Epithelial 5-HT4 Receptors as a Target for Treating Constipation and Intestinal Inflammation.

Because of their importance in the regulation of gut functions, several therapeutic targets involving serotonin-related proteins have been developed or repurposed to treat motility disorders, including serotonin transporter inhibitors, tryptophan hydroxylase blockers, 5-HT3 antagonists, and 5-HT4 agonists. This chapter focuses on our discovery of 5-HT4 receptors in the epithelial cells of the colon and our efforts to evaluate the effects of stimulating these receptors. 5-HT4 receptors appear to be expressed by all epithelial cells in the mouse colon, based on expression of a reporter gene driven by the 5-HT4 receptor promoter. Application of 5-HT4 agonists to the mucosal surface causes serotonin release from enterochromaffin cells, mucus secretion from goblet cells, and chloride secretion from enterocytes. Luminal administration of 5-HT4 agonists speeds up colonic motility and suppresses distention-induced nociceptive responses. Luminal administration of 5-HT4 agonists also decreases the development of, and improves recovery from, experimental colitis. Recent studies determined that the prokinetic actions of minimally absorbable 5-HT4 agonists are just as effective as absorbable compounds. Collectively, these findings indicate that targeting epithelial receptors with non-absorbable 5-HT4 agonists could offer a safe and effective strategy for treating constipation and colitis.

Protective actions of a luminally acting 5-HT4 receptor agonist in mouse models of colitis.

BACKGROUND: 5-hydroxytryptamine 4 receptors (5-HT4 Rs) are expressed in the colonic epithelium, and previous studies have demonstrated that luminal administration of agonists enhances motility, suppresses nociception, and is protective in models of inflammation. We investigated whether stimulation with a luminally acting 5-HT4 R agonist is comparable to previously tested absorbable compounds. METHODS: The dextran sodium sulfate (DSS), trinitrobenzene sulfonic acid (TNBS), and interleukin 10 knockout (IL-10KO) models of colitis were used to test the protective effects of the luminally acting 5-HT4 R agonist, 5HT4-LA1, in the absence and presence of a 5-HT4 R antagonist. The compounds were delivered by enema to mice either before (prevention) or after (recovery) the onset of active colitis. Outcome measure included disease activity index (DAI) and histological evaluation of colon tissue, and effects on wound healing and fecal water content were also assessed. KEY RESULTS: Daily enema of 5HT4-LA1 attenuated the development of, and accelerated recovery from, active colitis. Enema administration of 5HT4-LA1 did not attenuate the development of colitis in 5-HT4 R knockout mice. Stimulation of 5-HT4 Rs with 5HT4-LA1 increased Caco-2 cell migration (accelerated wound healing). Daily administration of 5HT4-LA1 did not increase fecal water content in active colitis. CONCLUSIONS AND INFERENCES: Luminally restricted 5-HT4 R agonists are comparable to absorbable compounds in attenuating and accelerating recovery from active colitis. Luminally acting 5-HT4 R agonists may be useful as an adjuvant to current inflammatory bowel disease (IBD) treatments to enhance epithelial healing.

Interplay of broccoli/broccoli sprout bioactives with gut microbiota in reducing inflammation in inflammatory bowel diseases.

Inflammatory Bowel Diseases (IBD) are chronic, reoccurring, and debilitating conditions characterized by inflammation in the gastrointestinal tract, some of which can lead to more systemic complications and can include autoimmune dysfunction, a change in the taxonomic and functional structure of microbial communities in the gut, and complicated burdens in a person's daily life. Like many diseases based in chronic inflammation, research on IBD has pointed towards a multifactorial origin involving factors of the person's lifestyle, immune system, associated microbial communities, and environmental conditions. Treatment currently exists only as palliative care, and seeks to disrupt the feedback loop of symptoms by reducing inflammation and allowing as much of a return to homeostasis as possible. Various anti-inflammatory options have been explored, and this review focuses on the use of diet as an alternative means of improving gut health. Specifically, we highlight the connection between the role of sulforaphane from cruciferous vegetables in regulating inflammation and in modifying microbial communities, and to break down the role they play in IBD.

