Pathogenesis Of Rejection Research Articles

The Role of the Endothelium during Antibody-Mediated Rejection: From Victim to Accomplice.

Antibody-mediated rejection (AMR) of solid organ transplants is characterized by the activation and injury of the allograft endothelium. Histological and transcriptomic studies have associated microvascular inflammation and endothelial lesions with the severity of rejection and poor graft outcomes. The allograft endothelium forms the physical barrier between the donor organ and the recipient; this position directly exposes the endothelium to alloimmune responses. However, endothelial cells are not just victims and can actively participate in the pathogenesis of rejection. In healthy tissues, the endothelium plays a major role in vascular and immune homeostasis. Organ transplantation, however, subjects the endothelium to an environment of inflammation, alloreactive lymphocytes, donor-specific antibodies, and potentially complement activation. As a result, endothelial cells become activated and have modified interactions with the cellular effectors of allograft damage: lymphocytes, natural killer, and myeloid cells. Activated endothelial cells participate in leukocyte adhesion and recruitment, lymphocyte activation and differentiation, as well as the secretion of cytokines and chemokines. Ultimately, highly activated endothelial cells promote pro-inflammatory alloresponses and become accomplices to AMR.

Pretransplant Numbers of CD16+ Monocytes as a Novel Biomarker to Predict Acute Rejection after Kidney Transplantation

Acute rejection is one of the major immunological determinants of kidney graft function and survival. Early biomarkers to predict rejection are lacking. Emerging evidence reveals a crucial role for the monocyte-macrophage lineage cells in the pathogenesis of rejection. We hypothesized that higher pre-transplant numbers of proinflammatory CD16+ monocytes can predict rejection. The study cohort consisted of 104 kidney transplant recipients (58 non-rejectors and 46 biopsy-proven rejectors), and 33 healthy individuals. Posttransplant median ± IQR follow up time was 14.7 (0.3-34) months. Pretransplantation blood samples were analyzed by flow cytometry for monocyte immunophenotypes. Groups were compared by Cox regression models for the occurrence of acute rejection. We documented a significantly increased absolute number of pretransplant CD16+ monocytes in patients who developed biopsy proven rejection after transplantation compared to non-rejectors and healthy individuals (Hazard Ratio [HR], 1.60; 95% Confidential Interval [CI], 1.28 to 2.00; p < 0,001 and HR, 1.47; CI, 1.18 to 1.82, p < 0,001). In parallel, significantly less absolute numbers of CD16- monocytes were observed at pretransplant time point in rejectors vs non-rejectors (HR, 0.74; CI, 0.58 to 0.94; p < 0,014). A higher pre-transplant number of CD16+ monocytes is significantly associated with a higher risk of acute rejection after kidney transplantation.

Compartmental Infiltration of Kidney Allograft With Monocyte-Macrophage Subtypes Defines the Type of Rejection

Rejection, regardless of the type and time, significantly worsens the graft function and survival. Emerging evidence has revealed a crucial role for the cells of monocyte-macrophage lineage in the pathogenesis of rejection. Here, we studied monocyte-macrophage compartmental infiltration of 62 kidney transplant biopsies with the diagnosis of 1) acute antibody mediated rejection (aABMR, n = 9), 2) chronic antibody mediated rejection (cABMR, n = 13), 3) acute cellular rejection type I (ACR I, n = 11), 4) acute cellular rejection type II (ACRII, n = 13), and 5) and 15 protocol biopsies from kidney transplants with stable function as controls. Next we studied the relationship between these findings and allograft function and survival in the long term. Immunohistochemical and immunofluorescent stainings were applied to study monocyte-macrophage infiltration. The intensity of infiltration was quantified by ImageJ analysis and laser scan confocal microscopy. Infiltrating monocytes were characterized by double staining with CD14 and CD16. Infiltrating macrophages were identified by expression of CD68, CD80, CD163 as follows: CD68 + CD80 (M1 type) and CD68 + CD163 (M2 type). Histopathological data was analyzed and correlated to eGFR, creatinine and proteinuria levels at the time of biopsy, 3, 6 and 12 months post-rejection. Overall, the presence of CD68 + macrophages in kidney biopsies was significantly associated with rejection compared to stable patients regardless of histopathological subtype (p = <0,01). Overall, the presence of CD68 + CD163+ macrophages was signigicantly associated with a lower eGFR at the time of biopsy, and at 3, 6 and 12 months after rejection (p < 0,001). Glomerular infiltration by classical and intermediate monocytes and and the presence of non-classical monocytes in the interstitium were significantly associated with rejection regardless of rejection subtype (p = <0,01). cABMR was characterized by glomerular and tubulointerstitial CD68+ macrophage infitration, and glomerular and tubulointerstitial classical and intermediate monocyte distribution as compared to aABMR (p = <0,05). ACRI and ACRII were charactherized by vascular and tubulointerstitial distribuation of CD68+ cells as compared to aABMR rejection type (p = <0,05). In sharp contrast to T cells, the presence of CD68 + macrophages in a kidney transplant biopsy is significantly associated with rejection regardless of histopathological subtype. There are significant compartmental differences in the distribution of different macrophage and monocyte subtypes between aABMR and cABMR, as well as between ACR I/II and aABMR. These findings could be of interest for the histopathological classifcation of rejection type. Future clarification of the pathogenetical role and the cell specific markers could help detect new rejection biomarkers.

