Background: The ante situm-technique allows for luxation of the liver in front of the situs, enabling the experienced HPB-surgeon to resect liver malignancies invading the hepatic veins/inferior vena cava. Single-center experience with ante-situm liver resection over the past 10 years is presented. Methods: Retrospective analysis on patients receiving ante situm liver resection comprising demographical and basic clinical data as well as perioperative courses. Results: Ante situm liver resection was performed on 7 patients suffering from primary (n=5) or secondary (n=2) liver tumors. Patients received trisegmentectomy (n=4), extended left hemihepatectomy (n=1) or atypical liver resection (segments 8/4a; n=2) combined with dissection of the suprahepatic vena cava/liver veins. Venous reconstruction was performed as reinsertion of liver veins (n=3) or vascular replacement with allogeneic donor veins (n=3) or PTFE-graft (n=1) in total vascular occlusion (mean of 29.6 min.). Severe morbidity defined as Dindo Clavien >3a was found in 3 patients; one patient died due to small-for-size syndrome. Mean length of stay at ICU and hospital were 9.0 and 34.3 days, respectively. R0-resection was achieved in 5 cases (twice a R1-situation). Disease-free survival was 24.6 months with an overall survival of 34.4 months. Conclusion: Ante situm liver resection offers the opportunity to achieve surgical cure in otherwise unresectable tumors. Nevertheless, this approach remains challenging with need for complex vascular reconstruction.

Background: ALPPS has been described as a promising method to increase the resectability of liver tumors. Optimal timing of hepatectomy at the point of sufficient hypertrophy of the future liver remnant (FLR) is essential for the morbidity and mortality of this procedure. The recently developed LiMAx-test can be applied to monitor postoperative liver function and hence might represent a useful tool for decision-making regarding the timing of the second step of ALPPS. Methods: A 73-year-old female patient presented with metachronous colorectal liver metastasis comprising the complete right liver lobe as well as segment IV. Due to an insufficient FLR (19.3%) and a low FLR: body weight ratio (0.28%) the decision was made to perform ALPPS. Results: Despite a formally sufficient increase of the FLR to 30.8% within 7 days after the first step of ALPPS, the liver function was seen to only slowly increase as expressed by a LiMAx value of 245 μg/h/kg. By means of the LiMAx-test, sufficient increase of liver function eventually was detected by postoperative day 11 (LiMAx value of 371 μg/h/kg; FLR 35.2%) so that second step of ALPPS was performed with no signs of liver failure during further clinical course. Conclusion: Applying LiMAx during ALPPS, we have found a significant difference between increase in volume and function of the FLR. LiMAx hence might proof valuable for application in two-stage liver resection to avoid postoperative small-for-size syndrome.

Introduction: Recently the ALPPS-procedure was developed to increase the resectability of marginally resectable or locally unresectable liver tumors. This study focused on the application of ALPPS in patients with advanced colorectal liver metastases (CRLM) and preoperative chemotherapy to further define suitable indications for this novel therapeutic concept. Methods: Retrospective analysis was performed on six patients suffering from advanced CRLM having received extensive preoperative chemotherapy. Analyses comprised demographical and basic clinical data, the perioperative courses as well as short- and long-term outcomes. Results: All patients presented with bilobular CRLM and a mean time of preoperative chemotherapy of 6 months (FOLFOX or FOLFORI based). Extended right hemihepatectomy was performed in all cases, 4 patients additionally received atypical resections in segments II/III. Mean future liver remnant was 397.8 cm3 (range: 121–753 cm3) prior to step 1, with an increase to 808 cm3 (average increase of 67.9%, range: 32.5–94.1%) before completion of ALPPS. The interval between step I and II was 11 days (range: 9–14 days). Severe morbidity (Dindo Clavien >3a) occurred in 2 of the 6 patients. One patient died due to right hepatic artery thrombosis after step I. Following completion of ALPPS the mean disease-free survival was 174.0 days (range: 77–268 days) with an overall survival of 17 months (range: 5–31 months) at last follow-up. Conclusion: Despite extensive preoperative chemotherapy the ALPPS-procedure seems to result in adequate liver hypertrophy, successfully preventing postoperative small-for-size syndrome. However, there might be a high risk of tumor recurrence in patients with an aggressive tumor biology.

