The role of antibody-mediated rejection (AMR) after liver transplantation (LT) remains controversial. Chronic AMR (cAMR) is often subclinical and may be missed without surveillance biopsies (svLbx). Transcriptome analyses have previously characterized molecular changes in T cell-mediated rejection (TCMR) after solid organ transplantation. We aimed to identify molecular signatures of cAMR after LT. Indication and surveillance biopsies from two prospective institutional biorepositories were screened. We performed bulk RNA sequencing on liver biopsies (discovery cohort: n = 71; Hannover validation cohort: n = 58; Barcelona validation cohort: n = 29). Downstream analyses explored the molecular features of cAMR, clinical TCMR, and subclinical TCMR, compared to no histological rejection. Nineteen percent of LT recipients with donor-specific antibodies had cAMR in the training cohort. Of these patients, 57% had normal/near normal liver enzymes, with cAMR detected only by svLbx. cAMR was associated with subsequent cirrhosis in 40-50% of cases and exhibited differentially expressed genes (DEGs) uniquely enriched in pathways related to fibrogenesis, complement activation, and TNFα signaling. In contrast, clinical TCMR was not associated with recurrent cirrhosis and showed DEGs enriched in antigen presentation, interferon signaling, and T cell receptor signaling. Subclinical TCMR was molecularly almost indistinguishable from no histological rejection. DEG profiles showed a high degree of concordance between the discovery and validation cohorts. We report a distinct transcriptomic profile of cAMR after LT, characterized by inflammatory and fibrogenic pathways, consistent with findings in other solid organ transplant settings. This molecular identity is associated with a high risk of liver graft failure. Chronic antibody-mediated rejection (AMR) is a recognized relevant cause of graft injury and unfavorable patient outcomes after kidney, heart and lung transplantation, but the role of chronic AMR after liver transplantation (LT) is controversial. Therefore, we aimed to characterize rejection phenotypes after LT on a molecular basis, as done in other solid organ transplant settings, to identify unique features of chronic AMR. Our findings identify chronic AMR as a molecularly distinct clinical phenotype of rejection after LT, often presenting with normal liver enzymes (validated in three independent cohorts) and associated with a recurrent cirrhosis rate of 40-50%. Our work supports the use of surveillance graft biopsies for the detection of a fibrosis-associated phenotype of chronic AMR. The molecular signature may be used in the future by researchers and clinicians to discriminate chronic AMR from graft injury of other causes.

The emerging zoonotic West Nile virus (WNV) has serious impact on public health. Thus, understanding the molecular basis of WNV infections in mammalian hosts is important to develop improved diagnostic and treatment strategies. In this context, the role of microRNAs (miRNAs) has been analyzed by several studies under different conditions and with different outcomes. A systematic comparison is therefore necessary. Furthermore, additional information from mRNA target expression data has rarely been taken into account to understand miRNA expression profiles under WNV infections. We conducted a meta-analysis of publicly available miRNA expression data from multiple independent studies, and analyzed them in a harmonized way to increase comparability. In addition, we used gene-set tests on mRNA target expression data to further gain evidence about differentially expressed miRNAs. For this purpose, we also studied the use of target information from different databases. We detected a substantial number of miRNA that emerged as differentially expressed from several miRNA datasets, and from the mRNA target data analysis as well. When using mRNA target data, we found that the targetscan databases provided the most useful information. We demonstrated improved miRNA detection through research synthesis of multiple independent miRNA datasets coupled with mRNA target set testing, leading to the discovery of multiple miRNAs which should be taken into account for further research on the molecular mechanism of WNV infections.

Detection of marker genes and other data analyses in single cell RNA-sequencing (scRNA-seq) experiments very much rely on the result of unsupervised clustering of cells. However, in 2-dimensional representation of clustering results, several cells appear as outliers or in the border area of a cluster suggesting that these cells may not adequately represent a particular cell type. We propose a novel and fast approach, scTrimClust, for identifying cells that may be interpreted of extreme specimens of their cell type. Identification is based on concave hulls build around each 2-dimensional cell cluster and the distance of each cell to the border area of its population. We study in two data examples, how cells with non-representative expression profile can influence the results of the analysis. We found that some sets of marker genes are little influenced by extreme cells while other sets are strongly modified, and must therefore be treated carefully. scTrimClust is also useful to compare the influence of other parameters of an scRNA-seq analysis, e.g. normalization or the clustering approach, on the results. We also provide a software implementation of scTrimClust.

Background&Aims: The role of antibody-mediated rejection (ABMR) after liver transplantation (LT) remains controversial. Chronic ABMR (cABMR) is often subclinical and potentially missed without surveillance biopsies (svLbx). Transcriptome analysis previously characterized molecular changes in T cell-mediated rejection (TCMR) after solid organ transplantation. We aimed to identify molecular cABMR signatures after LT. Methods: Indication and svLbx from two prospective institutional biorepositories were screened. We performed RNA-sequencing on liver biopsies (discovery cohort: n=71; Hannover validation cohort: n=58; Barcelona validation cohort: n=29). Downstream analyses explored the molecular features of cABMR, clinical (clinTCMR), subclinical TCMR (subTCMR) and chronic ductopenic rejection compared to no histological rejection (NHR). Results: Nineteen percent of LT recipients with donor-specific antibodies had cABMR in the training cohort. Fifty seven percent of cABMR patients had normal/near normal liver enzymes, recognized only by svLbx. CABMR was associated with subsequent cirrhosis development in 40-50% and displayed differentially expressed genes (DEGs) uniquely enriched in fibrogenesis-, complement activation- and TNFα-signaling-related pathways. ClinTCMR showed no such recurrent cirrhosis and DEGs uniquely enriched in antigen presentation-, interferon-signaling-, and T cell receptor-signaling-related pathways. SubTCMR was molecularly almost indistinguishable from NHR. There was a high degree of correlation in the DEGs between the training and two independent validation cohorts. Conclusions: We report transcriptome features of cABMR that is a unique molecular identity associated with inflammation and fibrogenesis as described in other solid organ transplantations and being linked to a high rate of liver graft failure. Impact and Implications: - Chronic antibody-mediated rejection (ABMR) is a recognized relevant cause of graft injury and unfavorable patient outcomes after kidney, heart and lung transplantation but the role of chronic ABMR after liver transplantation (LT) remains controversial, despite a recurrent cirrhosis rate of 40-50% in the present study. Therefore, we aimed to characterize rejection phenotypes after LT on a molecular basis, as done in other solids organ transplants, to identify unique features of chronic ABMR. - Our findings highlight chronic ABMR to be a molecularly distinct clinical phenotype of rejection after LT that often presents with normal liver enzymes validated in three independent cohorts. - Therefore, our work emphasizes the usage of surveillance graft biopsies for the detection of a fibrosis associated phenotype of chronic ABMR. The molecular signature may be used in the future by researchers and clinicians to discriminate chronic ABMR from graft injury of other causes.