Cytokine and lymphocyte activation during experimental acute pyelonephritis

We studied the cellular and humoral events which follow experimental acute pyelonephritis from P-fimbriated Escherichia coli to gain insight into the relationships among cells and specifically cytokines to determine how early events in untreated infection lead to renal damage. Cynomolgus (Macaca fascicularis) monkeys were studied after they were subjected to unilateral ureteral bacterial inoculation. We evaluated the blood for leukocytosis and studied lymphocyte subsets using flow cytometry and monoclonal antibodies to the subsets and serum, complement, cytokines and antibody titers. Interleukin-1, 2, and 6 and tumor necrosis factor (TNF) were assayed by enzyme-linked immunoadsorbent assay (ELISA), using monoclonal and polyclonal antibodies. Leukocytosis was marked and there were significant elevations in serum cytokines, interleukin-1, 2 and 6 with only small changes in the level of TNF. Interleukin-2 levels were sustained and may have upregulated the homing receptor for virgin lymphocytes. The studies illustrated the unique relationship between cytokines and lymphocytes and the response to bacterial infection, showing that the inflammatory response is regulated not only by cytokine activity but also by lymphocyte activation.     

细胞因子基因多态性与移植肾慢性排斥反应的关系

目的探讨供、受者细胞因子基因多态性与移植肾慢性排斥反应的关系。方法用序列特异引物聚合酶链反应（PCR-SSP）方法，对144例肾移植受者和65例部分供者进行5种细胞因子[肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）、白细胞介素10（IL-10）、转化生长因子β1（TGF-βt）、干扰素7（IFN-7）]基因型检测。结果TGF-β1。高分泌型的受者与中低分泌型受者相比，移植肾慢性排斥反应发生率明显升高，差异有统计学意义（P〈0．01）。TGF-β1高分泌型的供者与中低分泌型供者相比，移植肾慢性排斥反应发生率差异无统计学意义（P〉0．05）。TGF-β1，基因型为供者高分泌／受者高分泌组合时，移植肾慢性排斥反应发生率比所有其它基因型组合者高（P〈0．01）；而TGF-β1。基因型为供者中低分泌／受者中低分泌组合时，移植肾慢性排斥反应发生率比所有其它基因型组合者低（P〈0．01）。TNF-α、IL6、IL-10及IFN-γ的基因型与移植肾慢性排斥反应发生率的关系不明显。结论同时检测供、受者TGF-β1，基因多态性对预测移植肾慢性排斥反应发生率有指导意义。     

供者细胞因子基因多态性对肾移植受者急性排斥反应发生的影响

目的 研究供者的细胞因子和细胞因子受体基因多态性对肾移植受者急性排斥反应发生的影响.方法 (1)将126例肾移植受者分成急性排斥组和无排斥组,比较可能影响发生急性排斥反应的因素在两组中的分布情况;比较两组受者中供者的13种细胞因子及受体22个位点的基因型及部分细胞因子表达型的分布情况.(2)根据HLA-DR配型分成0～1个HLA-DR位点错配、HLA-DR完全错配两种情况,分别比较阳性基因多态性在急性排斥组和无排斥组中的分布情况.结果 (1)急性排斥组的HLA-DR错配数明显高于无排斥组;无排斥组供者的白细胞介素(IL)-1α889 C/C、IL-1Ra raspI 11100 T/T、IL-4Rd+1902 A/A、转化生长因子(TGF)-β1密码子10 C/C、IL-10-1082A/A、IL-10低表达型的频率明显较高,IL-12～1 188 A/A、IL-2-330 G/G、IL-10 GCC/ATA频率明显较低;(2)HLA-DR 0～1个位点错配时,两组供者中IL-1Rα msp I 11100 T/T、IL-4Rα+1902 A/A、TGF-β1密码子10 C/C、IL-2-330 G/G、IL-10低表达型频率明显不同,而在HLA-DR完全错配时,只有IL-12-1188 A/A表达频率明显不同.结论 供者的IL-1α-889 C/C、IL-1Rα msp I 11100 T/T、IL-4Rα+1902 A/A、TGF-β1,密码子10 C/C、IL-10-1082 A/A和IL-10低表达型是不发生肾移植急性排斥反应的遗传学指标,而IL-12-1188 A/A、IL-2-330 G/G则是发生急性排斥反应的遗传学危险因素.HLA-DR错配状况可干扰供者细胞因子基因多态性对急性排斥反应发生的影响.     

