Transcriptional profiling of stress response in cultured porcine islets.

Cell-based diabetes therapy may be achieved through xenotransplantation of adult porcine islets, but tissue quality and immunoreactivity barriers need to be overcome. Early identification and exclusion of irreversibly stressed and dying islets may improve transplant outcomes. We used oligonucleotide microarray and quantitative RT-PCR to identify molecular markers of physiological and immunological stress in porcine islets cultured under stress conditions of elevated glucose (16.7 mM), inflammatory cytokine addition (IL-1beta, TNF-alpha, and IFN-gamma), or both, for 48 h. Hyperglycemic conditions were associated with increased thioredoxin interacting protein and metabolic process mRNAs, as observed in rodent and primate species. Cytokine treatment increased expression of JAK-STAT pathway components, oxidative stress (transglutaminase 2), and beta cell dysfunction genes. Transglutaminase 2 induction is unique to porcine islets. Biomarkers involved in hyperglycemia and islet inflammation may serve as novel targets for improving and monitoring isolated porcine islet function and viability.

Mapping of genes expressed in activated porcine Peyer's patch.

To determine the chromosomal locations for genes expressed in porcine Peyer's patches, polymerase chain reaction-based mapping of expressed sequence tags (ESTs) isolated from a porcine Peyer's patch-specific cDNA library was performed across a 6500-rad swine radiation hybrid panel. A total of 116 ESTs were mapped with LOD scores >6.0, and another 11 ESTs had LOD scores between 5.0 and 6.0. Of these 127 ESTs, 63% matched known genes (

Gene discovery and expression profiling in porcine Peyer's patch.

Peyer's patches of the intestinal mucosa are essential for host defense and immune regulation in the enteric system. To better understand molecular mechanisms of Peyer's patch function, we have screened for differentially expressed genes specific to Peyer's patch. cDNA libraries were created from normal Peyer's patch, immune stimulated Peyer's patch, and pooled cDNA subtracted with fibroblast RNA. From the subtracted library, 3687 expressed sequence tags (ESTs), representing 2414 unique nucleotide sequences, were isolated, identified by BLAST searches against public databases, and spotted onto a microarray for gene expression profiling. Approximately 30% of these ESTs BLAST to genes of unknown function and 20% have no known homology in the public databases (novel genes). Of the novel genes, 70% are expressed in normal immune tissues by microarray analysis, suggesting that at least 371 of the unidentified EST sequences from the subtracted library are novel porcine genes and can now be further characterized to determine their function in the porcine Peyer's patch. We surmise that the products of these genes participate in biochemical and cellular functions related to the unique immunological and gastroenterological functions of the small intestine. The BLAST and gene ontology information for each of the subtracted library EST sequences, the normal and immune stimulated libraries, and the microarray are all valuable resources that will facilitate further examination of the biological function of porcine Peyer's patch tissue.

Evolution of porcine reproductive and respiratory syndrome virus during sequential passages in pigs.

Porcine reproductive and respiratory syndrome (PRRS) viruses are recognized as possessing a high degree of genetic and antigenic variability. Viral diversity has led to questions regarding the association of virus mutation and persistent infection in the host and has raised concerns vis-à-vis protective immunity, the ability of diagnostic assays to detect novel variants, and the possible emergence of virulent strains. The purpose of this study was to describe ongoing changes in PRRS virus during replication in pigs under experimental conditions. Animals were inoculated with a plaque-cloned virus derived from VR-2332, the North American PRRS virus prototype. Three independent lines of in vivo replication were maintained for 367 days by pig-to-pig passage of virus at 60-day intervals. A total of 315 plaque-cloned viruses were recovered from 21 pigs over the 367-day observation period and compared to the original plaque-cloned virus by virus neutralization assay, monoclonal antibody analysis, and sequencing of open reading frames (ORFs) 1b (replicase), 5 (major envelope protein), and 7 (nucleocapsid) of the genome. Variants were detected by day 7 postinoculation, and multiple variants were present concurrently in every pig sampled over the observation period. Sequence analysis showed ORFs 1b and 7 to be highly conserved. In contrast, sequencing of ORF 5 disclosed 48 nucleotide variants which corresponded to 22 amino acid variants. Although no epitopic changes were detected under the conditions of this experiment, PRRS virus was shown to evolve continuously in infected pigs, with different genes of the viral genome undergoing various degrees of change.