Effect of surrogate tolerogenesis on the vascular rejection of pig heart xenografts

BACKGROUND: Organ xenografts are fulminantly rejected by antibody-mediated vascular rejection. Surrogate tolerogenesis (ST), the induction of tolerance within the donor, is effective with aorta xenografts. This preliminary study assesses the effect of ST on preformed antibodies and rejection of porcine heart xenografts. METHODS: Tolerance to the donor pig was induced by infusing recipient marrow into fetal pigs. Later, pig splenocytes were transfused and heterotopic pig hearts transplanted using chimeric or nonchimeric pigs. Anti-pig antibodies were assessed. RESULTS: With ST alone, xenografts developed cellular rejection at 4-6 days, whereas control grafts developed vascular rejection at 3-4 days (cellular vs. vascular, P<0.03). There was a reduction in preformed antibodies (P<0.03). ST combined with moderate cyclosporine prevented rejection at 9+ and 25 days in sensitized recipients compared with vascular rejection at 0.5-2 days for controls (P<0.07). CONCLUSIONS: ST seems to provide protection against vascular rejection. The cellular rejection seems sensitive to cyclosporine.     

Pre-transplant analysis of accommodation in donor pigs

Accommodation could lead to xenograft acceptance without the need for severe immune suppression. Generally graft accommodation is appreciated in the sensitized recipient, after transplantation. By inducing accommodation in chimeric donors, however, the risk and cost of inducing accommodation in the recipient would be reduced. An indirect assay of accommodation in the donor pig is needed for screening donors prior to procurement of the xenograft. The resistance of peripheral blood lymphocytes to cytolysis by antibody and complement was assessed in chimeric pigs and compared with control pigs. Peripheral blood lymphocytes (PBL) from chimeric pigs demonstrated a wide range of cytolysis (0 to 85%, median 13%) whereas PBL from control pigs were consistently lysed with these conditions (86 to 99%, median 96.5%, P < 0.0001). Accommodation or reduction in cytolysis did not correlate with the amount of chimerism. A longitudinal study demonstrated persistent accommodation of the PBL for as long as 15 weeks, when the donors averaged 68 kg in weight. Accommodation has been induced by low levels of antibodies interacting with the target tissue. An ELISA for sheep IgG was developed and the serum from newborn pigs assessed. Sheep IgG (up to 4.6 microg/ml) was detected in four of seven piglets with chimerism detectable by flow cytometry and in one of four piglets with minimal chimerism, detectable only by PCR. Lymphocyte accommodation was observed in all pigs with detectable sheep IgG. Of four pigs without accommodation, none had sheep IgG. Three pigs without detectable sheep IgG also had accommodation, suggesting that factors other than sheep IgG may induce accommodation. Acute vascular rejection was not observed in the heterotopic heart transplants from six donors with PBL accommodation. Only one incident of moderate diffuse cellular rejection (grade 3) was observed.     

Chimeric pig hearts resist hyperacute rejection in ex vivo perfusion model

With surrogate tolerogenesis. the recipient immune system is engrafted within the donor pig before organ transplant. Chimeric pig hearts may resist hyperacute rejection by inducing accommodation. This hypothesis was tested using an ex vivo isolated piglet heart perfusion model. Processed sheep marrow was infused into fetal pigs at 45 days gestation. Heart explants from chimeric or nonchimeric pigs were suspended in a Langendorff apparatus and perfused with plasma from unsensitized sheep or sensitized sheep. Nonchimeric hearts perfused with plasma from unsensitized functioned for 240 min (N = 3). Nonchimeric hearts perfused with sensitized plasma deteriorated rapidly, functioning at 19+/-12 min (N = 6); Immunohistochemistry of heart graft revealed extensive deposition of IgG, IgM in the microvascular. In contrast, chimeric hearts perfused with sensitized plasma functioned for 183+/-46 min (N = 3)(p <.001); Deposition of IgG, IgM had substantially less. Heart grafts procured from chimeric pigs survived in the presence of antidonor IgG, IgM, and complement, demonstrating that chimeric pig hearts resist hyperacute rejection.     

