Cystic Fibrosis Saliva Research Articles

A mathematical model of ENaC and Slc26a6 regulation by CFTR in salivary gland ducts.

Cystic fibrosis (CF) is a genetic disease caused by the mutations of cystic fibrosis transmembrane conductance regulator (CFTR), the cystic fibrosis transmembrane conductance regulator gene. Cftr is a critical ion channel expressed in the apical membrane of mouse salivary gland striated duct cells. Although Cftr is primarily a Cl- channel, its knockout leads to higher salivary Cl- and Na+ concentrations and lower pH. Mouse experiments show that the activation of Cftr upregulates epithelial Na+ channel (ENaC) protein expression level and Slc26a6 (a 1Cl-:2[Formula: see text] exchanger of the solute carrier family) activity. Experimentally, it is difficult to predict how much the coregulation effects of CFTR contribute to the abnormal Na+, Cl-, and [Formula: see text] concentrations and pH in CF saliva. To address this question, we construct a wild-type mouse salivary gland model and simulate CFTR knockout by altering the expression levels of CFTR, ENaC, and Slc26a6. By reproducing the in vivo and ex vivo final saliva measurements from wild-type and CFTR knockout animals, we obtain computational evidence that ENaC and Slc26a6 activities are downregulated in CFTR knockout in salivary glands.NEW & NOTEWORTHY This paper describes a salivary gland mathematical model simulating the ion exchange between saliva and the salivary gland duct epithelium. The novelty lies in the implementation of CFTR regulating ENaC and Slc26a6 in a CFTR knockout gland. By reproducing the experimental saliva measurements in wild-type and CFTR knockout glands, the model shows that CFTR regulates ENaC and Slc26a6 anion exchanger in salivary glands. The method could be used to understand the various cystic fibrosis phenotypes.

Altered Carbohydrate Composition of Salivary Mucins from Patients with Cystic Fibrosis and the Adhesion of Pseudomonas aeruginosa

We compared the chemical composition of salivary mucin glycopeptides from cystic fibrosis (CF) and from non-CF subjects and the adhesion of Pseudomonas aeruginosa to these different salivary glycopeptides. Three pools of CF saliva, four pools of non-CF saliva, one individual CF saliva, and one individual non-CF saliva were studied. The soluble fraction of the saliva was treated with pronase, and gel filtration was performed to obtain high and low molecular mass salivary mucin glycopeptides. The yield of total glycopeptides was significantly higher from CF than from non-CF saliva. Furthermore, the chemical composition revealed a significantly higher sialic acid content in CF than in non-CF mucin glycopeptides, and higher sulfate and fucose content in CF than in non-CF high molecular mass glycopeptides. We studied the adhesion of a nonmucoid strain of P. aeruginosa (1244), its nonpiliated isogenic derivative, and a mucoid strain (M35) to salivary mucin glycopeptides from patients with CF and from non-CF subjects. The three strains bound significantly more to the CF salivary glycopeptides than to the corresponding non-CF salivary glycopeptides. The nonpiliated isogenic mutant of P. aeruginosa 1244 also bound to CF salivary glycopeptides, suggesting that the adhesion of P. aeruginosa could involve nonpilus adhesions. Furthermore, neuraminidase treatment of CF glycopeptides decreased the adhesion of P. aeruginosa 1244. Altogether these results suggested that differences in mucins may in part explain the specificity of P. aeruginosa for CF.

Role of cations and IgA in saliva-mediated aggregation of Pseudomonas aeruginosa in cystic fibrosis patients.

Oral colonization by Pseudomonas aeruginosa possibly precedes the pulmonary infection process in cystic fibrosis (CF) patients. As bacterial aggregates may play a role in establishment of pulmonary infections, involvement of IgA and cations in CF patient saliva-mediated aggregation of P. aeruginosa was investigated. For colonized patients, P. aeruginosa aggregation correlated with bacterial-specific and total salivary IgA. Cation or IgA depletion reduced P. aeruginosa aggregation by saliva from all patients. However, if cations were removed before IgA, and saliva was then reconstituted with calcium, only colonized patient saliva showed reduced aggregation. Aggregation by IgA-depleted saliva was augmented by reconstituting with original IgA. CF patient saliva-mediated aggregation of P. aeruginosa thus is cation-dependent and enhanced by bacterial-specific IgA. Characterizing the interactions among bacterial aggregating factor(s), cations, and antibodies in CF saliva will help clarify the link between P. aeruginosa oral colonization and pulmonary infections in CF patients.

