Chronic Allograft Vasculopathy Research Articles

Proliferation Signal Inhibitors in Transplantation: Questions at the Cutting Edge of Everolimus Therapy

While advances in immunosuppressive therapy have allowed dramatic improvements in the control of acute allograft rejection, there is still a need to improve long-term graft and patient survival rates following renal and heart transplantation. Among the recognized threats to long-term organ survival are chronic allograft dysfunction in the form of chronic allograft nephropathy and cardiac allograft vasculopathy, with long-term patient morbidity and mortality further compromised by higher than normal rates of posttransplant cardiovascular disease, infection, and malignancy. A growing body of evidence finds that the selection and dosing of immunosuppressive therapies can have great influence on long-term transplantation outcomes. Early evidence suggests that the proliferation signal inhibitors (PSIs), everolimus and sirolimus, might offer effective immunosuppressive activity together with antiproliferative effects that may address some of the unmet needs in the long-term therapeutic management of the posttransplant patient. This review summarizes the emerging evidence for employing PSI-based immunosuppression to seek a balance between the goals of maximizing graft and patient survival, while minimizing the risks of adverse events and long-term complications. Based on the proceedings of an international gathering of nephrologists, cardiologists and surgeons at the inaugural PSI Forum meeting “Proliferation signal inhibitors in transplantation: questions at the cutting edge,” this paper aims to provide both an evidence base and practical guidance for transplant physicians seeking to optimize their use of PSI treatment and highlights avenues of ongoing research into the clinical potential of this class of immunosuppressive therapy.

Mycophenolic Acid Inhibits Cell Proliferation and Extracellular Matrix Synthesis in Rat Vascular Smooth Muscle Cells Through Direct and Indirect Inhibition of Cellular Reactive Oxygen Species

Vascular smooth muscle cell (VSMC) proliferation and extracellular matrix (ECM) accumulation play important roles in the development and progression of chronic allograft vasculopathy. Although mycophenolic acid (MPA) inhibits activation of mesenchymal cells through cellular reactive oxygen species (ROS), the exact mechanisms involved in theses processes have not been clearly understood. This study explored the molecular mechanisms whereby MPA inhibits cellular ROS-mediated VSMC proliferation and ECM synthesis. Primary rat VSMCs were stimulated with platelet-derived growth factor (PDGF)-BB in the presence or absence of MPA 0.1-10 micromol/L or guanosine 100 micromol/L. Cell proliferation was assessed by methylthiazoletetrazolium and proliferating cell nuclear antigen expression, fibronectin secretion, and rac1 membrane translocation by Western blot analysis, total collagen synthesis by [(3)H]-proline incorporation, dichlorofluorescein-sensitive cellular ROS by confocal microscopy, and hydrogen peroxide (H(2)O(2)) concentration by iodometric analysis. MPA inhibited PDGF-induced VSMC proliferation, ECM synthesis, and cellular ROS, and these inhibitions were partially reversed by exogenous guanosine. MPA at dose inhibiting PDGF-induced VSMC activation inhibited rac1 membrane translocation, and this inhibition was fully recovered by exogenous guanosine. Additionally, MPA rapidly reduced H(2)O(2) concentration in vitro. The present study suggests that MPA inhibits PDGF-induced VSMC proliferation and ECM synthesis through inhibiting rac1-dependent cellular ROS and directly scavenging ROS. Both direct and indirect inhibition of cellular ROS would be the key mechanisms involved in the inhibitory effect of MPA in VSMCs.

