Strong expression of CD55 and CD59 completely protected porcine kidneys from hyperacute rejection and allowed a detailed analysis of xenograft rejection in the absence of immunosuppression. Coagulopathy appears to be a common feature of pig-to-baboon renal transplantation and represents yet another major barrier to its clinical application.

Incompatibility between pig thrombomodulin (TM) and primate thrombin is thought to be an important factor in the development of microvascular thrombosis in rejecting pig-to-primate xenografts. To examine this interaction at the molecular level, we cloned pig TM and measured its ability to bind human thrombin and act as a cofactor for the activation of human protein C and TAFI. The 579-residue pig TM protein showed approximately 69% sequence identity to human TM. Within the EGF domains necessary for binding of thrombin (EGF56), protein C (EGF4) and TAFI (EGF3), all of the amino acids previously identified as critical for the function of human TM, with the exception of Glu-408 in EGF5, were conserved in pig TM. Comparison of transfected cells expressing pig or human TM demonstrated that both proteins bound human thrombin and inhibited its procoagulant activity. However, pig TM was a poor cofactor for the activation of human protein C and TAFI, with domain swapping showing that EGF5 was the most important determinant of compatibility. Thus, while pig TM may be capable of binding thrombin generated in the vicinity of xenograft endothelium, its failure to promote the activation of human protein C remains a significant problem.

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.The instant blood-mediated inflammatory reaction (IBMIR) is a major obstacle to the engraftment of intraportal pig islet xenografts in primates. Higher expression of the galactose-a1,3-galactose (aGal) xenoantigen on neonatal islet cell clusters (NICC) than on adult pig islets may provoke a stronger reaction, but this has not been tested in the baboon model. Here, we report that WT pig NICC xenografts triggered profound IBMIR in baboons, with intravascular clotting and graft destruction occurring within hours, which was not prevented by anti-thrombin treatment. In contrast, IBMIR was minimal when recipients were immunosuppressed with a clinically relevant protocol and transplanted with NICC from aGal-deficient pigs transgenic for the human complement regulators CD55 and CD59. These genetically modified (GM) NICC were less susceptible to humoral injury in vitro than WT NICC, inducing significantly less complement activation and thrombin generation when incubated with baboon platelet-poor plasma. Recipients of GM NICC developed a variable anti-pig antibody response, and examination of the grafts 1 month after transplant revealed significant cell-mediated rejection, although scattered insulin-positive cells were still present. Our results indicate that IBMIR can be attenuated in this model, but long-term graft survival may require more effective immunosuppression or further donor genetic modification.

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Primary nonfunction of transplanted islets results in part from their sensitivity to reactive oxygen species (ROS) generated during the isolation and transplantation process. Our aim was to examine whether coexpression of antioxidant enzymes to detoxify multiple ROS increased the resistance of mouse islets to oxidative stress and improved the initial function of islet grafts. Islets from transgenic mice expressing combinations of human copper/zinc superoxide dismutase (SOD), extracellular SOD, and cellular glutathione peroxidase (Gpx-1) were subjected to oxidative stress in vitro. Relative viability after hypoxanthine/xanthine oxidase treatment was as follows: extracellular SOD ؉ Gpx-1 ؉ Cu/Zn SOD > extracellular SOD ؉ Gpx-1 > extracellular SOD > wild type. Expression of all three enzymes was the only combination protective against hypoxia/ reoxygenation. Islets from transgenic or control wildtype mice were then transplanted into streptozotocininduced diabetic recipients in a syngeneic marginal islet mass model, and blood glucose levels were monitored for 7 days. In contrast to single-and double-transgenic grafts, triple-transgenic grafts significantly improved control of blood glucose compared with wild type. Our results indicate that coexpression of antioxidant enzymes has a complementary beneficial effect and may be a useful approach to reduce primary nonfunction of islet grafts. Diabetes 54:2109 -2116, 2005

