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.     

The adrenergic system in lymphocytes from children with cystic fibrosis

Summary Several in vivo and in vitro studies have suggested that children suffering from cystic fibrosis (CF) might have a general defect of beta-adrenoceptors on the cell surface which might account for an unbalanced secretory process. In order to investigate if this view holds true, we determined the beta-adrenoceptor density and affinity on lymphocytes by means of radioligand studies using 125-iodo-cyano-pindolol (125-ICYP) in 20 children with CF. Cyclic AMP (cAMP) response was also investigated after specific beta-adrenoceptor stimulation with isoprenaline (IPN) and after direct stimulation of the adenylate cyclase with forskolin in lymphocytes. Children with CF and controls have identical numbers and affinities of beta-adrenoceptors on lymphocytes. The cyclic AMP response was identical in CF- and in age-matched control children regardless whether adenylate cyclase was stimulated directly or via beta-adrenoceptors. In conclusion, the data support the view that no general adrenoceptor or adenylate cyclase defect exists in CF. As several studies have found abnormal reactions to adrenergic stimuli in CF patients, we presume that there is a defect beyond the level of adrenergic receptors and cAMP which remains to be identified.Abbreviations BS/Ly binding sites per lymphocyte - BSA bovine serum albumin - cAMP cyclic AMP - CF Cystic Fibrosis - 125-ICYP 125-iodo-cyano-pindolol - IPN isoprenaline - SEM standard error of mean     

Iontophoretic beta-adrenergic stimulation of human sweat glands: possible assay for cystic fibrosis transmembrane conductance regulator activity in vivo.

With the advent of numerous candidate drugs for therapy in cystic fibrosis (CF), there is an urgent need for easily interpretable assays for testing their therapeutic value. Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) abolished beta-adrenergic but not cholinergic sweating in CF. Therefore, the beta-adrenergic response of the sweat gland may serve both as an in vivo diagnostic tool for CF and as a quantitative assay for testing the efficacy of new drugs designed to restore CFTR function in CF. Hence, with the objective of defining optimal conditions for stimulating beta-adrenergic sweating, we have investigated the components and pharmacology of sweat secretion using cell cultures and intact sweat glands. We studied the electrical responses and ionic mechanisms involved in beta-adrenergic and cholinergic sweating. We also tested the efficacy of different beta-adrenergic agonists. Our results indicated that in normal subjects the cholinergic secretory response is mediated by activation of Ca(2+)-dependent Cl(-) conductance as well as K(+) conductances. In contrast, the beta-adrenergic secretory response is mediated exclusively by activation of a cAMP-dependent CFTR Cl(-) conductance without a concurrent activation of a K(+) conductance. Thus, the electrochemical driving forces generated by beta-adrenergic agonists are significantly smaller compared with those generated by cholinergic agonists, which in turn reflects in smaller beta-adrenergic secretory responses compared with cholinergic secretory responses. Furthermore, the beta-adrenergic agonists, isoproprenaline and salbutamol, induced sweat secretion only when applied in combination with an adenylyl cyclase activator (forskolin) or a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine, aminophylline or theophylline). We surmise that to obtain consistent beta-adrenergic sweat responses, levels of intracellular cAMP above that achievable with a beta-adrenergic agonist alone are essential. beta-Adrenergic secretion can be stimulated in vivo by concurrent iontophoresis of these drugs in normal, but not in CF, subjects.     

Suppression of CFTR premature termination codons and rescue of CFTR protein and function by the synthetic aminoglycoside NB54

Certain aminoglycosides are capable of inducing "translational readthrough" of premature termination codons (PTCs). However, toxicity and relative lack of efficacy deter treatment with clinically available aminoglycosides for genetic diseases caused by PTCs, including cystic fibrosis (CF). Using a structure-based approach, the novel aminoglycoside NB54 was developed that exhibits reduced toxicity and enhanced suppression of PTCs in cell-based reporter assays relative to gentamicin. We examined whether NB54 administration rescued CFTR protein and function in clinically relevant CF models. In a fluorescence-based halide efflux assay, NB54 partially restored halide efflux in a CF bronchial epithelial cell line (CFTR genotype W1282X/F508del), but not in a CF epithelial cell line lacking a PTC (F508del/F508del). In polarized airway epithelial cells expressing either a CFTR-W1282X or -G542X cDNA, treatment with NB54 increased stimulated short-circuit current (I (SC)) with greater efficiency than gentamicin. NB54 and gentamicin induced comparable increases in forskolin-stimulated I (SC) in primary airway epithelial cells derived from a G542X/F508del CF donor. Systemic administration of NB54 to Cftr-/- mice expressing a human CFTR-G542X transgene restored 15-17% of the average stimulated transepithelial chloride currents observed in wild-type (Cftr+/+) mice, comparable to gentamicin. NB54 exhibited reduced cellular toxicity in vitro and was tolerated at higher concentrations than gentamicin in vivo. These results provide evidence that synthetic aminoglycosides are capable of PTC suppression in relevant human CF cells and a CF animal model and support further development of these compounds as a treatment modality for genetic diseases caused by PTCs.     

