Prolonged cardiac allograft survival using iodine 131 after human sodium iodide symporter gene transfer in a rat model.

BACKGROUND: Radioiodine is efficiently concentrated by tissues expressing the human sodium iodide symporter (hNIS). OBJECTIVE: To analyze the effects of iodine 131 on acute cardiac allograft rejection after ex vivo hNIS gene transfer in a rat model of cardiac allotransplantation. MATERIALS AND METHODS: Hearts from Brown Norway rats were perfused ex vivo either with UW (University of Wisconsin) solution (n = 9) or UW solution containing 1 x 10(9) pfu/mL of adenovirus 5 plus NIS (Ad-NIS) (n = 18). Donor hearts were transplanted heterotopically into the abdomen of Lewis rats, and recipients were treated on postoperative day 3 with either 15,000 microCi of (131)I or saline solution. The hearts were explanted when no longer beating, and were evaluated histologically for evidence of rejection and other changes. RESULTS: Grafts perfused with the Ad-NIS vector survived significantly longer in recipients injected with (131)I (mean [SD], 11.3 [1.9] days) compared with control animals not treated with (131)I (5.7 [0.65] days) (P < .001). Treatment with (131)I did not prolong graft survival in recipients of hearts that were not perfused with Ad-NIS (5.5 [1.0] vs 5.3 [0.8] days). In Ad-NIS (131)I-treated transplants, the level of myocardial damage on day 6 after surgery, when control hearts were rejected, was significantly lower (60.8 [28.0] vs 99.7 [0.8]; P < .05). CONCLUSION: Our findings indicate that (131)I, after NIS gene transfer, can effectively prolong cardiac allograft survival. To our knowledge, this is the first report of the use of NIS-targeted (131)I therapy in cardiac transplantation. Further studies are required to determine the mechanism of this effect and its potential for clinical application.

Expression of complement regulatory proteins in accommodated xenografts induced by anti-alpha-Gal IgG1 in a rat-to-mouse model.

Anti-graft antibodies are often associated with graft rejection. Under special conditions, grafts continue to function normally even in the presence of anti-graft antibodies and complement. This condition is termed accommodation. We developed a xenograft accommodation model in which baby Lewis rat hearts are transplanted into Rag/GT-deficient mice, and accommodation is induced by repeated i.v. injections of low-dose anti-alpha-Gal IgG(1). The accommodated grafts survived a bolus dose of anti-alpha-Gal IgG(1), while freshly transplanted second grafts were rejected. To study the mechanism of anti-alpha-Gal IgG(1)-mediated accommodation, both real-time PCR and immunohistochemical staining revealed elevated expression of DAF, Crry and CD59 in the accommodated grafts. In vitro exposure of rat endothelial cells to anti-alpha-Gal IgG(1) also induced the up-regulation of DAF, Crry and CD59, as revealed by Western blot analyses, and was associated with an acquired resistance to antibody and complement-mediated lysis in vitro. Collectively, these studies suggest that the up-regulation of complement regulatory proteins may abrogate complement-mediated rejection and permit the development of xenograft accommodation.

The structure of anti-Gal immunoglobulin genes in naïve and stimulated Gal knockout mice.

BACKGROUND: Naturally occurring antibodies (Nabs) that bind to terminal galactose alpha1,3-galactose carbohydrate structures (Gal) are present in humans and Old World monkeys but are negatively regulated in other mammalian species because they express Gal epitopes on their cell surfaces. A Gal knockout mouse (Gal-/-) model, generated by homologous disruption of alpha1,3-galactosyltransferase gene, is capable of producing natural anti-Gal Abs. METHODS: To study the genetic control of the anti-Gal response, we have generated anti-Gal hybridomas from Gal-/- mice and analyzed VH genes of anti-Gal Abs from naïve animals and from mice stimulated by rat heterotopic heart transplantation. RESULTS: Six immunoglobulin (Ig)M anti-Gal hybridomas derived from naïve Gal-/- mice exhibited anti-Gal binding activity with some cross-reactivity to related carbohydrate structures. These naïve anti-Gal Abs used five different VH genes in a germline configuration. Anti-Gal IgM hybridomas isolated after a rat heterotopic heart xenograft (4 days) utilized germline VH gene segments from the VH7183 family and exhibited less cross-reactivity. In contrast to mice 4 days after xenograft, we have predominantly isolated IgG anti-Gal hybridomas from mice 21 days after rat heterotopic heart xenografts, indicating an isotype switch. Nine of the IgG anti-Gal hybridomas secreted IgG3 subclass and one produced IgG1. Sequence analysis of the VH gene usage from the induced anti-Gal IgG antibodies demonstrated a restricted gene utilization (VHJ606-V14A). CONCLUSION: Our results demonstrate that the anti-Gal response in naïve Gal-/- mice is encoded by multiple germline progenitors. In response to a xenograft, the induced anti-Gal Abs exhibited a restricted gene usage and somatic mutations, indicating a positive selection.

