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Summary: Single-cell isolation techniques allow the investigation of physical and functional relationships between individual cells within a complex cell population. Here, we present a protocol for single-cell isolation from full-thickness intestinal tissue resections. We describe steps for pre-processing specimens, isolation of lamina propria and muscular layers, and red blood cell lysis. We then detail fixation of isolated cells and assessment of cell quality. The resulting cell suspension can be subjected to RNA sequencing on the 10× Chromium platform.For complete details on the use and execution of this protocol, please refer to Mukherjee et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

We aimed to test how the postbiotic butyrate impacts select gut bacteria, small intestinal epithelial integrity, and microvascular endothelial activation during acute ethanol exposure in mice and primary human intestinal microvascular endothelial cells (HIMECs). Supplementation during an acute ethanol challenge with or without tributyrin, a butyrate prodrug, was delivered to C57BL/6 mice. A separate group of mice received 3 days of clindamycin prior to the acute ethanol challenge. Upon euthanasia, blood endotoxin, cecal bacteria, jejunal barrier integrity, and small intestinal lamina propria dendritic cells were assessed. HIMECs were tested for activation following exposure to ethanol ± lipopolysaccharide (LPS) and sodium butyrate. Tributyrin supplementation protected a butyrate-generating microbe during ethanol and antibiotic exposure. Tributyrin rescued ethanol-induced disruption in jejunal epithelial barrier, elevated plasma endotoxin, and increased mucosal vascular addressin cell-adhesion molecule-1 (MAdCAM-1) expression in intestinal microvascular endothelium. These protective effects of tributyrin coincided with a tolerogenic dendritic response in the intestinal lamina propria. Lastly, sodium butyrate pre- and co-treatment attenuated the direct effects of ethanol and LPS on MAdCAM-1 induction in the HIMECs from a patient with ulcerative colitis. Tributyrin supplementation protects small intestinal epithelial and microvascular barrier integrity and modulates microvascular endothelial activation and dendritic tolerizing function during a state of gut dysbiosis and acute ethanol challenge.

The recent advent of sophisticated technologies like sequencing and mass spectroscopy platforms combined with artificial intelligence-powered analytic tools has initiated a new era of “big data” research in various complex diseases of still-undetermined cause and mechanisms. The investigation of these diseases was, until recently, limited to traditional in vitro and in vivo biological experimentation, but a clear switch to in silico methodologies is now under way. This review tries to provide a comprehensive assessment of state-of-the-art knowledge on omes, omics and multi-omics in inflammatory bowel disease (IBD). The notion and importance of omes, omics and multi-omics in both health and complex diseases like IBD is introduced, followed by a discussion of the various omics believed to be relevant to IBD pathogenesis, and how multi-omics “big data” can generate new insights translatable into useful clinical tools in IBD such as biomarker identification, prediction of remission and relapse, response to therapy, and precision medicine. The pitfalls and limitations of current IBD multi-omics studies are critically analyzed, revealing that, regardless of the types of omes being analyzed, the majority of current reports are still based on simple associations of descriptive retrospective data from cross-sectional patient cohorts rather than more powerful longitudinally collected prospective datasets. Given this limitation, some suggestions are provided on how IBD multi-omics data may be optimized for greater clinical and therapeutic benefit. The review concludes by forecasting the upcoming incorporation of multi-omics analyses in the routine management of IBD.

During the last few decades, major progress has been made in the treatment of the two major forms of inflammatory bowel disease (IBD): Crohn’s disease and ulcerative colitis. However, the success of the most advanced forms of therapy is at best 50%, and most patients lose responsiveness with time and need to switch to alternative medications. This denotes that a therapeutic ceiling has been reached and brand new approaches are badly needed. This commentary first briefly reviews current and recent therapeutic approaches for Crohn’s disease and ulcerative colitis, pointing out their limitations. This is followed by an objective evidence-based discussion of why the current approaches are far from optimal, and the commentary is concluded by proposing how to change IBD treatment based on the holistic concept of network medicine, and how to implement precision medicine for IBD using artificial intelligence-based multi-omics analyses.