Mycophenolic Acid Inhibits Oleic Acid–Induced Vascular Smooth Muscle Cell Activation by Inhibiting Cellular Reactive Oxygen Species

Vascular smooth muscle cell (VSMC) proliferation and matrix protein accumulation play important roles in the development and progression of chronic allograft vasculopathy. Mycophenolic acid (MPA) inhibits various types of mesenchymal cell proliferation and cellular reactive oxygen species (ROS) are involved in the anti-proliferative effect of MPA. In this study, we investigated the effects of MPA on oleic acid (OA)-induced VSMC proliferation and the role of ROS in this process. Primary VSMCs from Sprague-Dawley rats were stimulated with 100 microM OA, with or without MPA (0.1- 10 microM) or 5 mM N-acetylcysteine (NAC) for one hour prior to the addition of OA. Cell proliferation was measured by methylthiazoletetrazolium (MTT) assays, proliferating cell nuclear antigen (PCNA) expression, and fibronectin secretion by Western blot analysis, and dichlorofluorescein (DCF)-sensitive cellular ROS by fluorescence-activated cell scanning (FACS). OA (100 microM) increased cell proliferation, as measured by MTT (by 1.6-fold), PCNA expression, fibronectin secretion, and cellular ROS (by 1.6-fold). Treatment with MPA dose-dependently inhibited OA-induced VSMC proliferation, fibronectin secretion, and cellular ROS. Treatment with 5 mM NAC also inhibited OA-induced rat VSMC activation. These results suggest that MPA inhibits OA-induced VSMC proliferation and matrix protein synthesis partially by inhibiting cellular ROS.

Heart Retransplantation

Retransplants comprise only a small minority (3-4%) of heart transplants, however outcome following retransplantation is compromised. Risk factors for a poor outcome following retransplantation include retransplantation early (<6 months) after primary transplant, retransplantation for acute rejection or early allograft failure, and retransplantation in an earlier era. The incidence of rejection and infection is similar following primary transplant and retransplantation. The compromised outcomes and risk factors for a poor outcome are similar in adult and pediatric heart retransplantation. However, due to the short half-life of the transplanted heart, it is an expectation that patients transplanted in childhood may require retransplantation. Based on the data available and the opinion of the working group, indications for heart retransplantation are (i) chronic severe cardiac allograft vasculopathy with symptoms of ischemia or heart failure (should be considered) or asymptomatic moderate or severe left ventricular dysfunction (may be considered) or (ii) chronic graft dysfunction with symptoms of progressive heart failure in the absence of active rejection. Patients with graft failure due to acute rejection with hemodynamic compromise, especially <6 months post-transplant, are inappropriate candidates for retransplantation. In addition, guidelines established for primary transplant candidacy should be strictly followed.

