The main objective of the Grifols Chair for Translational Research is to foster and promote research studies aimed at characterizing the mechanisms underlying the presence of systemic inflammation in patients with decompensated cirrhosis and its role in the development of ACLF. In particular, the Grifols Chair is paying special attention to the mechanisms responsible for the therapeutic properties of albumin infusions in the prevention of organ failure(s) in these patients.
The EF Clif designed a full proposal of research activities for the 2018 period, which were focused on the therapeutic actions of albumin in vitro and the investigation of samples collected during clinical studies in which albumin infusion was employed as the main therapy. In broad terms, the 2018 proposal had three specific objectives:
- 1To establish new biology associated with the anti-inflammatory properties of albumin.
- 2To apply cutting-edge technology to understand the biological properties of albumin.
- 3To provide proof-of-concept of the “anti-inflammatory properties” of albumin in clinical studies.
> THE IMMUNOMODULATION EFFECTS OF ALBUMIN
PI: Joan Clària (Hospital Clínic, Barcelona, Spain).
Promoter: EF Clif
In vitro studies in immune cells.
Human serum albumin (HSA) is an effective therapy in patients with advanced liver disease at risk of developing acute-on-chronic liver failure (ACLF). Recent studies have demonstrated that HSA downplays systemic inflammation independently of its oncotic properties, although the mechanisms underlying these anti-inflammatory actions are unknown.
In the current study, we investigated the molecular mechanisms of HSA in peripheral innate immune cells exposed to a bacterial pathogen-associated molecular pattern molecule (PAMP).
Patients with AD receiving albumin therapy showed significantly reduced circulating levels of tumour necrosis factor (TNF) alpha, interleukin (IL) 6, IL-10 and granulocyte colony-stimulation factor (G-CSF). Similar anti-inflammatory effects were observed in vitro in PBMC and PMN stimulated with CpGs and incubated with HSA. No changes in phagocytosis and respiratory burst were detected, indicating that HSA therapy did not compromise leukocyte defensive mechanisms against pathogens. These findings suggest that HSA interferes with the leukocyte endosomal TLR signaling pathway, thus providing a mechanism for the anti-inflammatory properties of HSA infusions in patients with AD cirrhosis.
Ex vivo studies in precision-cut liver and renal tissue slices.
Albumin infusions reduce systemic inflammation and prevent organ failure(s) in patients with advanced chronic liver disease by mechanisms not completely defined. In the current study we performed in vivo (double transgenic humanized neonatal Fc receptor (FcRn)/albumin mice), ex vivo (precision-cut liver slices (PCLS)) and in vitro (hepatocytes) experiments to uncover the potential mechanisms implicated in the tissue protective actions of albumin.
Transgenic humanized FcRn/albumin cirrhotic mice receiving albumin showed reduced levels of hepatic F4/80 and caspase-3 immunostaining. In PCLS from wild-type mice, both preventive and therapeutic albumin administrations, significantly reduced TNF-alpha-induced caspase-3 activity and up-regulated the expression of tissue repair genes.
Reduction of caspase-3 activity in hepatocytes incubated with albumin was associated with reduced release of the cysteine protease cathepsin B from lysosomes and a lower expression of the apoptotic transcription factor CHOP.
Taken together, these findings uncover novel mechanisms by which albumin could protect organs from tissue damage, contributing to the understanding of why albumin infusions are useful in patients with advanced liver disease at risk of developing organ failures.
Translational studies based on samples obtained in interventional studies in patients with decompensated cirrhosis receiving intravenous human serum albumin (Albutein®, 20%).
We investigated the effect of albumin treatment (20% solution) on hypoalbuminemia, cardiocirculatory dysfunction, portal hypertension, and systemic inflammation in patients with decompensated cirrhosis with and without infections.
First, we assessed the effects of long-term (12 weeks) treatment with low- and high-albumin dosages (LAlbD, and HAlbD, respectively) on serum albumin, plasma renin, cardiocirculatory function, portal pressure and a large panel of cytokines in 18 non-infected patients (Pilot-PRECIOSA study). We also assessed the effect of short-term (one week) treatment with antibiotics alone versus antibiotics plus albumin on plasma cytokines in biobanking samples from 78 patients with bacterial infections included in a RCT (INFECIR-2 study).
Circulatory dysfunction and systemic inflammation were extremely unstable in many patients included in the pilot-PRECIOSA study. Long-term treatment with HAlbD but not with LAlbD was associated with normalization of serum albumin, improvement in circulation stability and left ventricular function, and suppression of keystone cytokines, without significant changes in portal pressure. The immunomodulatory effect of albumin observed in the Pilot-PRECIOSA study was confirmed in the INFECIR-2 study patients treated with albumin.
In summary, albumin treatment improves systemic inflammation and cardiocirculatory dysfunction in patients with decompensated cirrhosis. These effects may underlie the beneficial effects of albumin therapy on clinical outcomes in decompensated cirrhosis.
> IMMUNE CHECKPOINT RECEPTORS IN CIRRHOTIC PATIENTS WITH ACUTE DECOMPENSATION WITH AND WITHOUT ACLF.
