Abstracts of the Brazilian Purine Club Meeting : May 30 - July 2, 2015 Convention Center, Beach Hotel Maresias, City of São Sebastião - SP - Brazil.

Purinergic Signalling (2016) 12:343–410 DOI 10.1007/s11302-016-9503-x

ABSTRACT

Abstracts of the Brazilian Purine Club Meeting May 30 – July 2, 2015 Convention Center, Beach Hotel Maresias, City of São Sebastião - SP - Brazil

II. International Congress on Purinergic Signaling in South America V. Meeting of the Brazilian Purine Club May 30 – July 2, 2015 Convention Center, Beach Hotel Maresias, City of São Sebastião - SP - Brazil.

Guest Editors and Organizing Committee: Dr. Henning Ulrich, University of São Paulo Dr. Robson Coutinho Silva, Federal University of Rio de Janeiro Dr. Ana Lúcia Marques Ventura, Fluminense Federal University Dr. Zulma Felisbina da Silva Ferreira, University of São Paulo Dr. Flávia Sarmento Vieira, University of São Paulo

Sponsoring Funding Agencies: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Brazil

Additional Support: Alesco—Research for Life PeproTech Sinapse Biotecnologia Exxtend—Solução em Oligos Sarstedt Life Technologies Tissue Gnostics—Medical & Biotech Solutions

The Brazilian Purine Club and Introduction of the Event: The Brazilian Purine Club was founded in 2009 with the objective of gathering scientists interested in purinergic signaling in periodically organized scientific meetings. Four conferences held in the period from 2010 to 2013 in the Brazilian cities Àguas de Lindoia, Rio de Janeiro, Ouro Preto and Canela were successful considering the high scientific quality of the conferences, symposia and poster presentations and the ties formed between Brazilian students and researchers with their colleagues from abroad. New collaborations and student exchange were established, and the participation of the most renowned scientists in the field has given international visibility to this scientific community.

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Purinergic Signalling (2016) 12:343–410

The Brazilian society of purinergic signaling rapidly expands in view of new memberships, scientific publications and Ph.D. theses developed in the field. The II. International Congress on Purinergic Signaling in South America and V Brazilian Purine Cub Meeting was held from April 30 to May 2, 2015 in the coastal town Maresias in the State of São Paulo. The program was developed by the board of the Society (the President Dr. Henning Ulrich (University of São Paulo), the Vice-President Dr. Robson-Coutinho-Silva (Federal University of Rio de Janeiro) and the General Secretary Ana L. M. Ventura (Fluminese Federal University) with the support of the council and the local organizing committee headed by Dr. Zulma Felisbina da Silva Ferreira (University of São Paulo). Plenary lectures focused on biology and signaling of ectonucleotidases and P1 and P2 receptors and structural properties of these proteins. The conference was honored by an Opening Conference held by Prof. Geoffrey Burnstock, followed by six plenary lectures, 13 symposia, a session of oral communication and 88 presented posters. The VI. Meeting of the Brazilian Purine Club will be held from May 12–14, 2016 in the town of the João Pessoa in the State of Paraiba (information available at http://www.encontro2016.clubedepurinas.com.br/). The next of the biannual International Conferences (2014 in Bonn, Germany; 2016 in Vancouver, Canada) will be held in 2018 in Brazil.

The list of conferences and lectures are presented below:

Opening conference: Dr. Geoffrey Burnstock - Autonomic Neuroscience Centre, Royal Free and University College Medical School, London, UK. Against the odds: a personal story.

Conferences: Dr. Maria P. Abbracchio Department of Pharmacological and Biomolecular Sciences, University of Milan, Milan, Italy Purinergic regulation of adult central nervous system stem cells: is there a hope for novel neuro-reparative therapies? Dr. Joel Linden La Jolla Institute for Allergy and Immunology, La Jolla, California, USA Targeting myelod adenosine receptors for tumor immunotherapy Dr. Christa Müller PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry Institute, University of Bonn, Germany Chemical tools for exploring purinergic receptors and ectonucleotidases Dr. Gary Stacey Division of Plant Science, C.S. Bond Life Science Center, University of Missouri, Columbia, MO, USA How plants recognize and respond to extracellular ATP Dr. Attila Tárnok Department of Paediatric Cardiology, Heart Center, and Translational Centre for Regenerative Medicine (TRM), University of Leipzig, Germany Advancements in single cell analysis

Closing Conference: Dr. Henning Ulrich, University of São Paulo, Brazil. Purinergic signaling in neurogenesis and brain repair.

Lectures: Symposium 1—Purinergic signaling in immunity, stem and tumor cells Dr. Mariusz Ratajczak University of Louisville, USA Extracellular nucleotides and purinergic signaling as novel underappreciated pro-metastatic chemotactic factors for human lung cancer cells Dr. Elena Adinolfi University of Ferrara, Italy Oncogenic pathways activated by P2X7 receptor Dr. Guido Lenz Federal University of Rio Grande do Sul, Brazil Adenosine uptake and activation of autophagy are the main effectors of extracellular ATP toxicity in human cervical cancer cells Symposium 2—Purinergic signaling in central nervous system


Dr. Carla Tasca Federal University of Santa Catarina, Brazil Signaling pathways involved in the neuroprotective action of guanosine Dr. Geanne Matos de Andrade Federal University of Ceará, Brazil The role of P2 receptors in Parkinson’s disease Dr. Ana Sebastião University of Lisbon, Portugal Synapse-selective modulation of hippocampal GABAergic transmission by adenosine Dr. Rodrigo Cunha University of Coimbra, Portugal Role of adenosine A2A receptors in the control of mood-related neuropsychiatric diseases

Symposium 3—Purinergic signaling in diseases Dr. Célia Garcia University of São Paulo, Brazil Role of purinoceptor on malaria parasites development Dr. David Ojcius University of California, Merced, USA Effect of purinergic receptors on infection by Chlamydia trachomatis Dr. Manuella Kaster Federal University of Santa Catarina, Brazil Polymorphisms in adenosine receptors and major depressive disorder

Symposium 4—Purinergic signaling in pathological conditions: transporters and pores associated with purinergic signaling Dr. Schuichi Koizumi University of Yamanashi, Japan Gliotransmission by ATP and its pathophysiological consequences Dr. Maria José Fernandes Federal University of São Paulo, Brazil RNA-based strategy to study the role of P2X7 receptor in the temporal lobe epilepsy Dr. Gehard Dahl University of Miami, USA ATP release through pannexin channels

Symposium 5—Purinergic signaling in context of parasite infection Dr. Özlem Yilmaz University of Florida, USA Danger molecules ATP and adenosine signaling: at the crossroads of inflammation and pathogen persistence in the oral mucosa Dr. Tiana Tasca Federal University of Rio Grande do Sul, Brazil The purinergic signaling in Trichomonas vaginalis-host interaction Dr. Helle Praetorius Aarhus University, Denmark Bacterial RTX toxins allow acute ATP release from human erythrocytes directly through the toxin pore Dr. Claudia Lucia Martins Silva Federal University of Rio de Janeiro, Brazil Purinergic signaling contributes to schistosomiasis-related inflammation and endothelial dysfunction

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Dr. Maria Regina D’Império Lima University of São Paulo, Brazil P2X7 receptor signaling dives Th1 effector/effector memory cell differentiation in blood-stage Plasmodium chabaudi AS Malaria Symposium 6—Challenges on crosstalk between academia and industry on coffee research in Brazil Dr. Rui Prediger Federal University of Santa Catarina, Brazil The Brazilian Research looking for the effects of coffee on health and diseases Dr. Lisiane Porciúncula Federal University of Rio Grande do Sul, Brazil The impact of regular coffee consumption on learning and memory and age-related cognitive deficits Dr. Daniel Rial Federal University of Santa Catarina, Florianópolis, Brazil A2A receptor as a potential target in the control of dopamine-related function in the prefrontal cortex Dr. Nathan Herszkowicz Brazilian Association of Coffee Industry, Brazil The Programs of ABIC on Coffee Research—ABIC and coffee and health initiatives Dr. Rodrigo Cunha University of Coimbra, Portugal A personal experience of crosstalk between Academia and Industry on Coffee Research in Portugal

Symposium 7—Purinergic signaling in neural differentiation and brain diseases Dr. Álvaro Sebastian Serrano University of Madrid, Spain TNAP controls axonal growth of cortical neurons and its alteration triggers epileptic seizures Dr. Peter Illes University of Leipzig, Germany P2X7 receptor-mediated regulation of neural progenitor cell functions in the brain Dr. Eliana Scemes Albert Einstein College of Medicine, New York, USA Pannexin1 and P2X7 receptor in chronic pain: experimental and clinical evidence

Symposium 8—Biological functions of P2 receptors Dr. Patricia Castelucci University of São Paulo, Brazil Role of the P2X7 receptor in enteric neurons Dr. Paulo Correia-de-Sá University of Porto, Portugal Human bladder hyperactivity caused by UDP-sensitive P2Y6 receptors: on the role of ATP released from the urothelium via pannexin-1 hemichannel Dr. Zulma Silva Ferreira University of São Paulo, Brazil Purinergic signaling in the Immune-Pineal Axis Dr. Francisco Vázquez Cuevas National Autonomous University of Mexico, Mexico Functional expression of P2X7 receptors in the ovarian surface epithelium Symposium 9—Purinergic signaling in stem and cancer cells Dr. Márcia R. Wink Federal University of Health Science of Porto Alegre, Brazil Mesenchymal stem cells have differential capacity to metabolize extracellular nucleotides


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Dr. Cleber Trujillo University of California, San Diego, USA A model for neural development of Rett syndrome using human stem cells: the role of the neurotransmitter receptors Dr. Claudiana Lameu University of São Paulo, Brazil Purinergic signaling in neuroblastoma migration and metastasis (Oral Communication) MSc. Talita Glaser University of São Paulo, Brazil Spontaneous calcium oscillations are modulated by P2 purinergic receptors and control expression of neurogenesis related transcription factors (Oral Communication) Symposium 10—Purinergic signaling in the immune and cardiovascular system Dr. Jürgen Schrader University of Düsseldorf, Germany Purinergic signaling in T-cells and epicardial progenitor cells after myocardial infarction Dr. Debora Fior-Chadi University of São Paulo, Brazil Modulation of the nitrergic system by A1 and A2a receptors. Focus on the mechanisms involved in neural regulation of blood pressure Dr. Daniela Leal Federal University of Santa Maria, Brazil Effect of vitamin D supplementation on the purinergic system Dr. Hercules A. da Silva Souza Federal University of Rio de Janeiro, Brazil Inhibitors of the 5-lipoxygenase pathway activate pannexin1 channels in macrophages via the thromboxane receptor (Oral Communication) Symposium 11—Purinergic transmission modulating migration, cell death and survival Dr. Francisco Ciruela University of Barcelona, Spain Lighting up adenosine receptors in neuronal death Dr. Ana Ventura Fluminense Federal University, Brazil Involvement of nucleotides in the adhesion and migration of retinal glial cells in culture Dr. Carina R. Boeck Franciscano University Center, Santa Maria, Brazil Is adenosinergic system always neuroprotective? Dr. Francesco Di Virgilio University of Ferrara, Italy P2X7, a receptor with a split personality: an oncogene or an oncosuppressor gene?

Symposium 12—P2X7 receptors-associated channels Dr. Eleonora Kurtenbach Federal University of Rio de Janeiro, Brazil P2X7 purinergic signalling in a model of dilated cardiomiopathy induced by autoimmunity against muscarinic M2 receptors Dr. Pablo Pellegrin Murcia BioHealth Research Institute, Spain ATP release during tissue rejection: the role of P2X7 receptor and pannexin-1 Dr. Beáta Sperlágh Hungarian Academy of Sciences, Budapest, Hungary P2X7 receptors in neuroinflammation and psychiatric disorders

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Dr. Maria Teresa Miras Portugal University of Complutense de Madrid, Spain Neuroprotection mediated by P2Y13 and P2X7 nucleotide receptors and their signaling cascades Dr. Camila Marques da Silva Federal University of Rio de Janeiro, Brazil Thymine and thymidine photodamages can act as alarmins in a peritonitis mouse model (Oral communication) Symposium 13—Regulation of transporters for nucleosides and nucleotides Dr. Imogen R. Coe Ryerson University, Toronto, Canada The SLC29A Family of Nucleoside Transporters: New Modalities of Regulation and Therapeutics Dr. Alexandre Rodrigues Fluminense Federal University, Brazil Regulation of ENT1 in the chicken retina (Oral Communication) Dr. Cristina Lemos Universidade de Coimbra, Portugal Adenosine A2B receptor activation stimulates glucose uptake in the rodent forebrain (Oral Communication) Carlos André de Castro Herklotz Sinapse Biotecnologia Ltda, Brazil An Innovative Multibrand Supplier for the Brazilian Market - Quest for a Balanced Portfolio offer Oral Communications Dr. Claudia Benjamim Federal University of Rio de Janeiro, Brazil ADP Treatment Improves Wound Healing in Diabetic Mice Dr. Josiane Sabbadini Neves Federal University of Rio de Janeiro, Brazil Purinergic P2Y12 Receptor Activation in Eosinophils and the Schistosomal Host Response Dr. Thiago Moreira University of São Paulo, Brazil Independent purinergic mechanisms of central and peripheral chemoreception in the rostral ventrolateral medula Dr. Tamara Lah National Institute of Biology, Slovenia Interference of direct and indirect co-culture of glioblastoma and mesenchymal stem cells in expression levels of kininergic and purinergic componentes Dr. Rupert Ecker TissueGnostics GmbH, Austria Tissue Cytometry and Tissue Sociology – New Concepts to Look at Cells and Cellular Interaction in Tissue Sections

Abstracts Opening conference Against the odds: a personal story

Geoffrey Burnstock Autonomic Neuroscience Centre, University College Medical School, Rowland Hill Street, London NW3 2PF, UK This talk will tell my personal and scientific story from simple beginnings, through heavy opposition to acceptance and recognition. It will cover: my early years from a poor family, school days, war time, unsuccessful applications to study medicine, university BSc and PhD. Then, a postdoctoral appointment to Oxford University, Department of Pharmacology. Move to Australia in 1959 and became a Professor of Zoology and built an exciting research group. The purinergic signalling hypothesis was proposed in 1972, but rejected by most scientists for the next 20 years. I became the Head of Anatomy and Developmental Biology at University College London in 1975. The receptors for ATP and adenosine were cloned and characterised in the early 1990’s and this was the turning point in widespread acceptance of the purinergic hypothesis. Later involvement in the pathophysiology and therapeutic potential of purinergic signalling in collaboration with clinicians and the pharmaceutical industry.


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Guest conference 1

Purinergic regulation of adult central nervous system stem cells: is there a hope for novel neuro-reparative therapies?

Maria P. Abbracchio Laboratory of Molecular and Cellular Pharmacology of Purinergic Transmission, Department of Pharmacological and Biomolecular Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy In the CNS, neurogenesis and gliogenesis continue throughout life in both neurogenic niches (e.g., the subventricular zone, SVZ, of the lateral ventricles) and in the entire brain’s parenchyma, which is full of quiescent neural progenitors that are activated after injury. Two main types of adult stem-like cells have been identified: (i) reactive astrocytes, which remain within their lineage in vivo, but, as revealed by ex-vivo studies, re-acquire capacity for selfrenewal and can generate both neurons, astrocytes and oligodendrocytes (OLs) (Buffo et al. Proc. Natl. Acad. Sci. USA 105; 3581, 2008); (ii) NG2positive polydendrocytes (NG2 glia) that can differentiate to mature OLs participating to re-myelination after injury. Extracellular nucleotides (ATP, UTP, their break-down products and sugar nucleotides) are released at high amounts at the sites of CNS damage (Ulrich et al. Stem Cell Rev. 8; 755, 2012) and are key actors in regulating reparative responses via purinergic receptors (Abbracchio et al. Trends Neurosci. 32; 19, 2009). Concerning CNS niches, by using a conditional GLAST::CreERT2 Rosa YFP mouse model and an in vitro neurosphere assay, we have demonstrated that the P2Y receptor agonist ADPγS promotes the proliferation of SVZ neural progenitors and sustains their progression to neuroblasts, either directly or through the activation of parenchymal astrocytes (Boccazzi et al., Glia 62; 428, 2014). Concerning parenchymal NG2 glia, we have validated the P2Y- like receptor GPR17 expressed on these cells as a new target for remyelinating therapies (Lecca et al. PLoS One. 3(10):e3579, 2008; Ceruti et al. Brain.132; 2206, 2009; Fumagalli et al. J Biol Chem. 286;10593, 2011; Boda et al. Glia. 63; 271, 2015). In NG2 cells, GPR17 reaches its maximal expression peak at the stage of O4-positive immature OLs. Afterwards, GPR17 has to be downregulated, to allow cells to proceed to terminal differentiation, via both receptor desensitization/internalization (Fratangeli et al., J Biol Chem. 288; 5241, 2013; Daniele et al., Cell Signal. 26;1310, 2014) and post-transcriptional regulation mediated by microRNAs (miRN). In this respect, we have recently identified a new miRN potentially involved in the regulation of both GPR17 and other purinergic receptors on NG2 glia (Lecca D., Marangon D., Coppolino G.T., Abbracchio M.P., unpublished). Understanding how these mechanisms are dysregulated under neurodegenerative conditions will unveil novel purinergic-based neuroreparative strategies. Sponsored by Fondazione Italiana Sclerosi Multipla (FISM)2013/R-1 project to MPA. Symposium 1—Purinergic signaling in immunity, stem and tumor cells Extracellular nucleotides and purinergic signaling as novel underappreciated pro-metastatic chemotactic factors for human lung cancer cells

Gabriela Schneider1, Talita Glaser2, Henning Ulrich2 and Mariusz Z Ratajczak1 Stem Cell Institute at James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky, USA 2 Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil

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Background: One of the challenging problems of current radio-chemotherapy is recurrence and metastasis of cancer cells that survive initial treatment. We have proposed that one of the unwanted effects of radio-chemotherapy is release of phospho-sphingolipids in response to treatment and thus induction of pro-metastatic microenvironment in several organs (Mol Cancer Res 2013; 11:793). However, at the same time also intracellular nucleotides such as ATP and UTP are released from damaged (“leaky”) cells exerting chemotactic functions. Hypothesis: Based on these observations that, in addition to phospho-sphingolipids, also nucleotides (e.g., ATP and UTP) are released from damaged cells we hypothesized that these molecules if released into extracellular microenvironment may also direct chemotaxis and metastasis of cancer cells—alone or in synergy with phosphosphingolipids. Materials and Methods: Several complementary in vitro and in vivo approaches were employed to demonstrate a novel role of extracellular nucleotides (EXN) in a model of metastasis of lung cancer cells. In our studies we employed four large cell and two small cell lung cancer cell lines and a set of nucleotides including ATP, ADP, AMP, UTP, TTP, CTP, and GTP. Real time RT-PCR analysis of the expression of purinergic P1 and P2 receptors as well as chemotaxis, adhesion, proliferation, intracellular calcium flux and cell signaling studies in response to EXN stimulation were performed. Concentrations of ATP and UTP in several organs before and after radio-chemotherapy were measured by colorimetric kits. Purinergic receptor agonists and antagonists, inhibiting all or selected subtypes, were assayed in vitro and in vivo in pro-metastatic assays. Results: We observed that EXN accumulate in in several organs in response to radio-chemotherapy. RT-PCR analysis indicated that most of the P2X, P2Y and adenosine receptor subtypes are expressed in tested lung cancer cell lines. By employing in vitro migration assays, we found that, out of all the nucleotides tested, ATP, AMP and UTP have the strongest chemotactic activity for most of the human lung cancer cell lines correlating with phosphorylation of MAPKp42/44 and AKT. We also observed increased adhesion to fibronectin after stimulation with tested EXN, whereas proliferation was not affected by of EXN. More important, metastasis of lung cancer cells could be inhibited in immune-deficient mice in a presence of specific small molecule inhibitors of nucleotide receptors. Conclusions: Both systemic and local radio-chemotherapy leads to upregulation of EXN release in damaged tissues, and the side effect of such treatment is induction of an unwanted pro-metastatic microenvironment in different organs. Based on this data, EXN are novel pro-metastatic factors and inhibition of their pro-metastatic effects could become an important part of anti-metastatic treatment.

Oncogenic pathways activated by P2X7 receptor

E. Adinolfi Department of Morphology, Surgery and Experimental medicine, Section of Pathology, Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy

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Objectives/background: The P2X7 receptor for extracellular ATP recently emerged as regulator of cancer cell proliferation, energy production and migration (1). P2X7 expression has been demonstrated in a wide spectrum of tumour types including colon carcinoma, melanoma and neuroblastoma, all of which also showed P2X7-dependent growth in vivo (2). However, little is known about the intracellular events activated by P2X7 in cancer. Aim of our work was to identify the main biochemical ways responsible for P2X7 dependent cancer progression. Methods and results: The ability of P2X7 receptor to modulate intracellular calcium levels prompted us to investigate receptor-dependent PI3K/Akt pathway activation. In vitro and In vivo experiments in two models of neuroblastoma allowed us to demonstrate that P2X7 expression and activation positively influences the PI3K/Akt pathway. Moreover, P2X7 down-modulation, by either pharmacological blockade or silencing, reduced the activity of PI3K/Akt and increased that of GSK3β leading to a decrease in cellular glycogen stores. Up-modulation of GSK3β activity by P2X7 antagonism also caused a significant reduction in the levels of probably the best-known oncogene in neuroblastoma: MYC-N (3). Increased number of blood vessels in P2X7 positive tumours, let us hypothesise that P2X7 might activate the main pathway involved in cancer vascularization that is the HIF1α-VEGF axis. Indeed, this proved to be the case as intratumoral VEGF levels resulted increased in a P2X7-dependent fashion in experimental models of colon carcinoma, melanoma and neuroblastoma (3,−5). Moreover, administration of the VEGF blocking antibody Avastin reduced P2X7-dependent cancer growth (4). In the neuroblastoma model, P2X7 also affected HIF1α levels both in vitro and ex vivo (3). Conclusion: Taken together our data suggest that P2X7 receptor is an upstream regulator of the main signalling pathways involved in cancer growth, metabolic activity and angiogenesis, and a promising oncological therapeutic target. This work was supported by a grant of the Italian Association for Cancer Research (MFAG11630). References 1. Di Virgilio F. Cancer Res. 72(21):5441 2012 2. Adinolfi E. et al. Curr. Med. Chem, E pub ahead of print, 2014 3. Amoroso F. et al. Oncogene, Epub ahead of print, 2015 4. Adinolfi E. et al. Cancer Research 72(12):2957, 2012 5. Adinolfi E. et al. Cancer Research, Epub ahead of print, 2014

Adenosine uptake and activation of autophagy are the main effectors of extracellular ATP toxicity in human cervical câncer cells

Paola de Andrade Mello1, Eduardo Cremonese Filippi-Chiela 2, Aline Beckenkamp1, Franciele Kipper2, Marcia Rosângela Wink3, Guido Lenz2, and Andréia Buffon1 1 Laboratory of Biochemical and Cytological Analysis, Faculty of Pharmacy 2 Department of Biophysics and Center of Biotechnology, Federal University of Rio Grande do Sul, Brazil 3 Laboratory of Cell Biology, Federal University of Health Sciences of Porto Alegre, Porto Alegre, RS Brazil In cervical cancer, HPV infection and disruption of mechanisms involving cell growth, differentiation, and apoptosis are strictly linked with tumor progression and invasion. Tumor microenvironment is ATP and adenosine rich, suggesting a role for purinergic signaling in cancer cell growth and death. Here we investigate the effect of extracellular ATP on human cervical cancer cells. We find that extracellular ATP itself has a small cytotoxic effect, whereas adenosine formed from ATP degradation by ectonucleotidases is the main factor responsible for apoptosis induction. The level of P2X7 receptor seemed to define the main cytotoxic mechanism triggered by ATP, since ATP itself eliminated a small subpopulation of cells that express high P2X7 levels, probably through its activation. Corroborating these data, blockage or knockdown of P2X7 only slightly reduced ATP cytotoxicity. On the other hand, cell viability was almost totally recovered with dipyridamole, an adenosine transporter inhibitor. Moreover, ATP-induced apoptosis and signaling p53 increase, AMPK activation, and PARP cleavage as well as autophagy induction were also inhibited by dipyridamole. In addition, inhibition of adenosine conversion into AMP also blocked cell death, indicating that metabolization of intracellular adenosine originating from extracellular ATP is responsible for the main effects of the latter in human cervical cancer cells. Symposium 2—Purinergic signaling in central nervous system Signaling pathways involved in the neuroprotective action of guanosine

C.I. Tasca1, G. Poluceno1, K.A. Oliveira1,2, C.B.N. Mendes-de-Aguiar2 and T. Dal-Cim1 Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil 2 Departamento de Biologia Celular, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil

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Objectives/background: The endogenous nucleoside guanosine (GUO) has been revealed as an intercellular messenger implicated in relevant extracellular roles in the brain, such as modulation of glutamatergic transmission and neuronal protection against excitotoxic damage. Glial cells, namely the astrocytes, are involved in multiple cerebral functions that profoundly influence brain tissue viability during ischemia, as the maintenance of blood–brain barrier, cerebral blood flow regulation, ion homeostasis, regulation of physiological extracellular glutamate levels and they are also the main source of extracellular cerebral purines. This study aimed to evaluate the glioprotective role of GUO and the involvement of PI3K, MAPK and PKC signaling pathways in this effect in cultured astrocytes subjected to oxygen/glucose deprivation (OGD), an in vitro model of brain ischemia. Methods and results: Cultured astrocytes subjected to OGD showed a significant cell death. GUO treatment abolished OGD-induced cell death (from 10 to 500 μM). PI3K pathway inhibitor, LY294002 (10 μM), MEK inhibitor, PD98059 (10 μM), or PKC inhibitor cheleritrine (1 μM) prevented the protective effect afforded by GUO. GUO reduced the increased ROS production induced by OGD and this effect was partially blocked by the PI3K pathway inhibitor. Astrocytic cells subjected to OGD showed reduction of glutamate uptake. GUO abolished the reduction of glutamate uptake and this effect was inhibited by MEK inhibitor and PKC inhibitors. Immunocytochemical analysis of the glial glutamate transporter, Glt-1, showed OGD reduced the immunocontent of Glt-1 in astrocytic cells and GUO treatment prevented this effect.


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Conclusion: GUO reduces ROS production depending on PI3K and stimulates astrocytic glutamate uptake depending on MEK and PKC pathways activation in ischemic events. This astrocytic effect may contribute to microenvironment homeostasis and promote neuroprotection. Acknowledgments: CAPES-PVE 052/2012; CNPq; FAPESC-Pronex (NENASC). The role of P2 receptors in Parkinson’s disease

Geanne Matos de Andrade Federal University of Ceará, Brazil In Parkinson’s disease (PD) there is a progressive loss of dopaminergic neurons of the substantia nigra pars compacta (SN) projecting to the striatum. While the focus has been on the role of adenosine A2A receptors and their interaction with dopamine receptors in Parkinson’s disease, also P2 receptors have been implicated. Release of ATP from disrupted cells may cause cell death in neighboring cells expressing P2X7 receptors, leading to a necrotic volume increase. In the unilateral 6-OHDA rat model of PD, nigral P2X7 immunoreactivity was mainly found in microglia but also in astroglia. We showed that P2X7R antagonist, Brilliant Blue G (BBG), controlled the 6-OHDA-induced PD-like features in rats. BBG protected animals from 6-OHDA induced motor alterations and memory deficits, an effect mimicked by A438079 another P2X7R antagonist, the reduction of dopamine content in the striatum and SN, and the microgliosis and astrogliosis in the striatum. Also BBG prevented 6-OHDA-induced synaptosomal dysfunction and neurotoxicity. This suggests that P2X7R contribute to PD pathogenesis through a triple impact on synaptotoxicity, gliosis and neurotoxicity, highlighting the therapeutic potential of P2X7R antagonists in PD. Synapse-selective modulation of hippocampal GABAergic transmission by adenosine

A.M. Sebastião1, D.M. Rombo1, K.P. Lamsa2 and J.A. Ribeiro1 1 Instituto de Farmacologia e Neurociências e Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Portugal 2 Medical Research Council Anatomical Neuropharmacology Unit at Department of Pharmacology, Oxford University, UK Introduction: GABAA receptor (GABAAR)-mediated control of neuronal excitability has a tonic component, corresponding to the exocytic release of GABA generating inhibitory post-synaptic currents (IPSCs), and tonic inhibition, mediated by ambient GABA and extrasynaptic GABAAR. Based on the findings that adenosine A1 receptor (A1R) activation do not affect the frequency or amplitude of miniature IPSCs recorded from pyramidal neurons, it has been assumed until recently that adenosine A1R do not affect GABAergic transmission at the hippocampus. We decided to reevaluate this aspect investigating a possible influence of A1R upon tonic GABAergic inhibition. The possibility of selective influences of A1R upon GABAergic inputs to interneurons or pyramidal neurons was discriminated. Lastly, the influence of adenosine A2A receptors (A2AR) was also addressed. Methods and Results: Whole cell patch clamp recordings of phasic (miniature IPSCs and evoked IPSCs) and tonic GABAAR mediated currents from different subsets of interneurons and pyramidal neurons, as described in detail (Rombo et al., 2014a; b). We found that adenosine A1R at the hippocampus selectively modulate tonic inhibition mediated by activation of extrasynaptic GABAAR. The A1R-mediated modulation of tonic GABAergic currents is consistent in CA1 pyramidal cells, but present only in a specific population of postsynaptic CA1 GABAergic inhibitory interneurons know to be relevant for network oscillations, the CB1/CCK expressing interneurons. Accordingly, sustained A1R activity results in a decreased expression of GABAAR δ-subunit, a key component of extrasynaptic receptors mediating tonic GABAergic currents. Furthermore, we found that A2AR in nerve terminals of a subset of interneurons (those that express parvalbumin, known to be involved in network synchronization) enhance GABAergic inhibitory transmission between CA1 area interneurons leading to disinhibition of pyramidal cells. GABAergic inputs to excitatory neurons are also affected by A2AR activation. Worth noting, A2A receptor blockade robustly suppressed spontaneous interictal like events in an ex vivo model of epilepsy. Conclusions: These synaptic specific actions of adenosine upon GABAergic transmission, together with better known actions upon excitatory transmission (Dias et al., 2013), contribute fine-tune neuronal firing rate at the hippocampus. References Rombo DM et al. (2014a). Cereb Cortex. (ePub) Rombo DM et al. (2014b) Hippocampus. (ePub) Dias RB et al. (2013). Trends in Neuroscience 36:248–257 Role of adenosine A2A receptors in the control of mood-related neuropsychiatric diseases

Rodrigo Cunha CNC-Center for Neuroscience and Cell Biology & Fac.Medicine, Univ.Coimbra, Portugal Adenosine assists encoding information salience through a combined activation of inhibitory A1 and facilitatory A2A receptors (A2AR). This brain modulation system is imbalanced in stressful conditions, with increased A2AR density and decreased A1R density. Neuroprotection is afforded by repeated caffeine consumption (adenosine receptor antagonist), which prophylactically prevents depression, suicide ideation and memory deficits, namely in aging and Alzheimer’s disease. Using a mouse model of chronic unpredictable stress, and combining the use of selective A2AR antagonists (KW6002, 3 mg/kg, p.o.) with global and forebrain neuron-selective A2AR knockout mice, we concluded that neuronal A2AR control mood and memory deficits though a normalization of synaptic plasticity and A2AR blockade can actually therapeutically revert these installed aberrant phenotypes upon repeated stress. The mood/memory normalizing impact of neuronal A2AR blockade is further highlighted by the prevention of fear memory and amygdala synaptic plasticity, as gauged by the use of A2AR antagonists (SCH58261), global A2AR knockout mice or bilateral intra-amygdala injection of a lentivirus encoding a short hairpin RNA to downregulate A2AR (shA2AR), which reduced the acquisition and expression of fear responses to conditioned and unconditioned stimuli and dampened synaptic plasticity in excitatory synapses of the lateral amygdala. This paves the way to foster the therapeutic impact of A2AR to manage pathologies associated with abnormal encoding of aversive memory, in accordance with the association between A2AR polymorphisms and phobia or panic attacks in humans. Notably, repeated restraint stress (4 h daily for 2 weeks) increased the density of A2AR in

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the amygdala, mainly in glutamatergic synapses, and also bolstered synaptic plasticity in the excitatory synapses of the lateral amygdala. The bilateral amygdala injection of shA2AR attenuated the stress-induced aberrant synaptic plasticity in the amygdala and also attenuated the stress-induced increase of plasma corticosterone levels and prevented the anxiogenic-like and helpless-like behaviors caused by repeated stress, thus indicating that ablating amygdala A2AR is sufficient to prevent stress-induced behavioral and functional modifications associated with depressive-like conditions. Overall, this prompts targeting amygdala A2AR as a promising strategy to manage mood-related disorders. (Supported by DARPA, FCT, QREN, NARSAD, CAPES, CNPq-Ciência sem Fronteiras). Symposium 3—Purinergic signaling in diseases Role of purinoreceptor on malaria parasites development

Celia R. S. Garcia Departamento de Fisiologia, Instituto de Biociências, USP Brazil Malaria is a burden that affects roughly 500 million people each year. The complexity of parasite biology and its multiple forms represents a challenge to our understanding of its development. From the work of several labs, it is now accepted that Plasmodium senses the environment and exploits signaling pathways to modulate cellular functions. Decoding the molecular mechanism that enable the parasite to sense the environment will help us to develop new antimalarials. Among potential signaling molecules, we have investigated if ATP would affect the cycle of the human malaria parasite P. falciparum-infected Red blood Cells (RBCs). We have reported that removal of extracellular ATP from Plasmodium falciparum culture by either adding recombinant Apyrase from Schistosoma mansoni during parasite invasion or addition of purinergic receptor antagonist blocks parasite invasion. Likewise, similar experiments performed with rodent malaria parasites P. yoelli or P. berghei also impairs parasite development (Levano- Garcia et al., 2010; Cruz et al., 2012). To investigate the molecular mechanism of ATP action in parasite, we have used fluorescent Ca2+ probes and perform calcium measurements. We have reported cytosolic calcium changes in malaria parasites upon addition of ATP. Moreover, our lab have identified serpentine-like receptors in Plasmodium Genome database. To investigate their role in signaling as well as to test whether malarial proteins can couple with mammalian signaling machinery, we have expressed malarial protein in the HEK293T cell lines. Western blot using anti-FLAG antibodies detected Pf-SR like in HEK293T at the predicted molecular weight of ~80 kDa. The ~80 kDa band was clearly detected in immunoprecipitated fraction of HEK293T cells, both with anti-FLAG and anti- PfSR-like antibodies. The phosphorylation profile of HEK293T cells was altered upon co-transfection of PfSR-like as assessed by anti-phosphoserine and anti-phosphotyrosine antibodies. Indeed, HEK 293 T cells transfected with the codon-optimized receptor elicit a cytosolic calcium rise upon ATP addition that is 2-fold greater than empty vector transfected cells. Finally, the recent, generation of P. falciparum expressing genetically encoded calcium indicators (GECIs) represent an innovation in the study of calcium homeostasis and signaling within malaria parasites. The Plasmodium falciparum GCaMP, construction will provide new insight on the signaling pathways that controls parasite growth and development. Effect of purinergic receptors on infection by Chlamydia trachomatis

M.A. Pettengill1, C. Marques-da-Silva2, A.A. Abdul Sater1, P.M. Persechini2, R. Coutinho-Silva2 and D.M. Ojcius1 1 University of the Pacific, San Francisco, USA 2 Federal University of Rio de Janeiro, Rio de Janeiro, Brazil We have previously shown that millimolar concentrations of ATP inhibit irreversibly chlamydial infection in macrophages or epithelial cells through ligation of P2X7 receptors. P2X7 ligation leads to activation of a phospholipase D, which stimulates fusion between vacuoles harboring chlamydiae and host-cell lysosomes. Conversely, chlamydial infection dampens partially P2X7-mediated signaling in macrophages. We recently found that ATP is also released from Chlamydia trachomatis-infected epithelial cells. Moreover, further stimulation of the infected cells with micromolar extracellular ADP or ATP significantly impairs the growth of the bacteria in epithelial cells, with a profile characteristic of the involvement of P2X4 receptors. A specific role for P2X4 was confirmed using cells overexpressing P2X4. The chlamydiae remain viable and return to normal growth kinetics after removal of the extracellular stimulus, similar to responses previously described for persistence of chlamydial infection in epithelial tissues. We thus propose that ATP could have a direct effect on chlamydial infection through the action of different purinergic receptors on phagocytes or the preferred host cells of the pathogen, epithelial cells. Polymorphisms in adenosine receptors and major depressive disorder

M. Gazal2, F.N. Kauffman2, C. Wiener2, K. Jansen2, J.P. Oses2, L.D. Souza2, R.A. Silva2, D.C. Moreira3, D.R. Lara4, G. Ghisleni2 and M.P. Kaster1 1 Department of Biochemistry, Universidade Federal de Santa Catarina, Florianópolis, Brazil 2 Universidade Católica de Pelotas, Rio Grande do Sul, Brazil 3 Universidade Federal do Rio Grande do Sul, Brazil 4 Pontifícia Universidade Católica do Rio Grande do Sul, Rio Grande do Sul, Brazil The interest in the adenosinergic system in mood disorders stems from three lines of research: first, there is evidence that the consumption of caffeine might modify the mood profile both of volunteers as well as of psychiatric patients; secondly, there is evidence that different therapeutic strategies used to control mood disorders cause effects related to the adenosine modulation system; thirdly, there is evidence from animal models that the manipulation of adenosine receptors modifies behavioral responses considered relevant for mood function in humans. More recently, another line of evidence emerged indicating a possible role for the genetic alterations in the adenosinergic system in mood-related conditions. In fact, recent polymorphisms in multiple genes involved in adenosine metabolism and adenosine receptors were associated with an increased in vulnerability to psychiatric conditions, including depression, schizophrenia, psychosis, anxiety and phobia.


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The Diagnostic and Statistical Manual of Mental Disorders IV (DSM-IV) defines that the heritability of major depression is approximately 25–29 % for males and 42–49 % for females. The aim of our work was to identify a possible association between the 23155511C/T single nucleotide polymorphism (SNP) in exon 1 of the adenosine A2A receptor gene, ADORA2A (rs2298383), located in a potential promoter region, peripheral inflammatory cytokines (TNF-α, IL-1β and IL-6) and MDD in a southern Brazilian population. This work is part of a population-based study including 750 subjects (18 to 24 year-old) from the urban area of Pelotas, RS (Brazil). MDD diagnosis was made with the Mini International Neuropsychiatric Interview 5.0. Of the 750 subjects evaluated, we found 256 with MDD. Most of the MDD subjects were women (54 %), caucasian (78.9 %) and with a low use of psychiatric medication (8.3 %). No differences were detected according to diagnosis and genotypic distribution (χ2 = 0.211) or levels of TNF-α (control: 104.90 ± 15.41 vs MDD: 135.71 ± 25.94 pg/mL, p = 0.278), IL-1β (control: 9.09 ± 1.77 vs MDD: 15.40 ± 4.74 pg/mL, p = 0.14) or IL-6 (control: 20.17 ± 2.36 vs MDD: 18.15 ± 1.25 pg/mL, p = 0.45). However, after stratification by gender we observed an association between carriers of the T allele (C/T homo- and T/T heterozygotes) and protection against MDD in women (p < 0.05, Pearson’s chi-squared test). In addition, there was a tendency to increased TNF-α levels in MDD women vs control (63.72 ± 16.50 and 135.71 ± 25.94 pg/mL, respectively, p = 0.08, using Student’ t test). No changes were observed IL-1β and IL-6 levels in women according to diagnosis. In addition, two-way ANOVA revealed significant differences for the interaction between genotype and diagnosis in the levels of TNF-α (p < 0.05), but not IL-1β (p = 0.56) and IL-6 (p = 0.36). The post-hoc analysis indicated that in healthy control women the levels TNF-α were similar according to the genotype (CC: 60.05 ± 42.90 pg/mL, vs CT/TT 77.75 ± 23.64). However, the levels of TNF-α were significantly higher in MDD women with the CC genotype (241.32 ± 50.15 pg/mL) when compared to CT/TT genotype (91.95 ± 30.52 pg/mL). We concluded that there is an association between allele T of the ADORA2A SNP, lower levels of TNF-α and decreased risk for MDD in women. However, future studies should be necessary in order to investigate the potential mechanisms involved in the regulation of TNF-α production and secretion by A2AR receptors and their impact on psychiatric disorders. Symposium 4—Purinergic signaling in pathological conditions: transporters and pores associated with purinergic signaling Gliotransmission by ATP and its pathophysiological consequences

S. Koizumi1,3 and Y. Hirayama1,2 1 Department of Neuropharmacology, 2Department of Liaison Academy, Interdisciplinary Graduate School of Medicine, University of Yamanashi, Yamanashi 3 CREST, Japan Science and Technology, Tokyo, Japan Objectives / background: Preconditioning (PC) using a preceding sublethal ischemic insult is an attractive strategy for protecting neurons by inducing ischemic tolerance in the brain. Although the underlying molecular mechanisms have been extensively studied, almost all studies have focused on neurons. Here, using a middle cerebral artery occlusion (MCAO) model in mice, we show that astrocytes play an essential role in the induction of brain ischemic tolerance. Methods and results: All procedures were performed in accordance with the “Guiding Principles for the Care and Use of Animals in the Field of Physiologic Sciences” published by the Physiologic Society of Japan and with the previous approval of the Animal Care Committee of Yamanashi University (Chuo, Yamanashi, Japan, Approval No: A23-9). C57BL/6 J mice, 8–10 weeks old, were purchased from CLEA Japan (Shizuoka, Japan). P2X7 receptor knockout micewere kindly provided by Dr. Hiroshi Enaida (Saga University, Saga, Japan). MCAO was induced as previously described (Ikeda-Matsuo et al., PNAS, 2006). PC caused activation of glial cells without producing any noticeable brain damage. The spatiotemporal pattern of astrocytic, but not microglial, activation correlated well with that of ischemic tolerance. Interestingly, such activation in astrocytes lasted at least 8 weeks. Importantly, inhibiting astrocytes with fluorocitrate abolished the induction of ischemic tolerance. To investigate the underlying mechanisms, we focused on the P2X7 receptor as a key molecule in astrocyte-mediated ischemic tolerance. P2X7 receptors were dramatically upregulated in activated astrocytes. PC-induced ischemic tolerance was abolished in P2X7 receptor knockout mice. Moreover, our results suggest that hypoxia inducible factor-1α, a well-known mediator of ischemic tolerance, is involved in P2X7 receptor-mediated ischemic tolerance. Unlike previous reports focusing on neuron-based mechanisms, our results show that astrocytes play indispensable roles in inducing ischemic tolerance, and that upregulation of P2X7 receptors in astrocytes is essential (Hirayama et al., J Neurosci, in press). Conclusion: Upregulation of P2X7 receptors after PC is essential for the induction of ischemic tolerance. Although it is well established that neurons play an important role in the induction of ischemic tolerance, our findings suggest that glial cells may be more important. Thus, a better understanding of ischemic tolerance will require researchers to evaluate the combined contribution of neurons and glial cells. This new insight into the role of glial cells in the cell-autonomous and non-cell-autonomous mechanisms that mediate neuroprotection should promote the development of novel therapeutic strategies for brain ischemia. Acknowledgement: This study was supported by CREST (to SK), KAKENHI on Innovative Areas (25116512 & 25117003) (to SK) and on Challenging Exploratory Research (25670622) (to SK). RNAi-based strategy to study the role of P2X7 receptor in the temporal lobe epilepsy

M.J.S. Fernandes1, I.T.L. Cendes2, H. Ulrich3 and R.P. Amorim1 1 Disciplina de Neurociências, Departamento de Neurologia e Neurocirurgia da Universidade Federal de São Paulo (Unifesp), São Paulo, Brazil 2 Departamento de Genética Médica, Faculdade de Medicina da Unicamp, Campinas, SP, Brazil 3 Instituto de Química da Universidade de São Paulo (USP), São Paulo, Brazil Objectives: RNA interference (RNAi) is a powerful tool described by Andrew Fire and Craig Mello in 1998 used to silence gene expression. Doublestranded RNA (dsRNA) is processed into small inhibitory RNAs (siRNA) by enzymatic complex called Dicer. The functionally active siRNA can bind to specific target mRNA sequence and cause gene degradation or gene silencing by translational inhibition. In the present study, we used RNAi approach

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designed to silence mRNA of P2X7R to elucidate the role of these receptors in epileptogenesis. Previous studies have shown increased level of P2X7R in the hippocampus of rats subjected to pilocarpine model, located mainly in glial cells (Dona et al. Epilepsy Res 83, 157, 2009). Methods: Small interfering RNA (siRNA) was administered in vivo (icv), 6 h after SE. A short peptide derived from the rabies virus glycoprotein (RVG) fused to a nona-D-arginine residues (RVG-9dR) was used to deliver siRNA in the cell. A fluorescent oligonucleotide (BLOCK-ITTM) complexed to RVG-9dR was used to test the transfection agent. The P2X7R was assessed by western blot in hippocampal samples of rats from four studied groups: Saline-Saline, Saline-RNA, Pilo-Saline and Pilo-RNA. Neuronal death was analyzed by Fluoro-Jade B (FJ-B) histochemistry and hippocampal volume was analyzed 48 h after RNAi icv. Behavioral parameters as latence, frequency and severity of seizures were analyzed until 60 days after pilocarpineinduced SE. Results: Intense intracellular fluorescence indicated the presence of the BLOCK ITTM: RVG-9dR into the cell. Rats treated with siRNA target to mRNA of P2X7R showed decreased expression of P2X7 protein 48 h after injection in Saline-RNA (−43 %) and Pilo-RNA (−37 %) groups compared to the respective control groups. Less FJ-B dyed cell was observed in CA1 and CA3 of Pilo-RNA group compared to Pilo-Saline. The silencing of P2X7R in pilocarpine group reversed the increase in the hippocampal volume (edema) detected 48 h after SE onset in the hilus, dentate gyrus, CA1 and CA3. Furthermore, the silencing induced an increase in latency to the first spontaneous seizure and a reduction in the number of seizures when compared to Pilo-saline group. Conclusions: RNAi is a good tool to study the role of P2X7R in epileptogenesis in vivo. P2X7R silencing induced neuroprotection, reduced the hippocampal edema caused by SE and promoted antiepileptic effect in the pilocarpine model. The transfectant agent RVG-9dR binds mainly to nicotinic receptors in glial cells indicating that inhibition of P2X7R can interfere with epileptogenesis. Financial support: Fapesp, CNPq, INNT, CAPES. ATP release through pannexin channels

Gerhard Dahl University of Miami Medical School, Miami, FL 33136, USA Extracellular ATP serves as a signal for diverse physiological functions, including spread of calcium waves between astrocytes, control of vascular oxygen supply and control of ciliary beat in the airways. ATP can be released from cells by exocytosis, through specific channels or transporters or through compromised cell membranes. This talk focuses on channel mediated ATP release and its main enabler, Pannexin1 (Panx1). Although originally discovered as a gap junction protein, Panx1 appears to exclusively form nonjunctional membrane channels. Depending on the mode of stimulation, the Panx1 channel has large conductance (500 pS) and unselective permeability to molecules 90 % reductions in IL-10 expression in tumor associated macrophages, dendritic cells (DC), and myeloid-derived suppressor cells (MDSC). Myeloid deletion of A2ARs significantly increased CD44 expression on tumor-associated T cells and natural killer (NK) cells. Intratumoral injections of bladder tumors with the non-selective adenosine receptor antagonist theophylline (100 μl of 200 μM at 3 day intervals) reduced tumor volume by 60 % on day 21 (P < 0.001), but this response was primarily mediated by A2BR rather than A2AR blockade – possible because A2BRs are more highly expressed on myeloid than lymphoid cells.


