The History of Cystic Fibrosis by Dr James Littlewood OBE

Edited and produced by Daniel Peckham

 

Abnormal secretions and more “CF factors” - but little understanding of the pathogenesis

 

The scientists working on CF in the Seventies explored a wide variety of possible abnormalities which could be related to the eventual pathophysiology of the condition. These are confusing for the non-scientist but are reviewed in great detail by Boat and Dearborn in Chapter 3 of Lynn Taussig’s textbook (Cystic Fibrosis. Thieme-Stratton Inc. New York. 1984). However, by the end of the decade scientists were no nearer identifying the basic defect. Although, as observed by Boat and Dearborn, one of the most consistent observations related to altered water and/or electrolyte content of the secretions – an area where important progress would be made during the Eighties.

 

By the late Seventies three abnormal “CF factors” had been recognised in the serum of people with CF and their presence had been the subject a great deal of research and speculation - they were the Spock factor (Spock et al, 1967 below), the Mangos factor (Mangos et al, 1967 below) and the Lieberman factor (Lieberman et al, 1979 below). Spock reported ciliary dyskinesia of fresh water mussels, when observed under the microscope, when the serum of people with CF was added to the preparation. Much effort went into attempting to identify this “CF factor”. However, despite a great deal of research neither isoelectric focusing (Wilson et al, 1977 below) nor using gels from isoelectric focusing to raise antibodies (Manson & Brock, 1980 below) permitted accurate identification of the Spock factor.

 

Mangos and co-workers had reported a factor in saliva that inhibited resorption of sodium from the parotid ducts of rats (Mangos, 1967 above). Lieberman identified a lectin-like factor in the blood of people with CF that disappeared with antibiotic treatment and was postulated to stimulate excessive mucus production, reacting with its glycoprotein to cause its precipitation and increased viscosity (Lieberman et al, 1979 below). Dr Lynne Reid, former chairman of UK Research and Medical Advisory Committee, who had moved from London to Harvard, reviewed her extensive research into bronchial mucus and its glycoproteins in CF but was unable to draw any definite conclusions, which had a bearing on the aetiology (Reid, 1981 below).

 

In the second half of the decade the possibility of a deficiency of a proteolytic enzyme received considerable attention from Rao and Nadler (1972 and subsequently). Some of the varied reports of CF factors and wide variety of other abnormalities, many of which other workers were unable to repeat, are described and unfortunately indicate no one was anywhere near understanding the basic defect.

 

As Dr David Lawson had observed some years earlier - "there is as yet no wisp of smoke over the horizon of our knowledge" "we must deal with the problem as it is".

1972 Danes BS, Bearn AG. Oyster ciliary inhibition by cystic fibrosis culture medium. J Exp Med 1972; 136:1313-1317. [PubMed]
More data appeared on the cilia inhibition test considered to be due to the presence of ‘CF factor’ in patients’ serum.

 

1972 Schmoyer IR, Brooks SP, Fischer JF. Isolation and characterisation of a ciliary dyskinesia factor from cystic fibrosis heterozygous serum. Life Sci 1972; 11:1037. [PubMed]
This was the first isoelectric study to identify the ciliary dyskinesia factor that migrated as a single band - isoelectric point 8.54. Unfortunately the complexity of protein bands from serum made it impossible to use this method as a single step purification of the CF factor.

 

1972 Rao GJ, Nadler HL. Deficiency of trypsin-like activity in saliva of patients with cystic fibrosis. J Pediatr 1972; 80:573. [PubMed]
This finding raised the possibility of an enzyme deficiency of a proteolytic enzyme in people with cystic fibrosis. Plasma of both patients and heterozygotes also showed the deficiency of tryptic-like activity. The authors presented evidence that the enzymatic activity that hydrolyses a-N-benzoyl-L-arginine ethyl ester was markedly diminished in saliva of patients and less so in parents. Also there was reduced capacity of CF plasma activated with chloroform-ellajic acid to hydrolyse a-N-(p-toluenesulphonyl)-L-arginine methyl ester (Rao GJ et al. Science 1972; 177:619). Loss of activity was in esterase and corresponded to missing bands on isolelectric focussing (Rao GJ& Nadler HL. Pediatr Res 1974; 8:684-686.). Also the proteolytic nature of the missing activity was demonstrated (Rao GJ & Nadler HL. Pediatr Res 1975; 9:739-741.[PubMed]). Others confirmed these findings. Later Rao & Nadler reported similar results with the substrate 4-methyl-umbellifrylguanidine-benzoate (MGUB) using CF plasma (Rao GJ et al. Enzyme 1978; 23:314319. [PubMed]). Also there was decreased enzyme activity in amniotic fluid where the fetus had CF (Nadler & Walsh. Pediatrics 1980; 66:690-692.[PubMed]); but there were too many false positives and negatives for this test to be used in routine antenatal diagnosis.

