Olaf Guckelberger

Long-term outcome of liver transplants for chronic hepatitis C: a 10-year follow-up.

Background. Recurrence of hepatitis C (HCV) infection after orthotopic liver transplantation (OLT) in HCV-positive patients is almost universal. Severity of graft hepatitis increases during the long-term follow-up, and up to 30% of patients develop severe graft hepatitis and cirrhosis. However, there are still no clear predictors for severe recurrence. The aim of this study was to examine the 10-year outcome and risk factors for graft failure caused by HCV recurrence. Methods. In a prospective analysis, 234 OLTs in 209 HCV-positive patients with a median age of 53 years were analyzed. Immunosuppression was based on cyclosporine A or tacrolimus in different protocols. Predictors for outcome were genotype, viremia, donor variables, recipient demographics, postoperative immunosuppression, and human leukocyte antigen (HLA) compatibilities. Results. Actuarial 5-, and 10-year patient survival was 75.8% and 68.8%. Eighteen of 209 (8.7%) patients died because of HCV recurrence, which was responsible for 35.9% of the total 53 deaths. Significant risk factors for HCV-related graft failure in an univariate analysis were multiple steroid pulses, use of OKT3, and donor age greater than 40. However, in a multivariate analysis, multiple rejection treatments with steroids and OKT3 treatment proved to be significantly associated with HCV-related graft loss. Conclusions. The analysis of causes leading to graft failure in patients with HCV showed that HCV recurrence is responsible for one of three deaths in HCV-positive patients. Rejection treatment contributed significantly to an enhanced risk for HCV-related graft loss. New antiviral treatments, as well as adapted immunosuppressive protocols, will be necessary to further improve the outcome of HCV-positive patients after liver transplantation.

Assignment of ecto-nucleoside triphosphate diphosphohydrolase-1/cd39 expression to microglia and vasculature of the brain

Extracellular nucleotides are ubiquitous extracellular mediators that interact with and activate nucleotide type 2 (P2) receptors. These receptors initiate a wide variety of signalling pathways that appear important for functional associations between neurons and glial cells and for the regulation of blood flow, haemostatic and inflammatory reactions in the brain. Ectonucleotidases are extracellular nucleotide‐metabolizing enzymes that modulate P2 receptor‐mediated signalling by the regulated hydrolysis of these agonists. A considerable number of ectoenzyme species with partially overlapping substrate and tissue distributions have been described. Major candidates for expression in the brain are members of the ecto‐nucleoside triphosphate diphosphohydrolase (E‐NTPDase or CD39) family. The production of cd39–/–mice and specific reagents have enabled us to analyse the specific cellular distribution of NTPDase1 (CD39), the prototype member of the enzyme family, in the mouse brain. Using monospecific antibodies and enzyme histochemical staining, we have identified NTPDase1 as a major ectonucleotidase associated with both microglia and the endothelial and smooth muscle cells of the vasculature. NTPDase1 is not expressed by neurons and astrocytes. Additional unidentified ectonucleotidase functional activity is observed at lower levels throughout the brain parenchyma. NTPDase1 may regulate P2 receptor‐mediated functions of microglia as well as influence nucleotide signalling between neurons or astrocytes that are associated with multiple microglial ramifications. The expression of NTPDase1 by cerebrovascular endothelial and smooth muscle cells also suggests involvement in the regulation of blood flow and thrombogenesis.

Modulation of endothelial cell migration by extracellular nucleotides. Involvement of focal adhesion kinase and phosphatidylinositol 3-kinase - mediated pathways

Extracellular nucleotides bind to type-2 purinergic/pyrimidinergic (P2) receptors that mediate various responses, such as cell activation, proliferation and apoptosis, implicated in inflammatory processes. The role of P2 receptors and their associated signal transduction pathways in endothelial cell responses has not been fully investigated. Here, it is shown that stimulation of human umbilical vein endothelial cells (HUVEC) with extracellular ATP or UTP increased intracellular free calcium ion concentrations ([Ca(2+)](i)), induced phosphorylation of focal adhesion kinase (FAK), p130(cas) and paxillin, and caused cytoskeletal rearrangements with consequent cell migration. Furthermore, UTP increased migration of HUVEC in a phosphatidylinositol 3-kinase (PI3-K)-dependent manner. BAPTA or thapsigargin inhibited the extracellular nucleotide-induced increase in [Ca(2+)](i), a response crucial for both FAK phosphorylation and cell migration. Furthermore, long-term exposure of HUVEC to ATP and UTP, agonists of the G protein-coupled P2Y2 and P2Y4 receptor subtypes, caused upregulation of alpha(v) integrin expression, a cell adhesion molecule known to directly interact with P2Y2 receptors. Our results suggest that extracellular nucleotides modulate signaling pathways in HUVEC influencing cell functions, such as cytoskeletal changes, cellular adhesion and motility, typically associated with integrin-activation and the action of growth factors. We propose that P2Y2 and possibly P2Y4 receptors mediate those responses that are important in vascular inflammation, atherosclerosis and angiogenesis.