Dynamics of protein and fluid secretion from the major salivary glands of rat: relevance of research findings to clinically observed defective secretion in cystic fibrosis

Although there are indications of a defect in secretion of protein from exocrine cells in cystic fibrosis (CF), this remains an aspect of CF research that has not been adequately addressed. Using salivary glands of rat as model systems, and following the effects of parasympathetic and sympathetic autonomic nerve stimulation on these glands, we demonstrate the existence of three separate pathways through which secretion of protein can be evoked from serous and mucous exocrine cells. These pathways allow the secretion of proteins from the intracellular compartments in a constitutive, intermediate or regulated manner. The primary aspects of secretory profile including concentration and the degree of hydration of secreted material differ greatly between the pathways, are cell type specific, and presumably are a direct consequence of controlled changes in the levels of second messengers induced upon stimulation of these cells. As previously published reports suggest that only the beta-adrenergic regulated pathway is affected by CF, differences between the pathways in their secretory profiles may influence the development of lung disease, through disparate disturbances in the secretion of protein and fluid from serous and mucous cells of the submucosal glands that line the bronchiolar tree in humans. We gratefully acknowledge support from The Wellcome Trust and from The European Union Biomed II Programme.     

Role of sialic acid in saliva-mediated aggregation of Pseudomonas aeruginosa isolated from cystic fibrosis patients.

The mechanism of saliva-mediated aggregation of Pseudomonas aeruginosa in subjects with and without cystic fibrosis (CF) was investigated. Virtually all saliva from CF patients that we tested strongly agglutinated the Pseudomonas cells and was heat stable to 56 degrees C, whereas saliva from subjects without CF had a decreased aggregating ability and was heat sensitive. When saliva was treated with neuraminidase and proteases, and also when P. aeruginosa cells were treated with mixed gangliosides, there was a decrease in aggregating activities. However, neither the addition of the acid-hydrolyzed ganglioside nor the treatment of the P. aeruginosa cells by sugars had any effect on subsequent aggregating activities. Therefore, the release of sialic acid by enzymatic treatments of saliva, as well as the blockage of the sialic acid-binding sites on the cell wall by mixed gangliosides, resulted in the parallel loss of saliva-mediated aggregating activity of P. aeruginosa. The level of free sialic acid released by endogenous neuraminidase was higher in the saliva from CF patients than in that from the non-CF subjects examined. The increased aggregation of P. aeruginosa mediated by saliva from patients with CF seems to be directly related to the sialic acid content present, suggesting that this acid molecule acts as the salivary receptor for P. aeruginosa.     

Role of the amiloride-sensitive epithelial Na+ channel in the pathogenesis and as a therapeutic target for cystic fibrosis lung disease

Increased airway Na⁺ absorption mediated by the amiloride-sensitive epithelial Na⁺ channel (ENaC) is a basic defect in cystic fibrosis (CF) lung disease. Cystic fibrosis is one of the most common lethal hereditary diseases and is caused by mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene. The CFTR acts as a cAMP-dependent Cl⁻ channel and regulator of ENaC, and CFTR dysfunction causes impaired Cl⁻ secretion and increased Na⁺ absorption in the airways of CF patients. Evidence from in vitro studies suggested that increased Na⁺ absorption produces airway surface liquid (ASL) volume depletion and led to the generation of transgenic mice with airway-specific overexpression of ENaC to elucidate the role of this mechanism in the in vivo pathogenesis of lung disease. Studies of the pulmonary phenotype of βENaC-overexpressing mice demonstrated that increased airway Na⁺ absorption caused ASL depletion and reduced mucus transport, producing a CF-like lung disease with airway mucus plugging, chronic airway inflammation and pulmonary mortality. Further, recent pharmacological studies demonstrated that preventive, but not late, inhibition of increased airway Na⁺ absorption with the ENaC blocker amiloride reduced morbidity and mortality in this murine model of CF lung disease. These results support a critical role of ENaC in the in vivo pathogenesis of CF lung disease and suggest that amiloride may be an effective preventive therapy for CF patients.     