The role of antibodies in dysfunction of pig-to-baboon pulmonary transplants.

OBJECTIVE: Pulmonary transplantation has become the preferred treatment for end-stage lung disease, but application of the procedure is limited because of a paucity of donors. One way to solve donor limitations is to use animal organs as a donor source or xenotransplantation. The current barrier to pulmonary xenotransplantation is the rapid failure of the pulmonary xenograft. Although antibodies are known to play a role in heart and kidney xenograft rejection, their involvement in lung dysfunction is less defined. This project was designed to define the role of antibodies in pulmonary graft rejection in a pig-to-baboon model. METHODS: Orthotopic transgenic swine left lung transplants were performed in baboons depleted of antibodies by one of three techniques before transplantation: (1) ex vivo swine kidney perfusion, (2) total immunoglobulin-depleting column perfusion, and (3) ex vivo swine lung perfusion. Results were compared with those of transgenic swine lung transplants in unmodified baboons. RESULTS: All three techniques of antibody removal resulted in depletion of xenoreactive antibodies. Only pretransplantation lung perfusion improved pulmonary xenograft function compared with lung transplantation in unmodified baboons. CONCLUSIONS: The pathogenesis of pulmonary injury in a swine-to-primate transplant model is different from that in renal and cardiac xenografts. Depletion of antibodies alone does not have a beneficial effect and may actually be detrimental.

The role of anti-Galalpha1-3Gal antibodies in acute vascular rejection and accommodation of xenografts.

BACKGROUND: A major impediment to the transplanting of porcine organs into humans is the susceptibility of porcine organs to acute vascular rejection, which can destroy a vascularized xenograft over a period of hours to days. Acute vascular rejection of porcine-to-primate xenografts is thought to be triggered by binding of xenoreactive antibodies to the graft. We tested whether antibodies, binding to Galalpha1-3Gal epitopes in porcine tissue, initiate this phenomenon. METHODS AND RESULTS: Specific depletion of anti-Galalpha1-3Gal antibodies from the blood of baboons, using extracorporeal perfusion of separated plasma through columns of Sepharose beads covalently linked to the antigenic trisaccharide, Galalpha1-3Galbeta1-4GlcAc, averted the development of acute vascular rejection in porcine organs transgenic for human decay-accelerating factor and CD59. More importantly, after immunodepletion was stopped and Gala1-3Gal antibodies were allowed to return, these same organs continued to function and remained pathologically normal and thus seemed to achieve a state of accommodation. CONCLUSION: These results demonstrate that anti-Galalpha1-3Gal antibodies cause acute vascular rejection and suggest that depletion of these antibodies leads to accommodation of the donor cardiac xenograft and could supply an important model for additional study.

Human complement regulatory proteins protect swine lungs from xenogeneic injury.

BACKGROUND: Pulmonary xenotransplantation is not possible because of hyperacute lung injury, the pathogenesis of which is unknown. This study evaluates complement-dependent pathways of pulmonary injury during heterologous perfusion of swine lungs. METHODS: Lungs from unmodified swine and swine expressing human decay-accelerating factor and human CD59 (hDAF/hCD59 swine) were perfused with either human plasma or baboon blood. Pulmonary vascular resistance and static pulmonary compliance were measured serially, and swine lung tissue were examined by light microscopy. Complement activation was assessed by serial measurements of baboon plasma C3a-desArg concentrations. RESULTS: Perfusion of unmodified swine lungs with human plasma and baboon blood resulted in hyperacute lung injury within minutes of perfusion. However, function was preserved in swine lungs expressing human decay-accelerating factor and human CD59. In both study groups, xenogeneic perfusion with baboon blood resulted in at least a sevenfold increase in plasma C3a-desArg levels suggesting transient activation of complement. CONCLUSIONS: Lungs from swine expressing human decay-accelerating factor and human CD59 were resistant to injury during perfusion with human plasma and baboon blood, indicating that complement mediated some of the features of xenogeneic acute lung injury.

The role of antibodies in acute vascular rejection of pig-to-baboon cardiac transplants.

