Lesions Of Chronic Rejection Research Articles

Chronic lung allograft pathology lesions in two rat strain combinations.

BackgroundChronic lung allograft dysfunction remains an obstacle to long-term survival after lung transplantation. Two phenotypes have been described: obliterative bronchiolitis and restrictive allograft syndrome. Preclinical models are essential to analyze chronic lung allograft dysfunction pathophysiology.MethodsOrthotopic lung transplants from 38 Lewis into Fischer 344 (Lew→F344) and 67 Brown-Norway into Lewis (BN→Lew) rats were performed in our center in the last decade. We carefully reviewed and quantified all grafts with chronic rejection (40 cases) (18 Lew→F344, 22 BN→Lew) with the aim to investigate if histological changes of chronic lung allograft dysfunction could be also detected in rat grafts.ResultsAll animals showed human reminiscent histological lesions. Early chronic rejection lesions were detected in BN→Lew. End-stage chronic rejection with features of obliterative bronchiolitis was observed in 33% of Lew→F344; end-stage with restrictive allograft syndrome chronic rejection in 67% and 80% of Lew→F344 and BN→Lew, respectively. BN→Lew showed higher grades of endotheliitis, vascular fibrosis, and lower grades of lymphoid aggregates than Lew→F344 (P=0.007, P=0.043, P=0.004, respectively).ConclusionsChronic rejection lesions in rat lung allografts mimic those in humans. The frequent occurrence of restrictive allograft syndrome-like lesions in BN→Lew may be related to a higher degree of mismatch in this strain combination. These animal models could allow future mechanistic studies to better understand chronic lung allograft dysfunction pathogenesis.

Multi-gene technical assessment of qPCR and NanoString n-Counter analysis platforms in cynomolgus monkey cardiac allograft recipients

Quantitative gene expression profiling of cardiac allografts characterizes the phenotype of the alloimmune response, yields information regarding differential effects that may be associated with various anti-rejection drug regimens, and generates testable hypotheses regarding the pathogenesis of the chronic rejection lesions typically observed in non-human primate heart transplant models. The goal of this study was to assess interplatform performance and variability between the relatively novel NanoString nCounter Analysis System, ΔΔCT (relative) RT-qPCR, and standard curve (absolute) RT-qPCR utilizing cynomolgus monkey cardiac allografts. Methods for RNA isolation and preamplification were also systematically evaluated and effective methods are proposed. In this study, we demonstrate strong correlation between the two RT-qPCR methods, but variable and, at times, weak correlation between RT-qPCR and NanoString. NanoString fold change results demonstrate less sensitivity to small changes in gene expression than RT-qPCR. These findings appear to be driven by technical aspects of each platform that influence the conditions under which each technique is ideal. Collectively, our data contribute to the general effort to optimally utilize gene expression profiling techniques, not only for transplanted tissues, but for many other applications where accurate rank-order of gene expression versus precise quantification of absolute gene transcript number may be relatively valuable.

Chronic Rejection Lesions Are Decreased in IL-17 KO Mice after Orthotopic Lung Transplantation

<h3>Purpose</h3> Long term survival after lung transplantation (LTx) is hampered by chronic rejection, histologically characterized by obliterative bronchiolitis (OB). A murine model of orthotopic lung transplantation showing OB, lymphocytic bronchiolitis (LB) and abnormal parenchyma has previously been established in our lab (De Vleeschauwer et al. 2012). Interleukin-17 (IL-17) is identified as potential player in the onset of chronic rejection. Aim: To investigate the role of IL-17 by using IL-17 knock out (KO) mice in our model. <h3>Methods and Materials</h3> Allografts are donor Balb/c lungs transplanted in C57Bl6 (wild type) receptors. Knock out allografts are donor Balb/c lungs transplanted in C57Bl6 IL-17 KO receptors (kindly provided by J. Kolls and Y. Iwakura). Isografts have donor lungs from mice genetically identical to the receptors. All allograft receptors received daily 10 mg/kg cyclosporine and 1.6 mg/kg methylprednisolone. Histopathological examination (H&E staining) was performed 10 weeks after surgery. <h3>Results</h3> Ten weeks after LTx, isografts (n=4) had a completely normal lung histology. Allografts (n=4) showed OB, LB and abnormal parenchyma as previously established in the model. In the IL-17 KO allograft (n=4), however, no OB lesions could be identified. LB lesions were still present, but less pronounced and lung parenchyma was almost normal. [figure 1] <h3>Conclusions</h3> Chronic rejection lesions (OB) are decreased in IL-17 KO mice, hereby providing the ultimate proof of involvement of IL-17 in the onset of chronic rejection after lung transplantation.

