Clinical Presentation and Outcomes of Alpha-Gal Syndrome.

BACKGROUND AND AIMS: Alpha-gal syndrome (AGS) is an IgE-mediated allergic reaction to galactose-α-1,3-galactose, primarily linked with Lone Star tick bites in the United States. It presents with symptoms ranging from urticaria and gastrointestinal (GI) manifestations to delayed anaphylaxis following red meat consumption. We aimed to study AGS patients' clinical manifestations, diagnosis, and outcomes. METHODS: A retrospective chart review of patients who underwent serological testing for suspected AGS between 2014 and 2023 at Mayo Clinic was performed. Patients with positive serology were age and sex matched with those who tested negative. Clinical characteristics of seropositive cohort with and without GI symptoms were compared, and outcomes assessed. RESULTS: Of 1260 patients who underwent testing, 124 tested positive for AGS. They were matched with 380 seronegative control subjects. AGS patients reported a higher frequency of tick bites (odds ratio [OR], 26.0; 95% confidence interval [CI], 9.8-68.3), reported a higher prevalence of urticaria (56% vs 37%; P = .0008), and were less likely to have asthma (OR, 0.4; 95% CI, 0.3-0.7). They had a lower prevalence of heartburn (6% vs 12%; P = .03) and bloating (6% vs 13%; P = .03). A total of 47% had GI symptoms, and a higher proportion were female than those without GI symptoms (69% vs 35%; P = .002). During a mean follow-up of 27 months, 22 of 40 patients reported symptom resolution after avoiding red meat, and 7 were able to transition to regular diet. CONCLUSIONS: A diagnosis of AGS should be strongly considered in patients with a history of tick bites and clinical presentation of allergic or GI manifestations. Dietary intervention is effective in most but not all patients.

Transcriptomic and Metabolomic Correlates of Increased Colonic Permeability in Postinfection Irritable Bowel Syndrome.

BACKGROUND & AIMS: Postinfection irritable bowel syndrome (PI-IBS) is well-known epidemiologically; however, its physiological and molecular characteristics are not well studied. We aimed to determine the physiological phenotypes, colonic transcriptome, fecal microbiome, and metabolome in PI-IBS. METHODS: Fifty-one Rome III Campylobacter PI-IBS patients and 39 healthy volunteers (HV) were enrolled. Participants completed questionnaires, in vivo intestinal permeability, gastrointestinal transit, and rectal sensation. Fecal samples were collected for shotgun metagenomics, untargeted metabolomics, and sigmoid colonic biopsies for bulk RNAseq. Differential gene expression, differences in microbiota composition, and metabolite abundance were determined. Gene and metabolite clusters were identified via weighted gene correlation network analysis and correlations with clinical and physiological parameters determined. RESULTS: PI-IBS (59% female; 46 ± 2 years) and HV (64% female; 42 ± 2 years) demographics were comparable. Mean IBS-symptom severity score was 227; 94% were nonconstipation. Two- to 24-hour lactulose excretion was increased in PI-IBS, suggesting increased colonic permeability (4.4 ± 0.5 mg vs 2.6 ± 0.3 mg; P = .01). Colonic transit and sensory thresholds were similar between the 2 groups. Overall, expression of 2036 mucosal genes and 223 fecal metabolites were different, with changes more prominent in females. Fecal N-acetylputrescine was increased in PI-IBS and associated with colonic permeability, worse diarrhea, and negatively correlated with abundance of Collinsella aerofaciens. Histamine and N-acetylhistamine positively associated with 2- to 24-hour lactulose excretion. Eight weighted gene coexpression modules significantly correlated with phenotypes (sex, stool frequency, colonic permeability, transit). CONCLUSIONS: Campylobacter PI-IBS patients demonstrate higher colonic permeability, which associated with changes in polyamine and histamine metabolites. Female patients demonstrated greater molecular changes.

High-Dimensional Clustering of 4000 Irritable Bowel Syndrome Patients Reveals Seven Distinct Disease Subsets.

