Extracorporeal liver support by recirculating albumin dialysis: analysing the effect of the first clinically used generation of the MARSystem.

UNLABELLED: Albumin dialysis with the MARSystem is used in many hospitals to support excretory hepatic function in acute or acute on chronic liver failure. Potential pathogenic albumin bound substances accumulated in excretory liver insufficiency can be removed from patients blood by dialysis against albumin solution. A specific membrane enables the selective transport of albumin bound metabolites to the albumin containing dialysate compartment, where the loaded transport albumin is cleared and regenerated at the same time by adsorption columns and a second dialyser. Between 1993 and 1995 different membranes, set-ups and components in albumin dialysis were tested and led finally to the recirculating MARSystem with a modified polysulphone based membrane (P3/5S Gambro, Hechingen) and two adsorption columns (N350 and BR 350, ASAHI Medical Ltd.), which showed the best performance at this time. This first generation of MARSystems was used clinically between 1995 and 1998 with only minor changes in 15 patients with acute (n = 1) or acute deterioration of chronic liver disease in our department until the improved next generation of MARSystems has been available (MARS set and monitor, Teraklin AG, Rostock, Germany). Changes in blood tests pre/post during 95 single MARS treatments and in clinical status over treatment period were evaluated retrospectively. RESULTS: A significant decrease of albumin bound substances (average reduction during single MARS treatments: bilirubin -18%, bile acids -43.7%) as well as of water soluble metabolites (creatinine -32%, urea -31%) was observed. During extracorporeal therapy also a significant drop in platelets (- 15.4%) and a prolongation of activated prothrombin time (- 21%) was documented, whereas haemoglobin, WBC, electrolytes as well as transaminases and albumin were not affected significantly. CONCLUSION: Albumin dialysis with the first generation of MARS enables the removal of albumin bound and water soluble toxins. Unwanted side-effects and changes in laboratory tests are comparable to conventional haemodialysis (drop of platelets and prolongation of coagulation tests). The elimination of albumin bound and water soluble substances was accompanied by an improvement of clinical status.

Extracellular signal regulated kinases are key mediators of mitogenic signals in rat pancreatic stellate cells.

BACKGROUND: Pancreatic stellate cells (PSCs) have been implicated in pancreatic fibrosis as they synthesise increased amounts of extracellular matrix proteins in response to activation by profibrogenic mediators such as cytokines. AIMS: The purpose of this study was to analyse cytokine receptor stimulated signalling pathways involved in PSC activation. Using a rat culture model of PSCs, we have also tested the potential of the platelet derived growth factor (PDGF) antagonist trapidil and PD98059, a specific inhibitor of extracellular signal regulated kinase (ERK) activation, to suppress PSC growth. METHODS: Cultured PSCs were stimulated with PDGF, and the signal transduction pathways activated in response to the mitogen were analysed by immunoblotting, kinase assays, and electrophoretic mobility shift assays. Furthermore, comparison of signalling cascades activated in PSCs before and after completing transdifferentiation to alpha-smooth muscle actin expressing myofibroblasts was performed. Biological effects of PDGF, trapidil, and PD98059 were analysed by proliferation assays and correlated with molecular effects of the substances. RESULTS: PDGF induced rapid activation of Raf-1, ERKs 1 and 2, as well as AP-1 proteins. The transforming growth factor beta activated transcription factor Smad2 was found to be constitutively phosphorylated in PSCs of different transdifferentiation grades. Furthermore, the results indicate a correlation between ERK activities and induction of PSC activation. Trapidil efficiently inhibited both PDGF induced ERK activation and, in common with PD98059, PSC proliferation. CONCLUSIONS: Our data suggest that ERKs play a key role in the regulation of PSC growth and that inhibition of the ERK signalling pathway may become a strategy to prevent activation of these cells.

[Pancreatic leakage after pancreas resection. An analysis of 345 operated patients]. / Pankreasleckage nach Pankreasresektion. Eine Analyse von 345 operierten Patienten.