Chemokines and Chronic Heart Allograft Rejection

Early work reported that targeting the CXCR3 and CCR5 could delay, but not prevent, acute allograft rejection (1–6). However, it is unclear how targeting single or multiple chemokine receptors could affect chronic rejection. In this study, we have examined the importance of chemokine receptors in acute and chronic rejection using CCR2, CXCR3, CCR5 knockout mice and CXCR3/CCR5 double knockout mice with heart allografts. Transplanted heart grafts were harvested at or near cessation of palpable contractility or at 60 days for chronic rejection, bisected to create a four-chamber section to visualize the aortic anastomosis, where chronic allograft vasculopathy is most readily identified. The median survival times for B6, CCR2−/−, CCR5−/−, CXCR3−/−, and CXCR3−/− × CCR5−/− recipients of heart allografts from complete major histocompatibility complex mismatch Balb/c donors were 8, 9, 11, 12, and 14 days, respectively. The pathology of acute rejections was similar in all mice with a mononuclear infiltrates and myocyte necrosis, diagnostic of acute cellular rejection (Fig. 1A, D, G, J, and M).FIGURE 1.: Histopathology of acute and chronic rejection. Acute rejection of Balb/c allografts showing mononuclear cell infiltrates with myocyte necrosis (A, D, G, J, and M). Recipients are B6 (A), CCR2−/− (D), CCR5−/− (G) CXCR3−/− (J), and CXCR3−/− × CCR5−/− DKO (M). Histopathology of active chronic rejection of BM12 allograft showing coronary and intraparenchymal arteries infiltrated by mononuclear cells with endothelialitis (B, E, H, K, and N). Recipients are B6 (B), CCR2−/− (E), CCR5−/− (H), CXCR3−/− (K), and CXCR3−/− × CCR5−/− DKO (N). Histopathology of active chronic rejection of BM12 allograft showing donor aortas at the anastomotic site infiltrated by mononuclear cells with endothelialitis (C, F, I, L, and O, and R). Recipients are B6 (C), CCR2−/− (F), CCR5−/− (I), CXCR3−/− (L), and CXCR3−/− × CCR5−/− DKO (O). Hematoxylin/eosin stained slides. Original magnification, 400×.Using a well-established model of chronic rejection (7–9), hearts from donor BM12 mice were transplanted into B6, CCR2−/−, CCR5−/−, CXCR3−/−, and CXCR3−/− × CCR5−/− recipients. In both control and the KO animals, coronary arteries and/or intramural arteries contained intimal and medial mononuclear cell infiltrates with endothelialitis (Fig. 1B, E, H, K, and N) with similar infiltrates in the donor aorta adjacent to the aortic anastomotic site (Fig. 1C, F, I, L, and O). Morphometric analysis failed to identify any quantitative differences among animal groups. In addition, smooth muscle cell loss was present in the donor aortas. These histological findings are diagnostic of an active phase of chronic allograft vasculopathy. Little parenchymal inflammation was present in all chronic rejection recipients. Syngeneic allografts showed no intimal hyperplasia or inflammation in the coronary arteries or at the anastomotic site (data not shown). In this study, there was modest prolongation of acute heart allograft survival in the mice deficient for certain chemokine receptors and there was no effect on chronic rejection. Chemokines control the inflammatory infiltrate so that a modest delay in the recruitment of cells, especially CD4 cells, could have a modest effect on acute cellular rejection and chronic allograft vasculopathy (10–14). Because there is redundancy in the chemokine and chemokine receptor network deleting or targeting a single chemokine or receptor has only marginal effects in acute cellular rejection (15, 16). Additional prolongation observed in the DKO mice suggests that targeting multiple chemokine receptors may further delay rejection. Potent costimulatory blockade or conventional immunosuppressive strategies, which dramatically reduces parenchymal inflammation, is also often unable to reduce chronic rejection (17–21). Likewise in this BM12 model of chronic rejection using B6 and selected chemokine receptor knockout recipients, there is relatively little parenchymal inflammation with ongoing active chronic vasculopathy. Parenchymal infiltration, therefore, is probably not the trigger for the development of chronic allograft vasculopathy, which may come about by an inflammatory pathway distinct from that which recruits parenchymal inflammation. R. Neal Smith Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA Takuya Ueno Transplantation Unit, Surgical Services, Massachusetts General Hospital, Transplantation Research Center, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA Toshiro Ito Transplantation Unit, Surgical Services, Massachusetts General Hospital, Harvard Medical School, Boston, MA Katsunori Tanaka Transplantation Research Center, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA Susan P. Shea Transplantation Unit, Surgical Services, Massachusetts General Hospital, Harvard Medical School, Boston, MA Reza Abdi Transplantation Research Center, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA

Alloantibody-associated chronic rejection of MHC class I-disparate heart grafts is modulated by intravenous soluble donor alloantigen

Increasing evidence suggests that there may be a causal relationship between the development of donor-specific alloantibodies and chronic allograft vasculopathy (CAV). PVG.RT1 u rat heart allografts spontaneously undergo chronic rejection when transplanted into unmodified PVG.R8 congenic recipients that differ only at the classical MHC class I RT1.A locus. Here we show that development of vasculopathy in this experimental model is associated with production of a strong anti-A u antibody response. Perioperative intravenous administration of recombinant soluble RT1.A u heavy chain that is sequence identical to donor MHC class I, or chimaeric A u/a (donor/recipient) protein had a variable effect resulting generally in either sensitisation and accelerated rejection, or abrogation of alloantibody and attenuation of chronic rejection. These findings highlight the potential for soluble donor MHC class I alloantigen given at the time of heart transplantation to influence alloantibody production and graft outcome.

