Invariant natural killer T cells infiltrate intestinal allografts undergoing acute cellular rejection

Immunological responses in human intestinal allografts are poorly understood and accurate diagnosis of acute cellular rejection remains difficult. Here, human intestinal allografts were analyzed by multi-color quantitative fluorescent immunohistochemical morphometry in order to monitor the clinical course of rejection. Morphometry gave two-dimensional plots based on size and circularity, and identified phenotypes of individual cells infiltrating the allograft by fluorescent staining. Using this method, invariant TCRVα24(+) NKT (iNKT) cells were observed in the intestinal allograft during rejection. Because these were not identified in the normal donor intestine before surgery, this finding was considered to be a signature of acute cellular rejection of the intestinal allograft. Infiltrating iNKT cells released IL-4 and IL-5, Th2-related cytokines that antagonize the Th1 responses that induce acute cellular rejection. Histological observation suggested eosinophilic enteritis in the mucosa with elevation of IL-4 and IL-5. In conclusion, iNKT cells were recruited to the intestine; however, because higher levels of IL-4 and IL-5 may contribute to eosinophilic enteritis, timely steroid administration is recommended for allograft injury due to enteritis, as well as acute cellular rejection.     

Phenotypic analysis of lymphocytes infiltrating human cardiac allografts during acute rejection and the development of graft vascular disease

Abstract  Acute rejection (AR) and graft vascular disease (GDV) are processes mediated, at least in part, by cellular processes. Therefore, we cultured graft-infiltrating lymphocytes (GIL) from endomyocardial biopsies (EMB) taken during the first year after transplantation, determined their phenotypic composition, and correlated it to AR and GVD. We observed more often GIL growth from EMB with AR than from non-rejection EMB ( P = 0.02), but no difference was found between patients with and without GVD 1 year after transplantation. CD4 + cells were always more numerous than CD8 + cells, and no difference in phenotypic composition was found between AR and non-rejection EMB nor between EMB derived from patients with or without signs of GVD. In conclusion, AR is correlated with cell growth of EMB, but the development of GVD is not associated with AR, GIL growth from EMB, or their phenotypic composition.     

一氧化氮合成酶在大鼠小肠移植急性排斥反应中的作用

目的 观察神经型(nNOS)和诱导型一氧化氮合成酶(iNOS)在大鼠小肠移植急性排斥反应(AR)中作用.方法 行大鼠原位小肠移植.实验分为2组.1组:同系移植组(Lewis→Lewis,12例);2组:同种移植组(DA→Lewis,12例).观察术后生存时间.再灌注30 min、术后1、3、5、7d检测血清一氧化氮(NO)浓度;开腹行麦芽糖吸收实验;切取移植肠管,苏木素-伊红(HE)染色后光镜检查.免疫组织化学法观察移植肠nNOS和iNOS的活性.逆转录-聚合酶链反应(RT-PCR)法检测移植肠nNOS mRNA和iNOS mRNA的表达.结果 A组生存时间＞30 d.B组生存时间为(6.83±0.75)d.再灌注后A组nNOS染色与mRNA表达明显减弱,此后nNOS染色和mRNA表达分别于术后3、7d恢复正常.再灌注后A组iNOS染色与mRNA表达增强,此后逐渐减弱.与A组比较,术后3～7 d,B组nNOS染色减弱,iNOS染色增强,血清NO水平明显升高(P＜0.05),血糖吸收值显著降低(P＜0.01);术后5、7d,B组nNOS mRNA表达显著下降(P＜0.001),iNOS mRNA表达明显增强(P＜0.01).结论 在AR过程中,nNOS可能调节了iNOS的表达;nNOS的活性和表达与移植肠管的结构和吸收功能密切相关;iNOS的激活是加重组织损伤的重要因素之一.     

