Long-term Cardiac Allograft Survival Research Articles

Ligation of HLA class I molecules on smooth muscle cells with anti-HLA antibodies induces tyrosine phosphorylation, fibroblast growth factor receptor expression and cell proliferation.

The development of transplant atherosclerosis, a manifestation of chronic rejection, is the major obstacle to long-term survival of cardiac and renal allografts. The incidence of transplant atherosclerosis is increased in transplant recipients producing antidonor HLA antibodies following transplantation, suggesting that anti-HLA antibodies play a role in the pathogenesis of the disease. We have postulated that anti-HLA antibodies mediate the development of transplant atherosclerosis by binding to class I molecules on the endothelium and smooth muscle of the graft and transducing signals which stimulate cell proliferation. In this report we demonstrate that anti-HLA class I antibodies transduce signals in smooth muscle cells stimulating increased tyrosine phosphorylation of intracellular proteins and up-regulation of fibroblast growth factor (FGF) receptors. Antibody binding to class I molecules on smooth muscle cells is also accompanied by increased responsiveness to basic FGF and augmented cell proliferation. These findings may explain the increased occurrence of transplant atherosclerosis in recipients producing anti-donor HLA antibodies.

Synergism of the malononitrilamides 279 and 715 with cyclosporine A in the induction of long-term cardiac allograft survival.

We tested here the effects of malononitrilamide (MNA) 279 and MNA 715 (derivatives of A771726, the active metabolite of leflunomide) in monotherapy and in combination with cyclosporine A (CSA) on heterotopically transplanted rat cardiac allografts (BN) [Brown Norway Lewis]. Both MNAs (5-20 mg/kg) displayed a dose-dependent increase of efficacy. The combination of CSA (5 mg/kg) with the MNAs (5 and 10 mg/kg) showed a synergistic effect in the prolongation of allograft survival and in the induction of long-term allograft survival. To investigate the immunological mechanism responsible for long-term allograft survival, we transplanted second set (BN) and third party (Dark Agouti) skin allografts on the tail of long-term surviving rats. The cause for long-term allograft survival turned out to be a donor-specific tolerance. We formulate a hypothesis for the mechanism of the synergism of the combination MNA + CSA in the induction of tolerance.

Synergism of the malononitrilamides 279 and 715 with cyclosporine A in the induction of long-term cardiac allograft survival

Abstract We tested here the effects of malononitrilamide (MNA) 279 and MNA 715 (derivatives of A771726, the active metabolite of leflunomide) in monotherapy and in combination with cyclosporine A (CSA) on heterotopically transplanted rat cardiac allografts (BN) [Brown Norway Lewis]. Both MNAs (5–20 mg/kg) displayed a dose-dependent increase of efficacy. The combination of CSA (5 mg/kg) with the MNAs (5 and 10 mg/kg) showed a synergistic effect in the prolongation of allograft survival and in the induction of long-term allograft survival. To investigate the immunological mechanism responsible for long-term allograft survival, we transplanted second set (BN) and third party (Dark Agouti) skin allografts on the tail of long-term surviving rats. The cause for long-term allograft survival turned out to be a donor-specific tolerance. We formulate a hypothesis for the mechanism of the synergism of the combination MNA + CSA in the induction of tolerance.

