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The objective of this study was to evaluate the feasibility and safety of a hybrid liver support system with extracorporeal plasma separation and bioreactor perfusion in patients with acute liver failure (ALF) who had already fulfilled the criteria for high urgency liver transplantation (LTx). Eight patients (one male, seven female) were treated in terms of bridging to transplantation. The mean age was 36.5 yr (range 20 to 58). Etiology of liver failure was drug-related in two patients, hepatitis B infection in three patients, and unknown for three patients. The bioreactors were charged with primary liver cells from specific pathogen-free pigs. Cell viability varied between 91 and 98%. Continuous liver support treatment over a period of 8 to 46 h (mean 27.3 h) was safely performed and well-tolerated by all patients. No complications associated with the therapy were observed during the follow-up period. Thrombocytopenia was considered to be an effect of the plasma separation. Subsequently, all patients were transplanted successfully and were observed over at least 3 yr with an organ and patient survival rate of 100%. Screening of patient's sera for antibodies specific for porcine endogenous retroviruses (PERVs) showed no reactivity--either prior to application of the system, or after extracorporeal treatment. The results encourage us to continue the development of the technology, and further studies appear to be justified. The bioreactor technology has been integrated into a modular extracorporeal liver support (MELS) system, combining biologic liver support with artificial detoxification technology.

Porcine endogenous retroviruses (PERVs) pose a potential risk for xenotransplantation using pig cells, tissues or organs. A special threat comes from viruses generated by recombination between human-tropic PERV-A and ecotropic PERV-C. Serial passages of a recombinant PERV-A/C on human 293 cells resulted in increased infectious titers and a multimerization of transcription factor binding sites in the viral long terminal repeat (LTR). In contrast to the LTR, the sequence of the env gene did not change, indicating that the LTR represents the determinant of high infectivity.The virus was further propagated on human cells and characterized by different methods (titration, sequencing, infection experiments, electron microscopy).Further propagation on human 293 cells resulted in deletions and mutations in the LTR. In contrast to low-titer viruses, the high-titer virus was infectious for cells from non-human primates including chimpanzees. Scanning electron microscopy revealed clustering of budding virions at the cell surface of infected human cells and transmission electron microscopy indicated that the virus infects them via receptor-mediated endocytosis.After propagation of PERV on human cells without selection pressure, viruses with different LTR were generated. High titer PERV was shown to infect cells from non-human primates. The experiments performed here simulate the situation in vivo and give an extended characterization of human cell-adapted PERVs.

Transposon-based gene vectors have become indispensable tools in vertebrate genetics for applications ranging from insertional mutagenesis and transgenesis in model species to gene therapy in humans. The transposon toolkit is expanding, but a careful, side-by-side characterization of the diverse transposon systems has been lacking. Here we compared the Sleeping Beauty (SB), piggyBac (PB), and Tol2 transposons with respect to overall activity, overproduction inhibition (OPI), target site selection, transgene copy number as well as long-term expression in human cells. SB was the most efficient system under conditions where the availability of the transposon DNA is limiting the transposition reaction including hard-to-transfect hematopoietic stem/progenitor cells (HSCs), and the most sensitive to OPI, underpinning the need for careful optimization of the transposon components. SB and PB were about equally active, and both more efficient than Tol2, under nonrestrictive conditions. All three systems provided long-term transgene expression in human cells with minimal signs of silencing. Indeed, mapping of Tol2 insertion sites revealed significant underrepresentation within chromosomal regions with H3K27me3 histone marks typically associated with transcriptionally repressed heterochromatin. SB, Tol2, and PB constitute complementary research tools for gene transfer in mammalian cells with important implications for fundamental and translational research.

