Astroglial calcium signaling and homeostasis in tuberous sclerosis complex.

Tuberous Sclerosis Complex (TSC) is a multisystem genetic disorder characterized by the development of benign tumors in various organs, including the brain, and is often accompanied by epilepsy, neurodevelopmental comorbidities including intellectual disability and autism. A key hallmark of TSC is the hyperactivation of the mechanistic target of rapamycin (mTOR) signaling pathway, which induces alterations in cortical development and metabolic processes in astrocytes, among other cellular functions. These changes could modulate seizure susceptibility, contributing to the progression of epilepsy and its associated comorbidities. Epilepsy is characterized by dysregulation of calcium (Ca2+) channels and intracellular Ca2+ dynamics. These factors contribute to hyperexcitability, disrupted synaptogenesis, and altered synchronization of neuronal networks, all of which contribute to seizure activity. This study investigates the intricate interplay between altered Ca2+ dynamics, mTOR pathway dysregulation, and cellular metabolism in astrocytes. The transcriptional profile of TSC patients revealed significant alterations in pathways associated with cellular respiration, ER and mitochondria, and Ca2+ regulation. TSC astrocytes exhibited lack of responsiveness to various stimuli, compromised oxygen consumption rate and reserve respiratory capacity underscoring their reduced capacity to react to environmental changes or cellular stress. Furthermore, our study revealed significant reduction of store operated calcium entry (SOCE) along with strong decrease of basal mitochondrial Ca2+ concentration and Ca2+ influx in TSC astrocytes. In addition, we observed alteration in mitochondrial membrane potential, characterized by increased depolarization in TSC astrocytes. Lastly, we provide initial evidence of structural abnormalities in mitochondria within TSC patient-derived astrocytes, suggesting a potential link between disrupted Ca2+ signaling and mitochondrial dysfunction. Our findings underscore the complexity of the relationship between Ca2+ signaling, mitochondria dynamics, apoptosis, and mTOR hyperactivation. Further exploration is required to shed light on the pathophysiology of TSC and on TSC associated neuropsychiatric disorders offering further potential avenues for therapeutic development.

Getting everyone to agree on gene signatures for murine macrophage polarization in vitro.

Macrophages, key players in the innate immune system, showcase remarkable adaptability. Derived from monocytes, these phagocytic cells excel in engulfing and digesting pathogens and foreign substances as well as contributing to antigen presentation, initiating and regulating adaptive immunity. Macrophages are highly plastic, and the microenvironment can shaper their phenotype leading to numerous distinct polarized subsets, exemplified by the two ends of the spectrum: M1 (classical activation, inflammatory) and M2 (alternative activation, anti-inflammatory). RNA sequencing (RNA-Seq) has revolutionized molecular biology, offering a comprehensive view of transcriptomes. Unlike microarrays, RNA-Seq detects known and novel transcripts, alternative splicing, and rare transcripts, providing a deeper understanding of genome complexity. Despite the decreasing costs of RNA-Seq, data consolidation remains limited, hindering noise reduction and the identification of authentic signatures. Macrophages polarization is routinely ascertained by qPCR to evaluate those genes known to be characteristic of M1 or M2 skewing. Yet, the choice of these genes is literature- and experience-based, lacking therefore a systematic approach. This manuscript builds on the significant increase in deposited RNA-Seq datasets to determine an unbiased and robust murine M1 and M2 polarization profile. We now provide a consolidated list of global M1 differentially expressed genes (i.e. robustly modulated by IFN-γ, LPS, and LPS+ IFN-γ) as well as consolidated lists of genes modulated by each stimulus (IFN-γ, LPS, LPS+ IFN-γ, and IL-4).

Genetic deletion of astrocytic calcineurin B1 prevents cognitive impairment and neuropathology development in acute and chronic mouse models of Alzheimer's disease.

