Endogenous CCL2 neutralization restricts HIV-1 replication in primary human macrophages by inhibiting viral DNA accumulation

Macrophages are key targets of HIV-1 infection. We have previously described that the expressionof CC chemokine ligand 2 (CCL2) increases during monocyte differentiation to macrophages and it is furtherup-modulated by HIV-1 exposure. Moreover, CCL2 acts as an autocrine factor that promotes viral replication ininfected macrophages. In this study, we dissected the molecular mechanisms by which CCL2 neutralization inhibitsHIV-1 replication in monocyte-derived macrophages (MDM), and the potential involvement of the innate restrictionfactors protein sterile alpha motif (SAM) histidine/aspartic acid (HD) domain containing 1 (SAMHD1) and apolipoproteinB mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family members.Results:CCL2 neutralization potently reduced the number of p24 Gag+cells during the course of either productive orsingle cycle infection with HIV-1. In contrast, CCL2 blocking did not modify entry of HIV-1 based Virus Like Particles, thusdemonstrating that the restriction involves post-entry steps of the viral life cycle. Notably, the accumulation of viralDNA, both total, integrated and 2-LTR circles, was strongly impaired by neutralization of CCL2. Looking for correlates ofHIV-1 DNA accumulation inhibition, we found that the antiviral effect of CCL2 neutralization was independent of themodulation of SAMHD1 expression or function. Conversely, a strong and selective induction of APOBEC3A expression,to levels comparable to those of freshly isolated monocytes, was associated with the inhibition of HIV-1 replicationmediated by CCL2 blocking. Interestingly, the CCL2 neutralization mediated increase of APOBEC3A expression was typeI IFN independent. Moreover, the transcriptome analysis of the effect of CCL2 blocking on global gene expressionrevealed that the neutralization of this chemokine resulted in the upmodulation of additional genes involved in thedefence response to viruses.Conclusions:Neutralization of endogenous CCL2 determines a profound restriction of HIV-1 replication in primaryMDM affecting post-entry steps of the viral life cycle with a mechanism independent of SAMHD1. In addition, CCL2blocking is associated with induction of APOBEC3A expression, thus unravelling a novel mechanism which mightcontribute to regulate the expression of innate intracellular viral antagonistsin vivo. Thus, our study may potentially leadto the development of new therapeutic strategies for enhancing innate cellular defences against HIV-1 and protecting macrophages from infection

Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from Yeast

<p>Abstract</p> <p>Background</p> <p>The ability of cytosine deaminase (CD) to convert the antifungal agent 5-fluorocytosine (5-FC) into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU) raised considerable interest in this enzyme to model gene or antibody – directed enzyme-prodrug therapy (GDEPT/ADEPT) aiming to improve the therapeutic ratio (benefit versus toxic side-effects) of cancer chemotherapy. The selection and characterization of a human monoclonal antibody in single chain fragment (scFv) format represents a powerful reagent to allow in <it>in vitro </it>and <it>in vivo </it>detection of CD expression in GDEPT/ADEPT studies.</p> <p>Results</p> <p>An enzymatic active recombinant CD from yeast (yCD) was expressed in E. coli system and used as antigen for biopanning approach of the large semi-synthetic ETH-2 antibody phage library. Several scFvs were isolated and specificity towards yCD was confirmed by Western blot and ELISA. Further, biochemical and functional investigations demonstrated that the binding of specific scFv with yCD did not interfere with the activity of the enzyme in converting 5-FC into 5-FU.</p> <p>Conclusion</p> <p>The construction of libraries of recombinant antibody fragments that are displayed on the surface of filamentous phage, and the selection of phage antibodies against target antigens, have become an important biotechnological tool in generating new monoclonal antibodies for research and clinical applications. The scFvH5 generated by this method is the first human antibody which is able to detect yCD in routinary laboratory techniques without interfering with its enzymatic function.</p

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

The transcription factors ATF3 and EN1 as regulators of the profibrotic effects of TGF beta signaling in the pathogenesis of Systemic Sclerosis [Korrigierte Version]