INTERFERON-GAMMA (IFN??) IS CRITICAL FOR REGULATING THE PROLIFERATION OF ALLOANTIGEN-ACTIVATED T CELLS

25 We have observed that B7-CD28 T cell costimulation blockade with CTLA4Ig, administered two days after transplantation, induces longterm cardiac allograft survival in wildtype (IFNγ+/+) mice but fails to do so in IFNγ gene-knockout (IFNγ-/-) recipients or in IFNγ+/+ mice treated with IFNγ-neutralizing antibody[mean survival time = 51±15 (n=8; 2/8 grafts survived >100 days), 16±2 (n=7), and 10±1 (n=3) days, respectively]. In this study, we investigated the mechanisms by which endogenous IFNγ facilities induction of longterm allograft survival by analysing thein vivo proliferation and apoptosis of alloantigen-activated T cells in IFNγ+/+ and IFNγ-/- mice. IFNγ+/+ or IFNγ-/- BALB/c (H-2d) mice were injected in the footpads with 1 × 107 allogeneic C3H/He(H-2k) splenocytes on days 0 and 4. Mice were either left untreated or received 200µg CTLA4Ig i.p. on day 2. Lymph node T cells were isolated on day 6 and analysed for DNA replication (BrdU uptake) and for apoptosis (TUNEL) by flow cytometry. Control mice were injected with syngeneic splenocytes. Results: Alloantigen-induced in vivo T cell proliferation was markedly increased in IFNγ-/- mice. Moreover, CTLA4Ig failed to completely inhibit T cell proliferation in the absence of IFNγ: Table Similarly, exaggerated in vitro proliferation of IFNγ-/- splenocytes was observed in primary MLRs. Superantigen-induced Vβ8+ T cell expansion was also significantly increased in IFNγ-/- mice. On the other hand, alloantigen-inducedin vivo T cell apoptosis was neither impaired nor increased in the absence of IFNγ: Table Results shown are representative of 3 to 4 independent experiments. Exaggerated proliferation in the absence of IFNγ was observed in both the CD4 and CD8 T lymphocyte compartments.Conclusions: (1) IFNγ is critical for limiting in vivo expansion of alloreactive T lymphocytes, possibly by regulating their proliferation. This essential function of IFNγ may account for failure to induce longterm allograft survival in IFNγ-deficient mice.(2) IFNγ is not required for activation-induced apoptosis of alloreactive T lymphocytes.

IL4KO MICE FAIL TO DEVELOP LONG-TERM ALLOGRAFT SURVIVAL

671 Introduction: This study examines the role of IL4 in long-term cardiac allograft survival initiated by short-term treatment with gallium nitrate (GN) or the anti-CD4 mAb, GK1.5. Methods: DBA/2 cardiac allografts were implanted heterotopically into IL4-intact or IL4-KO C57BL/6 (B6) recipients which were then treated peri-transplant with GK1.5, or for 28 days with GN. Graft survival was monitored by transabdominal palpation. Immune function in these allograft recipients was assessed at >60 days post transplant by testing for donor-reactive alloantibody, and donor-reactive DTH responses. Results: DBA/2 heterotopic cardiac allografts are not rejected by IL4-intact C57BL/6 (B6) recipients if the animals are rendered functionally tolerant by transient treatment with GN or with GK1.5. Most(78%) IL4-intact cardiac allograft recipients produce donor-reactive IgG alloantibody. None of these animals exhibits donor-reactive DTH responses. The lack of donor-reactive DTH responses is due to active down regulation, evidenced by the linked unresponsiveness DTH assay. In this assay, splenocytes from long term allograft recipients interfere with DTH responses to an unrelated antigen, tetanus toxoid (TT), but only if both the donor antigen and the TT are locally provided. Unlike IL4-intact mice, IL4-KO B6 recipients reject DBA/2 cardiac allografts after the immunosuppressive effect of GN or GK1.5 dissipates. IL4-KO mice also exhibit donor-reactive DTH when challenged via the allo-MHC-restricted pathway, and are unable to down-regulate TT DTH responses in the linked unresponsiveness DTH assay. Like the IL4-intact recipients, IL4-KO graft recipients produce donor-reactive IgG alloantibody. Conclusions: Immunosuppression with GN or GK1.5 induces an alternative pattern of allosensitization which allows donor-reactive Ab production but not acute rejection or alloreactive DTH. The latter is due to the generation of a DTH regulator. Our data indicate that IL4 is required for the induction and/or maintenance of this alternative allosensitization process.

A MOLECULAR MECHANISM FOR THE DIRECT ANTIPROLIFERATIVE EFFECT OF MALONONITRILAMIDES (MNAs) ON VASCULAR SMOOTH MUSCLE CELLS.