Alzheimer's disease (AD) represents an urgent yet unmet challenge for modern society, calling for exploration of innovative targets and therapeutic approaches. Astrocytes, main homeostatic cells in the CNS, represent promising cell-target. Our aim was to investigate if deletion of the regulatory CaNB1 subunit of calcineurin in astrocytes could mitigate AD-related memory deficits, neuropathology, and neuroinflammation. We have generated two, acute and chronic, AD mouse models with astrocytic CaNB1 ablation (ACN-KO). In the former, we evaluated the ability of ß-amyloid oligomers (AßOs) to impair memory and activate glial cells once injected in the cerebral ventricle of conditional ACN-KO mice. Next, we generated a tamoxifen-inducible astrocyte-specific CaNB1 knock-out in 3xTg-AD mice (indACNKO-AD). CaNB1 was deleted, by tamoxifen injection, in 11.7-month-old 3xTg-AD mice for 4.4 months. Spatial memory was evaluated using the Barnes maze; ß-amyloid plaques burden, neurofibrillary tangle deposition, reactive gliosis, and neuroinflammation were also assessed. The acute model showed that ICV injected AßOs in 2-month-old wild type mice impaired recognition memory and fostered a pro-inflammatory microglia phenotype, whereas in ACN-KO mice, AßOs were inactive. In indACNKO-AD mice, 4.4 months after CaNB1 depletion, we found preservation of spatial memory and cognitive flexibility, abolishment of amyloidosis, and reduction of neurofibrillary tangles, gliosis, and neuroinflammation. Our results suggest that ACN is crucial for the development of cognitive impairment, AD neuropathology, and neuroinflammation. Astrocyte-specific CaNB1 deletion is beneficial for both the abolishment of AßO-mediated detrimental effects and treatment of ongoing AD-related pathology, hence representing an intriguing target for AD therapy.

Extracellular nicotinamide phosphoribosyltransferase (eNAMPT) neutralization counteracts T cell immune evasion in breast cancer.

BACKGROUND: Nicotinamide phosphoribosyltransferase (NAMPT) is a key intracellular enzyme that participates in nicotinamide adenine dinucleotide (NAD) homeostasis as well as a released cytokine (eNAMPT) that is elevated in inflammatory conditions and in cancer. In patients with breast cancer, circulating eNAMPT is elevated and its plasma levels correlate with prognosis and staging. In light of this, we investigated the contribution of eNAMPT in triple negative mammary carcinoma progression by investigating the effect of its neutralization via a specific neutralizing monoclonal antibody (C269). METHODS: We used female BALB/c mice injected with 4T1 clone 5 cells and female C57BL6 injected with EO771 cells, evaluating tumoral size, spleen weight and number of metastases. We injected two times a week the anti-eNAMPT neutralizing antibody and we sacrificed the mice after 28 days. Harvested tumors were analyzed by histopathology, flow cytometry, western blot, immunohistochemistry, immunofluorescence and RNA sequencing to define tumor characteristics (isolating tumor infiltrating lymphocytes and tumoral cells) and to investigate the molecular mechanisms behind the observed phenotype. Moreover, we dissected the functional relationship between T cells and tumoral cells using three-dimensional (3D) co-cultures. RESULTS: The neutralization of eNAMPT with C269 led to decreased tumor size and reduced number of lung metastases. RNA sequencing and functional assays showed that eNAMPT controlled T-cell response via the programmed death-ligand 1/programmed cell death protein 1 (PD-L1/PD-1) axis and its neutralization led to a restoration of antitumoral immune responses. In particular, eNAMPT neutralization was able to activate CD8+IFNγ+GrzB+ T cells, reducing the immunosuppressive phenotype of T regulatory cells. CONCLUSIONS: These studies indicate for the first time eNAMPT as a novel immunotherapeutic target for triple negative breast cancer.

Do France, Germany, and Italy agree on the added therapeutic value of medicines?

