BACKGROUND:Further expansion of the donor pool with ischemically damaged kidneys will be predicated on the ability to develop prognostic testing. Using a well-established canine autotransplantation injury model, we assessed whether actual restoration of renal metabolism by ex vivo warm perfusion could be used to predict the status of an organ before transplantation. METHODS:Kidneys were subjected to 30 min of warm ischemia followed by 24 hr of static storage in ViaSpan at 4 degrees C. After warm ischemia and static storage the kidneys were transitioned to 3 hr of warm perfusion using Exsanguinous Metabolic Support technology. During this period, parameters indicative of renal metabolism and vascular function were used to predict outcomes prospectively. Parameters included measures of oxidative metabolism, perfusion characteristics, and vascular condition. A Viability Score (VS) was calculated as the sum of the three parameters mentioned above. Results were grouped by a VS>2 and a VS<2. RESULTS:A clear association between the severity and duration of graft dysfunction and the VS was observed. Organs with a VS>2 had a significantly milder period of acute tubular necrosis, with both a less severe rise in serum creatinine (mean of 4.4 vs. 11 mg/dl) and a shorter recovery period (mean of 8 vs. 18 days) than those with a VS<2. CONCLUSIONS:Results indicate the possibility of utilizing warm perfusion to evaluate kidneys before transplantation. The VS developed demonstrated efficacy in classifying the severity of the acute tubular necrosis and the occurrence of primary nonfunction, offering a sensitive assay for prospective organ testing.

Purpose- Prognostic testing represents the basis for expansion of the donor pool with ischemically damaged kidneys. We evaluated if actual restoration of renal metabolism by ex vivo warm perfusion could be used to predict the status of an organ before transplantation. Using a canine autotransplantation model, we assessed the potential of prognostic testing during ex vivo warm perfusion with EMS technology. Methods- Kidneys were subjected to 30 minutes of warm ischemia (WI) followed by 24 hours of static storage in ViaSpan at 4C. Following WI and static storage the kidneys were transitioned to three hours of EMS perfusion at 30C. During this period parameters indicative of renal metabolism and function were employed to predict outcomes prospectively. Parameters included measures of innate metabolic capacity, perfusion characteristics, oxidative metabolism and the condition and barrier function of the vasculature. A Viabilty Index (VI) was calculated as the sum of the four parameters mentioned above. Results- A VI in the range of 2.5 to 2.9 was associated with mild acute tubular necrosis (ATN) (3 dogs); mean peak serum creatinine value was 3.5mg/dL and mean time to normalization of the serum chemistries was 7.3 days. Similarly, a VI in the range of 2.2 to 2.45 was associated with a moderate ATN (4 dogs); mean peak serum creatinine value was 5.6mg/dL and a mean time to normalization of the serum chemistries was 14.3 days. When the calculated VI was in the range of 1.5 to 2.00, the ATN was severe. In the two dogs with a severe ATN, the mean peak serum creatinine was 10.3mg/dL. In one case the calculated VI was negative. The dog was symptomatic of uremia and was euthanized on day 7 post-transplant with a serum creatinine of 12.5mg/dL. The results indicate the possibility of utilizing warm temperature perfusion to evaluate kidneys prior to transplantation. The VI used was able to identify and classify the severity of the ATN and the occurrence of primary non-function, offering asensitive assay for prospective organ testing.

Purpose- Ischemia, both warm and cold, inhibit cellular metabolism. This inhibition of metabolism has been postulated to influence the severity of the reperfusion injury. Reestablishing renal metabolism prior to implantation could potentially ameliorate posttransplant graft function. Using a canine autotransplantation model combining both warm and cold ischemia, the impact of EMS perfusion was evaluated. Methods- kidneys were exposed to 30 minutes of warm ischemia followed by 24 hours static storage in Viaspan at 4°C. Kidneys in the control group were reimplantated after the period of hypothermic preservation; kidneys in the experimental group were transitioned to 3 hours of EMS perfusion at 30C prior to reimplantation. Contralateral nephrectomy was perfomed before reperfusion of the preserved kidney. Posttransplant renal function was assessed by serum creatinine and survival. Results- EMS perfused kidneys had lower 24 hours postttransplant serum creatinine values than control kidneys (mean of 3.1 mg/dL vs 4.0 mg/dL). The survival rate for EMS perfused kidneys was 90 % (9/10) vs 73%(8/11) of the control kidneys. These results provide evidence that ex vivo restoration of cellular metabolism reduces damage seen upon actual reperfusion from the cold, thereby improving posttransplant graft function and survival.

