[Show abstract][Hide abstract] ABSTRACT: The instant blood-mediated inflammatory reaction (IBMIR) is a major obstacle to the engraftment of intraportal pig islet xenografts in primates. Higher expression of the galactose-α1,3-galactose (αGal) xenoantigen on neonatal islet cell clusters (NICC) than on adult pig islets may provoke a stronger reaction, but this has not been tested in the baboon model. Here, we report that WT pig NICC xenografts triggered profound IBMIR in baboons, with intravascular clotting and graft destruction occurring within hours, which was not prevented by anti-thrombin treatment. In contrast, IBMIR was minimal when recipients were immunosuppressed with a clinically relevant protocol and transplanted with NICC from αGal-deficient pigs transgenic for the human complement regulators CD55 and CD59. These genetically modified (GM) NICC were less susceptible to humoral injury in vitro than WT NICC, inducing significantly less complement activation and thrombin generation when incubated with baboon platelet-poor plasma. Recipients of GM NICC developed a variable anti-pig antibody response, and examination of the grafts 1 month after transplant revealed significant cell-mediated rejection, although scattered insulin-positive cells were still present. Our results indicate that IBMIR can be attenuated in this model, but long-term graft survival may require more effective immunosuppression or further donor genetic modification.
American Journal of Transplantation 05/2014; 14(6). DOI:10.1111/ajt.12722 · 6.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Xenotransplantation of porcine organs holds promise of solving the human organ donor shortage. The use of α-1,3-galactosyltransferase knockout (GTKO) pig donors mitigates hyperacute rejection, while delayed rejection is currently precipitated by potent immune and hemostatic complications. Previous analysis by our laboratory suggests that clotting factor VIII (FVIII) inhibitors might be elicited by the structurally restricted xenoantibody response which occurs after transplantation of either pig GTKO/hCD55/hCD59/hHT transgenic neonatal islet cell clusters or GTKO endothelial cells.MethodsA recombinant xenoantibody was generated using sequences from baboons demonstrating an active xenoantibody response at day 28 after GTKO/hCD55/hCD59/hHT transgenic pig neonatal islet cell cluster transplantation. Rhesus monkeys were immunized with GTKO pig endothelial cells to stimulate an anti-non-Gal xenoantibody response. Serum was collected at days 0 and 7 after immunization. A two-stage chromogenic assay was used to measure FVIII cofactor activity and identify antibodies which inhibit FVIII function. Molecular modeling and molecular dynamics simulations were used to predict antibody structure and the residues which contribute to antibody-FVIII interactions. Competition ELISA was used to verify predictions at the domain structural level.ResultsAntibodies that inhibit recombinant human FVIII function are elicited after non-human primates are transplanted with either GTKO pig neonatal islet cell clusters or endothelial cells. There is an apparent increase in inhibitor titer by 15 Bethesda units (Bu) after transplant, where an increase greater than 5 Bu can indicate pathology in humans. Furthermore, competition ELISA verifies the computer modeled prediction that the recombinant xenoantibody, H66K12, binds the C1 domain of FVIII.Conclusions
The development of FVIII inhibitors is a novel illustration of the potential impact the humoral immune response can have on coagulative dysfunction in xenotransplantation. However, the contribution of these antibodies to rejection pathology requires further evaluation because “normal” coagulation parameters after successful xenotransplantation are not fully understood.
