Search

Endomyocardial biopsies (EMB) gene expression profiling is a promising companion tool for rejection diagnosis. Targeted molecular profiling combined with reproducible formalin-fixed paraffin-embedded (FFPE) EMB-based technology has the potential to change the current paradigm of cardiac rejection diagnosis. We aimed to develop and validate targeted gene expression diagnostic models of cardiac rejection. We performed a multicenter, retrospective study, building a reference set of 610 FFPE-EMB collected between 2011 and 2021 and representative of the landscape of rejection (antibody-mediated rejection-AMR, n=189; acute cellular rejection-ACR, n=289; non-rejection, n=114). Nanostring nCounter technology with BHOT panel, a targeted consensus panel of 770 genes, was used to analyze tissue gene expression. Differential expression and elastic net approach were applied to identify key AMR and ACR-related transcripts. An ensemble model was used to build molecular classifiers of AMR and ACR. Their performance was evaluated in an independent validation set (n=117). Overall, the molecular analysis of 592 (97%) biopsies successfully passed all quality control and normalization steps. The molecular profiles of both ACR and AMR included typical transcripts reflecting the rejection-related pathophysiology. In the validation cohort, the AMR and ACR models accurately identified rejection. The diagnostic accuracy was 81.89% for AMR (ROC-AUC=0.844, Brier score=0.143) and 77.58% for ACR (ROC-AUC=0.812, Brier score=0.176). Principal component analysis based on the molecular scores showed the detection of 72% of variance (Figure) allowing the histo-molecular contextualization of the reference set samples according to the molecular scores. Applying a targeted gene set on FFPE EMB, we developed robust molecular-based models of cardiac rejection, showing high diagnostic accuracy for pathology-defined diagnoses. [ABSTRACT FROM AUTHOR]

Tissue-based gene expression approaches have been developed as a companion tool of routine pathology to refine the diagnosis of cardiac rejection. Whole-transcriptome profiling defined molecular phenotypes of rejection, but its clinical application remains limited. The consensus Banff Human Organ Transplant (BHOT) panel facilitate cost-effective and reproducible expression analysis of solid organ allografts. We aimed at validating the targeted panel as a proxy to whole-transcriptome analysis. To assess the BHOT panel reliability in detecting gene expression pattern associated with antibody-mediated rejection (AMR), we performed in silico analysis projecting the BHOT panel on already published microarray data of 142 heart transplant biopsies (Circulation, PMID:28148598). We compared the targeted and whole-transcriptome molecular profiles in AMR-biopsies (n=71) and non-AMR matched-control (non-rejection or acute cellular rejection, n=71) by performing differential expression, pathway, and network analyses. Out of the 30 most differentially expressed genes between AMR and non-AMR identified in whole transcriptome analysis, two-thirds (n=19) were included in the BHOT panel (Figure). These genes covered major immune pathways and key cells involved in the physiology of AMR, including IFNG inducible genes, NK cells, monocytes-macrophages, and B-cells. Major pathways related to AMR and graft injury identified in the whole transcriptome analysis were all identified in the targeted BHOT panel analysis: antigen processing cross presentation (q=2.86E-15), lymphoid and non-lymphoid immune interaction (q=1.00E-22), endothelial activation (q=6.80E-10). The targeted molecular signature of AMR based on the B-HOT panel detected relevant AMR-related genes and pathways. The targeted panel can be used as a proxy to whole-transcriptome to analyze the molecular profile of heart allograft rejection. [ABSTRACT FROM AUTHOR]

