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abnormal states; and their findings provide strongevidence that these changes depend not only on theseverity but even more on the type of the underlyingacid-base disturbance. 8-10

The degree of protection enjoyed by spinal fluid insevere metabolic acidosis is impressive. As the blood-bicarbonate concentration (and pH) falls, two pro-tective mechanisms seem to come into play. First,perhaps as a direct consequence of increased cerebralblood-flow, the normal pCO2 gap between blood andspinal fluid narrows. Second and more important, thenormal bicarbonate gradient between the two com-

partments is actually reversed. POSNER and PLUMexamine and dismiss several possible explanations.They do not believe that an uptake of

" fixed " acids

by the brain tissue can account for this reversal; nordo they accept a significant time lag in the diffusionout of bicarbonate from the spinal fluid into the blood.The underlying change, they suggest, must involve adefinite re-setting of the transport mechanism whichregulates the movement of bicarbonate ions across theblood/spinal-fluid barrier-a hypothesis supported byexperimental as well as by clinical evidence."-15 (Evenunder physiological conditions one must postulatesome form of active bicarbonate transport as distinctfrom passive diffusion, since the electrochemical

gradient between blood and spinal fluid would favoura higher, not a lower, spinal-fluid concentration.)

In contrast to the chain of clinical and chemicalevents in metabolic acidosis, respiratory acidosis is

accompanied by a widening of the pH gap and anearly and often dramatically sudden deterioration inmental state. There is still no general agreement aboutthe immediate cause of this encephalopathy-whetherit is the result of increased H-1--ion activity, a directanaesthetic action of CO2, or hypoxia - but its onset

can coincide with a blood pH of 7-1 or even 7-2.15The relative importance of blood pH and spinal-fluidpH in regulating ventilation remains controversial. 916-18

More detailed studies are needed before the clinicallessons can be more than tentative; but they are stillworth stating. From the point of view of cerebral

function, excessive zeal in correcting chronic or slowlyprogressive metabolic acidosis can be positively harmful,inducing an actual depression of spinal-fluid pH. Evenin acute metabolic acidosis the brain is in less immediate

danger than other organs-notably the heart-butthis too represents a hazard. Such patients can diefrom cardiovascular collapse before there is any cloud-ing of consciousness, still regarded by some as a

dependable warning sign. On the other hand, confusion8. Posner, J. B., Swanson, A. G., Plum, F. Archs Neurol. 1965, 12, 479.9. Posner, J. B., Plum, F. New Engl. J. Med. 1967, 277, 605.

10. Lee, J. E., Plum, F., Posner, J. B. J. clin. Invest. 1967, 46, 1083.11. Kibler, R. F., O’Neill, R. P., Robin, E. D. ibid. 1964, 43, 431.12. Roos, A. Am. J. Physiol. 1965, 209, 1233.13. Pontén, U. Acta neurol. scand. 1966, 42, 455.14. Bulger, R. J., Schrier, R. W., Arend, W. P., Swanson, A. G. New

Engl. J. Med. 1966, 277, 433.15. Fischer, V. J., Christianson, L. C. J. appl. Physiol. 1963, 18, 712.16. Mitchell, R. A. ibid. 1965, 20, 443.17. Pappenheimer, J. R., Fend, V., Heisey, S. R., Held, D. Am. J. Physiol.

1965, 208, 436.18. Pappenheimer, J. R. Harvey Lect. 1965-66, p. 71.

and coma appear early in acute respiratory acidosisand require for their relief prompt and effectiveventilation. Yet even in such an emergency there isneed for caution. In patients with longstandingrespiratory impairment a compensatory but severe

metabolic alkalosis develops; and the reversal of thisslow process may take days or even weeks. When anacute crisis supervenes in such an illness, too vigorousand too successful a relief of the respiratory acidosismay precipitate the patient into acute metabolic

alkalosis, perhaps a no less dangerous state.

