- Email: [email protected]
Developments in Cystic Fibrosis Research
307
Because most of the children born with serious cardiac malformations who do not survive die in the first few weeks or months of life, open-heart surgery in babies has not surprisingly become the highpriority technical challenge. In older children -the natural survivors-surgical treatment tends to be less urgent. Stimulated by the remarkable results from BARRATT-BOYES and his colleagues,! who reported that 33 of 37 infants under 10 kg. (25 of them aged eight days to twelve months) had survived intracardiac surgery under deep hypothermia with surface cooling and limited cardiopulmonary bypass, many centres are now
pursuing
a
much
more
aggressive
of surgery in infancy. 2-4,19, 20 The mortality is still somewhat high (up to 30% or more, and worst under the age of three months), but it will undoubtedly improve as it has for older children and adults during the past ten or fifteen years. The arguments for and against infant surgery are already beginning. Most babies with Fallot’s tetralogy are in no great danger in the neonatal period. Why take unnecessary risks when there is neither urgency nor even need to do so ?, say the conservatives: because, even if life is not threatened, early correction allows normal physical development throughout childhood and relieves the adverse effect of a severely handicapped child on the family situation, say the more adventurous. Obviously, if early correction is to become standard practice, it will have to achieve a lower mortality than either correction at a later stage or the combined mortality of early palliation followed by correction at a later stage. No judgment can yet be made; but if all the skills and resources can be concentrated in a relatively few specialist centres, so that intracardiac repair in babies does not become the next status symbol in cardiac surgery, there seems little doubt about the eventual outcome.
policy
Developments in Cystic Fibrosis Research ALTHOUGH cystic fibrosis (c.F.) has long been known to arise from the defect of an autosomal recessive gene (or genes), the identity of the defective gene(s) remains a complete mystery. Nor can any biochemical abnormality be regarded as primary, though ion transport, glycoprotein structure, and membrane permeability have all been under suspicion. A possible lead was the discovery, some years ago, of a humoral factor in cystics and heterozygotes which altered the motility of rabbit tracheal cilia and also inhibited the resorption of sodium in Breckenridge, I. M., Oelert, H., Graham, G. R., Stark, J., Waterston, D. J., Bonham-Carter, R. E. J. thorac. cardiovasc. Surg. 1973, 65, 58. 20. Malm, J. ibid. p. 64. 19.
in retrograde fashion.1,2 this factor was hampered by the nonquantitative nature of the ciliary effect and the technical difficulties of sodium-transport assay; but cilia from the gills of oysters and fresh-water mussels give With these tissues the a more quantitative assay. test measures cessation of ciliary motion (whereas the rabbit tracheal assay measures ciliary asynchrony); but seasonal variations in response are a difficulty.33 Despite the problems, information is emerging which may eventually lead to an understanding of the rat
parotid glands perfused
Work
on
pathogenesis ofc.F. Early attempts at fractionation on gel filtration columnssuggested that the anticiliary factor is adsorbed to several serum-proteins. BOWMAN’S group reported a consistent association of the factor with IgG-containing fractions4 and ScHMOYER et al.,5 by isoelectric focusing, showed that anticiliary activity is associated with several proteins of high isoelectric point.5 No pure sample has yet been obtained, but the factor is probably a low-molecularweight protein which tends to piggy-back " on other proteins, especially IgG during fractionation. CoNOVER et al. have now modified the original SPOCK assay for anticiliary activity and claim that their method reliably distinguishes normal control serum from that of heterozygotes and c.F. patients. (Since the test does not distinguish clearly between the heterozygote and homozygote, it is unlikely to be useful in antenatal screening.) In their investigation, all c.F. and heterozygote sera were positive, while 27 of the 29 normal subjects tested were negative.6 The same workers7 have observed anticiliary activity in the medium of lymphoid cell-lines cultured from c.F. patients and heterozygotes but not in that "
