New Methods of Treatment for Renal Allotransplant Using the Baboon as a Primate Experimental Model

Vol. 102, Nov. Printed in U.S.A.

T1rn:JoUR.NAL OP UROLOGY

Copyright © 1969 by The Williams & Wilkins Co.

KEW METHODS OF TREAT~V[ENT FOR RENAL ALLOTRANSPLANTS USING THE BABOON AS A PRIMATE EXPERil\/fENTAL MODEL J. N. DEKLERK, G. P. MURPHY*, J . .J. W. VANZYL, ,T. A. VANZYL, H. W. WEBER, H. D. BREDE

AND

w. w.

SCOTT

From lhe University of Stellenbosch and the Karl Bremer Hospital, Bellville, Cape Province, South Africa and the James Buchanan Brady Urological Institute, Johns Hopkins Hospital, Baltimore, Maryland

treatment was given to 8 animals post-renal allO·· transplantation; these animals served as controls. Biological immunosuppression was given to 136 anirnals.t Therefore, a total of 235 grafts was evaluated. Fourteen animals did not receive a graft but were given one of the drugs to serve as unoperated controls. Since renal allografts were performed on animals of different groups from different geographic locations, less inbreeding and na-tural immunological compatibility occurred, in contrast to more in-bred animal groups obtained elsewhere.'' 2 All animals were compatibly matched in terms of their human ABO blood groups. 1 Details of the operative techniques, method of anesthesia and postoperative study have been reported. 1 • 2 Some animals received the aorta and vena cava with 2 kidneys, while in other animals of suitable size, single kidneys were successfully used with direct anastomosis of the renal artery and vein. Detailed renal functional tests, biochemical determinations and hematological and serological studies were completed in all experimental groups preoperatively and at weekly intervals postoperatively. The methods of these determinations have been described previously. 1 , 2 • 4 Endogenous creatinine clearance for glomerular filtration rate (GFR) and radiohippuran measurement for renal plasma flow (RPF) were perforrnecl as previously clescribed. 3 Five closes of subcellular kidney cell fractions (SKCF) were used. 4 The animals included in this

Herein is reported the effects of various types of immunosuppression in a sub-human primate, the baboon. The comparative base line data in untreated renal allotransplanted or intact baboons have been described previously. 1 - 3 These studies have shown the close similarity of baboon renal rejection to that observed in man.1 • 2 MATERIALS AND METHODS

Renal allotransplants were completed in male and female Chacma baboons, cared for at the Stellenbosch-Johns Hopkins Primate Project at the Karl Bremer Hospital. Postoperatively 91 animals received chemical drug treatment. t No Accepted for publication .July 1, 1968. Editor's note. Publication delayed due to foreign pasta.I complications. Read at annual meeting of American Urological Association, Miami Bea.ch, Florida, May 13-16, 1968.

Supported in part by the James Buchanan Brady Urological Institute of The Johns Hopkins HospitaJ, The University of Stellenbosch, The Council for Scientific and Industrial Research, The Cape Provincial Administration and the \Vestern Province Blood Transfusion Service. Requests for reprints: Dr. J. N. deKlerk, 1029 Medipark, Cape Town, South Africa or Dr. G. P. Murphy, Roswell Park Memorial Institute, Buffalo. New York. *Current address: Department of Urology, Roswell Park Memorial Institute, Buffalo, New York 14203 1 Murphy, G. P., Weber, H. W., Brede, H. D., Retief, F. P., Retief, C. P., van Zyl, J. A. and van Zyl, .J. J. W.: The significance of human ABO blood groups in the survival of untreated baboon renal allografts. Amer. Surgeon, in press, 1969. 2 Weber, I-L W., Brede, H. D., Retief, F. P., Retief, C. P., van Zyl, J. A., van Zyl, J. J. W. and i\forphy, G. P.: Morphological and functional alterations noted after baboon renal allotransplantation. J. Urol., 101: 465, 1969. 3 Johnston, G. S., van Heerden, P. D., van Zyl., J. A., van Zyl, J. ,T. W., Retief, C. P. and Murphy, G. P.: Renal function studies in the intact baboon. [nvest. Urol., 6: J.25, 1968. t l'dethods of chemical immunosuppression: Chloroquine (chloroquin), 5 mg./kg. Quinacrine (metacrine), 6 mg./kg. Thalidomide, 10 to 100 mg./kg. Imuran, 1 or 3 mg./kg., q.d. Hydrocortisone, 60 mg., q.cl. Cyclophosphamide, 3 to 70 mg./kg.

