供体与受体移植前预处理延长大鼠移植心脏成活时间

目的：用供体品系大鼠皮肤致敏方法及腹腔内异位心脏移植模型研究雷公藤多式供体预处理及供体脾细胞受体胸腹内注射诱导免疫耐受对移植心脏存活影响及二种方法的协同效果。方法：SD大鼠作供体,Wistar大鼠作受体,所有受体在心脏移植前均无用供体皮肤致敏,A组作单纯心脏移植,B组作经供体预处理后的供心移植,C组用供体脾细胞胸腺内注射诱导耐受后作心脏移植,D组考察供受体两种预处理方法的协同效果。结果：各处理组供心存活时间均校对照组明显延长,C,D二组病检见淋巴细胞浸润及心肌坏死明显减轻,但微血管病变较严重。结论：两种方法均能明显延长移植供心成活时间,并有良好的协同效果,经胸腺途径诱导的耐受能明显抑制细胞免疫,但对急性血管排斥没有预防作用。     

Monoclonal Antibody Treatment to Prolong the Secondary Cardiac Allograft Survival in Alloantigen‐primed Mice

We have previously shown that costimulation blockade using a combination of monoclonal antibodies (mAbs) – CTLA4Ig, antibodies to CD154, LFA‐1, and OX40L – can induce tolerance of cardiac allografts in mice with adoptively transferred CD4⁺ memory T cells [ 1 ]. However, the effect of costimulatory blockade in secondary allograft rejection has not been studied. B6 mice that rejected BALB/c skin grafts for more than 4 weeks (defined as alloantigen‐primed mice) were used as recipients. The recipient mice were treated with the mAbs to CD154, LFA‐1, OX40L, and CD122 on days 0, 2, 4, and 6 after the secondary transplantation of BALB/c heart. The mean survival time (MST) of secondary cardiac allografts in rats treated with antibodies to CD154 and LFA‐1 (2‐antibodies approach) and those treated with antibodies to CD154, LFA‐1, OX40L, and CD122 (4‐antibodies approach) was greater than that of the controls (MST = 6.7 days, 22.2 days, and 3.2 days, respectively). The 4‐antibodies approach prevented lymphocytic infiltration in the grafts, inhibited memory T‐cells proliferation in the spleen, increased IL‐10 secretion in the serum, and enhanced the expression of CD4⁺ Foxp3⁺ regulatory T cells (Tregs) in spleen. Expression levels of alloreactive antibodies were high in the recipient mice of experimental and control groups. Inhibiting the memory T cells by costimulation blockade extended allograft survival in secondary transplant models but could not induce tolerance of graft. Alloreactive antibodies may participate in alloresponse and play an important role in secondary cardiac allograft rejection.     

Monoclonal Antibody Treatment (Anti-CD4 and Anti-Interleukin-2 Receptor) combined with Cyclosporin A has a Positive but not Simple Dose-Dependent Effect on Rat Renal Allograft Survival

The use of monoclonal antibodies (MoAbs) in experimental and clinical organ transplantation is of increasing interest since this treatment seems to offer an opportunity for specific immunomodulation. In a rat kidney allograft model, Cyclosporin A (CyA) treatment (12.5 mg/kg/d, day 0-14) was combined with murine anti-rat CD4 (MRC OX-38) and murine anti-rat IL-2R (MRC OX-39) MoAbs at doses of 100 or 300 micrograms/kg/d (day 0-7) and plasma concentrations of the murine MoAb were determined. In both groups receiving combined treatment with CyA and MoAb, graft survival was prolonged to an average of 65 days, compared to a graft survival of 9-10 days in non-treated recipients. Further, the data showed a beneficial effect of CyA + MoAb treatment versus CyA alone (graft survival 32 days). The threefold increased MoAb dose did not seem to improve graft survival or function. Treatment with OX-38 + OX-39 at a dose of 100 micrograms/kg/d each resulted in plasma levels of 280 ng/ml 14 days after transplantation. Corresponding values after the administration of 300 micrograms/kg/d were 1800 ng/ml in graft recipients as well as controls. These findings indicate that the effect of MoAbs in complex organ transplantation models is not simply dose dependent and that in vitro assays are of limited value in predicting the effect of a given MoAb when used in vivo. The determination of MoAb plasma levels, however, may be a useful tool in defining optimal MoAb administration and to monitor therapeutically effective plasma levels.     

Prolonged Allograft Survival In Newcastle Disease Virus-Treated Mice

There was persistent lymphocytopenia and prolonged survival of C57Bl skin allografts when recipient BALB/c mice were injected daily with Newcastle disease virus.     

