Exogenous Agonists Research Articles

Induced pluripotent stem cell-derived conditional medium promotes Leydig cell anti-apoptosis and proliferation via autophagy and Wnt/β-catenin pathway.

Leydig cell transplantation is a better alternative in the treatment of androgen‐deficient males. The main purpose of this study was to investigate the effects of induced pluripotent stem cell‐derived conditioned medium (iPS‐CM) on the anti‐apoptosis, proliferation and function of immature Leydig cells (ILCs), and illuminate the underlying mechanisms. ILCs were exposed to 200 μmol/L hydrogen peroxide (H2O2) for 24 hours with or without iPS‐CM treatments. Cell apoptosis was detected by flow cytometric analysis. Cell proliferation was assessed using cell cycle assays and EdU staining. The steroidogenic enzyme expressions were quantified with Western blotting. The results showed that iPS‐CM significantly reduced H2O2‐induced ILC apoptosis through down‐regulation of autophagic and apoptotic proteins LC3‐I/II, Beclin‐1, P62, P53 and BAX as well as up‐regulation of BCL‐2, which could be inhibited by LY294002 (25 μmol/L). iPS‐CM could also promote ILC proliferation through up‐regulation of β‐catenin and its target proteins cyclin D1, c‐Myc and survivin, but was inhibited by XAV939 (10 μmol/L). The level of bFGF in iPS‐CM was higher than that of DMEM‐LG. Exogenous bFGF (20 ng/mL) or Wnt signalling agonist lithium chloride (LiCl) (20 mmol/L) added into DMEM‐LG could achieve the similar effects of iPS‐CM. Meanwhile, iPS‐CM could improve the medium testosterone levels and up‐regulation of LHCGR, SCARB1, STAR, CYP11A1, HSD3B1, CYP17A1, HSD17B3 and SF‐1 in H2O2‐induced ILCs. In conclusion, iPS‐CM could reduce H2O2‐induced ILC apoptosis through the activation of autophagy, promote proliferation through up‐regulation of Wnt/β‐catenin pathway and enhance testosterone production through increasing steroidogenic enzyme expressions, which might be used in regenerative medicine for future.

T160. TREATMENT OF CLOZAPINE-ASSOCIATED OBESITY AND DIABETES WITH EXENATIDE (CODEX) IN ADULTS WITH SCHIZOPHRENIA: A RANDOMISED CONTROLLED TRIAL

BackgroundClozapine is the most effective anti-psychotic for treatment refractory schizophrenia, but causes significant metabolic disturbances including obesity and type 2 diabetes. The metabolic adverse reactions may be mediated in part by clozapine induced dysregulation of Glucagon-like-peptide-1 (GLP-1). GLP-1 is an intestinal epithelial derived peptide, released with ingestion of food, that triggers satiety, reduces glucagon production, promotes insulin production and slows gut motility. Clozapine has been shown to interfere with GLP-1 function in animal models, leading to metabolic dyregulation including obesity and preference for high calorie meals. Administration of exogenous GLP-1 agonists such as exenatide to animals have been shown to counter this effect of clozapine. Exenatide subcutaneous weekly injections may assist obese people on clozapine lose weight.MethodsThis randomised, controlled, open-label, pilot trial aimed to evaluate the effect of exenatide on weight loss among clozapine-treated obese adults who have schizophrenia, with or without stable diabetes. Twenty-eight out-patients were randomised to once weekly extended release sub-cutaneous exenatide or treatment as usual for 24 weeks. This trial examined the safety, tolerability and acceptability of exenatide among obese people with schizophrenia on clozapine, with an evaluation of change in weight, glycaemic control, psychosis severity and metabolic parameters.ResultsAll 28 participants completed the study. (Exenatide=14 (3 T2DM), control=14 (2 T2DM)). Six people on exenatide achieved >5% weight loss, compared to only 1 control (p=0.029). Mean weight loss was greater for exenatide than control at week 24 (-5.29kg vs -1.12kg, p=0.015) as were. BMI (-1.78 vs -0.39 p=0.019), fasting glucose (-0.34 vs 0.39, p=0.036) and HbA1c (-0.21 vs 0.03, p=0.004). There was no significant difference for other metabolic syndrome components. There were higher rates of transient nausea (n=8), vomiting (n=7) and diarrhoea (n=7) in the exenatide group.DiscussionExenatide is a promising therapeutic agent for glycaemic control and weight loss in clozapine-treated people with obesity. These results suggest good tolerability and a consistent and favourable pattern of weight loss effects with exenatide. GLP-1 agonists could assist in reducing the cardio-metabolic associated morbidity and mortality secondary to clozapine.

