Subclinical Rejection in Stable Positive Crossmatch Kidney Transplant Patients: Incidence and Correlations

We reviewed 116 surveillance biopsies obtained approximately 1, 3, 6 and 12 months posttransplantation from 50 +XM live donor kidney transplant recipients to determine the frequency of subclinical cell-mediated rejection (CMR) and antibody-mediated rejection (AMR). Subclinical CMR was present in 39.7% of the biopsies at 1 month and >20% at all other time points. The presence of diffuse C4d on biopsies obtained at each time interval ranged from 20 to 30%. In every case, where histological and immunohistological findings were diagnostic for AMR, donor-specific antibody was found in the blood, challenging the long-held belief that low-level antibody could evade detection due to absorption on the graft. Among clinical factors, only recipient age was associated with subclinical CMR. Clinical factors associated with subclinical AMR were recipient age, positive cytotoxic crossmatch prior to desensitization and two mismatches of HLA DR 51, 52 and 53 alleles. Surveillance biopsies during the first year post-transplantation for these high-risk patients uncover clinically occult processes and phenotypes, which without intervention diminish allograft survival and function.     

Ultra-Late Antibody-Mediated Rejection 30 Years After a Living-Related Renal Allograft

Antibody-mediated renal allograft rejection has become increasingly recognized and more clearly defined through the use of flow cytometry cross-matching and the deposition of C4d in renal allograft biopsies. All of the cases reported thus far have developed an antibody within 10 years of transplantation, and many lacked HLA and/or donor specificity. The present patient developed an anti-HLA donor-specific antibody between the 22nd and 30th year after a living-related renal transplant. At the 30th year post-transplantation, she experienced a rise in the serum creatinine from 0.7 to 1.9 mg/dL associated with transplant biopsy C4d deposition in peritubular capillaries and glomeruli. After the replacement of azathioprine with mycophenolate mofetil, and six apheresis treatments followed by two infusions of IVIG, the renal function stabilized at 1.9 mg/dL, 33 years after transplantation. Antibody-mediated rejection must be considered as a possible cause or renal allograft dysfunction at all time periods after transplantation.     

Humoral Rejection of Organ Allografts

In recent years, the deleterious clinical consequences of recipient de novo alloantibody production against HLA antigens from human organ allografts have been extensively investigated. In kidney transplantation, the identification of the complement C4d fragment in peritubular capillaries as a specific marker for humoral rejection has helped to define and characterize distinct clinical alloantibody-mediated syndromes. This knowledge is relevant for patient management as new therapeutic strategies to remove and control anti-donor antibody production, particularly in the setting of acute humoral rejection, have been reported. For recipients of nonrenal organ allografts such as heart transplant recipients, de novo anti-HLA alloantibody may also be important, although more studies are needed before clear guidelines can be proposed.     

Local Complement C3 Expression is Upregulated in Humoral and Cellular Rejection of Renal Allografts

Evidence on the role of the complement system in transplantation pathology has been accelerated by the discovery of C4d as an in situ marker of antibody-mediated rejection. However, a local or systemic source of complement expression during acute rejection is under discussion. Thus, we quantitatively analyzed local RNA expression of complement component C3 as a pivotal molecule in active humoral and cellular rejection of renal allografts. After laser microdissection, real-time RT-PCR was performed for C3 using RNA extracted from tubuli and glomeruli of 68 paraffin-embedded renal allograft biopsies. Protocol and indication biopsies with signs of humoral and/or cellular rejection were investigated. Quantitative expression analysis of cytokines (IFN gamma, MCP-1, IL2, IL8) potentially influencing local C3 expression was performed. We observed a significant increase in median expression level of C3 mRNA in tubuli of C4d-positive indication biopsies, and in tubuli from indication biopsies with signs of T-cell-mediated cellular rejection. Highest expression levels were found in C4d-positive indication biopsies with signs of cellular rejection. Biopsies with upregulated C3 showed increased IFN gamma expression, suggesting allograft-infiltrating T-cells as potential stimulus for local C3 expression. Therefore, locally synthesized complement component C3 contributes to both humoral and cellular rejection, with tubular epithelial cells being a major source.     

