急性排斥移植肾组织嗜酸性粒细胞浸润及C4d沉积

目的：通过观察移植肾PTC C4d沉积和嗜酸性粒细胞浸润与AHR的关系,探讨两者之间的相关性及其在诊断急性体液排斥中的作用。方法：采用间接免疫荧光法检测移植肾组织C4d沉积。26例移植肾PTC C4d弥漫性强阳性染色患者为C4d阳性组,而30例移植肾PTC C4d阴性患者为对照组。光镜下记数移植肾组织嗜酸性粒细胞数。结果：（1）26例C4d阳性患者中经病理诊断为AHR为13例,AHR＋ACR为8例,ACR1为3例,ACR2为2例。30例C4d阴性患者中,AHR为1例,AHR＋ACR为2例,ACR1为21例,ACR2为6例。与C4d阴性组相比,C4d阳性组难治性排斥患者较多,常需进一步干预性措施,且较多的患者对抗排斥治疗无反应,移植肾失功率高（7/26）。（2）C4d阳性组移植肾浸润嗜酸性粒细胞数亦明显高于C4d阴性组,两组相比差异显著（P〈0.01）。C4d阳性无细胞性排斥组移植肾间质浸润嗜酸性粒细胞数与C4d阳性＋ACR组相比差异无显著性（P〉0.05）。C4d阳性ACR组移植肾间质浸润嗜酸性粒细胞数明显高于C4d阴性ACR组（P〈0.05）。结论：移植肾PTC C4d沉积与嗜酸性粒细胞浸润密切相关,两者在肾移植AHR诊断过程中起重要作用。     

C4d/CD34 double-immunofluorescence staining of renal allograft biopsies for assessing peritubular capillary C4d positivity

Immunofluorescence detection of the complement split product C4d along peritubular capillaries in renal allograft biopsies is the mainstay for the diagnosis of antibody-mediated rejection. The extent of peritubular capillary C4d positivity may have significant clinical ramifications; however, peritubular capillary density in the renal cortex is often difficult to assess with single-channel immunofluorescence. In this study, we report a C4d/CD34 double-immunofluorescence staining protocol for renal allograft frozen sections that allows rapid and sensitive detection of C4d positivity, as well as improved accuracy in estimating the C4d-positive fraction of peritubular capillaries. In addition, this method aids in determining whether C4d-positive structures correspond to peritubular capillaries or whether they represent common mimics of peritubular capillaries such as tubular basement membranes. C4d/CD34 double immunofluorescence provides rapid, convenient, and low-cost implementation for laboratories currently utilizing single-channel C4d immunofluorescence.     

C3d and the septal microvasculature as a predictor of chronic lung allograft dysfunction

Studies have shown a potential role for humoral rejection in the evolution of lung graft dysfunction, apparently based on antibodies without human leukocyte antigen specificity. The correlation between extent of C4d deposition with clinical status further illustrates the importance of humoral immunity. Our study examines the potential value of C3d as a further diagnostic adjunct. C3d deposition was examined in lung allograft specimens using frozen tissue indirect direct immunofluorescence (IIF) and avidin biotin immunohistochemical applied to paraffin embedded tissue. Intermediate/extensive amounts of C3d using IIF and immunohistochemical (IH) methodologies correlated with chronic graft dysfunction; IIF C3d deposition was associated with septal and bronchial wall fibrosis (p < 0.0001). Weak/absent amounts of IIF and IH C3d correlated with clinical stability (p < 0.0001). Higher levels of C3d by IH were more sensitive than by IIF as a bronchiolitis obliterans syndrome determinant. C3d and C4d deposition using immunofluorescence and IH were correlated (p < 0.00001). C3d deposition appears prognostically significant. Higher tissue expression of C3d mark chronic graft dysfunction/persistent graft failure following transplantation. The close correlation between C3d and C4d lends credence to the role of humoral allograft rejection as a pulmonary graft dysfunction contributing factor. C3d by IH manifests higher sensitivity but similar specificity compared to C3d by IIF.     

