The Mucus Binding Factor Is Not Necessary for Lacticaseibacillus rhamnosus CRL1505 to Exert Its Immunomodulatory Activities in Local and Distal Mucosal Sites.

Both viable and non-viable orally administered Lacticaseibacillus rhamnosus CRL1505 modulate immunity in local (intestine) and distal (respiratory) mucosal sites. So, intestinal adhesion and colonization are not necessary for this probiotic strain to exert its immunomodulatory effects. In this work, a mucus-binding factor knockout CRL1505 strain (ΔmbfCRL1505) was obtained and the lack of binding ability to both intestinal epithelial cells and mucin was demonstrated in vitro. In addition, two sets of in vivo experiments in 6-week-old Balb/c mice were performed to evaluate ΔmbfCRL1505 immunomodulatory activities. (A) Orally administered ΔmbfCRL1505 prior to intraperitoneal injection of the Toll-like receptor 3 (TLR3) agonist poly(I:C) significantly reduced intraepithelial lymphocytes (CD3+NK1.1+CD8αα+) and pro-inflammatory mediators (TNF-α, IL-6 and IL-15) in the intestinal mucosa. (B) Orally administered ΔmbfCRL1505 prior to nasal stimulation with poly(I:C) significantly decreased the levels of the biochemical markers of lung tissue damage. In addition, reduced recruitment of neutrophils and levels of pro-inflammatory mediators (TNF-α, IL-6 and IL-8) as well as increased IFN-ß and IFN-γ in the respiratory mucosa were observed in ΔmbfCRL1505-treated mice when compared to untreated control mice. The immunological changes induced by the ΔmbfCRL1505 strain were not different from those observed for the wild-type CRL1505 strain. Although it is generally accepted that the expression of adhesion factors is necessary for immunobiotics to induce their beneficial effects, it was demonstrated here that the mbf protein is not required for L. rhamnosus CRL1505 to exert its immunomodulatory activities in local and distal mucosal sites. These results are a step forward towards understanding the mechanisms involved in the immunomodulatory capabilities of L. rhamnosus CRL1505.

Chemical structures of oligosaccharides in milks of the American black bear (Ursus americanus americanus) and cheetah (Acinonyx jubatus).

