The histone demethylase Jumonji domain-containing protein 3 (JMJD3) regulates fibroblast activation in systemic sclerosis.

OBJECTIVES: Systemic sclerosis (SSc) fibroblasts remain activated even in the absence of exogenous stimuli. Epigenetic alterations are thought to play a role for this endogenous activation. Trimethylation of histone H3 on lysine 27 (H3K27me3) is regulated by Jumonji domain-containing protein 3 (JMJD3) and ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) in a therapeutically targetable manner. The aim of this study was to explore H3K27me3 demethylases as potential targets for the treatment of fibrosis. METHODS: JMJD3 was inactivated by small interfering RNA-mediated knockdown and by pharmacological inhibition with GSKJ4. The effects of targeted inactivation of JMJD3 were analysed in cultured fibroblasts and in the murine models of bleomycin-induced and topoisomerase-I (topoI)-induced fibrosis. H3K27me3 at the FRA2 promoter was analysed by ChIP. RESULTS: The expression of JMJD3, but not of UTX, was increased in fibroblasts in SSc skin and in experimental fibrosis in a transforming growth factor beta (TGFβ)-dependent manner. Inactivation of JMJD3 reversed the activated fibroblast phenotype in SSc fibroblasts and prevented the activation of healthy dermal fibroblasts by TGFβ. Pharmacological inhibition of JMJD3 ameliorated bleomycin-induced and topoI-induced fibrosis in well-tolerated doses. JMJD3 regulated fibroblast activation in a FRA2-dependent manner: Inactivation of JMJD3 reduced the expression of FRA2 by inducing accumulation of H3K27me3 at the FRA2 promoter. Moreover, the antifibrotic effects of JMJD3 inhibition were reduced on knockdown of FRA2. CONCLUSION: We present first evidence for a deregulation of JMJD3 in SSc. JMJD3 modulates fibroblast activation by regulating the levels of H3K27me3 at the promoter of FRA2. Targeted inhibition of JMJD3 limits the aberrant activation of SSc fibroblasts and exerts antifibrotic effects in two murine models

M.A.T. - Modello di Accessibilità di Trasporto

Il modello MAT valuta gli attuali livelli di accessibilità regionale e di connessione interregionale ai fini dell'utilizzazione turistica delle risorse del territorio. La parte innovativa del modello è relativa alla misurazione dell'accessibilità entro un sistema complesso di valorizzazione e fruizione di risorse turistiche che comprendono fatti ambientali, culturali, architettonici, naturalistic

“Strumenti e metodologie per la gestione del sistema stradale urbano - Instruments and methodology for the management of the urban road system”,

Il Nuovo Codice della strada ha posto l'attenzione sugli strumenti della pianificazione dei trasporti di ambito comunale e provinciale imponendo ai comuni con più di 30.000 abitanti e alle provincie di adottare rispettivamente i Piani urbani del traffico ed i Piani del traffico per la viabilità extraurbana. Il decreto del Ministero dei lavori pubblici contenente le direttive per la redazione, adozione ed attuazione dei Piani urbani di traffico ha stabilito i contenuti di tali strumenti al fine di condurre il pianificatore ad occuparsi di tutte le componenti della mobilità e a definire con precisione le soluzioni da adottare in armonia con i piani urbanistici. Il livello di dettaglio richiesto alle analisi e alle proposte d'intervento rende necessari studi anche puntuali dei problemi che possono essere affrontati più agevolmente avvalendosi di uno strumento metodologico ed operativo contenente indirizzi, linee guida, metodi di analisi e di valutazione, schemi progettuali e quant'altro necessario a progettare il sistema stradale urbano come elemento integrato del sistema plurimodale dei trasporti e del sistema territoriale esistente e di progetto. Il volume, che raccoglie i risultati del lavoro svolto dall'Unità operativa dell'Università della Basilicata nell'ambito del Progetto Finalizzato Trasporti 2 del Cnr, si basa su di un'ampia ricerca bibliografica, i cui risultati, raccolti ed analizzati criticamente dagli autori, vengono utilizzati per definire tecniche e metodologie d'intervento. Attraverso una trattazione la più possibile semplice e completa si è cercato di offrire a quanti operano per la redazione e l'attuazione dei documenti di pianificazione urbana e dei trasporti uno strumento d'uso immediato, per la definizione di soluzioni generali e puntuali, con l'auspicio di contribuire a colmare il ritardo che ancora oggi si riscontra nell'applicazione delle conoscenze scientifiche alla programmazione e progettazione degli elementi del sistema urbano integrato dei trasporti

Intracellular human antibody fragments recognizing the VP35 protein of Zaire Ebola filovirus inhibit the protein activity

