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.     

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.     

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.     

Essential Role of Sphingosine-1-Phosphate Receptor 1-Bearing CD8+CD44+CCR7+ T Cells in Acute Skin Allograft Rejection

A subset of naturally formed sphingosine‐1‐phosphate receptor 1 (S1P1)‐bearing CD8⁺CD44⁺CCR7⁺ memory T cells has been identified in transplant recipient BALB/c (H‐2^d) mice. The frequency of this subset of memory T cells is significantly increased in the spleen, lymph nodes and skin grafts in the recipient BALB/c mice during acute skin allograft rejections. The immune‐reconstitution with CD8⁺CD44⁺CCR7⁺S1P1⁺ memory T cells facilitates acute skin allograft rejection in SCID mice. Being Th1‐polarized and cytotoxic, CD8⁺CD44⁺CCR7⁺S1P1⁺ memory T cells proliferate and differentiate immediately into effectors upon encountering allo‐antigens. A siRNA against S1P1 inhibits CD8⁺CD44⁺CCR7⁺S1P1⁺ memory T cell‐mediated acute skin allograft rejection in SCID mice by means of knocking‐down S1P1‐expression. CCL21 mutant (CCL21‐ΔCT) has been used to compete with wild‐type CCL21 in the course of binding to CCR7. Combined administration of siRNA S1P1 and CCL21‐ΔCT significantly prolongs the survival of skin allograft in the recipient BALB/c mice by means of inhibiting accumulation of CD8⁺CD44⁺CCR7⁺S1P1⁺ memory T cells in the spleen and the skin grafts. Our data provide direct evidence that S1P1 and CCR7 are involved in the proliferation and trafficking of CD8⁺CD44⁺CCR7⁺S1P1⁺ memory T cells. S1P1 may serve as a functional marker for CD8⁺CD44⁺CCR7⁺ memory T cells. Targeting CD8⁺CD44⁺CCR7⁺S1P1⁺ T cells may be a useful strategy to prolong the survival of allograft transplant.     

Acute Humoral Rejection in Hepatitis C-Infected Renal Transplant Recipients Receiving Antiviral Therapy

The use of interferon-alpha (IFN) in hepatitis C (HCV)-infected renal recipients has been associated with acute rejection and graft loss. We reviewed our recent experience in HCV (+) renal recipients treated with antiviral therapy for biopsy proven chronic hepatitis C. Twelve HCV (+) recipients who recently received antiviral therapy were analyzed. Post-treatment sera were tested for donor-specific human leukocyte antigen (HLA) antibodies (DSA). Within 6 months of initiating antiviral therapy, two of 12 patients (17%) developed acute rejection, which was characterized as acute humoral rejection (de novo DSA in serum and C4d deposits in peritubular capillaries). Both progressed to graft failure. Nine of the remaining 10 patients tested did not have DSA. The use of IFN was associated with severe acute humoral rejection (C4d +, DSA +). The recognition of IFN-associated acute humoral rejection in this series may explain the high rate of graft loss reported previously in renal recipients receiving IFN.     

Acute Humoral Rejection in an ABO Compatible Combined Liver–Kidney Transplant—The Kidney Is Not Always Protected

Combined liver–kidney transplantation has become a common practice for the treatment of patients with concurrent end-stage renal disease and end-stage liver disease. Liver transplantation in the setting of multiorgan transplantation is thought to have a protective effect against humoral rejection even when a positive crossmatch is obtained prior to surgery. In most centers, a pre liver–kidney transplant crossmatch is rarely performed because of the known immunoprotective effect of the liver allograft. In this report, a case of acute humoral rejection in the kidney allograft after a combined liver–kidney transplant is described. Although humoral rejection was treated using plasmapheresis, intravenous immunoglobulin and rituximab, the kidney required 3 months to recover function and finally progressed to chronic allograft nephropathy. A heightened index of suspicion for acute humoral rejection of the renal allograft is necessary when performing combined liver–kidney transplants to highly sensitized patients due to previous organ transplants.     