Complex pattern of the myelo-monocytic differentiation antigens MRP8 and MRP14 during chronic airway inflammation.

One of the characteristics of cystic fibrosis is the presence of the so-called cystic fibrosis antigen in the plasma of patients. The CF-antigen has been shown to consist of the two calcium-binding proteins MRP8 and MRP14. In the present study we investigate first whether elevated plasma titers of MRP8 and MRP14 are linked to the primary defect of CF or are rather a result of chronic airway inflammation; and second, whether the known complexes of these proteins may have in vivo relevance during inflammation. By employing the ELISA technique we measured MRP8 and MRP14 levels in the plasma of patients suffering from CF or nonspecific chronic bronchitis (CB) and of healthy controls, in sputum of CF and CB patients, and in saliva of CF patients and healthy controls, respectively. We found elevated plasma concentrations of both proteins in CF and CB patients compared to healthy controls. Levels correlated significantly with systemic and local signs of disease activity (i.e. c-reactive protein (CRP), daily sputum production). MRP8 and MRP14 both were found in high amounts at similar concentrations in sputum of CF and CB patients and, to a lesser extent, in saliva of CF patients and healthy donors. After covalent cross-linking at least three different complexes composed of MRP8 and MRP14 with approximate molecular weights of about 25, 35 and 48 kDa were detected in all samples. From this we conclude that the elevated plasma levels of MRP8 and MRP14 in CF and CB are the result of inflammatory processes. Further, possible biological functions of these proteins seem to be associated with complexed forms of MRP8 and MRP14 rather than with individual proteins.     

Activation by extracellular nucleotides of chloride secretion in the airway epithelia of patients with cystic fibrosis.

BACKGROUND. Cystic fibrosis is characterized by abnormal electrolyte transport across the epithelia of the airways. In particular, there is excessive sodium absorption and deficient chloride secretion. Drugs that block excessive sodium absorption may provide clinical benefit in cystic fibrosis, but there are no available therapeutic agents to improve chloride secretion. In vitro studies in cultured human-airway epithelia indicate that triphosphate nucleotides (ATP and UTP) induce chloride secretion through apical-membrane purinergic receptors. METHODS. We tested the ability of nucleotides to induce chloride secretion in vivo in 9 normal subjects and 12 patients with cystic fibrosis by measuring responses of nasal transepithelial potential difference (PD) to superfusion of nucleotides. Changes in transepithelial bioelectric properties and the permeability of the apical membrane to chloride in response to extracellular (apical) UTP were determined with ion-selective microelectrodes in cultured nasal epithelia. RESULTS. ATP and UTP induced chloride secretion in vivo in both groups. At their maximal effective concentrations of 10(-4) M, ATP and UTP were more effective chloride secretagogues in the patients with cystic fibrosis (mean [+/- SE] change in PD, -19.8 +/- 1.4 mV and -15.0 +/- 1.7 mV, respectively) than in the normal subjects (-6.9 +/- 0.6 mV and -8.1 +/- 0.9 mV, respectively). Microelectrode studies established that extracellular UTP stimulated a larger increase in PD and chloride secretory current in epithelial cells from patients with cystic fibrosis than in cells from normal subjects, by actions localized to the apical membrane. CONCLUSIONS. Extracellular nucleotides are effective in vivo chloride secretagogues in the nasal epithelia of patients with cystic fibrosis. The equipotency of ATP and UTP suggests that the effect is mediated by P2 nucleotide receptors. Selected nucleotides, such as UTP or nucleotide analogues, should be investigated as therapeutic agents for lung disease in cystic fibrosis.     