148 Introduction: Despite advances in immunosuppression, growth factor mediated chronic rejection in the form of graft vascular disease (GVD) and obliterative bronchiolitis (OB) remains one of the major factors limiting long-term cardiac and pulmonary allograft survival. Using animal models of GVD, it has been demonstrated in vivo, that leflunomide (LFM) prevented the formation of neointinal hyperplasia, characterized by proliferation of vascular smooth muscle cells in the vessel wall. MNAs belong to the derivatives of LFM's primary metabolite A77 1726 and have been shown to bind specifically to dehydroorotate-dehydrogenase, thereby inhibiting the de novo pyrimidine biosynthesis. They block T- and B-cell proliferation and were found to prevent and reverse acute allograft rejection and effectively prolonged xenograft survival. This led us to hypothesize, that MNAs also may have antiproliferative activities not only on the immune cells of the recipient but also on the mesenchymal cells in the donor organ. The aim of this study was to investigate the effects of the MNAs (HMR 1279 or HMR 1715) on the PDGF stimulated cell proliferation of smooth muscle cells. As a reference compound A77 1726 was used. Methods: Embryonic thoracic aorta smooth muscle cells from DB 1 X rats (A10 cells from the ATCC) were used in these experiments. They were incubated with the test substances for 24 hrs before PDGF addition and thereafter additionally for 3 days. Cell numbers were quantitated with the sulforhodamine B assay. To determine an appropriate(untoxic) drug concentration range, first simple IC50 estimations were carried out and a concentration range between 1.6 and 40 μM was chosen. In some experiments, uridine (1 mM) was added as a putative antagonist.Results: The MNAs (HMR 1279 or HMR 1714 and A77 1726) dose-dependently inhibited the proliferation of PDGF stimulated rat smooth muscle cells with an IC50 of approximately 3 μM. At low concentrations (1.6 μM) the order of potency was HMR 1279 > A77 1726> HMR 1715, and at 40 μM there was a complete inhibition with all three compounds. The antiproliferative effect (up to 8 μM of the MNAs) could be reversed by the simultaneous incubation with 1 mM uridine.Conclusion: While the MNAs HMR 1279 or HMR 1715 inhibit the proliferation of PDGF-stimulated rat smooth muscle cells in a dose-dependent manner, these effects can be antagonized by the addition of exogenous uridine, suggesting that suppression of pyrimidine biosynthesis may be involved in the mechanism of action of these immunosuppressive compounds.

INTERFERON-GAMMA (IFNγ) IS CRITICAL FOR REGULATING THE PROLIFERATION OF ALLOANTIGEN-ACTIVATED T CELLS.

25 We have observed that B7-CD28 T cell costimulation blockade with CTLA4Ig, administered two days after transplantation, induces longterm cardiac allograft survival in wildtype (IFNγ+/+) mice but fails to do so in IFNγ gene-knockout (IFNγ-/-) recipients or in IFNγ+/+ mice treated with IFNγ-neutralizing antibody [mean survival time = 51±15 (n=8; 2/8 grafts survived >100 days), 16±2 (n=7), and 10±1 (n=3) days, respectively]. In this study, we investigated the mechanisms by which endogenous IFNγ facilitates induction of longterm allograft survival by analysing the in vivo proliferation and apoptosis of alloantigen-activated T cells in IFNγ+/+ and IFNγ-/- mice. IFNγ+/+ or IFNγ-/- BALB/c (H-2d) mice were injected in the footpads with 1×107 allogeneic C3H/He (H-2k) splenocytes on days 0 and 4. Mice were either left untreated or received 200μg CTLA4Ig i.p. on day 2. Lymph node T cells were isolated on day 6 and analysed for DNA replication (BrdU uptake) and for apoptosis (TUNEL) by flow cytometry. Control mice were injected with syngeneic splenocytes. Results: Alloantigen-induced in vivo T cell proliferation was markedly increased in IFNγ-/- mice. Moreover, CTLA4Ig failed to completely inhibit T cell proliferation in the absence of IFNγ: TableSimilarly, exaggerated in vitro proliferation of IFNγ-/- splenocytes was observed in primary MLRs. Superantigen-induced Vβ8+ T cell expansion was also significantly increased in IFNγ-/- mice. On the other hand, alloantigen-inducedin vivo T cell apoptosis was neither impaired nor increased in the absence of IFNγ: TableResults shown are representative of 3 to 4 independent experiments. Exaggerated proliferation in the absence of IFNγ was observed in both the CD4 and CD8 T lymphocyte compartments. Conclusions: (1) IFNγ is critical for limiting in vivo expansion of alloreactive T lymphocytes, possibly by regulating their proliferation. This essential function of IFNγ may account for failure to induce longterm allograft survival in IFNγ-deficient mice. (2) IFNγ is not required for activation-induced apoptosis of alloreactive T lymphocytes.

