Dorian Araneda

OVERCOMING SEVERE RENAL ISCHEMIA: THE ROLE OF EX VIVO WARM PERFUSION

BACKGROUND The ability to effectively utilize kidneys damaged by severe (2 hr) warm ischemia (WI) could provide increased numbers of kidneys for transplantation. The present study was designed to examine the effect of restoring renal metabolism after severe WI insult during ex vivo warm perfusion using an acellular technology. After warm perfusion for 18 hr, kidneys were reimplanted and evaluated for graft function. METHODS Using a canine autotransplant model, kidneys were exposed to 120 min of WI. They were then either reimplanted immediately, hypothermically machine perfused (4 degrees C) for 18 hr with Belzers solution, or transitioned to 18 hr of warm perfusion (32 degrees C) with an acellular perfusate before implantation. RESULTS Warm perfused kidneys with 120 min of WI provided life-sustaining function after transplantation, whereas the control kidneys immediately reimplanted or with hypothermic machine perfusion did not. The mean peak serum creatinine in the warm perfused kidneys was 3.7 mg/dl, with the mean peak occurring on day 2 and normalizing on day 9 posttransplant. CONCLUSIONS These results indicate that 18 hr of ex vivo warm perfusion of kidneys is feasible. Furthermore, recovery of renal function during warm perfusion is demonstrated, resulting in immediate function after transplantation. The use of ex vivo warm perfusion to recover function in severe ischemically damaged kidneys could provide the basis for increasing the number of transplantable kidneys.

The development of a posttransplant TLI treatment strategy that promotes organ allograft acceptance without chronic immunosuppression.

The effectiveness of fractionated TLI in promoting allograft tolerance without chronic drug therapy is well established experimentally. TLI has not been widely applied in clinical transplantation, largely due to logistic and medical limitations of pretransplant TLI conditioning. Potential use of posttransplant TLI (PT-TLI) has not been critically evaluated. In this study we investigated PT-TLI in combination with rabbit antithymocyte globulin (RATG) in the absence of chronic immunosuppressive drugs as a strategy for inducing long-term kidney allograft acceptance. Recipients were studied with and without infusion of DR-CD3- donor bone marrow cells (DBMC). Normal rhesus monkeys, which received no pretransplant treatment, underwent bilateral intrinsic nephrectomy and splenectomy at the time of transplantation. RATG was administered daily for 3-5 consecutive days beginning on day 0. A total dose of 500-625 cGy of fractionated TLI was given in 4-5 treatments at 125 cGy per day beginning on day +1. Whereas neither PT-TLI nor RATG monotherapy induced long-term graft acceptance in splenectomized recipients, the combination of these modalities was remarkable in promoting long-term allograft acceptance without immunosuppressive drug therapy. Of 4 recipients given RATG x 5, splenectomy and PT-TLI, all were free of acute rejection. Of these, 3/4 had graft survivals greater than 100 days, 2/4 were greater than 150 days and 1/4 greater than 365 days. Of 5 recipients given this treatment plus an infusion of DR-CD3-DBMC, 4/5 grafts survived greater than 150 days and 3/5 still have normal functioning grafts at greater than 365 days. PT-TLI accentuated and prolonged changes in PBL subpopulations that were initially affected by RATG. Depressed levels of CD3+ cells were present for 4-5 months, with large numbers of circulating CD4+CD3- and especially CD8+ CD3- cells. In addition, antibody responses to RATG and alloantigens were suppressed in PT-TLI-treated recipients. Overall, the combination of PT-TLI, splenectomy, and RATG was found to be effective in promoting long-term allograft acceptance without chronic immunosuppressive drug therapy. The infusion of DR-CD3- DBMC in recipients treated with PT-TLI, RATG and splenectomy appeared to further increase the incidence of stable long-term survivors. If infectious complications can be improved, this approach may provide a clinically applicable posttransplant treatment strategy for inducing functional allograft tolerance.

