Background: We aimed to examine the relationship between disease symptoms and disease phenotype in a large Canadian cohort of persons with Crohn’s disease (CD). Methods: Adults (n=1515) with CD from 14 Canadian centers participated in the Mind And Gut Interactions Cohort (MAGIC) between 2018 and 2023. Disease activity was measured using the 24-item IBD Symptom Inventory-Short-Form (IBDSI-SF). We compared the symptoms commonly associated with active versus inactive disease, and explored symptoms patterns in relation to disease phenotype, based on the Montreal Classification. To assess psychological status the Generalized Anxiety Disorder-7 and Patient Health Questionnaire-9 were used. Results: The mean disease duration was 15.6±11.8 years. The 5 most common symptoms were similar for those with active disease, although at higher prevalence (89% to 98%) versus those with inactive disease (47% to 79%), and included fatigue, diarrhea, gas, bloating, and urgency. The intensity of symptoms was higher in those with active than inactive IBDSI-SF scores. The rank order and relative distribution of the symptoms and intensity of the symptoms reported were similar between those with different disease phenotypes B1, B2, and B3 and L1, L2, and L3. Persons with active IBDSI-SF had a higher prevalence of anxiety (24.6%) and depression (38.2%) versus persons with inactive IBDSI-SF (6.3% and 8%, respectively) Conclusions: Individuals with CD with active and inactive disease by IBDSI, experience similar symptoms, but the prevalence of symptoms and their intensity is greater in persons with active IBDSI. Persons with inactive IBDSI report many symptoms. There was no difference in symptom reporting by disease behavior or location.

BACKGROUND:The Inflammation, Microbiome, and Alimentation: Gastro-Intestinal and Neuropsychiatric Effects Strategy for Patient Oriented Research Network (IMAGINE) has conducted a 5-year multicenter prospective observational cohort study, Mind And Gut Interactions Cohort (MAGIC) in 14 centers across Canada from 2018 to 2022. Herein, we investigated the relationship between ulcerative colitis (UC) phenotypes, demographics and other relevant outcomes, and symptom reporting. METHODS:At baseline, participants answered surveys assessing disease activity, medications and complementary therapies, lifestyle factors, psychological status, and comorbidities. UC phenotypes were classified by the Montreal Classification. Herein, we describe the association between phenotypes and demographics, medications used, comorbidities, and symptoms experienced in adults with UC. The Inflammatory Bowel Disease Symptom Inventory (IBDSI) was used to assess symptoms. RESULTS:The maximal extent phenotypic distribution based on chart review was E1 (proctitis) n=261 (14.5%), E2 (left-sided colitis) n=671 (37.2%), and E3 (subtotal or pancolitis) n=794 (44.0%). More males had E3. Different phenotypes did not lead to differences in the use of complementary therapies. There was greater likelihood of primary sclerosing cholangitis but a lower likelihood of hypertension in E3. Among the 25 different symptoms queried in the IBDSI, there was no difference across phenotypes, except among persons with overall active IBDSI, there was more waking for urges for bowel movements in persons with E3. CONCLUSIONS:Overall, there was no difference in symptom reporting based on extent of UC except for cohort with overall active IBDSI there were some differences in nocturnal waking based on disease extent.