INTERFERON-GAMMA (IFNγ) IS REQUIRED FOR LONGTERM ALLOGRAFT SURVIVAL INDUCED BY COMBINED ADMINISTRATION OF DONOR-SPECIFIC SPLENOCYTE TRANSFUSION(DSST) AND CTLA4Ig.

433 Administration of donor splenocytes or bone marrow enhances the salutary effect of B7-CD28 costimulation blockade on allograft survival in rodents. Because IFNγ has important immunoregulatory functions, we addressed whether this cytokine plays a role in DSST+CTLA4Ig-mediated immunosuppression by comparing the survival of fully allogeneic cardiac or skin grafts in wildtype (IFNγ+/+) recipients to that in IFNγ gene-knockout(IFNγ-/-) mice. DSST (1 × 107 donor splenocytes) was administered i.v. at the time of transplantation. CTLA4Ig (200μg) was administered i.p. in a single dose two days after transplantation. Cardiac rejection was defined as cessation of palpable heart beat and was confirmed histologically. Skin rejection was defined as >90% necrosis of the graft(trunk skin). Results: DSST+CTLA4Ig-mediated prolongation of cardiac allograft survival (C3H/He → BALB/c) was significantly greater in IFNγ+/+ than IFNγ-/- recipients:TableThese data are consistent with our previous findings that endogenous IFNγ facilitates induction of longterm cardiac allograft survival in mice treated with CTLA4Ig alone. Similarly, DSST+CTLA4Ig-mediated prolongation of skin allograft survival(BALB/c → C57BL/6), although modest, was significantly greater in IFNγ+/+ than IFNγ-/- recipients:TableConclusions: (1) Endogenous IFNγ does not hinder but rather facilitates induction of longterm allograft survival in mice treated with donor-specific splenocyte transfusion and CTLA4Ig. (2) These preliminary data suggest that IFNγ plays an essential regulatory role in alloimmunity. Because cyclosporine inhibits IFNγ production, cyclosporine could abrogate the potential clinical utility of donor blood, bone marrow, or splenocyte administration to recipients of solid organ allografts.

Induction of allograft nonresponsiveness after intrathymic inoculation with donor class I allopeptides. II. Evidence for persistent chronic rejection despite high levels of donor microchimerism.

We have recently demonstrated that three synthetic peptides corresponding to the donor class I RT1.Aa molecule induce long-term survival of cardiac allografts in the PVG.R8-to-PVG.1U rat strain combination (disparate for one isolated class I, RT1.A, molecule) when presented to the recipient immune system in the thymus. Long-term graft survivors had measurable levels of donor-reactive alloantibodies in their serum. In this study, we examined long-term allografts for the presence of chronic rejection and donor microchimerism to assess whether this regimen of immune modulation establishes true tolerance and whether this tolerance is dependent upon the presence of donor-recipient microchimerism. Histological examination of long-term heart grafts (>100 days) demonstrated chronic rejection, including a mild degree of myocardial infiltration by mononuclear cells, mild to moderate myocardial fibrosis, and various vascular changes ranging from focal intimal thickening to total vascular lumen blockade due to smooth muscle cell proliferation. In contrast, long-term syngeneic hearts transplanted under similar experimental conditions lacked these pathological manifestations. Donor microchimerism was analyzed using the polymerase chain reaction with a pair of oligonucleotides specific for the donor class I RT1.Aa gene and genomic DNA harvested from various tissues from graft recipients. We detected high levels of donor microchimerism in the heart, kidney, liver, skin, bone marrow, thymus, and lymph nodes of long-term graft recipients. Donor microchimerism was also detected in unmanipulated control graft recipients at rejection (7 days) and in intrathymically manipulated recipients that rejected allografts in a delayed fashion (12-82 days). These data clearly demonstrate that intrathymic inoculation of donor class I allopeptides induces long-term graft survival but does not prevent chronic rejection. Allograft rejection occurred despite high levels of donor microchimerism, providing direct evidence that donor-recipient microchimerism is not sufficient for the prevention of acute or chronic rejection in this model.

