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Glucose-6-phosphatase (G6Pase) is a multicomponent system located in the endoplasmic reticulum comprising a catalytic subunit and transporters for glucose-6-phosphate, inorganic phosphate, and glucose. We have recently cloned a novel gene that encodes an islet-specific G6Pase catalytic subunit-related protein (IGRP) (Ebert et al., Diabetes 48:543-551, 1999). To begin to investigate the molecular basis for the islet-specific expression of the IGRP gene, a series of truncated IGRP-chloramphenicol acetyltransferase (CAT) fusion genes were transiently transfected into the islet-derived mouse betaTC-3 and hamster insulinoma tumor cell lines. In both cell lines, basal fusion gene expression decreased upon progressive deletion of the IGRP promoter sequence between -306 and -66, indicating that multiple promoter regions are required for maximal IGRP-CAT expression. The ligation-mediated polymerase chain reaction footprinting technique was then used to compare trans-acting factor binding to the IGRP promoter in situ in betaTC-3 cells, which express the endogenous IGRP gene, and adrenocortical Y1 cells, which do not. Multiple trans-acting factor binding sites were selectively identified in betaTC-3 cells that correlate with regions of the IGRP promoter identified as being required for basal IGRP-CAT fusion gene expression. The data suggest that hepatocyte nuclear factor 3 may be important for basal IGRP gene expression, as it is for glucagon, GLUT2, and Pdx-1 gene expression. In addition, binding sites for several trans-acting factors not previously associated with islet gene expression, as well as binding sites for potentially novel proteins, were identified. [ABSTRACT FROM AUTHOR]

This review presents mechanistic studies performed in vitro and in animal models, as well as data obtained in patients that contribute to a better understanding of the impact of nutrients interacting with the gut microbiota on metabolic and behavioural alterations linked to obesity. The gut microbiota composition and function are altered in several pathological conditions including obesity and related diseases i.e. non-alcoholic fatty liver diseases (NAFLD). The gut-liver axis is clearly influenced by alterations of the gut barrier that drives inflammation. In addition, recent papers propose that specific metabolites issued from the metabolic cooperation between the gut microbes and host enzymes, modulate inflammation and gene expression in the liver. This review illustrates how dietary intervention with prebiotics or probiotics influences host energy metabolism and inflammation. Indeed, intervention studies are currently underway in obese and NAFLD patients to unravel the relevance of the changes in gut microbiota composition in the management of metabolic and behavioural disorders by nutrients interacting with the gut microbiota. In conclusion, diet is among the main triggers of NAFLD and the gut microbiota is modified accordingly, underlining the importance of the concomitant study of the nutrients and microbial impact on liver health and metabolism, in order to propose innovative, clinically relevant, therapeutic approaches. [ABSTRACT FROM AUTHOR]

Background and Purpose: Although exogenous nitric oxide (NO) clearly modifies platelet function, the role and the source of endogenous NO in vivo remain undefined. In addition, endothelial NO synthase (NOS-3) critically regulates vessel tone but its role in modulating platelet function is unclear. In this paper we have investigated the roles of endogenous NO and NOS-3 in regulating platelet function in vivo and determined the functional contribution made by platelet-derived NO.Experimental Approach: We used a mouse model for directly assessing platelet functional responses in situ in the presence of an intact vascular endothelium with supporting in vitro and molecular studies.Key Results: Acute NOS inhibition by N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME) enhanced platelet aggregatory responses to thrombin and platelets were shown to be regulated primarily by NO sources external to the platelet. Elevation of endogenous NOS inhibitors to mimic effects reported in patients with cardiovascular diseases did not enhance platelet responses. Platelet responsiveness following agonist stimulation was not modified in male or female NOS-3(-/-) mice but responses in NOS-3(-/-) mice were enhanced by L-NAME.Conclusions and Implications: Platelets are regulated by endogenous NO in vivo, primarily by NO originating from the environment external to the platelet with a negligible or undetectable role of platelet-derived NO. Raised levels of endogenous NOS inhibitors, as reported in a range of diseases were not, in isolation, sufficient to enhance platelet activity and NOS-3 is not essential for normal platelet function in vivo due to the presence of bioactive NO following deletion of NOS-3. [ABSTRACT FROM AUTHOR]

Descending neural pathways in the mammalian auditory system are known to modulate the function of the peripheral auditory system. These pathways include the medial olivocochlear (MOC) efferent innervation to outer hair cells (OHCs) and the acoustic reflex pathways mediating middle ear muscle (MEM) contractions. Based on measurements in humans (Marks and Siegel, companion paper), we applied a sensitive method to attempt to differentiate MEM and MOC reflexes using contralateral acoustic stimulation in mice under different levels of anesthesia. Separation of these effects is based on the knowledge that OHC-generated transient evoked otoacoustic emissions (TEOAE) are delayed relative to the stimulus, and that the MOC reflex affects the emission through its innervation of OHC. In contrast, the MEM-mediated changes in middle ear reflectance alter both the stimulus (with a short delay) and the emission. Using this approach, time averages to transient stimuli were evaluated to determine if thresholds for a contralateral effect on the delayed emission, indicating potential MOC activation, could be observed in the absence of a change in the stimulus pressure. This outcome was not observed in the majority of cases. There were also no statistically significant differences between MEM and putative MOC thresholds, and variability was high for both thresholds regardless of anesthesia level. Since the two reflex pathways could not be differentiated on the basis of activation thresholds, it was concluded that the MEM reflex dominates changes in TEOAEs induced by contralateral noise. This result complicates the identification of purely MOC-induced changes on OAEs in mice unless the MEM reflex is inactivated surgically or pharmacologically. [ABSTRACT FROM AUTHOR]

The role of innate immunity and macrophage recruitment to the inner ear after hair cell injury is a subject where little is known. In this paper, we demonstrate recruitment of monocytes and macrophages to the inner ear after kanamycin. We also examined the effect of fractalkine receptor (CX3CR1) deletion in kanamycin ototoxicity. We observed more functional and structural damage in CX3CR1 null mice compared to wild-type and heterozygous littermates. In order to determine if increased susceptibility to kanamycin resulted from CX3CR1 deletion from cochlear leukocytes, we created bone marrow chimeras by transplanting CX3CR1-null bone marrow into wild-type mice whose native bone marrow was ablated by lethal irradiation. These mice were then treated with kanamycin sulfate. Auditory brainstem responses (ABR), hair cell counts, and numbers of macrophages recruited to the cochlea were recorded in irradiated mice that received either wild-type, CX3CR1 heterozygous, or CX3CR1 knockout bone marrow. A strong correlation was present between numbers of macrophages and hair cell death in recipients transplanted with CX3CR1 null marrow. No correlation between macrophage number and hair cell loss was present in mice transplanted with wild-type or CX3CR1 heterozygous marrow. We suggest that CX3CR1 plays a role in modulating the detrimental effects of cochlear macrophages after kanamycin ototoxicity. Our data point to the possibility that CX3CR1-deficient cochlear macrophages exacerbate kanamycin ototoxicity while CX3CR1-expressing monocytes do not. [ABSTRACT FROM AUTHOR]

Background: The effect of glycoengineering a membrane specific anti-carcinoembryonic antigen (CEA) (this paper uses the original term CEA for the formally designated CEACAM5) antibody (PR1A3) on its ability to enhance killing of colorectal cancer (CRC) cell lines by human immune effector cells was assessed. In vivo efficacy of the antibody was also tested.Methods: The antibody was modified using EBNA cells cotransfected with beta-1,4-N-acetylglucosaminyltransferase III and the humanised hPR1A3 antibody genes.Results: The resulting alteration of the Fc segment glycosylation pattern enhances the antibody's binding affinity to the FcgammaRIIIa receptor on human immune effector cells but does not alter the antibody's binding capacity. Antibody-dependent cellular cytotoxicity (ADCC) is inhibited in the presence of anti-FcgammaRIII blocking antibodies. This glycovariant of hPR1A3 enhances ADCC 10-fold relative to the parent unmodified antibody using either unfractionated peripheral blood mononuclear or natural killer (NK) cells and CEA-positive CRC cells as targets. NK cells are far more potent in eliciting ADCC than either freshly isolated monocytes or granulocytes. Flow cytometry and automated fluorescent microscopy have been used to show that both versions of hPR1A3 can induce antibody-dependent cellular phagocytosis (ADCP) by monocyte-derived macrophages. However, the glycovariant antibody did not mediate enhanced ADCP. This may be explained by the relatively low expression of FcgammaRIIIa on cultured macrophages. In vivo studies show the efficacy of glycoengineered humanised IgG1 PR1A3 in significantly improving survival in a CRC metastatic murine model.Conclusion: The greatly enhanced in vitro ADCC activity of the glycoengineered version of hPR1A3 is likely to be clinically beneficial. [ABSTRACT FROM AUTHOR]