Long-term success in xenotransplantation is currently hampered by acute vascular rejection. The inciting cause of acute vascular rejection is not yet known; however, a variety of observations suggest that the humoral immune response of the recipient against the donor may be involved in the pathogenesis of this process. Using a pig-to-baboon heterotopic cardiac transplant model, we examined the role of antibodies in the development of acute vascular rejection. After transplantation into baboons, hearts from transgenic pigs expressing human decay-accelerating factor and CD59 underwent acute vascular rejection leading to graft failure within 5 d; the histology was characterized by endothelial injury and fibrin thrombi. Hearts from the transgenic pigs transplanted into baboons whose circulating antibodies were depleted using antiimmunoglobulin columns (Therasorb, Unterschleisshein, Germany) did not undergo acute vascular rejection in five of six cases. Biopsies from the xenotransplants in Ig-depleted baboons revealed little or no IgM or IgG, and no histologic evidence of acute vascular rejection in the five cases. Complement activity in the baboons was within the normal range during the period of xenograft survival. In one case, acute vascular rejection of a xenotransplant occurred in a baboon in which the level of antidonor antibody rose after Ig depletion was discontinued. This study provides evidence that antibodies play a significant role in the pathogenesis of acute vascular rejection, and suggests that acute vascular rejection might be prevented or treated by therapies aimed at the humoral immune response to porcine antigens.

Complement-mediated pulmonary xenograft injury: studies in swine-to-primate orthotopic single lung transplant models.

BACKGROUND: The pathogenesis of acute pulmonary xenograft injury has not yet been determined. The present study evaluates the role of complement in mediating pulmonary xenograft dysfunction by using cobra venom factor (CVF) to deplete recipient complement and transgenic swine, which express human regulators of complement activation (human decay-accelerating factor [hDAF] and hCD59). METHODS: Fifteen orthotopic lung transplants were performed as follows: group I, swine-to-swine (n=5); group II, unmodified swine-to-baboon (n=3); group III, unmodified swine-to-(CVF treated) baboon (n=3); and group IV, hCD59/hDAF swine-to-baboon (n=4). Left pulmonary artery flow and pulmonary vascular resistance were measured at 30-min intervals. Serial lung biopsies were examined by light microscopy and immunofluorescence. The activation of complement was quantified by measurement of baboon plasma CH50 and C4 functional activity. RESULTS: Group II xenotransplants ceased functioning within 30 min of reperfusion. Histopathologic ab normalities included erythrocyte/platelet aggregates and hemorrhagic pulmonary edema. Groups I and IV showed excellent function throughout. hDAF/hCD59 lungs (group IV) showed trace venular fibrin plugs and moderate loss of alveolar architecture. Pretreatment with CVF (group III) was ineffective in preventing xenograft injury. CONCLUSIONS: These results characterize the fundamental features of discordant pulmonary xenotransplantation. Correction of the known defects in the regulation of heterologous complement activation was partially effective in preventing pulmonary xenograft dysfunction, suggesting that complement mediates, in part, some of the features of acute lung injury after discordant lung xenotransplantation.

Glycosyl phosphatidylinositol anchor.

Recently, we and others demonstrated the unique potential for glycosyl phosphatidylinositol (GPI) anchored proteins to transfer from one cell membrane to another in a process we termed 'painting'. The GPI-anchored proteins were shown to transfer intact and functional. The full significance of this phenomenon has yet to be fully realized, but implications exist in many areas including disease transmission (prions), cell protection (endothelial cells), and senescence (erythrocytes). It is of interest to note that cells exhibiting limited or no biosynthetic capacity (spermatozoa and erythrocytes) have been implicated thus far in cell-cell transfer of GPI-linked molecules. This observation demonstrates the potential for GPI-linked proteins to be 'painted' onto cells which otherwise may be incapable of expressing exogenous proteins. We show in this paper that GPI-linked CD59 and decay-accelerating factor will transfer intact from erythrocytes to endothelial cells in transgenic mice. We also demonstrate that the transfer process occurs under physiological conditions using several experimental models including organ and bone marrow transplantation. We detail the procedure to effect transfer of GPI-linked proteins from one cell type to another in either an in vivo or in vitro system.

Swine lungs expressing human complement-regulatory proteins are protected against acute pulmonary dysfunction in a human plasma perfusion model.

