Matthias Löhr

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Abstract: Recognition of Helicobacter pylori as an important factor in genesis of gastric adenocarcinoma lead to a large number of studies concerning potential role of Helicobacter spp. in the development of extragastric digestive malignancies. The serological studies indicated possible localizations in the digestive system being from interest in enlightening Helicobacter spp. carcinogenic potential. The PCR obtruded itself as a gold standard in proving existence of actual correlation. In this review, the authors have examined studies conducted in the last 10 years examining Helicobacter spp. correlation with extragastric digestive carcinogenesis. Studies have been observed in four groups referring to hepatic carcinoma, bile duct cancer, pancreatic cancer, and colon cancer. The results of these researches have shown that there is a strong correlation between Helicobacter spp. colonization and primary liver tumors as well as bile duct tumors, whereas conclusions made by authors examining pancreatic cancer are contradictory and demands further investigation. No correlation between Helicobacter spp. and colon cancer have been proven. The PCR subtype most widely used in studies included in this review was nested PCR, whereas genes targeted most frequently for amplification are 16S rDNA of Helicobacter spp. and UreA gene or cagA gene of H. pylori. During the last 10 years PCR has proven itself as a sovereign method for Helicobacter spp. diagnostic in extragastric organs in the digestive system. Knowledge and experiences obtained in this domain could be encouraging for researchers in analogous fields of interest.

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Abstract: To determine the normal function of the Coxsackievirus and Adenovirus Receptor (CAR), a protein found in tight junctions and other intercellular complexes, we constructed a mouse line in which the CAR gene could be disrupted at any chosen time point in a broad spectrum of cell types and tissues. All knockouts examined displayed a dilated intestinal tract and atrophy of the exocrine pancreas with appearance of tubular complexes characteristic of acinar-to-ductal metaplasia. The mice also exhibited a complete atrio-ventricular block and abnormal thymopoiesis. These results demonstrate that CAR exerts important functions in the physiology of several organs in vivo.

Abstract: Since the 1990s, autoimmune pancreatitis (AIP) has been increasingly recognized as a special type of chronic pancreatitis. Clinically, AIP patients commonly present with jaundice, weight loss and abdominal pain. Radiologically, there is diffuse or focal enlargement of the pancreas, often with narrowing of the pancreas and bile duct. Histologically, AIP is characterized by periductal infiltrates of lymphocytes and plasma cells, cellular fibrosis and obliterative phlebitis. Recently, two types of AIP were distinguished and called type 1 and type 2 AIP. They share the symptomatology and some histopathological features such as periductal lymphoplasmacytic infiltrate and storiform fibrosis, but differ in a particular duct change, called granulocytic epithelial lesion, which characterizes type 2 AIP. In addition, type 2 AIP usually has none or very few (<10 cells/ high power field (hpf)) IgG4-positive plasma cells. Clinically, type 1 AIP patients are roughly 10 years older than type 2 AIP patients, show a slight preponderance of men and reveal extrapancreatic manifestations of IgG4 associated systemic disease in approximately 50% of the cases. Type 2 AIP patients frequently show an association with inflammatory bowel disease and usually lack serological elevation of IgG4. The main differential diagnosis of AIP is pancreatic ductal adenocarcinoma. In many instances the diagnosis of AIP can be made by imaging together with serological markers. In difficult cases, particularly in type 2 AIP, the diagnosis has to be established by core needle biopsy. Both type 1 and type 2 AIP patients respond to steroid treatment, but a significant proportion of type 1 AIP patients suffer from recurrence of the disease. We describe the clinical and histological features of the two types of AIP as well as the treatment. In connection with pathogenesis and diagnosis, recent findings on immune complex deposition, autoantibodies in AIP and relevant patents are discussed.

Abstract: To achieve the goal of developing international consensus diagnostic criteria (ICDC) for autoimmune pancreatitis (AIP).

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Abstract: Nodal and Activin belong to the TGF-β superfamily and are important regulators of embryonic stem cell fate. Here we investigated whether Nodal and Activin regulate self-renewal of pancreatic cancer stem cells. Nodal and Activin were hardly detectable in more differentiated pancreatic cancer cells, while cancer stem cells and stroma-derived pancreatic stellate cells markedly overexpressed Nodal and Activin, but not TGF-β. Knockdown or pharmacological inhibition of the Nodal/Activin receptor Alk4/7 in cancer stem cells virtually abrogated their self-renewal capacity and in vivo tumorigenicity, and reversed the resistance of orthotopically engrafted cancer stem cells to gemcitabine. However, engrafted primary human pancreatic cancer tissue with a substantial stroma showed no response due to limited drug delivery. The addition of a stroma-targeting hedgehog pathway inhibitor enhanced delivery of the Nodal/Activin inhibitor and translated into long-term, progression-free survival. Therefore, inhibition of the Alk4/7 pathway, if combined with hedgehog pathway inhibition and gemcitabine, provides a therapeutic strategy for targeting cancer stem cells.

Abstract: BACKGROUND: Endoscopic retrograde cholangiopancreatography (ERCP) is widely used for young patients, but ERCP and endoscopic sphincterotomy in particular are reported to be associated with increased complication and mortality rates. This study aimed to calculate mortality and to identify risk factors for death within 90 days after ERCP for nonmalignant disease. METHODS: From the Swedish Hospital Discharge Registry, the authors identified all individuals in Stockholm County who had undergone in-patient ERCP during 1990-2003. Among these individuals, they excluded those recorded in the Swedish Cancer Registry as having a diagnosis of malignancy in the liver, pancreas, or bile ducts. Cases, defined as patients who had died within 90 days after the procedure, were identified by cross-linkage to the causes of death registry. Control subjects were randomly sampled from the same cohort. The medical records were studied to discern risk factors for death after ERCP. RESULTS: The mortality rate was 1.6%. Advanced age, severe comorbidity, high complexity of the procedure, and occurrence of a complication were associated with death within 90 days, whereas a previous cholecystectomy or the simultaneous performance of an endoscopic sphincterotomy reduced the risk. CONCLUSIONS: Old age and comorbidity are the main risk factors for death after ERCP, but a complex procedure or the occurrence of a complication also seems to increase short-term mortality. The performance of a sphincterotomy may reduce the risk of death, possibly by facilitating adequate drainage. A previous cholecystectomy also may decrease the risk of death after ERCP.

Abstract: BACKGROUND: Paclitaxel embedded in cationic liposomes (EndoTAG�-1; ET) is an innovative agent targeting tumor endothelial cells. This randomized controlled phase II trial evaluated the safety and efficacy of ET in combination with gemcitabine (GEM) in advanced pancreatic cancer (PDAC). PATIENTS AND METHODS: Chemotherapy-naive patients with locally advanced or metastatic disease were randomly assigned to receive weekly GEM 1000 mg/m(2) or GEM plus twice-weekly ET 11, 22 or 44 mg/m(2) for 7 weeks. After a safety run-in of 100 patients, a second cohort continued treatment. End points included overall survival (OS), progression-free survival (PFS), tumor response and safety. RESULTS: Two hundred and twelve patients were randomly allocated to the study and 200 were treated (80% metastatic, 20% locally advanced). Adverse events were manageable and reversible. Transient thrombocytopenia and infusion reactions with chills and pyrexia mostly grade 1 or 2 occurred in the ET groups. Disease control rate after the first treatment cycle was 43% with GEM and 60%, 65% and 52% in the GEM + ET cohorts. Median PFS reached 2.7 compared with 4.1, 4.6 and 4.4 months, respectively. Median OS was 6.8 compared with 8.1, 8.7 and 9.3 months, respectively. CONCLUSIONS: Treatment of advanced PDAC with GEM + ET was generally well tolerated. GEM + ET showed beneficial survival and efficacy. A randomized phase III trial should confirm this positive trend.

Abstract: The objective of this study was to clarify the clinical and pathophysiological characteristics of autoimmune pancreatitis (AIP) and its subtypes (lymphoplasmacytic sclerosing pancreatitis [LPSP] and idiopathic duct-centric pancreatitis [IDCP]) seen around the world.

Abstract: A sustained imbalance of pancreatic proteases and their inhibitors seems to be important for the development of chronic pancreatitis (CP). Mesotrypsin (PRSS3) can degrade intrapancreatic trypsin inhibitors that protect against CP. Genetic variants that cause higher mesotrypsin activity might increase the risk for CP.

Abstract: This study was designed to investigate the clinical performance of the Access GI Monitor (Beckman Coulter) on the UniCel DxI 800, a method for CA19-9 antigen determination, and to compare with CA19-9 assay on the AxSYM system (Abbott).

Abstract: The Swedish Registry for Gallstone Surgery and ERCP (GallRiks) is the first nationwide Web-based quality registry for gallstone surgery and ERCP in the world. In this article we report data from 11,074 ERCPs performed in 2007 and 2008.

Abstract: In spite of the increasing knowledge of the molecular pathology of pancreatic ductal adenocarcinoma (PDAC), treatment of this tumor still remains an unresolved problem. Thus, the identification of 'novel' genes involved in pancreatic tumor progression is essential for early diagnosis and new treatment regimens of PDAC. Ankyrin-B (ANK2) was identified as being overexpressed in PDAC in a previous study by our group. ANK2 overexpression has been described in several tumors; however, the function of ANK2 in pancreatic carcinoma has not been elucidated.

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Abstract: Gemcitabine is widely used as first-line chemotherapeutic drug in the treatment of pancreatic cancer. Our previous experimental chemotherapy studies have shown that treatment of human pancreatic carcinoma cells with 5-fluorouracil (5-FU) alters the cellular transporter expression profile and that modulation of the expression of multidrug resistance protein 5 (MRP5; ABCC5) influences the chemoresistance of these tumor cells. Here, we studied the influence of acute and chronic gemcitabine treatment on the expression of relevant uptake and export transporters in pancreatic carcinoma cells by reverse transcription-polymerase chain reaction (RT-PCR), quantitative RT-PCR, and immunoblot analyses. The specific role of MRP5 in cellular gemcitabine sensitivity was studied by cytotoxicity assays using MRP5-overexpressing and MRP5-silenced cells. Exposure to gemcitabine (12 nM for 3 days) did not alter the messenger RNA (mRNA) expression of MRP1, MRP3, MRP5, and equilibrative nucleoside transporter 1 (ENT1), whereas high dosages of the drug (20 microM for 1 hour) elicited up-regulation of these transporters in most cell lines studied. In cells with acquired gemcitabine resistance (up to 160 nM gemcitabine), the mRNA or protein expression of the gemcitabine transporters MRP5 and ENT1 was upregulated in several cell lines. Combined treatment with 5-FU and gemcitabine caused a 5- to 40-fold increase in MRP5 and ENT1 expressions. Cytotoxicity assays using either MRP5-overexpressing (HEK and PANC-1) or MRP5-silenced (PANC1/shMRP5) cells indicated that MRP5 contributes to gemcitabine resistance. Thus, our novel data not only on drug-induced alterations of transporter expression relevant for gemcitabine uptake and export but also on the link between gemcitabine sensitivity and MRP5 expression may lead to improved strategies of future chemotherapy regimens using gemcitabine in pancreatic carcinoma patients.

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Abstract: Confocal laser endomicroscopy (CLE) is rapidly emerging as a valuable tool for gastrointestinal endoscopic imaging. Fluorescent contrast agents are used to optimize imaging with CLE, and intravenous fluorescein is the most widely used contrast agent. Fluorescein is FDA-cleared for diagnostic angiography of the retina. For these indications, the safety profile of fluorescein has been well-documented; however, to date, fluorescein is not cleared for use with CLE.

Abstract: Helicobacter pylori has been suggested to be involved in pancreatic diseases, namely autoimmune pancreatitis and pancreatic carcinoma. We investigated the presence of conserved sequences of Helicobacter in pancreatic tissue and pancreatic juice from patients with chronic nonautoimmune and autoimmune pancreatitis as well as pancreatic ductal adenocarcinoma (PDAC).

Abstract: Background & Aims: Little is known about the etiology and antigenicity of autoimmune pancreatitis (AIP), a recently-identified pancreatic disease. Methods: We investigated gene and protein expression with microarray and proteomic techniques in human autoimmune pancreatitis to determine new target antigens and understand the pathomechanism of the disease. This was supplemented by serum analyses of patients with autoimmune and alcohol-induced chronic pancreatitis. This was supplemented by serum analyses of patients with autoimmune and alcohol-induced chronic pancreatitis. C57BL/6 mice infected with LP-BM5 murine leukemia virus served as an animal model for AIP. Results: Several pancreatic proteases (anionic trypsinogen, cationic trypsinogen, mesotrypsinogen, (pro)elastase 3B) and regenerating protein I showed distinct downregulation in AIP in comparison to alcohol-induced chronic pancreatitis while cathepsin B, calprotectin and several chemokines were upregulated. Immunohistochemistry demonstrated a reduction in the number of acinar cells identified by their staining for trypsin. In the pancreas of the AIP animals we found very similar protein patterns in the pancreas and a reduction of trypsinogen, chymotrypsinogen and (pro)elastase. Immunohistochemistry demonstrated a reduction in the number of acinar cells identified by their staining for trypsin. Western blot confirmed these findings. Concurring with these results, the patients displayed elevated titers of autoantibodies against trypsinogen. Conclusions: The protease deficiency with concomitant autoantibodies against key pancreatic antigens points to a selective immune process directed against pancreatic acini. They may be used in further diagnostic studies.

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Abstract: BACKGROUND: Pancreatic enzyme preparations are a life-saving substitution for a pivotal physiological function of the entire organism that is impaired in chronic pancreatitis, cystic fibrosis and other diseases with exocrine pancreatic insufficiency. Pancreatic enzyme preparations, generically called pancreatin, are not alike. Rather, they present a broad variety of pancreatin composition. AIM: The properties of a set of commercially available pancreatin preparations were investigated in light of the physiological tasks such enzymes must fulfill during the normal digestive process. METHODS: Measurements of size, surface, acid resistance, release of enzymes, pharmacokinetics and batch consistency were undertaken. RESULTS: Although all pancreatin preparations contain the declared lipase units and are acid-stable, a wide variation was observed in the particle size (pyloric passage), specific surface area and release kinetics of lipase activity at pH 6 (duodenum). CONCLUSION: At present, available pancreatin preparations vary widely with respect to investigated parameters, which may have consequences for facilitating optimal digestion.

Abstract: Background: Confocal laser endomicroscopy (CLE) is rapidly emerging as a valuable tool for gastrointestinal endoscopic imaging. Fluorescent contrast agents are used to optimize imaging with CLE, and intravenous fluorescein is the most widely used contrast agent. Fluorescein is FDA-cleared for diagnostic angiography of the retina. For these indications, the safety profile of fluorescein has been well-documented; however, to date, fluorescein is not cleared for use with CLE. Aims: To estimate the rate of serious and total adverse events attributable to intravenous fluorescein when used for gastrointestinal CLE. Methods: We performed a cross sectional survey of 16 International Academic Medical Centers with active research protocols in CLE that involved intravenous fluorescein. Centers using I.V. fluorescein for CLE who were actively monitored for adverse events were included. Results: Sixteen centers performed 2272 gastrointestinal CLE procedures. The most common dose of contrast agent was 2.5-5ml of 10% sodium fluorescein. No serious adverse events were reported. Mild adverse events occurred in 1.4% of individuals, including nausea/vomiting, transient hypotension without shock, injection site erythema, diffuse rash, and mild epigastric pain. The limitation is that only immediate post procedure events were actively monitored. Conclusions: Use of intravenous fluorescein for gastrointestinal CLE appears to be safe with few acute complications.