Antibody-Mediated Rejection in Human Cardiac Allografts: Evaluation of Immunoglobulins and Complement Activation Products C4d and C3d as Markers

Antibody-mediated rejection (AMR) in human heart transplantation is an immunopathologic process in which injury to the graft is in part the result of activation of complement and it is poorly responsive to conventional therapy. We evaluated by immunofluorescence (IF), 665 consecutive endomyocardial biopsies from 165 patients for deposits of immunoglobulins and complement. Diffuse IF deposits in a linear capillary pattern greater than 2+ were considered significant. Clinical evidence of graft dysfunction was correlated with complement deposits. IF 2+ or higher was positive for IgG, 66%; IgM, 12%; IgA, 0.6%; C1q, 1.8%; C4d, 9% and C3d, 10%. In 3% of patients, concomitant C4d and C3d correlated with graft dysfunction or heart failure. In these 5 patients AMR occurred 56-163 months after transplantation, and they responded well to therapy for AMR but not to treatment with steroids. Systematic evaluation of endomyocardial biopsies is not improved by the use of antibodies for immunoglobulins or C1q. Concomitant use of C4d and C3d is very useful to diagnose AMR, when correlated with clinical parameters of graft function. AMR in heart transplant patients can occur many months or years after transplant.     

Emerging Concepts and Controversies in Renal Pathology: C4d-Negative and Arterial Lesions as Manifestations of Antibody-Mediated Transplant Rejection

The consensus classification of antibody-mediated rejection (AMR) of renal allografts developed at the Sixth Banff Conference on Allograft Pathology, in 2001, identified three findings necessary for the diagnosis of active AMR: histologic evidence, antibodies against the graft, and capillary C4d deposition. Morphologic and molecular studies have noted evidence of microvascular injury, which, in the presence of donor-specific antibodies (DSAs) but the absence of C4d deposition, is associated with development of transplant glomerulopathy and graft loss. Recent studies suggest that intimal arteritis may in some cases be a manifestation of DSA-induced graft injury. These newly recognized lesions of AMR have now been incorporated into a revised Banff diagnostic schema.     

Antibody-Mediated Rejection of a Pancreas Allograft

The role of antibody-mediated rejection (AMR) in pancreas transplantation is poorly understood. Here, we report on a patient who developed AMR of his pancreas allograft after receiving a simultaneous pancreas-kidney transplant. Pre-operative enhanced cytotoxicity and flow cytometry T-cell crossmatches were negative; B-cell crossmatches were not performed as per institutional protocol. The patient's post-operative course was significant for elevated serum amylase levels and development of hyperglycemia approximately 1 month after transplantation. A pancreatic biopsy at this time showed no cellular infiltrate but strong immunofluorescent staining for C4d in the interacinar capillaries. Analysis of the patient's serum identified donor-specific HLA-DR alloantibodies. He received intravenous immunoglobulin (IVIg), rituximab and plasmapheresis, and his pancreatic function normalized. We conclude that clinically significant AMR can develop in a pancreas allograft and recommend that pancreatic biopsies be assessed for C4d deposition if the patient has risk factors for AMR and/or the pathologic evidence for cell-mediated rejection is underwhelming.     

Analysis of Renal Transplant Protocol Biopsies in ABO-Incompatible Kidney Transplantation

Numerous studies have shown that protocol biopsies have predictive power. We retrospectively examined the histologic findings and C4d staining in 89 protocol biopsies from 48 ABO-incompatible (ABO-I) transplant recipients, and compared the results with those of 250 controls from 133 ABO-compatible (ABO-C) transplant recipients given equivalent maintenance immunosuppression. Others have shown that subclinical rejection (borderline and grade I) in ABO-C grafts decreased gradually after transplantation. In our study, however, subclinical rejection in the ABO-I grafts was detected in 10%, 14% and 28% at 1, 3 and 6–12 months, respectively. At 6–12 months, mild tubular atrophy was more common in the ABO-C grafts whereas the incidence of transplant glomerulopathy did not differ between the two groups (ABO-C: 7%; ABO-I: 15%; p = 0.57). In the ABO-I transplants, risk factors for transplant glomerulopathy in univariate analysis were positive panel reactivity (relative risk, 45.0; p < 0.01) and a prior history of antibody-mediated rejection (relative risk, 17.9; p = 0.01). Furthermore, C4d deposition in the peritubular capillaries was detected in 94%, with diffuse staining in 66%. This deposition, however, was not linked to antibody-mediated rejection. We conclude that, in the ABO-I kidney transplantation setting, detection of C4d alone in protocol biopsies might not have any diagnostic or therapeutic relevance.     

