Glomerular monocytes predict worse outcomes after acute renal allograft rejection independent of C4d status

BACKGROUND: Both peritubular capillary (PTC) C4d deposition and macrophage/monocyte (MO) infiltration in acute rejection (AR) have separately been shown to be associated with reduced graft survival and recently were demonstrated to be closely correlated in AR. Whether MO infiltration is an independent predictor of graft outcome is uncertain. METHODS: All patients with biopsy-proven AR (over a 3-year period) were included (N= 96). All biopsies (N= 121) were graded according to the Banff 97 criteria and immunohistochemically stained for C4d and MO (CD68). The primary outcome was glomerular filtration rate (GFR) <30 mL/min 1-year posttransplant as estimated by the Modified Diet in Renal Disease (MDRD) Formula. Secondary outcomes at 2 and 4 years' posttransplant were also explored. A variety of clinical and biopsy variables were statistically analyzed to establish univariate predictors of graft outcome. Stepwise multivariate logistic regression modeling was applied to determine independent predictors of outcomes. RESULTS: There was a close correlation between PTC C4d and glomerular MO infiltration (P < 0.001). Univariate predictors of primary outcome (GFR <30 mL/min 1-year posttransplant) included mean glomerular MO count > or =1.0 MO/glomerulus (P= 0.014), female sex (P= 0.02), higher peak (P= 0.005), and pretransplant (P= 0.003) panel-reactive antibody levels, cadaveric donor (P= 0.006), transplant glomerulitis (P= 0.004), and longer cold ischemic time (CIT) (P= 0.002). Mean MO/glomerulus > or =1.0 [OR 10.3 (1.23, 87.1)], female sex [OR 5.27(1.31, 21.1)], and CIT [OR 1.14 (1.06, 1.25)] were identified as independent predictors of adverse graft outcome. Furthermore, mean MO/glomerulus > or =1.0 independently predicted poor renal function at 2 years [OR 3.89 (1.19, 12.70)] and 4 years [OR 4.03 (1.22, 13.28)] posttransplant. CONCLUSION: The results demonstrate that glomerular MO infiltration is an independent predictor of worse outcomes posttransplant following acute renal allograft rejection.     

Endothelial C4d deposition is associated with inferior kidney allograft outcome independently of cellular rejection.

BACKGROUND: Capillary deposition of complement split product C4d has been suggested to be a valuable marker for humoral rejection. In this retrospective study we evaluated the clinical impact of C4d deposition in renal allografts with special emphasis on associations between C4d staining patterns and histological features of acute rejection. METHODS: One hundred and two allograft biopsies obtained from 61 kidney transplants (1-532 days after transplantation; median 14 days) were examined by immunohistochemistry on routine paraffin sections using a novel anti-C4d polyclonal antibody (C4dpAb). RESULTS: Fourty-two of 102 biopsies showed endothelial C4d deposits in peritubular capillaries (PTC). Histopathological analysis revealed a significantly lower frequency of positive C4d staining in biopsies with rather than in those without acute cellular rejection defined by the Banff grading schema (P<0.01). For clinical evaluation, patients were classified according to C4d staining in allografts (C4d(PTC) positive in at least one biopsy, n=31 vs C4d(PTC) negative in all biopsies, n=30). C4d(PTC) positive patients had significantly higher serum creatinine levels than C4d negative patients. Even in the absence of morphological evidence for rejection, differences in serum creatinine levels between C4d(PTC) positive and negative recipients were significant (6 months: 2.01+/-0.75 vs 1.41+/-0.27 mg/dl; 12 months: 1.95+/-0.60 vs 1.36+/- 0.34 mg/dl; 18 months: 1.98+/-0.50 vs 1.47+/-0.31 mg/dl; P<0.05). All patients with rejection resistant to conventional therapy (n=4) were in the C4d(PTC) positive subgroup. All recipients with panel reactive antibodies (PRA) >50% (n=8) were C4d(PTC) positive. CONCLUSIONS: Our data indicate that endothelial C4d deposition is associated with inferior graft outcome. We provide evidence that this immunohistochemical finding and its clinical impact are not associated with morphological signs of cellular rejection.     