of cells cultured from control lines. Addition of the mitogen phytohxmagglutinin accelerated the rate of appearance of anticiliary factor in the c.F. and heterozygote lines, but control cell-lines remained negative. Addition of anti-human-IgG antiserum to the medium removed the anticiliary factor-which supports BOWMAN’S suggestion4 that the factor is bound to immunoglobulins in solution. These data are in general agreement with an extensive study by DANES et al.,8-10 who detected anticiliary activity, Spock, A., Heick, H. M. C., Cross, H., Logan, W. S. Pediat. Res. 1967, 1, 173. 2. Mangos, J., McSherry, N. ibid. 1967, 2, 378. 3. Besley, G. T., Patrick, A. D., Norman, A. P. J. med. Genet. 1969, 6, 278. 4. Bowman, B. H., Lockhart, L. H., McCombs, M. L. Science, 1970, 167, 871. 5. Schmoyer, I. R., Fischer, J. F., Brooks, S. P. Biochem. biophys. Res. Commun. 1972, 46, 1923. 6. Conover, J. H., Bonforte, R. J., Hathaway, P., Paciuc, S., Conod, E. J., Hirschhorn, K., Kopel, F. B. Pediat. Res. 1973, 7, 220. 7. Conover, J. H., Beratis, N. G., Conod, E. J., Ainbender, E., Hirschhorn, K. ibid. p. 224. 8. Danes, B. S., Beam, A. G. J. exp. Med. 1972, 136, 1313. 9. Bowman, B. H., Barnett, D. R., Matalon, R., Danes, B. S., Beam, A. G. Proc. natn. Acad. Sci. U.S.A. 1973, 70, 548. 10. Danes, B. S., Litwin, S. D., Hütteroth, T. H., Cleve, H., Bearn, A. G. J. exp. Med. 1973, 137, 1538. 1.
308
measured with the oyster-gill assay, in the serum of most but not all c.F. homozygotes and heterozygotes; anticiliary activity was present also in the medium of skin-fibroblast cultures from c.F. patients and heterozygotes, but not from normal controls. When c.F. cell culture was carried out in a protein-free medium, the anticiliary activity appeared to be associated with a low-molecular-weight, negatively charged molecule which contained no uronic acid, and was heat and pH labile. The factor was seen to bind IgG (seemingly to the heavy chains) and &bgr;microglobulin, but not IgM, IgA, or IgD. The presence of anticiliary factor in such diverse tissues as lymphocytes and fibroblasts is additional evidence that cystic fibrosis may not be simply a disorder of the exocrine glands. More likely, the factor is a normal cellular or extracellular constituent which is overproduced or ineffectively catabolised in c.F. An important question is: Does the factor modify exocrine function, giving rise to the symptoms, or is it just a secondary manifestation of the disease ? A potentially relevant experiment has lately been reported by DOGGETT et al. 11 Prompted by an earlier observation that heparin inhibits the anticiliary activities of c.F. serum and saliva, these workers gave intravenous heparin continuously to c.F. patients, whereupon there was a striking decrease in the oyster-cilia inhibitory activity of the parotid saliva and also a decrease in serum-amylase. Unfortunately, it is not yet known whether heparin affects the parotid secretion or serum-amylase in normal individuals. The same workers show that when the ciliostatic activity of c.F. saliva is inhibited by precipitation with heparin, amylase but not IgG or IgA is found in the precipitate, suggesting that in solution amylase is co-precipitated with the factor. One hopes that, eventually, a pure, well-characterised substance will be isolated for tests on exocrine tissue.
