Development of a self-limiting model of methotrexate-induced mucositis reinforces butyrate as a potential therapy.

Gastrointestinal mucositis is a complication of anticancer treatment, with few validated in vitro systems suitable to study the complex mechanisms of mucosal injury. Therefore, we aimed to develop and characterize a chemotherapeutic-induced model of mucositis using 3D intestinal organoids. Organoids derived from mouse ileum were grown for 7 days and incubated with different concentrations of the chemotherapeutic agent methotrexate (MTX). Metabolic activity, citrulline levels and cytokine/chemokine production were measured to determine the optimal dosage and incubation time. The protective effects of folinic acid on the toxicity of MTX were investigated by pre-treating organoids with (0.0005-50 µg/mL) folinic acid. The impact of microbial-derived short-chain fatty acids was evaluated by supplementation with butyrate in the organoid model. MTX caused a dose-dependent reduction in cell metabolic activity and citrulline production that was salvaged by folinic acid treatment. Overall, MTX causes significant organoid damage, which can be reversed upon removal of MTX. The protective effect of folinic acid suggest that the organoids respond in a clinical relevant manner. By using the model for intervention, it was found that prophylactic treatment with butyrate might be a valuable strategy for prophylactic mucositis prevention.

Elemental diet decreases inflammation and improves symptoms in adult eosinophilic oesophagitis patients.

BACKGROUND: Eosinophilic oesophagitis (EoE) is a chronic disease, driven by food allergens. Elemental diets are effective for the management of children with EoE, but studies on the effect of elemental diets in adults are scarce and poor palatability challenges dietary adherence. AIM: To assess the effects of an elemental diet (Neocate, Nutricia, Utrecht, the Netherlands) on the inflammation, symptoms and endoscopic signs in adult EoE patients. METHODS: In this prospective study, 21 patients with active EoE, confirmed by biopsies showing ≥15 eosinophils per microscopic high power field (HPF) and symptoms of oesophageal dysfunction were included. Patients underwent endoscopy before and 4 weeks after diet. Histological disease activity (peak eosinophil count/HPF), and endoscopic signs were scored by physicians. Symptoms and adherence to the diet were evaluated by questionnaires. Serum total IgE levels and total eosinophil counts were determined and the expression of inflammatory cytokines was analysed by qPCR. RESULTS: In total, 17 (81%) of the patients completed the diet, of whom 12 (71%) showed complete histological response (≤15 eosinophils/HPF) and 4 (24%) showed partial histological response (≥50% reduction of baseline eosinophil count). Peak eosinophil counts decreased significantly after the diet from 40 to 9 per HPF (P ≤ 0.001). A marked improvement in endoscopic signs was observed. Symptoms decreased significantly in all subjects, and 15 patients (88%) became completely asymptomatic (P ≤ 0.001). In 14 patients (82%), blood eosinophil count and serum IgE decreased (P ≤ 0.05). CONCLUSION: Elemental diet reduces eosinophilic inflammation and induces clinical remission in adult patients with eosinophilic oesophagitis.

Potential mechanisms explaining why hydrolyzed casein-based diets outclass single amino acid-based diets in the prevention of autoimmune diabetes in diabetes-prone BB rats.

BACKGROUND: It remains controversial whether avoidance of dietary diabetogenic triggers, such as cow's milk proteins, can prevent type 1 diabetes in genetically susceptible individuals. Here, different extensive casein hydrolysates (HC) and single amino acid (AA) formulations were tested for their effect on mechanisms underlying autoimmune diabetes pathogenesis in diabetes-prone BioBreeding rats. Intestinal integrity, gut microbiota composition and mucosal immune reactivity were studies to assess whether these formulations have differential effects in autoimmune diabetes prevention. METHODS: Diabetes-prone BioBreeding rats received diets in which the protein fraction was exchanged for the different hydrolysates or AA compositions, starting from weaning until the end of the experiment (d150). Diabetes development was monitored, and faecal and ileal samples were collected. Gut microbiota composition and cytokine/tight junction mRNA expression were measured by quantitative polymerase chain reaction. Cytokine levels of ileum explant cultures were measured by ELISA, and intestinal permeability was measured in vivo by lactulose-mannitol assay. RESULTS: Both HC-diet fed groups revealed remarkable reduction of diabetes incidence with the most pronounced effect in Nutramigen®-fed animals. Interestingly, AA-fed rats only showed delayed autoimmune diabetes development. Furthermore, both HC-fed groups had improved intestinal barrier function when compared with control chow or AA-fed animals. Interestingly, higher IL-10 levels were measured in ileum tissue explants from Nutramigen®-fed rats. Beneficial gut microbiota changes (increased Lactobacilli and reduced Bacteroides spp. levels) were found associated especially with HC-diet interventions. CONCLUSIONS: Casein hydrolysates were found superior to AA-mix in autoimmune diabetes prevention. This suggests the presence of specific peptides that beneficially affect mechanisms that may play a critical role in autoimmune diabetes pathogenesis.

