Monoclonal antibodies targeting ST2L Domain 1 or Domain 3 differentially modulate IL-33-induced cytokine release by human mast cell and basophilic cell lines.

The cell-surface receptor ST2L triggers cytokine release by immune cells upon exposure to its ligand IL-33. To study the effect of ST2L-dependent signaling in different cell types, we generated antagonist antibodies that bind different receptor domains. We sought to characterize their activities in vitro using both transfected cells as well as basophil and mast cell lines that endogenously express the ST2L receptor.

Lateral clustering of TLR3:dsRNA signaling units revealed by TLR3ecd:3Fabs quaternary structure.

Toll-like receptor 3 (TLR3) recognizes dsRNA and initiates an innate immune response through the formation of a signaling unit (SU) composed of one double-stranded RNA (dsRNA) and two TLR3 molecules. We report the crystal structure of human TLR3 ectodomain (TLR3ecd) in a quaternary complex with three neutralizing Fab fragments. Fab15 binds an epitope that overlaps the C-terminal dsRNA binding site and, in biochemical assays, blocks the interaction of TLR3ecd with dsRNA, thus directly antagonizing TLR3 signaling through inhibition of SU formation.

Novel antagonist antibody to TLR3 blocks poly(I:C)-induced inflammation in vivo and in vitro.

Toll-like receptor 3 (TLR3) binds and signals in response to dsRNA and poly(I:C), a synthetic double stranded RNA analog. Activation of TLR3 triggers innate responses that may play a protective or detrimental role in viral infections or in immune-mediated inflammatory diseases through amplification of inflammation. Two monoclonal antibodies, CNTO4685 (rat anti-mouse TLR3) and CNTO5429 (CDRs from CNTO4685 grafted onto a mouse IgG1 scaffold) were generated and characterized.

Secretion of the human Toll-like receptor 3 ectodomain is affected by single nucleotide polymorphisms and regulated by Unc93b1.

The innate immune receptor Toll-like receptor 3 (TLR3) can be present on the surface of the plasma membranes of cells and in endolysosomes. The Unc93b1 protein has been reported to facilitate localization of TLR7 and 9 and is required for TLR3, -7, and -9 signaling. We demonstrate that siRNA knockdown of Unc93b1 reduced the abundance of TLR3 on the cell surface without altering total TLR3 accumulation.

TLR3 activation stimulates cytokine secretion without altering agonist-induced human small airway contraction or relaxation.

Respiratory infections exacerbate chronic lung diseases promoting airway inflammation and hyperreactivity. Toll-like receptor 3 (TLR3) recognizes viral double-stranded (ds) RNA such as polyinosinic-polycytidylic acid [poly(I:C)] and stimulates innate immune responses. The objective of this study was to test the hypothesis that dsRNA promotes lung inflammation and alters airway responsiveness to cholinergic and beta-adrenergic receptor agonists in human lung slices.

Long-term activation of TLR3 by poly(I:C) induces inflammation and impairs lung function in mice.

Background: The immune mechanisms associated with infection-induced disease exacerbations in asthma and COPD are not fully understood. Toll-like receptor (TLR) 3 has an important role in recognition of double-stranded viral RNA, which leads to the production of various inflammatory mediators. Thus, an understanding of TLR3 activation should provide insight into the mechanisms underlying virus-induced exacerbations of pulmonary diseases.

Single-stranded oligonucleotides can inhibit cytokine production induced by human toll-like receptor 3.

Toll-like receptor 3 (TLR3) can signal the production of a suite of cytokines and chemokines in response to double-stranded RNA (dsRNA) ligands or the dsRNA mimic poly(I-C). Using a human embryonic kidney 293T cell line to express human TLR3, we determined that poly(I-C)-induced signal could be significantly inhibited by single-stranded DNAs (ssDNAs), but not ssRNA or dsDNA. The ssDNA molecules that down-modulated TLR3 signaling did not affect TLR4 and do not require the hypomethylated CpG motif found in TLR9 ligands.

