Milk Oligosaccharides From Different Cattle Breeds Influence Growth-Related Characteristics of Intestinal Cells

Oligosaccharides are present in human milk (HMO) in large amounts and in a high variety: Among other functions they are considered to influence the gut microbiota and gut maturation in infants. Due to the large volume of milk available bovine milk oligosaccharides (BMO) may be an alternative source of functional ingredients to potentially mimic HMO functions. Thus, we investigated direct effects of bovine milk oligosaccharides (BMO) from different cattle breeds on proliferation, differentiation and apoptosis in transformed (HT-29 and Caco-2) and non-transformed human intestinal cells (HIE cells). We observed a profound growth-inhibition effect induced by all BMO isolates in HT-29, Caco-2, and HIE cells in a dose-dependent manner. The effects varied not only between cell lines, i.e., HT-29 and Caco-2 cells were more sensitive than HIE cells, but also between the cattle breeds. Regarding the induction of differentiation, BMO induced differentiation only in HIE cells without affecting apoptosis. Cell cycle analysis via flow cytometry showed that growth inhibition was associated with a G2/M arrest in all cell lines. Expression levels detected by quantitative real-time RT-PCR revealed that this G2/M arrest was associated with changes in mRNA expression levels of cyclin A and B. Cyclin-dependent kinase inhibitors p21cip1 and p27kip1 and the tumor suppressor p53 were only enhanced in HIE cells necessary for arresting cells in the G2/M phase and induction of differentiation. In HT-29 and Caco-2 cells, a loss of p53 expression failed to induce G2/M associated induction of differentiation. The HIE cell specific differentiation induced by BMO was a result of influencing the phosphorylation states of EGFR (epidermal growth factor receptor) and MAP kinase, i.e., ERK1/2 (extracellular signal-regulated kinase 1/2), p38-α, and Akt2 phosphorylation. These results suggest that BMO inhibited intestinal cell proliferation and altered cell cycle dynamics by affecting corresponding regulator genes and mitogen-activated protein kinase signaling. As the development and maturation of digestive and absorptive processes depends on gut differentiation processes, our in vitro experiments show that breed-specific BMO are natural substances influencing various parameter which may be important in vivo in gastrointestinal development. This, however, needs to be proven in future studies

Bacterial Species Associated with Highly Allergenic Plant Pollen Yield a High Level of Endotoxins and Induce Chemokine and Cytokine Release from Human A549 Cells

none10siSensitization to pollen allergens has been increasing in Europe every year. Most studies in this field are related to climate change, phenology, allergens associated with different pollens, and allergic disorders. As a plant microhabitat, pollen is colonized by diverse microorganisms, including endotoxin-producing bacteria which may contribute to pollen allergy (pollinosis). Therefore, bacteria isolated from high allergenic and low allergenic plant pollen, as well as the pollen itself with all microbial inhabitants, were used to assess the effect of the pollen by measuring the endotoxins lipopolysaccharides (LPS) and lipoteichoic acid (LTA) concentrations and their effect on chemokine and cytokine release from transwell cultured epithelial A549 cells as a model of epithelial lung barrier. High allergenic pollen showed a significantly higher level of bacterial endotoxins; interestingly, the endotoxin level found in the bacterial isolates from high allergenic pollen was significantly higher compared to that of bacteria from low allergenic pollen. Moreover, bacterial LPS concentrations across different pollen species positively correlated with the LPS concentration across their corresponding bacterial isolates. Selected bacterial isolates from hazel pollen (HA5, HA13, and HA7) co-cultured with A549 cells induced a potent concentration-dependent release of the chemokine interleukin-8 and monocyte chemotactic protein-1 as well as the cytokine TNF-alpha and interleukin-2 to both apical and basal compartments of the transwell model. This study clearly shows the role of bacteria and bacterial endotoxins in the pollen allergy as well as seasonal allergic rhinitis.Ambika Manirajan, Binoy; Hinrichs, Ann-Kathrin; Ratering, Stefan; Rusch, Volker; Schwiertz, Andreas; Geissler-Plaum, Rita; Eichner, Gerrit; Cardinale, Massimiliano; Kuntz, Sabine; Schnell, SylviaAmbika Manirajan, Binoy; Hinrichs, Ann-Kathrin; Ratering, Stefan; Rusch, Volker; Schwiertz, Andreas; Geissler-Plaum, Rita; Eichner, Gerrit; Cardinale, Massimiliano; Kuntz, Sabine; Schnell, Sylvi

Influence of Plasma-Isolated Anthocyanins and Their Metabolites on Cancer Cell Migration (HT-29 and Caco-2) In Vitro: Results of the ATTACH Study

Cancer mortality is mainly due to metastasis. Therefore, searching for new therapeutic agents suppressing cancer cell migration is crucial. Data from human studies regarding effects of anthocyanins on cancer progression, however, are scarce and it is unclear whether physiological concentrations of anthocyanins and their metabolites reduce cancer cell migration in vivo. In addition, interactions with chemotherapeutics like 5-fluorouracil (5-FU) are largely unknown. Thus, we combined a placebo-controlled, double-blinded, cross-over study with in vitro migration studies of colon cancer cell lines to examine the anti-migratory effects of plasma-isolated anthocyanins and their metabolites (PAM). Healthy volunteers (n = 35) daily consumed 0.33 L of an anthocyanin-rich grape/bilberry juice and an anthocyanin-depleted placebo juice for 28 days. PAM were isolated before and after intervention by solid-phase extraction. HT-29 and Caco-2 cells were incubated with PAM in a Boyden chamber. Migration of HT-29 cells was significantly inhibited by PAM from juice but not from placebo. In contrast, Caco-2 migration was not affected. Co-incubation with 5-FU and pooled PAM from volunteers (n = 10), which most effectively inhibited HT-29 migration, further reduced HT-29 migration in comparison to 5-FU alone. Therefore, PAM at physiological concentrations impairs colon cancer cell migration and may support the effectiveness of chemotherapeutics