[Show abstract][Hide abstract] ABSTRACT: Background
Promising developments in porcine islet xenotransplantation could resolve the donor pancreas shortage for patients with type 1 diabetes. Using α1,3-galactosyltransferase gene knockout (GTKO) donor pigs with multiple transgenes should extend xenoislet survival via reducing complement activation, thrombus formation, and the requirement for exogenous immune suppression. Studying the xenoantibody response to GTKO/hCD55/hCD59/hHT islets in the pig-to-baboon model, and comparing it with previously analyzed responses, would allow the development of inhibitory reagents capable of targeting conserved idiotypic regions.Methods
We generated IgM heavy and light chain gene libraries from 10 untreated baboons and three baboons at 28 days following transplantation of GTKO/hCD55/hCD59/hHT pig neonatal islet cell clusters with immunosuppression. Flow cytometry was used to confirm the induction of a xenoantibody response. IgM germline gene usage was compared pre- and post-transplant. Homology modeling was used to compare the structure of xenoantibodies elicited after transplantation of GTKO/hCD55/hCD59/hHT pig islets with those induced by GTKO and wild-type pig endothelial cells without further genetic modification.ResultsIgM xenoantibodies that bind to GTKO pig cells and wild-type pig cells were induced after transplantation. These anti-non-Gal antibodies were encoded by the IGHV3-66*02 (Δ28%) and IGKV1-12*02 (Δ25%) alleles, for the immunoglobulin heavy and light chains, respectively. IGHV3-66 is 86.7% similar to IGHV3-21 which was elicited by rhesus monkeys in response to GTKO endothelial cells. Heavy chain genes most similar to IGHV3-66 were found to utilize the IGHJ4 gene in 85% of V-D regions analyzed. However, unlike the wild-type response, a consensus complementary determining region 3 was not identified.Conclusions
Additional genetic modifications in transgenic GTKO pigs do not substantially modify the structure of the restricted group of anti-non-Gal xenoantibodies that mediate induced xenoantibody responses with or without immunosuppression. The use of this information to develop new therapeutic agents to target this restricted response will likely be beneficial for long-term islet cell survival and for developing targeted immunosuppressive regimens with less toxicity.
[Show abstract][Hide abstract] ABSTRACT: BackgroundB-cell depletion significantly extends survival of α-1,3-galactosyltranferase knockout (GTKO) porcine organs in pig-to-primate models. Our previous work demonstrated that the anti-non-Gal xenoantibody response is structurally restricted. Selective inhibition of xenoantigen/xenoantibody interactions could prolong xenograft survival while preserving B-cell-mediated immune surveillance.Methods
The anti-idiotypic antibody, B4N190, was selected from a synthetic human phage display library after enrichment against a recombinant anti-non-Gal xenoantibody followed by functional testing in vitro. The inhibitory small molecule, JMS022, was selected from the NCI diversity set III using virtual screening based on predicted xenoantibody structure. Three rhesus monkeys were pre-treated with anti-non-Gal-specific single-chain anti-idiotypic antibody, B4N190. A total of five monkeys, including two untreated controls, were then immunized with GTKO porcine endothelial cells to initiate an anti-non-α-1,3-Gal (non-Gal) xenoantibody response. The efficacy of the inhibitory small molecule specific for anti-non-Gal xenoantibody, JMS022, was tested in vitro.ResultsAfter the combination of in vivo anti-id and in vitro small molecule treatments, IgM xenoantibody binding to GTKO cells was reduced to pre-immunization levels in two-thirds of animals; however, some xenoantibodies remained in the third animal. Furthermore, when treated with anti-id alone, all three experimental animals displayed a lower anti-non-Gal IgG xenoantibody response compared with controls. Treatment with anti-idiotypic antibody alone reduced IgM xenoantibody response intensity in only one of three monkeys injected with GTKO pig endothelial cells. In the one experimental animal, which displayed reduced IgM and IgG responses, select B-cell subsets were also reduced by anti-id therapy alone. Furthermore, natural antibody responses, including anti-laminin, anti-ssDNA, and anti-thyroglobulin antibodies were intact despite targeted depletion of anti-non-Gal xenoantibodies in vivo indicating that selective reduction of xenoantibodies can be accomplished without total B-cell depletion.Conclusions
This preliminary study demonstrates the strength of approaches designed to selectively inhibit anti-non-Gal xenoantibody. Both anti-non-Gal-specific anti-idiotypic antibody and small molecules can be used to selectively limit xenoantibody responses.