Peritubular capillary basement membrane changes in chronic renal allograft rejection: Comparison of light microscopic and ultrastructural observations

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

Peritubular capillaries in chronic renal allograft rejection: a quantitative ultrastructural study

Peritubular capillaries (PCs) with a circumferentially multilayered basement membrane have been suggested as an ultrastructural indicator of chronic renal allograft rejection (CR). The authors validated this lesion as a marker for CR, by analyzing its quantitative features, specificity, and sensitivity in 169 renal biopsy specimens. The mean number of circumferential layers (PCcirc) and the incidences of the grades (mild: 2 to 4, moderate: 5 to 6, severe: 7 or more layers) were investigated in biopsy specimens involving CR (CR(Bx), n = 46), acute rejection (n = 11), normal kidneys (n = 20), psoriatics treated with cyclosporine (n = 13), renal transplants with chronic cyclosporine toxicity (n = 12), native kidney diseases (NKD, n = 56), and transplant nephrectomies attributable to CR (Cr(nephr), n = 11). CR was diagnosed with regard to the clinical features and the presence of intimal fibrosis in 41 biopsy specimens or transplant glomerulopathy in 35 biopsy specimens (cg; identified only by electron microscopy in 10 cases). NKD included chronic glomerulonephritis, chronic tubulointerstitial nephritis, benign nephrosclerosis, thrombotic microangiopathy, diabetic nephropathy, and renal disease in elderly patients (median age, 72 years). All PCs around glomeruli were sampled (median, 14 profiles per case). PCs with a moderate/ severe lesion appeared as serrated profiles with a thick, ribbon-like basement membrane layer in semithin plastic sections. The numbers of circumferentially multilayered PCs were significantly characteristic of CR (PCcirc in CR(Bx): 2.87+/-1.83 SD; range, 0 to 7.36; P < .001 v other groups). A severe lesion occurred exclusively in CR (in 12% of the PCs in CR(Bx), and in 38% in CRnephr). A moderate lesion was observed in 0.6% of the PCs in NKD, 16% in CR(Bx), and 21% in CRnephr. Three or more PCs with a moderate lesion were encountered only in CR. A mild lesion was not suggestive of CR at all. In CR(Bx), 27 cases showed a severe lesion or 3 or more PCs with a moderate lesion (cpc; sensitivity: 59%). Four of the 27 cases lacked cg. The cumulative incidence of cpc and cg was 85%. In transplants with cyclosporine toxicity, the presence of cpc verified the coexistence of CR in 7 specimens. In conclusion, cpc is a specific marker of CR. The incidence of cpc increases as CR progresses. The lesion may be caused by a low-grade rejection injury to the PCs. Careful analysis of semithin sections promotes the better sampling of cpc. An ultrastructural demonstration of cpc and cg defines CR more precisely than does light microscopic evaluation per se.     

Comparative study for the detection of peritubular capillary C4d deposition in human renal allografts using different methodologies