Peritubular capillary basement membrane changes in chronic renal allograft rejection

Marked peritubular capillary basement membrane (PTCBM) multilayering, the ultrastructural feature of chronic antibody-mediated rejection (ABMR) of kidney allografts, was found to correspond histologically to PTCs with thickened BMs; such PTCs have been suggested as a novel histological marker of chronic rejection. We investigated whether scoring of PTCBM thickening can substitute the ultrastructural search for PTCBM multilayering. The thickening was graded in PAS- and Jones-stained sections in 110 biopsies from recipients with a late dysfunction, all examined ultrastructurally for transplant capillaropathy (≥3PTCs with ≥5BM layers). Grade 0 indicated no thickening. Grade 1 and grade 2 were assigned when the PTCBMs were as thick as or thicker than those of the non-atrophic tubules, and duplication/chain-like lamination of the PTCBM was noted in ≤3 or ≥4 high-power fields, respectively. The series was enrolled in subgroups of those with and those without histopathological lesions of chronic rejection. Fifty-six biopsies displayed lesions of chronic ABMR. Transplant capillaropathy was demonstrated in 40 biopsies. Grade 2 thickening furnished a substantial interobserver concordance rate (κ = 0.803) and correlated with the transplant capillaropathy. Jones staining performed somewhat better in scoring than PAS staining. Grade 2 thickening was verified in 35 biopsies involving chronic ABMR, and in one control biopsy (sensitivity 61.4%, specificity 0.98). Grade 1 thickening was not suggestive of chronic ABMR at all. In conclusion, grade 2 thickening can be regarded as the histopathological lesion of chronic ABMR; however, electron microscopy remains the gold standard in the assessment of PTCBM changes.

Overlapped glomerular lesions of chronic rejection and recurrent lupus nephritis in transplanted kidney: a case report

We describe a renal transplant recipient with systemic lupus erythematosus (SLE) who showed continuous proteinuria and low complement levels without clinical evidence of active SLE. Her first renal allograft biopsy, performed nine yr and eight months after transplantation, revealed unusual histological change of glomeruli, and it initially led us to make a contradictory diagnosis based on light and electron microscopic examinations. Diffuse global double- or multi-contour glomerular basement membrane was caused by chronic endothelial injury owing to chronic rejection, and mesangial proliferation associated with mesangial electron-dense deposit was a histological change characteristic of recurrent lupus nephritis (RLN). Immunofluorescence study displayed weak mesangial staining of IgM and C1q. We concluded that this case presented overlapped chronic rejection and RLN. Because both transplant nephropathy and lupus nephritis present constellations of various histologies, it is difficult to diagnose their overlap. Complete morphologic studies with both immunofluorescence and electron microscopic evaluations in addition to microscopic examination should be performed to elucidate complex histological findings.

Contribution of Electron Microscopy to the Final Diagnosis of Renal Biopsies in Egyptian Patients

