Characterization of intestinal O-glycome in reactive oxygen species deficiency.

Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation resulting from an inappropriate inflammatory response to intestinal microbes in a genetically susceptible host. Reactive oxygen species (ROS) generated by NADPH oxidases (NOX) provide antimicrobial defense, redox signaling and gut barrier maintenance. NADPH oxidase mutations have been identified in IBD patients, and mucus layer disruption, a critical aspect in IBD pathogenesis, was connected to NOX inactivation. To gain insight into ROS-dependent modification of epithelial glycosylation the colonic and ileal mucin O-glycome of mice with genetic NOX inactivation (Cyba mutant) was analyzed. O-glycans were released from purified murine mucins and analyzed by hydrophilic interaction ultra-performance liquid chromatography in combination with exoglycosidase digestion and mass spectrometry. We identified five novel glycans in ileum and found minor changes in O-glycans in the colon and ileum of Cyba mutant mice. Changes included an increase in glycans with terminal HexNAc and in core 2 glycans with Fuc-Gal- on C3 branch, and a decrease in core 3 glycans in the colon, while the ileum showed increased sialylation and a decrease in sulfated glycans. Our data suggest that NADPH oxidase activity alters the intestinal mucin O-glycans that may contribute to intestinal dysbiosis and chronic inflammation.

Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using Genetically Encoded Calcium Indicators.

Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory activity, actin dynamics, inflammatory responses, stem cell proliferation, and many other uncharacterized cellular functions. As such, mapping calcium signaling dynamics within the intestinal epithelium can provide insight into homeostatic cellular processes and unveil unique responses to various stimuli. Human intestinal organoids (HIOs) are a high-throughput, human-derived model to study the intestinal epithelium and thus represent a useful system to investigate calcium dynamics. This paper describes a protocol to stably transduce HIOs with genetically encoded calcium indicators (GECIs), perform live fluorescence microscopy, and analyze imaging data to meaningfully characterize calcium signals. As a representative example, 3-dimensional HIOs were transduced with lentivirus to stably express GCaMP6s, a green fluorescent protein-based cytosolic GECI. The engineered HIOs were then dispersed into a single-cell suspension and seeded as monolayers. After differentiation, the HIO monolayers were infected with rotavirus and/or treated with drugs known to stimulate a calcium response. An epifluorescence microscope fitted with a temperature-controlled, humidified live-imaging chamber allowed for long-term imaging of infected or drug-treated monolayers. Following imaging, acquired images were analyzed using the freely available analysis software, ImageJ. Overall, this work establishes an adaptable pipeline for characterizing cellular signaling in HIOs.

Utility of Combined Use of Transabdominal Ultrasonography and Fecal Immunochemical Test Examinations in Ulcerative Colitis.

This study examined the utility of the combined use of transabdominal ultrasonography (TUS) and fecal immunochemical testing (FIT) to detect mucosal inflammation, vis-a-vis the Mayo endoscopic subscore (MES), in ulcerative colitis (UC). Sixty-three UC patients who underwent TUS and FIT were retrospectively enrolled. For TUS, the colon was divided into five segments, and the bowel wall thickness was measured and evaluated. The accuracy of FIT (> 100 ng/ml) in detecting mucosal inflammation (MES>0) was 0.93, whereas that of TUS (BWT>2 mm) in each segment was 0.84-0.97. The combined use of TUS and FIT may be helpful in noninvasive treatment strategies.

Evaluation of Mucosal Healing in Ulcerative Colitis by Fecal Calprotectin vs. Fecal Immunochemical Test: A Systematic Review and Meta-analysis.

