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Background & Aims:CD4+CD25+Foxp3+regulatory T cells (Tregs) are essential to maintain immunological tolerance and have been shown to promote liver allograft tolerance in both rodents and humans. Low-dose IL-2 (LDIL-2) can expand human endogenous circulating Tregsinvivo, but its role in suppressing antigen-specific responses and promoting Treg trafficking to the sites of inflammation is unknown. Likewise, whether LDIL-2 facilitates the induction of allograft tolerance has not been investigated in humans. Methods:We conducted a clinical trial in stable liver transplant recipients 2–6 years post-transplant to determine the capacity of LDIL-2 to suppress allospecific immune responses and allow for the complete discontinuation of maintenance immunosuppression (ClinicalTrials.govNCT02949492). One month after LDIL-2 was initiated, those exhibiting at least a 2-fold increase in circulating Tregs gradually discontinued immunosuppression over a 4-month period while continuing LDIL-2 for a total treatment duration of 6 months. Results:All participants achieved a marked and sustained increase in circulating Tregs. However, this was not associated with the preferential expansion of donor-reactive Tregs and did not promote the accumulation of intrahepatic Tregs. Furthermore, LDIL-2 induced a marked IFNγ-orchestrated transcriptional response in the liver even before immunosuppression weaning was initiated. The trial was terminated after the first 6 participants failed to reach the primary endpoint owing to rejection requiring reinstitution of immunosuppression. Conclusions:The expansion of circulating Tregs in response to LDIL-2 is not sufficient to control alloimmunity and to promote liver allograft tolerance, due, at least in part, to off-target effects that increase liver immunogenicity. Our trial provides unique insight into the mechanisms of action of immunomodulatory therapies such as LDIL-2 and their limitations in promoting alloantigen-specific effects and immunological tolerance. Clinical Trials Registration:The study is registered at ClinicalTrials.gov (NCT02949492). Impact and implications:The administration of low-dose IL-2 is an effective way of increasing the number of circulating regulatory T cells (Tregs), an immunosuppressive lymphocyte subset that is key for the establishment of immunological tolerance, but its use to promote allograft tolerance in the setting of clinical liver transplantation had not been explored before. In liver transplant recipients on tacrolimus monotherapy, low-dose IL-2 effectively expanded circulating Tregs but did not increase the number of Tregs with donor specificity, nor did it promote their trafficking to the transplanted liver. Low-dose IL-2 did not facilitate the discontinuation of tacrolimus and elicited, as an off-target effect, an IFNγ-orchestrated inflammatory response in the liver that resembled T cell-mediated rejection. These results, supporting an unexpected role for IL-2 in regulating the immunogenicity of the liver, highlight the need to carefully evaluate systemic immunoregulatory strategies with investigations that are not restricted to the blood compartment and involve target tissues such as the liver.

Molecular characterization of radical prostatectomy specimens after systemic therapy may identify a gene expression profile for resistance to therapy. This study assessed tumor cells from patients with prostate cancer participating in a phase II neoadjuvant docetaxel and androgen deprivation trial to identify mediators of resistance. Transcriptional level of 93 genes from a docetaxel-resistant prostate cancer cell lines microarray study was analyzed by TaqMan low-density arrays in tumors from patients with high-risk localized prostate cancer (36 surgically treated, 28 with neoadjuvant docetaxel + androgen deprivation). Gene expression was compared between groups and correlated with clinical outcome. VIM, AR and RELA were validated by immunohistochemistry. CD44 and ZEB1 expression was tested by immunofluorescence in cells and tumor samples. Parental and docetaxel-resistant castration-resistant prostate cancer cell lines were tested for epithelial-to-mesenchymal transition (EMT) markers before and after docetaxel exposure. Reversion of EMT phenotype was investigated as a docetaxel resistance reversion strategy. Expression of 63 (67.7%) genes differed between groups (P < 0.05), including genes related to androgen receptor, NF-κB transcription factor, and EMT. Increased expression of EMT markers correlated with radiologic relapse. Docetaxel-resistant cells had increased EMT and stem-like cell markers expression. ZEB1 siRNA transfection reverted docetaxel resistance and reduced CD44 expression in DU-145R and PC-3R. Before docetaxel exposure, a selected CD44+ subpopulation of PC-3 cells exhibited EMT phenotype and intrinsic docetaxel resistance; ZEB1/CD44+ subpopulations were found in tumor cell lines and primary tumors; this correlated with aggressive clinical behavior. This study identifies genes potentially related to chemotherapy resistance and supports evidence of the EMT role in docetaxel resistance and adverse clinical behavior in early prostate cancer. Mol Cancer Ther; 13(5); 1270–84. ©2014 AACR.

PDF - 265K, Biochemical-relapse analysis according to gene expression levels of markers differentially expressed in treated vs non-treated patients.

PDF - 72K, Genes expression values by qRT-PCR in treated patients vs non-treated patients; TLDA: TaqMan Low Density Arrays; FDR: False Discovery Rate.

PDF - 475K, Radiological-progression analysis according to gene expression levels of markers differentially expressed in treated vs non-treated patients.

PDF - 35K, Legends for Supplementary Figures 1 and 2.

