- Email: [email protected]
ASSESSMENT OF HUMAN RENAL TRANSPLANTATION
1040
readily available in the Jimi Valley. Two of the salt springs still exist-one of them has recently been obliterated by a landslide. Water from the salt springs at Tumbi contained 15,800 salt parts per million with an iodine content of 4-4 parts per million, giving an iodine/salt ratio of 1/3600, whilst the Sangen spring contained 19,500 parts of salt per million with 6 parts of iodine per million and an iodine/salt ratio of 1/3250 (table n). There seems no doubt, therefore, that salt rich in iodine was available and used by the Jimi people prior to European contact and the cessation of its use resulted from European influence. The degree of salt iodisation introduced in various countries ranges from 1 part per 10,000 to 1 part iodine per 200,000,18 so that the salt available to the inhabitants of the Jimi Valley was appreciably richer in iodine than that usually commercially available. It is impossible to estimate the amount of traditional salt which would have been consumed daily by the inhabitants of the valley. Presumably, because of the scarcity of the commodity, it would be less than optima119. However, 100 mg. of the traditional salt certainly provided 15-20 jug. of iodine per day-a minimum requirement which has been regarded as critical to the appearance of endemic cretinism in a goitre region." A similar phenomenon relating to endemic goitre was observed in the canton of Vaud in Switzerland, where salt from a mine at Bex, which was naturally iodised at 1 part per 100,000, was consumed 21 It could be argued that the increasing prevalence of endemic cretinism in the Jimi Valley was the result of the cessation of tribal fighting. Warfare was likely to create a state of social instability which would militate against the survival of severely neurologically disabled individuals. Furthermore, the influence of mission teaching, Government laws concerning homicide, and the availability of such effective medical agents as penicillin and antimalarials would also increase the survival of the disabled. It is, however, a fact that in West New Guinea, in the Mulia Valley, endemic cretinism was observed to be common in the context of a traditional New Guinea culture unaffected by external contact.2 Missionaries noted cretinism in adults in a society where warfare was still a prominent component. The chronological events also suggest that the prevalence of endemic cretinism had changed before medical services were
that an alteration in the type of salt used by the people has been of paramount importance in disrupting a delicate ecological balance, with disastrous consequences.
Requests for reprints should be addressed to P. 0. D. P.. London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT. REFERENCES 1. 2. 3. 4.
5. 6.
7.
Querido, A. in Endemic Goiter. P.A.H.O. scientific publication no. 193, 1968, p. 85. Stanbury, J. B. in Human Development and the Thyroid Gland (edited by J. B. Stanbury and R. L. Kroc). New York, 1972. McCarrison, R. Lancet, 1908, ii, 1275. Stanbury, J. B., Ermans, A. M., Hetzel, B. S., Pretell, E. A., Querido, A. Wld Hlth Org. Chron. 1974, 28, 220. Greenwald, I. Tex. Rep. Biol. Med. 1957, 15, 874. Stott, H., Bhatia, B. B., Lal, R. S., Rai, K. C. Indian J. med. Res. 1930, 18, 1059. Stanbury, J. B., Brownell, G. L., Riggs, D. S., Perinetti, H.,
Itoiz, J., del Castillo, E. B. Harvard University monograph in medicine and public health no. 12, 1954, p. 86. 8. Wespi, H. J. Schweiz. med. Wschr. 1945, 75, 625. 9. Costa, A., Cottino, F., Mortara, M., Vogliazzo, U. Panminerva med. 1964, 6, 250. 10. Koenig, M. P., Veraguth, P. in Advances in Thyroid Research, 1961, p. 294. 11. Kicic, M., Milutinovic, P., Djordjevic, S., Ramzin, S. ibid. p. 301. 12. Norris, H. Med. Times, Lond. 1847, 17, 257. 13. Pharoah, P. O. D. in Endemic Cretinism. Institute of Human Biology Papua New Guinea monograph series no. 2, 1971, p. 109. 14. Buttfield, I. H. ibid. p. 94. 15. Pharoah, P. O. D., Buttfield, I. H., Hetzel, B. S. Lancet, 1971, i, 308. 16. Pharoah, P. O. D., Buttfield, I. H., Hetzel, B. S. in Human Development and the Thyroid Gland (edited by J. B. Stanbury and R. L. Kroc). New York, 1972. 17. Pharoah, P. O. D. M.D. thesis, University of London, 1972. 18. Matovinovic, J., Ramalingaswami, V. Wld Hlth Org. Monogr. Ser. 1960, no. 44, p. 385. 19. Clarke, W. C. Place and People: an Ecology of a New Guinean Community. Canberra, 1971. 20. Querido, A. in Endemic Cretinism. Institute of Human Biology Papua New Guinea monograph series no. 2, 1971, p. 30. 21. Eggenberger, H., Messerli, F. M. Transactions of the Third International Goiter Conference, 1938, p. 64. 22. Choufer, J. C., van Rhijn, M., Querido, A. J. clin. Endocrin. Metab. 1965, 25, 385.
ASSESSMENT OF HUMAN RENAL TRANSPLANTATION R. M. R. BARNES G. B. WILLIAMS H. E.
DE
G. D. PEGRUM E. M. GORDON WARDENER
Departments of Hœmatology, Surgery, and Medicine, Charing Cross Hospital Medical School, Fulham Palace Road, Hammersmith, London W6
improved. An alternative possibility concerned the infanticide of affected infants, but so far as can be determined this practice, which was common in the Jimi Valley and elsewhere in traditional Papua New Guinean communities, involved only the offspring of multiple births. Besides, the Jimi people are unable to accurately diagnose cretinous children in the immediate post-partum months. They deny that affected children were ever deliberately killed, and their present behaviour involves much solicitude and attention to the infants’ welfare, suggesting that such children have never been regarded as a disgrace or embarrassment. Although the epidemic occurrence of neurological damage in the Jimi Valley coincided with a variety of ecological changes, the overall evidence suggests
renal-transplant unit was established work with an existing hæmodialysis unit; initially it was decided to adopt a policy of close HL-A matching. Most of the recipients had been on long-term hæmodialysis and had received multiple transfusions in the past. Those patients who had not developed antibodies on this regimen had the best prognosis. It is suggested that good matching and prior exposure to blood-transfusion without antibody production are both factors indicating a favourable outcome in renal transplantation. Rejection episodes were also infrequent, and although several tests were used to determine early rejection none was entirely satisfactory. Estimation of urinary fibrinogen-degradation products generally gave the most helpful Sum ary
results.