抗体芯片在慢性肾脏病患者尿中多种细胞因子同步检测中的初步应用

目的 应用抗体芯片技术检测慢性肾脏病(CKD)患者尿液中细胞因子的水平，并探讨其临床意义。方法 研究对象共10例，7例为CKD患者，全部符合 K/DOQI指南中CKD的诊断标准，并且有肾脏病理诊断。依据GFR水平以及CKD分期，将7例患者分为两组:Ⅰ组:GFR≥80 ml•min-1•(1.73 m2)-1(CKD1~2期)共4例； Ⅱ组:GFR≤40 ml•min-1•(1.73 m2)-1(CKD3~5期)共3例。另选取3例性别、年龄相匹配的健康人作为正常对照。应用Raybiotech人类细胞因子抗体芯片，同时检测各组尿液中20种细胞因子水平的变化。结果 与正常对照组相比，CKD患者共有15种因子的水平发生了显著变化。单核细胞趋化蛋白(MCP)-1、RENTES、金属蛋白酶组织抑制物(TIMP)-1、肿瘤坏死因子(TNF)-α、血管内皮生长因子(VEGF)、E-选择素(seletin)、Fas、细胞间黏附分子(ICAM)-1、白细胞介素(IL)-2、基质金属蛋白酶(MMP)-2、转化生长因子(TGF)-β和血小板源生长因子(PDGF)-BB的水平同正常对照组相比升高了2~5倍，其中尿MCP-1， RANTES， TIMP-1， TNF-α和 VEGF的水平随着GFR的下降而进一步升高。VCAM-1 和PDGF-BB的水平同正常对照组相比有所下降。在芯片所包含的20种细胞因子中，MMP-9的水平变化尤其显著，同正常对照组相比，CKDⅠ组是正常对照组的492.8倍，CKDⅡ组是正常对照组的198.7倍。结论 首次应用抗体芯片技术，证实CKD患者尿液中细胞因子表达水平同正常对照组有明显差异，并且与疾病所处阶段有一定的关系。     

The biologic significance of the mixed lymphocyte kidney culture in humans

The mixed lymphocyte kidney culture (MLKC) in humans has been studied in normal and abnormal clinical conditions. Human renal cortical cells were extracted by collagenase treatment from the kidneys of "normal" heart-beating cadaver organ donors (n = 13), patients with end-stage renal disease (ESRD) at pretransplant bilateral nephrectomy and splenectomy (n = 13), and from irreversibly rejected renal allografts at the time of graft nephrectomy (n = 5). Proliferation of peripheral blood T lymphocytes of 2-DR-mismatched volunteers occurred in response to kidney cortical cells extracted from each of the 3 donor categories in a reaction termed the allogeneic mixed lymphocyte kidney culture. Additionally, splenic T cells from cadavers and patients with ESRD were seen to react to their autologous kidney cells. The renal cortical cells extracted from ESRD kidneys were more stimulatory in the allogeneic and autologous MLKC responses than those extracted from "normal" cadaver kidneys even when the ESRD kidneys were 99% depleted of passenger T and B lymphocytes by treatment with monoclonal antibodies T11 and B1. In order to help define the antigens operative in the MLKC, we pretreated stimulating lymphocytes and renal cortical cells with anti-class II monoclonal antibodies. The allogeneic mixed lymphocyte reaction and MLKC were inhibited ca. 80% and 30%, respectively. The autologous MLKC was unaffected by this treatment. To further support that tissue-specific immune mechanisms were operative in the reaction, experiments were performed with infiltrating lymphocytes isolated from the ESRD kidneys, which were seen to generate a proliferative response when stimulated with autologous cortical cells. However, the response of these same infiltrating lymphocytes when stimulated with allogeneic lymphocytes (MLR), was markedly weaker than the response of the patients' autologous spleen cells. In addition, two kidneys were obtained at rejection from recipients that had received grafts from HLA-MLR-identical sibling donors. A lymphoproliferative reaction of recipient peripheral blood T lymphocytes occurred in response to (donor) renal cortical cells, but not to donor peripheral blood lymphocytes. In contrast, infiltrating (recipient) kidney lymphocytes responded to the kidney cortical cells and to donor peripheral blood lymphocytes. Moreover, peripheral blood T lymphocytes of the HLA-identical donor responded to his own kidney cortical cells, which were isolated from the rejected recipient kidney, and did not respond to recipient peripheral blood lymphocytes. Finally, a "normal" cadaveric kidney was fortuitously available at the same time that a rejected transplant (cadaver)     

