Protein Arginine Deiminases: a novel mechanism of leukocyte trafficking under physiological and pathological conditions

Protein arginine deiminases (PADs) are a class of Ca2+ dependent cysteine hydrolases that catalyze a protein post-translational modification known as citrullination, where a positive protein-bound arginine residue is converted into a neutral unconventional citrulline residue. The reaction results in a shift of protein charge from positive to neutral, which dramatically impacts the structure, conformation, and function of proteins as well as intra/intermolecular interactions. Citrullination represents an important regulatory mechanism affecting several physiological cellular processes, such as gene expression, cell differentiation, apoptosis, and inflammatory immune responses. Among the PAD family members, PAD2 and PAD4, which are the only isoforms expressed by immune cells, have attracted considerable interest due to their role in producing neutrophil extracellular traps (NETs) and playing a detrimental role in a wide range of diseases such as Rheumatoid Arthritis, Ulcerative Colitis, Systemic Lupus Erythematosus, Multiple Sclerosis, and Alzheimer's Disease. Pharmacological PAD inhibition has brought beneficial therapeutic results in animal models of these pathologies since it leads to an amelioration of the disease-dependent inflammation and reduces immune cell accumulation and tissue damage. However, how PAD2 and PAD4 isoforms directly affect immune cell trafficking in the context of inflammation has not yet been studied. Hence, this project investigated the role of PAD-dependent citrullination in leukocyte adhesive activity under physiological and pathological conditions. We first treated neutrophils and lymphocytes with different concentrations of BB-Cl-amidine, a pan PAD inhibitor, and GSK199, which induces PAD4 specific blockade, and we studied the effect of cell treatment on \uf0622- integrin-dependent adhesion. Our data showed that PAD inhibitors reduce integrin activation and binding to endothelial ligands upon stimulation in vitro with chemokines, suggesting a role for PADs in rapid leukocyte adhesion mediated by \uf0622 integrins. In addition,, we demonstrated that BB-Cl-amidine also blocked lymphocyte \uf0621-integrin-dependent adhesion on VCAM-1 counterligand, suggesting that PADs are involved in controlling signaling pathways common to both \uf0622 and \uf0621 integrin activation. To exclude potential unspecific effects exerted by PAD inhibitors, we confirmed the data obtained with the pharmacological approach and showed that mRNA silencing of PADs blocks integrin-dependent adhesion in vitro, further confirming the involvement of PADs in leukocyte adhesion. To characterize more in detail the role of PADs in adhesion induction, we studied LFA-1 conformational changes triggered by chemoattractants and found that treatment with BB-Cl-amidine and GSK-199 completely prevented integrin transition to more extended conformations, suggesting a role for PADs in integrin affinity increase leading to leukocyte adhesion. Considering the function of PAD2 and PAD4 in NET release and our previous data demonstrating a role for neutrophils in animal models of Alzheimer's disease, we next investigated the effect of PAD blockade in 3xTg-AD mice, which develop both amyloid and tau pathologies. We treated the 3xTg-AD mice with BB-Cl-amidine, and the results from behavioural tests showed that PAD inhibition significantly rescues spatial working memory and associative learning compared to untreated control mice. Histopathological analysis confirmed the beneficial effects of PAD inhibitors since microglial activation, amyloid accumulation, and tau phosphorylation were strongly reduced after PAD blockade. Finally, we quantified brain-infiltrating leukocytes in BB-Cl-amidine treated mice, reporting that treated animals show reduced accumulation of neutrophils, CD4+ cells, and B lymphocytes compared to untreated mice, suggesting a role for PADs in leukocyte trafficking into the brain in AD mice. In conclusion, this PhD project demonstrated that PAD2 and PAD4 are key elements controlling leukocyte adhesion and that PAD-dependent citrullination is required for integrin-mediated adhesion under physiological and pathological conditions. Moreover, we reported that PADs are involved in disease pathogenesis in 3xTg-AD mice and that therapeutic blockade of PADs ameliorates cognition and reduces neuropathology, suggesting that PAD inhibitors may offer a novel therapeutic strategy for AD

Partial correction of immunodeficiency by lentiviral vector gene therapy in mouse models carrying Rag1 hypomorphic mutations

