肝星状细胞对同种异体胰岛移植物的保护作用

目的 研究小鼠肝星状细胞对同种异体胰岛移植物的保护作用.方法 将糖尿病小鼠随机分为三组,分别为糖尿病组、单纯胰岛移植组及与肝星状细胞共同移植组.单纯移植组于肾被膜下移植入同种异体胰岛300个;共同移植组移植入肝星状细胞(3×105个)与同种异体胰岛(300个)混合物.分别于移植术后监测受体小鼠血糖值及正常血糖维持时间;血糖正常一周后移植组及糖尿病组小鼠分别采血检测血清中TGF-β,TNF-α,IL-1β,IFN-γ的含量,同时取出移植物进行免疫组织化学检测.结果 术后共同移植组受体正常血糖维持时间为(23.75±8.96)d,单纯胰岛移植组受体正常血糖维持时间为(11.9±6.92)d,差异有统计学意义(P<0.05);三组受体血清中TNF-α、IL-1β、IFN-γ含量差异无统计学意义(P>0.05),共同移植组受体血清中TGF-β含量为(2292.31±5.87)pg/ml,单纯移植组为(1246.55±38.91)pg/ml,两组比较差异有统计学意义(P<0.05);病理学结果显示共同移植组胰岛素表达量大,并且在移植物周围有生物包膜形成.结论 在同种异体移植模型中,肝星状细胞可能通过高分泌TGF-β、局部形成包囊等方式保护胰岛移植物并延长其存活时间.     

经hCTLA4-Ig基因转染的猪胰岛细胞在小鼠体内的存活与功能状况研究

目的研究将人细胞毒T淋巴细胞相关抗原4免疫球蛋白（hCTLA4-Ig）基因转染至猪胰岛细胞后，再将其移植至小鼠体内的存活及功能状况。方法采用腺相关病毒载体（AAV）介导hCTLA4-Ig基因体外转染新生猪胰岛细胞（NIPs），再将转基因的NIPs移植至构建了人免疫系统的SCID糖尿病小鼠左肾被膜下。逆转录聚合酶链（RT-PCR）反应和免疫荧光染色法检测转染后hCTLA4-Ig基因的表达状况；观察小鼠移植转基因NIPs后的生存时间；移植物免疫组织化学分析及酶联免疫（ELISA）法测定受者血清中细胞因子的水平。结果NIPs经转染后，可检测到hCTLA4-Ig基因及其蛋白表达，且葡萄糖刺激胰岛素释放试验与未转染的对照组比较，差异无统计学意义（P〉0．05）；糖尿病小鼠经转基因的NIPs移植后，生存时间及血糖维持正常的平均时间分别为（72．5±30．6）d和（59．1±24．0）d，较未转染的对照组（26．9±6．9）d和（12．7±3．3）d显著延长（P〈0．01）；免疫组织化学染色检测移植后不同时间（15、30、90d）的移植物组织，可见转基因细胞移植部位有完整的胰岛细胞，而对照组胰岛细胞破坏、消失，周围可见较多的炎性细胞浸润；且转基因细胞移植的小鼠血清白细胞介素2（IL-2）、γ干扰素（IFN-γ）、肿瘤坏死因子α（TNF-α）的水平明显低于对照组（P〈0．05）。结论AAv介导hCTLA4-Ig基因体外转染猪胰岛细胞后再移植至受者体内，可提高受者的免疫耐受水平，延长胰岛细胞在异种受者体内的存活时间，而胰岛细胞的内分泌功能不受明显影响。     