Introduction: Colorectal cancer (CRC) is the second leading cause of cancer-related deaths, but currently available noninvasive screening programs have achieved only a modest decrease in mortality. MicroRNAs (miRNA) play an important role in a wide array of biological processes and are commonly dysregulated in neoplasia. We aimed to evaluate the feasibility of fecal miRNAs as biomarkers for colorectal neoplasia screening.Materials and Methods: Total RNA was extracted from freshly collected stool samples from 8 healthy volunteers and 29 samples collected via fecal occult blood testing from subjects with normal colonoscopies, colon adenomas, and CRCs. miRNA expression analyses were done with TaqMan quantitative reverse transcription-PCR for a subset of miRNAs. Illumina miRNA microarray profiling was done to evaluate the differences in expression patterns between normal colonic mucosa tissues and stool samples from healthy subjects.Results: We efficiently extracted miRNAs from stool specimens using our developed protocol. Data from independent experiments showed high reproducibility for miRNA extraction and expression. miRNA expression patterns were similar in stool specimens among healthy volunteers, and reproducible in stool samples that were collected serially in time from the same individuals. miRNA expression profiles from 29 patients showed higher expression of miR-21 and miR-106a in patients with adenomas and CRCs compared with individuals free of colorectal neoplasia.Conclusion: Our data indicate that miRNAs could be extracted from stool easily and reproducibly. The stools of patients with colorectal neoplasms have unique and identifiable patterns of miRNA expression.Impact: Fecal miRNAs may be an excellent candidate for the development of a noninvasive screening test for colorectal neoplasms. Cancer Epidemiol Biomarkers Prev; 19(7); 1766–74. ©2010 AACR.

Supplementary Table 1 from Fecal MicroRNAs as Novel Biomarkers for Colon Cancer Screening

Background & Aims Patients with acute-on-chronic liver failure (ACLF) present a systemic hyperinflammatory response associated with increased circulating levels of small-molecule metabolites. To investigate whether these alterations reflect inadequate cell energy output, we assessed mitochondrial morphology and central metabolic pathways with emphasis on the tricarboxylic acid (TCA) cycle in peripheral leukocytes from patients with acutely decompensated (AD) cirrhosis, with and without ACLF. Methods The study included samples from patients with AD cirrhosis (108 without and 128 with ACLF) and 41 healthy individuals. Leukocyte mitochondrial ultrastructure was visualized by transmission electron microscopy and cytosolic and mitochondrial metabolic fluxes were determined by assessing NADH/FADH2 production from various substrates. Plasma GDF15 and FGF21 were determined by Luminex and acylcarnitines by LC-MS/MS. Gene expression was analyzed by RNA-sequencing and PCR-based glucose metabolism profiler array. Results Mitochondrial ultrastructure in patients with advanced cirrhosis was distinguished by cristae rarefication and swelling. The number of mitochondria per leukocyte was higher in patients, accompanied by a reduction in their size. Increased FGF21 and C6:0- and C8:0-carnitine predicted mortality whereas GDF15 strongly correlated with a gene set signature related to leukocyte activation. Metabolic flux analyses revealed increased energy production in mononuclear leukocytes from patients with preferential involvement of extra-mitochondrial pathways, supported by upregulated expression of genes encoding enzymes of the glycolytic and pentose phosphate pathways. In patients with ACLF, mitochondrial function analysis uncovered break-points in the TCA cycle at the isocitrate dehydrogenase and succinate dehydrogenase level, which were bridged by anaplerotic reactions involving glutaminolysis and nucleoside metabolism. Conclusions Our findings provide evidence at the cellular, organelle and biochemical levels that severe mitochondrial dysfunction governs immunometabolism in leukocytes from patients with AD cirrhosis and ACLF. Lay summary Patients at advanced stages of liver disease have dismal prognosis due to vital organ failures and the lack of treatment options. In this study, we report that the functioning of mitochondria, which are known as the cell powerhouse, is severely impaired in leukocytes of these patients, probably as a consequence of intense inflammation. Mitochondrial dysfunction is therefore a hallmark of advanced liver disease.

Background and aims: Ductular reaction (DR) expands in chronic liver diseases and correlates with disease severity. Besides its potential role in liver regeneration, DR plays a role in the wound-healing response of the liver, promoting periductular fibrosis and inflammatory cell recruitment. However, there is no information regarding its role in intrahepatic angiogenesis. In the current study we investigated the potential contribution of DR cells to hepatic vascular remodeling during chronic liver disease. Approach and results: In mouse models of liver injury, DR cells express genes involved in angiogenesis. Among angiogenesis-related genes, the expression of Slit2 and its receptor Roundabout 1 (Robo1) was localized in DR cells and neoangiogenic vessels, respectively. The angiogenic role of the Slit2-Robo1 pathway in chronic liver disease was confirmed in ROBO1/2-/+ mice treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine, which displayed reduced intrahepatic neovascular density compared to wild-type mice. However, ROBO1/2 deficiency did not affect angiogenesis in partial hepatectomy. In patients with advanced alcohol-associated disease, angiogenesis was associated with DR, and up-regulation of SLIT2-ROBO1 correlated with DR and disease severity. In vitro, human liver-derived organoids produced SLIT2 and induced tube formation of endothelial cells. Conclusions: Overall, our data indicate that DR expansion promotes angiogenesis through the Slit2-Robo1 pathway and recognize DR cells as key players in the liver wound-healing response., Supported by grants from Fondo de Investigación Sanitaria Carlos III (FIS), cofinanced by Fondo Europeo de Desarrollo Regional (FEDER), Unión Europea, “Una manera de hacer Europa” (FIS PI20/00765, PI17/00673, to P.S.-B; FIS 18-PI18/00862, to I.G and M.C); from the National Institute on Alcohol Abuse and Alcoholism (1U01AA026972-01 and AGAUR 2017-SGR-01456, to P.S.-B.); and from the European Foundation for Alcohol Research (EA1653, to P.S.-B.). M.C. is funded by the Ramon y Cajal program from the Ministerio de Ciencia e Innovación RYC2019-026662-I. P.G. is funded by the Agencia de Gestió d'Ajuts Universitaris i de Recerca 2014 SGR 708, Centro de Investigaciónen Red Enfermedades Hepáticas y Digestivas (CIBERehd), and Institució Catalana de Recerca i Estudis Avançats. S.A. received a grant from the Ministerio de Educación, Cultura y Deporte (FPU17/04992). B.A.-B. is funded by the Instituto de Salud Carlos III (FI16/00203)

