Covid19 vaccination-associated portal vein thrombosis-An interdisciplinary clinical challenge.

Despite one of the largest vaccination campaigns in human history, the COVID-19 pandemic has not been yet defeated. More than 10 billion doses of COVID-19 vaccine have been administered worldwide. AstraZeneca's Vaxzevria (ChAdOx1 nCoV-19 / AZD1222) was approved as the first viral vector-based vaccine in the EU on 29 January 2021. Thromboembolic events are a rare complication of vaccination with ChAdOx1 nCoV-19 in the context of, now known as vaccine-induced immune thrombotic thrombocytopenia (VITT), with an incidence of 1.5-3 in 100,000 vaccinations. VITT is clinically as well as pathophysiologically comparable to heparin-induced thrombocytopenia. Illustrated by a fulminant patient case, a multidisciplinary step-by-step guideline was developed for the recognition, diagnosis, and management of patients with severe acute portosplanchic venous thrombosis with mesenteric ischemia due to vaccine-induced immunogenic thrombotic thrombocytopenia.

Bile duct damage after cold storage of deceased donor livers predicts biliary complications after liver transplantation.

BACKGROUND & AIMS: The aim of this study was to examine the development of biliary epithelial damage between organ retrieval and transplantation and its clinical relevance for patients. METHODS: Common bile duct samples during donor hepatectomy, after cold storage, and after reperfusion were compared to healthy controls by hematoxylin and eosin (H&E) staining and immunofluorescence for tight junction protein 1 and Claudin-1. A bile duct damage score to quantify biliary epithelial injury was developed and correlated with recipient and donor data and patient outcome. RESULTS: Control (N=16) and donor hepatectomy bile ducts (N=10) showed regular epithelial morphology and tight junction architecture. After cold storage (N=37; p=0.0119), and even more after reperfusion (N=62; p=0.0002), epithelial damage, as quantified by the bile duct damage score, was markedly increased, and both tight junction proteins were detected with inappropriate morphology. Patients with major bile duct damage after cold storage had a significantly increased risk of biliary complications (relative risk 18.75; p<0.0001) and graft loss (p=0.0004). CONCLUSIONS: In many cases, the common bile duct epithelium shows considerable damage after cold ischemia with further damage occurring after reperfusion. The extent of epithelial damage can be quantified by our newly developed bile duct damage score and is a prognostic parameter for biliary complications and graft loss. Possibly, in an intraoperative histological examination, this bile duct damage score may influence decision-making in transplantation surgery.

Improved outcome after 'bottom-up' immunosuppression in liver transplant recipients with preoperative renal impairment.

BACKGROUND: Most patients with high MELD scores have impaired renal function prior to transplantation. PATIENT AND METHODS: A retrospective case control study was conducted with initial low immunosuppression, which was increased when patients rejected or were clinically stable beyond day 30 ('bottom-up'). RESULTS: Thirty patients with impaired renal function were included. Fifteen were treated with de novo cyclosporine A (CsA; group A), and 15 had 'bottom-up' immunosuppression (group B). Baseline renal function was similar: serum creatinine (SCr) median 1.8 mg/dl (range: 1.5-4.0 mg/dl; group A) versus 2.4 mg/dl (range: 1.5-4.0 mg/dl; group B; p = 0.24). The requirement for renal replacement therapy was significantly lower in group B (p = 0.032). Ten received 'bottom-up' immunosuppression [4 CsA/1 sirolimus (Sir) 'on demand' after rejection, 5 Sir (stable)] beyond day 30. By months 6 and 12 (1.6 mg/dl vs. 1.2 mg/dl), SCr values were significantly better in group B (p = 0.006). Renal function in group B did not differ between patients receiving CsA or Sir. Overall complication rates, survival and biopsy-proven acute rejection were similar, although BANFF scores were higher in group B (p = 0.004). CONCLUSION: Successful implementation of 'bottom-up' immunosuppression in liver transplant recipients with high lab-MELD scores and renal dysfunction at the time of transplantation has the potential to substantially improve short- and long-term outcomes.

AAV plasmid DNA simplifies liver-directed in vivo gene therapy: comparison of expression levels after plasmid DNA-, adeno-associated virus- and adenovirus-mediated liver transfection.

