RESUMO
Arabinoxylans of various structures and sources have shown to possess the ability to induce a range of immune responses in different cell types in vitro and in vivo. Although the underlying mechanisms remain to be fully established, several studies point towards the involvement of activation of pattern recognition receptors (PRRs). Activation of specific PRRs (i.e., Dectin-1 and CR3) has also been shown to play a key role in the induction of a non-specific memory response in innate immune cells, termed 'trained innate immunity'. In the current study, we assessed whether arabinoxylans are also able to induce trained innate immunity. To this end, a range of arabinoxylan preparations from different sources were tested for their physicochemical properties and their capacity to induce innate immune training and resilience. In human macrophages, rice and wheat-derived arabinoxylan preparations induced training and/or resilience effects, the extent depending on fiber particle size and solubility. Using a Dectin-1 antagonist or a CR3 antibody, it was demonstrated that arabinoxylan-induced trained immunity in macrophages is mainly dependent on Dectin-1b. These findings build on previous observations showing the immunomodulatory potential of arabinoxylans as biological response modifiers and open up promising avenues for their use as health promoting ingredients.
Assuntos
Imunidade Inata , Lectinas Tipo C , Macrófagos , Xilanos , Humanos , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Receptores de Reconhecimento de Padrão , Xilanos/farmacologiaRESUMO
Pathogenesis of C. difficile in the intestine is associated with the secretion of toxins which can damage the intestinal epithelial layer and result in diseases such as diarrhoea. Treatment for C. difficile infections consists of antibiotics which, however, have non-specific microbiocidal effects and may cause intestinal dysbiosis which results in subsequent health issues. Therefore, alternative treatments to C. difficile infections are required. We investigated whether different black soldier fly- and mealworm-derived fractions, after applying the INFOGEST digestion protocol, could counteract C. difficile toxin A-mediated barrier damage of small intestinal Caco-2 cells. Treatment and pre-treatment with insect-derived fractions significantly (p < 0.05) mitigated the decrease of the transepithelial electrical resistance (TEER) of Caco-2 cells induced by C. difficile toxin A. In relation to these effects, RNA sequencing data showed an increased transcription of cell junctional and proliferation protein genes in Caco-2 cells. Furthermore, the transcription of genes regulating immune signalling was also increased. To identify whether this resulted in immune activation we used a Caco-2/THP-1 co-culture model where the cells were only separated by a permeable membrane. However, the insect-derived fractions did not change the basolateral secreted IL-8 levels in this model. To conclude, our findings suggest that black soldier fly- and mealworm-derived fractions can attenuate C. difficile induced intestinal barrier disruption and they might be promising tools to reduce the symptoms of C. difficile infections.
Assuntos
Toxinas Bacterianas/toxicidade , Proliferação de Células/genética , Enterotoxinas/toxicidade , Insetos , Junções Intercelulares/genética , Mucosa Intestinal/fisiologia , Intestino Delgado/citologia , Transcrição Gênica , Animais , Células CACO-2 , Clostridioides difficile , Técnicas de Cocultura , Besouros , Dípteros , Células Epiteliais/fisiologia , Humanos , Imunidade/genética , Imunomodulação , Proteínas de Insetos/farmacologia , Mucosa Intestinal/citologia , Intestino Delgado/fisiologia , Macrófagos , RNA-Seq , Células THP-1RESUMO
Beta-glucans enable functional reprogramming of innate immune cells, a process defined as "trained immunity", which results in enhanced host responsiveness against primary (training) and/or secondary infections (resilience). Trained immunity holds great promise for promoting immune responses in groups that are at risk (e.g. elderly and patients). In this study, we modified an existing in vitro model for trained immunity by actively inducing monocyte-to-macrophage differentiation using M-CSF and applying continuous exposure. This model reflects mucosal exposure to ß-glucans and was used to study the training effects of a variety of soluble or non-soluble ß-glucans derived from different sources including oat, mushrooms and yeast. In addition, trained immunity effects were related to pattern recognition receptor usage, to which end, we analyzed ß-glucan-mediated Dectin-1 activation. We demonstrated that ß-glucans, with different sources and solubilities, induced training and/or resilience effects. Notably, trained immunity significantly correlated with Dectin-1 receptor activation, yet Dectin-1 receptor activation did not perform as a sole predictor for ß-glucan-mediated trained immunity. The model, as validated in this study, adds on to the existing in vitro model by specifically investigating macrophage responses and can be applied to select non-digestible dietary polysaccharides and other components for their potential to induce trained immunity.
Assuntos
Ativação de Macrófagos/imunologia , Macrófagos/imunologia , beta-Glucanas/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Humanos , Ativação de Macrófagos/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/imunologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/efeitos dos fármacosRESUMO
Inhibition of maltase, sucrase, isomaltase and glucoamylase activity by acarbose, epigallocatechin gallate, epicatechin gallate and four polyphenol-rich tea extract from white, green, oolong, black tea, were investigated by using rat intestinal enzymes and human Caco-2 cells. Regarding rat intestinal enzyme mixture, all four tea extracts were very effective in inhibiting maltase and glucoamylase activity, but only white tea extract inhibited sucrase and isomaltase activity and the inhibition was limited. Mixed-type inhibition on rat maltase activity was observed. Tea extracts in combination with acarbose, produced a synergistic inhibitory effect on rat maltase activity. Caco-2 cells experiments were conducted in Transwells. Green tea extract and epigallocatechin gallate show dose-dependent inhibition on human sucrase activity, but no inhibition on rat sucrase activity. The opposite was observed on maltase activity. The results highlighted the different response in the two investigated model systems and show that tea polyphenols are good inhibitors for α-glucosidase activity.
