bluuebunny/crossref_metadata_2025 huggingface dataset:
A45 ASSESSING THE ROLE OF MICROBIOTA DYSBIOSIS ON INTESTINAL TUFT CELL FUNCTIONS UPON GIARDIA INFECTION
Authors: Sosnowski O , Allain T , McKay D , Buret A G
Published: February 2024

Background

Enteric tuft cells can detect and respond to intestinal parasitic infection by modulating host responses to aid parasite clearance, yet the extent of tuft cell functions upon other infections remains to be elucidated. Previous work in our lab showed that the enteric protozoan parasite Giardia induced tuft and goblet cell hyperplasia, yet Giardia colonization in Pou2f3-/- (tuft cell-deficient) mice was impaired. Not only can Giardia modulate the gut microbiota and lead to microbial dysbiosis, but the microbiota itself can influence Giardia colonization. Interestingly, certain microbiota-derived products can activate tuft cells in the gut. In this study, we investigated the role of microbiota dysbiosis in the crosstalk between Giardia and tuft cells.

Aims

We aim to explore the bidirectional effects between tuft cells and Giardia infection by investigating the role of 1) the host microbiota influenced by the presence or absence of tuft cells in altering Giardia infectivity, and 2) Giardia modified microbiota in contributing to tuft cell activity.

Methods

Fecal matter from 6–8-week-old C57Bl/6 and Pou2f3-/- mice were administered to polyethylene glycol (PEG) treated C57Bl/6 mice via oral gavage for 7 days, followed by gavage with 5x104Giardia muris trophozoites. Duodenal parasite burden was assessed 11 days after infection. Small intestinal (SI) microbiota was collected from 5–7-week-old C57Bl/6 and Pou2f3-/- mice following 11 days of G. muris infection and transplanted into PEG treated C57Bl/6 and Pou2f3-/- recipients. 11 days post-transfer, SI tuft and goblet cell hyperplasia was assessed in recipient mice by immunohistochemistry, and Dclk1, Atoh1, and Muc2 gene expression was quantified by RT-qPCR. Parameters of gut motility were evaluated.

Results

FMT with Pou2f3-/- mouse fecal matter prior to Giardia infection led to improved weight gain during infection compared to FMT from C57Bl/6 mice. Giardia modified microbiota (GMM) did not induce tuft or goblet cell hyperplasia 11 days after transfer. Jejunal Dclk1, Atoh1, and Muc2 were no different after GMM compared to control microbiota (CM) transfer. Ileal Atoh1 or Muc2 were upregulated in mice receiving GMM from C57Bl/6 or Pou2f3-/- mice, respectively. Intestinal transit time and stool water content was altered in mice which received GMM from Pou2f3-/- mice compared to CM.

Conclusions

The resident microbiota in tuft cell deficient mice may in part contribute to limiting Giardia infection. Giardia modified microbiota does not substantially contribute to tuft cell hyperplasia or related gene expression alterations at 11 days after microbiota transfer. The mechanistic contributors to tuft cell hyperplasia during Giardia infection and how a lack of tuft cells can lead to impaired colonization does not appear to be primarily driven by microbiota alterations and remains to be fully defined.

Funding Agencies

NSERC
A46 METABOLIC OUTCOMES 12-MONTHS POST-BARIATRIC SURGERY: SLEEVE VERSUS ROUX-EN-Y GASTRIC BYPASS
Authors: Bharatselvam S , Schwenger K , Ghorbani Y , Fischer S , Jackson T , Okrainec A , Allard J P
Published: February 2024

Background

Obesity and its related comorbidities, such as type 2 diabetes (T2D), hyperlipidemia, cardiovascular disease (CVD), and non-alcoholic fatty liver disease (NAFLD) are on the rise. When lifestyle modifications, such as diet and exercise, are ineffective at producing sustained weight loss and improvements in obesity-related comorbidities, other interventions may be recommended. Bariatric surgery is considered in those who have a body mass index (BMI) over 40 kg/m2 or with comorbid conditions and a BMI between 35-40 kg/m2. Two commonly used procedures within this category of surgery are Roux-en-Y gastric bypass (RYGB), the gold standard for weight loss; and laparoscopic sleeve gastrectomy (LSG). Though both surgeries are effective at inducing weight loss, more information is needed on their comparative effectiveness in improving clinical and biochemical outcomes in patients with T2D, hyperlipidemia, and NAFLD in real-world practice.

Aims

Therefore, our objective was to determine the overall effectiveness of RYGB versus LSG at 12-months post-surgery and specifically assess their responses in those with hyperlipidemia, T2D and NAFLD. We chose to analyze both glucose and lipid parameters along with anthropometric measures as they not only provide insight into improvements in comorbid outcomes of obesity but also little-to-no prospective studies compare all of these outcomes in one study using the same cohort.

Methods

This is a prospective cross-sectional and cohort study of 142 patients who underwent bariatric surgery. Clinical and biochemical data were collected at baseline, prior to surgery and 12-months post-bariatric surgery. A liver biopsy was collected during surgery to diagnose NAFLD. The main outcome was 12-month changes in lipid parameters, mainly total cholesterol, between types of surgery.

Results

107 individuals underwent RYGB (75.4%) and 35 underwent LSG (24.6%). Both groups were similar at baseline except for the higher waist circumference and proportion of males undergoing LSG. At 12-months post-surgery, RYGB versus LSG resulted in a significantly lower BMI (pampersand:003C0.0001), triglycerides (pampersand:003C0.05), total and LDL cholesterol (pampersand:003C0.005). Interestingly, ALT was significantly lower in those who underwent LSG (pampersand:003C0.005). In subgroup analysis for determining the effect of the type of surgery in those with comorbidities of obesity, RYGB was superior at improving lipid-related parameters in those with hyperlipidemia with a significant decrease in total cholesterol -0.48 (-0.84, 0.34) (pampersand:003C0.05) whereas LSG was superior at reducing ALT in those with NAFLD with significant decreases in total cholesterol and LDL.

