Antonio Manca

Increase in interleukin-8 production from circulating neutrophils upon antibiotic therapy in cystic fibrosis patients

BACKGROUND It is not known whether antibiotic therapy for lung disease in cystic fibrosis (CF) has an influence on circulating polymorphonuclear neutrophil (PMN) function and apoptosis. PATIENTS AND METHODS Blood PMNs were obtained from 14 CF patients before and after antibiotic treatment for an acute exacerbation, and from 10 healthy controls. PMNs were evaluated for production of reactive oxygen species (ROS) by spectrophotometry, of cytokines in the conditioned medium by ELISA, and apoptotic response by cytofluorimetry. RESULTS ROS and interleukin (IL)-8 were produced at higher levels by CF PMNs pre-therapy than control PMNs under basal conditions. IL-8 levels further increased after therapy. Early apoptotic response was higher in CF PMNs pre-therapy than in control PMNs, and this pattern did not change after antibiotic treatment. CONCLUSIONS Circulating PMNs are primed in CF acute patients. Further studies are needed to consider PMN-produced IL-8 as a biomarker to evaluate response to antibiotic therapy in CF patients.

Evaluation of Genome-Wide Expression Profiles of Blood and Sputum Neutrophils in Cystic Fibrosis Patients Before and After Antibiotic Therapy

In seeking more specific biomarkers of the cystic fibrosis (CF) lung inflammatory disease that would be sensitive to antibiotic therapy, we sought to evaluate the gene expression profiles of neutrophils in CF patients before treatment in comparison with non-CF healthy individuals and after antibiotic treatment. Genes involved in neutrophil-mediated inflammation, i.e. chemotaxis, respiratory burst, apoptosis, and granule exocytosis, were the targets of this study. Microarray analysis was carried out in blood and airway neutrophils from CF patients and in control subjects. A fold change (log) threshold of 1.4 and a cut-off of p<0.05 were utilized to identify significant genes. Community networks and principal component analysis were used to distinguish the groups of controls, pre- and post-therapy patients. Control subjects and CF patients before therapy were readily separated, whereas a clear distinction between patients before and after antibiotic therapy was not possible. Blood neutrophils before therapy presented 269 genes down-regulated and 56 up-regulated as compared with control subjects. Comparison between the same patients before and after therapy showed instead 44 genes down-regulated and 72 up-regulated. Three genes appeared to be sensitive to therapy and returned to “healthy” condition: phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), hydrogen voltage-gated channel 1 (HVCN1), and β-arrestin 1 (ARRB1). The up-regulation of these genes after therapy were confirmed by real time PCR. In airway neutrophils, 1029 genes were differentially expressed post- vs pre-therapy. Of these, 30 genes were up-regulated and 75 down-regulated following antibiotic treatment. However, biological plausibility determined that only down-regulated genes belonged to the gene classes studied for blood neutrophils. Finally, it was observed that commonly expressed genes showed a greater variability in airway neutrophils than that found in blood neutrophils, both before and after therapy. These results indicate more specific targets for future interventions in CF patients involving respiratory burst, apoptosis, and granule exocytosis.

Phenotypic expression of genotype-phenotype correlation in cystic fibrosis patients carrying the 852del22 mutation.

Currently, more than 1,000 mutations have been identified in the cystic fibrosis transmembrane regulator (CFTR) gene. While some mutations are common worldwide, the majority are restricted in certain ethnic groups. We have found that in Southern Italy, the 852del22 mutation is well represented with a frequency of 3.5%. We have screened, by reverse dot blot, denaturing gradient gel electrophoresis (DGGE), and gene sequencing, the entire coding regions of CFTR gene in 371 consecutive cystic fibrosis (CF) patients from Southern Italy and have identified 17 patients carrying rare genotypes, among which 13 [6 M; median age 21.7 years (range: 4.5–47.7 years)] carry the 852del22 mutation. To assess the phenotypic expression of CF in patients with the 852del22 mutations we have compared these patients with a group of age and gender matched patients homozygous for the ΔF508 mutation [n = 34; 19 M; median age 19.9 years (range: 3.8–34.6 years)]. Overall, we found no difference in terms of complications, patient survival (17.6% vs. 30.7%; P = NS), estimated time needed to develop a severe lung disease (22.1 vs. 24.5 years; P = NS), nutritional status, and rate of infection or colonization by most common pathogens between patients in the two groups. Finally, we have found that a late diagnosis was associated with a poor outcome (severe lung disease) regardless of genotype. Our data show that 852del22 mutation results in a phenotypic expression of disease as severe as that determined by the more typical ΔF508 and, as in the latter case, there is no strict genotype/phenotype correlation.

A large deletion causes apparent homozygosity for the D1152H mutation in the cystic fibrosis transmembrane regulator (CFTR) gene

We report the case of a patient with an apparent homozygosity for the D1152H mutation located in exon 18 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The parents had no personal history of cystic fibrosis (CF) and referred to our laboratory after the diagnosis of fetal bowel hyperechogenicity. The proband presented with meconium ileus and normal sweat chloride test. Sequencing of the CFTR exon 18 together with quantitative genomic assays, such as real-time PCR and the multiplex ligation probe amplification (MLPA) techniques, were performed and revealed that the father was heterozygous for the D1152H mutation and the mother carried a large deletion of the CFTR gene encompassing the genomic sequence including the same mutation. The child inherited D1152H from his father and the large deletion of the CFTR gene from his mother. We suggest that D1152H likely acts as a mild mutation with a dominant effect on the severe deletion of exon 18, considering that after 3 years of clinical examinations the child shows no classical signs and symptoms of CF. Not testing for large deletions in subjects with apparent homozygosity for a mutated CFTR allele could lead to the misidentification of CFTR mutation carrier status.

