Setup of multidisciplinary team discussions for patients with cholangiocarcinoma: current practice and recommendations from the European Network for the Study of Cholangiocarcinoma (ENS-CCA).

BACKGROUND: Cholangiocarcinomas (CCAs) are a rare group of malignancies characterized by dismal prognosis. There are currently no standardized guidelines for multidisciplinary teams (MDTs) in CCAs. MATERIAL AND METHODS: An online survey was built with the aim of defining the current practice of MDTs in CCAs and identifying possible areas of improvement, providing minimum standards of practice for an ideal CCA MDT. Analysis of the replies regarding current and ideal MDT practice was carried out by calculating weighted average (WA) of likelihood of every item. The survey was shared with members of the European Network for the Study of Cholangiocarcinoma and other medical centers with expertise in biliary tract cancer part of the EURO-CHOLANGIO-NET (European Cholangiocarcinoma Network: https://eurocholangionet.eu/) COST Action CA18122 initiative. RESULTS: The role of the MDT coordinator was a recognized priority in an ideal well-functioning MDT (WA 3.31/4), together with providing minimum clinical information before the meeting to secure adequate case preparation (WA 3.54/4). Optimal frequency of MDT meetings was weekly according to 76.92% of the participants; 73.06% believed that ideally all newly diagnosed patients and each new treatment should be discussed, although that happened only in less than half of the MDTs (46.15%) in current practice. Most participants stated that they always (46.15%) or often (50.00%) used guidelines, mainly international (61.00%) (European and American), followed by national/local (39.00%). We defined the ideal setup of a CCA MDT, identifying specialists whose presence is mandatory with WA >3.0 (oncologist, clinician responsible for patient's care, surgeon, diagnostic and interventional radiologist, hepatologist, pathologist, endoscopist and gastroenterologist) and those whose presence would be recommended with a WA <3.0 (palliative care, nurse, dietitian, basic researcher, psychologist and social worker). CONCLUSIONS: Our identified minimum requirements should be taken into account at the time of CCA MDT setup and quality assessment.

S-adenosyl-L-methionine (SAMe) halts the autoimmune response in patients with primary biliary cholangitis (PBC) via antioxidant and S-glutathionylation processes in cholangiocytes.

S-adenosyl-L-methionine is an endogenous molecule with hepato-protective properties linked to redox regulation and methylation. Here, the potential therapeutic value of SAMe was tested in 17 patients with PBC, a cholestatic disease with autoimmune phenomena targeting small bile ducts. Nine patients responded to SAMe (SAMe responders) with increased serum protein S-glutathionylation. That posttranslational protein modification was associated with reduction of serum anti-mitochondrial autoantibodies (AMA-M2) titers and improvement of liver biochemistry. Clinically, SAMe responders were younger at diagnosis, had longer duration of the disease and lower level of serum S-glutathionylated proteins at entry. SAMe treatment was associated with negative correlation between protein S-glutathionylation and TNFα. Furthermore, AMA-M2 titers correlated positively with INFγ and FGF-19 while negatively with TGFß. Additionally, cirrhotic PBC livers showed reduced levels of glutathionylated proteins, glutaredoxine-1 (Grx-1) and GSH synthase (GS). The effect of SAMe was also analyzed in vitro. In human cholangiocytes overexpressing miR-506, which induces PBC-like features, SAMe increased total protein S-glutathionylation and the level of γ-glutamylcysteine ligase (GCLC), whereas reduced Grx-1 level. Moreover, SAMe protected primary human cholangiocytes against mitochondrial oxidative stress induced by tBHQ (tert-Butylhydroquinone) via raising the level of Nrf2 and HO-1. Finally, SAMe reduced apoptosis (cleaved-caspase3) and PDC-E2 (antigen responsible of the AMA-M2) induced experimentally by glycochenodeoxycholic acid (GCDC). These data suggest that SAMe may inhibit autoimmune events in patients with PBC via its antioxidant and S-glutathionylation properties. These findings provide new insights into the molecular events promoting progression of PBC and suggest potential therapeutic application of SAMe in PBC.

Non-canonical Wnt signalling regulates scarring in biliary disease via the planar cell polarity receptors.