Conclusions

RYGB leads to greater reductions in BMI and lipid parameters, especially in those with hyperlipidemia, whereas LSG showed greater improvements in liver enzymes in those with NAFLD.

Funding Agencies

CIHR
A48 PARENT-REPORTED HEALTH RELATED QUALITY OF LIFE CARING FOR CHILDREN WITH PEDIATRIC FEEDING DISORDERS IN ALBERTA
Authors: Lang A , Chui A , Cohen J , Steinke V , Moland M , Turner J
Published: February 2024

Background

Children with pediatric feeding disorders (PFD), including those requiring tube feeding, need significant care. Caregiver burden includes time, cost, as well as worry over patient morbidity and mortality (1). This project used a validated health-related quality of life (HRQoL) tool for caregivers of children with PFD: the Feeding/Swallowing Impact Survey (FS-IS). The FS-IS has three subscales related to daily activities, worry, and feeding concerns, scored 1-5 with 5 being the most adverse. Prior studies have shown both the total sub-scale scores and the total score correlate with generic quality of life instruments for pediatric caregivers (2). Data was obtained from a provincial quality improvement initiative, the Alberta Provincial Pediatric Eating and Swallowing (PEAS) Project.

Aims

1. To determine the HRQoL reported by caregivers of children with PFD in Alberta. 2. To determine if tube-feeding is associated with worse reported HRQoL.

Methods

Caregivers receiving feeding and swallowing services in Alberta were offered the opportunity to complete the FS-IS online or by phone at baseline and 12 months later. All demographic data was self-reported.

Results

In total 52 caregivers completed the surveys once and 16 at follow-up. Average baseline FSIS was 3.1 (range 1.2-4.7) and at follow up was 3.0 (range 1.5-4.2). There was no difference between the scores of those completing at both time points (p=0.78). At all time points, only 15% of the cohorts reported scores ≤2. As shown in Table 1, children who were tube-fed were older with a longer duration of PFD symptoms, however, HRQoL scores were not different to children not ever tube-fed.

Conclusions

Caregivers of children with PFD face challenges, which do not abate over time. Surprisingly, in our cohort, this is independent of the need for tube feeding and of the duration of PFD symptoms, although we acknowledge the small sample size of those who were never tube-fed. Longitudinal follow-up studies and strategies to improve quality of life are needed.

* FS-IS Global Score (1=never to 5=almost always)

Funding Agencies

Alberta Health Services Maternal Newborn Child & Youth Strategic Clinical Network
A49 AN UNEXPECTED CASE OF SEVERE PROTEIN-LOSING ENTEROPATHY IN A TODDLER
Authors: Podgorny P , Kortbeek S , Lopushinsky S , Boctor D
Published: February 2024

Background

Protein-losing enteropathies (PLE) are characterized by excess loss of serum proteins into the gastrointestinal tract, resulting in hypoproteinemia, peripheral edema, and related respiratory and cardiac sequelae. PLE should be suspected in patients with hypoproteinemia not explained by protein malnutrition, impaired hepatic protein synthesis, or renal losses, and can occur as a consequence of erosive or non-erosive gastrointestinal diseases, and conditions that alter lymphatic flow.

Aims

To present a case of intestinal malrotation with chronic volvulus presenting as PLE.

Methods

In this report, we describe a pediatric patient presenting with PLE ultimately found to have intestinal malrotation with chronic volvulus.

Results

A healthy 1-year-old male presented with acute non-bloody diarrhea and peripheral edema on a background of recurrent colicky abdominal pain and non-bilious emesis. He had significant electrolyte derangements at presentation including hypokalemia, hyponatremia, hypomagnesemia, and metabolic acidosis, with additional laboratory findings of hypoalbuminemia (11g/L), hypogammaglobulinemia (IgA 0.11g/L, IgG 0.77g/L, IgM 0.28g/L), lymphopenia (0.4x10E9/L), and fat-soluble vitamin deficiency (Vitamin A 0.5umol/L, 25-Hydroxyvitamin D 18.4nmol/L, Vitamin E 6umol/L, INR 1.7). In addition to abdominal imaging, he underwent endoscopic evaluation which did not identify an underlying etiology. As his presentation was consistent with PLE, a high-protein, low-fat diet was initiated. Despite dietary adherence for 16 weeks, the PLE features persisted with ongoing severe hypoalbuminemia (20g/L).

Given a suspicion of intestinal lymphangiectasia, a capsule endoscopy was arranged. In the interim, the patient had recurrence of abdominal pain and emesis prompting a repeat abdominal ultrasound which demonstrated swirling of the mesenteric vessels in the midline. An upper gastrointestinal series identified a short mesenteric root with the duodenojejunal junction and cecum in proximity. Urgent laparotomy confirmed a 720-degree volvulus of the small bowel around the mesenteric root, with an abnormally positioned duodenojejunal flexure and cecum. This was presumed to be chronic given the absence of acute vascular compromise. The patient underwent a Ladd procedure. He had complete resolution of symptoms and PLE features on a liberalized diet.

Conclusions

This report identifies a case of intestinal malrotation with chronic volvulus presenting as PLE, highlighting an atypical presentation of intestinal malrotation outside the neonatal period. Awareness of these atypical presentations is important to facilitate prompt diagnosis and treatment, thereby reducing the rate of complications.

Funding Agencies

None
A51 MICROBIALLY-MEDIATED IMPAIRMENT OF VAGAL AFFERENT NEURONAL EXCITABILITY IS OREXIN RECEPTOR DEPENDENT
Authors: Sachdev S , Tashtush A , Alward T , Reed D E , Lomax A E
Published: February 2024

Background

An impairment of vagally-mediated satiety signalling has been implicated in the caloric imbalance that leads to weight gain during obesity. Previous studies have suggested that a reduction in the excitability of vagal afferent neurons with cell bodies in nodose ganglia (NG) was responsible, but the cellular mechanisms are unclear. Host and bacterially derived mediators present in the small intestine and stool provide a physiologically relevant model to help elucidate the role luminal mediators play in modulating vagal afferent neuronal excitability.