大鼠胰岛转染人血红素氧合酶-1基因对同种胰岛移植的影响

目的探讨大鼠胰岛转染人血红素氧合酶-1(HO-1)基因在胰岛移植中的潜在应用价值。方法采用携带人HO-1基因的腺病毒对新分离的SD大鼠胰岛细胞进行转染;对体外培养的胰岛细胞使用重组人肿瘤坏死因子α及放线菌酮诱导凋亡。使用流式细胞术检测凋亡率;每只链脲霉素诱导的糖尿病模型大鼠门静脉内置入约1200个胰岛当量的胰岛后,观察糖尿病大鼠血糖及体重变化;免疫组织化学法检测肝内移植胰岛细胞的胰岛素、HO-1蛋白表达及表达CD3抗原的淋巴细胞浸润情况。结果转染人HO-1基因的胰岛细胞凋亡率明显低于对照组(P<0.05);单纯胰岛细胞移植后移植物存活时间为(5.33±4.18)d,转染人HO-1基因的胰岛细胞移植后移植物存活时间为(10.56±4.33)d,两组比较,P<0.05;转染人HO-1基因的胰岛细胞培养48 h即见人HO-1蛋白表达;肝内移植7 d后移植物有人HO-1蛋白表达;移植胰岛周边及胰岛内淋巴细胞浸润程度较单纯胰岛移植组明显减轻。结论大鼠胰岛转染人HO-1基因能够增加体外培养胰岛细胞的抗凋亡能力,并能延长体内移植胰岛的存活时间,减轻淋巴细胞对胰岛的浸润。     

Th1/Th2细胞因子对器官移植排斥反应的影响

目的:研究Th1/Th2细胞因子对同种异系小鼠心脏移植存活时间的影响.方法:使用野生型BALB/c小鼠作为供体,野生型B6小鼠、IL-4基因去除B6小鼠及IFN-γ基因去除B6小鼠作为受体,行腹部异位小鼠心脏移植.部分IL-4基因去除小鼠、IFN-γ基因去除小鼠联合应用α-半乳糖神经酰胺(α-galactosylceramide,α-GalCer),以获得更强的Th1/Th2偏移.比较移植物存活时间.向野生型、IL-4基因去除及IFN-γ基因去除B6小鼠腹腔内注射供体小鼠脾细胞,提取受体小鼠脾脏CD8<'8>T细胞行淋巴细胞毒试验.结果:IFN-γ基因去除组小鼠的移植物存活时间为(6.40±0.55)d,联合应用α-GalCer组移植物存活时间为(8.00±1.15)d.IL-4基因去除组小鼠的移植物存活时间为(8.00±1.00)d,联合应用α-GalCer组移植物存活时间为(8.60±1.34)d.淋巴细胞毒试验显示IFN-γ基因去除小鼠的CD8+T细胞毒性明显增强.结论:Th1/Th2细胞因子与排斥反应之间并不存在简单的对应关系.     

IFN-γ对小鼠心脏移植免疫耐受的影响

目的探讨γ干扰素(IFN-γ)对阻断共刺激信号CD40-CD40配体所诱导的小鼠心脏移植免疫耐受的作用。方法移植心脏和脾脏取自行心脏移植后的同种同系受体和同种异系受体(包括接受和未接受抗CD40配体抗体治疗),使用实时定量RT-PCR方法测定IFN-γ的表达。比较野生型小鼠和IFN-γ基因去除小鼠受体移植物的存活时间。同时比较接受MR-1治疗的野生型小鼠受体和IFN-γ基因去除小鼠受体的CD4+T细胞混合淋巴细胞反应和CD8+T细胞的细胞毒性。结果发生排斥反应的移植物较免疫耐受移植物表达更高的IFN-γ。IFN-γ基因去除小鼠受体如未接受免疫抑制治疗,移植物存活时间无明显延长,较之野生型小鼠受体反而有所缩短。使用MR-1在野生型小鼠受体中诱导出移植物的长期存活,但在IFN-γ基因去除小鼠受体中却无类似效果。IFN-γ缺失使T细胞的增殖活性及细胞毒性增强。结论在阻断共刺激信号CD40-CD40配体所诱导的移植免疫耐受中,IFN-γ能够促进免疫耐受状态的形成。     

