bluuebunny/crossref_metadata_2025_split huggingface dataset:
A29 HOPX LABELS A COLONIC STEM CELL THAT CONTRIBUTES TO COLONIC REGENERATION BUT NOT COLONIC TUMORS
Authors: Iablokov V , Good H J , Shin A E , Fazio E N , Loggie J W , Zhang L , Asfaha S 
Published: February 2020

Abstract

Background
Colorectal cancer is the 2nd leading cause of cancer death in Canada. In rapidly dividing tissues such as the intestine or colon, only long-lived, multipotent, self-renewing tissue stem cells have longevity to accumulate mutations and serve as the cellular origin of cancer. In the small intestine, genetic fate mapping studies have demonstrated that there are at least two principal stem cell pools: actively cycling, crypt base cells expressing Lgr5, and quiescent cells situated above the crypt base. Clevers and colleagues have previously shown that Lgr5-expressing cells can give rise to cancer upon mutation. Interestingly, when Lgr5+ stem cells are selectively “killed”, intestinal integrity remains intact and other stem cells restore homeostasis. To determine whether another stem cell population can give rise to cancer in the colon, we examined whether the atypical homeobox protein Hopx, marks stem cells in the colon and whether these cells can give rise to colon cancer.

Aims
In the present study, we aim to determine whether Hopx-expressing cells are colonic stem cells that contribute to gut healing and can give rise to colonic tumours following the loss of APC.

Methods
To determine whether Hopx expressing cells show stemness, we crossed Hopx-CreERT mice to R26-TdTomato reporter mice. We then conducted genetic lineage tracing studies in the colon during homeostasis and following dextran sodium sulphate (DSS)-induced colitis. To test the function of Hopx expressing cells, Hopx-CreERT mice were also crossed to R26DTR mice and treated with diphtheria toxin (DT) following tamoxifen. These mice were then exposed to either normal drinking water or DSS to determine the role of Hopx+ cells in colonic regeneration. To test whether Hopx expressing cells can serve as a cellular origin for colon cancer, Hopx-CreERT mice were crossed to Apcf/f (floxed) mice.

Results
Consistent with the labeling of a stem cell, following tamoxifen, Hopx+ cells expressing tdTomato expanded to lineage trace full colonic crypts within 7 days, and labelling was persistent for greater than 6 months. Interestingly, ablation of Hopx+ cells with DT did not alter weight, histological damage or survival during normal homeostasis, however, Hopx+ cell ablation in mice treated with DSS resulted in increased histological damage. Surprisingly, loss of APC in Hopx-expressing cells did not induce colonic adenomas even after 8 months following tamoxifen administration.

Conclusions
These findings prove that Hopx expressing cells identify a novel colonic stem cell pool that is redundant in homeostasis, but in the context of injury, are essential for epithelial regeneration. Interestingly, Hopx+ cells do not have the capacity to give rise to colorectal adenomas upon loss of the APC gene.

Funding Agencies
CIHR
A33 THE ROLE OF A HOMOZYGOUS PROTEIN CODING VARIANT OF EPS8 IN THE PATHOGENESIS OF PEDIATRIC IBD
Authors: Long K , Warner N , Pan J , Guo C , Muise A 
Published: February 2020

Abstract

Background
The rate of onset for Inflammatory Bowel Disease (IBD) is rising worldwide—most significantly in the pediatric population. The pathogenesis of the disease involves a complicated interaction between the environment and genetics. Recent findings suggest that there is a broad range of rare, single-gene mutations that correlate with the IBD phenotype in children. Some of these single-gene defects can disrupt the epithelial barrier, which alters intestinal immune homeostasis. Epidermal Growth Factor Receptor Kinase Substrate 8 (EPS8) is crucial for the stabilization of F-actin by capping and bundling the barbed end. Previous literature shows that loss of expression of EPS8 disrupts the polymerization of F-actin in intestinal microvilli resulting in shortened brush borders. We identified a pediatric IBD patient with a homozygous missense EPS8 mutation(c.2099C&gt;T, I700T) in the actin-binding domain. The patient presented with pancolitis, colonic strictures and other IBD-like symptoms at 6 weeks of age. Currently, the functional role of EPS8 in the pathogenesis of very early-onset IBD remains elusive.

Aims
1)To investigate if the patient with EPS8 mutation has microvilli disorganization.
2)To determine EPS8 localization in patient samples and cell models.
3)To clarify the mechanism of how the EPS8 mutation affects F-actin in vitro.
Ultimately, we want to clarify how the mutation of EPS8 might contribute to the onset of pediatric IBD.

