P2X7 Receptor Function in Bone-Related Cancer

Modulation of tumor microenvironment by different mediators is central in determining neoplastic formation and progression. Among these molecules extracellular ATP is emerging as a good candidate in promoting cell growth, neovascularization, tumor-host interactions, and metastatization. This paper summarizes recent findings on expression and function of P2X7 receptor for extracellular ATP in primary and metastatic bone cancers. Search of mRNA expression microchip databases and literature analysis demonstrate a high expression of P2X7 in primary bone tumors as well as in other malignancies such as multiple myeloma, neuroblastoma, breast, and prostate cancer. Evidence that P2X7 triggers NFATc1, PI3K/Akt, ROCK, and VEGF pathways in osteoblasts promoting either primary tumor development or osteoblastic lesions is also reported. Moreover, P2X7 receptor is involved in osteoclast differentiation, RANKL expression, matrix metalloproteases and cathepsin secretion thus promoting bone resorption and osteolytic lesions. Taken together these data point to a pivotal role for the P2X7 receptor in bone cancer biology

The shed P2X7 receptor is an index of adverse clinical outcome in COVID-19 patients

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

Extracellular ATP is increased by release of ATP-loaded microparticles triggered by nutrient deprivation

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

GATEKEEPER’s Strategy for the Multinational Large-Scale Piloting of an eHealth Platform: Tutorial on How to Identify Relevant Settings and Use Cases

Background: The World Health Organization’s strategy toward healthy aging fosters person-centered integrated care sustained by eHealth systems. However, there is a need for standardized frameworks or platforms accommodating and interconnecting multiple of these systems while ensuring secure, relevant, fair, trust-based data sharing and use. The H2020 project GATEKEEPER aims to implement and test an open-source, European, standard-based, interoperable, and secure framework serving broad populations of aging citizens with heterogeneous health needs. Objective: We aim to describe the rationale for the selection of an optimal group of settings for the multinational large-scale piloting of the GATEKEEPER platform. Methods: The selection of implementation sites and reference use cases (RUCs) was based on the adoption of a double stratification pyramid reflecting the overall health of target populations and the intensity of proposed interventions; the identification of a principles guiding implementation site selection; and the elaboration of guidelines for RUC selection, ensuring clinical relevance and scientific excellence while covering the whole spectrum of citizen complexities and intervention intensities. Results: Seven European countries were selected, covering Europe’s geographical and socioeconomic heterogeneity: Cyprus, Germany, Greece, Italy, Poland, Spain, and the United Kingdom. These were complemented by the following 3 Asian pilots: Hong Kong, Singapore, and Taiwan. Implementation sites consisted of local ecosystems, including health care organizations and partners from industry, civil society, academia, and government, prioritizing the highly rated European Innovation Partnership on Active and Healthy Aging reference sites. RUCs covered the whole spectrum of chronic diseases, citizen complexities, and intervention intensities while privileging clinical relevance and scientific rigor. These included lifestyle-related early detection and interventions, using artificial intelligence–based digital coaches to promote healthy lifestyle and delay the onset or worsening of chronic diseases in healthy citizens; chronic obstructive pulmonary disease and heart failure decompensations management, proposing integrated care management based on advanced wearable monitoring and machine learning (ML) to predict decompensations; management of glycemic status in diabetes mellitus, based on beat to beat monitoring and short-term ML-based prediction of glycemic dynamics; treatment decision support systems for Parkinson disease, continuously monitoring motor and nonmotor complications to trigger enhanced treatment strategies; primary and secondary stroke prevention, using a coaching app and educational simulations with virtual and augmented reality; management of multimorbid older patients or patients with cancer, exploring novel chronic care models based on digital coaching, and advanced monitoring and ML; high blood pressure management, with ML-based predictions based on different intensities of monitoring through self-managed apps; and COVID-19 management, with integrated management tools limiting physical contact among actors. Conclusions: This paper provides a methodology for selecting adequate settings for the large-scale piloting of eHealth frameworks and exemplifies with the decisions taken in GATEKEEPER the current views of the WHO and European Commission while moving forward toward a European Data Space

Correction to: Two years later: Is the SARS-CoV-2 pandemic still having an impact on emergency surgery? An international cross-sectional survey among WSES members

