DNA concentrations in BAL fluid of cystic fibrosis patients with early lung disease: influence of treatment with dornase alpha.

Recombinant DNase (dornase alpha) was shown to improve lung function and reduce pulmonary exacerbations in cystic fibrosis (CF) patients, but its effects on DNA concentrations in the lower airways remain unclear at the present time. As part of the Bronchoalveolar Lavage in the Evaluation of Anti-Inflammatory Treatment (BEAT) Study, a multicenter open study to evaluate the evolution of inflammation in CF patients with early lung disease and its modulation by dornase alpha treatment, we studied DNA concentrations in the bronchoalveolar lavage (BAL) fluid of 48 CF patients with mild lung disease. After the initial BAL, 29 patients received daily treatment with 2.5 mg of dornase alpha; 19 patients served as controls. BAL was repeated after 18 months in all patients. Mean BAL fluid DNA concentrations were not different between groups at baseline (mean +/- SD, 14.1 +/- 6.9 microg/ml for controls, and 17.6 +/- 11.2 microg/ml for the dornase alpha group), but higher than previously reported for infants with CF. A weak but positive correlation (P <0.01) was observed between the percentage of neutrophils in BAL fluid and DNA levels. On reassessment after 18 months, the percentage of neutrophils was not different between the two groups, but DNA had increased in controls, whereas decreased levels were observed in treated patients (P <0.03, t-test). DNA concentrations increased by more than 10 microg/ml in 7 of 19 controls compared to 2 of 29 CF patients treated with rhDNase (P=0.01, Fisher's test). Therefore, treatment with dornase alpha over 18 months reduces DNA load in BAL fluid, which may have a positive effect on the clearance of lower airway secretions.

FRG2, an FSHD candidate gene, is transcriptionally upregulated in differentiating primary myoblast cultures of FSHD patients.

BACKGROUND: Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) is associated with partial deletion of the subtelomeric D4Z4 repeat array on chromosome 4qter. This chromosomal rearrangement may result in regional chromatin relaxation and transcriptional deregulation of genes nearby. METHODS AND RESULTS: Here we describe the isolation and characterisation of FRG2, a member of a chromosomally dispersed gene family, mapping only 37 kb proximal to the D4Z4 repeat array. Homology and motif searches yielded no clues to the function of the predicted protein. FRG2 expression is undetectable in all tissues tested except for differentiating myoblasts of FSHD patients, which display low, yet distinct levels of FRG2 expression, partly from chromosome 4 but predominantly originating from its homologue on chromosome 10. However, in non-FSHD myopathy patients only distantly related FRG2 homologues are transcribed, while differentiating myoblasts from healthy controls fail to express any member of this gene family. Moreover, fibroblasts of FSHD patients and control individuals undergoing forced Ad5-MyoD mediated myogenesis show expression of FRG2 mainly originating from chromosome 10. Luciferase reporter assays show that the FRG2 promoter region can direct high levels of expression but is inhibited by increasing numbers of D4Z4 repeat units. Transient transfection experiments with FRG2 fusion-protein constructs reveal nuclear localisation and apparently FRG2 overexpression causes a wide range of morphological changes. CONCLUSION: The localisation of FRG2 genes close to the D4Z4 repeats on chromosome 4 and 10, their transcriptional upregulation specifically in FSHD myoblast cultures, potential involvement in myogenesis, and promoter properties qualify FRG2 as an attractive candidate for FSHD pathogenesis.

Llama-derived phage display antibodies in the dissection of the human disease oculopharyngeal muscular dystrophy.

Functional analysis of the estimated 30,000 genes of the human genome requires fast and reliable high-throughput methods to study spatio-temporal protein dynamics. To explore the suitability of heavy-chain antibodies (HCAbs) for studying mechanisms underlying human disease, we used oculopharyngeal muscular dystrophy (OPMD) as a paradigm for the expanding group of protein aggregation disorders that is characterized by subcellular dislocalization and aggregation of mutant protein. OPMD is caused by a moderate alanine expansion in the poly-A binding protein nuclear 1 (PABPN1) and is associated with intranuclear PABPN1 deposition exclusively in muscle. An experimental approach was designed in which the primary sequence of the PABPN1 gene was employed for generating a prokaryotic expression construct that permitted its expression in the host Escherichia coli. The purified product was used for immunization of a llama as well as for the selection of an antigen-specific antibody fragment from the derived phage display library. This single-domain antibody was able to recognize the native gene product in mammalian cell lines and in human muscle tissue by immunocytochemical, immunohistochemical and immunoblot analysis. Our results suggest that phage display derived heavy-chain antibodies can be used in proteomics to study the localization and function of hypothetical gene products, relevant to human disease.

Multiple transcripts generated by the DCAMKL gene are expressed in the rat hippocampus.

We have recently cloned a novel Doublecortin CaMK-like kinase (rDCAMKL) cDNA, and a related cDNA called CaMK-related peptide (CARP) from the rat hippocampus. These genes are structurally highly similar to the human DCAMKL-1 gene and doublecortin, a gene associated with X-linked lissencephaly and subcortical band heterotopia. Here we report on the genomic organization of the murine DCAMKL gene and its products. Our results show that DCAMKL and CARP are alternative splice products of the same gene. The DCAMKL gene also generates three alternatively-spliced rDCAMKL transcripts of which we have cloned the corresponding cDNAs and which potentially generate different DCAMKL proteins. In situ hybridization experiments show that the different rDCAMKL transcripts are all expressed in the adult rat hippocampus. We conclude that alternative splicing of the DCAMKL gene may generate different but similar proteins in the adult rat hippocampus thereby regulating different but overlapping aspects of DCAMKL controlled neuronal plasticity.