Physicochemical Characterization and Biological Activity of Intrinsic Factor in Cystic Fibrosis

Absorption of crystalline labeled cobalamin is strongly decreased in cases of cystic fibrosis. In order to determine if this is due to an alteration or a lack of activation of intrinsic factor by proteases, the physicochemical properties and biological activity of intrinsic factor have been studied. Intrinsic factor was purified 800-fold from stimulated gastric juice of cystic fibrosis patients with a yield of 64.2%. Cystic fibrosis intrinsic factor had an estimated Mr of 57,000 in SDS-polyacrylamide gel electrophoresis. Its carbohydrate content resembled that of normal human intrinsic factor, except that the ratio fucose/sialic acid was higher (6.1 and 1.6, respectively) and that the content in N-acetylgalactosamine was decreased. The same alterations in carbohydrate composition were observed for Hc purified from cystic fibrosis saliva. Purified intrinsic factor from cystic fibrosis gastric juice was biologically active in vitro in the presence of ileal solubilized receptor as well as in vivo (Schilling test). The fate of iodinated cystic fibrosis intrinsic factor in guinea pig ileum studied by high-resolution radioautography was similar to that of normal intrinsic factor. In conclusion, despite modifications of the carbohydrate content of the molecule, the biological activity of intrinsic factor is not altered in cases of cystic fibrosis. The malassimilation of crystalline cobalamin observed in cystic fibrosis is due to a mechanism independent from intrinsic factor secretion.

Physicochemical Characterization and Biological Activity of Intrinsic Factor in Cystic Fibrosis

Absorption of crystalline labeled cobalamin is strongly decreased in cases of cystic fibrosis. In order to determine if this is due to an alteration or a lack of activation of intrinsic factor by proteases, the physicochemical properties and biological activity of intrinsic factor have been studied. Intrinsic factor was purified 800‐fold from stimulated gastric juice of cystic fibrosis patients with a yield of 64.2%. Cystic fibrosis intrinsic factor had an estimated Mr of 57,000 in SDS‐polyacrylamide gel electrophoresis. Its carbohydrate content resembled that of normal human intrinsic factor, except that the ratio fucose/sialic acid was higher (6.1 and 1.6, respectively) and that the content in N‐acetylgalactosamine was decreased. The same alterations in carbohydrate composition were observed for He purified from cystic fibrosis saliva. Purified intrinsic factor from cystic fibrosis gastric juice was biologically active in vitro in the presence of ileal solubilized receptor as well as in vivo (Schilling test). The fate of iodinated cystic fibrosis intrinsic factor in guinea pig ileum studied by high‐resolution radioautography was similar to that of normal intrinsic factor. In conclusion, despite modifications of the carbohydrate content of the molecule, the biological activity of intrinsic factor is not altered in cases of cystic fibrosis. The malassimilation of crystalline cobalamin observed in cystic fibrosis is due to a mechanism independent from intrinsic factor secretion.

Ribonuclease in different types of saliva from cystic fibrosis patients.

The flow rate, the activity of ribonuclease (RNAase) and the concentration of protein were determined in whole saliva and in parotid and submandibular saliva from patients with cystic fibrosis (CF) and from healthy controls, both before and after stimulation of the salivary secretion. Lower flow rates were found in all types of saliva from CF-patients than in control saliva. Increased activity of RNAase was found in CF saliva, both before and after stimulation of the secretion. The concentration of protein was also increased, but to a lesser degree. No correlation was found between either RNAase activity or protein concentration and severity of the disease or age of the children. It is therefore unlikely that these disturbances are secondary to progression of the disease. Considerable variations in RNAase activity and protein concentration were observed, especially within the CF group, and therefore despite the significant increase in these variables in CF their estimation is of limited diagnostic value.

Effect of human saliva and serum on ion transport by dog tracheal mucosa. Comparison of normal subjects with cystic fibrosis patients.