新生猪Sertoli细胞与大鼠胰岛细胞联合移植延长大鼠移植胰岛的存活时间

目的 探讨新生猪Sertoli细胞(NPSCs)与大鼠胰岛细胞联合移植对延长大鼠同种异体胰岛移植物存活时间的作用及其主要的机制.方法 将糖尿病Wistar大鼠随机分为3组.(1)单纯移植组(n=6):单纯移植1500个胰岛当量(IEQ)的SD大鼠胰岛细胞至糖尿病大鼠的左肾包膜下;(2)分侧移植组(n=4):将1500个IEQ的SD大鼠胰岛细胞移植到糖尿病大鼠的左肾包膜下,同时将1×10~7个NPSCs移植到糖尿病大鼠的右肾包膜下;(3)混合移植组(n=6):将1500个IEQ的SD大鼠胰岛细胞和1×10~7个NPSCs混合移植到糖尿病大鼠的左肾包膜下.移植后每天监测各组的血糖水平,以了解移植物的存活时间;移植后发生排斥反应时获取移植物标本,行HE和免疫组织化学染色,观察移植物中CD3~+T淋巴细胞浸润情况及细胞凋亡调控基因(Bcl-2)和血红素氧合酶1(HO-1)基因的表达水平.结果 单纯移植组、分侧移植组和混合移植组胰岛移植物存活时间分别为(5.7±1.0)d、(5.3±0.5)d和(16.3±1.4)d,混合移植组存活时间较其他两组显著延长(P<0.05).单纯移植组移植区可见大量淋巴细胞浸润,主要为CD3+T淋巴细胞;混合移植组移植区Bcl-2基因呈高表达;各组移植区HO-1基因均有表达,差异不明显.结论 NPSCs与大鼠胰岛细胞联合移植可以延长大鼠同种异体胰岛移植物的存活时间,其机制可能与NPSCs抑制移植物淋巴细胞浸润、促进Bcl-2基因高表达的局部免疫调节作用有关.     

输注胰岛抗原特异性Treg细胞延长同系NOD小鼠移植胰岛的存活时间

目的 探讨输注胰岛抗原特异性调节性T淋巴细胞(Treg细胞)对非肥胖糖尿病(NOD)小鼠同系胰岛移植物存活时间的影响.方法·以未成熟树突状细胞(imDC)联合谷氨酸脱羧酶-65在体外诱导童贞T淋巴细胞分化成胰岛抗原特异性Treg细胞.以已发生糖尿病的NOD小鼠为受者,将分离得到的尚未进展为糖尿病的NOD小鼠的胰岛(500胰岛当量)移植至受者的肾包膜下,对照组不行移植,只观察血糖变化;单纯胰岛移植组只进行胰岛移植,不输注胰岛抗原特异性Treg细胞;实验组于术前1d静脉输注1×106个胰岛抗原特异性Treg细胞,然后进行胰岛移植.术后检测受者的血糖,以判断移植胰岛的存活时间,观察胰岛移植物的病理学变化.结果 对照组血糖持续高于11.1 mmol/L;单纯胰岛移植组小鼠的血糖于术后1～2 d降至正常,到7～17d时开始陆续升高,并维持在术前水平,移植物存活时间为(12.2±2.6)d;实验组小鼠的血糖于术后1～2 d降至正常,至第27天开始有小鼠血糖升高超过11.1 mmol/L,第43天时,所有小鼠的血糖均超过11.1mmol/L,移植物的存活时间为(35.2±4.3)d,明显长于单纯胰岛移植组(P＜0.01).单纯胰岛移植组的移植胰岛有明显的淋巴细胞浸润,并伴有胰岛细胞严重破坏,胰岛素染色未见完整的胰岛存在,仅有极少量残存的分泌胰岛素的胰岛细胞;实验组第15天时移植胰岛形态完整,仅有少量淋巴细胞浸润,分泌胰岛素的胰岛大量存在.结论 体外诱导产生的胰岛抗原特异性Treg细胞可以延缓自身免疫系统对移植胰岛的破坏,明显延长NOD小鼠移植胰岛的存活时间.     

