ANTILYMPHOCYTE-SERUM PREPARATIONS IN TREATMENT OF RENAL-ALLOGRAFT REJECTION

16 If this is so, phosphorylated intermediates derived from the metabolism of fructose and glycerol might compete with F.D.P. for non-specific phosphatase activity, with resultant hypoglycaemia. It should be noted that the diagnosis of hereditary fructose intolerance is excluded not only by the enzymatic assays reported in table i, but by certain clinical features. 15 This child has never exhibited any intolerance to fructose-containing foods; indeed she enjoys eating fruits and sweets. Furthermore, she has several carious teeth. Fasting hypoglycaemia, a prominent feature of this child’s clinical problem, is not encountered in hereditary fructose intolerance. Moreover, we would not expect glycerol to precipitate hypoglycaemia in a patient with hereditary fructose intolerance, since glycerol enters the metabolic pathway below hepatic fructose-1-phosphate aldolase, the enzyme which is deficient in that disease. The period of fasting required to produce hypoglycaemia in this child seems to have increased as she has grown older. However, fasting alone is still sufficient to produce both profound hypoglycaemia and metabolic acidosis. The metabolic acidosis seems to result primarily from the accumulation of elevated levels of ketone bodies and lactate in her extracellular fluid, and it responds promptly to the administration of glucose. There is a certain similarity here to bovine ketosis.16 Fig. 1 suggests that we do not need to invoke a renal component to account for the metabolic acidosis in this patient. However, Enser et a1.17 have demonstrated the immunological and electrophoretic identity of hepatic and renal F.D.pase activity in the rabbit. Although no direct measurement of renal fructose diphosphatase activity has been made in this patient, it is possible that renal gluconeogenesis is also impaired. In view of current speculation concerning the role of renal gluconeogenesis in the regulation of renal ammonia production, it might be expected that this child would have impaired urinary ammonium excretion in response to an acid load. Blood-urea-nitrogen, creatinine clearance, fasting electrolytes, and arterial pH are normal in this patient after an overnight fast. After the administration of an acute ammoniumchloride load (100 mg. per kg),18 the patient’s plasmacarbon-dioxide fell from 23-9 to 20-3 meq. per litre. She was able to decrease her urinary pH to 4-6, and urinary titratable acidity increased appropriately, reaching a maximum of 111 eq. per minute per 1-73 sq. m. The rate of urinary ammonium excretion did not exceed 45 eq. per minute per 1-73 sq. m.; at its maximum, it did not account for more than 30% of the total hydrogen-ion excretion. This would seem to be an inadequate response, but sufficient data are not yet available to permit definitive conclusions concerning renal gluconeogenesis and ammonium production in this child. The deficiency of hepatic F.D.pase in this child may be inherited, in view of the similar clinical presentation of her sibling who died. Inherited defects of the

phosphatase activity.

unique to gluconeogenesis would be expected fasting hypoglycxmia. This has long been recognised with regard to glucose-6-phosphatase deficiency. Our observations suggest that an inherited enzymes to

result in

deficiency of hepatic F.D.Pase activity is the basis for a previously unrecognised disease, characterised by

episodes

of

fasting hypoglycaemia and metabolic

acidosis. Dr. Barbara I. Brown, Washington University School of Medicine, did the glycogen assays, and we thank her for permission We thank Dr. R. Kaye, Dr. M. Genel, Dr. to use these data. M. Paolini, and Dr. A. Nagle for allowing us to investigate this patient; Dr. S. Segal and Dr. E. R. Froesch for important suggestions and criticisms; Mrs. V. Jackson for skilful technical assistance; and Dr. P. Holtzapple for the needle liver biopsy. This research was supported by U.S. Public Health Service grants AM-HD13518, FR00240, AM04722 and GM06405. Requests for reprints should be addressed to L. B., Children’s Hospital of Philadelphia, 1740 Bainbridge Street, Philadelphia,

