CD40分子在肾癌组织中的表达研究

目的探讨肾癌组织中CD40分子的表达与癌发生浸润转移的关系。方法用免疫组织化学二步法（Envision法）对甲醛固定石蜡包埋的肾细胞癌组织（32例）和癌旁组织（10例）中CD40的表达情况进行研究，并分析CD40分子表达水平与肾癌临床分期、病理分级和发生淋巴结转移的相关性。结果CD40在肾细胞癌组织中表达的阳性率为87．5％，与肾癌癌旁组织组相比，具有显著性差异（P〈0．01）；CD40的阳性过度表达与肿瘤临床分期、病理组织学分级和淋巴结转移显著相关（P〈0．05）。结论CD40分子在肾癌中的异常表达可为肾癌的诊断、治疗及指导预后提供实验依据，并为进一步研究肾癌的生物学，尤其是为抑制Fas和TNFR介导的细胞凋亡与基因治疗肾细胞癌打下基础。     

CD40‐Independent Help by Memory CD4 T Cells Induces Pathogenic Alloantibody But Does Not Lead to Long‐Lasting Humoral Immunity

CD40/CD154 interactions are essential for productive antibody responses to T‐dependent antigens. Memory CD4 T cells express accelerated helper functions and are less dependent on costimulation when compared with naïve T cells. Here, we report that donor‐reactive memory CD4 T cells can deliver help to CD40‐deficient B cells and induce high titers of IgG alloantibodies that contribute to heart allograft rejection in CD40?/? heart recipients. While cognate interactions between memory helper T and B cells are crucial for CD40‐independent help, this process is not accompanied by germinal center formation and occurs despite inducible costimulatory blockade. Consistent with the extrafollicular nature of T/B cell interactions, CD40‐independent help fails to maintain stable levels of serum alloantibody and induce differentiation of long‐lived plasma cells and memory B cells. In summary, our data suggest that while CD40‐independent help by memory CD4 T cells is sufficient to induce high levels of pathogenic alloantibody, it does not sustain long‐lasting anti‐donor humoral immunity and B cell memory responses. This information may guide the future use of CD40/CD154 targeting therapies in transplant recipients containing donor‐reactive memory T cells.

     

Impact of patterns of proteinuria on renal allograft function and survival: a prospective cohort study

Suhail SM, Kee TSY, Woo KT, Tan HK, Yang WS, Chan CM, Foo MWY, Li HH, Siddique MM, Wong KS. Impact of patterns of proteinuria on renal allograft function and survival: a prospective cohort study.
Clin Transplant 2011: 25: E297–E303. © 2011 John Wiley & Sons A/S. Abstract: Background: Proteinuria is an important complication in renal transplant recipients. The aim of this prospective study was to evaluate the long‐term impact of transplant proteinuria patterns on allograft function and survival. Methods: We analyzed urinary protein of a cohort of 83 renal transplants with proteinuria ≥0.5 g/d by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and radial immunogel diffusion assay. After initial stratification and analysis, the cohort was followed up for 16 yr. The graft outcome and survival were analyzed using Cox regression model to determine their association with different patterns of initial transplant proteinuria. Results: Group with predominantly glomerular (middle‐ and high‐molecular‐weight with or without low‐molecular‐weight) proteinuria (61%) had higher serum creatinine (p < 0.001) than the group with predominantly tubular (low‐molecular‐weight) proteinuria (39%). The incidences of chronic graft dysfunction and graft loss had increased in the glomerular proteinuria group (p < 0.001, hazard ratio 3.6, 95% confidence interval 1.7–7.5 and p < 0.001, hazard ratio 4.9, 95% confidence interval 1.9–12.1, respectively). Patient death did not differ (p = 0.434, hazard ratio 1.5, 95% confidence interval 0.5–4.5). Conclusion: Proteinuria in renal transplants can be differentiated into glomerular and tubular types based on molecular weight. Glomerular proteinuria is associated with significant increase in graft dysfunction and graft loss.     

