Effects of Lactobacillus rhamnosus GG supplementation on cow's milk allergy in a mouse model

<p>Abstract</p> <p>Background</p> <p>Cow's milk allergy (CMA) is one of the most prevalent human food-borne allergies, particularly in infants and young children from developed countries. Our study aims to evaluate the effects of <it>Lactobacillus rhamnosus </it>GG (LGG) administration on CMA development using whole cow's milk proteins (CMP) sensitized Balb/C mice by two different sensitization methods.</p> <p>Methods</p> <p>LGG supplemented mice were either sensitized orally with CMP and cholera toxin B-subunit (CTB) as adjuvant, or intraperitoneally (IP) with CMP but without the adjuvant. Mice were then orally challenged with CMP and allergic responses were accessed by monitoring hypersensitivity scores, measuring the levels of CMP-specific immunoglobulins (IgG1, IgG2a and IgG) and total IgE from sera, and cytokines (IL-4 and IFN-γ) from spleen lysates.</p> <p>Results</p> <p>Sensitization to CMP was successful only in IP sensitized mice, but not in orally sensitized mice with CMP and CTB. Interestingly, LGG supplementation appeared to have reduced cow's milk allergy (CMA) in the IP group of mice, as indicated by lowered allergic responses.</p> <p>Conclusions</p> <p>Adjuvant-free IP sensitization with CMP was successful in inducing CMA in the Balb/C mice model. LGG supplementation favourably modulated immune reactions by shifting Th2-dominated trends toward Th1-dominated responses in CMP sensitized mice. Our results also suggest that oral sensitization by the co-administration of CMP and CTB, as adjuvant, might not be appropriate to induce CMA in mice.</p

Cell Walls of Saccharomyces cerevisiae Differentially Modulated Innate Immunity and Glucose Metabolism during Late Systemic Inflammation

BACKGROUND: Salmonella causes acute systemic inflammation by using its virulence factors to invade the intestinal epithelium. But, prolonged inflammation may provoke severe body catabolism and immunological diseases. Salmonella has become more life-threatening due to emergence of multiple-antibiotic resistant strains. Mannose-rich oligosaccharides (MOS) from cells walls of Saccharomyces cerevisiae have shown to bind mannose-specific lectin of Gram-negative bacteria including Salmonella, and prevent their adherence to intestinal epithelial cells. However, whether MOS may potentially mitigate systemic inflammation is not investigated yet. Moreover, molecular events underlying innate immune responses and metabolic activities during late inflammation, in presence or absence of MOS, are unknown. METHODS AND PRINCIPAL FINDINGS: Using a Salmonella LPS-induced systemic inflammation chicken model and microarray analysis, we investigated the effects of MOS and virginiamycin (VIRG, a sub-therapeutic antibiotic) on innate immunity and glucose metabolism during late inflammation. Here, we demonstrate that MOS and VIRG modulated innate immunity and metabolic genes differently. Innate immune responses were principally mediated by intestinal IL-3, but not TNF-α, IL-1 or IL-6, whereas glucose mobilization occurred through intestinal gluconeogenesis only. MOS inherently induced IL-3 expression in control hosts. Consequent to LPS challenge, IL-3 induction in VIRG hosts but not differentially expressed in MOS hosts revealed that MOS counteracted LPS's detrimental inflammatory effects. Metabolic pathways are built to elucidate the mechanisms by which VIRG host's higher energy requirements were met: including gene up-regulations for intestinal gluconeogenesis (PEPCK) and liver glycolysis (ENO2), and intriguingly liver fatty acid synthesis through ATP citrate synthase (CS) down-regulation and ATP citrate lyase (ACLY) and malic enzyme (ME) up-regulations. However, MOS host's lower energy demands were sufficiently met through TCA citrate-derived energy, as indicated by CS up-regulation. CONCLUSIONS: MOS terminated inflammation earlier than VIRG and reduced glucose mobilization, thus representing a novel biological strategy to alleviate Salmonella-induced systemic inflammation in human and animal hosts

Reduction of salmonella-induced enteric and systemic inflammation by mannan-oligosaccharide prebiotic through improvement of innate defense mechanism

