Progetti PRIN

The Glyoxalase pathway in cystic fibrosis: assessment of its role in the disease for the identification of novel targets to treat oxinflammation.

Finanziatore: MUR
Responsabile Scientifico: ANTOGNELLI Cinzia
Finanziamento: € 87.600 €
Durata:2023-2025

Summary: Cystic fibrosis (CF) is a genetic autosomal recessive disease caused by a defect in the CF transmembrane regulator (CFTR) gene. It is a multi-organ disease that affects the glandular secretion system and is mainly associated with a reduced lung function. Chronic inflammation, oxidative stress (OxS) and abnormal susceptibility to infections are pathogenic traits of CF, causing severe tissue damage. Notably, a strong interplay between inflammation and OxS occurs, inducing one another in a precise feedforward loop that aggravates tissue impairment. Although of great interest, anti-inflammatory therapies have shown limited efficacy in CF patients. Therefore, the identification of specific compounds that can reduce both OxS and inflammation (oxinflammation) could be an appealing approach to elaborate novel treatment strategies in this devastating disease. To this aim, the identification of novel pathways underpinning oxinflammation is mandatory. In this project the role of the glyoxalase pathway, consisting of glyoxalase 1 (Glo1) and glyoxalase 2 (Glo2), will be investigated. Why? Since the glyoxalase pathway is involved in the removal of the potent pro-oxinflammatory and glycating agent methylglyoxal (MG), it could represent a potential target to dampen MG-dependent oxinflammation in CF. Importantly, we have recently observed that the expression and activity of Glo1 are defective in murine and human bronchial epithelial cells models of CF upon challenge with Aspergillus fumigatus, a common pathogen in CF, and this is associated with pathogenic inflammation. Moreover, we have shown that anakinra, the recombinant version of the interleukin-1 receptor antagonist and an activator of indoleamine 2,3 dioxygenase (IDO)1, enzyme involved in immune tolerance and dysfunctional in CF, is able to restore Glo1 mRNA expression in CF mice upon aspergillosis, significantly mitigating CF pathogenic inflammation. Finally, in preliminary studies, we observed a concomitant Glo1 down- and Glo2 up-regulation in IDO1 knockout mice infected with A. fumigatus, as compared to A. fumigatus-infected WT mice. These data suggest a possible link between IDO1 and the glyoxalase pathway to be intriguingly explored in CF. The role of Glo2 in CF has never been investigated before. In order to dissect the role of the glyoxalase pathway in CF for the identification of novel targets to treat oxinflammation, we will take advantage of in vitro (CFBE41o-cells transduced with p.Phe508del-CFTR or WT-CFTR), in vivo (CftrF508del and C57BL/6 mice) and ex vivo (HBE cells from CF patients and controls) CF models and controls, both in the absence or presence of A. fumigatus conidia infection, and use Glo1, Glo2 and IDO1 modulators, including bioactive natural compounds. Our goal will be to identify a novel druggable IDO1/Glo1-Glo2-based patho-mechanism to elaborate safe and low-cost pharmacological therapies to dampen CF pathogenic oxinflammation.

The microbiome in Breast cAnceR therapy and potential for pRobIOtics to improve treatment outcome.

Acronyme: BARRIO
Finanziatore: MUR
Responsabile Scientifico: ARISTEI Cynthia
Finanziamento: € 100.231
Durata: 2023-2025

Summary: Breast cancer (BC) is the most frequent type of cancer in women. It is considered strictly linked to different risk factors such as familial history, eating habits, general lifestyle, and hormone replacement. Furthermore, genetics plays a role in a minority of cases. Interestingly, not all cases may be related to the known listed above risk factors. Therefore, in recent years, several groups of researchers have investigated a correlation between patient microbiome and BC. Altered composition of gut and breast microbiome represent a new risk factor in the occurrence of BC and may hinder therapeutic treatment. Moreover, the role of microbiome in the development and maintenance of inflammation, estrogen metabolism, and epigenetic alterations is under investigation. However, studies need to be conducted to exploit the role of microbiome in Radiation Therapy (RT) in BC. The success of BC RT may be dependent on both the microbiome niche in breast tissue as well as in the gut. - Hypothesis Since the sensitivity and outcome of BC therapy is strictly related to the environment, this may be associated with the microbial signature. Modulation of the microbial signature may impact the outcome of BC therapy. Thus, the aims of this proposal are: 1. To identify: a. the microbial signature in BC patient subtypes in a cohort of early stage BC patients b. microbial changes induced during BC treatment c. microbial changes during the evolution of the cancer itself post-RT 2. To correlate the metagenomic signatures with immune profiles of the mammary breast tissue 3. To establish patient-derived organoids, containing immune cells from the tumor microenvironment for validation of the results obtained in the human cohort 4. To establish a mouse model of BC undergoing RT and administered with probiotic and polyphenol treatments to investigate potential in improving BC therapy outcomes - Experimental Design This study employs a two-pronged approach (Fig. 1). A cohort study will be established with BC patients focused on women undergoing breast-conserving therapy. We aim to study both the gut and breast tissue microbiome and understand how BC radiotherapy may impact on the microbiome through the collection of fecal samples, and the 'nipple aspirate fluid' (NAF) during the course of therapy. In addition, tumor specimens from BC patients collected before commencement of therapy will be used for immune signature profiling and the generation of organoids to establish a BC-tumoroids biobank. Healthy breast tissue from women undergoing elective reduction mammaplasty will be used as control for comparison of tumor specimens. Concurrent animal studies will be done to establish a suitable mouse BC model to investigate the potential of probiotic and polyphenol treatment in improving BC radiation therapy outcomes.

Extracellular Vesicles In Slowing down the Ticking clock of Aging

Finanziatore: MUR
Responsabile Scientifico: BELLEZZA Ilaria
Finanziamento: € 130.451
Durata: 2023-2025

Summary: Age-associated chronic diseases are placing a tremendous burden on healthcare systems. The dissection of mechanisms driving aging and the development of novel therapeutics are urgent needs. Aging is characterised by the loss of stem cells (SCs) regenerative capacity which impairs the ability to respond to stress. However, physiological aging, which does not always result in age-associated diseases, differs from accelerated aging, which compromise tissue functions later in life. Thus, a cellular model recapitulating accelerated SCs aging will permit to delineate the molecular signature of age-associated diseases. In this scenario, oral mucosa derived epithelial SCs (OMESCs) from patients affected by EEC syndrome, represent a valuable resource since they undergo an accelerated aging process. All cells, including SCs, produce and secrete extracellular vesicles (EVs), membrane-enclosed particles devoted to cell-cell communication. EVs cargo composition is affected by the aging process. Our project aims to analyse EVs derived from SCs at different aging stages and their potential to control the behaviour of immune, endothelial and SCs to identify molecular pathways to be tackled for assuring healthy aging. To reach these goals, the project will be divided in 4 work packages (WP) developed by two research units (RUs) based in University of Perugia and University of Padua. Fondazione Banca degli Occhi del Veneto ONLUS, providing OMESCs cells for functional analyses and their conditioned medium for EVs isolation, will be involved as subunit. In WP1 we will perform the analysis of OMESC at different aged stages and the characterization of their EVs. We will obtain a deep miRNomic and proteomic characterization of EVs from OMESCs at different stages of aging. In WP2 we will elucidate whether OMESC-EVs differentially alter inflammatory responses and endothelial cell fitness. In WP3 we will define the role of OMESC-EVs on corneal keratinocytes fitness. In WP4 we aim to identify molecular candidates for the observed effects, paving the way for the identification EVs-carried molecules that can be recognized as targets for the prevention of aging-associated diseases. This project is expected to give important insights into the involvement of EVs in the mechanisms of aging induced by the accelerated epithelial SC aging. The novelty of this project resides in the molecular and functional characterization of EVs isolated from a model of prematurely aged SCs. The extensive functional portrait of the EVs from our cell models, will contribute to understand the effects on target cells which, together with the deep molecular characterization of the EVs cargo will be fundamental to define the molecular cargo selectively included in EVs during the aging process. The discovery of the EVs molecular signature of aging may allow the identification of druggable pharmacological targets and propose EVs administration as cell-free option to prevent accelerated epithelial aging.

