Arun R. Pandiri

Polg2 is essential for mammalian embryogenesis and is required for mtDNA maintenance

Mammalian mitochondrial DNA (mtDNA) is replicated by the heterotrimeric Pol γ comprised of a single catalytic subunit, encoded by Polg, and a homodimeric accessory subunit encoded by the Polg2 gene. While the catalytic subunit has been shown to be essential for embryo development, genetic data regarding the accessory subunit are lacking in mammalian systems. Here, we describe the generation of heterozygous (Polg2(+/-)) and homozygous (Polg2(-/-)) knockout (KO) mice. Polg2(+/-) mice are haplosufficient and develop normally with no discernable difference in mitochondrial function through 2 years of age. In contrast, the Polg2(-/-) is embryonic lethal at day 8.0-8.5 p.c. with concomitant loss of mtDNA and mtDNA gene products. Electron microscopy shows severe ultra-structural defects and loss of organized cristae in mitochondria of the Polg2(-/-) embryos as well as an increase in lipid accumulation compared with both wild-type (WT) and Polg2(+/-) embryos. Our data indicate that Polg2 function is critical to mammalian embryogenesis and mtDNA replication, and that a single copy of Polg2 is sufficient to sustain life.

Global Gene Profiling of Spontaneous Hepatocellular Carcinoma in B6C3F1 Mice: Similarities in the Molecular Landscape with Human Liver Cancer

Hepatocellular carcinoma (HCC) is an important cause of morbidity and mortality worldwide. Although the risk factors of human HCC are well known, the molecular pathogenesis of this disease is complex, and in general, treatment options remain poor. The use of rodent models to study human cancer has been extensively pursued, both through genetically engineered rodents and rodent models used in carcinogenicity and toxicology studies. In particular, the B6C3F1 mouse used in the National Toxicology Program (NTP) two-year bioassay has been used to evaluate the carcinogenic effects of environmental and occupational chemicals, and other compounds. The high incidence of spontaneous HCC in the B6C3F1 mouse has challenged its use as a model for chemically induced HCC in terms of relevance to the human disease. Using global gene expression profiling, we identify the dysregulation of several mediators similarly altered in human HCC, including re-expression of fetal oncogenes, upregulation of protooncogenes, downregulation of tumor suppressor genes, and abnormal expression of cell cycle mediators, growth factors, apoptosis regulators, and angiogenesis and extracellular matrix remodeling factors. Although major differences in etiology and pathogenesis remain between human and mouse HCC, there are important similarities in global gene expression and molecular pathways dysregulated in mouse and human HCC. These data provide further support for the use of this model in hazard identification of compounds with potential human carcinogenicity risk, and may help in better understanding the mechanisms of tumorigenesis resulting from chemical exposure in the NTP two-year carcinogenicity bioassay.

Hepatocellular Carcinomas in B6C3F1 Mice Treated with Ginkgo biloba Extract for Two Years Differ from Spontaneous Liver Tumors in Cancer Gene Mutations and Genomic Pathways

Ginkgo biloba leaf extract (GBE) has been used for centuries in traditional Chinese medicine and today is used as an herbal supplement touted for improving neural function and for its antioxidant and anticancer effects. Herbal supplements have the potential for consumption over extended periods of time, with a general lack of sufficient data on long-term carcinogenicity risk. Exposure of B6C3F1 mice to GBE in the 2-year National Toxicology Program carcinogenicity bioassay resulted in a dose-dependent increase in hepatocellular tumors, including hepatocellular carcinoma (HCC). We show that the mechanism of hepatocarcinogenesis in GBE exposed animals is complex, involving alterations in H-ras and Ctnnb1 mutation spectra, WNT pathway dysregulation, and significantly altered gene expression associated with oncogenesis, HCC development, and chronic xenobiotic and oxidative stress compared to spontaneous HCC. This study provides a molecular context for the genetic changes associated with hepatocarcinogenesis in GBE exposed mice and illustrates the marked differences between these tumors and those arising spontaneously in the B6C3F1 mouse. The molecular changes observed in HCC from GBE-treated animals may be of relevance to those seen in human HCC and other types of cancer, and provide important data on potential mechanisms of GBE hepatocarcinogenesis.

Isolation and Characterization of an Adventitious Avian Leukosis Virus Isolated from Commercial Marek's Disease Vaccines

