Mouse Heart Transplantation Model Research Articles

Delayed Anti-CD3 Therapy in a Mouse Heart Transplant Model Induced Tolerance and Long-Term Survival of Allograft: Achieving Tolerance

Goto R, You S, Zaitsu M, Chatenoud L, Wood KJ. Delayed anti-CD3 therapy results in depletion of alloreactive T cells and the dominance of Foxp3(+) CD4(+) graft infiltrating cells. Am. J. Transplant. 13(7), 1655-1664 (2013). Humanized Fc receptor nonbinding anti-CD3 monoclonal antibodies have been tested in patients with autoimmune diseases with the goal of inducing immune tolerance. However, the timing of drug administration may be an important determinant of the biologic effects, since not all T cells are equally affected, and there may be different subsets of cells involved during the evolution of immune responses. The study by Goto et al. showed that delayed administration of anti-CD3 therapy was more effective in depleting alloreactive T cells than administration at the time of transplant, and resulted in long-term survival of the graft by promoting infiltration of CD4 Tregs into the graft.

Double Deficiency for RORγt and T-bet Drives Th2-Mediated Allograft Rejection in Mice

Although Th1, Th2, and Th17 cells are thought to be major effector cells in adaptive alloimmune responses, their respective contribution to allograft rejection remains unclear. To precisely address this, we used mice genetically modified for the Th1 and Th17 hallmark transcription factors T-bet and RORγt, respectively, which allowed us to study the alloreactive role of each subset in an experimental transplant setting. We found that in a fully mismatched heterotopic mouse heart transplantation model, T cells deficient for T-bet (prone to Th17 differentiation) versus RORγt (prone to Th1 differentiation) rejected allografts at a more accelerated rate, indicating a predominance of Th17- over Th1-driven alloimmunity. Importantly, T cells doubly deficient for both T-bet and RORγt differentiated into alloreactive GATA-3-expressing Th2 cells, which promptly induced allograft rejection characterized by a Th2-type intragraft expression profile and eosinophilic infiltration. Mechanistically, Th2-mediated allograft rejection was contingent on IL-4, as its neutralization significantly prolonged allograft survival by reducing intragraft expression of Th2 effector molecules and eosinophilic allograft infiltration. Moreover, under IL-4 neutralizing conditions, alloreactive double-deficient T cells upregulated Eomesodermin (Eomes) and IFN-γ, but not GATA-3. Thus, in the absence of T-bet and RORγt, Eomes may salvage Th1-mediated alloimmunity that underlies IL-4 neutralization-resistant allograft rejection. We summarize that, whereas Th17 cells predictably promote allograft rejection, IL-4-producing GATA-3(+) Th2 cells, which are generally thought to protect allogeneic transplants, may actually be potent facilitators of organ transplant rejection in the absence of T-bet and RORγt. Moreover, Eomes may rescue Th1-mediated allograft rejection in the absence of IL-4, T-bet, and RORγt.

Immunosuppressive Effects of DTCM-G, a Novel Inhibitor of the mTOR Downstream Signaling Pathway

A newly developed compound, 3-[(dodecylthiocarbonyl)methyl]-glutarimide (DTCM-G), has been shown to inhibit nuclear translocation of c-Fos/c-Jun in a murine macrophage cell line. Herein, we studied the immunosuppressive properties and potency of DTCM-G. Using purified mouse T cells, the in vitro effects of DTCM-G on activation, cytokine production, proliferation, and cell cycle progression were assessed, and a possible molecular target of DTCM-G was investigated. In a BALB/c (H-2(d)) to C57BL/6 (H-2(d)) mouse heart transplantation model, transplant recipients were administered DTCM-G, a calcineurin inhibitor (tacrolimus), and a nuclear factor-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ). Treatment drugs were administered daily for 14 days after transplantation. Alloimmune responses were assessed in addition to graft survival time. After anti-CD3+anti-CD28 monoclonal antibody stimulation, DTCM-G significantly suppressed proliferation, interferon-γ production, and cell cycle progression of activated T cells but not CD25 expression or interleukin-2 production. These effects were accompanied by inhibition of 70-kDa S6 protein kinase phosphorylation, a downstream kinase of the mammalian target of rapamycin. The addition of tacrolimus and DHMEQ to DTCM-G resulted in a robust inhibition of T-cell proliferation. In vivo combination therapy of DTCM-G plus either tacrolimus or DHMEQ significantly suppressed alloreactive interferon-γ-producing precursors and markedly prolonged cardiac allograft survival. Furthermore, combination of all three agents markedly inhibited alloimmune responses and permitted long-term cardiac allograft survival. DTCM-G inhibits T cells by suppressing the downstream signal of mammalian target of rapamycin. DTCM-G in combination with tacrolimus and DHMEQ induces a strong immunosuppressive effect in vivo.