Genotype-phenotype correlation in cystic fibrosis patients bearing [H939R;H949L] allele

Cystic fibrosis (CF) is caused by CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations. We ascertained five patients with a novel complex CFTR allele, with two mutations, H939R and H949L, inherited in cis in the same exon of CFTR gene, and one different mutation per patient inherited in trans in a wide population of 289 Caucasian CF subjects from South Italy. The genotype-phenotype relationship in patients bearing this complex allele was investigated. The two associated mutations were related to classical severe CF phenotypes.

Mycobacterium abscessus in patients with cystic fibrosis: Low impact of inter-human transmission in Italy

No outbreak of M. abscessus among Italian CF patients despite the presence of a widespread circulating clone http://ow.ly/oM9l30cs6M0

CFTR‐dependent chloride efflux in cystic fibrosis mononuclear cells is increased by ivacaftor therapy

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) potentiator ivacaftor (Kalydeco®) improves clinical outcome in G551D cystic fibrosis (CF) patients. Here, we have investigated whether ivacaftor has a clinical impact on non‐G551D gating mutations and function of circulating leukocytes as well.

The novel complex allele [A238V;F508del] of the CFTR gene: clinical phenotype and possible implications for cystic fibrosis etiological therapies.

Few mutations in cis have been annotated for F508del homozygous patients. Southern Italy patients who at a first analysis appeared homozygous for the F508del mutation (n=63) or compound heterozygous for the F508del and another mutation in the cystic fibrosis transmembrane conductance regulator gene (n=155) were searched for the A238V mutation in exon 6. The allelic frequency of the complex allele [A238V;F508del] was 0.04. When the whole data set was used (comprised also of 56 F508del/F508del and 34 F508del/other mutation controls), no differences reached the statistical significance in the clinical parameters, except chloride concentrations which were lower in [A238V;F508del]/other mutation compared with F508del/other mutation (P=0.03). The two study groups presented less complications than the control groups. Within the minimal data set (34 F508del/F508del, 27 F508del/other mutation, 4 [A238V;F508del]/F508del cases and 5 [A238V;F508del]/other mutation cases); that is, presenting all the variables in each patient, forced expiratory volume in 1 s and forced vital capacity presented a trend to lower levels in the study groups in comparison with the F508del/F508del group, and C-reactive protein approximated statistically significant higher levels in the [A238V;F508del]/other mutation as compared with F508del/F508del patients (P=0.09). The analysis of statistical dependence among the variables showed a significant anticorrelation between chloride and body mass index in the [A238V;F508del]/other mutation group. In conclusion, the complex allele [A238V;F508del] seems to be associated with less general complications than in the control groups, on the other hand possibly giving a worse pulmonary phenotype and higher systemic/local inflammatory response. These findings have implications for the correct recruitment and clinical response of F508del patients in the clinical trials testing the new etiological drugs for cystic fibrosis.

The role of post-transplantation cyclosporine treatment in the course of cystic fibrosis pulmonary disease: a case report.

We describe the case of a 44-year-old female cystic fibrosis (CF) patient (R334W/852del22) who presented symptoms of prolonged acute respiratory infections and recurrent episodes of pneumonia. Computed tomography (CT) scan images of the chest showed that the patient presented airway and parenchymal changes throughout both lungs. She also had decreased lung function performances. In March 2004, she underwent live-related donor renal transplant and started an immunosuppressive therapy with cyclosporine. CT scan images taken respectively 2 and 6 years after transplantation documented a progressive significant size reduction of structural lung damages in both lungs and clinical signs and symptoms of improvements.

ATYPICAL CYSTIC FIBROSIS ASSOCIATED WITH COMPLEX ALLELE: DIAGNOSTIC AND MANAGEMENT DILEMMAS

trans with a 5 thymidines (5T) sequence within the intron 8 Splice Variant (IVS8) region. 5T is responsible of an exacerbated skipping of exon 9, decreasing the functional product levels. It has been demonstrated that 5T phenotypic expression is influenced by another adjacent polymorphic region, constituted by 9 to 13 TG repeats. In particular, 5T/TG13 combination was found only in affected subjects. Case Report: We report a case regarding two sisters, aged 23 and 18 years, carriers of the F508del mutation associated with the 5T/TG13 combination. DHPLC investigation did not detect a second mutation. Both sisters present mild pulmonary symptoms started in puberty, bronchiectasis, pancreatic sufficiency and border-line chloride values at the sweat test. However, they differ because the elder patient has more evident bronchiectasis, and she also presents pansinusitis, a positive sputum culture and a slightly reduced FEV1. Conclusions: The natural history of non classic CF is poorly understood. It may be asymptomatic for years but a significant lung involvement may occur, as seen in the elder sister. This finding suggests that a prevention therapy could be necessary also in mild, non classic CF and an early diagnosis may prevent the organ deterioration, as in the younger sister. Early diagnosis may be supported by TG repeats testing in individuals carrying the 5T variant; in fact, our report confirms that the presence of 5T allele, in trans with a severe CFTR mutation, is associated with non classic CF and that TG13 variant acts as a real mild mutation, enhancing the 5T penetrance and determining the onset of a mild symptomatology in all patients.