The impact of work-related stress on medication errors in Eastern Region Saudi Arabia.

OBJECTIVE: To examine the relationship between overall level and source-specific work-related stressors on medication errors rate. DESIGN: A cross-sectional study examined the relationship between overall levels of stress, 25 source-specific work-related stressors and medication error rate based on documented incident reports in Saudi Arabia (SA) hospital, using secondary databases. SETTING: King Abdulaziz Hospital in Al-Ahsa, Eastern Region, SA. PARTICIPANTS: Two hundred and sixty-nine healthcare professionals (HCPs). MAIN OUTCOME MEASURES: The odds ratio (OR) and corresponding 95% confidence interval (CI) for HCPs documented incident report medication errors and self-reported sources of Job Stress Survey. RESULTS: Multiple logistic regression analysis identified source-specific work-related stress as significantly associated with HCPs who made at least one medication error per month (P < 0.05), including disruption to home life, pressure to meet deadlines, difficulties with colleagues, excessive workload, income over 10 000 riyals and compulsory night/weekend call duties either some or all of the time. Although not statistically significant, HCPs who reported overall stress were two times more likely to make at least one medication error per month than non-stressed HCPs (OR: 1.95, P = 0.081). CONCLUSION: This is the first study to use documented incident reports for medication errors rather than self-report to evaluate the level of stress-related medication errors in SA HCPs. Job demands, such as social stressors (home life disruption, difficulties with colleagues), time pressures, structural determinants (compulsory night/weekend call duties) and higher income, were significantly associated with medication errors whereas overall stress revealed a 2-fold higher trend.

TLR3-specific double-stranded RNA oligonucleotide adjuvants induce dendritic cell cross-presentation, CTL responses, and antiviral protection.

Maturation of dendritic cells (DC) to competent APC is essential for the generation of acquired immunity and is a major function of adjuvants. dsRNA, a molecular signature of viral infection, drives DC maturation by activating TLR3, but the size of dsRNA required to activate DC and the expression patterns of TLR3 protein in DC subsets have not been established. In this article, we show that cross-priming CD8α(+) and CD103(+) DC subsets express much greater levels of TLR3 than other DC. In resting DC, TLR3 is located in early endosomes and other intracellular compartments but migrates to LAMP1(+) endosomes on stimulation with a TLR3 ligand. Using homogeneous dsRNA oligonucleotides (ONs) ranging in length from 25 to 540 bp, we observed that a minimum length of ∼90 bp was sufficient to induce CD86, IL-12p40, IFN-ß, TNF-α, and IL-6 expression, and to mature DC into APC that cross-presented exogenous Ags to CD8(+) T cells. TLR3 was essential for activation of DC by dsRNA ONs, and the potency of activation increased with dsRNA length and varied between DC subsets. In vivo, dsRNA ONs, in a size-dependent manner, served as adjuvants for the generation of Ag-specific CTL and for inducing protection against lethal challenge with influenza virus when given with influenza nucleoprotein as an immunogen. These results provide the basis for the development of TLR3-specific adjuvants capable of inducing immune responses tailored for viral pathogens.

Dimerization of Toll-like receptor 3 (TLR3) is required for ligand binding.

TLR3 (Toll-like receptor 3) recognizes dsRNA, a potent indicator of viral infection. The extracellular domain of TLR3 dimerizes when it binds dsRNA, and the crystal structure of the dimeric complex reveals three sites of interaction on each extracellular domain, two that bind dsRNA and one that is responsible for dimer formation. The goal of this study was to determine which amino acid residues are essential for forming a stable receptor·ligand complex and whether dimerization of TLR3 is required for dsRNA binding. Using a novel ELISA to analyze dsRNA binding by mutant TLR3 constructs, we identified the essential interacting residues and determined that the simultaneous interaction of all three sites is required for ligand binding. In addition, we show that TLR3 is unable to bind dsRNA when dimerization is prevented by mutating residues in the dimerization site or by immobilizing TLR3 at low density. We conclude that dimerization of TLR3 is essential for ligand binding and that the three TLR3 contact sites individually interact weakly with their binding partners but together form a high affinity receptor·ligand complex.