Background & Aims: Acute decompensation (AD) of cirrhosis is a heterogeneous clinical entity associated with moderate mortality. In some patients, this condition develops quickly into the more deadly acute-on-chronic liver failure (ACLF), in which other organs such as the kidneys or brain fail. The aim of this study was to characterize the blood lipidome in a large series of patients with cirrhosis and identify specific signatures associated with AD and ACLF development. Methods: Serum untargeted lipidomics was performed in 561 patients with AD (518 without and 43 with ACLF) (discovery cohort) and in 265 patients with AD (128 without and 137 with ACLF) in whom serum samples were available to perform repeated measurements during the 28-day follow-up (validation cohort). Analyses were also performed in 78 patients with AD included in a therapeutic albumin trial (43 patients with compensated cirrhosis and 29 healthy individuals). Results: The circulating lipid landscape associated with cirrhosis was characterized by a generalized suppression, which was more manifest during AD and in non-surviving patients. By computing discriminating accuracy and the variable importance projection score for each of the 223 annotated lipids, we identified a sphingomyelin fingerprint specific for AD of cirrhosis and a distinct cholesteryl ester and lysophosphatidylcholine finger-print for ACLF. Liver dysfunction and infections were the prin-cipal net contributors to these fingerprints, which were dynamic and interchangeable between patients with AD whose condition worsened to ACLF and those who improved. Notably, blood lysophosphatidylcholine levels increased in these patients after albumin therapy. Conclusions: Our findings provide insights into the lipid land-scape associated with decompensation of cirrhosis and ACLF progression and identify unique non-invasive diagnostic bio-markers of advanced cirrhosis. Lay summary: Analysis of lipids in blood from patients with advanced cirrhosis reveals a general suppression of their levels in the circulation of these patients. A specific group of lipids known as sphingomyelins are useful to distinguish between patients with compensated and decompensated cirrhosis. Another group of lipids designated cholesteryl esters further distinguishes patients with decompensated cirrhosis who are at risk of developing organ failures. (c) 2021 The Authors. Published by Elsevier B.V. on behalf of European Association for the Study of the Liver.

The inaccuracy of the current prognostic algorithms and the potential changes in the therapeutic management of localized ccRCC demands the development of an improved prognostic model for these patients. To this end, we analyzed whole-transcriptome profiling of 26 tissue samples from progressive and non-progressive ccRCCs using Illumina Hi-seq 4000. Differentially expressed genes (DEG) were intersected with the RNA-sequencing data from the TCGA. The overlapping genes were used for further analysis. A total of 132 genes were found to be prognosis-related genes. LASSO regression enabled the development of the best prognostic six-gene panel. Cox regression analyses were performed to identify independent clinical prognostic parameters to construct a combined nomogram which includes the expression of CERCAM, MIA2, HS6ST2, ONECUT2, SOX12, TMEM132A, pT stage, tumor size and ISUP grade. A risk score generated using this model effectively stratified patients at higher risk of disease progression (HR 10.79; p < 0.001) and cancer-specific death (HR 19.27; p < 0.001). It correlated with the clinicopathological variables, enabling us to discriminate a subset of patients at higher risk of progression within the Stage, Size, Grade and Necrosis score (SSIGN) risk groups, pT and ISUP grade. In summary, a gene expression-based prognostic signature was successfully developed providing a more precise assessment of the individual risk of progression.

Background and aim: Injury to hepatocyte mitochondria is common in metabolic dysfunction-associated fatty liver disease. Here, we investigated whether changes in the content of essential fatty acid-derived lipid autacoids affect hepatocyte mitochondrial bioenergetics and metabolic efficiency. Approach and results: The study was performed in transgenic mice for the fat-1 gene, which allows the endogenous replacement of the membrane omega-6-polyunsaturated fatty acid (PUFA) composition by omega-3-PUFA. Transmission electron microscopy revealed that hepatocyte mitochondria of fat-1 mice had more abundant intact cristae and higher mitochondrial aspect ratio. Fat-1 mice had increased expression of oxidative phosphorylation complexes I and II and translocases of both inner (translocase of inner mitochondrial membrane 44) and outer (translocase of the outer membrane 20) mitochondrial membranes. Fat-1 mice also showed increased mitofusin-2 and reduced dynamin-like protein 1 phosphorylation, which mediate mitochondrial fusion and fission, respectively. Mitochondria of fat-1 mice exhibited enhanced oxygen consumption rate, fatty acid β-oxidation, and energy substrate utilization as determined by high-resolution respirometry, [1-14 C]-oleate oxidation and nicotinamide adenine dinucleotide hydride/dihydroflavine-adenine dinucleotide production, respectively. Untargeted lipidomics identified a rich hepatic omega-3-PUFA composition and a specific docosahexaenoic acid (DHA)-enriched lipid fingerprint in fat-1 mice. Targeted lipidomics uncovered a higher content of DHA-derived lipid autacoids, namely resolvin D1 and maresin 1, which rescued hepatocytes from TNFα-induced mitochondrial dysfunction, and unblocked the tricarboxylic acid cycle flux and metabolic utilization of long-chain acyl-carnitines, amino acids, and carbohydrates. Importantly, fat-1 mice were protected against mitochondrial injury induced by obesogenic and fibrogenic insults. Conclusion: Our data uncover the importance of a lipid membrane composition rich in DHA and its lipid autacoid derivatives to have optimal hepatic mitochondrial and metabolic efficiency.

