Pilot study on the relationship between albumin administration and changes in the transcriptomic profile. An investigation in patients from the PREDICT Study receiving albumin as treatment for several complications during clinical course.

Observational Study Richard Moreau (EF Clif. Paris, France)
Joan Clària (Hospital Clínic Barcelona-IDIBAPS; EF Clif. Barcelona, Spain)
Emmanuel Weiss (INSERM. Paris, France)

Promoter: EF Clif
Start date: 01/01/2020
End date: 30/04/2021

The objective of the study is to use whole-blood RNA sequencing (RNA-seq) data obtained in patients of the PREDICT study, to investigate the effects of intravenous albumin on circulating immune cells from patients with severe forms of acutely decompensated cirrhosis.

Thirty-seven patients without shock have been investigated using whole-blood RNA sequencing (RNA-seq), that was performed twice in 18 patients (before and after albumin administration), and once in 19 patients not receiving albumin. Whole-blood RNA-seq data were obtained once in 10 healthy subjects. We uncovered that those patients who had not received albumin had a generalized decrease in RNA signatures of lymphoid cells and that albumin electively abrogated the decline in RNA signatures of B cells. These findings prompted us to perform additional studies in 31 new patients with acutely decompensated assigned to either whole-blood RNA-seq (to explore the effects of albumin in patients with septic shock), bulk RNA-seq in peripheral blood mononuclear cells (PBMCs), blood flow cytometry, or single-cell RNA-seq (scRNA-seq) in PBMCs). Results have been used to write a first report that has been submitted for publication.

ALBIN. Mechanisms of action of ALbumin in patients with cirrhosis and Bacterial INfections.

Prospective cohort study Javier Fernández (Hospital Clínic Barcelona-IDIBAPS; EF Clif. Barcelona, Spain)

Promoter: EF Clif
Start date: 01/07/2020
End date: 31/07/2022

Prospective cohort multicentre study, aimed at gaining knowledge of the mechanisms of action of albumin in cirrhosis. Fifty patients will be included: 25 with SBP receiving albumin and 25 with non-SBP infections, but sepsis who will not receive albumin. Inflammatory markers, blood metabolomics and lipidomics, whole-blood and single-cell transcriptomics and mitochondrial function will be evaluated. The study is currently active in two centres, Hospital Clinic and Hospital del Mar. Until date, only 3 patients have been included, all three in the non-albumin group.

Identification of albumin receptors and albumin endocytic trafficking in human monocytes.

Basic translational research Loredana Saveanu (INSERM. Paris, France)
Joan Clària (Hospital Clínic Barcelona-IDIBAPS; EF Clif. Barcelona, Spain)
Sophie Lotersztajn (INSERM. Paris, France)

Promoter: EF Clif
Start date: 01/06/2019
End date: 31/12/2020

Human albumin (HSA) is currently used in the treatment of chronic liver diseases, where, as recently demonstrated, it promotes an anti-inflammatory response. By combining cellular biology and immunology methods, we aim to identify the cell population that up-take the most efficiently the HSA in human blood, the HSA receptors and their intracellular trafficking and signalling.

By performing multiparametric flow cytometry experiments on peripheral mononuclear blood cells, we identified a cell population that is, by far, the most efficient in HSA internalization. After feeding these cells with HSA, we immunoprecipitated the HSA and by quantitative mass-spectrometry we identified an HSA receptor protein candidate. We are currently validating these data and we have set-up, in human primary cells, the depletion of HSA receptor candidate by lentivirus-coded specific shRNA. In parallel, using cell lines "similar" to HSA receptive primary cells, we identified the endocytosis mechanisms involved in HSA up-take. These results remain to be validated in primary cells, from healthy people and, if possible, from chronic liver disease patients.

Definition of the utility of immune checkpoint receptors as therapeutic targets in ACLF.

Translational research Shilpa Chokshi (Foundation for Liver Research. London, United Kingdom)

Promoter: EF Clif
Start date: 01/09/2018
End date: 31/10/2021

Acute on Chronic Liver Failure (ACLF) is an immunological paradox. It is a hyperinflammatory state at with coexisting profound immunoparesis. Immune checkpoints and their soluble forms have been shown in many contexts, to modulate autoimmunity and responses to infection or malignancy. We, therefore, aimed to explore their role in initiation and propagation of the pathological immune response in ACLF.

We have measured levels of several immune checkpoints in the CANONIC cohort and, also in a smaller longitudinal cohort. Several are elevated with increasing ACLF grade, and we show higher levels of some of these factors can discriminate clinical outcomes. We also demonstrate that ACLF grade increases markers of gut translocation, suggesting that this contributes to the pathological state that different patterns of soluble immune checkpoints associate with different predisposition and injury in ACLF. We also state that levels of some galectins -important immune checkpoints- are elevated in ACLF liver tissue at the transcriptional level and are also produced by stressed liver tissue in an explant mode and that levels of immune checkpoints are dynamic in ACLF. Coincubation and FACS experiments from this longitudinal cohort have shown that blockade of some factors can affect the immune response in vitro. The laboratory experiments are complete, and analysis of this exciting data is being finalized and a publication prepared.

Albuminomics: identification of metabolites and lipids bound to albumin in patients with decompensated cirrhosis, patients with ACLF and healthy subjects.