Background: The SARS-CoV-2 pandemic is still ongoing and a major challenge for health care services worldwide. In the first WSES COVID-19 emergency surgery survey, a strong negative impact on emergency surgery (ES) had been described already early in the pandemic situation. However, the knowledge is limited about current effects of the pandemic on patient flow through emergency rooms, daily routine and decision making in ES as well as their changes over time during the last two pandemic years. This second WSES COVID-19 emergency surgery survey investigates the impact of the SARS-CoV-2 pandemic on ES during the course of the pandemic. Methods: A web survey had been distributed to medical specialists in ES during a four-week period from January 2022, investigating the impact of the pandemic on patients and septic diseases both requiring ES, structural problems due to the pandemic and time-to-intervention in ES routine. Results: 367 collaborators from 59 countries responded to the survey. The majority indicated that the pandemic still significantly impacts on treatment and outcome of surgical emergency patients (83.1% and 78.5%, respectively). As reasons, the collaborators reported decreased case load in ES (44.7%), but patients presenting with more prolonged and severe diseases, especially concerning perforated appendicitis (62.1%) and diverticulitis (57.5%). Otherwise, approximately 50% of the participants still observe a delay in time-to-intervention in ES compared with the situation before the pandemic. Relevant causes leading to enlarged time-to-intervention in ES during the pandemic are persistent problems with in-hospital logistics, lacks in medical staff as well as operating room and intensive care capacities during the pandemic. This leads not only to the need for triage or transferring of ES patients to other hospitals, reported by 64.0% and 48.8% of the collaborators, respectively, but also to paradigm shifts in treatment modalities to non-operative approaches reported by 67.3% of the participants, especially in uncomplicated appendicitis, cholecystitis and multiple-recurrent diverticulitis. Conclusions: The SARS-CoV-2 pandemic still significantly impacts on care and outcome of patients in ES. Well-known problems with in-hospital logistics are not sufficiently resolved by now; however, medical staff shortages and reduced capacities have been dramatically aggravated over last two pandemic years

Colorectal Cancer Stage at Diagnosis Before vs During the COVID-19 Pandemic in Italy

IMPORTANCE Delays in screening programs and the reluctance of patients to seek medical attention because of the outbreak of SARS-CoV-2 could be associated with the risk of more advanced colorectal cancers at diagnosis. OBJECTIVE To evaluate whether the SARS-CoV-2 pandemic was associated with more advanced oncologic stage and change in clinical presentation for patients with colorectal cancer. DESIGN, SETTING, AND PARTICIPANTS This retrospective, multicenter cohort study included all 17 938 adult patients who underwent surgery for colorectal cancer from March 1, 2020, to December 31, 2021 (pandemic period), and from January 1, 2018, to February 29, 2020 (prepandemic period), in 81 participating centers in Italy, including tertiary centers and community hospitals. Follow-up was 30 days from surgery. EXPOSURES Any type of surgical procedure for colorectal cancer, including explorative surgery, palliative procedures, and atypical or segmental resections. MAIN OUTCOMES AND MEASURES The primary outcome was advanced stage of colorectal cancer at diagnosis. Secondary outcomes were distant metastasis, T4 stage, aggressive biology (defined as cancer with at least 1 of the following characteristics: signet ring cells, mucinous tumor, budding, lymphovascular invasion, perineural invasion, and lymphangitis), stenotic lesion, emergency surgery, and palliative surgery. The independent association between the pandemic period and the outcomes was assessed using multivariate random-effects logistic regression, with hospital as the cluster variable. RESULTS A total of 17 938 patients (10 007 men [55.8%]; mean [SD] age, 70.6 [12.2] years) underwent surgery for colorectal cancer: 7796 (43.5%) during the pandemic period and 10 142 (56.5%) during the prepandemic period. Logistic regression indicated that the pandemic period was significantly associated with an increased rate of advanced-stage colorectal cancer (odds ratio [OR], 1.07; 95%CI, 1.01-1.13; P = .03), aggressive biology (OR, 1.32; 95%CI, 1.15-1.53; P < .001), and stenotic lesions (OR, 1.15; 95%CI, 1.01-1.31; P = .03). CONCLUSIONS AND RELEVANCE This cohort study suggests a significant association between the SARS-CoV-2 pandemic and the risk of a more advanced oncologic stage at diagnosis among patients undergoing surgery for colorectal cancer and might indicate a potential reduction of survival for these patients