Systemic sclerosis (SSc) is a complex heterogeneous autoimmune disease with unknown etiology. It has three major pathologic hallmarks: vasculopathy, immune activation and extensive fibrosis that is an excessive accumulation of extracellular matrix proteins released from activated fibroblasts and destroys the physiological tissue architecture and function of the involved organs. Particularly, diffuse cutaneous SSc (dcSSc) has a highly increased risk of morbidity and mortality. Unfortunately, no targeted treatment is approved for fibrosis in SSc and there is a great medical need for new anti-fibrotic therapies for SSc. In this thesis, we evaluated the anti-fibrotic effects of targeting ATF3 and EN1 transcription factors. Both molecules have a distinct contribution to the disease’s pathogenesis and each one could serve as a potential target for the treatment of fibrosis in SSc. In the first part of this thesis, we evaluated the role of ATF3 in skin fibrosis as the most commonly affected tissue. Furthermore, we showed that ATF3 is a crucial regulator of TGF-β signaling in SSc. ATF3-deficient fibroblasts are less responsive to TGF-β with impaired production of extracellular matrix components such as collagens and activation of Smad-dependent transcription, reduced activity in reporter assays and decreased levels of TGF-β target genes. On the contrary, overexpression of ATF3 amplified the effect of TGF-β on fibroblast activation. Given that TGF-β stimulates the expression of ATF3 in a Smad3-dependent manner, ATF3 may represent an endogenous enhancer of canonical TGF-β signaling and its overexpression may thus contribute to the aberrant activation of TGF-β signaling in SSc. Furthermore, ATF3 deletion ameliorated fibrosis in several complementary mouse models of SSc. In the second part of this thesis, we investigated the role of EN1 in the pathogenesis of SSc. We demonstrated that EN1 regulates TGF-β signaling which is the core pathway in fibrotic diseases. Inactivation of EN1 signaling reduced the TGF-β mediated activation of fibroblasts and production of collagens and target genes. Moreover, fibroblast-specific knockout of EN1 ameliorated fibrosis in several mouse models

The transcription factors ATF3 and EN1 as regulators of the profibrotic effects of TGF beta signaling in the pathogenesis of Systemic Sclerosis

Systemic sclerosis (SSc) is a complex heterogeneous autoimmune disease with unknown etiology. It has three major pathologic hallmarks: vasculopathy, immune activation and extensive fibrosis that is an excessive accumulation of extracellular matrix proteins released from activated fibroblasts and destroys the physiological tissue architecture and function of the involved organs. Particularly, diffuse cutaneous SSc (dcSSc) has a highly increased risk of morbidity and mortality. Unfortunately, no targeted treatment is approved for fibrosis in SSc and there is a great medical need for new anti-fibrotic therapies for SSc. In this thesis, we evaluated the anti-fibrotic effects of targeting ATF3 and EN1 transcription factors. Both molecules have a distinct contribution to the disease’s pathogenesis and each one could serve as a potential target for the treatment of fibrosis in SSc. In the first part of this thesis, we evaluated the role of ATF3 in skin fibrosis as the most commonly affected tissue. Furthermore, we showed that ATF3 is a crucial regulator of TGF-β signaling in SSc. ATF3-deficient fibroblasts are less responsive to TGF-β with impaired production of extracellular matrix components such as collagens and activation of Smad-dependent transcription, reduced activity in reporter assays and decreased levels of TGF-β target genes. On the contrary, overexpression of ATF3 amplified the effect of TGF-β on fibroblast activation. Given that TGF-β stimulates the expression of ATF3 in a Smad3-dependent manner, ATF3 may represent an endogenous enhancer of canonical TGF-β signaling and its overexpression may thus contribute to the aberrant activation of TGF-β signaling in SSc. Furthermore, ATF3 deletion ameliorated fibrosis in several complementary mouse models of SSc. In the second part of this thesis, we investigated the role of EN1 in the pathogenesis of SSc. We demonstrated that EN1 regulates TGF-β signaling which is the core pathway in fibrotic diseases. Inactivation of EN1 signaling reduced the TGF-β mediated activation of fibroblasts and production of collagens and target genes. Moreover, fibroblast-specific knockout of EN1 ameliorated fibrosis in several mouse models. The results of both parts of the thesis might have clinical implications. Targeted inactivation of ATF3 and EN1 may thus offer potential to interfere with persistent TGF-β signaling and aberrant fibroblast activation in fibrotic diseases

La pianificazione ambientale di una infrastruttura di trasporto: un modello di analisi multiobiettivo

La ricerca esamina il problema della valutazione di impatto ambientale, proponendo l'applicazione di un originale modello multi-obiettivo, costruito dagli autori, alla valutazione ambientale di un tronco autostradale in Sicilia. La memoria espone i problemi di stima delle variabili ambientali e la loro valutazione quantitativa e discute sull'affidabilit\ue0 del risultato e sulla stabilit\ue0 della soluzione trovata, applicando un algoritmo informatico di variazione dei pesi nel rispetto di una scala ordinale di importanza degli stessi

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