A
to
1041
Introduction WHEN in 1969 a renal-transplant unit was established to work in conjunction with an existing hxmodialysis unit, it was decided to adopt a policy of close For this we cooperated in the HL-A matching. London Transplant Group,1 originally comprising 21 centres, to obtain the best possible matching. Our results in this respect have been reported as part of a wider assessment of the value of HL-A
matching.2-4 Lately less stringent HL-A matching
was
attempted
and thus we had an opportunity to compare results when the operative procedure and aftercare remained the same but our donor and recipient selection had changed. We discuss the assessment of kidney function in the immediate post-transplant period, the value
pre-transplant screening for’ cytotoxic antibodies, monitoring of immunological function during immunosuppression, and the diagnosis and discrimination of clinical rejection episodes. of
Patients and Methods
Thirty-six patients received cadaveric renal transplants, received kidneys from a parent. One of the thirty-six patients received 2 cadaveric transplants. Lymphocytotoxic cross-matching was negative in all cases. Transplanted patients have been followed for at least 6 months, and sometimes for as long as 4! years. A standard regimen of immunosuppression was used throughout, with azathioprine (’ Imuran’) starting at 3 mg. per kg. and being reduced to a maintenance dose of 75-150 mg. per day and methylprednisolone (’Solu-Medrol’) 1-5 mg. per kg. for 2 days followed by prednisone which was reduced to 10-40 mg. per day. Rejection episodes were treated with methylprednisolone (500 mg. daily) Tor 3 days while the patient was receiving heparin. In some cases the graft was irradiated (200 rad.x3) locally and actinomycin D was given (200 p,g. per day) for 3-4 days. Renal function was assessed routinely by blood urea, creatinine and creatinine clearance, renography, and and five
arteriography where necessary. In the immediate posttransplant period poorly functioning kidneys were always assessed by open biopsy. Histological examination of frozen sections always included examination by immunofluorescence, electron microscopy, and light microscopy. Transplant nephrectomy was performed if this became necessary.
Pre-transplant Procedures Tissue typing was performed using a modification of the microcytotoxicity technique of Terasaki,1 using sera supplied by the London Hospital and the national organmatching service, Bristol. The same technique, with suitable controls, was applied to lymphocytotoxic crossmatching before transplantation, but in this case was assessed after a standard incubation-time of 2 hours and again after prolonged incubation of at least 4 hours to detect the presence of weaker antibodies.
cytotoxicity
Antibody screening.-Since 1970, sera from patients awaiting transplantation have been examined for cytotoxic antibodies. In 1972 this screening became an established monthly routine and sera were tested against a panel of lymphocytes, both fresh and stored (liquid nitrogen), from at least fifteen normal donors selected to cover 30 HL-A specificities. A cytotoxic kill of 30 % or more was regarded as positive.5 All samples received were routinely screened for redblood-cell antibodies by the blood-transfusion department.
Post-transplant Procedures Urinary fibrinogen-degradation products (F.D.P.) were measured in urine samples taken in the early morning and estimated by tanned red blood-cell hsemagglutination inhibition6 using the Wellcome F.D.P. kit. Normal values are <1 1 ttg. per ml. Some samples were also treated the by staphylococcal-clumping techniqueusing kits (Hoechst). Cytotoxic mononuclear cells.-Cytotoxic killing by peripheral-blood leucocytes separated on ’Ficol ’/’ Triosil ’ gradients,8 using cultured Chang human liver cells labelled with chromium-51
as
target cells,
was
assessed.9
performed over an 18-hour period in the absence presence (antibody-induced cytotoxicity) or of rabbit (spontaneous cytotoxicity) anti-Chang antibody. Latterly this assay has been modified as a microtechnique. D.N.A. synthesis.-The method was based on that used for assessment of mixed lymphocyte cultures.1o 0’25 /.LCi I4C-thymidine was added to triplicate cultures of peripheral-blood leucocytes and incubated for 18 hours in an atmosphere of 5 % carbon dioxide in air. Incorporation of isotope was terminated by addition of ice-cold phosphate-buffered saline solution, the red blood-cells were lysed with Zaponin’, and the culture was fixed with 5 % glutaraldehyde. Non-incorporated isotope was removed by washing 3 times. Counts are expressed as
Assays
were
c.p.m. per 106 viable cells.
Results The level of primary non-function (P.N.F.) was low and it was always possible to return the patients to hoemodialysis. The use of an open-biopsy technique TABLE I-AMOUNT OF BLOOD RECEIVED IN RELATION TO CYTOTOXIC ANTIBODIES AND KIDNEY SURVIVAL
1042 that this decision could be made at an earlier before the patient became severely ill. Neverstage theless, one patient, who was a diabetic, died at this early stage from overwhelming infection. Open biopsy also provides an opportunity to identify ureteric obstruction masquerading as P.N.F. or rejection. Good HL-A matching in the first twenty-seven transplants (only three recipients, excluding living related donors, received donor kidneys with a net histocompatibility has meant that rejection episodes have ratio 11 of <05) been sporadic. The overall survival at 2 years was 67%, and, excluding two proven P.N.F. and three cases of probable recurrence of original disease, this figure reaches 85%. meant
Efject ofPrior Blood-transfusions The amount of blood received is indicated in table I, which shows that there was no significant correlation between previous blood-transfusions without the presence of antibodies and good graft survival (two-07). Nevertheless, the best results were achieved in those patients in whom there was a long interval between the last transfusion and kidney transplantation.