Cardiac xenotransplantation: progress toward the clinic

BACKGROUND: Animal organs could satisfy the demand for solid organ transplants, which currently exceeds the limited human donor supply. Hyperacute rejection, the initial immune barrier to successful xenotransplantation, has been overcome with pig donors transgenic for human complement regulatory proteins. Delayed xenograft rejection, thought to be mediated by anti-pig antibodies predominantly to Gal antigens, is currently regarded as the major barrier to successful xenotransplantation. A median graft survival of 90 days in the life-supporting position is considered a reasonable initial standard for consideration of entry to the clinic. METHODS: A series of 10 heterotopic heart transplants from CD46 transgenic pigs to baboons was completed. Immunosuppression consisted of splenectomy, Rituximab (Anti-CD20), tacrolimus, sirolimus, corticosteroids, and TPC. Thymoglobulin (Rabbit Anti-Thymocyte Globulin) was used to treat putative rejection episodes. RESULTS: Median graft survival was 76 days (range 56-113 days, n = 9). Only three grafts were lost to rejection. The remaining grafts lost were due to recipient mortality with baboon cytomegalovirus (BCMV) being the major cause (n = 4). No cellular infiltrates were present as a manifestation of rejection. Three hearts showed chronic graft vasculopathy. CONCLUSIONS: The median survival of 76 days in this group of heterotopic porcine-to-baboon cardiac xenografts represents a major advance over the median 27-day survival reported in the literature. Cellular rejection may not constitute a direct major barrier to xenotransplantation. A median survival of 90 days may be achievable with better control of BCMV infection. If further studies in the orthotopic position replicate these outcomes, criteria considered appropriate for clinical application of cardiac xenotransplantation would be approached.     

Cardiac Xenotransplantation

Heart failure is an important medical and public health problem. Although medical therapy is effective for many people, the only definitive therapy is heart transplantation, which is limited severely by the number of donors. Mechanical devices presently are used as “bridges” to transplantation. Their widespread use may solve the donor shortage problem, but at present, mechanical devices are limited by problems related to blood clotting, power supply, and foreign body infection. Cardiac xenotransplantation using animal donors is a potential biologic solution to the donor organ shortage. The immune response, consisting of hyperacute rejection, acute vascular rejection, and cellular rejection, currently prevents clinical xenotransplantation. Advances in the solution of these problems have been made using conventional immunosuppressive drugs and newer agents whose use is based on an understanding of important steps in xenoimmunity. The most exciting approaches use tools of molecular biology to create genetically engineered donors and to induce states of donor and recipient bone marrow chimerism and tolerance in xenogeneic organ recipients. The successful future strategy may use a combination of a genetically engineered donor and a chimeric recipient with or without nonspecific immunosuppressive drugs.     

Xenotransplantation: where are we in 2008?

Xenotransplantation holds promise to solve the ever increasing shortage of donor organs for allotransplantation. In the last 2 decades, major progress has been made in understanding the immunobiology of pig-into-(non)human primate transplantation and today we are on the threshold of the first clinical trials. Hyperacute rejection, which is mediated by pre-existing anti-alpha Gal xenoreactive antibodies, can in non-human primates be overcome by complement- and/or antibody-modifying interventions. A major step forward was the development of genetically engineered pigs, either transgenic for human complement regulatory proteins or deficient in the alpha1,3-galactosyltranferase enzyme. However, several other immunologic and nonimmunologic hurdles remain. Acute vascular xenograft rejection is mediated by humoral and cellular mechanisms. Elicited xenoreactive antibodies play a key role. In addition to providing B cell help, xenoreactive T cells may directly contribute to xenograft rejection. Long-term survival of porcine kidney- and heart xenografts in non-human primates has been obtained but required severe T and B cell immunosuppression. Induction of xenotolerance, e.g. through mixed hematopoietic chimerism, may represent the preferred approach, but although proof of principle has been delivered in rodents, induction of pig-to-non-human primate chimerism remains problematic. Finally, it is now clear that innate immune cells, in particular macrophages and natural killer cells, can mediate xenograft destruction, the determinants of which are being elucidated. Chronic xenograft rejection is not well understood, but recent studies indicate that non-immunological problems, such as incompatibilities between human procoagulant and pig anticoagulant components may play an important role. Here, we give a comprehensive overview of the currently known obstacles to xenografting: immune and non-immune problems are discussed, as well as the possible strategies that are under development to overcome these hurdles.     

