Diagnostic accuracy and interobserver agreement of cholangioscopy for indeterminate biliary strictures: A single-center experience.

BACKGROUND AND STUDY AIMS: Characterization of indeterminate biliary strictures (IDBS) still represents a major challenge. Digital single-operator cholangioscopy (DSOC) could potentially overcome limits of conventional biopsy and brush sampling. The aim of this study was to compare diagnostic accuracy of visual evaluation and DSOC-guided biopsies to conventional trans-papillary sampling techniques and to evaluate the inter-observer agreement (IOA) on visual diagnosis. PATIENTS AND METHODS: All consecutive patients undergoing DSOC-guided biopsy after conventional sampling techniques for IDBS during a six-year period were retrospectively evaluated. Final diagnosis was based on histological evaluation of the surgical specimen if available or a clinical follow-up of at least 6 months. For IOA, 20-second DSOC clips were retrospectively reviewed by 6 experts and 6 trainees and classified according to the Monaco Classification. RESULTS: Thirty-five patients underwent DSOC for IDBS in the study period; 14 patients (F = 9) with a median age of 64 years (range 53-76) met the study aim. After DSOC, strictures location was changed in three patients (additional yield of 21.4 %). Intraductal DSOC-guided biopsy were technically successful in all cases, with an adequacy of 92.8 %. No adverse events were recorded. Final diagnosis was benign disease in five cases and cholangiocarcinoma in the others. For IOA, 29 videos were evaluated with almost perfect agreement for final diagnosis (kappa 0.871; agreement 93.1, p <0.001), although overall accuracy of DSOC visual finding was 73.6 % and 64.4 % for experts and trainees, respectively. CONCLUSIONS: DSOC could improve diagnostic accuracy for IDBS, since it showed high sensitivity for visual finding and high specificity for DSOC guided-biopsy. Visual diagnosis seems reliable for diagnosis using the Monaco Classification.

Impact of Pregnancy on Weight Loss After Endoscopic Sleeve Gastroplasty.

PURPOSE: Obesity and pregnancy are strictly related: on the one hand, obesity-one of the most common comorbidities in women of reproductive age-contributes to infertility and obesity-related pregnancy complications, whereas pregnancy is a condition in which, physiologically, the pregnant woman undergoes weight gain. Endoscopic sleeve gastroplasty (ESG) may be used for the treatment of obesity in women of childbearing age. MATERIALS AND METHODS: A retrospective analysis was conducted to evaluate weight trajectories, the evolution of obesity-related comorbidities, and lifestyle modification in women who became pregnant after ESG. A comparison was made between childbearing-age women who became pregnant after ESG and non-pregnant women. RESULTS: A total of 150 childbearing-age women underwent ESG at a large tertiary medical center. Of these, 11 patients (33.4 ± 6.2 years) became pregnant after the procedure, following a mean time interval of 5.5 ± 3.9 months. Three women (two affected by polycystic ovary syndrome) reported difficulty getting pregnant before undergoing ESG. The mean preconception BMI was 31.9±4.0 kg/m2 (-7.24 ± 4.0 kg/m2 after ESG). Total body weight loss (TBWL, %) was 18.08 ± 8.00, 11.00 ± 11.08, and 12.08 ± 8.49, at the beginning of pregnancy, at the delivery, and at the first follow-up (19.6 ± 7.8 months after ESG). TBWL of at least 5% was achieved before pregnancy in all patients (73% reached a TBWL ≥ 10%). No significant differences in weight loss and QoL were found between the pregnancy and non-pregnancy groups up to 24 months after ESG. CONCLUSIONS: Endoscopic sleeve gastroplasty allows for adequate weight loss before and after pregnancy in patients with obesity.

Liquid Biopsy of Bile based on Targeted Mass Spectrometry for the Diagnosis of Malignant Biliary Strictures.

