Bile proteome reveals biliary regeneration during normothermic preservation of human donor livers
Ontology highlight
ABSTRACT:
INSTRUMENT(S): NextSeq 500
ORGANISM(S): Homo sapiens
SUBMITTER: Eugene Berezikov
PROVIDER: E-MTAB-13501 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Liver transplantation is the only treatment option for patients with end-stage liver disease. However, a persistent disparity remains between supply and demand for donor organs suitable for transplantation, resulting in a waiting list mortality of up to 20%. This has resulted in an increasing use of high-risk grafts from suboptimal, extended criteria donors, including livers from donation after circulatory death (DCD) donors, and donors with significant comorbidities such as advanced age, diabetes mellitus and livers with high fat content. While cold storage remains sufficient for livers considered optimal for transplantation, ECD grafts are more vulnerable to cold ischemic damage, resulting in many of these grafts being rejected. As an alternative to cold storage, ex-situ normothermic machine perfusion (NMP) has become an increasingly popular method for preservation, transportation and assessment of ECD livers prior to transplantation. This allows for functional assessment of the graft and provides the opportunity to assess key processes that can be used to determine organ viability. The use of both hepatocellular and biliary viability criteria in selection of suboptimal livers from ECD donors has led to the successful transplantation of these grafts with a low incidence of post-transplant cholangiopathies. However, the mechanisms of biliary injury, and recovery thereof, following static cold storage remains poorly understood. Insight into these mechanisms may further improve preservation and selection of ECD livers. Therefore, we performed an in-depth proteomics analysis of bile samples collected from human ECD livers during NMP to identify potential mechanisms linked to biliary injury, function and regeneration over the course of the perfusion, and to assess whether biliary protein profiles can distinguish between transplantable and non-transplantable grafts.
2023-11-21 | PXD046355 | Pride
Project description:Background and Aims: Liver transplantation provides an effective cure for end-stage liver disease but is hampered by a severe organ shortage. Normothermic machine perfusion (NMP) of donor livers allows dynamic preservation in addition to viability assessment prior to transplantation. Little is known about the injury and repair mechanisms induced during NMP. Therefore, we examined gene and protein expression changes in a cohort of discarded human livers during NMP, stratified by liver viability. Approach and Results: 6 human livers from donation after circulatory death (DCD) underwent 12 hours of NMP, of which 3 met viability criteria. We applied bulk transcriptomics to evaluate differences in gene expression relating to injury, repair, and regenerative responses among livers based on viability. Viable livers demonstrated robust activation of innate immunity after 3 hours of NMP followed by enrichment of pro-repair and pro-survival mechanisms. Nonviable livers demonstrated delayed and persistent enrichment of innate immune responses. Viable livers demonstrated effective induction of autophagy, the cellular repair and homeostasis pathway, compared to nonviable livers. Enrichment of pro-survival signaling was also broader in these livers. Conclusions: NMP of discarded DCD human livers results in ischemia-reperfusion injury, but importantly activates autophagy as a means of cellular repair. More pronounced activation of autophagy was seen in livers that met viability criteria for transplantation. Therapeutic targeting of the autophagy mechanism may allow rehabilitation of nonviable livers for transplantation.
2022-01-25 | GSE165568 | GEO
Project description:Liver are frequently declined for transplantation due to the donor age. It is still unclear how donor age affects the graft quality. Normothermic machine perfusion (NMP) has been proposed as a useful assessment tool prior to transplantation. We aimed to compare the performance of young and elderly rat liver grafts in a small animal NMP model. Grafts from 24 rats were procured, either 3 or 12 months old and perfused for 6 hours at 37°C or stored on ice as a reference group (n=6/group). Livers in both NMP groups cleared lactate and produced bile, with similar pressure, bile production, and pH levels. However, older rat livers showed higher peak transaminase and urea levels. Proteomic analysis revealed differences in protein composition, particularly higher levels of proteins related to oxidoreductase and catalytic activity in older livers. Older age appeared to increase susceptibility to oxidative stress in the livers.