Investigating the role of GBAR1, FXR and RORγt receptors in the pathogenesis of inflattory bowel diseases (IBDs) for the discovery of new therapeutic options

Finanziatore: MUR
Responsabile Scientifico: BIAGIOLI Michele
Finanziamento: € 120.000
Durata: 2023-2025

Summary: In the last decades the onset of several chronic inflammatory disorders is increased in the worldwide population due to unhealthy lifestyles, genetic predisposition or immune system malfunction. Inflammatory bowel disease (IBD), encompassing ulcerative colitis (UC) and Crohn’s disease (CD), is a chronic condition that displays relapsing-remitting chronic inflammation of the gastrointestinal tract. Although the aetiology of IBD remains largely unknown, its pathogenesis is associated with dysregulated immune responses that drives a persistent inflammatory state within the intestinal mucosa. The clinical course of this disease has shown to be variable and often difficult to manage. Furthermore, IBD increases long-term risk of developing colitis-associated cancer. Currently, there is no definitive treatment for this disease therefore the discovery of new targets, involved in the inflammatory process, and new chemical entities able to interact with them represents an important starting point for our proposed drug discovery program. Several evidences suggest that gastrointestinal homeostasis is strongly dependent on nuclear receptors (NRs) and G protein-coupled receptors (GPCRs) functions, resulting potential drug targets for therapeutic interventions in patients with IBD. These receptors, such as PPAR, VDR, FXR, RORγt and GPBAR1, play a variety of roles ranging from nutrient absorption and transport, sensing of microbial metabolites, to regulation of immune response and intestinal cell integrity. The present research program is focused on the development of a novel polypharmacology strategy for the treatment of IBD based on the restoration gut anti-inflammatory pathways by targeting some NRs and GPCRs. In particular, we will focus our attention on the discovery of new GPBAR1, RORγt and FXR modulators involved in gastrointestinal inflammation. The proposed project has a multidisciplinary and multi-target architecture combining integrated and complementary expertise in computational methods (virtual screening, molecular dynamics), chemical synthesis and experimental pharmacology evaluations in vitro and in vivo.

Translating Genetically Encoded Engineered Proteins Into Therapeutic Strategies For Brain Aging And Related Cognitive Decline

Finanziatore: MUR
Responsabile Scientifico: BOCCARDI Virginia
Finanziamento: € 53.768
Durata: 2023-2025

Summary: Aging represents the major risk factor for most chronic diseases, including dementia. At the cellular level, aging is characterized by the accumulation of molecular damages leading to the so-called Age-Related Changes (ARCs). Brain ARCs can lead to cognitive decline, which have been demonstrated to strongly correlate with alterations of functional and structural plasticity at glutamatergic synapses, mostly hosted at dendritic spines. In this context, cellular senescence driven by mitochondrial and bioenergetic defects and telomere biology impairment may play a detrimental role. Two proteins, i.e., LIMK1 and CREB, are hubs on which converge critical molecular pathways involved in synaptic functions and cellular bioenergetics, thus being central for neuronal plasticity and memory. Remarkably, the expression of both LIMK1 and CREB was significantly reduced with age; thus, strategies to correct their functional impairment might help in rescuing cognitive and learning functions. This project gathers research groups entirely composed of young researchers with complementary expertise and skills in the fields of cellular and molecular neuroscience to validate a toolbox of genetically encoded engineered proteins (GEEPs), whose activity can be controlled in neurons to harness spine plasticity and modulation of transcriptional factors required for memory formation, thus counteracting their age-related loss. The viability of this approach is supported by our preliminary results obtained by engineering LIMK1, a kinase critically involved in actin filament dynamics. Spatiotemporal GEE-LIMK1 activation in organotypic hippocampal slice cultures induced long-term enlargement of dendritic spines and boosted glutamatergic synaptic transmission. Similarly, we developed GEE-CREB to regulate the transcription of specific genes required for memory formation that, in our preliminary data, contrasted some aspects of the neuronal senescence. The proposed strategies are potentially important for both brain research and therapy allowing the control of neuronal activity by targeting cells with exogenous inert proteins that are engineered to respond selectively to a small-molecule agonist. Moreover, the use of a safe and well-tolerated clinically approved agonist such as rapamycin that i) crosses the blood-brain barrier, ii) activates the engineered proteins and iii) has well-known slow-aging beneficial effects, represents an additional strength of our proposal. To achieve our goal, we will test the ability of GEEPs to boost intracellular signaling regulating synaptic plasticity in living neurons (AIM1). Then, we will validate, in vivo, the therapeutic potential of GEEPs in aged mice characterized by cognitive deficits and synaptopathy (AIM2). Since brain aging still lacks efficacious pharmacological treatments, our study will boost further research into next-generation genetically encoded drugs as novel therapeutic strategies for brain aging and related cognitive decline.

New therapeutic approaches in Pediatric Immune Thrombocytopenia: role of miRNA loaded MSC-derived MVs

Finanziatore: MUR
Responsabile Scientifico: BRUSCOLI Stefano
Finanziamento: € 100.500
Durata: 2023-2025

Summary: Immune thrombocytopenia (ITP) is the most common childhood piastrinopenia, caused by immune platelets destruction. It is characterized by immune system and inflammatory response compromise, determined by a prevalence of M1 pro-inflammatory macrophages and Th1 lymphocytes, and also mesenchymal stromal cells (MSCs) impairment. MSCs have immunosuppressive and anti-inflammatory properties. ITP MSCs are strongly impaired. Glucocorticoids (GCs) are the mainstay therapy for pediatric ITP with long term side effects. GCs regulate innate and adaptive immunity cells and also MSCs. GCs effects are in part mediated by Glucocorticoid-Induced Leucine Zipper (GILZ). MSCs produce secretome, constituted by cytokines, proteins, and extracellular vesicles (EVs). EVs contain several molecules, among them miRNAs, and are involved in several physiological and pathological conditions. MSCs-derived EVs (microvesicles (MVs) and exosomes) have been tested in inflammatory or autoimmune diseases. Our study aims to identify a novel and safety therapeutic strategy for pediatric ITP using MSC-derived MVs loaded with miRNA involved in ITP pathogenesis. We will enroll newly diagnosed ITP patients and healthy donors (CTR) to obtain MSCs, lymphocytes and macrophages. We will isolate and characterize MVs from CTR-MSCs and ITP-MSCs secretome and obtain in vitro co-cultures (CTR and ITP-MSCs-derived MVs with ITP lymphocytes and macrophages). We will analyze cytokines release, lymphocytes viability, cell cycle progression and phenotype switch, and macrophages phenotypes. We expect to observe an alteration of MSC-derived MVs in their immunomodulating and anti-inflammatory properties in pediatric ITP. We will perform a small-RNA sequencing of CTR and ITP MSC-derived MVs to identify differentially expressed miRNAs, potentially responsible for observed functional alterations and we will transfect these MVs with identified miRNAs (miRNA-loaded MVs). They will be used to realize co-cultures with ITP macrophages and lymphocytes, and then we will evaluate viability, cell cycle progression, cytokine release and phenotype switch on lymphocytes and macrophages. We expect to observe an amelioration of immune and inflammatory profiles. We will also compare miRNA loaded MSC-derived MVs effects with ITP standard therapy. We will perform in vivo experiments on ITP passive murine model, injecting miRNA-loaded MVs and then monitoring disease progression. We will test whether combinatory therapy with GCs and miRNA-loaded MVs will induce an improvement of the parameters for the remission of the disease. Moreover, we will test whether deletion or over-expression of GILZ in the ITP mouse model affects the MSC-derived MVs composition. We expect that miRNAs expression in MSC-derived MVs is regulated by GCs and dependent by GILZ. This study will be the first one investigating the role of miRNA-loaded MVs in pediatric ITP.

Translating Genetically Encoded Engineered Proteins Into Therapeutic Strategies For Brain Aging And Related Cognitive Decline

Finanziatore: MUR
Responsabile Scientifico: CELLINI Barbara
Finanziamento: € 72.144
Durata: 2023-2025

Summary: Kidney stones, most of which are formed by calcium oxalate, affect up to 10% individuals worldwide. Oxalate is a metabolic end-products excreted by urine. High urinary oxalate concentrations lead to hyperoxaluria, which thus represents a major risk factor for kidney stones. The amount of urinary oxalate depends on both endogenous production and exogenous absorption by dietary intake, both of which could be impaired leading to pathological conditions. Indeed, increased endogenous oxalate formation, due inherited deficits of enzymes involved in the hepatic metabolism of the oxalate precursor glyoxylate, leads to Primary hyperoxaluria (PH). On the other hand, increased intestinal absorption of exogenous oxalate, e.g. due to fat malabsorption, leads to secondary hyperoxaluria (SH). In other cases hyperoxaluria is idiopathic and causes recurrent kidney stones or contributes to the onset of end-stage kidney disease (ESKD) in other pathologies. Thus, the identification of genetic biomarkers influencing the onset and/or the progression of hyperoxaluria represents an unmet medical need. Our project combines molecular, preclinical, and clinical approaches to (i) investigate the biochemical effects of genetic variations on proteins involved in oxalate metabolism, (ii) unravel their association with the onset and/or evolution of hyperoxaluria, and (iii) pave the way for new therapies in patients at risk. By whole exon sequencing analyses of different cohorts of patients we will investigate if genetic variations of genes involved in hepatic oxalate metabolism (AGXT1, AGXT2, GRHPR, HOGA1, and HAO1), exogenous oxalate transport (SLC26A1, SLC26A3, SLC26A6, SLC4A1) or both, could represent risk factors for the onset of SH, modulate severity and progression of PH, and affect the onset of idiopathic hyperoxaluria and ESKD, respectively. We will perform a detailed analysis of the effects of the identified genetic variations at protein level with multiple approaches ranging from analyses in silico and experiments in purified proteins to cellular models mimicking the natural environment of each protein. We will then pave the way for the development of a new treatment approach based on the use of an available engineered form of oxalate decarboxylase (EngOxDC), showing highly improved stability under physiological conditions. We will test the ability of EngOxDC to degrade both exogenous and endogenous oxalate, using an in-vivo model of SH or ex-vivo models of PH, respectively. The complementary background of the participating units and their previous collaboration, along with the expertise in the field and solid preliminary data, guarantee project feasibility. The expected results will have important relapses for a better understanding of oxalate metabolism and inter-individual variability in hyperoxaluria, the identification of patients at risk, and the development of new treatment strategies that could prevent or mitigate the symptoms.

