In vivo expansion of co-transplanted T cells impacts on tumor re-initiating activity of human acute myeloid leukemia in NSG mice.

Human cells from acute myeloid leukemia (AML) patients are frequently transplanted into immune-compromised mouse strains to provide an in vivo environment for studies on the biology of the disease. Since frequencies of leukemia re-initiating cells are low and a unique cell surface phenotype that includes all tumor re-initiating activity remains unknown, the underlying mechanisms leading to limitations in the xenotransplantation assay need to be understood and overcome to obtain robust engraftment of AML-containing samples. We report here that in the NSG xenotransplantation assay, the large majority of mononucleated cells from patients with AML fail to establish a reproducible myeloid engraftment despite high donor chimerism. Instead, donor-derived cells mainly consist of polyclonal disease-unrelated expanded co-transplanted human T lymphocytes that induce xenogeneic graft versus host disease and mask the engraftment of human AML in mice. Engraftment of mainly myeloid cell types can be enforced by the prevention of T cell expansion through the depletion of lymphocytes from the graft prior transplantation.

Xenotransplantation of human unrestricted somatic stem cells in a pig model of acute myocardial infarction.

BACKGROUND: Stem cell therapy may help restore cardiac function after acute myocardial infarction (AMI), but the optimal therapeutic cell type has not been identified. METHODS: We examined the effects of CD34-/CD45- human unrestricted somatic stem cells (USSCs) in pigs (n = 30) with an AMI created by a 90-min occlusion of the left anterior descending coronary artery. Pigs were randomly assigned to receive either USSCs (302 ± 23 × 10(6) cells) or phosphate-buffered saline via 15 NOGA-guided transendocardial injections 10 days after AMI. Cyclosporine A (10 mg/kg orally, twice a day) was started in all pigs 3 days before control or cell treatment. Cardiac function was assessed by echocardiography before injection and at 4 and 8 weeks after treatment. Serum titers for pig IgG antibodies against USSCs were also measured at these time points and before AMI. RESULTS: Compared with control pigs, USSC-treated pigs showed no significant differences in any of the functional parameters examined. USSC-treated pigs showed variable increases in anti-USSC IgG antibody titers in the blood and chronic inflammatory infiltrates at the cell injection sites. Immunohistochemical studies of the injection sites using human anti-mitochondrial antibodies failed to detect implanted USSCs. CONCLUSIONS: We conclude that human USSCs did not improve cardiac function in a pig model of AMI. Cell transplantation in a xenogeneic setting may obscure the benefits of stem cell therapy.

Suppression of NF-kappaB p65 expression attenuates delayed xenograft rejection.

BACKGROUND: Delayed xenograft rejection (DXR) involves type II vascular endothelial cell (VEC) activation including upregulation of pro-inflammatory genes, which contributes to infiltration into the graft and a complex process of cytokine production. Approaches to prevent DXR have shown limited success. In this study, we modified heart donors using siRNA in an attempt to attenuate DXR and to improve xenograft survival in the mouse-to-rat heterotopic heart transplant model. METHODS: siRNA technology was used to inhibit NF-kappaB p65 gene expression in vivo in mice. After the donor was transfected with siRNA, the effects of NF-kappaB siRNA on DXR and expression of NF-kappaB and pro-inflammatory genes were evaluated in a concordant mouse-to-rat cardiac xenograft model. RESULTS: Treatment of NF-kappaB siRNA prolonged median heart graft survival time in the recipient rats from 1.7 days in a PBS control group to 5.4 days in the NF-kappaB siRNA-treated group (P < 0.05). Compared with normal mouse hearts, the NF-kappaB p65 mRNA relative levels following siRNA injection in the donors decreased significantly (approximately 70% reduction) in grafts harvested 12 h after transplantation. The mRNA levels of VCAM-1, ICAM-1, and interleukin-1 displayed a similar reduction. Histological evaluation using light and electron microscopy showed that damage of endothelial cells after NF-kappaB siRNA treament occured at a later time. CONCLUSION: Transfection of NF-kappaB p65 siRNA in donor animals can delay the emergence of DXR. This treatment may be used as part of strategies to minimize the complex and multi-faceted rejection responses in vascularized xenografts.

Anti-CD2 producing pig xenografts effect localized depletion of human T cells in a huSCID model.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Characterization and quantification of porcine circulating endothelial cells.