Einleitung: Salinomycin (Sal) ist ein Polyether-Antibiotikum, dessen Potential in der Tumortherapie in diversen soliden und nicht-soliden Malignomen nachgewiesen werden konnte. Unlängst konnte die Effektivität von Sal gegen das HCC in vitro und in vivo gezeigt werden. Der Wirkmechanismus ist jedoch noch weitestgehend unverstanden.

Background: Transplantation of bone marrow derived adult stem cells (BMC) improves cardiac function after acute myocardial infarction (MI). However, the Cell Population mediating myocardial recovery and the fate of the transplanted cells are still controversial.Aims: We determined the effects of Sea-1(+) c-kit(+) lin(-) haematopoietic BMC on cardiac function after MI and the cell fate after transplantation.Methods: Sca-1(+) c-kit(+) lin(-) BMC of male donor C57BL/6 mice were transplanted by intravenous injection into syngenic females after permanent MI. LV dimensions and function were determined by echocardiography and cardiac magnetic resonance imaging, transplanted BMC were identified by Y chromosome DNA in situ hybridization.Results: BMC treatment completely prevented LV dilation (LV enddiastolic volume BMC 70 +/- 16 mu l vs. control 122 +/- 41 mu l; p < 0.05) and improved fractional shortening (BMC 22.9 +/- 8% vs. control 15.4 +/- 8.4%; p < 0.05) and ejection fraction (BMC 68.2 +/- 6.6% vs. control 52 +/- 14.3%, p < 0.05) as early as 3 days after transplantation, but did not decrease infarct size (BMC 27 +/- 6% vs. control 28 +/- 7%, p=n.s.). After 4 weeks, only sporadic cells of male origin were identified in infarcted hearts (< 0.01% of periinfarct cells).Conclusion: Intravenous injection of sca-1(+) c-kit(+) lin(-) BMC after MI improves LV dimensions and function without evidence for long term engraftment.

O271 Aims: We could recently introduce in a rat system less-toxic BMT conditioning using only an anti-CD45 mAb. As severe side effect in fully MHC mismatch setting risk of aplasia was observed and most likely provoked by an atypical GvH reaction on bone marrow level. In this study, unspecific immunosuppression (IS) was tested as addition to anti-CD45 mAb conditioning for establishment of a safe and tolerizing BMT protocol. Methods: The model consisted of LEW.1W (RT1u, RT7a) recipients, LEW.7B (RT1l,RT7b) BM donors, a single injection of anti-CD45 mAb (7.5 mg/kg BW) detecting RT7a as conditioning and orally applicated cyclosporine A (CsA 5 or 15 mg/kg BW) or sirolimus (1 or 2 mg/kg BW) as IS for 14 days. Animals were monitored for clinical status and haematological parameters. They were tested for multilineage chimerism at day 20 and day 100 by FACS. Long-term survivors received skin grafts of BM donor- and 3rd party-type on day 100. Results: Only animals of high-dosage sirolimus (2 mg/kg BW) treated group showed reproducible stable chimerism (9 of 12) compared to all other groups (no IS: 1 of 9, CsA 5: none of 9, CsA 15: 1 of 9, sirolimus 1: 1 of 9). Thereby, initial leukocyte depletion by anti-CD45 mAb as parameter for conditioning extent was highly critical for BMT success. Animals, which did not became stable chimeras, showed transient chimerism but suffered mainly from severe aplasia syndrome and died between days 29 to 70 (no IS: 7 of 9, CsA 5: 9 of 9, CsA 15: 6 of 9, sirolimus 1: 5 of 9, sirolimus 2: 3 of 12). GvHD signs were only seen in high-dosage CsA treated animals (2 of 7). The attempt to stabilize BM engraftment by prolongation of high-dosage CsA treatment (28 days) even aggravated GvHD symptoms (4 of 6) like thymic selection disturbance by thymic irradiation did in high-dosage sirolimus group (4 of 4). Moreover, analysis of persisting anti-CD45 mAb coated thymocytes suggested reduced thymocyte selection progress. Long-term lineage-typing of stable chimeras from high-dosage sirolimus group revealed mainly high-graded mixed chimerism in lymphoid and fully chimerism in myeloid lineages. Stable chimeras tolerated skin grafts only of donor-type (> 100 days), while acutely rejecting 3rd party control grafts like none chimeric survivors rejecting both graft-types. Conclusions: Addition of high-dosage sirolimus to isolated anti-CD45 mAb BMT conditioning reduced significantly risk of fatal aplasia in allogeneic BMT. This is in contrast to CsA, where the combination of intrathymically persisting anti-CD45 mAb and CsA seems to disturb TC selection processes leading to severe GvHD without stabilizing HSC engraftment. Without any clinical signs for GvHD the combination of anti-CD45 mAb and sirolimus in allogeneic BMT is promising for generation of stable mixed chimerism and tolerance induction.