OBJECTIVES: The Health Technology Assessment (HTA) of medicines is performed separately at the country level with some differences, but Italy, France, and Germany have implemented price and reimbursement systems strongly focused on the Added Therapeutic Value (ATV). This study investigates the level of agreement on ATV assessments by Agenzia Italiana del Farmaco (AIFA), Haute Autorité de Santé (HAS), and Gemeinsamer Bundesausschuss (G-BA). METHODS: A database was created collecting all information about drugs with innovativeness status requests in Italy from July 2017 to December 2022 and populated with the corresponding HAS and G-BA ATV assessments. The primary comparative analysis was conducted by grouping the ATV ratings into "higher added value" and "lower or no added value", while a secondary analysis considered the Italian innovativeness status as a criterion to include the quality of evidence assessment. The concordance between ATV assessments was investigated through percentage agreement and unweighted Cohen k-value. RESULTS: 189 medicines/indications were included. The greatest agreement was found when comparing G-BA versus HAS (82 percent; k = 0.61, substantial agreement). Lower levels of agreements were observed for AIFA versus HAS and AIFA versus G-BA (respectively 52 percent; k = 0.17 and 57 percent; k = 0.25). The secondary analysis led to a reconciliation to moderate agreement for AIFA versus HAS (72 percent; k = 0.45) and AIFA versus G-BA (74 percent; k = 0.47). CONCLUSIONS: A high degree of concordance between HTA organizations is reached when considering jointly ATV and quality of evidence, suggesting that the system is extensively mature to make a Joint Clinical Assessment, avoiding duplications and reducing access inequalities.

Inhibition of NHE1 transport activity and gene transcription in DRG neurons in oxaliplatin-induced painful peripheral neurotoxicity.

Oxaliplatin (OHP)-induced peripheral neurotoxicity (OIPN), one of the major dose-limiting side effects of colorectal cancer treatment, is characterized by both acute and chronic syndromes. Acute exposure to low dose OHP on dorsal root ganglion (DRG) neurons is able to induce an increase in intracellular calcium and proton concentration, thus influencing ion channels activity and neuronal excitability. The Na+/H+ exchanger isoform-1 (NHE1) is a plasma membrane protein that plays a pivotal role in intracellular pH (pHi) homeostasis in many cell types, including nociceptors. Here we show that OHP has early effects on NHE1 activity in cultured mouse DRG neurons: the mean rate of pHi recovery was strongly reduced compared to vehicle-treated controls, reaching levels similar to those obtained in the presence of cariporide (Car), a specific NHE1 antagonist. The effect of OHP on NHE1 activity was sensitive to FK506, a specific calcineurin (CaN) inhibitor. Lastly, molecular analyses revealed transcriptional downregulation of NHE1 both in vitro, in mouse primary DRG neurons, and in vivo, in an OIPN rat model. Altogether, these data suggest that OHP-induced intracellular acidification of DRG neurons largely depends on CaN-mediated NHE1 inhibition, revealing new mechanisms that OHP could exert to alter neuronal excitability, and providing novel druggable targets.

The endoplasmic reticulum stress and unfolded protein response in Alzheimer's disease: A calcium dyshomeostasis perspective.

Protein misfolding is prominent in early cellular pathology of Alzheimer's disease (AD), implicating pathophysiological significance of endoplasmic reticulum stress/unfolded protein response (ER stress/UPR) and highlighting it as a target for drug development. Experimental data from animal AD models and observations on human specimens are, however, inconsistent. ER stress and associated UPR are readily observed in in vitro AD cellular models and in some AD model animals. In the human brain, components and markers of ER stress as well as UPR transducers are observed at Braak stages III-VI associated with severe neuropathology and neuronal death. The picture, however, is further complicated by the brain region- and cell type-specificity of the AD-related pathology. Terms 'disturbed' or 'non-canonical' ER stress/UPR were used to describe the discrepancies between experimental data and the classic ER stress/UPR cascade. Here we discuss possible 'disturbing' or 'interfering' factors which may modify ER stress/UPR in the early AD pathogenesis. We focus on the dysregulation of the ER Ca2+ homeostasis, store-operated Ca2+ entry, and the interaction between the ER and mitochondria. We suggest that a detailed study of the CNS cell type-specific alterations of Ca2+ homeostasis in early AD may deepen our understanding of AD-related dysproteostasis.