Antibody Screening Over the past 2 years all our potential recipients have been screened for red and white blood-cell antibodies. Red blood-cell screening has revealed a high frequency of cold antibodies, particularly with anti-N and anti-L specificities (see table II). White-bloodcell antibodies have only been found occasionally. This type of screening is probably useful when the transfusion requirement is high; however, we have adopted a minimal transfusion policy in our hsemodialysis unit. Thus many patients receive no blood before transplantation, and this technique can no longer be used to identify possible high or low
responders
subsequent graft. of Graft Rejection In general, an increase in serum-creatinine and a fall in creatinine clearance were accepted as evidence of rejection, unless another factor such as obstruction could be identified. In an attempt to predict a rejection episode, several immunological techniques, including D.N.A. synthesis in separated lymphocytes, cytotoxic killing by mononuclear cells, and estimation of urinary F.D.P.S, have been used. D.N.A. Synthesis Regular monitoring of the patient’s lymphocytes by this technique was performed; in the early stages, mainly on individuals who in general had a smooth course. Increases of D.N.A. synthesis in the leucocytes were likely to occur with incidental infection, and levels were particularly high in virus infections (fig. 1). to a
Prediction
TABLE II-ANTIBODY SCREENING
*
5
patientshad received no transfusions but developed cold antibodies
red blood-cells. t Includes 4 patients with R.B.c. = red blood-cell. to
rejected transplants.
1-"C-thymidine incorporation by peripheral-blood leucocytes from 3 transplant patients without complications (a) and 2 (b and c) with infections shown. Consecutive observations were made between grafting and 6-12 months after transplantation.
Fig.
Estimation of D.N.A. synthesis, which we hoped might be used to predict rejection, was discontinued.
Cytotoxic Killing by Mononuclear Cells Spontaneous non-specific cytotoxicity of peripheralblood mononuclear cells for 5lCr-labelled Chang cells, a human polyploid cell-line, and killing in the presence of anti-Chang antibody at high dilution seemed to increase in the 2-3 month period after transplantation, with values persistently higher than normal (fig. 2). One patient was treated for a clinically diagnosed rejection episode (fig. 2a), and cytotoxicity values dropped markedly, presumably as a result of steroid treatment. However, low cytotoxic values may occur for other reasons-i.e., some were associated with surgical intervention for ureteric obstruction (fig. 2b Urinary F.D.P. In two patients (fig. 3aand b) with high urinary F.D.P.S there was no clinical problem in diagnosing transplant rejection and eventually transplant nephrectomy was performed. Retrospective analysis of urinary F.D.P. indicates that abnormally raised levels may be also found in conditions
episodes.
associated with transplant rejection Fig. 3d shows the results in one patien: not
who had two
clinically diagnosed rejection episodes.
1043
Fig. 2-Cytotoxicity values obtained in intervals after transplantation.
2
patients (a and b)
at
2 x 105 peripheral-blood mononuclear cells were incubated 20/1 ratio with’61Cr-1abelled target (Chang) cells for 18 hours 37°C. · 8 antibody-induced cytotoxicity; 0 - - - -0 spontaneous cytotoxicity.
at at
the first of which was treated without biopsy. Both were associated with normal urinary F.D.P., and subsequently he was shown to have ureteric obstruction. Varied levels may also be associated with urinary obstruction and after operative manoeuvres involving the kidney and ureters (see fig. 3a and c).
Discussion of tissue typing in renal transplantation may depend on the population involved. Serological determinants are not the only important factor, since HL-A matching in North America does not achieve improved graft survival. 12 In the less heterogeneous population of Europe, well-matched kidneys have been shown to be of definite advamage.2-4,13 The role of lymphocyte determinants 14 and immunological 11 responsiveness controlled by immune-response genes may be additional important influencing factors, but we do not as yet know how these interrelate. ’ The degree of responsiveness is difficult to determine without a challenge which might well affect a subsequent graft. We had an opportunity to make certain observations in this respect since the initial recipients had received many transfusions in the past. Prior transfusions in the absence of any detectable antibodies seem to indicate a good prognosis, and this accords with previous studies,",16 but others have demonstrated that previous transfusions may impair graft survival.11,18 Possibly, repeated transfusion of small quantities of blood, as in our patients, many
Fig. 3-Urinary F.D.P. in 4 Tests
were
transplant patients (a, b,
c, and
d).
performed daily after transplantation.
The influence
months before transplantation is more successful in determining those who areimmunologically poor responders. Since transfusion is now a rare event in hxmodialysis units, this opportunity of selecting patients is no longer available. Cold antibodies have been identified in patients who have not received blood but who are on regular dialysis. Some technique is urgently required to provide a stimulus to potential recipients for assessing their responsiveness without sensitisation. Skin sensitisation, which we have not used, may well provide such evidence, but the risk of sensitisation leading to accelerated rejection remains. A test described by Webster et a1.19 may prove helpful in this situation. Recognition of a rejection episode remains difficult. Many techniques have been used, including cutaneous sensitivity to soluble transplantation antigens, inhibition of macrophage migration, and estimation of urinary /3-glycosidases. None of these were reliable. At the beginning of the present study we monitored D.N.A. synthesis in lymphocytes as was suggested by
1044
Parker
et
al.23
Our results showed that any infective
episode, but particularly viral infections, were liable to produce a pronounced increase in D.N.A. synthesis, and autoradiographs demonstrated that this activity occurred predominantly in peripheral-blood lymphocytes. Although viral infection and accelerated graft rejection 24 are associated, the method is clearly open to misinterpretation. Lymphocytes activated in mixed lymphocyte culture have been shown to kill 5lCr-labelled Chang cells non-specifically in vitro 25 It was therefore interesting to study cytotoxic killing by peripheral-blood mononuclear cells from transplant patients, particularly during rejection episodes when host lymphocytes may be activated in vivo 26 In at least two patients followed repeatedly for 2-3 months several interesting findings emerged. High levels of cytotoxic killing occurred in the post-transplant period, despite continuous immunosuppression with prednisone and azathioprine, but in one patient given vigorous therapy for rejection, cytotoxic killing, both spontaneous and antibody-induced, was markedly reduced. Mellstedt and Holm,27 using a similar system, demonstrated that antibody-induced cytotoxicity was depressed in myeloma patients treated with cortisone and Alkeran. However, reduced values for cytotoxic killing may also be associated with surgical intervention. The relevance and usefulness of this system require further investigation. The predictive value of urinary F.D.P. estimations in the diagnosis of transplant rejection has been reported by others.28,29 We feel that " rejection episodes " diagnosed by a clinical assessment when F.D.P.s are not increased should be treated cautiously; these rejection episodes are often due to other pathological conditions or recurrence of the initial disease. In addition, the increased F.D.P. levels occasionally associated with non-immunological conditions, such as urinary obstruction, have not received enough attention. Despite these reservations, the technique is relatively simple for repeated sequential assays, using commercially available kits, and should be considered for providing supportive evidence of graft rejection. The early transplant recipients who had been on haemodialysis for a long time did better than more recent transplants with a shorter period on dialysis. It is tempting to postulate that good HL-A matching and a high proportion of " non-responders " to multiple blood-transfusions were included in our first renal allografts. 2-year survival-rate overall in twentyseven patients was 67%, and excluding P.N.F. and recurrence of original disease it is 85%, which compares favourably with reported 2-year survival data.4.12 Four patients died as a result of transplantation but not due to rejection. Irreversible rejection or P.N.F. was treated by transplant nephrectomy, and these patients were returned to haemodialysis. Thus a policy of establishing a patient on home dialysis before transplantation is of great value should the transplant fail. We thank Dr Roussel from Hoechst Pharmaceuticals for
supplying the staphylococcal clumping F.D.P. test kits. Requests for reprints should be addressed to G. D. P., Department of Hxmatology, Charing Cross Hospital (Fulham), Fulham Palace Road, London W6 8RF.