t Methods of biological immunosuppression, specific globulins: Hyper-immune serum, 15 ml., i.v RNA-ase (RNA),7 300 mg., i.m. Anti baboon goat , globulin (ABGG), 5 mg./kg. Anti baboon goat albumin a, /3 globulins (ABGAB) 600 mg. P globulin, alpha, beta baboon-goat, 180 mg. B globulin, alpha, beta, ba,boon-ververt monkey, 80 mg, X globulin, alpha, beta baboon-ververtgoat, 80 mg. 4 Brede, H. D. and Murphy, G. P.: Serologica,l methods applied to baboon renal allotransplan tation. S. Afr, Med . .J., 42: 22, 1969.

532

of the tepon did not exhihit g:ross thrornbosic at the anastomotic site of the tPc:hniel11 failure at autopsy. Thus, renaJ lunetion t,f:Sts and survival mt.BS thfc basis for the rrsults. histoevaluation was done. The principles and rnactices of the South African Animal 1Nelfare Society and .American Ka t10nal for Animal }\if eclical Research were obstrved throughout the studies RESULTS

Su,rvfra.l wi:th conventional chem:ical treatment The 14 animals given hydroc:ortisone lived 7 .Z ± ,54 clays. The 7 animals given 1 mg. per kg, irnuran survived 7 .6 ± 2.2 while the 8 animals gi1°en 3 mg. per kg. im11ran lived 7 .6 ± 1.7 days. The cornbined group treated with imurnn lived 7 .6 ± l .S clays. Animals treated \1·ith ;:: n-1g. per kg. cyclophospharnide survived 9.7 .± '.l.4 None of the animals treated one dose of the higher do8e schedule with rng. ver survived more than 7 Tbe mean surviYal rnte was 4.7 ± 2.9 results these studies view of the wrn not be discm,sed farther Survfoai with new chemfral treatment. Animals receiving .5 mg. per kg. chloroquine daily for the firnt nAcrrmc,vO·hs•n week lived 11.8 ± 3,9 Animals receiving .5 mg. per every other for the Arnt no,st,oner:1t1.ve week sun·ived 10.8 ± 7 All anima.18 treated with

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survived a n,can of 11.5 ± 3.7 renal functional cleclirrn wa:o morn severe than that observed in other untreated renal allotrans .. planted cont.;·ol animals J). 1 • 2 Concomitant, reduction in osmolai· clearance was nuwcL The pereentage of sodiurn reabsorption, a more proximal tubular J:unction,L 2 waf irnpain,d ieos severely in 1,hose animals receiving less chloroquine. Four animals quinacrine survived a mean of 12.2 ±. 6.1 post-renal aUotra.nsplantation. One of tlrn 3 control animals died. No significant or pre-mortem alterations in blood hematological values were observed. The postoperative reduction in the Hurviving animals of RPF, creatinine clearance and osrnolar clearance watS less severe in the animals treated with quinacrine (fig. 2). In fact, during the first postoperative week the levels of RPF and GFR ,vere significantly better (P < .01) than m untreated controls. Survival rate ill thalidomide-treated anirnals was related to the concentration of clrug adrninist,ered. Anirn.al,,. treated with one dose mg. per at rate of 21.2 ± ! 0.7 significant < compared to untreated controls ..Animals given repeated doses (HJ mg:. per kg, Several t.irneS nr.QT,,n,o>"> had a mean survival mte of 11 .J ± 2.7 Animals given tbe 1ngllest drug concentration (100 rng. per several Limes ,.c,,•,ww,,ni',,.,,,h, had