UNLABELLED: Pulmonary transplantation is currently limited by the number of suitable cadaver donor lungs. For this reason, pulmonary xenotransplantation is currently being investigated. OBJECTIVE: Our goal was to assess the role of complement in pulmonary xenograft dysfunction. METHODS: The pulmonary function of swine expressing human decay accelerating factor and human CD59 (n = 6) was compared with that of the lungs from nontransgenic (control) swine (n = 6) during perfusion with human plasma. RESULTS: After 2 hours of perfusion, the pulmonary vascular resistance was 1624 +/- 408 dynes.sec.cm-5 in control lungs and 908 +/- 68 dynes.sec.cm-5 in transgenic lungs (p < 0.05). Control lungs had a venous oxygen tension of 271 +/- 23 mm Hg with a ratio of venous oxygen tension to inspired oxygen fraction of 452 +/- 38 at 2 hours of perfusion; transgenic lungs had a venous oxygen tension of 398 +/- 11 mm Hg and a ratio of venous oxygen tension to inspired oxygen fraction of 663 +/- 18 (p < 0.05). Control lungs showed a decrease of 79.8% +/- 3.7% in static pulmonary compliance by 2 hours, versus a 12.0% +/- 8.1% decrease by the transgenic lungs (p < 0.05). The control lungs also developed 561.7 +/- 196.2 ml of airway edema over 2 hours, in contrast to 6.5 +/- 1.7 ml in transgenic lungs (p < 0.05). CONCLUSION: Lungs from swine expressing human decay accelerating factor and human CD59 functioned better than nontransgenic swine lungs when perfused with human plasma. These results suggest that complement activation is involved in producing acute pulmonary xenograft dysfunction and demonstrate that lungs from swine expressing human decay accelerating factor and human CD59 are protected against pulmonary injury when perfused with human plasma.

Transgenic pigs expressing human CD59 and decay-accelerating factor produce an intrinsic barrier to complement-mediated damage.

We characterize a line of transgenic pigs that express the human complement-regulatory proteins human CD59 and human decay-accelerating factor. These genes, under the control of heterologous promoters, are expressed in a variety of organs, including the vasculature of the heart, kidney, and liver. We demonstrate that moderate levels of these gene products are sufficient to protect peripheral blood cells from human or baboon complement. Using pig to baboon heterotopic heart transplants, we show that expression of these proteins is sufficient to block the complement-mediated damage that is the hallmark of such xenografts, when nontransgenic organs are used. These results indicate that there is significant species specificity of intrinsic complement regulatory protein function. This specificity is evident in transgenic organs in which low levels of human CD59 and human decay-accelerating factor expression significantly effect the humoral immune response that causes xenograft rejection. This result suggests that transgenic organs with high levels of human complement-regulatory protein expression will be sufficient to alleviate the humoral immunological barriers that currently block the use of xenogeneic organs for human transplantation.

Humoral responses to pig-to-baboon cardiac transplantation: implications for the pathogenesis and treatment of acute vascular rejection and for accommodation.

Organs transplanted between phylogenetically-disparate species, such as from the pig into the primate, are subject to hyperacute and acute vascular rejection. Hyperacute rejection of a porcine organ by a primate is thought to be initiated by the binding of xenoreactive natural antibodies to Galalpha1-3Gal expressed on the endothelial lining of blood vessels in the xenograft. The factor(s) which initiates acute vascular rejection is uncertain; however, there is some evidence implicating xenoreactive antibodies. The nature of the humoral response which might contribute to acute vascular rejection of a porcine organ was investigated in baboons which received a porcine cardiac xenograft plus immunosuppression with methylprednisolone, azathioprine, and cyclosporine. Following rejection and surgical removal of the xenografts, the serum concentration of xenoreactive antibodies increased in untreated animals but in immunosuppressed animals was similar to the concentration in preimmune serum. The antibodies in the sensitized recipients were specific for Galalpha1-3Gal (70-95%) and other determinants (5-30%). However, cross-blocking studies showed that, following xenotransplantation, the immunosuppressed baboons had no detectable IgM or IgG directed against "new" endothelial antigens. These results indicate that antibodies made by immunosuppressed individuals in response to xenotransplantation are much like xenoreactive natural antibodies and suggest that acute vascular rejection might in some cases be addressed by therapeutic strategies aimed at those antibodies.

Spatial and temporal regulation of a lacZ reporter transgene in a binary transgenic mouse system.

