猪—猴异种血管移植免疫反应初探

目的 探讨猪－猴异种血管移植超急性排斥反应（HAR）的机理。方法 猪股静脉原位异种移植于恒河猴,发生HAR后通过免疫组化检测移植血管IgG、IgG、C3及C4的沉积。结果 大量IgM、C3和C4沉积于移植静脉内皮, 未发现IgG沉积于移植血管内皮。结论 猪－猴异种移植HAR是由异种自然抗体IgM与异抗原特异结合启动,进而以经典途径激活补体系统而发生。     

补体在猪到猴异种心脏移植中的作用及机理

目的 探讨补体在异种大动物猪到猴心脏移植排斥反应中的作用及机理.方法 以梅山猪为供者,中国猕猴为受者,行异种腹腔异位心脏移植.随机将受者分为3组.A组(5只):为空白对照组,受者心脏移植后不作任何处理.B组(5只):为照射预处理组,受者于心脏移植前28 d、即1.5个月龄时接受60Coγ3 Gy全身剂量照射,其余同A组.C组(8只):为照射+胸腺注射预处理组,心脏移植前21 d,将供者的脾细胞(按照5×107个/只的数量)注入受者的两侧胸腺内,其余同B组.观察心脏移植术后各组移植心的存活时间;猪对猴单向混合淋巴细胞培养的刺激效应;采用双抗体夹心法检测补体C3和CD46的血清浓度;通过流式细胞术检测受者外周血细胞表面IgM、IgG阳性细胞百分比水平.结果 A、B、C三组移植心的存活时间分别为:(36.6±5.8)h、(65.6±6.5)h和(91.1±22.8)h,C组移植心的存活时间明显延长,与A组比较,P＜0.01,与B组比较,P＜0.05.C组在猪对猴单向混合淋巴细胞反应中的刺激效应较A、B组明显下降(P＜0.01).B、C组移植前补体水平(C3)无明显变化,但随着IgM、IgG水平的上升,发生排斥反应时C3和CD46水平显著降低.C组猕猴特异性抗猪抗体IgM及IgG的上升速度均较A、B组明显延缓.结论 对受者进行异种胸腺注射联合全身照射预处理在抑制T淋巴细胞免疫及体液免疫方面有重要作用,但无法抑制异种排斥反应中补体的激活,补体通过经典途径参与了延迟性异种排斥反应的发生.     

水苏糖对模拟异种心脏移植超急性排斥反应的抑制作用

目的观察水苏糖对猪到人异种心脏移植超急性排斥反应的抑制作用。方法以人血液体外灌注猪游离心脏为基础，模拟猪到人异种心脏移植的超急性排斥反应模型。实验分为A、B两组：即分别用人血液灌注及人血液加水苏糖灌注猪离体心脏。观察灌注后两组心脏的跳动时间；体外灌注1h后，对两组灌注心脏进行免疫组织化学（测定IgG及IgM的沉积）及病理学分析。结果A组灌注心脏平均跳动时间为（9．5±2．5）min；B组灌注心脏平均跳动时间为（46．8±8．1）min，其中有1个心脏在灌注的1h内一直跳动；两组心脏跳动时间比较，差异有统计学意义（P〈0．01）。A组心肌间质呈弥漫性出血、水肿，血管扩张，内皮细胞肿胀、坏死；免疫组织化学检查显示心肌血管内皮组织中有IgG及IgM沉积。B组心肌间质未见出、凝血和坏死，血管内皮细胞未见肿胀；免疫组织化学检测未见IgG及IgM沉积。结论水苏糖对异种心脏移植超急性排斥反应具有抑制作用。     

猪到人异种心脏移植超急性排斥反应实验模型的建立：人血体外灌注猪冠状动脉系统

目的建立一种猪到人异种心脏移植超急性排斥反应实验模型。方法 8只广西巴马猪正中切口锯开胸骨进胸,肝素化,完整剪取猪心脏,用4℃生理盐水反复冲洗各心腔及心脏表面,用改良圣托马斯液经主动脉灌注,冲洗冠状动脉系统后,建立人血液体外灌注猪游离心脏冠状动脉系统,记录各模型心脏搏动的持续时间,1h后对灌注心脏进行免疫组织化学（测定IgG及IgM的沉积）及病理学分析。结果 8个人血液体外灌注猪心脏冠状动脉系统实验模型均成功建立,灌注心脏平均搏动时间为（9.25±1.90）min;心肌间质弥漫性出血、水肿,血管扩张,内皮细胞肿胀、坏死;心肌血管内皮细胞有IgG及IgM沉积。结论通过人血液体外灌注猪心脏冠状动脉系统建立的猪到人异种心脏移植实验模型能模拟超急性排斥反应,操作简单,容易复制。     