Exsanguinous metabolic support perfusion--a new strategy to improve graft function after kidney transplantation.

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.

Prolongation Of Xenograft Survival After Combination Therapy With 15-deoxyspergualin And Total-lymphoid Irradiation In The Hamster-to-rat Cardiac Xenograft Model

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 allografts. 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<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.

PRETRANSPLANTATION PROGNOSTIC TESTING ON DAMAGED KIDNEYS DURING EX VIVO WARM PERFUSION 1

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°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.

Development of suppressor lymphocytes during acute rejection of rat cardiac allografts and preservation of suppression by anti-IL-2-receptor monoclonal antibody

Suppressor acivity was investigated in rats undergoing acute rejection of heterotopic cardiac allografts. Spleen cells were harvested at 7 days from LEW rats rejecting (LEWxBN) F1 heart grafts and fractionated into their T, T suppressor/cytotoxic, and t helper subpopulations. Transfer of alloimmune unseparated spleen

Phenotype, activation status, and suppressor activity of host lymphocytes during acute rejection and after cyclosporine-induced unresponsiveness of rat cardiac allografts.

Serial changes in phenotype, cell cycle, and functional behavior of lymphocyte subpopulations occurring both during acute rejection in unmodified hosts and in long-surviving heterotopic cardiac allografts in rats treated with cyclosporine (CsA) were studied. Using flow cytometry, RNA and DNA content of cells was examined during various phases of cell activation. In animals acutely rejecting their grafts, numbers of cells infiltrating the grafts and in host spleen in G1 phase (higher RNA content) increased, starting from day 3, and peaked by 5–6 days posttransplantation, and numbers of cells in S/G2/M phase (higher DNA content) remained stable. Similar, although slightly delayed changes were noted in CsA-treated recipients. The ratio of T helper (Th) to T suppressor/cytotoxic (Ts/c) phenotype cells infiltrating acutely rejecting grafts by day 3, was 1.6; it inverted abruptly to 0.7 by days 5–6, suggesting a preponderance of Ts/c during the later stages of allograft rejection. Ratio inversion occurred slightly later in host spleen and later in peripheral blood. Similarly, treatment with CsA produced a transient depression of Th, with recovery of the Th:Ts/c ratio during weeks 3–4 following transplantation. Adoptive transfer studies were then performed to investigate the functional significance of the T cell subsets. Survival of test grafts was prolonged significantly (ca. 14 days, P<0.001) when cells infiltrating grafts and spleen were transferred during inversion of Th:Ts/c; before that period, test graft survival was shortened in a second-set manner. These experiments suggest that suppressor cells may be responsible for resolution of acute rejection, as well as for host unresponsiveness seen after CsA treatment, and they represent an important homeostatic host mechanism following immunological stimulation.

Cyclosporine and Experimental Skin Allografts: Long-Term Survival in Rats Treated with Low Maintenance Doses

Although cyclosporine (CsA) is a powerful immunosuppressive agent in organ transplantation, its efficacy in skin transplantation has not been examined completely. We have tested it as primary immunosuppression in a rat skin allograft model. Histoincompatible Brown-Norway skin grafts are rejected in untreated Lewis hosts within 9 ± 1 days but survive for 22 ± 3, 34 ± 2, or 41 ± 8 days after 7, 14, or 21 days of CsA treatment (15 mg/kg per day subcutaneously), respectively (p < 0.001). Animals treated daily for 4 weeks died from drug toxicity; however, an initial 2-week course followed by a low maintenance dose (15 mg/kg every fourth day) produced indefinite (>150 days) graft acceptance without side effects. The long-surviving grafts were supple, grew long hair, and showed normal histology. When the drug was stopped at any time during this maintenance period, early signs of rejection (hair loss, epidermal breakdown, and localized ulceration) occurred, which could be reversed completely by a short CsA “pulse” (15 mg/kg per day for 7 days). These experimental data support the potential application of CsA immunosuppression in human skin allotransplantation.

