The cannabinoid receptor 1 (CB 1 ) and CB 2 cannabinoid receptors, associated with drugs of abuse, may provide a means to treat pain, mood, and addiction disorders affecting widespread segments of society. Whether the orphan G-protein coupled receptor GPR55 is also a cannabinoid receptor remains unclear as a result of conflicting pharmacological studies. GPR55 has been reported to be activated by exogenous and endogenous cannabinoid compounds but surprisingly also by the endogenous non-cannabinoid mediator lysophosphatidylinositol (LPI). We examined the effects of a representative panel of cannabinoid ligands and LPI on GPR55 using a ␤-arrestingreen fluorescent protein biosensor as a direct readout of agonist-mediated receptor activation. Our data demonstrate that AM251 and SR141716A (rimonabant), which are cannabinoid antagonists, and the lipid LPI, which is not a cannabinoid receptor ligand, are GPR55 agonists. They possess comparable efficacy in inducing ␤-arrestin trafficking and, moreover, activate the G-protein-dependent signaling of protein kinase C␤II. Conversely, the potent synthetic cannabinoid agonist CP55,940 acts as a GPR55 antagonist/partial agonist. CP55,940 blocks GPR55 internalization, the formation of ␤-arrestin GPR55 complexes, and the phosphorylation of ERK1/2; CP55,940 produces only a slight amount of protein kinase C␤II membrane recruitment but does not stimulate membrane remodeling like LPI, AM251, or rimonabant. Our studies provide a paradigm for measuring the responsiveness of GPR55 to a variety of ligand scaffolds comprising cannabinoid and novel compounds and suggest that at best GPR55 is an atypical cannabinoid responder. The activation of GPR55 by rimonabant may be responsible for some of the off-target effects that led to its removal as a potential obesity therapy.The CB 1 2 and CB 2 cannabinoid receptors comprise a twomember subfamily of G-protein-coupled receptors (GPCRs) that are notable as the targets of the tetrahydrocannabinol (THC) derivatives found in marijuana. More recently CB 1 receptors along with other GPCRs have been promoted as therapeutic pharmacological targets in the billion dollar weight loss market for controversial drugs such as rimonabant (SR141716A) and Fen-phen. Thus, an important utility of cannabinoid family receptors to society appears to arise from their role in regulating a broad spectrum of addiction-based behaviors, and the addition of new members to the cannabinoid receptor family may have social and economic implications that reach far beyond the initial scientific discovery. As a consequence, the re-classification of an orphan GPCR as a cannabinoid family member should be done with caution requiring strict criteria of receptor activation by THC derivatives or endogenous cannabinoid compounds and a widespread agreement of the results by the scientific community.Marijuana, one of the most widely abused substances (1), mediates many of its psychotropic effects by targeting CB 1 receptors in the central nervous system, but studies with CB 1 and CB 2 kno...

Mucosal associated invariant T (MAIT) cells are important for immune defense against infectiousMucosal associated invariant T (MAIT) cells are innate-like T cells expressing a semi-invariant T cell receptor (TCR) of Vα 7.2-Jα 33 chain and a limited array of Vβ 2 or Vβ 13 chain in humans 1 . Circulating and tissue-infiltrating MAIT cells can be characterized by expressing Vα 7.2 segment with either CD161 or IL-18Rα on cell surface 2,3 . MAIT cells are preferably resident in the intestinal mucosa and liver in humans [4][5][6] . In contrast to conventional T cells that recognize specific antigen peptides, MAIT cells can recognize and respond to microbial vitamin B metabolites in the major histocompatibility complex class I (MHC I)-related molecule (MR1) restricted manner [7][8][9][10] . Co-cultured with bacterium-infected antigen presenting cells (APC), activated MAIT cells can produce diverse cytokines, including interferon-γ (IFN-γ ), tumor necrosis factor-α (TNF-α ) and interleukin-17A (IL-17A) 3,11,12 .It was considered that MAIT cells acquired memory phenotypes after birth and accumulated in the lamina propria of intestinal mucosa in a manner depending on B cells and the commensal flora 13 . However, a recent study in the human fetus indicates that MAIT cells can acquire memory phenotypes before birth, independent of established commensal flora 14 . Previous studies have shown the importance of MAIT cells in host defense against various infectious pathogens 15-18 . Notably, accumulative MAIT cells protect from TNBS-induced colitis in rodents 19 and inflammatory bowel disease in humans 20 . Therefore, MAIT cells in the intestinal lamina propria may be natural protectors from infection and inflammation.Colorectal cancer (CRC) is one of the most common malignant tumors worldwide. The pathogenesis of CRC is attributed to epithelial genetic mutations, impaired mucosal integrity, disordered microbiota and inflammation 21 . CRC usually disrupts the mucosal homeostasis and barrier function. Its development and progression