Glomerular monocyte/macrophage influx correlates strongly with complement activation in 1-week protocol kidney allograft biopsies

BACKGROUND: The specific role of monocytes/macrophages (MO) in kidney graft rejection is not yet fully elucidated. In a recent protocol biopsy study of living-donor recipients, we demonstrated massive capillary influx of MO, associated with severe complement activation and acute rejection (AR) 1 week after transplantation [Sund et al.]. To gain further insight into the possible relationship between MO and complement activation, we analyzed glomerular and interstitial MO in these biopsies. METHODS: Twenty-seven protocol biopsies were stained with antibodies to calprotectin (L1) and CD68 as markers for MO. Cells were counted as an average number per glomerulus and as an average number per defined visual field in the interstitium. Polymorphonuclear leukocytes (PMN) were counted in glomeruli and interstitium by light microscopy. Baseline specimens from 10 of the patients served as controls. The results were compared with data on deposition of complement from the foregoing study, and with histopathologic and clinical data on AR. RESULTS: Cases with diffuse C4d deposition in peritubular capillaries consistent with acute antibody-mediated rejection (AbAR) (n = 4) had significantly higher numbers of intraglomerular MO than the other protocol biopsies (L1: median 20.7 vs 3.6, p = 0.0002; CD68: median 10.1 vs. 2.0, p = 0.0008). With a cut-off of 10 L1-positive and 6 CD68-positive MO, the specificity for the diagnosis of AbAR was 96% and 91%, respectively. The number of interstitial MO was significantly higher in patients with AR than in those without, but in contrast to glomerular MO, interstitial MO could not discriminate between complement positive and negative AR. The number of glomerular and interstitial PMNs was significantly higher in the AbAR group than in the other protocol biopsies. CONCLUSIONS: The strong correlation between complement activation and early glomerular influx of MO in the kidney allograft suggests a causal relationship between these 2 events. At 1 week after transplantation, a number of 10 L1-positive and 6 CD68-positive MO per glomerulus indicates AbAR.     

Focal peritubular capillary C4d deposition in acute rejection.

BACKGROUND: Diffuse peritubular capillary (PTC) C4d deposition has been shown to be associated with relatively poor graft outcome. The significance of focal PTC C4d staining in the early post-transplant period is uncertain. METHODS: Sixty-five biopsies from 53 patients with acute rejection were graded (Banff '97 criteria), stained for C4d, monocytes and T cells, and divided into three groups according to PTC C4d: (i) focal C4d (F) (14 biopsies, 14 patients), (ii) diffuse C4d (D) (23 biopsies, 15 patients) and (iii) no C4d (N) (28 biopsies, 24 patients). The three groups were compared with respect to a variety of biopsy and clinical parameters including outcome. RESULTS: The incidence of transplant glomerulitis and glomerular monocyte infiltration were significantly greater in F (64% and 2.0+/-2.0) and D (57% and 3.4+/-2.0) than in N (11% and 0.2+/-0.2). A significantly higher proportion of F (93%) demonstrated acute cellular rejection (Banff '97 grade > or = 1A) than did D (35%). The F and D groups included significantly more females (50 and 67%, respectively) than did N (21%). The percentage of patients with a second or third transplant was higher in F (29%) and D (40%) than in N (8%) (P = 0.0589). The proportion of patients with glomerular filtration rate < 30 ml/min at 12, 24 and 48 months was higher in the D and F groups than in the N, and there was a statistically significant increasing trend in odds of this outcome occurring at 48 months across the three groups (D > F > N group) (P = 0.0416). CONCLUSION: The results suggest that the biopsy findings and clinical course in patients with focal PTC C4d staining are similar to those associated with diffuse C4d.     

Polymorphs infiltrate glomeruli in mesangial IgA glomerulonephritis

During episodes of macroscopic hematuria, patients with IgA nephropathy commonly have polymorphonuclear neutrophils (PMNs) as well as fibrin and mononuclear cells in glomerular capillaries. We quantitated PMN and macrophage infiltration in glomeruli of 54 patients with IgA nephropathy in whom renal biopsies were obtained within 30 days of macroscopic hematuria. Control biopsies (N = 22) were from patients with IgA nephropathy and urinary erythrocyte counts below 50,000/ml. PMN monocyte/macrophages were quantitated using the monoclonal antibodies FMC10 and HAM56, respectively. In patients with heavy hematuria, 45.9 +/- 3.4% (mean +/- SE) of glomeruli were positive for PMNs (control 10.5 +/- 2.8%, P = 0.001) with a mean PMN count/glomerulus of 1.10 +/- 0.20 (control 0.13 +/- 0.03, P = less than 0.001). 65.6 +/- 9.7%. Of the glomeruli were positive for monocytes in the heavy hematuria group (control 40.0 +/- 8.4, P less than 0.05) with the mean monocyte count per glomerulus being 1.6 +/- 0.2 (control 0.6 +/- 0.1, P less than 0.01). We conclude that, in the acute phase of mesangial IgA nephropathy, PMN and monocytes are present and presumably participate in glomerular injury.     

