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Eugenol, cinnamaldehyde and D-limonene, the main components of natural essential oils, are endowed with antioxidant and anti-inflammatory properties which allow them to induce beneficial effects on intestinal, cardiac and neuronal levels. In order to characterize their pharmacokinetic profiles and aptitude to permeate in the central nervous system after intravenous and oral administration to rats, new analytical procedures, easily achievable with HPLC-UV techniques, were developed. The terminal half-lives of these compounds range from 12.4 ± 0.9 (D-limonene) and 23.1 ± 1.6 min (cinnamaldehyde); their oral bioavailability appears relatively poor, ranging from 4.25 ± 0.11% (eugenol) to 7.33 ± 0.37% (cinnamaldehyde). Eugenol evidences a marked aptitude to permeate in the cerebrospinal fluid (CSF) of rats following both intravenous and oral administrations, whereas cinnamaldehyde appears able to reach the CSF only after intravenous administration; limonene is totally unable to permeate in the CSF. Eugenol was therefore recruited for in vitro studies of viability and time-/dose-dependent dopamine release in neuronal differentiated PC12 cells (a recognized cellular model mimicking dopaminergic neurons), evidencing its ability to increase cell viability and to induce dopamine release according to a U-shaped time-course curve. Moreover, concentration-response data suggest that eugenol may induce beneficial effects against Parkinson’s disease after oral administration.

Geraniol is an acyclic monoterpene alcohol with well-known anti-inflammatory and antimicrobial properties which has shown eubiotic activity towards gut microbiota (GM) in patients with irritable bowel syndrome (IBS). Methods: Fifty-six IBS patients diagnosed according to Rome III criteria were enrolled in an interventional, prospective, multicentric, randomized, double-blinded, placebo-controlled trial. In the treatment arm, patients received a low-absorbable geraniol food supplement (LAGS) once daily for four weeks. Results: Patients treated with LAGS showed a significant reduction in their IBS symptoms severity score (IBS-SSS) compared to the placebo (195 vs. 265, p = 0.001). The rate of responders according to IBS-SSS (reduction ≥ 50 points) was significantly higher in the geraniol vs placebo group (52.0% vs. 16.7%, p = 0.009) mainly due to the IBS mixed subtype. There were notable differences in the microbiota composition after geraniol administration, particularly a significant decrease in a genus of Ruminococcaceae, Oscillospira (p = 0.01), a decreasing trend for the Erysipelotrichaceae and Clostridiaceae families (p = 0.1), and an increasing trend for other Ruminococcaceae taxa, specifically Faecalibacterium (p = 0.09). The main circulating proinflammatory cytokines showed no differences between placebo and geraniol arms. Conclusion: LAGS was effective in treating overall IBS symptoms, together with an improvement in the gut microbiota profile, especially for the IBS mixed subtype.

Diet is a matter of interest in the pathogenesis and management of Crohn’s Disease (CD). Little is known about CD children’s dietary habits. Our aim was assessing the quality and the amount of nutrient intake in a group of CD pediatric patients. Data were compared with those of healthy subjects (HS). In total, 20 patients (13 males) and 48 HS (24 males) aged 4–18 years were provided with a food diary to fill out for one week. Winfood software performed the bromatological analysis, providing data about intakes of proteins and amino acids, fatty acids, carbohydrates, cholesterol, fibers, minerals, vitamins, and polyphenols. Estimates of the antioxidant activity of foods and of the dietetic protein load were also calculated. The diet of CD patients was poorer in fibers, polyphenols, vitamin A, beta-carotene, and fatty acids, and richer in animal proteins, vitamin B12, and niacin. PRAL was higher in CD patients’ diets, while ORAC was higher in HS. No significant differences were observed in carbohydrate and other macro- and micronutrient consumptions. CD dietary habits seem to reflect the so-called Western diet, possibly involved in CD pathogenesis. Furthermore, analysis of dietary habits allows for prevention of nutritional deficiencies and timely correction through education and supplementation.

