Beavers, CD

Bier, M

Merkel, FK

Merriman, WG

Starzl, TE

Wilson, C

D-Scholarship@Pitt

Modification of Xenograft Response by Selective Plasmapheresis By F. K. MERKEL, M. BIER, C. D. BEAVERS, W. C. Mbooiy[A~I C. WILSON AND T. E. STARZL HYPERACUTE REJEcrION of human renal allografts in ABO compatible cases was first reported by TerasakU Kissmeyer-kielsonI~ Williams,3 and Starzl-l have further documented this problem, which has now been encountered in many active-transplant centers. Hyperacute rejection is initiated by preformed antidonor antibodies and may result in the destruction of the transplant within minutes of vasculariza-tion. A characteristic of this reaction is thrombotic occlusion of the microvascula-tureK~··; . The rejection of xenografts in widely divergent species, such as the pig to dog combination, is similar to hyperacute re-jection in humans. Naturally occurring heterospecific antibodies apparently cause rejection of renal xenografts within 6-15 minutes.6 The following experiments were designed to determine whether the xeno-graft response could be modified by selec-tive plasmapheresis ( SPF ) , an electro-phoretic process which can extract gamma globulin from the circulating blood. ~fA TERIALS «M> METHODS The SPF apparatus was developed by Bier in From the Departments of Surgery, University of Colorado ;\.[edical Center, The Denver General Hospital, and the Denver Veterans Administration Hospital, Denver, Colo.; the Tucson Veterans Ad-ministration Hospital, Tucson, Ariz.; and the De-partment of Experimental Pathology, Scripps Clinic and Research Foundation, La Jolla, Calif. Supported in part by Public Health Service Con-tract PH 43-68-621, and Public Health Service Grants Ai 04152, AI-AM 08898, AM 12148, AM 07772, RR 00051 and RR 00069, and by grants from the American Diabetes Association, the Colo-rado Heart Association, and the Eli Lilly Co. 1957.1 Two plastic endplates, containing ~ electrodes, surround a series of spacers, Illakfaa a cell pack much like some in use for hem~ I An external buffer carries away the toxic pr~ formed at the endplates and makes contact .... the cell. Temperature, pH, hemolysis, audt.w i shifts are controlled by an internal huier.' n. . membranes between the spacers are heavy ~ erated cellulose except between the hlood and the globulin channel, where a Millipore filter is UIed. ' A gradient of 6-8 volts! cm. results in giobaliaf extraction and prevents clogging of the MiIlipar. filter by the red cells. The apparatus is attadied ' to unanesthetized, heparinized mongrel doSJ by silastic extracorporeal shunts placed in the oedt vessels. Three dogs underwent SPF for approximateJ, four hours on two successive days and receiftd porcine renal xenografts toward the end of the second SPF (Fig. 1). One dog received the graft 1ST DAY 2No DAY 4 hours selective ~ 4 hours selective plasmapheresis pig to dog renal xenograft Fig. I.-Experimental model. After two 4-hour periods of selective plasmapheresis on 2 successive days, pig-to-dog xenograft is carried out and selective plasmapheresis is continued. .'534 TRANSPLANTATION PRQCEEDINGS, VOL. III, No.1 (MARCH), 1971 n ~tafcfCAno:--: Of XE:--:OCRAFT RESPONSE " •• ,I .• '::=:=---tfaKI~ S p r :;"'q_ J ~ I' r o 10 10 Ji) •• ;; ~K- ,: 8·' ~ 100 tlO 110 LJO 110 ISO 160 PO 110 DDDD~~EKy . Fig. 2.-Survival of pig-to-dog renal xeno-grafts. Untreated pig-to-dog xenografts rejected in 10 minutes. Electrodialysis (a process similar to selective plasmapheresis but without the ~1illipore filter essential for globulin extraction) and SPF with a 2-hour delay failed to prolong graft survival significantly. SPF dogs rejected their xenografts in 120, 90, and 180 minutes, extending survival by as much as eighteen times normal. two hOllrs after cessation of the second SPF. Elec-trodialysis was carried out on two dogs instead of SPF and followed by the transplant. In electro-dialysis. the same apparatus is employed, without the Millipore filter compartment. The difference is that no globulin extraction occurs. The onset of rejection was determined by a mottled appearance of the kidney and was judged complete when urine production ceased. Blood ;amples were taken at the beginning and end of ~ach SPF and at close intervals during and after the transplantation. These were analyzed for 'ormed blood elements, plasma hemoglobin, serum )roteins, fibrinogen, and complement. Biopsies for Fig. 3.-Fluorescent micrographs taken from PF dog 3. Stain is for fibrin. Samples A and ~ were taken from the pig xenograft at 30 linutes and sample C was at 60 minutes. Sec-ion D is dog's own kidney at 60 minutes, 'ibrin deposits seen in xenograft are type seen 1 unmodified recipients. It is not possible to uantiate this reaction. 1500 1000 soo (g._ globul1n)O "'91 Oogll--009'2 ••••••• 009') -------535 . : I t o I 2 3 4 Fig. 4.-Effect of setective plasmapheresis on gamma globulin. light and fluorescent microscopy were taken at the first sign of rejection and at selected periods until rejection was complete. RESULTS Pig-to-dog renal xenografts without SPF rejected within 10 minutes. Xenografts in SPF recipients survived with urine output of 120, 90 and 180 minutes. Mottling oc-curred about 30-45 minutes after grafting and increased in amount until urine output ceased, when the kidneys were solid purple in color. In the dog that had a 2-hour delay between SPF and transplantation, rejection occurred within 15 minutes. If electrodialy-sis was substituted for SPF, the xenograft response was equally rapid (Fig. 2). Oog,1 --009'2 ••••••• Oog'3 -----Fig. 5.