Supplementation with Bifidobacterium longum Bar33 and Lactobacillus helveticus Bar13 mixture improves immunity in elderly humans (over 75 years) and aged mice.

OBJECTIVE: Aging induces several physiologic and immune changes. The usefulness of probiotics in ameliorating age-related disorders remains largely unexplored. The aim of this study was to evaluate the effectiveness of a Bifidobacterium longum Bar33 and Lactobacillus helveticus Bar13 mixture in improving the physiologic status and immunity of older adults (over 75 years). Furthermore, the possible role of such mixture in ameliorating gut immunity in aged mice was investigated. METHODS: A randomized, double-blind, placebo-controlled trial was conducted with 98 adults (84.6 ± 7.8 y), supplemented for 30 d with a biscuit containing a probiotic mixture of B. longum Bar33 and L. helveticus Bar13 (1:1), or no probiotics, as placebo. Blood was collected for analysis of biochemical parameters, lymphocyte subpopulations, natural killer activity, and cytokine release. Aged Balb/c mice received the same probiotic mixture or placebo daily for 28 d, then blood and intestinal lymphocyte subpopulations were analyzed. RESULTS: The probiotic mixture ameliorated immune response in older adults by increasing naive, activated memory, regulatory T cells, B cells, and natural killer activity and decreasing memory T cells compared with placebo (P < 0.05). The biochemical parameters did not change after probiotic supplementation. In the gut of old mice, the two probiotics modulated cells crucial for gut immune homeostasis by increasing regulatory T (Treg and Tr1) and decreasing γδ T cells compared with control mice (P < 0.05). In addition, B cells increased in the gut and blood of probiotic-treated mice. CONCLUSION: Results from the present study data indicated that B. longum Bar33 and L. helveticus Bar13 improve immune function at intestinal and peripheral sites in aging.

Differential protection by cell wall components of Lactobacillus amylovorus DSM 16698Tagainst alterations of membrane barrier and NF-kB activation induced by enterotoxigenic F4+ Escherichia coli on intestinal cells.

BACKGROUND: The role of Lactobacillus cell wall components in the protection against pathogen infection in the gut is still largely unexplored. We have previously shown that L. amylovorus DSM 16698T is able to reduce the enterotoxigenic F4+ Escherichia coli (ETEC) adhesion and prevent the pathogen-induced membrane barrier disruption through the regulation of IL-10 and IL-8 expression in intestinal cells. We have also demonstrated that L. amylovorus DSM 16698T protects host cells through the inhibition of NF-kB signaling. In the present study, we investigated the role of L. amylovorus DSM 16698T cell wall components in the protection against F4+ETEC infection using the intestinal Caco-2 cell line. METHODS: Purified cell wall fragments (CWF) from L. amylovorus DSM 16698T were used either as such (uncoated, U-CWF) or coated with S-layer proteins (S-CWF). Differentiated Caco-2/TC7 cells on Transwell filters were infected with F4+ETEC, treated with S-CWF or U-CWF, co-treated with S-CWF or U-CWF and F4+ETEC for 2.5 h, or pre-treated with S-CWF or U-CWF for 1 h before F4+ETEC addition. Tight junction (TJ) and adherens junction (AJ) proteins were analyzed by immunofluorescence and Western blot. Membrane permeability was determined by phenol red passage. Phosphorylated p65-NF-kB was measured by Western blot. RESULTS: We showed that both the pre-treatment with S-CWF and the co- treatment of S-CWF with the pathogen protected the cells from F4+ETEC induced TJ and AJ injury, increased membrane permeability and activation of NF-kB expression. Moreover, the U-CWF pre-treatment, but not the co-treatment with F4+ETEC, inhibited membrane damage and prevented NF-kB activation. CONCLUSIONS: The results indicate that the various components of L. amylovorus DSM 16698T cell wall may counteract the damage caused by F4+ETEC through different mechanisms. S-layer proteins are essential for maintaining membrane barrier function and for mounting an anti-inflammatory response against F4+ETEC infection. U-CWF are not able to defend the cells when they are infected with F4+ETEC but may activate protective mechanisms before pathogen infection.

Lactobacillus amylovorus inhibits the TLR4 inflammatory signaling triggered by enterotoxigenic Escherichia coli via modulation of the negative regulators and involvement of TLR2 in intestinal Caco-2 cells and pig explants.