The TLR3 signaling complex forms by cooperative receptor dimerization.

Toll-like receptors (TLRs) initiate immune responses by recognizing pathogen-associated molecules, but the molecular basis for recognition is poorly understood. In particular, it is unclear how receptor-ligand interactions lead to the initiation of downstream signaling. Here, we describe the mechanism by which TLR3 recognizes its ligand, double-stranded RNA (dsRNA), and forms an active signaling complex. We show that dsRNA binds saturably, specifically, and reversibly to a defined ligand-binding site (or sites) on the TLR3 ectodomain (TLR3ecd). Binding affinities increase with both buffer acidity and ligand size. Purified TLR3ecd protein is exclusively monomeric in solution, but through a highly cooperative process, it forms dimers when bound to dsRNA, and multiple TLR3ecd dimers bind to long dsRNA strands. The smallest dsRNA oligonucleotides that form stable complexes with TLR3ecd (40-50 bp) each bind one TLR3ecd dimer, and these are also the smallest oligonucleotides that efficiently activate TLR3 in cells. We conclude that TLR3 assembles on dsRNA as stable dimers and that the minimal signaling unit is one TLR3 dimer.

The toll-like receptor 3:dsRNA signaling complex.

Toll-like receptors (TLRs) recognize conserved molecular patterns in invading pathogens and trigger innate immune responses. TLR3 recognizes dsRNA, a molecular signature of most viruses via its ectodomain (ECD). The TLR3-ECD structure consists of a 23 turn coil bent into the shape of a horseshoe with specialized domains capping the N and C-terminal ends of the coil. TLR3-ECDs bind as dimeric units to dsRNA oligonucleotides of at least 45 bp in length, the minimal length required for signal transduction. X-ray analysis has shown that each TLR3-ECD of a dimer binds dsRNA at two sites located at opposite ends of the TLR3 "horseshoe" on the one lateral face that lacks N-linked glycans. Intermolecular contacts between the C-terminal domains of two TLR3-ECDs stabilize the dimer and position the C-terminal residues within 20-25 A of each other, which is thought to be essential for transducing a signal across the plasma membrane in intact TLR3 molecules. Interestingly, in TLRs 1, 2 and 4, which bind lipid ligands using very different interactions from TLR3, the ligands nevertheless promote the formation of a dimer in which the same two lateral surfaces as in the TLR3-ECD:dsRNA complex face each other, bringing their C-termini in close proximity. Thus, a pattern is emerging in which pathogen-derived substances bind to TLR-ECDs, thereby promoting the formation of a dimer in which the glycan-free ligand binding surfaces face each other and the two C-termini are brought in close proximity for signal transduction.

Predicting Toll-like receptor structures and characterizing ligand binding.

Toll-like receptor (TLR) ligand-binding domains comprise 18-25 tandem copies of a 24-residue motif known as the leucine-rich repeat (LRR). Unlike other LRR proteins, TLRs contain significant numbers of non-consensus LRR sequences, which makes their identification by computer domain search programs problematic. Here, we provide methods for identifying non-consensus LRRs. Using the location of these LRRs, hypothetical models are constructed based on the known molecular structures of homologous LRR proteins. However, when a hypothetical model for TLR3 is compared with the molecular structure solved by x-ray crystallography, the solenoid curvature, planarity, and conformations of the LRR insertions are incorrectly predicted. These differences illustrate how non-consensus LRR motifs influence TLR structure. Since the determination of molecular structures by crystallography requires substantial amounts of protein, we describe methods for producing milligram amounts of TLR3 extracellular domain (ECD) protein. The recombinant TLR3-ECD previously used to solve the molecular structure of TLR3-ECD has also been used to study the binding of TLR3-ECD to its ligand, double-stranded RNA (dsRNA). In the last section, we describe the preparation of defined TLR3 ligands and present methods for characterizing their interaction with TLR3-ECD.

Induction of dendritic cell maturation by pertussis toxin and its B subunit differentially initiate Toll-like receptor 4-dependent signal transduction pathways.

