E40 glutenase detoxification capabilities of residual gluten immunogenic peptides in in vitro gastrointestinal digesta of food matrices made of soft and durum wheat.

Gluten degrading enzymes, which are commonly referred to as "glutenases," represent attractive candidates for the development of a pharmacological treatment of gluten related disorders, such as coeliac disease (CeD). Endoprotease-40 (E40), a novel glutenase secreted by the actinomycete Actinoallomurus A8 and recombinantly produced in S. lividans TK24, was shown to be active at pH 3 to 6 (optimum pH 5), resistant to pepsin and trypsin degradation, able to destroy immunotoxicity of both gliadin 33-mer peptide and whole proteins and to strongly reduce the response of specific T cells when added to gliadin in in vitro gastrointestinal digestion. This study aims to functionally assess the capabilities of Endoprotease-40 (E40) to detoxify residual gluten immunogenic peptides in gastrointestinal digesta of food matrices made of soft and durum wheat. The INFOGEST harmonized protocols were applied to the multicompartmental model of simulated human gastrointestinal digestion, for the quantitative assessment of residual gluten in liquid (beer) and solid (bread and pasta) foods, made of either soft or durum wheat. Proteomic and immunological techniques, and functional assays on intestinal T cell lines from celiac disease patients were used to identify gluten-derived immunogenic peptide sequences surviving in gastric and gastrointestinal digesta after the addition of E40 at increasing enzyme: wheat proteins ratios. During the gastric phase (2 h incubation time), the addition of E40 demonstrated an extensive (≥ 95%) dose-dependent detoxification of whole gluten in real food matrices. Overall, the residual gluten content was found at, or even below, the 20 ppm gluten-free threshold for soft and durum wheat-based food. Furthermore, unlike in untreated gastrointestinal digesta, none of the immunodominant α-gliadin peptides survived in E40-treated digesta. Traces of ω- and γ-gliadin derived immunogenic peptides were still detected in E40-treated digesta, but unable to stimulate celiac-intestinal T cells. In conclusion, E40 is a promising candidate for the oral enzymatic therapy of CeD, as a stand-alone enzyme being efficient along the complete gastrointestinal digestion of gluten.

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.

Silver sucrose octasulfate (IASOS™) as a valid active ingredient into a novel vaginal gel against human vaginal pathogens: in vitro antimicrobial activity assessment.

This in vitro study assessed the antimicrobial properties of a novel octasilver salt of Sucrose Octasulfate (IASOS) as well as of an innovative vaginal gel containing IASOS (SilSOS Femme), against bacterial and yeast pathogens isolated from human clinical cases of symptomatic vaginal infections. In BHI and LAPT culture media, different ionic silver concentrations and different pHs were tested. IASOS exerted a strong antimicrobial activity towards all the pathogens tested in both culture media. The results demonstrated that salts and organic compounds present in the culture media influenced IASOS efficacy only to a moderate extent. Whereas comparable MBCs (Minimal Bactericidal Concentrations) were observed for G. vaginalis (10 mg/L Ag+), E. coli and E. aerogenes (25 mg/L Ag+) in both media, higher MBCs were found for S. aureus and S. agalactiae in LAPT cultures (50 mg/L Ag+ versus 25 mg/L Ag+). No minimal concentration totally inhibiting the growth of C. albicans was found. Nevertheless, in both media at the highest ionic silver concentrations (50-200 mg/L Ag+), a significant 34-52% drop in Candida growth was observed. pH differently affected the antimicrobial properties of IASOS against bacteria or yeasts; however, a stronger antimicrobial activity at pH higher than the physiological pH was generally observed. It can be therefore concluded that IASOS exerts a bactericidal action against all the tested bacteria and a clear fungistatic action against C. albicans. The antimicrobial activity of the whole vaginal gel SilSOS Femme further confirmed the antimicrobial activity of IASOS. Overall, our findings support IASOS as a valid active ingredient into a vaginal gel.

Phytoestrogens/insoluble fibers and colonic estrogen receptor ß: randomized, double-blind, placebo-controlled study.