Inflammation derived from pathogen infection involves the activation of toll-like receptor (TLR) signaling. Despite the established immunomodulatory activities of probiotics, studies relating the ability of such bacteria to inhibit the TLR signaling pathways are limited or controversial. In a previous study we showed that Lactobacillus amylovorus DSM 16698T, a novel lactobacillus isolated from unweaned pigs, protects the intestinal cells from enterotoxigenic Escherichia coli (ETEC) K88 infection through cytokine regulation. In the present study we investigated whether the ability of L. amylovorus to counteract the inflammatory status triggered by ETEC in intestine is elicited through inhibition of the TLR4 signaling pathway. We used the human intestinal Caco-2/TC7 cells and intestinal explants isolated from 5 week-old crossbreed Pietrain/Duroc/Large-White piglets, treated with ETEC, L. amylovorus or L. amylovorus cell free supernatant, either alone or simultaneously with ETEC. Western blot analysis showed that L. amylovorus and its cell free supernatant suppress the activation of the different steps of TLR4 signaling in Caco-2/TC7 cells and pig explants, by inhibiting the ETEC induced increase in the level of TLR4 and MyD88, the phosphorylation of the IKKα, IKKß, IκBα and NF-κB subunit p65, as well as the over-production of inflammatory cytokines IL-8 and IL-1ß. The immunofluorescence analysis confirms the lack of phospho-p65 translocation into the nucleus. These anti-inflammatory effects are achieved through modulation of the negative regulators Tollip and IRAK-M. We also found that L. amylovorus blocks the up-regulation of the extracellular heat shock protein (Hsp)72 and Hsp90, that are critical for TLR4 function. By using anti-TLR2 antibody, we demonstrate that TLR2 is required for the suppression of TLR4 signaling activation. These results may contribute to develop therapeutic interventions using L. amylovorus in intestinal disorders of piglets and humans.

Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 induce different age-related metabolic profiles revealed by 1H-NMR spectroscopy in urine and feces of mice.

Age-related dysbioses of intestinal microbiota and decline in the overall metabolic homeostasis are frequently found in the elderly. Probiotic supplementation may represent a way to prevent or reduce the senescence-associated metabolic disorders. The present study evaluated the metabolic impact of Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 supplementation in relation to age by analyzing urine and feces metabolic profiles using (1)H-nuclear magnetic resonance spectroscopy and multivariate analysis. Adult (3 mo old) and aged (16 mo old) mice received an oral supplementation of the 2 probiotics (1 × 10(9) colony-forming units/d each) or phosphate buffered saline (control) daily for 30 d. Urine and feces were collected for 48 h before the end of the study. Partial least squares-discriminant analysis showed that the urinary discriminant metabolites for the probiotic treatment included higher dimethylglycine in adult and aged mice, lower sarcosine and nicotinate in adult mice, higher N-methylnicotinamide in adult mice and lower N-methylnicotinamide in aged mice compared with their controls. These results indicate a probiotic-induced modulation of homocysteine and NAD metabolism pathways, which have important implications because these pathways are involved in essential cellular processes that can be altered in senescence. The probiotic supplementation also modified the fecal metabolic profiles, inducing in both adult and aged mice higher 4-hydroxyphenylacetate and lower xylose in treated mice compared with their control mice, whereas valerate was greater in treated adult mice and lower in treated aged mice compared with their controls. The ANOVA simultaneous component analysis on urinary and fecal metabolic profiling showed an age × treatment interaction (P < 0.05), confirming the age-related modulation of the metabolic response to probiotic supplementation. The results suggest that L. acidophilus and B. lactis may prevent or reduce age-related metabolic dysfunction.

Impact of organic and conventional carrots on intestinal and peripheral immunity.

BACKGROUND: Studies on health effects of organic (ORG) products are still limited and often contradictory. We have investigated the impact of ORG and conventional (CV) carrots from two consecutive harvest years on mouse peripheral and intestinal immunity. RESULTS: Danish carrots (Bolero variety) were grown in three ORG (O1, O2 and O3) and one CV cropping system (D-CV). Italian carrots (Maestro and Excelso varieties) were grown in one ORG and one CV field for each variety. Immune phenotypes of blood, spleen and intestinal lymphocytes, and cytokine serum levels were analyzed in mice fed the different carrots for 30 days. Principal component analysis (PCA) was performed in mice fed the Danish carrots. The consumption of the 'more organic' O2 and O3 carrots induced some changes in lymphocyte populations, including an increase in regulatory T cells. In Italian carrots more differences between ORG and CV were observed in the first as compared to the second year. No relevant differences were observed in cytokine secretion. PCA showed a clear separation among mice fed the O1, O2, O3 and D-CV carrots. CONCLUSIONS: Although a great variability was observed between the two years, an immune stimulation was found after the ORG carrot consumption.