Increases in non-communicable and auto-immune diseases, with a shared etiology of defective autophagy and chronic inflammation, have motivated research both on natural products in drug discovery fields and on the interrelationship between autophagy and inflammation. Within this framework, the tolerability and protective effects of a wheat-germ spermidine (SPD) and clove eugenol (EUG) combination supplement (SUPPL) were investigated on inflammation status (after the administration of lipopolysaccharide (LPS)) and on autophagy using human Caco-2 and NCM460 cell lines. In comparison to the LPS treatment alone, the SUPPL + LPS significantly attenuated ROS levels and midkine expression in monocultures, as well as occludin expression and mucus production in reconstituted intestinal equivalents. Over a timeline of 2–4 h, the SUPPL and SUPPL + LPS treatments stimulated autophagy LC3-11 steady state expression and turnover, as well as P62 turnover. After completely blocking autophagy with dorsomorphin, inflammatory midkine was significantly reduced in the SUPPL + LPS treatment in a non-autophagy-dependent manner. After a 24 h timeline, preliminary results showed that mitophagy receptor BNIP3L expression was significantly downregulated in the SUPPL + LPS treatment compared to the LPS alone, whereas conventional autophagy protein expression was significantly higher. The SUPPL shows promise in reducing inflammation and increasing autophagy to improve intestinal health.

Candida spp. are commensal organisms of the skin, mucous membranes, gastrointestinal tract, blood, and vagina of animals and humans. In recent decades, the incidence of human fungal infections has increased, with Candida spp. (mainly C. albicans) infections being the most frequent, and the treatment of fungal infections is still a clinical challenge. Colonization of the human gastrointestinal tract by Candida spp. is significant because infections (e.g., candidemia and vulvovaginal candidiasis) frequently arise from commensal microorganisms. The aim of this study was to test in vitro the antifungal activity and the eventual synergistic effect of five pure components of essential oils: cinnamaldehyde, α-pinene, limonene, eucalyptol, and eugenol. These compounds were tested on 18 Candida strains (15 C. albicans, 2 C. glabrata, and 1 C. lusitaniae) derived from a culture collection of vaginal clinical strains. Methods: Fungistatic activity was evaluated using the disk diffusion method. For fungicidal activity, microdilution and time–kill curve protocols were set up. The checkerboard method was chosen to evaluate a possible synergistic effect of these compounds when mixed. Results: Cinnamaldehyde and eugenol gave the best results, inhibiting all the Candida strains and showing a highly additive effect (FICI 0.625). The cinnamaldehyde inhibition zone (IZ), MIC, and MFC means were 69 mm, 50.05 mg/L, and 109.26 mg/L respectively. Cinnamaldehyde led to the total loss of viable Candida cells within 4 h. Eugenol IZ, MIC, and MFC means were 35.2 mm, 455.42 mg/L, and 690.09 mg/L, respectively. Eugenol led to the total loss of viable fungal cells within 1 h. Treatment with α-pinene inhibited 88.9% of Candida strains, with an IZ mean of 21.2 mm, a MIC mean of 195.41 mg/L, and a MFC mean of 251.27 mg/L; this compound led to the total loss of viable fungal cells only after 24 h. Limonene inhibited only 33.3% of the tested strains and eucalyptol did not produce an inhibition halo, so these compounds were not tested further. Conclusions: These characteristics, together with the well-known safety of cinnamaldehyde and eugenol for human use, make these two natural compounds the perfect candidates for the treatment of candidiasis. This was a pilot study, the purpose of which was to evaluate the best composition of a mixture to be used against intestinal and vulvovaginal candidiasis; in vivo studies are needed to confirm these very encouraging results.

