Autism Detection in Children by Combined Use of Gaze Preference and the M-CHAT-R in a Resource-Scarce Setting.

Most children with autism spectrum disorder (ASD), in resource-limited settings (RLS), are diagnosed after the age of four. Our work confirmed and extended results of Pierce that eye tracking could discriminate between typically developing (TD) children and those with ASD. We demonstrated the initial 15 s was at least as discriminating as the entire video. We evaluated the GP-MCHAT-R, which combines the first 15 s of manually-coded gaze preference (GP) video with M-CHAT-R results on 73 TD children and 28 children with ASD, 36-99 months of age. The GP-MCHAT-R (AUC = 0.89 (95%CI: 0.82-0.95)), performed significantly better than the MCHAT-R (AUC = 0.78 (95%CI: 0.71-0.85)) and gaze preference (AUC = 0.76 (95%CI: 0.64-0.88)) alone. This tool may enable early screening for ASD in RLS.

Inhibition of Inflammatory and Neuropathic Pain by Targeting a Mu Opioid Receptor/Chemokine Receptor5 Heteromer (MOR-CCR5).

Chemokine release promotes cross-talk between opioid and chemokine receptors that in part leads to reduced efficacy of morphine in the treatment of chronic pain. On the basis of the possibility that a MOR-CCR5 heteromer is involved in such cross-talk, we have synthesized bivalent ligands (MCC series) that contain mu opioid agonist and CCR5 antagonist pharmacophores linked through homologous spacers (14-24 atoms). When tested on lipopolysaccharide-inflamed mice, a member of the series (MCC22; 3e) with a 22-atom spacer exhibited profound antinociception (i.t. ED50 = 0.0146 pmol/mouse) that was 2000× greater than morphine. Moreover, MCC22 was ~3500× more potent than a mixture of mu agonist and CCR5 antagonist monovalent ligands. These data strongly suggest that MCC22 acts by bridging the protomers of a MOR-CCR5 heteromer having a TM5,6 interface. Molecular simulation studies are consistent with such bridging. This study supports the MOR-CCR5 heteromer as a novel target for the treatment of chronic pain.

Putative kappa opioid heteromers as targets for developing analgesics free of adverse effects.

It is now generally recognized that upon activation by an agonist, ß-arrestin associates with G protein-coupled receptors and acts as a scaffold in creating a diverse signaling network that could lead to adverse effects. As an approach to reducing side effects associated with κ opioid agonists, a series of ß-naltrexamides 3-10 was synthesized in an effort to selectively target putative κ opioid heteromers without recruiting ß-arrestin upon activation. The most potent derivative 3 (INTA) strongly activated KOR-DOR and KOR-MOR heteromers in HEK293 cells. In vivo studies revealed 3 to produce potent antinociception, which, when taken together with antagonism data, was consistent with the activation of both heteromers. 3 was devoid of tolerance, dependence, and showed no aversive effect in the conditioned place preference assay. As immunofluorescence studies indicated no recruitment of ß-arrestin2 to membranes in coexpressed KOR-DOR cells, this study suggests that targeting of specific putative heteromers has the potential to identify leads for analgesics devoid of adverse effects.

The δ opioid receptor agonist SNC80 selectively activates heteromeric µ-δ opioid receptors.

Coexpressed and colocalized µ- and δ-opioid receptors have been established to exist as heteromers in cultured cells and in vivo. However the biological significance of opioid receptor heteromer activation is less clear. To explore this significance, the efficacy of selective activation of opioid receptors by SNC80 was assessed in vitro in cells singly and coexpressing opioid receptors using a chimeric G-protein-mediated calcium fluorescence assay, SNC80 produced a substantially more robust response in cells expressing µ-δ heteromers than in all other cell lines. Intrathecal SNC80 administration in µ- and δ-opioid receptor knockout mice produced diminished antinociceptive activity compared with wild type. The combined in vivo and in vitro results suggest that SNC80 selectively activates µ-δ heteromers to produce maximal antinociception. These data contrast with the current view that SNC80 selectively activates δ-opioid receptor homomers to produce antinociception. Thus, the data suggest that heteromeric µ-δ receptors should be considered as a target when SNC80 is employed as a pharmacological tool in vivo.

N-naphthoyl-beta-naltrexamine (NNTA), a highly selective and potent activator of µ/kappa-opioid heteromers.