P2X7 receptor orchestrates multiple signalling pathways triggering inflammation, autophagy and metabolic/trophic responses

P2X7 receptor is an ion channel activated by extracellular adenosine trisphosphate (eATP) that attracted increasing attention for its role in immune reactions, neurobiology and oncology. As receptor for an extracellular ligand, P2X7 activates a series of intracellular signalling pathways mainly via alterations of the ion permeability, but also through formation of a large unselective pore and direct interaction with other proteins. Here we wish to give an overview on the main biochemical paths initiated by P2X7 activation by revising recent and established literature on P2X7-triggered signalling cascades leading to cell death, inflammatory and immune response activation, proliferation and metabolism modulation. We will focus on the well-known P2X7 inflammasome/NF-kB and pro-apoptotic networks but also cover P2X7-activated emerging autophagic, pyroptotic and proliferative-oncogenic pathways, like beclin-1/LC3-II, caspase-11, Akt and VEGF axes

Purinergic signalling in autoimmunity: A role for the P2X7R in systemic lupus erythematosus?

Purinergic signalling plays a crucial role in immunity and autoimmunity. Among purinergic receptors, the P2X7 receptor (P2X7R) has an undisputed role as it is expressed to high level by immune cells, triggers cytokine release and modulates immune cell differentiation. In this review, we focus on evidence supporting a possible role of the P2X7R in the pathogenesis of systemic lupus erythematosus (SLE). Keywords: Extracellular ATP, P2X7R, Systemic lupus erythematosus, NLRP3 inflammasome, Interleukin-1Î

Elementi di Immunoterapia

L’introduzione dei farmaci di origine biologica ha rivoluzionato la terapia di molte patologie neoplastiche o su base infiammatoria cronica. Nonostante il costo a volte molto elevato di questi trattamenti (e le inevitabili ricadute su Sistemi Sanitari Nazionali), la disponibilità di farmaci ad alta selettività per uno specifico bersaglio cellulare ha accresciuto enormemente l’efficacia del nostro armamentario terapeutico. Ciò non implica in nessun modo un “pensionamento anticipato” dei farmaci di sintesi chimica ma piuttosto il loro affiancamento con nuove molecole di origine biologica caratterizzate da alta specificità e selettività, e perciò in grado di interagire con un determinato bersaglio molecolare con grande, virtualmente assoluta, precisione. L’impiego di molecole immunomodulatorie di origine biologica ha subito una vigorosa accelerazione a partire dal 1997, quando l’anticorpo monoclonale rituximab (diretto contro il determinante di superficie CD20, espresso dai linfociti B nelle fasi iniziali del differenziamento) ricevette dalle Autorità regolatorie statunitensi (FDA) l’autorizzazione per il trattamento di neoplasie del sistema linfopoietico ed, in seguito, di leucemie e linfomi a cellule B e di alcune patologie autoimmuni. L’impetuoso sviluppo delle terapie immunobiologiche si è basato, da un lato, sui progressi delle tecniche di biologia molecolare e di ingegnerizzazione cellulare e dall’altro su un radicale mutamento di paradigma nella terapia dei tumori. Infatti, il perfezionamento delle tecniche di manipolazione di cellule ed anticorpi ha permesso di produrre molecole ricombinanti virtualmente specifiche per qualsiasi bersaglio cellulare e, grazie all’introduzione delle tecniche di umanizzazione degli anticorpi monoclonali, ha ridotto sostanzialmente il rischio di reazioni avverse. Inoltre, le nuove terapie antitumorali hanno progressivamente spostato l’enfasi dalla cellula neoplastica al contesto in cui si sviluppa la neoplasia (il microambiente tumorale), di cui il sistema immunitario è un componente fondamentale. Per usare una perifrasi, si sta rapidamente affermando una nuova concezione della lotta contro i tumori basata su una più approfondita comprensione dell’”ecologia” del tumore, cioè dei rapporti che il tumore stabilisce con il microambiente che lo circonda e, più in generale, con l’organismo che lo ospit

ALTERED EXPRESSION AND FUNCTION OF P2X7 RECEPTOR IN PATIENTSAFFECTED BY SYSTEMIC LUPUS ERYTHEMATOSUS (SLE)