What Can Animal Models Teach Us About the Potential Safety and Efficacy of Regulatory Macrophage Therapy in Patients?

Cell-based immunosuppressive therapy is very much in its infancy, but with the inception of The ONE Study, a coordinated effort to bring such treatments to clinical trial is being made; therefore, we must ask, how great are the benefits of regulatory cell therapy in transplantation expected to be? Adoptive transfer of transplant tolerance with isolated populations of regulatory cell types is reliably achieved in homologous animal models, but whether such observations can be extrapolated to patients hinges on proving the equivalence between the animal and human cells in question. Our work centers on human regulatory macrophages (M reg) as a means of attenuating responses against alloantigen. The mouse proves a convenient model to test the combined effect of M reg treatment and conventional immunosuppression, but is only clinically relevant if mouse and human M regs are functional counterparts. M regs from both species arise from equivalent populations of monocytes under directly analogous culture conditions and share many morphological features and express unique markers, distinguishing them from other macrophages. Mouse and human M regs profoundly inhibit allogeneic T cell proliferation in co-culture, through deletion and phagocytosis of activated T cells, and a direct suppressive activity. Yet, differences between mouse and human M regs exist, and weighing these differences against the likenesses is necessarily subjective, so gene expression profiling by microarray was adopted as an unbiased approach to evaluating their true similarity. In co-clustering analyses, M regs from the two species grouped together when the entire set of orthologous genes was considered, so identifying human M regs as the closest counterpart of mouse M regs.[Figure]In a mouse heart transplant model, infusion of donor-strain M regs prior to transplantation significantly prolonged allograft survival in unconditioned, non-immunosuppressed, fully-allogeneic recipients, whereas recipient and third party-derived M regs were not effective. Co-treatment with M regs and rapamycin for 10-days post-transplant enhanced the effect of M regs compared to M reg or rapamycin treatment alone. After injection, M regs from CD45.2+ B6 mice or CD45.2+ BALB/c mice were detectable in the lung, blood, liver and spleen of CD45.1+ B6 recipients for up to two weeks. Donor-derived human M regs were administered to two renal transplant recipients 7-days prior to transplantation. Both patients were minimised to low-dose tacrolimus monotherapy within 24 weeks of transplantation and subsequently maintained excellent graft function. To assess their fate after infusion, M regs were labelled with Indium111-oxine for tracking in serial whole-body SPECT studies, which showed most M regs remained viable and trafficked from the pulmonary vasculature via the blood to liver, spleen and bone marrow.

New Approach to Selective Targeting of Dendritic Cells by Sirna, Resulting in Long Term Graft Survival in Mouse Heart Transplantation Model

New Approach to Selective Targeting of Dendritic Cells by Sirna, Resulting in Long Term Graft Survival in Mouse Heart Transplantation Model

Early immune activation of donor organs after brain death in a mouse heart transplantation model

Early immune activation of donor organs after brain death in a mouse heart transplantation model

Administration of anti-interleukin-6 monoclonal antibody prolongs cardiac allograft survival

