765

least two papovaviruses, one closely resembling SV40,have been found in brain cultures from cases of

progressive multifocal leucoencephalopathy.23Early batches of Sabin oral vaccine also contained

SV40, but the infectivity of SV40 by mouth is low 24and a parenteral route is more likely to be implicated.The suggestion that Salk vaccine might have con-

tained killed measles virus raises the further possi-bility that immune responses to this virus might havebeen modified. The speculation that an imbalancein the immune reaction to measles virus arose andSV40 was introduced at the same time may be

justified in the light of current concepts,25,6 especiallysince immune-complex deposition has been demon-strated in S.S.P .E.18

Further investigation into the composition of theSalk vaccine used in New Zealand is clearlyimpossible. However, the evidence presented here

strongly implicates it as a factor initiating the develop-ment of S.S.P.E. in New Zealand and lends supportto the two-virus theory of aetiology. A similar asso-ciation between the administration of Salk vaccineshould be sought in other countries and alternativesources of infection by SV40 or other papovavirusesmust be investigated.We thank the National Health Institute, Wellington, and

Dr William Hamilton for their cooperation. D. M. B. isa recipient of a grant from the Auckland Medical ResearchFoundation.

REFERENCES

1. Jabbour, J. T., Duenas, D. A., Sever, J. L., Krebs, H. M., Horta-Barbosa, L. J. Am. med. Ass. 1972, 220, 959.

2. New Zealand Yearbook, 1957.3. New Zealand Census, 1956, vol. 2.4. Connolly, J. H., Allen, I. V., Hurwitz, L. J., Millar, J. H. D.

Lancet, 1967, i, 542.5. New Zealand Department of Health Annual Report. 1957, p. 18.6. ibid. 1958, p. 16.7. ibid. 1963, p. 5.8. Sweet, B. H., Hilleman, M. R. Proc. Soc. exp. Biol. Med. 1960,

105, 420.9. Gerber, P., Hottle, G. A., Grubbs, R. E. ibid. 1961, 108, 205.

10. Rapp, F. Ann. Rev. Microbiol. 1969, 23, 293.11. Ruckle, G. Fedn Proc. 1956, 15, 610.12. Meyer, H. M., Brooks, B. E., Douglas, R. D., Rogers, N. G.

Am. J. Dis. Child. 1962, 103, 307.13. Girardi, A. J., Warren, J., Goldman, C., Jeffries, B. Proc. Soc.

exp. Biol. Med. 1958, 98, 18.14. Dawson, J. R. Am. J. Path. 1933, 9, 7.15. Payne, F. E., Baublis, J. V., Itabashi, H. H. New Engl. J. Med.

1969, 281, 585.16. Horta-Barbosa, L., Fucillo, D. A., London, W. T., Jabbour, J. T.,

Zeman, W., Sever, J. L. Proc. Soc. exp. Biol. Med. 1969, 132, 272.17. Payne, F. E., Baublis, J. V. Perspect. med. Virol. 1971, 7, 179.18. Dayan, A. D., Stokes, M. I. Br. med. J. 1972, ii, 374.19. Koprowski, H., Barbanti-Brodano, G., Katz, M. Nature, 1970,

225, 1045.20. Barbanti-Brodano, G., Oyanagi, S., Katz, M., Koprowski, H.

Proc. Soc. exp. Biol. Med. 1970, 134, 230.21. Miller, D., ter Meulen, V., Katz, M., Koprowski, H. Lab. Invest.

1971, 25, 337.22. Bech, V., von Magnus, P. Acta path. microbiol. scand. 1958, 42, 75.23. Weiner, L. P., Johnson, R. T., Herndon, R. M. New Engl. J. Med.

1973, 288, 1103.24. Melnick, J. L., Stinebaugh, S. Proc. Soc. exp. Biol. Med. 1962,

109, 965.25. Oldstone, M. B. A., Dixon, F. J. J. exp. Med. 1969, 129, 483.26. Sjögren, H. O., Hellström, I., Bansal, S. C., Hellström, K. E.

Proc. natn. Acad. Sci. U.S.A. 1971, 68, 1372.

Preliminary Communication

ASSOCIATION OF CYSTIC-FIBROSIS

FACTOR TO METACHROMASIA OF THE

CULTURED CYSTIC-FIBROSIS FIBROBLAST

B. SHANNON DANES

Department of Medicine, Cornell University MedicalCollege, New York, N.Y. 10021, U.S.A.

Summary An association between the presenceof cystic-fibrosis-factor activity

(C.F.F.A.) and metachromasia observed in some cul-tured C.F. fibroblasts has been demonstrated throughcell mixing. Correction of this mutant phenotype(metachromasia and presence of C.F.F.A.) occurred

only if there was cell-to-cell contact (i.e., metaboliccooperation) with normal fibroblasts. No correctionoccurred if metachromatic, C.F.F.A.-positive C.F.

fibroblasts were mixed with ametachromatic, C.F.F.A.-negative C.F. fibroblasts thus supporting geneticheterogeneity within this clinically defined disorder.