Pretransplant Numbers of CD16+ Monocytes as a Novel Biomarker to Predict Acute Rejection After Kidney Transplantation: A Pilot Study.

Acute rejection is one of the major immunological determinants of kidney graft function and survival. Early biomarkers to predict rejection are lacking. Emerging evidence reveals a crucial role for the monocyte/macrophage lineage cells in the pathogenesis of rejection. We hypothesized that higher pretransplant numbers of proinflammatory CD16+ monocytes can predict rejection. The study cohort consisted of 104 kidney transplant recipients (58 with no rejection and 46 with biopsy-proven rejection) and 33 healthy persons. Posttransplant median follow-up time was 14.7 mo (interquartile range 0.3-34 mo). Pretransplantation blood samples were analyzed by flow cytometry for monocyte immunophenotypes. Groups were compared by Cox regression models for the occurrence of acute rejection. We documented a significantly increased absolute number of pretransplant CD16+ monocytes in patients who developed biopsy-proven rejection after transplantation compared with those with no rejection (hazard ratio [HR] 1.60, 95% CI 1.28-2.00, p < 0.001) and healthy persons (HR 1.47, 95% CI 1.18-1.82, p < 0.001). In parallel, significantly fewer absolute numbers of CD16- monocytes were observed at pretransplant time points in rejectors versus nonrejectors (HR 0.74, 95% CI 0.58-0.94, p < 0,014). A higher pretransplant number of CD16+ monocytes is significantly associated with a higher risk of acute rejection after kidney transplantation.

Dual Role of Natural Killer Cells on Graft Rejection and Control of Cytomegalovirus Infection in Renal Transplantation.

Allograft rejection constitutes a major complication of solid organ transplantation requiring prophylactic/therapeutic immunosuppression, which increases susceptibility of patients to infections and cancer. Beyond the pivotal role of alloantigen-specific T cells and antibodies in the pathogenesis of rejection, natural killer (NK) cells may display alloreactive potential in case of mismatch between recipient inhibitory killer-cell immunoglobulin-like receptors (KIRs) and graft HLA class I molecules. Several studies have addressed the impact of this variable in kidney transplant with conflicting conclusions; yet, increasing evidence supports that alloantibody-mediated NK cell activation via FcγRIIIA (CD16) contributes to rejection. On the other hand, human cytomegalovirus (HCMV) infection constitutes a risk factor directly associated with the rate of graft loss and reduced host survival. The levels of HCMV-specific CD8+ T cells have been reported to predict the risk of posttransplant infection, and KIR-B haplotypes containing activating KIR genes have been related with protection. HCMV infection promotes to a variable extent an adaptive differentiation and expansion of a subset of mature NK cells, which display the CD94/NKG2C-activating receptor. Evidence supporting that adaptive NKG2C+ NK cells may contribute to control the viral infection in kidney transplant recipients has been recently obtained. The dual role of NK cells in the interrelation of HCMV infection with rejection deserves attention. Further phenotypic, functional, and genetic analyses of NK cells may provide additional insights on the pathogenesis of solid organ transplant complications, leading to the development of biomarkers with potential clinical value.