C4d沉积在抗体介导的慢性排斥反应中的临床意义

目的 探讨移植肾肾小管周围毛细血管补体片段C4d沉积与抗体介导的慢性排斥反应的病理形态、移植肾功能及预后的关系.方法 应用免疫组织化学技术检测77例肾移植受者移植肾肾小管周围毛细血管中C4d的沉积情况,根据检测结果分为C4d阳性组(35例)和C4d阴性组(42例).检测并比较C4d阳性和阴性组受者移植肾的病理形态结构、移植肾功能及预后.结果 与C4d阴性组比较,C4d阳性组受者移植肾肾小管萎缩和肾小球基底膜增生分层的例数明显增多,差异有统计学意义(P＜0.05).C4d阳性组受者的血肌酐水平在移植肾穿刺后12个月时较C4d阴性组受者明显升高,分别为(379.1±260.2)μmol/L和(260.5±175.3)μmol/L,差异有统计学意义(P＜0.05).C4d阳性组受者移植肾穿刺明确C4d阳性后1年内移植肾存活率为62.9%(22/35),而阴性组受者为83.3%(35/42),两组比较,差异有统计学意义(P＜0.05).结论 在抗体介导的慢性排斥反应中,移植肾C4d沉积常见的病理学改变为肾小管萎缩和肾小球基底膜增生分层,C4d阳性抗体介导的体液性慢性排斥反应加快了移植肾功能丧失的进展速度,使C4d阳性受者的移植肾功能丧失率升高,导致存活率降低.     

Monocytes/macrophages in renal allograft rejection

Monocytes/macrophages (MO) have long been recognized to be involved in renal allograft rejection. Monocytes/macrophages have been detected in the glomerular, vascular, and tubulointerstitial compartments during rejection. The recent demonstration that peritubular capillary deposition of complement split factor C4d, a marker for antibody-mediated rejection, is associated with relatively marked MO infiltration of the allograft during acute rejection is a significant development in our understanding of the role of the MO in rejection. High levels of MO in rejecting allografts have been associated with severe rejection, and glomerular MO infiltration in particular has been shown to be an indicator of poor graft outcome.     

移植肾动脉内膜炎的细胞类型及其与管周毛细血管C4d沉积的关系

目的 研究同种异体肾移植术后急性血管性排斥患者动脉内膜炎的细胞类型及其与管周毛细血管（PTC）C4d沉积的关系。 方法 共纳入20例自2006年6月至2008年6月在本中心行同种异体肾移植手术后肾功能异常且病理证实为急性血管性排斥受者的21份肾活检组织。免疫组化方法检测同一活检标本连续切片内T细胞表面标记物CD3、巨噬细胞表面标记物CD68及体液性排斥特异性标记物C4d的表达情况。根据PTC C4d的沉积情况将患者分为C4d阳性（C4d+）组和C4d阴性（C4d-）组,定量计数中动脉内膜阳性细胞数,并计算每个中动脉横切面的平均细胞数。 结果 急性血管性排斥患者动脉内膜都以巨噬细胞浸润为主;C4d+组和C4d-组患者的动脉内膜都以巨噬细胞浸润为主,与浸润的T淋巴细胞数相比差异均有统计学意义[（12.45±9.86）个/ 中动脉横切面比（3.91±3.03）个/中动脉横切面,P = 0.007;（3.47±1.89）个/中动脉横切面比（1.45±1.37）个/中动脉横切面,P = 0.006],且C4d+组患者动脉内膜巨噬细胞浸润数量显著多于C4d-组（P = 0.007）。 结论 肾移植术后急性血管性排斥患者动脉内膜以巨噬细胞浸润为主,且与PTC C4d的沉积有相关性。C4d+患者动脉内膜巨噬细胞数显著多于C4d-患者。     

Peritubular capillary C4d deposition and renal outcome in post-transplant IgA nephropathy