Lactobacillus rhamnosus GG and Bifidobacterium animalis MB5 induce intestinal but not systemic antigen-specific hyporesponsiveness in ovalbumin-immunized rats.

Probiotics may modulate the host immune response by mechanisms not yet fully understood. We evaluated the modulation of intestinal and systemic antigen-specific immune response by Lactobacillus rhamnosus GG (LGG) or Bifidobacterium animalis MB5 in tolerized and immunized rats. Three groups of rats received orally LGG, B. animalis, or PBS (control) for 28 d. Each group was divided into two subgroups of tolerized or immunized rats receiving orally ovalbumin (OVA; 7 mg) or PBS on d 7, 9, and 11. All rats were immunized with OVA (300 µg) on d 14 and 21. In tolerized rats, the OVA-induced proliferative response of mesenteric lymph nodes (MLN) and spleen cells did not differ from control, indicating that the two probiotics maintained the tolerance. LGG and B. animalis in immunized rats reduced the OVA-induced proliferative response in MLN (P < 0.01) but not in spleen, whereas the proliferative response to anti-CD3 and concanavalin A of MLN and spleen cells as well as the delayed-type hypersensitivity reaction were not affected by probiotic treatment, indicating OVA-specific hyporesponsiveness restricted to intestinal immunity. This hyporesponsiveness was associated with CD4+CD25+Foxp3+ T cell expansion (P < 0.01) and increased IL-10 and TGFß after LGG (P < 0.05), and increased apoptosis after B. animalis (P < 0.001) in MLN. In conclusion, we report a novel activity of LGG and B. animalis in inducing OVA-specific hyporesponsiveness in MLN of OVA-immunized rats that can be useful for a therapeutic strategy to prevent undesirable reactions to immunogenic antigens in the gut.

Prevention of TNBS-induced colitis by different Lactobacillus and Bifidobacterium strains is associated with an expansion of gammadeltaT and regulatory T cells of intestinal intraepithelial lymphocytes.

BACKGROUND: Probiotics may protect against inflammatory bowel disease through regulation of lamina propria lymphocytes (LPLs) function. Data are lacking on possible involvement of intraepithelial lymphocytes (IELs). The aim of this study was to investigate whether different probiotic mixtures prevented gut inflammatory disease and the role of both IELs and LPLs. METHODS: BALB/c mice received 2 probiotic mixtures orally for 3 weeks, as Mix1 (Lactobacillus acidophilus and Bifidobacterium longum), or Mix2 (Lactobacillus plantarum, Streptococcus thermophilus, and Bifidobacterium animalis subsp. lactis). Colitis was induced by intrarectal administration of trinitrobenzene sulfonic acid (TNBS). Probiotics in stools were analyzed by real-time polymerase chain reaction (PCR). Colon subpopulations of IELs and LPLs were assayed by flow cytometry. Serum cytokines were measured by cytometric bead array (CBA). RESULTS: All probiotics colonized the intestine. The 2 mixtures prevented the TNBS-induced intestinal damage, and Mix1 was the most effective. The Mix1 protection was associated with a reduction in CD4(+) cells of IELs and LPLs, an increase in gammadeltaT cells of IELs, and a decrease in gammadeltaT cells of LPLs. An expansion of T regulatory (Treg) cells of IELs was induced by Mix1 and Mix2. Both probiotic mixtures inhibited tumor necrosis factor (TNF)-alpha and monocyte chemotactic protein (MCP)-1 production and upregulated interleukin (IL)-10. In addition, Mix1 prevented the TNBS-induced increase of IL-12 and interferon (IFN)-gamma. CONCLUSIONS: The 2 probiotic mixtures were able to prevent the TNBS-induced colitis; the L. acidophilus and B. longum mixture was the most effective. Other than an involvement of LPLs, our results report a novel importance of the IELs population in probiotic protection.