[Show abstract][Hide abstract] ABSTRACT: Differential protein glycosylation in the donor and recipient can have profound consequences for transplanted organs, as evident in ABO-incompatible transplantation and xenotransplantation. In this study, we investigated the impact of altered fucosylation on graft acceptance by using donor mice overexpressing human α1,2-fucosyltransferase (HTF). Skin and heart grafts from HTF transgenic mice were rapidly rejected by otherwise completely matched recipients (median survival times 16 and 14 days, respectively). HTF skin transplanted onto mice lacking T and B cells induced an natural killer cell-mediated innate rejection crisis that affected 50-95% of the graft at 10-20 days. However, in the absence of adaptive immunity, the residual graft recovered and survived long-term (>100 days). Experiments using "parked" grafts or MHC class II-deficient recipients suggested that indirect rather than direct antigen presentation plays a role in HTF skin graft rejection, although the putative antigen(s) was not identified. We conclude that altered glycosylation patterns on donor tissue can trigger a powerful rejection response comprising both innate and adaptive components. This has potential implications for allotransplantation, in light of increasing recognition of the variability of the human glycome, and for xenotransplantation, where carbohydrate remodeling has been a lynchpin of donor genetic modification.
American Journal of Transplantation 02/2014; DOI:10.1111/ajt.12634 · 6.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Xenotransplantation using pigs as donors offers the possibility of eliminating the chronic shortage of donor kidneys, but there are several obstacles to be overcome before this goal can be achieved. Preclinical studies have shown that, while porcine renal xenografts are broadly compatible physiologically, they provoke a complex rejection process involving preformed and elicited antibodies, heightened innate immune cell reactivity, dysregulated coagulation, and a strong T cell-mediated adaptive response. Furthermore, the susceptibility of the xenograft to proinflammatory and procoagulant stimuli is probably increased by cross-species molecular defects in regulatory pathways. To balance these disadvantages, xenotransplantation has at its disposal a unique tool to address particular rejection mechanisms and incompatibilities: genetic modification of the donor. This review focuses on the pathophysiology of porcine renal xenograft rejection, and on the significant genetic, pharmacological, and technical progress that has been made to prolong xenograft survival.Kidney International advance online publication, 2 October 2013; doi:10.1038/ki.2013.381.
Kidney International 10/2013; 85(2). DOI:10.1038/ki.2013.381 · 8.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glutaraldehyde fixation does not guarantee complete tissue biocompatibility in current clinical bioprosthetic heart valves (BHVs). Particularly, circulating anti-αGal human antibodies increase significantly from just 10 days after a BHV implantation. The inactivation of such epitope should be mandatory to meet the requirements for a perspectively safe clinical application; nevertheless, its quantitative assessment in commercially available BHVs has never been carried out.
In this investigation, seven different models of BHVs were tested. The number of epitopes was determined with reference to a standard αGal source by an ELISA test. The presence of xenoantigen was subsequently confirmed by immunofluorescence analysis. Porcine tissue, knockout for the αGal epitopes, was used as negative control.
Epic™ valve was the only model among those tested, in which the αGal antigen appeared to be completely shielded. Composite Trifecta™ valve exhibited conflicting results: cusps of bovine pericardial tissue were devoid of reactive αGal epitopes, while the stent cover strip of porcine pericardium still maintained 30% of active antigens originally present in native tissue. All other tested BHVs express an αGal amount not significantly different from that exhibited by porcine Mosaic(®) valve (5.2 ± 0.6 × 10(10) each 10 mg of tissue).
For the first time, the quantitative evaluation of the αGal epitope in heart valve bioprostheses, already in clinical practice for about 40 yrs, was finally determined. Such quantification might provide indications of biocompatibility relevant for the selection of bioprosthetic devices and an increase in the confidence of the patient. It might become a major quality control tool in the production and redirection of future investigation in the quest for αGal-free long-lasting substitutes.
[Show abstract][Hide abstract] ABSTRACT: We investigated whether graft produced anti-human CD2, mediated by adenovirus (Adv) transduction of pig neonatal islet cell clusters (pNICC), would protect xenografts in a humanized mouse model from immune attack and whether such immunosuppression would remain local.
