Tracking the immunopathological response to Pseudomonas aeruginosa during respiratory infections

Repeated cycles of infections, caused mainly by Pseudomonas aeruginosa, combined with a robust host immune response and tissue injury, determine the course and outcome of cystic fibrosis (CF) lung disease. As the disease progresses, P. aeruginosa adapts to the host modifying dramatically its phenotype; however, it remains unclear whether and how bacterial adaptive variants and their persistence influence the pathogenesis and disease development. Using in vitro and murine models of infection, we showed that P. aeruginosa CF-adaptive variants shaped the innate immune response favoring their persistence. Next, we refined a murine model of chronic pneumonia extending P. aeruginosa infection up to three months. In this model, including CFTR-deficient mice, we unveil that the P. aeruginosa persistence lead to CF hallmarks of airway remodelling and fibrosis, including epithelial hyperplasia and structure degeneration, goblet cell metaplasia, collagen deposition, elastin degradation and several additional markers of tissue damage. This murine model of P. aeruginosa chronic infection, reproducing CF lung pathology, will be instrumental to identify novel molecular targets and test newly tailored molecules inhibiting chronic inflammation and tissue damage processes in pre-clinical studies

IL-17A impairs host tolerance during airway chronic infection by Pseudomonas aeruginosa

Resistance and tolerance mechanisms participate to the interplay between host and pathogens. IL-17-mediated response has been shown to be crucial for host resistance to respiratory infections, whereas its role in host tolerance during chronic airway colonization is still unclear. Here, we investigated whether IL-17-mediated response modulates mechanisms of host tolerance during airways chronic infection by P. aeruginosa. First, we found that IL-17A levels were sustained in mice at both early and advanced stages of P. aeruginosa chronic infection and confirmed these observations in human respiratory samples from cystic fibrosis patients infected by P. aeruginosa. Using IL-17a(-/-) or IL-17ra(-/-) mice, we found that the deficiency of IL-17A/IL-17RA axis was associated with: i) increased incidence of chronic infection and bacterial burden, indicating its role in the host resistance to P. aeruginosa; ii) reduced cytokine levels (KC), tissue innate immune cells and markers of tissue damage (pro-MMP-9, elastin degradation, TGF-β1), proving alteration of host tolerance. Blockade of IL-17A activity by a monoclonal antibody, started when chronic infection is established, did not alter host resistance but increased tolerance. In conclusion, this study identifies IL-17-mediated response as a negative regulator of host tolerance during P. aeruginosa chronic airway infection

A distinguishing gene signature shared by tumor-infiltrating Tie2-expressing monocytes, blood "resident" monocytes, and embryonic macrophages suggests common functions and developmental relationships

We previously showed that Tie2-expressing monocytes (TEMs) have nonredundant proangiogenic activity in tumors. Here, we compared the gene expression profile of tumor-infiltrating TEMs with that of tumor-associated macrophages (TAMs), spleen-derived Gr1(+)Cd11b(+) neutrophils/myeloid-derived suppressor cells, circulating "inflammatory" and "resident" monocytes, and tumor-derived endothelial cells (ECs) by quantitative polymerase chain reaction-based gene arrays. TEMs sharply differed from ECs and Gr1(+)Cd11b(+) cells but were highly related to TAMs. Nevertheless, several genes were differentially expressed between TEMs and TAMs, highlighting a TEM signature consistent with enhanced proangiogenic/tissue-remodeling activity and lower proinflammatory activity. We validated these findings in models of oncogenesis and transgenic mice expressing a microRNA-regulated Tie2-GFP reporter. Remarkably, resident monocytes and TEMs on one hand, and inflammatory monocytes and TAMs on the other hand, expressed coordinated gene expression profiles, suggesting that the 2 blood monocyte subsets are committed to distinct extravascular fates in the tumor microenvironment. We further showed that a prominent proportion of embryonic/fetal macrophages, which participate in tissue morphogenesis, expressed distinguishing TEM genes. It is tempting to speculate that Tie2(+) embryonic/fetal macrophages, resident blood monocytes, and tumor-infiltrating TEMs represent distinct developmental stages of a TEM lineage committed to execute physiologic proangiogenic and tissue-remodeling programs, which can be co-opted by tumors

AAV6-mediated Systemic shRNA Delivery Reverses Disease in a Mouse Model of Facioscapulohumeral Muscular Dystrophy

Treatment of dominantly inherited muscle disorders remains a difficult task considering the need to eliminate the pathogenic gene product in a body-wide fashion. We show here that it is possible to reverse dominant muscle disease in a mouse model of facioscapulohumeral muscular dystrophy (FSHD). FSHD is a common form of muscular dystrophy associated with a complex cascade of epigenetic events following reduction in copy number of D4Z4 macrosatellite repeats located on chromosome 4q35. Several 4q35 genes have been examined for their role in disease, including FRG1. Overexpression of FRG1 causes features related to FSHD in transgenic mice and the FRG1 mouse is currently the only available mouse model of FSHD. Here we show that systemic delivery of RNA interference expression cassettes in the FRG1 mouse, after the onset of disease, led to a dose-dependent long-term FRG1 knockdown without signs of toxicity. Histological features including centrally nucleated fibers, fiber size reduction, fibrosis, adipocyte accumulation, and inflammation were all significantly improved. FRG1 mRNA knockdown resulted in a dramatic restoration of muscle function. Through RNA interference (RNAi) expression cassette redesign, our method is amenable to targeting any pathogenic gene offering a viable option for long-term, body-wide treatment of dominant muscle disease in humans

