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Extracellular nucleotides might influence aspects of the biology of reproduction in that ATP affects smooth muscle contraction, participates in steroidogenesis and spermatogenesis, and also regulates transepithelial transport, as in oviducts. Activation of cellular nucleotide purinergic receptors is influenced by four plasma membrane-bound members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family, namely NTPDase1, NTPDase2, NTPDase3, and NTPDase8 that differ in their ecto-enzymatic properties. The purpose of this study was to characterize the expression profile of the membrane-bound NTPDases in the murine female and male reproductive tracts by immunological techniques (immunolabelling, Western blotting) and by enzymatic assays, in situ and on tissue homogenates. Other than the expected expression on vascular endothelial and smooth muscle cells, NTPDase1 was also detected in Sertoli cells and interstitial macrophages in testes, in ovarian granulosa cells, and in apical cells from epididymal epithelium. NTPDase2 was largely expressed by cells in the connective tissue; NTPDase3 in secretory epithelia, and finally, NTPDase8 was not detected in any of the tissues studied here. In addition, NTPDase6 was putatively detected in Golgi-phase acrosome vesicles of round spermatids. This descriptive study suggests close regulation of extracellular nucleotide levels in the genital tract by NTPDases that may impact specific biological functions.

For patients with concomitant diabetes mellitus an increased perioperative mortality and morbidity in hepatic resections has repeatedly been described. Other studies, however, demonstrated equal outcome data in diabetic and non-diabetic patients. As patient populations were selected for underlying disease, conflicting results may reflect patient selection criteria rather than impact of diabetes mellitus on outcome measures. Therefore, a multivariate analysis in a largely unselected patient population has been performed to determine the independent prognostic value of diabetes mellitus in liver surgery. From a prospective database 633 adult patients undergoing hepatic resection without preceding major abdominal surgery or chemotherapy have been identified. Besides diabetes mellitus, demographic data, variables expressing the functional reserve of the liver, and parameters of surgical technique were analyzed for their impact on mortality and morbidity. 75 patients were diabetic (11.8 %) and 96 hepatic resections (15.2 %) were performed in cirrhotic patients. In the univariate analysis, concomitant diabetes was associated with an increased mortality compared to all non-diabetic patients (10.7 % vs. 5.3 %, p = 0.047). Diabetic patients, however, were also significantly older and presented a higher prevalence of liver cirrhosis. Multivariate modeling finally identified only age, albumin, cirrhosis, extent of surgery, and era of surgery as independent variables with an impact on perioperative mortality. Overall, complications were detected in diabetic and non-diabetic patients with a comparable frequency (44 % vs. 36 %, p = 0.179). Also, the length of in-hospital stay did not significantly differ between both groups (18.5 +/- 1.7 vs. 17.7 +/- 1.0 days, p = 0.119). Rates of postoperative renal impairment, prolonged ascites or pneumonia, however, were higher in diabetics than in other patients. Following established cardiopulmonary and surgical selection criteria, diabetes mellitus is not an independent risk-factor for perioperative mortality in hepatic resections. Although the overall postoperative morbidity was not different in diabetic and non-diabetic patients, a specific pattern of complications has been identified, mandating particular attention in the postoperative course of diabetic patients.

Increased prevalence of cardiovascular risk factors has been acknowledged in liver transplant recipients, and an increased incidence of cardiovascular events has been suspected. Individual risk determination, however, has not yet been established. Outpatient charts of 438 primary liver transplants have been reviewed, and suspected cardiovascular risk factors were correlated with cardiovascular events observed during a follow-up period of 10 yr. Receiver operation characteristics curve (ROC) analysis was performed to validate established cardiovascular risk scores. For calibration, the Hosmer-Lemeshow test was performed. A total of 303 of 438 patients were available for risk factor analysis at 6 months and demonstrated complete follow-up data (175 male, 128 female). A total of 40 of those 303 patients experienced fatal or nonfatal cardiovascular events (13.2%). In univariate analysis, age (P < 0.001), gender (P = 0.002), body mass index (P = 0.018), cholesterol (P = 0.044), creatinine (P = 0.006), diabetes mellitus (P = 0.017), glucose (0.006), and systolic blood pressure (P = 0.043), but not cyclosporine A (P = 0.743), tacrolimus (P = 0.870), or steroid medication (P = 0.991), were significantly associated with cardiovascular events. Multivariate analysis, however, identified only age, gender, and cholesterol as independent predictors. In ROC analysis, corresponding areas under the curve for Systematic Coronary Risk Evaluation Project (SCORE), Prospective Cardiovascular Münster Study (PROCAM), and Framingham risk scores (FRSs) were calculated with 0.800, 0.778, and 0.707, respectively. Calibration demonstrated an improved goodness of fit for PROCAM compared to SCORE risk calculations. In conclusion, SCORE and PROCAM proved to be valuable in discriminating our liver transplant recipients for their individual risk of cardiovascular events. Furthermore, calibrated PROCAM risk estimates are required to calculate the number of patients needed to treat in the setup of prospective intervention trials., (Copyright 2006 AASLD)

Biliary complications such as ischemic (type) biliary lesions frequently develop following liver transplantation, requiring costly medical and endoscopic treatment. If conservative approaches fail, re-transplantation is most often an inevitable sequel. Because of an increasing donor organ shortage and unfavorable outcomes in hepatic re-transplantation, efforts to prolong graft survival become of particular interest. From a series of 1685 liver transplants, we herein report on three patients who underwent partial hepatic graft resection for (ischemic type) biliary lesions. In all cases, left hepatectomy (Couinaud's segments II, III and IV) was performed without Pringle maneuver or mobilization of the right liver. All patients fully recovered postoperatively, but biliary leakage required surgical revision twice in one patient. At last follow-up, two patients presented alive and well. The other patient with persistent hepatic artery thrombosis (HAT), however, demonstrated progression of disease in the right liver remnant and required re-transplantation 13 months after hepatic graft resection. Including our own patients, review of the literature identified 24 adult patients who underwent hepatic graft resection. In conclusion, partial graft hepatectomy can be considered a safe and beneficial procedure in selected liver transplant recipients with anatomical limited biliary injury, thereby, preserving scarce donor organs.

Increased prevalence of cardiovascular risk-factors in liver transplant recipients compared with pretransplant and standard population data has been acknowledged. The impact of risk-profiles on cardiovascular event rates or death, however, has not yet been established. Here we evaluate the development of risk-factors during a prospective follow-up of 10 years in 302 patients and compare numbers of coronary events with data from the German Prospective Cardiovascular Münster (PROCAM)-Score population. Prevalence of overweight (17% vs. 27%), hypertension (70% vs. 80%), and diabetes (21% vs. 25%) increased from early to late after transplantation, while elevated serum cholesterol (64% vs. 37%) and triglycerides (40% vs. 21%) became less frequent. Cardiovascular risk-profiles favoring tacrolimus over ciclosporin A based immunosuppression early after transplantation converged over time. Increased risk-scores in liver transplant recipients matched with score standardized event rates in the PROCAM population (ratio: 1.11, 95% CI: 0.53-2.03), nine events were predicted for the transplant population and oppose 10 events observed. Thus, indicating a reflection of increased cardiovascular risk-profiles in corresponding numbers of cardiovascular events.

The aim of this study was to evaluate the success of steroid (PRED) withdrawal due to replacement by mycophenolate mofetil (MMF) in orthotopic liver transplant (OLT) recipients with autoimmune hepatitis (AIH). Thirty patients with AIH > 12 months after OLT randomized to receive either PRED and tacrolimus (TAC) or MMF and TAC were followed for 24 months. Withdrawal of steroids showed no difference regarding graft and patient survival. Also we demonstrated significantly lower glucose levels with lower HbA1c and a reduced need for insulin as well as a significantly lower serum cholesterol in the MMF group. Patients without steroids showed a lower incidence of osteopenia. Maintenance therapy in OLT patients with AIH may be performed safely using MMF instead of prednisone.

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.

Ectonucleoside triphosphate diphosphohydrolases (NTPDases) are membrane-bound ectoenzymes that hydrolyze extracellular nucleotides. We investigated the distribution of NTPDase1 and NTPDase2 in murine salivary gland and pancreas. Histochemistry and immunostaining (by both light and electron microscopy), combined with functional assays, were used to describe the localization patterns and enzyme activities in the organs of wild-type and NTPDase1/cd39-null mice. Pancreatic acinar cells and salivary gland acinar/myoepithelial cells were positive for NTPDase1 and NTPDase2. Ecto-ATPase activity was slightly higher in salivary glands. Ductal epithelial cells expressed ecto-ATPase activity but NTPDase1 and NTPDase2 expression were weak at best. ATPase activity was found in blood vessels of both tissues and its localization pattern overlapped with NTPDase1 staining. In these structures, NTPDase2 antibodies stained the basolateral aspect of endothelial cells and the supporting cells. Biochemical assays and histochemical staining showed relatively high levels of ATPase activity in both glands of cd39(-/-) mice. Our data therefore support a physiological role for NTPDase2 and other ectonucleotidases in the pancreas and salivary glands. Because NTPDase1 is expressed in non-vascular cell types, this finding suggests that NTPDase1 may have functions in the gastrointestinal tract that differ from those demonstrated in the vascular system.

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.

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.

Orthotopic liver retransplantation (re-OLT) is highly controversial. The objectives of this study were to determine the validity of a recently developed United Network for Organ Sharing (UNOS) multivariate model using an independent cohort of patients undergoing re-OLT outside the United States, to determine whether incorporation of other variables that were incomplete in the UNOS registry would provide additional prognostic information, to develop new models combining data sets from both cohorts, and to evaluate the validity of the model for end-stage liver disease (MELD) in patients undergoing re-OLT. Two hundred eighty-one adult patients undergoing re-OLT (between 1986 and 1999) at 6 foreign transplant centers comprised the validation cohort. We found good agreement between actual survival and predicted survival in the validation cohort; 1-year patient survival rates in the low-, intermediate-, and high-risk groups (as assigned by the original UNOS model) were 72%, 68%, and 36%, respectively (P <.0001). In the patients for whom the international normalized ratio (INR) of prothrombin time was available, MELD correlated with outcome following re-OLT; the median MELD scores for patients surviving at least 90 days compared with those dying within 90 days were 20.75 versus 25.9, respectively (P =.004). Utilizing both patient cohorts (n = 979), a new model, based on recipient age, total serum bilirubin, creatinine, and interval to re-OLT, was constructed (whole model chi(2) = 105, P <.0001). Using the c-statistic with 30-day, 90-day, 1-year, and 3-year mortality as the end points, the area under the receiver operating characteristic (ROC) curves for 4 different models were compared. In conclusion, prospective validation and use of these models as adjuncts to clinical decision making in the management of patients being considered for re-OLT are warranted.

Background: The number of registries is increasing, but few of them perform reliability audits by comparing the data contained in the database with data contained in hospital charts., Methods: The European Liver Transplant Registry (ELTR) cocoordinating committee appointed an independent team to check the reliability of data contained in ELTR. Centers were selected at random. Ten percent of each center's files were selected at random, and 25 items per file were checked during the site visits. The rates of completeness and inconsistencies and the agreement between ELTR and charts were established. We also assessed the correlation between the quality of data and the visited centers' activity., Results: Seven hundred thirty-four files from 21 centers have been audited between June 1998 and June 2001. The rate of ELTR completeness was 95%, and the rate of consistency between charts and ELTR was 98%. The agreement between the ELTR and charts review was very good for all conditions (kappa value < or =0.81). However, comparisons of rates between items indicated that specific items, mostly cause of death or graft failure and patient outcome, should be targeted for improvement. No significant correlation was found between the quality of data and the experience of visited centers. The mean (min-max) and median cost per audited file were EUR 60 (8-150) and EUR 44, respectively., Conclusion: The results of audit visits indicate that ELTR data are reliable, and the scientific results of ELTR can be considered credible and representative of liver transplantation in Europe. The method could serve as a model for auditing a registry.

Background: Human leukocyte antigen (HLA) compatibilities are beneficial in the setting of kidney transplantation but have demonstrated inconclusive results after liver transplantation. On the basis of recent controversial reports, the authors analyzed the impact of HLA matching in their patients after liver transplantation under modern immunosuppressive drug regimens and new HLA typing techniques with respect to outcome and adverse immunologic events., Methods: Data from 924 transplants with complete donor-recipient HLA typing were retrospectively analyzed. Immunosuppression was commenced as either cyclosporine A- or tacrolimus-based therapy in different protocols. The follow-up period ranged from 1 to 144.8 months (median, 66 months)., Results: The actuarial graft survival was 88% after 1 year and 78.7% after 5 years and was similar in tacrolimus- and cyclosporine A-treated patients. However, cyclosporine A-treated patients underwent significantly more rejection episodes. The number of HLA compatibilities had no influence on graft survival, whereas the number of acute rejections was significantly less in transplants with more HLA compatibilities (P<0.05). Graft survival tended to be improved in patients with chronic hepatitis B and more HLA compatibilities (P=0.05). In contrast, graft survival in transplants for primary sclerosing cholangitis was significantly impaired in the presence of one or two HLA-DR compatibilities (P<0.05). In addition, in autoimmune hepatitis, survival tended to be lower in the presence of more HLA compatibilities (P=0.1). Overall graft survival or frequency of adverse immunologic events was not influenced by any specific donor-recipient HLA allele., Conclusion: This study demonstrated fewer acute rejections in transplants with more HLA compatibilities. However, in liver transplantation, a more specific investigation of HLA typing may be necessary, because in some indications HLA antigens play a role in the nature of the disease. Therefore, recurrence of autoimmune disease may be more severe in patients sharing HLA antigens.

Nucleoside triphosphate diphosphohydrolases (NTPDases) are a recently described family of ectonucleotidases that differentially hydrolyze the gamma and beta phosphate residues of extracellular nucleotides. Expression of this enzymatic activity has the potential to influence nucleotide P2 receptor signaling within the vasculature. We and others have documented that NTPDase1 (CD39, 78 kd) hydrolyzes both triphosphonucleosides and diphosphonucleosides and thereby terminates platelet aggregation responses to adenosine diphosphate (ADP). In contrast, we now show that NTPDase2 (CD39L1, 75 kd), a preferential nucleoside triphosphatase, activates platelet aggregation by converting adenosine triphosphate (ATP) to ADP, the specific agonist of P2Y(1) and P2Y(12) receptors. We developed specific antibodies to murine NTPDase1 and NTPDase2 and observed that both enzymes are present in the cardiac vasculature; NTPDase1 is expressed by endothelium, endocardium, and to a lesser extent by vascular smooth muscle, while NTPDase2 is associated with the adventitia of muscularized vessels, microvascular pericytes, and other cell populations in the subendocardial space. Moreover, NTPDase2 represents a novel marker for microvascular pericytes. Differential expression of NTPDases in the vasculature suggests spatial regulation of nucleotide-mediated signaling. In this context, NTPDase1 should abrogate platelet aggregation and recruitment in intact vessels by the conversion of ADP to adenosine monophosphate, while NTPDase2 expression would promote platelet microthrombus formation at sites of extravasation following vessel injury. Our data suggest that specific NTPDases, in tandem with ecto-5'-nucleotidase, not only terminate P2 receptor activation and trigger adenosine receptors but may also allow preferential activation of specific subsets of P2 receptors sensitive to ADP (e.g., P2Y(1), P2Y(3), P2Y(12)) and uridine diphosphate (P2Y(6)).

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.

Background: Extracellular ATP 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 beta-galactosidase (Adbeta-gal) encoding genes were constructed. Hartley guinea pig coronary arteries were perfused ex vivo with University of Wisconsin solution containing 10(9) 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 Adbeta-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.

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 for S-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.

Background: There is increasing evidence showing that extracellular nucleosides [corrected] may be important mediators of vascular inflammation. Nucleoside [corrected] triphosphate diphosphohydrolase-1 (NTPDase-1, identical to CD39), the major vascular endothelial ectonucleotidase, is responsible for the hydrolysis of both extracellular ATP and ADP in the blood plasma to AMP. Studies were therefore conducted to evaluate the role of vascular NTPDase-1/cd39 in modulating platelet activation and vascular injury in cardiac xenografts., Materials and Methods: Cardiac xenografts from both wild-type and cd39 knockout mice (C57BL/6 x 129 Svj) were transplanted into Lewis rats. Alterations in cd39 mRNA transcripts and NTPDase activity expression were evaluated in wild-type grafts in untreated rats and then following complement depletion and immunosuppression. Rejection responses were studied with both mutant and wild-type grafts in the following models: presensitization with or without complement depletion, complement depletion alone, and with chronic immunosuppression to induce long-term graft survival., Results: NTPDase biochemical activity in wild-type xenografts rapidly decreased after transplantation but soon rebounded with graft survival. Elevated levels of cd39 mRNA with associated increases in NTPDase activity were observed in all long-term surviving wild-type grafts. Hyperacute xenograft rejection times were comparable in wild-type and mutant grafts but cd39-deficient grafts were subject to more rapid rejection and exhibited pronounced vascular injury in complement-depleted, presensitized rats. The cd39-deficient grafts in immunosuppressed recipients were subject to increased intravascular platelet sequestration and fibrin deposition; this resulted in focal myocardial infarction in long-term surviving mutant xenografts., Conclusions: Augmentation of NTPDase-1 activity may be an important adaptive response for graft survival. Our results suggest that NTPDase-1/cd39 influences pathways of vascular injury in cardiac xenografts.

Vascular ATP diphosphohydrolase/CD39 is an endothelial cell membrane protein with both ecto-ATPase and ecto-ADPase activities. Suppression of constitutive CD39 expression may result in elevated concentrations of ATP and ADP at the vascular interface that could predispose to thrombosis and inflammation. To study the effects of suppression of CD39 synthesis, stable 25-base antisense chimeric oligonucleotides targeting sequences at the 5' region of CD39 were designed. Transfection of these stable oligomers into cultured human endothelial cells resulted in dramatic decreases in levels of CD39 mRNA transcripts. Following transfection with antisense oligonucleotides, total ADPase activity fell from 26.0 +/- 3.1 in control cultures to 9.5 +/- 3.4 nmol of P(i) min(-1) (mg of protein)(-1) (p < 0.005); suppression of CD39 protein expression was also observed by Western blotting. Decreases in ATP diphosphohydrolase activity were associated with increases in concentrations of extracellular purine nucleotides released following stimulation of endothelial cells. Rates of initial hydrolysis of extracellular ATP released from purinergic agonist-stimulated endothelial cells decreased from 17.9 +/- 5.0 to 4.8 +/- 0.5 pmol min(-1) per 10(6) cells (p < 0.005) in antisense transfected cells. Therefore, CD39 regulates extracellular ATP concentrations and may be an important modulator of purinergic receptor activity in vascular endothelial cells.

The introduction of quadruple induction therapy after liver transplantation with the murine anti-interleukin-2 receptor (IL-2R) antibody (BT563) has decreased the incidence of serious side effects, such as tachycardia, hypertension, rash, fever and nausea since it does not lyse its target cell. To investigate the immunosuppressive efficacy of BT563, a placebo-controlled trial was performed and BT563 was added to the standard triple induction after liver transplantation. Forty consecutive recipients of primary orthotopic liver transplants (OLT) (median age 47 yr [range 18-65]) were randomized. All patients received triple immunosuppression with cyclosporine A (CyA), prednisolone (PRED) and azathioprine (AZA). In addition, 19 patients received BT563 (Biotest, Dreieich, Germany) at a dose of 10 mg/d from day 0 until day 12. The remaining 21 patients received a placebo infusion at the same days after transplantation. Minimal follow-up for all patients was 3 yr. Patient survival at 3 yr was 74% in the BT563 group and 90% in placebo group. Similar results were observed for graft survival. Two acute rejection episodes were detected in the BT563 group and 9 acute rejections (5 steroid-resistant) were observed in the placebo group (p < 0.034). The incidences of sepsis, pneumonia, cholangitis, urinary tract infections as well as cytomegalo-virus (CMV) infections were similar in both groups. Side effects of the BT563 therapy and/or post-transplant lymphoproliferative disease (PTLD) were not detected. Quadruple induction therapy with BT563 significantly reduces the incidence of rejection episodes after liver transplantation, while infectious complications and/or PTLD is not increased. Therefore, the anti-IL2 receptor antibody BT563 constitutes a safe and efficient addition to the immunosuppressive induction regimen following OLT.