SEROLOGICAL RESPONSES OF MULTIPLESCLEROSIS PATIENTS AND CONTROLS TO A VIRUS ISOLATED FROM A MULTIPLESCLEROSIS CASE GEORGE J. NEMO
TACOB A. BRODY
Neurological Diseases and Stroke, Bethesda, Maryland 20014, U.S.A.
National Institute of
DAVID J. WATERS
Multiple Sclerosis Research Center of the Wistar Institute and Department of Neurology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, U.S.A. and Hæmagglutination - inhibition hæmolysis-inhibition antibody titres to M.S. 6/94, a virus recently isolated from the brain of a multiple-sclerosis patient, were no different in multiple-sclerosis patients than in matched controls. Similarly, no differences were encountered in antibody to parainfluenza type 1 virus. Higher measles antibody titres were found among patients in this series. There was no evidence of cross-reactivity between measles antibody and the other viruses tested. The ætiological significance of the virus isolated from an M.S. patient remains in question.
Summary
DR BARNES AND OTHERS : REFERENCES
1. Festenstein, H., Oliver, R. T. D., Sachs, J. A., Burke, J. M., Adams, E., Divver, W., Hyams, A., Pegrum, G. D., Balfour, I. C., Moorhead, J. F. Lancet, 1971, ii, 225. 2. Oliver, R. T. D., Sachs, J. A., Festenstein, H., Pegrum, G. D., Moorhead, J. F., Balfour, I. C. ibid. 1972, ii, 1381. 3. Dausset, J., Festenstein, H. Transplant. Proc. 1973, 5, 1299. 4. Dausset, J., Hors, J., Busson, M., Festenstein, H., Oliver, R. T. D., Paris, A. M. I., Sachs, J. A. New Engl. J. Med. 1974, 290, 979. 5. Mittal, K. K., Mickey, M. R., Singal, D. P., Terasaki, P. I. Transplantation, 1968, 6, 913. 6. Merskey, C., Kleiner, G. J., Johnson, A. J. Blood, 1966, 28, 1. 7. Hawiger, J., Niewiarowsky, S., Gurewick, V., Thomas, D. P. J. Lab. clin. Med. 1970, 75, 93. 8. Boyum, A. Scand. J. clin. Lab. Invest. 1968, 21, suppl. 97. 9. MacLennan, I, C. M. Transplant. Rev. 1972, 13, 67. 10. Pegrum, G. D., Evans, C. A., Middleton, V. L., Balfour, I. C. Clin. exp. Immun. 1972, 12, 357. 11. Rapaport, F. T., Dausset, J. Science, 1970, 167, 1260. 12. Belzer, F. O., Perkins, H. A., Fortmann, J. L., Kountz, S. L., Salvatierra, O., Cochrum, K. C., Payne, R. Lancet, 1974, i, 774. 13. van Hoof, J. P., Schippers, H. M. A., van der Steen, G. J., van Rood, J. J. ibid. 1972, ii, 1385. 14. Bach, F. H., Widmer, M. B., Bach, M. L., Klein, J. J. exp. Med.
1972, 136, 1430. 15. Benacerraf, B., McDevitt, H. O. Science, 1972, 175, 273. 16. Opelz, G., Mickey, M. R., Terasaki, P. I. Lancet, 1972, i, 868. 17. Michielsen, P. Proc. Eur. Dialysis Transplant. Ass. 1966, 3, 162. 18. Dossetor, J. B., MacKinnon, K. J., Gault, M. H., MacLean, L. D. Transplantation, 1967, 5, 844. 19. Webster, A. D. B., Efter, T., Asherson, G. L. Br. med. J. 1974,
iii, 16. 20. Kahan, B. D., Mittal, K. K., Reisfield, R., Bergan, J. Surg, St. Louis, 1973, 74, 153. 21. Wood, R., Gray, A., Briggs, J., Bell, P. Transplantation, 1973, 16, 41. 22. Wellwood, J. M., Ellis, B. G., Hall, J. H., Robinson, D. R., Thompson, A. E. Br. med. J. 1973, ii, 261. 23. Parker, J. R., Ellis, F. G., Cameron, J. S., Ogg, C. S. Proc. Eur. Dialysis Transplant. Ass. 1970, 7, 331. 24. Lopez, C., Simmons, R. L., Mauer, M., Park, B., Najarian, J. S., Good, R. A. Transplant. Proc. 1973, 5, 803. 25. Balfour, I. C., Evans, C. A., Middleton, V. L., Pegrum, G. D. Clin. exp. Immun. 1972, 10, 67. 26. Hamburger, J., Dimitiu, A., Bankir, L., Debray-Sachs, M., Auvert, J. Nature, 1971, 232, 633. 27. Mellstedt, H., Holm, G. Clin. exp. Immun. 1973, 15, 309. 28. Clarkson, A. R., Morton, J. B., Cash, J. D. Lancet, 1970, ii, 1220 29. Hulme, B., Pitcher, P. M. ibid. 1973, i, 6.
