CD73 is a Critical Regulator of Hepatocytes in Homeostasis and Disease

The enzymatic dephosphorylation of 5´-nucleotides like adenosine 5´-monophosphate (AMP) is a key step in purine salvage and purinergic signaling. This reaction occurs inside the cell or in the extracellular space. Ecto-5´-nucleotidase (CD73) is the major enzyme catalyzing the formation of extracellular adenosine from AMP. CD73-generated adenosine controls tissue homeostasis and responses related to inflammation, ischemia, fibrosis, and cancer. However, the specific functions of CD73 in liver homeostasis and diseases have not been fully elucidated. The objectives of the study were to 1) define the cell-specific role of CD73 in the liver, 2) determine how hepatocyte CD73 regulates alcohol-induced liver injury, and 3) examine CD73 regulation in human hepatocellular carcinoma. We generated and characterized a hepatocyte-specific CD73 knockout mouse (CD73-LKO) model using serological, biochemical, and histological assays. We found that CD73-LKO mice developed spontaneous liver injury in a sex- and age-dependent manner, which was associated with hypoactivation of AMPK, the key regulator of cellular energy metabolism. Furthermore, CD73-generated extracellular adenosine mediated AMPK activity upon cellular uptake through nucleoside transporters. Given that hepatocyte CD73 and adenosine exerts basal hepatoprotection, we then determined its role in alcohol-induced liver injury, a leading risk factor for chronic liver diseases. The loss of hepatocyte CD73 predisposed mice to more severe liver injury. In contrast, alcohol exposure in WT mice resulted in mild injury, concomitant with CD73 induction and enhanced enzymatic activity. In vitro assays revealed a non-enzymatic CD73 binding protein function for bioactive lipids, which are generated upon alcohol exposure. These data suggest CD73-mediated dual protection in alcoholic liver. In humans, chronic alcohol-induced liver injury leads to hepatocellular carcinoma (HCC), the fourth leading cause of cancer-related deaths worldwide. Because hepatocyte CD73 is critical in normal and injured liver, we examined its regulation in HCC. CD73 was expressed in tumor and non-tumor liver tissues. However, tumor-specific CD73 altered N-linked glycosylation led to its cytoplasmic redistribution and a reduction in the enzymatic activity, suggesting a potential impairment in CD73-mediated liver protection. In conclusion, these studies provide further understanding of CD73 biology in the liver and has implications for the potential therapeutic use of CD73 in chronic liver diseases.Doctor of Philosoph

Simbiótico conteniendo bacillus coagulans LMG-S-24828 y prebióticos en la reducción de trastornos gastrointestinales secundarios al tratamiento farmacológico de enfermedades hematológicas crónicas. Estudio piloto

Poster [PC-353] Introducción: Algunos fármacos inhibidores de Tirosin-kinasa (ITK) utilizados en el tratamiento de la leucemia mieloide crónica, y Miglustat, un iminoazúcar empleado en enfermedades lisosomales, pueden producir trastornos gastrointestinales como diarrea, meteorismo y dolor abdominal. Estos efectos adversos disminuyen la calidad de vida relacionada con la salud y provocan abandonos del tratamiento. Algunos probióticos han demostrado mejoría de los síntomas mencionados en pacientes con trastornos funcionales digestivos. Hipótesis: El simbiótico conteniendo Bacillus coagulans LMG-S-24828 y prebióticos reduce los efectos adversos gastrointestinales asociados a la utilización de ITKs y Miglustat y mejora la adherencia al tratamiento. Objetivos: Evaluar el efecto de la administración controlada de dicho simbiótico durante un mes, sobre la calidad de vida relacionada con la salud gastrointestinal en pacientes tratados con ITKs y miglustat. Secundariamente, establecer si el simbiótico aporta ventajas en la adherencia a los tratamientos citados. Métodos: Ensayo clínico aleatorizado de diseño cruzado en el que a 9 pacientes en tratamiento con ITKs o Miglustat se les administró de forma ciega placebo o simbiótico en una dosis diaria, con una fase de “lavado” de dos meses entre la administración de cada uno. Se solicitó al paciente cumplimentar la versión validada en español del cuestionario de Calidad de Vida Gastrointestinal GIQLI (Gastrointestinal Quality of Life Index) antes de la primera dosis de cada producto y trascurrido un mes desde su inicio. Se evaluó la frecuencia de abandono del tratamiento en cada grupo. El análisis de los resultados se realizó por protocolo mediante el test no paramétrico de Mann-Whitney, considerando significación estadística las diferencias con p valor<0, 05. El protocolo fue aprobado por el Comité de Ética autonómico. Resultados: Inicialmente se reclutaron 11 pacientes procedentes de un único centro, de los que 9 (5 H/ 4M), edad media: 49 (29-79) finalizaron el estudio. Tras un mes en tratamiento con simbiótico observamos una diferencia estadísticamente significativa (p=0, 039) en la puntuación media del cuestionario GIQLI no alcanzada con placebo. Ningún paciente abandonó el tratamiento con ITKs/Miglustat, ni se observaron variaciones analíticas en los biomarcadores de la enfermedad. Conclusiones: En pacientes bajo tratamiento con ITKs o Miglustat el simbiótico mejoró significativamente los síntomas adversos gastrointestinales. Este beneficio no fue observado con placebo y no se relacionó con el grado de adherencia terapéutica

