输注供体骨髓细胞诱导大鼠小肠移植基因嵌合的研究

目的 探讨经门静脉途径输注供体骨髓细胞后,大鼠小肠移植中受体基因嵌合率的改变及可能机制.方法 建立大鼠异位节段小肠移植模型(供体为雄性BN,受体为雌性Lewis,各15只),并将其随机分为三组,对照组、FK506组、PV组(喂养FK506及经门静脉注射供体骨髓细胞组).应用原位荧光杂交定位检测以及实时定量聚合酶链反应技术分析供体雄性细胞的SRY基因在移植术后受体大鼠血液、肝脏及脾脏中的嵌合情况.结果 PV组受体雌性大鼠血液(3.03±0.16)%、肝脏(0.86±0.05)%、脾脏(4.08±0.12)%中供体SRY基因嵌合率均较FK506组(1.15±0.07)%、(0.21±0.02)%、(1.23±0.05)%、对照组(1.10±0.07)%、(0.22±0.02)%、(1.17±0.06)%明显增高(P＜0.01).结论 经门静脉系统输注供体骨髓细胞可建立稳定的混和嵌合体状态.     

Early intragraft inflammatory events of liver allografts leading to chronic rejection

In this retrospective study, we have investigated the early intragraft inflammatory events of 12 liver allografts leading to chronic rejection. The cytological findings and clinical follow-up were analyzed in detail. Nine patients underwent at least one typical lymphoid activation of acute rejection, and three of them were treated more than once. Diagnosis of rejection was based on biopsy histology, cytology and liver dysfunction. In addition to the acute rejections, cytological analysis demonstrated in 11 of 12 grafts an unidentified lymphoid episode that differed from that of rejection. These lymphoid responses were associated with viral infections; cytomegalovirus (CMV) infection in 10 of 12 patients, hepatitis C virus (HCV) infection in 2 of 12 patients, 1 combined with CMV, and hepatitis B virus (HBV) infection in 1 patient. Graft dysfunction was still seen at the end of the follow-up. Thus, intragraft inflammation caused either by acute rejection or by viral infections may be involved in the induction of chronic rejection.     

Restoration of tolerance to rat hepatic allografts by spleen-derived passenger leukocytes

The tolerance induced by orthotopic liver transplantation (OLT) in certain combinations of rat strains can be prevented by total body irradiation (TBI) of the donor. We demonstrate here that the intravenous inoculation of splenic leukocytes into irradiated donors before OLT could re-establish tolerance in association with a state of microchimerism detected in the recipients. When donor DA (RT1^a) strain rats were irradiated with 1000 rad 24 h before liver harvesting and subsequent liver implantation into PVG recipients, five out of six rats died from rejection in this normally tolerogenic OLT (DA-PVG) combination. Injection of 1.5x10⁸ splenic leukocytes from naive DA rats into the irradiated DA donor rats 24 h before OLT restored the tolerogenic potential of the liver allografts. Immunofluorescence assay revealed an increased number of donor (DA) type cells in the PVG recipient bearing a repopulated DA liver, compared to the PVG recipient of an irradiated liver. These results suggest that passenger leukocytes reconstituted by splenic leukocytes have the capacity to protect liver allografts.     

Donor B cells in splenic follicles of experimental pulmonary allograft recipients

Background After transplantation, passenger leukocytes move to lymphoid organs of the recipient. These cells appear to initiate allograft rejection, but they also might be involved in tolerance induction. Materials and methods Orthotopic left lung transplantation was performed in the Dark Agouti to Lewis rat strain combination with no immunosuppression. Recipient spleens were removed at intervals of 24 h until day 6 after transplantation. For comparison, spleens from renal allograft recipients were analysed. Donor-derived major histocompatibility complex (MHC) class II antigens were detected by monoclonal antibody OX76. In double-staining experiments with antibodies specific for leukocyte subpopulations, their localisation and identity was analysed. Results OX76-positive leukocytes were already detected in recipient spleens on day 1 post-transplantation. They increased in number until day 3 and decreased in number thereafter. Most of them were localised in splenic follicles and expressed the B cell variant of CD45R and IgG. Cell surface antigens typical for other leukocyte subpopulations were not detected. In the spleens of renal allograft recipients, only few donor-derived cells were seen. Conclusion After lung transplantation, numerous MHC class II-positive B cells migrate to the splenic follicles of the recipient. These cells might, in part, be responsible for immunologic differences observed between renal and pulmonary allografts. Best abstracts - Surgical Forum 2007     

Characterization,quantification, and localization of passenger T lymphocytes and NK cells in human liver before transplantation