It has been proposed that cystic fibrosis (CF) saliva or serum contains factors that alter ion transport in various tissues. Since CF frequently affects the lungs, and disturbances in epithelial ion transport has been proposed to contribute to the pathophysiology of pulmonary disease in CF, we investigated whether saliva or serum from CF patients could alter ion transport across airways epithelium. Canine tracheal mucosae were mounted in lucite chamber and perfused on both sides with Krebs-Henseleit solution at 37 degrees C and pH 7.4. Unidirectional fluxes of 22Na and 36Cl were measured in pairs of mucosal tissues under short circuit conditions. Saliva from 5 CF patients stimulated short circuit current (SCC) by 33 +/- 8 microA/cm2 and net Cl secretion by 1.10 +/- 0.33 muEq/cm2 X h (mean +/- SE; p less than 0.05). No change occurred in net Na absorption. Saliva from normal subjects raised SCC by 34 +/- 7 microA/cm2 and increased net Cl secretion by 1.29 +/- 0.46 muEq/cm2 X h (n = 9; p less than 0.02). Serum from both CF patients and control subjects briefly stimulated SCC. Exposure of tracheal mucosa to normal as well as CF saliva, such as by aspiration, or to serum, such as by transudation, stimulates Cl secretion, and therefore water secretion into airway lumen. There appears to be no difference in the effect exerted by CF saliva or serum and that of normal subjects. These findings argue against the presence of a specific circulating or secreted CF factor that could alter ion transport across respiratory epithelia in a way that might contribute to lung disease in CF.

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.

Specificity of an isolated salivary factor material to cystic fibrosis.

An oyster gill ciliostatic factor material has been isolated from the saliva of patients with cystic fibrosis (CF) by utilizing its ability to bind to alpha-amylase. It was quantitatively assayed by its ability to reversibly inhibit rabbit muscle glycogen debranching enzyme. The specificity of this CF factor material was investigated by comparing activities from the saliva of CF homozygotes (patients) varying in age, sex, and the severity of the disease; CF obligate heterozygotes (carriers); normal control subjects who had no family history of CF; non-CF asthmatic and allergic bronchitis patients; non-CF immunologically deficient patients with chronic respiratory problems; non-CF juvenile diabetic patients; non-CF pancreatic insufficiency patients; non-CF patients with obstructive liver cirrhosis; and non-CF patients with ectodermal dysplasia. The results show that the CF factor material isolated from CF saliva is specific to subjects with cystic fibrosis and is not associated with similar non-CE chronic disease states, nor is it produced as a result of an organ pathology associated with CF. There was no correlation between the amount of factor present in an individual CF homozygote sample and the severity of the disease. In the case of both the CF homozygote and heterozygote samples, there was also no correlation in either age or sex and the amount of factor present. The degree of inhibition produced by CF homozygotes compared to CF heterozygotes is characteristic of the autosomal recessive mode of inheritance of CF. This finding appears to associate the isolated CF factor material with the affected CF gene and suggests that the factor material is related in some way to the genetic lesion in CF.

Saliva from patients with cystic fibrosis inhibits amiloride-sensitive sodium transport.

A factor is present in the saliva and sweat from patients with cystic fibrosis (CF) which inhibits the reabsorption of sodium in the ducts of the salivary and sweat glands. Inasmuch as the physiology of sodium absorption is similar in salivary ducts and the distal colon, we have examined the sensitivity of the sodium absorption in the rat colon to saliva from CF patients. Sodium absorption by the rat colon, estimated as the shortcircuit current, was inhibited by saliva from both patients with CF and normal volunteers. However, only with CF saliva was the inhibition consistently proportional to the saliva concentration. Furthermore, the inhibitory activity of CF saliva was greater than the inhibition observed with saliva from age- and sex-matched controls (percentage of inhibition: CF = 20.1 +/- 2.2, and controls = 14.9 +/- 2.1; P < 0.02), and the inhibition of the colonic shortcircuit current was proportional to the activity measured by the ductal retrograde perfusion assay with the rat parotid gland (linear correlation coefficient = 0.634; P < 0.005). This latter assay is an accepted assay for CF factor activity. We conclude that the CF factor present in saliva probably interacts in a reversible manner with the amiloride-sensitive sodium transport system which is present in all sodium scavenging epithelia. The rat colon is a promising assay system for CF factor activity because the electrical measurements permit a rapid quantitative estimate of activity and a single piece of tissue can be used to measure the activity of several saliva samples.

