NOD1 in the interplay between microbiota and gastrointestinal immune adaptations.

Nucleotide-binding oligomerization domain 1 (NOD1), a pattern recognition receptor (PRR) that detects bacterial peptidoglycan fragments and other danger signals, has been linked to inflammatory pathologies. NOD1, which is expressed by immune and non-immune cells, is activated after recognizing microbe-associated molecular patterns (MAMPs). This recognition triggers host defense responses and both immune memory and tolerance can also be achieved during these processes. Since the gut microbiota is currently considered a master regulator of human physiology central in health and disease and the intestine metabolizes a wide range of nutrients, drugs and hormones, it is a fact that dysbiosis can alter tissues and organs homeostasis. These systemic alterations occur in response to gastrointestinal immune adaptations that are not yet fully understood. Even if previous evidence confirms the connection between the microbiota, the immune system and metabolic disorders, much remains to be discovered about the contribution of NOD1 to low-grade inflammatory pathologies such as obesity, diabetes and cardiovascular diseases. This review compiles the most recent findings in this area, while providing a dynamic and practical framework with future approaches for research and clinical applications on targeting NOD1. This knowledge can help to rate the consequences of the disease and to stratify the patients for therapeutic interventions.

Kinetic Approach to the Influence of Chia Flour on Glucose Bioaccessibility from Hydrothermally Treated Maize and Quinoa Starch.

Starch structure and bioactive ingredients play an implicit role in the control of glucose release at intestinal level reducing the risk of inadequate metabolic response(s). This study performs a comparative kinetic approach to glucose release from hydrothermally treated (HT) maize (MS) and quinoa (QS) starch. Besides, chia flour (CF) (20%, w/w) was added to evaluate its influence of on the apparent diffusion coefficients (Dapp) when subjected to simulated gastrointestinal digestion. Hepatocyte cultures were used to monitor mitochondrial enzymes activity (test MTT) to bioaccessible glucose concentrations. With an increasing temperature, Dapp for both QS and its mixtures with CF were kept unaltered, while those for MS were disrupted progressively affecting glucose bioaccessibility. Principal component analysis revealed differences between maize and quinoa starches, but common features in the corresponding mixtures with CF. Data indicated that quinoa starch helps controlling glucose release and that addition of CF decreased mitochondrial activity in presence of insulin.

Raspberry Polyphenolic Extract Regulates Obesogenic Signals in Hepatocytes.

The aim of this in vitro study was to examine the effect of raspberry polyphenolic extract on the immune-metabolic molecular mechanisms activated by obesity-related signals in hepatocytes (HB-8965®). Alterations in endosomal/lysosomal activity (neutral red uptake assay, NR), the expression of selected genes involved with lipid oxidation, and metabolism and inflammation processes in the liver were studied. Hepatocytes were treated with plasma collected from Wistar rats that were fed a high-fat diet (HF), raspberry polyphenolic extract (PP), serine-type protease inhibitors as an agonist of TLR4 (TD) or a combination of PP with HF or TD treatments. The PP added to the experimental treatments modulated hepatic immune-metabolic mechanisms through the upregulation of STAT1, ANGPTL4, and CD44, as well as considerably reducing the NR uptake and downregulation of COX-2 and the multifunctional protein AhR. The kinetic analysis of AhR expression revealed that HF-related molecular mechanisms activated AhR mRNA expression earlier than PP initiated the regulatory effect. In conclusion, PP might be considered a valuable dietary agent that regulates obesity-related signals in hepatocytes. Moreover, taking AhR kinetic behavior into consideration, it can be assumed that PP might modulate the severity of the HF-induced downstream metabolic signaling of AhR.

Inclusion of Whole Flour from Latin-American Crops into Bread Formulations as Substitute of Wheat Delays Glucose Release and Uptake.