The transgenic mouse system is a powerful tool for the study of gene function. However, when the analysis involves genes that are critical for the normal developmental process, the usefulness of transgenic mouse systems is limited (for review see Hanahan, 1989; Westphal and Gruss, 1989; Byrne et al., 1991). This is due to potential transgene interference with development in case of ectopic or high level expression. As a result, establishing permanent transgenic mouse lines expressing these types of genes has proven difficult. To circumvent these difficulties, a binary transgenic mouse system has been established, termed the Multiplex System (Byrne and Ruddle, 1989). This is a two-tiered gene activation system in which expression of the gene of interest occurs only in offspring carrying transgenes encoding both components: transactivator and transresponder. Transactivator lines contain the gene encoding the VP16 protein of herpes simplex virus. Transresponder lines harbour the gene of interest linked to the IE promoter which includes recognition sequences for the VP16 transactivator. Previously, the inducibility of a chloramphenicol acetyltransferase reporter gene in newborn offspring that carried both a transactivator and transresponder transgene (Byrne and Ruddle, 1989) has been shown. Moreover, it has been demonstrated that expression of the VP16 protein was not detrimental to development and that transactivation appeared to be tissue specific. Here, the potential of the system for the expression of transgenes in early mouse embryogenesis was examined, using the Escherichia coli beta-galactosidase gene as a reporter in the transresponder mouse strain. To direct expression of VP16, the murine Hoxc-8 promoter, which is known to be active during early development, was used. Embryos from crosses of transactivators to transresponders were isolated at different stages of development and stained for beta-galactosidase activity. Transactivation, as demonstrated by strong beta-galactosidase staining, could be detected as early as eight days of development. At all stages examined, the pattern of lacZ transresponder gene expression accurately reflected the activity of the Hoxc-8 promoter controlling VP16 expression. It is demonstrated that the Multiplex System can be used to express transresponder transgenes in a spatially and temporally defined manner in multiple cell types early during mouse embryogenesis.

Protection of xenogeneic cardiac endothelium from human complement by expression of CD59 or DAF in transgenic mice.

We investigated the ability of membrane-bound human complement regulatory proteins to control complement-driven humoral immune reactions on murine microvasculature. The human complement regulatory proteins CD59 and DAF were expressed using heterologous promoters in a variety of tissues in transgenic mice. Animals expressing these gene products are healthy and exhibit significant levels of endothelial cell expression of CD59 and DAF in cardiac muscle. Transgenic hearts perfused with human plasma exhibited profound reductions in the level of complement deposition compared with nontransgenic controls. We have also produced transgenic pigs that express these two human genes. Our results indicate that expression of complement regulatory proteins can control activation of complement and suggest that these proteins may have therapeutic applications in some inflammatory diseases and in the development of xenogeneic organs for human transplantation.

In vivo transfer of GPI-linked complement restriction factors from erythrocytes to the endothelium.

Many proteins are associated with the outer layer of the cell membrane through a posttranslationally added glycosyl phosphatidylinositol (GPI) anchor. The functional significance of this type of protein linkage is unclear, although it results in increased lateral mobility, sorting to the apical surface of the cell, reinsertion into cell membranes, and possibly cell signaling. Here evidence is presented that GPI-linked proteins can undergo intermembrane transfer in vivo. GPI-linked proteins expressed on the surface of transgenic mouse red blood cells were transferred in a functional form to endothelial cells in vivo. This feature of GPI linkage may be potentially useful for the delivery of therapeutic proteins to vascular endothelium.

Transgenic expression of human complement regulatory proteins in mice results in diminished complement deposition during organ xenoperfusion.

Complement activation is an essential step in the hyperacute rejection of a vascularized xenograft. Endothelial cell-associated complement regulatory proteins limit complement activation in most settings, but are not able to limit the extensive complement activation that occurs in xenografts, at least in part due to their species specificity. To overcome this problem we and others have sought to express human complement regulatory proteins in the organs of potential donor animals. As an initial step toward evaluating this concept we tested organs from transgenic mice expressing human CD59 and/or decay-accelerating factor (DAF) in two in vitro perfusion systems for the ability to control activation of heterologous complement. In the first system, mouse hearts were perfused on a Langendorff circuit with 50% human plasma. Immunopathologic analysis of heart biopsies revealed deposition of human IgG, IgM, and C4 in both control and transgenic organs. The hearts from mice transgenic for human CD59 had substantially less and in some cases no membrane attack complex (MAC) and hearts from CD59/DAF transgenic mice had substantially less or no C5b and MAC. In the second system, mouse hearts were perfused with baboon blood through arterial lines inserted into baboons. Immunopathologic analysis of serial biopsies revealed the deposition of IgG, IgM, and C4 in control and transgenic hearts. Compared with controls, less MAC was deposited in many CD59-expressing hearts and less C5b and MAC in DAF-expressing hearts. These results demonstrate that human complement regulatory proteins expressed in a xenogeneic organ are able to contribute to the control of complement activation in that organ and support the concept that expression of these human molecules would help protect a xenogeneic organ transplanted into a human.