Background: In kidney transplantation, molecular diagnostics may be a valuable approach to improve the precision of the diagnosis. Using next-generation sequencing (NGS), we aimed to identify clinically relevant archetypes., Methods: We conducted an Illumina bulk RNA sequencing on 770 kidney biopsies (540 kidney recipients) collected between 2006 and 2021 from 11 European centers. Differentially expressed genes were determined for 11 Banff lesions. An ElasticNet model was used for feature selection, and 4 machine learning classifiers were trained to predict the probability of presence of the lesions. NGS-based classifiers were used in an unsupervised archetypal analysis to different archetypes. The association of the archetypes with allograft survival was assessed using the iBox risk prediction score., Results: The ElasticNet feature selection reduced the number of the genes from a range of 859-10 830 to a range of 52-867 genes. NGS-based classifiers demonstrated robust performances (precision-recall area under the curves 0.708-0.980) in predicting the Banff lesions. Archetypal analysis revealed 8 distinct phenotypes, each characterized by distinct clinical, immunological, and histological features. Although the archetypes confirmed the well-defined Banff rejection phenotypes for T cell-mediated rejection and antibody-mediated rejection, equivocal histologic antibody-mediated rejection, and borderline diagnoses were reclassified into different archetypes based on their molecular signatures. The 8 NGS-based archetypes displayed distinct allograft survival profiles with incremental graft loss rates between archetypes, ranging from 90% to 56% rates 7 y after evaluation (P < 0.0001)., Conclusions: Using molecular phenotyping, 8 archetypes were identified. These NGS-based archetypes might improve disease characterization, reclassify ambiguous Banff diagnoses, and enable patient-specific risk stratification., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Atherosclerotic lesions mainly form in arterial areas exposed to low shear stress (LSS), where endothelial cells express a senescent and inflammatory phenotype. Conversely, areas exposed to high shear stress (HSS) are protected from plaque development. Endothelial extracellular vesicles (EVs) have been shown to regulate inflammation and senescence, and therefore play a crucial role in vascular homeostasis. Whilst previous studies have shown links between hemodynamic forces and EV release, the effects of shear stress on the release and uptake of endothelial EVs remains elusive. We aim to decipher the interplay between these processes in endothelial cells exposed to atheroprone or atheroprotective shear stress. Confluent HUVECs were exposed to LSS or HSS for 24 h. Large and small EVs were isolated from conditioned medium by centrifugation and size exclusion chromatography. They were characterised by TEM, Western blot, tunable resistive pulse sensing, flow cytometry and proteomics. Uptake experiments were performed using fluorescently-labelled EVs and differences between groups were assessed by flow cytometry and confocal microscopy. We found that levels of large and small EVs in conditioned media were fifty and five times higher in HSS than in LSS conditions, respectively. In vivo and in vitro uptake experiments revealed greater EV incorporation by cells exposed to LSS conditions. Additionally, endothelial LSS-EVs have a greater affinity for HUVECs than HSS-EVs or EVs derived from platelets, erythrocytes and leukocytes. Proteomic analysis revealed that LSS-EVs were enriched in adhesion proteins (PECAM1, MCAM), participating in EV uptake by endothelial cells. LSS-EVs also carried mitochondrial material, which may be implicated in elevating ROS levels in recipient cells. These findings suggest that shear stress influences EV biogenesis and uptake. Given the major role of EVs and shear stress in vascular health, deciphering the relation between these processes may yield innovative strategies for the early detection and treatment of endothelial dysfunction., (© 2024 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Recently, interest in transcriptomic assessment of kidney biopsies has been growing. This study investigates the use of NGS to identify gene expression changes and analyse the pathways involved in rejection. An Illumina bulk RNA sequencing on the polyadenylated RNA of 770 kidney biopsies was conducted. Differentially-expressed genes (DEGs) were determined for AMR and TCMR using DESeq2. Genes were segregated according to their previous descriptions in known panels (microarray or the Banff Human Organ Transplant (B-HOT) panel) to obtain NGS-specific genes. Pathway enrichment analysis was performed using the Reactome and Kyoto Encyclopaedia of Genes and Genomes (KEGG) public repositories. The differential gene expression using NGS analysis identified 6,141 and 8,478 transcripts associated with AMR and TCMR. While most of the genes identified were included in the microarray and the B-HOT panels, NGS analysis identified 603 (9.8%) and 1,186 (14%) new specific genes. Pathways analysis showed that the B-HOT panel was associated with the main immunological processes involved during AMR and TCMR. The microarrays specifically integrated metabolic functions and cell cycle progression processes. Novel NGS-specific based transcripts associated with AMR and TCMR were discovered, which might represent a novel source of targets for drug designing and repurposing., Competing Interests: Author PG was employed by GenoSplice. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Cortes Garcia, Giarraputo, Racapé, Goutaudier, Ursule-Dufait, de la Grange, Adoux, Raynaud, Couderau, Mezine, Dagobert, Bestard, Moreso, Villard, Halleck, Giral, Brouard, Danger, Gourraud, Rabant, Couzi, Le Quintrec, Kamar, Morelon, Vrtovsnik, Taupin, Snanoudj, Legendre, Anglicheau, Budde, Lefaucheur, Loupy and Aubert.)

