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Dengue virus cocirculates globally as four serotypes (DENV1 to -4) that vary up to 40% at the amino acid level. Viral strains within a serotype further cluster into multiple genotypes. Eliciting a protective tetravalent neutralizing antibody response is a major goal of vaccine design, and efforts to characterize epitopes targeted by polyclonal mixtures of antibodies are ongoing. Previously, we identified two E protein residues (126 and 157) that defined the serotype-specific antibody response to DENV1 genotype 4 strain West Pac-74. DENV1 and DENV2 human vaccine sera neutralized DENV1 viruses incorporating these substitutions equivalently. In this study, we explored the contribution of these residues to the neutralization of DENV1 strains representing distinct genotypes. While neutralization of the genotype 1 strain TVP2130 was similarly impacted by mutation at E residues 126 and 157, mutation of these residues in the genotype 2 strain 16007 did not markedly change neutralization sensitivity, indicating the existence of additional DENV1 type-specific antibody targets. The accessibility of antibody epitopes can be strongly influenced by the conformational dynamics of virions and modified allosterically by amino acid variation. We found that changes at E domain II residue 204, shown previously to impact access to a poorly accessible E domain III epitope, impacted sensitivity of DENV1 16007 to neutralization by vaccine immune sera. Our data identify a role for minor sequence variation in changes to the antigenic structure that impacts antibody recognition by polyclonal immune sera. Understanding how the many structures sampled by flaviviruses influence antibody recognition will inform the design and evaluation of DENV immunogens. IMPORTANCE Dengue virus (DENV) is an important human pathogen that cocirculates globally as four serotypes. Because sequential infection by different DENV serotypes is associated with more severe disease, eliciting a protective neutralizing antibody response against all four serotypes is a major goal of vaccine efforts. Here, we report that neutralization of DENV serotype 1 by polyclonal antibody is impacted by minor sequence variation among virus strains. Our data suggest that mechanisms that control neutralization sensitivity extend beyond variation within antibody epitopes but also include the influence of single amino acids on the ensemble of structural states sampled by structurally dynamic virions. A more detailed understanding of the antibody targets of DENV-specific polyclonal sera and factors that govern their access to antibody has important implications for flavivirus antigen design and evaluation.

INTRODUCTION Intravenous (IV) fluid bags made of polyvinyl chloride (PVC) often contain the plasticizer di(2-ethylhexyl) phthalate (DEHP) to make the PVC flexible. Phthalate esters have been reported to inhibit neuronal nicotinic acetylcholine receptors, which are sensitive to many inhaled anesthetics. This raises the possibility that DEHP might modulate the function of other cys-loop receptors, such as gamma-amino butyric acid type A (GABA(A)) and glycine receptors, and that DEHP-plasticized PVC might interfere with electrophysiologic studies of anesthetic mechanisms on those receptors. METHODS alpha(1)beta(2) GABA(A) and alpha(1) glycine receptors were expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamping. We then measured the effect of buffers from IV bags containing DEHP-plasticized PVC, and of buffers saturated with DEHP, on agonist-induced currents. RESULTS Agonist-induced currents from glycine receptors were enhanced by buffers from IV bags containing DEHP-plasticized PVC by 291.9% +/- 84.5% (mean +/- se) and from saturated solutions of DEHP by 70.8% +/- 16.7%. Agonist-induced currents from alpha(1)beta(2) GABA(A) receptors were inhibited by buffers from IV bags containing DEHP-plasticized PVC by 19.3% +/- 3.2% and by 31.7% +/- 7.0% from buffers saturated with DEHP. CONCLUSIONS The plasticizer DEHP modulates the function of both GABA(A) and glycine receptors. DEHP contamination can confound the results of electrophysiologic studies of anesthetic mechanisms on these receptors if DEHP-plasticized PVC is present in the experimental apparatus.