To investigate the effects of anti-IL-6 monoclonal antibody (anti-IL-6 mAb) on acute allograft rejection and the potential mechanisms in a mouse heart transplantation model. Heterotopical heart graft model was performed. The anti-IL-6 mAb was administered to recipient mice after cardiac grafting. Results were compared with administration of anti-IL-17 mAb or anti-IL-6 mAb+anti-IL-17 mAb (the 'double' treatment). The cardiac allograft survival was monitored by daily palpation in combination with histological evaluation. Quantitative polymerase chain reaction assay, mixed lymphocyte reaction, and flow cytometric analysis were employed to determine the mRNA expression of pro-inflammatory cytokines, allogeneic T-cell proliferation, and the proportion of CD4(+) CD25(+) Foxp3(+) regulatory T cells in graft-infiltrating lymphocytes and splenocytes of recipients, respectively. The results showed that the cardiac allograft survival in anti-IL-6 mAb-treated mice was prolonged significantly when compared with that of the untreated or anti-IL-17 mAb-treated mice. Meanwhile, the 'double-treated' did not prolong graft survival significantly when compared with those treated with anti-IL-6 mAb. The increase of graft survival induced by anti-IL-6 mAb was associated with reduced transcript levels for IFN-γ and IL-17, accompanied by a dramatic reduction of T-cell proliferation capacity to alloantigen stimuli and a higher proportion of Treg cells. Thus, anti-IL-6 mAb may be protective against acute rejection after cardiac transplantation through suppressing the activation of effector T cells and promoting the induction of Treg cells.

ICOS-Ig combined with CsA induces long term survival of cardiac allografts in mouse

Objective To study the synergistic effect of ICOS-Ig combined with cyclosporine (CsA) on mouse heart transplantation and explore its therapeutic potential. Methods ICOS-Ig fusion protein was generated by fusing the extracellular portion of human ICOS and Fc portion of human IgG. To investigate the effect of ICOS-Ig on T-cell proliferation in vitro, ICOS-Ig or IgG was added to the primary MLR cultures (BALB/c spleen T cells as responder cells and irradiated C57BL/6 spleen cells as stimulator cells). The cells responsiveness rates were detected by 3H-TdR methods. Then the T cells of each group in primary MLR were cultured as responder cells for secondary MLR, and irradiated C57BL/6 (donor) or C3H (third party) spleen cells as stimulator cells. To study the effect of ICOS-Ig on T-cell proliferation in vivo, CFSE-labeled C57BL/6 spleen cells were transferred to irradiated BALB/c mice. Mice were then treated with IgG, ICOS-Ig or CsA. Seventy two hours after transfer, the spleen cells of the mice were harvested for the detection of CD4 +CFSE + and CD8 +CFSE + by FACS. C57BL/6 mouse underwent transplantation of the hearts of BALB/c mouse and were then randomly divided into five equal groups: no treatment group, control IgG treated group (250 μg i.p. d2, 4, 6), ICOS-Ig treated group (250 μg i.p. d2, 4, 6), CsA treated group (10 mg/kg i.p. d0-6), ICOS-Ig combined with CsA group. The cardiac allograft survival was monitored by daily palpation. Results In primary MLR, ICOS-Ig inhibited T-cell proliferation, (inhibition ratio 58±8.2% in 50 μg/ml). In secondary MLR, ICOS-Ig specifically inhibited donor spleen cells, which suggested ICOS-Ig could induce donor-specific hyporesponsiveness. In the CFSE dye assay, CD4 +CFSE + and CD8 +CFSE + in ICOS-Ig and CsA group was stronger than those in control group, which showed ICOS-Ig and CsA could inhibit the proliferation of allo-reactive T cells in vivo. In mouse heart transplantation model, survival was significantly prolonged in animals treated with ICOS-Ig or CsA as compared with controls. Moreover, ICOS-Ig combined with CsA group had even longer engraftment (>100 d) than ICOS-Ig or CsA used alone. In histological examination, it was found that there were congestions and edemas in no treatment and IgG treated recipients, together with a lot of inflammatory cells infiltrated. Allogeneic hearts from ICOS-Ig and/or CsA immunized recipients revealed milder histological changes. It was revealed in mechanical analysis that splenic T cells from recipients also exhibited depressed mixed leukocyte reactions (MLR) and cytotoxic lymphocyte reactions (CTL). Conclusion These data suggest that ICOS-Ig combined with CsA induces a long-term survival of mouse cardiac allografts, whereas monotherapy is less effective in this regard. Thus, ICOS-Ig combined with CsA treatment may be a novel regimen to combat allograft rejection.