A mouse anti-human CD2 Ab (CD2hb11) previously generated by us was genetically engineered to produce chimeric and humanized versions. The three forms of CD2hb11 were named dilimomab (mouse), diliximab (chimeric) and dilizumab (humanized). All 3 forms of CD2hb11 Ab were tested for their ability to bind CD3+ human T cells and to inhibit a human anti-pig xenogeneic mixed lymphocyte reaction (MLR). They were administered systemically in a humanized mouse model in order to test their ability to deplete human CD3+ T cells and whether they induced a cytokine storm. An adenoviral vector expressing diliximab was generated for transduction of pNICC. Humanized mice were transplanted with either control-transduced pNICC or diliximab-transduced pNICC and human T cells within grafts and spleens were enumerated by flow cytometry.
Dilimomab and diliximab inhibited a human anti-pig xenogeneic response but dilizumab did not. All 3 forms of CD2hb11 Ab bound human T cells in vitro though dilimomab and diliximab exhibited 300-fold higher avidity than dilizumab. All 3 anti-CD2 Abs could deplete human CD3+ T cells in vivo in a humanized mouse model without inducing upregulation of activation markers or significant release of cytokines. Humanized mice transplanted with diliximab-transduced pNICC afforded depletion of CD3+ T cells at the graft site leaving the peripheral immune system intact.
Local production of a single Ab against T cells can reduce graft infiltration at the xenograft site and may reduce the need for conventional, systemic immunosuppression.
[Show abstract][Hide abstract] ABSTRACT: Modulation of purinergic signaling, which is critical for vascular homeostasis and the response to vascular injury, is regulated by hydrolysis of proinflammatory ATP and/or ADP by ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD-1; CD39) to AMP, which then is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine. We report here that compared with littermate controls (wild type), transgenic mice expressing human ENTPDase-1 were resistant to the formation of an occlusive thrombus after FeCl(3)-induced carotid artery injury. Treatment of mice with the nonhydrolyzable ADP analog, adenosine-5'-0-(2-thiodiphosphate) trilithium salt, Ado-5'-PP[S], negated the protection from thrombosis, consistent with a role for ADP in platelet recruitment and thrombus formation. ENTPD-1 expression decreased whole-blood aggregation after stimulation by ADP, an effect negated by adenosine-5'-0-(2-thiodiphosphate) trilithium salt, Ado-5'-PP[S] stimulation, and limited the ability to maintain the platelet fibrinogen receptor, glycoprotein α(IIb)/β(3), in a fully activated state, which is critical for thrombus formation. In vivo treatment with a CD73 antagonist, a nonselective adenosine-receptor antagonist, or a selective A(2A) or A(2B) adenosine-receptor antagonist, negated the resistance to thrombosis in transgenic mice expressing human ENTPD-1, suggesting a role for adenosine generation and engagement of adenosine receptors in conferring in vivo resistance to occlusive thrombosis in this model. In summary, our findings identify ENTPDase-1 modulation of purinergic signaling as a key determinant of the formation of an occlusive thrombus after vascular injury.
American Journal Of Pathology 05/2012; 181(1):322-33. DOI:10.1016/j.ajpath.2012.03.024 · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: CD39 (ectonucleoside triphosphate diphosphohydrolase-1; ENTPD-1) rapidly hydrolyzes ATP and ADP to AMP; AMP is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine, an anti-thrombotic and cardiovascular protective mediator. While expression of human CD39 in a murine model of myocardial ischemia/reperfusion (I/R) injury confers cardiac protection, the translational therapeutic potential of these findings requires further testing in a large animal model. To determine if transgenic expression of CD39 reduces infarct size in a swine model of myocardial ischemia/reperfusion injury, transgenic pigs expressing human CD39 (hCD39) were generated via somatic cell nuclear transfer and characterized. Expression of hC39 in cardiac tissue was confirmed by immunoblot and immunohistochemistry. Myocardial I/R injury was induced by intracoronary balloon inflation in the left anterior descending (LAD) artery for 60 min followed by 3 hours of reperfusion. The ischemic area was delineated by perfusion with 5% phthalo blue and the myocardial infarct size was determined by triphenyl tetrazolium chloride (TTC) staining. During ischemia, the rate-pressure product was significantly lower in control versus hCD39-Tg swine. Following reperfusion, compared to littermate control swine, hCD39-Tg animals displayed a significant reduction in infarct size (hCD39-Tg: 17.2 ± 4.3% vs. Control: 44.7 ± 5.2%, P=0.0025). Our findings demonstrate for the first time that the findings in transgenic mouse models translate to large animal transgenic models and validate the potential to translate CD39 into the clinical arena to attenuate human myocardial ischemia/reperfusion injury.
Journal of Molecular and Cellular Cardiology 01/2012; 52(5):958-61. DOI:10.1016/j.yjmcc.2012.01.002 · 5.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Galactosyl-transferase knockout (GT-KO) pigs represent the latest major progress to reduce immune reactions in xenotransplantation. However, their organs are still subject to rapid humoral rejection involving complement activation requiring the ongoing development of further genetic modifications in the pig. In a pig-to-baboon renal transplantation setting, we have used donor pigs that are not only GT-KO, but also transgenic for human CD55 (hCD55), hCD59, hCD39, and fucosyl-transferase (hHT). We studied kidney xenograft survival, physiological and immunologic parameters, xenogeneic rejection characteristics, as well as viral transmission aspects among two groups of baboons: control animals (n = 2), versus those (n = 4) treated with a cocktail of cyclophosphamide, tacrolimus, mycophenolate mofetil, steroids, and a recombinant human C1 inhibitor. Whereas control animals showed clear acute humoral rejection at around day 4, the treated animals showed moderately improved graft survival with rejection at around 2 weeks posttransplantation. Biopsies showed signs of acute vascular rejection (interstitial hemorrhage, glomerular thrombi, and acute tubular necrosis) as well as immunoglobulin (Ig)M and complement deposition in the glomerular and peritubular capillaries. The low level of preformed non-Gal-α1.3Gal IgM detected prior to transplantation increased at 6 days posttransplantation, whereas induced IgG appeared after day 6. No porcine endogenous retrovirus (PERV) transmission was detected in any transplanted baboon. Thus, surprisingly, organs from the GT-KO, hCD55, hCD59, hCD39, and hHT transgenic donors did not appear to convey significant protection against baboon anti-pig antibodies and complement activation, which obviously continue to be significant factors under a suboptimal immunosuppression regimen. The association, timing, and doses of immunosuppressive drugs remain critical. They will have to be optimized to achieve longer graft survivals.
[Show abstract][Hide abstract] ABSTRACT: Modulation of purinergic signaling is critical to myocardial homeostasis. Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD-1; CD39) which converts the proinflammatory molecules ATP or ADP to AMP is a key regulator of purinergic modulation. However, the salutary effects of transgenic over expression of ENTPD-1 on myocardial response to ischemic injury have not been tested to date. Therefore we hypothesized that ENTPD-1 over expression affords myocardial protection from ischemia-reperfusion injury via specific cell signaling pathways. ENTPD-1 transgenic mice, which over express human ENTPDase-1, and wild-type (WT) littermates were subjected to either ex vivo or in vivo ischemia-reperfusion injury. Infarct size, inflammatory cell infiltrate and intracellular signaling molecule activation were evaluated. Infarct size was significantly reduced in ENTPD-1 versus WT hearts in both ex vivo and in vivo studies. Following ischemia-reperfusion injury, ENTPD-1 cardiac tissues demonstrated an increase in the phosphorylation of the cellular signaling molecule extracellular signal-regulated kinases 1/2 (ERK 1/2) and glycogen synthase kinase-3β (GSK-3β). Resistance to myocardial injury was abrogated by treatment with a non-selective adenosine receptor antagonist, 8-SPT or the more selective A(2B) adenosine receptor antagonist, MRS 1754, but not the A(1) selective antagonists, DPCPX. Additionally, treatment with the ERK 1/2 inhibitor PD98059 or the mitochondrial permeability transition pore opener, atractyloside, abrogated the cardiac protection provided by ENTPDase-1 expression. These results suggest that transgenic ENTPDase-1 expression preferentially conveys myocardial protection from ischemic injury via adenosine A(2B) receptor engagement and associated phosphorylation of the cellular protective signaling molecules, Akt, ERK 1/2 and GSK-3β that prevents detrimental opening of the mitochondrial permeability transition pore.
