bluuebunny/crossref_metadata_2025_split huggingface dataset:
ELEVATED ADENOMATOUS POLYPOSIS COLI IN GOBLET CELLS IS ASSOCIATED WITH INFLAMMATION IN MOUSE AND HUMAN COLON
Authors: Gomez Christian , Neufeld Kristi 
Published: January 2021

Abstract
Adenomatous Polyposis Coli (APC) functions as an essential colon tumor suppressor. Roles for APC in other disease states such as Inflammatory Bowel Disease (IBD) remain less defined. In the process of characterizing Apc protein in gastrointestinal tissues from human patients with active IBD, we found a subset of goblet cells with elevated Apc staining intensity. While normal colon tissue was only sparsely populated with these “APChigh” goblet cells, tissue from patients with Ulcerative Colitis and Crohn’s Disease contained entire regions where almost every goblet cell was APChigh. In the descending colon, the APChigh goblet cells were prominent in patients with active Ulcerative Colitis but were mostly lacking in normal samples. In mice, APChigh cells were observed only in the small intestines and proximal/medial colons. Upon induction of colitis with Dextran Sodium Sulfate (DSS), the APChigh goblet cells remained in the proximal and medial colon, but also appeared in the distal colon. To better understand the link between APC expression, goblet cells, and inflammation, cultured human colon cells derived from normal tissue were depleted of APC and RNAseq analysis was performed. APC depletion resulted in reduced expression of mRNAs encoding IL1 signaling pathway components IL1b and IL1R, known regulators of Muc2 expression. Muc2 is the main constituent of goblet cell mucus, which is secreted to form a protective barrier for the epithelial cells lining the large intestine. We found that treating cancer cells lacking wild-type APC with IL1b resulted in increased IL1R and MUC2 expression. Induction of full-length APC in these cells led to a similar phenotype. Combining IL1β treatment with APC induction led to an increase of MUC2 expression greater than expected for additive affects, suggesting that APC sensitized these cells to IL1 signaling. These results illuminate a novel role for APC in goblet cells under conditions of inflammation. We postulate that the higher amount of APC in a subset of goblet cells might sensitize them to IL-1 signaling. During times of inflammation, such as active Ulcerative Colitis, goblet cells with higher APC levels would be more sensitive to IL-1-induced MUC2 expression than other goblet cells, perhaps protecting the intestinal lining from further barrier breaches.
INCREASED CATHEPSIN EXPRESSION CORRELATES WITH SARS-COV-2 INFECTION IN HUMAN IBD ENTEROIDS
Authors: Alvarado David , Castro Maria Gomez , Sonnek Naomi , Cui Xueyang , Ding Siyuan , Ciorba Matthew 
Published: January 2021

Abstract

Introduction
Coronavirus Disease 2019 (COVID-19) is an ongoing public health crisis that has sickened or precipitated death in millions. The etiologic agent of COVID-19, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), infects the intestinal epithelium, with viral RNA shed in the stool, and can induce GI symptoms similar to the human inflammatory bowel diseases (IBD). An international surveillance epidemiology study, SECURE-IBD, reported that the standardized mortality ratio trends higher in IBD patients (1.5–1.8) and that 5-aminosalicylic acid (5-ASA) therapy correlates with poor outcome. Together these data indicate patients with IBD may represent a particularly vulnerable population during this COVID-19 pandemic.

Methods
Published datasets GSE75214 and GSE16879 were downloaded and expression levels of select genes were querried using RStudio. Primary human ileal spheroids (enteroids), derived from healthy donors and patients with Crohn’s disease (CD), were grown on 2D transwells until confluent. Cells were then differentiated for 3d before infection with a modified vesicular stomatitis virus expressing the SARS-CoV-2 spike protein (VSV-SARS-CoV-2) and green fluorescent protein (GFP) for 1 h at a multiplicity of infection (MOI) of ~0.5. Healthy enteroids were treated with 10 ng/ml of human Tumor Necrosis Factor alpha (TNF-α) for 24h before infection via the basolateral reservoir or 5-ASA 5h before infection via the apical reservoir. 24h after infection, cells were processed for immunofluorescence or RNA expression of select genes by qRT-PCR.