Early life exposure to broccoli sprouts confers stronger protection against enterocolitis development in an immunological mouse model of inflammatory bowel disease.

Crohn's Disease (CD) is a presentation of Inflammatory Bowel Disease (IBD) that manifests in childhood and adolescence, and involves chronic and severe enterocolitis, immune and gut microbiome dysregulation, and other complications. Diet and gut-microbiota-produced metabolites are sources of anti-inflammatories which could ameliorate symptoms. However, questions remain on how IBD influences biogeographic patterns of microbial location and function in the gut, how early life transitional gut communities are affected by IBD and diet interventions, and how disruption to biogeography alters disease mediation by diet components or microbial metabolites. Many studies on diet and IBD use a chemically induced ulcerative colitis model, despite the availability of an immune-modulated CD model. Interleukin-10-knockout (IL-10-KO) mice on a C57BL/6 background, beginning at age 4 or 7 weeks, were fed a control diet or one containing 10% (w/w) raw broccoli sprouts, which was high in the sprout-sourced anti-inflammatory sulforaphane. Diets began 7 days prior to, and for 2 weeks after inoculation with Helicobacter hepaticus, which triggers Crohn's-like symptoms in these immune-impaired mice. The broccoli sprout diet increased sulforaphane in plasma; decreased weight stagnation, fecal blood, and diarrhea associated; and increased microbiota richness in the gut, especially in younger mice. Sprout diets resulted in some anatomically specific bacteria in younger mice, and reduced the prevalence and abundance of pathobiont bacteria which trigger inflammation in the IL-10-KO mouse, for example; Escherichia coli and Helicobacter. Overall, the IL-10-KO mouse model is responsive to a raw broccoli sprout diet and represents an opportunity for more diet-host-microbiome research.

Early life exposure to broccoli sprouts confers stronger protection against enterocolitis development in an immunological mouse model of inflammatory bowel disease.

IMPORTANCE: To our knowledge, IL-10-KO mice have not previously been used to investigate the interactions of host, microbiota, and broccoli, broccoli sprouts, or broccoli bioactives in resolving symptoms of CD. We showed that a diet containing 10% raw broccoli sprouts increased the plasma concentration of the anti-inflammatory compound sulforaphane and protected mice to varying degrees against disease symptoms, including weight loss or stagnation, fecal blood, and diarrhea. Younger mice responded more strongly to the diet, further reducing symptoms, as well as increased gut bacterial richness, increased bacterial community similarity to each other, and more location-specific communities than older mice on the diet intervention. Crohn's disease disrupts the lives of patients and requires people to alter dietary and lifestyle habits to manage symptoms. The current medical treatment is expensive with significant side effects, and a dietary intervention represents an affordable, accessible, and simple strategy to reduce the burden of symptoms.

Prokinetic actions of luminally acting 5-HT4 receptor agonists.

BACKGROUND: 5-HT4 receptor (5-HT4 R) agonists exert prokinetic actions in the GI tract, but non-selective actions and potential for stimulation of non-target 5-HT4 Rs have limited their use. Since 5-HT4 Rs are expressed in the colonic epithelium and their stimulation accelerates colonic propulsion in vitro, we tested whether luminally acting 5-HT4 R agonists promote intestinal motility. METHODS: Non-absorbed 5-HT4 R agonists, based on prucalopride and naronapride, were assessed for potency at the 5-HT4 R in vitro, and for tissue and serum distribution in vivo in mice. In vivo assessment of prokinetic potential included whole gut transit, colonic motility, fecal output, and fecal water content. Colonic motility was also studied ex vivo in mice treated in vivo. Immunofluorescence was used to evaluate receptor distribution in human intestinal mucosa. KEY RESULTS: Pharmacological screening demonstrated selectivity and potency of test agonists for 5-HT4 R. Bioavailability studies showed negligible serum detection. Gavage of agonists caused faster whole gut transit and colonic motility, increased fecal output, and elevated fecal water content. Prokinetic actions were blocked by a 5-HT4 R antagonist and were not detected in 5-HT4 R knockout mice. Agonist administration promoted motility in models of constipation. Evaluation of motility patterns ex vivo revealed enhanced contractility in the middle and distal colon. Immunoreactivity for 5-HT4 R is present in the epithelial layer of the human small and large intestines. CONCLUSIONS AND INFERENCES: These findings demonstrated that stimulation of epithelial 5-HT4 Rs can potentiate propulsive motility and support the concept that mucosal 5-HT4 Rs could represent a safe and effective therapeutic target for the treatment of constipation.