Salmonella Enteritidis has become a major burden in human health due to emergence of multiple-antibiotic resistant strains that has made antibiotic treatment difficult. Today, there is urgency to develop efficacious alternatives to the utilization of sub-therapeutic antibiotics in poultry. We, therefore, hypothesized that, in contrast to sub-therapeutic antibiotics, mannose-rich oligosaccharides (MOS), a natural prebiotic, would improve intestinal innate defense mechanisms in healthy and Salmonella-infected chickens, and abrogate S. Enteritidis invasion of intestinal epithelium, thus mitigating Salmonella-induced intestinal and systemic inflammation. In contrast to virginiamycin (VIRG) and bacitracin (BACT) antibiotics, dietary MOS (0.2%) significantly improved intestinal innate defensive mechanisms in chickens raised under good sanitary conditions, as demonstrated by increased development of mucin-secreting goblet cells that ultimately secretes higher amounts of mucins, and microflora enrichment with beneficial bacteria, especially bifidobacteria. Moreover, MOS was equally effective as VIRG and BACT in the control of intestinal E. coli. But, at higher (0.5%) than the recommended (0.2%) dosage, MOS conferred no additional intestinal health benefits. To examine whether MOS may control S. Enteritidis through improvement of host's innate defense mechanisms, young chicks were deliberately infected with S. Enteritidis. Interestingly, whereas control (CTL) chicks suffered from drowsiness, diarrhea, starvation, exfoliation of epithelial cells and damaged villi integrity, such classic signs of Salmonella-induced enteric inflammation were abrogated by MOS and VIRG. However, IL 12 down-regulation by MOS revealed that MOS terminated S. Enteritidis-induced enteric inflammation earlier than VIRG. Whereas VIRG relied mostly on its bactericidal properties, MOS significantly improved host's mucins-mediated defense mechanism against S. Enteritidis. MOS increased secretions of neutral and acidic mucins through increased numbers of neutral and acidic goblet cells, rather than increased mucins-secreting capacity of goblet cells, as indicated by non-differential MUC 2 expressions between infected chicks fed VIRG and MOS. MUC 1 down-regulation by MOS indicated that MOS more significantly reduced S. Enteritidis damage of epithelial cells than VIRG. Evidently, intestinal villi were longer and healthier in S. Enteritidis-infected chicks fed MOS than VIRG. S. Enteritidis invasion of the intestinal epithelium triggers extra-intestinal infections and systemic inflammation. A Salmonella LPS-induced systemic inflammation chicken model and microarray analysis approach was employed to determine whether MOS, in contrast to VIRG, may potentially mitigate systemic inflammation, and reduce glucose mobilization during late systemic inflammation. MOS inherently induced IL 3 expression in non-challenged control hosts. However, consequent to LPS challenge, IL 3 was induced in VIRG hosts but not differentially expressed in MOS hosts, therefore revealing that MOS counteracted LPS's detrimental inflammatory effects. Indeed, the lower energy demands of LPS-challenged birds fed MOS were sufficiently met through TCA citrate-derived energy, as indicated by ATP citrate synthase (CS) up-regulation. Contrastingly, VIRG host's elevated energy requirements increased gene expressions for intestinal gluconeogenesis (PEPCK) and liver glycolysis (ENO2). In conclusion, this study revealed the mechanisms by which MOS, in contrast to VIRG, i) enhanced host's intestinal innate defense mechanisms against Salmonella; ii) terminated Salmonella-induced intestinal and systemic inflammation earlier; iii) and modulated innate immunity to markedly reduce glucose mobilization during late systemic inflammation. Therefore, MOS represents a biological strategy that can prevent or treat S. Enteritidis infections in poultry and humans, without posing the risk of developing antibiotic-resistance.Le