The milk oligosaccharides were studied for two species of the Carnivora: the American black bear (Ursus americanus, family Ursidae, Caniformia), and the cheetah, (Acinonyx jubatus, family Felidae, Feliformia). Lactose was the most dominant saccharide in cheetah milk, while this was a minor saccharide and milk oligosaccharides predominated over lactose in American black bear milk. The structures of 8 neutral saccharides from American black bear milk were found to be Gal(ß1-4)Glc (lactose), Fuc(α1-2)Gal(ß1-4)Glc (2'-fucosyllactose), Gal(α1-3)Gal(ß1-4)Glc (isoglobotriose), Gal(α1-3)[Fuc(α1-2)]Gal(ß1-4)Glc (B-tetrasaccharide), Gal(α1-3)[Fuc(α1-2)]Gal(ß1-4)[Fuc(α1-3)]Glc (B-pentasaccharide), Fuc(α1-2)Gal(ß1-4)[Fuc(α1-3)]GlcNAc(ß1-3)Gal(ß1-4)Glc (difucosyl lacto-N-neotetraose), Gal(α1-3)Gal(ß1-4)[Fuc(α1-3)]GlcNAc(ß1-3)Gal(ß1-4)Glc (monogalactosyl monofucosyl lacto-N-neotetraose) and Gal(α1-3)Gal(ß1-4)GlcNAc(ß1-3)Gal(ß1-4)Glc (Galili pentasaccharide). Structures of 5 acidic saccharides were also identified in black bear milk: Neu5Ac(α2-3)Gal(ß1-4)Glc (3'-sialyllactose), Neu5Ac(α2-6)Gal(ß1-4)GlcNAc(ß1-3)[Fuc(α1-2)Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (monosialyl monofucosyl lacto-N-neohexaose), Neu5Ac(α2-6)Gal(ß1-4)GlcNAc(ß1-3)[Gal(α1-3)Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (monosialyl monogalactosyl lacto-N-neohexaose), Neu5Ac(α2-6)Gal(ß1-4)GlcNAc(ß1-3){Gal(α1-3)Gal(ß1-4)[Fuc(α1-3)]GlcNAc(ß1-6)}Gal(ß1-4)Glc (monosialyl monogalactosyl monofucosyl lacto-N-neohexaose), and Neu5Ac(α2-6)Gal(ß1-4)GlcNAc(ß1-3){Gal(α1-3)[Fuc(α1-2)]Gal(ß1-4)[Fuc(α1-3)]GlcNAc(ß1-6)}Gal(ß1-4)Glc (monosialyl monogalactosyl difucosyl lacto-N-neohexaose). A notable feature of some of these milk oligosaccharides is the presence of B-antigen (Gal(α1-3)[Fuc(α1-2)]Gal), α-Gal epitope (Gal(α1-3)Gal(ß1-4)Glc(NAc)) and Lewis x (Gal(ß1-4)[Fuc(α1-3)]GlcNAc) structures within oligosaccharides. By comparison to American black bear milk, cheetah milk had a much smaller array of oligosaccharides. Two cheetah milks contained Gal(α1-3)Gal(ß1-4)Glc (isoglobotriose), while another cheetah milk did not, but contained Gal(ß1-6)Gal(ß1-4)Glc (6'-galactosyllactose) and Gal(ß1-3)Gal(ß1-4)Glc (3'-galactosyllactose). Two cheetah milks contained Gal(ß1-4)GlcNAc(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (lacto-N-neohexaose), and one cheetah milk contained Gal(ß1-4)Glc-3'-O-sulfate. Neu5Ac(α2-8)Neu5Ac(α2-3)Gal(ß1-4)Glc (disialyllactose) was the only sialyl oligosaccharide identified in cheetah milk. The heterogeneity of milk oligosaccharides was found between both species with respect of the presence/absence of B-antigen and Lewis x. The variety of milk oligosaccharides was much greater in the American black bear than in the cheetah. The ratio of milk oligosaccharides-to-lactose was lower in cheetah (1:1-1:2) than American black bear (21:1) which is likely a reflection of the requirement for a dietary supply of N-acetyl neuraminic acid (sialic acid), in altricial ursids compared to more precocial felids, given the role of these oligosaccharides in the synthesis of brain gangliosides and the polysialic chains on neural cell adhesion.

Chemical characterization of the milk oligosaccharides of some Artiodactyla species including giraffe (Giraffa camelopardalis), sitatunga (Tragelaphus spekii), deer (Cervus nippon yesoensis) and water buffalo (Bubalus bubalis).

Mammalian milk/colostrum usually contains oligosaccharides along with the predominant disaccharide lactose. It has been found that the number and identity of these milk oligosaccharides varies among mammalian species. Oligosaccharides predominate over lactose in the milk/colostrum of Arctoidea species (Carnivora), whereas lactose predominates over milk oligosaccharides in Artiodactyla including cow, sheep, goat, camel, reindeer and pig. To clarify whether heterogeneity of a variety of milk oligosaccharides is found within other species of Artiodactyla, they were studied in the milk of giraffe, sitatunga, deer and water buffalo. The following oligosaccharides were found: Neu5Ac(α2-3)[GalNAc(ß1-4)]Gal(ß1-4)Glc (GM2 tetrasaccharide), and Gal(α1-3)Gal(ß1-4)Glc (isoglobotriose) in giraffe milk; Neu5Ac(α2-3)Gal(ß1-4)Glc (3'-SL), Neu5Ac(α2-6)Gal(ß1-4)Glc (6'-SL), Gal(α1-4)Gal(ß1-4)Glc (globotriose) and isoglobotriose in sitatunga colostrum; Gal(ß1-3)Gal(ß1-4)Glc (3'-GL), Gal(ß1-6)Gal(ß1-4)Glc (6'-GL), isoglobotriose, Gal(ß1-4)GlcNAc(ß1-3)Gal(ß1-4)Glc (lacto-N-neotetraose, LNnT), Gal(ß1-4)Glc-3'-O-SO3 (3'-O-lactose sulphate) in deer milk; 3'-GL, isoglobotriose and Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc (3',3″-digalactosyllactose, DGL) in water buffalo colostrum. Thus it was shown that the milk oligosaccharides are heterogeneous among these Artiodactyla species.

Chemical structures of oligosaccharides in milk of the raccoon (Procyon lotor).