Paneth and intestinal stem cells preserve their functional integrity during worsening of acute cellular rejection in small bowel transplantation

Graft survival after small bowel transplantation remains impaired due to acute cellular rejection (ACR), the leading cause of graft loss. Although it was shown that the number of enteroendocrine progenitor cells in intestinal crypts was reduced during mild ACR, no results of Paneth and intestinal stem cells localized at the crypt bottom have been shown so far. Therefore, we wanted to elucidate integrity and functionality of the Paneth and stem cells during different degrees of ACR, and to assess whether these cells are the primary targets of the rejection process. We compared biopsies from ITx patients with no, mild, or moderate ACR by immunohistochemistry and quantitative PCR. Our results show that numbers of Paneth and stem cells remain constant in all study groups, whereas the transit‐amplifying zone is the most impaired zone during ACR. We detected an unchanged level of antimicrobial peptides in Paneth cells and similar numbers of Ki‐67⁺ IL‐22R⁺ stem cells revealing cell functionality in moderate ACR samples. We conclude that Paneth and stem cells are not primary target cells during ACR. IL‐22R⁺ Ki‐67⁺ stem cells might be an interesting target cell population for protection and regeneration of the epithelial monolayer during/after a severe ACR in ITx patients.     

外周血淋巴细胞穿孔素和颗粒酶B在肾移植急性排斥诊断中的价值

目的　探讨外周血淋巴细胞 (PBLs)中穿孔素 (P)和颗粒酶B (GB)表达水平在同种异体肾移植急性排斥诊断中的价值。　方法　定量RT PCR方法测定 10例肾移植患者移植前后PBLs中P和GB的表达情况 ,并对 3例患者急性排斥反应前后的P和GB表达情况进行对比分析。　结果　 10例患者肾移植前后P的相对表达量分别为 2 3 5± 42和 2 16± 3 5 ,GB分别为 62± 2 3和 5 7± 2 6,差异均无显著性意义 (P >0 .0 5 )。 3例发生急性排斥反应后P、GB相对表达量分别为 193 2± 3 2 6和 489± 5 7,均显著高于发生排斥反应前 (P <0 .0 0 1)。　结论　定量RT PCR测定外周血淋巴细胞中穿孔素和颗粒酶B表达可以较敏感预测肾移植急性排斥反应的发生 ,具有临床诊断参考价值     

Role of resident macrophages in the immunologic response and smooth muscle dysfunction during acute allograft rejection after intestinal transplantation.

Resident muscularis macrophages initiate an inflammatory cascade during ischemia/reperfusion that is associated with dysmotility and the activation of immunologic processes. We hypothesized that these muscularis macrophages may also play a potential immunologic role for acute allograft rejection in intestinal transplantation. Orthotopic SBTx (BN‐Lew) was performed without immunosuppression. Animals were sacrificed 7 days after SBTx. The role of resident macrophages was evaluated by transplantation of macrophage‐depleted and gadolinium chloride‐treated gut. Leukocyte infiltration was investigated in muscularis whole mounts by immunohistochemistry. Mediator mRNA expression was determined by Real‐Time‐RT‐PCR. Apoptosis was evaluated by TUNEL. Smooth muscle contractility was assessed in a standard organ bath. In comparison to vehicle‐treated grafts, macrophage‐depleted grafts exhibited significantly lower mediator mRNA peak expression (IL‐6, IL‐2, IL‐10, MCP‐1, iNOS, TNFα, IFNγ, FasL), leukocyte infiltrates (ED1‐ and ED2 positive monocytes and macrophages, neutrophils, CD4⁺ and CD8⁺ lymphocytes), apoptosis rates and an improved histologic rejection grading. Vehicle‐treated grafts showed a 77% decrease in smooth muscle contractility compared to naïve controls, while macrophage‐depleted gut exhibited only a 51% decrease in contractile activity. Transplantation of macrophage‐depleted gut attenuates the functionally relevant molecular and cellular immunologic response within the graft muscularis in acute allograft rejection. Resident macrophages participate in initiating these processes.     