Methods
A rare, damaging mutation in EPS8 was identified in a very early-onset IBD patient by Whole Exome Sequencing. From patient-derived colon biopsy samples, the localization of EPS8 and actin was visualized by immunofluorescence (IF) microscopy. The morphology of the patient’s intestinal microvilli was assessed using transmission electron microscopy (TEM). EPS8 expression was measured using western blot. An F-actin polymerization assay was performed comparing purified WT and mutant EPS8 protein to assess the rate of polymerization from monomeric G-actin to F-actin.

Results
IF microscopy of the colonic sections revealed lower co-localization of EPS8 and actin on the apical surfaces, compared to IBD and normal controls. Closer examination of the cell structure using TEM showed disruption of the microvilli in the EPS8 mutant, but not in IBD or normal controls. Western blots showed no differences in protein expression between wildtype and mutant EPS8 in HEK293T cells. F-actin polymerization assay revealed differences in the rate of G-actin to F-actin polymerization between wildtype and mutant EPS8.

Conclusions
The patient with EPS8 mutation had microvilli disorganization. EPS8 localized differently in the patient colon tissues compared to normal and IBD control samples. The EPS8 mutation may affect F-actin polymerization, which may lead to disruption of the microvilli.

Funding Agencies
CIHR
A35 MICROBIAL PROTEOLYTIC SIGNATURE IN ULCERATIVE COLITIS INDUCES AN INFLAMMATORY SIGNATURE IN MICROBIOTA-HUMANIZED MICE
Authors: Galipeau H J , Turpin W , Caminero Fernandez A , Santiago A , Libertucci J , Bermudez-Brito M , Armstrong S , Bedrani L , Croitoru K , Verdu E 
Published: February 2020

Abstract

Background
Altered gut microbiota composition has been associated with inflammatory bowel diseases (IBD) including ulcerative colitis (UC), but causality and bacterially-driven mechanisms, are unclear. Proteases within the gastrointestinal tract play a critical role in maintaining homeostasis and are tightly regulated by anti-proteases. Host-derived proteolytic imbalances have been described in IBD, including UC, however, the role of intestinal microbiota as a source of proteases and anti-proteases has largely been ignored.

Aims
To study microbial proteolytic activity and intestinal microbiota profiles in a cohort of individuals at-risk for IBD, and in those individuals that develop UC at follow-up.

Methods
Fecal samples were collected from healthy individuals at-risk for IBD and who went on to develop UC (pre-UC; n=14) and again after UC diagnosis (post-UC, n=10). Fecal samples from matched at-risk individuals that did not develop UC were used as healthy controls (n=52). Overall fecal proteolytic and elastolytic activity was measured. We performed metagenomics sequencing in 4 UC subjects (pre and post) and 4 matched HC using Illumina Hi-Seq from stool DNA. To investigate bacterial origin and functional significance, pregnant germ-free (GF) mice were colonized with a fecal sample from a selected UC subject (pre and post) and a matched HC. Naturally colonized litters were followed for 12 weeks, after which proteolytic activities and signs of inflammation were measured.

Results
Fecal proteolytic and elastase activity was increased in pre- and post-UC samples compared to HCs. Metagenomics revealed over 20k genes were significantly different between HC and pre-UC samples, and of these, 440 related to proteases and peptidases. Increased fecal proteolytic activity, higher lipocalin levels, and increased colonic polymorphonuclear cells in colonic H&E sections was observed in pre- and post-UC colonized mice compared to HC colonized mice. Mice colonized with pre-UC microbiota showed increased mRNA expression of genes linked to immunological disease, antimicrobial and inflammatory responses (ie. Tlr2, Tlr5, Nod2, and Il1b) as compared to HC colonized mice.

Conclusions
These results suggest increased fecal proteolytic activity is observed prior to the onset and clinical diagnosis of UC in patients at-risk for IBD, and upon transfer to mice born from colonized GF dams, low-grade inflammation develops. These pathways could be developed as novel non-invasive biomarkers to monitor at-risk populations. Submitted on behalf of the CCC-GEM Project consortium. Supported by CCC GIA to EF Verdu

Funding Agencies
CCC
A43 β-DEFENSINS AS MARKERS OF INTESTINAL DYSBIOSIS: THE NATURE OF CHANGES IN β-DEFENSINS IS DEPENDENT ON THE PROCESS UNDERLYING THE INDUCTION OF DYSBIOSIS
Authors: Saqib Z , De Palma G , Lu J , Bercik P , Collins S M 
Published: February 2020

Abstract

Background
Dysbiosis may be defined as a change in the microbial composition or function that results in altered host function. Defensins are antimicrobial peptides, are part of innate immunity, and are important in host defense and maintaining homeostasis. Dysbiosis is a putative mechanism underlying the expression of many functional GI disorders like Irritable Bowel Syndrome (IBS) for which no biomarkers exist. Previous studies have revealed increased β-defensin (β-Def) levels in IBS patients, most likely due to changes in the microbiota.