BACKGROUNDS: Immunological staining of the transplanted kidney for C4d in peritubular capillaries (C4d(PTC)) has emerged as a useful method to detect antibody-mediated rejection in situ. In this retrospective study, we evaluated the prevalence of C4d(PTC) deposition in allograft renal biopsies diagnosed of IgA nephropathy (IgAN) and analysed its clinical significance. METHOD: Sixty-six biopsy specimens of post-transplant IgAN, which were obtained to evaluate azotemia and/or heavy proteinuria, were examined by immunohistochemical staining of the paraffin sections with polyclonal antibody for C4d. RESULTS: C4d was stained positively in peritubular capillaries in 16 (24%) of the 66 cases. The C4d(PTC)-negative (n=50) and C4d(PTC)-positive groups (n=16) were not different in recipient gender, age, donor age, type of donor (living vs. cadaveric), interval from transplantation to graft biopsy (41.6+/- 21.8 vs. 48.3+/-26.1 months) and post-biopsy follow-up period (60.3+/-23.3 vs. 56.9+/-25.4 months). During the follow-up period, 12 of 50 (24%) although the incidence of graft failure was not different by the C4d deposition in peritubular capillaries, intervals from renal biopsy to graft failure tended to be shorter in C4d(PTC)-positive cases than C4d(PTC)-negative cases. In Kaplan-Meier analysis, the renal allograft function of the C4d(PTC)-positive group deteriorated more rapidly than that of the C4d(PTC)-negative group (p<0.05). Histologically, the C4d(PTC)-positive group had findings suggestive of acute cellular rejection more commonly than the C4d(PTC)-negative group (p<0.01). CONCLUSIONS: Evidence of humoral rejection, as demonstrated by C4d(PTC) deposition, was concurrently present in significant portions of post-transplant IgAN biopsy specimens and was associated with more rapid deterioration of renal function. These results suggest that C4d(PTC) positivity needs to be determined at the time of biopsy even in cases of post-transplant glomerulonephritis and immunosuppression may need to be modified accordingly.     

移植肾小球炎的细胞类型及其与管周毛细血管C4d沉积的关系

目的 研究同种异体肾移植术后急性排斥患者肾小球炎的细胞类型及其与管周毛细血管（PTC）C4d沉积的关系。 方法 2006年6月至2009年6月在本中心行同种异体肾移植手术后,肾功能异常且病理证实为急性排斥并发小球炎受者51例的肾活检组织为研究对象。免疫组化方法检测同一活检标本连续切片内T细胞表面标记物CD3、巨噬细胞表面标记物CD68、体液性排斥特异性标记物C4d、细胞毒T淋巴细胞标记物颗粒酶B及调节性T淋巴细胞标记物Foxp3的表达情况。根据PTC有无C4d沉积将受者分为C4d阳性组和C4d阴性组,定量计数小球内阳性细胞数,并计算每个小球的平均细胞数。 结果 C4d阳性与C4d阴性的急性排斥受者小球内浸润的炎细胞数分别为17.79±7.70和8.17±3.80,差异有统计学意义（P < 0.01）。C4d阳性急性排斥受者小球内以巨噬细胞浸润为主,与C4d阴性者差异有统计学意义（13.73±7.03 比2.57±1.22,P < 0.01）。C4d阴性急性排斥受者小球内以T淋巴细胞为主,与C4d阳性者差异有统计学意义（5.60±2.81比4.05±2.60,P = 0.023）。C4d阴性与C4d阳性急性排斥受者小球内T淋巴细胞都以细胞毒细胞为主 （3.37±2.34比4.27±2.41,P = 0.141）,而没有调节性T淋巴细胞的浸润。 结论 肾移植术后急性排斥患者小球内炎细胞浸润总数与PTC的C4d沉积有关。C4d阳性患者小球炎细胞数多于C4d阴性患者。C4d阳性患者小球炎细胞以巨噬细胞浸润为主;C4d阴性患者小球炎细胞以T淋巴细胞浸润为主。两组患者小球内T淋巴细胞都以细胞毒细胞为主。     