A Peripheral Blood Diagnostic Test for Acute Rejection in Renal Transplantation

Monitoring of renal graft status through peripheral blood (PB) rather than invasive biopsy is important as it will lessen the risk of infection and other stresses, while reducing the costs of rejection diagnosis. Blood gene biomarker panels were discovered by microarrays at a single center and subsequently validated and cross‐validated by QPCR in the NIH SNSO1 randomized study from 12 US pediatric transplant programs. A total of 367 unique human PB samples, each paired with a graft biopsy for centralized, blinded phenotype classification, were analyzed (115 acute rejection (AR), 180 stable and 72 other causes of graft injury). Of the differentially expressed genes by microarray, Q‐PCR analysis of a five gene‐set (DUSP1, PBEF1, PSEN1, MAPK9 and NKTR) classified AR with high accuracy. A logistic regression model was built on independent training‐set (n = 47) and validated on independent test‐set (n = 198)samples, discriminating AR from STA with 91% sensitivity and 94% specificity and AR from all other non‐AR phenotypes with 91% sensitivity and 90% specificity. The 5‐gene set can diagnose AR potentially avoiding the need for invasive renal biopsy. These data support the conduct of a prospective study to validate the clinical predictive utility of this diagnostic tool.     

Acute Antibody-Mediated Rejection Following Heart Transplantation

Acute antibody-mediated rejection (AMR) in heart transplantation is often associated with hemodynamic compromise, and is associated with increased mortality and development of accelerated transplant coronary artery disease (TCAD). The diagnosis of AMR has historically been controversial and outcomes with aggressive immunosuppressive therapy including plasmapheresis and cyclophosphamide are poor. Advances in diagnostic techniques like the demonstration of immunopathologic evidence for antibody-mediated rejection by deposition of the complement split product C4d in tissue and detection of anti-HLA antibodies by flow cytometry will assist in further characterizing AMR. Immunosuppression targeting B-lymphocytes and use of m-TOR inhibitors to alter the predilection to develop TCAD and improve survival in AMR remains to be proven.     

Determinants of Poor Graft Outcome in Patients with Antibody-Mediated Acute Rejection

This study analyzes the incidence and course of antibody-mediated rejection (AMR) in a cohort of 237 renal transplant patients followed for 30 +/- 20 months. Among these, 32 patients were considered to be at risk for AMR and received intravenous immunoglobulin (IVIg), either as preconditioning (Group A, n = 18) or at the time of transplant (Group B, n = 14). The prevalence of AMR was 27.8% in Group A, 57.1% in Group B and 3.9% in the remainder of the population. Although graft loss remains greater among AMR than for acute cellular rejection (ACR) or the overall transplant population, we have identified a good outcome group (GFR > 15 mL/min/1.73 m(2)) (n = 13), whose renal function at the end of follow-up was comparable to that of the general transplant population. The factors associated with bad outcome are: (1) immunologic: presence and/or persistence of donor-specific anti-HLA antibodies post-transplantation and (2) histologic: neutrophilic glomerulitis, peritubular capillary dilatation with neutrophil infiltrates and interstitial edema at the time of first biopsy; and at the time of late biopsy (3-6 months): lesions of vascular rejection, and monocyte/macrophage infiltrates in glomeruli and dilated peritubular capillaries. Persistence of C4d does not predict outcome. This study outlines for the first time the immunologic and histologic profiles of AMR patients with poor prognosis.     

CD44分子在肾移植患者急性排斥反应肾组织中的表达及意义

目的:探讨CD44分子与肾移植急性排斥反应的关系。方法:回顾分析2005年7月～2009年5月间肾移植术后穿刺病理活检证实为急性排斥反应患者28例的CD44在移植肾组织中的表达。结果:28例急性排斥反应肾组织中有25例CD44呈阳性表达,阳性率为89.29%;10例慢性排斥反应肾组织中有3例阳性表达,阳性率为30%;30例正常肾脏组织中5例CD44分子的阳性表达,阳性率为16.7%;两两比较,结果差异有统计学意义(P0.05)。结论:CD44与其与配体的相互作用在移植肾急性排斥反应中可能起到重要作用,CD44分子有可能成为一个特异性和敏感性都较好的早期诊断急性排斥的预测因子。     

Characterization of Urinary Peptide Biomarkers of Acute Rejection in Renal Allografts

