Adenosine Concentration Research Articles

Metabolic Aspects of Adenosine Functions in the Brain

Adenosine, acting both through G-protein coupled adenosine receptors and intracellularly, plays a complex role in multiple physiological and pathophysiological processes by modulating neuronal plasticity, astrocytic activity, learning and memory, motor function, feeding, control of sleep and aging. Adenosine is involved in stroke, epilepsy and neurodegenerative pathologies. Extracellular concentration of adenosine in the brain is tightly regulated. Adenosine may be generated intracellularly in the central nervous system from degradation of AMP or from the hydrolysis of S-adenosyl homocysteine, and then exit via bi-directional nucleoside transporters, or extracellularly by the metabolism of released nucleotides. Inactivation of extracellular adenosine occurs by transport into neurons or neighboring cells, followed by either phosphorylation to AMP by adenosine kinase or deamination to inosine by adenosine deaminase. Modulation of the nucleoside transporters or of the enzymatic activities involved in the metabolism of adenosine, by affecting the levels of this nucleoside and the activity of adenosine receptors, could have a role in the onset or the development of central nervous system disorders, and can also be target of drugs for their treatment. In this review, we focus on the contribution of 5′-nucleotidases, adenosine kinase, adenosine deaminase, AMP deaminase, AMP-activated protein kinase and nucleoside transporters in epilepsy, cognition, and neurodegenerative diseases with a particular attention on amyotrophic lateral sclerosis and Huntington’s disease. We include several examples of the involvement of components of the adenosine metabolism in learning and of the possible use of modulators of enzymes involved in adenosine metabolism or nucleoside transporters in the amelioration of cognition deficits.

Adenosine-regulated mechanisms in the pathogenesis of ventilation disorders in patients with pulmonary tuberculosis

Uncovering involvement of the purinergic system in the pathogenesis of ventilation disorders (VD) may provide additional information about the pathophysiological mechanisms leading to the development of VD in pulmonary tuberculosis (PT). The aim was to identify a relationship between the parameters of adenosine metabolism, inflammatory response and altered ventilation metabolism in PT patients. Materials and methods. Obstructive and mixed PT patients were assigned to subgroups with/without VD for assessing adenosine deaminase activity (ADA-1, 2) in serum, mononuclear cells, neutrophils; ecto-5’-nucleotidase (ecto-5’-NT); CD26 (dipeptidyl peptidase-4, DPP-4), phagocyte oxidative burst measured by NO generation. Results. PT patients showed decreased ADA-1 and CD26 (DPP-4), but increased ADA-2. Elevated intracellular adenosine concentration was found in mononuclear cells in patients lacking VD, whereas patients with mixed and obstructive VD — had it in neutrophils. Mononuclear cells of patients with PT lacking VD as well as with obstructive VD type had decreased NO3– concentration. Neutrophil hyperactivity was recorded in all groups of PT patients. Patients with PT lacking VD as well as with mixed VD type showed that the parameters of external respiration were associated with activity of extra-/intracellular ADA, whereas obstructive VD was caused by excessive formation of serum adenosine. Changes in respiratory function in PT were associated with decreased level of serum NO radicals, impaired nitrogen-dependent bactericidal phagocyte activity, and overproduced neutrophil oxygen radicals. Conclusion. Purinergic regulation is involved in regulating inflammatory and compensatory processes in PT patients as well as impaired ventilation efficiency. The most severe respiratory disorders observed in PT patients with mixed VD type are associated with the most prominent changes in nucleotidase activity, particularly ecto-ADA-2 and DPP-4/CD26.

Identification of Allosteric Inhibitors of CD73 Using Three‐Phrase Computational Protocol for Drug Discovery