Malononitrilamides 715 and 279 Prolong Rat Cardiac Allograft Survival, Reverse Ongoing Rejection, Inhibit Allospecific Antibody Production and Interact Positively with Cyclosporin

A77 1726 is a malononitrilamide (MNA) and the active metabolite of leflunomide, which has been extensively investigated and shown to be a potent immunosuppressive drug. However, the half-life of A77 1726 is about 15–18 days in humans and leflunomide is therefore currently being developed for the treatment of autoimmune disease and not for transplantation. Search for analogues has led to the discovery of MNA 715 and 279, derivatives of A77 1726. Previous experimental experience of these compounds is still limited. The aim of the present study was to verify the efficacy of these MNAs concerning prevention and reversal of rejection, inhibition of antibody production and interaction with cyclosporin A (CsA). Heterotopic cardiac transplantation in DA to PVG rats was used. Subgroups of rats were given either CsA, MNA 715 or MNA 279 for 10 days, starting at either day 0 or day 4, or received no treatment. Titres of allospecific immunoglobulin M (IgM) and immunoglobulin G (IgG) were quantified by flow cytometry. Ten days of induction with MNA 715 or 279 produced significantly longer graft survival than in controls. Treatment from day 4 onwards, when acute rejection was established, rescued all grafts. Allospecific production of IgM or IgG was absent during MNA induction and was suppressed in animals receiving a rescue course of MNA. The transplant model was potentiated by addition of the immunomodulator quinolone-3-carboxamide (Linomide®), which eliminates the effect of CsA and other immunosuppressants. The combined treatment with MNA and CsA was successful in overcoming the challenge of Linomide, demonstrating the additive effects of the two drugs. In conclusion, MNA 715 and 279 were shown to be potent immunosuppressants, preventing and reversing acute allograft rejection, inhibiting and suppressing allospecific antibody production, and the drugs interacted positively with CsA.     

Effects of Monoclonal Anti-T Cell Antibodies on Rat Cardiac Allografts

Monoclonal antibodies reactive with different T lymphocyte antigens were administered to rats receiving heart allografts. Ox 19 antibodies (directed to the rat Ly 1 equivalent) and Ox 8 antibodies (directed to the rat CD8 equivalent) both prolonged graft survival, whereas W3/25 (anti-CD4), Ox 6 (anti-Ia), and W3/13 (anti-pan T) antibodies did not affect graft rejection. Immunohistological studies were carried out on spleen and graft specimens in order to analyse further the mechanisms behind the prolongation of graft survival. The observed almost complete absence of Ox 8-reactive cells in the spleen after treatment with Ox 8 antibodies corroborates earlier observations that injection of moderate amounts of Ox 8 antibodies leads to complete elimination of suppressor/cytotoxic T cells from peripheral lymphoid organs and blood. The present data on graft survival therefore both support the notion that suppressor/cytotoxic T cells are involved in graft rejection, and suggest that these cells are not the only ones involved. An unexpected and as yet unexplained finding was that Ox 8-reactive molecules were found in large numbers on various inflammatory cells as well as on certain myocytes in the grafted hearts that had experienced a prolonged graft survival due to treatment with Ox 8 or Ox 19 antibodies.     

Recognition of Rat Dendritic Cells by a Monoclonal Antibody

A mouse monoclonal IgM antibody reactive with dendritic cells (DC) from the Brown Norway (BN) rat was prepared. This antibody (1F119) binds to a membrane-bound antigen present on DC from thoracic duct lymph, spleen, thymus, and lymph node. The antigen is present only in low density on 5% of splenic macrophages (M phi) and absent from peritoneal M luminal diameter. In situ, the antibody exhibits a strong reactivity towards DC in the thymic medulla, whereas no reaction is observed with cortical cells. Furthermore, cells positive for 1F119 can be identified in T-cell areas of spleen, lymph node, and Peyers' patches. 1F119 was genetically restricted in that a strong reactivity was found with DC from rats of the RT1n and RT1u haplotypes, an intermediate reactivity with the RT1c haplotype, only a weak reactivity with the RT1l and RT1b haplotypes, and no reactivity with the RT1a and RT1k haplotypes. The relatively weak reactivity of 1F119 with respect to the RT1l haplotype also appeared from a weak binding of 1F119 to DC from Lewis rats, as was assessed by FACS analysis. This result was comparable to the binding of OX3 (RT1.Bl and RT1.Bu) to DC from BN rats. Studies performed on thymus sections of recombinant rat strains indicate that 1F119, despite its apparent specificity for DC, reacts with a polymorphic RT1.B product.     