Bakery formulations limiting glucose availability for uptake without compromising product quality are required. Herein, bread formulations containing whole flour from Amaranthus hypochondriacus (AB), Chenopodium quinoa (QB), Salvia hispanica L (ChB) or wheat (WWB) were compared to white bread (WB) for glycaemic index (GI) in fasted animals. The hepatic expression (mRNA) of PPAR-γ receptor as key regulator in substrate fractionation towards energy expenditure was monitored. GIs were associated to fluxes of glucose release (FGluc) and metabolic response (MTT assay) of HepG2 cells. ChB (19.7%) and AB (13.5%) decreased GI to a higher extent than QB (2.7%), but all increased expression of PPARγ in relation to WB. FGluc (AB> > ChB, WWB, WB > QB) showed a reciprocal relationship with the area under curve (AUC) in vivo, and decreased MTT conversion values (WB > WWB, ChB, AB, QB) by HepG2 cells. Thus, inclusion of latin-american crops (LAcs) reducing GI, without compromising bread quality, could help preventing metabolic diseases.

Kojibiose ameliorates arachidic acid-induced metabolic alterations in hyperglycaemic rats.

Herein we hypothesise the positive effects of kojibiose (KJ), a prebiotic disaccharide, selected for reducing hepatic expression of inflammatory markers in vivo that could modulate the severity of saturated arachidic acid (ARa)-induced liver dysfunction in hyperglycaemic rats. Animals were fed daily (20 d) with ARa (0·3 mg) together or not with KJ (22 mg approximately 0·5 %, w/w diet). Glucose, total TAG and cholesterol contents and the phospholipid profile were determined in serum samples. Liver sections were collected for the expression (mRNA) of enzymes and innate biomarkers, and intrahepatic macrophage and T-cell populations were analysed by flow cytometry. ARa administration increased the proportion of liver to body weight that was associated with an increased (by 11 %) intrahepatic macrophage population. These effects were ameliorated when feeding with KJ, which also normalised the plasmatic levels of TAG and N-acyl-phosphatidylethenolamine in response to tissue damage. These results indicate that daily supplementation of KJ significantly improves the severity of ARa-induced hepatic alterations.

Impact of α-amylase during breadmaking on in vitro kinetics of starch hydrolysis and glycaemic index of enriched bread with bran.

Nowadays, the use of enzymes has become a common practice in the bakery industry, as they can improve dough quality and texture of final product. However, the use of α-amylases could have a negative effect in the glycaemic load of product, due to the released sugars from the starch hydrolysis that are not used by yeasts during the fermentation process. This study evaluated the effect of the addition of α-amylase in bakery products with bran on in vitro kinetics of starch hydrolysis. The use of flour with a high degree of extraction or high bran amount could decrease the GI even with the inclusion of α-amylase in the formulation. It should be taken into account the amount of bran and α-amylase when formulating breads in order to obtain products with lower GI than white bread. However, the fact that kinetics of starch hydrolysis remained unaltered indicates that the use of α-amylase in bread-making processes could provide technological advantages improving quality of breads without markedly changes in their glycaemic index.

Oral administration of Bifidobacterium longum CECT 7347 ameliorates gliadin-induced alterations in liver iron mobilisation.

Coeliac disease is an autoimmune disorder triggered by gluten intake, causing intestinal inflammation and mucosal damage commonly associated with the malabsorption of nutrients and ferropenic anaemia. The present study evaluates the effects of the oral administration of Bifidobacterium longum CECT 7347 on gliadin-mediated alterations in hepatic Fe deposition and Hb concentration, liver transferrin receptor (TfR)-2, IL-6, TNF-α and hepcidin (Hamp) expression (mRNA), and active hepcidin peptide production by liquid chromatography­MS/MS. Weanling rats, sensitised or not with interferon (IFN)-γ, were fed with gliadins and/or the bifidobacterial strain. Gliadin feeding increased hepatic Fe deposition; however, only gliadin-fed sensitised animals showed lower Hb concentrations than the controls. TfR2 expression decreased after gliadins were fed to both sensitised and non-sensitised animals,and restored by the administration of B. longum. These observations were accompanied by increases in IL-6 expression levels in all the treatment groups; however, TNF-α expression only increased significantly in animals fed gliadins alone or together with B. longum if they had previously been sensitised with IFN-γ. Liver expression levels of Hamp diminished in all cases to the lowest values in animals sensitised with IFN-γ after being fed with gliadins and/or bifidobacteria. In these animals, plasma Hamp active peptide concentrations significantly increased when compared with the controls. Significant correlations were calculated between Hamp expression and liver Fe contents (liver Fe = 1/0·0032 + 0·032 x Hamp(exp)), and Hb concentrations (Hb = 11·49 + 10·13 x (Hamp(exp))1/2). These data indicate that oral administration of B. longum ameliorates gliadin-mediated perturbations in liver Fe deposition and mobilisation.