Detection of peritubular capillary (PTC) C4d deposition in tissue sections of renal allograft biopsies became an important aid in the diagnosis of antibody-mediated rejection. Pathologists in many major transplant centers now routinely stain renal allograft biopsies for C4d. Currently, there are 3 commercially available antibodies. Two of these antibodies are monoclonal and are usually used with either a 3- or a 2-step indirect immunofluorescence (IF) methodology on frozen sections. A polyclonal antibody is used on formalin-fixed, paraffin-embedded tissue section with an immunoperoxidase detection system. The goal of our study was to compare these antibodies and methodologies in our renal allograft biopsy material. Twenty renal allograft biopsies with diffuse or focal PTC C4d staining, using immunofluorescence methods on frozen sections, were selected for this study. These biopsies were tested with the 3 commercially available anti-C4d antibodies (Biogenesis, Brentwood, Calif, cat no. 222-8004; Quidel Corporation, Santa Clara, Calif, cat no. A213; and ALPCO Diagnostic, Windham, NH, cat no. 004-BI-RC4D). Both monoclonal antibodies (Biogenesis and Quidel) were tested with a 3- and a 2-step indirect IF method on frozen sections. The polyclonal antibody (ALPCO) was applied to formalin-fixed paraffin sections using immunoperoxidase methodology. In selected cases, the polyclonal antibody was tested on frozen sections with a 3-step indirect IF method. To exclude possible false-negative staining with the IF method, we selected 10 additional biopsies that showed PTC margination of inflammatory cells, but were C4d-negative or only focally positive, and tested them with the ALPCO antibody on paraffin sections. We have found that all methodologies and antibodies tested provided adequate results with only minor differences between them. Perhaps the most sensitive method is the 3-step indirect IF on frozen sections using one of the monoclonal antibodies. We prefer the 2-step indirect IF method with the Quidel monoclonal antibody because of its simplicity, quick turnaround time, and relatively low cost. The advantages and disadvantages of the individual methodologies are discussed.     

Portal capillary C4d deposits and increased infiltration by macrophages indicate humorally mediated mechanisms in acute cellular liver allograft rejection

Almost no data exist concerning the role of antibody-mediated mechanisms in human acute cellular liver allograft rejection (ACR). Therefore, the aim of this study was to determine whether ACR is associated with depositions of complement split products and increased infiltration by B-lymphocytes, plasma cells and macrophages. A total of 35 liver biopsy specimens (ACR n=22, controls n=13) were analyzed by immunohistochemical single and double staining. The average numbers of CD20⁺, CD38⁺ and CD68⁺ cells per portal tract were established while the presence of C4d and C3d deposits was evaluated semiquantitatively. Significantly greater numbers of CD20⁺ (P=0.029) and CD38⁺ (P=0.014) cells were found in the ACR specimens than in the control specimens. Additionally, 50% of patients diagnosed with ACR showed C4d deposits along portal capillaries, which was associated with a significantly increased portal infiltration by macrophages (P=0.007). Taken together these results support the involvement of humorally mediated mechanisms in some cases of ACR.     

补体片段C4d与肾移植抗体介导排异反应的病理诊断

同种异体肾移植排异无非是以T细胞介导的细胞性排异和以抗体介导的体液性排异两种形式.在组织学上虽然对肾移植急性细胞性排异已有较好的认识,但对抗体介导的体液排异由于病变不典型,缺乏足够的认识和共识[1].近10年来,补体片段C4d在移植肾毛细血管上沉积与抗体介导排异的相关性研究备受瞩目[2-4],2007年发表的Banff分类也将C4d免疫阳性染色作为诊断急、慢性抗体介导排异的标准之一[5].我们就移植肾抗体介导排异、补体片段C4d及C4d在排异诊断中的意义做一综述如下.     

局灶节段性肾小球硬化症C4d表达的特点及意义

目的研究不同类型的原发性局灶节段性肾小球硬化症（FSGS）中C4d表达的特点及意义。方法采用免疫组化染色方法,并结合PASM—C4d套染和免疫组化双标染色方法,观察了97例FSGS中C4d在肾小球上沉积部位和强度,及其与病理类型之间的关系。结果88例（90．7％）FSGS的肾小球C4d阳性表达,C4d主要沉积在FSGS肾小球硬化病变处。细胞型、顶端型、塌陷型及非特殊型FSGS中均表现为硬化病变处血管袢C4d不均匀粗线状沉积,系膜区散在多少不等的颗粒状沉积;顶端型和非特殊型为节段沉积,细胞型和塌陷型为球性沉积。门部型FSGS主要在门部有C4d颗粒状沉积,未见血管袢沉积。结论补体经典途径激活参与了FSGS的发病机制;硬化病变处血管袢C4d不均匀粗线状沉积是细胞型、塌陷型、非特殊型和顶端型FSGS重要的免疫病理形态特点之一。     