Quantification and localization of the main lymphocyte populations were studied in the livers of normal (n=8) and brain dead (n=8) subjects. Cytometric analysis performed on mononuclear cell suspensions obtained from liver biopsies was compared to an automatic image analysis of immunostained sections. The overall number of liver associated lymphocytes was in the usual range of peripheral blood content (2 to 9 · 10⁹ cells). Phenotypic analysis showed predominant NK and CD8+ cells that highly expressed class II antigen and CD25 and CD69 activation markers. Quantitative mapping of these activated lymphocytes revealed their preferential localization in the portal tract and the perisinusoidal area as compared to the pericentrolobular zone, especially in donor livers. This strategic localization could suggest a possible early cooperation between donor lymphocytes and initial infiltrating cells from the recipient and could explain the special immunological status of allografted livers.     

Establishment of chimerism in donor liver with recipient-type bone marrow cells prior to liver transplantation produces marked suppression of allograft rejection in rats

Abstract In this study, we investigated whether establishment of chimerism in donor liver with recipient-type bone marrow cells (BMCs) prior to liver transplantation could prolong the liver allograft survival. Donor female ACI rats were inoculated with recipient-type BMCs of male LEW rats via the portal vein, with or without irradiation as cytoablation, followed by intramuscular administration of FK506 for 5 days. At 1–2 months later, livers were harvested and transplanted into naive female LEW rats. No immunosuppressants were used. Chimerism in donor rats was confirmed by primers specific for the sex determinant Y chromosome of rats. With livers from rats pretreated with recipient-type BMCs, survival of liver allografts was significantly extended, irrespective of irradiation. These results showed that modification of the donor liver by intraportal injection of recipient-type BMCs and concomitant administration of FK506 prior to liver transplantation prolonged liver allograft survival in rats.     

Suppressed alloreactivity and mixed chimerism in rats with accepted cardiac allografts by intrathymic injection of donor bone marrow cells

Intrathymic injection of donor bone marrow cells (ITBMCs) at the time of transplantation and treatment with antilymphocyte serum (ALS) permitted the indefinite survival of Brown Norway (BN, RT1ⁿ) rat heart grafts in 6 out of 8 Lewis (LEW, RT1^l) rat recipients. LEW recipients with long-surviving BN heart grafts (LSGs) also accepted additional BN heart grafts without further immunosuppression, though they rejected Piebald Virol Glaxo (PVG, RT1^c) rat heart grafts in the usual fashion. In the in vitro study, the proliferative response of the lymphocytes from LEW recipients with LSGs remained suppressed when they were stimulated by BN spleen cells, but not when stimulated by PVG cells. Bone marrow cells (BMCs) from LEW rats with LSGs showed strong, nonspecific, suppressive effects on the proliferative response in the mixed lymphocyte culture reaction, suggesting that one of the possible explanations for tolerance might be the involvement of a suppressor mechanism. Received: 7 August 1996 Received after revision: 12 February 1997 Accepted: 17 February 1997     

The characterization of reconstituted passenger leukocytes on the induction of tolerance in rat liver transplantation

The tolerance induced by orthotopic liver transplantation [DA (RT1^a) rats to PVG (RT1^c) rats] can be prevented by total body irradiation of the donor rat. Reconstitution of the irradiated donor with DA splenic leukocytes reintroduces this tolerance. To investigate the major histocompatibility complex (MHC) specificity of passenger leukocytes, irradiated DA donors were reconstituted by third-party BN (RT1ⁿ) splenic leukocytes. The reconstitution with BN splenocytes re-established DA-specific tolerance in PVG recipients, as confirmed by subsequent DA cardiac allografting, while BN hearts were rejected with second-set tempo. To determine which cell components play an important role in re-establishing liver graft tolerance, DA splenic leukocytes were further purified into three types: T, B, and adherent cells. Only “T-cell-enriched” preparations restored liver graft tolerance in three out of five PVG recipients. These results suggest that passenger leukocytes of differing MHC types can help to induce liver-specific tolerance and that T cells in the liver graft may be essential to regulate tolerance induction. Received: 31 December 1996 Received after revision: 14 April 1997 Accepted: 15 April 1997     

