Plasmodium falciparum-activated chloride channels are defective in erythrocytes from cystic fibrosis patients

An inwardly rectifying anion channel in malaria-infected red blood cells has been proposed to function as the "new permeation pathway" for parasite nutrient acquisition. As the channel shares several properties with the cystic fibrosis transmembrane conductance regulator (CFTR), we tested their interrelationship by whole-cell current measurements in Plasmodium falciparum-infected and uninfected red blood cells from control and cystic fibrosis (CF) patients. A CFTR-like linear chloride conductance as well as a malaria parasite-induced and a shrinkage-activated endogenous inwardly rectifying chloride conductance with properties identical to the malaria-induced channel were all found to be defective in CF erythrocytes. Surprisingly, the absence of the inwardly rectifying chloride conductance in CF erythrocytes had no gross effect on in vitro parasite growth or new permeation pathway activity, supporting an argument against a close association between the Plasmodium-activated chloride channel and the new permeation pathway. The functional expression of CFTR in red blood cells opens new perspectives to exploit the erythrocyte as a readily available cell type in electrophysiological, diagnostic, and therapeutic studies of CF.     

Detection of cystic fibrosis heterozygotes using a modified loading with bromide

Summary A three day loading with sodium bromide was performed in 19 healthy controls, 16 definite cystic fibrosis heterozygotes, and 14 homozygote patients with cystic fibrosis. After three days sodium, potassium, chloride, and bromide were determined in serum and sweat. Using a multivariate discriminant analysis two nonelementary functions with the features sodium index, bromide index, potassium index, and chloride in sweat allowed us to identify the three groups. The reclassification of the probands, however, was correct in only 69.3%. A second analysis with the groups of controls and heterozygotes only resulted in a unidimensional nonelementary function with the features potassium index, bromide/sodium in sweat, and sodium index. The reclassification of the probands was correct in 91.4%. The discriminant values were 8.28±0.96 in the controls and 11.67±1.03 in the heterozygotes with an overlap of twice the standard deviations between 9.61 and 10.20.     

Detection of cystic fibrosis homozygotes and heterozygotes by serum isoelectrofocusing

Summary It is suggested that the majority of individuals with the cystic fibrosis (CF) gene have a unique serum protein (CFP) which can be demonstrated by means of isoelectrofocusing (IEF) in thin layer polyacrylamide gels. We have found the CFP in 90% of patients with cystic fibrosis, in approximately 80% of individuals heterozygous for the CF gene, and in 8% of normal control individuals. We conclude that CFP is a useful marker for the CF gene.     

Antioxidant status in erythrocytes of cystic fibrosis children.

Activities of superoxide dismutase, catalase and glutathione peroxidase in erythrocytes of cystic fibrosis children were studied in order to estimate the severity of their deficiency. Our results point to increased susceptibility of erythrocytes of cystic fibrosis subjects to oxidative injury and indicate that the antioxidant status of patients should be carefully monitored.     

Further evidence for abnormal protein kinase C regulation of macromolecule secretion in fibroblasts from cystic fibrosis patients

In comparison to skin fibroblasts from normal subjects, those from patients with cystic fibrosis (CF): (1) bound [20-³H] phorbol 12,13-dibutyrate (PDBu) with a higher affinity (K_d=25.8 vs 12.8 nM respectively) but expressed a similar number of total phorbol ester binding sites (about 2.5 pmol PDBu bound/mg of protein); (2) exhibited a faster and higher response to 4-phorbol 12-myristate 13-acetate (PMA) for the stimulation of [³⁵S]-labelled glycoconjutate release, but were equally sensitive to the synergistic effect of A23187 on this process; and (3) secreted glycoconjugates with similar [³⁵S]-sulfate and [¹⁴C]-leucine to [¹⁴C]-glucosamine labelling ratios. Taken together, these results provide further evidence for abnormal protein kinase C (PKC) regulation of macromolecule secretion in CF disease.Abbreviations BSA Bovine serum albumin - DBcAMP Dibutyryl cyclic AMP - DMEM Dulbecco's modified Eagle's medium - DMSO Dimethylsulfoxide - LDH Lactate dehydrogenase - PBS Phosphate-buffered saline - PDBu 4-phorbol 12,13-dibutyrate - 4-PDD 4-phorbol 12,13-didecanoate - PMA 4-phorbol 12-myristate 13-acetate - TCA Trichloroacetic acid     

Glycosylation of sputum mucins is altered in cystic fibrosis patients

Cystic fibrosis (CF) is characterized by chronic lung infection and inflammation, with periods of acute exacerbation causing severe and irreversible lung tissue damage. We used protein and glycosylation analysis of high-molecular mass proteins in saline-induced sputum from CF adults with and without an acute exacerbation, CF children with stable disease and preserved lung function, and healthy non-CF adult and child controls to identify potential biomarkers of lung condition. While the main high-molecular mass proteins in the sputum from all subjects were the mucins MUC5B and MUC5AC, these appeared degraded in CF adults with an exacerbation. The glycosylation of these mucins also showed reduced sulfation, increased sialylation, and reduced fucosylation in CF adults compared with controls. Despite improvements in pulmonary function after hospitalization, these differences remained. Two CF children showed glycoprotein profiles similar to those of CF adults with exacerbations and also presented with pulmonary flares shortly after sampling, while the remaining CF children had profiles indistinguishable from those of healthy non-CF controls. Sputum mucin glycosylation and degradation are therefore not inherently different in CF, and may also be useful predictive biomarkers of lung condition.     