Aims

We hypothesize that the microbiota of obese individuals and mice produce mediators that impair NG neuron excitability and satiety in mice.

Methods

Perforated patch clamp was used to measure the excitability of NG neurons following exposure to human and mouse fecal supernatants (FS), mouse jejunal supernatants (JS), and mice serum samples. Human FS were from ampersand:003E 5 healthy human donors or FS from ampersand:003E 5 obese donors. Mice FS, JS and serum samples were collected from ampersand:003E 5 obese mice fed a high-fat diet and ampersand:003E 5 control mice fed a normal diet.

Results

NG neurons incubated in FS from obese participants were significantly less excitable (rheobase was 30% higher and action potential discharge at 2x rheobase was 50% lower) than NG neurons exposed to FS from non-obese participants. NG neurons incubated in FS or JS from obese mice were also significantly less excitable (rheobase was 65% higher and action potential discharge at 2x rheobase was 50% lower) than NG neurons incubated with FS or JS from control mice. Lastly, NG neurons incubated with obese mouse serum were significantly less excitable (rheobase was 50% higher and action potential discharge at 2x rheobase was 80% lower) than NG neurons incubated with serum from control mice.

We then attempted to identify mediators that may account for this inhibitory effect by using receptor antagonists that block GABA, ghrelin, and orexin signalling. Ghrelin and GABA receptor antagonists did not block the inhibitory effect of obese patients’ FS on NG neurons but the orexin receptor 1 antagonist (SB-334867;10µM) did. Following this, we incubated the orexin receptor 1 antagonist (SB-334867;10µM) on NG neurons incubated with mouse FS and JS and observed a similar blocking of inhibitory effects back to control values.

Conclusions

These findings suggest that the gut luminal contents of obese mice and humans contain an orexin receptor agonist that inhibits satiety and may contribute to over-eating.

Funding Agencies

CIHRNSERC
A53 MOLECULAR MECHANISMS FOR A ROLE OF SHP-2 IN TLR4 SIGNALING IN INTESTINAL EPITHELIAL CELLS
Authors: Chabot V , Lévesque D , Boisvert F , Rivard N
Published: February 2024

Background

Shp-2 gene polymorphisms are associated with greater susceptibility to ulcerative colitis. How Shp-2 contributes to disease susceptibility is however unclear. Intestinal epithelial cell (IEC)-specific deletion of Shp-2 results in severe colitis in mice (Shp-2IEC-KO). We found important alterations in microbiota composition, namely increased Enterobacteria of the Proteobacteria phylum and decreased Firmicutes levels, that preceded clinical signs of inflammation. Antibiotherapy or epithelial KO of Myd88 inhibits colitis in Shp-2IEC-KO mice. Notably, feces from Shp-2IEC-iKO mice display much more LPS-like bioactivity than do feces from controls (unpublished data). These observations support a role for TLR4/Myd88-bacterial recognition in initiation of colitis in Shp-2IEC-KO mice.

Aims

The aim of the present study was to identify the molecular mechanisms by which Shp-2 may regulate TLR4 signaling in IEC.

Methods

1) The regulation of Shp-2 phosphorylation was analysed in IEC-6 cells stimulated or not with 10 ug/ml of LPS, a TLR4 ligand. 2) We mapped the Shp-2-dependent interactome in LPS-stimulated IEC-6 cells by an APEX2 proximity assay used in combination with mass spectrometry (MS) analysis. Cells stably expressing APEX2-tagged Shp-2 were generated. Addition of doxycycline results in expression of “physiological” levels of Shp-2;APEX2 construct. Proximity biotinylation labeling of Shp-2 by APEX2 was performed after 10 min stimulation with LPS and labeled candidates were analyzed by MS.

Results

1) Upon stimulation of IEC-6 cells with LPS, Shp-2 protein is rapidly phosphorylated (Y542, Y580) and potentially activated. This increased phosphorylation is abolished by pretreatment with N-acetyl cystein, a commonly used antioxydant. 2) Using Prostar R package, the three replicates of MS data were filtered for the minimum quantification of 66,66% of each protein in at least one condition. They were normalized within conditions using VSN normalization, then the Partially Observed Values (POV) and the Missing in Entire Column (MEC) values were imputed respectively with SLSA and DetQuantile (Quantile 1%, Factor 0,2). A differential analysis between the LPS and non-treated conditions identified 334 proteins enriched with a FC of ± ≥2,5 with a p-value inferior then 0,05. Among the top Shp-2 interactors, we found Tak1 (ampersand:003E3,5X change) and Tab1 (ampersand:003E3,0X change), two proteins involved in NF-kB signaling. Decreased interaction with the TLR effectors Myd88 (ampersand:003C2,5X change) and Irak1 (ampersand:003C3,2X change) was also noted after LPS treatment.

Conclusions

These data suggest for the first time that Shp-2 may regulate NF-kB signaling downstream of TLR4 in IEC. Thus, SHP-2 may contribute to the maintenance of mucosal tolerance to microbiota in the colon.