P955 Aims: Limited compatibility for MHC class II between allogeneic bone marrow and organ graft is sufficient for tolerance by chimerism after TC ablative BMT conditioning. This seems to enable for promising GvHD free tolerance protocol by molecular chimerism transducing organ graft identical MHC class II genes into autologous BMC. Here, tolerance potential of BM grafts sharing only MHC class II alloantigens with secondarily heart grafts (HTx) were investigated in reduced TC ablative BMT conditioning supported by sirolimus. Methods: Total body irradiation (TBI) dosage dependent destruction of leukocyte subsets in lymphoid tissues was determined using immunohistology and FACS. MHC class II disparate BMT was performed using LEW.1AR2 recipients, LEW.1AR1 donors, dosage titrated TBI (10 to 6 Gy) and supportive sirolimus for 14 days (2mg/kg p.o every 2 days). BMT recipients were monitored for clinical status, TC reconstitution and chimerism using FACS. Spleen and thymus analysis was performed on days 14 and 60 to detect engraftment of allogeneic respectively mature antigen presenting cells (APC). On day 60, clinically stable animals received HTx sharing only MHC class II with BM donors or 3rd party ones. HTx were histologically analyzed for chronic rejection and graft infiltrating cells (GIC) after acceptance of 100 days. Spleen seeded TCs of HTx tolerating chimeras were tested for separated CD4 and CD8 mediated alloreactivity using CFSE-based MLR. Results: On day 3 after TBI thymus and spleen showed highly destruction of lymphoid structure after 8 and 6 Gy. Although marked reduction of absolute cell numbers was provoked by both dosages, a significant less destruction of CD4+ and CD8+ TCs in blood and spleen as well as CD4+/CD8+ and CD8+ thymocytes after 6 Gy TBI was given. Compared to exclusively TBI conditioned BMT recipients, which became stable chimeras only after 10 Gy, BMT recipients of sirolimus supported groups showed high-graded chimerism after 8 Gy and a prolonged, but mainly unstable chimerism after 6 Gy TBI. In stable chimeras, allogeneic mature APCs could be detected in relevant fractions in thymus and spleen, which was not possible in transient chimeras. MHC class II sharing HTx were tolerated from stable chimeras of 8 Gy TBI plus sirolimus group, but were significantly prolonged rejected by low-graded chimeras of 6 Gy TBI plus sirolimus group compared to 3rd party grafts. Tolerated HTx did not revealed significantly increased obliterative arteriopathy nor CD25+ TC GICs compared to tolerance controls. In HTx tolerating chimeras as well CD4+ TC as CD8+ TC alloreactivity against HTx donor was abolished. Conclusions: Sirolimus enables for stable MHC class II disparate BM engraftment even after reduced TC ablative conditioning. To garantuee stable and tolerizing APC engraftment a marked TC ablation is still necessary, since only high-graded chimeras are tolerant for limited MHC class II compatible HTx. The interaction of central CD4+ TC clone tolerization and HTx antigens is sufficient to abolish antigen-specific TC mediated alloreactivity despite preceded incomplete TC ablation.