Multiple reports implicated the function of G protein-coupled receptor (GPR)-30 with nongenomic effects of estrogen, suggesting that GPR30 might be a G-protein coupled estrogen receptor. However, the findings are controversial and the expression pattern of GPR30 on a cell type level as well as its function in vivo remains unclear. Therefore, the objective of this study was to identify cell types that express Gpr30 in vivo by analyzing a mutant mouse model that harbors a lacZ reporter (Gpr30-lacZ) in the Gpr30 locus leading to a partial deletion of the Gpr30 coding sequence. Using this strategy, we identified the following cell types expressing Gpr30: 1) an endothelial cell subpopulation in small arterial vessels of multiple tissues, 2) smooth muscle cells and pericytes in the brain, 3) gastric chief cells in the stomach, 4) neuronal subpopulations in the cortex as well as the polymorph layer of the dentate gyrus, 5) cell populations in the intermediate and anterior lobe of the pituitary gland, and 6) in the medulla of the adrenal gland. In further experiments, we aimed to decipher the function of Gpr30 by analyzing the phenotype of Gpr30-lacZ mice. The body weight as well as fat mass was unchanged in Gpr30-lacZ mice, even if fed with a high-fat diet. Flow cytometric analysis revealed lower frequencies of T cells in both sexes of Gpr30-lacZ mice. Within the T-cell cluster, the amount of CD62L-expressing cells was clearly reduced, suggesting an impaired production of T cells in the thymus of Gpr30-lacZ mice.

Islet cells from pig could be used as an alternative to the current treatment of diabetic patients. However, xenotransplantation from pig to humans may be associated with the risk of transmission of porcine endogenous retroviruses (PERVs) that are present in the genome of all pigs and infect human cells in vitro. Although transplantation of pig islet cells for treatment of diabetes may be not accompanied by immunosuppression that may facilitate virus survival, since islets will be used encapsulated, it is nevertheless of importance to study whether islet cells release PERVs able to infect human cells during co-incubation.Isolated islets from German landrace pigs were incubated with highly susceptible human 293 cells for one week. In order to prevent microchimerism 293 cells were made neomycin-resistant (293(neo+)), that allows the elimination of pig cells by a selection medium. The infection of 293(neo+ )target cells was analysed by PCR using PERV-specific primers up to fi ve weeks after co-cultivation. In addition, expression of viral mRNA in pig islet cells was studied by RT-PCR analysis, the expression of viral protein by FACS analysis.Despite the presence of numerous PERV proviruses in the genome of all pigs, no expression of PERV was observed in German landrace pig islet cells, neither as mRNA, nor as protein, nor as viral particles.Islet cells from German landrace pigs do not express PERVs and may therefore be used for breeding genetically modified pigs suitable for xenotransplantation and treatment of diabetes.

This study represents a long-term follow-up of human patients receiving pig islet xenotransplantation. Eighteen patients had been monitored for up to 9 years for potentially xenotic pig viruses: pig endogenous retrovirus, pig cytomegalovirus, pig lymphotropic herpesvirus, and pig circovirus type 2. No evidence of viral infection was found.

Objective: Porcine endogenous retroviruses (PERVs) pose a risk for xenotransplantations using pig materials as they are present in the genome of all pigs and are able to infect human cells in vitro. Until recently, transmission of PERVs in vivo was only described in severe combined immunodeficient (SCID) and nude mice inoculated with PERV-producing cells. However, in this series of experiments microchimerism could not be excluded. To overcome this problem, the risk of PERV infection was addressed in a similar way but using cell-free inoculation of mouse cells in vitro and SCID mice in vivo. Methods: Mouse cell lines and primary cells were incubated in vitro with PERV-A, with a recombinant PERV-A/C and with PERV-B. Provirus integration was assessed by PCR. Reverse transcriptase activity was measured in the cell supernatants. SCID mice were inoculated in vivo with cell-free virus at high titers. Results: None of the mouse cell lines and primary cells could be infected by PERV and no provirus integration was observed in different organs of the inoculated SCID mice. Conclusion: The data indicate that PERV-A, PERV-A/C and PERV-B could not infect different mouse cells. These data correlate with the recent finding that mouse cells lack a functional receptor for PERV-A. Although the receptor for PERV-B is still unknown, these data suggest that previously reported PERV transmissions to SCID and nude mice in vivo might be due to microchimerism or pseudotyping with murine viruses and indicate that normal mice are an inappropriate model for the study of PERV infection and pathogenesis.