负载供肾抗原的致耐受性树突状细胞延长大鼠移植肾存活时间

目的探讨负载供肾抗原的致耐受性受者树突状细胞(DCs)对移植肾存活时间的影响。方法以BN大鼠为供者,Lewis大鼠为受者,Wistar大鼠为无关供者。将受者的骨髓分离培养,未经其它处理得到的DCs,将其作为DCs1;DCs1经CTLA-4Ig基因重组的复制缺陷型腺病毒(AdvCTLA-4Ig)转染后作为DCs2;DCs1先经BN大鼠供肾抗原负载后再进行AdvCTLA-4Ig转染,作为DCs3。将受者随机分为DCs1、DCs2、DCs3及对照组,每组6只。术前24h,前3组分别注入1×106个DCs1、DCs2和DCs3,对照组不注射DCs。肾移植后观察各组移植肾存活时间,术后第20d检测受者脾细胞对负载供肾抗原及无关供者肾脏抗原DCs的反应。结果DCs1、DCs2,DCs3和对照组的移植肾存活时间分别为(8.1±0.8)d、(8.8±0.9)d、(62.1±13.7)d和(8.6±0.9)d。DCs1、DCs2组与对照组比较,差异无统计学意义(P>0.05),DCs3组与对照组比较,移植肾存活时间显著延长(P<0.01)。肾移植术后,DCs3组脾细胞对负载供肾抗原的DCs反应明显低于对照组(P<0.01),而对负载无关供者抗原的DCs反应与对照组比较,差异无统计学意义(P>0.05)。结论负载供肾抗原的受者DCs经CTLA-4Ig基因修饰后具有致耐受性,可以抑制受者对供者抗原的免疫反应,明显延长移植肾的存活时间。     

Comparative histopathology of hepatic allografts and xenografts in the nonhuman primate

Abstract: Liver transplantation was performed in the following groups: Group 1, baboon-to-baboon allografting (n = 8) (control group); Group 2, ABO-compatible vervet monkey-to-baboon xenografting (n = 8); Group 3, ABO-incompatible vervet monkey-to-baboon xenografting (n = 6); Group 4, pig-to-baboon xenografting (n = 2); and Group 5, pig-to-rhesus monkey xenografting (n = 6). Immunosuppressive therapy (cyclosporine, cyclophosphamide, and methylprednisolone) was begun 2–7 days before liver transplantation (LTx) and continued indefinitely after LTx. The liver grafts were biopsied pre-LTx and subsequently post-LTx at approximately 1 hr, 2–3 hr, 7–10 days, 20–30 days, 60 days, 120 days, and at euthanasia or spontaneous death. There were 19 successful LTxs with grafts functioning from one hour to 123 days. No pig liver (Groups 4 and 5) survived more than 5.5 hr, as there was an immediate severe vascular response after reperfusion, typical of hyperacute rejection (congestion and hemorrhage). Vascular rejection was not seen in allografts (Group l), but early mild-to-moderate congestion and neutrophil infiltration were present in concordant xenografts (Groups 2 and 3), which were associated with moderate deposition of immunoglobulin, C3, and fibrinogen. Lymphoid cell infiltration, bile duct damage, and portal vein endothelialitis in the portal zones occurred later in both allografts (Group 1) and concordant xenografts (Groups 2 and 3), developing earlier in the presence of ABO-incompatibility (Group 3). In concordant xenografts it was usually followed by fibrosis.     

Analyses of Peripheral Blood Mononuclear Cells in Operational Tolerance After Pediatric Living Donor Liver Transplantation

Operational tolerance (graft acceptance in an immunosuppression (IS)-free environment) after living-donor liver transplantation (LDLT) could occur by our elective protocol in some patients. There is, nevertheless, no reliable parameter to monitor patients who may discontinue IS without a risk of rejection. To identify such parameters, we systemically phenotyped peripheral blood mononuclear cells from operationally tolerant patients. An increase was observed in the frequency of CD4+CD25high+ cells, B cells and Vdelta1/Vdelta2 gammadeltaT-cells ratio in operationally tolerant patients (Gr-tol; n = 12), compared with those from age-matched volunteers (Gr-vol; n = 24) or patients on IS (Gr-IS; n = 19). The frequency of NK cells was decreased in Gr-tol, compared with those in Gr-IS or Gr-vol. The frequency of NKT cells was decreased after LDLT, compared with that in Gr-vol. Although the contribution of those subsets to the tolerant state remains elusive, the results may provide important clues for reliable indicators of tolerance after LDLT.     

