A Pilot Study on Donor Human Milk Microbiota: A Comparison with Preterm Human Milk Microbiota and the Effect of Pasteurization.

Human milk (HM) is the best feeding option for preterm infants; however, when mother's own milk (MOM) is not available, pasteurized donor human milk (DHM) is the best alternative. In this study, we profiled DHM microbiota (19 samples) using 16S rRNA amplicon sequencing and compared its compositional features with the MOM microbiota (14 samples) from mothers who delivered prematurely (PT-MOM). As a secondary study aim, we assessed the specific effect of pasteurization on the characteristics of the DHM microbiota. DHM showed significantly higher alpha diversity and significant segregation from PT-MOM. Compositionally, the PT-MOM microbiota had a significantly higher proportion of Staphylococcus than DHM, with Streptococcus tending to be and Pseudomonas being significantly overrepresented in DHM compared with the PT-MOM samples. Furthermore, pasteurization affected the HM microbiota structure, with a trend towards greater biodiversity and some compositional differences following pasteurization. This pilot study provided further evidence on the HM microbial ecosystem, demonstrating that the DHM microbiota differs from the PT-MOM microbiota, possibly due to inherent differences between HM donors and mothers delivering prematurely, and that pasteurization per se impacts the HM microbiota. Knowledge about HM microbiota needs to be acquired by investigating the effect of DHM processing to develop strategies aimed at improving DHM quality while guaranteeing its microbiological safety.

Consumption of Yeast-Fermented Wheat and Rye Breads Increases Colitis and Mortality in a Mouse Model of Colitis.

BACKGROUND: Cereals are known to trigger for wheat allergy, celiac disease and non-celiac wheat sensitivity (NCWS). Inflammatory processes and intestinal barrier impairment are suspected to be involved in NCWS, although the molecular triggers are unclear. AIMS: We were interested if different bread types influence inflammatory processes and intestinal barrier function in a mouse model of inflammatory bowel disease. METHODS: Epithelial caspase-8 gene knockout (Casp8ΔIEC) and control (Casp8fl) mice were randomized to eight groups, respectively. The groups received different diets for 28 days (gluten-free diet, gluten-rich diet 5 g%, or different types of bread at 50 g%). Breads varied regarding grain, milling and fermentation. All diets were isocaloric. RESULTS: Regardless of the diet, Casp8ΔIEC mice showed pronounced inflammation in colon compared to ileum, whereas Casp8fl mice were hardly inflamed. Casp8fl mice could tolerate all bread types. Especially yeast fermented rye and wheat bread from superfine flour but not pure gluten challenge increased colitis and mortality in Casp8ΔIEC mice. Hepatic expression of lipopolysaccharide-binding protein and colonic expression of tumor necrosis factor-α genes were inversely related to survival. The bread diets, but not the gluten-rich diet, also decreased colonic tight junction expression to variable degrees, without clear association to survival and inflammation. CONCLUSIONS: Bread components, especially those from yeast-fermented breads from wheat and rye, increase colitis and mortality in Casp8ΔIEC mice highly susceptible to intestinal inflammation, whereas control mice can tolerate all types of bread without inflammation. Yet unidentified bread components other than gluten seem to play the major role.

Necrotizing Enterocolitis: Overview on In Vitro Models.

Necrotizing enterocolitis (NEC) is a gut inflammatory disorder which constitutes one of the leading causes of morbidity and mortality for preterm infants. The pathophysiology of NEC is yet to be fully understood; several observational studies have led to the identification of multiple factors involved in the pathophysiology of the disease, including gut immaturity and dysbiosis of the intestinal microbiome. Given the complex interactions between microbiota, enterocytes, and immune cells, and the limited access to fetal human tissues for experimental studies, animal models have long been essential to describe NEC mechanisms. However, at present there is no animal model perfectly mimicking human NEC; furthermore, the disease mechanisms appear too complex to be studied in single-cell cultures. Thus, researchers have developed new approaches in which intestinal epithelial cells are exposed to a combination of environmental and microbial factors which can potentially trigger NEC. In addition, organoids have gained increasing attention as promising models for studying NEC development. Currently, several in vitro models have been proposed and have contributed to describe the disease in deeper detail. In this paper, we will provide an updated review of available in vitro models of NEC and an overview of current knowledge regarding its molecular underpinnings.

A Khorasan wheat-based diet improves systemic inflammatory profile in semi-professional basketball players: a randomized crossover pilot study.