Seminal plasma stimulates cytokine production in endometrial epithelial cell cultures independently of the presence of leucocytes

We examined the effects of normal and leucocyte-positive semen on cytokine expression in endometrial epithelial cell cultures. Cytokines in pooled seminal plasma (SP) samples of men with normal semen parameters without (n = 9) and with leucocytospermia (LCS) (n = 9) were determined. Cultures of epithelial endometrial cells of women (n = 7) in the secretory phase were incubated with 10% SP for 24 h. Cytokines in culture supernatants and mRNA concentrations in the cultured cells were determined. Mean concentrations of interleukin (IL)-1β and transforming growth factor (TGF)-β1 were significantly higher (P < 0.05) in culture media with SP of men with normal semen parameters and LCS compared with control. Accordingly, a significant increase (P < 0.05) in mRNA concentrations (amol ml⁻¹) of IL-1β and TGF-β1 could be detected. The mean TGF-β1 and IL-1β concentrations in the culture supernatant with 10% SP from patients with normal semen parameters were 24-fold (P < 0.05) and 2-fold higher respectively when compared with control. The mean protein concentration of TGF-β1 measured in the supernatant with SP of men with LCS was not significantly reduced as compared with the supernatant with normal SP. In conclusion, our experiments support the concept of an endometrial sensitation effect by semen.     

冻存的亲属活体供肾者的骨髓源性树突状细胞介导受者淋巴细胞反应

目的 探讨冻存的供者骨髓源性树突状细胞(DC)介导肾移植受者淋巴细胞反应的可行性.方法 肾移植前取供者骨髓,分离单个核细胞,液氮冻存备用.术后1、3、6、9个月,取冻存的供者骨髓细胞分离CD34+细胞,并培养DC,检测各时期DC活细胞的比率;同时,分别以供者DC和外周血淋巴细胞(PBL)作为刺激细胞,以术前未冻存过的供者骨髓源性DC作为对照,观察健康志愿者以及受者淋巴细胞对冻存不同时间的供者DC和PBL刺激的反应.结果术前每10 ml骨髓中可分离骨髓单个核细胞(BMMC)的数量平均为6.8 × 107个,免疫磁珠分选获得CD34+细胞数为(4.10±0.58)×105个.冻存不同时间的BMMC复苏后,所分离到的CD34+细胞的比率并无明显差异.术后1个月时,BMMC、CD34+细胞存活率均大幅明显下降,以后呈缓慢进行性下降,BMMC的下降幅度大于CD34+细胞.DC的活细胞比率维持在93.2%～94.8%.肾移植术后,各组DC对健康志愿者和受者淋巴细胞的刺激能力均显著强于PBL(P＜0.05);在术后各时间观察点,健康志愿者淋巴细胞对DC刺激的反应波动在12 067±1190至14 238±1220(P＞0.05),受者淋巴细胞对DC刺激的反应波动较大,在9490±1386～15 233±1098.结论 供者骨髓源性DC具有稳定的细胞活力和刺激能力,其作为术后长时间内介导肾移植受者淋巴细胞反应的刺激原,明显优于供者外周淋巴细胞,术前仅需一次性取少量骨髓冻存,术后可随时且多次用于监测.