développement des souches de S. Enteritidis résistantes aux antibiotiques compromet le traitement de la salmonellose avec des antibiotiques thérapeutiques dans la santé humaine. Donc, il y a urgence de développer des remplaçants efficaces aux antibiotiques de croissance chez la volaille. Notre hypothèse était que contrairement aux antibiotiques sous-thérapeutiques, l'utilisation d'oligosaccharides riches en mannose (MOS), un prébiotique naturel, améliorerait les mécanismes de défense intestinale innés chez les poulets sains et infectés par la Salmonelle, prévenant ainsi l'invasion de l'épithélium intestinal par S. Enteritidis et atténuant de ce fait l'inflammation intestinale et systémique induite par la Salmonelle. Contrairement aux antibiotiques tels que la virginiamycine (VIRG) et la bacitracine (BACT), le MOS (0.2%) a amélioré les mécanismes de défense intestinale innés chez les poulets, tel que démontré par la prolifération des cellules à gobelet sécrétant ainsi plus de mucines, et l'enrichissement de la flore microbienne avec des bactéries bénéfiques, particulièrement bifidobacteria. Le MOS était aussi efficace que la VIRG et BACT dans le contrôle intestinal d'E. coli. Mais, à une dose plus élevée (0.5%) que le dosage recommandé (0.2%), le MOS n'a démontré aucun bénéfice additionnel dans la santé intestinale. Afin d'examiner si le MOS pourrait mieux contrôler le S. Enteritidis intestinale tout en améliorant les mécanismes de défense innés, des poussins ont été infectés avec S. Enteritidis. Les poussins infectés alimentés avec la diète témoin ont souffert de somnolence, diarrhée, perte d'appétit, exfoliation des cellules épithéliales et endommagement des villosités, tandis que ces symptômes ont été abrogés par le MOS et la VIRG. Cependant, une réduction dans l'expression de IL 12 par le MOS relatif à la VIRG démontra que le MOS termina l'inflammation induite par S. Enteritidis plus tôt. Le MOS augmenta la sécrétion des mucines neutres et acides en augmentant les nombres de cellules à gobelet neutres et acides plutôt que par une sécrétion accrue de mucines par chaque cellule à gobelet; l'expression de MUC 2 était inchangé entre les poussins infectés et alimentés avec la VIRG ou MOS. En revanche une diminution dans l'expression du MUC 1 induite par le MOS indiqua que le MOS a réduit les dommages des cellules épithéliales causés par S. Enteritidis. Les villosités intestinales étaient plus longues et saines chez les poussins infectés et alimentés avec le MOS que la VIRG. L'invasion de l'épithélium intestinal par S. Enteritidis cause l'inflammation systémique. Pour déterminer si le MOS, contrairement à la VIRG, pourrait atténuer l'inflammation systémique et réduire la mobilisation de glucose pendant la phase avancée de l'inflammation, nous avons utilisé un modèle d'inflammation systémique et des analyses de microréseaux d'ADN. Le MOS induit l'expression d'IL 3 chez les poulets non soumis au LPS. Mais, suite à l'injection de LPS, l'expression d'IL 3 a été augmentée chez les poulets alimentés avec la VIRG que le MOS, indiquant que le MOS a aboli les effets inflammatoires du LPS. En effet, une augmentation dans l'expression de l'ATP citrate de synthase (CS) indiqua que les demandes énergétiques inférieures des poulets injectés avec du LPS et alimentés avec le MOS ont été suffisamment satisfaites par l'énergie dérivée par le cycle de l'acide citrique. Par contre, les besoins énergétiques plus élevés chez les poulets alimentés avec la VIRG ont entrainé une augmentation de l'expression des gènes de la néoglucogenèse intestinale (PEPCK) et de la glycolyse du foie (ENO2). En conclusion, cette étude a démontré les mécanismes par lesquels le MOS, contrairement à la VIRG, i) a augmenté les mécanismes de défense intestinaux innés contre la Salmonelle; ii) a mis fin à l'inflammation intestinale et systémique causée par la salmonelle plus tôt; iii) et a réduit la mobilisation du glucose pendant une phase avancée dans l'inflammation