The global coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to unprecedented social and economic consequences. The risk of morbidity and mortality due to COVID-19 increases dramatically in the presence of coexisting medical conditions, while the underlying mechanisms remain unclear. Furthermore, there are no approved therapies for COVID-19. This study aims to identify SARS-CoV-2 pathogenesis, disease manifestations, and COVID-19 therapies using network medicine methodologies along with clinical and multi-omics observations. We incorporate SARS-CoV-2 virus-host protein-protein interactions, transcriptomics, and proteomics into the human interactome. Network proximity measurement revealed underlying pathogenesis for broad COVID-19-associated disease manifestations. Analyses of single-cell RNA sequencing data show that co-expression of ACE2 and TMPRSS2 is elevated in absorptive enterocytes from the inflamed ileal tissues of Crohn disease patients compared to uninflamed tissues, revealing shared pathobiology between COVID-19 and inflammatory bowel disease. Integrative analyses of metabolomics and transcriptomics (bulk and single-cell) data from asthma patients indicate that COVID-19 shares an intermediate inflammatory molecular profile with asthma (including IRAK3 and ADRB2). To prioritize potential treatments, we combined network-based prediction and a propensity score (PS) matching observational study of 26,779 individuals from a COVID-19 registry. We identified that melatonin usage (odds ratio [OR] = 0.72, 95% CI 0.56-0.91) is significantly associated with a 28% reduced likelihood of a positive laboratory test result for SARS-CoV-2 confirmed by reverse transcription-polymerase chain reaction assay. Using a PS matching user active comparator design, we determined that melatonin usage was associated with a reduced likelihood of SARS-CoV-2 positive test result compared to use of angiotensin II receptor blockers (OR = 0.70, 95% CI 0.54-0.92) or angiotensin-converting enzyme inhibitors (OR = 0.69, 95% CI 0.52-0.90). Importantly, melatonin usage (OR = 0.48, 95% CI 0.31-0.75) is associated with a 52% reduced likelihood of a positive laboratory test result for SARS-CoV-2 in African Americans after adjusting for age, sex, race, smoking history, and various disease comorbidities using PS matching. In summary, this study presents an integrative network medicine platform for predicting disease manifestations associated with COVID-19 and identifying melatonin for potential prevention and treatment of COVID-19.

Background & Aims: Crohnâs Disease (CD) is a chronic inflammatory disease of the gastrointestinal tract. Fibrosis, a serious complication of CD, occurs when activated intestinal fibroblasts deposit excessive amounts of extracellular matrix (ECM) in affected areas. A major component of the ECM is high-molecular-weight hyaluronan (HA) that, when depolymerized to low-molecular-weight fragments, becomes proinflammatory and profibrotic. Mechanisms for HA degradation are incompletely understood, but the novel protein KIAA1199 recently was discovered to degrade HA. We hypothesized that KIAA1199 protein is increased in CD colon fibroblasts and generates HA fragments that foster inflammation and fibrosis. Methods: Fibroblasts were isolated from explants of surgically resected colon tissue from CD and nonâinflammatory bowel disease control (ND) patients. Protein levels and tissue distribution of KIAA1199 were assessed by immunoblot and immunostaining, and functional HA degradation was measured biochemically. Results: Increased levels of KIAA1199 protein were produced and deposited in the ECM by cultured CD fibroblasts compared with controls. Treatment of fibroblasts with the proinflammatory cytokine interleukin (IL) 6 increased deposition of KIAA1199 in the ECM. CD fibroblasts also produce significantly higher levels of IL6 compared with controls, and antibody blockade of IL6 receptors in CD colon fibroblasts decreased the level of KIAA1199 protein in the ECM. Colon fibroblasts degrade HA, however, small interfering RNA silencing of KIAA1199 abrogated that ability. Conclusions: CD fibroblasts produce increased levels of KIAA1199 primarily through an IL6-driven autocrine mechanism. This leads to excessive degradation of HA and the generation of proinflammatory HA fragments, which contributes to maintenance of gut inflammation and fibrosis. Keywords: Crohnâs Disease, Fibrosis, Hyaluronan, KIAA1199