IntroductionRecombination activating genes (RAG) 1 and 2 defects are the most frequent form of severe combined immunodeficiency (SCID). Patients with residual RAG activity have a spectrum of clinical manifestations ranging from Omenn syndrome to delayed-onset combined immunodeficiency, often associated with granulomas and/or autoimmunity (CID-G/AI). Lentiviral vector (LV) gene therapy (GT) has been proposed as an alternative treatment to the standard hematopoietic stem cell transplant and a clinical trial for RAG1 SCID patients recently started. However, GT in patients with hypomorphic RAG mutations poses additional risks, because of the residual endogenous RAG1 expression and the general state of immune dysregulation and associated inflammation.MethodsIn this study, we assessed the efficacy of GT in 2 hypomorphic Rag1 murine models (Rag1F971L/F971L and Rag1R972Q/R972Q), exploiting the same LV used in the clinical trial encoding RAG1 under control of the MND promoter.Results and discussionStarting 6 weeks after transplant, GT-treated mice showed a decrease in proportion of myeloid cells and a concomitant increase of B, T and total white blood cells. However, counts remained lower than in mice transplanted with WT Lin- cells. At euthanasia, we observed a general redistribution of immune subsets in tissues, with the appearance of mature recirculating B cells in the bone marrow. In the thymus, we demonstrated correction of the block at double negative stage, with a modest improvement in the cortical/medullary ratio. Analysis of antigenspecific IgM and IgG serum levels after in vivo challenge showed an amelioration of antibody responses, suggesting that the partial immune correction could confer a clinical benefit. Notably, no overt signs of autoimmunity were detected, with B-cell activating factor decreasing to normal levels and autoantibodies remaining stable after GT. On the other hand, thymic enlargement was frequently observed, although not due to vector integration and insertional mutagenesis. In conclusion, our work shows that GT could partially alleviate the combined immunodeficiency of hypomorphic RAG1 patients and that extensive efficacy and safety studies with alternative models are required before commencing RAG gene therapy in thesehighly complex patients

An ELF Phonopragmatic Approach to the Analysis of 'Migration Movies' in Pedagogic Contexts: Linguacultural Dimensions of Scripted Interactions

This paper introduces a novel phonopragmatic approach to the analysis of a corpus of ‘migration movies’ employed as teaching material in university courses of ELF for intercultural mediators. The pragmatic implications of the dialogic cues by a number of ELF-speaking characters in films will be explored both qualitatively, in terms of conversation moves and acts occurring within specific contexts of intercul- tural communication, and quantitatively, through a phonopragmatic investigation of the acoustic analysis of speech, and of the phonological segmentation into intonation units and acoustic variations. The objective is to investigate the extent to which the illocutionary and perlocutionary dimensions of the movie interactions can actually find cross-cultural ‘phonopragmatic’ realizations accounting for linguacultural differences in the expression and recognition of conversational presuppositions in the different ELF varieties used by the characters in the selected movies. More specifically, some new moves and acts will be identified to justify cross-cultural miscommunication due to semantic inaccessibility and cultural unavailability in ELF interactions, together with their phonopragmatic realizations in conversation which are here explored by applying a number of prosodic parameters aimed at cue disambiguation in different ELF varieties, such as: vowel sounds in different morpho-syntactic positions and their duration; pitch and duration of tonic syllables and of syllables preceding syntactic boundaries; pause duration at phrase boundaries and their influence on syllabic duration. The relevance of this approach to the teaching of ELF to intercultural mediators will also be discussed

An ELF phonopragmatic approach to the analysis of ‘migration movies’ in pedagogic contexts: linguacultural dimensions of scripted interactions

This paper introduces a novel phonopragmatic approach to the analysis of a corpus of `migration movies' employed as teaching material in university courses of ELF for intercultural mediators. The pragmatic implications of the dialogic cues by a number of ELF-speaking characters in lms will be explored both qualitatively, in terms of conversation moves and acts occurring within specic contexts of intercultural communication, and quantitatively, through a phonopragmatic investigation of the acoustic analysis of speech, and of the phonological segmentation into intonation units and acoustic variations. The objective is to investigate the extent to which the illocutionary and perlocutionary dimensions of the movie interactions can actually nd cross-cultural `phonopragmatic' realizations accounting for linguacultural dierences in the expression and recognition of conversational presuppositions in the dierent ELF varieties used by the characters in the selected movies. More specically, some new moves and acts will be identied to justify cross-cultural miscommunication due to semantic inaccessibility and cultural unavailability in ELF interactions, together with their phonopragmatic realizations in conversation which are here explored by applying a number of prosodic parameters aimed at cue disambiguation in dierent ELF varieties, such as: vowel sounds in dierent morpho-syntactic positions and their duration; pitch and duration of tonic syllables and of syllables preceding syntactic boundaries; pause duration at phrase boundaries and their in uence on syllabic duration. The relevance of this approach to the teaching of ELF to intercultural mediators will also be discussed

ELF Reformulations of Italian 'Lingua Franca' Uses in the Subtitling of the Migration Movie Lamerica

