Fiorentina Ascenzioni

Dysfunctional CFTR Alters the Bactericidal Activity of Human Macrophages against Pseudomonas aeruginosa

Chronic inflammation of the lung, as a consequence of persistent bacterial infections by several opportunistic pathogens represents the main cause of mortality and morbidity in cystic fibrosis (CF) patients. Mechanisms leading to increased susceptibility to bacterial infections in CF are not completely known, although the involvement of cystic fibrosis transmembrane conductance regulator (CFTR) in microbicidal functions of macrophages is emerging. Tissue macrophages differentiate in situ from infiltrating monocytes, additionally, mature macrophages from different tissues, although having a number of common activities, exhibit variation in some molecular and cellular functions. In order to highlight possible intrinsic macrophage defects due to CFTR dysfunction, we have focused our attention on in vitro differentiated macrophages from human peripheral blood monocytes. Here we report on the contribution of CFTR in the bactericidal activity against Pseudomonas aeruginosa of monocyte derived human macrophages. At first, by real time PCR, immunofluorescence and patch clamp recordings we demonstrated that CFTR is expressed and is mainly localized to surface plasma membranes of human monocyte derived macrophages (MDM) where it acts as a cAMP-dependent chloride channel. Next, we evaluated the bactericidal activity of P. aeruginosa infected macrophages from healthy donors and CF patients by antibiotic protection assays. Our results demonstrate that control and CF macrophages do not differ in the phagocytic activity when infected with P. aeruginosa. Rather, although a reduction of intracellular live bacteria was detected in both non-CF and CF cells, the percentage of surviving bacteria was significantly higher in CF cells. These findings further support the role of CFTR in the fundamental functions of innate immune cells including eradication of bacterial infections by macrophages.

Gene Therapy Progress and Prospects: Episomally maintained self-replicating systems

The use of nonviral gene therapy vectors has been hampered by low level of transfection efficiency and lack of sustained gene expression. Episomal self-replicating systems may overcome these hurdles through their large packaging capacity, stability and reduced toxicity. This article reviews three classes of episomal molecules that have been tested with possible therapeutic genes: (1) self-replicating circular vectors, containing the Epstein–Barr virus (EBV) elements oriP and EBNA1; (2) small circular vectors containing scaffold/matrix attachment regions (S/MARs) as cis-acting elements to maintain the episomal status of the vector; (3) chromosomal vectors, based on the functional elements of the natural chromosomes. The studies reported validate the use of episomal vectors to obtain stable and prolonged gene expression, although reveal some limitations that necessitate additional work.

Gene and cell therapy for cystic fibrosis: From bench to bedside

Clinical trials in cystic fibrosis (CF) patients established proof-of-principle for transfer of the wild-type cystic fibrosis transmembrane conductance regulator (CFTR) gene to airway epithelial cells. However, the limited efficacy of gene transfer vectors as well as extra- and intracellular barriers have prevented the development of a gene therapy-based treatment for CF. Here, we review the use of new viral and nonviral gene therapy vectors, as well as human artificial chromosomes, to overcome barriers to successful CFTR expression. Pre-clinical studies will surely benefit from novel animal models, such as CF pigs and ferrets. Prenatal gene therapy is a potential alternative to gene transfer to fully developed lungs. However, unresolved issues, including the possibility of adverse effects on pre- and postnatal development, the risk of initiating oncogenic or degenerative processes and germ line transmission require further investigation. Finally, we discuss the therapeutic potential of stem cells for CF lung disease.

The number of vertebrate repeats can be regulated at yeast telomeres by Rap1‐independent mechanisms

The number of telomeric DNA repeats at chromosome ends is maintained around a mean value by a dynamic balance between elongation and shortening. In particular, proteins binding along the duplex part of telomeric DNA set the number of repeats by progressively limiting telomere growth. The paradigm of this counting mechanism is the Rap1 protein in Saccharomyces cerevisiae. We demonstrate here that a Rap1‐independent mechanism regulates the number of yeast telomeric repeats (TG1–3) and of vertebrate repeats (T2AG3) when TEL1, a yeast ortholog of the human gene encoding the ATM kinase, is inactivated. In addition, we show that a T2AG3‐only telomere can be formed and maintained in humanized yeast cells carrying a template mutation of the gene encoding the telomerase RNA, which leads to the synthesis of vertebrate instead of yeast repeats. Genetic and biochemical evidences indicate that this telomere is regulated in a Rap1‐independent manner, both in TEL1 and in tel1Δ humanized yeast cells. Altogether, these findings shed light on multiple repeat‐counting mechanisms, which may share critical features between lower and higher eukaryotes.

Functional human CFTR produced by a stable minichromosome.

Artificial chromosomes have been claimed to be the ideal vector for gene therapy, but their use has been hampered by an inability to produce stable and well designed molecules. We have used a structurally defined minichromosome to clone the human cystic fybrosis transmembrane conductance regulator (CFTR) locus. To guarantee the presence of the proper regulatory elements, we used the 320 kb yeast artificial chromosome (YAC) 37AB12 with the intact CFTR gene and upstream sequences. The resulting minichromosome was analyzed for the presence of the entire CFTR gene and for its functional activity by molecular and functional methods. We have identified clones showing the presence of both the transcript and the CFTR protein. Moreover, in the same clones, a chloride secretory response to cAMP was detected. Mitotic and molecular stability after prolonged growth without selection demonstrated that the constructs were stable. This is the first example of a structurally known minichromosome made to contain an active therapeutic gene.