Although the work on anticiliary factor has given much valuable information, this has not been the only area of fruitful inquiry. Several groups have hypothesised that the basic defect is in the movement of ions and water through the exocrine glands.12,13 Calcium ions, in particular, have been implicated. Calcium concentrations in exocrine secretions are known to be raised in C.F.,14,15 and lately BLOMFIELD et al.16 have proposed that the increase in parotidsaliva calcium is due to association of extra calcium with the secretory granule. Increased calcium-binding
has also been reported 17 but is more In general, efforts to demonstrate doubt.1s open defective transport activity in c.F. tissues have given inconsistent results. Defective erythrocyte ion-pump
by
to
activities have been reported by some groups but not by others. BENKE et al.19 found no difference in the transport activities of c.F. and normal cultured fibroblasts. 19 Inhibition of c.F. and heterozygote serum on sugar uptake by rat jejunal tissue has been reported,2O but TAUSSIG and GARDNER 21 could not reproduce it. ARVANITAKIS et al.22now claim thatC.F. serum consistently inhibited sugar uptake into rat enterocytes-an effect which was eliminated by a diamine-oxidase preparation, suggesting that substances such as the polyamines (putrescine, spermine, spermidine, &c.) might be involved. One of these, spermidine, was indeed increased in the blood of c.F. patients and heterozygotes. The possible involvement of polyamines in c.F. is a new finding, and one of great potential. Polyamines are known to be associated with growth, membrane stability, and macromolecular biosynthesis. They are present in high concentrations in almost all living cells. But, to explain the sugar-transport-inhibition findings of ARVANITAKIS et al., one must postulate that the serum factor is some unknown metabolite of the polyamines, since these substances themselves are confined to the cellular fraction of blood. The relation of the serum sugar-transport inhibition to anticiliary activity is unknown. As more insight is gained into the biochemical background of cystic fibrosis, the probability increases that the disease is genetically heterogeneous. DANES and BEARN 23 were the first to express this concept, when they described two groups, one positive and one negative for cultured-cell metachromasia, who appeared to breed true in families. More lately, CoNOVER et al. 24 have shown that these two populations are segregated also on the basis of serum-hexosaminidase levels. C.F. patients and heterozygotes who give a positive metachromasia test have low levels of the enzyme and predominantly the B isoenzyme, while the group with a negative metachromasia test have high serum-enzyme levels and predominantly the A isoenzyme. c.F. may therefore not be a single entity but rather a group of closely related genetic abnormalities with similar pathological consequences. One hopes that from these varied laboratory investigations a unified theory of pathogenesis will emerge. 17.
Doggett, R. G., Harrison, F. M., Patrick, T. A. Nature New Biol. 1973, 243, 249. 12. Johansen, P. G., Anderson, C. M., Hadorn, B. Lancet, 1968, i, 455. 13. Gibson, L. E., Matthews, W. J., Minihan, P. T. ibid. 1970, ii, 189. 14. Chernick, W. S., Barbero, G. J., Parkins, F. M. J. Pediat. 1961, 59, 11.
890.
Mandel, I. D., Kutscher, A., Denning, C. R., Thomson, R. H., Zagarelli, E. V. Am. J. Dis. Child. 1967, 113, 431. 16. Blomfield, J., Warton, K. L., Brown, J. M. Archs Dis. Childh. 1973, 48, 267. 15.
c.F. serum
18. 19. 20. 21. 22. 23. 24.