The receptor for advanced glycation end products (RAGE) is a multiligand receptor that has been shown to contribute to the pathogenesis of diabetes, atherosclerosis, and neurodegeneration. However, its role in asthma and allergic airway disease is largely unknown. These studies use a house dust mite (HDM) mouse model of asthma/allergic airway disease. Respiratory mechanics were assessed and compared between wild-type and RAGE knockout mice. Bronchovascular architecture was assessed with quantitative scoring, and expression of RAGE, immunoglobulins, and relevant cytokines was assessed by standard protein detection methods and/or quantitative RT-PCR. The absence of RAGE abolishes most assessed measures of pathology, including airway hypersensitivity (resistance, tissue damping, and elastance), eosinophilic inflammation, and airway remodeling. IL-4 secretion, isotype class switching, and antigen recognition are intact in the absence of RAGE. In contrast, normal increases in IL-5, IL-13, eotaxin, and eotaxin-2 production are abrogated in the RAGE knockouts. IL-17 indicates complex regulation, with elevated baseline expression in RAGE knockouts, but no induction in response to allergen. Treatment of WT mice with an inhibitor of RAGE markedly reduces inflammation in the HDM model, suggesting that RAGE inhibition may serve as a promising therapeutic strategy. Finally, the results in the HDM model are recapitulated in an ovalbumin model of asthma, suggesting that RAGE plays a role in asthma irrespective of the identity of the allergens involved.

INTRODUCTION: Tolerance is observed for a variety of central nervous system depressants including ethanol, which is an anesthetic, but has not been convincingly demonstrated for a potent halogenated volatile anesthetic. Failure to demonstrate tolerance to these agents may be the result of inadequate exposure to anesthetic. In this study, we exposed Xenopus laevis tadpoles to surgical anesthetic concentrations of isoflurane for 1 wk. METHODS: Xenopus laevis tadpoles were produced by in vitro fertilization, and exposed to isoflurane (0.59%, 0.98%, 1.52%) or oxygen for 1 wk starting from the time of fertilization. RESULTS: Changes in anesthetic EC 50 were small and not in a consistent direction. Control animals had an anesthetic EC 50 of 0.594% ± 0.003% isoflurane. Tadpoles exposed to 1.52% isoflurane had a lower EC 50 than controls (by 16%), whereas tadpoles raised under 0.59% and 0.98% isoflurane had higher EC 50 S than control (by 4.7% and 7.4%, respectively). CONCLUSION: We provide the first description of week-long exposures of vertebrates to surgical anesthetic concentrations of isoflurane, and the first report of such exposures in developing vertebrates. Tolerance to isoflurane does not occur in developing Xenopus laevis tadpoles. Taken together with studies in other organisms, the development of tolerance to ethanol but not isoflurane suggests that mechanisms shared by these drugs probably do not account for the development of tolerance.

We tested the hypothesis that two metabolites that are elevated in ketosis (beta-hydroxybutyric acid, and acetone) modulate ion channels in a manner similar to anesthetics and produce anesthesia in animals.alpha1beta2gamma2sgamma-aminobutyric acid type A (GABA(A)), alpha1 glycine, NR1/NR2A N-methyl-d-aspartate, and two pore domain TRESK channels were expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamping. The effect of beta hydroxybutyric acid and acetone on channel function was measured. The anesthetic effects of these drugs were measured in X. laevis tadpoles.Both beta hydroxybutyric acid and acetone enhanced glycine receptor function in the concentration range that is obtained in ketoacidosis in humans. Beta hydroxybutyric acid also enhanced GABA(A) receptor function at these concentrations. Both acetone and beta-hydroxybutyric acid anesthetized tadpoles, with an EC50 for acetone of 264 +/- 2 mM (mean +/- se) and for beta-hydroxybutyric acid of 151 +/- 11 mM at pH 7.0. Acetone enhanced GABA(A) receptors at concentrations of 50 mM and above. Inhibition of TRESK channel function was seen with 100 mM acetone or larger concentration. N-methyl-D-aspartate receptor function was inhibited at concentrations of acetone of 200 mM and larger.Beta hydroxybutyric acid and acetone are anesthetics. Both ketone bodies enhance inhibitory glycine receptors at concentrations observed clinically in ketoacidosis. In addition, beta-hydroxybutyric acid enhances GABA(A) receptor function at these concentrations. Subanesthetic concentrations of these drugs may contribute to the lethargy and impairment of consciousness seen in ketoacidosis.