Sertoli细胞诱导大鼠肝内胰岛移植物免疫豁免的实验研究

目的 探究睾丸Sertoli细胞能否对肝内共移植的胰岛移植物提供免疫豁免作用以及共移植的睾丸Sertoli细胞最佳数量。方法将同种大鼠胰岛及不同数量的睾丸Sertoli细胞同时移植于糖尿病受体的肝内,观察移植物存活情况、胰岛功能、并检测移植物内胰岛素和Fas配体(FasL)表达以及浸润淋巴细胞凋亡情况。结果单纯胰岛移植组平均存活期为(5.6±0.8)d,同时与胰岛细胞在肝内共移植的睾丸细胞数增加至1×107个时,平均存活期为(41.4±4.61)d,明显延长(P<0.05),胰岛移植物中有大量表达FasL的睾丸细胞和表达胰岛素的胰岛细胞.在移植物周围有大量浸润的淋巴细胞凋亡。结论睾丸Sertoli细胞与胰岛细胞同时在肝内共移植,通过诱导局部豁免而延长胰岛移植物的存活时间,且同时共移植1×107个Sertoli细胞时效果最好。     

激活型Akt1转染大鼠胰岛联合免疫抑制剂在异种胰岛移植中的应用

目的 探讨腺病毒载体介导激活性Akt1基因(Adv-CA-Akt1)转染大鼠胰岛对异种移植胰岛功能和存活的影响.方法 以BALB/C糖尿病小鼠为受体,分离纯化雄性Wistar大鼠胰岛,体外培养,Adv-CA-Akt1转染后异种胰岛移植.受体小鼠分3组,实验组:Adv-CA-Akt1转染的大鼠胰岛体外培养24 h,小鼠肾被膜下移植,并口服环孢素A(CsA)30 mg·kg-1·d-1;CsA组:未转染胰岛移植,同剂量环孢素口服;对照组:单纯胰岛移植.每只接受300胰岛当量(IEQs)移植.检测术后血糖,移植物存活时间及组织病理学.结果 实验组和CsA组术后2 d血糖即降至正常,胰岛功能存活时间分别为(21.0±3.65)d和(9.0±2.54)d,而对照组血糖短暂下降后再次升高,胰岛功能存活时间(4.2±2.6)d.实验组小鼠生存时间为(31.0±5.67)d比CsA组(17.0±3.35)d和对照组(10.0±1.52)d明显延长,三组比较差异有统计学意义(P<0.05);胰岛素免疫组化染色实验组.肾被膜下见较多有功能胰岛细胞团,而CsA组和对照组胰岛素染色阳性细胞数减少.结论 Adv-CA-Akt1转染大鼠胰岛联合应用免疫抑制剂,可提高胰岛功能,延长异种胰岛移植物存活时间.     

Fas配体表达对同种胰岛移植的影响

目的探究睾丸细胞FasL表达能否对共移植的胰岛移植物提供免疫豁免作用以及胰岛细胞FasL基因转染对同种胰岛移植的影响.方法将同种大鼠胰岛及睾丸细胞同时移植于糖尿病受体,重组腺病毒AdV-FasL感染胰岛细胞后移植,观察移植物存活情况、胰岛功能,并检测移植物内浸润淋巴细胞以及基因转染胰岛细胞凋亡情况.结果单纯移植胰岛组平均存活期为(6.3±0.56)?d.与胰岛细胞同时移植的睾丸细胞数增加至1×107时,存活期大于50?d(P＜0.05).表达FasL的睾丸细胞在移植物内诱导浸润淋巴细胞凋亡.FasL基因转染组出现排斥加速,存活期缩短至(3.4±0.24)?d.FasL转染的胰岛细胞在移植后24h见FasL表达,48h表达增强,移植后FasL转染胰岛细胞凋亡.结论表达FasL的睾丸细胞与胰岛同时移植可诱导活化的淋巴细胞凋亡,使胰岛移植物获得免疫豁免、存活期延长,但通过FasL基因转染使胰岛细胞直接表达FasL引起胰岛细胞凋亡和粒细胞浸润,导致排斥加速.     

