Publications

Lower urinary tract symptoms (LUTS) are prevalent among the aging population, and current medications offer limited efficacy. Improved therapies require a better understanding of the mechanisms underlying LUTS. Abnormal extracellular ATP levels and altered purinergic contractility have been reported in patients with LUTS, suggesting dysregulation of the purinergic pathways. CD39/ENTPD1 is a major regulator of purinergic metabolism, and patients with ENTPD1 loss-of-function mutations exhibit bladder hypomotility and incontinence, suggesting critical roles for ENTPD1 in bladder function. We have tested this hypothesis in an overexpression mouse model with human ENTPD1 knock-in (hCD39TG), in a hemizygous Entpd1 mouse model (Entpd1+/-) mimicking LUTS patients with impaired ATP hydrolysis, and in a homozygous Entpd1-deleted mouse model (Entpd1-/-) mimicking the complete ENTPD1 loss-of-function observed in patients. We have demonstrated that ENTPD1 dysregulation leads to profound abnormalities in bladder voiding phenotypes and urodynamics, along with impaired BSM contractility and altered purinergic receptor signaling. Furthermore, we have found that modulation of downstream P2Y12/adenosine A2b receptor signaling partially or fully restores normal bladder function. Finally, we have identified expression of ENTPD1 and associated purinergic proteins in human BSM cells, underscoring the critical role of ENTPD1 in human bladder function and highlighting its translational potential for the treatment of LUTS.

BACKGROUND & AIMS: Within the Liver Imaging Reporting and Data System (LI-RADS), LI-RADS Malignant (LR-M) lesions remain diagnostically challenging: imaging indicates malignancy but often fails to distinguish hepatocellular carcinoma (HCC) from intrahepatic cholangiocarcinoma (iCCA). Established serum-based tools such as GALAD are optimized for HCC but are not designed to resolve entity ambiguity within LR-M. In this pilot proof-of-concept study, we investigated whether small extracellular vesicle (EV) integrated with routine serological markers could resolve LR-M lesion situation.

METHODS: A rare LR-M cohort (HCC = 25, iCCA = 25) was evaluated using LASSO logistic regression and principal component analysis (PCA) to prioritize informative biomarkers. Hybrid models combined CD9+CD133/2+ and CD81+CD133/2+ EVs with alkaline phosphatase, CRP, CA19-9, and optional AFP. Models were internally validated using an 80:20 train-test split and assessed with bootstrap and Monte-Carlo perturbation (±5-20%).

RESULTS: Individual markers demonstrated limited discrimination (AUROC ≤ 0.82). Hybrid logistic regression models showed moderate internal discrimination (AUROC 0.86 with AFP; 0.91 without AFP). Translation into additive scoring systems using ROC-Youden-derived cut-offs yielded high internal AUROC estimates (e.g., 0.95-0.96), though these remain internally validated. A simplified five-point PRISM score retained comparable discriminatory performance (AUROC ∼0.91). In exploratory survival analysis among iCCA patients (n = 25), those with above-median CD9+CD133/2+ EV levels (n = 13) had shorter overall survival (median 91 vs. 389 days; HR 2.80, 95% CI 1.16-6.74; p = 0.005).

CONCLUSIONS: Integrated EV and serological profiling may enable minimally invasive differentiation between HCC and iCCA within LR-M lesions. By transforming ML-guided model discovery into a clinically interpretable paper-and-pencil additive score, we illustrate a translational pathway from computational discovery to practical application.

IMPACT AND IMPLICATIONS: This study provides a scientific rationale for integrating small extracellular vesicle (EV) phenotyping with conventional serum biomarkers to improve minimally invasive differentiation between hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA) in LR-M lesions. By translating a machine learning (ML)_derived hybrid model into a simple additive scoring system, we demonstrate how ML can yield clinically interpretable tools that bridge computational discovery and bedside application. The findings are particularly relevant for hepatologists, oncologists, and radiologists managing patients in whom imaging remains indeterminate and biopsy carries procedural risk. The five-point model can be applied using routine laboratory values, providing a paper-and-pencil diagnostic aid that requires no specialized software or hardware. While external multicenter validation is necessary given the cohort size, this approach illustrates how transparent, interpretable machine learning can support precision diagnostics in primary liver cancer.

Infection with Mycobacterium tuberculosis can cause diverse lesions, such as necrotic pneumonia, which can contribute to tuberculosis progression and transmission between individuals. Despite advances in understanding the role of ATP-gated P2RX7 ion channels in the development of severe forms of the disease, the regulation of this important signaling pathway remains unclear. Herein, we show that the ectonucleotidase CD39 plays an essential regulatory role in tuberculosis progression by preventing lung tissue damage, bacterial dissemination, and excessive inflammatory responses. Mechanistically, through its enzymatic activity on the cellular surface, CD39 protects infected macrophages from undergoing necrotic death mediated by P2RX7 activation. Cell-intrinsic CD39 expression also hinders the establishment of other myeloid cells, such as neutrophils, in the infected lung. We proposed that, by protecting infected macrophages from P2RX7-mediated cell death and bacterial dissemination in the lung tissue, CD39 prevents the development of necrotic lesions. Altogether, these findings uncover a significant role for CD39 as an essential component of the molecular regulation underlying the development of severe tuberculosis.

