The main objective of the present study was to determine whether PBP2 could be recognized by DCs as a danger signal and LY2157299 TGF-beta inhibitor therefore increase its immunogenic properties. We therefore analyzed DC phenotypic maturation upon PBP2 treatment. We observed that PBP2 induced expression of CD80, CD86, CD40 and MHC class II on mouse bone marrow-derived DC (BMDC) (Figure 1A-C) in a dose and time-dependent manner (Figure 1B and Figure S1). Importantly, PBP2 also increased the expression of CD40, CD80, CD86, HLA-DR and the maturation marker CD83 in human monocyte-derived DCs (Figure 1D). Although endotoxin-free reagents were used during the purification process, it was critical to control for potential endotoxin contamination in the recombinant PBP2 preparations. PBP2 preparations were directly evaluated for the presence of bacterial endotoxin using the highly sensitive Limulus Amebocyte Lysate (LAL) assay (CAMBREX). The detection limit of the assay was 0.01 EU/ml. Maximum detectable endotoxin was at 0.314 EU/ml in a 10 mg/ml PBP2 sample. The minimum LPS concentration required to induce DC maturation under our experimental conditions in the present study was 1 EU/ml, three times greater than the maximal amount of LPS detectable in PBP2. To further address an eventual role of LPS contamination, we performed two different approaches. First, DCs were stimulated with PBP2 in the presence of the endotoxin-neutralizing antibiotic polymyxin B (PMB) and DC phenotype was assessed as described above. As expected, LPSinduced phenotype was completely abolished with PMB. In clear contrast, PBP2-induced mature phenotype was not affected by PMB Lapatinib treatment in mouse and human DCs (Figure 1C-D). Secondly, we observed that PBP2 degradation using trypsin completely abolished its effect on DC phenotype (Figure 1E). Taken together, we showed that PBP2 induces human and mouse DC maturation and that this effect is not due to endotoxin contamination. Most experiments were performed using affinity chromatography purified PBP2 as stated in MATERIALS AND METHODS.