Effect of liver transplantation on islet allografts: up-regulation of Fas ligand and apoptosis of T lymphocytes are associated with islet graft tolerance

BACKGROUND: Liver allografts in spontaneously tolerant strain combinations can protect other organs of the same donor origin from rejection and reverse ongoing rejection in previously placed grafts. The aims of this study were to examine whether liver allografts have the same protective effect on islet allografts and to investigate the underlying mechanisms. METHODS: PVG islets were transplanted beneath the kidney capsule of streptozotocin-induced diabetic DA rats with or without liver allografting. The cellular infiltrate, and the extent of apoptosis and of Fas ligand (FasL) expression in the islet grafts were evaluated on days 2, 4, and 7 after transplantation by means of immunostaining and the in situ terminal deoxynucleotide transferase-mediated dUTP nick end labeling assay. Donor and recipient mixed lymphocyte reactions (MLR) were determined at 7 days or 100 days after islet transplantation. RESULTS: Islet allografts transplanted alone were rapidly rejected within 5-8 days. Rejection was delayed, but not prevented, when islets were transplanted simultaneously with the liver. Liver transplantation 1 month before islet transplantation resulted in long-term survival (>100 days) of islet grafts in three of seven animals, whereas the other four died of liver rejection with functional islet grafts. Liver transplantation on day 4 after islet grafting reversed ongoing islet rejection and led to indefinite islet graft survival in three of seven cases. There was a progressive increase of cellular infiltration in all of the islet allografts, but the intensity of the infiltrate did not correlate with the outcome of the islet allografts. Islet rejection was characterized by an early dominance of monocytes/macrophages and CD25+ T cells in the infiltrates, a high incidence of apoptotic beta cells in grafts, and a sensitized status in the MLR. Tolerance of islet allografts was associated with increased numbers of dendritic cells in the graft infiltrates, up-regulation of FasL, and prominent apoptosis of alloreactive leukocytes in the islet grafts, as well as donor-specific MLR suppression in long-term survivors. CONCLUSIONS: These results demonstrate that the extent of the protective effect of liver transplantation on islet allografts varies with the time of liver grafting, ranging from delay in islet rejection to complete islet acceptance. Islet graft tolerance induced by liver transplantation is the result of an immune process that involves up-regulation of Fas ligand expression on, and apoptosis of, islet graft infiltrating lymphocytes.     

Sequential transplantation induces islet allograft tolerance

Liver transplantation (LT) in some rat strain combinations can induce tolerance to other organ grafts of liver donor strain. The aim of the study was to examine the effect of LT on islet allografts. Islets were isolated by collagenase digestion and purified by dextran-gradient separation. Islets from two donor animals (>2,000) were transplanted under the left kidney capsule of streptozotocin-induced diabetic rats. The liver was implanted orthotopically. The results showed that concurrent LT and islet transplantation (IT) prolonged survival of islet allografts modestly, but islet allografts survived indefinitely when LT was performed before or after IT. LT delayed or reversed ongoing islet graft rejection. IT could trigger rejection in otherwise tolerated liver grafts. We conclude that sequential IT-LT or LT-IT can induce islet allograft tolerance or liver rejection and that the mechanisms involved may differ from those involved with LT simultaneously with either IT or vascularized organ grafts.     

Multiple Combination Therapies Involving Blockade of ICOS/B7RP-1 Costimulation Facilitate Long-Term Islet Allograft Survival

In recent years a series of novel costimulatory molecules have been identified, including inducible costimulator (ICOS). In a fully major histocompatibility complex (MHC)-mismatched mouse model of islet transplantation, we demonstrate that while monotherapy with CTLA4-Ig, CD40 ligand monoclonal antibody (CD40L mAb) or rapamycin each improves islet allograft survival, graft rejection eventually develops. Immunohistologic analysis of rejected grafts revealed increased ICOS expression, suggesting a role for this costimulatory molecule as an alternate pathway for T-cell activation. The combination of a blocking anti-ICOS mAb with each of the above therapies resulted in significantly improved islet allograft survival, confirming the importance of ICOS signaling in islet allograft rejection. Mechanistic studies conducted in mice treated with anti-ICOS mAb and rapamycin demonstrated a lack of donor-specific immunological tolerance and an absence of regulatory T-cell activity. However, a dramatic effect was seen on acute anti-donor responses whereby anti-ICOS mAb and rapamycin significantly reduced the initial expansion and function of alloreactive T cells. These data demonstrate that blockade of the ICOS/B7RP-1 pathway has potential therapeutic benefit given its role in enhancing islet allograft survival and regulating acute alloresponses in vivo.     