readily available in the Jimi Valley. Two of the salt springs still exist-one of them has recently been obliterated by a landslide. Water from the salt springs at Tumbi contained 15,800 salt parts per million with an iodine content of 4-4 parts per million, giving an iodine/salt ratio of 1/3600, whilst the Sangen spring contained 19,500 parts of salt per million with 6 parts of iodine per million and an iodine/salt ratio of 1/3250 (table n). There seems no doubt, therefore, that salt rich in iodine was available and used by the Jimi people prior to European contact and the cessation of its use resulted from European influence. The degree of salt iodisation introduced in various countries ranges from 1 part per 10,000 to 1 part iodine per 200,000,18 so that the salt available to the inhabitants of the Jimi Valley was appreciably richer in iodine than that usually commercially available. It is impossible to estimate the amount of traditional salt which would have been consumed daily by the inhabitants of the valley. Presumably, because of the scarcity of the commodity, it would be less than optima119. However, 100 mg. of the traditional salt certainly provided 15-20 jug. of iodine per day-a minimum requirement which has been regarded as critical to the appearance of endemic cretinism in a goitre region." A similar phenomenon relating to endemic goitre was observed in the canton of Vaud in Switzerland, where salt from a mine at Bex, which was naturally iodised at 1 part per 100,000, was consumed 21 It could be argued that the increasing prevalence of endemic cretinism in the Jimi Valley was the result of the cessation of tribal fighting. Warfare was likely to create a state of social instability which would militate against the survival of severely neurologically disabled individuals. Furthermore, the influence of mission teaching, Government laws concerning homicide, and the availability of such effective medical agents as penicillin and antimalarials would also increase the survival of the disabled. It is, however, a fact that in West New Guinea, in the Mulia Valley, endemic cretinism was observed to be common in the context of a traditional New Guinea culture unaffected by external contact.2 Missionaries noted cretinism in adults in a society where warfare was still a prominent component. The chronological events also suggest that the prevalence of endemic cretinism had changed before medical services were
that an alteration in the type of salt used by the people has been of paramount importance in disrupting a delicate ecological balance, with disastrous consequences.
Requests for reprints should be addressed to P. 0. D. P.. London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT. REFERENCES 1. 2. 3. 4.
5. 6.
7.
Querido, A. in Endemic Goiter. P.A.H.O. scientific publication no. 193, 1968, p. 85. Stanbury, J. B. in Human Development and the Thyroid Gland (edited by J. B. Stanbury and R. L. Kroc). New York, 1972. McCarrison, R. Lancet, 1908, ii, 1275. Stanbury, J. B., Ermans, A. M., Hetzel, B. S., Pretell, E. A., Querido, A. Wld Hlth Org. Chron. 1974, 28, 220. Greenwald, I. Tex. Rep. Biol. Med. 1957, 15, 874. Stott, H., Bhatia, B. B., Lal, R. S., Rai, K. C. Indian J. med. Res. 1930, 18, 1059. Stanbury, J. B., Brownell, G. L., Riggs, D. S., Perinetti, H.,
Itoiz, J., del Castillo, E. B. Harvard University monograph in medicine and public health no. 12, 1954, p. 86. 8. Wespi, H. J. Schweiz. med. Wschr. 1945, 75, 625. 9. Costa, A., Cottino, F., Mortara, M., Vogliazzo, U. Panminerva med. 1964, 6, 250. 10. Koenig, M. P., Veraguth, P. in Advances in Thyroid Research, 1961, p. 294. 11. Kicic, M., Milutinovic, P., Djordjevic, S., Ramzin, S. ibid. p. 301. 12. Norris, H. Med. Times, Lond. 1847, 17, 257. 13. Pharoah, P. O. D. in Endemic Cretinism. Institute of Human Biology Papua New Guinea monograph series no. 2, 1971, p. 109. 14. Buttfield, I. H. ibid. p. 94. 15. Pharoah, P. O. D., Buttfield, I. H., Hetzel, B. S. Lancet, 1971, i, 308. 16. Pharoah, P. O. D., Buttfield, I. H., Hetzel, B. S. in Human Development and the Thyroid Gland (edited by J. B. Stanbury and R. L. Kroc). New York, 1972. 17. Pharoah, P. O. D. M.D. thesis, University of London, 1972. 18. Matovinovic, J., Ramalingaswami, V. Wld Hlth Org. Monogr. Ser. 1960, no. 44, p. 385. 19. Clarke, W. C. Place and People: an Ecology of a New Guinean Community. Canberra, 1971. 20. Querido, A. in Endemic Cretinism. Institute of Human Biology Papua New Guinea monograph series no. 2, 1971, p. 30. 21. Eggenberger, H., Messerli, F. M. Transactions of the Third International Goiter Conference, 1938, p. 64. 22. Choufer, J. C., van Rhijn, M., Querido, A. J. clin. Endocrin. Metab. 1965, 25, 385.
ASSESSMENT OF HUMAN RENAL TRANSPLANTATION R. M. R. BARNES G. B. WILLIAMS H. E.
DE
G. D. PEGRUM E. M. GORDON WARDENER
Departments of Hœmatology, Surgery, and Medicine, Charing Cross Hospital Medical School, Fulham Palace Road, Hammersmith, London W6
improved. An alternative possibility concerned the infanticide of affected infants, but so far as can be determined this practice, which was common in the Jimi Valley and elsewhere in traditional Papua New Guinean communities, involved only the offspring of multiple births. Besides, the Jimi people are unable to accurately diagnose cretinous children in the immediate post-partum months. They deny that affected children were ever deliberately killed, and their present behaviour involves much solicitude and attention to the infants’ welfare, suggesting that such children have never been regarded as a disgrace or embarrassment. Although the epidemic occurrence of neurological damage in the Jimi Valley coincided with a variety of ecological changes, the overall evidence suggests
renal-transplant unit was established work with an existing hæmodialysis unit; initially it was decided to adopt a policy of close HL-A matching. Most of the recipients had been on long-term hæmodialysis and had received multiple transfusions in the past. Those patients who had not developed antibodies on this regimen had the best prognosis. It is suggested that good matching and prior exposure to blood-transfusion without antibody production are both factors indicating a favourable outcome in renal transplantation. Rejection episodes were also infrequent, and although several tests were used to determine early rejection none was entirely satisfactory. Estimation of urinary fibrinogen-degradation products generally gave the most helpful Sum ary
results.
