Blood and alveolar lymphocyte subsets in pulmonary cytomegalovirus infection after lung transplantation

BACKGROUND: Cytomegalovirus (CMV) pneumonitis has been shown to be associated with lymphocytic alveolitis after lung transplantation. In the present study, we investigated a series of bronchoalveolar (BAL) and blood samples, collected in the absence of rejection or acute infectious episodes. in order -1: to evaluate intra-alveolar cell population changes concomitant with CMV replication and -2: to reappraise the value of cell population analysis in the management of patients after lung transplantation. METHODS: We used flow cytometry to investigate modifications of lymphocyte subpopulations related to pulmonary cytomegalovirus infections in blood and BAL samples from a series of 13 lung transplant recipients. After exclusion of samples obtained during pulmonary rejection, bronchiolitis obliterans or acute bacterial infection, 48 blood and BAL samples were retained for analysis: 17 were CMV positive by shell-vial assay and 31 were CMV negative in blood and BAL. RESULTS: Our results demonstrate that pulmonary CMV infection is associated with a significant increase in the total lymphocyte population in BAL samples, but with minor modifications of the various lymphocyte subpopulations and a significantly higher absolute number of B lymphocytes in blood samples. CONCLUSIONS: Cytomegalovirus pulmonary infection is accompanied by only minor changes in BAL lymphocyte subpopulations. The study of BAL lymphocyte subpopulations therefore appears to be of limited clinical value in the diagnosis of pulmonary CMV infection. However, increased blood B-lymphocytes seems to be a clinical feature associated with CMV infection

In vitro prediction of stop-codon suppression by intravenous gentamicin in patients with cystic fibrosis: a pilot study

BACKGROUND: Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which acts as a chloride channel activated by cyclic AMP (cAMP). The most frequent mutation found in 70% of CF patients is F508del, while premature stop mutations are found in about 10% of patients. In vitro aminoglycoside antibiotics (e.g. gentamicin) suppress nonsense mutations located in CFTR permitting translation to continue to the natural termination codon. Pharmacologic suppression of stop mutations within the CFTR may be of benefit to a significant number of patients. Our pilot study was conducted to determine whether intravenous gentamicin suppresses stop codons in CF patients and whether it has clinical benefits. METHODS: A dual gene reporter system was used to determine the gentamicin-induced readthrough level of the most frequent stop mutations within the CFTR in the French population. We investigated readthrough efficiency in response to 10 mg/kg once-daily intravenous gentamicin perfusions in patients with and without stop mutations. Respiratory function, sweat chloride concentration, nasal potential difference (NPD) and CFTR expression in nasal epithelial cells were measured at baseline and after 15 days of treatment. RESULTS: After in vitro gentamicin incubation, the readthrough efficiency for the Y122X mutation was at least five times higher than that for G542X, R1162X, and W1282X. In six of the nine patients with the Y122X mutation, CFTR immunodetection showed protein at the membrane of the nasal epithelial cells and the CFTR-dependent Cl(- )secretion in NPD measurements increased significantly. Respiratory status also improved in these patients, irrespective of the gentamicin sensitivity of the bacteria present in the sputum. Mean sweat chloride concentration decreased significantly and normalised in two patients. Clinical status, NPD and sweat Cl(- )values did not change in the Y122X patients with no protein expression, in patients with the other stop mutations investigated in vitro and those without stop mutations. CONCLUSION: Suppression of stop mutations in the CFTR gene with parenteral gentamicin can be predicted in vitro and is associated with clinical benefit and significant modification of the CFTR-mediated Cl(- )transport in nasal and sweat gland epithelium

Mucoviscidose et différence de potentiel nasal transépithéliale (intérêt diagnostique des tests pharmacologiques et relations avec le phénotype)

PARIS6-Bibl.Pitié-Salpêtrie (751132101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Therapeutic Approaches for Patients with Cystic Fibrosis Not Eligible for Current CFTR Modulators

Cystic fibrosis is a severe autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding the CFTR protein, a chloride channel expressed in many epithelial cells. New drugs called CFTR modulators aim at restoring the CFTR protein function, and they will benefit many patients with cystic fibrosis in the near future. However, some patients bear rare mutations that are not yet eligible for CFTR modulators, although they might be amenable to these new disease-modifying drugs. Moreover, more than 10% of CFTR mutations do not produce any CFTR protein for CFTR modulators to act upon. The purpose of this review is to provide an overview of different approaches pursued to treat patients bearing mutations ineligible for CFTR modulators. One approach is to broaden the numbers of mutations eligible for CFTR modulators. This requires developing strategies to evaluate drugs in populations bearing very rare genotypes. Other approaches aiming at correcting the CFTR defect develop new mutation-specific or mutation-agnostic therapies for mutations that do not produce a CFTR protein: readthrough agents for nonsense mutations, nucleic acid-based therapies, RNA- or DNA-based, and cell-based therapies. Most of these approaches are in pre-clinical development or, for some of them, early clinical phases. Many hurdles and challenges will have to be solved before they can be safely translated to patients