T227. THE METABOTROPIC GLUTAMATE RECEPTOR SUBTYPE 1 REGULATES STRIATAL DOPAMINE RELEASE VIA AN ENDOCANNABINOID-DEPENDENT MECHANISM: IMPLICATIONS FOR THE TREATMENT OF SCHIZOPHRENIA

BackgroundClinical and preclinical studies suggest that selective activators of the muscarinic M4 receptor have exciting potential as a novel approach for treatment of schizophrenia. M4 reduces striatal dopamine (DA) though release of endocannabinoids (eCB), providing a mechanism for local effects on DA signaling in the striatum. M4 signals through Gαi/o and does not couple to Gαq/11 or induce calcium (Ca++) mobilization. This raises the possibility that M4-induced eCB release and inhibition of DA release may require co-activation of another receptor that activates Gαq/11. If so, this receptor could provide a novel target that may be more proximal to inhibition of DA release. Interestingly, the group 1 metabotropic glutamate (mGlu) receptors (mGlu1 and Glu5), couple to Gαq/11 and activate eCB signaling in multiple brain regions.MethodsWe tested the hypothesis that M4-induced reductions in DA release and subsequent antipsychotic-effect requires co-activation of group 1 mGlu receptors. The effect of M4 activation on electrically-evoked DA release in striatal slices was assessed using fast-scan cyclic voltammetry (FSCV) in the absence or presence of selective negative allosteric modulators (NAMs) of group 1 mGlu receptor subtypes. To evaluate the potential role of mGlu1, we determined the effects of a selective mGlu1 positive allosteric modulators (PAMs) on striatal DA release and antipsychotic-like activity in rodent models that are dependent on increased DA transmission. Since reductions in DA signaling, including D1 signaling have been implicated in reduced motivation, we also determined the effects of an mGlu1 PAM, M4 PAM, and the typical antipsychotic haloperidol on motivational responding in a progressive ratio (PR) schedule.ResultsWe now present exciting new data in which we found that activation of mGlu1 through application of exogenous agonists or selective stimulation of thalamostriatal afferents induces a reduction of striatal DA release and that selective mGlu1 PAMs have robust antipsychotic-like effects in rodent models. Interestingly, our studies also suggest that mGlu1 activation is required for M4 PAM-induced inhibition of DA release and antipsychotic-like effects. However, in contrast to available antipsychotic agents, the present results and previous studies suggest that mGlu1 and M4 PAMs reduce DA signaling through local release of an eCB from striatal SPNs and activation of CB2 receptors on neighboring DA terminals to reduce DA release. While these studies suggest that the effects of M4 PAMs on DA release require activation of mGlu1, we have also found that these targets have important differences. Most notably, M4 PAMs also directly inhibits D1 signaling in D1-SPN terminals in the substatnia nigra pars reticulata (SNr). Unlike M4, mGlu1 does not directly inhibit DA D1 receptor signaling and does not induce behavioral changes that could be associated with negative symptoms.DiscussionOur findings are especially interesting in light of recent findings that multiple loss of function single nucleotide polymorphisms (SNPs) in the human gene encoding mGlu1 (GRM1) are associated with schizophrenia, and points to GRM1/mGlu1 as a gene within the “druggable genome” that could be targeted for treatment of schizophrenia. Recent clinical imaging studies suggesting that symptoms in schizophrenia patients are associated with selective increases in striatal DA signaling and while extrastriatal regions display hypo-dopaminergic function; thus, mGlu1 and M4 PAMs may provide a mechanism for selective inhibition of DA release in striatal regions that are important for antipsychotic efficacy, without further disruptions in extrastriatal DA signaling.

Parathyroid Hormone-Related Peptide Elicits Peripheral TRPV1-dependent Mechanical Hypersensitivity.

Bone metastasis in breast, prostate and lung cancers often leads to chronic pain, which is poorly managed by existing analgesics. The neurobiological mechanisms that underlie chronic pain associated with bone-metastasized cancers are not well understood, but sensitization of peripheral nociceptors by tumor microenvironment factors has been demonstrated to be important. Parathyroid hormone-related peptide (PTHrP) is highly expressed in bone-metastasized breast and prostate cancers, and is critical to growth and proliferation of these tumors in the bone tumor microenvironment. Previous studies have suggested that PTHrP could sensitize nociceptive sensory neurons, resulting in peripheral pain hypersensitivity. In this study, we found that PTHrP induces both heat and mechanical hypersensitivity, that are dependent on the pain-transducing transient receptor potential channel family vanilloid, member-1 (TRPV1), but not the mechano-transducing TRPV4 and TRPA1 ion channels. Functional ratiometric Ca2+ imaging and voltage-clamp electrophysiological analysis of cultured mouse DRG neurons show significant potentiation of TRPV1, but not TRPA1 or TRPV4 channel activation by PTHrP. Interestingly, PTHrP exposure led to the slow and sustained activation of TRPV1, in the absence of any exogenous channel agonist, and is dependent on the expression of the type-1 parathyroid hormone receptor (PTH1), as well as on downstream phosphorylation of the channel by protein kinase C (PKC). Accordingly, local administration of specific small-molecule antagonists of TRPV1 to mouse hindpaws after the development of PTHrP-induced mechanical hypersensitivity led to its significant attenuation. Collectively, our findings suggest that PTHrP/PTH1-mediated flow activation of TRPV1 channel contributes at least in part to the development and maintenance of peripheral mechanical pain hypersensitivity, and could therefore constitute a mechanism for nociceptor sensitization in the context of metastatic bone cancer pain.