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)

Background and aims Tolerance is transplantation's holy grail, as it denotes allograft health without immunosuppression and its toxicities. Our aim was to determine, among stable long-term pediatric liver transplant recipients, the efficacy and safety of immunosuppression withdrawal to identify operational tolerance. Approach and results We conducted a multicenter, single-arm trial of immunosuppression withdrawal over 36-48 weeks. Liver tests were monitored biweekly (year 1), monthly (year 2), and bimonthly (years 3-4). For-cause biopsies were done at investigators' discretion but mandated when alanine aminotransferase or gamma glutamyltransferase exceeded 100 U/L. All subjects underwent final liver biopsy at trial end. The primary efficacy endpoint was operational tolerance, defined by strict biochemical and histological criteria 1 year after stopping immunosuppression. Among 88 subjects (median age 11 years; 39 boys; 57 deceased donor grafts), 33 (37.5%; 95% confidence interval [CI] 27.4%, 48.5%) were operationally tolerant, 16 were nontolerant by histology (met biochemical but failed histological criteria), and 39 were nontolerant by rejection. Rejection, predicted by subtle liver inflammation in trial entry biopsies, typically (n = 32) occurred at ≤32% of the trial-entry immunosuppression dose and was treated with corticosteroids (n = 32) and/or tacrolimus (n = 38) with resolution (liver tests within 1.5 times the baseline) for all but 1 subject. No death, graft loss, or chronic, severe, or refractory rejection occurred. Neither fibrosis stage nor the expression level of a rejection gene set increased over 4 years for either tolerant or nontolerant subjects. Conclusions Immunosuppression withdrawal showed that 37.5% of selected pediatric liver-transplant recipients were operationally tolerant. Allograft histology did not deteriorate for either tolerant or nontolerant subjects. The timing and reversibility of failed withdrawal justifies future trials exploring the efficacy, safety, and potential benefits of immunosuppression minimization.

Reprogramming of immunosuppressive tumor-associated macrophages (TAMs) presents an attractive therapeutic strategy in cancer. The aim of this study was to explore the role of macrophage CD5L protein in TAM activity and assess its potential as a therapeutic target. Monoclonal antibodies (mAbs) against recombinant CD5L were raised by subcutaneous immunization of BALB/c mice. Peripheral blood monocytes were isolated from healthy donors and stimulated with IFN/LPS, IL4, IL10, and conditioned medium (CM) from different cancer cell lines in the presence of anti-CD5L mAb or controls. Subsequently, phenotypic markers, including CD5L, were quantified by flow cytometry, IF and RT-qPCR. Macrophage CD5L protein expression was studied in 55 human papillary lung adenocarcinoma (PAC) samples by IHC and IF. Anti-CD5L mAb and isotype control were administered intraperitoneally into a syngeneic Lewis Lung Carcinoma mouse model and tumor growth was measured. Tumor microenvironment (TME) changes were determined by flow cytometry, IHC, IF, Luminex, RNAseq and RT-qPCR. Cancer cell lines CM induced an immunosuppressive phenotype (increase in CD163, CD206, MERTK, VEGF and CD5L) in cultured macrophages. Accordingly, high TAM expression of CD5L in PAC was associated with poor patient outcome (Log-rank (Mantel-Cox) test p = 0.02). We raised a new anti-CD5L mAb that blocked the immunosuppressive phenotype of macrophages in vitro. Its administration in vivo inhibited tumor progression of lung cancer by altering the intratumoral myeloid cell population profile and CD4 + T-cell exhaustion phenotype, thereby significantly modifying the TME and increasing the inflammatory milieu. CD5L protein plays a key function in modulating the activity of macrophages and their interactions within the TME, which supports its role as a therapeutic target in cancer immunotherapy. For a full list of funding bodies, please see the Acknowledgements.

Due to the high vulnerability of the pancreas to ischemia-reperfusion injury, choices regarding preservation solution markedly affect pancreas transplant success. A retrospective single-center analysis of 380 pancreas transplants (2000–2019) was performed to correlate current preservation solutions with transplant outcomes. Early graft failure requiring transplantectomy within 30 days post-transplant occurred in 7.5% for University of Wisconsin (UW) group (n = 267), 10.8% of Celsior (CS) group (n = 83), 28.5% of Histidine-Tryptophan-Ketoglutarate (HTK) group (n = 7), and none for Institut Georges Lopez-1 (IGL-1) group (n = 23). The most common causes of technical failures in this cohort included abdominal hemorrhage (8.4%); graft pancreatitis (3.7%); fluid collections (2.6%); intestinal complications (6.6%); and vascular thrombosis (20.5%). Although IGL-1 solution provided lower surgical complication rates, no significant differences were found between studied groups. Nevertheless, HTK solution was associated with elevated pancreatitis rates. The best graft survival was achieved at 1 year using UW and IGL-1, and at 3 and 5 years using IGL-1 (p = 0.017). There were no significant differences in patient survival after a median follow-up of 118.4 months. In this setting therefore, IGL-1 solution appears promising for perfusion and organ preservation in clinical pancreas transplantation, compared to other commonly used solutions., This work was supported by grant from Ministerio de Ciencia, Innovación y Universidades, reference PID2019-104130RB-I00 awarded to EF-P.

Helicobacter pylori infects 4.4 billion individuals worldwide and is considered the most important etiologic agent for peptic ulcers and gastric cancer. Individual response to H. pylori infection is complex and depends on complex interactions between host and environmental factors. The pathway towards gastric cancer is a sequence of events known as Correa’s model of gastric carcinogenesis, a stepwise inflammatory process from normal mucosa to chronic-active gastritis, atrophy, metaplasia and gastric adenocarcinoma. This study examines gastric clinical specimens representing different steps of the Correa pathway with the aim of identifying the expression profiles of coding- and non-coding RNAs that may have a role in Correa’s model of gastric carcinogenesis. We screened for differentially expressed genes in gastric biopsies by employing RNAseq, microarrays and qRT-PCR. Here we provide a detailed description of the experiments, methods and results generated. The datasets may help other scientists and clinicians to find new clues to the pathogenesis of H. pylori and the mechanisms of progression of the infection to more severe gastric diseases. Data is available via ArrayExpress., Measurement(s) gene expression • ncRNA_gene • miRNA Technology Type(s) Microarray • small RNA sequencing assay Factor Type(s) H. pylori infection stage • Intestinal metaplasia Sample Characteristic - Organism Homo sapiens Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.12349334