Expression of CD39 by Human Peripheral Blood CD4+CD25+ T Cells Denotes a Regulatory Memory Phenotype

We have shown that CD39 and CD73 are coexpressed on the surface of murine CD4⁺Foxp3⁺ regulatory T cells (Treg) and generate extracellular adenosine, contributing to Treg immunosuppressive activity. We now describe that CD39, independently of CD73, is expressed by a subset of blood‐derived human CD4⁺CD25⁺CD127lo Treg, defined by robust expression of Foxp3. A further distinct population of CD4⁺CD39⁺ T lymphocytes can be identified, which do not express CD25 and FoxP3 and exhibit the memory effector cellular phenotype. Differential expression of CD25 and CD39 on circulating CD4⁺ T cells distinguishes between Treg and pathogenic cellular populations that secrete proinflammatory cytokines such as IFNγ and IL‐17. These latter cell populations are increased, with a concomitant decrease in the CD4⁺CD25⁺CD39⁺ Tregs, in the peripheral blood of patients with renal allograft rejection. We conclude that the ectonucleotidase CD39 is a useful and dynamic lymphocytes surface marker that can be used to identify different peripheral blood T cell‐populations to allow tracking of these in health and disease, as in renal allograft rejection.     

Transplant Acceptance Following Anti‐CD4 Versus Anti‐CD40L Therapy: Evidence for Differential Maintenance of Graft‐Reactive T Cells

Inductive therapy with anti‐CD4 or anti‐CD40L monoclonal antibodies (mAb) leads to long‐term allograft acceptance but the immune parameters responsible for graft maintenance are not well understood. This study employed an adoptive transfer system in which cells from mice bearing long‐term cardiac allografts following inductive anti‐CD4 or anti‐CD40L therapy were transferred into severe combined immunodeficiency (SCID) allograft recipients. SCID recipients of cells from anti‐CD4‐treated mice (anti‐CD4 cells) did not reject allografts while those receiving cells from anti‐CD40L‐treated mice (anti‐CD40L cells) did reject allografts. Carboxyfluorescein succinimidyl ester (CFSE) labeling of transferred cells revealed that this difference was not associated with differential proliferative capacities of these cells in SCID recipients. Like cells from naïve mice, anti‐CD40L cells mounted a Th1 response following transfer while anti‐CD4 cells mounted a dominant Th2 response. Early (day 10) T‐cell priming was detectable in both groups of primary allograft recipients but persisted to day 30 only in recipients treated with anti‐CD4 mAb. Thus, anti‐CD40L therapy appears to result in graft‐reactive T cells with a naïve phenotype while anti‐CD4 therapy allows progression to an altered state of differentiation. Additional data herein support the notion that anti‐CD40L mAb targets activated, but not memory, cells for removal or functional silencing.     

Prognostic value of cytotoxic T-lymphocytes and CD40 in biopsies with early renal allograft rejection

After renal transplantation, different immunological and non-immunological factors lead to long-term allograft deterioration. Acute rejection episodes are one risk factor for chronic renal allograft dysfunction (CRAD). Following the current Banff classification the histological grade in acute rejection episodes is of limited prognostic value, therefore, additional morphological surrogate markers would be helpful. We investigated the biopsies of 91 patients with early acute rejection episodes for the immunohistochemical expression of key molecules (perforin, granzyme B, TIA-1, CD40) in the T cell-mediated rejection process. Staining results were correlated to long-term allograft outcome. Patients with greater than 2% of granzyme B or greater than 25% of CD40-positive cells in the interstitial infiltrate showed significantly shorter allograft survival. Patients with a CD40-positive vascular rejection or greater than 2% of granzyme B-positive cells in the interstitial infiltrate were significantly correlated with an earlier onset of CRAD. Our findings provide potential morphological surrogate markers in biopsies with early acute rejection episodes after renal transplantation. These could become part of combined clinical and histological algorithms, allowing patient-specific risk estimation and customized therapy options to be made.     

