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Despite the advent of numerous targeted therapies in clinical practice, anthracyclines, including doxorubicin (DOX), continue to play a pivotal role in breast cancer (BC) treatment. DOX directly disrupts DNA replication, demonstrating remarkable efficacy against BC cells. However, its non-specificity toward cancer cells leads to significant side effects, limiting its clinical utility. Interestingly, DOX can also enhance the antitumor immune response by promoting immunogenic cell death in BC cells, thereby facilitating the presentation of tumor antigens to the adaptive immune system. However, the generation of an adaptive immune response involves highly proliferative processes, which may be adversely affected by DOX-induced cytotoxicity. Therefore, understanding the impact of DOX on dividing T cells becomes crucial, to deepen our understanding and potentially devise strategies to shield anti-tumor immunity from DOX-induced toxicity. Our investigation focused on studying DOX uptake and its effects on human lymphocytes. We collected lymphocytes from healthy donors and BC patients undergoing neoadjuvant chemotherapy (NAC). Notably, patient-derived peripheral blood mononuclear cells (PBMC) promptly internalized DOX when incubated in vitro or isolated immediately after NAC. These DOX-treated PBMCs exhibited significant proliferative impairment compared to untreated cells or those isolated before treatment initiation. Intriguingly, among diverse lymphocyte sub-populations, CD8 + T cells exhibited the highest uptake of DOX. To address this concern, we explored a novel DOX formulation encapsulated in ferritin nanocages (FerOX). FerOX specifically targets tumors and effectively eradicates BC both in vitro and in vivo. Remarkably, only T cells treated with FerOX exhibited reduced DOX internalization, potentially minimizing cytotoxic effects on adaptive immunity. Our findings underscore the importance of optimizing DOX delivery to enhance its antitumor efficacy while minimizing

Aggressive hepatocellular carcinoma (HCC) overexpressing Angiopoietin-2 (ANG-2) (a protein linked with angiogenesis, proliferation, and epithelial–mesenchymal transition (EMT)), shares 95% of up-regulated genes and a similar poor prognosis with the proliferative subgroup of intrahepatic cholangiocarcinoma (iCCA). We analyzed the pro-invasive effect of ANG-2 and its regulator vascular endothelial growth factor (VEGF) on HCC and CCA spheroids to uncover posUsible common ways of response. Four cell lines were used: Hep3B and HepG2 (HCC), HuCC-T1 (iCCA), and EGI-1 (extrahepatic CCA). We treated the spheroids with recombinant human (rh) ANG-2 and/or VEGF and then observed the changes at the baseline, after 24 h, and again after 48 h. Proangiogenic stimuli increased migration and invasion capability in HCC- and iCCA-derived spheroids and were associated with a modification in EMT phenotypic markers (a decrease in E-cadherin and an increase in N-cadherin and Vimentin), especially at the migration front. Inhibitors targeting ANG-2 (Trebananib) and the VEGF (Bevacizumab) effectively blocked the migration ability of spheroids that had been stimulated with rh-ANG-2 and rh-VEGF. Overall, our findings highlight the critical role played by ANG-2 and the VEGF in enhancing the ability of HCC- and iCCA-derived spheroids to migrate and invade, which are key processes in cancer progression.

The liver’s unique characteristics have a profound impact on the priming and maintenance of adaptive immunity. This review delves into the cellular circuits that regulate adaptive immune responses in the liver, with a specific focus on hepatitis B virus infection as an illustrative example. A key aspect highlighted is the liver’s specialized role in priming CD8+ T cells, leading to a distinct state of immune hyporesponsiveness. Additionally, the influence of the liver’s hemodynamics and anatomical features, particularly during liver fibrosis and cirrhosis, on the differentiation and function of adaptive immune cells is discussed. While the primary emphasis is on CD8+ T cells, recent findings regarding the involvement of B cells and CD4+ T cells in hepatic immunity are also reviewed. Furthermore, we address the challenges ahead and propose integrating cutting-edge techniques, such as spatial biology, and combining mouse models with human sample analyses to gain comprehensive insights into the liver’s adaptive immunity. This understanding could pave the way for novel therapeutic strategies targeting infectious diseases, malignancies, and inflammatory liver conditions like metabolic dysfunction-associated steatohepatitis and autoimmune hepatitis.

I neutrofili sono le cellule dell'immunità innata che rispondono per primi al danno tissutale o all'infezione. Tuttavia, nonostante il loro ruolo consolidato nelle infezioni, il meccanismo molecolare che media il loro reclutamento non è ancora stato descritto. Infatti, la migrazione dei neutrofili verso il sito infetto è stata associata per anni alla segnalazione del riconoscimento dei patogeni mediata dai PRR (Pattern Recognition Receptor). Di recente, questa idea è stata messa in discussione e sono anche state scoperte nuove sottopopolazioni di neutrofili nel sistema vascolare in condizioni normali, sebbene un chiaro ruolo funzionale, specialmente nelle infezioni, non sia ancora stato stabilito. Inoltre, la meccano-trasduzione è stata riconosciuta come un nuovo attore importante nella risposta ai batteri gram-negative, con o senza il contributo dei PRR. Pertanto, l'obiettivo del nostro progetto è stato di definire in modo molecolare il reclutamento dei neutrofili durante le infezioni e di studiare funzionalmente le sottopopolazioni dei neutrofili nel sito infetto. A questo scopo, abbiamo utilizzato un modello di infezione cutanea con funghi C. (Candida) albicans e batteri Gram-positivi e Gram-negativi, S. (Staphylococcus) aureus e P. (Pseudomonas) aeruginosa, in topi WT (Wild-Type) e topi carenti di recettori per le interleuchine e la segnalazione PRR. Abbiamo identificato i neutrofili come Ly6GhighCD11bhigh e le loro sottopopolazioni come CD62LhighCXCR4- per i neutrofili “fresh” (giovani) e CD62L-/lowCXCR4+ per quelli “aged” (invecchiati) attraverso la citofluorimetria multiparametrica. Per studiare la cascata molecolare che media il reclutamento dei neutrofili, abbiamo quantificato la produzione di mediatori pro-infiammatori mediante qPCR, ELISA e citofluorimetria. Abbiamo scoperto che il reclutamento dei neutrofili è definito cineticamente da diversi mediatori a monte, che però convergono tutti su MyD88. Abbiamo identificato due fasi del reclutamento de, Neutrophils are the cells of innate immunity that first respond to tissue damage or infection. However, regardless of their established role in infection, the exact molecular mechanism of recruitment has not yet been described. In fact, neutrophil migration to infected site has been linked for years to signaling from pathogen recognition mediated by PRRs (Pattern Recognition Receptors). Recently this idea has been challenged and new neutrophil subpopulations have been discovered in the vasculature at steady state, although a clear functional role, especially in infection, for these subpopulations has not yet been established. Moreover, mechano-sensing has been established as a new player in the response to gram-negative bacteria3, with or without PRRs contribution. Thus, the goal of our project is to molecularly define neutrophil recruitment during infection and to functionally study neutrophil subpopulations at the infected site. To this purpose, we made use of a skin infection model with fungi, C. (Candida) albicans, and Gram-positive and negative bacteria, S. (Staphylococcus) aureus and P. (Pseudomonas) aeruginosa, in WT (Wild-Type) and mice deficient for interleukin receptors and PRR signaling. We identified neutrophils as Ly6Ghigh/+Ly6CintCD11b+ and their subpopulations as CD62L+/highCXCR4low for fresh and CD62L-CXCR4+ for aged ones through multi-parametric flow-cytometry. To study the molecular cascade mediating neutrophil recruitment, in addition to the presence of neutrophils in the infected tissues, we quantified the production of pro-inflammatory mediators through qPCR, ELISA and flow-cytometry. We discovered that neutrophil recruitment is kinetically defined by different upstream mediators, all converging to MyD88. In fact, we identified two “waves” of neutrophil recruitment, and we could mechanistically define the first one. We found that it occurs during the first 6 hours, and it is mediated by an LTB4 (Leukotriene B4)- IL-1 axis, irrespective of the ty