Indefinite survival of rat islet allografts following infusion of donor bone marrow without cytoablation

We have tested the effect of donor bone marrow cell (DBMC) infusion on the survival of pancreatic islet allografts in the rat, without the use of cytoablative recipient conditioning. Lewis and diabetic Brown Norway rats were used as donors and recipients, respectively. Donor islets were placed beneath the left renal capsule. Infusion of DBMC and temporary immunosuppression followed by delayed islet transplantation resulted in indefinite survival of all islet grafts (MST >180 days). Control animals demonstrated recurrent hyperglycemia (islet allografts rejection). Donor bone marrow derived cells were detected in the spleen and cervical lymph nodes of BN recipients of LEW bone marrow but not in the recipients of islet transplants alone. Second set full thickness skin grafts were performed in normal BN and in recipients of a previously successful ITX. Donor specific skin grafts were accepted in the animals that had received DBMC 40 days before the islet allograft, while animals receiving DBMC at the time of the islet allograft rejected the donor specific skin graft similarly to the controls. However, these animals did not reject a second set donor-specific islet transplant. The results indicate that radiation conditioning of the recipients was not necessary to induce microchimerism and graft acceptance in this rodent model of islet allotransplantation.     

Graft survival and cytokine production profile after limbal transplantation in the experimental mouse model

Limbal transplantation or limbal stem cell (LSC) transfer represents the only way to treat severe ocular surface damage or LSC deficiency. However, limbal allografts are promptly rejected in spite of extensive immunosuppressive therapy. To characterize immune response after limbal transplantation, we established an experimental model of limbal transplantation in the mouse. Syngeneic, allogeneic and xenogeneic (rat) limbal grafts were grafted orthotopically in BALB/c mice and graft survival was evaluated. The presence of graft donor cells and the expression of IL-2, IL-4, IL-10, IFN-γ and inducible nitric oxide synthase (iNOS) mRNA in the grafts were detected by real-time PCR. While syngeneic grafts survived permanently, allografts were rejected in 9.0±1.8 days and xenografts in 6.5±1.1 days. The manifestation of clinical symptoms of rejection correlated with the disappearance of donor cells in the graft and in the recipient cornea. Intragraft expression of iNOS mRNA and distinct expression patterns of Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-10) cytokines were detected during rejection of limbal allografts and xenografts. The limbal graft rejection was prevented with anti-CD4, but not anti-CD8 monoclonal antibody therapy. The results indicate that limbal grafts do not enjoy immune privilege of the eye and are promptly rejected by Th1 (allografts) or by a combined Th1 and Th2 (xenografts) type of immune response involving CD4+ cells and iNOS expression. Targeting this pathway may be an effective way to prevent and treat limbal graft rejection.     