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Conclusions: Blocking adenosine receptors on T cells (mostly A2A) can activate cells to increase immune rejection of tumors under some circumstances, but strong T cell activation also produces activation-induced cell death that can lead to reduced T cell numbers in tumors and accelerated tumor growth. In contrast, blocking or deleting adenosine receptors (A2A and A2B) in myeloid antigen presenting cells changes the tumor microenvironment to facilitate moderate T cell and NK cell activation and more effectively killing of tumor cells. Hence myeloid cells may be preferable to lymphoid cells as targets for adenosine-receptor based tumor immunotherapy. Supported by grant R01-HL37942 from the National Institutes of Health. Symposium 5—Purinergic signaling in context of parasite infection Danger molecules ATP and adenosine signaling: at the crossroads of inflammation and pathogen persistence in the oral mucosa

Özlem Yilmaz University of Florida, College of Dentistry and the emerging Pathogens Institute, Gainesville, Florida, USA As a key danger signal (DS) molecule, adenosine-5′-triphosphate (ATP), has become well recognized for its major participation in immune activation and regulation of inflammation. ATP released from inflamed tissues acts through ionotropic purinergic receptors, notably P2X7 receptor, to activate specific proinflammatory signaling cascades. ATP-P2X7 coupling has lately been shown to limit the ability of pathogens to establish successful intracellular infections. While the pro-inflammatory features of ATP, especially for controlling of intracellular infections, have been increasingly characterized, a less appreciated component of host purinergic signaling is another DS molecule, adenosine. Adenosine, a metabolite of extracellular ATP has been shown to play a nonredundant role in downregulating inflammation and the anti-inflammatory nature of adenosine during infection remains largely unexplored. Antiinflammatory effect of adenosine is carried out via G-protein coupled receptors belonging to the P1 superfamily, including A1, A2a, A2b, and A3 subtypes. The latest findings also highlight the significance of purinergic signaling in the human oral cavity. Plasma-derived gingival crevicular fluid, which bathes the interface of gingival epithelia and oral biofilms, as well as saliva are both found to contain markedly high levels of purines during the gingival inflammation through unbiased metabolomics profiling. Our own recent studies on human oral mucosa, in particular, gingival epithelial cells, reveal novel key roles for the ATP and adenosine signaling in the context of persistent pathogen and host interaction. We showed that opportunistic oral pathogen, P. gingivalis, effectively subverts gingival epithelial cell death mediated by ATP-P2X7 during the infection. Furthermore, the microorganism modulates ATP-induced reactive-oxygen-species (ROS) formed through P2X7 / NADPH-oxidase/mitochondria interactome, and inhibits ATP-mediated oxidative stress via the bacterial secreted effector, a nucleoside-diphosphate-kinase (NDK) homolog. Our new data demonstrates P. gingivalis can attenuate ATP-mediated IL-1β secretion through the NDK enzyme. Intriguingly, our studies in parallel displayed that activation of A2a receptors with A2a receptor specific agonists results in elevated intracellular P. gingivalis replication, which also correlates with significantly higher levels of cAMP during the infection. Further, A2a receptor antagonism and knockdown via RNA interference significantly reduced metabolically active intracellular P. gingivalis. In summary, these findings point that successful opportunistic pathogen survival in the epi-mucosal tissues are at least partially mediated by P2X7 and A2a receptor signaling networks, which seem to also closely involve down-regulation of pro-inflammatory responses. The purinergic signaling in Trichomonas vaginalis-host interaction

Tiana Tasca Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil Trichomonas vaginalis, a parasitic protozoan, is the etiologic agent of trichomoniasis, the most prevalent non-viral sexually transmitted disease worldwide. The spectrum of clinical trichomoniasis in women ranges from the asymptomatic state to flagrant vaginitis and may cause major health consequences, including atypical pelvic in inflammatory disease, cervical and prostate cancers, low-weight and premature birth. Importantly, the disease is also a co-factor in promoting transmission of HIV. Metronidazole and tinidazole, both 5-nitroimidazoles, are two drugs of choice recommended by Food and Drug Administration (FDA, USA) for the treatment of trichomoniasis. However, drug-resistant isolates of T. vaginalis have been reported and therapeutic alternatives are being researched. The investigation of biochemical aspects of the parasite and its relationship with the host can help to clarify some mechanisms of trichomoniasis pathogenesis. Adenosine 5′-triphosphate (ATP) plays a crucial role in many extracellular functions and can act as damage-associated molecular pattern (DAMPs) performing a proinflammatory function in the microenvironment of damaged cells. In the other hand, adenosine may revert some of effects induced by extracellular ATP through immunosuppressive modulation. Both ATP and adenosine play their effects by binding to specific receptors named purinoceptors, P2 and P1, respectively. The regulation of this cell signaling can be attributed to enzymes called ectonucleotidases: the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family (NTPDase 1–8) and ecto-5′-nucleotidase (CD73). In sequence to ecto-5′-nucleotidase activity, adenosine deaminase (ADA) catalyses the conversion of adenosine to inosine. In parasites the purinergic system represents an important mechanism of escape of host immune response by ATP degradation and adenosine production, and in this manner modulating immune response. Moreover, in spite of adenosine importance in limiting the inflammatory response, pathogens may scavenge adenosine for growth from host cell because these organisms lack the ability to synthesize the purine ring de novo. NTPDase, ecto-5′-nucleotidase and ADA activities have already been characterized in T. vaginalis trophozoites. Our group have shown the importance of these enzymes for the parasite, since in an environment with low concentrations of adenosine the enzymes NTPDase and ecto-5′-nucleotidase provide the nucleoside necessary for the parasite growth. In addition, adenine nucleotides (ATP, ADP and ATPγS) as well as ATP enzymatic hydrolysis were not decisive for NO release by T. vaginalis-stimulated neutrophils. Unlike ATP, adenosine and inosine decreased significantly the NO levels, revealing the immunossupressive effect of adenosine – promoted by A2A activation and the importance of ecto-5′-nucleotidase activity of T. vaginalis during the establishment of trichomoniasis. Considering the high concentration of extracellular ATP at the infection site, the purinergic system represents a primordial form of chemical intercellular signaling where the parasite’s ectonucleotidases play an important role. The enzymes hydrolyze ATP producing adenosine which will be uptake and employed for the parasite growth and moreover, the anti-inflammatory effects of the nucleoside can contribute to the effectiveness of infection. In this context, the enzymes may be

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considered pathogenic markers in the identification of T. vaginalis isolates as well as future possible adjuncts on diagnosis and interesting targets of new alternatives for the treatment of trichomoniasis. Bacterial RTX toxins allow acute ATP release from human erythrocytes directly through the toxin pore

Helle A. Praetorius Department of Biomedicine, MEMBRANES, Aarhus University, Denmark Objectives / background: ATP is as an extracellular signalling molecule able to amplify the cell lysis inflicted by certain bacterial toxins including the two RTX toxins α-hemolysin (HlyA) from E. coli and leukotoxin A (LtxA) from Aggregatibacter actinomycetemcomitans. Inhibition of P2X receptors completely blocks the RTX toxin-induced haemolysis over a larger concentration range. It is, however, at present not known how the ATP that provides the amplification is released from the attacked cells. Methods and results: We have shown that both HlyA and LtxA trigger acute release of ATP from human erythrocytes that preceded and were not caused by cell lysis. This early ATP-release did not occur via previously described ATP-release pathways in the erythrocyte. Both HlyA and LtxA were capable of triggering ATP release in the presence of the pannexin 1 blockers carbenoxolone and probenecid, and the HlyA-induced ATP–release was found to be similar in erythrocytes from pannexin 1 wild type and knockdown mice. Moreover, inhibition of voltage dependent anion channel (VDAC) had no effect on ATP-release by either of the toxins. Finally, we showed that both HlyA and LtxA were able to release ATP from ATP-loaded lipid (POPC) vesicles devoid of any erythrocyte channels or transporters. Again we were able to show that this happened in a non-lytic fashion, using calcein-containing vesicles as controls. These data show that both toxins incorporate into lipid vesicles and allow ATP to be released. Conclusion: We suggest that both toxins cause acute ATP-release by letting ATP pass the toxin pores in both human erythrocytes and artificial membranes. Perspective: These findings may have significant implications for the general biological effects of bacterial pore-formers. It implies that no matter what cell type is attacked by the toxin, ATP release will automatically follow. Since P2 receptors are widely distributed in mammalian cells this would entail that the purinergic signalling system will be activated in tissues exposed to cytolysin producing bacteria. Acknowledgment of financial We are grateful to Novozymes for supplying Novicidin. The project was financially supported by: The Danish Council for Independent Research, Medical Sciences, Novo Nordisk Fonden, the Lundbeck Foundation, and the Danish Research Foundation (inSPIN). Purinergic signaling contributes to Schistosomiasis-related inflammation and endotelial dysfunction

Claudia Lucia Martins Silva Pharmacology and Inflammation Research Program, Biomedical Sciences Institute, Federal University of Rio de Janeiro, RJ, Brazil ATP is released into the extracellular environment by several mammalian cell types and modulates physiological or pathophysiological events such as vascular dysfunction. Extracellular ATP acts as an autocrine or paracrine mediator, through the activity of different P2 purinoceptor subtypes. Moreover, ectonucleotidases (NTPDases) are involved in the metabolism of extracellular nucleotides, controlling their availability for purinoceptor activation. Both G protein-coupled P2Y receptors (P2YR) and ion-channel P2X receptors (P2XR) are expressed in endothelial cells (ECs) and immune cells, where receptor activation triggers different signaling cascades, including immune cell migration, vasodilation and EC apoptosis. The main purinoceptor subtypes expressed on mammalian ECs are P2Y1R, P2Y2R and P2X4R, although vessel- and species-specific differences in receptor subtypes exist. As shown by ours and other research groups, alterations in vascular P2 receptor signaling and NTPDases function contribute to endothelial dysfunction. In the P2Y1R/apolipoprotein E (ApoE) double knockout mice monocyte diapedesis is reduced as compared to single knockout mice (ApoE−/−). Analysis of this double knockout model showed that P2Y1R contributes to the expression of endothelial adhesion molecules that mediate leucocyte recruitment during inflammation. In an intestinal inflammation model caused by schistosomiasis, our data show that increased expression of mesenteric EC NTPDase 2 triggers higher levels of extracellular ADP, a full agonist of P2Y1R, with subsequent endothelial P2Y1R-mediated monocyte adhesion. Short-term activation of endothelial P2X7R stimulates endothelial nitric oxide (NO) synthase activity, resulting in NO production and vasodilation. In the same inflammation model, basal P2X7R expression is reduced by higher than normal levels of TGF-β, resulting in a reduced NO synthesis. In conclusion, our group and others have produced considerable evidence in support of the notion that purinergic signaling contributes to vascular dysfunction in inflammatory diseases. Financial support: CNPq, FAPERJ P2X7 receptor signaling drives Th1 effector/effector memory cell differentiation in blood-stage Plasmodium chabaudi AS malaria

E.M. Salles1, M.N. Menezes1, H.B. da Silva1, F.S. Vieira1, S.I. Castillo-Méndez1, A.A. Cassado1, E.P. Amaral1, J.M. Alvarez1, R. Coutinho-Silva2,3 and M.R. D’Império-Lima1 1 Departamento de Imunologia, Universidade de São Paulo, São Paulo, Brazil 2 Programa de Imunobiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil 3 Instituto Nacional de Ciência e Tecnologia para Pesquisa Translacional em Saúde e Meio Ambiente da Região Amazônica, Rio de Janeiro, Brazil Objectives/background: P2X7 receptor (P2X7R) is a sensor of extracellular ATP, a damage signal released during tissue injury that provides for ion exchange and promotes cell activation and death. We investigated whether the P2X7R signaling is required for T helper 1 (Th1) cell differentiation during experimental blood-stage malaria.


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Methods and results: To evaluate whether the P2X7R contributes to protection against blood stages of P. chabaudi AS (PcAS), the disease progression was analyzed in C57BL/6 (B6) and P2X7R−/− mice. The parasitemia and clinical parameters (i.e. anemia, weight loss and hypothermia) developed similarly in both female groups up to day 7 p.i., but after this period the animals that lacked the P2X7R had difficulty in controlling the parasitemia and restoring the clinical parameters to the baseline in naïve mice. The disease was further aggravated in infected P2X7R−/− males that showed 60 % mortality. To investigate whether the rupture of infected red blood cells (iRBCs) leads to ATP release into the extracellular milieu, ATP serum levels were evaluated in B6 mice at days 4 and 5 p.i. with synchronized parasites, before and after erythrocyte re-invasion. Significantly higher ATP serum levels were observed after the rupture of iRBCs. Because the sensing of environment eATP by the P2X7R is thought to amplify the TCR signal during the immune synapsis, we focused our analysis on CD4+ T cells that have a central role in the development of protective immunity against PcAS malaria. To determine whether the increased ATP serum levels engage the P2X7R in blood CD4+ T cells, P2X7R-associated pore formation was evaluated by ethidium bromide (EB) uptake. On days 4 and 5 p.i., the percentages of EB-stained B6 CD4+ T cells increased after iRBC rupture, an effect that was not observed for P2X7R−/− CD4+ T cells. The inefficient parasite control in acutely and chronically infected P2X7R−/− mice was associated with low production of IFN-γ but high production of IgG antibodies. Furthermore, the expression of T-bet transcriptional factor in Th1 effector/effector memory cells induced by infection critically depended on the P2X7R signaling in CD4+ T cells. Indeed, P2X7R−/− CD4+ T cells differentiated in infected CD4−/− mice, in comparison to B6 CD4+ T cells, showed a bias towards the follicular T (Tfh) helper cell lineage over Th1 memory cell lineage. This bias was evidenced on day 30 p.i. by the high numbers per spleen of P2X7R−/− CD4+ cells expressing the Bcl-6 transcriptional factor in PD-1+CXCR5+ Tfh cells. Conclusion: This study provides a new insight into malaria immunology by showing that recognition of ATP, a damage signals, by the P2X7R is crucial for the differentiation of Th1 effector/effector memory cells during Plasmodium infection. It also demonstrates that P2X7R-mediated balance between Tbet and Bcl-6 transcriptional factors tunes the cellular and humoral immunity in malaria. Financial support: Fapesp and CNPq. Symposium 6—Challenges on crosstalk between academia and industry on coffee research in brazil The Brazilian research looking for the effects of coffee on health and disease

Rui Daniel Prediger Department of Pharmacology, Federal University of Santa Catarina, Florianópolis, Brazil Convergent epidemiological and pre-clinical data suggest that the blockade of adenosine A2A receptors (A2AR) by caffeine or selective antagonists may confer neuroprotection against the underlying neuron degeneration, and influence the onset and progression of age-related deficits, neurodegenerative and neurological diseases. The present presentation attempts to highlight recent clinical and experimental researches from Brazilian groups indicating the main effects of the regular consume of coffee on health and diseases. Support: CNPq, FAPESC, UFSC. The impact of regular coffee consumption on learning and memory age-related cognitive deficits

Lisiane de Oliveira Porciúncula Department of Biochemistry, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil The cognitive enhancer properties of caffeine, the main component of coffee, have been debated over the years. Acute caffeine promoted improvement of recognition memory in adult animals. In adolescent rodents caffeine enhances recognition memory. Chronic caffeine was able to prevent age-related memory decline and mnemonic deficits in an experimental model of sporadic dementia. These effects of caffeine were accompanied by modifications in synaptic proteins from different brain areas. Support: CNPq, FAPERGS, CAPES, PROPESQ/UFRGS. A2A receptor as a potential target in the control of dopamine-related function in the prefrontal cortex

Daniel Rial Department of Pharmacology, Federal University of Santa Catarina, Florianópolis, Brazil The prefrontal cortex (PFC) plays important roles in mood and executive functions involving decision-making, attention and working memory. PFC-related functions are modulated by the dopaminergic system (encompassing D1- and D2-like receptors) and their pharmacological manipulation is exploited to manage some neuropsychiatric conditions associated with abnormal PFC functioning. The adenosinergic system, namely adenosine A2A receptors (A2AR) antagonists, also control PFC- related behavioral responses and A2AR agonists have been proposed as potential anti-psychotic drugs. The main goal of this presentation is to contextualize new data about the interplay between the A2AR and D2Rs and possible implications to PFC-related pathologies. Support: CNPq, FCT, NARSAD and DARPA. The programs of ABIC on coffee research—ABIC and coffee and health initiatives

Nathan Herszkowicz Executive Director ABIC, Brazil

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Dr. Darcy Lima, MD Ph.D in Medicine, University of London, and professor at the Federal University of Rio de Janeiro, was dedicated to research the benefits of coffee to health for over 20 years, and got the support of the Brazilian Coffee Roasting Association (ABIC) since the early 90’s. To prevent some diseases such as alcoholism, depression and drug use due to its components, such as caffeine, chlorogenic acids and derivatives, was the first targets. The results include the creation, in 1999, of the Coffee Institute at Vanderbilt University, in Nashville, USA. Supporting Dr. Darcy as a communicator of health benefits on drinking coffee, ABIC starts a broad campaign to consumers education in Brazil, as well as a wide dissemination of researches results to the medical community trough the distribuition of Medical Newsletters. Another initiative of ABIC has led to create the COFFEE AND HEART PROJECT at the—INCOR – Heart Institute in Sao Paulo. Coffee at Breakfast and Health in School is also a program directed to bring the benefits of daily coffee drinking habit to children in schools. A personal experience of crosstalk between academia and industry on coffee research in Portugal

Rodrigo Cunha CNC-Center for Neuroscience and Cell Biology & Fac. Medicine, Univ. Coimbra, Portugal The partnership between our group and the Portuguese consortium of Coffee producers (AICC - Associação Industrial e Comercial do Café) will be described, as well as its extension to the International Coffee Organization. It will also be discussed our relation with the largest Portuguese Coffee distributor (Delta Café) to establish a long-term research grant agreement counterweighted by the group’s involvement in the scientific support of the activities of the Grupo de Inovação Nabeiro in coordination with the Institute for Scientific Information on Coffee (ISIC). This should illustrate that the crosstalk between academia and industry should begin based on a perceived need felt by the industry; then it evolves into partnerships largely dictated by the needs and interests of the players, with different solutions implemented for interaction with different partners. Support: DARPA, Santa Casa da Misericórdia, QREN, NARSAD, CNPq-Ciência sem Fronteiras. Guest conference 3 Chemical tools for exploring purinergic receptors and ectonucleotidases

C.E. Müller PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, Bonn, Germany Membrane receptors activated by purines are subdivided into three distinct families: nucleotide P2 receptors (P2Y and P2X receptor subfamilies), adenosine P1 receptors (A1, A2A, A2B, A3), and adenine P0 receptors. Purinergic signalling is modulated by ectonucleotidases that are often coexpressed with purine receptors catalyzing the degradation of extracellular nucleotides.1 Our group has been interested in the identification and development of potent and subtype-selective ligands—as tool compounds and potential drugs— for the various purine receptor subtypes as well as ectonucleotidases. Based on the development of novel assays for compound screening and characterization2 we identified and optimized several classes of ectonucleotidase inhibitors, especially for ecto-5′-nucleotidase (CD73) and nucleotide pyrophosphatase1 (NPP1).3 Moreover we recently discovered potent allosteric P2X receptor antagonists. Another focus of interest are the Gs-proteincoupled adenosine receptor subtypes A2A and A2B. All of the investigated membrane proteins have considerable potential as drug targets, e.g. for the treatment of inflammation, neurodegeneration, and cancer. References 1. Zimmermann et al. Purinergic Signal. 8; 437, 2012; Flögel et al. Science Transl. Med. 4; 146ra108, 2012; Augusto et al. J. Neurosci. 33; 11390, 2013 2. Lee & Müller, Electrophoresis 35; 855, 2014; Fiene et al. Analyst 140; 140, 2015 3.Lee et al. Biochem. Pharmacol. 93; 171, 2015. Chang et al. J. Med. Chem. 57; 10080, 2014 Symposium 7—Purinergic signaling in neural differentiation and brain diseases TNAP controls axonal growth of cortical neurons and its alteration triggers epileptic seizures

A. Sebastián-Serrano1,4, T. Engel5, L. de Diego-García1,4, C. Martínez-Frailes1,4, JL. Millán6, L. Olivos-Oré2,4, M. Arribas-Blázquez4,2, C. Perez Diaz3, AR. Artalejo2,4, M.T. Miras-Portugal1,4, D. Henshall5 and M. Díaz-Hernández1,4 1 Department of Biochemistry and Molecular Biology 2 Department of Pharmacology, 3Department of Medicine and Animal Surgery, Veterinary School, Complutense University of Madrid, Avda. Puerta de Hierro S/N, 28040 Madrid, Spain 4 Instituto de Investigación Sanitaria del Hospital Clínico San Carlos, IdISSC, Madrid, Spain 5 Department of Physiology & Medical Physics, Royal College of Surgeons in Ireland, Ireland 6 Sanford Children’s Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, California, USA Objectives / background: Tissue-nonspecific alkaline phosphatase (TNAP) is one of the four isozymes in humans and mice that have the capacity to hydrolyze phosphate groups from a wide spectrum of physiological substrates. Among these, TNAP degrades substrates implicated in neurotransmission. Transgenic mice lacking TNAP activity display the characteristic skeletal and dental phenotype of infantile hypophosphatasia, as well as spontaneous epileptic seizures and die around 10 days after birth. This physiopathology, linked to the expression pattern of TNAP in the central nervous system


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(CNS) during embryonic and postnatal stages, suggests an important role for TNAP in neuronal development and synaptic function, situating it as a good target to be explored for the treatment of neurological diseases. Methods and results: using primary culture of hippocampal neurons we demonstrated that TNAP plays an essential role for establishing neuronal circuits. The pro-neuritic effect induced by TNAP, which results in axonal length increase, is due to its enzymatic hydrolysis of extracellular ATP at the surrounding area of the axonal growth cone. In this way, the activation of ionotropic P2X7 receptor is prevented and as a consequence there is no inhibition of axonal growth. The existence of a close functional interrelation between both purinergic elements is finally supported by the fact that both elements may control, in a reciprocal way, the expression level of the other. Employing TNAP null transgenic mice, we corroborate the defects related to the axonal growth in cortical neurons at early postnatal stages and also the downregulation of P2X7R in the absence of this ectonucleotidase. Morphological studies of the TNAP−/− dentate gyrus, showed changes in the cytoarchitecture of the structure that could be related with the decrease of P2X7R expression and function. Conclusion: The expression of P2X7 and its ligand availability is regulated by TNAP in axonal growth events and during the development of dentate gyrus. Acknowledgements: this work has been funded by Ministerio de Ciencia e Innovación (BFU-2012-31195; BFU2011-24743).

P2X7 receptor-mediated regulation of neural progenitor cell functions in the brain

Peter Illes and Patrizia Rubini Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, 04107 Leipzig, Germany Neurogenesis requires the balance between the proliferation of newly formed progenitor cells and subsequent death of surplus cells. RTPCR and immunocytochemistry demonstrated the presence of ATP-sensitive P2X7 receptor (R) mRNA and immunoreactivity in cultured neural progenitor cells (NPCs) prepared from the adult mouse subventricular zone (SVZ). Whole-cell patch-clamp recordings showed a marked potentiation of the inward current responses both to ATP and the prototypic P2X7R agonist dibenzoyl-ATP (Bz-ATP) at low Ca2+ and zero Mg2+ concentrations (low X2+) in the bath medium. The Bz-ATP-induced currents reversed their polarity near 0 mV; in NPCs prepared from P2X7−/− mice, Bz-ATP failed to elicit membrane currents. The general P2X/P2Y receptor antagonist PPADS and the P2X7R selective antagonists Brilliant Blue G and A-438079 strongly depressed the effect of Bz-ATP. Long-lasting application of Bz-ATP induced an initial current, which slowly increased to a steady-state response. In combination with the determination of YO-PRO uptake, these experiments suggest the dilation of a receptor-channel and/or the recruitment of a dye-uptake pathway. Ca2+-imaging by means of Fura-2 revealed that in a Mg2+-deficient bath medium Bz-ATP causes [Ca2+]i transients fully depending on the presence of external Ca2+. The MTT test documented a concentration-dependent decrease in cell viability by Bz-ATP treatment. Correspondingly, Bz-ATP led to an increase in active caspase 3 immunoreactivity, indicating a P2X7R-controlled apoptosis. In acute SVZ brain slices of transgenic Tg(nestin/EGFP) mice, patch-clamp recordings identified P2X7Rs at NPCs with pharmacological properties identical to those of their cultured counterparts; nestin is a marker of type-1 and type-2 NPCs. Further, NPCs in the dentate gyrus (DG) of hippocampal brain slices of Tg(nestin/EGFP) mice responded to Bz-ATP and ATP in a manner similar to NPCs in SVZ brain slices of the same type of animal. In short, current responses to ATP and its structural analogue were largely increased when the normal extracellular medium was changed to a low X2 + −containing one. Concentration-response curves for the two agonists revealed a high potency of Bz-ATP in comparison to that of its mother compound. Excised patches still reacted to Bz-ATP confirming that the agonist effect has arisen at the cell under investigation rather than on neighbouring cells releasing an unknown signalling molecule. An inhibitor cocktail blocking AMPA, NMDA, and GABAA receptors as well as voltage-sensitive sodium channels (CNQX, AP-5, gabazine, tetrodotoxin) also failed to interfere with the effect of Bz-ATP. In conclusion, NPCs localized in both neurogenic niches of the adult brain (SVZ, DG) are endowed with P2X7Rs. We suggest that the apoptotic/necrotic P2X7Rs at these cells may be of particular relevance during pathological conditions which lead to increased ATP release and thus could counterbalance the ensuing excessive cell proliferation.

Pannexin1 and P2X7 recptor in chronic pain: experimental and clinical evidence

E. Scemes1, R. Hanstein1, D.C. Spray1 and R.B. Lipton2 1 Department of Neuroscience, Albert Einstein College of Medicine. Bronx, NY, USA 2 Department of Neurology, Albert Einstein College of Medicine. Bronx, NY, USA Pannexin1 (Panx1), a gap junction family of proteins, forms large conductance plasma membrane channels that has been shown to be the P2X7 receptor associated pore. In the nervous system, Panx1 is expressed in neurons and glial cells. Panx1 serves as a pathway for the diffusion of ATP that contributes to purinergic signaling mechanisms involved in a variety of physio(patho)logical situations, including chronic pain. Using a variety of approaches including pharmacological tools and transgenic mice lacking Panx1 and P2X7 receptor, evidence were obtained indicating a critical role of P2X7 receptor—Panx1 module in allodynia using a mouse model of orofacial pain. Evidence that ATP signaling contributes to the development of hyperalgesia/allodynia (Suadicani et al., 2010 Neuron Glia Biol 6:43) is supported by the findings demonstrating that orofacial hypersensitivity is abrogated or significantly attenuated in mice lacking P2X7R or Panx1. Initial screening of candidate genes in a human cohort of older adults identified non-synonimus polymorphisms on the human PANX1 gene likely involved in chronic pain. Statistical analysis suggests that at least one PANX1 SNP is associated with pain intensity/interference. These initial results are in agreement with a previous study indicating that genetically modified P2X7 pore affects chronic pain sensitivity in mice and humans (Sorge et al. 2012 Nat Med 18:595).

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Symposium 8—Biological functions of P2 receptors Role of the P2X7 receptor in the enteric neurons

Patricia Castelucci Department of Anatomy, University of São Paulo, Brazil Adenosine 5′-triphosphate (ATP) is a known co-transmitter in the nervous system and a ligand of the P2X receptor family, which consists of seven known receptor subunits (P2X1-7; Abbracchio et al. 2009). P2X receptors play an important role in synaptic transmission within neural pathways mediating intestinal motility (Bian et al. 2000; Galligan et al. 2000; Galligan 2002). The P2X7 receptor is known to mediate cell inflammation, apoptosis and proliferation (Burnstock 2013; Volonté et al. 2012). Immunohistochemical studies have documented the distribution of P2X receptors in the enteric nervous systems of the guinea pig (Castelucci et al. 2002; Hu et al. 2001; Poole et al. 2002; Van Nassauw et al. 2002; Vulchanova et al. 1996; Xiang et al. 2005), rat (Xiang and Burnstock, 2004a, 2004b; Yu et al. 2010) and mouse (Giaroni et al. 2002; Ruan et al. 2005). Inflammatory bowel diseases (IBD) are chronic diseases that affect the gastrointestinal tract these diseases include ulcerative colitis and Crohn’s disease (Kawada et al. 2007). Changes in neurotransmitters and neuropeptides produced by enteric neurons with IBD (Boyer et al. 2005; Lomax et al. 2005; Sharkey and Kroese, 2001) have also been observed. The expression of the P2X7 receptor has been studied in enteric neurons in both malnutrition (Girotti et al. 2013) and ischemia (Palombit et al. 2013). In addition, studies have observed the presence of P2X7 receptor in the glial cell of the rat gastrointestinal tract (Vanderwinden et al. 2003). However, the chemical code of the P2X7 receptor in ulcerative colitis has not been verified. The present work analyzed the effects of ulcerative colitis on neurons immunoreactive (IR) for the P2X7 receptor by specifically examining the expression of nitric oxide synthase (NOS), choline acetyltransferase (ChAT), Calbindin and Calretinin. Additionally, the neuronal density and somatic size of enteric glial cells in the myenteric plexus of the rat distal colon were analyzed in a colitis model. The colocalization of the P2X7 receptor-immunoreactive (IR) cells was observed in the myenteric plexus with NOS-, ChAT-, Calbindin-, Calretinin- and HuC/D-IR neurons and S100β-IR cells in the control, sham and colitis groups. The neuronal density (cell bodies/cm2) decreased in the myenteric plexus by 11, 18, 34, 22, and 60 % in the P2X7 receptor, NOS-, ChAT-, Calbindin- and, Calretinin neurons, respectively. In addition, the densities (cell bodies/cm2) of HuC/D-IR neurons and S100β-IR enteric glial cells decreased by 33 and 29 %, respectively. The profile areas were reduced by 6.8 and 21 % in NOS- and ChAT-IR neurons, respectively. There was also a 20 % increase of Calbindin-IR neurons. Morphological changes were observed, such as increased neutrophils, disintegration of the intestinal epithelium and goblet cells and decreased collagen. This study demonstrated that colitis differentially affects P2X7 receptor-expressing enteric neurons based on their chemical codes and may cause changes in morphology and motility. Support: FAPESP, CNPq Human bladder hyperactivity caused by UDP-sensitive P2Y6 receptors: on the role of ATP release from the urothelium via pannexin1 hemichannel

Isabel Silva1,2, Fátima Ferreirinha1,2, Miguel Silva-Ramos3 and Paulo Correia-de-Sá1,2 1 Laboratório de Farmacologia e Neurobiologia, 2Center for Drug Discovery and Innovative Medicines (MedInUP), Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto, Portugal 3 Serviço de Urologia, Centro Hospitalar do Porto (CHP), Porto, Portugal Objectives / background: Changes in the bladder sensory system during urine storage may contribute to persistent detrusor overactivity in patients with bladder outlet obstruction. Deregulation of purinergic signaling through the abnormal production, release and metabolism of ATP or altered expression of various P2 purinoceptors is a common feature of many urological diseases (e.g. interstitial cystitis, neurogenic bladder, outlet obstruction). We demonstrated that urinary ATP may be a dynamic biomarker of detrusor activity in overactive bladder syndromes (Silva-Ramos et al., 2013, PLoS One 8:e64696). Besides the influence of ionotropic P2X3 receptors localized in suburothelial sensory nerves, we showed that UDP-sensitive P2Y6 receptors increases the voiding frequency indirectly by releasing ATP from the urothelium via pannexin-1 hemichannels in the rat (Timóteo et al., 2014, Biochem Pharmacol. 87:371–9). Co-expression of P2Y6 and P2X3 in the urothelium has been shown by confocal microscopy (Carneiro et al., 2014, Br J Pharmacol. 171:3404–19). Therefore, we sought it was important to investigate the crosstalk between these two receptor subtypes regarding the release of signaling molecules (ATP and [3H]ACh) from the human urothelium. Methods and results: Human bladder samples were collected from cadaveric organ donors and patients with benign prostatic hyperplasia (BPH). All procedures were approved by the Ethics Committees of CHP-HGSA and ICBAS-UP. The ATP/[3H]ACh ratio was 5-fold higher in urothelial strips from BPH patients compared to control men. The selective P2Y6 receptor agonist, PSB0474 (100 nM), augmented by a similar proportion ATP and [3H]ACh release from the urothelium of both groups of individuals; the facilitatory effect of PSB0474 (100 nM) was prevented by MRS2578 (50 nM) and by carbenoxolone (10 μM), which selectively block the P2Y6 receptor and pannexin-1 hemichannels, respectively. Blockade of P2X3 receptors with A317491 (100 nM) also attenuated release facilitation by PSB0474 in control men, but not in BPH patients. Immunolocalization studies showed that P2Y6 and P2X3 receptors are present in choline acetyltransferase (ChAT) positive urothelial cells. However, in contrast to P2Y6 staining, immunoreactivity against ChAT and P2X3 decreased in the urothelium of BPH patients. Conclusion: Data indicate that activation of UDP-sensitive urothelial P2Y6 receptors triggers the release of ATP (as well as ACh) via pannexin-1 hemichannels, which may subsequently activate P2X3 receptors on neighboring urothelial cells and suburothelial sensory nerves to increase bladder activity. Thus, selective blockade of urothelial P2Y6 receptors might have therapeutic relevance to control persistent storage symptoms in BPH patients. Work supported by FCT (FEDER funding, PTDC/SAU-OSM/104369/2008 and Pest-OE/SAU/UI215/2014), Assoc. Port. Urologia and Univ. Porto / Caixa Geral de Depósitos. IS is in receipt of a PhD fellowship by FCT (SFRH/BD/88855/2012).


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Purinergic signaling in the immune-pineal axis

Z.S. Ferreira Laboratório de Cronofarmacologia, Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil The immune-pineal axis is defined as the switch of melatonin (MEL) synthesis from the pinealocytes to immune-competent cells. It is activated during the mounting of an inflammatory response and promotes a transient facilitation of leukocytes migration to the site of lesion, and a subsequent local synthesis of MEL that contributes to the resolution phase of the inflammatory response. The MEL synthesis by the pineal gland is driven by the two co-transmitters of the sympathetic nervous system (noradrenaline and ATP). It involves acetylation of serotonin and subsequent methylation of N-acetylserotonin (NAS) by the enzymes arylalkylamine-N-acetyltransferase (AA-NAT) and acetylserotonin-methyltransferase (ASMT), respectively. Both in pinealocytes and immunecompetent cells, the effect of pathogen- or damage-associated molecular patterns (PAMPs or DAMPs) is mediated by NFkB. The cell-specific nuclear translocation of NFkB dimers provided or not with transactivating domains will result in activation or inhibition of Aa-nat transcription (Markus et al., Int. J. Mol. Sci. 14:10979, 2013). Besides the co-transmitter role, ATP also acts as a DAMP, when activates P2X7 receptors (P2X7R). When ATP and analogs act as co-transmitter, the receptors activated are P2Y1, which lead to an increase in intracellular calcium via activation of phospholipase C. Although activation of P2Y1 leads to an increase in NAS, it reduced the final output of MEL. The increase in the precursor NAS is due to a calcium-mediated increase in the activity of AA-NAT, while the reduction of MEL synthesis is mediated by the decrease in the transcription of the gene and activity of the enzyme ASMT. The end product of ATP metabolism, adenosine, induces melatonin synthesis (Gharib et al., Neurosci. Lett 106: 345,1989). It is noteworthy that the expression of the genes and the activity of the enzymes that metabolize ATP have a daily variation, being more active at nighttime, while the activity of the enzyme that metabolize adenosine did not vary along the day. Therefore, our data clearly show that purinergic system presents a daily adaption for regulating physiological pineal gland activity. In pathophysiological condition cell disruption provides a higher amount of extracellular ATP. Then, we investigated whether higher concentrations of ATP could stimulate P2X7 receptors in the pineal gland, and provide a mechanism for understanding its role in defense responses. ATP (0.01–3 mM) and BzATP (30– 300 μM), a selective agonist for P2X7R reduced the content of beta-adrenoceptors-induced synthesis due to a decrease in the transcription of the gene and protein synthesis of ASMT, strongly suggesting that activation of P2X7R could play a role in the decrease of melatonin synthesis when the immune-pineal axis is activated. To evaluate if activation of P2X7R could induce melatonin synthesis by immune-competent cells, we tested the RAW 264.7 cell line macrophages. The basal melatonin content of these cells was below the detection limit. ATP (0.01–3 mM) increased MEL content in the supernatant up to 74.6 ± 6.6 pg/mL (n = 3). The selective P2X7R antagonist A438079 blocks AA-NAT phosphorylation, which is necessary for the activation of the enzyme. Conclusion: Our data show that ATP-stimulation of P2X7R suppresses pinealocytes MEL synthesis and promotes the synthesis of this indoleamine by macrophages. Therefore, high concentrations of ATP released as a damage signal activates the immune-pineal axis. Support: CAPES, FAPESP (2013/13691-1), CNPq. Functional expression of P2X7 receptors in the ovarian surface epithelium

A. Cruz-Rico, A.S. Martínez-Ramírez, E. Garay, R.O. Arellano and F.G. Vázquez Cuevas Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Mexico Objectives/Background: Ovarian surface epithelium (OSE) is the layer surrounding the ovary of mammals, and it has been proposed that it plays a role in the ovarian architectural changes that enable oocyte expulsion in each ovulatory cycle. Ovarian carcinoma is thought to originate in the OSE because this structure undergoes constant organizational changes. In the present study we investigate the functional expression of the P2X7 receptor, an ion channel operated by extracellular ATP that displays a broad spectrum of cellular functions, ranging from apoptosis to cell proliferation and tumorigenesis, in the OSE of mouse and in human carcinoma. Methods and results: In slices of ovary analyzed by immunofluorescence, P2X7 receptor expression was observed in the OSE, and the immunoreactivity colocalized with the marker cytokeratin 18. In OSE cells in culture, stimulation with 10 μM BzATP induced a dose-dependent increase in the cytoplasmic Ca2+ levels (EC50 = 1.2 ± 0.8 μM) to a maximum of 366 ± 13 % of the basal level. This response was sustained in the presence of the agonist but exhibited clear kinetic differences with responses elicited by 10 μM ATP; BzATP also induced uptake of YO-PRO dye. To study the response of the P2X7 receptor in OSE in situ, we injected purinergic substances intrabursally and analyzed the induction of apoptosis by TUNEL 24 h after. On the dioestrous and proestrus days of the estrous cycle the injection of BzATP induced TUNEL-positive cells; this effect was prevented by the antagonist A438079, suggesting that P2X7 receptor expression is under hormonal regulation. As mentioned, OSE cells are proposed to have a role in carcinogenesis. To analyze the expression of P2X7 in ovarian carcinoma, biopsies were analyzed by immunofluorescence. Samples of different carcinoma subtypes showed strong P2X7 expression, which was specifically enhanced in the OSE layer. The carcinoma cell lines SKOV-3 and CAOV-3 also showed P2X7 receptor expression. Stimulation of the P2X7 receptor with 10 μM BzATP did not induce cell death; moreover, BzATP elicited increases in the phosphorylation levels of the ERK and AKT kinases. Conclusion: the P2X7 receptor is expressed in the OSE layer, and its expression level depends on the day of the estrous cycle. Human carcinoma biopsies express high levels of this receptor. The P2X7 receptor can induce cell death in healthy mouse OSE cells but not in OSE-derived carcinoma cells. The P2X7 receptor is a modulator of the equilibrium between death and proliferation in OSE cells, and that it has a role in the pathological state. Supported by grants of CONACyT (166725) and PAPIIT-UNAM (IN205114) to V-CFG.