Thixotropic solutions enhance viral-mediated gene transfer to airway epithelia

Adenovirus-mediated gene transfer to airway epithelia is inefficient in part because its receptor is absent on the apical surface of the airways. Targeting adenovirus to other receptors, increasing the viral concentration, and even prolonging the incubation time with adenovirus vectors can partially overcome the lack of receptors and facilitate gene transfer. Unfortunately, mucociliary clearance would prevent prolonged incubation time in vivo. Thixotropic solutions (TS) are gels that upon a vigorous shearing force reversibly become liquid. We hypothesized that formulating recombinant adenoviruses in TS would decrease virus clearance and thus enhance gene transfer to the airway epithelia. We found that clearance of virus-sized fluorescent beads by human airway epithelia in vitro and by monkey trachea in vivo were markedly decreased when the beads were formulated in TS compared with phosphate-buffered saline (PBS). Adenovirus formulated in TS significantly increased adenovirus-mediated gene transfer of a reporter gene in human airway epithelia in vitro and in murine airway epithelia in vivo. Furthermore, an adenovirus encoding the cystic fibrosis transmembrane regulator (CFTR) gene (AdCFTR) formulated in TS was more efficient in correcting the chloride transport defect in cystic fibrosis airway epithelia than AdCFTR formulated in PBS. These data indicate a novel strategy to augment the efficiency of gene transfer to the airways that may be applicable to a number of different gene transfer vectors and could be of value in gene transfer to cystic fibrosis (CF) airway epithelia in vivo.     

Validation of nasal potential difference measurements in gut-corrected CF knockout mice

Attempts at correcting the nasal potential difference (PD) in cystic fibrosis (CF) mice have long been used in preclinical gene and small molecule therapy development. However, in general, CF mice suffer from intestinal disease, are runted, and have high mortality rates; they are therefore difficult to work with, especially if large numbers are required. Because of this, large-scale PD studies in CF mice have not been performed. Working with CF mice has become substantially easier after the generation of the gut-corrected CF-knockout mouse. Fatty acid-binding promoter (FABp)-mediated expression of CFTR in the gut, but not the airways, prevents the intestinal disease of the CF knockout mouse. This model has given us the unique opportunity to systematically study PDs in large numbers of CF mice. The nose, but not the lungs, of these animals mimic the bioelectric defect seen in humans. We have therefore assessed the bioelectrics of the respiratory epithelium comparing FABp-CF and wild-type mice. The large body of data gathered in CF and wild-type mice allowed us, for the first time, to establish power calculations that should inform sample sizes required in gene and small molecule therapy development. In addition, we address the important issues of intra-animal variability as well as intra- and inter-operator variability for scoring the traces, and the effect of age and sex on nasal PD in CF mice. These data should allow a more informed use of CF animals in future studies.     

Effects of chronic furosemide treatment on rat exocrine glands

It has been suggested that a defective chloride transport is the primary cellular basis for the disease cystic fibrosis (CF). Therefore, the effects of chronic furosemide treatment on the structure and function of rat exocrine glands were investigated. X-ray microanalysis of the submandibular gland showed an increase in the cellular Ca and Mg concentrations, and a decrease in the cellular Cl concentration. Transmission electron microscopy showed intracellular accumulation of mucus and the presence of mucus in acinar and ductal lumina. The volume of saliva secreted by the submandibular gland after pilocarpine stimulation was markedly reduced in furosemide-treated animals; the salivary concentrations of Na and Ca were higher, and that of K was lower, than in control animals. The protein concentration in submandibular saliva was not significantly affected. The response of the submandibular gland to isoproterenol stimulation was reduced in furosemide-treated animals. In the parotid gland, chronic furosemide treatment caused an accumulation of immature zymogen granules in the acinar cells and a decrease in the cellular Cl concentration. In the pancreas, the acinar lumen was dilated and completely filled with secretory material, and the acinar cells contained less Na and somewhat less Cl than in control animals. The chronically furosemide-treated rat shows a number of parallels with other animal models for CF, in particular the chronically reserpinized rat. There is also agreement with the human disease itself.     

Respiratory syncytial virus infection in a murine model of cystic fibrosis

Viral respiratory infections play an important role in the development and progression of pulmonary disease in cystic fibrosis (CF). The CF mouse model provides a tool to examine the relationship between the cystic fibrosis transmembrane conductance regulator (CFTR) defect and lung disease. This work investigates the cellular response to a common viral pathogen, respiratory syncytial virus (RSV) in the lung of CF mice. RSV was administered by intranasal inoculation of CFTR(tm1Unc)-Tg(FABPCFTR)1Jaw/J (CFTR-/-) and control mice. At day 5 post infection, viral titers, bronchoalveolar fluid nitrate levels (BALF) cell and differential counts, histology and studies on airway mechanics were performed. CFTR-/- mice had an impaired ability to clear RSV. This was associated with an exaggerated inflammatory response (increased lymphocytes and neutrophils) in BALF of RSV-infected CFTR-/- mice and a decreased ability to generate nitric oxide (NO) (measured as BAL nitrate). Lung histopathology of RSV-infected CFTR-/- mice demonstrated increased inflammation compared to RSV (-) CFTR-/- and control mice (regardless of RSV treatment). The airway response to methacholine was increased by RSV infection in CF mice when compared to controls. The CFTR-/- mouse exhibits an aberrant response to RSV infection. This model should be useful in providing further mechanistic information on the biology of respiratory viruses in mammalian models, and provide new insights into the pathogenesis of airway inflammation in patients with CF.     