INTRODUCTION: Complications after pancreatic resections remain frequent despite a decreasing mortality. Pancreatic leakages represent a relevant part of those complications but data on risk factors for their occurrence are rare. We analyzed our experience with incidence, clinical course, and risk factors of pancreatic leakage in a large patient group. METHODS: We analyzed the prospectively documented perioperative data of 345 patients with pancreatic resections carried out between 1994 and 2001. Main indications for surgery were chronic pancreatitis (57%) and malignant tumors (37%). The following operations were performed: Whipple's operation 15%, pylorus-preserving pancreaticoduodenectomy 53%, duodenum-preserving pancreatic head resection 19%, and distal pancreatic resection 13%. Risk factors were analyzed using uni- and multivariate methods. RESULTS: Postoperative mortality and complication rate were 2.9% and 41%, respectively. A pancreatic leakage occurred in 9.9%. In the majority of patients, pancreatic leakage was asymptomatic and controlled by prolonged drainage. However, one fourth of the patients with pancreatic leakage required reoperation. The mortality of pancreatic leakage was 12%. No patient with chronic pancreatitis died as a consequence of pancreatic leakage. Impaired preoperative renal function was the only risk factor for the occurrence of postoperative pancreatic leakage. CONCLUSIONS: Although easily managed in the majority of cases, pancreatic leakage still represents a relevant postoperative complication after pancreatic resection, especially in patients with malignant disease. Because of an increased risk of developing pancreatic leakage, an impaired renal function should be considered specifically in the perioperative management of the patients.

[Choledochal cysts in adult patients--presentation and clinical course]. / Choledochuszysten im Erwachsenenalter--klinische Erscheinungsformen und Verlauf.

HISTORY: A 35-year-old male was admitted with recurrent acute pancreatitis of unknown origin. He was found to have a cystic lesion in the upper abdomen believed to be a pancreatic pseudocyst (patient 1). A 59-year-old female with a choledochal cyst developed acute pancreatitis (patient 2). A 32-year-old male who had been operated upon for a choledochal cyst during childhood was admitted for upper abdominal pain, fever and vomitus (patient 3). INVESTIGATIONS AND DIAGNOSIS: Patient 1 was found to have a choledochal cyst type IV a according to TODANI. Patient 2 was diagnosed to have a choledochal cyst type Ib according to TODANI. The cyst was believed to contain a bile duct carcinoma. In patient 3, sonography showed an advanced Klatskin tumour with infiltration of the portal vein, the hepatic artery and the liver. Bile cytology confirmed the carcinoma. In all patients cholestasis was found. Pancreatic enzymes were elevated in patients 2 and 3. TREATMENT AND COURSE: Patient 1 underwent left hemihepatectomy and received a biliodigestive anastomosis. In patient 2 the choledochal cyst was resected while undergoing pylorus-preserving resection of the pancreatic head. The tumour suspected was confirmed and R0 resected. Patient 3 presented with an incurable bile duct carcinoma. He died 3 months later from multiple lung emboli. CONCLUSION: Choledochal cysts are associated with a 20 fold increase in the incidence of bile duct carcinoma.

Cytokine mRNA levels and lymphocyte infiltration in pancreatic tissue during experimental chronic pancreatitis induced by dibutyltin dichloride.

There is little information available regarding the role of inflammatory cells in the pathogenesis of chronic pancreatitis. Therefore, we analyzed the local cytokine profile and infiltrating lymphocytes in a rat model of chronic pancreatitis. Experimental pancreatitis was induced by a single intravenous application of dibultyltin dichloride (DBTC). During a time course of two months we observed the mRNA expression of cytokines using competitive RT-PCR. Lymphocytes were characterized by immunohistochemistry, FACS analysis, and the lymphocyte proliferation test. IL-1beta, IL-6, IL-5, and IL-10 were immediately up-regulated in the acute phase of disease, while lymphocyte-restricted expression of IL-2, IL-2R, and IFN-y was only found in the chronic course. Among the infiltrating lymphocytes, CD4+ cells dominated, but during the chronic process there was an increase of CD8+ cells, resulting in a reduced CD4/CD8 ratio. Mitogen-induced activation of isolated mesenteric lymph node cells increased during the chronic inflammation. Our results suggest that in experimental pancreatitis acute inflammatory reactions are followed by a T-lymphocyte-mediated process.