Abstract Background Irritable bowel syndrome (IBS) is characterized by abdominal pain and altered bowel habits. IBS is more common in females. Furthermore, food and stress are two common triggers of abdominal pain in IBS patients. A low FODMAP diet (LFD) reduces abdominal pain in a subgroup of IBS patients, although the mechanism behind this is unclear. We hypothesize that a LFD improves abdominal pain by altering neuroactive gut luminal mediators. Aims 1. Explore the effect of a LFD on IBS symptoms and gut luminal mediator-induced neuronal activity. 2. Investigate whether stress alters IBS symptoms and luminal mediator-induced neuronal activity. 3. Examine sex differences in dorsal root ganglion (DRG) neuron sensitivity to luminal mediators. Methods Six female IBS patients followed a LFD for six weeks. Participants donated stool samples and completed the IBS Symptom Severity Scale (IBS-SSS) and the Depression, Anxiety, and Stress Scale (DASS-21) questionnaires. Male and female C57Bl6 mouse DRG neurons were incubated with IBS patient and healthy control (HC) fecal supernatant (FS). Capsaicin-induced Ca2+ influx was quantified to measure neuronal activity. N refers to number of mice. Results Overall, IBS FS collected prior to the LFD caused a 17% increase in neuronal activity compared to HC FS (N=32 mice, p<0.05). This finding was sex dependent, as only DRG neurons from female mice exhibited an IBS FS-induced increase in neuronal activity (N=18, p<0.005). There was no difference in neuronal activity caused by IBS FS versus HC FS in neurons from male mice (N=16, p>0.05). FS collected from patients after a LFD did not alter neuronal activity compared to FS collected before the LFD (N=32, p>0.05). However, when solely analyzing neurons from female mice, LFD FS-induced neuronal activity was lower than IBS FS-treated neuronal activity (N=16, p<0.05). Four IBS patients reported a clinically significant improvement in symptoms (>50-point reduction in IBS-SSS scores) during the LFD. Of these, two had a >50% reduction in IBS-SSS scores and their LFD FS reduced neuronal activity compared to their IBS FS (N=10, p<0.05). The two others had modest symptom improvement on the LFD; their LFD FS had similar effect on neuronal activity compared to their IBS FS (N=11, p>0.05). Additionally, in two IBS patients, periods of severe stress (DASS-21 stress sub-scale score >26) corresponded to high IBS-SSS scores; FS from these time points increased neuronal activity (N=10, p<0.05). Conclusions In a subgroup of IBS patients, a LFD improves symptoms and reduces the excitatory effects of luminal mediators on sensory neuronal activation. Stress appears to increase the excitatory effects of luminal mediators and exacerbates IBS symptoms. Finally, neurons from female mice may be more sensitive to pro-nociceptive luminal mediators, potentially contributing to abdominal pain in female IBS patients. Funding Agencies CIHR

Abdominal pain poses a significant challenge for individuals with inflammatory bowel disease (IBD). Despite current treatments that target inflammation, IBD-associated abdominal pain often persists even in the absence of inflammation, negatively impacting patients’ quality of life. This persistence suggests that factors other than inflammation may be contributing to the pathology of IBD. Our previous research suggests that bacterial proteases can directly influence the excitability of dorsal root ganglia neurons, many of which are pain-sensing. Building on this, we hypothesize that proteases, both of host and bacterial origin, play a role in pain modulation during the active and remission phases of IBD. The effects of fecal supernatants (FS) from patients with active or remissive IBD and healthy volunteers, on pain-sensing neurons were assessed using ex-vivo single-unit afferent nerve recordings from mouse colons. A protease inhibitor cocktail (PIC; 1:1000) and a protease-activated receptor (PAR)-2 antagonist (GB83; 10µM) were independently applied in the bioassay to determine whether these inhibited the excitatory effect of the FS. In addition, the participant FS were tested for proteolytic cleavage of the N-terminal domain of protease-activated receptor PAR2 using a novel enzymatic assay. FS from healthy volunteers [N=5] had no effect on afferent nerve excitability (p >0.05). FS from active IBD patients [N=15] increased action potential discharge from colonic afferent nerves by 85% (p< 0.0001) and selectively increased the activation of high-threshold units, which are putative nociceptors, by 44% (p< 0.01). A protease inhibitor cocktail and PAR2 antagonist both independently inhibited the excitatory effects of IBD FS (p >0.05) on afferent nerve activity. In contrast, FS from IBD patients in remission [N=15], did not excite colonic afferent nerves (p >0.05). Interestingly, these findings were found to be consistent when IBD was split into disease subtypes: Crohn’s disease and ulcerative colitis. Furthermore, when normalized to total protein content, active disease yielded significantly greater PAR2 cleavage activity (p< 0.05), while the remission samples were not significantly different than the healthy participants (p >0.05). This PAR2 cleavage activity was found to be correlated to neuronal excitation (R2=0.4721, p< 0.001). Our findings suggest that active IBD leads to the generation of luminal mediators, including proteases acting on PAR2, that activate visceral nociceptive neurons. These luminal mediators are less abundant when inflammation is in remission. These data suggest that targeting proteases could offer a promising therapeutic approach for pain management in IBD.

