Small airway deposition of dornase alfa during exacerbations in cystic fibrosis; a randomized controlled clinical trial.

INTRODUCTION: Small airway obstruction is important in the pathophysiology of cystic fibrosis (CF) lung disease. Additionally, many CF patients lose lung function in the long term as a result of respiratory tract exacerbations (RTEs). No trials have been performed to optimize mucolytic therapy during a RTE. We investigated whether specifically targeting dornase alfa to the small airways improves small airway obstruction during RTEs. METHODS: In a multi-center, double-blind, randomized controlled trial CF patients hospitalized for a RTE and on maintenance treatment with dornase alfa were switched to a smart nebulizer. Patients were randomized to small airway deposition (n = 19) or large airway deposition (n = 19) of dornase alfa for at least 7 days. Primary endpoint was forced expiratory flow at 75% of forced vital capacity (FEF75 ). MAIN RESULTS: Spirometry parameters improved significantly during admission, but the difference in mean change in FEF75 between treatment groups was not significant: 0.7 SD, P = 0.30. FEF25-75 , FEV1 , nocturnal oxygen saturation and diary symptom scores also did not differ between groups. CONCLUSIONS: This study did not detect a difference if inhaled dornase alfa was targeted to small versus large airways during a RTE. However, the 95% confidence interval for the change in FEF75 was wide. Further studies are needed to improve the effectiveness of RTE treatment in CF.

Improved treatment response to dornase alfa in cystic fibrosis patients using controlled inhalation.

Better treatment of obstructed small airways is needed in cystic fibrosis. This study investigated whether efficient deposition of dornase alfa in the small airways improves small airway obstruction. In a multicentre, double-blind, randomised controlled clinical trial, cystic fibrosis patients on maintenance treatment with 2.5 mL dornase alfa once daily were switched to a smart nebuliser and randomised to small airway deposition (n = 24) or large airway deposition (n = 25) for 4 weeks. The primary outcome parameter was forced expiratory flow at 75% of forced vital capacity (FEF(75%)). FEF(75%) increased significantly by 0.7 sd (5.2% predicted) in the large airways group and 1.2 sd (8.8% pred) in the small airways group. Intention-to-treat analysis did not show a significant difference in treatment effect between groups. Per-protocol analysis, excluding patients not completing the trial or with adherence <70%, showed a trend (p = 0.06) in FEF(75%) Z-score and a significant difference (p = 0.04) between groups in absolute FEF(75%) (L · s(-1)) favouring small airway deposition. Improved delivery of dornase alfa using a smart nebuliser that aids patients in correct inhalation technique resulted in significant improvement of FEF(75%) in children with stable cystic fibrosis. Adherent children showed a larger treatment response for small airway deposition.

PTX3 genetic variations affect the risk of Pseudomonas aeruginosa airway colonization in cystic fibrosis patients.

Cystic fibrosis (CF) is a common life-threatening autosomal recessive disorder in the Caucasian population, and the gene responsible is the CF transmembrane conductance regulator (CFTR). Patients with CF have repeated bacterial infection of the airways caused by Pseudomonas aeruginosa (PA), which is one of the predominant pathogen, and endobronchial chronic infection represents a major cause of morbidity and mortality. Pentraxin 3 (PTX3) is a gene that encodes the antimicrobial protein, PTX3, which is believed to have an important role in innate immunity of lung. To address the role of PTX3 in the risk of PA lung colonization, we investigated five single nucleotide polymorphisms of PTX3 gene in 172 Caucasian CF patients who were homozygous for the F508del mutation. We observed that PTX3 haplotype frequencies were significantly different between patients with PA colonization, as compared with noncolonized patients. Moreover, a protective effect was found in association with a specific haplotype (odds ratio 0.524). Our data suggest that variations within PTX3 affect lung colonization of Pseudomonas in patients with CF.

Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice.

It is often challenging for the clinician interested in cystic fibrosis (CF) to interpret molecular genetic results, and to integrate them in the diagnostic process. The limitations of genotyping technology, the choice of mutations to be tested, and the clinical context in which the test is administered can all influence how genetic information is interpreted. This paper describes the conclusions of a consensus conference to address the use and interpretation of CF mutation analysis in clinical settings. Although the diagnosis of CF is usually straightforward, care needs to be exercised in the use and interpretation of genetic tests: genotype information is not the final arbiter of a clinical diagnosis of CF or CF transmembrane conductance regulator (CFTR) protein related disorders. The diagnosis of these conditions is primarily based on the clinical presentation, and is supported by evaluation of CFTR function (sweat testing, nasal potential difference) and genetic analysis. None of these features are sufficient on their own to make a diagnosis of CF or CFTR-related disorders. Broad genotype/phenotype associations are useful in epidemiological studies, but CFTR genotype does not accurately predict individual outcome. The use of CFTR genotype for prediction of prognosis in people with CF at the time of their diagnosis is not recommended. The importance of communication between clinicians and medical genetic laboratories is emphasized. The results of testing and their implications should be reported in a manner understandable to the clinicians caring for CF patients.