Immunological unresponsiveness in chimeric miniature swine following MHC-mismatched spleen transplantation

BACKGROUND: In rodents, spleen allotransplantation (SpTx) induces tolerance. We investigated the induction of chimerism and donor-specific unresponsiveness following pig SpTx. METHODS: Thirteen pigs underwent splenectomy (day 0); all received a blood transfusion. In 11/13 pigs, SpTx was performed across a MHC class I (n=1) or full (n=10) barrier; two control pigs received no SpTx. All pigs were monitored for chimerism, and anti-donor immune responses, including suppressor assays. Four pigs (two asplenic controls and two with SpTx) underwent delayed donor-matched kidney transplantation without immunosuppression. RESULTS: Six of the 11 spleen grafts were lost from rejection (n=5) or splenic vein thrombosis (n=1), and five remained viable. All 11 SpTx recipients developed multilineage chimerism, but chimerism was rapidly lost if the graft failed. Two control pigs showed <6% blood chimerism for 4 and 11 days only. Pigs with functioning spleen grafts had multilineage chimerism in blood, thymus and bone marrow for at least 2-6 months, without graft-versus-host disease. These pigs developed in vitro donor-specific hyporesponsiveness and suppression. In 2 pigs tolerant to the spleen graft, donor MHC-matched kidney grafts survived for >4 and >7 months in the absence of exogenous immunosuppression; in two asplenic pigs, kidney grafts were rejected on days 4 and 15. CONCLUSIONS: Successful SpTx can result in hematopoietic cell engraftment and in vitro donor-specific unresponsiveness, enabling prolonged survival of subsequent donor-matched kidney grafts without immunosuppression.     

The immunological hurdles to cardiac xenotransplantation

The main hurdle to clinical application of cardiac xenotransplantation is the immune response of the recipient against the graft. Although all xenografts arouse an intense immune response, the effect of that response depends very much on whether the graft consists of isolated cells or an intact organ, such as the heart. Intact organs, which are transplanted by primary vascular anastomosis, are subject to severe vascular injury owing to the reaction of immune elements with the endothelial lining of donor blood vessels. Vascular injury leads to hyperacute rejection, acute vascular rejection, and chronic rejection. The immunological basis for these types of rejection and potential therapies, which might be used to avert them, are discussed.     

Cellular rejection of fetal pancreas grafts: Differences between alio- and xenograft rejection

Abstract: Hyperacute rejection (HAR) is the major immunologic problem with vascularized xenografts between discordant donor/recipient combinations but does not occur in neovascularized grafts of organ-cultured fetal pig pancreas in either mice or cynomolgus monkeys. However, a form of cell-mediated acute rejection with quite different histopathologic features does occur with kinetics that are similar to acute cellular rejection of fetal pancreas allografts in non-immunosuppressed MHC-mismatched mice. Xenograft rejection is dominated by non-lymphoid cells, mostly eosinophils, that appear some days after transplantation. In contrast, in mouse allografts, mononuclear cells are the dominant population throughout the rejection process^. The rejected allograft site rapidly resolves to form a mature non-irifiltrated scar whereas the infiltrate in the xenograft site remains for weeks and forms a large granuloma before its eventual resolution. There are also differences in the intra-graft cytokine profile in the graft site between alio- and xenografts during acute rejection with an early predominance of IL-5 and TNF-α and an absence of TNF-γ in the xenografts. Immunosuppression with a depleting anti-CD4 mAb shows that xenograft rejection is more dependent on CD4+ve T cells but xenografts are more difficult to maintain with conventional immunosuppression that is often effective for allografts. Limited studies in primates have shown that the histopathology of fetal pig pancreas rejection is similar to that seen in mice but occurs at a faster tempo. Thus, although HAR may not be a problem in rejection of neovascularised xenografts, a vigorous form of cellular rejection is present that may require different immunosuppression than is usually used for the I control of allograft rejection.     

