Franco Zacchello

ExGen 500 is an efficient vector for gene delivery to lung epithelial cells in vitro and in vivo

Nonviral vectors might represent a safe alternative to adenovirus for gene therapy of lung disorders, in particular cystic fibrosis (CF). Cationic lipids have been shown to correct the CF defect both in vitro and in vivo, but more efficient vectors are needed to improve the low gene transfer efficiency. Here, we show that the cationic polymer ExGen 500, a linear polyethylenimine derivative, is more efficient than cationic lipids in transferring reporter genes to lung epithelial cells in vitro. In vivo ExGen 500 was able to mediate gene transfer into both newborn and adult rabbit lungs with comparable efficiencies. The best levels of transfection were obtained using neutral complexes. Under such conditions, luciferase activities corresponding to about 103 RLU/10 s/mg of protein were reproducibly obtained 2 days after transfection throughout the four lung lobes of newborn and adult rabbits. A nlslacZ reporter gene showed transfected cells around the lumen of large and small bronchi. No signs of acute toxicity (inflammation, cellular infiltration etc) were detected by direct histopathological analysis. Within 1 week after instillation, transgene expression decreased by two orders of magnitude.

Cysteinyl leukotrienes and 8-isoprostane in exhaled breath condensate of children with asthma exacerbations

Background: Cysteinyl leukotrienes (Cys-LTs) and isoprostanes are inflammatory metabolites derived from arachidonic acid whose levels are increased in the airways of asthmatic patients. Isoprostanes are relatively stable and specific for lipid peroxidation, which makes them potentially reliable biomarkers for oxidative stress. A study was undertaken to evaluate the effect of a course of oral steroids on Cys-LT and 8-isoprostane levels in exhaled breath condensate of children with an asthma exacerbation. Methods: Exhaled breath condensate was collected and fractional exhaled nitric oxide (FENO) and spirometric parameters were measured before and after a 5 day course of oral prednisone (1 mg/kg/day) in 15 asthmatic children with an asthma exacerbation. Cys-LT and 8-isoprostane concentrations were measured using an enzyme immunoassay. FENO was measured using a chemiluminescence analyser. Exhaled breath condensate was also collected from 10 healthy children. Results: Before prednisone treatment both Cys-LT and 8-isoprostane concentrations were higher in asthmatic subjects (Cys-LTs, 12.7 pg/ml (IQR 5.4–15.6); 8-isoprostane, 12.0 pg/ml (9.4–29.5)) than in healthy children (Cys-LTs, 4.3 pg/ml (2.0–5.7), p=0.002; 8-isoprostane, 2.6 pg/ml (2.1–3.0), p<0.001). After prednisone treatment there was a significant decrease in both Cys-LT (5.2 pg/ml (3.9–8.8), p=0.005) and 8-isoprostane (8.4 pg/ml (5.4–11.6), p=0.04) concentrations, but 8-isoprostane levels remained higher than in controls (p<0.001). FENO levels, which fell significantly after prednisone treatment (p<0.001), did not correlate significantly with either Cys-LT or 8-isoprostane concentrations. Conclusion: After a 5 day course of oral prednisone there is a reduction in Cys-LT and 8-isoprostane levels in EBC of children with an asthma exacerbation, although 8-isoprostane levels remain higher than in controls. This finding suggests that corticosteroids may not be fully effective in reducing oxidative stress in children with an exacerbation of asthma.

Cx26 deafness: mutation analysis and clinical variability.

Mutations in the gap junction protein connexin 26 (Cx26) gene (GJB2) seem to account for many cases of congenital sensorineural hearing impairment, the reported prevalence being 34-50% in autosomal recessive cases and 10-37% in sporadic cases. The hearing impairment in these patients has been described as severe or profound. We have studied 53 unrelated subjects with congenital non-syndromic sensorineural hearing impairment in order to evaluate the prevalence and type of Cx26mutations and establish better genotype-phenotype correlation. Mutations in the Cx26 gene were found in 53% of the subjects tested, 35.3% of the autosomal recessive and 60% of the sporadic cases in our series. Three new mutations were identified. The hearing deficit varied from mild to profound even in 35delG homozygotes within the same family. No evidence of progression of the impairment was found. Alterations of the Cx26 gene account for a large proportion of cases of congenital non-syndromic sensorineural deafness, so it seems appropriate to extend the molecular analysis even to subjects with mild or moderate prelingual hearing impairment of unknown cause.

Polyethylenimine shows properties of interest for cystic fibrosis gene therapy

Before being considered for a cystic fibrosis (CF) gene therapy trial, any gene delivery agent must be able to show that it produces low levels of toxicity as well as being able to protect the DNA from nuclease degradation. Here we show that complexes of linear polyethylenimine (L-PEI) and DNA can repeatedly be administered to animals (up to 21 consecutive days) without eliciting an immune response against PEI/DNA particles or inducing toxic side effects due to accumulation of PEI in the lungs. However, the host response to the exogenous protein resulted in some decrease of expression. PEI-mediated transfection was unaffected by treatment of the complexes with DNase (frequently used to reduce the viscosity of lung secretions in CF patients). Taken together, these properties make L-PEI a valuable vector for gene therapy of CF.