AIM: To assess the safety and effect of the supplementation of a patented blend of dietary phytoestrogens and insoluble fibers on estrogen receptor (ER)-ß and biological parameters in sporadic colonic adenomas. METHODS: A randomized, double-blind placebo-controlled trial was performed. Patients scheduled to undergo surveillance colonoscopy for previous sporadic colonic adenomas were identified, and 60 eligible patients were randomized to placebo or active dietary intervention (ADI) twice a day, for 60 d before surveillance colonoscopy. ADI was a mixture of 175 mg milk thistle extract, 20 mg secoisolariciresinol and 750 mg oat fiber extract. ER-ß and ER-α expression, apoptosis and proliferation (Ki-67 LI) were assessed in colon samples. RESULTS: No adverse event related to ADI was recorded. ADI administration showed a significant increases in ER-ß protein (0.822 ± 0.08 vs 0.768 ± 0.10, P = 0.04) and a general trend to an increase in ER-ß LI (39.222 ± 2.69 vs 37.708 ± 5.31, P = 0.06), ER-ß/ER-α LI ratio (6.564 ± 10.04 vs 2.437 ± 1.53, P = 0.06), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (35.592 ± 14.97 vs 31.541 ± 11.54, P = 0.07) and Ki-67 (53.923 ± 20.91 vs 44.833 ± 10.38, P = 0.07) approximating statistical significance. A significant increase of ER-ß protein (0.805 ± 0.13 vs 0.773 ± 0.13, P = 0.04), mRNA (2.278 ± 1.19 vs 1.105 ± 1.07, P < 0.02) and LI (47.533 ± 15.47 vs 34.875 ± 16.67, P < 0.05) and a decrease of ER-α protein (0.423 ± 0.06 vs 0.532 ± 0.11, P < 0.02) as well as a trend to increase of ER-ß/ER-α protein in ADI vs placebo group were observed in patients without polyps (1.734 ± 0.20 vs 1.571 ± 0.42, P = 0.07). CONCLUSION: The role of ER-ß on the control of apoptosis, and its amenability to dietary intervention, are supported in our study.

ERß expression in normal, adenomatous and carcinomatous tissues of patients with familial adenomatous polyposis.

OBJECTIVES: The APC gene mutation triggers familial adenomatous polyposis (FAP) and approximately 80% of sporadic colorectal cancers. FAP summarizes the natural history of colorectal cancer because low- and high-grade dysplastic lesions and adenocarcinoma are simultaneously present in the same patients free from individual and environmental variability factors. Estrogen receptor beta (ERß) has recently been suggested as the most likely mediator of estrogen-related anti-carcinogenic effects in Apc(Min-/+) mice and humans. In this study we assessed the ERß expression in the intestinal mucosa of FAP patients to verify its possible involvement in tumor progression in colorectal cancer. MATERIAL AND METHODS: ERß and ERα expression, cell proliferation (Ki-67) and apoptosis (TUNEL), were evaluated on archival biopsy material from six patients with FAP who underwent colectomy. RESULTS: A progressive significant decrease of ERß expression was observed in the different stages of the disease as compared to normal mucosa (p < 0.001). Interestingly, a decreased ERß expression was directly correlated with apoptosis (r = 0.76, p < 0.001), and inversely correlated with cell proliferation (r = 0.54, p < 0.05). CONCLUSIONS: ERß expression is related to the severity of the disease, supporting the role of ERß as a relevant biomarker of tumor progression and possible chemopreventive target in patients at risk of colonic neoplasia.

Dietary-induced ERbeta upregulation counteracts intestinal neoplasia development in intact male ApcMin/+ mice.

Most sporadic colorectal cancers (CRCs) develop through the adenoma-carcinoma sequence pathway and are initiated by adenomatous polyposis coli (APC) gene mutations. Estrogen receptor beta (ERbeta) is recognized to progressively reduce its expression in adenomatous and carcinomatous tissues in humans. Moreover, ERbeta deficiency enhances small intestinal tumorigenesis in rodents. In the Apc(Min/+) mouse model, we evaluated intestinal polyp development and ERbeta expression plus other biological parameters influencing tumor growth (epithelial cell proliferation, apoptosis and migration) following the addition of a combination of the ERbeta-selective agonist silymarin (SIL) and/or lignin (LIG) to a high-fat/low-fiber diet. Forty-five Apc(Min/+) mice were divided in four groups: animals fed on the tumorigenic high-fat/low-fiber diet, the tumorigenic diet supplemented with SIL (0.02%) or purified LIG (6.24%) or SIL (0.005%) + LIG (6.24%). In these animals, we assessed polyp number and volume and their degree of dysplasia together with ERbeta messenger RNA (mRNA) and protein levels and epithelial cell proliferation, migration and apoptosis. The latter group of parameters was evaluated in normal and adenomatous mucosa and the results compared with those found in wild-type (WT) mice fed on the control diet. The addition of SIL or LIG to the diet and even more the specific combination of the two significantly counteracted intestinal tumorigenesis and increased ERbeta mRNA and protein levels. Cell proliferation and apoptosis were rebalanced and cell migration accelerated, restoring values similar to those observed in WT animals. Our results further support a protective effect of ERbeta in CRC suggesting the use of the combination of SIL-LIG as a potential approach against CRC development.