The Oral Immune System: Dynamics of Salivary Immunoglobulin Production in the Inbred Mouse

In the oral cavity, salivary immunoglobulins (Igs) are the principal mediators of specific immunity. Using carbachol to stimulate saliva flow, we investigated, in a kinetic study, individual variations in salivary Ig concentrations in 23 adult BALB/c mice using an enzyme-linked immuno-sorbant assay (ELISA). It appeared that salivary Ig concentrations are highly variable in individual mice (IgA: 3-81 μg/mL; IgG: 0-2.9 μg/mL; IgM: 0.002-0.14 μg/mL). In individual mice stimulated at different times over a 3 week period there are considerable variations both in salivary Ig concentrations and in their respective ratio. Broad variations were also found in the levels of specific IgA and IgG antibodies to three indigenous oral murine bacteria. Present data thus indicate that among genetically identical mice of the same age and sex, sharing identical diet, there is considerable heterogeneity in salivary Igs. As this heterogeneity was mimicked at the cellular level in major and minor salivary gland-associated B-cells, it appears that antibody dynamics in the oral cavity could reflect the adaptive capacity of the oral immune system to local antigenic challenge.     

Fenretinide corrects newly found ceramide deficiency in cystic fibrosis

Chronic and persistent lung infections cause the majority of morbidity and mortality in patients with cystic fibrosis (CF). Galactosyl ceramide has been previously shown to be involved in Pseudomonas internalization. Therefore, we assessed ceramide levels in the plasma of patients with CF and compared them to healthy volunteers using high-performance liquid chromatography followed by mass spectrometry. Our results demonstrate that patients with CF display significantly lower levels of several ceramide sphingolipid species, specifically C14:0, C20:1, C22:0, C22:1, and C24:0 ceramides, and dihydroxy ceramide (DHC16:0). We report that Cftr-knockout mice display diminished ceramide levels in CF-related organs (lung, pancreas, ileum, and plasma) compared with their littermate controls. Since it has been previously reported that in vitro treatment with fenretinide induced ceramide in neuroblastoma cell lines, we decided to test this drug in vivo using our Cftr-knockout mice in an attempt to correct this newly identified defect in ceramide levels. We demonstrate that treatment with fenretinide is able to increase ceramide concentrations in CF-related organs. We further assessed the biological effect of fenretinide on the ability of Cftr-knockout mice to combat lung infection with P. aeruginosa. Our data show dramatic improvement in the ability of Cftr-knockout mice to control P. aeruginosa infection. Overall, these findings not only document a novel deficiency in several ceramide species in patients with CF, but also demonstrate a pharmacologic means to correct this defect in Cftr-knockout mice. Our data provide a strong rationale for clinical intervention that may benefit patients with CF suffering from CF lung disease.     

Non-CFTR chloride channels likely contribute to secretion in the murine small intestine

While most cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-knockout animals die due to intestinal obstruction before or at the time of weaning, a subpopulation of these animals are long living and exhibit a milder phenotype. The decreased severity of intestinal disease in these mildly affected CF mice is related to the expression of non-CFTR genetic modifiers. The identity of these genetic modifiers is not known, but we hypothesize that they may complement CFTR function as a chloride channel in this tissue. To assess the contribution of non-CFTR chloride channels to chloride secretion across the small intestine of CF mice with mild disease, we measured the basal transepithelial potential difference across this tissue as well as the secretory response to agonists of the cAMP and the calcium-mediated signaling pathways. Chloride secretion across the small intestine of mildly affected CF mice was not stimulated by forskolin or by carbachol. The absence of CFTR is thus not compensated by the activity of a distinct, cAMP- or calcium-activated chloride channel at the apical surface of the intestinal epithelium. On the other hand, a basal chloride secretion across the intestinal epithelium was present in these animals, and we hypothesize that this activity may be linked to improved survival of these animals.     

Mucosal immunity to Pseudomonas aeruginosa alginate in cystic fibrosis.