Background. Extracellular ATP (eATP) is one of the most diffuse danger associated molecular patterns (DAMPs) released actively through specific mechanisms from intact cells, or passively from damaged or dying cells¹. An implication for eATP has been found in SLE². Among receptors for eATP, P2X7R is deeply involved in inflammatory and immune processes and its activation drives different intracellular pathways such as NLRP3-inflammasome activation, IL-1β maturation and release, IL-6 and TNF-α production, regulation of lymphocyte proliferation and cell apoptosis³. Previous studies pointed out a possible relationship between P2X7R signaling pathways and SLE pathogenesis4 5. A marked inflammatory condition characterize serositis, that are among the most common manifestations of SLE, and chloroquine is one of the main drugs employed. Objectives. The aim of this study was to investigate P2X7R expression and activity in SLE. Methods 48 SLE patients, and 20 healthy control (HC) subjects were enrolled. Among SLE patients, 16 (SLE-S) presented, and 32 (SLE-NS) did not present history of serositis. All subjects gave written informed consent to peripheral venous blood withdrawal after approval by the local ethic committee. Plasma samples were used to measure IL-1β, IL-6 and TNF-α levels by ELISA. Mononuclear cells were isolated from blood samples by Ficoll gradient sedimentation and employed as follow: i) assessment of IL-1β, IL-6 and TNF-α release after stimulation with lipopolysaccharide (LPS) and/or Benzoyl ATP (BzATP); ii) evaluation of P2X7R mRNA expression by RT-PCR; iii) measurement of P2X7R activity as BzATP-induced increase of intracellular Ca²⁺ concentration using the Fura2/AM probe. Results In SLE patients respect to HC, plasma IL-1β levels were unmodified whereas IL-6 was higher, resulting significantly increased in SLE-S. Monocytes isolated from SLE patients released lower quantities of IL-1β after stimulation with BzATP, whereas the release of both IL-6 and TNF-α was significantly augmented in SLE-NS respect to both HC and SLE-S subjects after all types of stimulation. RT-PCR showed reduced P2X7R and augmented NLRP3 mRNA expression in SLE patients. Accordingly, P2X7R activity was significantly reduced in all SLE patients and did not appear to be influenced by a chloroquine pre-treatment. Conclusion In SLE patients, compared to HC subjects, we found reduced P2X7R mRNA expression, increased NLRP3 mRNA, as a possible compensating mechanism, and correspondingly, significantly lower BzATP-induced intracellular Ca²⁺ increase, without an apparent influence by chloroquine, one of the drugs most diffusely used for SLE treatment. The in vitro IL-1β release was reduced, whereas plasma IL-1β was unaltered, indicating an alternative source, other than monocytes, of this cytokine. Conversely, IL-6 and TNF-α levels were increased in vitro, and IL-6 was present in plasma at higher levels. The possible consequences of reduced P2X7R, mainly on cytokines network deregulation and lymphocyte proliferation, will be further investigated as well as the role of IL-6 and TNF-α as possible therapeutic targets. References [1] Giuliani, A.L. et al. Extracellular nucleotides and nucleosides as signalling molecules. Immunol Lett, doi:10.1016/j.imlet.2018.11.006 (2018). [2] Magna, M. & Pisetsky, D.S. The Role of Cell Death in the Pathogenesis of SLE: Is Pyroptosis the Missing Link? Scand J Immunol 82, 218-224, (2015). [3] Di Virgilio, F. et al. The P2X7 Receptor in Infection and Inflammation. Immunity 47, 15-31, (2017). [4] Di Virgilio, F. & Giuliani, A.L. Purinergic signalling in autoimmunity: A role for the P2X7R in systemic lupus erythematosus? Biomed J 39, 326-338, (2016). [5] Faliti, C.E. et al. P2X7 receptor restrains pathogenic Tfh cell generation in systemic lupus erythematosus. J Exp Med, (2019). Disclosure of Interests Anna Lisa Giuliani: None declared, Federica Furini: None declared, Alessandra Bortoluzzi: None declared, Marcello Govoni: None declared, Francesco Di Virgilio Consultant for: FDV is a member of the Scientific Advisory Board of Biosceptre Ltd, a UK-based biotech Company involved in the development of P2X7R-targeted therapeutics