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Innovation of cancer therapy has received a dramatic acceleration over the last fifteen years thanks to the introduction of the novel immune checkpoint inhibitors (ICI). On the other hand, the conspicuous scientific knowledge accumulated in purinergic signaling since the early seventies is finally being transferred to the clinic. Several Phase I/II clinical trials are currently underway to investigate the effect of drugs interfering with purinergic signaling as stand-alone or combination therapy in cancer. This is supporting the novel concept of "purinergic immune checkpoint" (PIC) in cancer therapy. In the present review we will address a) the basic pharmacology and cell biology of the purinergic system; b) principles of its pathophysiology in human diseases; c) implications for cell death, cell proliferation and cancer; d) novel molecular tools to investigate nucleotide homeostasis in the extracellular environment; e) recent developments in the pharmacology of P1, P2 receptors and related ecto-enzymes; f) P1 and P2 ligands as novel diagnostic tools; g) current issues in PIC-based anti-cancer therapy. This review will provide an appraisal of the current status of purinergic signaling in cancer and will help identify future avenues of development., Competing Interests: Declaration of competing interest F.D.V. is a member of the Scientific Advisory Board (SAB) of Biosceptre Ltd. (UK), and a consultant at Breye Therapeutics (Denmark), and at Crosslink Therapeutics Inc. (USA). Francesco Di Virgilio is a member of the Scientific Advisory Board (SAB) of Biosceptre Ltd. (UK), and a consultant at Breye Therapeutics (Denmark) and Crosslink Inc. (USA). The other Authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Microparticles (MPs) are secreted by all cells, where they play a key role in intercellular communication, differentiation, inflammation, and cell energy transfer. P2X7 receptor (P2X7R) activation by extracellular ATP (eATP) causes a large MP release and affects their contents in a cell-specific fashion. We investigated MP release and functional impact in microglial cells from P2X7R-WT or P2X7R-KO mice, as well as mouse microglial cell lines characterized for high (N13-P2X7RHigh) or low (N13-P2X7RLow) P2X7R expression. P2X7R stimulation promoted release of a mixed MP population enriched with naked mitochondria. Released mitochondria were taken up and incorporated into the mitochondrial network of the recipient cells in a P2X7R-dependent fashion. NLRP3 and the P2X7R itself were also delivered to the recipient cells. Microparticle transfer increased the energy level of the recipient cells and conferred a pro-inflammatory phenotype. These data show that the P2X7R is a master regulator of intercellular organelle and MP trafficking in immune cells., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Physiological Society.)

ATP is a ubiquitous extracellular messenger released in a wide number of pathophysiological conditions. ATP is known to be present in minute amounts in the extracellular space in healthy tissues and in the blood, and to modulate a multiplicity of cell responses. Cell culture systems are widely used to explore purinergic signaling. We show here that currently used fetal bovine sera contain ATP in the 300-1300 pmol/L range. Serum ATP is associated with albumin as well as with microparticle/microvesicle fraction. Serum microparticles/microvesicles affect in vitro cell responses due to their content of miRNAs, growth factors, and other bioactive molecules. ATP is likely to be one of these bioactive factors found in a variable amount in sera of different commercial sources. ATP in serum supports ATP-dependent biochemical reactions such as the hexokinase-dependent phosphorylation of glucose to glucose 6-phosphate, and affects purinergic signaling. These findings show that cells growing in vitro in serum-supplemented media are exposed to varying levels of extracellular ATP, and thus to varying degrees of purinergic stimulation., (© 2023. The Author(s).)

The discovery of the P2X7 receptor (P2X7R, originally named P2Z) in immune cells, its cloning, and the identification of its role in a multiplicity of immune-mediated diseases raised great hopes for the development of novel and more potent anti-inflammatory medicaments. Unfortunately, such hopes were partially deluded by the unsatisfactory results of most early clinical trials. This failure substantially reduced the interest of the pharmaceutical and biotech industries in the clinical development of P2X7R-targeted therapies. However, recent findings ushered in a second life for the P2X7R in diagnostic medicine. New P2X7R radioligands proved to be very reliable tools for the diagnosis of neuroinflammation in preclinical and clinical studies, and detection and measurement of free P2X7 receptor (or P2X7 subunit) in human blood suggested its potential use as a circulating marker of inflammation. Here we provide a brief review of these novel developments.

