP) in dogs. By using genomic DNA from dogs of various breeds with and without ITP, sequencing of PCR products encompassing all coding regions and exon-intron boundaries for these 3 genes revealed 4 single-nucleotide polymorphisms in ITGA2B resulting in amino acid polymorphisms in the canine genome, 3 previously reported and 1 newly identified (Gly[GGG]/Arg[AGG] at amino acid position 576 of ITGA2B. Of 16 possible ITGA2B protein alleles resulting from unique combinations of the 4 polymorphic amino acids, 5 different protein isoforms were present in homozygous dogs and explain all of the genotype combinations in heterozygous dogs. There was no amino acid polymorphism or protein isoform that was specific for a particular breed or for the diagnosis of ITP.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3750671PMC
August 2013

Evidence of an oncogenic gammaherpesvirus in domestic dogs.

Virology 2012 Jun 8;427(2):107-17. Epub 2012 Mar 8.

Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104-6010, USA.

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.
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http://dx.doi.org/10.1016/j.virol.2012.02.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4592777PMC
June 2012

CD40 ligand is necessary and sufficient to support primary diffuse large B-cell lymphoma cells in culture: a tool for in vitro preclinical studies with primary B-cell malignancies.

Leuk Lymphoma 2012 Jul 3;53(7):1390-8. Epub 2012 Feb 3.

Masonic Cancer Center, University of Minnesota, Minneapolis, MN 5455, USA.

Established cell lines are utilized extensively to study tumor biology and preclinical therapeutic development. However, they may not accurately recapitulate the heterogeneity of their corresponding primary disease. B-cell tumor cells are especially difficult to maintain under conventional culture conditions, limiting access to samples that faithfully represent this disease for preclinical studies. Here, we used primary canine diffuse large B-cell lymphoma to establish a culture system that reliably supports the growth of these cells. CD40 ligand, either expressed by feeder cells or provided as a soluble two-trimeric form, was sufficient to support primary lymphoma cells in vitro. The tumor cells retained their original phenotype, clonality and known karyotypic abnormalities after extended expansion in culture. Finally, we illustrate the utility of the feeder cell-free culture system for comparable assessment of cytotoxicity using dog and human B-cell malignancies. We conclude that this system has broad applications for in vitro preclinical development for B-cell malignancies.
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http://dx.doi.org/10.3109/10428194.2011.654337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3727651PMC
July 2012

CD40-activated B cell cancer vaccine improves second clinical remission and survival in privately owned dogs with non-Hodgkin's lymphoma.

PLoS One 2011 31;6(8):e24167. Epub 2011 Aug 31.

Department of Clinical Studies, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

Cell-based active immunotherapy for cancer is a promising novel strategy, with the first dendritic cell (DC) vaccine achieving regulatory approval for clinical use last year. Manufacturing remains arduous, especially for DC vaccines, and the prospect of using cell-based immunotherapy in the adjuvant setting or in combination with chemotherapy remains largely untested. Here, we used a comparative oncology approach to test the safety and potential efficacy of tumor RNA-loaded, CD40-activated B cells in privately owned dogs presenting with non-Hodgkin's lymphoma (NHL), a clinical scenario that represents not only a major problem in veterinary medicine but also a bona fide spontaneous animal model for the human condition. When administered to NHL dogs in remission after induction chemotherapy, CD40-B cells electroporated ex vivo with autologous tumor RNA safely stimulated immunity in vivo. Although chemotherapy plus CD40-B vaccination did not improve time-to-progression or lymphoma-specific survival compared to dogs treated with chemotherapy alone, vaccination potentiated the effects of salvage therapy and improved the rate of durable second remissions as well as subsequent lymphoma-specific survival following salvage therapy. Several of these relapsed dogs are now long-term survivors and free of disease for more than a year. Overall, these clinical and immunological results suggest that cell-based CD40 cancer vaccination is safe and synergizes with chemotherapy to improve clinical outcome in canine NHL. More broadly, our findings underscore the unique value of clinical investigations in tumor-bearing companion animals.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0024167PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3164165PMC
April 2012

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.

Clin Cancer Res 2011 Jul 24;17(14):4661-71. Epub 2011 May 24.

Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6010, USA.

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.
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http://dx.doi.org/10.1158/1078-0432.CCR-10-3310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3273413PMC
July 2011

Evaluation of equine peripheral blood apheresis product, bone marrow, and adipose tissue as sources of mesenchymal stem cells and their differentation potential.

Am J Vet Res 2011 Jan;72(1):127-33

Department of Clinical Studies-New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA 19348, USA.

Objective: To evaluate effects of apheresis on mesenchymal stem cells (MSCs) and compare those MSCs with MSCs obtained from adipose tissue or bone marrow (BM).

Sample Population: Samples obtained from 6 adult horses.

