Expression of human thrombomodulin on porcine endothelial cells can reduce platelet aggregation but did not reduce activation of complement or endothelium - an experimental study.

Clinical xenotransplantation will only be feasible when present limitations can be controlled sufficiently. Activation of endothelium and complement as well as coagulopathy and thrombotic microangiopathy (TMA) is important barriers. Transgenic expression of hTBM on porcine endothelial cells is a reasonable approach to reduce activation of haemostasis. Endothelial cells from wild-type pigs as well from pigs expressing hTBM alone or in combination with hCD46 and knockout of the alpha-1,3,-galactosyltransferase (GTKO) were perfused with platelet-rich plasma in a microfluidic flow chamber. Platelet aggregation and activation, coagulation, complement and endothelial cell activation were assessed. Perfusion of wild-type porcine aortic endothelial cells (PAEC) resulted in distinct platelet aggregation. Expression of hTBM in either mono-transgenic or triple-transgenic (GTKO/hCD46/hTBM) PAEC showed significantly reduced or absent platelet aggregation. Flow cytometric analysis of platelets showed an increased CD62P expression in wild-type PAEC and significantly reduced expression in mono- or triple-transgenic PAEC. Activation of coagulation measured by TAT occured in WT PAEC and was clearly reduced in hTBM and GTKO/hCD46/hTBM PAEC. Activation of complement and endothelial cells was only reduced in GTKO/hCD46/hTBM but not in PAEC expressing hTBM alone. Expression of hTBM was able to prevent activation of coagulation and platelet aggregation in mono- and triple-transgenic PAEC, while activation of complement and endothelial cells was not reduced in mono-transgenic PAEC.

Investigation of the influence of xenoreactive antibodies on activation of complement and coagulation in an ex vivo perfusion animal study using porcine kidneys.

During pig-to-primate xenotransplantation or perfusion of porcine organs with human blood, a xenogeneic coagulopathy with consecutive development of thrombotic microangiopathy (TMA) can be observed. The aim of this study was to elucidate the influence of the reduction of xenoreactive natural antibodies on the coagulopathy using an ex vivo perfusion system. Thirteen perfusion experiments using landrace wild-type porcine kidneys were performed in three different experimental groups: autologous, xenogeneic, and immunoadsorption. During and after perfusion, blood and tissue samples were collected to assess markers of coagulation, complement, inflammation, and endothelial activation. Immunoadsorption prior to perfusion did not prolong perfusion time (174 min ±28) compared to xenogeneic (182 min ±22) experiments, whereas autologous perfusion was possible for maximum of 240 min in all experiments. Activation of coagulation was similar comparing perfusions after immunoadsorption (D-Dimer 24 186 µg/l ±5813; TAT 566 µg/l ±34) to xenogeneic (D-Dimer 22 175 µg/l ±7826, TAT 600 µg/l ±0) experiments. But antibody-mediated complement activation was reduced in the immunoadsorption group. TNF-alpha and markers of endothelial cell activation were lower in the immunoadsorption group compared to the xenogeneic experiments. In this ex vivo perfusion model, we observed that marked removal of xenogeneic antibodies can reduce complement activation via the classical pathway as well as endothelial cell activation and inflammation. Immunoadsorption cannot prevent the activation of the terminal complement cascade and coagulation.

Predicting an HLA-DPB1 expression marker based on standard DPB1 genotyping: Linkage analysis of over 32,000 samples.

The risk of acute graft-versus-host disease (GvHD) after hematopoietic stem cell transplantation is increased with donor-recipient HLA-DPB1 allele mismatching. The single-nucleotide polymorphism (SNP) rs9277534 within the 3' untranslated region (UTR) correlates with HLA-DPB1 allotype expression and serves as a marker for permissive HLA-DPB1 mismatches. Since rs9277534 is not routinely typed, we analyzed 32,681 samples of mostly European ancestry to investigate if the rs9277534 allele can be reliably imputed from standard DPB1 genotyping. We confirmed the previously-defined linkages between rs9277534 and 18 DPB1 alleles and established additional linkages for 46 DPB1 alleles. Based on these linkages, the rs9277534 allele could be predicted for 99.6% of the samples based on DPB1 genotypes (99.99% concordance). We demonstrate that 100% prediction accuracy could be achieved if the prediction utilized exon 3 sequence information. DPB1 genotyping based on exon 2 data alone allows no unambiguous rs9277534 allele prediction but was estimated to maintain 99% accuracy for samples of European descent. We conclude that DPB1 genotyping is sufficient to infer the DPB1 expression marker rs9277534 with high accuracy. This information could be used to select donors with permissive HLA-DPB1 mismatches without directly screening for rs9277534.

