High mobility group box 1 (HMGB1), originally described as a DNA-binding protein, can also be released extracellularly and functions as a late mediator of inflammatory responses. Although recent reports have indicated that the receptor for advanced glycation end products (RAGE) as well as Toll-like receptor (TLR)2 and TLR4 are involved in cellular activation by HMGB1, there has been little evidence of direct association between HMGB1 and these receptors. To examine this issue, we used fluorescence resonance energy transfer (FRET) and immunoprecipitation to directly investigate cell surface interactions of HMGB1 with TLR2, TLR4, and RAGE. FRET images in RAW264.7 macrophages demonstrated association of HMGB1 with TLR2 and TLR4 but not RAGE. Transient transfections into human embryonic kidney-293 cells showed that HMGB1 induced cellular activation and NF-Bdependent transcription through TLR2 or TLR4 but not RAGE. Coimmunoprecipitation also found interaction between HMGB1 and TLR2 as well as TLR4, but not with RAGE. These studies provide the first direct evidence that HMGB1 can interact with both TLR2 and TLR4 and also supply an explanation for the ability of HMGB1 to induce cellular activation and generate inflammatory responses that are similar to those initiated by LPS. fluorescence resonance energy transfer; receptor of advanced glycation end products HIGH MOBILITY GROUP BOX 1 (HMGB1) protein, originally described as a DNA-binding protein that stabilizes nucleosomes and facilitates transcription, can also be released extracellularly by monocytes and macrophages stimulated by LPS, TNF-␣, or IL-1 (2, 44). Extracellular HMGB1 has been demonstrated to participate in inflammatory processes, including delayed endotoxin lethality and acute lung injury (1,44,46), and also appears to be involved in pathophysiological processes associated with cellular necrosis, such as acetaminophen-induced liver injury (34).Although HMGB1 and LPS appear to initiate similar intracellular events, including activation of kinases such as p38, ERK1/2, and Akt and transcriptional factors including NF-B, that lead to production of proinflammatory cytokines, gene arrays demonstrated differences in expression profiles with each of these stimuli (12, 30). Unlike LPS, which primarily increased the activity of IKK-␤, HMGB1 exposure resulted in activation of both IKK-␣ and IKK-␤ (31). In addition, culture of neutrophils lacking Toll-like receptor (TLR)4 with HMGB1, but not with LPS, still resulted in enhanced nuclear translocation of NF-B (31). Such results suggest that the receptors interacting with HMGB1 and leading to cellular activation and gene transcription are likely to be distinct from TLR4, which is responsible for LPS-induced responses (40). Recent data indicate that HMGB1 interacts not only with TLR4 but also with TLR2 and the receptor for advanced glycation end products (RAGE) (31, 46). In particular, a decrease in NF-B-dependent reporter gene expression after transfection with dominantnegative constructs to TLR2, TLR4, or both, demonstr...

Summary.  Background: Soluble thrombomodulin is a promising therapeutic natural anticoagulant that is comparable to antithrombin, tissue factor pathway inhibitor and activated protein C. Objectives: We conducted a multicenter, double‐blind, randomized, parallel‐group trial to compare the efficacy and safety of recombinant human soluble thrombomodulin (ART‐123) to those of low‐dose heparin for the treatment of disseminated intravascular coagulation (DIC) associated with hematologic malignancy or infection. Methods: DIC patients (n = 234) were assigned to receive ART‐123 (0.06 mg kg−1 for 30 min, once daily) or heparin sodium (8 U kg−1  h−1 for 24 h) for 6 days, using a double‐dummy method. The primary efficacy endpoint was DIC resolution rate. The secondary endpoints included clinical course of bleeding symptoms and mortality rate at 28 days. Results: DIC was resolved in 66.1% of the ART‐123 group, as compared with 49.9% of the heparin group [difference 16.2%; 95% confidence interval (CI) 3.3–29.1]. Patients in the ART‐123 group also showed more marked improvement in clinical course of bleeding symptoms (P = 0.0271). The incidence of bleeding‐related adverse events up to 7 days after the start of infusion was lower in the ART‐123 group than in the heparin group (43.1% vs. 56.5%, P = 0.0487). Conclusions: When compared with heparin therapy, ART‐123 therapy more significantly improves DIC and alleviates bleeding symptoms in DIC patients.

