Evaluation of nucleosome concentrations in healthy dogs and dogs with cancer.

INTRODUCTION: Nucleosomes consist of small fragments of DNA wrapped around a histone octamer core. Diseases such as cancer or inflammation lead to cell death, which causes fragmentation and release of nucleosomes into the blood. The Nu.Q™ technology measures circulating nucleosome levels and exploits the different compositions of cancer derived nucleosomes in blood to detect and identify cancer even at early stages. The objectives of this study are to identify the optimal sample type for the Nu.Q™ H3.1 assay and to determine if it can accurately detect nucleosomes in the blood of healthy canines as well as those with cancer. MATERIALS AND METHODS: Blood samples from healthy canine volunteers as well as dogs newly diagnosed with lymphoma were used. The blood was processed at a variety of times under a variety of conditions to determine the most reliable sample type and conditions, and to develop an appropriate processing strategy to ensure reliably accurate results. RESULTS: Nucleosomes could be detected using a variety of sample collection and processing protocols. Nucleosome signals were highest in EDTA plasma and serum samples and most consistent in plasma. Samples should be processed within an hour of collection. Experiments showed that samples were able to withstand several freeze thaw cycles. Processing time and tcollection tube type did affect nucleosome detection levels. Finally, significantly elevated concentrations of nucleosomes were seen in a small cohort of dogs that had been newly diagnosed with lymphoma. CONCLUSIONS: When samples are collected and processed appropriately, the Nu.Q™ platform can reliably detect nucleosomes in the plasma of dogs. Further testing is underway to validate and optimize the Nu.Q™ platform for veterinary use.

Early production of type I interferon during West Nile virus infection: role for lymphoid tissues in IRF3-independent interferon production.

Infection of cells with flaviviruses in vitro is reduced by pretreatment with small amounts of type I interferon (IFN-alpha/beta). Similarly, pretreatment of animals with IFN and experiments using mice defective in IFN signaling have indicated a role for IFN in controlling flavivirus disease in vivo. These data, along with findings that flavivirus-infected cells block IFN signaling, suggest that flavivirus infection can trigger an IFN response. To investigate IFN gene induction by the very first cells infected during in vivo infection with the flavivirus West Nile virus (WNV), we infected mice with high-titer preparations of WNV virus-like particles (VLPs), which initiate viral genome replication in cells but fail to spread. These studies demonstrated a brisk production of IFN in vivo, with peak levels of over 1,000 units/ml detected in sera between 8 and 24 h after inoculation by either the intraperitoneal or footpad route. The IFN response was dependent on genome replication, and WNV genomes and WNV antigen-positive cells were readily detected in the popliteal lymph nodes (pLN) of VLP-inoculated mice. High levels of IFN mRNA transcripts and functional IFN were also produced in VLP-inoculated IFN regulatory factor 3 null (IRF3(-/-)) mice, indicating that IFN production was independent of the IRF3 pathways to IFN gene transcription, consistent with the IFN type produced (predominantly alpha).

Mutations in West Nile virus nonstructural proteins that facilitate replicon persistence in vitro attenuate virus replication in vitro and in vivo.

West Nile virus (WNV) infections in vertebrates are generally acute but persistent infections have been observed. To investigate the ability of WNV to produce persistent infections, we forced subgenomic WNV replicons to replicate within a cell without causing cell death. Detailed analyses of these cell-adapted genomes revealed mutations within the nonstructural protein genes NS2A (D73H, M108K), NS3 (117Kins), NS4B (E249G) and NS5 (P528H). WNV replicons and WNVs harboring a subset of NS2A or NS3 mutations showed a reduction in genome replication, a reduction in antigen accumulation, a decrease in cytopathic effect, an increased ability to persist in cell culture and/or attenuation in vivo. Taken together, these data indicate that WNV with a defect in replication and an increased potential to persist within the host cell can be generated by point mutations at multiple independent loci, suggesting that persistent viruses could arise in nature.