8-Isoprostane in nasally exhaled breath condensate in different pediatric lung diseases

OBJECTIVE: Increased levels of 8-isoprostane were found in various human lung diseases suggesting 8-isoprostane as a marker of pulmonary oxidative stress in vivo. The exact role in pediatric lung diseases has not been defined yet. The goal of this study was to clarify the role of 8-isoprostane in nasally exhaled breath condensate as possible marker of oxidative stress in children with different lung diseases. METHODS: Levels of 8-isoprostane were measured in nasally exhaled breath condensate of 29 cystic fibrosis patients, 19 children with a history of wheezing episodes, 8 infants with acute respiratory tract infection and 53 healthy subjects using a specific enzyme immunoassay. RESULTS: Levels of 8-isoprostane did neither discriminate between different disease groups nor correlate with lung function in cystic fibrosis patients. CONCLUSIONS: Levels of 8-isoprostane in nasally exhaled breath condensate do not reflect oxidative stress in children with different lung diseases.

Assessment of bronchodilator responsiveness in preschool children using forced oscillations

BACKGROUND: The forced oscillation technique (FOT) requires minimal patient cooperation and is feasible in preschool children. Few data exist on respiratory function changes measured using FOT following inhaled bronchodilators (BD) in healthy young children, limiting the clinical applications of BD testing in this age group. A study was undertaken to determine the most appropriate method of quantifying BD responses using FOT in healthy young children and those with common respiratory conditions including cystic fibrosis, neonatal chronic lung disease and asthma and/or current wheeze. METHODS: A pseudorandom FOT signal (4-48 Hz) was used to examine respiratory resistance and reactance at 6, 8 and 10 Hz; 3-5 acceptable measurements were made before and 15 min after the administration of salbutamol. The post-BD response was expressed in absolute and relative (percentage of baseline) terms. RESULTS: Significant BD responses were seen in all groups. Absolute changes in BD responses were related to baseline lung function within each group. Relative changes in BD responses were less dependent on baseline lung function and were independent of height in healthy children. Those with neonatal chronic lung disease showed a strong baseline dependence in their responses. The BD response in children with cystic fibrosis, asthma or wheeze (based on both group mean data and number of responders) was not greater than in healthy children. CONCLUSIONS: The BD response assessed by the FOT in preschool children should be expressed as a relative change to account for the effect of baseline lung function. The limits for a positive BD response of -40% and 65% for respiratory resistance and reactance, respectively, are recommended.

Large deletions in the CFTR gene: clinics and genetics in Swiss patients with CF

Cystic fibrosis (CF) is the most common life-shortening autosomal recessive disorder in Caucasians, and is associated with at least one mutation on each CF transmembrane conductance regulator (CFTR) allele. Some patients, however, with only one identifiable point mutation carry on the other allele, a large deletion that is not detected by conventional screening methods. The overall frequency of large deletions in patients with CF is estimated to be 1-3%. Using the CFTR Multiplex Ligation dependent Probe Amplification Kit (MRC-Holland, Amsterdam, Netherlands) that allows the exact detection of copy numbers from all 27 exons in the CFTR gene, we screened 50 patients with only one identified mutation for large deletions in the CFTR gene. Each detected deletion was confirmed using our real-time polymerase chain reaction (PCR) assay and deletion-specific PCR reactions using junction fragment primers. We detected large deletions in eight patients (16%). These eight CF alleles belong to four different deletion types (CFTRindel2, CFTRdele14b-17b, CFTRdele17a-17b and CFTRdele 2-9) whereof the last is novel. Comparing detailed clinical data of all these patients with CF and the molecular genetic findings, we were able to elaborate criteria for deletion screenings and possible genotype-phenotype associations. In conclusion, we agree with other authors that deletion screenings should be implemented in routine genetic diagnostics of CF.

Sweat testing practice in Swiss hospitals

AIMS: To determine whether the current practice of sweat testing in Swiss hospitals is consistent with the current international guidelines. METHODS: A questionnaire was mailed to all children's hospitals (n = 8), regional paediatric sections of general hospitals (n = 28), and all adult pulmonology centres (n = 8) in Switzerland which care for patients with cystic fibrosis (CF). The results were compared with published "guidelines 2000" of the American National Committee for Clinical Laboratory Standards (NCCLS) and the UK guidelines of 2003. RESULTS: The response rate was 89%. All 8 children's hospitals and 18 out of 23 answering paediatric sections performed sweat tests but none of the adult pulmonology centres. In total, 1560 sweat tests (range: 5-200 tests/centre/year, median 40) per year were done. 88% (23/26) were using Wescor systems, 73% (19/26) the Macroduct system for collecting sweat and 31% (8/26) the Nanoduct system. Sweat chloride was determined by only 62% (16/26) of all centres; of these, only 63% (10/16) indicated to use the recommended diagnostic chloride-CF-reference value of >60 mmol/l. Osmolality was measured in 35%, sodium in 42% and conductivity in 62% of the hospitals. Sweat was collected for maximal 30-120 (median 55) minutes; only three centres used the maximal 30 minutes sample time recommended by the international guidelines. CONCLUSIONS: Sweat testing practice in Swiss hospitals was inconsistent and seldom followed the current international guidelines for sweat collection, analyzing method and reference values. Only 62% were used the chloride concentration as a diagnostic reference, the only accepted diagnostic measurement by the NCCLS or UK guidelines.