-Effect of selective plasmapheresis on fibrinogen. Effect appears more variable than with gamma globulin. ;:t:r. !l:!I"" l. ..... '" ::10 -'(:" .... D~:~:IK -• .... ';1 .... ~D :: 11 p ~ -l' 5,16 lOa 200 (C'H50 )100 I a 4 (hours) h .. 2 I i I il 4 I I i OSlO is 20 is lO (minutes) A • r,nal artery SImple Y • rtnl1 y,ln Simple .' Fig. 6.-Elfect of selective plasmapheresis in complement. Note return to normal within 12 hours of SPF. Within 5 minutes of xeno-grafting, marked arterial-venous differences in complement could be detected across trans-plant, persisting for at least 30 minutes. This is in striking contrast to control grafts, where differences rarely persist for more than a few minutes. Light and fluorescent microscopy of the grafts in the SPF treated dogs showed fibrin deposits in the glomerular capillaries iden-tical to that in the rapidly rejecting non-treated controls (Fig. 3). The hematoxylin and eosin light microscopy revealed an in-creasing polymorphonuclear leucocytic in-filtrate with time. The abnormalities in the xenografts in treated and untreated re-cipients were the same, the only difference being the times for )lIese changes to de-velop. After 4 hours ~he SPF caused 60 per cent reductions of gamma globulin concentra-tions in the recipients with recovery to 74 per cent of pretreatment values overnight. At the end of a second SPF, globulin levels were about 25% (Fig. 4). Fibrinogen (Fig. 5) and complement (Fig. 6) were similarly effected. Platelets were reduced 40 per cent hy SPF (Fig. 7), Red cell destruction was considerable, with the result of a rising --- --~K- --:MERKEL ET .\L. !~oKooo 1'1 .... 1.' ct.)O Fig. 7.-Effect of selective plasmapheresis on platelets. Depression of platelet count by SPF apparatus is substantial, but it is clear that across xenograft significant arterial-venOUs differences in platelets can be measured that also persist much longer than in untreated con-trols. plasma hemoglobin. \Vhite cells were not depleted. During rejection by the conditioned or treated recipients, arterial-venous dif-ferences across the grafts of platelets and complement were detectable for 30-70 minutes. In the treated animals, the degree of prior gamma globulin reduction by SPF did not correlate well with the duration of graft survival. . DISCUSSION SPF provides an electrical means for modifying the xenograft response. It is clear that the present apparatus is a prototype and must be modified to obtain better and more consistent results. Nevertheless, sig-nificant prolongation of pig-to-dog renal xenografts was obtained, although even-tually the rejection went to completion. This points up the possible use of SPF for more detailed study of the xenograft response since a reaction occurring within 5-15 minutes is stretched out to several hours. The graft prolongation by SPF was most likely by globulin depletion. In the control studies using electrodialysis, but omitting the \Iillipore filter compartment which is ~[nar·fCK-y qll~ OF XEXOGRAFT RESPOl\SE essential for globulin extraction, nonspecific changes occurred in fibrinogen and comple-ment, but no graft protection resulted. In combination with other modes of immuno-therapy. SPF may eventually aid in the prevention of hyperacute rejection. COXCU'SIOXS SPF prolongs the response to procine renal xenografts in dogs. The effect of SPF 537 is prohably a result of globulin depletion and/ or reduction of complement. The use-fulness of this process in the study and pre-vention of hyperacute rejection has heen suggested. AChkltibadMb~q We wish to thank Drs. Charles Zudoski and Scott Clark, Miss Joyce Trembath, ~frK Paul Tay-lor, Mr. Yosh Arai, ~1rsK Isabelle Snow, and Mrs. Ruth Gibbs for their assistance. REFERENCES 1. Terasaki. P. I., ~farchioroI T. L., and Starzl, r. E.: In Histocompatibility Testing. Washington J.C., :\ational Acad. Sci.-;'\ational Res. Council, 1965, pp. 83-96. 2. Kissmeyer-Nielson, F., Peterson, V. P., Olson, ; .. and Fieldborg, 0.: Lancet 11 :6-62, 1966. 3. Williams, G. :\1., Lee, H. M., Weymouth, i. F., Harlan. W. R., Holden, K. R., Stanley, G. vi., Millington, C. 5., and Hume, D. M.: Surgery 12. 204, 196i. 4. StaTZl. T. E., Lerner, R. A., Dixon, R. J .. :;roth, C. C., Brettschneider, L., and Terasaki, P. 1.: ~ew Eng. J. :\Ied. 2i8:642, 1968. 5. Simpson, K.. Bunch, D. L., Amemiya, H., Boehmig, H. J., Wilson, C. B., Dixon, F. J., Co-burg, A. J., Hathaway, W:'E., Giles, G. R., and Starzl, T. E.: Surgery 68:77, 1970. 6. Linn, B. S., Jenson, J. A., Porteil, P., and Snyder, G. B.: J. Surg. Res. 8:211, 1968. i. Bier, M.: Science 125: 1684, 1957. 8. Bier, M., Beavers, C. E., Merriman, W. G., Merkel, F. K., Eiseman, B., and Starz!, T. E.: Trans. Amer. Soc. Artif. Int. Organs 16:325, 1970. ~;KCErI :!tIIl .. : ........ ::to :~ ~Dt>DDDDDD ;::a... ~ '--K:~K :., 

Modification of xenograft response by selective plasmapheresis.

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Modification of xenograft response by selective plasmapheresis

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Modification of xenograft response by selective plasmapheresis.

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