Abstract: Background: Intestinal barrier function failure from ischemia/reperfusion (I/R) and acute hypoxia has been implicated as a critical determinant in the predisposition to intestinal inﬂammation and a number of inﬂam- matory disorders. Here, we identified the role of Adenosine A2B receptor (A2BAR) in the regulation of intestinal barrier function under I/R and acute hypoxic conditions. Methods: C57BL/6J mice were used, and were randomized into three groups: Sham, I/R, IR+PSB1115 (a specific A2BAR antagonist) groups. After surgery, the small bowel was harvested for immunohistochemical staining, RNA and protein content, and intestinal permeability analyses. Using an epithelial cell culture model, we investigated the inﬂuence of hypoxia on the epithelial function, and the role of A2BAR in the expressions of tight junction and epithelial permeability. The expressions of Claudin-1, occludin and ZO-1 were detected by RT-PCR and Western-Blot. Epithelial barrier function was assessed with transepithelial resistance (TER). Results and conclusions: The A2BAR antagonist, PSB1115, significantly increased tight junction protein expression after intestinal I/R or acute hypoxia conditions. PSB1115 also attenuated the disrupted distribu- tion of TJ proteins. Furthermore, inhibition of A2BAR attenuated the decrease in TER induced by I/R or acute hypoxic conditions, and maintained intestinal barrier function. Antagonism of A2BAR activity improves intestinal epithelial structure and barrier function in a mouse model of intestinal I/R and a cell model of acute hypoxia. These findings support a potentially destructive role for A2BAR under intestinal I/R and acute hypoxic conditions.
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Fig. 6. Antagonism of agonist-stimulated cAMP Glosensor responses by increasing concentrations of adenosine receptor antagonists. Competition curves for (a) NECA (10 mM), (b) adenosine (10 l M), (c) BAY 60-6583 (1 l M) or (d) CGS 21680 (1 l M) stimulated cAMP responses following 30 min pre-incubation with XAC, PSB 603, ZM 241385 or SCH 58261. Data represent mean ± S.E.M of the peak luminescence response in individual experiments carried out in triplicate. Data are expressed as% of the peak luminescence response to each agonist obtained in the absence of antagonist for (a) seven (PSB), six (XAC), five (ZM 241385) or four (SCH 58261) separate experiments performed in triplicate. The residual response following 10 mM PSB 603 pre-incubation is significantly different to that seen with following stimulation with vehicle alone (1 way ANOVA, ***
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23. Krishnan M, Jayaraj RL, Megala J, Elangovan N. Antioxidant mediated antiulcer effect of Eupatorium triplinerve Vahl against acetic acid induced ulcerative colitis in mice. Biomedicine and Aging Pathology. 2014;4(2):153-60. 24. Klotz KN, Hessling J, Hegler J, Owman C, Kull B, Fredholm BB, et al. Comparative pharmacology of human adenosine receptor subtypes characterization of stably transfected receptors in CHO cells. Naunyn- Schmiedeberg’s Archives of Pharmacology. 1997;357(1):1-9.
after peripheral nerve injury [73,74] and protection of retinal ganglia cells was compromised in LIF knock-out mice after lens injury . Finally, LIF was shown to limit demyelination in an experimental autoimmune enceph- alomyelitis mouse model  and has become a promin- ent therapeutic candidate for multiple sclerosis . We have previously shown that LIF can protect cortical as well as hippocampal neurons against glutamate-induced excitotoxicity . Here we show that LIF coming from the supernatant of NECA-treated astrocytes has the same protective effect. Indeed, astrocytes produce several other cytokines and neurotrophic factors including IL-6, NGF, brain-derived neurotrophic factor, neurotrophin-3, S- 100β protein and TGFβ , that might help neurons to cope with excitotoxic stress. Accordingly, conditioned media from astrocyte cultures protected cortical neurons against glutamate (data not shown). In order to confine the neuroprotective effect of astrocytic factors that are released in response to NECA treatment, we had to re- fresh astrocyte culture medium prior to NECA treatment and testing supernatant on glutamate-stressed neurons. This, together with LIF neutralization, indicates that LIF produced by astrocytes after adenosine receptor stimulation is necessary to witness neuronal protec- tion. Our results provide further evidence for a role of adenosine in neuronal protection. Indeed, it has been shown that adenosine can protect neurons dur- ing hypoxia [76,77], ischemia [78,79] and excessive neuronal activity [80,81]. This adenosine protection is often mediated through the A 1 receptor subtype [82-