Early acute cellular rejection: no effect on late hepatic allograft function in man

Whereas early acute cellular rejection, even if successfully treated, seems to have an impact on late function and survival of kidney and heart transplants, little quantitative data are available on its effect(s) on liver transplants. Routine liver function tests, the functioning liver cell mass (galactose elimination capacity) and microsomal metabolic capacity (aminopyrine breath test) were determined prospectively in 37 consecutive patients 1 year after liver transplantation. Of these, 19 (7 females and 12 males, 32–69 years of age) had previously required treatment for at least one biopsy proven acute cellular rejection episode occuring a median 7 days after grafting, while 18 (6 females and 12 males, 30–67 years of age) had not. The functioning liver cell mass and microsomal metabolic capacity were both within normal limits for the majority of patients and did not differ significantly between patients with and without previous acute cellular rejection episodes. In contrast to other solid organ transplants, early acute cellular rejection episodes do not affect late function of liver allografts in man. Received: 1 July 1998 Received in revised form 5 October 1999 Accepted: 12 October 1998     

Paclitaxel saves rat heart allografts from rejection by inhibition of the primed anti-donor humoral and cellular immune response: implications for transplant patients with cancer

Paclitaxel is an anti-neoplastic drug that was recently shown also to have immunosuppressive properties in naïve rat heart transplant recipients. Here, we tested whether paclitaxel could also effectively reverse an ongoing immune response in transplant recipients. We therefore used a model in which Lewis rat recipients receiving ACI rat heterotopic heart allografts were: (1) untreated, or treated with either (2) paclitaxel or (3) cyclosporine, starting 5 days after transplantation. Allograft survival was determined in one group, and in a second group cytotoxic T-lymphocyte (CTL) responses were determined and serum anti-donor cytotoxic antibody levels were measured. Results showed that paclitaxel was as effective as cyclosporine in saving recipients from imminent allograft rejection. Immunologically, paclitaxel reduced the allogeneic-CTL response, but most impressively, the cytotoxic antibody response was nearly eliminated in saved recipients. Therefore, paclitaxel's immunosuppressive properties, along with its known effectiveness against a wide variety of tumors, makes it potentially useful for the simultaneous treatment of rejection and neoplasms in cases of transplant-related cancer.     

Dendritic Cell Subset Ratio in Peripheral Blood Correlates with Successful Withdrawal of Immunosuppression in Liver Transplant Patients

Human dendritic cell (DC) subsets appear to play distinct roles in the induction and regulation of immune responses. While monocytoid DC (DC1) induce T-helper (Th) 1-type responses, plasmacytoid DC (DC2) have been reported to selectively induce Th2 responses. In blood, their precursors (p) can be identified as HLA-DR+ lineage- cells that are further characterized as CD11c+ CD123-/lo (IL-3Ralpha-/lo) (pDC1) or as CD11c- CD123hi (pDC2) by rare event, flow cytometric analysis. We compared the incidences of pDC1 and pDC2 in peripheral blood mononuclear cell populations isolated from normal healthy controls and from 3 groups of clinically stable liver transplant patients. Group A had been successfully withdrawn from immunosuppression, whereas group B were undergoing prospective drug weaning and on minimal anti-rejection therapy. In group C, drug withdrawal had either failed or never been attempted and patients were on maintenance immunosuppression. Assessment of DC subsets and the pDC2 : pDC1 ratio showed good intra-and interassay reproducibility. Compared with patients in group C, those in groups A and B demonstrated a significantly higher relative incidence of pDC2 and a lower incidence of pDC1 - similar to those values observed in normal healthy controls. Moreover, the pDC2 : pDC1 ratio was significantly higher in patients undergoing (successful) weaning and in those off immunosuppression compared with patients on maintenance immunosuppression.     

Prevention of allograft rejection by in vitro generated tolerogenic dendritic cells

Achieving immunological tolerance in transplantation has been a long sought-after goal since the 1960s. It is, therefore, interesting that the dendritic cells (DC), which are classically known as the most potent stimulators of T cell activation, are now also considered putative tools for tolerance induction. In line with this, much work has been performed using DC for vaccination and immune stimulation. Recently, great interest has been generated regarding the ability of DC to act as immune regulatory cells. Specific subsets of DC or immature DC (iDC) appear to be responsible for maintaining self-tolerance. In this review we will highlight our efforts at elucidating the contribution of DC in transplant tolerant in mice. Specifically, four strategies will be outlined that are currently being used for the generation of DC that have tolerogenic properties in the prevention of allograft rejection. The present study demonstrates that modulated iDC with blunted T cell stimulatory or antigen presentation abilities can afford transplant tolerance by minimizing T cell activation and proinflammatory cytokine production. Moreover, in an alternate strategy, normally matured DC have also been modulated such that alloreactive T cells are specifically targeted for deletion.     