BACKGROUND: Endothelial damage is a critical step in the development of (xeno) transplantation-related and cardiovascular pathology. In humans, the amount of circulating endothelial cells (CEC) correlates to disease intensity and functions as a valuable damage marker. While (xeno) transplantation and cardiovascular research is regularly performed in porcine models, the paucity of antibodies against porcine endothelium epitopes hinders the use of CEC as damage marker. OBJECTIVE: This study aimed to develop a method for porcine CEC detection using anti-human antibodies against porcine endothelium epitopes. METHODS: Human umbilical vein endothelial cells (HUVEC, control) and their swine equivalent (SUVEC) were used to assess the cross-species immunoreactivity of fluorescently labeled anti-human CD31/CD51/CD54/CD62E/CD105/CD106/CD144/CD146/PAL-E/lectin-1/vWF antibodies by isotype-controlled fluorescence-activated cell sorting (FACS) and confocal microscopy. Next, reactivity was ascertained with mature porcine kidney-derived endothelial cells (PKEC), and a FACS-based whole blood CEC quantification method was employed using osmotic erythrolysis and CD105 and CD146 double staining after CD45 exclusion. RESULTS: Of the 21 assayed antibodies, the MEM-229 clone of CD105 and P1H12 clone of CD146 showed immunoreactivity with SUVEC and PKEC. Double staining showed baseline porcine CEC count of 673.1 ± 551.4 CEC/ml, while the first 7.5 ml of drawn blood (representative of vascular damage) contained 1118 ± 661.4 CEC/ml (n = 14, P = 0.04). A second experiment (n = 5) including CD45 exclusion identified only 14.5 ± 10.8% double-positive CD105-146 events per ml blood. CONCLUSION: Porcine endothelium can be specifically labeled using anti-human CD146 and CD105 antibodies. These antibodies can therefore be used for the identification and quantification of CEC in porcine whole blood by FACS after osmotic erythrolysis.

Polymorphic Sirpa is the genetic determinant for NOD-based mouse lines to achieve efficient human cell engraftment.

Current mouse lines efficient for human cell xenotransplantation are backcrossed into NOD mice to introduce its multiple immunodeficient phenotypes. Our positional genetic study has located the NOD-specific polymorphic Sirpa as a molecule responsible for its high xenograft efficiency: it recognizes human CD47 and the resultant signaling may cause NOD macrophages not to engulf human grafts. In the present study, we established C57BL/6.Rag2(nullIl2rgnull) mice harboring NOD-Sirpa (BRGS). BRGS mice engrafted human hematopoiesis with an efficiency that was equal to or even better than that of the NOD.Rag1(nullIl2rgnull) strain, one of the best xenograft models. Consequently, BRGS mice are free from other NOD-related abnormalities; for example, they have normalized C5 function that enables the evaluation of complement-dependent cytotoxicity of antibodies against human grafts in the humanized mouse model. Our data show that efficient human cell engraftment found in NOD-based models is mounted solely by their polymorphic Sirpa. The simplified BRGS line should be very useful in future studies of human stem cell biology.

IL-15 activated human peripheral blood dendritic cell kill allogeneic and xenogeneic endothelial cells via apoptosis.

IL-15 is a pleotropic cytokine, which plays an important role in natural killer (NK) cell activity, T cell proliferation, and T cell cytotoxic activity. Dendritic cells (DCs) are the major antigen presenting cells in the immune system and presumed to play an important role in immune recognition of allo and xenotransplantation. We showed that IL-15 activated human peripheral blood DC is cytotoxic to human and porcine aortic endothelial cells. Unlike DCs, CD14+ monocytes show no cytotoxicity against the endothelial cells. This cytotoxic potential of IL-15 activated DC against endothelial cells is dose dependent and increases significantly upon treatment of endothelial cells with inflammatory cytokines like TNF-α or IFN-γ. The cytotoxic potential of IL-15 activated DC is associated with apoptosis of endothelial cells, as indicated by the increased Annexin V staining, caspase activation and loss of mitochondrial membrane potential. Further it was observed that DC mediated cytotoxicity against endothelial cell is mediated via granzyme B possibly secreted by the activated DCs.

Mechanisms of xenogeneic baboon platelet aggregation and phagocytosis by porcine liver sinusoidal endothelial cells.