Differential Effects of Activated Human Renal Epithelial Cells on T-Cell Migration

BackgroundRenal tubular epithelial cells (TECs) are one of the main targets of inflammatory insults during interstitial nephritis and kidney transplant rejection. While Th1 cells are know to be essential in the pathogenesis of rejection, the role of Th17 is still under debate. We hypothesize that TECs modulate the outcome of rejection process by production of distinct chemokines and cytokines that determine the attraction of different T-cell subsets. Therefore, we studied differential effects of activated human renal epithelial cells on T-cell migration.MethodsHuman primary TECs were stimulated by IFN-γ and TNF-α in vitro. Chemokines and cytokines produced by activated TECs were measured using Luminex or ELISA. Chemotaxis assay was performed using activated peripheral blood mononuclear cells composed of CD4+CXCR3+ and CD4+CCR6+ T cells migrating towards stimulated and unstimulated TECs.ResultsWhile activated TECs secreted abundant amounts of the pro-inflammatory cytokines IL-6 and IL-8, the T helper cell differentiation cytokines IL-1β, IL-12p70, IL-23 or TGF-β1 were not produced. The production of Th1 chemokines CXCL9, CXCL10 and CCL5 were significantly upregulated after TEC stimulation. In contrast, Th17 chemokine CCL20 could not be detected. Finally, activated TECs attracted significantly higher numbers of CD4+CXCR3+ T cells as compared to unstimulated TECs. No migration of CD4+CCR6+ T cells could be observed.ConclusionActivated primary renal tubular epithelial cells do not attract Th17 cells nor produce cytokines promoting Th17 cell differentiation in our experimental system mimicking the proinflammatory microenvironment of rejection.

Green Tea Epigallo-Catechin-Galleate Ameliorates the Development of Obliterative Airway Disease

ABSTRACTLung transplantation has the worst outcome compared to all solid organ transplants due to chronic rejection known as obliterative bronchiolitis (OB). Pathogenesis of OB is a complex interplay of alloimmune-dependent and -independent factors, which leads to the development of inflammation, fibrosis, and airway obliteration that have been resistant to therapy. The alloimmune-independent inflammatory pathway has been the recent focus in the pathogenesis of rejection, suggesting that targeting this may offer therapeutic benefits. As a potent anti-inflammatory agent, epigallo-catechin-galleate (EGCG), a green tea catechin, has been very effective in ameliorating inflammation in a variety of diseases, providing the rationale for its use in this study in a murine heterotopic tracheal allograft model of OB. Mice treated with EGCG had reduced inflammation, with significantly less neutrophil and macrophage infiltration and significantly reduced fibrosis. On further investigation into the mechanisms, inflammatory cytokines keratinocyte (KC), interleukin-17 (IL-17), and tumor necrosis factor-α (TNF-α), involved in neutrophil recruitment, were reduced in the EGCG-treated mice. In addition, monocyte chemokine monocyte chemoattractant protein-1 (MCP-1) was significantly reduced by EGCG treatment. Antifibrotic cytokine interferon-γ–inducible protein-10 (IP-10) was increased and profibrotic cytokine transforming growth factor-β (TGF-β) was reduced, further characterizing the antifibrotic effects of EGCG. These findings suggest that EGCG has great potential in ameliorating the development of obliterative airway disease.

The role of antibodies in transplantation.

For the past 40 years, T cells have been considered the primary threat to the survival of allografts. However, antibodies can induce severe vascular disease of organ transplants, and this disease, particularly "antibody-mediated" rejection, has become a major clinical challenge. Not only do antibodies cause rejection, the rejection caused by antibodies resists treatment by conventional drug regimens. On the other hand, antibodies can induce a condition in which grafts seemingly resist antibody-mediated injury, which is accommodation. In this communication, we discuss the role of antibodies in the diagnosis and pathogenesis of rejection and accommodation, and suggest what we considered the major gaps in knowledge and directions research into this subject might productively take.