OBJECTIVE: Pertussis toxin (PT) has the capacity to activate dendritic cells (DCs) for the augmentation of cell-mediated immune responses. To investigate the mechanism(s) by which PT activates DCs, we investigated the effects of PT and its B-oligomer (PTB) on the maturation of human and mouse DCs and determined whether PT could act as a pathogen-associated molecular pattern to activate one of the Toll-like receptors (TLRs). METHODS: The effects of PT and PTB on the maturation of human and mouse DCs were analyzed in terms of surface marker expression, cytokine production, antigen-presenting capacity, and intracellular signaling. The participation of TLR4 in PT-induced signaling was determined by comparing the effect of PT on DCs derived from TLR4-deficient and wild-type mice, as well as by measuring PT-induced NF-kappaB activation in HEK293 cells transiently transfected to express various TLRs. RESULTS: Although both promoted phenotypic and functional maturation DCs, however, unlike PT that induced DC production of interleukin (IL)-6, tumor necrosis factor-alpha, IL-12, and interferon-inducible protein, PTB was capable of stimulating the production of interferon-inducible protein. Bone marrow-derived DCs from C3H/HeJ mice with defective TLR-4 alleles were unresponsive to PT and PTB, whereas DCs from C3H/HeN mice responded. In addition, PT induced NF-kappaB activation and IL-8 production in HEK293 cells transfected with a combination of TLR4 and MD2 but not in nontransfected or TLR2-transfected HEK293 cells. Comparison of the patterns of cytokine induction and intracellular signaling events in DCs treated by PT and PTB revealed that although PT, like lipopolysaccharide, triggered both MyD88-dependent and -independent pathways, PTB preferentially triggered MyD88-independent pathways. Interestingly, mouse splenocyte proliferation in response to PT and PTB was only partially dependent on TLR4. CONCLUSION: The data identify PT as another pathogen-associated molecular pattern that induces DC maturation in a TLR4-dependent manner. Unlike PT, which triggers both MyD88-dependent and -independent pathways, PTB only triggers the MyD88-independent pathway in DCs.

The molecular structure of the TLR3 extracellular domain.

Toll-like receptors (TLRs), type I integral membrane receptors, recognize pathogen associated molecular patterns (PAMPs). PAMP recognition occurs via the N-terminal ectodomain (ECD) which initiates an inflammatory response that is mediated by the C-terminal cytosolic signaling domain. To understand the molecular basis of PAMP recognition, we have begun to define TLR-ECD structurally. We have solved the structure of TLR3-ECD, which recognizes dsRNA, a PAMP associated with viral pathogens. TLR3-ECD is a horseshoe-shaped solenoid composed of 23 leucine-rich repeats (LRRs). The regular LRR surface is disrupted by two insertions at LRR12 and LRR20 and 11 N-linked carbohydrates. Of note, one side of the ECD is carbohydrate-free and could form an interaction interface. We have shown that TLR3-ECD binds directly to pI:pC, a synthetic dsRNA ligand, but not to p(dI):p(dC). Without a TLR3-dsRNA complex structure, we can only speculate how ligand binds. Analysis of the unliganded structure reveals two patches of basic residues and two binding sites for phosphate backbone mimics, sulfateions, that may be capable of recognizing ligand. Mutational and co-crystallization studies are currently underway to determine how TLR3 binds its ligand at the molecular level.

Controlling the Toll road to dendritic cell polarization.

The activation of dendritic cells (DC) via Toll-like receptors (TLRs) plays a decisive role in shaping the outcome of primary immune responses. Following TLR engagement by microbial products, DC migrate from peripheral tissues to lymphoid organs and up-regulate major histocompatibility complex and costimulatory molecules, acquiring the unique capacity to prime pathogen-specific, naïve T cells. In addition, DC determine the character of the ensuing immune response by secreting cytokines that drive the development of T cells into T helper cell type 1 (Th1), Th2, or T regulatory effector cells. Three major factors influence the pattern of cytokines released by DC and accordingly, the Th balance: the lineage to which DC belong; the maturation stimulus; and inflammatory mediators present at the site of infection. A major focus of this review is the capacity of DC to integrate these factors and elicit distinct classes of immune responses.

