James Z. Appel

Pig kidney transplantation in baboons: anti-Gal(alpha)1-3Gal IgM alone is associated with acute humoral xenograft rejection and disseminated intravascular coagulation

BACKGROUND Kidneys harvested from miniature swine or pigs transgenic for human decay-accelerating factor (hDAF) were transplanted into baboons receiving an anti-CD154 monoclonal antibody (mAb) and either a whole body irradiation (WBI)- or cyclophosphamide (CPP)-based immunosuppressive regimen. METHODS Group 1 baboons (n=3) underwent induction therapy with WBI and thymic irradiation, pretransplantation antithymocyte globulin, and immunoadsorption of anti-Gal(alpha)1-3Gal (Gal) antibody (Ab). After transplantation of a miniature swine kidney, maintenance therapy comprised cobra venom factor, mycophenolate mofetil, and an anti-CD154 mAb (for 14-28 days). In group 2 (n=2), WBI was replaced by CPP in the induction protocol. Group 3 (n=3) animals received the group 2 regimen, but underwent transplantation with hDAF pig kidneys. RESULTS Group 1 and 2 animals developed features of disseminated intravascular coagulation (DIC), with reductions of fibrinogen and platelets and increases of prothrombin time, partial thromboplastin time, and fibrin split products. Graft survival was for 6-13 days. Histology showed mild acute humoral xenograft rejection (AHXR) of the kidneys, but severe rejection of the ureters. Group 3 animals developed features of DIC in two of three cases during the fourth week, with AHXR in the third case. Graft survival was for 28 (n=1) or 29 (n=2) days. Histology of day 15 biopsy specimens showed minimal focal mononuclear cellular infiltrates, with predominantly CD3+ cells. By days 28 and 29, kidneys showed mild-to-moderate features of AHXR. In all groups, the humoral response was manifest by reappearance of anti-Gal IgM below baseline level, with no or low return of anti-Gal IgG. All excised kidneys showed IgM deposition, but no complement and no or minimal IgG deposition. No baboon showed a rebound of anti-Gal Ab immediately after excision of the graft, and anti-Gal Ab increased over pretransplantation levels only when anti-CD154 mAb was discontinued. CONCLUSIONS DIC was observed with WBI- or CPP-based therapy, and after miniature swine or hDAF kidney transplantation. AHXR+/-DIC was observed in all recipients even in the absence of complement and no or low levels of anti-Gal IgG, but was significantly delayed in the hDAF recipients. These results confirm our earlier observation that CD154 blockade prevents T cell-dependent sensitization in baboons to pig antigens, but that baseline natural anti-Gal Ab production is not inhibited. We suggest that IgM deposition, even in the absence of IgG and complement, leads to endothelial cell activation with the development of DIC, even when there are only minimal histologic changes of AHXR.

Adult porcine islet transplantation in baboons treated with conventional immunosuppression or a non-myeloablative regimen and CD154 blockade

The aim of the present study was to assess the survival of adult porcine islets transplanted into baboons receiving either (1) conventional triple drug immunosuppressive therapy or (2) a non‐myeloablative regimen and an anti‐CD154 monoclonal antibody (mAb) aimed at tolerance‐induction.

Role of flow cytometry to define unacceptable HLA antigens in lung transplant recipients with HLA-specific antibodies