 

Unfortunately studies in at least five other laboratories failed to confirm these findings relating to a proteolytic enzyme deficiency. (This account is based on pp 50-51 of Taussig’s Cystic Fibrosis by Boat & Dearborn which provides a very readable account of the research at the time)

 

1973 Wilson GB, Jahn TL, Fonseca JR. Demonstration of serum protein differences in cystic fibrosis by isolelectric focusing in thin-layer polyacrylamide gels. Clin Chem Acta 1973; 49:79. [PubMed]
Isoelectric focusing on whole serum confirmed there was a band at 8.41 in CF but not in non-CF serum.

 

1974 Conover JH, Conod EJ, Hirschorn K. Studies on ciliary dyskinesia factor in cystic fibrosis. IV: Its possible identification as anaphylatoxin (C3a)-IgG complex. Life Sci 1974; 14:253. [PubMed]
There was active release of lysosomal enzymes from granulocytes by CF serum. The authors queried whether there was a basic defect of an enzyme which normally inactivates a family of specific proteins including CF factors. This suggestion was supported by Nadler (1975).

 

1976 Holsi P, Erickson RP, Vogt E. Prospects of prenatal diagnosis of cystic fibrosis: induction of biochemical abnormalities in fibroblasts from patients with cystic fibrosis by urinary glycoproteins. Biochem Bioph Res Co 1977; 73:209.
Suggested leakage of lysosomal enzymes was occurring resulting in increased intracellular alkaline phosphatase. This could be induced in amniotic fluid cells by Tamm-Horsfall protein (a urinary glycoprotein). Seven fold inductions could discriminate between heterozygotes and homozygotes. Unfortunately this was another finding that others could not confirm (Riordan JR et al. CF Club abstracts 1978. p86; Aitken & Hoogeveen J Med Genet 1980; 17:187).

 

It was hoped that the test could predict a CF fetus but it did not stand up to reliability and the basis of the reaction was never explained according to David Brock (1993).

 

1976 Wilson GB, Fudenberg HH. Studies on cystic fibrosis using isoelectric focusing. II. Demonstration of deficient proteolytic cleavage of alpha2-macroglobulin in cystic fibrosis plasma. Pediatr Res 1976; 10:87-96. [PubMed]
The first report of abnormal interactions between proteases and alpha 2-macroglobulin which could preclude protection against protease activity that recognises small peptides. This could lead to an inability to degrade small peptides some of which may have biological activity and act as “CF Factors”.


A number of workers were unable to confirm these findings. So deficiency of protease activity or alpha 2-macroglobulin alterations was not convincingly related to CF pathophysiology. However, Wilson published more work in this area (Wilson et al,1977 below).

 

1977 Wilson GB, Monsher MT, Fudenberg HH. Studies of cystic fibrosis using isoelectric focusing. III. Correlation between cystic fibrosis protein and ciliary dyskinesia activity in serum shown by modified rabbit tracheal bioassay. Pediatr Res 1977; 11:143-146.[PubMed]
Using a modified rabbit tracheal bioassay the ciliary dyskinesia factor (CDF) could be detected in the sera of all 31 CF homozygotes or obligate heterozygotes whereas 13 of 14 normal control sera were non-reactive. These results were considered to confirm the association of CDF with cystic fibrosis; although the serum from some patients with bronchial asthma also caused ciliary dyskinesia. Isoelectric focusing showed that the presence or absence of CF protein corresponded with that of dyskinesia activity in all sera tested except for the active samples from seven asthma patients, which were negative for CF protein.

 

The authors suggested that CF protein and ciliary dyskinesia factor may be identical or closely related markers for the CF gene, and suggested that the activity detected by their rabbit tracheal bioassay in sera from patients with asthma and other diseases probably was caused by a substance different from a CF-specific ciliary dyskinesia factor.
Unfortunately attempts of other workers to repeat these findings met with variable success. The last publication on the subject from Wilson in 1984 indicated that heparinised plasma may be a more satisfactory fluid with which to work (Wilson GB et al. Clin Genet 1984; 26:331-338.[PubMed).