Soluble tumor necrosis factor-receptors are not a useful marker of acute allograft rejection: a study in patients with renal or cardiac allografts

In this study, we investigated soluble tumor necrosis factor receptor (sTNF-R) levels in plasma of patients with either a kidney or cardiac allograft when clinical suspicion of acute rejection was raised. In plasma of patients with acute renal graft rejection, the sTNF-R levels were strongly enhanced (20–150 ng/ml) as compared to plasma of patients with stable renal function. Following successful treatment of the rejection, a gradual decline in sTNF-R levels occurred with improving renal function, and an inverse correlation between creatinine clearance and sTNF-R was found. To determine whether the increase was caused by an accumulation of constitutively released sTNF-R and lack of clearance by the kidney, or whether the immunological process of the rejection caused the enhancement, we measured sTNF-R in patients suffering from acute cardiac graft rejection but with predominantly stable kidney function. Rejection of a cardiac graft did not lead to a significant enhancement of sTNF-R levels. However, treatment with ATG or OKT3 did cause enhanced sTNF-R levels, followed by a decline that reached starting values after 7 days. These results provide evidence that the immune reaction that occurs during rejection of a graft does not per se induce discernible changes in sTNF-R levels, whereas that induced by ATG or OKT3 does. Thus, sTNF-R levels are not a reliable marker in transplant recipient monitoring.     

Selective treatment of early acute rejection after liver transplantation: Effects on liver,infection rate,and outcome

To evaluate the results of selective treatment of biopsy-proven mild acute rejection episodes, we retrospectively studied 1-week liver biopsies of 103 patients with a primary liver graft in relation to liver function tests. The overall incidence of rejection was 35 %. In four patients the biopsy showed histological features consistent with rejection; in 27 patients it showed mild acute rejection (grade 1), and in 5 patients it showed moderate acute rejection (grade 2). Study group 1 consisted of 19 untreated patients with grade 1 rejection and group 2 of 8 treated patients with grade 1 rejection. At 30 and 90 days, no differences in liver function tests were found. The infection rate, histology after 1 year, and survival in the two groups did not differ. It may, therefore, be concluded that withholding treatment in histologically proven mild acute rejection is possible in selected patients based on histological, biochemical, and clinical criteria. This may reflect the functional diversity of morphologically similar lymphocytic infiltrates observed in graft biopsies showing features of mild acute rejection.Liver Transplant Group     

Selective treatment of early acute rejection after liver transplantation: effects on liver, infection rate, and outcome

Abstract To evaluate the results of selective treatment of biopsy-proven mild acute rejection episodes, we retrospectively studied 1-week liver biopsies of 103 patients with a primary liver graft in relation to liver function tests. The overall incidence of rejection was 35 %. In four patients the biopsy showed histological features consistent with rejection; in 27 patients it showed mild acute rejection (grade 1), and in 5 patients it showed moderate acute rejection (grade 2). Study group 1 consisted of 19 untreated patients with grade 1 rejection and group 2 of 8 treated patients with grade 1 rejection. At 30 and 90 days, no differences in liver function tests were found. The infection rate, histology after 1 year, and survival in the two groups did not differ. It may, therefore, be concluded that withholding treatment in histologically proven mild acute rejection is possible in selected patients based on histological, biochemical, and clinical criteria. This may reflect the functional diversity of morphologically similar lymphocytic infiltrates observed in graft biopsies showing features of mild acute rejection.     

Reproducibility of fine-needle aspiration biopsy in the diagnosis of acute rejection of renal allografts

BACKGROUND: Although it has been used as a diagnostic tool in many renaltransplant centres the reproducibility of fine-needle aspirationbiopsy (FNAB) has not been critically evaluated. METHODS: In the present study material sequentially obtained from 15patients (177 aspirations) over a 3-month period following renaltransplantation was evaluated by two independent observers.Intraobserver reproducibility was studied through the analysisof two evaluations by observer 1 performed 8 months apart. Interobserverreproducibility was calculated comparing the second evaluationof observer 1 with the evaluation of observer 2. All evaluationswere performed blindly. Slides and protocols of individual patientswere chronologically arranged and thus interpreted. Representation,total corrected increment (TCI), accuracy, and the percent agreementfor the diagnosis of acute rejection were evaluated. RESULTS: No intraobserver statistically significant differences wereobserved. Differences observed in interobserver evaluation ofTCI either during, or out of acute rejection episodes, wellas the representative quality of the sample and accuracy, werenot statistically significant. The percent of agreement forthe presence or absence of acute rejection was 77.5% for theintraobserver comparison and 76.7% for the interobserver evaluation.Both values showing either a non-significant value for the differenceor a statistical significance for the concordance. CONCLUSION: We concluded that FNAB is an accurate method with fairly goodintra- and interobserver reproducibility for the diagnosis ofacute rejection of renal allografts.     

