DEFINITION OF AN IN VITRO MODEL OF HUMAN MONOCYTE ACTIVATION REPRESENTATIVE OF THE DEFENSIVE INFLAMMATORY RESPONSE

La reazione di difesa innata/infiammatoria \ue8 attivata in risposta a patogeni esterni o a segnali provenienti dal tessuto danneggiato. I monociti/macrofagi hanno un ruolo chiave nell\u2019inizio e risoluzione della infiammazione per mezzo di differenti programmi di attivazione. Infatti i macrofagi possono adottare in vivo una variet\ue0 di fenotipi diversi che dipendono dai cambiamenti del microambiente tissutale, esibendo un continuum di stati funzionali diversi. Inoltre i monociti del sangue periferico non sono una popolazione omogenea ma differiscono nei loro fenotipi e funzioni. Nonostante l\u2019esplosivo aumento di informazioni sull\u2019argomento, molte questioni sono ancora aperte riguardo la caratterizzazione fenotipica e funzionale dei monociti/macrofagi, e il loro ruolo durante l\u2019omeostasi e l\u2019infiammazione. La maggior parte dei dati provengono da studi sul topo e molti immunologi fanno ancora affidamento su modelli di topo malgrado la distanza evolutiva e le differenze tra i sistemi immuni murino e umano. Nel tentativo di capire le questioni di cui sopra e di dirigere gli sforzi verso una immunobiologia basata sull\u2019uomo, il fine di questo lavoro \ue8 stato quello di costruire e validare un modello umano della risposta di difesa innata/infiammatoria in vitro che ricapitolasse le differenti fasi della reazione infiammatoria, dal reclutamento e inizio, allo sviluppo e risoluzione dell\u2019infiammazione e conseguente ripristino della omeostasi. Il modello \ue8 basato su monociti umani primari del sangue esposti in coltura a cambiamenti sequenziali delle condizioni microambientali (chemiochine, citochine, temperatura, molecole di derivazione batterica, ecc.) per 48 h. L\u2019analisi al citofluorimetro ha dimostrato che la popolazione monocitaria utilizzata era rappresentativa dell\u2019eterogeneit\ue0 monocitaria cos\uec come presente nella circolazione sanguigna. Tutte le fasi della risposta infiammatoria sono state definite mediante analisi trascrittomica effettuata con U133Plus 2.0 GeneChip (Affymetrix). I risultati sono stati confrontati e integrati con profili trascrizionali pubblicamente disponibili di monociti/macrofagi, raccolti e annotati in un database ad hoc. Il profilo trascrittomico di alcuni fattori trascrizionali e fattori correlati con l\u2019infiammazione sono stati confermati e validati mediante qPCR e ELISA. La \u201ccluster analysis\u201d ha rivelato cluster ampi e distinti che comprendono geni con un chiaro andamento che ben descrivono le differenti fasi dell\u2019infiammazione. Per ottenere maggiori indicazioni sul ruolo biologico dei geni differenzialmente espressi durante la risposta infammatoria, ciascun cluster \ue8 stato analizzato con la GSEA (Gene Set Enrichment Analysis). I set di geni identificati dalla GSEA correlati con il profilo di espressione dei differenti cluster ha rivelato che la fase infiammatoria era arricchita di pathway infiammatorie mentre la fase anti-infiammatoria, cos\uec come quella di risoluzione, di pathway relative al metabolismo, al ciclo cellulare e al riarrangiamento genico. Inoltre confrontando le liste dei geni differenzialmente espressi tra monociti e macrofagi M1 e tra monociti e macrofagi M2 estratte dal meta-database, \ue8 stato dimostrato che i monociti trattati in vitro secondo il modello mostrano un profilo M1 durante la fase infiammatoria e M2 durante la risoluzione. L\u2019espressione genica dei fattori trascrizionali e di quelli relativi alla infiammazione rispecchiavano il profilo di espressione ottenuto con microarray. In conclusione i dati di microarray e