CD32 inhibition and high dose of rhFVIII suppress murine FVIII-specific recall response by distinct mechanisms in vitro.

Development of neutralising antibodies (inhibitors) against factor VIII (FVIII) is a frequent and severe complication of replacement therapy in haemophilia A. Previous data from haemophilia A mouse model demonstrates that both CD32 inhibition and high doses of rhFVIII prevent the differentiation of FVIII-specific memory B cells (MBCs) into antibody secreting cells (ASCs). Here, cellular targets responsible for the suppression of ASC formation by means of CD32 inhibition and high dose of rhFVIII were analysed. We investigated apoptosis on FVIII-specific MBCs using a pan caspases inhibitor, and screened for defects in rhFVIII presentation by analysing T cell release of Th1- and Th2-cytokines in vitro. Although high dose of rhFVIII suppressed ASC formation, cytokine response was not affected. Upon re-stimulation of splenocytes with high dose of rhFVIII, prevention of apoptosis fully restored the FVIII-specific recall response. In contrast, genetic deletion or inhibition of CD32 significantly altered Th1- and Th2-response. CD32 blockade and inhibition of apoptosis resulted in a partial rescue of FVIII-specific ASCs. Normal cytokine secretion could not be restored. In conclusion, suppression of FVIII-specific recall response by CD32 and high doses of rhFVIII is mediated by distinct mechanisms. High dose of rhFVIII induces apoptosis in FVIII-specific MBCs but does not influence FVIII-specific T cell response. CD32 blockade, however, may suppress the FVIII-specific recall response by two ways: i) increasing apoptosis of FVIII-specific MBCs and ii) disturbing FVIII-specific T cell response by modulating presentation of rhFVIII to CD4+ T cells in vitro.

Effect of TNF-alpha blockade on coagulopathy and endothelial cell activation in xenoperfused porcine kidneys.

BACKGROUND: Following pig-to-primate kidney transplantation, endothelial cell activation and xenogenic activation of the recipient's coagulation eventually leading to organ dysfunction and microthrombosis can be observed. In this study, we examined the effect of a TNF-receptor fusion protein (TNF-RFP) on endothelial cell activation and coagulopathy utilizing an appropriate ex vivo perfusion system. METHODS: Using an ex vivo perfusion circuit based on C1-Inhibitor (C1-Inh) and low-dose heparin administration, we have analyzed consumptive coagulopathy following contact of human blood with porcine endothelium. Porcine kidneys were recovered following in situ cold perfusion with Histidine-tryptophan-ketoglutarate (HTK) organ preservation solution and were immediately connected to a perfusion circuit utilizing freshly drawn pooled porcine or human AB blood. The experiments were performed in three individual groups: autologous perfusion (n = 5), xenogenic perfusion without any further pharmacological intervention (n = 10), or with addition of TNF-RFP (n = 5). After perfusion, tissue samples were obtained for real-time PCR and immunohistological analyses. Endothelial cell activation was assessed by measuring the expression levels of E-selectin, ICAM-1, and VCAM-1. RESULTS: Kidney survival during organ perfusion with human blood, C1-Inh, and heparin, but without any further pharmacological intervention was 126 ± 78 min. Coagulopathy was observed with significantly elevated concentrations of D-dimer and thrombin-antithrombin complex (TAT), resulting in the formation of multiple microthrombi. Endothelial cell activation was pronounced, as shown by increased expression of E-selectin and VCAM-1. In contrast, pharmacological intervention with TNF-RFP prolonged organ survival to 240 ± 0 min (max. perfusion time; no difference to autologous control). Formation of microthrombi was slightly reduced, although not significantly, if compared to the xenogenic control. D-dimer and TAT were elevated at similar levels to the xenogenic control experiments. In contrast, endothelial cell activation, as shown by real-time PCR, was significantly reduced in the TNF-RFP group. CONCLUSION: We conclude that although coagulopathy was not affected, TNF-RFP is able to suppress inflammation occurring after xenoperfusion in this ex vivo perfusion model.

Kidneys From α1,3-Galactosyltransferase Knockout/Human Heme Oxygenase-1/Human A20 Transgenic Pigs Are Protected From Rejection During Ex Vivo Perfusion With Human Blood.