Background and purpose: Nitroxyl (HNO) is emerging as an important regulator of vascular tone as it is potentially produced endogenously and dilates conduit and resistance arteries. This study investigates the contribution of endogenous HNO to endothelium-dependent relaxation and hyperpolarization in resistance arteries. Experimental approach: Rat and mouse mesenteric arteries were mounted in small vessel myographs for isometric force and smooth muscle membrane potential recording. Key results: Vasorelaxation to the HNO donor, Angeli's salt, was attenuated in both species by the soluble guanylate cyclase inhibitor (ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one), the voltage-dependent K+ channel inhibitor, 4-aminopyridine (4-AP) and the HNO scavenger,l-cysteine. In mouse mesenteric arteries, nitric oxide (NO) synthase inhibition (withl-NAME, Nω-Nitro-L-arginine methyl ester) markedly attenuated acetylcholine (ACh)-mediated relaxation. Scavenging the uncharged form of NO (NO•) with hydroxocobalamin (HXC) or HNO withl-cysteine, or 4-AP decreased the sensitivity to ACh, and a combination of HXC andl-cysteine reduced ACh-mediated relaxation, as didl-NAME alone. ACh-induced hyperpolarizations were significantly attenuated by 4-AP alone and in combination withl-NAME. In rat mesenteric arteries, blocking the effects of endothelium-derived hyperpolarizing factor (EDHF) (charybdotoxin and apamin) decreased ACh-mediated relaxation 10-fold and unmasked a NO-dependent component, mediated equally by HNO and NO•, as HXC andl-cysteine in combination now abolished vasorelaxation to ACh. Furthermore, ACh-evoked hyperpolarizations, resistant to EDHF inhibition, were virtually abolished by 4-AP. Conclusions and implications: The factors contributing to vasorelaxation in mouse and rat mesenteric arteries are NO• = HNO > EDHF and EDHF > HNO = NO• respectively. This study identified HNO as an endothelium-derived relaxing and hyperpolarizing factor in resistance vessels. British Journal of Pharmacology (2009) 157, 540–550; doi:10.1111/j.1476-5381.2009.00150.x; published online 26 March 2009 This article is commented on by Martin, pp. 537–539 of this issue and is part of a themed section on Endothelium in Pharmacology. For a list of all articles in this section see the end of this paper, or visit: [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) 'pulsed' with an appropriate antigen may elicit an antitumour immune response in mouse models. However, while attempting to develop a DC immunotherapy protocol for the treatment of breast cancer based on the tumour-associated MUC1 glycoforms, we found that unpulsed DCs can affect tumour growth. Protection from RMA-MUC1 tumour challenge was achieved in C57Bl/6 MUC1 transgenic mice by immunising with syngeneic DCs pulsed with a MUC1 peptide. However, unpulsed DCs gave a similar level of protection, making it impossible to evaluate the effect of immunisation of mice with DCs pulsed with the specific peptide. Balb/C mice could also be protected from tumour challenge by immunisation with unpulsed DCs prior to challenge with murine mammary tumour cells (410.4) or these cells transfected with MUC1 (E3). Protection was achieved with as few as three injections of 50,000 naïve DCs per mouse per week, was not dependent on injection route, and was not specific to cell lines expressing human MUC1. However, the use of Rag2-knockout mice demonstrated that the adaptive immune response was required for tumour rejection. Injection of unpulsed DCs into mice bearing the E3 tumour slowed tumour growth. In vitro, production of IFN-gamma and IL-4 was increased in splenic cells isolated from mice immunised with DCs. Depleting CD4 T cells in vitro partially decreased cytokine production by splenocytes, but CD8 depletion had no effect. This paper shows that naïve syngeneic DCs may induce an antitumour immune response and has implications for DC immunotherapy preclinical and clinical trials. [ABSTRACT FROM AUTHOR]

The coxsackievirus and adenovirus receptor (CAR) is a membrane glycoprotein with a cytoplasmic domain, a transmembrane domain and an extracellular region consisting of two immunoglobulin-like domains, an amino-terminal immunoglobulin variable (IgV)-related domain (D1), which is distal to the cell surface, and a proximal IgC2 domain (D2). The coxsackievirus and adenovirus receptor has been shown to exhibit tumour suppression activity in human bladder and prostate cancer cells. In the current paper, we demonstrate that CAR is a tumour suppressor in glioma cells and that the extracellular D2 domain is not required for this inhibitory effect. This finding provides a biological basis for the observation that expression of CAR is downregulated in malignant glioma cells. This suggests that strategies to redirect adenoviruses to achieve CAR-independent infection will be necessary to realise the full potential of adenoviral vectors for cancer gene therapy. [ABSTRACT FROM AUTHOR]

Thrombopoietin (TPO) and its receptor (MPL) are important regulators of megakaryopoiesis. MPL belongs to a cytokine receptor superfamily. To date, all constitutively active MPL mutants have been artificially constructed with amino acid substitutions in the transmembrane domain or extracellular domain of the protein, and they activate signal transduction pathways in Ba/F3 cells that can also be activated by the normal MPL. In this paper, we report a novel spontaneously occurring mutation of MPL, with an amino acid substitution of Trp(508) to Ser(508) in the intracellular domain of MPL, that induces the factor-independent growth of Ba/F3 cells. Examination of intracellular signaling pathways demonstrated that the mutant MPL protein constitutively activates three distinct signaling pathways, SHC-Ras-Raf-MAPK/JNK, JAK-STAT, and PI3K-Akt-Bad. [ABSTRACT FROM AUTHOR]

Hypoxia in solid tumours has been correlated with poor prognosis and resistance to radiation and chemotherapy. Hypoxia is also a strong stimulus for gene expression. We previously proposed a gene therapy approach which exploits the presence of severe hypoxia in tumours for the induction of therapeutic genes. Hypoxic cells are known to have a reduced metabolic rate, transcription and translation. These facts may prevent gene transfer and therefore warranted further investigation. In this paper the feasibility of gene delivery in vitro under tumour conditions was demonstrated. DNA was delivered in vitro using a peptide-mediated non-viral system. Across a range of oxygen tensions and mammalian cell lines (including human tumour and endothelial cells) it was shown that hypoxic cells could be transfected. Transfection efficiencies varied depending on the level of hypoxia, cell characteristics and gene promoters used. An in vitro model of hypoxia/reoxygenation, designed to mimic the variable nature of tumour hypoxia, showed that hypoxic preconditioning and reoxygenation alone did not reduce transfection efficiency significantly; only chronic anoxia reduced transfection. The fact that neither intermediate hypoxia nor intermittent anoxia significantly reduced transfection is promising for future hypoxia-targeted gene therapy strategies. [ABSTRACT FROM AUTHOR]

Microbially mediated mechanisms of human decomposition begin immediately after death, and are a driving force for the conversion of a once living organism to a resource of energy and nutrients. Little is known about post-mortem microbiology in cadavers, particularly the community structure of microflora residing within the cadaver and the dynamics of these communities during decomposition. Recent work suggests these bacterial communities undergo taxa turnover and shifts in community composition throughout the post-mortem interval. In this paper we describe how the microbiome of a living host changes and transmigrates within the body after death thus linking the microbiome of a living individual to post-mortem microbiome changes. These differences in the human post-mortem from the ante-mortem microbiome have demonstrated promise as evidence in death investigations. We investigated the post-mortem structure and function dynamics of Staphylococcus aureus and Clostridium perfringens after intranasal inoculation in the animal model Mus musculus L. (mouse) to identify how transmigration of bacterial species can potentially aid in post-mortem interval estimations. S. aureus was tracked using in vivo and in vitro imaging to determine colonization routes associated with different physiological events of host decomposition, while C. perfringens was tracked using culture-based techniques. Samples were collected at discrete time intervals associated with various physiological events and host decomposition beginning at 1h and ending at 60 days post-mortem. Results suggest that S. aureus reaches its highest concentration at 5-7 days post-mortem then begins to rapidly decrease and is undetectable by culture on day 30. The ability to track these organisms as they move in to once considered sterile space may be useful for sampling during autopsy to aid in determining post-mortem interval range estimations, cause of death, and origins associated with the geographic location of human remains during death investigations. [ABSTRACT FROM AUTHOR]

Calcitonin gene-related peptide (CGRP)-antagonizing drugs represent a major advance in migraine treatment. However, up to 50% of patients do not benefit from monoclonal antibodies against CGRP or its receptor. Here, we test the hypothesis that a closely related peptide, pituitary adenylate cyclase-activating peptide (PACAP-38), works independently of CGRP and thus might represent a new, alternative drug target. To understand differences in CGRP- and PACAP-mediated migraine pain, we used mouse models of provoked migraine-like pain based on multiple stimulations and subsequent measurement of tactile sensitivity response with von Frey filaments. Genetically modified mice lacking either functional CGRP receptors (Ramp1 knockout) or TRPA1 channels (Trpa1 knockout) were used together with CGRP-targeting antibodies and chemical inhibitors in wild-type mice (ntotal = 299). Ex vivo myograph studies were used to measure dilatory responses to CGRP and PACAP-38 in mouse carotid arteries. PACAP-38 provoked significant hypersensitivity and dilated the carotid arteries independently of CGRP. In contrast, glyceryl trinitrate-induced hypersensitivity is dependent on CGRP. Contrary to previous results with the migraine-inducing substances glyceryl trinitrate, cilostazol and levcromakalim, PACAP-38-induced hypersensitivity worked only partially through inhibition of ATP-sensitive potassium channels. Using multiple migraine-relevant models, these findings establish the PACAP-38 pathway as distinct from other migraine provoking pathways such as CGRP and glyceryl trinitrate. PACAP antagonism may therefore be a novel therapeutic target of particular interest in patients unresponsive to CGRP-antagonizing drugs. [ABSTRACT FROM AUTHOR]

The axon initial segment is a specialized compartment of the proximal axon of CNS neurons where action potentials are initiated. However, it remains unknown whether this domain is assembled in sensory dorsal root ganglion neurons, in which spikes are initiated in the peripheral terminals. Here we investigate whether sensory neurons have an axon initial segment and if it contributes to spontaneous activity in neuropathic pain. Our results demonstrate that myelinated dorsal root ganglion neurons assemble an axon initial segment in the proximal region of their stem axon, enriched in the voltage-gated sodium channels Nav1.1 and Nav1.7. Using correlative immunofluorescence and calcium imaging, we demonstrate that the Nav1.7 channels at the axon initial segment are associated with spontaneous activity. Computer simulations further indicate that the axon initial segment plays a key role in the initiation of spontaneous discharges by lowering their voltage threshold. Finally, using a Cre-based mouse model for time-controlled axon initial segment disassembly, we demonstrate that this compartment is a major source of spontaneous discharges causing mechanical allodynia in neuropathic pain. Thus, an axon initial segment domain is present in sensory neurons and facilitates their spontaneous activity. This study provides a new insight in the cellular mechanisms that cause pathological pain and identifies a new potential target for chronic pain management. [ABSTRACT FROM AUTHOR]

Recent advances in genetic analysis have significantly helped in progressively attenuating the heritability gap of obesity and have brought into focus monogenic variants that disrupt the melanocortin signaling. In a previous study, next-generation sequencing revealed a monogenic etiology in ∼50% of the children with severe obesity from a consanguineous population in Pakistan. Here we assess rare variants in obesity-causing genes in young adults with severe obesity from the same region. Genomic DNA from 126 randomly selected young adult obese subjects (BMI 37.2 ± 0.3 kg/m2; age 18.4 ± 0.3 years) was screened by conventional or augmented whole-exome analysis for point mutations and copy number variants (CNVs). Leptin, insulin, and cortisol levels were measured by ELISA. We identified 13 subjects carrying 13 different pathogenic or likely pathogenic variants in LEPR, PCSK1, MC4R, NTRK2, POMC, SH2B1, and SIM1. We also identified for the first time in the human, two homozygous stop-gain mutations in ASNSD1 and IFI16 genes. Inactivation of these genes in mouse models has been shown to result in obesity. Additionally, we describe nine homozygous mutations (seven missense, one stop-gain, and one stop-loss) and four copy-loss CNVs in genes or genomic regions previously linked to obesity-associated traits by genome-wide association studies. Unexpectedly, in contrast to obese children, pathogenic mutations in LEP and LEPR were either absent or rare in this cohort of young adults. High morbidity and mortality risks and social disadvantage of children with LEP or LEPR deficiency may in part explain this difference between the two cohorts. [ABSTRACT FROM AUTHOR]