Known agonists of the orphan receptor GPR35 are kynurenic acid, zaprinast, 5-nitro-2-(3-phenylproplyamino) benzoic acid, and lysophosphatidic acids. Their relatively low affinities for GPR35 and prominent off-target effects at other pathways, however, diminish their utility for understanding GPR35 signaling and for identifying potential therapeutic uses of GPR35. In a screen of the Prestwick Library of drugs and drug-like compounds, we have found that pamoic acid is a potent GPR35 agonist. Pamoic acid is considered by the Food and Drug Administration as an inactive compound that enables longacting formulations of numerous drugs, such as the antihelminthics oxantel pamoate and pyrantel pamoate; the psychoactive compounds hydroxyzine pamoate (Vistaril) and imipramine pamoate (Tofranil-PM); and the peptide hormones triptorelin pamoate (Trelstar) and octreotide pamoate (OncoLar). We have found that pamoic acid induces a G i/o -linked, GPR35-mediated increase in the phosphorylation of extracellular signal-regulated kinase 1/2, recruitment of ␤-arrestin2 to GPR35, and internalization of GPR35. In mice, it attenuates visceral pain perception, indicating an antinociceptive effect, possibly through GPR35 receptors. We have also identified in collaboration with the Sanford-Burnham Institute Molecular Libraries Probe Production Center new classes of GPR35 antagonist compounds, including the nanomolar potency antagonist methyl-5-[(tertbutylcarbamothioylhydrazinylidene)methyl]-1-(2,4-difluorophenyl)pyrazole-4-carboxylate (CID2745687). Pamoic acid and potent antagonists such as CID2745687 present novel opportunities for expanding the chemical space of GPR35, elucidating GPR35 pharmacology, and stimulating GPR35-associated drug development. Our results indicate that the unexpected biological functions of pamoic acid may yield potential new uses for a common drug constituent.

The postinjury inflammatory response in the CNS leads to neuronal excitotoxicity. Our previous studies show that a major component of this response, the inflammatory cytokine tumor necrosis factor ␣ (TNF␣), causes a rapid increase in AMPA glutamate receptors (AMPARs) on the plasma membrane of cultured hippocampal neurons. This may potentiate neuron death through an increased vulnerability to AMPAR-dependent excitotoxic stress. Here, we test this hypothesis with an in vitro lactose dehydrogenase death assay and examine in detail the AMPAR surface delivery time course, receptor subtype, and synaptic and extrasynaptic distribution after TNF␣ exposure. These data demonstrate that surface levels of glutamate receptor 2 (GluR2)-lacking Ca 2ϩ -permeable AMPARs peak at 15 min after TNF␣ treatment, and the majority are directed to extrasynaptic sites. TNF␣ also induces an increase in GluR2-containing surface AMPARs but with a slower time course. We propose that this activity contributes to excitotoxic neuron death because TNF␣ potentiation of kainate excitotoxicity is blocked by a Ca 2ϩ -permeable AMPAR antagonist [NASPM (1-naphthyl acetyl spermine)] and a specific phosphoinositide 3 kinase (PI3 kinase) inhibitor (LY294,002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one]) previously shown to block the TNF␣-induced increase in AMPAR surface delivery. This information forms the basis for future in vivo studies examining AMPAR-dependent potentiation of excitotoxic neuron death and dysfunction caused by TNF␣ after acute injury and during neurodegenerative or neuropsychiatric disorders.

The aim of this study was to assess the change of IL-37 concentrations in rheumatoid arthritis (RA) patients under Disease-modifying anti-rheumatic drug (DMARD) therapy, and to establish a correlation between Interleukin-37 and pro-inflammatory cytokines in plasma and disease activity. The plasma level of IL-37 was determined using ELISA in 50 newly diagnosed RA patients and 30 healthy controls (HC). Plasma levels of IL-17A, IL-6 and TNF-α were measured using flow a cytometric bead array assay. We found that the concentrations of IL-37, as well as IL-17A, IL-6 and TNF-α, were higher in plasma of RA patients compared to HCs. Compared to patients who did not respond to DMARD treatment, treatment of patients responsive to DMARDs resulted in down-regulation of IL-17A, IL-6 and TNF-α expression. The plasma level of the anti-inflammatory cytokine IL-37 was also decreased in drug responders after DMARD treatment. The plasma level of IL-37 in RA patients was positively correlated with pro-inflammatory cytokines (IL-17A, TNF-α) and disease activity (CRP, DAS28) in RA patients. IL-37 expression in RA and during DMARD treatment appears to be controlled by the level of pro-inflammatory cytokines. This results in a strong correlation between plasma levels of IL-37 and disease activity in RA patients.