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Abstract: Nerve growth factor (NGF), a survival factor for neurons enforces pain by sensitizing nociceptors. Also in the pancreas, NGF was associated with pain and it can stimulate the proliferation of pancreatic cancer cells. Hepatic stellate cells (HSC) respond to NGF with apoptosis. Transforming growth factor (TGF)-beta, one of the strongest pro-fibrogenic activators of pancreatic stellate cells (PSC) induced NGF and its two receptors in an immortalized human cell line (ihPSC) and primary rat PSC (prPSC) as determined by RT-PCR, western blot, and immunofluorescence. In contrast to HSC, PSC expressed both NGF receptors, although p75(NTR) expression was weak in prPSC. In contrast to ihPSC TGF-beta activated both Smad signaling cascades in prPSC. NGF secretion was diminished by the activin-like kinase (ALK)-5 inhibitor SB431542, indicating the predominant role of ALK5 in activating the NGF system in PSC. While NGF did not affect proliferation or survival of PSC it induced expression of Inhibitor of Differentiation-1. We conclude that under conditions of upregulated TGF-beta, like fibrosis, NGF levels will also increase in PSC which might contribute to pancreatic wound healing responses.

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Abstract: We investigated the therapeutic efficiency of sulfonate-modified polyvinyl alcohol beads loaded with doxorubicin, irinotecan or mitoxantrone in vitro and in vivo in a model of experimental peritoneal carcinomatosis (PC). In vitro, cell proliferation was efficiently impaired by doxorubicin drug eluting bead (DEB) treatment while mitoxantrone DEBs were less effective than. Irinotecan showed little effect for both DEBs and free drug. Apoptosis was not different between free mitoxantrone and the DEB form while more apoptosis induction was observed in cells incubated with free doxorubicin and irinotecan. Experimental PC was produced in mice. The therapeutic efficiency of either mitoxantrone and doxorubicin DEB or free drugs were compared. Mice were treated either once on day 12 or by 3 repetitive applications on days 7, 10 and 12. Mice treated by DEBs showed less weight loss and mortality. Therapeutic effect was determined by measuring tumor volume and tumor load on the day 15 after tumor inoculation. For the single application on the day 12, an advantage could be observed for the free drugs. After 3 repeated injections of both free and mitoxantrone DEB no difference in tumor load or tumor volume could be observed. Least tumor load and tumor volume was observed in mice that received 3 repeated injections of doxorubicin DEB. No animal survived 3 injections of free doxorubicin. We conclude that bead encapsulation of chemotherapeutic drugs may show the advantage of less toxicity in peritoneal spread of colorectal cancer.

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Abstract: BACKGROUND: Reduction in angiotensin-converting enzyme (ACE) activity has been shown to attenuate pancreatic stellate cell activation and pancreatic fibrosis and suggested as a potential treatment for chronic pancreatitis. The ACE gene insertion/deletion (I/D) polymorphism in intron 16 accounts for nearly half the variation in serum ACE levels. This study determined the frequency of the I/D polymorphism in patients with acute and chronic pancreatitis. METHODS: In total, 887 patients (346 with alcoholic, 443 with nonalcoholic, and 98 with acute pancreatitis) were enrolled, and were compared with 1294 healthy controls. Genotyping of the I/D polymorphism was performed by PCR or melting curve analyses. RESULTS: No significant differences were found in the prevalence of the ACE-deletion genotype frequencies when patients with alcoholic (27.5%), nonalcoholic (26.4%), and acute pancreatitis (32.7%) were compared with controls (26.9%). Likewise, allele frequencies of the ACE deletion polymorphism were not significantly different in patients with alcoholic (53.8%), nonalcoholic (50.6%), and acute pancreatitis (54.1%) and controls (52.7%). CONCLUSION: The I/D polymorphism of the ACE gene was not found to be associated with acute and chronic pancreatitis.

Abstract: BACKGROUND: Pancreatic cancer is characterized by high resistance to chemotherapy. Such chemoresistance can be mediated by multidrug resistance proteins (MRPs), breast cancer resistance protein (BCRP), and MDR1 P-glycoprotein. However, the contribution of individual MRP isoforms to chemoresistance in pancreatic carcinoma is unclear. We studied ATP-binding cassette (ABC) transporter expression in human pancreatic carcinoma cell lines as compared to primary pancreatic duct cells, and analyzed the MRP expression profile in 5-fluorouracil-resistant cells. METHODS: Transporter expression was analyzed by quantitative and qualitative RT-PCR, by immunoblot, and chemoresistance by cytotoxicity assay. RESULTS: Primary pancreatic duct cells expressed MRP1, MRP3, MRP4, and MRP5, but not MRP2 mRNA. The established carcinoma cell lines expressed MRP1, MRP4, and MRP5, most of them also MRP2, MRP3, MRP7, and BCRP, but none contained detectable amounts of MRP6, MRP8, or MRP9 mRNA. Immunoblot analyses demonstrated presence of MRP1, MRP4, and MRP5 protein in all, but MRP3 and BCRP protein only in some of these cells. Compared to parental Capan-1 cells, Capan-1 cells with acquired chemoresistance towards 5-fluorouracil showed an upregulated mRNA and protein expression of MRP3, MRP4, and MRP5. In addition, silencing of MRP5 by RNA interference resulted in enhanced sensitivity of parental Capan-1 cells towards 5-fluorouracil cytotoxicity. CONCLUSION: MRP3, MRP4, and MRP5 are upregulated in 5-fluorouracil-resistant cells, and MRP5 contributes to 5-FU resistance in pancreatic carcinoma cells.

Abstract: OBJECTIVES: Chronic pancreatitis (CP) and pancreatic adenocarcinoma (pCA) are associated with risk factors such as alcohol intake and tobacco smoking. Microsomal epoxide hydrolase (EPHX1) is a phase II detoxifying enzyme capable of tobacco-borne toxicant inactivation. We studied the role of the EPHX1 c.337T>C (p.Y113H) variant, whichleads to altered enzyme activity, in pancreatic diseases. METHODS: We genotyped 2391 patients by melting curve analysis. We enrolled 367 patients with pCA, 341 patients with alcoholic CP (aCP), 431 patients with idiopathic CP or hereditary pancreatitis, 192 patients with acute pancreatitis, and 679 controls of German descent. We replicated data in 77 patients with aCP and 304 controls from The Netherlands. RESULTS: In German patients with aCP, Y113 was more common than in controls (allele frequencies, 0.73 vs 0.68; risk ratio, 1.21 [95% confidence interval, 1.05-1.39]). However, we could not confirm this association in the Dutch population (allele frequencies, 0.62 vs 0.68, P=not significant). In total, Y113 frequency was 0.71 in aCP and 0.68 in controls (P = not significant). Allele frequencies did not differ in the other disease groups (acute pancreatitis, 0.69; idiopathic CP or hereditary pancreatitis, 0.68; pCA, 0.68; and control, 0.68). CONCLUSIONS: The EPHX1 Y113H variant is not associated with pancreatic diseases indicating that EPHX1 does not play a significant role in the initiation of pancreatic inflammation or cancer.

Abstract: Autoimmune pancreatitis has been established as a special entity of pancreatitis. It is an enigmatic disease since it is adding an autoimmune etiology to the existing causes of pancreatitis. Morphological hallmarks of the disease are narrowing of the pancreatic duct system and the bile duct by periductal lymphoplasmocytic inflammation. This results in many cases in obstructive jaundice due to a mass-forming lesion in the pancreatic head mimicking pancreatic ductal adenocarcinoma. Therefore, patients will frequently undergo surgery. Histopathologically, the disease can be diagnosed by IgG4-positive plasma cells. Serologically, patients may present with elevated serum IgG and IgG4 levels. Other autoantibodies are also described. Association with other autoimmune manifestations in a wide range of organs is frequent. Autoimmune pancreatitis will respond to steroid treatment, which is of specific importance because pancreatic cancer is one of its clinical differential diagnoses. It is important to positively diagnose autoimmune pancreatitis, especially if the bile ducts are affected, since cholangitis may be or become a prominent problem before or after surgery.

Abstract: Chronic pancreatitis is a persistent inflammatory disease of the pancreas, in which the digestive protease trypsin has a fundamental pathogenetic role. Here we have analyzed the gene encoding the trypsin-degrading enzyme chymotrypsin C (CTRC) in German subjects with idiopathic or hereditary chronic pancreatitis. Two alterations in this gene, p.R254W and p.K247_R254del, were significantly overrepresented in the pancreatitis group, being present in 30 of 901 (3.3%) affected individuals but only 21 of 2,804 (0.7%) controls (odds ratio (OR) = 4.6; confidence interval (CI) = 2.6-8.0; P = 1.3 x 10(-7)). A replication study identified these two variants in 10 of 348 (2.9%) individuals with alcoholic chronic pancreatitis but only 3 of 432 (0.7%) subjects with alcoholic liver disease (OR = 4.2; CI = 1.2-15.5; P = 0.02). CTRC variants were also found in 10 of 71 (14.1%) Indian subjects with tropical pancreatitis but only 1 of 84 (1.2%) healthy controls (OR = 13.6; CI = 1.7-109.2; P = 0.0028). Functional analysis of the CTRC variants showed impaired activity and/or reduced secretion. The results indicate that loss-of-function alterations in CTRC predispose to pancreatitis by diminishing its protective trypsin-degrading activity.

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Abstract: Background: Despite a recognized association between venous thromboembolic events (VTE) and cancer, little is known about the strength and the features of this association. We performed a meta-analysis in order to clarify this issue. Methods: We retrieved data from 40 reports published between 1982 and 2007: 12 contained cancer risk estimates for patients with either idiopathic or secondary VTE versus subjects without VTE and 17 for patients with idiopathic versus secondary VTE. We also pooled risk estimates from 4 cohort studies to assess the association between VTE and specific forms of cancer and conducted a proportional incidence study, based on the remaining 28 reports which did not provided risk estimates. Results: The pooled relative risk of cancer was 3.2 (95%CI 2.4-4.5) for patients with any form of VTE versus no VTE, 2.7 (95%CI 1.9-3.9) for patients with idiopathic versus no VTE and 3.8 (95%CI 2.6-5.4) for patients with idiopathic versus secondary VTE. In the pooled cohort studies, relative risks for VTE vs. no VTE were significantly elevated for cancer of the ovary (RR 7.0), pancreas (RR 6.1), liver (RR 5.6), blood (4.2), brain (RR 3.8), kidney (RR 3.4), lung (3.1), colon (2.9) and esophagus (2.1). In the proportional incidence study cancer of the pancreas, colon and blood were significantly more frequently observed than in the general population. Conclusions: Overall we found a three-fold excess risk of occult cancer in patients with VTE. The risk varies according to tumor site and is highest for cancer of the ovary, pancreas and liver.

Abstract: AIM: To determine the effect of Prepacol, a combination of sodium phosphate and bisacodyl, on transit and quality of capsule endoscopy (CE). METHODS: Fivety two consecutive patients were included in this prospective study. CE was performed following a 12 h fasting period. Twenty six patients were randomized for additional preparation with Prepacol. The quality of CE was assessed separately for the proximal and the distal small bowel by 3 experienced endoscopists on the basis of a graduation which was initially developed with 20 previous CE. RESULTS: Preparation with Prepacol accelerated small bowel transit time (262 +/- 55 min vs 287 +/- 97 min), but had no effect on the quality of CE. Visibility was significantly reduced in the distal compared to the proximal small bowel. CONCLUSION: The significantly reduced visibility of CE in the distal small bowel allocates the need for a good preparation. Since Prepacol has no beneficial effect on CE the modality of preparation and the ideal time of application remains unclear. Further standardized examinations are necessary to identify sufficient preparation procedures and to determine the impact of the volume of the preparation solution.

Abstract: Pancreatic ductal adenocarcinoma is characterized by an abundant desmoplastic stroma. Interactions between cancer and stromal cells play a critical role in tumour invasion, metastasis and chemoresistance. Therefore, we hypothesised that gene expression profile of the stromal components of pancreatic carcinoma is different from chronic pancreatitis and reflects the interaction with the tumour. We investigated the gene expression of eleven stromal tissues from pancreatic ductal adenocarcinoma, nine from chronic pancreatitis and cell lines of stromal origin using the Affymetrix U133 GeneChip set. The tissue samples were microdissected, the RNA was extracted, amplified, and labelled using a repetitive in vitro transcription protocol. Differentially expressed genes were identified and validated using quantitative RT-PCR and immunohistochemistry. We found 255 genes to be over-expressed and 61 genes to be under-expressed within the stroma of pancreatic carcinoma compared to the stroma of chronic pancreatitis. Analysis of the involved signal transduction pathways revealed a number of genes associated with the Wnt pathway of which the differential expression of SFRP1 and WNT5a was confirmed using immunohistochemistry. Moreover, we could demonstrate that WNT5a expression was induced in fibroblasts during co-cultivation with a pancreatic carcinoma cell line. The identified differences in the expression profile of stroma cells derived from tumour compared to cells of inflammatory origin suggest a specific response of the tissue surrounding malignant cells. The over-expression of WNT5a, a gene involved in the non canonical Wnt signalling and chondrocyte development might contribute to the strong desmoplastic reaction seen in pancreatic cancer.

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Abstract: BACKGROUND: Several classification systems of chronic pancreatitis have been proposed to provide a basis for treatment and research. All of these previous classifications were designed at the height of pancreatic research of their respective times; thus, each represented the most current knowledge available to pancreatologists at the time. However, none of these classifications provide simultaneously a simple standardized system for the clinical classification of chronic pancreatitis according to etiology, clinical stage, and severity of the disease, nor are they consistently useful for directing clinical practice and comparing interinstitutional data. Thus, we aimed to develop a new classification system of chronic pancreatitis to provide a framework for studying the interaction of various risk factors on the course of the disease. METHODS: We reviewed the literature on the clinical course of all different forms of chronic pancreatitis, and we reviewed all previous classification systems of the disease. This approach provided a basis for the development of a new and unifying classification of chronic pancreatitis. RESULTS: We established the M-ANNHEIM multiple risk factor classification system based on the current knowledge of acute and chronic pancreatitis. This classification allows patients to be categorized according to the etiology, clinical stage, and severity of their disease. The severity of pancreatic inflammation was assessed using a scoring system that takes into account the clinical symptoms and treatment options of chronic pancreatitis. Finally, four hypothetical patients were categorized according to the M-ANNHEIM classification system to provide examples of its applicability in clinical practice. CONCLUSIONS: The M-ANNHEIM multiple risk factor classification system is simple, objective, accurate, and relatively noninvasive, and it incorporates etiology, different stages of the disease, and various degrees of clinical severity. This new classification system will be helpful for investigating the impact and interaction of various risk factors on the course of the disease and will facilitate the comparison and combination of interinstitutional data.

Abstract: The prognosis of peritoneal spread from gastrointestinal cancer and subsequent malignant ascites is poor, and current medical treatments available are mostly ineffective. Targeted chemotherapy with intraperitoneal prodrug activation may be a beneficial new approach. L293 cells were genetically modified to express the cytochrome P450 enzyme 2B1 under the control of a cytomegalovirus immediate early promoter. This CYP2B 1 enzyme converts ifosfamide to its active cytotoxic compounds. The cells are encapsulated in a cellulose sulfate formulation (Capcell; Bavarian Nordic, Martinsried, Germany). Adult Balb/c mice were inoculated intraperitoneally (i.p.) with 1 x 10(6) colon cancer cells, previously transfected with GFP to emit a stable green fluorescence, by injection into the left lower abdominal quadrant. Two or five day's later animals were randomly subjected to either i.p. treatment with ifosfamide alone or ifosfamide combined with microencapsulated CYP2B1 expressing cells. Peritoneal tumour volume and tumour viability were assessed 10 days after tumour inoculation by means of fluorescence microscopy, spectroscopy and histology. Early i.p. treatment with ifosfamide and CYP2B1 cells resulted in a complete response. Treatment starting on day five and single-drug treatment with ifosfamide resulted in a partial response. These results suggest that targeted i.p. chemotherapy using a combination of a prodrug and its converting enzyme may be a successful treatment strategy for peritoneal spread from colorectal cancer. In summary, by using GFP-transfected colon 26 tumour cells in mice we established a well reproducible animal model of metastatic peritoneal cancer. Fluorescent imaging of GFP-transfected tumour was used to demonstrate tumour distribution in the peritoneal cavity and to estimate tumour growth and tumour response to treatment in this model. The application of Capcell and ifosfamide into the peritoneal cavity is a safe and well tolerated procedure in animal models and may help to target chemotherapeutic agents specifically at metastatic peritoneal cancer.