Type 2 diabetes (T2D) causes an increased risk of morbidity and mortality in response to viral infection. T2D is characterized by hyperglycemia and is typically associated with insulin resistance and compensatory hyperinsulinemia. CD8 T cells express the insulin receptor, and previously, we have shown that insulin is able to directly modulate effector CD8 T-cell function. We therefore hypothesized that memory CD8 T-cell responsiveness in the context of T2D is negatively impacted by hyperinsulinemia or hyperglycemia. Using a mouse model for T2D, we could show that memory CD8 T-cell function was significantly reduced in response to rechallenge by viral infection or with melanoma cells. Basal insulin injection of mice increased GLUT-1 expression and glucose uptake in memory CD8 T-cell precursors early after infection, which was prevented when these cells were deficient for the insulin receptor. However, neither insulin injection nor insulin receptor deficiency resulted in a difference in metabolism, memory formation, cytokine production, or recall responses of memory CD8 T cells compared with controls. Importantly, in context of obesity, insulin receptor deficiency on CD8 T cells did not affect the functional capacity of memory CD8 T cells. In contrast, we could show in vitro and in vivo that hyperglycemia significantly impairs the antiviral capacity of memory CD8 T cells. Our findings indicate that obesity impairs the memory CD8 T-cell response against viral infection and cancer through the detrimental effects of hyperglycemia rather than hyperinsulinemia. [ABSTRACT FROM AUTHOR]

Background: Dietary interventions targeting the gut microbiota have been proposed as innovative strategies to improve obesity-associated metabolic disorders. Increasing physical activity (PA) is considered as a key behavioral change for improving health. We have tested the hypothesis that changing the PA status during a nutritional intervention based on prebiotic supplementation can alter or even change the metabolic response to the prebiotic. We confirm in obese subjects and in high-fat diet fed mice that performing PA in parallel to a prebiotic supplementation is necessary to observe metabolic improvements upon inulin.Methods: A randomized, single-blinded, multicentric, placebo-controlled trial was conducted in obese participants who received 16 g/day native inulin versus maltodextrin, coupled to dietary advice to consume inulin-rich versus -poor vegetables for 3 months, respectively, in addition to dietary caloric restriction. Primary outcomes concern the changes on the gut microbiota composition, and secondary outcomes are related to the measures of anthropometric and metabolic parameters, as well as the evaluation of PA. Among the 106 patients who completed the study, 61 patients filled a questionnaire for PA before and after intervention (placebo: n = 31, prebiotic: n = 30). Except the dietitian (who provided dietary advices and recipes book), all participants and research staff were blinded to the treatments and no advices related to PA were given to participants in order to change their habits. In parallel, a preclinical study was designed combining both inulin supplementation and voluntary exercise in a model of diet-induced obesity in mice.Results: Obese subjects who increased PA during a 3 months intervention with inulin-enriched diet exhibited several clinical improvements such as reduced BMI (- 1.6 kg/m2), decreased liver enzymes and plasma cholesterol, and improved glucose tolerance. Interestingly, the regulations of Bifidobacterium, Dialister, and Catenibacterium genera by inulin were only significant when participants exercised more. In obese mice, we highlighted a greater gut fermentation of inulin and improved glucose homeostasis when PA is combined with prebiotics.Conclusion: We conclude that PA level is an important determinant of the success of a dietary intervention targeting the gut microbiota.Trial Registration: ClinicalTrials.gov, NCT03852069 (February 22, 2019 retrospectively registered). [ABSTRACT FROM AUTHOR]

Background: Targeting glutamine metabolism is previously indicated as a potential and attractive strategy for gastric cancer (GC) therapy. However, the underlying mechanisms responsible for the modification of glutamine metabolism in GC cells have not been fully elucidated. Accordingly, the current study sought to investigate the physiological mechanisms of RUNX3-mediated circDYRK1A in glutamine metabolism of GC.Methods: Firstly, GC tissues and adjacent normal tissues were obtained from 50 GC patients to determine circDYRK1A expression in GC tissues. Next, the binding affinity among RUNX3, circDYRK1A, miR-889-3p, and FBXO4 was detected to clarify the mechanistic basis. Moreover, GC cells were subjected to ectopic expression and knockdown manipulations of circDYRK1A, miR-889-3p, and/or FBXO4 to assay GC cell malignant phenotypes, levels of glutamine, glutamic acid, and α-KG in cell supernatant and glutamine metabolism-related proteins (GLS and GDH). Finally, nude mice were xenografted with GC cells to explore the in vivo effects of circDYRK1A on the tumorigenicity and apoptosis.Results: circDYRK1A was found to be poorly expressed in GC tissues. RUNX3 was validated to bind to the circDYRK1A promoter, and circDYRK1A functioned as a miR-889-3p sponge to up-regulate FBXO4 expression. Moreover, RUNX3-upregulated circDYRK1A reduced levels of glutamine, glutamic acid, and α-KG, and protein levels of GLS and GDH, and further diminished malignant phenotypes in vitro. Furthermore, in vivo experimentation substantiated that circDYRK1A inhibited the tumorigenicity and augmented the apoptosis in GC.Conclusion: In conclusion, these findings highlighted the significance and mechanism of RUNX3-mediated circDYRK1A in suppressing glutamine metabolism in GC via the miR-889-3p/FBXO4 axis. [ABSTRACT FROM AUTHOR]

Study Question: How does ectopic endometrial stromal cell (Ecto-ESC)-derived extracellular vesicular Legumain pseudogene 1 (EV-LGMNP1), a newly identified pseudogene of Legumain (LGMN), contribute to M2-phenotype macrophage polarization, and does it predict recurrence in patients with ovarian endometriosis (EMs)?Summary Answer: EV-LGMNP1, which is abundant in Ecto-ESCs and serum from ovarian EMs, can direct macrophages towards an M2 phenotype by upregulating LGMN expression and is a promising biomarker for predicting ovarian EMs recurrence.What Is Known Already: Extracellular vesicles (EVs) can mediate cell-to-cell crosstalk to promote disease progression via cargo molecule transport. Recently, LGMNP1, a newly identified pseudogene of LGMN, has been reported to promote cancer progression by upregulating LGMN. LGMN is a well-studied protein that can induce M2-like polarization.Study Design, Size, Duration: An in vitro study was conducted with Ecto-ESCs isolated from ectopic endometrial samples, collected from two patients with ovarian EMs (diagnosed by laparoscopy and histological analysis). A clinical retrospective cohort study of 52 ovarian EMs patients and 21 controls with available preoperative serum samples was carried out (2013-2017). The follow-up period ended either at the time of recurrence or on 31 December 2018.Participants/materials, Setting, Methods: Ecto-ESC-derived EVs (EV/Ecto-ESCs) were characterized by nanoparticle tracking analysis, transmission electron microscopy and western blotting. EV internalization by THP-1 cells, which are the most widely used primary human macrophages model, was detected by fluorescence labelling. After EV treatment, THP-1 cell polarization was detected by quantitative real-time PCR (qRT-PCR) and western blot analyses of CD86 (M1-related marker) and CD206 (M2-related marker). LGMNP1 mRNA expression level in EVs from both primary ectopic endometrioc stromal cells and serum was examined using qRT-PCR. Additionally, the expression of LGMN, the downstream target gene of LGMNP1, in THP-1 cells was evaluated using qRT-PCR and western blotting. Kaplan-Meier and multivariate Cox regression analyses were applied to evaluate the independent predictive factors of EMs recurrence-free survival. A novel nomogram model based on serum EV-LGMNP1 was then formulated to predict EMs recurrence.Main Results and the Role Of Chance: In vitro assays demonstrated that EV/Ecto-ESCs drove macrophages towards an M2-like phenotype. Moreover, LGMNP1 contributed to EV/Ecto-ESC-induced M2 macrophage polarization by upregulating LGMN mRNA expression levels. Clinically, serum EV-LGMNP1 was more highly expressed in recurrent EMs patients than in controls and EMs patients without recurrence. Survival analysis and our novel nomogram reconfirmed that serum EV-LGMNP1 was a novel promising and meaningful non-invasive biomarker for predicting EMs recurrence.Large Scale Data: N/A.Limitations, Reasons For Caution: In vitro experiments were only performed on samples from two patients with ovarian endometriosis, and a larger sample size is needed. ESCs isolated from the eutopic endometrium of EMs and non-EMs patients should be studied in the future. Additionally, in vitro experiments should be performed using endometrial epithelium cells and further in vivo experiments, such as using mice endometriotic models to investigate whether EV/Ecto could induce M2 macrophage polarization, should be conducted. Moreover, multicentre, large-sample data are needed to validate our predictive nomogram model.Wider Implications Of the Findings: Our study provides novel insights into the mechanism of M2 polarization involved in ovarian EMs progression mediated by an 'EV-shuttled pseudogene LGMNP1' mode. In addition, serum EV-LGMNP1 may serve as a novel non-invasive biomarker for predicting recurrence, providing a new therapeutic target for ovarian EMs.Study Funding/competing Interest(s): This project was supported by funding from the National Natural Science Foundation of China (81971361), the Natural Science Foundation of Shanghai Science and Technology (19ZR1406900), the Shanghai 'Rising Stars of Medical Talent' Youth Development Program (AB83030002019004), the Clinical Research Plan of SHDC (SHDC2020CR4087), the Shanghai Municipal Health Commission (202040498), the Research and Innovation Project of the Shanghai Municipal Education Commission (2019-01-07-00-07-E00050) and the Clinical Research Plan of SHDC (SHDC2020CR1045B). There are no competing interests to declare. [ABSTRACT FROM AUTHOR]