Arsenic Trioxide Attenuated the Rejection of Major Histocompatibility Complex Fully-Mismatched Cardiac Allografts in Mice

We investigated the effects of arsenic trioxide (As 2O 3) on allogeneic immune response using a mouse heart transplantation model. Mice were randomly divided into 4 groups of 6 animals each. The control group received phosphate-buffered saline (PBS); the As 2O 3-treated group, intraperitoneal (IP) injection of As 2O 3 (1 mg/kg) from days −3 to 10 after heart transplantation. The cyclosporine (CsA)-treated group was given a subtherapeutic dose of CsA (10 mg/kg) IP, and the As 2O 3 plus CsA-treated group, a combined protocol of As 2O 3 and CsA. Six days after transplantation, cardiac allografts were harvested for immunohistology and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The survival of the allografts was significantly improved among the As 2O 3-treated group compared with the control group (17.2 ± 1.9 vs 8.0 ± 0.9 days; P < .05). A marked prolongation (28.6 ± 6.0 days) of graft survival was achieved by the combined protocol compared with the CsA-treated group (9.6 ± 3.0 days; P < .05) or the As 2O 3-treated group. Allografts of As 2O 3-treated and As 2O 3 plus CsA-treated mice showed a changing pattern of Th1/Th2 cytokine mRNA expression. Allograft rejection was apparently alleviated by low-dose As 2O 3, and particularly when combined with a subtherapeutic CsA dose.

Altered Distribution of H60 Minor H Antigen-Specific CD8 T Cells and Attenuated Chronic Vasculopathy in Minor Histocompatibility Antigen Mismatched Heart Transplantation in Cxcr3−/− Mouse Recipients

Chemokine-chemokine receptor interactions and the subsequent recruitment of T lymphocytes to the graft are believed to be among the initial events in the development of acute and chronic rejection of heart transplants. We sought to determine the role of chemokine receptor Cxcr3 on the development of acute and chronic rejection in a multiple minor Ag mismatched mouse heart transplant model. The frequencies and kinetics of immunodominant H60 (LTFNYRNL) miHA-specific CD8 T cells in wild-type or Cxcr3-/- C57BL/6 recipients were monitored using MHC class I tetramer after BALB/b donor hearts were transplanted. Acceptance of grafts, severity of rejection, and infiltration of T cells were not altered in Cxcr3-/- recipients. However, graft survival was moderately prolonged in Cxcr3-/- recipient mice undergoing acute rejection. Analyses of splenocytes, PBLs, and graft-infiltrating cells revealed increased alloreactive T cells (H60-specific CD8 T cells) in the peripheral blood and spleen but not in the graft. Adoptively transferred Cxcr3-/- CD8 T cells in the BALB/b heart-bearing B6 scid mice showed retention of alloreactive CD8 T cells in the blood but less infiltration into the graft. Cxcr3-/- recipients with long-term graft survival also showed a marked decrease of CD8+ T cell infiltration and reduced neo-intimal hyperplasia. These data indicate that Cxcr3 plays a critical role in the trafficking as well as activation of alloreactive T cells. This role is most eminent in a transplant model when a less complex inflammatory milieu is involved such as a well-matched graft and chronic rejection.

Analysis of the Origin and Population Dynamics of Cardiac Progenitor Cells in a Donor Heart Model

Cardiac progenitor (stem) cells have recently been detected in and isolated from the myocardium of neonatal and adult mice, rats, and humans; however, the precise origin and characterization of these cells remain unclear. Using a heterotopic mouse heart transplantation model, we investigated the origin and population dynamics of cardiac progenitor cells. Donor hearts from wild-type C57/BL6 female mice were transplanted into green fluorescent protein (GFP)-transgenic C57/BL6 male mice. The donor hearts were collected 0, 2, 4, 8, and 12 weeks after transplantation. We used quantitative flow cytometry to analyze the number and origin of stem cells in the donor hearts and immunostaining to evaluate the time-related changes in their characteristics. Extracardiac GFP-positive stem cells immigrated into the donor hearts soon after transplantation. Immunostaining revealed that these GFP-positive stem cells in the donor hearts gradually lost expression of the hematopoietic markers of CD45 and CD34 and shifted to express the cardiac-specific transcription factors GATA-4 and NKx2.5. A few of the GFP-positive cells in the donor hearts finally acquired the mature cardiac phenotype in the absence of cell fusion with donor cardiomyocytes. Our discovery provides the first evidence that extracardiac stem cells may be of bone marrow origin, from which they can transform into cardiac progenitor cells in response to myocardial environment cues.

