Cystic Fibrosis Homozygotes Research Articles

Protein Abnormalities in Adult Respiratory Distress Syndrome, Tuberculosis, and Cystic Fibrosis Sera

Crossed immunoelectrophoresis (X-IEP) revealed several abnormalities in serum proteins from patients with adult respiratory distress syndrome (ARDS), tuberculosis (TB), and cystic fibrosis (CF). The two quite different kinds of pulmonary disease, one acute (ARDS) and the other chronic (TB and CF) exhibited serum changes specific for each disease and abnormalities associated with inflammation and pathogenesis, in general. In ARDS sera, most proteins were extremely low, presumably due to leakage into the lungs through damaged tissue, while the acute-phase proteins, orosomucoid, alpha 1-antitrypsin, alpha 1-antichymotrypsin, and haptoglobin, were markedly high when compared to the overall protein pattern. The extremely high alpha 1-antichymotrypsin values were not seen in corresponding TB and CF sera. Numerous TB patients had elevated alpha 1-antitrypsin, alpha 1-antichymotrypsin, and haptoglobin, but only the alpha 1-antitrypsin population mean was significantly different from normal. Gc-globulin, ceruloplasmin, and beta-lipoprotein were higher and alpha 1-lipoprotein and inter-alpha-trypsin inhibitor lower than normal. All other quantitative serum changes were not statistically significant. Surprisingly, all TB patients belonged to the Gc-1-1 genotype in contrast to the Gc-1-1, Gc-1-2, Gc-2-2 polymorphisms of the other populations. CF homozygote sera revealed statistically significant increases in the acute-phase proteins, alpha 1-antitrypsin, alpha 1-antichymotrypsin, and haptoglobin, while orosomucoid, transferrin, IgA, and IgG tended to be higher than normal. The tendency for higher levels of transferrin indicated possible iron deficiency in some patients. In contrast, prealbumin, alpha 1-lipoprotein, and inter-alpha-trypsin inhibitor were significantly depressed in CF patients. CF heterozygotes shared the decrease of alpha 1-lipoprotein with the patients while exhibiting small but significant depressions of alpha 2-macroglobulin and IgG. Though not statistically significant, lowered concentrations of alpha 1-antitrypsin were evident for the heterozygotes.

Monoclonal antibodies to cystic fibrosis antigen

A series of monoclonal antibodies has been prepared against a granulocyte antigen previously shown to be present in the serum of individuals homozygous or heterozygous for the cystic fibrosis (CF) gene. The specificity of the monoclonals was established by their ability to remove the antigen (CF antigen) from serum. The monoclonals defined at least two epitope regions on CF antigen, thus permitting the establishment of a two-site sandwich enzyme-linked immunosorbent assay (ELISA). Using the ELISA significant differences in levels of CF antigen were demonstrated in serum samples from CF homozygotes, CF heterozygotes and normal controls.

Increased monocyte chemiluminescence in cystic fibrosis patients and in their parents.

We examined the chemiluminescence response of peripheral blood monocytes from patients with cystic fibrosis (CF) and their asymptomatic parental carriers of the CF gene to three different types of stimulation. We found that monocytes from both patients and carriers have increased luminol-dependent chemiluminescence in the first 25 min after stimulation by adherence to glass. These results are consistent with the hypothesis that monocytes from both CF heterozygotes and homozygotes respond to adhesion with increased oxygen radical formation. The increased adherence-induced monocyte chemiluminescence of the parental carriers did not vary with age or length of exposure of the parents to a child with CF. Also, repeated exposure to medications and respiratory secretions of CF patients was not associated with an increase in adherence-induced monocyte chemiluminescence of their nonbiologically related caretakers. Thus, this observed increase in chemiluminescence is not simply secondary to the medications or respiratory dysfunction seen in the patients with CF. Patients with other types of obstructive lung disease did not show increased adherence-induced monocyte chemiluminescence. We conclude that increased early phase adherence-induced monocyte chemiluminescence occurs in patients with cystic fibrosis and the obligate carriers of the CF gene independent of environmental influences.