Background: Cross-species immunological incompatibilities have hampered pig-to-human xenotransplantation, but porcine genome engineering recently enabled the first successful experiments. However, little is known about the immune response after the transplantation of pig kidneys to human recipients. We aimed to precisely characterise the early immune responses to the xenotransplantation using a multimodal deep phenotyping approach., Methods: We did a complete phenotyping of two pig kidney xenografts transplanted to decedent humans. We used a multimodal strategy combining morphological evaluation, immunophenotyping (IgM, IgG, C4d, CD68, CD15, NKp46, CD3, CD20, and von Willebrand factor), gene expression profiling, and whole-transcriptome digital spatial profiling and cell deconvolution. Xenografts before implantation, wild-type pig kidney autografts, as well as wild-type, non-transplanted pig kidneys with and without ischaemia-reperfusion were used as controls., Findings: The data collected from xenografts suggested early signs of antibody-mediated rejection, characterised by microvascular inflammation with immune deposits, endothelial cell activation, and positive xenoreactive crossmatches. Capillary inflammation was mainly composed of intravascular CD68 + and CD15 + innate immune cells, as well as NKp46 + cells. Both xenografts showed increased expression of genes biologically related to a humoral response, including monocyte and macrophage activation, natural killer cell burden, endothelial activation, complement activation, and T-cell development. Whole-transcriptome digital spatial profiling showed that antibody-mediated injury was mainly located in the glomeruli of the xenografts, with significant enrichment of transcripts associated with monocytes, macrophages, neutrophils, and natural killer cells. This phenotype was not observed in control pig kidney autografts or in ischaemia-reperfusion models., Interpretation: Despite favourable short-term outcomes and absence of hyperacute injuries, our findings suggest that antibody-mediated rejection in pig-to-human kidney xenografts might be occurring. Our results suggest specific therapeutic targets towards the humoral arm of rejection to improve xenotransplantation results., Funding: OrganX and MSD Avenir., Competing Interests: Declaration of interests AD and DA are employees of Revivicor. All other authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The molecular refinement of the diagnosis of heart allograft rejection based on whole-transcriptome analyses faces several hurdles that greatly limit its widespread clinical application. The targeted Banff Human Organ Transplant gene panel (B-HOT, including 770 genes of interest) has been developed to facilitate reproducible and cost-effective gene expression analysis of solid organ allografts. We aimed to determine in silico the ability of this targeted panel to capture the antibody-mediated rejection (AMR) molecular profile using whole-transcriptome data from 137 heart allograft biopsies (71 biopsies reflecting the entire landscape of histologic AMR, 66 non-AMR control biopsies including cellular rejection and non-rejection cases). Differential gene expression, pathway and network analyses demonstrated that the B-HOT panel captured biologically and clinically relevant genes (IFNG-inducible, NK-cells, injury, monocytes-macrophage, B-cell-related genes), pathways (interleukin and interferon signaling, neutrophil degranulation, immunoregulatory interactions, endothelial activation) and networks reflecting the pathophysiological mechanisms underlying the AMR process previously identified in whole-transcriptome analysis. Our findings support the potential clinical use of the B-HOT-gene panel as a reliable proxy to whole-transcriptome analysis for the gene expression profiling of cardiac allograft rejection., Competing Interests: MM is the chairman of the Board of Trustees of the Banff Foundation for Allograft Pathology and receives consultancy honoraria from CSL Behring and Novartis as a central review pathologist for clinical trials. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Giarraputo, Coutance, Aubert, Fedrigo, Mezine, Zielinski, Mengel, Bruneval, Duong van Huyen, Angelini and Loupy.)

COVID-19 and infectious diseases have been included in strategic development goals (SDG) of United Nations (UN). Severe form of COVID-19 has been described as an endothelial disease. In order to better evaluate Covid-19 endotheliopathy, we characterized several subsets of circulating endothelial extracellular vesicles (EVs) at hospital admission among a cohort of 60 patients whose severity of COVID-19 was classified at the time of inclusion. Degree of COVID-19 severity was determined upon inclusion and categorized as moderate to severe in 40 patients and critical in 20 patients. We measured citrated plasma EVs expressing endothelial membrane markers. Endothelial EVs were defined as harboring VE-cadherin (CD144+), PECAM-1 (CD31 + CD41-) or E-selectin (CD62E+). An increase in CD62E + EV levels on admission to the hospital was significantly associated with critical disease. Moreover, Kaplan-Meier survival curves for CD62E + EV level showed that level ≥ 88,053 EVs/μL at admission was a significant predictor of in hospital mortality (p = 0.004). Moreover, CD62E + EV level ≥ 88,053 EV/μL was significantly associated with higher in-hospital mortality (OR 6.98, 95% CI 2.1-26.4, p = 0.002) in a univariate logistic regression model, while after adjustment to BMI CD62E + EV level ≥ 88,053 EV/μL was always significantly associated with higher in-hospital mortality (OR 5.1, 95% CI 1.4-20.0, p = 0.01). The present findings highlight the potential interest of detecting EVs expressing E-selectin (CD62) to discriminate Covid-19 patients at the time of hospital admission and identify individuals with higher risk of fatal outcome., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)