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Crassostrea virginica, the oyster native to the gulf coast, has served as a source of ciliated epithelium for studies on the inhibitory factor in cystic fibrosis. In these studies, protein molecules with biological activity, obtained from serum, urine, saliva, and cells from cystic fibrosis homozygotes and heterozygotes, were detected, purified, and characterized. Properties of these factors are similar to those detected by other ciliated systems in mussel gills and rabbit trachea. Although none of the ciliated assays in their present stage of development offer a reliable means for heterozygote screening or for prenatal diagnosis, they represent powerful tools for characterizing biologically active molecules related to cystic fibrosis. The importance of purifying and characterizing the various cystic fibrosis factors described by several laboratories is based on evidence that their biological activities observed in vitro mimic some of the expressions of the disease observed in vivo and that the concentration of the mucociliary inhibitor in serum and fibroblast medium preparations from cystic fibrosis heterozygotes is approximately one-half of that in serum and fibroblast medium preparations from homozygotes.

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.

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.

The cystic fibrosis ciliary inhibitor (CFCI) has been partially purified from serum and plasma of cystic fibrosis (CF) homozygotes and heterozygotes, and from media of cultured fibroblasts derived from cystic fibrosis genotypes. Characterization and comparison of fractions containing the CFCI were carried out by polyacrylamide gel electrophoresis. Gel electrophoresis confirmed previous molecular weight estimations of 4,500 to 11,000 for the CFCI and provided an estimate of the number of proteins present in the fractions. Low molecular weight proteins from serum and media were combined with IgG preparations. No specific binding to IgG by the media fraction containing the CFCI could be demonstrated by the techniques employed. There was decreased binding of the low molecular weight serum fraction containing CFCI to native IgG molecules from cystic fibrosis patients as compared to IgG from normal individuals. However, IgG from CF individuals demonstrated increased binding of the cfci-containing low molecular weight serum fraction after gel filtration in the presence of guanidinium chloride. This suggests: 1) that very low concentrations of CFCI are present in media fractions; and 2) that native CF IgG cannot bind the low molecular weight CFCI fractions to the same degree as native IgG from normals or CF IgG that has been dissociated from non-covalently bound components.