Abstract: Intraductal papillary mucinous tumor (IPMN) of the pancreas is a rare pancreatic tumor characterized by intraductal proliferation of mucin producing cells with hypersecretion of mucin that leads to cystic dilatation of the involved ducts. The usual clinical presentation is recurrent episodes of pancreatitis due to hypersecretion of mucin and temporary obstruction of the main pancreatic duct.

Abstract: Pancreatic cancer ranks 4th as a cause of cancer mortality and in approximately 5% to 10% of patients, this lethal tumor develops before age 50. We used age-, sex-, and country-specific cancer incidence and mortality data to describe the burden of early onset pancreatic cancer (EOPC) worldwide. We also reviewed the current published evidence on smoking and genetic factors associated with EOPC. We found an excess of EOPC resulting in a substantial number of years-of-life-lost in countries from Central and Eastern Europe. Worldwide, the proportion of EOPC is strongly correlated with lung cancer mortality (R(2) = 0.53), suggesting that approximately half of the variation in the proportion of EOPC could be explained by smoking. The unusual pattern of the incidence of pancreatic cancer by gender and race supports the primary role of smoking in the etiology of EOPC: the excess male-to-female rate ratio, attributable mainly to smoking, gradually approaches unity with increasing age. Moreover, male-to-female rate ratios are greater in blacks than in whites only in younger patients. Published studies also identified genetic alterations involved either alone or in association with smoking in the development of EOPC. In conclusion, EOPC constitutes only 1% to 5% of the total deaths from pancreatic cancer worldwide, but is responsible for 20% to 30% of the total number of years-of-life-lost caused by the disease. Smoking and genetic mutations are the major identified risk factors and seem to be even more important for EOPC than for PC in older age groups.

Abstract: Pancreatic carcinoma is a devastating disease with the worst prognosis of all solid tumors; the only cure is surgery. The vast majority of patients are inoperable at the time of diagnosis and require palliative treatment. With a median survival time oscillating around 6 months, indicating an almost complete resistance to conventional cytotoxic and radiation therapy, there is ample room for improvement. Therefore, pancreatic carcinoma has been used to trial many new substances and novel concepts. All aspects of palliative antitumor treatment will be presented in detail and discussed. Finally, some outlooks are given into the future of pancreatic cancer treatment.

Abstract: PURPOSE: The homozygous p.C282Y variant of the HFE gene is a major risk factor for hereditary hemochromatosis, a disorder of iron metabolism resulting in progressive iron accumulation in a variety of organs including the pancreas. Heterozygosity of p.C282Y and p.H63D may increase susceptibility to chronic liver and pancreatic disease. This study determines the frequencies of p.C282Y and p.H63D alterations in patients with chronic pancreatitis and pancreatic adenocarcinoma. METHODS: In total, 958 patients (349 with alcoholic pancreatitis, 343 with idiopathic pancreatitis, 64 with familial chronic pancreatitis, 34 with acute pancreatitis, and 168 with pancreatic adenocarcinoma) were enrolled and compared with 681 healthy and 100 alcoholic controls. Furthermore, 45 parent-offspring trios were included for segregation analysis. Genotyping of p.C282Y and p.H63D was performed by restriction fragment length polymorphism or melting curve analyses. RESULTS: No significant differences were found in heterozygosity for p.C282Y and p.H63D when patients with alcoholic (8.0/21.5%), idiopathic (7.3/24.5%), or familial (9.8/23.0%) pancreatitis, or pancreatic adenocarcinoma (5.4/28.6%) were compared with healthy (6.2/24.8%) and alcoholic (7.0/25.0%) controls. Neither genotype was associated with the presence of secondary diabetes mellitus in patients with chronic pancreatitis. CONCLUSION: Although hemochromatosis is associated with pancreatic pathology, the p.C282Y and p.H63D variants do not play a significant role in the pathogenesis of chronic pancreatitis or pancreatic adenocarcinoma.

Abstract: Pancreatic carcinoma has an extremely bad prognosis due to lack of early diagnostic markers and lack of effective therapeutic strategies. Recently, we have established an in vitro model recapitulating the first steps in the carcinogenesis of the pancreas. SV40 large T antigen-immortalized bovine pancreatic duct cells formed intrapancreatic adenocarcinoma tumors on k-ras(mut) transfection after orthotopic injection in the nude mouse pancreas. Here we identified genes and proteins differentially expressed in the course of malignant transformation using reciprocal suppression subtractive hybridization and 2D gel electrophoresis and mass spectrometry, respectively. We identified 34 differentially expressed genes, expressed sequence tags, and 15 unique proteins. Differential expression was verified for some of the genes or proteins in samples from pancreatic carcinoma. Among these genes and proteins, the majority had already been described either to be influenced by a mutated ras or to be differentially expressed in pancreatic adenocarcinoma, thus proving the feasibility of our model. Other genes and proteins (e.g., BBC1, GLTSCR2, and rhoGDIalpha), up to now, have not been implicated in pancreatic tumor development. Thus, we were able to establish an in vitro model of pancreatic carcinogenesis, which enabled us to identify genes and proteins differentially expressed during the early steps of malignant transformation.

Abstract: Recent evidence suggests that Runt-related transcription factors play a role in different human tumours. In the present study, the localisation of the Runt-related transcription factor-2 (Runx2), its transcriptional activity, as well as its regulation of expression was analysed in human pancreatic ductal adenocarcinoma (PDAC). Quantitative real-time PCR and immunohistochemistry were used for Runx2 expression and localisation analysis. Runt-related transcription factor-2 expression was silenced using specific siRNA oligonucleotides in pancreatic cancer cells (Panc-1) and immortalised pancreatic stellate cells (IPSCs). Overexpression of Runx2 was achieved using a full-length expression vector. TGF-beta1, BMP2, and other cytokines were assessed for their potential to regulate Runx2 expression. There was a 6.1-fold increase in median Runx2 mRNA levels in PDAC tissues compared to normal pancreatic tissues (P<0.0001). Runt-related transcription factor-2 was localised in pancreatic cancer cells, tubular complexes, and PanIN lesions of PDAC tissues as well as in tumour-associated fibroblasts/stellate cells. Coculture of IPSCs and Panc-1 cells, as well as treatment with TGF-beta1 and BMP2, led to increased Runx2 expression in Panc-1 cells. Runt-related transcription factor-2 overexpression was associated with decreased MMP1 release as well as decreased growth and invasion of Panc-1 cells. These effects were reversed by Runx2 silencing. In conclusion, Runx2 is overexpressed in PDAC, where it is regulated by certain cytokines such as TGF-beta1 and BMP2 in an auto- and paracrine manner. In addition, Runx2 has the potential to regulate the transcription of extracellular matrix modulators such as SPARC and MMP1, thereby influencing the tumour microenvironment.

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Abstract: Pancreatic ductal adenocarcinoma remains an extremely aggressive malignancy that is virtually therapy-resistant and has therefore one of the worst prognoses of all human cancers. The focus of research, which had been placed mostly on genetic and epigenetic alterations of the cancer cells themselves, has shifted gradually towards the microenvironment. The cancer microenvironment consists of various components, including fibroblasts, endothelial cells, immune cells, and endocrine cells, that interact with each other and the cancer cells in a complex fashion. This interplay has implications for pancreatic cancer cell growth, migration and invasion, angiogenesis, and immunological recognition of cancer cells. Evidence is accumulating that the cancer microenvironment plays an active role in disease progression, and efforts are being made to target this interplay between cancer cells and host cells to improve the outcome of this deadly disease.

Abstract: Early-stage chronic pancreatitis may be undetected as a clinical entity. However, it may carry a definite risk for subsequent secondary damage, depending on the etiology of the disease. Therefore, the most important question is whether indeed the patient in question does have early-stage chronic pancreatitis rather than oligosymptomatic advanced-stage chronic pancreatitis. This can be easily determined by appropriate imaging such as abdominal computed tomography. For early changes, endoscopic ultrasound is superior to any other technique. Endosonography may also tell about anatomical obstacles (e.g., papillary stenosis, pancreas divisum) that may be treated to prevent progression of the disease. Treatment options at this stage are endoscopic for the most part. Depending on the etiology and familiar/hereditary background of the given patient, one must look further into molecular markers. Such markers may give an estimate on the progression or dynamics of the disease in the future and include mutations in the cationic (PRSS1) and anionic (PRSS2) trypsinogen genes as well as mutations in the serine protease (SPINK1) or cystic fibrosis (CFTR) genes. Admitted ly, these are not markers of early-stage chronic pancreatitis but must be investigated if and when such pathogenesis is suspected. Further, rare forms of chronic pancreatitis, such as autoimmune pancreatitis, which can be cured by appropriate medical treatment with steroids, must be excluded. Markers for autoimmune pancreatitis are elevated serum IgG, especially IgG4, and autoantibodies to carbonic anhydrase (type II) and lactoferrin. It is noteworthy that these markers, present in almost every Asian patient with autoimmune pancreatitis, are mostly lacking in Caucasian populations of patients with autoimmune pancreatitis.

Abstract: A disintegrin and metalloproteinase (ADAM) molecules are known for their unique potential to combine adhesion, proteolysis, and signaling. To understand the role of ADAM17/tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE) in pancreatic ductal adenocarcinoma (PDAC), we investigated its expression, function, and in vitro regulation. ADAM17/TACE mRNA was expressed in 3 of 10 normal pancreatic tissues, 6 of 8 samples from patients with chronic pancreatitis, 10 of 10 PDAC tissues, and 9 of 9 pancreatic cancer cell lines, but it was absent in primary duct epithelial cells. Immunohistochemical staining revealed positive cancer cells in 8 of 10 PDACs but no staining of ducts in normal pancreas. ADAM17/TACE was found in 0 of 16 pancreatic intraepithelial neoplasia (PanIN)-1A lesions, 1 of 30 PanIN-1B lesions, 2 of 13 PanIN-2 lesions but, in 13 of 15 PanIN-3 lesions, associated with PDAC. Western blot, flow cytometry, and confocal microscopy analyses showed the aberrant expression of ADAM17/TACE protein in pancreatic cancer cell lines. The proteolytic activity of ADAM17/TACE, assessed by the release of TNF-alpha, was inhibited by TNF-alpha protease inhibitor. ADAM17/TACE gene silencing using small interfering RNA technique in vitro reduced invasion behavior dramatically, whereas proliferation was unaffected. Furthermore, ADAM17/TACE mRNA expression was down-regulated in pancreatic cancer cells arrested in G2-M phase as well as in a time-dependent manner after TNF-alpha and interleukin-6 incubation. In conclusion, our findings provide evidence of aberrant expression of the proteolytically active ADAM17/TACE in advanced precursor lesions (PanIN-3) and PDAC while identifying its critical involvement in the invasion process.

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Abstract: Gene therapy offers an elegant alternative to toxic chemotherapy regimens, mostly without severe side effects. Gene therapy for cancer was one of the first applications. Following the enthusiasm in the early 1990 s, a more rationale view is preferred today. This general review looks at the tools of gene therapy and their principle elements (vector, promoter, targeting, therapeutic genes). The principles of gene therapy such as gene-directed enzyme prodrug therapy (GDEPT) and gene-directed tumor vaccination are explained. Furthermore, published protocols and clinical studies in gastrointestinal oncology are reviewed. Finally, an outlook is given on the latest developments, some of them going beyond the realm of conventional gene therapy.

Abstract: Wireless capsule enteroscopy, being a novel, painless investigative technique, is reported to be significantly superior to push enteroscopy in its ability to find bleeding abnormalities in the small intestine. Here we report a case of acute jejunal obstruction following wireless capsule endoscopy. The patient had a 1-month history of gastrointestinal bleeding of unknown source. Further evaluation including gastroscopy and colonoscopy, angiography and computed tomography (angio-CT), and radio-labeled erythrocytes scan failed to reveal a source of bleeding. Therefore, wireless capsule enteroscopy was performed. Before capsule endoscopy, there was no clinical or imaging evidence of strictures or stenosis. At readmission it could be shown that there were two inflamed strictures of the small intestine. The capsule was detected at a stricture of the small intestine detected by abdominal ultrasonography and conventional computed tomography. The patient underwent a medical treatment with steroidal and other anti-inflammatory drugs for a total of 23 days and was discharged without complaints. Acute laparotomy after readmission with jejunal ileus proofed the capsule occluding two highly inflamed jejunal stenosis caused by Crohn disease. The present case demonstrates the potential for complications when wireless capsule enteroscopy is performed in the presence of intestinal strictures. Any history of inflammatory bowel disease, abdominal irradiation, cancer, obstruction, and abdominal surgery must be elicited in detail and may exclude the use of wireless capsule enteroscopy.

Abstract: The prognosis of peritoneal spread from gastrointestinal cancer and subsequent malignant ascites is poor, and current medical treatments available are mostly ineffective. Targeted chemotherapy with intraperitoneal prodrug activation may be a beneficial new approach. L293 cells were genetically modified to express the cytochrome P450 enzyme 2B1 under the control of a cytomegalovirus immediate early promoter. This CYP2B1 enzyme converts ifosfamide to its active cytotoxic compounds. The cells are encapsulated in a cellulose sulfate formulation (Capcell). Adult Balb/c mice were inoculated intraperitoneally with 1 x 10(6) colon 26 cancer cells, previously transfected with GFP to emit a stable green fluorescence, by injection into the left lower abdominal quadrant. Two or five day's later animals were randomly subjected to either i.p. treatment with ifosfamide alone or ifosfamide combined with microencapsulated CYP2B1-expressing cells. Peritoneal tumor volume and tumor viability were assessed 10 days after tumor inoculation by means of fluorescence microscopy, spectroscopy and histology. Early i.p. treatment with ifosfamide and CYP2B1 cells resulted in a complete response. Treatment starting on day 5 and single-drug treatment with ifosfamide resulted in a partial response. These results suggest that targeted i.p. chemotherapy using a combination of a prodrug and its converting enzyme may be a successful treatment strategy for peritoneal spread from colorectal cancer.

Abstract: OBJECTIVE: To report the effect of regional combination chemotherapy in a cohort of patients with inoperable pancreatic carcinoma treated with or without low-dose warfarin. MATERIAL AND METHODS: A retrospective analysis was performed on 180 patients with pancreatic carcinoma. Patients received one of seven regimens of chemotherapy. Unrelated to the type of chemotherapy, some patients received 1.25 mg warfarin daily. The primary end-point was median survival. RESULTS: Treatment with warfarin resulted in improved median survival from the start of regional therapy (warfarin versus no warfarin: 5.0 versus 2.3 months, n = 111 versus 69; p < 0.0001). This effect was not dependent on the type of chemotherapy used. Among the seven regimens examined, the one consisting of regional gemcitabine and mitomycin-C with systemic gemcitabine was associated with the longest median survival of 5.1 months from the start of regional therapy (p = 0.006) and 12.7 months from diagnosis. This regimen combined with warfarin was associated with improved median survival (7.1 months, n = 32). CONCLUSIONS: Treatment with low-dose warfarin improved survival irrespective of the chemotherapy received. Of the regimens examined, the combination of regional gemcitabine and mitomycin-C with systemic gemcitabine was associated with the longest survival time. Survival was increased further by the addition of warfarin. These data provide a rationale, based on safety and efficacy, for a definitive study on the use of warfarin and combined regional and systemic chemotherapy in patients with pancreatic carcinoma.

Abstract: Gene therapy offers an elegant alternative to toxic chemotherapy regimens, mostly without severe side effects. Gene therapy for cancer was one of the first applications. Following the enthusiasm in the early 1990 s, a more rationale view is preferred today. This general review looks at the tools of gene therapy and their principle elements (vector, promoter, targeting, therapeutic genes). The principles of gene therapy such as gene-directed enzyme prodrug therapy (GDEPT) and gene-directed tumor vaccination are explained. Furthermore, published protocols and clinical studies in gastrointestinal oncology are reviewed. Finally, an outlook is given on the latest developments, some of them going beyond the realm of conventional gene therapy.

