Serum and Urine Metabolomic Profiling of Newly Diagnosed Treatment-Naïve Inflammatory Bowel Disease Patients.

BACKGROUND AND AIMS: Inflammatory bowel disease (IBD) is a prevalent chronic noncurable disease associated with profound metabolic changes. The discovery of novel molecular indicators for unraveling IBD etiopathogenesis and the diagnosis and prognosis of IBD is therefore pivotal. We sought to determine the distinctive metabolic signatures from the different IBD subgroups before treatment initiation. METHODS: Serum and urine samples from newly diagnosed treatment-naïve IBD patients and age and sex-matched healthy control (HC) individuals were investigated using proton nuclear magnetic resonance spectroscopy. Metabolic differences were identified based on univariate and multivariate statistical analyses. RESULTS: A total of 137 Crohn's disease patients, 202 ulcerative colitis patients, and 338 HC individuals were included. In the IBD cohort, several distinguishable metabolites were detected within each subgroup comparison. Most of the differences revealed alterations in energy and amino acid metabolism in IBD patients, with an increased demand of the body for energy mainly through the ketone bodies. As compared with HC individuals, differences in metabolites were more marked and numerous in Crohn's disease than in ulcerative colitis patients, and in serum than in urine. In addition, clustering analysis revealed 3 distinct patient profiles with notable differences among them based on the analysis of their clinical, anthropometric, and metabolomic variables. However, relevant phenotypical differences were not found among these 3 clusters. CONCLUSIONS: This study highlights the molecular alterations present within the different subgroups of newly diagnosed treatment-naïve IBD patients. The metabolomic profile of these patients may provide further understanding of pathogenic mechanisms of IBD subgroups. Serum metabotype seemed to be especially sensitive to the onset of IBD.

Long Non-Coding RNA Signatures in the Ileum and Colon of Crohn's Disease Patients and Effect of Anti-TNF-α Treatment on Their Modulation.

Biological therapies only benefit one-third of patients with Crohn's disease (CD). For this reason, a deeper understanding of the mechanisms by which biologics elicit their effect on intestinal mucosa is needed. Increasing evidence points toward the involvement of long noncoding RNAs (lncRNAs) in the pathogenesis of CD, although their role remains poorly studied. We aimed to characterize lncRNA profiles in the ileum and colon from CD patients and evaluate the effect of anti-TNF-α treatment on their transcription. Terminal ileum and left colon samples from 30 patients (active CD = 10, quiescent CD = 10, and healthy controls (HCs) = 10) were collected for RNA-seq. The patients were classified according to endoscopic activity. Furthermore, biopsies were cultured with infliximab, and their transcriptome was determined by Illumina gene expression array. A total of 678 differentially expressed lncRNAs between the terminal ileum and left colon were identified in HCs, 438 in patients with quiescent CD, and 468 in patients with active CD. Additionally, we identified three new lncRNAs in the ileum associated with CD activity. No differences were observed when comparing the effect of infliximab according to intestinal location, presence of disease (CD vs. HC), and activity (active vs. quiescent). The expression profiles of lncRNAs are associated with the location of intestinal tissue, being very different in the ileum and colon. The presence of CD and disease activity are associated with the differential expression of lncRNAs. No modulatory effect of infliximab has been observed in the lncRNA transcriptome.

Inflammatory macrophages reprogram to immunosuppression by reducing mitochondrial translation.

Acute inflammation can either resolve through immunosuppression or persist, leading to chronic inflammation. These transitions are driven by distinct molecular and metabolic reprogramming of immune cells. The anti-diabetic drug Metformin inhibits acute and chronic inflammation through mechanisms still not fully understood. Here, we report that the anti-inflammatory and reactive-oxygen-species-inhibiting effects of Metformin depend on the expression of the plasticity factor ZEB1 in macrophages. Using mice lacking Zeb1 in their myeloid cells and human patient samples, we show that ZEB1 plays a dual role, being essential in both initiating and resolving inflammation by inducing macrophages to transition into an immunosuppressed state. ZEB1 mediates these diverging effects in inflammation and immunosuppression by modulating mitochondrial content through activation of autophagy and inhibition of mitochondrial protein translation. During the transition from inflammation to immunosuppression, Metformin mimics the metabolic reprogramming of myeloid cells induced by ZEB1. Mechanistically, in immunosuppression, ZEB1 inhibits amino acid uptake, leading to downregulation of mTORC1 signalling and a decrease in mitochondrial translation in macrophages. These results identify ZEB1 as a driver of myeloid cell metabolic plasticity, suggesting that targeting its expression and function could serve as a strategy to modulate dysregulated inflammation and immunosuppression.