Mucosal healing has been considered a treatment goal for patients with inflammatory bowel disease. To compare the accuracy of fecal immunochemical test and fecal calprotectin in the judgment of mucosal healing in ulcerative colitis, a meta-analysis was performed. We searched the PubMed, Cochrane Library, Web of Science, and Embase for the studies on fecal immunochemical test and fecal calprotectin predicting mucosal healing in ulcerative colitis. The comprehensive sensitivity, specificity, diagnostic odds ratio, positive likelihood ratio, and negative likelihood ratio were calculated to evaluate the accuracy. By analyzing 22 publications, we found that the combined sensitivity and specificity of fecal immunochemical test were 0.87 (95% CI, 0.80-0.92) and 0.73 (95% CI, 0.62-0.81), respectively. The combined sensitivity and specificity of fecal calprotectin were 0.76 (95% CI, 0.70-0.80) and 0.80 (95% CI, 0.76-0.84), respectively. The area under the curve values of the fecal immunochemical test and fecal calprotectin summary receiver operating characteristic (SROC) curves were 0.88 and 0.85, respectively. Consequently, fecal immunochemical test had higher sensitivity in predicting mucosal healing in ulcerative colitis patients, while fecal calprotectin had higher specificity. Compared with fecal calprotectin, fecal immunochemical test was more accurate in judging mucosal healing in ulcerative colitis.

High-throughput Raman spectroscopy allows ex vivo characterization of murine small intestinal intra-epithelial lymphocytes (IEL).

T cells are considered to be critical drivers of intestinal inflammation in mice and people. The so called intra-epithelial lymphocyte (IEL) compartment largely consist of T cells. Interestingly, the specific regulation and contribution of IELs in the context of inflammatory bowel disease remains poorly understood, in part due to the lack of appropriate analysis tools. Powerful, label-free methods could ultimately provide access to this cell population and hence give valuable insight into IEL biology and even more to their disease-related functionalities. Raman spectroscopy has demonstrated over the last few years its potential for reliable cell characterization and differentiation, but its utility in regard to IEL exploration remains unknown. To address this question experimentally, we utilized a murine, T cell-driven experimental model system which is accepted to model human gut inflammation. Here, we repopulated the small intestinal IEL compartment (SI IELs) of Rag1-deficient mice endogenously lacking T cells by transferring naïve CD4+ T helper cells intraperitoneally. Using multivariate statistical analysis, high-throughput Raman spectroscopy managed to define a cell subpopulation ex vivo within the SI IEL pool of mice previously receiving T cells in vivo that displayed characteristic spectral features of lymphocytes. Raman data sets matched flow cytometry analyses with the latter identifying T cell receptor (TCR)αß+ CD4+ T cell population in SI IELs from T cell-transferred mice, but not from control mice, in an abundance comparable to the one detected by Raman spectroscopy. Hence, in this study, we provide experimental evidence for high-throughput Raman spectroscopy to be a novel, future tool to reliably identify and potentially further characterize the T cell pool of small intestinal IELs ex vivo.

Three-Dimensional-Printed Electrochemical Multiwell Plates for Monitoring Food Intolerance from Intestinal Organoids.

Common symptoms of food intolerance are caused by chemical components within food that have a pharmacological activity to alter the motility of the gastrointestinal tract. Food intolerance is difficult to diagnose as it requires a long-term process of eliminating foods that are responsible for gastrointestinal symptoms. Enterochromaffin (EC) cells are key intestinal epithelium cells that respond to luminal chemical stimulants by releasing 5-HT. Changes in 5-HT levels have been shown to directly alter the motility of the intestinal tract. Therefore, a rapid approach for monitoring the impact of chemicals in food components on 5-HT levels can provide a personalized insight into food intolerance and help stratify diets. Within this study, we developed a three-dimensional (3D)-printed electrochemical multiwell plate to determine changes in 5-HT levels from intestinal organoids that were exposed to varying chemical components found in food. The carbon black/poly-lactic acid (CB/PLA) electrodes had a linear range in physiological concentrations of 5-HT (0.1-2 µM) with a limit of detection of 0.07 µM. The electrodes were stable for monitoring 5-HT overflow from intestinal organoids. Using the electrochemical multiwell plate containing intestinal organoids, increases in 5-HT were observed in the presence of 0.1 mM cinnamaldehyde and 10 mM quercetin but reduction in 5-HT levels was observed in 1 mM sorbitol when compared to control. These changes in the presence of chemicals commonly found in food were verified with ex vivo ileum tissue measurements using chromatography and amperometry with boron-doped diamond electrodes. Overall, our 3D electrochemical multiwell plate measurements with intestinal organoids highlight an approach that can be a high-throughput platform technology for rapid screening of food intolerance to provide personalized nutritional diet.

