423

management. It may be argued that any investigationwhich raises suspicion of the presence of a cerebralneoplasm is of value. If, however, this results in aproportion of patients being subjected to further

investigations which are uncomfortable, not withouthazard, often require a general anaesthetic, and areexpensive, only to find that no tumour is present, itsvalue is lessened. An investigation which permits ahigh percentage of definite diagnoses with few equi-vocal results is certainly preferable. This the EMIscanner achieves.

A further advantage is that the C.T. scan enabled apositive diagnosis to be made in a very high percentageof the non-tumour cases. Cerebral atrophy and infarc-tion cause recognisable abnormalities, although, as

mentioned previously, it is not always possible with asingle examination to distinguish an infarct from atumour.

Comparison of the EMI scanner with scinti-

graphy, angiography, and pneumoencephalography isdifficult since each aims to answer somewhat different

questions. Scintigraphy may reveal a lesion, but itis seldom precisely informative about its pathology ordimensions and it cannot detect hydrocephalus or

cerebral atrophy. Pneumoencephalography identifiestumours indirectly by their effect on the ventricularsystem and subarachnoid space and only occasionallyprovides a specific diagnosis. It is particularly valu-able for identifying neoplasms in the posterior fossaand the perisellar region, two areas where c.T. scanninghas definite limitations. However, until the advent ofc.T. scanning, pneumoencephalography was the onlysatisfactory radiological method for demonstratingcerebral atrophy. Angiography may reveal a tumourdirectly by the presence of abnormal vasculature orindirectly by the displacement of vessels, and suchanatomical details may be of great value to the surgeon.Angiography also permits estimation of ventricular

size, it may give evidence of infarction, and is the onlyavailable method of showing disease of the blood-vessels themselves. Against the advantages of everyinvestigation must be weighed the disadvantages andhazards, and this is especially true of pneumo-encephalography and angiography, which carry a smallmorbidity. The EMI scanner clearly possesses veryconsiderable advantages. It is neither uncomfortablenor hazardous and can be readily used for outpatients.A positive diagnosis can be obtained in a very highpercentage of patients as it records much more definiteand detailed information than can be obtained fromother screening procedures. Furthermore, manypatients do not require subsequent contrast radiology,either because a firm diagnosis can be made on thebasis of the c.T. scan or the surgeon is provided withsufficient information to proceed to craniotomy. Asmall number of patients in the present series wereoperated on without contrast radiology. The surgeonwill, however, often need to know the source andextent of the blood-supply of a tumour before embark-ing upon craniotomy, and at present this can only beclearly shown by angiography.Decision about the most appropriate tomographic

" slices" in a particular case demands medical super-vision. In addition, the avoidance of artefacts and

equivocal results requires meticulous attention to

technique by an experienced radiographer. Experi-ence has shown that the interpretation of the polaroidpictures may sometimes be clarified by reference to

the digital print-out. It would be tragic for such apromising diagnostic method to acquire a reputationfor inaccuracy because of carelessness in its use and inthe interpretation of its results.

The very ease with which an EMI scan may be

performed may well pose problems. Other writershave stressed the wasteful and uninformed use of theskull X-ray,6 electroencephalogram,’ and isotope brainscan.s In our experience about three-quarters ofisotope scans show no abnormality and, even takinginto account the value of a negative result, about aquarter cannot be justified. It- would be unfortunateif the EMI scanner were similarly employed to

examine a large number of patients in whom a

proper clinical assessment makes the presence ofa lesion unlikely. Such a development would be awasteful use of a method of investigation which hasenormous potential for positive diagnosis.We thank EMI Limited for permission to publish fig. 1,

and for their support of our research programme and inparticular for a grant to J. G. Invaluable radiographic helpwas provided by Miss V. Fullom, and secretarial assistance byMiss B. Laatz and Miss S. Andrews.

Requests for reprints should be addressed to J. B.

REFERENCES

1. Hounsfield, G. N. Br. J. Radiol. 1973, 46, 1016.2. Ambrose, J. ibid. p. 1023.3. New, P. F. J., Scott, W. R., Schnur, J. A., Davis, K. R., Taveras,

J. M. Radiology, 1974, 110, 109.4. Baker, H. L., Campbell, J. K., Houser, W. D., Reese, D. F.,

Sheedy, P. F., Holman, C. B., Kurland, R. L. Proc. Mayo Clin.1974, 49, 17.

