Biochemical characterization of the cystic fibrosis transmembrane conductance regulator in normal and cystic fibrosis epithelial cells.

Affinity-purified polyclonal antibodies, raised against two synthetic peptides corresponding to the R domain and the C terminus of the human cystic fibrosis transmembrane conductance regulator (CFTR), were used to characterize and localize the protein in human epithelial cells. Employing an immunoblotting technique that ensures efficient detection of large hydrophobic proteins, both antibodies recognized and approximately 180-kDa protein in cell lysates and isolated membranes of airway epithelial cells from normal and cystic fibrosis (CF) patients and of T84 colon carcinoma cells. Reactivity with the anti-C terminus antibody, but not with the anti-R domain antibody, was eliminated by limited carboxypeptidase Y digestion. When normal CFTR cDNA was overexpressed via a retroviral vector in CF or normal airway epithelial cells or in mouse fibroblasts, the protein produced had an apparent molecular mass of about 180 kDa. The CFTR expressed in insect (Sf9) cells by a baculovirus vector had a molecular mass of about 140 kDa, probably representing a nonglycosylated form. The CFTR in epithelial cells appears to exist in several forms. N-glycosidase treatment of T84 cell membranes reduces the apparent molecular mass of the major CFTR band from 180 kDa to 140 kDa, but a fraction of the T84 cell CFTR could not be deglycosylated, and the CFTR in airway epithelial cell membranes could not be deglycosylated either. Moreover, wheat germ agglutinin absorbs the majority of the CFTR from detergent-solubilized T84 cell membranes but not from airway cell membranes. The CFTR in all epithelial cell types was found to be an integral membrane protein not solubilized by high salt or lithium diiodosalicylate treatment. Sucrose density gradient fractionation of crude membranes prepared from the airway epithelial cells, previously surface-labeled by enzymatic galactosidation, showed a plasma membrane localization for both the normal CFTR and the CFTR carrying the Phe508 deletion (delta F 508). The CFTR in all cases co-localized with the Na+, K(+)-ATPase and the plasma membrane calcium ATPase, while the endoplasmic reticulum calcium ATPase and mitochondrial membrane markers were enriched at higher sucrose densities. Thus, the CFTR appears to be localized in the plasma membrane both in normal and delta F 508 CF epithelial cells.     

Different O-glycosylation of respiratory mucin glycopeptides from a patient with cystic fibrosis

The O-linked oligosaccharides from three fractions of highly glycosylated mucin glycopeptides obtained from sputum of a patient with cystic fibrosis were characterized and compared regarding size, composition, sequence and when possible linkage positions. Neutral and sialic acid-containing glycans were permethylated and analyzed by high-temperature GC-MS and MALDI-MS, showing more than 60 different oligosaccharides with a size of up to 15 monosaccharide units. Some of the observed oligosaccharides are novel for respiratory secretions, one being a trifucosylated heptasaccharide with the proposed structure: Fuc-Gal-4(Fuc-3)GlcNAc-(Fuc-)Gal-3GalNAcol. The glycosylation of two of the glycopeptide fractions was similar with regard to the neutral and sialylated oligosaccharides despite their different origins from the sol or gel phase. Analysis of the sulfated oligosaccharides by FAB-MS/MS indicated that the gel fraction contained C-6 linked sulfate groups while the two sol fractions also contained C-3 linked sulfate. The results suggest the presence of different glycosylated mucin domains, probably originating from different mucin glycoforms and/or apoproteins in the airway of cystic fibrosis patients.     

Third component of complement in cystic fibrosis.

In a study of C3 levels and phenotypes in 64 cystic fibrosis (CF) patients, 92 CF parents, 64 normal siblings, and 126 healthy controls, significant elevations of mean C3 levels were found in CF patients, their parents, and in one genetic sub-group of their siblins (SS females). C3 concentration in CF patients correlated with the degree of clinical impairment as measured by Shwachman-Kulczycki (S-K) score. No significant differences were found in the prevalences of C3 phenotypes or the S and F gene frequencies among the groups studied.     

Isolation and characterization of microparticles in sputum from cystic fibrosis patients

Background

Microparticles (MPs) are membrane vesicles released during cell activation and apoptosis. MPs have different biological effects depending on the cell from they originate. Cystic fibrosis (CF) lung disease is characterized by massive neutrophil granulocyte influx in the airways, their activation and eventually apoptosis. We investigated on the presence and phenotype of MPs in the sputum, a rich non-invasive source of inflammation biomarkers, of acute and stable CF adult patients.

Methods

Spontaneous sputum, obtained from 21 CF patients (10 acute and 11 stable) and 7 patients with primary ciliary dyskinesia (PCD), was liquefied with Sputasol. MPs were counted, visualized by electron microscopy, and identified in the supernatants of treated sputum by cytofluorimetry and immunolabelling for leukocyte (CD11a), granulocyte (CD66b), and monocyte-macrophage (CD11b) antigens.