Blockade of the CD40-CD40 ligand pathway potentiates the capacity of donor-derived dendritic cell progenitors to induce long-term cardiac allograft survival.

Failure of costimulatory molecule-deficient donor dendritic cells (DCs) to induce indefinite allograft acceptance may be a result of the 'late" up-regulation of these molecules on the DCs after interaction with host T cells. Ligation of CD40 on antigen-presenting cells by its cognate ligand CD40L is thought to induce expression of CD80 (B7-1) and CD86 (B7-2). We examined the influence of anti-CD40L monoclonal antibody (mAb) on the capacity of donor-derived DC progenitors to induce long-term allograft survival. High purity DC progenitors were grown from B10 (H2b) mouse bone marrow in granulocyte-macrophage colony-stimulating factor and transforming growth factor beta1 (TGFbeta1). Mature DC were propagated in granulocyte-macrophage colony-stimulating factor and interleukin-4. Their phenotype was characterized by flow cytometric analysis and their function by mixed leukocyte reactivity. Anti-donor cytotoxic T lymphocyte activity in grafts and spleens of vascularized heart allograft recipients was also assessed. The TGFbeta3-cultured cells were (1) DEC 205-positive, MHC class II-positive, CD80dim, CD86dim, and CD40dim, (2) poor stimulators of naive allogeneic T-cell proliferation, and (3) able to prolong significantly B10 cardiac allograft survival in C3H (H2k) recipients when given (2 x 10[6] i.v.) 7 days before organ transplantation (median survival time [MST] 26 days vs. 12 days in controls, and 5 days in interleukin-4 DC-treated animals). Their allostimulatory activity was further diminished by addition of anti-CD40L mAb at the start of the mixed leukocyte cultures. Anti-CD40L mAb alone (250 microg/mouse, i.p.; day -7) did not prolong cardiac graft survival (MST 12 days). In contrast, TGFbeta-cultured DCs + anti-CD40L mAb extended graft survival to a MST of 77 days, and inhibited substantially the anti-donor cytotoxic T lymphocyte activity of graft-infiltrating cells and host spleen cells assessed 8 days after transplant. The CD40-CD40L pathway appears important in regulation of allogeneic DC-T-cell functional interaction in vivo; its blockade increases markedly the potential of costimulatory molecule-deficient DCs of donor origin to induce long-lasting allograft survival.

Long-term survival of solid organ allografts by brief anti-lymphocyte function-associated antigen-1 monoclonal antibody monotherapy.

Strategies targeting lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18) and intercellular adhesion molecule-1 (ICAM-1) have previously been shown to produce long-term survival of solid organ allografts in animals only when both CD11a and ICAM-1 are targeted for a brief (6-7 days) time or when extended (14 weeks) treatment with anti-CD11a monoclonal antibody (mAb) is administered. We show that recipient pretreatment followed by a brief (13 days) treatment course with high-dose anti-CD11a mAb alone produces long-term survival of cardiac allografts in the rigorous, nonprimarily vascularized heart allograft model in mice. This treatment regimen induces specific unresponsiveness in our model. In recipients bearing long-term beating cardiac grafts after treatment with anti-CD11a mAb, there still exists a high frequency of potentially antigen-reactive T cells in isolated peripheral blood lymphocyte fractions. Therefore, clonal deletion does not appear to explain the induction of specific unresponsiveness by treatment with anti-CD11a mAb in this model. These findings support the further investigation of the use of high-dose anti-LFA-1 mAb monotherapy in the pre- and early postoperative period to promote solid organ allograft survival.