NAMPT and NAPRT serum levels predict response to anti-TNF therapy in inflammatory bowel disease.

Background: Nicotinamide phosphoribosyltransferase (NAMPT) and nicotinic acid phosphoribosyl transferase (NAPRT) are key intracellular enzymes that participate in the biosynthesis on NAD but have also been shown to be released as proinflammatory cytokines. A number of reports have shown that circulating NAMPT is increased in serum of patients with inflammatory disorders, including inflammatory bowel diseases (IBD), while nothing is known regarding circulating NAPRT and the presence of both cytokines in IBD patient stools. In the present study, we evaluated eNAMPT and eNAPRT levels in a large cohort of IBD patients not on biological therapy and in a subset that then was prescribed biologics. Methods: We conducted a retro-perspective study on 180 patients, of which 111 underwent subsequent biological treatment (adalimumab, vedolizumab, and ustekinumab). We analyzed eNAMPT and eNAPRT concentrations in serum and faces of IBD patients, correlating them with response to biologics. Results: We now report that eNAMPT and eNAPRT are significantly increased in both serum and stools of IBD patients. NAMPT and NAPRT levels correlate with disease severity, with C reactive protein and with serum IL-6 levels. Importantly, levels of NAMPT in patients starting treatment with adalimumab correlate with response failure at three months: patients with levels above 4 ng/ml were significantly less likely to obtain benefit. Serum NAMPT as a biomarker of response yields a sensitivity of 91% and a specificity of 100%. Conclusion: The present work strongly suggests that a prospective trial evaluating eNAMPT and eNAPRT levels in relation to response to biologicals in IBD should be initiated.

Immortalized hippocampal astrocytes from 3xTg-AD mice, a new model to study disease-related astrocytic dysfunction: a comparative review.

Alzheimer's disease (AD) is characterized by complex etiology, long-lasting pathogenesis, and cell-type-specific alterations. Currently, there is no cure for AD, emphasizing the urgent need for a comprehensive understanding of cell-specific pathology. Astrocytes, principal homeostatic cells of the central nervous system, are key players in the pathogenesis of neurodegenerative diseases, including AD. Cellular models greatly facilitate the investigation of cell-specific pathological alterations and the dissection of molecular mechanisms and pathways. Tumor-derived and immortalized astrocytic cell lines, alongside the emerging technology of adult induced pluripotent stem cells, are widely used to study cellular dysfunction in AD. Surprisingly, no stable cell lines were available from genetic mouse AD models. Recently, we established immortalized hippocampal astroglial cell lines from amyloid-ß precursor protein/presenilin-1/Tau triple-transgenic (3xTg)-AD mice (denominated as wild type (WT)- and 3Tg-iAstro cells) using retrovirus-mediated transduction of simian virus 40 large T-antigen and propagation without clonal selection, thereby maintaining natural heterogeneity of primary cultures. Several groups have successfully used 3Tg-iAstro cells for single-cell and omics approaches to study astrocytic AD-related alterations of calcium signaling, mitochondrial dysfunctions, disproteostasis, altered homeostatic and signaling support to neurons, and blood-brain barrier models. Here we provide a comparative overview of the most used models to study astrocytes in vitro, such as primary culture, tumor-derived cell lines, immortalized astroglial cell lines, and induced pluripotent stem cell-derived astrocytes. We conclude that immortalized WT- and 3Tg-iAstro cells provide a non-competitive but complementary, low-cost, easy-to-handle, and versatile cellular model for dissection of astrocyte-specific AD-related alterations and preclinical drug discovery.

Immortalized Alzheimer's Disease Astrocytes: Characterization of Their Proteolytic Systems.