PRaTo: A web-tool to select optimal primer pairs for qPCR

An essential pre-requisite to perform sound quantitative real-time polymerase chain reaction (qPCR) assays is to design outstanding primer pairs. This means they must have a good efficiency and be not prone to produce multiple amplicons or primer dimer products. To circumvent these issues, several softwares are available to help primer design. Although satisfactory computer-aided primer design tools are available for standard PCR, less efforts were done to provide specific methods for selection of optimal primer pairs for qPCR. We have developed PRaTo a web-based tool that enables checking and ranking of primers pairs for their attitude to perform optimally and reliably when used in qPCR experiments. PRaTo is available at http://prato.daapv.unipd.it

On the Optimal Operation Frequency to Minimize Phase Noise in Integrated Harmonic Oscillators

This brief aims at finding the most appropriate frequency of operation of an integrated harmonic oscillator in order to maximize its spectral purity. To achieve this, a simple, yet accurate, scalable model is developed for the LC tank, that tracks the dependence of the parasitics on the inductance value. Using an ultra-scaled CMOS digital technology as a case study, the frequencies around 5 GHz are singled out as the sweet spot to minimize the phase noise

Computational Intelligence Methods for Bioinformatics and Biostatistics - 11th International Meeting, CIBB 2014, Cambridge, UK, June 26-28, 2014, Revised Selected Papers

This book constitutes the thoroughly refereed post-conference proceedings of the 11th International Meeting on Computational Intelligence Methods for Bioinformatics and Biostatistics, CIBB 2014, held in Cambridge, UK, in June 2014. The 25 revised full papers presented were carefully reviewed and selected from 44 submissions. The papers focus problems concerning computational techniques in bioinformatics, systems biology, medical informatics and biostatistics

Identification of the arabidopsis RAM/MOR signalling network: Adding new regulatory players in plant stem cell maintenance and cell polarization

none8siBackground and Aims The RAM/MOR signalling network of eukaryotes is a conserved regulatory module involved in co-ordination of stem cell maintenance, cell differentiation and polarity establishment. To date, no such signalling network has been identified in plants. Methods Genes encoding the bona fide core components of the RAM/MOR pathway were identified in Arabidopsis thaliana (arabidopsis) by sequence similarity searches conducted with the known components from other species. The transcriptional network(s) of the arabidopsis RAM/MOR signalling pathway were identified by running in-depth in silico analyses for genes co-regulated with the core components. In situ hybridization was used to confirm tissue-specific expression of selected RAM/MOR genes. Key Results Co-expression data suggested that the arabidopsis RAM/MOR pathway may include genes involved in floral transition, by co-operating with chromatin remodelling and mRNA processing/post-transcriptional gene silencing factors, and genes involved in the regulation of pollen tube polar growth. The RAM/MOR pathway may act upstream of the ROP1 machinery, affecting pollen tube polar growth, based on the co-expression of its components with ROP-GEFs. In silico tissue-specific co-expression data and in situ hybridization experiments suggest that different components of the arabidopsis RAM/MOR are expressed in the shoot apical meristem and inflorescence meristem and may be involved in the fine-tuning of stem cell maintenance and cell differentiation. Conclusions The arabidopsis RAM/MOR pathway may be part of the signalling cascade that converges in pollen tube polarized growth and in fine-tuning stem cell maintenance, differentiation and organ polarity.restrictedZermiani, Monica; Begheldo, Maura; Nonis, Alessandro; Palme, Klaus; Mizzi, Luca; Morandini, Piero; Nonis, Alberto; Ruperti, BenedettoZermiani, Monica; Begheldo, Maura; Nonis, Alessandro; Palme, Klaus; Mizzi, Luca; Morandini, Piero; Nonis, Alberto; Ruperti, Benedett

Evaluation of candidate reference genes for qPCR in maize

Quantitative real-time PCR (qPCR) is a powerful tool to measure gene expression levels. Accurate and reproducible results are dependent on the correct choice of the reference genes for data normalization. To date, screenings evaluating candidate reference gene stability for expression studies in maize have not been reported. In the present work, we analyzed the expression patterns of 12 genes in a set of 20 maize samples, obtained from different tissues of plants grown at various experimental conditions. Using genormPLUS, NormFinder and BestKeeper algorithms, the expression stability of three \u201cclassical\u201d reference genes, such as ACT, TUB and 18S rRNA, and the newly identified candidates, was assessed. With respect to the algorithms, our results showed similar performance among genormPLUS, NormFinder and BestKeeper in evaluating the suitability of reference genes. Our data therefore showed that the currently and widely used reference genes for data normalization in maize were not the most stable expressed transcripts. Five of the new putative reference genes (CUL, FPGS, LUG, MEP and UBCP) exhibited the highest expression stability according to all algorithms. In conclusion, with this study, we provide a list of validated reference genes and their relative primer sequences to conduct reliable qPCR experiments in maize

Effect of the number of removed lymph nodes on prostate cancer recurrence and survival: evidence from an observational study

Abstract Background The aim of this article is to analyze the effect on biochemical recurrence and on overall survival of removing an extensive number of pelvic lymph nodes during prostate cancer surgery. The lack of evidence from randomized clinical trials to address this specific question has hampered the ability to determine the true effect of the number of nodes removed. Results Our analysis is based on a large observational study, and this can lead unadjusted estimates to be very sensitive to confounding bias due to the different prognosis of individuals. We assess the effect of the number of lymph nodes removed by means of an Inverse Probability Weighting adjustment based on a Poisson regression model, and by a Doubly-robust adjustment. Conclusions Our findings suggest that a large number of nodes removed is associated with a significant improvement in time to biochemical recurrence. However, it appears to have no impact on overall survival