Competitive inhibition of photoaffinity labelling of P-glycoprotein by anticancer drugs and modulators including S9788.

The affinity of the multidrug resistance modulator S9788 to interact with P-glycoprotein was characterized by its ability to inhibit the photoaffinity labelling of plasma membranes of multidrug resistant chinese hamster ovary B30 cells by iodomycin. This iodinated analogue of daunomycin specifically photolabels P-glycoprotein in membrane vesicles as well as in intact cells. The multidrug resistance reversing agents verapamil and cyclosporin and the cytotoxic drugs vinblastine and daunomycin which are known to be recognized by P-glycoprotein competed with iodomycin for its binding site on P-glycoprotein. Vinblastine and cyclosporin bound with high affinity, S9788 and verapamil with medium affinity to P-glycoprotein.

Cystic fibrosis: the impact of analytical technology for genotype-phenotype studies.

The generalized exocrinopathy cystic fibrosis (CF) is the most common severe genetic disease in Caucasian populations. A panel of more than 700 chromosomes from German and Turkish CF patients was screened for disease-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene by chemical cleavage of mismatch, single strand conformation polymorphism, restriction analysis and direct sequencing of genomic DNA amplified by polymerase chain reaction. Besides the major 3-bp deletion, delta F508 that was found on 73% of German CF chromosomes, more than 50 other missense, nonsense, frame-shift, and splice-site mutations have already been identified. In general, a CFTR mutation is linked with a single 10-marker haplotype which indicates that in most cases a particular mutation spread from a common ancestor. The comparison of mutation genotypes with the disease phenotype emphasized the causative role of the type and localization of the CFTR mutation for clinical course and prognosis. Pancreatic status and the risk of colonization of airways with opportunistic pathogens are genetically determined. Most patients who are harbouring mutations in the nucleotide binding folds were suffering from severe CF disease. Mild or even aberrant forms of CF were observed for many missense mutations located in the putative transmembrane domains or for mutations that are expected to result in a truncated protein of half of wild-type CFTR.

Strategies of Pseudomonas aeruginosa to colonize and to persist in the cystic fibrosis lung.

The early stage of the colonization of Pseudomonas aeruginosa in cystic fibrosis (CF) was investigated. Most CF patients, once they have become colonized, harboured genomically related P. aeruginosa strains in their respiratory tract over long periods. Unrelated CF patients were colonized with different strains; however, within families cross-infection between CF siblings frequently took place. Adherence tests with buccal epithelial cells demonstrated that the epithelial cell layer of the oropharynx remained intact for up to 2 years after the onset of colonization with P. aeruginosa. Among the constituents of the microcolony the bronchial mucins were determined to be the major binding targets of CF isolates of P. aeruginosa. The time course of antibody formation to outer membrane antigens of P. aeruginosa in CF serum was analysed by Western immunoblots. Lipopolysaccharide and protein H induced the first systemic immune response.

Membrane permeability of Pseudomonas aeruginosa to 4-quinolones.

The outer membrane-disorganizing agent polymyxin B nonapeptide sensitized Pseudomonas aeruginosa strains 2-40fold to ciprofloxacin, norfloxacin, and ofloxacin, and 80-200fold to nalidixic acid. Sensitized P. aeruginosa strains were nearly as susceptible to nalidixic acid as Escherichia coli strains. The data suggest that the outer membrane permeability is of critical importance for the antipseudomonal activity of 4-quinolones. The low penetration of the outer membrane is the major cause of the nalidixic acid resistance of P. aeruginosa.

Immune response in cystic fibrosis to outer membrane proteins of Pseudomonas aeruginosa.