The EMT factor ZEB1 paradoxically inhibits EMT in BRAF-mutant carcinomas.

Despite being in the same pathway, mutations of KRAS and BRAF in colorectal carcinomas (CRCs) determine distinct progression courses. ZEB1 induces an epithelial-to-mesenchymal transition (EMT) and is associated with worse progression in most carcinomas. Using samples from patients with CRC, mouse models of KrasG12D and BrafV600E CRC, and a Zeb1-deficient mouse, we show that ZEB1 had opposite functions in KRAS- and BRAF-mutant CRCs. In KrasG12D CRCs, ZEB1 was correlated with a worse prognosis and a higher number of larger and undifferentiated (mesenchymal or EMT-like) tumors. Surprisingly, in BrafV600E CRC, ZEB1 was associated with better prognosis; fewer, smaller, and more differentiated (reduced EMT) primary tumors; and fewer metastases. ZEB1 was positively correlated in KRAS-mutant CRC cells and negatively in BRAF-mutant CRC cells with gene signatures for EMT, cell proliferation and survival, and ERK signaling. On a mechanistic level, ZEB1 knockdown in KRAS-mutant CRC cells increased apoptosis and reduced clonogenicity and anchorage-independent growth; the reverse occurred in BRAFV600E CRC cells. ZEB1 is associated with better prognosis and reduced EMT signature in patients harboring BRAF CRCs. These data suggest that ZEB1 can function as a tumor suppressor in BRAF-mutant CRCs, highlighting the importance of considering the KRAS/BRAF mutational background of CRCs in therapeutic strategies targeting ZEB1/EMT.

Transcriptomics and translatomics identify a robust inflammatory gene signature in brain endothelial cells after ischemic stroke.

Vascular endothelial function is challenged during cerebral ischemia and reperfusion. The endothelial responses are involved in inflammatory leukocyte attraction, adhesion and infiltration, blood-brain barrier leakage, and angiogenesis. This study investigated gene expression changes in brain endothelial cells after acute ischemic stroke using transcriptomics and translatomics. We isolated brain endothelial mRNA by: (i) translating ribosome affinity purification, enabling immunoprecipitation of brain endothelial ribosome-attached mRNA for translatome sequencing and (ii) isolating CD31+ endothelial cells by fluorescence-activating cell sorting for classical transcriptomic analysis. Both techniques revealed similar pathways regulated by ischemia but they showed specific differences in some transcripts derived from non-endothelial cells. We defined a gene set characterizing the endothelial response to acute stroke (24h) by selecting the differentially expressed genes common to both techniques, thus corresponding with the translatome and minimizing non-endothelial mRNA contamination. Enriched pathways were related to inflammation and immunoregulation, angiogenesis, extracellular matrix, oxidative stress, and lipid trafficking and storage. We validated, by flow cytometry and immunofluorescence, the protein expression of several genes encoding cell surface proteins. The inflammatory response was associated with the endothelial upregulation of genes related to lipid storage functions and we identified lipid droplet biogenesis in the endothelial cells after ischemia. The study reports a robust translatomic signature of brain endothelial cells after acute stroke and identifies enrichment in novel pathways involved in membrane signaling and lipid storage. Altogether these results highlight the endothelial contribution to the inflammatory response, and identify novel molecules that could be targets to improve vascular function after ischemic stroke.

Metabolomics as a tool to predict the risk of decompensation or liver-related death in patients with compensated cirrhosis.