Rationale and Objectives: The authors correlate computed tomography (CT) findings in biphasic spiral technique with histopathology in patients with hepatocellular carcinoma (HCC) who had undergone liver resection (LR) or orthotopic liver transplantation (OLT)., Methods: Preoperative biphasic spiral CT findings in 33 consecutive patients (23 men, 10 women, aged 43-74 years; LR group: n = 17; OLT group; n = 16) with liver cirrhosis and HCC were reviewed retrospectively by consensus of two radiologists and correlated with pathology from liver specimens., Results: Of the 16 patients in the OLT group with 1 to 5 confirmed HCC lesions (total lesions: 29; mean lesion diameter: 2 cm; range: 0.6-5.0 cm), CT before OLT depicted 22 lesions in 15 patients (sensitivity for lesions with a diameter of 0.5-1.0 cm, 20%; for lesions 1.1-2.0 cm, 82%; and for lesions 2.1-3.0 cm and > 3.0 cm, 86% and 100%, respectively). Among the 17 patients in the LR group (total lesions: 21; mean lesion diameter: 5.4 cm; range: 1.0-11.0 cm), CT detected 18 lesions. Lesion-by-lesion sensitivity, as correlated with pathology, was calculated at 76% and 86% in the OLT and LR groups, respectively (overall sensitivity, 80%). The diameter of CT detected lesions, compared with liver specimens, corresponded in 90% of lesions (maximum deviation, 15%). Characteristic CT findings of HCC included unenhanced hypoattenuating focal liver lesions (32 lesions), with hyperattenuation (38 lesions) in the arterial phase of contrast material administration., Conclusions: Biphasic spiral CT for preoperative HCC detection correlated with pathology in 80%, thus proving this technique to represent a sensitive imaging modality for pretherapeutic evaluation of HCC.

Background: Endothelial cells are known to be an early target of preservation/reperfusion injury and acute rejection, whereas the extracellular matrix (ECM) may also play an equally important role in the sequelae of both events., Methods: Syngeneic and allogeneic rat small bowel transplantations (SBTX) were performed after 6 hr of preservation. Animals were subsequently killed at defined time points for determination of ECM parameters within the graft and in plasma., Results: Laminin levels were significantly increased 20 min after reperfusion (syngeneic SBTX: 357+/-65.9 ng/ml; allogeneic SBTX: 361+/-79.6 ng/ml; P< or =0.01). After syngeneic transplantation, laminin levels normalized by postoperative day (POD) 7, whereas there was a rejection-induced increase after allogeneic SBTX (POD 7: 179+/-60.1 ng/ml; POD 9: 333+/-13.6 ng/ml; P< or =0.01 vs. syngeneic SBTX). This increase was accompanied by an increase in tumor necrosis factor-alpha levels at POD 9. Hyaluronic acid levels were significantly elevated after 24 hr (syngeneic SBTX: 1086+/-176 microg/L; allogeneic SBTX: 918+/-108 microg/L; P< or =0.01). After syngeneic SBTX, hyaluronic acid levels normalized by POD 7, whereas persistently higher levels were observed after allogeneic SBTX. Immunohistochemistry confirmed early changes (20 min after reperfusion) at the ECM. Anti-laminin and anti-CD44 staining normalized at POD 5 after syngeneic SBTX. After allogeneic SBTX, rejection-specific changes were evident with anti-laminin staining commencing on POD 5 and progressing until POD 9. At similar time points, increased expression of fibronectin- and interferon-gamma-positive material was evident., Conclusions: The ECM can be considered to be an early target of preservation/reperfusion injury and acute rejection. Plasma parameters reliably reflected the changes observed within the graft. Laminin and hyaluronic acid levels may be used as indicators of initial graft function. Furthermore, the increase in laminin levels was an early indicator of acute rejection. Determination of these parameters may significantly improve monitoring after SBTX.

Recurrence-free survival (RFS) in patients with small hepatocellular carcinoma (HCC) after orthotopic liver transplantation (OLT) was analyzed. From 1988 until 1996, 725 OLTs were performed in 669 patients. In 52 adults, HCC was confirmed histologically. OLT was limited to patients with small (< 5 cm) HCC with a maximum number of three nodules. Actuarial survival for these 52 patients at 1 and 5 years is 88% and 71%. RFS was defined as time until death without recurrence time until follow up with a diagnosis of recurrence, or, in patients without recurrence, time of last follow up. Overall, the 5-year RFS was 60%. Five-year RFS was less for bilobar compared to unilobar tumors (36% vs 70%), less for stage IVa tumors (UICC) compared to stage I-III tumors (17% vs 71%), and less for multiple compared to solitary tumors (54% vs 67%). In conclusion, potential cure may be achieved in more than 50% of all transplanted patients.