Journal of Molecular and Cellular Cardiology 09/2011; 51(6):927-35. DOI:10.1016/j.yjmcc.2011.09.003 · 5.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acute humoral xenograft rejection (AHXR) is an important barrier to xenograft survival. Human tumor necrosis factor-α (hTNF-α) is one of the essential mediators of AHXR and induces activation of porcine endothelial cells (PECs), resulting in upregulation of major histocompatibility complex molecules, adhesion molecules, and proinflammatory chemokines. We investigated whether introduction of a soluble human tumor necrosis factor receptor I-Fc (shTNFRI-Fc) fusion gene can suppress activation of PECs and, more importantly, produced shTNFRI-Fc transgenic pigs.
The shTNFRI-Fc gene expression vector was constructed and inserted into PECs. The inhibitory effects of shTNFRI-Fc were tested by luciferase assay, reverse-transcriptase polymerase chain reaction, and flow cytometry. A shTNFRI-Fc transgenic pig was generated by somatic cell nuclear transfer. The expression of shTNFRI-Fc in the transgenic pig was evaluated by PCR, western blot, enzyme-linked immunosorbent assay, and immunohistochemistry. The inhibitory effects of shTNFRI-Fc in the serum obtained from the transgenic pig were also tested.
In comparison with control green fluorescent protein, shTNFRI-Fc protein showed much stronger inhibitory effects on NF-κB activation in the HEK293-NF-κB-luciferase reporting cell line, expression of chemokines and adhesion molecules in PECs, and TNF-α-mediated cytotoxicity. We successfully generated shTNFRI-Fc transgenic pig. Sera obtained from the transgenic pig inhibited induction of chemokines, and E-selectin in PECs stimulated with Human TNF-α.
We have generated transgenic pigs producing shTNFRI-Fc protein that can inhibit TNF-α-mediated activation of PECs. Because TNF-α is an important mediator of xenograft rejection, the use of xenografts that can produce shTNFRI-Fc proteins de novo could be an effective approach in overcoming a considerable component of the xenograft rejection process, especially AHXR.
[Show abstract][Hide abstract] ABSTRACT: Deletion of the α1,3-galactosyltransferase (GalT) gene in pigs has removed a major xenoantigen but has not eliminated the problem of dysregulated coagulation and vascular injury. Rejecting GalT knockout organ xenografts almost invariably show evidence of thrombosis and platelet sequestration, and primate recipients frequently develop consumptive coagulopathy. This review examines recent findings that illuminate potential mechanisms of this current barrier to successful xenotransplantation.
The coagulation response to xenotransplantation differs depending on the type of organ and quite likely the distinct vasculatures. Renal xenografts appear more likely to initiate consumptive coagulopathy than cardiac xenografts, possibly reflecting differential transcriptional responses. Liver xenografts induce rapid and profound thrombocytopenia resulting in recipient death within days due to bleeding; ex-vivo data suggest that liver endothelial cells and hepatocytes are responsible for platelet consumption by a coagulation-independent process.It has been proposed that expression of recipient tissue factor on platelets and monocytes is an important trigger of consumptive coagulopathy. Finally, pigs transgenic for human anticoagulants and antithrombotics are slowly but surely coming on line, but have not yet been rigorously tested to date.
Successful control of coagulation dysregulation in xenotransplantation may require different combinatorial pharmacological and genetic strategies for different organs.
Current opinion in organ transplantation 04/2011; 16(2):214-21. DOI:10.1097/MOT.0b013e3283446c65 · 2.38 Impact Factor