An immunohistochemical evaluation of C4d deposition in pediatric inflammatory liver diseases

C4d is a marker of the activated complement cascade used to assess the humoral component of rejection, mostly in kidney allograft transplants. The role of C4d deposition has recently been addressed in hepatic allograft but has never been tested in a series of inflammatory liver diseases without previous liver transplantation. The aim of this study was to compare the immunohistochemistry profile of C4d deposition in a pediatric population, between a cohort of patients with autoimmune hepatitis (AIH) and a series of patients with chronic viral hepatitis B or C. Immunohistochemical analysis was performed on 64 liver biopsies. C4d deposition was observed in 25 (83%) of 30 AIH biopsies examined, in 6 (40%) of 15 hepatitis C biopsies, and in 17 (89%) of 19 hepatitis B biopsies. No expression of C4d was observed in 4 noninflammatory liver specimens used as negative controls. In the AIH group, a staining of the periportal sinusoids was often observed, as well as focal periductal reinforcement. Centrolobular vein staining was observed in the 3 hepatitis groups with a higher frequency in viral hepatitis B biopsies. Regardless of the etiology, lymphoid aggregates demonstrated an accentuation of the staining. These results confirm a role for a humoral immune response in pediatric autoimmune as well as in viral hepatitis. The relative ratios of positive cases imply that this immunostaining does not represent a strong diagnostic criterion in the differentiation between viral hepatitis and AIH. However, differences in the pattern of the staining were observed, depending on the etiology of the disease. The high prevalence of C4d reactivity in viral hepatitis strongly suggests that C4d does not represent a useful marker in the differentiation between acute rejection and viral hepatitis relapse in liver transplants.     

Regulatory T cells as modulators of chronic allograft dysfunction

Chronic allograft dysfunction (CAD) in solid organ transplantation is a principal cause of patient morbidity and late allograft loss. The pathogenesis of CAD is largely secondary to chronic damage by the adaptive immune system and long-term immunosuppression. Manipulating these factors may be possible with the use of regulatory T cells (Treg), which have the ability to suppress specific immune responses and therefore potentially remove the need for immunosuppressive drugs. Studies of CAD in experimental models have demonstrated the capacity for both mouse and human Treg cellular therapy to prevent the development of some manifestations of CAD. Furthermore, a role for Treg has been demonstrated in clinically tolerant transplant patients. Certain immunosuppressive therapies are also proving to be 'Treg friendly' and may be helpful in promoting Treg while maintaining other immunosuppressive activity. With this in mind, monitoring for biomarkers of operational tolerance with tailored immunosuppressive therapy or controlled weaning in conjunction with Treg cellular therapy may be a useful strategy to pursue.     

Peritubular capillary control of proximal tubule reabsorption in the rat.

Changes in peritubular capillary hydrostatic and oncotic pressures, which probably affect net interstitial pressure and, thus, the force on fluid movement across the tubule basement membrane, can modulate absolute proximal reabsorption rate (APR). To examine the relationship between APR and net interstitial pressure, we measured peritubular capillary hydrostatic and oncotic pressure, single nephron filtration rate, APR, absolute distal reabsorption (ADR), and tubular hydrostatic pressure in hydropenic, saline-loaded, and plasma-loaded rats. Net interstitial pressure in saline loading was estimated from subcapsular hydrostatic pressure and lymph protein concentration measurements. The surface area-hydraulic conductivity product of the peritubular capillary network was estimated from these measurements with a model of capillary fluid exchange in which fluid uptake was defined to be APR plus ADR. The estimated value was assumed to remain constant in all three states, and was then used to estimate net interstitial pressure in hydropenic and plasma-loaded rats. APR and net interstitial pressure correlated strongly, a finding consistent with the hypothesized role for net interstitial pressure in regulating proximal reabsorption.     