Palmitoylation Targets CD39/Endothelial ATP Diphosphohydrolase to Caveolae

Ectonucleotidases influence purinergic receptor function by the hydrolysis of extracellular nucleotides. CD39 is an integral membrane protein that is a prototype member of the nucleoside 5′-triphosphate diphosphohydrolase family. The native CD39 protein has two intracytoplasmic and two transmembrane domains. There is a large extracellular domain that undergoes extensive glycosylation and can be post-translationally modified by limited proteolysis. We have identified a potential thioester linkage site forS-acylation within the N-terminal region of CD39 and demonstrate that this region undergoes palmitoylation in a constitutive manner. The covalent lipid modification of this region of the protein appears to be important both in plasma membrane association and in targeting CD39 to caveolae. These specialized plasmalemmal domains are enriched in G protein-coupled receptors and appear to integrate cellular activation events. We suggest that palmitoylation could modulate the function of CD39 in regulating cellular signal transduction pathways.

Beneficial effects of CD39/ecto-nucleoside triphosphate diphosphohydrolase-1 in murine intestinal ischemia-reperfusion injury.

CD39 (ecto-nucleoside triphosphate diphosphohydrolase-1; E-NTPDase-1), is highly expressed on quiescent vascular endothelial cells and efficiently hydrolyzes extracellular ATP and ADP to AMP and ultimately adenosine. This action blocks extracellular nucleotide-dependent platelet aggregation and abrogates endothelial cell activation. However, CD39 enzymatic activity is rapidly lost following exposure to oxidant stress. Modulation of extracellular nucleotide levels may therefore play an important role in the pathogenesis of vascular injury. Acute ischemic injury of the bowel is a serious medical condition characterized by high mortality rates with limited therapeutic options. Here we evaluate the effects of cd39-deletion in mutant mice and the use of supplemental NTPDase or adenosine in influencing the outcomes of intestinal ischemia-reperfusion. Wild-type, cd39-null, or hemizygous cd39-deficient mice were subjected to intestinal ischemia. In selected animals, 0.2 U/g apyrase (soluble NTPDase) was administered prior to re-establishment of blood-flow. In parallel experiments adenosine/amrinone was infused over 60 min during reperfusion periods. Survival rates were determined, serum and tissue samples were taken. Intravital videomicroscopy and studies of vascular permeability were used to study platelet-endothelial cell interactions and determine capillary leakage. In wild-type animals, ischemia reperfusion injury resulted in 60% mortality within 48 hours. In mutant mice null or deficient for cd39, ischemia reperfusion-related death occurred in 80% of animals. Apyrase supplementation protected all wild-type animals from death due to intestinal ischemia but did not fully protect cd39-null and cd39-hemizygote mice. Adenosine/amrinone treatment failed to improve survival figures. In wild type mice, platelet adherence to postcapillary venules was significantly decreased and vascular integrity was well preserved following apyrase administration. In cd39-null mice, ischemia-reperfusion induced marked albumin leakage indicative of heightened vascular permaeability when compared to wild-type animals (p=0.04). Treatment with NTPDase or adenosine supplementation abrogated the increased vascular permeability in ischemic jejunal specimens of both wild-type mice and cd39-null. CD39 activity modulates platelet activation and vascular leak during intestinal ischemia reperfusion injury in vivo. The potential of NTPDases to maintain vascular integrity suggests potential pharmacological benefit of these agents in mesenteric ischemic injury.

Biliary reconstruction using a side-to-side choledochocholedochostomy with or without T-tube in deceased donor liver transplantation: a prospective randomized trial.

