An imminent need for veterinary medical educators: are we facing a crisis?

A potential emerging shortage of veterinary medical educators requires the profession to acknowledge and understand the factors leading to this outcome. Expanding class sizes within existing schools and colleges of veterinary medicine and the expected expansion of new programs seeking AVMA-Council of Education accreditation have heightened the need to address an impending shortage of veterinary medical educators. A solution-oriented approach that accurately projects educator workforce needs and identifies factors contributing to the shortage requires effective collaboration across various partnering organizations to develop innovations in pedagogy and educational delivery methods. The veterinary profession must also identify and reduce disincentives that deter students and post-DVM trainees from pursuing careers in education. Finally, efforts at the state and federal level are critical to advocate for financial support and incentives for expansion of the veterinary medical educator workforce. Through these collective approaches and partnerships, the veterinary medical educator workforce can be strengthened to overcome obstacles for educating the next generation of veterinarians to meet societal needs.

Characterization of New Zealand White Rabbit Gut-Associated Lymphoid Tissues and Use as Viral Oncology Animal Model.

Rabbits have served as a valuable animal model for the pathogenesis of various human diseases, including those related to agents that gain entry through the gastrointestinal tract such as human T cell leukemia virus type 1. However, limited information is available regarding the spatial distribution and phenotypic characterization of major rabbit leukocyte populations in mucosa-associated lymphoid tissues. Herein, we describe the spatial distribution and phenotypic characterization of leukocytes from gut-associated lymphoid tissues (GALT) from 12-week-old New Zealand White rabbits. Our data indicate that rabbits have similar distribution of leukocyte subsets as humans, both in the GALT inductive and effector sites and in mesenteric lymph nodes, spleen, and peripheral blood. GALT inductive sites, including appendix, cecal tonsil, Peyer's patches, and ileocecal plaque, had variable B cell/T cell ratios (ranging from 4.0 to 0.8) with a predominance of CD4 T cells within the T cell population in all four tissues. Intraepithelial and lamina propria compartments contained mostly T cells, with CD4 T cells predominating in the lamina propria compartment and CD8 T cells predominating in the intraepithelial compartment. Mesenteric lymph node, peripheral blood, and splenic samples contained approximately equal percentages of B cells and T cells, with a high proportion of CD4 T cells compared with CD8 T cells. Collectively, our data indicate that New Zealand White rabbits are comparable with humans throughout their GALT and support future studies that use the rabbit model to study human gut-associated disease or infectious agents that gain entry by the oral route.

Animals Used in Research and Education, 1966-2016: Evolving Attitudes, Policies, and Relationships.

Since the inception of the Association of American Veterinary Medical Colleges (AAVMC), the use of animals in research and education has been a central element of the programs of member institutions. As veterinary education and research programs have evolved over the past 50 years, so too have societal views and regulatory policies. AAVMC member institutions have continually responded to these events by exchanging best practices in training their students in the framework of comparative medicine and the needs of society. Animals provide students and faculty with the tools to learn the fundamental knowledge and skills of veterinary medicine and scientific discovery. The study of animal models has contributed extensively to medicine, veterinary medicine, and basic sciences as these disciplines seek to understand life processes. Changing societal views over the past 50 years have provided active examination and continued refinement of the use of animals in veterinary medical education and research. The future use of animals to educate and train veterinarians will likely continue to evolve as technological advances are applied to experimental design and educational systems. Natural animal models of both human and animal health will undoubtedly continue to serve a significant role in the education of veterinarians and in the development of new treatments of animal and human disease. As it looks to the future, the AAVMC as an organization will need to continue to support and promote best practices in the humane care and appropriate use of animals in both education and research.

Acetylation of the c-MYC oncoprotein is required for cooperation with the HTLV-1 p30(II) accessory protein and the induction of oncogenic cellular transformation by p30(II)/c-MYC.