Identification of cystic fibrosis homozygotes and heterozygotes by isoelectric focusing of serum proteins.

Screening of 44 cystic fibrosis homozygotes, 17 heterozygotes and 36 normal controls, by identification of cystic fibrosis protein (CFP), was performed on sera using an improved isoelectric focusing technique. CFP was observed in 91% of homozygotes, 88% of heterozygotes and 8% of normal controls tested. Partial purification of CFP by chromatofocussing indicates that CFP has a molecular weight of about 52,000 u. It is speculated that CFP is a normal serum protein that exhibits a quantitative difference in concentration between CF homozygotes, heterozygotes and normals.

PRENATAL DIAGNOSIS IN CYSTIC FIBROSIS

Cystic fibrosis is the most common autosomal recessive genetic disorder in the Caucasian population (1:2000-1:4000) (Warwick, W. J. (1978) Helv. Paediatr. Acta 33, 117-125). This defect is characterized by chronic obstructive pulmonary disease, pancreatic exocrine insufficiency and abnormally high perspiration electrolytes in most patients (Talamo et al. (1985) In: The metabolic basis of inherited diseases, pp. 1887-1917). The elevated electrolyte level provides the most reliable diagnostic test for cystic fibrosis homozygotes. Although prospects for cystic fibrosis patients have improved, genetically homozygous cystic fibrosis is effectively a lethal disease. Because of the seriousness of the disease, many families with one affected child desire a prenatal diagnosis when a second pregnancy occurs. Despite extensive research, the biochemical basis of cystic fibrosis remains unknown. Secondary effects on microvillar enzymes allow second trimester diagnosis (17-18 weeks of gestation (Brock, D. H. J. (1983) Lancet II, 941-943). First trimester prenatal diagnosis for cystic fibrosis became possible with DNA technology. Application of polymorphic marker loci to problems of prenatal diagnosis and carrier-testing is discussed.

Prenatal diagnosis of cystic fibrosis.

Cystic fibrosis (CF) is one of the most common recessively inherited genetic diseases in Caucasian populations. It was first formally described as a distinct disorder in 1938 (Andersen, 1938), and has subsequently been the subject of intensive clinical and laboratory investigations. Despite the barrage of experimental work on the disorder, the primary protein lesion in CF remains unknown. The medical literature is awash with reports on significant biochemical and physiological abnormalities in CF homozygotes, but few of these have turned out to be consistently reproducible and none has yet led to a consensus on an area of metabolism that might be the source of an aberrant protein or enzyme. A possible exception to this statement is recent work by Quinton and colleagues (Quinton, 1983; Bijman and Quinton, 1984) identifying a chloride ion transport abnormality in epithelial cells dissected from the ducts of isolated sweat glands. Though these observations are physiological rather than biochemical, they have begun to make sense of the pathology of CF and may soon lead to identification of a primary protein abnormality.

Plasma and submandibular saliva lysosomal enzymes in cystic fibrosis

This study determined in a blind fashion the activity levels and thermostability properties of two lysosomal hydrolytic enzymes, acid phosphatase and α-mannosidase, in plasma samples from 25 cystic fibrosis (CF) patients and 25 age- and sex-matched normal controls. Mean α-mannosidase activity (3.2 ± 1.0 mU/ml) and acid phosphatase activities (6.5 ± 2.9 mU/ml) in CF patients were not significantly different from those found in normal individuals (2.8 ± 0.7 and 7.6 ± 3.4 mU/ml, respectively). Using stringent conditions no differences in thermostability properties of these enzymes were found between plasma from CF patients as compared to that of normal controls. When activity levels of these enzymes and of four additional hydrolytic enzymes, α- l-fucosidase, α-galactosidase, α-glucosidase and β-galactosidase, were determined in submandibular saliva, no significant differences in enzyme levels between CF and age- and sex-matched controls were noted nor were thermostability differences found. Our data do not support the concept that altered properties of these enzymes are useful as markers for detection of CF homozygotes and heterozygotes, nor the hypothesis that the defect underlying this disease is a deficiency of post-translational modification of glycoproteins leading to their miscompartmentalization and qualitative alteration.