小肠移植急性排斥反应中bcl-2及bax表达的意义

目的探讨bcl-2及bax的异常表达与小肠移植急性排斥反应的关系。方法选用近交系F344/N和封闭群Wistar/A大鼠建立同种异基因小肠移植模型,并随机分为同基因移植组、异基因移植组、异基因加普乐可复(FK506)治疗组和对照组,用免疫组织化学技术检测36只大鼠术后移植肠组织中bcl-2及bax在移植肠组织中的表达。结果异基因组术后第3天,bcl-2的表达显著低于对照组(P<0.05),并随着移植天数的增加,差异更加具有显著性意义(P<0.01);bax的表达与对照组比较差异无显著性意义(P>0.05);FK506治疗组bcl-2及bax表达与对照组比较,差异均无显著性意义(P>0.05)。结论移植肠组织内bcl-2表达水平可作为早期诊断急性排斥反应的一个有参考意义的指标。     

Alteration of intestinal intraepithelial lymphocytes after massive small bowel resection

BACKGROUND: Intraepithelial lymphocytes (IEL) comprise the inner most layer of the gut immune system, and play a critical role in protecting the host from enteric organisms. Massive small bowel resection (MSBR) is one such clinical condition where patients are at particularly high risk for the development of such enteric infectious complications. Because of this, we hypothesized that the IEL may change significantly after the formation of a MSBR. To address this, a mouse model of MSBR was created and the acute phenotypic and functional characteristics of the IEL were studied. MATERIALS AND METHODS: Mice underwent a 70% mid-small bowel resection. After 7 days, IEL were isolated and analyzed for phenotypic changes by flow cytometry. IEL cytokine expression was performed with semiquantitative polymerase chain reaction techniques. To assess the functional significance of these changes, IEL proliferative response was assessed in vitro.Results. MSBR led to significant decreases in specific IEL subpopulations: CD 44+ (used as a marker of cell maturity); CD 8alphabeta+ (marker of thymic derivation), and CD 69+ (marker of T cell activation). Compared with controls, IEL TNF-alpha mRNA expression increased 84%, while IL-2 and IL-10 mRNA expression decreased by 69 and 72%, respectively. Spontaneous proliferation of IEL in the MSBR group was significantly higher than controls, however, proliferation failed to increase with T cell stimulation.Conclusion. These changes suggest a shift to a more immature and possibly less activated cell population. It is possible that such alterations may play an important role in the increase in enterically derived infections in patients with MSBR.     

Increased apoptosis is specific for acute rejection in rat small bowel transplant

Apoptosis has been associated with several events in solid organ transplantation, including ischemia/reperfusion (IR) injury and acute rejection. To determine whether apoptosis-profiles may distinguish these two conditions, we analyzed apoptosis rates in a rat orthotopic small bowel transplant (SBT) model. SBT was performed in Lewis rats with either freshly harvested or preserved (4 h, in UW at 4 degrees C) syngeneic and allogeneic (Brown-Norway) grafts. Bowel samples were collected 2 h after reperfusion and on small bowel transplant postoperative days (POD) 1, 4, and 7. Apoptosis was detected by measuring levels of histone-associated DNA fragments and caspase 3 expression, and by determining apoptotic body counts. All markers measured 2 h after reperfusion increased profoundly in association with preservation. After a significant decrease on POD 1, apoptosis rates rose again between POD 4 and 7 only in allogeneic grafts. This distinct second increase in apoptosis may be an early and specific sign of acute rejection.     