A
to
1041
Introduction WHEN in 1969 a renal-transplant unit was established to work in conjunction with an existing hxmodialysis unit, it was decided to adopt a policy of close For this we cooperated in the HL-A matching. London Transplant Group,1 originally comprising 21 centres, to obtain the best possible matching. Our results in this respect have been reported as part of a wider assessment of the value of HL-A
matching.2-4 Lately less stringent HL-A matching
was
attempted
and thus we had an opportunity to compare results when the operative procedure and aftercare remained the same but our donor and recipient selection had changed. We discuss the assessment of kidney function in the immediate post-transplant period, the value
pre-transplant screening for’ cytotoxic antibodies, monitoring of immunological function during immunosuppression, and the diagnosis and discrimination of clinical rejection episodes. of
Patients and Methods
Thirty-six patients received cadaveric renal transplants, received kidneys from a parent. One of the thirty-six patients received 2 cadaveric transplants. Lymphocytotoxic cross-matching was negative in all cases. Transplanted patients have been followed for at least 6 months, and sometimes for as long as 4! years. A standard regimen of immunosuppression was used throughout, with azathioprine (’ Imuran’) starting at 3 mg. per kg. and being reduced to a maintenance dose of 75-150 mg. per day and methylprednisolone (’Solu-Medrol’) 1-5 mg. per kg. for 2 days followed by prednisone which was reduced to 10-40 mg. per day. Rejection episodes were treated with methylprednisolone (500 mg. daily) Tor 3 days while the patient was receiving heparin. In some cases the graft was irradiated (200 rad.x3) locally and actinomycin D was given (200 p,g. per day) for 3-4 days. Renal function was assessed routinely by blood urea, creatinine and creatinine clearance, renography, and and five
arteriography where necessary. In the immediate posttransplant period poorly functioning kidneys were always assessed by open biopsy. Histological examination of frozen sections always included examination by immunofluorescence, electron microscopy, and light microscopy. Transplant nephrectomy was performed if this became necessary.
Pre-transplant Procedures Tissue typing was performed using a modification of the microcytotoxicity technique of Terasaki,1 using sera supplied by the London Hospital and the national organmatching service, Bristol. The same technique, with suitable controls, was applied to lymphocytotoxic crossmatching before transplantation, but in this case was assessed after a standard incubation-time of 2 hours and again after prolonged incubation of at least 4 hours to detect the presence of weaker antibodies.
cytotoxicity
Antibody screening.-Since 1970, sera from patients awaiting transplantation have been examined for cytotoxic antibodies. In 1972 this screening became an established monthly routine and sera were tested against a panel of lymphocytes, both fresh and stored (liquid nitrogen), from at least fifteen normal donors selected to cover 30 HL-A specificities. A cytotoxic kill of 30 % or more was regarded as positive.5 All samples received were routinely screened for redblood-cell antibodies by the blood-transfusion department.
Post-transplant Procedures Urinary fibrinogen-degradation products (F.D.P.) were measured in urine samples taken in the early morning and estimated by tanned red blood-cell hsemagglutination inhibition6 using the Wellcome F.D.P. kit. Normal values are <1 1 ttg. per ml. Some samples were also treated the by staphylococcal-clumping techniqueusing kits (Hoechst). Cytotoxic mononuclear cells.-Cytotoxic killing by peripheral-blood leucocytes separated on ’Ficol ’/’ Triosil ’ gradients,8 using cultured Chang human liver cells labelled with chromium-51
as
target cells,
was
assessed.9
performed over an 18-hour period in the absence presence (antibody-induced cytotoxicity) or of rabbit (spontaneous cytotoxicity) anti-Chang antibody. Latterly this assay has been modified as a microtechnique. D.N.A. synthesis.-The method was based on that used for assessment of mixed lymphocyte cultures.1o 0’25 /.LCi I4C-thymidine was added to triplicate cultures of peripheral-blood leucocytes and incubated for 18 hours in an atmosphere of 5 % carbon dioxide in air. Incorporation of isotope was terminated by addition of ice-cold phosphate-buffered saline solution, the red blood-cells were lysed with Zaponin’, and the culture was fixed with 5 % glutaraldehyde. Non-incorporated isotope was removed by washing 3 times. Counts are expressed as
Assays
were
c.p.m. per 106 viable cells.
Results The level of primary non-function (P.N.F.) was low and it was always possible to return the patients to hoemodialysis. The use of an open-biopsy technique TABLE I-AMOUNT OF BLOOD RECEIVED IN RELATION TO CYTOTOXIC ANTIBODIES AND KIDNEY SURVIVAL
1042 that this decision could be made at an earlier before the patient became severely ill. Neverstage theless, one patient, who was a diabetic, died at this early stage from overwhelming infection. Open biopsy also provides an opportunity to identify ureteric obstruction masquerading as P.N.F. or rejection. Good HL-A matching in the first twenty-seven transplants (only three recipients, excluding living related donors, received donor kidneys with a net histocompatibility has meant that rejection episodes have ratio 11 of <05) been sporadic. The overall survival at 2 years was 67%, and, excluding two proven P.N.F. and three cases of probable recurrence of original disease, this figure reaches 85%. meant
Efject ofPrior Blood-transfusions The amount of blood received is indicated in table I, which shows that there was no significant correlation between previous blood-transfusions without the presence of antibodies and good graft survival (two-07). Nevertheless, the best results were achieved in those patients in whom there was a long interval between the last transfusion and kidney transplantation.