Results

Electron microscopy revealed that sputum MPs were in the 100-500 nm range and did not contain bacteria, confirming microbiological tests. CF sputa contained higher number of MPs in comparison with PCD sputa. Levels of CD11a⁺-and CD66b⁺-, but not CD11b⁺-MPs were significantly higher in CF than in PCD, without differences between acute and stable patients.

Conclusions

In summary, MPs are detectable in sputa obtained from CF patients and are predominantly of granulocyte origin. This novel isolation method for MPs from sputum opens a new opportunity for the study of lung pathology in CF.     

The disulphide-bond content and rheological properties of intestinal mucins from normal subjects and patients with cystic fibrosis.

The disulphide/thiol (S-S/SH) content and rheological properties of highly purified small-intestinal mucins from normal (N) subjects and patients with cystic fibrosis (CF) were investigated. (1) An assay was developed to measure free SH groups (before reduction) and total SH content (after reduction) using 4,4'-dipyridyl disulphide. S-S bonds were calculated by difference. Experimental values for the S-S and SH contents of well-characterized proteins obtained with the assay showed good agreement with expected values. (2) The S-S and free SH contents of nine N and six CF mucins were variable: 44.4 +/- 5.4 nmol of S-S and 4.3 +/- 1.1 nmol of free SH per mg of N mucin and 31.7 +/- 7.6 nmol of S-S and 7.5 +/- 3.7 nmol of SH per mg of CF mucin. N and CF mucins were not statistically different. In most mucins, approximately 90% of the SH groups were involved in S-S bonds. (3) Gels were reconstituted from the same N and CF mucins at concentrations between 8 and 25 mg/ml and their rheological properties were assessed by using a magnetic microrheometer. (4) Once formed, mucin gels were stable and maintained the same mechanical properties over a long period of time (3-14 days). (5) The rheological profiles of both N and CF samples did not vary with the concentration of mucin present and were characteristic of weak, visco-elastic gels. (6) Although variations were seen in the visco-elastic properties of individual mucins, no systematic differences were detected between N and CF preparations. (7) There was no apparent correlation between the rheological properties of a mucin and its S-S/SH content.     

Characteristics of rodent intestinal mucin Muc3 and alterations in a mouse model of human cystic fibrosis

Human mucin MUC3 and rodent Muc3 are widely assumed to represent secretory mucins expressed in columnar and goblet cells of the intestine. Using a 3'-oligonucleotide probe and in situ hybridization, we observed expression of rat Muc3 mostly in columnar cells. Two antibodies specific for COOH-terminal epitopes of Muc3 localized to apical membranes and cytoplasm of columnar cells. An antibody to the tandem repeat (TR) sequence (TTTPDV)3, however, localized to both columnar and goblet cells. On CsCl gradients, Muc3 appeared in both light- and heavy-density fractions. The lighter species was immunoreactive with all three antibodies, whereas the heavier species reacted only with anti-TR antibody. Thus Muc3 is expressed in two forms, a full-length membrane-associated form found in columnar cells (light density) and a carboxyl-truncated soluble form present in goblet cells (heavy density). In a mouse model of human cystic fibrosis, both soluble Muc3 and goblet cell Muc2 were increased in amount and hypersecreted. Thus Muc2 and Muc3 contribute to the excess intestinal luminal mucus of cystic fibrosis mice.     

Cell-mediated immunity in patients with cystic fibrosis.

Leucocytes from 26 patients with cystic fibrosis (CF) and 18 healthy controls were investigated by migration inhibition induced by a variety of antigens. In patients with CF cell-mediated immunity was found to human lung and pancreatic tissue extracts as well as to Aspergillus fumigatus, Pseudomonas aeruginosa, and food antigens but not to brain, heart, or kidney. Those patients with the severest form of the disease had the greatest impairment of cell-mediated immunity, but this impairment could be reversed by steroid treatment. Cell-mediated cytotoxicity may also be concerned in the pathogenesis of CF.     

Protease deficiency in plasma of patients with cystic fibrosis. Reduced reaction of 4-methylumbelliferylguanidinobenzoate with plasma of patients with cystic fibrosis.

Protease activity in plasma is assayed using 4-methylumbelliferylguanidinobenzoate. The assay is modified by carrying out the reaction in the presence and absence of benzamidine, a competitive inhibitor of trypsin-like proteases. The parameters of the assay are described in detail. Using this assay, our earlier demonstration of a deficiency of protease activity in plasma of patients with cystic fibrosis is confirmed. The activity, corrected for the nonspecific hydrolysis of 4-methylumbelliferylguanidinobenzoate by benzamidine, is expressed as nanomoles of 4-methylumbelliferone released per milliliter plasma. Under standard conditions, the activity in plasma activated with chloroform-ellagic acid was 127.2 +/- 23.1 in 7 controls, 70.4 +/- 11.7 in 11 obligate heterozygotes, and 48.7 +/- 16.6 in 12 patients with cystic fibrosis. Identical results were obtained when unactivated plasma was used. These data demonstrate that the judicious use of specific inhibitors such as benzamidine might be useful in assaying low levels of protease activity in crude systems.     