Abstract: Chronic pancreatitis is a common inflammatory disease of the pancreas. Mutations in the genes encoding cationic trypsinogen (PRSS1) and the pancreatic secretory trypsin inhibitor (SPINK1) are associated with chronic pancreatitis. Because increased proteolytic activity owing to mutated PRSS1 enhances the risk for chronic pancreatitis, mutations in the gene encoding anionic trypsinogen (PRSS2) may also predispose to disease. Here we analyzed PRSS2 in individuals with chronic pancreatitis and controls and found, to our surprise, that a variant of codon 191 (G191R) is overrepresented in control subjects: G191R was present in 220/6,459 (3.4%) controls but in only 32/2,466 (1.3%) affected individuals (odds ratio 0.37; P = 1.1 x 10(-8)). Upon activation by enterokinase or trypsin, purified recombinant G191R protein showed a complete loss of trypsin activity owing to the introduction of a new tryptic cleavage site that renders the enzyme hypersensitive to autocatalytic proteolysis. In conclusion, the G191R variant of PRSS2 mitigates intrapancreatic trypsin activity and thereby protects against chronic pancreatitis.

Abstract: Ductal pancreatic adenocarcinoma is a dismal disease, having the worst prognosis of all solid tumors. While genomics and transcriptomics have provided a wealth of data, no contribution has been made to clinical medicine in terms of diagnostic or prognostic markers. Hope lies in yet another novel technology, proteomics. Conceptually, proteomics bears the advantage of incorporating both posttranslational modifications as well as host factors. This is thought to be important in factors influencing survival such as chemoresistance. This tutorial review discusses the state of the art in pancreatic cancer proteomics in light of technical developments. At this moment, proteomics is still at the beginning in clinical application. First results, however, suggest some hope for the development of a new understanding of the molecular biology in pancreatic cancer yielding into very specific markers of disease or allowing a rational and individualized therapy.

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Abstract: The term autoimmune pancreatitis (AIP) describes a nonalcoholic, chronic lymphoplasmocytic pancreatitis. The lymphoplasmocytic infiltration is characterized by periductal localization of predominantly CD4-positive T cells, fibrosis, and acinar atrophy, frequently resulting in stenosis of the main pancreatic and distal common bile ducts. Imaging studies often reveal a diffuse narrowing of the pancreatic main duct and swelling of the pancreatic head wrongly suggesting the presence of a malignant tumor. Clinical signs include mild abdominal pain, jaundice, recurrent episodes of acute pancreatitis, and even new-onset diabetes mellitus. Additionally, AIP can be associated with other autoimmune diseases such as Sjogren's syndrome, primary sclerosing cholangitis, chronic inflammatory bowel diseases, and retroperitoneal fibrosis. Serological markers include autoantibodies and increased levels of gamma globulin and especially IgG4. Steroids seem to be effective in improving clinical symptoms as well as in the resolution of pancreatic and bile duct narrowing. This distinguishes AIP from other forms of pancreatitis and from pancreatic neoplasms. Further studies of the underlying pathophysiologic mechanisms, prognosis, and new diagnostic tools are needed to provide adequate and effective treatment in the future. In this article, we summarize the current knowledge about AIP and present 17 cases that underwent surgical resection at our institution from 2003 to 2004.

Abstract: AIM: To study expression of tissue factor (TF) in pancreatic cancer and its role in the development of thromboembolism. METHODS: TF expression was studied in eight human pancreatic carcinoma cell lines by Northern blot and indirect immunofluorescence. Expression of alternatively spliced TF (asTF) was assessed by RT-PCR. In addition, TF expression was determined by immunofluorescence in pancreatic tissues of 19 patients with pancreatic adenocarcinoma (PCa), 9 patients with chronic pancreatitis (CP) and 20 normal controls. Plasma samples (30 PCa-patients, 13 CP-patients and 20 controls) were investigated for soluble TF levels and coagulation activation markers [thrombin-antithrombin III complex (TAT), prothrombin fragment 1 + 2 (F1 + 2)]. RESULTS: All pancreatic carcinoma cell lines expressed TF (8/8) and most of them expressed asTF (6/8). TF expression at the protein level did not correlate with the differentiation of the carcinoma cell line. All but two pancreatic cancer tissue samples stained positive for TF (17/19). In all samples of CP weak staining was restricted to pancreatic duct cells, whereas only a few subendothelial cells were positive in 9/20 of normal controls. TF and TAT levels in PCa patients were significantly elevated compared to controls whereas elevated F1 + 2 levels did not reach statistical significance compared to controls. In CP patients TAT and F1 + 2 levels proved to be significantly elevated compared to controls, although TAT elevation was less pronounced than in PCa patients. CONCLUSION: We conclude that in addition to the upregulated expression of TF on the cell membrane, soluble TF might contribute to activation of the coagulation system in pancreatic cancer.

Abstract: Local therapy of pancreatic cancer with microencapsulated CYP2B1-producing cells and ifosfamide showed an effect both on the primary tumor and on distant metastatases. This possibly represents a consequence of the activation of immune response. Other studies have demonstrated that local tumor irradiation leads to the activation of the intratumoral lymphocyte infiltration. The aim of our study was to investigate the efficacy of the combined therapy with low-dose irradiation, ifosfamide and CYP2B1-producing cells. Syngenic pancreatic cancer was induced in 38 Lewis-rats by subcutaneous inoculation of 1 x 10(6) (DSL6A) tumor cells. Microencapsulated CYP2B1-producing cells were injected peritumorally 10--12 weeks after tumor implantation. Animals were randomized to the following groups: 1) control (NaCl, 1 ml i.p.), 2) ifosfamide (50 mg/kg, i.p., (3x/week), 3) local irradiation with 5 Gy and 4) ifosfamide plus irradiation. The tumor growth was monitored for 3 weeks. The tumor infiltration with CD4+, CD8+, NK-cells, microvessel density and proliferation rates were investigated by immunohistochemistry. Cytokine plasma level for TNF-alpha were measured by ELISA. Seven of 9 animals in the group of combined therapy showed an objective response to the therapy. The therapy with ifosfamide or radiation alone showed 5 and 3 responders, respectively. The mean tumor volume was significantly reduced after combined ifosfamide plus radiation therapy in the first week, whereas monotherapy with ifosfamide or radiation significantly decreased tumor growth earliest after 2 and 3 weeks, respectively. The high plasma level of TNF-alpha in the control group was significantly reduced after combined ifosfamide/irradiation treatment. The lymphocyte infiltration and tumor proliferation were not significantly different between the groups. Microvascular density was significantly increased after ifosfamide and ifosfamide plus irradiation therapy. The combination of ifosfamide/CYP2B1-producing cells and irradiation showed an earlier therapeutical effect on the growth of rat pancreatic cancer than the irradiation or ifosfamide alone. There was no evidence of late activation of lymphocyte infiltration and PCNA-positive tumor cells.

Abstract: Molecular analyses have demonstrated mutations in the K-ras gene at codon 12 in the majority of pancreatic ductal adenocarcinomas (PDACs). In order to determine whether the K-ras mutation rate increases parallel to the grade of dysplasia in duct lesions, we performed a meta-analysis of the studies published between 1988 and 2003 that provide information on K-ras mutations in hyperplastic and dysplastic duct lesions in the pancreas. The described duct lesions were reclassified according to the nomenclature for pancreatic intraepithelial neoplasia (PanIN), and the molecular methods for detecting K-ras were reviewed. In PanIN lesions from pancreata of patients with PDAC, there was a stepwise increase in K-ras mutations that correlated with the grade of dysplasia of the PanIN lesion. K-ras mutations were found in 36%, 44%, and 87% of PanIN-1a, 1b, and 2-3 lesions, respectively (trend statistic P <.001). Mutation-enriched polymerase chain reaction (PCR) resulted in higher rates of K-ras mutations in PanIN than plain PCR did. The incidence of K-ras mutations in PanIN lesions associated with chronic pancreatitis (CP) or normal pancreas was low (around 10%). In CP, K-ras mutations were only found after a disease duration of 3 years. The correlation of the incidence of K-ras mutations with the grade of dysplasia in PanIN and the occurrence of these mutations in CP with a duration of more than 3 years underlines the importance of this genetic change for the development of PDAC.

Abstract: BACKGROUND: Endoscopic management of chronic pancreatitis (CP), especially pancreatic stent placement, has made tremendous advances. However, good clinical results are hampered by rapid occlusion. The objective of this study was to understand mechanisms and materials that cause stent occlusion. METHODS: The clogging material of 50 lyophilized pancreatic endoprostheses (length 8.5 cm, range 5-14 cm, diameter 7-11F) from patients with CP was completely removed and weighed. Protein solubilization was achieved at pH 8.0 by using sodium dodecyl sulfate (SDS) and 2-mercaptoethanol in the presence of proteasome inhibitors. Proteins were separated by using a SDS-polyacrylamide gel electrophoresis. Protein identification was performed by the Western blot technique, as well as by mass spectrometry. Insoluble components were examined by polarized light microscopy and after staining (periodic acid-Schiff [PAS]). RESULTS: Clogging material was found in 49 prostheses, mainly at the duodenal flap (80%). More than a third of the prostheses contained visible calcium carbonate calculi. Light microscopy and PAS staining showed plant debris (80%), crystals (73.5%), and mucopolysaccharides (100%). The dry weight of clogging material (18 +/- 13 mg, range 3-72 mg) correlated significantly with the stent diameter ( p = 0.029) but not with any other stent- or patient-related criteria. Albumin, its degradation products, and lithostathine were identified as the main proteinaceous components. CONCLUSIONS: Almost all pancreatic stents had clogging material, predominantly located at the duodenal flap, which contained plant material, mucopolysaccharides, and crystals, as well as visible calcium carbonate calculi. Albumin and lithostathine may play an important role in the development of stent occlusion.

Abstract: BACKGROUND: Strokes are an important cause of morbidity and mortality in young adults. However, in most cases the cause of the stroke remains unclear. Anderson-Fabry disease is an X-linked recessive lysosomal storage disease resulting from deficient alpha-galactosidase and causes an endothelial vasculopathy followed by cerebral ischaemia. To determine the importance of Fabry disease in young people with stroke, we measured the frequency of unrecognised Fabry disease in a cohort of acute stroke patients. METHODS: Between February, 2001, and December, 2004, 721 German adults aged 18 to 55 years suffering from acute cryptogenic stroke were screened for Fabry disease. The plasma alpha-galactosidase activity in men was measured followed by sequencing of the entire alpha-GAL gene in those with low enzyme activity. By contrast, the entire alpha-GAL gene was genetically screened for mutations in women even if enzyme activity was normal. FINDINGS: 21 of 432 (4.9%) male stroke patients and seven of 289 (2.4%) women had a biologically significant mutation within the alpha-GAL gene. The mean age at onset of symptomatic cerebrovascular disease was 38.4 years (SD 13.0) in the male stroke patients and 40.3 years (13.1) in the female group. The higher frequency of infarctions in the vertebrobasilar area correlated with more pronounced changes in the vertebrobasilar vessels like dolichoectatic pathology (42.9%vs 6.8%). INTERPRETATION: We have shown a high frequency of Fabry disease in a cohort of patients with cryptogenic stroke, which corresponds to about 1.2% in young stroke patients. Fabry disease must be considered in all cases of unexplained stroke in young patients, especially in those with the combination of infarction in the vertebrobasilar artery system and proteinuria.

Abstract: BACKGROUND: Pancreatic cancer is one of the leading causes of cancer-related death. Using DNA gene expression analysis based on a custom made Affymetrix cancer array, we investigated the expression pattern of both primary and established pancreatic carcinoma cell lines. METHODS: We analyzed the gene expression of 5 established pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-1, Capan-2 and HPAF II) and 5 primary isolates, 1 of them derived from benign pancreatic duct cells. RESULTS: Out of 1,540 genes which were expressed in at least 3 experiments, we found 122 genes upregulated and 18 downregulated in tumor cell lines compared to benign cells with a fold change >3. Several of the upregulated genes (like Prefoldin 5, ADAM9 and E-cadherin) have been associated with pancreatic cancer before. The other differentially regulated genes, however, play a so far unknown role in the course of human pancreatic carcinoma. By means of immunohistochemistry we could show that thymosin beta-10 (TMSB10), upregulated in tumor cell lines, is expressed in human pancreatic carcinoma, but not in non-neoplastic pancreatic tissue, suggesting a role for TMSB10 in the carcinogenesis of pancreatic carcinoma. CONCLUSION: Using gene expression profiling of pancreatic cell lines we were able to identify genes differentially expressed in pancreatic adenocarcinoma, which might contribute to pancreatic cancer development.

Abstract: Pancreatic biopsy is an invasive diagnostic method that is only performed when all other diagnostic measures for establishing the diagnosis of a tumorous lesion of the pancreas have failed. Because of the advances in modern imaging techniques, fine needle biopsy of the pancreas guided by ultrasonography, computer tomography or endosonography has become a reliable method that allows the diagnosis of ductal adenocarcinoma or any of the other, rarer pancreatic tumors with high sensitivity and specificity. Complications are rare, particularly with the endosonographically guided biopsy. A new biopsy indication is the demonstration of certain markers or gene mutations that are needed for the initiation of special treatments, e.g. EGFR-Cetuximab.

Abstract: The enteric nervous system in vertebrates is the most complex part of the peripheral nervous system. Concerning chemical coding, ultrastructure and neuronal circuits, it is more similar to the central than to the peripheral nervous system. Its networks, the myenteric and submucous plexus are integrated in the gut wall. The enteric nervous system is a system of high plasticity, which not only changes during pre- and postnatal development, but also with disease or changing dietary habits. The Aim of this study was to elucidate changes in protein expression during the first two postnatal weeks in the rat myenteric plexus. Colonic and duodenal myenteric plexus from newborn (P1) and fourteen-day old (P14) Sprague-Dawley rats was isolated following a procedure that combines enzymatic digestion and mechanical agitation. The neuronal tissue was collected and processed for two-dimensional gel electrophoresis (2-DE). The obtained 2-D gels were stained with silver for image analysis or with colloidal Coomassie for subsequent protein identification. Gels from the various samples showed a high degree of consistence concerning protein-spots found in all preparations. Nevertheless, there was a number of proteins that were clearly detected in one sample but not, or only in significantly smaller amounts in the other. Several differentially expressed proteins in the postnatal myenteric plexus were identified with MALDI-TOF mass spectrometry. Especially stathmin, polyubiquitin and heterogeneous nuclear ribonucleoprotein seem to play an important role in pre- and postnatal development. 2-DE combined with mass spectrometry can help to identify pathological relevant proteins in the enteric nervous system, and so deliver a valuable tool for the early diagnosis of also central nervous system diseases by using biopsies from the gut.

Abstract: Tissue fibrosis is one of the characteristics of chronic pancreatitis and pancreatic adenocarcinoma. Activated pancreatic stellate cells (PSC) play a central role in this process. However, analysis of the molecular mechanisms leading to PSC activation is hampered by the lack of an established human PSC line. To overcome this problem, we immortalized and characterized primary human PSC. The cells were isolated by the outgrowth method and were immortalized by transfection with SV40 large T antigen and human telomerase (hTERT). Primary human PSC served as controls. An immortalized line, RLT-PSC, was analyzed for the expression of stellate cell markers. Moreover, the effects of transforming growth factor beta 1(TGFbeta1) or platelet-derived growth factor stimulation and of cultivation on basement membrane components or N-acetylcysteine (NAC) treatment on gene and protein expression and proliferation were analyzed. Immortal RLT-PSC cells retained the phenotype of activated PSC proven by the expression of alpha-smooth muscle actin (alphaSMA), vimentin, desmin and glial fibrillary acidic protein (GFAP). TGFbeta1 treatment upregulated the expression of alphaSMA, collagen type I (Col I), fibronectin and TGFbeta1. Incubation of RLT-PSC cells and primary human activated PSC on Matrigel plus NAC treatment resulted in a deactivated phenotype as evidenced by a decrease of alphaSMA, connective tissue growth factor and Col I expression and by a decreased proliferation of the cells. Moreover, this treatment restored the ability of the cells to store vitamin A in cytoplasmic vesicles. In conclusion, we have established an immortal pancreatic stellate cell line, without changing the characteristic phenotype. Importantly, we were able to demonstrate that besides soluble factors, the matrix surrounding PSC plays a pivotal role in the maintenance of the activation process of PSC. Cultivation of activated PSC on a reconstituted basement membrane plus treatment with NAC was able to deactivate the cells, thus pointing to the possibility of an antifibrosis therapy in chronic pancreatitis.