Results of Gal‐Knockout Porcine Thymokidney Xenografts

Clinical transplantation for the treatment of end‐stage organ disease is limited by a shortage of donor organs. Successful xenotransplantation could immediately overcome this limitation. The development of homozygous α1,3‐galactosyltransferase knockout (GalT‐KO) pigs removed hyperacute rejection as the major immunologic hurdle to xenotransplantation. Nevertheless, GalT‐KO organs stimulate robust immunologic responses that are not prevented by immunosuppressive drugs. Murine studies show that recipient thymopoiesis in thymic xenografts induces xenotolerance. We transplanted life‐supporting composite thymokidneys (composite thymus and kidneys) prepared in GalT‐KO miniature swine to baboons in an attempt to induce tolerance in a preclinical xenotransplant model. Here, we report the results of seven xenogenic thymokidney transplants using a steroid‐free immunosuppressive regimen that eliminated whole‐body irradiation in all but one recipient. The regimen resulted in average recipient survival of over 50 days. This was associated with donor‐specific unresponsiveness in vitro and early baboon thymopoiesis in the porcine thymus tissue of these grafts, suggesting the development of T‐cell tolerance. The kidney grafts had no signs of cellular infiltration or deposition of IgG, and no grafts were lost due to rejection. These results show that xenogeneic thymus transplantation can support early primate thymopoiesis, which in turn may induce T‐cell tolerance to solid organ xenografts.     

The prospects for renal xenotransplantation

Summary: Xenotransplantation of non-human organs into human recipients has long been proposed as a possible strategy to overcome the acute shortage of donor organs. However, vascular organ transplants to humans from phylogenetically disparate species such as the pig are not currently possible due to a rapid rejection process termed hyperacute rejection. This process is initiated by the binding of host pre-formed 'natural antibodies' to the donor vascular endothelium, activation of the host complement system and activation or injury of the donor endothelial cells, leading to intravascular coagulation and loss of the graft due to ischaemic necrosis within minutes to hours of engraftment. Prevention of natural antibody binding and complement activation is viewed as paramount to preventing hyperacute rejection. Even if hyperacute rejection can be prevented, further barriers to successful discordant xenografts such as delayed xenograft rejection and a donor-directed cell-mediated rejection process will still represent major obstacles. This review examines recent advances being made in the various areas of xenograft research and the potential clinical application of pig-to-human xenografts that these strategies may bring.     

骨髓输注减轻大鼠肺移植急性排斥反应的研究

目的 研究供体骨髓细胞输注减轻大鼠肺移植急性排斥反应。方法 应用Cuff技术改进大鼠原位单肺移植模型，同时输注供体骨髓细胞，采集受体鼠外周血动态监测嵌合；存活4周大鼠杀死后切取移植肺进行排斥反应分级，切取脾应用免疫组织化学方法检测组织嵌合。结果 流式细胞术在骨髓输注组外周血中检测到明显的嵌合，在脾脏中得到组织嵌合。骨髓输注组移植肺急性排斥反应明显减轻。结论 肺移植同时输注供体骨髓细胞，可以产生显著的供体细胞与受体细胞嵌合，有效地抑制大鼠肺移植急性排斥反应。     

Immunobiology of xenogeneic cornea grafts in mouse eyes. I. Fate of xenogeneic cornea tissue grafts implanted in anterior chamber of mouse eyes

BACKGROUND: The cornea is an immune privileged tissue that, when grafted orthotopically, forms the anterior surface of the immune privileged anterior chamber. We have recently reported that allogeneic cornea fragments implanted in the anterior chamber of mouse eyes resist immune rejection, although such graft fragments are rejected outside the eye. We wished to determine the extent to which xenogeneic cornea fragments placed in the eyes of normal mice are vulnerable to immune rejection. METHODS: Guinea pig corneas, deprived surgically of epithelium, were cut into fragments and inserted into the anterior chamber of eyes of BALB/c and severe combined immune deficient (SCID) mice, adjacent to the central cornea of the recipient. The fate of the grafts was assessed clinically by biomicroscopy and histologically for 8 weeks postimplantation. RESULTS: The majority of guinea pig cornea fragments devoid of epithelium came to rest with the raw stroma adjacent to recipient endothelium. These fragments remained clear for the 8-week observation interval in both BALB/c and severe combined immune deficient mice. Clear grafts displayed viable guinea pig keratocytes and endothelial cell layers for 4 weeks. The endothelium was then replaced by murine cells by 8 weeks. A minority of guinea pig cornea fragments were oriented with donor endothelium adjacent to recipient endothelium. Although these grafts in severe combined immune deficient eyes eventually acquired an endothelial layer that faces the anterior chamber and remained clear, similar fragments in BALB/c eyes became opaque, failed to acquire a proper lining of endothelium that faces the anterior chamber, and incited an inflammatory reaction in adjacent recipient cornea. CONCLUSIONS: Immune privilege is afforded to xenografts of guinea pig cornea placed as stromal: endothelial cell fragments in the anterior chamber of mouse eyes, but only if the surface of the fragments that faces the anterior chamber is promptly covered with corneal endothelium. The possible roles of corneal endothelium in promoting immune privilege of corneal xenografts are discussed.     

