Recent success in pancreatic islet allotransplantation has raised expectations but has equally highlighted the acute shortage of donor tissue. The use of xenogeneic tissue would help to address this shortage; however, strong cellular immunity limits the application of this approach. T-cell responses to xenogeneic tissues involve recognition of intact species-mismatched major histocompatibility complex (MHC) molecules, the direct pathway, and xenogeneic proteins presented as peptides by responder-type MHC molecules, the indirect pathway. In this study, we exploited the species difference to selectively and sequentially inhibit direct and indirect xenoresponses after transplantation of porcine islets into mice. Selective inhibition of the direct response was achieved using porcine CTLA4-Ig, which binds preferentially to pig versus mouse B7 molecules. Selective inhibition of the indirect response was achieved using murine CTLA4-Ig, which binds preferentially to mouse B7 molecules. Administration of porcine CTLA4-Ig alone caused modest prolongation of islet survival. Injection of murine CTLA4-Ig alone had a minimal effect. However, the injection of the porcine fusion protein early and the murine homolog late after grafting led to permanent survival of the porcine islets, in the absence of any other immunosuppression. These results suggest that a similar approach could have clinical utility in porcine islet xenotransplantation.
The narrow therapeutic window of cyclosporine A (CsA) means its use is controlled by pharmacokinetic monitoring. However, pharmacokinetics do not always reflect the functional effects of a drug--its pharmacodynamics, such as vasoconstriction. We developed a technique for measuring renal blood flow and used a pig model to determine whether CsA-induced renal vasoconstriction could be detected, thus offering a tool for pharmacodynamic therapeutic drug monitoring. This has been shown to differentiate acute rejection from acute tubular necrosis. Power Doppler intensitometry was used to assess relative vascular volume, and the renal arteriovenous transit time was determined with an intravenous microbubble bolus. Measurements were taken before and at intervals after an intravenous bolus of CsA (10 mg/kg). There was no correlation between index and CsA concentration. Lack of detectable effect after CsA administration to high concentrations suggests that this technique may be able to differentiate CsA toxicity from acute rejection.
ABSTRACT. A feature of the tolerance that has been described in experimental models is that it can be transferred by CD4+ T cells to a naive recipient. Described is a novel approach to induce indirect pathway regulatory T cells in a rat model that exploits the natural processing and presentation of major histocompatability complex (MHC) molecules as peptide by the MHC class II molecules of the same cell. Dendritic cells (DC) coexpressing donor (AUG) and recipient (LEW) MHC molecules were rendered tolerogenic by treatment with dexamethasone. After injection into LEW animals followed by a single low dose of CTLA4-Ig, T cells were rendered unresponsive to indirectly presented AUG alloantigens, but retained direct pathway responsiveness to fully allogeneic AUG cells. The T cells from the DC-injected rats were unresponsive to (LEW × AUG)F1 stimulator cells, suggesting the presence of indirect pathway regulatory cells whose activity depended on the presence of LEW MHC molecules. Depletion of CD25+ cells from the responder population led to a marked increase in proliferation, and the T cells from the DC-injected rats inhibited the response of naive LEW T cells to (LEW × AUG)F1, but not to AUG, stimulator cells, further indicating indirect pathway-mediated regulation. Most importantly, pretreatment of LEW rats with the dexamethasone-treated DC led to the indefinite survival of AUG kidney grafts after a short course of cyclosporin to inhibit the early direct pathway response. Similarly treated AUG DC had no effect, confirming the privileged status of F1 cells in the induction of indirect pathway regulation.
The pig is generally regarded as likely to be the preferred donor animal in xenotransplantation. Although many hurdles remain to be cleared, it would be useful to be able to manipulate porcine endothelium genetically, among other reasons, to test approaches in the modulation of inflammation. However, as a nondividing cell, it is less easy to manipulate.The authors performed in vivo and in vitro gene transfection experiments using as an adjunct the DNA-binding agent 4',6'-diamidino-2-phenylindole (DAPI), which protects DNA from degradation.The introduction of DAPI into a liposomal transfection system was able to increase in vitro transfection efficiency of both endothelial and vascular smooth muscle cells from the pig, even in the presence of small amounts of serum. This last observation encouraged the authors to use this system in vivo in porcine carotid arteries. In this model, the authors were also able to demonstrate a high degree of transfection efficiency using DAPI, which seemed to work by protecting DNA from degradation.The authors believe this technique may allow them to address many biological questions relating to intervening in vascular disease, inflammation, and immune responses in the context of transplantation and beyond.
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