CD40在大鼠腹膜间皮细胞的表达及其功能研究

目的 研究腹膜间皮细胞CD40的表达。调节因素及其活化后与细胞间黏附分子1（ICAM-1）分泌的相关性。方法 分离，培养大鼠腹膜间皮细胞，用IFN-γ，TNF-α，IL-1刺激24h，通过逆转录-多聚酶链反应（RT-PCR）及流式细胞仪（FACS）检测分析间皮细胞CD40表达。通过CD40单克隆抗体（CD40mAb)活化间皮细胞CD40，用FACS检测分析间皮细胞ICAM-1的表达。结果 间皮细胞结构性表达低水平的CD40。IFN-γ，IL-1可显著增加间皮细胞表面CD40mRNA及其蛋白的表达。而以IFN-γ增幅最大，TNF-α对间皮细胞CD40mRNA及其蛋白的表达无显著增加作用。用IFN-γ增加间皮细胞CD40表达的同时，加入CD40mAb活化CD40受体可显著增强间皮细胞ICAM-1的表达。结论 腹膜间皮细胞功能性表达CD40，其与腹腔中阳性CD40配体（CD40L^ )细胞相互作用。可能在腹腔局部防御中发挥重要作用，其结果对于限制炎症的发生和发展具有积极意义。     

Left versus right deceased donor renal allograft outcome

It has been suggested that the left kidney is easier to transplant than the right kidney because of the longer length of the left renal vein, facilitating the formation of the venous anastomosis. There are conflicting reports of differing renal allograft outcomes based on the side of donor kidney transplanted (left or right).We sought to determine the effect of side of donor kidney on early and late allograft outcome in our renal transplant population. We performed a retrospective analysis of transplanted left–right deceased donor kidney pairs in Ireland between January 1, 1998 and December 31, 2008. We used a time to death-censored graft failure approach for long-term allograft survival and also examined serum creatinine at different time points post-transplantation. All outcomes were included from day of transplant onwards. A total of 646 transplants were performed from 323 donors. The incidence of delayed graft function was 16.1% in both groups and there was no significant difference in acute rejection episodes or serum creatinine from 1 month to 8 years post-transplantation. There were 47 death-censored allograft failures in the left-sided group compared to 57 in the right-sided group ( P  = 0.24). These observations show no difference in renal transplant outcome between the recipients of left- and right-sided deceased donor kidneys.     

肾小管上皮细胞转分化在慢性移植肾肾病中的作用

目的探讨肾小管上皮细胞-肌成纤维细胞转分化在慢性移植肾肾病(CAN)的发生、发展中的作用。方法36例移植肾穿刺标本分为正常组(N组),CAN组(C组),依Banff 97标准将C组按CANⅠ、Ⅱ、Ⅲ级分为C_1、C_2、C_3组,每组9例。免疫组化染色半定量分析比较α-平滑肌肌动蛋白(α-SMA)、波形蛋白(VIM)、角质蛋白(CK_(AEI/AE3))和转化生长因子β1(TGF-β1)在各组中的表达,线性相关分析TGF—β1和α-SMA、VIM、CK_(AE1/AE3)表达的相关性。结果C_1、C_2、C_3组α-SMA表达积分分别为0．95±0．07、1．78±0．12、2．42±0．31,VIM表达积分分别为0．74±0．05、1．31±0．18、2．34±0．25,组间呈递增趋势,均明显高于N组α-SMA表达积分0．07±0．02、VIM表达积分0．09±0．02(P＜0．05)；移植肾组织中TGF-β1表达与α-SMA、VIM呈正相关(r分别为0．73、0．68,P＜0．05),而与CKAE1/AE3表达呈负相关(r=-0．71,P＜0．05)。结论肾小管上皮细胞-肌成纤维细胞转分化参与了CAN的发生、发展,而局部肾组织中TGF-β1的表达上调可能是介导肾小管上皮细胞转分化的重要原因。     

Different mechanisms of cardiac allograft rejection in wildtype and CD28-deficient mice.

Although CD28 blockade results in long-term cardiac allograft survival in wildtype mice, CD28-deficient mice effectively reject heart allografts. This study compared the mechanisms of allogeneic responses in wildtype and CD28-deficient mice. Adoptive transfer of purified CD28-deficient T cells into transplanted nude mice resulted in graft rejection. However, this model demonstrated that the allogeneic T cell function was severely impaired when compared with wildtype T cells, despite similar survival kinetics. Cardiac allograft rejection depended on both CD4+ and CD8+ T cell subsets in CD28-deficient mice, whereas only CD4+ T cells were necessary in wildtype recipients. These results suggested that CD8+ T cells were more important in CD28-deficient than wildtype mice. In addition to the CD8+ T cell requirement, allograft rejection in CD28-deficient mice was dependent on a sustained presence of CD4+ T cells, whereas it only required the initial presence of CD4+ T cells in wildtype mice. Taken together, these data suggest that CD4+ T cells from CD28-deficient mice have impaired responses to alloantigen in vivo, thus requiring long-lasting cooperation with CD8+ T cell responses to facilitate graft rejection. These results may help to explain the failure to promote graft tolerance in some preclinical and clinical settings.     

零点活检对活体肾移植术后受体肾功能的预测价值

目的探讨活体肾移植供肾零点活检对受体术后1年内移植肾功能的预测价值。方法 149例活体肾移植受者,根据是否同意活检和活检是否发现异常分为3组：未活检组（63例）,活检正常组（58例）和活检异常组（28例）。受体术后平均随访8个月,比较3组间受体术后移植肾功能恢复情况。结果供肾零点活检异常率为33%,其中肾小管炎7例,肾小管萎缩5例,肾小球硬化8例,肾小球钙化3例,肾小球玻璃样变3例,肾间质炎7例,肾间质纤维化1例,系膜增生2例以及小动脉玻璃样变2例（部分病例有一种以上病理改变）。供者年龄与移植前零点活检异常相关（P〈0.05）。从术后1个月之后至术后1年内,活检异常组各时间点受体血清肌酐均高于未活检组和活检正常组（均为P〈0.05）;术后3个月,活检异常组各时间点受体肾小球滤过率均低于未活检组和活检正常组（P〈0.05）,但术后1年内3组各随访时间点的血尿素氮比较差异无统计学意义（P〉0.05）。术后6个月内重复测量趋势分析显示,与活检正常组比较,活检异常组的血清肌酐和肾小球滤过率的变化趋势差异有统计学意义（均为P〈0.05）,活检异常组的血清肌酐与未活检组比较差异亦有统计学意义（P〈0.05）。结论活体供肾零点活检结果对术后1年内特别是术后6个月内移植肾功能有预测价值,具有临床实用性。     

系统性红斑狼疮患者外周血单个核细胞 CD40及CD40配体分子表达的研究

目的探讨CD40及CD40配体(CD40L)分子在活跃期SLE患者外周血单个核细胞(PBMC)上的表达及在SLE发病中的可能作用机制.方法采用流式细胞仪检测13例初发活跃期及13例复发活跃期SLE患者PBMC表面CD40及CD40L分子的表达.结果初发及复发活跃期SLE患者PBMC表达CD40分子(25.94±10.10)%及(27.34±12.94)%,均较正常对照组(13.99±5.26)%明显升高(P＜0.001);初发活跃期SLE患者PBMC表达CD40L分子(1.74±1.66)%较正常对照组(0.91±0.39)%亦明显升高(P＜0.05).初发与复发活跃期SLE患者之间比较PBMC表达CD40及CD40L分子无显著性差异.结论活跃期SLE患者外周血PBMC存在CD40及CD40L分子的异常表达,提示CD40及CD40L分子在SLF患者T、B细胞之间的信号传导及SLE发病机制中可能起着重要的作用.     

IL-17 expression as a possible predictive parameter for subclinical renal allograft rejection

In the present study we have tried to establish the role of IL-17 in subclinical renal allograft rejection. In this animal model, renal grafts from BN (RT1n) were transplanted heterotopically into LEW (RT1l) rats. As controls, LEW grafts were transplanted into LEW rats. The histopathological examination demonstrated that the changes in the allograft kidney on day 2 were similar to those ranked as borderline changes according to the Banff classification scale. On day 2, the serum level of blood urea nitrogen (BUN) and creatinine were the same as on day 1. The examination of allograft cytokines mRNA showed that IL-17 mRNA expressed earlier on the second postoperative day, peaked at day 5, and then declined, becoming almost undetectable at day 9, when most rats died. IL-17 antigen was also proven, by histochemical staining, to be expressed early, however we could not find the same early appearance on other Th1/Th2 cytokines. In human renal biopsy samples, the IL-17 antigen could be found scattered around in the borderline changed rejected renal allografts without evidence of a serum creatinine increase, but was undetectable both in normal controls and in renal transplant tissue without signs of rejection. IL-17 mRNA was detected in the mononuclear cells of the urinary sediment of patients suffering from borderline subclinical rejection. From the above results we can hypothesize that IL-17 could serve as a predictive parameter for borderline subclinical renal allograft rejection in the future.     

Renal allograft rupture is associated with rejection or acute tubular necrosis, but not with renal vein thrombosis.

BACKGROUND: Whereas rejection was reported to be the most common cause of renal allograft rupture (RAR) in the pre-cyclosporin era, renal vein thrombosis (RVT) is purported to be the main cause of RAR in patients taking cyclosporin. The extremely low incidence of RVT in our series (0.11%) prompted us to analyse our collective with regard to RAR. METHOD: Between 1974 and 1999, 1811 renal transplants were performed. Patients with RAR, defined as a tear of the renal capsule and parenchyma, were identified and possible underlying factors studied. RESULTS: RAR was diagnosed in nine male and five female recipients (0.8%) with a median age of 36 years. Immunosuppression consisted of azathioprine and prednisolone in seven patients and of cyclosporin-based therapy in the seven others. At exploration five grafts were removed immediately: three because of irreversible rejection, one because of deep wound infection, and one with a twisted renal vein. Six of the nine salvaged kidneys have been functioning after a mean observation time of 45 months. In the pre-cyclosporin era RAR was associated with acute rejection in five out of seven cases as compared with only three of the seven on cyclosporin treatment. Core biopsies might have been the cause in three cases. CONCLUSION: RAR is a rare complication after renal transplantation. Acute rejection still represents the most frequent cause of RAR in the cyclosporin era.     

Soluble CD30 correlates with clinical but not subclinical renal allograft rejection

Soluble CD30 (sCD30) has been proposed as a promising noninvasive biomarker for clinical renal allograft rejection, but its diagnostic characteristics regarding detection of subclinical rejection have not been assessed. We investigated sCD30 in 146 consecutive kidney allograft recipients under tacrolimus–mycophenolate‐based immunosuppression having 250 surveillance biopsies at 3 and 6 months as well as 52 indication biopsies within the first year post‐transplant. Allograft histology results were classified as (i) acute Banff score zero or interstitial infiltrates only, (ii) tubulitis t1, (iii) tubulitis t2‐3 and (iv) isolated vascular compartment inflammation. sCD30 correlated well with the extent of clinical (P < 0.0001), but not subclinical tubulointerstitial rejection (P = 0.06). To determine diagnostic characteristics of sCD30, histological groups were assigned to two categories: no relevant inflammation (i.e. acute Banff score zero and interstitial infiltrates only) versus all other pathologies (tubulitis t1‐3 and isolated vascular compartment inflammation). For clinical allograft inflammation, AUC was 0.87 (sensitivity 89%, specificity 79%; P = 0.0006); however, for subclinical inflammation, AUC was only 0.59 (sensitivity 50%, specificity 69%; P = 0.47). In conclusion, sCD30 correlated with clinical, but not subclinical renal allograft rejection limiting its clinical utility as a noninvasive rejection screening biomarker in patients with stable allograft function receiving tacrolimus–mycophenolate‐based immunosuppression.     

阻断OX40和CD40协同共刺激信号延长小鼠胰岛移植物存活时间

目的 研究阻断OX40/OX40L和CD40/CD154L协同共刺激通路对小鼠胰岛移植物存活的影响及其机制.方法 以DBA/2小鼠为供者,C57BL/6小鼠为受者,制作胰岛移植模型.受鼠分为4组.(1)对照组,注射IgG; (2)抗OX40组,注射抗OX40L单克隆抗体;(3)抗CD154组,注射抗CD154单克隆抗体;(4)联合治疗组,注射抗OX40L单克隆抗体和抗CD1 54单克隆抗体.记录各组胰岛移植物平均存活时间(MST).将CD154敲除小鼠处死,取其脾脏T淋巴细胞,体外检测活化T淋巴细胞表面OX40的表达;在活化T淋巴细胞中加入不同浓度的抗OX40L单克隆抗体,体外检测T淋巴细胞增殖情况.结果 对照组胰岛移植物MST为19 d,抗CD154组胰岛移植物MST为48 d(P＜0.05);抗OX40组胰岛移植物MST为22 d,与前两组相比较,差异无统计学意义(P＞0.05);联合治疗组胰岛移植物MST＞ 150 d,高于另外3组(P＜0.05).66％的胞表达OX40,较初始T淋巴细胞的表达率高(2％,P＜0.05);加入抗OX40L单克隆抗体后,T淋巴细胞增殖受抑制且呈剂量依赖性.结论 阻断OX40/OX40L和CD40/CD154L双通路可诱导小鼠胰岛移植物长期存活, 其发挥作用的关键机制是抑制了T淋巴细胞的增殖.     

Elimination of donor CD47 protects against vascularized allograft rejection in mice

CD47 is a ubiquitously expressed transmembrane glycoprotein that plays a complex role in regulation of cell survival and function. We have previously shown that the interspecies incompatibility of CD47 plays an important role in triggering rejection of cellular xenografts by macrophages. However, the role of CD47 in solid organ transplantation remains undetermined. Here, we explored this question in mouse models of heart allotransplantation. We observed that the lack of CD47 in donor hearts had no deleterious effect on graft survival in syngeneic or single MHC class I‐mismatched recipients, in which both wild‐type (WT) and CD47 knockout (CD47 KO) mouse hearts survived long term with no sign of rejection. Paradoxically, elimination of donor CD47 was beneficial for graft survival in signal MHC class II‐ and class I‐ plus class II‐mismatched combinations, in which CD47 KO donor hearts showed significantly improved survival compared to WT donor hearts. Similarly, CD47 KO donor hearts were more resistant than WT hearts to humoral rejection in α1,3‐galactosyltransferase‐deficient mice. Moreover, a significant prolongation of WT allografts was observed in recipient mice treated with antibodies against a CD47 ligand thrombospondin‐1 (TSP1) or with TSP1 deficiency, indicating that TSP1‐CD47 signaling may stimulate vascularized allograft rejection. Thus, unlike cellular transplantation, donor CD47 expression may accelerate the rejection of vascularized allografts.     

Evaluation of renal allograft function by Doppler spectrum analysis

A decreased renal function is rather common after renal transplantation. The causes of this decreased function are diverse and difficult to differentiate. Yet, duplex examination, and especially quantitative Doppler spectrum analysis of the blood velocities in the renal artery, may be an effective method for differentiating between some of these causes. Forty-five renal transplant recipients were included in this preliminary study. Doppler spectra were recorded from the renal artery to the allograft. Parameters were derived from every Doppler spectrum in order to characterize each spectrum. Renal allograft function was evaluated on the basis of a number of clinical parameters. A significant correlation was found between the clinical parameters and the Doppler spectrum parameters indicative for changes in the peripheral resistance. Patients with a normal renal allograft function showed Doppler spectra with a high diastolic flow, typical of a vascular bed with a low peripheral resistance. Patients with a decreased renal allograft function caused by a stenosis in the renal artery could be distinguished by a low peak velocity and a low pulsatility index. A decreased allograft function caused by allograft rejection or cyclosporin nephrotoxicity also led to characteristic arterial flow disturbances. In these cases, the peripheral resistance was increased, and this was primarily reflected in a decrease in the diastolic blood velocity. We conclude that quantitative analysis of the blood velocities in the renal artery by Doppler spectrum analysis seems to be a useful, noninvasive diagnostic tool that discriminates between some of the causes of a decreased renal allograft function.