In this study on milk saccharides of the raccoon (Procyonidae: Carnivora), free lactose was found to be a minor constituent among a variety of neutral and acidic oligosaccharides, which predominated over lactose. The milk oligosaccharides were isolated from the carbohydrate fractions of each of four samples of raccoon milk and their chemical structures determined by 1H-NMR and MALDI-TOF mass spectroscopies. The structures of the four neutral milk oligosaccharides were Fuc(α1-2)Gal(ß1-4)Glc (2'-fucosyllactose), Fuc(α1-2)Gal(ß1-4)GlcNAc(ß1-3)Gal(ß1-4)Glc (lacto-N-fucopentaose IV), Fuc(α1-2)Gal(ß1-4)GlcNAc(ß1-3)Gal(ß1-4)GlcNAc(ß1-3)Gal(ß1-4)Glc (fucosyl para lacto-N-neohexaose) and Fuc(α1-2)Gal(ß1-4)GlcNAc(ß1-3)[Fuc(α1-2)Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (difucosyl lacto-N-neohexaose). No type I oligosaccharides, which contain Gal(ß1-3)GlcNAc units, were detected, but type 2 saccharides, which contain Gal(ß1-4)GlcNAc units were present. The monosaccharide compositions of two of the acidic oligosaccharides were [Neu5Ac]1[Hex]6[HexNAc]4[deoxy Hex]2, while those of another two were [Neu5Ac]1[Hex]8[HexNAc]6[deoxy Hex]3. These acidic oligosaccharides contained α(2-3) or α(2-6) linked Neu5Ac, non reducing α(1-2) linked Fuc, poly N-acetyllactosamine (Gal(ß1-4)GlcNAc) and reducing lactose.

Exopolysaccharides from Lactobacillus delbrueckii OLL1073R-1 modulate innate antiviral immune response in porcine intestinal epithelial cells.

Previous studies demonstrated that the extracellular polysaccharides (EPSs) produced by Lactobacillus delbrueckii OLL1073R-1 (LDR-1) improve antiviral immunity, especially in the systemic and respiratory compartments. However, it was not studied before whether those EPSs are able to beneficially modulate intestinal antiviral immunity. In addition, LDR-1-host interaction has been evaluated mainly with immune cells while its interaction with intestinal epithelial cells (IECs) was not addressed before. In this work, we investigated the capacity of EPSs from LDR-1 to modulate the response of porcine IECs (PIE cells) to the stimulation with the Toll-like receptor (TLR)-3 agonist poly(I:C) and the role of TLR2, TLR4, and TLR negative regulators in the immunoregulatory effect. We showed that innate immune response triggered by TLR3 activation in porcine IECs was differentially modulated by EPS from LDR-1. EPSs treatment induced an increment in the expression of interferon (IFN)-α and IFN-ß in PIE cells after the stimulation with poly(I:C) as well as the expression of the antiviral factors MxA and RNase L. Those effects were related to the reduced expression of A20 in EPS-treated PIE cells. EPS from LDR-1 was also able to reduce the expression of IL-6 and proinflammatory chemokines. Although further in vivo studies are needed, our results suggest that these EPSs or a yogurt fermented with LDR-1 have potential to improve intestinal innate antiviral response and protect against intestinal viruses.

Identification of sialyl oligosaccharides including an oligosaccharide nucleotide in colostrum of an addax (Addax nasomaculatus) (Subfamily Antelopinae).

Mammalian milk/colostrum usually contains milk oligosaccharides along with the predominant lactose. Although milk oligosaccharides of a variety of Bovidae species including cow, sheep and goat have been characterized, those of the addax, an Antelopinae species of the Bovidae, have not as yet been clarified. In this study, several sialyl oligosaccharides were purified from a sample of addax colostrum and characterized as follows: Neu5Ac(α2-8)Neu5Ac(α2-3)Gal(ß1-4)Glc, Neu5Gc(α2-8)Neu5Gc(α2-3)Gal(ß1-4)Glc, Neu5Ac(α2-3)Gal(ß1-4)Glc, Neu5Ac(α2-6)Gal(ß1-4)GlcNAc, Neu5Gc(α2-3)Gal(ß1-4)Glc, Neu5Gc(α2-6)Gal(ß1-4)Glc, Neu5Gc(α2-6)Gal(ß1-4)GlcNAc. In addition, an oligosaccharide nucleotide Neu5Gc(α2-6)Gal(ß1-4)GlcNAcα1-UDP was characterized. Molecular species of a variety of sialyl oligosaccharides found in milk and colostrum of these Bovidae were compared.