Fitzpatrick, D. F., Landon, E. J., James, V. Nature New Biol. 1973, 235, 173. Smith, Q. T., Shapiro, B. L., Hamilton, M. J., Warwick, W. J. ibid. 1972, 240, 56. Benke, P. J., Erbstoeszer, M., Pitot, H. C. Lancet, 1972, i, 182. Brown, G. A., Oshin, A., Goodchild, M. C., Anderson, C. M. ibid. 1971, ii, 639. Taussig, L. M., Gardner, J. D. ibid. 1972, i, 1367. Arvanitakis, S. N., Mangos, J. A., McSherry, N. R., Rennert, O. M., Lapointe, D. Pediat. Res. 1973, 7, 336 (abstr.). Danes, B. S., Beam, A. G. J. exp. Med. 1969, 129, 775. Conover, J. H., Conod, E., Hirschhorn, K. Lancet, 1973, i, 1122
Because most of the children born with serious cardiac malformations who do not survive die in the first few weeks or months of life, open-heart surgery in babies has not surprisingly become the highpriority technical challenge. In older children -the natural survivors-surgical treatment tends to be less urgent. Stimulated by the remarkable results from BARRATT-BOYES and his colleagues,! who reported that 33 of 37 infants under 10 kg. (25 of them aged eight days to twelve months) had survived intracardiac surgery under deep hypothermia with surface cooling and limited cardiopulmonary bypass, many centres are now
pursuing
a
much
more
aggressive
of surgery in infancy. 2-4,19, 20 The mortality is still somewhat high (up to 30% or more, and worst under the age of three months), but it will undoubtedly improve as it has for older children and adults during the past ten or fifteen years. The arguments for and against infant surgery are already beginning. Most babies with Fallot’s tetralogy are in no great danger in the neonatal period. Why take unnecessary risks when there is neither urgency nor even need to do so ?, say the conservatives: because, even if life is not threatened, early correction allows normal physical development throughout childhood and relieves the adverse effect of a severely handicapped child on the family situation, say the more adventurous. Obviously, if early correction is to become standard practice, it will have to achieve a lower mortality than either correction at a later stage or the combined mortality of early palliation followed by correction at a later stage. No judgment can yet be made; but if all the skills and resources can be concentrated in a relatively few specialist centres, so that intracardiac repair in babies does not become the next status symbol in cardiac surgery, there seems little doubt about the eventual outcome.
policy
Developments in Cystic Fibrosis Research ALTHOUGH cystic fibrosis (c.F.) has long been known to arise from the defect of an autosomal recessive gene (or genes), the identity of the defective gene(s) remains a complete mystery. Nor can any biochemical abnormality be regarded as primary, though ion transport, glycoprotein structure, and membrane permeability have all been under suspicion. A possible lead was the discovery, some years ago, of a humoral factor in cystics and heterozygotes which altered the motility of rabbit tracheal cilia and also inhibited the resorption of sodium in Breckenridge, I. M., Oelert, H., Graham, G. R., Stark, J., Waterston, D. J., Bonham-Carter, R. E. J. thorac. cardiovasc. Surg. 1973, 65, 58. 20. Malm, J. ibid. p. 64. 19.
in retrograde fashion.1,2 this factor was hampered by the nonquantitative nature of the ciliary effect and the technical difficulties of sodium-transport assay; but cilia from the gills of oysters and fresh-water mussels give With these tissues the a more quantitative assay. test measures cessation of ciliary motion (whereas the rabbit tracheal assay measures ciliary asynchrony); but seasonal variations in response are a difficulty.33 Despite the problems, information is emerging which may eventually lead to an understanding of the rat
parotid glands perfused
Work
on
