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.

Shortcomings in predicting postnatal renal function using prenatal urine biochemistry in fetuses with congenital hydronephrosis.

Ten fetuses with hydronephrosis underwent one to seven urine sampling procedures at 23 to 36 weeks' gestation to evaluate renal function. Postnatally, the infants' renal function was assessed by a combination of serum creatinine measurement, ultrasonography and renal scintigraphy. Six infants had pyelo-ureteric junction obstruction, two had megabladder with megaureter, and two had vesico-ureteric reflux. All infants had normal serum creatinine levels at the time of postnatal follow-up, but five of the seven with unilateral involvement had moderate or severe renal damage. Abnormal urinary electrolyte concentrations were found antenatally in only two of them. For the three infants with bilateral hydronephrosis, postnatal evaluation showed moderately or severely damaged kidneys despite prenatal evidence of normal biochemical indexes. Fetal urine electrolyte measurement may be accurate in the diagnosis of renal dysplasia, but its sensitivity is poor in predicting moderate renal dysfunction.

Subtotal but not unilateral nephrectomy induces hyperplasia and protooncogene expression.

It is generally accepted that renal compensatory growth after unilateral nephrectomy (Uni) is due to prominent hypertrophy with no involvement of protooncogenes. Neither the balance between hypertrophy and hyperplasia nor the expression of the early-growth-related genes has been studied after subtotal nephrectomy (Nx). The occurrence of cystic tubular dilatations after Nx may suggest an excessive cell proliferation in this model. We measured DNA, RNA, and protein content, number of nuclei per tubular section, as well as c-fos, c-jun, c-myc, c-H-ras, c-sis, and c-erb-B2 protooncogene expression in kidneys taken at time of surgery and 2, 7, and 14 days after sham operation (control rats), Uni, or Nx. After Uni, hyperplasia was greater than expected (+79% for DNA at day 14) and was associated with moderate hypertrophy (+11% for protein/DNA ratio). After Nx, compensatory growth was due only to hyperplasia (+117% for DNA at day 14), with unchanged protein/DNA ratio (vs. Uni, P < 0.02). The greater hyperplasia after Nx was confirmed by nuclei counting. The protooncogene mRNA expression was constantly absent in control and Uni rats, whereas that of c-fos and c-jun genes was detected in Nx rats at day 14 with a 2- to 12-fold increment. The c-fos and c-jun protein levels were also increased at that time in Nx rats. This suggests the following: 1) the cellular events following Uni and Nx are not the same, and 2) the late protooncogene expression in Nx exclusively could favor a particular type of cell proliferation possibly more related with cystic formation than with actual compensatory growth.

Acute pyelonephritis as a cause of hyponatremia/hyperkalemia in young infants with urinary tract malformations.

Obstructive uropathy causes tubular resistance to aldosterone and severe metabolic imbalance may be precipitated by an episode of pyelonephritis. In the last 3 years we investigated 52 episodes of pyelonephritis (positive urine culture, elevated C reactive protein, fever, elevated neutrophil count) in 50 children between 15 days and 15 months of age. Ultrasonography voiding cystography and renal scintiscan were performed in all cases and i.v. urography in some. A salt-losing syndrome with hyponatremia and hyperkalemia (Na < 125 meq/liter; K > 6.3 meq/liter) was observed in 17 infants < 3 months, accompanied by plasma aldosterone concentration of 5000 to 23,000 pg/ml (normal value, < 1000 pg/ml). All these children had a severe urinary tract (UT) malformation (ureteropelvic junction stenosis in 7 cases, vesicoureteral reflux in 7, posterior urethral valves in 2, double system in 1). Thirteen infants < 3 months, 7 with no urinary tract malformations, did not have electrolyte imbalance. Pyelonephritis was diagnosed in 20 other patients ages 4 to 15 months, including 16 with severe UT malformations; 4 had normal UTs. We conclude that a salt-losing syndrome with tubular resistance to aldosterone can occur during pyelonephritis in young infants with congenital UT malformation, that the risk diminishes considerably or disappears after 3 months of age and that in the absence of UT malformation pyelonephritis does not cause acute sodium loss of clinical relevance.

Congenital renal damage associated with primary vesicoureteral reflux detected prenatally in male infants.

To assess the course of vesicoureteral reflux, we performed cystography, renal scintigraphy, and urography in all neonates with the prenatal diagnosis of renal pelvic dilation and revealed the presence of primary reflux (grades I to V) in 27 cases. Higher grades of reflux were associated with congenital renal damage, as shown by reduced tracer uptake during scintigraphy. Reflux was diagnosed more frequently in male infants (male/female ratio, 6:1), in many of whom bladder abnormalities were found by cystography. In another group of seven infants, in whom the reflux was associated with other urologic abnormalities, there was no sex prevalence. We conclude that severe primary reflux associated with hydronephrosis usually affects male infants and may be due to abnormal embryologic development of the male urethra, and that the kidney damage is primary and not the result of urinary tract infections. This pattern differs from that of vesicoureteric reflux diagnosed at an older age, which is observed most commonly in female patients.