Early Graft CAD Due to Virus

Accelerated diffuse coronary atherosclerosis is the main limitation of long-term cardiac allograft survival. This study from Stanford University compared the early and late appearance of accelerated graft coronary artery disease in 569 consecutive heart transplant recipients. One hundred thirty-nine patients developed angiographically apparent graft CAD during follow-up and were …

Predictive model to assess risk for cardiac allograft vasculopathy: An intravascular ultrasound study

This study was performed to assess the influence and interdependence of immunologic and nonimmunologic risk factors in the development of cardiac allograft vasculopathy. Another primary objective was to establish a clinically useful model for risk assessment of cardiac allograft vasculopathy that would facilitate identifying those heart transplant recipients likely to have severe intimal proliferation and thereby at greater risk for adverse clinical events. To our knowledge, no comprehensive intravascular ultrasound study has assessed the relative influences of both nonimmunologic and immunologic factors in the development of cardiac allograft vasculopathy, currently the major limitation to long-term cardiac allograft survival. Using a computer-assisted model of stepwise logistic regression, immunologic and nonimmunologic risk factors were evaluated to help identify the development of severe intimal thickening in 101 subjects who underwent intravascular ultrasound. Prospective validation of the findings was performed in a separate consecutive cohort of 37 heart transplant recipients, and the accuracy of this model to predict a relative risk > 1 for the development of severe intimal hyperplasia was assessed. Significant independent predictors of severe intimal hyperplasia in this model included a donor age > 35 years, a first-year mean biopsy score > 1 (a measure not only of severity of rejection, but also of frequency of insidious rejection) and hypertriglyceridemia at two incremental levels of risk (150 to 250 mg/dl [1.70 to 2.83 mmol/liter] and > 250 mg/dl [2.83 mmol/liter]). Based on the absence (0) or presence (1) of these factors, 12 individual categories of risk were ascertained with increasing relative risks and predicted probabilities for severe intimal hyperplasia. Prospective validation of this model revealed a sensitivity and specificity of 70% and 90%, respectively, and the positive and negative predictive values were 85% and 80%, respectively. Additionally, subjects with severe intimal thickening had a four-fold higher cardiac event rate than those without severe intimal proliferation on intravascular ultrasound. This study establishes a clinically useful predictive model that can be applied to individual heart transplant recipients to assess their risk for developing significant cardiac allograft vasculopathy and, thus, aids in the identification of patients at risk for cardiac events in whom closer surveillance and risk factor modification may be warranted.

Assessment of transplant arteriopathy by intracoronary two‐dimensional ultrasound imaging and coronary flow velocity

Transplant coronary arteriopathy is the major impediment to the long-term survival of cardiac allografts. This report highlights two-dimensional imaging and Doppler flow to assess transplant coronary arteriopathy.

Donor-Specific Unresponsiveness to Murine Cardiac Allografts Induced by Intrathymic-Soluble Alloantigens Is Dependent on Alternate Pathway of Antigen Presentation