Funding Agencies

CIHR
A54 SHP2 FUNCTION IN NORMAL AND APC-MUTANT INTESTINAL EPITHELIAL CELLS
Authors: Côté-Biron A , Gagné-Sansfaçon J , Rivard N
Published: February 2024

Background

Somatic SHP2 gain-of-function mutations were found in some colorectal tumours. However, its function in colorectal epithelium remains to be determined. SHP2 is a tyrosine phosphatase necessary for RAS/MAPK pathway activation by most, if not all, tyrosine kinase receptors, as well as by GPCRs and cytokine receptors. SHP2 can also regulate the PI3K, Jak/Stat, RhoA and Hippo pathways in different cell settings. As a result, SHP2 can control cellular processes including growth, migration and differentiation. Previously, we observed that SHP2 levels are enhanced in human colorectal tumours, notably in late adenomas. Likewise, SHP2 silencing inhibited ERK activation and tumour properties of established human CRC cell lines. Hence, these findings support a model in which SHP2 activation promotes tumorigenesis in the intestinal epithelium. But how?

Aims

To analyze the exact cellular and molecular mechanisms underlying SHP2 pro-oncogenic function in intestinal epithelial cells.

Methods

The impact of SHP2 pharmaco-inhibition (RMC-4550 and/or SHP099) on intracellular signaling, cell cycle, proliferation and survival was evaluated in three complementary models: 1) HIEC-6 cells, normal human fetal intestinal crypt-like cells; 2) organoids derived from normal mouse small intestine or ApcMin/+ tumors; 3) Caco-2/15 cells, colorectal cancer cells with APC mutation.

Results

1- Pharmaco-inhibition of SHP2 blocks proliferation of HIEC-6 cells (loss of pRb phosphorylation, decreased EdU staining) and induces cell enlargement and darkening of the outside of perinuclear area. This phenotype was reminiscent of cellular senescence, and we indeed observed increased senescence-associated β-galactosidase activity (SA-β-Gal) and INK4A gene expression. 2- As expected, SHP2 inhibition in normal mouse organoids resulted in a reduction in the number of protrusions and EdU positive cells. RNA sequencing was performed, and gene set enriched revealed significant down regulation of several Ras signaling-dependant genes including cell cycle regulated genes (E2F targets, G2/M checkpoint) and Myc targets 48h after SHP2 inhibition when compared to DMSO treated organoids. Genes associated with PI3K signaling were also diminished in SHP099 treated organoids but no significant modulation of genes associated with Wnt/b-catenin signaling was observed. 3- Notably, SHP2 pharmaco-inhibition in Caco-2/15 cells as well as APC-mutant organoids rapidly induced cell death associated with increased expression of stem cell markers.

Conclusions

These results suggest that SHP2 is required for proliferation of normal IEC and protects against senescence. Notably, its inhibition restrains the proliferative and tumorigenic potential conferred by APC inactivation. Overall, our data suggest that SHP2 could represent a potential target to counter colorectal tumour initiation.

Funding Agencies

CIHRFRQS
A55 FROM FETAL REVERSION TO INTESTINAL TUMORIGENESIS: PROBING MOLECULAR REGULATORS IN THE TGFß AND HIPPO PATHWAYS
Authors: Czubik J , Javkar T , Gregorieff A
Published: February 2024

NOT PUBLISHED AT AUTHOR’S REQUEST

Funding Agencies

None
A56 EVALUATING THE DYNAMICS OF CYP3A11 AND REG3G MODULATION BY IL-22
Authors: Delanne-Cuménal A , Flannigan K , Hirota S
Published: February 2024

Background

IL-22 is a cytokine produced by a number of immune cell populations, including type 3 innate lymphoid cells. We have shown previously that IL-22 suppresses the expression Cyp3a11 in the mouse intestine, a gene that encodes a drug metabolizing enzyme that accounts for roughly 70% of total drug metabolism. In addition, IL-22 induces the expression of Reg3g, the gene that encodes an antimicrobial C-type lectin that has bactericidal activity against Gram+ bacteria in the small intestine. Modulation of these proteins can influence both the oral bioavailability of drugs and microbial composition.

Aims

The aim of this study was to define the existence of reversible modulation of IL-22 on Cyp3a11 and Reg3g expression in the intestinal epithelium.

Methods

In vitro enteroids from each part of the mouse small intestine were cultured in 3D in the presence or absence of IL-22 for 5 days. Cyp3a11 and Reg3g transcript expression was evaluated by qPCR followed by protein analysis to define their modulation at varying timepoints after IL-22 treatment cessation.

Results

IL-22 reduced Cyp3a11 expression, an effect that normalized the day after treatment cessation. However, 4 days after treatment cessation there was rebound hyperexpression of Cyp3a11 in duodenal enteroids, which normalized the following day. IL-22 reduced the expression and activity of Cyp3a11 which normalized soon after stopping IL-22 treatment. In contrast, IL-22 induced the expression of Reg3g, an effect that lasted up to 5 days after treatment cessation. The effect of IL-22 on Cyp3a11 expression and activity was rapidly reversed in contrast to the effect on Reg3g which was more prolonged.

Conclusions

IL-22 suppresses the expression of Cyp3a11 and induces the expression of Reg3g. The reversible nature of these effects is different between both, with the increased expression of Reg3g 5 days after IL-22 treatment cessation. These data suggest that the regulation of gene expression, protein or enzyme activity by IL-22 may exhibit varying temporal effects and should be considered when assessing its role in intestinal mucosal biology.

Funding Agencies

This work was funded by the Dr. Lloyd Sutherland Chair in IBD/GI Research and the Weston Foundation.
A57 LONG-TERM EXPOSURE TO ANTICANCER TREATMENTS INCREASES AUTOPHAGY IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA
Authors: Hamilton M , Douchin J , Gaudette B , Giroux V
Published: February 2024

Background

The most common type of esophageal cancer, esophageal squamous cell carcinoma (ESCC), has a 5-year survival rate of only 15%. This low survival rate is to some extent attributed to a high relapse rate, which is, in part, linked to the presence of cancer stem cells (CSC). CSC are a subpopulation of tumor cells that show reduced sensitivity to conventional anticancer therapies, increased potency and self-renewal capacities. With that in mind, ESCC cell lines were exposed in a prolonged manner to anticancer treatments (radiotherapy, 5-FU chemotherapy, and combined therapy) in our laboratory. As expected, an increase in the proportion of CSC (CD44highCD24high) was observed by flow cytometry following the treatments. In pursuit of innovative therapeutic approaches, we conducted an in-depth investigation of the proteomic profiles of these cell lines by mass spectrometry (MS), seeking to unveil novel biological mechanisms. Interestingly, many proteins associated with metabolism and autophagy were modulated. Several alterations were observed by a metabolomic approach as well, namely an increase in intracellular lactate concentration. Recently, a novel role for lactate has been described: lactylation, which is a post-translational modification. Lactylome of those cell lines revealed alteration in proteins related to autophagy and those results led to my research project.

