cytidylyl-3--5--guanosine and Cystic-Fibrosis

Pulmonary infections with Burkholderia cepacia complex organisms contribute significantly to morbidity and mortality in patients with cystic fibrosis (CF), partially due to the intense inflammatory response of the host to the presence of bacteria and their byproducts. In the present study we show that Burkholderia genomes contain a large number of immunostimulatory CpG motifs. This is mainly because of their large genome size. This suggests that DNA from Burkholderia sp. has the potential to cause significant inflammatory response. Whether this contributes significantly to the airway inflammation often observed in infected CF patients remains to be determined.

Topics: Animals; Burkholderia cepacia; Burkholderia Infections; CpG Islands; Cystic Fibrosis; Dinucleoside Phosphates; DNA, Bacterial; Genome, Bacterial; Humans; In Vitro Techniques; Mice; Sequence Analysis, DNA

Since unmethylated CpG motifs are more frequent in DNA from bacteria than vertebrates, and the unmethylated CpG motif has recently been reported to have stimulatory effects on lymphocytes, we speculated that bacterial DNA may induce inflammation in the lower respiratory tract through its content of unmethylated CpG motifs. To determine the role of bacterial DNA in lower airway inflammation, we intratracheally instilled prokaryotic and eukaryotic DNA in C3H/HeBFEJ mice and performed whole lung lavage 4 h after the exposure. Heat denatured, single stranded Escherichia coli genomic DNA (0.06 ng endotoxin/microg DNA) was compared to heat denatured, single stranded calf thymus DNA (0.007 endotoxin/microg DNA). 10 microg of bacterial DNA, in comparison to 10 microg of calf thymus DNA, resulted in a fourfold increase in the concentration of cells (P = 0.0002), a fivefold increase in the concentration of neutrophils (P = 0.0002), a 50-fold increase in the concentration of TNF-alpha (P = 0.001), and a fourfold increase in the concentration of both IL-6 (P = 0.0003) and macrophage inflammatory protein-2 (P = 0.0001) in the lavage fluid. Importantly, instillation of 0.60 ng of E. coli LPS resulted in a negligible inflammatory response. To test whether the stimulatory effects of bacterial DNA are due to its unmethylated CpG dinucleotides, we methylated the bacterial DNA and also prepared 20 base pair oligonucleotides with and without CpG motifs. In comparison to instillation of untreated bacterial DNA, methylation of the bacterial DNA resulted in a significant reduction in the concentration of cells and cytokines in the lower respiratory tract. Moreover, oligonucleotides containing embedded unmethylated CpG motifs resulted in inflammation in the lower respiratory tract that was indistinguishable from that observed with untreated bacterial DNA. In contrast, oligonucleotides without the embedded CpG motifs or with embedded but methylated CpG motifs resulted in significantly less inflammation in the lower respiratory tract. The possible relevance of these data to human disease was shown by extracting and analyzing DNA in sputum from patients with cystic fibrosis (CF). Approximately 0.1 to 1% of this sputum DNA was bacterial. Intratracheal instillation of highly purified CF sputum DNA caused acute inflammation similar to that induced by bacterial DNA. These findings suggest that bacterial DNA, and unmethylated CpG motifs in particular, may play an important pathogenic ro

Topics: Animals; Base Sequence; Bronchoalveolar Lavage Fluid; Carrier State; Chemokine CXCL2; Chemotactic Factors; Conserved Sequence; Cystic Fibrosis; Cytokines; Dinucleoside Phosphates; DNA Primers; DNA, Bacterial; Humans; Inflammation; Interleukin-6; Lung; Male; Mice; Mice, Inbred C3H; Monokines; Neutrophils; Polymerase Chain Reaction; Pseudomonas aeruginosa; Pseudomonas Infections; Sputum; Tumor Necrosis Factor-alpha

Search for mutations in a cystic fibrosis patient, compound heterozygous for 1717-1G-->A and another uncharacterized molecular defect, revealed the presence of a de novo R1066H mutation on the affected chromosome of paternal origin. Three additional rare mutations (R1066C, R1066S and R1066L), occurring at the CpG dinucleotide at position 3328-3329 of the cystic fibrosis transmembrane conductance regulator gene, have so far been reported. The identification of a R1066H de novo mutation further suggests that this dinucleotide may constitute a mutational hotspot.

Topics: Adolescent; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Dinucleoside Phosphates; Fathers; Haplotypes; Heterozygote; Humans; Italy; Male; Point Mutation

To determine whether a relationship exists between DNA methylation and CFTR gene expression, we investigated the methylation status of CpG sites in the mouse and human CFTR promoters. Tissues and previously characterized cell lines that vary with respect to CFTR expression were selected for analysis using the methylation sensitive restriction endonuclease Hha I. We find that CpG sites are not methylated in high and low CFTR-expressing cell lines, whereas in the very low or non-CFTR-expressing cell lines, the CpG sites are partially or completely methylated. However, none of these sites were methylated in any of the tissues examined irrespective of the state of CFTR expression. Therefore, we conclude that the CFTR promoter belongs to the class of CpG-rich promoters in which the associated CpG sites are not methylated in tissues and that an inverse correlation between methylation and CFTR expression can only be found in cell lines.

Topics: 3T3 Cells; Animals; Base Sequence; Cell Line; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Dinucleoside Phosphates; DNA Primers; Humans; Membrane Proteins; Methylation; Mice; Molecular Sequence Data; Promoter Regions, Genetic; Tumor Cells, Cultured

A total of 75 non-delta F508 chromosomes from 59 German cystic fibrosis patients was screened for mutations in exon 11 of the cystic fibrosis (CFTR) gene. These Caucasian patients were found to possess an identical haplotype background for two common mutations (G551D, R553X) consistent with their being identical by descent. However, a different R553X associated haplotype found in American black patients was suggestive of recurrent mutation, a postulate supported by the location of the R553X alteration in a hypermutable CpG dinucleotide. Likelihood estimates for recurrent mutation and identity by descent were compared and strongly supported the hypothesis of recurrent R553X mutation. The ability to distinguish between these two alternatives provides an indication of whether or not the search for mutations should be restricted to chromosomes with similar haplotypes.

Topics: Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Dinucleoside Phosphates; Exons; Female; Gene Frequency; Genotype; Haplotypes; Humans; Male; Membrane Proteins; Mutation; Pedigree; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Restriction Mapping

The probe pCS.7, isolated from an HpaII tiny fragments (HTF) island and tightly linked to the mutation causing cystic fibrosis (CF), detects a polymorphism with the rare-cutter restriction enzyme BssHII. In a single digest, the resulting restriction fragment length polymorphism (RFLP) cannot be detected by either conventional or pulse-field gel electrophoresis. A double digest with BssHII in conjunction with a six-cutter restriction enzyme that does not recognize a site containing a CpG dinucleotide enables the probe to be used routinely for prenatal diagnosis and carrier exclusion. This strategy can be used to identify polymorphisms in HTF islands.

Topics: Alleles; Cystic Fibrosis; Deoxyribonuclease HpaII; Deoxyribonucleases, Type II Site-Specific; Dinucleoside Phosphates; Female; Genetic Markers; Humans; Male; Pedigree; Polymorphism, Genetic; Polymorphism, Restriction Fragment Length