Alzheimer's disease (AD) is a progressive neurodegeneration with dysfunctions in both the ubiquitin-proteasome system (UPS) and autophagy. Astroglia participation in AD is an attractive topic of research, but molecular patterns are partially defined and available in vitro models have technical limitations. Immortalized astrocytes from the hippocampus of 3xTg-AD and wild-type mice (3Tg-iAstro and WT-iAstro, respectively) have been obtained as an attempt to overcome primary cell line limitations and this study aims at characterizing their proteolytic systems, focusing on UPS and autophagy. Both 26S and 20S proteasomal activities were downregulated in 3Tg-iAstro, in which a shift in catalytic subunits from constitutive 20S proteasome to immunoproteasome occurred, with consequences on immune functions. In fact, immunoproteasome is the specific complex in charge of clearing damaged proteins under inflammatory conditions. Parallelly, augmented expression and activity of the lysosomal cathepsin B, enhanced levels of lysosomal-associated membrane protein 1, beclin1, and LC3-II, together with an increased uptake of monodansylcadaverine in autophagic vacuoles, suggested autophagy activation in 3Tg-iAstro. The two proteolytic pathways were linked by p62 that accumulated in 3Tg-iAstro due to both increased synthesis and decreased degradation in the UPS defective astrocytes. Treatment with 4-phenylbutyric acid, a neuroprotective small chemical chaperone, partially restored proteasome and autophagy-mediated proteolysis in 3Tg-iAstro. Our data shed light on the impaired proteostasis in 3Tg-iAstro with proteasome inhibition and autophagic compensatory activation, providing additional validation of this AD in vitro model, and propose a new mechanism of action of 4-phenylbutyric acid in neurodegenerative disorders.

The Predictive Role of Extracellular NAPRT for the Detection of Advanced Fibrosis in Biopsy-Proven Non-Alcoholic Fatty Liver Disease.

Intrahepatic oxidative stress is a key driver of inflammation and fibrogenesis in non-alcoholic fatty liver disease (NAFLD). We aimed to investigate the role of extracellular Nicotinamide phosphoribosyltransferase (eNAMPT) and extracellular nicotinic acid phosphoribosyltransferase (eNAPRT) for the detection of advanced fibrosis. eNAMPT and eNAPRT were tested in 180 consecutive biopsy-proven NAFLD patients and compared with liver stiffness (LS) and the FIB-4 score. eNAMPT was similarly distributed across fibrosis stages, whereas eNAPRT was increased in patients with advanced fibrosis (p = 0.036) and was associated with advanced fibrosis (OR 1.08, p = 0.016). A multiple stepwise logistic regression model containing significant variables for advanced fibrosis (eNAPRT, type 2 diabetes, age, male sex, ALT) had an area under the curve (AUC) of 0.82 (Se 89.6%, Sp 67.3%, PPV 46.7%, NPV 93.8%) when compared to that of LS (0.79; Se 63.5%, Sp 86.2%, PPV 66.0%, NPV 84.8%) and to that of the FIB-4 score (0.73; Se 80.0%, Sp 56.8%, PPV 44.9%, NPV 86.6%). The use of eNAPRT in clinical practice might allow for the better characterization of NAFLD patients at higher risk of disease progression.

Identification of novel aza-analogs of TN-16 as disrupters of microtubule dynamics through a multicomponent reaction.

Despite novel biological targets emerging at an impressive rate for anticancer therapy, antitubulin drugs remain the backbone of numerous oncological protocols and their efficacy has been demonstrated in a wide variety of adult and pediatric cancers. In the present contribution, we set to develop analogs of a potent but neglected antitubulin agent, TN-16, originally discovered via modification of tenuazonic acid (3-acetyl-5-sec-butyltetramic acid). To this extent, we developed a novel multicomponent reaction to prepare TN-16, and then we applied the same reaction for the synthesis of aza-analogs. In brief, we prepared a library of 62 novel compounds, and three of these retained nanomolar potencies. TN-16 and the active analogs are cytotoxic on cancer cell lines and, as expected from antitubulin agents, induce G2/M cell cycle arrest. These agents lead to a disruption of the microtubules and an increase in α-tubulin acetylation and affect in vitro polymerization, although they have a lesser effect in cellular tubulin polymerization assays.