Synthesis of an allergy inducing tetrasaccharide "4P-X".

4P-X (ß-D-galactopyranosyl-(1 â†’ 4)-ß-D-galactopyranosyl-(1 â†’ 6)-[ß-D-galactopyranosyl-(1 â†’ 4)]-ß-D-glucopyranose) is included in galacto-oligosaccharides (GOSs) produced by ß-galactosidase derived from Bacillus circulans. 4P-X has been known to induce particularly strong allergies. High purity 4P-X is essential for use as a standard to quantify the amount of 4P-X in GOSs; however, the isolation of high purity 4P-X has never been reported. In this study, we achieved the synthesis of 4P-X by a combination of organic and enzymatic chemical syntheses in a short time. This is the first report of isolated, high purity 4P-X.

Isolation of lactic acid bacteria bound to the porcine intestinal mucosa and an analysis of their moonlighting adhesins.

The adhesion of lactic acid bacteria (LAB) to the intestinal mucosa is one of the criteria in selecting for probiotics. Eighteen LAB were isolated from porcine intestinal mucin (PIM): ten strains of Lactobacillus, six strains of Weissella, and two strains of Streptococcus. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for phosphate-buffered saline (PBS) extracts from the LAB, many bands were detected in half of the samples, while a few and/or no clear bands were detected in the other half. All six of the selected LAB showed adhesion to PIM. L. johnsonii MYU 214 and MYU 221 showed adhesion at more than 10%. W. viridescens MYU 208, L. reuteri MYU 213, L. mucosae MYU 225, and L. agilis MYU 227 showed medium levels of adhesion at 5.9-8.3%. In a comprehensive analysis for the adhesins in the PBS extracts using a receptor overlay analysis, many moonlighting proteins were detected and identified as candidates for adhesins: GroEL, enolase, and elongation factor Tu in MYU 208; peptidase C1, enolase, formyl-CoA transferase, phosphoglyceromutase, triosephosphate isomerase, and phosphofructokinase in MYU 221; and DnaK, enolase, and phosphoglycerate kinase in MYU 227. These proteins in the PBS extracts, which included such things as molecular chaperones and glycolytic enzymes, may play important roles as adhesins.

Immunoregulatory effects triggered by immunobiotic Lactobacillus jensenii TL2937 strain involve efficient phagocytosis in porcine antigen presenting cells.

BACKGROUND: Immunobiotic Lactobacillus jensenii TL2937 modulates porcine mononuclear phagocytes from Peyer's patches (PPMPs) and induces a differential production of pro- and anti-inflammatory cytokines in response to Toll-like receptor (TLR)-4 activation. In view of the important role played by phagocytosis in the activation of antigen presenting cells (APCs), the aim of the present work was to examine the interaction of TL2937 with porcine PPMPs focusing on phagocytosis. In addition, this study aimed to investigate whether the effects of L. jensenii TL2937 in porcine blood monocyte-derived dendritic cells (MoDCs) are similar to those found in PPMPs considering that MoDCs do not recapitulate all functions of mucosal APCs. RESULTS: Studies showed a high ability of porcine CD172a(+) PPMPs to phagocytose L. jensenii TL2937. Interestingly, our results also revealed a reduced capacity of the non-immunomodulatory L. plantarum TL2766 to be phagocytosed by those immune cells. Phagocytosis of L. jensenii TL2937 by porcine PPMPs was partially dependent on TLR2. In addition, we demonstrated that TL2937 strain was able to improve the expression of IL-1ß, IL-12 and IL-10 in immature MoDCs resembling the effect of this immunobiotic bacterium on PPMPs. Moreover, similarly to PPMPs those immunomodulatory effects were related to the higher capacity of TL2937 to be phagocytosed by immature MoDCs. CONCLUSIONS: Microbial recognition in APCs could be effectively mediated through ligand-receptor interactions that then mediate phagocytosis and signaling. For the immunobiotic strain TL2937, TLR2 has a partial role for its interaction with porcine APCs and it is necessary to investigate the role of other receptors. A challenge for future research will be advance in the full understanding of the molecular interactions of immunobiotic L. jensenii TL2937 with porcine APCs that will be crucial for the successful development of functional feeds for the porcine host. This study is a step in that direction.

