Dual blockade of the PI3K/Akt/mTOR pathway inhibits posttransplant Epstein-Barr virus B cell lymphomas and promotes allograft survival.

Posttransplant lymphoproliferative disorder (PTLD) is a serious complication of organ transplantation that often manifests as Epstein-Barr virus (EBV)-associated B cell lymphomas. Current treatments for PTLD have limited efficacy and can be associated with graft rejection or systemic toxicities. The mTOR inhibitor, rapamycin, suppresses tumor growth of EBV+ B cell lymphoma cells in vitro and in vivo; however, the efficacy is limited and clinical benefits of mTOR inhibitors for PTLD are variable. Here, we show constitutive activation of multiple nodes within the PI3K/Akt/mTOR pathway in EBV+ PTLD-derived cell lines. Inhibition of either PI3K or Akt, with specific inhibitors CAL-101 and MK-2206, respectively, diminished growth of EBV+ B cell lines from PTLD patients in a dose-dependent manner. Importantly, rapamycin combined with CAL-101 or MK-2206 had a synergistic effect in suppressing cell growth as determined by IC50 isobolographic analysis and Loewe indices. Moreover, these combinations were significantly more effective than rapamycin alone in inhibiting tumor xenograft growth in NOD-SCID mice. Finally, both CAL-101 and MK-2206 also prolonged survival of heterotopic cardiac allografts in C57BL/6 mice. Thus, combination therapy with rapamycin and a PI3K inhibitor, or an Akt inhibitor, can be an efficacious treatment for EBV-associated PTLD, while simultaneously promoting allograft survival.

Vaccination and Host Marek's Disease-Resistance Genotype Significantly Reduce Oncogenic Gallid alphaherpesvirus 2 Telomere Integration in Host Birds.

Marek's disease (MD) is an infectious disease characterized by lymphomas and high mortality in susceptible chickens. The causative and ubiquitous alpha-herpesvirus known as MD virus (MDV) integrates into host telomeres during early infection through latency, known to be an important phase for oncogenic transformation. Herein, we sought to determine the influence of vaccination and host genetics on the temporal dynamics of MDV-host genome interactions. We studied integration profiles using 2 MD vaccines that vary in protective efficacy in 2 genetic lines that differ in MD resistance/susceptibility. Virus integration of both oncogenic MDV and vaccine strains was observed in both MD susceptible and resistant birds, however, the lines differed in their dynamic telomere-integration profiles. Notably, the resistant host genotype exhibited a smaller percentage of replicating cells with the virus telomere-integrated only phenotype as compared to the susceptible genotype. Vaccination with Rispens, the most protective MD vaccine, also reduced the establishment of the virus telomere-integrated only phenotype, suggesting a significant role of the phenotype in MD lymphoma development. The effect of Rispens vaccination was most dramatic in the susceptible genotype. These results suggest important connections between vaccinal immunity, MDV telomere integration, virus-induced oncogenesis, and virus-host genome interactions in the context of host genetics and disease susceptibility.

Comparative cytogenomics of poultry: mapping of single gene and repeat loci in the Japanese quail (Coturnix japonica).

Well-characterized molecular and cytogenetic maps are yet to be established in Japanese quail (Coturnix japonica). The aim of the current study was to cytogenetically map and determine linkage of specific genes and gene complexes in Japanese quail through the use of chicken (Gallus gallus) and turkey (Meleagris gallopavo) genomic DNA probes and conduct a comparative study among the three genomes. Chicken and turkey clones were used as probes on mitotic metaphase and meiotic pachytene stage chromosomes of the three species for the purpose of high-resolution fluorescence in situ hybridization (FISH). The genes and complexes studied included telomerase RNA (TR), telomerase reverse transcriptase (TERT), 5S rDNA, 18S-5.8S-28S rDNA (i.e., nucleolus organizer region (NOR)), and the major histocompatibility complex (MHC). The telomeric profile of Japanese quail was investigated through the use of FISH with a TTAGGG-PNA probe. A range of telomeric array sizes were confirmed as found for the other poultry species. Three NOR loci were identified in Japanese quail, and single loci each for TR, TERT, 5S rDNA and the MHC-B. The MHC-B and one NOR locus were linked on a microchromosome in Japanese quail. We confirmed physical linkage of 5S rDNA and the TR gene on an intermediate-sized chromosome in quail, similar to both chicken and turkey. TERT localized to CJA 2 in quail and the orthologous chromosome region in chicken (GGA 2) and in turkey (MGA 3). The cytogenetic profile of Japanese quail was further developed by this study and synteny was identified among the three poultry species.

Marek's disease herpesvirus vaccines integrate into chicken host chromosomes yet lack a virus-host phenotype associated with oncogenic transformation.

Marek's disease (MD) is a lymphotropic and oncogenic disease of chickens that can lead to death in susceptible and unvaccinated host birds. The causative pathogen, MD virus (MDV), a highly oncogenic alphaherpesvirus, integrates into host genome near the telomeres. MD occurrence is controlled across the globe by biosecurity, selective breeding for enhanced MD genetic resistance, and widespread vaccination of flocks using attenuated serotype 1 MDV or other serotypes. Despite over 40 years of usage, the specific mechanism(s) of MD vaccine-related immunity and anti-tumor effects are not known. Here we investigated the cytogenetic interactions of commonly used MD vaccine strains of all three serotypes (HVT, SB-1, and Rispens) with the host to determine if all were equally capable of host genome integration. We also studied the dynamic profiles of chromosomal association and integration of the three vaccine strains, a first for MD vaccine research. Our cytogenetic data provide evidence that all three MD vaccine strains tested integrate in the chicken host genome as early as 1 day after vaccination similar to oncogenic strains. However, a specific, transformation-associated virus-host phenotype observed for oncogenic viruses is not established. Our results collectively provide an updated model of MD vaccine-host genome interaction and an improved understanding of the possible mechanisms of vaccinal immunity. Physical integration of the oncogenic MDV genome into host chromosomes along with cessation of viral replication appears to have joint signification in MDV's ability to induce oncogenic transformation. Whereas for MD vaccine serotypes, a sustained viral replication stage and lack of the chromosome-integrated only stage were shared traits during early infection.