2024-08-09 | PXD045394 | Pride
Project description:Bile has gained increasing attention for its potential to reflect the overall health of the biliary system. Proteomic analysis of bile could deepen our understanding of the molecular pathophysiology of biliary disease and injury and provides a potential source of diagnostic biomarkers. With the emergence of normothermic machine perfusion (NMP) prior to liver transplantation, bile samples can be easily collected and analyzed. However, the unique composition of bile, including high concentrations of endogenous detergents and salts, can make application of proteomics challenging. In this study, we systematically evaluated a variety of available trypsin-digestion methods to optimize proteomics analysis of bile obtained from human NMP livers. Bile was collected from 12 human donor livers that were accepted for transplantation after NMP viability assessment. We performed tryptic digestion using 6 different methods: in-gel, in-solution, S-Trap, SMART, EasyPep, and filter-aided sample purification, with or without an additional precipitation step prior to digestion. Untargeted, data-independent acquisition proteomics was performed using an Exploris 480 mass spectrometer coupled with a FAIMS device. The methods were judged for total protein IDs, variation, and specific protein characteristics to determine the most optimal method. A total of 4650 unique proteins were identified across all samples and all methods. Methods involving precipitation identified substantially more proteins (4500 vs 3815, respectively). In-gel crude was the exception, identifying a comparable number of proteins to precipitated in-gel digest. We found minimal differences in the mass, cellular component, or hydrophobicity of identified proteins between methods. Intra-method variability was notably diverse, with in-gel, in-solution, and EasyPep outperforming other methods with considerably less variation. Age-related biological comparisons revealed significant disparities in protein abundances between methods, but younger donors consistently showed upregulation of metabolic-related processes compared to older donors. Older donors showed upregulation of immune response and cell cycle-related processes. The digestion method used for proteomic analysis of bile can heavily influence the results obtained. A method of protein purification appears to be essential for sufficient quality mass spectrometry runs and subsequent data. Sample processing speed, cost, cleanliness, and reproducibility should be carefully considered when selecting a protein digestion method for this difficult sample type.
2024-08-27 | PXD051062 | Pride
Project description:Backgroud: among strategies to limit ischemia/reperfusion (IR) injuries in transplantation, cell therapy using stem cells to condition/repair transplanted organ appears promising. We hypothesized that using a cell therapy based on exosomes derived from Urine Progenitor Cells (UPCs) during hypothermic and normothermic machine perfusion can prevent IR-related kidney damages. Methods: we isolated and characterized porcine UPCs and their exosomes (Exo). Then these were used in an ex vivo preclinical porcine kidney preservation model. Kidneys were subjected to warm ischemia (32min) then preserved by Hypothermic Machine Perfusion (HMP) for 24h before 5h of Normothermic Machine Perfusion (NMP). Three groups were performed (n=5-6): Group 1 (G1): HMP/vehicle + NMP/vehicle, Group 2 (G2): HMP/Exo + NMP/vehicle, Group 3 (G3): HMP/Exo + NMP/Exo. Results: porcine UPCs were successfully isolated from urine and fully characterized as well as their exosomes which were found of expected size/phenotype. RNA-seq performed on kidney biopsies showed different profiles between G1 and G3 with regulation of potential IR targets of Exo therapy. Conclusions: we showed the feasibility/efficacy of UPC-exosomes for hypothermic/normothermic kidney conditioning prior to transplantation, paving the way of combining machine perfusion with exosome-based cell therapy for organ conditioning.
2024-06-01 | GSE255005 | GEO
Project description:We performed in-depth serial immune cell mapping of human donor livers before and after normothermic machine perfusion (NMP) as well as after reperfusion to specifically investigate the source of immunomodulatory cytokines/chemokines and the dynamics of immune activation at the single-cell level.
2023-02-15 | GSE216584 | GEO
Project description:Normothermic machine perfusion (NMP) has been successfully implemented in clinical routine of liver transplantation over the past years. However, little is known about the mechanisms how NMP impacts on the transcriptome of a human donor liver. We herein examined gene expression profiles in transplanted and non-transplanted livers over NMP time. 50 livers subjected to NMP were included in this study. 30 were transplanted after a maximum of 20 hours (h) perfusion, while 15 were discarded due to poor performance. Biopsies were collected befor eNP (PRE), 1h, 6h, 12h, 20h of NMP and after reperfusion. Next-generation sequencing was applied in liver biopsies to assess differential gene expression over perfusion time. Perfusate samples were collected regularly to monitor liver function. Comparison in differential gene expression between PRE and 20h NMP showed 415 upregulated and 727 downregulated genes. Most significantly upregulated genes were associated with extra cellular matrix organization, cell growth/differentiation processes and cytokine signaling. A set of genes were identified which were significantly differentially expressed and important for classification of non-transplanted vs transplanted biopsies, especially at 12 and 20h of NMP. A 7-gene-signature showed good separation already at 6H NMP, thereby CD274 (PD-L1) expression was ponted out as most important.
2024-08-29 | GSE263614 | GEO
Project description:Acute rejection remains an important risk factor affecting the survival of recipients following transplantation. Bone marrow mesenchymal stem cells (BMMSCs) are used in the treatment of organ transplantation due to their immunomodulatory ability. BMMSCs were isolated from rat bone marrow and modified with the adenovirus for heme oxygenase 1 (HO-1) gene. Saline solution, BMMSCs or HO-1/BMMSCs were perfused into the donor liver in vitro using a normothermic machine perfusion (NMP) system, followed by liver transplantation. The liver grafts were collected at 7 days post-transplantation. Gene chip technology was used to detect differential gene expression.