Background & Aims: Cathelicidin (LL-37 in human and mCRAMP in mice) represents a family of endogenous antimicrobial peptides with anti-inflammatory effects. LL-37 also suppresses collagen synthesis, an important fibrotic response, in dermal fibroblasts. Here, we determined whether exogenous cathelicidin administration modulates intestinal fibrosis in two animal models of intestinal inflammation and in human colonic fibroblasts. Methods: C57BL/6J mice (n = 6 per group) were administered intracolonically with a trinitrobenzene sulphonic acid (TNBS) enema to induce chronic (6â7 weeks) colitis with fibrosis. We administered mCRAMP peptide (5 mg/kg every 3 day, week 5â7) or cathelicidin gene (Camp)-expressing lentivirus (107 infectious units week 4) intracolonically or intravenously, respectively. We then infected 129Sv/J mice with Salmonella typhimurium orally to induce cecal inflammation with fibrosis. Camp-expressing lentivirus (107 infectious units day 11) was administered intravenously. Results: TNBS-induced chronic colitis was associated with increased colonic collagen (col1a2) mRNA expression. Intracolonic cathelicidin (mCRAMP peptide) administration or intravenous delivery of lentivirus-overexpressing cathelicidin gene significantly reduced colonic col1a2 mRNA expression in TNBS-exposed mice compared with vehicle administration. Salmonella infection also caused increased cecal inflammation associated with collagen (col1a2) mRNA expression that was prevented by intravenous delivery of Camp-expressing lentivirus. Exposure of human primary intestinal fibroblasts and human colonic CCD-18Co fibroblasts to transforming growth factor-β1 (TGF-β1) and/or insulin-like growth factor 1 induced collagen protein and mRNA expression, which was reduced by LL-37 (3â5 μM) through a MAP kinase-dependent mechanism. Conclusions: Cathelicidin can reverse intestinal fibrosis by directly inhibiting collagen synthesis in colonic fibroblasts. Keywords: Antimicrobial Peptide, Collagen, Inflammatory Bowel Disease

The pathogenesis of Crohn's disease (CD) and ulcerative colitis (UC), the two main forms of inflammatory bowel disease (IBD), is still unclear, but both autoimmune and immune-mediated phenomena are involved. Autoimmune phenomena include the presence of serum and mucosal autoantibodies against intestinal epithelial cells in either form of IBD, and against human tropomyosin fraction five selectively in UC. In addition, perinuclear antineutrophil cytoplasmic antibodies (pANCA) are common in UC, whereas antibodies against Saccharomyces cerevisiae (ASCA) are frequently found in CD. Immune-mediate phenomena include a variety of abnormalities of humoral and cell-mediated immunity, and a generalized enhanced reactivity against intestinal bacterial antigens in both CD and UC. It is currently believed that loss of tolerance against the indigenous enteric flora is the central event in IBD pathogenesis. Various complementary factors probably contribute to the loss of tolerance to commensal bacteria in IBD. They include defects in regulatory T-cell function, excessive stimulation of mucosal dendritic cells, infections or variants of proteins critically involved in bacterial antigen recognition, such as the products of CD-associated NOD2/CARD15 mutations.

The precise etiology and mechanisms of inflammatory bowel disease (IBD) are still unclear. Nevertheless, several concepts are gaining acceptance and constitute the basis for a better understanding of its pathogenesis and for improved therapy. The association of Crohn’s disease (CD) and ulcerative colitis (UC) with ‘western’ lifestyle is well recognized, and is considered a reason for the increasing frequency of CD and UC in countries with previously low incidence. Proposed linkages of CD and UC with particular human leukocyte antigen haplotypes suggest a genetic predisposition, but no uniform or consistent patterns have emerged. Similarly, the study of susceptibility or disease markers has not offered reproducible results. The search for specific infectious agents is being pursued, and the measles virus is presently considered a possible culprit. A true explosion has occurred in the area of animal models, and a large number of chemically or genetically induced experimental colitides are at hand. Immunological factors continue to dominate the bulk of basic research in IBD. This area is vast and complex, and autoantibodies, immune, epithelial and mesenchymal cells, lipid mediators, cytokines, neuropeptides and oxygen metabolites are under investigation. Finally, other factors whose role in IBD is uncertain, including smoking and possible abnormalities of intestinal permeability or mucus composition, continue to receive attention. These extensive and varied efforts are yielding some profits, and new forms of therapy are being devised. Based mostly on studies of soluble mediators, a number of novel immunosuppressive and highly specific blocking agents are being developed and undergoing clinical trials.

