Towards the selection of chickens resistant to Salmonella and Campylobacter infections.

Resistance to infection with enteric pathogens such as Salmonella and Campylobacter can be at many levels and include both non-immune and immune mechanisms. Immune resistance mechanisms can be specific, at the level of the adaptive immune response, or non-specific, at the level of the innate immune response. Whilst we can extrapolate to some degree in birds from what is known about immune responses to these pathogens in mammals, chickens are not "feathered mice", but have a different repertoire of genes, molecules, cells and organs involved in their immune response compared to mammals. Fundamental work on the chicken's immune response to enteric pathogens is therefore still required. Our studies focus particularly on the innate immune response, as responses of heterophils (the avian neutrophil equivalent) from commercial birds, and macrophages from inbred lines of chickens, correlate with resistance or susceptibility to Salmonella infection with a variety of Salmonella serovars and infection models. We work on two basic resistance mechanisms - resistance to colonization with Salmonella or Campylobacter, and resistance to systemic salmonellosis (or fowl typhoid). To map genes involved in resistance to colonization with Salmonella and Campylobacter, we are using a combination of expression quantitative trait loci (eQTLs) from microarray studies, allied with whole genome SNP arrays (WGA), a candidate gene approach and analysis of copy number variation across the genome. For resistance to systemic salmonellosis, we have refined the location ofa novel resistance locus on Chromosome 5, designated SAL1, using high density SNP panels, combined with advanced back-crossing of resistant and susceptible lines. Using a 6th generation backcross mapping population we have confirmed and refined the SAL1 locus to 8-00 kb of Chromosome 5. This region spans 14 genes, including two very striking functional candidates; CD27-binding protein (Siva) and the RAC-alpha serine/threonine protein kinase homologue, AKT1.

Integrated immunogenomics in the chicken: deciphering the immune response to identify disease resistance genes.

Resistance to infection takes place at many levels, and involves both non-specific and specific immune mechanisms. The chicken has a different repertoire of immune genes, molecules, cells and organs compared to mammals. To understand the role of any disease resistance gene(s), it is therefore important to understand these different repertoires, and the bird's response to a particular pathogen. Our studies focus on the innate immune response, as responses of macrophages from inbred lines of chickens, and heterophils from commercial birds, correlate with resistance or susceptibility to Salmonella infection with a variety of Salmonella serovars and infection models. To map disease resistance genes, we are using a combination of expression quantitative trait loci (eQTLs) from microarray studies, allied with whole genome SNP arrays (WGA) and a candidate gene approach. There are over 500 human genes with the Gene Ontology term "innate immunity". We have identified over 400 of these genes in the chicken genome, and are actively identifying informative SNPs in them. The segregation of 6000 WGA SNPs across all of our inbred lines was also assessed, which should yield approximately 900 informative SNPs for a cross between any two lines. The initial focus of these studies is on mapping resistance genes in our inbred lines, but the studies will be extended to commercial flocks.

LY303511 amplifies TRAIL-induced apoptosis in tumor cells by enhancing DR5 oligomerization, DISC assembly, and mitochondrial permeabilization.

Certain classes of tumor cells respond favorably to TRAIL due to the presence of cell surface death receptors DR4 and DR5. Despite this preferential sensitivity, resistance to TRAIL remains a clinical problem and therefore the heightened interest in identifying compounds to revert tumor sensitivity to TRAIL. We recently demonstrated that the phosphatidylinositide-3-kinase (PI3K) inhibitor, LY294002, and its inactive analog LY303511, sensitized tumor cells to vincristine-induced apoptosis, independent of PI3K/Akt pathway. Intrigued by these findings, we investigated the effect of LY303511 on TRAIL-induced apoptosis in HeLa cells. Preincubation of cells with LY30 significantly amplified TRAIL signaling as evidenced by enhanced DNA fragmentation, caspases 2, 3, 8, and 9 activation, and reduction in the tumor colony formation. This increase in TRAIL sensitivity involved mitochondrial membrane permeabilization resulting in the egress of cytochrome c and second mitochondrial activator of caspase/direct IAP-binding protein with low PI, cleavage of X-linked inhibitor of apoptosis protein, and activation of caspase 9. We link this execution signal to the ability of LY30 to downregulate cFLIP(S) and oligomerize DR5, thus facilitating the signaling of the death initiating signaling complex. The subsequent exposure to TRAIL resulted in processing/activation of caspase 8 and cleavage of its substrate, the BH3 protein Bid. These data provide a novel mechanism of action of this small molecule with the potential for use in TRAIL-resistant tumors.

Resolution of acylated dipeptide stereoisomers by capillary electrophoresis using sulfobutylether derivatized beta-cyclodextrin.

The separation of enantiomerically and diastereomerically related stereoisomers of acylated Asp-Phe dipeptides was explored using capillary electrophoresis (CE). This series of dipeptides included the alpha-L,L parent compound and the three other potential Asp containing stereoisomers (alpha-D,D, alpha-L,D, and alpha-D,L), as well the four possible isoAsp containing stereoisomers (beta-L,L, beta-D,D, beta-L,D and beta-D,L). The separation of these substances was explored using both neutral and charged cyclodextrins as the stereoisomer selector added to the running electrolyte. The major experimental parameters investigated included pH, the cyclodextrin type, and the cyclodextrin concentration. Due to differences in the pKa values of the carboxylic acid groups, adjustment of the separation buffer to between pH 3.0 and 4.0 provided for sufficient electrophoretic mobility differences to result in excellent separations of the diastereomerically related peptides in this pH region. The resolution of the enantiomerically related peptide stereoisomers was accomplished using low concentrations (1 mM) of the anionic cyclodextrin derivative, sulfobutylether-beta-cyclodextrin (SBE-beta-CD). This negatively charged cyclodextrin was found to be superior for the resolution of the enantiomerically related peptides as compared to native beta-cyclodextrin or the neutral derivatives, dimethyl beta-cyclodextrin and hydroxypropyl beta-cyclodextrin. An alternative approach using anionic or neutral surfactants in conjunction with the SBE-beta-CDs was also explored and found to be successful but problematic.

Double ventricular parasystole. Supernormal phase of conduction as a mechanism of intermittent parasystole. Report of a case.

A rare case of spontaneous double ventricular parasystole was studied in depth, together with a critical review of similar cases in the literature. The discussion was focused on 1) the variation of the shortest interectopic interval (SIEI), 2) entrance block and its failure, 3) supernormality as a mechanism of intermittence, and 4) effects of lidocaine and atropine on such an arrhythmia. In double ventricular parasystole a greater than usual variation in the SIEI tended to occur in one of the two parasystolic groups. If, however, such variations were too great in the face of otherwise parasystolic rhythm, presence of intermittence was confirmed. A temporary loss of the entrance block was deemed primarily responsible for the intermittency. That is to say, invasion, discharge, and resetting of one parasystolic focus by another parasystolic impulse during the supernormal phase of the ventricle was considered the cause of an intermittence. In a strict sense, this is the first report in the literature in which the supernormality was clearly indicated as one mechanism of intermittent ventricular parasystole. The advantage of the concept of double ventricular parasystole as compared to single parasystole in defining such a mechanism is stressed.