慢性移植肾失功的透析时机及影响因素

背景：移植肾功能丧失后要及时转入透析以继续维持患者生命,并为部分患者的再移植作准备。目前国内有关移植肾功能丧失后透析时机的报道尚不多见。 目的：探讨慢性移植肾失功患者开始透析的时机及其影响因素。 方法：调查2005年7月至 2012年12月上海长征医院肾移植康复病房收治的肾移植后慢性移植肾失功患者98例,回顾性分析其临床资料,主要包括内生肌酐清除率、血肌酐、尿毒症症状及并发症,比较不同血液透析时机患者间的差别,分析影响透析时机的因素。 结果与结论：透析前87%的患者有明显尿毒症症状,78%出现心、脑并发症,31.6%需急诊透析。全部移植肾失功患者进入血液透析时的内生肌酐清除率为(5.94±0.63) mL/min,9例患者内生肌酐清除率> 10 mL/min,51例患者内生肌酐清除率为5-10 mL/min,38例患者内生肌酐清除率< 5 mL/min。其中合并丙肝患者进入血液透析治疗时的内生肌酐清除率明显比非丙肝患者高(P < 0.05)。结果显示,开始透析的慢性移植肾失功患者有明显尿毒症并发症,多数透析时机较迟,尤其是丙肝患者。健康宣教、医疗保健是影响透析时机的关键因素。     

Peritubular capillary loss is associated with chronic tubulointerstitial injury in human kidney: altered expression of vascular endothelial growth factor

Chronic tubulointerstitial injury (CTI) including tubular atrophy and interstitial fibrosis represents one major determinant for the progression of chronic renal disease regardless of cause. Although peritubular capillaries (PTCs) are essential to maintain the normal structure and function of renal tubules, little is known about the role of PTCs in the development of CTI. The integrity of PTCs seems to be regulated by growth factors. Vascular endothelial cell growth factor (VEGF) has recently been recognized as a potent regulator of angiogenesis, vascular survival, and vascular permeability. Knowledge of the role of VEGF in renal disease is still rudimentary, and its role in CTI has not been explored. We analyzed the morphologic changes of PTCs and correlated them with other morphologic parameters of CTI in 32 human kidneys with various types of chronic tubulointerstitial disease. The VEGF expression was immunohistochemically evaluated. Compared with normal kidney, PTC loss (41% to 55% of control) and reduced size of PTCs (55% to 88% of control) were noted in kidneys with CTI. The PTC density was positively correlated with the proximal tubular density (r = 0.66, P <.0001), proximal tubular size (r = 0.54, P <.001), and negatively correlated with interstitial volume (r = -0.84, P <.0001). Compared with normal kidney, where podocytes were the only cell type that constantly expressed VEGF, an interesting pattern of increased VEGF expression by renal tubules, especially morphologically intact or hypertrophic ones, was shared by all cases with CTI. Loss of VEGF in sclerotic glomeruli was noted. PTC injury is pathogenetically linked to tubular atrophy, tubular loss, and interstitial fibrosis in human kidneys with CTI and might be a key factor for the progression of chronic tubulointerstitial disease. The characteristic and uniform pattern of altered VEGF expression in kidneys with CTI may result from ischemia induced by PTC loss and represent a protective mechanism against further PTC injuries. HUM PATHOL 31:1491-1497.     