l\u2019analisi cinetica dei fattori infiammatori e anti-infiammatori validano il modello in vitro proposto, modello che consente di descrivere la sequenza tempo-dipendente e coordinata degli eventi relativi alla infiammazione.The innate/inflammatory defensive reaction is activated in response to foreign pathogens or signals from damaged tissue. Monocytes/macrophages are key players in the initiation and resolution of inflammation by different activation programmes. Indeed in vivo macrophages can adopt a variety of different phenotypes depending on changes in the tissue microenvironment displaying a continuum of diverse functional states. Moreover peripheral blood monocytes are not a homogeneous population but differ in their phenotypes and functions. In spite of the explosive growth of data, many issues are still open about the phenotypic and functional characterization of monocytes/macrophages, and their role during the homeostasis and in inflammatory conditions. The great majority of the data originates from studies in mice and many immunologists still rely on mouse models despite the evolutionary distance and the differences between the murine and human immune systems. In an attempt to understanding the above issues, and to direct efforts in human immunobiology, the aim of this work was to build and validate a human model of innate/inflammatory defence response in vitro that recapitulates the different phases of the inflammatory reaction, from recruitment and initiation, to development and resolution of inflammation, and re-establishment of homeostasis. The model is based on human primary blood monocytes exposed in culture to sequential changes of microenvironmental conditions (chemokines and cytokines, temperature, bacterial-derived molecules, etc.) for 48 h. The flow cytometrical analysis has shown that the monocyte population used is representative of the monocyte heterogeneity as present in the circulation. All phases of the inflammatory response were profiled by transcriptomic analysis carried out with U133Plus 2.0 GeneChip (Affymetrix). Results were compared and integrated with publicly available transcriptional profiles of monocyte/macrophages, collected and annotated in an ad hoc database. The transcriptomic profiling of some transcriptional and inflammatory-related factors were confirmed and validated by qPCR and by ELISA. The \u201ccluster analysis\u201d revealed broad distinct clusters comprising genes with a clear behaviour that well described the different phases of inflammation. To gain more insight into the biologic role of the genes that are differentially expressed during the inflammatory response, each cluster was subjected to gene set enrichment analysis (GSEA). The gene sets identified by GSEA correlated with the expression profile of different clusters revealed that the inflammatory phase was enriched in inflammatory pathways while the anti-inflammatory phase, as well as the resolution phase, in pathways related to metabolism, cell cycle, and gene rearrangement. Moreover, by comparing the lists of differentially expressed gene between monocytes vs. M1 macrophages and vs. M2 macrophages extracted from the meta-database, it was shown that monocytes treated in vitro according to model resemble M1 during the inflammatory phase and M2 during the resolution. The gene expression of transcriptional and inflammatory-related factors matched with the expression profile obtained with microarrays. In conclusion the microarray data and the kinetical analysis of inflammatory and anti-inflammatory factors validate the proposed in vitro model of the inflammatory response, and allowed describing the time-dependent and coordinated sequence of inflammation-related events