A three-phase computational protocol for drug discovery has been developed that includes 1) 3D shape characterization of binding sites of a protein to predict optimum performance with selected molecular docking programs; 2) Implementation of predictive algorithms for pharmacokinetic properties and ADME profiling for targeted searches in the ZINC15 database; 3) Flexible molecular docking/dynamics protocols of small molecule/protein complexes for drug discovery. Focusing on cancer immunotherapy to implement this computational approach, the allosteric site of ecto-5’-nucleotidase (CD73), a regulator of the extracellular concentration of adenosine, is targeted for large scale small molecule searches. CD73 which hydrolyzes AMP to adenosine, is a GPI-anchored cell surface protein cleaved and activated in the extracellular environment as a homodimer. CD73 exhibits higher expression on lymphocytes, endothelial, and epithelial cells during a stress response and his been reported activated for various types of cancer. CD73 participates in blocking antitumor responses and enhances tumor growth and metastasis by producing high levels of adenosine in the extracellular environment. Protein coordinates of CD73 were downloaded for these from Rutgers Consortium of Structural Biology (RCSB) Protein Data Bank (PDB) and the potential inhibitory molecules from the ZINC15 Database. AutoDock Vina was selected as the ideal docking program for the target based on comparing predicting binding constants with actual Ki's and RMSD analysis of the co-crystallized ligand with the re-docked ligand. Once molecules with high affinity predicted scores were identified from docking, molecular dynamics studies were performed on the highest scoring protein/small molecule complexes. Studies were carried out using GROMACS, implemented with AMBER force field. Dissociation constants (Ki's) for the protein-ligand complex were calculated throughout MD runs. Conformational changes in the binding sites were observed visually and quantitified with PCA analysis. Novel hydrogen bonding networks and hydrophobic networks of residues in the 3D structures were identified to connect the active sites to the allosteric binding site using a MD protocol.

Release of adenosine-induced immunosuppression: Comprehensive characterization of dual A2A/A2B receptor antagonist

Immunosuppression is one of the main mechanisms facilitating tumor expansion. It may be driven by immune checkpoint protein expression, anti-inflammatory cytokine secretion or enhanced metabolic enzyme production, leading to the subsequent build-up of metabolites such as adenosine. Under physiological conditions, adenosine prevents the development of tissue damage resulting from a prolonged immune response; the same mechanism might be employed by tumor tissue to promote immunosuppression. Immune cells expressing A2A and A2B adenosine receptors present in an adenosine-rich environment have suppressed effector functions, such as cytotoxicity, proinflammatory cytokine release, antigen presentation and others, making them inert to cancer cells. This study was designed to investigate the dual antagonist potential of SEL330-639 to abolish adenosine-driven immunosuppression. SEL330-639 has slow dissociation kinetics. It inhibits cAMP production in human CD4+ cells, CD8+ cells and moDCs, which leads to diminished CREB phosphorylation and restoration of antitumor cytokine production (IL-2, TNFα, IL-12) in multiple primary human immune cells. The aforementioned results were additionally validated by gene expression analysis and functional assays in which NK cell line cytotoxicity was recovered by SEL330-639. Adenosine-driven immunosuppression is believed to preclude the effectiveness of immune checkpoint inhibitor therapies. Hence, there is an urgent need to develop new immuno-oncological strategies. Here, we comprehensively characterize SEL330-639, a novel dual A2A/A2B receptor antagonist effective in both lymphoid and myeloid cell populations with nanomolar potency. Due to its tight binding to the A2A and A2B receptors, this binding is sustained even at high adenosine concentrations mimicking the upper limit of the range of adenosine levels observed in the tumor microenvironment.

ENTPD1 (CD39) Expression Inhibits UVR-Induced DNA Damage Repair through Purinergic Signaling and Is Associated with Metastasis in Human Cutaneous Squamous Cell Carcinoma

UVR and immunosuppression are major risk factors for cutaneous squamous cell carcinoma (cSCC). Regulatory T cells promote cSCC carcinogenesis, and in other solid tumors, infiltrating regulatory T cells and CD8+ T cells express ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1) (also known as CD39), an ectoenzyme that catalyzes the rate-limiting step in converting extracellular adenosine triphosphate (ATP) to extracellular adenosine (ADO). We previously showed that extracellular purine nucleotides influence DNA damage repair. In this study, we investigate whether DNA damage repair is modulated through purinergic signaling in cSCC. We found increased ENTPD1 expression on T cells within cSCCs when compared with the expression on T cells from blood or nonlesional skin, and accordingly, concentrations of derivative extracellular adenosine diphosphate (ADP), adenosine monophosphate (AMP), and ADO are increased in tumors compared with those in normal skin. Importantly, ENTPD1 expression is significantly higher in human cSCCs that metastasize than in those that are nonmetastatic. We also identify in a mouse model that ENTPD1 expression is induced by UVR in an IL-27-dependent manner. Finally, increased extracellular ADO is shown to downregulate the expression of NAP1L2, a nucleosome assembly protein we show to be important for DNA damage repair secondary to UVR. Together, these data suggest a role for ENTPD1 expression on skin-resident T cells to regulate DNA damage repair through purinergic signaling to promote skin carcinogenesis and metastasis.