Immune and Microbial Profiles as Novel determinants of Tumor Progression of Oral Squamous Cell Carcinomas - IMProNT-OSCC

Finanziatore: MUR
Responsabile Scientifico: DI SANTE Gabriele
Finanziamento: € 64.000
Durata: 2023-2025

Summary: Even if they are pretty homogeneous as for histology (more than 90% are squamous cell carcinomas, SCC), head and neck cancers, altogether the 6th most frequent malignancy worldwide, are a very heterogeneous group of tumors. The most relevant heterogeneity factor is primary site. In the present project, we focus on SCC of the oral cavity (OSCC), whose prognosis slowly slightly improved in the last decades, in association with refinements in surgery, which remains the mainstay of treatment, and multimodality treatments. However, differently from oropharyngeal SCC, with an inclusion of molecular markers, and in particular HPV, in the diagnostic work up and staging, predictors presently in use for OSCC are only “classical” clinical and histopathological ones. This configures an unmet need of additional predictors in OSCC. The oral cavity harbors one the most diverse microbiota including more than 770 species of bacteria. Some of them have been associated with certain cancers, as is the case of Fusobacterium nucleatum (Fn), a species highly represented in the oral cavity, in colorectal cancer (CRC). An association of Fn also with OSCC has been reported. However, when it comes to prognostic prediction, the Fn role seems opposite. In fact, recently two different groups independently reported consistent results concerning a protective role of Fn in patients with OSCC, with a better prognosis and a markedly reduced nodal metastatic potential in cases with a higher representation of Fn in tumors. These findings support a role of microbiota, and of Fn in particular, in influencing the interaction of immune cells with tumor cells, which has been postulated to play a pivotal role in OSCC progression. Indeed, a novel histopathologic classifications of OSCC has been proposed based on the assessment of the inflammatory and lymphocytic cell infiltrates as a part of their grading system (immunoscore), with a promising prognostic value in early mobile tongue cancer. The goal of the present project is to look for novel reliable risk factors, predictors and prognostic markers by defining the role of microbiota and associated immune profile as determinants of progression of OSCC. We aim at these targets by defining changes in microbiota among precancerous lesions, small and bulky OSCC, and normal mucosa of the same areas, to obtain information about its role as cause or consequence of tumor progression. Patients and controls will be enrolled, evaluated, treated and followed up according to the main international guidelines, in a multidisciplinary setting, with a standardized prospective data collection. Swabs, blood, primary tumor and lymph node samples will be obtained. We will evaluate the impact of oral microbiota on the immune system in OSCC patients. Finally, we will analyze correlation of microbiota and associated immune responses with known relevant clinical and histopathological parameters and ultimately with prognosis.

Cellular and molecular mechanisms governing immune tolerance to protein and gene replacement therapy inhemophilia (TolerEight)

Finanziatore: MUR
Responsabile Scientifico: FALLARINO Francesca
Finanziamento: € 116.000
Durata: 2023-2025

Summary:Protein and Gene Therapies (GT) are innovative treatment options for numerous diseases, with several products recently receiving market authorization. The hosts immune response to delivered proteins, vectors, and transgenes, however, remains a major obstacle, with knowledge of the major pathways regulating this response, lacking. To overcome current obstacles in this field, a joint effort by expert scientists from various disciplines is needed. Hemophilia A (HA), a hemorrhagic disorder mainly caused by F8 gene mutations, is being extensively approached by protein and GT and represents an ideal model to address these issues. One of the most challenging complications (>35–40%) is the development of inhibitory alloantibodies against the infused FVIII protein, and the immune response to viral vectors and F8 transgene following GT. The TolerEight project, based on the collaboration of 2 scientific teams with interdisciplinary expertise in gene therapy and immune response in HA, aims to join forces to conduct an ambitious research program that will lead to the identification of the key cellular and molecular mechanisms involved in long term acceptance of advanced therapies for HA. TolerEight will achieve this by taking advantage of state-of-the-art methodologies, innovative tools, and in vivo preclinical models, including new GT vectors targeting F8 to specific tissues or cells, Tg mouse models for HA expressing trace amounts of residual FVIII, and innovative conditional mouse models devoid of selected cells and immune cellular pathways. Our interdisciplinary approach aims at deciphering intra-and intercellular signaling synergies involved in immune tolerance to the successful and durable FVIII therapies. Specifically, Tolereight aims at: i. defining the role of antigen-presenting cells (APCs) and checkpoint pathways in the immune response to FVIII following FVIII protein replacement and GT; ii. dissecting the impact of residual FVIII expression in anti-FVIII immune responses following protein and gene replacement therapy; iii. to translate the experimental findings to HA patients. If successful, even in part, the project will provide a roadmap that will advance both basic and translational knowledge on the major molecular and cellular determinants of immune responses to FVIII protein and gene replacement therapies for HA. This in turn will help in predicting the immune response and favor immune tolerance tailored to FVIII patients. Further, these findings may also offer hope to many other genetic disorders in the development and optimization of future innovative therapeutic strategies, with significant benefits for both patients and national healthcare systems.

Impact of SARS-CoV-2 variability on antiviral agents

Finanziatore: MUR
Responsabile Scientifico: FRANCISCI Daniela
Finanziamento: € 37.279
Durata: 2023-2025

Summary:Despite massive worldwide vaccination, SARS-CoV-2 infections still occur at a high pace. Repeated vaccine doses protect most individuals from severe disease, however protection from infection is suboptimal. Thus, viral spread remains sustained by both unvaccinated and vaccinated individuals, with morbidity and mortality driven by local differences in vaccine coverage. Factors contributing to the limited success of vaccination include (i) rapid waning of natural and artificial immunity to coronaviruses, (ii) newly emergent SARS-CoV-2 variants with increased adaptation to humans and (iii) use of obsolete vaccine preparations based on the ancestral Wuhan wild type viral strain from which subsequent SARS-CoV-2 variants have substantially diverged. Indeed, SARS-CoV-2 has shown a striking ability to evolve into newly divergent variants which may become dominant in a matter of weeks, challenging the strategy of updating vaccines to cope with virus variability. Antiviral treatments have become available, mostly in the last few months, as an eagerly awaited complement to vaccination. These include monoclonal antibodies (mAbs) and direct acting antivirals (DAAs), both to be administered within a few days from diagnosis to people at risk of progression to severe disease. In addition, one mAb cocktail has been approved as pre-exposure prophylaxis in high-risk subjects who are unlikely to mount an adequate immune response to COVID-19 vaccination or for whom COVID-19 vaccination is not recommended. In light of SARS-CoV-2 variability and evolution, it is crucial to define the activity of mAbs and DAAs against current and future viral variants as well as the potential for viral resistance to such treatments. This proposal aims at investigating the following areas: 1. Activity of mAbs and DAAs vs. SARS-CoV-2 variants in vitro 2. Genetic barrier to resistance to mAbs and DAAs in vitro 3. Emergent resistance to mAbs and DAAs in vivo 4. Persistence of mAb activity in vivo over time The first two activities will be performed at the University of Siena (UNISI) where prototype viral variants, anti-SARS-CoV-2 DAAs and mAbs are available and dedicated lab assays have been developed. Activities 3 and 4 will be performed through observational studies led by researchers at the University of Padua (UNIPD) and Perugia (UNIPG) who have been extensively involved in SARS-CoV-2 clinical research. Importantly, the three academic groups have been collaborating for more than one year in this research area, thus connections functional to the conduct of the study are well established. Gaining knowledge in the above areas is fundamental to define the role of anti-SARS-CoV-2 treatment as a complement of the continued global vaccination campaign. In addition, the combined investigation of natural and drug-selected virus variability will be an extraordinarily effective way to define the viral genetic space and the potential for virus evolution in the upcoming years.