Incidence of latent mesangial IgA deposition in renal allograft donors in Japan

BACKGROUND: Mesangial immunoglobulin A (IgA) deposition is incidentally encountered in asymptomatic individuals, but its precise frequency and significance had not been clarified. The background of the latent IgA deposition is related to the epidemiology and pathogenesis of IgA nephropathy. METHODS: Zero-hour allograft biopsies were performed in 510 renal transplantations (446 living donors, and 64 cadaveric donors) at the Kidney Center of Tokyo Women's Medical University. Mesangial IgA and C3 deposition were analyzed immunohistochemically, and the frequency and clinicopathologic features of mesangial IgA deposition were investigated. RESULTS: Mesangial IgA deposition was present in 82 (16.1%) of the total 510 allografts with no statistical difference between living donors (72/446, 16.1%) and cadaveric donors (10/64, 15.6%) or between blood-related donors (66/392, 16.8%) and nonblood-related donors (16/110, 14.5%). Mesangial C3 deposition was present in 16 (19.5%) of the 82 allografts with mesangial IgA deposition. The grade of hematuria in IgA(+) donors was significantly higher than IgA(-) donors (1.30 +/- 1.17 vs. 0.86 +/- 0.89, P = 0.025). Histologic investigation of IgA(+) allografts revealed the frequency of mesangioproliferative glomerulonephritis (PGN) was significantly higher in IgA(+)/C3(+) allografts (8/16, 50%) than in IgA(+)/C3(-) allografts (11/66, 16.7%) (P = 0.0084). Moreover, the number of infiltrated macrophages to glomerulus (cells/glomerular cross section) was significantly higher in the IgA(+)/C3(+) allografts than in IgA(+)/C3(-), IgA(-)/C3(+) and IgA(-)/C3(-) allografts (1.10 +/- 0.62 vs. 0.61 +/- 0.42, P = 0.0008; 0.47 +/- 0.34, P = 0.023; and 0.37 +/- 0.23, P = 0.002, respectively). CONCLUSION: The latent mesangial IgA deposition was a relatively common phenomenon in the healthy Japanese donors. This phenomenon was associated with mild degree of microhematuria, mesangial proliferation and glomerular macrophage infiltration in some of the affected individuals, especially with combined IgA and C3 deposition.     

Intracoronary E-/L-selectin blockade reduces neutrophil infiltration in heart transplantation

BACKGROUND: This study examined the effect of local intracoronary delivery of a unique monoclonal antibody (mAb) to both E- and L-selectin (EL-246) on neutrophil infiltration after global ischemia during cardiac transplantation. METHODS: In 12 ovine heart transplants, allograft coronary arteries were locally perfused with EL-246 (n = 6), or isotype-matched control antibodies (n = 2) or saline (n = 4). At 24 hours posttransplant, myocardium was analyzed for neutrophil infiltration and myocardial water content. RESULTS: The mean number of intramyocardial neutrophils per area (PMN/hpf) was greatly reduced in the allografts perfused with EL-246 (3.45 +/- 0.4 PMN/hpf), compared with an average 6.5 +/- 0.97 PMN/hpf in control hearts (p = 0.004). Peripheral leukocyte counts were unaffected; myocardial water content was not significantly reduced. CONCLUSIONS: Local perfusion of cardiac allografts with blocking antibody EL-246 before reperfusion significantly reduced the neutrophilic infiltration that occurs early after transplantation. Prohibiting neutrophil-endothelial adhesion and transmigration may be useful in decreasing neutrophil-dependent post-reperfusion injury in transplantation and routine cardiac surgery.     

C4d and C3d Staining in Biopsies of ABO- and HLA-Incompatible Renal Allografts: Correlation with Histologic Findings

Biopsies of ABO-incompatible and positive crossmatch (HLA-incompatible) renal allografts were retrospectively examined to compare results of C4d and C3d staining, and the correlation between such staining and histologic findings suggestive of antibody-mediated rejection (AMR). A total of 75 biopsies (55 protocol, 17 for graft dysfunction, 3 for other indications) of 24 ABO-incompatible grafts and 244 biopsies (103 protocol, 129 for graft dysfunction, 12 for other indications) of 66 HLA-incompatible grafts were examined; all were stained for C4d and approximately 40% for C3d. In ABO-incompatible grafts, 80% of protocol biopsies and 59% performed for graft dysfunction showed C4d staining in peritubular capillaries (PTC); this staining was not correlated with neutrophil margination in PTC. In HLA-incompatible grafts, PTC C4d was present in 26% of protocol biopsies and 60% of biopsies for graft dysfunction; 92% of biopsies with >1+ (0-4+ scale), diffuse PTC C4d had > or =1+ margination and/or thrombotic microangiopathy (TMA), compared with 12% of C4d-negative biopsies. C3d was somewhat more predictive of margination than C4d in ABO-incompatible, but not HLA-incompatible, grafts. In summary, while PTC C4d deposition indicates probable AMR in biopsies of HLA-incompatible grafts, including protocol biopsies, there is no histologic evidence that C4d deposition is correlated with injury in most ABO-incompatible grafts.     