Defining a Clopidogrel Response Cut-Off Value Using Light Transmission Aggregometry Before Pipeline Embolization of Unruptured Intracranial Aneurysms.

The choice of appropriate antiplatelet therapy before the Pipeline Embolization Device (PED) placement is usually guided by platelet function testing such as light transmission aggregometry (LTA). In this study, we aimed to define the optimal threshold LTA value for clopidogrel responsiveness to predict the risk of postprocedural thromboembolic complications and to help guide appropriate antiplatelet regimen. A prospectively maintained database at an academic neurosurgical center in the United States was retrospectively analyzed from 2014 to 2017 to identify patients with unruptured intracranial aneurysms treated with the PED. Clinical and radiographic data were analyzed to identify thromboembolic complications in the context of platelet function testing performed by LTA. A total of 95 procedures were performed for PED placement to treat 110 unruptured intracranial aneurysms. Thromboembolic complications were encountered in 4 (4.2%) of these patients. After stratifying the complication rate based on the maximal extent of platelet aggregation after administration of an exogenous platelet agonist, a marked increase in thromboembolic events was observed in patients with LTA values greater than 50%. When LTA was dichotomized based on this value, patients with an LTA value less than 50% had a thromboembolic complication rate of 1.3% (1/80), compared with 20% (3/15) for those with LTA values ≥50% (P= 0.001). We observed the greatest increase in the rate of thromboembolic complications with LTA values of ≥50%. This can serve as an appropriate cut-off value for determining the clopidogrel response in patients undergoing endovascular treatment with the PED.

Aryl hydrocarbon receptor signaling modulates antiviral immune responses: ligand metabolism rather than chemical source is the stronger predictor of outcome

The aryl hydrocarbon receptor (AHR) offers a compelling target to modulate the immune system. AHR agonists alter adaptive immune responses, but the consequences differ across studies. We report here the comparison of four agents representing different sources of AHR ligands in mice infected with influenza A virus (IAV): TCDD, prototype exogenous AHR agonist; PCB126, pollutant with documented human exposure; ITE, novel pharmaceutical; and FICZ, degradation product of tryptophan. All four compounds diminished virus-specific IgM levels and increased the proportion of regulatory T cells. TCDD, PCB126 and ITE, but not FICZ, reduced virus-specific IgG levels and CD8+ T cell responses. Similarly, ITE, PCB126, and TCDD reduced Th1 and Tfh cells, whereas FICZ increased their frequency. In Cyp1a1-deficient mice, all compounds, including FICZ, reduced the response to IAV. Conditional Ahr knockout mice revealed that all four compounds require AHR within hematopoietic cells. Thus, differences in the immune response to IAV likely reflect variances in quality, magnitude, and duration of AHR signaling. This indicates that binding affinity and metabolism may be stronger predictors of immune effects than a compound’s source of origin, and that harnessing AHR will require finding a balance between dampening immune-mediated pathologies and maintaining sufficient host defenses against infection.

Opposing Cholinergic and Serotonergic Modulation of Layer 6 in Prefrontal Cortex.

Prefrontal cortex is a hub for attention processing and receives abundant innervation from cholinergic and serotonergic afferents. A growing body of evidence suggests that acetylcholine (ACh) and serotonin (5-HT) have opposing influences on tasks requiring attention, but the underlying neurophysiology of their opposition is unclear. One candidate target population is medial prefrontal layer 6 pyramidal neurons, which provide feedback modulation of the thalamus, as well as feed-forward excitation of cortical interneurons. Here, we assess the response of these neurons to ACh and 5-HT using whole cell recordings in acute brain slices from mouse cortex. With application of exogenous agonists, we show that individual layer 6 pyramidal neurons are bidirectionally-modulated, with ACh and 5-HT exerting opposite effects on excitability across a number of concentrations. Next, we tested the responses of layer 6 pyramidal neurons to optogenetic release of endogenous ACh or 5-HT. These experiments were performed in brain slices from transgenic mice expressing channelrhodopsin in either ChAT-expressing cholinergic neurons or Pet1-expressing serotonergic neurons. Light-evoked endogenous neuromodulation recapitulated the effects of exogenous neurotransmitters, showing opposing modulation of layer 6 pyramidal neurons by ACh and 5-HT. Lastly, the addition of 5-HT to either endogenous or exogenous ACh significantly suppressed the excitation of pyramidal neurons in prefrontal layer 6. Taken together, this work suggests that the major corticothalamic layer of prefrontal cortex is a substrate for opposing modulatory influences on neuronal activity that could have implications for regulation of attention.