Aims
We examined the hypotheses that: 1) Changes in β-Def are dependent on the manner in which dysbiosis is induced, and that 2) the direction of the change in β-Def depends on how dysbiosis was induced.

Methods
We used 4 models of experimentally induced dysbiosis to determine changes in fecal β-Def and to characterize the microbiota composition before and during the induction of dysbiosis. We used: 1) an antimicrobial cocktail (AC) in water; 2) a high-fat/ high-sugar diet (HFHSD); 3) a high salt diet (HSD) that we previously showed to induce a pro-inflammatory microbiota; and 4) mild restraint stress (MRS). All studies were performed in C57/BL6 mice except studies using MRS that were performed in NIH Swiss mice. In the AC or dietary studies, we employed a one-week intervention preceded by one-week baseline and recovery periods. In MRS studies, mice comparisons were made between a control and a stressed group. Stool samples were collected every 24 hours and were assayed for fecal β-Def levels analysis by an ELISA and microbial composition by 16S gene profiling.

Results
Exposure to AC or dietary change, but not MRS, resulted in significant decreases in fecal β-Def. Additionally, bacterial composition and diversity profiles were different in all mice except MRS mice (control vs. MRS males: p=0.414; control vs. MRS females: p=0.96). In contrast, mice exposed to the HSD revealed a significant increase in β-Def during treatment compared to baseline in both males (p=0.025) and females (p= 0.0019). The AC mice showed the largest changes and significant correlations between changes in β-Def levels and bacterial diversity (males: p=0.013, r=0.6; females: p=0.007, r=0.6) and richness (males: p=0.0008, r=0.70; females: p=0.003, r=0.62). However, no significant correlations were found between specific bacteria and β-Def levels in the HFHSD group.

Conclusions
We conclude that directional changes in fecal β-Def levels are dependent on the manner in which dysbiosis is induced. The use of β-Def as a biomarker requires comparisons with baseline levels obtained during remission in order to identify dysbiosis presence in microbiota-associated chronic GI conditions like IBS. Such an approach will identify patient subgroups that may benefit from microbiota-directed therapies.

Funding Agencies
CIHR
A52 BUTYRATE SUPPLEMENTATION AMELIORATES DAMAGE CAUSED BY ENTERIC CITROBACTER RODENTIUM INFECTION
Authors: Celiberto L S , Healey G , Xu J , Xia L , Vallance B 
Published: February 2020

Abstract

Background
Patients with inflammatory bowel disease (IBD) often display a dysbiotic microbiome as well as a defective intestinal mucus layer, which appears thinner and more penetrable than the mucus layer of healthy subjects. Tributyrin (TB), a prodrug of butyric acid, has shown beneficial effects in models of IBD due to its anti-inflammatory effects. We previously showed that mice lacking the major intestinal mucin Muc2 (Muc2-/-) or lacking the “Core1” enzyme responsible for glycosylating Muc2 (C1galt1-/-) were highly susceptible to infection by Citrobacter rodentium, a murine model of intestinal inflammation.

Aims
The study explored the role of gut mucus in providing host defense against C. rodentium, as well as the effects of TB supplementation in the prevention of mucosal damage in this model.

Methods
Six to ten week old wildtype (WT), Muc2-/-, flox control (C1galt1f/f) and C1galt1-/- mice were infected with C. rodentium (∼2.5 × 108 CFU) by oral gavage. For TB supplementation experiments, mice received 100µL of TB or glycerol as a control by oral gavage every other day starting on day 1 post infection. Mice were monitored daily throughout the experiment and were euthanized at day 6 of infection. Several tissues of interest were collected to verify bacterial colonization in the gut and at systemic sites as well as histological tissue damage. Cecal contents were collected for the analysis of short chain fatty acids, while blood was collected by cardiac puncture after oral gavage with FITC-dextran to measure intestinal permeability.

