Johanna Laukkarinen

Autophagy is needed for the growth of pancreatic adenocarcinoma and has a cytoprotective effect against anticancer drugs

BACKGROUND AND AIM Autophagy is a regulated process of degradation and recycling of cellular constituents. The role of autophagy in pancreatic cancer is still not clear. Some studies indicate that in pancreatic cancer autophagy exerts cytoprotective effects, whereas others suggest that autophagy positively contributes to cell death by enhancing cytotoxicity of anticancer drugs. The aim of this study was to investigate the role of autophagy in pancreatic cancer, and to provide insights into new strategies for treatment. MATERIALS AND METHODS Pancreatic cancer cell lines PANC-1 and BxPC-3 were treated with anticancer drugs (5-fluorouracil or gemcitabine) alone and in combination with autophagy inhibitors (chloroquine or wortmannin). Biopsy samples were retrieved from patients from pancreatic normal tissue and adenocarcinoma. Western blot of microtubule-associated protein 1 light chain 3 (LC3)-II was performed to investigate the degree of autophagy and cell proliferation was assessed by a crystal violet assay. RESULTS Autophagy was active in PANC-1 cells under basal conditions. Autophagy was significantly induced in pancreatic ductal adenocarcinoma compared to healthy pancreatic tissue in patients. Inhibition of autophagy by chloroquine suppressed the growth of PANC-1 and BxPC-3. Autophagy was markedly increased after treatment with 5-fluorouracil or gemcitabine. Inhibition of autophagy by chloroquine potentiated the inhibition of cell proliferation of PANC-1 and BxPC-3 by 5-fluorouracil and gemcitabine. CONCLUSIONS Our results with pancreatic cancer cell lines and human pancreatic adenocarcinoma suggest that autophagy contributes to pancreatic cancer cell growth. Autophagy has a cytoprotective effect against 5-fluorouracil and gemcitabine in pancreatic cancer cells. Combination therapy of these anticancer drugs and chloroquine should be investigated.

Biocompatibility of a New Bioabsorbable Radiopaque Stent Material (BaSO4 Containing Poly-L,D-Lactide) in the Rat Pancreas

Background/Aim: During recent years, we have been developing bioabsorbable biliary stents with promising experimental results. In developing pancreatic stents before long-term experiments, the acute toxicity to the pancreas of a bioabsorbable, radiopaque polylactide (PLA 96-barium sulfate, BaSO4) stent material was investigated. Methods: The pancreas of 65 Sprague-Dawley rats was exposed either to radiopaque stent material [PLA 96 with 25% (w/w) of BaSO4], radiolucent stent material (PLA 96), or inert steel by inserting a 5-mm-long (diameter 0.3 mm) fiber/stick of material into the pancreas after laparotomy under general anesthesia. Pancreatic tissue specimens and blood samples were taken after 1, 3, 7, and 21 days for histological examination and amylase activity measurements. Samples were also taken from 5 baseline (control) rats without exposing to any materials. Results: The baseline serum amylase activity was normal, and no histological changes in the pancreas were observed. A significant increase (mean ± SE) in the serum amylase activity was observed only on day 1 in the animals having radiopaque stent material (PLA 96-BaSO4; 5,845 ± 1,135 U/l), steel (4,946 ± 667 U/l), or radiolucent stent material (PLA 96; 7,684 ± 667 U/l) inserted. There was slightly more acinar cell necrosis on day 7 in the steel group than in the radiopaque stent (PLA 96-BaSO4) group (p = 0.028). Conclusions: Radiopaque stent material (PLA 96-BaSO4) was not more toxic than the reference steel material in the rat pancreas during the 21-day observation period and is thus applicable for further in vivo experiments when developing pancreatic bioabsorbable stents.