Symposium 9—Purinergic signaling in stem and cancer cells Mesenchymal stem cells have differential capacity to metabolize extracellular nucleotides

Márcia Wink Fundação Universidade Federal de Ciências da Saúde de Porto Alegre, UFCSPA, Brazil Mesenchymal stem cells (MSCs) have shown a great potential for cell-based therapy and many different therapeutic purposes. Despite the recent advances in the knowledge of MSCs biology, their biochemical and molecular properties are still poorly defined. Ecto-nucleoside triphosphate

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diphosphohydrolases (E-NTPDases) and ecto-5′-nucleotidase (eNT/CD73) are widely expressed enzymes that hydrolyze extracellular nucleotides, generating an important cellular signaling cascade. Currently, studies have evidenced the relationship between the purinergic system and the development, maintenance, and differentiation of stem cells. The objective of this study is to identify the NTPDases and eNT/CD73 and compare the levels of nucleotide hydrolysis on MSCs isolated from different murine tissues (bone marrow, lung, vena cava, kidney, pancreas, spleen, skin, and adipose tissue). MSCs from all tissues investigated expressed the ectoenzymes at different levels. In MSCs from pancreas and adipose tissue, the hydrolysis of triphosphonucleosides was significantly higher when compared to the other cells. The diphosphonucleosides were hydrolyzed at a higher rate by MSC from pancreas when compared to MSC from other tissues. The differential nucleotide hydrolysis activity and enzyme expression in these cells suggests that MSCs play different roles in regulating the purinergic system in these tissues. Overall MSCs are an attractive adult-derived cell population for therapies, however, the fact that ecto-nucleotide metabolism can affect the microenvironment, modulating important events, such as immune response, makes the assessment of this metabolism an important part of the characterization of MSCs to be applied therapeutically. A model for neural development of Rett syndrome using human stem cells: the role of the neurotrasmitter receptors

Cleber Trujillo University of California, San Diego, USA Autism spectrum disorders (ASD) are complex neurodevelopmental diseases in which different combinations of genetic mutations may contribute to the phenotype. Using Rett syndrome (RTT) as an ASD genetic model, we developed a culture system using induced pluripotent stem cells (iPSCs) from RTT patients’ fibroblasts. RTT patients’ iPSCs are able to undergo X-inactivation and generate functional neurons. Neurons derived from RTT-iPSCs had fewer synapses, reduced spine density, smaller soma size, altered calcium signaling and electrophysiological defects when compared to controls. Our data uncovered early alterations in developing human RTT neurons. Finally, we can RTT neurons to test the effects of drugs in rescuing synaptic defects. Our data provide evidence of an unexplored developmental window, before disease onset, in RTT syndrome where potential therapies could be successfully employed. Our model recapitulates early stages of a human neurodevelopmental disease and represents a promising cellular tool for drug screening, diagnosis and personalized treatment. More broadly, using cut-edge technologies like genome editing and the generation of a human chimeric brains by transplanting MeCP2-mutant-derived NPCs into a mouse brain allow us to understand the maturation of human neurons and generating unparalleled insights into the disease progression. Human-mouse chimeric brains could ultimately serve as a more predictive preclinical translational model for studying disease mechanisms. Purinergic signaling in neuroblastoma migration and metastasis (Oral communication)

Henning Ulrich1, Mariusz Z. Ratajczak2, Gabriela Schneider2, Enéas Galdini Ferrazoli1, Talita Glaser1, Micheli M. Pillat1 and Claudiana Lameu1,2 1 Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, SP, Brazil 2 Stem Cell Institute, James Brown Cancer Center, University of Louisville, KY, United States Background: Bone marrow metastasis is present in approximately 350,000 patients that die annually in the U.S. alone. While the stromal cell-derived factor-1 (SDF-1)-CXCR4 axis is known to exert chemotaxis in bone metastasis, such functions have not been studied for the interaction of SDF-1 with the kinin and purinergic system. Methods: Assays of intracellular Ca2+ mobilization, real time PCR, adhesion, proliferation and chemotaxis of neuroblastoma cells in vitro and in vivo were used. Results: We show here that bradykinin augments chemotactic responsiveness of three neuroblastoma cells line for physiological SDF-1 and ATP concentration. In line with the recruitment of cancer cells to the bone-marrow expression of bradykinin precursors and kinin-B2 receptors was upregulated in the bone marrow of irradiated mice. Bradykinin promoted adhesion, SDF-1-induced Ca2+ mobilization as well as resensitization and expression of P2X7 purinergic receptors. Immunodeficient nude/nude mice transplanted with neuroblastoma cells pretreated with BK revealed significantly higher metastasis to the bone marrow than observed in animals engrafted with untreated cells. Animals receiving Brilliant Blue G (BBG), an antagonist of the P2X7 receptor, did not show metastasis to bone marrow and liver or metastasis rates were drastically reduced. Conclusion: Advances have been made to understand the interrelationship between the kinin and the purinergic systems in the metastasis of neuroblastoma cells opening novel perspectives for therapy. Financial support: FAPESP and CNPq. Spontaneous calcium oscilations are modulated by P2 purinergic receptors and control expression of neurogenesis related transcription factors (Oral communications)

Talita Glaser1, Hiromi Shimojo2, Ana Regina Geciauskas Lage Castillo1, Ryoichiro Kageyama2 and Henning Ulrich1 1 University of São Paulo, Brazil 2 Kyoto University, Japan Background: Purinergic receptors have been attributed with developmental functions including gastrulation and neural differentiation. Upon activation many P2 purinergic receptors trigger intracellular calcium transients controlling different cell processes. Since spontaneous calcium signaling is involved in neural differentiation, we induced neuronal differentiation of mouse neural stem cells and embryonic stem cells (ESC) with retinoic acid to study the influence of P2 receptors activity on spontaneous calcium transients and consequently on expression of transcription factors related to neurogenesis. Principal findings: First of all, during different moments of neural differentiation we characterized, by time lapse imaging of fluorescent calcium indicator, the different patterns of spontaneous calcium transients (waves and spikes) and reported that both frequency and amplitude increased along differentiation. Cells treated with inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor (Xestospongin C) showed spikes but not waves, indicating that waves depend exclusively on Ca2+ release from the endoplasmic reticulum (ER) and IP3R activation. Cells treated with various P2 receptor subtype modulators (Bz-ATP/P2X7R agonist, 2SUTP/P2Y2R agonist, MRS2179/ P2Y1R antagonist) increased frequency and amplitude of calcium transients, mainly spikes in NSC and ESC differentiated for 8 days. Data obtained by luminescence time lapse imaging of stable transfected cells with Mash1 or Ngn2 promoter-protein fusion with a luciferase reporter


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showed an increase of Mash1 and Ngn2 expression due to activation of P2Y2R or inhibition of P2Y1R. Expression of these transcription factors decreased following P2X7R activation. Furthermore, cells imaged in presence of extracellular calcium chelator (EGTA) or following ER-Ca2+ store depletion by thapsigargin diminished Mash1 and Ngn2 expression, indicating that both are regulated by calcium signaling. Conclusion: Altogether these data suggest that P2X7, P2Y2 and P2Y1 purinergic receptors modulates spontaneous calcium oscillations during neural differentiation and consequently change the expression pattern of Mash1 and Ngn2, thus controlling the cell fate decision towards neuronal phenotypes. Acknowledgements: This research is supported by FAPESP and CNPq.

Symposium 10—Purinergic signaling in the immune and cardiovascular system Purinergic signaling in T-cells and epicardial progenitor cells after myocardial infarction

J. Schrader, N. Borg, J. Hesse, D. Friebe and X. Ding Institute of Molecular Cardiology, Heinrich-Heine-University of Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany Myocardial infarction (MI) leads to the invasion of immune cells to the site of injury and at the same time induces the formation of epicardium derived cells (EPDC) which recapitulate an embryonic program to stimulate cardiac regeneration. Immune cells and EPDC are both characterized by high expression of CD73 and this study explored their functional relevance. After ischemia/reperfusion (I/R) we found upregulation of CD73 expression and activity on infiltrating T-lymphocytes. I/R in a global CD73 knockout resulted in a prolonged cardiac inflammatory response, enhanced fibrosis, and immature scar formation. Using T-cell specific CD73−/− mice we surprisingly found ventricular impairment and immature scar formation to be identical to that of the global CD73 knockout. To gain further mechanistic insights, T-cells were isolated from control and T-cell specific CD73−/− mice hearts subjected to I/R. We found that lack of CD73-derived adenosine triggers the formation of pro-inflammatory and pro-fibrotic cytokines (IL-17; IFN-γ), which may serve as key signals for the observed changes in phenotype. Gene expression analysis revealed that I/R induces on cardiac T-cells the de-novo expression of the A2B adenosine receptor which was not expressed on respective blood cell controls. EPDC are of mesenchymal origin and CD73 is well known to be a key marker enzyme. We succeeded in culturing EPDC from the rat heart and found that they not only have high CD73 activity but also rapidly degrade ATP via ADP and AMP to adenosine (HPLC analysis). Surprisingly, NAD is equally well hydrolyzed to adenosine involving ADP-ribose and AMP. Thus, CD73 is the critical bottleneck of both degradation pathways. Quantitative RT-PCR showed that EPDC express a distinct set of both adenosine (A2A > A2B > A3 > A1) and ATP (P2X4 > P2X7 > P2X5; P2Y2 > P2Y4 > P2Y12) receptors. As to the endogenous source of nucleotides, we found quinacrine (ATP store marker) and Bodipy ATP (fluorescent ATP analog) localized to vesicular structures suggesting ATP-storing granules. In line with this hypothesis, elevating intracellular Ca++ by ionomycin caused the release of ATP which could be blocked by brefeldin A. Pharmacologic stimulation of A2B but not A2A receptors greatly stimulated the release of IL-6 and VEGF. In summary our findings demonstrate that adenosine formed by CD73 on T-cells orchestrates the healing process after MI most likely involving A2B receptors. EPDC can release nucleotides from vesicular structures, avidly degrade ATP as well as NAD via CD73 to adenosine which acts on A2B receptors to release IL-6 and VEGF. CD73 on T-cell and EPDC are therefore crucial in modulating the cardiac cytokine milieu after MI. Modulation of the nitreegic system by A1 and A2A receptors. Focus on the mechanisms involved in neural regulation of blood pressure

Debora Fior-Chadi1, Daniel C. Carrettiero2, João Paulo P. Matsumoto1 and Maisa A. Costa1 1 University of São Paulo, Brazil 2 Federal University of ABC, Brazil Adenosine and nitric oxide act on the fine-tuning regulation of neural cardiovascular control in the nucleus tractus solitarius (NTS). The mechanisms by which adenosine interferes in the dynamics of nitrergic neurotransmission, is not completely understood. Thus, changes in the interaction between these systems may be especially relevant for individuals predisposed to hypertension. The interaction between the adenosinergic and nitrergic systems in cell culture from the dorsomedial medulla oblongata of Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) will be discussed based on the quantification of nitrite levels, RT-PCR analysis and RNA interference. Adenosine A1 (A1R) and A2a receptor (A2aR) agonists induced a concentrationdependent decrease and increase of nitrite and nNOS mRNA levels in cultured cells from WKYand SHR, respectively. These effects in nitrite levels were attenuated by the administration of the A1R and A2aR selective antagonists, CPT and ZM 241385. Furthermore, knockdown of A1R and A2aR showed an increase and decrease of nNOS mRNA levels, respectively. Pretreatment with the nonselective inhibitor of NOS, L-NAME, abolished nitriteincreased levels triggered by CGS 21680 in WKY and SHR cells. It was also shown that the cAMP-PKA pathway is involved in A1R and A2aR mediated decrease and increase in nitrite levels in SHR and WKY cells. In summary, the results highlight the influence of adenosine on nitric oxide levels in cultured cells from dorsal medulla oblongata of WKY and SHR rats. Effect of vitamin D supplementation on the purinergic system

Daniela Bitencourt Rosa Leal Pós-Graduação em Ciências Biológicas: Bioquímica Toxicológica, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, RS, Brazil Objectives/background: The deficiency of vitamin D, a hormone known to play an important role in calcium homeostasis, has been related to several autoimmune diseases, including rheumatoid arthritis (RA) and type 1 diabetes. This fact is due to its immunomodulatory role, inhibiting the proliferation of T lymphocytes, especially Th1 lymphocytes as well as in the production and action of cytokines. The purinergic signalling system plays an important role in

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the modulation of inflammatory and immune responses through extracellular biomolecules such as adenine nucleotides and its derivative adenosine nucleoside, whose extracellular concentrations are controlled by the activity of ectoenzymes such as ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) and ecto-adenosine deaminase (E-ADA) present on the surface of various cells. Based on these principles, the effect of vitamin D3 was evaluated on the activity of E-NTPDase and E-ADA enzymes in lymphocytes from animals with arthritis induced by complete Freund’s adjuvant (CFA) and on oxidative stress biomarkers in cerebral cortex, lipid metabolism, ectonucleotidases activities in platelets and on memory of streptozotocin-induced diabetic rats. Methods and Results: Hematological and biochemical parameters, including serum concentrations of vitamin D, were determined. The results showed that adult Wistar rats with induced arthritis treated with 120 IU/day of VD3 or vitamin D3 lipid-core nanocapsules formulation at a dose of 15.84 IU/day for 15 days had a reduction in arthritis score, thermal hyperalgesia and paw edema. At the same time, histological analyzes showed that both formulations were able to reduce inflammatory changes induced by CFA together with an increase in the serum levels of 25(OH)D. The activity of E-NTPDase in lymphocytes from rats that developed RA was increased in comparison with control group, whereas the activity of E-ADA was decreased and this effect was reversed with VD3 treatment. This vitamin had a protective effect against cerebral cortex damage promoted by oxidative stress in diabetic rats, evidenced by the ability of this compound in preventing the lipid peroxidation in this structure and even modifications in the activity of enzymes important to cognitive processes. Such protection has resulted in improved memory performance in rats treated with VD3. Additionally, it has shown to have potential in preventing hyperlipidemia and platelet hyperreactivity in the diabetic state by avoiding the increased activity of ectonucleotidases. Conclusion: VD3 is capable of modulating the activity of ectoenzymes in platelets and lymphocytes and to improve memory performance, suggesting that it could be used as a complementary therapeutic agent for the treatment of autoimmune diseases. Supported by: CAPES, CNPq, FAPERGS and UFSM. Inhibitors of the 5-lipoxygenase pathway activate pannexin1 channels in macrophages via the thromboxane receptor (Oral communication)

H.A. Da Silva-Souza1,2,3, M.N. Lira1, N.K. Patel3, D.C. Spray3, P.M. Persechini1,2 and E. Scemes3 1 Institute of Biophysics Carlos Chagas Filho – Federal University of Rio de Janeiro –UFRJ; Rio de Janeiro –RJ –Brazil 2 National Institute of Science and Technology of Translational Research in Health and Environment of the Amazon Region– INPeTAm, Brazil 3 Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York, USA Objectives / background: A multitude of environmental signaling molecules influence monocyte and macrophage innate and adaptive immune responses, including ATP and prostanoids. Interestingly, we have previously described that 5-LOX inhibitors induced the influx of cationic dyes in macrophages independently of P2X7 receptor activation and that such influx did not occur after inhibiting the activity of cyclooxygenases, enzymes that transform arachidonic acid (AA) into prostanoids. Thus these recent studies raise the possibility that the production of prostanoids resultant from the blockade of the 5-LOX pathway activates a permeation pathway with properties (influx of dye and ATP release) similar to those described for pannexin-1 (Panx-1). Here, we provide evidence that Panx-1 is a component of this pathway and present the intracellular signaling molecules linking the thromboxane (TP) receptor to Panx1-mediated dye influx and ATP release. Methods and Results: Using pharmacological tools and transgenic mice deficient in Panx-1, we have used fluorescent dye uptake assays in thioglycollateelicited murine peritoneal macrophages and we also have measured the release of ATP in these cells using the luciferin/luciferase assay with a luminometer. Our results show that two 5-LOX pathway inhibitors induce ATP release and influx of dye in a Panx-1-dependent manner. Electrophysiological recordings performed in wild-type (WT) and Panx1-deficient macrophages confirmed that these 5-LOX pathway inhibitors activate currents characteristic of Panx-1 channels. WT and Panx-1 peritoneal macrophages were used to quantify the levels of Panx-1 transcripts (qRT-PCR). We found that the mechanism by which Panx-1 channels are activated under this condition involves activation of the TP receptor that is mediated by the cAMP/PKA pathway. This is to our knowledge the first evidence for the involvement of Panx-1 in the TP receptor signaling pathway. Future studies aimed to clarify the contribution of this TPPanx-1 signaling network to macrophage immune responses are likely to be important for targeting inflammatory and autoimmune diseases. Conclusion: We conclude that macrophages release ATP in response to 5-LOX inhibitors have important implications for the understanding of the interplay between purinergic and prostanoid signaling in the biology of monocytes. Financial Suport: CNPq, FAPERJ, INCT-INPeTAm and Fogarty Training Grant. Guest conference 4 How plants recognize and respond to extracellular ATP

Gary Stacey Division of Plant Science, C.S. Bond Life Science Center, University of Missouri, Columbia, MO, USA Adenosine triphosphate (ATP) is well known as an energy currency in most organisms. However, ATP also acts as a signal molecule when it is released into the extracellular matrix. While the function of extracellular ATP (eATP) signaling in animals has been extensively studied, the role of eATP as a signal molecule in plants has been identified relatively recently. Exogeneous ATP application induces measurable physiological effects on plant growth, development and stress responses. In animals, secreted ATP is recognized by the two classes of plasma membrane receptors, P2X and P2Y, functioning as ligand-gated ion channels and G-protein coupled receptors, respectively. However, utilizing the sequences of these animal receptors to search the available plant genomes failed to identify eATP receptor candidates. However, the use of animal-based pharmacological reagents suggests that plants may possess non-canonical P2-like receptors. Recently, we identified the first ATP receptor in plants, doesn’t respond to nucleotides, DORN1. This protein consists of an extracellular lectin domain, a transmembrane domain and an intracellular serine/threonine kinase domain. DORN1 is distinct from the P2Y and P2X purinoreceptors found in animals and, hence, it represents the founding member of a new family of purinoreceptors, termed P2K (k = kinase). More recent work in the laboratory has focused on two areas: 1. studies to understand the function of the DORN1 receptor and 2. studies to define the role of extracellular ATP signaling in the context of the broader response of plants to stress. For example, molecular modeling of the DORN1 extracellular domain suggested key residues as being involved in ATP binding. These residues were mutated and were shown to be essential for ATP binding. Likewise, we have begun to explore the DORN1 receptor complex.


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The data suggest that ATP is released from plants in response to stress agents. For example, wounding causes the release of ATP. This ATP then acts as a DAMP (damage associated molecular pattern) to induce downstream stress response pathways. We are exploring the connection between extracellular ATP and the response of plants to a variety of stresses. References 1. Choi, J., Tanaka, K. et al. (2014) Identification of a plant receptor for extracellular ATP. Science Vol. 343 no. 6168 pp. 290–294 2. Cao, Y., Tanaka, K. et al. (2014) Extracellular ATP is a central signaling molecule in plant stress responses. Curr. Op. Plant Biol. 20: 82–87 3. Choi, J., Tanaka, K. et al. (2014) Extracellular ATP, a danger signal, is recognized by DORN1 in Arabidopsis. Biochem. J. (in press) 4. Tanaka, K., Choi, J. et al. 2014. Extracellular ATP as a damage associated molecular pattern (DAMP) signal in plants. Frontiers in Plant Science (in press, available online) Symposium 11 – Purinergic transmission modulating migration, cell death and survival Lighting up adenosine receptors in neuronal death

Francisco Ciruela1,2 and Víctor Fernández-Dueñas1 Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina, IDIBELL, Universitat de Barcelona, 08907 L’Hospitalet de Llobregat, Spain 2 Department of Physiology, Faculty of Sciences, University of Ghent, 9000 Gent, Belgium 1

Parkinson’s disease (PD) is a neurodegenerative condition of the central nervous system characterized by brandykinesia, tremor and rigidity. These symptoms are secondary to the death of dopaminergic neurons within the substantia nigra and the concomitant lost of dopaminergic projections to and dopamine content in the striatum. In the last decade it has been demonstrated that dopaminergic and adenosinergic transmission systems are functionally and physically interconnected. Indeed, we recently demonstrated a direct receptor-receptor—i.e. dopamine D2 receptor (D2R) and adenosine A2A receptor (A2AR)—interaction in native conditions. Thus, by means of different complementary experimental approaches (i.e. immunoelectron microscopy, proximity ligation assay and TR-FRET), we unambiguously identified native D2R/A2AR oligomer in rat striatum. Importantly, in a PD animal model (i.e. the unilaterally 6-hydroxydopamine lesioned rat) the amount of D2R/A2AR oligomer was reduced in the lesioned hemisphere when compared to the control opposite one. Overall, these results provide definitive evidence for a native D2R/A2AR oligomer alteration in experimental parkinsonism, thus put into the context the rationale of using A2AR antagonists in PD pharmacotherapy. This work was supported by grants SAF2014-55700-P, PCIN-2013-019-C03-03 and PIE14/00034 from Ministerio de Economía y Competitividad/ Instituto de Salud Carlos III, ICREA Academia-2010 from the Catalan Institution for Research and Advanced Studies and SBO-140028 from Agentschap voor Innovatie door Wetenschap en Technologie. Involvement of nucleotides in the adhesion and migration of retinal glial cells in culture

A.L.M. Ventura1, T. Martins Silva1, G.R. França1, H. Ulrich2, I.M. Ornelas1 and E.C. Loiola1 1 Departamento de Neurobiologia, Programa de Neurociências, Universidade Federal Fluminense, Niterói, RJ, Brazil 2 Departamento de Bioquímica, Universidade de São Paulo, São Paulo, SP, Brazil ATP is an emerging signaling molecule that via activation of P2 receptors modulates the development of the retina. Previous data have shown that while activation of P2Y1 receptors induces the proliferation of glial/bipolar progenitors in culture via stimulation of phospholipase C, PKC, MAP kinases and the PI3K/Akt pathway, activation of P2X7 receptors induces the death of cultured developing retinal neurons. In the present study, we show that activation of nucleotide receptors also regulates the adhesion and migration of retinal glial cells in culture. When retinal cell cultures are mechanically scratched, only dividing and migrating 2 M6 positive glial cells grow over the empty area. Apyrase, Suramin and Reactive Blue 2 (RB-2), but not MRS 2179 or PPADS, significantly attenuate glial growth toward the scratched area, indicating that receptors other than P2Y1 are involved in glial growth. These receptors likely are UTP-sensitive receptors since UTPγS but not ADPβS antagonizes apyrase-induced inhibition of glial growth in the scratched cultures. Glial proliferation at the border of the scratch is not affected by nucleotides as no decrease in PCNA+ cells is observed at the border of the scratch in apyrase-treated cultures. Glial cytoplasm protrusions are smaller and unstable in apyrase-treated cultures showing less organized actin filaments and alfa-tubulin-labeled microtubules parallel to scratch. Very few vinculin-labeled adhesion sites are noticed in the treated cultures. Phosphorylation of both Akt and ERKs is observed in UTP-treated retinal cultures, a response that is inhibited by the SRC inhibitor 1 and the PI3K blocker LY294002. Both blockers and the FAK inhibitor PF573228 also decrease glial growth in scratched cultures. Regulation of glia adhesion and migration by nucleotides is indicated by data showing that RB-2 decreases dissociated glial cell attachment to fibronectin-coated dishes and migration through transwell membranes. Additional data reveal that developing retinal glial cells do express UTP-sensitive P2Y2 receptors that under activation with the selective agonist MRS 2768 induce intracellular calcium increase in glial cells at the border of the scratched area. Supported by: FAPERJ, CNPq, PROPPi-UFF, CAPES. Is adenosinergic system always neuroprotective?

C.R. Boeck Programa de Pós-graduação em Nanociências, Centro Universitário Franciscano, Santa Maria/RS, Brazil Cellular tolerance or resistance can be achieved by interventions before or after injury through potential toxic agents used in low stimulus or dose. Brain tolerance evoked by pre- or postconditioning is a neuroprotective strategy that can afford attenuation of motor, cognitive, emotional, or memory deficits

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aroused following a brain damage. Glutamate receptors are protagonists of the adjacent cellular mechanisms contributing to damage as well as tolerance. Adenosinegic system has been reported as a fundamental endogenous neuroprotective system in the adjacent cellular mechanisms contributing to the development of brain tolerance. Ecto-nucleotidases and adenosine receptors are associated to protection elicited by adenosine, but in specific stimulus they contribute to the cell damage. When and how is adenosine neuroprotective? “NAADP modulates calcium signaling and autophagy in cell death”. Calcium signaling alterations, oxidative stress, and autophagy in aging

Rodrigo Portes Ureshino, Katiucha Karolina Rocha, Guiomar Silva Lopes, Cláudia Bincoletto and Soraya Soubhi Smaili Department of Pharmacology, Federal University of São Paulo, Rua Três de Maio, n.100, São Paulo/SP 04044-020, Brazil Aging is a multi-factorial process that may be associated with several functional and structural deficits which can evolve into degenerative diseases. In this review, we present data that may depict an expanded view of molecular aging theories, beginning with the idea that reactive oxygen species (ROS) are the major effectors in this process. In addition, we have correlated the importance of autophagy as a neuroprotective mechanism and discussed a link between age-related molecules, Ca2+ signaling, and oxidative stress. There is evidence suggesting that alterations in Ca2+ homeostasis, including mitochondrial Ca2+ overload and alterations in electron transport chain (ETC.) complexes, which increase cell vulnerability, are linked to oxidative stress in aging. As much as Ca2+ signaling is altered in aged cells, excess ROS can be produced due to an ineffective coupling of mitochondrial respiration. Damaged mitochondria might not be removed by the macroautophagic system, which is hampered in aging by lipofuscin accumulation, boosting ROS generation, damaging DNA, and, ultimately, leading to apoptosis. This process can lead to altered protein expression (such as p53, Sirt1, and IGF-1) and progress to cell death. This cycle can lead to increased cell vulnerability in aging and contribute to an increased susceptibility to degenerative processes. A better understanding of Ca2+ signaling and molecular aging alterations is important for preventing apoptosis in age-related diseases. In addition, caloric restriction, resveratrol and autophagy modulation appear to be predominantly cytoprotective, and further studies of this process are promising in age-related disease therapeutics. P2X7, a receptor with a split personality: an oncogene or an oncosuppressor gene?

F. Di Virgilio Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Italy The P2X7 receptor (P2X7R or P2RX7, according to gene nomenclature) is a member of the P2 receptor family. This receptor has been widely associated to cytotoxicity and release of pro-inflammatory cytokines (1). However, it now clear that the P2X7R has a strong promoting effect on cell proliferation. This has led to investigation of P2X7R contribution to tumor progression (2). While there is little doubt that P2X7R overexpression by cancer cells greatly accelerates tumor growth, and that accordingly systemic administration of P2X7R blockers inhibit growth and metastases formation (3), it has also been shown that P2X7R blockade increases proliferation in a model of inflammationpromoted colon cancer (4). Finally, investigation of the role played in tumor-host interaction by the P2X7R expressed on host immune cells, has provided another intriguing observation as tumor growth in genetically-deleted mice is greatly accelerated relative to wt (3, 4). These apparently contrasting findings raise while on one hand highlight the role of the P2X7R in cancer, on the other hand stress the need to thoroughly understand its role on both the host immune cells and the cancer cells. References 1. Di Virgilio F. Curr Med Chem. 2015: 22;866–77. 2. Adinolfi E, Raffaghello L, Giuliani AL, Cavazzini L, Capece M, Chiozzi P, Bianchi G, Kroemer G, Pistoia V, Di Virgilio F. Cancer Res. 2012; 72:2957–69. 3. Adinolfi E, Capece M, Franceschini A, Falzoni S, Giuliani AL, Rotondo A, Sarti AC, Bonora M, Syberg S, Corigliano D, Pinton P, Jorgensen NR, Abelli L, Emionite L, Raffaghello L, Pistoia V, Di Virgilio F. Cancer Res. 2015; 75:635–644. 4. Hofman P, Cherfils-Vicini J, Bazin M, Ilie M, Juhel T, Hébuterne X, Gilson E, Schmid-Alliana A, Boyer O, Adriouch S, Vouret-Craviari V. Cancer Res. 2015, in press.

Symposium 12—P2X7 receptors-associated channels P2X7 purinergic signalling in a model of dilated casdiomiopathy induced by autoimmunity against muscarinic M2 receptors

Camila Guerra Martinez1, Daniel Zamith2, Marcia Gracindo da Silva1, Izaíra Trincani Brandão3, Celio Lopes Silva3, Bruno Lourenço Diaz1, Pedro Muanis Persechini1 and Eleonora Kurtenbach1 1 Instituto de Biofísica Carlos Chagas Filho, UFRJ, Brazil 2 Instituto de Microbiologia Prof. Paulo de Goes, UFRJ, Brazil 3 Departamento de Bioquímica e Imunologia, FMRP, USP, Brazil In the 90’s, several authors described that patients with Dilated Cardiomyopathy (DCM) presented antibodies against M2AChR that reproduce the negative chronotropic effect of muscarinic agonists. In addition, a study with 104 DCM patients demonstrated that those positive for anti-M2AChR (40 %) displayed a higher incidence of atrial fibrillation when compared with anti-M2AChR-negative patients. Besides the influence of auto-antibodies, the involvement of IFN-γ and IL-17 in autoimmune DCM and their severity had also been showed. In the heart purinergic P2X7 receptors influence electrical conduction, coronary circulation and response to ischemia. In the immune system they can trigger pro-inflammatory responses and the development of some cardiac autoimmune diseases. In this work we studied the involvement of P2X7 in heart function


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and in the development of autoimmune DCM provoked by antibodies anti-M2AChR. To this, seven-week-old female C57BL6/J wild type and purinergic P2X7 receptor knockout mice (P2X7−/−) mice were immunized with the pcDNA3-hM2, a DNA plasmid carrying the entire M2AChR cDNA sequence. We previously showed that Balb-C mice submitted to the same protocol developed anti-M2AChR–associated DCM that mimicked important functional and cellular characteristics of human DCM (Gimenez et al., 2005). Surprisingly pre-immunized P2X7−/− mice displayed an increased heart rate and ST segment depressions, but similar exercise performance when compared to age-matched wild type (WT) animals. After immunization with pcDNA3 plasmid containing M2AChR cDNA sequence (M2AChR animals), WT mice efficiently produced anti-M2AChR antibodies, while P2X7−/− mice showed a latter attenuated production. Despite this, M2AChR P2X7−/− developed some features of DCM, such as a left ventricle cavity enlargement and a decreased exercise tolerance, also observed in WT animals. Additionally, we showed that immunized M2AChR WT mice showed an increase in IL-1β, IFNγ and IL17 levels in the heart tissue, while M2AChR P2X7−/− mice produced the lowest amounts of IL-1β and IL-17 and the highest amounts of IFNγ. These results pointed to previously unnoticed roles of P2X7 receptors in cardiovascular and immune systems, and underscored the participation of IL-17 and IFNγ in the progress of autoimmune DCM. All animal procedures were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (approved protocol IBCCF041). Financial support: CNPq/Doenças Negligenciadas, FAPERJ/Doenças Negligenciadas and Instituto Nacional para Pesquisa Translacional em Saúde e Ambiente na Região Amazônica - INPETAM, CNPq/MCT. ATP release during tissue rejection: the role of P2X7 receptor and pannexin-1

M. Barberà-Cremades1, J. Amores-Iniesta1, C.M. Martínez1, A. Baroja-Mazo1 and P. Pelegrín1 Unidad de Inflamación y Cirugía Experimental, Hospital Clínico Universitario Virgen de la Arrixaca, Instituto Murciano de Investigación Biosanitaria (IMIB)-Arrixaca, 30120 Murcia, Spain 1

Background & objectives: Extracellular ATP acting on P2X7 receptors activates the NLRP3 inflammasome and the subsequent release of IL-1beta. P2X7 receptor activation requires high concentrations of ATP, which in vivo are found after severe tissue injury or stress. ATP activating the P2X7 receptor is considered a danger signal aimed to restore homeostasis by eliciting an inflammatory response. P2X7 receptor function has been implicated in different pathophysiological scenarios, including fever, contact hypersensitivity, graft-vs-host disease, lung inflammation or irritable bowel syndrome. Our recent work has investigated the release of ATP and P2X7 receptor signalling in allogeneic graft rejection, where immunity develops recognizing non-self antigens in the absence of microbes. Methods and results: Male C57BL/6 and Balb/c mice (from 8–12 weeks) were used for in vivo models of skin transplantation after Hospital Virgen Arrixaca ethics committee approval. Ear skin from either C57BL/6 or Balb/c was transplanted to the back of C57BL/6 mice to respectively get a syngeneic or allogeneic immune response. In vivo extracellular ATP was measured using HEK293-pmeLuc bioluminescence probe after 3, 7 and 14 days after transplantation. We found that extracellular ATP is accumulated in response to allogeneic skin, but not syngeneic skin grafts. The release of ATP occurred before the adaptive immune response is established (day 3) and the allogeneic tissue is destroyed and rejected. The mechanism for ATP release in vivo in response to allogeneic skin transplantation was highly dependent on macrophages acting as antigen presenting cells and involved the hemichannel pannexin-1, but not the NLRP3 inflammasome. Mechanistically, we found that the activation of the alloantigen presentation pathway in macrophages was responsible to induce the release of ATP by the opening of pannexin-1 hemichannels and then positively amplified via P2X7 receptor, culminating with NLRP3 inflammasome activation. In vivo, P2X7 receptor deficiency or specific antagonism with A438079 reduced the levels of ATP release and delayed the rejection of allogeneic skin grafts. Conclusion: All this data suggest that P2X7 receptor controls the production of endogenous danger signals that modulate the immune system after the recognition of “non-pathogenic” and “non-self” signals. Acknowledgment: This work was supported by the European Research Council Consolidator grant (DangerATP) and instituto salud carlos III-FEDER grants (PS09/00120 and PI13/00174) to PP. JA-I was supported by Sara Borrell fellowship from the instituto salud carlos III. P2X7 receptors in neuroinflammation and psychiatric disorders

Beáta Sperlágh Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary Objectives / background: The ATP-sensitive homomeric P2X7 receptor (P2X7R) has received particular attention as a potential drug target because of its widespread involvement in inflammatory diseases as a key regulatory element of the inflammasome complex. However, it has only recently become evident that P2X7Rs also play a pivotal role in central nervous system (CNS) pathology. Now a plethora of experimental data indicate that inhibition of P2X7Rs alter responsiveness in animal models of CNS disorders, accompanied by neuroinflammation such as stroke, neurotrauma, epilepsy, neuropathic pain, multiple sclerosis, amyotrophic lateralsclerosis, Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. Moreover, recent studies suggest that P2X7Rs regulate the pathophysiology of psychiatric disorders, including mood disorders. Methods and results: We have examined the role of P2X7Rs in various animal models of depression, bipolar disorder and schizophrenia. Our data show that P2X7 receptor deficient mice (P2rx7 −/−) displayed lack of behavioral despair in the forced swim test (FST), decreased immobility in the tail suspension test (TST) and an attenuated anhedonia in the sucrose preference test (SPT) consistent with an antidepressant phenotype. Moreover, they also exhibited decreased responsiveness in an animal model of mania i.e. in the amphetamine induced hyperactivity test (AH). These alterations were reproduced through systemic treatments with P2rx7 antagonists. The activation of P2rx7 resulted in the concentration-dependent release of [3H]glutamate in P2rx7+/+ but not P2rx7−/− mice, and the NR2B subunit mRNA and protein was upregulated in the hippocampus of P2rx7−/− mice. The hippocampal brain-derived neurotrophic factor (BDNF) expression is also proved to be under the regulation of P2rx7. This effect was dependent on the activation of NMDA and non-NMDA receptors but not on Group I metabotropic glutamate receptors (mGluR1,5). An increased 5-bromo-2-deoxyuridine (BrdU) incorporation was also observed in the dentate gyrus derived from P2rx7−/− mice. Basal level of 5-HT was increased, whereas the 5HIAA/5-HT ratio was lower in the hippocampus of P2rx7−/− mice, which accompanied the increased uptake of [3H]5-HT and an elevated number of [3H]citalopram binding sites. These neurochemical changes might serve as an explanation to the mood stabilizing behavioral phenotype found in the absence of P2rx7. In addition we found that both the frequency and time of social interactions were elevated, whereas phencyclidine (PCP)-induced hyperlocomotion, and stereotype behavior was alleviated in P2rx7−/− mice. These changes were accompanied with alterations of gene expression in the prefrontal cortex. Conclusion: Our data demonstrate the prominent role of P2X7Rs in the regulation of normal and pathological behavior, and implicating P2X7R as a potential drug target in a variety of psychiatric disorders.

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Neuroprotection mediated by P2Y13 and P2X7 nucleotide receptors and their signaling cascades

M.T. Miras-Portugal, F. Ortega, R. Gomez-Villafuertes M.J. Queipo, J. Gualix, J.I. Diaz-Hernandez, E.G Delicado and R. Pérez-Sen Department of Biochemistry, Veterinary Faculty, Complutense University of Madrid, Spain In adult brains, ionotropic and metabotropic purinergic receptors are widely expressed in neurons and glial cells, playing different roles according to the physiological or pathological situation. Cerebellar granule neurons express several types of nucleotide receptors, with the metabotropic P2Y13 and the ionotropic P2X7 being the most relevant in this model. Stimulation of these receptors results in ERK1/2 signaling activation and protection against glutamate excitotoxicity (Ortega et al. Neuropharmacology. 2011, 61, 1210–21). The nucleotidic agonists 2MeSADP (2-methylthioadenosine-5′-diphosphate) for P2Y13 and BzATP (2′(3′)-O-(4-benzoylbenzoyl)adenosine-5′-triphosphate) for P2X7 receptors were able to increase around two-fold the levels of ERK1/2 phosphorylation. These effects were sensitive to the inhibitory action of the antagonists MRS-2211 and A-438079, specific for P2Y13 and P2X7 receptors, respectively. Although both receptor subtypes shared the same pattern of transient ERK1/2 phosphorylation, they differed in the intracellular cascades coupled to them, being PI3K-dependent for P2Y13 and calcium/calmodulin kinase II (CaMKII)-dependent for P2X7. In addition, P2X7, NMDA and BDNF receptors converge on GSK3 phosphorylation and cooperate to promote survival, thus indicating that they share similar survival routes with trophic factors (Ortega F. et al. Cell Mol Life Sci. 2010, 67: 1723–33). Neuroprotection against oxidative stress was also found in granule neurons by activation of P2Y13 receptor that induces up-regulation of the cytoprotective protein heme oxygenase-1 through the transcription factor Nrf2 (Espada S. et al. Free Radic Biol Med. 2010, 49: 416–26). Other compromising situations for neuronal survival are those related with genotoxic stress mediated by exposure to cisplatin or UV radiation that induce caspase-3-dependent apoptotic cell death by phosphorylation of p38 proapoptotic protein (Morente V. et al., Biochim Biophys Acta. 2014, 1843: 1886–98). Once more P2Y13 plays a leading role in neuroprotection involving an increase in the expression of a dual protein phosphatase DUSP2, able to de-phosphorylate its substrate p38 to maintain neuronal survival. Conclusion: Neuroprotection is a wide concept and purinergic P2 receptors can play a leading role protecting from a large variety of neuronal insults. New signaling pathways, some of them reported for the first time to be coupled to P2 purinergic receptors, have been here described for P2Y13 and P2X7. Acknowledgments: This work has been supported by research grants from Ministerio de Ciencia e Innovación (BFU2011-24743), the Spanish Ion Channel Initiative (CSD2008-00005), Grupos CAM-BRADE S2013/ICE-2958; and Fundación Marcelino Botín. Thymine and thymidine photodamages can act as alarmins in a peritonitis mouse model (Oral communication)

C. Marques da Silva1., F.S. Vieira2, J.W. Souza3, C. Bandeira-Melo4, C. Lage3 and R. Coutinho-Silva1 1 Laboratório de Imunofisiologia, IBCCF, UFRJ, Brazil 2 Departamento de Imunologia, ICB, USP, Brazil 3 Laboratório de Radiações em Biologia, IBCCF, UFRJ, Brazil 4 Laboratório de Inflamação, IBCCF, UFRJ, Brazil Ultraviolet (UV) radiation is known as an important factor disrupting immune homeostasis. Cellular exposure to UV light can ensue both inflammatory and anti-inflammatory responses. Purinic and pyrimidinic bases in DNA strongly absorb UV light. Thymidine Dimers are produced when adjacent thymidine residues are covalently linked by exposure to UV radiation. Dimerized thymines may be replicated as a single base, which results in frameshift mutations whenever they are not repaired before replication. It was already shown that one of the inflammatory effects is due to expression and release of the alarmin high mobility group box 1 (HMGB1) after acute and chronic UV irradiation of the skin (Johnson et al. Arch Dermatol Res. 305(9) 2013). Alarmins are endogenous molecules that act as potent pro-inflammatory mediators when they are released by cells or accumulate extracellularly. Here, we show that thymine is one of the possible molecules released after UV radiation and can also act by itself as a novel class of alarmins. Methods and Results: Peritonitis was performed by intraperitoneal injection of thymine, thymidine dimers, ATP or vehicle at different time points and different concentrations in both sexes of BALB/c mice between 8 and 12 weeks old. After peritoneal wash, cells were counted, processed and fixed to proceed citospyn (staining with Panotico Rapido Kit), immune phenotyping (staining cells with anti-CD3, anti-GR1/Ly6, anti-CD11b/F4-80 antibodies conjugated with different fluorocromes), reactive oxygen species production (by fluorescent probe CM-H2DCFDA) and lipid vesicles staining (Osmium staining). The supernatants were processed to cytokines (ELISA), leukotrienes (EIA), and nitric oxide measurements (Greiss reaction). Our results showed that thymine and thymidine photodamages induce an augment of 40.3 ± 12.9 and 154.7 ± 3.1 (×105) cells over vehicle injection alone. Also, there was no cell death difference between treatments. Trough flow cytometry analysis we observed that most of the dimers recruited cells were neutrophils, with an augment of 141 ± 12.1 % over vehicle, and timine augmented in about 99 % Monocytes/macrophages and about 33 % lymphocytes and neutrophils. The ROS production was augmented in both thymine and its dimers, 2748 ± 483 and 2536 ± 515 MIF, conversely, nitric oxide was not detected in any of the experimental situations. Since cell recruitment was such a noticeable data we wondered which was the recruiting agent released during peritonitis, and turned out to be leukotriene B4, with an improve of 200 % release over vehicle treated mice in timine and its dimers. Conclusion: In the peritonitis model, UV damage products thymine and thymidine were shown to be pro inflammatory agents orchestrating several steps of acute inflammation. Our results shed some light on its mechanisms, possibly due to the release of a new class of UV-induced alarmins. Support: PIBIC/CNPq, CAPES, FAPERJ. Guest conference 5 Advancements in single cell analysis

Attila Tárnok Department of Paediatric Cardiology, Heart Center, and Translational Centre for Regenerative Medicine (TRM), University of Leipzig, Germany In the emerging field of high-content single cell analysis for systems biology and cytomics polychromatic (>8-colors) analysis becomes increasingly important. Polychromatic flow cytometry (FCM) combined with systems-biology tools provide deep insights into cell biological processes, disease cause and development,


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as well as therapy response, improving individualized medicine. State of the art standard FCM instruments can measure up to 15 parameters simultaneously and can thereby quantify expression and localization of various cell constituents in millions of cells from a single sample. Optimized polychromatic panels are developed for combining identification of all major leukocyte subsets with measurement of cell surface activation markers. New fluorescent dyes allow combination of up to 22 different markers. This is even surpassed by mass cytometry (CyTOF) that can detect up to 100 cell constituents in a single run. Image cytometry (IC) can even increase the complexity of single cell analysis and measure virtually anything stainable in a cell. This approach is termed hyperchromatic and different components suitable to be combined for achieving this task will be presented. For cell analysis microscopic IC is ideal as it is nonconsumptive, i.e. the analyzed sample is fixed on the slide and can be reanalyzed after treatment. In an overview, various approaches for hyperchromatic cytometry are demonstrated for a commercial ic: 1) polychromatic cytometry, 2) iterative restaining (restaining with same fluorochrome and reanalysis), 3) differential photobleaching (differentiating fluorochromes by different photostability), 4) photoactivation (activating fluorescent dyes), and 5) photodestruction. Based on the ability to relocate cells immobilized on the IC identical cells can be subsequently reanalyzed and data collected on the single cell level after each manipulation step. With intelligent combination of the above techniques hyperchromatic cytometry allows to quantify and analyze virtually all components on the identical cell. The information gained per specimen is only limited by the number of available antibodies and by sterical hindrance. The high level of complexity of the obtained data requires new approaches for (automated) data analysis and data visualization. Several new tools allow automated recognition of different cell types and their visualization exists and will be discussed. Symposium 13 – Regulation of transporters for nucleosides and nucleotides The SLC29A family of nucleoside transporters: new modalities of regulation and therapeutics

I.R. Coe Department of Chemistry and Biology, Ryerson University, Toronto, Ontario, Canada Purine nucleosides, such as adenosine, rely on membrane transport proteins to cross cell membranes. The SLC28 and SLC29 transporter families are responsible for the uni- or bi-directional flux of nucleosides and nucleoside analog drugs used in a wide variety of clinical settings. The contribution of members of the SLC29 family (the ENTs) to purinergic signaling is well established (Rose et al. AJP 298: H771, 2010; Grenz et al. JCI 122:693, 2012) and the clinical relevance of presence and activity of ENT1 and ENT2 in the uptake of nucleoside analog drugs is clear. However, the factors and mechanisms that regulate the ENTs are still poorly understood and we cannot yet apply our understanding of ENT regulation to improving or enhancing nucleoside analog drug efficacy. To address this gap in our understanding, we have used a variety of biochemical, physiological and pharmacological approaches to investigate the function and regulation of ENTs. Our data show that ENTs exist in a continual dynamic flux within the cell and are regulated at the plasma membrane by glycosylation, protein-protein interactions and the formation of homo- and hetero-complexes. Moreover, ENT1 is functionally integrated into, and regulated by, more extensive cellular signaling pathways, including, but not limited to purinergic pathways, while ENT2 splice variants regulate the location and function of ENT2, impacting cellular proliferation in a novel and previously un-described manner. We are also investigating the role of ENTs in novel combination therapies, to address clinical situations where ENT expression is low and therefore drug efficacy is compromised. Taken together, our data suggest that ENTs are integral members of the purinome and are involved in basic cellular homeostasis. ENTs are regulated in complex ways that need to be fully understood in order to optimize nucleoside analog drug therapies. Funding for the studies presented here is provided by Natural Science & Engineering Council of Canada (NSERC) and by Ryerson University. Regulations of ENT1 in the chicken retina (Oral communication)

Alexandre dos Santos Rodrigues Department of Neurobiology, Graduate Program of Neurosciences, Institute of Biology, Fluminense Federal University (Niteroi - Rio de Janeiro - Brazil) The purinome is a rich complex of proteins and cofactors which are involved in fundamental aspects of cellular homeostasis and cellular responses. Nucleoside Transporters (NTs) are membrane proteins involved in regulation of nucleoside flux across cellular membranes. The SLC29 family comprises the equilibrative nucleoside transporters (ENTs), which are widely distributed in human tissues but poorly understood in terms of their regulation. They are clinically important because they are the route of entry for nucleoside analog drugs, which are widely used in anti-cancer, anti-viral and anti-parasite therapies. Adenosine (Ado) is an important neuromodulator in the central nervous system (CNS), regulating synaptic transmission and plasticity, cell proliferation, differentiation, cellular repair processes and neurodegeneration. Glial cells play fundamental roles in the CNS, such as regulation of neuronal migration, neuroprotection and control of extracellular glutamate concentrations. ENT1 and ENT2 play key roles in the rapid removal of extracellular Ado, which can be found in neurons and astrocytes. Our previous work showed that nucleoside transporters (NTs) are present in cultures of chick retinal cells, but little is known about the mechanisms regulating adenosine transport in chicken retina and also in other cellular systems. We have been working trying to achieve a better understanding of how these transporters, especially ENT1, can be regulated in the retina, using different approaches. Understanding the regulation of ENT1 will provide insights into the role of the NTs in the purinome. These observations are additionally important since they may provide new approaches to enhancing the therapeutic efficacy of nucleoside analog drugs transported by ENT1. Supported by CAPES, CNPq, FAPERJ and PRONEX-MCT. Adenosine A2B receptor activation stimulates glucose uptake in the rodent forebrain (Oral communication)

Cristina Lemos, Bárbara S. Pinheiro, Rui O. Beleza, Ricardo J. Rodrígues, Rodrigo A. Cunha, Daniel Rial and Attila Koffalvi Center for Neurosciences and Cell Biology, Coimbra University, Portugal Objectives / background: A2B receptors (A2BR) are Gs/q protein-coupled receptors with low affinity for adenosine and an elusive role in the brain. We have recently reported that A2BRs are present in hippocampal glutamatergic terminals and are responsible for the control of A1R-mediated inhibition of

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synaptic transmission (Gonçalves et al., 2015, Eur J Neurosci). Sincet intense neuronal activity leads to the consumption of large amounts of ATP, this can generate high extracellular (synaptic) levels of adenosine, which may activate A2BRs. Here, we aimed at investigating the hypothesis that A2BR activation stimulates glucose transport in the hippocampus to help to replenish ATP stores. Methods and results: We monitored the spatiotemporal accumulation of 2-NBDG, a fluorescent glucose analogue in hippocampal slices of A2BR knockout C57Bl/6 male mice and their wild-type (WT) littermates (8–10 week-old, weighting 35–40 g), and also measured 3H-deoxyglucose uptake in mouse cortical neuronal and astrocytic cultures. The A2BR agonist, BAY60-6583 (300 nM) triggered an immediate increase in the 2-NBDG signal 26.70 ± 10.64 (mean ± S.E.M.), P < 0.05) which remained stable for over 10 min. This effect of BAY60-6583 was absent either upon pharmalogical blockade of the A2BR with MRS1754 (200 nM), or in A2BR knockout mice. A2BR activation with BAY60-6583 (300 nM) also triggered a rapid [3H]DG transport into neurons and astrocytes in culture, in a fashion sensitive to the pretreatment with MRS1754 (200 nM). Conclusion: we report here that A2BR activation can stimulate hippocampal glucose transport, which is expected to gain physiological importance under a recurrent recruitment of the circuitry. This prompts new studies to confirm the physiological significance of our findings, with the future aim of developing a new class of nootropics to manage cerebral hypometabolism. (Supported by FCT, QREN, Santa Casa da Misericódia, CNPq-Ciência sem Fronteiras). An innovative multi brand supplier for the Brazilian market—quest for a balanced portfolio offer

Carlos André de Castro Herklotz Sinapse Biotecnologia Ltda, Brazil The life science research reagents supplies in Brazil has a restrictions imposed by governmental regulatory agencies. Even “research only” clearly stated products must pass through the same regulation of human use collateral products on importation processes. Those policies significantly impacts on price, delivery time, but mainly on the variety of products offered in Brazil. We bring our testimonial of more than 20 years distributing small to medium size companies and its product ranges in Brazil. On our presentation, we emphasize our strategic choices in a new model for a multi-brand product offer. The constant quest for innovative, cost benefit, high performance products and the continuous learning and improving of our team are some elements we combine to provide a very complete and dynamic portfolio offer for the Brazilian science community.