Background and Purpose: Ilexgenin A is a triterpenoid from ShanLv Cha with beneficial effects on metabolic homeostasis. We investigated whether ilexgenin A could inhibit hepatic de novo fatty acid synthesis via the interfering with SREBP1 maturation.Experimental Approach: The effects of Ilexgenin A on CRTC2 translocation and SREBP1 maturation were investigated in the liver of fasted mice and hepatocytes exposed to saturated fatty acids. The effect of Iilexgenin A on hepatic lipid accumulation was also observed in high-fat diet fed mice.Key Results: Sec23A and Sec31A are two subunits of COPII complex and their interaction is essential for the processing of SREBP1 maturation. Ilexgenin A activates AMPK by reducing cellular energy and preventing cytoplasmic CRTC2 to compete with Sec23A for binding to Sec31A under nutrient-rich conditions. Consequently, ilexgenin A impaired COPII-dependent SREBP1 maturation via disrupting Sec31A-Sec23A interaction, leading to the inhibition of de novo fatty acid synthesis in the liver. In contrast, mTORC1 phosphorylated Ser136 of CRTC2, facilitating the formation of Sec31A-Sec23A interaction to promote SREBP1 maturation, whereas this action was reversed by ilexgenin A in an AMPK-dependent manner. Ilexgenin A protected CRTC2 function and restrained hepatic lipogenic response in high fat diet-fed mice, providing in vivo evidence to support the beneficial effects of ilexgenin A on lipid metabolism.Conclusions and Implications: Ilexgenin A activated AMPK and restrained CRTC2 to the cytoplasm to prevent SREBP1 maturation via impairing COPII function in the liver. This suggests that CRTC2 might be a potential target for pharmacological intervention to prevent hepatic lipid deposition.Linked Articles: This article is part of a themed issue on Preclinical Models for Cardiovascular disease research (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.5/issuetoc. [ABSTRACT FROM AUTHOR]

Background: Long noncoding RNA NEAT1 has been implicated in glioma progression. However, the effect of NEAT1 on glycolysis of glioma cell and the potential mechanism remain unclear.Methods: In vitro experiments, including CCK-8, colony formation, ECAR, and lactate detection assays were performed to evaluate the effect of NEAT1 on proliferation and glycolysis of glioma cell. RNA pulldown and RIP assays were performed to identify the interaction between NEAT1 and PGK1. Truncated mutation of NEAT1 and PGK1 was used to confirm the specific interactive domains between NEAT1 and PGK1. Animal studies were performed to analyze the effect of NEAT1/PGK1 on glioma progression.Results: NEAT1 knockdown significantly suppressed the proliferation and glycolysis of glioma cells. NEAT1 could specifically interact with PGK1, which promotes PGK1 stability. Hairpin A of NEAT1 is essential for interaction with M1 domain of PGK1. Depletion of NEAT1 markedly inhibited tumor growth in mice, while PGK1 could reverse this effect. Higher expression of NEAT1 was associated with poor overall survival of GBM patients.Conclusions: NEAT1 over expression promotes glioma progression through stabilizing PGK1. NEAT1/PGK1 axis is a candidate therapeutic target for glioma treatment. [ABSTRACT FROM AUTHOR]

Cerebral oedema develops after anoxic brain injury. In two models of asphyxial and asystolic cardiac arrest without resuscitation, we found that oedema develops shortly after anoxia secondary to terminal depolarizations and the abnormal entry of CSF. Oedema severity correlated with the availability of CSF with the age-dependent increase in CSF volume worsening the severity of oedema. Oedema was identified primarily in brain regions bordering CSF compartments in mice and humans. The degree of ex vivo tissue swelling was predicted by an osmotic model suggesting that anoxic brain tissue possesses a high intrinsic osmotic potential. This osmotic process was temperature-dependent, proposing an additional mechanism for the beneficial effect of therapeutic hypothermia. These observations show that CSF is a primary source of oedema fluid in anoxic brain. This novel insight offers a mechanistic basis for the future development of alternative strategies to prevent cerebral oedema formation after cardiac arrest. [ABSTRACT FROM AUTHOR]

Objective: The aim of this study was to research the role and underlying mechanism of miR-195 involved in pancreatic β-cell dedifferentiation induced by hyperlipemia in type 2 diabetes mellitus.Methods: High-fat-diet-induced obese C57BL/6J mice and palmitate-stimulated Min6 cells were used as the models of β-cell dedifferentiation in vivo and in vitro, respectively. The expression of miR-195 and insulin secretion during β-cell dedifferentiation were measured. Also, the influence of regulated miR-195 expression on β-cell dedifferentiation was examined. Meanwhile, the IRS-1/2/Pi3k/Akt pathway and mitofusin-2 (Mfn2) expression were investigated during β-cell dedifferentiation.Results: MiR-195 was upregulated during lipotoxicity-induced β-cell dedifferentiation in both in vivo and in vitro experiments, and miR-195 functionally contributed to lipotoxicity-induced β-cell dedifferentiation. Furthermore, miR-195 inhibited IRS-1/2/Pi3k/Akt pathway activation, which accompanied β-cell dedifferentiation. Mfn2, a target of miR-195, was found to be downregulated and was associated with increased mitochondrial production of reactive oxygen species during β-cell dedifferentiation. Instructively, inhibition of miR-195, at least partially, reversed the downregulation of Mfn2, restored IRS-1/2/Pi3k/Akt pathway activation, and prevented β-cell dedifferentiation.Conclusions: MiR-195 promoted β-cell dedifferentiation through negatively regulating Mfn2 expression and inhibiting the IRS-1/2/Pi3k/Akt pathway, providing a promising treatment for type 2 diabetes mellitus. [ABSTRACT FROM AUTHOR]

Objective: The aim of this study was to assess gut microbiota modifications after exercise in humans and animal models with obesity or type 2 diabetes and their role in exercise-induced weight loss.Methods: A systematic search of six databases was conducted on July 31, 2021. The extracted data on body fat or body weight from human and animal studies were analyzed using random-effects meta-analysis.Results: A total of 28 studies were included, with all studies reporting exercise-induced gut microbiota modifications; however, the modified taxa varied among studies. Proteobacteria was the only taxa reported to be altered by exercise in more than one human and one animal study. Taxa belonging to Firmicutes were the most responsive to exercise in humans and mice, whereas Proteobacteria taxa were the most responsive to exercise in rats. A meta-analysis was conducted to examine the weight-lowering effect of exercise based on data subgrouped by altered or unaltered α-diversity or β-diversity. The association between the weight-lowering effect of exercise and altered β-diversity was observed in humans with obesity but not in animals.Conclusions: These findings suggest that gut microbiota modifications contribute to exercise-induced weight loss in obesity; however, their precise contributions, especially those of taxon-level variations, remain to be investigated. [ABSTRACT FROM AUTHOR]

Background: Methamphetamine (MA) is a non-selective monoamine releaser and thus releases serotonin (5-HT), norepinephrine (NE) and dopamine (DA) from corresponding nerve terminals into synapses. DOI ((±)-2, 5-dimethoxy-4-iodoamphetamine) is a direct-acting serotonergic 5-HT2A/C receptor agonist and induces the head-twitch response (HTR) via stimulation of 5-HT2A receptor in mice. While more selective serotonin releasers such as d-fenfluramine evoke the HTR, monoamine reuptake blockers (e.g., cocaine) suppress the DOI-evoked HTR via indirect stimulation of serotonergic 5-HT1A- and adrenergic ɑ2-receptors. Since the induction of HTR by DOI is age-dependent, we investigated whether: (1) during development MA can evoke the HTR by itself, and (2) acute pretreatment with either the selective 5-HT2A receptor antagonist EMD 281014 or low-doses of MA can: (i) modulate the DOI-induced HTR in mice across postnatal days 20, 30 and 60, and (ii) alter the DOI-induced c-fos expression in mice prefrontal cortex (PFC). To further explore the possible modulatory effect of MA on DOI-induced HTR, we investigated whether blockade of inhibitory serotonergic 5-HT1A- or adrenergic ɑ2-receptors by corresponding selective antagonists (WAY 100635 or RS 79948, respectively), can prevent the effect of MA on DOI-induced HTR during aging.Results: Although neither EMD 281014 nor MA by themselves could evoke the HTR, acute pretreatment with either EMD 281014 (0.01, 0.05 and 0.1 mg/kg, i.p.) or MA (1, 2.5, 5 mg/kg, i.p.), dose-dependently suppressed the DOI-induced HTR across ages. While WAY 100635 significantly reversed the inhibitory effect of MA in 20- and 30-day old mice, RS 79948 failed to significantly counter MA's inhibitory effect. Moreover, DOI significantly increased c-fos expressions in several PFC regions. EMD 281014 prevented the DOI-induced increases in c-fos expression. Despite the inhibitory effect of MA on DOI-induced HTR, MA alone or in combination with DOI, significantly increased c-fos expression in several regions of the PFC.Conclusion: The suppressive effect of MA on the DOI-evoked HTR appears to be mainly due to functional interactions between the HTR-inducing 5-HT2A receptor and the inhibitory 5-HT1A receptor. The MA-induced increase in c-fos expression in different PFC regions may be due to MA-evoked increases in synaptic concentrations of 5-HT, NE and/or DA. [ABSTRACT FROM AUTHOR]

Anemia is a significant complication of chronic inflammation and may be related to dysregulated activities among erythroblastic island (EBI) macrophages. GM-CSF was reported to be upregulated and attracted as a therapeutic target in many inflammatory diseases. Among EBIs, we found that the GM-CSF receptor is preferentially and highly expressed among EBI macrophages but not among erythroblasts. GM-CSF treatment significantly decreases human EBI formation in vitro by decreasing the adhesion molecule expression of CD163. RNA-sequence analysis suggests that GM-CSF treatment impairs the supporting function of human EBI macrophages during erythropoiesis. GM-CSF treatment also polarizes human EBI macrophages from M2-like type to M1-like type. In addition, GM-CSF decreases mouse bone marrow (BM) erythroblasts as well as EBI macrophages, leading to a reduction in EBI numbers. In defining the molecular mechanism at work, we found that GM-CSF treatment significantly decreases the adhesion molecule expression of CD163 and Vcam1 in vivo. Importantly, GM-CSF treatment also decreases the phagocytosis rate of EBI macrophages in mouse BM as well as decreases the expression of the engulfment-related molecules Mertk, Axl, and Timd4. In addition, GM-CSF treatment polarizes mouse BM EBI macrophages from M2-like type to M1-like type. Thus, we document that GM-CSF impairs EBI formation in mice and humans. Our findings support that targeting GM-CSF or reprogramming EBI macrophages might be a novel strategy to treat anemia resulting from inflammatory diseases. [ABSTRACT FROM AUTHOR]