BACKGROUND AND AIMS: Patients with compensated cirrhosis with clinically significant portal hypertension (CSPH: HVPG > 10 mm Hg) have a high risk of decompensation. HVPG is, however, an invasive procedure not available in all centers. The present study aims to assess whether metabolomics can improve the capacity of clinical models in predicting clinical outcomes in these compensated patients. APPROACH AND RESULTS: This is a nested study from the PREDESCI cohort (an RCT of nonselective beta-blockers vs. placebo in 201 patients with compensated cirrhosis and CSPH), including 167 patients for whom a blood sample was collected. A targeted metabolomic serum analysis, using ultra-high-performance liquid chromatography-mass spectrometry, was performed. Metabolites underwent univariate time-to-event cox regression analysis. Top-ranked metabolites were selected using Log-Rank p -value to generate a stepwise cox model. Comparison between models was done using DeLong test. Eighty-two patients with CSPH were randomized to nonselective beta-blockers and 85 to placebo. Thirty-three patients developed the main endpoint (decompensation/liver-related death). The model, including HVPG, Child-Pugh, and treatment received ( HVPG/Clinical model ), had a C-index of 0.748 (CI95% 0.664-0.827). The addition of 2 metabolites, ceramide (d18:1/22:0) and methionine (HVPG/Clinical/Metabolite model), significantly improved the model's performance [C-index of 0.808 (CI95% 0.735-0.882); p =0.032]. The combination of these 2 metabolites together with Child-Pugh and the type of treatment received (Clinical/Metabolite model) had a C-index of 0.785 (CI95% 0.710-0.860), not significantly different from the HVPG-based models including or not metabolites. CONCLUSIONS: In patients with compensated cirrhosis and CSPH, metabolomics improves the capacity of clinical models and achieves similar predictive capacity than models including HVPG.

Aged lipid-laden microglia display impaired responses to stroke.

Microglial cells of the aged brain manifest signs of dysfunction that could contribute to the worse neurological outcome of stroke in the elderly. Treatment with colony-stimulating factor 1 receptor antagonists enables transient microglia depletion that is followed by microglia repopulation after treatment interruption, causing no known harm to mice. We tested whether this strategy restored microglia function and ameliorated stroke outcome in old mice. Cerebral ischemia/reperfusion induced innate immune responses in microglia highlighted by type I interferon and metabolic changes involving lipid droplet biogenesis. Old microglia accumulated lipids under steady state and displayed exacerbated innate immune responses to stroke. Microglia repopulation in old mice reduced lipid-laden microglia, and the cells exhibited reduced inflammatory responses to ischemia. Moreover, old mice with renewed microglia showed improved motor function 2 weeks after stroke. We conclude that lipid deposits in aged microglia impair the cellular responses to ischemia and worsen functional recovery in old mice.

microRNA-based signatures obtained from endometrial fluid identify implantative endometrium.