Bread supplemented with amaranth (Amaranthus cruentus): effect of phytates on in vitro iron absorption.

The objective of the present study was to evaluate the effect of the bread supplemented with whole amaranth flour (0, 20 and 40%) on iron bioavailability using Caco-2 cells model. The phytate and lower myo-inositol phosphates content in in vitro bread digests were measured by high pressure liquid chromatography. The breads made with amaranth showed significant increase of soluble phytates levels (up to 1.20 µmol/g in dry matter for the 40% of substitution) in comparison with controls, which have not detectable values. A negative correlation among phytate and Fe availability was found when increased levels of amaranth.Ferritin concentration was found 2.7- and 2.0-fold higher (P<0.05) in cultures exposed to 20% and 40% of amaranth formulated bread samples, respectively, compared to control bread. The soluble phytate/Fe molar ratio explained the whole amaranth flour-mediated inhibitory effect associated to the limitation of available Fe; however, the use up to 20% of amaranth in bread formulation appears as a promising strategy to improve the nutritional value of bread, as indicated by the ferritin concentrations quantified in cell cultures. Higher proportion of amaranth flour increased Fe concentration although there was not detected any increase in Fe uptake.

NOD1-Targeted Immunonutrition Approaches: On the Way from Disease to Health.

Immunonutrition appears as a field with great potential in modern medicine. Since the immune system can trigger serious pathophysiological disorders, it is essential to study and implement a type of nutrition aimed at improving immune system functioning and reinforcing it individually for each patient. In this sense, the nucleotide-binding oligomerization domain-1 (NOD1), one of the members of the pattern recognition receptors (PRRs) family of innate immunity, has been related to numerous pathologies, such as cancer, diabetes, or cardiovascular diseases. NOD1, which is activated by bacterial-derived peptidoglycans, is known to be present in immune cells and to contribute to inflammation and other important pathways, such as fibrosis, upon recognition of its ligands. Since immunonutrition is a significant developing research area with much to discover, we propose NOD1 as a possible target to consider in this field. It is relevant to understand the cellular and molecular mechanisms that modulate the immune system and involve the activation of NOD1 in the context of immunonutrition and associated pathological conditions. Surgical or pharmacological treatments could clearly benefit from the synergy with specific and personalized nutrition that even considers the health status of each subject.

Beyond classic concepts in thyroid homeostasis: Immune system and microbiota.

It has long been known that thyroid hormones have implications for multiple physiological processes and can lead to serious illness when there is an imbalance in its metabolism. The connections between thyroid hormone metabolism and the immune system have been extensively described, as they can participate in inflammation, autoimmunity, or cancer progression. In addition, changes in the normal intestinal microbiota involve the activation of the immune system while triggering different pathophysiological disorders. Recent studies have linked the microbiota and certain bacterial fragments or metabolites to the regulation of thyroid hormones and the general response in the endocrine system. Even if the biology and function of the thyroid gland has attracted more attention due to its pathophysiological importance, there are essential mechanisms and issues related to it that are related to the interplay between the intestinal microbiota and the immune system and must be further investigated. Here we summarize additional information to uncover these relationships, the knowledge of which would help establish new personalized medical strategies.

Graphene Particles Interfere with Pro-Inflammatory Polarization of Human Macrophages: Functional and Electrophysiological Evidence.

The interaction of two types of fragmented graphene particles (30-160 nm) with human macrophages is studied. Since macrophages have significant phagocytic activity, the incorporation of graphene particles into cells has an effect on the response to functional polarization stimuli, favoring an anti-inflammatory profile. Incubation of macrophages with graphene foam particles, prepared by chemical vapor deposition, and commercially available graphene nanoplatelet particles does not affect cell viability when added at concentrations up to 100 µg mL-1 ; macrophages exhibit differential quantitative responses to each type of graphene particles. Although both materials elicit similar increases in the release of reactive oxygen species, the impact on the transcriptional regulation associated with the polarization profile is different; graphene nanoplatelets significantly modify this transcriptomic profile. Moreover, these graphene particles differentially affect the motility and phagocytosis of macrophages. After the incorporation of both graphene types into the macrophages, they exhibit specific responses in terms of the mitochondrial oxygen consumption and electrophysiological potassium currents at the cell plasma membrane. These data support the view that the physical structure of the graphene particles has an impact on human macrophage responses, paving the way for the development of new mechanisms to modulate the activity of the immune system.