Cardiac fibrosis, a well-known major pathological process that ultimately leads to heart failure, has attracted increasing attention and focus in recent years. A large amount of research indicates that long noncoding RNAs (lncRNAs) play an important role in cardiac fibrosis, but little is known about the specific function and mechanism of the lncRNA NEAT1 in the progression of cardiac fibrosis to heart failure. In the present study, we have demonstrated that the lncRNA NEAT1 is upregulated in patients with heart failure. Similarly, the expression of Neat1 was also increased in the left ventricular tissue of transverse aortic constriction (TAC) surgery mice and cardiac fibroblasts treated with TGF-β1. Further, gain-of-function and loss-of-function experiments showed that silencing of Neat1 attenuated cardiac fibrosis, while overexpression of Neat1 with adenovirus significantly aggravated the in vitro progression of fibrosis. With regard to the underlying mechanism, our experiments showed that Neat1 recruited EZH2 to the promoter region of Smad7 through physical binding of EZH2 to the promoter region, as a result of which Smad7 expression was inhibited and the progression of cardiac fibrosis was ultimately exacerbated. We found that the introduction of shNeat1 carried by adeno-associated virus-9 significantly ameliorated cardiac fibrosis and dysfunction caused by TAC surgery in mice. Overall, our study findings demonstrate that the lncRNA Neat1 accelerates the progression of cardiac fibrosis and dysfunction by recruiting EZH2 to suppress Smad7 expression. Thus, NEAT1 may serve as a target for the treatment of cardiac fibrosis. [ABSTRACT FROM AUTHOR]

Background: Although an immunosuppressive tumor microenvironment (TME) is key for tumor progression, the molecular characteristics associated with the immunosuppressive TME remain unknown in triple-negative breast cancer (TNBC). Our previous functional proteomic study of TNBC tumors identified that C-JUN N-terminal kinase (JNK) pathway-related molecules were enriched in a cluster associated with the inflammatory pathway. However, the role of the JNK pathway in the TNBC TME is still unclear.Methods: Transcriptomic analysis was conducted using The Cancer Genome Atlas datasets. The effect of JNK-IN-8, a covalent pan-JNK inhibitor, on TNBC tumor growth, lung metastasis, and the TME was measured in TNBC syngeneic mouse models (n = 13 per group). Tumor (n = 43) or serum (n = 46) samples from TNBC patients were analyzed using multiplex immunohistochemistry or Luminex assay. All statistical tests were 2-sided.Results: CIBERSORT analysis revealed that TNBC patients with high phosphorylated JNK level (n = 47) had more regulatory T cell (Treg) infiltration than those with a low phosphorylated JNK level (n = 47) (P = .02). Inhibition of JNK signaling statistically significantly reduced tumor growth (P < .001) and tumor-infiltrating Tregs (P = .02) while increasing the infiltration of CD8+ T cells in TNBC mouse models through the reduction of C-C motif ligand 2 (CCL2). Tumor-associated macrophages were the predominant cells secreting CCL2, and inhibition of JNK signaling reduced CCL2 secretion of human primary macrophages. Moreover, in patients with TNBC (n = 43), those with high levels of CCL2+ tumor-associated macrophages had more Treg and less CD8+ T cell infiltration (P = .04), and the serum CCL2 level was associated with poor overall survival (hazard ratio = 2.65, 95% confidence interval = 1.29 to 5.44, P = .008) in TNBC patients (n = 46).Conclusions: The JNK/C-JUN/CCL2 axis contributes to TNBC aggressiveness via forming an immunosuppressive TME and can offer novel therapeutic strategies for TNBC. [ABSTRACT FROM AUTHOR]

Background and Purpose: Maternal infections are one of the main causes of adverse developmental outcomes including embryonic resorption and preterm labour. In this study a mouse model of inflammation-associated preterm delivery was developed, and used to study the relationship between nitric oxide (NO) and prostaglandins (PGs).Experimental Approach: The murine model of preterm labour was achieved by assaying different doses of bacterial lipopolysaccharides (LPS). Once established, it was used to analyse uterine levels of prostaglandins E(2) and F(2α) (by radioimmunoassay), cyclooxygenases (COX) and NOS proteins (by Western blot) and NO synthase (NOS) activity. Effects of inhibitors of COX and NOS on LPS-induced preterm labour were also studied. In vitro assays with a nitric oxide donor (SNAP) were performed to analyse the modulation of prostaglandin production by NO.Key Results: Lipopolysaccharide increased uterine NO and PG synthesis and induced preterm delivery. Co-administration of meloxicam, a cyclooxygenase-2 inhibitor, or aminoguanidine, an inducible NOS inhibitor, prevented LPS-induced preterm delivery and blocked the increase in PGs and NO. Notably, the levels of NO were found to determine its effect on PG synthesis; low concentrations of NO reduced PG synthesis whereas high concentrations augmented them.Conclusions and Implications: An infection-associated model of preterm labour showed that preterm delivery can be prevented by decreasing PG or NO production. NO was found to have a dual effect on PG synthesis depending on its concentration. These data contribute to the understanding of the interaction between NO and PGs in pregnancy and parturition, and could help to improve neonatal outcomes. [ABSTRACT FROM AUTHOR]

Background and Purpose: Insulin-induced Na(+) retention in the distal nephron may contribute to the development of oedema/hypertension in patients with type 2 diabetes. This response to insulin is usually attributed to phosphatidylinositol-3-kinase (PI3K)/serum and glucocorticoid-inducible kinase 1 (SGK1) but a role for protein kinase B (PKB) has been proposed. The present study therefore aimed to clarify the way in which insulin can evoke Na(+) retention.Experimental Approach: We examined the effects of nominally selective inhibitors of PI3K (wortmannin, PI103, GDC-0941), SGK1 (GSK650394A) and PKB (Akti-1/2) on Na(+) transport in hormone-deprived and insulin-stimulated cortical collecting duct (mpkCCD) cells, while PI3K, SGK1 and PKB activities were assayed by monitoring the phosphorylation of endogenous proteins.Key Results: Wortmannin substantially inhibited basal Na(+) transport whereas PI103 and GDC-0941 had only very small effects. However, these PI3K inhibitors all abolished insulin-induced Na(+) absorption and inactivated PI3K, SGK1 and PKB fully. GSK650394A and Akti-1/2 also inhibited insulin-evoked Na(+) absorption and while GSK650394A inhibited SGK1 without affecting PKB, Akti-1/2 inactivated both kinases.Conclusion and Implications: While studies undertaken using PI103 and GDC-0941 show that hormone-deprived cells can absorb Na(+) independently of PI3K, PI3K seems to be essential for insulin induced Na(+) transport. Akti-1/2 does not act as a selective inhibitor of PKB and data obtained using this compound must therefore be treated with caution. GSK650394A, on the other hand, selectively inhibits SGK1 and the finding that GSK650394A suppressed insulin-induced Na(+) absorption suggests that this response is dependent upon signalling via PI3K/SGK1. [ABSTRACT FROM AUTHOR]

Background and Purpose: Cannabinoid CB(1) receptor antagonists reduce food intake and body weight, but clinical use in humans is limited by effects on the CNS. We have evaluated a novel cannabinoid antagonist (AM6545) designed to have limited CNS penetration, to see if it would inhibit food intake in rodents, without aversive effects.Experimental Approach: Cannabinoid receptor binding studies, cAMP assays, brain penetration studies and gastrointestinal motility studies were carried out to assess the activity profile of AM6545. The potential for AM6545 to induce malaise in rats and the actions of AM6545 on food intake and body weight were also investigated.Key Results: AM6545 binds to CB(1) receptors with a K(i) of 1.7 nM and CB(2) receptors with a K(i) of 523 nM. AM6545 is a neutral antagonist, having no effect on cAMP levels in transfected cells and was less centrally penetrant than AM4113, a comparable CB(1) receptor antagonist. AM6545 reversed the effects of WIN55212-2 in an assay of colonic motility. In contrast to AM251, AM6545 did not produce conditioned gaping or conditioned taste avoidance in rats. In rats and mice, AM6545 dose-dependently reduced food intake and induced a sustained reduction in body weight. The effect on food intake was maintained in rats with a complete subdiaphragmatic vagotomy. AM6545 inhibited food intake in CB(1) receptor gene-deficient mice, but not in CB(1)/CB(2) receptor double knockout mice.Conclusions and Implications: Peripherally active, cannabinoid receptor antagonists with limited brain penetration may be useful agents for the treatment of obesity and its complications. [ABSTRACT FROM AUTHOR]

Background and Purpose: Excessive production of nitric oxide (NO) by inducible NO synthase (iNOS) is thought to underlie the vascular dysfunction, systemic hypotension and organ failure that characterize endotoxic shock. Plasma levels of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are raised in animal models and humans with endotoxic shock and correlate with the associated cardiovascular dysfunction. Since both NO and natriuretic peptides play important roles in cardiovascular homeostasis via activation of guanylate cyclase-linked receptors, we used mice lacking natriuretic peptide receptor (NPR)-A (NPR1) to establish if natriuretic peptides contribute to the cardiovascular dysfunction present in endotoxic shock.Experimental Approach: Wild-type (WT) and NPR-A knockout (KO) mice were exposed to lipopolysaccharide (LPS) and vascular dysfunction (in vitro and in vivo), production of pro-inflammatory cytokines, and iNOS expression and activity were evaluated.Key Results: LPS-treated WT animals exhibited a marked fall in mean arterial blood pressure (MABP) whereas NPR-A KO mice maintained MABP throughout. LPS administration caused a greater suppression of vascular responses to the thromboxane-mimetic U46619, ANP, acetylcholine and the NO-donor spermine-NONOate in WT versus NPR-A KO mice. This differential effect on vascular function was paralleled by reduced pro-inflammatory cytokine production, iNOS expression and activity (plasma [NO(x)] and cyclic GMP).Conclusions and Implications: These observations suggest that NPR-A activation by natriuretic peptides facilitates iNOS expression and contributes to the vascular dysfunction characteristic of endotoxic shock. Pharmacological interventions that target the natriuretic peptide system may represent a novel approach to treat this life-threatening condition. [ABSTRACT FROM AUTHOR]