Background/Aims: Adult bone marrow contains progenitors capable of generating hepatocytes. Here a new liver failure model is introduced to assess whether bone marrow-derived progeny contribute to liver regeneration after acute hepatotoxic liver failure.Methods: Retrorsine was used to inhibit endogenous hepatocyte proliferation, before inducing acute liver failure by carbon tetrachloride. Bone marrow chimeras were generated before inducing liver failure to trace bone marrow derived cells. Therefore, CD45 and major histocompatibility complex (MHC) class I dimorphic rat models were applied.Results: Early after acute liver failure a multilineage inflammatory infiltrate was observed, mainly consisting of granulocytes. In long-term experiments small numbers of CD90(+)/CD45(-) cells of donor origin occurred in clusters associated with portal triads. Bone marrow cell infusion was not able to enhance liver regeneration. Cellular hypertrophy was the predominant way of liver mass regeneration in models applying retrorsine.Conclusions: Retrorsine pretreatment did not affect sensitivity for carbon tetrachloride. A multilineage inflammatory infiltrate was observed in rats whether pretreated with retrorsine or not. Few donor cells co-expressing CD90 (THY 1) were present in recipient livers, which may resemble donor-derived hematopoietic progenitors or oval cells. No other donor cells within liver parenchyma were detected. This is in contrast to other cell infusion models of acute cell death. (C) 2003 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Spender-spezifische Toleranz wird bei der Transplantation solider Organe durch eine simultane oder vorangegangene Knochenmarktransplantation (KMT) vom selben Spender garantiert. Bislang schließt die systemische Toxizität zur Verfügung stehender KMT-Protokolle (Ganzkörperbestrahlung und zytotoxische Medikamente) die klinische Testung der allogenen KMT bei nicht malignen Grunderkrankungen weitestgehend aus. Im Rattenmodell wurde hier ein neuartiges und selektiv wirkendes Konditionierungsprotokoll bestehend aus einem teil-depletierenden Alloantikörper gegen CD45 für die allogene KMT getestet. KM-Empfänger waren LEW.1W Ratten (RTlu, RT7a), Spendertiere LEW.7B Ratten (RT11, RT7b). Das Konditionierungsprotokoll bestand aus einer zweimaligen Injektion (Tag — 1 und + 3) des anti-RT7.1 monoklonalen Antikörpers (mAk), der das RT7a-Antigen detektiert (eine polymorphe Determinate, die bei allen CD45-Isoformen präsent ist). Komplett MHC-inkompatibles Knochenmark (1 x 10E8 Zellen) wurde appliziert; die Kontrollgruppe erhielt lediglich den Antikörper. Die Tiere wurden ab Tag + 5 wöchentlich auf allogenen Chimärismus im peripheren Blut als Zeichen von Knochenmarkengraftment per FACS getestet. Spender-spezifische Toleranz wurde durch Hauttransplantationen ab Tag + 50 und durch gemischte Lymphozytenkultur an Tag +100 bestätigt. Sequentielle FACS-Analysen zeigten, dass der anti-RT7.1 mAk absolute T- und NK-Zellzahlen in Blut und Knochenmark prompt reduzierte. Zellzahlen für B-Lymphozyten, Granulozyten sowie deren Vorläufer undulierten in beiden Kompartimenten mit niedrigen Werten auf Knochenmarksebene während der ersten Woche nach mAk-Injektion. Vier Wochen nach der KMTwiesen alle Empfängertiere einen gemischten Chimärismus auf. Ein stabiler gemischter Chimärismus zwischen 20 und 90% allogener Leukozyten im Empfängerblut wurde bei 11 von 15 Tieren erzielt. Diese Langzeitchimären akzeptierten spender-spezifische Hauttransplantate (> 100 Tage), während Drittantigen tragende Transplantate (BN Spenderratten) akut abgestoßen wurden (9 + / — 1 Tage). KMT-Kontrolltiere zeigten spätestens 60 Tage nach der Konditionierung mit anti-RT7.1 mAk ohne Zellapplikation eine komplette autologe Rekonstitution des hämatopoetischen Systems. Die selektive Manipulation des Leukozytensystems durch diesen anti-CD45 mAk erlaubt in der Ratte permanentes Engraftment von komplett MHC-inkompatiblen Knochenmark. Die systemische Toxizität von Ganzkörperbestrahlung oder zytotoxischen Substanzen konnte bei diesem erfolgreichen KMT-Konditionierungsprotokoll eliminiert werden. In dieser experimentellen Anordnung garantiert die Erzielung von stabilem gemischten Chimärismus spender-spezifische Toleranz für solide Organtransplantate durch Aufhebung der spender-spezifischen Alloreaktivität im Empfänger.