Lectin-like factor and co-factor in serum from cystic fibrosis heterozygotes

Previous attempts to use an assay for a serum lectin-like factor as a carrier test for the cystic fibrosis gene have not been successful because food Intake and effects of serum storage have interfered. A revised method for detecting the lectin-like factor employs the serum IgM fraction, rather than whole serum, separated on an S-300 Sephacryl gel filtration column. Elevated lectin titers were found in 95 percent of 43 obligate heterozygotes, 89 percent of patients with cystic fibrosis, 64 percent of siblings of patients with cystic fibrosis, and 5 percent of 60 control subjects. Lectin activity was shown to be associated with the IgM fraction of serum and to result from the binding of a low-molecular-weight co-factor to IgM. Unbound cofactor was also detected in a serum fraction from 81 percent of the heterozygotes and 88 percent of the cystic fibrosis homozygotes, and in a urine fraction from nine patients with cystic fibrosis who had positive serum lectin activity, but not in any urine sample from heterozygous subjects. The presence of co-factor in the urine of cystic fibrosis homozygotes suggests higher serum levels of this agent that spill over into the urine. Intravenous antibiotic therapy removes the lectin and co-factor from blood and urine for variable periods of time, suggesting a beneficial effect of antibiotics in cystic fibrosis apart from their antibacterial action. Detection of the lectin and co-factor are useful in screening for cystic fibrosis carriers, although the somewhat subjective biologic assay requires much experience to achieve reliability.

Identification of the major 4-methylumbelliferyl p-guanidinobenzoate-hydrolyzing plasma protein in cystic fibrosis: implication for intrauterine and heterozygote detection.

Measurement of 4-methylumbelliferyl p-guanidinobenzoate (MUGB)-hydrolyzing activity in the plasma of normal controls, cystic fibrosis (CF) heterozygotes, and CF homozygotes did not support previously reported (35) differences in MUGB-hydrolyzing activity. We identified human plasma albumin as the major source of MUGB-hydrolyzing activity by comparison of our plasma results to those obtained with physiologic concentrations of commercial albumin samples. Substantiating evidence was obtained from gel filtration experiments and correlation of albumin levels in CF plasma with MUGB titers. We found essentially no proteolytic activity towards dinitrophenylprotamine sulfate associated with commercial albumin samples. It appears that the reaction between human albumin and MUGB represents a weak esterase activity, perhaps involving the acylation of a specific site(s) on the protein. Hypoalbuminemia has been documented (8) in some CF patients. Low albumin concentrations, indicated by MUGB titers less than 190 nmole methylumbelliferone/ml plasma, were found in 42% of CF homozygotes, 6% of heterozygotes, and 4% of controls. Gel filtration studies of a normal amniotic fluid supernatant indicated that albumin was the major MUGB-hydrolyzing substance in this fluid. We conclude that MUGB abnormalities are not associated with the basic gene defect in CF and cannot be used as the basis of a test for intrauterine or heterozygote detection.

Cystic fibrosis-like changes in saliva of healthy persons subjected to anaerobic exercise

The biochemical composition of saliva secreted by healthy persons and by heterozygotes and homozygotes of cystic fibrosis at rest and by healthy persons subjected to aerobic or anaerobic effort were compared. In the saliva from cystic fibrosis homozygotes at rest substantial increases of the activity of ribonuclease ( p < 0.001) and of the concentrations of protein ( p < 0.001), lactate ( p < 0.001), sodium ( p < 0.001), potassium ( p < 0.01) and calcium ( p < 0.05) were found in comparison with saliva from healthy persons at rest. In the saliva from cystic fibrosis heterozygotes at rest similar but less pronounced changes were seen. After anaerobic exercise these biochemical parameters were increased in the saliva of healthy persons and mimicked the values of cystic fibrosis saliva. However, after aerobic effort no changes other than a slightly increased ribonuclease activity were seen in the saliva of healthy persons. This indicates that salivary glands of cystic fibrosis patients, at rest, are in the same state of lactate acidosis and energy depletion as these glands are in healthy persons after anaerobic work.

