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Subcellular compartmentalisation and the intracellular movement of nuclear receptors are major regulatory steps in executing their transcriptional function. Though significant progress has been made in understanding these regulatory processes in cultured mammalian cells, such results have rarely been confirmed within cells of a living mammal. This article describes a simple, time-efficient approach to study the nuclear versus cytoplasmic accumulation of nuclear receptors and the regions of nuclear receptor proteins that govern subcellular trafficking within hepatocytes of live mice. Pregnane X receptor, a xenobiotic-activated member of the nuclear receptor family, was used to exemplify the approach. Using dual-labeled wild-type and mutant PXR expression constructs, we outline their in vivo delivery, simultaneous cellular expression, visualization and categorical classification within hepatocytes of live mice. Using this approach, we identified three mutants that had an altered subcellular distribution in the presence and absence of a PXR ligand. This novel in vivo method complements the current cell culture-based experimental systems in protein subcellular localisation studies.

Background: T-cell apoptosis is an important regulatory mechanism in transplant tolerance. The aim of this study was to identify specific apoptotic molecules important for tolerance induction., Methods: Mice expressing the human Bcl-2 molecule in T cells or Bim -/- mice were used as islet allograft or rat islet xenograft recipients and treated with CTLA4-Fc and MR1 costimulation blockade., Results: hBcl-2 transgenic mice and Bim -/- accepted islet allografts and rat islet xenografts for more than 100 days, similar to wildtype controls. Changes in the dose of the CTLA4-Fc and MR1 did not lead to differences in graft survival and there were no differences in the percentage of CD4+ T cells expressing Fas, CD25, or undergoing apoptosis., Conclusions: Inhibition of the passive cell death pathway in T cells did not block tolerance induction, suggesting that the mechanism by which apoptosis regulates the alloimmune response is more complex than first thought.

Background: Costimulatory molecule blockade with antibody-based immunosuppressive agents has been shown to prolong the survival of many types of allograft. The effects were evaluated of local costimulatory molecule blockade with different CTLA4-Ig constructs and of systemic, short-term treatment with an anti-CD28 monoclonal antibody on orthotopic corneal allograft survival in the rat., Methods: Adult Fischer-344 rats underwent Wistar-Furth orthotopic corneal grafts. The rats were treated with two different CTLA4-fusion proteins administered intraocularly in the perioperative period, or systemically with anti-CD28 monoclonal antibody JJ319. Corneal graft survival was determined by daily slit-lamp examination. The day of rejection was defined as the first postoperative day on which the iris margin was no longer clearly visible through the corneal graft., Results: Local administration of CTLA4-fusion protein with mutated immunoglobulin constant region domains via a single perioperative intraocular injection prolonged corneal graft survival modestly but significantly (P < 0.05), in contrast to a CTLA4-fusion protein with wild-type immunoglobulin domains, which had no effect on graft survival (P > 0.5). Systemic short-term administration of 400 microg total of an anti-CD28 monoclonal antibody also prolonged corneal graft survival significantly (P < 0.05) and was more effective than systemic administration of 2 mg total of CTLA4-fusion protein (P < 0.05)., Conclusions: Local administration of CTLA4-fusion protein with mutated (non-functional) immunoglobulin domains or systemic administration of anti-CD28 monoclonal antibody can prolong corneal allograft survival in the rat.