Intestinal and peripheral immune response to MON810 maize ingestion in weaning and old mice.

This study evaluated the gut and peripheral immune response to genetically modified (GM) maize in mice in vulnerable conditions. Weaning and old mice were fed a diet containing MON810 or its parental control maize or a pellet diet containing a GM-free maize for 30 and 90 days. The immunophenotype of intestinal intraepithelial, spleen, and blood lymphocytes of control maize fed mice was similar to that of pellet fed mice. As compared to control maize, MON810 maize induced alterations in the percentage of T and B cells and of CD4(+), CD8(+), gammadeltaT, and alphabetaT subpopulations of weaning and old mice fed for 30 or 90 days, respectively, at the gut and peripheral sites. An increase of serum IL-6, IL-13, IL-12p70, and MIP-1beta after MON810 feeding was also found. These results suggest the importance of the gut and peripheral immune response to GM crop ingestion as well as the age of the consumer in the GMO safety evaluation.

Health, probiotics, and inflammation.

Probiotic bacteria are normal inhabitants of microflora and may confer several benefits, including prevention against intestinal inflammation. However, the exact mode of action of probiotics is still largely unknown. The first line of defense against the entry of pathogens is represented by the gut membrane barrier and probiotics may prevent pathogen-induced membrane damage by inhibiting pathogen adhesion and maintaining the correct organization of the tight junction and cytoskeleton proteins. The gut immune system should not only protect the mucosa against pathogens, but also avoid hypersensitivity reactions to food proteins and normal microflora. Failure of induction or maintenance of oral tolerance has been postulated to be a cause of food allergy. Feeding probiotic bacteria may prevent or ameliorate the onset of allergic disease and the associated inflammatory reactions through mechanisms involving modulation of T regulatory cells. Breakdown in tolerance toward intestinal bacteria is a primary cause of inflammatory bowel disease. Recent studies have shown that probiotics may ameliorate experimental colitis in mice by inducing interleukin 10 and interleukin 10-dependent T regulatory cells. In this article, an update of the anti-inflammatory activity of different probiotics and of the more accredited mechanisms underlying such activities are reported.

Zinc deficiency induces membrane barrier damage and increases neutrophil transmigration in Caco-2 cells.

Zinc may contribute to the host defense by maintaining the membrane barrier. In this study, we questioned whether zinc deficiency affects the membrane function and junctional structure of intestinal epithelial cells, causing increased neutrophil migration. We used the Caco-2 cell line grown in control (C), zinc-deficient, or zinc-replete medium until differentiation. Zinc deprivation induced a decrease of transepithelial electrical resistance and alterations to tight and adherens junctions, with delocalization of zonula occludens (ZO-1), occludin, beta-catenin, and E-cadherin. Disorganization of F-actin and beta-tubulin was also found in zinc deficiency. These changes were associated with a loss of the amounts of ZO-1, occluding, and beta-tubulin. In addition, zinc deficiency caused a dephosphorylation of occludin and hyperphosphorylation of beta-catenin and ZO-1. Disruption of membrane barrier integrity led to increased migration of neutrophils. In addition, zinc deficiency induced an increase in the secretion of interleukin-8, epithelial neutrophil activating peptide-78, and growth-regulated oncogene-alpha, alterations that were not found when culture medium was replete with zinc. These results provide new information on the critical role played by dietary zinc in the maintenance of membrane barrier integrity and in controlling inflammatory cell infiltration.

The novel porcine Lactobacillus sobrius strain protects intestinal cells from enterotoxigenic Escherichia coli K88 infection and prevents membrane barrier damage.