UNLABELLED: Multiple modifications of the porcine genome are required to prevent rejection after pig-to-primate xenotransplantation. Here, we produced pigs with a knockout of the α1,3-galactosyltransferase gene (GGTA1-KO) combined with transgenic expression of the human anti-apoptotic/anti-inflammatory molecules heme oxygenase-1 and A20, and investigated their xenoprotective properties. METHODS: The GGTA1-KO/human heme oxygenase-1 (hHO-1)/human A20 (hA20) transgenic pigs were produced in a stepwise approach using zinc finger nuclease vectors targeting the GGTA1 gene and a Sleeping Beauty vector coding for hA20. Two piglets were analyzed by quantitative reverse-transcription polymerase chain reaction, flow cytometry, and sequencing. The biological function of the genetic modifications was tested in a (51)Chromium release assay and by ex vivo kidney perfusions with human blood. RESULTS: Disruption of the GGTA1 gene by deletion of few basepairs was demonstrated in GGTA1-KO/hHO-1/hA20 transgenic pigs. The hHO-1 and hA20 mRNA expression was confirmed by quantitative reverse-transcription polymerase chain reaction. Ex vivo perfusion of 2 transgenic kidneys was feasible for the maximum experimental time of 240 minutes without symptoms of rejection. CONCLUSIONS: Results indicate that GGTA1-KO/hHO-1/hA20 transgenic pigs are a promising model to alleviate rejection and ischemia-reperfusion damage in porcine xenografts and could serve as a background for further genetic modifications toward the production of a donor pig that is clinically relevant for xenotransplantation.

Recombinant human antithrombin prevents xenogenic activation of hemostasis in a model of pig-to-human kidney transplantation.

BACKGROUND: Xenogenic activation of hemostasis (XAH) represents a major hurdle for the transplantation of discordant animal organs into humans as it results in thrombotic microangiopathy (TMA). We have previously shown that recombinant human-activated protein C (rhAPC) mitigates XAH and TMA in an ex vivo model of pig-to-human kidney transplantation. However, the use of rhAPC may not be feasible in a perioperative setting due to possible bleeding complications. METHODS: Here, we explored the effects of another natural inhibitor of coagulation, human recombinant antithrombin (rhAT), in comparison with rhAPC. Unmodified porcine kidneys (n = 25) were perfused ex vivo with porcine blood, human blood, or human blood supplemented with rhAPC or rhAT. Surrogate parameters of organ survival, markers of XAH (D- Dimer, thrombin-antithrombin complex [TAT], fibrinogen, antithrombin activity, plasminogen), endothelial cell and platelet activation (E-selectin, P-selectin), platelet function tests and histological signs of TMA were evaluated. RESULTS: Perfusion was feasible for > 240 min in all experiments with autologous porcine blood, but limited to 126 ± 78 min with human blood due to increased vascular resistance. Addition of rhAT protected from TMA and allowed for perfusion times > 240 min. In addition, there were less signs of XAH with reduced release of P-selectin and overexpression of E-selectin, whereas the progressive loss of platelet function, observed during discordant perfusion, was prevented. The effect of rhAT was dose-dependent with maximum protection obtained at 3 IU/ml. CONCLUSION: In conclusion, in this ex vivo model of discordant xenotransplantation, rhAT reduced XAH and prevented TMA in doses that appear feasible for use in clinical or preclinical transplantation settings.

The Clubroot Pathogen (Plasmodiophora brassicae) Influences Auxin Signaling to Regulate Auxin Homeostasis in Arabidopsis.

The clubroot disease, caused by the obligate biotrophic protist Plasmodiophora brassicae, affects cruciferous crops worldwide. It is characterized by root swellings as symptoms, which are dependent on the alteration of auxin and cytokinin metabolism. Here, we describe that two different classes of auxin receptors, the TIR family and the auxin binding protein 1 (ABP1) in Arabidopsis thaliana are transcriptionally upregulated upon gall formation. Mutations in the TIR family resulted in more susceptible reactions to the root pathogen. As target genes for the different pathways we have investigated the transcriptional regulation of selected transcriptional repressors (Aux/IAA) and transcription factors (ARF). As the TIR pathway controls auxin homeostasis via the upregulation of some auxin conjugate synthetases (GH3), the expression of selected GH3 genes was also investigated, showing in most cases upregulation. A double gh3 mutant showed also slightly higher susceptibility to P. brassicae infection, while all tested single mutants did not show any alteration in the clubroot phenotype. As targets for the ABP1-induced cell elongation the effect of potassium channel blockers on clubroot formation was investigated. Treatment with tetraethylammonium (TEA) resulted in less severe clubroot symptoms. This research provides evidence for the involvement of two auxin signaling pathways in Arabidopsis needed for the establishment of the root galls by P. brassicae.