Bile holds biomarkers of malignant biliary strictures (MBS) but is unsuited for automated analyzers used in routine diagnostic laboratories. Selected reaction monitoring (SRM) is a flexible high-throughput analytical approach based on targeted mass spectrometry (MS) already implemented in clinical settings. We tested the hypothesis that SRM could be used to quantify cancer biomarkers in human bile. An SRM-based assay was developed to simultaneously quantify up to 37 peptides from 13 bile proteins in a developmental cohort of 15 patients (MBS, n = 8; benign biliary stricture or obstruction (BBS), n = 7). The most reliable biomarkers were then absolutely quantified by SRM in a verification cohort of 67 patients (MBS, n = 37; BBS, n = 30). The diagnostic performances of single and combined biomarkers were assessed. In the developmental cohort, SRM-based analysis revealed six protein biomarkers with significantly higher peptide ratios (endogenous vs. standard) in bile from MBS vs. BBS. In the verification cohort, five of these biomarkers proved good diagnostic ability (individual receiver operating characteristic-area under the receiver operating characteristic curve (ROC-AUC) up to 0.889, accuracies from 67.8% to 83.1%). Combining bile biomarkers and serum CA19-9 in 2 panels allowed differentiating MBS from BBS with up to 0.929 ROC-AUC and 89.8% accuracy. In this study, a newly developed SRM-based assay proved able to simultaneously quantify multiple biomarkers in bile samples. The combination of bile biomarkers with serum CA19-9 was highly accurate for the diagnosis of MBS. Liquid biopsy of bile based on targeted MS is eligible to support MBS diagnosis in clinical practice.

Challenging biliary strictures: pathophysiological features, differential diagnosis, diagnostic algorithms, and new clinically relevant biomarkers - part 1.

It is frequently challenging to make the correct diagnosis in patients with biliary strictures. This is particularly important as errors may have disastrous consequences. Benign-appearing strictures treated with stents may later be revealed to be malignant and unnecessary surgery for benign strictures carries a high morbidity rate. In the first part of the review, the essential information that clinicians need to know about diseases responsible for biliary strictures is presented, with a focus on the most recent data. Then, the characteristics and pitfalls of the methods used to make the diagnosis are summarized. These include serum biomarkers, imaging studies, and endoscopic modalities. As tissue diagnosis is the only 100% specific tool, it is described in detail, including techniques for tissue acquisition and their yields, how to prepare samples, and what to expect from the pathologist. Tricks to increase diagnostic yields are described. Clues are then presented for the differential diagnosis between primary and secondary sclerosing cholangitis, IgG4-related sclerosing cholangitis, cholangiocarcinoma, pancreatic cancer, autoimmune pancreatitis, and less frequent diseases. Finally, algorithms that will help to achieve the correct diagnosis are proposed.

Molecular basis of senescence transmitting in the population of human endometrial stromal cells.

Hormone-regulated proliferation and differentiation of endometrial stromal cells (ESCs) determine overall endometrial plasticity and receptivity to embryos. Previously we revealed that ESCs may undergo premature senescence, accompanied by proliferation loss and various intracellular alterations. Here we focused on whether and how senescence may be transmitted within the ESCs population. We revealed that senescent ESCs may induce paracrine senescence in young counterparts via cell contacts, secreted factors and extracellular vesicles. According to secretome-wide profiling we identified plasminogen activator inhibitor -1 (PAI-1) to be the most prominent protein secreted by senescent ESCs (data are available via ProteomeXchange with identifier PXD015742). By applying CRISPR/Cas9 techniques we disclosed that PAI-1 secreted by senescent ESCs may serve as the master-regulator of paracrine senescence progression within the ESCs population. Unraveled molecular basis of senescence transduction in the ESCs population may be further considered in terms of altered endometrial plasticity and sensitivity to invading embryo, thus contributing to the female infertility curing.

Pre-fractionation of Noncirculating Biological Fluids to Improve Discovery of Clinically Relevant Protein Biomarkers.

Nowadays, significant difficulties remain in the diagnosis and/or prognosis of many diseases, leading to an unsatisfactory patient management and a counterproductive increase in time and costs. It is therefore crucial to bridge the gap between basic and applied research by complying with clinical requirements, notably from the design stage of the experimental workflow. In this chapter we provide key suggestions for selecting appropriate biological samples and reducing pre-analytical and analytical variabilities to improve the discovery of clinically relevant protein biomarkers.