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mean survival rate of 7 .7 ± 2.2 days. There were various renal functional alterations observed in the groups given different doses of thalidomide post-renal allotransplantation (fig. 3). The postoperative decline in renal function expressed in terms of the level of elevation of urea nitrogen and depression of osmolar clearance, 24-hour urinary output, creatinine clearance, RPF and percentage tubular reabsorption of sodium, was in direct relation to the drug dose. Animals

receiving only one dose of 10 mg. per kg. thalidomide did best and their renal function after 2 weeks was comparable to, or better than, that of untreated control animals. However, even this was not sustained since all these animals died from renal rejection by 33 days post-renal allotransplantation. Biological immunosuppression. Untreated animals survived 9.7 ± 5.6 days. Six animals with .02 cc SKCF survived 12 ± 4 days and 13 ani-

nmls with 2 SKC.F survived 20 ± 21 One of t,be animals lin•d 89 before renal rejectioll and death. Five animals with 20 cc SKCF survived 7 ± 4 and a,11 died with thrombosis of the renal not associated with technical failure. Six animal::; with 0.2 cc SKCF survived 16 ± g Six animals pretreated 4 weeb with 0.02 ec SKCF survived 7.0 ± 5.8 post-renal allotmnsplanta tion. ·with the exception of the group receiving 20 cc SKCF, all animals in the present group died 1vith gross evidence of renal The prolonged survirnl rates in 2 cc and 0.2 e;c SKCF groups were statistically significant <: .05).

Renal function resu.lts seen in SKCf.Lreated animals in the vanous Observations

groups (0.2 cc and 2 ce; RPF up to 3 weeks These im, levels were also associated with better levels of GFR and statistically significant pro .. longation of survival rate. ,m,rni,u,.,, t,o un .. treated controls," 2 the 2 cc and 0.2 ce Sl{CF groups had better levels of renal function at, 2 weeks. At 3 week, RPF in the 2 SKCF groups was

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also higher (figs. 4 and 5). Osmolar clearance and net tubular reabsorption of solute free water in all SKCF groups progressively fell postoperatively. Tubular reabsorption of sodium _was within normal range in all groups until 2 to 3 weeks post-renal allotransplantation when significant salt loss was noted in the 2 cc and 0.2 cc SKCF groups. Tubular failure was not noted in those animals surviving 3 weeks postoperatively. However, animals in the .02 cc SKCF group did have significant tubular salt loss at this time. The alterations in tubular handling of salt and GFR noted in SKCF animals were not previously observed in other untreated baboon renal allo-· grafts. 1 • 2 Decrease in tubular sodium handling was a terminal event and better levels of RPF and GFR were maintained for longer periods in untreated animals. Specific globulins. Comparable control untreated animals survived 9.7 ± 5.6 days. RNA-protein treated animals lived 7 .0 ± 2 days. ABGG animals survived 13 ± 7 days; hyper-immune serum treated animals lived 8.9 ± 3.2 days and ABGAB animals died after 10.5 ± 4.4 days. P globulin animals were all dead within 6 days. The mean survival was 2.2 ± 1.4. days. One P globulin plus antihistamine-treated animal survived more than 10 days post-renal allotransplantation. A mean :c;urvival rate of 6.9 ± 4.4 days was observed in this group. Therefore, the P globulin was quite toxic. Two unoperated animals were given doses similar to those receiving renal allotransplants. One animal died after receiving a P globulin injection. The remaining P globulin unoperated animals, ·with or without antibiotic therapy, all survived the injection. X globulin animals were all dead within 8 days post-renal allotransplantation except for one animal which survived 15 days. The mean survival rate was .5.3 ± 3.4 days. B globulin animals lived 6.4 ± 3.5 days. These limited results preclude further comment. PATHOLOGY

Chemically immunosuppressed animals. In the imrnunosuppressed groups the histopathological findings were essentially similar to the untreated control group but differed in degree. 2 Diminution of characteristic vascular and cellular infiltrations was most notable in the chloroquine, quinacrine and thalidomide groups. Subcellular kidney cell fractions. In this series the antigenicity of SKCF and their possible efficacy as immunosuppressants were tested.

