Own Research Lines

Line 1: Biological Markers and Induction of Operational Tolerance in Liver Transplantation

Liver transplant tolerance is defined as the ability of a transplanted patient to accept the graft in the absence of immunosuppression (IS) while maintaining intact liver function. The liver is considered an immunoprivileged organ, with approximately 40% of liver transplant recipients potentially achieving tolerance. Identifying markers and/or pathways that enhance our understanding of this phenomenon could improve the lives of more patients suffering from the severe side effects of immunosuppressive drugs. Dr. Baroja's group studies transplant tolerance through two distinct approaches:

1.1. Identification of Biological Markers.
This line focuses on the identification of biological markers (RNA, miRNA, protein expression, circulating DNA) and immunological markers (cellular populations) in both peripheral blood and liver tissue. The goal is to determine which patients are more likely to become tolerant before IS withdrawal and to understand the pathways involved in immunotolerance. This approach has yielded excellent results, such as establishing the essential role of FOXP3-expressing regulatory T cells (Tregs) in this phenomenon, alongside other key players like B cells, NK cells, γδTCR cells, and liver iron metabolism.

1.2. Induction of a Pro-Tolerant Environment.
Another approach to tolerance is creating an environment that favors it. Clinical trials involving in vitro-expanded Treg infusion or replacing conventional immunosuppressive drugs with rapamycin derivatives—based on the differential effects of rapamycin on human Tregs and effector T cells (Teffs)—are some examples. Dr. Baroja’s group focuses on studying compounds or pathways that differentially affect Tregs and Teffs to enhance the Treg:Teff ratio.

Line 2: Danger Signals (DAMPs) in Liver Grafts Induced by Ischemia as a Predictive Value to Improve Organ Donation Quality in Liver Transplantation

In the past decade, the "Danger Hypothesis" has gained prominence, linking innate and adaptive immune system activation. The innate system detects "danger signals," known as DAMPs (Danger-Associated Molecular Patterns), and responds by creating a pro-inflammatory environment. These DAMPs, typically intracellular molecules that become extracellular upon tissue damage—such as ischemia/reperfusion injury—can influence transplant outcomes and even contribute to early rejection events.

2.1. Correlation Between Intra-Graft DAMPs After Ischemia and Transplant Outcomes in the Medium and Long Term.
This subline aims to correlate direct DAMP quantification with inflammasome activation in the human monocytic/macrophage cell line THP-1. Liver inflammation is assessed through immunofluorescence on biopsy samples to detect inflammasome proteins (IL1b, Casp1, ASC) and correlate findings with classical inflammation markers and ischemia/reperfusion injury quantification.

2.2. Differential DAMP Profiles in Grafts From Brain-Dead vs. Circulatory-Death (DCD) Donors.

2.3. Designing Potential Treatments to Inhibit DAMP-Mediated Transplant Deterioration.
New pharmacological inhibitors are explored to target various levels of the inflammasome activation pathway, including:

  • Signaling inhibitors (e.g., P2X7 inhibitors)
  • Complex formation inhibitors (e.g., NLRP3 inhibitors or oligomerization blockers)
  • Cytokine release inhibitors (e.g., punicalagin)

Line 3: Use of Organ Preservation Solution (OPS) Collected After Cold Ischemia as a Biomarker for Donor Organ Quality and Liver Transplantation Outcomes

3.1. Detection and Analysis of Biomarkers in OPS.
Expanding on the previous research line, this study seeks new biomarkers through a holistic approach, incorporating metabolomics, proteomics, microbiome analysis, miRNAs, extracellular vesicles, oxidative stress markers, iron metabolism, and more. The goal is to predict post-transplant evolution using a minimally invasive method: collecting OPS after cold ischemia. This could help prevent adverse post-transplant events, which still affect a significant percentage of patients despite advances in immunosuppression.

3.2. In Vitro Functional Studies.
Various cell types (macrophages, hepatocytes, cholangiocytes, endothelial cells, etc.) are cultured with OPS or specific compounds identified as correlated with transplant outcomes (miRNAs, vesicles, metabolites). Each cell type undergoes a tailored analysis, including gene expression changes, activation markers, and protein secretion.

3.3. Complementary Analysis of Liver Biopsies.
Two liver biopsies are collected from each donor organ:

  • T1 (pre-cold ischemia)
  • T2 (post-cold ischemia)

Samples are preserved in PaxGene Tissue Containers and embedded in paraffin. This fixation method enables comprehensive analyses, including histology (assessment of ischemia/reperfusion injury severity), immunofluorescence, gene expression (RNA, miRNA), DNA (metagenomic studies), metabolomics, and protein expression (Western blot or antibody arrays). These assays are performed as needed to complement previous findings.

Line 4: Identification of Molecular Signatures Involved in Hepatic Artery Thrombosis and Biliary Complications After Liver Transplantation

Hepatic artery thrombosis and biliary tract complications are among the leading causes of morbidity and mortality in liver transplant recipients. While these complications have traditionally been attributed to surgical technique, their underlying molecular mechanisms remain unclear. The primary objective of this project is to identify the molecular pathways implicated in these post-transplant complications and propose new therapeutic strategies to reduce their incidence.

To achieve this, samples are collected from the distal hepatic artery and bile duct of both the donor organ and recipient. These samples undergo differential gene expression, metabolomic, and proteomic analyses to uncover potential molecular drivers of these complications.

Line 5: Influence of Hepatic Microbiota on Liver Transplant Outcomes

The gut-liver axis, connected through the portal vein, makes the study of hepatic microbiota highly relevant in liver transplantation. A murine skin transplant model has demonstrated that commensal microbiota present in tissue can induce rejection. Hepatic microbiota, originating from the gut microbiota, can be characterized through metagenomic sequencing of liver tissue samples, potentially linking it to transplant outcomes.

5.1. Influence of diet in liver microbiota. Mouse model feed with different diet to study how this impact in the liver microbiota.

5.2. Rat Liver Transplantation Model.
A rat liver transplantation model will be established to study the effect of hepatic microbiota on transplant outcomes. This involves dietary modifications (standard, high-fiber, high-fat, ketogenic diets), fecal microbiota transplantation (FMT) between groups, and antibiotic treatments. The impact of these interventions on hepatic microbiota composition and transplant evolution will be assessed.

5.2. Hepatic Microbiota in Liver Transplant Patients.
Building on previous research lines, this study aims to identify microbiome signatures correlating with poorer transplant outcomes. Additionally, donor-recipient microbiota compatibility will be analyzed for its potential influence on transplantation success.