pathogenesis ofc.F. Early attempts at fractionation on gel filtration columnssuggested that the anticiliary factor is adsorbed to several serum-proteins. BOWMAN’S group reported a consistent association of the factor with IgG-containing fractions4 and ScHMOYER et al.,5 by isoelectric focusing, showed that anticiliary activity is associated with several proteins of high isoelectric point.5 No pure sample has yet been obtained, but the factor is probably a low-molecularweight protein which tends to piggy-back " on other proteins, especially IgG during fractionation. CoNOVER et al. have now modified the original SPOCK assay for anticiliary activity and claim that their method reliably distinguishes normal control serum from that of heterozygotes and c.F. patients. (Since the test does not distinguish clearly between the heterozygote and homozygote, it is unlikely to be useful in antenatal screening.) In their investigation, all c.F. and heterozygote sera were positive, while 27 of the 29 normal subjects tested were negative.6 The same workers7 have observed anticiliary activity in the medium of lymphoid cell-lines cultured from c.F. patients and heterozygotes but not in that "
of cells cultured from control lines. Addition of the mitogen phytohxmagglutinin accelerated the rate of appearance of anticiliary factor in the c.F. and heterozygote lines, but control cell-lines remained negative. Addition of anti-human-IgG antiserum to the medium removed the anticiliary factor-which supports BOWMAN’S suggestion4 that the factor is bound to immunoglobulins in solution. These data are in general agreement with an extensive study by DANES et al.,8-10 who detected anticiliary activity, Spock, A., Heick, H. M. C., Cross, H., Logan, W. S. Pediat. Res. 1967, 1, 173. 2. Mangos, J., McSherry, N. ibid. 1967, 2, 378. 3. Besley, G. T., Patrick, A. D., Norman, A. P. J. med. Genet. 1969, 6, 278. 4. Bowman, B. H., Lockhart, L. H., McCombs, M. L. Science, 1970, 167, 871. 5. Schmoyer, I. R., Fischer, J. F., Brooks, S. P. Biochem. biophys. Res. Commun. 1972, 46, 1923. 6. Conover, J. H., Bonforte, R. J., Hathaway, P., Paciuc, S., Conod, E. J., Hirschhorn, K., Kopel, F. B. Pediat. Res. 1973, 7, 220. 7. Conover, J. H., Beratis, N. G., Conod, E. J., Ainbender, E., Hirschhorn, K. ibid. p. 224. 8. Danes, B. S., Beam, A. G. J. exp. Med. 1972, 136, 1313. 9. Bowman, B. H., Barnett, D. R., Matalon, R., Danes, B. S., Beam, A. G. Proc. natn. Acad. Sci. U.S.A. 1973, 70, 548. 10. Danes, B. S., Litwin, S. D., Hütteroth, T. H., Cleve, H., Bearn, A. G. J. exp. Med. 1973, 137, 1538. 1.
308
measured with the oyster-gill assay, in the serum of most but not all c.F. homozygotes and heterozygotes; anticiliary activity was present also in the medium of skin-fibroblast cultures from c.F. patients and heterozygotes, but not from normal controls. When c.F. cell culture was carried out in a protein-free medium, the anticiliary activity appeared to be associated with a low-molecular-weight, negatively charged molecule which contained no uronic acid, and was heat and pH labile. The factor was seen to bind IgG (seemingly to the heavy chains) and &bgr;microglobulin, but not IgM, IgA, or IgD. The presence of anticiliary factor in such diverse tissues as lymphocytes and fibroblasts is additional evidence that cystic fibrosis may not be simply a disorder of the exocrine glands. More likely, the factor is a normal cellular or extracellular constituent which is overproduced or ineffectively catabolised in c.F. An important question is: Does the factor modify exocrine function, giving rise to the symptoms, or is it just a secondary manifestation of the disease ? A potentially relevant experiment has lately been reported by DOGGETT et al. 11 Prompted by an earlier observation that heparin inhibits the anticiliary activities of c.F. serum and saliva, these workers gave intravenous heparin continuously to c.F. patients, whereupon there was a striking decrease in the oyster-cilia inhibitory activity of the parotid saliva and also a decrease in serum-amylase. Unfortunately, it is not yet known whether heparin affects the parotid secretion or serum-amylase in normal individuals. The same workers show that when the ciliostatic activity of c.F. saliva is inhibited by precipitation with heparin, amylase but not IgG or IgA is found in the precipitate, suggesting that in solution amylase is co-precipitated with the factor. One hopes that, eventually, a pure, well-characterised substance will be isolated for tests on exocrine tissue.
