James H. Conover

Studies on ciliary dyskinesia factor in cystic fibrosis. I. Bioassay and heterozygote detection in serum.

Extract: We have modified Spocks rabbit tracheal bioassay to the extent that it is reproducible and reliable for the detection of the cystic fibrosis (CF) gene in single or double dose from serum. Three critical modifications were: (1) maintaining a 37°G temperature throughout screening and bioassay, (2) setting high standards of tissue selection to be used for bioassay, (3) quick manipulations during the bioassay. These alterations have eliminated the necessity of concentrating euglobulins from sera of CF carriers in order to detect a ciliary dyskinesia factor (CDF). In addition, the time necessary to detect a CDF response was reduced to 3 to 6 minutes in every serum sample from CF homozygotes and obligate heterozygotes. In general, sera from metachromasia negative CF affected and carrier subjects elicited a ciliotoxic response, as opposed to the more commonly observed ciliary dyskinesia seen in metachromasia positive CF individuals and their parents.Speculation: It is our hypothesis that the CDF is a normal cellular product, and that the defect in CF is in the production or release of a factor inhibiting the CDF.

Studies on Ciliary Dyskinesia Factor in Cystic Fibrosis. III. Skin Fibroblasts and Cultured Amniotic Fluid Cells

Extract: The cell-free media from cultured skin fibroblasts derived from 16 healthy subjects, 13 homozygotes, 6 obligate heterozygotes, and 1 potential heterozygote for cystic fibrosis (CF) were investigated for ciliary dyskinesia factor (CDF) by a modified rabbit tracheal bioassay. Fluid was obtained for study from cultures during growth phase, 24 and 48 hr after feeding, before confluence had been reached. A positive CDF response was demonstrated in the media of all homozygous and heterozygous for CF skin fibro-blast cultures, after incubation of the culture fluid with human immunoglobulin G (IgG). No CDF response was observed before the addition of IgG. Although there was a tendency of the cell-free media obtained from the CF homozygous fibroblast cultures to elicit a stronger CDF response than the media from heterozygous cultures, no consistent distinction between homozygous and heterozygous fibroblast cultures could be obtained. The fluid of 15 out of 16 fibroblast cultures derived from normal donors did not demonstrate CDF activity before or after the addition of IgG. The media from cultured amniotic fluid cells obtained from a pregnant woman homozygous for CF and from four pregnancies derived from heterozygous parents demonstrated CDF activity in the presence of IgG. The media of 10 out of 11 control amniotic fluid cell cultures were CDF negative. In the one CDF-positive control culture a positive CDF response was also elicited in the maternal serum. Media from CF fibroblast cultures, to which IgG had been added, lost their CDF activity after treatment with anti-IgG. The culture fluid from confluent fibroblast and amniotic fluid cell cultures of all types of subjects, including control subjects, inhibited the ciliary activity of rabbit tracheal explants without addition of IgG. No CDF activity was observed in the amniotic fluid of a fetus affected with CF. Amicon filtration experiments demonstrated that a molecule with molecular weight between 1,000 and 10,000 produced by CF fibroblast cultures causes ciliary dyskinesia in the presence of IgG. The findings suggest that cultured skin fibroblasts and amniotic fluid cells that carry the CF gene in a homozygous or heterozygous state produce a small molecule, preciliary dyskinesia factor (pre-CDF), which is activated in the presence of IgG, as tested with the rabbit bioassay. It appears that this activation of pre-CDF could be reversed by its removal from the IgG.Speculation: Cells from all individuals produce a pre-CDF, which is a normal product important for rapid changes in electrolyte flux at the cell membrane. This molecule is normally rapidly inactivated by an appropriate enzyme. The genetic defect in CF is in the reduced activity of this enzyme, which results in abnormalities at the cell membrane because of excess of pre-GDF, and, after activation of pre-CDF to CDF by IgG, in interference with bronchial ciliary function.

The Biologic Activities of Cystic Fibrosis Serum. I. The Effects of Cystic Fibrosis Sera and Calcium Ionophore A 23187 on Rabbit Tracheal Explants

Summary: An ionophore A23187-induced increase in membrane permeability to calcium ions in culture medium produced a rabbit tracheal mucociliary response indistinguishable from that caused by cystic fibrosis (CF) sera on three different occasions. Specific chelation of calcium ions with EGTA in the basal medium Eagle (BME) media with no additive or in native CF sera abolished the mucociliary disturbances in all cases. Increased membrane permeability to calcium may be important in the production of the mucociliary response by CF serum factor(s) in the tracheal assay system.Speculation: CF serum factor(s) may be acting on the cultured rabbit tracheal explant cellular membranes to produce altered permeability to ions. This alteration in membrane permeability may be promoting a loss of intracellular communication and cellular injury. Such changes on the cellular level may be related to the pathophysiology of this genetic disorder.