Extracellular Vesicles in Bile as Markers of Malignant Biliary Stenoses.

BACKGROUND & AIMS: Algorithms for diagnosis of malignant common bile duct (CBD) stenoses are complex and lack accuracy. Malignant tumors secrete large numbers of extracellular vesicles (EVs) into surrounding fluids; EVs might therefore serve as biomarkers for diagnosis. We investigated whether concentrations of EVs in bile could discriminate malignant from nonmalignant CBD stenoses. METHODS: We collected bile and blood samples from 50 patients undergoing therapeutic endoscopic retrograde cholangiopancreatography at university hospitals in Europe for CBD stenosis of malignant (pancreatic cancer, n = 20 or cholangiocarcinoma, n = 5) or nonmalignant (chronic pancreatitis [CP], n = 15) origin. Ten patients with CBD obstruction due to biliary stones were included as controls. EV concentrations in samples were determined by nanoparticle tracking analyses. The discovery cohort comprised the first 10 patients with a diagnosis of pancreatic cancer, based on tissue analysis, and 10 consecutive controls. Using samples from these subjects, we identified a threshold concentration of bile EVs that could best discriminate between patients with pancreatic cancer from controls. We verified the diagnostic performance of bile EV concentration by analyzing samples from the 30 consecutive patients with a diagnosis of malignant (pancreatic cancer or cholangiocarcinoma, n = 15) or nonmalignant (CP, n = 15) CBD stenosis. Samples were compared using the Mann-Whitney test and nonparametric Spearman correlation analysis. Receiver operating characteristic area under the curve was used to determine diagnostic accuracy. RESULTS: In both cohorts, the median concentration of EVs was significantly higher in bile samples from patients with malignant CBD stenoses than controls or nonmalignant CBD stenoses (2.41 × 1015 vs 1.60 × 1014 nanoparticles/L in the discovery cohort; P < .0001 and 4.00 × 1015 vs 1.26 × 1014 nanoparticles/L in the verification cohort; P < .0001). A threshold of 9.46 × 1014 nanoparticles/L in bile best distinguished patients with malignant CBD from controls in the discovery cohort. In the verification cohort, this threshold discriminated malignant from nonmalignant CBD stenoses with 100% accuracy. Serum concentration of EVs distinguished patients with malignant vs patients with nonmalignant CBD stenoses with 63.3% diagnostic accuracy. CONCLUSIONS: Concentration of EVs in bile samples discriminates between patients with malignant vs nonmalignant CBD stenosis with 100% accuracy. Further studies are needed to confirm these findings. Clinical Trial registration no: ISRCTN66835592.

De-regulated expression of the BRG1 chromatin remodeling factor in bone marrow mesenchymal stromal cells induces senescence associated with the silencing of NANOG and changes in the levels of chromatin proteins.

Stem cells have a peculiar chromatin architecture that contributes to their unique properties, including uncommitted status, multi/pluripotency and self-renewal. We analyzed the effect of the de-regulation of the SWI/SNF chromatin remodeling complex in mesenchymal stromal cells (MSC) through the silencing and up-regulation of BRG1, which is the ATPase subunit of the complex. The altered expression of BRG1 promoted the senescence of MSC with suppression of the NANOG transcription, which is part of the transcriptional circuitry governing stem cell functions. To gain insight on the way NANOG was silenced, we evaluated how the de-regulated BRG1 expression affect the binding of activators and repressors on the NANOG promoter. We found 4 E2F binding motifs on NANOG promoter, which can be occupied by RB1 and RB2/P130. These are members of the retinoblastoma gene family. In MSC with a silenced BRG1, the relative binding of the 2 retinoblastoma proteins increased, and this was associated with the recruitment of DNMT1. This induced the methylation of CpG on the NANOG promoter. Opposingly, when a high level of BRG1 was present, the same E2F binding motifs were docking sites for BRG1, which induced chromatin compaction without CpG methylation but with increased histone deacetylation, associated with the presence of HDAC1 on E2F binding sites. Besides the sharp regulation of the NANOG expression, we evidenced, through proteomic analysis, that the de-regulation of the SWI/SNF function affected the expression of histones and other nuclear proteins involved in "nuclear architecture," suggesting that BRG1 may act as global regulator of gene expression.