Introduction: The pathophysiology of the Corona Virus Disease 2019 (COVID-19) is incompletely known. A robust inflammatory response caused by viral replication is a main cause of the acute lung and multiorgan injury observed in critical patients. Inflammasomes are likely players in COVID-19 pathogenesis. The P2X7 receptor (P2X7R), a plasma membrane ATP-gated ion channel, is a main activator of the NLRP3 inflammasome, of the ensuing release of inflammatory cytokines and of cell death by pyroptosis. The P2X7R has been implicated in COVID-19-dependent hyperinflammation and in the associated multiorgan damage. Shed P2X7R (sP2X7R) and shed NLRP3 (sNLRP3) have been detected in plasma and other body fluids, especially during infection and inflammation., Methods: Blood samples from 96 patients with confirmed SARS-CoV-2 infection with various degrees of disease severity were tested at the time of diagnosis at hospital admission. Standard haematological parameters and IL-6, IL-10, IL-1β, sP2X7R and sNLRP3 levels were measured, compared to reference values, statistically validated, and correlated to clinical outcome., Results: Most COVID-19 patients included in this study had lymphopenia, eosinopenia, neutrophilia, increased inflammatory and coagulation indexes, and augmented sNLRP3, IL-6 and IL-10 levels. Blood concentration of sP2X7R was also increased, and significantly positively correlated with lymphopenia, procalcitonin (PCT), IL-10, and alanine transaminase (ALT). Patients with increased sP2X7R levels at diagnosis also showed fever and respiratory symptoms, were more often transferred to Pneumology division, required mechanical ventilation, and had a higher likelihood to die during hospitalization., Conclusion: Blood sP2X7R was elevated in the early phases of COVID-19 and predicted an adverse clinical outcome. It is suggested that sP2X7R might be a useful marker of disease progression., Competing Interests: Author FDV is a member of the Scientific Advisory Board of Biosceptre Ltd, a biotech Company involved in the development of anti-P2X7 antibodies, and a Consultant with Axxam SpA. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Vultaggio-Poma, Sanz, Amico, Violi, Ghisellini, Pizzicotti, Passaro, Papi, Libanore, Di Virgilio and Giuliani.)

Identifying the subcellular localization of a protein within a cell is often an essential step in understanding its function. The main objective of this report was to determine the presence of the P2X7 receptor (P2X7R) in healthy human cells of skeletal system, specifically osteoblasts (OBs), chondrocytes (Chs) and intervertebral disc (IVD) cells. This receptor is a member of the ATP-gated ion channel family, known to be a main sensor of extracellular ATP, the prototype of the danger signal released at sites of tissue damage, and a ubiquitous player in inflammation and cancer, including bone and cartilaginous tissues. Despite overwhelming data supporting a role in immune cell responses and tumor growth and progression, a complete picture of the pathophysiological functions of P2X7R, especially when expressed by non-immune cells, is lacking. Here we show that human wild-type P2X7R (P2X7A) was expressed in different samples of human osteoblasts, chondrocytes and intervertebral disc cells. By fluorescence microscopy (LM) and immunogold transmission electron microscopy we localized P2X7R not only in the canonical sites (plasma membrane and cytoplasm), but also in the nucleus of all the 3 cell types, especially IVD cells and OBs. P2X7R mitochondrial immunoreactivity was predominantly detected in OBs and IVD cells, but not in Chs. Evidence of subcellular localization of P2X7R may help to i. understand the participation of P2X7R in as yet unidentified signaling pathways in the joint and bone microenvironment, ii. identify pathologies associated with P2X7R mislocalization and iii. design specific targeted therapies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Penolazzi, Notarangelo, Lambertini, Vultaggio-Poma, Tarantini, Di Virgilio and Piva.)