This study extends the finding that intrathymic (IT) inoculation of uv-B irradiated donor spleen cells (SC) or soluble alloantigens (Ag) induces peripheral tolerance to organ allografts in the rat to the murine cardiac allograft model. In our initial experiment, we showed that IT inoculation of uv-B irradiated SC combined with transient immunosuppression of the recipient with either sublethal TBI or ALS on Day-7 led to donor-specific, long-term cardiac allograft survival (>300 days) in the completely mismatched A/J-to-C57BL/6 mice combination. To test our hypothesis that peripheral tolerance induced by IT injection of donor Ag is dependent on presentation of the foreign MHC molecule by thymic APC to T cell precursors, we examined the effect of IT injection of donor APC-free-soluble Ag inoculum obtained from 3 M KCl extracts of purified MHC class I resting T cells on cardiac allograft survival in the A/J-to-C3H mice combination. The results showed that IT injection of the optimal dose of 500 μg soluble Ag combined with 0.5 ml ALS on Day-7 led to donor-specific permanent graft survival (>200 days). This finding could not be reproduced by intravenous administration of soluble Ag, thus confirming the privileged position of the thymus in tolerance induction. To further define the role of host APC in allorecognition, we studied the presentation of soluble Ag by responder APC in MLR. The results showed that primed T cells obtained from A/J skin graft-sensitized C3H T cells specifically developed alloreactivity to A/J-soluble Ag in MLR. Addition of anti-Ia mAb blocked the proliferative response, thus confirming that primed T cells can respond to soluble Ag presented by responder-type APC. This study suggests that induction of peripheral tolerance by IT inoculation of soluble Ag is reproducible in the murine cardiac allograft model, where tolerance is dependent on the Ag dose and the indirect pathway of Ag presentation. This approach of immunologic manipulation of the adult thymus may have clinical therapeutic applications.

936-89 Extent of Coronary Myointimal Proliferation and its Relationship to Resistance Vessel Function in the Cardiac Allograft

Transplant coronary arteriopathy is the major obstacle to long-term survival of cardiac allografts and is characterized by myointimal proliferation involving both epicardial conduit vessels and intra myocardial resistance vessels. To evaluate the relationship between conduit disease and resistance vessel integrity, 70 coronary arteries in 58 transplant patients were studied with a 3.5 Fr 30 MHz intravascular ultrasound (IVUS) probe and an 0.018” doppler guidewire. Epicardial conduit disease was characterized by the presence or absence of IVUS discernible intimal thickening, (>500 microns or <500) micronsl in either a diffuse (trilaminar appearance along whole course of studied segment) or a focal (with areas of vessel free of intimal thickening) distribution. Resistance vessel function was assessed by measuring coronary flow reserve (CFR = maximal hypermic/resting blood flow velocity) using intracoronary adenosine (18 mg). (means ± SD): None(n = 12) Diffuse > 500(n = 19) Diffuse < 500(n = 18) Focal > 500(n = 7) Focal < 500(n = 14) CFR 3.1 ± 0.5 3.3 ± 0.4 37 ± 0.7 23 ± 0.6 * 2.8 ± 4 * p < 0.01 vs diffuse There were no differences in the CFR between arteries with (n = 58) and without (n = 12) intimal thickening (3.2 ± 0.6 vs 3.1 ± 0.5; P = NS) or based on the maximal thickness of the intima (<500 microns, 3.2 ± 8 vs >500 microns 3.0 ± 0.7, P = NS). Extensive myointimal thickening can occur without diminishment of the CFR. Focal intimal disease is associated with a significantly more profound effect on microvascular function then classically described diffuse disease. This suggests that typical epicardial transplant arteriopathy can occur without involvement of small vessels.

Fetal liver haematopoietic cells and tolerance to organ allografts

Fetal liver haematopoietic cells were used to induce neonatal tolerance to cardiac allografts. Newborn mice were injected with fetal or newborn haematopoietic liver cells in two fully allogeneic strain combinations. There was no clinical evidence of graft-versus-host disease. Long-term survival of subsequent cardiac allografts occurred in both strains without immunosuppressants. Unresponsiveness was found not be be donor-specific with prolongation of third-party allografts as well as donor-type grafts. These findings have important implications for inducing tolerance in paediatric organ transplantation.