Results
VSV-SARS-CoV-2 was able to infect both healthy and CD enteroids as determined by co-staining of GFP, indicative of virus infection, and the viral receptor ACE2. However, levels of GFP fluorescence did not correlate with ACE2 expression in CD enteroids. A subset of CD enteroids exhibited enhanced protease expression (TMPRSS2, TMPRSS4, CTSL), each of which correlated with higher viral RNA levels (P=0.04, P=0.002, P=0.006, respectively). In Vero E6 cells, 5-ASA inhibited the replication of a clinical isolate of SARS-CoV-2 in a concentration-dependent manner. Treating healthy enteroids with 5-ASA did not have any effect on viral proliferation, while TNF-α pretreatment reduced viral RNA. 5-ASA treatment caused a reduction of ACE2 and an increase in CTSL expression.

Conclusions
Host proteases, particularly the lysosomal protease CTSL, contribute to the infection of CD enteroids and may represent novel therapeutic targets in patients with IBD and COVID-19. 5-ASA modulates the expression of several epithelial genes relevant to SARS-CoV-2 infection, yet does not alter viral replication in healthy enteroids.
PROTON PUMP INHIBITORS (PPI) INDUCES COLONIC TIGHT JUNCTION BARRIER (TJ) DYSFUNCTION VIA AN UPREGULATION OF TJ PORE FORMING CALUDIN-2 PROTEIN
Authors: Nighot Meghali , Nighot Prashant , Ma Thomas 
Published: January 2021

Abstract

Background
Proton pump inhibitors (PPIs) are highly effective antagonists of gastric acid secretion and are widely used to treat a number of gastroesophageal disorders, including peptic ulcer disease, GERD, and Barrett’s esophagus. PPI-induced elevation in intra-gastric pH and subsequent alterations of gastrointestinal physiology are thought to cause undesired effects on the entire GI tract. Defective intestinal Tight Junction (TJ) barrier is an important pathogenic factor for intestinal inflammation. Claudin-2 is a pore forming TJ protein whose overexpression causes selective increase in TJ permeability to small molecules. Claudin-2 expression is known to be up-regulated in intestinal inflammation and inflammation-associated colon carcinogenesis. The effect of PPI on Intestinal TJ barrier is not known. Aim: The aim of the present study was to study the effect of PPI on Intestinal TJ permeability.

Methods
A cell culture model of filter grown human intestinal epithelial Caco-2 monolayers, was used to study intestinal epithelial TJ barrier function. The mouse colonic permeability was measured by Ussing chambers studies. Western blot (WB) and Immunofluorescence (IF) was used to study the protein expression of claudin-2 in Caco-2 cells and in mouse colon. Cell surface biotinylation was used to study intracellular trafficking of TJ proteins.

Results
PPIs, caused a concentration- and time-dependent decrease in transepithelial resistance (TER), and increase in urea flux in filter-grown Caco-2 cells. Further studies on intestinal TJ revealed that PPI caused a significant increase in pore forming TJ protein claudin-2 protein level (western blot), but not mRNA (RT-PCR) in Caco-2 monolayers. Claudin-2 was co-localized to its known intercellular trafficking vesicles, clathrin pits. PPI treatment caused an increase in expression and junctional localization of claudin-2 and a decrease in its cytoplasmic co-localization with clathrin. Similarly, pulse-chase assay showed that the claudin-2 half-life was increased by PPI. In cell surface biotinylation assay, the rate of claudin-2 endocytosis was significantly reduced by PPI. In ex-vivo, Ussing chamber studies, PPI administration for 45 days resulted in a significant decrease in murine colonic TER and increase in urea flux. Immunofluorescence and Western blot analysis showed that PPI also caused an increase in TJ protein claudin-2 expression in mice colonic mucosa.

Conclusion
Our results suggest that the PPIs induces colonic TJ permeability via increasing pore forming claudin-2 levels. PPIs affect intracellular trafficking to reduce claudin-2 degradation and increased its junctional localization.
CLONAL EPIGENETIC MOSAICISM: MECHANISM FOR CROHN’S DISEASE HETEROGENEITY
Authors: Gupta Saumya , Bortvin Andrew , Vinogradova Svetlana , Shivdasani Ramesh , Gimelbrant Alexander 
Published: January 2021