Background and Purpose: Gram-negative bacteria contain ligands for Toll-like receptor (TLR) 4 and nucleotide oligomerization domain (NOD) 1 receptors. Lipopolysaccharide (LPS) activates TLR4, while peptidoglycan products activate NOD1. Activation of NOD1 by the specific agonist FK565 results in a profound vascular dysfunction and experimental shock in vivo.Experimental Approach: Here, we have analysed a number of pharmacological inhibitors to characterize the role of key signalling pathways in the induction of NOS2 following TLR4 or NOD1 activation.Key Results: Vascular smooth muscle (VSM) cells expressed NOD1 mRNA and protein, and, after challenge with Escherichia coli or FK565, NOS2 protein and activity were induced. Macrophages had negligible levels of NOD1 and were unaffected by FK565, but responded to E. coli and LPS by releasing increased NO and expression of NOS2 protein. Classic pharmacological inhibitors for NF-kappaB (SC-514) and mitogen-activated protein kinase (SB203580, PD98059) signalling pathways inhibited responses in both cell types regardless of agonist. While TLR4-mediated responses in macrophages were specifically inhibited by the pan-caspase inhibitor z-VAD-fmk and the PKC inhibitor Gö6976, NOD1-mediated responses in VSM cells were inhibited by the Rip2 inhibitor PP2.Conclusions and Implications: Our findings suggest a selective role for NOD1 in VSM cells, and highlight NOD1 as a potential novel therapeutic target for the treatment of vascular inflammation. [ABSTRACT FROM AUTHOR]

Background and Purpose: The present study evaluated the role of CB(2) receptors in the regulation of depressive-like behaviours. Transgenic mice overexpressing the CB(2) receptor (CB2xP) were challenged with different types of acute and chronic experimental paradigms to evaluate their response in terms of depressive-like behaviours.Experimental Approach: Tail suspension test (TST), novelty-suppressed feeding test (NSFT) and unpredictable chronic mild stress tests (CMS) were carried out in CB2xP mice. Furthermore, acute and chronic antidepressant-like effects of the CB(2) receptor-antagonist AM630 were evaluated by means of the forced swimming test (FST) and CMS, respectively, in wild-type (WT) and CB2xP mice. CB(2) gene expression, brain-derived neurotrophic factor (BDNF) gene and protein expressions were studied in mice exposed to CMS by real-time PCR and immunohistochemistry, respectively.Key Results: Overexpression of CB(2) receptors resulted in decreased depressive-like behaviours in the TST and NSFT. CMS failed to alter the TST and sucrose consumption in CB2xP mice. In addition, no changes in BDNF gene and protein expression were observed in stressed CB2xP mice. Interestingly, acute administration of AM630 (1 and 3 mg x kg(-1), i.p.) exerted antidepressant-like effects on the FST in WT, but not in CB2xP mice. Chronic administration of AM630 for 4 weeks (1 mg x kg(-1); twice daily, i.p.) blocked the effects of CMS on TST, sucrose intake, CB(2) receptor gene, BDNF gene and protein expression in WT mice.Conclusion and Implications: Taken together, these results suggest that increased CB(2) receptor expression significantly reduced depressive-related behaviours and that the CB(2) receptor could be a new potential therapeutic target for depressive-related disorders. [ABSTRACT FROM AUTHOR]

Background and Purpose: A new class of heterotricyclic glutamate analogues recently was generated by incorporating structural elements of two excitotoxic marine compounds, kainic acid and neodysiherbaine A. Rather than acting as convulsants, several of these 'IKM' compounds markedly depressed CNS activity in mice. Here, we characterize the pharmacological profile of the series with a focus on the most potent of these molecules, IKM-159.Experimental Approach: The pharmacological activity and specificity of IKM compounds were characterized using whole-cell patch clamp recording from neurons and heterologous receptor expression systems, in combination with radioligand binding techniques.Key Results: The majority of the IKM compounds tested reduced excitatory synaptic transmission in neuronal cultures, and IKM-159 inhibited synaptic currents from CA1 pyramidal neurons in hippocampal slices. IKM-159 inhibited glutamate-evoked whole-cell currents from recombinant GluA2- and GluA4-containing alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors most potently, whereas kainate and NMDA receptor currents were not reduced by IKM-159. Antagonism of steady-state currents was agonist concentration dependent, suggesting that its mechanism of action was competitive, although it paradoxically did not displace [(3)H]-AMPA from receptor binding sites. IKM-159 reduced spontaneous action potential firing in both cultured hippocampal neurons in control conditions and during hyperactive states in an in vitro model of status epilepticus.Conclusions and Implications: IKM-159 is an AMPA receptor-selective antagonist. IKM-159 and related nitrogen heterocycles represent structurally novel AMPA receptor antagonists with accessible synthetic pathways and potentially unique pharmacology, which could be of use in exploring the role of specific populations of receptors in neurophysiological and neuropathological processes. [ABSTRACT FROM AUTHOR]

Background and Purpose: Current therapies for muscular dystrophy are based on corticosteroids. Significant side effects associated with these therapies have prompted several studies aimed at identifying possible alternative strategies. As inflammation and defects of nitric oxide (NO) generation are key pathogenic events in muscular dystrophies, we have studied the effects of combining the NO donor isosorbide dinitrate (ISDN) and the non-steroidal anti-inflammatory drug ibuprofen.Experimental Approach: alpha-Sarcoglycan-null mice were treated for up to 8 months with ISDN (30 mg.kg(-1)) plus ibuprofen (50 mg.kg(-1)) administered daily in the diet. Effects of ISDN and ibuprofen alone were assessed in parallel. Drug effects on animal motility and muscle function, muscle damage, inflammatory infiltrates and cytokine levels, as well as muscle regeneration including assessment of endogenous stem cell pool, were measured at selected time points.Key Results: Combination of ibuprofen and ISDN stimulated regeneration capacity, of myogenic precursor cells, reduced muscle necrotic damage and inflammation. Muscle function in terms of free voluntary movement and resistance to exercise was maintained throughout the time window analysed. The effects of ISDN and ibuprofen administered separately were transient and significantly lower than those induced by their combination.Conclusions and Implications: Co-administration of NO and ibuprofen provided synergistic beneficial effects in a mouse model of muscular dystrophy, leading to an effective therapy. Our results open the possibility of immediate clinical testing of a combination of ISDN and ibuprofen in dystrophic patients, as both components are approved for use in humans, with a good safety profile. [ABSTRACT FROM AUTHOR]

Background and Purpose: AE9C90CB (N- [(1R, 5S, 6R)-3-azabicyclo [3.1.0] hex-6-ylmethyl]-2-hydroxy-N-methyl-2, 2-diphenylacetamide), a novel muscarinic receptor antagonist, was synthesized for the treatment of overactive bladder. Here we describe the in vitro and in vivo profiles of AE9C90CB for action in bladder over salivary gland and compare it with four agents already in clinical use (tolterodine, oxybutynin, solifenacin and darifenacin).Experimental Approach: Radioligand binding assay and isolated tissue-based functional assay were used to evaluate affinity, potency, and receptor subtype selectivity of compounds. Inhibition of carbachol-induced increase in intravesicular pressure and salivary secretion were measured in anaesthetized rabbits to assess the functional selectivity.Key Results: In vitro radioligand binding study using human recombinant muscarinic receptors showed that AE9C90CB had greater affinity for M(3) muscarinic receptors with pKi of 9.90 +/- 0.11 and was 20-fold more selective for M(3) than for M(2) muscarinic receptors. AE9C90CB exhibited an unsurmountable antagonism on rat bladder strips (pK(B), 9.13 +/- 0.12). In anaesthetized rabbits after intravenous administration, AE9C90CB dose dependently inhibited carbachol-induced increase in intravesicular pressure and salivary secretion, and exhibited functional selectivity for urinary bladder over salivary gland which was ninefold better than that of oxybutynin.Conclusions and Implications: We have identified AE9C90CB, a compound exhibiting moderate selectivity for M(3) over M(2) receptors but greater selectivity for urinary bladder over salivary gland than oxybutynin, tolterodine, solifenacin and darifenacin. Therefore, AE9C90CB may be a promising compound for the treatment of overactive bladder with reduced potential to cause dry mouth than currently available antimuscarinic drugs. [ABSTRACT FROM AUTHOR]

Background and Purpose: Arteries from hypertensive subjects are reportedly hyperresponsive to 5-hydroxytryptamine (5-HT), but it remains unclear whether this is true in chronic type 2 diabetes. We have assessed responses to 5-HT shown by mesenteric arteries from type 2 diabetic ob/ob mice (27-32 weeks old) and have identified the molecular mechanisms involved.Experimental Approach: Contractions of mesenteric rings to 5-HT were examined in vitro. Activation of mesenteric RhoA, Rho kinase and Src was measured by Western blotting or by modified enzyme-linked immunosorbent assay.Key Results: Concentration-dependent contractions to 5-HT were greater in mesenteric rings from the ob/ob than in those from the age-matched control ('Lean') group. In each group, there was no significant change in the 5-HT-induced contractions after inhibition of nitric oxide synthase (with N(G)-nitro-L-arginine), of cyclooxygenase (with indomethacin) or of protein kinase C (with chelerythrine). However inhibition of the MEK/ERK pathway (with PD98059) decreased the response to 5-HT. Although the diabetes-related enhancement of the 5-HT response was preserved with each of these inhibitors, enhancement was abolished by a Rho kinase inhibitor (Y27632) and by Src kinase inhibitors (PP1 analogue or Src kinase inhibitor I). 5-HT-induced activation of RhoA, Rho kinase and Src kinase in mesenteric arteries was greater in the ob/ob than in the Lean group, but the expression of RhoA, Rho kinase isoforms and Src did not differ between these groups.Conclusions and Implications: These results suggest that the enhancement of 5-HT-induced contraction in mesenteric arteries from ob/ob mice may be attributable to increased activation of RhoA/Rho kinase and Src kinase. [ABSTRACT FROM AUTHOR]