异种肝细胞移植排斥机理的探讨

目的：探讨异种肝细胞排斥反应的机理，为治疗异种肝细胞移植排斥反应提供理论依据。方法，用D－氨基半乳糖腹腔内注射制成肝功能衰竭大鼠模型，并在其脾内植入豚鼠肝细胞，通过免疫组化方法，用抗大鼠IgM抗体和抗大鼠IgG抗体，抗大鼠CD4和抗大鼠CD8抗体与因排斥反应在大鼠脾 内可能产生的IgM,IgG抗体和CD4，CD8淋巴细胞结合，在移植后不同时间取受鼠脾脏标本，检测其内是否有IgM,IgG抗体和CD4，CD8淋巴细胞存在。结果：移植后12hIgM抗体存在，移植后24h大鼠脾内出现IgG抗体，同时有CD4^=和CD8^＋淋巴细胞，且在移植后1周大鼠脾内均可见上述4种物质。结论：体液和细胞免疫均参与异种肝细胞移植的排斥反应。     

清除补体避免猪到猕猴异种心脏移植超急性排斥反应的实验研究

目的 观察纯化的眼镜蛇毒因子(CVF)对猪到狱猴异种心脏移植超急性排斥反应的影响。方法 以幼猪为供者，施行猪到狱猴腹腔内异位心脏移植，实验组(n＝4)使用CVF完全清除受者体内补体，对照组(n＝5)不使用CVF，两个组术后均采用环抱素A、甲泼尼龙和环磷酰胺抑制排斥反应，通过检测血清C3、C4水平及总补体活性验证CVF的效果，移植心停跳时切取移植心进行病理检查。结果 在使用CVF后，实验组血清C3降为0，总补体活性CH50值也几乎为0，末发现明显毒副反应，移植猪心存活时间平均为lld，最长达13d，病理学提示均发生了延迟性异种排斥反应；对照组3个移植心在移植后60min内发生超急性排斥反应，另2个分别存活22h及6d。结论 纯化的CVF有良好的清除补体的作用，且末见明显副作用；使用CVF可克服猪到狱猴异种心脏移植超急性排斥反应的发生。     

猕猴预致敏后肾移植加速性排斥反应的免疫学及病理学特点

目的 观察猕猴预致敏后肾移植加速性排斥反应的免疫学及病理学变化特点.方法 建立猕猴皮肤预致敏后肾移植加速性排斥反应模型(供、受者各3只).检测3只受者皮肤移植预致敏前、后及肾移植后血清内供者特异性抗体的变化.并在发生排斥反应时对移植肾进行免疫组织化学(测定补体、抗体的沉积及各类型淋巴细胞浸润情况)及病理学分析.结果 3只受者均发生了加速性排斥反应.其中2只受者在预致敏后血清中供者特异性抗体明显增加,对供者的淋巴毒反应明显升高;肾移植后受者血清中供者特异性抗体及针对供者的淋巴毒进一步升高.苏木精-伊红染色显示排斥反应的移植肾内有明显的动脉坏死、血栓形成、间质出血、中性粒细胞浸润;免疫组织化学及荧光染色显示移植肾内有大量的补体、抗体沉积(主要为IgG),而各种类型的淋巴细胞浸润少见.另1只受者体内的供者特异性抗体及对供者淋巴毒反应的升高程度不如前2只明显,病理学变化以肾小管损伤为主.结论 皮肤移植预致敏可以诱导受者产生程度不等的预存抗体,导致大多数移植肾在术后早期发牛主要南抗体和补体介导的严重的急性体液性排斥反应.     