BACKGROUND: Baboons receiving xenogeneic livers from wild type and transgenic pigs survive less than 10 days. One of the major issues is the early development of profound thrombocytopenia that results in fatal hemorrhage. Histological examination of xenotransplanted livers has shown baboon platelet activation, phagocytosis and sequestration within the sinusoids. In order to study the mechanisms of platelet consumption in liver xenotransplantation, we have developed an in vitro system to examine the interaction between pig endothelial cells with baboon platelets and to thereby identify molecular mechanisms and therapies. METHODS: Fresh pig hepatocytes, liver sinusoidal and aortic endothelial cells were isolated by collagenase digestion of livers and processing of aortae from GTKO and Gal+ MGH-miniature swine. These primary cell cultures were then tested for the differential ability to induce baboon or pig platelet aggregation. Phagocytosis was evaluated by direct observation of CFSE labeled-platelets, which are incubated with endothelial cells under confocal light microscopy. Aurintricarboxylic acid (GpIb antagonist blocking interactions with von Willebrand factor/vWF), eptifibatide (Gp IIb/IIIa antagonist), and anti-Mac-1 Ab (anti-α(M)ß(2) integrin Ab) were tested for the ability to inhibit phagocytosis. RESULTS: None of the pig cells induced aggregation or phagocytosis of porcine platelets. However, pig hepatocytes, liver sinusoidal and aortic endothelial cells (GTKO and Gal+) all induced moderate aggregation of baboon platelets. Importantly, pig liver sinusoidal endothelial cells efficiently phagocytosed baboon platelets, while pig aortic endothelial cells and hepatocytes had minimal effects on platelet numbers. Anti-MAC-1 Ab, aurintricarboxylic acid or eptifibatide, significantly decreased baboon platelet phagocytosis by pig liver endothelial cells (P<0.01). CONCLUSIONS: Although pig hepatocytes and aortic endothelial cells directly caused aggregation of baboon platelets, only pig liver endothelial cells efficiently phagocytosed baboon platelets. Blocking vWF and integrin adhesion pathways prevented both aggregation and phagocytosis.

Porcine bioprosthetic heart valves: The next generation.

There have been significant advances in organ xenotransplantation (cross-species transplantation), especially in the development of genetically engineered pigs, but clinical trials of solid organ transplants are still a time away. However, there is a form of pig-to-human xenotransplantation that has been taking place since the 1960s-bioprosthetic heart valve (BHV) replacement. Recently, there has been increasing evidence that, despite glutaraldehyde fixation of BHVs, there is a significant immune reaction to the valves, leading to calcification, rapid structural deterioration, and failure, particularly in young patients who have a more vigorous immune system and metabolism than the elderly. However, it is the young patients who would most benefit from such BHVs because these avoid the complications associated with the lifelong anticoagulation required with mechanical valves. In this review, we examine pathologic and immunohistochemical reports of failed BHVs that suggest that there is an immune response to these valves. Small animal studies that link the development of calcification and BHV failure to the immune response are reviewed. We draw parallels between the problems of glutaraldehyde-fixed tissue xenotransplantation and those currently being faced in live organ xenotransplantation. Finally, we discuss the advances being made in the production of genetically modified pigs and the evidence that these pigs may become a source of BHVs that can be used worldwide to treat valvular heart disease in children and young adults (for whom there is no ideal valve replacement in existence today). The design of a BHV that is resistant to the host's immune response would be a major step forward in cardiac surgery.

Costimulation blockade in pig artery patch xenotransplantation - a simple model to monitor the adaptive immune response in nonhuman primates.