Immortalized bovine pancreatic duct cells become tumorigenic after transfection with mutant k-ras.

Mutation of the K-ras gene is thought to be an early and important event in pancreatic carcinogenesis. In order to study the role of this molecular alteration in the transition from the normal to the neoplastic pancreatic cell, bovine pancreatic duct cells were first immortalized by SV40 large T antigen (Ag) complementary (c)DNA transfection and then transfected with a mutated K-ras gene. As did primary duct cells, the immortalized duct cells (more than 100 passages) expressed cytokeratins, carbonic anhydrase type-II, cystic fibrosis transmembrane conductance regulator (CFTR), and multidrug resistance (mdr). They grew as a single layer after transplantation under plastic domes and formed three-dimensional structures resembling ducts when grown on Matrigel. Cell growth was stimulated by insulin, epidermal growth factor (EGF), transforming growth factor (TGF)-alpha, but cells did not respond to gastrin and CCK-8. They did not form colonies in soft agar nor did they form tumors in nude mice. Immortalized cells transfected with mutated K-ras acquired the ability to form tumors after orthotopic injection into the nude mouse pancreas. It is concluded that SV 40 immortalized bovine pancreatic

p53 and K-ras mutations in pancreatic juice samples from patients with chronic pancreatitis.

BACKGROUND: Mutations in p53 and ras genes are frequent in pancreatic carcinoma. Several ras mutations are consistently detected in the pancreatic juice from patients with chronic pancreatitis. The p53 gene mutations have been detected occasionally in chronic pancreatitis tissue. It was the aim of this study to evaluate the presence and clinical significance of p53 and ras mutations in clinical pancreatic juice samples from patients with chronic pancreatitis. METHODS: Pancreatic juice was obtained from 66 patients with chronic pancreatitis and no evidence of pancreatic carcinoma (51 men, 15 women; age 17-86 years [mean 49.6 +/- 12.9]). Patients were followed prospectively for 26 +/- 3 (4-54) months. Detection of p53 gene mutations was by temperature gradient gel electrophoresis (TGGE) and single strand conformation polymorphism (SSCP) for exons 5-8. Analysis of ras mutations was performed by SSCP/polymerase chain reaction, restriction fragment length polymorphism/polymerase chain reaction. All mutations were confirmed by sequencing. RESULTS: Five of 66 (7.5%) pancreatic juice samples contained p53 mutations, and ras mutations were detected in 6 cases (9%). Cytology was negative in all cases. No pancreatic carcinoma developed during follow-up and neither cancer cells nor preneoplastic lesions could be detected histologically in resected specimens. Although no correlation between p53 mutations and duration of pancreatitis or drinking habits was found, K-ras mutations correlated with both heavy smoking and severity of the disease. CONCLUSION: p53 and ras mutations can be detected in a minority of pancreatic juice samples from patients with chronic pancreatitis in the absence of malignancy.

Microencapsulated cell-mediated treatment of inoperable pancreatic carcinoma.

Pancreatic cancer can seldom be resected, and chemotherapy has only a limited effect on survival or tumour load. We did a phase I/II trial in 14 patients with pancreatic cancer to assess the safety of local activation of low-dose ifosfamide. We encapsulated genetically modified allogeneic cells, which expressed a cytochrome P450 enzyme, in cellulose sulphate and delivered them by supraselective angiography to the tumour vasculature. These cells locally activated systemically administered ifosfamide. The tumours of four patients regressed after treatment, and those of the other ten individuals who completed the study remained stable. Median survival was doubled in the treatment group by comparison with historic controls, and 1-year survival rate was three times better. Further studies of this cell-therapy-based treatment combined with chemotherapy for inoperable pancreatic cancer are warranted.