IL-8 and pH values in exhaled condensate after antibiotics in cystic fibrosis children.

Interleukin (IL)-8 is a major factor in inflammatory response and the IL-8 levels in exhaled breath condensate (EBC) may be used as a marker of airway inflammation. Airway acidification is implicated in the pathophysiology of obstructive airway diseases and pH EBC values have been used as a marker of airway acidification. The aim of our study is to investigate whether IL-8 and pH levels in EBC of cystic fibrosis (CF) children with respiratory exacerbations change after antibiotic treatment. Lung function, IL-8 and pH EBC values were measured in fifteen CF children (mean age 11 years) with acute exacerbation before (T0) and after two weeks (T1) of antibiotic treatment. IL-8 and pH values were compared by paired t-test. A p less than 0.05 was considered significant. IL-8 EBC levels decreased after antibiotic treatment (T0 0.36+/-0.03pg/ml vs T1 0.28+/-0.03pg/ml; p=0.03) and pH values increased (T0 7.36+/-0.09 vs T1 7.61+/-0.08; p=0.04). Results suggest possible application of EBC as a non-invasive tool to monitor efficacy of antibiotic treatment in CF patients.

Effects of azithromycin on the expression of ATP binding cassette transporters in epithelial cells from the airways of cystic fibrosis patients.

Induction of ATP Binding Cassette (ABC) proteins involved in chloride transport has been proposed as a possible mechanism of the beneficial effects of azithromycin (AZM) in cystic fibrosis (CF) patients. This study focused on the effects of AZM on mRNA and protein expression of Multidrug Resistance-associated Protein 1 (MRP1) and Multidrug Resistance Protein 1 (MDR1) by real-time quantitative PCR, flow cytometry and gene reporter assays in two CF and two isogenic non-CF airway epithelial cell lines. We detected higher levels of MRP1 and lower levels of MDR1 mRNA in CF versus non-CF cells while both proteins were not differentially expressed. After AZM treatment we found modest differences in MRP1 and MDR1 mRNA expression while protein levels were unaffected. The ability of AZM to regulate MRP1 promoter transcriptional activity was excluded by gene reporter assays. Our data do not support the hypothesis of induction of ABC transporters by AZM.

Human respiratory cells from nasal polyps as a model for gene transfer by non-viral cationic vectors.

The influence of cell differentiation and proliferation on cationic vector mediated gene transfer into the explant-outgrowth cell culture from nasal polyps was investigated. Respiratory cells were categorized into two groups based on the expression of cytokeratin filaments (CKs), which were used as differentiation markers. Outgrowths grown for 2 weeks expressed similar levels of CKs 14, 13 and 18 showing a de-differentiated phenotype, while outgrowths cultured for 4 weeks presented very high levels of CK 13, high CK 14 and low CK 18 expression and were squamous differentiated. De-differentiated cells presented higher proliferation indexes than squamous cells. Gene transfer levels, as evaluated using a quantitative reporter gene (firefly luciferase), were significantly higher in the 2- than in the 4-week-old outgrowths. Cationic vector transfected respiratory cells were located both proximally and distally to the explant, as shown by enzymatic staining of beta-galactosidase-positive cells. Respiratory cell outgrowths from nasal polyps can be considered a suitable model to study gene transfer protocols in vitro.

Bacterial infections and inflammation in the lungs of cystic fibrosis patients.

The aim of this review is to describe the role of respiratory epithelial cells in processes that contribute to the pathogenesis of lung disease in patients with cystic fibrosis.

A pilot survey of cystic fibrosis clinical manifestations in CFTR mutation heterozygotes.

