M. Winkler

Productive infection of primary human endothelial cells by pig endogenous retrovirus (PERV)

Abstract: The potential risk of viral transmission in the setting of xenotransplantation has gained major attention. Different porcine cell types have been shown to release retroviral particles, which are infectious for human cell lines in vitro. However, there are only a few data on whether PERV (pig endogenous retrovirus) is able to infect primary human cells. In this study we have analyzed endothelial cells, vascular fibroblasts, mesangial cells, mononuclear cells, hematopoetic stem cells and bone marrow stromal cells for PERV transmission. We now provide evidence for primary human endothelial cells, vascular fibroblasts, and mesangial cells to be susceptible to PERV transmission. PERV infection was productive in endothelial cells and mesangial cells. Our data confirm and extend former reports concerning the PERV infection of human cells. The PERV infection of different primary human cells represents further significant evidence for a viral risk during xenotransplantation. In this context, special attention should be directed towards productive infection of human endothelial cells: in the setting of xenotransplantation this cell type will have close contact with porcine cells and PERV particles.

Acute vascular rejection is associated with systemic complement activation ina pig-to-primate kidney xenograft model

Abstract: The introduction of h‐DAF transgenic porcine organs into pre‐clinical pig‐to‐primate discordant xenotransplantation has led to complete and reliable abrogation of hyperacute xenograft rejection (HAR). Despite additional heavy immunosuppression however, most xenografts are still lost due to acute vascular rejection (AVR), with current treatment protocols being of only limited value. In a life‐supporting model of pig‐to‐primate kidney transplantation, unmodified (n = 8) or h‐DAF‐transgenic (n = 9) porcine kidneys were transplanted into cynomolgus monkeys under cyclophosphamide (CyP), cyclosporine and low‐dose steroid immunosuppression. Longest recipient survival was 11 days in the control group and 68 days in the h‐DAF transgenic group. Stable initial graft function with recipient survival > 4 days was generated in eight animals (two controls and six transgenics). In these animals, plasma complement levels were analyzed during ongoing AVR. Compared with baseline levels, a two‐fold increase in C3a levels and a four‐fold increase in sC5b‐9 levels were measured. In parallel to systemic complement activation, increased deposition of C3 and C5b‐9 along with massive staining for recipient IgM immunoglobulins was detected in the xenografts on immunohistochemistry. We conclude that acute vascular xenograft rejection of porcine kidneys in cynomolgus monkeys is associated with classical pathway complement activation following binding of induced recipient anti‐porcine antibodies. This complement activation can be observed despite membrane bound expression of human complement regulators in the porcine xenografts. Therefore, additional short‐term fluid phase complement inhibition seems necessary for the future development of protocols designed for treatment of AVR in the pig‐to‐primate combination.

Analysis of potential porcine endogenous retrovirus (PERV) transmission in a whole‐organ xenotransplantation model without interfering microchimerism

Abstract The question whether porcine xenografts can lead to porcine endogenous retrovirus (PERV) infection of recipients is critical for the evaluation of the safety of pig-to-man xenotransplantation. Unfortunately, polymerase chain reaction (PCR)-based analysis of potential PERV infections in nonhuman-primate whole-organ xenotransplantation models is hampered by false positive results due to chimeric porcine cells. To avoid the inherent analytical problem of xenomicrochimerism, we developed a non-life-supporting pig-to-primate kidney xenotransplantation model: porcine kidneys were transplanted, whereas the functioning recipient kidneys remained in situ. Subsequent to rejection (after 2 hours to 15 days), xenografts were removed, and recipients remained alive for up to 287 days. Immunosuppressive therapy based on cyclophosphamide, cyclosporine, and steroids was maintained for 28 days after transplantation. Using appropriate PCR assays, xenochimerism was found in tissue samples and partly even in peripheral blood leukocytes (PBLs) while the porcine kidneys were in situ. After graft removal, xenochimerism was no longer detectable, thus allowing analysis for possible PERV transmission.

Human CMV immediate‐early enhancer: a useful tool to enhance cell‐type‐specific expression from lentiviral vectors

Lentiviral vectors are attractive delivery tools for gene therapy, especially in terminally differentiated target cells. While restriction of gene expression to specific cell populations is of particular importance, highly efficient cell‐type‐specific gene expression after viral gene transfer so far has been hampered by low levels of transgene expression.

No Evidence for Infection of Human Embryonic Stem Cells by Feeder Cell–Derived Murine Leukemia Viruses

Until recently, culture and expansion of nondifferentiated human embryonic stem cells (hESCs) depended on coculture with murine embryonic fibroblasts. Because mice are known to harbor a variety of pathogens, such culture conditions implicate the risk of xenozoonoses. Among these pathogens, endogenous retroviruses, including murine leukemia viruses (MuLVs), are of special importance. It is well known that some strains cause pathogenic (e.g., leukemic) effects and that xenotropic, polytropic, and amphotropic MuLVs are able to infect human cells.