There are few publications studying the impact and cost benefit relationship of electron microscopy in the diagnosis of glomerulopathies in routine service. The aim of this study is to assess the contribution of EM to the final diagnosis of renal glomerular diseases in Egyptian patients. Retrospective evaluation of 120 renal biopsy specimens received for primary diagnosis at EM center of Ain Shams university Specialized hospital, Cairo Egypt during 2007 in the knowledge of light microscopic, immunofluorescence and electron microscopic findings. It was found that EM was essential for diagnosis in 25% of renal biopsies, corresponding to 100% of hereditary glomerulopathies and 23.5% of other glomerulopathies, It was useful to the diagnosis in 41.67% of the cases, confirming the preliminary diagnosis. In 33.33% of cases EM was considered unhelpful in diagnosis. It's concluded that the importance of EM has not decreased during the last years. New glomerular diseases and variants can be diagnosed only by EM as fibrillary glomerulonephritis and immunotactoid glomerulopathy. Routine evaluation of allograft biopsies should include EM to achieve better recognition of capillary lesions of chronic rejection. EM provides useful diagnostic information in about 66% of native renal biopsies. Kidney biopsy protocols should include EM in all biopsy cases. If electron microscopy cannot be performed routinely on all such biopsies, tissue should be reserved for EM studies.

Preemptive CD20+ B cell depletion attenuates cardiac allograft vasculopathy in cyclosporine-treated monkeys

Chronic rejection currently limits the long-term efficacy of clinical transplantation. Although B cells have recently been shown to play a pivotal role in the induction of alloimmunity and are being targeted in other transplant contexts, the efficacy of preemptive B cell depletion to modulate alloimmunity or attenuate cardiac allograft vasculopathy (CAV) (classic chronic rejection lesions found in transplanted hearts) in a translational model has not previously been described. We report here that the CD20-specific antibody (alphaCD20) rituximab depleted CD20+ B cells in peripheral blood, secondary lymphoid organs, and the graft in cynomolgus monkey recipients of heterotopic cardiac allografts. Furthermore, CD20+ B cell depletion therapy combined with the calcineurin inhibitor cyclosporine A (CsA) prolonged median primary graft survival relative to treatment with alphaCD20 or CsA alone. In animals treated with both alphaCD20 and CsA that achieved efficient B cell depletion, alloantibody production was substantially inhibited and the CAV severity score was markedly reduced. We conclude therefore that efficient preemptive depletion of CD20+ B cells is effective in a preclinical model to modulate pathogenic alloimmunity and to attenuate chronic rejection when used in conjunction with a conventional clinical immunosuppressant. This study suggests that use of this treatment combination may improve the efficacy of transplantation in the clinic.

Anti-CD28 Antibodies Modify Regulatory Mechanisms and Reinforce Tolerance in CD40Ig-Treated Heart Allograft Recipients

Blockade of CD40-CD40 ligand (CD40L) costimulation has been shown to synergize with that of CTLA4/CD28-B7 to promote transplant tolerance. To date, however, CD28-B7 interactions have been prevented using B7-blocking reagents like CTLA4-Ig that inhibit CD28-B7 together with CTLA4-B7 interactions. In this study, we have tested anti-CD28 Abs to prevent selectively CD28-B7 interactions while preserving CTLA4-B7 in addition to CD40-CD40L blockade. In the LEW.1W to LEW.1A rat combination, interfering with CD40-CD40L interactions by CD40Ig administration through gene transfer resulted in indefinite heart allograft survival due to the appearance of clonotypic CD8+CD45RClow regulatory T cells that were capable of transferring the tolerant state to naive animals. However, cardiac transplants in these recipients systematically developed chronic rejection lesions. Whereas anti-CD28 Ab monotherapy only delayed acute rejection and failed to induce tolerance, coadministration of anti-CD28 Abs and CD40Ig resulted in the long-term acceptation of allografts without chronic rejection lesions in 60% of the recipients, reduced the level of intragraft mRNA transcripts for cytokines and immune factors, and fully abrogated alloantibody production. In addition, the nature of regulatory cells was modified: the CD8+CD45RClow clonotypic T cells described in the CD40Ig-treated animals could not be found in cotreated animals, and the other CD8+CD45RClow cells had no regulatory activity and a different cytokine expression profile. Instead, in cotreated recipients we found IDO-dependent non-T cells with regulatory activity in vitro. Thus, the addition of a short-term anti-CD28 treatment with CD40Ig resulted in decreased heart allograft chronic rejection lesions, complete inhibition of Ab production, and modified regulatory mechanisms.