Background. Antidonor HLA-specific antibodies have been associated with hyperacute rejection and primary graft failure in lung transplant recipients. Thus, transplant candidates with HLA-specific antibodies generally undergo prospective crossmatching to exclude donors with unacceptable HLA antigens. However, the need to perform a prospective crossmatch limits the donor pool and is associated with increased waiting list times and mortality. A virtual crossmatch strategy using flow cytometry, which enables precise determination of HLA-specific antibody specificity, was compared to prospective crossmatching in sensitized lung transplant candidates. Methods. In all, 341 lung transplant recipients were analyzed retrospectively (April 1992 to July 2003). Sixteen patients with HLA-specific antibodies underwent transplantation based on flow cytometric determination of antibody specificity and 10 underwent prospective crossmatching. Results. Freedom from bronchiolitis obliterans syndrome (BOS) at three years was similar in those undergoing a virtual crossmatch, those undergoing prospective crossmatching, and those without HLA-specific antibodies (80.4%±13.4, 85.7%±13.2, and 73.8%±2.8, respectively, P=0.88). Three-year survival was also comparable (87.5%±8.3, 70.0%±14.5, and 78.5%±2.4, respectively, P=0.31). Elimination of prospective crossmatching for sensitized patients was associated with a significant decrease in time on the waiting list (P<0.01) and in waiting list mortality (P<0.05). All 16 patients undergoing a virtual crossmatch had negative retrospective crossmatches. Conclusions. By carefully determining the specificity of HLA-specific antibodies, flow cytometry methodologies enable the prediction of negative crossmatch results with up to 100% accuracy, enabling the determination of appropriateness of donors. Using this virtual crossmatch strategy, crossmatching can be safely omitted prior to lung transplantation, thereby decreasing waiting list time and mortality rates for candidates with HLA-specific antibodies.

Characterization of the innate immune response to chronic aspiration in a novel rodent model

BackgroundAlthough chronic aspiration has been associated with several pulmonary diseases, the inflammatory response has not been characterized. A novel rodent model of chronic aspiration was therefore developed in order to investigate the resulting innate immune response in the lung.MethodsGastric fluid or normal saline was instilled into the left lung of rats (n = 48) weekly for 4, 8, 12, or 16 weeks (n = 6 each group). Thereafter, bronchoalveolar lavage specimens were collected and cellular phenotypes and cytokine concentrations of IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-gamma, TNF-alpha, and TGF-beta were determined.ResultsFollowing the administration of gastric fluid but not normal saline, histologic specimens exhibited prominent evidence of giant cells, fibrosis, lymphocytic bronchiolitis, and obliterative bronchiolitis. Bronchoalveolar lavage specimens from the left (treated) lungs exhibited consistently higher macrophages and T cells with an increased CD4:CD8 T cell ratio after treatment with gastric fluid compared to normal saline. The concentrations of IL-1alpha, IL-1beta, IL-2, TNF-alpha and TGF-beta were increased in bronchoalveolar lavage specimens following gastric fluid aspiration compared to normal saline.ConclusionThis represents the first description of the pulmonary inflammatory response that results from chronic aspiration. Repetitive aspiration events can initiate an inflammatory response consisting of macrophages and T cells that is associated with increased TGF-beta, TNF-alpha, IL-1alpha, IL-1beta, IL-2 and fibrosis in the lung. Combined with the observation of gastric fluid-induced lymphocyitic bronchiolitis and obliterative bronchiolitis, these findings further support an association between chronic aspiration and pulmonary diseases, such as obliterative bronchiolitis, pulmonary fibrosis, and asthma.

Inhibition of platelet aggregation in baboons: therapeutic implications for xenotransplantation

Methods: Drugs tested in these experiments were aurintricarboxylic acid (ATA, von Willebrand Factor‐GPIb inhibitor), fucoidin (a selectin‐inhibitor), 1‐benzylimidazole (1‐BI, thromboxane synthase antagonist), prostacyclin (PGI2, endothelial stabilizer), heparin (thrombin antagonist), nitroprusside sodium or nicotinamide (NPN or NA, both NO‐donors), and eptifibatide (EFT, GPIIb/IIIa receptor antagonist). These were infused intravenously to nine baboons. Coagulation parameters and platelet counts were monitored and baboons were observed for adverse side‐effects. The efficacy of these agents in inhibiting platelet aggregation was assayed in a platelet aggregometer.

