Western Diet-Induced Metabolic Alterations Affect Circulating Markers of Liver Function before the Development of Steatosis

Since nutrition might have a significant impact on liver function, we analyzed the early effect of Western-type diet on hepatic tissue and lipid and drug metabolism in Wistar-Kyoto rats (n = 8); eight rats fed with a standard diet were used as controls. Histological analysis of liver tissue was performed, and plasma biochemical parameters were measured. Plasma concentration of six bile acids was determined by ultra-liquid chromatography-tandem mass spectrometry UHPLC-MS/MS. Hepatic gene expressions of enzymes involved in drug and lipid metabolism were assessed by means of real-time reverse transcription (qRT)-PCR. Liver of rats fed with a Western diet did not show macroscopic histological alterations, but number and diameter of lipid droplets increased, as well as DGAT1, GPAT4, SCD, FASN and SREBP2 expression. Furthermore, Western diet-fed animals showed an increase in the activation of hepatic stellate cells and macrophage number in liver tissue, as well as a significant increase in AST and bilirubin levels (p < 0.01), and in the LDL:HDL cholesterol ratio (p < 0.001). Plasma chenodeoxycholic acid concentration increased significantly, whereas cholic acid decreased (p < 0.05), and cytochrome P450 genes were generally downregulated. Significant changes in hepatic lipid and drug metabolism are early induced by the Western diet, prior to steatosis development. Such changes are associated with a peculiar alteration in circulating bile acids, which could represent an early marker of non-alcoholic fatty liver disease (NAFLD) development

Familial occurrence of systemic and cutaneous mastocytosis in an adult multicentre series

n/

Assessment of p.Phe508del-CFTR functional restoration in pediatric primary cystic fibrosis airway epithelial cells

© 2018 Sutanto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background Mutations in the cystic fibrosis transmembrane regulator (CFTR) gene can reduce function of the CFTR ion channel activity and impair cellular chloride secretion. The gold standard method to assess CFTR function of ion transport using the Ussing chamber requires a high number of airway epithelial cells grown at air-liquid interface, limiting the application of this method for high throughput screening of potential therapeutic compounds in primary airway epithelial cells (pAECs) featuring less common CFTR mutations. This study assessed an alternative approach, using a small scale halide assay that can be adapted for a personalized high throughput setting to analyze CFTR function of pAEC. Methods Pediatric pAECs derived from children with CF (pAEC CF ) were established and expanded as monolayer cultures, before seeding into 96-well plates for the halide assay. Cells were then transduced with an adenoviral construct containing yellow fluorescent protein (eYFP) reporter gene, alone or in combination with either wild-type CFTR (WT-CFTR) or p.Phe508-del CFTR. Four days post transduction, cells were stimulated with forskolin and genistein, and assessed for quenching of the eYFP signal following injection of iodide solution into the assay media. Results Data showed that pAEC CF can express eYFP at high efficiency following transduction with the eYFP construct. The halide assay was able to discriminate functional restoration of CFTR in pAEC CF treated with either WT-CFTR construct or the positive controls syntaxin 8 and B-cell receptor-associated protein 31 shRNAs. Significance The current study demonstrates that the halide assay can be adapted for pediatric pAEC CF to evaluate restoration of CFTR function. With the ongoing development of small molecules to modulate the folding and/or activity of various mutated CFTR proteins, this halide assay presents a small-scale personalized screening platform that could assess therapeutic potential of molecules across a broad range of CFTR mutations

Cuban Brown Propolis Interferes in the Crosstalk between Colorectal Cancer Cells and M2 Macrophages

Tumor-associated macrophages (TAMs), primarily the M2 phenotype, are involved in the progression and metastasis of colorectal cancer (CRC). Cuban brown propolis (Cp) and its main component Nemorosone (Nem) displays an antiproliferative effect on different cancer cells, including CRC cell lines. However, whether Cp and Nem could exploit its effect on CRC cells by targeting their relationship with TAMs remains to be elucidated. In this study, we differentiated the human monocytic THP-1 cells to M2 macrophages and confirmed this transition by immunofluorescence (IF) staining, qRT-PCR and zymography. An MTT assay was performed to determine the effect of Cp and Nem on the viability of CRC HT-29 cells co-cultured with M2 macrophages. Furthermore, the migration and invasion abilities of HT-29 cells were determined by Transwell assays and the expression levels of epithelial&ndash;mesenchymal transition (EMT) markers were analyzed by IF staining. We demonstrated that Cp and Nem reduced the viability of M2 macrophages and, accordingly, the activity of the MMP-9 metalloprotein. Moreover, we demonstrated that M2 macrophages produce soluble factors that positively regulate HT-29 cell growth, migration and invasion. These M2-mediated effects were counteracted by Cp and Nem treatments, which also played a role in regulating the expression of the EMT markers E-cadherin and vimentin. Taken together, our results indicate that Nem contained in Cp interferes in the crosstalk between CRC cells and TAMs, by targeting M2 macrophages

