Deoxycholate induces COX-2 expression via Erk1/2-, p38-MAPK and AP-1-dependent mechanisms in esophageal cancer cells.

BACKGROUND: The progression from Barrett's metaplasia to adenocarcinoma is associated with the acquirement of an apoptosis-resistant phenotype. The bile acid deoxycholate (DCA) has been proposed to play an important role in the development of esophageal adenocarcinoma, but the precise molecular mechanisms remain undefined. The aim of this study was to investigate DCA-stimulated COX-2 signaling pathways and their possible contribution to deregulated cell survival and apoptosis in esophageal adenocarcinoma cells. METHODS: Following exposure of SKGT-4 cells to DCA, protein levels of COX-2, MAPK and PARP were examined by immunoblotting. AP-1 activity was assessed by mobility shift assay. DCA-induced toxicity was assessed by DNA fragmentation and MTT assay. RESULTS: DCA induced persistent activation of the AP-1 transcription factor with Fra-1 and JunB identified as the predominant components of the DCA-induced AP-1 complex. DCA activated Fra-1 via the Erk1/2- and p38 MAPK while Erk1/2 is upstream of JunB. Moreover, DCA stimulation mediated inhibition of proliferation with concomitant low levels of caspase-3-dependent PARP cleavage and DNA fragmentation. Induction of the anti-apoptotic protein COX-2 by DCA, via MAPK/AP-1 pathway appeared to balance the DCA mediated activation of pro-apoptotic markers such as PARP cleavage and DNA fragmentation. Both of these markers were increased upon COX-2 suppression by aspirin pretreatment prior to DCA exposure. CONCLUSION: DCA regulates both apoptosis and COX-2-regulated cell survival in esophageal cells suggesting that the balance between these two opposing signals may determine the transformation potential of DCA as a component of the refluxate.

Activation of human NK cells by the bacterial pathogen-associated molecular pattern muramyl dipeptide.

Muramyl dipeptide (MDP) is a bacterial pathogen associated molecular pattern derived from both Gram-positive and -negative bacteria. It is a specific ligand for nuclear oligomerization domain 2, a pattern recognition receptor best characterized for its role in immunosurveillance in the gut. In this study, we demonstrate that human peripheral blood NK cells express nuclear oligomerization domain 2 and respond to MDP. NK cells naturally internalize MDP leading to direct cell activation, including signaling through NFkappaB: characterized by p50/p65 heterodimers at early stimulations times and sustained activation of p50 homodimers. Moreover, MDP synergizes with IFN-alpha and IL-12 to activate NK cells and stimulate IFN-gamma secretion, suggesting a role for accessory cells in induction of an optimal NK cell response. Although IL-12 costimulation leads to a greater IFN-gamma response by NK cells, higher levels of CD69 in response to MDP are induced in the presence of IFN-alpha, suggesting that different pathogen-induced cytokine profiles will affect downstream NK cell responses. In contrast, MDP alone or in combination with either IFN-alpha or IL-12 only poorly increases NK cell cytotoxicity. In summary, this report identifies MDP as a bacterial pathogen associated molecular pattern that activates human NK cells.

Active immunization of hamsters against Clostridium difficile infection using surface-layer protein.

Clostridium difficile is the leading cause of infectious antibiotic-associated diarrhoea, particularly among the elderly. Its surface-layer protein (SLP) was tested as a vaccine component in a series of immunization and challenge experiments with Golden Syrian hamsters, combined with different systemic and mucosal adjuvants. Some regimens were also tested in a nonchallenge BALB/c mouse model, enabling closer monitoring of the immune response. None of the regimens conferred complete protection in the hamster model, and antibody stimulation was variable within regimens, and generally modest or poor. Mice displayed stronger antibody responses to SLP compared with hamsters. Two hamsters of five given SLP with Ribi (monophosphoryl lipid A and synthetic trehalose dicorynomycolate) survived the challenge, as did two of three given SLP with Ribi and cholera toxin. This modest trend to protection is interpreted with caution, because the survivors had low anti-SLP serum antibody titres. The hamsters were an outbred line, and subject to more genetic variability than inbred animals; however, BALB/c mice also showed strongly variable antibody responses. There is a clear need for better adjuvants for single-component vaccines, particularly for mucosal delivery. The hamster challenge model may need to be modified to be useful in active immunization experiments with SLP.