水苏糖在猪到猴异种心脏移植超急性排斥反应中的作用（附视频）

目的观察水苏糖在猪到猕猴异位心脏移植超急性排斥反应中的作用。方法采用猪到猕猴腹腔内心脏异位移植模型。根据是否经水苏糖处理分为实验组及对照组,每组3只猕猴,观察移植心脏移植后24h内持续搏动时间和组织形态学变化。结果对照组移植心脏持续搏动时间分别为5、15、20min;心肌间质弥漫性出血、水肿,血管扩张,内皮细胞肿胀、坏死。实验组移植心脏持续搏动时间分别为50min、97min和24h;心肌间质未见出血、坏死,血管内皮细胞未见肿胀。结论水苏糖可能对猪到猕猴异位心脏移植的超急性排斥反应有抑制作用。     

供体骨髓间充质干细胞干预非协调性异种肝移植免疫排斥反应的实验研究

目的研究骨髓间充质干细胞(MSCs)对非协调性异种肝移植排斥反应的免疫干预作用。方法密度梯度离心法分离培养豚鼠MSCs,流式细胞仪检测其膜表面CD34、CD45、CD44和CD29的表达,脂肪细胞诱导液培养诱导MSCs向脂肪细胞分化。豚鼠MSCs输注大鼠后检测不同时相免疫球蛋白IgG、IgM、IgA以及补体C3、C4的浓度变化。豚鼠和大鼠各30只随机分为3组,所有大鼠受体经环磷酰胺预处理,A组为MSCs输注组;B组为生理盐水输注组;C组为地塞米松输注组。各组行豚鼠大鼠原位肝脏移植手术,观察受体的存活情况及排斥反应情况。结果MSCs膜表面CD34阴性、CD45阴性、CD44阳性、CD29阳性。脂肪细胞诱导液培养后细胞内出现脂滴沉着。MSCs细胞悬液输注受体后各个时相的免疫球蛋白IgG、IgM和补体C3较输注前有显著降低(P<0.01),而IgA和补体C4与输注前相比无明显变化(P>0.05),输注后的免疫球蛋白IgG、IgM和补体C3各个时相点之间的浓度变化无显著差异(P>0.05)。肝脏移植模型A组受体存活时间为(431±27)min,较B组和C组的存活时间[(148±16)min、(141±22)min]显著延长(P<0.01)。A组受体超急性排斥反应发生较迟,程度轻。结论MSCs的鉴定可根据细胞形态,膜表面标志物和分化能力来确定,MSCs可干预异种肝脏移植的超急性排斥反应的发生。     

单核细胞、NK细胞和T细胞在猪-猕猴延迟性异种移植排斥反应中的作用

目的观察单核细胞、NK细胞和T细胞在猪-猕猴延迟性异种移植排斥反应(DXR)中的作用。方法建立湖北白猪-云南猕猴的腹腔异位心脏移植模型，实验分为2组：对照组(n=5)，不使用中华眼睛蛇毒因(Y-CVF)；实验组(n=4)应用Y-CVF完全清除受者体内补体。2组受体猴均采用环孢素A(CsA)，环磷酰胺(CTX)和甲基强的松龙(MP)三联免疫抑制治疗。免疫组织化学方法检测移植心组织中细胞间黏附分子(ICAM)-1、肿瘤坏死因子(TNF)-α、单核细胞、NK细胞和T细胞的表达。结果对照组3个移植心在15～60min内发生超急性排斥反应(HAR)，另2个分别存活22h及6d，移植心均未见明显的炎性细胞浸润及ICAM-1和TNF-α的表达。实验组移植心存活时间分别为8、10、13和13d，移植物浸润细胞中可见大量的单核细胞(50％)，少量的NK细胞(8％～10％)，CD4^ T细胞(15％)和C08^ T细胞(25％)。移植物血管内皮细胞表面出现ICAM-1的表达上调，移植物间质中出现TNF-α的表达增加。结论单核细胞、NK细胞和T细胞介导的移植物损伤，在应用Y-CVF处理的猪-猕猴DXR发生中发挥重要作用。     

Acute vascular rejection is associated with systemic complement activation in a pig-to-primate kidney xenograft model