Mass spectrometric characterization of circulating and functional antigens derived from piperacillin in patients with cystic fibrosis

A mechanistic understanding of the relationship between the chemistry of drug Ag formation and immune function is lacking. Thus, mass spectrometric methods were employed to detect and fully characterize circulating Ags derived from piperacillin in patients undergoing therapy and the nature of the drug-derived epitopes on protein that can function as an Ag to stimulate T cells. Albumin modification with piperacillin in vitro resulted in the formation of two distinct haptens, one formed directly from piperacillin and a second in which the dioxopiperazine ring had undergone hydrolysis. Modification was time and concentration dependent, with selective modification of Lys(541) observed at low concentrations, whereas at higher concentrations, up to 13 out of 59 lysine residues were modified, four of which (Lys(190), Lys(195), Lys(432), and Lys(541)) were detected in patients' plasma. Piperacillin-specific T lymphocyte responses (proliferation, cytokines, and granzyme B release) were detected ex vivo with cells from hypersensitive patients, and analysis of incubation medium showed that modification of the same lysine residues in albumin occurred in situ. The antigenicity of piperacillin-modified albumin was confirmed by stimulation of T cells with characterized synthetic conjugates. Analysis of minimally modified T cell-stimulatory albumin conjugates revealed peptide sequences incorporating Lys(190), Lys(432), and Lys(541) as principal functional epitopes for T cells. This study has characterized the multiple haptenic structures on albumin in patients and showed that they constitute functional antigenic determinants for T cells.     

Surface enzymes in cultured fibroblasts from cystic fibrosis patients.

Membrane function was examined in cultured cells from cystic fibrosis patients by assaying several enzymes on intact skin fibroblasts attached to culture dishes. This technique required few cells and minimized disruption of cellular organization. Comparison of enzyme activities of intact and broken cells showed that 12% of total glucose-6-phosphate dehydrogenase, a cytoplasmic enzyme, was measurable using intact cells, while all adenosine monophosphatase was measurable using intact cells. Alkaline paranitrophenylphosphatase activity was divided between the cell surface and interior. Substrate competition experiments indicated that substrate specificities for adenosine monophosphatase and paranitrophenylphosphatase activities were different. Adenosine monophosphatase activities of 2 control and 2 cystic fibrosis strains fluctuated similarly during the cell culture cycle. The apparent Km values relative to adenosine monophosphate were similar in all strains. A chromatographic fraction of serum from a cystic fibrosis patient that was inhibitory to oyster ciliary activity had no effect on adenosine monophosphatase activity of normal fibroblasts. Furthermore, fractions of media from cystic fibrosis homozygote and heterozygote fibroblast cultures were not inhibitory to adenosine monophosphatase activities of intact normal fibroblasts or of part iculate fractions prepared from them. In light of previous studies that showed that factors from cystic fibrosis serum of culture medium disrupted specific membrane activities, it is proposed that the cystic fibrosis factor interacts with the plasma membrane, interfering most conspicuously with the protein functions that are sensitive to changes in their membrane environment.     

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.     

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.     

Meconium ileus equivalent in older patients with cystic fibrosis.

Meconium ileus equivalent is one of the lesser known manifestations of cystic fibrosis, and occurs most often in older patients. With the improved overall survival of patients with cystic fibrosis, one would expect to see this condition more often in the future. Of 53 patients attending our cystic fibrosis clinic for adolescents and adults, eight had experienced a total of 25 episodes of meconium ileus equivalent. Recurrent attacks occurred in seven patients, of whom four had at least four separate, well documented episodes: these episodes were associated with obvious recognised precipitating factors in only three patients. All patients responded to appropriate medical treatment with acetylcysteine or sodium diatrizoate, including three who had previously undergone surgery for meconium ileus equivalent elsewhere. Controversies remain, however, concerning the role of prophylactic medical management with acetylcysteine, sodium diatrizoate, and pancreatic supplements.     

Rat small intestinal mucin: isolation and characterization of a water-soluble mucin fraction

A water-soluble fraction of sialoglycoprotein containing sulfate was isolated from the mucosal scrapings of the rat small intestine without prior treatment with proteolytic enzymes. Chromatography of the water-soluble mucin on a DEAE-cellulose column gave three main fractions: a major carbohydrate-rich fraction containing sulfate (IGP-A), one high in protein content, and a third with a composition similar to the starting material. Fraction IGP-A was resolved into two components by ultracentrifugation and disc-gel electrophoresis. The higher molecular-weight species of IGP-A was separated from the second component by Sepharose-4B chromatography. These two glycoprotein fractions designated IGP-A₁ and IGP-A₂ had the same chemical composition as IGP-A.     

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.