Nucleotides play a crucial role in extracellular signaling across species boundaries. All the three kingdoms of life (Bacteria, Archea and Eukariota) are responsive to extracellular ATP (eATP) and many release this and other nucleotides. Thus, eATP fulfills different functions, many related to danger-sensing or avoidance reactions. Basically all living organisms have evolved sensors for eATP and other nucleotides with very different affinity and selectivity, thus conferring a remarkable plasticity to this signaling system. Likewise, different intracellular transduction systems were associated during evolution to different receptors for eATP. In mammalian evolution, control of intracellular ATP (iATP) and eATP homeostasis has been closely intertwined with that of Ca 2+ , whether in the extracellular milieu or in the cytoplasm, establishing an inverse reciprocal relationship, i.e. high extracellular Ca 2+ levels are associated to negligible eATP, while low intracellular Ca 2+ levels are associated to high eATP concentrations. This inverse relationship is crucial for the messenger functions of both molecules. Extracellular ATP is sensed by specific plasma membrane receptors of widely different affinity named P2 receptors (P2Rs) of which 17 subtypes are known. This confers a remarkable plasticity to P2R signaling. The central nervous system (CNS) is a privileged site for purinergic signaling as all brain cell types express P2Rs. Accruing evidence suggests that eATP, in addition to participating in synaptic transmission, also plays a crucial homeostatic role by fine tuning microglia, astroglia and oligodendroglia responses. Drugs modulating the eATP concentration in the CNS are likely to be the new frontier in the therapy of neuroinflammation. This article is part of the Special Issue on 'Purinergic Signaling: 50 years'., Competing Interests: Declaration of competing interest FDV is a member of the Scientific Advisory Board of Biosceptre Ltd, a biotech Company involved in the development of anti-P2X7 antibodies, and a Consultant with Axxam SpA. The other authors declare no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Chagas disease represents a major public health concern in several Latin American countries, including Bolivia., Methods: We present a longitudinal serosurvey for Trypanosoma cruzi antibodies among a cohort of 120 school-age children from rural communities in the Bolivian Chaco at three time points between 2017 and 2019. Serum samples extracted from dry blood spots collected on filter paper were tested for T. cruzi antibodies by enzyme-linked immunosorbent assay and rapid diagnostic test., Results: T. cruzi antibodies were detected in 7/120 (5.8%), 8/120 (6.7%) and 11/120 (9.2%) samples in 2017, 2018 and 2019, respectively. An average incidence of 1.76 per 100 person-years was observed., Conclusions: Our findings support the persistence of vector-borne T. cruzi transmission in this area, highlighting the need for strengthening multidisciplinary efforts against Chagas disease., (© The Author(s) 2022. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.)

The Bolivian Chaco is a semiarid region with a low population density, situated in the southeast part of the Plurinational State of Bolivia. Here, despite the improvements of the last 15 years, poverty remains high in rural areas, where social vulnerability is widespread. The Guaraní ethnic group often lives in isolated communities with a low standard of hygiene and sanitation. This epidemiological scenario favors the spread of transmissible diseases, including several parasitic infections belonging to the neglected tropical diseases (NTDs) group. In this area, a long-standing research activity, built upon the synergism between local and foreign institutions, has been established since the late 1980s and helps to fill in the knowledge gap about the epidemiology dynamics of soil-transmitted helminths, vector-borne parasites, and other parasitic diseases. A 35-year history of cooperation programs in parasitology research has contributed to informing local health authorities of the NTD burden in the Bolivian Chaco and, ultimately, supports local healthcare providers in the management of parasitic diseases.

Objectives: The aim of this study was to perform two cross-sectional surveys on the fecal carriage of CTX-M-producing Enterobacterales in school-aged children from rural areas of the Bolivian Chaco (2016 vs 2019)., Methods: A total of 757 fecal samples were collected from school-aged children living in nine indigenous communities (n=337, 2016; n=420, 2019). After a first passage onto MacConkey agar (MCA), samples were plated onto MCA plus cefotaxime 2 µg/mL (MCA-CTX), and a loopful of the bacterial growth was used as a template for the detection of group 1, 2, 8/25, and 9 bla CTX-M variants by multiplex reverse transcriptase polymerase chain reaction . Positive samples were tested again for detecting, identifying, and characterizing CTX-M-positive isolates., Results: Growth onto MCA-CTX was obtained with 208 samples (27.5%; 62/337, 2016; 146/420, 2019), of which 201 (96.6%) were positive for bla CTX-M genes. Overall, a relevant increase of fecal carriage of CTX-M-producing Enterobacterales was observed in the study period: 17,5% (59/337) in 2016 compared with 33,8% (142/420) in 2019, p<0.01. Nonetheless, the relative group distribution of CTX-M groups remained stable, with group 1 being the prevalent, followed by group 9 and group 8/25. Group 2 was not detected., Conclusions: The present study demonstrated an alarming spread of CTX-M enzymes in rural areas of the Bolivian Chaco, where antibiotics consumption is limited. Further studies are encouraged to better understand the dissemination dynamics of such relevant resistance determinants., Competing Interests: Declaration of Competing Interest The authors have no competing interests to declare., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Extracellular ATP (eATP) and P2 receptors are novel emerging regulators of T-lymphocyte responses. Cellular ATP is released via multiple pathways and accumulates at sites of tissue damage and inflammation. P2 receptor expression and function are affected by numerous single nucleotide polymorphisms (SNPs) associated with diverse disease conditions. Stimulation by released nucleotides (purinergic signalling) modulates several T-lymphocyte functions, among which energy metabolism. Energy metabolism, whether oxidative or glycolytic, in turn deeply affects T-cell activation, differentiation and effector responses. Specific P2R subtypes, among which the P2X7 receptor (P2X7R), are either up- or down-regulated during T-cell activation and differentiation; thus, they can be considered indexes of activation/quiescence, reporters of T-cell metabolic status and, in principle, markers of immune-mediated disease conditions.