The cytochrome P450 aromatase lacking exon 5 is associated with a phenotype of nonclassic aromatase deficiency and is also present in normal human steroidogenic tissues

OBJECTIVE: The previously described c655G>A mutation of the human cytochrome P450 aromatase gene (P450aro, CYP19) results in aberrant splicing due to disruption of a donor splice site. To explain the phenotype of partial aromatase deficiency observed in a female patient described with this mutation, molecular consequences of the c655G>A mutation were investigated. DESIGN: To investigate whether the c655G>A mutation causes an aberrant spliced mRNA lacking exon 5 (-Ex5), P450aro RNA was analysed from the patient's lymphocytes by reverse transcription polymerase chain reaction (RT-PCR) and by splicing assays performed in Y1 cells transfected with a P450aro -Ex5 expression vector. Aromatase activity of the c655G>A mutant was predicted by three dimensional (3D) protein modelling studies and analysed in transiently transfected Y1 cells. Exon 5 might be predicted as a poorly defined exon suggesting a susceptibility to both splicing mutations and physiological alternative splicing events. Therefore, expression of the -Ex5 mRNA was also assessed as a possibly naturally occurring alternative splicing transcript in normal human steroidogenic tissues. PATIENTS: An aromatase deficient girl was born with ambiguous genitalia. Elevated serum LH, FSH and androgens, as well as cystic ovaries, were found during prepuberty. At the age of 8.4 years, spontaneous breast development and a 194.6 pmol/l serum oestradiol level was observed. RESULTS: The -Ex5 mRNA was found in lymphocytes of the P450aro deficient girl and her father, who was a carrier of the mutation. Mutant minigene expression resulted in complete exon 5 skipping. As expected from 3D protein modelling, -Ex5 cDNA expression in Y1 cells resulted in loss of P450aro activity. In addition, the -Ex5 mRNA was present in placenta, prepubertal testis and adrenal tissues. CONCLUSIONS: Alternative splicing of exon 5 of the CYP19 gene occurs in the wild type (WT) as well as in the c655G>A mutant. We speculate that for the WT it might function as a regulatory mechanism for aromatization, whereas for the mutant a relative prevalence of the shorter over the full-length protein might explain the phenotype of partial aromatase deficiency.

Evidence of oligogenic inheritance in nephronophthisis

Nephronophthisis is a recessive cystic renal disease that leads to end-stage renal failure in the first two decades of life. Twenty-five percent of nephronophthisis cases are caused by large homozygous deletions of NPHP1, but six genes responsible for nephronophthisis have been identified. Because oligogenic inheritance has been described for the related Bardet-Biedl syndrome, we evaluated whether mutations in more than one gene may also be detected in cases of nephronophthisis. Because the nephrocystins 1 to 4 are known to interact, we examined patients with nephronophthisis from 94 different families and sequenced all exons of the NPHP1, NPHP2, NPHP3, and NPHP4 genes. In our previous studies involving 44 families, we detected two mutations in one of the NPHP1-4 genes. Here, we detected in six families two mutations in either NPHP1, NPHP3, or NPHP4, and identified a third mutation in one of the other NPHP genes. Furthermore, we found possible digenic disease by detecting one individual who carried one mutation in NPHP2 and a second mutation in NPHP3. Finally, we detected the presence of a single mutation in nine families, suggesting that the second recessive mutation may be in another as yet unidentified NPHP gene. Our findings suggest that oligogenicity may occur in cases of nephronophthisis.