Abstract：

Objective To explore the feasibility of mediating recipient lymphocyte reaction with donor dendritic cells (DCs) in renal allograft recipients. Methods Donor bone marrow monocytes (BMMCs) were isolated and cryopreserved in liquid nitrogen before kidney transplantation. At 0 day, 1month,3 month, 6 month and 9 month post-operation, CD34+ cells which were isolated from frozen BMMCs and cultured into DCs as well as the peripheral blood lymphocytes (PBLs) of donors were used as the stimulating cells to the PBLs of recipients and healthy volunteers. The number of viable DCs from frozen- and room temperature-preserved BMMCs was counted and the reactions of recipients'and healthy volunteers' lymphocytes to DCs and donor PBLs were measured. Results 6. 8 × 107BMMCs were isolated from each 10 ml of donor bone marrow on average while (4. 10 ± 0. 58) × 105CD34+ cells were isolated by magnetic active cell sorting (MACS). There was no significant difference in the isolating rate of recovered CD34+ cells at each observation point postoperatively. The percentage of viable BMMCs and CD34+ was decreased significantly at 1 month after surgery, then, decreased slowly and progressively. The decreasing rate of BMMCs was higher than CD34+. The rate of viable DCs was maintained stable (93. 2%-94. 8% ) in each group. The reactions of recipients' and healthy volunteers' lymphocytes to DCs were stronger than those to donor PBLs (P＜0. 05). The reactions of healthy volunteers' lymphocytes to DCs were maintained stable while those of recipients' were fluctuating. Conclusion Bone marrow-derived DCs are superior to PBLs in mediating long-term lymphocyte reaction after kidney transplantation due to their stable viability and stimulating ability to lymphocytes. Only once collection of a small quality of bone marrow of donors is needed to meet the demand of immune monitoring at any time after transplantation.     

供者DC介导的淋巴细胞反应在肾移植后个体化免疫抑制治疗中的作用

目的 探讨根据供者骨髓源性树突状细胞(DC)介导肾移植受者淋巴细胞反应指导肾移植术后免疫抑制剂个体化应用的价值。方法 2008年1月至2010年1月间接受亲属活体供肾移植者30例,根据药物剂量调整依据的不同分为试验组和对照组,每组各15例,免疫抑制方案同为他克莫司(Tac)+吗替麦考酚酯(MMF)+皮质激素。试验组术后根据受者淋巴细胞对供者DC的反应强度,结合血Tac、MMF浓度调整药物剂量;对照组术后仅根据血药浓度调整药物剂量。术后每月检查肝功能、肾功能、血常规、尿常规、血糖,随访时间为1年。结果 随访期内试验组急性排斥反应的发生率为13.3％,对照组为46.7％(P＜0.05);试验组和对照组的感染发生率分别为6.7％和40.0％(P＜0.05);试验组和对照组不良反应的总体发生率分别为13.3％和46.7％(P＜0.05)。两组各时间点的血清肌酐的差异无统计学意义(P＞0.05)。结论 利用供者骨髓源性DC介导肾移植受者淋巴细胞反应结合治疗药物血药浓度监测作为免疫监测指标,指导肾移植术后免疫抑制剂个体化应用,较仅利用血药浓度监测更全面、准确。     

Serial mixed lymphocyte culture responses in pigs after liver or kidney auto- or allotransplantation

In order to investigate further the peculiarly limited response of the transplanted pig liver to rejection, this study investigated serial mixed lymphocyte culture (MLC) responses and suppression of phytohaemagglutinin (PHA)-stimulated lymphocytes by serum from transplanted animals. The responses of lymphocytes from liver allografted pigs were compared with those from sham-operated, liver-autografted or kidney auto- or allografted animals for up to 22 weeks postoperatively. Lymphocytes from operated animals were added to cultures of 2 'universal' donors which were not anaesthetised at any time and which remained lymphocyte donors for the period of each operated pig's survival. After liver allograft, a sustained stimulation of MLC responses was noted which contrasted with the minimal random stimulatory activity seen in the other groups. The response was not seen in lymphocytes from animals subjected to liver autograft. There was a surprising variability in the week-to-week MLC tests of individual animals for which no cause could be found. The serum from liver auto- and allografted pigs had suppressive action on the PHA stimulation of lymphocytes. There was no correlation between the length of survival and the intensity of the pre-operative MLC response. Whilst the biochemical data of liver allografted pigs showed evidence of ongoing hepatocyte damage, the histological changes were less obvious especially in the long-surviving group. This study highlights again the ability of unimmunosuppressed pigs to accept liver allograft and seems to associate this with sustained hepatocyte damage and stimulation of the MLC.     