BACKGROUND AND AIMS: Irritable bowel syndrome (IBS) is a pain disorder classified by bowel habits, disregarding other factors that may influence the clinical course. The aim of this study was to determine if IBS patients can be clustered based on clinical, dietary, lifestyle, and psychosocial factors. METHODS: Between 2013 and 2020, the Mayo Clinic Biobank surveyed and received 40,291 responses to a questionnaire incorporating Rome III criteria. Factors associated with IBS were determined and latent class analysis, a model-based clustering, was performed on IBS cases. RESULTS: We identified 4021 IBS patients (mean 64 years; 75% women) and 12,063 controls. Using 26 variables separating cases from controls, the optimal clustering revealed 7 latent clusters. These were characterized by perceived health impairment (moderate or severe), psychoneurological factors, and bowel dysfunction (diarrhea or constipation predominance). Health impairment clusters demonstrated more pain, with the severe cluster also having more psychiatric comorbidities. The next 3 clusters had unique enrichment of psychiatric, neurological, or both comorbidities. The bowel dysfunction clusters demonstrated less abdominal pain, with diarrhea cluster most likely to report pain improvement with defecation. The constipation cluster had the highest exercise score and consumption of fruits, vegetables, and alcohol. The distribution of clusters remained similar when Rome IV criteria were applied. Physiologic tests were available on a limited subset (6%), and there were no significant differences between clusters. CONCLUSIONS: In this cohort of older IBS patients, 7 distinct clusters were identified demonstrating varying degrees of gastrointestinal symptoms, comorbidities, dietary, and lifestyle factors. Further research is required to assess whether these unique clusters could be used to direct clinical trials and individualize patient management.

Serine proteases as luminal mediators of intestinal barrier dysfunction and symptom severity in IBS.

OBJECTIVE: The intestinal lumen contains several proteases. Our aim was to determine the role of faecal proteases in mediating barrier dysfunction and symptoms in IBS. DESIGN: 39 patients with IBS and 25 healthy volunteers completed questionnaires, assessments of in vivo permeability, ex vivo colonic barrier function in Ussing chambers, tight junction (TJ) proteins, ultrastructural morphology and 16 s sequencing of faecal microbiota rRNA. A casein-based assay was used to measure proteolytic activity (PA) in faecal supernatants (FSNs). Colonic barrier function was determined in mice (ex-germ free) humanised with microbial communities associated with different human PA states. RESULTS: Patients with IBS had higher faecal PA than healthy volunteers. 8/20 postinfection IBS (PI-IBS) and 3/19 constipation- predominant IBS had high PA (>95th percentile). High-PA patients had more and looser bowel movements, greater symptom severity and higher in vivo and ex vivo colonic permeability. High-PA FSNs increased paracellular permeability, decreased occludin and increased phosphorylated myosin light chain (pMLC) expression. Serine but not cysteine protease inhibitor significantly blocked high-PA FSN effects on barrier. The effects on barrier were diminished by pharmacological or siRNA inhibition of protease activated receptor-2 (PAR-2). Patients with high-PA IBS had lower occludin expression, wider TJs on biopsies and reduced microbial diversity than patients with low PA. Mice humanised with high-PA IBS microbiota had greater in vivo permeability than those with low-PA microbiota. CONCLUSION: A subset of patients with IBS, especially in PI-IBS, has substantially high faecal PA, greater symptoms, impaired barrier and reduced microbial diversity. Commensal microbiota affects luminal PA that can influence host barrier function.

Human cryptosporidiosis caused by Cryptosporidium tyzzeri and C. parvum isolates presumably transmitted from wild mice.

We report a case of severe human cryptosporidiosis caused by Cryptosporidium tyzzeri and C. parvum with an unusually high frequency of liquid stools. Wild mice were the most likely source of infection, demonstrating the potential for wild-mouse-borne Cryptosporidium to infect humans and highlighting the health risks associated with synantropic rodents.

Gut microbial ß-glucuronidases regulate host luminal proteases and are depleted in irritable bowel syndrome.