BACKGROUND/OBJECTIVES: Khorasan wheat is an ancient grain with widely acclaimed beneficial effects on human health. The objective of the study was to examine the effect of a Khorasan-based diet on the wellbeing and inflammatory profile of young athletes. RESULTS: We conducted a randomized, single-blinded crossover trial involving 20 male young athletes. The participants were randomly assigned to consume products (pasta, bread, biscuits and crackers) made either with Khorasan (KAMUT® brand) or modern semi-whole-grain wheat for 4-weeks with a 4-week washout period before the crossover. Laboratory analyses and fitness tests were performed both at the beginning and end of each diet period. The consumption of Khorasan products was associated with a significant reduction of monocyte chemoattractant protein-1 (MCP-1; mean reduction: -36.15 pg/mL; -25.67%) while the consumption of modern wheat was not associated with significant differences in Interleukin-8 (IL-8) or Interleukin-1 receptor antagonist (IL-1ra). The consumption of the Khorasan-based diet also resulted in a significant improvement in self-rated health status. No statistically significant differences in any athletic performance parameter were observed between the two diets. CONCLUSION: The present results suggest that a Khorasan-based diet could be effective in reducing the inflammatory status in young athletes. © 2019 Society of Chemical Industry.

Dietary geraniol ameliorates intestinal dysbiosis and relieves symptoms in irritable bowel syndrome patients: a pilot study.

BACKGROUND: (Trans)-3,7-Dimethyl-2,6-octadien-1-ol, commonly called geraniol (Ge-OH), is an acyclic monoterpene alcohol with well-known anti-inflammatory and antimicrobial properties. Ge-OH is a non-toxic compound classified as Generally Recognized As Safe (GRAS) by the US Food and Drug Administration and the European Food Security Agency. METHODS: Ge-OH was orally administered at a maximum daily dose of 8 mg kg(- 1) body weight for four weeks in a delayed release formulation capable of reaching the colon. Fecal microbiota and blood cytokines were analyzed before and after Ge-OH treatment, as well as IBS symptomatology by using Visual Analogue Scale (VAS-IBS). RESULTS: The results show that orally administered Ge-OH is a powerful modulator of the intestinal microbial ecosystem, capable of leading to increased relative abundances of Collinsella and especially Faecalibacterium, a well-known health-promoting butyrate producer consistently found to be decreased in IBS patients. Moreover, Ge-OH strongly improved the clinical symptoms of colitis by significantly reducing the score recorded by the VAS-IBS questionnaire. Clinical improvement was associated with a significant reduction in the circulating MIP-1ß, a chemokine found to be increased in several IBS patients. CONCLUSION: Ge-OH could be a powerful component for food supplement targeted to the treatment of IBS patients. TRIAL REGISTRATION: ISRCTN47041881 , retrospectively registered on 19th July 2018.

Geraniol Pharmacokinetics, Bioavailability and Its Multiple Effects on the Liver Antioxidant and Xenobiotic-Metabolizing Enzymes.

Geraniol is a natural monoterpene showing anti-inflammatory, antioxidant, neuroprotective and anticancer effects. No pharmacokinetic and bioavailability data on geraniol are currently available. We therefore performed a systematic study to identify the permeation properties of geraniol across intestinal cells, and its pharmacokinetics and bioavailability after intravenous and oral administration to rats. In addition, we systematically investigated the potential hepatotoxic effects of high doses of geraniol on hepatic phase I, phase II and antioxidant enzymatic activities and undertook a hematochemical analysis on mice. Permeation studies performed via HPLC evidenced geraniol permeability coefficients across an in vitro model of the human intestinal wall for apical to basolateral and basolateral to apical transport of 13.10 ± 2.3 × 10-3 and 2.1 ± 0.1â‹…× 10-3 cm/min, respectively. After intravenous administration of geraniol to rats (50 mg/kg), its concentration in whole blood (detected via HPLC) decreased following an apparent pseudo-first order kinetics with a half-life of 12.5 ± 1.5 min. The absolute bioavailability values of oral formulations (50 mg/kg) of emulsified geraniol or fiber-adsorbed geraniol were 92 and 16%, respectively. Following emulsified oral administration, geraniol amounts in the cerebrospinal fluid of rats ranged between 0.72 ± 0.08 µg/mL and 2.6 ± 0.2 µg/mL within 60 min. Mice treated with 120 mg/kg of geraniol for 4 weeks showed increased anti-oxidative defenses with no signs of liver toxicity. CYP450 enzyme activities appeared only slightly affected by the high dosage of geraniol.