Patients with cystic fibrosis commonly acquire chronic pulmonary infection with alginate-producing Pseudomonas aeruginosa. The infection remains localized at the mucosal surfaces of the airways. Using enzyme-linked immunosorbent assays immunoglobulin concentrations and titers of specific antibodies to purified P. aeruginosa alginate and to P. aeruginosa sonicated antigens were measured in tears, saliva, sputum and serum. CF patients had significantly higher concentrations of IgG, IgA and SIgA in serum and saliva than controls. They also had significantly higher levels of specific antibodies to alginate and sonicated antigen in secretions and serum. Local production of IgA, IgG and IgM antibodies to P. aeruginosa was demonstrated. Only a minor proportion of specific IgA antibodies were present as secretory IgA in tears, saliva and sputum. The ratio of alginate-specific SIgA to specific monomeric IgA in sputum was significantly lower than the similar ratio in saliva, whereas the same ratio for specific P. aeruginosa sonicate antigens was found in saliva and sputum.     

Pancreatic polypeptide in cystic fibrosis

Patients with cystic fibrosis (CF) and their normal siblings and parents were studied for protein meal-stimulated pancreatic polypeptide (PP) secretion. Patients with CF who had exocrine pancreatic insufficiency did not respond to a protein meal, whereas patients with CF and normal pancreatic function presented relatively well-preserved basal and elevated postmeal PP levels. The siblings responded with relatively lower PP levels compared with the control subjects. Plasma insulin levels were also investigated, and showed sluggish initial-phase but relatively well-preserved insulin responses in the patients with CF. Immunocytochemical and morphometric studies were made of pancreata obtained at autopsy from patients with CF. In the patients younger than 7 years of age, well-preserved islet tissue was disclosed by islet-area morphometry with normal and/or below-normal PP cell counts, whereas patients older than 9 years had relatively less insulin-containing islet tissue and scanty PP cells. Absent PP secretory response in patients with CF who had exocrine pancreatic insufficiency may suggest defects in the PP secretory mechanism. Abnormal PP secretion may be used as an indirect index of pancreatic damage in CF.     

Whole, submandibular, and parotid saliva-mediated aggregation of Pseudomonas aeruginosa in cystic fibrosis.

The aggregation of mucoid and nonmucoid Pseudomonas aeruginosa by submandibular, parotid, and whole saliva from patients with cystic fibrosis (CF) and non-CF subjects was investigated. There were significant differences (P less than 0.01) in aggregation of mucoid and nonmucoid variants of P. aeruginosa by submandibular and whole saliva from CF patients and non-CF subjects. However, the differences in the parotid secretion were not as pronounced. Patients with CF who were colonized with P. aeruginosa demonstrated a significantly higher (P less than 0.05) percent aggregation of the mucoid variants by the submandibular secretion and of both mucoid and nonmucoid variants by whole saliva, compared with corresponding secretions from patients with CF not colonized with this pathogen. The parotid saliva aggregation activity was not markedly different for the two groups with CF. From patients with CF, whole saliva demonstrated a higher percent P. aeruginosa aggregation than did the submandibular saliva. In non-CF subjects, however, the percent aggregation of P. aeruginosa by submandibular saliva was higher than that by whole saliva. Our results indicate that the sero-mucous products of the submandibular gland have a more significant role in P. aeruginosa aggregation than the serous secreting parotid cells and that the submandibular secretion is possibly responsible for the differences in oral colonization by this pathogen in subjects with and without CF.     

Cystic fibrosis: Studies with the oyster ciliary assay

Bioassays using ciliary systems have detected a factor or factors in cystic fibrosis (CF) sera and tissue culture medium derived from CF cells. The typical shortcomings of an assay measuring biological activity have been studied, and the means to overcome the weaknesses of the oyster gill cilia assay have been established. The presence of the cystic fibrosis mucociliary inhibitor (CFMI) in experimental fractions may be determined by accepting data from only those assays in which authentic CF and normal (non-CF) fractions give defined reactions, by measuring the reaction of each sample at least three times, and by examining each experimental sample at a protein concentration greater than the minimum established in this study. The relative concentrations of the CFMI present in the first steps of purification of serum and medium have been calculated in terms of units of inhibition. Generally, the units of inhibition present in serum and medium fractions from heterozygotes are close to one-half of that in fractions from homozygous sources. Analogous fractions concentrated from a normal (non-CF) source never inhibited mucociliary activity, even when tested at nearly 100 times the CF concentration. Ciliary assays utilizing oyster gills are essential for monitoring fractionation procedures aimed at purifying the CFMI, and have been shown to be capable and reliable enough to do so.