Abstract: Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis. To improve the prognosis, novel molecular markers and targets for earlier diagnosis and adjuvant and/or neoadjuvant treatment are needed. Recent advances in human genome research and high-throughput molecular technologies make it possible to cope with the molecular complexity of malignant tumors. With DNA array technology, mRNA expression levels of thousand of genes can be measured simultaneously in a single assay. As several studies using microarrays in PDAC have already been published, this review attempts to compare the published data and therefore to validate the results. In addition, the applied techniques are discussed in the context of pancreatic malignancies.

Abstract: Proteomics represents a novel methodological approach to investigate the expression of all proteins by a cell or organism in its entireness, similar to global strategies for DNA (genomics) and RNA (transcriptomics). This review focuses on the history of protein analysis, which made up the golden age of pancreatic physiology, the current methodology for proteomics (2D gel electrophoresis, mass spectrometry) and the few published experiences with proteomics in the field of pancreatology until now. Finally, potential applications of proteomics for the pancreas, in concert with other techniques, are cited.

Abstract: Small cell carcinoma (SCC) of the pancreas is a rare malignancy with an extremely poor prognosis. We present the case of a 74-year-old man with a 2-month history of upper abdominal discomfort who was diagnosed with SCC of the pancreas tail, involvement of peripancreatic and mesenteric lymph nodes and multiple liver metastases (extended disease). A CT scan and a positive somatostatin receptor scintigraphy showed no evidence of a primary lung tumour. The diagnosis of a SCC was confirmed by biopsy. Local tumour control could be achieved by gemcitabine once a week and a long-acting somatostatin analogue once a month, but liver metastasis showed progress. Thus, 5-fluorouracil on a weekly basis was started. The patient died 8 months after diagnosis and had not been hospitalised in the meantime.

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Abstract: BACKGROUND: The paper discusses the non-invasive (tubeless) pancreatic function tests used to diagnose exocrine pancreatic insufficiency (EI). Studies evaluating the diagnostic validity of these tests are integrated into a meta-analysis, provided that they comply with the following criteria: The sensitivity (Ss) of a test has to be calculated by comparing it with an invasive function test which is accepted as the gold standard of pancreatic function diagnostics. Furthermore, the test must differentiate between slight (sl), moderate (md) and severe (sv) EI. For assessment of the specificity (Sp), the control group should not contain healthy persons but rather patients with other gastrointestinal diseases and a normal pancreatic function. In the statistical evaluation, each study was weighted according to the number of persons included. RESULTS: Tests (n = sum of persons included in all analysed studies): Fecal chymotrypsin: Ss (n = 169) 54 % (sl EI), 53 % (md EI), 89 % (sv EI), Sp (n = 202) 74 %. NBT-PABA test: Ss (n = 394) 49 % (sl EI), 64 % (md EI), 72 % (sv EI), Sp (n = 218) 83 %. Pancreolauryl test: Ss (n = 320) 63 % (sl EI), 76 % (md EI), 94 % (sv EI), Sp (n = 171) 85 %. Fecal elastase-1: Ss (n = 307) 54 % (sl EI), 75 % (md EI), 95 % (sv EI), Sp (n = 347) 79 %. Additional tests discussed but not included in the meta-analysis were fecal fat, (13)C breath tests, amino acid consumption test, serum tests. CONCLUSION: None of the non-invasive pancreatic function tests is sensitive enough to diagnose reliably a slight to moderate exocrine pancreatic insufficiency.

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Abstract: Claudin-4 has been identified as an integral constituent of tight junctions and has been found to be highly expressed in pancreatic cancer. The aim of the present study was to elucidate the effect of claudin-4 on growth and metastatic potential in pancreatic cancer cells, as well as the regulation of claudin-4 by oncogenic pathways. Claudin-4 was stably overexpressed in SUIT-2 pancreatic cancer cells, and its effect on invasion and growth in vitro was examined by using two-chamber invasion assays, soft agar assays, and fluorescence-activated cell sorter analysis. Claudin-4 localization was characterized by light and electron microscopy, and pulmonary colonization was analyzed in vivo after injection of claudin-4 overexpressing cells into the tail vein of nude mice. Overexpression of claudin-4 was associated with significantly reduced invasive potential in vitro and inhibited colony formation in soft agar assays. In vivo, tail vein-injected claudin-4 overexpressing cells formed significantly less pulmonary metastases in comparison with mock-transfected cells. These effects were not caused by changes in proliferation, cell cycle progression, or matrix metalloproteinase gelatinolytic activity, but were paralleled by increased cell contact formation. Moreover, proinvasive transforming growth factor beta was able to down-regulate claudin-4 in PANC-1 cells. Inhibition of Ras signaling by using dominant-negative Ras and specific inhibitors of both downstream effectors mitogen-activated protein/extracellular signal-regulated kinase kinase and phosphatidylinositol 3'-kinase also decreased claudin-4 expression. Our findings identify claudin-4 as a potent inhibitor of the invasiveness and metastatic phenotype of pancreatic cancer cells, and as a target of the transforming growth factor beta and Ras/Raf/extracellular signal-regulated kinase pathways.

Abstract: INTRODUCTION: K-ras mutations are present in most ductal adenocarcinomas (DACs) of the pancreas and may also be found in ductal precursor lesions and even in normal ductal epithelium. The question is addressed whether mutated K-ras interferes with the regulation of apoptosis or proliferation. METHODOLOGY: In 50 Whipple resection specimens, tissue adjacent to DACs was histologically screened for ductal lesions that were classified as pancreatic intraepithelial neoplasia (PanIN) according to WHO criteria. PanIN lesions were microdissected and analyzed for K-ras mutations by means of a nested PCR. Apoptosis was identified by the TUNEL method. Proliferation and the expression of p53 and Bcl-2 were immunohistochemically determined. RESULTS: On average, 30% of PanIN-1A and B lesions showed mutated K-ras. In PanIN-2 and PanIN-3 lesions, the rate of mutated K-ras increased to 45% and 56%, respectively. Apoptosis was present only in 2 of 26 PanIN-3 lesions. There was a gradual increase in proliferative activity from PanIN-1 to PanIN-3. p53 expression was found in 11% of PanIN-2 and 44% of PanIN-3 lesions. Bcl-2 expression was lacking in PanIN lesions of all grades. In invasive DACs, the apoptotic rate correlated with the degree of tumor differentiation and proliferation, with grade 3 carcinomas showing the highest apoptotic rate. CONCLUSION: In view of the discrepancy between the considerable rate of K-ras mutations in PanIN-1 and PanIN-2 lesions and the lack of apoptosis and Bcl-2 expression, coupled with very low p53 immunoreactivity, it is unlikely that mutated K-ras affects the apoptotic activity in low grade PanINs. Instead, K-ras mutations may have an effect on proliferation in PanIN-1 and PanIN-2.

Abstract: BACKGROUND: The therapeutic efficacy of intratumoral instillation of genetically engineered, CYP2B1-expressing, microencapsulated cells in combination with ifosfamide had been previously demonstrated in xenografted human pancreatic ductal carcinomas [Gene Ther 1998;5:1070-1078]. Prior to a clinical study, the feasibility of an intra-arterial application of microencapsulated cells to the pancreas and its consequences to the organ had to be evaluated. MATERIAL AND METHODS: Microencapsulated, CYP2B1-producing cells were instilled both in vivo (transfemoral angiographical access) and in vitro (perfusion model) in the splenic lobe of the pig pancreas. In vivo, animals were monitored clinically for 7 days, then treated with ifosfamide and sacrificed. In vitro, ifosfamide was administered intra-arterially. RESULTS: In all animals, 100 microcapsules could be instilled safely via the femoral route without clinical, biochemical or histological signs of pancreatitis. Histological examination revealed partial obstruction of small arteries by the capsules, without causing any parenchymal damage. In vitro, instillation reduced blood flow by half. Ifosfamide, also in combination with the capsules, did not add any damage to the pancreas. CONCLUSION: Intra-arterial instillation of microencapsulated cells to the pig pancreas is feasible and safe. Neither pancreatitis, foreign body reactions nor circulatory disturbances were observed. Clinical application of this genetically enhanced chemotherapeutic method seems possible.

Abstract: The early diagnosis of pancreatic cancer, as well as distinguishing between chronic pancreatitis and malignant pancreatic disease, remains still a clinical problem. Presently, there is no specific tumor marker for diagnosing pancreatic cancer. Mucin-associated marker like CA 19-9 are the most widely available pancreatic cancer tumor marker, but its value as a screening marker is limited by its reduced specificity. Mucins (MUCs) are heavily glycosylated, high molecular weight glycoproteins with an aberrant expression profile in various malignancies. This review considers briefly the potential use of the mucin expression pattern in diagnosis of pancreatic neoplasm. The overview will point out the present knowledge about changes in the mucin gene expression in pancreatic intraepithelial neoplasia (PanINs) as precursor lesions and in pancreatic adenocarcinoma, compared to normal pancreas and chronic pancreatitis and the potential role for differentiating chronic pancreatitis from pancreatic cancer.Furthermore, the potential use of MUCs in the diagnosis and differentiation of intraductal papillary-mucinous neoplasm's (IPMNs) will be discussed.

Abstract: BACKGROUND: The use of ifosfamide for conventional chemotherapy of pancreatic tumors is limited by systemic toxicity caused, in part, by its activation by liver-specific cytochrome enzymes and the subsequent systemic distribution of metabolites. Activation at the tumor site, on the other hand, should reduce systemic toxicity and provide more effective local therapeutic concentrations. The results of a phase I/II open, single-arm clinical trial of local intratumor activation of ifosfamide are presented. METHODS: Treatment involved angiographic implantation of encapsulated allogeneic cells, genetically modified to overexpress the enzyme cytochrome P-450 2B1, into the vasculature leading to the tumor. The cells are protected from immune rejection and locally activate systemically administered ifosfamide to antitumor metabolites. RESULTS: Of the 14 patients treated, 4 showed tumor regression and the other 10 patients showed stable disease, i.e., no further tumor growth. The median survival of the patient cohort was twice that of the control group. The 1-year survival was 36%, i.e., threefold that of the control group (twice that reported for gemcitabine). As far as clinical benefit was concerned, 4 of the 13 evaluable patients reported improvements in pain assessment at the last observation, with 6 remaining unchanged (4 of these experienced no pain) and 3 patients experiencing slightly greater pain. Using a worst case scenario, 50% experienced a clinical benefit, which rose to 71% of patients if a best case scenario was applied. The best predictor of clinical benefit was lack of pain, whereas significant weight loss possibly predicts a poor clinical benefit. CONCLUSIONS: The data obtained from this phase I/II clinical trial are promising and suggest that further clinical trials are warranted.

Abstract: There are a large number of stable pancreatic ductal carcinoma cell lines that are used by researchers worldwide. Detailed data about their differentiation status and growth features are, however, often lacking. We therefore attempted to classify commonly used pancreatic carcinoma cell lines according to defined cell biological criteria. Twelve pancreatic ductal adenocarcinoma cell lines were cultured as monolayers and spheroids and graded according to their ultrastructural features. The grading system was based on the integrity of membrane structures and on the presence of mucin granules, cell organelles, nuclear and cellular polymorphism, cell polarity, and lumen formation. On the basis of the resulting scores the cell lines were classified as well, moderately, or poorly differentiated. In addition, immunocytochemistry was performed for the markers cytokeratin 7, 8, 18, 19, carcinoembryonic antigen, MUC1 MUC2, MUC5, and MUC6. The population doubling time of monolayer cultures, determined by a tetrazolium salt based proliferation assay was correlated with the ultrastructural grade. The grading of the ultrastructural features of the monolayers, and particularly of the spheroids, revealed that Capan-1 and Capan-2 cells were well differentiated; Colo357, HPAF-2, Aspc-1, A818-4, BxPc3, and Panc89 cells were moderately differentiated and PancTu-I, Panc1, Pt45P1, and MiaPaCa-2 cells poorly differentiated. Membrane-bound MUC1 staining was a characteristic of well differentiated cell lines. The population doubling time of the monolayer cultures was related to the differentiation grade. No relationship was found between the p53, K-ras, DPC4/Smad4, or p16(INK4a) mutation status and the grade of differentiation. We conclude that the proposed ultrastructural grading system combined with the proliferative activity provides a basis for further comparative studies of pancreatic ductal adenocarcinoma cell lines.

Abstract: The case of a 35-year-old male with portal vein erosion and acute life-threatening abdominal hemorrhage as a complication of acute pancreatitis is reported. Erosion of arterial walls is the most common complication of pancreatitis and pancreatic pseudocysts, and erosion of the portal vein leading to a fistula between the pancreas and the portal vein is very rare. Diagnosis was made by multislice spiral computed tomography, and nonsurgical treatment was elected, leading to complete recoverage.

Abstract: INTRODUCTION: In recent years a type of chronic pancreatitis has been described that is clearly distinct from alcoholic chronic pancreatitis. It is characterized by its special pathology, immunologic features, clinical presentation, and steroid responsiveness. Because of its histologic hallmarks, i.e., ductal and periductal infiltration by lymphocytes, plasma cells, and granulocytes, it has been called duct-destructive chronic pancreatitis. The frequent association of this type of pancreatitis with other autoimmune diseases such as Sjogren's disease and a number of other immune phenomena has led to the concept that it is an autoimmune disease. Hence, the term autoimmune pancreatitis has been introduced and will be used in this review. AIMS: This review focuses on the pathology and related clinical and immunologic features of this new type of pancreatitis. CONCLUSIONS: As the ability to diagnose autoimmune pancreatitis on the basis of clinical, imaging, and laboratory findings improves, it seems likely that fewer patients with this diagnosis will undergo resection. Thus, there is a need to accumulate and study additional retrospective series of patients undergoing resection because of mass-forming chronic pancreatitis.

Abstract: In a search for new molecular markers of pancreatic ductal adenocarcinoma (PDAC), we compared the gene expression profiles of seven pancreatic carcinomas and one carcinoma of the papilla Vateri with those of duct cells from three non-neoplastic pancreatic tissues. In addition, the human pancreatic duct cell line and five PDAC cell lines (AsPC-1, BxPC-3, Capan-1, Capan-2, HPAF) were examined. RNA was extracted from microdissected tissue or cultured cell lines and analysed using a custom-made Affymetrix Chip containing 3023 genes, of which 1000 were known to be tumour associated. Hierarchical clustering revealed 81 differentially expressed genes. Of all the genes, 26 were downregulated in PDAC and 14 were upregulated in PDAC. In PDAC cell lines versus normal pancreatic duct cells, 21 genes were downregulated and 20 were upregulated. Of these 81 differentially expressed genes, 15 represented human genes previously implicated in the tumourigenesis of PDAC. From the genes that were so far not known to be associated with PDAC tumorigenesis, we selected ADAM9 for further validation because of its distinct overexpression in tumour tissue. Using immunohistochemistry, the over-expressed gene, ADAM9, was present in 70% of the PDACs analysed. In conclusion, using microarray technology we were able to identify a set of genes whose aberrant expression was associated with PDAC and may be used to target the disease.

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Abstract: A small number of patients exist with carcinoma of the pancreas with an inoperable but not metastasized tumor. Prospective randomized studies defined the standard of combined radiochemotherapy during the early 1980s for these patients. Since then, new drugs have shown considerable activity and in parallel improvements in radiotherapy treatment planning and delivery have been achieved. Therefore, it is time to ask whether patients with locally advanced, inoperable pancreatic cancer without metastases should still be irradiated or not. This review summarizes the current literature on combined radiochemotherapy for locally advanced carcinoma of the pancreas. Median survival times of 10-11 months and 1-year survival rates of about 40% can be achieved with modern radiochemotherapy regimens.