抗体诱导的移植免疫耐受依赖于B细胞来源的TGF-β的调节

目的 研究抗CD45RB抗体或联合抗Tim-1抗体诱导的移植免疫耐受中转化生长因子(TGF)-β的调节作用。方法  建立小鼠同种异体心脏移植以及胰岛移植模型,前者用抗CD45RB抗体和(或)抗TGF-β抗体治疗,后者用抗CD45RB抗体联合抗Tim-1抗体治疗并加用或不用抗TGF-β抗体,观察移植物长期存活时间。分离胰岛移植物长期耐受的受体B细胞,过继输注至胰岛细胞移植模型小鼠体内,经抗TGF-β抗体治疗后观察移植胰岛的存活情况。在体外将经双抗体治疗14 d的移植有胰岛细胞的受体B细胞与脂多糖(LPS)混合刺激培养过夜,采用流式细胞仪检测CD19⁺LAP⁺细胞的比例。结果  抗CD45RB抗体治疗后有6/9的同种异体小鼠心脏长期存活,但若加用抗TGF-β抗体治疗则所有移植心脏均被排斥。在小鼠胰岛细胞移植模型中,抗CD45RB抗体联合抗Tim-1抗体治疗后有90%移植物长期耐受,但加用抗TGF-β抗体后则无1例移植物长期存活。B细胞输注治疗后,8/9的胰岛移植物不被排斥,而加用抗TGF-β抗体后则移植物很快被排斥。B细胞体外刺激培养,流式细胞术检测显示CD19⁺LAP⁺细胞在B细胞中的比例显著增高(P < 0.0001)。结论  抗CD45RB抗体或联合抗Tim-1抗体诱导的移植免疫耐受需要TGF-β,其可能受调节性B细胞的调节。     

CTLA4-Ig-modified dendritic cells inhibit lymphocyte-mediated alloimmune responses and prolong the islet graft survival in mice

The induction of antigen specific tolerance is critical for prevention and treatment of allograft rejection. In this study, we transfected CTLA4-Ig gene into dendritic cells (DCs), and investigated their effect on inhibition of lymphocyte activity in vitro and induction of immune tolerance on pancreatic islet allograft in mice. An IDDM C57BL/6 murine model induced by streptozotocin is as model mouse. The model mice were transplanted of the islet cells isolated from the BALB/c mice to their kidney capsules, and injected of CTLA4-Ig modified DCs (mDCs). The results showed that mDCs could significantly inhibit T lymphocyte proliferation and induce its apoptosis; whereas, unmodified DCs (umDCs) promoted the murine lymphocyte proliferation. Compared with injection of umDCs and IgG1 modified DCs, the injection of mDCs prolonged IDDM mice's allograft survival, and normalized their plasma glucose (PG) levels within 3 days and maintained over 2 weeks. The level of IFN-gamma was lower and the level of IL-4 was higher in mDCs treated recipient mice than that in control mice, it indicated that mDCs led to Th1/Th2 deviation. After 7 days of islet transplantation, HE stain of the renal specimens showed that the islets and kidneys were intact in structure, and islet cells numbers are increased in mDCs treated mice. Our studies suggest that DCs expressing CTLA4-Ig fusion protein can induce the immune tolerance to islet graft and prolong the allograft survival through the inhibition of T cell proliferation in allogeneic mice.     

alpha4 integrin in islet allograft rejection.

BACKGROUND: Adhesion molecules are involved in multiple steps of the continuum of allograft rejection. We studied the effects of blockade of the interactions between alpha4 integrin and its ligands, vascular cell adhesion molecule-1 (VCAM-1) and fibronectin, on allograft survival. METHODS: Streptozotocin-induced diabetic CBA (H-2k) mice received islet transplants from BALB/c (H-2d) donors. Recipient mice were treated with antibodies against alpha4 integrin (PS/2), VCAM-1 (MK 2.7), and a peptide corresponding to the binding site of alpha4 integrin on fibronectin (connecting segment 1 peptide, CS1-peptide). Graft function was measured by daily tail vein blood glucose levels, with rejection defined as the return of hyperglycemia. Graft-bearing kidneys were removed for immunohistochemical analysis. RESULTS: Treatment with anti-alpha4 integrin antibody, anti-VCAM-1 antibody, or with CS1-peptide led to long-term survival of islet allografts. Recipients with long-surviving islet grafts did not show tolerance, in that they rejected a second donor-type islet allograft. Although both anti-alpha4 integrin antibody and CS1-peptide completely abolished cellular infiltration of the islet graft 7 days after transplantation, anti-VCAM-1-treated recipients showed a dense peri-islet infiltrate of activated, alpha4 integrin+, cytotoxic T cells. CONCLUSIONS: These data show that alpha4 integrin is critically important to allograft rejection. Anti-VCAM-1 antibody appears to prevent rejection without qualitatively affecting either T cell activation or migration to the graft. Conversely, anti-alpha4 integrin antibody and CS1-peptide may prevent islet allograft rejection by altering either T cell activation or lymphocyte trafficking. Blocking interactions between alpha4 integrin and its ligands may provide novel forms of immunosuppression.     