The Potential Role of T-Cell Memory Distribution as Predisposing Factor for Rejection in Heart Transplant Recipients

CD4 T cells play a significant role in the pathogenesis of rejection, providing help to alloreactive CD8 and B cells, however, the exact contribution of each memory compartment in vivo has not been defined. They are also important for the maintenance of tolerance due to regulatory activity of specialized subsets. In this study, we assessed changes in frequencies of functionally distinct lymphocyte subsets of peripheral blood (PBLs) in 26 heart transplant recipients (HT) in association with rejection episodes. Patients who developed rejection (n = 7), namely Grade 3B (n = 1), 3A (n = 4), or 2 (n = 2), in comparison with those with stable graft function displayed at baseline (pre-HT) higher percentages of naive (CCR7+CD45RA+) CD4 T cells (median 48 vs 36.6%; P = .035) and lower percentages of central memory (CCR7+CD45RA-) CD4 T cells (33.3 vs 46.5%; P = .035). At 30 days post-HT, CD4/CD127(low)FoxP3+ T cells were significantly reduced among patients with rejection episodes (0.84 vs 2.15%; P = .042). CD8 final effector T cells were increased at 90 days post-HT among those patients who experienced rejection (TEM2: 60.8 vs 31.9%; P < .1), at the expense of shrinking CD8 central memory compartment (TCM: 8.6 vs 12.9%; P = .046). The potential role of T-cell memory distribution should be further evaluated in HT patients as possible markers to discriminate patients at risk for rejection.

Activated Effector and Memory T Cells Contribute to Circulating sCD30: Potential Marker for Islet Allograft Rejection

T-cell activation up-regulates CD30 resulting in an increase in serum soluble CD30 (sCD30). CD4+ T cells, a major source for sCD30, play a significant role in the pathogenesis of rejection. In this study, sCD30 was measured pre- and posttransplant in mouse islet allograft models and human islet allograft recipients. sCD30 was measured by ELISA in diabetic C57BL/6, CD4Knockout (KO) and CD8KO islet allograft recipients. sCD30 increased significantly prior to rejection (1.8 +/- 1 days) in 80% of allograft recipients. Sensitization with donor splenocytes, or a second graft, further increased sCD30 (282.5 +/- 53.5 for the rejecting first graft vs. 374.6 +/- 129 for the rejecting second graft) prior to rejection suggesting memory CD4+ T cells contribute to sCD30. CD4KO failed to reject islet allograft and did not demonstrate sCD30 increase. CD8KO showed elevated (227 +/- 107) sCD30 (1 day) prior to rejection. High pretransplant sCD30 (>20 U/ml) correlated with poor outcome in human islet allograft recipients. Further, increase in sCD30 posttransplant preceded (3-4 months) loss of islet function. We conclude that sCD30 is released from activated CD4 T cells prior to islet allograft rejection and monitoring sCD30 can be a valuable adjunct in the follow-up of islet transplant recipients.

Genetics and genomics in human lung transplantation

Lung transplantation is the only effective treatment for many advanced lung diseases. However, long-term survival after transplantation remains relatively poor, thus limiting the application of lung transplantation to patients with end-stage disease only. Acute and chronic rejection is the main reason for allograft failure. Attempts to treat or prevent rejection have been stymied by our incomplete understanding of the mechanisms leading to this devastating complication and the lack of representative animal models. A systems-biology approach to lung transplantation with the use of genomics and gene expression profiling has led to new insights into the pathogenesis of rejection, by elucidating the mechanisms of T-cell activation and uncovering the role of B cells and innate immunity. Systems-biology approaches, such as genetics and genomics, may allow minimally invasive diagnosis of rejection and permit individually tailored immunosuppressive regimens. Herein we review the emerging application of genomics and genetics to human lung transplantation and highlight the tremendous potential for these approaches to enhance clinical practice and augment our understanding of basic transplant biology.