5. Ethier, R., Sherwin, A., Taylor, S., Leppik, I., Thompson, C.First International C.A.T. Symposium, Montreal, 1974 (in thepress).

6. Bull, J. W. D., Zilkha, K. J. Br. med. J. 1968, iv, 569.7. Matthews, W. B. J. R. Coll. Physns, 1973, 7, 207.8. Claveria, L. E., Du Boulay, G., Zingesser, L. Proc. R. Soc. Med.

1973, 66, 823.

PROSPECTIVE, CONTROLLED TRIAL OFCYCLOPHOSPHAMIDE THERAPY IN

CHILDREN WITH THE NEPHROTIC

SYNDROME

Report of the International Study of Kidney Diseasein Children*

Summary This therapeutic trial was designed tocompare the efficacy of cyclophos-

phamide with that of prednisone in two categories ofchildren with the nephrotic syndrome: patients whocontinue to have proteinuria after 8 weeks of pred-nisone therapy (early non-responders) and patientswho respond initially but who subsequently have

multiple exacerbations of their disease (frequentrelapsers). The results in early non-responders indi-cate that the proportion of patients who lost their

proteinuria was similar in both treatment groups.However, proteinuria subsided earlier in those whoreceived cyclophosphamide. The results in frequent* The writing committee consisted of Dr A. SPITZER, Dr G.

GORDILLO-P., Prof. 1. B. HOUSTON, and Dr L. B. TRAVIS. -

424

relapsers confirm previous reports that cyclophos-phamide is more effective than prednisone in

inducing a lasting remission. This study also revealsthat frequent relapsers are much less likely to relapsewhile receiving cyclophosphamide than they are

during intermittent therapy with prednisone. More-

over, the rate of subsequent relapses seems to belower in frequent relapsers who received cyclophos-phamide than in those treated exclusively with

prednisone.

Introduction

TREATMENT of children with the nephrotic syn-drome with adrenocortical steroids is accompanied bydiuresis and disappearance of abnormal proteinuriain a high percentage of patients. The regularity withwhich these events occur warrants their interpretationas a response to the treatment. In the International

Study of Kidney Disease in Children (I.S.K.D.C.),84-4% of 463 children with the nephrotic syndrometreated with prednisone shortly after the clinicalonset responded by the end of 8 weeks (early respon-ders). Since the treatment was so effective andevidence of drug toxicity was either absent or mini-mal, this regimen was considered satisfactory forinitial therapy. However, 24-2% of the patients(28 6% of early responders) subsequently had frequentexacerbations of proteinuria (frequent relapsers), eachrequiring restitution of therapy. These children,therefore, are at risk of periodic bouts of suboptimalhealth and significant steroid toxicity. The desire to

improve the poor outcome of the patients who con-tinue to have proteinuria after prednisone therapyand to avoid steroid toxicity in patients who relapse’frequently has stimulated the use of a variety of otherdrugs, notably cytotoxic compounds. Controlledstudies have provided strong evidence that cytotoxicagents are very effective in patients with frequentrelapses, and uncontrolled observations have sugges-ted that cytotoxic agents were effective in treatingpatients who did not respond to steroids..

The present therapeutic trial was designed to answertwo questions: is cyclophosphamide, in combinationwith intermittent prednisone, superior to intermittentprednisone alone in inducing a response in patientswho still have proteinuria after 8 weeks of steroid

therapy; and is cyclophosphamide superior to inter-mittent prednisone in preventing or decreasing the

frequency of subsequent relapses in frequent relapsers?The results in early non-responders indicate that

addition of cyclophosphamide to intermittent pred-nisone did not increase the likelihood that protein-uria would disappear. However, in patients in whomproteinuria did disappear (late responders) cyclophos-phamide hastened the time of response, suggestingeither a different mechanism of action or a higherpotency than prednisone. The great majority of lateresponders had histological renal lesions which are

generally considered to have a good prognosis.The results in frequent relapsers confirm previous

reports that cyclophosphamide is more effective thanprednisone in inducing a lasting remission. More-

over, among patients who do relapse, the frequencyof relapses was reduced as a result of a single courseof cyclophosphamide.