Background: Important phylogenetic differences between pig and human tissues prevent xenotransplantation from becoming a clinically feasible option. Humans lack the galactose-alpha1,3-galactose (alphaGal) epitope on endothelial cell surfaces and therefore have preformed anti-alphaGal antibodies. The role of these antibodies in rejection of non-vascular xenografts remains controversial. This study investigated the role of anti-alphaGal antibodies in rejection of non-vascularized alphaGal+/+ grafts in alphaGal -/- mice., Methods: alphaGal +/+ and alphaGal -/- pancreatic islets were transplanted under the renal capsule of streptozotocin-induced diabetic (1) alphaGal -/- mice and (2) alphaGal +/+ mice. alphaGal -/- recepients were immunized with rabbit red blood cell membranes (RRBCs) to produce elevated anti-alphaGal antibody levels., Results: Six of the 18 alphaGal -/- mice rejected the alphaGal +/+ grafts within 68 days whereas indefinite graft survival was achieved in the control groups. Animals with surviving islet grafts were challenged with alphaGal +/+ skin grafts. Although all alphaGal +/+ skin grafts were rejected within 58 days, the islet grafts remained intact. This observation correlated with the level of alphaGal expression (which was very low on islets compared to skin) rather than the actual titre of anti-alphaGal antibody., Discussion: The results suggest that the level of alphaGal expression plays an important role in graft survival. Therefore, its removal is important in the development of a pig islet donor for future clinical therapy.

Macrophages have been proposed as the major effector cell in T cell-mediated xenograft rejection. To determine their role in this response, NOD-SCID mice were transplanted with fetal pig pancreas (FPP) before reconstitution with CD4(+) T cells from BALB/c mice. Twelve days after CD4(+) T cell reconstitution, purified macrophages (depleted of T cells) were isolated from CD4(+) T cell-reconstituted FPP recipient mice and adoptively transferred to their nonreconstituted counterparts. After adoptive macrophage transfer, FPP recipient mice transferred with macrophages from CD4(+) T cell-reconstituted mice demonstrated xenograft destruction along with massive macrophage infiltration at day 4 and complete graft destruction at day 8 postmacrophage transfer. By contrast, FPP recipients that received macrophages from nonreconstituted mice showed intact FPP xenografts with few infiltrating macrophages at both days 4 and 8 after macrophage transfer. The graft-infiltrating macrophages showed increased expression of their activation markers. Depletion of endogenous macrophages or any remaining CD4(+) T cells did not delay graft rejection in the macrophage-transferred FPP recipients, whereas depletion of transferred macrophages with clodronate liposomes prevented graft rejection. Our results show that macrophages primed by FPP and activated by CD4(+) T cells were attracted from the peripheral circulation and were capable of specific targeting and destruction of FPP xenografts. This suggests that in xenograft rejection, there are macrophage-specific recognition and targeting signals that are independent of those received by T cells.

Previously, we demonstrated that combination CTLA4-Fc and anti-CD40L mAb treatment results in tolerance to concordant, cellular islet xenografts. The aim of this study was to determine its effectiveness in a model of fetal pig pancreas (FPP) xenotransplantation. Survival of FPP fragment grafts were compared to the survival of rat islet or cardiac xenografts following short term CTLA4-Fc and anti-CD40L mAb treatment. Rat islet and FPP fragment grafts survived long-term. However, rat cardiac grafts were rejected by 52-91 days. Both rat islet and FPP grafts showed similar histology with intact islet structures and adjacent 'nests' of lymphocytes. Concordant vascularised rat hearts showed extensive polymorphonuclear infiltrate, concentric vasculitis and a perivascular infiltrate predominantly of CD8+ T cells. This suggests that this therapy is effective for prolonging islet xenografts and demonstrates that the cellular mechanism of rejection for vascularised and non-vascularised xenografts are different.