Clinical interest in the human gut microbiota has increased considerably, because of the increasing number of studies linking the human intestinal microbiota and microbiome to an ever increasing number of non-communicable diseases. Many attempts at modulating the gut microbiota have been made using probiotics and prebiotics. However, there are other avenues that are still little explored from a clinical point of view that appear promising to obtain modifications of the microbial ecology and biological activities connected to the microbiome. This chapter summarizes all in vitro, in vivo and clinical studies demonstrating the possibility to positively modulate the intestinal microbiota by using probiotics, foods (and prebiotics), essential oils, fungus and officinal plants. For the future, clinical studies investigating the ability to modify the intestinal microbiota especially by using foods, officinal and aromatic plants or their extracts are required. More knowledge in this field is likely to be of clinical benefit since modulation of the microbiome might support the therapy of most non-communicable diseases in the future.

The search for new fiber supplements that can claim to be “prebiotic” is expanding fast, as the role of prebiotics and intestinal microbiota in well-being has been well established. This work explored the prebiotic potential of a novel fiber plus D-Limonene supplement (FLS) in comparison to fructooligosaccharides (FOS) over distal colonic fermentation with the in vitro model MICODE (multi-unit in vitro colon gut model). During fermentation, volatilome characterization and core microbiota quantifications were performed, then correlations among volatiles and microbes were interpreted. The results indicated that FLS generated positive effects on the host gut model, determining: (i) eubiosis, (ii) increased abundance of beneficial bacteria, as Bifidobacteriaceae, (iii) production of beneficial compounds, as n-Decanoic acid, (iv) reduction in detrimental bacteria, as Enterobaceteriaceae, (v) reduction in detrimental compounds, as skatole. The approach that we followed permitted us to describe the prebiotic potential of FLS and its ability to steadily maintain the metabolism of colon microbiota over time. This aspect is two-faced and should be investigated further because if a fast microbial turnover and production of beneficial compounds is a hallmark of a prebiotic, the ability to reduce microbiota changes and to reduce imbalances in the productions of microbial metabolites could be an added value to FLS. In fact, it has been recently demonstrated that these aspects could serve as an adjuvant in metabolic disorders and cognitive decline.

Essential oils (EOs) are a complex mixture of hydrophobic and volatile compounds synthesized from aromatic plants, commonly present in the human diet. In recent years, many in vitro studies have suggested possible anticancer properties of single EO compounds, on colorectal cancer (CRC) cells. However, the majority of these studies did not compare the effects of these compounds on normal and cancer colon cells. By using NCM-460, a normal human mucosal epithelial cell line, Caco-2, a human colon epithelial adenocarcinoma cell line, and SW-620, colon cancer cells derived from lymph node metastatic site, we identified cinnamaldehyde, derived from cinnamon EO and eugenol, derived from bud clove EO, as compounds with a specific anticancer action selectively targeting the transformed colonic cells. Both cinnamaldehyde (75 µM) and eugenol (800 µM), after 72 h of treatment, were capable to induce apoptosis, necrosis and a cell cycle slowdown in Caco-2 and in SW-620, but not in NCM-460 cells. If associated with a targeted delivery to the colon, these two compounds could prove effective in the prevention or treatment of CRC.

Several studies showed that D-Limonene can improve metabolic parameters of obese mice via various mechanisms, including intestinal microbiota modulation. Nevertheless, its effective doses often overcome the acceptable daily intake, rising concerns about toxicity. In this study we administered to C57BL/6 mice for 84 days a food supplement based on D-Limonene, adsorbed on dietary fibers (FLS), not able to reach the bloodstream, to counteract the metabolic effects of a high-fat diet (HFD). Results showed that daily administration of D-Limonene (30 and 60 mg/kg body weight) for 84 days decreased the weight gain of HFD mice. After 84 days we observed a statistically significant difference in weight gain in the group of mice receiving the higher dose of FLS compared to HFD mice (35.24 ± 4.56 g vs. 40.79 ± 3.28 g, p &lt, 0.05). Moreover, FLS at both doses tested was capable of lowering triglyceridemia and also fasting glycemia at the higher dose. Some insights on the relevant fatty acid changes in hepatic tissues were obtained, highlighting the increased polyunsaturated fatty acid (PUFA) levels even at the lowest dose. FLS was also able to positively modulate the gut microbiota and prevent HFD-associated liver steatosis in a dose-dependent manner. These results demonstrate that FLS at these doses can be considered non-toxic and could be an effective tool to counteract diet-induced obesity and ameliorate metabolic profile in mice.