Molecular profiling of the Phytophthora plurivora secretome: a step towards understanding the cross-talk between plant pathogenic oomycetes and their hosts.

The understanding of molecular mechanisms underlying host-pathogen interactions in plant diseases is of crucial importance to gain insights on different virulence strategies of pathogens and unravel their role in plant immunity. Among plant pathogens, Phytophthora species are eliciting a growing interest for their considerable economical and environmental impact. Plant infection by Phytophthora phytopathogens is a complex process coordinated by a plethora of extracellular signals secreted by both host plants and pathogens. The characterization of the repertoire of effectors secreted by oomycetes has become an active area of research for deciphering molecular mechanisms responsible for host plants colonization and infection. Putative secreted proteins by Phytophthora species have been catalogued by applying high-throughput genome-based strategies and bioinformatic approaches. However, a comprehensive analysis of the effective secretome profile of Phytophthora is still lacking. Here, we report the first large-scale profiling of P. plurivora secretome using a shotgun LC-MS/MS strategy. To gain insight on the molecular signals underlying the cross-talk between plant pathogenic oomycetes and their host plants, we also investigate the quantitative changes of secreted protein following interaction of P. plurivora with the root exudate of Fagus sylvatica which is highly susceptible to the root pathogen. We show that besides known effectors, the expression and/or secretion levels of cell-wall-degrading enzymes were altered following the interaction with the host plant root exudate. In addition, a characterization of the F. sylvatica root exudate was performed by NMR and amino acid analysis, allowing the identification of the main released low-molecular weight components, including organic acids and free amino acids. This study provides important insights for deciphering the extracellular network involved in the highly susceptible P. plurivora-F. sylvatica interaction.

Characterization of the bile and gall bladder microbiota of healthy pigs.

Bile is a biological fluid synthesized in the liver, stored and concentrated in the gall bladder (interdigestive), and released into the duodenum after food intake. The microbial populations of different parts of mammal's gastrointestinal tract (stomach, small and large intestine) have been extensively studied; however, the characterization of bile microbiota had not been tackled until now. We have studied, by culture-dependent techniques and a 16S rRNA gene-based analysis, the microbiota present in the bile, gall bladder mucus, and biopsies of healthy sows. Also, we have identified the most abundant bacterial proteins in the bile samples. Our data show that the gall bladder ecosystem is mainly populated by members of the phyla Proteobacteria, Firmicutes, and Bacteroidetes. Furthermore, fluorescent in situ hybridization (FISH) and transmission electron microscopy (TEM) allowed us to visualize the presence of individual bacteria of different morphological types, in close association with either the epithelium or the erythrocytes, or inside the epithelial cells. Our work has generated new knowledge of bile microbial profiles and functions and might provide the basis for future studies on the relationship between bile microbiota, gut microbiota, and health.

Bile proteome in health and disease.

The study of bile proteins could improve the understanding of physiological processes involved in the regulation of the hepato-biliary system. Researchers have tried for years to investigate the bile proteome but, until recently, only a few tens of proteins were known. The advent of proteomics, availing of large-scale analytical devices paired with potent bioinformatic resources, lately allowed the identification of thousands of proteins in bile. Nevertheless, the knowledge of their role in the hepato-biliary system still represents almost a "blank page in the book of physiology." In this review, we first guide the reader through the historical phases of the analysis of bile protein content, emphasizing the recent progresses achieved through the use of proteomic techniques. Thereafter, we deeply explore the involvement of bile proteins in health and disease, with a particular focus on the discovery of biomarkers for biliary tract malignancies.

Proximal fluid proteomics for the discovery of digestive cancer biomarkers.