Monocyte implication in renal allograft dysfunction

Macrophages are involved in the development and progression of kidney fibrosis. The aim of this study was to analyse the phenotype of circulating monocytes and their ability to predict kidney allograft dysfunction in living kidney transplant recipients. Whole blood samples from 25 kidney recipients and 17 donors were collected at five time‐points. Monocyte phenotype was analysed by flow cytometry, and interleukin (IL)‐10 and soluble CD163 by enzyme‐linked immunosorbent assay. One week after transplantation, surface CD163 and IL‐10 levels increased significantly from baseline [2·99 ± 1·38 mean fluorescence intensity (MFI) to 5·18 ± 2·42 MFI for CD163; 4·5 ± 1·46 pg/ml to 6·7 ± 2·5 pg/ml for IL‐10]. This CD163 increase correlated with 4‐month creatinine levels (r = 0·4394, P = 0·04). However, soluble CD163 decreased significantly from baseline at 1 week (797·11 ± 340·45 ng/ml to 576·50 ± 293·60 ng/ml). CD14⁺CD16^– monocytes increased at 4 months and correlated positively with creatinine levels at 12 and 24 months (r = 0·6348, P = 0·002 and r = 0·467, P = 0·028, respectively) and negatively with Modification of Diet in Renal Disease (MDRD) at 12 months (r = 0·6056, P = 0·003). At 4 months, IL‐10 decreased significantly (P = 0·008) and correlated positively with creatinine at 2 years (r = 0·68, P = 0·010) and with CD14⁺CD16^– monocytes at 4 months (r = 0·732, P = 0·004). At 24 h, levels of human leucocyte antigen D‐related declined from 12·12 ± 5·99 to 5·21 ± 3·84 and CD86 expression decreased from 2·76 ± 1·08 to 1·87 ± 0·95. Both markers recovered progressively until 12 months, when they decreased again. These results indicate that monitoring monocytes could be a promising new prognostic tool of graft dysfunction in renal transplant patients.     

Complement split product C4d deposition in placenta in systemic lupus erythematosus and pregnancy‐induced hypertension

Systemic lupus erythematosus (SLE) and pregnancy‐induced hypertension (PIH) are related to premature delivery and intrauterine growth restriction (IUGR), and share histological findings of the placenta. Association with complement dysregulation has been reported in pregnancy for both disorders. The purpose of this study was to investigate the utility of C4d immunohistochemistry for placentas with SLE‐ and PIH‐associated pregnancy. C4d staining was performed on paraffin‐embedded tissue of placentas from 26 patients with SLE, 26 with PIH, and 25 control cases. We used the H‐score with a range of 0–300 for the evaluation of C4d immunoreactivity. Placentas of SLE and PIH cases showed a higher H‐score than control cases (average, SLE, 38.3 (P < 0.05); PIH, 17.8; control, 1.68), with linear staining on the membrane of syncytiotrophoblast. C4d‐high groups comprised 50% (12/26) of SLE and 35% (9/26) of PIH cases, with H‐scores ranging 14–270 and 15–170. C4d‐high groups were significantly associated with low‐placental weights and low birth weight in both SLE and PIH (P < 0.05), and lower gestational age (P < 0.05) in PIH cases. These results suggest that C4d might be utilized as a biomarker evaluating the subsequent risk for IUGR and disease control during the gestation period in these patients.     

神经轴突导向因子1裸质粒转染对5/6肾切除模型大鼠肾脏管周毛细血管网的影响

背景：神经轴突导向因子1作为血管内皮细胞的促有丝分裂原,能够促进血管内皮细胞的迁移和增殖,发挥诱导血管新生的作用。 目的：观察神经轴突导向因子1裸质粒转染对5/6肾切除大鼠模型残余肾功能的保护及对肾小管周围毛细血管网的影响。 方法：将30只SD大鼠随机等分为假手术组、模型组和治疗组。模型组和治疗组大鼠切除左侧肾脏上、下极各1/3,1周后切除右肾,建立残肾模型。模型组和治疗组大鼠于切除右肾的同时在左肾静脉分别注射空质粒IRES2-EGFP和pCMV6-XL5-Netrin- 1-IRES2-EGFP。 结果与结论：与模型组相比,治疗组大鼠血尿素氮、血肌酐水平降低,肾间质纤维化程度减轻,肾小管周围毛细血管网的密度增加,肾小管胞浆中神经轴突导向因子1 蛋白表达增加。提示神经轴突导向因子1裸质粒转染能明显改善5/6肾切除模型大鼠的肾功能,减轻残肾组织的病理损害和肾间质纤维化面积,增加肾小管周围毛细血管网密度、降低缺氧诱导因子1α表达,从而改善肾间质小管缺氧状态。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程