Effects of hypoxia-inducible factor-1alpha expression and C4d deposition in implantation biopsies on renal allograft

Upregulation of hypoxia-inducible factor-1alpha (HIF-1alpha) in response to ischemic states has been suggested to have a role in the development of chronic allograft nephropathy. Deposition of C4d in the peritubular capillaries of renal allografts has been reported to be a sensitive marker of acute humoral rejection. The purpose of this study was to determine the effects of HIF-1alpha expression and C4d deposition in implantation biopsies of renal allografts. Implantation biopsies and 22 rejection proved biopsies were performed in 54 renal transplant recipients between December 1996 and July 1999. The mean follow-up was 82.8 months. Immunohistochemical studies were performed using a mouse monoclonal antibody for HIF-1alpha expression and a rabbit polyclonal antibody for C4d detection. HIF-1alpha was demonstrated in 19 of 54 implantation biopsies (35%), and C4d deposition in one (1.9%). The HIF-1alpha-positive group included a higher percentage of deceased donor organs (66.4% vs 17.1%; P = .002) and longer mean cold ischemia times (261.3 +/- 231 vs 103 +/- 40 min; P = .008) compared with the HIF-1alpha-negative group. The relative risks (95% confidence intervals) of expression of HIF-1alpha for allograft rejection, chronic allograft nephropathy, and graft loss were 1.53 (0.82-2.87), 0.61 (0.06-5.50), and 2.45 (0.62-9.85). The C4d-positive patient developed acute accelerated rejection on postoperative day 4. In the present study, the expression of HIF-1alpha showed a significant correlation with the use of a deceased donor kidney and with cold ischemia time. However, there were no significant effects on the prognosis for a graft after implantation of a kidney with HIF-1alpha expression.     

The presence and prognostic importance of glomerular macrophage infiltration in renal allografts

AIM: The purpose of this study was to analyze the role of intraglomerular macrophage infiltration in human renal allografts by examining biopsies from kidney grafts that were dysfunctional after transplantation. METHODS: Eighty-three patients (58 men, 25 women) of a mean age of 30.2 +/- 1.4 years were evaluated. In all cases, biopsy specimens were examined for the presence of macrophage infiltration in the glomeruli. The infiltration of these cells was evaluated immunohistochemically using monoclonal antibody CD68, which labels macrophage cytoplasm. 10 renal allograft biopsies with normal histopathology were used as control group. The CD68-positive macrophages in all glomeruli were counted and the glomerular macrophage index (GMI) was calculated. RESULTS: Of the 83 patients, 40 showed acute rejection (AR), 33 showed chronic rejection (CR) and 10 showed cyclosporin A (CsA) toxicity. Only the biopsies of 28 patients stained positive for CD68 in the glomeruli. Neither patients with CsA toxicity nor controls showed intraglomerular macrophages. The CD68-positive group consisted of 7/33 CR and 21/40 AR patients. We observed intraglomerular macrophages in only 6 of the 20 AR cases that responded to steroid therapy (mean GMI 0.3 +/- 0.1) and in 15 of the 20 steroid-resistant AR cases (mean GMI 1.7 +/- 1.2; p < 0.01). The outcome of grafts that contained intraglomerular macrophages was significantly worse than the outcomes of other grafts noticed during the follow-up. CONCLUSION: We conclude that the presence of glomerular macrophages can be considered a marker for rejection and is a valuable additional criterion of rejection in the histological examination of renal allograft biopsies. The presence of intraglomerular macrophages indicates that the outcome of the graft will be significantly worse than that of grafts without intraglomerular macrophage infiltration.     

Capillary deposition of complement split product C4d in renal allografts is associated with basement membrane injury in peritubular and glomerular capillaries: a contribution of humoral immunity to chronic allograft rejection

Endothelial deposition of the complement split product C4d is an established marker of antibody-mediated acute renal allograft rejection. A contribution of alloantibody-dependent immune reactions to chronic rejection is under discussion. In this study, the association of immunohistochemically detected endothelial C4d deposition in peritubular capillaries (PTC) with morphologic features of chronic renal allograft injury was investigated in a large study cohort. C4d deposits in PTC were detected in 73 (34%) of 213 late allograft biopsies performed in 213 patients more than 12 mo after transplantation (median, 4.9 yr) because of chronic allograft dysfunction. Endothelial C4d deposition was found to be associated with chronic transplant glomerulopathy (CG) (P < 0.0001), with basement membrane multilayering in PTC (P = 0.01), and with an accumulation of mononuclear inflammatory cells in PTC (P < 0,001). Furthermore, C4d deposits in PTC (in biopsies with normal glomerular morphology) were associated with development of CG in follow-up biopsies. Other morphologic features of chronic allograft nephropathy (with exception of tubular atrophy) were not associated with C4d deposits in PTC. Analyses of previous and follow-up biopsies revealed that C4d deposits may occur de novo and may also disappear at any time after transplantation. In conclusion, the data suggest that complement activation in renal microvasculature, indicating humoral alloreactivity, contributes to chronic rejection characterized by chronic transplant glomerulopathy and basement membrane multilayering in PTC.     

