Search

Enterotoxigenic Escherichia coli (ETEC) infection is of global importance because of its high morbidity and mortality rate in humans and animals. ETEC has been the principal agent causing the traveler's diarrhea in humans and post-weaning diarrhea in pigs. Intestinal innate immunity is the first line of defense in enteric bacterial infections. The pathogenicity of ETEC is due to the fimbriae and heat labile (LT) and heat stable (ST) toxins. LT has two subunits A and B. Porcine intestinal epithelial cells (IPEC-J2) are a suitable in vitro model for studying ETEC infections. This study was conducted to analyze the changes in the gene expression of proinflammatory cytokines and chemokines upon stimulation of IPEC-J2 cells with different strains of ETEC and LT toxins for 3 hrs. We used wild type ETEC strains with K88ac fimbriae 3030-2 (O157:K87 LT+, STb+), 2534-86 (O8:K87: NM: LT-I+, STb+), 1836-2 (LT_, ST-, astA+), and G58-1, a non pathogenic, non-fimbriated, wild-type (O101:K28: NM, LT_), at multiplicity of infection 10:1. LT and LTb were used at 100 ng/ml. G58-1 and cells with media alone served as negative controls. The changes in the gene expression of proinflammatory cytokines such as IL-1α, IL-1β, TNF-α, IL-6 and chemokine, IL-8, were quantified relative to porcine specific cyclophilin-A. We found that the 3030-2 strain decreased IL-6 and increased TNF-α expression at 3h. Strain 1836-2 increased IL-8 and LTb decreased IL-8 gene expressions. Further investigations will help in understanding more about enteric mucosal innate immune responses to ETEC, which may potentially contribute to its prevention and treatment. [ABSTRACT FROM AUTHOR]

We describe a surgical method for ileal resection and anastomosis in newborn germfree piglets that was undertaken to establish a model that can be used for immunologic research and other applications. A preliminary experiment indicated that neonatal piglets with resection of approximately 60 cm of their ileum (removal of approximately 90% of the continuous ileal Peyer patches; group A) and those in which the ileum was transected (group B) could be maintained germfree for 35 d, colonized with defined gut flora, and maintained in a clean room until 70 d of age. In the final study, 12 piglets (4 each for groups A and B and 4 untreated controls), were monitored for postoperative feeding behavior, malaise, evidence for contamination with pathogenic bacteria, and weight gain. All surgical animals were free from incidental contamination from pathogens and environmental organisms with atypical colony types for 35 d. Two piglets in group B died postoperatively (1 during the preliminary experiment and 1 during the final study). Control (group C) piglets gained significantly more weight than did those in group A. These studies demonstrated that surgical resection of the ileal Peyer patches under germfree conditions can be accomplished successfully without compromising piglet health or introducing pathogens and with only a modest reduction in weight gain.

The MHC class II transactivator (CIITA), the master regulator of MHC class II (MHC II) expression, is a co-activator that controls MHC II transcription. Human B lymphocytes express MHC II constitutively due to persistent activity of CIITA promoter III (pIII), one of the four potential promoters (pI-pIV) of this gene. Although increases in MHC II expression in B cells in response to cytokines have been observed and induction of MHC II and CIITA by IFN-gamma has been studied in a number of different cell types, the specific effects of IFN-gamma on CIITA expression in B cells have not been studied. To investigate the regulation of CIITA expression by IFN-gamma in B cells, RT-PCR, in vivo and in vitro protein/DNA binding studies, and functional promoter analyses were performed. Both MHC II and CIITA type IV-specific RNAs increased in human B lymphocytes in response to IFN-gamma treatment. CIITA promoter analysis confirmed that pIV is IFN-gamma inducible in B cells and that the GAS and IRF-E sites are necessary for full induction. DNA binding of IRF-1 and IRF-2, members of the IFN regulatory factor family, was up-regulated in B cells in response to IFN-gamma and increased the activity of CIITA pIV. In vivo genomic footprint analysis demonstrated proteins binding at the GAS, IRF-E and E box sites of CIITA pIV. Although CIITA pIII is considered to be the hematopoietic-specific promoter of CIITA, these findings demonstrate that pIV is active in B lymphocytes and potentially contributes to the expression of CIITA and MHC II in these cells.