Nucleotides are released from all cells through regulated pathways or as a result of plasma membrane damage or cell death. Outside the cell, nucleotides act as signalling molecules triggering multiple responses via specific plasma membrane receptors of the P2 family. In the nervous system, purinergic signalling has a key function in neurotransmission. Outside the nervous system, purinergic signalling is one of the major modulators of basal tissue homeostasis, while its dysregulation contributes to the pathogenesis of various disease, including inflammation and cancer. Pre-clinical and clinical evidence shows that selective P2 agonists or antagonists are effective treatments for many pathologies, thus highlighting the relevance of extracellular nucleotides and P2 receptors as therapeutic targets., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Adenosine triphosphate (ATP) is the key energy intermediate of cellular metabolic processes and a ubiquitous extracellular messenger. As an extracellular messenger, ATP acts at plasma membrane P2 receptors (P2Rs). The levels of extracellular ATP (eATP) are set by both passive and active release mechanisms and degradation processes. Under physiological conditions, eATP concentration is in the low nanomolar range but can rise to tens or even hundreds of micromoles/L at inflammatory sites. A dysregulated eATP homeostasis is a pathogenic factor in several chronic inflammatory diseases, including type 2 diabetes mellitus (T2DM). T2DM is characterized by peripheral insulin resistance and impairment of insulin production from pancreatic β-cells in a landscape of systemic inflammation. Although various hypoglycemic drugs are currently available, an effective treatment for T2DM and its complications is not available. However, counteracting systemic inflammation is anticipated to be beneficial. The postulated eATP increase in T2DM is understood to be a driver of inflammation via P2X7 receptor (P2X7R) activation and the release of inflammatory cytokines. Furthermore, P2X7R stimulation is thought to trigger apoptosis of pancreatic β-cells, thus further aggravating hyperglycemia. Targeting eATP and the P2X7R might be an appealing novel approach to T2DM therapy.

Rationale : Caloric restriction improves the efficacy of anti-cancer therapy. This effect is largely dependent on the increase of the extracellular ATP concentration in the tumor microenvironment (TME). Pathways for ATP release triggered by nutrient deprivation are largely unknown. Methods: The extracellular ATP (eATP) concentration was in vivo measured in the tumor microenvironment of B16F10-inoculated C57Bl/6 mice with the pmeLuc probe. Alternatively, the pmeLuc-TG-mouse was used. Caloric restriction was in vivo induced with hydroxycitrate (HC). B16F10 melanoma cells or CT26 colon carcinoma cells were in vitro exposed to serum starvation to mimic nutrient deprivation. Energy metabolism was monitored by Seahorse. Microparticle release was measured by ultracentrifugation and by Nanosight. Results : Nutrient deprivation increases eATP release despite the dramatic inhibition of intracellular energy synthesis. Under these conditions oxidative phosphorylation was dramatically impaired, mitochondria fragmented and glycolysis and lactic acid release were enhanced. Nutrient deprivation stimulated a P2X7-dependent release of ATP-loaded, mitochondria-containing, microparticles as well as of naked mitochondria. Conclusions : Nutrient deprivation promotes a striking accumulation of eATP paralleled by a large release of ATP-laden microparticles and of naked mitochondria. This is likely to be a main mechanism driving the accumulation of eATP into the TME., Competing Interests: Competing Interests: FDV is a member of the Scientific Advisory Board of Biosceptre Ltd, a biotech Company involved in the development of anti-P2X7 antibodies, and a consultant with Axxam SpA. The other authors declare no conflict of interest., (© The author(s).)