 

1978 Breslow JL, Epstein J, Fontaine JH, Forbes GB. Enhanced dexamethasone resistance in cystic fibrosis cells: potential use for heterozygote detection and prenatal diagnosis. Science 1978; 201: 180. [PubMed]
Epstein and colleagues reported CF cells had increased resistance to both ouabain (1977) and steroids and later cyclic AMP (1978) and a number of other drugs. Disagreement came from Kurz et al who could not confirm the findings (Science 1979; 206:1317) and also Liedtke et al (NEJM 1981; 304:974).

 

Despite their initial encouraging results in eight patients with CF, later these authors were unable to confirm the suggested defect in sodium transport in the CF fibroblasts (Breslow JL & McPherson N Eng J Med 1981; 305:98).

 

1979 Lieberman J, Costea NV, Jakulis VJ, Kaneshiro W. Detection of a lectin in the blood of cystic fibrosis homozygotes and heterozygotes. Clin Res 1979; 27: 507A . Trans Assoc Am Physicians 92; 121. [PubMed]
Jack Lieberman of Los Angeles published a number of papers around this time on a lectin that had similar properties to the CF protein but its presence was not confirmed by di Sant’Agnese et al, (Monogr Pediatr 1981; 14:1). Lieberman stated, in a presentation in 1981 that 25 years of study had led to the discovery of a “lectin-like activity” in the blood of all CF homozygotes and 99% of heterozygotes (“Discovery of a CF lectin: climax of 25 years of research in cystic fibrosis”. In: 1000 years of Cystic Fibrosis. Warren Warwick, (Ed). pp 49-57) . The lectin was removed by high doses of intravenous antibiotics within three days. Lieberman postulated that "the lectin stimulates excessive mucus production reacting with the mucous glycoprotein to cause its precipitation and increased mucous viscosity”.

The significance of the lectin was never thoroughly validated and the few subsequent publications concerned the presence of the lectin in the urine as a diagnostic test (Lieberman J, Schwartzman MN, 1990. [PubMed]) and finally the presence of the lectin in amniotic fluid for antenatal diagnosis (Lieberman J. 1991). [PubMed]

Fig. 1: David Brock

1979 Brock DJ, Hayward C. Methylumbelliferyl-guanidinobenzoate reactive proteases and prenatal diagnosis of cystic fibrosis. Lancet 1979; i: 1245-1246. [PubMed]
The titration of trypsin like proteases in cell free amniotic fluid against an artificial substrate, 4methylumbelliferyl-guanidinobezoate which had been proposed as a means of antenatal diagnosis by Nadler et al (Lancet 1980; ii: 96-97; Nadler HL, Walsh MMJ. Pediatrics 1980; 66:690-692.[PubMed] ) looked hopeful but could not be reproduced in other centres resulting in an unacceptable number of false negative and false positive results (Tummler B et al. Clin Chem Acta 1982; 125:219-232.[PubMed]).

 

Professor David Brock (1936-2004) (figure 1) of Edinburgh was a distinguished molecular biologist and pioneered antenatal screening for various conditions – notably spina bifida and later cystic fibrosis. He discovered the association of high alphfetoprotein in the amniotic fluid of unborn fetuses with spina bifida. He was a prolific writer and editor. As a result of his work in the field both ante-natal diagnosis and prenatal CF screening and diagnosis were offered to families in Scotland – one of the few areas offering this service (Mennie ME et al.1992 below; Livingstone J et al. 1994 below). The prenatal screening service was subsequently withdrawn.

 

1979 Feigal RJ, Shapiro BL. Altered intracellular calcium in fibroblasts from patients with cystic fibrosis. Pediatr Res 1979; 13:764. [PubMed]
Skin fibroblasts from patients with CF, obligate heterozygotes and age- and sex-matched controls were used in matched pair experiments measuring 45Ca exchange into and efflux from the cells over time. CF cell lines and heterozygote cell lines exhibit increased 45Ca exchange when compared with their respective controls; thought likely attributable to an altered capacity of one or more of the intracellular Ca sequestering organelles.


The finding of an altered Ca pool size in both CF and particularly heterozygote cells suggested that altered Ca metabolism was related to the basic gene defect in CF. The increased intracellular Ca was due to increased uptake by the mitochondria. The authors speculated that the high calcium levels may be related to increased viscosity of the secretions (Feigal RJ, Shapiro BL. Nature 1979; 278:276-277.[PubMed]). .