A cystic fibrosis (CF) heterozygote incidence higher than in the general population has been repeatedly reported in conditions which include clinical features found in CF, like pancreatitis, disseminated bronchiectasis, and allergic bronchopulmonary aspergillosis. Some cases may be explained by an unidentified compound heterozygosity; others could be manifesting heterozygotes. This study was aimed at detecting the incidence of CF-related clinical features in a population of carriers. A group of 261 obligate heterozygotes (mean age, 44 years) and a control group, composed of 201 individuals negative for a standard mutation panel (mean age, 36 years), were surveyed for possibly CF-related conditions (asthma, bronchiectasis, pneumothorax, allergic bronchopulmonary aspergillosis, sinusitis, nasal polyps, gallstones, liver cirrhosis, diabetes, pancreatitis, bone fractures, plus hypertension). A questionnaire was administered, and the accuracy of the statements was evaluated by phone interviews. There was no difference between heterozygotes and controls, with the exception of hypertension (carriers 28/261, controls 7/201, p = 0.004), and, in males, nasal polyps (carriers 7/126, controls 0/102, p value = 0.0178), and, again, hypertension (carriers 17/126, controls 5/102, p value = 0.0407). To avoid age bias, 126 heterozygotes matched to controls of the same gender and age were separately processed: these two groups showed no significant differences. CF-related clinical manifestations in obligate CFTR mutation heterozygotes are not more represented than in individuals with a low risk of being carriers.

Biodistribution and transgene expression with nonviral cationic vector/DNA complexes in the lungs.

Biodistribution of nonviral cationic vector/DNA complexes was studied after systemic or intratracheal administration to the lungs and correlated with transgene expression. Intravenous injection in C57Bl/6 mice gave maximal and significant luciferase expression in the lungs with the cationic polymer PEI 22K/DNA complexes at the highest ratios of positive/negative charges versus DNA alone. While DOTAP/DNA complexes with high charge ratio determined lower but still significant luciferase activity versus uncomplexed DNA, GL-67A and PEI 25K mediated negligible luciferase expression. Labelled PEI 22K and DOTAP complexes were evenly distributed in the alveolar region, where GFP expression was revealed, while PEI 25K and GL-67A complexes were not detected, suggesting a different interaction of these complexes with the plasma membrane of endothelial cells. Following an intratracheal injection, the highest and significant levels of transfection were obtained with slightly positive PEI complexes as compared with DNA alone, whereas cationic lipid-based vectors, DOTAP and GL-67A, gave not significant luciferase activity. Both types of polyplexes gave similar levels of lung luciferase expression by targeting different airway cell populations. PEI 25K complexes determined high levels of GFP in the bronchial cells, confirming confocal data on fluorescent complexes internalization. PEI 22K complexes gave mainly high GFP signal in the distal tract of the bronchial tree, where tagged complexes were recovered. Fluorescent lipid complexes were found in aggregates in the lumen of bronchi totally (DOTAP) or partially (GL-67A) co-localizing with surfactant protein A. Results indicated that cationic polymers could overcome the surfactant barrier which inhibited airway cell transfection mediated by cationic lipids.

Restoration of bacterial killing activity of human respiratory cystic fibrosis cells through cationic vector-mediated cystic fibrosis transmembrane conductance regulator gene transfer.

In vitro and in vivo studies have demonstrated that gene transfer of the CFTR (cystic fibrosis transmembrane conductance regulator) cDNA into human respiratory cells through nonviral vectors can occur safely and can be done repeatedly. Although functional evaluation of CFTR in cystic fibrosis (CF) patients enrolled in phase I clinical trials using cationic liposomes has shown a partial correction of nasal potential difference, a biological assay indicating a therapeutic relevance of CFTR gene transfer is still missing. Our aims were to study the induction of killing activity toward Pseudomonas aeruginosa (PA) in CF cells by cationic vector-mediated CFTR gene transfer and to use this assay as a therapeutic end point. Luciferase expression and GFP FACS analysis were used to evaluate the optimal vector and the efficiency of gene transfer into non-CF human respiratory cells growing from nasal polyp explants at the air-liquid interface. To prove that transgenic CFTR was expressed in CF cell cultures under the same experimental conditions, a specific RT-PCR was performed. Challenge of the outgrowths with a known amount of PA showed a bacterial clearance activity by non-CF respiratory cells, while in the case of CF cells it even resulted in bacterial growth. Cationic vector-mediated CFTR cDNA determined the recovery of bacterial clearance activity only under those conditions yielding 5% or more of GFP-positive cells. The results shown in this study might be helpful in considering cationic vectors as therapeutic nonviral vectors for transferring CFTR into human CF respiratory cells, as well as for restoring the bacterial killing activity defective in cystic fibrosis.

Immunity to poliomyelitis, diphtheria and tetanus in pediatric patients before and after renal or liver transplantation.