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Abstract: Until recently, 5-fluorouracil was the most widely used treatment for non-resectable pancreatic cancer. This treatment, however, only resulted in a median survival time of approximately 4 months. In the last few years, gemcitabine has rapidly become the new treatment benchmark, due more to its superior clinical benefit rather than to it conferring an increased median survival (approximately 5-6 months). Thus, the outlook for patients with pancreatic cancer is still relatively bleak. A number of new treatment options are presently being investigated. Some of these are combination therapies involving gemcitabine and other chemotherapeutic agents or radiation. Other novel treatment strategies are also already being evaluated in clinical studies. Some of the more promising treatments in development are discussed and evaluated in this article.

Abstract: BACKGROUND: The etiology of tumors arising in the biliary tract remains unclear. Several previous studies have detected Helicobacter pylori organisms in bile from patients with gallstones or cholecystitis. The objective of this study was to determine whether there is an association between H. pylori in bile and biliary tract carcinoma. METHODS: The authors used polymerase chain reaction (PCR) assays to detect the presence of H. pylori in the stomach and bile from 89 patients: Sixty-three disease free patients had biliary calculi, 15 patients had carcinoma of the biliary tract, and 11 patients had neither gallstones nor carcinoma. Bile was considered to contain H. pylori only if the results of PCR determinations were positive in two or more samples assayed independently in two separate laboratories. RESULTS: There was a strong association between the presence of H. pylori in the stomach and in the bile (P < or = 0.01). Biliary H. pylori was associated with age but not with gender, and it was associated strongly with the clinical diagnosis. Patients with gallstones were 3.5 times as likely to have H. pylori in the bile compared with patients in a control group (95% confidence interval [95%CI], 0.8-15.8; P = 0.100), and H. pylori was 9.9 times more frequent in patients with biliary tract carcinoma compared with patients in the control group (95%CI, 1.4-70.5; P = 0.022). CONCLUSIONS: There is a strong association between biliary tract carcinoma and H. pylori in bile. If these results are confirmed by prospective studies, H. pylori may be responsible for a significant proportion of malignant biliary tract disease.

Abstract: BACKGROUND: Infections are frequent complications and determine clinical course and outcome in severe pancreatitis. A novel animal model was used to assess minimal transit time of bacterial translocation (BT) across the gut mucosa in vivo using green fluorescent protein-transfected Escherichia coli and intravital video microscopy. METHODS: Three hours after induction of acute pancreatitis by i.p. injection of 40 microg/kg cerulein, 0.5 ml of a suspension of green fluorescent protein-transfected E. coli were injected into the lumen of a small bowel reservoir formed by ligature in anesthetized Wistar rats. Translocation of E. coli was assessed by intravital microscopy. Animals were sacrificed 5 h after induction of pancreatitis. RESULTS: BT across the mucosa and into the muscularis propria took a mean +/- SD of 36.4 +/- 8 min and 80.9 +/- 9.5 min, respectively, in sham animals. Pancreatitis resulted in a significantly shorter minimal transit time across the mucosa (16.4 +/- 4.9 min, p = 0.007) and into the muscularis propria (47.7 +/- 2.5 min, p = 0.001). E. coli were detected on frozen cross-sections and on bacteriological examination of pancreatic tissue in animals with acute pancreatitis but not in controls. Discussion: Intravital microscopy of fluorescent bacteria is a new approach towards studying BT in vivo. Minimal transit time of BT serves as a novel functional aspect of mucosal barrier function during acute pancreatitis. The observation of fluorescent bacteria translocating from the small bowel lumen into the pancreas provides substantial experimental proof for the gut-origin-hypothesis of infectious complications in pancreatitis.

Abstract: BACKGROUND: Conventional chemotherapy of pancreatic carcinoma is only marginally effective. This is in part due to the severity of side effects following systemic administration of the cytostatic drug. The aim was to create a therapeutic tool allowing the targeting of the conversion site of a cytotoxic prodrug to the site of the tumor. This was realized by transfection of the CYP2B1 gene, the major ifosfamide-converting P450 enzyme, in cells with subsequent microencapsulation and administration of these microcapsules to or into the tumor. The enzyme activity (resorufin assay) remained stable for weeks in vitro and in vivo within the microencapsulated CYP2B1-expressing cells. We demonstrated a significant antitumor effect of the intratumorally injected capsules against xenotransplanted human pancreatic carcinomas in the nude mouse. Angiographic experiments in the pig confirmed the feasibility of an intraarterial placement of the capsules into the pancreas. A clinical protocol was established and approved. PATIENTS, MATERIAL AND METHODS: L293 cells were transfected with the CYP2B1 gene, microencapsulated (diameter 0.7 mm) under GCP conditions and packed sterile. Patients with confirmed inoperable adenocarcinoma of the pancreas underwent angiography, and capsules were injected into a vessel leading into the tumor. The patients were monitored for 48 h to exclude allergic reactions or pancreatitis. A day later, ifosfamide was administered for three consecutive days to be repeated on days 21-23. The patients were followed up for 5 months. RESULTS: A total of 17 patients were enrolled. The patients tolerated the procedure without any complications. No allergic reactions or pancreatitis were encountered. Chemotherapy was uneventful. All patients had stable disease, and two patients a partial remission. The median survival was 44 weeks which compared favorably with that of a historical control group (22 weeks). CONCLUSIONS: The intraarterial administration of microcapsules for targeted chemotherapy was well tolerated. Control of local tumor growth was achieved.

Abstract: BACKGROUND/AIMS: Analysis of gene expression is dependent on normalization using housekeeping genes. However, many of these housekeeping genes (e.g. GAPDH, beta-actin) are upregulated in chronic pancreatitis and pancreatic cancer, and cannot be used for normalization. For this reason we tried to identify a housekeeping gene useful for expression analysis in pancreatic diseases. METHODS: RNA isolated from various tissues and states of disease was subjected to reverse transcription and subsequently amplified by PCR using primers for GAPDH and for the ribosomal highly basic 23-kDa (rb 23-kDa, RPL13A) protein. RESULTS: As anticipated, expression of GAPDH varied markedly in the different tissues, whereas the expression of rb 23-kDa was constant in all samples investigated. CONCLUSION: We recommend the use of the ribosomal highly basic 23-kDa protein as a standard for normalization at least for the pancreas and the prostate.

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Abstract: The identification of pancreatic proteins is generally hampered by the high content and activity of proteases produced by this organ. The aim of this work was the development of a protocol for the analysis of pancreatic juice by two-dimensional (2-D) gel electrophoresis allowing consistent and reproducible protein analysis encompassed by high-resolution protein 2-D maps and subtle protein spot recognition without substantial losses due to proteases. Immobilized pH gradient (IPG) strips were used for the first dimension, the second dimension was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, the key step was the sample preparation technique. Improvements were achieved by using several protease inhibitors (phenylmethylsulfonyl fluoride, aprotinin, L-1-chloro-3-[4-tosyl-amido]-7-amino-2-heptanine (TLCK)-HCI, Complete) to prevent degradation of the proteins. The application of different pH-ranges was a valuable step for getting an overview of the expressed protein pattern. These investigations resulted in well-resolved 2-D maps with a high reproducibility.

Abstract: BACKGROUND & AIMS: Recently, several members of the claudin family have been identified as integral constituents of tight junctions. Using expression profiling, we previously found claudin-4 to be overexpressed in pancreatic cancer. Because claudin-4 has been described as a receptor for the cytotoxic Clostridium perfringens enterotoxin (CPE), we investigated the effect of CPE on pancreatic cancer cells. METHODS: Expression of claudin-4 was analyzed by Northern blots. In vitro toxicity of CPE was determined by trypan blue exclusion and the (86)Rb-release assay. The in vivo effect of CPE was studied in claudin-4-expressing nude mouse xenografts of the Panc-1 cell line. RESULTS: Expression analyses showed that claudin-4 was overexpressed in most pancreatic cancer tissues and cell lines and several other gastrointestinal tumors. CPE led to an acute dose-dependent cytotoxic effect, restricted to claudin-4-expressing cells and dependent on claudin-4 expression levels. Furthermore, transforming growth factor beta was identified as a negative modulator of both claudin-4 expression and susceptibility to CPE. In vivo, intratumoral injections of CPE in Panc-1 xenografts led to large areas of tumor cell necrosis and significant reduction of tumor growth. CONCLUSIONS: Our findings suggest that targeting claudin-4-expressing tumors with CPE represents a promising new treatment modality for pancreatic cancer and other solid tumors.

Abstract: Proliferation of fibrotic tissue (desmoplasia) is one of the hallmarks of several epithelial tumors including pancreatic adenocarcinoma. This tissue reaction may be deleterious or advantageous to the host or tumor. In a systematic analysis, we identified two growth factors expressed by human pancreatic carcinoma cells that are positively correlated with the ability to induce fibroblast proliferation both in vitro and in vivo, i.e., transforming growth factor (TGF)-beta1 and fibroblast growth factor-2. Here we demonstrate that the overexpression of TGF-beta1 induced up-regulation of matrix proteins and growth factors in the TGFbeta1-transfected pancreatic tumor cells. Furthermore, transfection of PANC-1 cells induces the same change in fibroblasts in either cocultivation experiments or when they are grown in conditioned medium from TGF-beta1-transfected PANC-1 cells. TGF-beta1-transfected pancreatic tumor cells induced a rich stroma after orthotopical transplantation in the nude mouse pancreas. The transfer of a single growth factor, TGF-beta1, conveys the ability to induce a fibroblast response similar to that seen in desmoplasia in human pancreatic adenocarcinoma. This effect cannot only be attributed to direct effects of TGF-beta1 but also results from the up-regulation of several other factors including collagen type I, connective tissue growth factor, and platelet-derived growth factor.

Abstract: The K-ras, p53, p16 and DPC4 genes are among those most frequently altered in pancreatic ductal carcinoma. We analyzed 22 cell lines for alterations in these genes by direct sequence analysis and methylation-specific polymerase chain reaction. These cell lines showed mutations in K-ras and p53 at frequencies of 91% and 95%, respectively. Alterations in p16INK4a were found in all cases and included nine homozygous deletions, seven mutations and promoter methylation in six cases. Eight cell lines (36%) had an alteration of DPC4, including one mutation and seven homozygous deletions. The most typical mutational profile involved K-ras, p53, and p16INK4a, concurrently aberrated in 20 cases (91%). Eight cell lines had alterations in all four genes. Inactivation of DPC4 was always accompanied by alteration of all of the other three genes. This comprehensive data regarding the cumulative genetic alterations in pancreatic carcinoma cell lines will be of great value for studies involving drug sensitivity or resistance that may be associated with inactivation of a particular gene or molecular pathway.

Abstract: CD44 is an integral cell-surface glycoprotein. Overexpression of the CD44 standard (CD44st) and its variants (CD44v) has been implicated in transformation and progression of many cancer types. Here, we investigated expression of CD44st, CD44v3-7, CD44v7/8, and v10 in five human pancreatic tumor cell lines and normal human pancreatic duct cells transfected with the SV40 large T antigen. CD44st and its variant proteins were quantified using immunocytochemistry and flow cytometry. CD44v7 was expressed at low levels, whereas CD44st, CD44v3, CD44 v4, CD44v, and CD44v6 were expressed at moderate levels in all pancreatic tumor cell lines. In contrast, CD44v7/8 and CD44v10 were expressed at very low levels in two out of the five pancreatic tumor cell lines. Overall, staining of CD44st and CD44 variants was significantly weaker compared to another surface molecule, ICAM-1, reported to be overexpressed in pancreatic cancer cells. Furthermore, the SV40 large T transfected duct cells showed only a weak staining for CD44st, CD44v5, and CD44v6. To determine a possible mechanism for the regulation of surface expression of CD44st, v5 and v6, we incubated Panc-1 cells with bFGF, TGF-beta1, EGF, TNFalpha, and IFNgamma. Only IFNgamma affected the CD44 expression by down-regulation of CD44v6. The constitutive expression of CD44 variants seems to be associated with the malignant state of invasive carcinoma.

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Abstract: The quantitative comparison of spot patterns relies heavily on protein stains that do provide an appropriate dynamic range. Unfortunately most spot picking robot devices are still limited to nonfluorescent protein stains and the appropriate equipment is still quite expensive. These problems are solved by the application of a newly developed "GelMarker" that combines a spot picking robot device and a UV scanner. The "GelMarkers" are detectable in both the visible and UV range of light and permit the comparison of gel pictures taken by such different devices. The application of these "GelMarkers" together with the transformation of spot coordinates by using a "spot matching" procedure allows the automated excision of selected protein spots. The obtained picking accuracies are as good as those obtainable from visible stained gels due to the shape stability of the gels even over a longer time period.

Abstract: 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

Abstract: BACKGROUND AND AIMS: Programmed cell death via the Fas receptor/Fas Ligand and DR4, DR5/TRAIL plays a major role in tumor escape and elimination mechanisms. It also promises to be an effective therapy alternative for aggressive tumors, as has been recently shown for colon, breast, and lung cancer cells. We attempted to clarify the role of these molecules in aggressivity of pancreatic carcinomas and to identify possible pathways as targets for therapy. METHODS: Five pancreatic cell lines were investigated for the expression of FasL/Fas, DcR3, DR4, DR5/TRAIL, DcR1, DcR2, and other death pathways related molecules such as Bax, bcl-xL, bcl-2, FADD, and caspase-3 by flow cytometry, immunoblotting, and RT/PCR, both semiquantitative and real time (TaqMan). The susceptibility of these cell lines to apoptosis mediated by recombinant TRAIL was investigated. The effect of therapeutic agents (gemcitabine) on their susceptibility to TRAIL induced apoptosis was studied as well. RESULTS: Pancreatic adenocarcinomas expressed high levels of apoptosis-inducing receptors and ligands. They showed differential susceptibility to cell death induced by TRAIL, despite expressing intact receptors and signaling machineries. Treatment with commonly used therapeutic agents did not augment their susceptibility to apoptosis. This could be explained by the fact that they expressed differentially high levels of decoy receptors, as well as molecules known as inhibitors of apoptosis. CONCLUSIONS: The data suggest that pancreatic carcinoma cells have developed different mechanisms to evade the immune system. One is the expression of nonfunctional receptors, decoy receptors, and molecules that block cell death, such as bcl2 and bcl-xL. The second is the expression of apoptosis-inducing ligands, such as TRAIL, that could induce cell death of immune cells. The success in treating malignant tumors by recombinant TRAIL might apply to some but not all pancreatic tumors because of their differential resistance to TRAIL-induced cell death.

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Abstract: 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.

Abstract: 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.

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Abstract: This review addresses the history of the ras oncogene, the techniques used to detect molecular alterations in the ras oncogene, and the application of polymerase chain reaction (PCR)-based methods to determine point mutations in clinical samples of patients with pancreatic diseases, namely pancreatic carcinoma and chronic pancreatitis. The frequency of ras mutations in pancreatic carcinoma is high, ranging from 70 to almost 100%. The frequence of ras mutations in chronic pancreatitis, either in pancreatic tissue or pancreatic secretions, vary between 0 and 100%. This wide range in part may be owing to differences in sampling, DNA extraction, or PCR method. The meaning of a k-ras mutation is under debate. Taking into account the positivity of ductal hyperplasias in normal pancreas and ras mutations in normal appearing duct cells, this molecular finding may not mean anything. In contrast, ras mutations are associated with smoking, one acknowledged risk factor for pancreatic carcinoma. The need for large prospective cohort studies is emphasized.

Abstract: 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.