Generation and Characterization of Functional Cardiomyocytes from Rhesus Monkey Embryonic Stem Cells

Embryonic stem cells (ESCs) from mice and humans (hESCs) have been shown to be able to efficiently differentiate toward cardiomyocytes (CMs). Because murine ESCs and hESCs do not allow for establishment of pre‐clinical allogeneic transplantation models, the aim of our study was to generate functional CMs from rhesus monkey ESCs (rESCs). Although formation of ectodermal and neuronal/glial cells appears to be the default pathway of the rESC line R366.4, we were able to change this commitment and to direct generation of endodermal/mesodermal cells and further differentiation toward CMs. Differentiation of rESCs resulted in an average of 18% of spontaneously contracting embryoid bodies (EBs) from rESCs. Semiquantitative reverse transcription‐polymerase chain reaction analyses demonstrated expression of marker genes typical for endoderm, mesoderm, cardiac mesoderm, and CMs, including brachyury, goosecoid, Tbx‐5, Tbx‐20, Mesp1, Nkx2.5, GATA‐4, FOG‐2, Mlc2a, MLC2v, ANF, and α‐MHC in rESC‐derived CMs. Immunohistological and ultrastructural studies showed expression of CM‐typical proteins, including sarcomeric actinin, troponin T, titin, connexin 43, and cross‐striated muscle fibrils. Electrophysiological studies by means of multielectrode arrays revealed evidence of functionality, electrical coupling, and β‐adrenergic signaling of the generated CMs. This is the first study demonstrating generation of functional CMs derived from rESCs. In contrast to hESCs, rESCs allow for establishment of pre‐clinical allogeneic transplantation models. Moreover, rESC‐derived CMs represent a cell source for the development of high‐throughput assays for cardiac safety pharmacology.

Conversion from cyclosporin to FK 506 after liver transplantation

Thirty-seven liver-grafted patients with steroid-resistant acute or chronic graft rejection or with cyclosporin-related complications were converted from CyA to FK 506. The clinical outcome of the patients primarily depended on the degree of liver dysfunction present at initiation of FK 506 treatment. In patients switched to FK 506 for treatment of acute or early chronic graft rejection, CyA nephrotoxicity, or CyA malabsorption, the FK 506 therapy was associated with a clear improvement in the clinical course. In contrast, in patients with advanced chronic graft rejection, a lower response rate to the conversion in immunosuppression was observed. The lower response rate was associated with a higher patient mortality. These studies demonstrate that FK 506 represents a valuable alternative immunosuppressant for liver-grafted patients. The conversion from CyA to FK 506 should take place before serious — and potentially irreversible — disturbances in liver function are observed.

Influence of bile on cyclosporin absorption from microemulsion formulation in primary liver transplant recipients

We analysed the absorption, after oral application, of a new galenic form of cyclosporin A (CyA-NOF) in liver-grafted patients (n=12) during the 1st week (days 2–4) after transplantation. Pharmacokinetic profiling was performed with an open or clamped T tube in situ or with the T tube absent. The pharmacokinetic parameters of CyA-NOF were influenced by T tube clamping and bile diversion. The highest AUC, Cmax and earliest Tmax values were found in patients without a T tube in situ, indicating that absorption of CyA-NOF in patients during the early course after liver transplantation is not bile-independent. CyA-NOF, at a dose of 7.5 mg/kg, was enterally absorbed with appropriate AUC and Cmax levels. Patients receiving a starting dose of 7.5 mg/kg were successfully maintained on CyA-NOF during the subsequent clinical course.

Evaluation of the Pro-Trac tacrolimus monoclonal whole-blood enzyme-linked immunosorbent assay for monitoring of tacrolimus levels in patients after kidney, heart, and liver transplantation.

In a prospective study, we evaluated a novel enzyme-linked immunosorbent assay (ELISA) (Pro-Trac) for determining tacrolimus (FK 506) concentrations in whole blood. Results obtained by the ELISA were compared with those obtained either by microparticle enzyme immunoassay (MEIA) or by high-performance liquid chromatography/mass spectrometry (HPLC-MS). The lower limit of quantitation of the ELISA was 0.5 microgram/L. The within-series coefficient of variation (CV) was < 11%. For spiked blood samples containing different concentrations of tacrolimus, interassay CV was 23.6% at 2.5 micrograms/L; however, at 15 and 60 micrograms/L, interassay CV was 44.9 and 50.8%, respectively. In crossover studies including blood samples from patients after liver, heart, or kidney transplantation, ELISA results correlated with those of the HPLC-MS (r = 0.73) as well as with those generated by MEIA (r = 0.82). The ELISA and MEIA showed 52.3 and 56.2% cross-reactivity with 15-O-demethyltacrolimus, respectively, but only 5.0 and 5.4% cross-reactivity with 13-O-demethyltacrolimus. We conclude that if assay precision in the upper range is improved, the Pro-Trac ELISA might be a valuable alternative to the MEIA for therapeutic drug monitoring of tacrolimus.

Neurological examinations after liver transplantation concerning patients under corticosteroid immunosuppression and either FK 506 or cyclosporin

Abstract To study the neurological sequelae in liver transplanted recipients, 25 patients were followed up between 5 and 30 months after transplantation and another 14 patients were seen before and after transplantation. Physical examination took special note of tremor and polyneuropathy; additionally, patients estimated concentration and memory, tremor, paraesthesias and sleep disturbances on a self‐rating scale. Tremor was reported to be preexistent in 50% of the later FK 506 and cyclosporin group and only temporarily rose afterwards. Twenty‐eight percent complained of tremor and 20 % said that it interfered mildly with daily activity. Only 2 of 39 patients showed new signs of polyneuropathy. Concentration and memory improved significantly after transplantation. In the second group of patients, MRI, EEG, lumbar puncture and neuropsychological tests were done just before and routinely after transplantation, revealing numerous preexisting neurological deficits with only singular changes afterwards.