Essential oils (EOs) are a complex mixture of hydrophobic and volatile compounds synthesized from aromatic plants, most of them commonly used in the human diet. In recent years, many studies have analyzed their antimicrobial, antioxidant, anti-inflammatory, immunomodulatory and anticancer properties in vitro and on experimentally induced animal models of colitis and colorectal cancer. However, there are still few clinical studies aimed to understand their role in the modulation of the intestinal pathophysiology. Many EOs and some of their molecules have demonstrated their efficacy in inhibiting bacterial, fungi and virus replication and in modulating the inflammatory and oxidative processes that take place in experimental colitis. In addition to this, their antitumor activity against colorectal cancer models makes them extremely interesting compounds for the modulation of the pathophysiology of the large bowel. The characterization of these EOs is made difficult by their complexity and by the different compositions present in the same oil having different geographical origins. This review tries to shift the focus from the EOs to their individual compounds, to expand their possible applications in modulating colon pathophysiology.

Polyphenols display health-promoting properties linked to their biological activities. They are initially absorbed in the small intestine, then they are largely metabolized in the colon, whereupon they are able to exert systemic effects. The health-promoting properties of polyphenols have led to the development of food supplements, which are also largely consumed by healthy people, even if data on their safety are still yet lacking. In the present paper, the content of gallic acid and ferulic acid was analyzed in two supplements, and shown to be higher than the relative contents found in fruit and flour. To evaluate the effects of these phenolic compounds on epithelial intestinal tissue, gallic and ferulic acids were added to a new in vitro model of the intestinal wall at different concentrations. The effects on viability, proliferation and migration of these compounds were respectively tested on three different cell lines (Caco2, L929 and U937), as well as on a tridimensional intestinal model, composed of a mucosal layer and a submucosa with fibroblasts and monocytes. Results indicated that gallic and ferulic acids can exert toxic effects on in vitro cell models at high concentrations, suggesting that an excessive and uncontrolled consumption of polyphenols may induce negative effects on the intestinal wall.

The life-threatening sequela of hemorrhagic colitis induced by Shiga toxins (Stx)-producing Escherichia coli (STEC) infections in humans is hemolytic uremic syndrome (HUS), the main cause of acute renal failure in early childhood. The key step in the pathogenesis of HUS is the appearance of Stx in the blood of infected patients because these powerful virulence factors are capable of inducing severe microangiopathic lesions in the kidney. During precocious toxemia, which occurs in patients before the onset of HUS during the intestinal phase, Stx bind to several different circulating cells. An early response of these cells might include the release of proinflammatory mediators associated with the development of HUS. Here, we show that primary human monocytes stimulated with Shiga toxin 1a (Stx1a) through the glycolipid receptor globotriaosylceramide released larger amounts of proinflammatory molecules (IL-1β, TNFα, IL-6, G-CSF, CXCL8, CCL2, CCL4) than Stx1a-treated neutrophils. The mediators (except IL-1β) are among the top six proinflammatory mediators found in the sera from patients with HUS in different studies. The molecules appear to be involved in different pathogenetic steps of HUS, i.e. sensitization of renal endothelial cells to the toxin actions (IL-1β, TNFα), activation of circulating monocytes and neutrophils (CXCL8, CCL2, CCL4) and increase in neutrophil counts in patients with poor prognosis (G-CSF). Hence, a role of circulating monocytes in the very early phases of the pathogenetic process culminating with HUS can be envisaged. Impairment of the events of precocious toxemia would prevent or reduce the risk of HUS in STEC-infected children.