Novel player in thromboinflammation: the multi faced role of proline-rich tyrosinekinase Pyk2

Finanziatore: MUR
Responsabile Scientifico: LA STARZA Roberta
Finanziamento: € 96.141
Durata: 2023-2025

Summary: Besides their role in haemostasis and thrombosis, blood platelets are the first sentinels against microbial infection and rapid responders to pathogen entry with important functions in primary immunity. However, when their reactions go beyond physiologic functions they give rise to thromboinflammation (TI), a pathological condition in which inflammation and thrombosis reinforce each other. TI is the major determinant of severe sepsi-associated complications, like disseminated intravascular coagulation and organ failure, which correlate with high morbidity and mortality. TI is started by complex interactions between platelets, leukocytes, endothelial cells (EC), and the innate immune system. The unravelling of the molecular mechanisms of these interactions is crucial for the development of novel therapeutic strategies to reduce sepsis-associated mortality. While several studies have explored the receptors involved in these interactions, little information is available on the intracellular signalling pathways that regulate these events, and some clues suggest that the proline-rich tyrosine kinase Pyk2 may play a central and multifaceted role. We have previously shown that Pyk2 is a crucial intracellular mediator of platelet activation and thrombus formation. Pyk2 is also involved in acute and chronic inflammation and preliminary unpublished data by the proponents indicate a role of Pyk2 also in TI-driven systemic infection. In this project we plan to get deeper insights into the mechanisms of generation and progression of TI in sepsis focusing on the roles of Pyk2 in platelets, leukocytes and EC and in their reciprocal crosstalk. To this aim two Research Units, with complementary and integrated expertise and previous successful collaborations, will investigate the role of Pyk2 in TI using a translational approach that conjugates cellular biochemistry and cell biology experiments, in vivo mouse models, and clinical studies. The project will be divided into 3 workpackages aiming to: 1) explore the role of Pyk2-dependent signalling in platelets and in their crosstalk with leukocytes and EC. The generation of proinflammatory and prothrombotic mediators during TI will be analyzed. This WP will be carried out using advanced biochemical, cell biology and cell imaging techniques, and will take advantage of validated Pyk2-specific pharmacological inhibitors and selective miRNA. 2) investigate the role of Pyk2 in two complementary murine models of sepsis in vivo: endotoxemia and cecal ligation and puncture models. This WP will be realized using Pyk2 knockout mice and ad hoc generated chimeric mice. 3) clarify the contribution of Pyk2 in human sepsis-associated TI by analyzing Pyk2 expression and activation in platelets from septic patients Our results will help to identify novel therapeutic targets for the improvement of sepsis-related outcomes and the prevention of sepsis-associated thrombotic complications.

Development of a new antibacterial nano-filled composite material for dental treatment of vulnerable patients: biological, mechanical and microbiological characterization

Finanziatore: MUR
Responsabile Scientifico: MARINUCCI Lorella
Finanziamento: € 62.500
Durata: 2023-2025

Summary:Low socio-economic status is related to sanitary vulnerability, particularly in relation to oral diseases. Dental caries, the most common non-communicable disease, are more severe in vulnerable populations. To protect and treat these patients, specialized intervention strategies have been established to achieve good results quickly and with low-cost materials. These materials can be easily used even with non-cooperative patients, such as Minimally Invasive Treatments (MITs). However, composite resins are the most widely used restorative materials in conservative dentistry for the restoration of therapeutic cavities after caries removal. The composition of these materials has undergone various modifications over the years to improve their mechanical and aesthetic properties. However, given the possibility of recurrent caries due to bacterial infiltration at the margin between the restoration and the tooth, it is important to evaluate the addition of antibacterial molecules to the composition of these restorative materials. This would improve the condition of socio-economically vulnerable patients, who are more prone to secondary caries, as well as "special needs" patients. This study aims to determine the optimal concentration of silver-based agents to obtain a composite that combines good antimicrobial and mechanical properties.

Investigating DNA methyltransferase 3A (DNMT3A) gene mutation for its essentiality in acute myeloid leukemia (AML) maintenance, associated-pharmacogenomic profile and potentiality in human AML development

Finanziatore: MUR
Responsabile Scientifico: MARTELLI Maria Paola
Finanziamento: € 112.000
Durata: 2023-2025

Summary:DNMT3A mutations (mDNMT3A) are one of the most frequent genetic alterations of the epigenetic modifiers in acute myeloid leukemia (AML). mDNMT3A are found in age-related clonal hematopoiesis, act as loss-of-function (LOF) mutations and are pre-leukemic genetic lesions. In murine models, knock-out (KO) of DNMT3A in hematopoietic stem cells (HSCs) confers in vivo immortality, with a self-renewal potential far exceeding that of normal HSCs, and a gradual and focal losses of DNA methylation in regions associated with self-renewal genes, recapitulating the effects of mDNMT3A in human diseases. However, human models of mDNMT3A-driven leukemogenesis are lacking. Due to its role in AML development, great efforts are made to develop drugs targeting mDNMT3A, although its value as therapeutic target needs to be proven. In this setting, our project is timely and addresses key unanswered questions: i) can DNMT3A mutant be considered a valid therapeutic target in AML?; ii) is DNMT3A mutation associated with a specific pharmacogenomic profile?; iii) how DNMT3A LOF condition the normal human HSCs? Aiming at validating DNMT3A as target for therapy, we have recently applied CRISPR-based genome editing to correct the DNMT3A mutated gene into wild-type n the OCI-AML3 AML cell line, carrying DNMT3A-R882C mutation, and found no significant differences between the edited and parental clones in terms of leukemia growth, either in vitro or in vivo in xenograft immunocompromised mice, suggesting that leukemia disengages from the mutation. Therefore, our hypothesis is that whilst on one side mDNMT3A may condition acquisition of new mutations, AML onset and response to drugs, on the other side they may be not essential for maintenance of AML, once this has established. The project is organized in 4 main work packages (WP). In WP1, CRISPR-based genome editing will be used to correct mDNMT3A in different human AML cell lines. Cell lines models will be then functionally studied both in vitro and in vivo and deeply characterized. In particular, methylation, transcriptomic and epigenomic/epitrascriptomic profiles will be analyzed using standard approaches and third-generation long-read sequencing (LRS). Results will be validated in 3 patient-derived xenograft (PDX) models of DNMT3A-mutated AML by CRISPR-based DNMT3A KO followed by re-expression of DNMT3A wild-type by lentiviral infection. In WP2, we will screen the above models for FDA-approved/epigenetic drugs libraries to identify an eventual mDNMT3A-associated pharmacogenomic profile. In WP3, CRISPR-based DNMT3A KO (mimicking DNMT3A LOF mutations) will be performed on human HSCs, and cells will be serially transplanted in mice and studied overtime in order i) to dissect time-dependent changes induced by DNMT3A KO in human hematopoiesis, ii) to capture the eventual spontaneous onset of secondary mutations, and iii) to evaluate whether human AML develops. In WP4, results will be validated in a cohort of AML patients.

Potential role of urinary microRNAs as diagnostic and prognostic biomarkers for Renal Cell Carcinoma

Finanziatore: MUR
Responsabile Scientifico: MEARINI Ettore
Finanziamento:€ 136.650
Durata: 2023-2025

Summary:Renal cell carcinoma (RCC) represents about 3-5% of all tumors, and Western countries account for 50% of the global incidence. In 2020, over 430,000 cases of RCC were diagnosed worldwide and about 180,000 people died from this disease. About 70% of RCCs showed clear cell RCC (ccRCC) as a widespread histological type. Currently, due to lack of symptoms at early stages, about 60% of RCC are diagnosed occasionally by imaging and about 20% are metastatic already at diagnosis, affecting patient survival. Recently, it has been proved that microRNAs (miRNAs) can be a potential marker for several types of cancer including RCC. MiRNAs are a class of small non-coding single-strand RNAs inhibiting RNA translation and promoting RNA degradation. Interestingly, the role of miRNAs in cancer and their stability in biological fluids make them promising biomarkers for cancer diagnosis and prognosis. In 2020, we have published a new method for the diagnosis of ccRCC based on the evaluation of urinary miR-122, miR-1271, and miR-15b in a discovery cohort. Afterward, these promising results have been also confirmed in an independent validation cohort that were published in 2022. This project aims to improve the above-mentioned method for diagnosis and prognosis by evaluating in the urine (1) the levels of miR-122, miR-1271, and miR-15b and other three urinary miRNA (miR-15a, miR-210, miR-let-7b) in ccRCC patients and healthy subjects by the use of Reverse Transcription and quantitative real-time PCR (RT-qPCR); (2) the levels of other miRNAs selected by Microarray Assay in different subtypes of RCC. The enrollment will include 300 patients in 18 months. The recruited population will include 160 patients with any histological RCC, 60 patients with benign renal mass, and 80 healthy controls. Each subject will withdraw urine in the routine visits. The urine collection from patients will be performed before surgery, 5 and 25 days after surgery, and then at 3, 6, 12 e 18 months of follow-up. To further improve the method and identify a specific panel of miRNAs associated with different renal cancer histological subtypes, Microarray Assay will be carried out in urine samples from patients with these tumor subtypes. Finally, the best miRNAs, selected by Microarray Assay, will be validated by RT-qPCR on a larger number of patients. The selected panel of miRNAs in the urine will be included in an algorithm for the following purposes: 1) to make the diagnosis of RCC; 2) to correlate the miRNA levels with tumor staging including the presence of metastasis, 3) to correlate the miRNA levels with the diagnosis of local and distant recurrence during the follow-up. Therefore, the final objective of the project is to develop a new diagnostic and prognostic tool for RCC that would be inexpensive (RT-qPCR-based) and could be performed on easily available biological materials through non-invasive techniques.