Numerous G protein-coupled receptors (GPCRs) have been shown to form heteromeric receptors in cell-based assays. Among the many heteromers reported in the opioid receptor family are µ/κ, κ/δ, and µ/δ. However, the in vivo physiological and behavioral relevance for the proposed heteromers have not yet been established. Here we report a unique example of a ligand, N-naphthoyl-ß-naltrexamine (NNTA) that selectively activates heteromeric µ/κ-opioid receptors in HEK-293 cells and induces potent antinociception in mice. NNTA was an exceptionally potent agonist in cells expressing µ/κ-opioid receptors. Intriguingly, it was found to be a potent antagonist in cells expressing only µ-receptors. In the mouse tail-flick assay, intrathecal (i.t.) NNTA produced antinociception that was ~100-fold greater than by intracerebroventricular (i.c.v.) administration. The κ-antagonist, norBNI, decreased the i.t. potency, and the activity was virtually abolished in µ-opioid receptor knockout mice. No tolerance was induced i.t., but marginal tolerance (3-fold) was observed via the i.c.v. route. Moreover, NNTA produced neither significant physical dependence nor place preference in the ED50 dose range. Taken together, this work provides an important pharmacologic tool for investigating the in vivo functional relevance of heteromeric µ/κ-opioid receptors and suggests an approach to potent analgesics with fewer deleterious side effects.

A κ Opioid Pharmacophore Becomes a Spinally Selective κ-δ Agonist When Modified with a Basic Extender Arm.

We have explored the concept of a molecular extender arm attached to a κ opioid agonist pharmacophore 3 (ICI-199,441) in an effort to potentially interact with a complementary group on a neighboring opioid receptor. The molecular arm containing a terminal amine group was lengthened incrementally from 11 up to 18 atoms. Increasing the number of atoms in the arm produced virtually no change in the mouse intracerebroventricular (i.c.v.) antinociceptive potency. In contrast, the intrathecal (i.t.) potency of 6 (KDA-16) with a 16-atom arm was dramatically increased, as reflected by its antinociceptive i.c.v./i.t. ED50 ratio of ∼130. Further lengthening led to a decreased ED50 ratio. In vivo selective antagonist studies of KDA-16 revealed that κ and δ opioid receptors were responsible for the greatly enhanced i.t. potency. Calcium release experiments in HEK-293 cells suggested that KDA-16 selectively activate κ-δ heteromers. These data are consistent with the reported possible presence of heteromeric κ-δ opioid receptors in mouse spinal cord but not in the brain. The use of a molecular extender arm may be useful for developing spinally selective analgesics.

Induced association of mu opioid (MOP) and type 2 cholecystokinin (CCK2) receptors by novel bivalent ligands.

Both mu-opioid (MOP) and type 2 cholecystokinin (CCK2) receptors are present in areas of the central nervous system that are involved in modulation of pain processing. We conducted bioluminescence resonance energy transfer (BRET) studies on COS cells coexpressing MOP and CCK2 receptors to determine whether receptor heterodimerization is involved in such modulation. These studies revealed the absence of constitutive or monovalent ligand-induced heterodimerization. Heterodimerization of MOP and CCK2 receptors therefore is unlikely to be responsible for the opposing effects between morphine and CCK in the CNS. However, association was induced, as indicated by a positive BRET signal, on exposure of the cells to bivalent ligands containing mu-opioid agonist and CCK2 receptor antagonist pharmacophores linked through spacers containing 16-22 atoms but not with a shorter (9-atom) spacer. These studies demonstrate for the first time that an appropriately designed bivalent ligand is capable of inducing association of G-protein-coupled receptors. The finding that opioid tolerance studies with these ligands in mice showed no correlation with the BRET data is consistent with the absence of association of MOP and CCK2 receptors in vivo.

Deployable, field-sustainable, reverse transcription-polymerase chain reaction assays for rapid screening and serotype identification of dengue virus in mosquitoes.

Dengue virus universal and serotype 1 to 4 fluorogenic probe hydrolysis, reverse transcription (RT)-polymerase chain reaction (PCR) assays and positive-control RNA template were freeze-dried in a thermally stable, hydrolytic enzyme-resistant format and deployed for testing in a dengue fever-endemic region of Thailand. The study site presented austere testing conditions. Field-collected Aedes aegypti mosquitoes spiked with inoculated A. aegypti mosquitoes and individual and pooled, field-collected, A. aegypti, A. albopictus, and Culex tritaeniorhynchus mosquitoes were used for RT-PCR assay evaluations. For dengue virus-inoculated A. aegypti mosquitoes and spiked samples, in vitro sensitivity and specificity results for all five assays were concordant with indirect fluorescent antibody assay results. A single pool of field-collected, female, A. aegypti mosquitoes was identified as dengue virus positive. Cross-reactivity was not observed across heterologous serotypes, mosquito vectors, or human DNA. The limit of detection was >7 to < or =70 genomic equivalents. Sample processing and analysis required <2 hours. These results show promise of field-formatted RT-PCR reagents for rapid, sensitive, specific dengue virus screening and serotype identification in mosquitoes under field-deployed conditions.