Inflammatory bowel diseases (IBD) are currently considered multifactorial pathologies in which various combined environmental factors act on a genetic background, giving rise to a chronic inflammation of the gastrointestinal tract. Among the various environmental factors, it now seems clear that the diet plays the major role in IBD onset and progression. Several clinical studies have attempted to understand the impact of diet in the development and progression of these diseases in order to establish useful guidelines for their management. However, the modest and sometimes contradictory results did not lead to the definition of shared dietary suggestions. On the other hand, food fads and recommendations based on anecdotal episodes are often followed by IBD patients to improve their diet. This review provides a critical overview of existing data on the role of diet as a risk factor for IBD. The methodology used was that of analyzing the results of clinical studies conducted on diet and IBD over the last 12 years through PubMed, as well as analyzing the most relevant studies on nutrients and their possible roles in IBD through the knowledge of the mechanisms by which they can modulate the microbiota or the intestinal physiology.

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.

Although ancient, heritage, and modern wheat varieties appear rather similar from a nutritional point of view, having a similar gluten content and a comparable toxicity linked to their undigested gluten peptide, whenever the role of ancient end heritage wheat grains has been investigated in animal studies or in clinical trials, more anti-inflammatory effects have been associated with the older wheat varieties. This review provides a critical overview of existing data on the differential physiological responses that could be elicited in the human body by ancient and heritage grains compared to modern ones. The methodology used was that of analyzing the results of relevant studies conducted from 2010 through PubMed search, by using as keywords “ancient or heritage wheat”, “immune wheat” (protein or peptides), and immune gluten (protein or peptides). Our conclusion is that, even if we do not know exactly which molecular mechanisms are involved, ancient and heritage wheat varieties have different anti-inflammatory and antioxidant proprieties with respect to modern cultivars. It is, therefore, reasonable to assume that the health proprieties attributed to older cultivars could be related to wheat components which have positive roles in the modulation of intestinal inflammation and/or permeability.

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 x 10−3 and 2.1 ± 0.1 x 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 minutes. 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 minutes. Mice treated with 120 mg/kg of geraniol for four 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.

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-1β, 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.

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.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.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 (P0.001) and CCL5 (P = 0.003) levels post dive. No cases of decompression sickness occurred.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.

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.

Colitis is the term used for chronic inflammatory bowel diseases at substantially increased risk of developing a form of colorectal cancer (CRC) known as colitis-associated cancer. In our study we synthesized core-shell polymeric micelles obtained by self-assembly of block copolymers for high efficiency delivery of anti-inflammatory and anti-cancer compounds to colonocytes and colon mucosa. We achieved an efficient intracellular delivery of these hydrophobic compounds (prednisone, retinoic acid and doxorubicin) to cultured colonocytes without cellular toxicity. The efficacy of retinoic acid and doxorubicin administration was significantly increased using these nanosized carriers. Moreover, these polymeric micelles have been shown to overcome the multidrug resistance efflux mechanism effectively delivering doxorubicin to multidrug-resistant colon cancer cells. These nanocarriers are also suitable for selective in vivo delivery of lipophilic drugs by enema administration to the inflamed colon tissue, specifically targeting the inflamed mucosa. From the Clinical Editor This team of investigators studied polymeric micelles as highly efficient drug delivery systems enabling intracellular delivery of hydrophobic compounds (prednisone, retinoic acid, and doxorubicin) to cultured colonocytes without cellular toxicity, also demonstrating beneficial in vivo effects.

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-β 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.

(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.

Colorectal cancer (CRC) is the second leading cause of cancer-related mortality in Western countries. Although the aberrant expression of several microRNAs (oncomiRs) is associated with CRC progression, the molecular mechanisms of this phenomenon are still under investigation. Here we show that miR-101 expression is differentially impaired in CRC specimens, depending on tumour grade. miR-101 re-expression suppresses cell growth in 3D, hypoxic survival and invasive potential in CRC cells showing low levels of miR-101. Additionally, we provide molecular evidence of a bidirectional regulatory mechanism between miR-101 expression and important CRC pro-malignant features, such as inflammation, activation of the Wnt/β-catenin signalling pathway and epithelial-mesenchymal transition (EMT). We then propose that up-regulated miR-101 may function as a tumour suppressor in CRC and that its pharmacological restoration might hamper the aggressive behaviour of CRC in vivo. MiR-101 expression may also represent a cancer biomarker for CRC diagnosis and prognosis.

