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Objective: In heart transplantation, there is a critical need for development of biomarkers to noninvasively monitor cardiac allografts for immunologic rejection or injury. Exosomes are tissue-specific nanovesicles released into circulation by many cell types. Their profiles are dynamic, reflecting conditional changes imposed on their tissue counterparts. We proposed that a transplanted heart releases donor-specific exosomes into the recipient's circulation that are conditionally altered during immunologic rejection. We investigated this novel concept in a rodent heterotopic heart transplantation model., Materials and Methods: Full major histocompatibility mismatch (BALB/c [H2-K d ] into C57BL/6 [H2-K b ]) heterotopic heart transplantation was performed in 2 study arms: Rejection (n = 64) and Maintenance (n = 28). In the Rejection arm, immunocompetent recipients fully rejected the donor heart, whereas in the Maintenance arm, immunodeficient recipients (C57BL/6 Prkdc SCID ) accepted the allograft. Recipient plasma exosomes were isolated and a donor heart-specific exosome signal was characterized on the nanoparticle detector for time-specific profile changes using anti-H2-K d antibody quantum dot., Results: In the Maintenance arm, allografts were viable throughout follow-up of 30 days, with histology confirming absence of rejection or injury. Time course analysis (days 1, 2, 3, 4, 5, 7, 9, 11, 15, and 30) showed that total plasma exosome concentration (P = .157) and donor heart exosome signal (P = .538) was similar between time points. In the Rejection arm, allografts were universally rejected (median, day 11). Total plasma exosome quantity and size distribution were similar between follow-up time points (P = .278). Donor heart exosome signals peaked on day 1, but significantly decreased by day 2 (P = 2 × 10 -4 ) and day 3 (P = 3.3 × 10 -6 ), when histology showed grade 0R rejection. The receiver operating characteristic curve for a binary separation of the 2 study arms (Maintenance vs Rejection) demonstrated that a donor heart exosome signal threshold < 0.3146 was 91.4% sensitive and 95.8% specific for diagnosis of early acute rejection., Conclusions: Transplant heart exosome profiling enables noninvasive monitoring of early acute rejection with high accuracy. Translation of this concept to clinical settings might enable development of a novel biomarker platform for allograft monitoring in transplantation diagnostics., (Copyright © 2018 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

The long-lived plasma cells, which develop after alloantigen sensitization, produce donor specific alloantibodies (DSAs) that generate a positive serum cross-match and preclude transplantation. Bortezomib, a proteasome inhibitor, is being investigated in clinical desensitization protocols, however preclinical studies in a transplant model are nonexistent. We hypothesized that sustained treatment with only a proteasome inhibitor would eliminate plasma cells and reduce DSA over time. Cardiac allografts were transplanted into murine recipients. Eight weeks after allograft rejection the proteasome inhibitor, bortezomib, was injected intravenously twice weekly for 60 days. Serum alloantibody responses were assayed using flow cross-match. Total and alloreactive plasma cell numbers were enumerated using flow cytometry and ELISPOT. All recipients of cardiac allografts rejected their graft promptly within 16 days and demonstrated alloantibody by flow cross-match. DSA was sustained in the control mice while mice treated with bortezomib had sustained elimination of DSA and a marked reduction in plasma cell population. Also, bortezomib was associated with an increased level of BLyS. Within a murine model, proteasome inhibition can eliminate alloantibody secreting plasma cells, and reduce alloantibody. Cessation of bortezomib is not associated with return of DSA., (© 2013.)

Background: Systemic administration of soluble recombinant fusion protein of cytotoxic T lymphocyte antigen 4 (CTLA4Ig) induces blockade of the CD28/B7 costimulatory pathway and promotes survival of allogeneic and xenogeneic grafts. We tested the efficacy of local expression of CTLA4Ig gene in the myocardium, induced by transduction with a recombinant adenovirus encoding the CTLA4Ig gene, on the survival of rat cardiac allografts., Methods: The donor hearts were perfused ex vivo with recombinant adenovirus encoding CTLA4Ig cDNA (AdCTLA4Ig) via intra-aorta coronary artery before transplantation. The distribution and duration of CTLA4Ig transgene expression in the myocardium was assessed by reverse transcriptase polymerase chain reaction (RT-PCR) or in situ RT-PCR after transplantation., Results: In situ RT-PCR demonstrated abundant expression of CTLA4Ig transgene in the endo-myocardium of AdCTLA4Ig-perfused cardiac grafts. Lewis and Brown Norway cardiac allografts transduced with AdCTLA4Ig survived indefinitely in nonimmunosuppressed Wistar Furth recipients. However, donor-strain skin grafts were rejected by long-term recipients of cardiac allografts, which also triggered the rejection of the primary heart grafts., Conclusions: A single ex vivo intra-aortic infusion of recombinant adenovirus encoding the CTLA4Ig gene induced efficient transduction of the endo-myocardium and promoted the permanent survival of cardiac allografts in nonimmunosuppressed hosts. Despite the beneficial effect of local immunosuppression on cardiac allograft survival, the strategy failed to promote a state of donor-specific peripheral tolerance.