Identification and validation of novel drug targets to treat Cystic Fibrosis: modifiers of the trafficking defect of DF508-CFTR

Abnormal retention of the CFTR ΔF508 mutated protein in lung epithelial cells underlies the pathology in a large proportion of individuals with cystic fibrosis (CF). A drug discovery alliance between CFFT and Galapagos was initiated with the aim to identify novel genes which upon shRNAmediated knockdown were able to efficiently restore ΔF508 CFTR activity. The final goal of this program is to prioritize these targets for entry into drug discovery. BioFocus DPI’s proprietary adenoviral shRNA libraries totaling 11,334 viruses against the human druggable genome were screened in a highthroughput functional assay in a human cystic fibrosis bronchial epithelial 2009 Cystic Fibrosis Conference 216 cell line (CFBE41o-). A total of 354 hits were identified and confirmed in the primary assay. Validation of the hits included a lack of cytotoxicity of the shRNA, expression of target in airway epithelial cells, identification of a second shRNA against the same target, and cell surface expression by biotinylation of CFTR ΔF508 upon shRNA-mediated knock-down of the target. Most importantly, these shRNAs restored functional activity of the mutant channel in primary bronchial epithelial cells from ΔF508/ΔF508 CF patients in transepithelial CFTR mediated current assays (Table 1). There was a very strong correlation between fully glycosylated band C expression in the CFBE41o- cells and functional activity in the primary cells of CF patients. We will present an overview of the target discovery and validation program, resulting in a portfolio of 19 novel drug targets to treat CF. The identity of these novel targets also sheds light on the biology of trafficking of CFTR. For example, these data point to a role of TGF-beta signaling and inflammatory mediators in airways in the regulation of CFTR trafficking. Acknowledgments: We thank Drs. Bill Guggino, Ineke Braakman, Kevin Foskett, John Hanrahan and Hugo De Jonge for helpful discussions. We acknowledge the support of Cystic Fibrosis Foundation Therapeutics

Primary biliary cholangitis: perception and expectation of illness

Unlabelled: An important tool to explore personal experience of symptoms, treatment and clinical outcome is stratification of illness perception in patients affected by PBC. Aim: To assess the perception of illness in a cohort of Italian patients with PBC. Methods: Between June and December 2019, a specific questionnaire was administered to a pool of 210 patients from 7 tertiary Italian centers, in order to identify and assess the patient's past history, symptoms and their impact on the quality of life, follow-up, treatment and perceived satisfaction of patients toward the provided care. Results: Fatigue, pruritus, and abdominal discomfort and sicca syndrome were present in 50.4%, 45%, 30.4% and 28.5% of patients, fatigue having the most impacting the daily-life. After a consultation with a specialist, the diagnosis of PBC was met within 18 months for 143 patients. Patients were mostly concerned about possible health problems that occur and in 25% of cases, symptoms had a negative impact on their life. Eighty percent of patients said they were satisfied with efficacy and tolerability of treatment, while 26% requested an improvement in the relationship with the specialist. Conclusions: The results highlight the importance of both promoting timely referral to the specialist and facilitating communication between healthcare professionals and patients

Primary biliary cholangitis: Perception and expectation of illness in Italian patients

none14noneFloreani, Annarosa; Scaffidi, Michela; Coco, Barbara; Giannini, Edoardo Giovanni; Labanca, Sara; Bonaiuto, Emanuela; De Martin, Sara; Invernizzi, Pietro; Carbone, Marco; Alvaro, Domenico; Bragazzi, Maria Consiglia; Calvaruso, Vincenza; Cossiga, Valentina; Cazzagon, NoraFloreani, Annarosa; Scaffidi, Michela; Coco, Barbara; Giannini, Edoardo Giovanni; Labanca, Sara; Bonaiuto, Emanuela; De Martin, Sara; Invernizzi, Pietro; Carbone, Marco; Alvaro, Domenico; Bragazzi, Maria Consiglia; Calvaruso, Vincenza; Cossiga, Valentina; Cazzagon, Nor