Chemical characterization of milk oligosaccharides of the tiger quoll (Dasyurus maculatus), a marsupial.

Milk oligosaccharides were separated from the carbohydrate fraction of milk of the tiger quoll a species of marsupial that is closely related to the eastern quoll, Dasyurus viverrinus. They were characterized by (1)H - nuclear magnetic resonance spectroscopy and matrix - assisted laser desorption/ionization time-of-flight mass spectrometry. The following oligosaccharides were identified; Gal(ß1-3)Gal(ß1-4)Glc, Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc, Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc, Gal(ß1-3)Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc, Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-3)Gal(ß1-4)Glc, Gal(ß1-3)[Gal(ß1-3)Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc, Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc, Neu5Ac(α2-3) Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc, Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc with an α(2-3)Neu5Ac linked to ß(1-4)Gal residue of either branch of Gal(ß1-4)GlcNAc(ß1-6) units, and Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc with a ß(1-3) linked Gal and an α(2-3) linked Neu5Ac. In addition, larger oligosaccharides were characterized as follows; Gal(ß1-3){Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)GlcNAc(ß1-6)}Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc and Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-3){Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)GlcNAc(ß1-6)}Gal(ß1-4)Glc and their α(2-3) linked Neu5Ac derivatives.

Characterization of two novel sialyl N-acetyllactosaminyl nucleotides separated from ovine colostrum.

The milk/colostrum of some mammalian species is known to contain sugar nucleotides including uridine diphosphate (UDP) oligosaccharides in addition to lactose and milk oligosaccharides, but the detailed structures of these UDP oligosaccharides have not so far been clarified. In this study we isolated two UDP-sialyl N-acetyllactosamines from ovine colostrum and characterized them using (1)H-NMR and MALDI-TOFMS spectroscopies. Their structures were found to be Neu5Gc(α2-3)Gal(ß1-4)GlcNAcα1-UDP and Neu5Gc(α2-6)Gal(ß1-4)GlcNAcα1-UDP.

Immunobiotic Bifidobacteria Strains Modulate Rotavirus Immune Response in Porcine Intestinal Epitheliocytes via Pattern Recognition Receptor Signaling.

In this work, we aimed to characterize the antiviral response of an originally established porcine intestinal epithelial cell line (PIE cells) by evaluating the molecular innate immune response to rotavirus (RVs). In addition, we aimed to select immunomodulatory bacteria with antiviral capabilities. PIE cells were inoculated with RVs isolated from different host species and the infective titers and the molecular innate immune response were evaluated. In addition, the protection against RVs infection and the modulation of immune response by different lactic acid bacteria (LAB) strains was studied. The RVs strains OSU (porcine) and UK (bovine) effectively infected PIE cells. Our results also showed that RVs infection in PIE cells triggered TLR3-, RIG-I- and MDA-5-mediated immune responses with activation of IRF3 and NF-κB, induction of IFN-ß and up-regulation of the interferon stimulated genes MxA and RNase L. Among the LAB strains tested, Bifidobacterium infantis MCC12 and B. breve MCC1274 significantly reduced RVs titers in infected PIE cells. The beneficial effects of both bifidobacteria were associated with reduction of A20 expression, and improvements of IRF-3 activation, IFN-ß production, and MxA and RNase L expressions. These results indicate the value of PIE cells for studying RVs molecular innate immune response in pigs and for the selection of beneficial bacteria with antiviral capabilities.

Chemical characterization of milk oligosaccharides of the common wombat (Vombatus ursinus).