Human conjunctival epithelial cells lack lipopolysaccharide responsiveness due to deficient expression of MD2 but respond after interferon-gamma priming or soluble MD2 supplementation.

Inflammatory responses to Gram-positive and Gram-negative bacterial cell wall components are initiated by Toll-like receptor 2 (TLR2) and TLR4, respectively. Therefore, the existence of functionally active TLR2 and TLR4 in human conjunctival epithelial cells (HCEC) are critical for the effective host defense against bacterial infections in the eye. We examined the ability of HCEC to respond to TLR4 ligand, lipopolysaccharide (LPS), or TLR2 ligands, lipoteichoic acid (LTA) and peptidoglycan (PGN) using the Chang conjunctival epithelial cell line and the primary conjunctival epithelial cell line (IOBA-NHC) as in vitro models. Incubation of Chang cells with LPS (1 to 1,000 ng/ml) failed to stimulate IL-6 production where as stimulation with LTA or PGN resulted in marked increases in IL-6 production. Semi-quantitative RT-PCR and immunofluorescence analyses showed that Chang cells express TLR2 and TLR4 mRNA and proteins. However, these cells expressed little or no mRNA encoding MD2, an accessory molecule required for TLR4 signaling. Incubation of Chang epithelial cells with interferon-gamma (IFNgamma), but not TNF-alpha, stimulated MD2 mRNA expression and restored LPS responsiveness. In addition, when Chang cell cultures were supplemented with soluble MD2, LPS was able to stimulate IL-6 production. The lack of LPS response, deficient expression of MD2, and induction of MD2 expression and LPS response after IFNgamma priming, were also evident in IOBA-NHC cells. These results demonstrate that HCEC lack LPS responsiveness due to deficient expression of MD2 and that the response can be restored by IFN-gamma priming or MD2 supplementation.

The structural biology of Toll-like receptors.

The membrane-bound Toll-like receptors (TLRs) trigger innate immune responses after recognition of a wide variety of pathogen-derived compounds. Despite the wide range of ligands recognized by TLRs, the receptors share a common structural framework in their extracellular, ligand-binding domains. These domains all adopt horseshoe-shaped structures built from leucine-rich repeat motifs. Typically, on ligand binding, two extracellular domains form an "m"-shaped dimer sandwiching the ligand molecule bringing the transmembrane and cytoplasmic domains in close proximity and triggering a downstream signaling cascade. Although the ligand-induced dimerization of these receptors has many common features, the nature of the interactions of the TLR extracellular domains with their ligands varies markedly between TLR paralogs.

Structural basis of toll-like receptor 3 signaling with double-stranded RNA.

Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA), a molecular signature of most viruses, and triggers inflammatory responses that prevent viral spread. TLR3 ectodomains (ECDs) dimerize on oligonucleotides of at least 40 to 50 base pairs in length, the minimal length required for signal transduction. To establish the molecular basis for ligand binding and signaling, we determined the crystal structure of a complex between two mouse TLR3-ECDs and dsRNA at 3.4 angstrom resolution. Each TLR3-ECD binds dsRNA at two sites located at opposite ends of the TLR3 horseshoe, and an intermolecular contact between the two TLR3-ECD C-terminal domains coordinates and stabilizes the dimer. This juxtaposition could mediate downstream signaling by dimerizing the cytoplasmic Toll interleukin-1 receptor (TIR) domains. The overall shape of the TLR3-ECD does not change upon binding to dsRNA.

Neisseria meningitidis type C capsular polysaccharide inhibits lipooligosaccharide-induced cell activation by binding to CD14.