Down modulation of human TLR3 function by a monoclonal antibody.

Toll-like receptors are a family of pattern-recognition receptors that contribute to the innate immune response. Toll-like receptor 3 (TLR3) signals in response to foreign, endogenous and synthetic ligands including viral dsRNA, bacterial RNA, mitochondrial RNA, endogenous necrotic cell mRNA and the synthetic dsRNA analog, poly(I:C). We have generated a monoclonal antibody (mAb CNTO2424) that recognizes the extracellular domain (ECD) of human TLR3 in a conformation-dependent manner.

Identification and characterization of novel bone marrow myeloid DEC205+Gr-1+ cell subsets that differentially express chemokine and TLRs.

Bone marrow-derived immunomodulatory cytokines impart a critical function in the regulation of innate immune responses and hemopoiesis. However, the source of immunomodulatory cytokines in murine bone marrow and the cellular immune mechanisms that control local cytokine secretion remain poorly defined. Herein, we identified a population of resident murine bone marrow myeloid DEC205(+)CD11c(-)B220(-)Gr1(+)CD8alpha(-)CD11b(+) cells that respond to TLR2, TLR4, TLR7, TLR8, and TLR9 agonists as measured by the secretion of proinflammatory and anti-inflammatory cytokines in vitro.

Effects of single nucleotide polymorphisms on Toll-like receptor 3 activity and expression in cultured cells.

Recognition of double-stranded RNA by Toll-like receptor 3 (TLR3) will increase the production of cytokines and chemokines through transcriptional activation by the NF-kappaB protein. Over 136 single-nucleotide polymorphisms (SNPs) in TLR3 have been identified in the human population. Of these, four alter the sequence of the TLR3 protein.

Biochemical and functional analyses of the human Toll-like receptor 3 ectodomain.

The structure of the human Toll-like receptor 3 (TLR3) ectodomain (ECD) was recently solved by x-ray crystallography, leading to a number of models concerning TLR3 function (Choe, J., Kelker, M. S.

A rapid and efficient method for generating anti-variable region monoclonal antibodies using type-1 interferons as immune modulators.

The generation of anti-variable region monoclonal antibodies (mAbs) against therapeutic antibodies is essential in the pharmacokinetic/pharmacodynamic (PK/PD) assessments of the drugs in clinical study samples. Sandwich EIA and other methods are typically employed to achieve sensitivity and selectivity for the PK/PD analyses. These assays usually require generation of mAb reagents that bind specifically to the drug in non-competing pair combinations.

Healthcare practice ISOlation: articulating systems that drift toward higher quality.

Can higher quality healthcare practices emerge from the development and use of a loosely integrated set of intranets? This paper examines that question through a series of empirical examples that draw on data from one healthcare organization that was part of a multi-year, multi-industry study of intranets. Some of this organization's intranets link clinical practice guidelines to healthcare delivery, in ways that mimic manufacturing sector efforts to improve product quality and reliability. Others act as staging grounds and information repositories for coordinating responses to new rulings and changing interpretations of how to implement recently enacted healthcare legislation.

Structural and functional analyses of the human Toll-like receptor 3. Role of glycosylation.

Toll-like receptors (TLRs) play critical roles in bridging the innate and adaptive immune responses. The human TLR3 recognizes foreign-derived double-stranded RNA and endogenous necrotic cell RNA as ligands. Herein we characterized the contribution of glycosylation to TLR3 structure and function.

Managing managed care: the enabling role of IS/IT.

In their conceptualization of the principal/agent relationship, Jensen and Meckling were not referring to a knowledge worker (KM) agent. Agency theory is extended to the context of a KW agent to identify a key role for IS/IT in facilitating the monitoring function. To test this, the dynamics of a critical principal-KW agent relationship in the health-care industry are investigated It is demonstrated that IS/IT goes beyond just alleviating the agency problem to enabling the KW agent to perform "self-monitoring".