For many years the P2X7 receptor (P2X7R) was considered the prototypic cytolytic receptor due to its ability to cause dramatic changes in plasma membrane permeability, eventually leading to cell death. However, later studies revealed that controlled P2X7R activation has beneficial effects on cell metabolism and nowadays our perception of the physiological role of this receptor has radically changed. Some of the biochemical pathways underlying the trophic effect of the P2X7R are being unveiled, thus disclosing an unanticipated role of P2X7Rs in mitochondrial and glycolytic metabolism. We provide here an update of the effects of the P2X7R on cell energy metabolism., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Chronic Chagas cardiomyopathy (CChC) is the most common cause of death related to Chagas disease (CD). The aim of this study was to assess the feasibility of a combined rapid diagnostic test (RDT) and electrocardiographic (ECG) screening in a remote rural village of the Bolivian Chaco, with a high prevalence of CChC., Methods: Consecutive healthy volunteers > 15 years were enrolled in the community of Palmarito (municipality of Gutierrez, Santa Cruz Department, Bolivia) in February 2019. All patients performed an RDT with Chagas Stat-Pak ® (CSP, Chembio Diagnostic System, Medford, NY, USA) and an ECG by D-Heart ® technology, a low-cost, user-friendly smartphone-based 8-lead Bluetooth ECG. RDTs were read locally while ECGs were sent to a cardiology clinic which transmitted reports within 24 h from recording., Results: Among 140 people (54 men, median age 38(interquartile range 23-54) years), 98 (70%) were positive for Trypanosoma cruzi infection, with a linear, age-dependent, increasing trend ( p < 0.001). Twenty-five (18%) individuals showed ECG abnormalities compatible with CD. Prevalence of ECG abnormalities was higher in infected individuals and was associated with higher systolic blood pressure and smoking. Following screening, 22 (16%) individuals underwent clinical evaluation and chest X-ray and two were referred for further evaluation. At multivariate analysis, positive CSP results (OR = 4.75, 95%CI 1.08-20.96, p = 0.039) and smoking (OR = 4.20, 95%CI 1.18-14.92, p = 0.027) were independent predictors of ECG abnormalities. Overall cost for screening implementation was <10 $., Conclusions: Combined mobile-Health and RDTs was a reliable and effective low-cost strategy to identify patients at high risk of disease needing cardiologic assessment suggesting potential future applications.

We performed a cross-sectional parasitological survey to assess the prevalence of protozoa and helminth infection among 275 school-age children (SAC) living in rural and peri-urban areas located in different ecological zones of the Department of La Paz, Bolivia. Public health activities for the control of STH, based on the biannual administration of mebendazole to preschool and school children are implemented at national level since 1986. We found an overall prevalence of 82.2% for intestinal parasites, including protozoa (80%) and helminths (23.3%). Blastocystis and Entamoeba coli were the most prevalent protozoa (found in 44% and 20.7% of the SAC enrolled); as for helminths, Ascaris lumbricoides and Hymenolepis nana were diagnosed in 14.5% and 3.3% of the children, respectively, followed by Trichuris trichiura 1.4%, Enterobius vermicularis 1.4%, Strongyloides stercoralis 0.7% and hookworms 0.7%. Molecular characterization of Blastocystis positive samples evidenced three different subtypes (ST1, ST2, ST3) highlighting the risk of transmission also from animal reservoir. We found a significant difference in the distribution of intestinal parasitic infection (IPIs) by ecological zone (44/74. 59% in Andean highlands, 94/170, 88% in tropical lowlands and 88/94, 94% in the Yungas, p  < 0.001). Access to potable water (OR 0.1 95%CI 0.02-0.5, p  = 0.004) and the habit of boiling drinking water (OR 0.3, 95% CI 0.2-0.7, p  = 0.004) showed an independent association with a lower risk of all IPIs and STHs, respectively. The very low prevalence of STH infections of moderate heavy intensity demonstrate that periodical deworming has been successful in reducing the morbidity due to these parasites, however the high prevalence of protozoa demonstrate that sanitation is still problematic and there is a relevant contamination of the environment with human faeces. Significant efforts are still needed to reduce IPIs transmission and to improve health and sanitation in this area., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

It is increasingly appreciated that ion channels have a crucial role in tumors, either as promoters of cancer cell growth, or modulators of immune cell functions, or both. Among ion channels, P2X receptors have a special status because they are gated by ATP, a common and abundant component of the tumor microenvironment. Furthermore, one P2X receptor, i.e. P2X7, may also function as a conduit for ATP release, thus fuelling the increased extracellular ATP level in the tumor interstitium. These findings show that P2X receptors and extracellular ATP are indissoluble partners and key regulators of tumor growth, and suggest the exploitation of the extracellular ATP-P2X partnership to develop innovative therapeutic approaches to cancer., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Basal expression of the P2X7 receptor (P2X7R) improves mitochondrial metabolism, Adenosine 5'-triphosphate (ATP) synthesis, and overall fitness of immune and non-immune cells. We investigated P2X7R contribution to energy metabolism and subcellular localization in fibroblasts (mouse embryo fibroblasts and HEK293 human fibroblasts), mouse microglia (primary brain microglia, and the N13 microglia cell line), and heart tissue. The P2X7R localizes to mitochondria, and its lack (1) decreases basal respiratory rate, ATP-coupled respiration, maximal uncoupled respiration, resting mitochondrial potential, mitochondrial matrix Ca 2+ level, (2) modifies expression pattern of oxidative phosphorylation enzymes, and (3) severely affects cardiac performance. Hearts from P2rx7 -deleted versus wild-type mice are larger, heart mitochondria smaller, and stroke volume, ejection fraction, fractional shortening, and cardiac output, are significantly decreased. Accordingly, the physical fitness of P2X7R-null mice is severely reduced. Thus, the P2X7R is a key modulator of mitochondrial energy metabolism and a determinant of physical fitness., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Physiological Society.)