Striking differences were found. Only positive alterations will be mentioned. The group receiving 2 cc SKCF intramuscularly had a hiµ;h incidence of p;raft thrombosis, but large renal infarcts were not encountered. The rejection reaction was much milder than in the control untreated group. Four of 13 baboons had no endovasculitis, 3 of these lived more than 10 days postoperatively; whereas all baboons of the control group surviving more than 10 days had endovasculitis. Fibrinoid necrosis was common but was not as severe as in the control group. The interstitial infiltration also was not as dense as in the control animals. The number of mast and eosinophilic cells infiltrating the rejected kidneys was smaller than in the control group. N ecrotizing arteries were not observed and glomerular fibrinoid necrosis was found only once; whereas this was observed in 30 per cent of the control animals surviving more than 10 days. The average postoperative survival time was prolonged to 20 days. Ten baboons received 0.2 cc SKCF intramuscularly. One baboon had an infarctecl autolytic graft so that histopathological details could not be identified. Thrombosis was found frequently. The histopathological changes were similar to those in the 2 cc SKCF group in that they were less extensive and less severe than in the control group. The average postoperative survival time was prolonged to 16 days. Since SKCF had proved its antigenicity and its dose-related enchancing properties it was attempted to use antisera against SKCF in ordE:r to improve the post-transplant survival time. The results were generally negative. SEROLOGICAL RESULTS

Serological studies for estimation of effects on chemical substances in allotransplantation include the observation of immunosuppressive properties. These may be specific in the form of the depression of heterohemagglutinin or of heterohemolysin titers, or may be unspecific, as shown by alterations in the hemolytic complement content. Sera of baboons were regularly examined for these ;3 substances. Chloroquine, quinacrine. The chemical immunosuppressive remedies chloroquine (chloroquin) and quinacrine (mepacrine) produced similar effects: 1) The heterohemagglutinins did not rise after transplantation, they did not surpass standard values and were relatively constant. 2)

E1Frnted re11al allotransplantation 111 baJmons and ultin1ate.ly readtdJ values below · they were de the ;,iandard n,··"""Pn in the quinac:rine series. 3) FreA hemolytic complement levels did not surpa8~ dilution titers of 1: H\ ; the lowest and highest titers \Vi thin normal limits of .intact animals, One of the 16 baboon:, treated with chloroquine-; reaeted with rheumatoid factor after 7 days of treatment. This may be mterpreted as due to the pre8enee of gamma globulin antibodies. Since in this case heteroagglutinins (gamma globulin) were not in enlarged quantities, another n1ecbanisrn has to be postulated for the explanation of the reaction. Pike and Schulze found that gamma. globulin from a. number of different species reaets with rheurnatoid and that the reactivity is variable: tl1is seerned to indicate a multiplicity of rh,mmatoid factors." AH these rhet1matoicl faetors are a, family of gamma-NI globulins, some of whir;h have specificity for determined antigenic grou,ps present un the heavy chains of gamma-G globuln1. 0 that it wac; possible to separate into 3 fragments clegebtion an.d that one fragment, the Fe, whrch contains ouly fragments of heavy chains, has no antibody but does carry the antigenic deterrn.inant.s which enilble it to combine wit.b rheumatoid factor.,; .A explanation of the rheumatoid factor reaction may be seen m thee appearance of gamma-G dtain S(·t free by breakage of cells atta,ck(od by antibodie,; of tr1e gamma-G elass. The C-reaetive reaction is another sign of acute phase cell reactions were not animals, Howobse1·ved in ever, .in ihe found: one ma,toid fact,or -reaction and another allotraus

th.is vva:3 ]in.:dt,ecl th8 a.si:?.oeiated of bvth ren,c\clieo. Neither drug lw.d any comi.n.fi11etiCf on. {he

boons treated with l mg. per kg_ imumn had heterobemagglutinins, sins and complement titers equal to untreated animals. It seems that this dose waii too low to produce immunos11ppression in baboons. However, this dose level wa.s not without other toxic effects. In the group treated witb 3 rng. per kg. every day the heteroagglutinins and het~ erohernolysins were suppressed. The heterohemag ~ glutinins 1vern suppressed to a greater degree th8,n the het.erohemolysins. Thern 11·as no infl.uern:e on the complement.. Hydrocortisone, 60 mg, intramuscularly every other did not influence the post-transplani. complement levels or the heterohemolysins level postoperatively. Imuran suppressed the format1011 of heterohernagglutinins in rena.1 ailotransplanted baboons, Baboons treated with a single dose of phospbaniicle of 70 rng. per did not survive long to allow serological exammation, 1.'he second eyclophospharnicle group Lreated w1th 3 rng. per kg. every clay showed a

tlrn and the cornplernent wa~ not influenccxl.