Abstract Commercial Mareks disease (MD) vaccines produced by two manufacturers were tested for possible contamination with avian leukosis virus (ALV). Samples of MD vaccines manufactured by two companies (A and B) were received from a breeder company; samples were also received directly from vaccine company B. Using virus isolation tests, samples initially tested positive for subgroup E (endogenous) ALV. However, upon repassage, the vaccines also tested positive for exogenous ALV. The isolated exogenous ALV proved to be a subgroup A virus, as determined by flow cytometry using polyclonal chicken antibodies specific for various subgroups of ALV, and by DNA sequencing of the envelope glygoprotein (gp85). The exogenous ALV isolated from MD vaccines was inoculated in chickens from ADOL lines 15I5 × 71 and 0 to determine its pathogenicity and compare it with that of Rous-associated-virus–1 (RAV-1), the prototype strain of ALV-A. Each chicken from each line was inoculated with approximately 10,000 infectious units of RAV-1 or the ALV-A isolated from vaccines termed B-39 virus at 7th day of embryonation. At hatch, and at 4, 8, and 16 wk of age, chickens were tested for viremia and cloacal shedding; chickens were also observed for ALV-induced tumors within 16 wk of age. Viremia and cloacal shedding results suggest that chickens from both lines were susceptible to infection with either virus. Within 16 wk of age, the proportion of ALV tumors induced by strain B-39 in line 0 and line 15I5 × 71 chickens was 0% and 12%, respectively, compared with 62% and 67% in chickens inoculated with RAV-1. The data indicate that commercial MD vaccines produced by two manufacturers were contaminated with endogenous subgroup E and an exogenous subgroup A ALV. Further, data from biological characterization suggest that the ALV-A isolated from commercial MD vaccines is of low oncogenicity, compared with that of RAV-1. GenBank accession numbers: The gp85 gene sequences of ALV isolated from commercial Mareks disease vaccines have been deposited in GenBank and assigned the following accession numbers: A46 subgroup A, DQ412726 ; B53 subgroup A, DQ412727; A46 subgroup E, DQ412728; B53 subgroup E, DQ412729.

Molecular and Biological Characterization of a Naturally Occurring Recombinant Subgroup B Avian Leukosis Virus with a Subgroup J–Like Long Terminal Repeat

Abstract Infection of broiler chickens with subgroup J avian leukosis virus (ALV) results in the induction of myeloid tumors. However, although egg-type chickens are susceptible to infection with ALV-J, the tumor incidence is very low, and on rare occasions the tumors observed are of the myeloid lineage. We recently described the isolation of an ALV (AF115-4) from commercial egg-type chickens suffering from myeloid leukosis. AF115-4 was initially identified as an ALV-J isolate based on PCR analysis of the long terminal repeat (LTR). However, further characterization of the viral envelope indicated that the virus is recombinant with subgroups B envelope and J LTR. Here we further characterize this recombinant virus at both the molecular and biological levels. We show that the AF115-4 isolate expresses a recombinant envelope glycoprotein encoded by a subgroup B gp85 region and a subgroup E gp37 region. The host range of AF115-4 was analyzed using cells resistant to infection by subgroups A/B, J, or E; this shows that no ALV-J was present in the isolates obtained from the affected chickens. Additional antigenic characterization of AF115-4 using chicken sera specific for subgroups B or J indicated that no ALV-J was present in the samples examined. Inoculation of AF115-4 into ALV-susceptible 15I5 × 71 chickens resulted in the induction of lymphoid leukosis but not the expected myeloid leukosis affecting the commercial chickens. These results suggest that differences in the genetic makeup of the chickens from which AF115-4 was isolated and the line 15I5 × 71 used in the present experiments may be responsible for the observed differences in pathogenicity. In addition, the results suggest that ALV-J continues to evolve by recombination, generating new viruses with different pathological properties.

Epibulbar melanoma in a foal.

A case of epibulbar melanoma in a 6-month-old, gelded, chestnut Hanoverian foal is reported. The location and clinical appearance upon initial presentation led to the tentative diagnosis of staphyloma or a congenital mass of unknown origin. An attempt was made to surgically excise the mass under general anesthesia, but due to its infiltrative nature and intraoperative appearance, most, but not all was removed without compromising the integrity of the globe. Histopathological evaluation revealed a multinodular to packeted, poorly demarcated, unencapsulated, infiltrative exophytic melanocytic neoplasm composed of bundles and nests of plump spindloid to polygonal heavily pigmented epithelioid neoplastic cells interspersed with pigment-laden macrophages within a fine fibrovascular stroma. Upon examination after enucleation, neoplastic cells were found to infiltrate into the lateral cornea, sclera and the choroid. This is a unique case of an epibulbar melanoma with choroidal invasion in a foal. Based on the sudden onset and rapid growth as well as the histological evidence of invasion, well-differentiated features, heavy pigmentation, and no apparent mitoses, this neoplasm was considered to be a low-grade malignant melanoma. At 14 months after excision there is no evidence of recurrence.

ATP Binding Cassette Transporter G1 Deletion Induces IL-17–Dependent Dysregulation of Pulmonary Adaptive Immunity