Intrathymic inoculation of allogeneic cells after systemic administration of antilymphocyte serum in adult experimental animals has produced donor-specific tolerance to cardiac allografts. We investigated whether thymic injection of allogeneic cells without antilymphocyte serum in neonatal Lewis rats (day 1 of life) with immature immune systems also produced tolerance to cardiac grafts. Intrathymic or intraperitoneal injection of 5 x 10(7) Lewis (control) or Lewis-Brown Norway (allogeneic) spleen cells in Lewis neonates was followed by heterotopic cardiac transplantation using Lewis, Lewis-Brown Norway, or Wistar Furth (third-party allograft) hearts at 6 to 8 weeks of age. Graft survival was prolonged with both intraperitoneal and intrathymic allogeneic cells. Recipients of cells by the intrathymic route had longer graft survival, and 2 of 5 animals achieved permanent graft acceptance (longer than 100 days). As expected, Lewis isografts survived indefinitely, whereas third-party Wistar Furth allografts were rejected in the usual time frame. Intrathymic introduction of allogeneic cells in a neonatal recipient with an immature immune system can produce donor-specific tolerance to a subsequent graft without the need for a systemic immunosuppression regimen, even transiently.

"Actively acquired tolerance" to foreign cells was described in 1953 by Medawar and colleagues and formed the basis for subsequent efforts in organ transplantation. We have applied these historic principles of intrauterine immune manipulation in a vascularized cardiac allograft model. Allogeneic Lewis-Brown Norway (LBN) splenocytes (0 to 5 x 10(7) cells) were injected intraperitoneally into each fetus of a pregnant Lewis rat at day 14 to 16 of gestation, when T cells are "educated" to distinguish self from foreign. This manipulation causes fetal attrition inversely proportional to the number of cells injected. Heterotopic transplantation using an LBN heart was carried out in each surviving fetus at 6 to 8 weeks of age. Untreated Lewis rats rejected LBN hearts within 7.6 days. Rats receiving LBN splenocytes in utero demonstrated prolongation of graft survival proportional to the number of cells given. Surviving animals exposed to 5 x 10(7) allogeneic cells in utero (n = 4) had graft survivals of 24 to more than 150 days (mean, 88.0 days), significantly longer than control animals (6 to 10 days; mean, 7.6 days; p < 0.02). Significant prolongation of cardiac allograft survival and in some cases complete tolerance can be achieved by intrauterine exposure to allogeneic cells at a critical period of immunologic development. Because many serious cardiac defects amenable to treatment by cardiac transplantation can be detected by ultrasound early in gestation, treatment based on this strategy may become useful in pediatric heart transplantation.

Permanent tolerance to an experimental cardiac allograft can be achieved by pretransplantation intrathymic inoculation of donor-specific lymphoid cells. We studied the effects of intrathymic inoculation of xenogeneic cells and intravenous cobra venom factor in a rodent model of cardiac xenotransplantation. Lewis rats underwent intraabdominal heterotopic heart transplantation with Syrian hamster donors. In untreated animals, mean graft survival time was 3 days. Five rats had 1 ml of antilymphocyte serum administered intraperitoneally. One day later, 2.5 x 10(7) hamster spleen cells were inoculated into the thymus under direct vision. Twenty-one days after antilymphocyte serum was given, heterotopic heart transplantation with a hamster donor was carried out. In all cases, rejection was accelerated and occurred between 20 minutes and 1 day after transplantation. Mean graft survival time was 5.2 hours (p < 0.0001 versus control). Six animals treated with antilymphocyte serum and intrathymic xenogeneic cells had 0.5 ml of cobra venom factor, a complement antagonist, administered intravenously 3 hours before transplantation and every other day thereafter. Mean graft survival was 3 days, which was not different from the response of naive animals. Animals treated with antilymphocyte serum only had no prolongation of graft survival (mean survival time 3 days, p = not significant). Animals treated with cobra venom factor alone (n = 5) before transplantation and on alternate days subsequently had mild graft prolongation with a mean survival time of 4 days (p = 0.0133). In contrast to experimental allograft models, intrathymic inoculation of xenogeneic cells produces hyperacute rejection in these naturally concordant species. The administration of cobra venom factor abrogates the hyperacute response, but the combination of cobra venom factor and intrathymic inoculation does not produce long-term graft survival.

Donor-specific unresponsiveness is an important goal of heart transplantation research. Work by other investigators has shown that intrathymic transplantation of pancreatic islet cells not only leads to factor permanent acceptance of the intrathymic graft but tolerance to subsequent extrathymic islet cell transplants. We applied this concept to a model of heart allotransplantation in the rat. Recipient Lewis rats were treated with 1 ml of antilymphocyte serum and 5 x 10(7) Lewis-Brown Norway spleen cells injected into the thymus under direct vision. Twenty-one days later, heterotopic heart transplantation was performed with Lewis-Brown Norway (allograft) and Wistar-Furth (third-party allograft) donors. Control Lewis recipients received no treatment, antilymphocyte serum alone, or antilymphocyte serum plus intrathymic syngeneic Lewis spleen cells. Another group of animals received intravenous Lewis-Brown Norway cells before transplantation with Lewis-Brown Norway heart donors. Untreated control animals had heart graft survival of 6 to 10 days (mean, 7.6 days) and 7 to 9 days (mean, 7.8 days) for Lewis-Brown Norway (n = 5) and Wistar-Furth (n = 5) donors, respectively. Antilymphocyte serum alone (n = 10) failed to prolong survival of Lewis-Brown Norway grafts (mean, 10.7 days; p = not significant). Antilymphocyte serum plus intrathymic Lewis cells (n = 5) did not prolong survival of a subsequent Lewis-Brown Norway graft (mean, 9.2 days; p = not significant). Survival of Wistar-Furth third-party allografts (n = 10) was not prolonged by intrathymic Lewis-Brown Norway cells (mean survival, 13.9 days; p = not significant).(ABSTRACT TRUNCATED AT 250 WORDS)