Methods

DefinitionsThe ncphrotic syndrome.- To be eligible for the trial

patients had to have heavy proteinuria and hypo-albuminaemia (defined below), to be older than twelve weeksand younger than sixteen years, to have not been treatedfor their disease with adrenocortical steroids or other agentsthought to have a similar effect, and to have no evidenceof underlying diseases or exposure to agents associated withthe nephrotic syndrome.

Hypoalbuminaemia.-2’5 g. or less per 100 ml. serum.

Heavy proteinuria.-40 mg. or more per hour per sq.m.determined quantitatively on an overnight collection.

TpoMM.—Demonstration of a protein-free urine on

three consecutive examinations on three separate daysduring the course of not more than seven days. Protein-free urine was one containing 4 mg. or less per hour persq.m. determined either quantitatively on an overnightcollection or semiquantitatively on the first voided morningspecimen. Semiquantitative testing with sulphosalicylicacid or ’Albustix’ was interpreted as follows: negativeor trace, less than 4 mg. per hour per sq.m.; 2-4+,more than 4 mg. per hour per sq.m.

Relapse.-Demonstration of protein-positive urine(more than 4 mg. per hour per sq.m.) on at least threeconsecutive examinations on three separate days duringthe course of not more than seven days. If proteinuriareappeared in the presence of infection or if proteinuriawas minimal, the investigator decided if and when relapsehas occurred.

Early responder.-A patient who responded during theinitial eight weeks of prednisone therapy (for dosage see

Trial Procedure).Early non-responder.-A patient who has not responded

during the initial eight weeks of prednisone therapy.Late responder.-An early non-responder who responded

subsequently. In contrast to early responders in whomsubsidence of proteinuria was interpreted as a responseto treatment, the terms " respond " and " responder " inthis group of patients might .not describe a cause andeffect relationship.

Frequent relapser.-An early responder who had twcrelapses within six months of the initial response or foU!

relapses within any twelve-month period.

Trial Procedure

Renal biopsies were done before therapy was startedand were interpreted independently by four pathologistswho were not aware of the clinical course.

All patients were treated initially with prednisone, givendaily in divided doses for 4 weeks in a dosage of 60 mg.per sq.m. per day, followed by a 4-week course of inter-mittent prednisone, given in a dosage of 40 mg. per sq.m.per day in divided doses on 3 consecutive days out of 7.

Early non-responders were allocated randomly either toan experimental group (cyclophosphamide plus intermittentprednisone) or to a control group (intermittent prednisone).Cyclophosphamide was given initially in a dosage of5 mg. per kg. per day which was generally sufficient to

induce leukopenia (3000-5000 white blood-cells perc.mm.). The dosage was then decreased to 1-3 mg. perkg. per day in an attempt to keep the white-blood- cell-count in the leukopenic range. If the count fell below1000 per c.mm. the drug was discontinued, and reintro-duced only when the count had risen above 1800 perc.mm. The total duration of the therapeutic trial wa,

90 days. Supportive therapy (diuretics, dietary alterationsand antibiotics) was given at the discretion of the inw5(1

gator.Frequent relapsers were allocated randomly either to a.

experimental group (cyclophosphamide) or to a contro

425

group (intermittent prednisone). In both groups, therecurrence that qualified a patient as a frequent relapserwas treated with daily prednisone until the urine was

protein-free for 3 days. The control group then received

prednisone intermittently (in the dosage schedule describedabove) for 180 days. The experimental group was givencyclophosphamide for 42 days using the regimen describedfor early non-responders. During the first 10 days of

cyclophosphamide treatment, the patients in the experi-mental group received prednisone in a daily dosage of10 mg. per sq.m. per day. Relapses in the control groupduring the treatment period were managed with dailyprednisone until response, followed by a return to inter-mittent prednisone. In the experimental group, relapsesduring the 42 days of cyclophosphamide administrationwere treated with daily prednisone until response occurred.After the treatment periods, relapses in both groups weretreated with daily prednisone until response occurred,followed by 4 weeks of intermittent prednisone. To assessthe effect of a single course of cyclophosphamide on thefrequency of subsequent relapses no alkylating or cytotoxicdrugs were permitted during the succeeding 2 years.The condition of the patients was thoroughly assessed