Questa tesi è il risultato di un programma di dottorato condotto in due istituzioni accademiche, l'Università di Milano-Bicocca in Italia e l'Università della California di Los Angeles (UCLA) negli Stati Uniti. Le fasi di questo programma sono culminate in due progetti distinti, ognuno dei quali contribuisce a una comprensione più approfondita di aspetti critici nel campo dell'immunologia e dell'epigenomica. Il primo progetto, sviluppato presso l'Università degli Studi di Milano-Bicocca, mira a decifrare l'eterogeneità e la funzionalità delle sottopopolazioni di cellule dendritiche (DC) nel contesto delle infezioni umane e dei tumori attraverso l'applicazione di tecnologie a singola cellula. Dopo una descrizione completa delle DCs e un'introduzione alla tecnologia di single-cell RNA sequencing e alle analisi computazionali, il capitolo 3 è una versione dell'articolo "Marongiu L, Protti G, et al. Maturation signatures of conventional dendritic cell subtypes in COVID-19 suggest direct viral sensing. Eur J Immunol. 2022”, dove abbiamo caratterizzato le signature trascrizionali delle DC nel sangue periferico di pazienti COVID-19 e donatori sani, tramite l’analisi di dataset di scRNA-seq. I capitoli 4 e 5 ampliano ulteriormente la nostra comprensione delle sottopopolazioni di DC, focalizzandosi sul loro ruolo nel microambiente tumorale. Specificamente, il capitolo 4 si focalizza sulla caratterizzazione funzionale delle DC3s, una sottopopolazione recentemente identificata, descrivendo il loro ruolo nello sviluppo e nella progressione del cancro polmonare non a piccole cellule (NSCLC). I risultati derivati da questa ricerca hanno il potenziale di migliorare la nostra comprensione della funzionalità delle DC nei tumori umani, fungendo così da preziosa risorsa per identificare potenziali bersagli e biomarcatori per rifinire le tecniche di immunoterapia. Il capitolo 5 ha una prospettiva più ampia, focalizzandosi sullo studio della composizione, abbondanza e attivaz, This dissertation represents the outcome of a comprehensive doctoral program conducted into two academic institutions, the University of Milano-Bicocca in Italy and the University of California Los Angeles (UCLA) in the United States. The phases of this program have culminated in two distinct projects, each contributing to a deeper understanding of critical facets in the fields of immunology and epigenomics. The first project, developed at the University of Milano-Bicocca, aims at deciphering the heterogeneity and functionality of dendritic cell (DC) subpopulations in the context of human infections and tumours through the application of cutting-edge single-cell technologies. After a comprehensive description of DCs and an introduction to single-cell RNA sequencing technology and computational analysis, chapter 3 is a version of the article titled “Marongiu L, Protti G, et al. Maturation signatures of conventional dendritic cell subtypes in COVID-19 suggest direct viral sensing. Eur J Immunol. 2022” where we have delved into the transcriptional signatures of DCs in the peripheral blood of COVID-19 patients and healthy donors. This investigation was underpinned by the analysis of in-house and publicly available datasets of single-cell RNA sequencing, unravelling distinct DC subpopulations and their maturation signatures in response to SARS-CoV-2 infection. Chapters 4 and 5 further advance our understanding of DC subsets within the tumour microenvironment. Specifically, Chapter 4 focuses on the functional characterization of DC3s, a recently identified subset. Our study describes their role in the development and progression of non-small cell lung cancer (NSCLC) through an extensive analysis of cancer samples spanning from early to advanced stages. The insights derived from this research hold the potential to enhance our comprehension of DC subsets' roles in human tumours, thereby serving as a valuable resource for identifying potential targets and biomarkers to refin

Breast cancer (BC) is the most frequent cancer and the second leading cause of death in women. A typical feature of BC cells is the metabolic shift toward increased glycolysis, which has become an interesting therapeutic target for metabolic drugs such as metformin (MET). Recently, the administration of the antihypertensive syrosingopine (SYRO) in combination with MET has shown a synergistic effect toward a variety of cancers. However, a fundamental need remains, which is the development of in vivo biomarkers that are able to detect early clinical response. In this study, we exploited a triple-negative murine BC cell line (4T1) and a metastatic ER+ murine BC cell line (TS/A) in order to investigate, in vivo, the early response to treatment, based on MET and/or SYRO administration, evaluating [18F]FDG and [18F]FLT as potential biomarkers via PET/CT. The study provides evidence that SYRO plus MET has a synergistic effect on tumor growth inhibition in both 4T1 and TS/A experimental models and has showed the highest efficacy on the TNBC xenograft mice (4T1) via the expression reduction in the lactate transporter MCT4 and in the epithelial–mesenchymal transition biomarker Snail, promoting its potential application in therapy settings. In addition, the selective reduction in the [18F]FLT tumor uptake (at 7 dd), observed in the SYRO plus MET treated mice in comparison with the vehicle group, suggests that this radiotracer could be potentially used as a biomarker for the early detection of therapy response, in both evaluated xenografts models.

Metabolic dysfunction-associated fatty liver disease (MAFLD), the hepatic component of the metabolic syndrome caused by insulin resistance, is a major public health problem, affecting about the 25 % of the general population in Western countries. Morbidity and mortality of MAFLD patients is increased primarily due to cardiovascular disease (CVD). Liver fibrosis, the byproduct of hepatic repair, is the main determinant of MAFLD progression and the strongest predictor for overall mortality. Since the mechanistic relationship between MAFLD, fibrosis, insulin resistance and the cardiometabolic risk is far to be clear, deciphering the functional link of hepatic fibrogenesis with genetic factors and hypercoagulability in MAFLD-associated CVD may hold translational potential for risk profiling and innovative therapeutic targeting.

Oncolytic viruses (OVs) are promising therapeutics for tumors with a poor prognosis. An OV based on herpes simplex virus type 1 (oHSV-1), talimogene laherparepvec (T-VEC), has been recently approved by the Food and Drug Administration (FDA) and by the European Medicines Agency (EMA) for the treatment of unresectable melanoma. T-VEC, like most OVs, is administered via intratumoral injection, underlining the unresolved problem of the systemic delivery of the oncolytic agent for the treatment of metastases and deep-seated tumors. To address this drawback, cells with a tropism for tumors can be loaded ex vivo with OVs and used as carriers for systemic oncolytic virotherapy. Here, we evaluated human monocytes as carrier cells for a prototype oHSV-1 with a similar genetic backbone as T-VEC. Many tumors specifically recruit monocytes from the bloodstream, and autologous monocytes can be obtained from peripheral blood. We demonstrate here that oHSV-1-loaded primary human monocytes migrated in vitro towards epithelial cancer cells of different origin. Moreover, human monocytic leukemia cells selectively delivered oHSV-1 to human head-and-neck xenograft tumors grown on the chorioallantoic membrane (CAM) of fertilized chicken eggs after intravascular injection. Thus, our work shows that monocytes are promising carriers for the delivery of oHSV-1s in vivo, deserving further investigation in animal models.