Antibody Screening Over the past 2 years all our potential recipients have been screened for red and white blood-cell antibodies. Red blood-cell screening has revealed a high frequency of cold antibodies, particularly with anti-N and anti-L specificities (see table II). White-bloodcell antibodies have only been found occasionally. This type of screening is probably useful when the transfusion requirement is high; however, we have adopted a minimal transfusion policy in our hsemodialysis unit. Thus many patients receive no blood before transplantation, and this technique can no longer be used to identify possible high or low
responders
subsequent graft. of Graft Rejection In general, an increase in serum-creatinine and a fall in creatinine clearance were accepted as evidence of rejection, unless another factor such as obstruction could be identified. In an attempt to predict a rejection episode, several immunological techniques, including D.N.A. synthesis in separated lymphocytes, cytotoxic killing by mononuclear cells, and estimation of urinary F.D.P.S, have been used. D.N.A. Synthesis Regular monitoring of the patient’s lymphocytes by this technique was performed; in the early stages, mainly on individuals who in general had a smooth course. Increases of D.N.A. synthesis in the leucocytes were likely to occur with incidental infection, and levels were particularly high in virus infections (fig. 1). to a
Prediction
TABLE II-ANTIBODY SCREENING
*
5
patientshad received no transfusions but developed cold antibodies
red blood-cells. t Includes 4 patients with R.B.c. = red blood-cell. to
rejected transplants.
1-"C-thymidine incorporation by peripheral-blood leucocytes from 3 transplant patients without complications (a) and 2 (b and c) with infections shown. Consecutive observations were made between grafting and 6-12 months after transplantation.
Fig.
Estimation of D.N.A. synthesis, which we hoped might be used to predict rejection, was discontinued.
Cytotoxic Killing by Mononuclear Cells Spontaneous non-specific cytotoxicity of peripheralblood mononuclear cells for 5lCr-labelled Chang cells, a human polyploid cell-line, and killing in the presence of anti-Chang antibody at high dilution seemed to increase in the 2-3 month period after transplantation, with values persistently higher than normal (fig. 2). One patient was treated for a clinically diagnosed rejection episode (fig. 2a), and cytotoxicity values dropped markedly, presumably as a result of steroid treatment. However, low cytotoxic values may occur for other reasons-i.e., some were associated with surgical intervention for ureteric obstruction (fig. 2b Urinary F.D.P. In two patients (fig. 3aand b) with high urinary F.D.P.S there was no clinical problem in diagnosing transplant rejection and eventually transplant nephrectomy was performed. Retrospective analysis of urinary F.D.P. indicates that abnormally raised levels may be also found in conditions
episodes.
associated with transplant rejection Fig. 3d shows the results in one patien: not
who had two
clinically diagnosed rejection episodes.
1043
Fig. 2-Cytotoxicity values obtained in intervals after transplantation.
2
patients (a and b)
at
2 x 105 peripheral-blood mononuclear cells were incubated 20/1 ratio with’61Cr-1abelled target (Chang) cells for 18 hours 37°C. · 8 antibody-induced cytotoxicity; 0 - - - -0 spontaneous cytotoxicity.
at at
the first of which was treated without biopsy. Both were associated with normal urinary F.D.P., and subsequently he was shown to have ureteric obstruction. Varied levels may also be associated with urinary obstruction and after operative manoeuvres involving the kidney and ureters (see fig. 3a and c).
Discussion of tissue typing in renal transplantation may depend on the population involved. Serological determinants are not the only important factor, since HL-A matching in North America does not achieve improved graft survival. 12 In the less heterogeneous population of Europe, well-matched kidneys have been shown to be of definite advamage.2-4,13 The role of lymphocyte determinants 14 and immunological 11 responsiveness controlled by immune-response genes may be additional important influencing factors, but we do not as yet know how these interrelate. ’ The degree of responsiveness is difficult to determine without a challenge which might well affect a subsequent graft. We had an opportunity to make certain observations in this respect since the initial recipients had received many transfusions in the past. Prior transfusions in the absence of any detectable antibodies seem to indicate a good prognosis, and this accords with previous studies,",16 but others have demonstrated that previous transfusions may impair graft survival.11,18 Possibly, repeated transfusion of small quantities of blood, as in our patients, many
Fig. 3-Urinary F.D.P. in 4 Tests
were
transplant patients (a, b,
c, and
d).
performed daily after transplantation.
The influence
months before transplantation is more successful in determining those who areimmunologically poor responders. Since transfusion is now a rare event in hxmodialysis units, this opportunity of selecting patients is no longer available. Cold antibodies have been identified in patients who have not received blood but who are on regular dialysis. Some technique is urgently required to provide a stimulus to potential recipients for assessing their responsiveness without sensitisation. Skin sensitisation, which we have not used, may well provide such evidence, but the risk of sensitisation leading to accelerated rejection remains. A test described by Webster et a1.19 may prove helpful in this situation. Recognition of a rejection episode remains difficult. Many techniques have been used, including cutaneous sensitivity to soluble transplantation antigens, inhibition of macrophage migration, and estimation of urinary /3-glycosidases. None of these were reliable. At the beginning of the present study we monitored D.N.A. synthesis in lymphocytes as was suggested by
1044
Parker
et
al.23
Our results showed that any infective
episode, but particularly viral infections, were liable to produce a pronounced increase in D.N.A. synthesis, and autoradiographs demonstrated that this activity occurred predominantly in peripheral-blood lymphocytes. Although viral infection and accelerated graft rejection 24 are associated, the method is clearly open to misinterpretation. Lymphocytes activated in mixed lymphocyte culture have been shown to kill 5lCr-labelled Chang cells non-specifically in vitro 25 It was therefore interesting to study cytotoxic killing by peripheral-blood mononuclear cells from transplant patients, particularly during rejection episodes when host lymphocytes may be activated in vivo 26 In at least two patients followed repeatedly for 2-3 months several interesting findings emerged. High levels of cytotoxic killing occurred in the post-transplant period, despite continuous immunosuppression with prednisone and azathioprine, but in one patient given vigorous therapy for rejection, cytotoxic killing, both spontaneous and antibody-induced, was markedly reduced. Mellstedt and Holm,27 using a similar system, demonstrated that antibody-induced cytotoxicity was depressed in myeloma patients treated with cortisone and Alkeran. However, reduced values for cytotoxic killing may also be associated with surgical intervention. The relevance and usefulness of this system require further investigation. The predictive value of urinary F.D.P. estimations in the diagnosis of transplant rejection has been reported by others.28,29 We feel that " rejection episodes " diagnosed by a clinical assessment when F.D.P.s are not increased should be treated cautiously; these rejection episodes are often due to other pathological conditions or recurrence of the initial disease. In addition, the increased F.D.P. levels occasionally associated with non-immunological conditions, such as urinary obstruction, have not received enough attention. Despite these reservations, the technique is relatively simple for repeated sequential assays, using commercially available kits, and should be considered for providing supportive evidence of graft rejection. The early transplant recipients who had been on haemodialysis for a long time did better than more recent transplants with a shorter period on dialysis. It is tempting to postulate that good HL-A matching and a high proportion of " non-responders " to multiple blood-transfusions were included in our first renal allografts. 2-year survival-rate overall in twentyseven patients was 67%, and excluding P.N.F. and recurrence of original disease it is 85%, which compares favourably with reported 2-year survival data.4.12 Four patients died as a result of transplantation but not due to rejection. Irreversible rejection or P.N.F. was treated by transplant nephrectomy, and these patients were returned to haemodialysis. Thus a policy of establishing a patient on home dialysis before transplantation is of great value should the transplant fail. We thank Dr Roussel from Hoechst Pharmaceuticals for
supplying the staphylococcal clumping F.D.P. test kits. Requests for reprints should be addressed to G. D. P., Department of Hxmatology, Charing Cross Hospital (Fulham), Fulham Palace Road, London W6 8RF.