BACKGROUND: Successful liver gene therapy depends on efficient gene transfer techniques and long-lasting gene expression after successful transfer. Over the last decades, important progress has been made with the introduction of viral vectors using animal models, although their use is hampered by a complex and costly preparation compared to the simple and cost-effective preparation of plasmid DNA. These problems become even more critical when considering the application of viral vectors in human gene therapy and gene therapy trials. In a previous study, we were able to show that the hydrodynamics-based gene transfer of plasmid-DNA, containing the adeno-associated-virus specific inverted terminal repeats (AAV-ITR), prolongs gene expression in the liver, although it remained unclear whether plasmid gene transfer could achieve similar expression levels compared to viral-vector gene transfer. METHODS: Rat livers were transfected in-vivo with AAV-ITR-containing plasmid-DNA using a modified hydrodynamics-based procedure. Expression levels were monitored thereafter and compared with expression levels after viral-vector gene transfer. RESULTS: A high and stable long-term expression was achieved after in vivo transfection of rat livers with AAV-ITR-containing plasmids. The expression course resembled that after AAV-mediated gene transfer, and the expression was at least as high, and lasted as long, compared to recombinant AAV-mediated gene transfer. CONCLUSIONS: We consider AAV-ITR-containing plasmids as a simple and cost-effective alternative to recombinant viral vectors, especially for liver-directed gene therapy in rodents. With ongoing progress in gene transfer methods for naked DNA, these plasmids may also become a successful alternative to recombinant viral vectors in human gene therapy.

"Rescue allocation offers" in liver transplantation: is there any reason to reject "unwanted" organs?

To increase the number of transplanted organs, the Eurotransplant foundation uses a so-called "rescue-organ-allocation" procedure for organs that had been rejected by at least three consecutive transplant centers for medical reasons. The transplant center that finally accepts such an organ can then freely choose a patient from its own waiting list, without being bound to regular allocation criteria. Almost 30% of deceased donor livers are now allocated through this process in the Eurotransplant region. We report our results of 38 "rescue-allocation" livers (RA livers) transplanted at our institution (2003-2007), compared to a group of 115 regularly allocated organs within the same period. From our data, RA livers have the same results as regularly allocated livers. Type and frequency of postoperative morbidity did not differ between both groups, though the analysis of subgroups showed a tendency toward reduced survival of RA livers in patients with viral hepatitis. Interestingly, the Donor Risk Index (DRI) showed no difference between RA livers and regularly allocated livers. Although preliminary due to small numbers, we conclude that RA livers can be safely transplanted without increased mortality or morbidity. However, no donor specific criteria which would justify rejecting a RA liver were found. This highly challenges the applicability of the RA procedure in its current form.

High volume naked DNA tail-vein injection restores liver function in Fah-knock out mice.

BACKGROUND: Despite pharmaceutical treatment with NTBC (2-2-nitro-4-fluoromethylbenzoyl-1,3-cyclohexanedione), a high incidence of liver malignancies occur in humans and mice suffering from hereditary tyrosinemia type 1 (HT1) caused by mutation of the fumarylacetoacetate hydrolase (fah) gene. METHODS: To evaluate the efficacy of a definitive treatment for HT1, we transfected fah knockout mice with naked plasmid DNA using high volume tail-vein injection. This approach was chosen to reduce the occurrence of insertional mutagenesis that is frequently observed when using other (retro-)viral vectors. To prolong gene expression, the fah gene was cloned between adeno-associated virus (AAV)-specific inverted terminal repeats (ITRs). RESULTS: All animals treated with high volume plasmid DNA injections could be successfully weaned off NTBC and survived in the long term without any further pharmacological support. Up to 50% fah positive hepatocytes were detected in livers of naked plasmid DNA-treated animals and serum liver function tests approximated those of wild-type controls. CONCLUSIONS: Naked plasmid DNA transfection offers a promising alternative treatment for HT1. Minimizing side-effects makes this approach especially appealing.

Adult Living Donor Liver Transplantation with ABO-Incompatible Grafts: A German Single Center Experience.

Adult living donor liver transplantations (ALDLTs) across the ABO blood group barrier have been reported in Asia, North Americas, and Europe, but not yet in Germany. Several strategies have been established to overcome the detrimental effects that are attached with such a disparity between donor and host, but no gold standard has yet emerged. Here, we present the first experiences with three ABO-incompatible adult living donor liver transplantations in Germany applying different immunosuppressive strategies. Four patient-donor couples were considered for ABO-incompatible ALDLT. In these patients, resident ABO blood group antibodies (isoagglutinins) were depleted by plasmapheresis or immunoadsorption and replenishment was inhibited by splenectomy and/or B-cell-targeted immunosuppression. Despite different treatments ALDLT could safely be performed in three patients and all patients had good initial graft function without signs for antibody-mediated rejection (AMR). Two patients had long-term graft survival with stable graft function. We thus propose the feasibility of ABO-incompatible ALDLT with these protocols and advocate further expansion of ABO incompatible ALDLT in multicenter trials to improve efficacy and safety.

Targeting of interleukin-10 is superior to cytotoxic T-lymphocyte associated antigen 4 with human immunoglobulin G(1) for the prevention of chronic allograft deterioration in organ transplantation.