Although the work on anticiliary factor has given much valuable information, this has not been the only area of fruitful inquiry. Several groups have hypothesised that the basic defect is in the movement of ions and water through the exocrine glands.12,13 Calcium ions, in particular, have been implicated. Calcium concentrations in exocrine secretions are known to be raised in C.F.,14,15 and lately BLOMFIELD et al.16 have proposed that the increase in parotidsaliva calcium is due to association of extra calcium with the secretory granule. Increased calcium-binding
has also been reported 17 but is more In general, efforts to demonstrate doubt.1s open defective transport activity in c.F. tissues have given inconsistent results. Defective erythrocyte ion-pump
by
to
activities have been reported by some groups but not by others. BENKE et al.19 found no difference in the transport activities of c.F. and normal cultured fibroblasts. 19 Inhibition of c.F. and heterozygote serum on sugar uptake by rat jejunal tissue has been reported,2O but TAUSSIG and GARDNER 21 could not reproduce it. ARVANITAKIS et al.22now claim thatC.F. serum consistently inhibited sugar uptake into rat enterocytes-an effect which was eliminated by a diamine-oxidase preparation, suggesting that substances such as the polyamines (putrescine, spermine, spermidine, &c.) might be involved. One of these, spermidine, was indeed increased in the blood of c.F. patients and heterozygotes. The possible involvement of polyamines in c.F. is a new finding, and one of great potential. Polyamines are known to be associated with growth, membrane stability, and macromolecular biosynthesis. They are present in high concentrations in almost all living cells. But, to explain the sugar-transport-inhibition findings of ARVANITAKIS et al., one must postulate that the serum factor is some unknown metabolite of the polyamines, since these substances themselves are confined to the cellular fraction of blood. The relation of the serum sugar-transport inhibition to anticiliary activity is unknown. As more insight is gained into the biochemical background of cystic fibrosis, the probability increases that the disease is genetically heterogeneous. DANES and BEARN 23 were the first to express this concept, when they described two groups, one positive and one negative for cultured-cell metachromasia, who appeared to breed true in families. More lately, CoNOVER et al. 24 have shown that these two populations are segregated also on the basis of serum-hexosaminidase levels. C.F. patients and heterozygotes who give a positive metachromasia test have low levels of the enzyme and predominantly the B isoenzyme, while the group with a negative metachromasia test have high serum-enzyme levels and predominantly the A isoenzyme. c.F. may therefore not be a single entity but rather a group of closely related genetic abnormalities with similar pathological consequences. One hopes that from these varied laboratory investigations a unified theory of pathogenesis will emerge. 17.
Doggett, R. G., Harrison, F. M., Patrick, T. A. Nature New Biol. 1973, 243, 249. 12. Johansen, P. G., Anderson, C. M., Hadorn, B. Lancet, 1968, i, 455. 13. Gibson, L. E., Matthews, W. J., Minihan, P. T. ibid. 1970, ii, 189. 14. Chernick, W. S., Barbero, G. J., Parkins, F. M. J. Pediat. 1961, 59, 11.
890.
Mandel, I. D., Kutscher, A., Denning, C. R., Thomson, R. H., Zagarelli, E. V. Am. J. Dis. Child. 1967, 113, 431. 16. Blomfield, J., Warton, K. L., Brown, J. M. Archs Dis. Childh. 1973, 48, 267. 15.
c.F. serum
18. 19. 20. 21. 22. 23. 24.
Fitzpatrick, D. F., Landon, E. J., James, V. Nature New Biol. 1973, 235, 173. Smith, Q. T., Shapiro, B. L., Hamilton, M. J., Warwick, W. J. ibid. 1972, 240, 56. Benke, P. J., Erbstoeszer, M., Pitot, H. C. Lancet, 1972, i, 182. Brown, G. A., Oshin, A., Goodchild, M. C., Anderson, C. M. ibid. 1971, ii, 639. Taussig, L. M., Gardner, J. D. ibid. 1972, i, 1367. Arvanitakis, S. N., Mangos, J. A., McSherry, N. R., Rennert, O. M., Lapointe, D. Pediat. Res. 1973, 7, 336 (abstr.). Danes, B. S., Beam, A. G. J. exp. Med. 1969, 129, 775. Conover, J. H., Conod, E., Hirschhorn, K. Lancet, 1973, i, 1122