Mice with genetic deletion of the cholesterol transporter ATP binding cassette G1 (ABCG1) have pulmonary lipidosis and enhanced innate immune responses in the airway. Whether ABCG1 regulates adaptive immune responses to the environment is unknown. To this end, Abcg1+/+ and Abcg1−/− mice were sensitized to OVA via the airway using low-dose LPS as an adjuvant, and then challenged with OVA aerosol. Naive Abcg1−/− mice displayed increased B cells, CD4+ T cells, CD8+ T cells, and dendritic cells (DCs) in lung and lung-draining mediastinal lymph nodes, with lung CD11b+ DCs displaying increased CD80 and CD86. Upon allergen sensitization and challenge, the Abcg1−/− airway, compared with Abcg1+/+, displayed reduced Th2 responses (IL-4, IL-5, eosinophils), increased neutrophils and IL-17, but equivalent airway hyperresponsiveness. Reduced Th2 responses were also found using standard i.p. OVA sensitization with aluminum hydroxide adjuvant. Mediastinal lymph nodes from airway-sensitized Abcg1−/− mice produced reduced IL-5 upon ex vivo OVA challenge. Abcg1−/− CD4+ T cells displayed normal ex vivo differentiation, whereas Abcg1−/− DCs were found paradoxically to promote Th2 polarization. Th17 cells, IL-17+ γδT cells, and IL-17+ neutrophils were all increased in Abcg1−/− lungs, suggesting Th17 and non-Th17 sources of IL-17 excess. Neutralization of IL-17 prior to challenge normalized eosinophils and reduced neutrophilia in the Abcg1−/− airway. We conclude that Abcg1−/− mice display IL-17–mediated suppression of eosinophilia and enhancement of neutrophilia in the airway following allergen sensitization and challenge. These findings identify ABCG1 as a novel integrator of cholesterol homeostasis and adaptive immune programs.

Nonproliferative and Proliferative Lesions of the Gastrointestinal Tract, Pancreas and Salivary Glands of the Rat and Mouse

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) project is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature and diagnostic criteria for nonproliferative and proliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature and diagnostic criteria for classifying lesions in the digestive system including the salivary glands and the exocrine pancreas of laboratory rats and mice. Most lesions are illustrated by color photomicrographs. The standardized nomenclature, the diagnostic criteria, and the photomicrographs are also available electronically on the Internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous and age related lesions as well as lesions induced by exposure to test items. Relevant infectious and parasitic lesions are included as well. A widely accepted and utilized international harmonization of nomenclature and diagnostic criteria for the digestive system will decrease misunderstandings among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

Effect of Diluting Marek's Disease Vaccines on the Outcomes of Marek's Disease Virus Infection When Challenged with Highly Virulent Marek's Disease Viruses

Abstract Dilution of Mareks disease (MD) vaccines is a common practice in the field to reduce the cost associated with vaccination. In this study we have evaluated the effect of diluting MD vaccines on the protection against MD, vaccine and challenge MD virus (MDV) kinetics, and body weight when challenged with strains Md5 (very virulent MDV) and 648A (very virulent plus MDV) by contact at day of age. The following four vaccination protocols were evaluated in meat-type chickens: turkey herpesvirus (HVT) at manufacturer-recommended full dose; HVT diluted 1∶10; HVT + SB-1 at the manufacturer-recommended full dose; and HVT + SB-1 diluted 1∶10 for HVT and 1∶5 for SB-1. Vaccine was administered at hatch subcutaneously. One-day-old chickens were placed in floor pens and housed together with ten 15-day-old chickens that had been previously inoculated with 500 PFU of either Md5 or 648A MDV strains. Chickens were individually identified with wing bands, and for each chicken samples of feather pulp and blood were collected at 1, 3, and 8 wk posthatch. Body weights were recorded at 8 wk for every chicken. Viral DNA load of wild-type MDV, SB-1, and HVT were evaluated by real time-PCR. Our results showed that dilution of MD vaccines can lead to reduced MD protection, reduced relative body weights, reduced vaccine DNA during the first 3 wk, and increased MDV DNA load. The detrimental effect of vaccine dilution was more evident in females than in males and was more evident when the challenge virus was 648A. However, lower relative body weights and higher MDV DNA load could be detected in chickens challenged with strain Md5, even in the absence of obvious differences in protection.

Subgroup J Avian Leukosis Virus Neutralizing Antibody Escape Variants Contribute to Viral Persistence in Meat-Type Chickens

Abstract We have previously demonstrated a high incidence of chickens with persistent viremia even in the presence of neutralizing antibodies (V+A+) against the inoculated parental virus in commercial meat-type chickens inoculated at hatch with subgroup J avian leukosis virus (ALV J) field isolates. In this study, we used an ALV J molecular clone, ADOL pR5-4, to determine the role of neutralizing antibody (NAb) escape mutants in maintaining a high incidence of viral persistence, namely, V+A+ infection profile in commercial meat-type chickens. Chickens were housed as a flock in a pen or housed in isolation in solitary Horsfall-Bauer units for testing for NAb escape variants. The emergence of NAb escape variants was evaluated by sequential autologous virus neutralization (VN) (between virus and antibody from the same sampling period) and heterologous VN (between virus and antibody from preceding and succeeding sampling periods). Sequential virus isolates and corresponding antisera from 18 chickens were examined by VN matrix. In all chickens, autologous virus isolates were not neutralized by corresponding antisera. However, some of these resilient autologous virus isolates were neutralized by antibodies from subsequent sampling intervals. Nucleotide sequence analysis of consecutive isolates from three individually housed chickens with V+A+ infection profile revealed distinct changes within the envelope region, suggesting viral evolution to escape the host immune response. These results demonstrate that the emergence of antibody escape variants in commercial meat-type chickens contributes to ALV J persistence.