Mutation analysis of NPHP6/CEP290 in patients with Joubert syndrome and Senior-Løken syndrome

BACKGROUND: Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease that constitutes the most common genetic cause of renal failure in the first three decades of life. Using positional cloning, six genes (NPHP1-6) have been identified as mutated in NPHP. In Joubert syndrome (JBTS), NPHP may be associated with cerebellar vermis aplasia/hypoplasia, retinal degeneration and mental retardation. In Senior-Løken syndrome (SLSN), NPHP is associated with retinal degeneration. Recently, mutations in NPHP6/CEP290 were identified as a new cause of JBTS. METHODS: Mutational analysis was performed on a worldwide cohort of 75 families with SLSN, 99 families with JBTS and 21 families with isolated nephronophthisis. RESULTS: Six novel and six known truncating mutations, one known missense mutation and one novel 3 bp pair in-frame deletion were identified in a total of seven families with JBTS, two families with SLSN and one family with isolated NPHP.

Ciprofloxacin-induced hematuria

We used ciprofloxacin, a quinolone-derivative, to treat a lung infection due to Pseudomonas aeruginosa in an adult cystic fibrosis patient. On three different occasions the use of ciprofloxacin was associated with the development of an asymptomatic hematuria with red blood cell casts. The mechanism responsible for this hematuria is presently unknown, but clinicians should be aware of this potential adverse effect of ciprofloxacin.

[The mandatory diaper peek in newborn girls]

We present a 3 day old girl born at term presenting with an interlabial, cystic mass. Pregnancy, delivery and routine antenatal screening were unremarkable. The smooth lesion was located in the anterior half of the vulva, covered with thin vessels. The medially displaced urethra and the medio-posteriorly displaced hymen were identified. Voiding was not impaired. We discuss the differential diagnosis of vulvar masses in the newborn girl. A thorough clinical examination must be the standard of care of pediatric examinations in infants.

Ligation dependent allele specific quantification (LASQ) of CFTR cDNA on the LightCycler using MLPA hybridization probes

BACKGROUND: As for Cystic Fibrosis (CF) and many other hereditary diseases there is still a lack in understanding the relationship between genetic (e.g. allelic) and phenotypic diversity. Therefore methods which allow fine quantification of allelic proportions of mRNA transcripts are of high importance. METHODS: We used either genomic DNA (gDNA) or total RNA extracted from nasal cells as starting nucleic acid template for our assay. The subjects included in this study were 9 CF patients compound heterozygous for the F508del mutation and each one F508del homozygous and one wild type homozygous respectively. We established a novel ligation based quantification method which allows fine quantification of the allelic proportions of ss and ds CFTR cDNA. To verify reliability and accuracy of this novel assay we compared it with semiquantitative fluorescent PCR (SQF-PCR). RESULTS: We established a novel assay for allele specific quantification of gene expression which combines the benefits of the specificity of the ligation reaction and the accuracy of quantitative real-time PCR. The comparison with SQF-PCR clearly demonstrates that LASQ allows fine quantification of allelic proportions. CONCLUSION: This assay represents an alternative to other fine quantitative methods such as ARMS PCR and Pyrosequencing.

Diversity of the basic defect of homozygous CFTR mutation genotypes in humans

BACKGROUND: Knowledge of how CFTR mutations other than F508del translate into the basic defect in cystic fibrosis (CF) is scarce due to the low incidence of homozygous index cases. METHODS: 17 individuals who are homozygous for deletions, missense, stop or splice site mutations in the CFTR gene were investigated for clinical symptoms of CF and assessed in CFTR function by sweat test, nasal potential difference and intestinal current measurement. RESULTS: CFTR activity in sweat gland, upper airways and distal intestine was normal for homozygous carriers of G314E or L997F and in the range of F508del homozygotes for homozygous carriers of E92K, W1098L, R553X, R1162X, CFTRdele2(ins186) or CFTRdele2,3(21 kb). Homozygotes for M1101K, 1898+3 A-G or 3849+10 kb C-T were not consistent CF or non-CF in the three bioassays. 14 individuals exhibited some chloride conductance in the airways and/or in the intestine which was identified by the differential response to cAMP and DIDS as being caused by CFTR or at least two other chloride conductances. DISCUSSION: CFTR mutations may lead to unusual electrophysiological or clinical manifestations. In vivo and ex vivo functional assessment of CFTR function and in-depth clinical examination of the index cases are indicated to classify yet uncharacterised CFTR mutations as either disease-causing lesions, risk factors, modifiers or neutral variants.