STUDY QUESTION: Is it possible to use free and extracellular vesicle-associated microRNAs (miRNAs) from human endometrial fluid (EF) samples as non-invasive biomarkers for implantative endometrium? SUMMARY ANSWER: The free and extracellular vesicle-associated miRNAs can be used to detect implantative endometrium in a non-invasive manner. WHAT IS KNOWN ALREADY: miRNAs and extracellular vesicles (EVs) from EF have been described as mediators of the embryo-endometrium crosstalk. Therefore, the analysis of miRNA from this fluid could become a non-invasive technique for recognizing implantative endometrium. This analysis could potentially help improve the implantation rates in ART. STUDY DESIGN, SIZE, DURATION: In this prospective study, we first optimized different protocols for EVs and miRNA analyses using the EF of a setup cohort (n = 72). Then, we examined differentially expressed miRNAs in the EF of women with successful embryo implantation (discovery cohort n = 15/validation cohort n = 30) in comparison with those for whom the implantation had failed (discovery cohort n = 15/validation cohort n = 30). Successful embryo implantation was considered when pregnancy was confirmed by vaginal ultrasound showing a gestational sac 4 weeks after embryo transfer (ET). PARTICIPANTS/MATERIALS, SETTING, METHODS: The EF of the setup cohort was obtained before starting fertility treatment during the natural cycle, 16-21 days after the beginning of menstruation. For the discovery and validation cohorts, the EF was collected from women undergoing frozen ET on Day 5, and the samples were collected immediately before ET. In this study, we compared five different methods; two of them based on direct extraction of RNA and the other three with an EV enrichment step before the RNA extraction. Small RNA sequencing was performed to determine the most efficient method and find a predictive model differentiating between implantative and non-implantative endometrium. The models were confirmed using quantitative PCR in two sets of samples (discovery and validation cohorts) with different implantation outcomes. MAIN RESULTS AND THE ROLE OF CHANCE: The protocols using EV enrichment detected more miRNAs than the methods based on direct RNA extraction. The two most efficient protocols (using polymer-based precipitation (PBP): PBP-M and PBP-N) were used to obtain two predictive models (based on three miRNAs) allowing us to distinguish between an implantative and non-implantative endometrium. The first Model 1 (PBP-M) (discovery: AUC = 0.93; P-value = 0.003; validation: AUC = 0.69; P-value = 0.019) used hsa-miR-200b-3p, hsa-miR-24-3p and hsa-miR-148b-3p. Model 2 (PBP-N) (discovery: AUC = 0.92; P-value = 0.0002; validation: AUC = 0.78; P-value = 0.0002) used hsa-miR-200b-3p, hsa-miR-24-3p and hsa-miR-99b-5p. Functional analysis of these miRNAs showed strong association with key implantation processes such as in utero embryonic development or transforming growth factor-beta signaling. LARGE SCALE DATA: The FASTQ data are available in the GEO database (access number GSE178917). LIMITATIONS, REASONS FOR CAUTION: One important factor to consider is the inherent variability among the women involved in the trial and among the transferred embryos. The embryos were pre-selected based on morphology, but neither genetic nor molecular studies were conducted, which would have improved the accuracy of our tests. In addition, a limitation in miRNA library construction is the low amount of input RNA. WIDER IMPLICATIONS OF THE FINDINGS: We describe new non-invasive protocols to analyze miRNAs from small volumes of EF. These protocols could be implemented in clinical practice to assess the status of the endometrium before attempting ET. Such evaluation could help to avoid the loss of embryos transferred to a non-implantative endometrium. STUDY FUNDING/COMPETING INTEREST(S): J.I.-P. was supported by a predoctoral grant from the Basque Government (PRE_2017_0204). This study was partially funded by the Grant for Fertility Innovation (GFI, 2011) from Merck (Darmstadt, Germany). It was also supported by the Spanish Ministry of Economy and Competitiveness MINECO within the National Plan RTI2018-094969-B-I00, the European Union's Horizon 2020 research and innovation program (860303), the Severo Ochoa Centre of Excellence Innovative Research Grant (SEV-2016-0644) and the Instituto de Salud Carlos III (PI20/01131). The funding entities did not play any role in the study design, collection, analysis and interpretation of data, writing of the report or the decision to submit the article for publication. The authors declare no competing interests.

Neddylation tunes peripheral blood mononuclear cells immune response in COVID-19 patients.

The COVID-19 pandemic caused by SARS-CoV-2 has reached 5.5 million deaths worldwide, generating a huge impact globally. This highly contagious viral infection produces a severe acute respiratory syndrome that includes cough, mucus, fever and pneumonia. Likewise, many hospitalized patients develop severe pneumonia associated with acute respiratory distress syndrome (ARDS), along an exacerbated and uncontrolled systemic inflammation that in some cases induces a fatal cytokine storm. Although vaccines clearly have had a beneficial effect, there is still a high percentage of unprotected patients that develop the pathology, due to an ineffective immune response. Therefore, a thorough understanding of the modulatory mechanisms that regulate the response to SARS-CoV-2 is crucial to find effective therapeutic alternatives. Previous studies describe the relevance of Neddylation in the activation of the immune system and its implications in viral infection. In this context, the present study postulates Neddylation, a reversible ubiquitin-like post-translational modification of proteins that control their stability, localization and activity, as a key regulator in the immune response against SARS-CoV-2. For the first time, we describe an increase in global neddylation levels in COVID-19 in the serum of patients, which is particularly associated with the early response to infection. In addition, the results showed that overactivation of neddylation controls activation, proliferation, and response of peripheral blood mononuclear cells (PBMCs) isolated from COVID-19 patients. Inhibition of neddylation, and the subsequent avoidance of activated PBMCs, reduces cytokine production, mainly IL-6 and MCP-1 and induce proteome modulation, being a critical mechanism and a potential approach to immunomodulate COVID-19 patients.

Long-lasting adaptive immune memory specific to SARS-CoV-2 in convalescent coronavirus disease 2019 stable people with HIV.