INTRODUCTION

SINCE the demonstration that cultured skin fibro-blasts from most patients with cystic fibrosis (c.F.)showed cytoplasmic metachromasia,l it has beenassumed that such metachromasia reflected theabnormal genotype of the c.F. cultured fibroblast.

However, the causal relationship between the meta-chromatic staining and C.F. is unknown. Ciliaryinhibitory activity (C.F.F.A.) has been demonstrated inbody fluids of c.F. patients.2-5 The recognition ofC.F.F.A. in serum-free medium from C.F. skin-fibro-

blast cultures 4,6 has further suggested that the cul-tured cell was capable of expressing its abnormal

genotype. Moreover, only in c.F. cultures showingmetachromasia could this activity be demonstrated.’These observations raised the possibility that there

might be an association between the presence ofC.F.F.A. and metachromasia observed in the culturedc.F. fibroblast.

MATERIALS AND METHODS

Skin-fibroblast lines were established from nine C.F.

patients with serum-c.F.F.A. and nine c.F. patients andnine healthy controls without serum-C.F.F.A. by the oystercilia test. After 1 month in culture (two to four sub-cultures by trypsinisation) cell lines were trypsinisedinto suspension and approximately 105 cells were

inoculated into 75 sq. cm. flasks (Falcon Plastics, Oxnard,California) containing a microcoverslip (3 X 3 mm.) forhistochemical study. Excess glass had to be avoidedbecause c.F.F.A. could not be consistently detected in

glass culture flasks.6 The medium used was Waymouth’s"

special " medium 8 with 10% by volume of fetal calfserum. After the cells had formed an adherent non-confluent monolayer (usually 12 hours), the cultureswere washed three times with warmed balanced-saltsolution, and medium without serum was added (pH 7’5).

After 72 hours, the cells on the coverslip were stainedwith toluidine-blue 0 for metachromasia,7 and a sampleof the medium was assayed for c.F.F.A. using the oyster-cilia test.3

For cell-interaction experiments, cell lines were

trypsinised into suspension, and approximately 104 cellswere suspended into 5 ml. of Waymouth’s special mediumwith serum and inoculated into either the inside or out-side well of a Falcon microdiffusion dish which containeda microcoverslip for a histochemical preparation. Afterthe cells had adhered, the culture was washed with salinesolution and medium without serum was added. When

766

cell mixing was desired, 5 ml. volumes of two such cell

suspensions were mixed in a plastic test tube, and themixture was then introduced into both wells of the micro-diffusion dish, thus giving the same cell concentration inboth the separate and mixture experiments. After thecultures were incubated in a humidified atmosphere of

5 % carbon dioxide in air at 37 °C for 72 hours, the cover-slip preparations were stained for metachromasia andthe media assayed for C.F.F.A.

RESULTS

Cultured c.F. skin fibroblasts used in these experi-ments were classed into two groups: positive (meta-chromatic, C.F.F.A. positive) and negative (ameta-chromatic, C.F.F.A. negative) (table). When positivec.F. cells were grown in used media from either

INFLUENCE OF CELL INTERACTION ON THE CULTURED CYSTIC-

FIBROSIS FIBROBLAST

* Cultured skin fibroblasts derived from nine healthy controls (NL)and nine c.F. patients whose cultured fibroblasts were positive (+)and nine whose fibroblasts were negative (-) for metachrom-asia and C.F.F.A. were tested in both separation and mixture experi-ments. Cell concentration of each line tested was 10’ cells per micro-diffusion dish.

normal or negative c.F. cells, metachromasia per-sisted and C.F.F.A. was detected after 24 hours’ incuba-tion. If normal or negative c.F. cells were grown inused medium from a positive culture, the cells didnot show metachromasia and C.F.F.A. could not bedetected after 3 hours of culture.When the positive fibroblasts were grown in the

same dish with normal or negative c.F. fibroblastsbut separated by a plastic barrier so only the mediumwas in common, metachromasia and C.F.F.A. persisted(table). If the positive C.F. cells were mixed withnormal cells before inoculation into the culture

dish, the metachromasia and C.F.F.A. could no longerbe demonstrated. If the ratio of positive c.F./normalcells exceeded 2/1 in such mixtures, this reversalin metachromasia and C.F.F.A. did not occur. How-

ever, if the positive c.F. cells were mixed with

negative c.F. cells, the metachromasia and C.F.F.A.

persisted even if the initial inoculum contained twiceas many negative to positive cells.

