EPEC strain E22 infecting rabbits appeared as an appropriate model to study the immune response since it is not a modified strain, it is an E. coli species (unlike Citrobacter) that shares the LEE pathogenicity island found in human EPEC strains. This strain produces signs and symptoms in rabbits [33] that reflect the effects caused by EPEC strains in

human infection. E22 can also reproduce EPEC pathogenesis in epithelial cell lines [34]. Therefore, infection with E22 is a valuable resource to develop coordinated in vitro and in vivo EPEC pathogenesis studies. Here, we performed an integral analysis of pathogen recognition, signalling pathway activation, and cytokine production by studying virulence factors that might define check details the epithelial inflammatory response against EPEC infection. We analysed the reaction of the intestinal epithelial cell line HT-29 to EPEC virulence factors during the infection with strains

E2348/69 and E22, the latter being considered as an atypical EPEC, because of the lack of bundle forming pilus (BFP). We evaluated the effects of EPEC intimate adherence (intimin and T3SS) during the proinflammatory response by FliC activation. Our experiments focused on TLR5 expression and subcellular Caspase activation localization, ERK1/2 and NF-κB activation, and synthesis and secretion of cytokines [IL-1β, IL-8 and tumour necrosis factor alpha (TNF-α)]. Bacterial strains.  Except for E22 isogenic fliC mutant, E22 wild type and the other isogenic mutant strains (Table 1) were kindly donated by Eric Oswald (INRA-ENVT). Strains were preserved at −70 °C in LB with 10% glycerol. most For each experiment, bacteria were inoculated in LB and incubated overnight at 37 °C. Before cell interaction, the overnight cultures were activated in minimum essential medium (MEM) without foetal bovine serum (FBS) and without antibiotics and incubated for 2 h at 37 °C. The construction of fliC mutant.  EPEC E22 fliC gene was interrupted by a kanamycin resistance cassette using the

Lambda Red recombinase system [35]. The kanamycin resistance gene was amplified from pKD4 by PCR with fliC-FRT-sense primers (5′-CAG TCT GCG CTG TCG AGT TCT ATC GAG CGT CTG TCT TCT GGC TGT GTA GGC TGG AGC TGC TT) and fliC-FRT-antisense primers (5′-TAC GTC GTT GGC TTT TGC CAG TAC GGA GTT ACC GGC CTG CAT ATG AAT ATC CTC CTT AG). The product was treated with DpnI and introduced into E22 WT carrying pKD46. Colonies containing the fliC::Km interrupted gene (referred to as E22ΔfliC) were selected as previously described [35]. Specific interruption of the fliC gene was confirmed by PCR. Absence of FliC was also confirmed by protein purification by acid hydrolysis and ultracentrifugation [36]. The proteins were concentrated with UltraFree filters (Millipore, Billerica, MA, USA) and analysed in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE).

tuberculosis, and tetanic toxoid. Analysis of the specific immune response to mycobacterial antigens in comparison to the NS culture revealed an increase in spot-forming cells both in RR and RR/HIV when cells were stimulated with ML [Fig. 2a,b; RR NS = 135 (30–260) versus ML = 830 (50–5380); P < 0·01; RR/HIV NS = 202·5 (40·0–2560) versus ML = 2260 (50·0–7380); P < 0·05]. The ML p38 peptide

did not modulate the frequency of IFN-γ-producing cells after 48 hr of culture in the PBMCs of the different groups tested. ML peptide p69, which induces a T CD8 response, increased the this website frequency of IFN-γ-producing cells in the PBMCs of RR patients when compared with NS cells [Fig. 2a,b; RR NS = 140 (50–250) versus Tamoxifen mouse p69 = 830 (390–1000); P < 0·05]. However, no significant differences were observed between the PBMCs of RR/HIV stimulated or not with p69 (Fig. 2a,b). In addition, an increase in IFN-γ production in both RR and RR/HIV cells stimulated in vitro with p69 was also observed in contrast to cells in the HC group under the same conditions [Fig. 2b; HC 370 (70–650) versus RR/HIV 830 (250–1960); P < 0·05]. Although M. tuberculosis stimulation induced spots in both RR and RR/HIV cells, there

were no significant differences when compared with unstimulated cells or the HC group. Tetanus toxoid induced an increase in IFN-γ production only in the HC group when compared with NS cells (Fig 2a,b). As expected, PHA stimulation induced a greater number of spots in the HC, RR and RR/HIV groups when compared with the NS cells (Fig. 2a,b). HIV infection induces Axenfeld syndrome significant immunological impairment, resulting in the increased expression of activation markers such as CD38 and HLA-DR in CD8+ T cells. This increased expression has been associated with particular clinical outcomes.[24] The next step was to evaluate whether ML stimulation modulates the activation of the immune system in RR/HIV co-infected patients. For this purpose, cellular activation parameters were investigated by analysing the surface expression

markers CD25, CD69 and CD38 in both CD4 and CD8 T cells in the PBMC cell culture after stimulation with irradiated ML for 24 hr. As observed in Fig. 3(a), ML increased CD4+ CD69+ T-cell frequencies in the HC and RR groups but not in the RR/HIV patients that presented a greater percentage of CD4+ CD69+ cells in the NS cell culture regardless of ML stimulus [Fig. 3a,b; HC NS = 2·78 (1·57–5·42) versus ML = 9·33 (4·97–17·43), P < 0·01; RR NS = 2·27 (0·57–8·72) versus ML = 10·39 (7·27–18·87), P < 0·01]. Although ML did not affect the expression of CD4+ T-cell activation markers in RR/HIV patients, an increase in CD8+ CD69+ T-cell frequencies in ML-stimulated cells was observed in this group compared with the NS cells [Fig. 4a,b; NS = 13·90 (5·16–22·80) versus ML = 44·49 (21·69–56·90), P < 0·05].