Oral communications ADP treatment improves wound healing in diabetic mice

Paula A. Borges, Janaína L. Georgii, Janaína F. Barros, Ariane R. Brogliato, José Roberto Meyer, Robson Coutinho-Silva, Josiane S. Neves and Claudia Farias Benjamim Federal University of Rio de Janeiro, Brazil Objectives and Backgrounds: Chronics wounds are a health problem worldwide, which affect 6.5 millions patients in USA. Such problem in association with high global prevalence of diabetes, reflects the increase in diabetic ulcers. Considering the absence of an effective treatment for chronic wound, we investigated the possible beneficial effects of purinergic agonists in tissue repair. In the present work we explore the role of ADP in healing process of skin chronic wounds in diabetic mice. Methods and results: In this study, Diabetes Mellitus was induced by a single intravenous Alloxan injection (65 mg/Kg) 8 h after fast, in Swiss male mice (20–24 g). Mice were then separated in four groups and treated topically once a day per 14 days as following: a) control group: saline; b) ADP group, 30 μM of ADP was applied on wound beds; c) clopidogrel + saline: clopidogrel was given by gavage; and d) clopidogrel + ADP. Wounds contraction was measured at days 3, 7, 10, and 14 days after skin lesions. Wounds were collected at day 7 after wounding, formalin fixed and paraffin-embedded. Skin sections were stained with HE (for granulation tissue), Sirius Red (for collagen), modified Sirius Red (for eosinophil), Alcian Blue (for mast cell), or immunostained for α-actin, laminin, macrophages, VEGF and TGF-α. Wounds were also collected 7 days after wounding, homogenized and MPO activity assay (for neutrophil quantification), ELISA (TNF-α, IL-10 e IL-13) and CBA (IL-6, IL10, MCP-1, IFN-γ, TNF, IL-12p70) were performed. Our data showed that ADP was able to accelerate the wound healing of diabetic mice, which resembles the healing of non-diabetic mice. Clopidogrel treatment, a P2Y12 receptor antagonist, prevented the lesion closure in diabetic mice treated with ADP. Interestingly, clopidogrel worsened the lesion closure even in non-diabetic mice. Others nucleotides as adenosine, ATP, AMP5′ and AMP3′ at 30 μM did not accelerate the lesion healing, as observed for ADP. Through histological analysis it was observed that ADP treatment improved the granulation tissue formation, collagen synthesis and increased the recruitment of eosinophils and neutrophils, and the population of mast cells on the seventh day after the lesion. ADP stimulated the release of the cytokine IFN-γ on the third day and the IL-10 and IL-13 on the seventh day after the lesion. In addition, at day 7 after lesion, ADP was effective in increasing the differentiation of myofibroblasts and the expression of laminin, VEGF and TGF-α. Still in this time point, ADP seemed to increase the arginase+ cells and to reduce iNOS+ cells, which implies in the increase of M2 macrophages in the wound after ADP. Conclusion: Our results suggest that ADP accelerates the wound healing in diabetic mice partially via recruitment of inflammatory cells to the lesion. We expect that ADP may become a new treatment for chronic injuries. Financial support: CAPES, CNPq and FAPERJ. Purinergic P2Y12 receptor activation in eosinophils and the schistosomal host response

Josiane Sabbadini Neves Federal University of Rio de Janeiro, Brazil


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Background: Identifying new target molecules through which eosinophils activate and secrete their stored proteins may be highly significant for understanding the pathophysiology of host immune responses to parasites and may reveal new therapeutic targets for the control of eosinophilic disorders. We have recently reported the expression of the purinergic P2Y12 receptor (P2Y12R) in human eosinophils; however, its functional role in this cell type and its involvement in eosinophilic inflammation remain unknown. In this study, we investigated functional roles of P2Y12R in isolated human eosinophils and in a murine model of eosinophilic inflammation induced by Schistosoma mansoni (S. mansoni) infection. Methodology/Principal Findings: Eosinophils were isolated from the blood of healthy donors by negative immunomagnetic selection (license number 190/09 HUCFF). Functionally, we found that adenosine 5′-diphosphate (ADP) induced isolated eosinophils to secrete eosinophil peroxidase (EPO) via a mechanism dependent on P2Y12R activation. In contrast, ADP did not interfere with human eosinophil apoptosis or chemotaxis in vitro, as assessed by flow cytometry and a transwell system, respectively. In vivo, C57Bl/6 mice were percutaneously infected with 60 cercariae of the BH strain of S. mansoni (license number DFBCICB043 - CEUA/UFRJ). Analyses performed 55 days post infection revealed that P2Y12R blockade reduced the area of the hepatic granuloma and modulated the eosinophilic inflammatory infiltrate without affecting the Th2 response and inhibited collagen deposition and IL-13/IL-4 production in the liver without altering the parasite oviposition. Furthermore, P2Y12R inhibition increased blood eosinophilia (2-fold increase), whereas it decreased the eosinophil count in the bone marrow, as assessed by blood smears and cytospin analyses, respectively. Conclusions/Significance: Taken together, our results suggest that P2Y12R has an important role in eosinophil EPO secretion and in establishing the inflammatory response in the course of a S. mansoni infection. Independent purinergic mechanisms of central and peripheral chemoreception in the rostral ventrolateral medulla

T.S. Moreira1, A.C. Takakura1, C.R. Sobrinho1 and B.F. Barna1 1 Department of Physiology and Biophysics, Institute of Biomedical Science, University of São Paulo, 05508–000, São Paulo, SP, Brazil 2 Department of Pharmacology, Institute of Biomedical Science, University of São Paulo, 05508–000, São Paulo, SP, Brazil Objectives/Background: Central and peripheral chemoreceptors sense changes in CO2/H+ and/or O2 and communicate this information to cardiorespiratory centers to regulate breathing and sympathetic outflow to ensure adequate ventilation-perfusion matching in tissues. Despite the importance of this reflex, the cellular and neurotransmitter basis for integration of this information in the brainstem is unclear. It has been shown that excitatory drive from peripheral chemoreceptors is relayed through the retrotrapezoid region (RTN) located in the ventrolateral medulla, a region also known to contribute to central chemoreception. Evidence also suggests that purinergic signaling in this region can influence both respiratory and sympathetic drive, possibly by activation of P2Y1-receptors (P2Y1-R). Therefore, we hypothesize that P2Y1-R represent the molecular basis for central and peripheral chemoreceptor integration at the level of the RTN region. Methods and Results: Here, we use in vivo and in vitro recording techniques to examine the role of P2Y1-R’s expressed by RTN region neurons in central and peripheral chemoreception. In vivo, unilateral injection of MRS2365, a P2Y1-R specific agonist, into the RTN region of anesthetized adult rats increased mean arterial pressure by 23 ± 1 mmHg (compared to 4 ± 1 mmHg in control experiments) and increased phrenic nerve amplitude and frequency by 59 ± 6 and 53 ± 5 %, respectively. Conversely, application of the specific P2Y1-R antagonist MRS2179 decreased peripheral chemoreceptor mediated activation of breathing and blood pressure but did not affect CO2-responsivness. In vitro, loose-patch recordings of neurons in slices from rats (P7-12) show that baseline activity and CO2/H+−sensitivity of chemoreceptors was unaffected by MRS2179 or MRS2365. Conversely, MRS2179 decreased baseline activity of non-chemosensitive neurons by 42 % and MRS2365 increased activity of these cells by 630 %. After recording, a subset of CO2-insensitive P2Y1-expressing cells were filled with biocytin for later immunohistochemical characterization; 4 of 6 cells were immunoreactive for tyrosine hydroxylase and the transcription factor phox2b, suggesting they are C1 cells. Conclusions: These data indicate that i) P2Y1-R are preferentially expressed by C1 cells and contribute to peripheral chemoreceptor modulation of breathing and blood pressure; ii) P2Y1-R are not expressed by ventral surface chemoreceptors and do not contribute to CO2-responsivness in vivo or in vitro; iii) P2-R targeted by CO2-evoked ATP release remain undetermined. These results suggest that purinergic signaling contributes to cardiorespiratory integration at the level of the RTN region. Financial support: FAPESP (09/54888-7 and 13/10573-8); CAPES and CNPq/PROEX. Interference of direct and indirect co-colture of gliobastoma and mesenchymal stem cells in expression levels of kininergic and purinergic components

Mateja Delač2, Mona das Neves Oliveira1, Barbara Breznik1, Micheli M. Pillat2, Talita Glaser2, Henning Ulrich2 and Tamara T. Lah1 1 National Institute of Biology, Slovenia 2 University of São Paulo, Brazil Bone marrow derived mesenchymal stem cells (BM-MSCs) display high tropism towards tumours, including the most malignant brain tumour glioblastoma (GBM). However, the molecular mechanisms of their interactions are still not clear (Motaln and Lah, 2015). Previously, we confirmed that BM-MSC impair proliferation, invasion and senescence of several GBM lines (Schichor et al., 2011; Motaln et al., 2012). Transciptomics analyses revealed that the activation of bradykinin (BK) and its receptors (kinin-B1 and B2-receptors) may be involved upon the intercellular interactions. We show here that direct and indirect co-culture of GBM and MSC are also affecting the expression rates of kinin receptors and CD73 (5′ectonucleotidase), a key enzyme of the purinergic system, metabolizing 5′-AMP into adenosine and thereby determining activity levels of adenosine receptors. Upon direct co-culture for 72hs, down-regulation of kinin-B1 and B2-receptors in MSC occur, while receptor expression is enhanced in U87 glioma cells, suggesting functions of this receptor in invasiveness and immunomodulatory responses. Similarly, CD73 expression levels diminished in MSC in direct co-culture, while expression rates of this enzyme is augmented in U87 cells, being in line with their increased invasiveness, as observed previously (Schichor et al., 2011). Indirect co-culture, where no direct interactions between cell surfaces of U87 and MSC occur, but the exchange of soluble factors is possible, expression rates of kinin-B1 and B2 receptors did not change, while again in U87 cells an increase in receptor expression was observed. Reduction of CD73 expression in MSC was less prominent in indirect co-culture, when compared to direct co-culture, however it increased in

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U87 cells to similar levels as those observed in conditions of direct co-culture. Further aims of this work lies in the investigation of interrelationships between the kininergic system and CD73, being used as a prognostic marker of glioblastoma malignancy. Motaln and Lah, Proteins and Peptide Letters (in press,2015); Motaln et al. Cell Transplantation 2012, 1529–45; Schichor et al. Exp. Neurology 2012, 234, 208–19.

Tissue cytometry and tissue sociology—new concepts to look at cells and cellular interactions in tissue sections

Rupert Ecker TissueGnostics GmbH, Austria Determining the in-situ immune status of diseased organs or quantify coexpression of molecules on the single-cell level has mostly been subject to visual estimation, or—at best—to manual counting for decades. Hence, experts usually had the choice of the “least of evils” between guessing and endless (manual) counting. In tumor immunology, infiltrating inflammatory cells need to be phenotypically characterized on a quantitative basis. To better understand the function of inflammatory cells in tumor development, type and number of inflammatory cells and their proximity to glandular/tumor structures have to be analyzed in-situ and correlated with disease state. Using TissueFAXS™ Cytometry the time-consuming and error-prone human evaluation of stained histological sections can be approached with an observer-independent and reproducible technology platform, offering a high degree of automation, paired with user interaction at relevant points of the analytical workflow. This platform can be applied as a means of tissue cytometry for both immunofluorescence and immunohistochemistry. The TissueFAXS Cytometry platform can be used to determine e.g. the immune response in situ, measure proliferation, apoptosis, cytokine expression, signalling molecules, and others. It can do end-point assays as well as live-cell imaging and time-kinetic experiments. But TissueFAXS Cytometry also promotes tissue cytometry to a new level of quality, where complex cellular interactions can be addressed on the single-cell level but still in histological context. A cell in tissue plays a defined role as being a member of a small cellular subunit within a larger organic structure. The development and differentiation of a given cell is defined by the parent cell and the local cell neighborhood and environment. Consequently, the analysis of cells in their local environment is of major importance. At TissueGnostics we understand cells not just as a “bunch of biomolecules” but as ACTING ENTITIES that communicate and interact with each other—just like humans talk to and interact with each other. Consequently, we here introduce the concept of “Tissue Sociology”. While in “Sociology” researchers investigate the behavior of and interactions between individual human beings, state-of-the-art image cytometry software permits a similar approach on the single cell level! We can analyze locations of single cells in tissue context, perform “neighborhood analyses” and quantitate cellular interactions in various ways. Different cells might not be just next to each other “by chance”, but any cellular presence, absence, or distribution will have both a “specific cause and meaning”. From a conceptual and scientific point of view we invite you to evaluate and consider this novel path of research to better understanding cancer cells, their immunocompetent antagnosists, and the interactions between them!

Closing conference Purinergic signaling in neurogenesis and brain repair

Henning Ulrich University of São Paulo, Brazil Recent data have provided evidence for the participation of purinergic receptors participate in development and tissue regeneration. Here, we have explored the participation of P2 receptors in neural differentiation processes. Using pluripotent mouse embryonal carcimoma and embryonic stem cells as models for early neuroectodermal differentiation, we showed that P2Y1, P2Y2 and P2X7 receptors participate in the maintenance of ESC pluripotency and proliferation as well as in the increase of the number of glial cells. While P2Y2 and P2X2 receptor expression was necessary for differentiation into neuronal lineages, P2X7 receptor expression needed to be down-regulated for cells undergoing neurogenesis. In agreement, expression levels of Mash-1, determining the neuronal fate of differentiating cells, declined when neural stem cells (NSC) had been exposed to the P2X7 receptor agonist Bz-ATP, as revealed by time-lapse imaging. P2Y2 receptor stimulation by 2-thio-UTP, on the other hand, induced Mash-1 expression. Both purinergic receptor agonists, Bz-ATP and 2-thio-UTP, promoted specific cytosolic calcium spike patterns; however, P2Y2 receptor activity resulted in higher calcium transients, supposedly related to Mash-1 activation and neuronal fate specification. P2X7 receptor inhibition presents a promising strategy for prevention of neuronal cell death. Our above-mentioned data let to suggest that P2X7 receptor expression in adult NSC could prevent these cells from neurogenesis for brain repair. An animal model of Parkinson’s disease was used to further address this question. For this purpose, unilateral hemisphere lesions of the nigrostriatal pathway of adult male Sprague–Dawley rats were induced by stereotactic injection of 6-hydroxydopamine (6-OHDA). One week after lesion, the animals presented rotational behavior when challenged with apomorphine. Treatment with Brilliant Blue-G (BBG), an antagonist of P2X7 receptor, had beneficial functional effects. Animals injected with 6-OHDA, which in the following received BBG (n = 6) during 7 days at a 50 mg/kg dose, showed a statistically significant decrease in the number of rotations per minute (13 to 4, p < 0.05), whereas animals receiving only saline did not reveal any significant improvements in rotational tests (11 to 8, p < 0.05). In agreement, levels of regeneration of dopaminergic neurons in BBG-treated animals, as revealed by anti-tyrosine hydroxylase staining, were twice as high than those observed for the saline-treated control group. The here shown results demonstrate the importance of P2X7 receptor inhibition for neurogenesis and regeneration in brain diseases with possible therapeutic applications. Support: Grants and student fellowships awarded by FAPESP, CNPq and CAPES (Brazil).


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Poster session Adenosine receptor signaling

01 A2A adenosine receptor activation modulates Src kinase activity in cAMP manner dependent Pâmella Silva, Mariana Pereira, Elisa Vardiero and Roberto Paes-de-Carvalho Universidade Federal Fluminense, Brazil Src kinase is a member of the family of tyrosine kinases involved in many developmental processes such as cell proliferation, differentiation and survival. The phosphorylation sites which regulate Src activity include tyrosine 416 (Tyr416), which results in Src activation by autophosphorylation, and tyrosine 527 (Tyr527) which promotes Src inhibition mediated by C-terminal Src kinase (Csk) phosphorylation. Thus, Src activity is regulated by phosphorylation levels at Tyr527 and Tyr416 residues. Adenosine, a neuromodulator in the CNS, activates different metabotropic receptors, including the A2a receptor which is coupled to Gs protein and cyclic AMP production. Our previous results showed that A2a receptor activation decreases Src phosphorylation at Tyr416. Thus, our objective here was to evaluate the signaling pathways involved in the regulation of Src activity by A2a receptor activation in retinal cultures. Retinal cultures were stimulated with CGS21680 (A2a agonist) for 5 min and p-Src levels were evaluated by Western Blot. We observed a decrease in pSrc (Tyr416) induced by A2a receptor stimulation that was blocked by ZM241385 (antagonist A2a) and SQ22536 (adenylyl cyclase inhibitor). In addition, A2a receptor activation promoted an increase in p-Src (Tyr 527) and p-Csk induced by A2a receptor stimulation. Similar results were observed in retinas from 9-day-old chick embryos where CGS21680 promoted a decrease of p-Src (Tyr416) and an increase of p-Csk. These results demonstrate that stimulation of A2a receptors induces a decrease of p-Src and this effect involves the cAMP pathway. Our working hypothesis is that activation of PKA by A2a receptors leads to Csk activation and Tyr527 phosphorylation, with a consequent decrease of Tyr416 phosphorylation and Src activity. Financial Support: CNPq, CAPES, FAPERJ, PRONEX-MCT. 02 Activation of the adenosine A1 receptor regulates phosphorylation of ERK and CREB in cultures of suprachiasmatic nucleus cells R. Brito1, R. Paes-de-Carvalho1, K.C. Calaza1 and V. Ramkumar2 Federal University Fluminense, Brazil 2 Southern Illinois University School of Medicine, USA

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Objective: Adenosine is a nucleoside that plays many important roles in the CNS by activating metabotropic receptors: A1, A2A, A2B and A3. A1 and A3. The suprachiasmatic nucleus (SCN) controls the circadian rhythms of the body by processing the light information received the environment. Some studies show that glutamate has the ability to increase the phosphorylation of ERK (P-ERK) and CREB (P-CREB) in the SCN. However, there is no information on the role of adenosine. Thus, the aim of this study is to evaluate the role of activation of the A1 receptor (A1R) in the activity of ERK and CREB in SCN. Methodology: SCN immortalized cells of rats (SCN2.2 cells) were grown in MEM medium. Cells, in cultures with 70 % confluence, were pretreated for 30 min with adenosine deaminase and treated with drugs of interest. After treatment, cells were processed for western blot (WB). Results: We found, by using WB approach, that SCN cells show A1R. These receptors are functional because the treatment with 100 nM R-PIA (agonist A1R) increased P-ERK, both 10 and 30 min after treatment (Control:100 n = 6; 5 min:120.1 ± 11.5 n = 5; 10 min:159.1 ± 19.8 n = 4 ***p < 0.001; 30 min:142.2 ± 13.2 n = 3 *p < 0.05; 60 min:131 ± 1.8 n = 4; 120 min:127.4 ± 1.9 n = 5). Exposure to R-PIA for 10 min also increased P-CREB (Control:100 n = 7; 5 min:117.7 ± 17 n = 7; 10 min:177.2 ± 22.4 n = 4 **p < 0.01; 30 min:102.4 ± 14.3 n = 6; 60 min:114.2 ± 10.1 n = 7; 120 min:124.5 ± 13.5 n = 7). So, we establish the time of treatment to 10 min in all subsequent experiments. The concentration curve of R-PIA showed maximal effect at 1 μM for P-ERK (Control:100 n = 5; 100nM:205 ± 28.8 n = 5; 500nM:390.3 ± 40.9 n = 3 * < p0.05; 1 μM:485.8 ± 102.9 n = 4 ***p < 0.001; 5 μM:385.3 ± 57.6 n = 5 **p < 0.01; 10 μM:466.0 ± 56.0 n = 5***p < 0.001) and P-CREB (Control:100 n = 4; 100nM:169.8 ± 17.5 n = 4 **p < 0.01; 500nM:148.0 ± 12.4 n = 3; 1 μM:172.1 ± 9.1 n = 3 **p < 0.01; 5 μM:153.1 ± 21.3 n = 2, 10 μM:229.1 ± 4.1 n = 3 ***p < 0.001). The effect of R-PIA in the P-ERK was inhibited by DPCPX (A1R antagonist), but not by ZM (A2AR antagonist) (Control:100 n = 9; R-PIA:149.3 ± 8 n = 6 * < p0.05; DPCPX:107.7 ± 8.4 n = 5; DPCPX+R-PIA:99.5 ± 9.1 n = 5; ZM:99.9 ± 5.2 n = 6; ZM+R-PIA:141.2 ± 13.8 n = 6 **p < 0.01). On the other hand, both antagonists blocked the P-CREB induced by R-PIA. PD (an inhibitor of MEK) inhibited the P-ERK (Control:100 n = 4, R-PIA:176.5 ± 20.8 n = 3 ***p < 0.001, PD:4.0 ± 1.5 n = 4, PD+R-PIA:6.9 ± 3.2 n = 4) and P-CREB (Control:100 n = 4; R-PIA:138.8 ± 7.1 n = 3**p < 0.01; PD:62 ± 3.0 n = 3, PD+R-PIA:38.0 ± 11.7 n = 3) induced by R-PIA. A specific blocker of CREB was able to block phosphorylation induced by R-PIA (Control n = 4; R-PIA n = 4 * < p0.05; CREBi10μM n = 3; CREBi10μM + R-PIA n = 3; CREBi20μM n = 4; CREBi20μM + R-PIA n = 5). Conclusion: A1 receptors are expressed and functional in the SCN cells, since activation with R-PIA was able to increase the P-ERK. Interestingly, the same treatment is able to induce the P-CREB. Thus, adenosinergic system can be of great importance in the control of circadian rhythm in the SCN. Financial Support: CNPq, CAPES. 03 Adenosine A2B receptor activation stimulates glucose uptake in the rodent forebrain Cristina Lemos, Bárbara S. Pinheiro, Rui O. Beleza, Ricardo J. Rodrígues, Rodrigo A. Cunha, Daniel Rial and Attila Koffalvi Center for Neurosciences and Cell Biology, Coimbra University, Portugal

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Objectives / background: A2B receptors (A2BR) are Gs/q protein-coupled receptors with low affinity for adenosine and an elusive role in the brain. We have recently reported that A2BRs are present in hippocampal glutamatergic terminals and are responsible for the control of A1R-mediated inhibition of synaptic transmission (Gonçalves et al., 2015, Eur J Neurosci). Sincet intense neuronal activity leads to the consumption of large amounts of ATP, this can generate high extracellular (synaptic) levels of adenosine, which may activate A2BRs. Here, we aimed at investigating the hypothesis that A2BR activation stimulates glucose transport in the hippocampus to help to replenish ATP stores. Methods and results: We monitored the spatiotemporal accumulation of 2-NBDG, a fluorescent glucose analogue in hippocampal slices of A2BR knockout C57Bl/6 male mice and their wild-type (WT) littermates (8–10 week-old, weighting 35–40 g), and also measured 3H-deoxyglucose uptake in mouse cortical neuronal and astrocytic cultures. The A2BR agonist, BAY60-6583 (300 nM) triggered an immediate increase in the 2-NBDG signal 26.70 ± 10.64 (mean ± S.E.M.), P < 0.05) which remained stable for over 10 min. This effect of BAY60-6583 was absent either upon pharmalogical blockade of the A2BR with MRS1754 (200 nM), or in A2BR knockout mice. A2BR activation with BAY60-6583 (300 nM) also triggered a rapid [3H]DG transport into neurons and astrocytes in culture, in a fashion sensitive to the pretreatment with MRS1754 (200 nM). Conclusion: we report here that A2BR activation can stimulate hippocampal glucose transport, which is expected to gain physiological importance under a recurrent recruitment of the circuitry. This prompts new studies to confirm the physiological significance of our findings, with the future aim of developing a new class of nootropics to manage cerebral hypometabolism. (Supported by FCT, QREN, Santa Casa da Misericódia, CNPq-Ciência sem Fronteiras). 04 Adenosine receptors expression and cell death in copper-induced inflammation in zebrafish larvae Fernanda Fernandes Cruz, Carlos Eduardo Leite, Bruna Haas Drago, Talita Carneiro Brandão Pereira, Mauricio Reis Bogo, Carla Denise Bonan, Ana Maria Oliveira Battastini, Maria Martha Campos and Fernanda Bueno Morrone PUCRS, Brazil INTRODUCTION: Zebrafish (Danio rerio) is becoming a popular model organism in biomedical research (Kalueff, Trends Pharmacol Sci. V. 35(2), P. 63, 2014). Adenosine receptors are a family of G-coupled-protein receptors divided into four subtypes: A1, A2A, A2B, and A3 (Burnstock, Curr Top Med Chem., V. 4(8), P. 793, 2004). For the A2A, were described two genes in zebrafish, identified as A2A.1 and A2A.2 (Boehmler, Gene Expr Patterns, V. 9(3), P. 144, 2009). Adenosine signaling is involved on tissue protection and repair from excessive inflammatory responses (Jacobson, Nat Rev Drug Discov, V.5(3), P.247, 2006). Copper is a heavy metal that can induce the production of reactive oxygen species and oxidative stress, and previous studies demonstrated its effect in purinergic signaling (Rosemberg, Toxicology, V. 236, P. 132, 2007). OBJECTIVES: Study the effect of copper in cell death and in adenosine receptors expression in zebrafish larvae. METHODS AND RESULTS: Determination of cell death: 7 dpf larvae were exposed to 10 μM CuSO4 for 1 h and treated for 30 min with acridine orange. The animals were anesthetized with tricaine to carry out the pictures, and tissue labeling was evaluated by optical microscopy. Cell death of neuromasts was visually confirmed in the trunk region and in the skull of the larvae. Molecular Analysis: Using qRT-PCR, the expression of adenosine receptors was determined 4 and 24 h after 10 μM CuSO4 exposure. It was required a pool of 15 larvae per group (n = 4). Statistical comparison was performed by one-way ANOVA followed by Tukey’s test, and p < 0.05 was considered as significant. Animals exposed to copper showed an increase in A1, A2A.1, A2A.2 e A2B receptors gene expression during 4 h (14.2 ± 5.2; 3.7 ± 1.16; 1.5 ± 0.31; 1.54 ± 0.23, respectively), and 24 h (17.42 ± 6.62; 3.69 ± 0.88; 1.69 ± 0.16; 1.7 ± 0.11, respectively). The P value for each receptor gene expression analysis were 0.0008; 0.0005; 0.0010 and P < 0.0001 for A1, A2A.1, A2A.2 e A2B receptors, respectively. All protocols were approved by the Institutional Animal Care Committee (09/00135, CEUA–PUCRS). CONCLUSION: Our results suggest that adenosine signaling is involved in inflammation induced by copper in zebrafish larvae. FINANCIAL SUPORT: PUCRSINFRA, CNPq, FAPERGS/PRONEX. FAPERGS/CNPq n. 008/2009 (PRONEX). 05 Alkaline phosphatase and adenosine receptors in embryonic stem cell biology Patrícia Pereira Lopes Martins1, Renata Pereira Beco1, Talita Glaser1, Angélica Regina Cappelari2, Ana Maria Battastini2 and Henning Ulrich1 University of Sao Paulo, Brazil 2 Federal University of Rio Grande do Sul, Brazil 1

Background: ATP, adenosine and further purines (purinergic signaling) modulate many processes of cell biology including proliferation and differentiation. The extracellular concentration of ATP and adenosine is modulated by ectonucleotidases, which hydrolyses ATP into adenosine (ADO). In view of that we explored the alkaline phosphatase (ALPL) functions in embryonic stem cell (ESC) biology, because these are pluripotent cells that proliferate indefinitely and their differentiation resemble the early embryo development. Methods and results: First, we observed that among ectonucleotidases expressed by ESC, CD73 and ALPL revealed altered expression patterns upon spontaneous differentiation. Based on these results, undifferentiated (+LIF) and differentiated (−LIF) ESCs were subjected to culture in the absence or presence of 1 mM levamisole (ALPL inhibitor) and 5 μM αβ methylene-ADP (CD73 inhibitor). Real-time PCR studies indicated that ADA, ENPP1 and ENTDP5 were highly expressed in undifferentiated cells and that the inhibition of ALPL induced differentiation (decreased OCT4 and Nanog expression) and increased expression of CD73. Cells treated with ALPL inhibitor showed also ENPP1, ENTDP6 and P1 receptor subtypes (A1 and A2A receptors) lower expression levels than +LIF cells. Under these conditions, we also observed by cell cycle analysis that inhibition of ALPL resulted in a decrease in the number of cells at S and G2/M phases, while the CD73 inhibitor did not affect cell cycle distribution. An enzymatic essay and nucleotides/nucleosides quantification by HPLC indicate basal concentration of ATP in differentiated cells higher than in other conditions while the level of all the other products of this pathway is higher in undifferentiated cells. There was also detected a high activity of ADA in undifferentiated cells, since the formation of INO was the highest and corroborated by real time PCR experiments. Conclusion: In summary, ALPL and ADA participate in maintenance of ESC pluripotency and proliferation. Inhibiting ALPL activity is sufficient to differentiate these cells even in the presence of LIF, known for helping to maintain pluripotency, by controlling the availability of ADO in the extracellular milieu and then activity of P1 receptor subtypes.


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Acknowledgments: This project is financial supported by FAPESP and CNPq. 06 Caffeine protects against memory loss induced by cannabidiol in adult zebrafish (Danio rerio) Luiza Reali Nazario, Régis Antoniolli Júnior, Katiucia Marques Capiotti, Carla Denise Bonan and Rosane Souza da Silva PUCRS, Brazil Background and Objectives: Cannabidiol (CBD) has low affinity for cannabinoid receptors and agonistic properties to 5-HT receptors. Interaction between cannabinoid and purinergic systems has been proposed through the indirect agonistic effect of CBD on adenosine receptors, by increasing adenosine levels as a response to nucleoside transport. The purpose of this study is evaluate CBD properties on memory and the effects of pre-treatment with adenosine receptor blockers using adult zebrafish. Materials and Methods: Adult fishes (4 months) were weighted to adjust the CBD dose (0.1, 0.5, 5.0 and 10 mg/Kg) into 10 μL via intraperitoneal injection. Previously to injection the animal were anesthetized with tricaine immersion (100 mg/L). The inhibitory avoidance task we analyze the effect of CBD gave 1 h before or after training on memory and was registered by the comparison of the latency to cross chambers during training and task sections as a measure of memory retention (protocol Blank et al. Neurobiol Learn Mem. BRA Nov;92(4):529–34, 2009). Blockage of adenosine receptor was performed through a chronic pretreatment with caffeine (~20 mg/L - 4 months) and an acute co-treatment with specific antagonists (DPCPX (6 mg/ L) or ZM241385 (6 mg/L) – 1 h). The control group for CBD was the vehicle Tween 80 2 % and for the antagonists DMSO 1 %. On memory assessment, T test was used to compare latency on training versus latency on test section (comparison inside the groups), while One Way ANOVA was used to compare the latency between groups. Results: CBD given before training section at the doses 0.1, 0.5 and 10 mg/Kg did not affect the memory retention, while CBD 5 mg/Kg decreased the latency on test session affecting memory retention (Training latency versus test latency, p > 0.05). When CBD was given after training section, the doses 0.1, 0.5 and 10 mg/Kg did not affect the memory retention, while CBD 5 mg/Kg affected the memory parameter (Training latency versus test latency, p > 0.05). The previous long-term caffeine treatment was not able to alter CBD (5 mg/Kg) effects on memory, when CBD was given prior to training, while caffeine was protective against the memory disruption promoted by CBD (5 mg/Kg), when given after training (p = 0.0003). The impairment on memory exerted by CBD 5 mg/Kg given after training section was prevented by the co-administration of DPCPX or ZM241385 (Training latency versus test latency, p < 0.0001 and p = 0.0074, respectively). Conclusions: Here we showed that CBD affected memory formation in a specific dose (5 mg/kg). Chronic exposure to caffeine appears as a protective way to avoid the non-desired effects of high doses of CBD, at least on memory consolidation. Prevention by co-treatment with specific adenosine receptors antagonist indicated that adenosine can exert effects on CBD action on memory. Financial support: FAPERGS, CNPq. 07 Dipyridamole and EHNA prevented scopolamine-induced memory impairment in zebrafish Josiane Woutheres Bortolotto1,2, Gabriela Madelana de Mello2, Giana de Paula Cognato3, Mônica R. Vianna2 and Carla Denise Bonan2 1 Unicruz, Brazil 2 PUCRS, Brazil 3 UFPel, Brazil Introduction and Aim: Reduction in acetylcholine (ACh) content at the neuronal synapses contributes to memory decline. Drugs, such as scopolamine, that antagonize cholinergic transmission, have profound amnesic effects in a variety of learning paradigms. Conversely, studies showed that adenosine, an important neuromodulator, is involved in formation of memory and other cognitive processes. Extracellular adenosine levels are dynamically controlled by the action of ectonucleotidases, involved in adenosine production by ATP degradation, and equilibrative and concentrative nucleoside transporters. In addition, adenosine may be deaminated by adenosine deaminase (ADA), a cytosolic enzyme that is also found in the external cell surface already described in zebrafish. Thus, considering the involvement of adenosine signaling in cognition and behavior, in this study we investigated the effects of acute exposure to dipyridamole (inhibitor of nucleoside transporters) and EHNA (inhibitor of adenosine deaminase) in a model of cognitive deficit induced by scopolamine in zebrafish. Methods and results: Adult zebrafish were anesthetized with tricaine (100 mg/L) and were submitted to intraperitoneal administration of dipyridamole (5 mg/kg) or EHNA (100 μg/Kg). Control groups were submitted to vehicle administration (saline or DMSO 1 %). After the treatments, the animals were placed in a tank containing scopolamine (200 μM) or unchlorined water during 1 h. For behavioral analysis, zebrafish were individually trained and tested for inhibitory avoidance task and the latency of training and tested groups were analyzed. Locomotion and social interaction were also evaluated. Training and test latencies for each group were compared by Wilcoxon matched pairs test. Latencies of multiple groups were compared using Kruskal– Wallis and Mann–Whitney U tests. Exploratory assessment and social interaction data were analyzed via one-way analysis of variance (ANOVA) followed by Tukey’s HSD test. For all comparisons, p < 0.05 was considered significant. Results and Conclusion: Our results showed that zebrafish exposed to scopolamine presented a lower latency in the test session (7.9 ± 2.2 s p < 0.05, n = 12) when compared to control (39.4 ± 8.9 s; n = 12). However, zebrafish submitted to dipyridamole (18.1 ± 3.8 s; p < 0.05; n = 12) or EHNA (51.6 ± 10.2 s p < 0.05; n = 12) prevented amnesic effects of scopolamine treatment. No significant differences in locomotion were found in animals that received any treatments when compared to the control group. Social interaction also did not present significant changes between experimental groups, indicating that social interaction was not altered by either scopolamine or modulators of adenosine signaling. Therefore, these findings demonstrated that a possible increase of adenosine levels induced by inhibition of nucleoside transport or adenosine catabolism may prevent scopolamineinduced memory deficits.