Metabolism of Milk Oligosaccharides in Preterm Pigs Sensitive to Necrotizing Enterocolitis

Human milk oligosaccharides (HMO) are major components of breast milk that may have local effects in the gastrointestinal tract and systemic functions after being absorbed, both depending on their metabolism. Using preterm pigs, we investigated the metabolic fate of HMO in three experiments with two different HMO blends. In addition, we examined effects on the colonic microbiota in the presence or absence of necrotizing enterocolitis (NEC). Thus, preterm pigs (n = 112) were fed formula without or with HMO supplementation (5–10) g/L of a mixture of 4 (4-HMO) or >25 HMO (25-HMO) for 5 (Experiment 1 and 2) or 11 days (Experiment 3). Individual HMO were quantified in colon contents and urine using MALDI-TOF-MS (matrix-assisted laser desorption ionization mass spectrometry) and HPAEC-PAD (high-performance anion-exchange chromatography with pulsed amperometric detection). Microbial colonization was analyzed by sequencing of 16S rRNA gene tags. Intestinal permeability was measured by lactulose to mannitol ratio in urine. HMO supplemented to formula were detected in urine and colon contents in preterm piglets after 5 and 11 days in all three experiments. The amount of HMO excreted via the gut or the kidneys showed large individual variations. Microbial diversity in the colon changed from high levels of Firmicutes (dominated by Clostridium) at day 5 (Exp 2) to high levels of Proteobacteria dominated by Helicobacter and Campylobacter at day 11 (Exp 3). Colonic microbiota composition as well as HMO excretion pattern varied greatly among piglets. Interestingly, the 5-day supplementation of the complex 25-HMO blend led to low concentrations of 3-fucosyllactose (FL) and lacto-N-fucopentaose (LNFP) I in colonic contents, indicating a preferred utilization of these two HMO. Although the interpretation of the data from our piglet study is difficult due to the large individual variation, the presence of Bifidobacteria, although low in total numbers, was correlated with total HMO contents, and specifically with 2′FL levels in colonic content. However, early supplementation of formula with HMO did not affect NEC incidence

Logging and soil nutrients independently explain plant trait expression in tropical forests

We acknowledge financial support by the UK Natural Environment Research Council (NE/K016253/1), with additional support through an ERC Advanced Investigator Award to YM (GEM-TRAIT; 321131). We are indebted to the Sabah Biodiversity Council, Yayasan Sabah, the Maliau Basin and Danum Valley Management Committees, the Institute for Tropical Biology and Conservation at the University of Malaysia, Sabah, and the Sabah Forest Research Centre at Sepilok. We thank Glen Reynolds and the South East Asia Rainforest Research Partnership (SEARRP). This study was supported by funding from the Sime Darby Foundation to the Stability of Altered Forest Ecosystems (SAFE) Project. This project would not have been possible without the indispensable support from dozens of research assistants in Sabah. The support from Laura Kruitbos, Unding Jami, Lisa P. Bentley, Benjamin Blonder, Puikiat Hoo, Palasiah Jotan, Alexander Shenkin and Chun Xing Wong is gratefully acknowledged. We thank Bernadus Bala Ola, Bill McDonald, Alexander Karolus and MinSheng Khoo for species identification. This publication is a contribution from the UK NERC-funded Biodiversity And Land-use Impacts on Tropical Ecosystem Function (BALI) consortium (http://bali.hmtf.info) through its Human Modified Tropical Forests thematic programme.Peer reviewedPostprin

PEPT1-Mediated Cefixime Uptake into Human Intestinal Epithelial Cells Is Increased by Ca(2+) Channel Blockers

Ca(2+) channel blockers like nifedipine have been shown to increase the oral bioavailability of β-lactam antibiotics, such as cefixime, in humans. The molecular mode of action of Ca(2+) channel blockers on β-lactam absorption, however, has not yet been defined. Using the Caco-2 human intestinal epithelial cell line, we assessed whether alterations in intracellular free Ca(2+) ion (Ca(2+)(in)) concentrations by Ca(2+) channel blockers or by Ca(2+) ionophores affect [(14)C]cefixime absorption. Reduction of Ca(2+)(in) levels by Ca(2+) channel blockers (nifedipine, verapamil, diltiazem, or bepridil) at concentrations of 100 μM led to 35 to 50% increases in the cellular uptake of 1 mM [(14)C]cefixime. Increases in Ca(2+)(in) levels by Ca(2+) ionophores, on the other hand, led to 40% reductions in [(14)C]cefixime absorption. Nifedipine increased the V(max) of cefixime transport by 67%, whereas the K(m) of cefixime transport remained unaffected. By measuring the pH in Caco-2 cells loaded with the pH-sensitive fluorescent dye 2′,7′-bis(2-carboxyethyl)-5-(6)-carboxyfluorescein, we show that cefixime transport mediated by the intestinal H(+)-coupled peptide transporter PEPT1 leads to intracellular acidification. This acid load was reduced by nifedipine, although the Ca(2+) channel blocker increased the level of H(+) and cefixime cotransport. Increases in Ca(2+)(in) levels by ionomycin enhanced the decline in intracellular pH induced by cefixime alone, although ionomycin reduced the level of H(+) and cefixime cotransport. In conclusion, our studies demonstrate that alterations of Ca(2+)(in) levels, e.g., by Ca(2+) channel blockers, affect pH regulatory systems, such as apical Na(+) and H(+) exchange, and thereby alter the H(+) gradient that serves as the driving force for uptake of β-lactams into intestinal epithelial cells