Adenosine triphosphate (ATP) is one of the main biochemical components of the tumor microenvironment (TME), where it can promote tumor progression or tumor suppression depending on its concentration and on the specific ecto-nucleotidases and receptors expressed by immune and cancer cells. ATP can be released from cells via both specific and nonspecific pathways. A non-regulated release occurs from dying and damaged cells, whereas active release involves exocytotic granules, plasma membrane-derived microvesicles, specific ATP-binding cassette (ABC) transporters and membrane channels (connexin hemichannels, pannexin 1 (PANX1), calcium homeostasis modulator 1 (CALHM1), volume-regulated anion channels (VRACs) and maxi-anion channels (MACs)). Extracellular ATP acts at P2 purinergic receptors, among which P2X7R is a key mediator of the final ATP-dependent biological effects. Over the years, P2 receptor- or ecto-nucleotidase-targeting for cancer therapy has been proposed and actively investigated, while comparatively fewer studies have explored the suitability of TME ATP as a target. In this review, we briefly summarize the available evidence suggesting that TME ATP has a central role in determining tumor fate and is, therefore, a suitable target for cancer therapy.

Previous data from our laboratory show that expression of the P2X7 receptor (P2X7R) is needed for amyloid β (Aβ)-stimulated microglia activation and IL-1β release in vitro and in vivo. We also showed that Aβ-dependent stimulation is inhibited by the dihydropyridine nimodipine at an intracellular site distal to the P2X7R. In the present study, we used the N13 microglia cell line and mouse primary microglia from wt and P2rx7-deleted mice to test the effect of nimodipine on amyloid β (Aβ)-dependent NLRP3 inflammasome expression and function, and on mitochondrial energy metabolism. Our data show that in microglia Aβ causes P2X7R-dependent a) NFκB activation; b) NLRP3 inflammasome expression and function; c) mitochondria toxicity; and these changes are fully inhibited by nimodipine. Our study shows that nimodipine is a powerful blocker of cell damage caused by monomeric and oligomeric Aβ, points to the mitochondria as a crucial target, and underlines the permissive role of the P2X7R.

The P2X7 receptor (P2X7R) is a key pro-inflammatory plasma membrane receptor responsible for NLRP3 inflammasome activation and IL-1β release. Various inflammatory plasma membrane receptors (e.g., IL-1 type I receptor, TNF type I and II receptors, IL-2 receptor) are shed under different pathophysiological conditions. In the present study, we show that the full length P2X7R is released into circulation in patients as well as in healthy subjects. Blood levels of shed P2X7R (sP2X7R) correlate to those of the inflammatory marker C reactive protein (CRP). Blood sP2X7R ranged from 16.74 to 82.17 ng/L, mean ± SE 40.97 ± 3.82 ( n = 26) in healthy subjects, from 33.1 to 484.0 ng/L, mean ± SE 114.78 ± 12.22 ( n = 45) in patients with CRP <3 mg/L, and from 63.65 to 1092.3 ng/L, mean ± SE 204.2 ± 30.94 ( n = 42) in patients with CRP >3 mg/L. sP2X7R in plasma was largely associated to microvesicles/microparticles. Peripheral blood monocytes from healthy subjects released sP2X7R upon stimulation with the semi-selective P2X7R agonist benzoyl ATP. These data show that the P2X7R can be released into circulation, and that its blood levels increase in various disease conditions.

Water borne diarrheal pathogens might accumulate in river water and cause contamination of drinking and irrigation water. The La Paz River basin, including the Choqueyapu River, flows through La Paz city in Bolivia where it is receiving sewage, and residues from inhabitants, hospitals, and industry. Using quantitative real-time PCR (qPCR), we determined the quantity and occurrence of diarrheagenic Escherichia coli (DEC), Salmonella enterica, Klebsiella pneumoniae, Shigella spp. and total enterobacteria in river water, downstream agricultural soil, and irrigated crops, during one year of sampling. The most abundant and frequently detected genes were gapA and eltB, indicating presence of enterobacteria and enterotoxigenic E. coli (ETEC) carrying the heat labile toxin, respectively. Pathogen levels in the samples were significantly positively associated with high water conductivity and low water temperature. In addition, a set of bacterial isolates from water, soil and crops were analyzed by PCR for presence of the genes blaCTX-M, blaKPC, blaNDM, blaVIM and blaOXA-48. Four isolates were found to be positive for blaCTX-M genes and whole genome sequencing identified them as E. coli and one Enterobacter cloacae. The E. coli isolates belonged to the emerging, globally disseminated, multi-resistant E. coli lineages ST648, ST410 and ST162. The results indicate not only a high potential risk of transmission of diarrheal diseases by the consumption of contaminated water and vegetables but also the possibility of antibiotic resistance transfer from the environment to the community., Competing Interests: The authors have declared that no competing interests exist.