Lactobacilli have a potential to overcome intestinal disorders; however, the exact mode of action is still largely unknown. In this study, we have used the intestinal porcine intestinal IPEC-1 epithelial cells as a model to investigate a possible protective activity of a new Lactobacillus species, the L. sobrius DSM 16698(T), against intestinal injury induced by enterotoxigenic Escherichia coli (ETEC) K88 infection and the underlying mechanisms. Treatment of infected cells with L. sobrius strongly reduced the pathogen adhesion. L. sobrius was also able to prevent the ETEC-induced membrane damage by inhibiting delocalization of zonula occludens (ZO)-1, reduction of occludin amount, rearrangement of F-actin, and dephosphorylation of occludin caused by ETEC. RT-PCR and ELISA experiments showed that L. sobrius counteracted the ETEC-induced increase of IL-8 and upregulated the IL-10 expression. The involvement of IL-8 in the deleterious effects of ETEC was proven by neutralization of IL-8 with a specific antibody. A crucial role of IL-10 was indicated by blockage of IL-10 production with neutralizing anti-IL-10 antibody that fully abrogated the L. sobrius protection. L. sobrius was also able to inhibit the internalization of ETEC, which was likely favored by the leaking barrier. The protective effects were not found with L. amylovorus DSM 20531(T) treatment, a strain derived from cattle waste but phylogenetically closely related to L. sobrius. Together, the data indicate that L. sobrius exerts protection against the harmful effects of ETEC by different mechanisms, including pathogen adhesion inhibition and maintenance of membrane barrier integrity through IL-10 regulation.

Probiotic bacteria Bifidobacterium animalis MB5 and Lactobacillus rhamnosus GG protect intestinal Caco-2 cells from the inflammation-associated response induced by enterotoxigenic Escherichia coli K88.

Probiotic bacteria may provide protection against intestinal damage induced by pathogens, but the underlying mechanisms are still largely unknown. We investigated whether Bifidobacterium animalis MB5 and Lactobacillus rhamnosus GG (LGG) protected intestinal Caco-2 cells from the inflammation-associated response induced by enterotoxigenic Escherichia coli (ETEC) K88, by inhibiting pathogen attachment to the cells, which is the first step of ETEC pathogenicity, and regulating neutrophil recruitment, a crucial component of inflammation. A partial reduction of ETEC adhesion was exerted by probiotics and their culture supernatant fractions either undigested or digested with proteases. ETEC viability was unaffected by the presence of B. animalis, LGG or their supernatant fractions in the culture medium, indicating an absence of probiotic bactericidal activity. Probiotics and their supernatant fractions, either undigested or digested with proteases, strongly inhibited the neutrophil transmigration caused by ETEC. Both B. animalis and LGG counteracted the pathogen-induced up regulation of IL-8, growth-related oncogene-alpha and epithelial neutrophil-activating peptide-78 gene expression, which are chemokines essential for neutrophil migration. Moreover, the probiotics prevented the ETEC-induced increased expression of IL-1beta and TNF-alpha and decrease of transforming growth factor-alpha, which are regulators of chemokine expression. These results indicate that B. animalis MB5 and LGG protect intestinal cells from the inflammation-associated response caused by ETEC K88 by partly reducing pathogen adhesion and by counteracting neutrophil migration, probably through the regulation of chemokine and cytokine expression.

Altered expression, localization, and phosphorylation of epithelial junctional proteins in celiac disease.

We aimed to study the expression and localization of the molecular components of enterocyte junctions in celiac disease together with the level of tyrosine phosphorylation, a phenomenon known to affect their cellular distribution and function, and to explore the influence of proinflammatory cytokines. Duodenal biopsy specimens from patients with celiac disease and control subjects were used for immunoprecipitation, immunoblotting, and immunolocalization by using antioccludin, anti-zonula occludens (ZO)-1, anti-E-cadherin, anti-beta-catenin, and antiphosphotyrosine antibodies. The same procedures were carried out on filter-grown Caco-2 cells incubated in the absence or presence of interferon g and tumor necrosis factor a. In active celiac disease, the absence of a phosphorylated ZO-1 and the extensive phosphorylation of beta-catenin might be responsible for the absence of membranous localization of occludin and E-cadherin, respectively. The in vitro system showed an influence of the cytokines on the assembly of these complexes that proved the opposite to celiac samples as far as tight junctions were concerned because the presence of a phosphorylated ZO-1 enables occludin to localize in the membrane.

Novel approach for food safety evaluation. Results of a pilot experiment to evaluate organic and conventional foods.