Immunonutritional contribution of gut microbiota to fatty liver disease.

INTRODUCTION: Non-alcoholic fatty liver disease (NAFLD) is indisputably the most widespread liver disease worldwide, leading to a significant increase in patient morbidity, mortality, and health care utilization. The gut microbiota and its genome (microbiome) have emerged as a novel modulator of the immunometabolic processes that NAFLD implies, but microbiota-targeted interventions have resulted both astounding and at the same time unsuccessful. The most relevant alteration appears to be the overgrowth of Gram-negative bacteria, characterized by an increased ratio of Firmicutes to Bacteroidetes, although current evidence indicates species- and strain-specific effects influencing energy harvest, the host's innate and adaptive immune systems, and epigenetic regulation as determinants of the immunomodulatory milieu in NAFLD. The genera Lactobacillus and Bifidobacterium deserve special attention since many of their probiotic strains are marketed for human consumption, even more so when considering that, in conjunction with prebiotics, they are potential modulators of gut microbiota composition and/or metabolic activity. Here, a better understanding of the major intestinal microbial factors with a detrimental or preventive role in NAFLD, and of the dynamic interplay between gut microbiome and host factors, appears crucial in defining the exposome for the prevention and treatment of NAFLD and associated diseases such as metabolic syndrome, type-2 diabetes, and obesity.

INTRODUCCIÓN: La esteatosis hepática no alcohólica (NAFLD, por sus siglas en inglés) es indiscutiblemente la patología hepática más extendida a escala mundial y conlleva un aumento significativo de la utilización de la atención médica de los pacientes, así como de la morbilidad y la mortalidad. La microbiota intestinal y su genoma (microbioma) se han revelado como uno de los factores moduladores de los procesos inmunometabólicos subyacentes que desencadenan la NAFLD. Las intervenciones dirigidas a modificar la composición y/o la actividad de la microbiota han resultado sorprendentes y, al mismo tiempo, infructuosas. La disbiosis más relevante en la patología es un aumento de la proporción entre Firmicutes y Bacteroidetes. La evidencia actual indica que los efectos específicos de la especie y la cepa influyen en el resultado funcional de la microbiota sobre el metabolismo de los nutrientes, la rama innata y la adaptativa del sistema inmune, y la regulación epigenética del genoma humano en relación al NAFLD. Los géneros Lactobacillus y Bifidobacterium merecen especial atención ya que muchas cepas probióticas de estos géneros se comercializan para consumo humano, e incluso más si se considera que, junto con los prebióticos, son moduladores potenciales de la composición de la microbiota intestinal y/o su actividad metabólica. En este contexto, una mejor comprensión de los principales factores microbianos con papel perjudicial o preventivo en la NAFLD, y de la interacción dinámica entre el microbioma intestinal y los factores del huésped, parece crucial para definir el exposoma de la prevención y el tratamiento de la NAFLD y sus enfermedades asociadas, como el síndrome metabólico, la diabetes de tipo 2 y la obesidad.

Different responses of Fe transporters in Caco-2/HT29-MTX cocultures than in independent Caco-2 cell cultures.

The human intestinal epithelium is composed of several cell types, mainly enterocytes and goblet (mucin-secreting) cells. This study compares the cellular response of Fe transporters in Caco-2, HT29-MTX, and Caco-2/HT29-MTX co-culture models for Fe bioavailability. Caco-2 cells in vitro differentiate into enterocyte-like cells and HT29-MTX cell lineage into a mucin-secreting cellular population. Cell cultures were exposed to digests of Fe+3, Fe+3/ascorbic acid, cooked fish (high-available Fe) or white beans (low-available Fe). Cell responses as shown by mRNA expression of the main Fe transporters, DMT1 and DcytB, and cell ferritin formation were monitored. In Caco-2/HT29-MTX co-cultures, the mucin layer lowered the pool of free Fe to diffuse towards the cell brush border membrane of enterocytes, which was accompanied of an upregulation of DMT1 mRNA expression. In contrast, cultures exposed to digests of fish or white beans showed no significant differences in the regulation of Fe transporters.