Infecciones más comunes en el paciente trasplantado

Organ transplantation has become one of the most important areas of medical research and, at present, is still the only therapeutical tool for several diseases. However, there are a number of factors related to transplantation, like immunosuppression and prolonged neutropenia that affect the incidence of infection. These infections are somehow peculiar to transplant recipients. In fact, there are infectious diseases that only occur in immunodepression situations and, moreover, clinical expression of these infectious diseases can be quite different from that in immunocompetent patients. Besides these aspects, some infections, due to the high prevalence described, must be considered for prevention strategies because they continue to be a principal cause of morbidity and mortality, either due to direct effects or to their implication in the pathogenesis of rejection. These strategies commence before transplantation by active immunization through vaccine administration to the patient and to people in the milieu and continue after transplantation with prophylaxis or pre-emptive therapy. The importance of infectious diseases in the evolution and prognosis of transplant recipients gives a special meaning to the understanding of associated infections, their clinical expression and ways of prevention and treatment.

Mast cells and c‐Kit expression in liver allograft rejection

The pathogenesis of rejection following liver transplantation is not fully understood. It has been postulated that mast cells may play a role in acute and chronic rejection of a number of other solid organ grafts. The aim of this study was to assess the possible role of mast cells and c-Kit+ cells in acute and chronic liver allograft rejection. Biopsy specimens from (i) 'time zero' grafts with a minimal degree of perfusion injury (controls), (ii) transplanted livers with different grades of acute rejection, and (iii) transplanted livers with end-stage chronic rejection, were stained immunohistochemically using monoclonal anti-mast cell tryptase and polyclonal anti-c-Kit antibodies. Tryptase- and c-Kit-positive cell densities were assessed by image analysis. Tryptase-positive mast cell densities (P<0.001) were strongly correlated with acute liver allograft rejection grades and chronic liver allograft rejection. Furthermore, a similarly strong relationship was found between c-Kit+ cell densities and increasing rejection grade (P<0.001). Tryptase- and c-Kit-positive mast cells form part of the inflammatory infiltrate in both acute and chronic liver allograft rejection, and may be important effector cells in these processes.

Viral Prophylaxis in Organ Transplant Patients

AbstractFor Abstract see ChemInform Abstract in Full Text.

THE PROGNOSTIC VALUE OF CIRCULATING SOLUBLE CELL ADHESION MOLECULES IN CARDIAC ALLOGRAFT RECIPIENTS

A283 Aims: The aim of this study is to test the hypothesis that plasma soluble cell adhesion molecules, which may play an important role in the pathogenesis of rejection and accelerated coronary artery disease (CAD), are related to subsequent clinical outcome in heart transplant recipients. Methods: We measured circulating levels of two soluble cell adhesion molecules (vascular cell adhesion molecule-1 [VCAM-1] and intercellular adhesion molecule-1 [ICAM-1]) in 82 asymptomatic, apparently healthy cardiac allograft recipients with left ventricular ejection fraction >50%. Major adverse clinical events (death, graft failure, severe infections, rejection, worsening heart failure, and angiographically documented CAD) during a median follow-up period of 682 days were determined. Results: The concentrations of the sVCAM-1 and sICAM-1 were significantly associated with one another. Significantly higher sVCAM-1 and sICAM-1 levels were noted in those patients who had major adverse clinical events during the follow-up period. By survival analysis, sICAM-1 was a significant predictor for development of CAD and sVCAM-1 was a significant predictor for development of non-CAD adverse events. Conclusions: These findings indicate that the levels of sVCAM-1 and sICAM-1 are related to clinical outcomes.