Prostanoid biosynthesis in patients with cystic fibrosis

The urinary excretion rate (ng/h/1.73 m²) of prostanoids was determined with a capillary gas-liquid chromatographic mass spectrometric method in 19 patients with cystic fibrosis (CF) aged 1–29 years. Patients with CF showed an increased excretion of prostaglandin E₂ metabolites (PGE-M) and thromboxane B₂ and its metabolites at all ages. An imbalance in the excretion pattern of thromboxane B₂ metabolites also suggested a relative impairment of β-oxidation. There was no increased excretion of dinor-6-keto-PGF_1α, indicating normal prostacyclin biosynthesis. No correlation was found to genotype, clinical score, lung function or bacterial colonization but a significant negative relation was found between the main prostanoids in the urine and serum phospholipid levels of essential fatty acids. The results show that, contrary to the generally accepted decrease of prostanoid excretion in essential fatty acid deficiency, patients with CF increase their production parallel to the development of the deficiency. Since prostanoid synthesis is rate limited by arachidonic acid release, our data support a previously presented hypothesis about a pathological regulation of the release of arachidonic acid in CF.     

Altered intracellular pH regulation in neutrophils from patients with cystic fibrosis

Cystic fibrosis (CF) is a condition characterized by neutrophil-mediated lung damage and bacterial colonization. The physiological basis for reported functional alterations in CF neutrophils, including increased release of neutrophil elastase, myeloperoxidase, and oxidants, is unknown. These processes are, however, regulated by intracellular pH (pH(i)). We demonstrate here that pH(i) regulation is altered in neutrophils from CF patients. Although resting pH(i) is similar, pH(i) after acid loading and activation (N-formyl-methionyl-leucyl-phenylalanine and phorbol 12-myristate 13-acetate) is more acidic in CF cells than in normal cells. Furthermore, patients with non-CF-related bronchiectasis handle acid loading and activation in a fashion similar to subjects with normal neutrophils, suggesting that chronic pulmonary inflammation alone does not explain the difference in pH(i). This is further supported by data showing that normal neutrophils exposed to the CF pulmonary milieu respond by increasing pH(i) as opposed to decreasing pH(i) as seen in activated CF neutrophils. These pH(i) differences in activated or acid-loaded CF neutrophils are abrogated by ZnCl(2) but not by amiloride and bafilomycin A(1), suggesting that passive proton conductance is abnormal in CF. In addition, DIDS, which inhibits HCO(3)(-)/Cl(-) exchange, causes alkalinization of control but not of CF neutrophils, suggesting that anion transport is also abnormal in CF neutrophils. In summary, we have shown that pH(i) regulation in CF neutrophils is intrinsically abnormal, potentially contributing to the pulmonary manifestations of the condition.     

Unfractionated heparin reduces the elasticity of sputum from patients with cystic fibrosis

Mucus obstruction of the airway in patients with cystic fibrosis (CF) reduces lung function, invites infection, and limits delivery of inhaled drugs including gene therapy vectors to target cells. Not all patients respond to presently available mucolytics, and new approaches are needed. Our objectives were to investigate the in vitro effects of unfractionated heparin (UFH) on the morphology and rheology of sputum and the effect of UFH on diffusion of 200-nm nanospheres through sputum from adult CF patients. Confocal laser scanning microscopy was used to image fluorescently stained actin and DNA components of CF sputum, and atomic force microscopy was used to image isolated DNA networks. The viscoelasticity of CF sputum was measured using dynamic oscillatory rheometry. Nanosphere diffusion was measured through CF sputum using a Boyden chamber-based assay. Actin-DNA bundles in CF sputum were disaggregated by UFH at concentrations of 0.1-10 mg/ml, and UFH enhanced the endonuclease activity in sputum from patients on dornase alfa therapy. UFH significantly reduced the elasticity and yield stress, but not the viscosity, of CF sputum from patients not receiving dornase alfa therapy. Heparin dose-dependently significantly increased the diffusion of nanospheres through CF sputum from patients not on dornase alfa therapy from 10.5 +/- 2.5% at baseline to 36.9 +/- 4.4% at 10 mg/ml but was more potent, with maximal effect at 0.1 mg/ml, in patients who were on dornase alfa therapy. Thus the mucoactive properties of UFH indicate its potential as a new therapeutic approach in patients with cystic fibrosis.     