Intrahepatic cholangiocarcinoma (iCCA) is a rare neoplasm of the bile ducts with a low survival rate, whose incidence is continuously increasing and is associated with a rich and varied tumor microenvironment (TME). Although the main mutations characterizing iCCA are known, there are several unresolved issues regarding the processes leading to the accumulation of mutations in the normal cholangiocyte. The inflammatory mediators and the molecular pathways involved in cholangiocarcinogenesis, which regulate the transition from normal to dysplastic cells, resulting in neoplastic cholangiocytes, are poorly understood. Moreover, once the tumor is established, it is unclear which effects of the interaction between the tumor and TME constituent cells, in particular cancer-associated fibroblasts (CAFs), are responsible for stimulating the malignant behavior of iCCA. In this review, we described the main mutations affecting the bile ducts leading to iCCA development as well as the putative inflammatory mediators and morphogenetic pathways involved in the establishment of the malignant transition of the bile ducts. We also described the main signaling pathways involved in TME-tumor cell interactions, with particular emphasis on the effect of CAFs in cancer. Finally, we wanted to analyze possible new therapeutic approaches aimed at modifying the composition of TME and the possible role of immunotherapy in improving the treatment of this cancer.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy, deriving from the neoplastic transformation of hepatocytes, most often forced to long-lasting regeneration by the cirrhotic background. HCC is an extremely aggressive tumor with still limited effective treatments, and is characterized by the presence of a very complex and multifaceted tumor microenvironment (TME). Among the variety of cell types populating the TME of HCC, cancer-associated fibroblasts (CAFs) are the most prevalent. CAFs are a specific population of fibroblasts in a persistent state of activation, with a high level of heterogeneity, partly dependent on a wide range of cell origin, which are endowed with a repertoire of functions, profoundly modulating the biology of the tumor. Given the close relationship of HCC with cirrhosis, CAFs are paradigmatic of the role played by activated fibroblasts in promoting the evolution of a chronic, non-resolving, fibro-inflammatory condition towards a neoplastic disease and its aggressive phenotype. In this review, we will discuss the most recent findings regarding the interplay of CAFs with the tumoral epithelial compartment, with the multiple cell elements of the TME (macrophages, neutrophils, myeloid-derived suppressor cells, vascular cells), and with the extracellular matrix. Finally, we will address the translational value of CAF manipulation in HCC to unveil possible ameliorations for the treatment of a still worrisome disease.

Cholangiocarcinoma (CCA) is a rare malignancy that develops at any point along the biliary tree. CCA has a poor prognosis, its clinical management remains challenging, and effective treatments are lacking. Therefore, preclinical research is of pivotal importance and necessary to acquire a deeper understanding of CCA and improve therapeutic outcomes. Preclinical research involves developing and managing complementary experimental models, from in vitro assays using primary cells or cell lines cultured in 2D or 3D to in vivo models with engrafted material, chemically induced CCA or genetically engineered models. All are valuable tools with well-defined advantages and limitations. The choice of a preclinical model is guided by the question(s) to be addressed; ideally, results should be recapitulated in independent approaches. In this Consensus Statement, a task force of 45 experts in CCA molecular and cellular biology and clinicians, including pathologists, from ten countries provides recommendations on the minimal criteria for preclinical models to provide a uniform approach. These recommendations are based on two rounds of questionnaires completed by 35 (first round) and 45 (second round) experts to reach a consensus with 13 statements. An agreement was defined when at least 90% of the participants voting anonymously agreed with a statement. The ultimate goal was to transfer basic laboratory research to the clinics through increased disease understanding and to develop clinical biomarkers and innovative therapies for patients with CCA.

The hepatocyte nuclear factor 1 beta (HNF1B) gene is involved in the development of specialized epithelia of several organs during the early and late phases of embryogenesis, performing its function mainly by regulating the cell cycle and apoptosis pathways. The first pathogenic variant of HNF1B (namely, R177X) was reported in 1997 and is associated with the maturity-onset diabetes of the young. Since then, more than 230 different HNF1B variants have been reported, revealing a multifaceted syndrome with complex and heterogenous genetic, pathologic, and clinical profiles, mainly affecting the pediatric population. The pancreas and kidneys are the most frequently affected organs, resulting in diabetes, renal cysts, and a decrease in renal function, leading, in 2001, to the definition of HNF1B deficiency syndrome, including renal cysts and diabetes. However, several other organs and systems have since emerged as being affected by HNF1B defect, while diabetes and renal cysts are not always present. Especially, liver involvement has generally been overlooked but recently emerged as particularly relevant (mostly showing chronically elevated liver enzymes) and with a putative relation with tumor development, thus requiring a more granular analysis. Nowadays, HNF1B-associated disease has been recognized as a clinical entity with a broader and more variable multisystem phenotype, but the reasons for the phenotypic heterogeneity are still poorly understood. In this review, we aimed to describe the multifaceted nature of HNF1B deficiency in the pediatric and adult populations: we analyzed the genetic, phenotypic, and clinical features of this complex and misdiagnosed syndrome, covering the most frequent, unusual, and recently identified traits.

Cholangiocarcinoma remains an aggressive and deadly malignancy that is often diagnosed late. Intrinsic tumour characteristics and the growth pattern of cancer cells contribute to the challenges of diagnosis and chemoresistance. However, establishing an early and accurate diagnosis, and in some instances identifying targetable changes, has the potential to impact survival. Primary sclerosing cholangitis, a chronic cholangiopathy prodromal to the development of a minority of cholangiocarcinomas, poses a particular diagnostic challenge. We present our diagnostic and theranostic approach to the initial evaluation of cholangiocarcinomas, focusing on extrahepatic cholangiocarcinoma. This involves a multipronged strategy incorporating advanced imaging, endoscopic methods, multiple approaches to tissue sampling, and molecular markers. We also provide an algorithm for the sequential use of these tools. (c) 2023 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

La calcineurina (Cn) è una Serina/Treonina-fosfatasi responsabile della defosforilazione di una numerosa serie di substrati tra cui il fattore trascrizionale NFAT. L’asse calcineurina-NFAT è noto essere in grado di regolare molteplici processi tumorigenici quali proliferazione, sopravvivenza, transizione epitelio-mesenchimale e metastatizzazione e pertanto si presenta come ottimo bersaglio per lo studio dei meccanismi di tumorigenesi. In particolare, è stato studiato il ruolo dell’asse calcineurina-NFAT nei tumori “freddi”, in quanto refrattari alle immunoterapie moderne. Pertanto bersagliare questo asse potrebbe offrire nuove opportunità terapeutiche. Per gli studi sono state utilizzate delle linee cellulari tumorali murine geneticamente modificate per avere un blocco costitutivo dell’interazione tra calcineurina ed NFAT. L’inibizione farmacologica dell’attività enzimatica della calcineurina con farmaci quali Ciclosporina A e Tacrolimus è noto causare un effetto citostatico. È stato dunque verificato che il blocco dell’interazione calcineurina-NFAT non impattasse la crescita ed il ciclo cellulare, che avrebbe potuto introdurre un ulteriore bias nel modello. Per verificare gli effetti globali di questo blocco dell’interazione Cn-NFAT sullo stato delle cellule coltivate in 2D ed in 3D è stato effettuato un sequenziamento degli RNA in bulk. Dall’analisi dei dati sono emerse come alterate diverse vie di segnalazione cellulare, a seconda delle condizioni di coltura, ed in particolare la via della transizione epitelio-mesenchimale, le vie infiammatorie e le vie coinvolte nella fosforilazione ossidativa. Essendo noto il ruolo di NFAT nella regolazione del microambiente tumorale, sono stati anche indagati gli effetti dell’inibizione dell’asse Cn-NFAT in vivo, in modelli murini di tumori sottocutanei. Sorprendentemente, è stato osservato un aumento dell’infiltrato intratumorale di cellule T CD8+. Un’ulteriore analisi dei dati di sequenziamento dell’RNA ha evidenzia, Calcineurin is a serine-threonine phosphatase that can dephosphorylate a large number of substrates, among which there is the transcription factor NFAT. My research activity has focused on investigating the role of calcineurin (Cn) in “cold” tumors, and in particular the role of the Cn-NFAT axis. This axis is known to play various protumorigenic roles in tumors and its modulation could prove beneficial towards the development of novel therapies, since cold tumors are notoriously refractory to immunotherapy. We employed genetically engineered tumor cell lines to study the impact of a constitutive inhibition of the Cn-NFAT interaction both in vitro and in vivo. We investigated the impact that this inhibition has in vitro by confirming that there is no cytostatic effect that could confound ulterior results and no alterations in the cell cycle. We also confirmed that growth wasn’t altered in vivo. We performed an RNA sequencing on cells cultivated both in 2D and in 3d in order to determine the global expression variations that this inhibition induced in our cell line models. Based on the data analysis we identified several pathways that were altered based on the specific cultivation techniques we used. From this we could infer that the Cn-NFAT axis plays a role in several cell processes such as epithelial-to-mesenchymal transition and also oxidative phosphorylation. Since NFAT is known to contribute to the tumor microenvironment, we investigated whether there are any alterations in the immune infiltrate in vivo. Surprisingly we found that in our model there was an enhanced recruitment of CD8+ T cells. Further analysis of the RNAseq data revealed that the impairment of the Cn-NFAT axis induced an increased production of T cell chemotactic factors such as cytokines and chemokines. Indeed, the inhibition of these pathways in vivo abrogated the T cell recruitment. We then sought to understand why this increase in the CD8+ T cell infiltrate did not result in alterations in c