Aims

Investigate the role of lactylation in autophagy of ESCC.

Methods

Human ESCC cell lines (including the cell lines exposed to anticancer treatments) will be used as cell culture models. Cell and organoid cultures, mass spectrometry, qPCR, WB, and proliferation assay were used.

Results

MS analysis of proteins related to autophagy (i.e. RAB1B, CK2, SNX6 et GABARAP) suggests differential regulation of autophagy in cell lines exposed to prolonged treatments. A strong contribution of the 5-FU treatment to the autophagic modification is observable in the double-treated cells. Thus, we confirmed an increase in the LC3BII/I ratio by WB in the cell lines exposed to anticancer treatments. LC3BII/I WB used in combination with Bafilomycin A1 treatment for inhibition of autophagosome and lysosome fusion is a commonly used read-out for autophagic flux. To further study the link between lactylation, autophagy and CSC, we screened 10 human ESCC cell lines for their LC3BII/I ratio. Four cell lines were selected: TE1, TE2, HCE4 and TE5. Using Bafilomycin A1, we confirmed that the autophagic flux in those cell lines was not blocked. Those cell lines will now be used to perform lactylome analysis of sorted CSC cells.

Conclusions

In brief, our results showed that the autophagic flux is increased in cells exposed to prolonged treatments. This suggests that autophagy could play a role in ESCC response to treatment.

Funding Agencies

CIHRTRIANGLE, Chaires de recherche du Canada, CRCHUS, Université de Sherbrooke
A59 EXPLORING THE ROLE OF ARID1A IN COLONIC HOMEOSTASIS AND REGENERATION
Authors: Lei D , Loe A , Kim T
Published: February 2024

Background

Recent advances in cancer genome analysis have revealed frequently mutated epigenetic regulators as a novel feature in cancer development. Within this context, the Arid1a (AT-rich interactive domain-containing protein 1A) gene, a subunit of the BAF chromatin remodeling complex, has emerged as a frequently mutated gene in many cancers. Additionally, repeated colonic injury caused by diseases such as inflammatory bowel disease (IBD), is also a major risk factor leading to colorectal cancer. Therefore, understanding the connection between colonic epigenetic regulation and its role in response to injury is particularly important for understanding the pathobiology of colorectal cancer.

Aims

Aim 1: Exploring the role of Arid1a during colonic homeostasis.

Mouse model with Arid1a conditionally knocked out in the colon will be analysed at different time points to test whether Arid1a is required during colonic homeostasis.

Aim 2: Determining the role of Arid1a during colonic injury and regeneration.

Mice will be exposed to a known colitis model to induce colonic injury. Arid1a will be deleted after injury to find out its role during regeneration and recovery from the injury.

Methods

To examine the effect of Arid1a loss on colonic homeostasis, mice between 6-10 weeks old were injected with 2mg/20g tamoxifen for three days to induce Arid1a knockout using the VilCreERT2; Arid1afl/fl mousseline. Mice were then euthanised using CO2 chamber and analysed 10 days, 3 weeks, and 8 weeks post Arid1a deletion. Histological analysis was performed to examine morphological changes in the colon. To examine the effect of Arid1a on injury caused by colitis, mice were treated with dextran sulfate sodium (DSS) to cause colitis-like injury. Then,

Arid1a is deleted by injecting 2mg/20g tamoxifen. Changes in histology and specific cell types will be examined both in the short- and long-term.

Results

To test whether Arid1a loss alone causes morphological changes in the colon, I compared the crypt lengths between VilCreERT2; Arid1afl/fl mutant mice with their littermate controls and saw that there was no difference in both morphology and length of the crypts. My results show that

Arid1a loss in the colon after injury resulted in impaired regeneration characterized by the prolonged loss of goblet cells and cryptal structure in the distal colon. In the long term, Arid1a loss before or after DSS treatment both lead to tumor formation, suggesting that Arid1a is required for colonic regeneration and repair while suppressing tumorigenesis.

Conclusions

Based on my results, I conclude that Arid1a deletion alone does not cause significant morphological changes in the colon, but this loss affects colonic regeneration and recovery compared to colons with Arid1a intact. In the long term, the dysregulated colonic regeneration may lead to tumorigenesis.

Funding Agencies

University of Toronto Department of Molecular Genetics
A61 ELUCIDATING INTESTINAL EPITHELIAL INFLAMMASOME MECHANISMS THAT REPAIR THE MUCOSAL BARRIER DURING ENTERIC INFECTION
Authors: Ranger A , Goyal S , Girardin S E
Published: February 2024

Background

To protect the gastrointestinal tract from infection, intestinal epithelial cells (IECs) lining the mucosa possess cytosolic complexes called inflammasomes which detect pathogenic insult and rapidly initiate inflammation to stop infectious spread. Active inflammasomes mediate crucial effector functions such as epithelial cell extrusion, pyroptotic cell death, and the release of interleukin (IL)-18 and prostaglandin E2 (PGE2). Recent literature has shown that IL-18 and PGE2 mediate intestinal repair following epithelial damage from DSS-induced colitis. Exogenous administration of IL-18 in inflammasome deficient mice improves survival and reduces pathology. PGE2 activates intestinal cellular reprogramming to drive intestinal wound healing. Whether inflammasome derived IL-18 and PGE2 mediate epithelial repair during enteric infection is unclear.