Responses of blood mononucleated cells and clinical outcome of non-celiac gluten sensitive pediatric patients to various cereal sources: a pilot study.

Non-celiac gluten sensitivity (NCGS) is a clinical entity recently documented by the scientific community in pediatric patients. Nevertheless, its triggering mechanisms remain largely unsettled. We studied 11 children with NCGS who were diagnosed based on a clear-cut relationship between wheat consumption and development of symptoms, after excluding celiac disease (CD) and wheat allergy, matched with 18 children with active CD. Sixteen pediatric patients were also enrolled as controls. Cultured peripheral blood mononucleated cells (PBMCs) obtained from NCGS, CD and control patients were cultured in the presence of wheat proteins extracted from ancient and modern cultivars. Results demonstrated that wheat proteins induced an overactivation of the proinflammatory chemokine CXCL10 in PBMC from NCGS pediatric patients and that this overexpression also depended on the wheat cultivar from which proteins were extracted. Proteins from modern wheat cultivar activated CXCL10 to a greater extent than those extracted from ancient wheat genotypes.

A comparative evaluation of two decompression procedures for technical diving using inflammatory responses: compartmental versus ratio deco.

INTRODUCTION: The aim of this study was to compare two decompression procedures commonly adopted by technical divers: the ZH-L16 algorithm modified by 30/85 gradient factors (compartmental decompression model, CDM) versus the 'ratio decompression strategy' (RDS). The comparison was based on an analysis of changes in diver circulating inflammatory profiles caused by decompression from a single dive. METHODS: Fifty-one technical divers performed a single trimix dive to 50 metres' sea water (msw) for 25 minutes followed by enriched air (EAN50) and oxygen decompression. Twenty-three divers decompressed according to a CDM schedule and 28 divers decompressed according to a RDS schedule. Peripheral blood for detection of inflammatory markers was collected before and 90 min after diving. Venous gas emboli were measured 30 min after diving using 2D echocardiography. Matched groups of 23 recreational divers (dive to 30 msw; 25 min) and 25 swimmers were also enrolled as control groups to assess the effects of decompression from a standard air dive or of exercise alone on the inflammatory profile. RESULTS: Echocardiography at the single 30 min observation post dive showed no significant differences between the two decompression procedures. Divers adopting the RDS showed a worsening of post-dive inflammatory profile compared to the CDM group, with significant increases in circulating chemokines CCL2 (P = 0.001) and CCL5 (P = 0.006) levels. There was no increase in chemokines following the CDM decompression. The air scuba group also showed a statistically significant increase in CCL2 (P < 0.001) and CCL5 (P = 0.003) levels post dive. No cases of decompression sickness occurred. CONCLUSION: The ratio deco strategy did not confer any benefit in terms of bubbles but showed the disadvantage of increased decompression-associated secretion of inflammatory chemokines involved in the development of vascular damage.

Can supplementation of phytoestrogens/insoluble fibers help the management of duodenal polyps in familial adenomatous polyposis?

Familial adenomatous polyposis (FAP) is an autosomal dominant inherited disorder, and prophylactic colectomy has been shown to decrease the incidence of colorectal cancer (CRC). Duodenal cancer and desmoids are now the leading causes of death in FAP. We evaluate whether 3 months of oral supplementation with a patented blend of phytoestrogens and indigestible insoluble fibers (ADI) help the management of FAP patients with ileal pouch-anal anastomosis (IPAA). In a prospective open label study, we enrolled 15 FAP patients with IPAA and duodenal polyps who underwent upper gastrointestinal endoscopy at baseline and after 3 months of treatment. The primary endpoint was the change in gene expression in polyp mucosa, whereas the secondary endpoint was the reduction in polyp number and size. After 3 months of ADI treatment, all patients showed a reduction in the number and size of duodenal polyps (P = 0.021). Analysis of the expression of CRC promoting/inhibiting genes in duodenal polyps biopsies demonstrated that different CRC-promoting genes (PCNA, MUC1 and COX-2) were significantly downregulated, whereas CRC-inhibiting genes (ER-ß and MUC2) were significantly upregulated after ADI treatment. In conclusion, ADI proved to be safe and effective, and its long-term effects on FAP patients need further investigation. Judging from the results we observed on COX-2 and miR-101 expression, the short-term effects of ADI treatment could be comparable with those obtained using COX-2 inhibitors, with the advantage of being much more tolerable in chronic therapies and void of adverse events.