Pennsylvania 19146. REFERENCES

Pontremoli, S., Grazi, E. in Carbohydrate Metabolism and its Disorders (edited by F. Dickens, P. J. Randle, and W. J. Whelan); vol. I, p. 259. New York, 1968. 2. Sols, A. ibid. p. 62. 3. Washko, M. W., Rice, E. W. Clin. Chem. 1961, 7, 542. 4. Novak, M. J. Lipid Res. 1965, 6, 431. 5. Bessman, S. T., Anderson, M. Fedn Proc. 1957, 16, 154. 6. Technical Bulletin 826-UV, Sigma Chemical Co., St. Louis. 7. Technical Bulletin 726-UV, Sigma Chemical Co., St. Louis. 8. Henry, R. J. (editor) Clinical Chemistry: Principles and Techniques; p. 278. New York, 1964. 9. Dryer, R. L., Tammes, A. R., Routh, J. I. J. biol. Chem. 1957, 225, 177. 10. Soeldner, J. S., Slone, D. Diabetes, 1965, 14, 771. 11. Estabrook R. W., Williamson, J. R., Frenkel, R., Maitrá, P. K. in Methods in Enzymology (edited by S. P. Colowick and N. O. Kaplan); vol x, p. 474. New York, 1967. 12. Wolf, H. P. in Methods of Enzymatic Analysis (edited by H. U. Bergmeyer); p. 732. New York, 1965. 13. Heinz, F., Lamprecht, W. Z. Physiol. Chem. 1967, 348, 855. 14. Heinz, F., Lamprecht, W., Kirsch, J. J. clin. Invest. 1968, 47, 1826. 15. Froesch, E. R. in The Metabolic Basis of Inherited Disease (edited by J. B. Stanbury, J. B. Wyngaarden, and D. S. Fredrickson; p. 124. New York, 1966. 16. Grunder, H. D. Dt. tierärztl. Wschr. 1961, 68, 401. 17. Enser, M., Shapiro, S., Horecker, B. L. Archs Biochem. Biophys. 1969, 129, 377. 18. Wrong, O., Davies, H. E. F. Q. Jl Med. 1959, 28, 249. 1.

ANTILYMPHOCYTE-SERUM PREPARATIONS IN TREATMENT OF RENAL-ALLOGRAFT REJECTION A. D. MEE

D. B. EVANS Addenbrooke’s

Hospital, Cambridge

Seven renal-homograft patients with rejection episodes were treated with antilymphocytic serum (A.L.S.) or antilymphocytic globulin (A.L.G.) besides steroids and azathioprine. Only two patients overcame the rejection episode.

Summary

These results were no better than would have been expected had antilymphocytic preparations not been added to the steroids and azathioprine. Thrombocytopenia was seen in most patients receiving A.L.S. or A.L.G. and frequently limited the course of treatment. Introduction THE management of rejection is

a major problem in Early diagnosis and prompt effective treatment are essential and may frequently save a transplanted organ. High doses of steroids are established practice in antirejection therapy as also are the maintenance of adequate levels of azathioprine and

transplanted patients.

17 of 0-5 g. while on day 15 the 24-hour volume had fallen to 16 ml. Rejection was diagnosed and antirejection therapy, as described above, was commenced. A.L.S. was given intramuscularly, 10 ml. per day on 14 of 23 consecutive days-therapy being stopped on days when there was a low platelet-count. After 4 days of antirejection therapy the urine volume was over 1 litre per day, but this improvement was not sustained, and on day 38, when A.L.s. was stopped, the urine volume had fallen to 420 ml. per day containing 4-2 g. urea. The platelet-count had fallen from 240,000 to