Although the surgical treatment of hilar cholangiocarcinoma represents the only potentially curative option, survival figures remain low over the long term. After hilar and partial hepatic resections for hilar cholangiocarcinoma, loco-regional tumor recurrence appears as the primary site of failure. From April 1992 to April 1996, 14 patients underwent extended bile duct resections. Extended bile duct resections combine total hepatectomy, partial pancreatoduodenectomy, and liver transplantation in an attempt to eradicate the entire biliary tract without dissecting the hepatoduodenal ligament. The postoperative 60-day mortality rate was 14% (n = 2). The rate of curative resections was 93% (13 of 14 extended bile duct resections). One- and 4-year survival rates after curative resections were 56% and 30%, respectively. The rate of curative resections increased by combining total hepatectomy, partial pancreatoduodenectomy, and liver transplantation, i.e., extended bile duct resection. However, survival figures have not improved accordingly. Therefore, this extended surgical procedure has to be implemented with caution and possibly not without modifications (e.g., multimodal treatment).

Background: Although conventional immunosuppression after liver transplantation consists of cyclosporine A (CsA), steroids, and azathioprine, recently introduced protocols entail CsA-based quadruple induction protocols or tacrolimus-based combinations. These protocols aim to reduce the rejection rate and the considerable morbidity related to the side effects of additional immunosuppressive treatment, but have not yet been analyzed regarding their long term de novo neoplastic risk., Methods: From September 1988 to May 1994, 500 liver transplantations were performed in 458 patients. The median follow-up was 50 months (range, 0.3-97 months) for all patients. Conventional triple therapy was implemented in 25 patients, CsA-based quadruple induction therapy using an antilymphocyte globulin preparation (ATG) in 190 patients, an interleukin-2 receptor antibody (BT563) in 141 patients, and tacrolimus-based dual or triple immunosuppression in 102 patients. The different protocols were evaluated in four randomized and two nonrandomized prospective trials., Results: De novo neoplasias were detected in 33 patients (7.2%) and were comprised of lymphomas (n = 7), skin malignancies (n = 8 lesions in 7 patients), intraepithelial neoplasias of the cervix uteri (n = 7), breast carcinoma (n = 3), lung carcinoma (n = 3), and other malignancies (n = 6). The incidence of de novo neoplasias did not differ in the different trial arms. Only a positive T-crossmatch and a low CD4+/CD8+ ratio in patients receiving CsA-based immunosuppression demonstrated a significant correlation with the development of a de novo tumor in a multivariant logistic regression analysis., Conclusions: The development of de novo neoplastic diseases after liver transplantation with the use of CsA-based quadruple induction protocols or tacrolimus-based regimens for immunosuppresion was assessed over the long term. Recently introduced immunosuppressive protocols did not alter the posttransplant de novo tumor rate. Patients with a low CD4+/CD8+ ratio during CsA-based therapy or a positive T-crossmatch were identified to be at an increased risk for the development of a de novo malignancy.

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.

In a retrospective study the records of 302 adult patients (167 male, 135 female) after orthotopic liver transplantation (OLT) with a minimum follow-up of 6 months (median follow-up 18 months, maximum 5 yr) were reviewed with regard to cardiovascular risk factors. In 197 patients data concerning the occurrence of arterial hypertension, hyperglycemia, or hypercholesterolemia prior to OLT were available. We found a highly significant increase of cardiovascular risk factors following OLT. Obesity was found in 17.4% of male and 22.2% of female recipients after OLT. Hypercholesterolemia was evident in 66.2% of liver graft recipients. Hypertriglyceridemia occurred in 49.7% of all male patients. In females there was a significantly different prevalence of hypertriglyceridemia comparing patients with a follow-up period up to 2 yr and more than 2 yr (50% vs. 24.6%, p = 0.018). Nearly 45% of all patients met the criteria for arterial hypertension, with a slight increase in male patients beyond the second year of survival (p = 0.094). Hyperglycemia had a significantly higher frequency in male than in female patients (30.5% vs. 10.4%, p < 0.005). Furthermore we observed a clear trend towards reduction of occurrence of hyperglycemia more than 24 months after OLT, but not reaching statistical significance. No correlation was detected when serum levels of triglycerides, and cholesterol, body-mass-index, and arterial blood pressure were compared with applied dosages of immunosuppressive agents [cyclosporin A (CyA), tacrolimus, and prednisolone]. Only decreasing tacrolimus application was significantly correlated with decreasing glucosemia (p = 0.041). Patients receiving tacrolimus instead of CyA as primary immunosuppressant showed a significantly lower prevalence of hypercholesterolemia. Even hypertension, hyperglycemia, hypertriglyceridemia, and obesity had a lower occurrence in patients treated with tacrolimus although not reaching statistical significance.