Proliferation signal inhibitors in cardiac transplantation

Standard immunosuppression after cardiac transplantation includes a calcineurin inhibitor in combination with mycophenolate mofetil or azathioprine and corticosteroids. These agents have led to excellent outcomes but have shortcomings in terms of efficacy and toxicity. A new class of immunosuppressants, proliferation signal inhibitors, may meet some of these shortcomings. The efficacy of the available proliferation signal inhibitors - sirolimus and its derivative everolimus - has been compared with azathioprine in three randomized clinical trials. Sirolimus or everolimus use was associated with lower rates of acute rejection and reduced development of chronic allograft vasculopathy. Sirolimus was not found to be superior to mycophenolate mofetil in a randomized trial. Proliferation signal inhibitors have been reported to be effective in refractory recurrent acute rejection. Nonrandomized studies have demonstrated that proliferation signal inhibitor-based immunosuppression enables recovery from renal dysfunction secondary to calcineurin inhibitor treatment. Proliferation signal inhibitor-based treatment is associated with a lower risk of malignancy than calcineurin inhibitor-based regimens. Proliferation signal inhibitors have significant adverse effects that may limit widespread use. Proliferation signal inhibitors are important new immunosuppressive agents that have added considerably to the armamentarium allowing further tailored immunosuppression to individualize patient care after heart transplantation.

341: Monocenter study of percutaneous coronary interventions in 154 cardiac transplant recipients with chronic allograft vasculopathy

Purpose: Chronic Allograft Vasculopathy (CAV) is a complication of heart transplantation (HTx). Percutaneuous Coronary Intervention (PCI) is a method of choice for local revascularization used increasingly in transplant recipients with CAV.

Immunosuppressive Therapy in Older Cardiac Transplant Patients

Cardiac transplantation has become an established intervention for end-stage heart disease. Clinical outcomes in older cardiac transplant patients have improved over the last decade and are almost similar to those in younger patients. Nevertheless, morbidity and mortality due to infections, cancer and chronic allograft vasculopathy remain problematic. On the other hand, older transplant patients seem to have lower incidences of acute rejection episodes than younger patients. Conventional immunosuppression with calcineurin-inhibiting drugs, azathioprine and corticosteroids is responsible for a number of adverse effects. Although these adverse effects can also be seen in younger patients, tolerance to these agents seems to decrease with increasing age. In particular, diabetes mellitus, osteoporosis and chronic renal insufficiency are associated with higher morbidity and mortality in older cardiac transplant patients. As the elderly become an ever-increasing segment of the cardiac transplant population, new and innovative immunosuppressive strategies will have to be developed and applied.Currently, the availability of new immunosuppressive drugs means more individualised immunosuppressive protocols can be used. New antibodies for induction therapy, a choice between ciclosporin and tacrolimus, and the advent of mycophenolate mofetil as well as proliferation signal inhibitors (everolimus, sirolimus) have changed immunosuppressive protocols dramatically. Therefore, a generalised protocol for all patients has been replaced by individualised immunosuppression depending on the patient group. Moreover, protocols can be modified during follow-up depending on the individual patient's requirements and problems. Hypertension and hyperlipidaemia could be influenced by the selection of tacrolimus over ciclosporin, and weaning of corticosteroids might have a positive impact on osteoporosis or diabetes. There is also no clear evidence that tacrolimus is associated with a higher risk for new onset of diabetes. Chronic renal insufficiency can be managed with calcineurin inhibitor-free immunosuppression consisting of mycophenolate mofetil and proliferation signal inhibitors. Both everolimus and sirolimus also seem to have a protective effect against the onset of graft vasculopathy and some sorts of cancer after cardiac transplantation. As a general rule, however, older cardiac transplant patients should be treated with lower doses and fewer immunosuppressive drugs to avoid over-immunosuppression.