the addition of actinomycin C or D. In this unit, we have also used antilymphocytic serum (A.L.S.) containing immune gamma-globulin and, more recently, pure antilymphocytic globulin (A.L.G.) in a small proportion of cases (seven patients), as an adjunct to steroids and azathioprine and as a substitute for actinomycin C. We describe these cases here and compare kidney-survival rate in the A.L.S. and A.L.G. treated group with the kidney-survival rate in a larger group receiving no antilymphocytic serum preparation. All patients received grafts from unrelated cadaver donors. Methods Since May, 1968, prospective tissue typing has been utilised for each transplant. Transplants in the period May, 1967, to May, 1968, have been retrospectively tissue typed. Most patients have received kidneys with at least one or two major antigen mismatches. At the time of transplantation the patients are given hydrocortisone 500 mg. daily for 2 days, then 75 mg. prednisolone daily until 1 litre of urine is passed in 24 hours. The dose is then reduced to 50 mg. daily with further reductions of 5 mg. per day every month to a minimum of 10-15 mg. per day. Azathioprine 5 mg. per kg. body-weight is given in the first 24 hours. Thereafter the dosage varies according to renal function, but in general the maximum tolerated dose is given. Details are reported elsewhere.1 The early diagnosis of rejection may present difficulties, especially in those patients who are oliguric after transplantation. Failure of diuresis may be due to ischaemic renal tubular damage, infection, rejection, or obstruction. Renal biopsy is a useful diagnostic procedure in these cases. In those patients where a diuresis has been achieved, a deterioration of function is suggestive of rejection, hence the importance of a daily determination of blood and urine biochemistry. The following symptoms, signs, biochemical, and hsematological changes may be seen in the presence of rejection: swelling and tenderness of the transplanted organ, fever and/or tachycardia, reduction of 24-hour urine volume, 24-hour urea content of urine, and 24-hour osmolar excretion in urine, changes in urea and creatinine clearance, reduction in platelet-count, proteinuria, and elevation of blood-pressure. Chisholm et al. have suggested that reduction in urea and osmolar excretion are the most useful measures for the early detection of rejection. Percutaneous needle biopsy of the transplanted organ is confirmatory where there is doubt. We have found very little morbidity with needle biopsy and this procedure is now routine.3 When the diagnosis of rejection has been established, antirejection therapy is instituted: prednisolone 200 mg. is given daily for 3 days. The dosage is then reduced by 25 mg. per day every 2 or 3 days. The azathioprine dosage is unchanged (usually 1-2 mg. per kg. per day, depending on renal function). Actinomycin C 200 g. daily has been given intravenously to most of the patients for 3 days. In the seven cases described here, A.L.S. or A.L.G. was given instead of actinomycin C or after a short course of actinomycin C. The early cases received 10 ml. per day of A.L.s. (Prof. M. F. A. Woodruff), containing 1-3 g. immune gamma-globulin, intramuscularly. Later cases received 20 ml. per day of A.L.G. (Prof. W. Brendel) intravenously. In all cases an intradermal test dose was given first and where there was any reaction, antihistamines were administered with the A.L.G. ’

Case-reports Case1 This

during A.L.S. treatment. Multiple complications including massive gastrointestinal bleeding and subdural haemorrhage occurred, and the patient died on day 40. 65,000 per

c.mm.

Case 2 This 38-year-old

man was

given

a

kidney transplant

On day 3 he passed 620 ml. of urine with a urea content of 4 g. Over the next 2 days urine and urea outputs fell to 24-hour values of 300 ml. and 2-7 g., respectively. Antirejection therapy was started including A.L.S., 10 ml. intramuscularly on 8 of 11 consecutive days. At the end of this period, the urine volume had reached 2220 ml. per 24 hours with 29 g. of urea. The creatinine clearance was 23 ml. per minute. Unlike all the other cases treated with A.L.S., the platelet-count was substantially unaffected. The absolute lymphocyte-count had actually increased (850 per c.mm. to 1550 per c.mm.). The kidney still functions well 21/2 years later. Case 3 A 36-year-old woman, given a transplant on Aug. 23, 1967, had still not produced urine by postoperative day 12. Renal transplant biopsy at this time showed some evidence of tubular regeneration, and cellular infiltration of the interstitial tissue (probably immune in nature). Steroids in antirejection doses were started, and, since there was no response after 2 days, A.L.S. was given at doses of 10 ml. intramuscularly on 9 consecutive days. At the end of this time a 24-hour urine volume of 1110 ml. containing 5-6 g. of urea had been achieved. Kidney function did not, however, improve despite further antirejection therapy (including one injection of A.L.S., discontinued when the platelets fell from 54,000 to 15,000 per c.mm.). The patient developed uncontrollable, generalised bleeding, became comatose, and died. At necropsy the kidney showed the changes of severe on

Aug. 17,

1967.