Abstract
Crohn’s disease is a chronic heterogenous disorder with patients showing complex phenotypes and highly variable response to therapies. GWAS studies have identified hundreds of genetic factors associated with the disease but the precise mechanism with which these genes cause variable phenotype in the disease are unknown. We observed an autosomal analog of X-chromosome inactivation causes clonally stable epigenetic mosaicism in intestinal organoids generated from mice. This epigenetic mosaicism was detected by measuring allele specific expression in organoids grown from different individuals and from spatially different regions of the mouse intestine. This epigenetic mechanism has been observed in more than 25% of autosomal genes in mouse and in human cell lines and is called autosomal monoallelic expression (MAE). Allelic imbalance in MAE genes is clonally stable, persisting for multiple organoid passages and is regulated partially by DNA methylation. Our findings suggest how non-genetic variation leads to formation of developmental clones (or patches) within an individual that could then cause a wide range of functional diversity, thereby driving phenotypic variability. This will point the ways to affect epigenetic differences in intestinal tissue for disease prevention.
PRO-INFLAMMATORY MOLECULAR AND INFLAMMATORY MECHANISMS OF SULFUR METABOLISM IN IBD-RELEVANT CLOSTRIDIA SPECIES
Authors: Suarez Gabriel , Liu Bo , Herzog Jeremy , Sartor Ryan 
Published: January 2021

Abstract
Sulfur metabolism is emerging as a signature of IBD gut microbiota. Overrepresentation of sulfur-reducing bacteria (SRB) in IBD results in SRB-derived epithelial toxic H2S production that can overwhelm the body’s detoxification capacity, leading to impaired cellular respiration by inhibiting oxygen binding to mitochondrial cytochrome-c-oxidase. Butyrate potently inhibits SRBs and H2S, yet IBD patients have reduced short chain fatty acid (SCFA) production. More critically, H2S blocks butyrate oxidation, the primary energy source of colonocytes; butyrate oxidation deficiency is a defining characteristic of IBD. Since cysteine is the preferred substrate for H2S production by SRBs, a cysteine-rich environment provided by either a high protein diet or local intestinal mucus degradation promotes ideal conditions for SRB establishment and proliferation. SRBs can catabolize other sulfur-containing compounds critical for immune homeostasis and cellular health, such as taurine-conjugated bile acids and the “master antioxidant” glutathione, leading to further toxic H2S production. However, the molecular underpinnings of sulfur metabolism by specific bacterial genera is understudied in IBD. Results: Using a combination of in-vivo and in-vitro screening to detect the relative induction of interleukin 10 (IL-10) and interferon g (IFNg) by 19 resident bacterial strains isolated from a healthy human donor, we identified 4 bacterial strains that induce a low IL-10/IFNg ratio. These 4 strains (low group), but not 3 bacterial strains that induce a high IL-10/IFNg ratio, induce colitis in selectively colonized gnotobiotic Il10-/- mice (Fig.1A). Two of these 4 disease-inducing strains, Clostridium perfringens (A12) and Clostridium bolteae (B6), produce high concentrations of H2S in monoassociated mice (Fig.1B). In-vitro H2S production by these strains is dependent on cysteine (Fig.1C). C. perfringens and C. bolteae each induce colitis in monoassociated Il10-/- mice (Fig.1D). We are dissecting the sulfur metabolic pathways in C. perfringens and C. bolteae and their contribution to inflammatory processes by interrupting key genes predicted to contribute to H2S production, cysteine catabolism and bile acid metabolism. We will use these mutants in both in-vitro and in-vivo Il10 -/- gnotobiotic mice models to characterize their metabolic and inflammatory profiles. We have created several mutants using Targetron gene editing, including the dissimilatory sulfate reductase (Δdsr), a putative sulfonate membrane transporter (ΔssuA), anaerobic sulfite reductase (ΔasrA) and bile salt hydrolase (Δbsh). Conclusions: H2S producing bacterial strains can induce experimental colitis. Our planned mechanistic studies will determine the metabolic routes for H2S production by specific aggressive bacteria to guide novel therapeutic or dietary interventions to improve IBD prognosis.
A NOVEL LANGERIN EXPRESSING TYPE 2-CONVENTIONAL DENDRITIC CELL IS SIGNIFICANTLY DECREASED IN CROHN’S DISEASE
Authors: Doyle Chloe , Bertram Kirstie , Rhodes Jake , Cunningham Anthony , Ctercteko Grahame , Byrne Scott , Harman Andrew 
Published: January 2021