There is evidence that organic food often contains relatively high amounts of natural toxic compounds produced by fungi or plants, whereas corresponding conventional food tends to contain more synthetic toxins such as pesticide residues, but only a few studies have evaluated the impact of their consumption on health. This study proposes a novel approach to evaluate the potential health risk of organic compared to conventional food consumption, that is, the assay of sensitive markers of cell function in vulnerable conditions. The markers utilized were intestinal and splenic lymphocyte proliferative capacity and liver acute-phase reaction, both responding to the presence of toxins. The vulnerable conditions in which body defenses can be less efficient were weaning and protein-energy malnutrition. This study reports the results of a pilot experiment on one sample of eight varieties of organically and conventionally grown wheat. Weaned rats were assigned to two groups fed conventional (CV) or organic (ORG) wheat for 30 days. Each group was divided in two subgroups of well-nourished (WN) or protein-energy-malnourished (PEM) rats. For each rat, the lymphocyte proliferation was assayed by [(3)H]thymidine incorporation after stimulation of cells with a mitogen, in a culture medium containing either commercial fetal calf serum (FCS) or the corresponding rat serum (RS) to mimic the in vivo proliferative response. The acute-phase proteins (albumin, transthyretin, transferrin, ceruloplasmin, retinol-binding protein) were measured in plasma by Western blotting and immunostaining with specific antibodies. The proliferative response of lymphocytes cultured with FCS and the amount of acute-phase proteins of rats fed the ORG wheat sample, either WN or PEM, did not differ from those of rats fed the CV wheat sample. However, the proliferative response of lymphocytes cultured with RS was inhibited in PEM-CV compared with PEM-ORG. The content of mycotoxins was highest in the organic sample, and therefore the immunotoxic effect was probably due to other contaminants in the CV wheat. In conclusion, these results indicate that the conventional wheat sample tested represented a higher risk for lymphocyte function than the wheat sample organically grown, at least in vulnerable conditions.

The mycotoxin fumonisin B1 alters the proliferation and the barrier function of porcine intestinal epithelial cells.

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium verticillioides (formerly F. moniliforme), a fungus that commonly contaminates maize. FB1 causes toxicological effects in laboratory and domestic animals including pigs. Because the gastrointestinal tract represents the first barrier met by exogenous food compounds, the purpose of this study was to investigate the effects of FB1 on IPEC-1, a porcine intestinal epithelial cell line. We first verified that low concentrations of FB1 did not exert any cytotoxic effect on IPEC-1. Indeed, significant LDH release was only observed for FB1 concentrations greater than 50 and 700 microM on proliferating and nonproliferating cells, respectively. We then demonstrated that FB1 inhibits proliferation of IPEC-1. Fluorescence-activated cell sorting (FACS) analysis of the cell cycle indicated that FB1 blocks the proliferation of intestinal cells in the G0/G1 phase. Similar results were obtained with LLC-PK1, a renal porcine epithelial cell line, which is considered to be a good model for studying FB1 in vitro effects. We have also assessed the effects of FB1 on the integrity of the barrier formed by the intestinal epithelium. We demonstrated that FB1 decreases the transepithelial electrical resistance (TEER) of IPEC-1 in a time- and dose-dependent manner. This effect was only noticed after a long exposure (8-12 days of treatment). FB1 induced the TEER decrease independently of the cell differentiation stage, and this effect was partially reversible. Taken together, our data indicate that FB1 alters the proliferation and the barrier function of intestinal cells. These results may have implications for humans and animals consuming FB1-contaminated food or feed.

Zinc oxide protects cultured enterocytes from the damage induced by Escherichia coli.

There is some evidence that zinc oxide (ZnO) protects against intestinal diseases. However, despite the suggestions that ZnO may have an antibacterial effect, the mechanisms of this protective effect have not yet been elucidated. We investigated the potential benefits of ZnO in protecting intestinal cells from damage induced by enterotoxigenic Escherichia coli (ETEC, strain K88) and the related mechanisms, using human Caco-2 enterocytes. Cell permeability, measured as transepithelial electrical resistance (TEER), was unaffected by 0.01 and 1 mmol/L ZnO treatments and moderately increased by 5 mmol/L ZnO, compared with untreated cells. Transfer of (14)C-inulin was slightly increased by 5 mmol/L ZnO compared with untreated cells; transfer was unaffected by lower concentrations. The TEER and (14)C-inulin transfer were lower in ETEC-infected cells than in uninfected cells. Treatment of ETEC exposure with 0.2 mmol/L ZnO prevented disruption of membrane integrity. The ETEC was able to adhere to enterocytes and, to some extent, invade the cells. The ZnO treatment reduced bacterial adhesion and blocked bacterial invasion. The ETEC infection upregulated the expression of the inflammatory cytokines interleukin-8, growth-related oncogene-alpha and tumor necrosis factor-alpha, and reduced that of the anti-inflammatory cytokine transforming growth factor-beta, compared with uninfected cells. The addition of 0.2 or 1 mmol/L ZnO counteracted the alteration of cytokine mRNA levels caused by ETEC. The protective effects of ZnO were not due to any antibacterial activity, because the viability of ETEC grown in a medium containing ZnO was unaffected. In conclusion, ZnO may protect intestinal cells from ETEC infection by inhibiting the adhesion and internalization of bacteria, preventing the increase of tight junction permeability and modulating cytokine gene expression.