Adenovirus-Mediated Heme Oxygenase Transfer Inhibits Graft Arteriosclerosis in Rat Aortic Transplants

Purpose Heme oxygenase-1 (HO-1) has been implicated in graft protection because its induction is associated with long-surviving allografts. The aim of this study was to analyze the efficacy of adenovirus-mediated HO-1 overexpression to inhibit graft arteriosclerosis and estimate whether the protective role correlated with nuclear factor κB (NF = κB) expression and grafts apoptosis. Methods Aortic transplantation was performed using inbred Brown-Norway (BN) rats (RT 1n male) as donors and Lewis rats (RT 1l male) as recipients. The experiments were divided into four groups: group A, Lewis–Lewis ( n = 6), no treatment; group B, BN–Lewis ( n = 6), no treatment; group C, BN–Lewis ( n = 6), donor aorta perfused with buffer containing 4 × 10 10 pfu Ad-Null (the noncoding adenoviral vector); and group D, BN–Lewis ( n = 6), donor aorta perfused with buffer containing 4 × 10 10 pfu Ad-HO-1 (adenoviral vector coding for HO-1). All grafts were harvested at 60 days after transplantation. Reverse transcriptase polymerase chain reaction was performed to detect the expression of HO-1 gene. Hematoxylin and eosin staining was used to detect the morphology and cytology of the transplanted vessels. Intimal thickening was detected by Masson staining. Immunohistochemistry was used to detect the localization and expression of HO-1 and NF-κB and TUNEL assays to detect the apoptosis of the grafts. Results Gene transfer of HO-1 grafts using an adenoviral vector resulted in the expression of HO-1 protein in endothelium and adventitia. We observed that the intimal thickness in Ad-HO-1–treated aortas (11.11 ± 0.92 μm) was significantly thinner compared with untreated (85.20 ± 6.90 μm) or Ad-null treated (87.20 ± 6.20 μm) aortas ( P < .01). Immunohistology showed that treatment with Ad-HO-1 resulted in a significant reduction in leukocyte infiltration and a decreased number of vascular smooth muscle cells in the intima, compared with Ad-null–treated aortas. The levels of NF-κB and the number of apoptotic cells in Ad-HO-1–treated aortas showed significantly lower compared with Ad-null–treated arotas ( P < .05). Conclusion HO-1 prevented the development of graft arteriosclerosis in the rat aortic transplant model. The protective role of HO-1 in chronic rejection lesions seemed to correlate with downregulation of the expression of NF-κB and inhibition of apoptosis in the grafts.

Revisiting tolerance induction by donor cell priming

Purpose of review Donor specific transfusion is one of the most potent approaches to induce tolerance to vascularized organs and has been extensively studied in the rat. Following donor specific transfusion, both immune activation and suppressive mechanisms involving T cells, dendritic cells and immunosuppressive cytokines have been described in tolerant recipients. Recent Findings Recent data on the donor specific transfusion effect have led to the development of new concepts concerning this tolerance induction phenomenon, particularly concerning the role of the transplant itself and the coexistence of both regulatory T cells able to transfer tolerance and chronic rejection lesions in the long-term surviving grafts. Summary Reviewing these mechanisms would help in the quest to improve strategies to achieve tolerance in the clinic.

An MHC Class II Disparity Raises the Threshold for Tolerance Induction in Pulmonary Allografts in Miniature Swine

Objectives The mechanisms and treatment of chronic rejection in pulmonary allotransplantation remain elusive. We have induced robust tolerance to class I-disparate lung allografts in miniature swine using an intensive 12-day course of tacrolimus. Here, we tested whether a tolerant state can be induced in swine receiving fully mismatched lung allografts. Methods Orthotopic left lung allografts were performed using MHC class I-disparate (group 1: n = 3) or fully disparate (group 2: n = 6) donors. The recipients received a 12-day postoperative course of tacrolimus (continuous intravenous infusion; target level = 35–50 ng/mL) as their only immunosuppression. Results All swine in group 1 maintained their grafts long term without developing any lesions of chronic rejection (>497, >432, >451 days). These recipients exhibited donor-specific hyporesponsiveness in cell-mediated lymphocytotoxity (CML) and mixed lymphocyte reaction (MLR) assays. In group 2, five of the six recipients maintained their grafts long term (sacrificed on postoperative days 515, 389, 429, 481, and 438 with viable grafts). Isolated lesions of obliterative bronchiolitis were occasionally seen on biopsy, and donor-specific hyporesponsiveness on assays was consistently observed. The remaining recipient rejected its graft on day 103 with histologic findings of obliterative bronchiolitis. Conclusions We report long-term graft acceptance without chronic immunosuppression in five of six recipients across a full MHC disparity, albeit with some evidence of obliterative bronchiolitis. These data suggest that the class II disparity inherent in a fully mismatched transplant increases the requirement for tolerance induction.