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08 Function of adenine receptors and interactions with adenosine A1 receptors in the rat cingulated cortex Karen Nieber, Kathrin Sichert and Marcus Bloßfeld University of Leipzig, Germany The aim of the present study was to evaluate the effect of adenine on the synaptic transmission on pyramidal cells of the rat cingulate cortex and to investigate an interaction with adenosine A1 receptors (A1Rs). Therefore, mRNA expression was investigated in tissue probes of the rat cingulate cortex. Functional investigations were done electrophysiologically by intracellular recordings on pyramidal cells in layer V of the cingulate cortex in rat brain slices. Postsynaptic potentials (PSPs) were evoked by electrical stimulation with a bipolar electrode in layer I. Adenine (1 mM) depressed reversibly the PSPs but had no influence on membrane potential and input resistance. Both the NMDA and non-NMDA component of the PSPs were inhibited suggesting an influence on presynaptic glutamate release. In further experiments the interaction with A1Rs was tested. Adenine as well as the selective A1R agonist N6cyclopentyladenosine CPA was superfused successively without washout, each in a concentration that induced maximum inhibition in individual experiments. After preincubation of adenine (1 mM) the influence of CPA (10 μM) was significantly reduced compared with the CPA effect when given alone. In contrast, after preincubation of 100 mM adenine a low concentration of CPA (1 nM) induced an increase of the PSPs. In further experiments the selective A1R antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) was superfused 5 min before application of adenine. DPCPX (0.1 μM) did not influence the adenine induced PSP inhibition. The current results confirm the functional expression of adenine receptors in the rat cingulate cortex and suggest an interaction between adenine and adenosine A1R. 09 Genetic stability and tumorigenicity in potential cell therapy Heidrun Holland University of Leipzig, TRM, Germany Cell therapy raises the question whether the application of these cell products to humans is safe. Consensus of the Medicines Agencies in Europe is: “In conclusion, on the basis of the state of art, conventional karyotyping can be considered a valuable and useful technique to analyse chromosomal stability during preclinical studies.”(Barkholdt et al., 2013) Therefore, a potential tumorigenic risk by genetic instable or changed cells should be analyzed prior to cell therapy. Trypsin-Giemsa staining (GTG banding) was performed on mRNA-iPS cells and the parental fibroblasts to show the presence or absence of genetic instabilities or chromosomal aberrations according to the ISCN 2013 recommendations (Committee for the International System for Human Cytogenetic Nomenclature). The mRNA-iPS cells showed a tendency to reduce numerical and structural chromosomal aberrations. In a preclinical study, we analyzed 408 chondrocyte samples (100 adherent cultures and 308 spheroids) from seven donors using GTG-banding, spectral karyotyping (SKY), and locus-specific fluorescence in situ hybridization (FISH). For at least 3 passages, our genetic analyses revealed no significant chromosomal abnormalities using these techniques [for example fragile site fra(4)(q31)- only single event in passage 3 of one donor]. Clonal occurrence of polyploid metaphases and endomitoses were identified with increasing cultivation time (passage 4–10). Y-chromosomal losses were obtained in the two male donors with increasing frequency during the cultivation time. One donor showed trisomy of chromosomes 1,7,8,12, and translocation of chromosomes 7 and 9, which are also described for extraskeletal myxoid chondrosarcoma. However, a combination of different genetic techniques is useful to increase the knowledge and experience of potential cell therapeutics and to shape the safety of cell therapy of (combined) ATMPs. 10 Guanosine administration induces anxiolytic-like effects in rats: proposal for a novel mechanism of action for a multi-target drug Roberto Farina de Almeida1, Samanta Oliveira Loureiro1, Anelise Reis Gaya1, Gisele Hansel1, Eduardo Rigon Zimmer1, Marcelo Ganzella2 and Diogo Souza1 1 UFRGS, Brazil 2 Max Planck Institute for Biophysical Chemistry Anxiety disorders represent the most common worldwide psychiatric diseases but nowadays there is no satisfactory strategy to their treatment without adverse effects. Accumulating evidence indicates that excitatory synaptic processes are linked to the neurobiology and treatment of anxiety. New studies are pointing that drugs that modulate brain glutamatergic and adenosinergic neurotransmission were demonstrated to be targets for innovative and effective anxiolytic drugs. In this way, several findings support the hypothesis that the nucleoside guanosine (GUO) can finely modulate both glutamate (GLU) and adenosine (ADO) systems in the brain. In this line, we previously demonstrated that a systemic administration of guanosine-5′monophosphate (GMP) induces a similar anxiolytic-like effects to benzodiazepine in rats, however the mechanism of such effect remained unclear. Thus, the present study aimed to investigate the putative anxiolytic potential of guanosine (GUO), as well as to elucidate the underlying mechanisms, with particular emphasis on the glutamatergic and adenosinergic neurotransmission systems. Our results showed that systemic administration of GUO induced anxiolytic-like effects in the Round Open Field (ROF) and in the Elevated Plus-Maze (EPM) tasks, enhanced ADO and decreased GLU level in the cerebrospinal fluid (CSF). Meanwhile, the GUO-induced anxiolytic-like effects in the EPM task and the decrement in CSF GLU levels were abolished by pre-treatment with caffeine (CAF, a non-specific ADO receptor antagonist). Moreover, looking for elucidate the exact GUO mechanism of action, our in vitro investigations indicated that GUO itself was capable of decreasing K+−stimulated GLU release from hippocampal synaptosome, an effect (a) similar and not influenced by CCPA, a specific adenosine agonist, (b) fully reversed by DPCPX, a specific ADO A1 receptor antagonist, and (c) not influenced by the ZM241385, a specific A2a receptor antagonist. Combining these findings, we hypothesize that systemic administration of GUO


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promotes increases in CSF ADO levels and that the ADO consequently binding to ADO A1 receptors will decrease GLU release in the hippocampus; together, these neuromodulatory effects induce anxiolytic-like behavior. Through this work, we have made advances in the discovery of the molecular targets and signaling pathways recruited by GUO Financial Support: This study was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), INCT para Excitotoxicidade e Neuroproteção, FAPERGS and by the FINEP research grant: “Rede Instituto Brasileiro de Neurociência (IBN-Net)” #01.06.0842-00. 11 Guanosine promotes neuroprotection against oxidative and bioenergetic damage in a model of cerebral ischemia in vitro Carla Inês Tasca, Daniel Tonial Thomaz, Débora L Scheffer, Alexandra Latini and Tharine Dal-Cim UFSC, Brazil Oxygen and glucose deprivation (OGD) during cerebral ischemia produces a rapid decrease of neuronal ATP and collapse of ion gradients. The failure of Na+/K+−ATPase leads to membrane depolarization, an increase in intracellular Ca2+, and an enhance in reactive oxygen and nitrogen species synthesis. These events trigger neuronal death. In recent years, our research group has studied the effects of guanosine (GUO), an endogenous nucleoside, as a neuroprotective agent during cerebral ischemia events. Thus, the aim of this study was to investigate the neuroprotective effect of GUO on oxidative damage and bioenergetics in hippocampal slices of rats subjected to OGD, a model of cerebral ischemia in vitro. Male adult Wistar rats (60–90 days) were used for the experiments after approval by the local Ethical Committee for Animal Research. Hippocampal slices were subject to 15 min of OGD followed by 120 or 180 min of reoxygenation. GUO (100 μM) was added during reoxygenation period. Nitric oxide (NO) and peroxynitrite (ONOO-) production were measured by fluorescent probes. The intracellular ATP and extracellular lactate levels were determined enzymatically. Hippocampal slices subjected to OGD undergo a significant decrease in cell viability and GUO incubation during reoxygenation reverses this decrease in cell viability. In this study we observed that OGD induces an increase in NO levels and GUO was able of attenuate this increase (C: 100 ± 3.9; GUO: 102.4 ± 4.9; OGD: 121.5 ± 4.9; OGD + GUO: 102.1 ± 4.2 %). Furthermore, OGD raised the levels of ONOO-, whereas GUO reduces this increment (C: 100 ± 11.1; GUO: 101.4 ± 6.4; OGD: 142.1 ± 6.1; OGD + GUO: 115.7 ± 9.3 %). Intracellular levels of ATP decreased significantly in hippocampal slices incubated under OGD. GUO was not able to recover the ATP levels after 120 min of reoxygenation (C: 2.0 ± 0.2; GUO: 1.9 ± 0.2; OGD: 0.8 ± 0.1; OGD+GUO: 0.9 ± 0.2 μM), but after 180 min of reoxygenation GUO was capable to restore ATP levels (C: 1.9 ± 0.3; GUO: 1.9 ± 0.2; OGD: 0.7 ± 0.2; OGD+GUO: 1.5 ± 0.1 μM). Similarly, after 120 min of reoxygenation, the extracellular levels of lactate decline in hippocampal slices incubated under OGD and GUO was not able to recover the lactate levels (C: 0.60 ± 0.07; GUO: 0.60 ± 0.05; OGD: 0.23 ± 0.03; OGD+GUO: 0.18 ± 0.06 nmol/L), however after 180 min of reoxygenation, lactate levels were partially restored when hippocampal slices were incubated with GUO (C: 0.75 ± 0.05; GUO: 0.75 ± 0.03; OGD: 0.23 ± 0.04; OGD + GUO: 0.46 ± 0.06 nmol/L). In conclusion, our results showed that GUO can afford protection to hippocampal slices subjected to OGD preventing the development of oxidative stress and modulating the cellular energy metabolism. 12 Inosine and combination of caffeine/inosine present antigenotoxic effects in face of methylmercury genotoxicity Sérgio José Macedo Júnior, Amanda Fernades de Oliveira, Vanessa Benitez, Adair Roberto Soares dos Santos and Alcíbia Helena de Azevedo Maia Universidade Federal de Santa Catarina, Brazil Introduction: Methylmercury exposure (MeHg) promotes damage to the genetic material (DNA) and parameters such as formation of micronuclei (MN) have been widely used as biomarkers of its genotoxicity. The objective was to investigate the antigenotoxic effect of inosine, caffeine and the interaction between these treatments, upon MN formation in primary cultures of human lymphocytes exposed to MeHg. Methods: 500 μL of whole blood obtained from healthy human were added to 3.9 mL of RPMI culture medium, 1 ml of fetal bovine serum, 0.1 ml of phytohemagglutinin (1 mg/ml), 60 μl of penicillin (5 mg/ml) and 25 μl of streptomycin (1 mg/ml). Cells were incubated at 37 °C. Twenty hours later cells were treated with DMSO (0.25 %) or MeHg chloride 10 μM (diluted in 0.25 % DMSO) in combination with inosine 5 μM, caffeine 5 μM or both (concentration previously determined in pilot studies). After 24 h at 37 °C, cytokinesis blockage was performed by adding 30 μL of cytochalasin B (1 mg/ml). After 28 h at 37 °C, cells were treated with sodium citrate hypotonic solution (1 %) and a fixing solution (acetic acid: methanol 1: 3, 3 cycles) at room temperature. After that, cells were arranged on slides and stained with Giemsa solution diluted in water (1:15). The number of micronuclei in 1,000 binucleated cells were determined using optical microscopy. Results were expressed as mean ± S.E.M. Statistical analysis was performed by two-way ANOVA followed by Bonferroni post hoc test. Results: MeHg chloride (10 μM) significantly induced MN formation in primary cultures of human lymphocytes when compared to non-exposed group (mean = 4.63 ± 0.32 vs 1.83 ± 1.09; p < 0.05). Inosine (5 μM) significantly prevent MN formation induced by MeHg chloride (mean = 2.37 ± 0.72 vs 4.63 ± 0.32; p < 0.05) different from that observed with caffeine (5 μM) (3.250 ± 0.48 vs 4.63 ± 0.32). Moreover, combination of inosine (5 μM) and caffeine (5 μM) significantly prevented MN formation induced by MeHg chloride (1.12 ± 0.47 vs. 4.63 ± 0.32; p < 0.01). Conclusion: Inosine was able to prevent MN formation induced by MeHg, unlike caffeine. However, when caffeine was used in combination with inosine, it can be observed a possible synergistic effect of the treatments. Financial support: Capes/CNPq. 13 Inosine binds to A1 adenosine receptor to induce pain relief in mice Francisney P Nascimento1,2, Sérgio José Macedo-Junior1, Fabricio A Pamplona1,3, Jana Sawynok2, Adair R S Santos1 1 UFSC, Brazil 2 Dalhousie University 3 Instituto D’Or Aim of Investigation. Inosine is a purinergic metabolite of adenosine. Originally, inosine was considered to be an inactive metabolite, it is now recognized to be biologically active and can produce several physiologic actions. Recently, our research group showed inosine effectiveness in several

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preclinical models of nociception. In order to understand the mechanisms of inosine on pain, this study investigated the involvement of adenosine A1 receptor (A1R) on this effect. Methods. We used C57/BL-6 mice wild-type and A1R knockout with 20–30 g. To evaluate pain behavior the formalin pain test (injection of 2 % solution in hindpaw) was used. Pain behavior was evaluated in animals pretreated with vehicle or DPCPX (A1R selective antagonist) before inosine administration. In another cohort of animals, before inosine administration and pain evaluation they received intrathecal (i.t.) injections of antisense oligonucleotide (AS-ODN) for A1R or mismatch oligonucleotide (MM-ODN) for 5 consecutive days. Further, we carried out an assay of specific binding of inosine with A1R of rat brain. Results. DPCPX was able to block the antinociception induced by inosine when given by intraperitoneal (i.p.) and i.t. route, in both flinches and licking/ biting behavior. Peripherally, DPCPX blocked inosine effect only in licking/biting behavior, but not in flinches behavior. In A1R knockout animals inosine was not able to induce antinociception when it was given by i.p. and i.t. route, but it was effective when given peripherally (paw injection). The knockdown gene of A1R prevented the analgesic effect of inosine. The immunohistochemical assay confirmed that AS-ODN treatment decreased by 61 % the A1R expression compared with control group (MM-ODN treatment) in the dorsal horn of the spinal cord. Furthermore, in a specific binding assay of inosine with A1R, inosine displaced the A1R antagonist 3H- DPCPX with IC50 of 35.9 nM and Ki value of 18 nM. Conclusions. We now demonstrate that the blockade of A1R through pharmacological (administration of A1R selective antagonist) or genetic (AS-ODN and A1R knockout animals) tools blocks the antinociception induced by inosine. However, peripherally, inosine action seems mainly to involve other mechanisms. In addition, we show that inosine does not present effect when given to animals with reduced expression of A1R in the spinal cord. Also, inosine binds to A1R. Taken together, we can conclude that the systemic analgesic effect of inosine depends exclusively of A1 receptor. Financial support: CNPq, UFSC, Dalhousie University. 14 Modulation of the nitrergic system by A1 and A2A receptors. Focus on the mechanisms involved in neural regulation of blood pressure Debora R Fior Chadi1, Daniel C Carrettiero2, João Paulo P Matsumoto1 and Maisa A Costa1 1 University of São Paulo, Brazil 2 Federal University of ABC, Brazil Adenosine and nitric oxide act on the fine-tuning regulation of neural cardiovascular control in the nucleus tractus solitarius (NTS). The mechanisms by which adenosine interferes in the dynamics of nitrergic neurotransmission, is not completely understood. Thus, changes in the interaction between these systems may be especially relevant for individuals predisposed to hypertension. The interaction between the adenosinergic and nitrergic systems in cell culture from the dorsomedial medulla oblongata of Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) will be discussed based on the quantification of nitrite levels, RT-PCR analysis and RNA interference. Adenosine A1 (A1R) and A2a receptor (A2aR) agonists induced a concentrationdependent decrease and increase of nitrite and nNOS mRNA levels in cultured cells from WKYand SHR, respectively. These effects in nitrite levels were attenuated by the administration of the A1R and A2aR selective antagonists, CPT and ZM 241385. Furthermore, knockdown of A1R and A2aR showed an increase and decrease of nNOS mRNA levels, respectively. Pretreatment with the nonselective inhibitor of NOS, L-NAME, abolished nitriteincreased levels triggered by CGS 21680 in WKY and SHR cells. It was also shown that the cAMP-PKA pathway is involved in A1R and A2aR mediated decrease and increase in nitrite levels in SHR and WKY cells. In summary, the results highlight the influence of adenosine on nitric oxide levels in cultured cells from dorsal medulla oblongata of WKY and SHR rats. 15 Participation of adenosine A2B receptor activation in right ventricular adaptation to pulmonary arterial hypertension Bragança, B. Paulo Correia-de-Sá, Rodrigues, T., M. Gonçalves, F. Ferreirinha and A.P. Fontes-Sousa, ICBAS - Universidade do Porto, Portugal Objectives/background: Pulmonary arterial hypertension (PAH) is a progressive disease that overloads the right heart by increasing pulmonary vascular resistance. Adenosine reacts against stressful conditions through the activation of four receptors (A1AR, A2AAR, A2BAR and A3AR). Maladaptive responses of pulmonary A2BAR have been identified in PAH by favouring proinflammatory and fibrotic responses. The role of ARs on right heart adaptation to PAH is unknown. In this study, we investigated the cardiac effects of AR agonists and antagonists in a PAH rat model. Methods and results: PAH was induced in male Wistar rats by a single subcutaneous injection of monocrotaline (60 mg/kg; MCT group); control animals received the same volume of saline (CTRL group). Myographic recordings (spontaneously beating atria and electrically paced right ventricle (RV) strips) and immunolocalization studies were performed 21 to 25 days after monocrotaline administration. In spontaneously beating atria, the non-selective P1 receptor agonist, NECA (0.01–100 μM), caused negative chronotropic (IC50, 6.74 ± 0.08 in CTRL vs 6.59 ± 0.09 in MCT, P > 0.05) and inotropic (IC50, 6.0 ± 0.1 in CTRL vs 5.9 ± 0.1 in MCT, P > 0.05) responses in both experimental groups. Likewise, R-PIA (0.001–1 μM, a selective A1AR agonist) was equally effective in reducing chronotropy and inotropy in both groups of animals. DPCPX (10 nM, a selective A1AR antagonist) prevented the atrial effects of NECA and R-PIA. In paced RV strips, NECA (0.01–10 μM) and R-PIA (0.001–1 μM) caused a small negative inotropic effect at higher concentrations tested. Selective activation of A2AAR, A2BAR and A3AR with CGS 21680 (0.003–1 μM), BAY 60–6583 (0.001–1 μM) and 2-Cl-IB-MECA (0.001–1 μM), respectively, did not change the spontaneous atrial activity and the amplitude of RV strips contractions. Blockade of A2BAR with PSB 603 (100 nM) unveiled a positive inotropic response of NECA (0.01–10 μM) only in RV strips of MCT animals. The RV myocardium of PAH animals exhibited increased amounts of A2BAR immunoreactive cell infiltrates in interstitial spaces. Subsets of these A2BAR positive cells also expressed CD11b (macrophage marker) and vimentin (fibroblast marker). Conclusion: The retaliatory action of adenosine to decrease atrial frequency and inotropy is not modified in PAH. In PAH animals, the A2BR may contribute to decrease RV contractility, since its blockade uncovered a positive inotropic effect of the adenosine analogue, NECA. Extensive fibrosis and interstitial infiltration with A2BAR-positive macrophages in the RV myocardium of PAH animals suggest that adenosine may control the release of pro-fibrotic inhibitory mediators and contribute to remodelling and mechanical adaptation of RV to pressure overload in PAH patients.


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Work supported by FCT (FCOMP-01-0124-FEDER-028726 - FEDER, COMPETE - PTDC/DTPFTO/0802/2012 and PEst-OE/SAU/UI0215/2014). 16 Purinergic expression in rat brain after lithium treatment Maria Carolina Bittencourt Gonçalves, Juliana C. Corrêa, Henning Ulrich and Denise Yamamoto University of São Paulo, Brazil Background: From the last decades, lithium is being largely used for reversing and avoiding recurrent manic crisis in patients with Bipolar Disorder (BD), which is a multifaceted and multifactorial disorder characterized by oscillating changes in mood between mania and depression. Even though, little is known about the etiology of BD or about the mechanisms involved on lithium (and others mood stabilizers) use for treating/preventing mania. Furthermore, the physiopathology of BD shows the involvement of different signaling pathways, including the purinergic system (Machado-Vieira et al. Prog. Neuropsychopharmacol. Biol. Psychiatry, 57:117–31, 2015). The role of purinergic system in BD is still debatable. The presented work evaluated the purinergic genes expression in hippocampus and prefrontal cortex of healthy rats treated with lithium chloride. Methods: Purinergic receptors expression was analyzed in prefrontal cortex and hippocampus of adult rat on day 7 following treatment with lithium chloride (LiCl) compared to saline control (NaCl). All following procedures with animals were in agreement with protocols approved by the local Ethics’ Committee. Adult Sprague–Dawley rats (age 100–120 days; weight 350–500 g) were subjected to repeated intraperitoneal (I.P.) injections twice per day for a period of 7 days with 47.5 mg/kg LiCl (n = 7) or 0.9 % NaCl (n = 7). Prefrontal cortexes and hippocampus of treated adult rats were collected two hours after the last treatment and RNA was isolated followed by cDNA synthesis for RT-qPCR analysis. Results: Expression of adenosine A1 (21.6 %****), A2a (71.0 %*) and A3 (53.5 %*) receptors was significantly decreased after LiCl treatment compared to vehicle (NaCl, set as 100 %). In addition, we observed a significant decrease in ionotropic P2X2 (54.4 %*), P2X3 (63.6 %*), P2X4 (52.9 %**), P2X5 (47.1 %**), P2X6 (50.9 %*), P2X7 (48.6 %**) and metabotropic P2Y4 (36.5 %***), P2Y12 (70.5 %*), P2Y13 (58.1 %*), P2Y14 (29.3 %***) receptors gene expression in comparison to the control group (set as 100 %). (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Conclusion: Downregulation of purinergic receptor expression in rat brain indicates a possible compensatory effects to treatment with lithium, an inhibitor of ectonucleotidases. Further studies are necessary for elucidating underlying mechanisms. Financial support: CAPES, CNPq, FAPESP. 17 Regulation of NMDA receptor subunits expression by activation of A2A adenosine receptors in retinal cultures Olga Grichtchouk, Ivan Domith and Roberto Paes-de-Carvalho Fluminense Federal University, Brazil Objectives: NMDA receptors are important players in several CNS functions including neurogenesis and synaptic maturation. However, glutamate excitotoxicity appears to be primarily mediated by overactivation of NMDA receptors and changes in NMDA receptor channel function, subunit expression, traffic and cellular localization can contribute to a variety of neuropathological conditions. NMDA-type glutamate receptors are tetrameric receptors in which two N1 subunits are obligatory for a functional receptor but different combinations of subunits N2(A-D) and/or N3 are possible and provide different receptor properties. Adenosine is a purine nucleoside which exerts a neuromodulatory function in the CNS, inhibiting for example the release of glutamate at the synaptic cleft. Previous data from our group demonstrated the neuroprotective effect of adenosine on NMDA receptor-mediated cell death in retinal cultures, an effect which was obtained after chronic activation of A2a adenosine receptor by selective agonists. This raised the possibility that A2a receptor activation modulates NMDA receptor expression and traffic in retinal cultures. Therefore, the aim of our study is to examine if chronic treatment of retinal cultures with A2a receptor agonists modifies the expression and localization of NMDA receptor subunits. Methods: Mixed retinal cultures composed by neurons and glial cells were obtained from 8-day-old (E8) White Leghorn chicken embryos. Cultures were treated in their first day (C1) with 100nM CGS21680 or DPMA, selective A2a receptor agonists, for 72 or 48 h, respectively. Cell extracts were obtained and Western blotting was used to analyze expression of NMDA receptor subunits. Additionally, after 72 h of CGS21680 treatment, immunocytochemistry was carried out without Triton X-100 detergent so that the specific antibody could bind only in the extracellular epitope. N2B subunit was analyzed using confocal microscopy. Results: Cell cultures treated with DPMA (100nM) demonstrated an increase of 89.8 % ± 17.6 % (n = 10) in N2B subunit expression and a decrease of 70.3 % ± 7.0 % (n = 7) in N1 subunit expression. Immunocytochemistry results showed that cultures treated with CGS21680 (100nM) demonstrated an increase 148.2 % ± 12.6 % (n = 2) of N2B subunits localized at the cell surface. Conclusion: Chronic treatment with A2a adenosine receptor agonists promotes a reduction of NMDA receptor N1 subunits expression, and an increase of N2B subunits expression in retinal cultures. The Increase of N2B subunits expression is followed by an increase in their localization at the cell surface, suggesting that adenosine regulates NMDA receptor expression and traffic in cultures of retinal cells. Financial support: Faperj, CNPq, Capes and Proppi/UFF. 18 Signaling pathways involved in the antidepressant-like effect elicited by inosine Filipe Marques Gonçalves, Vivian Binber Neis, Débora Kurrle Rieger Venske, Tanara Vieira Peres, Ana Lúcia Severo Rodrigues, Manuella Pinto Kaster and Rodrigo Bainy Leal

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Departamento de Bioquímica – UFSC, Brazil Aim: Inosine is a purine nucleoside, which is formed by the breakdown of adenosine. It was recently shown that inosine administration produces antidepressant-like effects in mice through activation of both A1 and A2A adenosine receptors. The present study was designed to investigate the involvement of PKA, PI3K/Akt, ERK 1/2, CAMKII and GSK-3β in the antidepressant-like effect of inosine in the tail suspension test (TST). In addition, we attempt to verify if inosine administration can modify intracellular targets associated with antidepressant response, such as CREB phosphorylation and the immunocontent of BDNF and β-catenin. Methods: Male adult Swiss mice (6–10 animals per group) were pretreated with H-89 (PKA inhibitor, 1 ug/site, i.c.v.), wortmanin (PI3K/Akt inhibitor, 0.1 ug/site, i.c.v.), U0126 (MEK/ERK inhibitor, 5 ug/site, i.c.v.) or KN-62 (CAMKII inhibitor, 1 ug/site, i.c.v.) and after 15 min, an active dose of inosine (10 mg/kg, i.p.) was administered. In another set of experiments, mice received a subeffective dose of inosine (0.1 mg/kg, i.p.) and 15 min later, a subeffective dose of AR-A014418 (GSK-3β inhibitor, 0.001 μg/site, i.c.v.) was administered. Thirty minutes after inosine administration, the immobility time was evaluated in the TST and the locomotor activity was assessed in the open field test (OFT) in 6–min sessions. For western-blott analysis animals were euthanized and the hippocampus was isolated 30, 60 or 120 min after inosine administration (10 mg/kg, i.p.). Appropriate control animals were used in all experiments. Results: The antidepressant-like effect of inosine in the TST was prevented by the pretreatment of mice with H-89, wortmanin, U0126 and KN-62 (P0.05, two-way ANOVA followed by Newman-Keuls test). Furthermore, the administration of a subeffective dose of AR-A014418, in combination with a subeffective dose of inosine reduced the immobility time in the TST (P0.05, two-way ANOVA followed by Newman-Keuls test). None of the compounds, alone or in combination, produced significant effects on the locomotor activity in the OFT (P0.05, two-way ANOVA). However, inosine was not able to modify CREB phosphorylation or β-catenin and BDNF immunocontent in mice hippocampus in any of the time points evaluated (P0.05 as revealed by Students’t test). Conclusion: Our findings provide evidence that the antidepressant-like effect of inosine in the TST involves the activation of PKA, PI3K/Akt, ERK 1,2, CAMKII and the inhibition of GSK-3β. These results contribute to better elucidate the mechanisms underlying the antidepressant-like effect of inosine and the modulation of purinergic signaling in mood disorders. Financial support: FINEP, Rede Instituto Brasileiro de Neurociência (IBN-Net), CNPq and CAPES. 19 The adenosine facilitation of synaptic plasticity in cortiço-striatal synapses is co-operated by the pre-synaptic and astrocytic A2A receptors Daniel Rial1, Nélio Gonçalves2, Francisco Gonçalves2, Joana Real2, Henrique Silva2, Rui D.S. Prediger1, Jiang-Fan Chen3, Detlev Boison4, Ângelo R. Tomé2 and Rodrigo A. Cunha2 1 Universidade Federal de Santa Catarina, Brazil 2 Universidade de Coimbra, Portugal 3 Neuropharmacology Lab, Boston University, USA 4 Legacy Research Institute, Portland, USA Background: Adenosine A2A receptors (A2AR) control numerous striatal functions (locomotion, habits, addiction). A2AR are mainly located in striatal medium spiny neurons (MSN), which are driven by cortical glutamatergic projections and modulated by dopaminergic inputs. Objective and Methods: We now tested if and how A2AR controlled cortico-striatal plasticity, by comparing the amplitude of long-term potentiation (LTP) in cortico-striatal slices (% of basal), in the absence and in the presence of the selective A2AR antagonist, SCH58261 (50 nM) in 5 mouse lines (all male mice weighing 35–45 g): wild type (WT), global A2AR knockout (g-KO), striatum-A2AR-KO (st-KO with selective A2AR elimination in MSN), forebrain-A2AR-KO mice (fb-KO, with A2AR elimination in forebrain glutamatergic and GABAergic neurons) and GFAP-A2AR-KO mice (ast-KO with A2AR in astrocytes). Results: In WT, LTP amplitude was 127 ± 4 % and SCH58261 inhibited LTP by −57 ± 4 % (n = 4), which was unaffected by 1 μM atropine (n = 4), excluding the involvement of cholinergic modulation. To gauge the cellular site of action of A2AR, we found that the effect of SCH58261 on LTP (n = 4) was: 1-blunted in g-KO; 2-preserved in st-KO (−58 ± 5 %); 3-attenuated in fb-KO (−24 ± 7 %); 4-attenuated in WT mice injected in the pre-motor cortex with lentivirus expressing an sh-RNA to down-regulate A2AR in glutamatergic terminals (−18 ± 1 %); 5-blunted in ast-KO. Accordingly, the blockade of astrocytic glutamate uptake with DL-TBOA (50 μM) attenuated the effect of SCH58261 on LTP (−24 ± 12 %, n = 4) in WT and st-KO (−13 ± 4 %, n = 4) mice and blunted the effect of SCH58261 in fb-KO (n = 4). Conclusion: Taken together, our results indicate a co-operative control of LTP in cortico-striatal synapses by the presynaptic and astrocytic A2AR. Funding Support: CAPES, DARPA, FCT and CNPq. 20 Unexpected role of A1 adenosine receptors: enhancement of vasa vasorum endothelial cell barrier function via GI and AKT-dependent pathways Evgenia Gerasimovskaya1, Umapathy Siddaramappa2, Elzbieta Kaczmarek3, Laimute Taraseviciene1, Martin Lapel1, Philip Weston1, Miguel Fragoso1, Kurt R Stenmark1 and Alexander D Verin2 1 University of Colorado Denver, USA 2 Vascular biology Center, Georgia Regents University, USA 3 Department of Surgery, Harvard Medical School, BIDMC, USA Objectives/Background: The vasa vasorum (VV) is a microcirculatory network that provides oxygen and nutrients to the adventitia and media of large blood vessels. Previously, we have demonstrated that angiogenic expansion of pulmonary artery (PA) adventitia VV in the chronically hypoxic hypertensive calves was accompanied by accumulation of circulating progenitor and inflammatory cells in the PA adventitia. These pathophysiological responses suggest rapidly proliferating vasa vasorum endothelial cells (VVEC) exhibit increased permeability for circulating blood cells and macromolecules. Since extracellular adenosine has been implicated in the regulation of vascular permeability under hypoxic and inflammatory conditions, we


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aimed to determine the differences in a barrier function of VVEC isolated from control and chronically hypoxic animals and to establish a role of adenosine receptors (ARs) in VVEC barrier regulation. Methods and Results: VVEC were isolated from the pulmonary arteries of control (Denver altitude) or chronically hypoxic (2-week hypobaric hypoxia, PB = 430 Hg) neonatal calves. Using quantitative RT-PCR we demonstrated that VVEC express A1R, A2AR, A2BR and A3R, and that A1R expression decreased by 2–3 fold (p < 0.05; n = 4) in VVEC isolated from chronically hypoxic animals (VVEC-Hx) compared to controls (VVEC-Co). We further showed that adenosine significantly enhanced barrier function in VVEC-Co, measured by transendothelial electrical resistance (TER), and to lesser extent in VVEC-Hyp. By using an A1R specific agonist (CCPA, 1uM), antagonist (PSB36, 1nM), as well as siRNA, we demonstrated that A1Rs are mostly responsible for adenosine-induced enhancement in VV barrier function. A barrier protective effect of adenosine was attenuated by pretreatment with pertussis toxin (PTx), inhibitors of PI3K and Akt, and cytochalasin B, suggesting the involvement of Gi proteins, PI3K-Akt pathway, and actin cytoskeleton remodeling. Importantly, A1R expression was demonstrated in the VV of pulmonary arteries of Sprague Dawley (SD) rats, indicating that VV localization of A1R is not restricted to bovine VV. Intravenous co-injection of Rodamine-conjugated Griffonia Simplicifolia lectin (1.4 mg/kg, 40 min) and 150 kDa FITC-conjugated dextran (1.25 mg/mkg, 40 min) in SD rats followed by two photon confocal microscopy of dissected pulmonary arteries enabled visualization of the VV network and characterization of its permeability. This model shows a great promise for the in vivo validation of the protective role A1R agonists in VV barrier integrity. Conclusion: In summary, we demonstrate for the first time that A1Rs enhance VVEC barrier function through the activation of Gi/PI3K/Akt pathway and actin cytoskleton remodeling. We propose that A1Rs can be recognized as a vascular bed-specific and novel therapeutic target to regulate vasa vasorum barrier function and pathologic vascular remodeling in chronic hypoxia. Acknowledgment: NIH/R01 HL086783 to EVG. Ectonucleotidases 21 6-hydroxydopamine alters the ATP metabolism and ADAasi mRNA levels in brain of adult zebrafish (Danio rerio) Stefani Altenhofen1, Josiane Woutheres Bortolotto1, Gabriela Madalena de Melo1, Giana de Paula Cognato2, Carlos Eduardo Leite1, Luiza W. Kist1, Maurício Reis Bogo1 and Carla Denise Bonan1 1 PUCRS, Brazil 2 UFPel, Brazil Background and objectives: Parkinson’s disease (PD), the second most common neurodegenerative disorder is a motor disease associated to the degeneration of striatal dopaminergic neurons. 6-hydroxydopamine (6-OHDA) is a neurotoxic synthetic compound used in scientific research to induce Parkinsonism in experimental animals, such as mice, rats and zebrafish, in order to develop and test new medicines and treatments for PD. Studies have shown the neurodegeneration observed in PD also affects other neurotransmitter systems, including purinergic signaling. This system is characterized by the action of ATP and adenosine on purinoreceptor P2 and P1, respectively. The levels of these molecules are regulated especially by the Nucleoside triphosphate diphosphohydrolases (NTPDases) and ecto-5′-nucleotidase, which constitute the extracellular cascade for ATP hydrolysis to adenosine. Adenosine can be subsequently deaminated to inosine by the action of adenosine deaminase (ADA). Thus, this study evaluated the effects of 6-OHDA exposure on ectonucleotidase activities, ATP metabolism and ectonucleotidase and ADA mRNA levels in brain of adult zebrafish Methods and results: Male zebrafish received one i.p. injection with saline (vehicle) or 6-OHDA (25 mg/Kg or 50 mg/Kg), before each injection, the fish were anaesthetized with 100 mg/tricaine solution (Ethics Committee PUCRS: 11/00257-CEUA). Six days after 6-OHDA i.p exposure, brain membranes were prepared and NTPDase, ecto-5′-nucleotidase, and ADA activities were determined (J Neurochem 61:1685, 1993; Comp Biochem Physiol B Biochem Mol Biol 139:203, 2004), as well as ATP and its metabolites by HPLC (J Chromatogr 199:345–354, 1980) and ectonucleotidase and ADA mRNA levels. NTPDases and ecto-5′-nucleotidase activities and ATP catabolism showed no changes in zebrafish brain membrane after 6-OHDA treatment. However, ADP levels presented a different profile during 180 min of analysis. Firstly, the treatment with 25 mg/Kg 6-OHDA presented a decline in 30 and 60 min and reached the control levels after 120 min of analysis. However, treatment with 50 mg/Kg 6-OHDA increased ADP levels in 120 and 180 min when compared to control group. The treatment with 6-OHDA, in both doses tested, promoted a significant decrease in AMP levels compared to control group. Additionally, adenosine and inosine levels were also evaluated and presented a significant enhancement after treatment with 6-OHDA when compared to control group during 180 min of analysis. The RT-qPCR analysis showed a significant decrease in ADAasi isoform mRNA levels in fish treated with 25 mg/kg and 50 mg/kg of 6-OHDA compared to control group. However, RT-qPCR analysis showed no significant changes in gene expression of other genes analyzed. Conclusion: The findings reinforce the involvement of purinergic signaling in Parkinsonism induce by 6-OHDA, contributing for the development of new therapies. 22 Adenine nucleotide metabolism in lymphocytes of patients with hepatitis C Paulo Guilherme Schimites, Maria Emilha Basso, Thiago Luiz Sponchiado, Mariana Blankenheim Stoever, Claudia de Mello Bertoncheli, Daniela Ferreira Passos, Jamile Fabbrin Gonçalves, Lívia Gelain Castilhos and Daniela Bitencourt Rosa Leal Department of Microbiology and Parasitology - CCS / UFSM, Brazil Objectives / background: Hepatitis C is an important public health concern and a major cause of chronic hepatic disease. The etiologic agent is the hepatitis C virus (HCV) which is transmitted mainly by some invasive medical procedures, sexual intercourse and needle sharing. The HCV targets the hepatocytes where it remains invisible to the innate immune system despite high viral loads and chronic inflammation. Hepatocyte damage leads to the release of adenine nucleotides into the extracellular environment; these nucleotides interact with purinergic receptors or are degraded by ectoenzymes. The ectoenzymes modulate the inflammatory response by degrading these nucleotides in order to maintain homeostasis. This work aims to investigate the E-NTPDase activity in lymphocytes of patients with hepatitis C. Methods: Thirteen patients with hepatitis C and twenty one control individuals were selected for analysis. Lymphocytes from peripheral human blood collected with EDTA were isolated and separated by density gradient. Protein was measured by the Comassie Blue method according to Bradford using

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serum albumin as standard. NTPDase activity was determined as described by Leal et al., which measures the amount of inorganic phosphate released by colorimetric assay. The lymphocytes were suspended in saline solution and added to the reaction medium and pre-incubated. The substrate (ATP or ADP) was added to start the reaction. The released inorganic phosphate was assayed by the method of Chan et al. Controls were carried out by adding the enzyme preparation after TCA addition to correct for nonenzymatic nucleotide hydrolysis. All samples were run in triplicate and specific activity is reported as nmol Pi released/min/mg of protein. This study was approved by the UFSM Ethics Committee (approval number 832.436). Data were analysed by t-test and P < 0.05 were considered significant. Data are represented as mean ± SEM. Results: An increase of 44.2 % in the E-NTPDase enzyme activity, was observed in patients with hepatitis C (70.75 ± 6.564; N = 13) when compared with control individuals (39.48 ± 4.703; N = 21) using ATP as substrate. When ADP was used as substrate, an increase of 53.17 % was found in patients with hepatitis C (87.00 ± 5.874; N = 13) compared to control individuals (40.74 ± 3.605; N = 21). In both cases the differences were considered significant (P < 0.001). Conclusion: HCV infection promotes hepatocyte damage with consequent liberation of nucleotides in extracellular medium. Therefore, the increased ENTPDase activity in lymphocytes of patients with hepatitis C, observed in this study, suggests an increase in ATP and ADP hydrolysis as a compensatory mechanism to decrease the inflammatory process. Financial support: self sponsored. 23 CD39 and CD73 in human visceral leishmaniasis B.G. Macêdo, R.F. Peixoto, L.V. Silva and T.S.L. Keesen Biotechnology Centre of Federal University of Paraíba, Brazil Objectives/Background: Leishmaniasis is an infectious disease caused by several species of protozoa of the genus Leishmania. Among the clinical forms of the disease, the most severe is visceral leishmaniasis (VL), whose main symptoms include fever, hepatosplenomegaly and anemia, and can be fatal if not treated correctly. Immune response to infection by Leishmania species may be associated to both exacerbation of infection in immune system and/or control of the disease. Inappropriate activation of the immune system can lead to unacceptable levels of collateral tissue damage and the development of various pathophysiological conditions, such as visceral leishmaniasis. In visceral leishmaniasis the severity of disease is associated with high IL-10 production by immune cells as monocytes and T cells. CD39 and CD73 are molecules related to regulation of immune response and inflammation control; however its role in visceral leishmaniasis are not determined yet. Thus, our goal was to characterize activated CD4 T cells expressing CD25 in human visceral leishmaniasis and evaluate CD39 and CD73 and the IL-10 cytokine produced by them. Methods and results: To reach this aim, whole blood from endemic control (CTL-n = 3) and visceral leishmaniasis patients (VL- n = 3) were collected. CD4 T expressing or not the CD25 were evaluated after specific Leishmania antigen stimulation (SLA) and Phytohemagglutinin (PHA) as a positive stimulation control. Furthermore, CD4 T cells were labeled to differentiate CD25 positive or negative subpopulations (CD4+CD25+ and CD4+CD25-) and within each subpopulations the level of IL-10, CD39 and CD73 were determined using flow cytometry. This study was approved by the Ethics Committee at Lauro Wanderley Hospital of Federal University of Paraíba (CAAE: 17813013.8.0000.5183). Our results showed that: 1) CD4+CD25+ activated T cells produced higher IL-10 in VL patients than CTL; 2) CD4+CD25- T cells have a slight contribution of IL-10 production in disease 3) CD4+CD25+ T cells from VL patients without stimulation showed an increase of CD39 and CD73 expression, compared to CTL (p < 0.05); 4) SLA induced a decrease of both CD39 and CD73 expression in CD4+CD25+ T cells in VL patients (p < 0.05); 5) Lastly, CD4+CD25- T cells from VL patients presented higher expression of CD39 and CD73 when compared to CTL, before stimulation. In contrast, SLA not induced changes in the expression of these markers in CD4+CD25- T cells. Conclusion: Our data suggest that active visceral leishmaniasis may be involved in the positive modulation of CD39 and CD73 molecules and that this expression may correlate with typical immunosuppression observed in this disease. Acknowledgments: CNPq, Fapesq-PB. 24 Characterization of NTPDASE3 overexpression and ecto-5′-nucleotidase/CD73 knockout clones of T24 bladder cancer cell line Liliana Rockenbach1, Fabrícia Dietrich1, Anna Caroline Avila da Rocha1, Alain Tremblay2, Julie Pelletier2, Jean Sévigny2 and Ana Maria Oliveira Battastini1 1 PPGCB:Bioquímica, ICBS, UFRGS, Porto Alegre, RS, Brazil 2 Centre de recherche du CHU, Université Laval, Québec, QC, Canada Objectives/background: Bladder cancer is the second most common malignancy of geniturinary tract. The current treatments for this malignancy are not efficient enough to avoid the tumor recurrence and progression. For this reason, researches continues to look for new therapeutic targets that could end in more effective treatments for this cancer. In vitro and in vivo experiments have already shown that healthy urothelium did not express ecto-5′-NT/CD73, whose expression is high in bladder cancer. Inversely, NTPDase3 is expressed in healthy urothelium and its expression is low in bladder cancer. Due these evidences of the involvement of purinergic enzymes in bladder cancer progression, the aim of this study is to better investigate the involvement and consequences of the lack of ecto-5′-NT/CD73 and overexpression of NTPDase3 for malignancy of T24 bladder cancer cell line, starting by clones characterization. Methods: T24 human bladder cancer cell line was transfected with lipofectamine using pcDNA3 containing geneticine resistance gene for NTPDase3 overexpression or using CRISPR/Cas system to the knockout of ecto-5′-NT/CD73. Cells were grown in RPMI culture medium with 10 % fetal bovine serum plus penicillin/streptomycin or geneticine (to NTPDase3 clones) and maintained in 5%CO2/95 % air at 37 °C. The different clones of each transfection were isolated and analyzed for enzymatic activity. Then, the best clones were choosen and the efficacy of the tranfsections was confirmed by protein expression and repeating enzymatic activity. To determine the enzyme specific activity, the phosphate released was measured by Malachite Green and the protein by Coomassie Blue. The protein expression was measured by flow cytometry.