Procedures: Samples of blood from a peripheral vein, adipose tissue, and BM aspirate were obtained from each horse. Samples were processed via apheresis of blood and techniques reported elsewhere for adipose tissue and BM. Cultures were maintained until adherence and subsequently were subjected to differentiation protocols to evaluate adipogenic, osteoblastogenic, and chondrogenic potential.

Results: Apheresis product had a significantly higher mononuclear percentage, higher platelet count, and lower RBC count, compared with values for peripheral blood. No cell adherence to the tissue culture plates was detected for the apheresis product. Adherence was detected for 6 of 6 adipose-derived and 4 of 6 BM-derived samples. Variations in efficiency were detected for differentiation of adipose- and BM-derived cells into adipocytes, chondrocytes, and osteoblasts.

Conclusions And Clinical Relevance: Apheresis was able to concentrate mononuclear cells and reduce RBC contamination. However, the apheresis product was unable to adhere to the tissue culture plates. In matched horses, adipose- and BM-derived MSCs were capable of producing lipids, glycosaminoglycan, and mineral. The BM was vastly superior to adipose tissue as a source of MSCs with osteoblastogenic potential in matched horses. Additional studies will be necessary to optimize apheresis techniques for horses before peripheral blood can be considered a suitable source for multipotential cells for use in cell-based treatments.
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http://dx.doi.org/10.2460/ajvr.72.1.127DOI Listing
January 2011

Generation and validation of canine single chain variable fragment phage display libraries.

Vet Immunol Immunopathol 2011 Jan 7;139(1):27-40. Epub 2010 Aug 7.

Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, 19106, USA.

Single chain variable region fragments (scFvs) are composed of an immunoglobulin (Ig) variable heavy (VH) and variable light (VL) chain joined by a flexible serine-glycine linker. They represent the smallest antibody fragments that maintain antigen specificity and they hold significant potential for therapeutic antigen targeting in vivo. Here we report on the design and validation of a series of degenerate primers that amplify the recombined variable regions of canine Ig heavy and light chain genes from lymphocyte cDNA. We show that these VH and VL amplicons can be randomly combined by a flexible linker using splicing by overlap extension PCR to form scFv constructs that can be expressed on the surface of M13 bacteriophage. To demonstrate that scFvs with specificity for previously encountered antigens are contained within these scFv phage display libraries we used simple panning procedures to isolate canine parvovirus (CPV) specific scFvs from a library made from the splenocytes of a dog immunized against CPV. These studies reveal the feasibility of this approach for generating diverse canine scFv libraries and pave the way toward future studies to isolate canine antigen-specific scFv of interest that may be tested as targeting agents for the treatment of infectious, inflammatory and neoplastic diseases in the dog.
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http://dx.doi.org/10.1016/j.vetimm.2010.07.026DOI Listing
January 2011

Clinical and clinicopathologic effects of plateletpheresis on healthy donor dogs.

Transfusion 2008 Oct 28;48(10):2214-21. Epub 2008 Jun 28.

Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6010, USA.

Background: The safety and feasibility of plateletpheresis using a commercially available apheresis system (COBE Spectra, Gambro BCT) were evaluated in donor dogs, with characterization of its clinical and clinicopathologic effects.

Study Design And Methods: Fourteen adult dogs (18-27.7 kg) underwent a plateletpheresis procedure. Complete blood counts were obtained at baseline, 2 hours after apheresis, and daily for 1 week. Blood was collected every 15 minutes for acid-base and electrolyte analysis and measurement of serum citrate concentration. Dogs were monitored by continuous electrocardiogram and indirect blood pressure measurement. All dogs received prophylactic calcium (Ca) supplementation (10% Ca gluconate infusion at 15 mL/hr [139.5 mg Ca ion/hr]; the rate was increased based on serial measurement of ionized Ca [iCa] concentration).

Results: A high-quality platelet concentrate (PC) was collected, with a mean total yield of 3.3 x 10(11) platelets (PLTs). The mean donor PLT count decreased from 356 x 10(9) to 159 x 10(9) per L after apheresis. The procedure was generally well tolerated, with no evidence of hypotension. Serum citrate concentration progressively increased, causing the ionized magnesium concentration to decrease by 45 percent and iCa to decrease to less than 1 mmol per L (mean baseline, 1.2 mmol/L) in 10 dogs, despite receiving 0.9 mg of Ca ion per mL acid-citrate-dextrose formula A. Lip licking was noted in 3 dogs, and generalized tremors and ventricular ectopy were noted in 1 dog.

Conclusion: Canine plateletpheresis using the COBE Spectra is a feasible option for production of a PC. Hypocalcemia, however, is a potential serious adverse effect of plateletpheresis in dogs. Ca supplementation is recommended to limit clinical signs of hypocalcemia during the procedure.
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http://dx.doi.org/10.1111/j.1537-2995.2008.01803.xDOI Listing
October 2008

TRAF6-dependent mitogen-activated protein kinase activation differentially regulates the production of interleukin-12 by macrophages in response to Toxoplasma gondii.