Objective:The biliary anastomosis is still one of the major causes for morbidity after orthotopic liver transplantation. The optimal method of reconstruction remains controversial. The aim of the study was to assess biliary complications after liver transplantation using a choledochocholedochostomy with or without a temporary T-tube. Background Data:Several reports have suggested that biliary reconstruction without T-tube is a safer method with a lower rate of biliary complications compared with T-tube insertion. Methods:A total of 194 recipients of deceased donor liver grafts were randomized. In group 1 the biliary reconstruction was performed by side-to-side choledochocholedochostomy with (n = 99) and in group 2 (n = 95) without a T-tube. The T-tube was removed after 6 weeks. Results:The overall biliary complication rate was significantly increased in group 2 (P < 0.0005). Biliary leaks occurred in 5 patients in group 1 and in 9 patients in group 2 (5.05% vs. 9.47%; P = 0.2756 ns). Anastomotic strictures of the bile duct were seen in 7 patients in group 1 and in 8 patients in group 2 (7.07% vs. 8.42%; P = 0.7923 ns). Two of the patients in group 1 and 5 patients in group 2 developed an ischemic type biliary lesion (2.02% vs. 5.26%; P = 0.2716 ns). The rate of reoperations was comparable in both groups. The rate of invasive interventions was higher in the group without T-tubes (9% vs. 18%, P = ns), as was the rate of cholangitis (5% vs. 11%. P = ns) and pancreatitis (4% vs. 14%, P = 0.0218). No complications after removal of the T-tube were observed. Conclusion:This study is a large prospective randomized trial to assess biliary complications that occur following liver transplantation, after anatomizing the bile duct with or without T-tubes. A significant increased rate of complications in the group without T-tube insertion was observed. In summary, our results indicate that the usage of T-tubes is safe and an excellent tool for the quality control of biliary anastomoses.

Twenty‐Year Longitudinal Follow‐Up After Orthotopic Liver Transplantation: A Single‐Center Experience of 313 Consecutive Cases

With excellent short‐term survival in liver transplantation (LT), we now focus on long‐term outcome and report the first European single‐center 20‐year survival data. Three hundred thirty‐seven LT were performed in 313 patients (09/88–12/92). Impact on long‐term outcome was studied and a comparison to life expectancy of matched normal population was performed. A detailed analysis of 20‐years follow‐up concerning overweight (HBMI), hypertension (HTN), diabetes (HGL), hyperlipidemia (HLIP) and moderately or severely impaired renal function (MIRF, SIRF) is presented. Patient and graft survival at 1, 10, 20 years were 88.4%, 72.7%, 52.5% and 83.7%, 64.7% and 46.6%, respectively. Excluding 1‐year mortality, survival in the elderly LT recipients was similar to normal population. Primary indication (p < 0.001), age (p < 0.001), gender (p = 0.017), impaired renal function at 6 months (p < 0.001) and retransplantation (p = 0.034) had significant impact on patient survival. Recurrent disease (21.3%), infection (20.6%) and de novo malignancy (19.9%) were the most common causes of death. Prevalence of HTN (57.3–85.2%, p < 0.001), MIRF (41.8–55.2%, p = 0.01) and HBMI (33.2–45%, p = 0.014) increased throughout follow‐up, while prevalence of HLIP (78.0–47.6%, p < 0.001) declined. LT has conquered many barriers to achieve these outstanding long‐term results. However, much work is needed to combat recurrent disease and side effects of immunosuppression (IS).

A prospective randomized trial comparing interleukin-2 receptor antibody versus antithymocyte globulin as part of a quadruple immunosuppressive induction therapy following orthotopic liver transplantation.

BACKGROUND Quadruple immunosuppressive induction therapy has been shown to markedly reduce the incidence of acute rejection episodes without increasing the incidence of infectious complications after liver transplantation. However, the use of polyclonal antibody preparations (e.g. antithymocyte globulin [ATG]) is associated with side effects such as fever and tachycardia. To evaluate the efficacy and the safety of a monoclonal antibody directed against the interleukin-2 receptor (BT563) in comparison with ATG as part of a quadruple induction regimen, a prospective, randomized study was conducted. METHODS Eighty consecutive adult recipients of primary orthotopic liver transplants were randomized to receive either BT563 (10 mg/day; days 0-12; n=39) or ATG (5 mg/kg/day; days 0-6; n=41) in addition to the standard immunosuppressive protocol consisting of cyclosporine, and prednisolone, and azathioprine. RESULTS Patients treated with BT563 had a significantly lower incidence of steroid-sensitive rejection episodes (3 vs. 11; P<0.025) and also significantly fewer drug-related side effects (4 vs. 18, P<0.038) when compared with patients treated with ATG. The incidence of infectious complications was not different between the two groups. Patient survival did not differ significantly between the two groups (84.6% at 1, 2, and 3 years in the BT563 group and 90.2% at 1 year and 87.8% at 2 and 3 years for the ATG group). Analysis of graft function showed an advantage for the BT563 group in terms of postoperative bilirubin levels. However, no differences were observed in long-term follow-up between the two groups. CONCLUSIONS Our results indicate that treatment with anti-interleukin-2 receptor antibody as part of quadruple induction therapy after orthotopic liver transplantation is safe and effective and shows fewer steroid-sensitive rejection episodes as well as fewer side effects when compared with quadruple induction therapy including ATG.