Intestinal proteases mediate digestion and immune signalling, while increased gut proteolytic activity disrupts the intestinal barrier and generates visceral hypersensitivity, which is common in irritable bowel syndrome (IBS). However, the mechanisms controlling protease function are unclear. Here we show that members of the gut microbiota suppress intestinal proteolytic activity through production of unconjugated bilirubin. This occurs via microbial ß-glucuronidase-mediated conversion of bilirubin conjugates. Metagenomic analysis of faecal samples from patients with post-infection IBS (n = 52) revealed an altered gut microbiota composition, in particular a reduction in Alistipes taxa, and high gut proteolytic activity driven by specific host serine proteases compared with controls. Germ-free mice showed 10-fold higher proteolytic activity compared with conventional mice. Colonization with microbiota samples from high proteolytic activity IBS patients failed to suppress proteolytic activity in germ-free mice, but suppression of proteolytic activity was achieved with colonization using microbiota from healthy donors. High proteolytic activity mice had higher intestinal permeability, a higher relative abundance of Bacteroides and a reduction in Alistipes taxa compared with low proteolytic activity mice. High proteolytic activity IBS patients had lower fecal ß-glucuronidase activity and end-products of bilirubin deconjugation. Mice treated with unconjugated bilirubin and ß-glucuronidase-overexpressing E. coli significantly reduced proteolytic activity, while inhibitors of microbial ß-glucuronidases increased proteolytic activity. Together, these data define a disease-relevant mechanism of host-microbial interaction that maintains protease homoeostasis in the gut.

Post-infection Irritable Bowel Syndrome.

Epidemiologic data support that acute gastrointestinal infection is one of the strongest risk factors for development of irritable bowel syndrome (IBS). Risk of post-infection IBS (PI-IBS) seems to be greater with bacterial and protozoal than viral enterocolitis. Younger individuals, women, and those with severe enterocolitis are more likely to develop PI-IBS. Disease mechanisms in animal models and humans involve chronic perturbation of intestinal microbiome, epithelial and neuronal remodeling, and immune activation. These mechanisms can lead to luminal (increased proteolytic activity, altered bile acid composition) and physiologic (increased permeability, transit changes, and visceral hypersensitivity) alterations that can mediate PI-IBS symptoms.

Colonic expression of Ace2, the SARS-CoV-2 entry receptor, is suppressed by commensal human microbiota.

Infection with severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Angiotensin-converting enzyme 2 (Ace2) is expressed in the gastrointestinal (GI) tract and a receptor for SARS-CoV-2, making the GI tract a potential infection site. This study investigated the effects of commensal intestinal microbiota on colonic Ace2 expression using a humanized mouse model. We found that colonic Ace2 expression decreased significantly upon microbial colonization. Humanization with healthy volunteer or dysbiotic microbiota from irritable bowel syndrome (IBS) patients resulted in similar Ace2 expression. Despite the differences in microbiota, no associations between α-diversity, ß-diversity or individual taxa, and Ace2 were noted post-humanization. These results highlight that commensal microbiota play a key role in regulating intestinal Ace2 expression and the need to further examine the underlying mechanisms of this regulation.

Intestinal barrier dysfunction in irritable bowel syndrome: a systematic review.

BACKGROUND AND AIM: Irritable bowel syndrome (IBS) is a complex and heterogeneous disorder. Sensory, motor and barrier dysfunctions are the key physiological endophenotypes of IBS. Our aim is to review studies evaluating barrier dysfunction in adults and children with IBS, as well as to link those changes with IBS symptomatology and quality of life. METHODS: A comprehensive and systematic review of multiple databases was performed up to March 2020 to identify studies comparing intestinal permeability in IBS patients with healthy controls. Both in vivo and in vitro studies were considered. RESULTS: We identified 66 studies, of which 27 used intestinal probes to quantify barrier function. The prevalence of barrier dysfunction differed between PI-IBS (17-50%), IBS-D (37-62%) and IBS-C (4-25%). At a group level, permeability was increased compared with healthy controls in IBS-D (9/13 studies) and PI-IBS (4/4 studies), but only a minority of IBS-C (2/7 studies) and not in the only IBS-M study. All four studies in children with IBS demonstrated loss of barrier function. A heterogeneous set of tight junction genes were found to be altered in small and large intestines of adults with IBS, but these have not been evaluated in children. Positive associations were identified between barrier dysfunction and bowel disturbances (6/9 studies), abdominal pain (9/13 studies), overall symptom severity (1/6 studies), depression and anxiety (1/1 study) and quality of life (1/4 studies). Fecal slurry or supernatants of IBS patients were found to induce barrier disruption in animal models (5/6 studies). CONCLUSIONS: Barrier dysfunction is present in a significant proportion of adult and all pediatric IBS studies, especially in the IBS-D and PI-IBS subtype. The majority of studies indicated a positive association between loss of barrier function and symptoms such as abdominal pain and changes in the bowel function.

Campylobacter jejuni genotypes are associated with post-infection irritable bowel syndrome in humans.