Flow limitation in infants with bronchopulmonary dysplasia and respiratory function at school age

Bronchopulmonary dysplasia is associated with abnormalities in lung function during infancy, yet many infants recover with no respiratory problems in the long term. We therefore did a longitudinal study of pulmonary function in 18 children with moderate to severe bronchopulmonary dysplasia. Forced expiratory volume in 1 s (FEV1) and forced mid-expiratory flow (FEF25-75) at school age were lower than normal in 15 of 18 children, and both showed a significant positive correlation with the maximal flow at functional residual capacity (Vmax(FRC)) at 24 months of age (r=0.68 and 0.85, respectively). Our results suggest that assessment of respiratory function during infancy can help to identify children with bronchopulmonary dysplasia at risk of incomplete recovery of respiratory function during childhood.

Reference values of exhaled nitric oxide for healthy children 6-15 years old

Nitric oxide (NO) can be detected in human exhaled air, and its endogenous production is increased in patients with asthma. It may provide a noninvasive means for measuring airway inflammation. The aim of this study was to establish reference values for exhaled NO concentrations in a large number of healthy school‐age children. We measured exhaled NO levels in 159 white healthy children (88 girls, 71 boys, age range 6–15 years) recruited from two public schools of Padua, Italy. Exhaled NO levels in exhaled gas were measured by a tidal breathing method with a chemiluminescence analyzer, and NO steady‐state levels were recorded. Nasal NO levels were measured by direct sampling from the nose during mouth breathing.

Safety and success of exhaled breath condensate collection in asthma

Background: Exhaled breath condensate (EBC) is a rapidly expanding area of research to study airway inflammation through the detection of volatile and non-volatile substances in the airways. Aims: To determine the safety and feasibility of EBC procedure in a group of children with asthma of varying severity. Methods: In a cross sectional study of children aged 4–17 years, 18 healthy and 91 asthmatic children (69 in stable condition and 22 with asthma exacerbation) underwent the EBC procedure. Outcomes assessed included completion of the procedure, decrease in FEV1, change in fractional exhaled nitric oxide (FENO), and adverse effects. No pretreatment with β2 agonists was given. All children were able to successfully complete the EBC procedure. Results: Median fall in FEV1 after the procedure was −1% (IQR −3.5, 1.8) in asthmatics and was comparable to that observed in healthy children. In only one asthmatic child did the drop in FEV1 exceed 12%. No significant changes in FENO were observed after EBC. Conclusion: This study suggests that EBC is a simple and well tolerated method for evaluating biological samples from the lower airway. The procedure was safe in children with asthma exacerbation, and the success rate was 100% in children aged 4 years and above.

Gas exchange during exercise in obese children

Twenty-three obese children, aged 9 to 14 years, ranging in percentage overweight from 26% to 83% (median 51.6%±16.3%), and 37 normal-weight children, matched for sex, age and height, performed a maximal exercise test on a treadmill. Cardiorespiratory performance was assessed by determination of the ventilatory anaerobic threshold (VAT) expressed in ml O2/min per kg and as a percent of maximal oxygen uptake (% VO2max). VAT and VO2max related to body weight were significantly lower (P<0.01) in the obese than in the normal-weight children. VAT % VO2max was similar in the two groups. A significant correlation was found between VAT and VO2max both in the obese (r=0.85) and in the control groups (r=0.79). The habitual level of physical activity was lower in the obese subjects compared to the control subjects (P<0.001). In conclusion our study shows that physical fitness of overweight children is quantitatively lowered and that it can be assessed by VAT. VAT does not require a maximal test and is particularly useful in the ergometric study of subjects with exercise intolerance.

IN-VITRO PRODUCTION OF HIV-SPECIFIC ANTIBODY IN CHILDREN AT RISK OF AIDS

To improve on the early diagnosis of human immunodeficiency virus (HIV) infection, 37 children born to HIV-infected mothers and 22 controls were investigated for in-vitro synthesis of IgG antibody directed against HIV components. For 14 of 16 infected children western blot showed HIV-specific IgG in the supernatants of cultures of their peripheral blood lymphocyte cultures. HIV-specific IgG synthesis was detected in cultures from 4 out of 17 seropositive children aged under 15 months with no clinical or laboratory evidence of infection. No HIV-specific IgG production was observed in cultures from 4 uninfected children or 22 controls. The results show that the demonstration of in-vitro production of HIV-specific IgG may help in the early diagnosis of HIV infection in children.

Progressive neuropathy and recurrent myoglobinuria in a child with long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency

From the Department of Metabolism, Bambino GesO Childrens Hospital, lstituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy, the Department of Pediatrics, University of Padua, Padua, Italy, the Department of Clinical Chemistry, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland, and the Division of Endocrinology and Diabetes, Childrens Hospital of Philadelphia, Philadelphia, Pennsylvania