PI: Shilpa Chokshi (Institute of Hepatology, Foundation for Liver Resarch, London, UK)
Promoter: EF Clif
Acute-on-chronic liver failure (ACLF) is characterised by a suprainflammatory state that coexists with profound immune paralysis. Individual immunoregulatory checkpoint receptors (CR) can direct functional activation and/or inhibition of both innate and adaptive immunity. We have shown that bacterial translocation drives hyperexpression of inhibitory-CRs in Alcoholic Hepatitis but their role in ACLF is unknown.
We measured serum levels of 15 known stimulatory and inhibitory CRs, bacterial translocation and gut barrier integrity in 511 patients with decompensated cirrhosis included in the CANONIC Study; 334 with acute decompensation (AD) and 177 with ACLF at inclusion. Both soluble immune-activatory-CR CD40 and immunosuppressive-CR BTLA increased significantly with presence of ACLF and ACLF grade, but we saw no changes in PD1, PDL1 and CTLA4. Serum CD80 also increased with ACLF grade.
This was correlated with bacterial translocation and gut permeability across ACLF grades. Bacteremia during admission for all patients was also associated with increases in both DLac and ZO1. One-year transplant-free survival analysis demonstrated that increased survival was associated with lower serum BTLA, CD40 and CD80.
In this study, we demonstrate for the first time the association of both inhibitory and stimulatory immune checkpoints with the paradoxical ‘hyperinflammatory immunodeficient’ state observed. Our analysis also reveals that intestinal permeability and bacterial translocation are related to ACLF severity and together with serum levels of checkpoint receptors may discriminate between clinical outcomes.
> METABOLOMICS UNCOVERS A UNIQUE BLOOD METABOLITE FINGERPRINT OF PATHOPHYSIOLOGICAL RELEVANCE IN PATIENTS WITH ACLF
PI: Richard Moreau
Promoter: EF Clif
Acute-on-chronic liver failure (ACLF) which develops in patients with acutely decompensated cirrhosis, is characterized by intense systemic inflammation associated with organ failure(s). Because cell metabolism alterations which are associated with organ failures are unknown, we aimed to analyze blood metabolome in patients with and without ACLF. Along these lines, we performed untargeted metabolomics using liquid chromatography coupled to high-resolution mass spectrometry in serum from 334 patients with AD (acute decompensation without ACLF), 179 with ACLF, 20 with compensated cirrhosis, and 29 healthy subjects.
Our results of a specific blood metabolite fingerprint which intensity increased across ACLF grades, and was similar in patients with kidney failure and in those without, indicating that it was not a simple reflect of decreased kidney excretion but rather an alteration of cell metabolism. These findings indicate that ACLF is distinguishable from AD by a blood metabolite fingerprint capturing blood metabolite accumulation in ACLF and witnessing profound multiorgan alterations in major metabolic pathways, in particular depressed energy production, which may partake into the development of organ failures. In addition, metabolites accumulated in blood may contribute to the severity via toxic effects.
> GENOME-WIDE ASSOCIATION ANALYSES (GWAS) IDENTIFY NOVEL GENETIC VARIANTS ASSOCIATED WITH THE ACLF SYNDROME
PI: Joan Clària
Promoter: EF Clif
The aim of this study was the identification of genetic variants associated with ACLF syndrome that might influence its clinical course and severity.
The study was performed in 829 patients with decompensated cirrhosis recruited in the CANONIC study (a multicenter observational investigation involving 1343 hospitalized patients with cirrhosis from February to September 2011 in 29 liver units within 8 European countries), which were genotyped using the Infinium® Global Screening Array (Illumina, San Diego, CA).
This array has a very rich up-to-date content, includes 692.368 SNPs and is suitable for GWAS including rare variants, while also containing clinically relevant content.
Prior to association analysis, a validation of the data was performed. All Caucasian patients according to the database were selected and the remaining patients were analysed by IBD with HAPMAP data. The genotypes with a MAF of less than a 1% were excluded from the sample. SNPs with a Hardy-Weinberg analysis p-value of less than 0.001 were also excluded. Some markers with locus missingness were also excluded.
Finally, we checked the sex concordance with the database. The final database available for GWAS analysis included 806 individuals and 500.953 SNPs. The association between each single nucleotide polymorphism (SNP) and traits relevant to the ACLF syndrome was tested using linear regression, adjusted for age, gender and ethnicity.
The traits included in the analysis were ACLF, organ dysfunctions or organ failure, mortality (28-day, 90-day and 6-month), markers of systemic inflammation (cytokines, CRP and WBC count), oxidative stress (HNA2, irreversible oxidized albumin) and macrophage activation (sCD206 and sCD163). The association analysis was performed using PLINK software. Continuous variables were normalized if needed. Additional permutation tests were performed to confirm associations and to avoid false positives.
The SNPs were considered significantly associated when the Bonferroni adjusted p-value was less than 5.0x10-8. Following this procedure, GWAS analysis identified 17 candidate SNPs strongly associated with clinical and laboratory features of the ACLF syndrome. SNPs were disease-related and associated with higher ACLF prevalence and severity, worse prognostic scores and higher prevalence of bacterial infections.