Calcineurin Signalling in Astrocytes: From Pathology to Physiology and Control of Neuronal Functions.

Calcineurin (CaN), a Ca2+/calmodulin-activated serine/threonine phosphatase, acts as a Ca2+-sensitive switch regulating cellular functions through protein dephosphorylation and activation of gene transcription. In astrocytes, the principal homeostatic cells in the CNS, over-activation of CaN is known to drive pathological transcriptional remodelling, associated with neuroinflammation in diseases such as Alzheimer's disease, epilepsy and brain trauma. Recent reports suggest that, in physiological conditions, the activity of CaN in astrocytes is transcription-independent and is required for maintenance of basal protein synthesis rate and activation of astrocytic Na+/K+ pump thereby contributing to neuronal functions such as neuronal excitability and memory formation. In this contribution we overview the role of Ca2+ and CaN signalling in astroglial pathophysiology focusing on the emerging physiological role of CaN in astrocytes. We propose a model for the context-dependent switch of CaN activity from the post-transcriptional regulation of cell proteostasis in healthy astrocytes to the CaN-dependent transcriptional activation in neuroinflammation-associated diseases.

Protein synthesis inhibition and loss of homeostatic functions in astrocytes from an Alzheimer's disease mouse model: a role for ER-mitochondria interaction.

Deregulation of protein synthesis and ER stress/unfolded protein response (ER stress/UPR) have been reported in astrocytes. However, the relationships between protein synthesis deregulation and ER stress/UPR, as well as their role in the altered homeostatic support of Alzheimer's disease (AD) astrocytes remain poorly understood. Previously, we reported that in astrocytic cell lines from 3xTg-AD mice (3Tg-iAstro) protein synthesis was impaired and ER-mitochondria distance was reduced. Here we show that impaired protein synthesis in 3Tg-iAstro is associated with an increase of p-eIF2α and downregulation of GADD34. Although mRNA levels of ER stress/UPR markers were increased two-three-fold, we found neither activation of PERK nor downstream induction of ATF4 protein. Strikingly, the overexpression of a synthetic ER-mitochondrial linker (EML) resulted in a reduced protein synthesis and augmented p-eIF2α without any effect on ER stress/UPR marker genes. In vivo, in hippocampi of 3xTg-AD mice, reduced protein synthesis, increased p-eIF2α and downregulated GADD34 protein were found, while no increase of p-PERK or ATF4 proteins was observed, suggesting that in AD astrocytes, both in vitro and in vivo, phosphorylation of eIF2α and impairment of protein synthesis are PERK-independent. Next, we investigated the ability of 3xTg-AD astrocytes to support metabolism and function of other cells of the central nervous system. Astrocyte-conditioned medium (ACM) from 3Tg-iAstro cells significantly reduced protein synthesis rate in primary hippocampal neurons. When added as a part of pericyte/endothelial cell (EC)/astrocyte 3D co-culture, 3Tg-iAstro, but not WT-iAstro, severely impaired formation and ramification of tubules, the effect, replicated by EML overexpression in WT-iAstro cells. Finally, a chemical chaperone 4-phenylbutyric acid (4-PBA) rescued protein synthesis, p-eIF2α levels in 3Tg-iAstro cells and tubulogenesis in pericyte/EC/3Tg-iAstro co-culture. Collectively, our results suggest that a PERK-independent, p-eIF2α-associated impairment of protein synthesis compromises astrocytic homeostatic functions, and this may be caused by the altered ER-mitochondria interaction.

Implications of Oncology Trial Design and Uncertainties in Efficacy-Safety Data on Health Technology Assessments.