Low frequency of p53 and ras mutations in bile of patients with hepato-biliary disease: a prospective study in more than 100 patients.

The diagnosis of biliary disease, namely malignant disorders, is frequently hampered by the inconclusive cytology. We investigated prospectively the frequency of molecular changes in p53 and ras compared with cytology in patients with primary or secondary hepato-biliary disease. We investigated 118 consecutive patients, aged 24-89 with the following clinical diagnoses: choledocho/cholecystolithiasis (28), cholangiocellular carcinoma (21), gall bladder tumor (8), liver metastasis (3), autoimmune disease (8), chronic pancreatitis (16), pancreatic carcinoma (11), papillary disease (4), hepatic cirrhosis (6), cholangitis (2), anomalies (2), and normal (9). Bile was aspirated during routine endoscopic retrograde cholangio pancreatography (ERCP) or percutaneous transhepatic cholangiography (PTC). DNA was prepared freshly from a native aliquot. p53 mutations were detected by polymerase chain reaction (PCR) for exons 5 through 8 followed by TGGE. PCR for ras mutations was performed as RFLP-PCR with sequencing. In four cases, mutations in p53 could be found in exons 6 and 7. Twenty-two samples showed ras mutations; ras mutations were found in choledocholithiasis (4/28), bile duct (5/21), gall bladder (3/8) and pancreatic (1/11) carcinoma, liver metastasis (3/3), ulcerative colitis (2/3), PSC (1/2), and chronic pancreatitis (1/16). Cytology was clearly positive in seven cases, suspicious in three other, inconclusive in six, and negative in the rest. The molecular analysis resulted in a sensitivity of 33% and specificity of 87%, respectively, for the diagnosis of a malignant condition. PCR for p53 and ras mutations may aid the diagnosis of primary and secondary (metastatic) hepatobiliary disease if a malignant condition of the bile ducts and the liver is suspected and cytology is inconclusive or negative. However, the incidence of p53 and ras mutations in bile seems less frequent than in other malignant conditions of the gastrointestinal tract and the pancreas and lower than in tissue, leaving a poor sensitivity and specificity. Nevertheless, the presence of a p53 and/or ras mutation per se supports a clinical suspicion of malignancy, even when the conventional cytology is negative or inconclusive.

[Anisakiasis of the stomach--a case report from Germany]. / Anisakiasis des Magens--ein Fallbericht aus Deutschland.

Anisakiasis or "herring worm disease" is one of the most important parasitic diseases of the gastrointestinal tract in Japan. In 1988 Lorenz and Warzok published 8 cases of intestinal anisakiasis in Eastern Germany. In 1988 Spehn et al. reported a case of gastric anisakiasis in an AIDS patient. Here, we describe a case of gastric anisakiasis in Germany with an impressive serious clinical course. The symptoms--acute abdominal cramps, severe chest pain, diarrhoea, sub-febrile temperatures and leucocytosis--followed 4 h after consumption of raw herring, which was homemade pickled in vinegar. The conventional and the endoscopic ultrasonography showed a thickened gastric wall made of mainly thickened submucosa. The larvae of Anisakis in the gastric mucosa were found and extracted endoscopically. Acute and severe abdominal pain after eating raw fish is an indication for early gastroscopy. The endoscopical extraction of possible larvae is the only effective therapy, as anthelmintics against nematodes (mebendazole, albendazole, thiabendazole) are ineffective.

Liver support by extracorporeal blood purification: a clinical observation.