Abstract
Crohn’s disease is a chronic relapsing auto-inflammatory condition of the gastrointestinal tract that primarily affects young individuals with an increasing incidence rate. There is still no cure for Crohn’s disease and current treatment-options are limited to controlling inflammatory molecules such as TNF. Unfortunately, these biologics can produce significant side effects and not all patients respond to anti-TNF treatment. New treatments are therefore urgently needed. Targeting the cells and molecules of the immune system still represents the most promising approach which is why we have conducted an in-depth study of the immune cells and molecules associated with a healthy intestinal immune system and compared that to what is happening in Crohn’s disease.
Our group has privileged access to large human ileum explants as well as draining lymph node samples isolated from patients undergoing colorectal surgery to remove Crohn’s affected tissue. We have designed and optimised enzymatic tissue digestion protocols to isolate these cells in an immature state with minimal receptor cleavage. We have also developed high-parameter flow and mass cytometry panels to comprehensively identify and characterise all known subsets of mononuclear phagocytes (MNP), innate lymphoid cells, mucosal-associated invariant T cells, natural killer cells and T and B lymphocytes.
We have discovered that the newly described Langerin+ type 2-conventional dendritic cell (cDC2) is significantly decreased in Crohn’s affected ileum compared to healthy ileum. Furthermore, we have shown this decrease corresponds with lower TGF-b levels, a known driver of Langerin expression. After 7 days of co-culture, sorted Langerin+ cDC2 induced significantly higher levels of IL-17 and IL-22 in allogenic naïve CD4+ T cells compared to other MNP subsets, including cDC2 which did not express Langerin. These differentiated T cells expressed high levels of RORt and aryl hydrocarbon receptor (AHR) – transcription factors that are associated with CD4+ T helper 17 cells, implying that they may play a crucial role in intestinal barrier repair and regeneration that is absent in Crohn’s disease. Together these results suggest that Langerin+ cDC2 may have an anti-inflammatory role in human tissue and their reduction in Crohn’s disease may contribute to the pathogenesis of this disease, highlighting a potential therapeutic target for Crohn’s disease.
NEUTROPHILS ALTER DSB REPAIR PATHWAY IN INFLAMED MUCOSA TO PROMOTE COLON CARCINOGENESIS
Authors: Bui Triet , Butin-Israeli Veronika , Hanauer Stephen , Sebag Julien , Sumagin Ronen 
Published: January 2021

Abstract
Due to exacerbated inflammation and recurring tissue injury patients with Inflammatory Bowel Disease (IBD) are at higher risk of developing colorectal cancer (CRC). Although, PMN infiltration of the intestinal mucosa is a hallmark of IBD, and is associated with tissue injury, the contribution of PMNs to CRC and more so to IBD associated CRC is not known. We recently showed that PMNs can acutely exacerbate tissue injury and promote genomic instability by promoting miR-23a and miR-155-dependent accumulation of double-strand breaks (DSBs) and inhibition of DSB-repair by homologous recombination (HR). We now demonstrate that in chronic gut inflammation and recurring epithelial injury, as seen in IBD, via similar miRNA-dependent mechanism, PMNs alter DNA damage responses (DDR) to promote CRC progression. In murine model of Colitis-associated CRC (Azoxymethane, AOM/Dextran sodium sulfate, DSS) and human CRC xenografts, intratumoral PMNs intriguingly, functional dualism, suppressing tumor onset, but promoting tumor cell survival and growth in progressive tumors. The observed PMN effects were mediated by persistent suppression of HR-mediated DSB repair. HR suppression by PMNs, initially resulted in elevated replication stress and increased tumor cell apoptosis, however, longer term, altered DDR transcriptional profile and facilitated the upregulation of DSB repair by non-homologous end-joining (NHEJ). NHEJ upregulation enhanced CRC progression and tumor cell survival. PMN depletion, CRSPER-mediated deletion of miR-155 responsive sequence in RAD51 (preserves HR activity) or inhibition of NHEJ by small molecule inhibitors increased tumor cell death and diminished tumor development. Collectively, our data define a novel link between PMN-mediated mucosal injury and colon carcinogenesis.
OBSTRUCTED LYMPHATIC TRANSPORT AND LEAKAGE DRIVEN BY MESENTERIC TERTIARY LYMPHOID ORGANS IS A FEATURE OF CROHN’S DISEASE MOUSE MODEL
Authors: Czepielewski Rafael , Erlich Emma , Onufer Emily , Young Shannon , Kim Ki-Wook , Wang Peter , Bala Shashi , Hsieh Chyi-Song , Zinselmeyer Bernd , Davis Michael , Randolph Gwendalyn 
Published: January 2021