Functional assessment of immune markers of graft rejection: a comprehensive study in live-related donor renal transplantation

A better understanding of the immunobiological processes and predictors of graft rejection holds promise for the development of potential therapeutic strategies and also individualization of immunosuppression. The objective of this study is to analyze the clinical relevance of immune parameters such as antidonor antihuman leukocyte antigen (anti-HLA) antibodies, monitoring of cytokines and their receptors on the graft outcome following live-related donor renal transplantation. Flow cytometry-based methods were used to detect antidonor antibodies (flow cytometry crossmatch, FCXM) and intracellular cytokines. Enzyme-linked immunosorbent assay (ELISA) methods were employed to detect anti-HLA class I and class II antibodies and quantitative serum-soluble interleukin-2 receptor (sIL-2R) levels. The data revealed that patients with HLA class I-specific IgG antibody experienced higher acute rejection (AR) episodes at 1 yr in comparison to the antibody negative group (82% vs. 56%, p = 0.01). On the contrary, donor-specific class II antibodies (B+) did not have any influence on the graft survival. However, 15 recipients having both T- and B-cell antidonor antibodies (T+B+) had significantly poor graft survival (60%) as compared to the antibody-negative group (T-B-, 82%, p = 0.05). Additionally, patients having non-donor but HLA-specific antibodies (FCXM-/ELISA+) had poor graft survival as compared to the antibody-negative group (64% vs. 88%, p < 0.05). Further, patients undergoing AR episodes had significantly higher expression of IFN-gamma-producing T cells (19.16 +/- 7.4% median 17.50) as compared to their pre-transplant levels (5.68 +/- 1.63%, Median 5.20) and the non-rejecter group (5.97 +/- 4.39%, median 4.3, p = 0.0004). Similarly sIL-2 was significantly increased in AR episodes during the first month of transplantation (292 +/- 131.5 pmol/L) as compared to those with well-functioning grafts (p = 0.01) and healthy controls (p = 0.001). Evaluation of antidonor antibodies by flow cytometry is found to be relatively more sensitive and a better predictor of graft outcome. Further monitoring of cytokine expression profile of primed peripheral T-helper cells and quantitative analysis of sIL-2R offer additional valuable diagnostic and prognostic tools for follow-up of transplant subjects and a better alternative for functional assessment of immunosuppression.     

Consideration of IL-2, IFN-γ and IL-4 expression and methylation levels in CD4+ T cells as a predictor of rejection in kidney transplant

Kidney transplantation is the certain treatment for the end-stage-kidney disease patients. However after transplantation, allograft rejection or graft dysfunction are serious problems which the patients can be encountered. In several studies new biomarkers to predict rejection episodes tried to be evaluated and cytokines are thought to be one of these biomarkers. Additionally, epigenetic regulation of the cytokine genes can be an opportunity to detect the graft survival or dysfunction that lead to rejection. In this study, we aimed to detect the expression levels and methylation profile of cytokines IL-2, IL-4 and IFN-γ to follow the clinical situation of the patients. 25 kidney transplant patients were included in our study group and peripheral blood samples were collected before and 6 months after transplantation. CD4+ T cells were separated by using magnetic separation system and expression levels are detected by qPCR while methylation profile analysis was performed by pyrosequencing. According to our study we noticed that all of the patients with allograft rejection have increased expression levels of IFN-γ. When methylation profile of the CpGs in the promotor region of IFN-γ is evaluated, +128CpG was found as methylated when compared with +122. In conclusion, epigenetic mechanisms can effect several processed in renal transplantation and further studies with higher numbers of patients are needed to detect new biomarkers for prediction of allograft rejection.