Differences between Cystic Fibrosis and Normal Saliva α-Amylase as a Function of Age and Sex

Summary: α-Amylase activity and protein concentration of cystic fibrosis (CF), obligate heterozygote (H), and normal (N) saliva have been evaluated with respect to genotype, age, sex and clinical status. “Factors” present in CF saliva had no detectable effect on salivary amylase activity under reaction conditions optimized for both competitive and noncompetitive inhibition. Tris [tris-(hydroxymethyl)aminomethane] inhibited amylase activity with a Ki = 59 ± 10 mM, but CF “factors” had no detectable effect on either substrate or Tris binding to α-amylase.α-Amylase concentration and protein content of CF saliva had bimodal distributions. Thirty-six percent of the CF patients were found to be amylase hypersecretors, with amylase concentration 3 or more SD above the N mean. Amylase hypersecretion was marginally correlated with both genetic factors (with parent values, P = 0.075) and clinical status (with Shwachman scores, P = 0.068). All the hypersecretors were in the 14–24 year age group. Amylase concentration for this age was significantly higher in CF samples (mean ± SD), 82 ± 37 starch units/ml (n = 12), than in N samples, 33 ± 17 starch units/ml (n = 13), as measured by the Student f-test (P < 0.001). Salivary protein content of 14− to 24-year-old CF males was elevated compared to all other groups [1.57 ± 0.76 (n = 5) vs. 0.63 ± 0.47 (n = 20); Student t-test, P < 0.005]. Mean specific activity of male saliva dropped sharply as age increased from 10–15 years, from over 200 to less than 75 starch units/mg protein. Mean normal female specific activity values were essentially constant [97 ± 50 (n = 18) starch units/mg protein] from 5–30 years of age. CF male specific activity values decreased similarly to the N male values from preadolescence to adolescence, but the mean CF female values peaked later, at 17 years, with a maximum of 171 ± 58 (n = 4). This is significantly higher than all the other groups in the 16− to 20-year age range: 63 ± 30 (n = 9), P < 0.005. These data emphasize the need for age-and sex-matched controls in all studies of salivary composition.Speculation: We were unable to detect any effect of a salivary CF factor on the enzymatic activity of α-amylase. Hypersecretion of salivary α-amylase was observed in 36% of CF patients. This may reflect alterations in secretory function basic to CF, or be secondary to the disease process and result from decreased secretion from the mucous salivary glands or deterioration of pancreatic function.

Effect of saliva from cystic fibrosis patients and from normal subjects on red blood cell sodium transport.

Saliva, whether taken from patients with cystic fibrosis or from normal subjects, caused an increase in red blood cell Na+ efflux (in the presence or absence of ouabain) of 19-29% as compared with non-saliva controls. However, there was no significant difference between the effects of cystic fibrosis saliva and normal saliva.

Quantitation of submandibular proteins resolved from normal individuals and children with cystic fibrosis

Submandibular secretions collected from children with cystic fibrosis (CF) showed increased protein concentration (milligrams/milliliter) and increased amylase specific activity (units/milligram of protein) relative to normal secretions. These differences between normal (N) and CF secretions were as follows: protein, 1.25 ± 0.51 (N), 1.75 ± 0.35 (CF) ( P < 0.02); and amylase, 58 ± 18 (N), 80 ± 19 (CF) ( P < 0.001). To determine the basis for elevated protein in CF saliva, several major proteins resolved by polyacrylamide disc gel electrophoresis were quantitated by densitometry. These included four phosphoproteins (PP), serum albumin, an acid phosphatase-containing fraction, amylase, and an unidentified protein referred to as PI-7.1. Together, these proteins comprise greater than 75% of the total protein in the secretion. Differences in individual protein concentrations (milligrams/milliliter) resolved from normal and CF secretions, respectively, were as follows: PP 2, 0.02 ± 0.01, 0.03 ± 0.02 (NS, not significant); PP 3, 0.06 ± 0.04, 0.05 ± 0.03 (NS); acid phosphatase fraction, 0.06 ± 0.04, 0.12 ± 0.07 ( P < 0.05); amylase, 0.09 ± 0.04, 0.27 ± 0.16 ( P < 0.01); and pI-7.1, 0.04 ± 0.02, 0.13 ± 0.08 ( P < 0.02). Amylase, the most significant contributor to the elevated protein, comprised 26% of the total protein of normal secretions and 38% of the total protein of CF secretions. Thus, our results do not support the concept of a generalized increase in all organic components in CF submandibular secretions but, rather, increases in specific proteins, namely amylase, component pI-7.1, and an acid phosphatase-containing fraction.