BACKGROUND: Ebola hemorrhagic fever is caused by the Ebola filovirus (EBOV), which is one of the most aggressive infectious agents known worldwide. The EBOV pathogenesis starts with uncontrolled viral replication and subversion of both the innate and adaptive host immune response. The multifunctional viral VP35 protein is involved in this process by exerting an antagonistic action against the early antiviral alpha/beta interferon (IFN-α/β) response, and represents a suitable target for the development of strategies to control EBOV infection. Phage display technology permits to select antibodies as single chain Fragment variable (scFv) from an artificial immune system, due to their ability to specifically recognize the antigen of interest. ScFv is ideal for genetic manipulation and to obtain antibody constructs useful for targeting either antigens expressed on cell surface or intracellular antigens if the scFv is expressed as intracellular antibody (intrabody) or delivered into the cells. RESULTS: Monoclonal antibodies (mAb) in scFv format specific for the EBOV VP35 were isolated from the ETH-2 library of human recombinant antibodies by phage display technology. Five different clones were identified by sequencing, produced in E.coli and expressed in CHO mammalian cells to be characterized in vitro. All the selected scFvs were able to react with recombinant VP35 protein in ELISA, one of the scFvs being also able to react in Western Blot assay (WB). In addition, all scFvs were expressed in cell cytoplasm as intrabodies; a luciferase reporter gene inhibition assay performed in A549 cells showed that two of the scFvs can significantly hamper the inhibition of the IFN-β-induced RIG-I signaling cascade mediated by EBOV VP35. CONCLUSION: Five antibodies in scFv format recognize an active form of EBOV VP35 in ELISA, while one antibody also recognizes VP35 in WB. Two of these scFvs were also able to interfere with the intracellular activity of VP35 in a cell system in vitro. These findings suggest that such antibodies in scFv format might be employed to develop therapeutic molecules able to hamper EBOV infections

The glutathione S-transferase inhibitor 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol overcomes the MDR1-P-glycoprotein and MRP1-mediated multidrug resistance in acute myeloid leukemia cells.

There has been an ever growing interest in the search for new anti-tumor compounds that do not interact with MDR1-Pgp and MRP1 drug transporters and so circumvent the effect of these proteins conferring multidrug resistance (MDR) and poor prognosis in AML patients. We have investigated the cytotoxic activity of the strong glutathione S-transferase (GST) inhibitor 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) on AML (HL60) cell lines. METHODS: Functional drug efflux studies and cell proliferation assays were performed on both sensitive and MDR AML (HL60) cells after incubation with NBDHEX. Moreover, the mode of cell death (apoptosis vs. necrosis) as well as the correlation between NBDHEX susceptibility and GST activity or Bcl-2 expression was investigated. RESULTS: NBDHEX is not a substrate of either MDR1-Pgp or MRP1 efflux pumps; in fact, it is not only cytotoxic toward the parental HL60 cell line, but also overcomes the MDR phenotype of its HL60/DNR and HL60/ADR variants. CONCLUSIONS: The data herein reported show that NBDHEX mediates efficient killing of both MDR1-Pgp and MRP1 over-expressing AML cells. Therefore, this drug can potentially be used as an effective agent for treating MDR in AML patients

A rare case of IgM-Multiple Myeloma

IgM Myeloma

Development of a novel human phage display-derived anti-LAG3 scFv antibody targeting CD8+ T lymphocyte exhaustion

Background: Lymphocyte-activation gene (LAG)3 is a 498 aa transmembrane type I protein acting as an immune inhibitory receptor. It is expressed on activated lymphocytes, natural killer cells and plasmacytoid dendritic cells. In activated lymphocytes, LAG3 expression is involved in negative control of cell activation/proliferation to ensure modulation and control of immune responses. In view of its deregulated expression in tumor-infiltrating lymphocytes, LAG3, together with the additional immune checkpoint inhibitors CTLA4 and PD1, is considered a major target in order to reverse the immunosuppression typically mounting in oncologic diseases. Since many patients still fail to respond to current immune checkpoints-based therapies, the identification of new effective immune inhibitors is a priority in the ongoing fight against cancer. Results: We identified a novel human single-chain variable fragment (scFv) Ab against a conformational epitope of LAG3 by in vitro phage display technology using the recombinant antigen as a bait. This scFv (referred to as F7) was characterized in terms of binding specificity to both recombinant antigen and human LAG3-expressing cells. It was then rebuilt into an IgG format pre-optimized for clinical usage, and the resulting bivalent construct was shown to preserve its ability to bind LAG3 on human cells. Next, we analyzed the activity of the anti-LAG3 scFvF7 using two different antigen-specific CD8+ T lymphocyte clones as target cells. We proved that the reconstituted anti-LAG3 F7 Ab efficiently binds the cell membrane of both cell clones after peptide-activation. Still more significantly, we observed a striking increase in the peptide-dependent cell activation upon Ab treatment as measured in terms of IFN-γrelease by both ELISA and ELISPOT assays. Conclusions: Overall, the biotechnological strategy described herein represents a guiding development model for the search of novel useful immune checkpoint inhibitors. In addition, our functional data propose a novel candidate reagent for consideration as a cancer treatment