Abstract: The prognosis of pancreatic cancer is poor, and current medical treatment is mostly ineffective. The aim of this study was to design a new treatment modality in an animal model system. We describe here a novel treatment strategy employing a mouse model system for pancreatic carcinoma. Embryonal kidney epithelial cells were genetically modified to express the cytochrome P450 subenzyme 2B1 under the control of a cytomegalovirus (CMV) immediate early promoter. This CYP2B1 gene converts ifosfamide to its active cytotoxic compounds, phosphoramide mustard, which alkylates DNA, and acrolein, which alkylates proteins. The cells were then encapsulated in a cellulose sulphate formulation and implanted into preestablished tumors derived from a human pancreatic tumor cell line. Intraperitoneal administration of low-dose ifosfamide to tumor bearing mice that received the encapsulated cells results in partial or even complete tumor ablation. Such an in situ chemotherapy strategy utilizing genetically modified cells in an immunoprotected environment may prove useful for solid tumor therapy in man.

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Abstract: BACKGROUND/AIMS: Elevated levels of serum markers of extracellular matrix, i.e., the amino-terminal procollagen-III-peptide, hyaluronic acid and laminin are found in various diseases. The study aims to examine a panel of these parameters in patients with acute pancreatitis and correlate them with the course and severity of the disease. METHODOLOGY: We prospectively examined the time-course of procollagen-III-peptide, hyaluronic acid and laminin in 24 consecutive patients with acute necrotizing (n = 13) or edematous (n = 11) pancreatitis. Patients with chronic pancreatitis with (n = 10) or without (n = 17) acute pain, and 6 patients in complete remission after an episode of acute pancreatitis as well as healthy individuals served as controls. In addition, serum levels of collagen VI and undulin were followed in 10 and 9 patients with acute necrotizing pancreatitis, respectively. RESULTS: The serum concentrations of procollagen-III-peptide, hyaluronic acid and laminin were significantly higher at the onset of acute necrotizing pancreatitis compared to edematous pancreatitis and the controls. They returned to almost normal levels during the course of the disease when the patient recovered, but remained elevated in patients with a lethal course. Laminin allowed us to discriminate between patients with necrotizing pancreatitis from all other forms of pancreatitis on admission (specificity 82%, sensitivity 92%, PPV 86%, NPV 90%). Collagen type VI levels were 2-3-fold higher in sera of patients with acute pancreatitis than in healthy controls, whereas the results for undulin were inconclusive. CONCLUSIONS: Since markers of matrix metabolism (especially laminin) are differently elevated in acute necrotizing versus edematous pancreatitis, we suggest that they might be used as parameters for the outcome.

Abstract: CASE REPORT: We describe the case of a 43-year-old patient with a Burkitt lymphoma in the small intestine, who initially presented with abdominal discomfort, weight loss, constipation and neurological symptoms. DIAGNOSIS: In terms of differential diagnosis other inflammatory and tumorous diseases had to be considered. Non-Hodgkin lymphomas are common malignant afflictions of the GI-tract. The total body tumor burden is the principal determinant of prognosis. Serum lactate dehydrogenase (LDH) level is one parameter that reflects the tumor burden. On the other hand abdominal mass, bone marrow and central nervous system involvement are negative prognostic factors. Lymphomas can invade in the CNS at any time during the course of disease. This is generally associated with a poor prognosis if not treated immediately. TREATMENT: High doses of cyclophosphamid and MTX have been shown successful in the treatment of Burkitt lymphoma. Almost all relapses occur on therapy or shortly after termination of treatment. Therefore, criteria are needed to select patients of higher and lesser risk to protect the latter from the further intensification of therapy.

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Abstract: BACKGROUND: Development of fibrosis characterizes chronic pancreatitis. As it results from deposition of extracellular matrix (ECM) proteins such as hyaluronan (HA) or laminin, the release of these ECM components into blood or pancreatic secretion may be enhanced during the course of chronic pancreatitis. PATIENTS, MATERIAL AND METHODS: Using immunoassays for HA and laminin, the concentration for these ECM components was measured in pancreatic juice and serum samples from 20 patients with and 20 patients without chronic pancreatitis. Pancreatic calculi of varying size and weight obtained from 13 patients with chronic pancreatitis were also examined for the content of ECM components. Tissue samples from normal pancreas and those showing chronic pancreatitis were investigated immunocytochemically with an antibody to HA synthetase (HAS). RESULTS: After stimulation with secretin high levels of ECM components were found in the initial washout period in chronic pancreatitis patients as well as in controls. HA levels, however, were seen seven times higher in patients with chronic pancreatitis (mean +/- SEM; 734 +/- 301 vs. 95 +/- 15 microg/l; p < 0.01). Serum HA levels correlated with the duration of chronic pancreatitis and with the levels of HA in pancreatic juice. HA and laminin were detected in the supernatants of pancreatic calculi (laminin 0.70 +/- 0.30 U/l; HA 275 +/- 85 microg/l). Immunocytochemically, strong staining for HAS was found in the duct epithelium and in centroacinar cells of chronic pancreatitis specimens. CONCLUSION: Demonstration of increased amounts of HA in pancreatic juice of chronic pancreatitis patients stimulated with secretin suggests enhanced production of this ECM component in the chronically inflamed pancreas. The source of HA appears to be the pancreatic ductal epithelium.

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Abstract: 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.

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Abstract: Microencapsulation, as a tool for immunoisolation for allogenic or xenogenic implants, is a rapidly growing field. However most of the approaches are based on alginate/polylysine capsules, despite this system's obvious disadvantages such as its pyrogenicity. Here we report a different encapsulation system based on sodium cellulose sulfate and polydiallyldimethyl ammonium chloride for the encapsulation of mammalian cells. We have characterized this system regarding capsule formation, strength and size of the capsules as well as viability of the cells after encapsulation. In addition, we demonstrate the efficacy of these capsules as a "microfactory" in vitro and in vivo. Using encapsulated hybridoma cells we were able to demonstrate long-term release of antibodies up to four months in vivo. In another application we could show the therapeutic relevance of encapsulated genetically modified cells as an in vivo activation center for cytostatic drugs during tumor therapy.

Abstract: CONCLUSION: Preparation of DNA from pancreatic juice for subsequent polymerase chain reaction (PCR) is difficult, but manageable. The protocol presented offers a simple and fast solution. This method might be applicable to other complicated samples, such as saliva, would secretions, or stool washings. BACKGROUND: Of all the biological samples used for PCR amplification, pancreatic juice is the most problematic because of the presence of potential inhibitory substances and the amount of nucleases. This demands a DNA preparation procedure that is suitable for routine diagnostic PCR, and is therefore efficient and safe. This is particularly true for pancreatic juice obtained during routine endoscopy. METHODS: We describe here a simple method utilizing modified phenol/chloroform extraction and precipitation directly from native pancreatic juice suitable for diagnostic PCR applications, such as oncogenes. RESULTS: DNA could be prepared in quantitative amounts from routine endoscopic specimens. DNA could also be prepared from samples kept several days at room temperature.

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Abstract: The use of xenogenic or genetically engineered cell types in bioartificial liver support systems requires separation methods between the patients' blood and the liver support bioreactors that guarantee the sufficient transfer of pathophysiologically relevant substances but prevent complications. The present paper describes a new membrane separation system that is nearly impermeable to proteins but enables the exchange of water soluble and protein bound toxins by a special membrane and a recycled protein containing dialysate. Because the full range of toxins in hepatic failure has still not been identified, the value of this membrane separation method was evaluated clinically. Thirteen patients suffering from life threatening hepatic failure who had not responded to state of the art therapy were treated with this device, the molecular adsorbent recycling system (MARS). The overall survival rate was 69%. All patients showed positive response to the therapy, indicating that the presented membrane separator combines therapeutic effectivity with the highest safety criteria for the patient by cutting the exchange of substances below the level of proteins.

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Abstract: BACKGROUND/AIMS: The gastrointestinal peptide gastrin might be a stimulatory effector of the growth of human pancreatic cancer cell lines. Several authors suggest that this effect is mediated by CCKB receptors. However, no studies have examined human pancreatic cells for CCKB receptor expression. The study was designed to evaluate cell lines from human pancreatic cancer (n = 6), or normal pancreatic ducts (n = 2), pancreatic tumor and control tissues (n = 9) for CCKB receptor expression. METHODOLOGY: RNA from cell lines and tissues was subject to DNAse digestion, reverse transcription and amplification using CCKB receptor cDNA sequence specific oligonucleotides via polymerase chain reaction (PCR). After confirmation of CCKB receptor sequence, the products were examined using southern blot analysis. The relative expression was determined by photodensitometrical quantification and normalization to the housekeeping gene beta-actin. RESULTS: Six pancreatic tumor cell lines expressed the CCKB receptor. Amplification of CCKB receptor cDNA from 2 cell lines derived from non-malignant human pancreatic duct cell lines resulted in products with a significantly lower copy number. Tissues like stomach and brain served as positive controls. CONCLUSIONS: These results provide evidence that most human pancreatic cancer cell lines of ductal origin express CCKB receptor mRNA. Malignant pancreatic tissue may overexpress these receptors in comparison with normal tissue.

Abstract: BACKGROUND: Modern pancreatin preparations consist of enteric-coated microspheres to protect the enzymes from gastric acid. There are, however, no clinical trials comparing different sizes of pancreatin microspheres with regard to fat excretion and fat intake. AIM: To prove both equivalent efficacy and safety of conventional pancreatin microspheres and smaller pancreatin minimicrospheres in patients with exocrine insufficiency due to chronic pancreatitis. METHODS: In this prospective, randomized, double-blind, multicentre, crossover trial, patients with a stool fat excretion of > 7.5 g/day during a placebo period were randomly assigned either to the minimicrosphere/microsphere treatment sequence or vice versa. The primary end-point was the coefficient of fat absorption, which was calculated from fat excretion and fat intake during the course of a standardized diet. Stool weight, clinical symptoms and the safety of the preparations were also evaluated. RESULTS: Thirty-seven patients entered the study, of whom 23 fulfilled the criteria for the crossover period. In the per protocol analysis (n=18), the 90% confidence intervals for the coefficient of fat absorption of both crossover periods lay entirely within the equivalence range (P=0.02). The intention-to-treat analysis revealed similar results, but the equivalence range was slightly missed (P=0.07). Similar results were obtained for the secondary parameters and the reported adverse events. CONCLUSIONS: Pancreatin minimicrospheres have been shown to be equally effective as microspheres in improving the coefficient of fat absorption in patients with exocrine insufficiency due to chronic pancreatitis.

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Abstract: BACKGROUND/AIMS: Chronic pancreatitis is histologically characterized by an extended fibrosis and infiltration of leukocytes. We intended to differentiate the infiltration to evaluate the inflammatory process. METHODS: Samples of tissues of normal pancreas (NP, n = 12), of chronic pancreatitis (CP, n = 7), and pancreatic tissues surrounding pancreatic carcinoma (CA, n = 7) were investigated by immunohistochemical staining using the APAAP technique. RESULTS: In normal pancreas, mononuclear cells (47.1 +/- 26.0 cells/mm2) were observed with a predominance of macrophages (56.3%) and T lymphocytes (31.3%) which were differentiated in CD8+ lymphocytes (9.3 +/- 7.2 cells/ mm2) and CD4+ lymphocytes (6.7 +/- 3.2 cells/mm2). Rarely, plasma cells (5.3%) and B lymphocytes (7.1%) could be detected. In pancreatic tissue of patients with CP and in CA there was a significant increase of mononuclear cells to 264.4 +/- 120.3 cells/mm2 and 284.3 +/- 67.8 cells/mm2, respectively. In both diseases percentages of T lymphocytes (CP: 50.5%; CA: 48.1%) were higher than in normal controls. CD4+/CD8+ ratio of 0.77 in CP and 0.82 in CA demonstrated a predominance of CD8+ cells compared to the peripheral blood. In NP and CA, nearly all T lymphocytes expressed CD45R0 identifying memory cells, while only 58% of T lymphocytes were CD45R0 positive in CP. CONCLUSION: Our data suggest that the investigated cases of CP were of a common inflammatory type rather than due to an autoimmunological reaction. CD8+ T lymphocytes were the predominant T cell subset in the inflammatory infiltrates in both CP and CA.

Abstract: The prognosis of pancreatic adenocarcinoma is poor and current treatment ineffective. A novel treatment strategy is described here using a mouse model system for pancreatic cancer. Cells that have been genetically modified to express the cytochrome P450 2B1 enzyme are encapsulated in cellulose sulphate and implanted into pre-established tumours derived from human pancreatic cells. Cytochrome P450 2B1 converts the chemotherapeutic agent ifosfamide to toxic metabolites. Administration of ifosfamide to tumour-bearing mice that were recipients of implanted encapsulated cells results in partial or even complete tumour ablation. These results suggest that in situ chemotherapy with genetically modified cells in an immunoprotected environment may prove useful for application in man.

Abstract: The prognosis of pancreatic adenocarcinoma is poor and current treatment is for the most part ineffective. We describe here a novel treatment strategy using a mouse model system for pancreatic cancer. Human embryonic epithelial cells have been genetically modified to express the cytochrome P450 2B1 enzyme under the control of a CMV immediate-early promoter. This CYP2B1 gene converts oxazaphosphorines (ifosfamide or cyclophosphamide) to their active cytotoxic compounds, phosphoramide mustard, which alkylates DNA, and acrolein, which alkylates proteins. A number of assays were performed to demonstrate the CYP2B1 gene function as well as toxic effects on neighbouring cells (bystander effect). The cells were then encapsulated in a cellulose sulphate formulation shown to be well tolerated in the pancreas of immunocompetent mice, and injected 1 cm away from pre-established tumours derived from a human pancreatic tumour cell line (PaCa-44). Intraperitoneal administration of low-dose ifosfamide to tumour bearing mice that received the encapsulated cells results in partial or even complete tumour ablation. Such an in situ chemotherapy strategy utilizing genetically modified cells in an immunoprotected environment may prove useful for solid tumour therapy in man.

Abstract: BACKGROUND: The predominance of secretory IgA (S-IgA) in intestinal secretions compared with blood is well established, but concentrations of this protein in pancreatic juice and its origin, especially in chronic pancreatitis, are unknown. AIMS: To investigate the role of S-IgA in chronic pancreatitis. PATIENTS: Twenty one patients with chronic pancreatitis (group I), three patients with proven malignancies (group II), and 12 patients without pancreatic disease (group III). METHODS: Pure human pancreatic juice was collected endoscopically in four fractions after consecutive stimulation with secretin and cholecystokinin (CCK). Samples were analysed for S-IgA, protein, trypsinogen, and proteolytic activity. RESULTS: The S-IgA level was significant increased in fraction 1 of pancreatic juice of group I (1210 (1411) ng/ml) compared with controls (33 (70) ng/ml). Protein concentrations and trypsinogen content were lower in group I than in the other groups. Proteolytic activity could be observed in 53% of all 133 pancreatic juice samples, but in 87% of fraction 1. In pancreatic tissue of three patients with chronic pancreatitis both IgA and secretory component were detected by immunohistology. Expression of the secretory component by human pancreatic epithelial cells was increased in patients with chronic pancreatitis compared with normal controls. The concentration of S-IgA in pancreatic juice did not correlate with the serum S-IgA level. In contrast, serum levels of S-IgA were decreased in patients with chronic pancreatitis. CONCLUSION: There are high levels of S-IgA in human pancreatic juice following chronic inflammation and a protective role is suggested for this immunoglobulin.