Impact of Salinomycin on human cholangiocarcinoma: induction of apoptosis and impairment of tumor cell proliferation in vitro.

BACKGROUND: Cholangiocarcinoma (CC) is a primary liver cancer with increasing incidence worldwide. Despite all efforts made in past years, prognosis remains to be poor. At least in part, this might be explained by a pronounced resistance of CC cells to undergo apoptosis. Thus, new therapeutic strategies are imperatively required. In this study we investigated the effect of Salinomycin, a polyether ionophore antibiotic, on CC cells as an appropriate agent to treat CC. Salinomycin was quite recently identified to induce apoptosis in cancer stem cells and to overcome apoptosis-resistance in several leukemia-cells and other cancer cell lines of different origin. METHODS: To delineate the effects of Salinomycin on CC, we established an in vitro cell culture model using three different human CC cell lines. After treatment apoptosis as well as migration and proliferation behavior was assessed and additional cell cycle analyses were performed by flowcytometry. RESULTS: By demonstrating Annexin V and TUNEL positivity of human CC cells, we provide evidence that Salinomycin reveals the capacity to break apoptosis-resistance in CC cells. Furthermore, we are able to demonstrate that the non-apoptotic cell fraction is characterized by sustainable impaired migration and proliferation. Cell cycle analyses revealed G2-phase accumulation of human CC cells after treatment with Salinomycin. Even though apoptosis is induced in two of three cell lines of CC cells, one cell line remained unaffected in regard of apoptosis but revealed as the other CC cells decreased proliferation and migration. CONCLUSION: In this study, we are able to demonstrate that Salinomycin is an effective agent against previously resistant CC cells and might be a potential candidate for the treatment of CC in the future.

Influence of in situ steam formation by radio frequency heating on thermodesorption of hydrocarbons from contaminated soil.

Thermal desorption of a wide spectrum of organic contaminants, initiated by radio frequency (RF) heating, was studied at laboratory and pilot-plant scales for an artificially contaminated soil and for an originally contaminated soil from an industrial site. Up to 100 °C, moderate desorption rates were observed for light aromatics such as toluene, chlorobenzene, and ethylbenzene. Desorption of the less volatile contaminants was greatly enhanced above 100 °C, when fast evaporation of soil-water produced steam for hydrocarbon stripping (steam-distillation, desorption rates increased by more than 1 order of magnitude). For hydrocarbons with low water solubility (e.g., aliphatic hydrocarbons), the temperature increase above 100 °C after desiccation of soil again led to a significant increase of the removal rates, thus showing the impact of hydrocarbon partial pressure. RF heating was shown to be an appropriate option for thermally enhanced soil vapor extraction, leading to efficient cleaning of contaminated soils.

Occurrence of perfluorinated compounds (PFCs) in drinking water of North Rhine-Westphalia, Germany and new approach to assess drinking water contamination by shorter-chained C4-C7 PFCs.