before admission into the study and every 3 months there-after. Daily semiquantitative measurements of proteinin the urine were performed by the parents or patientsthroughout the entire period of observation. The infor-mation obtained was transcribed on specially designedforms which were forwarded to the I.S.K.D.C. centraloffice for computer coding, storage, and evaluation.The statistical analyses were done by Fisher’s t-test,

the chi-square test, or the difference between two pro-portions.’

Results

Between April, 1970, and June, 1972, 228 patients(158 male and 70 female) were found to fulfil the

requirements for admission into the study.

Early Non-responders33 children z5 % ) qualified for the trial as early

non-responders. 18 of them were treated with cyclo-phosphamide and 15 were continued on intermittentprednisone (table l). 10 of those treated with the

alkylating agent (56%) and 6 of those treated withsteroids (40%) became protein-free, a difference thatwas not statistically significant. Moreover, histologi-cal examination of the renal tissue revealed that 9 ofthe 16 patients who responded during the admini-stration of either of the two drugs had " minimalchanges ". Of the remaining patients who responded2 had proliferative glomerulonephritis, 1 pure mesan-

gial hypercellularity, 2 focal segmental sclerosis, 1focal sclerosis, and in 1 the histology was unknown.

TABLE I-EARLY NON-RESPONDERS

TABLE II-EARLY NON-RESPONDERS: INTERVAL BETWEEN BEGINNING

OF TREATMENT AND RESPONSE

The interval from the onset of the treatment to thetime of response was significantly shorter in the cyclo-phosphamide than in the prednisone-treated group(table 11).

Frequent Relapsers63 patients (27-6%) qualified for the drug trial as

frequent relapsers. 10 of them were lost to follow-

up or treated incorrectly and were excluded from theanalysis. Of the remaining 53 patients, 27 were allo-cated to cyclophosphamide and 26 to prednisonetherapy. The proportion of patients who relapsedover an average period of observation of twenty-twomonths was 48 % among those who were treated withcyclophosphamide and 88 % among those who receivedprednisone. (table ill). The average length of the firstremission among the patients who relapsed followingallocation to the trial was 120::t:39 (S.D.) days in thecyclophosphamide-treated group and 81 ± 17 days inthe prednisone-treated patients. The difference is

statistically significant (P<0-001). Although 13 (48%)of the patients who received cyclophosphamide eventu-ally relapsed again, the frequency of subsequentrelapses was significantly lower among them thanthat observed among the patients who received

prednisone.

TABLE III-FREQUENT RELAPSERS: RELAPSE AFTER ALLOCATION TOTRIAL

* Relapses per patient per year.

The proportion of patients who relapsed duringtherapy was significantly higher in the prednisonethan in the cyclophosphamide-treated group whenanalysed at 42 days (duration of cyclophosphamidetherapy) (table iv).The immediate side-effects of cyclophosphamide -

therapy included temporary alopecia of various degrees <<*

(50% of the patients) and mild cystitis (4% of

patients). Observations in regard to the integrity of

TABLE IV-FREQUENT RELAPSERS: INTERVAL BETWEEN ALLOCATIONTO TRIAL AND RELAPSE

Percentages are cumulative.

426

the gonads are not yet available since 84 % of the

patients who received cyclophosphamide are still

prepubertal.Discussion

The results presented in this prospective, controlledtrial on the effect of cyclophosphamide in childrenwith the nephrotic syndrome offer new informationon the treatment of patients who continue to haveproteinuria after 8 weeks of treatment with predni-sone. Besides confirming previous reports of the

therapeutic efficacy of cyclophosphamide in frequentrelapsers, the study also provides data on some

characteristics of the clinical response and permitssuggestions about the relative value of different thera-peutic regimens. Finally, the results serve as a basisfor attempting to establish guidelines regarding theuse of cyclophosphamide in children with the