The number of patients diagnosed with chronic bile duct disease is increasing and in most cases these diseases result in chronic ductular scarring, necessitating liver transplantation. The formation of ductular scaring affects liver function; however, scar-generating portal fibroblasts also provide important instructive signals to promote the proliferation and differentiation of biliary epithelial cells. Therefore, understanding whether we can reduce scar formation while maintaining a pro-regenerative microenvironment will be essential in developing treatments for biliary disease. Here, we describe how regenerating biliary epithelial cells express Wnt-Planar Cell Polarity signalling components following bile duct injury and promote the formation of ductular scars by upregulating pro-fibrogenic cytokines and positively regulating collagen-deposition. Inhibiting the production of Wnt-ligands reduces the amount of scar formed around the bile duct, without reducing the development of the pro-regenerative microenvironment required for ductular regeneration, demonstrating that scarring and regeneration can be uncoupled in adult biliary disease and regeneration.

Prevalence and Factors Associated With Fatigue in Patients With Inflammatory Bowel Disease: A Multicentre Study.

BACKGROUND AND AIMS: The aims of this study were to determine the prevalence of fatigue in patients with inflammatory bowel disease [IBD], to identify the factors associated with fatigue and its severity, to assess the impact of fatigue on quality of life [QoL], and to evaluate the relationship between fatigue and sleep disorders. METHODS: This was a prospective multicentre study conducted at 22 Spanish centres. Consecutive patients followed at IBD Units were included. Fatigue was evaluated with the Fatigue Severity Scale [FSS] and the Fatigue Impact Scale [FIS]. Quality of life and sleep quality were assessed using the IBD Questionnaire-Short Form [IBDQ-9] and the Pittsburgh Sleep Quality Index [PSQI], respectively. RESULTS: A total of 544 consecutive adult IBD patients were included [50% women, mean age 44 years, 61% Crohn's disease]. The prevalence of fatigue was 41% (95% confidence interval [CI] = 37-45%). The variables associated with an increased risk of fatigue were: anxiety [OR = 2.5, 95% CI = 1.6-3.7], depression [OR = 2.4, 95% CI = 1.4-3.8], presence of extraintestinal manifestations [EIMs] [OR = 1.7, 95% CI = 1.1-2.6], and treatment with systemic steroids [OR = 2.8, 95% CI = 1.4-5.7]. The presence of EIMs [regression coefficient, RC = 8.2, 95% CI = 2.3-14.2], anxiety [RC = 25.8, 95% CI = 20.0-31.5], depression [RC = 30.6, 95% CI = 24.3-37.0], and sleep disturbances [RC = 15.0, 95% CI = 9.3-20.8] were associated with severity of fatigue. Patients with fatigue had a significantly decreased IBDQ-9 score [p < 0.001]. CONCLUSIONS: The prevalence of fatigue in IBD patients is remarkably high and has a negative impact on QoL. Therapy with systemic steroids is associated with an increased risk of fatigue. The severity of fatigue is associated with anxiety, depression, sleep disorders, and the presence of EIMs. Fatigue was not associated with anaemia, disease activity or anti-TNF therapy.

Differential effects of FXR or TGR5 activation in cholangiocarcinoma progression.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is an aggressive tumor type affecting cholangiocytes. CCAs frequently arise under certain cholestatic liver conditions. Intrahepatic accumulation of bile acids may facilitate cocarcinogenic effects by triggering an inflammatory response and cholangiocyte proliferation. Here, the role of bile acid receptors FXR and TGR5 in CCA progression was evaluated. METHODS: FXR and TGR5 expression was determined in human CCA tissues and cell lines. An orthotopic model of CCA was established in immunodeficient mice and tumor volume was monitored by magnetic resonance imaging under chronic administration of the specific FXR or TGR5 agonists, obeticholic acid (OCA) or INT-777 (0,03% in chow; Intercept Pharmaceuticals), respectively. Functional effects of FXR or TGR5 activation were evaluated on CCA cells in vitro. RESULTS: FXR was downregulated whereas TGR5 was upregulated in human CCA tissues compared to surrounding normal liver tissue. FXR expression correlated with tumor differentiation and TGR5 correlated with perineural invasion. TGR5 expression was higher in perihilar than in intrahepatic CCAs. In vitro, FXR was downregulated and TGR5 was upregulated in human CCA cells compared to normal human cholangiocytes. OCA halted CCA growth in vivo, whereas INT-777 showed no effect. In vitro, OCA inhibited CCA cell proliferation and migration which was associated with decreased mitochondrial energy metabolism. INT-777, by contrast, stimulated CCA cell proliferation and migration, linked to increased mitochondrial energy metabolism. CONCLUSION: Activation of FXR inhibits, whereas TGR5 activation may promote, CCA progression by regulating proliferation, migration and mitochondrial energy metabolism. Modulation of FXR or TGR5 activities may represent potential therapeutic strategies for CCA.

MicroRNAs and extracellular vesicles in cholangiopathies.

Cholangiopathies encompass a heterogeneous group of disorders affecting biliary epithelial cells (i.e. cholangiocytes). Early diagnosis, prognosis and treatment still remain clinically challenging for most of these diseases and are critical for adequate patient care. In the past decade, extensive research has emphasized microRNAs (miRs) as potential non-invasive biomarkers and tools to accurately identify, predict and treat cholangiopathies. MiRs can be released extracellularly conjugated with lipoproteins or encapsulated in extracellular vesicles (EVs). Research on EVs is also gaining attention since they are present in multiple biological fluids and may represent a relevant source of novel non-invasive biomarkers and be vehicles for new therapeutic approaches. This review highlights the most promising candidate miRs and EV-related biomarkers in cholangiopathies, as well as their relevant roles in biliary pathophysiology. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen. RESEARCH STRATEGY: PubMed search (April 2017) was done with the following terms: "microRNA", "miRNA", "miR", "extracellular vesicles", "EV", "exosomes", "primary biliary cholangitis", "primary biliary cholangitis", "PBC", "primary sclerosing cholangitis", "PSC", "cholangiocarcinoma", "CCA", "biliary atresia", "BA", "polycystic liver diseases", "PLD", "cholangiopathies", "cholestatic liver disease". Most significant articles in full-text English were selected. The reference lists of selected papers were also considered.

New Advances in Polycystic Liver Diseases.

Polycystic liver diseases (PLDs) include a heterogeneous group of congenital disorders inherited as dominant or recessive genetic traits; they are manifested alone or in association with polycystic kidney disease. Ductal plate malformation during embryogenesis and the loss of heterozygosity linked to second-hit mutations may promote the dilatation and/or development of a large number (> 20) of biliary cysts, which are the main cause of morbidity in these patients. Surgical procedures aimed to eliminate symptomatic cysts show short-term beneficial effects, but are not able to block the disease progression. Therefore, liver transplantation is the only curative option. Intense studies on the molecular mechanisms involved in the pathogenesis of PLDs have resulted in different clinical trials, some of them with promising outcomes. Here the authors summarize the key aspects of PLD etiology, pathogenesis, and therapy, highlighting the most recent advances and future research directions.

Mechanisms of Resistance to Chemotherapy in Gastric Cancer.

Although surgical resection is the standard curative therapy for gastric cancer, these tumors are often diagnosed at an advanced stage, when surgery is not recommended. Alternative treatments such as radiotherapy and chemotherapy achieve only very modest results. There is therefore an urgent need to advance in this field of oncologic gastroenterology. The poor response of gastric cancer to chemotherapy is usually due to a combination of mechanisms of chemoresistance (MOC), which may include a reduction in drug uptake (MOC-1a), enhanced drug efflux (MOC-1b), a reduced proportion of active agents in tumor cells due to a reduction in pro-drug activation or an enhancement in drug inactivation (MOC-2), changes in the expression/function of the molecular targets of anticancer drugs (MOC-3), an enhanced ability of cancer cells to repair anticancer drug-induced DNA damage (MOC-4), and decreased expression/function of pro-apoptotic factors or up-regulation of anti-apoptotic genes (MOC-5). Two major goals of modern pharmacology aimed at overcoming this situation are the prediction of a lack of response to chemotherapy and the identification of the underlying mechanisms accounting for primary or acquired refractoriness to anticancer drugs. These are important issues if we are to select the best pharmacological regime for each patient and develop novel strategies to overcome chemoresistance. The present review reports updated information regarding the mechanisms of chemoresistance (from MOC-1 to MOC-5) in gastric cancer, the advances made in the prediction of the failure of chemotherapeutic treatment, and novel strategies based on gene therapy currently being developed to treat these tumors.

Evaluation of the Effect of 1,3-Bis(4-Phenyl)-1H-1,2,3-Triazolyl-2-Propanolol on Gene Expression Levels of JAK2-STAT3, NF-κB, and SOCS3 in Cells Cultured from Biopsies of Mammary Lesions.

Breast cancer is the most frequent neoplasia in women and is responsible for approximately 13.8% of deaths per year for this gender. It has been suggested that JAK2, STAT3, and NF-κB gene expression is involved in this type of cancer. The objective of the present study was to determine the effect of bistriazole in these signaling pathways in patients with breast cancer and benign mammary lesions. The inhibitory concentration 50 of bistriazole was calculated in cell cultures of patients with benign lesions, Probit = 4.6 µM with IC = 95%. The study was performed by examining 63 women who submitted to mammary biopsies. Biopsies of the mammary lesions were performed, gene expression was determined, and cells were cultured in the presence of 4.6 µM bistriazole. We found that breast cancer is related to age greater than 50 (P ≤ 0.01), being overweight (P ≤ 0.023) and having a waist circumference larger than 80 cm (P ≤ 0.01). The gene expression of JAK2, STAT3, and NF-κB was higher in groups of patients with breast cancer, while SOCS3 expression was lower. After being exposed to bistriazole, the expression of JAK2 and STAT3 decreased, and the expression of SOCS3 and NF-κB increased. In conclusion, this molecule in development has an effect on the gene expression of JAK3 and STAT3; nevertheless, the lack of change in NF-κB indicates that it is not a regulator of inflammation, and therefore, more studies should be performed.

Polycystic liver in the adult (PLA) in Spain: analysis of a structured survey analyzing the experience and attitude of gastroenterologists in Spain.

BACKGROUND: Polycystic liver in the adult (PLA) is a rare disease characterized by chronic liver enlargement. OBJECTIVE: To analyse gastroenterologists´ involvement in, experience with, and attitude toward diagnosing, monitoring, andtreating patients with PLA in Spain. METHODS: Each of seven study coordinators contacted 15 specialists in their geographic area about participating in the study via an online structured survey. RESULTS: Of the 105 clinics contacted, 88 completed the questionnaire, with a mean of 3 patients being followed per practice, although 6 clinics were following more than 20 patients with PLA. Patients were being followed mainly by the Department of Hepatology (81 %) and/or the Department of Gastroenterology (33 %). The majority of patients were diagnosed (98 %) and monitored (97 %) using liver ultrasound. When diagnosed, 76 % of patients were under 50 years of age, females predominating.The primary treatment objective for the patients was symptomatic management. Pharmacotherapy was prescribed by 28 % of physicians: Somatostatin analogues, primarily, followed by mTOR inhibitors. One-third of the clinics indicated that they had patients who had undergone liver transplant and/or surgery. CONCLUSIONS: Ultrasound is the diagnosing and monitoring method of choice. Among the clinics using pharmacotherapy for symptomatic management, somatostatin analogues were the drugs of choice. These clinics´ infrequent use of invasive procedures suggests that they perceive the various invasive techniques as not very effective.

Linezolid-induced lactic acidosis in two liver transplant patients with the mitochondrial DNA A2706G polymorphism.

Mitochondrial toxicity has been recently suggested to be the underlying mechanism of long-term linezolid-associated toxicity in patients with 16S rRNA genetic polymorphisms. Here, we report for the first time two cases of lactic acidosis due to long-term linezolid exposure in liver transplant recipients who presented an A2706G mitochondrial DNA polymorphism.

Development of new assays and improved procedures for the purification of recombinant human chymase.

Chymase mediates a major alternative way of angiotensin II production from angiotensin I beside angiotensin converting enzyme in the final step of the renin-angiotensin system. This enzyme is also involved in other physio-pathological processes such as angiogenesis, atherosclerosis and inflammation. Several purification attempts of natural or recombinant chymase were reported in the literature. Most of these reports were not successful in obtaining the recombinant enzyme in a highly active form and in large quantity. In the present study, we describe a facile route for the purification of the human recombinant chymase. Chymase being produced as inactive prochymase, to be cathepsin C-activated, newly raised anti-chymase Ig were used to follow the purification. In order to complete the available tools for the search of chymase inhibitors, we developed and assessed a new 96-well plate based assay for the measurement of enzyme activity, as well as a low throughput, HPLC-based one. The assays used an original derivative of angiotensin I, or the native hormone. Chymase was produced in CHO cells and appropriately matured. The amount of enzyme obtained at the end of the process is compatible with the medium-throughput screening (up to 10,000 points per day), about 800 microg x L(-1) of culture medium with a specific activity of 6.16 mmol of angiotensin I cleaved per minute per mg of protein. All the biological and technical tools are now available for the discovery of new classes of chymase inhibitors.

Anticardiolipin antibodies are not associated with rheumatic heart disease.

The aim of this study was to examine the prevalence of anticardiolipin antibodies in rheumatic valve heart disease. Serum samples of 31 consecutive patients with rheumatic heart disease and documented valve involvement, as well as six patients with acute rheumatic fever were tested for IgG anticardiolipin antibodies by a validated ELISA. No anticardiolipin antibodies were found when a cut-off point set at mean +/- 5 s.d. was applied. We can conclude that anticardiolipin antibodies are not present in rheumatic heart disease patients and, as suggested by several observations, these antibodies do not appear to have a pathogenic role in this particular disease.

Absence of antiphospholipid/co-factor antibodies in Takayasu arteritis.

UNLABELLED: There are anecdotal reports and small series describing the presence of anticardiolipin antibodies in patients with Takayasu Arteritis. This communication describes a systematic study searching for non-organ specific autoantibodies which includes antinuclear antibodies, anticardiolipin and anti-beta(2) GP(1) antibodies in a cohort of 28 Mexicans with angiographic definitive diagnostic of Takayasu Arteritis. MATERIAL AND METHODS: Twenty-eight consecutive patients, who fulfilled classification and diagnostic criteria for Takayasu Arteritis and had a diagnostic panaortogram, were bled to study the presence of circulating autoantibodies in a cross-sectional design. RESULTS: There were no antinuclear antibodies, although a few sera had faint cytoplasm fluorescent deposit and reacted with cell extract. We did not recognize a distinct pattern. Also, there was no IgG nor IgM anticardiolipin antibodies nor anticofactor antibodies of clinical interest. DISCUSSION AND CONCLUSIONS: The presence of circulating non-organ specific autoantibodies is not a characteristic feature in Takayasu Arteritis when strict diagnostic criteria are applied. The occasional presence of such immune markers could be due to technical differences in sample management, less strict diagnosis or biological variability in certain cases, but has no diagnostic value.

Evaluation of adenosine deaminase activity in the Mycobacterium tuberculosis culture supernatants.

BACKGROUND: Adenosine deaminase (ADA) catalyzes hydrolytic and irreversible deamination of deoxyadenosine into deoxyinosine and of adenosine into inosine, and is related to lymphocytic proliferation and differentiation. The measurement of ADA activity in body fluids is a useful tool in the evaluation of mycobacterial infections. Elevated ADA activity has been found in pleural effusions of patients with pleural tuberculosis relative to those from patients with nontuberculous pleural diseases, and is mainly associated with cellular host factors such as monocyte-macrophages or lymphocytes. In contrast, there is little information about ADA activity measurement in mycobacteria culture supernatants. METHODS: We evaluated ADA activity as described by Giusti in the culture supernatants of eight Mycobacterium tuberculosis isolates. RESULTS: Mycobacteria culture supernatants did not display any ADA activity. CONCLUSIONS: This result supports the notion that Mycobacterium tuberculosis is not the source of ADA activity. However, increased ADA activity in biological fluids from tuberculosis patients might be due to the interaction of the mycobacterium with host factors.

On the correct determination of reference values for serum antibodies against pigeon serum antigen using a group of healthy blood donors.