Zinc deficiency suppresses the development of oral tolerance in rats.

Oral tolerance is a specific immune unresponsiveness to food antigens to prevent hypersensitivity reactions. We investigated whether zinc deficiency affects oral tolerance. Rats were fed a control (C) or zinc-deficient (ZD) diet, or pair-fed (PF) to ZD rats for 28 d. Beginning on d 7, rats were administered ovalbumin (OVA) orally to induce tolerance, or PBS 3 times/wk, and were then immunized by OVA injection. The proliferation of mesenteric lymph node (MLN) and spleen lymphocytes after in vitro OVA stimulation and the delayed-type hypersensitivity were higher in OVA-fed ZD than in OVA-fed C rats and not different between OVA- and PBS-fed ZD rats, indicating a suppression of tolerance. Lymphocyte proliferation did not differ between PF and C rats. Expressions of cytokines involved in oral tolerance, i.e., interleukin (IL)-4, IL-10 and transforming growth factor-beta, were higher in OVA- than in PBS-fed C rats, but not in ZD rats. Apoptosis was higher in OVA- than in PBS-fed C rats but not different between OVA- and PBS-fed ZD rats. Inflammation and ulcerations that were not present in ZD rats on d 7 (ZD(7)) developed in OVA- or PBS-fed ZD rats. Compared with ZD(7) rats, tumor necrosis factor-alpha and cytokine-induced neutrophil chemoattractant were higher in OVA- and PBS-fed ZD rats, whereas interferon-gamma increased only in OVA-fed ZD rats. In conclusion, zinc deficiency suppresses oral tolerance through dysregulation of cytokine expression and lack of antigen-specific clonal deletion. We suggest that abrogation of tolerance may lead to development of mucosal inflammation and damage.

Efecto preventivo de la lecitina poliinsaturada sobre las alteraciones de las lipoproteinas del plasma de ratas alimentadas con una dieta hipercolesterolemica / Preventive effects of polyunsaturated leccthin an the alterations of plasma lipoproteins of rats on a hypercholesrolemia diet

Se diseñó un experimento para conocer cuál de los componentes de un concentrado proteico de vicia faba previno las alteraciones producidas por una dieta hipercolesterolémica en las lipoproteínas del plasma de ratas. Se observó que la lecitina poliinsaturada evitaba en gran medida las modificaciones provocadas po una dieta hipercolesterolémica sobre la distribución del colesterol y la proteína en 5 fracciones lipoproteicas. Este fosfolipido mantuvo los índices aterogénicos en valores cercanos a los encontrados en animales que consumieron una dieta estándar. La saponina mostró un efecto limitado sobre los indicadores estudiados. Se demuestra que la lecitina poliinsaturada es la que ejerce el efecto preventivo producido por el concentrado proteico de vicia faba

Efecto preventivo de la lecitina poliinsaturada sobre las alteraciones de las lipoproteinas del plasma de ratas alimentadas con una dieta hipercolesterolemica / Preventive effects of polyunsaturated leccthin an the alterations of plasma lipoproteins of rats on a hypercholesrolemia diet

Se diseñó un experimento para conocer cuál de los componentes de un concentrado proteico de vicia faba previno las alteraciones producidas por una dieta hipercolesterolémica en las lipoproteínas del plasma de ratas. Se observó que la lecitina poliinsaturada evitaba en gran medida las modificaciones provocadas po una dieta hipercolesterolémica sobre la distribución del colesterol y la proteína en 5 fracciones lipoproteicas. Este fosfolipido mantuvo los índices aterogénicos en valores cercanos a los encontrados en animales que consumieron una dieta estándar. La saponina mostró un efecto limitado sobre los indicadores estudiados. Se demuestra que la lecitina poliinsaturada es la que ejerce el efecto preventivo producido por el concentrado proteico de vicia faba