BACKGROUND: CD154 blockade-based immunosuppression successfully prevents both humoral and cellular adaptive immune responses in baboons receiving α1,3-galactosyltransferase gene-knockout (GTKO) pig organs. Using a GTKO pig artery transplantation model in baboons, we evaluated the efficacy of CD28/B7 costimulatory pathway blockade in comparison with CD154 blockade. METHODS: Baboons received artery patch grafts from GTKO pigs, with no (Group1), anti-CD154mAb-based (Group2), or CTLA4-Ig-based (Group3) immunosuppressive therapy. Anti-pig IgM and IgG antibody and cellular responses were monitored. Xenografts were immunohistologically evaluated for antibody and complement deposition, and cellular infiltration. RESULTS: Group1 baboons developed increased IgM and IgG antibody and cellular responses against GTKO antigens. In Group2, anti-CD154mAb alone prevented the development of both IgM and IgG antibody and cellular responses,but not cellular infiltration of the graft. In the single baboon that received anti-thymocyte globulin (ATG) + mycophenolate mofetil (MMF) + anti-CD154mAb, cellular infiltration of the graft was not seen. In Group3, CTLA4-Ig with ATG + MMF inhibited the cellular proliferative response to pig antigens but did not prevent the IgG response or cellular infiltration. CONCLUSIONS: (i) Artery patch transplantation is a simple model to monitor the adaptive immune response to xenografts; (ii) anti-CD154mAb prevents sensitization but not cellular infiltration (but, without anticoagulation, may result in early thrombosis of a pig xenograft); (iii) although in only one baboon, the addition of ATG and MMF prevents cellular infiltration and (iv) replacement of anti-CD154mAb by CTLA4-Ig (at the doses used), even in combination with ATG and MMF, prevents the cellular proliferative response to GTKO pig antigens but is insufficient to prevent the development of anti-pig antibodies.

Increased human tumor necrosis factor-α levels induce procoagulant change in porcine endothelial cells in vitro.

BACKGROUND: Intravascular thrombosis and systemic coagulation abnormalities are major hurdles to successful xenotransplantation and are signs of acute humoral rejection. Increased expression of tissue factor (TF) is associated with the development of microvascular thrombosis in xenografts. To develop an effective strategy to prevent accelerated coagulation in xenografts, we investigated the mechanism by which porcine endothelial cells (PECs) become procoagulant after contact with human blood. METHODS: The changes in TF mRNA levels and activity in PECs after incubation with 20% human serum or human bioactive molecules, including C5a, tumor necrosis factor-α (TNFα) and interleukin (IL)-1α, were evaluated using real-time PCR and the factor Xa chromogenic assay, respectively. The procoagulant changes in PECs by these agonists were evaluated by measuring the coagulation time of human citrated plasma suspended with PECs pretreated with each agonist. TF expression and coagulation times were also assessed in PECs transfected with short interfering RNA (siRNA) designed to knock down porcine TF. We also examined the production of proinflammatory cytokines in human whole-blood or plasma after contact with PECs, which were screened using the cytometric bead array system. TNFα levels were measured using ELISA in whole-blood after contact with PECs, with or without the addition of xenoreactive antibodies or C1 esterase inhibitor. RESULTS: Porcine TF mRNA and activity in PECs were up-regulated in response to human TNFα and IL-1α but were not affected by C5a or 20% human serum. Up-regulation of TF expression by human TNFα or IL-1α shortened PEC-induced coagulation time, while siRNA-mediated knockdown of TF expression prolonged coagulation time. The incubation of PECs with human whole-blood led to a significant increase in human TNFα levels in the blood, which was promoted by the addition of xenoreactive antibodies and prevented by C1 esterase inhibitor. CONCLUSIONS: Human TNFα level increases in human blood after contact with PECs, which is attributed to xenoreactive antibody binding and subsequent complement activation. Human TNFα induces procoagulant changes in PECs with increased TF expression. This study suggests that human TNFα may be one of the mediators linking complement activation with procoagulant changes in the xenoendothelium.

Phytohemagglutinin-activated human T cells induce lethal graft-versus-host disease in cyclophosphamide and anti-CD122 conditioned NOD/SCID mice.

Graft-versus-host disease (GVHD) is a common complication after allogeneic hematopoietic stem cell transplantation. Much of our knowledge regarding GVHD comes from experiments on the mouse hematopoietic system due to ethical and technical constraints. Thus, in vivo GVHD models of the human immune system are required. In this study, we report an effective and reliable protocol for xenogeneic GVHD (xeno-GVHD) model induction using NOD/SCID mice, in which mice underwent a conditioning regimen consisting of intraperitoneal injection of cyclophosphamide and anti-CD122, followed by transfusion of phytohemagglutinin-activated human peripheral blood mononuclear cells containing 1 × 107 T cells, which has not been reported previously. The present model can be utilized to study human immune cell function in vivo and elucidate the mechanisms underlying the pathogenesis of human GVHD. In addition, this model system can help researchers to rapidly determine whether proposed therapeutic strategies for GVHD are efficient in vivo and will elucidate the underlying mechanisms of drugs and cells to be investigated. Furthermore, such a protocol will undoubtedly be very helpful to laboratories that have no available sources of irradiation.