Background. The compounding damage of warm ischemia (WI) followed by cold preservation is a major barrier in renal transplantation. Although the relative effect of WI is not yet well understood, therapeutic strategies have mostly focused on minimizing the pathology seen upon reperfusion from the cold.Our study was designed to examine the effect of restoration of renal metabolism by warm perfusion on graft survival and to investigate the compounding damage of WI. Methods. Using a known critical canine autotransplantation model (1), kidneys were exposed to 30 min WI followed by 24 hr cold storage in Viaspan. They were then either reimplanted directly or first transitioned to 3 hr of warm perfusion with an acellular perfusate before reimplantation. Contralateral kidneys were subjected to 0, 30, or 60 min WI; 24 hr cold storage, and 3 hr warm perfusion. Results. Transplanted kidneys that were warm perfused before reimplantation had both lower 24 hr posttransplant serum creatinine (median of 3.2 vs. 4.1 mg/dl) and lower peak serum creatinine (median of 4.95 vs. 7.1 mg/dl). Survival rate for warm perfused kidneys was 90% (9/10) vs. 73% (8/11). In the contralateral kidneys, metabolism was affected by the compounding damage of WI. Renal oxygen and glucose consumption diminished significantly, whereas vascular resistance and lactate dehydrogenase-release rose significantly with increasing WI. Conclusions. The results demonstrate a reduction of reperfusion damage by an acellular ex vivo restoration of renal metabolism. Furthermore, data from the contralateral kidneys substantiates the relative role of WI on metabolism in renal transplantation.

193 The Golden Syrian hamster (HAM) to Lewis (LEW) rat combination is a useful model for studying immune responses to concordant solid organ xenografts. The rejection of hamster cardiac xenografts by Lewis rat recipients has been prevented only by immunosuppressive or cytotoxic drugs, administered continuously or at high dose. We hypothesized that peritransplant antilymphocyte serum (ALS) would deplete circulating lymphocytes and that posttransplant Rapamycin (RAPA) would restrict xenoreactive T and B cell proliferation. We also tested the effect of donor bone marrow (BM), administered in the posttransplant period to counter donor-specific xenoreactive repopulating cells. Methods: HAM hearts were transplanted heterotopically into the abdomen of LEW rats. ALS (6 ml/kg s.c) was injected on d-1, 0, and + 1. RAPA (a gift from Dr. Seghal, Wyeth-Ayerst) (2-3 mg/kg in Tween80/CMC) was injected i.p. every other day posttx for 7 doses. HAM donor BM (2-3 × 103 cells) was infused i.v. on d + 5. No treatment was administered after day 13 in any of the groups. α-HAM serum antibody (Ab) titer was analyzed by flow cytometry with HAM lymph nodes and fluorescent secondary Ab. Results: Untreated LEW rats (CTRL) and rats treated with only RAPA rejected HAM heart xenografts by 4 days (MST 4 ± 0.5d, n = 5; MST 4 ± Od, n = 4). ALS alone and ALS & HAM BM were only weakly effective (MST 8 ± 0.5d, n = 5; MST 8 ± 0.8d, n = 6) at prolonging xenograft survival. However, the combination of ALS & RAPA acted synergistically to significantly extend MST to 16 ± 0.6d (n = 3) (p < 0.001 vs CTRL, RAPA, ALS or ALS & BM). Furthermore, the addition of HAM donor BM to this protocol further increased MST to 22 ± 3d (n = 4), over a week after the cessation of immunosuppression (p < 0.001 vs ALS & RAPA). ALS & RAPA (w/ and w/out BM) caused a more severe, prolonged depression of circulating lymphocytes than ALS alone, that reached a mean nadir of 250 cells/µl (< 5 % pretx levels) and remained significantly below normal at the time of rejection. α-HAM Ab titers at rejection were significantly suppressed in all groups receiving RAPA (undetectable to 1:2) vs. all other groups (1:50 to >1:100). Conclusions: RAPA, a clinically acceptable drug, in combination with ALS and donor BM has effectively suppressed serum antidonor antibody levels and enabled remarkable prolongation of xenograft survival. These results are exceptional in the absence of irradiation, chronic or high dose drug therapy and suggest that these agents will play an important role in future immunosuppressive xenotransplantation protocols.

These studies argue strongly against the widespread use of skin grafts for rejection testing. Primarily vascularized grafts of a clinically grafted organ would seem to be much more relevant to future studies. These results suggest that the elimination of either class I or class II antigens in grafts or techniques to modulate these antigens or reduce the degree of incompatibility of these antigens by tissue typing should be actively pursued for solid organ grafting in the future.