The human T-cell leukemia retrovirus type-1 (HTLV-1) p30(II) protein is a multifunctional latency-maintenance factor that negatively regulates viral gene expression and deregulates host signaling pathways involved in aberrant T-cell growth and proliferation. We have previously demonstrated that p30(II) interacts with the c-MYC oncoprotein and enhances c-MYC-dependent transcriptional and oncogenic functions. However, the molecular and biochemical events that mediate the cooperation between p30(II) and c-MYC remain to be completely understood. Herein we demonstrate that p30(II) induces lysine-acetylation of the c-MYC oncoprotein. Acetylation-defective c-MYC Lys→Arg substitution mutants are impaired for oncogenic transformation with p30(II) in c-myc(-/-) HO15.19 fibroblasts. Using dual-chromatin-immunoprecipitations (dual-ChIPs), we further demonstrate that p30(II) is present in c-MYC-containing nucleoprotein complexes in HTLV-1-transformed HuT-102 T-lymphocytes. Moreover, p30(II) inhibits apoptosis in proliferating cells expressing c-MYC under conditions of genotoxic stress. These findings suggest that c-MYC-acetylation is required for the cooperation between p30(II)/c-MYC which could promote proviral replication and contribute to HTLV-1-induced carcinogenesis.

Animal models of bovine leukemia virus and human T-lymphotrophic virus type-1: insights in transmission and pathogenesis.

Bovine leukemia virus (BLV) and human T-lymphotrophic virus type-1 (HTLV-1) are related retroviruses associated with persistent and lifelong infections and a low incidence of lymphomas within their hosts. Both viruses can be spread through contact with bodily fluids containing infected cells, most often from mother to offspring through breast milk. Each of these complex retroviruses contains typical gag, pol, and env genes but also unique, nonstructural proteins encoded from the pX region. These nonstructural genes encode the Tax and Rex regulatory proteins, as well as novel proteins essential for viral spread in vivo. Improvements in the molecular tools to test these viral determinants in cellular and animal models have provided new insights into the pathogenesis of each virus. Comparisons of BLV and HTLV-1 provide insights into mechanisms of spread and tumor formation, as well as potential approaches to therapeutic intervention against the infections.

Comparative host protein interactions with HTLV-1 p30 and HTLV-2 p28: insights into difference in pathobiology of human retroviruses.

BACKGROUND: Human T lymphotropic virus type-1 (HTLV-1) and type 2 (HTLV-2) are closely related human retroviruses, but have unique disease associations. HTLV-1 is the causative agent of an aggressive T-cell leukemia known as adult T-cell leukemia (ATL), HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), and other inflammatory diseases. HTLV-2 infection has not been clearly associated with any disease condition. Although both viruses can transform T cells in vitro, the HTLV-1 provirus is mainly detected in CD4+ T cells whereas HTLV-2 is mainly detected in CD8+ T cells of infected individuals. HTLV-1 and HTLV-2 encode accessory proteins p30 and p28, respectively, which share partial amino acid homology and are required for viral persistence in vivo. The goal of this study was to identify host proteins interacting with p30 and p28 in order to understand their role in pathogenesis. RESULTS: Affinity-tag purification coupled with mass spectrometric (MS) analyses revealed 42 and 22 potential interacting cellular partners of p30 and p28, respectively. Of these, only three cellular proteins, protein arginine methyltransferase 5 (PRMT5), hnRNP K and 60 S ribosomal protein L8 were detected in both p30 and p28 fractions. To validate the proteomic results, four interacting proteins were selected for further analyses using immunoblot assays. In full agreement with the MS analysis two cellular proteins REGγ and NEAF-interacting protein 30 (NIP30) selectively interacted with p30 and not with p28; heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1) bound to p28 and not to p30; and PRMT5 interacted with both p30 and p28. Further studies demonstrated that reduced levels of PRMT5 resulted in decreased HTLV-2 viral gene expression whereas the viral gene expression of HTLV-1 was unchanged. CONCLUSION: The comparisons of p30 and p28 host protein interaction proteome showed striking differences with some degree of overlap. PRMT5, one of the host proteins that interacted with both p30 and p28 differentially affected HTLV-1 and HTLV-2 viral gene expression suggesting that PRMT5 is involved at different stages of HTLV-1 and HTLV-2 biology. These findings suggest that distinct host protein interaction profiles of p30 and p28 could, in part, be responsible for differences in HTLV-1 and HTLV-2 pathobiology. This study provides new avenues of investigation into mechanisms of viral infection, tropism and persistence.