Failure of rat kidney nephron components to induce allogeneic lymphocytes to proliferate in mixed lymphocyte kidney cell culture

Rat kidney glomerular mesangial, glomerular epithelial, and tubular epithelial cells were isolated in virtually pure form and studied in mixed lymphocyte kidney cell culture (MLKC) for their ability to induce allogeneic spleen lymphocyte proliferation. The responses were compared with proliferative responses induced by allogeneic endothelial cells or spleen lymphocytes in the same strain combination. Stimulator cells were treated or left untreated with gamma-interferon, known to increase the major histocompatibility complex class II (and class I) expression of the stimulator cells. The results demonstrate that the nephron components are not able to induce lymphoproliferation in the MLKC. In contrast, the endothelial cells of rat heart were potent inducers of lymphoproliferation in mixed lymphocyte endothelial cell cultures (MLEC), as were allogeneic spleen cells. Although the kidney parenchymal cells have been shown to be immunogenic in vivo, the present finding suggests that they are unable to function as antigen-presenting cells on their own.     

供、受者细胞因子基因多态性预测肾移植效果的意义

目的 评价供受者细胞因子基因多态性预测移植肾急性排斥反应的意义。方法 采用序列特异引物聚合酶链反应(PCR-SSP)方法,对144例肾移植受者和65例供者进行细胞因子INF-α,IL-10,IL-6,TGF-β及IFN-γ基因型检测。结果 受者TNF-α或IL-10为高分泌型时移植肾急性排斥反应发生率与受者为低分泌型者相比有极显著性差异(P<0.01)。供者TNF-α或IL-1O为高分泌型时移植肾急性排斥反应发生率与供者为低分泌型者相比无显著性差异(P>0.05))。受者高分泌/供者高分泌TNF-α基因型组合的受者急性排斥反应发生率比所有其它基因型组合者高(P<0.01),而受者低分泌/供者低分泌TNF-α基因型组合的受者急性排斥反应发生率比所有其它基因型组合者低(P<0.01)。结论 同时检测供受者TNF-α基因型能为我们更加合理地选择肾移植供受者提供一种有效手段。     

肾移植受者的细胞因子及其受体单核苷酸多态性与术后感染的相关性

目的探讨肾移植受者的某些细胞因子及其受体基因多态性与术后感染的相关性。方法采用自行研制的细胞因子及其受体单核苷酸多态性检测芯片,分析129例肾移植受者的肿瘤坏死因子(TNF-α)、白细胞介素10(IL-10)、转化生长因子(TGF-β1)、IL-4、IL-6及其受体的21个位点的基因多态性分布情况,并按照患者术后是否发生感染进行分组比较。结果5种细胞因子及其受体单核苷酸多态性在感染组和非感染组中分布明显不同,分别是基因型IL-6R(-183GG、G/A)、IL-10(-824C/T,-597C/A)及TNF-α(-308GG、G/A);等位基因为IL-10R1(1112G/A)、IL-6R(-183G/A)、IL-4R(1902A/G)、TNF-α(-308G/A)及TGF-β1( 869T/C)。结论基因型IL-6R(-183GG)、IL-10(-824C/T,-597C/A)及TNF-α(-308GG),等位基因IL-4R(1902A)、IL-6R(-183G)、IL-10R1(1112G)、TNF-α(-308G)及TGF-β1( 869C)是肾移植后感染的易患因素;而基因型IL-6R(-183G/A)和TNF-α(-308G/A)可能为移植后感染的非易患因素。     