This paper enquires into the scripted ELF variation adopted in the English subtitling of Lamerica (Gianni Amelio, 1994). The target script reformulates the original interactions through Lingua-franca Italian by means of hybridization processes between spoken Italian lingua franca uses and written ELF rendering that are seen as enabling/failing to realize the complex unequal encounters in contexts of specialized (legal-bureaucratic) communication between low-status Albanians and high-status Italians. The analysis of the spoken interactions rendered into ELF subtitling involves three different, yet complementary dimensions of analysis: the register dimension in the perspective of Halliday’s (1978) functional approach and of van Dijk’s (1980) processes of deletion, construction and generalization in rendering the original social interactions into ELF subtitling, respecting the technical limits and facilitating accessibility and acceptability of culture-bound concepts either between the participants in the interaction and in the international audience of the subtitled movie; the phonopragmatic dimension (Sperti, 2014) of the spoken interactions to explore the socio-pragmatic processes accounting for illocutionary and perlocutionary implications (Searle, 1983), and the rendering of such dimensions characterizing the cross-cultural unequal encounters into equivalent written forms of ELF; and the functional dimension involving the standard and scripted ELF variations used in the subtitles, analysed through the application of a causal model of translation (Chesterman, 2000; Bogucki, 2011), enquiring into the cognitive and pragmatic features of the translator’s retextualizations, characterised by relevant lexical and syntactic choices in the attempt to render the participants’ status asymmetries. ELF in this case may thus represent a new hybrid mode of spoken lingua franca rendered into written forms in situations of difficult intercultural communication due to power/status asymmetries between the participants

ELF Reformulations of Italian ‘Lingua Franca’ Uses in the Subtitling of the Migration Movie Lamerica

This paper enquires into the scripted ELF variation adopted in the English subtitling of Lamerica (Gianni Amelio, 1994). The target script reformulates the original interactions through Lingua-franca Italian by means of hybridization processes between spoken Italian lingua franca uses and written ELF rendering that are seen as enabling/failing to realize the complex unequal encounters in contexts of specialized (legal-bureaucratic) communication between low-status Albanians and high-status Italians. The analysis of the spoken interactions rendered into ELF subtitling involves three different, yet complementary dimensions of analysis: the register dimension in the perspective of Halliday’s (1978) functional approach and of van Dijk’s (1980) processes of deletion, construction and generalization in rendering the original social interactions into ELF subtitling, respecting the technical limits and facilitating accessibility and acceptability of culture-bound concepts either between the participants in the interaction and in the international audience of the subtitled movie; the phonopragmatic dimension (Sperti, 2014) of the spoken interactions to explore the socio-pragmatic processes accounting for illocutionary and perlocutionary implications (Searle, 1983), and the rendering of such dimensions characterizing the cross-cultural unequal encounters (in terms of intonational and prosodic variations, emotional and attitudinal conveyance, paralinguistic and visual information) into equivalent written forms of ELF; and the functional dimension involving the standard and scripted ELF variations used in the subtitles, analysed through the application of a causal model of translation (Chesterman, 2000; Bogucki, 2011), enquiring into the cognitive and pragmatic features of the translator’s retextualizations, characterised by relevant lexical and syntactic choices in the attempt to render the participants’ status asymmetries. ELF in this case may thus represent a new hybrid mode of spoken lingua franca rendered into written forms in situations of difficult intercultural communication due to power/status asymmetries between the participants

Blockchain e proprietà intellettuale: funzioni, opportuntà e sfide

Il capitolo analizza le possibili utilizzazioni e le funzioni della Blockchain nella gestione dei diritti di proprietà intellettuale ed industriale, riflettendo sull’idoneità della tecnologia come valido strumento per migliorare l’efficienza dei sistemi di tutela della proprietà intellettuale ed interrogandosi sui correlati rischi. Per far ciò, dopo una breve introduzione alle caratteristiche di Blockchain e Distributed Ledger Technologies (paragrafo I), il paragrafo II si concentra sulla Blockchain come strumento di registrazione e prova, considerando la sua possibile utilizzazione sia con riferimento ai diritti non soggetti a registrazione (come il diritto d’autore) sia con riferimento ai diritti soggetti al procedimento di registrazione (come marchi, brevetti e design industriale). Il paragrafo III è dedicato ad esaminare l’utilizzo di tale tecnologia per la circolazione e la gestione dei diritti di proprietà intellettuale, approfondendo i profili legati all’esercizio dell’autonomia contrattuale attraverso Blockchain e Smart Contracts. Il paragrafo IV, infine, considera alcune delle principali funzioni della Blockchain per l’enforcement dei diritti di proprietà intellettuale e industriale. Sullo sfondo, la domanda principale è se e fino a che punto la gestione della proprietà intellettuale possa beneficiare di una tale automazione.The chapter focuses on the possible uses and opportunities of the Blockchain Technology within the Intellectual Property (IP) management. The goal of the chapter is twofold: (i) the first aim is to explore the role of the Blockchain as a valuable tool for enhancing the efficiency of the IP protection system, while at the same time (ii) the second aim is to examine and to assess the limitations and the risks connected with a pervasive use of such technology. In order to pursue these goals, this paper starts with an overview of the main features of the Blockchain and the Distributed Ledger Technologies (Part 1). Then, Part 2 focuses on the use of the Blockchain as a tool to make IP registration and evidence more efficient both for not registrable IP rigths (such as copyright) and registered IP rights (such as patents, trademarks and industrial designs). Part 3 looks at the use of Blockchain and smart contracts within the IP licensing, questioning if they may bring more room for private ordering and trigger a further disintermediation in IP. Finally, Part 4 examines the use of the Blockchain as a tool to enforce IPRs. The final question is whether and to what extent the IP management might benefit by such level of automation