Evidence for ischemia induced host-derived bone marrow cell mobilization into cardiac allografts

Mobilized bone marrow stem cells (BMSC) exhibit high degree of plasticity and participate in the repair process in the event of myocardial damage. In this study, we verified the proportional contribution of recipient BMSC in the repair process and identified their specific surface markers. Wild-type (WT) donor female heart was transplanted into abdominal cavity of male rat (Group I). In some of recipient animals, infarction was created by LAD occlusion (Group II). Two weeks later, transplanted female hearts were harvested for histological analysis of the mobilized cells. C-kit, CD31, Ki67 and Y-chromosome were used as markers to identify mobilized cells in the female hearts. Y-chromosome positive cells were found in the donor female cardiac allografts. Acute myocardial infarction (AMI) of recipient heart induced migration of progenitor cells into the lesions of chronic rejection in the allograft. Donor ventricular mass reduction was more pronounced in Group I. Endothelial progenitor cells induced by AMI from male recipient extensively migrated into the cardiac allograft. SDF-1 mRNA levels significantly increased (peak level at 24 h after AMI) in recipient heart. CXCR4 was strongly expressed in the transplanted hearts around the perivascular area. Spontaneous mobilization of hematopoietic progenitor cells (HPCs) occurred in cardiac allografts after creating recipient heart AMI and was detectable until 2 weeks. These data suggests that CXCR4 overexpression enhances vascularization in the damaged myocardium and SDF-1/CXCR4 axis seems particularly important in progenitor cell chemotaxis, homing, engraftment and retention in damaged myocardium. In addition, BMSC attracted to the site of ischemic injury participate in cardiac repair.

Clinical Operational Tolerance after Kidney Transplantation

Induction of allograft-specific tolerance and the detection of a "tolerance" state in recipients under immunosuppression with long-term stable graft function are major challenges in transplantation. Clinical "operational tolerance," defined as stable and acceptable graft function without immunosuppression for years, is a rare event. There is no report on the clinical history of such patients. In this article, we report on the medical history of 10 kidney recipients who display an immunosuppressive drug-free "operational tolerance" for 9.4 +/- 5.2 years. Clinical factors that may favor such a tolerant state are underlined. Firstly, most of the patients interrupted immunosuppression over a long time period (until 4 years), which mimics the procedure of intentional immunosuppression weaning following liver transplantation. Secondly, donor age was younger (median 25 years) than the one of the general transplanted population, suggesting that graft quality is one of the conditions favoring "operational tolerance." Moreover, the "operationally tolerant" recipients may be 'low responders' to blood transfusions (PRA 6 +/- 5.4%, six blood transfusions). We also show that "operational tolerance" occurs in the presence of anti-donor class II antibodies, as assessed in two patients. Finally, two patients degraded their renal function 9 to 13 years after treatment withdrawal, however only one presented histological lesions of chronic rejection.