Combination Effect of Mycophenolate Mofetil with Mizoribine on Cell Proliferation Assays and in a Mouse Heart Transplantation Model

Mycophenolate mofetil (MMF) is a highly potent immunosuppressant that suppresses the proliferation of T and B cells by the uncompetitive inhibition of inosine monophosphate dehydrogenase (IMPDH). Consequently, under MMF immunosuppression, good graft survival has been achieved in clinical organ transplantation, although MMF shows adverse gastrointestinal effects. Mizoribine (MZ) also inhibits IMPDH in a competitive manner and is used clinically for organ transplantation in Japan as an immunosuppressant with fewer adverse gastrointestinal effects. Therefore, in this study we investigated the synergistic effects on in vitro and in vivo assays of mice of a combination of MMF with MZ immunosuppression. Mixed lymphocyte reaction (MLR) assay and a mouse heart transplantation model were used to evaluate the immunosuppressive effect of MMF with MZ. The median-effect principle and a combination index (CI) were employed to determine synergism, an additive effect, or antagonism. Combination of MMF with MZ resulted in mild synergistic effects in the inhibition of MLR (CI = 0.854-1.143). In the mouse heart transplantation model, C57BL/6 recipients who received a BALB/c heart under the combination of MMF and MZ at 40 + 40 or 80 + 80 mg/kg/day showed a strong synergistic prolongation of graft survival with 19.7 +/- 18.9 (P = 0.0012, CI = 0.438) and 78.4 +/- 36.9 days (P = 0.0002, CI = 0.317), respectively, compared with recipients treated with MMF or MZ monotherapy. CONCLUSIONS.: The combination of MMF and MZ showed mild synergistic effects in the inhibition of MLR and strong synergistic effects in a mouse heart transplantation model.

Novel technique for blood circuit reconstruction in mouse heart transplantation model

The surgical technique of heterotopic abdominal heart transplantation in mice, a standard model for transplantation immunology research, is very challenging restricting the widespread use of this model. A novel technique for the blood circuit reconstruction in heterotopic abdominal heart transplantation was developed. The new method makes use of the suprahepatic inferior vena cava instead of the pulmonary artery of the donor heart for the anastomosis to the infrarenal vena cava of the recipient. The sutures for the double vessel anastomoses were completed within 20.8+/-1.3 min, which was significantly shorter than 27.5+/-1.3 min in the traditional method. Similarly, the heart rebeating time postoperation (1.1+/-0.2 min) was significantly shortened as compared to 2.1+/-0.4 min using the traditional method. This novel technique facilitated abdominal heart transplantation in mice with reduced warm ischemia and grafted heart rebeating time.

Various Costimulatory Pathways Are Essential for Induction of Regulatory Cells by Intratracheal Delivery of Alloantigen

Background We previously reported that intratracheal delivery of alloantigen induced regulatory cells in a mouse heart transplantation model. We investigated the roles of costimulatory pathways in the induction of regulatory cells by intratracheal delivery of alloantigen. Methods CBA (H-2 k) mice were pretreated with intratracheal delivery of splenocytes (1 × 10 7) from C57BL/10 (H-2 b) mice and administration of monoclonal antibodies (mAb) specific for programmed death (PD)-1 and its ligands, programmed death-ligand (PD-L)1 and PD-L2, CD70, CD134 ligand (CD134L), CD153, CD137L, or receptor activator of nuclear factor-κB (NF-κB) (RANK). Seven days later, naive CBA mice underwent adoptive transfer of splenocytes (5 × 10 7) from the pretreated CBA mice and transplantation of C57BL/10 heart. Results Adoptive transfer of splenocytes from CBA mice that had been pretreated with intratracheal delivery of C57BL/10 splenocytes significantly prolonged the survival of C57BL/10 allograft (median survival time [MST], 68 days) as compared with adoptive transfer from untreated CBA mice (MST, 12 days). Concomitant administration of control immunoglobulin (Ig)G, anti-PD-L2 mAb, or anti-CD137L along with intratracheal delivery did not significantly affect the prolongation (MST, 72, 68, and 65 days, respectively). In contrast, anti-PD-1, anti-PD-L1, anti-CD70, anti-CD134L, anti-CD153, or anti-RANK mAb abrogated the prolongation induced by adoptive transfer from the pretreated mice with intratracheal delivery (MST, 18, 17, 16, 14, 10, and 18 days, respectively). Conclusion The PD-1/PD-L1, CD27/CD70, CD134/CD134L, CD30/CD153, and tumor necrosis factor (TNF)–related activation-induced cytokine (TRANCE)/RANK interactions are independently required for generation of regulatory cells by intratracheal delivery of alloantigen.