Basic research Joan Clària (Hospital Clínic Barcelona-IDIBAPS; EF Clif. Barcelona, Spain)
Michael Rothe (Lipidomix. Berlin, Germany)
Óscar Yanes (Universitat Rovira i Virgili. Reus, Spain)

Promoter: EF Clif
Start date: 01/01/2020
End date: 31/12/2020

The goal of this project was to identify which metabolites and bioactive lipid mediators circulate free or bound to albumin, in the vascular bed of patients with acutely decompensated cirrhosis. To achieve this goal, plasma samples were processed suing immunoaffinity columns, able to separate the albumin-enriched fraction from the albumin-depleted fraction.

The metabolomic and lipidomic profiles of the albumin-enriched and albumin-depleted fractions were obtained by LC-MS/MS. Metabolomic analysis still needs further tuning but the lipidomic analysis have revealed a specific signature of lipids not bound by albumin. Within this signature, we have identified a small set of bioactive lipid mediators, which have the ability to modulate the immune system in the systemic circulation.

Effects of albumin on human neutrophil function in patients suffering from AD and ACLF.

Translational Study Daniel Irimia (Massachusetts General Hospital. Boston, USA)

Promoter: EF Clif
Start date: 01/04/2020
End date: 31/03/2021

Albumin levels are low in patients with chronic liver failure. Here, we are testing the hypothesis that albumin levels affect neutrophil function, the key white blood cells that protect against infections. If this is true, increasing the levels of albumin in chronic liver failure patients may help restore the natural defences against microbes.

We started working on the project with a 4 months-delay due to the pandemic. On the four tasks of the project, we completed the first two, representing approx. 66% of the proposed work. For these tasks, we performed a through characterization of the effect of albumin on the neutrophil antimicrobial functions, using neutrophils from healthy donors, with and without stimulation. For this purpose, we employed a recently reported assay, named neutrophil swarming. In parallel work, we determined using the same swarming assay that the neutrophils from patients with cirrhosis are impaired in their antimicrobial activities. The remaining work will leverage these two streams of knowledge and determine with high precision if albumin has an effect on patients’ neutrophils, in the context of chronic liver failure.

Autophagy as a mediator of HSA anti-inflammatory effects.

Basic Translational Study Sophie Lotersztajn (INSERM. Paris, France)
Loredana Saveanu (INSERM. Paris, France)
Joan Clària (Hospital Clínic Barcelona-IDIBAPS; EF Clif. Barcelona, Spain)

Promoter: EF Clif
Start date: 22/07/2020
End date: 31/12/2021

Autophagy is a major player in the regulation of anti-inflammatory response. Based on our preliminary data, this project will investigate whether human serum albumin (HSA) triggers autophagy in blood cells at different stages of chronic liver diseases, and whether the anti-inflammatory properties of HSA rely on autophagy activation and explore the underling molecular mechanisms.

Our pilot studies demonstrate that HSA enhances particular pathways of autophagy in specific blood cell sub-types from both, healthy subjects and patients with cirrhosis. We are further validating these data in a larger cohort, and explore the early signalling pathways, leading to autophagy activation upon HSA treatment. Current studies also combine pharmacological and lentivirus-coded specific shRNA approaches, to identify the molecular mechanisms leading to autophagy activation by HAS. These concern the identification of cellular receptors and the intracellular trafficking mechanisms that -upon HSA treatment- are able to trigger autophagy in peripheral blood cells from both, healthy and cirrhotic patients.

Effects of albumin in a human model of dermal inflammation.

Experimental medicine Allastair O'Bryen (UCL, Royal Free Hospital. London, United Kingdom)

Promoter: EF Clif
Start date: 31/12/2020
End date: 31/08/2021

Chronic liver disease is associated with profound immunological changes that leave patients highly susceptible to infection. Albumin, the large circulating plasma protein, has been shown to have anti-inflammatory effects and we shall investigate this, using a human blister model of inflammation.

As England is in lockdown, recruiting healthy volunteers for the blister models is not currently possible. We have, therefore, investigated possible lipid mediators that may be modified by albumin infusions by performing analyses on existing plasma and blister samples from patients with decompensated cirrhosis and correlating this with albumin treatment and measured circulating levels.

Protein and lipid inflammatory signatures in circulating exosomes in patients with ACLF.

Basic translational research Maria Rosa Sarrias (Germans Trias i Pujol Research Institute (IGTP). Badalona, Spain)

Promoter: EF Clif
Start date: 15/07/2019
End date: 30/06/2021

The objective of this study is to compare the lipid mediator signature and the content of immune-resolving proteins in exosomes from ACLF patients with full-blown systemic inflammation, with that of exosomes from patients with mere acute decompensation, compensated cirrhosis and healthy subjects.

Extracellular vesicles (EVs) were isolated from plasma by Size-Exclusion Chromatography (SEC). The EV were defined as positive for EV markers CD9 and CD5L, and their size, amount and morphology characterized by NTA and cryo-EM, respectively. Then, EVs were analysed by LC-MS/MS to characterize the profile of pro-resolving lipids. The results suggest a loss of pro-resolving lipids and CD5L protein content in EVs from ACLF vs decompensated cirrhotic patients. Moreover, in vitro analyses on macrophage activity showed that CD5L signalling affects lipid mediator content, and viceversa, providing for the first time, a link between this anti-inflammatory protein and pro-resolving lipid signalling in liver pathology.