Evaluation of purified lignin and mannanoligosaccharides as alternatives to antibiotic growth promoters in poultry production

The potential of lignin and mannanoligosaccharides (MOS), as alternatives to antibiotic growth promoters was evaluated in broilers. Dietary treatments included: (1) negative control (CTL-, antibiotic free); (2) positive control (CTL+, 11 mg/kg virginiamycin); (3) MOS (diet 1 + Bio-Mos: 0.2% to 21 d and 0.1% thereafter); (4) LL (diet 1 + 1.25% Alcell lignin); (5) HL (diet 1 + 2.5% Alcell lignin). Bodyweight and feed conversion were not different when broilers were fed the CTL+, MOS, LL or HL diet. Birds fed MOS or LL had increased jejunum villi height (P &lt; 0.05) and greater goblet cell number per villus (P &lt; 0.05) when compared to those fed the CTL+ diet. MOS and LL increased (P &lt; 0.05) the cecal populations of Lactobacilli and Bifidobacteria when compared to CTL+ fed birds. However, Lactobacilli and Bifidobacteria loads were lowest (P &lt; 0.05) in birds fed the CTL+ or HL diet. Litter E. coli load was reduced (P &lt; 0.05) when birds were fed MOS than when fed the CTL+ diet, but comparable to LL or HL fed birds. In birds challenged with pathogenic strains of E. coli (O2 and O88 serotypes) and fed the MOS or HL diet, the cecal population of total E. coli was lower (P &lt; 0.05) than those fed the CTL+ diet; LL fed birds tended to have lower E. coli load than CTL+ fed birds. In summary, birds fed the MOS or LL diet had comparative advantage over CTL+ fed birds as evidenced by increased cecal populations of Lactobacilli and Bifdobacteria, increased villi height and greater goblet cell number in the jejunum, lower E. coli load in the litter, and lower cecal population of E. coli after an in vivo challenge with pathogenic strains of E. coli. Therefore, MOS and lignin could be regarded as natural alternatives to antibiotic growth promoters in poultry production.Key words. Antibiotics, mannanoligosaccharides, lignin, gut health, broilers

Genes identified as differentially expressed due to LPS within antibiotic-fed hosts¹.

1<p>Hosts fed antibiotic (VIRG): LPS-challenged v/s non-challenged controls; The complete raw data have been deposited in the Gene Expression Omnibus (GEO) database, <a href="http://www.ncbi.nlm.nih.gov/projects/geo" target="_blank">www.ncbi.nlm.nih.gov/projects/geo</a> (accession no. GSE28959).</p>2<p>+: up-regulated genes by LPS; −: down-regulated genes by LPS.</p

Schematic illustration of LPS effects on glucose metabolism between MOS- and VIRG-fed hosts.

<p>(i) LPS caused no major intestinal metabolic activities in MOS-fed hosts; (ii) in absence of liver glucose mobilization, <i>KCNA3</i> was up-regulated, whereas <i>ENO2</i> and <i>UGP2</i> down-regulation reduced glycolysis and glycogen synthesis, respectively; (iii) <i>CS</i> up-regulation increased TCA cycle-derived energy from high liver pyruvate; (iv) <i>ACLY</i>, <i>ME</i> and <i>FAS</i> down-regulations inhibited liver fatty acid biosynthesis; whereas PRKAG2 up-regulation inhibited fatty acid and cholesterol biosynthesis.</p

Schematic illustration of VIRG effects on glucose metabolism between control and LPS-challenged hosts.

<p>(i) LPS increased intestinal gluconeogenesis by up-regulating <i>PEPCK</i>; (ii) mobilized glucose increased liver glycolytic activities through <i>ENO2</i> up-regulation; (iii) <i>CS</i> down-regulation reduced utilization of glycolytic substrates by the TCA cycle for energy; (iv) <i>ACLY</i>, <i>ME</i> and <i>FAS</i> up-regulations increased liver fatty acid biosynthesis from high liver citrate.</p

Schematic illustration of MOS effects on glucose metabolism between control and LPS-challenged hosts.

<p>(i) LPS triggered no major intestinal metabolic activities; (ii) in absence of glucose mobilization, liver glucose uptake and transport were repressed by <i>DIO2</i> down-regulation and <i>KCNA3</i> up-regulation, respectively; (iii) glycolysis and glycogen synthesis were coordinately reduced by <i>ENO2</i> and <i>UGP2</i> down-regulation, respectively; (iv) <i>CS</i> up-regulation increased TCA-derived energy from high liver pyruvate; (v) <i>PRKAG2</i> up-regulation inhibited fatty acid and cholesterol biosynthesis.</p

RT-qPCR validation of microarray data.

<p>LPS up-regulated liver <i>ACLY</i>, <i>ME</i> and <i>FAS</i> among VIRG-fed hosts (<i>A</i>). But, LPS down-regulated these genes in MOS- than VIRG-fed host (<i>B</i>). Intestinal <i>PEPCK</i> was up-regulated among control hosts fed MOS (<i>C</i>), and by LPS among VIRG-fed hosts (<i>D</i>). Data are presented as mean ± SEM (<i>n</i> = 6). *, <i>P</i><0.01 by Scheffe's <i>t</i> test.</p