Few studies have considered the safety, efficacy and appropriateness of vaccinations in pediatric patients before and after solid organ transplantation. The aim of this study was to evaluate the immune status after primary vaccination to diphtheria, tetanus and poliomyelitis in pediatric patients before and after hepatic transplantation and to poliomyelitis in pediatric patients before and after renal transplantation. All the patients had received a complete primary immunization schedule for diphtheria and tetanus and poliomyelitis. Immunity to the three polioviruses was evaluated in 56 patients with renal transplant, 27 on chronic dialysis and 33 controls and in 39 patients with hepatic transplant, 25 with chronic hepatic failure and their 36 controls. Immunity to diphtheria and tetanus was evaluated in 52 liver transplant patients, 29 children with chronic hepatic failure and 54 healthy children. Renal transplant patients were less protected and had lower antibody geometric mean titers than healthy controls for polioviruses 1 and 2. Whereas, protection in the children liver transplant patients was similar to that in their controls. Patients with chronic hepatic failure had higher antibody geometric mean titers to diphtheria and polioviruses 1 and 3 than their control group. Immunosuppression after transplantation has a negative influence on the immune status after primary vaccination in children with renal transplant. Whereas children with chronic hepatic failure have higher antibodies than a normal population. When possible, it could be advisable to individualize immunization schedules in patients at high risk.

Duration of immunity to diphtheria and tetanus in young kidney transplant patients.

A considerable proportion of patients with renal transplant, evaluated many years after transplant, lack protective diphtheria antibody levels, despite primary immunization, but maintain immunity to tetanus. These patients respond to a diphtheria and tetanus booster but the duration of the response is uncertain. This study was undertaken to assess if protective antibodies evoked by primary immunization are lost quickly after transplantation, and whether the extent of the immune response to a booster influences the persistence of protective antibodies. We studied 15 patients (group 1) immediately after renal transplant and 35 patients with renal transplant for 6 +/- 4 yr who received a diphtheria and tetanus booster (group 2). Six patients (40%) of group 1 lost protective diphtheria antibodies a median time of 6.5 months after transplant. Thirty-three patients of group 2 responded to the booster with normal diphtheria antibody titers (> 1 IU/mL) in 22 cases and with low titers in 11. Four of the latter lacked immunity to diphtheria at 12 months follow-up. All patients with normal immunity maintained protective levels of diphtheria antibodies. The low responders had a creatinine clearance of 50 +/- 20 mL/min/1.73 m2. Tetanus immunity was maintained in almost all patients of both groups. In conclusion, renal transplant patients had an accelerated loss of diphtheria antibodies in the early post-transplant period. Response to a diphtheria booster identified a group at particular risk, namely the low responders, who may require frequent booster doses. This group had significantly poorer renal function than the normal responders.

Epidemiology and cost analysis of varicella in Italy: results of a sentinel study in the pediatric practice. Italian Sentinel Group on Pediatric Infectious Diseases.

BACKGROUND: Describing the epidemiology of varicella is relevant to the development of specific prevention strategies and to building up of economic models evaluating the cost:efficiency ratios of these strategies. AIM: Our study was designed to describe the epidemiology of chickenpox among Italian children and to assess the resulting economic and health burden on the country. METHODS: Thirty-nine Italian pediatricians participated in a sentinel network on pediatric infectious diseases representing a total pediatric population of 30 168 children. Each case of varicella observed from January through December, 1997, was recorded. Economic analysis was conducted from the societal point of view. All costs were broken down into two groups: direct and indirect costs. RESULTS: A total of 1599 cases of varicella were reported among children 0 to 14 years old. There were 1266 primary cases (mean age, 4.5 +/- 2 years) and 333 secondary cases (mean age, 3.6 +/- 3.2 years). The global incidence of chickenpox was 51.01/1000/year. Complications were seen in 56 cases (3.5%). Drugs were prescribed in 672 cases. A group of adults (364 susceptible and 193 with uncertain status) were exposed to primary cases. Seventy (12.5%) were eventually infected among whom there were 4 pregnant women. For pediatric patients an average cost of $146.90 (250 400 lire) was estimated; this is largely accounted for by indirect costs. CONCLUSIONS: The epidemiology of varicella in Italy is consistent with that found in previous studies in industrialized countries. Severe complications did not occur in our population. We believe that the health arguments in favor of universal vaccination of children > 18 months of age do not differ in our own country from those of other industrialized nations. Our data could now be incorporated into pharmacoeconomic models to establish cost-efficient strategies for Italy.

Hypotonic saline infusion alters the renal response to amino acids in men.