A mechanism of anesthesia has recently been proposed which predicts that coreleased neurotransmitters may modulate neurotransmitter receptors for which they are not the native agonist in a manner similar to anesthetics.We tested this prediction by applying acetylcholine to a NR1/NR2A N-methyl-d-aspartate receptor, glycine to a wild-type alpha(1)beta(2) and anesthetic-resistant alpha(1)(S270I)beta(2) gamma-amino-butyric acid (GABA) type A receptor, and GABA to a homomeric alpha(1) wild type and anesthetic-resistant alpha(1) S267I glycine receptor. Receptors were expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamping.We found inhibition of N-methyl-d-aspartate receptor function by acetylcholine, enhancement of glycine receptor function by GABA, and enhancement of GABA type A receptor function by glycine. As expected of compounds with anesthetic activity, GABA showed far less potentiation (enhancement) of the function of the anesthetic-resistant S267I glycine receptor than that of the wild-type receptor. Glycine potentiated the function of wild-type GABA type A receptors but inhibited the function of the anesthetic-resistant S270I GABA type A receptor.These results show that neurotransmitters that are coreleased onto anesthetic-sensitive receptors may modulate the function of receptors for which they are not the native agonist via an anesthetic-like mechanism. These findings lend support to a recent theory of anesthetic action.

A recent theory of anesthesia predicts that some endogenous compounds should have anesthetic properties. This theory raises the possibility that metabolites that are profoundly elevated in disease may also exert anesthetic effects. Because in pathophysiologic concentrations, ammonia reversibly impairs memory, consciousness, and responsiveness to noxious stimuli in a manner similar to anesthetics, we investigated whether ammonia had anesthetic properties.The effect of ammonia was studied on alpha1beta2 and alpha1beta2gamma2s gamma-amino butyric acid type A, alpha1 glycine, and NR1/NR2A N-methyl-D-aspartate receptors, and the two-pore domain potassium channel TRESK. Channels were expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamping. The immobilizing effect of ammonia in rats was evaluated by determining the reduction in isoflurane minimum alveolar concentration produced by IV infusion of ammonium chloride. The olive oil-water partition coefficient was measured to determine whether free ammonia (NH3) followed the Meyer-Overton relation.Ammonia positively modulated TRESK channels and glycine receptors. No effect was seen on alpha1beta2 and alpha1beta2gamma2s gamma-amino butyric acid type A receptors or NR1/NR2A N-methyl-d-aspartate receptors. Ammonia reversibly decreased the requirement for isoflurane, with a calculated immobilizing EC50 of 1.6 +/- 0.1 mM NH4Cl. The Ostwald olive oil-water partition coefficient for NH3 was 0.018. At a pH of 7.4, and at the anesthetic EC50, the NH3 concentration in bulk olive oil is 0.42 muM, approximately five orders of magnitude less than observed by anesthetics that follow the Meyer-Overton relation.These findings support the hypothesis that ammonia has anesthetic properties. Bulk oil concentration did not predict the potency of ammonia.

Background Single nucleotide polymorphisms in the human gene for the receptor for advanced glycation end-products (RAGE) are associated with an increased incidence of asthma. RAGE is highly expressed in the lung and has been reported to play a vital role in the pathogenesis of murine models of asthma/allergic airway inflammation (AAI) by promoting expression of the type 2 cytokines IL-5 and IL-13. IL-5 and IL-13 are prominently secreted by group 2 innate lymphoid cells (ILC2s), which are stimulated by the proallergic cytokine IL-33. Objective We sought to test the hypothesis that pulmonary RAGE is necessary for allergen-induced ILC2 accumulation in the lung. Methods AAI was induced in wild-type and RAGE knockout mice by using IL-33, house dust mite extract, or Alternaria alternata extract. RAGE's lung-specific role in type 2 responses was explored with bone marrow chimeras and induction of gastrointestinal type 2 immune responses. Results RAGE was found to drive AAI by promoting IL-33 expression in response to allergen and by coordinating the inflammatory response downstream of IL-33. Absence of RAGE impedes pulmonary accumulation of ILC2s in models of AAI. Bone marrow chimera studies suggest that pulmonary parenchymal, but not hematopoietic, RAGE has a central role in promoting AAI. In contrast to the lung, the absence of RAGE does not affect IL-33–induced ILC2 influx in the spleen, type 2 cytokine production in the peritoneum, or mucus hypersecretion in the gastrointestinal tract. Conclusions For the first time, this study demonstrates that a parenchymal factor, RAGE, mediates lung-specific accumulation of ILC2s.