Multi-language transfer learning for low-resource legal case summarization

Analyzing and evaluating legal case reports are labor-intensive tasks for judges and lawyers, who usually base their decisions on report abstracts, legal principles, and commonsense reasoning. Thus, summarizing legal documents is time-consuming and requires excellent human expertise. Moreover, public legal corpora of specific languages are almost unavailable. This paper proposes a transfer learning approach with extractive and abstractive techniques to cope with the lack of labeled legal summarization datasets, namely a low-resource scenario. In particular, we conducted extensive multi- and cross-language experiments. The proposed work outperforms the state-of-the-art results of extractive summarization on the Australian Legal Case Reports dataset and sets a new baseline for abstractive summarization. Finally, syntactic and semantic metrics assessments have been carried out to evaluate the accuracy and the factual consistency of the machine-generated legal summaries

Different Regulation of Interleukin-1 Production and Activity in Monocytes and Macrophages: Innate Memory as an Endogenous Mechanism of IL-1 Inhibition

Production and activity of interleukin (IL)-1β are kept under strict control in our body, because of its powerful inflammation-promoting capacity. Control of IL-1β production and activity allows IL-1 to exert its defensive activities without causing extensive tissue damage. Monocytes are the major producers of IL-1β during inflammation, but they are also able to produce significant amounts of IL-1 inhibitors such as IL-1Ra and the soluble form of the decoy receptor IL-1R2, in an auto-regulatory feedback loop. Here, we investigated how innate immune memory could modulate production and activity of IL-1β by human primary monocytes and monocyte-derived tissue-like/deactivated macrophages in vitro. Cells were exposed to Gram-negative (Escherichia coli) and Gram-positive (Lactobacillus acidophilus) bacteria for 24 h, then allowed to rest, and then re-challenged with the same stimuli. The presence of biologically active IL-1β in cell supernatants was calculated as the ratio between free IL-1β (i.e., the cytokine that is not bound/inhibited by sIL-1R2) and its receptor antagonist IL-1Ra. As expected, we observed that the responsiveness of tissue-like/deactivated macrophages to bacterial stimuli was lower than that of monocytes. After resting and re-stimulation, a memory effect was evident for the production of inflammatory cytokines, whereas production of alarm signals (chemokines) was minimally affected. We observed a high variability in the innate memory response among individual donors. This is expected since innate memory largely depends on the previous history of exposure or infections, which is different in different subjects. Overall, innate memory appeared to limit the amount of active IL-1β produced by macrophages in response to a bacterial challenge, while enhancing the responsiveness of monocytes. The functional re-programming of mononuclear phagocytes through modulation of innate memory may provide innovative approaches in the management of inflammatory diseases, as well as in the design of new immunization strategies. In this respect, the interindividual variability in innate memory suggests the need of a personalized assessment

Circulating levels of IL-1 family cytokines and receptors in Alzheimer's disease: new markers of disease progression?

BACKGROUND: Although the mechanisms underlying AD neurodegeneration are not fully understood, it is now recognised that inflammation could play a crucial role in the initiation and progression of AD neurodegeneration. A neuro-inflammatory network, based on the anomalous activation of microglial cells, includes the production of a number of inflammatory cytokines both locally and systemically. These may serve as diagnostic markers or therapeutic targets for AD neurodegeneration. METHODS: We have measured the levels of the inflammation-related cytokines and receptors of the IL-1 family in serum of subjects with AD, compared to mild cognitive impairment (MCI), subjective memory complaints (SMC), and normal healthy subjects (NHS). Using a custom-made multiplex ELISA array, we examined ten factors of the IL-1 family, the inflammation-related cytokines IL-1α, IL-1β, IL-18, and IL-33, the natural inhibitors IL-1Ra and IL-18BP, and the soluble receptors sIL-1R1, sIL-1R2, sIL-1R3, and sIL-1R4. RESULTS: The inflammatory cytokines IL-1α and IL-1β, their antagonist IL-1Ra, and their soluble receptor sIL-1R1 were increased in AD. The decoy IL-1 receptor sIL-1R2 was only increased in MCI. IL-33 and its soluble receptor sIL-1R4 were also significantly higher in AD. The soluble form of the accessory receptor for both IL-1 and IL-33 receptor complexes, sIL-1R3, was increased in SMC and even more in AD. Total IL-18 levels were unchanged, whereas the inhibitor IL-18BP was significantly reduced in MCI and SMC, and highly increased in AD. The levels of free IL-18 were significantly higher in MCI. CONCLUSIONS: AD is characterised by a significant alteration in the circulating levels of the cytokines and receptors of the IL-1 family. The elevation of sIL-1R4 in AD is in agreement with findings in other diseases and can be considered a marker of ongoing inflammation. Increased levels of IL-1Ra, sIL-1R1, sIL-1R4, and IL-18BP distinguished AD from MCI and SMC, and from other inflammatory diseases. Importantly, sIL-1R1, sIL-1R3, sIL-1R4, and IL-18BP negatively correlated with cognitive impairment. A significant elevation of circulating sIL-1R2 and free IL-18, not present in SMC, is characteristic of MCI and disappears in AD, making them additional interesting markers for evaluating progression from MCI to AD

Transcriptomic profiling of the development of the inflammatory response in human monocytes in vitro.