Most digestive malignancies have asymptomatic course, often progressing to poor outcome stages. Surgical resection usually represents the only potentially curative option but a prior assumption of the malignant nature of the lesion is mandatory to avoid exposing patients to unnecessary risks. Unfortunately, currently available diagnostic tools lack accuracy in many cases, consequently more reliable markers are needed to improve detection of malignant lesions. In this challenging context, fluids surrounding digestive malignancies represent a valuable source for the search of new potential biomarkers and proteomic tools offer the opportunity to achieve this goal. The new field of proximal fluid proteomics is thus emerging in the arena of digestive cancer biomarker discovery. In the present review, the state-of-the-art of proteomic investigations aimed at identifying new cancer biomarkers in fluids surrounding gastrointestinal malignancies is summarized. A comprehensive catalog of proteomic studies in which potential cancer biomarkers from gastrointestinal fluids have been identified and assessed for their diagnostic performances is also provided. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

Bile carcinoembryonic cell adhesion molecule 6 (CEAM6) as a biomarker of malignant biliary stenoses.

Differentiating malignant from nonmalignant biliary stenoses is challenging. This could be facilitated by the measurement of cancer biomarkers in bile. We aimed at (i) identifying new cancer biomarkers by comparative proteomic analysis of bile collected from patients with a malignant or benign biliary stenosis (exploratory phase) and (ii) verifying the accuracy of the newly identified potential biomarkers for discriminating malignant versus nonmalignant biliary stenoses in a larger group of patients (confirmation phase). Overall, 66 proteins were found overexpressed (ratio>1.5) in at least one cancer condition using proteomic analysis and 7 proteins were increased in all malignant/nonmalignant disease comparisons. Preliminary screening by immunoblot highlighted carcinoembryonic cell adhesion molecule 6 (CEAM6), a cell surface protein overexpressed in many human cancers, as an interesting candidate biomarker. ELISA subsequently confirmed CEAM6 as a potential bile biomarker for distinguishing malignant from benign biliary stenoses with a receiver operating characteristic (ROC) area under the curve (AUC) of 0.92 (specificity 83%, sensitivity 93%, positive predictive value 93%, and negative predictive value 83%). No significant difference in serum CEAM6 level was found between malignant and nonmalignant samples. Combining bile CEAM6 and serum CA19-9 in a panel further improved diagnostic accuracy for malignant stenoses (AUC 0.96, specificity 83%, sensitivity 97%, positive predictive value 93%, and negative predictive value 91%). CEAM6 measurement in bile could be clinically useful to discriminate between malignant and nonmalignant causes of biliary stenosis. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

An integrated approach for comparative proteomic analysis of human bile reveals overexpressed cancer-associated proteins in malignant biliary stenosis.

Proteomics is a key tool in the identification of new bile biomarkers for differentiating malignant and nonmalignant biliary stenoses. Unfortunately, the complexity of bile and the presence of molecules interfering with protein analysis represent an obstacle for quantitative proteomic studies in bile samples. The simultaneous need to introduce purification steps and minimize the use of pre-fractionation methods inevitably leads to protein loss and limited quantifications. This dramatically reduces the chance of identifying new potential biomarkers. In the present study, we included differential centrifugation as a preliminary step in a quantitative proteomic workflow involving iTRAQ labeling, peptide fractionation by OFFGEL electrophoresis and LC-MS/MS, to compare protein expression in bile samples collected from patients with malignant or nonmalignant biliary stenoses. A total of 1267 proteins were identified, including a set of 322 newly described bile proteins, mainly belonging to high-density cellular fractions. The subsequent comparative analysis led to a 5-fold increase in the number of quantified proteins over previously published studies and highlighted 104 proteins overexpressed in malignant samples. Finally, immunoblot verifications performed on a cohort of 8 malignant (pancreatic adenocarcinoma, n=4; cholangiocarcinoma, n=4) and 5 nonmalignant samples (chronic pancreatitis, n=3; biliary stones, n=2) confirmed the results of proteomic analysis for three proteins: olfactomedin-4, syntenin-2 and Ras-related C3 botulinum toxin substrate 1. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

An integrated approach based on multiplexed protein array and iTRAQ labeling for in-depth identification of pathways associated to IVF outcome.