The introduction of h-DAF transgenic porcine organs into pre-clinical pig-to-primate discordant xenotransplantation has led to complete and reliable abrogation of hyperacute xenograft rejection (HAR). Despite additional heavy immunosuppression however, most xenografts are still lost due to acute vascular rejection (AVR), with current treatment protocols being of only limited value. In a life-supporting model of pig-to-primate kidney transplantation, unmodified (n=8) or h-DAF-transgenic (n=9) porcine kidneys were transplanted into cynomolgus monkeys under cyclophosphamide (CyP), cyclosporine and low-dose steroid immunosuppression. Longest recipient survival was 11 days in the control group and 68 days in the h-DAF transgenic group. Stable initial graft function with recipient survival >4 days was generated in eight animals (two controls and six transgenics). In these animals, plasma complement levels were analyzed during ongoing AVR. Compared with baseline levels, a two-fold increase in C3a levels and a four-fold increase in sC5b-9 levels were measured. In parallel to systemic complement activation, increased deposition of C3 and C5b-9 along with massive staining for recipient IgM immunoglobulins was detected in the xenografts on immunohistochemistry. We conclude that acute vascular xenograft rejection of porcine kidneys in cynomolgus monkeys is associated with classical pathway complement activation following binding of induced recipient anti-porcine antibodies. This complement activation can be observed despite membrane bound expression of human complement regulators in the porcine xenografts. Therefore, additional short-term fluid phase complement inhibition seems necessary for the future development of protocols designed for treatment of AVR in the pig-to-primate combination.     

Pathogenesis and pathology of delayed xenograft rejection in pig-to-rhesus monkey cardiac transplantation

It has been recognized that delayed xenograft rejection (DXR) is the major barrier to the acceptance of xenotransplantation after overcoming hyperacute rejection. OBJECTIVES: This study sought to investigate the pathogenesis and pathology of delayed xenograft rejection following pig-to-rhesus monkey heart xenotransplantation. METHODS: Heterotopic xenogeneic heart transplants in the abdominal cavity were performed using piglet donors to four monkey recipients. Complete complement depletion was achieved in the recipients with repetitive doses of high-activity cobra venom factor (Y-CVF). The recipients were immunosuppressed with a combination of cyclosporine, cyclophosphamide, and steroids. Sera were analyzed for C3 and C4 levels and complement activity and anti-pig endothelial xenoantibody. The grafts were examined histopathologically and immunohistochemically for C3, C4;C5b-9, IgM, IgG, tumor necrosis factor-alpha (TNF-alpha), intercellular adhesion molecule-1(ICAM-1), CD57(NK cells), CD68 (macrophages), CD4, and CD8. RESULTS: Xenografts survived 8, 10, 13, and 13 days respectively, all developing DXR. Venous thrombosis was the outstanding feature within DXR xenografts, complicated by interstitial edema, local hemorrhage, myocardial necrosis, and mild to moderate cellular infiltration. The serum C3 levels and complement activity decreased to almost 0 from the day of transplantation due to treatment with Y-CVF. The C4 level began to decrease 2 to 4 days before the cardiac xenografts lost their function. Anti-pig endothelial xenoantibody also decreased after transplantation, slightly increasing during DXR. All rejected xenografts showed C3, C4, C5b-9, IgG, and IgM deposits to various degrees. Large numbers of macrophages (50% of total leukocytes) infiltrated the entire xenograft with a few natural killer cells (8% to 10%), as well as some CD4+ T cells (15%) and CD8+ T cells (25%). Upregulation of ICAM-1 on graft endothelial cells and TNF-alpha in the interstitium were also demonstrated in the rejected heart. CONCLUSION: Both humoral and cell-mediated immunologic reactions may play important roles in the pathogenesis of DXR. Besides C3, C4, C5b-9, IgM, and IgG destroying the xenograft, NK cells, macrophages, and CD4+ and CD8+ T cells may further aggravate the development of DXR.     