rejection. Case 4 This

25-year-old woman received a kidney transplant on Aug. 23, 1967, and passed 660 ml. of urine in the first 24 hours. However, the urea content was only 1 g. per 24 hours. The daily urine volume rapidly declined and rejection was diagnosed on the 13th postoperative day when the urine volume was nil and the blood-pressure had risen from 120/80 to 210/120 mm. Hg. The possibility of renal-artery stenosis causing a similar sequence of events was considered; however, renal biopsy confirmed rejection. The steroid dosage was increased on day 13, and on day 15 there had been a small response (viz., 250 ml. in 24 hours). We felt that A.L.S. might hasten the recovery of function and it was started in a dose of 10 ml. per day intramuscularly, on day 15. It was given daily on 14 of 15 consecutive days, at the end of which period the 24-hour urine volume 3690 ml. with a urea content of 30 g. The creatinine clearance was 10 ml. per minute and the blood-pressure 180/150 mm. Hg. Platelets had fallen during treatment from 354,000 to 56,000 per c.mm. The kidney maintains good function 21/2 years later.

was

Case 5

received a kidney transplant onl July 25, 1967. The graft was slow to function. By day 13, the 24-hour urine volume was only 90 ml. with a urea content:

40-year-old

man

kidney transplant on Oct. 10, had passed no urine. Biopsy showed evidence of rejection. The steroid dosage was 9

days after receiving

1967, this 26-year-old

a

man

18 increased and A.L.S. started simultaneously, 10 ml. being given intramuscularly daily for 8 days. The 24-hour urine volume was only 30 ml. after this course of treatment and the platelets had fallen from 260,000 to 65,000 per c.mm. Steroids and azathioprine were continued without A.L.S., and on day 25, 1 litre was passed. Peak function was attained on day 30 when 2640 ml. of urine with 12-3 g. A steady deterioration in the transplant urea were passed. then occurred and the transplant was removed on day 38. This patient has since been successfully retransplanted. Case 6 This man, aged 35, was transplanted on April 16, 1969. Up to the 16th postoperative day the maximum 24-hour urine output was 80 ml. On day 16 a renal biopsy was taken and this showed moderate immune reaction. Antirejection therapy (steroids, azathioprine, and actinomycin C) was started and, because renal function had not improved by day 20, actinomycin C was stopped and A.L.G. was substituted: 20 ml. of A.L.G. in 300 ml. of 5% dextrose were given intravenously daily for 3 days. It was then discontinued, the platelets having fallen from 331,000 to 47,000 per c.mm., and the patient having had a febrile reaction to the last two infusions. Biopsy on day 26 showed necrosis of the parenchyma of the kidney with mononuclear infiltration consistent with a severe immune reaction. The transplanted organ was removed on day 32.

Case 7 This 33-year-old man was transplanted on April 28, 1969. After an anuric period of some days the kidney began to function and on day 21 he passed 1340 ml. containing 11 4 g. urea. However, function began to deteriorate and on day 25 the 24-hour volume was 710 ml. Urea output was 7-3 g. A renal biopsy showed moderate immune reaction. Antirejection therapy (steroids, azathioprine and actinomycin C) was started and 4 days later the 24-hour figures were 1580 ml. of urine and 18-2 g. of urea. At this time A.L.G. was added to the regimen (20 ml. in 5% dextrose given intravenously daily for 3 days) replacing the actinomycin C which was stopped. There was little change in the function of the kidney. As the platelets fell during these 3 days from 208,000 to 84,000 per c.mm. A.L.G. was stopped. Coincidentally the function of the graft rapidly deteriorated, and biopsy on day 39 showed substantial immune reaction. Transplant nephrectomy was performed. Results

95

kidney grafts involving eighty-two patients had been done in this unit up to Oct. 1, 1969. In fortysix cases rejection has been diagnosed and treatment instituted. Thirty-nine cases received steroids, azathioprine, and, occasionally, actinomycin C according to the regimen described previously. Twentyfive of these overcame the rejection episode (i.e., about The 66 °o recovered satisfactory renal function). seven patients discussed in this paper have received the same standard regimen of steroids and azathioprine plus A.L.s. or A.L.G. In this much smaller group RESULTS IN TREATMENT OF

REJECTION

WITH A.L.S. OR A.L.G.

have recovered satisfactory renal function (i.e., 29%). However, in four of these cases A.L.S. or A.L.G. was added 2-4 days after the steroids had been increased, because there had been little or no improveIn this respect it may be said ment in renal function. that the group receiving antilymphocytic preparations presented greater problems than the group not receiving A.L.S. or A.L.G. Therefore the results may have been expected to be poorer had antilymphocytic preparations not been given. The fact that the results were indeed poorer in this group suggests that A.L.S. or A.L.G. made no significant difference to the expected two

outcome

(see table).