P2X7 Receptors in Neurodegeneration: Potential Therapeutic Applications From Basic to Clinical Approaches.

Purinergic receptors play important roles in central nervous system (CNS), where the bulk of these receptors are implicated in neuroinflammatory responses and regulation of cellular function of neurons, microglial and astrocytes. Within the P2X receptor family, P2X7 receptor is generally known for its inactivity in normal conditions and activation by moderately high concentrations (>100 μM) of extracellular adenosine 5′-triphosphate (ATP) released from injured cells as a result of brain injury or pathological conditions. Activation of P2X7R contributes to the activation and proliferation of microglia and directly contribute to neurodegeneration by provoking microglia-mediated neuronal death, glutamate-mediated excitotoxicity, and NLRP3 inflammasome activation that results in initiation, maturity and release of the pro-inflammatory cytokines and generation of reactive oxygen and nitrogen species. These components of the inflammatory response play important roles in many neural pathologies and neurodegeneration disorders. In CNS, expression of P2X7R on microglia, astrocytes, and oligodendrocytes are upregulated under neuroinflammatory conditions. Several in vivo studies have demonstrated beneficial effects of the P2X7 receptor antagonists in animal model systems of neurodegenerative diseases. A number of specific and selective P2X7 receptor antagonists have been developed, but only few of them have shown efficient brain permeability. Finding potent and selective P2X7 receptor inhibitors which are also CNS penetrable and display acceptable pharmacokinetics (PK) has presented challenges for both academic researchers and pharmaceutical companies. In this review, we discuss the role of P2X7 receptor function in neurodegenerative diseases, the pharmacological inhibition of the receptor, and PET radiopharmaceuticals which permit non-invasive monitoring of the P2X7 receptor contribution to neuroinflammation associated with neurodegeneration.

Safety and Efficacy of Aminophylline in Intrathecal Methotrexate-related Neurological Toxicity in Large Pediatric Oncology Centre.

Children with acute lymphoblastic leukemia, receiving intrathecal methotrexate (IT-MTX), develop acute and subacute neurological dysfunction in 3-15% of cases. Altered level of consciousness, seizure, and stroke-like manifestations are among the most common presentations. MRI of the brain in an early stage is consistent with white matter leukoencephalopathy. There are no specific guidelines for the treatment of such a syndrome. An elevated cerebrospinal fluid (csf) adenosine concentration causes vasodilatation in the brain and leads to cerebral ischemia, which may be reduced by aminophylline infusion. The study is a retrospective data analysis in which electronic data records of 30 patients, collected from September 2017 - August 2019. The primary objective of the study was to evaluate the safety and efficacy of aminophylline in IT-MTX induced neurotoxicity. Aminophylline infusion was used in 30 patients with IT-MTX neurotoxicity. Twenty-five patients (83.33%) showed dramatic improvement of neurologic signs and symptoms within 48 hours. Aminophylline was, hence, coined as a salvageable therapy. No noticeable side effects were observed during treatment with aminophylline infusion. Key Words: Intrathecal methotrexate, Neurotoxicity, Children, Cancer, Aminophylline.