Dietary Geraniol by Oral or Enema Administration Strongly Reduces Dysbiosis and Systemic Inflammation in Dextran Sulfate Sodium-Treated Mice.

(Trans)-3,7-Dimethyl-2,6-octadien-1-ol, commonly called geraniol (Ge-OH), is an acyclic monoterpene alcohol with well-known anti-inflammatory, antitumoral, and antimicrobial properties. It is widely used as a preservative in the food industry and as an antimicrobial agent in animal farming. The present study investigated the role of Ge-OH as an anti-inflammatory and anti-dysbiotic agent in the dextran sulfate sodium (DSS)-induced colitis mouse model. Ge-OH was orally administered to C57BL/6 mice at daily doses of 30 and 120 mg kg((-1)) body weight, starting 6 days before DSS treatment and ending the day after DSS removal. Furthermore, Ge-OH 120 mg kg((-1)) dose body weight was administered via enema during the acute phase of colitis to facilitate its on-site action. The results show that orally or enema-administered Ge-OH is a powerful antimicrobial agent able to prevent colitis-associated dysbiosis and decrease the inflammatory systemic profile of colitic mice. As a whole, Ge-OH strongly improved the clinical signs of colitis and significantly reduced cyclooxygenase-2 (COX-2) expression in colonocytes and in the gut wall. Ge-OH could be a powerful drug for the treatment of intestinal inflammation and dysbiosis.

Human cord blood-derived platelet lysate enhances the therapeutic activity of adipose-derived mesenchymal stromal cells isolated from Crohn's disease patients in a mouse model of colitis.

INTRODUCTION: Due to their immunomodulatory properties, mesenchymal stromal cells (MSCs) have been used for auto-immune disease treatment. Crohn disease (CD) and ulcerative colitis are two major inflammatory bowel diseases (IBDs), resulting from pathological immune responses to environmental or microbial antigens. Preclinical and clinical studies have suggested that MSC-based cellular therapy hold promising potential for IBD treatment. However, open issues include the selection of the proper cell dose, the source and the optimal route of administration of MSCs for more effective results. Platelet lysate has gained clinical interest due to its efficacy in accelerating wound healing. Thus, we propose to combine the administration of MSCs with a human umbilical cord blood-derived platelet lysate (hCBPL) as a novel strategy to improve MSC-based therapy for IBD resolution. METHODS: Colitis was induced in 8-week-old C57BL/6J mice by daily oral administration of dextran sulphate sodium (DSS) (1.5 % w/v in tap water) for 9 days. MSCs were isolated from adipose tissue of CD patients (adCD-MSCs), expanded in proliferation medium, resuspended in hCBPL or PBS and administrated via enema for three times (1 × 10(6) cells/mouse/time) every other day starting on day +7 from DSS induction. The colitis evolution was evaluated by daily monitoring of body weight, stool consistency and bleeding. Histopathological analysis was performed. Inflammatory cytokine plasma levels were determined. adCD-MSCs stained with lipophilic membrane dye Nile Red, were injected in DSS mice as described above. Colon section of mice sacrificed 24 hours after last cell administration, were analyzed by confocal microscopy. RESULTS: We found that adCD-MSCs could be easily isolated and expanded from CD patients. Upon injection, adCD-MSCs exerted a therapeutic effect on DSS-induced colitis. Moreover, hCBPL increased adCD-MSCs efficacy by significantly reducing colitis scores, extension of the colon inflamed area and plasma levels of inflammatory mediators. Finally, Nile Red staining of MSCs is very efficient, stable and does not impair their vitality and function. Nile Red-labelling was clearly detected in the colitic area of adCD-MSCs injected mice and it was significantly brighter in the colon sections of mice that had received adCD-MSCs/hCBPL. CONCLUSIONS: In summary, with this study we propose a novel and promising adCD-MSC/hCBPL-based therapy for refractory IBDs.

Responses of peripheral blood mononucleated cells from non-celiac gluten sensitive patients to various cereal sources.