OBJECTIVE: While the course of natural immunization specific to SARS-CoV-2 has been described among convalescent coronavirus disease 2019 (COVID-19) people without HIV (PWOH), a thorough evaluation of long-term serological and functional T- and B-cell immune memory among people with HIV (PWH) has not been reported. METHODS: Eleven stable PWH developing mild ( n  = 5) and severe ( n  = 6) COVID-19 and 39 matched PWOH individuals with mild (MILD) ( n  = 20) and severe (SEV) ( n  = 19) COVID-19 infection were assessed and compared at 3 and 6 months after infection for SARS-CoV-2-specific serology, polyfunctional cytokine (interferon-γ [IFN-γ], interleukin 2 [IL-2], IFN-γ/IL-2, IL-21) producing T-cell frequencies against four main immunogenic antigens and for circulating SARS-CoV-2-specific immunoglobulin G (IgG)-producing memory B-cell (mBc). RESULTS: In all time points, all SARS-COV-2-specific adaptive immune responses were highly driven by the clinical severity of COVID-19 infection, irrespective of HIV disease. Notably, while a higher proportion of mild PWH showed a higher decay on serological detection between the two time points as compared to PWOH, persistently detectable IgG-producing mBc were still detectable in most patients (4/4 (100%) for SEV PWH, 4/5 (80%) for MILD PWH, 10/13 (76.92%) for SEV PWOH and 15/18 (83.33%) for MILD PWOH). Likewise, SARS-CoV-2-specific IFN-γ-producing T-cell frequencies were detected in both PWH and PWOH, although significantly more pronounced among severe COVID-19 (6/6 (100%) for SEV PWH, 3/5 (60%) for MILD PWH, 18/19 (94.74%) for SEV PWOH and 14/19 (73.68%) for MILD PWOH). CONCLUSIONS: PWH develop a comparable short and long-term natural functional cellular and humoral immune response than PWOH convalescent patients, which are highly influenced by the clinical severity of the COVID-19 infection.

Epigenome-Wide DNA Methylation Profiling of Normal Mucosa Reveals HLA-F Hypermethylation as a Biomarker Candidate for Serrated Polyposis Syndrome.

Serrated polyposis syndrome (SPS) is associated with a high risk for colorectal cancer. Intense promoter hypermethylation is a frequent molecular finding in the serrated pathway and may be present in normal mucosa, predisposing to the formation of serrated lesions. To identify novel biomarkers for SPS, fresh-frozen samples of normal mucosa from 50 patients with SPS and 19 healthy individuals were analyzed by using the 850K BeadChip Technology (Infinium). Aberrant methylation levels were correlated with gene expression using a next-generation transcriptome profiling tool. Two validation steps were performed on independent cohorts: first, on formalin-fixed, paraffin-embedded tissue of the normal mucosa; and second, on 24 serrated lesions. The most frequently hypermethylated genes were HLA-F, SLFN12, HLA-DMA, and RARRES3; and the most frequently hypomethylated genes were PIWIL1 and ANK3 (Δß = 10%; P < 0.05). Expression levels of HLA-F, SLFN12, and HLA-DMA were significantly different between SPS patients and healthy individuals and correlated well with the methylation status of the corresponding differentially methylated region (fold change, >20%; r > 0.55; P < 0.001). Significant hypermethylation of CpGs in the gene body of HLA-F was also found in serrated lesions (Δß = 23%; false discovery rate = 0.01). Epigenome-wide methylation profiling has revealed numerous differentially methylated CpGs in normal mucosa from SPS patients. Significant hypermethylation of HLA-F is a novel biomarker candidate for SPS.

Coding and non-coding co-expression network analysis identifies key modules and driver genes associated with precursor lesions of gastric cancer.