Le cellule dendritiche (DC) e i monociti sono cellule immunitarie innate coinvolte nel riconoscimento di patogeni e molecole associate a danno, nello scatenare l’immunità adattativa e nel mantenimento dell’omeostasi. Queste cellule sono collettivamente chiamate cellule del sistema fagocitico mononucleare (MPS), e la loro origine ed il loro sviluppo nell’uomo richiedono ancora studio. I recettori delle citochine determinanti l’origine (LDCR) sono fattori di crescita con importante ruolo nel differenziamento e nella sopravvivenza delle cellule ematopoietiche. Questi recettori sono presenti anche su cellule mature, suggerendone un ruolo post-differenziamento. Il legame tra DC e monociti è stato qui dimostrato usando CSF1R, un LDCR conosciuto in precedenza come espresso solo su macrofagi, ma ora caratterizzato anche su tutte le cellule del MPS. Il MPS c’è stato analizzato in diversi contesti patologici, per caratterizzarne lo stato di sviluppo e attivazione. Le DC e i monociti sono stati studiati in due diversi vaccini antimalarici. Entrambi i vaccini contengono lo stesso antigene del Plasmodium falciparum, la proteina AMA1. Uno dei vaccini si basa su vettori virali, mentre l’altro contiene la proteina ricombinante somministrata con un adiuvante lipidico. Il MPS è risultato modulato, con percentuali di cellule diverse a seconda del vaccino somministrato. Inoltre, il vaccino a vettore virale ha causato una maggiore attivazione delle cellule rispetto al vaccino proteico. L’espressione di LDCR e proteine di superficie nelle cellule CSF1R+ è stata modificata in maniera dipendente dalla formulazione dei vaccini. Le DC hanno un importante ruolo anche nelle malattie infettive. Nella pandemia globale di COVID-19, capire come il MPS è modulato dal virus SARS-CoV-2 era cruciale, specialmente per gli anziani e le persone con patologie pre-esistenti. Due gruppi di pazienti COVID del Sud Africa sono stati studiati, ed erano presenti coinfezioni da HIV e tubercolosi, trattament, Dendritic cells (DCs) and monocytes are innate immune cells involved in recognising pathogens or damage-associated molecules, triggering adaptive immune responses, and maintaining homeostatic conditions. DCs and monocytes are collectively referred to as Mononuclear Phagocyte System (MPS) cells, and their origin and development in humans still need elucidating. Lineage-determining cytokine receptors (LDCRs) are growth factors with a pivotal role in haematopoietic cell differentiation and survival. These receptors are retained on mature cell surface, suggesting an active role after differentiation. Here, the link between DCs and monocytes at a developmental level was assessed at steady state using CSF1R, a LDCR previously known as expressed only on macrophages, but now proved to be on all cells of the MPS. The MPS was further studied across multiple diseases, to assess its development or effector state. DCs and monocytes were assessed in two distinct anti-malarial vaccine challenges. Both vaccines targeted the same blood-stage Plasmodium falciparum antigen apical membrane protein 1 (AMA1), involved in erythrocyte invasion by malaria. One vaccine formulation was based on a prime-boost viral delivery of the antigen, while the other vaccine consisted of the recombinant protein administered with a liposome-based adjuvant system. The MPS was greatly affected and reshaped, with cell fluctuations in percentages depending on the vaccine formulation. The viral-vectored vaccine also seemed to activate cells more effectively compared to the protein vaccine. LDCR and surface marker expression in CSF1R+ cells was also affected by the different vaccine vector components. DCs also play a role in infectious diseases. In the global pandemic of COVID-19, understanding how the MPS is modulated by the SARS-CoV-2 virus was crucial, especially for elderly people and individuals with underlying conditions. Two South African COVID patients’ cohorts were investigated, where HIV and Tb coinfec

Le cellule CIK (Cytokine Induced Killer) sono cellule T citotossiche indotte da citochine che esprimono marcatori fenotipici delle cellule Natural Killer (NK) ed esibiscono una elevata capacità citotossica. Diversi studi clinici hanno dimostrato che le cellule CIK hanno un minimo livello di alloreattività e hanno attività terapeutica, quest’ultima solo in contesti con bassi livelli di malattia. Per questo motivo, nuove strategie che aumentino l’attività terapeutica delle cellule CIK sono necessarie. Nella prima parte del mio progetto ho confrontato l’attività in vitro di due strategie in grado di aumentare la specificità delle cellule CIK. La prima strategia è modificare geneticamente le cellule CIK per esprimere un recettore chimerico CAR, usando il sistema trasposonico Sleeping Beauty. Abbiamo confrontato due costrutti CAR anti-CD19, ricalcando la struttura di prodotti già utilizzati nella pratica clinica, uno il CAR anti-CD19 Tisagenlecleucel (Novartis) e il secondo il CAR del protocollo clinico CARCIK-CD19 (Fondazione Matilde Tettamanti Menotti De Marchi Onlus). La seconda strategia è la combinazione di cellule CIK con l’anticorpo bispecifico Blinatumomab, CD3xCD19, il quale agisce da “ponte” tra le cellule CIK CD3+ e le cellule tumorali CD19+. Abbiamo studiato l’attività in vitro delle due tipologie di cellule CARCIK e delle cellule CIK+Blinatumomab in termini di citotossicità, proliferazione indotta dal legame con l’antigene, rilascio di citochine e attivazione intracellulare di NFAT e NF-kB. I dati ottenuti suggeriscono che tutte e tre le strategie per migliorare la specificità delle cellule CIK sono funzionali e che l’aggiunta dell’anticorpo bispecifico Blinatumomab è comparabile, se non in alcuni casi più efficiente, delle CARCIK in vitro. Il confronto in vivo in modelli murini dell’attività di queste cellule è in corso e sarà fondamentale per completare la valutazione della loro attività. La seconda parte del progetto si basa sui risultat, Cytokine Induced Killer (CIK) cells are CD3+CD56+ double positive T cells that have acquired phenotypic markers of natural killer (NK) cells and show non-restricted cytotoxicity against different antigens. Clinical trials have demonstrated CIK cells therapeutic activity only in low tumor burden context, so strategies to increase the therapeutic efficacy of CIK cells are required. In the first part of my project, I compared the activity of two different methods to improve CIK cells anti-tumor efficacy and specificity against CD19. One method was to genetically engineer CIK cells to express a chimeric antigen receptor (CAR), using stable transfection of a Sleeping Beauty transposon plasmid. Using this transposon system, two anti-CD19 CARs, recapitulating the structure of the clinical anti-CD19 CAR Tisagenlecleucel (Novartis), originally inserted in a lentivirus, and the CAR-CD19 used in current clinical CARCIK-CD19 protocol (Fondazione Matilde Tettamanti Menotti De Marchi Onlus) were compared. The second strategy investigated was to combine CIK cells with Blinatumomab, an anti-CD3xCD19 bispecific antibody, which can bridge CD19+ targets with CD3+ CIK, perhaps in a similar way to CD19-CAR, but without genetic modification. The activity of the different CARs or Blinatumomab were compared in vitro in terms of CIK cells proliferation, cytokines release, cytotoxicity and NFAT and NF-kB signaling against two CD19+ target cell lines. The data obtained suggest that the addition of Blinatumomab to CIK cultures may be as efficacious, if not more, as the CAR gene transduction in vitro. Comparisons in vivo in a mouse model are in progress. Future results will complete this work and verify whether specific in vitro and in vivo activities correlate, and if so in what manner. The second part of the project is based on the knowledge gained from the anti-CD19 CARs, but is focused on a novel tumor target antigen for CAR-CIK therapy, a tumor microenvironment protein, refered to here as