The Effect of Platelet-Derived Growth Factor Ligands in Rat Cardiac Allograft Vasculopathy and Fibrosis

Background In chronic rejection, parenchymal fibrosis and cardiac allograft vasculopathy (CAV) characterized by neointimal growth are the leading causes of graft loss for heart transplant recipients. During alloimmune responses a variety of cytokines, adhesion proteins, and growth factors, such as platelet-derived growth factor (PDGF), are up-regulated. The PDGF family (AA, AB, BB, CC, DD), which acts mainly on connective tissue cells, is considered to be a potent mitogenic and chemotactic factor for fibroblasts and vascular smooth muscle cells. In this study, we evaluated the effects of PDGF ligands in chronic rejection. Methods Heterotopic heart transplantations were performed between fully major histocompatability complex–mismatched Dark Agouti to Wistar Furth rats receiving cyclosporine immunosuppression. Allograft coronary arteries were perfused with a recombinant adeno-associated virus (AAV) encoding enhanced green fluorescence protein (EGFP) as a control gene or PDGF-A, -B, -C, -D. Allografts were harvested at 100 days for morphometric analysis of CAV and fibrosis. Results AAV-mediated transgene expression was detected by EGFP immunoreactivity across the graft section (at 100 days) in AAV EGFP-perfused allografts. AAV PDGF-A, -C, and -D perfusion resulted in accelerated CAV and fibrosis. In contrast, AAV PDGF-B perfusion did not induce arteriosclerotic changes or fibrosis in cardiac allografts. Conclusions AAV PDGF-A, -C, and -D overexpression accelerated the development of chronic rejection, whereas PDGF-B did not. Our results suggested that more targeted therapy with monoclonal antibodies blocking the active sites of PDGF-A, -C, and -D may produce beneficial effects on heart transplant survival.

Carvedilol Inhibits Platelet-Derived Growth Factor-Induced Extracellular Matrix Synthesis by Inhibiting Cellular Reactive Oxygen Species and Mitogen-Activated Protein Kinase Activation

Vascular smooth muscle cell (VSMC) proliferation, migration, and extracellular matrix (ECM) synthesis are major pathologic features of chronic allograft vasculopathy. Carvedilol, an anti-hypertensive agent, might be an effective agent for preventing the development and progression of chronic allograft vasculopathy, since it can inhibit VSMC proliferation and migration. The present study was designed to examine the effect of carvedilol on platelet-derived growth factor (PDGF)-induced ECM synthesis in rat VSMCs. Furthermore, we evaluated whether carvedilol inhibits PDGF-induced cellular reactive oxygen species (ROS) and the activation of mitogen-activated protein kinase (MAPK). Primary cultured rat VSMCs were stimulated with PDGF-BB (10 ng/ml) in the presence or absence of carvedilol, and the effects of carvedilol were compared with those of ROS or MAPK inhibitors. Fibronectin secretion, proliferating cell nuclear antigen (PCNA) expression, and each MAPK activation were determined by Western blot analysis, total collagen synthesis by [3H]-proline incorporation, and cellular ROS by flow cytometry. PDGF significantly increased PCNA expression, fibronectin secretion, total collagen synthesis, cellular ROS, and MAPK activation in rat VSMCs. Carvedilol at doses that inhibited PDGF-induced cell proliferation, inhibited ECM synthesis, cellular ROS, or subsequent MAPK activation. Structurally different anti-oxidants and extracellular signal-regulated protein kinase or p38 MAPK inhibitor effectively inhibited PDGF-induced fibronectin secretion and total collagen synthesis. These results suggest that carvedilol inhibits PDGF-induced VSMC proliferation and matrix protein synthesis by inhibiting cellular ROS and the subsequent activation of MAPK. Thus the targeted inhibition of cellular ROS and MAPK might provide an effective therapeutic strategy to treat chronic allograft vasculopathy.

Appraisal of the extent of chronic allograft vasculopathy in animal models: Proposition of a standardized micromorphometric method

Arterial obstruction due to intimal myproliferation is an outcome used for investigating chronic allograft vasculopathy in animal models. Since harvested tissue may be distorted while processing, discrepancy may happen in the measure of the obstruction. We propose a standardized micromorphometric method for calculating the percentage of obstruction with eliminating the variability in the shape of the vessel cross-section. The mathematical adjustment avoids the overestimation of the percentage of obstruction and improves accuracy.