8'llbceUular cell rr,,n,,m,.,"' SKCF wa:'i first injected into 6 rabbits. After ;j administered over ,l weeh, the rabbits produced antibodies against SKCF i.11 form of fixing antibodies, sheep red cells henmggJutiarn"' and the original antigen antibodies. The latter formed up to 6 line~ in agar gel plai.es ( 6 per cent purified agar), hut not one line was significant of a baboon .speeific antibody. Baboons were treaied in gronpf with SKCF, but all these values have be regarded as masked, since 8l(CF absorbs heterohernolys1ns, heterohernaggluti11ins and complement linked to the

iVL L.: of in with anhriris serum, ,J. ImmunoL, 91: J. W : Re:wtion uf of with globulin. Proc . Soc.

the Iormat1011 of heternantibodi,:s, Interest m the frai:,tions u-f globulin was 3timuia,ted work of otl1ern. 'Mowbrnv, J. F, iJ,nd anLibody p/ocluctfor, by I1nn1un0l, 11: 421, 1966, Bondevik, IL, Inhibition of aliograft isolated from human

19Gl

1967.

588 Our anti-baboon goat albumin alpha beta globulin fraction was tested on 12 baboons. investigation showed a dear suppressinn of heterohemagglutinin:o and heterohemolysins, but no influence on the complement system. In other trials we eliminated albumin from the fraction and increased the close per baboon. This fraction was called P globulin for our own internal laboratory use. The 16 animals receiving this preparation died so suddenly that serological tests could not be performed. Bacteriological investigation showed a high incidence of endogenous infoc-especially with the common latent Salmonella sundsvall infection, with Staphylococcus aureus, Proteus mirabilis and Clostridium perfringens. Inoculations of guinea pigs without P-fraction showed similar results with endogenous infections, but they only occurred after 10 or more days. Rabbits, on the other hand, were very resistant. They survived 5 mL P-fraction intravenously for 5 weeks and showed only intermit short periods of inactivity. At present we can say that 150 mg. a.nti.-baboon goat alpha beta globulin per ba.boon acts as a strong immunodepressor which activates latent, and endogenous iDfections and, therefore, has to be combined with broacl.. spectrum antibiotics. The other preparations used did not differ markedly. DISC'CSSION

Significant prolonga.tion in chloroquine or quinacrine animals was not seen. Although the meari survivals in them drug-treated groups were all longer, there were no "'" t«, 1·,r,o significant differences from the heterogenic untreated con· trols ± 5.6 _ Functional studies ,.how a.ssociated decreases, e.g. RPF and GFR, which are more severe than those seen in untreated controls. This may be accepted as evidence of renal The death of un;mpports t.his it i.s unknown to what extem other extra.renal e.g. may have in the present series. This has been noted in humans 10 Both chloroquine and treatments ,rnm•,o,c,µ,i lymphocytosis " Mowbray, J I(: Ability of iarge doses of an a.lpha-2 plasma protein fraction to inhibit antibody production Immunology, 6: 217, 1963. 10 Alving, A, S., Eichelbergel', L., Crnige, B., Jr., Sones, R., .Jr., \Vhorton, C, JV[. and Pullman, T. N.: Studies on the chronic toxicity of chloroquine (SN-7618). J. Clin. lnvest., 27: 60, 1948.

to a limited degree. However, peripheral evider1ce of renal rejections was not abolished as a cornmonly boon renal allografts. 1 , The effects of chloroquine and quinacrine on DNA and RNA metabolism in general are responsible for the observed catabolic alterations in protein metabolism. 1 H Thus, it ,rns found that chloroquine a.nd quinacrine are toxic imrnunosuppressive agents in baboons, perhaps more so than in humans. Chloroquine is also toxic at similar dose levels to chimpanzees. 16 A recent report, has confirmed that quinacrine can retard the rate of skin allograft rejection in rabbits, but does not change tissue antigenicity_17 This result seems to confirm the present experimental observations. Thalidomide, by all the experimental criteria used, is a toxic immunosuppressive agent. The depression in postoperative renal function was less at the lower dose range. Survival was significantly extended in those animals a single 10 mg. per kg. dose of thalidomide. However, other biochemical, enzymatic and histological evidence of hepatic and all aL1imals cortisone and cyclophosphamide all failed to prolong renal allogra.ft survival in the baboon. In fact, by all available criteria these agents were directly nephrotoxic. The severe impairrnent in postoperative renal function and rnetabolic status can only be interpreted in this light. Untreated baboons given simi.lar closes of these drugs also had some renal impairment. Imuran is also toxic at similar dose levels in the rhesus u S, N, and I{,_ L Inhibition ,)f DNA RNA polymerase reactions by chloro. quine. Proc. Nat. Acad, Sci., 54: 521, 1965. 12 Ciak, ,J. and Hahn, F, E.: Chloroquine: mode of action. Science, 151: 347, 1966, ·· " Goldberg, I. H,: l\fode of action of antibiotics. IL Drugs affecting nucleic acid and protein synAmer. J. M.ed., 39: 1965. N. B. &.nd L K: lieaetion and quina.criue and m11~.,·1n1:, J. La,b, 60: D.