Rat‐to‐mouse small bowel xenotransplantation: A novel model for studying acute vascular and hyperacute xenograft rejection and xenogenic cell migration

Kiyochi H, Kellersmann R, Blömer A, Garcia BM, Zhang Z, Zhong R and Grant DR. Rat-to-mouse small bowel xenotransplantation: A novel model for studying acute vascular and hyperacute xenograft rejection and xenogenic cell migration. Xenotransplantation 1999; 6: 00-00. ©Munksgaard, Copenhagen Abstract: The present study was undertaken to establish a rat-to-mouse vascularized small bowel xenotransplantation model to study acute vascular and hyperacute xenograft rejection, and xenogenic cell migration. Lewis rat small bowel grafts were transplanted heterotopically to group 1, Balb/c mice, and group 2, Balb/c mice pre-sensitized with a donor spleen cell injection. The grafts were examined by serial pathology and flow cytometry. In group 1, acute vascular rejection was present by the 5th post-operative day (POD). Immunohistology showed a strong endothelial deposition of IgG, IgM and C3, associated with a minimal lymphocytic infiltrate. There was a vigorous cell migration from the recipient to the graft, in which recipient origin cells comprised 80.1± 6.9% of the graft mesenteric lymph node by POD 3. However, there was almost no cell migration from the graft to the recipient. The intestinal xenografts in the group 2 showed massive hemorrhage, fibrin deposition, vascular congestion and thrombosis 60 min after transplantation. IgG and C3 were present on the endothelium as early as 1 min after reperfusion. The vigorous humorally-mediated vascular damage and rapid elimination of donor cells seen with intestinal xenograft rejection are distinct from the usual picture of allograft rejection. Hyperacute rejection can be induced by recipient pre-sensitization with donor spleen cells. The potential advantages of studying xenotransplantation in this model include: (1) the wide range of immunologic reagents available for mice; (2) the opportunity to study the progression of vascular damage easily by performing serial biopsies in the same animal; and (3) the opportunity to study, in vivo, two-way cellular response by examining cell trafficking in the mesenteric lymph nodes.     

Predictors of renal transplant histology at three months.

BACKGROUND: The quality of a damaged kidney, the complexity of the surgery, and the events in the first weeks after transplantation, such as delayed graft function (DGF) and acute rejection, may influence its histological appearance and long-term survival. The aim of this study was to evaluate the importance of these factors in predicting renal allograft histology at 3 months. METHODS: Prospective, protocol kidney biopsy specimens (n=112), obtained 3 months after transplantation, were scored for chronic damage by the Banff schema and evaluated by multivariate analysis against donor factors, implantation histology, prior recipient sensitization, ischemia, perioperative factors, and subsequent clinical events, such as DGF and acute rejection. RESULTS: Adequate samples were obtained in 102 of 112 biopsies and classified as chronic Banff grade 0 (n=22), grade I (n=56), grade II (n=23), or grade III (n=1). Acute Banff scores were minimal. DGF occurred in 49% and was the strongest predictor of tubulointerstitial damage at 3 months. DGF correlated with acute tubular necrosis on the implantation biopsy specimen and with the number of acute rejection episodes; DGF also correlated with the Banff grades of chronic glomerulitis, chronic interstitial fibrosis, and tubular atrophy scores (P<0.05-0.001) in the 3-month biopsy specimen. By multivariate analysis, chronic tubular atrophy was independently predicted by the presence of vascular disease in the donor biopsy specimen, DGF, and vascular rejection occurring within the first 3 months (P<0.05-0.001). Chronic interstitial fibrosis was unrelated to fibrosis in the donor biopsy specimen but was independently predicted by DGF, donor age, and vascular rejection (P<0.05-0.001). Vascular disease in the donor biopsy specimen correlated with chronic intimal thickening (r=0.36, P<0.01) and arteriolar hyalinosis score (r=0.54, P<0.001) on the 3-month biopsy specimen. Banff chronic intimal vascular thickening was independently predicted by donor biopsy specimen vascular grade, prior vascular rejection episodes, and renal cold ischemia time (P<0.05-0.01). There were no correlates with the mean cyclosporine (CsA) dose, blood levels, diagnosis of CsA toxicity, or cellular rejection within the first 3 months. CONCLUSIONS: This study has demonstrated that the quality of the donor organ at implantation was strongly predictive of subsequent renal histology in grafts functioning at 3 months. Vascular rejection and DGF had a significant long-term effect on graft damage, but cellular rejection and simple measures of CsA exposure did not.     

