Toll-like receptors (TLRs) are receptors of the innate immune system responsible for recognizing pathogen-associated molecular patterns. TLR2 seems to be the most promiscuous TLR receptor able to recognize the most diverse set of pathogenassociated patterns. Its promiscuity has been attributed to its unique ability to heterodimerize with TLRs 1 and 6 and, most recently, to its association with CD36 in response to diacylated lipoproteins. Thus, it seems that TLR2 forms receptor clusters in response to different microbial ligands. In this study we investigated TLR2 cell surface heterotypic interactions in response to different ligands as well as internalization and intracellular trafficking. Our data show that TLR2 forms heterodimers with TLR1 and TLR6 and that these heterodimer pre-exist and are not induced by the ligand. Upon stimulation by the specific ligand, these heterodimers are recruited within lipid rafts. In contrast, heterotypic associations of TLR2/6 with CD36 are not preformed and are ligand-induced. All TLR2 receptor clusters accumulate in lipid rafts and are targeted to the Golgi apparatus. This localization and targeting is ligand-specific. Activation occurs at the cell surface, and the observed trafficking is independent of signaling.

Keywords: sepsis, serum proteins, LPS, LTABacterial cell wall components, such LPS and LTA, are potent initiators of an inflammatory response that can lead to septic shock. The advances in the past were centered around membrane-bound receptors and intracellular events, but our understanding of the initial interactions of these bacterial components with serum proteins as they enter the bloodstream remain unclear. In this study we identified several serum proteins, which are involved in the innate recognition of bacterial products. Using affinity chromatography and mass spectrometry we performed proteomic analysis of LPS-and LTA-binding serum proteins. We isolated proteins from normal serum that can interact with LPS and LTA. Fluorescent binding experiments and cytokine assays revealed that serum proteins, such as apolipoprotein, LDL, transferrin and holotransferrin could neutralize LPS/LTA binding as well as the subsequent inflammatory response, suggesting that serum proteins modulate LPS/LTA-induced responses. When compared with the proteomic profile of serum from septic patients it was shown that these proteins were in lower abundance. Investigation of serum proteins in 25 critically ill patients with a mortality rate of 40% showed statistically higher levels of these proteins in survivors.Patients surviving sepsis had statistically significant higher levels of apolipoprotein, albumin, LDL, transferrin and holotransferrin than individuals that succumbed, suggesting that these proteins have an inhibitory effect on LPS/LTAinduced inflammatory responses and in their absence there might be an augmented inflammatory response in sepsis.

Mannan-binding lectin (MBL) deficiency is determined by MBL gene polymorphisms and is associated with an increased infection risk. To clarify the role of MBL in Allo-SCT, 131 recipients-donors were analysed. MBL genotypes were determined by PCR and heteroduplex analyses, MBL serum levels by ELISA, and MBL oligomers by western blotting. MBL levels o400 ng/ml were associated with increased susceptibility to fungal pneumonia (7/12 vs 35/111; P ¼ 0.04, adjusted P ¼ 0.002), HSV/VZV (7/12 vs 26/111; P ¼ 0.03), CMV reactivation and acute GVHD. Donor genotypes had no influence. Pre-SCT MBL levels corresponded to recipients' genotypes (Po0.001), changed significantly post-SCT, but were not influenced by donors' genotypes. MBL oligomer profiles were similar pre-/post-SCT. Cultured CD34 þ cells were found not to synthesise MBL. In conclusion, low MBL levels pre-transplant predisposed patients to sepsis, fungal and viral infection. Donors' MBL genotypes did not influence infection rates. Prospective studies should clarify the importance of MBL as a prelude for MBL replacement after SCT.

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