Le neoplasie mieloidi sono un gruppo di malattie caratterizzate da un'origine clonale mieloide comune. In ogni singolo gruppo di queste malattie il decorso clinico è eterogeneo e difficile da prevedere, in particolare per la Leucemia Mieloide Acuta (LMA) e per la Mielofibrosi (MF). Recentemente, l'identificazione di mutazioni molecolari mediante sequenziamento ad alto rendimento (NGS) ha notevolmente migliorato la nostra conoscenza della genetica molecolare di LAM e MF e ha messo in evidenza le mutazioni geniche e il loro significato prognostico. La mia attività di dottorato mirava a definire meglio il ruolo delle alterazioni geniche nel predire il decorso clinico delle malattie e guidare le strategie terapeutiche. In questo contesto, abbiamo definito il profilo mutazionale in una coorte di 221 LAM a cariotipo normale (NK) arruolate in uno studio clinico prospettico randomizzato. In questa coorte di NK-LAM, mediante analisi multivariata, il raggiungimento della remissione completa è stato influenzato negativamente dalla mutazione SRSF2. Le alterazioni di FLT3 (FLT3-ITD) e U2AF1 erano associate a un peggioramento della sopravvivenza globale (OS) e della sopravvivenza libera da malattia (DFS) (p <0,05). I pazienti positivi per FLT3-ITD sottoposti a alloHSCT avevano una probabilità di sopravvivenza simile a quella negativa per FLT3-ITD. Inoltre, con l'obiettivo di implementare la definizione di AML secondaria, abbiamo analizzato una coorte prospettica di 413 pazienti con AML concentrandoci su una signature mutazionale cromatina-spliceosoma (CS). Le mutazioni CS sono state identificate nel 17,6% dei casi de novo clinicamente definiti, mostrando caratteristiche cliniche più vicine alla sAML. Gli esiti clinici in questo gruppo erano avversi e paragonabili a quelli dei pazienti con sAML. AlloHSCT ha migliorato i risultati sia nei pazienti sAML che CS-AML. Questi dati supportano una definizione molecolare di sAML, con implicazioni per un trattamento ottimizzato. Inoltr, Myeloid malignancies are a group of diseases characterized by a common myeloid clonal origin. In every single group of these diseases the clinical course is heterogeneous difficult to predict, particularly for Acute Myeloid Leukemia (AML) and for Myelofibrosis (MF). Recently, the identification of molecular mutations by high throughput sequencing (NGS) has dramatically improved our knowledge of AML and MF molecular genetics and shed new light on the prognostic significance gene mutations. My PhD activity aimed to better define the role of genes alterations to predict the clinical course of the diseases and guide therapeutic strategies. In this context, we defined the mutational profile in a cohort of 221 normal karyotype (NK) AML enrolled into a prospective randomized clinical trial. In this cohort ok NK-AML, by multivariate analysis, the achievement of complete remission was negatively affected by the SRSF2 mutation. Alterations of FLT3 (FLT3-ITD) and U2AF1 were associated with a worse overall (OS) and disease-free survival (DFS) (p < 0.05). FLT3-ITD positive patients who underwent alloHSCT had a survival probability similar to FLT3-ITD negative. Moreover, aiming to implement the definition of secondary AML, we analyzed a prospective cohort of 413 AML patients focusing on a chromatin-spliceosome (CS) mutational signature. CS mutations were identified in 17.6% of clinically defined de novo cases, showing clinical characteristics closer to sAML. Clinical outcomes in this group were adverse and comparable to those of sAML patients. AlloHSCT improved outcomes in both sAML and CS-AML patients. These data support a molecular definition of sAML, with implications for optimized treatment. Furthermore, we evaluated the post alloHSCT outcome of MF patients enrolled into a multicentric perspective clinical trial. NGS allowed us to classify patients by MIPSS scoring systems, which also consider the prognostic value of HMR mutations, obtaining that the great part of them result

HCV infection could have extrahepatic manifestations due to an aberrant immune response. HCV/HIV co-infection increases such persistent immune activation. Aim of the present study is to describe the evolution of inflammatory markers used in clinical practice, mixed cryoglobulinemia (MC) and autoantibody reactivity in co-infected individuals who achieved sustained virological response (SVR) after DAA treatment. This prospective, observational study included all HIV/HCV co-infected subjects who started any DAA regimen from 2015 to 2020. Samples for laboratory measurements (ferritin, C reactive protein, C3 and C4 fractions, rheumatoid factor, MC, anti-thyroglobulin Ab, anti-thyroid peroxidase Ab, ANCA, ASMA, anti-LKM, anti-DNA, AMA, ANA, T CD4+ and CD8+ cell count, and CD4/CD8 ratio) were collected at baseline, after 4 weeks, at end of treatment, and at SVR12. The analysis included 129 individuals: 51.9% with a F0–F3 fibrosis and 48.1% with liver cirrhosis. Cryocrit, C3 fraction, and rheumatoid factor significantly improved at week 4; ferritin, anti-thyroglobulin Ab, and C4 fraction at EOT; total leukocytes count at SVR12. MC positivity decreased from 72.8% to 35.8% (p <.001). T CD4+ cell slightly increased at SVR12, but with an increase also in CD8+ resulting in stable CD4/CD8 ratio. Autoantibody reactivity did not change significantly. ANA rods and rings positivity increased from 14.8% to 28.6% (p =.099): they were observed in three subjects without exposure to RBV. DAA therapy may lead to improvement in inflammatory markers and MC clearance but without significant changes in autoantibodies reactivity and CD4/CD8 ratio over a follow up of 12 weeks.