2021-03-02 | GSE167980 | GEO
Project description:Background and Aims: Liver transplantation is a successful treatment for patients with liver failure. However, organ shortage results in over 11% of patients loosing their chance of a transplant due to worsening of their liver disease. Ischemia/reperfusion injury (IRI) is the major cause leading to graft loss after liver transplantation. Therefore, novel methods of organ-preservation have been developed in recent years to minimize IRI. One such device is the OrganOx metra, a normothermic machine perfusion (NMP) device providing oxygen and nutrition to allow aerobic metabolism and minimizing IRI. OrganOx metra has presented with several advantages compared to conventional cold storage (CS). We aimed to confirm the impact of NMP on reducing IRI and to define the underling mechanisms. Methods: We compared 12 NMP with 27 CS-preserved livers by performing gene microarray, immunoprofiling of hepatic lymphocytes and immunochemistry staining of liver tissues for assessing necrosis, platelet deposition and neutrophil infiltration, and the status of steatosis after NMP or CS pre- and post-reperfusion. Results: Recipients receiving NMP grafts showed significantly lower peak AST levels than those receiving CS grafts. NMP altered gene-expression profiles of liver tissue from pro-inflammation to pro-healing and regeneration. NMP also reduced the number of IFN- and IL-17 producing T cells and enlarged CD4posCD25highCD127negFOXP3pos Treg pool. NMP liver tissues showed less necrosis and apoptosis in the parenchyma and fewer neutrophils and platelet infiltration compared to CS liver tissues. Conclusion: Reduced IRI in NMP recipients was the consequence of the combination of inhibiting inflammation and promoting graft regeneration.
2019-04-10 | GSE112713 | GEO
Project description:There is a ever widening gap between the availability of donor livers suitable for transplantation and liver transplant demand of a growing waiting list. To expand the pool of liver grafts, donation after circulatory death is increasingly being used. Livers procured after circulatory death are exposed to a harmful period of warm Ischemia (WI) during the donation process, reducing post-transplant results significantly. Additionally, the conventional technique of static cold storage does not protect grafts that suffered WI. New dynamic preservation strategies by means of Normothermic Machine Perfusion (NMP) showed enhanced preservation of these type of grafts, but it is unknown how NMP influences liver cell biology during perfusion. Hepatocytes and cholnagiocytes have been shown to release Extracellular Vesicles (EVs) in the systemic circulation and bile, respectively. We hypothesized that EVs are released during liver NMP as well, in perfusate and bile. EVs have been separated from perfusate and bile during NMP of porcine livers, and the RNA cargo of the EVs has been sequenced to gain insights on liver cell biology during machine perfusion. Pigs were randomly allocated to undergo liver procurement either immediately after the start of surgery (no-WI, n=5) or after a period of WI of 60-minutes (clamping of thoracic aorta; WI-60, n=5). All livers (n=10) were cooled down and flushed out with ice-cold preservation solution, according to standard clinical practice. Porcine blood was collected and washed with a cell saver to obtain packed red blood cells for NMP. The hepatic artery, portal vein, and inferior vena cava were cannulated and after a period of about 2-hours of cold storage, NMP was started. All liver underwent NMP for 6-hours. Packed red blood-based perfusate samples were taken at 1, 3, and 6h of NMP; whereas, the bile produced the first 3-hours and the last 3-hours was pooled in 2 samples. Perfusate and bile samples were processed immediately for EVs separation with charge-based precipitation in a mixture of protamine and polyethylene glycol. The total RNA was extracted from EVs. Libraries of mRNA and of small RNA were prepared and analyzed for quality and size range, and sequenced on an Illumina HiSeq4000 instrument. Count-based differential expression analysis was done with R-based Bioconductor package DESeq2, and the following comparisons were performed: - Unpaired (between groups) comparison - perfusate samples WI-60 vs. no-WI - bile samples WI-60 vs. no-WI - Paired (withing group) comparison - no-WI - perfusate sample 1h vs. perfusate sample 3h - perfusate sample 1-3h vs. perfusate sample 6h - bile sample 1-3h vs. bile sample 3-6h - perfusate samples vs. bile samples - WI-60 - bile sample 1-3h vs. bile sample 3-6h - perfusate samples vs. bile samples Results were adjusted for multiple testing with the Benjamini-Hochberg procedure, to control for false discovery rate. Additionally, the function of the transcripts identified in the EVs of both groups and of the differentially expressed transcripts was investigated with gene ontology enrichment analysis for the domains \\"biological process\\" and \\"pathways\\".
2024-09-27 | E-MTAB-11420 | biostudies-arrayexpress