BACKGROUND:Many patients with irritable bowel syndrome (IBS) believe gluten or wheat triggers their symptoms. We compared symptomatic responses to wheat and gluten with gluten-free sham challenge in patients with IBS who previously perceived benefit from a gluten-free diet. METHODS:We conducted this randomised, double-blind, sham-controlled crossover study at McMaster University Medical Centre, ON, Canada. Eligible participants were adults aged 18 years or older who met Rome IV criteria for IBS and had previously self-reported improvement on a gluten-free diet, which was implemented for at least 3 weeks before enrolment. Eligible participants were randomly assigned (1:1:1:1:1:1) to receive one of six sequences of wheat, gluten, and sham (containing gluten and wheat free flour) in three periods of 7 days, separated by 14-day washout periods. Randomisation was done using the randomizeBE package in R and the cereal bars were designed to have the same appearance, taste, and smell to maintain blinding. The primary outcome was worsening of IBS symptoms of at least 50 points on the IBS Symptom Severity Score (IBS-SSS) after dietary challenges. Outcome and safety analyses were done in all patients who completed all three challenges. This trial was registered with ClinicalTrials.gov, NCT03664531. FINDINGS:Between Nov 15, 2018, and June 19, 2023, we assessed 101 people for eligibility. 72 people were excluded due to ineligibility (n=15), refusing screening (n=42), and refusing participation (n=15). 29 participants were enrolled and randomly assigned to wheat-gluten-sham (n=5), wheat-sham-gluten (n=5), gluten-wheat-sham (n=5), gluten-sham-wheat (n=5), sham-wheat-gluten (n=5), and sham-gluten-wheat (n=4). One participant in the wheat-sham-gluten group completed the first challenge but withdrew without providing a reason. In the 28 patients completing the study, there were no statistically significant differences in the proportion of participants with a worsening of IBS-SSS of at least 50 points after wheat (11 [39%] of 28 participants, risk difference vs sham 0·11; 95% CI -0·16 to 0·35) or gluten (ten participants [36%], 0·07; -0·19 to 0·32) versus sham (eight participants [29%]). Adverse events were reported in 26 (93%) of 28 patients after wheat, 26 patients (93%) after gluten, and 26 patients (93%) after sham. Study emergent adverse events were similar between challenges (five [18%] of 28 participants after wheat, five [18%] after gluten, and seven [25%] after sham). No patients reported severe adverse events. INTERPRETATION:IBS patients with self-perceived gluten sensitivity reacted similarly to gluten, wheat, and sham challenges. These findings suggest that expectations played a major role in symptom generation, and that only some of these patients could benefit from gluten or wheat restriction. Identifying this subset of patients while destigmatising wheat and gluten in the remaining ones should be considered for effective management of patients with IBS. FUNDING:Canadian Digestive Health Foundation, Society for the Study of Celiac Disease.