The systemic humoral immune response in cystic fibrosis (CF) to outer membrane (OM) proteins of Pseudomonas aeruginosa was investigated as a function of the time of colonization by immunoblotting. OM proteins were prepared from bacteria grown in ion-sufficient, magnesium-depleted, and iron-deficient media. The location of proteins F, H, and I on the blots was verified by monoclonal antibodies. Proteins H2 and H1 were differentiated by the overexpression of H1 under magnesium depletion. Iron-regulated membrane proteins (IRMPs) were recognized by their overproduction under iron limitation. Plasma samples from 43 CF patients and ten healthy adults were analyzed after preadsorption with lipopolysaccharide (LPS). Within the first year of colonization, only two to six specific plasma antibodies to OM proteins were produced. After a strong increase during the second year, long-lasting levels were seen in the majority of patients. Large variations of the immune response were noted among the patients. The number of specific antibodies to different OM proteins correlated with the severity of the course of lung disease. At maximum 38 immunostained bands were observed. Proteins H and I were the earliest antigens amongst the major OM proteins. During the second year, antibodies directed to protein F became detectable. IRMPs which indicate the growth of P. aeruginosa under iron deprivation were only recognized by plasma samples from chronically colonized CF patients with advanced lung disease.

Interaction of polymyxin B nonapeptide with anionic phospholipids.

The interaction of polymyxin B nonapeptide (PMBN) and polymyxin B (PMB) with the anionic phospholipids phosphatidylserine (PS), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidic acid (DPPA), and 1:1 mixtures (w/w) of DPPA and distearoylphosphatidylcholine (DSPC) was studied by calorimetry, electron spin resonance, and fluorescence spectrometry, electron microscopy, and fusion and leakage assays. The phase transition temperatures of DPPA and DPPG were very similar when bound to PMB or PMBN, indicating that the lipids are in a similar state when bound to the cationic peptides. Both PMB and PMBN caused the interdigitation of DPPG bilayers, suggesting that the penetration of hydrophobic side chains from a peptide bound electrostatically on the surface is sufficient to induce this phenomenon. Stopped-flow experiments revealed that PMBN and PMB induced the fusion of small unilamellar PS and large unilamellar DPPA-DSPC vesicles. The aggregation of vesicles was found to be diffusion-controlled process; the subsequent fusion took place with a frequency of 10(2)-(5 X 10(2] s-1 for small vesicles and 1-100 s-1 for large vesicles. The freeze-fracture replicas of the PMB-treated vesicles displayed 12-50-nm depressions on several superimposed bilayers, indicating the formation of stable lipid-PMB domains. Since the incubation with PMBN produced similar depressions only if the specimens were fixed, PMBN-induced domain formation seems to be a reversible rapid process. The differences in the phospholipid-peptide interactions are correlated with the differences in the physiological action of the antibiotic PMB and the nonbactericidal PMBN on the cell envelope of Gram-negative bacteria.

[Local immunization against P. aeruginosa with an outer-membrane-protein vaccine]. / Lokale Immunisierung gegen P. aeruginosa mit einer Outer-Membrane-Protein (OMP)-Vakzine.

Averting initial colonization of the respiratory tract with P. aeruginosa would be of great benefit for patients with cystic fibrosis (CF). Our approach to this problem is mucosal immunization with a vaccine prepared from the OMP fraction of a PAO-1 strain of P. aeruginosa. Sprague-Dawley rats were given 5 intragastric doses of the vaccine on 5 consecutive days and an intranasal booster dose 21 days later. Immunized animals developed high titers of OMP-specific IgG antibodies in serum and a specific IgA response in bronchioalveolar and small intestinal lavage samples, all determined by ELISA. When challenged 7 days after the booster (day 28) by intratracheal injection of live bacteria of a heterologous strain of P. aeruginosa the immunized rats showed enhanced bronchopulmonary bacterial clearance compared to nonimmunized controls, as indicated by bacterial counts from homogenized lung tissue taken 4 hrs after challenge. Thus, mucosal immunization with OMP vaccines might hinder initial colonisation of the lungs with P. aeruginosa.

Localization of the iodomycin binding site in hamster P-glycoprotein.

