JOURNAL OF SURGICAL RESEARCH 72, 84–88 (1997)ARTICLE NO. JR975145

Phase-Directed Therapy and Cardiac Xenograft Survival1

Hua Lin, M.D., Brian R. Gastman, M.D., Ru-Qi Wei, M.D., Steven L. Kunkel, Ph.D.,David Gordon, M.D., and Steven F. Bolling, M.D.2

Thoracic Surgery Research Laboratory, Departments of Surgery and Pathology,University of Michigan Medical Center, Ann Arbor, Michigan 48109

Submitted for publication January 22, 1997

sponses. Only once this is resolved, can xenograft sur-Xenotransplant rejection is facilitated not only by T vival be an attainable goal.

cell upregulation but also by endothelial activation Therapy directed at each specific phase of xenorejec-and B cell/antibody mechanisms, which standard im- tion may improve xenograft survival. The standardmunosuppression is unable to overcome and xenore- therapy for allografts, cyclosporine (CsA), alters cellu-jection ensues. However, therapy directed specifically lar response, acting on T cells by inhibiting IL-2 produc-at each phase of xenorejection may improve xenograft tion [2–4]. Cyclophosphamide (Cyc) abrogates humoralsurvival. To study this we used a heterotopic cardiac responses by interacting with B-lymphocytes and de-xenotransplant model (Syrian hamster to Lewis rat). creasing serum antibody titers [5, 6]. However, CsA orControls had no immunotherapy. Xenorecipients re- Cyc alone have not been shown to significantly alterceived cyclosporine to restrict T cellular response/de-

rejection time in concordant xenomodels [4, 7]. Re-velopment or cyclophosphamide, an antiproliferative,cently, TNF has been implicated as a major cytokineto reduce xenoreactive clones and antibody/comple-involved in EC activation. Conversely, anti-TNF hasment injury, or anti-TNF antibody to alter cytokinebeen shown to prolong allograft survival by decreasingcascades and endothelial activation/inflammation.TNF levels and downregulating cytokine productionFurther xenorecipients received combinations. While[8–10]. Therefore, we investigated directed combina-single modalities alone did not enhance survival, com-tions of CsA with Cyc and anti-TNF antibody in pro-binations appeared to be at least additive in vivo, sug-longing concordant cardiac xenograft survival.gesting that therapy directed at specific phases of

xenorejection may prove useful. q 1997 Academic Press

MATERIALS AND METHODS

Cardiac transplantation. Inbred male Lewis rats (150 to 200 g)INTRODUCTIONwere used as recipients and male Golden Syrian hamsters (100 to 150g) were used as donors. The neck heterotopic heart xenotransplantTransplantation is accepted therapy for end-stagetechnique, without ischemia or reperfusion, was accomplished with

cardiac failure and returns patients to an acceptable near 100% survival and has been reported previously [9]. Xenograftlife. However, due to organ shortages, only 60% of pa- survival was assessed by daily palpation and confirmed by EKG.

The day of rejection was defined as the day of cessation of palpabletients on heart transplant waiting lists eventually un-heartbeat and was verified by histopathology.dergo transplantation. Donor organ shortage is likely

Immunosuppressive agents. Cyclosporine A was obtained fromto worsen, as the UNOS waiting list grew more thanSandoz Pharmaceuticals, East Hanover, New Jersey. Cyclophospha-300% over the past 4 years [1]. Owing to this allograft mide was obtained from Adria Laboratories, Columbus, Ohio, and

shortage, xenotransplantation as an alternative has anti-TNF antibody, raised against recombinant human TNF injectedinto rabbits, was kindly provided by S. Kunkel, Ph.D. The prepara-gained attention. Xenograft rejection differs from allo-tion and specificity of the anti-TNF sera have been described pre-graft rejection, as one must overcome not only the cellu-viously [10].lar rejection of allografts but also humoral responses as

IgM measurement. Levels of IgM in rat serum were determinednatural preformed antibodies exist against xenografts,using an ELISA technique. Goat anti-rat IgM was incubated on mi-

and, secondly, endothelial cell (EC) activation, as xeno- crotiter plates at a concentration of 10 mg/ml in PBS. The platesgraft endothelium is a major site of host immune re- were washed at 47C with PBS containing 1% BSA and 0.04% Azide.