DJ',TA 101: 335, J963 16 Hickrrrnn, R L, Gochenour, W, S., Jr o.nd Ma.rsha.11, J_ D,, Jr.: Drug resistant Plasmodium faleiparum in th~ chimpanzee. Milit. Med., voL suppl. 19, p. 935, 1966. Harrisori, H. N., Stein, S. F. and Ahie, M ..J : lmmunosuppression without lymphocyte depletion using quinacrine HCL Clin. Res., l5: 294, 1967. l 8 Murphy, G. P .. Johnston, G. S., Mira.nd, E

ha.s other toxic effeets in the benefit of such drugs in prerenal ::tllotransplant function was undetectable in these baboons. Cortisone prepara. used as uu,,~UICIH agents in the dog, prolonged renal survival rate in one study. 20 Steroid use in humans has usually been in combination with other modes of therapy. 21 Thus, the failure to prolong survival rate in this with hydrocortisone may be 1nore comparable to that seen in other primates. Imuran-treated animals exhibited more severe nephrotoxic features post-renal allotransplantation. Unoperated control irnuran animals cl.id not do so to the same degree. Therefore the post· state of renal insufficiency in wme of these animals may accentuate the deleterious effects of imuran. Such has been the case in humans treated with irnuran while in renal insufficiency and azot,emia.U The drug-related toxic effects in these instances have been attributed to direct, bone marrow The hernatoeffects of iro.uran ancl eye.lo· phosphamicle were also without benefit, as deter-· rnined in the present studies. The n"""""i-.,,.n of effec1jve RPF and GF:R noted in the SKC.F groups was associated with statistically significant prolongation in posttra11splant survival rates. The kidney eelJ fraetions were effective on a dose-related basis. Reasons for the doserelated basis of kidney cell fractions are likely a reflection of the type of preparation used and the of different subthe widespread restances presenL. Yor nal thrornbo.si, noted with the large dose SKCF could be attributed to a biological conFurther purifica-modification uf these re1ml subcellufar fractions could improve the beneficial 3urvivaL This form of effect on however, deserves further re-evah.rntion ,vith different mudificatirms of SKCY pos·

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The failun, of some of the globulins to renal survival rate does 11ot infer that these sub .. sLances lack 1rrm1un,osui:m1·essnrc The lack of sucr:ess ,vith mdividual globulin preparations is in part attributable to speeific effecte,. The additional factors of infection and decreased immunity are also of considerv.·· tion as causes of early death. Hyper--immune serum and anti-baboon goat albumin a, (3 globulms (ABGAB) were also failures. The decreases in survival rate associated with renal insufficiency and protein elcctrophoretic changes were gener .. ally similar to RN.A.se treated animals, The reasons elaborated for the failurP of these agents are also believed to be .similar, although features of irnmunosuppression were evident. Anti-baboon goat I globulin, all available criteria, is an effective irnrnunosuppressive agent" The failure to indefinitely prolong survival rate may be limited by the fact tha,t the animaJs received only one close of the preparation. Pretreatment was not evaluated. At present it is not apparent what deleterious effects long-term SKGF or A.EGG administration may have. T_;nlike P, X and I3 globulins, ABGG was not toxic and no deaths could be attributed to the itself. Thme results, notwithstanding the present series of experirnents, have adequately demonstrated that: 1) baboon kidney cells are potent antigens; 2) subcellular fract,ions of renal tissue or globulins isolated from other goats or monkeys inJectecl with kidney cells, a,re .1m1nunosuppressive agents find 3) this form of on a dose-related basis can renal allotransplant survival. From a histopathological closes of 2 cc and 0.2 cc hyper-immune serurn, ABGG and .ABGAB were treated with these reaction than untreated control baboons. The rnode of act10n of the aJorementioned ,1nti2era. is not k:no\117n with HoYve.ver, the mode of action of SKCF in intramuscular doses 0.2 first a massive the of desensitization second of the recipient There is the third of neutralization of absorption of the reciprnnt's a11· tibodies the a.llografts. by an antigenic overload can be ruled out with