We describe the molecular cloning of a receptor tyrosine kinase from a cell line (LK63) derived from a case of human pre-B-cell leukemia. We have previously shown that a monoclonal antibody (IIA4) raised against LK63 recognized a glycosylated, cell-surface 135-kDa molecule (HEK), which displayed tyrosine kinase activity in vitro. The (5), platelet-derived growth factor (6), colony-stimulating factor 1 (7), and stem cell factor (8). The recent isolation of lineagespecific (e.g., flk-2) (9) and developmentally regulated (e.g., CEK 5) (10) PTKs highlights an emerging role for this group of molecules in differentiation and development. As part of our study of human B-cell growth and differentiation, we have raised a number of monoclonal antibodies (mAbs) against early B-cell populations. One of these, designated IIIA4, has been shown to recognize a 135-kDa molecule (HEK) on the surface ofthe pre-B-cell leukemic cell line LK63 (11). Glycosylation studies demonstrated an estimated molecular mass of 95 kDa for the HEK core protein.By indirect immunofluorescence, the human T-cell line JM also expressed this antigen, but a wide range of other lymphoid and myeloid cell lines were negative. Fluorescence-activated cell sorter analysis of normal human lymphoid and bone marrow cells showed no detectable HEK expression. However, HEK expression was observed on a small number of fresh hemopoietic tumor specimens, raising the possibility that IIIA4 recognized the product of a sporadically overexpressed gene. Immunoprecipitated HEK protein was shown to have tyrosine kinase activity in vitro. These data suggested that IIIA4 recognized a membraneassociated PTK. We used the IIIA4 mAb to affinity purify HEK protein from LK63 cells. Amino acid sequencing demonstrated a unique N terminus and PTK-related internal sequences.In this report, we describe the isolation and sequence determination of a 4.5-kilobase (kb) cDNA encoding the HEK receptor tyrosine kinase (RTK)

We report here our experience regarding the production of double or homozygous Gal knockout (Gal KO) pigs by breeding and somatic cell nuclear transfer (SCNT). Large White x Landrace female heterozygous Gal KO founders produced using SCNT were mated with Hampshire or Duroc males to produce a F1 generation. F1 heterozygous pigs were then bred to half-sibs to produce a F2 generation which contained Gal KO pigs. To determine the viability of mating Gal KO pigs with each other, one female F2 Gal KO pig was bred to a half-sib and subsequently a full-sib Gal KO. F1 and F2 heterozygous females were also mated to F2 Gal KO males. All three types of matings produced Gal KO pigs. To produce Gal KO pigs by SCNT, heterozygous F1s were bred together and F2 fetuses were harvested to establish primary cultures of Gal KO fetal fibroblasts. Gal KO embryos were transferred to five recipients, one of which became pregnant and had a litter of four piglets. Together our results demonstrate that Gal KO pigs can be produced by breeding with each other and by SCNT using Gal KO fetal fibroblasts.

Xenotransplantation from pigs has been advocated as a solution to the perennial shortage of donated human organs and tissues. CRISPR/Cas9 has facilitated the silencing of genes in donor pigs that contribute to xenograft rejection. However, the generation of modified pigs using second-generation nucleases with much lower off-target mutation rates than Cas9, such as FokI-dCas9, has not been reported. Furthermore, there have been no reports on the use of CRISPR to knock protective transgenes into detrimental porcine genes. In this study, we used FokI-dCas9 with two guide RNAs to integrate a 7.1 kilobase pair transgene into exon 9 of the GGTA1 gene in porcine fetal fibroblasts. The modified cells lacked expression of the αGal xenoantigen, and secreted an anti-CD2 monoclonal antibody encoded by the transgene. PCR and sequencing revealed precise integration of the transgene into one allele of GGTA1, and a small deletion in the second allele. The cells were used for somatic cell nuclear transfer to generate healthy male knock-in piglets, which did not express αGal and which contained anti-CD2 in their serum. We have therefore developed a versatile high-fidelity system for knocking transgenes into the pig genome for xenotransplantation purposes.