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ABSTRACT: Previous studies have demonstrated that the activation of adenosine A2B receptors increases adenosine 3'-cyclic monophosphate (cAMP) accumulation in rat skeletal muscle cells, which is probably an important yet unknown mechanism contributing to the regulation of skeletal muscle functions. via rat L6 skeletal muscle cells, it has been elucidated further potential molecular signaling responsible for adenosine A2B receptor modulations via quantitative real-time PCR assays (probe- based). The results of the present study has shown for the first time that NECA alters the expression of PGC-1α significantly in both one week and 19 hours starved skeletal muscle cells (P<0.05 and P<0.01, respectively) (2.5 and -0.58 fold change compared to vehicle, respectively). Adenosine A2B receptors mediate NECA-modulated PGC-1α mRNA gene expression in skeletal muscle cells. To our knowledge, this is the first study demonstrating an induction of PGC-1α gene by CGS 21680 significantly (P<0.001) (around 1.8 fold change compared to vehicle). In the current study, NECA (10 μM) increases NR4A1 and NR4A3 mRNA gene expression significantly (P<0.05) (around 2.7 and 5.2 -fold change to vehicle, respectively), which are blocked by a selective adenosine A2B receptor antagonist, PSB 603. This current study identifies the adenosine A2B receptors as a significant regulator of PGC-1α, NR4A1 and NR4A3 mRNA gene expression in skeletal muscle, thereby pointing to its therapeutic potential. In summary, it has been observed the selective, potentially functional expression of adenosine A2 receptors in skeletal muscle cells. Whether adenosine A2 receptor mediated functional responses play a role in skeletal muscle pathophysiology is yet to be elucidated.
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and cancer patients is mediated by tumor-derived inflammatory factors and growth factors that accumulate in the tumor environment [24, 26]. MDSCs in turn produce immunosuppressive and pro-angiogenic factors which promote further recruitment of MDSCs into tumor lesions. Hypoxia induces production of adenosine, which directly inhibits T cells functions and induces Tregs in the tumor microenvironment via A2A receptor activation [6, 47]. Recent evidence has emerged that adenosine/adenosine receptors pathways have a critical role in controlling MDSC numbers in tumors. A positive feedback between hypoxia, adenosine and MDSC has been also delineated. MDSCs produce extracellular adenosine  that can in turn regulate the expansion/recruitment of additional MDSCs within tumor microenvironment through the A2B receptor [13, 22]. A similar positive feedback exists between hypoxia, VEGF and MDSC . In this study, we show that A2B stimulation promotes intratumoral VEGF production from endothelial cells, which express A2B receptor, while melanoma cells do not. In addition, our data indicate that MDSC, which accumulated into tumor lesions upon A2B stimulation, contribute to release VEGF (Figure 7). We are currently investigating the possible role of tumor-associated fibroblasts, which are abundant in
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Others have shown that ADO, per se, also inhibits T cell proliferation and reduces the synthesis of IL-2 [36, 56, 72, 73], while can increase the numbers of T regulatory cells through an increase in IL-10 at the inflamed site, which can inhibit the activity of effector T cells . Moreover, the inhibition of T cell proliferation by ADO has been shown to occur via activation of the A2b receptor [75, 76]. Related to the role of A3 receptors in inflammation there is still a controversial debate. Some studies found that A3 receptor activation can improve inflammatory mediators release and degranulation in mast cells, suggesting a pro-inflammatory role [77, 78]. Other works have shown that its activation can suppress TNF-α release from LPS-stimulated macrophages and superoxide production and chemotaxis of neutrophils besides its com- petence to suppress the proliferation T cells [34, 78–81]. Interestingly, a recent study indicates a common associ- ation of the A3 receptor with potent analgesic effect in rodent models of chronic pain . Maybe tick saliva’s ADO signs via A3 receptor in the early phase of feeding, so it can get its blood meal properly by blocking pain.
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Confocal microscopy immunofluorescence analysis was performed on frozen sections of melanoma tissue or fibroblasts isolated from either melanoma or skin tissues. Frozen sections of melanoma tissue, fixed in 4% paraformaldehyde (PFA), were incubated with 20% goat serum containing 0.5% TritonX-100 for 30 minutes at room temperature to block nonspecific binding sites. Fibroblasts plated on poly-lysine-coated slides were washed twice in PBS, fixed in 4% PFA, and nonspecific binding sites were blocked using 20% goat serum or 5% bovine serum albumin (BSA) for 30 minutes at room temperature, as appropriate. Slides were stained overnight with the following primary antibodies: anti-FAPα (H-56) or anti-FGF-2 (147) (1:100; Santa Cruz Biotechnology) were detected with Alexa Fluor® 488 or 555 Goat Anti-Rabbit IgG (H+L) (1:5000) secondary antibodies (Life Technologies, Italy); anti- CXCL12 (1:100; Santa Cruz Biotechnology) (clone P-159X) was detected with the secondary antibody Alexa Fluor® 488 Goat Anti-Mouse IgG (H+L) (1:5000); anti- vimentin (E-5) (1:500; Santa Cruz Biotechnology) or anti- fibronectin (A17) (1:200; Abcam) were detected with the secondary antibody Alexa Fluor® 555 Goat Anti-Mouse IgG (H+L) (1:5000); anti-CXCR4 (clone L276F12) (1:100; BioLegend) was detected with Alexa Fluor® 555 Goat Anti-Rat IgG (H+L) (1:5000); FITC-conjugated anti-CD31 (MEC 13.3) (1:100, BioLegend) was also used. Primary antibody for A2B receptor: A2B-R (N-19) (1:50; Santa Cruz Biotechnology) was detected with the secondary antibodies DyLight™ 549-conjugated AffinePure Donkey anti-goat IgG (H+L) (Jackson ImmunoResearch Lab, UK). DAPI was used to counterstain nuclei. In all staining experiments, isotype-matched IgG and omission of the primary Ab were used as controls. Slides were observed using a Zeiss LSM 710 Laser Scanning Microscope (Carl Zeiss MicroImaging GmbH). Samples were vertically scanned from the bottom of the coverslip with a 63 × or 40 x (1.40 NA) Plan-Apochromat oil-immersion objective. Images were generated with Zeiss ZEN Confocal Software (Carl Zeiss MicroImaging GmbH).
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reuptake into proximal tubule cells by equilibrative nucleotide transporter 1, which can be inhibited by dipyridamole. The work suggests that adenosine A2B receptor agonists and inhibition of equilibrative nucleoside transporters by dipyridamole may have therapeutic potential in ischemic acute kidney injury, a condition for which there are currently no specific therapeutic interventions.
Adenosine-mediated enhancement of ciliary beat frequency is due to A2B receptor activation. (A) Histogram showing that ciliary beat frequency enhancement was not eliminated by pre-incubation with a cocktail of CNT and ENT inhibi- tors (1 mM phloridzin, 100 μM dipyridamole, 10 μM NBMPR; n = 5). (B) Summary histogram showing that ciliary beat fre- quency did not increase in response to a no drug control (Cont., n = 10), nor selective concentrations of the A 2A agonist CGS 21680 (100 nM, n = 5), the A 1 agonist 2'MeCCPA (100 nM, n = 5), nor the A 3 agonist IB-MECA (100 nM, n = 5). There was no significant difference between the no drug control and CGS 21680, 2'MeCCPA, or IB-MECA. (C) Histogram demonstrating that the response to 1 μM NECA was significantly reduced by the A 2B antagonist MRS 1754 (100 nM, n = 9) and eliminated by
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COS-7 cells that possess A2b receptors were used in view of the difficulty of transfecting T84 cells. It has been shown previously that the A2b receptors in COS- 7 cells, like those in the T84 cells, positively couple to adenylate cyclase (31). Stimulation of COS-7 cells with adenosine thus results in increased levels of intracellu- lar cAMP, and this forms the major signaling pathway induced by adenosine via the A2b receptor. In addition, COS 7 cells secrete IL-6 (unstimulated: 36.8 ± 3.5; adenosine 100 µM: 155.5 ± 25.0 pg/ml). As seen in Fig- ure 4b, adenosine induced approximately a 40-fold increase in CAT activity in the cells transfected with the full-length IL-6 promoter construct (+15 to –435). Mutations in either the region of the ATF/CREB or NF–IL-6 elements of the IL-6 promoter abolished responsiveness to adenosine (Figure 4b) while muta- tion of the NF- κ B site did not affect the CAT activity in response to adenosine. Such data imply that both the ATF/CREB and the NF–IL-6 sites are important for adenosine induced IL-6 secretion.
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Infection by human herpesvirus 8 (HHV-8) is associated with the development of Kaposi’s sarcoma (KS). Since regression of KS can be achieved by treatment of the patients with alpha interferon (IFN- a ), we analyzed the effects of IFN- a or anti-IFN- a antibodies (Ab) on HHV-8 latently infected primary effusion lymphoma- derived cell lines (BCBL-1 and BC-1) and on peripheral blood mononuclear cells (PBMC) from patients with all forms of KS and from at-risk subjects. IFN- a inhibited in a dose-dependent manner the amplification of HHV-8 DNA in BCBL-1 cells induced to lytic infection with tetradecanoyl phorbol acetate (TPA). This effect was associated with the inhibition of the expression of HHV-8 nut-1 and kaposin genes that are induced early and several hours, respectively, after TPA treatment. In addition, IFN- a inhibited virus production and/or release from BCBL-1 cells. Inhibition of nut-1 and kaposin genes by IFN- a was also observed in BC-1 cells induced with n-butyrate. Conversely, the addition of anti-IFN- a Ab to TPA-induced BCBL-1 cells resulted in a larger number of mature enveloped particles and in a more extensive cytopathic effect due to the neutral- ization of the endogenous IFN produced by these cells. IFN was also produced by cultured PBMC from HHV-8-infected individuals, and this was associated with a loss of viral DNA during culture. However, the addition of anti-IFN- a Ab or anti-type I IFN receptor Ab promoted the maintenance of HHV-8 DNA in these cells that was associated with the detection of the latency-associated kaposin RNA. Finally, the addition of IFN- a reduced the HHV-8 load in PBMC. Thus, IFN- a appears to have inhibitory effects on HHV-8 persistent infection of PBMC. These results suggest that, in addition to inhibiting the expression of angiogenic factors that are key to KS development, IFN- a may induce KS regression by reducing the HHV-8 load and/or inhibiting virus reactivation.
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The adenosine receptors were identified as described earlier 27 , with modifications. Muller cells were seeded (50,000/well) onto six-well tissue culture plates contai n- ing sterile coverslips in a medium containing low gl ucose (5 mM) and high glucose (25 mM) and allowed to attach and proliferate in the respective medium for 2 days. The cells were fixed with cold methanol for 10 min at –20C, rinsed twice with PBS, blocked at room temperature with 0.5% Tween (v/v) and 2% BSA (w/v) in PBS (blocking solution) for 1 h and rinsed twice with PBS. The cells were incubated overnight with primary antibodies (anti- A1 receptor, anti-A2A receptor, anti-A2B receptor and anti-A3 receptor) in blocking solution 1 : 100. Goat anti- actin antibodies were used as positive control . The fol- lowing day, cells were washed twice with PBS and incu- bated for 1 h in a dark room with secondary antibodies conjugated with fluorescein isothiocyanate (FITC) in blocking solution at 1 : 200. After rinsing twice with PBS, the coverslips were mounted with antifading medium (ProLong Gold, Life Technologies, USA).
Western blot analysis of the netrin-1 receptor protein For biochemical studies, the eyeballs were removed after neck dislocation on P12, P17, and P21, and the animals with weight >6 g were used. The retinas were dissected and placed in cold PBS on ice, carefully soaked with a piece of filter paper, and collected in 1.5-ml test tubes and weighed. They were dissected and lysed in radio- immunoprecipitation assay (RIPA) lysis buffer (Pierce, Rockford, IL, USA). Protein concentrations were de- termined with a protein assay reagent (Pierce, Rockford, IL, USA), using the bicinchoninic acid assay method. For each sample, 100 μg protein was fractionated using 10% sodium dodecyl sulfate polyacrylamide gel electro- phoresis, and transferred onto polyvinylidene mem- branes (Pierce). The membranes were incubated with anti-UNC5B (1:100), anti-UNC5C (1:100), anti-UNC5D (1:100), anti-DCC (1:50), anti-A2b(1:100), or anti-neogenin (1:100) for 2 hours at room temperature, then incu- bated with a specific secondary antibody (horseradish peroxidase-conjugated anti-immunoglobulin G). Bind- ing was detected using an enhanced chemiluminescence (ECL) method.
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During normal pregnancy, extravillous trophoblast cells invade the maternal decidua, replace the vascular endothelium and become embedded into the arterial walls. The mechanisms involved in the invasion of trophoblast cells during implantation are not fully understood . One goal of this study was to understand the interaction between trophoblast and endothelial cells. There are very complex mechanisms involved, including various processes e.g. proliferation, migration and the activation of different signals. Therefore, we used an co-culture assay of tropho- blast integration into an endothelial cell monolayer, instead of an in vitro invasion monoculture assay. In this study, we found that activation of the adenosine A 2B receptor leads to
Our former studies have shown that hIFN-a2b from IFN-transformed B. longum is expressed mainly as a mature secretory cytokine and that serum hIFN-a2b level can be enhanced in the mice that have been admini- strated orally with B. longum [12,13]. As we know, the expression of hIFN-a2b in IFN-transformed B. longum is mainly induced by L-arabinose, which is a component of biopolymers such as hemicellulose and pectin . The administration of IFN-transformed B. longum has been demonstrated to increase the serum and intestinal IFN- a2b level and we hypothesize that IFN expression by this bifidobacteria in mice might be induced persistently by L-arabinose in MRS or by the administered food and then enter the blood circulation by gastrointestinal absorption . In this study, we compared mice either treated with saline and control B. longum mice respec- tively. The Mx1 mRNA levels, which represent the local tissue IFN concentration, were increased significantly in cardiac tissues in the BIFN group, which suggested that IFN-transformed B. longum can increase the level of active type 1 IFN locally. Further study is needed to ascertain how to control the expression of IFN stably in gut and whether these bacteria affect the microbial flora.
A key therapeutic goal in the treatment of IPF is to reduce tissue fibrosis, which may be achieved by enhan- cing signals that counteract fibrotic processes, and/or in- hibit signals and mediators that promote fibrosis. There are at present two drugs that are now approved for use in the treatment of IPF; nintedanib and pirfenidone. Nintedanib inhibits multiple receptor tyrosine kinases (RTK)  and pirfenidone has broad anti-inflammatory/ anti-transforming growth factor β (TGFβ) activity [5, 6]. Although both nintedanib and pirfenidone have been shown to be efficacious in slowing the progression of disease [7–10], their non-specific action leads to severe adverse effects such as diarrhoea, dyspnoea, vomiting and weight loss, that are dose limiting thereby restricting their use to the more severe patients. Research to iden- tify alternative pathways in IPF treatment is essential to enable the development of safer, more efficacious and better tolerated drugs.
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DISCUSSION: In the present study, stable adenosine analogue modulates metabolic gene expression levels in cultures of rat skeletal muscle over a period of one-several hours. This effect is apparently mediated by at least A2B receptor subtypes and involves the activation of genes encoding proteins well known for their role in the regulation of glycolysis, glucose oxidation, insulin resistance and metabolism. The present pharmacological study is based on the use of a selective adenosine A2A receptor agonist, CGS 21680, and selective antagonist of adenosine A2B receptors, PSB 603. Hence, the lack of effect of CGS 21680 provides compelling evidence for the involvement of adenosine A2B receptors in the action of NECA. The results of the present study have shown for the first time that stable adenosine analogue alters the expression of genes involved in glycolysis (HKII and PFK). Moreover, this is the first study demonstrating that selective adenosine A2B receptor antagonist/inverse agonist, PSB 603 alters the expression of genes involved in glycolysis (HKII and PFK), and insulin resistance/glucose oxidation (PDK4). To our knowledge, this is the first study demonstrating an alteration of PFK gene by selective adenosine A2A receptors agonist, CGS 21680.
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adenosine receptor–knockout/reporter gene–knock-in (A 2B AR-knockout/reporter gene– knock-in) mouse model and showed receptor gene expression in the vasculature and macrophages, the ablation of which causes low-grade inflammation compared with age-, sex-, and strain-matched control mice. Augmentation of proinflammatory cytokines, such as TNF-a, and a consequent downregulation of IkB-a are the underlying mechanisms for an observed upregulation of adhesion molecules in the vasculature of these A 2B AR-null mice. Intriguingly, leukocyte adhesion to the vasculature is significantly increased in the A 2B AR- knockout mice. Exposure to an endotoxin results in augmented proinflammatory cytokine levels in A 2B AR-null mice compared with control mice. Bone marrow transplantations indicated that bone marrow (and to a lesser extent vascular) A 2B ARs regulate these processes. Hence, we identify the A 2B AR as a new critical regulator of inflammation and vascular adhesion primarily via signals from hematopoietic cells to the vasculature, focusing attention on the receptor as a therapeutic target.
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To examine whether the effect of adenosine is to increase the expression of HSPC genes or the number of HSPCs, we added an adenosine receptor agonist and antagonist to Tg(cmyb: eGFP; flk1:mcherry) embryos, in which HSCs and progenitors are marked by coexpression of both fluorescent proteins (Yue et al., 2012). We found that the number of HSPCs in these embryos was significantly increased after NECA treatment (4.4 ± 0.4/somite vs. 3.6 ± 0.2/somite [con]) and significantly decreased after CGS15943 exposure (3.0 ± 0.2/somite; Fig. 1, D–H). These data indicate that adenosine increases HSPC numbers rather than acting on gene expression alone. There- fore, adenosine regulates definitive hematopoiesis during de- velopment, and likely via an effect on an adenosine receptor.
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