Urinary miR‐210 as a Mediator of Acute T‐Cell Mediated Rejection in Renal Allograft Recipients

MicroRNAs (miRNAs) are small ribonucleotides regulating gene expression. Circulating miRNAs are remarkably stable in the blood. We tested whether miRNAs are also detectable in urine and may serve as new predictors of outcome in renal transplant patients with acute rejection. We profiled urinary miRNAs of stable transplant patients and transplant patients with acute rejection. The miR‐10a, miR‐10b and miR‐210 were strongly deregulated in urine of the patients with acute rejection. We confirmed these data in urine of a validation cohort of 62 patients with acute rejection, 19 control transplant patients without rejection and 13 stable transplant patients with urinary tract infection by quantitative RT‐PCR. The miR‐10b and miR‐210 were downregulated and miR‐10a upregulated in patients with acute rejection compared to controls. Only miR‐210 differed between patients with acute rejection when compared to stable transplant patients with urinary tract infection or transplant patients before/after rejection. Low miR‐210 levels were associated with higher decline in GFR 1 year after transplantation. Selected miRNAs are strongly altered in urine of the patients with acute renal allograft rejection. The miR‐210 levels identify patients with acute rejection and predict long‐term kidney function. Urinary miR‐210 may thus serve as a novel biomarker of acute kidney rejection.     

Subclinical Acute Antibody-Mediated Rejection in Positive Crossmatch Renal Allografts

Subclinical antibody-mediated rejection (AMR) has been described in renal allograft recipients with stable serum creatinine (SCr), however whether this leads to development of chronic allograft nephropathy (CAN) remains unknown. We retrospectively reviewed data from 83 patients who received HLA-incompatible renal allografts following desensitization to remove donor-specific antibodies (DSA). Ten patients had an allograft biopsy showing subclinical AMR [stable SCr, neutrophil margination in peritubular capillaries (PTC), diffuse PTC C4d, positive DSA] during the first year post-transplantation; 3 patients were treated with plasmapheresis and intravenous immunoglobulin. Three patients had a subsequent rise in SCr and an associated biopsy with AMR; 5 others showed diagnostic or possible subclinical AMR on a later protocol biopsy. One graft was lost, while remaining patients have normal or mildly elevated SCr 8-45 months post-transplantation. However, the mean increase in CAN score (cg + ci + ct + cv) from those biopsies showing subclinical AMR to follow-up biopsies 335 +/- 248 (SD) days later was significantly greater (3.5 +/- 2.5 versus 1.0 +/- 2.0, p = 0.01) than that in 24 recipients of HLA-incompatible grafts with no AMR over a similar interval (360 +/- 117 days), suggesting that subclinical AMR may contribute to development of CAN.     

Detection of Acute Tubulointerstitial Rejection by Proteomic Analysis of Urinary Samples in Renal Transplant Recipients

This study investigates proteomic analysis of urinary samples as a non-invasive method to detect acute rejection of renal allografts. Capillary electrophoresis coupled to mass spectrometry (CE-MS) was used to analyze urinary samples in 19 patients with different grades of subclinical or clinical acute rejection (BANFF Ia to IIb), 10 patients with urinary tract infection and 29 patients without evidence of rejection or infection. A distinct urinary polypeptide pattern identified 16 out of 17 cases of acute tubolointerstitial rejection, but was absent in two cases of vascular rejection. Urinary tract infection resulted in a different polypeptide pattern that allowed to differentiate between infection and acute rejection in all cases. Potentially confounding variables such as acute tubular lesions, tubular atrophy, tubulointerstitial fibrosis, calcineurin inhibitor toxicity, proteinuria, hematuria, allograft function and different immunosuppressive regimens did not interfere with test results. Blinded analysis of samples with and without rejection showed correct diagnosis by CE-MS in the majority of cases. Detection of acute rejection by CE-MS offers a promising non-invasive tool for the surveillance of renal allograft recipients. Further investigation is needed to establish polypeptide patterns in vascular rejection and to explore whether changes in the urinary proteome occur before the onset of histologically discernible rejection.     

Acute Humoral Rejection of Human Lung Allografts and Elevation of C4d in Bronchoalveolar Lavage Fluid

Antibody-mediated rejection is well established for renal allografts but remains controversial for lung allografts. Cardinal features of antibody-mediated rejection in renal allografts include antibodies to donor human leukocyte antigen (HLA) and evidence for antibody action, such as complement activation demonstrated by C4d deposition. We report a lung allograft recipient with circulating antibodies to donor HLA who failed treatment for acute cellular rejection but responded to therapy for humoral rejection. To address the second criteria for antibody-mediated rejection, we determined whether complement activation could be detected by measuring C4d in bronchoalveolar lavage fluid (BALF) by ELISA. Airway allergen challenge of asthmatics activates the complement pathway; therefore, we used BALF from asthmatics pre- and post-allergen challenge to measure C4d. These controls demonstrated that ELISA could detect increases in C4d after allergen challenge. BALF from the index patient had elevated C4d concomitant with graft dysfunction and anti-donor HLA in the absence of infection. Analysis of BALF from 25 additional lung allograft recipients showed that C4d concentrations >100 ng/mL were correlated with anti-HLA antibodies (p = 0.006), but were also observed with infection and in asyptomatic patients. The findings support the occurrence of anti-HLA-mediated lung allograft rejection and suggest that C4d measurement in BALF may be useful in diagnosis.     

