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.

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.

Betaxolol kinetics in hypertensive children with normal and abnormal renal function.

The kinetic and clinical profile of betaxolol--a beta 1-selective blocker with 80% to 90% bioavailability and a 16 to 20 hr t1/2--were studied in ten children aged 5 to 13 yr with chronic renal hypertension and mild to severe renal failure. An IV dose of 20 mg of betaxolol per 1.73 m2 body surface area (BSA) was followed by six daily oral doses. Six patients were maintained on combination therapy and four on betaxolol alone; two of these were newly treated. After the intravenous dose, t1/2 (mean +/- SE) was 19.9 +/- 1.7 hr, total clearance 0.30 +/- 0.03 l/kg/hr, and volume of distribution 8.2 +/- 0.7 l/kg. Clearance adjusted for BSA was 7.9 +/- 0.6 l/hr. The t1/2 correlated linearly to serum creatinine levels. After the last dose, peak concentration was 97.4 +/- 7.6 ng/ml, and t1/2 19.4 +/- 2.7 hr. Betaxolol 24-hr blood levels were twice as high as after the first dose. Blood pressure was reduced in two newly treated patients and two patients on combination therapy; previous responses were maintained in the others. The maximum effect was reached after the first dose and was maintained throughout the study week. Our results indicate that betaxolol disposition in children aged 5 to 13 yr with different degrees of renal failure is of the same order as that in young healthy adults, implying that there may be a higher rate of non-renal clearance. Renal failure-induced modification led to a doubling of the t1/2 in the most severe cases, again as in adult renal patients. There is an antihypertensive effect.

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.

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.

Counterimmunoelectrophoresis and latex particle agglutination in the etiologic diagnosis of presumed bacterial pneumonia in pediatric patients.

A commercial latex agglutination (LA) kit (Wellcogen, Wellcome Diagnostics) used to detect bacterial polysaccharide antigens (Haemophilus influenzae type b and Streptococcus pneumoniae) was compared with a modified counterimmunoelectrophoresis technique and blood culture for etiologic diagnosis of presumptive bacterial pneumonia requiring hospitalization in 60 infants and children. Serum, urine and either sputum or nasopharyngeal secretions were collected during the first 5 days of therapy for antigen detection. Blood culture was positive in 6 of 52 (11.5%) of cases. Antigens were detected by counterimmunoelectrophoresis and/or LA in 13 of 60 (21.7%) serum samples, 2 of 16 (12.5%) unconcentrated urine samples, 19 of 42 (45.2%) urine samples concentrated 25-fold and 21 of 45 (46.7%) sputum or nasopharyngeal secretions. Antibiotic treatment for 5 days did not affect the antigen detection rate. Counter-immunoelectrophoresis was more sensitive than LA in serum and urine but not in sputum. However, because false positive reactions were frequently obtained with LA on nasopharyngeal secretions of an age-matched control group, this test appears unreliable.

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.

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.

N-acetyl-beta-D-glucosaminidase (NAG) and NAG isoenzymes in children with upper and lower urinary tract infections.

The use of N-acetyl-beta-D-glucosaminidase (NAG) to diagnose the site of urinary tract infection was studied in pediatric patients. Differentiation between upper and lower tract infections (UTI) was based on clinical grounds and on elevated erythrocyte sedimentation rate, C-reactive protein and fever. NAG excretion expressed as nmol X h-1 X mg-1 of urinary creatinine was higher in children with upper UTI (mean +/- SE 906 +/- 236) than in those with lower UTI (145 +/- 23) or healthy children (151.6 +/- 10) (p less than 0.01 by Duncan's test). In children with upper UTI, NAG excretion fell in parallel with the remission due to antibiotic treatment. This however was not seen in children treated with aminoglycosides. A specific and significant elevation (p less than 0.01) of the B isoenzyme of NAG was documented in children with upper UTI but not in those with lower UTI (B form in upper UTI 49.2% +/- 3.9 versus 21.9 +/- 3.3 in lower UTI; healthy children 18.9 +/- 3.4). The percentage of B isoenzyme excreted was high in two children with upper UTI but was low total NAG urinary excretion, suggesting that the quantification of isoenzymes offers further specificity in diagnosis. We conclude that the measurement of NAG and its isoenzymes in children with UTI provides useful information in the diagnosis of the site of infection.