EFFECTS OF SUCROSE LOAD ON THE BIOCHEMICAL COMPOSITION OF BLOOD, SALIVA AND URINE FROM CYSTIC FIBROSIS HOMOZYGOTES AND HETEROZYGOTES AND HEALTHY CONTROLS

Ceder, O., Bardoń, A., Hellsing, K. and Kollberg, H. (Department of Pediatrics, University Hospital, Umeå, Sweden, Department of Clinical Chemistry, University Hospital, Uppsala, Sweden, Department of Biochemistry, Institute of Sport, Warsaw, Poland and Department of Pediatrics, University of Kuwait, Safat, Kuwait). Effects of sucrose load on the biochemical composition of blood, saliva and urine from cystic fibrosis homozygotes and heterozygotes, and healthy controls. Acta Paediatr Scand, 1983; Suppl. 309:33. A disturbance in the metabolism of carbohydrates has been suggested to be responsible for many of the clinical symptoms in cystic fibrosis. To study this, the biochemical compositions of blood, mixed saliva and urine from homozygotes and heterozygotes for cystic fibrosis and healthy children and adults were compared before and after an oral load of sucrose. In blood, the fasting values of glucose, pyruvate and lactate were similar in the four groups. In saliva, the fasting concentrations of protein, sodium, chloride and potassium were significantly increased for homozygotes. Heterozygote saliva values were intermediate, although not significantly different from those of controls. The urinary concentrations of sodium, chloride, potassium and calcium were also significantly increased in the homozygotes. Again, heterozygotes values were intermediate and did not differ significantly from control values. Sucrose intake caused greater increases in the blood levels of glucose, lactate and pyruvate in the homozygotes and heterozygotes than in their respective control groups. Reversal of the increased concentrations occurred most quickly in the homozygotes and 150 min after sucrose intake the glucose and lactate concentrations were significantly lower in this group than in control children. In saliva significantly lower concentrations of sodium, chloride and potassium were found in the homozygotes and heterozygotes 30 and 90 min after sucrose intake than in the fasting state. Thus, their salivary concentrations of electrolytes became closer to those of controls. In controls sucrose intake caused an increase in the activity of ribonuclease in the saliva while the electrolyte values were unaffected. Limited effects of the sucrose intake were seen on the urinary values in the four groups.

Relative ion permeability of normal and cystic fibrosis nasal epithelium.

The raised transepithelial electric potential difference (PD) across respiratory epithelia in cystic fibrosis (CF) has suggested an abnormality in ion permeation. We characterized this abnormality further by measuring in the nasal epithelia of CF and normal subjects the concentration-PD relationship for amiloride, an inhibitor of cell Na+ permeability, and PD responses to superfusion with solutions of different composition. Amiloride was more efficacious in the CF subjects but the ED50 was not different from that of normals (approximately 2 X 10(-6) M). Na+ replacement by choline induced effects similar to those of amiloride, i.e. a greater depolarization in CF subjects. A 10-fold increase in the K+ concentration of the perfusate induced a small (less than 10 mV) depolarization in both subject populations. When Cl- in the perfusate was replaced by gluconate or SO2-(4) the nasal PD of normal subjects hyperpolarized (lumen became more negative) by approximately 35 mV. A significantly smaller response (less than 17 mV) was induced in CF homozygotes but not in heterozygotes (38 mV). The smaller response of CF subjects appears to reflect an absolute decrease in luminal surface Cl- permeability because pretreatment with amiloride did not increase the response to Cl- free solution (7 mV). Accordingly, three abnormalities (decreased Cl- permeability, raised PD, greater amiloride efficacy) have been identified in CF respiratory epithelia. Whereas "excessive" active Na+ transport can account for these abnormalities and the dessication of airway surface liquid, it is possible that a lower lumenal cell membrane Cl- permeability and inhibition of a potential path of Cl- secretion can also explain the observations.