Encapsulated Neisseria meningitidis can invade mucosal barriers and cause systemic diseases. Activation of the innate immune system by conserved meningococcal molecules such as lipooligosaccharides (LOS) is essential for the generation of an effective host immune response. Here we show that the type C capsular polysaccharide of N. meningitidis (MCPS) inhibited LOS-induced interleukin-6 and TNF-alpha secretion from monocytes, and blocked the maturation of dendritic cells induced by LOS, while the capsular polysaccharide from group B streptococcus type III and t(4-hydroxy-3-nitrophenyl) acetyl (NP)-Ficoll had no such effect. MCPS also inhibited the LOS-induced NF-kappaB activation and phosphorylation of signalling molecules such as ERK1/2, p38 and Jun N-terminal kinase. In a direct binding assay, MCPS manifested a concentration-dependent binding to recombinant lipoprotein binding protein and CD14, the two members of the LOS receptor complex. In addition, the binding of LOS to CD14 and lipopolysaccharide binding protein was inhibited by MCPS. We established that MCPS binding to CD14 is responsible for the inhibition of LOS-mediated cell activation because MCPS inhibition of LOS was reversed when access amounts of CD14 were added to culture media of HEK293 cells expressing TLR4 and MD-2, and the magnitude of recovery in LOS stimulation correlated with the increase in CD14 concentration. These results suggest a new virulence property of meningococcal capsular polysaccharides.

Dendritic cell modulation by mast cells controls the Th1/Th2 balance in responding T cells.

The cytokines secreted by pathogen-activated human dendritic cells (DC) are strongly regulated in vitro by histamine, a major component of mast cell granules, ultimately modulating the capacity of the DC to polarize naive T cells. Because DC and mast cells are located in close proximity in peripheral compartments, we hypothesized that mast cell products would influence the maturation of DC and hence the Th balance of an immune response in vivo. In this study, we show that specific mast cell degranulation stimuli, given s.c. in mice with Ag and adjuvant, produce effector T cells that proliferate to Ag but secrete dramatically reduced levels of IFN-gamma and increased amounts of IL-4 compared with control T cells primed in the absence of a mast cell stimulus. Immunization with Ag and adjuvant in the presence of a degranulation stimulus also resulted in the accumulation of DC in the draining lymph nodes that had reduced capacity to induce Ag-specific Th1 cells, in comparison with DC from mice lacking a degranulation stimulus. Therefore, by acting upon DC at sites of inflammation, mast cells play a critical role in determining the polarity of Ag-specific T cell responses in vivo.

The dsRNA binding site of human Toll-like receptor 3.

Pathogen recognition by Toll-like receptors (TLRs) initiates innate immune responses that are essential for inhibiting pathogen dissemination and for the development of acquired immunity. The TLRs recognize pathogens with their N-terminal ectodomains (ECD), but the molecular basis for this recognition is not known. Recently we reported the x-ray structure for unliganded TLR3-ECD; however, it has proven difficult to obtain a crystal structure of TLR3 with its ligand, dsRNA. We have now located the TLR3 ligand binding site by mutational analysis. More than 50 single-residue mutations have been generated throughout the TLR3-ECD, but only two, H539E and N541A, resulted in the loss of TLR3 activation and ligand binding functions. These mutations locate the dsRNA binding site on the glycan-free, lateral surface of TLR3 toward the C terminus and suggest a model for dsRNA binding and TLR3 activation.

Cytoplasmic targeting motifs control localization of toll-like receptor 9.

Toll-like receptors (TLRs) are essential for host defense. Although several TLRs reside on the cell surface, nucleic acid recognition of TLRs occurs intracellularly. For example, the receptor for CpG containing bacterial and viral DNA, TLR9, is retained in the endoplasmic reticulum. Recent evidence suggests that the localization of TLR9 is critical for appropriate ligand recognition. Here we have defined which structural features of the TLR9 molecule control its intracellular localization. Both the cytoplasmic and ectodomains of TLR9 contain sufficient information, whereas the transmembrane domain plays no role in intracellular localization. We identify a 14-amino acid stretch that directs TLR9 intracellularly and confers intracellular localization to the normally cell surface-expressed TLR4. Truncation or mutation of the cytoplasmic tail of TLR9 reveals a vesicle localization motif that targets early endosomes. We propose a model whereby modification of the cytoplasmic tail of TLR9 results in trafficking to early endosomes where it encounters CpG DNA.