Prognostic indicators for endomyocardial biopsies: intragraft IL-2 and IFN-gamma are associated with poor longterm survival of cardiac allografts

Currently the diagnosis of cardiac allograft rejection depends on morphologic analysis of paraffin sections of endomyocardial biopsies. The clinical decision of when lo treat rejection, especially if the patient is well, remains a difficult decision. We questioned if expression of cytokines and other activation molecules was predictive of a given rejection episode leading to fata! rejection vs. longterm survival. Hence, a rat model of cardiac allograft rejection was established whereby a clinically relevant protocol of peri-transplant injection of donor spleen cells, plus a single injection of cyclosporin 2 days post-transplant, induced longterm allograft survival (>90 d). whereas unirealed controls rejected by day 7. We undertook immunoperoxidase localization of various leucocyte populations, cytokines and markers of cytokine-induced immune activation. Whilst H&E staining of paraffin sections at day 7 post-Tx showed grade Illb rejection for both groups, the results of immunoperoxidase staining were markedly different: Group No Rx Spleen Cells plus CsA (day 7) (day 7) (day 93) IL-2R (CD25) 21 ± 3 1 ± 1 ** 0 MHC Class 11+ cells 57 ± 11 21 ± 14 * 30 ± 8 PCNA 26 ± 4 5 ± 1 ** not done IL-2 10 ± 5 0 ** 0 IFN-gamma 7 ± 5 1 ± 1 ** 0 * p ** p Moreover, uprcgulation of endothelial ICAM-1 expression and downregulation of thrombomodulin were seen in untreated but not treated groups. Thus, despite identical grading of cardiac rejection using standard criteria applied to H&E-slained paraffin sections, immunohistologic demonstration of key pro-inflammatory cytokines IL-2 and IFN-gamma, as well as the activalion molecules IL-2R and PCNA (proliferating cell nuclear antigen), allowed clearcut prediction of those cardiac allografts which would result in acute rejection versus those likely lo achieve longterm survival. This approach may (a) improve the accuracy of diagnosis of clinical cardiac allograft rejection. (b) predict Ihe outcome of a rejection episode, and (c) increase overall survival rales and decrease mortality from infection, by decreasing immunosuppressive requirements in allografts found lo show prognostic indicators of longterm survival.

Immunomodulation of Kidney and Heart Transplants by Anti-idiotypic Antibodies

To explore the possibility that circulating HLA antigens from the graft and anti-anti-HLA (anti-idiotypic) antibodies influence the long-term survival of renal and cardiac allografts, analysis of 330 renal allograft recipients and 174 recipients of cardiac allografts was conducted. Anti-donor-HLA antibodies (Ab1) present before or after transplantation are associated with graft failure, whereas irrelevant anti-HLA antibodies had no impact on actuarial graft survival. Ab1 may be uncovered by dissociation of immune complexes and depletion of soluble antigens with monoclonal antibody-coated magnetic beads. Of the 421 sera tested from 65 heart recipients, 97 showed Ab1 before depletion and 178 after depletion; similar rise in positive sera was seen in 39 renal transplant recipients. Three distinct patterns of appearance of Ab1 and Ab2 (anti-Ab1 antibody) were recognized. Patients with cyclic variations of Ab1 in association with Ab2 had 100% graft survival, whereas patients with cyclic variations of Ab1 but no detectable Ab2 had 2-year graft survival of 36% for kidneys and 71% for hearts. Presence of Ab1 in all sera after transplantation led to 47% and 56% 2-year renal and heart allograft survival, respectively.

Specific Immunosuppression in Cardiac Allografting Using Antithymocyte Sera and Soluble Transplantation Antigen

Two techniques of specific immunosuppression, using antithymocyte globulin (ATG) alone and in combination with soluble transplantation antigen (STA) prior to treatment, were studied in heterotopic outbred canine cardiac allografts. Both techniques resulted in prolonged survival of dogs having cardiac allografts with a low incidence of infections and drug toxicity complications as compared to animals treated with Prednisone and Imuran. This study demonstrates the ability of short-course ATG treatment to produce long-term (greater than 2 to 3 months) cardiac allograft survival without chronic immunosuppression. Combined ATG and STA pretreatment gave the longest allograft survival (mean, 93 days) in a small group of animals. This improved allograft survival, associated with a reduction in infections and drug toxicity complications, suggests that these techniques of specific immunosuppression may have the potential for improving survival and reducing the complications of immunosuppressive therapy following cardiac transplantation.