We previously demonstrated that 4.7 kDa and 4.4 kDa peptides are useful in diagnosing acute rejection in renal transplant recipients. The aim of this study was to characterize these polypeptides and assess their potential as biomarkers. The polypeptides were identified as human beta-Defensin-1 (4.7 kDa) and alpha-1-antichymotrypsin (4.4 kDa), by tandem mass spectrometry and ProteinChip immunoassay. The urinary abundance of both polypeptides, assessed using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), revealed a reduction in beta-Defensin-1 while alpha-1-antichymotrypsin increased in patients with rejection (p < 0.05) compared with clinically stable transplants. The area under the curve (AUC) for the receiver operator characteristic (ROC) curve for the diagnosis of rejection for the ratio of both peptides combined was 0.912. Longitudinal analysis confirmed a reduction in beta-Defensin-1 with a reciprocal increase in alpha-1-antichymotrypsin as rejection developed. The difference in urinary beta-Defensin-1 levels quantified by radioimmunoassay was 176.8 +/- 122.3 pg/mL in stable patients compared with 83.2 +/- 52.2 pg/mL in patients with acute rejection, with an ROC AUC of 0.749 (p < 0.01). Immunohistochemistry (IHC) confirmed reduced beta-Defensin-1 expression in the renal parenchyma of patients experiencing acute rejection. In conclusion, the ratio of beta-Defensin-1 and alpha-1-antichymotrypsin excretion in the urine is a novel, potentially useful candidate biomarkers of acute rejection.     

Early Ultrastructural Changes in Renal Allografts: Correlation With Antibody‐Mediated Rejection and Transplant Glomerulopathy

Transplant glomerulopathy (TG) is associated with antibody‐mediated renal allograft rejection (AMR) and reduced graft survival. Histologically, TG is typically seen >1 year posttransplantation. However, ultrastructural changes including glomerular endothelial swelling, subendothelial widening and early glomerular basement membrane duplication are associated with development of TG but appear much earlier. We examined the specificity of these changes for AMR, and whether these are inevitably associated with development of TG. Of 98 for cause renal allograft biopsies carried out within 3 months of transplantation with available serologic data, 17 showed C4d‐positive AMR and 16 had histologic changes of AMR and donor‐specific antibodies (DSA), but no C4d. All three ultrastructural changes were seen in 11 of 17 biopsies with C4d‐positive AMR, 8 of 16 with histologic changes of AMR and DSA but no C4d, and 0 of 65 without histologic changes of AMR and/or DSA (p < 0.0001 for both of the former groups vs. the latter). Twenty patients with positive DSA (18 with histologic changes of AMR and 11 C4d‐positive) had ≥1 follow‐up biopsy; eight developed overt TG 3.5–30 months posttransplantation. Among the 18 patients with DSA and histologic changes of AMR, 11 C4d‐positive and 7 C4d‐negative, treatment for AMR after the early biopsy significantly reduced subsequent development of overt TG.     

The Spectrum of Antibody-Mediated Renal Allograft Injury: Implications for Treatment

Improvements in anti-HLA antibody detection and diagnostic criteria have increased recognition of antibody-mediated rejection (AMR) following renal transplantation. Therapy of acute AMR is directed toward rapidly lowering circulating donor-specific antibody (DSA) activity. Despite reversal of acute renal dysfunction, however, antibody-secreting plasma cells in spleen and bone marrow are not depleted by treatment and circulating DSA commonly remains detectable in peripheral blood. Sequential ultrastructural studies of renal allografts during acute AMR show progression of microvascular endothelial abnormalities from necrosis and apoptosis to glomerular and peritubular capillary basement membrane duplication, termed transplant glomerulopathy (TG), a manifestation of chronic AMR. Additionally, long-term exposure to anti-HLA antibodies (particularly against class II antigens) is associated with shortened allograft survival and TG even in the absence of documented acute AMR. The association of TG with prior acute AMR and with circulating DSA provides evidence that antibody-mediated allograft injury exists as a spectrum of renal injury. Although effective therapy is available for acute AMR, allografts remain at risk for chronic AMR and shortened survival. The optimum approach to treatment for chronic AMR remains to be determined.     

Association of CD20+ Infiltrates with Poorer Clinical Outcomes in Acute Cellular Rejection of Renal Allografts

We undertook a study to ascertain the relationship between the presence of CD20-positive B-lymphocytes in renal allografts undergoing acute cellular rejection and graft survival. We identified 27 patients transplanted between January 1, 1998 and December 31, 2001, with biopsy-proven Banff 1-A or Banff 1-B rejection in the first year after transplantation, and stained the specimens for CD20 and C4d. At least 4 years of follow-up data were available for each patient studied. Six patients had CD20-positive B-cell clusters in the interstitium, and 21 patients were negative for CD20 infiltrates. The CD20-positive group was significantly more likely to have steroid-resistant rejection and reduced graft survival compared to CD20-negative controls. This study supports prospective identification of CD20-positive B-cell clusters in biopsy-proven rejection and offers a therapeutic rationale for a trial of monoclonal anti-CD20 antibody in such patients.     