BACKGROUND: Genetic manipulation of the allograft is an attractive approach to prevent the graft against chronic deterioration through stable expression of immunomodulatory or protective genes. However, the best strategy for prevention of chronic allograft deterioration remains unclear. METHODS: The efficacies of adeno-associated viral vector-mediated stable expression of indoleamine 2,3-dioxygenase (IDO), cytotoxic T-lymphocyte associated antigen 4 with human immunoglobulin G(1) (CTLA4Ig) or interleukin-10 (IL-10) in the prevention of chronic allograft deterioration were compared in a rat heart transplantation model. RESULTS: Transduction of grafts with IL-10 significantly prolonged allograft survival, whereas transduction of grafts with IDO did not improve graft survival compared to controls. Analysis of long-term survived heart allografts showed that both CTLA4Ig and IL-10 could significantly reduced the T cells and macrophage infiltration. However, stable expression of CTLA4Ig failed to prevent the development of transplant arteriosclerosis. By contrast, IL-10 suppressed the development of transplant arteriosclerosis effectively. The suppressive effects of IL-10 in preventing the development of chronic allograft deterioration were associated with lower transcript levels of transforming tumor growth factor beta 1 and macrophage migration inhibitory factor in the graft. In addition, higher transcript levels of heme oxygenase-1 were found in IL-10-transduced allograft. CONCLUSIONS: Targeting on IL-10 is superior to CTLA4Ig or IDO for the treatment of chronic allograft deterioration.

Dosing of rapamycin is critical to achieve an optimal antiangiogenic effect against cancer.

Rapamycin has antiangiogenic activity against tumors. This has been discussed while addressing the problem of cancer in organ transplantation. Here we investigated effective dosing schedules against tumors and angiogenesis. Growth of established CT-26 colon adenocarcinoma tumors was measured in Balb/c mice treated with total equivalent rapamycin doses (1.5 mg/kg/day) given once a day, once every 3 days, or by continuous infusion. Tumors were most inhibited with continuous rapamycin infusion, and less by bolus dosing. Interestingly, however, continuous dosing produced the lowest rapamycin blood levels (15 ng/ml). As rapamycin-sensitive p70S6-kinase intracellular signaling is critical for angiogenesis, p70S6-kinase activation was measured in endothelial cells by Western blotting. Maximal p70S6-kinase inhibition occurred from 1-5 ng/ml rapamycin. These same rapamycin concentrations optimally blocked vessel-sprouting from cultured aortic rings. Therefore, low-level rapamycin dosing most effectively controls tumors in mice. Importantly, antiangiogenic rapamycin levels are compatible with immunosuppressive doses, supporting its potential use in transplant patients with cancer.

Blockage of 2-deoxy-D-ribose-induced angiogenesis with rapamycin counteracts a thymidine phosphorylase-based escape mechanism available for colon cancer under 5-fluorouracil therapy.

PURPOSE: Colorectal neoplasms remain a leading cause of cancer-related deaths. A recognized weakness of conventional 5-fluorouracil (5-FU) therapy relates to expression of the intracellular enzyme, thymidine phosphorylase (TP). Although TP promotes 5-FU cytotoxicity, TP-derived 2-deoxy-D-ribose (dRib) counterproductively stimulates tumor angiogenesis. Here, the newly discovered antiangiogenic drug rapamycin was combined with 5-FU to counteract the potential escape mechanism of dRib-induced angiogenesis. EXPERIMENTAL DESIGN: Orthotopic tumor growth was assessed in rapamycin and 5-FU-treated BALB/c mice with TP-expressing CT-26 colon adenocarcinoma cells. To examine liver metastasis, green-fluorescent protein-transfected CT-26 cells were visualized by fluorescence microscopy after intraportal injection. Cell counting and Ki67 staining were used to determine in vitro and in vivo cell expansion, respectively. In vitro angiogenic effects of dRib were assessed with endothelial cell migration and aortic ring assays. Western blotting detected dRib effects on p70/S6 kinase activation. RESULTS: Rapamycin treatment of mice bearing orthotopic tumors inhibited tumor growth more than did 5-FU, and mice treated with both drugs typically developed no tumors. In the liver metastasis assay, combination therapy blocked metastatic expansion of solitary tumor cells. Interestingly, complex drug activities were suggested by tumor-cell proliferation being more sensitive to 5-FU than to rapamycin in vitro, but more sensitive to rapamycin in vivo. With regard to angiogenesis, dRib-induced endothelial cell migration and aortic ring formation were completely abrogated by rapamycin, correlating with blockage of dRib-induced p70/S6 kinase activation in endothelial cells. CONCLUSIONS: Inhibition of dRib-induced angiogenesis with rapamycin counteracts a potential TP-based escape mechanism for colorectal cancer under 5-FU therapy, introducing a novel, clinically feasible, combination treatment option for this common neoplasm.