BIOLUMINOMETRIC ASSAY OF CELL ATP DETECTION UNDER STRESS CONDITIONS AND IN THE ASSESSMENT OF CLEANNESS OF THE FOOD PRODUCING COMPANY

Detection and tracking of microorganism’s behavior in natural environments and under stressed conditions have been nowadays accelerated by the requirement for the risk assessment associated with environmental contamination. Luminescence-based techniques, for they cost efficiency, fastness, reliability and reproducibility are particularly appropriate for such studies. We used bioluminescence sensing assay to determine ATP levels in the yeast Schizosaccharomyce pombe. Determination of adenosine concentration per cell was assessed in the yeast cells counted prior to the experiment. To test the methodology, model organism was exposed to life-threatening concentration of heavy metals cadmium and nickel for three hours. Afterwards, changes in the ATP levels were detected and compared to the control. Strikingly, our results revealed that such conditions lead to considerable increase in the production of ATP by mitochondria compared to untreated cells. Normal level of ATP in the control increased from the basal 9.92x10-16 M to 2.90x10-15 M after Cd exposure and to 7.58x10-15 M upon Ni treatment. The methodology was subsequently used for assessment of cleanness of margarine producing company devices after regular sanitation procedure. Detection of only negligible amount of the ATP signal confirmed good cleaning strategy in the tested food processing company.

Simultaneous inhibition of CD73 and IL-6 molecules by siRNA-loaded nanoparticles prevents the growth and spread of cancer

High concentrations of adenosine and interleukin (IL)-6 in the tumor microenvironment have been identified as one of the leading causes of cancer growth. Thus, we decided to inhibit the growth of cancer cells by inhibiting the production of adenosine and IL-6 in the tumor environment at the same time. For this purpose, we used chitosan-lactate-PEG-TAT (CLP-TAT) nanoparticles (NPs) loaded with siRNA molecules against CD73, an adenosine-producing enzyme, and IL-6. Proper physicochemical properties of the produced NPs led to high cell uptake and suppression of target molecules. Administration of these NPs to tumor-bearing mice (4T1 and CT26 models) greatly reduced the size of the tumor and increased the survival of the mice, which was accompanied by an increase in anti-tumor T lymphocyte responses. These findings suggest that combination therapy using siRNA-loaded CLP-TAT NPs against CD73 and IL-6 molecules could be an effective treatment strategy against cancer that needs further study.

A bifunctional electrochemical aptasensor based on AuNPs-coated ERGO nanosheets for sensitive detection of adenosine and thrombin

A simple and sensitive bifunctional electrochemical aptasensor for detection of adenosine and thrombin has been developed using gold nanoparticles–electrochemically reduced graphene oxide (AuNPs-ERGO) composite film-modified electrode. Firstly, the reduced graphene oxide film and AuNPs were sequentially immobilized on glassy carbon electrode (GCE) surface. Secondly, thrombin aptamer was immobilized on the modified electrode. Finally, adenosine aptamer was hybridized with it to serve as a recognition element and methylene blue (MB) as electrochemical signal indicator. In the presence of adenosine or thrombin, the sensor recognized it and a conformational change was induced in aptamer, resulting in decrease of the peak current of MB. The linear relation between concentration of adenosine or thrombin and peak current of MB allowed quantification of them. Thanks to the special electronic characteristic of AuNPs-ERGO composite film, sensitivity of sensor was greatly improved. Under optimal conditions, the proposed aptasensor presented an excellent performance in a linear range of 25 nM to 750 nM for adenosine and 0.5 nM to 10 nM for thrombin. Detection limits were estimated to be 8.3 nM for adenosine and 0.17 nM for thrombin, respectively. Moreover, dual-analyte detection of adenosine and thrombin was achieved without potentially increasing the complexity and cost of the assay.

Relation between coffee consumption and risk of seizure‐related respiratory dysfunction in patients with drug‐resistant focal epilepsy