A novel technique for hepaticojejunostomy for nondilated bile ducts: a purse-string anastomosis with an intra-anastomotic biodegradable biliary stent

In non-dilated bile ducts, performing a well-functioning hepaticojejunal anastomosis (HJ) may be challenging. We investigated a novel technique for small-caliber HJ: a purse-string anastomosis with an intra-anastomotic biodegradable stent. HJ was performed randomly either conventionally with interrupted sutures without any stent (n = 5; conventional) or using the novel purse-string technique with a 4-mm caliber polylactide-barium sulfate biodegradable biliary stent (n = 4; pursestring + stent) in minipigs with bile ducts 3.5-4.0 mm in caliber. The anastomosis creation time was not different in the groups. In the conventional group 2 complications occurred: 1 early anastomotic leakage, and 1 late anastomotic stricture. The remaining animals (3/5 in conventional, and 4/4 in purse-string + stent group) had normal liver histology and function, and developed no signs of complications during the 6-month follow-up. All biodegradable stents disappeared by 3 months. At 6 months, the HJ caliber was smaller in the conventional (5 [1-9] mm) than in the purse-string + stent group (12 [4-15] mm; P < .05). We conclude that this novel HJ technique is easy and safe to perform, and ensures a well-functioning anastomosis in nondilated bile ducts.

Abstinence after First Acute Alcohol-Associated Pancreatitis Protects Against Recurrent Pancreatitis and Minimizes the Risk of Pancreatic Dysfunction

AIMS To determine the recurrence of pancreatitis and subsequent pancreatic function in patients who stop drinking after the first episode of alcohol-associated pancreatitis. METHODS Of a total of 118 patients suffering from their first alcohol-associated pancreatitis, 18 (all men, age median 47 (27-71) years) met the inclusion criterion for abstinence during follow-up. The criterion for abstinence was alcohol consumption <24 g per 2 months (self-estimated), which is in line with questionnaires eliciting alcohol consumption and dependency (Alcohol Use Disorders Identification Test < 8 and Short Alcohol Dependence Data < 9). Recurrent attacks of acute pancreatitis were studied. Smoking, body mass index and laboratory tests detecting heavy consumption of alcohol were recorded. Blood and faecal tests were studied to assess endocrine and exocrine pancreatic function. RESULTS During a mean follow-up time of 5.15 (1.83-9.13) years and a total of 92.7 patient-years, there were no recurrent attacks of acute pancreatitis among the 18 abstainers. Two patients had diabetes prior to and one was diagnosed immediately after the first episode of acute pancreatitis. One patient had impaired glucose metabolism at 2 years. Two patients had low insulin secretion in glucagon-C-peptide test, one at 4 years and the other at 5 years. Only one patient (6%) maintained low elastase-1 activity during the abstinence follow-up. Of the 100 non-abstainers, 34% had at least one recurrence during the follow-up. CONCLUSION Regardless of the mediator mechanisms of acute alcoholic pancreatitis, abstinence after the first episode protects against recurrent attacks. Pancreatic dysfunction is also rare among abstinent patients.

European evidence-based guidelines on pancreatic cystic neoplasms

Evidence-based guidelines on the management of pancreatic cystic neoplasms (PCN) are lacking. This guideline is a joint initiative of the European Study Group on Cystic Tumours of the Pancreas, United European Gastroenterology, European Pancreatic Club, European-African Hepato-Pancreato-Biliary Association, European Digestive Surgery, and the European Society of Gastrointestinal Endoscopy. It replaces the 2013 European consensus statement guidelines on PCN. European and non-European experts performed systematic reviews and used GRADE methodology to answer relevant clinical questions on nine topics (biomarkers, radiology, endoscopy, intraductal papillary mucinous neoplasm (IPMN), mucinous cystic neoplasm (MCN), serous cystic neoplasm, rare cysts, (neo)adjuvant treatment, and pathology). Recommendations include conservative management, relative and absolute indications for surgery. A conservative approach is recommended for asymptomatic MCN and IPMN measuring <40 mm without an enhancing nodule. Relative indications for surgery in IPMN include a main pancreatic duct (MPD) diameter between 5 and 9.9 mm or a cyst diameter ≥40 mm. Absolute indications for surgery in IPMN, due to the high-risk of malignant transformation, include jaundice, an enhancing mural nodule >5 mm, and MPD diameter >10 mm. Lifelong follow-up of IPMN is recommended in patients who are fit for surgery. The European evidence-based guidelines on PCN aim to improve the diagnosis and management of PCN.