Mechanisms of human T-lymphotropic virus type 1 transmission and disease.

Human T-lymphotrophic virus type-1 (HTLV-1) infects approximately 15-20 million people worldwide, with endemic areas in Japan, the Caribbean, and Africa. The virus is spread through contact with bodily fluids containing infected cells most often from mother to child through breast milk or via blood transfusion. After prolonged latency periods, approximately 3-5% of HTLV-1 infected individuals will develop either adult T-cell leukemia/lymphoma, or other lymphocyte-mediated disorders such as HTLV-1-associated myelopathy/tropical spastic paraparesis. The genome of this complex retrovirus contains typical gag, pol, and env genes, but also unique nonstructural proteins encoded from the pX region. These nonstructural genes encode the Tax and Rex regulatory proteins, as well as novel proteins essential for viral spread in vivo such as p30, p12, p13 and the antisense-encoded HTLV-1 basic leucine zipper factor (HBZ). While progress has been made in knowledge of viral determinants of cell transformation and host immune responses, host and viral determinants of HTLV-1 transmission and spread during the early phases of infection are unclear. Improvements in the molecular tools to test these viral determinants in cellular and animal models have provided new insights into the early events of HTLV-1 infection. This review will focus on studies that test HTLV-1 determinants in context to full-length infectious clones of the virus providing insights into the mechanisms of transmission and spread of HTLV-1.

Distinct transformation tropism exhibited by human T lymphotropic virus type 1 (HTLV-1) and HTLV-2 is the result of postinfection T cell clonal expansion.

Human T lymphotropic virus type 1 (HTLV-1) and HTLV-2 are related but pathogenically distinct viruses. HTLV-1 mainly causes adult T cell leukemia, while HTLV-2 is not associated with leukemia. In vitro, HTLV-1 and HTLV-2 predominantly transform CD4(+) and CD8(+) T cells, respectively: the genetic determinant maps to the viral envelope. Herein, we investigate whether this transformation tropism occurs during initial infection or subsequently during the cellular transformation process. Since most individuals are chronically infected at the time of detection, we utilized an established rabbit model to longitudinally measure the early HTLV-1 and HTLV-2 infection and replication kinetics in purified CD4(+) and CD8(+) T cells. HTLV-1 and HTLV-2 were detected in both CD4(+) and CD8(+) T cells within 1 week postinoculation. In HTLV-1-infected rabbit CD4(+) T cells, proviral burden and tax/rex mRNA expression peaked early, and expression levels were directly proportional to each other. The late expression of the antisense transcript (Hbz or Aph-2) correlated directly with a late proviral burden peak in HTLV-1- or HTLV-2-infected rabbit CD8(+) T cells, respectively. This study provides the first in vivo evidence that these viruses do not exhibit cellular preference during initial infection. We further evaluated the transformation tropism of HTLV-1 and HTLV-2 over a 9-week period using in vitro cell growth/immortalization assays. At the early weeks, both HTLV-1 and HTLV-2 showed proportionate growth of CD4(+) and CD8(+) T cells. However, beyond week 5, the predominance of one particular T cell type emerged, supporting the conclusion that transformation tropism is a postinfection event due to selective clonal expansion over time.

Conference highlights of the 15th International Conference on Human Retrovirology: HTLV and related retroviruses, 4-8 June 2011, Leuven, Gembloux, Belgium.

The June 2011 15th International Conference on Human Retrovirology: HTLV and Related Viruses marks approximately 30 years since the discovery of HTLV-1. As anticipated, a large number of abstracts were submitted and presented by scientists, new and old to the field of retrovirology, from all five continents. The aim of this review is to distribute the scientific highlights of the presentations as analysed and represented by experts in specific fields of epidemiology, clinical research, immunology, animal models, molecular and cellular biology, and virology.

Recombinant human T-cell leukemia virus types 1 and 2 Tax proteins induce high levels of CC-chemokines and downregulate CCR5 in human peripheral blood mononuclear cells.

Human T-cell leukemia viruses types 1 (HTLV-1) and 2 (HTLV-2) produce key transcriptional regulatory gene products, known as Tax1 and Tax2, respectively. Tax1 and Tax2 transactivate multiple host genes involved in cellular immune responses within the cellular microenvironment, including induction of genes encoding expression of CC-chemokines. It is speculated that HTLV Tax proteins may act as immune modulators. In this study, recombinant Tax1 and Tax2 proteins were tested for their effects on the viability of cultured peripheral blood mononuclear cells (PBMCs), and their ability to induce expression of CC-chemokines and to downregulate the level of CCR5 expression in PBMCs. PBMCs obtained from uninfected donors were cultured in a range of Tax1 and Tax2 concentrations (10-100 pM), and supernatant fluids were harvested at multiple time points for quantitative determinations of MIP-1α/CCL3, MIP-1ß/CCL4, and RANTES/CCL5. Treatment of PBMCs with Tax1 and Tax2 proteins (100 pM) resulted in a significant increase in viability over a 7-d period compared to controls (p<0.01). Both Tax1 and Tax2 induced high levels of all three CC-chemokines over the dosing range compared to mock-treated controls (p<0.05). The gated population of lymphocytes treated with Tax2, as well as lymphocytes from HTLV-2-infected donors, showed a significantly lower percentage of CCR5-positive cells compared to those of uninfected donors and from mock-treated lymphocytes, respectively (p<0.05). These results suggest that Tax1 and Tax2 could promote innate immunity in the extracellular environment during HTLV-1 and HTLV-2 infections via CC-chemokine ligands and receptors.

Molecular determinants of human T-lymphotropic virus type 1 transmission and spread.

Human T-lymphotrophic virus type-1 (HTLV-1) infects approximately 15 to 20 million people worldwide, with endemic areas in Japan, the Caribbean, and Africa. The virus is spread through contact with bodily fluids containing infected cells, most often from mother to child through breast milk or via blood transfusion. After prolonged latency periods, approximately 3 to 5% of HTLV-1 infected individuals will develop either adult T-cell leukemia/lymphoma (ATL), or other lymphocyte-mediated disorders such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The genome of this complex retrovirus contains typical gag, pol, and env genes, but also unique nonstructural proteins encoded from the pX region. These nonstructural genes encode the Tax and Rex regulatory proteins, as well as novel proteins essential for viral spread in vivo such as, p30, p12, p13 and the antisense encoded HBZ. While progress has been made in the understanding of viral determinants of cell transformation and host immune responses, host and viral determinants of HTLV-1 transmission and spread during the early phases of infection are unclear. Improvements in the molecular tools to test these viral determinants in cellular and animal models have provided new insights into the early events of HTLV-1 infection. This review will focus on studies that test HTLV-1 determinants in context to full length infectious clones of the virus providing insights into the mechanisms of transmission and spread of HTLV-1.

Efficacy of novel histone deacetylase inhibitor, AR42, in a mouse model of, human T-lymphotropic virus type 1 adult T cell lymphoma.

Human T-lymphotropic virus type 1 (HTLV-1) causes a variety of forms of adult T-cell leukemia/lymphoma (ATL), a refractory CD4+/CD25+ T-cell malignancy. Novel approaches to treat ATL patients are required due to the resistance of ATL to conventional chemotherapies. Histone deacetylase inhibitors (HDACi), which induce histone hyperacetylation leading to chromatin remodeling and reactivation of transcriptionally repressed genes have shown efficacy against a variety of cancers. Herein, we tested if valproic acid and the novel orally bioavailable HDACi, AR-42 reduced the proliferation of ATL cell lines by promoting apoptosis and histone hyperacetylation. Both compounds were cytotoxic and elicited a dose dependent increase in cytochrome C and cleaved Poly (ADP-ribose) polymerase (PARP) indicating the induction of cell death by apoptosis and promoted acetylation of histone H3 in both MT-2 and C8166 cell lines. We then evaluated the effects of AR-42, for survival in an ATL NOD/SCID mouse model. A dietary formulation of AR-42 prolonged survival of ATL engrafted mice compared to controls. Our data provide new directions for the treatment of ATL and support the further development of AR-42 against HTLV-1-associated lymphoid malignancies.

Human T-lymphotropic virus type 1 p30 interacts with REGgamma and modulates ATM (ataxia telangiectasia mutated) to promote cell survival.

Human T-lymphotropic virus type 1 (HTLV-1) is a causative agent of adult T cell leukemia/lymphoma and a variety of inflammatory disorders. HTLV-1 encodes a nuclear localizing protein, p30, that selectively alters viral and cellular gene expression, activates G(2)-M cell cycle checkpoints, and is essential for viral spread. Here, we used immunoprecipitation and affinity pulldown of ectopically expressed p30 coupled with mass spectrometry to identify cellular binding partners of p30. Our data indicate that p30 specifically binds to cellular ATM (ataxia telangiectasia mutated) and REGγ (a nuclear 20 S proteasome activator). Under conditions of genotoxic stress, p30 expression was associated with reduced levels of ATM and increased cell survival. Knockdown or overexpression of REGγ paralleled p30 expression, suggesting an unexpected enhancement of p30 expression in the presence of REGγ. Finally, size exclusion chromatography revealed the presence of p30 in a high molecular mass complex along with ATM and REGγ. On the basis of our findings, we propose that HTLV-1 p30 interacts with ATM and REGγ to increase viral spread by facilitating cell survival.

Early spatial and temporal events of human T-lymphotropic virus type 1 spread following blood-borne transmission in a rabbit model of infection.

Human T-lymphotropic virus type 1 (HTLV-1) infection causes adult T-cell leukemia/lymphoma (ATL) and is associated with a variety of lymphocyte-mediated disorders. HTLV-1 transmission occurs by transmission of infected cells via breast-feeding by infected mothers, sexual intercourse, and contaminated blood products. The route of exposure and early virus replication events are believed to be key determinants of virus-associated spread, antiviral immune responses, and ultimately disease outcomes. The lack of knowledge of early events of HTLV-1 spread following blood-borne transmission of the virus in vivo hinders a more complete understanding of the immunopathogenesis of HTLV-1 infections. Herein, we have used an established animal model of HTLV-1 infection to study early spatial and temporal events of the viral infection. Twelve-week-old rabbits were injected intravenously with cell-associated HTLV-1 (ACH-transformed R49). Blood and tissues were collected at defined intervals throughout the study to test the early spread of the infection. Antibody and hematologic responses were monitored throughout the infection. HTLV-1 intracellular Tax and soluble p19 matrix were tested from ex vivo cultured lymphocytes. Proviral copy numbers were measured by real-time PCR from blood and tissue mononuclear leukocytes. Our data indicate that intravenous infection with cell-associated HTLV-1 targets lymphocytes located in both primary lymphoid and gut-associated lymphoid compartments. A transient lymphocytosis that correlated with peak virus detection parameters was observed by 1 week postinfection before returning to baseline levels. Our data support emerging evidence that HTLV-1 promotes lymphocyte proliferation preceding early viral spread in lymphoid compartments to establish and maintain persistent infection.

Adult T-cell leukemia/lymphoma development in HTLV-1-infected humanized SCID mice.