Aliment Pharmacol Ther 2011; 34: 982–993 Summary Background Erosive reflux disease (ERD) and non-erosive reflux disease (NERD) are often regarded as part of the spectrum of the same disease. Aim To elucidate molecular features that characterise NERD and ERD at the protein level. Methods A total of 56 consecutive subjects were enrolled: 10 healthy subjects, 24 with NERD and 22 with ERD. Eight specimens were taken from macroscopically normal mucosa at 5 cm of gastro-oesophageal junction. Four were processed for the proteins extraction and four for evaluation using haematoxylin-eosin and immunohistochemistry. We used shotgun proteomics to identify tentative disease molecular features for ERD or NERD. Candidate distinctive proteins were verified using immunohistochemistry. Results Shotgun proteomics analysis revealed 33 differentially expressed proteins in NERD vs. ERD samples, involved in cellular proliferation, keratinisation, stress responses and sugar metabolism. Based on a gene ontology meta-analysis, seven of them were further analysed using Western blotting (WB) and four also using immunohistochemistry. We identified novel candidate disease molecular features for GERD and few distinctive proteins to discriminate NERD and ERD. In particular, Transitional Endoplasmic Reticulum ATPase (TER ATPase), GAPDH, Alpha 1 Acid Glycoprotein 1, Annexin A1, Calmodulin and 14-3-3 proteins were confirmed at WB analysis. Conclusions Non-erosive reflux disease and ERD are distinct disease entities at the protein level. This study proposes an array of candidate biomarkers possibly useful to discriminate between NERD and ERD.

Background: Cyclooxygenase-2 (COX-2) overexpression is strongly associated with colorectal tumourigenesis. It has been demonstrated that the chronic use of non-steroidal anti-inflammatory drugs (COX inhibitors) partially protects patients from colorectal cancer (CRC) development and progression but induces severe cardiovascular side effects. New strategies for selective COX-2 blockade are required. Methods: We developed an improved technique, based on RNA interference (RNAi), to gain a selective COX-2 silencing in CRC cells by a tumour-dependent expression of anti-COX-2 short-hairpin RNA (shCOX-2). Anti-COX-2 shRNA-expressing vectors were delivered in CRC cells (in vitro) and in colon tissues (ex vivo) using engineered Escherichia coli strains, capable of invading tumour cells (InvColi). Results: A highly tumour-dependent shCOX-2 expression and a significant COX-2 silencing were observed in CRC cells following InvColi strain infection. Cyclooxygenase-2 silencing was associated with a strong reduction in both proliferative and invasive behaviour of tumour cells. We also demonstrated a pivotal role of COX-2 overexpression for the survival of CRC cells after bacterial infection. Moreover, COX-2 silencing was achieved ex vivo by infecting colon tissue samples with InvColi strains, leading to anti-inflammatory and anti-tumour effects. Conclusion: Our RNAi/InvColi-mediated approach offers a promising tool for a highly selective COX-2 blockade in vitro and in vivo.

Aliment Pharmacol Ther 2010; 32: 603–611 Summary Background Cannabinoid (CB) receptors have been located in brain areas involved in the triggering of TLESRs as well as in the nodose ganglion from which vagal afferents emanate. The distribution of CB1 receptors has been investigated in the human gastrointestinal mucosa, as expression of inflammatory process. Aim To evaluate the CB1 expression in oesophageal mucosa. Methods A total of 87 consecutive subjects were enrolled: 10 controls, 39 NERD and 38 erosive oesophagitis. Eight specimens were taken from macroscopically normal mucosa. Five were processed by haematoxylin–eosin, MIB1/CB1 evaluation and three for the RNA and proteins extraction. Results The mean MIB1-LI value was 31% and 22% in NERD and ERD patients, respectively, compared to 68% in the healthy subjects. Mean CB1mRNA/GUSB mRNA value of the controls was 0.66, while in GERD patients, it was 0.28. In NERD and ERD, the mean values of CB1/GUSB were 0.38 and 0.17, respectively, with highly significant differences between the NERD vs. ERD groups. Semi-quantitative analysis of CB1 expression, performed with WB, shows in NERD patients a higher CB1 receptor expression than ERD patients. Conclusions With this study, we showed for the first time the presence of CB1 receptors in the human oesophageal epithelium.

