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.     

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.     

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.     

Ultrastructural Examination of Glomerular and Tubular Changes in Renal Allografts with Acute Rejection

Acute rejection is the most important threat to transplanted kidneys in the early phase after transplantation. With the advances in renal transplant surgery and immunosuppressive therapies, one-year graft survival rates reached 90%, but long-term graft survival did not improve to a similar degree. To prevent acute rejection more effectively and decrease the risk of chronic nephropathy development, the pathogenesis and effects of acute rejection on renal grafts should be further explored.

This study aimed to examine the glomerular and tubular changes ultrastructurally. Tissues were obtained from 11 renal allografts with acute rejection, fixed in 1% Osmium tetra oxide embedded in Epon. The changes in glomerular basement membrane, podocyte, mesangium, and proximal tubules were examined by electron microscope.

Tubular changes such as tubular basement membrane multi-lamellation, MN and PMN cells in peritubular capillaries, tubular vacuolization, mitochondrial changes (increase in number, alterations in cristae organization, or cristae effacement), and infiltration of tubular epithelium by MN cells (mainly lymphocytes) were found statistically significant (p < 0.01) when compared to those of control group. Some forms of endothelial injury (swelling of endothelial cells or fenestrae loss) were also statistically significant (p < 0.01).

Acute rejection is an important predictor of long-term graft survival, and there may be no clinical clue to make diagnosis easier. Therefore ultrastructural changes may help solve this problem together with molecular studies.     

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。     

Paclitaxel Impairs Endothelial Cell Adhesion But Not Cytokine-Induced Cellular Adhesion Molecule Expression

Local delivery of antiproliferative agents using drug-eluting stents has become a productive area of research for preventing in-stent restenosis. Recently, the microtubule stabilizing drug paclitaxel has been used to coat stents. While the actions of paclitaxel on smooth muscle are well documented, effects on endothelial cells (ECs) are largely unknown. Nevertheless, restoration of EC function is a critical step in repairing the vascular lesion. We assessed the effects of paclitaxel by examining three events that are critical in controlling the severity of vascular injury: (1) adhesion of ECs to matrix proteins, (2) EC migration, and (3) cytokine-stimulated cellular adhesion molecule (CAM) expression on the surface of ECs. Paclitaxel inhibited both EC adhesion and migration of ECs; however, it had no effect on tumor necrosis-stimulated CAM expression on ECs. The mechanisms of paclitaxel action on matrix adhesion and migration are not clear, but protein kinase C and myosin light chain kinase do not appear to play a role as they are unaffected by treatment of the cells with paclitaxel. On the other hand, the MAP kinase ERK1/2 is modestly inhibited by paclitaxel. While paclitaxel-coated endovascular stents may prevent smooth muscle proliferation, their attenuation of EC migration and adhesion to the lesion coupled with an inability to reduce cytokine-induced CAM expression on ECs may limit their effectiveness.     

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.     

Indoleamine 2,3-Dioxygenase and Foxp3 Expression in Skin Rejection of Human Hand Allografts

Human hand transplantation is complicated by skin rejection. To better define the characteristics of infiltrating cells, biopsies from human hand transplants have been investigated for expression of Foxp3 and indoleamine 2,3-dioxygenase (IDO), a key regulatory enzyme to induce T-lymphocyte unresponsiveness. A total of 104 skin biopsies taken from three bilateral hand transplant recipients over 6 years posttransplant were assessed by hematoxylin-eosin histology (graded 1-4b) and immunohistochemistry for IDO and Foxp3 according to a three-grade classification and correlated with the grade of rejection as well as time after transplantation. Overall, rejection ranged between grades 0 and 4a with an average score of 0.94. IDO was expressed in the endothelium independent of rejection. Upon rejection, IDO staining within the cellular infiltrate was significantly increased. Foxp3 in regulatory T cells was mainly found in samples undergoing severe rejection. Expression of IDO and Foxp3 compared well to each other, although the overall expression of Foxp3 was lower when compared to IDO. An increased expression of IDO as well as Foxp3 during rejection late after transplantation was observed. Characteristics of the cellular infiltrate indicate tolerogenic properties of a proportion of the cells and therefore a tendency toward self-limitation of the alloimmune response during skin rejection after hand transplantation.     

Acute Rejection of Small Intestine Allografts Is Associated With Increased Expression of Toll-like Receptors

Although outcomes after intestinal transplantation have steadily improved owing to advances in immunosuppressive therapy, operative techniques, and postoperative medical management, rejection of the intestinal allograft continues to be a major clinical problem and constitutes the primary reason for graft loss. Although the adaptive immune system has been the major focus of investigation regarding regulation of rejection of the intestinal allograft, the role of the innate immune system has recently become of increased interest. We hypothesized that microbial products of the microflora associated with the intestinal allograft may engage the Toll-like receptor pathway of the innate immune system to potentiate alloimmune responses and rejection of the allograft. To investigate this, we established a murine model for orthotopic intestinal transplantation and allograft rejection. Using this model, we show that the expression of Toll-like receptor 2 is increased 50-fold and the expression of Toll-like receptor 4 is increased 200-fold during rejection of the allograft. We then performed survival studies that showed increased survival of mice, which had the Toll-like receptor knocked out. These preliminary studies suggest an important role for in innate immune system in acute rejection of the small intestinal allografts, and as such represents an emerging and promising area of investigation.     

