Axel Doenecke

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.

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.

A cell penetrating heme oxygenase protein protects heart graft against ischemia/reperfusion injury

Ischemia/reperfusion (I/R) injury is an unavoidable barrier that significantly affects outcome of solid organ transplantation. Here, we establish a protein transduction system to extend graft preservation time and to prevent I/R injury in heart transplantation. We generated a recombinant heme oxygenase-1 (HO-1) protein containing a modified protein transduction domain (PTD). PTD could cross cover cell membrane and carry target molecule to parenchymal cells of cold-preserved heart grafts. The newly generated PTD-HO-1 protein localized mainly in subcellular membrane organelle and nucleus after delivery that significantly prolonged cold preservation of heart grafts. This effect was associated with significantly less endothelial cell activation, less neutrophil and macrophage infiltration in PTD-HO-1-transduced heart grafts after reperfusion as compared with controls. In addition, transduction of PTD-HO-1 protein to heart graft significantly suppressed the I/R injury-associated myocardiocyte apoptosis. The infarct areas of heart graft after I/R injury were significantly reduced after PTD-HO-1 protein treatment. We show here for the first time that PTD can maintain its biological activities during cold preservation. Transduction of cell penetrating HO-1 protein significantly prolongs the cold preservation time and protects the graft from the I/R injury. This approach represents a novel method for the improvement of the overall outcome of organ transplantation.

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.

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.

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.

“Rescue allocation offers” in liver transplantation: Is there any reason to reject “unwanted” organs?

Abstract 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.

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

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.

Glucose-6-phosphate dehydrogenase deficiency: a contraindication for living donor liver transplantation?

Living donor liver transplantation (LDLT) is a wellestablished treatment option for patients suffering from end-stage liver disease with acceptable outcome for the recipient and minimal risk for the donor, given that a suitable donor can be found [1]. But many potential living donors are excluded because of medical reasons, one of which is Glucose-6-phosphate dehydrogenase deficiency (G6PDD) [2]. Glucose-6-phosphate dehydrogenase (G6PD) is a ubiquitous enzyme that plays a key role in the metabolism of glucose and is essential for protecting red blood cells from oxidative stress [3]. G6PDD is the most common enzyme deficiency, and it has been estimated that more than 400 million people worldwide are deficient in this enzyme [4]. The highest frequencies are detected in Africa, Asia, the Mediterranean region, and in the Middle East; owing to recent migrations, however, the disorder is also found in North and South America and in northern European countries. The X-linked, hereditary genetic defect is caused by mutations in the G6PD gene, which results in protein variants with different levels of enzyme activity, that are associated with a broad range of biochemical and clinical phenotypes. The most common clinical manifestations are neonatal jaundice and acute haemolytic anaemia, which are triggered by oxidative stress [3,5]. Infection, oxidative drugs and ingestion of fava beans are probably the most typical causes of haemolysis in people with G6PDD [3]. Moreover, several clinical disorders, such as pneumonia, viral hepatitis, diabetes, myocardial infarction, and, most important from our point of view, surgical stress have been reported to precipitate haemolysis in individuals with G6PD defect [6–8]. The precise mechanism by which increased sensitivity to oxidative damage leads to haemolysis is not fully understood; furthermore, the exact sequence of events once an exogenous trigger factor is present is also unknown [3]. However, severe consequences of fulminant haemolysis with consecutive haemolytic anaemia associated with G6PDD, such as renal failure, disseminated intravascular coagulation, immunodeficiency and pancreatitis might occur and have been described previously. Therefore, the most important consideration for patients with G6PDD is the avoidance of oxidative stress [8], thus explaining the fact that so far, to our knowledge, there is no published experience in liver resection, particularly in the setting of partial liver donation for LDLT. Therefore, here we report for the first time, a case from a living-donor with G6PDD who donated the right liver lobe for his mother suffering from HCC in chronic hepatitis C cirrhosis. A 57-year-old female from the Middle East with suspected hepatocellular carcinoma (HCC) in cirrhosis because of chronic hepatitis C was referred to our department for assessment of possible LDLT. In her home country, donation after brain death was uncommon. For LDLT, the 25-year-old son of the patient was intended to act as the donor. It was known that the potential donor had G6PDD, but so far he never had shown any clinical manifestations of the enzyme deficiency. Complete blood count and liver function tests were within normal range. Further evaluation [1] including liver biopsy showed no other contraindications. As, of four potential donors he was the only one with matching blood group, it was decided to proceed with LDLT despite G6PDD by a multidisciplinary team. LDLT was performed as described elsewhere [1], but including the middle hepatic vein with the graft. Surgery and the postoperative course were uneventful for both, the donor and recipient. Liver function tests of the donor showed early postoperative increase of bilirubin to 8.43 mg/dl (unconjugated bilirubin, 4.75 mg/dl; conjugated bilirubin, 3.68 mg/dl), but returned to normal range within 1 week (Fig. 1). Although this increase in bilirubin is unusual in a patient after normal liver resection, we have observed a similar increase in serum bilirubin in almost all our donors for LDLT – a phenomenon not explained yet. Liver function tests of the recipient were also uneventful. Histopathology of the explant showed a HCC of 2 cm diameter in gross cirrhosis. Both recipient and donor were discharged on the 18th postoperative day in good health and liver function tests within normal range. Today, 3 years after the transplantation, they are in good health without signs of liver dysfunction or recurrence of HCC in the recipient.