Abstract
Pioneer reports of Crohn’s disease (CD) suggested that impaired lymphatic flow might drive its pathogenesis but remains unsettled. Nodules of tertiary lymphoid organs (TLO) are found in association with collecting lymphatic vessels (CLVs) of the mesentery that normally conducts lymph outflow from the intestine. Whether TLOs affect lymph transport is unknown. In the TNFΔARE mouse model of Crohn’s-like ileitis, TLOs are found in valves regions. Using lymphatic reporters and photoconversion to study cell trafficking from the intestine, our findings indicated that TLOs halts immune cells traveling from the inflamed ileum to the lymph node, effectively trapping DCs, B, and T cells, and impacting the development of microbe tolerogenic regulatory T cells. Lymphatic transport defects were intrinsic to the CLVs because the soluble fluorescent tracer’s passage through TLO was also blocked. Lymph blockage promoted retrograde lymphatic flow returning towards the gut wall due to incapable valves. Moreover, significant lymph leakage was found, specifically at the TLOs. Neutralizing anti-TNF mAb treatment into TNFΔARE mice is ineffective in eliminating TLOs or restoring lymphatic trafficking when administered in female mice with advanced disease. In males, the therapy was able to restore forward flow to the lymph node. However, even in the presence of TNF inhibition, both sexes demonstrated TLO lymph leakage. Thus, mesenteric TLOs that form during chronic ileitis drive broadly impaired lymph transit of molecules and cells from the intestine that is only partially reversible by neutralizing the cytokine cascade underlying the disease and establish a perennial tissue alteration.
ALBUMIN-BASED NANOPARTICLES FOR DRUG COMBINATION IN INFLAMMATORY BOWEL DISEASE
Authors: Zhang Sufeng , Cho Won Joon , Jin Amy , Korzenik Josh , Lennerz Jochen , Traverso Carlo 
Published: January 2021

Abstract
Despite recent therapeutic advances in the treatment of inflammatory bowel disease (IBD), the off-target systemic side effects of small-molecule drugs or biologics remain problematic. Targeting the site of intestinal inflammation offers an approach to maximize potential therapeutic benefits while minimizing systemic side effects. Importantly, drug combination provides the possibility of tackling IBD through different pathways that could improve the treatment outcome. Here we introduced an inflammation-targeting drug delivery system utilizing albumin-based nanoparticles (NPs) for drug combination with relevance for IBD treatment. The pathophysiological features of the inflamed mucosa in IBD include the disrupted mucosal barrier and increased intestinal permeability; inflammation of the colonic mucosa is also accompanied by an in-situ secretion and accumulation of positively charged proteins at the site of inflammation 1, 2. We formulated the NPs using human serum albumin (HSA) with heparin coating (termed as HEP-HSA NPs) to increase the negative surface charge to interact with the accumulated positively charged proteins at the inflamed mucosa. Both small-molecule drugs and biologics were chosen to be encapsulated in these NPs, including budesonide (BUD), vancomycin (Vanco), and granulocyte macrophage colony-stimulating factor (GM-CSF). As an example of drug combination, we loaded BUD and GM-CSF simultaneously in the HEP-HSA NPs. The dual-drug loaded HEP-HSA NPs exhibited a higher anti-inflammatory effect than both of the single-drug loaded NPs in vitro and selectively bound to the inflamed intestine after enema administration in vivo in a murine model of colitis. Importantly, analyses of the physicochemical characteristics and targeting capacities of these NPs indicate that HEP coating modulates NP binding to the inflamed intestine, providing a foundation for future development of inflammation-targeting NP formulations 3. Furthermore, by analyzing disease parameters in individual mice, we also investigated the correlation between NP targeting and disease activity in the inflamed colon, which provides an approach to further our understanding on the underlying interactions between NPs and the biological interface in colitis.
References:
1. Tirosh B, Khatib N, Barenholz Y, et al. Transferrin as a luminal target for negatively charged liposomes in the inflamed colonic mucosa. Mol Pharm 2009;6:1083–91.
2. Zhang S, Ermann J, Succi MD, et al. An inflammation-targeting hydrogel for local drug delivery in inflammatory bowel disease. Sci Transl Med 2015;7:300ra128.
3. Zhang S, Cho WJ, Jin AT, et al. Heparin-Coated Albumin Nanoparticles for Drug Combination in Targeting Inflamed Intestine. Adv Healthc Mater 2020;9:e2000536.