Previous structural characterizations of marsupial milk oligosaccharides have been performed in the tammar wallaby, red kangaroo, koala, common brushtail possum and the eastern quoll. To clarify the homology and heterogeneity of milk oligosaccharides among marsupial species, which could provide information on their evolution, the oligosaccharides of wombat milk carbohydrate were characterized in this study. Neutral and acidic oligosaccharides were isolated from the carbohydrate fractions of two samples of milk of the common wombat and characterized by (1) H-nuclear magnetic resonance spectroscopy. The structures of six neutral saccharides were found to be Gal(ß1-4)Glc (lactose), Gal(ß1-3)Gal(ß1-4)Glc (3'-galactosyllactose), Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc (3',3"-digalactosyllactose), Gal(ß1-3)Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc, Gal(ß1-3)Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (galactosyl lacto-N-novopentaose I) and Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (lacto-N-novooctaose), while those of six acidic saccharides were Neu5Ac(α2-3)Gal(ß1-3)Gal(ß1-4)Glc. (sialyl 3'-galactosyllactose), Neu5Ac(α2-3)Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc (sialyl 3',3"-digalactosyllactose), Neu5Ac(α2-3)Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (sialyl lacto-N-novopentaose a), Gal(ß1-3)[Neu5Ac(α2-3)Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (sialyl lacto-N-novopentaose c), Neu5Ac(α2-3)Gal(ß1-3)Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc,, Neu5Ac(α2-3)Gal(ß1-3)Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc and Gal(ß1-3)Gal(ß1-3)[Neu5Ac(α2-3)Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc. In addition, small amounts of sulfated oligosaccharides but no oligosaccharides containing Neu5Gc or α(2-6) linked Neu5Ac were detected.

Abundances of Triacylglycerol Positional Isomers and Enantiomers Comprised of a Dipalmitoylglycerol Backbone and Short- or Medium-chain Fatty Acids in Bovine Milk Fat.

Bovine milk fat (BMF) is composed of triacylglycerols (TAG) rich in palmitic acid (P), oleic acid (O), and short-chain or medium-chain fatty acids (SCFAs or MCFAs). The composition and binding positions of the fatty acids on the glycerol backbone determine their physical and nutritional properties. SCFAs and MCFAs are known to characteristically bind to the sn-3 position of the TAGs in BMF; however, there are very few non-destructive analyses of TAG enantiomers binding the fatty acids at this position. We previously reported a method to resolve the enantiomers of TAGs, binding both long-chain saturated fatty acid and unsaturated fatty acid at the sn-1 and 3 positions, in palm oil, fish oil, and marine mammal oil using chiral HPLC. Here, we further developed a method to resolve several TAG enantiomers containing a dipalmitoyl (PP) glycerol backbone and one SCFA (or MCFA) in BMF. We revealed that the predominant TAG structure in BMF was homochiral, such as 1,2-dipalmitoyl-3-butyroyl-sn-glycerol. This is the first quantitative determination of many TAG enantiomers, which bind to a SCFA or MCFA, in BMF was evaluated simultaneously. Furthermore, the results indicated that the amount ratios of the positional isomers and enantiomers of TAGs consisting of a dipalmitoyl (PP) glycerol backbone and SCFA (or MCFA), resembled the whole TAG structures containing the other diacylglycerol backbones consisting of P, O, myristic acid, and/or stearic acid in BMF.

Chemical characterization of milk oligosaccharides of the eastern quoll (Dasyurus viverrinus).

Structural characterizations of marsupial milk oligosaccharides have been performed in four species to date: the tammar wallaby (Macropus eugenii), the red kangaroo (Macropus rufus), the koala (Phascolarctos cinereus) and the common brushtail possum (Trichosurus vulpecula). To clarify the homology and heterogeneity of milk oligosaccharides among marsupials, the oligosaccharides in the carbohydrate fraction of eastern quoll milk were characterized in this study. Neutral and acidic oligosaccharides were separated and characterized by (1)H-nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The structures of the neutral oligosaccharides were Gal(ß1-3)Gal(ß1-4)Glc (3'-galactosyllactose), Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc (3",3'-digalactosyllactose), Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (lacto-N-novopentaose I), Gal(ß1-3)Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (galactosyl lacto-N-novopentaose I), Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-3)Gal(ß1-4)Glc (galactosyl lacto-N-novopentaose II), Gal(ß1-3)[Gal(ß1-3)Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (galactosyl lacto-N-novopentaose III) and Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (lacto-N-novooctaose). The structures of the acidic oligosaccharides detected are Neu5Ac(α2-3)Gal(ß1-4)Glc (3'-sialyllactose), Gal(ß1-3)(O-3-sulfate)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (lacto-N-novopentaose I sulfate a), Gal(ß1-3)[Gal(ß1-4)(O-3-sulfate)GlcNAc(ß1-6)]Gal(ß1-4)Glc (lacto-N-novopentaose I sulfate b), Neu5Ac(α2-3)Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (sialyl lacto-N-novopentaose a), Gal(ß1-3)[Neu5Ac(α2-3)Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (sialyl lacto-N-novopentaose c), Neu5Ac(α2-3) Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc, and Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc with an α(2-3) Neu5Ac linked to ß(1-4)Gal residue of either branch of Gal(ß1-4)GlcNAc(ß1-6) units. The most predominant oligosaccharides in the carbohydrate fraction of mid-lactation milk were found to be lacto-N-novopentaose I and lacto-N-novooctaose, i.e., branched oligosaccharides that contain N-acetylglucosamine. The predominance of these branched oligosaccharides, rather than of a series of linear ß(1-3) linked galacto oligosaccharides, appears to be the main feature of the eastern quoll milk oligosaccharides that differentiates them from those of the tammar wallaby and the brushtail possum.