The SIgA system and hypersensitivity in patients with cystic fibrosis.

A number of investigations have been used for the first time to examine the secretory IgA (SIgA) system in different body fluids from patients with cystic fibrosis (CF). Free J-chain was detected in all the sputum specimens examined. The isolated free secretory component (SC) from CF sputum differed in electrophoretic mobility from the SC isolated from normal human colostrum. In addition the free SC from some CF saliva formed precipitin lines of partial identity with normal human saliva or colostrum. A higher proportion of CF sera (33%) than of normal sera (10%) contained free SC. These investigations suggest that there may be some defect in the synthesis and/or the assembly of the SIgA immunoglobulins, which if confirmed, may help to explain the impaired Type I and Type III allergic manifestations in patients with CF.

Parotid gland function in children with cystic fibrosis and child control subjects.

Parotid saliva was collected from 22 children with cystic fibrosis (CF) and 21 control children. Stimulation was by 0.1 ml 5% citric acid on the tongue every 15 sec over a period of 10 min, and samples were collected for 2-min intervals. Stimulated saliva showed a lag period of low amylase release, and an activated period of higher amylase release. The major difference between CF and child control parotid saliva was in the elevated calcium concentrations in CF subjects. Flow rate and amylase, although higher in the CF group, were not raised to a statistically significant level. There were higher mean values for sodium and inorganic phosphate in CF children but statistical significance was minimal. Potassium values were almost identical in CF and control saliva. In the activated parotid saliva samples of both control and CF subjects there were positive correlations between flow rate and calcium, flow rate and sodium, anylase and calcium, and sodium and calcium, and negative correlations between sodium and potassium. The parotid function test has no value as a diagnostic test for cystic fibrosis, as even calcium values show too much overlap with control saliva to be of diagnostic worth.

Polyamines in cystic fibrosis.

Polyamine concentrations have been measured in blood samples from a series of cystic fibrosis (CF) patients and control subjects. Analyses were made by a fluorometric method, in which the amines were reacted with dansyl chloride and separated by thin layer chromatography on Kieselguhr. There was no significant difference between the polyamine concentrations of blood samples from CF patients and control subjects. Also, the spermidine-spermine ratio was similar in both groups. Polyamines were also analyzed in exocrine fluids, and although none could be detected in either CF or control sweat or saliva, significant amounts of spermine and spermidine were present in all samples of bile and duodenal fluids. It thus seems likely that the biliary route is a significant pathway in the human for excretion of polyamines. The observation that polyamines are excreted in considerable concentrations via the bile in children may be of interest in view of the reports of elevated urinary polyamine concentrations in neoplastic states. Since measurable amounts of spermine and spermidine were detected in all six bile preparations available for this study, analysis of these substances in bile may be of benefit in particular cases.

Hypersecretion of zymogen granules in the pathogenesis of cystic fibrosis.

In the submandibular saliva of 10 cystic fibrosis subjects and 10 controls the turbidity and elevated calcium, protein, and amylase concentrations of the cystic fibrosis secretions, and precipitation of calcium and phosphate in a ratio consistent with hydroxyapatite have been confirmed. By electron microscopy the centrifuged deposits of the cystic fibrosis saliva were seen to be composed predominantly of round or oval subcellular corpuscles. By comparison with submandibular gland, these corpuscles have been identified as inclusion bodies (spherules) from within zymogen granules. Hydroxyapatite crystals formed on standing in the cystic fibrosis saliva. Polyacrylamide gel disc electrophoresis of the cystic fibrosis centrifuged deposits showed five bands, one of which, band 4, was more prominent in the deposit than in the supernatant gels. Comparisons have been made between these results and other studies and have shown (1) elevated calcium and protein in cystic fibrosis exocrine secretions; (2) simultaneous secretion of calcium and enzymes from salivary glands, stomach, and pancreas; and (3) increased salivary secretion of calcium and protein in response to parasympathomimetic and sympathomimetic drugs. Hypersecretion of calcium-containing zymogen granules is postulated as the cause of obstruction in cystic fibrosis.