Studies on ciliary dyskinesia factor in cystic fibrosis. IV. Its possible identification as anaphylatoxin (C3a)-IgG complex

Abstract Presumptive evidence indicates that the substance responsible for the pathophysiologic conditions of cystic fibrosis (CF) is a complement component, C3a, also known as anaphylatoxin. The known biochemical and physiological properties of C3a, together with the behavior of this purified substance when associated with IgG in our tracheal bioassay, compare favorably with those of molecular species which we have separated from sera and cell cultures of CF homozygotes and heterozygotes. Sera from normal healthy subjects, previously inactive by bioassay for ciliary dyskinesia factor (CDF), were converted to a CDF positive state by incubation with epsilon-amino-caproic-acid (EACA). EACA is a known inhibitor of the carboxypeptidase-B-like anaphylatoxin inactivator, and is the methof of choice for accumulating anaphylatoxins in normal blood. Incubation of EACA-treated normal sera and two fresh CDF-positive CF sera with carboxypeptidase-B produced reversion in all instances to a CDF negative, normal state. It is proposed that anaphylatoxin inactivator, a carboxypeptidase-B-like enzyme, is defective or deficient in cystic fibrosis and that this deficiency is the primary gene defect.

Studies on Ciliary Dyskinesia Factor in Cystic Fibrosis. II. Short Term Leukocyte Cultures and Long Term Lymphoid Lines

Extract: The cell-free medium obtained after 48 hour culture of phytohemagglutinin (PHA)-stimulated leukocytes of three cystic fibrosis (CF) subjects and two carriers, contained a ciliary dyskinesia factor (CDF) detected by a modified rabbit tracheal bioassay. Positive CDF responses began to be observed in supernates of parallel non-stimulated cultures of leukocytes from these same CF affected and carrier individuals by 72 hours of culture. Cell-free media from leukocyte cultures of four normal donors did not produce a positive CDF response with or without PHA over a period of 6 days.The cell-free medium of long-term lymphoid lines derived from three CF affected and four CF carrier subjects demonstrated a positive CDF response, while media from similar lymphoid lines derived from normal subjects contained no CDF. Distinction between homozygous and heterozygous lymphoid lines was not always possible, although there was a tendency of the homozygous lines to give an earlier CDF response than the heterozygous ones. Addition of rabbit anti-human IgG to the CDF positive cell line supernates resulted in the elimination of their ability to elicit a positive CDF response in each instance.Speculation: CDF is a manifestation of a normal cellular product which is a small molecule bound to IgG. The resulting complex represents the CDF detected by bioassay. The defect in CF is in the production or release of a factor inhibiting the CDF.

Demonstration of human leukocyte degranulation induced by sera from homozygotes and heterozygotes for cystic fibrosis.

Extract: The ability of εamino caproic acid (EACA)-treated normal serum and of cystic fibrosis (CF)-affected and carrier sera to promote the release of lysosomal enzymes from sensitized human polymorphonuclear leukocytes (PMN) was assessed through the measurement of β-glucuronidase and myeloperoxidase acitivity after exposure of these cells to the various test sera. This study was initiated to extend the analogies between preciliary dyskinesia factor (pre-CDF), separated from the cell-free media of cultures derived from CF homozygous and heterozygous individuals, and C3a anaphylatoxin. The extent of lysosomal degranulation of human PMN exposed to fresh untreated sera of each of five controls, seven CF homozygotes, and eight heterozygotes, as expressed by the amount of β-glucuronidase released, was 7.84% (± 0.934) for control sera, 14.01% (±1.79) for CF-affected sera, and 10.61% (±1.43) for heterozygous sera. The difference between CF homozygotes and control subjects is significant (P <0.001), as is the difference between CF-affected and carrier individuals (0.001 < P < 0.005) and between control subjects and carriers (0.001 < P < 0.005), when β-glucuronidase release is measured. Analogously, values of myeloperoxidase released by the three groups studied reflect differences similar to those of β-glucuronidase. However, the differences between control subjects and CF heterozygous individuals are not significant. Treatment of these sera with 1 M EACA gave values for β-glucuronidase and myeloperoxidase release which are slightly reduced when compared with those obtained with fresh, untreated samples. EACA apparently reduces the activity of β-glucuronidase released from PMN. Amicon filtration studies of these serum samples demonstrated that degranulating ability and the presence of ciliary dyskinesia, as assessed by rabbit tracheal bioassay, are not always associated. Therefore, the relationship between pre-CDF and the degranulator activity in native CF-affected and carrier sera is unclear, in part because of the limitations inherent in the test systems employed.Speculation: The pathophysiology of CF can be explained by excessive degranulation of exocrine glandular cells, resulting in inspissation of their ducts. The finding of degranulator molecules in CF sera allows for a test of this hypothesis. The possibility exists that this degranulating activity, as well as the molecules responsible for ciliary dyskinesia, whether they are the same or different molecular species, may represent an excess of normal products. These molecules, related to C3a anaphylatoxin and/or kinins, are present in excess in CF because of the deficiency of an enzyme which normally controls their level by inactivation.