Hydroxysafflor Yellow A Shows Protection against PPARγ Inactivation in Nitrosative Neurons.

Peroxynitrite-mediated nitrosative stress in the brain has been associated with various neurodegenerative disorders. Recent evidence highlights peroxisome proliferator-activated receptor γ (PPARγ) as a critical neuroprotective factor in neurodegenerative diseases. Here, we observed the effect of the herb hydroxysafflor yellow A (HSYA) during nitrosative stress in neurons and investigated the mechanism based on PPARγ protection. We found that a single exposure of primary neurons to peroxynitrite donor SIN-1 caused neuronal injury, which was accompanied by the increase of PPARγ nitration status and lack of activation of the receptor, as measured by PPARγ DNA-binding activity, by agonist (15d-PGJ2 or rosiglitazone) stimulation. The crucial role of PPARγ in neuronal defense against nitrosative stress was verified by showing that pretreatment with 15d-PGJ2 or rosiglitazone attenuated SIN-1-induced neuronal injury but pretreatment with GW9662, a PPARγ antagonist, aggravated SIN-1-induced neuronal injury. The addition of HSYA not only inhibited SIN-1-induced neuronal damage but prevented PPARγ nitrative modification and resumed PPARγ activity stimulated by either 15d-PGJ2 or rosiglitazone. Furthermore, HSYA also showed the ability to rescue the neuroprotective effect of 15d-PGJ2 or rosiglitazone when the agonists were coincubated with SIN-1. Finally, in vivo experiments demonstrated that the administration of HSYA also efficiently blocked PPARγ nitration and loss of activity in the SIN-1-injected hippocampus and reversed the increased neuronal susceptibility which was supported by the inhibition of Bcl-2 protein downregulation induced by SIN-1. The results suggest that HSYA protects neurons from nitrosative stress through keeping PPARγ as a functional receptor, allowing a more effective activation of this neuroprotective factor by the endogenous or exogenous agonist. Our findings provide new clues in understanding the role of the neuroprotective potential of the herbal HSYA.

In vitro and in vivo pharmacological characterization of ASP8477: A novel highly selective fatty acid amide hydrolase inhibitor

Although exogenous agonists for cannabinoid (CB) receptors are clinically effective for treating chronic pain, global activation of brain CB receptors causes frequent central nervous system (CNS) side-effects. Fatty acid amide hydrolase (FAAH) is a primary catabolic enzyme for anandamide (AEA), an endogenous CB. Recently, we discovered a novel FAAH inhibitor, 3-pyridyl 4-(phenylcarbamoyl)piperidine-1-carboxylate (ASP8477). In vitro studies demonstrated that ASP8477 inhibited human FAAH-1, FAAH-1 (P129T) and FAAH-2 activity with IC50 values of 3.99, 1.65 and 57.3nM, respectively. ASP8477 at 10µM had no appreciable interactions with 65 different kinds of receptors, ion channels, transporters and enzymes, including CB1 and CB2 receptors and monoacylglycerol lipase. In adolescent rats, orally administered ASP8477 (0.3–10mg/kg) elevated AEA concentrations in both plasma and brain. In a capsaicin-induced secondary hyperalgesia model, a pretreatment with ASP8477 significantly improved mechanical allodynia and thermal hyperalgesia at 0.3–3mg/kg p.o. ASP8477 also significantly improved mechanical allodynia in an L5/L6 spinal nerve ligation neuropathic pain model, with an ED50 value of 0.63mg/kg, and in a streptozotocin-induced diabetic neuropathy model at 3 and 10mg/kg p.o. Furthermore, ASP8477 significantly attenuated the reduction in rearing events at 1 and 3mg/kg p.o. in a monoiodoacetic acid-induced osteoarthritis model. Importantly, ASP8477 had no significant effect on motor coordination up to 30mg/kg p.o. These results indicate that ASP8477 is a potent, selective, and oral active FAAH inhibitor with activity in the CNS, with the potential to be a new analgesic agent with a wide safety margin.

Endomorphin-2 Decreases Excitatory Synaptic Transmission in the Spinal Ventral Horn of the Rat.