Advanced application of porcine intramuscular adipocytes for evaluating anti-adipogenic and anti-inflammatory activities of immunobiotics.

We previously established a clonal porcine intramuscular preadipocyte (PIP) line and we were able to establish a protocol to obtain functional mature adipocytes from PIP cells. We hypothesized that both PIP cells and mature adipocytes are likely to be useful in vitro tools for increasing our understanding of immunobiology of adipose tissue, and for the selection and study of immunoregulatory probiotics (immunobiotics) able to modulate adipocytes immune responses. In this study, we investigated the immunobiology of PIP cells and mature adipocytes in relation to their response to TNF-α stimulation. In addition, we evaluated the possibility that immunobiotic microorganisms modify adipogenesis and immune functions of porcine adipose tissue through Peyer's patches (PPs) immune-competent cells. We treated the porcine PPs immune cells with different probiotic strains; and we evaluated the effect of conditioned media from probiotic-stimulated immune cells in PIP cells and mature adipocytes. The Lactobacillus GG and L. gasseri TMC0356 showed remarkable effects, and were able to significantly reduce the expression of pro-inflammatory factors and negative regulators (A20, Bcl-3, and MKP-1) in adipocytes challenged with TNF-α. The results of this study demonstrated that the evaluation of IL-6, and MCP-1 production, and A20 and Bcl-3 down-regulation in TNF-α-challenged adipocytes could function as biomarkers to screen and select potential immunobiotic strains. Taking into consideration that several in vivo and in vitro studies clearly demonstrated the beneficial effects of Lactobacillus GG and L. gasseri TMC0356 in adipose inflammation, the results presented in this work indicate that the PIP cells and porcine adipocytes could be used for the screening and the selection of new immunobiotic strains with the potential to functionally modulate adipose inflammation when orally administered.

4-O-Acetyl-sialic acid (Neu4,5Ac2) in acidic milk oligosaccharides of the platypus (Ornithorhynchus anatinus) and its evolutionary significance.

Monotremes (echidnas and platypus) retain an ancestral form of reproduction: egg-laying followed by secretion of milk onto skin and hair in a mammary patch, in the absence of nipples. Offspring are highly immature at hatching and depend on oligosaccharide-rich milk for many months. The primary saccharide in long-beaked echidna milk is an acidic trisaccharide Neu4,5Ac2(α2-3)Gal(ß1-4)Glc (4-O-acetyl 3'-sialyllactose), but acidic oligosaccharides have not been characterized in platypus milk. In this study, acidic oligosaccharides purified from the carbohydrate fraction of platypus milk were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and (1)H-nuclear magnetic resonance spectroscopy. All identified structures, except Neu5Ac(α2-3)Gal(ß1-4)Glc (3'-sialyllactose) contained Neu4,5Ac2 (4-O-acetyl-sialic acid). These include the trisaccharide 4-O-acetyl 3'-sialyllactose, the pentasaccharide Neu4,5Ac2(α2-3)Gal(ß1-4)GlcNAc(ß1-3)Gal(ß1-4)Glc (4-O-acetyl-3'-sialyllacto-N-tetraose d) and the hexasaccharide Neu4,5Ac2(α2-3)Gal(ß1-4)[Fuc(α1-3)]GlcNAc(ß1-3)Gal(ß1-4)Glc (4-O-acetyl-3'-sialyllacto-N-fucopentaose III). At least seven different octa- to deca-oligosaccharides each contained a lacto-N-neohexaose core (LNnH) and one or two Neu4,5Ac2 and one to three fucose residues. We conclude that platypus milk contains a diverse (≥ 20) array of neutral and acidic oligosaccharides based primarily on lactose, lacto-N-neotetraose (LNnT) and LNnH structural cores and shares with echidna milk the unique feature that all identified acidic oligosaccharides (other than 3'-sialyllactose) contain the 4-O-acetyl-sialic acid moiety. We propose that 4-O-acetylation of sialic acid moieties protects acidic milk oligosaccharides secreted onto integumental surfaces from bacterial hydrolysis via steric interference with bacterial sialidases. This may be of evolutionary significance since taxa ancestral to monotremes and other mammals are thought to have secreted milk, or a milk-like fluid containing oligosaccharides, onto skin surfaces.