Caffeine is an antagonist of the adenosine pathway, which is involved in regulation of breathing. Extracellular concentrations of adenosine are increased in the immediate aftermath of a seizure. Seizure-related overstimulation of adenosine receptors might promote peri-ictal apnea. However, the relation between caffeine consumption and risk of seizure-related respiratory dysfunction in patients with drug-resistant focal epilepsy remains unknown. We performed a cross-sectional analysis of data collected in patients included in the SAVE study in Lyon's epilepsy monitoring unit at the Adult Epilepsy Department of the Lyon University Hospital between February 2016 and October 2018. The video-electroencephalographic recordings of 156 patients with drug-resistant focal epilepsy included in the study were reviewed to identify those with ≥1 focal seizure (FS), valid pulse oximetry (SpO2 ) measurement, and information about usual coffee consumption. This latter was collected at inclusion using a standardized self-questionnaire and further classified into four groups: none, rare (≤3 cups/week), moderate (4 cups/week to 3 cups/day), and high (≥4 cups/day). Peri-ictal hypoxemia (PIH) was defined as SpO2 < 90% for at least 5s occurring during the ictal period, the post-ictal period, or both. Ninety patients fulfilled inclusion criteria, and 323 seizures were analyzed. Both the level of usual coffee consumption (p=.033) and the level of antiepileptic drug withdrawal (p=.004) were independent risk factors for occurrence of PIH. In comparison with FS in patients with no coffee consumption, risk of PIH was four times lower in FS in patients with moderate consumption (odds ratio [OR]=.25, 95% confidence interval [CI]=.07-.91, p=.036) and six times lower in FS in patients with high coffee consumption (OR=.16, 95% CI=.04-.66, p=.011). However, when PIH occurred, its duration was longer in patients with moderate or high consumption than in those with no coffee consumption (p=.042). Coffee consumption may be a protective factor for seizure-related respiratory dysfunction, with a dose-dependent effect.

Changed signals of blood adenosine and cytokines are associated with parameters of sleep and/or cognition in the patients with chronic insomnia disorder

ObjectivesThis study aimed to investigate whether plasma levels of adenosine, adenosine deaminase (ADA), and certain cytokines change in patients with chronic insomnia disorder (CID), and if so, whether these alterations are associated with poor sleep quality and cognitive dysfunction. MethodsFifty-five CID patients were selected for the study, along with fifty-five healthy controls (HC) matched to the patients according to their basic data. All subjects completed sleep, emotion, and cognition assessments, with some CID patients also completing an overnight polysomnography. The plasma level of adenosine was measured using liquid chromatography–tandem mass spectrometry, while ADA level was quantified using a quantitative sandwich enzyme-linked immunosorbent assay. Levels of cytokines, including IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, TNF-α, and IFN-γ, were measured using Luminex liquid chip technology. ResultsCID patients had a lower adenosine level, and higher levels of ADA and some of the cytokines (IL-1β, IL-2, IL-6, IL-10 and TNF-α) compared with controls. In the CID group, plasma concentrations of adenosine were negatively correlated with Pittsburgh Sleep Quality Index scores, while concentrations of IL-1β, IL-6 and TNF-α were positively correlated with these scores. Concentrations of IL-1β and TNF-α were negatively correlated with scores on the Chinese-Beijing Version of the Montreal Cognitive Assessment. Moreover, levels of IL-1β, TNF-α, IL-6, and IL-2 were positively correlated with memory test errors by CID patients after controlling for confounding factors. ConclusionsThe reduced adenosine and elevated cytokine levels of CID patients were associated with the severity of insomnia and/or cognitive dysfunction.

Inhibition of adenosine kinase attenuates myocardial ischaemia/reperfusion injury.

Increased adenosine helps limit infarct size in ischaemia/reperfusion‐injured hearts. In cardiomyocytes, 90% of adenosine is catalysed by adenosine kinase (ADK) and ADK inhibition leads to higher concentrations of both intracellular adenosine and extracellular adenosine. However, the role of ADK inhibition in myocardial ischaemia/reperfusion (I/R) injury remains less obvious. We explored the role of ADK inhibition in myocardial I/R injury using mouse left anterior ligation model. To inhibit ADK, the inhibitor ABT‐702 was intraperitoneally injected or AAV9 (adeno‐associated virus)—ADK—shRNA was introduced via tail vein injection. H9c2 cells were exposed to hypoxia/reoxygenation (H/R) to elucidate the underlying mechanisms. ADK was transiently increased after myocardial I/R injury. Pharmacological or genetic ADK inhibition reduced infarct size, improved cardiac function and prevented cell apoptosis and necroptosis in I/R‐injured mouse hearts. In vitro, ADK inhibition also prevented cell apoptosis and cell necroptosis in H/R‐treated H9c2 cells. Cleaved caspase‐9, cleaved caspase‐8, cleaved caspase‐3, MLKL and the phosphorylation of MLKL and CaMKII were decreased by ADK inhibition in reperfusion‐injured cardiomyocytes. X‐linked inhibitor of apoptosis protein (XIAP), which is phosphorylated and stabilized via the adenosine receptors A2B and A1/Akt pathways, should play a central role in the effects of ADK inhibition on cell apoptosis and necroptosis. These data suggest that ADK plays an important role in myocardial I/R injury by regulating cell apoptosis and necroptosis.