Transfert de gènes dans les cellules épithéliales des voies aériennes à l'aide de dérives de polylysine (application à la mucoviscidose)

PARIS5-BU Méd.Cochin (751142101) / SudocPARIS-BIUM (751062103) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Trafic intracellulaire de complexes plasmide/ polymères cationiques glycosylés dans des cellules épithéliales des voies aériennes humaines (application à la thérapie génique de la mucoviscidose)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

New treatments targeting the basic defects in cystic fibrosis

Cystic fibrosis (CF) is a monogenic autosomal recessive disorder affecting around 75,000 individuals worldwide. It is a multi-system disease but the main morbidity and mortality is caused by chronic lung disease. Due to newborn screening, a multidisciplinary approach to care and intensive symptomatic treatment, the prognosis has dramatically improved over the last decades and there are currently more adults than children in many countries. However, CF is still a very severe disease with a current median age of life expectancy in the fourth decade of life. The disease is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene which encodes the CFTR protein, a protein kinase A-activated ATP-gated anion channel that regulates the transport of electrolytes such as chloride and bicarbonate. More than 2000 mutations have been reported, although not all of these have functional consequences. An enormous research effort and progress has been made in understanding the consequences of these mutations on the CFTR protein structure and function, and this has led to the approval of two new drug therapies that are able to bind to defective CFTR proteins and partially restore their function. They are mutation-specific therapies and available at present for specific mutations only. They are the first personalized medicine for CF with a possible disease-modifying effect. A pipeline of other compounds is under development with different mechanisms of action. It is foreseeable that new combinations of compounds will further improve the correction of CFTR function. Other strategies including premature stop codon read-through drugs, antisense oligonucleotides that correct the basic defect at the mRNA level or gene editing to restore the defective gene as well as gene therapy approaches are all in the pipeline. All these strategies are needed to develop disease-modifying therapies for all patients with CF

Sugar-Mediated Uptake of Glycosylated Polylysines and Gene Transfer into Normal and Cystic Fibrosis Airway Epithelial Cells

International audienceWe have examined the membrane lectin expressed by immortalized normal and cystic fibrosis (CF) airway epithelial cells, using fluorescein-labeled neoglycoproteins; the uptake of plasmid DNA using fluoresceinylated glycoplexes (plasmid/glycosylated polylysine complexes); and the efficiency of gene transfer when glycosylated polylysines and glycosylated, partially gluconoylated polylysines were used as vectors. The most efficient uptake of neoglycoproteins by normal and CF cells was obtained with mannosylated BSA (bovine serum albumin). Similarly, the most efficient uptake of plasmid DNA was obtained with glycoplexes bearing alpha-D-Man residues. Surprisingly, glycoplexes bearing alpha-D-Man residues were poorly efficient for gene transfer into normal and CF cells. The highest luciferase activity was achieved with lactosylated polylysine- and beta-D-GlcNAc-substituted gluconoylated polylysine as vectors. Gene transfer efficiency obtained with gluconoylated polylysine bearing beta-D-GlcNAc residues was similar to that observed with polyethylenimine (PEI; 25 and 800 kDa) and 10-fold higher than that observed with lipofectin and LipofectAMINE. These results suggest that the transfection efficiency with glycoplexes is not determined only by the specificity of the lectin expressed at the cell surface membrane but also by intracellular trafficking of the glycoplexes, which could be mediated by lectins present inside the cells

Cystic fibrosis gene therapy: key questions and prospects

Cystic fibrosis is a monogenic disorder with significant morbidity and mortality, despite advances in conventional treatment. It is a good candidate for gene therapy and this field has progressed rapidly since the cystic fibrosis transmembrane conductance regulator gene was cloned. We will review the specific questions to address for successful cystic fibrosis gene therapy, such as the extra- and intracellular barriers to airway gene transfer, the target cells and the endpoints to assess efficacy. We will discuss recent advances in viral and nonviral gene transfer agents, delivery techniques and novel strategies to enhance airway gene transfer and expression