Chronic Aspiration of Gastric Fluid Induces the Development of Obliterative Bronchiolitis in Rat Lung Transplants

Long‐term survival of a pulmonary allograft is currently hampered by obliterative bronchiolitis (OB), a form of chronic rejection that is unique to lung transplantation. While tracheobronchial aspiration from gastroesophageal reflux disease (GERD) has clinically been associated with OB, no experimental model exists to investigate this problem. Using a WKY‐to‐F344 rat orthotopic left lung transplant model, the effects of chronic aspiration on pulmonary allograft were evaluated. Recipients received cyclosporine with or without 8 weekly aspirations of gastric fluid into the allograft. Six (66.7%) of 9 allografts with aspiration demonstrated bronchioles with surrounding monocytic infiltrates, fibrosis and loss of normal lumen anatomy, consistent with the development of OB. In contrast, none of the allografts without aspiration (n = 10) demonstrated these findings (p = 0.002). Of the grafts examined grossly, 83% of the allografts with chronic aspiration but only 20% without aspiration appeared consolidated (p = 0.013). Aspiration was associated with increased levels of IL‐1α, IL‐1β, IL‐6, IL‐10, TNF‐α and TGF‐β in BAL and of IL‐1α, IL‐4 and GM‐CSF in serum. This study provides experimental evidence linking chronic aspiration to the development of OB and suggests that strategies aimed at preventing aspiration‐related injuries might improve outcomes in clinical lung transplantation.

Mechanisms of thrombotic microangiopathy following xenogeneic hematopoietic progenitor cell transplantation.

Introduction. Attempts to induce tolerance though mixed hematopoietic chimerism in the discordant pig-to-baboon xenotransplantation model are sometimes complicated by a potentially fatal thrombotic microangiopathy in the recipient baboons. This state develops immediately after the infusion of porcine mobilized peripheral blood leukocytes, containing progenitor cells (PBPC). In our study, we examined the interaction of infused porcine PBPC with recipient platelets in vivo in baboons and investigated the underlying mechanisms using an in vitro model. Methods. Two naïve baboons and six baboons preconditioned with irradiation and immunosuppression that received porcine PBPC were evaluated in vivo. The interaction of porcine and baboon PBPC with baboon platelets was investigated by an in vitro platelet aggregation assay. Fresh and cryopreserved PBPC were evaluated as well as PBPC obtained from growth-factor mobilized and unmobilized pigs. Furthermore, cellular subsets of PBPC were assessed for potential to induce platelet aggregation. Immunohistochemical staining was performed on platelet-leukocyte aggregates and potential inhibition of aggregation with anti-P-selectin and anti-CD154 mAbs, or eptifibatide (a GPIIb/IIIa receptor antagonist), was tested. Results. All baboons that received porcine PBPC rapidly developed marked thrombocytopenia (<20,000/&mgr;l), elevated serum lactate dehydrogenase (>1,500U/liter), schistocytosis, and platelet aggregates on blood smear. Three baboons died (two untreated and one preconditioned), and substantive platelet aggregates containing porcine leukocytes were observed in the microvasculature of lungs and kidneys. In vitro, porcine, but not baboon, PBPC induced aggregation of baboon platelets in a dose-dependent manner. Immunohistological examination of these aggregates confirmed the incorporation of porcine leukocytes. Cryopreserved PBPC caused less aggregation than fresh PBPC, and growth-factor-mobilized PBPC induced less aggregation than unmobilized PBPC. Aggregation was fully abrogated by the addition of eptifibatide, and modulated by anti-P-selectin and anti-CD154 monoclonal antibodies that recognize adhesion receptors on activated platelets. Purified fractions (granulocytes, CD2+, and CD− cells) of porcine PBPC did not initiate aggregation, whereas addition of exogenous porcine PBPC membranes (erythrocytes, dead cells, and/or platelets) to the purified fractions exacerbated the aggregation response. Conclusions. These data indicate that porcine PBPC mediate aggregation of baboon platelets. This process likely contributes to the thrombotic microangiopathy observed after PBPC transplantation in the pig-to-baboon model. Eptifibatide can fully abrogate platelet aggregation induced by porcine PBPC in vitro. Purification of the progenitor cells from porcine PBPC and/or treatment of baboons with eptifibatide may be beneficial.