Seasonal changes in adenosine kinase in tanycytes of the Arctic ground squirrel (Urocitellus parryii)

Hibernation is a seasonal strategy to conserve energy, characterized by modified thermoregulation, an increase in sleep pressure and drastic metabolic changes. Glial cells such as astrocytes and tanycytes are the brain metabolic sensors, but it remains unknown whether they contribute to seasonal expression of hibernation. The onset of hibernation is controlled by an undefined endogenous circannual rhythm in which adenosine plays a role through the activation of the A1 adenosine receptor (A1AR). Seasonal changes in brain levels of adenosine may contribute to an increase in A1AR sensitivity leading to the onset of hibernation. The primary regulator of extracellular adenosine concentration is adenosine kinase, which is located in astrocytes. Using immunohistochemistry to localize and quantify adenosine kinase in Arctic ground squirrels’ brain collected during different seasons, we report lower expression of adenosine kinase in the third ventricle tanycytes in winter compared to summer; a similar change was not seen in astrocytes. Moreover, for the first time, we describe adenosine kinase expression in tanycyte cell bodies in the hypothalamus and in the area postrema, both brain regions involved in energy homeostasis. Next we describe seasonal changes in tanycyte morphology in the hypothalamus. Although still speculative, our findings contribute to a model whereby adenosine kinase in tanycytes regulates seasonal changes in extracellular concentration of adenosine underling the seasonal expression of hibernation.

Optimization of In Vitro Culture Conditions for Production of Cordyceps bassiana Spores (Ascomycetes) and the Effect of Spore Extracts on the Health of Caenorhabditis elegans.

Cordyceps bassiana, an entomogenous fungus, has been used as a dietary therapeutic in traditional Chinese medicine for several millennia. However, naturally occurring C. bassiana is notably scarce in the field, especially at the spore stage. To date, there are few comprehensive studies on the biological characteristics and pharmacological attributes of C. bassiana spores. In this study, an isolate of C. bassiana (Bs1112) was identified as the teleomorph of Beauveria bassiana using morphology and rDNA internal transcribed spacer sequences. In vitro solid substrate fermentation parameters were evaluated; a rice substrate with 0.01% K+ achieved the maximum spore yield. High-performance liquid chromatography analysis of the bioactive components of C. bassiana spore powder (BC-CBSP), which was extracted using alcohol, showed that concentrations of adenosine were higher than concentrations of inosine, uridine, and cordycepin. The pharmacological impact of BC-CBSP was elucidated in a live animal model, Caenorhabditis elegans, at a range of concentrations from 0.145 to 14.5 mg/mL. The reproductive rate, frequency of head swinging and body bending (indicative of C. elegans locomotion), and lifespan of C. elegans were determined and compared with untreated populations. At the lowest concentration of BC-CBSP, reproduction was slowed but the number of eggs was not reduced. However, at both of the higher concentrations (1.45 and 14.5 mg/mL), BC-CBSP reduced the total number of eggs produced during the 8-day observation period. Furthermore, 0.145 mg/mL of BC-CBSP significantly increased the frequency of head swinging/body bending and the lifespan of C. elegans. This is the first report on the pharmacological effects of C. bassiana spores. Results suggest that low concentrations of BC-CBSP have no negative effects on the reproduction rate of C. elegans but could enhance nervous system activity and extend their lifespan.

Ticagrelor alleviates sepsis-induced myocardial injury via an adenosine-dependent pathway in a mouse sepsis model.