Non-celiac gluten sensitivity (NCGS) is still an undefined syndrome whose triggering mechanisms remain unsettled. This study aimed to clarify how cultured peripheral blood mononucleated cells (PBMC) obtained from NCGS patients responded to contact with wheat proteins. Results demonstrated that wheat protein induced an overactivation of the proinflammatory chemokine CXCL10 in PBMC from NCGS patients, and that the overactivation level depends on the cereal source from which proteins are obtained. CXCL10 is able to decrease the transepithelial resistance of monolayers of normal colonocytes (NCM 460) by diminishing the mRNA expression of cadherin-1 (CDH1) and tight junction protein 2 (TJP2), two primary components of the tight junction strands. Thus, CXCL10 overactivation is one of the mechanisms triggered by wheat proteins in PBMC obtained from NCGS patients. This mechanism is activated to a greater extent by proteins from modern with respect to those extracted from ancient wheat genotypes.

Cyclooxygenase-2 silencing for the treatment of colitis: a combined in vivo strategy based on RNA interference and engineered Escherichia coli.

Nonpathogenic-invasive Escherichia coli (InvColi) bacteria are suitable for genetic transfer into mammalian cells and may act as a vehicle for RNA Interference (RNAi) in vivo. Cyclooxygenase-2 (COX-2) is overexpressed in ulcerative colitis (UC) and Crohn's disease (CD), two inflammatory conditions of the colon and small intestine grouped as inflammatory bowel disease (IBD). We engineered InvColi strains for anti-COX-2 RNAi (InvColi(shCOX2)), aiming to investigate the in vivo feasibility of a novel COX-2 silencing strategy in a murine model of colitis induced by dextran sulfate sodium (DSS). Enema administrations of InvColi(shCOX2) in DSS-treated mice led to COX-2 downregulation, colonic mucosa preservation, reduced colitis disease activity index (DAI) and increased mice survival. Moreover, DSS/InvColi(shCOX2)-treated mice showed lower levels of circulating pro-inflammatory cytokines and a reduced colitis-associated shift of gut microbiota. Considering its effectiveness and safety, we propose our InvColi(shCOX2) strategy as a promising tool for molecular therapy in intestinal inflammatory diseases.

Longitudinal analysis of inflammation and microbiota dynamics in a model of mild chronic dextran sulfate sodium-induced colitis in mice.

AIM: To characterize longitudinally the inflammation and the gut microbiota dynamics in a mouse model of dextran sulfate sodium (DSS)-induced colitis. METHODS: In animal models, the most common method used to trigger colitis is based on the oral administration of the sulfated polysaccharides DSS. The murine DSS colitis model has been widely adopted to induce severe acute, chronic or semi-chronic colitis, and has been validated as an important model for the translation of mice data to human inflammatory bowel disease (IBD). However, it is now clear that models characterized by mild intestinal damage are more accurate for studying the effects of therapeutic agents. For this reason, we have developed a murine model of mild colitis to study longitudinally the inflammation and microbiota dynamics during the intestinal repair processes, and to obtain data suitable to support the recovery of gut microbiota-host homeostasis. RESULTS: All plasma cytokines evaluated, except IL-17, began to increase (P < 0.05), after 7 d of DSS administration. IL-17 only began to increase 4 d after DSS withdrawal. IL-1ß and IL-17 continue to increase during the recovery phase, even when clinical signs of colitis had disappeared. IL-6, IL-10 and IFN-γ reached their maxima 4 d after DSS withdrawal and decreased during the late recovery phase. TNFα reached a peak (a three- fold increase, P < 0.05), after which it slightly decreased, only to increase again close to the end of the recovery phase. DSS administration induced profound and rapid changes in the mice gut microbiota. After 3 d of DSS administration, we observed a major reduction in Bacteroidetes/Prevotella and a corresponding increase in Bacillaceae, with respect to control mice. In particular, Bacteroidetes/Prevotella decreased from a relative abundance of 59.42%-33.05%, while Bacillaceae showed a concomitant increase from 2.77% to 10.52%. Gut microbiota rapidly shifted toward a healthy profile during the recovery phase and returned normal 4 d after DSS withdrawal. Cyclooxygenase 2 expression started to increase 4 d after DSS withdrawal (P < 0.05), when dysbiosis had recovered, and continued to increase during the recovery phase. Taken together, these data indicated that a chronic phase of intestinal inflammation, characterized by the absence of dysbiosis, could be obtained in mice using a single DSS cycle. CONCLUSION: Dysbiosis contributes to the local and systemic inflammation that occurs in the DSS model of colitis; however, chronic bowel inflammation is maintained even after recovery from dysbiosis.