Analysis of TLR4 polymorphic variants: new insights into TLR4/MD-2/CD14 stoichiometry, structure, and signaling.

TLR4 is the signal-transducing receptor for structurally diverse microbial molecules such as bacterial LPS, respiratory syncytial virus fusion (F) protein, and chlamydial heat shock protein 60. Previous studies associated two polymorphic mutations in the extracellular domain of TLR4 (Asp(299)Gly and Thr(399)Ile) with decreased LPS responsiveness. To analyze the molecular basis for diminished responsiveness, site-specific mutations (singly or coexpressed) were introduced into untagged and epitope (Flag)-tagged wild-type (WT) TLR4 expression vectors to permit a direct comparison of WT and mutant signal transduction. Coexpression of WT TLR4, CD14, and MD-2 expression vectors in HEK293T cells was first optimized to achieve optimal LPS-induced NF-kappaB reporter gene expression. Surprisingly, transfection of cells with MD-2 at high input levels often used in the literature suppressed LPS-induced signaling, whereas supraoptimal CD14 levels did not. Under conditions where WT and polymorphic variants were comparably expressed, significant differences in NF-kappaB activation were observed in response to LPS and two structurally unrelated TLR4 agonists, chlamydial heat shock protein 60 and RSV F protein, with the double, cosegregating mutant TLR4 exhibiting the greatest deficiency. Overexpression of Flag-tagged WT and mutant vectors at input levels resulting in agonist-independent signaling led to equivalent NF-kappaB signaling, suggesting that these mutations in TLR4 affect appropriate interaction with agonist or coreceptor. These data provide new insights into the importance of stoichiometry among the components of the TLR4/MD-2/CD14 complex. A structural model that accounts for the diminished responsiveness of mutant TLR4 polymorphisms to structurally unrelated TLR4 agonists is proposed.

The molecular structure of the Toll-like receptor 3 ligand-binding domain.

Innate immunity is the first line of defense against invading pathogens. Toll-like receptors (TLRs) act as sentinels of the innate immune system, sensing a variety of ligands from lipopolysaccharide to flagellin to dsRNA through their ligand-binding domain that is composed of leucine-rich repeats (LRRs). Ligand binding initiates a signaling cascade that leads to the up-regulation of inflammation mediators. In this study, we have expressed and crystallized the ectodomain (ECD) of human TLR3, which recognizes dsRNA, a molecular signature of viruses, and have determined the molecular structure to 2.4-A resolution. The overall horseshoe-shaped structure of the TLR3-ECD is formed by 23 repeating LRRs that are capped at each end by specialized non-LRR domains. The extensive beta-sheet on the molecule's concave surface forms a platform for several modifications, including insertions in the LRRs and 11 N-linked glycans. The TLR3-ECD structure indicates how LRR loops can establish distinct pathogen recognition receptors.

Measurement of intercellular conjugates by flow cytometry.

Analysis of fluorescently labeled cellular aggregates can be accomplished quickly and accurately with the technique of flow cytometry. With a dual-laser flow cytometer, the excitation and emission spectra of fluorophores used to label cells are widely separated, and there is no spillover of emission from one fluorophore into the detector measuring emission from the other. Consequently, conjugates can be measured by a dual-laser instrument when the extent of labeling two cell types with fluorophores are very different. The more commonly available single-laser cytometers can also be used to measure multicellular conjugates, but due to overlaps in emission spectra, the extent of labeling cells with fluorophores must be controlled much more carefully when the single-laser machines are used. This unit describes the labeling of cells and analysis of conjugates with either dual-laser or single laser flow cytometers.

Production of bispecific antibodies.

Bispecific antibodies (bsAbs) contain two different binding specificities within a single molecule and can specifically bind two different molecules together. BsAbs can be produced by chemically cross-linking purified antibodies or Fab fragments with reducible disulfide bonds or nonreducible thioether bonds, both of which are described in this unit. Protocols are also presented for producing BsAbs by fusing two antibody-producing hybridomas that can be selected for based on drug resistance, or by double labeling with fluorochromes and FACS. Support protocols describe screening and purification of bsAbs.