The emergence of high-throughput protein quantification methodologies has enabled the comprehensive characterization by longitudinal and cross-sectional studies of biological fluids under physiological and pathological conditions. In particular, the simultaneous investigation of cytokines and growth factors signaling pathways and their associated downstream effectors by integrated multiplexed approaches offers a powerful strategy to gain insights into biological networks and processes in living systems. A growing body of research indicates that bioactive molecules of human reproductive fluids, including human follicular fluid (hFF), may affect oocyte quality, fertilization and embryo development, thus potentially influencing the physiopathology of pregnancy-related conditions. In this work, an iTRAQ labeling strategy has been complemented with a multiplexed protein array approach to analyze hFFs with the aim to investigate biological processes and pathways related to in vitro fertilization (IVF) outcome. The iTRAQ labeling strategy lead to the quantification of 89 proteins, 30 of which were differentially expressed in hFFs with successful compared to unsuccessful IVF outcome. The targeted study, based on multiplexed antibody protein arrays, allowed the simultaneous quantification of 27 low abundance proteins, including growth factors, chemokines and cytokines endowed with pro- and anti-inflammatory activity. A significant number of differentially regulated proteins were involved in biological functions related to blood coagulation, acute phase response signaling and complement system. Overall, the present results provide an integrated overview of protein changes in hFFs associated to IVF outcome, thus improving current knowledge in reproductive medicine and fertility research.

Analysis of secreted proteins.

Most biological processes including growth, proliferation, differentiation, and apoptosis are coordinated by tightly regulated signaling pathways, which also involve secreted proteins acting in an autocrine and/or paracrine manner. In addition, extracellular signaling molecules affect local niche biology and influence the cross-talking with the surrounding tissues. The understanding of this molecular language may provide an integrated and broader view of cellular regulatory networks under physiological and pathological conditions. In this context, the profiling at a global level of cell secretomes (i.e., the subpopulations of a proteome secreted from the cell) has become an active area of research. The current interest in secretome research also deals with its high potential for the biomarker discovery and the identification of new targets for therapeutic strategies. Several proteomic and mass spectrometry platforms and methodologies have been applied to secretome profiling of conditioned media of cultured cell lines and primary cells. Nevertheless, the analysis of secreted proteins is still a very challenging task, because of the technical difficulties that may hamper the subsequent mass spectrometry analysis. This chapter describes a typical workflow for the analysis of proteins secreted by cultured cells. Crucial issues related to cell culture conditions for the collection of conditioned media, secretome preparation, and mass spectrometry analysis are discussed. Furthermore, an overview of quantitative LC-MS-based approaches, computational tools for data analysis, and strategies for validation of potential secretome biomarkers is also presented.

Epithelial cells as active player in fibrosis: findings from an in vitro model.

Kidney fibrosis, a scarring of the tubulo-interstitial space, is due to activation of interstitial myofibroblasts recruited locally or systemically with consecutive extracellular matrix deposition. Newly published clinical studies correlating acute kidney injury (AKI) to chronic kidney disease (CKD) challenge this pathological concept putting tubular epithelial cells into the spotlight. In this work we investigated the role of epithelial cells in fibrosis using a simple controlled in vitro system. An epithelial/mesenchymal 3D cell culture model composed of human proximal renal tubular cells and fibroblasts was challenged with toxic doses of Cisplatin, thus injuring epithelial cells. RT-PCR for classical fibrotic markers was performed on fibroblasts to assess their modulation toward an activated myofibroblast phenotype in presence or absence of that stimulus. Epithelial cell lesion triggered a phenotypical modulation of fibroblasts toward activated myofibroblasts as assessed by main fibrotic marker analysis. Uninjured 3D cell culture as well as fibroblasts alone treated with toxic stimulus in the absence of epithelial cells were used as control. Our results, with the caveats due to the limited, but highly controllable and reproducible in vitro approach, suggest that epithelial cells can control and regulate fibroblast phenotype. Therefore they emerge as relevant target cells for the development of new preventive anti-fibrotic therapeutic approaches.

Proteomic analysis of podocyte exosome-enriched fraction from normal human urine.