Background & Aims We recently showed that a bacterial infection can break oral tolerance to food and lead to immunoglobulin E (IgE)-dependent mast cell activation and food-induced abdominal pain, which could constitute an important pathogenic mechanism in postinfectious irritable bowel syndrome (IBS). Here, we investigated whether similar immune mechanisms in response to psychological stress lead to food-evoked pain signaling, and thus potentially explain the pathophysiology in a larger group of patients with IBS. Methods Mice were exposed to ovalbumin (OVA) during water avoidance stress (WAS) and re-exposed to OVA 5 weeks later. Nociception was evaluated by visceromotor responses and afferent nerve recordings to intestinal distension, and patch-clamp recordings of sensory neurons incubated with intestinal supernatants. The role of IgE and type 2 immunity was evaluated using pharmacologic and genetic approaches. Results Re-exposure to OVA increased pain signaling in the colon and small intestine only in mice exposed to OVA during WAS, in the absence of systemic allergy. OVA-induced increases in pain responses depended on mast cells, IgE, and signal transducer and activator of transcription 6 signaling. Notably, incubation of sensory neurons with ileum and colon supernatants from WAS/OVA+OVA mice lowered their threshold of excitability. Finally, treatment with histamine receptor H1 antagonist pyrilamine blocked the increased sensory neuron excitability, and reduced ileal afferent nerve firing to distension in WAS/OVA+OVA mice. Conclusions Psychological stress induces a type 2 immune response to food antigens, with IgE-mediated mast cell activation and increased pain signaling in the small intestine and colon in response to food. These findings may explain the potential role of psychological stress in food-induced symptoms in IBS.

Background Improved pain management is an identified top five priority for the treatment of patients suffering from inflammatory bowel disease. Although conventional opioids and NSAIDs can provide pain relief, their side effects pose serious health risks. We developed a unique pH-sensitive analgesic, NFEPP, that is only active at sites of injury/inflammation without affecting healthy tissues, e.g. brain and gut. Preclinical studies using parenteral routes(s.c. or i.v.) demonstrate that NFEPP exhibits potency comparable to fentanyl, but without the risks of addiction or severe side effects like respiratory depression. The duration of action of NFEPP is ~2 hrs, which would require continuous infusion or frequent applications to treat a painful disorder. To broaden the potential clinical applications, strategies are needed to increase its half-life and to understand its efficacy compared to conventional treatments. Aims To examine the therapeutic index(TI) of NFEPP and determine if a high index could be exploited to extend its duration of action, and to compare the analgesic efficacy of NFEPP with a standard NSAID, diclofenac. Methods Acute colitis was induced in C57BL/6 mice by administering 2.5% dextran sulfate sodium(DSS) in drinking water for five days. Following s.c. injections of vehicle, NFEPP, fentanyl or diclofenac, visceromotor responses (VMRs) to colorectal distension were measured using telemetric transmitters inserted in the abdominal wall muscles. To assess cardiorespiratory side effects, mice were monitored via pulse oximetry. Results NFEPP(0.4 mg s.c.) and fentanyl(0.4 mg s.c.) exhibited similar analgesic effects on VMR but fentanyl markedly inhibited O2 saturation (~10%; p<0.05), causing hypoxia. NFEPP at 50X’s this analgesic dose(20 mg/kg s.c.) had no effect on O2 saturation, suggesting a very high TI. We therefore examined whether the duration of action of NFEPP could be extended into the range of other oral analgesics by increasing the dose (doubling the dose increases T1/2 by ~<1, 1st order kinetics). NFEPP 0.4 mg/kg s.c. was tested at 2 hrs and 1.6 mg/kg s.c. at 6 hrs. NFEPP (1.6 mg/kg s.c.) caused a 37.8% (p<0.05) reduction in VMR responses after 6 hrs, similar to the effect of NFEPP 0.4 mg/kg at 2 hrs (36.8% reduction in VMR; p<0.01). Comparator studies of the efficacy of NFEPP(0.4 mg/kg s.c.; ~EC75 dose) and diclofenac(30 mg/kg s.c., ~EC100 dose) demonstrated a 54.2% (p<0.001) and 49.7% (p<0.01) reduction in VMR, respectively. Conclusions NFEPP has a very high TI compared to fentanyl, and this property could potentially be exploited to increase its half-life in oral and/or patch formulations by increasing the dose without risk of respiratory depression. NFEPP has similar or greater analgesic efficacy compared to NSAIDs and could potentially be an alternative for some clinical indications. Funding Agencies CIHR