After detection of perfluorooctanoate (PFOA) in drinking water at concentrations up to 0.64 microg/l in Arnsberg, Sauerland, Germany, the German Drinking Water Commission (TWK) assessed perfluorinated compounds (PFCs) in drinking water and set for the first time worldwide in June 2006 a health-based guide value for safe lifelong exposure at 0.3 microg/l (sum of PFOA and perfluorooctanesulfonate, PFOS). PFOA and PFOS can be effectively removed from drinking water by percolation over granular activated carbon. Additionally, recent EU-regulations require phasing out use of PFOS and ask to voluntarily reduce the one of PFOA. New and shorter-chained PFCs (C4-C7) and their mixtures are being introduced as replacements. We assume that some of these "new" compounds could be main contributors to total PFC levels in drinking water in future, especially since short-chained PFCs are difficult to remove from drinking water by common treatment techniques and also by filtration over activated carbon. The aims of the study were to summarize the data from the regularly measured PFC levels in drinking water and in the drinking water resources in North Rhine-Westphalia (NRW) for the sampling period 2008-2009, to give an overview on the general approach to assess PFC mixtures and to assess short-chained PFCs by using toxicokinetic instead of (sub)chronic data. No general increase of substitutes for PFOS and PFOA in wastewater and surface water was detected. Present findings of short-chained PFC in drinking waters in NRW were due to extended analysis and caused by other impacts. Additionally, several PFC contamination incidents in drinking water resources (groundwater and rivers) have been reported in NRW. The new approach to assess short-chained PFCs is based on a ranking of their estimated half-lives for elimination from the human body. Accordingly, we consider the following provisional health-related indication values (HRIV) as safe in drinking water for lifelong exposure: perfluorobutanoate (PFBA) 7 microg/l, perfluoropentanoate (PFPA) 3 microg/l, perfluorohexanoate (PFHxA) 1 microg/l, perfluoroheptanoate (PFHpA) 0.3 microg/l, perfluorobutanesulfonate (PFBS) 3 microg/l, perfluoropentanesulfonate (PFPS) 1 microg/l, perfluorohexanesulfonate (PFHxS) 0.3 microg/l and perfluoroheptanesulfonate (PFHpS) 0.3 microg/l. For all PFCs the long-term lowest maximal quality goal (general precautionary value, PVg) in drinking water is set to -0.1 microg/l.

[Effects of fasting and endurance training on energy metabolism and physical fitness in obese patients]. / Einfluss von therapeutischem Fasten und Ausdauertraining auf den Energiestoffwechsel und die körperliche Leistungsfähigkeit Adipöser.

BACKGROUND: Buchinger therapeutic fasting, based on a low caloric (<500 kcal/d) fluid diet (juice-broth), is a traditional natural-healing strategy to reduce body weight and cure cardiometabolic complications in obese patients. Although there are some anecdotal reports about a serious protein loss during this type of fasting, there are no validated data that support this claim. OBJECTIVE: Is Buchinger fasting associated with a critical protein loss and is this loss aggravated by additional endurance training which might lead to cardiac symptoms? PATIENTS AND METHODS: A therapeutic concept for 'complex re-conditioning of obese patients', a combination of Buchinger fasting and endurance training was tested in obese patients for 28 days. Parameters of energy, carbohydrate, lipid, and protein metabolism were studied in several subgroups either with or without endurance training of defined intensity. RESULTS: Additional endurance training resultet in a greater loss of body mass (12.2 +/- 3.2 vs. 10.4 +/- 2.2 kg; p < 0.001) and fat mass (8.1 +/- 1.6 vs. 5.9 +/- 1.3 kg; p < 0.001), and in improved lipid utilisation, physical efficiency, and a smaller decrease in metabolic rate per kilogram fat-free mass (-7.6 +/- 12.4 vs. -14.3 +/- 12.2%; p < 0.001). Without training, total protein loss over 28 days was about 1,000 and 650 g for men and women, respectively. With training, there was an additional protein loss of 130 g / 28 d (p < 0.01) in men. CONCLUSIONS: Endurance training is an important, safe and necessary component of a 28-day Buchinger fasting therapy.

Effects of pharmacological intervention on coagulopathy and organ function in xenoperfused kidneys.

BACKGROUND: Following pig to primate kidney transplantation, xenogenic activation of the coagulation (XAC) system of the recipient eventually leading to organ dysfunction and disseminated intravascular coagulation (DIC) can be observed. METHODS: Using an ex-vivo perfusion circuit based on low-dose heparin-mediated anticoagulation and exogenous complement inhibition by C1- Inhibitor (C1-Inh), we have analysed XAC following contact of human blood with porcine endothelium. Porcine kidneys (n = 23) were recovered following in situ cold perfusion with histidine-tryptophan-ketoglutarate (HTK) solution and were connected to a perfusion circuit utilizing freshly drawn pooled human AB blood. RESULTS: Kidney survival during organ perfusion with human blood, CI-Inh, heparin but without any further pharmacological intervention was 126 +/- 78 min. XAC was observed with significantly elevated levels of D-dimer and thrombin antithrombin complexes (TAT). Pharmacological intervention with nitroprusside and prostacycline resulted in increased organ survival (220 +/- 28 min and 180 +/- 85 min respectively) but failed to inhibit XAC. In contrast, addition of activated protein C (APC) significantly reduced the increase in D-dimer and TAT and prolonged organ survival to 240 min (+/-0). On histology, no remarkable signs of XAC were observed. CONCLUSIONS: We conclude that exogenous APC is able to reduce XAC in this ex vivo perfusion model.