Although immunotherapy research to date has focused largely on T cells, there is mounting evidence that tumour-infiltrating B cells and plasma cells (collectively referred to as tumour-infiltrating Blymphocytes (TIL-Bs)) have a crucial, synergistic role in tumour control. In many cancers, TIL-Bs have demonstrated strong predictive and prognostic significance in the context of both standard treatments and immune checkpoint blockade, offering the prospect of new therapeutic opportunities that leverage their unique immunological properties. Drawing insights from autoimmunity, we review the molecular phenotypes, architectural contexts, antigen specificities, effector mechanisms and regulatory pathways relevant to TIL-Bs in human cancer. Although the field is young, the emerging picture is that TIL-Bs promote antitumour immunity through their unique mode of antigen presentation to T cells; their role in assembling and perpetuating immunologically 'hot' tumour microenvironments involving T cells, myeloid cells and natural killer cells; and their potential to combat immune editing and tumour heterogeneity through the easing of self-tolerance mechanisms. We end by discussing the most promising approaches to enhance TIL-B responses in concert with other immune cell subsets to extend the reach, potency and durability of cancer immunotherapy.

Background Hepatocyte nuclear factor 1B (HNF1B) is a member of the homeodomain-containing family of transcription factors located on 17q12. HNF1B deficiency is associated with a clinical syndrome (kidney and urogenital malformations, maturity-onset diabetes of the young, exocrine pancreatic insufficiency) and to an underdiagnosed liver involvement. Differently from HNF1A, the correlation between hepatocellular carcinoma (HCC) and germline HNF1B deficiency has been poorly evaluated. Case report Here, we report a novel case of a syndromic HNF1B-deficient paediatric patient that developed HCC with unique histopathological features characterised by neoplastic syncytial giant cells, which was observed only in one additional case of paediatric cholestatic liver disease of unknown origin. Conclusions Our case highlights the influence of HNF1B deficiency in liver disease progression and its putative association with a rare yet specific HCC histotype. We hypothesised that HCC could be secondary to the repressive effect of HNF1B variant on the HNF1A transcriptional activity.

Cholangiocarcinoma (CCA), a neoplasm burdened by a poor prognosis and currently lacking adequate therapeutic treatments, can originate at different levels of the biliary tree, in the intrahepatic, hilar, or extrahepatic area. The main risk factors for the development of CCA are the presence of chronic cholangiopathies of various etiology. To date, the most studied prodromal diseases of CCA are primary sclerosing cholangitis, Caroli's disease and fluke infestations, but other conditions, such as metabolic syndrome, nonalcoholic fatty liver disease and obesity, are emerging as associated with an increased risk of CCA development. In this review, we focused on the analysis of the pro-inflammatory mechanisms that induce the development of CCA and on the role of cells of the immune response in cholangiocarcinogenesis. In very recent times, these cellular mechanisms have been the subject of emerging studies aimed at verifying how the modulation of the inflammatory and immunological responses can have a therapeutic significance and how these can be used as therapeutic targets.

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent liver disease worldwide, and affects 25% of the population in Western countries. NAFLD is the hepatic manifestation of the metabolic syndrome, linked to insulin resistance, which is the common pathogenetic mechanism. In approximately 40% of NAFLD patients, steatosis is associated with necro-inflammation and fibrosis, resulting in nonalcoholic steatohepatitis (NASH), a severe condition that may progress to cirrhosis and liver cancer. Although the hepatocyte represents the main target of the disease, involvement of the bile ducts occurs in a subset of patients with NASH, and is characterized by ductular reaction and activation of the progenitor cell compartment, which incites portal fibrosis and disease progression. We aim to dissect the multiple biological effects that adipokines and metabolic alterations exert on cholangiocytes to derive novel information on the mechanisms driven by insulin resistance, which promote fibro-inflammation and carcinogenesis in NASH.

Cholangiocarcinoma (CCA), an aggressive tumor originating from both intra-and extra-hepatic biliary cells, represents an unmet need in liver oncology, as treatment remains largely unsatisfactory. A typical feature of CCA is the presence of a complex tumor microenvironment (TME) composed of neoplastic cells, a rich inflammatory infiltrate, and cancer-associated fibroblasts and desmoplastic matrix that makes it extremely chemoresistant to traditional chemotherapeutic drugs. In this review, we describe the cell populations within the TME, in particular those involved in the innate and adaptive immune response and how they interact with tumor cells and with matrix proteins. The TME is crucial for CCA to mount an immune escape response and is the battlefield where molecularly targeted therapies and immune therapy, particularly in combination, may actually prove their therapeutic value.

Data on the vertical transmission rate of COVID-19 in pregnancy are limited, although data reporting mother-fetal transmission in the second trimester of pregnancy are controversial. We described a case of second-trimester twin stillbirth in a woman with SARS-CoV-2 infection in which placental and fetal markers of infection were detected, despite the absence of respiratory syndrome. The patient developed clinical chorioamnionitis and spontaneously delivered 2 stillborn infants. Placental histology and immunohistochemistry demonstrated SARS-CoV-2 infection mostly within the syncytiotrophoblast, and fetal autopsy showed the development of interstitial pneumonia. Our findings demonstrated that in utero vertical transmission is possible in asymptomatic pregnant women with SARS-CoV-2 infection and that infection can lead to severe morbidity in the second trimester of pregnancy.

Background & Aims: Cholangiocarcinoma (CCA) comprises a heterogeneous group of malignant tumors associated with dismal prognosis. Alterations in post-translational modifications (PTMs), including NEDDylation, result in abnormal protein dynamics, cell disturbances and disease. Herein, we investigate the role of NEDDylation in CCA development and progression. Methods: Levels and functions of NEDDylation, together with response to pevonedistat (NEDDylation inhibitor) or CRISPR/Cas9 against NAE1 were evaluated in vitro, in vivo and/or in patients with CCA. The development of preneoplastic lesions in Nae1+/- mice was investigated using an oncogene-driven CCA model. The impact of NEDDylation in CCA cells on tumor-stroma crosstalk was assessed using CCA-derived cancer-associated fibroblasts (CAFs). Proteomic analyses were carried out by mass-spectrometry. Results: The NEDDylation machinery was found overexpressed and overactivated in human CCA cells and tumors. Most NEDDylated proteins found upregulated in CCA cells, after NEDD8-immunoprecipitation and further proteomics, participate in the cell cycle, proliferation or survival. Genetic (CRISPR/Cas9-NAE1) and pharmacological (pevonedistat) inhibition of NEDDylation reduced CCA cell proliferation and impeded colony formation in vitro. NEDDylation depletion (pevonedistat or Nae1+/- mice) halted tumorigenesis in subcutaneous, orthotopic, and oncogene-driven models of CCA in vivo. Moreover, pevonedistat potentiated chemotherapy-induced cell death in CCA cells in vitro. Mechanistically, impaired NEDDylation triggered the accumulation of both cullin RING ligase and NEDD8 substrates, inducing DNA damage and cell cycle arrest. Furthermore, impaired NEDDylation in CCA cells reduced the secretion of proteins involved in fibroblast activation, angiogenesis, and oncogenic pathways, ultimately hampering CAF proliferation and migration. Conclusion: Aberrant protein NEDDylation contributes to cholangiocarcinogenesis by promoting cell

The prognosis of cholangiocarcinoma remains poor in spite of the advances in immunotherapy and molecular profiling, which has led to the identification of several targetable genetic alterations. Surgical procedures, including both liver resection and liver transplantation, still represent the treatment with the best curative potential, though the outcomes are significantly compromised by the early development of lymph node metastases. Progression of lymphatic metastasis from the primary tumor to tumor-draining lymph nodes is mediated by tumor-associated lymphangiogenesis, a topic largely overlooked until recently. Recent findings highlight tumor-associated lymphangiogenesis as paradigmatic of the role played by the tumor microenvironment in sustaining cholangiocarcinoma invasiveness and progression. This study reviews the current knowledge about the intercellular signaling and molecular mechanism of tumor-associated lymphangiogenesis in cholangiocarcinoma in the hope of identifying novel therapeutic targets to halt a process that often limits the success of the few available treatments.