Interleukin-10 but not Transforming Growth Factor-β is Essential for Generation and Suppressor Function of Regulatory Cells Induced by Intratracheal Delivery of Alloantigen

We previously reported that intratracheal delivery of alloantigen-induced regulatory cells in mouse heart-transplantation model. Here, we investigated roles of interleukin (IL)-10 and transforming growth factor (TGF)-beta in induction and effector phases of the regulatory cells. CBA mice were pretreated with intratracheal delivery of C57BL/10 splenocytes and administration of neutralizing anti-IL-10 or anti-TGF-beta monoclonal antibody (mAb). Seven days after the pretreatment, naive CBA mice (secondary recipients) were given adoptive transfer of splenocytes from the pretreated mice and underwent heart grafting from C57BL/10 mice. To determine roles of these cytokines in the effector phase of the regulatory cells, anti-IL-10 or anti-TGF-beta mAb was administered weekly into the secondary recipients after the adoptive transfer. Adoptive transfer of splenocytes from CBA mice that had been pretreated with intratracheal delivery of C57BL/10 splenocytes significantly prolonged the survival of C57BL/10 allograft (median survival time [MST] 68 days) as compared with adoptive transfer from untreated CBA mice (MST 12 days). In the induction phase, anti-IL-10 mAb abrogated development of the regulatory cells that afforded prolonged allograft survival in the secondary recipients (MST 20 days), whereas anti-TGF-beta mAb did not abrogate it (MST 88 days). In the effector phase, anti-IL-10 mAb abrogated prolonged allograft survival afforded by adoptive transfer of the regulatory cells in the secondary recipients (MST 27 days), whereas anti-TGF-beta mAb did not abrogate suppressor function of the regulatory cells (MST 53 days). IL-10 but not TGF-beta was required for generation and suppressor function of the regulatory cells induced by intratracheal delivery of alloantigen.

Effects of a Short Course of Leflunomide on T-Independent B-Lymphocyte Xenoreactivity and on Susceptibility of Xenografts to Acute or Chronic Rejection

Leflunomide is a novel immunosuppressive agent with promising activity for xenotransplantation. It is not clear yet which mechanisms of action of leflunomide are responsible for that. In a hamster-to-C57BL/6 nude mouse heart transplantation model, a 2-week course of leflunomide was used after transplantation or for pretreating donors. Nontolerant B lymphocytes were transferred to recipients after transplantation of first or second xenogeneic heart grafts that were transplanted with or without leflunomide treatment. Hamster xenogeneic hearts transplanted into athymic C57BL/6 nude mice receiving leflunomide did not induce immunoglobulin (Ig) M xenoantibodies (XAb) and survived without signs of chronic rejection. Second xenogeneic hearts transplanted 4 weeks after withdrawal of leflunomide survived without induction of XAb but developed chronic vascular lesions. After injection of naive B lymphocytes at 6 weeks after grafting a first or second hamster heart, only in the latter case were XAb induced. These were deposited in, and provoked acute rejection of, only the second grafts. Pretreatment of donors with leflunomide decreased the ex vivo xenoantibody deposition on the xenogeneic heart endothelia. A short posttransplant course of leflunomide induces T-independent B-lymphocyte xenotolerance. Leflunomide treatment also influences xenoantigen expression, as nontolerant B lymphocytes provoke IgM XAb formation and rejection of only second xenografts (transplanted without leflunomide) and not of first xenografts (transplanted with leflunomide treatment). The ex vivo experiments that show that XAb deposition is decreased in leflunomide-pretreated xenografts further confirm this. The latter may also explain the resistance of first and not second xenografts against chronic rejection.