Objective. High mobility group box chromosomal protein 1 (HMGB-1), a nuclear DNA binding protein, was recently rediscovered as a new proinflammatory cytokine. The purpose of this study was to demonstrate HMGB-1 expression in vivo and to identify the role of HMGB-1 in the pathogenesis of rheumatoid arthritis (RA).Methods. HMGB-1 concentrations in synovial fluid (SF) and serum from RA and osteoarthritis (OA) patients were measured by immunoblot analysis. The protein's specific receptor, receptor for advanced glycation end products (RAGE), was examined in SF macrophages (SFMs). We measured levels of proinflammatory cytokines released by SFMs treated with HMGB-1 via enzyme-linked immunosorbent assay and used soluble RAGE (sRAGE) to block the release of tumor necrosis factor ␣ (TNF␣). Immunohistochemical analysis and immunofluorescence assay were employed to examine localization of HMGB-1 in RA synovium and its translocation in SFMs after TNF␣ stimulation.Results. HMGB-1 concentrations were significantly higher in SF of RA patients than in that of OA patients. SFMs expressed RAGE and released TNF␣, interleukin-1␤ (IL-1␤), and IL-6 upon stimulation with HMGB-1. HMGB-1 was found in CD68-positive cells of RA synovium, and TNF␣ stimulation translocated HMGB-1 from the nucleus to the cytosol in SFMs. Blockade by sRAGE inhibited the release of TNF␣ from SFMs.Conclusion. HMGB-1 was more strongly expressed in SF of RA patients than in that of OA patients, inducing the release of proinflammatory cytokines from SFMs. HMGB-1 plays a pivotal role in the pathogenesis of RA and may be an original target of therapy as a novel cytokine.High mobility group box chromosomal protein 1 (HMGB-1) has 219 residues in its primary amino acid sequence, and there is Ͼ98% sequence identity between the HMGB-1 of rodents and that of humans (1-6). In most cells, HMGB-1 is located in the nucleus. It is an abundant, highly conserved cellular protein and is widely known as a nuclear DNA binding protein that stabilizes nucleosome formation (7,8), facilitates gene transcription, and regulates the activity of steroid hormone receptors (9,10). However, it has been reported that HMGB-1 might be translocated from the nucleus to the cytosol and then released extracellularly.A previous study demonstrated that extracellular HMGB-1 induces the production of proinflammatory cytokines in macrophages (11). When released by activated monocytes, it participates in the development of lethality and activates downstream cytokine release. Furthermore, like other cytokine mediators of endotoxemia, HMGB-1 activates proinflammatory cytokine re-

This study was performed to examine the putative role of high mobility group box (HMGB) protein in the pathogenesis of acute lung injury (ALI). Observations were made (1) in 21 patients who were septic with ALI and 15 patients with normal lung function and (2) in a mouse model 24 hours after intratracheal instillation of lipopolysaccharide (LPS). The concentrations of HMGB1 were increased in plasma and lung epithelial lining fluid of patients with ALI and mice instilled with LPS. LPS-induced ALI was mitigated by anti-HMGB1 antibody. Although this protein was not detected in the plasma of control humans or mice, the concentrations of HMGB1 in lung epithelial lining fluid or in bronchoalveolar lavage fluid were unexpectedly high. The nuclear expression of HMGB1 was apparent in epithelial cells surrounding terminal bronchioles in normal mice, whereas its nuclear and cytoplasmic expression was observed in alveolar macrophages in LPS-instilled mice. Lung instillation of HMGB2 did not cause as much inflammation as HMGB1. Extracellular HMGB1 may play a key role in the pathogenesis of clinical and experimental ALI. However, its expression in normal airways is noteworthy and suggests that it also plays a physiologic role in the lung.