High-dose standard-of-care chemotherapy is the only option for triple-negative breast cancer (TNBC) patients, which eventually die due to metastatic tumors. Recently, metronomic chemotherapy (mCHT) showed advantages in treating TNBCs leading us to investigate the anti-metastatic and anti-angiogenic potential of metronomic 5-Fluorouracil plus Vinorelbine (5-FU+VNR) on endothelial cells (ECs) and TNBCs in comparison to standard treatment (STD). We found that 10-fold lower doses of 5-FU+VNR given mCHT vs. STD inhibits cell proliferation and survival of ECs and TNBC cells. Both schedules strongly affect ECs migration and invasion, but in TNBC cells mCHT is significantly more effective than STD in impairing cell migration and invasion. The two treatments disrupt FAK/VEGFR/VEGF signaling in both ECs and TNBC cells. mCHT, and to a much lesser extent STD treatment, induces apoptosis in ECs, whereas it switches the route of cell death from apoptosis (as induced by STD) to autophagy in TNBC cells. mCHT-treated TNBCs-derived conditioned medium also strongly affects ECs’ migration, modulates different angiogenesis-associated proteins, and hampers angiogenesis in matrix sponge in vivo. In conclusion, mCHT administration of 5-FU+VNR is more effective than STD schedule in controlling cell proliferation/survival and migration/invasion of both ECs and TNBC cells and has a strong anti-angiogenic effect. Our data suggest that the stabilization of tumor growth observed in TNBC patients treated with mCHT therapy schedule is likely due not only to direct cytotoxic effects but also to anti-metastatic and anti-angiogenic effects.

La Sclerosi Multipla (MS) è una patologia autoimmune cronica che colpisce il sistema nervoso centrale (SNC) determinando demielinizzazione. I principali fenotipi patologici sono Recidivante-Remittente (RRMS) e Primariamente Progressiva (PPMS) – quest’ultima è la forme più grave. Ajami B. et al. hanno dimostrato che nel modello murino affetto da Encefalomielite (EAE) - il modello animale di MS- vi è una forte correlazione tra la presenza di monociti a livello del SNC ed il peggioramento dei sintomi motori tipici della malattia. Partendo da queste evidenze , per il nostro studio sono stati prelevati campioni di sangue da HC, RRMS e PPMS -tutte femmine- per poi isolare i monociti CD14+. Su tali campioni sono stati condotti esperimenti microarray e successivamente qRT-PCR. L’analisi bioinformatica ha evidenziato che gli RRMS si distribuivano formando due gruppi: un gruppo (RR1) si distribuiva similmente agli HC, l’altro (RR2) si distribuiva similmente ai PPMS. Dalla Gene Ontology è emerso che i processi biologici maggiormente deregolati erano quelli di Infiammazione e Colesterolo. In aggiunta a questi pazienti, è stata validata tramite qRT-PCR una seconda coorte di RRMS e PPMS. La validazione tramite qRT-PCR ha confermato che i geni coinvolti nella biosintesi del colesterolo sono deregolati nei pazienti di entrambe le coorti, ma con specifiche differenze basate sul paziente. Infatti, sia per gli RRMS che per i PPMS si sono potute apprezzare differenze nei livelli di espressione dello stesso gene anche tra i pazienti con lo stesso fenotipo clinico. Questa deregolazione a livello metabolico, ha permesso di ipotizzare che i monociti di questi pazienti possano avere un fenotipo concordante con la Trained Immunity (TI), recentemente scoperta. Per TI si intende la memoria dell’immunità innata: monociti venuti in contatto con uno stimolo primario conserverebbero memoria di tale “incontro” reagendo in maniera più violenta ad una seconda stimolazione come potrebbe essere, Multiple Sclerosis (MS) is a chronic autoimmune disease that affects the central nervous system (CNS) leading to demyelination. The main pathological phenotypes are Relapsing-Remitting (RRMS) and Primary Progressive (PPMS) - the latter being the most severe form. Ajami B. et al. have shown that in the mouse model affected by Encephalomyelitis (EAE) - the animal model of MS- there is a strong correlation between the presence of monocytes at the level of the CNS and the worsening of the motor symptoms typical of the disease. Based on this evidence, blood samples from HC, RRMS and PPMS -all female- were collected for our study and CD14+ monocytes were isolated. Microarray experiments were conducted on these samples and subsequently qRT-PCR was performed. Bioinformatics analysis showed that RRMS distributed in two groups: one group (RR1) had similar trend to HC, the other group (RR2) had similar trend to PPMS. Gene Ontology showed that the most deregulated biological processes were those of Inflammation and Cholesterol. In addition to these patients, a second cohort of RRMS and PPMS was validated by qRT-PCR. Validation by qRT-PCR confirmed that the genes involved in cholesterol biosynthesis were deregulated in patients of both cohorts, but with specific patient-based differences. In fact, for both RRMS and PPMS, differences in expression levels of the same gene could be appreciated even among patients with the same clinical phenotype. This deregulation at the metabolic level, allowed us to hypothesize that the monocytes of these patients may have a phenotype consistent with the recently discovered Trained Immunity (TI). By TI is meant the memory of innate immunity: monocytes come into contact with a primary stimulus would retain memory of such "encounter", reacting more violently - and in the case of Multiple Sclerosis in an autoimmune way - to a second stimulation as could be an inflammatory stimulus. The first stimulus may be either a vaccine or molecules such as meva

Activation of glial cells (reactive gliosis) and the purinergic pathway, together with metalloproteinase (MMP)-induced remodeling of the neural extracellular matrix (nECM), drive maladaptive changes in the spinal cord following peripheral nerve injury (PNI). We evaluated the effects on spinal maladaptive plasticity through administration of oxidized ATP (oxATP), an antagonist of P2X receptors (P2XR), and/or GM6001, an inhibitor of MMPs, in rats following spared nerve injury (SNI) of the sciatic nerve. With morpho-molecular techniques, we demonstrated a reduction in spinal reactive gliosis and changes in the neuro-glial-nECM crosstalk via expression remodeling of P2XR, nerve growth factor (NGF) receptors (TrkA and p75), and histone deacetylase 2 (HDAC2) after treatments with oxATP/GM6001. Altogether, our data suggest that MMPs and purinergic inhibition have a modulatory impact on key proteins in the neuro-glial-nECM network, acting at different levels from intracellular signaling to epigenetic modifications.