Cell Substrates for Virus VaccinesUNTIL a few years ago all virus vaccines were pro-

duced in or on living animals-rabies in rabbit brains,yellow fever in fertile hens’ eggs, and smallpox on theskin of sheep or calves. Apart from ensuring that thevaccine virus retained its attenuated characteristics orthat it had been successfully killed, few additional testswere applied to detect the presence of extraneous vir-uses. The development of successful cell-culture tech-niques on a large scale was a major advance, givinggreater production potential and reproducibility frombatch to batch. These techniques also increased the

ability to detect extraneous viruses and, for the first

time, vaccines produced on cell-cultures were examinedvery thoroughly for such contaminants. Monkey kidneybecame popular as a cell substrate 1 because it was aneasily accessible tissue that would grow poliomyelitisvirus. But in many respects it is unsuitable. Monkeysin the wild are infected with many viruses. B virus hasbeen known for many years and fortunately laboratorytests are adequate to detect it should it contaminate avaccine; but not all simian viruses can be detected. In1960 a new simian virus 40 remained as a live contamin-ant in some batches of killed vaccine.3 This virus,though oncogenic in hamsters, apparently has littleeffect in man, but how long will our good fortune last?The reports 4 5 of a haemorrhagic disease transmittedfrom monkeys to man have been a stern reminder thatwe should take a second look at the source of cell sub-strates for vaccine production.The possibility of vaccine contamination can be

largely avoided by using a cleaner tissue. Measles vac-cine has been produced on chick-embryo-fibroblast cell-cultures 6 obtained from chickens known to be free fromchick pathogens, including fowl leucosis viruses. Somemeasles vaccines have been grown on kidney culturesfrom dogs bred in a clean environment. Rubellavaccines are being produced, for experimental purposes,

1. Enders, J. F., Weller, T. H., Robbins, F. C. Science, N.Y. 1949, 109, 85.2. Sabin, A. B. J. clin. Invest. 1949, 28, 808.3. Sweet, B. H., Hilleman, M. R. Second International Conference on

Live Poliovirus Vaccines, 1960. Pan. Am. Hlth Org. sci. Publ. no. 504. The findings of Prof. G. A. Martini and Prof. R. Siegert, of the Uni-

versity of Marburg, are shortly to appear in the Deutsche MedizinischeWochenschrift.

5. Gordon Smith, C. E., Simpson, D. I. H., Bowen, E. T. W., Zlotnik, I.Lancet, 1967, ii, 1119.

6. Enders, J. F., Katz, S. L., Milovanovic, H. V., Holloway, A. New Engl.J. Med. 1960, 263, 153.

7. Musser, S. J., Slater, M. S. Am. J. Dis. Child. 1962, 103, 476.

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on cell-cultures derived from duck embryos 8 or rabbitkidneys. The use of these tissues from animals bred

under clean conditions is a big step forward and hasresulted in far fewer virus harvests being contaminatedwith extraneous viruses. But have we gone far

enough ?The work of HAYFLICK and MOORHEAD 9 has opened

up a new horizon in cell substrates, and during the pastthree years the prospects for commercial developmenthave been realised. Human lung tissue, obtained from aioetus after therapeutic abortion, is teased out in a

growth medium, and the cell population that developsis propagated for 45-50 passages before it ceases to beviable. At any stage of propagation the cells may befrozen in liquid nitrogen and held indefinitely awaitingexhaustive tests for the presence of contaminants beforethe cells may be used for vaccine production. Thus, forthe first time, it is possible to establish a " cell seedsubstrate " which is as important to vaccine productionas the " virus seed system " is regarded today. With-out being specific about the animal species used assource of tissue, two guiding principles can be suggested.Firstly, the cell cultures should be derived from normal

8. Hilleman, M. R. Clin. Pharmac. Ther. 1966, 7, 752.9. Hayflick, L., Moorhead, P. S. Exp. Cell. Res. 1961, 25, 585.

tissue and retain their characteristics of normalitythroughout their propagation; foetal tissue is preferablebecause of the decreased possibility of contamination.Secondly, the cells should be frozen and held until suchtime as exhaustive tests for contaminants have shown thesubstrate to be suitable for use.