Un simbiótico conteniendo bacillus coagulans LMG-S-24828 reduce los síntomas gastrointestinales secundarios al tratamiento farmacológico de enfermedades hematológicas crónicas. Estudio multicéntrico

PB-062 Introducción: La presencia de blastos permite diagnosticar, en muchas ocasiones, una neoplasia hematológica dentro de un amplio abanico de posibilidades diagnósticas, siendo el ejemplo clásico el de las leucemias agudas. Además, apoyando al examen morfológico, la citometría de flujo es de gran utilidad para el diagnóstico y la clasificación de estas neoplasias. Existen otras enfermedades no neoplásicas que se manifiestan con células inmaduras, por ello presentamos el caso de una lactante de 19 meses con hepatoesplenomegalia, presencia de blastos con diagnóstico inicial de leucemia aguda que no se confirma tras un estudio completo. Métodos y Resultados: Presentamos una lactante de 19 meses que acude por fiebre de 15 días de predominio nocturno, máximo 39,5ºC, distensión abdominal y desde dos semanas antes de ingreso coincidiendo con reinicio de fiebre, mayor aumento del perímetro abdominal. Se realiza ecografía abdominal con esplenomegalia de 10,7 cm, bazo accesorio de 1,6x1,5 cm. A la exploración se objetiva palidez cutánea, abdomen distendido, no doloroso y marcada hepatoesplenomegalia. ..

Development of multiplex PCR and multi-color fluorescent in situ hybridization (m-FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease

Abstract Background Several pathogens have been debated to play a role in inflammatory bowel disease (IBD) including Crohn’s disease (CD). None of these pathogens have been investigated together in same clinical samples. We developed a multiplex PCR and multi-color fluorescent in situ hybridization (m-FISH) protocols for simultaneous detection of CD-associated pathogens including Mycobacterium avium subspecies paratuberculosis (MAP), Klebsiella pneumoniae, and adherent-invasive Escherichia coli strain LF82. Methods The multiplex PCR is based on 1-h DNAzol® extraction protocol modified for rapid extraction of bacterial DNA from culture, blood, and intestinal biopsies. Oligonucleotide primers sequences unique to these pathogens were evaluated individually and in combinations using bioinformatics and experimental approaches. m-FISH was based on fluorescent-tagged oligonucleotides and confocal scanning laser microscopy (CSLM). Results Following several attempts, the concentration of the oligonucleotide primers and DNA templates and the PCR annealing temperatures were optimized. Multiplex PCR analyses revealed excellent amplification signal in trials where a single primer set and combinations of two and three primers sets were tested against a mixture of DNA from three different bacteria or a mixture of three bacterial cultures mixed in one tube before DNA extraction. Slides with individual and mixtures of bacterial cultures and intestinal tissue sections from IBD patients were tested by m-FISH and the CSLM images verified multiplex PCR results detected on 3% agarose gel. Conclusion We developed a 4-h multiplex PCR protocol, which was validated by m-FISH images, capable of detecting up to four genes from major pathogens associated with CD. The new protocol should serve as an excellent tool to support efforts to study multi-pathogens involved in CD and other autoimmune disease

Tumorâ Selective Altered Glycosylation and Functional Attenuation of CD73 in Human Hepatocellular Carcinoma

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151913/1/hep41410_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151913/2/hep41410.pd

Prevalence of severe esophagitis in Spain. Results of the PRESS study (Prevalence and Risk factors for Esophagitis in Spain: A cross-sectional study)