In order to show the complexity of translating from Latin to French in Middle-Ages, this article focuses on the term libertas. The pivotal question for medieval translators is how to render the different meanings (theological, ethical, political) of libertas. Three medieval authors as Denis Foulechat, Evrart de Tremaugon and Nicole Oresme find personal solutions, handling the French of their own age. But are their personal choices consistent with the meaning of the word that they are translating? A creative translator can help his readers and enrich the language they speak, but he can also alter what he is translating

Probiotics appear to be beneficial in inflammatory bowel disease, but their mechanism of action is incompletely understood. We investigated whether probiotic-derived sphingomyelinase mediates this beneficial effect.Neutral sphingomyelinase (NSMase) activity was measured in sonicates of the probiotic L. brevis (LB) and S. thermophilus (ST) and the non-probiotic E. coli (EC) and E. faecalis (EF). Lamina propria mononuclear cells (LPMC) were obtained from patients with Crohn's disease (CD) and Ulcerative Colitis (UC), and peripheral blood mononuclear cells (PBMC) from healthy volunteers, analysing LPMC and PBMC apoptosis susceptibility, reactive oxygen species (ROS) generation and JNK activation. In some experiments, sonicates were preincubated with GSH or GW4869, a specific NSMase inhibitor. NSMase activity of LB and ST was 10-fold that of EC and EF sonicates. LB and ST sonicates induced significantly more apoptosis of CD and UC than control LPMC, whereas EC and EF sonicates failed to induce apoptosis. Pre-stimulation with anti-CD3/CD28 induced a significant and time-dependent increase in LB-induced apoptosis of LPMC and PBMC. Exposure to LB sonicates resulted in JNK activation and ROS production by LPMC. NSMase activity of LB sonicates was completely abrogated by GW4869, causing a dose-dependent reduction of LB-induced apoptosis. LB and ST selectively induced immune cell apoptosis, an effect dependent on the degree of cell activation and mediated by bacterial NSMase.These results suggest that induction of immune cell apoptosis is a mechanism of action of some probiotics, and that NSMase-mediated ceramide generation contributes to the therapeutic effects of probiotics.

Quando si usa il termine laico, occorre sempre rispondere ad alcune domande implicite: laico rispetto a chi? Quando? Un percorso tra le varie classificazioni che si possono trovare nei testi medievali. Nella convinzione che la struttura stessa della classificazione sia importante, l'articolo procede sulla base di un criterio quantitativo, facendo riferimento al numero di classi o generi identificati, a partire dalla divisione più semplice. Una passaggio dal semplice al complesso alla luce della centralità del binomio concettuale di unità-differenza. When we use the term laico, we must ever answer to some implicit questions: laico compared to whom? When? A path among the various classifications that we can find in medieval texts. Persuaded that the very structure of the classification is important, the paper goes on, according to a quantitative criterion, by reference to the number of identified classes or genres, starting from the simplest partition. A transition from the simple to the complex in the light of the centrality of two-faced concept of unit-difference.

Necessità pratiche e riflessioni teoriche accompagnano il lavoro di Oresme, nella consapevolezza che la traduzione delle grandi opere è uno strumento fondamentale per la grandezza di un popolo.

L'articolo intende evidenziare la strategia adottata da Ildegarda di Bingen per divulgare le visioni ricevute. Si mette allora in luce la strada dell'annullamento della personalità che riduce Ildegarda a uno specchio che deve riflettere le parole e le immagini di cui è stata testimone. L'esperienza di Ildegarda si dimostra essenzialmente visiva e parzialmente auditiva, con rarissimi accenni alla percezione di altri sensi.L'analisi del testo mette in evidenza una profonda sfiducia nella parola umana ritenuta strutturalmente incapace di inadatta a comunicare l'esperienza visionaria. Per questo motivo assume importanza l'adozione di immagini miniate, disegnate dalla stessa Ildegarda, al fine di supplire ai limiti del linguaggio degli uomini.