Already some vaccines prepared in human diploid cellswi-38 are on trial. In Yugoslavia 10 400,000 childrenhave been given oral poliomyelitis vaccine and 25,000have been injected with measles vaccine, and in Russia 113000 children have been injected with measles vaccine.Although the vaccine is not licensed in the U.S.A., anenteric-coated capsule containing adeno-4 vaccine 12

prepared on this tissue has been given to 300,000 mem-bers of the Armed Forces without ill-effect during2-3 years’ surveillance.At a recent cell culture meeting at the National Insti-

tutes of Health in Bethesda much support was given fora move towards the establishment of exhaustively testedand fully characterised cell substrates in order to placefuture vaccine production on a

" cell seed system".10. Ikic, D. Meeting on Cell Cultures for Virus Vaccine Production,

Washington, November, 1967 (in the press).11. Andzaparidze, O. G. ibid.12. Chanock, R. M., Ludwig, W., Huebner, R. J., Cate, T. C., Lian-Wei

Chu. J. Am. med. Ass. 1966, 195, 445.

Annotations

PACIFARINS

THE long story of human and animal epidemics hasoften suggested a link between nutritional state andresistance to infection. The trouble is that neither in thefield nor in the laboratory has any clearcut evidence of sucha relation been demonstrated.1 Following the brilliantideas of Topley,2 laboratory models of epidemic diseasewere intensively studied; but though many interestingresults emerged, no special effects of diet could bediscerned. Very little was discovered that seemed tohave any practical importance for public health. Webster 3for instance, showed that, by selective breeding, strainsof mice could be built up which were either totallysusceptible or totally resistant to salmonella infection.But any hope of practical application was dispelled whenhe showed that total resistance to infection by one agentcould be combined with great susceptibility to another.4All this ingenious work seemed to reach an impasse.Schneider,5 however, now suggests that in human popu-lations diet may powerfully influence resistance or sus-ceptibility to4nfectious disease. In Webster’s inbredmouse strains no difference in effect on resistance or

susceptibility to Salmonella typhimurium could bedetected between a semisynthetic diet and a " natural

"

diet of ground whole wheat and dried milk. But whenunselected stock of Webster Swiss mice was used, the" natural " diet was found to improve the survival-rate.Complicated experimental models had to be set up todetermine the nature of the resistance factor. A richsource was found in egg-white, but only when this hadundergone some microbial fermentation. An aerobacter1. Scrimshaw, N., Taylor, C. E., Gordon, J. E. Am. J. med. Sci. 1959,

237, 367.2. Topley, W. W. C. Lancet, 1919, ii, 1, 45, 91.3. Webster, L. T., Hodes, H. L. J. exp. Med. 1939, 70, 193.4. Webster, L. T., Hodes, H. L. ibid. 1937, 65, 261.5. Schneider, H. A. Science, N.Y. 1967, 158, 597.

was the most effective and will now produce the sal-monella-resistance factor (S.R.F.) on a simple syntheticmedium. S.R.F. has been partially purified and has someunusual physiochemical properties. Its effectiveness inthe mouse salmonella situation seems remarkable, 200-400parts per 1,000,000,000 in the diet raising the survival-rate from 10% to 90%. S.R.F. is synthesised by avirulentsalmonehx but is not detectable in the media on whichvirulent salmonehx of the same type are cultured. It isnot required by mice or salmonehm for growth or main-tenance, so it is not by these criteria a vitamin; and, sinceit has no detectable bactericidal or bacteriostatic effect,it can hardly be termed an antibiotic. Precisely how itworks is unknown.

Schneider 5 suggests that the S.R.F. may be an exampleof a class of biological agents that have long interestedstudents of marine ecology. It has been suggested that,in ocean waters, various ecological exclusions and suc-cessions are determined by minute traces of organiccompounds-" ecological ectocrines "-produced by pre-vious inhabitants. Schneider has suggested that theS.R.F. might be termed a " pacifarin

" and that it may bethe first-recognised member of a group of highly potentbiological substances which may be of prime importancein infectious disease. Thus the importance of the dietmay lie not in the known content but in the consumption,in some natural foodstuffs, of pacifarins which have beenproduced by microbial action. If Schneider is correct

the implications are enormous, and other workers willcertainly want to investigate .the " pacifarins". Some,however, will be interested in the fact that dietary effectscould be demonstrated in this animal model only when thepre-bred stock was abandoned. So much attention hasbeen devoted to establishing pure lines of animals thatit is useful to be reminded that their artificiality mayobscure the more haphazard natural situations in whichdisease arises.

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6. Lucas, C. E. Biol. Rev. 1947, 22, 270.