Crystal Structure of the TLR4-MD-2 Complex with Bound Endotoxin Antagonist Eritoran

SummaryTLR4 and MD-2 form a heterodimer that recognizes LPS (lipopolysaccharide) from Gram-negative bacteria. Eritoran is an analog of LPS that antagonizes its activity by binding to the TLR4-MD-2 complex. We determined the structure of the full-length ectodomain of the mouse TLR4 and MD-2 complex. We also produced a series of hybrids of human TLR4 and hagfish VLR and determined their structures with and without bound MD-2 and Eritoran. TLR4 is an atypical member of the LRR family and is composed of N-terminal, central, and C-terminal domains. The β sheet of the central domain shows unusually small radii and large twist angles. MD-2 binds to the concave surface of the N-terminal and central domains. The interaction with Eritoran is mediated by a hydrophobic internal pocket in MD-2. Based on structural analysis and mutagenesis experiments on MD-2 and TLR4, we propose a model of TLR4-MD-2 dimerization induced by LPS

Toll-Like Receptor 4 Decoy, TOY, Attenuates Gram-Negative Bacterial Sepsis

Lipopolysaccharide (LPS), the Gram-negative bacterial outer membrane glycolipid, induces sepsis through its interaction with myeloid differentiation protein-2 (MD-2) and Toll-like receptor 4 (TLR4). To block interaction between LPS/MD-2 complex and TLR4, we designed and generated soluble fusion proteins capable of binding MD-2, dubbed TLR4 decoy receptor (TOY) using ‘the Hybrid leucine-rich repeats (LRR) technique’. TOY contains the MD-2 binding ectodomain of TLR4, the LRR motif of hagfish variable lymphocyte receptor (VLR), and the Fc domain of IgG1 to make it soluble, productive, and functional. TOY exhibited strong binding to MD-2, but not to the extracellular matrix (ECM), resulting in a favorable pharmacokinetic profile in vivo. TOY significantly extended the lifespan, when administered in either preventive or therapeutic manners, in both the LPS- and cecal ligation/puncture-induced sepsis models in mice. TOY markedly attenuated LPS-triggered NF-κB activation, secretion of proinflammatory cytokines, and thrombus formation in multiple organs. Taken together, the targeting strategy for sequestration of LPS/MD-2 complex using the decoy receptor TOY is effective in treating LPS- and bacteria-induced sepsis; furthermore, the strategy used in TOY development can be applied to the generation of other novel decoy receptor proteins

Self-assembled adipose-derived mesenchymal stem cells as an extracellular matrix component- and growth factor-enriched filler

The clinical application of mesenchymal stem cells (MSCs) is attracting attention due to their excellent safety, convenient acquisition, multipotency, and trophic activity. The clinical effectiveness of transplanted MSCs is well-known in regenerative and immunomodulatory medicine, but there is a demand for their improved viability and regenerative function after transplantation. In this study, we isolated MSCs from adipose tissue from three human donors and generated uniformly sized MSC spheroids (∼100 µm in diameter) called microblocks (MiBs) for dermal reconstitution. The viability and MSC marker expression of MSCs in MiBs were similar to those of monolayer MSCs. Compared with monolayer MSCs, MiBs produced more extracellular matrix (ECM) components, including type I collagen, fibronectin, and hyaluronic acid, and growth factors such as vascular endothelial growth factor and hepatocyte growth factor. Subcutaneously injected MiBs showed skin volume retaining capacity in mice. These results indicate that MiBs could be applied as regenerative medicine for skin conditions such as atrophic scar by having high ECM and bioactive factor expression

Serum Neopterin Concentration and Impaired Glucose Metabolism: Relationship With β-Cell Function and Insulin Resistance

Aim: The purpose of this study was to measure the serum neopterin according to glucose metabolism and to evaluate neopterin as a predictor of type 2 diabetes (T2D) in a hospital-based cohort.Methods: A 75-g oral glucose tolerance test (OGTT) was performed by people who visited the outpatient clinic in Seoul National University Bundang Hospital for suspected abnormal glucose tolerance or a strong family history of T2D. Neopterin was measured using an enzyme-link immunosorbent assay with baseline samples from the OGTT.Results: Neopterin was measured in 184 participants. Indices related to glucose metabolism, such as the HOMA-IR, disposition index, etc. were calculated based on the results of the OGTT. The classifications for the 184 participants were: 24 (13%) had NGT, 89 (48.4%) prediabetes, and 60 (38.6%) T2D. Neopterin increased with deterioration of glucose metabolism (0.55 ± 0.25 vs. 0.58 ± 0.27 vs. 0.67 ± 0.27 ng/ml, p = 0.041; NGT, prediabetes, and T2D, respectively). Neopterin also correlated with fasting plasma glucose, 30-min and 120-min glucose of OGTT and HbA1c (r = 0.251, 0.259, 0.184, and 0.270, all p < 0.05). The HOMA-IR and disposition index correlated with neopterin (r = 0.291 and −0.170, respectively, both p < 0.05). When combined with C-peptide level, neopterin was as powerful as HOMA-IR in predicting future T2D.Conclusion: Serum neopterin appears to be related to impaired insulin secretion and insulin resistance in the development of T2D. Further investigation of the relationship between neopterin and glucose metabolism would be helpful to understand the pathophysiology for the development of T2D