Diagnosis of cystic fibrosis homozygotes and heterozygotes from plasma and fibroblast cultures. A three‐generation family study

The diagnosis of cystic fibrosis (CF) homozygotes and heterozygotes and of individuals without the CF gene, based on differences in the thermal stability of acid phosphatase and alpha-mannosidase, is reported. The residual activities at 36.5 degrees C and 41.3 degrees C were below 10% of the activity in unheated samples for homozygotes, 40-50% for heterozygotes and above 90% for normals. The intracellular alkaline phosphatase and extracellular beta-hexosaminidase activities after treatment with heparin and gammaglobulin were 500% and 200%, respectively, of the activities without this treatment in CF homozygotes, whereas for heterozygotes and normals the values were the same after treatment as before. Pedigrees of four CF families, covering 2-3 generations, are presented. The possible use of these tests as diagnostic tools is further discussed.

Differences in mucus-stimulating serum fractions of cystic fibrosis patients and controls.

In nature, the urn cell complexes which swim in the coelomic fluid of the marine invertebrate, Sipunculus nudus, produce "tails" of mucus in response to bacterial pathogens. Since they produce measurable tails of mucus in vitro, suspensions of urn cell complexes provide a bioassay for mucus-stimulating substances (MSS) in biological fluids, including several human body fluids. Heat-activated seawater dilutions of human serum contain MSS. Serum from 87 cystic fibrosis (CF) homozygotes, 60 obligate heterozygotes, and 45 controls were fractionated on a Sephadex G-200 gel filtration column. After subsequent heating for 4 min at 85 degrees C, the fractions of all normal sera showed two characteristic peaks of MSS activity. The pattern differed in heated serum fractions of CF patients, in that the second peak was lacking in 59% of individual tests. The pattern was intermediate in heterozygote sera. Of the 36 CF serum fractions which did have two peaks of activity, 89% had the predominant activity in peak 1. The frequency of single peaks of activity increased with patient age, from 33% in those under 10 years to 75% in those over 16. The molecular weight of peak 1 is about 75,000 daltons, of peak 2 about 30,000. One may speculate that the frequent lack of peak 2 serum components may be associated with the inability of most CF patients to produce normal mucus following respiratory infection.

Lysosomal enzyme secretion by cystic fibrosis fibroblasts is normal

Lysosomal enzyme secretion by skin fibroblasts derived from cystic fibrosis homozygotes and normal donors of the same range of age, sex and culture passage number were compared. No differences were detected between these groups in their rates of spontaneous lysosomal enzyme release. Incubation of either normal or cystic fibrosis fibroblasts with cystic fibrosis serum did not alter these secretion rates. The lysosomal enzyme secretion of both normal and cystic fibrosis fibroblasts responded similarly to incubation with NH 4Cl, monensin, A23187, trifluoperazine or Cytochalasin B. It was concluded that lysosomal enzyme hypersecretion, which has previously been suggested to occur in cystic fibrosis, is unlikely to provide a reliable diagnostic test for the disease.

NADH dehydrogenase in cystic fibrosis.

It has been claimed that the pH profile of mitochondrial NADH dehydrogenase in cultured skin fibroblasts can be used to distinguish cystic fibrosis homozygotes, heterozygotes and normal controls. This phenomenon has been examined in more readily accessible white blood cell preparations. The relative changes of activity of white cell NADH dehydrogenase in phosphate buffer at pH 8.0,8.3 and 8.6 were too small and too random to be useful in identifying the genotype of the donor.

Mucin release from rabbit tracheal epithelium in response to sera from normal and cystic fibrosis subjects.