There is an urgent need for analgesics to treat pain that lacks the serious side effects of existing drugs, such as conventional opioids and nonsteroidal anti-inflammatory drugs. Most side effects arise from the non-selective actions of these drugs at sites where the pain is not generated because of the ubiquitous expression of the drug targets in the body regardless of the underlying disease. In this narrative review, we explore 2 mechanistic approaches focusing on visceral nociceptive neurons that have the potential to limit side effects while preserving efficacy. Strategy 1 demonstrates how mechanistic pain studies underlying a specific disorder, such as irritable bowel syndrome, can identify targets specifically upregulated in that condition. We discuss recent findings regarding 2 neuroactive mediators, histamine and proteases, including novel intestinal sources, signalling pathways, and intracellular synergistic actions that could serve as potential therapeutic targets. Strategy 2 examines how acidic microenvironments unique to the sites of inflammation where pain is generated, such as in inflammatory bowel disease, can be exploited. pH-sensitive analgesics have been developed that inhibit μ-opioid receptors at sites of inflammation where tissue pH is low, ie, 6.5, while showing no activity at other sites where tissue pH is normal, ie, 7.4. Collectively, these studies highlight the value of investigating the mechanisms underlying specific disorders, which can lead to novel biomarkers and therapeutic strategies that can enhance the specificity of the new therapies.

Background: Irritable bowel syndrome (IBS) is a chronic abdominal pain disorder that affects women twice as often as men. While luminal mediators of both host and bacterial origin have been implicated in modulating abdominal pain in IBS patients, gonadal hormones have also been shown to influence pain signaling. Estrogen has been identified as a pronociceptive mediator that can modulate central and peripheral neural pathways. Given this, we hypothesized that the estrous cycle modulates sensory neuronal excitability, thereby altering the sensitivity to luminal mediators and this contributes to the female predominance of IBS. Aim: Identify the impact of the estrous cycle on nociceptive signaling and compare the effects of fecal supernatants (FS) from male and female IBS patients on abdominal pain pathways. Methods: Current clamp recordings measured rheobase and voltage clamp measured voltage-gated Na+ current in thoracolumbar dorsal root ganglia (DRG) neurons. FS from male and female IBS patients reporting high levels of abdominal pain were used. FS were perfused through male, female, and ovariectomized murine colonic preparations while performing extracellular colonic afferent nerve recordings to measure changes in action potential frequency during spontaneous firing and in response to colonic distension. Phase of estrous cycle in female mice was determined through analysis of vaginal swabs. Ovariectomies were performed 4 weeks prior to assays. Results: Current clamp recordings revealed an increase in excitability due to a 20% reduction in rheobase in DRG neurons taken from proestrus/estrus female mice compared to males, metestrus/diestrus females and ovariectomized females (p < 0.05). Voltage-gated Na+ current density was increased by 40% in neurons from proestrus/estrus mice compared to metestrus/diestrus female and male mice (p < 0.01). Extracellular afferent nerve recordings revealed that FS from female IBS patients reporting high abdominal pain (N=3) increased afferent nerve discharge (p < 0.05) in proestrus/estrus female mice by 70%. Single unit analysis of nociceptive axons showed that their activation was increased over 50% following FS perfusion. H owever, this excitatory effect was abolished in ovariectomized mice. Interestingly, FS from male IBS patients reporting high abdominal pain (N=4) had no effect in male mice while female IBS patient FS (N = 6) increased afferent nerve discharge (p< 0.05). Conclusion: This work suggests that the estrous cycle impacts abdominal pain signaling, which may contribute to the female predominance of IBS. This work is funded by CIHR and The Weston Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