Blockchain e propriet\ue0 intellettuale: funzioni, opportunt\ue0 e sfide

Il capitolo analizza le possibili utilizzazioni e le funzioni della Blockchain nella gestione dei diritti di propriet\ue0 intellettuale ed industriale, riflettendo sull\u2019idoneit\ue0 della tecnologia come valido strumento per migliorare l\u2019efficienza dei sistemi di tutela della propriet\ue0 intellettuale ed interrogandosi sui correlati rischi. Per far ci\uf2, dopo una breve introduzione alle caratteristiche di Blockchain e Distributed Ledger Technologies (paragrafo I), il paragrafo II si concentra sulla Blockchain come strumento di registrazione e prova, considerando la sua possibile utilizzazione sia con riferimento ai diritti non soggetti a registrazione (come il diritto d\u2019autore) sia con riferimento ai diritti soggetti al procedimento di registrazione (come marchi, brevetti e design industriale). Il paragrafo III \ue8 dedicato ad esaminare l\u2019utilizzo di tale tecnologia per la circolazione e la gestione dei diritti di propriet\ue0 intellettuale, approfondendo i profili legati all\u2019esercizio dell\u2019autonomia contrattuale attraverso Blockchain e Smart Contracts. Il paragrafo IV, infine, considera alcune delle principali funzioni della Blockchain per l\u2019enforcement dei diritti di propriet\ue0 intellettuale e industriale. Sullo sfondo, la domanda principale \ue8 se e fino a che punto la gestione della propriet\ue0 intellettuale possa beneficiare di una tale automazione.The chapter focuses on the possible uses and opportunities of the Blockchain Technology within the Intellectual Property (IP) management. The goal of the chapter is twofold: (i) the first aim is to explore the role of the Blockchain as a valuable tool for enhancing the efficiency of the IP protection system, while at the same time (ii) the second aim is to examine and to assess the limitations and the risks connected with a pervasive use of such technology. In order to pursue these goals, this paper starts with an overview of the main features of the Blockchain and the Distributed Ledger Technologies (Part 1). Then, Part 2 focuses on the use of the Blockchain as a tool to make IP registration and evidence more efficient both for not registrable IP rigths (such as copyright) and registered IP rights (such as patents, trademarks and industrial designs). Part 3 looks at the use of Blockchain and smart contracts within the IP licensing, questioning if they may bring more room for private ordering and trigger a further disintermediation in IP. Finally, Part 4 examines the use of the Blockchain as a tool to enforce IPRs. The final question is whether and to what extent the IP management might benefit by such level of automation

Clinical Significance of Germline Pathogenic Variants among 51 Cancer Predisposition Genes in an Unselected Cohort of Italian Pancreatic Cancer Patients

Multigene germline panel testing is recommended for Pancreatic Cancer (PC) patients; however, for non-BRCA1/2 genes, the clinical utility is unclear. A comprehensive multi-gene assessment in unselected Italian PC patients is missing. We evaluated the prevalence and impact of Pathogenic Variants (PV) in 51 PC susceptibility genes in a real-world series of 422 Italian PC patients unselected for Family History (FH), compared the clinical characteristics and conducted survival analyses. 17% of patients had PVs (70/422), mainly in BRCA1/2 (4.5%, all CDKN2A (4.5%, all >50 y), ATM (2.1%). PV carriers were younger (64 vs. 67; p = 0.02) and had more frequent personal/FH of PC, melanoma and breast/ovarian cancer (all p p = 0.090), comprising ATM carriers (HR 0.33; p = 0.054). In the oxaliplatin-treated subset, PV carriers showed better control of the disease, although this was not statistically significant (67% vs. 56%). CDKN2A, BRCA2 and ATM were the most frequently altered genes. ATM PVs were positively associated with OS in 41% of PV carriers, 60% of whom carried CDKN2A,BRCA2 or ATM PVs, had negative FH and would have been missed by traditional referral. Thus, CDKN2A and ATM should be added to BRCA1/2 testing regardless of FH