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.     

Interleukin 1 and Chronic Rejection: Possible Genetic Links in Human Heart Allografts

Chronic rejection is a leading cause of graft loss in thoracic transplant recipients. Studies on the pathogenesis of chronic rejection have suggested a contributory role for certain cytokines and growth factors. The activity of these mediators is subject to genetic variation if a polymorphism alters expression, or function, of the ligand or its receptor. Here we have asked if certain cytokine and growth factor gene polymorphisms correlate with chronic rejection in recipients of thoracic allografts. In a retrospective analysis of 179 recipients of thoracic organ transplants (128 heart; 36 heart-lung; and 15 lung), polymorphisms in 8 genes that influence the inflammatory process, namely IL1B, IL1R1, IL1RN, IL6, IL10, TNFA, TGFB1 and FCGRIIA, were examined. Genotypic data from recipients who had either died or been re-transplanted as a result of chronic rejection (n = 96) were then compared to those of recipients who had a functioning graft for more than 11 years (n=83). In the heart graft recipients, only those polymorphisms that influenced expression of the IL1 receptor antagonist gene had a significant correlation with graft survival, with homozygosity for the IL1RN*1 allele being associated with rejection. The alternative, less frequent IL1RN alleles emerged as genomic predictors of long-term allograft survival. This association was especially strong when IL1 region haplotypes were considered, particularly when analysis was confined to heart transplant recipients who had had multiple acute rejection episodes (OR>20). This case-control study indicates that gene polymorphisms which influence IL1 bioactivity also influence the progression of chronic rejection in heart grafts.     

活化白细胞黏附分子在IgA肾病中的表达及意义

目的:探讨活化白细胞黏附分子(CD166)在IgA肾病中的表达及与IgA肾病临床病理特征的关系.方法:用免疫组化二步法检测CD166蛋白在79例IgA肾病患者肾组织的表达.结果:CD166在IgA肾病患者肾小管、间质普遍表达,按Lee分级的级别增加逐渐增高.与单纯性血尿患者(A组)相比,混合蛋白尿、血尿患者(B组)肾小管间质CD166表达明显增高.肾小管间质CD166表达与患者病理分级、小管间质病变、单个核细胞浸润程度及血清C反应蛋白(CRP)水平正相关.结论:CD166在IgA肾病中表达异常,在IgA肾病小管、间质病变及疾病进展中起一定作用.     

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.     

Acute Humoral Rejection of Renal Allografts in CCR5–/– Recipients

Increasing detection of acute humoral rejection (AHR) of renal allografts has generated the need for appropriate animal models to investigate underlying mechanisms. Murine recipients lacking the chemokine receptor CCR5 reject cardiac allografts with marked C3d deposition in the parenchymal capillaries and high serum donor-reactive antibody titers, features consistent with AHR. The rejection of MHC-mismatched renal allografts from A/J (H-2^a) donors by B6.CCR5^–/– (H-2^b) recipients was investigated . A/J renal allografts survived longer than 100 days in wild-type C57BL/6 recipients with normal blood creatinine levels (28 ± 7 μmol/L). All CCR5^–/– recipients rejected renal allografts within 21 days posttransplant (mean 13.3 ± 4 days) with elevated creatinine (90 ± 31 μmol/L). The rejected allografts had neutrophil and macrophage margination and diffuse C3d deposition in peritubular capillaries, interstitial hemorrhage and edema, and glomerular fibrin deposition. Circulating donor-reactive antibody titers were 40-fold higher in B6.CCR5^–/– versus wild-type recipients. Depletion of recipient CD8 T cells did not circumvent rejection of the renal allografts by CCR5-deficient recipients. In contrast, μMT^–/–/CCR5^–/– recipients, incapable of producing antibody, did not reject most renal allografts. Collectively, these results indicate the rapid rejection of renal allografts in CCR5^–/– recipients with many histopathologic features observed during AHR of human renal allografts.     

MP84 Expression in the Urine Specimens of Acute Rejection Renal Transplant Recipients

MP84 is a novel protein synthesized in response to all cytokines. This antigen is expressed only in stimulated mesangial cells and decreased kidney sections, but not in the normal kidney sections (1,2). This study was performed to determine the excretion of MP84 in the urine of renal transplant recipients with acute rejection. Six persons with renal transplant acute rejection and 10 healthy persons were included. Two urine specimens from each person were collected. Dot-blot assay was performed. It was shown that 12 urine specimens from 6 persons with acute rejection revealed MP84 in the matrix dot-blot assay while there was no staining for MP84 in the urine specimens of healthy persons. This could be due to the immunological alteration during the acute rejection which could lead to autocrine and paracrine secretion of growth factors and then the excretion of MP84 in the urine. The mechanism of MP84 secretion is not clear.