Liver failure associated with excretory insufficiency and jaundice results in an endogenous accumulation of toxins involved in the impairment of cardiovascular, kidney, and cerebral function. Moreover, these toxins have been shown to damage the liver itself by inducing hepatocellular apoptosis and necrosis, thus creating a vicious cycle of the disease. We report a retrospective cohort study of 26 patients with acute or chronic liver failure with intrahepatic cholestasis (bilirubin level > 20 mg/dL) who underwent a new extracorporeal blood purification treatment. A synthetic hydrophilic/hydrophobic domain-presenting semipermeable membrane (pore size < albumin size, 100-nm thick) was used for extracorporeal blood detoxification using dialysis equipment. The opposite side was rinsed with ligandin-like proteins as molecular adsorbents that were regenerated online using a chromatography-like recycling system (molecular adsorbent recirculating system [MARS]). Bile acid and bilirubin levels, representing the previously described toxins, were reduced by 16% to 53% and 10% to 90% of the initial concentration by a single treatment of 6 to 8 hours, respectively. Toxicity testing of patient plasma onto primary rat hepatocytes by live/dead fluorescence microscopy showed cell-damaging effects of jaundiced plasma that were not observed after treatment. Patients with a worsening of Child-Turcotte-Pugh (CTP) index before the treatments showed a significant improvement of this index during a period of 2 to 14 single treatments with an average of 14 days. After withdrawal of MARS treatment, this improvement was sustained in all long-term survivors. Ten patients represented a clinical status equivalent to the United Network for Organ Sharing (UNOS) status 2b (group A1), and all survived. Sixteen patients represented a clinical status equivalent to UNOS status 2a, and 7 of these patients survived (group A2), whereas 9 patients (group B) died. We conclude that in acute excretory failure caused by a chronic liver disease, this treatment provides a therapy option to remove toxins involved in multiorgan dysfunction secondary to liver failure.

Improvement of hepatorenal syndrome with extracorporeal albumin dialysis MARS: results of a prospective, randomized, controlled clinical trial.

In hepatorenal syndrome (HRS), renal insufficiency is often progressive, and the prognosis is extremely poor under standard medical therapy. The molecular adsorbent recirculating system (MARS) is a modified dialysis method using an albumin-containing dialysate that is recirculated and perfused online through charcoal and anion-exchanger columns. MARS enables the selective removal of albumin-bound substances. A prospective controlled trial was performed to determine the effect of MARS treatment on 30-day survival in patients with type I HRS at high risk (bilirubin level, > or =15 mg/dL) compared with standard treatment. Thirteen patients with cirrhosis with type I HRS were included from 1997 to 1999. All were Child's class C, with Child-Turcotte-Pugh scores of 12.4 +/- 1. 0, United Network for Organ Sharing status 2A, and total bilirubin values of 25.7 +/- 14.0 mg/dL. Eight patients were treated with the MARS method in addition to hemodiafiltration (HDF) and standard medical therapy, and 5 patients were in the control group (HDF and standard medical treatment alone). None of these patients underwent liver transplantation or received a transjugular intrahepatic portosystemic shunt or vasopressin analogues during the observation period. In the MARS group, 5.2 +/- 3.6 treatments (range, 1 to 10 treatments) were performed for 6 to 8 hours daily per patient. A significant decrease in bilirubin and creatinine levels (P <.01) and increase in serum sodium level and prothrombin activity (P <.01) were observed in the MARS group. Mortality rates were 100% in the control group at day 7 and 62.5% in the MARS group at day 7 and 75% at day 30, respectively (P <.01). We conclude that the removal of albumin-bound substances with the MARS method can contribute to the treatment of type I HRS.

The anti-proliferative agent jasplakinolide rearranges the actin cytoskeleton of plant cells.