Viral Prophylaxis in Organ Transplant Patients

Viral pathogens have emerged as the most important microbial agents having deleterious effects on solid organ transplant (SOT) recipients. Antiviral chemoprophylaxis involves the administration of medications to abort transmission of, avoid reactivation of, or prevent progression to disease from, active viral infection. Cytomegalovirus (CMV) is the major microbial pathogen having a negative effect on SOT recipients. CMV causes infectious disease syndromes, augments iatrogenic immunosuppression and is commonly associated with opportunistic superinfection. CMV has also been implicated in the pathogenesis of rejection. Chemoprophylactic regimens for CMV have included oral aciclovir (acyclovir) at medium and high doses, intravenous and oral ganciclovir, and the prodrugs valaciclovir (valacyclovir) and valganciclovir. CMV prophylactic strategies should be stratified, with the highest-risk patients receiving the most 'potent' prophylactic regimens. Herpes simplex virus (HSV) reactivation in SOT recipients is more frequent, may become more invasive, takes longer to heal, and has greater potential for dissemination to visceral organs than it does in the immunocompetent host. Prophylactic regimens for CMV are also effective chemoprophylaxis against HSV; in the absence of CMV prophylaxis, aciclovir, valaciclovir or famciclovir should be used as HSV prophylaxis in seropositive recipients. Primary varicella-zoster virus (VZV) after SOT is rare and most commonly seen in the paediatric transplant population because of VZV epidemiology. Zoster occurs in 5-15% of patients, usually after the sixth post-transplant month. Prophylactic regimens for zoster are neither practical nor cost effective after SOT because of the late onset of disease and low proportion of affected individuals. All SOT recipients should receive VZV immune globulin after contact with either varicella or zoster. Epstein-Barr virus has its most significant effect in SOT as the precipitating factor in the development of post-transplant lymphoproliferative disorders. Antiviral agents that could be effective are the same as those used for CMV, but indications for and effectiveness of prophylaxis are poorly established. Hepatitis B virus (HBV) and hepatitis C virus (HCV) are important pathogens in the SOT population as indications for transplantation. So-called 'prophylaxis' for recurrent HBV and HCV after liver transplantation is controversial, suppressive rather than preventive, and potentially lifelong. Influenza infection after SOT is acquired by person-to-person contact. During epidemic periods of influenza, transplant populations experience a relatively high frequency of infection, and influenza may affect immunosuppressed SOT recipients more adversely than immunocompetent individuals. Antiviral medications for prevention of influenza are administered as post-exposure prophylaxis to SOT recipients, in addition to yearly vaccine, in circumstances such as influenza epidemics and nosocomial outbreaks, and after exposure to a symptomatic individual during 'flu season'.

Diagnostic value of peripheral blood T-cell activation and soluble IL-2 receptor for acute rejection in liver transplantation

Background: T-cells play an important role in the pathogenesis of rejection. Monitoring T-cells activation markers in peripheral blood may contribute to diagnosis of acute rejection after liver transplantation (LTX). Methods: Lymphocyte subset distribution, expression of T-cell activation markers (flow cytometry), concentration of soluble (s) interleukin-2 receptor (IL-2R) (solid phase chemiluminescence immunoassay), and liver enzymes as well as bilirubin were prospectively tested in peripheral blood samples of LTX patients with ( n=69) and without acute rejection ( n=50). Acute rejection was assessed by standard criteria including liver biopsies. Results: Intra-individual monitoring of immune parameters revealed an up-regulation of IL-2 receptor (CD25) expression on CD4 and CD8 T-cells together with increases in sIL-2R levels in patients with acute rejections. Measuring sIL-2R levels resulted in highest diagnostic efficiency (>85%). This level of diagnostic efficiency was not reached by any other marker tested. From all conventional markers of hepatocellular integrity and function, alkaline phosphatase reached the highest level of diagnostic efficiency with 70%. Conclusions: Monitoring of up-regulation of the IL-2/IL-2R pathway represents a useful tool for assessment of acute rejection after LTX.

Beneficial effects of targeting CCR5 in allograft recipients.

The chemokine receptor, CCR5, and its three high-affinity ligands, macrophage inflammatory protein- (MIP) 1alpha, MIP-1beta, and regulated on activation normal T cell expressed and secreted (RANTES), are expressed by infiltrating mononuclear cells during the rejection of clinical and experimental organ allografts, although the significance of these molecules in the pathogenesis of rejection has not been established. We studied intragraft events in four allograft models. First, we studied cardiac transplants in fully MHC-mismatched mice that were deficient in CCR5 or two of its ligands, MIP-1alpha or RANTES. Second we tested the effects of a neutralizing rat anti-mCCR5 monoclonal antibody on allograft survival. Third we assessed whether a subtherapeutic course of cyclosporine would potentiate enhance survival in CCR5-deficient recipients. Finally, we tested the effect of targeting CCR5 in a class II-mismatched model. Whereas mice deficient in expression of MIP-1alpha or RANTES reject fully MHC-mismatched cardiac allografts normally, CCR5-/- mice, or CCR5+/+ mice treated with a neutralizing mAb to mCCR5, show enhanced allograft survival. MHC class II-disparate mismatched are permanently accepted in CCR5-/- but not CCR5+/+ recipients. Finally, the beneficial effects of targeting of CCR5 are markedly synergistic with the effects of cyclosporine, resulting in permanent engraftment without development of chronic rejection. We conclude that CCR5 plays a key role in the mechanisms of host T cell and macrophage recruitment and allograft rejection, such that targeting of CCR5 clinically may be of therapeutic significance.