Idiopathic aplastic anemia (AA) has 2 key characteristics: an autoimmune response against hematopoietic stem/progenitor cells and regulatory T-cells (Tregs) deficiency. We have previously demonstrated reduction in a specific subpopulation of Treg in AA, which predicts response to immunosuppression. The aims of the present study were to define mechanisms of Treg subpopulation imbalance and identify potential for therapeutic intervention. We have identified 2 mechanisms that lead to skewed Treg composition in AA: first, FasL-mediated apoptosis on ligand interaction; and, second, relative interleukin-2 (IL-2) deprivation. We have shown that IL-2 augmentation can overcome these mechanisms. Interestingly, when high concentrations of IL-2 were used for in vitro Treg expansion cultures, AA Tregs were able to expand. The expanded populations expressed a high level of p-BCL-2, which makes them resistant to apoptosis. Using a xenograft mouse model, the function and stability of expanded AA Tregs were tested. We have shown that these Tregs were able to suppress the macroscopic clinical features and tissue manifestations of T-cell–mediated graft-versus-host disease. These Tregs maintained their suppressive properties as well as their phenotype in a highly inflammatory environment. Our findings provide an insight into the mechanisms of Treg reduction in AA. We have identified novel targets with potential for therapeutic interventions. Supplementation of ex vivo expansion cultures of Tregs with high concentrations of IL-2 or delivery of IL-2 directly to patients could improve clinical outcomes in addition to standard immunosuppressive therapy.

BACKGROUND AND AIMS Portal hypertension is the main consequence of cirrhosis, responsible for the complications defining clinical decompensation. The only cure for decompensated cirrhosis is liver transplantation, but it is a limited resource and opens the possibility of regenerative therapy. We investigated the potential of primary human amniotic membrane-derived mesenchymal stromal (hAMSCs) and epithelial (hAECs) stem cells for the treatment of portal hypertension and decompensated cirrhosis. METHODS In vitro: Primary liver sinusoidal endothelial cells (LSECs) and hepatic stellate cells (HSCs) from cirrhotic rats (chronic CCl4 inhalation) were co-cultured with hAMSCs, hAECs or vehicle for 24 hours, and their RNA profile was analysed. In vivo: CCl4-cirrhotic rats received 4x106 hAMSCs, 4x106 hAECs, or vehicle (NaCl 0.9%) (intraperitoneal). At 2-weeks we analysed: a) portal pressure (PP) and hepatic microvascular function; b) LSECs and HSCs phenotype; c) hepatic fibrosis and inflammation. RESULTS In vitro experiments revealed sinusoidal cell phenotype amelioration when co-cultured with stem cells. Cirrhotic rats receiving stem cells, particularly hAMSCs, had significantly lower PP than vehicle-treated animals, together with improved liver microcirculatory function. This hemodynamic amelioration was associated with improvement in LSECs capillarization and HSCs de-activation, though hepatic collagen was not reduced. Rats that received amnion derived stem cells had markedly reduced hepatic inflammation and oxidative stress. Finally, liver function tests significantly improved in rats receiving hAMSCs. CONCLUSIONS This preclinical study shows that infusion of human amniotic stem cells effectively decreases PP by ameliorating liver microcirculation, suggesting that it may represent a new treatment option for advanced cirrhosis with portal hypertension.

© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ., Objective: Alcohol-related liver disease (ALD) ranges from never-decompensated ALD (ndALD) to the life-threatening decompensated phenotype, known as alcohol-related hepatitis (AH). A multidimensional study of the clinical, histological and molecular features of these subtypes is lacking. Design: Two large cohorts of patients were recruited in an international, observational multicentre study: a retrospective cohort of patients with ndALD (n=110) and a prospective cohort of patients with AH (n=225). Clinical, analytical, immunohistochemistry and hepatic RNA microarray analysis of both disease phenotypes were performed. Results: Age and mean alcohol intake were similar in both groups. AH patients had greater aspartate amino transferase/alanine amino transferase ratio and lower gamma-glutamyl transferase levels than in ndALD patients. Patients with AH demonstrated profound liver failure and increased mortality. One-year mortality was 10% in ndALD and 50% in AH. Histologically, steatosis grade, ballooning and pericellular fibrosis were similar in both groups, while advanced fibrosis, Mallory-Denk bodies, bilirubinostasis, severe neutrophil infiltration and ductular reaction were more frequent among AH patients. Transcriptome analysis revealed a profound gene dysregulation within both phenotypes when compare to controls. While ndALD was characterised by deregulated expression of genes involved in matrisome and immune response, the development of AH resulted in a marked deregulation of genes involved in hepatocyte reprogramming and bile acid metabolism. Conclusions: Despite comparable alcohol intake, AH patients presented with worse liver function compared with ndALD patients. Bilirubinostasis, severe fibrosis and ductular reaction were prominent features of AH. AH patients exhibited a more profound deregulation of gene expression compared with ndALD patients., This work was supported by the National Institute on Alcohol Abuse and Alcoholism (NIAAA 1U01AA021908-01, 1U01AA020821 and P50AA011999) and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK P30DK120531 and P30DK120515). PSwas supported by Fondo de Investigación Sanitaria Carlos III, cofinanced by Fondo Europeo de Desarrollo Regional (FEDER), Unión Europea, 'Una manera de hacer Europa' PI17/00673; PI20/00765 and Miguel Servet, CPII16/0004. MV-C is a recipient of the Juan Rodés JR19/00015. JA is a recipient of a grant from Agencia Estatal de Salud (PI20/01663).