This study was designed to test the alternative hypothesis to the T-cell cytotoxicity as a primary element in concordant xenograft rejection. Two sets of studies were done with one involving the known NK- and K-cell deficient Be mouse and another in which a normal mouse was induced with high levels (3 to 5 times normal) of LAK cell killing by a constant Osmolar Mini-Pump infusion of rIL-2. In the Be animals the CXR of skin and cardiac CXR grafts was slightly prolonged and the graft survived for a longer time than normal grafts, indicating the NK- and K-cell mechanisms are operative in CXR. These studies suggest that the NK and K cells act as ancillary mechanisms in cytotoxicity in CXR. In the second portion of these studies, the increasing of LAK cell activity by infusions of rIL-2 failed to delay rejection beyond that in the Be mouse but did delay rejection beyond controls. These results suggest that the NK- and K-cell killing acts as an ancillary mechanism in CXR. Because these animals had only a slight rise in ADCC shortly after transplantation with maximum titers of 1:256 in this model, it would be presumptive to assume that cell killing was not important in CXR because many xenograft models show extraordinarily high levels of ADCC after transplantation, especially in a late transplant period. As in human allografting, the vasculitis seen with ADCC antibody could be expected to represent a significant pathology long after transplantation and this mechanism of cytotoxicity involving NK and K cells may be important in a later phase after xenografting when chronic vasculitis develops in the long surviving xenografts. Techniques for immunomodulation of the NK and K activity are now being actively pursued in our laboratory.

These results indicate that both the choice of species combination as well as the choice of the donor organ studied can be crucial in the reproducibility, validity, and probably the relevance of xenograft testing. The vexatious results often encountered in a variety of mouse strains should lead to caution in the use of mouse models in general. Perhaps it is fair to say that the authors of mouse studies should indicate some particular reason for choice of the combination of rat-to-mouse transplant rather than mouse-to-rat as their primary model, or utilize the reciprocal species combination as a control. In regard to choice of organ xenograft transplant, the primarily vascularized heart xenograft is an excellent model. In contrast to skin, isolated pancreas islet, and some other xenograft organ models, the primarily vascularized heart model is a valid one and shows little variance in mean survival with a given treatment in the mouse. If a choice of rat-to-mouse or other species xenograft to mouse recipients is necessary or desirable, the primarily vascularized heart should be used as the organ of choice. In the emerging field of xenograft testing, the reproducibility, validity, and relevance of these xenograft models will be crucially important in deriving information useful for expansion of xenografting and to higher animals ultimately into clinical practice. There is little doubt that the rodent studies in both xenografts and allografts have provided a wealth of the immunological and surgical information that was obtained in a cost-effective manner with minimal ethical problems.(ABSTRACT TRUNCATED AT 250 WORDS)

Adequate immunosuppression remains a major obstacle to successful xenotransplantation, with early xenograft rejection appearing to be mediated by humoral factors. Total-lymphoid irradiation (TLI) and 15-deoxyspergualin (DOSP) have been shown to be effective immunosuppressive agents in allografs. In this study, TLI alone and in combination with DOSP and cyclosporine were evaluated in the hamster-to-rat heterotopic cardiac xenograft model. The animals were divided into four groups: group 1--control (n = 9); group 2--TLI alone, administered pretransplant at 125 cGy/day, four days per week, for three weeks (n = 12); group 3--TLI plus CsA at 10 mg/kg/day (n = 17); and group 4--TLI plus DOSP at 2.5 mg/kg/day (n = 10). Tissue sections were taken from rejected xenografts in all treatment groups for histological examination. Complement-dependent cytotoxicity assays were performed on the control group and also the TLI-DOSP group. The control animals were found to have a mean graft survival of 3.2 +/- 0.4 days. TLI alone (5.8 +/- 0.7 days) did not significantly improve graft survival in comparison with the control group. Combination of TLI with DOSP (26.3 +/- 5.9 days) results in significantly improved survival (P less than 0.05) in comparison with the control, TLI alone, and combination of TLI and CsA (13.6 +/- 8.6 days). Complement-dependent cytotoxicity assays revealed that control groups have low rat antihamster lymphocytotoxic antibody titer (1/1-1/10) prior to xenografting, and that these antibody titers show a precipitous rise to a level of 1/640-1/1280 by day 3, the time at which rejection occurred. This correlates with the histological findings of the rejected hearts showing a severe humoral type of rejection and no evidence of cellular rejection. In contrast, animals in the TLI-DOSP group had markedly lowered rat antihamster lymphocytotoxic antibody titers (1/20-1/40) on day 3, and these titers only increased to 1/160 at time of rejection. This correlates with the histological findings of a lesser degree of humoral rejection in the TLI-DOSP group. Combination therapy with TLI and DOSP results in a marked increase of survival in xenografts in this model not seen with any other drug combination studied in over 500 xenografts in our laboratory. This study indicates that TLI combined with DOSP results in prolonged suppression of the antixenograft antibody response. This combination of agents appears to have the potential to prevent early xenograft rejection.