PSenotherapeutics to prevent cognitive decline. A randomized study of metformin cycles in subjects with mild cognitive impairment

Finanziatore: MUR
Responsabile Scientifico: MECOCCI Patrizia
Finanziamento:€ 138.000
Durata: 2023-2025

Summary: Dementia is the main cause of disability in older adults, currently affecting about 50 million people worldwide with this number estimated to triple in the next 30 years. The preclinical stage, Mild Cognitive Impairment (MCI), has become a major research focus for prevention and treatment. The identification of pathways within the biology of aging represents an important endpoint for the development of novel effective disease-modifying drugs in the field. In recent years metformin (MET), which is the recommended first-line oral therapy for the treatment of type 2 diabetes (T2DM) after diet and lifestyle changes, has shown senotherapeutic properties as it prevents cellular senescence and related inflammation. Moreover MET use is associated with a reduced risk of cognitive impairment and dementia in T2DM and pre-clinical data suggest that MET could affect the pathogenesis of dementia beyond its efficacy on glycaemic control. Thus, the identification of key networks that are associated with MET senotherapeutic functions and prevention of cognitive decline is an area of intense research. We aim at investigating whether the MET intervention may attenuate or ameliorate cognitive, physical, and functional performances, and ultimately may positively affect molecules that may be influenced by the aging processes, and so evaluated as possible aging hallmarks, among older adults with MCI, overweight and impaired fasting glycemia. The primary objective of the study is to test the effect of MET compared to placebo on neuropsychological changes in verbal learning and memory using semantic cues, as measured by the Free and Cued Selective Reminding Test (FCSRT). The secondary objectives are to test the effect of MET versus placebo on changes in different cognitive domains, physical and functional performances. Then, as exploratory analyses, we aim to measure metabolic-related molecules, myokines, molecules associated with senescence, cytokines, and chemokines, evaluated in plasma before and after treatment with MET, and to test their association to the clinical variables. Participants are subjects with MCI, aging 55-80 years, overweight (BMI>25 kg/m2), or impaired fasting glycemia (100-125 mg/dl or HbA1c range 5.6-6.4%), recruited in two Geriatric Units. They will be randomly assigned 1:1 to intervention or placebo in double-blinded. The intervention consists of MET tablets extended-release, with a dosage of 500 or 1000 mg once daily as tolerated by participants. The intervention duration is 12 months. During the study period, all participants will attend three visits with comprehensive assessment (baseline, 6, 12 months) and they receive two clinical (month 1 and month 3) and telephone follow-ups for compliance, adherence, tolerability, and safety.

EFFECT OF HEAT-NOT-BURN TOBACCO (IQOS) VERSUS ELECTRONIC AND TOBACCO CIGARETTES ON THE ORAL HEALTH: A MULTIDISCIPLINARY APPROACH FOR A (POTENTIAL) PREVENTION STRATEGY

Finanziatore: MUR
Responsabile Scientifico: MIGLIORATI Graziella
Finanziamento:€ 75.520
Durata: 2023-2025

Summary: In recent years, the “heat-not-burn” systems, including IQOS, have grown in popularity as a healthier alternative to tobacco smoke (TS). With the increasing diffusion of electronic cigarettes (e-cig) and the growing consumer discontent due to the absence, or decrease, of the so-called “throat hit” (the physical sensation that one feels down his throat when the smoke flows from the mouth to the lungs), associated with e-cig use, the “heat-not-burn” devices are gaining market shares. For this reason, big tobacco multinationals have thought, in the recent past years, of investing much financial resources in this market. As such, their impact on human health is widely discussed among scientists, leading however to controversial and inconclusive results, thus boosting research to further investigation. In particular, their long-term biological and immunological effects are poorly known, especially in the oral cavity, the first target of exposure. The aim of this project is to compare the biological effects of IQOS with those of TS and e-cig in oral mucosa experimental models and ex-vivo samples. In particular, after performing the characterization of the qualitative and quantitative composition of IQOS and e-cig aerosols as well as of TS: - viability, proliferation, apoptosis, cell cycle, DNA damage, abnormal nuclear morphologies, epithelial-mesenchymal transition (EMT), methylglyoxal (MG)-dependent glycative stress (MGGS), oxidative stress (OS) and inflammation will be evaluated on human oral cell populations (keratinocytes, fibroblasts and osteoblasts) and dental pulp stem cells (DPSCs). In order to mimic a more realistic environment, the same end-points will be also evaluated in human dentin-pulp-like organoids from DPSCs that best reproduce the general tissue architecture, organization and the interconnections to the microenvironment. Finally, the levels of the major enzymes associated with MGGS and OS stress-responses will be evaluated as susceptibility factors to the exposure effects, using saliva and oral mucosa scrab from IQOS versus TS and e-cig users. - in parallel, the immunological effects of IQOS versus TS and e-cig aerosol exposure will be studied on T-cell (sub)populations isolated from peripheral blood and saliva of non-smokers. In particular, cytokines (i.e. IL-17A, IL-21, IL-22, IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ) and chemokine production will be assessed together with citrullinated vimentin. - a morphological/functional analysis of oral microvessels will be performed in patients by intraoral video-capillaroscopy to determine potential changes in microcirculation as a marker of oral and systemic diseases in IQOS smokers, non-smokers, TS and e-cig smokers. In the same cohorts, the qualitative and quantitative chemical composition of the exhaled will be studied and related to the TS, e-cig and IQOS aerosols obtained by the smoke machine.

The multi faceted facets of metabolic disease: unveil molecular mechanisms of impaired immunometabolicand cardiovascular response to target visceral infl ammati on with enhanced immunoregulatory cell therapy

Finanziatore: MUR
Responsabile Scientifico: MONDANELLI Giada
Finanziamento:€ 48.386
Durata: 2023-2025

Summary:Metabolic disease represents a cluster of pathological conditions that increased the risk of developing cardiovascular and metabolic clinical manifestations such as hypertension, heart disease, stroke and type 2 diabetes. It usually manifests with excess body fat, especially around the waist, known as visceral adiposity, abnormal cholesterol or triglyceride plasma levels, hyperglycemia and impaired insulin sensitivity as well as hypertension. Parallel to preferring healthier habits (eating and physical activity), current pharmacological strategies are aimed at reducing the classical risk factors (lipid lowering, anti-hypertensive and glycemic controlling therapies); however, metabolic disorders and their related comorbidities are still one of the main causes of hospitalization, disability and death worldwide with critical consequences on our society This evidence calls basic and experimental research to investigate novel, selected and more effective therapeutic targets by dissecting the molecular pathways involved in metabolic disease. Recent achievements in the filed have elucidated the role of inflammation and effector response mediated by immune cells in the onset and progression of systemic metabolic morbidities triggered by visceral fat accumulation, thus proposing immunomodulation as a complementary approach for the treatment of cardiometabolic diseases. This scenario paves the way to our proposal that will aim at investing the following main objectives: 1) depict the role of metabolic checkpoints on cellular energetic circuits to improve immuno-modulatory response; 2) tuning hypertensive response by vascular-immune-metabolic paths; 3) demonstrate the efficacy of an immunoregulatory cell therapy targeting visceral inflammation to improve metabolic dysfunction and hypertension. To pursue this ambitious goal, a multidisciplinary team with competences in immuno, vascular and metabolic biology has conceived an experimental plan aimed, firstly, to point out the molecular mechanisms responsible of immune alterations, focusing on the metabolic disturbances that impair the modulatory and tolerogenic functions of regulatory T cells (Treg) and dendritic cells, and their effect on vascular remodeling and hypertension. Unveiling these molecular mechanisms will set the stage for testing a cell therapy with functionally engineered Treg to target visceral inflammation as a therapeutic strategy to limit systemic spreading of effector response mediated by immune cells and thus protecting from disease progression. This project will put a step forward in the pharmacological management of metabolic disease, providing the evidence that the recent technological advancements in immunomodulatory therapy, based on cellular genetic manipulations, could be extended for the treatment of patients with metabolic disorders and characterized by an impaired balance between tolerogenic and effector response.