Of the two patients who did recover good renal function after A.L.S. therapy, one was given A.L.S. simultaneously with the increase in steroids, making evaluation of the contribution of A.L.S. to this kidney’s recovery difficult. Discussion A.L.G. can produce immunological depression in experimental situations,4-8 and it has been used clinically by Starzl and his colleagues 9,10 in a large series of renal allografts. Using related live donors with good histocompatibility Starzl et al. found that when A.L.G. was given 5 days before transplantation and continued for 4 months, kidney survival figures were notably improved, steroid doses cut by half, and azathioprine doses reduced. On withdrawal of A.L.G. from patients who had received kidneys from nonrelated cadaveric donors with poor histocompatibility; Starzl et al. noted a progressive deterioration in the graft. Referring to Starzl’s series, van Rood 11 comments that A.L.G. permitted reduction of steroids il1 cases where there was a good match for the HL-A antigens, but could not override strong histocompatibility differences in the group of non-related cadaveric donors. This latter group more closely resembles the group of transplants under discussion ir this paper, with histocompatibility differences in mosi cases of at least two major antigens. Starzl et al.10 hav( attempted to prevent late rejection in homografts witt histocompatibility differences of this order by usin increased doses of A.L.G. at the time of transplantatior in an effort to produce immune tolerance. However thrombocytopenia and lymphopenia have been limitin

factors.

Although there is

some disagreement about hov works 12,13 most workers agree that to be mos effective it should be started before grafting.9 In thi: unit we have not treated recipients with A.L.G. before transplantation: A.L.G. has been introduced into the scheme of treatment at some time after transplantation as an adjunct to steroids, in the treatment of rejectiox

A.L.G.

only. The side-effects of A.L.S. and A.L.G. are well known Serum sickness may occur. Features of this are poly. arthritis, serositis, and vasculitis. The vascular change! may involve many tissues including the transplantec organ.14 Anaphylaxis has been reported.9 Althougl serum sickness and anaphylaxis were not seen in ou patients, thrombocytopenia, often severe, developed it most cases, and frequently necessitated withdrawal o the antilymphocytic preparation. At least two patient had severe generalised bleeding probably aggravate< by the low platelet-courits. In 2 cases describe(

19

(nos. 6 and 7) the A.L.G. was known to have a high antithrombocyte titre. Preparations with a low antithrombocyte titre are now available.16 Lymphopenia was not a consistent finding. In the earlier cases A.L.s. was given intramuscularly, and pain and tenderness at More recently the injection site were problems. A.L.G. was given intravenously in 5% dextrose solution. Thrombosis of the veins utilised for the infusion in

was

patients (including liver-transplant recipient, not described here, treated with A.L.G.). The seen

some

a

dextrose solution may have been another factor contributing to the thrombosis. This is a small group of patients compared to the total number treated for rejection, and their rejection episodes seemed to be especially severe. Clearcut beneficial effects of the antilymphocyte preparations have not been demonstrated. We thank Prof. R. Y. Calne for permission to publish information about his patients, Dr. R. McMillan for his assistance and advice, and Mrs. J. Small for preparation of the typescript. Supplies of A.L.S. and A.L.G. were generously provided by Sir

Preliminary Communications EFFECT OF L-DOPA ON SEBORRHŒA OF PARKINSONISM

J. L. BURTON

Michael

Woodruff, F.R.S. (Edinburgh), and Prof. W. Brendel (Munich). Requests for reprints should be addressed to A. D. M. REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10.