Differential expression of tissue factor (TF) in calcineurin inhibitor-induced nephrotoxicity and rejection—implications for development of a possible diagnostic marker

Deposition of fibrin in the form of fibrinoid necrosis is a common feature of severe acute renal allograft rejection. The role of the coagulation system and its initiator tissue factor (TF) during this process is, however, still poorly understood. In this study, we analyzed the expression of TF in 88 renal transplants afflicted with different forms of rejection and calcineurin inhibitor-induced nephrotoxicity, to see whether there was differential expression of this protein. TF immunoreactivity was evaluated semiquantitatively in six different renal structures: the podocytes, Bowman epithelium, the endothelium of the glomeruli, the brush border of tubular cells, the thin ascending loop of Henle, and small arteries/arterioles. The TF expression of normal renal tissue ( n = 6) was restricted to the glomerular podocytes and Bowman epithelium, and to some extent the ascending loop of Henle. Renal allografts undergoing acute rejection (AR) of grades I–III, ( n = 13, n = 17 and n = 12, respectively) did not show any altered TF expression in the glomeruli or vascular endothelium. In the ascending loop of Henle, a reduced expression could be seen (ARI, p = 0.015; and ARII, p = 0.043). TF staining of the brush border of renal transplants undergoing acute cyclosporin A (CsA) nephrotoxicity ( n = 18) was significantly higher than in normal kidneys ( p = 0.0003), as well as in transplants undergoing various degrees of acute rejection (ARI, p = 0.027; ARII, p = 0.0012; and ARIII, p = 0.0001). Tubular brush border-expressed TF was also evident in 10 of 15 allografts suffering from chronic CsA nephrotoxicity, compared to 4 out of 13 cases with chronic allograft vasculopathy (CAV), but the increase was not statistically significant relative to normal kidneys. The majority of the grafts afflicted with either of the two chronic conditions displayed a TF-positive arterial endothelium (CAV, p = 0.0034; and chronic CsA nephrotoxicity, p = 0.0026) relative to controls. In conclusion, these results indicate that vascular TF expression is not altered during acute rejection, but may be of importance in chronic allograft nephropathy. Furthermore, TF immunoreactivity in the tubular brush border may be specific to acute CsA nephrotoxicity and might be used as a biomarker for this condition. Further studies are required to evaluate the possible role of brush border-expressed TF in the pathogenesis of CsA nephrotoxicity.

47. Ex Vivo Hypothermic Recirculatory Adenoviral Gene Transfer to the Transplanted Pig Heart

Background: Heart transplantation is the most efficient and successful therapeutic procedure for end stage heart failure, but several limitations such as primary graft failure, acute graft rejection and chronic allograft vasculopathy still remain to be overcome. To facilitate the application of adenoviral gene therapy in clinical heart transplantation, we developed an ex vivo hypothermic recirculatory adenoviral gene transfer method to the transplanted pig heart.

Sclerosing peritonitis after intestinal transplantation in children

Long-term graft dysfunction and/or graft loss after intestinal transplantation (ITx) is a significant concern. Sclerosing peritonitis (SP) is a manifestation of chronic allograft failure and its presence may also include classic arterial obliterative arteriopathy (OA) as in chronic rejection. We describe the clinical presentation and management of SP occurring after ITx in children. Case records of 121 children undergoing ITx from 1990 to 2003 were reviewed. Three children (2.4%) presented with SP of the intestine allograft at a mean time of 6.6 yr following ITx as follows: age at Tx (yr) 8.2, and 3.7, with indication for ITx being gastroschisis in two and midgut volvulus in one patient. Type of ITx was isolated intestine in one and liver/intestine in two patients. Gross findings of SP included fibrosis/strictures; microscopically SP showed fibrosis/serositis, and fibrous adhesions; one patient had evidence of chronic allograft vasculopathy. All patients presented with clinical signs and symptoms of bowel obstruction and gastrointestinal contrast studies confirmed distal ileal obstruction (DIO). Operative findings confirmed SP and DIO in all patients; all patients were initially treated with distal segmental intestine allograft resection and lysis of the fibrous peel. All three patients recovered, although two required repeat laparotomy, there is only one long-term survival. SP after ITx may be a different manifestation of long-term intestine allograft degeneration. Surgical resection appears to offer palliation.