Can human viruses infect porcine xenografts?

Millard AL, Mueller NJ. Can human viruses infect porcine xenografts? Xenotransplantation 2010; 17: 6–10. © 2010 John Wiley & Sons A/S. Abstract: Xenotransplantation exposes the recipient to known and unknown pathogens of the donor pig (donor‐derived xenosis). A major effort has been undertaken to minimize the risk of transmission from the donor using specialized breeding techniques. With the exception of endogenous retroviruses and porcine lymphotropic herpesvirus, exclusion of known pathogens was successful and has eliminated a majority of donor pathogens. In the recipient, enhanced replication of many pathogens will be stimulated by the immune responses induced by transplantation and by the immune suppression used to prevent graft rejection. Infection of the graft may occur with unpredictable consequences due to the cross‐species situation. Infectivity may be decreased as entry or replication is altered by missing receptors or inability to use the cellular machinery. Replication of organisms in the xenograft and the inability of the human host to respond to human pathogens in the context of a xenograft infection due to immune suppression, or the presentation of such pathogens in the context of pig instead of human major histocompatibility complex (MHC) could impair control of such infections. Recent data suggest that some human herpesviruses infections, such as human cytomegalovirus, may infect porcine tissue and are associated with a pro‐inflammatory phenotype. This review focuses on human or recipient‐derived pathogens and their potential harmful role in xenograft infection.     

Long-term survival of cardiac xenografts in fully xenogeneic (mouse --> rat) bone marrow chimeras

BACKGROUND: The shortage of human hearts remains a major barrier to the efficacy of heart transplantation for the treatment of end-stage heart disease. One potential solution to the supply problem would be the use of hearts from nonhuman donors (xenografts). We have established a model of mouse to rat xenogeneic bone marrow chimerism, and in this study we have hypothesized that such chimeric rats will accept both donor and recipient specific heart grafts while rejecting third-party mouse and rat grafts. We also investigated humoral responses in naive and chimeric rats with and without donor murine cardiac grafts. METHODS: Recipient Lewis rats (n = 22) were given 1100 cGy lethal total body irradiation and the same day received 300 x 10(6) donor B10.BR mouse bone marrow cells intravenously. Peripheral blood of surviving rats (n = 18) was typed at 4 weeks and then monthly thereafter. Donor and recipient specific and third-party heterotopic heart transplantations were performed at 6 to 8 weeks after reconstitution with bone marrow. RESULTS: Multilineage bone marrow chimerism was produced in all experimental animals with complete replacement of recipient marrow by donor cells. Murine donor and rat recipient strain hearts transplanted in chimeric rats survived indefinitely. Third-party rat and mouse hearts were rejected, though at a slower rate than bone marrow matched naive controls. High levels of antimouse antibodies were detected in rats with rejected hearts. These antibodies were absent in chimeric animals with long-term surviving heart grafts. CONCLUSIONS: Long-term multilineage bone marrow chimerism can be produced in a mouse --> rat bone marrow transplant model. Long-term survival of donor specific and recipient specific vascularized cardiac grafts can be produced in these chimeric animals. These animals are clinically normal but show signs of subclinical immunosuppression regimen as they reject third-party hearts later than naive animals. Our results suggest that antibodies also play a significant role in concordant xenograft rejection, and induction of bone marrow chimerism can overcome this barrier.     