BACKGROUND:There is compelling evidence that microbe-host interactions in the intestinal tract underlie many human disorders, including disorders of gut-brain interactions (previously termed functional bowel disorders), such as irritable bowel syndrome (IBS). Small intestinal bacterial overgrowth (SIBO) has been recognized for over a century in patients with predisposing conditions causing intestinal stasis, such as surgical alteration of the small bowel or chronic diseases, including scleroderma and is associated with diarrhea and signs of malabsorption. Over 20 years ago, it was hypothesized that increased numbers of small intestine bacteria might also account for symptoms in the absence of malabsorption in IBS and related disorders. This SIBO-IBS hypothesis stimulated significant research and helped focus the profession's attention on the importance of microbe-host interactions as a potential pathophysiological mechanism in IBS. PURPOSE:However, after two decades, this hypothesis remains unproven. Moreover, it has led to serious unintended consequences, namely the widespread use of unreliable and unvalidated breath tests as a diagnostic test for SIBO and a resultant injudicious use of antibiotics. In this review, we examine why the SIBO hypothesis remains unproven and, given the unintended consequences, discuss why it is time to reject this hypothesis and its reliance on breath testing. We also examine recent IBS studies of bacterial communities in the GI tract, their composition and functions, and their interactions with the host. While these studies provide important insights to guide future research, they highlight the need for further mechanistic studies of microbe-host interactions in IBS patients before we can understand their possible role in diagnosis and treatment of patient with IBS and related disorders.

Abdominal pain is a major symptom of diseases associated with microbial dysbiosis. Disruption of the gut microbiota with antibiotics increases visceral pain, and germ‐free mice are more prone to pain than conventionally‐raised mice. However, the mechanisms underlying microbial modulation of pain remain elusive. We hypothesized that disruption of the intestinal microbiota modulates the excitability of peripheral nociceptive neurons. Patch clamp electrophysiological recordings of dorsal root ganglion (DRG) neuron excitability were obtained from control mice and mice treated with the non‐absorbable antibiotic vancomycin (50 µg/ml in drinking water) for one week. Ten days prior to recording visceromotor response (VMR) telemetric transmitters were placed into the abdominal cavity of the mice and allowed to recover. VMR was measured by insertion of balloon catheter into the rectum under light anesthetization in both control and vancomycin treated mice, then distended to 80 mmHg and VMR recorded. Bacterial dysbiosis was verified by metagenomic analysis of stool microbial composition. Mice treated with vancomycin were more sensitive to colorectal distension in vivo (VMR increased by 70% at 80 mmHg compared to control), and DRG neurons from vancomycin‐treated mice were hyperexcitable in vitro compared to water‐treated controls (rheobase decreased by 30% relative to control). Interestingly, hyperexcitability of DRG neurons was not restricted to gut projecting neurons, suggesting a widespread effect of gut dysbiosis on pain pathways. Incubation of DRG neurons from naïve mice in serum from vancomycin‐treated mice increased neuron excitability (rheobase decreased by 30% relative to control), suggesting that microbial dysbiosis alters circulating mediators that influence nociception. Multiplex ELISA measurements did not detect any significant changes in serum cytokines or chemokines between vancomycin‐treated and control mice. The cysteine protease inhibitor E64 (30 nM) and the protease‐activated receptor 2 (PAR2) antagonist GB‐83 (10 µM) each blocked the increase in DRG neuron excitability in response to serum from vancomycin‐treated mice. Naïve DRG neurons incubated with fecal supernatants from vancomycin‐treated mice also exhibited increased excitability (rheobase decreased by 40% relative to control), but supernatants derived from colonic tissue failed to cause hyperexcitability. Overall, this data suggests that microbial dysbiosis within the gut alters pain sensitivity. This effect is not caused by inflammation or host derived factors, rather bacterially‐derived cysteine proteases activating PAR2 on DRG neurons.