Covered self-expanding metal stents may be preferable to plastic stents in the treatment of chronic pancreatitis-related biliary strictures: a systematic review comparing 2 methods of stent therapy in benign biliary strictures.

Background: Covered self-expanding metal stents (CSEMS) are being increasingly used in the endoscopic treatment of benign biliary strictures (BBS). There is no solid evidence yet to support their routine use. Goals: To evaluate feasibility, success rate, and complications of CSEMS compared with multiple plastic stents (PS) in BBS in a systematic review. Study: A systematic search of electronic databases (Medline, Scopus, and Embase) for studies published from 2000 to 2012 combined to hand-search of reference lists resulted 4977 articles. Out of 99 potentially relevant studies selected for full-text review, 12 CSEMS (376 patients) and 13 PS studies (570 patients) met the final inclusion criteria. A systematic review comparing the 2 methods was made using proportion meta-analysis. Results: A tendency to successful use of CSEMS in strictures related to chronic pancreatitis (CP) was shown: clinical success of 77% and 33% [95% confidence interval (CI), 61%-94% vs. 4%-63%, P=0.06] was achieved with CSEMS and PS at 12 months follow-up, respectively. There were no differences in the success rates of other etiologies except CP or in the early complications. In CSEMS, incidence of late adverse events was lower in CP-related strictures (3% vs. 67%, 95% CI, 0%-13% vs. 17%-99%, P=0.02). The median number of endoscopic retrograde cholangiopancreatographies was lower with CSEMSs: 1.5 versus 3.9 (P=0.002). Conclusions: Improved clinical success with fewer endoscopic sessions and corresponding complication rate may be achieved with CSEMS treatment compared with PS in BBS secondary to CP. In other BBS etiologies, this systematic review remains inconclusive.

Spyglass single-operator peroral cholangioscopy seems promising in the evaluation of primary sclerosing cholangitis-related biliary strictures

Abstract Early diagnosis of dysplastic changes and exclusion of cholangiocarcinoma (CCA) in patients with primary sclerosing cholangitis (PSC) remain a major clinical challenge. Although SpyGlass single-operator cholangioscopy (SOC) appears effective in diagnostics of indeterminate biliary strictures, there are only few studies on its safety in PSC-related strictures. Objective. The aim of this study was to assess the clinical feasibility of SOC and directed biopsies, flow cytometry, and brush cytology in PSC patients. Materials and methods. Eleven consecutive patients (median age 45 years, 5 females) undergoing SOC for progression of PSC in a single tertiary center were included in a prospective observational study. Results. Brush sample and directed biopsies were successfully acquired from strictures in all cases. Samples were adequate for cytological and histological diagnosis in 9 (82%) and 10 patients (91%), respectively. There were two cases of pancreatitis. In one patient, flow cytometry showed aneuploidy, which resulted in closer follow-up. Conclusions. SpyGlass SOC and directed biopsies seem to offer a feasible and promising method in evaluation of PSC-related strictures. However, the long-term prognostic value it adds to cytology and flow cytometry remains to be assessed in future trials.