The molecular and genetic factors induced by human T-lymphotropic virus type-1 (HTLV-1) that initiate adult T-cell leukemia/lymphoma (ATLL) remain unclear, in part from the lack of an animal model that accurately recapitulates leukemogenesis. HTLV-1-infected humanized nonobese diabetic severe combined immunodeficiency (HU-NOD/SCID) mice were generated by inoculation of NOD/SCID mice with CD34(+) hematopoietic progenitor and stem cells (CD34(+) HP/HSCs) infected ex vivo with HTLV-1. HTLV-1-HU-NOD/SCID mice exclusively developed CD4(+) T-cell lymphomas with characteristics similar to ATLL and elevated proliferation of infected human stem cells (CD34(+)CD38(-)) in the bone marrow were observed in mice developing malignancies. Purified CD34(+) HP/HSCs from HTLV-1-infected patient peripheral blood mononuclear cells revealed proviral integrations suggesting viral infection of human bone marrow-derived stem cells. NOD/SCID mice reconstituted with CD34(+) HP/HSCs transduced with a lentivirus vector expressing the HTLV-1 oncoprotein (Tax1) also developed CD4(+) lymphomas. The recapitulation of a CD4(+) T-cell lymphoma in HU-NOD/SCID mice suggests that HSCs provide a viral reservoir in vivo and act as cellular targets for cell transformation in humans. This animal model of ATLL will provide an important tool for the identification of molecular and cellular events that control the initiation and progression of the lymphoma and potential therapeutic targets to block tumor development.

The ARF tumor suppressor regulates bone remodeling and osteosarcoma development in mice.

The ARF tumor suppressor regulates p53 as well as basic developmental processes independent of p53, including osteoclast activation, by controlling ribosomal biogenesis. Here we provide evidence that ARF is a master regulator of bone remodeling and osteosarcoma (OS) development in mice. Arf(-/-) mice displayed increased osteoblast (OB) and osteoclast (OC) activity with a significant net increase in trabecular bone volume. The long bones of Arf(-/-) mice had increased expression of OB genes while Arf(-/-) OB showed enhanced differentiation in vitro. Mice transgenic for the Tax oncogene develop lymphocytic tumors with associated osteolytic lesions, while Tax+Arf(-/-) mice uniformly developed spontaneous OS by 7 months of age. Tax+Arf(-/-) tumors were well differentiated OS characterized by an abundance of new bone with OC recruitment, expressed OB markers and displayed intact levels of p53 mRNA and reduced Rb transcript levels. Cell lines established from OS recapitulated characteristics of the primary tumor, including the expression of mature OB markers and ability to form mineralized tumors when transplanted. Loss of heterozygosity in OS tumors arising in Tax+Arf(+/-) mice emphasized the necessity of ARF-loss in OS development. Hypothesizing that inhibition of ARF-regulated bone remodeling would repress development of OS, we demonstrated that treatment of Tax+Arf(-/-) mice with zoledronic acid, a bisphosphonate inhibitor of OC activity and repressor of bone turnover, prevented or delayed the onset of OS. These data describe a novel role for ARF as a regulator of bone remodeling through effects on both OB and OC. Finally, these data underscore the potential of targeting bone remodeling as adjuvant therapy or in patients with genetic predispositions to prevent the development of OS.

Cyclosporine-induced immune suppression alters establishment of HTLV-1 infection in a rabbit model.

Human T-lymphotropic virus type 1 (HTLV-1) infection causes adult T-cell leukemia and several lymphocyte-mediated inflammatory diseases. Persistent HTLV-1 infection is determined by a balance between host immune responses and virus spread. Immunomodulatory therapy involving HTLV-1-infected patients occurs in a variety of clinical settings. Knowledge of how these treatments influence host-virus relationships is not understood. In this study, we examined the effects of cyclosporine A (CsA)-induced immune suppression during early infection of HTLV-1. Twenty-four New Zealand white rabbits were split into 4 groups. Three groups were treated with either 10 or 20 mg/kg CsA or saline before infection. The fourth group was treated with 20 mg/kg CsA 1 week after infection. Immune suppression, plasma CsA concentration, ex vivo lymphocyte HTLV-1 p19 production, anti-HTLV-1 serologic responses, and proviral load levels were measured during infection. Our data indicated that CsA treatment before HTLV-1 infection enhanced early viral expression compared with untreated HTLV-1-infected rabbits, and altered long-term viral expression parameters. However, CsA treatment 1 week after infection diminished HTLV-1 expression throughout the 10-week study course. Collectively, these data indicate immunologic control is a key determinant of early HTLV-1 spread and have important implications for therapeutic intervention during HTLV-1-associated diseases.