Surfactant proteins SP-B and SP-C and their precursors in bronchoalveolar lavages from children with acute and chronic inflammatory airway disease

BACKGROUND: The surfactant proteins B (SP-B) and C (SP-C) are important for the stability and function of the alveolar surfactant film. Their involvement and down-regulation in inflammatory processes has recently been proposed, but their level during neutrophilic human airway diseases are not yet known. METHODS: We used 1D-electrophoresis and Western blotting to determine the concentrations and molecular forms of SP-B and SP-C in bronchoalveolar lavage (BAL) fluid of children with different inflammatory airway diseases. 21 children with cystic fibrosis, 15 with chronic bronchitis and 14 with pneumonia were included and compared to 14 healthy control children. RESULTS: SP-B was detected in BAL of all 64 patients, whereas SP-C was found in BAL of all but 3 children; those three BAL fluids had more than 80% neutrophils, and in two patients, who were re-lavaged later, SP-C was then present and the neutrophil count was lower. SP-B was mainly present as a dimer, SP-C as a monomer. For both qualitative and quantitative measures of SP-C and SP-B, no significant differences were observed between the four evaluated patient groups. CONCLUSION: Concentration or molecular form of SP-B and SP-C is not altered in BAL of children with different acute and chronic inflammatory lung diseases. We conclude that there is no down-regulation of SP-B and SP-C at the protein level in inflammatory processes of neutrophilic airway disease.

Cerebellar cleft: a form of prenatal cerebellar disruption

In contrast to malformations, cerebellar disruptions have attracted little interest in the literature. We draw attention for the first time to the hypothesis that cerebellar clefts are residual changes following a prenatal cerebellar insult, and represent disruptions. We reviewed the clinical records and MR findings of six patients with a cerebellar cleft, two of whom also had prenatal MRI at 24 weeks of gestation. The clefts were located in the left cerebellar hemisphere in five cases, in the right in one patient. Other typical findings included disorderly alignment of the cerebellar folia and fissures, irregular gray/white matter junction, and abnormal arborization of the white matter in all patients. The cerebellar cleft extended into the fourth ventricle in three cases, and in two children cystic cortical lesions were seen. Supratentorial schizencephaly was found in two patients. In two patients there was a documented fetal cerebellar hemorrhage at 24 weeks of gestation. We conclude that cerebellar clefts are residual changes resulting from a prenatal cerebellar insult and consequently represent disruptions rather than primary malformations. The supratentorial findings are also in agreement with an acquired lesion. The outcome in these children was variable, mainly depending of the presence of supratentorial lesions.

Activation of the epidermal growth factor receptor (EGFR) by a novel metalloprotease pathway

Neutrophil Elastase (NE) is a pro-inflammatory protease present at higher than normal levels in the lung during inflammatory disease. NE regulates IL-8 production from airway epithelial cells and can activate both EGFR and TLR4. TACE/ADAM17 has been reported to trans-activate EGFR in response to NE. Here, using 16HBE14o-human bronchial epithelial cells we demonstrate a new mechanism by which NE regulates both of these events. A high molecular weight soluble metalloprotease activity detectable only in supernatants from NE-treated cells by gelatin and casein zymography was confirmed to be meprin alpha by Western immunoblotting. In vitro studies demonstrated the ability of NE to activate meprin alpha, which in turn could release soluble TGFalpha and induce IL-8 production from 16HBE14o- cells. These effects were abrogated by actinonin, a specific meprin inhibitor. NE-induced IL-8 expression was also inhibited by meprin alpha siRNA. Immunoprecipitation studies detected EGFR/TLR4 complexes in NE-stimulated cells overexpressing these receptors. Confocal studies confirmed colocalization of EGFR and TLR4 in 16HBE14o- cells stimulated with meprin alpha. NFkappaB was also activated via MyD88 in these cells by meprin alpha. In bronchoalveolar lavage fluid from NE knock-out mice infected intra-tracheally with Pseudomonas aeruginosa meprin alpha was significantly decreased compared with control mice, and was significantly increased and correlated with NE activity, in bronchoalveolar lavage fluid from individuals with cystic fibrosis but not healthy controls. The data describe a previously unidentified lung metalloprotease meprin alpha, and its role in NE-induced EGFR and TLR4 activation and IL-8 production.

Astroblastoma in a child

BACKGROUND: Astroblastoma, an uncommon neuroepithelial tumor, typically presents in young adults as a well-circumscribed cortical or subcortical spherical mass. Astroblastoma may cause a diagnostic problem to anyone unfamiliar with its architectural and histological features. CASE HISTORY: We report the case of a 4-year-old boy who was referred for complaints of progressive deficits of balance and difficulty with walking during the previous 3 months. A large fronto-parietal cystic mass with solid mural nodule was discovered. Total removal of the tumor mass was performed, and a diagnosis of high grade (malignant) variant of astroblastoma was made. Postoperatively, the patient received radiation therapy, for a period of 11 weeks, followed by chemotherapy. He is in a good neurological recovery without any evidence of recurrence for 8 months. PROGNOSIS: The best treatment modality for astroblastoma is surgical resection if possible, whereas adjuvant therapy (radiotherapy and/or chemotherapy) can be considered in high-grade astroblastomas, with a close follow-up for all cases.