Monocytes/macrophages are key players in all phases of physiological and pathological inflammation. To understanding the regulation of macrophage functional differentiation during inflammation, we designed an in vitro model that recapitulates the different phases of the reaction (recruitment, initiation, development, and resolution), based on human primary blood monocytes exposed to sequential changes in microenvironmental conditions. All reaction phases were profiled by transcriptomic microarray analysis. Distinct clusters of genes were identified that are differentially regulated through the different phases of inflammation. The gene sets defined by GSEA analysis revealed that the inflammatory phase was enriched in inflammatory pathways, while the resolution phase comprised pathways related to metabolism and gene rearrangement. By comparing gene clusters differentially expressed in monocytes vs. M1 and vs. M2 macrophages extracted from an in-house created meta-database, it was shown that cells in the model resemble M1 during the inflammatory phase and M2 during resolution. The validation of inflammatory and transcriptional factors by qPCR and ELISA confirmed the transcriptomic profiles in the different phases of inflammation. The accurate description of the development of the human inflammatory reaction provided by this in vitro kinetic model can help in identifying regulatory mechanisms in physiological conditions and during pathological derangement

Full Geant4 and FLUKA Simulations of an e-LINAC for its Use in Particle Detectors Performance Tests

In this work we present the results of full Geant4 and FLUKA simulations and comparison with dosimetry data of an electron LINAC of St. Maria Hospital located in Terni, Italy. The facility is being used primarily for radiotherapy and the goal of present study is the detailed investigation of electron beam parameters to evaluate the possibility to use the e-LINAC (during time slots when it is not used for radiotherapy) to test the performance of detector systems in particular those designed to operate in space. The critical beam parameters are electron energy, profile and flux available at the surface of device to be tested. The present work aims to extract these parameters from dosimetry calibration data available at the e-LINAC. The electron energy ranges is from 4 MeV to 20 MeV. The dose measurements have been performed by using an Advanced Markus Chamber which has a small sensitive volume.Comment: 10 pages, 10 figures, 2 table

On the use of highly pixellated CMOS imagers to measure therapeutic beam profile

The characterization of high-intensity charged-particle and photon beams at medical accelerators is often a time-consuming task. In this work, we discuss the possibility to use highly segmented CMOS imagers as a way to measure the fluxes with high spatial precision and in a short time. Quite recently CMOS imagers, designed to collect visible light, have been used to detect ionizing radiation, either charged particles (electron, proton) or photons. These devices, due to the very low single pixel noise, have a very high detection efficiency, once the interaction between radiation and silicon has taken place, and act primarily as counting detectors. We will show how it is possible to extract a precise beam shape using as a test case a therapeutic electron beam delivered by an Elekta e-LINAC at the S. Maria Hospital in Terni (Italy), and as sensors commercial off-the-shelf (COTS) CMOS imagers

The Mycobacterial LysR-Type Regulator OxyS Responds to Oxidative Stress and Negatively Regulates Expression of the Catalase-Peroxidase Gene

Protection against oxidative stress is one of the primary defense mechanisms contributing to the survival of Mycobacterium tuberculosis in the host. In this study, we provide evidence that OxyS, a LysR-type transcriptional regulator functions as an oxidative stress response regulator in mycobacteria. Overexpression of OxyS lowers expression of the catalase-peroxidase (KatG) gene in M. smegmatis. OxyS binds directly with the katG promoter region and a conserved, GC-rich T-N11-A motif for OxyS binding was successfully characterized in the core binding site. Interestingly, the DNA-binding activity of OxyS was inhibited by H2O2, but not by dithiothreitol. Cys25, which is situated at the DNA-binding domain of OxyS, was found to have a regulatory role for the DNA-binding ability of OxyS in response to oxidative stress. In contrast, the other three cysteine residues in OxyS do not appear to have this function. Furthermore, the mycobacterial strain over-expressing OxyS had a higher sensitivity to H2O2.Thus, OxyS responds to oxidative stress through a unique cysteine residue situated in its DNA-binding domain and negatively regulates expression of the katG gene. These findings uncover a specific regulatory mechanism for mycobacterial adaptation to oxidative stress