Burkholderia cenocepacia strains isolated from cystic fibrosis patients are apparently more invasive and more virulent than rhizosphere strains

Given the widespread presence of Burkholderia cenocepacia in the rhizosphere it is important to determine whether rhizosphere strains are pathogenic for cystic fibrosis patients or not. Eighteen B. cenocepacia strains of rhizosphere and clinical origin were typed by multi-locus sequence typing (MLST) analysis and compared for their ability to invade pulmonary epithelial cells and their virulence in a mouse model of airway infection. Although there was great variability, clinical strains were the most invasive in vitro. Almost all the rhizosphere and two clinical strains were defined as non-invasive, six clinical strains as invasive, and two strains of both clinical and environmental origin as indeterminate. Exposure of murine airways to clinical strains caused higher acute mortality than that seen after challenge with rhizosphere strains. Furthermore, both clinical and environmental strains were able to persist in the lungs of infected mice, with no significant differences in bacterial loads and localization 14 days after challenge. DNA dot blot analyses of AHL synthase, porin and amidase genes, which play a role in B. cenocepacia virulence, showed that they were present in B. cenocepacia strains irrespective of their origin. Overall, our results suggest that rhizosphere strains do not differ from clinical strains in some pathogenic traits.

The basic N-terminal domain of TRF2 limits recombination endonuclease action at human telomeres

The stability of mammalian telomeres depends upon TRF2, which prevents inappropriate repair and checkpoint activation. By using a plasmid integration assay in yeasts carrying humanized telomeres, we demonstrated that TRF2 possesses the intrinsic property to both stimulate initial homologous recombination events and to prevent their resolution via its basic N-terminal domain. In human cells, we further showed that this TRF2 domain prevents telomere shortening mediated by the resolvase-associated protein SLX4 as well as GEN1 and MUS81, 2 different types of endonucleases with resolvase activities. We propose that various types of resolvase activities are kept in check by the basic N-terminal domain of TRF2 in order to favor an accurate repair of the stalled forks that occur during telomere replication.

Molecular and cytological analysis of a 5.5 Mb minichromosome

Mammalian artificial chromosomes (MACs) provide a new tool for the improvement of our knowledge of chromosome structure and function. Moreover, they constitute an alternative and potentially powerful tool for gene delivery both in cultured cells and for the production of transgenic animals. In the present work we describe the molecular structure of MC1, a human minichromosome derived from chromosome 1. By means of restriction and hybridization analysis, satellite‐PCR, in situ hybridization on highly extended chromatin fibres, and indirect immunofluorescence, we have established that: (i) MC1 has a size of 5.5 Mb; (ii) it consists of 1.1 Mb alphoid, 3.5 Mb Sat2 DNA, and telomeric and subtelomeric sequences at both ends; (iii) it contains an unusual region of interspersed Sat2 and alphoid DNAs at the junction of the alphoid and the Sat2 blocks; and (iv) the two alphoid blocks and the Sat2‐alphoid region bind centromeric proteins suggesting that they participate in the formation of a functional kinetochore.

Reactive-Oxygen-Species-Mediated P. aeruginosa Killing Is Functional in Human Cystic Fibrosis Macrophages

Pseudomonas aeruginosa is the most common pathogen for chronic lung infection in cystic fibrosis (CF) patients. About 80% of adult CF patients have chronic P. aeruginosa infection, which accounts for much of the morbidity and most of the mortality. Both bacterial genetic adaptations and defective innate immune responses contribute to the bacteria persistence. It is well accepted that CF transmembrane conductance regulator (CFTR) dysfunction impairs the airways-epithelium-mediated lung defence; however, other innate immune cells also appear to be affected, such as neutrophils and macrophages, which thus contribute to this infectious pathology in the CF lung. In macrophages, the absence of CFTR has been linked to defective P. aeruginosa killing, increased pro-inflammatory cytokine secretion, and reduced reactive oxygen species (ROS) production. To learn more about macrophage dysfunction in CF patients, we investigated the generation of the oxidative burst and its impact on bacterial killing in CF macrophages isolated from peripheral blood or lung parenchyma of CF patients, after P. aeruginosa infection. Our data demonstrate that CF macrophages show an oxidative response of similar intensity to that of non-CF macrophages. Intracellular ROS are recognized as one of the earliest microbicidal mechanisms against engulfed pathogens that are activated by macrophages. Accordingly, NADPH inhibition resulted in a significant increase in the intracellular bacteria survival in CF and non-CF macrophages, both as monocyte-derived macrophages and as lung macrophages. These data strongly suggest that the contribution of ROS to P. aeruginosa killing is not affected by CFTR mutations.

Development of an in vitro Assay, Based on the BioFilm Ring Test ® , for Rapid Profiling of Biofilm-Growing Bacteria

Microbial biofilm represents a major virulence factor associated with chronic and recurrent infections. Pathogenic bacteria embedded in biofilms are highly resistant to environmental and chemical agents, including antibiotics and therefore difficult to eradicate. Thus, reliable tests to assess biofilm formation by bacterial strains as well as the impact of chemicals or antibiotics on biofilm formation represent desirable tools for a most effective therapeutic management and microbiological risk control. Current methods to evaluate biofilm formation are usually time-consuming, costly, and hardly applicable in the clinical setting. The aim of the present study was to develop and assess a simple and reliable in vitro procedure for the characterization of biofilm-producing bacterial strains for future clinical applications based on the BioFilm Ring Test® (BRT) technology. The procedure developed for clinical testing (cBRT) can provide an accurate and timely (5 h) measurement of biofilm formation for the most common pathogenic bacteria seen in clinical practice. The results gathered by the cBRT assay were in agreement with the traditional crystal violet (CV) staining test, according to the κ coefficient test (κ = 0.623). However, the cBRT assay showed higher levels of specificity (92.2%) and accuracy (88.1%) as compared to CV. The results indicate that this procedure offers an easy, rapid and robust assay to test microbial biofilm and a promising tool for clinical microbiology.