Francesco Di Virgilio, one of the giants of purinergic signalling, died suddenly on September 22, 2024, which is an immense loss to so many friends and colleagues and especially to his beloved wife, Dorianna. Here, 1 year on, we pay tribute to him and his immense contribution to our field. Francesco graduated in medicine from the University of Padova, Italy, in 1979, with what became a lifelong interest in inflammation. He then held post-doctoral positions at University College London, Padova University, and Columbia University, New York, where he became acquainted with P2X7 receptors. He then returned to Padova as an Associate Professor of Molecular Pathology before moving to the University of Ferrara in 1992, where he set up a world-leading lab that studied the roles and underlying cellular mechanisms of the action of P2X7 receptors in inflammatory pathologies. Francesco published over 370 peer-reviewed articles, which have been cited > 35,000 times, giving him an H-index > 100. In addition, he filed several patents related to purinergic signalling. He also collaborated extensively, both within the University of Ferrara and worldwide, including in universities in the UK, Spain, Germany, the USA, and Brazil. Francesco was a man of great passion and intellect, who possessed scientific vision, intuition, and integrity, and he became the go-to world expert on P2X7 receptors and inflammation. We have lost a giant in the field and a dear friend, but he leaves behind an exceptional body of work, an outstanding legacy, and many friends who will miss him.

ENTPD5 and ENTPD6 are members of the CD39-ectonucleoside triphosphate diphosphohydrolase (CD39-ENTPD) family, which play an important role in modulating the purinergic signaling pathway. Most of the knowledge in this area has been obtained by studying CD39/ENTPD1, the prototype member of this family, and evaluating the translational potential by either treating inflammation directly with recombinant proteins or by using antagonists to elicit immune responses in cancer. ENTPD5 and ENTPD6, "orphan-type" ectonucleotidases, are understudied to date, although both are expressed at high levels in various tissues, where they appear involved in regulating signal transduction, cellular energy, and metabolism. ENTPD5 is abnormally overexpressed in several types of malignancies, including prostate, liver, lung, and ovarian cancers. ENTPD5 appears to promote protein glycosylation and folding in part by regulating UDP and UMP levels, thereby enhancing the survival and proliferation of somatic or cancer cells. As such, ENTPD5 has been considered a potential proto-oncogene and a therapeutic target in cancer treatment. In contrast, despite comparable functionality, the related ENTPD6 shows relatively stable expression across tissues in both normal and pathological conditions, with specific roles in cancer yet unclear. This review provides a comprehensive overview of these two understudied ectoenzymes, detailing their shared molecular structures and control of purinergic signal transduction. In addition, we explore different patterns of tissue and organelle expression of these ecto-enzymes and propose relevance to the modulation of cellular metabolism, as would be important in cancer. We review the sometimes conflicting evidence from experimental animal models and propose potential future clinical applications. This review offers insights into the roles of this distinct duo of ENTPD family members to support future basic and translational research in this field.

The purine molecule ATP plays a crucial role in essential cellular functions, including energy transfer and extracellular signaling. It also serves as a precursor for the structural components of nucleic acids, including DNA and RNA, as well as for cyclic AMP and various cofactors. ATP is continuously degraded in cells and in extracellular space, leading to the sequential formation of ADP, AMP, adenosine, inosine, and hypoxanthine. ATP degradation is counterbalanced by the coordinated action of de novo biosynthetic pathways and salvage mechanisms, which generate inosine-5'-monophosphate (IMP), a precursor of AMP, which is subsequently converted into ATP. The synthesis and overall metabolism of ATP is closely linked to immune cell function, and dysregulation of these metabolic pathways can lead to immunodeficiency or worsen inflammatory diseases. Several approved drugs targeting ATP metabolism, including methotrexate, azathioprine, and allopurinol, are widely used to modulate immune system activity in the treatment of cancer, autoimmune diseases, and in gout. Here, we highlight the role of intracellular ATP homeostasis in coordinating bioenergetics and metabolism of immune cells. We also discuss inherited and acquired disorders that result in compromised ATP metabolism and impact immunity, as well as reviewing novel therapeutic approaches to target purinergic pathways in immunodeficiency and inflammatory diseases.