Calne, R. Y., Evans, D. B., Herbertson, B. M., Joysey, V., McMillan, R., Maginn, R. R., Millard, P. R., Pena, J. R., Salaman, J. R., White, H. J. O., Withycombe, J. F. R., Yoffa, D. E. Br. med. J. 1968, ii, 404. Chisholm, G. D., Papadimitriou, M., Kulatilake, A. E., Shackman, R. Lancet, 1969, i, 904. Millard, P. R., Herbertson, B. M., Evans, D. B. ibid. 1970, i, 113. Woodruff, M. F. A., Anderson, N. F. Nature, Lond. 1963, 200, 702. Gray, J. G., Monaco, A. P., Russell, P. S. Surg. Forum, 1964, 15, 142. Monaco, A. P., Abbott, W. H., Othersen, H. B., Simmons, R. L., Wood, M. L., Flax, M. H., Russell, P. S. Science, 1966, 153, 1264. Levey, R. H., Medawar, P. B. Ann. N.Y. Acad. Sci. 1966, 129, 164. Levey, R. H., Medawar, P. B. Proc. natn. Acad. Sci. 1966, 56, 1130. Starzl, T. E., Marchioro, T. L., Hutchinson, O. E., Porter, K. A., Cerilli, C. J., Brettschneider, L. Transplantation, 1967, 5, 1100. Starzl, T. E., Brettschneider, L., Penn, I., Schmidt, R. W., Bell, P., Kashiwogi, B., Townsend, C. M., Putnam, C. W. Transplant. Proc. 1969, 1, 448.

11. 12. 13. 14. 15.

Rood, J. J. Lancet, 1969, i, 1142. Woodruff, M. F. A. Transplantation, 1967, 5, 1127. Medawar, P. B. Transplant. Proc. 1969, 1, 666. Russell, P., Monaco, A. P. Transplantation, 1967, 5, 1086. Brendel, W. Personal communication. van

of sebum excretion fell in all five patients. This (±S.E.) percentage decrease in sebum-excretion rate after L-dopa (29-7-4-5%) was highly significant Two patients volunteered the (t=6’6; n<0-01). information that their skin felt less greasy during treatment with L-dopa. Though only five patients were investigated, the results clearly show that treatment with L-dopa

rate

mean

SAM SHUSTER EFFECT OF L-DOPA ON SEBUM-EXCRETION RATE IN PARKINSONISM

University Department of Dermatology, University of Newcastle upon Tyne Sebum-excretion rates were measured in five patients with parkinsonism participating in a clinical trial of L-dopa. Doses were raised to the maximum tolerable levels (1·5-5 g. per day) and patients took this dose for 3 weeks. Sebumexcretion rates fell by 30% (range 17-42%).

Sum ary

INTRODUCTION

PATIENTS with parkinsonism have an increased rate of sebum excretion, 1,2 but the cause of this seborrhoea is unknown. L-dopa improves parkinsonism, so we investigated its effect on the rate of sebum excretion in such patients. PATIENTS AND METHODS

Five patients with

idiopathic parkinsonism who were participating in a Medical Research Council therapeutic trial of L-dopa were investigated before and after treatment. The sebum-excretion rate from forehead skin was measured by the method of Strauss and Pochi3 as modified by Cunliffe and Shuster.4 All the patients were receiving a constant dose of oral anticholinergic drugs before and during treat. ment with L-dopa. The L-dopa was started in low dosag( and gradually increased until the maximum tolerable dos( was reached for each patient. This dose varied from 1 -5-5 g

daily in the five patients studied. The second measuremen of the sebum-excretion rate was made when the patient! had been on the maximum dose for 3 weeks. RESULTS AND DISCUSSION

The rate of sebum excretion was increased in fom of the five patients before treatment as compared witt our normal data.4 After treatment with L-dopa th(

decreases the rate of sebum excretion in parkinsonism. Two of our patients remarked that the skin was less greasy, and this has been noticed in the clinical trials with L-dopa./i-7 The seborrhoea of parkinsonism is likely to be endocrine in origin, since patients with unilateral parkinsonism have bilateral seborrhoea (unpublished observation) and because there is no known neurotrophic stimulus to sebaceous secretion. There is now a good deal of evidence that the pituitary plays an important part in the control of sebum secretion in the rat,8-10 and it may be that L-dopa exerts its effect on sebum production by modifying pituitary secretion. Our findings are, therefore, of considerable theoretical interest in relation to the normal regulation of sebum secretion and because of their wider implications concerning endocrine control mechanisms. It remains to be seen whether L-dopa, at tolerable doses, decreases the seborrhoea of acne. We thank Prof. J. N. Walton and Dr. R. C.

Hughes

for per-