INHIBITION OF TERMINAL COMPLEMENT WITH A FUNCTIONALLY BLOCKING ANTI-C5 MONOCLONAL ANTIBODY PREVENTS ACUTE HUMORAL REJECTION AND ACCELERATED HUMORAL REJECTION IN A MOUSE CARDIAC ALLOGRAFT TRANSPLANTATION MODEL

O190* Aims: Antibody-mediated rejection including hyperacute rejection (HAR) or accelerated humoral rejection (ACHR) in presensitized recipients and de novo acute humoral rejection (AHR) in non-presensitized recipients cannot be prevented by anti-T cell therapy, leading to early graft loss. Complement has been shown to play an important role in antibody-mediated rejection. We previously reported that a functionally blocking anti-C5 monoclonal antibody (mAb) inhibited terminal complement activation and prevented HAR in a rat-to-presensitized mouse heart transplant model. The present study was undertaken to determine whether blocking terminal complement with anti-C5 mAb would prevent antibody-mediated rejection in mouse allograft models of AHR and ACHR. Methods: In the AHR model, C3H mouse hearts were heterotopically transplanted into BALB/c mice. In the ACHR model, BALB/c recipients were presensitized with C3H skin grafts one week prior to heart transplantation from same donor, a model designed to mimic HAR or ACHR in presensitized patients. Graft survival was determined by daily palpation. Complement activity and circulating anti-donor antibody levels were measured by a presensitized hemolytic assay and flow cytometry, respectively. In addition, antibody and complement deposition, as well as CD4 and CD8 cell infiltration in heart grafts were detected by immunohistochemistry. Results: AHR model: Allografts in untreated recipients were rapidly rejected at 8.0 ± 0.6 days. These grafts exhibited typical AHR, characterized by elevation of circulating anti-donor antibodies and antibody deposition, vasculitis, hemorrhage, fibrin deposition and thrombosis in the grafts. Administration of cyclosporine A (CsA, 15mg/kg, daily, S.C.) did not inhibit AHR, and grafts were rejected in 15.5 ± 1.1 days. Anti-C5 mAb monotherapy (40mg/kg/day, twice a week, I.P.) completely inhibited terminal complement activity and attenuated pathological changes of AHR, but did not prolong graft survival. In contrast, the combination of anti-C5 mAb and CsA successfully prevented AHR and achieved indefinite graft survival in all recipients (>100 days). This therapy completely inhibited terminal complement activity and prevented both cellular- and humoral-mediated rejection. Evaluation of the long-term surviving allografts revealed no pathology. ACHR model: After presensitization, anti-donor antibody levels, predominantly total IgG and IgG1, were rapidly elevated in BALB/c recipients. In untreated recipients, C3H heart grafts were rejected in 3 days by ACHR, characterized by severe hemorrhage, infarction and thrombosis. Immunohistochemistry showed massive antibody and complement deposition but minimal CD4+ and CD8+ cell infiltration in heart grafts. Treatment with CsA (15mg/kg, daily, S.C.) and/or cyclophosphamide (CyP, 40mg/kg, I.V., days 0 and 1) did not prevent ACHR and grafts were rejected in 3-4 days. Remarkably, the addition of anti-C5 mAb to the same dosing regimen of CsA and CyP effectively inhibited antibody-mediated rejection. These grafts have survived for more than 70 days and continue to demonstrate normal function and histology. Conclusions: The combination therapy of anti-C5 mAb and CsA achieves indefinite graft survival in a mouse allograft model of AHR and triple therapy of anti-C5 mAb, CsA and CyP effectively prevents ACHR in presensitized mouse recipients. These data suggest that inhibition of terminal complement using a novel anti-C5 mAb may be of value in the prevention of humoral rejection in clinical transplantation including presensitized recipients.