BACKGROUND: Advances in cancer medicines have resulted in tangible health impacts, but the magnitude of benefits of approved cancer medicines could vary greatly. Health Technology Assessment (HTA) is a multidisciplinary process used to inform resource allocation through a systematic value assessment of health technology. This paper reviews the challenges in conducting HTA for cancer medicines arising from oncology trial designs and uncertainties of safety-efficacy data. METHODS: Multiple databases (PubMed, Scopus and Google Scholar) and grey literature (public health agencies and governmental reports) were searched to inform this policy narrative review. RESULTS: A lack of robust efficacy-safety data from clinical trials and other relevant sources of evidence has made HTA for cancer medicines challenging. The approval of cancer medicines through expedited pathways has increased in recent years, in which surrogate endpoints or biomarkers for patient selection have been widely used. Using these surrogate endpoints has created uncertainties in translating surrogate measures into patient-centric clinically (survival and quality of life) and economically (cost-effectiveness and budget impact) meaningful outcomes, with potential effects on diverting scarce health resources to low-value or detrimental interventions. Potential solutions include policy harmonization between regulatory and HTA authorities, commitment to generating robust post-marketing efficacy-safety data, managing uncertainties through risk-sharing agreements, and using value frameworks. CONCLUSION: A lack of robust efficacy-safety data is a central problem for conducting HTA of cancer medicines, potentially resulting in misinformed resource allocation.

Functionalization of ß-cyclodextrin with a urea-based PSMA ligand and preliminary studies on targeting prostate cancer cells.

Targeted delivery of drugs into specific cancer cells is an effective way to enhance the efficacy and minimize the side effects of therapy. Prostate malignant cells overexpress the prostate-specific membrane antigen (PSMA), a membrane protein that may be a valid target for selective drug administration. To target prostate cancer cells, a ß-cyclodextrin perfunctionalised with dipeptide-like urea arms, a well-established mimic of a selective ligand against PSMA, is herein reported, to develop a multivalent drug delivery and targeting system. Firstly, fluorescein was used to validate the system on cells that express high levels of PSMA (prostate tumoral cells, LNCap) or very low levels of PSMA (non-tumoral cells, Hek293T). Then, the antineoplastic agent doxorubicin complexed with ß-cyclodextrin functionalized with PSMA-like ligand takes less time to induce cytotoxicity on LNCap cells compared to doxorubicin alone. This might represent a promising drug-delivery approach to selectively target prostate cancer cells.

Time-Trends of Drug-Drug Interactions among Elderly Outpatients in the Piedmont Region (Italy): A Population-Based Study.

Adverse drug reactions (ADRs) are a major health problem in the primary care setting, particularly among the elderly population. While the high frequency of ADRs in the elderly has several causes, a major and common determinant is polypharmacy, which can in turn increase the risk of drug-drug interactions (DDIs). In this paper, we analyzed the drugs prescriptions dispensed to elderly outpatients, to assess changes in the prevalence of selected DDIs in the period 2013−2019. Overall, about 15% of the patients aged >65 years were poly-treated. Among them, a decreasing trend in prevalence was observed for the majority of DDIs during the study period. This trend was particularly noticeable for DDIs involving fluoroquinolones and vitamin K antagonists, where a sharp reduction of over 40% was observed. On the opposite, a small increase in prevalence was observed for the association of antidiabetics and beta-blocking agents and for that of clopidogrel and PPIs. While the occurrence of most of the considered DDIs among poly-treated elderly decreased over time, the prevalence of some of them is still worrying. The complexity of the national drug formularies, as well as the increased number of prescribing actors that are involved, further urges the update of DDI lists to be used to monitor drug appropriateness and reduce avoidable ADRs.

CIC-39Na reverses the thrombocytopenia that characterizes tubular aggregate myopathy.