Anti-CD45 monoclonal antibodies (mAbs) are potentially powerful tools for the depletion of mature leukocytes. As their application for immunotherapy also depends on their effects on bone marrow (BM) progeny, the in vivo effects of an anti-CD45 mAb (anti-RT7(a) mAb) on BM precursor cells were analyzed in a rat model. Anti-RT7(a) mAb treatment was performed in LEW.1W (RT1(u) RT7(a)) rats with the use of different dosages. In addition, major histocompatibility complex (MHC)-congenic BM transplantation making use of a diallelic polymorphism (RT7(a)/RT7(b)) of rat CD45 was applied. Following injection of anti-RT7(a) mAb into normal LEW.1W rats, T cells were profoundly depleted in blood, lymph nodes, and spleen, whereas B cells were coated only by the antibody. Single injection of anti-RT7(a) mAb in a high dose induced a lethal aplastic syndrome with severe thrombocytopenia. Rescue of antibody-treated animals with BM from congenic LEW.1W-7B rats (RT1(u) RT7(b)) and transplantation of BM from LEW.1W rats pretreated with anti-RT7(a) mAb into sublethally irradiated LEW.1W-7B recipients revealed a profound effect of the mAb on progeny of myeloid and T-cell lineage. Following repeated antibody treatment of stable mixed chimeras (RT7(b)/RT7(a)), very few RT7(a)-positive B cells were still detectable after 6 months and their number declined during the subsequent year. These observations show that this anti-RT7(a) mAb effectively depletes mature T cells as well as BM precursor cells of myeloid, T-cell, and thrombocytic lineage after in vivo application. In contrast, mature B cells are not depleted, but precursors also appear to be eliminated. Overall, the findings suggest that the anti-RT7(a) mAb efficiently depletes early rat hematopoietic stem cells.

Gemischter allogener Chimärismus induziert durch Knochenmarktransplantation (KMT) ist assoziiert mit Toleranz für solide Organtransplantate, die syngen mit dem Knochenmarkinokulum sind. In dieser Studie wurde untersucht wie sich limitierte MHC-Kompatibilität zwischen Knochenmark und nachfolgendem Herztransplantat auf die Entwicklung von Transplantattoleranz auswirkt. Hierzu wurden verschiedene MHC kongene Rattenstämme auf LEW Hintergrund eingesetzt, um gemischte Knochenmarkschimären durch nicht-myeloablative KMT-Konditionierungsprotokolle (8 bzw. 9 Gy Ganzkörperbestrahlung) zu generieren. 50 Tage nach der KMT wurden Herzen transplantatiert, die komplett MHC-inkompatibel gegenüber den Empfängertieren, aber zu unterschiedlichem Ausmaß MHC-teilkompatibel gegenüber den Knochenmarkspendern waren. Für das Knochenmarkengraftment war vorrangig die Anzahl der MHC-Mismatche bedeutsam. Während die Chimärismusinzidenz im Setting mit kombinierten MHC-Inkompatibilitäten bei reduzierter Bestrahlungsdosis deutlich geringer war (8 Gy: 14% für haploidentisches Knochenmark, 17% bei kombiniertem Mismatch für ein MHC Klasse I und II Antigen), entwickelte sich stabiler Chimärismus im Falle eines isolierten MHC-Mismatches nahezu unabhängig vom MHC-Locus (8 Gy: 85% bei isoliertem MHC Klasse II Mismatch, 100% bei isoliertem MHC Klasse I Mismatch). Bezüglich der funktionellen Relevanz der MHC Alloantigene bei gemischtem Chimärismus wurde Toleranz für die allogenen Herztransplantate beobachtet, solange das MHC Klasse II Antigen zwischen Knochenmark- und Herzspender identisch war (mittleres Transplantatüberleben > 200 Tage). Dies Ergebnis wurde durch weitere Übereinstimmungen von MHC Klasse I Antigenen nicht signifikant beeinflußt. Im Gegensatz dazu wurde durch eine isolierte übereinstimmung von MHC Klasse I Antigenen keine signifikante Verlängerung der Herztransplantatfunktion im Vergleich zu bestrahlten, aber nicht einer KMT unterzogenen Tieren erzielt (mittleres Transplantatüberleben 11 Tage gegen 8 Tage). Zusammenfassend erlaubte sublethale Konditionierung ein sicheres Engraftment von allogenem Knochenmark, wenn die Inkompatibilität auf einen isolierten MHC-Locus beschränkt blieb (Klasse I oder Klasse II). Desweiteren bedeutete die Kompatibilität von MHC Klasse II Antigenen zwischen Knochenmark- und Organspendern den Schlüdssel zur Transplantattoleranz durch gemischten Chimärismus. Durch diese Ergebnisse wurden neue Strategien für klinisch anwendbare Toleranzinduktion basierend auf Allo-MHC-Gentransfer in autologe hämatopoetische Stammzellen unter immunologischen Gesichtspunkten bearbeitet und vorbereitet.

