Cell-based diabetes therapy requires an abundant cell source. Here, we report reversal of diabetes for more than 100 d in cynomolgus macaques after intraportal transplantation of cultured islets from genetically unmodified pigs without Gal-specific antibody manipulation. Immunotherapy with CD25-specific and CD154-specific monoclonal antibodies, FTY720 (or tacrolimus), everolimus and leflunomide suppressed indirect activation of T cells, elicitation of non-Gal pig-specific IgG antibody, intragraft expression of proinflammatory cytokines and invasion of infiltrating mononuclear cells into islets.

POU transcription factors are involved in transcriptional regulation during early embryonic development and cell differentiation. Oct-4, a member of this family, has been shown to be under strict regulation during murine development. The expression of Oct-4 correlates with the undifferentiated cell phenotype of the mouse preimplantation embryo. In this study, expression of a gene construct consisting of selected parts of the region upstream from the murine Oct-4 gene as promoter/enhancer, enhanced green fluorescent protein (EGFP) as reporter and the five exons of the murine Oct-4 gene (GOF18-delta PE EGFP) was evaluated in murine, porcine, and bovine preimplantation embryos. For comparison, expression of the endogenous Oct-4 gene was also analyzed in all three species by immunocytochemistry. The transgene construct was microinjected into zygotes cultured in vitro to various developmental stages. The EGFP fluorescence was visualized in developing embryos by excitation with blue light at different days following microinjection and showed similar expression patterns in all three species. Most embryos displayed a mosaic pattern of transgene expression. The EGFP fluorescence was not restricted to the inner cell mass (ICM) but was also seen in trophoblastic cells. An affinity-purified polyclonal antibody specific to Oct-4 was used for immunocytochemical analysis of in vivo- and in vitro-derived bovine and porcine blastocysts and also of in vivo-derived murine blastocysts. In the in vivo-derived murine embryos, Oct-4 protein was detectable in the ICM but not the trophectoderm, whereas in porcine and bovine blastocysts, derived in vivo or in vitro, Oct-4 protein was detected in both the ICM and the trophectoderm. Thus, in the two large animal species, Oct-4 expression from the endogenous gene was clearly not restricted to the pluripotent cells of the early embryo. These results show that Oct-4 regulation differs between these species and that the presence of Oct-4 protein may not be sufficient for selection of undifferentiated cell lines in domestic animals.

Pig-to-primate intraportal islet xenografts reverse diabetes and the majority of intraportally transplanted xenogeneic islets are not subject to hyperacute rejection. They undergo acute cellular rejection mediated by CD4+- and CD8+ T cells and macrophages.

BACKGROUND Radiofrequency (RF) has become an accepted energy source for myocardial ablation but may result in discontinuous lesions and nontargeted tissue injury. We examined the feasibility and safety of lesion formation using high-amplitude, bipolar pulsed electric fields delivered from a multielectrode array catheter. OBJECTIVE The purpose of this study was to compare duty-cycled radiofrequency ablation (RFA) to pulsed field ablation (PFA) in terms of acute electrical effects, 2-week lesion formation, and injury to nontargeted tissues. METHODS Intracardiac ablations were performed in 6 pigs using a circular pulmonary vein ablation catheter. The energy source for ablation delivery was randomized to deliver either PFA or RFA to 3 atrial endocardial sites. Bipolar pace capture and electrogram amplitude measurements were recorded at each site. Histopathology and necropsies were performed after 2 weeks. RESULTS The circular pulmonary vein ablation catheter was used to deliver pulsed electric fields to produce cardiac lesions without skeletal muscle stimulation. Evaluating all ablations in each site, electrogram amplitudes were reduced to ,0.5 mV in 67.5% of PFA vs 27.0% of RFA deliveries (P ,.001). Bipolar cardiac capture was lost after 100% vs 92.0% of PFA vs RFA (P 5 .005). At 2 weeks, PFA resulted in consistent transmural and homogeneous replacement fibrosis devoid of lingering myocyte "sequesters." RFA lesions showed a stronger inflammatory response extending to the epicardial fat, arterial injury, and thrombosis. Neither PFA nor RFA lesions showed endocardial thrombus. CONCLUSION Intracardiac PFA can be feasibly delivered from a circular catheter to create fibrotic lesions that have acute electrical effects, without injury to nontargeted tissue.

The Sleeping Beauty (SB) transposon system can integrate foreign sequences of DNA in the genome of mouse somatic cells eliciting long-term expression in vivo. This technology holds great promise for human gene therapy as a nonviral technology to deliver therapeutic genes. SB also provides a means to study the effects of defined genetic elements, such as oncogenes, on somatic cells in mice. Here, we test the ability of the SB transposon system to facilitate somatic integration of a transposon containing an activated NRAS oncogene in mouse hepatocytes to elicit tumor formation. NRAS oncogene-driven tumors developed when such vectors were delivered to the livers of p19Arf-null or heterozygous mice. Delivery of the NRAS transposon cooperates with Arf loss to cause carcinomas of hepatocellular or biliary origin. These tumors allowed characterization of transposon integration and expression at the single-cell level, revealing robust NRAS expression and both transposase-mediated and random insertion of delivered vectors. Random integration and expression of the SB transposase plasmid was also observed in one instance. In addition, studies using effector loop mutants of activated NRAS provide evidence that mitogen-activated protein kinase activation alone cannot efficiently induce liver carcinomas. This system can be used to rapidly model tumors caused by defined genetic changes.cancer ͉ gene therapy ͉ Nras ͉ p19Arf