Acute humoral rejection in kidney transplantation: II. Morphology, immunopathology, and pathologic classification

The incidence of acute humoral rejection (AHR) in renal allograft biopsies has been difficult to determine because widely accepted diagnostic criteria have not been established. C4d deposition in peritubular capillaries (PTC) of renal allografts has been proposed as a useful marker for AHR. This study was designed to test the relative value of C4d staining, histology, and serology in the diagnosis of AHR. Of 232 consecutive kidney transplants performed at a single institution from July 1995 to July 1999, all patients (n = 67) who developed acute rejection within the first 3 mo and had a renal biopsy with available frozen tissue at acute rejection onset, as well as posttransplant sera within 30 d of the biopsy, were included in this study. Hematoxylin and eosin and periodic acid-Schiff stained sections were scored for glomerular, vascular, and tubulointerstitial pathology. C4d staining of cryostat sections was done by a sensitive three-layer immunofluorescence method. Donor-specific antibodies (DSA) were detected in posttransplant recipient sera using antihuman-globulin-enhanced T cell and B cell cytotoxicity assays and/or flow cytometry. Widespread C4d staining in PTC was present in 30% (20 of 67) of all acute rejection biopsies. The initial histologic diagnoses of the C4d(+) acute rejection cases were as follows: AHR only, 30%; acute cellular rejection (ACR) and AHR, 45%; ACR (CCTT types 1 or 2) alone, 15%; and acute tubular injury (ATI), 10%. The distinguishing morphologic features in C4d(+) versus C4d(-) acute rejection cases included the following: neutrophils in PTC, 65% versus 9%; neutrophilic glomerulitis, 55% versus 4%; neutrophilic tubulitis, 55% versus 9%; severe ATI, 75% versus 9%; and fibrinoid necrosis in glomeruli, 20% versus 0%, or arteries, 25% versus 0%; all P < 0.01. Mononuclear cell tubulitis was more common in the C4d(-) group (70% versus 100%; P < 0.01). No significant difference between C4d(+) and C4d(-) acute rejection was noted for endarteritis, 25% versus 32%; interstitial inflammation (mean % cortex), 27.2 +/- 27% versus 38 +/- 21%; interstitial hemorrhage, 25% versus 15%; or infarcts, 5% versus 2%. DSA were present in 90% (18 of 20) of the C4d(+) cases compared with 2% (1 of 47) in the C4d(-) acute rejection cases (P < 0.001). The pathology of the C4d(+) but DSA(-) cases was not distinguishable from the C4d(+), DSA(+) cases. The C4d(+) DSA(-) cases may be due to non-HLA antibodies or subthreshold levels of DSA. The sensitivity of C4d staining is 95% in the diagnosis of AHR compared with the donor-specific antibody test (90%). Overall, eight grafts were lost to acute rejection in the first year, of which 75% (6 of 8) had AHR. The 1-yr graft failure rate was 27% (4 of 15) for those AHR cases with only capillary neutrophils versus 40% (2 of 5) for those who also had fibrinoid necrosis of arteries. In comparison, the 1-yr graft failure rates were 3% and 7%, respectively, in ACR 1 (Banff/CCTT type 1) and ACR 2 (Banff/CCTT type 2) C4d(-) groups. A substantial fraction (30%) of biopsy-confirmed acute rejection episodes have a component of AHR as judged by C4d staining; most (90%), but not all, have detectable DSA. AHR may be overlooked in the presence of ACR or ATI by histology or negative serology, arguing for routine C4d staining of renal allograft biopsies. Because AHR has a distinct therapy and prognosis, we propose that it should be classified separately from ACR, with further sub-classification into AHR 1 (neutrophilic capillary involvement) and AHR 2 (arterial fibrinoid necrosis).     