Establishment of stable multilineage hematopoietic chimerism and donor-specific tolerance without irradiation

BACKGROUND: Induction of tolerance to organ transplants will increase graft survival and decrease patient mortality and morbidity. Radiation-induced cytoreduction/ablation followed by donor hematopoietic cell reconstitution has been the most consistently successful approach to experimental tolerance induction. However, reluctance of clinicians to expose recipients to radiation has hampered its clinical application. METHODS: In the studies described, administration of polyclonal antilymphocyte serum (ALS), donor-specific bone marrow (DSBM) (150x10(6) cells), and sirolimus (24 mg/kg) in a completely mismatched murine model (B10.A donor, C57B/10 recipient) produced 100% indefinite (>250 days) skin graft survival. The level and character of donor-specific chimerism was evaluated with flow cytometry. RESULTS: Specific tolerance was confirmed by continued acceptance of primary and secondary donor-specific skin allografts and rejection of third-party grafts. The level and duration of chimerism induced was directly related to the dose of DSBM administered. Mice given 150x10(6) DSBM cells showed levels of 8-10% donor peripheral blood mononuclear cell chimerism by 30 days, and these levels persisted indefinitely (>250 days) in association with permanent tolerance of donor grafts. Eighty percent of donor chimeric cells were B lymphocytes (MHC class I and II positive, Fc receptor positive, CD45/B220 positive but negative for CD4, CD8 and Thy 1.2) and 20% were sorted in the macrophage monocyte population. CONCLUSIONS: These studies demonstrate for the first time that cytoreduction/ablation with ALS combined with sirolimus and reconstitution with donor bone marrow induces tolerance and chimerism in a completely mismatched murine combination. The use of ALS and sirolimus, currently employed therapies in clinical transplantation, and the lack of requirement for radiation make this tolerance protocol attractive for clinical application.     

Does endothelial chimerism correlate with renal allograft rejection?

BACKGROUND: The blood vessels of a transplanted organ are an interface between the donor and the recipient. The endothelium is believed to be a major target for graft rejection. After transplantation endothelial cells of a transplanted organ may be of recipient origin. OBJECTIVES: In this study we sought to determine whether endothelial chimerism correlates with graft rejection. METHODS: Biopsy samples from 34 renal transplants of female recipients who received kidneys from male donors were studied for the presence of endothelial cells of recipient origin. Formalin-fixed, paraffin-embedded tissue sections of renal biopsy samples were examined by fluorescence in situ hybridization (FISH) for the presence of endothelial cells containing two X chromosomes, using a biotinylated Y-chromosome probe and digoxigenin-labeled X-chromosome probe. RESULTS: The FISH methods identified endothelial cells of recipient origin. Endothelial chimerism was common, irrespective of rejection. Its presence was focal with these elements, coexisting in the biopsy. CONCLUSIONS: We observed no correlation between the percentage of recipient endothelial cells among vascular elements and the type of graft rejection (P > .05).     

Analysis of Acute Cellular Rejection Episodes in Recipients of Primary Intestinal Transplantation: A Single Center, 11-Year Experience

Intestinal transplantation has evolved over the years with major improvements in patient and graft survival. Acute cellular rejection of the intestine, however, still remains one of the most challenging aspects of postoperative management. We analyzed retrospectively collected data from 209 recipients of primary intestinal grafts at our institution over the past 11 years. A total of 290 episodes of biopsy-proven rejection requiring clinical treatment were analyzed. Rejection episodes doubled in length, on average, with each increasing grade (mild, moderate, severe). We observed increased incidence of overall rejection and particularly severe rejection in recipients of isolated intestinal and liver-intestine grafts in comparison with multivisceral grafts. Two rejection history variables had a significant negative impact on graft survival: the occurrence of a severe rejection episode and a rejection episode lasting >or=21 days. The lower incidence rate of severe rejection in recipients of multivisceral grafts might be due to a combination of increased donor lymphatic tissue and larger load of donor-derived immune competent cells present in the graft. The development of more effective monitoring and treatment protocols to prevent the occurrence of severe and/or lengthy rejection episodes is of critical importance for intestinal graft survival.