Background. Donor lymphocytes infused after organ transplantation can have strong immunoregulatory effects. Application of such protocols for transplant tolerance induction in a clinical setting will, however, require combination of specific immunomodulatory strategies with nonspecific immunosuppressive medication for safety reasons. The aim of this study was to analyze the effects of immunosuppressive treatment on tolerance induction protocols by posttransplantation donor lymphocyte infusion. Methods. The interaction of postoperative donor leukocyte infusion with different types, dosage, and timing of immunosuppressive drugs were studied in a rat model of heart transplantation. Results. Tolerance could be achieved if donor cell infusion was combined with delayed, but not immediate, low-dose cyclosporine treatment, and this was associated with activation and apoptosis of host lymphocytes. In contrast, combinations with an antibody against the interleukin 2 receptor led to long-term graft survival but severe chronic rejection, and combinations with high-dose cyclosporine or sirolimus led to acute rejection. Conclusions. Postoperative donor leukocyte infusion is a potential way for tolerance induction, but the type, dose, and timing of medication are highly critical for its efficacy.

Mesenchymal stem cells (MSCs) can be used for immunomodulation therapy after solid organ transplantation. Here, we focus on the immunoregulatory potential of combination therapies of MSCs and classic pharmacotherapy to mediate acceptance of solid organ grafts.To determine which drugs influence the immunosuppressive effect of MSCs, we assessed the interaction of MSCs and common clinical immunosuppresants (MMF, sirolimus [Srl], and ciclosporin A [CiA]) in a parent-into-F1 cell transfer model. In this model, the transfer of parental strain T cells into semi-allogeneic F1 recipients induces a graft-versus-host reaction (GvHR). Re-isolated CFSE-labelled T lymphocytes were analyzed by flow cytometry. These findings were compared to a fully allogeneic heart transplantation model.We found that MSC treatment alone had no significant effect on allograft survival of heterotopic heart grafts. However, MSCs combined with short-term mycophenolate mofetil (MMF) significantly prolonged graft survival. Quantitative analysis of three different MSC – drug combinations in the F1 model revealed, that only the MSC–MMF combination led to a super-additive immunosuppressive effect. We also investigated the effect of MMF and CiA on IFNγ production of stimulated lymphocytes and found that MMF left the expression of IFNγ unaffected, whereas CiA completely abolished the production of IFNγ.Our data show that the type of concurrent immunosuppression strongly influences the immunosuppressive effect of MSC, most likely through differential secretion of IFNγ. A regimen combining MSCs and MMF was most immunosuppressive.