CD20+ lymphocytes in renal allografts are associated with poor graft survival in pediatric patients

BACKGROUND: The presence of CD20+ lymphocyte renal allograft infiltrates has been associated with steroid-resistant rejection and poor graft survival. We quantified the number of CD20+ lymphocytes in renal allograft biopsies and correlated the results with graft survival. We also determined the relationships between CD20+ lymphocytes and acute cellular rejection versus antibody-mediated rejection. METHODS: We examined 45 biopsy samples from 31 pediatric patients biopsied for suspicion of rejection from November 2001 to November 2004. Immunohistochemical staining for CD20 and C4d was performed on all biopsies; CD20+ cell density per high-power field (hpf) was determined for each core. Patient graft status was followed postbiopsy and documented for graft survival or failure using the cutoff date of December 31, 2005. RESULTS: Patients with 2-10 and 11-100 CD20+ cells/hpf had worse graft survival in Kaplan-Meier analysis with a hazard ratio 4.56 (CI 1.07-19.35) two years postbiopsy compared to those with 0-1 cells/hpf (P = 0.02). The presence of CD20+ lymphocytes was significantly associated with acute cellular rejection (P = 0.0001) and not associated with antibody-mediated rejection (P = 0.16). Receiver-operating curve analysis confirmed > or =3 cells/hpf correlating with acute cellular rejection, yielding sensitivity 90% and specificity 76%. CONCLUSIONS: This study shows a significant 4.5-fold risk of graft failure at two years postbiopsy with presence of > or =2 CD20+ cells/hpf. Moreover, > or =3 CD20+ lymphocytes were highly associated with acute cellular rejection. They may be functioning as professional antigen-presenting cells in the graft. In steroid-refractory cellular rejections, therapies that target B cells may prolong graft survival.     

Urinary C4d does not correlate with C4d-staining in peritubular capillaries but reflects nonspecific glomerular injury

BACKGROUND: C4d-staining in peritubular capillaries (PTC) in allograft biopsies is a hallmark of antibody-mediated rejection (AMR). This study investigated whether urinary C4d correlates with C4d-staining in PTC, allowing for a noninvasive screening procedure. METHODS: Urine samples from 34 patients with diffuse C4d-staining in PTC (C4d-pos group) and 68 urine samples from patients with negative C4d-staining in PTC (C4d-negative group) matched 1:2 according to proteinuria and time posttransplant were compared regarding urinary C4d-levels. RESULTS: Overall, urinary C4d/creatinine-ratios (C4d/C) were not different between the C4d-positive and the C4d-negative group (P=0.55). Urinary C4d/C strongly correlated with total protein/creatinine-ratios (P/C) (r2=0.53; P<0.0001) and albumin/creatinine-ratios (r2=0.52; P<0.0001). C4d/C were not different between C4d-pos and C4d-negative patients with P/C>50 mg/mmol (P=0.57) and P/C<50 mg/mmol (P=0.65), respectively. However, in both the C4d-positive and the C4d-negative group urinary C4d/C were significantly higher in patients with P/C>50 mg/mmol than in patients with P/C<50 mg/mmol (P相似文献   

异体气管移植去抗原性的实验研究

目的探讨移植段气管去除上皮细胞和腺体细胞后异体、异位移植排斥反应的强弱。方法25只雄性SD大鼠作为供体,制备新鲜移植段气管、冷冻移植段气管和去上皮细胞移植段气管。取制备的新鲜移植段气管40个平均分为4组,分别用0、0.1、0.3和0.5mg/ml的蛋白酶溶液,4℃浸泡12h,根据镜下移植段气管上皮细胞和腺体细胞脱落情况及软骨细胞破坏程度确定蛋白酶的最佳浓度。另取30只雄性SD大鼠作受体,均分成3组,分别为:新鲜气管移植组(A组)、冷冻气管移植组(B组)及去上皮细胞移植组(C组),n=10。行左上腹旁正中切口,提出大网膜包绕各移植段气管,于21d后取出移植段气管,行组织学观察及淋巴细胞浸润测定。结果0.3mg/ml蛋白酶能去除移植段气管上皮细胞及腺体细胞,而对软骨细胞无明显损坏。异体植入大鼠腹腔的3组气管软骨均成活,血运建立,其中A组管腔内有肉芽组织,出现坏死、实变;B组有少量肉芽组织;C组管腔内无肉芽组织。A、B、C3组淋巴细胞浸润分别为29.16±2.69、15.17±2.19和11.56±0.87个/Hp,A组与B、C组比较及B组与C组比较,差异均有统计学意义(P<0.05),排斥反应强弱为:A组>B组>C组。结论0.3mg/ml蛋白酶,4℃,浸泡12h的移植段气管,能完全脱上皮细胞和腺体,对软骨细胞基本无损伤,与冷冻法比较去抗原作用更好。一期异体大网膜包裹异位移植后,血运重建且移植段气管成活。