Results
While WT and C1galt1f/f mice were only modestly susceptible to C. rodentium infection, Muc2-/- and C1galt1-/- mice displayed dramatically (100 fold) increased pathogen burdens, significantly greater intestinal macroscopic and histopathology scores, and heightened barrier disruption as compared to controls. Moreover, Muc2-/- and C1galt1-/- mice showed significantly lower levels of butyric acid as compared to control mice under baseline conditions. Interestingly, when supplemented with TB, Muc2-/- and C1galt1-/- proved less susceptible to C. rodentium infection, as indicated by reduced weight loss and clinical signs of colitis, while pathogen burdens were greatly reduced as was histological tissue damage, and epithelial barrier dysfunction. The same protection was conferred when TB was administered as a dietary supplementation, thus confirming its beneficial effect in protecting mice against C. rodentium infection.

Conclusions
These findings demonstrate that intestinal mucus controls host susceptibility to C. rodentium infection via control over butyrate levels, and highlight the need to explore the mechanisms by which gut mucus modulates the resident microbiota and its metabolites.

Funding Agencies
CCC, CIHR
A55 LACK OF EXPERIMENTAL REPRODUCIBILITY IN PRECLINICAL RESEARCH IS INFLUENCED BY THE NUTRITIONAL PROFILE OF STANDARD RODENT CHOWS
Authors: Tuck C J , De Palma G , Takami K , Brant B J , Caminero Fernandez A , Reed D E , Muir J , Gibson P , Winterborn A , Bercik P , Verdu E , Vanner S 
Published: February 2020

Abstract

Background
Lack of experimental reproducibility, particularly in studies investigating the microbiota, is of growing concern to the scientific community. Factors such as environment, stress, and sex have been identified as contributors, whereas dietary composition has received less attention.

Aims
To profile the use of standard rodent chows in research institutions, analyze the nutrient content of common rodent chows, and assess the impact of different chows on microbiota profiles.

Methods
A survey was conducted to evaluate which rodent chows are used by research institutions worldwide. Seven standard rodent chows were selected for analysis of ingredients, fermentable carbohydrate (FODMAP) and gluten content. FODMAP content was measured by high-performance liquid-chromatography and enzymatic assays, and gluten content was assessed by a commercial kit using G12 antibody. Forty C57BL/6 mice were randomized to 4 groups of 10 mice (5 male, 5 female). Group A was euthanized at baseline. Group B received the breeding institution chow (LabDiet 5066). Group C received ResearchDiets AIN93G (low FODMAP and gluten content). Group D received LabDiet 5001 (high FODMAP and gluten content). After 3 weeks, cecal contents were collected and analyzed by 16S rRNA gene sequencing; short- and branched-chain fatty acids (SCFA, BCFAs) content was also determined.

Results
Twenty-eight different types of rodent chows were reported to be used by 45 international survey respondents. Ingredient composition of the standard chows varied widely, resulting in differences in FODMAP and gluten content. Microbiota analysis of cecal contents revealed that gut microbiota composition was altered by the standard chows, as reflected by significantly different clusters in β-diversity analysis as well as by altered α-diversity. Microbiota diversity was also influenced by sex in Groups A and B (p&lt;0.01). Total SCFA levels were lower (p=0.03) and BCFA levels were higher (p=0.01) in Group C compared to Group B.

Conclusions
Our results suggest that the composition of the diet not only significantly impacts gut microbiota profiles and fermentation patterns but may also emphasize sex differences in preclinical studies, with major implications for the reproducibility of results across laboratories.

Funding Agencies
CIHRThe Canadian Nutrition Society
A60 THE INCIDENCE OF INFLAMMATORY BOWEL DISEASE IS DECREASING IN SASKATCHEWAN: A POPULATION-BASED COHORT STUDY
Authors: Osei J A , Peña-Sánchez J , Fowler S , Muhajarine N , Kaplan G G , Lix L M 
Published: February 2020

Abstract

Background
Canada has one of the highest inflammatory bowel disease (IBD) incidence rates worldwide, although within Canada rates vary. Evidence show increasing incidence rates of IBD in Ontario (i.e. adults aged 30–60), stable in Alberta and decreasing in Manitoba. Additionally, higher incident rates of IBD have been identified among urban regions compared to rural regions. There is limited data on the incidence of IBD in Saskatchewan.

Aims
The study objectives were to 1) estimate IBD incidence rates in Saskatchewan from 1999 to 2016, and 2) test for differences in IBD incidence rates for rural and urban regions of Saskatchewan.

Methods
A population-based study was conducted using linked provincial administrative health databases. Individuals age 18+ old with newly diagnosed Crohn’s disease (CD) or ulcerative colitis (UC) were identified using a validated case definition. Generalized linear models with a negative binomial distribution were used to estimate incidence rates and incidence rate ratios (IRR) adjusted for age group, sex, and rurality with 95% confidence intervals (95%CI).

