Orthotopic liver transplantation in human-immunodeficiency-virus-positive patients in Germany

Objectives: This summary evaluates the outcomes of orthotopic liver transplantation (OLT) of HIV-positive patients in Germany. Methods: Retrospective chart analysis of HIV-positive patients, who had been liver-transplanted in Germany between July 1997 and July 2011. Results: 38 transplantations were performed in 32 patients at 9 German transplant centres. The reasons for OLT were end-stage liver disease (ESLD) and/or liver failure due to hepatitis C (HCV) (n = 19), hepatitis B (HBV) (n = 10), multiple viral infections of the liver (n = 2) and Budd-Chiari-Syndrome. In July 2011 19/32 (60%) of the transplanted patients were still alive with a median survival of 61 months (IQR (interquartile range): 41-86 months). 6 patients had died in the early post-transplantation period from septicaemia (n = 4), primary graft dysfunction (n = 1), and intrathoracal hemorrhage (n = 1). Later on 7 patients had died from septicaemia (n = 2), delayed graft failure (n = 2), recurrent HCC (n = 2), and renal failure (n = 1). Recurrent HBV infection was efficiently prevented in 11/12 patients; HCV reinfection occurred in all patients and contributed considerably to the overall mortality. Conclusions: Overall OLT is a feasible approach in HIV-infected patients with acceptable survival rates in Germany. Reinfection with HCV still remains a major clinical challenge in HIV/HCV coinfection after OLT

Selection-Independent Generation of Gene Knockout Mouse Embryonic Stem Cells Using Zinc-Finger Nucleases

Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10−6. In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells

Assessment of metaphase chromosomes and pluripotency.

<p>(<b>A–C</b>) Chromosome analysis. Metaphase spreads of BK4-G3.16 cells were stained with Giemsa. A representative picture of ∼20 analyzed metaphase spreads per clone is shown. (<b>D–L</b>) Assessment of pluripotency. ZFN-treated BK4-G3.16 cells were injected subcutaneously into immunodeficient mice and teratomas removed after 4–8 weeks. Histological analysis using hematoxylin/eosin staining revealed tissues derived from ectoderm (D, G, J), mesoderm (E, H, K) and endoderm (F, I, L). Scale bars = 100 µm for (D–G) and 50 µm for (H–L).</p

Molecular characterization of individual ESC clones.

<p>(<b>A</b>) Schematic of <i>EGFP</i> locus in BK4-G3.16 cells. The primer binding sites and the lengths (in base pairs) of the generated PCR amplicons are indicated. (<b>B</b>) Genotyping. The genomic PCR amplicon encompassing target site E502 was digested with TaqI. The positions of the TaqI-resistant DNA fragment (228 bp) and a cleavage product (132 bp) are indicated. (<b>C, D</b>) Sequence of disrupted <i>EGFP</i> alleles. A genomic PCR expected to produce a 556-bp amplicon was evaluated by agarose gel electrophoresis (C) and the isolated DNA fragments sequenced (D). The positions of the expected (556 bp) and the additional DNA fragment (415 bp) are indicated in (C), the sequences of all alleles are shown in (D). The ZFN target site (subsites E502L and E502R) is highlighted in capital letters, the TaqI site in bold letters.</p