We investigated the effects of hypotonic saline-induced modifications of extracellular volume and sodium handling on the renal and metabolic response to amino acids (AA). Renal hemodynamics (Inutest, p-aminohippurate clearance), plasma AA, and glucagon levels, as well as urea and sodium excretion, were studied in seven adult volunteers infused for 2 h, on six separate occasions, according to the following protocols: 1) high-AA solution (300 mg. min-1. 1.73 m-2); 2) low-AA solution (150 mg. min-1. 1.73 m-2); 3) low AA + 2,000 ml/1.73 m2 of 0.23% saline solution; 4) high AA + 0. 23% saline; 5) high AA + 0.45% saline; and 6) 0.45% saline alone. The glomerular filtration rate (GFR) rise induced by the high-AA solution was similar to that induced by the low-AA solution (DeltaGFR = +24 +/- 6 and +20.2 +/- 7 ml. min-1. 1.73 m-2, respectively), whereas the plasma AA and glucagon levels and urea excretion rate increases were related to AA dose. The addition of 0. 23% saline to the low-AA solution and of 0.45% saline to the high-AA solution blunted the renal hemodynamic response (DeltaGFR = +6.6 +/- 10.1 and +11.4 +/- 8.3 ml. min-1. 1.73 m-2, respectively) without modifying the pattern of plasma AA and glucagon levels and urea excretion observed with the AA infusion alone. Urinary sodium excretion increased from baseline with each protocol and rose even further when saline was added to AA. A negative correlation (r = -0. 38, P < 0.05) was found between the changes from basal values in GFR and those in sodium excretion rate with high-AA infusion at different levels of sodium concentration. These data suggest that AA-induced hyperfiltration might be blunted by hypotonic saline infusion, possibly through an acute modification of renal sodium handling and extracellular volume.

Comparison between cationic polymers and lipids in mediating systemic gene delivery to the lungs.

Airway inflammation frequently found in congenital and acquired lung diseases may interfere with gene delivery by direct administration through either instillation or aerosol. Systemic delivery by the intravenous administration represents an alternative route of delivery that might bypass this barrier. A nonviral approach for transfecting various airway-derived cell lines in vitro showed that cationic polymers (PEI 22K and 25K) and lipids (DOTAP, GL-67/DOPE) are able to transfect with high efficiency the reporter genes firefly luciferase and E. coli lacZ. Notably, two properties predicted that cationic vectors would be useful for a systemic gene delivery approach to the lung: (1) transfection was not inhibited or increased when cells were incubated with cationic lipids or polymers in the presence of serum; and (2) cationic vectors protected plasmid DNA from DNase degradation. A single injection of DNA complexed to the cationic polymer PEI 22K into the tail vein of adult mice efficiently transfected primarily the lungs and to a lesser extent, heart, spleen, kidney and liver. The other vectors mediated lower to undetectable levels of luciferase expression in the lungs, with DOTAP > GL67/DOPE > PEI 25K > DOTMA/DOPE. A double injection protocol with a 15-min interval between the two doses of DOTAP/DNA complexes was investigated and showed a relevant role of the first injection in transfecting the lungs. A two log increase in luciferase expression was obtained either when the two doses were comprised of luciferase plasmid or when an irrelevant plasmid was used in the first injection. The double injection of luciferase/PEI 22K complexes determined higher transgene levels than a single dose, but a clear difference using an irrelevant plasmid as first dose was not observed. Using lacZ as a reporter gene, it was shown that only cells in the alveolar region, including type II penumocytes, stained positively for the transgene product.

Metabolic factors in the renal response to amino acid infusion.

In order to investigate the renal effects of amino acids (AA) with different metabolic fate, we compared the changes in glomerular and tubular function, nitrogen metabolism and glucoregulatory hormones in 7 volunteers during two infusions, one of a complete solution of amino acids (MIX-AA), which included five AA electively metabolized at the splanchnic level, and the other of a solution containing only essential AA (EAA), which escape splanchnic metabolism. MIX-AA increased GFR and RPF (from 104 +/- 6 to 122 +/- 13 and from 488 +/- 46 to 572 +/- 34 ml/min/1.73 m2), stimulated splanchnic metabolism as demonstrated by rises in urinary urea excretion (from 20.7 +/- 2 to 30.6 +/- 7.5 mg/min/1.73 m2) and the plasma glucagon/insulin ratio (from 21.4 +/- 13 to 26.7 +/- 15), and caused increases in fractional excretion of AA, FeNa and free-water clearance. During MIX-AA infusion significant correlations were observed between the individual values of GFR and the urea excretion rate (r = 0.66), and between GFR modifications (DeltaGFR) and the plasma glucagon/plasma insulin ratio (r = 0.40). No change in renal function, urea excretion and the glucagon/insulin ratio was observed with EAA. An intermediate splanchnic step (increased plasma glucagon/insulin ratio and ureagenesis) seems necessary in the pathway leading to the nonessential AA-induced rise in GFR; this might stimulate an ultimate intrarenal pathway (possibly involving the diluting segment) via a still undefined mechanism.