Chronic Humoral Rejection of Human Kidney Allografts Is Associated with MMP‐2 Accumulation in Podocytes and its Release in the Urine

Chronic humoral rejection (CHR) is an important cause of late graft failures following kidney transplantation. Overall, the pathophysiology of CHR is poorly understood. Matrix metalloproteinase‐2 (MMP‐2), a type IV collagenase, has been implicated in chronic kidney disease and allograft rejection in previous studies. We examined the presence of MMP‐2 in allograft biopsies and in the urine of kidney transplant recipients with CHR. MMP‐2 staining was detected by immunohistochemistry in podocytes for all CHR patients but less frequently in patients with other renal complications. Urinary MMP‐2 levels were also significantly higher in CHR patients (median 4942 pg/mL, N = 27) compared to non‐CHR patients (median 598 pg/mL, N = 65; p < 0.001). Elevated urinary MMP‐2 correlated with higher levels of proteinuria in both CHR and non‐CHR patients. Longitudinal analysis indicated that increase in urine MMP‐2 coincided with initial diagnosis of CHR as documented by the biopsies. Using an enzymatic assay, we demonstrated that MMP‐2 was present in its active form in the urine of patients with CHR. Overall, our findings associate MMP‐2 with glomerular injury as well as interstitial fibrosis and tubular atrophy observed in patients with CHR.     

Lack of Improvement in Renal Allograft Survival Despite a Marked Decrease in Acute Rejection Rates Over the Most Recent Era

Acute rejection is known to have a strong impact on graft survival. Many studies suggest that very low acute rejection rates can be achieved with current immunosuppressive protocols. We wanted to investigate how acute rejection rates have evolved on a national level in the U.S. and how this has impacted graft survival in the most recent era of kidney transplantation. For this purpose, we analyzed data provided by the Scientific Registry of Transplant Recipients regarding all adult first renal transplants between 1995 and 2000. We noted a significant decrease in overall acute rejection rates during the first 6 months, during the first year, and also in late rejections during the second year after transplantation. Despite this decrease in the rate of acute rejection, there was no significant improvement in overall graft survival; furthermore, we noted a statistically significant trend towards worse death-censored graft survival. There was also a trend for a greater proportion of rejection episodes to fail to recover to previous baseline function after treatment. Our data suggest that decreasing acute rejection rates between 1995 and 2000 have not led to an increase in long-term graft survival. Part of this discordance might be related to a higher proportion of acute rejections which have not resolved with full functional recovery in more recent years. However, the etiology of this concerning trend for worse death censored graft survival in recent years will warrant further investigation.     

IL‐17 Expression by Tubular Epithelial Cells in Renal Transplant Recipients with Acute Antibody‐Mediated Rejection

Acute rejection is still a common complication of kidney transplantation. IL‐17 is known to be associated with allograft rejection but the cellular source and the role of this cytokine remains unclear. We investigated IL‐17 graft expression in renal transplant recipients with acute antibody‐mediated rejection (ABMR), acute T‐cell‐mediated rejection (TCMR), interstitial fibrosis and tubular atrophy (IFTA) and acute tubular damage due to calcineurin‐inhibitor toxicity (CNI). In acute ABMR, tubular IL‐17 protein expression was significantly increased compared to TCMR, where most of the IL‐17⁺cells were CD4⁺graft infiltrating lymphocytes, IFTA and CNI control groups. The tubular expression of IL‐17 in acute ABMR colocalized with JAK2 phosphorylation and peritubular capillaries C4d deposition. In addition, IL‐17 tubular expression was directly and significantly correlated with the extension of C4d deposits. In cultured proximal tubular cells, C3a induced IL‐17 gene and protein expression along with an increased in JAK2 phosphorylation. The inhibition of JAK2 abolished C3a‐induced IL‐17 expression. The use of steroids and monoclonal antibodies reduced IL‐17 expression, JAK2 phosphorylation and C4d deposition in acute ABMR patients. Our data suggest that tubular cells represent a significant source of IL‐17 in ABMR and this event might be mediated by the complement system activation featuring this condition.     