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Results: T24 NTPDase3 overexpression resulted in 94 clones, among them the best ATPase activity was showed by the clones 69 (71.6 ± 29 nmol Pi/ min/mg protein) and 94 (59.8 ± 33 nmol Pi/min/mg protein) in contrast of wild type T24 and empty vector ATPase activities, 2.6 ± 1.6 e 1.7 ± 1.2 nmol Pi/min/mg protein (n = 4). In agreement, the percentage of positive cells in flow cytometry was 2.9 ± 0.7 %, 6 ± 3.8 %, 93 ± 1.5 % e 93 ± 2.4 % for respectively, T24, empty vector and clones 69 and 94 (n = 2). T24 ecto-5′-NT/CD73 knockout resulted in more than 100 clones, among them the best AMPase activity was showed by the clones 1.4-26 (0 ± 0.2 nmol Pi/min/mg protein) and 2.4-9 (11.6 ± 5 nmol Pi/min/mg protein) in contrast of wild type T24 activity, 23.3 ± 5 nmol Pi/min/mg protein (n = 4). In agreement, the percentage of positive cells in flow cytometry was 87.2 ± 6.6 %, 0 ± 0.6 % e 64.7 ± 20.6 % for respectively, T24 and clones 1.4-26 and 2.4-9 (n = 3). Conclusion: The results showed that the transformations in T24 bladder cancer cell line successful worked. More studies are necessary to describe the involvement and consequences of these purinergic alterations in T24 bladder cancer cell line malignancy. Financial support: CNPq, CAPES, FAPERGS and CIHR. 25 Development of capillary electrophoresis protocols for the selection of aptamers targeting CD73 Cheffer Arquimedes1, A Shala2, C.E Müller3, S.N. Krylov2, and H. Ulrich1 1 University of São Paulo, Brazil 2 York University, Canada 3 University of Bonn, Germany BACKGROUND: The metabolism of ATP into its metabolites ADP, AMP, and adenosine, and consequently, the regulation of purinergic signaling, is a tightly regulated process by a family of cell surface-located ecto-nucleotidases, including nucleoside triphosphate diphosphohydrolases (NTPDases) and ecto-5′nucleotidase (CD73). AMP is generated by the stepwise catabolism of ATP via the intermediate ADP in two reactions predominantly carried out by CD39 (NTPDase-1), whereas CD73 is mainly responsible for the conversion of AMP to adenosine. Adenosine, in turn, is able to activate receptors expressed in membranes, e.g. of immune system cells, promoting immunosuppressive effects, on cancer cells inducing proliferation, and on further cells inducing angiogenesis. This draws attention to CD73 as a potential therapeutic target for cancer therapy, in which immune system suppression is crucial for the initiation of malignant neoplasms and the progression of established tumors. Indeed, recent evidence suggests that CD73 inhibition reduces tumorigenesis and metastasis, as well as enhances the efficacy of conventional therapies. However, only very few CD73 inhibitors are currently available for pre-clinical trials. OBJECTIVES: Our goal was to develop capillary electrophoresis methods for the selection of aptamers targeting CD73. These oligonucleotides developed by an in vitro selection protocol show affinity and specificity comparable to those obtained with monoclonal antibodies, and are easily produced and optimized. Due to their lack of immunogenicity, they present a promising alternative to small molecules and antibodies. METHODS AND RESULTS: We combined molecular biology and capillary electrophoresis techniques for the development of selection protocols of aptamers against CD73. Tris and borate buffers at different concentrations were tested as running buffers. Although increasing Tris concentrations have had the positive effect of reducing the protein absorption onto the capillary walls, they also resulted in more time-consuming separations. The same was observed when we used ethylene glycol to decrease the electroosmotic flow. The best resolution between the DNA and target peaks was obtained with 50 mM and 100 mM Tris-acetate when the enzyme and a catalytic site-corresponding peptide were used as targets. After determining the aptamer collection window and performing one selection cycle against a target concentration of 5 nM, PCRs were carried out in order to determine the best number of amplification cycles, which was found to be 20. CONCLUSIONS: In summary, intermediate running buffer concentrations resulted in better shaped peaks, higher resolution and non-time-consuming capillary electrophoresis protocols for the separation of aptamers targeting CD73. The first selection cycle has already been performed, and this method will be further applied in order to obtain aptamers with higher specificity and affinity. Acknowledgments: FAPESP and CNPq. 26 E-NTPDASE and E-ADA activities and cytokines serum levels in patients with acute lymphoblastic leukemia Jader Betsch Ruchel1, Liliane Zimmermann de Oliveira1,2, Claudia de Mello Bertoncheli dos Santos1, Marina Sequeira1 and Daniela Bitencourt Rosa Leal1 1 LABIBIO/UFSM, Brazil 2 Hemato-Oncologia-HUSM, Brazil Aim: Acute lymphoblastic leukemia (ALL) is a neoplasia characterized by abnormal clonal proliferation of lymphoid precursor cells, which deregulates most of physiological processes e.g. the immune response. Adenine nucleotides and nucleoside modulate lymphocyte activity through purinergic receptors and enzymatic mechanisms thus regulating the immune and inflammatory responses. This study aims to evaluate E-NTPDase and ecto-adenosine deaminase (EADA) activities in lymphocytes and cytokine serum levels of ALL patients, in order to elucidate the immune processes occurring during this neoplasia. Methods and results: Newly diagnosed and untreated patients (D0), patients treated for 15 days (D15) and control subjects (C) were studied. Group C was paired with D0 and D15 according to age. This study was approved by the UFSM Ethics Committee. White blood cell counts (WBC) were evaluated in peripheral blood by electronic equipment. E-NTPDase (Leal et al., Biochimica et Biophysica Acta, 1721, 2005) and E-ADA (Giusti and Galanti, Wein View Chem, 315, 1984) enzymatic activities (expressed in nmol Pi released/min/mg protein and U/mg protein, respectively) were measured in peripheral lymphocytes of patients with ALL and control subjects by a colorimetric method. Cytokines serum levels (IL-2, IL-6, TNF-α, IFN-γ, IL-4, IL-17 and IL10) were determined by flow cytometry in D0 ALL patients (n = 7) and control subjects (n = 5). Parametric t-test and nonparametric Mann–Whitney and Wilcoxon tests were used for statistical analysis. Results were considered significant when P < 0.05 and were expressed as mean ± SD or SEM. Regarding the WBC counts (expressed in x1000; cells/μL), D0 (60.9, SD = 90.2) and C (7.7, SD = 2.4) differed significantly (P < 0.001). The same was observed when D0 was compared to D15 (1.6, SD = 1.0) (P < 0.01). There was no significant difference in the E-NTPDase activity in D0 for ATP hydrolysis compared to C or D15. As to ADP hydrolysis, a significant increase was observed (P < 0.05) when D0 (29.0, SEM = 5.0, n = 21) was

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compared to C (47.0, SEM = 6.8, n = 16); whereas there was no difference between D0 and D15. A greater activity of E-ADA was found in D0 (161.8, SEM = 50.4, n = 20) compared to C (53.3, SEM = 21.0, n = 16) (P < 0.05), and a lower deamination in D15 (47.5, SEM = 11.5, n = 8) compared to D0 (P < 0.05). Cytokine serum levels did not differ significantly between the groups. Conclusion: The changes found in E-NTPDase and E-ADA activities in ALL patients lymphocytes suggest a possible modulation in the purinergic system of leukemic lymphocytes, which can be reversed, in part, by treatment and reduction of the WBC. Financial Support: PROIC/UFSM, FAPERGS, CNPq, CAPES. 27 Exploring the effect of CD39 delection on radiotherapy induced immune response Marina Petersen Gehring PUCRS, Brazil Despite over a century of effort in the treatment of cancer, there has been little. For most of this time, cancer has been viewed as a disease of hyperproliferative, transformed cells and cancer treatments have been focused on their direct elimination. However, tumor mechanisms employed to evade immune rejection are rapidly being appreciated as a central feature of cancer. Therefore, there is a growing consensus that re-thinking therapeutic approaches to those focused on stimulating tumor rejection by the immune system is needed. Interestingly, there has been accelerating realization that radiotherapy is a potent enhancer of antitumor immunity. Recent clinical reports showed that combining radiotherapy with immunotherapy resulted in dramatic sustained clinical responses, providing profound proof of principle for further development of similar approaches. The CD39-CD73-adenosine pathway has been recognized as a critical immunosuppressive pathway within tumors and thus a promising therapeutic target in such approaches for oncology. We hypothesized that CD39 activity may constitute an important mechanism deployed by the tumor to protect it from ATP-induced cytotoxicity and immune cytotoxic cell attack and the causes the inhibition of the immune response. Thus, we propose to test whether CD39 deletion can improve radiotherapy generating a more robust antitumor immune response. Using CD39 knockout mice bearing-GL261 glioma cell line we observed that CD39 deletion combined to radiotherapy induced a significantly decrease of MDSC (myeloid derived suppressor cells) on the tumor compared to the WT irradiated mice. In addition, CD39 deletion combined to radiotherapy induced a significantly increase of CCR7 positive lymphocytes, macrophages and dendritic cells on the spleen relative to their WT counterparts. The CCR7 increased positive cells were also observed following Cyberknife irradiation of GL261 tumors on CD39KO mice. CCR7 regulates recirculation of a variety of immune cells, guiding their chemotactic homing to lymph nodes, tumor/infected tissues and target cells. An increase in the number of cells expressing this receptor indicates a greater amount of freshly mobilized immune cells available to differentiate in immune-effector. Lastly, we detected that CD39KO mice irradiated presented a significantly decrease of Tregs on the spleen and a significantly increase of IL-15, INF-γ, IL-1β, IL-17A GITR, CCL2 and CCL3 expression on the tumor when compared to WT mice irradiated. These robust responses observed in the CD39 knockout mice following irradiation of their tumors demonstrate a role for CD39 and purinergic signaling to improve radiotherapy-induced immune response. 28 Extracellular hydrolysis of adenine nucleotides in the blood serum of male sedentary individuals submitted to acute physical exercise Cesar Eduardo Jacintho Moritz, Bruno Costa Teixeira, Liliana Rockenbach, Alvaro Reischak-Oliveira, Emerson André Casali and Ana Maria Oliveira Battastini UFRGS, Porto Alegre/RS, Brazil Objectives/background: Purinergic singnalling is responsible for influencing physiological and pathological situations through hydrolysis of nucleotides by nucleotidases. Physical exercise could be a non pharmacolgical conduct in many diseases, however is unclear its importance in this extracelullar signaling. The aim of this study is analyze how is the behavior of these enzymes in blood serum of individuals exposed to acute physical exercise. Methods: Seven healthy sedentary male subjects (n = 7) with a mean age 26.1 ± 2.7 were selected. All procedures were approved by ethics committee of Universidade Federal do Rio Grande do Sul (n° 760.528) and the free consent was obtained from all individuals. Subjects performed an initial evaluation to collect clinical, anthropometric data and maximal oxygen (VO2MÁX) uptake were defined for ergospirometry system open-circuit gas analysis. Seven days after the evaluation, all volunteers performed 30 min of aerobic exercise on treadmill with 70 % of maximal heart rate. Blood samples were collected pre- and post-exercise by venipuncture and centrifuged to obtain serum. ATPase, ADPase and 5′-nucleotidase activities were quantified by the release of Pi released from ATP, ADP or AMP hydrolysis. Specific activity are expressed as nmol Pi/min/mg protein (Anal. Biochem. 157:375, 1986). Phosphodiesterase activity was assessed using p-Nph-5′-TMP as substrate and the specific activity are expressed as nmol p-nitrophenol/min/mg protein. (Thromb. Res. 91:83, 1998). Data are presented as mean ± S.E.M. Results: ATP (0.173 ± 0.014 vs. 0.535 ± 0.038), ADP (0.194 ± 0.038 vs. 0.736 ± 0.123) and AMP (0.260 ± 0.027 vs. 0.476 ± 0.077) hydrolysis increased from pre from post-exercise respectively. Phosphodiesterase activity was also increased by exercise (3.27 ± 1.01 vs. 5.70 ± 0.95). Conclusion: Physical exercise possibly exert modulatory effects in nucleotidasic activities. Since it is a preliminary result, we still need bigger sample size to better define what kind of modulatory effects are these. More studies are necessary to define effects of exercise in purinergic signaling. Financial support: CAPES / CNPq. 29 Interference of direct and indirect co-culture of gliobastoma and mesenchymal stem cells in expression levels of kininergic and purinergic components Mateja Delač1, Mona das Neves Oliveira2, Barbara Breznik1, Micheli M. Pillat2, Talita Glaser, Henning Ulrich2 and Tamara T. Lah1 1 National Institute of Biology, Slovenia 2 University of São Paulo, Brazil Bone marrow derived mesenchymal stem cells (BM-MSCs) display high tropism towards tumours, including the most malignant brain tumour glioblastoma (GBM). However, the molecular mechanisms of their interactions are still not clear (Motaln and Lah, 2015).


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Previously, we confirmed that BM-MSC impair proliferation, invasion and senescence of several GBM lines (Schichor et al., 2011; Motaln et al., 2012). Transciptomics analyses revealed that the activation of bradykinin (BK) and its receptors (kinin-B1 and B2-receptors) may be involved upon the intercellular interactions. We show here that direct and indirect co-culture of GBM and MSC are also affecting the expression rates of kinin receptors and CD73 (5′ectonucleotidase), a key enzyme of the purinergic system, metabolizing 5′-AMP into adenosine and thereby determining activity levels of adenosine receptors. Upon direct co-culture for 72hs, down-regulation of kinin-B1 and B2-receptors in MSC occur, while receptor expression is enhanced in U87 glioma cells, suggesting functions of this receptor in invasiveness and immunomodulatory responses. Similarly, CD73 expression levels diminished in MSC in direct co-culture, while expression rates of this enzyme is augmented in U87 cells, being in line with their increased invasiveness, as observed previously (Schichor et al., 2011). Indirect co-culture, where no direct interactions between cell surfaces of U87 and MSC occur, but the exchange of soluble factors is possible, expression rates of kinin-B1 and B2 receptors did not change, while again in U87 cells an increase in receptor expression was observed. Reduction of CD73 expression in MSC was less prominent in indirect co-culture, when compared to direct co-culture, however it increased in U87 cells to similar levels as those observed in conditions of direct co-culture. Further aims of this work lies in the investigation of interrelationships between the kininergic system and CD73, being used as a prognostic marker of glioblastoma malignancy. References Motaln and Lah, Proteins and Peptide Letters (in press,2015); Motaln et al. Cell Transplantation 2012, 1529–45; Schichor et al. Exp. Neurology 2012, 234, 208–19. 30 Measurement of E-NTPDASE and E-5′-nucleotidase enzymes activities in platelets on patients with cutaneous melanoma Margarete Dulce Bagatini1, Aline Mânica2, Alexsandra Martins Da Silva1, Cristiane Marolli1, Kelly Aparecida Zanella1, Marcelo Moreno3, Andréia Machado Cardoso2 and Vera Maria Morsch2 1 Universidade Federal da Fronteira Sul, Brazil 2 Universidade Federal de Santa Maria, Brazil 3 Universidade Comunitária da Região de Chapecó, Brazil Objectives: The cutaneous melanoma (CM) is formed from malignant transformation of melanocytes and involves environmental and genetic factors. The melanoma grows in different parts of the body through a combination of mitogenic and genotoxic effects on melanocytes and consequently invading the immune system (Scient, 2013: 1, 2013). The number of deaths caused by CM is excessively higher than other malignant cancers, thus it is a major public health concern (Arq Cat Med, 38: 14, 2009; SNRI Derm, 2013: 1, 2013). Nucleoside and adenine nucleotides mediate a variety of biological functions through extracellular signaling: development, regeneration, differentiation, proliferation, and cell death (Purin Sign., 3: 431, 2014). Enzymes that degrade adenine nucleotides, called ectonucleotidases play an important role in regulating the tumor microenvironment. This enzyme is involved in maintaining the balance between Adenosine triphosphate (ATP), Adenosine diphosphate (ADP), Adenosine monophosphate (AMP), and adenosine levels (Can Immun Immun., 63:. 1073, 2014). Thus, the aim of this study is to analyze the activity of the ectonucleotidases E-NTPDase and E-5′nucleotidase in platelets from patients with CM post-treatment and control patients. Method: This study was submitted to the UFFS Ethics Committee and approved under the following opinion: 822,782. The sample consisted of 23 patients with CM and 54 patients as control group. The confidence interval was 95 %, considering p-value 12 cells; ***p < 0.001) of the resting levels when cells were treated with 1 mM ATP, 100 μM Bz-ATP and 100 μM ATP, respectively. We analyzed labeled glutamate release in cultured glia using the same purinergic agonists and found an increase in the extracellular content of the amino-acid after 15 min. stimulation (3 mM ATP = 145 ± 10 %***, 100 μM Bz-ATP = 149 ± 13 %** and 100 μM ATP = 139 ± 6 %***; n > 5, **p < 0.01). Treatment of the cells with 1 mM glutamate induced increases in intracellular calcium with peak values representing 150 ± 5 % of the resting levels (n = 8; p < 0.001). While no change was detected in quinacrine stained, ATP-containing vesicles for at least 10 min in control cultures, treatment of the cells with 1 mM glutamate resulted in reduced intracellular levels of quinacrine fluorescence at the same time interval. Incubation of the cells for 5 min with 1 mM glutamate was also able to increase the levels of ATP in extracellular medium by 179 ± 15 % (p < 0.001, n = 19), a response that was blocked by the calcium chelator BAPTA-AM. Conclusion: These results suggest that cultured glial cells from chick embryo retina can release glutamate following activation of the calcium-inducing purinergic receptors. Furthermore, cultured Müller cells have ATP-containing vesicles that can be released in a calcium-dependent way by activation of glutamate receptors. Supported by: CNPq,Faperj,Proppi-UFF,Pronex-MCT,CAPES. 45 Bradykinin and purinergic signaling in brain diseases Y. Naaldijk1, J. Corrêa3, E.G. Ferrazoli4, M.C. Gonçalves4, C. Fabian1,2, T. Glaser3, C. Heine1, H. Holland1, C. Lameu3, S. Melzer1, A. Oliveira3, M.M. Pillat3, P.D. Negraes3, L. Sardá-Arroyo3, V. Savkovic1, J. Stachnik1, H.D.N. Souza3, A. Tarnok1, U. Sack1 and H. Ulrich3 1 Translational Centre of Regenerative Medicine (TRM) & Medical Faculty, Univ. Leipzig, Germany 2 Fraunhofer IZI, Leipzig, Germany

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3

Department. of Biochemistry, Institute of Chemistry, University of Sao Paulo, Neurology/Neuroscience Program, UNIFESP, São Paulo, Brazil

4

State of the art: Neurodegenerative diseases (e.g. ALS and Parkinson’s disease) and psychiatric disorders (e.g. bipolar disease and depression) present a common health problem. Recent studies have implicated functions of bradykinin acting through kinin-B2 receptors (B2BKR) and ATP acting through purinergic P2 receptors to regulate neurogenesis. B2BKR activity induces neurogenesis over gliogenesis of differentiating murine embryonic neural progenitor cells and promotes neuroprotection in an in vitro model of cerebral ischemia. Unpublished data from our group show the induction of adult hippocampal neurogenesis by bradykinin, and possible applications of this peptide in neuroprotection / neuroregeneration in a rat model of Parkinson’s disease. Purinergic metabotropic receptors participate in in vitro neurogenesis of pluripotent stem cells, while P2X7 receptors are related to gliogenesis. Subsequently, expression levels of these receptors are down-regulated following induction of neurogenesis. In agreement, chronic inhibition of the P2X7 receptor promotes neuroregeneration in the Parkinson’s disease animal model. Lithium chloride, a psychiatric medication, inhibiting degradation of ATP into adenosine, affects P1 and P2 subtype expression in rat hippocampus. Interrelationships exist between kinin- and ATP-signaling, i.e. differential regulation of purinergic P2 receptors in conditions of chronic inhibition of B2BKR or NO production, a principal signaling pathway induced by bradykinin, during neural differentiation. Our bilateral project (IQ-USP and TRM/Medical Faculty of Univ. Leipizig) aims to study the effects of the mentioned signaling systems on cellular therapies. Stem cell therapies may provide treatment options for brain diseases. A challenge is the immunological response of transplanted cells, which will be explored further. It is supposed that modulation of kinin and purinergic receptors supports differentiation and reduces immune responses. Future aims: We plan to investigate whether bradykinin and purinergic signaling represent targets for cellular therapies by stem cells. Common features will be worked out, using diverse stem and iPS cells regarding the interaction of kinin and purinergic receptors in cellular regeneration. In vivo and ex vivo models will implicate future clinical applications. Acknowledgement: BMBF, grant 1315883 to the TRM and 01DN13037. FAPESP (Proj. No. 2012/50393-6 and 2012/50880-4) and CNPq grants, Brazil. 46 Contribution of P2X7 receptor in immune response during chronic toxoplasmosis A. Moreira-Souza1,2,3, R.C. Vommaro2 and R. Coutinho-Silva1,3, Lab. Imunofisiologia, Universidade Federal do Rio de Janeiro, Brazil 2 Lab. Ultraestrutura Celular Hertha Meyer, Universidade Federal do Rio de Janeiro, Brazil 3 Instituto Nacional de Pesquisa Translacional em Saúde e Ambiente na Região Amazônica (INPeTAm/UFRJ), Rio de Janeiro, Brazil 1

Background: The purinergic receptor P2X7 is involved in several physiological and pathological events. P2X7 receptor was described to participate in the immune response against different intracellular pathogens such as Leishmania amazonensis, Chlamydia and the acute phase of toxoplasmosis (Coutinho-Silva & Ojcius, Microbes and Infection 14, 1271, 2012). Toxoplasma gondii is a protozoan parasite that infects homoeothermic host, and may cause serious impairment or death to immunocompromised individuals such as HIV patients, transplanted patients and pregnant women. In this work, we evaluated the contribution of P2X7 receptor for the immune response during the chronic phase of toxoplasmosis. Methods and Results: We used female C57BL/6 (WT) and P2X7 knockout (P2X7−/−) mice 6–8 weeks old, orally infected with 5 or 10 cysts of Me-49 strain T. gondii, and the survival was monitored. We observed that all P2X7−/−mice succumbed 8 week after infection with 5 cysts, while all WT mice survived. We also analyzed the effect of infection on two shock organs (brain and liver). For that, all animals groups were euthanized after 30 days to perform dosage of hepatic enzymes release. ALT and AST enzymes were evaluted in the liver, and found higher levels of both enzymes in WT mice when compared with P2X7−/− (5.6 ± 0.6, n = 8; 1 ± 0.2, n = 8, to ALT and 1.8 ± 0.4, n = 8; 0.8 ± 0.1, n = 8, to AST, in WT and P2X7−/−, respectively). We observed a larger number of cysts in the brain of P2X7−/− animals when compared with WT, (22 ± 8 and 2 ± 0.6, n = 6 to P2X7−/− and WT mice respectively. In addition, we found increased levels of pro-inflammatory cytokine IL-12 in brain extracts of both WT and P2X7−/− animals, Although the secretion was lower in brain of P2X7−/− mice (1.1 ± 0.06, n = 8; 0.8 ± 0.05, n = 6, to WT and P2X7−/−, respectively). The same effect also was observed in animals infected with 10 cysts of T. gondii. Conclusion: The P2X7 receptor participates in immune response against T. gondii during chronic toxoplasmosis. Financial Support: CNPq, FAPERJ, PRONEX, CAPES, INPeTAm/UFRJ. 47 Decreased UTP-induced signaling prolonged by activation of IGF-1 receptor in culture of chicken retinal cells Yago Côrtes Pinheiro Gomes, Mariana Souza Elysio, Erick Correia Loiola and Ana Lucia Marques Ventura Neurobiology Department, UFF, Niterói/RJ, Brazil Objectives: IGF-1 is a trophic factor involved in the proliferation of glial progenitor cells of the retina induced by ADP. This factor stimulates the expression of the CDK1 and with ADP induces progression of these cells through the cell cycle. These progenitors are also affected by UTP, wich stimulates its growth, adhesion and / or migration in cultures subjected to mechanical injury by a mechanism dependent of PI3K / Akt pathway. In this study, we investigated whether IGF-1 is capable of modulating the intracellular signaling induced by UTP in retinal cell cultures. Methods and Results: Retinal Cultures prepared from chick embryos of 7 days old were cultured for 24 h in MEM containing 5 % fetal bovine serum. After this period, the culture medium was removed and the cells cultured for 48 h in serum free medium. In the last 24 h, cultures were treated with or without 100 ng / ml IGF-1, washed and stimulated with 100 mM UTP for 5 min. The levels of phosphorylation of proteins were analyzed by western blot. Increased phosphorylation of Akt 217.7 ± 38.4 % (* p < 0.05, n = 3) was observed at 5 min after stimulation of the cultures with 100 mM UTP. However, pretreatment of the cultures for 24 h with 100 ng / ml IGF-1 decreased the phosphorylation of Akt induced by 100 uM UTP to 79.7 ± 7.7 % of control levels (** p < 0.01, n = 3) . Moreover, the reduction induced by IGF-1 was completely blocked by the joint addition of 25 uM LY 294002, a blocker of PI3K or 30 uM monodansyl-cadaverine a blocking endocytosis, suggesting that the decrease induced by IGF- 1 on phosphorylation of Akt dependent UTP is caused by activation


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of the IGF-1 receptor. On the other hand, no decrease in Akt phosphorylation induced by 100 ng / ml of IGF-1 was observed when cultures were pretreated for 24 h with 100 mM UTP, suggesting that the prolonged activation of Akt is not capable UTP regular mediated signaling of IGF-1 receptors. Conclusion: These results suggest that the prolonged activation of IGF-1 receptor is capable of inducing desensitization of UTP-stimulated signaling in chick retinal cell cultures. Financial support: CNPq, CAPES, Proppi-UFF. 48 Effect of irradiation on the expression of enzymes and receptors of purinergic system in T24 human bladder cancer cell line Patrícia Boni de Paula1, Fabrícia Dietrich2, Angélica Regina Cappellari3, Fernanda Bueno Morrone3 and Ana Maria Oliveira Battastini2 1 UFCSPA, Brazil 2 Departamento de Bioquímica UFRGS, Brazil 3 Faculdade de Farmácia PUCRS, Brazil Objectives/background: Bladder cancer is the second most common malignancy that affects the genitourinary tract and the seventh most common cancer among men in the world. The treatment of this disease varies according to the invasive nature of the tumor. For invasive bladder cancer, cystectomy remains the main treatment, but has been growing the interest in therapeutic approaches that aimed preserve the organ. In recent years, several studies have pointed out the existence of a relationship between bladder tumorigenesis and purinergic signaling. The purine and pyrimidine extracellular exert different effects in the body, which occur via purinoreceptores and are controlled by ectonucleotidases. Previous studies from our laboratory have shown that this system is altered in bladder cancer. Once the radiotherapy is also a treatment choice for bladder cancer and considering the involvement of the purinergic system in the progression of this malignancy, this study was developed in order to investigate possible changes in the profile of ectonucleotidases and the purinergic receptors expression (P1 and P2) after T24 human bladder cancer cell line irradiation. Methods: T24 cell line, derived from an invasive bladder tumor with metastatic potential, was cultured in RPMI culture medium supplemented with 10 % fetal bovine serum (FBS). T24 cells were seeded and after reaching semi-confluence the cultures were exposed to 4Gy irradiation. Control cultures, which not receive irradiation, also were performed. After 48 h of irradiation, the mRNA expression of ectonucleotidases and purinergic receptors was evaluated by PCR and Real time PCR. Results: It was observed a significant decrease in the NTPDase 5 (28.92 ± 0.04 %, n = 3) and in NPP1 (15.59 ± 0.09 %, n = 3) mRNA expression. The protocol of irradiation, induced a significant increase on the ecto-5′-nucleotidase (92.00 ± 0.13 %, n = 3). None alteration was observed in the expression of P1 receptors. However, a significant increase in the expression of P2X5 (35.16 ± 0.09 %, n = 4) and P2X6 receptors (128.42 ± 0.40 %, n = 3) was observed in the irradiated cells. 4Gy irradiation also caused a significant increase in P2Y2 (51.75 ± 0.06 %, n = 3), P2Y6 (95.70 ± 0.26 %, n = 3) and P2Y12 (31.54 ± 0.25 %, n = 4) and a significant decrease in P2Y1 (23.42 ± 0.02 %, n = 3). Conclusion: These preliminary results clearly show that irradiation leads to changes in purinergic signaling, altering some enzymes and some receptors of this system. Financial support: CNPq and Capes. 49 Extracellular ATP homeostasis and osmosis Marta Santos1,3, Pedro Persechini1,3 and Julieta Schachter1,3 1 Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil 2 Polo Xerém, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil 3 Instituto Nacional de Pesquisa Translacional em Saúde e Ambiente na Região Amazônica, Brazil Introduction: In several physiological and/or pathologic conditions, mammal cells are subjected to external osmotic changes, which affects the cellular volume. When subjected to hypotonic shock, cells swell but most of them can recover their original shape, a process known as RVD (regulatory volume decrease). In particular, macrophages can face changes in external osmolarity during the inflammatory response, and it is believed that this condition modifies their function. In the present work, we explore the possibility that RVD of macrophages is regulated by ATP molecules released in response to the hypoosmotic shock. Materials and Methods: RAW 273 macrophages were maintained in a medium with physiological osmolality (300 mOsm) containing (in mM): 155 NaCl, 2.7 KCl, 1.5 KH2PO4 2.5 Na2HPO4, 1 CaCl2, 1 MgSO4, 5 glucose. Osmotic shock was applied by exposing the cells to a similar solution of 150 mOsm containing a smaller concentrations of NaCl, adjusted with the use of a osmometer (Osmomat 300). Macrophage volumes were estimated by measuring the cross-section area of approximately 30 cells observed in an Olympus IX71 inverted light microscope using Image-Pro 6.3 software. Extracellular ATP was quantified using a Bioluminescence Assay kit (Promega). Apyrase (Sigma) was used to investigate the involvement of extracellular ATP with RVD and carbonoxolone (Sigma), and mefloquine (BioBlocks) were used to test the possible involvement of pannexin-1 in hypoosmotic shock-induced ATP release and RVD. Results: ATP is release in a nanomolar concentration by macrophages, starting approximately 2 min after the hypotonic shock. This time matches with the onset of volume decrease. Volume decrease, but not cell swelling was inhibited by 85 and 45 % when cells were preincubated for 30 min with the pannexin-1 inhibitors carbonoxolone (10 μM) or mefloquine (100 nM) respectively. The presence of apyrase (10 Un/mL) during osmotic shock reduced RVD by 95 %, suggesting that extracellular ATP is required to induce volume recovery. Conclusion: Our data suggest that ATP is released in response to hypoosmotic shock by a pannexin-1-dependent mechanism and that this nucleotide is required for the cell volume recovery characteristic of RVD. More studies are needed to establish the pathways of ATP release and its mechanism of action during the RVD process in macrophages.

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Financial support: CAPES, CNPq, FAPERJ and INPETAM –Instituto Nacional de Pesquisa Translacional em Saúde e Ambiente na Região Amazônica. 50 Inhibitors of the 5-lipoxygenase pathway activate pannexin1 channels in macrophages via the thromboxane receptor H.A. Da Silva-Souza1,2,3, M.N. Lira1, N.K. Patel3, D.C. Spray3, P.M. Persechini1,2 and E. Scemes3 1 Institute of Biophysics Carlos Chagas Filho – Federal University of Rio de Janeiro –UFRJ; Rio de Janeiro –RJ –Brazil 2 National Institute of Science and Technology of Translational Research in Health and Environment of the Amazon Region– INPeTAm, Brazil 3 Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York, USA Objectives / background: A multitude of environmental signaling molecules influence monocyte and macrophage innate and adaptive immune responses, including ATP and prostanoids. Interestingly, we have previously described that 5-LOX inhibitors induced the influx of cationic dyes in macrophages independently of P2X7 receptor activation and that such influx did not occur after inhibiting the activity of cyclooxygenases, enzymes that transform arachidonic acid (AA) into prostanoids. Thus these recent studies raise the possibility that the production of prostanoids resultant from the blockade of the 5-LOX pathway activates a permeation pathway with properties (influx of dye and ATP release) similar to those described for pannexin-1 (Panx-1). Here, we provide evidence that Panx-1 is a component of this pathway and present the intracellular signaling molecules linking the thromboxane (TP) receptor to Panx1-mediated dye influx and ATP release. Methods and Results: Using pharmacological tools and transgenic mice deficient in Panx-1, we have used fluorescent dye uptake assays in thioglycollate-elicited murine peritoneal macrophages and we also have measured the release of ATP in these cells using the luciferin/ luciferase assay with a luminometer. Our results show that two 5-LOX pathway inhibitors induce ATP release and influx of dye in a Panx-1-dependent manner. Electrophysiological recordings performed in wild-type (WT) and Panx1-deficient macrophages confirmed that these 5-LOX pathway inhibitors activate currents characteristic of Panx-1 channels. WT and Panx-1 peritoneal macrophages were used to quantify the levels of Panx-1 transcripts (qRT-PCR). We found that the mechanism by which Panx-1 channels are activated under this condition involves activation of the TP receptor that is mediated by the cAMP/PKA pathway. This is to our knowledge the first evidence for the involvement of Panx-1 in the TP receptor signaling pathway. Future studies aimed to clarify the contribution of this TP-Panx-1 signaling network to macrophage immune responses are likely to be important for targeting inflammatory and autoimmune diseases. Conclusion: We conclude that macrophages release ATP in response to 5-LOX inhibitors have important implications for the understanding of the interplay between purinergic and prostanoid signaling in the biology of monocytes. Financial Suport: CNPq, FAPERJ, INCT-INPeTAm and Fogarty Training Grant. 51 Innate mechanisms of macrophages induced by P2X7 receptor during in vitro cutaneous leishmaniasis S. P. Chaves1, V. R. Figliuolo2, B. Rossi-Bergmann2 and R. Coutinho-Silva2 UFRJ-Macaé, Brazil 2 UFRJ-IBCCF, Brazil 1

Objectives/background: Previously we have shown that purinergic receptor P2X7 (P2X7R) is overexpressed during the BALB/c mice infection with Leishmania amazonensis, and its physiologic agonist ATP has an antiamastigote activity in vitro (Microbes Infec., 11,842, 2009). In this work we evaluated the P2X7R role in the innate mechanisms response during the in vitro infection with L. amazonensis using peritoneal macrophages from C57Bl/6 P2X7R knock-out mice (KO) and wild-type (WT). Methods and results: KO and WT macrophages were plated and rested for 48 h/37o C/5 % CO2, then, they were infected during 4 h/34o C/5 % CO2 with promastigotes of L. amazonensis-GFP (5:1). The adherence/internalization of promastigotes was analyzed in plate fluorimeter. KO macrophages adhered/internalized fewer promastigotes (5284.7 ± 627.8) than WT macrophages (9297.5 ± 6.36) (n = 3). To evaluate the specificity of this result, phagocytosis of KO and WT macrophages in suspension was evaluated using cytometer and latex beads. KO macrophages had a lower capacity to phagocyte latex beads than WT (12.9 and 29.4 %, respectively) (n = 3). Also the leishmanial mechanisms, nitric oxide and oxygen reactive species, were tested in KO and WT macrophages, using Griess Assay and H2CFDA, respectively. P2X7R absence decreased both leishmanicidal mechanisms in KO macrophages compared to WT (NO (p ≤ 0.05) and ROS (p ≤ 0.001). Conclusion: Together these results indicate the importance of P2X7R in major in vitro innate mechanisms of macrophages as phagocyte activity and release of nitrogen and oxygen reactive species in the control of leishmania protozoan. 52 L. amazonensis elimination by P2X7 receptor and leukotriene B4 requires NLRP3 inflammasome activation Mariana Martins Chaves1, Débora A Sinflório1, Maria Bellio1, Dario S Zamboni2, Claudio Canetti1 and Robson Coutinho-Silva1 1 UFRJ, Brazil 2 USP, Brazil Objectives. P2X7 receptors activation via extracellular ATP (eATP) has been reported as an important molecule signal to intracellular parasites elimination. After activation, the P2X7 receptor can lead to apoptosis, and cytokines release such as IL-1β, among others mechanisms. Recently, it was demonstrated that IL-1β release, after NLRP3 inflammasome activation, participates of the resistance to Leishmania amazonensis (Lima-Junior et al., Nat Med, 19, 909, 2013). Furthermore, our group showed that L. amazonensis elimination through P2X7 receptor activation depends of leukotriene B4 (LTB4) production (Chaves et al., J Immunol, 192, 4765, 2014). Thus, we investigated whether L. amazonesis elimination by P2X7 receptor and LTB4 involves NLRP3 inflammasome activation. Methods and Results. Peritoneal macrophages from C57Bl/6 (C57Bl/6), P2X7 receptor (P2X7−/−) and NLRP3 (NLRP3−/−) deficient mice, 2–7 months, were infected or not with L. amazonensis at 10:1 MOI ratio. These were tested for parasitic load presence or absence of 500 μM ATP, 100 nM LTB4, or 100 pg/mL IL-


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1β for 30 min at 37 °C. After 24 h, macrophages were fixed and stained with Panotic kit and analysed by optical microscope. C57Bl/6 and P2X7−/− mice were infected in the footpad with 106 L. amazonensis promastigotes for 7 days. After, P2X7−/− mice were treated in infect footpad twice a week with 5 ng of LTB4 for 3 weeks and thickness paw and parasitic load were determined. Paw’s thickness was accompanied with thickness gauge and the parasitic load was established with limiting dilution assay (LDA). Nitric oxide (NO) levels were measured by Griess assay. The graphs were generated and analyzed using the GraphPad Prism 5.0. Our results showed that macrophages from NLRP3−/− mice treated with ATP and LTB4 did not decrease parasitic load (difference between mens 3.67 %; n = 4, 5.00 n = 3, respectively). However, when NLRP3−/− macrophages were treated with IL-1β, a decrease in parasitic load was noted (difference between mens 19.5 %; n = 3). Similarly, macrophages from P2X7−/− mice treated with IL-1β also decreased parasitic load (difference between mens 38.7 %, n = 3). Furthermore, P2X7−/− mice infected with L. amazonensis in the footpad treated with exogenous LTB4 showed more resistance to infection, because their footpad had lower parasite load (difference between means 1.1 × 109 ± 4.7 × 108 parasites; n = 8) and lower lesion (difference between means 37.94 ± 11.45 mm, n = 8) when compared to untreated P2X7−/−mice. This resistance does not seem to be via NO. Conclusion. Thereby, these data suggest the involvement of LTB4 in resistance of WT mice when compared with P2X7−/− mice, and that L. amazonensis elimination by P2X7 receptor mediated by LTB4 depends of NLRP3 activation. Financial Support: CNPq, CAPES, FAPERJ. 53 Nucleotide receptors modulate retinal glia adhesion and migration following scratch injury of monolayer cultures Thayane Martins Silva1, Guilherme Rapozeiro França1, Isis Moraes Ornelas1, Erick Correia Loiola1, Henning Ulrich2 and Ana Lucia Marques Ventura1 1 Federal Fluminense University, Brazil 2 University of São Paulo, Brazil Objectives: In the present work, we investigated if nucleotides regulate the growth of Müller cells in scratched chick embryo retinal monolayer cultures. Methods and results: When retinal cell cultures cultivated for 7 days were mechanically scratched, a significant growth of glial cells at the empty area was detected between the 1st and 3rd days after scratch and the area devoid of cells decreased by ~70 %. Incubation of cultures with 2.5 U/ml apyrase (APY) significantly attenuated the growth of glial cells and while in control cultures the area free of cells represented 31.4 ± 2.9 % of the original area after 3 days, in apyrase-treated cultures, it represented 74.8 ± 4.2 % of the original area, p < 0.001, n = 6. Incubation of cultures with the P2 antagonists, 100 μM Suramin and 40 μM Reactive Blue 2 (RB-2) and also the P2X7 receptor antagonists, 10 μM BBG and 200 μM ATPo, significantly attenuated the growth of glial cells, the area free of cells representing 68.2 ± 3.6, 52.2 ± 8.7, 76.2 ± 1.8 and 88.32 ± 7.8 % of the original area, respectively, p < 0.01 and p < 0.001, n ≥ 3, suggesting that nucleotide receptors are involved in the growth of glial cells. UTPγS (10 μM) but not ADPβS (50 μM) antagonized apyrase-induced growth inhibition in scratched cultures, suggesting the participation of UTP-sensitive receptors. In apyrase-treated cultures, glial cytoplasm protrusions were smaller and unstable. Actin filaments were less organized and alfa-tubulin-labeled microtubules were mainly parallel to scratch. In contrast to control cultures, very few vinculin-labeled adhesion sites could be noticed in these cultures. Treatment with 0.2 μM Src inhibitor-1, 10 μM LY294002, a PI3K/Akt inhibitor and 10 μM PF573228, a FAK inhibitor, also decreased glial growth over the scratch, the area free of cells representing 74.50 ± 2.1, 51.4 ± 5.4 and 64.55 ± 3.6 % of the original area, respectively, p < 0.05, p < 0.01, and p < 0.001, n ≥ 3. RB-2 decreased dissociated glial cell attachment to fibronectin-coated dishes (50 μM RB-2 = 37.5 ± 13.3 % inhibition and 100 μM RB-2 = 58.9 ± 4.0 % inhibition of the control values; p < 0.05 and p < 0.01, n = 3). Treatment with 100 μM RB-2 also decreased by 62.9 ± 6.6 % the migration of purified glial cells through transwell membranes, p < 0.01, n = 4. Treatment with the P2X7 receptor agonist, 100 μM Bz-ATP also inhibited the adhesion of glial cells by 34.59 ± 5.8 %, p < 0.001, n = 3. These data suggest that nucleotide receptors regulated adhesion and migration of glial cells. Conclusion: Mechanical scratch of retinal cell cultures induces growth of glial cells over the empty area through a mechanism that is dependent on activation of nucleotide receptors, SRC, PI3K and FAK. Financial support: Capes, CNPq, PROPPi-UFF, Faperj. 54 P2X7 purinergic receptor and endocytic trafficking in dendritic cells and macrophage Barbara Fortes1, Aline Costa2, Alexandre Morrot3 and Pedro Muanis Persechini1 1 IBCCF - UFRJ, Brazil 2 UEZO, Brazil 3 IMPPG, Brazil Dendritic cells (DCs) are the most effective antigen presenting cells at initiating T lymphocyte response. DCs express different purinergic receptors, in particular the activation of P2X7 receptor by extracellular ATP induces transmembrane transport mechanisms that lead to the entrance of organic molecules of up to 900 Da. This phenomenon has proved to be very complex and our group has recently demonstrated that in primary macrophages, there are at least two distinct mechanisms of transport, one for cations and other for anions. Objective: The objective of this study is to investigate the effects of extracellular nucleotides in the endocytic activity of macrophages and bone marrowderived dendritic cells, and contribution of endocytosis to the ATP-induced P2X7- dependent uptake of organic molecules. Methods: We used dendritic cells generated by treating murine bone marrow cells with GM-CSF and thioglycollate-elicited intraperitoneal macrophages. Cells were incubated with fluorescent dyes (calcein, ethidium bromide, sulforhodamine B and others) in the presence or not of different concentrations of ATP, ADP, UTP and ADP in DMEEM without serum, for 30 min at 37 °C, pH 7.4. Cells were then washed and incubated with 5 mM ATP for 10 min at 37 °C. Dye uptake was analyzed by flow cytometry using FACS-Calibur. Results: Extracellular nucleotides did not induce significant changes in dye uptake by endocytosis neither in dendritic cells nor in macrophages. When these cells were treated with 5 mM ATP after the endocytic step, we observed an increased fluorescence intensity in cells containing ethidium and a decreased fluorescence in cells containing calcein.

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Conclusion: Our results are consistent with the possibility that ATP can modulate the intracellular traffic of organic molecules taken up by endocytosis in macrophages and dendritic cells. Financial support: CNPq, FAPERJ. 55 P2X7 receptor deletion attenuates the sepsis-induced brain dysfunction M.G.J Andrade1, L.E.B Savio1, P.T. Santana1, J. Kolling2, A. Longoni2, A.T.S. Wyse2 and R. Coutinho-Silva1 1 Laboratory of Immunophysiology, Biophysics Institute Carlos Chagas Filho, Federal University of Rio de Janeiro, RJ, Brazil 2 Laboratory of Neuroprotection and Metabolic Diseases, Department of Biochemistry, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil Background and objectives: Sepsis is the primary cause of admission to intensive care units worldwide. This severe clinical condition is characterized by an uncontrolled, excessive, and systemic inflammation that affects distal organs, including the brain. Interestingly, cognitive deficits has been reported in sepsis survivors. Pro-inflammatory cytokines, such as IL-1β, and the oxidative stress have been associated with the pathophysiology of sepsis-induced cognitive impairment. Since P2X7 receptor activation induces the maturation and release of pro-inflammatory cytokines, such as IL-1β and IL-18, and the production of reactive nitrogen and oxygen species, herein, we sought to investigate the role of P2X7 receptor in sepsis-induced brain dysfunction. Methods and results: Male (8–10-week old) wild type (WT) or P2X7 receptor deficient (P2X7−/−) C57BL/6 mice (originally from the Jackson Laboratory, USA) weighing ~25 g were used in this study. For sepsis induction, mice underwent sham laparotomy or CLP induced by ligation and puncture of the cecum. The brains were harvested at 24 h after surgery. The IL-1β production in the cerebral cortex and hippocampus was measured by ELISA method. The production of reactive oxygen species (ROS) and nitrogen reactive species (RNS) was assessed through the 2′,7′-DCF oxidation. The antioxidant status was evaluated measuring the activities of superoxide-dismutase (SOD), catalase (CAT). Our results show that the genetic ablation of the P2X7 receptor attenuated the IL-1β production in the cerebral cortex and hippocampus from septic mice (n = 5 mice/group). The P2X7 deletion also reduced the oxidation of DCF in the hippocampus from CLP-induced mice, indicating a lower production of reactive species when compared to WT septic mice group (n = 5 mice/group). In addition, we observed an increase in SOD and CAT activities in cerebral cortex and hippocampus from P2X7 deficient septic mice in comparison to WT septic mice, suggesting that P2X7−/− mice present an enhanced adaptive capacity to oxidative stress (n = 5 mice/group). Conclusion: In summary, our results show that P2X7 receptor may contributes to sepsis-induced acute brain inflammation and damage. In addition, these findings support the notion that P2X7 receptor may represent a suitable therapeutic target for the development of treatments to reduce the brain damage during sepsis-associated encephalopathy. Financial support: CNPq, CAPES, FAPERJ, PRONEX. 56 P2X7 receptor signaling contributes to hepatic necrosis caused by blood-stage Plasmodium chabaudi AS malaria F.S. Vieira1, A.P. Freitas-do-Rosário1, E. Machado de Salles1, K.R. Bortoluci2, R. Coutinho-Silva3 and M.R. D’Império Lima1 University of São Paulo, Brazil 2 Federal University of São Paulo, Brazil 3 Federal University of Rio de Janeiro, Brazil 1

The intense activation of the immune system during the erythrocytic stage of Plasmodium is responsible for several syndromes associated with the disease. The purinergic P2X7 receptor detects extracellular ATP and their interaction induces inflammasome activation in macrophages and consequent production of proinflammatory cytokines and cell death. In malaria, ATP is released upon erythrocyte rupture and leukocyte activation. In the murine model of malaria caused by P. chabaudi AS, infected erythrocytes preferentially adhere to the liver endothelium. Therefore, it is believed that the rupture of parasitized erythrocytes (PE) and consequent release of ATP mainly occurs in that organ. Methods and Results: To study the role of P2X7R in hepatic necrosis, C57BL/6, P2X7R−/−, Nalp3−/−, ASC−/− and caspase 1/11−/− mice were infected intraperitoneally with 106 PE and 7 days later the livers were collected for histopathological analysis. Differently from infected C57BL/6 mice, infected P2X7R−/− mice did not show liver necrosis. Infected Nalp3 −/−, ASC−/− and caspase 1/11−/− mice presented smaller areas of liver necrosis if compared to infected C57BL/6 mice. In another approach, we infected C57BL/6 mice, which were treated with P2X7-antagonist Brilliant Blue-G (BBG) (45.5 mg/Kg) every 48 h for 5 days. Seven days after infection the livers were collected for histopathological analysis. Our results indicate that BBG administration prevents hepatic necrosis in C57BL/6 mice, and similar results were observed using DBA/2 mice treated with BBG. The inhibition of P2X7R in DBA/2 mice not only prevented the tissue damage but also had an impact on the survival of mice. Conclusions: These data demonstrate that the absence of P2X7 receptor or the administration of BBG ameliorate the severity of hepatic necrosis. Financial Support: FAPESP, CNPq. 57 P2X7 receptor signaling drives Th1 effector/effector memory cell differentiation in blood-stage Plasmodium chabaudi AS malaria Érika Machado de Salles1, Maria Menezes1, Henrique da Silva1, Flávia Sarmento Vieira1, Sheyla Castillo-Méndez1, Alexandra Cassado1, Eduardo Amaral1, José Maria Alvarez1, Robson Coutinho-Silva2 and Maria Regina D’Império Lima1 1 University of São Paulo, Brazil 2 Federal University of Rio de Janeiro, Brazil Objectives/background: P2X7 receptor (P2X7R) is a sensor of extracellular ATP, a damage signal released during tissue injury that provides for ion exchange and promotes cell activation and death. We investigated whether the P2X7R signaling is required for T helper 1 (Th1) cell differentiation during experimental blood-stage malaria.