BACKGROUND: Helicobacter pylori infection is the most important risk factor for gastric cancer (GC). Human gastric adenocarcinoma develops after long-term H. pylori infection via the Correa cascade. This carcinogenic pathway describes the progression from gastritis to atrophy, intestinal metaplasia (IM), dysplasia and GC. Patients with atrophy and intestinal metaplasia are considered to have precancerous lesions of GC (PLGC). H. pylori eradication and endoscopy surveillance are currently the only interventions for preventing GC. Better knowledge of the biology of human PLGC may help find stratification markers and contribute to better understanding of biological mechanisms. One way to achieve this is by using co-expression network analysis. Weighted gene co-expression network analysis (WGCNA) is often used to identify modules from co-expression networks and relate them to clinical traits. It also allows identification of driver genes that may be critical for PLGC. AIM: The purpose of this study was to identify co-expression modules and differential gene expression in dyspeptic patients at different stages of the Correa pathway. METHODS: We studied 96 gastric biopsies from 78 patients that were clinically classified as: non-active (n = 10) and chronic-active gastritis (n = 20), atrophy (n = 12), and IM (n = 36). Gene expression of coding RNAs was determined by microarrays and non-coding RNAs by RNA-seq. The WGCNA package was used for network construction, module detection, module preservation and hub and driver gene selection. RESULTS: WGCNA identified 20 modules for coding RNAs and 4 for each miRNA and small RNA class. Modules were associated with antrum and corpus gastric locations, chronic gastritis and IM. Notably, coding RNA modules correlated with the Correa cascade. One was associated with the presence of H. pylori. In three modules, the module eigengene (ME) gradually increased in the stages toward IM, while in three others the inverse relationship was found. One miRNA module was negatively correlated to IM and was used for a mRNA-miRNA integration analysis. WGCNA also uncovered driver genes. Driver genes show both high connectivity within a module and are significantly associated with clinical traits. Some of those genes have been previously involved in H. pylori carcinogenesis, but others are new. Lastly, using similar external transcriptomic data, we confirmed that the discovered mRNA modules were highly preserved. CONCLUSION: Our analysis captured co-expression modules that provide valuable information to understand the pathogenesis of the progression of PLGC.

A comprehensive assessment of long-term SARS-CoV-2-specific adaptive immune memory in convalescent COVID-19 Solid Organ Transplant recipients.

Long-term adaptive immune memory has been reported among immunocompetent individuals up to eight months following SARS-CoV-2 infection. However, limited data is available in convalescent patients with a solid organ transplant. To investigate this, we performed a thorough evaluation of adaptive immune memory at different compartments (serological, memory B cells and cytokine [IFN-γ, IL-2, IFN-γ/IL12 and IL-21] producing T cells) specific to SARS-CoV-2 by ELISA and FluoroSpot-based assays in 102 convalescent patients (53 with a solid organ transplants (38 kidney, 5 liver, 5 lung and 5 heart transplant) and 49 immunocompetent controls) with different clinical COVID-19 severity (severe, mild and asymptomatic) beyond six months after infection. While similar detectable memory responses at different immune compartments were detected between those with a solid organ transplant and immunocompetent individuals, these responses were predominantly driven by distinct COVID-19 clinical severities (97.6%, 80.5% and 42.1%, all significantly different, were seropositive; 84% vs 75% vs 35.7%, all significantly different, showed IgG-producing memory B cells and 82.5%, 86.9% and 31.6%, displayed IFN-γ producing T cells; in severe, mild and asymptomatic convalescent patients, respectively). Notably, patients with a solid organ transplant with longer time after transplantation did more likely show detectable long-lasting immune memory, regardless of COVID-19 severity. Thus, our study shows that patients with a solid organ transplant are capable of maintaining long-lasting peripheral immune memory after COVID-19 infection; mainly determined by the degree of infection severity.

Treatment With Simvastatin and Rifaximin Restores the Plasma Metabolomic Profile in Patients With Decompensated Cirrhosis.

Patients with decompensated cirrhosis, particularly those with acute-on-chronic liver failure (ACLF), show profound alterations in plasma metabolomics. The aim of this study was to investigate the effect of treatment with simvastatin and rifaximin on plasma metabolites of patients with decompensated cirrhosis, specifically on compounds characteristic of the ACLF plasma metabolomic profile. Two cohorts of patients were investigated. The first was a descriptive cohort of patients with decompensated cirrhosis (n = 42), with and without ACLF. The second was an intervention cohort from the LIVERHOPE-SAFETY randomized, double-blind, placebo-controlled trial treated with simvastatin 20 mg/day plus rifaximin 1,200 mg/day (n = 12) or matching placebo (n = 13) for 3 months. Plasma samples were analyzed using ultrahigh performance liquid chromatography-tandem mass spectroscopy for plasma metabolomics characterization. ACLF was characterized by intense proteolysis and lipid alterations, specifically in pathways associated with inflammation and mitochondrial dysfunction, such as the tryptophan-kynurenine and carnitine beta-oxidation pathways. An ACLF-specific signature was identified. Treatment with simvastatin and rifaximin was associated with changes in 161 of 985 metabolites in comparison to treatment with placebo. A remarkable reduction in levels of metabolites from the tryptophan-kynurenine and carnitine pathways was found. Notably, 18 of the 32 metabolites of the ACLF signature were affected by the treatment. Conclusion: Treatment with simvastatin and rifaximin modulates some of the pathways that appear to be key in ACLF development. This study unveils some of the mechanisms involved in the effects of treatment with simvastatin and rifaximin in decompensated cirrhosis and sets the stage for the use of metabolomics to investigate new targeted therapies in cirrhosis to prevent ACLF development.