Store-operated Ca2+-entry is a cellular mechanism that governs the replenishment of intracellular stores of Ca2+ upon depletion caused by the opening of intracellular Ca2+-channels. Gain-of-function mutations of the 2 key proteins of store-operated Ca2+-entry, STIM1 and ORAI1, are associated with several ultra-rare diseases clustered as tubular aggregate myopathies. Our group has previously demonstrated that a mouse model bearing the STIM1 p.I115F mutation recapitulates the main features of the STIM1 gain-of-function disorders: muscle weakness and thrombocytopenia. Similar findings have been found in other mice bearing different mutations on STIM1. At present, no valid treatment is available for these patients. In the present contribution, we report that CIC-39Na, a store-operated Ca2+-entry inhibitor, restores platelet number and counteracts the abnormal bleeding that characterizes these mice. Subtle differences in thrombopoiesis were observed in STIM1 p.I115F mice, but the main difference between wild-type and STIM1 p.I115F mice was in platelet clearance and in the levels of platelet cytosolic basal Ca2+. Both were restored on treatment of animals with CIC-39Na. This finding paves the way to a pharmacological treatment strategy for thrombocytopenia in tubular aggregate myopathy patients.

STIM1 and ORAI1 mutations leading to tubular aggregate myopathies are sensitive to the Store-operated Ca2+-entry modulators CIC-37 and CIC-39.

Gain-of-function mutations on STIM1 and ORAI1 genes are responsible for an increased store-operated calcium entry, and underlie the characteristic symptoms of three overlapping ultra-rare genetic disorders (i.e tubular aggregate myopathy, Stormorken syndrome, York platelet syndrome) that can be grouped as tubular aggregate myopathies. These mutations lead to a wide spectrum of defects, which usually include muscle weakness and cramps. Negative modulators of store-operated Ca2+-entry targeting wild-type STIM1 and ORAI1 have entered clinical trials for a different array of disorders, including pancreatitis, COVID-19, cancer, and autoimmune disorders and, while efficacy data is awaited, safety data indicates tolerability of this STIM1/ORAI1 mutations are amenable to pharmacological intervention. If this were so, given that there are no approved treatments or clinical trials ongoing for these rare disorders, it could be envisaged that these agents could also rehabilitate tubular aggregate myopathy patients. In the present contribution we characterized the Ca2+-entry patterns induced by eleven STIM1 and three ORAI1 mutations in heterologous systems or in patient-derived cells, i.e. fibroblasts and myotubes, and evaluated the effect of CIC-37 and CIC-39, two novel store-operated calcium entry modulators. Our data show that all STIM1 and ORAI1 gain-of-function mutations tested, with the possible exception of the R304Q STIM1 mutation, are amenable to inhibition, albeit with slightly different sensitivities, paving the way to the development of SOCE modulators in tubular aggregate myopathies.

Extracellular nicotinamide phosphoribosyltransferase boosts IFNγ-induced macrophage polarization independently of TLR4.

Nicotinamide phosphoribosyltransferase (NAMPT), alongside being a crucial enzyme in NAD synthesis, has been shown to be a secreted protein (eNAMPT), whose levels are increased in patients affected by immune-mediated disorders. Accordingly, preclinical studies have highlighted that eNAMPT participates in the pathogenesis of several inflammatory diseases. Herein, we analyzed the effects of eNAMPT on macrophage-driven inflammation. RNAseq analysis of peritoneal macrophages (PECs) demonstrates that eNAMPT triggers an M1-skewed transcriptional program, and this effect is not dependent on the enzymatic activity. Noteworthy, both in PECs and in human monocyte-derived macrophages, eNAMPT selectively boosts IFNγ-driven transcriptional activation via STAT1/3 phosphorylation. Importantly, the secretion of eNAMPT promotes the chemotactic recruitment of myeloid cells, therefore providing a potential positive feedback loop to foster inflammation. Last, we report that these events are independent of the activation of TLR4, the only eNAMPT receptor that has hitherto been recognized, prompting the knowledge that other receptors are involved.