SEROLOGICAL RESPONSES OF MULTIPLESCLEROSIS PATIENTS AND CONTROLS TO A VIRUS ISOLATED FROM A MULTIPLESCLEROSIS CASE GEORGE J. NEMO
TACOB A. BRODY
Neurological Diseases and Stroke, Bethesda, Maryland 20014, U.S.A.
National Institute of
DAVID J. WATERS
Multiple Sclerosis Research Center of the Wistar Institute and Department of Neurology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, U.S.A. and Hæmagglutination - inhibition hæmolysis-inhibition antibody titres to M.S. 6/94, a virus recently isolated from the brain of a multiple-sclerosis patient, were no different in multiple-sclerosis patients than in matched controls. Similarly, no differences were encountered in antibody to parainfluenza type 1 virus. Higher measles antibody titres were found among patients in this series. There was no evidence of cross-reactivity between measles antibody and the other viruses tested. The ætiological significance of the virus isolated from an M.S. patient remains in question.
Summary
DR BARNES AND OTHERS : REFERENCES
1. Festenstein, H., Oliver, R. T. D., Sachs, J. A., Burke, J. M., Adams, E., Divver, W., Hyams, A., Pegrum, G. D., Balfour, I. C., Moorhead, J. F. Lancet, 1971, ii, 225. 2. Oliver, R. T. D., Sachs, J. A., Festenstein, H., Pegrum, G. D., Moorhead, J. F., Balfour, I. C. ibid. 1972, ii, 1381. 3. Dausset, J., Festenstein, H. Transplant. Proc. 1973, 5, 1299. 4. Dausset, J., Hors, J., Busson, M., Festenstein, H., Oliver, R. T. D., Paris, A. M. I., Sachs, J. A. New Engl. J. Med. 1974, 290, 979. 5. Mittal, K. K., Mickey, M. R., Singal, D. P., Terasaki, P. I. Transplantation, 1968, 6, 913. 6. Merskey, C., Kleiner, G. J., Johnson, A. J. Blood, 1966, 28, 1. 7. Hawiger, J., Niewiarowsky, S., Gurewick, V., Thomas, D. P. J. Lab. clin. Med. 1970, 75, 93. 8. Boyum, A. Scand. J. clin. Lab. Invest. 1968, 21, suppl. 97. 9. MacLennan, I, C. M. Transplant. Rev. 1972, 13, 67. 10. Pegrum, G. D., Evans, C. A., Middleton, V. L., Balfour, I. C. Clin. exp. Immun. 1972, 12, 357. 11. Rapaport, F. T., Dausset, J. Science, 1970, 167, 1260. 12. Belzer, F. O., Perkins, H. A., Fortmann, J. L., Kountz, S. L., Salvatierra, O., Cochrum, K. C., Payne, R. Lancet, 1974, i, 774. 13. van Hoof, J. P., Schippers, H. M. A., van der Steen, G. J., van Rood, J. J. ibid. 1972, ii, 1385. 14. Bach, F. H., Widmer, M. B., Bach, M. L., Klein, J. J. exp. Med.
1972, 136, 1430. 15. Benacerraf, B., McDevitt, H. O. Science, 1972, 175, 273. 16. Opelz, G., Mickey, M. R., Terasaki, P. I. Lancet, 1972, i, 868. 17. Michielsen, P. Proc. Eur. Dialysis Transplant. Ass. 1966, 3, 162. 18. Dossetor, J. B., MacKinnon, K. J., Gault, M. H., MacLean, L. D. Transplantation, 1967, 5, 844. 19. Webster, A. D. B., Efter, T., Asherson, G. L. Br. med. J. 1974,
iii, 16. 20. Kahan, B. D., Mittal, K. K., Reisfield, R., Bergan, J. Surg, St. Louis, 1973, 74, 153. 21. Wood, R., Gray, A., Briggs, J., Bell, P. Transplantation, 1973, 16, 41. 22. Wellwood, J. M., Ellis, B. G., Hall, J. H., Robinson, D. R., Thompson, A. E. Br. med. J. 1973, ii, 261. 23. Parker, J. R., Ellis, F. G., Cameron, J. S., Ogg, C. S. Proc. Eur. Dialysis Transplant. Ass. 1970, 7, 331. 24. Lopez, C., Simmons, R. L., Mauer, M., Park, B., Najarian, J. S., Good, R. A. Transplant. Proc. 1973, 5, 803. 25. Balfour, I. C., Evans, C. A., Middleton, V. L., Pegrum, G. D. Clin. exp. Immun. 1972, 10, 67. 26. Hamburger, J., Dimitiu, A., Bankir, L., Debray-Sachs, M., Auvert, J. Nature, 1971, 232, 633. 27. Mellstedt, H., Holm, G. Clin. exp. Immun. 1973, 15, 309. 28. Clarkson, A. R., Morton, J. B., Cash, J. D. Lancet, 1970, ii, 1220 29. Hulme, B., Pitcher, P. M. ibid. 1973, i, 6.