Annarita Farina

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.

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.

Proteomic analysis of human bile from malignant biliary stenosis induced by pancreatic cancer.

Stenosis of the common bile duct may be either due to benign (chronic pancreatitis) or malignant (cholangiocarcinoma, pancreatic adenocarcinoma) conditions. The benign nature of the stricture should be first confirmed in order to ensure appropriate therapy. Therefore, the identification of markers allowing discrimination between malignant and benign biliary stenosis would be very valuable in clinical practice. To this intent, we performed a proteomic analysis of bile samples from patients having a biliary stenosis caused by pancreatic adenocarcinoma. Bile samples were collected during endoscopic retrograde cholangiopancreatography and purified using different methods. The extracted proteins were then analyzed by SDS-PAGE and LC-MS/MS. A total of 127 proteins were identified, 34 of which have not been previously detected in proteomic studies of bile. Among them, several proteins have been described as potential biomarkers of pancreatic cancer. We extended our investigation by studying the expression of some of these pancreatic cancer markers in bile samples collected from patients with various etiologies of biliary stenosis including pancreatic cancer, cholangiocarcinoma, chronic pancreatitis, as well as gallstone-induced stenosis. Our data showed a conspicuous overexpression of CEACAM6 and MUC1 (CA19-9) in pancreatic cancer and cholangiocarcinoma samples, according to the hypothesis that bile fluid collects cancer-associated protein leaking from the tumor microenvironment. These results underline the interest of using bile as a source of biomarkers for the diagnosis of malignant biliary stenosis.

Proteomic analysis of human bile and potential applications for cancer diagnosis.

Bile is a body fluid produced by the liver and drained by biliary ducts into the duodenum. It has two major functions: first, it contains bile acids, which are critical for the digestion of fats, and second, it is an excretory pathway for many endogenous and exogenous compounds. Proteomic analysis of bile is particularly difficult since this fluid contains high concentrations of various substances that strongly interfere with protein separation and identification techniques. Furthermore, owing to its deep location in the body, bile must be collected by surgical or endoscopic procedures. However, as was speculated for other body fluids, bile appears to be a promising sample for the discovery of disease biomarkers leaking from proximal tissues: the liver, pancreas or biliary tree. The interest in clinical proteomics was demonstrated by two studies that identified in bile potential biomarkers for two deadly and difficult to diagnose neoplasms, pancreatic cancer and cholangiocarcinoma.

Role of proteomics to differentiate between benign and potentially malignant pancreatic cysts

Pancreatic cystic neoplasms represent 10-15% of primary cystic masses of the pancreas. While pancreatic cysts are detected with an increasing frequency due to the use of advanced imaging modalities in clinical practice, the diagnosis of pancreatic cystic neoplasms remains unsatisfactory because available diagnostic techniques proved not sensitive enough so far. This study was designed to characterize the proteomic pattern of pancreatic cyst fluids obtained from various cystic lesions. Cyst fluids were collected by direct puncture during open surgery to avoid any possible contamination from other tissues. CEA, CA-19-9, and amylase concentrations were measured using specific immunoassays. After immunodepletion and fractionation by SDS-PAGE, proteins were digested and analyzed by LC-MS/MS. Specific histological lesions were found to be associated with distinct protein patterns. Interestingly, some of these proteins have been proposed as biomarkers of pancreatic cancer. Immunoblots allowed for verifying the differential expression in specific cyst fluids of two selected proteins, olfactomedin-4 and mucin-18. Finally, immunohistochemistry was performed to correlate these data with the expression pattern of olfactomedin-4 and mucin-18 in pancreatic cyst tissues. Results from this study indicate that proteomic analysis of cyst fluid could provide reliable candidates for developing new biomarkers for the preoperative management of malignant and premalignant pancreatic cysts.

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.

Temporal proteomic profiling of embryonic stem cell secretome during cardiac and neural differentiation

During recent years, increased efforts have focused on elucidating the pluripotency and self‐renewal of stem cells. Differentiation towards the different lineages has attracted significant attention given the potential use of stem cells in regenerative medicine. Embryonic stem cell differentiation is a complex process coordinated by strictly regulated extracellular signals that act in an autocrine and/or paracrine manner. Through secreted molecules, stem cells affect local niche biology and influence the cross‐talking with the surrounding tissues. Emerging evidence supports the hypothesis that fundamental cell functions, including proliferation and differentiation, are strictly regulated by the complex set of molecules secreted from cells. The understanding of this molecular language could largely increase our knowledge on pathways regulating stem cell differentiation. Here, we have used a proteomics platform to investigate the profile of proteins secreted during differentiation of murine embryonic stem cells. We have followed the dynamics of protein secretion by comparing the secretomes at different time points of murine embryonic stem cell cardiac and neural differentiation. In addition to previously reported molecules, we have identified many secreted proteins not described so far as released from embryonic stem cells nor shown to be differentially released during the process of cardiomyogenesis and neurogenesis.

A step further in the analysis of human bile proteome.

Bile was shown to collect proteins known as potential cancer biomarkers. Thorough proteomic analysis of bile is of particular interest to search for new, more sensitive and more specific, biomarkers of cancers affecting the biliary tract and surrounding organs, such as the pancreas and the liver. Therefore, extending the knowledge of the bile proteome is highly relevant, but this has proved technically difficult. In this study, we describe a strategy that circumvents problems related to the biochemical complexity of this sample and the presence of high concentrations of interfering substances. Bile collected from a patient suffering from a biliary stenosis caused by a pancreatic adenocarcinoma was fractionated by a differential centrifugation scheme, involving a stepwise increase in centrifugation speeds. Pellets and the final supernatant were further fractionated by polyacrylamide gel electrophoresis and proteins were in-gel digested prior to LC-MS/MS analysis. This approach allowed the identification of 445 unique proteins with at least two peptides (812 proteins if single-hit proteins were included), which represents a 3-fold increase in the knowledge of bile proteome. The subsequent literature comparison revealed that numerous biliary proteins identified in this sample were related to pancreas cancer. Immunoblot analysis of some known tumor markers revealed that they were preferentially associated with the soluble fraction rather than with pellets containing cellular components.

Proteomic profiling in an animal model of acute pancreatitis

Acute pancreatitis (AP) is an inflammatory disease of the pancreas, which evolves in approximately 20% of the patients to a severe illness associated with a high mortality rate. In this study, we performed a comparative proteomic analysis of pancreatic tissue extracts from rats with AP and healthy rodent controls in order to identify changes in protein expression related to the pathobiological processes of this disease. Pancreatic extracts from diseased and controls rats were analyzed by 2‐DE and MS/MS. A total of 125 proteins were identified from both samples. Comparative analysis allowed the detection of 42 proteins or protein fragments differentially expressed between diseased and control pancreas, some of them being newly described in AP. Interestingly, these changes were representative of the main pathobiological pathways involved in this disease. We observed activation of digestive proteases and increased expression of various inflammatory markers, including several members of the α‐macroglobulin family. We also detected changes related to oxidative and cell stress responses. Finally, we highlighted modifications of 14‐3‐3 proteins that could be related to apoptosis regulation. These results showed the interest of proteomic analysis to identify changes characterizing pancreatic tissue damage and, therefore, to highlight new potential biomarkers of AP.

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.