Urine results from a coordinated activity of glomerular and tubular compartments of the kidney. As a footprint of these cellular functional processes, urinary exosomes, and 40-80 nm membrane vesicles released after fusion with the plasma membrane into the extracellular environment by renal epithelial cells, are a source for identification of proteins and investigation of their role in the kidney. The aim of the present study was the identification of podocyte exosome proteins based on urine immunoabsorption using podocyte-specific CR1-immunocoated beads followed by proteomic analysis using LC MS/MS techniques. This methodology allowed the identification of 1195 proteins. By using a bioinformatic approach, 27 brain-expressed proteins were identified, in which 14 out of them were newly demonstrated to be expressed in the kidney at a mRNA level, and, one of them, the COMT protein, was demonstrated to be expressed in podocytes at a protein level. These results, attesting the reliability of the methodology to identify podocyte proteins, need now to be completed by further experiments to analyze more precisely their biological function(s) in the podocytes.

Autologous bone marrow mononuclear cell transplantation in patients with decompensated alcoholic liver disease: a randomized controlled trial.

OBJECTIVE: Impaired liver regeneration is associated with a poor outcome in patients with decompensated alcoholic liver disease (ALD). We assessed whether autologous bone marrow mononuclear cell transplantation (BMMCT) improved liver function in decompensated ALD. DESIGN: 58 patients (mean age 54 yrs; mean MELD score 19, all with cirrhosis, 81% with alcoholic steatohepatitis at baseline liver biopsy) were randomized early after hospital admission to standard medical therapy (SMT) alone (n = 30), including steroids in patients with a Maddrey's score ≥32, or combined with G-CSF injections and autologous BMMCT into the hepatic artery (n = 28). Bone marrow cells were harvested, isolated and reinfused the same day. The primary endpoint was a ≥3 points decrease in the MELD score at 3 months, corresponding to a clinically relevant improvement in liver function. Liver biopsy was repeated at week 4 to assess changes in Ki67+/CK7+ hepatic progenitor cells (HPC) compartment. RESULTS: Both study groups were comparable at baseline. After 3 months, 2 and 4 patients died in the BMMCT and SMT groups, respectively. Adverse events were equally distributed between groups. Moderate alcohol relapse occurred in 31% of patients. The MELD score improved in parallel in both groups during follow-up with 18 patients (64%) from the BMMCT group and 18 patients (53%) from the SMT group reaching the primary endpoint (p = 0.43 (OR 1.6, CI 0.49-5.4) in an intention to treat analysis. Comparing liver biopsy at 4 weeks to baseline, steatosis improved (p<0.001), and proliferating HPC tended to decrease in both groups (-35 and -33%, respectively). CONCLUSION: Autologous BMMCT, compared to SMT is a safe procedure but did not result in an expanded HPC compartment or improved liver function. These data suggest either insufficient regenerative stimulation after BMMCT or resistance to liver regenerative drive in patients with decompensated alcoholic cirrhosis. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN83972743.

Secretome profiling of differentiated neural mes-c-myc A1 cell line endowed with stem cell properties.

Neural stem cell proliferation and differentiation play a crucial role in the formation and wiring of neuronal connections forming neuronal circuits. During neural tissues development, a large diversity of neuronal phenotypes is produced from neural precursor cells. In recent years, the cellular and molecular mechanisms by which specific types of neurons are generated have been explored with the aim to elucidate the complex events leading to the generation of different phenotypes via distinctive developmental programs that control self-renewal, differentiation, and plasticity. The extracellular environment is thought to provide instructive influences that actively induce the production of specific neuronal phenotypes. In this work, the secretome profiling of differentiated neural mes-c-myc A1 (A1) cell line endowed with stem cell properties was analyzed by applying a shotgun LC-MS/MS approach. The results provide a list of secreted molecules with potential relevance for the functional and biological features characterizing the A1 neuronal phenotype. Proteins involved in biological processes closely related to nervous system development including neurites growth, differentiation of neurons and axonogenesis were identified. Among them, proteins belonging to extracellular matrix and cell-adhesion complexes as well as soluble factors with well established neurotrophic properties were detected. The presented work provides the basis to clarify the complex extracellular protein networks implicated in neuronal differentiation and in the acquisition of the neuronal phenotype. This article is part of a Special Issue entitled: An Updated Secretome.