The purpose of this study was to determine whether ticagrelor, a classic anti-platelet drug, has a therapeutic effect on sepsis-induced myocardial injury. The C57BL6J mice received oral ticagrelor (10, 25 and 50 mg/kg) for seven days after which cecum ligation and puncture (CLP) were performed. An adenosine-receptor antagonist (CGS15943) was administered two hours before CLP. After 24 h, cardiac function was measured using cardiac echocardiography, then the heart and blood were collected. Hematoxylin and eosin (HE) staining and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL staining) were used to observe pathological changes and cardiomyocyte apoptosis. Plasma concentration of TNF-α, IL-6 and adenosine and myocardial tissue levels of TNF-α and IL-6 were determined. Survival analysis was performed. Western blot was used to determine the expression of a signalling protein in the myocardial tissue. The HE and TUNEL staining showed less inflammatory cell infiltration and less cardiomyocyte apoptosis in the ticagrelor group. Cardiac echocardiography showed preserved heart function in the ticagrelor group. Plasma TNF-α, IL-6 and relative expression of TNF-α and IL-6 in myocardial tissue were significantly lower in the ticagrelor group. Plasma adenosine levels were significantly higher in the ticagrelor group. Adenosine-receptor antagonists significantly blocked the protective effect of ticagrelor. Ticagrelor reduced the mortality of sepsis mice, and this reduction was blocked by the adenosine-receptor antagonist. Western blot showed that ticagrelor activated the phosphorylation of AKT and mTOR. Adenosine-receptor antagonists inhibited the activation of AKT and mTOR. The protective effect of ticagrelor was dependent on adenosine-receptor activation, with downstream upregulation of phosphorylation of AKT and mTOR.

Study on adenosine loading capacity of porous nanosilica for application in drug delivery

Mesoporous silica nanoparticles (MSNs) are used as drug delivery materials because of their outstanding features such as large surface area, easy synthesis and high biocompability. In this study, inorganic mesoporous nanosilica material, MCM-41, was synthesized by sol-gel hydrothermal method using tetraethyl orthosilicate precursor (TEOS). The material structure and composition were analyzed by X-ray power diffraction (P-XRD), N2 adsorption isotherm, thermalgravimetric analysis (TGA) and Fourier-transform infrared spectroscopy (FT-IR). Its morphology was examined by scanning electron microscopy (SEM) and transmission electron microscope (TEM). The results showed that synthesized MCM-41 has a spherical shape, homogeneous with an average size of 100 nm. The specific surface area is 845 m2 g􀀀1, the pore size is approximately 35 Å. It has high thermal stability until 800oC. FT-IR result showed the formation of the Si-O-Si bond in the structure. The adenosine loading capacity of MCM-41 was investigated based on the influence of loading factors including adenosine concentration, time, solvent, and temperature. The kinetics and thermodynamics of the adsorption processes were also studied. The adenosine loading ability on MCM-41 is significant high, approximately 1699 mg g􀀀1. The kinetic and thermodynamic results showed that the drug adsorbed of MCM-41 occurred with fast rate and spontaneously. Moreover, the release profile of adenosine proved that the drug release process occurred quickly which is suitable for application in acute disease treatment.

TAMI-45. PHENOGENOMIC CHARACTERIZATION OF IMMUNOMODULATORY PURINERGIC SIGNALING IN GLIOBLASTOMA

Abstract INTRODUCTION Purinergic signaling plays critical roles in the regulation of tumor growth and anti-tumor immunity via autocrine/paracrine binding of metabolites to receptors on neoplastic and non-neoplastic populations. Extracellular purine concentrations are mediated by the ectonucleotidase enzymes CD39 and CD73, which catabolize ATP to adenosine. Within tumors such as glioblastoma, neoplastic, immune, and stromal cells expressing these enzymes may co-localize to generate immunosuppressive adenosine-rich environments. However, the composition, architecture, and phenotypic properties of these tumor ecosystems and their relationship to tumor genotype are poorly characterized. METHODS We quantified CD73 expression by immunohistochemistry in a cohort of CNS tumors [meningiomas(n=222), gliomas(n=244), ependymomas(n=44), medulloblastomas(n=24), and craniopharyngiomas(n=38)]. We used publicly-available single-cell RNA-seq data and 36-marker multiplexed tissue imaging (t-CyCIF) of 139 clinically and genomically annotated glioblastoma resections to characterize CD39 and CD73-expressing populations, define the immune architecture and tumor cell-states at single cell resolution, and identify markers of clinical outcome. We used mass spectrometry imaging (MALDI-MSI) to generate spatially-resolved quantification of purine metabolite levels in glioblastoma resections (n=10). RESULTS CD73 exhibited strong expression in a subset of gliomas and meningiomas but was typically not expressed in ependymomas or medulloblastomas. CD73 expression correlated with poor progression-free-survival in IDH-wildtype glioblastoma (p=0.04). scRNA-seq and t-CyCIF in glioblastoma showed CD73 expression in tumor cells, and CD39 expression in macrophages and endothelial cells. MALDI-MSI showed significantly greater adenosine concentrations (3.5-fold;p=0.04) in glioblastomas with high CD73 expression. scRNA-seq showed direct correlations between stem-like mRNA expression, proliferation, and CD73 expression in DIPG. CD73 expression significantly correlated with EGFR amplification, interferon signaling, and PD-L1 expression in glioblastoma. CONCLUSIONS Phenogenomic analysis of purinergic immunomodulatory signaling revealed significant interplay between CD73 activity and genotype, adenosine concentration, differentiation-state, clinical outcome, and possible interaction between CD39-positive macrophages and CD73-positive neoplastic cells. Anti-CD73 therapy may provide therapeutic benefits in glioblastoma by blunting immunosuppressive and oncogenic adenosine signaling.