Immunopathology of an hDAF transgenic pig model liver xenotransplant into a primate

BACKGROUND: hDAF transgenic pigs do not display the inherent hyperacute rejection reactions of pig-to-primate xenotransplants. The purpose of this study was to determine the immunopathologic phenomena following an hDAF transgenic pig hepatic orthotopic xenotransplant into a baboon. METHODS: Donor animals were unmodified pigs (n=4) and hDAF transgenic pigs (n=2). Recipient animals were baboons (Papio anubis). Liver biopsies were immunostained using monoclonal antibodies to C3, C5b-9, IgG, IgM, CD2, CD4, CD8, CD68, CD20, Bric 216, CD31, and fibrin, and polyclonal antibody to C4. RESULTS: hDAF transgenic grafts showed IgG, IgM, and C4 endothelial deposits. However, no fibrin, C3, or C5b9 deposits were observed after reperfusion. hDAF xenografts displayed CD31 staining in the portal spaces, perilobular areas, and at hepatic sinuisoidal levels. The baboon that lived for 4 days displayed either CD4 or CD8 T-cells periportal infiltrate. CONCLUSIONS: Future studies will seek to determine the physiologic role of CD31 hepatic sinusoidal expression in transgenic xenotransplants, and will also study the role of T-cell infiltrates in xenograft rejection.     

Pathologic characteristics of transplanted kidney xenografts

For xenotransplantation to become a clinical reality, we need to better understand the mechanisms of graft rejection or acceptance. We examined pathologic changes in α1,3-galactosyltransferase gene-knockout pig kidneys transplanted into baboons that were treated with a protocol designed to induce immunotolerance through thymic transplantation (n=4) or were treated with long-term immunosuppressants (n=3). Hyperacute rejection did not occur in α1,3-galactosyltransferase gene-knockout kidney xenografts. By 34 days, acute humoral rejection led to xenograft loss in all three xenografts in the long-term immunosuppression group. The failing grafts exhibited thrombotic microangiopathic glomerulopathy with multiple platelet-fibrin microthrombi, focal interstitial hemorrhage, and acute cellular xenograft rejection. Damaged glomeruli showed IgM, IgG, C4d, and C5b-9 deposition. They also demonstrated endothelial cell death, diffuse endothelial procoagulant activation with high expression of tissue factor and vWF, and low expression of the ectonucleotidase CD39. In contrast, in the immunotolerance group, two of four grafts had normal graft function and no pathologic findings of acute or chronic rejection at 56 and 83 days. One of the remaining kidneys had mild but transient graft dysfunction with reversible, mild microangiopathic glomerulopathy, probably associated with preformed antibodies. The other kidney in the immunotolerance group developed unstable graft function at 81 days and developed chronic xenograft glomerulopathy. In summary, the success of pig-to-primate xenotransplantation may necessitate immune tolerance to inhibit acute humoral and cellular xenograft rejection.     

Induction of anti-Forssman antibodies in the hamster-to-rat xenotransplantation model

BACKGROUND: In the hamster-to-rat heart xenotransplantation model, the serum response of the host contributes to determine whether the xenograft is accommodated or rejected. METHODS: To further characterize the serum response in this model, we compared anti-hamster antibodies found in naive LEW-1A rats, or in LEW-1A rats rejecting or accommodating a hamster heart, using a combination of cobra venom factor (CVF) and cyclosporin A (CsA) given for 10 days, and then CsA alone. RESULTS: Hamster hearts grafted into rat recipients contained IgG and IgA deposits to the same extent whether the xenograft was rejected or accommodated. Only immunoglobulins of the IgM isotype were found to be more abundant in recipients rejecting their graft. A significant part of this IgM response was directed toward the Forssman antigen, a sphingolipid present in the hamster but not in the rat. However, although anti-Forssman antibodies bind in situ to hamster tissues, this binding was not able to induce hyperacute rejection after antibody transfer. Furthermore, depletion of anti-Forssman antibodies from a rejecting serum did not modify its rejection properties. CONCLUSION: Unlike the pig-to-primate discordant xenotransplantation model, in which preexisting anti-carbohydrate antibodies are directly responsible for hyperacute rejection, in the concordant hamster-to-rat situation, the evoked IgM anti-Forssman carbohydrate antibodies do not appear to be the main cause of the vascular rejection.     