The Impact of Cytomegalovirus Infection and Disease on Rejection Episodes in Renal Allograft Recipients

Cytomegalovirus (CMV) infection and disease are potential risk factors for acute allograft rejection in renal transplant recipients. The present study specifically addresses this issue. From October 1994 to July 1997, 477 consecutive renal allograft recipients (397 first transplants and 80 retransplants) were included in the study. CMV infection (cytomegalovirus pp65 antigen in leukocytes) and disease (infection and clinical symptoms or signs of disease) were examined prospectively for 3 months. No CMV prophylaxis was given, and CMV disease was treated with intravenous (i.v.) ganciclovir. The retransplantation of four patients transplanted twice during the study and 22 patients receiving kidneys from human leucocyte antigen (HLA)-identical siblings were excluded from statistical analysis. Rejections were evaluated clinically [277(61%)] and 173 (38%) also had a biopsy verified rejection. CMV infection occurred in 64% of the patients and 24% experienced CMV disease. In a multiple time-dependent Cox analysis, CMV infection and CMV disease were independent significant predictors for clinical acute rejections, RR = 1.6 (1.1-2.5, p = 0.02) and RR = 2.5 (1.2-5.1, p = 0.01), respectively. Among 173 patients with biopsy verified rejection, 72% of the patients had tubulointerstitial rejection whereas 28% had a vascular rejection. CMV disease, but not CMV infection was a predictor of tubulointerstitial rejection, RR = 3.1 (1.1-9.3, p = 0.04). CMV infection and disease are independent risk factors for clinical acute rejection in kidney allograft recipients. CMV disease is an independent risk factor for biopsy verified acute tubulointerstitial rejection in kidney allograft recipients.     

Prolonged Hypothermia Causes Primary Nonfunction in Preserved Canine Renal Allografts due to Humoral Rejection

Canine kidney preservation models have historically used autotransplants to avoid the complications of rejection, although clinically all transplants are allografts. This study investigated the effects of preservation time and method on early kidney function in a canine allograft vs. autograft model. Kidneys were harvested from beagles, preserved by cold storage (CS) in UW solution for 0, 24 or 72 h, or by machine perfusion (MP) with Belzer MPS for 72 h. In some experiments 45 min of warm ischemia (WI) was performed in situ before harvest. Allograft recipients received steroid immunosuppression. Kidney function was assessed by serum creatinine and survival for 7 days. Allografts preserved for 0 and 24 h performed as well as autografts. Allografts preserved for 72 h by either CS or MP had a higher incidence of primary nonfunction (PNF) compared with autografts, as determined by survival (50% vs. 100%, p < 0.003). Primary nonfunction kidneys had thrombotic microangiopathy, vascular and peritubular capillary binding of IgM and complement C4d, and evidence of circulating donor-specific antibodies; all consistent with humoral rejection. These responses were dependent on hypothermia time and were not attributable to ischemia, immunosuppression, preservation solution, or cellular rejection. In conclusion, prolonged hypothermia can cause PNF in allografts owing to acute humoral rejection.     

Role of Integrin CD103 in Promoting Destruction of Renal Allografts by CD8+ T Cells

Infiltration of CD8(+)TCRalphabeta(+) T-effector populations (CD8 effectors) into graft epithelial compartments has long been recognized as a key lesion in progression of clinical renal allograft rejection. While the afferent phase of allograft immunity is increasingly well-defined, the efferent pathways by which donor-reactive CD8-effector populations access and ultimately destroy the graft renal tubules (rejection per se) have received remarkably little attention. This is an important gap in our knowledge of transplantation immunology, because epithelial compartments comprise the functional elements of most commonly transplanted organs including not only kidney, but also liver, lung, pancreas, and intestine. Furthermore, there is increasing evidence that attack of graft epithelial elements by CD8-effector populations not only causes short-term graft dysfunction but is also a major contributor to development of chronic allograft nephropathy and late graft loss, which now represent the salient clinical problems. Recent studies of the T-cell integrin, alpha(E)beta(7) (CD103), have provided insight into the mechanisms that promote interaction of CD8 effectors with graft epithelial compartments. The purpose of this communication is to review the known properties of the CD103 molecule and its postulated role in the efferent phase of renal allograft rejection.     

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.     

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。