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Methods and results: To evaluate whether the P2X7R contributes to protection against blood stages of P. chabaudi AS (PcAS), the disease progression was analyzed in C57BL/6 (B6) and P2X7R−/− mice. The parasitemia and clinical parameters (i.e. anemia, weight loss and hypothermia) developed similarly in both female groups up to day 7 p.i., but after this period the animals that lacked the P2X7R had difficulty in controlling the parasitemia and restoring the clinical parameters to the baseline in naïve mice. The disease was further aggravated in infected P2X7R−/− males that showed 60 % mortality. To investigate whether the rupture of infected red blood cells (iRBCs) leads to ATP release into the extracellular milieu, ATP serum levels were evaluated in B6 mice at days 4 and 5 p.i. with synchronized parasites, before and after erythrocyte re-invasion. After the rupture of iRBCs, we found higher ATP serum levels (1.5 times). Because the sensing of environment eATP by the P2X7R is thought to amplify the TCR signal during the immune synapsis, we focused our analysis on CD4+ T cells that have a central role in the development of protective immunity against PcAS malaria. To determine whether the increased ATP serum levels engage the P2X7R in blood CD4+ T cells, P2X7R-associated pore formation was evaluated by ethidium bromide (EB) uptake. On days 4 and 5 p.i., the percentages of EB-stained B6 CD4+ T cells increased after iRBC rupture, an effect that was not observed for P2X7R−/− CD4+ T cells. The inefficient parasite control in acutely and chronically infected P2X7R−/− mice was associated with low production of IFN-g; but high production of IgG antibodies. Furthermore, the expression of T-bet transcriptional factor in Th1 effector/effector memory cells induced by infection critically depended on the P2X7R signaling in CD4+ T cells. Indeed, P2X7R−/− CD4+ T cells differentiated in infected CD4−/− mice, in comparison to B6 CD4+ T cells, showed a bias towards the follicular T (Tfh) helper cell lineage over Th1 memory cell lineage. This bias was evidenced on day 30 p.i. by the high numbers per spleen of P2X7R−/− CD4+ cells expressing the Bcl-6 transcriptional factor in PD1+CXCR5+ Tfh cells. Registration number of Ethical Committe approval: 175. Conclusion: This study provides a new insight into malaria immunology by showing that recognition of ATP, a damage signals, by the P2X7R is crucial for the differentiation of Th1 effector/effector memory cells during Plasmodium infection. It also demonstrates that P2X7R-mediated balance between Tbet and Bcl-6 transcriptional factors tunes the cellular and humoral immunity in malaria. 58 P2X7 receptor stimulation induces melatonin production in Raw 264.7 murine macrophage cell line L. Dargenio-Garcia, R.P. Markus and Z.S. Ferreira Laboratório de Cronofarmacologia, Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil Objectives/Background: Melatonin is synthesized by acetylation of serotonin and subsequent methylation of N-acetylserotonin by the enzymes arylalkylamine-N-acetyltransferase (AA-NAT) and acetylserotonin-methyltransferase (ASMT), respectively (Simonneaux and Ribelayga Pharmacol Rev. 55: 325, 2003). This indolamine is able to modulate immune responses regulating the proliferation and the activity of immune competent cells (Pontes et al., J Pineal Res. 41: 136, 2006; Carrilo-Vico et al., Int. J. Mol. Sci. 14: 8638, 2013; Markus et al., Int J Mol Sci. 14: 10979, 2013). In macrophages, pathogen-associated molecular patterns (PAMPs), such as LPS, activate Aanat transcription, inducing melatonin synthesis, which leads to the expression of dectin-1, increasing phagocytosis (Muxel et al., PLos One 7: e52010, 1012, Pires-Lapa et al., J Pineal Res. 55: 240, 2013). Considering that purines act as a damage-associate molecular pattern (DAMP), which mimic PAMPs phenotypes, we evaluated whether activation of P2X7 receptor could induce melatonin synthesis in RAW 264.7 macrophages. Methods and Results: The expression of phospho-AA-NAT was determined in RAW 264.7 murine macrophage cell line (105 cells) stimulated or not with ATP (3 mM, 1 h) in the presence or absence of the selective P2X7 receptor antagonist A438079 (1 μM) and a nonselective P2 receptor antagonist (suramin, 100 μM) incubated 30 min before. Dose–response curves for melatonin was obtained from the supernatant of cells (2 × 106 cells) incubated for 3 h with ATP (1 μM – 3 mM). Immunocytochemistry detection of phospho-AA-NAT, was performed using anti-phospho-AA-NAT (Imuny 0451) primary antibody and anti-rabbit FITC (Sigma F7512) secondary antibody. Nuclear DNA was stained with DAPI. Images from independent experiments were acquired by a wide-field microscopy (Zeiss Axiovert Scope A1) and the fluorescence intensity was measured from 7 cells of three different randomly chosen fields, each in the same well. Melatonin content was determined by ELISA (IBL, Ge). The 50 % (n = 21) increase in phospho-AA-NAT expression induced by ATP (3 mM) was completely antagonized by A438079 (n = 6). Suramin did not block ATP effect (n = 6). In addition, an ATP dose-dependent increase in the content of melatonin in the medium was detected. In the absence of ATP no melatonin was detected, and the maximal concentration was produced by ATP 3 mM (74.65 ± 6.6 pg/ml, n = 3). Conclusion: Activation of P2X7 receptor by ATP leads to the expression of phospho-AA-NAT and the synthesis of melatonin, reinforcing that extracellular ATP may act as a danger signal. The fact that melatonin is being produced, suggest that the cell is entering in a regulatory phase, compatible with increasing phagocytic activity. Inasmuch, ATP-induced melatonin macrophage synthesis is probably relevant for restraining the inflammatory phase of P2X7 mediated response. Support: CAPES, FAPESP, CNPq. 59 P2X7 receptor-activation reduces Leishmania amazonensis infection in human macrophages Débora Alves Sinflório, Mariana Machado Chaves and Robson Coutinho Silva IBCCF – UFRJ, Brazil Objective/ background: P2X7 receptor is involved in the elimination of pathogens (Coutinho-Silva &Ojcius, Microbes Infect, 14, 1271, 2012). It has already been described that activation of P2X7 receptor can reduce the parasitic load of L. amazonensis in murine macrophages (Chaves et al., Microbes Infect., 11, 842, 2009; Chaves et al., J Immunol, 192, 4765, 2014). Thereby, we investigated whether L. amazonensis elimination by P2X7 receptor activation occurs in human macrophages. Methods and Results: THP-1 cells (ATCC® TIB-202 ™) were treated with 10 μg/ml PMA by 48 h to differentiate into human macrophages. Then the cells were infected with L. amazonensis in the ratio 10: 1 by 4 h at 37 °C and 5 % CO2. Twenty four hours after, infected macrophages were treated with 500 μM ATP by 30 min and/or 25 nM A740003 (specific inhibitor of the P2X7 receptor) by 30 min at 37 °C. After 24 h, macrophages were fixed and stained with Panotic kit to verify parasitic load by light microscope. The graphs were generated using the GraphPad Prism 5.0. Our data showed that ATP

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significantly reduced the parasite load (difference between means 67 ± 3 %, n = 3) in human macrophages. In addition, pre-treatment with A740003 (a specific inhibitor to P2X7 receptor) completely reversed the ATP effects (n = 3). Conclusion: Extracellular ATP, via activation of P2X7 receptors, is important in the controlling of Leishmania amazonensis infection in human macrophages. Financial Support: CNPq, FAPERJ, CAPES, INPeTAm. 60 P2X7R expression is not modified in stabilized bipolar disorder in patients Carolina de Moura Gubert, Gabriel Rodrigo Fries, Pâmela Ferrari, Mirela Paiva Vasconcelos-Moreno, Bárbara Tietböhl Martins Quadros dos Santos, Adam Fijtman, Márcia Kauer-Sant’Anna, Flávio Kapczinski and Ana Maria Oliveira Battastini Universidade Federal do Rio Grande do Sul, Brazil Background: Bipolar disorder (BD) is a chronic psychiatric illness that affects 2.4 % of the world population, and it is characterized by recurrent episodes of mania and depression interspersed with periods of remission of the symptoms, called euthymia. . Evidence points to increased levels of the pro-inflammatory cytokines in plasma and postmortem frontal cortex of BD patients when compared to healthy controls. Also it has been suggested that the purinergic system is implicated in the pathophysiology of central nervous system disroders, such as BD. For instance, the P2X7 receptor (P2X7R), an adenosine 5′-triphosphate (ATP)-binding ligand-gated ion channel of the purinergic system, when activated plays a key role in the modulation of the inflammatory response. In the present work, we aimed to assess the expression of P2X7R in blood samples of euthymic patients with BD and healthy controls in order to verify any relationship between the disorder and the mRNA levels of this receptor. Methods: Twenty five euthymic patients with BD and twenty two healthy controls were recruited. Approximately 2.5 mL peripheral blood were collected in RNA-stabilizing tubes from each subject and the total RNA was extracted using PAXgene™ blood RNA kit. The mRNA levels of P2X7R were measured by real-time RT-PCR, using the TaqMan® technology. The concentration of the pro- and anti-inflammatory cytokine, IL-6 and IL-10, respectively, was determined by flow cytometry using the BD™ Cytometric Bead Array (CBA) Human Enhanced Sensitivity Flex Set. Mann–Whitney U statistical test was performed to evaluate differences between groups. Ethical Committee approval: n° 100503. Results: Patients and healthy controls did not differ regarding age, gender, body mass index, smoking, years of education, or psychological scales. We found no difference in the P2X7R mRNA levels between patients and controls (p = 0.579) and no difference in IL-6 levels between groups (p = 0.083). On the other hand we found an increase of IL-10 levels in patients when compared to controls (p = 0.011). Conclusion: The majority of studies which report increased levels of pro-inflammatory cytokines in patients with BD evaluate subjects during acute manic or depressive episodes of the disorder. Accordingly, it is likely that no pro-inflammatory increase in cytokines was found since we assessed patients in euthymic period. The cytokines results suggest that patients were not in a pro-inflammatory state at the moment of blood collection. Hence, this might explain the lack of statistical difference in P2X7R expression in patients when compared to controls, given that this receptor is directly related to inflammation. More experiments are necessary to verify the amount of P2X7R protein and to identify other inflammatory markers in these samples. Also more studies are required to compare the P2X7R mRNA levels in acute and euthymic patients with BD. Supported by: CNPq, CAPES, FIPE – HCPA. 61 P2Y1 receptor modulates cell proliferation of rat retina in vivo Luana de Almeida Pereira1, Camila Feitosa Magalhães1, Marinna Repossi1, Alfred Sholl Franco2, Ana Lúcia Marques Ventura1 and Lucianne Fragel Madeira1 1 Universidade Federal Fluminense, Brazil 2 Universidade Federal do Rio de Janeiro, Brazil In vertebrates the histogenesis of the retina is guided by extrinsic and intrinsic mechanisms, in which adenine nucleotides have been widely studied as being important to many processes involved with nervous system development. Previous studies from our group demonstrated that exogenous ATP was able to regulate the proliferation of retinal progenitor cells in vitro via P2Y1 receptor, a G protein coupled receptor. Based on these results, the objective of this study was to evaluate the function of adenine nucleotides in vivo during the development of the retina of newborn rats. The intravitreal injection of PPADS, a P2 receptors inhibitor, decreased the number of proliferating cells labeled with BrdU in approximately 23 % at the concentration of 1000 μM in the P2 animals (3.676 ± 737.0) compared to control (4.409 ± 637.2). Furthermore, we identified the presence of P2Y1 receptor in all layers of the retina at P2 animals, including proliferating Ki-67 positive cells in neuroblast layer, suggesting that it is a possible candidate receptor for the action of adenine nucleotides on the neuroblasts proliferation. Indeed, intravitreal injection of MRS2179, a selective antagonist of P2Y1 receptor, at the concentration of 100 μM reduced approximately 21 % of number of proliferating cells labeled with Ki-67 at P2 animals (7.437 ± 908.3) compared to control (9.873 ± 720.8). Intravitreal injection ADPβ-S, a non-hydrolysable analog of ADP, at the concentration of 100 μM was able to induce an increase the number of BrdU positive cell in aproximately of 25 % at P2 animals (3.781 ± 1.302.0) compared to control (3.170 ± 669.8), while the injection ATP -S, a non-hydrolysable analog of ATP, at the concentration of 100 μM (3.318 ± 741.1) did not affect the proliferation rate compared to control (3.170 ± 669.8). However, in the presence of apyrase, an enzyme that hydrolyzes nucleotides, both ADPβ-S and ATP -S, at the same concentrations described above, increased the number of proliferating cells in about 30 % (3.225 ± 130.2) compared with apirase (2.221 ± 761), indicating that ADP and ATP can stimulate P2Y1 receptor. Increasing endogenous ATP at P4 animals by the treatment with ARL67156, an ectonucleotidase inhibitor at the concentration de 200 μM, stimulated the proliferation of retinal cells, increased the number of cells labeled with Ki-67 in approximately 50 % (6.436 ± 843.4) compared to control (3.682 ± 1.336.3). In addition, we observed an increase in the P2Y1 receptor expression during the first postnatal month, indicating other functions to P2Y1 during the development of the rat retina. Therefore, we concluded that adenine nucleotides


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modulated proliferation of neuroblasts during early stages of retinal development through P2Y1 receptor and probably changes in this modulation could affect the histogenesis of retina and consequently it function. Financial Support: Capes, CNPq, Proppi-UFF. 62 Participation of P2Y12 signaling during murine silicosis Marcos Vinícius Rangel Ferreira Tatiana Luna Gomes da Silva, P.T. Santana, M.N. Machado, W.A. Zin, C.M. Takiya and R. Coutinho-Silva IBCCF – UFRJ, Brazil Background and objectives: Silicosis is a fibrotic lung disease caused by the inhalation of silica particles, and is considered an occupational disease, given that these particles are present in the working environment of many mining and civil construction industries. The symptoms of the disease are caused by the intense inflammatory response, which is followed by intense fibrosis and partial loss of lung capacity. The inflammatory process of silicosis is characterized by production of pro-inflammatory cytokines, such as IL-1β and TNF-α and pro-fibrogenic factors such as TGF-β. P2Y12 is a member of the P2 receptor family, which consists of the ion-channel P2X and the G-protein-coupled P2Y receptors. P2Y12 receptor is activated by ADP, which can be released from a diversity of cells including immune cells. The participation of P2Y12 signaling during inflammatory process has been reported but is controversial. However, the participation of P2Y12 signaling during inflammatory lung disease was recently described (Paruchuri S et al. J Exp Med. 206; 2543, 2009). Here, we investigated the role of P2Y12 receptor during murine silicosis. Methods and results: BALB/c mice (25–30 g and 6–8 weeks) were divided into 3 groups (number of Ethical Committee approval IBCCF 154), Saline (n = 5), Silica (n = 5) and Silica treated with P2Y12 antagonist, Clopidogrel (n = 6). Mice were anesthetized and intratracheally injected with 0.10 mL of PBS or 6 mg of silica particles. The Clopidogrel group were treated 48/48 h with 20 mg/Kg after instillation. All animals were analyzed 14 days after saline or silica administration. Leukocyte infiltration and fibrosis were evaluated by lung histology stained with hematoxylin–eosin. Cytokines production were analyzed by ELISA and nitrite was detected by Griess test. Values are expressed as means ± SEM of at least five animals. + P £ 0.05 compared with control or silica. Silica group showed mono and polymorphonuclear cell infiltration in lung parenchyma. However, these were significantly reduced in images of lung histology of silica group treated with P2Y12 antagonist. Also, silica instillation induced significant increase in Nitrite (74 %), IL-1β (144 %), TNF-α (99 %) and TGF-β (2800 %) secretion when compared with control group. The group treated with P2Y12 antagonist showed 35 % less nitrite, 36 % less IL-1β, 49.6 % less TNF-α and 98.2 % less TGF-β when compared with silica group. Conclusion: Our findings show that the inhibition of P2Y12 signaling may improve some inflammatory parameters observed during murine silicosis. These data can represent a new therapeutic target for treatment of silicosis. Nevertheless, new experiments for understanding of mechanisms involved in this phenomenon are needed. Financial support: CAPES, FAPERJ and CNPq. 63 Participation of purinergic receptor P2X7 in acute kidney injury induced by sepsis A. S. Tamura1,2, P. T. Santana1,2, C. Caruso-Neves2 and R. Coutinho-Silva1 1 Laboratory of Immunophysiology – Federal University of Rio de Janeiro, Rio de Janeiro/RJ, Brazil 2 Laboratory of Biochemistry and Cellular Signalization – Federal University of Rio de Janeiro, Rio de Janeiro/RJ, Brazil Background: Sepsis is defined as the syndrome of systemic inflammatory response triggered by an infectious agent that may result in shock, multiple organs failure and death. The kidney is a key organ in the regulation of homeostasis of the organism and can be directly affected during sepsis with the commitment of renal activity by the development of acute kidney injury (AKI). The purinergic receptor P2X7 is activated by extracellular ATP and leads to inflammatory cascades in cells. Objectives: The aim of this work is to investigate the role of purinergic receptor P2X7 in acute kidney injury (AKI) induced by sepsis by the model of cecal ligation and puncture (CLP). Methods: In the present work, sepsis was induced in wild type (WT) and knockout for receptor P2X7 (P2X7KO) male mice of background C57BL/6 of 2 months old by CLP and 24 h later the kidneys, blood and urine were collected. In renal macerates the inflammatory cytokines IL-1β and TNF-α were measured by ELISA technique. From urine and blood samples renal function parameters were evaluated, such as: urinary proteinuria and UP:Cr, that is the ratio between the proteinuria and urinary creatinine. The protocols used in this work were previously approved by the ethics committee (CEUA code IBCCF154). Results: It was possible to observe that after 24 h from sepsis the levels of IL-1β and TNF-α increased in kidneys of wild type and knockout animals (*p < 0.05, n = 5). Also renal parameters are better comparing P2X7KO animals with WT animals, since proteinuria levels and UP:Cr were lower in P2X7KO animals (*p < 0.05, n = 5). Conclusion: From these set of data it is possible to suggest that P2X7 receptor contributes to exacerbation of worse renal function after sepsis and that this receptor may be involved with renal inflammatory mechanism. FUNDS: CNPq, CAPES, FAPERJ and INPeTAm. 64 Purinergic P2Y12 receptor activation in eosinophils and the schistosomal host response Josiane Sabbadini Neves Federal University of Rio de Janeiro, Brazil Background: Identifying new target molecules through which eosinophils activate and secrete their stored proteins may be highly significant for understanding the pathophysiology of host immune responses to parasites and may reveal new therapeutic targets for the control of eosinophilic disorders. We have recently reported the expression of the purinergic P2Y12 receptor (P2Y12R) in human eosinophils; however, its functional role in

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this cell type and its involvement in eosinophilic inflammation remain unknown. In this study, we investigated functional roles of P2Y12R in isolated human eosinophils and in a murine model of eosinophilic inflammation induced by Schistosoma mansoni (S. mansoni) infection. Methodology/Principal Findings: Eosinophils were isolated from the blood of healthy donors by negative immunomagnetic selection (license number 190/09 HUCFF). Functionally, we found that adenosine 5′-diphosphate (ADP) induced isolated eosinophils to secrete eosinophil peroxidase (EPO) via a mechanism dependent on P2Y12R activation. In contrast, ADP did not interfere with human eosinophil apoptosis or chemotaxis in vitro, as assessed by flow cytometry and a transwell system, respectively. In vivo, C57Bl/6 mice were percutaneously infected with 60 cercariae of the BH strain of S. mansoni (license number DFBCICB043 - CEUA/UFRJ). Analyses performed 55 days post infection revealed that P2Y12R blockade reduced the area of the hepatic granuloma and modulated the eosinophilic inflammatory infiltrate without affecting the Th2 response and inhibited collagen deposition and IL13/IL-4 production in the liver without altering the parasite oviposition. Furthermore, P2Y12R inhibition increased blood eosinophilia (2-fold increase), whereas it decreased the eosinophil count in the bone marrow, as assessed by blood smears and cytospin analyses, respectively. Conclusions/Significance: Taken together, our results suggest that P2Y12R has an important role in eosinophil EPO secretion and in establishing the inflammatory response in the course of a S. mansoni infection. 65 Purinergic signalling contributes to schistosomiasis-related inflammation and endothelial dysfunction C.L.M. Silva Pharmacology and Inflammation Research Program, Biomedical Sciences Institute, Federal University of Rio de Janeiro, Brazil Background: ATP is released into the extracellular environment by several mammalian cell types and modulates physiological or pathophysiological events such as vascular dysfunction. Extracellular ATP acts as an autocrine or paracrine mediator, through the activity of different P2 purinoceptor subtypes. Moreover, ectonucleotidases (NTPDases) are involved in the metabolism of extracellular nucleotides, controlling their availability for purinoceptor activation. Both G protein-coupled P2Y receptors (P2YR) and ion-channel P2X receptors (P2XR) are expressed in endothelial cells (ECs), vascular smooth muscle cells and immune cells, where receptor activation triggers different signaling cascades, including those leading to vasodilation or vasoconstriction, as well as immune cell migration and EC apoptosis. The main purinoceptor subtypes expressed on mammalian ECs are P2Y1R, P2Y2R, P2X4R and P2X7R, although vessel- and species-specific differences in receptor subtypes exist. Schistosomiasis is an intravascular parasitic disease related to EC dysfunction (Plos One 6; e23547, 2011). Results: In the murine model, P2Y1R participate in EC activation, which is essential for monocyte diapedesis. Analysis of P2Y1R knockout model showed that P2Y1R contributes to the expression of endothelial adhesion molecules that mediate leucocyte recruitment during inflammation (Circulation 123; 2404, 2011). In an intestinal inflammation murine model caused by schistosomiasis, our data showed that the increased expression of mesenteric EC NTPDase 2 triggers higher levels of extracellular ADP, a full agonist of P2Y1R, with subsequent endothelial P2Y1R-mediated monocyte adhesion to mesenteric EC monolayer that was higher than control group (P < 0.05). Pretreatment of EC with the antagonist of P2Y1R (MRS2179) blocked this effect. Short-term activation of endothelial P2X7R stimulates endothelial nitric oxide (NO) synthase activity, resulting in NO production, vasodilation and leukocyte adhesion inhibition. In the same inflammation model, basal P2X7R expression is reduced resulting in a reduced NO synthesis (Purinergic Signal. 9; 81, 2013). Conclusion: Endothelial P2Y1R signaling and NTPDase 2 expression are upregulated during schistosomiaisis favoring monocyte adhesion, while P2X7R-mediated NO production is downregulated. Both events are key features of endothelial dysfunction. The pharmacological targeting of purinergic signaling might be effective to reduce endothelial dysfunction in inflammatory diseases such as schistosomiasis. Financial support: CNPq, FAPERJ. 66 Purinergic signaling in neuroblastoma migration and metastasis Henning Ulrich1, Mariusz Z. Ratajczak2, Gabriela Schneider2, Enéas Galdini Ferrazoli1, Talita Glaser1, Micheli M. Pillat1 and Claudiana Lameu1,2 1 Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, SP, Brazil 2 Stem Cell Institute, James Brown Cancer Center, University of Louisville, KY, United States Background: Bone marrow metastasis is present in approximately 350,000 patients that die annually in the U.S. alone. While the stromal cell-derived factor-1 (SDF-1)-CXCR4 axis is known to exert chemotaxis in bone metastasis, no such functions have been studied for the interaction of SDF-1 with the kinin and purinergic system. Methods: Assays of intracellular Ca2+ mobilization, real time PCR, adhesion, proliferation and chemotaxis of neuroblastoma cells in vitro and in vivo were used. Results: We show here that bradykinin augments chemotactic responsiveness of three neuroblastoma cells line for physiological SDF-1 and ATP concentration. In line with the recruitment of cancer cells to the bone-marrow expression of bradykinin precursors and kinin-B2 receptors was upregulated in the bone marrow of irradiated mice. Bradykinin promoted adhesion, SDF-1-induced Ca2 + mobilization as well as resensitization and expression of P2X7 purinergic receptors. Immunodeficient nude/nude mice transplanted with neuroblastoma cells pretreated with BK revealed significantly higher metastasis to the bone marrow than observed in animals engrafted with untreated cells. Animals receiving Brilliant Blue G (BBG), an antagonist of the P2X7 receptor, did not show metastasis to bone marrow and liver or metastasis rates were drastically reduced. Conclusion: Advances have been made to understand the interrelationship between the kinin and the purinergic systems in the metastasis of neuroblastoma cells opening novel perspectives for therapy. Financial support: FAPESP and CNPq. 67 Mesenchymal stem cells up-regulate the expression of proteolytic enzymes in glioblastoma as part of their immunomodulatory-like response B. Breznik, H. Motaln, M. Golob, H. Ulrich and T. Lah Turnšek


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Department of Biochemistry, Faculty of Chemistry and Chemical Engineering, University of Ljubljana, Slovenia and Institute of Chemistry, Sao Paolo University, Sao Paolo, Brazil Objectives : The poor survival of patients with glioblastoma multiforme (GBM) is due to a single GBM cell invasion into surrounding healthy brain tissue, assisted by the tumour microenvironment in which the role of mesenchymal stem cells (MSCs) is still not well understood. MSCs become part of the tumour inflammatory microenvironment as they are recruited from bone marrow or endogenous tissue niches by tumor secreted cytokines and growth factors (Motaln et al., Cell Transpl. 2012; Motaln & Lah Protein-peptide Res. 2015). The intrinsic tumour tropic migratory behaviour of MSCs was demonstrated in several types of cancer including the most invasive brain tumour GBM. Recently, we reported on enhanced GBM invasion in vitro, when GBM cells were in direct interaction with MSCs. Thus the aim of this study were to elucidate the role of proteolytic enzymes in GBM invasion upon direct contact with bone marrow derived MSCs (Schichor et al., Exp Neurol.2012) Methods and Results: We evaluated the expression of various proteases linked to GBM invasion on gene (qRT-PCR) and protein levels (flow cytometry, immunocytochemistry, Western blot) in directly co-cultured MSCs and GBM cells (U87 dsRED). The invasion of MSCs and GBM cells in direct cocultures was analysed by 3D-spheroid-assay, using collagen type I, laminin and Matrigel as an embedding substrate. Our results implicate on the crucial role of urokinase-type plasminogen activator (uPA) in the direct cross-talk between MSCs and GBM cells. uPA was found to be up-regulated in MSC/GBM direct co-cultures both at gene and protein level, compared to GBM cells grown alone. Beside uPA, cathepsin B was significantly over-expressed in the conditioned medium of MSC/GBM direct co-cultures. Most relevant was the findings that MMP9 was upregulated in GBM cells directly co-cultured with MSCs, presumably resulting from BK signallingConclusion: Taken together, our findings suggest that direct contact between MSCs and GBM cells promotes the invasion of GBM cell through the activation of protease signalling cascade involved in degradation of the extracellular matrix proteinsThis study was supported by Slovenian Research Agency, PhD fellowship to BB and Porgrame P10245 to TTL and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil. 68 Pyrimidinergic receptors agonists reduces, in vivo, L. amazonensis infection Maria Luiza Thorstenberg, Camila Marques da Silva, Mariana M. Chaves, Vanessa R. Figliuolo, Luiz Eduardo Savio and Robson Coutinho-Silva UFRJ, Brazil Objective/ background: Leishmania is an obligate intramacrophage parasite which manipulates the host cells in other to survive. We have already shown that the nucleotide UTP acting though P2Y2 and P2Y4 receptors stimulates the microbicidal activation of macrophages against L. amazonensis (L.a) (Cel. Microb. 13:9, 2011). In the present study, we evaluated the microbicidal effects of 2-thio-UTP (a P2Y2 receptor agonist) on L.a-infected macrophages, and the effects of UTP treatment in the initial step of in L.a infection. Methods and results: BALB/c mice (2–3 months old) were infected in the footpad with 2 × 106 L.a promastigotes in 20 μl PBS. After 7 days, 6 doses of 10 mM UTP in 20 μl PBS were injected in the infected footpad over a range of 2 days. The UTP treatment decreased the lesion size (15 ± 5 mm n = 4) when compared to the PBS-injected mice (19 ± 7 mm n = 4). We also observed that UTP treatment reduced 53.4 % the parasitic load in footpad (3 ± 1 × 1011) and 60 % in lymph node (6 ± 2 × 108 n = 4) when compared to the PBS injected mice. Unlike the levels of NO did not showed statistics difference both to footpad and lymph node. Interestingly, in the analysis PCR, we observed an upregulation of 30 % of the P2Y2 receptor gene expression profile in L.a when compared to the PBS group. To have a better understanding of the involvement of UTP in the early events of infection we inoculated 2 × 106 L.a, after air pouch induction in BALB/c mice, followed by 1 mM UTP treatment. After 6 h of UTP treatment the cellularity increased 50 % in the cavity when compared with to PBS. The phenotypic analysis showed an increase of 19.5 % of neutrophils (GR1+F4/80+), and these cells augmented the ROS production (389.6 AU). However the level of NO did not show any difference between the groups as have been observed in L.a infected mice. In order to show specific involvement of P2Y2 receptors in L.a infection, peritoneal resident macrophages (2 × 105 cells per well) were adhered and infected to 2 × 106 L.a. The infectivity index were reduced in 50 % after treatment of 100 μM UTP (n = 2), in addition, the treatment with selective agonist 2-thio-UTP provide an reduction of 40 % for 25nM, 50nM, 100nM and 1 μM concentration of nucleotide (n = 2), when compared to the L.a untreated group (n = 2). Conclusions: Our findings support the involvement of pyrimidinergic receptors in host response against L. amazonensis infection. Financial support: CNPQ, FAPERJ, CAPES. 69 Role of EGFR in ERK, CREB and calcium signaling in P2Y-induced proliferation of retinal cells in culture Carolina Gomes Lopes, Flávia de Jesus Jaques, Erick Correia Loiola, Isis Moraes Ornelas and Ana Lucia Marques Ventura Neurobiology Department, UFF, Niterói, RJ, Brazil Objectives / background: ATP is involved in the proliferation of neural precursors in the developing chick retina. It can induce Ca2+ mobilization in this tissue, a response that declines in parallel with the decrease in mitotic activity during retinal development. Since ATPdependent cell proliferation can rely on trophic factors such as EGF, we investigated the influence of EGF receptors (EGFR) on P2Ydependent cell proliferation and calcium mobilization in cultured retinal cells. In addition, the effect of EGFR activation and blockade on ERK and CREB signaling pathways was investigated. Methods and results: Retinal cells obtained from retinas of 7-day-old chick embryos were cultured for 2 or 3 days. Cell proliferation was estimated by the incorporation of [3H]-thymidine. Phosphorylation of ERK and CREB was determined by western blot using specific antisera. Calcium imaging was carried out on confocal microscopy using the Fluo-3 AM calcium indicator. Cultures treated for 24 h with 250 μM ADP increased [3H]-thymidine incorporation (213 ± 15 % compared to control, p < 0.001, n = 9), an effect that was prevented by 5 μM AG1478, an EGF receptor inhibitor (91 ± 17 %

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compared to control, p < 0.01 compared to ADP, n = 5). Levels of p-ERK and p-CREB were also increased by treating the cells with 250 μM ADP (respectively 425 ± 71 %, p < 0.01 and 152 ± 1 %, p < 0.001 compared to control levels, n = 3) a response that was decreased by the incubation of the cultures with 10 μM AG1478 for 3 h (ERK: 195 ± 28 %, p < 0.05 compared to ADP, n = 3; and CREB: 80 ± 4 % compared to control, p < 0.001 compared to ADP, n = 3). In contrast to ADP, 100 ng/ml EGF did not induce cell proliferation (127 ± 10 %, n = 6), neither ERK (73 ± 9 %, n = 3) nor CREB (127 ± 7, n = 4) phosphorylation. Moreover, activation of nucleotide receptors induced transient increases in intracellular calcium concentration in the retina cell cultures. Peak values of the increases represented 208 ± 8 % of the resting levels when cells were treated with 250 μM ADP. The ADPinduced calcium responses were attenuated when cultures were treated with 5 μM AG1478 for 24 h (143 ± 5 % of the resting level, p < 0.001 compared to ADP, n = 59 cells). Conclusion: These results suggest that ADP-induced proliferation as well as ERK and CREB phosphorylation in retinal cultures are dependent on the activation of EGF receptors. Furthermore, since AG1478 attenuated ADP-dependent calcium responses, our results raise the possibility that EGF regulates nucleotide responses by affecting the expression of P2Y receptors in the retinal cultures. Supported by: CNPq, Faperj, Proppi-UFF, Pronex-MCT, CAPES. 70 Role of nucleoside diphosphate kinase during Porphyromonas gingivalis infection Cássio Luiz Coutinho Almeida da Silva1, Gabrielle da Costa Rocha1, Erivan Schanaider Ramos Júnior1, Ana Carolina Morandini1, Júlio Scharfstein1, Özlem Yilmaz2, David Ojcius3 and Robson Coutinho Silva1 1 UFRJ, Brazil 2 University of Florida, USA 3 University of California, USA Objectives/background: Porphyromonas gingivalis (Pg) are Gram-negative bacteria associated with periodontitis. One of Pg’s virulence factors is a homolog of nucleoside disphosphate kinase (NDK) enzyme, which is secreted by Pg. NDK cleaves extracellular ATP, suppressing ATP-induced P2X7-dependent apoptosis and ATP-induced reactive oxygen species (ROS) production via P2X7-NADPH-oxidase, and contributes to Pg persistence in gingival epithelial cells. Our group showed that ATP via P2X7 controls infections with Leishmania amazonensis and Toxoplasma gondii. The protective role of the P2X7 receptor involves IL-1B production. We investigated the role of NDK in Pg infection. Methods and results: Using the air pouch model, C57BL/6 mice (male, 8–12 weeks old) were injected with sterile air in the dorsum and infected with 109 Pg33277 or NDK-deficient Pg (PgNDK−/−). After 24 h, cells was enumerated by microscopy counts and both Pg strains induced inflammatory cells migration; moreover, Pg33277 infection induced more cell migration than PgNDK−/− (Sham, 1.7 + 0.2 × 106; Pg33277, 1.2 × 107 + 3.1 × 106, p = 0.003; PgNDK−/− 6.4 + 0.7 × 106, p = 0.001; n = 2 with 8–11 mice/group). Using flow cytometry, the predominant cell subtype recruited was neutrophils, followed by small numbers of lymphocytes and macrophages. Pg33277 induced more migration of neutrophils than PgNDK−/− (Pg33277, p = 0.02; PgNDK−/−, p = 0.0001; n = 1 with 4–5 mice/group). We also investigated whether NDK influenced the secretion of IL-1B by bone marrow derived macrophages (BMDM) infected with Pg (MOI 100:1). After 6 h, 18 h or 24 h of infection, cytokines were secreted only when 5 mM ATP was added to infected cells. PgNDK−/−induced less secretion of IL1B than Pg33277 (6 h: Pg33277, 43231 + 914.8 pg/ml, PgNDK−/−, 25653 + 482.5 pg/ml, n = 3 18 h: Pg33277, 62340 + 5725 pg/ml, PgNDK −/−, 31369 + 551.8 pg/ml, n = 1; 24 h: Pg33277, 12193 + 3311 pg/ml, PgNDK−/−, 5433 + 413.9 pg/ml, n = 1). Pro-IL-1B was detected in BMDM lysates and IL-1B in supernatants by Western blot after infection with Pg. By densitometry, 46 % less pro-IL-1B was detected in extracts and 46 % less IL-1B in supernatants of BMDM infected with PgNDK−/− than Pg33277 (n = 3). Further, BMDM were infected to evaluate infectivity in vitro using the antibiotic protection assay. Both strains infected similarly after 2 h (n = 3). Conclusions: Our data support the idea that Pg’s NDK is involved in the induction of the host immune response against infection with Pg. Financial support: CAPES, CNPq, FAPERJ. 71 Role of P2X7 receptor during Porphyromonas gingivalis infection Gabrielle da Costa Rocha1, Cássio Luiz Almeida da Silva1, Erivan Schanaider Ramos Júnior1, Ana Carolina de Faria Morandini1, Júlio Scharfstein1, Özlem Yilmaz2, David Ojcius3 and Robson Coutinho Silva1 1 Federal University of Rio de Janeiro, Brazil 2 University of Florida, USA 3 University of California, USA Objectives/background: Porphyromonas gingivalis (Pg) are anaerobic Gram-negative bacteria associated with periodontitis. Previous studies from our group showed the importance of the P2X7 receptor in control of intracellular pathogen infections, such as Chlamydia trachomatis, Leishmania amazonensis and Toxoplasma gondii. The ligation of ATP to the P2X7 receptor can induces different responses, for example activation of inflammasomes and release of IL-1B. Here, we investigated the role of P2X7 receptor during Pg infection in macrophages and in vivo models of infection. Methods and results: We treated Balb/c mice (male, 6–8 weeks of age) with antibiotics in drinking water during 10 days. Then, we infected orally with 109 Pg (strain 33277) three times in 2-day intervals and we analyzed P2X7 receptor expression in maxilla samples. The P2X7 receptor expression evaluated by Western blotting was increased in maxilla from Balb/c infected relative to sham-controls (n = 2 with 4–5 mice/group). In the air pouch model, we injected sterile air in the dorsum of C57BL/6 and P2X7−/−mice (male, 8–12 weeks old) and infected them with 109 Pg to evaluate cell recruitment. After 24 h, we quantified total cells by microscopy counts in Pg infection. The bacteria induced inflammatory cells migration in both mouse strains but C57BL/6 mice recruited more cells than P2X7−/− mice (C57BL/6 SHAM, 1.5 + 0.1 × 106, C57BL/6 infected, 7.7 + 2.1 × 106, p = 0.03; P2X7 −/− SHAM, 0.8 + 0.08 × 106, P2X7−/− infected 4.0 + 0.7 × 106, p = 0.006, n = 1 with 4–5 mice/group). We analyzed the recruited cells by flow cytometry after 24 h and observed that the predominant cell subtype was neutrophils followed by small numbers of lymphocytes and macrophages


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(n = 1 with 4–5 mice/group). Next, we quantified IL-1B secretion by ELISA in bone marrow derived macrophages (BMDM) from C57BL/6 or P2X7−/− mice infected with Pg in vitro (MOI of 100) treated or not with 5 mM ATP. We observed that the P2X7 receptor was required for IL-1B secretion after Pg infection (n = 3). In C57BL/6 BMDM, Pg induced cytokine secretion only when ATP was added to infected cells (n = 3). Conclusions: Our data support the hypothesis that the P2X7 receptor is required for the host immune response against Pg infection. Financial support: CAPES, CNPq, FAPERJ. 72 ROS production and IL-1β secretion mediates Toxoplasma elimination after P2X7 activation T. P. Rangel, A. Moreira-Souza, R. Miranda, R.C. Vommaro and R. Coutinho-Silva Federal University of Rio de Janeiro, Brazil Background: The P2X7 receptor participates in the immune response against different intracellular pathogens such as Leishmania amazonensis, Chlamydia P. gingivalis and T. gondii (Coutinho-Silva & Ojcius, Microbes and Infection 14, 1271, 2012). However, the P2X7 mediated mechanism in Toxoplasma elimination in macrophage is not completely understood. The protozoan Toxoplasma gondii is a parasite known to avoid lysosomal fusion, survive in the parasitophorous vacuole and actively reorganize vital organelles of the host cell to its surrounding in order to obtain the essential nutrients for its replication. After an intense lytic replicative cycle, the parasites exit the host cell and initiate new infections in the adjacent cells, causing irreversible damage to the tissue. Here we investigated the possible involvement of ROS induced by P2X7 activation as key component of host response against Toxoplasma infection in murine macrophages. Methods and Results: Experiments were performed using peritoneal macrophages (Mo) from female C57BL/6 mice, 6–8 weeks old. Cells were plated for 24 h, infected with tachyzoites of T. gondii (RH strain) in the ratio of 3:1 for 2 h. Then, infected cells were incubated with ROS inhibitors for 40 min and treated with 1 mM ATP or 1 nM IL-1B for 30 min. We analyzed the parasite load and IL-1B secretion 18 h after treatment and ROS production during 60 min after ATP stimulation, following the same protocol. We observed that ATP and IL-1B reduced parasite load in 68 ± 4 % and 78 ± 2 %, respectively. ROS inhibitors reverted ATP and IL-1B effects on parasite elimination 18 h after treatment. The P2X7-induced ROS production was time dependent starting 30 min with max-50 min after ATP treatment (1474 AU ± 26). In addition, anti oxidant treatment completely inhibited ROS production. ATP treatment induced IL-1B secretion by infected macrophage after 18 h (401.4 ± 25 pg/ml) and this effect was abolished by pretreatment with Mito-TEMPO (mitochondrial ROS inhibitor). Conclusion: Our results support the hypothesis that ROS is a key component of ATP-inducing Toxoplasma gondii elimination in infected macrophages. Financial Support: CNPq, FAPERJ, PRONEX, CAPES, INPeTAm/UFRJ. 73 Spontaneous calcium oscilations are modulated by P2 purinergic receptors and control expression of neurogenesis related transcription factors Talita Glaser1, Hiromi Shimojo2, Ana Regina Geciauskas Lage Castillo1, Ryoichiro Kageyama2 and Henning Ulrich1 São Paulo University, Brazil 2 Kyoto University, Japan 1