Background and Purpose: Lopinavir is extensively metabolized by cytochrome P450 3A (CYP3A) and is considered to be a substrate for the drug transporters ABCB1 (P-glycoprotein) and ABCC2 (MRP2). Here, we have assessed the individual and combined effects of CYP3A, ABCB1 and ABCC2 on the pharmacokinetics of lopinavir and the relative importance of intestinal and hepatic metabolism. We also evaluated whether ritonavir increases lopinavir oral bioavailability by inhibition of CYP3A, ABCB1 and/or ABCC2.Experimental Approach: Lopinavir transport was measured in Madin-Darby canine kidney cells expressing ABCB1 or ABCC2. Oral lopinavir kinetics (+/- ritonavir) was studied in mice with genetic deletions of Cyp3a, Abcb1a/b and/or Abcc2, or in transgenic mice expressing human CYP3A4 exclusively in the liver and/or intestine.Key Results: Lopinavir was transported by ABCB1 but not by ABCC2 in vitro. Lopinavir area under the plasma concentration - time curve (AUC)(oral) was increased in Abcb1a/b(-/-) mice (approximately ninefold vs. wild-type) but not in Abcc2(-/-) mice. Increased lopinavir AUC(oral) (>2000-fold) was observed in cytochrome P450 3A knockout (Cyp3a(-/-)) mice compared with wild-type mice. No difference in AUC(oral) between Cyp3a(-/-) and Cyp3a/Abcb1a/b/Abcc2(-/-) mice was observed. CYP3A4 activity in intestine or liver, separately, reduced lopinavir AUC(oral) (>100-fold), compared with Cyp3a(-/-) mice. Ritonavir markedly increased lopinavir AUC(oral) in all CYP3A-containing mouse strains.Conclusions and Implications: CYP3A was the major determinant of lopinavir pharmacokinetics, far more than Abcb1a/b. Both intestinal and hepatic CYP3A activity contributed importantly to low oral bioavailability of lopinavir. Ritonavir increased lopinavir bioavailability primarily by inhibiting CYP3A. Effects of Abcb1a/b were only detectable in the presence of CYP3A, suggesting saturation of Abcb1a/b in the absence of CYP3A activity. [ABSTRACT FROM AUTHOR]

Background and Purpose: The overexpression of epidermal growth factor receptor (EGFR) and its mutated variant EGFRvIII occurs in 50% of glioblastoma multiforme. We developed antibody fragments against EGFR/EGFRvIII for molecular imaging and/or therapeutic targeting applications.Experimental Approach: An anti-EGFR/EGFRvIII llama single-domain antibody (EG(2)) and two higher valency format constructs, bivalent EG(2)-hFc and pentavalent V2C-EG(2) sdAbs, were analysed in vitro for their binding affinities using surface plasmon resonance and cell binding studies, and in vivo using pharmacokinetic, biodistribution, optical imaging and fluorescent microscopy studies.Key Results: Kinetic binding analyses by surface plasmon resonance revealed intrinsic affinities of 55 nM and 97 nM for the monovalent EG(2) to immobilized extracellular domains of EGFR and EGFRvIII, respectively, and a 10- to 600-fold increases in apparent affinities for the multivalent binders, V2C-EG(2) and EG(2)-hFc, respectively. In vivo pharmacokinetic and biodistribution studies in mice revealed plasma half-lives for EG(2), V2C-EG(2) and EG(2)-hFc of 41 min, 80 min and 12.5 h, respectively, as well as a significantly higher retention of EG(2)-hFc compared to the other two constructs in EGFR/EGFRvIII-expressing orthotopic brain tumours, resulting in the highest signal in the tumour region in optical imaging studies. Time domain volumetric optical imaging fusion with high-resolution micro-computed tomography of microvascular brain network confirmed EG(2)-hFc selective accumulation/retention in anatomically defined tumour regions.Conclusions: Single domain antibodies can be optimized for molecular imaging applications by methods that improve their apparent affinity and prolong plasma half-life and, at the same time, preserve their ability to penetrate tumour parenchyma. [ABSTRACT FROM AUTHOR]

Background and Purpose: Natural and synthetic cannabinoids (CBs) induce deleterious or beneficial actions on neuronal survival. The Fas-associated protein with death domain (FADD) promotes apoptosis, and its phosphorylated form (p-FADD) mediates non-apoptotic actions. The regulation of Fas/FADD, mitochondrial apoptotic proteins and other pathways by CB receptors was investigated in the mouse brain.Experimental Approach: Wild-type, CB(1) and CB(2) receptor knock-out (KO) mice were used to assess differences in receptor genotypes. CD1 mice were used to evaluate the effects of CB drugs on canonical apoptotic pathways and associated signalling systems. Target proteins were quantified by Western blot analysis.Key Results: In brain regions of CB(1) receptor KO mice, Fas/FADD was reduced, but p-Ser191 FADD and the p-FADD/FADD ratio were increased. In CB(2) receptor KO mice, Fas/FADD was increased, but the p-FADD/FADD ratio was not modified. In mutant mice, cleavage of poly(ADP-ribose)-polymerase (PARP) did not indicate alterations in brain cell death. In CD1 mice, acute WIN55212-2 (CB(1) receptor agonist), but not JWH133 (CB(2) receptor agonist), inversely modulated brain FADD and p-FADD. Chronic WIN55212-2 induced FADD down-regulation and p-FADD up-regulation. Acute and chronic WIN55212-2 did not alter mitochondrial proteins or PARP cleavage. Acute, but not chronic, WIN55212-2 stimulated activation of anti-apoptotic (ERK, Akt) and pro-apoptotic (JNK, p38 kinase) pathways.Conclusions and Implications: CB(1) receptors appear to exert a modest tonic activation of Fas/FADD complexes in brain. However, chronic CB(1) receptor stimulation decreased pro-apoptotic FADD and increased non-apoptotic p-FADD. The multifunctional protein FADD could participate in the mechanisms of neuroprotection induced by CBs. [ABSTRACT FROM AUTHOR]

Background and Purpose: 3,4-methylenedioxymethamphetamine (MDMA) causes a persistent loss of dopaminergic cell bodies in the substantia nigra of mice. Current evidence indicates that such neurotoxicity is due to oxidative stress but the source of free radicals remains unknown. Inhibition of mitochondrial electron transport chain complexes by MDMA was assessed as a possible source.Experimental Approach: Activities of mitochondrial complexes after MDMA were evaluated spectrophotometrically. In situ visualization of superoxide production in the striatum was assessed by ethidium fluorescence and striatal dopamine levels were determined by HPLC as an index of dopaminergic toxicity.Key Results: 3,4-methylenedioxymethamphetamine decreased mitochondrial complex I activity in the striatum of mice, an effect accompanied by an increased production of superoxide radicals and the inhibition of endogenous aconitase. alpha-Lipoic acid prevented superoxide generation and long-term toxicity independent of any effect on complex I inhibition. These effects of alpha-lipoic acid were also associated with a significant increase of striatal glutathione levels. The relevance of glutathione was supported by reducing striatal glutathione content with L-buthionine-(S,R)-sulfoximine, which exacerbated MDMA-induced dopamine deficits, effects suppressed by alpha-lipoic acid. The nitric oxide synthase inhibitor, N(G)-nitro-L-arginine, partially prevented MDMA-induced dopamine depletions, an effect reversed by L-arginine but not D-arginine. Finally, a direct relationship between mitochondrial complex I inhibition and long-term dopamine depletions was found in animals treated with MDMA in combination with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.Conclusions and Implications: Inhibition of mitochondrial complex I following MDMA could be the source of free radicals responsible for oxidative stress and the consequent neurotoxicity of this drug in mice. [ABSTRACT FROM AUTHOR]

Background and Purpose: In various models vagus nerve activation has been shown to ameliorate intestinal inflammation, via nicotinic acetylcholine receptors (nAChRs) expressed on immune cells. As the alpha7 nAChR has been put forward to mediate this effect, we studied the effect of nicotine and two selective alpha7 nAChR agonists (AR-R17779, (-)-spiro[1-azabicyclo[2.2.2] octane-3,5'-oxazolidin-2'-one and GSK1345038A) on disease severity in two mouse models of experimental colitis.Experimental Approach: Colitis was induced by administration of 1.5% dextran sodium sulphate (DSS) in drinking water or 2 mg 2,4,6-trinitrobenzene sulphonic acid (TNBS) intrarectally. Nicotine (0.25 and 2.50 micromol.kg(-1)), AR-R17779 (0.6-30 micromol.kg(-1)) or GSK1345038A (6-120 micromol.kg(-1)) was administered daily by i.p. injection. After 7 (DSS) or 5 (TNBS) days clinical parameters and colonic inflammation were scored.Key Results: Nicotine and both alpha7 nAChR agonists reduced the activation of NF-kappaB and pro-inflammatory cytokines in whole blood and macrophage cultures. In DSS colitis, nicotine treatment reduced colonic cytokine production, but failed to reduce disease parameters. Reciprocally, treatment with AR-R17779 or GSK1345038A worsened disease and led to increased colonic pro-inflammatory cytokine levels in DSS colitis. The highest doses of GSK1345038A (120 micromol.kg(-1)) and AR-R17779 (30 micromol.kg(-1)) ameliorated clinical parameters, without affecting colonic inflammation. Neither agonist ameliorated TNBS-induced colitis.Conclusions and Implications: Although nicotine reduced cytokine responses in vitro, both selective alpha7 nAChR agonists worsened the effects of DSS-induced colitis or were ineffective in those of TNBS-induced colitis. Our data indicate the need for caution in evaluating alpha7 nAChR as a drug target in colitis. [ABSTRACT FROM AUTHOR]

Background and Purpose: Over three decades of research evaluating the biology of melanocortin (MC) hormones and synthetic peptides, activation of the MC type 1 (MC(1)) receptor has been identified as a viable target for the development of novel anti-inflammatory therapeutic agents. Here, we have tested a recently described selective agonist of MC(1) receptors, BMS-470539, on leucocyte/post-capillary venule interactions in murine microvascular beds.Experimental Approach: Intravital microscopy of two murine microcirculations were utilized, applying two distinct modes of promoting inflammation. The specificity of the effects of BMS-470539 was assessed using mice bearing mutant inactive MC(1) receptors (the recessive yellow e/e colony).Key Results: BMS-470539, given before an ischaemia-reperfusion protocol, inhibited cell adhesion and emigration with no effect on cell rolling, as assessed 90 min into the reperfusion phase. These properties were paralleled by inhibition of tissue expression of both CXCL1 and CCL2. Confocal investigations of inflamed post-capillary venules revealed immunostaining for MC(1) receptors on adherent and emigrated leucocytes. Congruently, the anti-inflammatory properties of BMS-470539 were lost in mesenteries of mice bearing the inactive mutant MC(1) receptors. Therapeutic administration of BMS-470539 stopped cell emigration, but did not affect cell adhesion in the cremasteric microcirculation inflamed by superfusion with platelet-activating factor.Conclusions and Implications: Activation of MC(1) receptors inhibited leucocyte adhesion and emigration. Development of new chemical entities directed at MC(1) receptors could be a viable approach in the development of novel anti-inflammatory therapeutic agents with potential application to post-ischaemic conditions. [ABSTRACT FROM AUTHOR]