Motor impairment is one of the serious side-effects of morphine, which is an exogenous agonist of the μ-opioid receptor (MOR) as well as a widely used analgesic drug in clinical practice for chronic pain treatment. Endomorphins (EMs, including EM-1 and EM-2), the most effective and specific endogenous agonists of the MOR, exert more potent analgesia in acute and neuropathic pain than other opiates, such as morphine. Although EMs had fewer side-effects comparing to other opiates, motor impairment was still one unwanted reaction which limited its clinical application. In order to prevent and treat the motor impairment, it is critical to reveal the neural mechanisms underlying such locomotion disorder. The purpose of the present study was to reveal the neural mechanisms underlying the effects of EM-2 on the activity of motoneurons in the spinal ventral horn. First, we examine the distribution of EM-2-immunoreactive (IR) primary afferent fibers and their synaptic connections with the motoneurons innervating the skeletal muscles of the lower limb revealed by sciatic nerve retrograde tracing. The results showed that EM-2-IR fibers and terminals were sparsely observed in lamina IX and they formed symmetric synaptic connections with the motoneurons within lamina IX of the spinal ventral horn. Then, whole-cell patch-clamp technique was used to observe the effects of EM-2 on the spontaneous excitatory postsynaptic current (sEPSC) of motoneurons in lamina IX. The results showed that EM-2 could decrease both the frequency and amplitude of the sEPSC of the motoneurons in lamina IX, which was reversed by the MOR antagonist CTOP. These results indicate that EM-2-IR fibers originated from primary afferent fibers form symmetric synaptic connections with motoneurons innervating skeletal muscles of the lower limbs in lamina IX of the spinal ventral horn and EM-2 might exert inhibitory effects on the activities of these motoneurons through both presynaptic and postsynaptic mechanisms.

Delayed Varenicline Administration Reduces Inflammation and Improves Forelimb Use Following Experimental Stroke

Pharmacological activation of the cholinergic anti-inflammatory pathway (CAP), specifically by activating α7 nicotinic acetylcholine receptors, has been shown to confer short-term improvements in outcome. Most studies have investigated administration within 24 hours of stroke, and few have investigated drugs approved for use in human patients. We investigated whether delayed administration of varenicline, a high-affinity agonist at α7 nicotinic receptors and an established therapy for nicotine addiction, decreased brain inflammation and improved functional performance in a mouse model of experimental stroke. CSF-1R-EGFP (MacGreen) mice were subjected to transient middle cerebral artery occlusion and administered varenicline (2.5 mg/kg/d for 7 days) or saline (n = 10 per group) 3 days after stroke. Forelimb asymmetry was assessed in the Cylinder test every 2 days after surgery, and structural lesions were quantified at day 10. Enhanced green fluorescent protein (EGFP) and growth associated protein 43 (GAP43) immunohistochemistry were used to evaluate the effect of varenicline on inflammation and axonal regeneration, respectively. Varenicline-treated animals showed a significant increase in impaired forelimb use compared with saline-treated animals 10 days after stroke. Varenicline treatment was associated with reduced EGFP expression and increased GAP43 expression in the striatum of MacGreen mice. Our results show that delayed administration of varenicline promotes recovery of function following experimental stroke. Motor function improvements were accompanied by decreased brain inflammation and increased axonal regeneration in nonpenumbral areas. These results suggest that the administration of an exogenous nicotinic agonist in the subacute phase following stroke may be a viable therapeutic strategy for stroke patients.

The Molecular Mechanistic Effects of Acupuncture in Endometriosis Management

Recent advances in neuroendocrinology and immunology have provided an entry to understand acupuncture. Here, we highlight these progresses and use endometriosis as an example to provide a perspective in how to effectively engage acupuncture into modern biomedicine. Endometriosis is a common chronic inflammatory disease in which endometrial tissue progressively grows outside the uterine cavity and into the pelvic region, including the fallopian tubes and ovaries. Also, estrogen promotes the endometriosis progression through on-site inflammation. Current treatments for endometriosis involve hormonal therapy and surgical procedures, however, they both may induce several side effects. Several studies have shown that the administration of exogenous dopamine agonists can significantly suppress endometriosis progression. Acupuncture is multivalent in which it can simultaneously stimulate the secretion of endogenous dopamine to modulate the GnRH neuroendocrine pathway in CNS and the COX-2 inflammatory pathway in PNS. Hence, acupuncture can smoothly manage endometriosis without the side effects of exogenous dopamine. The endometriosis management by acupuncture provides us an example of how to incorporate acupuncture into current biomedical systems. Acupuncture may provide a convenient, physiological method to regulate the neuroendocrine system in an integrative, systematic means.