Phosphorylation of S6 ribosomal protein at serine 235/236 is a marker of human heart transplantation

Antibody (Ab) ligation of HLA class I molecules on the surface of endothelial cells (EC) stimulates cell proliferation and survival pathways including activation of the PI3K/Akt pathway. We investigated phosphorylation of S6 ribosomal protein (p-S6RP), a downstream target of the PI3K/Akt/mTOR pathway in EC as a potential marker of Ab mediated-rejection(AMR) of heart allografts. Human aortic EC were treated with anti-HLA class I Ab and cell lysates were studied for phosphorylation of PI3K, Akt and S6 ribosomal protein by Western blot. Endomyocardial biopsies (n 65) from 35 heart transplant recipients (7 with AMR, 7 with acute cellular rejection (ACR)) were tested for the presence of p-S6RP using an anti-phospho serine 235/236 Ab. Diagnosis of AMR was based on histology and immunofluorescence or immunoperoxidase staining of paraffin-embedded tissue (CD68-positive intravascular macrophages). Diagnosis of ACR was based on ISHLT grading. The relationship between p-S6RP and rejection was analyzed by Fisher’s exact test.Treatment of EC with anti-class I Ab resulted in a time and dose dependent increase in phosphorylation of Akt, PI3K and the S6RP. To investigate these results in a relevant in vivo model we determined whether biopsy samples from transplant recipients diagnosed with AMR or ACR showed increased p-S6RP on the graft vasculature.Results showed show a significant association of p-S6RP staining with diagnosis of AMR (p 0.001). Four of 7 patients with ACR showed p-S6RPN staining (p NS). No association between p-S6RP staining with C4d deposition was observed. Study of the kinetics of p-S6RP staining showed that patients diagnosed with AMR display sustained positivity ( 144 days) compared to patients diagnosed with ACR ( 21days). These findings support the role of anti-class I Ab mediated signaling in AMR and indicate that phosphorylation of S6RP is a useful marker of AMR in cardiac allografts. Sustained p-S6RP is observed in patients with AMR supporting its role in chronic allograft vasculopathy.

CD70 Signaling Is Critical for CD28-Independent CD8+ T Cell-Mediated Alloimmune Responses In Vivo

The inability to reproducibly induce robust and durable transplant tolerance using CD28-B7 pathway blockade is in part related to the persistence of alloreactive effector/memory CD8(+) T cells that are less dependent on this pathway for their cellular activation. We studied the role of the novel T cell costimulatory pathway, CD27-CD70, in alloimmunity in the presence and absence of CD28-B7 signaling. CD70 blockade prolonged survival of fully mismatched vascularized cardiac allografts in wild-type murine recipients, and in CD28-deficient mice induced long-term survival while significantly preventing the development of chronic allograft vasculopathy. CD70 blockade had little effect on CD4(+) T cell function but prevented CD8(+) T cell-mediated rejection, inhibited the proliferation and activation of effector CD8(+) T cells, and diminished the expansion of effector and memory CD8(+) T cells in vivo. Thus, the CD27-CD70 pathway is critical for CD28-independent effector/memory CD8(+) alloreactive T cell activation in vivo. These novel findings have important implications for the development of transplantation tolerance-inducing strategies in primates and humans, in which CD8(+) T cell depletion is currently mandatory.