Results
In total, 4,908 newly diagnosed individuals with IBD were included. The average annual incidence rate of IBD decreased from 75 (95%CI 67–84) per 100,000 people in 1999 to 15 (95%CI 12–18) per 100,000 population in 2016. This decrease was evident in both UC (from 36/100,000 [95%CI 31–42] in 1999 to 6/100,000 [95%CI 4–8] in 2016) and CD (37/100,000 [95%CI 32–42] in 1999 to 8/100,000 [95%CI 6–10] in 2016). A significant decline of 6.9% (95%CI 6.2–7.6) in the average annual incidence of IBD was estimated between 1999 and 2016 (see Figure 1). Urban residents had a greater overall risk of IBD (IRR=1.19, 95%CI 1.11–1.27) than rural residents. This risk difference was statistically significant for CD (IRR=1.25, 95%CI 1.14–1.36), but not UC (IRR=1.08, 95%CI 0.97–1.19).

Conclusions
A decreasing trend in IBD incidence in Saskatchewan was identified after adjusting for age group, sex, and rural/urban region of residence. Around 150 new cases of IBD are still diagnosed annually in Saskatchewan, but this estimate is lower than estimates from other provinces. Urban dwellers have a 25% higher risk of CD onset compared to their rural counterparts. This finding could suggest the presence of specific risk factors in urban settings that require further investigation. Health care providers and decision-makers should plan IBD-specific health care programs taking into account these specific IBD rates in Saskatchewan.

Funding Agencies
College of Medicine, University of Saskatchewan
A65 VARIATION IN THE CARE OF CHILDREN WITH INFLAMMATORY BOWEL DISEASE: A CANGIEC POPULATION-BASED STUDY
Authors: Kuenzig E , Singh H , Bitton A , Kaplan G G , Carroll M W , Otley A , Stukel T A , Spruin S , Griffiths A , Mack D , Jacobson K , Nguyen G C , Targownik L , El-Matary W , Nasiri S , Benchimol E I 
Published: February 2020

Abstract

Background
Inflammatory bowel disease (IBD) is rising rapidly in Canadian children. These children require consistent high-quality specialized care to prevent long-term complications.

Aims
Evaluate variation in health services utilization and surgery rates across pediatric IBD centres in Ontario.

Methods
Incident cases of IBD &lt;16y (1999–2010), identified from health administrative data using a validated algorithm, were assigned to pediatric IBD centres based on location of IBD hospitalization, endoscopy and outpatient care. Children receiving IBD-specific care outside pediatric centres were also grouped. Frailty models, median hazard ratios (MHR), and Kendall’s t described variation in IBD-related ED visits, hospitalizations, and surgery 6–60 months after diagnosis, adjusting for age, sex, rural/urban household, and income. Mean diagnostic lag (time from first health system contact for an IBD symptom to final IBD diagnosis) and proportion of children with IBD care by gastroenterologists (GIs) at each centre were evaluated as centre-level predictors of variation.

Results
Of 2584 IBD cases, 73.4% were treated in a pediatric IBD centre. Between-centre differences accounted for 0.18% (MHR 1.06) and 0.41% (MHR 1.09) of variation in hospitalizations and ED visits, respectively. Children treated at centres where a higher proportion of children were cared for by GIs were more likely to be hospitalized (HR 2.09, 95% CI 1.26–3.45). Children treated at centres with a longer mean diagnostic lag were also more likely to be hospitalized (HR 1.01, 95% CI 1.003–1.02). ED visits were not associated with the proportion of children cared for by gastroenterologists or diagnostic lag. Among 1529 CD cases, 14.1% required intestinal resection; 1.79% of variation in the risk of surgery resulted from between-centre differences (MHR 1.20). Surgery was less common among patients at centres where more children were cared for by GIs (HR 0.24, 95% CI 0.07–0.84) and with a longer mean diagnostic lag (HR 0.98, 95% CI 0.97–0.99). After adjusting for these, between-centre differences accounted for 0.005% (MHR 1.01) of variation in care. Minimal variation was observed among the 11.0% of 872 UC cases requiring colectomy, with 0.37% of variation due to between-centre differences (MOR 1.09). Colectomy risk was not associated with GI care or diagnostic lag.

Conclusions
Variation in ED visits, hospitalizations, and surgery among children with IBD is small; however, centre-level differences in GI specialist care use and time to diagnosis were associated with hospitalization and surgery. It is essential to understand between-centre differences to reduce variation and ensure high-quality care.

Funding Agencies
CCC