Abstract 3644: Combination of OX40L fusion protein with an anti-tumor vaccine reduces lung metastasis and prolongs survival in a murine model of metastatic triple negative breast cancer

Abstract OX40 is a costimulatory receptor on activated T-cells that potentiates the proliferation, survival, and memory formation of the CD4+ and CD8+ immune populations upon its interaction with OX40L on antigen presenting cells. The engagement of OX40 on regulatory T-cells (Tregs) with its ligand has also been shown to inhibit their suppressive capabilities. The use of exogenous OX40L agonists for cancer immunotherapy is particularly attractive in combination with cancer vaccines in order to boost the immune activity of antigen specific T-cells and overcome self-tolerance and immunosuppressive mechanisms of the tumor. We investigated the efficacy of this strategy in the 4T1 murine mammary tumor model of metastatic triple-negative breast cancer by combining an OX40L fusion protein (OX40L-FP) with a poxvirus-based cancer vaccine (MVA-Twist-TRICOM) that induces T-cell responses specific to the tumor antigen Twist, a transcription factor highly expressed in lung metastases. OX40L-FP was administered 3 and 6 days after the prime and boost vaccinations to maximize its interaction with OX40 on activated T-cells. As single agents, OX40L-FP and MVA-Twist-TRICOM had minimal effect on the formation of lung metastases in 4T1 tumor-bearing mice. However, when combined, the two immunotherapies significantly decreased the number of metastatic colonies per lung, and prolonged survival after surgical resection of the primary tumor. Antibody depletion studies demonstrated that the observed anti-tumor effects were mediated by both CD4+ and CD8+ T-cells. Immune subset analysis revealed that the combination therapy increased the total number of CD4+Foxp3- and CD4+ effector memory T-cells in both the lung and periphery compared to the monotherapy controls. More importantly, the two agents synergistically induced greater CD4+ Twist-specific T-cell responses, as determined by ex vivo proliferation assays using T-cells isolated from the lung and spleen that were stimulated with Twist protein. Although the combination had no added effect on the number of total CD8+ and CD8+ effector memory T-cells in relation to the vaccine monotherapy, tetramer staining revealed a significant increase in CD8+ T-cells that were specific for Twist. We also observed a greater number of CD8+ T-cell memory precursors, as well as a greater number of IFNγ-producing CD8+ T-cells in both the lung and periphery. Tregs isolated from the lung and splenic tissue were found to be significantly less suppressive following the combination therapy, compared to those isolated from mice that received the OX40L-FP and MVA-Twist-TRICOM single agent treatments. This study provides the rationale for pairing OX40 agonists with cancer vaccines to stimulate CD4+ and CD8+ T-cell responses, while inhibiting Treg function, as a way to induce specific immunity and clearance of metastatic tumors. Citation Format: Anthony S. Malamas, Scott A. Hammond, Jeffrey Schlom, James W. Hodge. Combination of OX40L fusion protein with an anti-tumor vaccine reduces lung metastasis and prolongs survival in a murine model of metastatic triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3644. doi:10.1158/1538-7445.AM2017-3644

Inhibition of the endocannabinoid-regulating enzyme monoacylglycerol lipase elicits a CB1 receptor-mediated discriminative stimulus in mice

Substantial challenges exist for investigating the cannabinoid receptor type 1 (CB1)-mediated discriminative stimulus effects of the endocannabinoids, 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anandamide; AEA), compared with exogenous CB1 receptor agonists, such as Δ9-tetrahydrocannabinol (THC) and the synthetic cannabinoid CP55,940. Specifically, each endocannabinoid is rapidly degraded by the respective hydrolytic enzymes, monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH). Whereas MAGL inhibitors partially substitute for THC and fully substitute for CP55,940, FAAH inhibitors do not substitute for either drug. Interestingly, combined FAAH-MAGL inhibition results in full THC substitution, and the dual FAAH-MAGL inhibitor SA-57 serves as its own discriminative training stimulus. Because MAGL inhibitors fully substitute for SA-57, we tested whether the selective MAGL inhibitor MJN110 would serve as a training stimulus. Twelve of 13 C57BL/6J mice learned to discriminate MJN110 from vehicle, and the CB1 receptor antagonist rimonabant dose-dependently blocked its discriminative stimulus. CP55,940, SA-57, and another MAGL inhibitor JZL184, fully substituted for MJN110. In contrast, the FAAH inhibitor PF-3845 failed to substitute for the MJN110 discriminative stimulus, but produced a 1.6 (1.1–2.2; 95% confidence interval) leftward shift in the MJN110 dose-response curve. Inhibitors of other relevant enzymes (i.e., ABHD6, COX-2) and nicotine did not engender substitution. Diazepam partially substituted for MJN110, but rimonabant failed to block this partial effect. These findings suggest that MAGL normally throttles 2-AG stimulation of CB1 receptors to a magnitude insufficient to produce cannabimimetic subjective effects. Accordingly, inhibitors of this enzyme may release this endogenous brake producing effects akin to those produced by exogenously administered cannabinoids.