Background and Purpose: Amitriptyline is a tricyclic antidepressant that is also widely used to treat neuropathic pain in humans, but the mechanism of this anti-hyperalgesic effect is unknown. Microglia in the mouse spinal cord become activated in neuropathic pain, and expression of P2X4 receptors by these microglia is increased. Antisense RNA targeting P2X4 receptors suppresses the development of tactile allodynia in rats. This suggests that blockade of P2X4 receptors might be the mechanism by which amitriptyline relieves neuropathic pain.Experimental Approach: We expressed human, rat and mouse P2X receptors (P2X2, P2X4, P2X7) in human embryonic kidney cells and evoked inward currents by applying ATP. We compared the action of ATP on control cells and cells treated with amitriptyline.Key Results: Amitriptyline (10 microM), either applied acutely or by pre-incubation for 2-6 h, had no effect on inward currents evoked by ATP (0.3-100 microM) at human P2X4 receptors. At rat and mouse receptors, amitriptyline (10 microM) caused a modest reduction in the maximum responses to ATP, without changes in EC(50) values, but it had no effect at 1 microM. Amitriptyline also had no effects on currents evoked by ATP at rat P2X2 receptors, or at rat or human P2X7 receptors.Conclusion and Implications: The results do not support the view that amitriptyline owes its pain-relieving actions in man to the direct blockade of P2X4 receptors. [ABSTRACT FROM AUTHOR]

Background and Purpose: It is well known that adenine-based purines exert multiple effects on pain transmission. However, less attention has been given to the potential effects of guanine-based purines on pain transmission. The aim of this study was to investigate the effects of intraperitoneal (i.p.) and oral (p.o.) administration of guanosine on mice pain models. Additionally, investigation into the mechanisms of action of guanosine, its potential toxicity and cerebrospinal fluid (CSF) purine levels were also assessed.Experimental Approach: Mice received an i.p. or p.o. administration of vehicle (0.1 mM NaOH) or guanosine (up to 240 mg x kg(-1)) and were evaluated in several pain models.Key Results: Guanosine produced dose-dependent antinociceptive effects in the hot-plate, glutamate, capsaicin, formalin and acetic acid models, but it was ineffective in the tail-flick test. Additionally, guanosine produced a significant inhibition of biting behaviour induced by i.t. injection of glutamate, AMPA, kainate and trans-ACPD, but not against NMDA, substance P or capsaicin. The antinociceptive effects of guanosine were prevented by selective and non-selective adenosine receptor antagonists. Systemic administration of guanosine (120 mg x kg(-1)) induced an approximately sevenfold increase on CSF guanosine levels. Guanosine prevented the increase on spinal cord glutamate uptake induced by intraplantar capsaicin.Conclusions and Implications: This study provides new evidence on the mechanism of action of the antinociceptive effects after systemic administration of guanosine. These effects seem to be related to the modulation of adenosine A(1) and A(2A) receptors and non-NMDA glutamate receptors. [ABSTRACT FROM AUTHOR]

Background and Purpose: Changes in extracellular fluid osmolarity, which occur after tissue damage and disease, cause inflammation and maintain chronic inflammatory states by unknown mechanisms. Here, we investigated whether the osmosensitive channel, transient receptor potential vanilloid 4 (TRPV4), mediates inflammation to hypotonic stimuli by a neurogenic mechanism.Experimental Approach: TRPV4 was localized in dorsal root ganglia (DRG) by immunofluorescence. The effects of TRPV4 agonists on release of pro-inflammatory neuropeptides from peripheral tissues and on inflammation were examined.Key Results: Immunoreactive TRPV4 was detected in DRG neurones innervating the mouse hindpaw, where it was co-expressed in some neurones with CGRP and substance P, mediators of neurogenic inflammation. Hypotonic solutions and 4alpha-phorbol 12,13-didecanoate, which activate TRPV4, stimulated neuropeptide release in urinary bladder and airways, sites of neurogenic inflammation. Intraplantar injection of hypotonic solutions and 4alpha-phorbol 12,13-didecanoate caused oedema and granulocyte recruitment. These effects were inhibited by a desensitizing dose of the neurotoxin capsaicin, antagonists of CGRP and substance P receptors, and TRPV4 gene knockdown or deletion. In contrast, antagonism of neuropeptide receptors and disruption of TRPV4 did not prevent this oedema. TRPV4 gene knockdown or deletion also markedly reduced oedema and granulocyte infiltration induced by intraplantar injection of formalin.Conclusions and Implications: Activation of TRPV4 stimulates neuropeptide release from afferent nerves and induces neurogenic inflammation. This mechanism may mediate the generation and maintenance of inflammation after injury and during diseases, in which there are changes in extracellular osmolarity. Antagonism of TRPV4 may offer a therapeutic approach for inflammatory hyperalgesia and chronic inflammation. [ABSTRACT FROM AUTHOR]

Background and Purpose: In adults, neurogenesis persists in the hippocampus and the subventricular zone (SVZ), and this is important for learning and memory. Inhibitors of COX-2 suppress ischaemia-induced neurogenesis in the hippocampus. Here, we have determined the effects of COX-2 inhibitors on neurogenesis throughout the normal adult mouse brain.Experimental Approach: Young adult mice were treated with COX-2 inhibitors, and the proliferation of neural progenitor cells was measured in the SVZ and hippocampus. In addition, the local uptake of lentiviral vectors in the rostral migratory stream enabled the formation of new neurons in the olfactory bulb (OB) to be assessed.Key Results: The COX-2 inhibitor meloxicam suppressed progenitor cell proliferation in the SVZ and hippocampus. A significant decrease in the appearance of new neurons in the OB was also observed. Similar effects on progenitor proliferation in the SVZ were seen with nimesulide. The absence of COX-2 expression in the proliferating progenitors in vivo, and the lack of effect of the COX-2 inhibitors on the growth rate of a cultured progenitor cell line, suggest that the effect is indirect. The specific expression of COX-2 in resting microglia that closely associate with the proliferating progenitor cells provides for a possible site of action.Conclusions and Implications: Treatment with a COX-2 inhibitor results in a substantial inhibition of adult neurogenesis. Studies on human tissues are warranted in order to determine if this effect extends to humans, and whether inhibition of neurogenesis should be considered as an adverse effect of these drugs. [ABSTRACT FROM AUTHOR]

Background and Purpose: Activation of the proteinase-activated receptor-2 (PAR-2) induces scratching behaviour in mice. Here, we have investigated the role of kinin B(1) and B(2) receptors in the pruritogenic response elicited by activators of PAR-2.Experimental Approach: Scratching was induced by an intradermal (i.d.) injection of trypsin or the selective PAR-2 activating peptide SLIGRL-NH(2) at the back of the mouse neck. The animals were observed for 40 min and their scratching response was quantified.Key Results: I.d. injection of trypsin or SLIGRL-NH(2) evoked a scratching behaviour, dependent on PAR-2 activation. Mice genetically deficient in kinin B(1) or B(2) receptors exhibited reduced scratching behaviour after i.d. injection of trypsin or SLIGRL-NH(2). Treatment (i.p.) with the non-peptide B(1) or B(2)receptor antagonists SSR240612 and FR173657, respectively, prevented the scratching behaviour caused by trypsin or SLIGRL-NH(2). Nonetheless, only treatment i.p. with the peptide B(2)receptor antagonist, Hoe 140, but not the B(1)receptor antagonist (DALBK), inhibited the pruritogenic response to trypsin. Hoe 140 was also effective against SLIGRL-NH(2)-induced scratching behaviour when injected by i.d. or intrathecal (i.t.) routes. Also, the response to SLIGRL-NH(2) was inhibited by i.t. (but not by i.d.) treatment with DALBK. Conversely, neither Hoe 140 nor DALBK were able to inhibit SLIGRL-NH(2)-induced scratching behaviour when given intracerebroventricularly (i.c.v.).Conclusions and Implications: The present results demonstrated that kinins acting on both B(1) and B(2) receptors played a crucial role in controlling the pruriceptive signalling triggered by PAR-2 activation in mice. [ABSTRACT FROM AUTHOR]

Background and Purpose: Tetrandrine, a well-known naturally occurring calcium antagonist with anti-inflammatory, antioxidant and anti-fibrogenetic activities, has long been used clinically for treatment of cardiovascular diseases such as hypertension and arrhythmia. However, little is known about the effect of tetrandrine on cardiac hypertrophy. The aims of the present study were to determine whether tetrandrine could attenuate cardiac hypertrophy and to clarify the underlying molecular mechanisms.Experimental Approach: Tetrandrine (50 mg x kg(-1) x day(-1)) was administered by oral gavage three times a day for one week and then the mice were subjected to either chronic pressure overload generated by aortic banding (AB) or sham surgery (control group). Cardiac function was determined by echocardiography.Key Results: Tetrandrine attenuated the cardiac hypertrophy induced by AB, as assessed by heart weight/body weight and lung weight/body weight ratios, cardiac dilatation and the expression of genes of hypertrophic markers. Tetrandrine also inhibited fibrosis and attenuated the inflammatory response. The cardioprotective effects of tetrandrine were mediated by blocking the increased production of reactive oxygen species and the activation of ERK1/2-dependent nuclear factor-kappaB and nuclear factor of activated T cells that occur in response to hypertrophic stimuli.Conclusions and Implications: Taken together, our results suggest that tetrandrine can improve cardiac function and prevent the development of cardiac hypertrophy by suppressing the reactive oxygen species-dependent ERK1/2 signalling pathway. [ABSTRACT FROM AUTHOR]