Campylobacter enterocolitis may lead to post-infection irritable bowel syndrome (PI-IBS) and while some C. jejuni strains are more likely than others to cause human disease, genomic and virulence characteristics promoting PI-IBS development remain uncharacterized. We combined pangenome-wide association studies and phenotypic assays to compare C. jejuni isolates from patients who developed PI-IBS with those who did not. We show that variation in bacterial stress response (Cj0145_phoX), adhesion protein (Cj0628_CapA), and core biosynthetic pathway genes (biotin: Cj0308_bioD; purine: Cj0514_purQ; isoprenoid: Cj0894c_ispH) were associated with PI-IBS development. In vitro assays demonstrated greater adhesion, invasion, IL-8 and TNFα secretion on colonocytes with PI-IBS compared to PI-no-IBS strains. A risk-score for PI-IBS development was generated using 22 genomic markers, four of which were from Cj1631c, a putative heme oxidase gene linked to virulence. Our finding that specific Campylobacter genotypes confer greater in vitro virulence and increased risk of PI-IBS has potential to improve understanding of the complex host-pathogen interactions underlying this condition.

Measurement of novel intestinal secretory and barrier pathways and effects of proteases.

The epithelial lining of the gastrointestinal (GI) tract in conjunction with the enteric nervous system (ENS) plays an important role in mediating solute absorption and secretion. A dysregulated ionic movement across the epithelium can result in GI diseases that manifest as either watery diarrhea or constipation. Hirschsprung disease is an example of an ENS disorder characterized by absence of enteric ganglia in distal gut resulting in obstructive phenotype. Receptor rearranged during transfection (RET) gene variants are the most commonly recognized genetic associations with Hirschsprung disease. In this issue of Neurogastroenterology and Motility, Russell et al demonstrate that RET mediates colonic ion transport through modulation of cholinergic nerves. They go on to show inhibition of RET can attenuate accelerated transit in a rat model. Normalizing secretory and absorptive defects has been an attractive therapeutic strategy. In addition to the intrinsic regulation of secretory processes, luminal mediators like bile acids, short-chain fatty acids, and proteases can affect both secretion and barrier function of the intestinal epithelium. Elevated levels of proteases have been identified in a wide range of GI diseases including irritable bowel syndrome. Proteases are known to cause visceral hypersensitivity and barrier disruption in vitro and in animal models. The goals of this review are to describe fundamental concepts related to intestinal epithelial secretion, the utility of Ussing chambers to measure ionic mechanisms and to discuss examples of novel signaling pathways; namely the RET signaling cascade in secretomotor neurons and effects of luminal proteases on barrier and ionic secretion.

Glycoproteins and Gal-GalNAc cause Cryptosporidium to switch from an invasive sporozoite to a replicative trophozoite.

The apicomplexan parasite Cryptosporidium causes cryptosporidiosis, a diarrheal disease that can become chronic and life threatening in immunocompromised and malnourished people. There is no effective drug treatment for those most at risk of severe cryptosporidiosis. The disease pathology is due to a repeated cycle of host cell invasion and parasite replication that amplifies parasite numbers and destroys the intestinal epithelium. This study aimed to better understand the Cryptosporidium replication cycle by identifying molecules that trigger the switch from invasive sporozoite to replicative trophozoite. Our approach was to treat sporozoites of Cryptosporidium parvum and Cryptosporidium hominis, the species causing most human cryptosporidiosis, with various media under axenic conditions and examine the parasites for rounding and nuclear division as markers of trophozoite development and replication, respectively. FBS had a concentration-dependent effect on trophozoite development in both species. Trophozoite development in C. parvum, but not C. hominis, was enhanced when RPMI supplemented with 10% FBS (RPMI-FBS) was conditioned by HCT-8 cells for 3h. The effect of non-conditioned and HCT-8 conditioned RPMI-FBS on trophozoite development was abrogated by proteinase K and sodium metaperiodate pretreatment, indicating a glycoprotein trigger. Cryptosporidium parvum and C. hominis trophozoite development also was triggered by Gal-GalNAc in a concentration-dependent manner. Cryptosporidium parvum replication was greatest following treatments with Gal-GalNAc, followed by conditioned RPMI-FBS and non-conditioned RPMI-FBS (P<0.05). Cryptosporidium hominis replication was significantly less than that in C. parvum for all treatments (P<0.05), and was greatest at the highest tested concentration of Gal-GalNAc (1mM).