The P2X7 receptor [P2X7R or P2RX7 in National Center for Biotechnology Information (NCBI) gene nomenclature] is a member of the P2X receptor (P2XR) subfamily of P2 receptors (P2Rs). The P2X7R is an extracellular ATP-gated ion channel with peculiar permeability properties expressed by most cell types, mainly in the immune system, where it has a leading role in cytokine release, oxygen radical generation, T lymphocyte differentiation and proliferation. A role in cancer cell growth and tumor progression has also been demonstrated. These features make the P2X7R an appealing target for drug development in inflammation and cancer. The functional P2X7R, recently (partially) crystallized and 3-D solved, is formed by the assembly of three identical subunits (homotrimer). The P2X7R is preferentially permeable to small cations (Ca 2+ , Na + , K + ), and in most (but not all) cell types also to large positively charged molecules of molecular mass up to 900Da. Permeability to negatively charged species of comparable molecular mass (e.g., Lucifer yellow) is debated. Several highly selective P2X7R pharmacological blockers have been developed over the years, thus providing powerful tools for P2X7R studies. Biophysical properties and coupling to several different physiological responses make the P2X7R amenable to investigation by electrophysiology and cell biology techniques, which allow its identification and characterization in many different cell types and tissues. A careful description of the physiological features of the P2X7R is a prerequisite for an effective therapeutic development. Here we describe the most common techniques to asses P2X7R functions, including patch-clamp, intracellular calcium measurements, and membrane permeabilization to large fluorescent dyes in a selection of different cell types. In addition, we also describe common toxicity assays used to verify the effects of P2X7R stimulation on cell viability., (© 2019 Elsevier Inc. All rights reserved.)

The P2X7 receptor (P2X7R) is a ligand-gated plasma membrane ion channel belonging to the P2X receptor subfamily activated by extracellular nucleotides. General consensus holds that the physiological (and maybe the only) agonist is ATP. However, scattered evidence generated over the last several years suggests that ATP might not be the only agonist, especially at inflammatory sites. Solid data show that NAD + covalently modifies the P2X7R of mouse T lymphocytes, thus lowering the ATP threshold for activation. Other structurally unrelated agents have been reported to activate the P2X7R via a poorly understood mechanism of action: (a) the antibiotic polymyxin B, possibly a positive allosteric P2X7R modulator, (b) the bactericidal peptide LL-37, (c) the amyloidogenic β peptide, and (d) serum amyloid A. Some agents, such as Alu-RNA, have been suggested to activate the P2X7R acting on the intracellular N- or C-terminal domains. Mode of P2X7R activation by these non-nucleotide ligands is as yet unknown; however, these observations raise the intriguing question of how these different non-nucleotide ligands may co-operate with ATP at inflammatory or tumor sites. New information obtained from the cloning and characterization of the P2X7R from exotic mammalian species (e.g., giant panda) and data from recent patch-clamp studies are strongly accelerating our understanding of P2X7R mode of operation, and may provide hints to the mechanism of activation of P2X7R by non-nucleotide ligands.

La Paz River in Andean highlands is heavily polluted with urban run-off and further contaminates agricultural lowlands and downstream waters at the Amazon watershed. Agricultural produce at this region is the main source of vegetables for the major Andean cities of La Paz and El Alto. We conducted a 1 year study, to evaluate microbial quality parameters and occurrence of multiple enteropathogenic bacteria (Enterohemorrhagic E. coli-EHEC, Enteroinvasive E. coli or Shigella-EIEC/Shigella, Enteroaggregative E. coli-EAEC, Enteropathogenic E. coli-EPEC Enterotoxigenic E. coli-ETEC and Salmonella) and its resistance to 11 antibiotics. Four sampling locations were selected: a fresh mountain water reservoir (un-impacted, site 1) and downstream sites receiving wastewater discharges (impacted, sites 2-4). River water (sites 1-4, N = 48), and soil and vegetable samples (site 3, N = 24) were collected during dry (April-September) and rainy seasons (October-March). Throughout the study, thermotolerant coliform density values at impacted sites greatly exceeded the guidelines for recreational and agricultural water uses. Seasonal differences were found for thermotolerant coliform density during dry season in water samples nearby a populated and hospital compound area. In contrast to the un-impacted site, where none of the tested enteropathogens were found, 100 % of surface water, 83 % of soil and 67 % of vegetable samples at impacted sites, were contaminated with at least one enteropathogen, being ETEC and Salmonella the most frequently found. ETEC isolates displayed different patterns of toxin genes among sites. The occurrence of enteropathogens was associated with the thermotolerant coliform density. At impacted sites, multiple enteropathogens were frequently found during rainy season. Among isolated enteropathogens, 50 % were resistant to at least two antibiotics, with resistance to ampicillin, nalidixic acid, trimethoprim-sulfamethoxazole and tetracycline commonly present. Moreover, some Salmonella isolates were distinguished by their multi-resistance to ≥8 antibiotics, within soil and vegetable samples. Overall, this study demonstrates that La Paz River-an affluent of the Amazon macrobasin-is heavily polluted along the year with a high density of thermotolerant coliforms and is a reservoir of multiple antibiotic resistant enteropathogens, present in river water, soil and vegetables. These data highlight health risk associated with food and waterborne diseases at the region.