辅助性同种异体肝肠联合移植术后急性排斥反应的监测

目的评价猪同种异体辅助性肝肠联合移植术后排斥反应的监测方法。方法将50头杂交长白猪分为3组．A、B组各20头各完成10次猪辅助性带胰头及十二指肠的同种异体肝肠联合移植术．其中B组术后予以免疫抑制治疗：C组10头完成交互的同种异体节段性小肠移植术10例。术后1、3、5、7、14、21及30d经移植肠远端造口取小肠黏膜经常规处理后。分别在光镜和电镜下观察并进行排斥反应评分。结果术后A组出现排斥反应的中位时间为8（7～12）d．迟于c组的5（3～5）d（P〈0．05）。术后1周,A组的排斥反应评分为1．11±0．20。低于C组（2．56±0．18,P〈0．05）;但比B组高（O．20±0．13,P〈O．05）。A组移植术后中位存活时问为9（7～25）d,C组为12（7～20）d．而B组术后全部成活超过30d．与以上两组比较,P〈0.05．差异有统计学意义。结论移植术后排斥反应通过肠造口取材进行监测方便有效。     

肠内营养对长期禁食肠瘘患者肠黏膜上皮内淋巴细胞及黏液屏障功能的研究

目的研究肠内营养对长期禁食的肠外瘘患者肠黏膜上皮内淋巴细胞（iIEL）及黏液屏障功能的影响。方法对2003年7月至2004年5月收治的10例唇状小肠外瘘病例，分别于实施肠内营养支持前和实施第5天及第10天，在肠镜下经小肠外瘘口距瘘口15～20cm以活检钳取检肠黏膜，行常规苏木精-伊红染色计数iIEL，免疫组织化学染色测定CD8阳性淋巴细胞比例和黏蛋白基因2阳性细胞比例，特殊染色阿利新蓝（Alcian Blue，AB）结合法检测黏液层厚度。结果肠内营养实施5d后，各活检标本黏蛋白基因2阳性细胞比例及黏液层厚度较实施前显著增高（P〈0．05）；10d后，iIEL及CD8阳性淋巴细胞比例也较肠内营养实施前显著增多（P〈0．05）。结论实施肠内营养对于长期禁食的肠外瘘患者的肠道黏膜具有保护作用，并可增强肠黏膜的免疫功能。     

巨噬细胞在小肠移植急性排斥反应期的极化状态及意义

目的  探究小肠移植术后发生急性排斥反应（AR）时巨噬细胞极化状态的改变。 方法  将6只Brown Norway（BN）大鼠和24只Lewis大鼠分为假手术组（6只Lewis大鼠）、同基因组（Lewis→Lewis,供受体各6只）和异基因组（BN→Lewis,供受体各6只）。对各组大鼠术后7 d的移植肠组织进行苏木素-伊红（HE）染色和脱氧核糖核酸末端转移酶介导的 dUTP 缺口末端标记（TUNEL）法检测,观察其病理学表现和细胞凋亡情况;采用酶联免疫吸附试验（ELISA）检测血清中M1和M2型巨噬细胞极化相关细胞因子表达水平;利用免疫荧光技术检测各组移植肠组织中M1和M2型巨噬细胞表面标志物并进行共定位计数分析。 结果  HE染色和TUNEL检测结果显示假手术组与同基因组肠上皮形态结构正常,未见明显凋亡小体;异基因组大鼠术后7 d移植肠组织上皮层绒毛结构破坏严重,隐窝数量减少,凋亡小体增多,炎症细胞浸润肠壁全层,呈现中-重度AR。ELISA结果显示异基因组受体鼠血清中M1型巨噬细胞极化相关细胞因子肿瘤坏死因子（TNF）-α、干扰素（IFN）-γ和白细胞介素（IL）-12表达水平高于假手术组和同基因组,同基因组中M2型巨噬细胞极化相关细胞因子IL-10和转化生长因子（TGF）-β表达水平高于假手术组和异基因组,差异均有统计学意义（均为P<0.05）。免疫荧光结果显示异基因组移植肠组织中M1型巨噬细胞计数多于假手术组和同基因组,同基因组M2型巨噬细胞计数多于假手术组和异基因组,差异均有统计学意义（均为P<0.05）。 结论  小肠移植术后发生AR的移植物中,大量巨噬细胞浸润肠壁全层,以M1型为主并分泌大量促炎因子,调控巨噬细胞极化方向是治疗小肠移植术后AR的潜在方法。