Infect Immun 2004 Oct;72(10):5662-7

Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

The production of interleukin-12 (IL-12) is critical to the development of innate and adaptive immune responses required for the control of intracellular pathogens. Many microbial products signal through Toll-like receptors (TLR) and activate NF-kappaB family members that are required for the production of IL-12. Recent studies suggest that components of the TLR pathway are required for the production of IL-12 in response to the parasite Toxoplasma gondii; however, the production of IL-12 in response to this parasite is independent of NF-kappaB activation. The adaptor molecule TRAF6 is involved in TLR signaling pathways and associates with serine/threonine kinases involved in the activation of both NF-kappaB and mitogen-activated protein kinase (MAPK). To elucidate the intracellular signaling pathways involved in the production of IL-12 in response to soluble toxoplasma antigen (STAg), wild-type and TRAF6(-/-) mice were inoculated with STAg, and the production of IL-12(p40) was determined. TRAF6(-/-) mice failed to produce IL-12(p40) in response to STAg, and TRAF6(-/-) macrophages stimulated with STAg also failed to produce IL-12(p40). Studies using Western blot analysis of wild-type and TRAF6(-/-) macrophages revealed that stimulation with STAg resulted in the rapid TRAF6-dependent phosphorylation of p38 and extracellular signal-related kinase, which differentially regulated the production of IL-12(p40). The studies presented here demonstrate for the first time that the production of IL-12(p40) in response to toxoplasma is dependent upon TRAF6 and p38 MAPK.
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http://dx.doi.org/10.1128/IAI.72.10.5662-5667.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC517541PMC
October 2004

New lessons from old pathogens: what parasitic infections have taught us about the role of nuclear factor-kappaB in the regulation of immunity.

Immunol Rev 2004 Oct;201:48-56

Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA.

The nuclear factor-kappaB (NF-kappaB) family of transcription factors is activated by many infectious and inflammatory stimuli. This family regulates the expression of multiple genes, whose products include cytokines, chemokines, adhesion molecules, and antiapoptotic factors that are important components of the innate and adaptive immune response. A prominent role of NF-kappaB transcription factors in resistance to a variety of infectious diseases was revealed by studies with mice that lack individual family members. However, little is known about the basis for these effects or about the role of individual family members during a coordinated immune response to infection. Diverse parasites such as Toxoplasma gondii, Leishmania major, and Trichuris muris provide a unique opportunity to understand the role of the NF-kappaB system in the development of innate and adaptive immunity to these infections. The basis for resistance and susceptibility to these parasites is well understood, and studies using these experimental systems have provided unique insights into the role of NF-kappaB in the regulation of T-helper 1 cell (Th1) and Th2 type responses. It has become clear that NF-kappaB family members have cell lineage-specific functions and that their relative importance varies with type of infection as well as route of pathogen entry. Thus, studies with models of parasitic infection have revealed that individual NF-kappaB family members perform distinct, nonoverlapping, and biologically significant functions in the regulation of immunity and inflammation.
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http://dx.doi.org/10.1111/j.0105-2896.2004.00189.xDOI Listing
October 2004

T cell-intrinsic expression of c-Rel regulates Th1 cell responses essential for resistance to Toxoplasma gondii.

J Immunol 2004 Mar;172(6):3704-11

Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

The ability of many microbial and inflammatory stimuli to activate members of the Rel/NF-kappaB family of transcription factors is associated with the regulation of innate and adaptive responses required to control infection. Individual family members play distinct roles during different infectious and inflammatory responses. For example, c-Rel is essential for the production of IL-12 in response to LPS, but dispensable for IL-12 production in response to Toxoplasma Ag. To assess the role of c-Rel during immunity to the intracellular pathogen Toxoplasma gondii, wild-type (WT) and c-Rel(-/-) mice were infected with Toxoplasma and the immune response was analyzed. c-Rel(-/-) mice developed severe toxoplasmic encephalitis with increased numbers of parasites compared with WT controls and succumbed to infection within 5-8 wk. Although increased susceptibility of c-Rel(-/-) mice was associated with decreased T cell activation, proliferation, and production of IFN-gamma, these mice were able to generate Th1 effector cells that were present in the brain during chronic infection. In vitro mixing studies using WT and c-Rel(-/-) dendritic cells and WT and c-Rel(-/-) TCR transgenic T cells indicated that c-Rel(-/-) dendritic cells are defective in their ability to stimulate T cell responses. However, when c-Rel(-/-) T cells were transferred into T cell-deficient hosts, early defects in T cell activation, proliferation, and IFN-gamma production persisted, and these mice remained susceptible to infection. Together, these studies indicate that although c-Rel is an important regulator of innate immune responses, it also plays an important role in optimization and maintenance of adaptive T cell responses during infection.
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http://dx.doi.org/10.4049/jimmunol.172.6.3704DOI Listing
March 2004