In the sea urchin embryo, primary mesenchyme cells (PMCs) are committed early in development to direct skeletogenesis, provided that a permissive signal is conveyed from adjacent ectoderm cells. We showed that inhibition of extracellular matrix (ECM)-ectoderm cells interaction, by monoclonal antibodies (mAb) to Pl-nectin, causes an impairment of skeletogenesis and reduced expression of Pl-SM30, a spicule-specific matrix protein. When PMCs are experimentally removed, some secondary mesenchyme cells (SMCs) switch to skeletogenic fate. Here, for the first time we studied SMC transfating in PMC-less embryos of Paracentrotus lividus. We observed the appearance of skeletogenic cells within 10 h of PMCs removal, as shown by binding of wheat germ agglutinin (WGA) to cell surface molecules unique to PMCs. Interestingly, the number of WGA-positive cells, expressing also msp130, another PMC-specific marker, doubled with respect to that of PMCs present in normal embryos, though the number of SM30-expressing cells remained constant. In addition, we investigated the ability of SMCs to direct skeletogenesis in embryos exposed to mAbs to Pl-nectin after removal of PMCs. We found that, although phenotypic SMC transfating occurred, spicule development, as well as Pl-SM30-expression was strongly inhibited. These results demonstrate that ectoderm inductive signals are necessary for transfated SMCs to express genes needed for skeletogenesis.

Ozone depletion results in an increased flux of biologically damaging radiations reaching the earth. Although ultraviolet (UV) penetration is attenuated by the seawater, harmful effects can be still observed at low depths where sea urchin embryos are living. We have used Paracentrotus lividus embryos to study the impacts of UV radiation on their development. Blastula cultures were exposed to different doses of UVB (312 nm) radiations and the resulting endpoint effects were evaluated in terms of embryonic morphological abnormalities, variations in specific gene expression, and changes in the levels of stress proteins. We found that embryos were moderately sensitive to 50 J/m2 UVB radiation; an increase in the number of developmentally delayed and malformed embryos was detected when increasing doses, up to 1000 J/m2, were used. Major developmental defects, observed 24 and 48 h after exposure, consisted in the failure of skeleton elongation and patterning. Accordingly, we found a reduction in the number of primary mesenchyme cells that expressed Pl-SM30, a gene coding for one of the specific matrix proteins of the skeleton. The morphological effects observed 1, 24, and 48 h after exposure were correlated with a dose-dependent increase in the level and in the activation of two recognized stress markers, namely hsp70 and p38 MAPk, respectively, consistent with their role in mediating cellular response to stress and suggesting a function in embryo survival.

Pl-nectin is an ECM protein located on the apical surface of ectoderm cells of Paracentrotus lividus sea urchin embryo. Inhibition of ECM-ectoderm cell interaction by the addition of McAb to Pl-nectin to the culture causes a dramatic impairment of skeletogenesis, offering a good model for the study of factor(s) involved in skeleton elongation and patterning. We showed that skeleton deficiency was not due to a reduction in the number of PMCs ingressing the blastocoel, but it was correlated with a reduction in the number of Pl-SM30-expressing PMCs. Here, we provide evidence on the involvement of growth factor(s) in skeleton morphogenesis. Skeleton-defective embryos showed a strong reduction in the levels of expression of Pl-univin, a growth factor of the TGF-beta superfamily, which was correlated with an equivalent strong reduction in the levels of Pl-SM30. In contrast, expression levels of Pl-BMP5-7 remained low and constant in both skeleton-defective and normal embryos. Microinjection of horse serum in the blastocoelic cavity of embryos cultured in the presence of the antibody rescued skeleton development. Finally, we found that misexpression of univin is also sufficient to rescue defects in skeleton elongation and SM30 expression caused by McAb to Pl-nectin, suggesting a key role for univin or closely related factor in sea urchin skeleton morphogenesis.