Mechanism of the Prorelaxing Effect of Thyroxine on the Sphincter of Oddi

Background: Disturbances in the function of sphincter of Oddi (SO) may prevent normal bile flow and thus enhance the probability of common bile duct stone (CBDS) formation. Previously, we have shown increased prevalence of hypothyroidism in CBDS patients. Methods: In animal (pig) experiments, thyroxine (T 4 ) and triiodothyronine have a specific inhibitory effect on SO contractility, which raises the possibility that the lack of this prorelaxing effect in hypothyroidism could, at least in part, explain the increased prevalence of CBDS. The aims of the present study were to investigate, whether human SO reacts similarly to T 4 , and to study the mechanisms of the T 4 prorelaxing effect. Results: We found that T 4 had similar inhibitory effects on both human and pig SO contractions. The T 4 effect was dosedependent, and maximum was observed in 30 min. The maximal prorelaxing effect was achieved with 0.1 nM T 4 concentration, the effect of the physiological T 4 concentration (0.01 nM) being about half of the maximal effect. Addition of α -adrenoceptor antagonist phentolamine, β -adrenoceptor antagonist propranolol, nitric oxide (NO)-synthesis inhibitor L-NAME, nerve conductance blocker tetrodotoxin, or cyclooxygenase inhibitor diclofenac did not affect the T 4 -induced inhibition of contraction. Addition of transcription inhibitor actinomycin D or translation inhibitor cyclophosphamide partially reversed the T 4 -induced inhibition of contraction. Addition of K + channel blocker glibenclamide totally reversed the T 4 -induced inhibition of contraction. In Western blotting, the thyroid hormone receptor (TR) antibody recognized 53 kDa and 58 kDa proteins, corresponding to β 1 and β 2 isoforms of TR, in the human SO tissue. Conclusions: We conclude that T 4 has a direct prorelaxing effect on human SO that expresses TR β 1 and β 2. This effect is mediated through a transcriptional mechanism that requires new mRNA and protein synthesis and subsequently leads to the activation of K + channels.

Colorectal cancer and cholangiocarcinoma in patients with primary sclerosing cholangitis and inflammatory bowel disease

Abstract Objective. Inflammatory bowel disease (IBD) patients with concomitant primary sclerosing cholangitis (PSC) carry an increased risk of colorectal cancer (CRC) and cholangiocarcinoma (CCA). We evaluated the relative risk of these malignancies in IBD patients with PSC, who had been under regular surveillance. Material and methods. The survey involved a cohort of 51 patients with IBD and concomitant PSC. All patients had been under regular surveillance for a median of 19 years. The standardized incidence ratios (SIRs) of CRC and CCA were estimated between 1986 and 2007. Results. During the follow up, three patients (5.9%) developed CRC and five patients (9.8%) developed CCA. SIRs were 20.71 (95% confidence interval [CI]: 5.62–79.70) and 916.63 (95% CI: 297.88–2140.99), respectively. The median age at diagnosis of CRC was 39.5 years. All patients with PSC were <45 years of age at the time of detecting CRC and had other risk factors for CRC. The median age at the time of the CCA diagnosis was 54.0 years. Conclusion. Despite regular surveillance, the relative risks of CCA and CRC remained increased in patients with IBD and PSC. A rigorous endoscopic surveillance is maintained for all patients with PSC, but better indicators of the development of CCA are urgently needed.

A novel explant outgrowth culture model for mouse pancreatic acinar cells with long-term maintenance of secretory phenotype.

The development of in vitro models able to support the long-term viability and function of acinar cells is critical for exploring pancreatic pathophysiology. Despite considerable efforts, no long-term culture models for non-transformed pancreatic acini exist. Our aim was to develop and validate culture conditions for this purpose. An explant outgrowth culture design was established in which mouse pancreatic explants were cultured at the gas-liquid interphase. An enriched culture medium, pH 7.8, was employed to promote the selective outgrowth of acinar cells and to support their differentiated phenotype. After 7 days, the outgrown primary acinar cells were subcultured and maintained up to an additional 7 days as secondary monolayers on tissue culture plastic. Measurements of basal and caerulein-induced amylase secretion, phase-contrast microscopy and immunohistochemical analyses were used to characterize the cultures. Explants retained their pancreatic cytoarchitecture for 2 days in vitro. A triphasic dose response to caerulein was detected in 7-day primary cultures. The maximal rate of secretion was 1.2-fold versus basal (p=0.009) and 1.7-fold versus 1 pM caerulein (p=0.014). In secondary cultures the response was biphasic with maximal rates of secretion being 1.9-fold in 3- to 4-day cultures at 0.01 nM (p=0.049) and 2-fold in 6- to 7-day cultures at 0.1 nM (p=0.003). The present culture model provides a means to obtain functionally competent normal mouse acinar cells for long-term in vitro experimentation.