Adefovir dipivoxil treatment has significantly improved the outcome of chronic hepatitis B virus (HBV) infection. However, it remains largely unknown how immune system responds to the treatment. Chronic HBV patients were treated with adefovir dipivoxil and examined for serum HBV DNA loads, cytokines, and T helper (Th1) and 2 (Th2) cytokine producing T cells during 104 weeks of the treatment. Th1/Th2 cytokines producing T cells were significantly lower in chronic HBV patients as compared to normal individuals. Adefovir dipivoxil treatment led to the increase of Th1/Th2 cytokines producing T cells and serum cytokine levels in association with the decline of HVB DNA load. In contrast, Th1/Th2 cytokines producing T cells remained lower in one patient detected with adefovir dipivoxil resistant HBV A181T/V mutation. This study has established inverse correlation of the increase of Th1/Th2 immunity and the decline of HBV DNA load in chronic HBV patients during adefovir dipivoxil treatment.

Gastric cancer (GC) is one of the leading causes of cancer death in the world. The role of histone deacetylase 4 (HDAC4) in specific cell and tissue types has been identified. However, its biological roles in the development of gastric cancer remain largely unexplored. Quantitative real time PCR (qRT-PCR) and western blot were used to analyze the expression of HDAC4 in the clinical samples. siRNA and overexpression of HDAC4 and siRNA p21 were used to study functional effects in a proliferation, a colony formation, a adenosine 5′-triphosphate (ATP) assay and reactive oxygen species(ROS) generation, cell cycle, cell apoptosis rates, and autophagy assays. HDAC4 was up-regulated in gastric cancer tissues and several gastric cancer cell lines. The proliferation, colony formation ability and ATP level were enhanced in HDAC4 overexpression SGC-7901 cells, but inhibited in HDAC4 knockdown SGC-7901 cells. HDAC4 knockdown led to G0/G1 phase cell arrest and caused apoptosis and ROS increase. Moreover, HDAC4 was found to inhibit p21 expression in gastric cancer SGC-7901 cells. p21 knockdown dramatically attenuated cell proliferation inhibition, cell cycle arrest, cell apoptosis promotion and autophagy up-regulation in HDAC4-siRNA SGC-7901 cells. We demonstrated that HDAC4 promotes gastric cancer cell progression mediated through the repression of p21. Our results provide an experimental basis for understanding the pro-tumor mechanism of HDAC4 as treatment for gastric cancer.

Recent studies have demonstrated that the majority of endogenous cannabinoid type 1 (CB 1 ) receptors do not reach the cell surface but are instead associated with endosomal and lysosomal compartments. Using calcium imaging and intracellular microinjection in CB 1 receptor-transfected HEK293 cells and NG108-15 neuroblastoma ؋ glioma cells, we provide evidence that anandamide acting on CB 1 receptors increases intracellular calcium concentration when administered intracellularly but not extracellularly. The calcium-mobilizing effect of intracellular anandamide was dose-dependent and abolished by pretreatment with SR141716A, a CB 1 receptor antagonist. The anandamide-induced calcium increase was reduced by blocking nicotinic acid-adenine dinucleotide phosphate-or inositol 1,4,5-trisphosphate-dependent calcium release and abolished when both lysosomal and endoplasmic reticulum calcium release pathways were blocked. Taken together, our results indicate that, in CB 1 receptor-transfected HEK293 cells, intracellular CB 1 receptors are functional; they are located in acid-filled calcium stores (endolysosomes). Activation of intracellular CB 1 receptors releases calcium from endoplasmic reticulum and lysosomal calcium stores. In addition, our results support a novel role for nicotinic acid-adenine dinucleotide phosphate in cannabinoid-induced calcium signaling.Although most studies on the cannabinoid type 1 (CB 1 ) 3 receptor have focused on its localization to the plasma membrane, increasing evidence suggests that not only are CB 1 receptors highly expressed in intracellular compartments but also that these intracellular receptors are functional. An early study in N18TG2 neuroblastoma cells demonstrated localization of CB 1 receptors to nuclear membranes in cells not treated with agonists (1). More recent studies have suggested that the CB 1 receptor undergoes constitutive endocytosis in both neurons and transfected cells (2-4). Rozenfeld and Devi (5) found that the majority of the endogenous CB 1 receptors do not reach the cell surface but are instead associated with endosomal and lysosomal compartments. They further demonstrated that, during trafficking, the lysosomal CB 1 receptors may interact with G␣ i proteins. Another recent report indicated that the intracellular pool of CB 1 receptors does not contribute to cellsurface repopulation (6), and thus, it may have a distinct yet unknown function from the membrane CB 1 receptors.Most previous studies examining cannabinoids, including anandamide, have demonstrated a nonspecific effect in that non-transfected cells also showed a calcium response (7,8). One study showed that only the indole derivative WIN55,212-2 was able to increase intracellular calcium via CB 1 receptor coupling to G q/11 proteins (9). Many studies have shown that cannabinoids can inhibit a voltage-sensitive calcium channel present in neurons and neuroblastoma cells (e.g. Refs. 7, 10, and 11). In this study, we show that anandamide increases intracellular calcium concentration but only when injected ...