Role of Kupffer cells in the survival after rat liver transplantation with long portal vein clamping times

Applying the orthotopic rat liver transplantation (ORLT) model, postoperative survival has been shown to be mainly dependent on the portal vein clamping time (PVCT). It was hypothesized that prolonged intestinal congestion was responsible for the activation of Kupffer cells (KC) with overproduction of TNF, secondary to splanchnic endotoxin accumulation and release on reperfusion. The role of KCs was directly investigated in the context of long PVCTs by eliminating them (using liposome-encapsulated dichloromethylene diphosphonate), by preventing their activation (using a calcium channel blocker, nisoldipine) and by inhibiting TNF production (using thalidomide). Livers from different groups of rats were transplanted following 24-h cold preservation in the UW solution with long PVCTs (from 18–21 min). KCs depletion, preservation with nisoldipine and pretreatment with thalidomide significantly improved survival in conditions using long PVCTs. KC depletion and nisoldipine preservation had no effect on liver enzymes or pathological findings while lung injury was significantly improved. The present data confirm that, in the context of ORLT with long PVCTs, KCs are directly responsible for the systemic endotoxin-like shock syndrome and their effect is mediated through overproduction of TNF. Received: 30 August 1999 Revised: 18 May 2000 Accepted: 12 September 2000     

Prediction of hepatic graft viability before reperfusion: an analysis of effluent from porcine allografts

Rapid and reliable assessment of hepatic graft viability is important for successful orthotopic liver transplantation (OLTx). OLTx was performed in 11 pairs of pigs via a venovenous bypass. Six of these grafts were transplanted immediately (group A), while the other five were preserved in University of Wisconsin (UW) solution for 24 h and then transplanted (group B). All grafts were flushed with 300 ml of chilled (4°C) Ringer's lactate solution before reperfusion of the graft, when 20 ml of effluent from the graft was collected and the concentrations of ammonia, lactic acid, GOT, and LDH were measured. Four of the six pigs in group A survived longer than 3 days, while the other two pigs died of causes other than graft dysfunction. All five pigs in group B died either of hemoperitoneum or hemodynamic instability due to liver failure. The histology of postperfusion biopsies in group A showed minimal pathological changes, while the grafts in group B revealed moderate to severe ischemic injuries. Ammonia and lactic acid in the effluent of group B were significantly higher than those of group A (1511±216 vs 417±333 g/dl and 114.1±12.2 vs 91.4±12.2 mg/dl, respectively; P<0.05 in both cases). Before reperfusion, the rate of total adenine nucleotides in all of the substances in the graft, which were measured using high performance liquid chromatography (HPLC), inversely correlated with the ammonia levels in the effluent. We conclude that an analysis of the effluent, (i.e. the levels of ammonia and lactic acid), flushed from a hepatic graft before reperfusion could serve as a predictor of hepatic graft viability.     

Rapamycin-conditioned, alloantigen-pulsed dendritic cells promote indefinite survival of vascularized skin allografts in association with T regulatory cell expansion

Clinically-applicable protocols that promote tolerance to vascularized skin grafts may contribute to more widespread use of composite tissue transplantation. We compared the properties of alloantigen (Ag)-pulsed, rapamycin (Rapa)-conditioned and control bone marrow-derived host myeloid dendritic cells (DCs) and their potential, together with transient immunosuppression (anti-lymphocyte serum+cyclosporine), to promote long-term, vascularized skin graft survival in Lewis rats across a full MHC barrier. Both types of DCs expressed low levels of CD86, but Rapa DC expressed lower levels of MHC II and CD40 and were less stimulatory in MLR. While both Rapa and control DCs produced low levels of IL-12p70 and moderate levels of IL-6 and IL-10 following TLR ligation, Rapa DC secreted significantly lower levels of IL-6 and IL-10 in response to LPS. Donor Ag-pulsed Rapa DC, but not control DC, induced long-term skin graft survival (median survival time >133 days) when administered 7 and 14 days post-transplant. Circulating T cells in hosts with long-surviving grafts were hyporesponsive to donor alloAg stimulation, but proliferated in response to third-party stimulation and produced IFN-gamma and IL-10. When recipients of long-surviving grafts were challenged with skin grafts, donor but not third-party grafts were prolonged, suggesting underlying regulatory mechanisms. Both flow cytometry and immunohistochemical analysis revealed that donor Ag-pulsed Rapa DC infusion expanded CD4+ Foxp3+ Treg in recipients' spleens, graft-associated lymph nodes and the graft. These data demonstrate for the first time that pharmacologically-modified, donor Ag-pulsed host DC administered post-transplant can promote indefinite vascularized skin graft survival, associated with Treg expansion.