Laparoscopic surgery for kidney orthotopic transplant in the pig model.

BACKGROUND AND OBJECTIVES: Laparoscopic surgery has rapidly expanded in surgical practice with well-accepted benefits of minimal incision, less analgesia, better cosmetics, and quick recovery. The surgical technique for kidney transplantation has remained unchanged since the first successful kidney transplant in the 1950s. Over the past decade, there were only a few case reports of kidney transplantation by laparoscopic or robotic surgery. Therefore, the aim of this study is to develop a laparoscopic technique for kidney transplantation at the region of the native kidney. METHODS: After initial development of the laparoscopic technique for kidney transplant in cadaveric pigs, 5 live pigs (Sus scrofa, weighing 45-50 kg) underwent laparoscopic kidney transplant under general anesthesia. First, laparoscopic donor nephrectomy was performed, and then the kidney was perfused and preserved with cold Ross solution. The orthotopic auto-transplant was subsequently performed using the laparoscopic technique. The blood flow of the kidney graft was assessed using Doppler ultrasonography, and urine output was monitored. RESULTS: The laparoscopic kidney transplant was successful in 4 live pigs. Immediate urine output was observed in 3 pigs. The blood flow in the kidney was adequate, as determined using Doppler ultrasonography. CONCLUSION: It has been shown that laparoscopic kidney orthotopic transplant is feasible and safe in the pig model. Immediate kidney graft function can be achieved. A further study will be considered to identify the potential surgical morbidity and mortality after recovery in a pig model before translating the technique to clinical human kidney transplantation.

CD8-interaction mutant HLA-Cw3 molecules protect porcine cells from human natural killer cell-mediated antibody-dependent cellular cytotoxicity without stimulating cytotoxic T lymphocytes.

BACKGROUND: CD56+ human natural killer (NK) cells are the principal anti-pig cytotoxic effectors in vitro. Expression of certain human leukocyte antigen (HLA) class I molecules in porcine cells can inhibit NK cell-mediated natural cytotoxicity in serum-free medium, but had not been shown to inhibit antibody-dependent cellular cytotoxicity (ADCC) by CD16+ NK cells in the presence of human xenoreactive immunoglobulin G. Moreover, expression of HLA molecules might amplify the previously weak CD8+ cytotoxic T-lymphocyte (CTL) response against porcine cells. METHODS: A novel porcine B-lymphoblastoid cell line (13271) was stably transfected with HLA-Cw*0304 gene constructs encoding wild-type (wt) Cw3 or genetically modified Cw3 unable to interact with CD8 (Cw3-D227K). The Cw3 transfectants were used in limiting dilution assays to estimate the CTL precursor frequency in CD56-depleted human peripheral blood mononuclear cells (PBMC) obtained from eight unrelated donors. The 13271 transfectants were also used as targets for clonal and polyclonal NK cells in the presence and absence of human serum, to measure inhibition of ADCC. RESULTS: Expression of Cw3-wt in 13271 cells significantly increased the human CTL response compared with the empty-vector control transfectant, whereas no significant increase resulted from expression of CD8-interaction mutant Cw3-D227K molecules. The Cw3-D227K mutant was indistinguishable from Cw3-wt in its ability to inhibit both natural cytotoxicity and ADCC mediated by human NK clones that have the appropriate CD158b inhibitory receptor. CONCLUSIONS: Transgenic expression of HLA molecules in pig cells will likely amplify the CD8+ CTL response against the xenograft. Disruption of HLA-CD8 interaction could minimize this amplification without compromising NK-cell inhibition.

Genetically modified HLA class I molecules able to inhibit human NK cells without provoking alloreactive CD8+ CTLs.

Human NK cells are likely to be important effectors of xenograft rejection. Expression of HLA class I molecules by transfected porcine cells can protect them from human NK cell-mediated lysis; however, this strategy has the potential to augment the anti-graft response by recipient CD8(+) T cells recognizing foreign pig peptides presented by HLA. In this study we show that the introduction of a mutation (D227K) in the alpha(3) domain of HLA-Cw3 abrogates its recognition by CD8-dependent T cells but leaves intact its ability to function as an inhibitory ligand for NK cells. Such genetically modified molecules may have potential therapeutic applications in the prevention of delayed xenograft rejection and in the facilitation of allogeneic and xenogeneic bone marrow engraftment.

The receptor-bound N-terminal ectodomain of the amyloid precursor protein is associated with membrane rafts.

The soluble N-terminal ectodomain of amyloid precursor protein (sAPP), resulting from alpha-secretase-mediated proteolytic processing, has been shown to function as a growth factor for epithelial cells, including keratinocytes and thyrocytes. Extracellularly applied sAPP binds to a cell surface receptor and exhibits a patchy binding pattern reminiscent of that observed for raft proteins. Here we show that (i) the receptor-bound sAPP resides in a detergent-insoluble membrane microdomain which cofractionates in density gradients with cholesterol-rich membrane rafts and caveolae; (ii) the sAPP-binding microdomains are different from caveolae; and (iii) sAPP is capable of binding to isolated rafts and inducing tyrosine phosphorylation of some raft proteins. These observations suggest that a novel type of membrane raft is involved in sAPP signaling.