Aims

Using murine small intestinal organoids (mSIOs), we will investigate how IL-18 and PGE2 production by intestinal inflammasomes directly impacts IECs at both transcriptional and physiological levels. We hypothesize that inflammasome derived IL-18 and PGE2 will act on the intestinal epithelium resulting in transcriptional reprogramming that promotes tissue regeneration and barrier reinforcement.

Methods

WT and Nlrc4-/- mSIOs were stimulated with flagellin or FlaTox to activate the NAIP-NLRC4 inflammasome. The transcriptomic landscape was assessed with bulk-RNA sequencing and inflammasome activation was assessed by Western blot. IL-18 and PGE2 dependent gene targets were identified using Il-18-/- and indomethacin treated mSIOs by RT-qPCR.

Results

Bulk-RNA sequencing revealed an inflammasome activation dependent transcriptional signature in murine IECs upon NAIP-NLRC4 inflammasome activation. GO term analysis showed these genes to be involved in actin cytoskeleton rearrangement, epidermal growth factor signaling, and regulation of cell-cell adhesion. The expression of PGE2 gene targets were significantly reduced by indomethacin treatment while IL-18 gene targets remained unaffected in Il-18-/- IECs. FlaTox induced the same transcriptional changes but substantially amplified their expression. FlaTox but not flagellin led to a significant drop in Lgr5 (stem cells) and Lyz1 (Paneth cell) expression and an increase in Ly6a, a marker of fetal-like stem cells.

Conclusions

This data demonstrates that inflammasome signaling causes transcriptional reprogramming of IECs that activates pathways involved in maintaining epithelial barrier integrity. While IL-18 had no effects on gene expression at 4 hours, PGE2 paracrine signaling contributed to the expression of wound healing genes. Stronger inflammasome mediated damage with FlaTox amplified the repair response at the transcript level and we observed transient reprogramming of IECs into a fetal-like state suggesting active tissue regeneration.

Funding Agencies

CCC, CIHREPIC: Emerging & Pandemic Infections Consortium
A62 EXAMINING THE ACTIVATION OF XENOBIOTIC RECEPTORS PXR AND AHR IN COLONOIDS USING MICROBIAL METABOLITES AND CHEMICAL LIGANDS
Authors: Shenoda Eva I , Hirota Simon A
Published: February 2024

Background

The Aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR) are vital xenobiotic receptors activated by foreign substances, playing pivotal roles in regulating chemical metabolism. Traditionally, these receptors have been closely linked to their roles in mediating responses to toxic compounds. However, recent findings have revealed their newfound significance in maintaining gut homeostasis and regulating inflammatory processes.

Aims

We investigated the dual roles of AhR and PXR activation. We started with dosage response experiments to determine optimal treatment conditions, such as treatment concentration and exposure time. Subsequently, we compared transcriptomic responses induced by chemical ligands with those from microbial metabolites, aiming to understand how AhR and PXR activation can yield both adverse and beneficial effects.

Methods

Using mouse 3D colonoids and 2D monolayer cultures, PXR and AhR were activated using the microbial metabolites indole-3-propionic acid (IPA) and indole-3-pyruvic acid (IPyA), respectively. Concentrations were determined through dosage response experiments. Changes to PXR and AhR target gene expression were measured using qPCR. We compared the gene induction by IPA and IPyA to responses driven by the chemical ligands pregnenolone 16α-carbonitrile (PCN) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), respectively.

Results

In 3D cell culture, PCN induced PXR target genes Cyp2c55 and Abcb1a, while IPA had no significant effect. Cyp3a11 was absent. Both IPyA and TCDD induced AhR genes, Cyp1a1 and Cyp1b1, with varying induction, and Cyp1a2 showed no response. In 2D cell culture, PCN significantly increased Cyp2c55 gene expression, but neither PCN nor IPA significantly induced Abcb1a. In 3D culture, TCDD significantly induced Cyp1a1 and Cyp1b1, while IPyA only induced Cyp1a1, revealing differences in AhR gene induction between 2D and 3D cell culture models.

Conclusions

Our research reveals contrasting gene induction patterns following AhR and PXR activation through microbial metabolites and chemical ligands. These varied responses highlight the multifaceted roles these receptors play within the gut environment. This study enhances our understanding of xenobiotic receptors' contributions to maintaining gut homeostasis and regulating inflammatory processes, shedding light on the interplay between ligands and receptors.

Methods

First, we isolate the crypts of mice to create 3D organoids, were we have access to the basolateral side. To capture the true biological system, we convert the 3D culture to 2D. This enables us to explore both the apical and basolateral sides, retaining the crucial polarity of the cells.

Funding Agencies

CIHR
A64 HUMAN INTESINAL ORGANOIDS AS A MODEL FOR GASTROINTESTINAL FUNCTION AND TOXICITY SCREENING
Authors: Stahl M , Leung D , Anderson W , Conlin V , Eaves A , Louis S A , Conder R K
Published: February 2024

Aims

Drug-induced gastrointestinal (GI) toxicity is a common adverse event in clinical trials, with symptoms including diarrhea, ulceration, and inflammation resulting from impaired barrier function. Furthermore, the intestine is a key mediator of both drug absorption and metabolism. The human colon carcinoma cell line (Caco-2) is the most common model used in drug discovery, however, intestinal organoids provide a model that better recapitulates the self-renewal, cellular composition, and function of the intestinal epithelium.This study utilizes human intestinal organoids as a high-throughput model for evaluating intestinal function and toxicity in preclinical drug discovery.