We have developed an assay for the release of mucous glycoproteins secreted by rabbit tracheal mucosa to study the mucus-stimulating properties of cystic fibrosis (CF) serum. Glycoproteins are labeled to equilibrium with both [35]-sulfate and [3H]-glucosamine. The relative secretory rate (RSR) of these glycoproteins is assessed after incubation with 50% (v/v) CF and control sera. Serum from all sources significantly increased the RSR for incubation times ranging from 10 min to 4 h. CF serum produced a significantly greater RSR than control serum after 10 min exposure, but not after longer exposure intervals. After 10 min, RSR for [35SO4]-glycoproteins was 3.6 +/- 0.2 in the presence of sera from healthy individuals, and 2.3 +/- 0.4 in the presence of sera from control patients with lung disease. Heterozygote RSR values were usually higher than control values, but not different than CF homozygote values. The CF serum activity was reduced by freezing and thawing. Increasing the calcium content of incubation medium did not alter serum effects. Calcium inophore A23187 did not mimic CF serum activity. High molecular weight and albumin, but not IgG, fractions from Sephadex G-200 chromatography of CF and control sera generally contained high levels of mucin-release activity. No chromatographic pattern of activity was distinctive for CF.

Quantitative immunoassays for diagnosis and carrier detection in cystic fibrosis.

Quantitative immunoprecipitation and immunoradiometric assays have been developed for a protein present in the serum of cystic fibrosis homozygotes, and to a lesser extent in the serum of heterozygotes. When tested on a panel of sera from 14 cystic fibrosis patients, 29 heterozygotes and 23 controls, the immunoprecipitation assay allowed correct assignments to be made on 94% of occasions with one batch of antiserum and 95% with another. With the same panel of sera, the immunoradiometric assay allowed 94% correct assignments. It is suggested that such accuracy is the maximum that can be expected in the present state of knowledge of cystic fibrosis.

Cystic fibrosis. I. Fractionation of the mucociliary inhibitor from plasma.

A protein fraction present in low concentrations in plasma of cystic fibrosis homozygotes and heterozygotes has been identified by its biologic activity in ciliary preparations of gills from Crassostrea virginica, where it causes mucociliary inhibition. In the present study, mucociliary inhibition was shown to be associated with an IgG-rich fraction. The cystic fibrosis mucociliary inhibitor was obtained as a low molecular weight protein after dissociation from IgG in 5 M guanidinium chloride. Gel filtration of the dissociated ultrafiltrate fraction on Bio-Gel P-30 isolated the cystic fibrosis mucociliary inhibitor activity in a pool containing protein of 6,000 to 12,000 MW. The mucociliary inhibitor was heterogeneous upon isoelectric focusing, with inhibition demonstrated in a focused range of pH 4.6 to 6.1 and in a fraction focused near pH 9. Lectin-affinity chromatography of the low molecular weight proteins dissociated from the IgG fraction of a cystic fibrosis homozygote demonstrated that at least one molecular species of the mucociliary inhibitor contained a carbohydrate moiety, since inhibitory fractions were found both in the nonadsorbed fraction and in the fraction eluted with N-acetylglucosamine.

Serum concentrations of vitamin D-binding protein (group-specific component) in cystic fibrosis.

Vitamin D-binding protein (DBP) concentrations were determined in the sera of 90 cystic fibrosis homozygotes, 57 obligate heterozygotes, and 46 normal controls. Very significantly lower mean concentrations were found in the sera of CF homozygotes compared with both heterozygotes and controls (P less than 0.01, Wilcoxon Rank Sums Test). Subdivision of the samples by Gc phenotype showed that this relationship held true both in the Gc1 and Gc2-1 phenotypes. The small sample size of the Gc2 genotype makes the significance levels of limited usefulness, but the pattern of variation of DBP levels among CF homozygotes, heterozygotes, and controls was consistent with that observed for the Gc1 and Gc2-1 classes. Haptoglobin levels showed high coefficients of variation when compared among CF homozygotes, obligate heterozygotes, and controls, presumably because of nonspecific elevation in the acute-phase response. Alpha 2-macroglobulin levels were, if anything, slightly elevated in CF homozygotes compared with controls, while albumin levels showed no significant mean differences between these groups. Since the DBP concentration does not vary with age nor with levels of vitamin D and its metabolites, we interpret our results to mean that DBP levels are specifically decreased in cystic fibrosis, perhaps as the result of impaired glycosylation of the protein.