Peritubular Capillary Damage in Acute Humoral Rejection: An Ultrastructural Study on Human Renal Allografts

The ultrastructural features of peritubular capillary (PC) damage was studied in 12 kidney allografts with acute humoral rejection (AHR). AHR manifested in diffuse linear PC staining for C4d, and histology consistent with Banff grade III in 7 recipients and Banff grade II in 5. Allografts with acute tubular necrosis served as controls. First biopsies (post-transplantation day 16.2 +/- 2.2): The intra-capillary exudate comprised monocytes (59%), polymorphonuclears (14%), lymphocytes (12%) and not otherwise specified mononuclears (15%). Three patterns of focal PC endothelial injury were observed: lysis, an increased rate of apoptosis and fragmentation. No correlation was found between the respective damage types and the inflammatory cell types or the Banff grades. Controls revealed endothelial swelling, detachment from basement membrane and fragmentation. Follow-up biopsies: Monocytes transformed into macrophages intra-luminally. The reparative changes comprised endothelial cytoplasmic protrusions, binucleated endothelial cells and capillary sprouts. Early transplant capillaropathy and transplant glomerulopathy were noted in 2 recipients. Literature data indicate that lysis is mediated by anti-HLA alloantibodies; apoptosis, demonstrated first in the present study, may be induced by non-HLA-type anti-endothelial antibodies. Fragmentation is caused by ischemia. Ongoing endothelial injury leads to transplant capillaropathy and transplant glomerulopathy, the characteristic lesions of chronic rejection.

Inhibition of Chronic Rejection and Development of Tolerogenic T Cells after ICOS-ICOSL and CD40-CD40L Co-stimulation Blockade

: Blockade of the CD40-CD40L pathway results in long-term allograft survival but does not prevent chronic rejection. ICOS-ICOSL are members of the CD28-B7 family that play an important role in T-cell activation. : The authors analyzed the effect of single or combined treatment with an anti-ICOS monoclonal antibody and CD40 immunoglobulin (Ig) on acute and chronic rejection of heart allografts in rats. : Treatment with anti-ICOS resulted in a modest but significant prolongation of allograft survival. Treatment with CD40Ig resulted in long-term graft survival but the cardiac grafts developed chronic rejection lesions. Combined CD40Ig+anti-ICOS treatment led to indefinite graft survival in all recipients and a significant decrease of chronic rejection lesions compared with CD40Ig alone. Importantly, four of the seven CD40Ig+anti-ICOS-treated recipients showed a complete absence of chronic rejection lesions, whereas all of the CD40Ig-treated recipients showed chronic rejection. The CD40Ig+anti-ICOS group also showed significant decreased graft infiltration, decreased antidonor cytotoxic T-lymphocyte activity, and decreased alloantibodies compared with the CD40Ig-treated group. Adoptive transfer of splenocytes indefinitely prolonged allograft survival, whereas those depleted of T cells did not, suggesting the development of T-regulatory mechanisms. : These data indicate that the chronic rejection mechanisms that are CD40-CD40L independent are ICOS-ICOSL dependent. These results were obtained with conservation of cognate immune responses and development of tolerogenic T cells.

CD40L AND CD28 BLOCKADE INDUCES REGULATORY MECHANISM AND INHIBITS CHRONIC ALLOGRAFT REJECTION

P1037 Aims: Blockade of individual T cell costimulatory pathways may result in long-term allograft survival but tolerance is not obtained since chronic rejection is observed. To obtain transplantation tolerance, blockade of more than one costimulatory pathway is needed but the importance of different pathways and the optimal reagents to be used have not been fully defined. Methods: Heart transplantation was performed between MHC mismatched rats. CD40-CD154 interactions were blocked by gene transfer of CD40Ig. We interfered with CD28 costimulatory signals by administering an anti-CD28 Mab, which in contrast to CTLA4Ig, preserves T cell inhibitory signals through CTLA4. Results: CD40Ig treatment resulted in long-term (>120d, n=19) heart allograft survival and development of chronic rejection in all grafts whereas acute rejection occurred in the non-coding adenovirus-treated (7.6±1.6d, n=9) group. Treatment with anti-CD28 Mab resulted in a moderate but significant prolongation of allograft survival (17.4±1.7d, n=10). Cotreatment with both CD40Ig and anti-CD28Mab resulted in long term allograft survival (>120d, n=7) and a significant reduction in the percentage and degree of vessels with chronic rejection lesions, with 3/7 grafts completely free of lesions and therefore tolerant. Splenocytes from cotreated tolerant recipients, but not those with chronic rejection (CD40Ig or cotreated), showed completely suppressed MLR responses against 1st but not 3rd party donor cells. T cells purified from splenocytes of tolerant cotreated recipients showed normal MLR responses indicating that among total splenocytes, non T cells inhibited the proliferation of T cells. Splenocytes from tolerant recipients but not those with chronic rejection lesions inhibited naïve MLR, indicating the presence of active tolerogenic mechanisms. Transfer of splenocytes from CD40Ig-treated recipients to naïve sublethaly irradiated rats conferred long term allograft survival (>120d, n=4 vs. 17 d, n=2 in controls). In contrast, transfer of splenocytes from CD40Ig+anti-CD28 cotreated rats, either tolerant or with chronic rejection lesions, did not prolong allograft survival (17d, n=3). Conclusions: The association of CD40Ig with anti-CD28 Mab resulted in decreased chronic rejection lesions and tolerogenic mechanisms different to those seen with CD40Ig.