La fragilità è una sindrome geriatrica definita da un progressivo declino età-correlato di diverse facoltà fisiologiche, che si traduce in una ridotta funzionalità d’organo e in un’aumentata vulnerabilità in condizioni di stress. Fried e coll. propongono una definizione operativa delle fragilità. La letteratura riporta un’alta prevalenza di fragilità in individui affetti da insufficienza renale cronica. Tra questi soggetti, il rischio di sviluppare fragilità è aumentato di due/tre volte rispetto ai soggetti sani. Ad oggi, la definizione e la valutazione della fragilità in questi pazienti è ancora controversa. Tuttavia quello che si sa è che l’invecchiamento è un processo strettamente correlato alla ridotta capacità delle cellule staminali di auto-rinnovarsi e differenziarsi. Quest’alterazione delle funzioni delle cellule staminali può svolgere un ruolo chiave nella fisiopatologia delle malattie associate all’invecchiamento, inclusa la disfunzione renale. Il nostro gruppo ha identificato e isolato le cellule staminali renali adulte a partire da colture clonali di nefrosfere umane. All’interno di queste sono presenti cellule a diversa differenziazione e maturazione, tra queste anche le staminali identificate come PKHhigh/CD133+/CD24-, multipotenti e in grado di ripopolare scaffold renali decellularizzati. Date queste premesse l'obiettivo del progetto è valutare gli effetti di condizioni biologiche legate alla fragilità sul comportamento delle cellule staminali renali adulte umane e capire se tali condizioni sono in grado di esaurire il pool di cellule staminali e alterarne la funzione. In primo luogo abbiamo arruolato soggetti fragili, pre-fragili e non-fragili e giovani sani come controllo e raccolto i rispettivi plasmi e cellule mononucleate dal sangue periferico (PBMC). Sia nelle PBMC che nelle cellule staminali/progenitrici ematopoietiche circolanti abbiamo valutato il danno al DNA osservando una percentuale di cellule positive al danno statistic, Frailty is a geriatric syndrome that can be defined as an age-related progressive impairment of multiple physiological systems, resulting in a significantly reduced capacity to compensate for external stressors. Fried and colleagues proposed a phenotype characterization of frailty through five physical criteria, so this can be possible only after the onset of clinical manifestations without the possibility of a precocious diagnosis. Several studies report a high prevalence of frailty in both old and young individuals with kidney dysfunction, and this further increases with advancing age and progressive decline of renal function. Elderly individuals with chronic kidney disease (CKD) are two to three times more likely to be frail than those with normal renal function. However, the relationship between CKD and frailty is still unclear. The aging process can have adverse effects on stem cells; their self-renewal ability declines and their differentiation potential into the various cell types is altered. Aging-induced exhaustion and deterioration of stem cell pool and functions may play a key role in the pathophysiology of aging-associated diseases, including kidney dysfunction. Our group isolated a pure population of multipotent renal stem-like cells by a functional approach, taking advantage from the ability of renal stem cells (RSC) to grow as nephrospheres (NS). Investigating the expression of renal progenitor markers described in literature, our group identified in NS a homogeneous PKHhigh/CD133+/CD24- cell population displaying in vitro stem-cell properties, able to repopulate human decellularized renal scaffold and exhibiting multipotency. In this scenario, we tested whether in the organism of elderly and frail people there are biological conditions able to alter RSC behavior, justifying the high prevalence of chronic kidney dysfunction in the frail status and its severity. First, we recruited frail, pre-frail and non-frail subjects, and young subjects as controls

Different functional states determine glioblastoma (GBM) heterogeneity. Brain cancer cells coexist with the glial cells in a functional syncytium based on a continuous metabolic rewiring. However, standard glioma therapies do not account for the effects of the glial cells within the tumor microenvironment. This may be a possible reason for the lack of improvements in patients with high-grade gliomas therapies. Cell metabolism and bioenergetic fitness depend on the availability of nutrients and interactions in the microenvironment. It is strictly related to the cell location in the tumor mass, proximity to blood vessels, biochemical gradients, and tumor evolution, underlying the influence of the context and the timeline in anti-tumor therapeutic approaches. Besides the cancer metabolic strategies, here we review the modifications found in the GBM-associated glia, focusing on morphological, molecular, and metabolic features. We propose to analyze the GBM metabolic rewiring processes from a systems biology perspective. We aim at defining the crosstalk between GBM and the glial cells as modules. The complex networking may be expressed by metabolic modules corresponding to the GBM growth and spreading phases. Variation in the oxidative phosphorylation (OXPHOS) rate and regulation appears to be the most important part of the metabolic and functional heterogeneity, correlating with glycolysis and response to hypoxia. Integrated metabolic modules along with molecular and morphological features could allow the identification of key factors for controlling the GBM-stroma metabolism in multi-targeted, time-dependent therapies.

Benign biliary tumor are common lesions that are often an incidental finding in subjects who undergo medical imaging tests for other conditions. Most are true neoplasms while few result from reactive or malformative proliferation. Benign tumors have no clinical consequences, although the premalignant nature or potential for malignant transformation is of concern in some cases. The main practical problem for pathologists is the need to differentiate them from malignant biliary tumours, which is not always straightforward. Premalignant lesions of the bile duct have been described, although their incidence has been poorly characterized. These lesions include biliary mucinous cystic neoplasms, intraductal papillary neoplasms of the bile duct, and biliary intraepithelial neoplasia. In this article, histopathology of benign biliary tumors and biliary tumor precursors is discussed, with a focus on the main diagnostic criteria.

Fibropolycystic liver diseases (FPLDs) encompass a heterogeneous group of rare, congenital disorders of the biliary tree deriving from an abnormal embryogenesis of the primordial ductal plate caused by a genetically determined dysfunction of morphogenetic proteins expressed in the primary cilia of cholangiocytes ("ciliopathies"). Among this group, it is important to notice three different clusters of disease: polycystic liver diseases (PLDs), fibrocystic liver diseases (FLDs), and choledochal cysts (CCs). PLDs are characterized by the development of multiple (>20) fluid-filled biliary cysts widespread throughout liver parenchyma and disconnected from biliary tree. The natural history of PLDs is characterized by growth of cyst during adult age, leading to debilitating hepatomegaly. The ductal dysgenesis may affect, in FLDs, the biliary system at multiple levels, from the small intrahepatic bile ducts (congenital hepatic fibrosis) to the larger intrahepatic bile ducts (Caroli's disease or Caroli's syndrome, when Caroli's disease coexists with congenital hepatic fibrosis), leading to biliary microhamartomas and segmental bile duct dilations, accompanied by progressive fibrogenesis. These fundamental lesions are the hallmark of FLDs and are responsible for the major complications, such as portal hypertension, cholestasis, recurrent cholangitis, sepsis, and cholangiocarcinoma. CCs are congenital alterations resulting from ductal plate malformation, involving the largest intra or extrahepatic bile ducts that mainly affect a paediatric population. Early surgical intervention is pivotal to avoid complications related to obstructive frame, ab extrinseco compression, rupture or malignant evolution. FPLDs often associate with a spectrum of disorders affecting many organs, primarily the kidney, and thus they are collectively termed hepatorenal fibrocystic disease (HRFCD). Among them, the autosomal-dominant and recessive polycystic kidney disease (ARPKD) is the most frequentl

Cholangiocarcinoma (CCA) includes a group of different epithelial cancers with features of biliary tract differentiation arising from any tract of the biliary tree. Histologically, they usually are adenocarcinomas. It is a rare cancer accounting about 3% of all gastrointestinal malignancies. Based on its anatomical location, CCA is classified as intrahepatic (iCCA), perihilar (pCCA), and distal (dCCA). Currently, the three types of CCA are considered as distinct cancers since different pathogenic and management features. Cholangiocarcinomas are aggressive tumours, often diagnosed at advanced stages. Most cases are sporadic but conditions leading to chronic inflammation and cholestasis have been recognized as risk factors. Diagnosis needs a multimodal approach, mixing imaging, endoscopy, laboratory tests, including onco-biomarkers, and pathology. Surgical resection with histologically negative margins is the only curative treatment, although it is possible only for few patients; unfortunately, recurrence is frequent. Likewise, liver transplantation is an option for a small subset of selected patients suffering from pCCA. Generally, prognosis is poor for most patients. Desmoplastic nature, highly variable genetics, interaction with a rich tumour microenvironment, all contribute to the resistance of therapy. Advances in targeted-, radio- and immunotherapy will lead to improvement in survival.