DISCUSSION

Metachromasia is a non-specific staining reactiongiven by any macromolecule containing electro-

negative radicals and exhibiting a periodic negativesurface charged Substances such as metaphosphates,nucleic acids, polypeptides, mucopolysaccharides,and lipids show tissue metachromasia Culturedcells from the genetic mucopolysaccharidoses,lipidoses, and lipomucopolysaccharidoses stain

metachromatically, reflecting increased intracellularaccumulation of known metabolites. Fibroblastsfrom normal individuals have stained metachro-

matically when grown under adverse culture con-

ditions such as an alkaline pH.12 The basis for themetachromasia in the cultured cell with the c.F.

genotype has not been understood although it was

presumed to indicate that the genetic defect, as yetunknown, was being expressed by skin fibroblasts inculture.

The other abnormalities noted in c.F. cultures 7

have also been non-specific (variable mucopoly-saccharide content, increased glycogen storage,quantitative increase in lysosomes, decreased collagensynthesis) rather than identifying a specific cyto-plasmic defect.

C.F.F.A., assayed as the ability to stop oyster ciliarymovement,3 has been detected in serum-free mediumfrom actively growing c.F. skin-fibroblast cultureswhich showed metachromasia.7 C.F.F.A. was foundto be associated with a low-molecular-weight,negatively charged molecule that contained no

uronic acid, was heat and pH labile, and bound toIgG subclasses 1 and 2 .6 The relationship of theC.F.F.A. to the primary defect in c.F. has not beenestablished.

Based on the experiments reported here (see table),neither expression of the c.F. genotype in culture

(metachromasia or C.F.F.A.) could be altered by grow-ing c.F.F.A.-positive cells (1) in used medium fromnormal or c.F.F.A.-negative c.F. cultures or (2) separ-ated from the other cell line so only the medium wasin common. Since C.F.F.A. is lost when positive usedmedium is stored at 37°C for a few hours,6 such

lability probably precluded any significant influenceon cell function. This correction was distinctly differ-ent from that observed in the mucopolysaccharidoseswhere the mutant phenotype was corrected by incu-bation with used medium containing the secretionsfrom normal fibroblasts.13

Correction of c.F. mutant phenotype occurred onlywith intimate contact of the positive c.F. cells withnormal cells. Such correction requiring cell-to-cellcontact has been named metabolic cooperation."This process has been extensively studied in the

Lesch-Nyhan syndrome where metabolic cooperationbetween normal cells and mutant cells deficient in

hypoxanthine-guanine or adenine phosphoribosyltransferase may have been the result of transfer ofthe enzyme product, nucleotide, or nucleotide deriva-tive from normal to mutant cells. Separation of

Lesch-Nyhan cells from normal cells resulted in

prompt reversion to the mutant phenotype. How-ever, the molecular basis of metabolic cooperationin this metabolic defect is not known.

767

Several reports 15-17 have established the existence

in culture of two classes of c.F. fibroblasts, meta-chromatic and ametachromatic, which showed

intrafamilial agreement. However, there seemed tobe no clinical difference between the two classes.

Furthermore, although the metachromatic classcould be distinguished in cell culture by both stain-ing reaction and presence of C.F.F.A., the ametachro-matic class was indistinguishable from culturesderived from non-carriers. The mixing experimentsrevealed a distinct difference (table). When positivec.F. cells were mixed with normal cells, correction

(i.e., metabolic cooperation) occurred, whereas if

positive c.F. cells were mixed with negative c.F.

cells, no such correction occurred. These obser-vations supported genetic heterogeneity within cysticfibrosis.

This research was made possible through a grant fromthe National Foundation-March of Dimes.

REFERENCES

1. Danes, B. S., Beam, A. G. J. exp. Med. 1969, 129, 775.2. Spock, A., Heick, M. C., Cross, H., Logan, W. S. Pediat. Res. 1967,

1, 173.3. Bowman, B. H., Lockhart, L. H., McCombs, M. L. Science, 1969,

164, 325.4. Bowman, B. H., Barnett, D. R., Matalon, R., Danes, B. S., Bearn,

A. G. Proc. natn. Acad. Sci. U.S.A. 1973, 70, 548.5. Doggett, R. G., Harrison, G. M. Cystic Fibrosis Club Abstr. 1971,

10, 34.6. Danes, B. S., Litwin, S. D., Hütteroth, T. H., Cleve, H., Bearn,

A. G. J. exp. Med. 1973, 137, 1538.7. Danes, B. S., Bearn, A. G. ibid. 1972, 136, 1313.8. Gordon, L., Waymouth, C. Proc. Soc. exp. Biol. Med. 1965, 119,

287.9. Sylven, B. Acta histochem. 1958, suppl. 1, p. 79.

10. Barka, T., Anderson, P. J. Histochemistry, Theory, Practice andBibliography; p. 84. New York, 1963.

11. Matalon, R., Dorfman, A. Lancet, 1969, ii, 838.12. McKusick, V. A., Hussels, I. E., Howell, R. R., Neufeld, E. F.,

Stevenson, R. E. ibid. 1972, i, 993.13. Fratantoni, J. C., Hall, C. W., Neufeld, E. F. Science, 1969, 162, 570.14. Cox, R. P., Krauss, M. R., Balis, M. E., Dancis, J. Expl Cell Res.