Development of mature and functional human myeloid subsets in hematopoietic stem cell-engrafted NOD/SCID/IL2rγKO mice.

Although physiological development of human lymphoid subsets has become well documented in humanized mice, in vivo development of human myeloid subsets in a xenotransplantation setting has remained unevaluated. Therefore, we investigated in vivo differentiation and function of human myeloid subsets in NOD/SCID/IL2rγ(null) (NSG) mouse recipients transplanted with purified lineage(-)CD34(+)CD38(-) cord blood hematopoietic stem cells. At 4-6 mo posttransplantation, we identified the development of human neutrophils, basophils, mast cells, monocytes, and conventional and plasmacytoid dendritic cells in the recipient hematopoietic organs. The tissue distribution and morphology of these human myeloid cells were similar to those identified in humans. After cytokine stimulation in vitro, phosphorylation of STAT molecules was observed in neutrophils and monocytes. In vivo administration of human G-CSF resulted in the recruitment of human myeloid cells into the recipient circulation. Flow cytometry and confocal imaging demonstrated that human bone marrow monocytes and alveolar macrophages in the recipients displayed intact phagocytic function. Human bone marrow-derived monocytes/macrophages were further confirmed to exhibit phagocytosis and killing of Salmonella typhimurium upon IFN-γ stimulation. These findings demonstrate the development of mature and functionally intact human myeloid subsets in vivo in the NSG recipients. In vivo human myelopoiesis established in the NSG humanized mouse system may facilitate the investigation of human myeloid cell biology including in vivo analyses of infectious diseases and therapeutic interventions.

Cellular studies for in vitro modeling of xenogeneic immune responses.

Cellular studies are essential in the xenotransplantation field in order to investigate the cellular immune responses triggered by xenogeneic cells and identify the key molecules involved. A series of functional studies can be conducted with this purpose that include treatment with proinflammatory cytokines and xenogeneic cell-based assays that put together pig cells and human leukocytes such as monocytes, NK cells, and T cells. The choice of the pig cell type is critical to appropriately model the transplant setting of interest. Thus, pig endothelial cells are commonly used for studying the rejection process of vascularized organs. Treatment with cytokines allows studying the regulation of adhesion, costimulatory molecules, and receptors involved in triggering the immune response in an attempt to reproduce the more complex in vivo situation. The adhesion assays are used to determine the capacity of human leukocytes to adhere to porcine cells under various conditions. Furthermore, we describe coculture, costimulatory, and cytotoxicity assays for investigating the cellular and molecular mechanisms that take place during the xenogeneic immune response.

Thymic transplantation in pig-to-nonhuman primates for the induction of tolerance across xenogeneic barriers.

With the advent of knockout pigs for α1,3-galactosyltransferease (GalT-KO, which lack a cell-surface antigen to which humans have preformed antibodies), investigators have extended the survival of life-supporting xenorenal grafts. However, despite these increases, nonhuman primates transplanted with GalT-KO renal grafts are susceptible to anti-donor T-cell responses that are strong or stronger than allogeneic responses. In order to prevent rejection, recipients must be subjected to morbidly high levels of immunosuppression. For these reasons, our laboratory has attempted to develop novel methods of xenogeneic tolerance using vascularized porcine thymic grafts in order to reteach the recipient's immune system to accept the xenogeneic organ as self. These strategies, largely developed by Dr. Kazuhiko Yamada, involve the co-transplantation of a vascularized donor thymus with a kidney. This has been successfully done in two ways. The first method involves the preparation of a composite tissue "thymokidney" and the second utilizes the transplantation of an isolated vascularized thymic lobe. Both strategies involve the transplantation of fully vascularized thymic tissue at the time of xenotransplantation, a fact which is crucial for function of the thymic tissue immediately after xenografting and reeducation of recipient T-cells. These strategies have successfully induced tolerance across fully allogeneic models in miniature swine and prolonged graft survival in our pig-to-baboon model of life-supporting xenotransplantation to greater than 80 days with in vitro evidence of donor-specific unresponsiveness. Although it is too early for the development of clinical renal xenotransplantation protocols, this chapter describes the authors' unique experience with one of the most promising preclinical large-animal models of xenotransplantation. Furthermore, understanding the importance and measurement of T-cell responses in xenotransplantation is contingent upon a functional knowledge of these procedures.