Rabbit Anti-thymocyte Globulin Induction Therapy Does Not Prolong Survival After Lung Transplantation

BACKGROUND Lung transplant survival is limited by the development of bronchiolitis obliterans syndrome (BOS). The strongest risk factor for BOS is acute rejection (AR). We have previously shown that rabbit anti-thymocyte globulin (RATG) induction therapy is associated with a decrease in early AR. Thus, we hypothesized that RATG induction would translate to reduced BOS and improved long-term graft survival. METHODS Forty-four lung recipients were prospectively randomized to receive conventional immunosuppression with RATG induction (RATG group) or conventional immunosuppression alone (control group). End-points included graft survival, early and total acute rejection, BOS and treatment complications. RESULTS There was no difference in graft survival between the groups at 8 years (RATG: 36%; control: 23%; p = 0.48). The RATG group had fewer early rejections compared with the control group (5% vs 41%; p = 0.01). However, the overall rejection incidence did not differ (RATG: 62%; control: 68%; p = 0.52). There was a trend toward a delay in BOS onset among RATG subjects compared with control subjects (2,376 days vs 1,108 days; log rank, p = 0.15). There was no difference in the incidence of infections, but the RATG group had a higher rate of malignancies. CONCLUSIONS Our results suggest that alternative approaches to anti-thymocyte induction should be pursued to reduce BOS and prolong allograft survival.

Hepatitis B Core Antibody Positive Donors as a Safe and Effective Therapeutic Option to Increase Available Organs for Lung Transplantation

Background. The use of hepatitis B core antibody (HBcAb+) and hepatitis C antibody (HCV Ab+) positive donors represents one strategy to increase available donor organs, but this remains controversial because of concern for viral transmission to recipients. We hypothesized that isolated HBcAb+ donors represent minimal risk of viral transmission in vaccinated lung transplant (LTx) recipients. Methods. A retrospective study was performed of LTx recipients who received HBcAb+ or HCV Ab+ pulmonary allografts. We analyzed liver function studies, viral hepatitis screening tests, quantitative polymerase chain reaction for hepatitis B viral DNA (HBV DNA) and hepatitis C viral RNA (HCV RNA), freedom from bronchiolitis obliterans syndrome, acute rejection, and survival. Results. Between April 1992 and August 2003, 456 LTx operations were performed. Twenty-nine patients (HB group) received HBcAb+ allograft transplants with a median posttransplant follow-up of 24.5 months. Three critically ill patients (HC group) received HCV Ab+ allografts with a median follow-up of 21.5 months. One-year survival for the HB group is 83% versus 82% for all patients who received non-HB organs (P=0.36). No patient in the HB group developed clinical liver disease because of viral hepatitis, and all patients alive (n=21) at follow-up are, to date, HBV DNA and/or HBcAb negative. All patients in the HC group tested HCV RNA positive; one patient died of liver failure at 22 months. Conclusions. Risk of viral transmission with HCV Ab+ allografts seems high after LTx. However, the use of HBcAb+ pulmonary allografts in recipients with prior hepatitis B vaccination seems to be a safe and effective strategy to increase organ availability.

The Pig as a Source of Cardiac Xenografts

The inadequate availability of human donor hearts and other organs has inspired interest in the field of xenotransplantation. Historically, ten attempts to transplant animal hearts into human recipients have been reported. Those who received hearts from nonhuman primates (i.e., baboons and chimpanzees) survived rather longer than did those who received hearts from nonprimates (i.e., sheep and pigs). Nevertheless, current opinion is that the pig is the best candidate as a source of hearts for humans despite the considerable immunologic disparity between the two species. Pigs are available in large numbers and can be bred easily and rapidly. They grow to appropriate sizes and their cardiovascular system is similar to that of humans. Substantial knowledge has been accumulated regarding both genetic engineering and tolerance induction in pigs, two strategies that may help to overcome the existing immunologic barriers. Concern has been raised, however, with regard to the potential for the transfer of a porcine infection with the pig organ to the human recipient. This brief review addresses these and other aspects of the use of the pig as a source of hearts for patients with end‐stage cardiac disease.