BACKGROUND: The efficacy of granulocyte-colony stimulating factor (G-CSF) treatment for acute-on-chronic liver failure (ACLF) remains controversial. The aim of this study, which is a secondary analysis of the GRAFT study (NCT02669680), was to identify potential prognostic biomarkers in ACLF and to find markers that could be used to predict response to G-CSF treatment.

METHODS: Blood samples from 79 patients randomized in the GRAFT study to receive G-CSF (n = 40) or standard medical therapy (n = 39) were collected at different timepoints. Samples were analyzed for cells, cytokines, extracellular particles, cell-free DNA, and functional properties. An exploratory approach was taken, whereby the measured variables were subjected to univariate and multivariate Cox analyses, and the patients were stratified into clusters by unsupervised hierarchical clustering.

RESULTS: Patients with ACLF had increased plasma levels of pro-inflammatory cytokines and increased absolute levels of specific cell populations when compared to healthy controls. ROC curve analysis suggested that plasma VEGF-A levels at baseline (timepoint) are a prognostic factor of transplant-free survival (AUC: 0.70; 95%CI 0.56-0.84). Hierarchical clustering of circulating immune cell populations at baseline appeared to define a patient subset within the analyzed G-CSF-treated patients with improved median transplant-free survival (102 days vs 16 days; p = 0.002). In our study, a higher percentage of CD39+ lymphocytes serves as an independent predictor of unfavorable outcomes following G-CSF treatment (HR: 1.05, 95%CI: 1.01-1.09, p = 0.008).

CONCLUSION: VEGF-A appears to be a suitable biomarker for predicting the prognosis of patients with ACLF. With the help of hierarchical cell clusters, ACLF patients who could benefit from G-CSF therapy could be identified and selected prior to treatment. CD39 expression on lymphocytes seems suitable to allow stratification. Further testing and validation are necessary and could help to adapt treatment options for each individual patient.

Immunotherapies revolutionized cancer treatment, yet their efficacy remains constrained by the tumor's immunosuppressive microenvironment. Here, we evaluated whether combining CD39 blockade with other modalities of immunotherapy such as IL-2/anti-IL-2 complexes (IL-2cx) administration could further enhance T cell-mediated antitumor responses and improve tumor control. We demonstrated that CD39 deficiency in MC38 tumor-bearing CD39KO (Entpd1 null) mice decreases tumor growth. This better tumor growth control was associated with increased infiltration of PD-1High CD8+ T cells, expressing elevated levels of exhaustion markers and transcription factors such as TOX. This PD-1High CD8+ T cell subset also exhibited a higher frequency of IFN-γ-producing and cytotoxic (Granzyme B+, Perforin+) cells. In contrast, the less immunogenic B16F10-OVA model did not show significant differences in tumor growth; however, CD39KO mice displayed an increased frequency of antigen-specific, pre-exhausted (PD-1Int) CD8+ T cells, a population recognized as a key target of immunotherapy. Pharmacological CD39 blockade with POM-1, when combined with IL-2cx treatment to redirect IL-2 activity, enhanced the accumulation of pre-exhausted CD8+ T cells with cytotoxic potential, thereby improving tumor control. This combinatorial strategy also reshaped the tumor immune landscape by increasing activated NK cells, elevating Granzyme B expression in CD4+ T cells, and decreasing immunosuppressive M-MDSCs expressing CD39, CD38, and CD73. Collectively, our findings demonstrate that integrating purinergic pathway inhibition with IL-2-based immunotherapies can coordinately reprogram lymphoid and myeloid compartments, attenuate immunosuppressive mechanisms within the tumor microenvironment, and amplify antitumor immunity, providing a strong rationale for advancing this strategy toward clinical translation.

The concept of the intensive care unit (ICU) was developed around 70 years ago, and this has become essential for caring for a hospital's sickest patients. A commonality for many patients' critical illness is the systemic inflammatory state with multiple-organ injury precipitated by infectious causes and/or other pathophysiologic insults. Therapeutic modalities to address these complications remain unclear, and there are no FDA-approved drugs to treat these often-devastating clinical situations. Clinical deterioration may be associated with the release of "damage-associated molecular patterns" (DAMPs), such as extracellular adenosine triphosphate (eATP), from stressed and dying cells. Pharmacological dosing or boosting of CD73, an ectoenzyme that can convert pro-inflammatory adenosine monophosphate (AMP) to anti-inflammatory adenosine, in this setting of critical illness has been shown to have a survival benefit with decreased time in the hospital and ICU. Whether there are clinical benefits of extracellular nucleotide (eATP) scavenging over adenosine generation within the setting of inflammatory diseases remains unclear. Upcoming pre-clinical developments testing soluble forms of CD39 to hydrolyze eATP to AMP in sepsis and following cardiac surgery should clarify this question. We conclude by suggesting that exogenous CD39, CD73, and/or other ectonucleotidases may provide therapeutic benefits in critically ill patients.