Salivary phosphate-binding chewing gum reduces hyperphosphatemia in dialysis patients.

In uremic patients, hyperphosphatemia is associated with cardiovascular calcification and increased cardiovascular mortality. Despite the use of phosphate binders, only half of hemodialysis (HD) patients achieve recommended serum phosphate levels. A hyperphosphoric salivary content, which correlates linearly with serum phosphate, has been reported in HD patients. We hypothesized that binding salivary phosphate during periods of fasting in addition to using phosphate binders with meals could improve the treatment of hyperphosphatemia. We assessed the phosphate-binding capacity of the natural polymer chitosan by (31)P nuclear magnetic resonance and established that 10 and 20% (wt/vol) middle viscosity chitosan solutions bind 30 and 50% of the phosphate contained in PBS, respectively. Thirteen HD patients with serum phosphate levels >6.0 mg/dl despite treatment with sevelamer hydrochloride chewed 20 mg of chitosan-loaded chewing gum twice daily for 2 wk at fast in addition to their prescribed phosphate-binding regimen. Salivary phosphate and serum phosphate significantly decreased during the first week of chewing; by the end of 2 wk, salivary phosphate decreased 55% from baseline (73.21 +/- 19.19 to 33.19 +/- 6.53; P < 0.00001), and serum phosphate decreased 31% from baseline (7.60 +/- 0.91 to 5.25 +/- 0.89 mg/dl; P < 0.00001). Salivary phosphate returned to baseline by day 15 after discontinuing the chewing gum, whereas serum phosphate levels took 30 d to return to baseline. Parathyroid hormone and serum calcium concentrations were not affected by the gum. In conclusion, adding salivary phosphate binding to traditional phosphate binders could be a useful approach for improving treatment of hyperphosphatemia in HD patients.

Toward the definition of the mechanism of action of silymarin: activities related to cellular protection from toxic damage induced by chemotherapy.

Silymarin, the active extract from milk thistle, has been extensively used in patients with liver disease of different etiology. Although silymarin is a complex of 7 flavonolignans and polyphenols, silibinin is usually regarded as the most active component. In vitro and in vivo studies indicate that silymarin and silibinin protect the liver from oxidative stress and sustained inflammatory processes, mainly driven by Reactive Oxygen Species (ROS) and secondary cytokines. Oxidative stress and inflammation are also involved in cellular damage of many other tissues and their role in the development and toxic reactions in patients receiving cancer therapies is established. The protective effects of silymarin and silibinin, demonstrated in various tissues, suggest a clinical application in cancer patients as an adjunct to established therapies, to prevent or reduce their toxicity. Here we discuss the possible mechanism of the protective action of silymarin and silibinin, focusing on cancer therapies as agents causing cellular damage.

Aescin protection of human vascular endothelial cells exposed to cobalt chloride mimicked hypoxia and inflammatory stimuli.

Human vascular endothelial cells (HUVECs) were exposed to CoCl2 as an in vitro model of hypoxia. Expression of VCAM-1 (vascular cell adhesion molecule), reduction of PECAM-1 (platelet endothelial cell adhesion molecule) and cytoskeletal changes without alterations in cell viability were observed. HUVECs were also exposed to Escherichia coli lipopolysaccaride (LPS) as an in vitro model of inflammation: significant IL-6 release was measured. Pre-treatment of HUVECs with aescin prevented, in a concentration-dependent fashion (0.1-1 microM), the action of CoCl2 on VCAM-1 and PECAM-1, also preserving endothelial cell morphology. Furthermore, aescin pre-treatment reduced IL-6 release from LPS-activated vascular endothelium.