US-based Real-time Elastography for the Detection of Fibrotic Gut Tissue in Patients with Stricturing Crohn Disease

PURPOSE To assess whether ultrasonography (US)-based real-time elastography (RTE) can be used to detect gut fibrosis. MATERIALS AND METHODS In this institutional review board-approved, prospective, proof-of-concept study, unaffected and affected gut segments in 10 patients with Crohn disease (four women, six men; median age, 49 years) were examined pre-, intra-, and postoperatively with US, including RTE to assess strain. Disease activity was scored by using the Limberg index on the basis of (a) bowel wall thickness and (b) size and extent of Doppler signal. After surgical resection, strain of full gut wall segments was measured with direct tensiometry. Gut wall layers, fibrosis, and collagen content were quantified histologically. Aggregated data per patient, disease status, and available measurements were assessed with mixed-effects models. RESULTS Unaffected versus affected gut segments yielded higher RTE (mean ± standard deviation, 169.0 ± 27.9 vs 43.0 ± 25.9, respectively) and tensiometry (mean, 77.1 ± 21.4 vs 13.3 ± 11.2, respectively) values used to assess strain (both P < .001). There was good correlation between pre-, intra-, and postoperative RTE values of unaffected (intraclass correlation coefficient, 0.572) and affected (intraclass correlation coefficient, 0.830) segments. RTE was not associated with pre- or intraoperative Limberg scores (median, 1 vs 2; P = .255 and .382, respectively). Affected internal (median, 2011 vs 1363 μm; P = .011) and external (median, 929 vs 632 μm; P = .013) muscularis propria, serosa (median, 245 vs 64 μm; P = .019), and muscularis mucosae (median, 451 vs 80 μm; P = .031) were wider than unaffected segments. Width differences of internal muscularis propria and mucularis mucosae were associated with RTE-assessed strain (P = .044 and .012, respectively) and tensiometry-assessed strain (P = .006 and .014, respectively). Masson trichrome (median, 4 vs 0; P < .001) and elastica-van Gieson (median, 805 346 μm(2) vs 410 649 μm(2); P < .001) stains and western blotting (median, 2.01 vs 0.87; P = .009) demonstrated a higher collagen content in affected versus unaffected segments and were associated with RTE-assessed strain (both P < .001) and tensiometry-assessed strain (P < .001 and 0.025, respectively). CONCLUSION RTE can be used to detect fibrosis in human Crohn disease. Online supplemental material is available for this article.

Recombinant adenoviral mediated CD39 gene transfer prolongs cardiac xenograft survival.

Background. ExtracellularATP and ADP may be important mediators of vascular inflammation and thrombosis. Nucleoside triphosphate diphosphohydrolase (NTPDase or CD39) is a vascular ectoenzyme that hydrolyses ATP and ADP; however, this activity is lost during reperfusion injury. We show that the supplementation of NTPDase activity within xenograft vasculature using CD39 recombinant adenoviruses (AdCD39) has protective effects in vivo. Methods. Recombinant adenoviruses containing human CD39 or &bgr;-galactosidase (Ad&bgr;-gal) encoding genes were constructed. Hartley guinea pig coronary arteries were perfused ex vivo with University of Wisconsin solution containing 109 plaque-forming units of the recombinant adenovirus. Infected grafts were then implanted in the abdomen of complement depleted Lewis rats. Results. NTPDase activities decreased in all grafts within the first 24 hr and subsequently recovered only in those hearts infected with AdCD39. Immunohistological examination of AdCD39-infected grafts confirmed successful CD39 gene transfer into the endocardium and macrovasculature. Expression of CD39 modestly prolonged graft survival (90.2±5.4 hr, mean±SD, n=5) when compared with Ad&bgr;-gal-infected grafts (67.4±5.4 hr, P <0.005) and perfusion controls (66.4±5.2 hr;P <0.005). Conclusions. Recombinant adenoviral infection can induce expression of CD39 within cardiac xenografts and provide survival benefits in vivo. Our data show that ex vivo infection by recombinant adenovirus vectors can result in vascular expression of a potential therapeutic agent.