Combination of IDO1-PI3Kgamma/delta axis inhibition and tumor associated antigens DNA vaccination to cure pancreatic cancer

Finanziatore: MUR
Responsabile Scientifico: PALLOTTA Maria Teresa
Finanziamento:PALLOTTA Maria Teresa
Durata: 2023-2025

Summary:Pancreatic ductal adenocarcinoma (PDA) is still an urgent medical need with little advances in therapy and patient survival. New poly chemotherapy regimens recently employed have been shown to improve outcomes in metastatic patients and also represent an attractive neoadjuvant treatment strategy for locally advanced disease. Targeting the immune system is an active area of research and includes different approaches to improve host immune system fitness. Our preliminary data have demonstrated that DNA vaccination with the tumor associated antigen (TAA) alpha-enolase (ENO1) in association with a Phospho Inositide-3-Kinase (PI3K) gamma (g) and delta (d) subunits dual inhibitor reduced tumor burden in genetically engineered mice that spontaneously develop PDA. We have also shown in an orthotopic PDA mouse model that genetic inhibition of PI3Kg strengthens the anticancer immune response, especially in combination with ENO1 vaccination, and that humoral and cytotoxic specific anti-ENO1 antibody response plays a critical role in influencing the effect of therapy. We have also demonstrated that PI3Kd is recruited to indoleamine 2,3-dioxygenase 1 (IDO1) and has a role in mediating IDO1 signal transduction. In addition, our preliminary data suggest that also PI3Kg is involved in IDO1-mediated signaling events, although it cannot be directly bound to IDO1. Selective inhibitors of PI3Kg and PI3Kd significantly impaired TGFbeta (b)-mediated induction of Ido1 in plasmacytoid dendritic cells (pDCs), while IFNgamma (g) failed to induce IDO1 expression in PDA cell lines derived from PI3Kg deficient mice. These data suggest the existence of a functional relationship between IDO1 and PI3Kg/d in controlling immune response suppression. Therefore, co-administration of PI3Kg/d inhibitors with molecules capable of inhibiting IDO1 activity may increase the rate of success of immunotherapy in PDA. By employing orthotopically injected PDA mouse models, this project aims to i) investigate the mechanisms of action of the PI3Kg/d-IDO1 axis in the control of immune suppression in PDA; ii) select in vitro the more effective combination between PI3Kg/d and IDO1 inhibitors to unleash the immune response to PDA; iii) assess the in vivo effectiveness of the selected PI3Kg/d-IDO1 inhibitors to potentiate anti-tumor effect of therapeutic combination of DNA vaccination; iv)correlate peripheral and infiltrating immune cell subsets and the expression of IDO1 and PI3Kg/d with clinical parameters and response to therapy in mouse model. The accomplishment of this research project will provide information that will impact PDA clinical management and lead to the definition of novel immunotherapy protocol to be easily transferred to the clinical practice.

Assessment of clinical, genetic and biochemical determinants of cardiovascular risk to improve the treatment of Familial Hypercholesterolemia

Finanziatore: MUR
Responsabile Scientifico: PIRRO Matteo
Finanziamento:€ 72.332
Durata: 2023-2025

Summary:Familial Hypercholesterolemia (FH) is one of the most frequent genetic disease. Mutations in LDLR, APOB and PCSK9 genes are responsible for a severe phenotype characterized by very high levels of low-density lipoprotein-cholesterol (LDL-C) since birth, which leads to premature atherosclerotic cardiovascular disease. The aim of this project proposal is to investigate the clinical, genetic and biochemical contributors to the development of atherosclerosis in this patient’s population. Novel players, such as the gut microbiota and the metabolomics, will be explored. The results obtained in the human studies will be confirmed in the LDL-receptor deficient mouse. In this animal model the effects on the experimental atherosclerosis of the microbiota manipulations and the gene therapy will be evaluated. Identification of microbiota components that may reduce atherosclerosis development may lead to new probiotic formulations as well as to the identification of postbiotic that may improve patients’ conditions. Metabolomics may also lead to the identification of novel prognostic biomarkers. 150 Heterozygous FH (HeFH) and 100 Hypercholesterolemic non-FH hypercholesterolemic controls will be enrolled, genotyped for the genetic diagnosis of FH and evaluated for the clinical cardiovascular risk profile. Carotid, aortic and coronary atherosclerosis will be assessed by ultrasonography, echocardiography and coronary artery calcium (CAC) score. A Follow-up visit will be planned in both groups in order to perform a more detailed risk estimation of all hypercholesterolemic patients and an appropriate adjustment of treatment decisions. Among genetic factors, variants able to affect the levels of LDL-C and the atherogenic Lipoprotein (a) will be investigated. In FH patients selected for being outliers in terms of presence/absence of coronary atherosclerosis with a respectively lower/higher risk profile, an extensive genetic analysis of the about 25000 coding genes present in human exome will be performed in order to find novel genes responsible of protection /susceptibility respectively against/to the development of atherosclerosis. The metagenomic, metabolomic and target lipidomic analyses will be applied to both human and animal study populations to provide information on the contribution of a known byproduct of the gut microbiota the trimethyl amino oxide (TMAO) to the atherosclerosis in FH and will assess the role of other novel biochemical species as biomarkers of the atherosclerotic cardiovascular disease. The results obtained will expand the knowledge of the mechanisms underlying the development of the accelerated atherosclerosis in familial hypercholesterolemia and could be translated in the clinical practice. The final outcome of this study proposal will be the improvement of the therapeutic and clinical management of FH patients and the decision-making process in the perspective of increasing the cardiovascular event free survival of these patients.

ASP-MS: AntagonizingS100B as a Potential new tool to counteract Multiple Sclerosis processes

Finanziatore: MUR
Responsabile Scientifico: RENDE Mario
Finanziamento:€ 54.748
Durata: 2023-2025

Summary: The present proposal aims at conclusively establishing S100B as a key factor, and consequently a therapeutic target, in Multiple Sclerosis (MS), thus becoming the prerequisite for clinical studies. Based on the preliminary results, the rationale of this project is to deepen our understanding of S100B as one of the triggering factors for pathogenesis, with the final goal of highlighting its potential therapeutic use. S100B is a small EF-related Ca2+/ and Zn2+/binding protein, mainly synthesized by astrocytes. It exerts both intracellular and extracellular actions. While a clearly defined intracellular univocal function for this protein has not been delineated at present, astrocyte-secreted S100B is regarded to act as an autocrine, paracrine or even endocrine factor with concentration-dependent effects. In physiological conditions, astrocytes secrete S100B at nanomolar concentration, which exerts a neurotrophic action; under stress conditions, including nervous tissue inflammation, astrocytes secrete S100B at micromolar concentration, which has a neurotoxic effect, behaving as a DAMP molecule. Data have been reported indicating that S100B plays a key role in pathogenic processes - and S100B levels in biological fluids are a reliable biomarker - in a series of neural disorders. Interestingly, in many cases, over-expression/administration of the protein induces worsening of the disease, while its deletion/inactivation produces amelioration of these disorders. Therefore, it is reasonable to hypothesize that high concentrations of S100B may play a promoting role in MS, based on a number of correlative evidence: (i) elevated levels of S100B are detected in the cerebrospinal fluid (CSF and sera of MS patients in the acute phase, being reduced in the stationary phase of the disease); (ii) an increased expression of S100B is identified in both active demyelinating and chronic active MS plaques; (iii) in ex-vivo demyelinating models, a marked astrocytic elevation of S100B is observed upon demyelination, while inhibition of S100B action reduces demyelination and down-regulates the expression of inflammatory molecules. Our proposal aims at deepening the role of S100B and consequently its potential as therapeutic target, in MS, thus becoming the prerequisite for clinical studies. We will focus on selective blockade of S100B molecule using effective neutralizing anti-S100B antibody and S100B constitutive and conditional (restricted to astrocytes) gene ablation. These approaches will guarantee direct and diverse effects on S100B molecule, more specific and complete than those obtained by the drugs (PTM and AA) previously tested in EAE, thus deepening the mechanisms and pathological link between S100B and MS.

Toward a green toxicology: new approach methodologies to screen chemical-induced immunotoxicity (GREENTOX

Finanziatore: MUR
Responsabile Scientifico: RONCHETTI Simona
Finanziamento: € 87.935
Durata: 2023-2025

Summary: Green Toxicology is an emerging discipline that seeks to provide a framework for integrating the principles of toxicology into the design of safer chemicals using 21st century toxicology tools (e.g., including high-dimensional data, in vitro data, computational approaches). Inspired by the European Green Deal, and by the emerging needs to have safe chemicals, that while preserving efficacy of function show reducing toxicity, with this project we aim to develop new approach methodologies to assessed chemical-induced immunotoxicity, using selected per- and polyfluoroalkyl substances (PFAS) as a proof of principle. PFAS are a group of thousands of manmade chemicals widely used in consumer and industrial products due to their properties, e.g. oil and water repellency, resistance to high temperature, to which we are all exposed, and for the majority of which toxicological information are lacking. Animal and human studies as well as in vitro studies have shown negative effects on the immune system, including a reduced response to vaccines. In this project, effects of selected PFAS on the immune system will be assessed using in silico, in vitro and in vivo models. Focus will be on the immune system, as reduced response to vaccination is the critical endpoint identified in the last EFSA opinion (2020). In addition, effects of different PFAS will need in deep investigations, as it appears that there are differences due to chain length and functional groups. GREENTOX aims to fill some of the data gaps in the area of PFAS-induced immunotoxicity. We will focus on six PFAS, namely PFBS, PFBA, PFHxS, PFHxA, PFOS and PFOA. To include two short chain PFAS (C4) with short human half-life with two different functional groups (PFBS: 28 d; and PFBA: 3 d), one longer chain PFAS (C6) with short human half-life (PFHxA: 32 d) and one longer PFAS (C6) with long human half-life with a different functional group (PFHxS: > 5.3y), and two long chain PFAS (C8) and long half-life with two different functional groups (PFOS: > 3.4 y and PFOA: > 2.1 y). The latest (PFHxS, PFOS and PFOA) are three of the four PFAS that makes up approximately half of exposure to PFAS in humans (EFSA, 2020). The choice of these PFAS will allow to understand the role of chain length and functional groups on immunotoxicity, to rank them according to potency, and using in silico models to estimate human risks. In parallel to functional tests, RNAseq and miRNAs profile will be used to get information on the mechanisms underlying immunosuppressive effects. The project will be divided in six work packages, addressing in vitro, in silico and in vivo aspects. The participating units have long experience in studying in vitro and in vivo the effects on immune systems, and the collaboration with computational experts will guarantee the achievement of objectives. At the end, this project will establish new knowledge, and approaches to protect health from chemical-induced immunotoxicity.