Transplant Glomerulopathy: Subclinical Incidence and Association with Alloantibody

Transplant glomerulopathy (TG) usually has been described as part of a constellation of late chronic histologic abnormalities associated with proteinuria and declining function. The current study used both protocol and clinically-indicated biopsies to investigate clinical and subclinical TG, their prognosis and possible association with alloantibody. We retrospectively studied 582 renal transplants with a negative pre-transplant T-cell complement dependent cytotoxicity crossmatch. TG was diagnosed in 55 patients, 27 (49%) based on protocol biopsy in well-functioning grafts. The cumulative incidence of TG increased over time to 20% at 5 years. The prognosis of subclinical TG was equally as poor as TG diagnosed with graft dysfunction, with progressive worsening of histopathologic changes and function. Although TG was associated with both acute and chronic histologic abnormalities, 14.5% of TG biopsies showed no interstitial fibrosis or tubular atrophy, while 58% (7/12) of biopsies with severe TG showed only minimal abnormalities. TG was associated with acute rejection, pretransplant hepatitis C antibody positivity and anti-HLA antibodies (especially anti-Class II), with the risk increasing if the antibodies were donor specific. We suggest that subclinical TG is an under-recognized cause of antibody-mediated, chronic renal allograft injury which may be mechanistically distinct from other causes of nephropathy.     

Subclinical Rejection Phenotypes at 1 Year Post-Transplant and Outcome of Kidney Allografts

Kidney allograft rejection can occur in clinically stable patients, but long-term significance is unknown. We determined whether early recognition of subclinical rejection has long-term consequences for kidney allograft survival in an observational prospective cohort study of 1307 consecutive nonselected patients who underwent ABO-compatible, complement-dependent cytotoxicity-negative crossmatch kidney transplantation in Paris (2000–2010). Participants underwent prospective screening biopsies at 1 year post-transplant, with concurrent evaluations of graft complement deposition and circulating anti-HLA antibodies. The main analysis included 1001 patients. Three distinct groups of patients were identified at the 1-year screening: 727 (73%) patients without rejection, 132 (13%) patients with subclinical T cell-mediated rejection (TCMR), and 142 (14%) patients with subclinical antibody-mediated rejection (ABMR). Patients with subclinical ABMR had the poorest graft survival at 8 years post-transplant (56%) compared with subclinical TCMR (88%) and nonrejection (90%) groups (P<0.001). In a multivariate Cox model, subclinical ABMR at 1 year was independently associated with a 3.5-fold increase in graft loss (95% confidence interval, 2.1 to 5.7) along with eGFR and proteinuria (P<0.001). Subclinical ABMR was associated with more rapid progression to transplant glomerulopathy. Of patients with subclinical TCMR at 1 year, only those who further developed de novo donor-specific antibodies and transplant glomerulopathy showed higher risk of graft loss compared with patients without rejection. Our findings suggest that subclinical TCMR and subclinical ABMR have distinct effects on long-term graft loss. Subclinical ABMR detected at the 1-year screening biopsy carries a prognostic value independent of initial donor-specific antibody status, previous immunologic events, current eGFR, and proteinuria.     

The Significance of Subclinical Rejection and the Value of Protocol Biopsies

Subclinical rejection (SCR) is diagnosed by protocol histology with a maximal prevalence occurring early after transplantation, falling to low levels by 1 year. Needle-core biopsy is safe, and the histology obtained fairly reflects subclinical immune activity. Several studies have consistently shown that SCR is associated with chronic tubulointerstitial damage, subsequent renal dysfunction and reduced graft survival. SCR is effectively treated by pulse corticosteroid therapy, although increased baseline immunosuppression may be necessary. A single randomized clinical trial of biopsy and corticosteroid therapy demonstrated significantly improved early structural and functional outcomes, and a (nonsignificant) 17% risk reduction in 4-year graft survival. Three possible approaches include: no protocol biopsies (usually accompanied by powerful immunosuppression); biopsies only in high-risk recipients (who may be difficult to reliably predict) or universal screening protocol biopsy (comprehensive but limited by cost and resource utilization). The appropriate screening methodology for a transplant unit is both a clinical and an economic decision; influenced by the SCR prevalence and potential gains of treatment, against costs and resource utilization. Further trials to quantify the cost-benefit balance in a typical, heterogeneous recipient population using modern immunosuppression are required.