In mammals, 10 TLRs recognize conserved pathogen-associated molecular patterns, resulting in the induction of inflammatory innate immune responses. One of these, TLR9, is activated intracellularly by bacterial DNA and synthetic oligodeoxynucleotides (ODN), containing unmethylated CpG dinucleotides. Following treatment with CpG ODN, TLR9 is found in lysosome-associated membrane protein type 1-positive lysosomes, and we asked which intracellular compartment contains TLR9 before CpG exposure. Surprisingly, we found by microscopy and supporting biochemical evidence that both transfected and endogenously expressed human TLR9 is retained in the endoplasmic reticulum. By contrast, human TLR4 trafficked to the cell surface, indicating that endoplasmic reticulum retention is not a property common to all TLRs. Because TLR9 is observed in endocytic vesicles following exposure to CpG ODN, our data indicate that a special mechanism must exist for translocating TLR9 to the signaling compartments that contain the CpG DNA.

Background: The ATTRACT Trial previously reported that pharmacomechanical catheterdirected thrombolysis (PCDT) did not prevent the post-thrombotic syndrome (PTS) in patients with acute proximal deep vein thrombosis (DVT). In the current analysis, we examine the effect of PCDT in ATTRACT patients with iliofemoral DVT. Methods: Within a large multicenter randomized trial, 391 patients with acute DVT involving the iliac and/or common femoral veins were randomized to PCDT with anticoagulation versus anticoagulation alone (No-PCDT) and were followed for 24 months to compare short-term and long-term outcomes. Results: Between 6 and 24 months, there was no difference in the occurrence of PTS (Villalta scale ≥5 or ulcer: 49% PCDT versus 51% No-PCDT; risk ratio (RR)=0.95; 95% confidence interval (CI), 0.78-1.15; p=0.59). PCDT led to reduced PTS severity as shown by: lower mean Villalta and Venous Clinical Severity Scores [VCSS] (p<0.01 for comparisons at 6, 12, 18, and 24 months); and fewer patients with moderate-or-severe PTS (Villalta scale ≥10 or ulcer: 18% versus 28%; RR 0.65; 95% CI 0.45-0.94, p=0.021) or severe PTS (Villalta scale ≥15 or ulcer: 8.7% versus 15%; RR 0.57; 95% CI 0.32-1.01, p=0.048; and VCSS ≥8: 6.6% versus 14%; RR 0.46; 95% CI 0.24-0.87, p=0.013). From baseline, PCDT led to greater reduction in leg pain and swelling (p<0.01 for comparisons at 10 and 30 days) and greater improvement in venous diseasespecific QOL (VEINES-QOL unit difference 5.6 through 24 months, p=0.029), but no difference in generic QOL (p > 0.2 for comparisons of SF-36 mental and physical component summary scores through 24 months). In patients having PCDT versus No-PCDT, major bleeding within 10 days occurred in 1.5% versus 0.5% (p=0.32), and recurrent VTE over 24 months was observed in 13% versus 9.2% (p=0.21). Conclusions: In patients with acute iliofemoral DVT, PCDT did not influence the occurrence of PTS or recurrent VTE. However, PCDT significantly reduced early leg symptoms and, over 24 Comerota et al.

MD-2, a glycoprotein that is essential for the innate response to lipopolysaccharide (LPS), binds to both LPS and the extracellular domain of Toll-like receptor 4 (TLR4). Following synthesis, MD-2 is either secreted directly into the medium as a soluble, active protein, or binds directly to TLR4 in the endoplasmic reticulum before migrating to the cell surface. Here we investigate the function of the secreted form of MD-2. We show that secreted MD-2 irreversibly loses activity over a 24-h period at physiological temperature. LPS, but not lipid A, prevents this loss in activity by forming a stable complex with MD-2, in a CD14-dependent process. Once formed, the stable MD-2⅐LPS complex activates TLR4 in the absence of CD14 or free LPS indicating that the activating ligand of TLR4 is the MD-2⅐LPS complex. Finally we show that the MD-2⅐LPS complex, but not LPS alone, induces epithelial cells, which express TLR4 but not MD-2, to secrete interleukin-6 and interleukin-8. We propose that the soluble MD-2⅐LPS complex plays a crucial role in the LPS response by activating epithelial and other TLR4 ؉ /MD-2 ؊ cells in the inflammatory microenvironment. Lipopolysaccharide (LPS),1 a component of the outer membrane of Gram-negative bacteria, stimulates an exceptionally potent innate immune response in mammals that can result in septic shock and death (1). The LPS response is mediated by four proteins (2): LPS binding protein extracts single molecules from LPS micelles and transfers them to CD14, a glycosylphosphatidylinositol-anchored cell surface receptor that also exists as a serum protein. In turn, the CD14⅐LPS complex activates two proteins that comprise the essential signaling complex of the LPS response, Toll-like receptor 4 (TLR4) and MD-2 (3-6). TLR4 is a type I integral membrane glycoprotein and is one of 10 TLR paralogs that activate NF-B, mitogen-activated protein kinases, and other transducers of inflammatory signals in response to pathogen-specific structural motifs. MD-2, a small cysteine-rich glycoprotein, binds to the ectodomain of TLR4 (7) in the endoplasmic reticulum and then transits to the cell surface in an active TLR4⅐MD-2 complex. However, MD-2 is also secreted into the medium as a soluble, active protein (sMD-2) by primary cells such as immature dendritic cells (iDC), and by MD-2-transfected cell lines (8). The activity of sMD-2 was shown by its ability to bind to TLR4 and confer LPS responsiveness to cells that express TLR4 but lack MD-2 (8, 9). Forward genetic and gene deletion studies have demonstrated that both MD-2 and TLR4 are required for normal responsiveness to LPS in vitro and in vivo (3)(4)(5)10).Analyses of species specificity differences for various forms of LPS provide strong evidence that LPS interacts directly with the TLR4⅐MD-2 complex (11-16). However, the molecular events leading to LPS binding and TLR4 activation are only partially understood. Photoaffinity labeling (17) and binding (9,18,19) studies have shown that LPS binds directly to MD-2 and TLR4, and that binding of LPS to TLR4...

Worsening lower extremity arterial disease, assessed as ABI decline, occurred in 9.5% of this elderly cohort over 6 years and was associated with modifiable vascular disease risk factors.