Copper dyshomeostasis is responsible for the neurological symptoms observed in the genetically inherited copper-dependent disorders (e.g., Menkes' and Wilson's diseases), but it has been also shown to have an important role in neurodegenerative diseases such as Alzheimer disease, prion diseases, Parkinson's disease and amyotrophic lateral sclerosis. It is widely accepted that increased extracellular copper levels contribute to neuronal pathogenic process by increasing the production of dangerous radical oxygen species, but the existence of other molecular mechanisms explaining copper neurotoxicity has not been investigated yet. By using a cellular model based on hypothalamic GN11 cultured neurons exposed to copper supplementation and by analysing the cell conditioned media, we try here to identify new molecular events explaining the association between extracellular copper accumulation and neuronal damages. We show here that increased extracellular copper levels produce a wide complex of alterations in the neuronal extracellular environment. In particular, copper affects the secretion of molecules involved in the protection of neurons against oxidative stress, such as cyclophilin A (CypA), or of molecules capable of shifting neuronal cells towards a pro-inflammatory state, such as IL-1alpha, IL-12, Rantes, neutrophil gelatinase-associated lipocalin (NGAL) and secreted protein acidic and rich in cysteine (SPARC). Copper pro-inflammatory properties have been confirmed by using primary neurons.

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 × 106 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.

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 (InvColishCOX2), 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 InvColishCOX2 in DSS-treated mice led to COX-2 downregulation, colonic mucosa preservation, reduced colitis disease activity index (DAI) and increased mice survival. Moreover, DSS/InvColishCOX2-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 InvColishCOX2 strategy as a promising tool for molecular therapy in intestinal inflammatory diseases. 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 viva 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 Inv Coll 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.

none 10 no 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-1β 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. none De Fazio L; Cavazza E; Spisni E; Strillacci A; Centanni M; Candela M; Praticò C; Campieri M; Ricci C; Valerii MC. De Fazio L; Cavazza E; Spisni E; Strillacci A; Centanni M; Candela M; Praticò C; Campieri M; Ricci C; Valerii MC.

Cyclooxygenase-2 (COX-2) enzyme has been involved in the tumorigenesis and in the progression of colorectal cancer (CRC). The use of traditional nonsteroidal anti-inflammatory drugs (NSAIDs) or selective COX-2 inhibitors has been proposed for the prevention and the treatment of this relevant neoplastic disease. In the light of an innovative alternative to these pharmacological approaches, we review here the possible strategies to achieve a strong and selective inhibition of COX-2 enzyme by using the mechanism of RNA Interference (RNAi) targeted against its mRNA. Anti-COX-2 siRNA molecules (siCOX-2) can be generated in CRC cells from short hairpin RNA (shRNA) precursors, delivered in vitro by a retroviral expression system, and induce a significant and stable silencing of overexpressed COX-2 in human colon cancer cells. As a safer alternative to viral approach, nonpathogenic bacteria (E. coli) can be engineered to invade eukaryotic cells and to generate siCOX-2 molecules in cancer cells. Moreover, the involvement of miRNAs in COX-2 posttranscriptional regulation opens up the possibility to exploit an endogenous silencing mechanism to knockdown overexpressed COX-2. Thus, these recent strategies disclose new challenging perspectives for the development of clinically compatible siRNA or miRNA capable of selectively inhibiting COX-2 enzyme.