The pig analogue of CD59 protects transgenic mouse hearts from injury by human complement

BACKGROUND: It has been proposed that hyperacute rejection (HAR) of pig-to-primate vascularized xenografts is due in large part to ineffective regulation of recipient complement by pig complement regulatory proteins (CRPs), and indeed transgenic expression of human CRPs in pigs can prevent hyperacute rejection. However, at least one pig CRP (CD59) efficiently regulates human complement in vitro, suggesting that it is the level of expression of a particular CRP(s) rather than cross-species incompatibility that explains the HAR of porcine xenografts. We investigated the relative effectiveness of transgenically expressed pig and human CD59 in providing protection of mouse hearts from human complement in an ex vivo setting. METHODS: Transgenic mice expressing pig CD59 or human CD59 under the control of the human ICAM-2 promoter, which restricts expression in tissues to vascular endothelium, were used. Hearts from mice expressing similar levels of pig CD59 or human CD59 were perfused ex vivo with 10% human plasma and heart function was monitored for 60 min. Sections of perfused hearts were examined for deposition of the membrane attack complex (MAC). RESULTS: Control nontransgenic hearts (n=5) were rapidly affected by the addition of human plasma, with mean function falling to less than 10% of the initial level within 15 min. In contrast, hearts expressing either pig CD59 (n=6) or human CD59 (n=8) were protected from plasma-induced injury, maintaining 31 and 35% function, respectively, after 60 min of perfusion. MAC deposition was markedly reduced in both pig CD59 and human CD59 transgenic hearts compared to nontransgenic control hearts. CONCLUSIONS: When highly expressed on endothelium in transgenic mice, pig CD59 provided equivalent protection to human CD59 in a model of human complement-mediated xenograft rejection. Thus supranormal expression of endogenous porcine CRPs may be a feasible alternative to the expression of human CRPs in preventing HAR of pig-to-primate xenografts.     

The study of mitoxantrone as a potential immunosuppressor in transgenic pig renal xenotransplantation in baboons: comparison with cyclophosphamide

Mounting evidence suggests that delayed xenograft rejection (DXR) of discordant xenografts has a strong humoral component. To explore the possibility of targeting this humoral response more efficiently, we performed a preliminary study in baboons immunized against pig blood cells using the immunosuppressor mitoxantrone (Mx). The results from this study showed that, in comparison with cyclophosphamide (CyP), Mx induced a long-lasting depletion of circulating B cells within 6 days of its administration and delayed secondary anti-Gal antibody (Ab) responses to pig blood cell immunizations. Given these results, we next evaluated Mx in an in vivo model of pig to baboon renal xenotransplantation. We performed a series of renal xenotransplantations in baboons using human CD55-CD59 transgenic donor pigs. In the first group of baboons (Mx group; n = 4) Mx was administered 6 days prior to the day of transplantation, the objective being to perform the xenotransplantation in a context where the recipient would have few remaining circulating B cells and thus have an impaired capacity to mount an Ab response to the xenograft. We compared this group to a second group of baboons treated with CyP starting 1 day prior to transplantation (CyP group; n = 2). All baboons receiving Mx or CyP received an additional immunosuppression of cyclosporin A, mycophenolate mofetil and steroids. No hyperacute rejection was observed in either group but all xenografts underwent DXR. Mx did not show superiority to CyP in terms of graft survival with a mean survival time of 8 +/- 2 days compared with 9 days for both CyP-treated baboons. Neither CyP nor Mx decreased serum levels of pre-existing anti-Gal Abs but levels of these Abs decreased dramatically within 1 day of transplantation, likely reflecting their immediate trapping within the xenograft. Interestingly however, in contrast to CyP, Mx inhibited the return of anti-Gal immunoglobulin M (IgM) to the circulation, even at the time of rejection. Nevertheless, strong intragraft deposits of IgM, IgG and the activated complement complex C5b-9 were observed in biopsies at rejection. Furthermore, despite the expected profound depletion of circulating B cells by Mx within 6 days of its administration, biopsies from both groups at rejection displayed a mild B cell infiltrate accompanied by a strong macrophage and intermediate T-cell infiltration, the latter tending to be more abundant in Mx-treated animals. Our data show that in this particular model of pig to baboon xenotransplantation and at the dose used, Mx was not superior to CyP in conferring protection against rejection, despite its capacity to profoundly deplete circulating B cells and to inhibit anti-Gal Ab responses to xenografts. DXR was thus possible without the return of anti-Gal Abs and may have been mediated by the early fixation of pre-existing Abs with secondary complement activation. However, although Mx was not more efficient than CyP in controlling DXR, its capacity to deplete B cells and delay Ab recovery may be beneficial in the context of Gal knockout organ transplantation where the induced Ab response is likely to take precedence over the preformed response.