nephrotic syndrome.Most studies in early non-responders have not

been controlled,3 and in some histological diagnoseswere not available. The only other controlled thera-peutic trial in early non-responders was conductedby the I.S.K.D.C.,’ and showed that azathioprine plusintermittent prednisone had no advantage over inter-mittent steroids alone.The major observation in early non-responders is

that about half of them did become free of protein-uria subsequently. There was no difference in this

regard between patients who received intermittent

prednisone alone and those who received it in combi-nation with cyclophosphamide. It should be pointedout that 6-8% of the patients with minimal changeson renal biopsy continued to have proteinuria after8 weeks of prednisone administration; they accountedfor 50% of the early non-responders and for 60%of the early non-responders whose proteinuria subse-quently subsided. The effect of treatment on thecourse of the disease in these patients cannot beassessed; the only suggestion that either drug affec-ted the result was that the time of response was earlierin patients receiving cyclophosphamide. Only 3 ofthe late responders had a lesion (focal sclerosis) whichis considered to be resistant to therapy. Whether ornot the administration of cyclophosphamide influencesthe outcome in patients with focal sclerosis is beinginvestigated in a therapeutic trial conducted by theI.S.K.D.C.The results in frequent relapsers demonstrate a

clear superiority of cyclophosphamide over predni-sone in reducing the proportion of patients who

relapse subsequently, confirming the impressionsgained from uncontrolled observations 3,6,8-14 and the. "!’> evidence of controlled trials .15-11 However, severaladditional observations emerge from the present find-

ings. Cyclophosphamide seems to be more effectivethan prednisone in suppressing proteinuria during theadministration of the drug. Only 1 patient relapsedwhile on cyclophosphamide whereas 70% of thosewho eventually relapsed in the prednisone group didso during treatment. This finding suggests that inter-mittent prednisone is not effective in preventingrelapses in frequent relapsers. Conclusive evidencewould require a comparison of patients given inter-mittent steroids with untreated controls. However,even if 100% of untreated patients were to have

TABLE V-FREQUENT RELAPSERS TREATED WITH CYCLOPHOSPHAMIDE

subsequent relapses, clinical value of the treatmentwould be very questionable.The use of leukopenia as a biological marker for

cyclophosphamide administration generated differ-ences in the total number of days individual patientstook the drug, permitting a comparison between theactual number of days on treatment and the patternof relapse during the follow-up period (table v).Patients who relapsed received cyclophosphamide forfewer days and in slightly lower total dose than thosewho were still in remission at the end of the observa-tion. Although these differences are not statisticallysignificant the trend is strong, especially in regard todays on treatment, giving support to the finding ofBarratt et aJ.l8 that the length of treatment influencesthe duration of the remission. This is not incom-

patible with the observation of McCrory et al.,19 whocompared cyclophosphamide 2’5 mg. per kg. per dayfor 90 days with 5 mg. per kg. per day for 45 days,and found that the therapeutic outcome was similar inboth groups but toxic effects were greater in thoseon the higher daily dose. It seems, therefore, that tobe effective in frequent relapsers cyclophosphamideneed not induce leukopenia and that it becomes lesseffective when the duration of administration of the

drug falls below a critical level.Although differences in the therapeutic regimens

make comparisons with other series difficult, there isa suggestion that those trials in which steroids wereadministered together with cyclophosphamide 17,19resulted in longer average remissions than trials, suchas ours, in which cyclophosphamide was used essen-tially alone 15 About 80% of patients given bothcyclophosphamide and prednisone were still in re-

mission after 2 years of observation compared withabout 60% of those receiving cyclophosphamide only(see figure).

Actuarial analysis showing percentage of patients remainingin remission during 24-month period after treatment.

(a) McCrory et al.19 (b) Drummond et al." (c) This C.&egrave;.

(d) Barratt and Soothill.115 (e) Cameron. 29

427

The decision concerning whether or not cyclophos-phamide should be used in addition to prednisone inthe treatment of children with the nephrotic syndromemust rest upon the balance between therapeuticefficacy and toxicity of each of the two agents. Themost dangerous side-effect of cyclophosphamide seemsto be permanent suppression of reproductive cells,especially in the testis .20-21 Although such an effectcan occur even when the drug is administered beforepuberty,26-28 it would appear that the duration ofadministration correlates directly with the severityof the damage.