Optimal selection of high-risk patients with stage II colon cancer is crucial to ensure clinical benefit of adjuvant chemotherapy. Here, we investigated the prognostic value of genomic intratumor heterogeneity and aneuploidy for disease recurrence. We combined targeted sequencing, SNP arrays, fluorescence in situ hybridization, and immunohistochemistry on a retrospective cohort of 84 untreated stage II colon cancer patients. We assessed the clonality of copy-number alterations (CNAs) and mutations, CD8

INTRODUCTION: Colorectal cancer (CRC) is a potentially life-threatening complication of long-standing ulcerative colitis (UC). MicroRNAs (miRNA) are epigenetic regulators that have been involved in the development of UC-associated CRC. However, their role as potential mucosal biomarkers of neoplastic progression has not been adequately studied. METHODS: In this study, we analyzed the expression of 96 preselected miRNAs in human formalin-fixed and paraffin-embedded tissue of 52 case biopsies (20 normal mucosa, 20 dysplasia, and 12 UC-associated CRCs) and 50 control biopsies (10 normal mucosa, 21 sporadic adenomas, and 19 sporadic CRCs) by using Custom TaqMan Array Cards. For validation of deregulated miRNAs, we performed individual quantitative real-time polymerase chain reaction in an independent cohort of 50 cases (13 normal mucosa, 25 dysplasia, and 12 UC-associated CRCs) and 46 controls (7 normal mucosa, 19 sporadic adenomas, and 20 sporadic CRCs). RESULTS: Sixty-four miRNAs were found to be differentially deregulated in the UC-associated CRC sequence. Eight of these miRNAs were chosen for further validation. We confirmed miR-31, -106a, and -135b to be significantly deregulated between normal mucosa and dysplasia, as well as across the UC-associated CRC sequence (all P < 0.01). Notably, these miRNAs also confirmed to have a significant differential expression compared with sporadic CRC (all P < 0.05). DISCUSSION: UC-associated and sporadic CRCs have distinct miRNA expression patterns, and some miRNAs indicate early neoplastic progression. This work was funded by grants from the Instituto de Salud Carlos III (PI12/01481; PI19/01050). Project PI19/ 01050 is funded by Instituto de Salud Carlos III (ISCIII) and co funded by the European Union. CIBEREHD is funded by the Insti tuto de Salud Carlos III and Beca Marató de TV3 (201932-30). Parts of this work were also supported by the Xarxa de Bancs de Tumors de Catalunya sponsored by Pla Director d’Oncología de Catalunya (XBTC) and by the Hospital Clínics Premi Fi de Residència (G.J). None of the funding parties has been involved in collection, analysis, and interpretation of the data.

The molecular mechanisms driving the progression from early-chronic liver disease (CLD) to cirrhosis and, finally, acute-on-chronic liver failure (ACLF) are largely unknown. Our aim was to develop a protein network-based approach to investigate molecular pathways driving progression from early-CLD to ACLF.Transcriptome analysis was performed on liver biopsies from patients at different liver disease stages, including fibrosis, compensated cirrhosis, decompensated cirrhosis and ACLF, and control healthy livers. We created 9 liver-specific disease-related protein-protein interaction networks capturing key pathophysiological processes potentially related to CLD. We used these networks as a framework and performed gene set-enrichment analysis (GSEA) to identify dynamic gene profiles of disease progression.Principal component analyses revealed that samples clustered according to the disease stage. GSEA of the defined processes showed an upregulation of inflammation, fibrosis and apoptosis networks throughout disease progression. Interestingly, we did not find significant gene expression differences between compensated and decompensated cirrhosis, while ACLF showed acute expression changes in all the defined liver disease-related networks. The analyses of disease progression patterns identified ascending and descending expression profiles associated with ACLF onset. Functional analyses showed that ascending profiles were associated with inflammation, fibrosis, apoptosis, senescence and carcinogenesis networks, while descending profiles were mainly related to oxidative stress and genetic factors. We confirmed by qPCR the upregulation of genes of the ascending profile and validated our findings in an independent patient cohort.ACLF is characterized by a specific hepatic gene expression pattern related to inflammation, fibrosis, apoptosis, senescence and carcinogenesis. Moreover, the observed profile is significantly different from that of compensated and decompensated cirrhosis, supporting the hypothesis that ACLF should be considered a distinct entity.By using transjugular biopsies obtained from patients at different stages of chronic liver disease, we unveil the molecular pathogenic mechanisms implicated in the progression of chronic liver disease to cirrhosis and acute-on-chronic liver failure. The most relevant finding in this study is that patients with acute-on-chronic liver failure present a specific hepatic gene expression pattern distinct from that of patients at earlier disease stages. This gene expression pattern is mostly related to inflammation, fibrosis, angiogenesis, and senescence and apoptosis pathways in the liver.