A single injection of a depleting monoclonal antibody (mAb) against CD45 induces tolerance for fully MHC mismatched heart allografts in rats. The purpose of this study was to characterize the resulting state of tolerance. The experimental system consisted of LEW.1W (RT1u ) rats receiving a LEW (RT11) heart graft 1 day after a single injection of the anti-CD45 mAb. FACS analyses of PBMC and graft-infiltrating cells (GIC) were performed weekly. Cytokine patterns within grafts were sequentially analyzed by RT-PCR. Tolerant animals were challenged by secondary skin or heart transplants on day 100 (donor and third party). The anti-CD45 mAb particularly depleted T-cells (CD4+ T-cells more effectively than CD8+ T-cells). Massive reduction of CD4+ T-cells (< 1% of PBMC) was associated with tolerance for allogeneic heart grafts in 19 of 25 treated animals (MST > 200 days). The remaining six rats rejected heart allografts in a delayed fashion (MST 31 ± 7 days). CD4+ T-cells among GIC were primarly activated (CD25high ) on day 14 (> 40%), but the proportion of activated T-cells decreased considerably during peripheral T-cell reconstitution (> day 21). The proportion of activated CD8+ T-cells was significantly reduced (< 25%) compared with acute rejection (> 50%). The intragraft cytokine profile shifted from a Thi dominance on days 14 and 21 to a TI12 pattern (IL-4, IL-10) during tolerance stabilization (> day 21). Only secondary heart grafts of the donor strain were tolerated. In conclusion, this anti-CD45 mAb required an effective T-cell depletion to achieve tolerance, which was associated with a Th2 pattern of cytokine production.

Background. In clinical organ transplantation monoclonal antibodies (mAb) to different surface molecules of immunocompetent cells become integral parts of the immunosuppressive therapy. In this study, a mAb against the rat leukocyte common antigen CD45 (RT7) was tested for its immunosuppressive potency after a single perioperative injection. Methods. Binding and depleting properties of the anti-RT7 mAb were investigated by flow cytometry. In the fully major histocompatibility complex–disparate heart and skin transplantation models (LEW [RT1l] → LEW.1W [RT1u]), a single dose of anti-RT7 mAb (10 mg/kg) was administered intravenously (day −1). To characterize the long-term acceptance of heart allografts second set skin transplantation (day 100), mixed lymphocyte reaction studies (day 100) and reverse transcriptase-polymerase chain reaction analysis for intragraft cytokine expression (day 200) were performed. Results. The anti-RT7 mAb bound to nearly all hematopoietic lineage cells, but particularly T and NK cells, and profoundly depleted these cells in circulation and lymphoid tissues. Anti-RT7 mAb-treated rats showed long-term acceptance of heart allografts (>200 days; n=12), whereas untreated recipients rejected allografts by day 8 (n=6). In contrast to hearts, primary skin allograft survival was only moderately prolonged. Animals with stable heart allograft acceptance showed normal in vitro lymphocyte proliferation responses to donor and third party antigen. These recipients also acutely rejected second set donor-strain skin grafts without inducing rejection of persisting heart allografts. Reverse transcriptase-polymerase chain reaction analysis of intragraft cytokines showed up-regulation of Fas-ligand and IL-4 mRNA in long-surviving heart allografts. Conclusions. The findings demonstrate that a single injection of an anti-RT7 mAb in the rat can induce stable long-term acceptance of heart allografts by transient but profound T-cell depletion. Local immunoregulatory mechanisms seem to play a role for maintenance of long-term graft acceptance.

Objective. Organ allografts contain passenger leukocytes that are transferred to the recipient with the transplantation, but their functional relevance to the recipient's immune system is still controversial.Materials and Methods. To clarify the functional capacity of passenger leukocytes, we attempted to enhance their effect in rat heart allograft recipients by selective depletion of recipient leukocytes using a monoclonal antibody (mAb) against a recipient-specific allotype of CD45 (RT7(a)).Results. Although antibody treatment of the recipient alone led to profound lymphopenia and reversible myelosuppression, additional transplantation of an major histocompatibility complex-incompatible heart graft from an RT7(b) donor led to lethal aplastic anemia in the recipients. This lethal effect was completely abrogated by postoperative anti-CD3 treatment of the recipient and was partially abrogated or delayed by depletion of passenger leukocytes through additional anti-RT7(b) antibody treatment of the recipient or gamma -irradiation of the graft,Conclusions. The results suggest a role for both donor and recipient-type T cells for the induction of aplastic anemia in this model. The study shows that, under defined conditions, allogeneic passenger leukocytes in a heart graft can have a profound effect on the recipient's immune system and bone marrow, (C) 2001 International Society for Experimental Hematology. published by Elsevier Science Inc.