Conclusion

On the basis of this and other controlled trials thefollowing guidelines are recommended: cyclophos-phamide is not indicated in the treatment of earlynon-responders; it should not be used to treat fre-

quent relapsers unless patients seem to be in dangerof developing serious steroid toxicity; and if used inpatients with the nephrotic syndrome, cyclophos-phamide should be given together with intermittentsteroids in dosages of 2-3 mg. per kg. per 24 hoursand for no longer than 8 weeks.

The following directed and conducted the trial: Central Office,H. L. Barnett, C. M. Edelmann, Jr., I. Greifer, and A. Spitzer; regionaldirectors, G. C. Arneil, G. Gordillo-P., N. Hallman, O. Kobayashi,H. Ttddens, and L. B. Travis; regional coordinators, 1. B. Houston andR. H. Kuyten; pathologists, J. Bernstein, J. Churg, R. Habib, andR. H. R. White; consultants, J. S. Cameron, E. Lewis, and C. D. West;statistician, H. Levine; heads of participating clinics, G. C. Arneil(Glasgow), A. Fanconi (Winterthur), G. Gordillo-P. (Mexico City),A. B. Gruskin (Philadelphia), I. B. Houston (Manchester), O. Kobayashi(Nugata), R. H. Kuyten (Utrecht), E. Leumann (Zurich), J. F. Lewy(New York), M. I. McVicar (New York), J.-G. Mongeau (Montreal),M. A. Nash (New York), O. Oetliker (Bern), D. Santos (Mexico City),K. S. Scharer (Heidelberg), H. Stark (Petach Tikva), L. B. Travis(Galveston), Y. C. Tsao (Hong Kong), J. Vilska (Helsinki), C. D.West (Cmcmnati), and R. H. R. White (Birmingham).

This study was supported by the National Institutes ofHealth Research grant AM 14490-03, Kidney Foundation ofNew York, Kidney Disease Institute of the State of New York,National Kidney Foundation, U.K., and the John Rath Founda-tion.

Requests for reprints should be addressed to Dr Adrian

Spitzer, I.S.K.D.C., 1410 Pelham Pkway So., Bronx, N.Y.

10-161, U.S.A.

REFERENCES

1. Snedecor, G. W. Statistical Methods. Ames, Iowa, 1956.2. Churg, J., Habib, R., White, R. H. R. Lancet, 1970, i, 1299.3 Grupe, W. E., Heymann, W. Am. J. Dis. Child. 1966, 112, 448.4 White, R. H. R., Cameron, J. S., Trounce, J. R. Br. med. J. 1966,

ii, 853.5 Campbell, R. A., Jacinto, E. Y. J.-Lancet, 1967, 87, 149.6 Pachioli, R., Genova, R. Pediatrics, 1971, 47, 731.7 Abramowicz, M., Arneil, G. C., Barnett, H. L., Barron, B. A.,

Edelmann, C. M., Jr , Gordillo, P. G., Griefer, I., Hallman, N.,Kobayashi, O., Tiddens, H. A. Lancet, 1970, i, 959.

8 Coldbeck, J. H. Med. J. Aust. 1963, ii, 987.9. West, C. D , Hong, R., Holldan, N. H. J. Pediat. 1966, 68, 516.10 Etteldorf, J. N., Roy, S., Summit, R. L., Sweeney, M. J., Wall,

H. P., Berton, W. M. ibid. 1967, 70, 758.11 Drummond, K. N., Hillman, S. A., Marchesault, J. H. V., Feldman,

W Can. med Ass. J. 1968, 98, 524.12 Moncrieff, M. W., White, R. H. R., Ogg, C. S., Cameron, J. S.

Br. med. J. 1969, 1, 666.13 Dundon. J., Carroll, R., Dunleavy, M., Raftery, J. J. Irish med.

Ass. 1971, 64, 243.14 Moon, J. B., Chase, H. P. Jap. J. Pediat. 1971, 24, 1795.15 Barratt, T M., Soothill, J. F. Lancet, 1970 ii, 479.16. Tsao Y. C. Young, C. H. Archs Dis. Childh. 1971. 46 327.

