Expression and intracellular processing of chimeric and mutant CFTR molecules.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated chloride channel comprising two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs) and a unique regulatory (R) domain. The most frequent cystic fibrosis (CF) mutation, a deletion of Phe508 in NBD1, results in the retention of the DeltaF508 CFTR in the endoplasmic reticulum, as do many other natural or constructed mutations located within the first NBD. In order to further define the role of NBD1 in CFTR folding and to determine whether the higher frequency of mutations in NBD1 with respect to NBD2 results from its position in the molecule or is related to its primary sequence, we constructed and expressed chimeric CFTRs wherein NBD domains were either exchanged or deleted. Synthesis, maturation and activity of the chimeras were assessed by Western blotting and iodide efflux assay after transient or stable expression in COS-1 or CHO cells respectively. The data showed that deletion of NBD1 prevented transport of CFTR to the cytoplasmic membrane whereas deletion of NBD2 did not impair this process but resulted in an inactive chloride channel. On the other hand, substituting or inverting NBDs in the CFTR molecule impaired its processing. In addition, while the NBD1 R555K mutation is known to partially correct the processing of CFTR DeltaF508 and to increase activity of both wild-type and DeltaF508 individual channels, it showed no positive effect when introduced into the double NBD1 chimera. Taken together, these observations suggest that the proper folding process of CFTR results from complex interactions between NBDs and their surrounding domains (MSDs and/or R domain).

Prevalence of the delta F508 deletion of the cystic fibrosis gene in Belgian patients.

A sample of 107 Belgian cystic fibrosis patients has been tested for the presence of the delta F508 deletion. We have shown that 166 (78%) of the CF chromosomes presented the deletion, and that 97% of the deleted chromosomes and 50% of the non-deleted chromosomes presented the haplotype B (KM19-2/XV2c-1).

Carrier prediction of cystic fibrosis in 36 families by means of restriction fragment length polymorphism.

Transmission of cystic fibrosis (CF) was studied in 36 families with at least one affected and one unaffected child. DNA was prepared from peripheral leukocytes and submitted to restriction fragment length polymorphism (RFLP) analysis with two CF probes (pj3.11 and met). Twenty families were shown to be informative so that accurate predictions could be made of the status of the offspring. Sixteen were only partially informative. The allele frequency was similar to that originally reported except for one Msp I site detected with the pj3.11 probe, for which we found a significantly higher heterozygote frequency, making it more informative than expected in our population sample. Pedigree analysis demonstrated no obligate recombinant between CF and the polymorphic markers.

Alteration of the N-formyl-methionyl-leucyl-phenylalanine-induced response in cystic fibrosis neutrophils.

In order to determine whether cystic fibrosis neutrophils are affected in their secretory functions, lysosomal enzyme release and chemiluminescence (light emission from cells) were assayed in patients' cells and compared with those in normal control cells. We observed a decreased response of cystic fibrosis neutrophils in beta-glucuronidase release and chemiluminescence after stimulation by N-formyl-methionyl-leucyl-phenylalanine. There was no significant correlation of these results with the clinical score nor with the medical treatment. On the other hand, responses to the calcium ionophore A23187 and to opsonized zymosan showed no significant difference between normal and cystic fibrosis subjects in lysosomal enzyme release. N-formyl-methionyl-leucyl-phenylalanine receptor alterations did not seem involved in the observed effect as demonstrated by Scatchard plot analysis of N-formyl-methionyl-leucyl-phenylalanine binding to these receptors. These results clearly demonstrate a difference between normal and cystic fibrosis neutrophils in release and chemiluminescence responses to N-formyl-methionyl-leucyl-phenylalanine stimulation, a difference that might be located in the plasma membrane as both responses are membrane dependent.

Plasma and erythrocyte zinc, copper and selenium in cystic fibrosis.

Plasma and erythrocyte zinc, copper and selenium were measured in 20 cystic fibrosis children, aged 7 to 19 years. Mean plasma zinc and copper levels were not different from those in age-matched controls but very low zinc levels occurred sporadically. Plasma zinc concentrations were significantly lower in patients with moderate-to-severe growth retardation and with severe pulmonary disease as compared to patients without growth failure and with moderate pulmonary disease. Mean erythrocyte zinc (40.8 micrograms/g Hb +/- 9.2) and copper levels (3.56 micrograms/g Hb +/- 0.50) were very significantly increased (30.4 micrograms/g Hb +/- 5.2 and 2.73 micrograms/g Hb +/- 0.30 respectively, for age-matched controls). Mean plasma and erythrocyte selenium levels (63 ng/ml +/- 15 and 329 ng/g Hb +/- 86) were significantly lower than those in age-matched controls (82 ng/ml +/- 13 and 404 ng/g Hb +/- 116). The trace element concentrations in erythrocytes are discussed in relation to the activities of the copper- and zinc-containing enzyme superoxide dismutase and the seleno-enzyme glutathione peroxidase. We consider that more data on trace element metabolism in CF should be collected before specific supplementation is considered.

Erythrocyte and plasma trace element levels in clinical assessments : Zinc, copper, and selenium in normals and patients with Down's syndrome and cystic fibrosis.

On the basis of original investigations on zinc, copper, and selenium levels in plasma and erythrocytes of Down's syndrome (DS), cystic fibrosis (CF), and control subjects, the possible importance of erythrocytic trace element concentrations in clinical analysis is emphasized. Red blood cell levels of copper and zinc were found significantly increased in both groups of diseased patients as compared to age-matched controls, although plasma levels did not statistically differ. Plasma selenium levels were significantly lower in both investigated groups, but red blood cell levels were only decreased in CF and were not different from controls in DS. Significant differences were also found between zinc, copper, and selenium levels in erythrocytes of two control groups originating from distinct geographic areas, although plasma levels were not statistically different. Some factors likely to modify trace element concentrations in erythrocytes are examined and a more systematic determination of these levels is suggested for use in clinical analysis.

Study of the relation between the clinical pulmonary condition of children with cystic fibrosis and the lymphoblastic response to the antigen Pseudomonas aeruginosa.

This study was performed on four sibling pairs affected by cystic fibrosis. Of each sibling pair, one was more affected than the other. The results show that the lymphoblastic response to the antigen Pseudomonas aeruginosa of the most affected patient was strongly reduced in comparison to the response of the less affected one. The plasma from the most affected patient contains an inhibitory factor which reduces the lymphoblastic response of the less affected one. On the other hand, plasma from the less affected patient improves the lymphoblastic response of the most affected one (although not significantly). One notes a better lymphoblastic response when the most affected patient's lymphocytes are put in the presence of the less affected one's antigens and plasma. These findings suggest a phenomenon of lymphocyte tolerance in the most affected patient towards P. aeruginosa.

Study of helper and suppressor T-cells in cystic fibrosis.

The ratio between inducer and cytotoxic/suppressor subset T-cells was studied in 11 cystic fibrosis patients and 11 non-cystic fibrosis controls. No statistically significant difference was found between the two groups. It is suggested that the major immune deficiency in some patients suffering from cystic fibrosis is a state of tolerance to the same bacterial antigens such as Pseudomonas aeruginosa. Inhibitory factors are present in the serum of the most affected patients.

Nucleoside uptake in normal and cystic fibrosis fibroblasts in vitro.

Cultured skin fibroblasts derived from unrelated patients with diagnosed cystic fibrosis were compared with those from one normal individual in respect to thymidine and uridine uptake. The results obtained show that the two nucleosides are incorporated at the same rate by the three cell strains. Therefore, it appears that the reported abnormality [6] in nucleoside uptake in cystic fibrosis cells is not a general characteristic of these cells.