The Biologic Activities of Cystic Fibrosis Serum. II. Ultrastructural Aspects of the Effect of Cystic Fibrosis Sera and Calcium Ionophore A23187 on Rabbit Tracheal Explants

Summary: Ultrastructural and cytochemical observations indicate that both cystic fibrosis (CF) sera and calcium ionophore A23187 induce a swelling or an increase in the size and possibly the number of secondary lysosomes and an increase in mucus secretion in epithelium of the rabbit tracheal bioassay system. Extended incubation of the rabbit tracheal explants with either CF or control sera produces a cytotoxic effect on the tracheal epithelium, but only after the termination of the normal bioassay time period. Comparative ultrastructural study of the effect of both CF sera and calcium ionophore A23187 on the rabbit tracheal bioassay system indicates that increased membrane permeability to calcium may be important in the production of the ciliary dyskinesia response by CF serum factor(s) in the rabbit tracheal bioassay system.Speculation: CF serum factor(s) may be acting on the cultured rabbit tracheal explant epithelial membranes to produce altered membrane permeability to ions and to calcium in particular. This alteration in membrane function may be promoting the observed ultrastructural changes in the epithelial cells which include a swelling or increase in the number and size of secondary lysosomes and an increase in mucus secretion as well as ciliary dyskinesia. Alterations in membrane function and permeability may be related to the pathophysiology of this genetic disorder.

Persistence of Phosphoglucomutase (PGM) Polymorphism in Long-Term Lymphoid Lines

Summary Long-term lymphoid lines are useful in studying the relationship of an individuals genotype in vivo and in vitro. The persistence of the PGM1 phenotype in these established lines serves as a consistent, reliable genetic marker which can be used in a variety of studies and be related directly back to the cell line donor. This experimental advantage was heretofore not available in established heteroploid monolayer lines, or in diploid fibroblasts, which are not capable of continuous vigorous growth beyond a finite number of generations. We thank Dr. Daniel Stites for providing us with blood samples.

Separation of serum ciliary dyskinesia substances from cystic fibrosis subjects.

Purified serum immunoglobulin G (IgG), derived from eight cystic fibrosis (CF) and five carrier subjects, has been shown to be responsible for the mucociliary disturbances noted in the rabbit tracheal bioassay. A small molecular substance of less than 10,000 but greater than 1,000 daltons (by Amicon filtration) was found associated with gamma-globulin fractions isolated from sera of these same cystic patients and their parents. Once separated by PM-10 ultrafiltration, this small substance was unable to promote the ciliary dyskinesia response in eight of eight CF and five of five CF carrier individuals unless pooled purified human IgG was added. In addition, the IgG-rich fraction retained by PM-10 ultrafiltration was still able to promote the ciliary dyskinesia response in the bioassay, an event noted in our earlier work with whole serum. The size of the small serum substance and its association with IgG closely corresponds to that described for the oyster test system, as well as to that produced by cultured cells derived from homozygotes and heteroxygotes for this genetic disorder. The persistence of the ability to promote mucociliary disturbances by the IgG-rich retentate PM-10 fractions may be indicative of the ineffective molecular separation by the Amicon ultransfiltration apparatus or may represent another CF-related, IgG-associated substance not influenced by ultrafiltration. Speculation The genetic disturbance of CF can be explained by the presence of a molecule(s) which has an affinity for IgG, which in turn gives rise to the various physiologic facets of this disorder. This molecule(s) is present in CF only because of a deficiency of an enzyme which normally controls its level by inactivation.

Production of Human-Mosquito Somatic Cell Hybrids and their Response to Virus Infection

Somatic cell hybridization techniques have given cell biologists a unique opportunity to obtain specific information concerning cellular function and gene expression. Intra- and inter-species hybrids have been produced by means of biochemical selection (Littlefield, 1964), viral induction (Harris and Watkins, 1965), or a combination of these methods (Miggiano et al., 1969).