SYNERGISTIC EFFECT OF ICOS/B7RP-1 AND CD40/CD40L COSTIMULATION BLOCKADE IN THE INDUCTION OF ALLOGENEIC TOLERANCE

O461 Aims: Blockade of the CD40-CD40L pathway results in long-term allograft survival but does not prevent chronic rejection. Therefore new immunotherapies are needed to obtain tolerance. ICOS and its receptor, B7RP-1, are members of the CD28-B7 families which play an important role in T cell activation and survival. Methods: We used an adenovirus coding for CD40Ig (which blocks CD40-CD40L interactions) in a rat heart allograft transplantation model. We analyzed the effect of co-treatment with an anti-ICOS MAb and CD40Ig on chronic rejection. Results: CD40Ig and anti-ICOS treatment led to indefinite graft survival in all recipients (>120 days). Treatment with anti-ICOS resulted in a modest but significant prolongation of allograft survival (17±1.5 vs 7.6±1.6, p=0.002, n=4). Analysis of chronic rejection lesions at day 120 in the CD40Ig+anti-ICOS group (n=7) vs. the CD40Ig group (n=17) showed that the percentage of vessels occluded (15.2±6.6 vs. 33.7±4.4, p=0.017), degree of vessel occlusion (1.1±0.5 vs. 2.4±0.2, p=0.007) and vessel lesions (1±0.3 vs. 2.3±0.1, p<0.001) but not fibrosis were significantly lower. Importantly, 4 of the 7 CD40Ig+anti-ICOS-treated recipients showed complete absence of chronic rejection lesions whereas all CD40Ig-treated recipients showed signs of chronic rejection. The CD40Ig+anti-ICOS group showed decreased graft infiltration by TCRαβ+, CD4+ and CD8α+ cells as well as macrophages and mast cells. Recipients in the CD40Ig+anti-ICOS group displayed significantly inhibited anti-donor CTL activity. Alloantigenic proliferative responses of splenocytes in the CD40Ig+anti-ICOS group were strongly inhibited and were reversed by IL-2. CD40Ig treatment strongly but incompletely inhibited total IgG, Th2 and Th1-dependant alloantibody production. The residual total IgG and Th1 but not Th2 alloantibody were significantly reduced in the sera of recipients of CD40Ig+anti-ICOS treated allografts at day 120, suggesting an inhibition of Th1 responses. Conclusions: These data indicate that the chronic rejection mechanisms that are CD40-CD40L-independent are ICOS/B7RP-1-dependent. Operational tolerance can be obtained by simultaneous blockade of both costimulatory pathways.

The pathobiology of chronic allograft nephropathy: Immune-mediated damage and accelerated aging

Chronic allograft nephropathy includes chronic calcineurin nephrotoxicity, recurrent and de novo glomerulonephritis and a group of disorders with graft dysfunction of unknown etiology designated chronic rejection. Review of risk factors of the latter category show that the chronic rejection lesions emerge in organs that have undergone injury. Despite the relevance of nonalloantigen-dependent progression factors in the tissue injury, alloantigen-dependent factors predominate in the pathogenesis. Lately, B cell responses have received increasing interest in transplant rejection and include responses against both major histocompatibility complex (MHC) and tissue-specific antigens, mainly on the endothelium and in the glomeruli. These humoral responses are thought to be involved in the development of vascular and glomerular lesions. Furthermore, at the tissue level, markers of senescence are found in the tubular epithelium contributing to the lesions of tubular atrophy and interstitial fibrosis.