Congenital cystic adenomatoid malformation type 4.

A 9-day-old boy presented in respiratory distress and with failure to thrive. The chest X-ray showed a hyperlucent area of the left lung. A resection of the markedly emphysematous segment 2 of the left upper lobe was performed assuming the emphysematous tissue was due to congenital lobar emphysema (CLE). Histological examination of the lung tissue, however, revealed a pattern consistent with congenital cystic adenomatoid malformation (CCAM) type 4. The therapy for CLE as well as for CCAM is similar, i.e., resection of the emphysematous tissue. As far as the prognosis is concerned, it is important to diagnose the exact type of malformation in order to exclude associated anomalies, as well as the risk of development of malignancies in later life. The frequency of associated malformations of CCAM type 4 is unknown. Although the risk for development of malignancies from CCAM type 4 is not clear at the moment, the possible development of malignancies justifies prompt resection shortly after diagnosis, even in asymptomatic patients. A life-long follow-up in those patients who had a resection of CCAM in early childhood is recommended.

Pharmacokinetics of inhaled colistin in patients with cystic fibrosis.

OBJECTIVES: Inhaled colistin is commonly used in patients with cystic fibrosis (CF), but only limited data are available to define its pharmacokinetic profile. PATIENTS AND METHODS: We performed a multicentre study in 30 CF patients to assess sputum, serum and urine concentrations after a single dose of 2 million units of colistin administered by inhalation. In a subgroup of patients we also compared the efficacy of two different nebulizers for administration of inhaled colistin. RESULTS: Serum concentrations of colistin reached their maximum 1.5 h after inhalation and decreased thereafter. Serum concentrations were well below those previously reported for systemic application in all patients. A mean 4.3+/-1.3% of the inhaled dose was detected in urine. Elimination characteristics did not differ significantly from those previously reported for systemic application. A positive correlation was found between forced expiratory volume in 1 s (FEV1) in per cent predicted and both AUC and maximal colistin concentrations in serum (Cmax). Maximum sputum concentrations were at least 10 times higher than the MIC breakpoint for Pseudomonas aeruginosa proposed by the British Society for Antimicrobial Chemotherapy. Although sputum drug concentrations decreased after a peak at 1 h, the mean colistin concentrations were still above 4 mg/L after 12 h. No differences were seen in polymyxin E sputum concentrations, for CF patients between the two nebulizer systems. CONCLUSIONS: The low systemic and high local concentrations of colistin support the use of inhaled colistin in CF patients infected with P. aeruginosa.

Kainate-elicited seizures induce mRNA encoding a CaMK-related peptide: a putative modulator of kinase activity in rat hippocampus.

By means of differential display techniques, we have previously identified an mRNA transcript whose expression is highly induced in the rat hippocampus by kainate-elicited seizures. Here, we report the cloning of a corresponding cDNA encoding a 55-amino-acid, serine-rich peptide which contains four predicted phosphorylation sites. The peptide was designated CaMK-related peptide (CARP) as it shares significant amino acid sequence identity with part of a novel putative calcium/calmodulin-dependent kinase (CaMK-VI) that was also cloned in this study. It appears that CARP and CaMK-VI are derived from the same gene through differential splicing. Intriguingly, CARP also exhibits 64% amino acid sequence identity with the C-terminal part of human doublecortin, encoded by a recently identified gene which is mutated in patients with X-linked lissencephaly and the double-cortex syndrome. In addition, the structure of CARP resembles the autoinhibitory, serine-rich N-terminal domain of CaMK-IV, suggesting a possible modulatory role of CARP with respect to CaMK activity. Northern blot analysis and in situ hybridization experiments showed that CARP mRNA is specifically induced by kainate-elicited seizures in the dentate gyrus and in the pyramidal layers CA1 and CA2, but not in CA3. In contrast, kainate-induced seizures did not change the level of expression of the CaMK-VI gene. We propose that CARP induction leads to the modulation of kinase activity in specific subregions of the rat hippocampus, providing a negative feedback mechanism for seizure-induced kinases.

A genome-wide search for linkage to asthma. German Asthma Genetics Group.

Asthma is among the most frequent chronic diseases in childhood. Although numerous environmental risk factors have already been identified, the basis for familial occurrence of asthma remains unclear. Previous genome screens for atopy in British/Australian families and for asthma in different American populations showed inconsistent results. We report a sib pair study of a sample of 97 families, including 415 persons and 156 sib pairs. Following an extensive clinical evaluation, all participants were genotyped for 351 polymorphic dinucleotide markers. Linkage analysis for asthma identified four chromosomal regions that could to be linked to asthma: chromosome 2 (at marker D2S2298, P = 0.007), chromosome 6 (around D6S291, lowest P = 0.008), chromosome 9 (proximal to D9S1784, P = 0.007), and chromosome 12 (D12S351, P = 0.010). These linkage regions could be reproduced for all loci by analysis of total or specific immunoglobulin E (minimum P values at these regions were 0. 003, 0.001, 0.010, and 0.015, respectively).