Background: Purinergic receptors have been attributed with developmental functions including gastrulation and neural differentiation. Upon activation many P2 purinergic receptors trigger intracellular calcium transients controlling different cell processes. Since spontaneous calcium signaling is involved in neural differentiation, we induced neuronal differentiation of mouse neural stem cells and embryonic stem cells (ESC) with retinoic acid to study the influence of P2 receptors activity on spontaneous calcium transients and consequently on expression of transcription factors related to neurogenesis. Principal findings: First of all, during different moments of neural differentiation we characterized, by time lapse imaging of fluorescent calcium indicator, the different patterns of spontaneous calcium transients (waves and spikes) and reported that both frequency and amplitude increased along differentiation. Cells treated with inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor (Xestospongin C) showed spikes but not waves, indicating that waves depend exclusively on Ca2+ release from the endoplasmic reticulum (ER) and IP3R activation. Cells treated with various P2 receptor subtype modulators (Bz-ATP/ P2X7R agonist, 2SUTP/P2Y2R agonist, MRS2179/ P2Y1R antagonist) increased frequency and amplitude of calcium transients, mainly spikes in NSC and ESC differentiated for 8 days. Data obtained by luminescence time lapse imaging of stable transfected cells with Mash1 or Ngn2 promoter-protein fusion with a luciferase reporter showed an increase of Mash1 and Ngn2 expression due to activation of P2Y2R or inhibition of P2Y1R. Expression of these transcription factors decreased following P2X7R activation. Furthermore, cells imaged in presence of extracellular calcium chelator (EGTA) or following ER-Ca2+ store depletion by thapsigargin diminished Mash1 and Ngn2 expression, indicating that both are regulated by calcium signaling. Conclusion: Altogether these data suggest that P2X7, P2Y2 and P2Y1 purinergic receptors modulates spontaneous calcium oscillations during neural differentiation and consequently change the expression pattern of Mash1 and Ngn2, thus controlling the cell fate decision towards neuronal phenotypes. Acknowledgements: This research is supported by FAPESP and CNPq. 74 Study of candidate proteins to pore associated with P2X7 receptor in different cell types C. S. Oliveira1, A. V. P. Alberto1, M. S. Freitas2 and L. A. Alves1 Laboratório de Comunicação Celular – FIOCRUZ, Brazil 2 Centro Nacional de Ressonância Magnética Nuclear - UFRJ, Rio de Janeiro/RJ, Brazil 1

Aim: The P2X7R is a purinergic receptor, which differs from others subtypes due to its structural and pharmacological characteristics. When exposed for extended time or to high concentrations of its agonist (ATP), promotes an increase in membrane permeability, allowing the passage of molecules up to

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900 Da. There is a controversy among several authors that leave in doubt if this receptor needs a second protein for the pore formation and which protein could be. We select five pore-forming proteins: TRPV1, TRPA1, Connexins-43 (Cx-43), Pannexin-1 (Panx-1) and VDAC. We believe that different mechanisms and proteins could be associated with P2X7R, depending on the cell type and their microenvironment stimuli. In this context, our main goal is identify possible proteins that could be associated with the P2X7R pore in different cells and species. Methods and Results: We started with RT-PCR technique of cell lines: J774.G8, N2A, U373, U937, HEK-293 and primary cells from Wistar mouse and Swiss mice. We used different primers and PCR cycle for each target at different species. We observed that the P2X7, Panx-1 and Cx-43 are the most abundant and are present in all cell types except the absence of P2X7 in U373 cells and Panx-1 in mice macrophages and U373 cells. However, TRPV1 was seen at N2A and U937 cells and TRPA1 in and primary cells from mouse and mice and in J774.G8 cells. Regarding to the VDAC, it is present in mouse macrophages, J774.G8 and HEK-293 cells. The further steps, we verified if those proteins could be physically associated with the P2X7R. We coimmunoprecipitated the P2X7R of J774.G8 (with or without ATP), mice macrophages, HEK-293 and U937 cells (n = 3). The samples were applied in two separated 12.5 % bis/acrylamide gels: one destined to Mass Spectrometry and the other to Western Blot. At this point, we confirmed the presence of P2X7R, and observed several others proteins associated to P2X7R at different cell conditions, mainly when we exposed, J774.G8 cell, to 5 mM ATP. At this condition we found Hsp70, 75, and 90; alpha and beta tubulin; myosin Va; alpha, beta and gamma actin; malate and lactate dehydrogenase. Although U977 and HEK-293 had not received ATP treatment, we found several proteins associated to P2X7. Conclusion: We conclude that the P2X7R activated by extracellular ATP triggers the recruitment of variety different proteins. At this condition, we can suggest that maybe there is a conformational change, regardless of the numerous recruitment structural proteins. In addition, apparently, the pore-forming proteins (Panx-1, VDAC, Cx-43, TRPV1, TRPA1) are not physically associated with P2X7R. Financial Support: FIOCRUZ, CAPES and FAPERJ. 75 Study of effects of experimental ulcerative colitis on P2X7 receptor expression in submucosal neurons and enteric glial cells Marcos V. da Silva, Aline R. Marosti, Cristina E. Mendes, Kelly Palombit and Patricia Castelucci University of São Paulo, Brazil Objectives / background: The digestive tracts of ulcerative colitis and Crohn’s disease patients present with pathophysiological processes and intestinal necrosis. This study examined the P2X7 receptor and changes in the distal colon in submucosal neurons of rats with experimental ulcerative colitis. Methods and results: The analysis was performed in the distal colons of rats with ulcerative colitis induced by the administration of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS; colitis group). The survival time after colitis induction was 24 h. The treated animals were compared to sham rats injected with phosphate buffered saline and to animals with no intervention (control group). Tissues were prepared for immunohistochemical double-staining methods to examine P2X7 receptor, choline acetyltransferase (ChAT), Calbindin, Calretinin, anti-HuC/D (pan-neuronal) and S100β (pan-glial). Results: The colocalization of the P2X7 receptor-immunoreactive (IR) cells was observed in the submucosal plexus with Calbindin-, Calretinin- and HuC/D-IR neurons and S100β-IR cells in the control, sham and colitis groups. The neuronal density (cell bodies/cm2) decreased in the submucosal plexus by 23.4, 34.7, 11.7, and 33.4 %, in the P2X7 receptor, Calbindin-, Calretinin- and HuC/D-IR neurons, respectively. In addition, the densities (cell bodies/cm2) S100β-IR enteric glial cells decreased by 44.2 %. The profile areas were reduced by 12 % Calbindin- and 6 % Calretinin-IR neurons, respectively. Morphological changes were observed, such as increased neutrophils, disintegration of the intestinal epithelium and goblet cells and decreased collagen. Conclusion: This study demonstrated that colitis differentially affects P2X7 receptor-expressing submucosal neurons based on their chemical codes and may cause changes in morphology and glial cells. Sources of research support: São Paulo Research Foundation (FAPESP)/ Capes/CNPq. 76 Study of plants from Jurubatiba resting (RJ) on function of P2X7 receptor and its associated pore Eloisa Portugal Barros Silva Soares de Souza1,2, Leandro Machado Rocha1 and Robson Xavier Faria2 Universidade Federal Fluminense, Brazil 2 Fundação Oswaldo Cruz – RJ, Brazil

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Introduction: The study of medicinal plants has become an important source of research and scientific interest due to its value at the level of chemical structure and pharmacological properties. They have therapeutic potential based on the popular description, but there is no scientific confirmation about its mechanism of action. P2X7 receptor, a receptor family of purinergic P2 receptors activated by ATP, has been extensively studied in function of its involvement in processes of pain and inflammation. This receptor can be found in immune cells, and its activation leads to responses such as plasmatic membrane permeabilization, activation of phospholipase A2 and COX-2, additionally this channel may release IL1-B; independently of the COX pathway. In this context, natural products may be a significant source to obtain new P2X7 receptor inhibitors. The objective of this project is assess the plant species of Parque Nacional da Restinga de Jurubatiba related to P2X7 receptor and its subsequent involvement in the processes of pain and inflammation, thus contributing to the development of new medicines based on brazilian flora. Objectives: To study the functional properties and applications of the P2X7 receptor and its associated pore, evaluating stem extracts and plant leaves of the Restinga Jurubatiba, and its fractions, in the activity of the P2X7 receptor, and their toxicity in mammalian cells. Methods: Cell permeability assays using peritoneal macrophages from male mice Swiss-Webster with mass 30 g, whose protocols were approved by CEUA. U937 and J774G8 lineage cells were also used. Cells were incubated with the extract, ATP as agonist, Briliant Blue G (100nM) as the reference antagonist and propidium iodide (1 μM) as a marker, and the measurement was performed by flow cytometry. Toxicity test based on the release of lactate dehydrogenase and cell viability based on the reduction of resazurin. The plants


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were collected from Restinga de Jurubatiba, extracted by soaking and pre-purification with increasing polarity solvents, and serial dilutions for in vitro tests. The plants belong to families Lauraceae, Myrsinaceae and Myrtaceae. The species are not disclosed in the work, because they are under patent process. Results: We tested 8 extracts from 5 diferents plants, and two of them showed inhibition. In the dye uptake assay tested the fraction acetate leaves of a plant of the dunes. ATP induced a 48 % dye uptake compared to the positive control. This response was reduced to 20 % by BBG reference antagonist, and the 22 % tested extract. With respect to the toxicity after treatment with 12 extracts of 7 different plants by 1, 3, 6, 24 and 72 h in cell viability assay, it was observed that the compounds are maintained at levels comparable to the negative control over time. Conclusion: Based on these results it can be said that ATP has its dye uptake response partially blocked by the test compound. And the extracts do not show toxicity in times evaluated.

77 Study of the P2X7 receptor role in the pilocarpine epilepsy model using RNA interference R.P. Amorim1, M.G.L. Araújo1, J. Valero2, I.T.L. Cendes3, H. Ulrich4, J.O. Malva2 and M.J.S. Fernandes1 1 Universidade Federal de São Paulo, Brazil 2 Universidade de Coimbra, Portugal 3 Universidade Estadual de Campinas, Brazil 4 Universidade de São Paulo, Brazil Objectives: Cell signaling mediated by purinergic receptor P2X7 (P2X7R) has been suggested to be involved in epileptogenesis. Indeed, P2X7R activation is associated with increased intracellular calcium levels, excitotoxicity, activation of inflammatory cascades and cell death. The main aim of this work is to elucidate the role of P2X7R in epileptogenesis in vivo and to test the therapeutic potential of downregulating its expression by using RNA interference (RNAi). Methods: Pilocarpine, a cholinergic agonist, was injected systemically (370 mg/kg) in adult male Wistar rats. Small interfering RNA (siRNA) designed against P2X7R was administered intracerebroventricular (icv, into the lateral ventricles) in vivo, 6 h after Status epilepticus (SE) induced by pilocarpine. A short peptide derived from the rabies virus glycoprotein fused to a nona-D-arginine residue (RVG-9dR) was used to deliver siRNAs to the cells. A fluorescent oligonucleotide (BLOCK-ITTM) complexed to RVG-9dR was served to test intracellular delivery of the construct. Hippocampal P2X7R protein levels were assessed by western blotting. Four animal groups were used in this study: Saline-Vehicle, Saline-siRNA, Pilo-Vehicle and PilosiRNA (N = 5/group). Neuronal death was measured by Fluoro-Jade B (FJ-B) histochemistry and hippocampal volume was analyzed 48 h after RNAi icv (N = 3/group). Epileptic-behavioral parameters as latency, number and severity of seizures were evaluated until 60 days after pilocarpine-induced SE (N = 8/group). Ethical Committee Approved Protocol - CEP 0961/10. Results: Intense intracellular fluorescence indicated the presence of the BLOCK-ITTM: RVG-9dR into the hippocampal cells. Saline and pilocarpine rats treated with P2X7R siRNA showed a 43 % (p = 0.0002) and 37 % (p = 0.0008) reduction, respectively, in P2X7R protein levels 48 h after injection when compared to their respective control groups. Less FJ-B dying cells were observed in CA1 and CA3 of Pilo-siRNA group compared to Pilo-Vehicle. P2X7R silencing in pilocarpine group reversed the increase in the hippocampal volume (edema) detected 48 h after SE onset in the hilus (p = 0.0225), dentate gyrus suprapyramidal (p = 0.0308), CA1 (p < 0.0001) and CA3 (p < 0.0001). Furthermore, the silencing induced an increase in latency to the first spontaneous seizure (p = 0.0009) and a reduction in the number of seizures (p = 0.0002) when compared to Pilo-Vehicle group. Conclusions: Our results demonstrate that P2X7R has a direct role in epileptogenic-associated to cell death, edema and seizure occurrence. Furthermore, our data reveals the potential of P2X7R as a therapeutic target for the treatment of epilepsy. Financial support: Fapesp, CNPq, INNT, CAPES. 78 The blockade of P2Y1 receptors in vivo altered horizontal cell morphology of the rat retina L.A. Pereira1, C.F. Magalhães1, M.G. Repossi1, A. Sholl-Franco2, A.L.M. Ventura1 and L. Fragel-Madeira1 Departamento de Neurobiologia, Instituto de Biologia, Universidade Federal Fluminense, Niterói/RJ – Brazil 2 Instituto de Biofísica Carlos Chagas Filho, Universidade Federal Rio de Janeiro, Rio de Janeiro/RJ - Brazil 1

Objectives / background: The retina is a tissue member of the central nervous system, consists of six different types of neurons (cone and rods photoreceptors, horizontal, amacrine, bipolar and ganglion cells), astrocytes and Müller glia and is organized into nuclear and plexiform layers. Changes that occur during retinal development can cause alterations in its histology and consequently functioning. Adenine nucleotides are known to modulate neuroblasts proliferation at early stages of retinal development. Previous results of our group reported that intravitreal injection of MRS2179, a selective antagonist for P2Y1 receptor, at concentration of 50 and 100 μM reduced cell proliferation approximately 20 % in rats at 3-postnatal days (P3). The purpose of this study was evaluated how the reduction in the number of proliferating cells in the retina, induced by the blockade of P2Y1 receptors, could affect the histogenesis of this tissue. Methods and results: Lister hooded rats at P2 were anaesthetized by hypothermia followed by intravitreal injection of 50 μM MRS 2179 (1 μl) through a Hamilton microsyringe in each eye on the experimental group or 1 μl of phosphate buffered saline in control animals. After intravitreal injection, the animals were subjected to different survival times according to experiment. To evaluate the cell death we intravitreously injected MRS 2179 at concentration of 50 μM and the animals were subjected to survival of 3, 6, 12, 16 and 20 h. The treatment with MRS 2179, at the concentration of 50 μM for 20 h, decreased by 30 % the number of calbindin-positive horizontal cells and, apparently, there was also a reduction in the dendritic arborization of these cells. To exclude the possibility of cell death induction by this drug, we assessed the apoptotic profiles during the treatment period by neutral red but we did not identify any difference in the number of pyknotic profiles. Amacrine and rod photoreceptors cells were evaluated after 20, 48 and 72 h of treatment and did not change the cell number and morphology of these cellular populations.

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Conclusions: Our data suggest that adenine nucleotides could act during early stages of retinal development modulating the horizontal cells differentiation. Financial Support: Capes, FAPERJ, CNPq, Proppi-UFF. 79 The effect of the P2X7 receptor antagonist AZ10606120 in the pilocarpine-induced epilepsy model Michelle Gasparetti Leão Araújo, Rebeca Padrão Amorim, Marcus Vinicius Buri, José Eduardo Marques-Carneiro, Luis Fernando Sierra de Araújo, Gabriela de Melo Predebon, Vassiliki de Brito Patsis, Edgar Julian Paredes-Gamero and Maria José da Silva Fernandes Universidade Federal de São Paulo, Brazil Aims: P2X7 receptors (P2X7R) are increased in the hippocampus of rats subjected to temporal lobe epilepsy model and have been correlated with hyperexcitability and excitotoxity. In this way, P2X7R antagonist could represent an interesting tool to study the role of these receptors in the epileptogenesis. In this study, the antagonist AZ10606120 (AZ) was administered in rats subjected to pilocarpine-induced status epilepticus (SE) in order to determine the role of P2X7R on calcium mobilization and cell death resulting from SE. Methods: Adult male Wistar rats were used in the study. The AZ was administered in vivo intracerebroventricular (i.c.v.) at doses of 1, 2 and 3 μg, 30 min after SE. Twenty-four hours later, the groups Saline+ Vehicle, Saline+AZ, Pilo+Vehicle, Pilo+AZ were used to evaluate hippocampal cellular death by Fluoro Jade-B (FJ-B) histochemistry (n = 3/group) and the P2X7R protein level of hippocampus by Western Blot (n = 4/group). The dose that induced lower number of FJ-B positive cells was chosen for the study (3 μg). Behavioral parameters as mortality rate within the first 24 h after SE onset (n = 26, Pilo+AZ group; n = 30, Pilo+Vehicle group), latency, number and severity of seizures were analyzed 30 days after SE onset (n = 6/group). Calcium mobilization was assessed by calcium image using Fluo4 AM in hippocampal slices groups: Control+BzATP, Control+AZ+BzATP, Pilo+BzATP, Pilo+AZ+BzATP (n = 4/group). The AZ concentration that reduced the calcium uptake elicited by BzATP was defined in a pilot study using hippocampal slices of normal rats. The concentrations of 2.5, 5 and 10 μM were tested. Number of Ethical Comittee approval: CEP 0019/2012. Results: The 3 μg dose of AZ i.c.v. caused a reduction of FJ-B labeled cell in CA1, CA3 and hilus of dentate gyrus of Pilo+AZ group compared to Pilo+Vehicle. A reduced expression of P2X7R (−42 %, p = 0.0058) was detected in rats from Pilo+AZ group compared to Pilo+Vehicle group. The treatment with AZ reduced the mortality rate in the acute period (27 %; p = 0.045) compared to Pilo+Vehicle group (53 %), increased the number of seizures (p = 0.0004) but reduced the severity of them compared to vehicle (p < 0.0001). The concentration of 2.5 μM AZ caused an inhibition on the calcium concentration in hippocampal slices under BzATP stimuli (p < 0.0001). However, AZ did not alter calcium in hippocampal slices of Pilo group (Pilo+AZ+BzATP) compared to Pilo+BzATP group. Conclusion: Our data corroborate with previous data showing the involvement of the P2X7R on cell death and in the epileptogenesis. The AZ is a good tool to investigate the role of P2X7R in rat brain. Financial support: CNPq, Capes, FAPESP, CNPq-INNT. 80 The helper T cells subtypes from mesenteric lymph nodes are differentially sensitive to cell death induced by P2X7 receptor activation Vanessa Ribeiro Figliuolo da Paz1, Hanaa Safya2, Heitor S.P. de Souza3, Cláudia M.L.M. Coutinho4, Pierre Bobé2 and Robson Coutinho Silva1 IBCCF, UFRJ, Brazil 2 INSERM, UPS, France 3 HUCFF, UFRJ, Brazil 4 LITEB, FIOCRUZ/ UFF, Brazil 1

Objective: P2X7 receptors have its expression increased in T lymphocytes surface after activation of the TCR complex, concomitantly with the quick release of the nucleotide. ATP acts in an autocrine manner contributing to the T cells activation by inducing rapid Ca2+ influx. While high concentrations of ATP in the extracellular medium result in T cells death, low levels of this nucleotide stimulate proliferation of these cells. Therefore, we decided to study the cell death induced by P2X7 activation at different stages of T cell activation. Methods and Results: The mouse strains used were P2X7 knockout mice (KO) or P2X7 wild type (WT) C57BL/6. T cells from mesenteric lymph nodes were evaluated. The lymph nodes were dissociated and the number of viable cells was counted using Trypan Blue. The cells were incubated with 0.5 or 1.0 mM ATP for 30 min than immunostained with fluorescent antibodies recognizing CD90.1, CD4, CD45RB, CD25 and FoxP3 positive cells. The expression of CD45RB in T CD4 cells was analyzed by flow cytometry identifying activated and naïve T cells. The CD45RBhigh, int and low populations were taking in account. The functionality of P2X7 receptor was evaluated by YOPRO uptake and the ATP-induced cell death by 7-AAD dye incorporation. Our results have shown an inverse relationship between P2X7 sensitivity and CD45RB expression, in which activated T cell populations (CD45RBint and low) were more sensitive to ATP-induced YOPRO uptake. In turn, activated T cells were also more sensitive to cell death than naive T cells. Moreover, the TCD45RBlow population, previously reported as producer of TGF-β, contained 40 % of T CD4FoxP3+ cells, therefore regulatory T cells. Conclusion: The different Th cells sensitivity to cell death induced by P2X7 activation demonstrates the importance of this receptor during different phases (effective and regulatory) of acquired immune response. Financial support: CNPq, FAPERJ, CAPES. 81 The P2X7 purinergic receptor is a critical regulator of intestinal inflammation and colitis-associated colorectal cancer in mice Claudio Bernardazzi de Miranda Azevedo, Morgana T. Castelo-Branco, Raquel Coutinho, Beatriz Pêgo Damasceno, Joao Carlos Machado, Robson Coutinho-Silva and Heitor S.P. de Souza UFRJ, Brazil


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Background and aims: Inflammatory bowel diseases (IBD) have been associated with an increased risk for colorectal cancer, a phenomenon largely attributed to chronic intestinal inflammation. Previous studies from our group support a role for purinergic signaling mediated by the P2X7 receptor in intestinal homeostasis and inflammation. Therefore, in this study, we sought to investigate a possible role for the P2X7 receptor pathway in the development and progression of colitis-associated colorectal cancer (CAC). Methods: To mimic the human disease, colitis and CAC were chemically induced in male C57/BL6 and P2X7R knockout (KO) mice, by the treatment with the carcinogen azoxymethane (AOM) combined with dextran sodium sulfate (DSS) in three cycles. In a therapeutic protocol, wild-type mice were treated with intra-peritoneal injections of A-740003, a P2X7R-selective inhibitor, 1 h prior to the second and the third cycles of DSS. The effects of treatments were evaluated using follow-up video-endoscopy associated with an endoluminal ultrasound biomicroscopic (eUBM) system, and also by histologic scores, the quantification of T-cells and macrophages by immunoperoxidase, cytokines measurement in culture supernatants of colon explants, by cytometric bead array, and investigating nuclear factor-kappa B (NF-kappa B) and external mitogen activated protein kinase (pERK) activation. Results: Video-colonoscopy with eUBM revealed significantly more inflammation, with mucosal granulosity, ulcers, bleeding, wall thickness, and elevated lesions including tumors, in wild-type- compared with P2X7 KO-induced animals and with animals treated with A-740003, throughout the study. Histological assessment confirmed significantly more inflammation and tumors in wild-type- compared with P2X7 KO-induced animals, and with animals treated with A-740003. Increased accumulation of T cells and macrophages, served to characterize the chronic inflammatory process in the colon of induced animals. Colonic NF-kappa-B and Erk activation were significantly lower in A-740003-treated mice and in P2X7 KO-induced animals compared to wild-type induced mice. Cytokine measurements showed a remarkable increase in the levels of TNF-alpha and IL-6, with a reduction of IL10, in wild-type induced mice, whereas levels tended to stabilize after P2X7R blockade, and almost did not change in P2X7 KO mice. Conclusion: P2X7 KO mice are remarkably resistant to DSS-induced colitis and do not develop CAC. In wild-type mice, the therapeutic treatment with the selective P2X7R pharmacological blockage hampers the development of CAC. Taken together, these data suggest that the P2X7R-ATP pathway represents a key pro-tumorigenic player, which may contribute to different stages of initiation and progression of CAC in colitic mice. 82 The P2X7 receptor promotes glioma cell growth Letícia Scussel Bergamin1, Marina Capece2, Erica Salaro2, Ana Maria Oliveira Battastini1 and Francesco Di Virgilio2 1 Universidade Federal do Rio Grande do Sul, Brazil 2 University of Ferrara, Italy Glioblastoma multiforme is the most common and devastating primary brain tumor. Different signaling pathways, including the purinergic system and COX-2 overexpression, are involved in glioma progression. The P2X7 receptor is involved with tumorigenesis in several types of cancer. However, the role of this receptor in glioma cell death or proliferation is controversial. Aim: here, we investigated the role of P2X7 receptor in cell proliferation in vitro and in vivo in human glioma cell line (U138MG) and the role of P2X7 in the COX-2 expression. Methods: the human glioma cell line (U138MG) was transfected with P2X7 receptor and stable P2X7-policlone (U138-P2X7) and P2X7clone (U138-1 F18) were selected in the continuous presence of G418 sulfate (Geneticin). Expression of P2X7 and COX-2 in U138MG, policlone and clone cells were verified by qPCR analysis. The functionality of P2X7 receptor was assessed by quantifying cytosolic free calcium in a thermostat controlled PerkinElmer fluorimeter, using BzATP (300μM). For proliferation assay, cells were seeded and analyzed at the following time points: 0 h, 24 h, 48 h and 72 h with ImageJ. For in vivo experiments, U138MG and U138-1 F18 were subcutaneously inoculated into nude/nude mice, and the tumor size was assessed by in vivo caliper measurement. Animal procedures were approved by the University of Ferrara ethic committee and the Italian Ministry of Health in compliance with international laws and policies. Results: P2X7transfected cells showed a higher mRNA expression of this receptor (Mean ± SD: U138MG: 3.2 ± 3.4; U138-P2X7: 105 ± 51; U138-1 F18: 800 ± 258), as well as higher cytosolic free calcium levels ([Ca+2]i: U138MG: 0 nM; U138-P2X7: 111 nM; U138-1 F18: 1300 nM). U138P2X7 exhibited a proliferation increased of the 20, 40 and 60 % when compared to U138MG (24, 48 and 72 h, respectively). Furthermore, U138-1 F18 glioma cells generated larger in vivo tumors compared to U138MG cells (tumor size (mm): U138: 0.3 ± 0.6; U138-1 F18: 2.7 ± 1.0). Finally, we also found that P2X7 transfection causes COX-2 downregulation by qPCR (U138: 0.8 ± 0.1; U138-1 F18: 0.2 ± 0.1). Conclusion: although experiments are still in progress, the preliminary findings allow us to infer that P2X7 has an important role in the progression of glioma cells. Financial Support: This work was supported by CNPq, CAPES-PDSE (99999.002512/2014-09), AIRC, Telethon, the Italian Ministry of Education, the Italian Ministry of Heatlh and the NanoStroke Era-NET project. 83 The presence of anti-M2ACHR antibodies leads cardiac disarrangement at P2X7 purinergic knockout mice Camila Guerra Martinez, Marcia Gracindo Silva, Pedro Muanis Persechini and Eleonora Kurtenbach UFRJ, Brazil Aim: Dilated Cardiomyopathy (DCM) was associated with the presence of autoantibodies against M2AChR. Our previous results demonstrated that antiM2AChR antibodies induced morphological and functional cardiac disarrangements of WT mice immunized with pcDNA3-hM2. These WT immunized animals had an efficient production of anti-M2AChR antibodies at 5 weeks post-immunization, that was not accompanied by mice deficient in the purinergic receptors P2X7. These knockouts mice produced a very smaller amount of antibodies only at 25 weeks post-immunization. Even with this different kinetics of antibodies production, both groups of mice reproduced some features of DCM, like an increase at NPPA (Atrial Natriuretic Factor promoter) transcriptional levels, a dilatation of the left ventricle, and a decrease in exercise tolerance. Additionally, electrocardiogram analysis showed that P2X7−/− mice presented particular electrocardiographic disarrays, regardless of the immunization, showing a trend in the development of heart failure. Thus, this work aimed to evaluate the influence of the administration of high titers of anti-M2AChR from M2AChR wild-type mice on cardiac function of P2X7 −/− mice.

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Methods and results: WT and knockout mice for the P2X7 purinergic receptor (P2X7−/−) received three i.v. doses of a serum pool from wild type animals previously immunized with the plasmid pcDNA3-hM2 or with the empty pcDNA3 plasmid (control serum group). The four experimental groups were submitted to cardicac functional and molecular analysis (Ethical Committee approval: 005/14). Ten weeks post-serum transfer WT and P2X7 −/− mice recipients of control serum did not show any significant cardiac abnormalities. Meanwhile P2X7−/− recipients’ mice of control serum showed a depression in the ST segment in the electrocardiogram analysis at this time. WT and P2X7−/− mice recipients of anti-M2AChR antibodies showed a decrease in the RR interval and in the variation of the O2 consumption (deltaVO2); an increase in hearts’ volume and an increased the production of TNFa. Despite these similarities, knockout P2X7−/− mice that received serum containing high titer of anti-M2AChR presented unique characteristics of heart damage that were not observed in wild-type mice recipients of the same serum. Among these features we can highlighted, a significant increase in NPPA and significance alterations in the electrocardiogram. It is also worth to mention that these P2X7−/− mice anti-M2AChR recipients still presented the ST segment depression observed in their respective control. Conclusion: These data demonstrate that C57Bl6/J WT and P2X7 −/− mice presented some features of dilated cardiomyopathy after been submitted to anti-M2AchR antibodies transference. This challenge seems to be more severe to P2X7−/− mice. This reinforced our hypothesis that P2X7−/− mice could have a tendency to develop heart disease. Financial support: CAPES, CNPq and FAPERJ. 84 The quinovic acid glycosides purified fraction from uncaria tomentosa protects against hemorrhagic cystitis induced by cyclophosphamide in mice by down-regulating P2X7 receptor Fabrícia Dietrich1, Jerônimo Pietrobon Martins2, Samuel Kaiser3, Rodrigo Braccini Madeira Silva2, Maria Isabel Albano Edelweiss4, George González Ortega3, Fernanda Bueno Morrone2, Maria Martha Campos5 and Ana Maria Oliveira Battastini6 1 UFRGS, Brazil 2 Faculdade de Farmácia, PUCRS, Brazil 3 Faculdade de Farmácia, UFRGS, Brazil 4 Hospital de Clínicas de Porto Alegre, UFRGS, Brazil 5 Instituto de Toxicologia e Farmacologia, PUCRS, Brazil 6 Departamento de Bioquímica, UFRGS, Brazil Objectives/background: The purinergic system is involved in several pathophysiological events. Among purinergic receptors, P2X7 is involved in hemorrhagic cystitis (HE) induced by cyclophosphamide (CYP) and, therefore, can be considered a potential new therapeutic target. HE is an inflammatory condition of the bladder associated with the use of anticancer drugs such as CYP. Sodium 2-mercaptoethanesulfonate (Mesna) has been used to prevent the occurrence of HE, although this compound is not effective in established lesions. It has been demonstrated that Uncaria tomentosa is widely used in folk medicine for the treatment of numerous diseases, such as urinary tract disease. Considering the above, the objective of this study was to investigate the potential therapeutic effect of the quinovic acid glycosides purified fraction (QAPF) from U. tomentosa in the mouse model of CYPinduced HE. Methods: This study was approved by the Ethics Committee of Pontifícia Universidade Católica do Rio Grande do Sul (CEUA 11/00243). The quinovic acid glycosides purified fraction (QAPF) was obtained and suitably characterized by HPLC-PDA and UPLC/Q-TOF-MS analysis. Male Swiss mice (25 to 30 g) were used and the HE was induced by a single intraperitoneal injection of CYP (300 mg/kg). After the treatment with Mesna or QAPF, the bladder gross evaluation, the wet weight determination and histological analysis were performed. The visceral sensitivity was evaluated using von Frey filaments. Neutrophil recruitment to mouse bladder was measured by means of tissue myeloperoxidase activity. The IL-1β levels was measured in the homogenized badders. Finally, expression of the P2X7 purinergic receptor in bladder tissue was evaluated by immunohistochemical analysis. Results: Pretreatment with QAPF decreases CYP-induced nociceptive behavior scores (100 mg/kg decrease 36.53 ± 12.90 %) in the HE model, with an efficacy comparable to the reference compound Mesna (32.53 ± 11.61 %) (n = 5–14 animals). Treatment with QAPF also had a protective effect on HEinduced urothelial damage: 100 mg/kg inhibited 82.35 ± 8.23 % of edema, 51.73 ± 17.18 % of hemorrhage and 100 mg/kg reduced 47.35 ± 5.28 % of bladder wet weight (n = 12–13 animals). The same therapeutic strategy was able to control visceral pain, to inhibit neutrophil migration (45.76 ± 9.60 %) (n = 3–8 animals) and to decrease the tissue levels of IL-1β (40.18 ± 4.59 %) (n = 8–15 animals), most likely by down-regulating significantly P2X7 receptors (62.05 ± 6.85 %) (n = 3–6 animals). Conclusion: This research clearly demonstrates the promising anti-inflammatory properties of QAPF, supporting its use as complementary therapy. Here we show the potential involvement of P2X7 receptor in the therapeutic action of U. tomentosa. Financial support: CNPq, Capes and FINEP research grant “Implantação, Modernização e Qualificação de Estrutura de Pesquisa da PUCRS” (PUCRSINFRA) # 01.11.0014-00. 85 The use of agonists to P2X7R for the treatment of intracellular microorganisms: an innovative and promising therapy for MDR and XDR tuberculosis R.J. Soares-Bezerra, C.M. Silva, R.T. Pinho, T.C. Benévolo-de-Andrade, R.C. Bisaggio, M.O. Moraes and L.A. ALves Fiocruz - Rio de Janeiro - RJ – Brazil Objectives/background: Treatment for tuberculosis is effective with the use of proper antibiotics, but the number of drug-resistant cases are increasing. Drug resistance occurred in 630,000 cases of the 20 million patients in treatment worldwide in 2011, which demonstrates the necessity of finding new therapeutic approaches. Actually, several preclinical studies demonstrate the involvement of the P2X7 receptor (P2X7R) in the control of Mycobacterium tuberculosis (MTB) infection. Adenosine triphosphate (ATP), a natural agonist of P2X7, promotes MTB death and the induction of


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apoptosis in monocytes and macrophages infected with MTB via activation of the P2X7R by extracellular ATP. In addition, P2X7R activation in the presence of ATP increases the expression of major histocompatibility complex (MHC) class II by macrophages infected with Mycobacterium bovis (BCG) or MTB, which contributes to the generation of the antimicrobial immune response via T cells. We propose the use of P2X7 agonists in conjunction with low-molecular-weight anti-tuberculosis medicines for the treatment of multi-drug-resistant tuberculosis. Methods and results: Our methodology is based on the culture of both human and mouse mononuclear cell lineages. Infected, or not with BCG expressing GFP, using the spectrophotometric and fluorescence microscopic techniques as tools for analysis. As results, firstly, we observed the activity of the current first line drugs on BCG-GFP. The treatments using [1 μg/mL] of Isoniazid promoted an inhibition of 80 % of the growth of the bacillus, and the treatment with Rifampicin inhibited 70 %. Together, both antibiotics promoted a significant inhibition rate (90 %). We also observed the action of the extracellular ATP on the infection of J774.G8 and U937 cells with BCG-GFP, in a M.O.I of 5:1. The J774.G8 infected cells, when treated with each antibiotic [1 μg/mL] for 24 h, together or alone, submitted or not to an extra treatment with ATP [5 mM] for one more hour, demonstrate that, the treatments with the antibiotics plus ATP lead to an inhibition rate of 40 % of the infection regarding those without ATP. However, the treatment with ATP [5 mM] alone inhibited 60 %. Although when the same protocol was applied to the U937 cells, the additional treatments with ATP showed an increase in the inhibition rate of 50 % of the infection, it is important to point out that alone, the ATP inhibited 70 %. We also standardized a spectrophotometric methodology for the screening of new anti TB compounds. All experimental protocols were approved (protocol n° 535–09) by the Institutional Ethical Committee. Conclusions: Taken together, our results pointed to the applicability of a selective agonist for P2X7R as a promising therapy for intracellular infections, including the multi-drug-resistant cases of TB. Acknowledgment: CNPq and Faperj. 86 Thymine and thymidine photodamages can act as alarmins in a peritonitis mouse model C. Marques da Silva1, F.S. Vieira2, J.W. Souza3, C. Bandeira-Melo4, C. Lage3 and R. Coutinho-Silva1 Laboratório de Imunofisiologia, IBCCF, UFRJ, Brazil 2 Departamento de Imunologia, ICB, USP, Brazil 3 Laboratório de Radiações em Biologia, IBCCF, UFRJ, Brazil 4 Laboratório de Inflamação, IBCCF, UFRJ, Brazil

1

Ultraviolet (UV) radiation is known as an important factor disrupting immune homeostasis. Cellular exposure to UV light can ensue both inflammatory and anti-inflammatory responses. Purinic and pyrimidinic bases in DNA strongly absorb UV light. Thymidine Dimers are produced when adjacent thymidine residues are covalently linked by exposure to UV radiation. Dimerized thymines may be replicated as a single base, which results in frameshift mutations whenever they are not repaired before replication. It was already shown that one of the inflammatory effects is due to expression and release of the alarmin high mobility group box 1 (HMGB1) after acute and chronic UV irradiation of the skin (Johnson et al. Arch Dermatol Res. 305(9) 2013). Alarmins are endogenous molecules that act as potent pro-inflammatory mediators when they are released by cells or accumulate extracellularly. Here, we show that thymine is one of the possible molecules released after UV radiation and can also act by itself as a novel class of alarmins. Methods and Results: Peritonitis was performed by intraperitoneal injection of thymine, thymidine dimers, ATP or vehicle at different time points and different concentrations in both sexes of BALB/c mice between 8 and 12 weeks old. After peritoneal wash, cells were counted, processed and fixed to proceed citospyn (staining with Panotico Rapido Kit), immune phenotyping (staining cells with anti-CD3, anti-GR1/Ly6, anti-CD11b/F4-80 antibodies conjugated with different fluorocromes), reactive oxygen species production (by fluorescent probe CM-H2DCFDA) and lipid vesicles staining (Osmium staining). The supernatants were processed to cytokines (ELISA), leukotrienes (EIA), and nitric oxide measurements (Greiss reaction). Our results showed that thymine and thymidine photodamages induce an augment of 40.3 ± 12.9 and 154.7 ± 3.1 (×105) cells over vehicle injection alone. Also, there was no cell death difference between treatments. Trough flow cytometry analysis we observed that most of the dimers recruited cells were neutrophils, with an augment of 141 ± 12.1 % over vehicle, and timine augmented in about 99 % Monocytes/macrophages and about 33 % lymphocytes and neutrophils. The ROS production was augmented in both thymine and its dimers, 2748 ± 483 and 2536 ± 515 MIF, conversely, nitric oxide was not detected in any of the experimental situations. Since cell recruitment was such a noticeable data we wondered witch was the recruiting agent released during peritonitis, and turned out to be leukotriene B4, with an improve of 200 % release over vehicle treated mice in timine and its dimers. Conclusion: In the peritonitis model, UV damage products thymine and thymidine were shown to be pro inflammatory agents orchestrating several steps of acute inflammation. Our results shed some light on its mechanisms, possibly due to the release of a new class of UV-induced alarmins. Support: PIBIC/CNPq, CAPES, FAPERJ. 87 Tissue cytometry and tissue sociology – new concepts to look at cells and cellular interactions in tissue sections Rupert Ecker TissueGnostics GmbH, Austria Determining the in-situ immune status of diseased organs or quantify coexpression of molecules on the single-cell level has mostly been subject to visual estimation, or – at best – to manual counting for decades. Hence, experts usually had the choice of the “least of evils” between guessing and endless (manual) counting. In tumor immunology, infiltrating inflammatory cells need to be phenotypically characterized on a quantitative basis. To better understand the function of inflammatory cells in tumor development, type and number of inflammatory cells and their proximity to glandular/tumor structures have to be analyzed in-situ and correlated with disease state. Using TissueFAXS™ Cytometry the time-consuming and error-prone human evaluation of stained histological sections can be approached with an observer-independent and reproducible technology platform, offering a high degree of automation, paired with user interaction at relevant points of the analytical workflow. This platform can be applied as a means of tissue cytometry for both immunofluorescence and immunohistochemistry. The TissueFAXS Cytometry platform can be used to determine e.g. the immune response in situ, measure proliferation, apoptosis, cytokine expression, signalling molecules, and others. It can do end-point assays as well as live-cell imaging and time-kinetic experiments. But TissueFAXS Cytometry also promotes tissue cytometry to a new level of quality, where complex cellular interactions can be addressed on the single-cell level but still in histological context.

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A cell in tissue plays a defined role as being a member of a small cellular subunit within a larger organic structure. The development and differentiation of a given cell is defined by the parent cell and the local cell neighborhood and environment. Consequently, the analysis of cells in their local environment is of major importance. At TissueGnostics we understand cells not just as a “bunch of biomolecules” but as ACTING ENTITIES that communicate and interact with each other – just like humans talk to and interact with each other. Consequently, we here introduce the concept of “Tissue Sociology”. While in “Sociology” researchers investigate the behavior of and interactions between individual human beings, state-of-the-art image cytometry software permits a similar approach on the single cell level! We can analyze locations of single cells in tissue context, perform “neighborhood analyses” and quantitate cellular interactions in various ways. Different cells might not be just next to each other “by chance”, but any cellular presence, absence, or distribution will have both a “specific cause and meaning”. From a conceptual and scientific point of view we invite you to evaluate and consider this novel path of research to better understanding cancer cells, their immunocompetent antagnosists, and the interactions between them! 88 Training does not improve physical abilities in P2Y6 deficient mice L.G. Castilhos1,3, A.M. Cardoso1,3, D.B.R. Leal1, J. Pelletier2 and J. Sévigny2,3 Departamento de Microbiologia e Parasitologia, Centro de Ciências da Saúde, Universidade Federal de Santa Maria, Santa Maria-RS, Brazil 2 Centre de Recherche du CHU de Québec, CHUL, Québec, QC, Canada G1V 4G2 3 Département de Microbiologie-Infectiologie et d’Immunologie, Faculté de Médecine, Université Laval, Québec, QC, Canada G1V 0A6. 1

Objectives/background: During chronic exercises, the adaptation processes change the purinergic system wich is related to performance improvement. Until this moment nothing has been reported in the literature about P2Y6 receptor deficiency and adaptation to exercise. However, the aim of this study was to investigate if nucleotide signaling is involved in exercise adaptation using P2Y6 deficient mice. Methods and results: Wild Type and P2ry6−/− mice were divided in four groups, WT-sedentary, WT-swimming, P2ry6−/− sedentary and P2ry6−/− swimming. Trained mice were submitted to swimming endurance exercise protocol during 4 weeks, 5 days/week. Body weight load was attachet to the mice tail (1-3 %) during swimming protocol. The exercise capacity in this groups was evaluated by 2 tests: performance test (exhaustion time during swimming) using 10 % bw load attached on the tail, and lactate test before swimm (basal level) and after 3 min swimming using 0, 4 and 8 % of body weight boad. The grip test was realized to evaluate the mice strength using a force transducer equipped with a metal bar. This study was approved on Centre de Recherche du CHU de Québec under protocol number 2012–159. The results obtained for performance test showed increase on swimming time for WT-sw group (19.11 min; SEM = 1.2; n = 8; p < 0.001) when compared to WT-sed (7.84 min; SEM = 0.8; n = 7; p < 0.001). In P2ry6−/− sw group the performance test was also increased (12.18 min; SEM = 0.9; n = 8; p < 0.01) when compared to P2ry6−/− sed group (5.85 min; SEM = 1.2; n = 7; p < 0.01) but significantly less then in WT-sw mice (19.11 min; SEM = 1.2; n = 8; p < 0.001). For the lactate test, the lactate level increased in both WT-sw (6.84 mmol/l; SEM = 0.4; n = 8; p < 0.05) and P2ry6−/− sw groups (6.82 mmol/l; SEM 0.3; n = 8; p < 0.05) when compared to respectives sedentary groups, WT-sed (9.20 mmol/l; SEM = 0.3; n = 7; p < 0.05) and P2ry6−/− sed (9.31 mg/dl; SEM = 0.7; n = 7; p < 0.05). For the grip test, the grip strength did not change among groups, WT-sed (7.92 g/g; SEM = 0.3; n = 7), WT-sw (8.53 g/g; SEM = 0.1; n = 8), P2ry6−/− sed (8.12 g/g; SEM = 0.1; n = 7), P2ry6−/− sw (8.12 g/g; SEM = 0.3; n = 8). Conclusion: The preliminary results suggest P2Y6 receptor is involved in exercise adaptations more specifically on swimming performance. Now we are looking for the mechanism that lead this deficiency in P2ry6−/− mice and, why lactate levels was not correlate with this deficiency.

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