Synergistic suppression of pre-perfusion of donor livers with recipient serum and cobra venom factor treatment on hyperacute rejection following liver xenotransplantation / Supressão sinérgica da rejeição hiperaguda no xenotransplante hepático com uso da pre-perfusão dos fígados doadores tratados com soro do receptor e com fator veneno de cobra

PURPOSE: To investigate synergistic suppression of donor liver pre-perfusion with recipient serum (RS) and cobra venom factor (CVF) treatment on hyperacute rejection (HAR) following liver xenotransplantation. METHODS: Guinea-pigs (GP, n=24) and Sprague-Dawley rats (SD, n=24) were recruited. Before transplantation, serum was collected from SD rats and used for preparation of inactivated complements. GP and SD rats were randomly assigned into four groups (n=6), respectively: RS group, CVF group, RS+CVF group and control group. Orthotopic liver xenotransplantation was performed with modified two-cuff technique. The survival time and liver function of recipients, morphological and pathological changes in rat livers were investigated. RESULTS: There was no piebald like change in the recipient livers in all experiment groups. The survival time of recipients in all experiment groups was longer than that in control group (p<0.05). Moreover, the survival time in the RS+CVF group was markedly longer than that in the RS group (p<0.01) and CVF group (p<0.05). The serum ALT level in all experiment groups were lower than that in the control group (p<0.05). Furthermore, the ALT level in the RS+CVF group was significantly lower than that in the CVF group (p<0.05) and RS group (p<0.01). The histological damages were significantly improved when compared with the control group, and the histological damages in the RS+CVF group were milder than those in the remaining groups (p<0.05) CONCLUSION: Pre-perfusion of donor liver with recipient serum and cobra venom factor treatment can exert synergistic suppressive effects on the hyperacute rejection following liver xenotransplantation.

OBJETIVO: Investigar a supressão sinérgica da pré-perfusão do doador de fígado com soro do receptor (SR) e tratamento com fator veneno de cobra (FVC) na rejeição hiperaguda (RHA) após o xenotransplante de fígado. MÉTODOS: Foram utilizados Cobaias (GP, n=24) e ratos Sprague-Dawley (SD, n=24). Antes do transplante foram coletadas amostras de soro dos ratos SD e usados para a preparação dos complementos inativados. Cobaias GP e ratos SD foram randomicamente distribuídos em quatro grupos (n=6), respectivamente: grupo RS, grupo FVC, grupo SR+FVC e grupo controle. Xenotransplante ortotópico do fígado foi realizado com a técnica de dois cuffs modificados. Foram investigados o de tempo de sobrevida, a função hepática dos receptores e alterações morfopatológicas em fígados de ratos. RESULTADOS: Não houve alteração na coloração do parênquima dos fígados nos receptores. O tempo de sobrevida dos receptores em todos os grupos experimentais foi mais longo do que o grupo controle (p<0,05). Além disso, o tempo de sobrevida do grupo SR+ FVC foi marcadamente maior do que o grupo SR (p<0,01) e o grupo FVC (p<0,05). O nível sérico ALT foi menor em todos os grupos experimentais do que o grupo controle (p<0,05). O nível de ALT no grupo SR+ FVC foi significantemente menor do que no grupo FVC (p<0,05) e o grupo SR (p<0,01). As alterações histológicas foram significantemente melhoradas quando comparado com o grupo controle, e os danos histológicos no grupo SR+ FVC foram mais moderados do que nos grupos restantes (p<0,05). CONCLUSÃO: Pré-perfusão do fígado doador com soro do receptor e fator veneno de cobra pode exercer efeito supressor sinérgico da rejeição hiperaguda após xenotransplante de fígado.

A crucial role of IL-17 and IFN-γ during acute rejection of peripheral nerve xenotransplantation in mice.