Cyclosporine nephrotoxicity: how does it affect renal allograft function and transplant morphology?

Chronic CSA nephrotoxicity is the second most important diagnosis responsible for the late graft failure. CSA associated arteriolopathy (CAA) is a well-known lesion of chronic CSA nephrotoxicity. The clinicopathological characteristics and the significance of CSA nephrotoxicity have changed following reduction in CSA doses and implementation of monitoring of blood levels. Seventy-four CAA patients on CSA therapy were classified as functioning ( n = 30) or loss groups ( n = 44). There was no significant difference in severity of CAA. The concomitant lesion of chronic rejection, but not the severity of CAA, was the most important risk factor for graft loss. Among 54 recipients with focal segmental glomerulosclerosis lesions (FGS), 32 (59%) were diagnosed as CAA associated glomerulopathy (CAG). Eighteen of the 32 CAG patients lost their grafts upon follow-up. Decreasing the CSA dosage to maintain lower blood levels than the usually optimal concentrations, but not discontinuation of CSA, has been useful to retard the progression of graft dysfunction in half of 15 isolated pure CAG patients. Patients with increasing daily proteinuria exceeding 2 grams lost their graft function despite CSA reductions. CAA is not a specific lesion of chronic CSA nephrotoxicity; the FGS lesion is also a nonspecific lesion often seen in renal allografts. Isolated chronic CSA arteriolopathy of severe degree has a fairly good prognosis under controlled CSA therapy. The FGS lesion accompanying CAA is considered to be CSA-associated glomerulopathy. These data contribute to therapeutic plans for renal transplant patients during long-term CSA treatment.

Balanced expression of mitochondrial apoptosis regulatory proteins correlates with long-term survival of cardiac allografts.

Abnormal regulation of apoptosis is observed in ischemic injury and may contribute to the pathogenesis of atherosclerosis. However, its role in cardiac allograft vasculopathy (CAV), the fundamental lesion of chronic rejection (CR) in heart transplantation, remains uncertain. To clarify this issue, apoptosis was quantitated in myocardium and coronary arteries from 5 cardiac allograft donors (NL) and explanted hearts of 24 patients with ischemic cardiomyopathy (IsCM) and 15 patients with CR. Tissue samples were analyzed via end-labeling fragmented DNA [via deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)] and immunoblotting for activated caspase-3 and -9. Myocyte apoptosis assessed by TUNEL was similarly increased over NL (0.21%) in both the CR (0.88%; P < 0.01) and IsCM (0.88%; P < 0.01) groups. Activated caspase-9 levels were significantly higher in CR (14.7%) compared with IsCM (6.9%; P < 0.01) and NL (0%) groups, whereas activated caspase-3 levels were similarly elevated in both CR and IsCM (7.8 and 6.5% vs. 0% in NL; P < 0.01 and P < 0.05) groups. Expression of myocardial Bcl-2 and Bax was increased in CR compared with both NL (Bax, 4.3-fold; P < 0.01; Bcl-2, 5.9-fold; P < 0.01) and IsCM (IsCM: Bax, 2.2-fold; P < 0.05; Bcl-2, 3.2-fold; P < 0.01) groups. The rate of apoptosis and the Bcl-2/Bax ratio independently correlated to graft survival in CR (activation of caspase-9: r = 0.87; P < 0.01; Bcl-2/Bax: r = 0.57; P = 0.05). Compared with native atherosclerosis, coronary arteries with CAV showed more medial apoptosis (7.8-fold; P < 0.01) and higher Bcl-2 levels (5.1-fold; P < 0.01) with lower Bax levels (threefold; P < 0.05) in the intima. These results indicate that abnormal Bcl-2 and Bax expression in myocardium and coronary arteries of cardiac allografts with CR is distinct from that in IsCM and suggest that balancing Bcl-2 to Bax in transplanted hearts promotes long-term graft survival.