Early Inhibition of heme oxygenase enzymatic activity precipitates xenograft rejection.

514 Introduction: We have recently demonstrated that endothelial cell (EC) expression of heme oxygenase-1 (HO-1) is essential to insure accommodation of mouse hearts transplanted into rats treated with the complement inhibitor cobra venom factor (CVF) and Cyclosporin A (CyA). This study aimed to analyze the mechanism by which HO-1 contributes to suppress xenograft rejection and to investigate at what time after transplantation the expression of HO-1 is imperative to insure xenograft survival Methods: BALB/c mouse hearts were transplanted into Lewis rats treated with CVF (60 U/kg one day before transplantation and 20 U/kg at the time of transplantation) and CyA (15 mg/kg/daily starting at the time of transplantation). HO activity was inhibited by administration of Tin Protoporphyrin (SnPP, 30μM/kg/daily). Iron protoporphyrin (FePP, 30μM/kg/daily) was used as a control treatment Result: All mouse hearts transplanted into rats treated with CVF plus CyA survived long-term. Inhibition of HO activity by SnPP given on day −2, −1 and 0 (the day of transplantation) to the donor, and daily on day 0 to the time of rejection to the recipient, led to xenograft rejection 3 to 7 days after transplantation (Table 1).Table 1HO inhibition by SnPP given daily to the recipient from day 2 resulted in xenograft rejection in 3 of 7 cases, while inhibition of HO activity starting at later time points no longer induced xenograft rejection. Xenograft rejection under SnPP treatment was very similar to that of HO-1 deficient (knock-out) hearts: rejection was characterized by thrombosis of large coronary vessels, extensive myocardial infarction, cardiomyocyte apoptosis and infiltration by host macrophages and neutrophils. Thrombosis was detected in the xenograft aorta by 24 hours after transplantation, indicating that the pathogenesis of rejection may involve thrombosis of large coronary arteries, a decreased blood supply to the heart, anoxia and infarction. Conclusion: Early expression of HO-1 in the xenograft prevents injury leading to thrombosis of large coronary vessels, thus avoiding subsequent myocardial infarction and xenograft rejection. Inhibition of HO activity starting on day 2 following transplantation no longer precipitates rejection in all grafts, and later administration of SnPP does not lead to rejection. It is attractive to think that other mechanisms, such as the expression of other protective genes, exist to promote xenograft survival at those later times.

ANTIBODIES ELUTED FROM ACUTELY REJECTED RENAL ALLOGRAFTS BIND TO AND ACTIVATE HUMAN ENDOTHELIAL CELLS

164 Antibodies (Abs) were eluted from 25 renal allografts lost by acute"humoral" rejection in patients without anti-HLA antibodies, and from 2 kidneys lost by renal vein thrombosis as controls. All patients were under immunosuppression at the time of nephrectomy. Abs from 8 rejected kidney bound to cultured Human Umbilical Vein Endothelial Cells (HUVEC) as shown by FACS analysis and did not induce any cytotoxicity in a usual PRA assay. Incubation of HUVEC (4 hours at 37°C) with those Abs (2.5 mg/ml) led to up-regulation of mRNAs coding for VCAM (9 to 15 fold increases) and ELAM-1(20 to 30 fold increases), as compared to control Abs, as assessed by semi-quatitative RT-PCR normalized for GAPDH. Membrane expressions of the same molecules, as assessed by FACS analysis, were strikingly increased, with 80% of the cells expressing VCAM and 65% expressing ELAM-1 upon incubation. Thus, anti-endothelial cell antibodies, capable of activating human endothelial cells, can be recovered from acutely rejected kidneys and may play a direct role in the pathogenesis of rejection.