Methods

To assess toxicity, intestinal organoids were treated with two GI-toxic drugs, gefitinib and colchicine, and post-treatment cell viability was compared to Caco-2 control cultures. To assess barrier function, organoid-derived monolayers cultured in Transwell® plates were treated with colchicine and sapitinib, and post-treatment permeability was evaluated using the fluorescent low-permeability markers, 4 kDa FITC-dextran and Lucifer yellow.

Results

Human intestinal organoid cultures exhibited 14-fold greater sensitivity to gefitinib (average organoid IC50 = 0.82 μM, n = 3; Caco-2 IC50 =11.4 μM, n = 2) and 2,238 times greater sensitivity to colchicine (average organoid IC50 = 0.028 μM, n = 3; Caco-2 IC50 = 62.89 μM, n = 6) compared to Caco-2 controls. This 96-well assay was reproducible, producing comparable IC50 concentrations in repeated experiments with cells from the same and different donors (n = 3 donors). Organoid-derived monolayers exhibited ampersand:003E 10-fold (n = 3) increase in permeability to both sapitinib and colchicine compared to Caco-2 cultures, which displayed minimal responses to either drug treatment. Finally, organoid-derived monolayer cultures in transwell® plates can be utilized to quantify drug transport and metabolism by the intestinal epithelium. Using ileal organoid-derived monolayers and Caco-2 cultures, we quantified the active transport of methotrexate by the efflux transporter BCRP, calculating an efflux ratio of 11.46 from the basal to apical chambers. We also quantified CYP3A4 activity of ileal monolayers relative to Caco-2 cultures. Ileal monolayers converted midazolam to hydroxymidazolam ampersand:003E 50-fold (n = 2) more than Caco-2 cultures, which displayed minimal CYP3A4 activity below the limit of detection. Together, these data demonstrate that human intestinal organoids are a valuable tool for high-throughput GI toxicity screening and for evaluating intestinal function in preclinical drug discovery.

Conclusions

Together, these data demonstrate that human intestinal organoids are a valuable tool for high-throughput GI toxicity screening and for evaluating intestinal function in preclinical drug discovery.

Funding Agencies

None
A65 THE YAP-PER: CAN YAP/TAZ MEDIATE EPITHELIAL-IMMUNE CROSSTALK DURING PYLORIC METAPLASIA?
Authors: Sung J , Gregorieff A , Gruenheid S
Published: February 2024

Background

Gastric cancer is one of the leading causes of cancer-related deaths and its prevalence has been associated with infection by Helicobacter pylori. H. pylori persistence leads to chronic inflammation, which results in the elimination of acid-secreting parietal cells and the appearance of abnormal mucus-producing cells at the base of the gastric glands, known as spasmolytic polypeptide/trefoil factor 2-expressing metaplasia (SPEM) cells. The processes driving these alterations in epithelial cell fate during H. pylori infection and their function in regeneration of the gastric epithelium remain unclear. Previous work done in our lab have demonstrated that Hippo signalling, more specifically the transcriptional effectors yes-associated protein 1 (YAP) and transcription coactivator with PDZ-binding motif (TAZ), plays an essential role in controlling cell fate during gastric tissue regeneration in a chemical injury model. In the same model, loss of YAP/TAZ resulted in chronic immune cell infiltration in corpus glands, suggesting an immunomodulatory role for YAP and TAZ.

Aims

The objectives of this project is to characterize the immunomodulatory function of YAP/TAZ in an in vivo model of acute injury as well as an in vivo model of H. pylori infection.

Methods

Acute injury is induced by high-dose tamoxifen treatment in conditional knock-out (cKO) mice with YAP/TAZ deleted throughout the gastric epithelium. Subsequent immunophenotyping experiments such as flow cytometry and multiplex analysis are performed to characterize the role played by YAP/TAZ in regulating the immune response. For a more biologically relevant model, cKO mice are also infected with H. pylori, and similar immunophenotyping analyses are performed.

Results

Preliminary results showed that the loss of YAP/TAZ resulted in differential type II response as well as chronic B cell infiltration upon tissue injury.

Conclusions

This project aims to provide important insights on immune-epithelial cell interactions important for tissue regeneration and tumorigenesis as well as host-pathogen interactions implicated in H. pylori persistence.

Funding Agencies

CIHRFRQS
A66 EFFECTS OF HYMENOLEPIS DIMINUTA EXTRACELLULAR VESICLES ON MOUSE MACROPHAGES AND THEIR MITOCHONDRIA
Authors: Volk P , Allain T , Khan N , Wang A , Buret A G , McKay D
Published: February 2024

Background

In recent years, extracellular vesicles (EVs) have emerged as important means of intercellular communication and immune modulation between helminth and host. Earlier work revealed that excretory/secretory products from the rat tapeworm Hymenolepis diminuta (HES) have anti-inflammatory properties when applied to macrophages. In this study, we have investigated the immunomodulatory properties of H. diminuta EVs (HEVs) on macrophages.

Aims

1. To isolate and characterize bacterial contaminant-free H. diminuta EVs (Hd-EVs).

2. To determine the effects of Hd-EVs on murine macrophage metabolism and LPS-evoked cytokine production.

Methods

Adult H. diminuta were cultured for 6h in serum-free RMPI and Hd-EVs isolated from the conditioned medium using an exoEasy kit. Hd-EV protein content was measured using a micro-BCA kit, and size and quantity were measured using Nanosight tracking analysis. Murine bone marrow-derived macrophages or the human macrophage THP-1 cell line were treated with LPS (10 ng/ml) ± a crude H. diminuta extract (HdE), HES, or Hd-EVs for 24h. TNF-α levels in the culture medium was measured by ELISA. Macrophage oxidative phosphorylation (OX.PHOS) and glycolysis were measured in real-time using an Agilent Seahorse XF Analyzer.