Immunonutritional consequences of different serine-type protease inhibitors in a C57BL/6 hepatocarcinoma model.

Imbalances in innate immunity and the activity of innate immune cells are implicated in the development of hepatocellular carcinoma (HCC). Plant seeds are good sources of protease inhibitors, which can have a significant influence on human health disorders, especially in the field of cancer prevention. To elucidate the impact and preventive effects of immunonutritional serine-type protease inhibitors (STPIs) on HCC, it was used an established model of chemically induced liver injury. Injured livers induced Akt as well as hepatic infiltration of NKG2D + and CD74 + cells. Feeding STPIs reduced size and number of intrahepatic nodes of mononuclear. These animals showed an inverse association of the severity of HCC with bioactive hepcidin levels, which was significantly correlated with the hepatic myeloperoxidase activity. According to their origin, administration of STPIs significantly induce increased numbers of F4/80 + cells in injured livers that can be responsible for the biological effects detected on the parenchyma and inflammatory markers under DEN/TAA treatment. These findings can have direct implications in HCC immunotherapy where enhanced response(s) in inflammation-driven cancer patients could help promoting inflammation-driven processes and favor tumor growth. Altogether, this study demonstrates that oral administration of STPIs modulate innate immunity response influencing HCC aggressiveness and progression. These results represent a path forward to develop durable, long-lasting response against hepatocarcinoma and open a future research path in the development of coadjutant intervention strategies to pharmacological therapies.

Tolerability and Safety of a Nutritional Supplement with Potential as Adjuvant in Colorectal Cancer Therapy: A Randomized Trial in Healthy Volunteers.

Bioactive supplements display relevant therapeutic properties when properly applied according to validated molecular effects. Our previous research efforts established the basis to develop a dietary supplement based on a Rosmarinus officinalis supercritical extract. This was enriched in phenolic diterpenes (RE) with proven properties against signaling pathways involved in colon tumorigenesis, and shark liver oil rich in alkylglycerols (AKG) as a bioactive lipid vehicle to improve RE bioavailability and synergize with the potential therapeutic action of the extract. Herein, we have investigated the tolerability and safety of the supplement and the biological and molecular effects from an immuno-nutritional perspective. Sixty healthy volunteers participated in a six week, double-blind, randomized parallel pilot study with two study arms: RE-AKG capsules (CR) and control capsules (CC). Mean age (±SD) of volunteers was 28.32 (±11.39) and 27.5 (±9.04) for the control and the study groups, respectively. Safety of the CR product consumption was confirmed by analyzing liver profile, vital constants, and oxidation markers (LDLox in blood and isoprostanes and thromboxanes in urine). The following were monitored: (1) the phenotyping of plasmatic leukocytes and the ex vivo response of lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs); (2) expression of genes associated with immune-modulation, inflammation, oxidative stress, lipid metabolism, and tumorigenesis; and (3) the correlation of selected genetic variants (SNPs) with the differential responses among individuals. The lack of adverse effects on liver profile and oxidation markers, together with adequate tolerability and safe immunological adaptations, provide high-quality information for the potential use of CR as co-adjuvant of therapeutic strategies against colorectal cancer.

Inulin affects iron dialyzability from FeSO4 and FeEDTA solutions but does not alter Fe uptake by Caco-2 cells.

The in vitro effects of inulin on the fluxes of Fe (F(Fe)) and uptake by Caco-2 cells from FeSO4 and FeEDTA were evaluated. Cell ferritin formation was used as a measure of Fe uptake. Mitochondrial (MTT test) and lysosomal activities were monitored as biomarkers of the changes of cellular metabolism. Changes in mRNA expression of Fe transporters, DMT1 and Dcytb, were evaluated. Inulin decreased dialyzability and F(Fe) from FeSO4 solution, suggesting a mineral binding effect, but increased those from FeEDTA. Cultures exposed to FeEDTA solutions exhibited higher ferritin values and MTT conversion percentages. Regardless of Fe source, cell Fe uptake and mRNA expression of Fe transporters were similar with or without inulin, suggesting that inulin did not impair Fe uptake. These observations might indicate a faster cellular Fe internalization from FeEDTA solutions. From a physiological perspective, the decreased F(Fe) from FeSO4 might be reflected in a decreased Fe uptake.