The 2 most frequent human MLL hematopoietic malignancies involve either AF4 or AF9 as fusion partners; each has distinct biology but the role of the fusion partner is not clear. We produced Mll-AF4 knock-in (KI) mice by homologous recombination in embryonic stem cells and compared them with Mll-AF9 KI mice. Young Mll-AF4 mice had lymphoid and myeloid deregulation manifest by increased lymphoid and myeloid cells in hematopoietic organs. In vitro, bone marrow cells from young mice formed unique mixed pro-B lymphoid (B220 ؉ CD19 ؉ CD43 ؉ sIgM ؊ , PAX5 ؉ , TdT ؉ , IgH rearranged)/myeloid (CD11b/Mac1 ؉ , c-fms ؉ , lysozyme ؉ ) colonies when grown in IL-7-and Flt3 ligand-containing media. Mixed lymphoid/myeloid hyperplasia and hematologic malignancies (most frequently B-cell lymphomas) developed in Mll-AF4 mice after prolonged latency; long latency to malignancy indicates that Mll-AF4-induced lymphoid/myeloid deregulation alone is insufficient to produce malignancy. In contrast, young Mll-AF9 mice had predominately myeloid deregulation in vivo and in vitro and developed myeloid malignancies. The early onset of distinct mixed lymphoid/myeloid lineage deregulation in Mll-AF4 mice shows evidence for both "instructive" and "noninstructive" roles for AF4 and AF9 as partners in MLL fusion genes. The molecular basis for "instruction" and secondary cooperating mutations can now be studied in our Mll-AF4 model. IntroductionThe myeloid/mixed lymphoid leukemia gene (MLL) on human chromosome 11 was first described from a cell line derived from a patient with a hematologic malignancy that resulted from a reciprocal translocation involving chromosome 4. 1 MLL was subsequently shown to partner with many other genes to result in hematologic malignancy. 2 The fusion of MLL to AF4 family members, LAF4 and AF5, results in malignancies that are the most common and unique among the MLL fusion gene malignancies in that they are generally lymphoid or lymphoid/myeloid in type but rarely purely myeloid. They are also unique because of the high frequency in infants, extensive spread beyond the hematopoietic compartment, and a poor outcome with treatment. [3][4][5][6][7][8][9][10] In contrast to MLL-AF4, the fusion of MLL with most other partners, including the second most common partner AF9, 8 results in myeloid malignancies.To date, no murine model of MLL-AF4 translocation has been reported and thus neither the premalignant early events nor the eventual malignancies have been defined. In this study, we produced Mll-AF4 knock-in (KI) mice and compare them with Mll-AF9 mice developed previously. 11 The KI models, which have the advantage of having a single copy of the fusion gene in all stem/progenitor cells, permit control of bias introduced by a variable number of gene copies in the various progenitor populations in other models.The mechanisms for the association between the MLL partner gene and type of malignancy have not been elucidated. The MLL partner may be "instructive" in directing the selective expansion and transformation of cells t...

Background-Although distal embolization and the "no-reflow" phenomenon are well described in saphenous vein graft (SVG) interventions, the frequency, magnitude, and characterization of embolized debris have not been evaluated in routine coronary interventions. A unique embolus protection device described herein provides a means of containing and retrieving plaque material dislodged during percutaneous coronary interventions. This report details the first clinical experience of the effectiveness and safety of an emboli protection system in 11 SVG lesions and 15 native coronary artery lesions. Methods and Results-The AngioGuard Emboli Capture Guidewire (Cordis) consists of a PTCA wire with an expandable filter at the distal tip. The porous membrane permits normal distal blood flow, while trapping potential emboli by filtration. After crossing the lesion, the filter is expanded, and routine angioplasty is performed over the same wire. Emboli retrieval is achieved by collapsing the filter and retracting the emboli capture wire (ECW). In 26 patients, standard angioplasty was performed over the ECW; 20 of these 26 patients received a stent. Collected debris was sent for histopathological analysis. Plaque debris was retrieved after native coronary and SVG interventions in all cases. The ECW was positioned and retrieved without complications. No major adverse events occurred. Myocardial infarctions and no-reflow were not observed. Conclusions-The embolization of plaque fragments frequently occurs during coronary and SVG intervention. Distal embolization leading to microvascular obstruction and no-reflow could be successfully minimized by using the ECW.

Background Pulsed field ablation (PFA) has been identified as an alternative to thermal‐based ablation systems for treatment of atrial fibrillation patients. The objective of this Good Laboratory Practice (GLP) study was to characterize the chronic effects and safety of overlapping lesions created by a PFA system at intracardiac locations in a porcine model. Methods A circular catheter with nine gold electrodes was used for overlapping low‐ or high‐dose PFA deliveries in the superior vena cava (SVC), right atrial appendage (RAA), and right superior pulmonary vein (RSPV) in six pigs. Electrical isolation was evaluated acutely and chronic lesions were assessed via necropsy and histopathology after 4‐week survival. Acute and chronic safety data were recorded peri‐ and post‐procedurally. Results No animal experienced ventricular arrhythmia during PFA delivery, and there was no evidence of periprocedural PFA‐related adverse events. Lesions created in all anatomies resulted in electrical isolation postprocedure. Lesions were circumferential, contiguous, and transmural, with all converting into consistent lines of chronic replacement fibrosis, regardless of trabeculated or smooth endocardial surface structure. Ablations were non‐thermally generated with only minimal post‐delivery temperature rises recorded at the electrodes. There was no evidence of extracardiac damage, stenosis, aneurysms, endocardial disruption, or thrombus. Conclusion PFA deliveries to the SVC, RAA, and RSPV resulted in complete circumferential replacement fibrosis at 4‐week postablation with an excellent chronic myocardial and collateral tissue safety profile. This GLP study evaluated the safety and efficacy of a dosage range in preparation for a clinical trial and characterized the non‐thermal nature of PFA.