P-glycoprotein, the overexpression of which is a major cause for the failure of cancer chemotherapy in man, recognizes and transports a broad range of structurally unrelated amphiphilic compounds. This study reports on the localization of the binding site of P-glycoprotein for iodomycin, the Bolton-Hunter derivative of the anthracycline daunomycin. Plasma membrane vesicles isolated from multidrug-resistant Chinese hamster ovary B30 cells were photolabeled with [125I]iodomycin. After chemical cleavage behind the tryptophan residues, 125I-labeled peptides were separated by electrophoresis and high performance liquid chromatography. Edman sequencing revealed that [125I]iodomycin had been predominantly incorporated into the fragment 230-312 of isoform I of hamster P-glycoprotein. According to models based on hydropathy plots, the amino acid sequence 230-312 forms the distal part of transmembrane segment 4, the second cytoplasmic loop, and the proximal part of transmembrane segment 5 in the N-terminal half of P-glycoprotein. The binding site for iodomycin is recognized with high affinity by vinblastine and cyclosporin A.

Genetic determinants of airways' colonisation with Pseudomonas aeruginosa in cystic fibrosis.

Exocrine pancreatic insufficiency and lung infection with Pseudomonas aeruginosa are major features of cystic fibrosis (CF). This monogenic disease is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. 267 children and adolescents with CF who were regularly seen at the same centre were assessed for an association of the CFTR mutation genotype with exocrine pancreatic function and the age of onset of chronic colonisation with P aeruginosa. The major mutation delta F508 accounted for 74% of CF alleles; 33 further CFTR mutations had been detected on the CF chromosomes of the study population by June, 1992. With the exception of delta F508/R347P compound heterozygotes, patients of the same mutation genotype were either pancreas insufficient (PI) or pancreas sufficient (PS). The age-specific colonisation rates with P aeruginosa were significantly lower in PS than in PI patients. The missense and splice site mutations that are "mild" CF alleles with respect to exocrine pancreatic function were also "low risk" alleles for the acquisition of P aeruginosa. On the other hand, the proportion of P aeruginosa-positive patients increased most rapidly in the PI delta F508 compound heterozygotes who were carrying a termination mutation in the nucleotide binding fold-encoding exons. Pancreatic status and the risk of chronic airways' colonisation with P aeruginosa are predisposed by the CFTR mutation genotype and can be differentiated by the type and location of the mutations in the CFTR gene.

Infections with Pseudomonas aeruginosa in patients with cystic fibrosis.

The lung infection with Pseudomonas aeruginosa is regarded as one of the major causes of health decline in patients with cystic fibrosis (CF). The CF host response to the persistent bacterial antigen load in the endobronchiolar lumen is characterized by a pronounced humoral response, local production of cytokines, influx of neutrophils into the lung and a protease-protease inhibitor imbalance predominantly sustained by released neutrophil elastase. CF is an autosomal recessive disease, and we could demonstrate for our local patient population that the age-dependent risk to become chronically colonized with P. aeruginosa can be differentiated by the disease-causing CFTR mutation genotype. The age-specific colonisation rates were significantly lower in pancreas sufficient than in pancreas insufficient patients. P. aeruginosa is occasionally detected in throat swabs already in infancy or early childhood in most patients although there is a lapse of several years amenable to preventive measures such as vaccination until onset of persistent colonization. The epidemiology of the infection with P. aeruginosa was investigated by quantitative macrorestriction fragment pattern analysis. The distribution and frequency of clones found in CF patients match that found in other clinical and environmental aquatic habitats, but the over-representation of specific clones at a CF clinic indicates a significant impact of nosocomial transmission for the prevalence of P. aeruginosa-positive patients at a particular center. Most patients remain colonized with the initially acquired P. aeruginosa clone. According to direct sputum analysis the majority of patients is carrying a single clonal variant at a concentration of 10(7)-10(9) CFU. Co-colonization with other species or other clones is infrequent. Independent of the underlying genotype, the CF lung habitat triggers a uniform, genetically fixed conversion of bacterial phenotype. Most CFP, aeruginosa strains become non-motile, mucoid, LPS-, pyocin- and phage-deficient, secrete less virulence determinants and shift the production of cytokines evoked in neutrophils. On the other hand, other properties such as antimicrobial susceptibility or adherence to bronchial mucins remain highly variable reflecting the capacity of P. aeruginosa to adapt to ongoing changes in the CF lung habitat.