CD8 Lymphocytes are Sufficient for the Development of Chronic Rejection

The role of CD8 lymphocytes, in chronic rejection or cardiac allograft vasculopathy (CAV), is incompletely understood. The purposes of this study were to determine whether CD8 lymphocytes, in the absence of CD4 lymphocytes, are capable of causing the intimal lesions of CAV; and if so, to define the effector mechanism(s) of CD8 lymphocytes. We modified a previously characterized major histocompatibility complex class II mismatched murine model of CAV. Wild-type CD8 lymphocytes were transferred to nude mice followed by heterotopic heart transplantation. Recipient mice were then treated with a CD40 activating antibody, which is known to provide help for CD8 lymphocyte activation, in the absence of CD4 lymphocytes. Donor hearts were harvested on day 40 posttransplantation and analyzed for cellular infiltrates and intimal thickening. In separate experiments, isolated perforin -/-, Fas ligand (FasL) -/-, and interferon (IFN)-gamma -/- CD8 lymphocytes were transferred to nude mice followed by identical experimented protocol. With adaptive transfer of wild-type CD8 lymphocytes, the donor hearts were infiltrated with activated CD8 lymphocytes and displayed significant intimal lesions. Adoptive transfer of perforin -/- and FasL -/- CD8 lymphocytes to nude mice resulted in similar patterns of CD8 lymphocyte infiltration and similar severity of intimal lesions. The donor hearts from IFN-gamma -/- CD8 lymphocyte reconstituted recipients displayed minimal intimal lesions, although CD8 lymphocytes were present in the allografts. Unprimed CD8 lymphocytes in the absence of CD4 lymphocytes can cause intimal lesions of CAV. CD8 lymphocytes production of IFN-gamma, but not the perforin or the FasL-mediated cytotoxicity, is the critical step in the development of intimal lesions.

Mycobacterium xenopi infection after heart transplantation: An unreported pathogen

Mycobacterial infections are a well-known, potentially serious, albeit infrequent complication of solid-organ transplantation. Nontuberculous mycobacteria generally account for less than 50% of all such isolates in this patient population. Mycobacterium xenopi, an environmentally ubiquitous organism and common contaminant of hospital hot water systems, is a particularly uncommon isolate after transplantation and has never been reported in heart allograft recipients. We report the occurrence of cavitary M. xenopi infection in an immunocompromised heart transplant recipient in which all the diagnostic criteria of the American Thoracic Society were met. To our knowledge, this is the first such case in a heart transplant recipient described in the literature. Despite therapy, to which the isolates were sensitive in vitro, the patient developed extensive lung cavitation and nodules and succumbed 5 months later to allograft rejection, chronic allograft vasculopathy, and pneumonia.

Rapamycin has no effect on fibrosis-associated gene expression or extracellular matrix accumulation when administered to animals with established or early allograft vasculopathy

Background Most patients with functioning heart transplants have established and progressive chronic allograft vasculopathy, a fibroproliferative process for which there is no effective treatment. Coronary artery disease is characterized by histologic evidence of extracellular matrix accumulation (fibrosis). This study compares the effect of rapamycin administered to rats with established allograft vasculopathy on histologic indices of disease progression, extracellular matrix accumulation (fibrosis), and the expression of genes known to regulate extracellular matrix turnover in this model. Methods Lewis recipients of Fisher 344 rat thoracic to abdominal aorta transplants were administered rapamycin starting at 8, 12, and 16 weeks posttransplant or no treatment. Six grafts in each group were harvested at 24 weeks. Vascular remodeling and collagen accumulation (Sirius red) were measured by computerized histomorphometry of aortic sections. mRNA was extracted from frozen tissue, and the expression of fibrosis-associated genes was studied by means of semiquantitative reverse transcriptase-polymerase chain reaction. Results Rapamycin had no effect on the progression of early or established allograft vasculopathy with regard to intimal thickening, remodeling, extracellular matrix accumulation, or profibrotic gene expression, regardless of the time commenced. Conclusion The attenuation of the fibroproliferative response in rodents by rapamycin is not seen if the onset of rapamycin therapy is delayed.