Role of ve-cadherins, beta-1 integrins and endothelin-1 in endothelial dysfunction after cardiac transplantation

Introduction: Endothelial dysfunction is a major cause of chronic allograft vasculopathy in cardiac transplantation. The dysfunction of the endothelium in coronary vasculature can be due to the loss of endothelial contacts with the extracellular matrix, endothelial cell-to-cell contact. We investigated the expression of VE-cadherins, integrins and endothelin-1 in a heart transplant model in mice to determine if endothelial dysfunction is from the underexpression of these endothelial specific molecules. Methods: Nonvascularised neonatal heart allografts from C57 BL/6 (H-2b) were transplanted to ear pinna of Balb/c (H-2d) recipient mice. Isografts from C57 BL/6 (H-2b) mice were performed as control. Allo and isografts were harvested at P.O day 1,3,5,7 & immunostaining for endothelin-1, VE-cadherin and Beta-1 integrin was performed with monoclonal antibodies specific for each molecules. Results: In normal neonatal hearts, VE-cadherin and Beta-1 integrin were detected in endothelial cells only, while ET-1 was detected in both endothelial and myocardial cells. Following transplantation, VE-cadherin became undetectable until P.O day 7 in isografts and P.O.day 5 in allografts at which time it became detectable at low levels on endothelial cells. Beta-1 integrin showed a pattern similar to VE-cadherin, except that it became barely detectable in either iso or allografts after transplantation. ET-1 did not show any detectable changes in either allo or isografts after transplantation. Conclusion: VE-cadherin and Beta-1 integrin expression on endothelial cells is diminished after cardiac transplantation, while ET-1 expression continues after transplantation. These data provide molecular insights into the loss of vascular endothelial integrity in cardiac allografts.

Cardiac Unloading Alters Contractility and Calcium Homeostasis in Ventricular Myocytes

M. Ritter, Z. Su, S. Xu, J. Shelby and W. H. Barry. Cardiac Unloading Alters Contractility and Calcium Homeostasis in Ventricular Myocytes. Journal of Molecular and Cellular Cardiology (2000) 32, 577–584. Altered cardiac workload has an important effect on myocyte structure and function. Cardiac hypertrophy resulting from an increase in load has been studied extensively in the past. However, the effects of unloading and atrophy have recently become of more interest since devices for mechanical left ventricular unloading have been introduced into clinical practice for the treatment of patients with terminal heart failure, and a resulting improved cardiac and myocyte contractility have been reported. We used the heterotopic abdominal mouse heart transplant model in order to study the effects of 5 days of unloading on cell size (confocal microscopy), contractility (fractional shortening: video motion), calcium homeostasis ([Ca2+]itransients, SR Ca2+content); and L-type Ca2+and sodium/calcium exchanger currents (whole cell patch clamp technique). We found unloading caused decreased cell volume consistent with atrophy. An increased fractional shortening and [Ca2+]itransient were observed in myocytes from unloaded hearts as compared with controls. Transsarcolemmal ICa,Land INa/Cadensities, and SR Ca2+content were unaltered, as was membrane capacitance. A reduction in cell volume with mainteinance of internal and surface membrane areas, and/or a decrease in concentration of cellular protein Ca2+buffers, may contribute to the increase in the [Ca2+]itransient in this model.

Complement inhibition with an anti-C5 monoclonal antibody prevents hyperacute rejection in a xenograft heart transplantation model.

The present study was undertaken to determine whether anti-complement 5 (C5) monoclonal antibodies (mAb) prevent hyperacute rejection (HAR) in a rat-to-presensitized mouse heart transplantation model and whether these mAb, combined with cyclosporine (CsA) and cyclophosphamide (CyP), can achieve long-term graft survival. BALB/c mice were presensitized with 2x10(7) splenocytes from Lewis rats 14 days before grafting. Heart grafts from Lewis rats were heterotopically transplanted into BALB/c mice. Presensitized mice were treated with either anti-C5 mAb or a combination of anti-C5 mAb, CsA, and CyP. Controls included: presensitized mice with no treatment, presensitized mice treated with either CsA + CyP or IgG, and nonpresensitized mice with either no treatment or with CsA + CyP treatment. Although typical features of HAR were evident in the presensitized grafts, the mAb completely inhibited complement activation and successfully prevented HAR. Despite complement inactivation, the graft was rejected on postoperative day 6 with acute vascular rejection (AVR) also known as delayed xenograft rejection (DXR). Notably, this type of rejection cannot be effectively overcome by CsA and CyP. We conclude that (1) anti-C5 mAb prevents HAR, (2) AVR/DXR still occurs when HAR is prevented by complement inactivation, and (3) AVR/DXR cannot be overcome by conventional immunosuppression. These data suggest that anti-C5 mAb may be valuable for preventing HAR in future clinical xenotransplantation and that additional interventions may be required to address AVR/DXR.