Mild fetal hydronephrosis indicating vesicoureteric reflux.

The management of neonates with mild hydronephrosis diagnosed antenatally is still debated. Although some of these infants are normal, it is recognised that others will have mild obstruction of the ureteropelvic junction or vesicoureteric reflux (VUR). A prospective study was performed in all newborn infants with an antenatal diagnosis of mild hydronephrosis (47 babies, 62 kidneys) born over a two year period in order to assess the frequency of VUR. Voiding cystography in 14 patients with 21 renal units showed VUR. Two patients underwent surgery and the VUR resolved; the other 12 received medical treatment. Repeat cystography was scheduled for 12-18 months later, when a high rate of spontaneous cure was observed. The remaining patients were monitored by ultrasonography but only in one case did hydronephrosis deteriorate because of the presence of severe ureteropelvic junction obstruction. It is concluded that mild dilatation of the pelvis might be an expression of a potentially severe malformation such as VUR, and a careful follow up of these cases is mandatory.

Increased renal tubular Na-K-ATPase activity in Milan hypertensive rats in the prehypertensive period.

Milan hypertensive (MSH) rats develop hypertension around the 3rd-4th week of life and exhibit increased Na-pump activity in adulthood. The present study was performed to evaluate whether or not hypertension is preceded by an increase in Na-K-ATPase activity. Total and ouabain-sensitive ATPase activities were studied in single microdissected medullary thick ascending limb of Henle (mTAL) tubules from MHS, Milan normotensive (MNS) and Sprague-Dawley (SD) rats at 22-24, 26-28 and 45-60 days of age. Data are given as mean +/- SEM. Total and Na-K-ATPase activity exhibited a developmental pattern in MHS, MNS and SD rats. At 22-24 days no difference was seen between MHS and MNS animals. At 26-28 days MHS had a higher total and Na-K-ATPase activity than MNS (3031 + 171 vs 2471 + 178 pmol phosphate/mm tubule per hour, P less than 0.05; 2289 + 205 vs 1653 + 151, n = 10, P less than 0.05). At this age there was still no difference in mean arterial blood pressure (88 + 4 vs 86 + 3 mm Hg, n = 15). Adult MHS rats had higher blood pressure (140 + 9 vs 112 + 8 mm Hg, P less than 0.001) and higher total (3544 + 136 vs 2718 + 215 pmol phosphate/mm tubule per hour, n = 10, P less than 0.01) and Na-K-ATPase activity (2670 + 99 vs 1942 + 217 pmol phosphate/mm tubule per hour, n = 10, P less than 0.05) than adult MNS rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin 6 activity in infants and children with bacterial meningitis. The Collaborative Study on Meningitis.

Concentrations of interleukin 6 (IL-6) in cerebrospinal fluid (CSF) and serum of infants and children with bacterial meningitis were determined and correlations were sought with other indices of inflammation and with outcome. Forty-two patients ages 1 month to 15 years (mean, 2.5 years) were studied. IL-6 activity was detectable (greater than 50 units/ml) in 30 of 36 CSF samples collected at admission from patients with meningitis and in 1 of 23 controls with fever and normal CSF findings. Mean values were 36,000 units/ml (range, 151-156,000). IL-6 activity in CSF persisted during the first 5 days of illness. IL-6 concentrations at admission were not associated with clinical findings, CSF leukocyte, protein and glucose concentrations, serum C-reactive protein concentration and neurologic complications or sequelae. IL-6 was also detected in the serum of 3 of 14 patients with meningitis and in 0 of 7 controls with no infectious disease. The presence of IL-6 was not associated with bacteremia or with duration of fever before admission. The presence of IL-6 in the CSF of pediatric patients with bacterial meningitis is in accordance with available data on other cytokines and suggests their role as mediators of meningeal inflammation.

Increased sodium requirement following early postnatal surgical correction of congenital uropathies in infants.

Serum electrolyte equilibrium and plasma aldosterone concentrations were monitored in 19 infants who had severe obstructive uropathy or grade 5 vesico-ureteral reflux and were undergoing surgical correction in the first 2 months of life. Before surgery high plasma aldosterone levels were observed in 8 patients, but serum sodium and potassium concentrations were normal. Plasma concentrations of aldosterone were elevated in all patients during the week following surgery and 7 patients developed severe hyponatraemia, hyperkalaemia and weight loss despite very high plasma aldosterone concentrations. As a consequence 5 infants were infused with sodium chloride (4 mEq/kg per day) before and for 36 h after surgery; this prevented metabolic imbalance. We conclude that infants undergoing surgical correction of uropathies may require a high sodium intake to maintain electrolyte balance and adequate growth.