Saccharomyces cerevisiae as a superior host for overproduction of prokaryotic integral membrane proteins.

Integral membrane proteins (IMPs) constitute ~30% of all proteins encoded by the genome of any organism and Escherichia coli remains the first-choice host for recombinant production of prokaryotic IMPs. However, the expression levels of prokaryotic IMPs delivered by this bacterium are often low and overproduced targets often accumulate in inclusion bodies. The targets are therefore often discarded to avoid an additional and inconvenient refolding step in the purification protocol. Here we compared expression of five prokaryotic (bacterial and archaeal) IMP families in E. coli and Saccharomyces cerevisiae. We demonstrate that our S. cerevisiae-based production platform is superior in expression of four investigated IMPs, overall being able to deliver high quantities of active target proteins. Surprisingly, in case of the family of zinc transporters (Zrt/Irt-like proteins, ZIPs), S. cerevisiae rescued protein expression that was undetectable in E. coli. We also demonstrate the effect of localization of the fusion tag on expression yield and sample quality in detergent micelles. Lastly, we present a road map to achieve the most efficient expression of prokaryotic IMPs in our yeast platform. Our findings demonstrate the great potential of S. cerevisiae as host for high-throughput recombinant overproduction of bacterial and archaeal IMPs for downstream biophysical characterization.

Proton Stereotactic Radiosurgery for Brain Metastases: A Single-Institution Analysis of 370 Patients.

PURPOSE: To report the first series of proton stereotactic radiosurgery (SRS) for the treatment of patients with single or multiple brain metastases, including failure patterns, survival outcomes, and toxicity analysis. METHODS AND MATERIALS: This was a single-institution, retrospective study of 815 metastases from 370 patients treated with proton SRS between April 1991 and November 2016. Cumulative incidence estimates of local failure, distant brain failure, and pathologically confirmed radionecrosis and Kaplan-Meier estimates of overall survival were calculated. Fine and Gray and Cox regressions were performed to ascertain whether clinical and treatment factors were associated with the described endpoints. RESULTS: The median follow-up from proton SRS was 9.2 months. The 6- and 12-month estimates of local failure, distant brain failure, and overall survival were 4.3% (95% confidence interval [CI] 3.0%-5.9%) and 8.5% (95% CI 6.7%-10.6%), 39.1% (95% CI 34.1%-44.0%) and 48.2% (95% CI 43.0%-53.2%), and 76.0% (95% CI 71.3%-80.0%) and 51.5% (95% CI 46.3%-56.5%), respectively. The median survival was 12.4 months (95% CI 10.8-14.0 months) after proton SRS. The most common symptoms were low-grade fatigue (12.5%), headache (10.0%), motor weakness (6.2%), seizure (5.8%), and dizziness (5.4%). The rate of pathologically confirmed radionecrosis at 12 months was 3.6% (95% CI 2.0%-5.8%), and only target volume was associated on multivariate analysis (subdistribution hazard ratio 1.13, 95% CI 1.0-1.20). CONCLUSIONS: To the best of our knowledge, this is the first reported series of proton SRS for the management of brain metastases. Moderate-dose proton SRS is well tolerated and can achieve good local control outcomes, comparable to those obtained with conventional photon SRS strategies. Although proton SRS remains resource-intensive, future strategies evaluating its selective utility in patients who would benefit most from integral dose reduction should be explored.