TLR7/8-mediated activation of human NK cells results in accessory cell-dependent IFN-gamma production.

NK cells express receptors that allow them to recognize pathogens and activate effector functions such as cytotoxicity and cytokine production. Among these receptors are the recently identified TLRs that recognize conserved pathogen structures and initiate innate immune responses. We demonstrate that human NK cells express TLR3, TLR7, and TLR8 and that these receptors are functional. TLR3 is expressed at the cell surface where it functions as a receptor for polyinosinic acid:cytidylic acid (poly(I:C)) in a lysosomal-independent manner. TLR7/8 signaling is sensitive to chloroquine inhibition, indicating a requirement for lysosomal signaling as for other cell types. Both R848, an agonist of human TLR7 and TLR8, and poly(I:C) activate NK cell cytotoxicity against Daudi target cells. However, IFN-gamma production is differentially regulated by these TLR agonists. In contrast to poly(I:C), R848 stimulates significant IFN-gamma production by NK cells. This is accessory cell dependent and is inhibited by addition of a neutralizing anti-IL-12 Ab. Moreover, stimulation of purified monocyte populations with R848 results in IL-12 production, and reconstitution of purified NK cells with monocytes results in increased IFN-gamma production in response to R848. In addition, we demonstrate that while resting NK cells do not transduce signals directly in response to R848, they can be primed to do so by prior exposure to either IL-2 or IFN-alpha. Therefore, although NK cells can be directly activated by TLRs, accessory cells play an important and sometimes essential role in the activation of effector functions such as IFN-gamma production and cytotoxicity.

Passive immunisation of hamsters against Clostridium difficile infection using antibodies to surface layer proteins.

Clostridium difficile is a major cause of antibiotic-associated diarrhoea and the primary cause of pseudomembraneous colitis in hospitalised patients. We assessed the protective effect of anti-surface layer protein (SLP) antibodies on C. difficile infection in a lethal hamster challenge model. Post-challenge survival was significantly prolonged in the anti-SLP treated group compared with control groups (P=0.0281 and P=0.0283). The potential mechanism of action of the antiserum was shown to be through enhancement of C. difficile phagocytosis. This report indicates that anti-SLP antibodies can modulate the course of C. difficile infection and may therefore merit closer investigation for use as constituents of multi-component vaccines against C. difficile associated diarrhoea.

Sustained IL-12 signaling is required for Th1 development.

STAT4 is an essential transcription factor for Th1 cell development. IL-12 and IFN-alpha both activate STAT4, but with different kinetics. In this study we compared their capacities to drive differentiation of human naive Th cells toward the Th1 phenotype. The Th1-polarizing activity of IFN-alpha was much weaker than that of IL-12, correlating with a marked difference in the kinetics of STAT4 activation; the response to IL-12 was sustained (>48 h), whereas the response to IFN-alpha was transient (4 h). The continuous presence of IL-12 was required for sustained STAT4 activation. Similarly, optimal Th1 polarization was only achieved upon prolonged exposure to IL-12 and could not be induced by a transient IL-12 pulse. Furthermore, the cytokine IL-2 potentiated sustained IL-12/STAT4 responses through up-regulation of IL-12R expression and synergized with IL-12 in driving Th1 cell development. Transient IFN-alpha responses, on the other hand, were not prolonged by IL-2. IFN-alpha treatment induced down-regulation of IFN-alphabeta receptor subunit 1, rendering cells refractory to IFN-alpha, but did not trans-inhibit the IL-12/STAT4 response. These data indicate that sustained IL-12 signaling is essential for optimal Th1 cell development and that transient activation of STAT4 in response to IFN-alpha may explain the poor Th1-polarizing capacity of this cytokine. Collectively these data show that the duration of cytokine signaling is important for determining the biological response.