Resumen La meditación de atención plena es una técnica ampliamente usada en la actualidad, con origen en la cultura budista, que permite desarrollar la habilidad de dirigir la atención sin prejuicios a las experiencias del momento presente, tratando de disminuir el procesamiento autorreferencial narrativo o de experiencias y la prospección en el futuro. Los estudios en el área han demostrado la utilidad de la meditación de atención plena para aumentar el bienestar psicológico y físico en términos generales, pero los mecanismos neurofisiológicos asociadas a estos cambios aún están sin dilucidarse. En la presente revisión se recopila información de estudios que han utilizado neuroimagen y electroencefalograma para valorar los mismos, con énfasis en aquellos estudios que permiten discutir la propuesta de la meditación de atención plena como un estímulo que disminuye el procesamiento de tareas autorreferenciales y favorece los estados de tareas cognitivas y tareas asociativas sensoriales, incluida la interocepción. Estos estudios evidencian cambios estructurales y funcionales en sectores como corteza prefrontal, ínsula, el hipocampo, el giro cíngulo, la corteza visual primaria y el estriado, que son discutidos en este artículo a la luz del conocimiento actual. Abstract Mindfulness meditation is a technique widely used today, originating in Buddhist culture, which allows developing the ability to direct attention without prejudice to the experiences of the present moment, trying to reduce narrative or experience self-referential processing and prospecting in the future. Studies in the area have shown the usefulness of mindfulness meditation to increase psychological and physical well-being in general terms, but the neurophysiological mechanisms associated with these changes remain to be elucidated. In this review, information is collected from studies that have used neuroimaging and electroencephalography to assess them, with emphasis on those studies that allow discussing the proposal of mindfulness meditation as a stimulus that decreases the processing of self-referential tasks and favors states of cognitive tasks and sensory associative tasks, including interoception. These studies show structural and functional changes in sectors such as prefrontal cortex, insula, hippocampus, cingulate gyrus, primary visual cortex and striatum, which are discussed in this article in the light of current knowledge.

Chronic liver diseases are characterized by the expansion of ductular reaction (DR) cells and the expression of liver progenitor cell (LPC) markers. In alcoholic hepatitis (AH), the degree of DR expansion correlates with disease progression and short-term survival. However, little is known about the biological properties of DR cells, their impact on the pathogenesis of human liver disease, and their contribution to tissue repair. In this study, we have evaluated the transcriptomic profile of DR cells by laser capture microdissection in patients with AH and assessed its association with disease progression. The transcriptome analysis of cytokeratin 7-positive (KRT7(+)) DR cells uncovered intrinsic gene pathways expressed in DR and genes associated with alcoholic liver disease progression. Importantly, DR presented a proinflammatory profile with expression of neutrophil recruiting C-X-C motif chemokine ligand (CXC) and C-C motif chemokine ligand chemokines. Moreover, LPC markers correlated with liver expression and circulating levels of inflammatory mediators such as CXCL5. Histologically, DR was associated with neutrophil infiltration at the periportal area. In order to model the DR and to assess its functional role, we generated LPC organoids derived from patients with cirrhosis. Liver organoids mimicked the transcriptomic and proinflammatory profile of DR cells. Conditioned medium from organoids induced neutrophil migration and enhanced cytokine expression in neutrophils. Likewise, neutrophils promoted the proinflammatory profile and the expression of chemokines of liver organoids. CONCLUSION: Transcriptomic and functional analysis of KRT7(+) cells indicate that DR has a proinflammatory profile and promote neutrophil recruitment. These results indicate that DR may be involved in the liver inflammatory response in AH, and suggest that therapeutic strategies targeting DR cells may be useful to mitigate the inflammatory cell recruitment in AH.

BACKGROUND In cirrhosis, a decrease in hepatic venous pressure gradient (HVPG) > 10% after acute iv propranolol (HVPG response) is associated with a lower risk of decompensation and death. Only a part of patients are HVPG responders and there are no accurate non-invasive markers to identify them. We aimed at discovering metabolomic biomarkers of HVPG responders to propranolol. METHODS Sixty-six patients with cirrhosis and HVPG ≥ 10 mm Hg in whom the acute HVPG response to propranolol was assessed, were prospectively included. A targeted metabolomic serum analysis using ultrahigh-performance liquid chromatography coupled to mass spectrometry was performed. Different combinations of 2-3 metabolites identifying HVPG responders (HVPG reduction > 10%) were obtained by stepwise logistic regression. The best of these model (AUROC, Akaike criterion) underwent internal cross-validation and cut-offs to classify responders/non-responders was proposed. RESULTS A total of 41/66 (62%) patients were HVPG responders. Three hundred and eighty-nine metabolites were detected and 177 were finally eligible. Eighteen metabolites were associated to the HVPG response at univariate analysis; at multivariable analysis, a model including a phosphatidylcholine (PC(P-16:0/22:6)) and a free fatty acid (20:2(n-6), eicosadienoic acid) performed well for HVPG response, with an AUROC of 0.801 (0.761 at internal validation). The cut-off 0.629 was the most efficient for overall classification (49/66 patients correctly classified). Two cut-off values allowed identifying responders (0.688, PPV 84%) and non-responders (0.384, NPV 82%) with undetermined values for 17/66 patients. Clinical variables did not add to the model. CONCLUSIONS The combination of two metabolites helps at identifying HVPG responders to acute propranolol. It could be a useful non-invasive test to classify the HVPG response to propranolol.

Somatically acquired uniparental disomies (aUPDs) are frequent events in solid tumors and have been associated with cancer‐related genes. Studies assessing their functional consequences across several cancer types are therefore necessary. Here, we aimed at integrating aUPD profiles with the mutational status of cancer‐related genes in a tumor‐type specific manner. Using TCGA datasets for 1,032 gastrointestinal cancers, including colon (COAD), rectum (READ), stomach (STAD), esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC), we show a non‐random distribution of aUPD, suggesting the existence of a cancer‐specific landscape of aUPD events. Our analysis indicates that aUPD acts as a “second hit” in Knudson's model in order to achieve biallelic inactivation of tumor suppressor genes. In particular, APC, ARID1A and NOTCH1 were recurrently inactivated by the presence of homozygous mutation as a consequence of aUPD in COAD and READ, STAD and ESCC, respectively. Furthermore, while TP53 showed inactivation caused by aUPD at chromosome arm 17p across all tumor types, copy number losses at this genomic position were also frequent. By experimental and computationally inferring genome ploidy, we demonstrate that an increased number of aUPD events, both affecting the whole chromosome or segments of it, were present in highly aneuploid genomes compared to near‐diploid tumors. Finally, the presence of mosaic UPD was detected at a higher frequency in DNA extracted from peripheral blood lymphocytes of patients with colorectal cancer compared to healthy individuals. In summary, our study defines specific profiles of aUPD in gastrointestinal cancers and provides unequivocal evidence of their relevance in cancer., What's new? Somatically acquired uniparental disomies (aUPDs), in which two copies of a chromosome originate from the same parent, have been documented in various human cancers. Here, the authors examined the frequency of aUPDs in different gastrointestinal cancer types. Events involving aUPDs were found to occur at high incidence in gastrointestinal cancers and at increased frequency particularly in highly aneuploid genomes. The data also reveal a nonrandom distribution of aUPDs, with evidence of biallelic inactivation of tumor suppressor genes and activation of oncogenes in a tumor type‐specific manner. The findings suggest that aUPDs are functionally relevant in gastrointestinal malignancies.

Simple Summary We define the transcriptome and secretome of primary LSECs during the progression of cirrhosis, revealing specific molecular signatures, novel biomarkers and therapeutic targets for new disease-modifying treatments for patients with advanced chronic liver disease. Abstract The poor prognosis of chronic liver disease (CLD) generates the need to investigate the evolving mechanisms of disease progression, thus disclosing therapeutic targets before development of clinical complications. Considering the central role of liver sinusoidal endothelial cells (LSECs) in pre-neoplastic advanced CLD, the present study aimed at investigating the progression of CLD from an endothelial holistic perspective. RNAseq defined the transcriptome of primary LSECs isolated from three pre-clinical models of advanced CLD, during the progression of the disease, and from fresh human cirrhotic tissue. At each stage of the disease, the effects of LSECs secretome on neighboring cells and proteomic analysis of LSECs-derived extracellular vesicles (EVs) were also determined. CLD was associated with deep common modifications in the transcriptome of LSECs in the pre-clinical models. Pathway enrichment analysis showed predominance of genes related with pro-oncogenic, cellular communication processes, and EVs biogenesis during CLD progression. Crosstalk experiments revealed endothelial EVs as potent angiocrine effectors. The proteome of LSECs EVs showed stage-specific signatures, including over-expression of tropomyosin-1. Proof-of-principle experiments treating cirrhotic HSCs with recombinant tropomyosin-1 suggested de-activating effects. Our data provide the basis for discovering novel biomarkers and therapeutic targets for new disease-modifying treatments for patients with advanced CLD.

Intraductal papillary mucinous neoplasms (IPMN) are pancreatic cystic lesions that can develop into pancreatic ductal adenocarcinoma (PDAC). Although there is an increasing incidence of IPMN diagnosis, the mechanisms of formation and progression into invasive cancer remain unclear. MicroRNAs (miRNAs) are small non-coding RNAs, repressors of mRNA translation, and promising diagnostic biomarkers for IPMN and PDAC. Functional information on the role of early-altered miRNAs in this setting would offer novel strategies for tracking the IPMN-to-PDAC progression. In order to detect mRNAs that are likely to be under miRNA regulation in IPMNs, whole transcriptome and miRNome data from normal pancreatic tissue (n = 3) and IPMN lesions (n = 4) were combined and filtered according to negative correlation and miRNA-target prediction databases by using miRComb R package. Further comparison analysis with PDAC data allowed us to obtain a subset of miRNA-mRNA pairs shared in IPMN and PDAC. Functional enrichment analysis unravelled processes that are mainly related with cell structure, actin cytoskeleton, and metabolism. MiR-181a appeared as a master regulator of these processes. The expression of selected miRNA-mRNA pairs was validated by qRT-PCR in an independent cohort of patients (n = 40), and then analysed in different pancreatic cell lines. Finally, we generated a cellular model of HPDE cells stably overexpressing miR-181a, which showed a significant alteration of actin cytoskeleton structures accompanied by a significant downregulation of EPB41L4B and SEL1L expression. In situ hybridization of miR-181a and immunohistochemistry of EPB41L4B and SEL1L in pancreatic tissues (n = 4 Healthy, n = 3 IPMN, n = 4 PDAC) were also carried out. In this study, we offer insights on the potential implication of miRNA alteration in the regulation of structural and metabolic changes that pancreatic cells experience during IPMN establishment and that are maintained in PDAC.

Optimal selection of high-risk patients with stage II colon cancer is crucial to ensure clinical benefit of adjuvant chemotherapy after surgery. Here, we investigate the prognostic and predictive value of genomic intratumor heterogeneity and aneuploidy for disease recurrence. We combined SNP arrays, targeted next-generation sequencing, fluorescence in situ hybridization and inmunohistochemistry on a retrospective cohort of 84 untreated stage II colon cancer patients. We assessed the subclonality of copy-number alterations (CNAs) and mutations, CD8+ lymphocyte infiltration and their association with time to recurrence (TTR). Prognostic factors were included in machine learning analysis to evaluate their ability to predict individual relapse risk. Tumors from recurrent patients (N = 38) exhibited greater proportion of CNAs compared with non-recurrent (N = 46) (mean 31.3% vs. 23%, respectively; P = 0.014), which was confirmed in an independent cohort. Candidate chromosome-specific aberrations included the gain of the chromosome arm 13q (P = 0.02; HR, 2.67) and loss of heterozygosity at 17q22-q24.3 (P = 0.05; HR, 2.69), both associated with shorter TTR. CNA load positively correlated with intratumor heterogeneity (R = 0.52; P < 0.0001), indicating ongoing chromosomal instability. Consistently, subclonal copy-number heterogeneity was associated with elevated risk of relapse (P = 0.028; HR, 2.20), which we did not observe for subclonal mutations. The clinico-genomic model rated an area under the curve of 0.83, achieving a 10% incremental gain compared to clinicopathological markers. In conclusion, tumor aneuploidy and copy-number heterogeneity were predictive of a poor outcome in early-stage colon cancer, and improved discriminative performance in comparison to clinicopathological data.