Perinatal development of cytochrome P-450, NADPH-cytochrome c reductase and ethoxycoumarin deethylase in rat liver nuclear membranes.

Perinatal development of rat liver nuclear membrane enzymatic activities was investigated with respect to the metabolism of xenobiotica. The qualitative pattern observed was very close to that reported for microsomal enzymes during development. Cytochrome P-450, NADPH-cytochrome c reductase and ethoxycoumarin deethylase are already present in fetuses at 18 days of gestational age. Phenobarbital pretreatment appears to be effective as an inducing agent for all the enzymes studied, but only after birth. The pattern of induction of cytochrome P-450 showed a peak at the 38th day of life three times higher than basal values at that age. NADPH-cytochrome c reductase presented a constant elevation to about twice basal activity throughout the period taken into consideration. Ethoxycoumarin deethylase activity took only 17 days to reach the basal value observed later in adult animals. This enzyme proved highly inducible by phenobarbital (5-fold) early after birth but the increase dropped to 3-fold from the 24th day of life.

Perinatal development of styrene monooxygenase and epoxide hydrolase in rat liver microsomes and nuclei.

Nuclear enzymes were found to develop earlier than the corresponding microsomal activities. In fact styrene monooxygenase enzymatic activity at 18 days gestational age reached about half the values of adult animals, whereas fetal microsomal activity was only about 1/20 the adult level at the same age. In microsomes and nuclei the ontogenic development of epoxide hydrolase is slightly slower than styrene monooxygenase. This suggests that fetuses and newborn animals are exposed to higher risk of accumulation of styrene-7,8-oxide, a toxic and possibly teratogenic product of styrene monooxygenase.

Plasma glutamic acid levels in premature newborn.

24 premature, newborn infants were investigated for plasma glutamic acid (GA) levels before and after a normal milk feed, to ascertain if the ingestion of GA present in the milk could result in an increase of its plasma level. No increases were detected in plasma between 5 and 90 min after the feed. These results may be important in respect to the problem of the possible toxicity of monosodium glutamate (MSG) added to baby foods.

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.

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.

Prediction of the progression of renal failure in adult and in pediatric patients with malignant focal glomerulosclerosis.

A retrospective analysis of the progression of renal failure was performed in 6 children and 5 adults with idiopathic nephrotic syndrome unresponsive to steroids and immunosuppressants and with histological findings of focal glomerulosclerosis. We analyzed the linear regression of Ccr and of their logarithmic transformation vs time (months) starting from the time when the first abnormal value was observed (t = 0). The regression analysis was performed at three different times, when Ccr was: 30-20, 20-25 and 10-15 ml/min/1.73 sqm. Extrapolation of each of these lines on the x axis predicted when renal function would be zero. The difference in months, between the predicted and actual time of starting dialysis was prediction error. "r" values were always elevated and statistically significant for both linear and logarithmic regression; there was a large intersubject variability in the rate of loss of renal function but the mean prediction error at various levels of Ccr was within limits clinically acceptable. Moreover its magnitude did not significantly decrease by prolonging the time of observation. This indicates that in this disease the decay of Ccr enters a track which proceeds linearly or logarithmically after the onset of renal failure and no major deviation from the predicted line is to be expected. The good predictability in our study may be attributed to the fact that our patients were homogeneous for a number of factors which may be relevant to progression of the disease. It confirms the view supported by Gretz et al. [1983] that there is a need for stratification of patients in large cohort studies on the predictability of loss of renal function.

N-acetyl-beta-D-glucosaminidase (NAG) levels in amniotic fluid or urine in prenatal and postnatal life.

The isoenzyme pattern of N-acetyl-beta-D-glucosaminidase (NAG) was determined in the amniotic fluid of 75 women undergoing amniocentesis for genetic indications. In the amniotic fluid and in the newborn's urine the NAG was completely different from that in adult urine, indicating the presence of a fetal isoenzymatic form also present in the first stage of life.