Rat serum (100 ml) at 1:4000 dilution in the blocking buffer anddilutions of purified IgM standard, 40000 to 39 ng/ml in blockingbuffer, were added to individual wells. The plates were incubated1 Supported in part by an American Heart Association Grant

93008360. and washed. Goat anti-rat IgM conjugated to alkaline phosphataseat 1.6 mg/ml in Elisa Tris Buffer at pH 8.0 was added and reacted2 To whom reprint requests should be addressed at The University

of Michigan Hospitals, Section of Thoracic Surgery, 1500 E. Medical with 100 ml of 1 mg/ml p-nitrophenyl phosphate in Elisa carbonatebuffer at pH 9.8. The reaction was allowed to proceed for 60 min;Center Drive, 2120D Taubman Center, Box 0344, Ann Arbor, MI

48109. then absorbance at 405 nm was determined. Concentration of IgM

840022-4804/97 $25.00Copyright q 1997 by Academic PressAll rights of reproduction in any form reserved.

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85LIN ET AL.: DIRECTED THERAPY FOR XENOTRANSPLANTS

TABLE 1 7 days prior to transplant in triple combination therapy(45 vs 78 days).Xenograft Survival

Effect on IgM serum levels. A significant decreaseSurvival time Mean survival in the levels of IgM was noted with anti-TNF, Cyc, and

Group n (days) time { SE CsA. Using naive animals as matched controls (n Å 6),three animals with xenografts that were nonrejectingControl 5 3, 3, 3, 2, 3 2.8 { 0.2and treated with anti-TNF, Cyc, and CsA had IgM lev-CsA 5 6, 12, 18, 11, 11 11.6 { 1.9*els of 82–160% of that of naive controls (Fig. 1a), whileAnti-TNF 4 3, 3, 3, 3 3 { 0

Cyc0 4 4, 4, 4, 4 4 { 0* three rejecting xenorecipients (with other treatments)CsA / Anti-TNF 7 27, 28, 27, 30, 27 { 0.6*,† had IgM levels of 332–549% of that of controls (Fig.

26, 26, 25 1b), confirming xenoantibody attenuation and/or iso-CsA / Cyc0 4 28, 25, 28, 25 26.5 { 1.5*,†type switching. Late IgM levels at ú80 days showedCsA / Anti-TNF / Cyc0 4 43, 46, 40, 51 45.0 { 2.3*,†

CsA / Anti-TNF / Cyc07 5 27, ú80, ú86, 78.6 { 13.5*,†,‡ an increase in titers regardless of rejection status orú100, ú100 treatment regime.

Histological examination. Histological examinationNote. CsA, cyclosporin 15 mg/kg/day im q.d. 1 7 d, then q.o.d. 1of xenografts at rejection 03 days following transplant14 d, Anti-TNF 1 ml ip on the day of transplantation; Cyc, cyclophos-

phamide 75 mg/kg ip at Day 07; or cyclophosphamide 75 mg/kg ip in untreated animals revealed extensive thrombosis,at Day 0. úThe animal was sacrificed by experimental design with hemorrhage, and necrosis in the myocardium, with in-the donor hamster heart still beating. *P õ 0.05 vs control. † vs Cyc, terstitial edema and a mild mononuclear cell infiltra-‡ vs Cyc0. tion. With Cyc/CsA/anti-TNF therapy on Day 3, there

was minimal change in the xenografts. Following rejec-tion of a xenograft treated with CsA/anti-TNF on Day

in serum was determined by a comparison of absorbencies for serum 28, infiltration of mononuclear cells and fibrosis of thesamples with the IgM dilution series run on the same plate. myocardium were observed. However, with CsA/anti-

Experimental design. Eight groups of xenograft recipients were TNF in combination with Cyc, there was minimal histo-studied. These included controls and animals treated with CsA im logic change in a xenograft harvested on Day 102, as15 mg/kg/day, started at the day of transplant, which after 14 days

only scattered fibrosis was noted and most of the myo-was changed to 3 times/week; anti-TNF as a single injection of anti-TNF 1 ml ip on Day 0; or CyC (75 mg/kg) ip on the day of transplant, cardium was normal.all given alone or CsA and anti-TNF or CsA and CyC or all threedrugs with Cyc on Day 0 or 7 days before transplantation.

DISCUSSIONStatistical analysis. Data were analyzed for statistical signifi-cance by ANOVA or Student’s t test, where appropriate. Differenceswere considered to be statistically significant at a confidence limit Xenografts have long interested transplant surgeons.of 95% (Põ 0.05). Mean survival times for each group were compared

However, results of clinical xenografting have been dis-by analysis of variance, as appropriate. Animals received humanecare in compliance with the ‘‘Principles of Laboratory Animal Care’’ appointing, as the mechanism of xenorejection has notformulated by the National Society for Medical Research and the been satisfactorily defined [6]. Organs experimentally‘‘Guide for the Care and Use of Laboratory Animals’’ prepared by xenotransplanted between discordant species are re-the National Academy of Sciences and published by the National jected hyperacutely (within minutes to hours), whileInstitutes of Health (NIH Publication No. 80-23, 1978).

those transplanted between concordant species are re-jected in days.