Abstract: BACKGROUND/AIM: The suggestion that estimation of faecal elastase 1 is a valuable new tubeless pancreatic function test was evaluated by comparing it with faecal chymotrypsin estimation in patients categorised according to grades of exocrine pancreatic insufficiency (EPI) based on the gold standard tests, the secretin-pancreozymin test (SPT) and faecal fat analysis. METHODS: In 64 patients in whom EPI was suspected, the following tests were performed: SPT, faecal fat analysis, faecal chymotrypsin estimation, faecal elastase 1 estimation. EPI was graded according to the results of the SPT and faecal fat analysis as absent, mild, moderate, or severe. The upper limit of normal for faecal elastase 1 was taken as 200 micrograms/g, and for faecal chymotrypsin 3 U/g stool. Levels between 3 and 6 U/g stool for faecal chymotrypsin are usually considered to be suspicious for EPI. In this study, both 3 and 6 U/g stool were evaluated as the upper limit of normal. RESULTS: Exocrine pancreatic function was normal in 34 patients, of whom 94, 91, and 79% had normal faecal elastase 1 and faecal chymotrypsin levels (< 3 U/g and < 6 U/g) respectively. Thirty patients had EPI, of whom 53, 37, and 57% had abnormal faecal enzyme levels (differences not significant). When EPI was graded as mild, moderate, or severe, 63% of patients had mild to moderate EPI, and 37% had severe EPI. In the latter group, between 73 and 91% of patients had abnormal faecal enzymes. In the group with mild to moderate EPI, abnormal test results were obtained for both faecal enzymes in less than 50% of the patients (differences not significant). Some 40% of the patients had pancreatic calcifications. There were no significant differences for either faecal enzyme between the two groups with and without pancreatic calcifications. In 62% of the patients who underwent an endoscopic retrograde cholangiopancreatography (ERCP), abnormal duct changes were found. Again, there were no significant differences for either faecal enzyme between the two groups with abnormal and normal ERCP. CONCLUSION: Estimation of faecal elastase 1 is not distinctly superior to the traditional faecal chymotrypsin estimation. The former is particularly helpful only in detecting severe EPI, but not the mild to moderate form, which poses the more frequent and difficult clinical problem and does not correlate significantly with the severe morphological changes seen in chronic pancreatitis.

Abstract: Preparation of pancreatic duct epithelial cells from adult organs is possible by limited digestion and outgrowth of cells. These primary cells are mitotically active for only a short period. Therefore transfection with SV40 large-T antigen is one method to obtain an immortalized cell clone. Because the transfection efficacy of primary cells with conventional vectors is comparatively low, our aim was to develop conditions with improved transfection rates. Best transfection rates (approximately 6% of the resting cells) were obtained by using the BES buffered saline (BBS) calcium phosphate (Ca-P) coprecipitation technique at low pH. By using these optimized transfection parameters, primary cultures of human pancreatic duct epithelial cells were successfully transfected with the plasmid pSV3neo, bearing the large- and small-T antigen of SV40. A G 418 resistant clone (E4) was maintained in culture for 14 months before reaching terminal crisis.

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Abstract: We report the case of a 61-year-old woman, who suffered from abdominal pain, nausea, vomiting and fever. She had a past medical history of acute rheumatism, pyelonephritis and systemic scleroderma. Since 1971 she was hospitalized many times because of recurrent abdominal pain with increased serum amylase and lipase values. On admission, she was in distress and demonstrated clinical signs of acute pancreatitis. The link between systemic lupus erythematosus and acute pancreatitis is discussed in view of the reported cases of the world literature.

Abstract: BACKGROUND & AIMS: Regulatory mechanisms in chronic pancreatitis finally resulting in pancreatic fibrosis cannot be studied sufficiently in human pancreas. Results of a new pancreatitis model in rats suitable for investigation of the processes leading to pancreatic fibrosis are presented. METHODS: Experimental pancreatitis was induced by intravenous application of 8 mg/kg body wt dibutyltin dichloride. Pancreatitis was characterized by histology, serum parameters, and immunohistochemistry, detecting inflammatory cells. Gene expression of collagen type I and transforming growth factor beta1 was shown by Northern blot analysis. RESULTS: Dibutyltin dichloride induced an acute edematous pancreatitis within 24 hours. Extensive infiltration with mononuclear cells could be observed after day 7 followed by the development of fibrosis. Parallel to the cell infiltration, an upregulation of messenger RNA-encoding collagen type I and transforming growth factor beta1 could be shown. An active inflammatory process could be shown until the end of the observation period, i.e., 2 months. CONCLUSIONS: The findings suggest that dibutyltin dichloride-induced pancreatitis in rats is suitable to study cellular interactions and mediators involved in the development of pancreatic fibrosis.

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Abstract: BACKGROUND AND AIM: Complications in chronic pancreatitis, such as duct occlusion due to stenosis or stones can be treated by interventional endoscopic procedures. The benefit of such procedures and the effect on the clinical course is still under debate. Therefore, it was the aim of this study to analyze the effect of endoscopic interventional procedures in more detail with respect to technical and clinical benefit. PATIENTS AND METHODS: 100 patients with chronic pancreatitis (cP) were studied retrospectively. In 58 patients an indication for interventional endoscopic therapy was given (45 +/- 12 yrs; 46 M, 12 F; cP 1 degree: n = 1, cP II degree: n = 8, cP III degree: n = 49). The patients were allocated to three groups: stenosis (n = 18), pancreatic duct stones (n = 18) and stenosis and pancreatic duct stones (n = 20). In two patients no visualization of the main pancreatic duct was performed due to cholestasis that was treated primarily. In total, 295 endoscopic procedures were performed (EPT, duct dilatation, plastic endoprothesis, ESWL, mechanical lithotripsy). Technical success was 95%. All stenoses could be dilated or bridged by plastic stents. Fragmentation of main pancreatic duct stones was achieved in 92%. The overall complication rate of all 319 endoscopies (fever, bleeding, stent dislocation) was 15.8%. Five patients had to undergo surgery, however, not as a direct consequence of complications from or unability of endoscopic procedures. 86% of the patients reported complete pain relief after the endoscopic-interventional procedures and 62% during the follow-up interval (7.4 +/- 6.3 months). 59% of the patients with weight loss and 58% of the patients with initially stable weight experienced a weight gain following endoscopic-interventional therapy. Individual patients showed improvement of endocrine or exocrine pancreatic function. 82% of the patients did not require inhouse treatment or emergency admission to the hospital whereas three admissions on average were recorded prior to endoscopic interventional treatment. Therefore, we conclude that a subset of patients does benefit from endoscopic interventional therapy of complications of chronic pancreatitis. However, a controlled prospective study is still mandatory.

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Abstract: There has been a slow but steady improvement in the results of treatment for exocrine pancreatic cancer in the last decade. One obstacle to further improvement has been lack of standardization for reporting of results, from classification to surgery and follow-up. This four-day symposium was organized in order to bridge the clinical praxis of curatively intended radical pancreatic surgery to scientific evaluation of its results. The meeting comprised four workshops, three special lectures, and seven plenary sessions.

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Abstract: Extracellular matrix proteins (ECM) may influence cellular differentiation via their receptors, the integrins. We recently presented evidence that ductal adenocarcinomas of the pancreas are able to produce ECM in vitro and in vivo (Br J Cancer 1994;49:144-51). This study examines whether pancreatic carcinoma cells are able to interact with ECM by expressing functionally active integrins. In eight human pancreatic tumor cell lines (AsPC-1, BxPC-3, CAPAN-1 and -2, PANC, PaTu-2, -3, and -44) and six xenografted tumors RNA and protein expression of integrin subunits alpha 5 (fibronectin receptor), alpha 6 (laminin receptor), and alpha V (vitronectin receptor) was investigated. In addition, alpha 1-alpha 6 and alpha V as well as beta 1-beta 4 were studied by fluorescence-activated cell sorter analysis. Integrin function was tested by attachment assays. alpha 2, alpha 3, alpha 5, alpha 6, and alpha V as well as beta 1, beta 3, and beta 4 were expressed, at both the RNA and the protein level, by all pancreatic tumors in vitro and in vivo. The tumor cell lines showed dose dependent adhesion to collagen, fibronectin, and laminin. Integrin expression could be modulated in part by serum depletion. None of the tumors showed alpha 1, alpha 4, and beta 2. Tumor cell differentiation or production of ECM was not correlated with integrin expression. Pancreatic ductal adenocarcinomas express a certain pattern of functionally active integrins that enable interaction with most matrix proteins, e.g., collagen, fibronectin, and laminin. Pancreatic adenocarcinomas possess both the ligands and the receptors of the extracellular matrix network. We speculate that through both classes of molecules, the tumor cells may bind to fibroblasts and probably stimulate their growth. However, it remains unclear whether and how integrin-ECM interaction exerts an influence on tumor cell differentiation.

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Abstract: OBJECTIVE: To investigate the presence of extracellular matrix (ECM) proteins in human bile and gallstones and to determine whether they play a role in gallstone formation. METHODS: ECM components [procollagen-III-peptide (P-III-P), laminin, and hyaluronic acid] in bile from patients with (n = 22) and without (n = 6) gallstone disease were investigated by immunoassay. Bile, gallstones, and serum were assayed for extracellular matrix components in an additional 19 patients with gallstone disease and gallstones were analysed in a third set of 26 patients. The expression of hyaluronic acid synthetase in bile duct and gall bladder epithelia was investigated by immunocytochemistry. RESULTS: Hyaluronic acid levels were significantly elevated in hepatic and gall bladder bile, but not in the serum of patients with compared with those without gallstone disease (137 versus 81 micrograms/l, respectively; P < 0.05). No differences were found between hepatic and gall bladder bile. Procollagen-III-peptide and laminin were detected in the hepatic bile of patients in both groups. Laminin levels were higher in gall bladder bile than in serum in all patients and measurable amounts of hyaluronic acid were found in gallstones. The amount of hyaluronic acid was inversely correlated to the volume of the gallstone, i.e., the smallest gallstones contained the highest levels of hyaluronic acid. No procollagen-III-peptide or laminin was found in the gallstones. Immunocytochemistry of the epithelial cells of bile duct and gall bladder mucosa stained strongly for hyaluronic acid synthetase. CONCLUSIONS: Hyaluronic acid as a progenitor of ECM can be detected in bile and is significantly elevated in patients with gallstone disease. Small gallstones contain more hyaluronic acid than large stones, suggesting that hyaluronic acid may play a role in gallstone formation, particularly since it is produced by the epithelial lining of bile ducts and is found in gall bladder mucosa.

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Abstract: A pilot study was undertaken to test whether combining the polymerase chain reaction with restriction fragment length polymorphism is suitable for the routine diagnosis of carcinoma of the pancreas. The method makes it possible to recognize point mutations in codon 12 of the Ki-ras oncogene. 60 cytological specimens from the pancreatobiliary tract, the bronchopulmonary system (by bronchoalveolar lavage or from pleural effusion) and ascites were tested. Results from nine pancreas carcinoma cell lines served as control. A PCR product was successfully amplified in all cell lines and 47 of the clinical specimens. In all eight samples in which a mutated Ki-ras oncogene was demonstrated, there was at least a suspicion of malignancy by cytological examination. A mutation was also found in four of five pancreas carcinomas. But no mutation was found in one, clinically certain, case of pancreas carcinoma. The described method is an elegant and, most of all, rapid means to complement and optimize any cytological diagnosis.

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Abstract: Pancreatic ductal adenocarcinomas are characterised by a dense connective tissue reaction. To test the hypothesis that stroma components are synthesised and produced by the tumour cells themselves, eight cell lines as well as six xenografted tumours from human ductal adenocarcinomas of the pancreas were examined for the expression of extracellular matrix proteins (ECM), using cDNA probes and antibodies to collagen types I, III and IV, vitronectin, fibronectin, undulin and laminin. All tumour cell lines (CAPAN-1, CAPAN-2, AsPC-1, BxPC-3, PANC-1, PaCa-2, PaCa-3, PaCa-44) and xenografted human pancreatic tumours expressed at least one of the examined ECM at the RNA (collagen type IV > laminin = fibronectin = vitronectin > collagen type III > undulin > collagen type I) or protein level (collagen type IV = collagen type III > vitronectin > laminin > collagen type I = fibronectin > undulin). In nude mouse tumours expression of laminin and collagen I was most pronounced in well-differentiated carcinomas. In a few tumours, collagen type III, vitronectin and undulin were expressed on the luminal side of the neoplastic glands, suggesting loss of normal polar differentiation. Incubation with fetal calf serum modulated ECM RNA levels to a varying extent in all but one cell line (AsPC-1). The results suggest that human pancreatic ductal adenocarcinomas cells are capable of synthesising and producing extracellular matrix proteins in vitro and in vivo, but that the extent and pattern of ECM expression differs between the various tumours and conditions tested.

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Abstract: To establish a suitable control for pancreatic tumor cell lines, we have isolated and cultured primary human pancreatic duct cells from transplant donors. Duct cells were isolated by dissecting the main pancreatic duct and first-degree branches and enzymatic digestion. Aggregates of cells were cultured for 1 up to 5 weeks and monitored for changes in morphology and growth by phase contrast microscopy. Contaminating fibroblasts were mechanically removed from day 4 on and by cloning of epithelial cells. Cultured cells were characterized by phase contrast microscopy, electron microscopy, and immunofluorescence with antibodies against intermediate filaments (cytokeratins, vimentin, desmin), mucins (Du-Pan-2, CA 19-9), carbonic anhydrase II, acinar cell enzymes (amylase, lipase, trypsin), and islet cells. About 90% of the cultured cells could be identified as ductal epithelial cells by their expression of cytokeratins, mucins, and carbonic anhydrase II. These cells showed the ultrastructural features of duct cells. After 3-5 weeks of culture, most of the cultured cells showed co-expression of cytokeratins and vimentin in addition to duct cell markers. About 10% of cells were contaminating fibroblasts (vimentin positive, cytokeratin negative). The cultured normal human duct cells as the postulated cells of origin of the pancreatic adenocarcinoma may serve as a useful control for cultured pancreatic tumor cell lines.

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Abstract: The natural course of the classical symptoms of chronic pancreatitis, i.e. pain, exocrine and endocrine pancreatic insufficiency, was followed up in 335 patients over a median of 9.8 years (mean 11.3 +/- 8.3 years). Pain relief was not obtained in the majority of patients, even after a long-term observation of > 10 years, and severe exocrine/endocrine insufficiency, severe duct abnormalities and pancreatic calcifications developed. Alcohol abstinence failed to have a significant beneficial effect on pain. Pancreatic surgery led to pain relief immediately after operation, but later on the pain course between operated and nonoperated patients was not significantly different. Repeated exocrine pancreatic function tests in 143 patients showed that functional exocrine impairment came to a standstill (46%), or improved (11%). At the end of observation, 22% of 335 patients still had normal endocrine function and only 40% required insulin treatment. Alcohol abstinence had a significant beneficial effect on endocrine, but not on exocrine pancreatic insufficiency. Chronic pancreatitis led to a sharp increase in unemployment and retirement. Pancreatic carcinoma occurred in 3% and extrapancreatic carcinoma in 4%. The mortality rate within the observation period was 22%, pancreatitis-induced complications accounted for 13% of these deaths.

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Abstract: Human cytomegalovirus (HCMV) was recently demonstrated in the pancreas of about half the patients with type 2 diabetes mellitus in the absence of mumps, rubella or Coxsackie B virus. The present study addresses the question as to whether type 2 diabetes with an HCMV-positive pancreas differs from those with HCMV-negative pancreases with respect to age, sex, treatment, duration of disease, volume densities of B-cells and D-cells, mRNA levels of insulin and somatostatin, islet amyloid peptide deposits and major histocompatibility complex (MHC) class I and class II gene transcription, and protein expression. HCMV-positive type 2 diabetic patients showed a tendency towards a shorter duration of disease and significantly increased levels of MHC class II on RNA. In addition, expression of MHC class II product (HLA-DR) was identified in duct epithelial cells and/or islet cells in 9 diabetic pancreases and in 2 non-diabetic glands. No MHC class I expression could be detected. No other clinical differences between HCMV-positive and HCMV-negative glands were found. All 10 HCMV-positive diabetics showed a strong expression of MHC class II mRNA in the pancreas. By immunocytochemistry, 4 of 10 demonstrated expression on the islets; three of ten also expressed MHC DR beta on ductal cells. This finding might be related to the viral infection, as only 2 of the 9 HCMV-negative patients were HLA-DR beta positive and none of the non-diabetic controls showed increased levels of MHC class II mRNA. These data suggest that HCMV infection in the pancreas is associated with type 2 diabetes. However, no conclusions as to a role of this virus in the aetiopathology of type 2 diabetes can be drawn at present.

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