Supplemental inulin does not enhance iron bioavailability to Caco-2 cells from milk- or soy-based, probiotic-containing, yogurts but incubation at 37°C does.

The in vitro effects of supplemental inulin (4%) on iron (Fe) availability in two different probiotic-containing yogurts were examined. Milk or soy-based yogurts, with and without inulin, were incubated (37°C) for 48h or without any incubation before comparison by an in vitro gastrointestinal digestion/Caco-2 cell culture model was used to assess iron bioavailability. The dialysable Fe fraction, cell ferritin formation, and cell associated Fe were monitored. Supplemental inulin decreased dialysable Fe only in non-incubated milk-based yogurt. In both yogurts incubation by itself increased dialysable Fe, and inulin increased the latter only in soy-based yogurt. Cellular ferritin concentration were higher after exposure to non-incubated milk-based than soy-based yogurt, although, after incubation the latter induced the highest ferritin formation. These data suggest that inulin does not have a direct effect on Fe bioavailability in the small intestine, and that probiotic bacteria play an enhancing role on Fe bioavailability.

Cytotoxic effect of As(III) in Caco-2 cells and evaluation of its human intestinal permeability.

Inorganic arsenic has been classified as a carcinogen for humans (Group I). However, its transit across the human intestinal epithelium has not been characterized. Using Caco-2 cells, the thiol-redox balance and apparent permeability coefficients (P(app)) for As(III) in the apical to basolateral (AP-BL) and basolateral to apical (BL-AP) direction were evaluated. After As(III) exposure, GSH-induced synthesis was observed, increasing the GSH/GSSG ratio by elevating the As(III) concentration. The AP-BL permeabilities decreased as the As(III) concentrations increased, indicating the existence of a mediated transport mechanism. The (BL-AP)/(AP-BL) permeability ratios were higher than unity, suggesting the existence of a secretion process.

Inclusion of ancient Latin-American crops in bread formulation improves intestinal iron absorption and modulates inflammatory markers.

This study compares iron (Fe) absorption in Fe-deficient animals from bread formulations prepared by substitution of white wheat flour (WB) by whole wheat flour (WWB), amaranth flour (Amaranthus hypochondriacus, 25%) (AB) and quinoa flour (Chenopodium quinoa, 25%) (QB), or chia flour (Salvia hispanica L, 5%) (ChB). Hematological parameters of Fe homeostasis, plasmatic active hepcidin peptide production (LC coupled to Ms/Ms), and liver TfR-2 and IL-6 expression (RT-qPCR) were determined. The different bread formulations increased Fe content between 14% and 83% relative to white bread. Only animals fed with WWB, AB and ChB increased haemoglobin concentrations significantly. Feeding the different bread formulations did not increase hepcidin levels, but down-regulated transferrin receptor 2 (TfR2) (apart from WWB) and IL-6 (apart from QB) expression levels. Only AB and ChB had a significant influence on Fe bioavailability at the investigated level of substitution. The potential contribution of these flours would not differ considerably from that of WWB.

An approach to As(III) and As(V) bioavailability studies with Caco-2 cells.

Foods and drinking water are the main sources of human exposure to inorganic arsenic [As(III) and As(V)]. After oral ingestion, the intestinal epithelium is the first barrier to absorption of these species. A human intestinal cell line (Caco-2) was used to evaluate cell retention and transport of As(III) (15.6-156.0 microM) and/or As(V) (15.4-170.6 microM). Cell monolayer integrity, cell viability, membrane damage and effects on cell metabolism were evaluated. Only the highest concentrations assayed [As(III): 156.0 microM; As(V): 170.6 microM] produced a cytotoxic effect with different cellular targets: As(III) altered the permeability of tight junctions, and As(V) caused uncoupling of the respiratory chain. Retention and transport of As(III) was more efficient than that of As(V). After 4h of exposure to As(III) or As(V), monolayer retention percentages varied between 0.87-2.28% and 0.14-0.39%, respectively. Transepithelial transport was greater for As(III) (5.82-7.71%) than for As(V) (not detectable-1.55%). The addition of As(III) and As(V) jointly produced a transport rate similar to that observed when they were added independently.