1972, 74, 251.15. Danes, B. S. Proc. fifth int. cystic Fibrosis Conf. 1969, p. 67.16. Matalon, R., Dorfman, A. Biochem. Biophys. Res. Commun. 1968,

33, 954.17. Conover, J. H., Conod, E. J., Hirschhorn, K. Lancet, 1973, i, 1122.

Reviews of Books

The Greater Medical Profession

Edited by VERNON W. LIPPARD, M.D., and ELIZABETHPURCELL. London: H. K. Lewis (for the Royal Society ofMedicine). New York: Josiah Macy Foundation. 1973.

Pp. 253. E2.50 (E3 outside U.K.);$7.50.THE results of research into the aetiology of diseases and

of clinical trials of procedures designed to prevent, alleviate,or cure them bring considerable uniformity to the clinicalpractices of physicians and surgeons all over the world.Until recently, however, ethnocentrism dominated studiesof the operation, structure, and financing of medical-caresystems and of their influence on patient access, site oftreatment facilities, and utilisation of medical manpower.Indeed, it was widely assumed that substantial differencesin social goals, political systems, and organisation ofhealth services meant that countries had little to learn fromeach other. The profession and the Government in theU.S.A. were committed to free enterprise in medical care,to paying doctors on a fee-for-service basis, and to con-trolling patients’ access to health facilities by their capacityto pay or their eligibility through insurance cover. Whatcould they learn from Britain which had gone far along theroad to " socialising " its health-care system by givingcitizens free access to all health services ? Similarly,British doctors and Governments felt they had little tolearn from Americans about the organisation of healthcare.

Lately, however, a dialogue has begun. One reason forthe growing interest in the U.S.A. in communicating withcounterparts in Britain is the realisation that Governmentmust now provide and meet the cost of medical care for upto a third of its population. Since the hitherto despisedBritish system of health care does not seem (at least fromAmerica) to be going through a " crisis " (the term usedby Senator Edward Kennedy and many others to describethe situation in the United States), there is a refreshingwillingness to see whether any lessons can be learned fromthe British experience and what in that experience can betransplanted successfully to American soil. The British,too, have become a little less insular and complacent asthey see the costs of their own Health Service escalate andas they begin to appreciate that such issues as the approp-

riate use of technology, the demand for hospital treatment,the relative increase in specialised as against generalistmedical manpower, dependency on foreign medical

graduates, and the efficacy and efficiency of the hospitaland primary-care sectors are universal problems, not

exclusively those of the National Health Service.The subject of a Royal Society of Medicine/Josiah Macy

Foundation symposium held at the R.S.M. in June,1972, was the identification of practices hitherto performedexclusively by doctors which might equally well from thetechnical point of view be undertaken by other membersof the " greater medical profession ", the phrase coinedby Sir Theodore Fox (Lancet, 1956, ii, 779) to refer to allthose occupied in health care. Participants from both sidesof the Atlantic described the manpower problems in theircountries, the feasibility of delegating doctors’ functionsto other health workers, the emergence of new occupationalgroups such as the nurse practitioner and physicianassistant, the economic and educational implications ofchanging roles within the health centre and other medicalsettings, and, finally, the legal, cultural, and psychologicalconsequences of new forms of teamwork.How useful to non-participants is the account of the

symposium which includes not only the formal papersdelivered, but the spontaneous discussion which followedthem ? To those who were there, the volume is interestingin that it confirms the impression gained at the time-

namely, of considerable differences between the Americanand the British participants’ accounts of their problems.By and large, the Americans were agreed that they couldnot meet their health-care obligations to provide a moreeffective and less expensive form of primary care for all,including the poor in urban ghettos and rural backwaters,without the creation of new health occupations, inter-mediate between the traditional ones of doctor and nurse.Their concerns were with the repercussions of such adevelopment on the health-care system and on health-careconsumers. The British, on the other hand, were confidentthat future health-care delivery problems could be settledby reorganising the working relationships of existingoccupations, and that no

" new " occupation was required,but rather a restructuring of inter-staff hierarchies andtasks.

Like most collections, the quality of the individualpapers in this report varies. Some, including the prologueby Prof. C. T. Dollery, are well worth reading; but