In the present study, we have characterized the action of the natural cyclodepsipeptide jasplakinolide (JAS) on the cytoplasmic architecture, actin-based cytoplasmic motility, and the organization of the actin cytoskeleton in selected examples of green algae (Acetabularia, Pseudobryopsis and Nitella) and higher plant cells (Allium bulb scale cells and Sinapis root hairs). JAS was capable of influencing the actin cytoskeleton and inhibiting cytoplasmic streaming in a differential, cell type-specific manner. With the exception of Nitella, two consecutive responses were observed upon incubation with 2.5 microM JAS: In the first phase cytoplasmic streaming increased transiently alongside with minor modifications of the actin cytoskeleton in the form of adventitious actin spots and spikes appearing throughout the cell cortex in addition to the normal actin bundle system typical for each cell type. In the second phase, cytoplasmic streaming stopped and the actin cytoskeleton became heavily reorganized into shorter, straight, more and more randomly oriented bundle segments. JAS exerted severe long-term effects on the actin cytoskeleton when treatments exceeded 30min at a concentration of 2.5 microM. An in situ competition assay using equimolar concentrations of JAS and FITC-phalloidin suggested that JAS has a phalloidin-like action. Effects of JAS were significantly different from those of cytochalasin D with respect to the resulting degree of perturbance of cytoplasmic organization, the distribution of actin filaments and the speed of reversibility.

Does bile of patients with acute gallstone pancreatitis cause pancreatic inflammatory lesions? A study of the pancreatic toxicity of choledochal secretions collected at ERCP.

BACKGROUND: Bile flow into the pancreatic duct has been proposed as the cause of acute biliary pancreatitis. However, the pancreatic toxicity of choledochal bile from patients with acute gallstone pancreatitis has not been studied. METHODS: Bile was collected endoscopically from the common bile ducts of 21 patients with acute gallstone pancreatitis within 72 hours after the onset of disease. The bile samples were instilled into the pancreatic duct of rabbits, and light microscopic examination of the pancreas morphology was performed to assess the toxicity of human bile. Microbiologic quantitative analysis of the aerobic and anaerobic bacterial bile flora was performed. RESULTS: Bile of six patients with acute gallstone pancreatitis (29%) induced interstitial inflammation in the rabbit pancreas. Choledochal bile of these patients harbored more than 10(4) CFU/mL bacteria (Proteus vulgaris, n = 1; Klebsiella pneumoniae, n = 1; Escherichia coli, n = 2; enterococci, n = 2). After sterilization, the bile samples did not induce acute pancreatitis. In 15 patients (71%), bile did not cause acute pancreatitis in the rabbit pancreas. These choledochal secretions were sterile or contained less than 10(4) CFU/mL. CONCLUSIONS: Reflux of infected bile may be a potential cause of acute pancreatitis in the minority of patients with bacterobilia. In most patients with gallstone pancreatitis, bile is neither infected nor harmful to the pancreas, and its flow into the gland is unlikely to be the cause of inflammation.

Immortalized pancreatic duct cells in vitro and in vivo.

Although pancreatic adenocarcinoma has become one of the best characterized malignant diseases, severe diagnostic and therapeutic problems are still associated with this disease. The establishment of a molecular model of pancreatic carcinogenesis may provide tools that could result in earlier diagnosis of this disease and, in turn, improves prognosis. Since pancreatic adenocarcinoma seems to originate in epithelial cells in the pancreatic ducts, cultivation of native pancreatic duct epithelial cells (PDEC) is the initial step in the establishment of an in vitro model of pancreatic carcinogenesis. As these native cells survive only a short period in culture, the aim of this study was to establish a stable pancreatic duct cell line by immortalization with the SV40 large T antigen. Furthermore, initial steps in pancreatic carcinogenesis should possibly be imitated by additional transfections of mutated ki-ras and/or mutated p53 genes. By optimization of the isolation protocol and the culture medium, yield as well as proliferative activity of isolated PDEC was increased considerably. Transfection of SV40 large T antigen resulted in an increase in the proliferative lifetime of the isolated cells, but no real immortal phenotype was obtained. Moreover, one step in the transformation from the normal to the malignant phenotype was imitated successfully by additional transfection of mutated ki-ras.