Revisiting nicotine's role in the ageing brain and cognitive impairment.

Brain ageing is a complex process which in its pathologic form is associated with learning and memory dysfunction or cognitive impairment. During ageing, changes in cholinergic innervations and reduced acetylcholinergic tonus may trigger a series of molecular pathways participating in oxidative stress, excitotoxicity, amyloid-β toxicity, apoptosis, neuroinflammation, and perturb neurotrophic factors in the brain. Nicotine is an exogenous agonist of nicotinic acetylcholine receptors (nAChRs) and acts as a pharmacological chaperone in the regulation of nAChR expression, potentially intervening in age-related changes in diverse molecular pathways leading to pathology. Although nicotine has therapeutic potential, paradoxical effects have been reported, possibly due to its inverted U-shape dose-response effects or pharmacokinetic factors. Additionally, nicotine administration should result in optimum therapeutic effects without imparting abuse potential or toxicity. Overall, this review aims to compile the previous and most recent data on nicotine and its effects on cognition-related mechanisms and age-related cognitive impairment.

PCMT1 Ameliorates Neuronal Apoptosis by Inhibiting the Activation of MST1 after Subarachnoid Hemorrhage in Rats.

Mammalian sterile 20-like kinase 1 (MST1) is found to promote neuronal apoptosis. Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1), an anti-apoptosis factor, was recently identified as an MST1-interacting protein. This study aims to explore the potential role of PCMT1 in reducing MST1-induced neuronal apoptosis after subarachnoid hemorrhage (SAH) in rats. One hundred ninety-eight male Sprague-Dawley rats were used. An exogenous PCMT1 agonist, CGP 3466B, was injected subcutaneously 1h after the SAH induced by endovascular perforation. Chelerythrine or calyculin A was given immediately via intracerebroventricular administration after SAH. The SAH grade, Garcia score, and brain water content were measured at 24 and 72h after the SAH. Neuronal apoptosis was detected by an immunofluorescent assay. The expression levels of endogenous PCMT1, MST1, phospho-MST1 (p-MST1), cleaved MST1 (cl-MST1), and apoptosis-related proteins were studied by western blotting. The expression of PCMT1 and MST1 decreased, while the level of active caspase 3 increased in rats after SAH. CGP 3466B treatment improved neurobehavioral function, reduced brain water content, inhibited the activity of MST1, and relieved neuronal apoptosis. These neuroprotective effects were significantly weakened either through accelerating MST1 phosphorylation by calyculin A or increasing cl-MST1 by chelerythrine. PCMT1 inhibited neuronal apoptosis by reducing MST1 phosphorylation and the level of cl-MST1. PCMT1/MST1 pathway might be an alternative therapeutic target for alleviating early brain injury after SAH.

Toll‐like receptor 5 signaling restrains T‐cell/natural killer T‐cell activation and protects against concanavalin A–induced hepatic injury

The TLR5 signaling pathway plays an important role in T cell-mediated hepatic injury and may be exploited for therapeutic treatment of inflammatory liver diseases. (Hepatology 2017;65:2059-2073).

Sildenafil normalizes bowel transit in preclinical models of constipation.

Guanylyl cyclase-C (GC-C) agonists increase cGMP levels in the intestinal epithelium to promote secretion. This process underlies the utility of exogenous GC-C agonists such as linaclotide for the treatment of chronic idiopathic constipation (CIC) and irritable bowel syndrome with constipation (IBS-C). Because GC-C agonists have limited use in pediatric patients, there is a need for alternative cGMP-elevating agents that are effective in the intestine. The present study aimed to determine whether the PDE-5 inhibitor sildenafil has similar effects as linaclotide on preclinical models of constipation. Oral administration of sildenafil caused increased cGMP levels in mouse intestinal epithelium demonstrating that blocking cGMP-breakdown is an alternative approach to increase cGMP in the gut. Both linaclotide and sildenafil reduced proliferation and increased differentiation in colon mucosa, indicating common target pathways. The homeostatic effects of cGMP required gut turnover since maximal effects were observed after 3 days of treatment. Neither linaclotide nor sildenafil treatment affected intestinal transit or water content of fecal pellets in healthy mice. To test the effectiveness of cGMP elevation in a functional motility disorder model, mice were treated with dextran sulfate sodium (DSS) to induce colitis and were allowed to recover for several weeks. The recovered animals exhibited slower transit, but increased fecal water content. An acute dose of sildenafil was able to normalize transit and fecal water content in the DSS-recovery animal model, and also in loperamide-induced constipation. The higher fecal water content in the recovered animals was due to a compromised epithelial barrier, which was normalized by sildenafil treatment. Taken together our results show that sildenafil can have similar effects as linaclotide on the intestine, and may have therapeutic benefit to patients with CIC, IBS-C, and post-infectious IBS.