Mutation p.E92K is the primary cause of cystic fibrosis in Chuvashes

Molecular genetic study of the CFTR gene in cystic fibrosis patients from the Chuvash Republic is presented. We found linkage disequilibrium of the disease with 22-7-16-13 haplotype using intragenic markers. Major mutation p.E92K was revealed in chromosomes carrying this haplotype. The frequency of this mutation in Chuvash patients was 66.6%. Population study of the distribution of two mutations (p.E92K and F508del) of the CFTR gene revealed that their population frequency in heterozygous carriers was one per 37 subjects while calculated cystic fibrosis frequency in Chuvashia is one per 5420 newborns.     

Differential effects of glycolytic and oxidative metabolism blockers on the Na-K pump in erythrocytes of the frog, Rana temporaria

This study was undertaken to evaluate the effects of various metabolic blockers on the Na-K-pump activity and ATP content of frog erythrocytes. To eliminate K-C1 cotransport, the frog erythrocytes were incubated in nitrate media at 20 °C. Incubation of the red cells in a glucose-free medium for 2 h had no effect on cell ATP content and K⁺ influx measured as ⁸⁶Rb uptake for 60 min. The Na⁺-K⁺-pump activity was also unchanged in the frog erythrocytes incubated in a glucose-free medium containing 10 mM 2-deoxy-D-glucose or adenosine. Unexpectedly, the treatment of red cells with 1–2 mM glycolytic blocker iodoacetate produced a 2-fold increase in the ouabain-sensitive K⁺ influx. The cell ATP content declined by 9.4% after 2 h of cell incubation with iodoacetate. Incubation of the red cells for 90 min in the presence of 2 mM cyanide, 0.01 mM antimycin A or 5 mM azide resulted in a significant reduction in K⁺ influx by about 50%, 45% and 32%, respectively. The cell ATP content diminished over 60 min and 120 min of cell incubation with 2 mM cyanide by 15.6% and 31.7% of control levels, respectively. In time-course experiments, a 50% reduction in the K⁺ influx was observed when the frog erythrocytes were incubated for only 30 min in the presence of 2 mM cyanide. In contrast, 0.01–0.10 mM rotenone, a site I inhibitor, and 0.01 mM carbonyl cyanide m-chlorophenylhydrazone, an uncoupler of oxidative phosphorylation were without effect on K⁺ influx into frog erythrocytes. These results indicate that about one-half of the Na⁺ -K⁺-pump activity in frog erythrocytes is tightly functionally coupled to cytochromes via a separate “membrane-associated” ATP pool. Accepted: 12 July 1997     

Mannosylation of glycoproteins and dolichol derivatives in fibroblasts from patients with cystic fibrosis

Increased incorporation of mannose into endogenous glycoprotein fractions has been found in whole cell lysates and crude membrane preparations of cultured skin fibroblasts from patients with cystic fibrosis (1.3–2.3-times normal) when GDP[¹⁴C]mannose served as the mannosyl donor. In contrast, the incorporation of mannose from GDPmannose into lipid fractions containing dolichol phosphate and dolichol pyrophosphate oligosaccharides as well as the incorporation of mannose from dolichol phospho[³H]mannose into both glycoproteins and dolichol derivatives were not significantly different among cell preparations from patients with cystic fibrosis and normal controls. Mannosyltransferase activity toward exogenous glycoproteins as well as the activities of soluble and membranous α-mannosidase and β-mannosidase appeared to be normal and could not account for the observed differences. The altered incorporation of mannose into endogenous glycoprotein may reflect changes in glycosylation processes other than mannosylation.     

Superantigen types inStaphylococcus aureus isolated from patients with cystic fibrosis

The screening of 17 SAg genes of S. aureus isolated from the sputum of cystic fibrosis (CF) patients revealed that among 47 genetically different strains, 39 (83 %) carried SAg genes. Superantigens forming enterotoxin gene cluster were detected in 20 strains. The 2nd most common superantigen type was selk detected in 13 strains. In 9 strains, selk occurred together with the sea gene. Out of 74 strains recovered from nasal carriers, 56 (75 %) were found to carry SAg genes, 38 carried egc genes, while selk was detected in 5 strains. The predominant SAg types in both investigated S. aureus populations were egc and selk/sea, but selk gene frequency was significantly higher in the CF-derived strains.     

AP-PCR typing of Pseudomonas aeruginosa isolated from patients with cystic fibrosis

Arbitrarily Primed Polymerase Chain Reaction has been used for an epidemiological evaluation of 42 strains of P. aeruginosa isolated from nine cystic fibrosis patients during a three-year investigation period. The resistance patterns of the same strains have also been evaluated. The AP-PCR type fingerprinting was perfomed with primers 10514 and 208. Resistance was evaluated by the Minimal Inhibitory Concentration method. With 10514 eleven different genotypes could be evidenced, while with 208 only five of them could be detected. During the investigation period patients were always colonised by the same genotype. A possible correlation between resistance pattern and genotype with both primers has shown, within the same patient, a correspondence of about 20% for 10514 and a correspondence of only 10% for 208. Patients are colonised by one or two strains of P. aeruginosa and there is no relation between genotype and resistance pattern.