Background The current prevalence of esophagitis in southern Europe is unknown. In addition, the risk factors for reflux esophagitis are not fully understood. Objective The objective of this article is to assess the prevalence and risk factors for esophagitis in Spain. Methods A prospective, observational, cross-sectional, multicenter study (PRESS study) was conducted among 31 gastrointestinal endoscopy units throughout Spain. A total of 1361 patients undergoing upper gastrointestinal endoscopy were enrolled. Sociodemographic, clinical and treatment data were recorded. Results A total of 95% of patients were Caucasian and 52% were male (mean age: 5317 years). The most frequent symptoms prompting endoscopy were heartburn (40%), regurgitation (26%) and dysphagia (15%). Fifty-four percent of patients undergoing endoscopy were receiving proton pump inhibitor (PPI) treatment. Esophagitis (mainly mild-moderate) was present in 154 (12.4%) patients. The severe form was recorded in only 11 (0.8%) patients. Multivariate analysis results indicated that the likelihood of esophagitis was higher in men (OR=1.91, 95% CI=1.31-2.78), in patients with high GERD-Q scores (OR=1.256, 95% CI=1.176-1.343), weight increase (OR=1.014, 95% CI=1.003-1.025) and high alcohol consumption (OR=2.49, 95% CI=1.16-5.36). Conclusion Severe esophagitis is a rare finding in the Spanish population. Male gender, high GERD-Q score, weight increase and high alcohol consumption are main risk factors for its appearance

Indications and hemoglobin thresholds for red blood cell transfusion and iron replacement in adults with gastrointestinal bleeding: An algorithm proposed by gastroenterologists and patient blood management experts

Gastrointestinal (GI) bleeding is associated with considerable morbidity and mortality. Red blood cell (RBC) transfusion has long been the cornerstone of treatment for anemia due to GI bleeding. However, blood is not devoid of potential adverse effects, and it is also a precious resource, with limited supplies in blood banks. Nowadays, all patients should benefit from a patient blood management (PBM) program that aims to minimize blood loss, optimize hematopoiesis (mainly by using iron replacement therapy), maximize tolerance of anemia, and avoid unnecessary transfusions. Integration of PBM into healthcare management reduces patient mortality and morbidity and supports a restrictive RBC transfusion approach by reducing transfusion rates. The European Commission has outlined strategies to support hospitals with the implementation of PBM, but it is vital that these initiatives are translated into clinical practice. To help optimize management of anemia and iron deficiency in adults with acute or chronic GI bleeding, we developed a protocol under the auspices of the Spanish Association of Gastroenterology, in collaboration with healthcare professionals from 16 hospitals across Spain, including expert advice from different specialties involved in PBM strategies, such as internal medicine physicians, intensive care specialists, and hematologists. Recommendations include how to identify patients who have anemia (or iron deficiency) requiring oral/intravenous iron replacement therapy and/or RBC transfusion (using a restrictive approach to transfusion), and transfusing RBC units 1 unit at a time, with assessment of patients after each given unit (i.e., “don’t give two without review”). The advantages and limitations of oral versus intravenous iron and guidance on the safe and effective use of intravenous iron are also described. Implementation of a PBM strategy and clinical decision-making support, including early treatment of anemia with iron supplementation in patients with GI bleeding, may improve patient outcomes and lower hospital costs

Evaluation of a New Monoclonal Chemiluminescent Immunoassay Stool Antigen Test for the Diagnosis of Helicobacter pylori Infection: A Spanish Multicentre Study

The stool antigen test (SAT) represents an attractive alternative for detection of Helicobacter pylori. The aim of this study was to assess the accuracy of a new SAT, the automated LIAISON(R) Meridian H. pylori SA based on monoclonal antibodies, compared to the defined gold standard C-13-urea breath test (UBT). This prospective multicentre study (nine Spanish centres) enrolled patients >= 18 years of age with clinical indication to perform UBT for the initial diagnosis and for confirmation of bacterial eradication. Two UBT methods were used: mass spectrometry (MS) including citric acid (CA) or infrared spectrophotometry (IRS) without CA. Overall, 307 patients (145 naive, 162 with confirmation of eradication) were analysed. Using recommended cut-off values (negative SAT = 1.10) the sensitivity, specificity, positive predictive value, negative predictive value and accuracy were 67%, 97%, 86%, 92% and 91%, respectively, obtaining an area under the receiver operating characteristic (ROC) curve (AUC) of 0.85. Twenty-eight patients, including seven false positives and 21 false negatives, presented a discordant result between SAT and UBT. Among the 21 false negatives, four of six tested with MS and 11 of 15 tested with IRS presented a borderline UBT delta value. In 25 discordant samples, PCR targeting H. pylori DNA was performed to re-assess positivity and SAT accuracy was re-analysed: sensitivity, specificity, positive predictive value, negative predictive value, accuracy and AUC were 94%, 97%, 86%, 99%, 97% and 0.96, respectively. The new LIAISON(R) Meridian H. pylori SA SAT showed a good accuracy for diagnosis of H. pylori infection

Functional divergence in the role of N-linked glycosylation in smoothened signaling

The G protein-coupled receptor (GPCR) Smoothened (Smo) is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh) pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Gαi. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. Herein, we present a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, we found that all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Gαi, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, we postulate a profound shift in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice