Dihydrosphingolipids are associated with steatosis and increased fibrosis damage in non-alcoholic fatty liver disease.

Dihydrosphingolipids are lipids biosynthetically related to ceramides. An increase in ceramides is associated with enhanced fat storage in the liver, and inhibition of their synthesis is reported to prevent the appearance of steatosis in animal models. However, the precise association of dihydrosphingolipids with non-alcoholic fatty liver disease (NAFLD) is yet to be established. We employed a diet induced NAFLD mouse model to study the association between this class of compounds and disease progression. Mice fed a high-fat diet were sacrificed at 22, 30 and 40 weeks to reproduce the full spectrum of histological damage found in human disease, steatosis (NAFL) and steatohepatitis (NASH) with and without significant fibrosis. Blood and liver tissue samples were obtained from patients whose NAFLD severity was assessed histologically. To demonstrate the effect of dihydroceramides over NAFLD progression we treated mice with fenretinide an inhibitor of dihydroceramide desaturase-1 (DEGS1). Lipidomic analyses were performed using liquid chromatography-tandem mass spectrometry. Triglycerides, cholesteryl esters and dihydrosphingolipids were increased in the liver of model mice in association with the degree of steatosis and fibrosis. Dihydroceramides increased with the histological severity observed in liver samples of mice (0.024 ± 0.003 nmol/mg vs 0.049 ± 0.005 nmol/mg, non-NAFLD vs NASH-fibrosis, p < 0.0001) and patients (0.105 ± 0.011 nmol/mg vs 0.165 ± 0.021 nmol/mg, p = 0.0221). Inhibition of DEGS1 induce a four-fold increase in dihydroceramides improving steatosis but increasing the inflammatory activity and fibrosis. In conclusion, the degree of histological damage in NAFLD correlate with dihydroceramide and dihydrosphingolipid accumulation. LAY SUMMARY: Accumulation of triglyceride and cholesteryl ester lipids is the hallmark of non-alcoholic fatty liver disease. Using lipidomics, we examined the role of dihydrosphingolipids in NAFLD progression. Our results demonstrate that de novo dihydrosphingolipid synthesis is an early event in NAFLD and the concentrations of these lipids are correlated with histological severity in both mouse and human disease.

Transit-amplifying cells control R-spondins in the mouse crypt to modulate intestinal stem cell proliferation.

Intestinal epithelium regenerates rapidly through proliferation of intestinal stem cells (ISCs), orchestrated by potent mitogens secreted within the crypt niche. However, mechanisms regulating these mitogenic factors remain largely unknown. Here, we demonstrate that transit-amplifying (TA) cells, marked by unconventional prefoldin RPB5 interactor (URI), control R-spondin production to guide ISC proliferation. Genetic intestinal URI ablation in mice injures TA cells, reducing their survival capacity, leading to an inflamed tissue and subsequently decreasing R-spondin levels, thereby causing ISC quiescence and disruption of intestinal structure. R-spondin supplementation or restoration of R-spondin levels via cell death inhibition by c-MYC elimination or the suppression of inflammation reinstates ISC proliferation in URI-depleted mice. However, selective c-MYC and p53 suppression are required to fully restore TA cell survival and differentiation capacity and preserve complete intestinal architecture. Our data reveal an unexpected role of TA cells, which represent a signaling platform instrumental for controlling inflammatory cues and R-spondin production, essential for maintaining ISC proliferation and tissue regeneration.

Histone acetylation of bile acid transporter genes plays a critical role in cirrhosis.

BACKGROUND & AIMS: Owing to the lack of genetic animal models that adequately recreate key clinical characteristics of cirrhosis, the molecular pathogenesis of cirrhosis has been poorly characterized, and treatments remain limited. Hence, we aimed to better elucidate the pathological mechanisms of cirrhosis using a novel murine model. METHODS: We report on the first murine genetic model mimicking human cirrhosis induced by hepatocyte-specific elimination of microspherule protein 1 (MCRS1), a member of non-specific lethal (NSL) and INO80 chromatin-modifier complexes. Using this genetic tool with other mouse models, cell culture and human samples, combined with quantitative proteomics, single nuclei/cell RNA sequencing and chromatin immunoprecipitation assays, we investigated mechanisms of cirrhosis. RESULTS: MCRS1 loss in mouse hepatocytes modulates the expression of bile acid (BA) transporters - with a pronounced downregulation of Na+-taurocholate cotransporting polypeptide (NTCP) - concentrating BAs in sinusoids and thereby activating hepatic stellate cells (HSCs) via the farnesoid X receptor (FXR), which is predominantly expressed in human and mouse HSCs. Consistently, re-expression of NTCP in mice reduces cirrhosis, and genetic ablation of FXR in HSCs suppresses fibrotic marks in mice and in vitro cell culture. Mechanistically, deletion of a putative SANT domain from MCRS1 evicts histone deacetylase 1 from its histone H3 anchoring sites, increasing histone acetylation of BA transporter genes, modulating their expression and perturbing BA flow. Accordingly, human cirrhosis displays decreased nuclear MCRS1 and NTCP expression. CONCLUSIONS: Our data reveal a previously unrecognized function of MCRS1 as a critical histone acetylation regulator, maintaining gene expression and liver homeostasis. MCRS1 loss induces acetylation of BA transporter genes, perturbation of BA flow, and consequently, FXR activation in HSCs. This axis represents a central and universal signaling event in cirrhosis, which has significant implications for cirrhosis treatment. LAY SUMMARY: By genetic ablation of MCRS1 in mouse hepatocytes, we generate the first genetic mouse model of cirrhosis that recapitulates human features. Herein, we demonstrate that the activation of the bile acid/FXR axis in liver fibroblasts is key in cirrhosis development.

Comparison Between Real-time Ammonium and pH Measurement, Immunohistochemistry, and Histochemistry for the Diagnosis of Helicobacter pylori.

BACKGROUND: Helicobacter pylori (HP) infection has been implicated in several malignant and nonmalignant conditions. The confirmatory diagnosis of HP requires an endoscopic biopsy, followed by a rapid urease test, culture, and/or histopathologic examination using hemotoxylin and eosin, histochemical stains, or immunohistochemistry against HP. EndoFaster is a novel device that can perform real-time ammonium and pH measurements in gastric juice, allowing a diagnosis of HP during gastroduodenal endoscopy. GOAL: This study aimed to validate the accuracy of EndoFaster and to compare different histochemical and immunohistochemical techniques for the diagnosis of HP infection. STUDY: Consecutive patients who underwent upper endoscopy at our center were prospectively enrolled. During the endoscopy procedure, gastric juice was aspirated to perform an automatic analysis by EndoFaster and gastric biopsies were taken. Histologic sections were reviewed to assess the histopathologic features. The sensitivity, specificity, positive predictive value, and negative predictive value were calculated for the EndoFaster test, Diff-Quick (DQ), and immunohistochemistry against HP (anti-HP) using Warthin-Starry as the gold standard for HP detection. RESULTS: Overall, 80 patients were enrolled. In 19 cases (23,75%), histology was normal. In the remaining cases, varying degrees of inflammation were found. The sensitivity, specificity, positive predictive value, and negative predictive value were 73.33%, 86.00%, 75.86%, and 84.31% for the EndoFaster test using 67 ppm/mL as the positive threshold; 73.33%, 100%, 100%, and 86.21% for DQ; and 79.31%, 88.00%, 79.31%, and 88.00% for anti-HP, respectively. CONCLUSIONS: The EndoFaster test has good sensitivity and specificity for the diagnosis of HP during the gastroscopy procedure. DQ and anti-HP are excellent alternatives to Warthin-Starry for the detection of HP.

Inhibition of the IL-17A axis in adipocytes suppresses diet-induced obesity and metabolic disorders in mice.

Overnutrition causes obesity, a global health problem without any effective therapy. Obesity is characterized by low-grade inflammation, which predisposes individuals to metabolic syndrome via unknown mechanisms. Here, we demonstrate that abolishing the interleukin-17A (IL-17A) axis in mice by inhibition of RORγt-mediated IL-17A production by digoxin, or by ubiquitous deletion of IL-17 receptor A (Il17ra), suppresses diet-induced obesity (DIO) and metabolic disorders, and promotes adipose-tissue browning, thermogenesis and energy expenditure. Genetic ablation of Il17ra specifically in adipocytes is sufficient to completely prevent DIO and metabolic dysfunction in mice. IL-17A produced in response to DIO induces PPARγ phosphorylation at Ser273 in adipocytes in a CDK5-dependent manner, thereby modifying expression of diabetogenic and obesity genes, which correlates with IL-17A signalling in white adipose tissues of individuals with morbid obesity. These findings reveal an unanticipated role for IL-17A in adipocyte biology, in which its direct action pathogenically reprograms adipocytes, promoting DIO and metabolic syndrome. Targeting the IL-17A axis could be an efficient antiobesity strategy.

Onset of ulcerative colitis and complete response during the treatment of a metastatic colon cancer: case report and literature review.

Colorectal cancer is a common cancer worldwide. Several risk factors have been described, such as age, lifestyle and family history. Inflammatory bowel diseases (IBD) are a well-recognized risk factor for the development of colorectal cancer. However, the onset of an IBD de novo in the context of the treatment of a colorectal neoplasia has not been reported before, except in the context of the treatment with immunocheckpoint inhibitors. Fifty-nine-years old man diagnosed with a metastatic colorectal cancer who received conventional treatment with chemotherapy and an antiangiogenic inhibitor. The patient had a complete response with the therapy after few cycles. Nevertheless, during the treatment, the patient presented with rectal bleeding, and was diagnosed with ulcerative colitis. Although the treatment was discontinued, tumoral complete remission is maintained. The relevance of this case lies in the concurrence of the onset of an autoimmune disease and a complete response of the malignancy. The concurrence of these events has been described previously only with immunotherapy. There are not cases reported involving chemotherapy and antiangiogenic drugs. Other causes of colitis were ruled out due to the unusual presentation of the case.

Molecular Heterogeneity of High Grade Colorectal Adenocarcinoma.

High grade colorectal carcinomas (HG-CRCs), which comprise 15% of colorectal carcinomas, are underrepresented in reported molecular studies. Clinicopathological, immunohistochemical, and molecular features of 40 HG-CRCs are described. Moreover, glandular and solid areas of 25 tumors were separately analyzed. The expression of MLH1, PMS2, MSH2, MSH6, p53, E-cadherin, CDX2, CK20, CD8, PDL1, PAN-TRK, c-MET, SMARCB1, ARID1A, SMARCA2, and SMARCA4 was analyzed by immunohistochemistry. Promoter MLH1 methylation was analyzed in tumors with MLH1/PMS2 loss. Next-generation sequencing was used to screen 161 genes for hotspot mutations, copy number variations and gene fusions. In this series, 72.5% of HG-CRCs showed mismatch repair deficiency (MMRd). MMR deficient tumor and MMR proficient (MMRp) tumors showed striking molecular differences. Thus, whereas BRAF mutations were only observed in MMRd tumors, mutations in KRAS and TP53 were more frequent in MMR proficient tumors. Moreover, gene fusions (NTRK1 and MET) were detected only in MMRd tumors, whereas gene amplification (MYC, CCND1 and EGFR) predominated in MMRp/TP53-mutated tumors. Loss of expression of proteins involved in chromatin remodeling, such as ARID1A, was observed only in MMRd HG-CRCs, which also showed more frequently PD-L1 expression and a higher number of tumor infiltrating lymphocytes. The separate analysis of glandular and solid areas indicated that the clonal or subclonal nature of the molecular alterations also depended on MMR status. Mutations in genes such as TP53 and KRAS were always clonal in MMRp-CRCs but occurred as subclonal events in MMRd-CRCs. Gene amplification was implicated in the progression of MMRp tumors, but not in MMRd tumors, in which clonal diversity was due to accumulation of mutations in genes of different pathways such as NOTCH, MMR, or PIK3CA. In summary, intertumor and intratumor molecular heterogeneity in HG-CRCs is mainly due to MMR status.

Coxsackievirus B Type 4 Infection in ß Cells Downregulates the Chaperone Prefoldin URI to Induce a MODY4-like Diabetes via Pdx1 Silencing.

Enteroviruses are suspected to contribute to insulin-producing ß cell loss and hyperglycemia-induced diabetes. However, mechanisms are not fully defined. Here, we show that coxsackievirus B type 4 (CVB4) infection in human islet-engrafted mice and in rat insulinoma cells displays loss of unconventional prefoldin RPB5 interactor (URI) and PDX1, affecting ß cell function and identity. Genetic URI ablation in the mouse pancreas causes PDX1 depletion in ß cells. Importantly, diabetic PDX1 heterozygous mice overexpressing URI in ß cells are more glucose tolerant. Mechanistically, URI loss triggers estrogen receptor nuclear translocation leading to DNA methyltransferase 1 (DNMT1) expression, which induces Pdx1 promoter hypermethylation and silencing. Consequently, demethylating agent procainamide-mediated DNMT1 inhibition reinstates PDX1 expression and protects against diabetes in pancreatic URI-depleted mice . Finally, the ß cells of human diabetes patients show correlations between viral protein 1 and URI, PDX1, and DNMT1 levels. URI and DNMT1 expression and PDX1 silencing provide a causal link between enterovirus infection and diabetes.

Next generation sequencing to decipher concurrent loss of PMS2 and MSH6 in colorectal cancer.

BACKGROUND: Immunohistochemistry (IHQ) is commonly used for the detection of mismatch repair proteins deficiency (MMRD). One very infrequent abnormal pattern of MMR protein expression is the loss of PMS2 and MSH6, with intact expression of MLH1 and MSH2. CASE PRESENTATION: We review the frequency of this MMRD IHC pattern among 108 colorectal (CRCs) and 35 endometrial cancers in our files with loss of expression of at least one protein, and present two CRCs showing loss of PMS2 and MSH6 protein expression (1.9% of CRCs). NGS analysis of these tumours identified PMS2 mutations (R134* germline mutation in one tumour and M1R and c.1239delA somatic mutation in the other) as the primary event and somatic MSH6 mutation (c.3261dupC) as the secondary event in both tumours. CONCLUSIONS: This study suggests that Next Generation Sequencing (NGS) tumour analysis should be considered in the algorithm of Lynch syndrome screening to detect MMR gen somatic mutation in inconclusive cases.

Cardiac toxicity of immune-checkpoint inhibitors: a clinical case of nivolumab-induced myocarditis and review of the evidence and new challenges.

Immune checkpoint inhibitors have revolutionized cancer treatment due to their undeniable efficacy, but a range of new adverse events (AE) has emerged. In particular, cardiac toxicity is a potentially fatal AE, and introduces new challenges regarding its underlying molecular mechanisms of occurrence, optimal treatment and follow up, and prevention. We present a clinical case of a patient with advanced kidney cancer treated with nivolumab as a third line treatment. After four cycles, the patient developed nonspecific symptoms and was hospitalized, identifying a set of clinical, analytical and electrocardiographic alterations compatible with myocarditis. Despite the intensive support, the patient died and a necropsy study was performed. We present a detailed description of the clinical case including the pathological and molecular findings, and we conduct a review of the available evidence related to immune-mediated cardiac toxicity to offer some new highlights in the management of this AE.

URI is required to maintain intestinal architecture during ionizing radiation.

Ionizing radiation (IR) can cause gastrointestinal syndrome (GIS), a lethal disorder, by means of unknown mechanisms. We show that high-dose irradiation increases unconventional prefoldin RPB5 interactor (URI) levels in mouse intestinal crypt, but organ regeneration correlates with URI reductions. URI overexpression in intestine protects mice from radiation-induced GIS, whereas halving URI expression sensitizes mice to IR. URI specifically inhibits ß-catenin in stem cell-like label-retaining (LR) cells, which are essential for organ regeneration after IR. URI reduction activates ß-catenin-induced c-MYC expression, causing proliferation of and DNA damage to LR cells, rendering them radiosensitive. Therefore, URI labels LR cells which promote tissue regeneration in response to high-dose irradiation, and c-MYC inhibitors could be countermeasures for humans at risk of developing GIS.

Myeloid p38α signaling promotes intestinal IGF-1 production and inflammation-associated tumorigenesis.

The protein kinase p38α plays a key role in cell homeostasis, and p38α signaling in intestinal epithelial cells protects against colitis-induced tumorigenesis. However, little is known on the contribution of p38α signaling in intestinal stromal cells. Here, we show that myeloid cell-specific downregulation of p38α protects mice against inflammation-associated colon tumorigenesis. The reduced tumorigenesis correlates with impaired detection in the colon of crucial chemokines for immune cell recruitment. We identify insulin-like growth factor-1 (IGF-1) as a novel mediator of the p38α pathway in macrophages. Moreover, using genetic and pharmacological approaches, we confirm the implication of IGF-1 produced by myeloid cells in colon inflammation and tumorigenesis. We also show a correlation between IGF-1 pathway activation and the infiltration of myeloid cells with active p38α in colon samples from patients with ulcerative colitis or colon cancer. Altogether, our results uncover an important role for myeloid IGF-1 downstream of p38α in colitis-associated tumorigenesis and suggest the interest in evaluating IGF-1 therapies for inflammation-associated intestinal diseases, taking into consideration IGF-1 signaling and immune cell infiltration in patient biopsies.

Huge recurrent gastric neuroendocrine tumor: a second-line chemotherapeutic dilemma.

Chemotherapy is considered "state of the art" for the treatment of poorly differentiated neuroendocrine neoplasms. Unfortunately, there is no standard effective post-first-line treatment for relapsing high-grade gastroenteropancreatic neuroendocrine neoplasms. We report the case of a patient with a gastric neuroendocrine carcinoma stage IV, with massive gastrointestinal bleeding at diagnosis. After the first line of platin-based chemotherapy a major tumoral response was documented, but the patient relapsed after 4 months. A second line of chemotherapy treatment was given, with the FOLFOX regimen, and the patient has been free of progression for almost 2 years. There is no second-line standard treatment accepted for this type of carcinoma, but 5-fluorouracil combined with oxaliplatin showed interesting antitumor activity.

Huge recurrent gastric neuroendocrine tumor: a second-line chemotherapeutic dilemma

Chemotherapy is considered "state of the art" for the treatment of poorly differentiated neuroendocrine neoplasms. Unfortunately, there is no standard effective post-first-line treatment for relapsing high-grade gastroenteropancreatic neuroendocrine neoplasms. We report the case of a patient with a gastric neuroendocrine carcinoma stage IV, with massive gastrointestinal bleeding at diagnosis. After the first line of platin-based chemotherapy a major tumoral response was documented, but the patient relapsed after 4 months. A second line of chemotherapy treatment was given, with the FOLFOX regimen, and the patient has been free of progression for almost 2 years. There is no second-line standard treatment accepted for this type of carcinoma, but 5-fluorouracil combined with oxaliplatin showed interesting antitumor activity.

mTORC1 Inactivation Promotes Colitis-Induced Colorectal Cancer but Protects from APC Loss-Dependent Tumorigenesis.

Dietary habits that can induce inflammatory bowel disease (IBD) are major colorectal cancer (CRC) risk factors, but mechanisms linking nutrients, IBD, and CRC are unknown. Using human data and mouse models, we show that mTORC1 inactivation-induced chromosomal instability impairs intestinal crypt proliferation and regeneration, CDK4/6 dependently. This triggers interleukin (IL)-6-associated reparative inflammation, inducing crypt hyper-proliferation, wound healing, and CRC. Blocking IL-6 signaling or reactivating mTORC1 reduces inflammation-induced CRC, so mTORC1 activation suppresses tumorigenesis in IBD. Conversely, mTORC1 inactivation is beneficial in APC loss-dependent CRC. Thus, IL-6 blockers or protein-rich-diet-linked mTORC1 activation may prevent IBD-associated CRC. However, abolishing mTORC1 can mitigate CRC in predisposed patients with APC mutations. Our work reveals mTORC1 oncogenic and tumor-suppressive roles in intestinal epithelium and avenues to optimized and personalized therapeutic regimens for CRC.

Hepatocellular Carcinomas Originate Predominantly from Hepatocytes and Benign Lesions from Hepatic Progenitor Cells.

Hepatocellular carcinoma (HCC) is an aggressive primary liver cancer. However, its origin remains a debated question. Using human data and various hepatocarcinogenesis mouse models, we show that, in early stages, transformed hepatocytes, independent of their proliferation status, activate hepatic progenitor cell (HPC) expansion. Genetic lineage tracing of HPCs and hepatocytes reveals that, in all models, HCC originates from hepatocytes. However, whereas in various models tumors do not emanate from HPCs, tracking of progenitors in a model mimicking human hepatocarcinogenesis indicates that HPCs can generate benign lesions (regenerative nodules and adenomas) and aggressive HCCs. Mechanistically, galectin-3 and α-ketoglutarate paracrine signals emanating from oncogene-expressing hepatocytes instruct HPCs toward HCCs. α-Ketoglutarate preserves an HPC undifferentiated state, and galectin-3 maintains HPC stemness, expansion, and aggressiveness. Pharmacological or genetic blockage of galectin-3 reduces HCC, and its expression in human HCC correlates with poor survival. Our findings may have clinical implications for liver regeneration and HCC therapy.

Metabolic Inflammation-Associated IL-17A Causes Non-alcoholic Steatohepatitis and Hepatocellular Carcinoma.

Obesity increases hepatocellular carcinoma (HCC) risks via unknown mediators. We report that hepatic unconventional prefoldin RPB5 interactor (URI) couples nutrient surpluses to inflammation and non-alcoholic steatohepatitis (NASH), a common cause of HCC. URI-induced DNA damage in hepatocytes triggers inflammation via T helper 17 (Th17) lymphocytes and interleukin 17A (IL-17A). This induces white adipose tissue neutrophil infiltration mediating insulin resistance (IR) and fatty acid release, stored in liver as triglycerides, causing NASH. NASH and subsequently HCC are prevented by pharmacological suppression of Th17 cell differentiation, IL-17A blocking antibodies, and genetic ablation of the IL-17A receptor in myeloid cells. Human hepatitis, fatty liver, and viral hepatitis-associated HCC exhibit increased IL-17A correlating positively with steatosis. IL-17A blockers may prevent IR, NASH, and HCC in high-risk patients.