Identification and Evaluation of Novel MicroRNA Biomarkers in Plasma and Feces Associated with Drug-induced Intestinal Toxicity.

Gastrointestinal toxicity is dose limiting with many therapeutic and anticancer agents. Real-time, noninvasive detection of markers of toxicity in biofluids is advantageous. Ongoing research has revealed microRNAs as potential diagnostic and predictive biomarkers for the detection of select organ toxicities. To study the potential utility of microRNA biomarkers of intestinal injury in a preclinical toxicology species, we evaluated 3 rodent models of drug-induced intestinal toxicity, each with a distinct mechanism of toxicity. MiR-215 and miR-194 were identified as putative intestinal toxicity biomarkers. Both were evaluated in plasma and feces and compared to plasma citrulline, an established intestinal injury biomarker. Following intestinal toxicant dosing, microRNA changes in feces and plasma were detected noninvasively and correlated with histologic evidence of intestinal injury. Fecal miR-215 and miR-194 levels increased, and plasma miR-215 decreased in a dose- and time-dependent manner. Dose-dependent decreases in plasma miR-215 levels also preceded and correlated positively with plasma citrulline modulation, suggesting miR-215 is a more sensitive biomarker. Moreover, during the drug-free recovery phase, plasma miR-215 returned to predose levels, supporting a corresponding recovery of histologic lesions. Despite limitations, this study provides preliminary evidence that select microRNAs have the potential to act as noninvasive, sensitive, and quantitative biomarkers of intestinal injury.

Feline B7.1 and B7.2 proteins produced from swinepox virus vectors are natively processed and biologically active: potential for use as nonchemical adjuvants.

Costimulatory ligands, B7.1 and B7.2, have been incorporated into viral and DNA vectors as potential nonchemical adjuvants to enhance CTL and humoral immune responses against viral pathogens. In addition, soluble B7 proteins, minus their transmembrane and cytoplasmic domains, have been shown to block the down regulation of T-cell activation through blockade of B7/CTLA-4 interactions in mouse tumor models. Recently, we developed swinepox virus (SPV) vectors for delivery of feline leukemia antigens for vaccine use in cats [Winslow, B.J., Cochran, M.D., Holzenburg, A., Sun, J., Junker, D.E., Collisson, E.W., 2003. Replication and expression of a swinepox virus vector delivering feline leukemia virus Gag and Env to cell lines of swine and feline origin. Virus Res. 98, 1-15]. To explore the use of feline B7.1 and B7.2 ligands as nonchemical adjuvants, SPV vectors containing full-length feline B7.1 and B7.2 ligands were constructed and analyzed. Full-length feline B7.1 and B7.2 produced from SPV vectors were natively processed and costimulated Jurkat cells to produce IL-2, in vitro. In addition, we explored the feasibility of utilizing SPV as a novel expression vector to produce soluble forms of feline B7.1 (sB7.1) and B7.2 (sB7.2) in tissue culture. The transmembrane and cytoplasmic regions of the B7.1 and B7.2 genes were replaced with a poly-histidine tag and purified via a two-step chromatography procedure. Receptor binding and costimulation activity was measured. Although feline sB7.1-his and sB7.2-his proteins bound to the human homolog receptors, CTLA-4 and CD28, both soluble ligands possessed greater affinity for CTLA-4, compared to CD28. However, both retained the ability to partially block CD28-mediated costimulation in vitro. Results from these studies establish the use of SPV as a mammalian expression vector and suggest that full-length-vectored and purified soluble feline B7 ligands may be valuable, nonchemical immune-modulators.