Prolonged Survival of Implanting Rat Blastocysts in the Uterus of Congenitally Athymic Mice

Rat blastocysts were transferred to the uteri of either immunodeficient, congenitally athymic (nu/nu) 'nude' mice or immunocompetent, heterozygous (nu/+) mice. Histological analysis showed that there was a similar frequency of implanted blastocysts in the two types of mice, that blastocysts transferred to nude athymic mice outlived those in heterozygous controls by at least 3 days, and that blastocysts in nude mice showed delayed and abnormal development and were eventually rejected. These findings suggest that the T-cell deficiency in nude mice may prolong the survival of implanting blastocyst but that other immune or non-immune factors still limit their development.     

静息B细胞免疫诱导小鼠心肌移植物存活时间的延长

本文用小鼠耳后心肌移植作为模型观察静息B细胞在诱导延长移植物存活的作用。用C5 7BL/ 6新生小鼠的心脏移植给Balb/c成年雌性小鼠的耳后 ,受鼠在移植前 1周事先尾静脉注射C5 7BL/ 6静息B细胞 ,同时观察了用静息B细胞免疫后的Balb/c小鼠脾细胞对C5 7BL/ 6小鼠脾细胞的CTL活性。结果显示单独使用静息B细胞免疫组受鼠的同种移植物平均存活时间为 (16± 1 34 )d [空白对照组为 (10± 1 0 5 )d],单独注射阿霉素组心肌平均存活时间为 (14 6± 1 74)d ,静息B细胞预处理同时配合使用阿霉素组 ,心肌平均存活时间延长至 (2 7 8± 3 96 )d (P <0 0 0 1)。而活化B细胞组的小鼠排斥反应强烈 ,在移植后 8d之内全部排斥。静息B细胞免疫鼠对同种细胞的杀伤率 ,明显低于用活化B细胞免疫的对照组。     

抗Tac单克隆抗体对ConA诱导小鼠实验性肝损伤的影响

本研究采用ＣｏｎＡ诱导，建成小鼠肝损伤模型，并检测其血浆肿瘤坏死因子－α（ＴＮＦ－α）含量的变化，发现模型组鼠血浆ＴＮＦ－α含量为０．１９±０．０６ｎｇ／ｍｌ，而正常对照组未检出ＴＮＦ－α，二者比较有极显著差异（Ｐ＜０．００１）。预先注射抗Ｔａｃ单克隆抗体可减少ＣｏｎＡ注射后小鼠ＴＮＦ－α的产生，与模型组比较（分别为０．０３±０．０２和０．１９±０．０６），二者有极显著差异（Ｐ＜０．００１），而且注射抗Ｔａｃ单抗组无肝损伤发生。上述结果表明，ＣｏｎＡ诱导的肝损伤与ＴＮＦ－α等的介导有关，抗Ｔａｃ单克隆抗体对ＣｏｎＡ诱导的肝损伤有保护作用。     

大动物同种异体心脏移植供心存活期与IL-2、IL-10关系的研究

实验以大动物猪作为研究对象 ,使用胸腺内注射供体脾细胞和γ射线全身照射方法进行受体预处理 ,再行同种异体异位心脏移植 ,来评估在大动物实验中IL 2和IL 1 0与供心存活期的关系。结果表明在实验组预处理后第 2周 ,与照射前、照射 1周后以及单纯照射组相比混合淋巴细胞培养刺激效应有明显下降 (P <0 0 5 ) ,而单纯照射组无显著变化 (P >0 0 5 )。移植供心存活期实验组 (MST 5 6d )较空白组 (MST 2 4d )和照射组 (MST 2 2d )明显延长 (P <0 0 5 ) ,而空白组与单纯照射组比较无显著差异 (P >0 0 5 )。IL 2和IL 1 0检测的结果表明 ,实验组移植后 1～ 3dIL 2的水平明显低于对照组和空白组 (P<0 0 5 )。IL 1 0水平三组无显著差异 (P >0 0 5 )。以上结果表明胸腺内修饰和全身照射联合预处理受体 ,可以使受体产生短暂的免疫抑制。IL 2水平的变化与排斥反应的发生关系密切 ,它可以作为监测早期急性排斥反应发生的一个重要指标     

Depression of Peripheral Lymphocyte Proliferation and Hemolytic Antibody Production in Passively Enhanced Rat Renal Allograft Recipients

Specific suppression of renal allograft rejection in two strain combinations of rats by passive enhancement was associated with depression of both peripheral lymphocyte proliferation and hemolytic antibody production. Variations in these responses were related to allograft survival in individual rats. In general, these studies confirm the value of monitoring both the lymphocyte proliferation and hemolytic antibody responses for the prediction of subsequent rejection, and for evaluating the efficacy of the enhancement therapy.     