Results

Analysis of 14 Hd-EV samples revealed 4.1 X 109 - 5.48 X 1010 particles/mL, with Hd-EVs predominantly 100-350 nm in diameter and with a protein content of 64.9-181.6 ng/mL. HdE and HES reduced TNF-α production in LPS-activated macrophages by 39-76% (n=11), while Hd-EVs did not lower TNF-α production. Metabolic analysis suggests that HES and Hd-EVs can up-regulate macrophage glycolysis, while only Hd-EVs exposed macrophages showed increased OX.PHOS (n=6).

Conclusions

We have successfully isolated and begun to characterize EVs from adult H. diminuta. Despite the known abilities of HES, we unexpectedly noted that exposure to Hd-EVs did not result in a statistically significant decrease in macrophage TNF-α release. However, the increase in OX.PHOS macrophages treated with Hd-EVs is something to examine further. The next steps are to determine the contents and immunomodulatory activity of the Hd-EVs, thus advancing knowledge of how helminths can manipulate their hosts.

Funding Agencies

NSERC

Clinical Practice
A67 REDUCING INAPPROPRIATE GAMMA-GLUTAMYL TRANSFERASE TESTING FOR INPATIENTS: A QUALITY IMPROVEMENT INITIATIVE IN LAB WASTE REDUCTION APPLYING THE MODEL FOR CONTINUOUS IMPROVEMENT
Authors: Afzaal T , AlRamdan R , Bualbanat H , Howarth N C , Malhi G , Hudson D , ChinYee I , Teriaky A
Published: February 2024

Background

Review of the literature identifies a rising trend in laboratory testing, with over 30% of tests estimated to be inappropriately repeated. Laboratory overutilization increases healthcare costs, and can lead to overdiagnosis, overtreatment and negative health outcomes. Indications for repeat Gamma Glutamyl Transferase (GGT) testing in adults are limited, particularly repeat testing within the same admission.

Aims

Our aim was to reduce the inappropriate ordering of repeat GGT testing by 25% for all inpatients at the London Health Sciences Centre (LHSC) over a one-year study period.

Methods

An interprofessional team was created to help engage relevant stakeholders, collect baseline data and reassess the indications for GGT testing. A combination of root cause analysis tools, specifically the Ishikawa diagram and Pareto chart, were employed to identify potential factors contributing to the overutilization of GGT testing. After prioritizing potential solutions, intervention bundles were developed, and Plan-Do-Study-Act (PDSA) cycles were created to target correctable factors. In PDSA cycle #1, the process started by eliminating GGT as a laboratory testing option in the three most commonly used admission order care sets. Considering the hierarchy of intervention effectiveness, PDSA cycle #2 involved implementing a computerized Clinical Decision Support (CDS) system to restrict the reordering of GGT tests within 72 hours of the same admission.

Results

Baseline data showed that in 2022, a total of 62,542 GGT tests were ordered, with an average of approximately 5,200 GGT tests ordered per month. Of these, 16.4% were ordered through the top 3 most prevalent admission order care sets, and around 25% of all GGT tests were repeats within 72 hours of admission. Referring to Figure 1, PDSA cycle #1 yielded no significant reduction in GGT testing. PDSA cycle #2 successfully reduced the proportion of repeat GGT tests ordered by 12% within two months of implementation, leading to an estimated annualized cost savings of approximately $37,440.

Conclusions

Our results establish the effectiveness of CDS systems in reducing laboratory testing overutilization, suggesting their superiority to individual care set targeting interventions, and emphasize the potential for cost-effective CDS development in contemporary healthcare.

Funding Agencies

None
A68 PERCEPTIONS OF ADOLESCENTS AND YOUNG ADULTS WITH INFLAMMATORY BOWEL DISEASE REGARDING A BIOPSYCHOSOCIAL TRANSITION INTERVENTION: A QUALITATIVE STUDY
Authors: Allemang B , Browne M , Barwick M , Bollegala N , Fu N , Lee K , Miatello A , Nistor I , Dekker E , Anthony S J , Benchimol E I
Published: February 2024

Background

The transition from the pediatric to adult care healthcare system for adolescents and young adults (AYAs) with inflammatory bowel disease (IBD) is complex. This group requires support to prepare for and adapt to adult care due to the manifestation of the disease, expectations for self-management, and simultaneous life transitions. To address this need, a multi-site randomized controlled trial (RCT) is being conducted to evaluate the impact of a multimodal transition intervention on functional, clinical, and psychosocial outcomes for AYAs with IBD. An embedded qualitative study explored participants’ perspectives of the intervention's acceptability and appropriateness to support the translation of findings into practice.

Aims

Using in-depth individual qualitative interviews with a purposive sample of AYAs in both the intervention and control arms of the RCT, this qualitative study aimed to understand AYAs’ experiences preparing for and undergoing service transitions, developing self-management skills, and engaging with the intervention.

Methods

Semi-structured interviews were held via videoconference with 21 AYAs aged 16-18 years participating in the RCT (intervention arm n=11; control arm n=10) in Toronto, Canada. Purposive sampling was used to recruit AYAs with diverse demographic and clinical characteristics at different stages of the healthcare transition. Interviews were audio-recorded, transcribed verbatim, and analyzed using an inductive approach to reflexive thematic analysis.

Results

The analysis yielded four overarching themes related to 1) individual, familial, and system-level factors promoting transition readiness, 2) areas of strength and areas for growth regarding self-management skill development, 3) perceptions of the multimodal transition intervention, including barriers and facilitators to engagement, and 4) recommendations for future transition care, including refinement of the intervention. Findings to date indicate the criticality of considering AYAs’ support systems, learning styles, and communication preferences when designing and implementing transition interventions. The qualitative data analysis is ongoing, and the full results will be presented.

Conclusions

The perspectives of AYAs with IBD can inform the development and refinement of clinical interventions, ensuring they are tailored to recipients’ unique needs and preferences and promote engagement and skill development during pediatric-adult transitions.

Funding Agencies

CCCThe Leona M. and Harry B. Helmsley Charitable Trust