Plasticity of TRPV1‐dependent Neurotransmission in the Paraventricular Nucleus of db/db Mice

Transient receptor potential vanilloid type 1 (TRPV1) has been shown to play a role in the modulation of glucose homeostasis. Preautonomic, liver‐related neurons in the paraventricular nucleus (PVN) of the hypothalamus contribute to the regulation of sympathetic and parasympathetic outflow to the liver. Our previous findings demonstrated that liver‐related PVN neurons are controlled by TRPV1‐dependent excitatory neurotransmission. In this study, liver‐related PVN neurons were identified with a retrograde, trans‐neuronal viral tracer and whole‐cell patch‐clamp recordings were conducted. We tested the hypothesis that the TRPV1‐dependent synaptic control of liver‐related PVN neurons is reduced in db/db mice. Application of the exogenous TRPV1 agonist, capsaicin (1μM) increased the average frequency of miniature excitatory post‐synaptic currents (mEPSCs) in liver‐related PVN neurons from lean mice. In contrast, in db/db mice capsaicin application did not change the overall frequency of mEPSCs. Capsaicin did not alter the frequency of miniature inhibitory post‐synaptic currents (mIPSCs) neither in lean or db/db mice. We did not observe significant difference in the basal frequency of mEPSCs and mIPSCs in liver‐related PVN neurons of lean and db/db mice. On the other hand, the amplitude of mIPSCs was smaller in liver‐related PVN neurons of db/db mice compared with lean mice. GABAA‐mediated inhibitory tonic currents as bicuculline‐dependent inward shift of the holding current were also revealed in liver‐related PVN neurons. Our data did not show difference in the magnitude of tonic inhibition between the groups. Together, this study suggests that the TRPV1‐dependent control of liver‐related PVN neurons is diminished in db/db mice. These findings are consistent with our previous data demonstrating synaptic plasticity of hypothalamic autonomic circuits in hyperglycemic conditions, and further suggest dysregulation of neuronal activity at the level of the PVN in obese‐diabetic state.Support or Funding InformationSupported by NIDDK R01DK099598

Discrepancy between TRPA1 activation by reversible and irreversible electrophiles suggests involvement of cytosolic cofactors other than polyphosphates.

Transient Receptor Potential Ankyrin 1 (TRPA1) is a non‐selective cation channel that is expressed in a subset of nociceptive afferent sensory nerves. When activated, TRPA1 can evoke nociceptive responses (e.g. pain and defensive reflexes), as well as peripheral neurogenic inflammation. TRPA1 is activated by a host of endogenous (inflammatory mediators) and exogenous (pollutants, food) agonists. Many TRPA1 agonists are electrophilic compounds that covalently modify reactive cysteine residues on TRPA1, leading to activation of the channel. Electrophiles can bind through reversible or irreversible reactions based on the structure of the compound. Here, patch clamp techniques and calcium imaging have been used to characterize human TRPA1 (hTRPA1) channel activity (expressed in HEK293 cells) in the presence of reversible and irreversible electrophiles. Calcium imaging shows that both reversible electrophiles (e.g. allylisothiocyanate [AITC]) and irreversible electrophiles (e.g. iodoacetamide [IA] and N‐ethylmaleimide [NEM]) rapidly induce TRPA1 activation. In contrast, our patch clamp studies show a discrepancy between irreversible and reversible electrophilic TRPA1 activation. Whole cell patch clamp studies exhibit a reduction in the kinetics and magnitude of TRPA1 activation by IA and NEM compared to AITC. Furthermore, single channel patch clamp experiments display reduced open probability with IA and NEM compared to AITC. Both whole cell patch and excised patch clamp recordings were performed in the presence of 5mM polyphosphates. In preliminary gramicidin perforated patch recordings there was no difference in NEM and AITC‐evoked TRPA1 activation. Thus in intact cellular assays (Ca2+ imaging and gramicidin‐perforated patch) there is little difference in TRPA1 activation by reversible and irreversible electrophiles, whereas in dialyzed cellular assays (whole cell patch and excised patch) irreversible electrophiles evoked reduced TRPA1 activation compared to reversible electrophiles. The data suggests that cytosolic factors other than polyphosphates are important for TRPA1 activation by irreversible electrophilic ligands.Support or Funding InformationNHLBI R01 (HL119802) and Supplement (HL119802‐S1)