Background: Acute liver failure (ALF) remains a disease with high mortality. Bioartificial liver support systems, which combine living cells of the liver in an extracorporeal circuit, have been successfully used in first clinical trials. The shortage of human organs to be used for bioreactors and the lack of safe and effective human liver cell lines have resulted in pigs becoming an important hepatic cell source. However, using these cells may be associated with the risk of transmission of porcine endogenous retroviruses (PERVs). PERVs are present in the genome of all pigs and are able to infect human cells in vitro. However, it remains unclear whether PERVs infect transplant recipients in vivo and, if so, whether they are pathogenic. Objectives: To detect antibodies directed against specific epitopes from PERVs in seven individuals who were treated with porcine liver cell bioreactor therapy prior to liver transplantation. Methods : Sera from seven patients treated with a hybrid liver support system based on porcine liver cells for ALF who survived the treatment and were discharged from hospital were investigated for antibodies against PERV. For this in addition to methods already reported (Xenotransplantation (2001) 125), new immunological detection methods were developed. Results: PERV-specific antibodies were found in none of the patients using Western blot assays based on purified virus or recombinant viral core and envelope proteins or ELISA based on synthetic diagnostic peptides. Conclusion: The assays used are specific and sensitive, and correlated in their diagnostic value. The data indicate that no PERV infection had occurred in none of the patients treated with the CellModule bioreactor containing porcine cells.

The inducible costimulator receptor (ICOS) is a third member of the CD28 receptor family that regulates T cell activation and function. ICOS binds to a newly identified ligand on antigen presenting cells different from the CD152 ligands CD80 and CD86. We used soluble ICOSIg and a newly developed murine anti-human ICOS ligand (ICOSL) monoclonal antibody to further characterize the ICOSL during ontogeny of antigen presenting cells. In a previous study, we found that ICOSL is expressed on monocytes, dendritic cells, and B cells. To define when ICOSL is first expressed on myeloid antigen presenting cells, we examined ICOSL expression on CD34(+) cells in bone marrow. We found that CD34(bright) cells regardless of their myeloid commitment were ICOSL(-), whereas ICOSL was first expressed when CD34 expression diminished and the myeloid marker CD33 appeared. However, acute myeloid leukemia cells were ICOSL-negative, whereas among B-cell malignancies only some cases of the most mature tumors such as prolymphocytic leukemia and hairy cell leukemia were positive. Next, we investigated purified CD34(+) hematopoietic progenitor cells that did not constitutively express ICOSL but were induced to express ICOSL within 12 h after granulocyte/macrophage colony-stimulating factor/tumor necrosis factor alpha (TNF-alpha) stimulation. Interestingly, ICOSL was induced prior to CD80/CD86 induction on CD34(+) cells so that ICOSL was expressed in the absence of CD80/CD86. This suggests that ICOSL is an early differentiation marker along the monocytic/dendritic maturation pathway. Induction of ICOSL was dependent on TNF-alpha and was regulated via NF-kappa B as revealed by use of inhibitors specific for I kappa B alpha phosphorylation such as BAY 11-7082 and BAY 11-7085. The antigen presenting capacity of TNF-alpha stimulated CD34(+) cells was strongly inhibited by ICOSIg fusion proteins or by NF-kappa B inhibition. Thus, TNF-alpha-induced ICOSL expression seemed to be functionally important for the costimulatory capacity of CD34(+) hematopoietic progenitor cells.

Nature Genetics 39, 933–934 (2007); published online 27 July 2007; corrected after print 30 October 2007 In the version of this article initially published, the second author (Jens Hansen) should have been listed as an equal contributor with the first author. The last two authors (Harald von Melchner and Patricia Ruiz Noppinger) should have been listed as corresponding authors.