The toll-like receptor family protein RP105/MD1 complex is involved in the immunoregulatory effect of exopolysaccharides from Lactobacillus plantarum N14.

The radioprotective 105 (RP105)/MD1 complex is a member of the Toll-like receptor (TLR) family. It was reported that RP105/MD1 cooperates with the lipopolysaccharide (LPS) receptor TLR4/MD2 complex and plays a crucial role in the response of immune cells to LPS. This work evaluated whether RP105, TLR4 or TLR2 were involved in the immunoregulatory capacities of Lactobacillus plantarum N14 (LP14) or its exopolysaccharides (EPS). EPS from LP14 were fractionated into neutral (NPS) and acidic (APS) EPS by anion exchange chromatography. Experiments with transfectant HEK(RP105/MD1) and HEK(TLR2) cells demonstrated that LP14 strongly activated NF-κB via RP105 and TLR2. When we studied the capacity of APS to activate NF-κB pathway in HEK(RP105/MD1) and HEK(TLR4) cells; we observed that APS strongly stimulated both transfectant cells. Our results also showed that LP14 and APS were able to decrease the production of pro-inflammatory cytokines (IL-6, IL-8 and MCP-1) in porcine intestinal epithelial (PIE) cells in response to enterotoxigenic Escherichia coli (ETEC) challenge. In order to confirm the role of TLR2, TLR4 and RP105 in the immunoregulatory effect of APS from LP14, we used small interfering RNA (siRNA) to knockdown these receptors in PIE cells. The capacity of LP14 and APS to modulate pro-inflammatory cytokine expression was significantly reduced in PIE(RP105-/-) cells. It was also shown that LP14 and APS were capable of upregulating negative regulators of the TLR signaling in PIE cells. This work describes for the first time that a Lactobacillus strain and its EPS reduce inflammation in intestinal epithelial cells in a RP105/MD1-dependend manner.

Lactobacillus delbrueckii TUA4408L and its extracellular polysaccharides attenuate enterotoxigenic Escherichia coli-induced inflammatory response in porcine intestinal epitheliocytes via Toll-like receptor-2 and 4.

SCOPE: Immunobiotics are known to modulate intestinal immune responses by regulating Toll-like receptor (TLR) signaling pathways, which are responsible for the induction of cytokines and chemokines in response to microbial-associated molecular patterns. However, little is known about the immunomodulatory activity of compounds or molecules from immunobiotics. METHODS AND RESULTS: We evaluated whether Lactobacillus delbrueckii subsp. delbrueckii TUA4408L (Ld) or its extracellular polysaccharide (EPS): acidic EPS (APS) and neutral EPS (NPS), modulated the response of porcine intestinal epitheliocyte (PIE) cells against Enterotoxigenic Escherichia coli (ETEC) 987P. The roles of TLR2, TLR4, and TLR negative regulators in the immunoregulatory effects were also studied. ETEC-induced inflammatory cytokines were downregulated when PIE cells were prestimulated with both Ld or EPSs. Ld, APS, and NPS inhibited ETEC mediated mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) activation by upregulating TLR negative regulators. The capability of Ld to suppress inflammatory cytokines was diminished when PIE cells were blocked with anti-TLR2 antibody, while APS failed to suppress inflammatory cytokines when cells were treated with anti-TLR4 antibody. Induction of Ca²âº fluxes in TLR knockdown cells confirmed that TLR2 plays a principal role in the immunomodulatory action of Ld, while the activity of APS is mediated by TLR4. In addition, NPS activity depends on both TLR4 and TLR2. CONCLUSION: Ld and its EPS have the potential to be used for the development of anti-inflammatory functional foods to prevent intestinal diseases in both humans and animals.