Synergy between subtherapeutic doses of cyclosporine and immunobiological manipulations in rat heart graft recipients.

Cyclosporine in combination with other chemical or biological immunosuppressive modalities has been useful in clinical and experimental organ transplantation. In these studies, the efficacy of adjunctive subtherapeutic doses of CsA given to immunologically enhanced heart graft recipients or to animals treated with an anti ‐IL‐2 receptor monoclonal antibody (ART18) are described. Individually, the treatment entities are only partially effective. In rats undergoing active and passive enhancement alone, heart allograft survival was increased to 25±12 days in two‐thirds, indefinitely in one‐third. After ART18 treatment, grafts survive 21±1 days. Grafts are accepted permanently in animals receiving full‐dose CsA (15 mg/kg x7), but are rejected acutely (c. 7 days) when subtherapeutic doses (1.5 mg/kg ×7) are used. However, when subtherapeutic doses of CsA are given in combination with immunological enhancement or with interleukin‐2‐receptor‐targeted therapy, graft survival increases dramatically, with permanent or markedly prolonged engraftment occurring in all instances. In the early phases of host unresponsiveness, both enhancement and IL‐2R‐targeted therapy spare selectively T cells with suppressor activity in vivo; in enhanced animals, the W3/25+ subset is responsible for prolonged graft survival, the OX8+ fraction is responsible in ART18‐treated animals and in CsA‐treated animals. Both subpopulations show suppressor activity in the later stages of combination treatment. IL‐3 production is increased significantly in these states of unresponsiveness, an observation also noted during maintenance CsA treatment; this seems to correlate with suppressor activity. Immunoperoxidase studies of the graft infiltrates emphasize the synergistic effects of combination treatments. Thus, subtherapeutic doses of CsA plus biologic host manipulations produce greatly increased graft survival by affecting selectively different host immune mechanisms.

Restoration of allograft responsiveness in B rats. II. Requirements for lymphoid populations and lymphokine.

B rats, produced by lethal x-irradiation of thymectomized animals that are then reconstituted with bone marrow cells from syngeneic, thymectomized, and thoracic-duct-drained donors, are unable to reject histoin-compatible heterotopic cardiac allografts. In these studies, we have continued to investigate the role of immune competent cells and lymphokine in restoring acute allograft rejection in these animals, noting that adoptive transfer of 10(8) sensitized splenocytes (sSL) plus Interleukin-2-rich conditioned medium, IL-2 (CM) will reproducibly produce acute responsiveness toward long-standing, well-functioning, heart grafts. We have now noted that sensitized lymph node cells or thoracic duct lymphocytes can also reverse the unresponsive state, with IL-2 rich conditioned supernatants increasing the effectiveness of small numbers of cells from these populations in inducing allograft rejection. Furthermore, the major cellular components within the spleen have been separated using antibody techniques, and their individual role in the rejection reaction has been assessed. Although 10(8) sSL plus IL-2 (CM) can produce acute rejection in a time comparable to that occurring in unmodified recipients (n = 20, MST +/- SD = 8.4 +/- 1.3 days), neither the T cell fraction (6 x 10(7] of 10(8) SL + IL-2 (CM) (n = 10, MST +/- SD = 17.4 +/- 7.3 days), nor addition of the adherent cell fraction to the T cell component (MST +/- SD = 18 +/- 2 days) or addition of the B cell fraction (MST +/- SD = 15.5 +/- 2.1 days) could restore acute responsiveness. However, increasing the numbers of T cells to 10(8) and transferring concomitantly with IL-2 (CM) again produced acute rejection (n = 4, MST +/- SD = 9 +/- 1.1 days). These data suggest that T cells form the essential element of transfer in the B rat, although other cells present in SL appear necessary to provide an optimal milieu. Our experiments also suggest that this response is independent of the route of administration or of higher doses of IL-2. Finally, using a dual allograft model, it appears that graft destruction is determined by the specific sensitivity of transferred SL; IL-2 (CM) being a nonspecific factor in the response.