Clinicopathological and Molecular Prognostic Classifier for Intermediate/High-Risk Clear Cell Renal Cell Carcinoma.

The probability of tumor progression in intermediate/high-risk clear cell renal cell carcinoma (ccRCC) is highly variable, underlining the lack of predictive accuracy of the current clinicopathological factors. To develop an accurate prognostic classifier for these patients, we analyzed global gene expression patterns in 13 tissue samples from progressive and non-progressive ccRCC using Illumina Hi-seq 4000. Expression levels of 22 selected differentially expressed genes (DEG) were assessed by nCounter analysis in an independent series of 71 ccRCCs. A clinicopathological-molecular model for predicting tumor progression was developed and in silico validated in a total of 202 ccRCC patients using the TCGA cohort. A total of 1202 DEGs were found between progressive and non-progressive intermediate/high-risk ccRCC in RNAseq analysis, and seven of the 22 DEGs selected were validated by nCounter. Expression of HS6ST2, pT stage, tumor size, and ISUP grade were found to be independent prognostic factors for tumor progression. A risk score generated using these variables was able to distinguish patients at higher risk of tumor progression (HR 7.27; p < 0.001), consistent with the results obtained from the TCGA cohort (HR 2.74; p < 0.002). In summary, a combined prognostic algorithm was successfully developed and validated. This model may aid physicians to select high-risk patients for adjuvant therapy.

Randomized Controlled Trial Substudy of Cell-specific Mechanisms of Janus Kinase 1 Inhibition With Upadacitinib in the Crohn's Disease Intestinal Mucosa: Analysis From the CELEST Study.

BACKGROUND: Janus kinase (JAK) inhibition shows promise for treatment of patients with moderate to severe Crohn's disease. We aimed to provide mechanistic insights into the JAK1-selective inhibitor upadacitinib through a transcriptomics substudy on biopsies from patients with Crohn's disease from CELEST. METHODS: Seventy-four patients consented to this optional substudy. Ileal and colonic biopsies were collected during endoscopy at screening and week 12 or 16. RNA isolated from 226 samples was analyzed by RNAseq, with additional qPCR analysis. Additional biopsies from patients with Crohn's disease receiving anti-tumor necrosis factor (anti-TNF; n = 34) and healthy controls (n = 10) were used for qPCR. Single-cell RNAseq public profiles were used to evaluate treatment effects on specific cellular subsets, associations with endoscopic improvement, and indirect comparisons with the anti-TNF-treated cohort. RESULTS: In involved areas of mucosa with endoscopic remission after upadacitinib treatment, 1156 and 76 protein-coding genes were significantly regulated (false discovery rate < 0.05) at week 12/16 in colonic and ileal biopsies, respectively (60 overlapped), compared with baseline. Upadacitinib did not significantly affect transcriptomes of noninvolved intestinal areas. CELEST patients (mostly anti-TNF-refractory) showed baseline differences in gene expression compared with a separate cohort of biologic-naïve patients. Notably, upadacitinib reversed overexpression of inflammatory fibroblast and interferon-γ effector signature markers. CONCLUSIONS: Upadacitinib modulates inflammatory pathways in mucosal lesions of patients with anti-TNF-refractory Crohn's disease, including inflammatory fibroblast and interferon-γ-expressing cytotoxic T cell compartments. This substudy is the first to describe the molecular response to JAK1 inhibition in inflammatory bowel disease and differential effects relative to anti-TNF treatment. (Clinical trial identifier: NCT02365649).

SARS-CoV-2-specific serological and functional T cell immune responses during acute and early COVID-19 convalescence in solid organ transplant patients.