Definition of avalidated food supplement from controlled plantation of Mediterranean plants to counteract osteosarcopenia in elderly (PhytoMuscleBone

Finanziatore: MUR
Responsabile Scientifico: SORCI Guglielmo
Finanziamento:€ 159.316
Durata: 2023-2025

Summary:The concomitant loss of muscle mass (sarcopenia) and bone deterioration (osteoporosis) predispose elderly subjects to frailty, bone fractures and loss of independence, compromising life quality and leading to increased morbidity and mortality. Considering the increasing life expectancy in the Western countries, osteosarcopenia represents an urgent and still unresolved health problem against which no pharmacological drugs are available. Although physical exercise, together with an adequate diet, has shown positive results in osteosarcopenia, the reduced motor skills typical of elderly people limit the clinical use of exercise-based approaches. In recent years, the scientific interest in the use of medicinal plants to counteract the mechanisms underlining several pathological conditions has increased, and some herbal extracts have shown positive effects in maintaining muscle or bone homeostasis. The project aims to study and create a dietary supplement able to counteract the loss of muscle and bone mass associated with aging starting from plants typical of the Mediterranean area and cultivated under controlled conditions in order to enrich these species in metabolites active in conferring the best anti-osteosarcopenic power. Several Mediterranean plants will be selected for their content in functional and bioactive compounds, and herbal extracts will be obtained and progressively evaluated in the following in vitro models: i) C2C12 myotubes treated with different atrophying stimuli and human myotubes derived from sarcopenic subjects, as models of muscle atrophy; ii) RANKL-treated RAW 264.7 cells and human osteoclasts, as models of osteoclast differentiation, responsible for bone resorption; and iii) BMP2-treated C2C12 cells and human osteoblasts, as models of osteoblast differentiation and bone mineralization. The resulting most efficacious plants will be cultivated under different cultivation protocols (combining irrigation, inorganic fertilization inputs and balsamic times) and the relative herbal extracts will be re-evaluated in parallel on human sarcopenic myotubes, osteoclasts or osteoblasts. The best three herbal extracts in terms of anti-sarcopenic and anti-osteoporotic effects will be analysed for active metabolite content, and the mechanisms of action investigated. The three herbal extracts will be mixed together to obtain an herbal formulation and the analysis of the anti-osteosarcopenic power of the exosomes released by muscle and bone cells following treatment with the selected herbal formulation will be performed in vitro. Finally, the herbal formulation will be evaluated in terms of specific pathological scores, histological assessment and gene expression profiling in geriatric mice developing osteosarcopenia to assess its efficacy in vivo. Thus, we will define cultivation protocols to obtain a validated food supplement from Mediterranean plants useful as a natural polypill in counteracting osteosarcopenia in elderly subjects.

Demystifying mosquito sex: unraveling MOsquito SWARMs with labbased 3D video tracking (acronym MOSWARM

Finanziatore: MUR
Responsabile Scientifico: SPACCAPELO Roberta
Finanziamento: € 103.044
Durata: 2023-2025

Summary: A female mosquito mates once in her life. On the outcome of this single, poorly understood encounter hangs her lifetime fertility, and thus the size of the mosquito population; a key factor in the capacity of these insects to transmit life-threatening diseases such as malaria and dengue fever. It will also decide the success or failure of eagerly anticipated vector control strategies such as gene drive and sterile insect technique (SIT) which depend entirely on the ability of genetically-modified males to mate with wild-type females. One of the few things we know about mosquito sex is that for most medically important mosquito species it takes place in the context of male swarms; complex, apparently disordered, groupings of hundreds or thousands of individual males that form at dusk and in to which a female enters solely to mate. What happens once she enters the swarm is essentially unknown. How do males maintain a swarm? How does the swarm react to the presence of a female? Do females choose their mate? If so, what do females look for in a male? How do mosquitoes recognise and mate only with their own species even in the context of mixed swarms containing different species? These all remain open questions yet the answers are critical not only to better understand the mating biology of the most important vectors of malaria but to the safe and effective roll-out of potentially game-changing new strategies for vector control like gene drive. The aim of this proposal is to exploit two recent developments to break new ground in our understanding of mosquito swarming and courtship behaviour: (i) The unique large cage infrastructure developed in Research Unit 1 that allows robust natural swarming behaviour in a lab-based setting, and (ii) Cutting edge 3D imaging and analysis techniques, pioneered by Research Unit 2 that have proved capable of extracting biological meaning from other highly complex swarm systems. Building on pilot data generated from this ongoing collaboration, we will leverage the synergy in these two approaches to address three main objectives: 1. To define individual and group dynamics in male Anopheles gambiae swarms 2. To define details of precopulatory flight dynamics in males and females 3. To use this information to define the role of mosquito hearing in swarming and mating success

Overcoming T Cell impairment in CCL to boost CSR T cell function

Finanziatore: MUR
Responsabile Scientifico: SPORTOLETTI Paolo
Finanziamento:€ 53.610
Durata: 2023-2025

Summary: T cells engineered to express a chimeric antigen receptor (CAR-T) are undoubtedly among the most promising therapeutic modalities in oncology and in particular in hematology. Nevertheless, the efficacy of CAR-T cell treatment appears suboptimal, due to a number of factors, including manufacturing variables, CAR structure optimization and patient’s T-cell features of exhaustion and terminal differentiation. Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the western world, is an exceptional model to investigate mechanisms influencing CAR-T cell cytotoxicity, as CD19 CAR-T cell therapy is effective only in a minority of patients. Despite the progress obtained in the treatment thanks to the introduction of targeted therapies, CLL remains an incurable disease, whereby CAR T cells may represent a valuable option if efficacy can be improved. To this end, a full understanding of the in vivo mechanisms of the T-cell impairment is mandatory in order to design meaningful strategies towards their reversion. Along this line, it is interesting to note that the immunomodulation occurring under treatment with novel drugs appears to empirically improve the generation and efficacy of CD19-directed CAR-T cells from patients with CLL. Based on the existing background, the OBJECTIVES of our proposal are twofold, aiming at: 1) assessing the efficiency in the generation of the final CAR-T cell product from patients with CLL in the presence of novel targeted therapies; 2) identifying leukemia-specific modulators of CAR-T cell function that can be therapeutically exploited; The RESULTS we aim at achieving will help identify both T-cell intrinsic and extrinsic mechanisms of immunosuppression that are potentially targetable in order to improve anti-tumor efficacy of CAR-T cells. In addition, our results will help define the best timing for CAR-T cell administration during the natural history of the disease, based on the possible negative or positive interactions with approved targeted therapies. Finally, information on mechanisms of immunomodulation that may arise from the successful completion of this project may have crucial implications also in the management and prevention of infections, one of the main comorbidities that affect patients with CLL. In addition, the results will be swiftly transferred and tested in other B cell malignancies if not other oncological diseases, in the future, to assess their relevance and applicability to further improve the success of cellular therapies for the treatment of malignancies.