Patients who are receiving an organ transplant nowadays are sentenced to the life-long administration of immunosuppressive drugs, which have serious side effects. The reliable induction of donor-specific tolerance therefore remains a major goal in organ transplantation. Previously, we have developed a sublethal, non-myeloablative murine model in which permanent mixed, multilineage chimerism and donor-specific tolerance are established. Our model involves engraftment of fully allogeneic T cell depleted donor bone marrow cells in low dose irradiated and anti-CD3 treated major histocompatibility complex (MHC)-disparate recipient mice. To investigate whether vascularized organ grafts are accepted in our model, we performed heterotopic heart transplantations in our mixed chimeric mice. Chimeric mice permanently accepted hearts from the bone marrow donor (>130 days) and rapidly rejected third party-type allografts (median survival time 9 days). Untreated control recipient mice rejected both donor- and third party-type allografts. In addition, mice that accepted their cardiac grafts, donor-specific acceptance of skin grafts was observed. In conclusion, the establishment of stable mixed chimerism with this low-toxicity regimen resulted in permanent donor-specific acceptance of vascularized organ as well as skin grafts across a full MHC barrier.

Background: The RT7 antigen is part of the rat CD45 molecule. The CD45 molecule exerts costimulatory function for T cell activation. The aim of this study was to investigate the in vivo and in vitro effects caused by a monoclonal antibody detecting the RT7 antigen. Methods: Increasing dosages (0, 1, 10, 100 mu g/ml) of anti-RT7 monoclonal antibody (anti-RT7 mAb) were added to unspecific and allospecific mixed lymphocytes cultures (MLC). In vivo anti-RT7 mAb (10 mg/kg BW) was injected intravenously one day before heterotopic transplantation of MHC-incompatible heart grafts. On day 100 donor-specific and third party skin was grafted. 2 x 10(8) donor-specific activated lymphocytes were applicated as adoptive transfer. Results: Dosage dependent enhancement of lymphocyte proliferation was confirmed in MLC. A single intravenous application of anti-RT7 mAb induced a strong but transient T cell depletion persisting for 2 weeks. Thereby graft survival of tolerance to MHC-incompatible heart grafts (n = 9) was observed for more than 100 days. No graft rejection could be induced by adoptive transfer of strongly alloreactive lymphocytes on day 150 (n = 3). Acceptance of donor-specific skin grafts (n = 5, on day 100) was just slightly prolonged (< 18 days, control group < 11 days). Conclusions: A single injection of an anti-RT7 mAb induces a long-term survival of MHC-incompatible heart grafts but not skin grafts. The basis for long-term graft acceptance appears to be an immunoregulatory mechanism.

260 Previous studies demonstrated that posttransplant infusion of viable donor splenocytes (SC) alone could exert an active immunological effect to downregulate alloreactivity of recipient. This effect was donor-specific and might depend upon potential GvHR of infused SC. To induce long-term allograft survival, we investigated the effects of accompany use of Cyclosporine (CsA) (dose, timing) with posttranslant infusion of donor SC in a fully MHC-mismatched rat transplantation model. Methods: Cardiac allografts of LEW (RT1Al B/Dl Cl) were transplanted heterotopically into abdomens of LEW.1W (RT1Au B/Du Cu) recipients and divided into following groups. Group 1: no treatment; group 2: CsA 0.5mg/kg/day i.m., day0-5; group 3: CsA 0.5mg/kg/day i.m., day 6-10; group 4: donor SC 2×108 cells in 1 ml Ringer's solution, i.v. within 6 hours after transplantation (DSC i.v.); group 5: DSC i.v. plus CsA 0.5mg/kg/day i.m., day0-5; group 6: DSC i.v. plus CsA 0.5 mg/kg/day i.m., day 6-10; group 7: DSC i.v. plus CsA 0.5 mg/kg/day i.m., day 0-10. Graft function was detected by daily heart beat palpation. Complete cessation of cardiac pulsation was defined as rejection and was confirmed by histology. Results: See following table:TableConclusions: The findings suggest long-term allograft survival can be induced by posttransplant infusion of donor SC followed temporary use of CsA. The active suppressive effects of infused cells could be influenced by simultaneously use of CsA even in a low-dosage fashion. These support the hypothesis that early active immune interaction between donor and recipient lymphocytes is required for induction of long-term allograft unresponsiveness.