受者细胞因子基因多态性预测肾移植术后效果的意义

目的 探讨受者细胞因子基因多态性预测移植肾急性排斥反应的意义。方法 采用序列特异引物聚合酶链反应（PCR－SSCP）方法，检测144例肾移植受者细胞因子基因型。结果 TNF－α或IL－10高分泌基因型组急性排斥反应发生率高于中低分泌组，差异有极显著性意义（P＜0．001）。TNF－α、IL－10高分泌基因型组急性排斥反应发生率比其它基因型组合组高，差异有显著性意义（P＜0．05）。结论 受者TNF－α和IL－10基因型是移植肾急性排斥反应的决定性因素之一。     

Tea polyphenol inhibits allostimulation in mixed lymphocyte culture

Green tea polyphenols are known to protect allogenic donor tissues from acute rejection by their recipients. This immunosuppressive effect may be generated by a unique chemical property of the major component, epigallocatechin-o-gallate (EGCG), which can block specific cell surface molecules of the donor tissues. To test this hypothesis, we examined the effects of EGCG on the murine mixed lymphocyte reactions. EGCG treatment of stimulator cells significantly attenuated the proliferation of responder T cells. The proliferation did not recover upon the secondary stimulations by fresh untreated cells or exogenous IL-2. Flow cytometric analyses showed that EGCG treatment decreased the staining intensities of various cell surface molecules including MHC II, which plays a major role in antigen presentation, and B7.1, B7.2, and their ligand, CD28, which are required for costimulatory signals in T-cell activation. These results suggest that an anergic state of alloreactive T cells may be induced by either weakening of antigen signaling or blockage of costimulatory signals with EGCG. Other possible mechanisms behind the immunosuppressive effect and a potential use of EGCG treatment of donor tissues in transplantation medicine are discussed.     

Murine autogeneic mixed lymphocyte culture. Role of culture conditions

In an attempt to determine whether culture conditions significantly influence autoreactivity, we tested the effects of fetal calf, syngeneic, and autogeneic serum with and without 2-mercaptoethanol on the murine self-reactive mixed lymphocyte culture. We used both unfractionated and T-enriched lymph node cells as responders and unfractionated and B-enriched irradiated spleen cells as stimulators in a primary mixed lymphocyte culture (MLC). Using unfractionated cells as responders to various stimulator cell populations, we found excellent allogeneic reactions in all media tested but minimal or no self-reactivity in syngeneic of autogeneic serum. When using T-enriched lymph node cells as responders, allogeneic reactivity was excellent but self-reactivity was present only in the cultures supplemented with fetal calf serum and 2-mercaptoethanol. The possibility that the substances in fetal calf serum and/or 2-mercaptoethanol may be needed to enhance minimal, but biologically relevant, self-reactivity is discussed, as well as the possibility that the culture supplements may be inducing "nonspecific" self-reactivity.     

肾移植术后早期外周血淋巴细胞因子mRNA表达研究

目的研究肾移植受者术后早期免疫功能状态。方法采用实时荧光定量PCR检测32例肾移植受者术前当天、术后第3、7、14、28天外周血淋巴细胞有关细胞因子mRNA表达;对2例急性排斥反应（AR）患者予以附加时间点检测。结果手术前当天IL-2、IL-10、IFN-γ和TGF-β1 mRNA分别为0．171、0．121、0．062和0．128;术后第3天分别为0．161、0．165、0．059和0．134;术后第7天分别为0．149、0．148、0．054和0．129;术后第14天分别为0．153、0．135、0．056和0．136;术后第28天分别为0．188、0．165、0．060和0．146。术后第3天,IL-10、TGF-β1 mRNA表达均较术前增强,IL-10表达增强更为明显（P〈0．01）,至第7天两者表达逐渐减弱至正常水平;术后IL-2、IFN-γmRNA表达减弱,第7天降至最低水平,此后逐渐回升直至正常。2例早期AR患者IL-2、IL-10、IFN-γ、TGF-β1 mRNA表达于AR发生前后均增强,以IL-2 mRNA表达增强最为明显。结论实时荧光定量PCR检测肾移植术后早期外周血淋巴细胞因子mRNA表达,可以了解肾移植受者术后早期免疫功能状态。Th1和Th2类细胞因子均可能在AR的发生发展中发挥重要作用。