A quantitative mass spectrometry method to differentiate bovine and ovine heparins from pharmaceutical porcine heparin.

Heparin is a polysaccharide extracted from animal tissues and is used widely as an anticoagulant. In most countries, porcine intestine mucosa is the only legal source for producing heparin. It is challenging to differentiate heparins derived from porcine, ovine and bovine, especially when low amounts of ruminant heparin are adulterated into porcine heparin. Herein, we find that two marker saccharides, ΔUA2S-GlcNS6S-HexA2S (ΔISH) and ΔUA2S-GlcNAc6S (ΔIA), show significant differences in the basic building blocks of porcine heparin obtained from ruminant heparin. A quantitative mass spectrometry (MS) method was then established to selectively monitor these two marker saccharides. By using the ΔISH to ΔIA ratio, porcine heparin adulterated with a low amount of ruminant heparin (10 % ovine heparin or 5 % bovine heparin) can be differentiated. This represents a robust and sensitive method for ensuring the authenticity and safety of heparin drugs.

Efficient Heparin Recovery from Porcine Intestinal Mucosa Using Zeolite Imidazolate Framework-8.

Heparin is one of the most valuable active pharmaceutical ingredients, and it is generally isolated from porcine intestinal mucosa. Traditionally, different types of commercial resins are employed as an adsorbent for heparin uptake; however, using new, less expensive adsorbents has attracted more interest in the past few years to enhance the heparin recovery. Zeolite imidazolate framework-8 (ZIF-8), as a metal-organic framework (MOF) with a high surface area, porosity, and good stability at high temperatures, was selected to examine the heparin recovery. In this research, we demonstrate that ZIF-8 can recover up to ~70% (37 mg g-1) of heparin from porcine intestinal mucosa. A mechanistic study through kinetic and thermodynamic models on the adsorption revealed appropriate surface conditions for the adsorption of heparin molecules. The effect of different variables such as pH and temperature on heparin adsorption was also studied to optimize the recovery. This study is the first to investigate the usage of MOFs for heparin uptake.

Variability in the composition of porcine mucosal heparan sulfates.

Commercial porcine intestinal mucosal heparan sulfate (HS) is a valuable material for research into its biological functions. As it is usually produced as a side-stream of pharmaceutical heparin manufacture, its chemical composition may vary from batch to batch. We analysed the composition and structure of nine batches of HS from the same manufacturer. Statistical analysis of the disaccharide compositions placed these batches in three categories: group A had high GlcNAc and GlcNS, and low GlcN typical of HS; group B had high GlcN and GlcNS, and low GlcNAc; group C had high di- and trisulfated, and low unsulfated and monosulfated disaccharide repeats. These batches could be placed in the same categories based on their 1H NMR spectra and molecular weights. Anticoagulant and growth factor binding activities of these HS batches did not fit within these same groups but were related to the proportions of more highly sulfated disaccharide repeats.

Polyaminated pullulan, a new biodegradable and cationic pullulan derivative for mucosal drug delivery.

AIM: To prepare new polycationic pullulan derivatives exhibiting highly mucoadhesive and sustained drug release properties. METHODS: Hydroxy groups of pullulan were activated with mesyl chloride followed by conjugation with low-molecular weight polyamines. Pullulan-tris(2-aminoethyl)amine (Pul-TAEA) and pullulan-polyethyleneimine (Pul-PEI) were evaluated regarding swelling behaviour, mucoadhesive properties and potential to control drug release. RESULTS: Pul-TAEA and Pul-PEI exhibited excellent swelling properties at pH 6.8 showing 240- and 370-fold increase in weight. Compared to unmodified pullulan, Pul-TAEA and Pul-PEI displayed 5- and 13.3-fold increased dynamic viscosity in mucus. Mucoadhesion studies of Pul-TAEA and Pul-PEI on intestinal mucosa showed a 6- and 37.8-fold increase in tensile strength, and a 72- and 120-fold increase in mucoadhesion time compared to unmodified pullulan, respectively. Due to additional ionic interactions between cationic groups on polyaminated pullulans and an anionic model drug, a sustained drug release was achieved. CONCLUSIONS: Polyaminated pullulans are promising novel mucoadhesive excipients for mucosal drug delivery.

Independent effects of structural optimization and resveratrol functionalization on extracellular matrix scaffolds for bone regeneration.

Due to their natural biological activity and low immunogenicity, decellularized extracellular matrix (ECM) materials have aroused interest as potential scaffold materials in tissue engineering. Decellularized small intestinal submucosa (SIS) is one ECM biomaterial that can be easily sourced. In the present study, we tested whether the osteogenesis of SIS scaffolds was enhanced via structural optimization and resveratrol (RSV) functionalization and explored the independent effects of these modifications. We obtained SIS scaffolds with different pore structures by controlling the preparation concentration. The group with superior osteogenic properties was further RSV-functionalized via covalent immobilization. We conducted a series of in vitro and in vivo studies to explore the effects of these two optimization strategies on the osteogenic properties of SIS scaffolds. The results showed that pore structure and RSV functionalization significantly affected the osteogenic properties of SIS scaffolds. With a fabrication concentration of 1%, the SIS scaffolds had superior osteogenic properties. Through covalent coupling, RSV was successfully grafted onto SIS scaffolds, where it was slowly released. The most significant improvements in osteogenic properties were obtained with a coupling concentration of 1%. Furthermore, in in vivo experiments, vascular and new bone tissue formation was enhanced with RSV/SIS scaffolds compared with SIS scaffolds and the blank control group at 4 weeks after implantation. These findings indicate that the RSV/SIS scaffolds obtained via dual optimization strategies show promise as biomaterials in bone tissue engineering.

Impact of Intestinal Concentration and Colloidal Structure on the Permeation-Enhancing Efficiency of Sodium Caprate in the Rat.

In this work, we set out to better understand how the permeation enhancer sodium caprate (C10) influences the intestinal absorption of macromolecules. FITC-dextran 4000 (FD4) was selected as a model compound and formulated with 50-300 mM C10. Absorption was studied after bolus instillation of liquid formulation to the duodenum of anesthetized rats and intravenously as a reference, whereafter plasma samples were taken and analyzed for FD4 content. It was found that the AUC and Cmax of FD4 increased with increasing C10 concentration. Higher C10 concentrations were associated with an increased and extended absorption but also increased epithelial damage. Depending on the C10 concentration, the intestinal epithelium showed significant recovery already at 60-120 min after administration. At the highest studied C10 concentrations (100 and 300 mM), the absorption of FD4 was not affected by the colloidal structures of C10, with similar absorption obtained when C10 was administered as micelles (pH 8.5) and as vesicles (pH 6.5). In contrast, the FD4 absorption was lower when C10 was administered at 50 mM formulated as micelles as compared to vesicles. Intestinal dilution of C10 and FD4 revealed a trend of decreasing FD4 absorption with increasing intestinal dilution. However, the effect was smaller than that of altering the total administered C10 dose. Absorption was similar when the formulations were prepared in simulated intestinal fluids containing mixed micelles of bile salts and phospholipids and in simple buffer solution. The findings in this study suggest that in order to optimally enhance the absorption of macromolecules, high (≥100 mM) initial intestinal C10 concentrations are likely needed and that both the concentration and total dose of C10 are important parameters.

Patient-Reported Outcome and Clinical Scores Are Equally Accurate in Predicting Mucosal Healing in Ulcerative Colitis: A Prospective Study.

BACKGROUND: Optimal management of patients with ulcerative colitis (UC) requires the accurate, objective assessment of disease activity. AIMS: We aimed to determine how strong patient-reported outcomes, clinical scores and symptoms correlate with endoscopy and biomarkers for assessment of disease activity in patients with UC. METHODS: Consecutive patients with UC followed at the McGill University IBD Center and referred for endoscopy (surveillance or flare) were included prospectively between September 2018 and August 2020. Patient-reported outcome (PRO2), partial Mayo, Simple Clinical Colitis Activity Index (SCCAI), Mayo endoscopic subscore (MES) and Baron and Ulcerative Colitis Endoscopic Index of Severity (UCEIS) scores were calculated. C-reactive protein (CRP) and fecal calprotectin (FCAL) were collected. RESULTS: A total of 171 patients with UC [age: 49(IQR:38-61) years, female: 46.2%, 57.3% extensive disease, 42.7% on biologicals] were included prospectively. Rectal bleeding (RBS), stool frequency (SF) subscore of 0, or total PRO2 remission (RBS0 and SF ≤ 1), partial Mayo (≤ 2) and SCCAI (≤ 2.5) remission were similarly associated with mucosal healing defined by MES (0 or ≤ 1), Baron (0 or ≤ 1) or UCEIS (≤ 3) scores in ROC analysis (AUC:0.93-0.72). There was a moderate-to-strong agreement between MES Baron and UCEIS (K = 0.91-0.41). A UCEIS of ≤ 3 was identified as the best cutoff to clinical or endoscopic remission. Agreement between CRP and clinical remission or endoscopic healing (MES/Baron) was poor (K ~ 0.2), while agreement between FCAL and RBS-PRO2 or MES/Baron/UCEIS was moderate to strong (K = 0.44-0.70). CONCLUSIONS: Agreement between RBS, SF, PRO2, partial Mayo and SCCAI in predicting endoscopic healing was moderate to strong, while no clinically meaningful difference was found in accuracy across the scores and definitions. FCAL, but not CRP, was associated to clinical and endoscopic remission.

Fatty acid in colorectal cancer in adult and aged patients of both sexes.

PURPOSE: Colorectal cancer represents the second most common type of cancer in Serbia. Alteration of lipid metabolism begins early, and can represent a central hallmark in cancer evolution. Fatty acids have various important functions as building components of cell membranes, as signaling molecules in immune responses and also manage the general cancer signaling network. The purpose of this study was to investigate the difference of various fatty acids content between colorectal cancer and adjacent healthy intestinal tissue in adult and aged patients of both sexes. METHODS: 52 subjects participated in this study. Healthy colon mucosa and tumor tissue samples were obtained from patients previously diagnosed with colorectal carcinoma. Simplified method of Berstad et al was used for direct transesterification of total lipids in tumor and healthy mucosa tissue samples and separations of the methyl esters was carried out using a gas chromatograph equipped with a split/splitless injector and a flame ionization detector. RESULTS: 18 0, 18 1 n7, 20 3, 20 4, 20 5, 22 4, 22 5 22 6, SFA, PUFA, n6, n3 and AA/EPA were significantly higher in tumor tissue. On the other hand, 18 1 n9, 18 2, 18 3 n3, MUFA, n6/n3 were significantly higher in healthy tissue. CONCLUSIONS: Saturation index (SI) could be a valuable tool to delineate robust immune response and worse prognosis in patients with colorectal cancer. Our study demonstrated significant differences in fatty acid profiles between tumor tissue and healthy mucosa. Parameters, such as gender, age, stage and mucinous component didn't influence altered fatty acid content.

Highly sialylated mucin-type glycopeptide from porcine intestinal mucosa after heparin extraction: O-glycan profiling and immunological activity evaluation.

Mucins are the major proteins that distributed on the intestinal mucosa layer and protect the intestine from pathogens infection. The composition of intestinal mucin O-glycans can affect the health of the gastrointestinal tract in pigs. Porcine intestinal mucosa is widely used as the main raw material of heparin extraction. The heparin extraction residues rich in mucins were usually wasted. The structure of mucin derived O-glycans in porcine intestinal mucosa are currently unknown. In this study, we isolated the mucins from the heparin extraction residues and profiled the O-glycans. After heparin extraction, mucin was digested with trypsin, and separated by strong anion exchange chromatography. The mucin derived O-glycans were release by alkaline ß elimination, and analyzed by ultra high performance liquid chromatography-porous graphitized carbon-Fourier transform mass spectrometry (UPLC-PGC-FTMS/MS). Thirty five kinds of O-glycans were identified, most of which were Core 3-derived glycans. In particular, the O-glycans containing sialic acid Neu5Ac accounted for 71.93% of the total O-glycans, which were different from that of other species, including mouse intestine, fish intestine, and porcine colon. The high content sialylated mucin may explain its effect in biological processes. Furthermore, the immunological activity results indicated that the porcine intestinal mucin could promote phagocytosis and proliferation without any cytotoxic effects, which may aid in the development of immunomodulators.

A biomimetic hierarchical small intestinal submucosa-chitosan sponge/chitosan hydrogel scaffold with a micro/nano structure for dural repair.

The dura mater is an essential barrier to protect the brain tissue and the dural defects caused by accidents can lead to serious complications. Various materials have been applied to dural repair, but it remains a challenge to perfectly match the structure and properties of the natural dura mater. Small intestinal submucosa has been developed for dural repair because of its excellent biocompatibility and biological activity, but its application is tremendously limited by the rapid degradation rate. Chitosan has also been broadly investigated in tissue repair, but the traditional chitosan hydrogels exhibit poor mechanical properties. A nanofiber chitosan hydrogel can be constructed based on an alkaline solvent, which is equipped with surprisingly high strength. Therefore, based on the bilayer structure of the natural dura mater, a biomimetic hierarchical small intestinal submucosa-chitosan sponge/chitosan hydrogel scaffold with a micro/nano structure was fabricated, which possessed a microporous structure in the upper sponge and a nanofiber structure in the lower hydrogel. The degradation rate was remarkably reduced compared with that of the small intestinal submucosa in the enzymatic degradation experiment in vitro. Meanwhile, the chitosan nanofibers brought high mechanical strength to the bilayer scaffold. Moreover, the hierarchical micro/nano structure and the active factors in the small intestinal submucosa have a fantastic effect on promoting the proliferation of fibroblasts and vascular endothelial cells. The bilayer scaffold showed good histocompatibility in the experiment of in vitro subcutaneous implantation in rats. Thus, the biomimetic hierarchical small intestinal submucosa-chitosan sponge/chitosan hydrogel scaffold with micro/nano structure simulates the structure of the natural dura mater and possesses properties with excellent performance, which has high practical value for dural repair.

miR-144-3p inhibited the growth, metastasis and epithelial-mesenchymal transition of colorectal adenocarcinoma by targeting ZEB1/2.

miR-144-3p is aberrantly expressed in several types of human cancer and functions as a tumor suppressor by inhibiting metastasis. However, the clinical significance and biological function of miR-144-3p in colorectal adenocarcinoma (CRA) have yet to be elucidated. Here we reported that miR-144-3p expression level was significantly down-regulated in CRA tissues compared with matched noncancerous colorectal mucosae tissues. Low miR-144-3p expression was correlated with adverse clinicopathologic characteristics and poor prognosis of CRA patients. Cox regression analysis showed that low miR-144-3p expression was an independent risk factor for DFS and OS in CRA. In vitro and in vivo assays showed that miR-144-3p significantly inhibited proliferation, migration and invasion of CRA cells. In particular, miR-144-3p could suppress EMT process of CRA cells by regulating the cytoskeleton and EMT markers. Bioinformatics analysis indicated that EMT associated transcription factors ZEB1 and ZEB2 were potential targets of miR-144-3p, and miR-144-3p inhibited ZEB1 and ZEB2 expression and was negatively correlated with their expression in CRA. Finally, we confirmed that ZEB1 and ZEB2 down-regulation collaboratively mediated the inhibitory effect of miR-144-3p on proliferation, invasion and EMT of CRA cells. In conclusion, our study provided evidence that miR-144-3p could inhibit CRA cell proliferation, invasion and EMT by targeting ZEB1/2.

HIV infection drives interferon signaling within intestinal SARS-CoV-2 target cells.

SARS-CoV-2 infects epithelial cells of the human gastrointestinal (GI) tract and causes related symptoms. HIV infection impairs gut homeostasis and is associated with an increased risk of COVID-19 fatality. To investigate the potential link between these observations, we analyzed single-cell transcriptional profiles and SARS-CoV-2 entry receptor expression across lymphoid and mucosal human tissue from chronically HIV-infected individuals and uninfected controls. Absorptive gut enterocytes displayed the highest coexpression of SARS-CoV-2 receptors ACE2, TMPRSS2, and TMPRSS4, of which ACE2 expression was associated with canonical interferon response and antiviral genes. Chronic treated HIV infection was associated with a clear antiviral response in gut enterocytes and, unexpectedly, with a substantial reduction of ACE2 and TMPRSS2 target cells. Gut tissue from SARS-CoV-2-infected individuals, however, showed abundant SARS-CoV-2 nucleocapsid protein in both the large and small intestine, including an HIV-coinfected individual. Thus, upregulation of antiviral response genes and downregulation of ACE2 and TMPRSS2 in the GI tract of HIV-infected individuals does not prevent SARS-CoV-2 infection in this compartment. The impact of these HIV-associated intestinal mucosal changes on SARS-CoV-2 infection dynamics, disease severity, and vaccine responses remains unclear and requires further investigation.

Ultra-high Magnification Endocytoscopy and Molecular Markers for Defining Endoscopic and Histologic Remission in Ulcerative Colitis-An Exploratory Study to Define Deep Remission.

BACKGROUND: Endoscopic and histological remission are both important treatment goals in patients with ulcerative colitis (UC). We aimed to define cellular architecture, expression of molecular markers, and their correlation with endoscopic scores assessed by ultra-high magnification endocytoscopy (ECS) and histological scores. METHODS: Patients with UC (n = 29) were prospectively recruited. The correlation among ECS score (ECSS), Mayo endoscopic score (MES), and histological scores were determined. Area under curve were plotted to determine the best thresholds for ECSS that predicted histological remission by Robarts (RHI) and Nancy Histological Index (NHI).Soluble analytes relevant to inflammation were measured in serum and mucosal culture supernatants using ProcartaPlex Luminex assays and studied by partial least square discriminant analysis and logistic model. Mucosal RNA sequencing and bioinformatics analysis were performed to define differentially expressed genes/pathways. RESULTS: Endocytoscope scoring system correlated strongly with RHI (r = 0.89; 95% CI, 0.51-0.98) and NHI (r = 0.86; 95% CI, 0.42-0.98) but correlated poorly with MES (r = 0.28; 95% CI, 0.27-0.70). We identified soluble brain-derived neurotrophic factors (BDNF), macrophage inflammatory proteins (MIP-1 α) and soluble vascular cell adhesion molecule 1 (sVCAM-1) predicted histological remission. Mucosal biopsy cultures also identified sVCAM-1 associated with healed mucosa. RNA-seq analysis identified gene expressions shared between ECSS, RHI, or NHI defined healing. A number of gene expressions and pathways were identified including inflammation and metabolic and tumor suppressors that discriminated healed from nonhealed mucosa. CONCLUSIONS: Endocytoscopy represents an interesting tool that may sit between endoscopy and histology-but closer to the latter-identifying gene expression markers and pathways that are also identified by histology.

HER2 and APC Mutations Promote Altered Crypt-Villus Morphology and Marked Hyperplasia in the Intestinal Epithelium.

BACKGROUND AND AIMS: The Cancer Genome Atlas (TCGA) project has identified HER2 mutations or amplification in 7% of colon cancers. In addition to HER2 mutations, colon cancer patients also possess co-occurring mutations in genes such as APC. Here, we investigated the role of HER2 and APC mutations on the crypt-villus architecture of the intestinal epithelium, localization of secretory cells, and expression of intestinal stem cell markers. METHODS: We generated a HER2 transgenic mouse (HER2V777L Tg) possessing an activating mutation commonly found in colorectal cancer patients, HER2V777L, using transcription activator-like effector nucleases-based gene editing technology. We expressed the HER2V777L transgene in mouse small intestine and colon using Lgr5-Cre and Villin-Cre recombinases. In addition, we analyzed Lgr5-Cre; APCmin; HER2V777L Tg mice by morphologic and gene expression assays on intestinal sections and organoids derived from the epithelium. RESULTS: HER2V777L expression resulted in hypertrophic crypt formation with expanded zones of proliferation. Proximal intestinal villi showed increased abundance of multiple differentiated lineages including extensive intermediate cell differentiation, as evidenced by MUC2/MMP7 co-immunofluorescence and transmission electron microscopy. HER2V777L expression in the context of APC loss resulted in further enhancement and expansion of the proliferative crypt compartment. CONCLUSIONS: We established an epithelial intrinsic role for HER2V777L on enhanced cellular proliferation. Additionally, we determined that HER2 and APC mutations, when combined, promote enhanced proliferation of intestinal crypts.