RESULTS The objective of our study was to alter graft sur-vival of concordant cardiac xenotransplants by possi-

Xenograft survival results are shown in Table 1. bly modifying humoral rejection and EC activation,Single modality. The median graft survival in un- as well as cellular responses. We utilized the immu-

treated animals was 3 days. No differences in graft nosuppressive drug CsA due to its known anti-rejec-survival were seen in rats receiving a single dose of tion effects in allotransplantation. CsA acts primar-anti-TNF compared with controls. Survival between ily as an anti-IL-2 agent, blocking g-interferon andcontrol and Cyc treatment alone was also similar (3 depressing recruitment of monocytes. CsA wasvs 4 days). However, CsA alone did prolong xenograft shown to work synergistically in our xenotransplan-survival to 11 days. tation model, implying a CsA-mediated decrement in

cellular xenoresponse. However, CsA alone did notCombined modalities. The addition of anti-TNF toCsA significantly prolonged xenograft survival to 27 result in long-term xenoengraftment. This is not sur-

prising, as even rats rendered totally deficient in Tdays (Põ 0.05 vs single modality). The addition of Cycto CsA also significantly prolonged xenograft survival cells will reject a hamster heart [12–14].

Many studies demonstrate the presence of xenoreac-to 26 days (P õ 0.05 vs single modality). A furthersignificant prolongation in graft survival (45 days) was tive natural antibodies (XNA) against antigens pre-

sented by cells from genetically different species. In theseen in rats receiving a combination of Cyc and anti-TNF added to CsA. Interestingly, there was a signifi- hamster to rat model, the hamster heart is confronted

with preformed XNAs whose levels rapidly increase,cant difference between rats receiving Cyc at Day 0 vs

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86 JOURNAL OF SURGICAL RESEARCH: VOL. 72, NO. 1, SEPTEMBER 1997

FIG. 1. IgM in the serum of LEW rat recipients, as determined by ELISA. The percentage change of IgM was determined by comparingthe level of IgM in naive rat serum. (A) Serum IgM levels from nonrejecting rats treated with Cyc, CsA, and anti-TNF at 0, 3, and 80 daysposttransplant. (B) Serum IgM levels from three rats at the time of cardiac xenograft rejection, Day 4 and 3 and 26 days posttransplant,treated with Cyc alone, anti-TNF alone, and CsA/anti-TNF, respectively.

elicit complement, and destroy the graft within 3 days transplant in combination with CsA and anti-TNF in-creased xenograft survival two-fold versus the samein untreated recipients [15]. We used the antiprolifera-

tive drug, Cyc, to potentially reduce xenoreactive regimen with Cyc given at Day 0, indicating that thereexists a crucial time period for cytoxic antibodies toclones. Cyc is a cytotoxic drug that effects short-lived

lymphocytes in the peripheral lymphoid tissue with a affect a xenograft.Cyc was only given once in this model, which impliespreferential effect on B-lymphocytes, but it may also

depress protein turnover and surface antigen presenta- that depleting XNAs may be important early in xeno-graft rejection and that a later rise in XNAs, as noted,tion. Cyc has been shown to deplete spleens of periar-

teriolar lymphoid sheaths within 3 days of administra- may not be deleterious. This concept of humoral ‘‘ac-commodation’’ is described as an adaptation so thattion [16]. Cyc is active for 2 weeks after administration

with peak activity at 7 days. In our study we found eventually there is no antibody-mediated xenograft re-sponse [17], despite XNAs. This accommodation maya significant increase in xenograft survival with the

addition of Cyc administration. Furthermore, the tim- be due to the antibodies changing or receptor alterationor to reactions wherein ECs protect themselves againsting of Cyc was important. Cyc given 7 days prior to

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87LIN ET AL.: DIRECTED THERAPY FOR XENOTRANSPLANTS

injury from XNAs and/or complement [18]. Thomas [18] to define anti-TNF antibody’s exact role in xenograftsurvival.found that early graft success was correlated with

In conclusion, we altered rejection of concordant xe-lower titers of cytotoxic antibodies and complement.nograft cardiac transplants, using therapy specificallyHowever, late increases did not correlate with survivaldirected at phases of xenotransplant rejection, includ-or rejection, as no quantitative differences in antibodying T cell upregulation, endothelial activation/adhe-titers in early rejecting grafts were noted comparedsion, and B cell/antibody mechanisms. This approachwith late nonrejecting surviving grafts. Our study con-may have clinical relevance.firms this, as the long-term surviving grafts had low

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