Background: Our purpose was to determine if short-term inhibition of the CD40/CD40L and CD28/B7 costimulatory pathways was capable of inducing specific unresponsiveness to pancreatic islet xenografts and to ascertain the mechanism of tolerance induction., Methods: Diabetic B6AF1 mice were transplanted with Wistar or DA rat islets and were treated short term with CTLA4-Fc and anti-CD40L mAb (MR1)., Results: Coadministration of CTLA4-Fc with MR1, resulted in indefinite rat islet xenograft survival in mice. Tolerance was species but not strain specific as long-term surviving recipients rejected third party BALB/c islet allografts but accepted a second rat islet xenograft from the same or different donor strain. Tolerance induction was associated with a large leukocyte infiltrate that did not exhibit features of immune deviation as intragraft T cell-specific cytokine gene expression was globally reduced. In particular, interleukin-4 gene expression was markedly suppressed. There was a complete inhibition of anti-donor IgG, IgG1, and IgM antibody in the serum of CTLA4-Fc/MR1- treated animals. Tolerance induction was associated with increased CD4+ T cell apoptosis as there was an increased proportion of annexin-V staining and Fas expressing CD4+ T cells and a decrease in CD4+ T cell Bcl-2 expression in the grafts and draining lymph nodes of CTLA4-Fc/MR1-treated recipients., Conclusion: Combined costimulatory blockade was capable of producing tolerance to pancreatic islet xenografts. The induction of this tolerant state was associated with increased T cell apoptosis, whereas the maintenance phase of tolerance was associated with the accumulation of a large number of inactive lymphocytes within the graft.

Background: Xenograft rejection is a complex response in which macrophages and other effector cells are activated by CD4+ T cells. Initiation and regulation of this response is in part mediated by cytokines. In this study we test the hypothesis that xenograft destruction is an interleukin- (IL) 10 responsive, macrophage-mediated event., Methods: To study the effect of the systemic administration of IL-10 on pancreatic islet xenograft rejection, a fusion protein of IL-10/Fc was used. This immunoligand possesses the bioavailability of IL-10 and the long circulating t1/2 in vivo, characteristic of Ig. Wistar rat islets were transplanted into C57BL6 mice. IL-10/Fc was administered either immediately before transplantation or in the posttransplant period., Results: Both therapeutic protocols prolonged xenograft survival. Macrophage effector function was reduced in IL-10/Fc-treated mice, with a reduced macrophage infiltrate, reduced IL-12 and tumor necrosis factor-alpha gene expression and reduced serum NO2- levels. Although the number of T cells infiltrating islet grafts was not reduced, T cell effector function was inhibited in IL-10/Fc-treated animals with reduced interferon-gamma and IL-4 gene expression, reduced anti-donor cytotoxicity by recipient splenocytes and reduced anti-donor IgG1 antibody production. Ultimate rejection of the xenografts appears to be mediated by a CD4+ T cell dependent mechanism probably as a result of inadequate inhibition of IL-12 production by macrophages., Conclusion: IL-10/Fc prolonged rat pancreatic islet xenograft survival by inhibiting macrophage mediated immune responses. The effectiveness of this agent when administered pretransplant suggests it may have a role as an induction agent with potential clinical application.

Background: Islet xenografts have clinical potential, may avoid hyperacute rejection, and therefore are a good place to examine the cellular xenograft immune response. The aim of this study was to examine the cellular, humoral, and cytokine response in islet xenograft rejection and to determine the difference in the immune response with a different donor species., Methods: Two islet xenograft models (DA rat islets to B6AF1 mouse and canine islets to B6AF1 mouse) and a mouse syngeneic control model were examined histologically and by a semiquantitative polymerase chain reaction method., Results: There was significant up-regulation of all intragraft cytokines tested (interleukin [IL]-2, IL-4, IL-5, IL-10, and interferon-gamma) in both xenograft models compared with the controls. However, the dog islet grafts had higher levels of IL-4 and IL-5 gene expression than the rat islet grafts, which, conversely, had higher levels of interferon-gamma gene expression. These differences correlated with the histological and anti-donor antibody production differences between the two models. The dog to mouse model had an intense eosinophilic infiltrate and an early up-regulation of anti-donor antibody, whereas there was little eosinophilic infiltrate and a delayed anti-donor antibody up-regulation in the rat to mouse model., Conclusions: The mouse used different mechanisms to reject the rat and canine islets, suggesting that the immune response in islet xenograft rejection may be dependent on the species combination. It may not be possible to characterize the cellular xenograft rejection response in a bipolar manner as has been the case with humoral rejection response. Caution therefore needs to be taken before extrapolating the cellular immune responses seen in animal models to the clinical setting.