Protective Effect of Anti-P-Selectin Monoclonal Antibody in Lipopolysaccharide-Induced Lung Hemorrhage

Excessive leukocyte accumulation is involved in the pathogenesis of the sepsis-induced acute lung injury. Selectins are essential to the interaction between leukocytes and endothelial cells. In this report, we investigated the role of selectins in the severe lung injury induced by lipopolysaccharide (LPS). Significant lung hemorrhage was observed 24 h after the intravenous administration of LPS (1 mg/kg). First, we evaluated the effect of sialyl Lewis X-oligosaccharide (SLeX-OS), a derivative of sialyl Lewis X which is one of the ligands for E-, P- and L-selectins. The treatment with SLeX-OS (26.5 mg/kg iv bolus + 19.8 mg/kg iv infusion) resulted in a decrease of lung hemorrhage by 49.5% (P < 0.05 versus the control group). Second, we tested the effect of anti-P-selectin monoclonal antibody (MAb), PB1.3, to investigate the role of P-selectin. The bolus administration of PB1.3 at a dose of 5 mg/kg attenuated the lung hemorrhage by 74.6% (P < 0.05 versus the control group). In addition, we also detected an increase of soluble P-selectin in plasma 24 h after the injection of LPS. These results suggest that P-selectin has a substantial role in the pathogenesis of the lung injury induced by LPS.     

Effect of Cyclosporin A on the in Situ Inflammatory Response of Rat Renal Allograft Rejection

The impact of cyclosporin A (CyA) on a normal kidney parenchyma and on the in situ inflammatory response of rejection was investigated in normal DA rats and after transplantation of DA renal allografts to Lewis recipients. In a normal, non-transplanted DA kidney more than 80 mg/kg/day of CyA induced light-microscopic changes in the distal tubular cells of the renal cortex and outer medulla. These changes were not accompanied by any visible inflammation and were directly proportional to the dose of the drug and to the duration of drug administration. Treatment of a transplant recipient with 40 mg/kg/day of CyA abolished or at least efficiently reduced the in situ inflammatory response of rejection both as analysed from tissue sections and as quantified from the recovery of inflammatory cells after enzymatic digestion. It also reduced efficiently not only the number of T and B blast cells of the inflammatory infiltrate but also the number of other inflammatory cells, such as in situ lymphocytes, monocytes, and macrophages, and abolished or at least reduced the generation of (T) killer cells in situ and in the recipient spleen. These effects were inversely proportional to the time elapsed between grafting and initiation of treatment: although a complete suppression of all three features was obtained if the drug treatment was initiated already on the day of transplantation, a significant reduction of these functions was still found if the treatment was initiated later when the blastogenic response was already underway.     

抗人心肌肌球蛋自重链单克隆抗体的筛选与鉴定

用自制人心肌肌球蛋白作为哺抗原，按杂交瘤常规技术获得一株稳定的Ｇ４杂交瘤细胞株。单抗亚型为ＩｇＧ１型；用免疫印迹法对成人、胎儿、哺乳类动物及鲫鱼的心肌肌球蛋白提取液在２００ｋＤ处有一着色带；免疫组化ＡＢＣ法表明单抗与成人、胎儿、猪、兔、大白鼠的心肌细胞胞浆均有阳性反应，与血管平滑肌均呈阴性反应。该单克隆抗体可用于与心肌坏死病理过程有关的疾病的诊断。     

CD28/B7和CD134/CD134L共刺激通路阻断与免疫抑制剂联合治疗对移植心脏存活时间影响

目的探讨CD28-B7、CD134-CD134L共刺激信号通路阻断以及雷帕霉素、供体特异性脾细胞输注(DST)等联合治疗对心脏移植排斥反应的影响。方法将DA大鼠心脏移植到LEW大鼠的腹腔内,用CTLA4-Ig融合蛋白、CD134L抗体分别阻断CD28-B7、CD134-CD134L共刺激信号通路,以及雷帕霉素、DST等不同治疗组合观察对移植心脏存活时间的影响,并利用免疫组化方法观察炎症细胞浸润情况。结果CD134-CD134L、CD28-B7共刺激信号通路单独阻断和雷帕霉素单独治疗后心脏存活时间分别为7.6+0.5d、12+3.5d和19.1+6d;CD134-CD134L、CD28-B7共刺激信号通路阻断与雷帕霉素联合治疗心脏存活时间分别为25.2+2.2d和31.5+4.6d。CD134-CD134L、CD28-B7共刺激信号通路共同阻断与雷帕霉素、DST联合治疗心脏存活时间明显延长,为59.1±10.4d,另外有2例存活时间超过120d。结论单独雷帕霉素、CD134-CD134L和CD28-B7通路阻断对移植心脏炎症细胞浸润和组织坏死程度无明显改善,而两个共刺激通路同时阻断与雷帕霉素、DST联合治疗时改善效果最明显。