Novel Pathway of Adenosine Generation in the Lungs from NAD+: Relevance to Allergic Airway Disease.

Adenosine and uric acid (UA) play a pivotal role in lung diseases such as asthma and chronic obstructive pulmonary disease (COPD). In the present experiments, we measured adenosine synthesis from nicotinamide adenine dinucleotide (NAD+) in membranes prepared from wild type (WT) and CD38 knockout (CD38KO) mouse lungs, from cultured airway smooth muscle and epithelial cells, and in bronchoalveolar lavage fluid after airway challenge with epidemiologically relevant allergens. Adenosine was determined using an enzymatically coupled assay that produces ATP and is detected by luminescence. Uric acid was determined by ELISA. Exposure of cultured airway epithelial cells to Alternaria alternata extract caused significant nucleotide (NAD+ and ATP) release in the culture media. The addition of NAD+ to membranes prepared from WT mice resulted in faster generation of adenosine compared to membranes from CD38KO mice. Formation of adenosine from NAD+ affected UA and ATP concentrations, its main downstream molecules. Furthermore, NAD+ and adenosine concentrations in the bronchoalveolar lavage fluid decreased significantly following airway challenge with house-dust mite extract in WT but not in CD38KO mice. Thus, NAD+ is a significant source of adenosine and UA in the airways in mouse models of allergic airway disease, and the capacity for their generation from NAD+ is augmented by CD38, a major NADase with high affinity for NAD+. This novel non-canonical NAD+-adenosine-UA pathway that is triggered by allergens has not been previously described in the airways.

A phosphodiesterase 11 (Pde11a) knockout mouse expressed functional but reduced Pde11a: Phenotype and impact on adrenocortical function

Phosphodiesterases catalyze the hydrolysis of cyclic nucleotides and maintain physiologic levels of intracellular concentrations of cyclic adenosine and guanosine mono-phosphate (cAMP and cGMP, respectively). Increased cAMP signaling has been associated with adrenocortical tumors and Cushing syndrome. Genetic defects in phosphodiesterase 11A (PDE11A) may lead to increased cAMP signaling and have been found to predispose to the development of adrenocortical, prostate, and testicular tumors. A previously reported Pde11a knockout (Pde11a−/−) mouse line was studied and found to express PDE11A mRNA and protein still, albeit at reduced levels; functional studies in various tissues showed increased cAMP levels and reduced PDE11A activity. Since patients with PDE11A defects and Cushing syndrome have PDE11A haploinsufficiency, it was particularly pertinent to study this hypomorphic mouse line. Indeed, Pde11a−/− mice failed to suppress corticosterone secretion in response to low dose dexamethasone, and in addition exhibited adrenal subcapsular hyperplasia with predominant fetal-like features in the inner adrenal cortex, mimicking other mouse models of increased cAMP signaling in the adrenal cortex. We conclude that a previously reported Pde11a−/− mouse showed continuing expression and function of PDE11A in most tissues. Nevertheless, Pde11a partial inactivation in mice led to an adrenocortical phenotype that was consistent with what we see in patients with PDE11A haploinsufficiency.