Background: A diminished biodiversity of the intestinal flora has been reported in patients with IBD. Restoration of a normal flora is being discussed as an alternative treatment approach. Methods: We assessed safety and efficacy of fecal transplantation (FT) in moderately to severely, chronic active patients with UC (n = 5, f/m: 2/3, median Mayo score: 11) refractory to standard therapy. Immunosuppressive therapy was stopped prior FT. Fecal donors were healthy adults with normal bowel function who were screened for enteric pathogens and serologically for viral diseases. Donor stool was diluted in saline and administered via nasojejunal tube and enema. Adverse events and blood tests were regularly obtained during a followup of 12 weeks. H2-glucose breath test was performed to exclude bacterial overgrowth at wk 4 and wk 12 weeks. Clinical activity was assessed according to Mayo Score. Results: Patients completed an antibiotic (n = 5) and probiotic (n = 4) therapy for 5 to 10 days and a single bowel lavage before FT. FT was performed daily for 3 days (n = 4). In one patient there was a gap of 5 weeks between the first and the second FT due to fever >39oC and a >8-fold increase of C-reactive protein (CRP) after first FT. Altogether, median 23.8 g (range:16.7-g-25 g) and 20 g (range: 6 g-21.7 g) stool was administered via nasojejunal tube and enema, respectively. All of the patients reported on worsening of diarrhoea and fever during FT. Additionally, a temporary increase of CRP was observed. In patients (n = 2), who had a temperature >38oC blood cultures were taken, but no bacterial pathogen was detectable. Additionally, flatulance (n = 1) and vomiting (n = 1) were reported. In the follow-up period common cold (n = 3), itchiness (n = 1), erythema (n = 1), paraesthesia on the hip (n = 1), collapse (n = 1), and blisters on the tongue (n = 1) were reported. No serious adverse event occurred. Bacterial overgrowth was not detectable in any patient. In 2 patients a further deterioration of UC was observed. Although the the general well-being improved from poor to very well at week 12 in the other 3 patients, the median total Mayo score improved only from 11 to 9. In one patient there was an improvement of the Mayo endoscopic subscore from 3 to 2. Conclusions: In our experience FT might be safe but activates a temporary systemic immune response. Our preliminary data are less impressive with regard to efficacy after a minimum follow-up of 12 weeks.

Background The vaginal microbiota of healthy women consists of a wide variety of anaerobic and aerobic bacterial genera and species dominated by the genus Lactobacillus. The activity of lactobacilli helps to maintain the natural healthy balance of the vaginal microbiota. This role is particularly important during pregnancy because vaginal dismicrobism is one of the most important mechanisms for preterm birth and perinatal complications. In the present study, we characterized the impact of a dietary supplementation with the probiotic VSL#3, a mixture of Lactobacillus, Bifidobacterium and Streptococcus strains, on the vaginal microbiota and immunological profiles of healthy women during late pregnancy. Results An association between the oral intake of the probiotic VSL#3 and changes in the composition of the vaginal microbiota of pregnant women was revealed by PCR-DGGE population profiling. Despite no significant changes were found in the amounts of the principal vaginal bacterial populations in women administered with VSL#3, qPCR results suggested a potential role of the probiotic product in counteracting the decrease of Bifidobacterium and the increase of Atopobium, that occurred in control women during late pregnancy. The modulation of the vaginal microbiota was associated with significant changes in some vaginal cytokines. In particular, the decrease of the anti-inflammatory cytokines IL-4 and IL-10 was observed only in control women but not in women supplemented with VSL#3. In addition, the probiotic consumption induced the decrease of the pro-inflammatory chemokine Eotaxin, suggesting a potential anti-inflammatory effect on the vaginal immunity. Conclusion Dietary supplementation with the probiotic VSL#3 during the last trimester of pregnancy was associated to a modulation of the vaginal microbiota and cytokine secretion, with potential implications in preventing preterm birth. Trial registration ClinicalTrials.gov NCT01367470