Nerve injuries causing segmental loss require nerve grafting. However, autografts and allografts have limitations for clinical use. Peripheral nerve xenotransplantation has become an area of great interest in clinical surgery research as an alternative graft strategy. However, xenotransplant rejection is severe with cellular immunity, and Th1 cells play an important role in the process. To better understand the process of rejection, we used peripheral nerve xenografts from rats to mice and found that mononuclear cells expressing IFN-γ and IL-17 infiltrated around the grafts, and IFN-γ and IL-17 producing CD4+ and CD8+ T cells increased during the process of acute rejection. The changes of IL-4 level had no significant difference between xenotransplanted group and sham control group. The rejection of xenograft was significantly prevented after the treatment of IL-17 and IFN-γ neutralizing antibodies. These data suggest that Th17 cells contribute to the acute rejection process of peripheral nerve xenotransplant in addition to Th1 cells.

Ectopic expression of murine CD47 minimizes macrophage rejection of human hepatocyte xenografts in immunodeficient mice.

UNLABELLED: Macrophages play an important role in the rejection of xenogeneic cells and therefore represent a major obstacle to generating chimeric mice with human xenografts that are useful tools for basic and preclinical medical research. The signal inhibitory regulatory protein α (SIRPα) receptor is a negative regulator of macrophage phagocytic activity and interacts in a species-specific fashion with its ligand CD47. Furthermore, SIRPα polymorphism in laboratory mouse strains significantly affects the extent of human CD47-mediated toleration of human xenotransplants. Aiming to minimize macrophage activity and thus optimize human cell engraftment in immunodeficient mice, we lentivirally transduced murine CD47 (Cd47) into human liver cells. Human HepG2 liver cells expressing Cd47 were less frequently contacted and phagocytosed by murine RAW264.7 macrophages in vitro than their Cd47-negative counterparts. For the generation of human-mouse chimeric livers in immunodeficient BALB-ΔRAG/γ(c) -uPA (urokinase-type plasminogen activator) mice, freshly thawed cryopreserved human hepatocytes were transduced with a lentiviral expression vector for Cd47 using a refined in vitro transduction protocol immediately before transplantation. In vivo, Cd47-positive human primary hepatocytes were selectively retained following engraftment in immunodeficient mice, leading to at least a doubling of liver repopulation efficiencies. CONCLUSION: We conclude that ectopic expression of murine Cd47 in human hepatocytes selectively favors engraftment upon transplantation into mice, a finding that should have a profound impact on the generation of robust humanized small animal models. Moreover, dominance of ectopically expressed murine Cd47 over endogenous human CD47 should also widen the spectrum of immunodeficient mouse strains suitable for humanization.

Anti-human leukocyte antigen-DR (MHC class II) humanized monoclonal antibody, IMMU-114, suppresses human to bovine cellular responses.

UNLABELLED: The effect of an anti-human leukocyte antigen-DR (MHC class II) humanized monoclonal antibody, IMMU-114, against the human to bovine cellular response was investigated. METHODS: Human peripheral mononuclear cells (PBMCs) were cocultured with inactivated self-PBMCs (Self), bovine PBMCs with control antibody (Xeno), or bovine PBMCs with IMMU-114 (IMMU-114). Cellular responses were investigated by thymidine incorporation assay, CFSE (carboxyfluorescein diacetate succinimidyl ester)-mixed lymphocyte reaction, and cytokine production in culture medium. RESULTS: Thymidine incorporation rates at a 1:1 responder to stimulator ratio for Xeno + control antibody, Xeno + IMMU-114, Self + control antibody, and Self + IMMU-114 were 14201.3 ± 1968.4, 513.0 ± 49.5, 952.7 ± 128.7, and 423.3 ± 138.8 cpm, respectively (P = 0.032). Those at a 1:2 ratio were 6518.0 ± 690.1, 896.6 ± 92.9, 1051.0 ± 123.6, and 736.0 ± 35.6 cpm, respectively (P = 0.036). CFSE-mixed lymphocyte reaction demonstrated that the frequencies of CFSE-low, CD4(+), and CD25(+) activating T cells in Self, Xeno, and IMMU-114 were 0.27 ± 0.04%, 3.65 ± 0.53%, and 1.23 ± 0.15%, respectively (P = 0.027). Cytokine production in culture medium indicated that IMMU-114 decreased Th1-type cytokines, including interleukin-2, interferon-γ, and tumor necrosis factor-α. CONCLUSION: IMMU-114 effectively suppresses human to bovine cellular responses. The mechanism involves direct inhibition of the interaction between class II human leukocyte antigen-DR-positive cells and CD4(+) T cells, and indirect suppression of Th1 cytokine production.