Absence of coupling between release and biosynthesis of peptide hormones in insect neuroendocrine cells.

Adipokinetic hormone (AKH)-producing cells in the corpus cardiacum of the insect Locusta migratoria represent a neuroendocrine system containing large quantities of stored secretory peptides. In the present study we address the question whether the release of AKHs from these cells induces a concomitant enhancement of their biosynthesis. The effects of hormone release in vivo (by flight activity) and in vitro (using crustacean cardioactive peptide, locustamyoinhibiting peptide, and activation of protein kinase A and C) on the biosynthetic activity for AKHs were measured. The intracellular levels of prepro-AKH mRNAs, the intracellular levels of pro-AKHs, and the rate of synthesis of (pro-)AKHs were used as parameters for biosynthetic activity. The effectiveness of in vitro treatment was assessed from the amounts of AKHs released. Neither flight activity as the natural stimulus for AKH release, nor in vitro treatment with the regulatory peptides or signal transduction activators appeared to affect the biosynthetic activity for AKHs. This points to an absence of coupling between release and biosynthesis of AKHs. The strategy of the AKH-producing cells to cope with variations in secretory stimulation seems to rely on a pool of secretory material that is readily releasable and continuously replenished by a process of steady biosynthesis.

Intracisternal granules in the adipokinetic cells of locusts are not degraded and apparently function as supplementary stores of secretory material.

The intracisternal granules in locust adipokinetic cells appear to represent accumulations of secretory material within cisternae of the rough endoplasmic reticulum. An important question is whether these granules are destined for degradation or represent stores of (pro)hormones. Two strategies were used to answer this question. First, cytochemistry was applied to elucidate the properties of intracisternal granules. The endocytic tracers horseradish peroxidase and wheat-germ agglutinin-conjugated horseradish peroxidase were used to facilitate the identification of endocytic, autophagic, and lysosomal organelles, which may be involved in the degradation of intracisternal granules. No intracisternal granules could be found within autophagosomes, and granules fused with endocytic and lysosomal organelles were not observed, nor could tracer be found within the granules. The lysosomal enzyme acid phosphatase was absent from the granules. Second, biochemical analysis of the content of intracisternal granules revealed that these granules contain prohormones as well as hormones. Prohormones were present in relatively higher amounts compared with ordinary secretory granules. Since the intracisternal granules in locust adipokinetic cells are not degraded and contain intact (pro)hormones it is concluded that they function as supplementary stores of secretory material.

Differential location of peptide hormones in the secretory pathway of insect adipokinetic cells.

Immunoreactivity of granules containing secretory material in the adipokinetic cells of the insect Locusta migratoria was studied using antisera specific for the adipokinetic hormone-associated peptides (AAP) I, II and III. Immunocytochemical detection of these associated peptides represents a new strategy for studying the intracellular location of the adipokinetic hormones and their prohormones. Fixation with 2% glutaraldehyde and 2% formaldehyde with low-temperature embedding in Lowicryl HM20 allowed highly selective immunogold labelling of both secretory and intracisternal granules. All three associated peptides were co-localized in secretory granules. This indicates that also all three adipokinetic hormones can be co-localized in these granules, which was confirmed by experiments in which, after secretory stimulation, adipokinetic hormone III was released from the adipokinetic cells together with adipokinetic hormones I and II. The immunopositivity of the intracisternal granules was similar to that of the secretory granules, although with the exception that the intracisternal granules did not show any specific reaction with anti-AAP III. The presence of AAP I and AAP II in intracisternal granules indicates that these granules only function as stores of adipokinetic prohormones I and II and not of adipokinetic prohormone III. The observed differences in storage in intracisternal granules among the three adipokinetic prohormones suggest differences in physiological significance of the three adipokinetic hormones in L. migratoria.

Adipokinetic hormones. Coupling between biosynthesis and release.

During long-distance flight of migratory locusts, the dramatic energy demand of the flight muscles is controlled by three adipokinetic hormones (AKHs). These peptide hormones regulate the mobilization of lipid and carbohydrate stored in the fat body to serve as energy substrates for the flight muscles. Despite the relatively huge quantities of the three AKHs that are stored in the corpora cardiaca, flight induces a differential 2-4-fold increase in the mRNAs for the three hormones. Moreover, newly synthesized AKHs can be released only during a restricted period of time, suggesting that by far most of the stored hormones are physiologically inactive. This raises the question of how the biosynthetic activity in the AKH-producing cells is coupled to their secretory activity. The present review discusses the potential mechanisms by which generation and release of mixtures of bioactive neurohormones are controlled and how peptidergic neuroendocrine cells cope with variations in physiological stimulation, with the AKH-producing cells serving as a model system.