Do air pollution, social, environmental, and comorbidity variables, jointly affect the risk of SARS-CoV-2 infection and of severity of COVID-19 clinical manifestation? Collecting and processing multiple heterogeneous data over a distributed ‘data lake’ platform for advanced modelling

Finanziatore: MUR
Responsabile Scientifico: STRACCI  Fabrizio
Finanziamento:€ 100.880
Durata: 2025-2027

COVID-19 pandemic is a public health emergency worldwide, yet among different geographical areas it shows remarkable differences in incidence rate of infection, and of morbidity and death. In Italy the highest rate of SARS-COV-2 infection and of severe COVID-19 disease and deaths were observed in those regions with highest level of industrialization, and where transportation is higher. Conversely, other Italian regions where manufacturing plants and traffic are less prevalent showed substantially lower rates of infection and death Since in those regions degree of industrialization and traffic is largely mirrored by level of air pollution, it has been hypothesized that pollution may contribute to the spread and/or the severity of COVID-19, through 2 possible mechanisms: Acute: microparticles derived from fossil fuels might act as airborne carriers of virus; Chronic: exposure to microparticles and chemical pollutants might cause chronic lung injury, exacerbating the consequences of viral infection Recent studies have investigated this issue. However, they were mostly based on analyses simply comparing pollution data and COVID metrics. This approach is not satisfactory, as it ignores a variety of components that may influence rates of SARS-COV-2 diffusion and infection (e.g., atmospheric data, population density, urban vs rural environment, mobility, socio-economic conditions, etc..); at the same time, severity of COVID-19 disease and deaths are also influenced by many variables (age, gender, comorbidities, frailty, etc.) For these reasons, a thorough assessment of the relationship between air pollution and COVID-related health outcomes is much needed. It must take into account many complementary social, demographic, and health parameters. Furthermore, to be accurate this approach requires a high spatial resolution that only can be achieved through advanced geolocalization techniques. Putting together solid information about these various issues could provide substantial information about the epidemiology of the pandemic. It might also inform potentially relevant choices of public health and social organization. Due to its geographical differences in terms of incidence of COVID-19 and of air pollution, Italy is particularly suited to investigate these issues The ambitious task of putting together large sets of diverse data from multiple sources (pollutant concentrations, COVID-related metrics, social, demographics, health-related parameters), to be investigated at a very detailed geographical level, requires a strong, concerted effort that leverages different skills and requires high-power computer capability To this end, our project is designed to provide strong synergy between solid epidemiological and medical knowledge and powerful computational skills to deal with heterogeneous and complex data sets, and to model them accordingly. The research Units of this proposal have a proven track record in these various items.

Immune synapse imaging as a new tool for optimizing CAR T cells that resist tumor-mediated immune-suppression: focus on a dual CD30/PDL1 targeting strategy of Hodgkin lymphoma

Finanziatore: MUR
Responsabile Scientifico: TIACCI Enrico
Finanziamento:€ 92.906
Durata: 2025-2027

Chimeric antigen receptor (CAR) T cell therapy has provided a major breakthrough in exploiting the killing ability of cytotoxic T cells (CTL) by harnessing the tumor antigen specificity of monoclonal antibodies (mAb) to the CTL signaling machinery. Nonetheless, CAR T cell effectiveness can be hampered by various factors, including CAR design and CAR T cell exhaustion or suppression by the tumor microenvironment (TME). This is exemplified by failure of anti-CD30 CAR T cells in classic Hodgkin lymphoma (cHL), a CD30+tumor overexpressing also PDL1. Clinical responses to PD1 blockade in relapsed/refractory (r/r) cHL highlight the importance of the PD1/PDL1 immune-suppressive pathway in this lymphoma. Hence, combining in a single T-cell product an anti-CD30 CAR and a PDL1 blocker might overcome the cHL TME-dependent, PDL1-driven exhaustion of CD30 CAR T cells. Here we will implement such novel strategy: i) by engineering new proprietary anti-CD30 and anti PDL1 mAb into CTL cells for generating anti-CD30 CAR T cells also endowed with an anti-PDL1 chimeric receptor lacking a signaling domain; and ii) by using, as CAR signaling readout, an innovative platform for imaging the architecture of the immune synapsis (IS), the specialized signaling and secretory domain that forms at the CTL interface with its cognate cellular target. Indeed, emerging evidence shows that CAR T cells form immune synapses whose architecture can be affected by CAR design. To image IS architecture with high accuracy we will use the supported lipid bilayer (SLB) system, where defined combinations of freely-moving CAR ligands, integrins and co-stimulatory or inhibitory receptors are embedded in a lipid-coated support, forming surrogate target cells and allowing to visualize events occurring at the T cell-SLB contact in real time through super-resolution imaging (Task 1). This novel platform will inform the principles for an optimal CAR design (including spacer and linker length) for generating single CD30 and dual CD30/PDL1 CAR T cells that best perform in parallel standard cytotoxicity, proliferation and cytokine-release assays against cHL cell lines (Task 2). We will then apply the same imaging platform to assess the ability of dual CAR T cells to overcome cHL TME-mediated exhaustion driven by surface PDL1 and/or soluble cHL-derived immune-suppressive cytokines (Task 3). The project will capitalize on the highly complementary expertises of the Baldari lab in IS imaging and the Tiacci lab in cHL pathogenesis and in new mAb-based therapies against cHL. This project has strong application potential as it may lead to an innovative CAR T cell therapy for a tumor prevalent in the young and with limited treatment options in r/r cases. Additionally, by exploiting our image-based assay to evaluate IS architecture as an early predictor of T cell function, we may provide the biomedical community with a robust tool to inform CAR design for generating high-performance CAR T cells in general.

Mechanisms underlying therapeutic approaches in an alpha-synuclein-basedmodel of Parkinson's disease: from pharmacological to non-pharmacological strategies

Finanziatore: MUR
Responsabile Scientifico: TOZZI Alessandro
Finanziamento: € 95.155
Durata: 2023-2025

Summary:The study of Parkinson’s Disease (PD) has been drastically changed by the discovery of the pathological role of a-synuclein (a-syn). The misfolding and aggregation of this small synaptic protein constitute an early process in PD pathogenesis, deeply associated to the onset of neuroinflammation and synaptopathy. The mechanistic link between these three phenomena is, however, still obscure. A crucial contribution to the understanding of the pathogenetic cascade in PD has been made by the development of new animal models of the disease. In particular, intracerebrally-injected a-syn pre-formed fibrils (PFF) rodents recapitulate the key features of synucleinopathy spreading and the subsequent development of structural and functional changes, followed by motor manifestations. We aim to better trace the connections between a-syn aggregation and inflammation by taking advantage of two profoundly different therapeutic strategies, sharing the promise to have a disease modifying potential, tested in a-syn PFF rats. On one hand, exercise training is supposed to modulate neuroinflammation and neuronal plasticity by acting on brain-derived neurotrophic factor (BDNF) signaling, thus promoting an anti-inflammatory environment; on the other hand, a novel class of small molecules targeting a-syn aggregates will exert antiaggregating and disaggregating properties against a-syn deposits. In order to dissect how these two therapeutic tools will effectively change the natural progression of PD, we have conceived different therapeutic schedules, to be applied in PFF rats, consisting of a treadmill training protocol or the administration of novel small molecules targeting a-syn. Our groups will in fact assess the impact of those approaches on synucleinopathy and neuroinflammation by detecting a-syn aggregates, modifications of cytokines levels and morphological alterations of the glia. Furthermore, the groups will investigate and compare the effects of treadmill training and drugs on the recovery of normal synaptic function and plasticity via electrophysiological recording, and on motor and non-motor performance, via behavioral testing.

Effects of genomic profiles on thromboembolic risk in pati ents with locally advanced ormetastati c non-small-cell lung cancer

Finanziatore: MUR
Responsabile Scientifico: VERSO Melina
Finanziamento:€ 83.100
Durata: 2023-2025

Durata: 2023-2025Summary:The main hypothesis of this project is that genomic profile is a strong determinant of the prothrombotic state in patients with NSCLC, with major influence on the risk for VTE and on thromboembolic-related mortality. This hypothesis is supported by increasing body of evidence regarding the potential association between specific driver oncogenic mutations and thromboembolic events in NSCLC. By assessing plasma concentration of TF, TG and other markers of inflammation and platelet activation before the initiation of first or further line of anticancer therapies, our project has the potential to define the prothrombotic effect that is peculiar of individual genomic profile and distinguish the prothrombotic potential of oncogene expression profiling from that of target therapies. TF is the most important component of tumor procoagulant activity. Preclinical studies have suggested that several cancer genotypes, such as EGFR or KRAS mutation and ALK or ROS1 rearrangements, can induce overexpression of TF by cancer cells leading to thrombin generation and clot formation. It is likely that cancer genotype could play an important role in the genesis of hypercoagulability increasing the incidence of VTE in NSCLC patients. The measurements of plasmatic levels of other prothrombotic markers as IL 6, vWF, ADAMTS-13 activity and platelet aggregation test in patients with different genomic profile of NCSLC could clarify the pathway of relationship between cancer cells and prothrombotic state. From a clinical point of view, demonstrating an association between the oncogene profile and the risk of thromboembolic events could contribute to a better identification of categories of patients with high thromboembolic risk candidates to antithromboticprophylaxis. The main aim of this project is to assess the thromboembolic potential in patients with both oncogene-addicted and wild type NSCLC in terms of both biochemical markers and clinical events. Biochemical thromboembolic potential will be measured by the assessment of levels of plasma TF, TG, IL 6, vWF, ADAMTS-13 activity and by platelet activation and aggregation. Biochemical parameters will be measured at patient enrollment and after 3 months or in case of clinically overt VTE events. Clinical thromboembolic risk will bemeasured by the assessment of newly diagnosed VTE.