The description of protective humoral and T cell immune responses specific against SARS-CoV-2 has been reported among immunocompetent (IC) individuals developing COVID-19 infection. However, its characterization and determinants of poorer outcomes among the at-risk solid organ transplant (SOT) patient population have not been thoroughly investigated. Cytokine-producing T cell responses, such as IFN-γ, IL-2, IFN-γ/IL-2, IL-6, IL-21, and IL-5, against main immunogenic SARS-CoV-2 antigens and IgM/IgG serological immunity were tracked in SOT (n = 28) during acute infection and at two consecutive time points over the following 40 days of convalescence and were compared to matched IC (n = 16) patients admitted with similar moderate/severe COVID-19. We describe the development of a robust serological and functional T cell immune responses against SARS-CoV-2 among SOT patients, similar to IC patients during early convalescence. However, at the infection onset, SOT displayed lower IgG seroconversion rates (77% vs. 100%; p = .044), despite no differences on IgG titers, and a trend toward decreased SARS-CoV-2-reactive T cell frequencies, especially against the membrane protein (7 [0-34] vs. 113 [15-245], p = .011, 2 [0-9] vs. 45 [5-74], p = .009, and 0 [0-2] vs. 13 [1-24], p = .020, IFN-γ, IL-2, and IFN-γ/IL-2 spots, respectively). In summary, our data suggest that despite a certain initial delay, SOT population achieve comparable functional immune responses than the general population after moderate/severe COVID-19.

Serum transferrin as a biomarker of hepatocyte nuclear factor 4 alpha activity and hepatocyte function in liver diseases.

BACKGROUND: Serum transferrin levels represent an independent predictor of mortality in patients with liver failure. Hepatocyte nuclear factor 4 alpha (HNF4α) is a master regulator of hepatocyte functions. The aim of this study was to explore whether serum transferrin reflects HNF4α activity. METHODS: Factors regulating transferrin expression in alcoholic hepatitis (AH) were assessed via transcriptomic/methylomic analysis as well as chromatin immunoprecipitation coupled to DNA sequencing. The findings were corroborated in primary hepatocytes. Serum and liver samples from 40 patients with advanced liver disease of multiple etiologies were also studied. RESULTS: In patients with advanced liver disease, serum transferrin levels correlated with hepatic transferrin expression (r = 0.51, p = 0.01). Immunohistochemical and biochemical tests confirmed reduced HNF4α and transferrin protein levels in individuals with cirrhosis. In AH, hepatic gene-gene correlation analysis in liver transcriptome revealed an enrichment of HNF4α signature in transferrin-correlated transcriptome while transforming growth factor beta 1 (TGFß1), tumor necrosis factor α (TNFα), interleukin 1 beta (IL-1ß), and interleukin 6 (IL-6) negatively associated with transferrin signature. A key regulatory region in transferrin promoter was hypermethylated in patients with AH. In primary hepatocytes, treatment with TGFß1 or the HNF4α inhibitor BI6015 suppressed transferrin production, while exposure to TNFα, IL-1ß, and IL-6 had no effect. The correlation between hepatic HNF4A and transferrin mRNA levels was also seen in advanced liver disease. CONCLUSIONS: Serum transferrin levels constitute a prognostic and mechanistic biomarker. Consequently, they may serve as a surrogate of impaired hepatic HNF4α signaling and liver failure.

Multi-omic modelling of inflammatory bowel disease with regularized canonical correlation analysis.

BACKGROUND: Personalized medicine requires finding relationships between variables that influence a patient's phenotype and predicting an outcome. Sparse generalized canonical correlation analysis identifies relationships between different groups of variables. This method requires establishing a model of the expected interaction between those variables. Describing these interactions is challenging when the relationship is unknown or when there is no pre-established hypothesis. Thus, our aim was to develop a method to find the relationships between microbiome and host transcriptome data and the relevant clinical variables in a complex disease, such as Crohn's disease. RESULTS: We present here a method to identify interactions based on canonical correlation analysis. We show that the model is the most important factor to identify relationships between blocks using a dataset of Crohn's disease patients with longitudinal sampling. First the analysis was tested in two previously published datasets: a glioma and a Crohn's disease and ulcerative colitis dataset where we describe how to select the optimum parameters. Using such parameters, we analyzed our Crohn's disease data set. We selected the model with the highest inner average variance explained to identify relationships between transcriptome, gut microbiome and clinically relevant variables. Adding the clinically relevant variables improved the average variance explained by the model compared to multiple co-inertia analysis. CONCLUSIONS: The methodology described herein provides a general framework for identifying interactions between sets of omic data and clinically relevant variables. Following this method, we found genes and microorganisms that were related to each other independently of the model, while others were specific to the model used. Thus, model selection proved crucial to finding the existing relationships in multi-omics datasets.