17 Chiu, J., McLaine, P. N., Drummond, K. N. J. Pediat. 1973, 82,607.

18 Barratt, T. M., Cameron, J. S., Chantler, C., Ogg, C. S., Soothill,J S. Archs Dis. Childh. 1973, 48, 286.

INTERNATIONAL STUDY: REFERENCES&mdash;continued

19. McCrory, W. W., Shibuya, M., Lu, W. H., Lewy J. E. J. Pediat.1973, 82, 614.

20. Fairley, K. F., Barrie, J. U., Johnson, W. Lancet, 1972, i, 568.21. Kumar, R., Biggart, J. D., McEvoy, J., McGeown, M. G. ibid.

1972, i, 1212.22. Qureshi, M. S. A., Goldsmith, H. J., Pennington, J. H., Cox, P.

ibid. 1972, ii, 1290.23. Uldall, P. R., Kerr, D. N. S., Tacchi, D. ibid. 1972, i, 693.24. Cameron, J. S., Ogg, C. S. ibid. 1972, i, 1174.25. Greifer, I., Barnett, H. L. Kidney Int. 1972, 2, 352.26. Pennisi, A. J., Penny, R. Grushkin, C. M., Lieberman, E., Annual

meeting of the American Society of Nephrology, held in Washing-ton, D.C., in 1973; abstr. p. 82.

27. Penso, J., Lippe, B., Ehrlich, R., Smith, F. G. ibid. p. 83.28. Rapola, J., Koskimies, O., Huttunen, N. P., Floman, P., Vilska, J.,

Hallman, N. Lancet, 1973, 1, 98.29. Cameron, J. S. J. R. Coll. Physcns, 1971, 5, 301.

THYROID-STIMULATING

IMMUNOGLOBULINS IN GRAVES’ DISEASE

BERNARD REES SMITH REGINALD HALL

Endocrine Unit, Department of Medicine, andDepartment of Clinical Biochemistry, University of

Newcastle upon Tyne

Summary A receptor assay for T.S.H. using humanthyroid membranes and receptor-

purified 125I-labelled T.S.H. was used to study theinteraction of Graves’ immunoglobulins with thyroidmembranes. Immunoglobulins from twenty-two of

twenty-five patients with Graves’ disease clearly in-hibited the binding of T.S.H. to human thyroid mem-branes. Significant effects were also observed withone out of thirteen immunoglobulins from patientswith Hashimoto’s disease, and one out of six immuno-globulins from patients with thyroid cancer. Compari-son of the effects of Graves’ immunoglobulins on thebinding of T.S.H. to human and guineapig thyroidmembranes provided good evidence for the speciesspecificity of some thyroid-stimulating immunoglobu-lins.

Introduction

THE sera of patients with Graves’ disease have beenshown to contain at least two types of thyroid-stimu-lating immunoglobulins-long-acting thyroid stimula-ted 1 (L.A.T.S.) and L.A.T.S.-protector 2,3 (L.A.T.S.-P.).L.A.T.S. stimulated the thyroid activity of several

species,4 whereas L.A.T.S.-P. was specific for the humanthyroid.’

Both L.A.T.S. and L.A.T.S.-P. can bind particulate 2,6and soluble thyroid extracts.’,’,’ However, they repre-sent a small proportion of the total serum-immuno-globulins,7.9 and so far it has not been possible todevelop a reliable radio-receptor assay for thesematerials using labelled Graves’ immunoglobulins.

Receptor assays using thyroid membranes havebeen described for thyroid-stimulating hormone

(T.S.H.).10,11 Graves’ immunoglobulins have beenshown to inhibit the binding of T.S.H. to guineapig andhuman thyroid membranes,10-12 which suggested thatthe binding sites for Graves’ immunoglobulins wererelated to the T.S.H. receptor. Thus, it seemed possiblethat a radio-receptor assay for thyroid-stimulatingimmunoglobulins could be developed using the bind-ing of T.S.H. to thyroid membranes. In view of the

species specificity of thyroid-stimulating immuno-