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the 2 aforementioned doses of SKCF. An antigenic overload was attempted with 20 cc SKCF, intramuscularly. The result was a hyperacute rejection reaction. Desensitization was attempted with 0.02 cc SKCF given as preoperative treatment. In these animals the findings closely resembled those of the untreated control group. Therefore, we propose that SKCF works, if administered in the right concentration, as a neutralizing agent which absorbs isoantibodies against the graft and complement. This could explain why SKCF is of beneficial influence only if administered in small doses, enough to neutralize circulating antibodies without inducing an immunological reaction resulting in a further production of antibodies. Such a conception matches with the hypothesis of the existence of a natural antibody to protect vertebrates against strange tissue. To challenge these ideas it seems to be advisable to study the possible synergistic effects of combining small antigen injections with immunosuppressive agents. In our baboon series with the aforementioned drugs there was evidence for insufficient depression especially of the heterohemolysins and for inability to suppress the formation of complement. The last point seems to be of great importance, since complement as a whole and not only in form of certain components is an important factor of rejection reactions. If we consider our serological observations in untreated and in treated allotransplanted baboons,4 another factor is noticeable. This is the evidence of naturally occurring preformed heterohemolysins and heterohemagglutinins. Animals with high titers, such as 1: 32 or more and high free complement titers (1: 64 or more) show an inferior survival rate. These animals often undergo acute rejection due to antibodies which are circulating at the time of transplantation. From serological observations on 427 baboons we learned that better results can be expected by eliminating animals with elevated titers against heterohemagglutinins, heterohemolysins or of complement. These animals are presensitized in some other way, most probably with gramnegative bacteria which often share partial antigens with body cells. The policy of testing candidates for transplantation previously for hetero-antibodies and for a surplus of free complement should also be extended to human beings. From the serological

point of view it is likely that the acute rejections in these baboons were entirely due to circulating antibodies. The early rejections occurring during the first 10 days were largely cell mediated: whereas rejections occurring from 11 days onwards were produced by humoral and cellular mechanisms characterized by coating of arteries, glomerular capillaries and arteriolae with immunoglobulins and complement. The larger arteries showed fibrin thrombi. The late and insidious rejection is further characterized by accumulations of IgM and complement on the glomerular capillary basement membrane, leading to slow impairment of renal function. These are the 4 distinct rejection forms we ean perceive. They are the same as the ones observed by Porter in kidney transplanted human beings. 22 SU:M:MARY

Antimalarial agents, such as chloroquine and quinacrine as well as thalidomide, are effective immunosuppressive agents i.n baboon renal a.llotransplants. However, their merit is limited by dose-related nephrotoxicity. Conventional immunosuppressive chemical agents such as imuran or cyclophosphamide are nephrotoxic in the baboon and fail to prolong survival rate. Hydrocortisone was without beneficial effect in terms of renal function, serology, pathological changes, metabolism or survival rate. Baboon subcellnlar kidney cell fractions can, on a dose-related basis, prolong renal allotransplant survival rate, with associated benefit in measured renal function. Other features of hematological irnmunosuppression were observed. Baboon kidney cells are thus strong tissue antigens, Specific globulins prepared from goats or monkeys immunized with baboon subcellular kidney cell fractions were also isolated. These possess certain toxic as well as immunosuppressive effects. Anti-baboon goat 'Y globulin fractions significantly prolong renal allotransplant survival rate with measured benefits to renal function. Other preparations rich in a protein fractions were ineffective. Doctors R. Schoonees, J. H. Groenewald, J. A. van Zyl, P. D. R. van 1-Ieerdon and C. P. Retief assisted in portions of this study. 22 Porter, K. A.: Rejection in treated renal allografts. J. Clin. Path., 20: 518, 1967.