An enzymatic immunoassay was developed in order to evaluate the statistical distribution of IgG serum antibodies against pooled pigeon sera antigen in 102 healthy blood donors (HBD). A non-normal distribution was obtained as demonstrated by abnormal values of skewness (2.02) and kurtosis (6.50). A cut-off point (0.120) was determined from the mean plus 2 standard deviations of the optical density values obtained in the HBD group. This value was able to segregate 94% of subjects. However, when calculation of the mean less 2 SD was performed to delimit 95% of the samples, an aberrant negative value was obtained. In contrast, when the nonparametric method of percentile calculation was applied, an optical density value of 0.130 discriminated 97.5% of samples. In addition, the interval between p97.5 and p2.5 delimited 95% of samples. We conclude that when reference values and cut-off point are determined from an enzymatic immunoassay, careful analysis of the statistical distribution of reference values is necessary in order to avoid the inappropriate application of parametric procedures as demonstrated in this study for antibodies against pigeon serum antigens.

Cellular immune response to fractionated avian antigens by peripheral blood mononuclear cells from patients with pigeon breeder's disease.

Pigeon breeder's disease (PBD), a form of hypersensitivity pneumonitis caused by repeated inhalation of antigens of pigeon origin, is characterized by a diffuse inflammation of the lower respiratory tract. Although a variety of immunologic and nonimmunologic mechanisms have been described in the development of the disease, the pathogenesis is still far from clear. In this study we analyzed the T-lymphocyte proliferative response to a variety of avian antigens with use of peripheral blood mononuclear cells from 11 patients who had PBD and 10 healthy volunteers. We used a new method based on avian antigen-bearing nitrocellulose particles derived from Western blots to study the T-cell proliferative response to 15 antigenic fractions obtained from pigeon serum. With this technique, complex mixtures of antigens can be fractionated by polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and used for T-cell proliferation assays with selected antigenic determinants. A wide variety of responses were observed, and there were no reproducible patterns of reaction within either group. Nine of 10 healthy subjects responded to some soluble fractions. However, patients with PBD displayed the strongest response and responded to a significantly greater number of antigenic fractions. Fraction 2, representing a 220 kd molecular weight protein, was the only immunodominant antigen when both groups were compared; it was recognized by 73% of the patients with PBD and by only 20% of control subjects (p < 0.03). These findings show that T lymphocytes of patients with PBD recognize a wide range of bird proteins, which induce marked T-cell proliferation.

Detection of salivary and seric IgG and IgA antipooled pigeon sera activities in patients with pigeon breeder's disease.

Pigeon breeder's disease (PBD) is an interstitial lung disease induced by exposure to pigeon antigens. Search of antipigeon antigen antibodies (APSA) in serum or bronchoalveolar lavage is generally used for auxiliary diagnostic purposes. However, APSA can be present in a number of exposed but asymptomatic individuals as well as in patients with other interstitial lung diseases who live in areas where keeping pigeons is a common domestic habit. In this study, saliva was evaluated as an alternative means to serum for APSA detection by ELISA using pooled pigeon sera as antigen. Serum and saliva samples obtained from 17 patients with PBD, 14 with idiopathic pulmonary fibrosis (IPF), 19 asymptomatic relatives (AR) exposed to pigeon antigens, and 27 clinical healthy voluntary subjects (CHVS) were tested for IgG and IgA APSA. Our results showed that both fluids obtained from PBD patients exhibited a significantly higher specific IgG antibody activity compared to the other groups. Serum optical density (O.D.) values for PBD were 1.187 +/- 0.738 vs. 0.024 +/- 0.033, 0.255 +/- 0.471, and 0.204 +/- 0.346 for CHVS, AR and IPF, respectively (P < 0.05). Salivary O.D. for PBD were 0.801 +/- 0.447 vs 0.010 +/- 0.011, 0.104 +/- 0.151, and 0.22 +/- 0.447 (P < 0.05). In contrast, serum specific IgA did not discriminate between PBD and IPF patients. In addition, although the PBD group exhibited the highest values of IgA salivary APSA, high levels were also observed in saliva specimens from CHVS, a group of normal individuals who deny pigeon exposure. These findings suggest that measurement of IgG salivary APSA can play a role in the evaluation process of patients with pigeon breeder's disease.