Assuntos
Glicosídeo Hidrolases/antagonistas & inibidores , Intestinos/enzimologia , Extratos Vegetais/química , Polifenóis/farmacologia , Chá/química , Acarbose/farmacologia , Animais , Células CACO-2 , Catequina/análogos & derivados , Catequina/farmacologia , Glucana 1,4-alfa-Glucosidase/antagonistas & inibidores , Inibidores de Glicosídeo Hidrolases/farmacologia , Humanos , Cinética , Oligo-1,6-Glucosidase/antagonistas & inibidores , Ratos , Sacarase/antagonistas & inibidores , alfa-Glucosidases/efeitos dos fármacosRESUMO
The immunomodulatory properties of non-digestible polysaccharides (NDPs) have been recognized in in vitro and in vivo studies. The latter mostly demonstrated altered frequencies and inflammatory status of immune cells as clinical parameters. Most of the NDP activity will be exerted in the intestine where they can directly interact with macrophages. The predominant macrophage phenotype in the intestine is M2-like, with M1-like macrophages arising during inflammation. Here, we investigated transcriptional and functional impact on these macrophage phenotypes by NDP-treatment (i.e. yeast-derived soluble ß-glucan (yeast-ßG), apple-derived RG-I (apple-RGI), shiitake-derived ß-glucan (shiitake-ßG) or wheat-derived arabinoxylan (wheat-AX)). Wheat-AX, and to a lesser extent shiitake-ßG and apple-RGI but not yeast-ßG, reduced endocytosis and antigen processing capacity of M1- and M2-like macrophages. Moreover, the NDPs, and most notably wheat-AX, strongly induced transcription and secretion of a unique set of cytokines and chemokines. Conditioned medium from wheat-AX-treated M2-like macrophages subsequently demonstrated strongly increased monocyte recruitment capacity. These findings are in line with clinically observed immunomodulatory aspects of NDPs making it tempting to speculate that clinical activity of some NDPs is mediated through enhanced chemoattraction and modifying activity of intestinal immune cells.
Assuntos
Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Triticum/química , Xilanos/farmacologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Endocitose/efeitos dos fármacos , Humanos , Inflamação/tratamento farmacológico , Lentinula/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Xilanos/análise , beta-Glucanas/farmacologiaRESUMO
PURPOSE: Epidemiological and intervention studies have attempted to link the health effects of a diet rich in fruits and vegetables with the consumption of polyphenols and their impact in neurodegenerative diseases. Studies have shown that polyphenols can cross the intestinal barrier and reach concentrations in the bloodstream able to exert effects in vivo. However, the effective uptake of polyphenols into the brain is still regarded with some reservations. Here we describe a combination of approaches to examine the putative transport of blackberry-digested polyphenols (BDP) across the blood-brain barrier (BBB) and ultimate evaluation of their neuroprotective effects. METHODS: BDP was obtained by in vitro digestion of blackberry extract and BDP major aglycones (hBDP) were obtained by enzymatic hydrolysis. Chemical characterization and BBB transport of extracts were evaluated by LC-MSn. BBB transport and cytoprotection of both extracts was assessed in HBMEC monolayers. Neuroprotective potential of BDP was assessed in NT2-derived 3D co-cultures of neurons and astrocytes and in primary mouse cerebellar granule cells. BDP-modulated genes were evaluated by microarray analysis. RESULTS: Components from BDP and hBDP were shown to be transported across the BBB. Physiologically relevant concentrations of both extracts were cytoprotective at endothelial level and BDP was neuroprotective in primary neurons and in an advanced 3D cell model. The major canonical pathways involved in the neuroprotective effect of BDP were unveiled, including mTOR signaling and the unfolded protein response pathway. Genes such as ASNS and ATF5 emerged as novel BDP-modulated targets. CONCLUSIONS: BBB transport of BDP and hBDP components reinforces the health benefits of a diet rich in polyphenols in neurodegenerative disorders. Our results suggest some novel pathways and genes that may be involved in the neuroprotective mechanism of the BDP polyphenol components.
Assuntos
Barreira Hematoencefálica/metabolismo , Fármacos Neuroprotetores/farmacologia , Extratos Vegetais/farmacologia , Polifenóis/farmacologia , Rubus/metabolismo , Animais , Células Cultivadas , Cromatografia Líquida , Humanos , Técnicas In Vitro , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Fármacos Neuroprotetores/metabolismo , Extratos Vegetais/metabolismo , Reação em Cadeia da Polimerase , Polifenóis/metabolismoRESUMO
More understanding of the risk-benefit effect of the glycoalkaloid tomatine is required to be able to estimate the role it might play in our diet. In this work, we focused on effects towards intestinal epithelial cells based on a Caco-2 model in order to analyze the influence on the cell monolayer integrity and on the expression levels of genes involved in cholesterol/sterol biosynthesis (LDLR), lipid metabolism (NR2F2), glucose and amino acid uptake (SGLT1, PAT1), cell cycle (PCNA, CDKN1A), apoptosis (CASP-3, BMF, KLF6), tight junctions (CLDN4, OCLN2) and cytokine-mediated signaling (IL-8, IL1ß, TSLP, TNF-α). Furthermore, since the bioactivity of the compound might vary in the presence of a food matrix and following digestion, the influence of both pure tomatine and in vitro digested tomatine with and without tomato fruit matrix was studied. The obtained results suggested that concentrations <20 µg/mL of tomatine, either undigested or in vitro digested, do not compromise the viability of Caco-2 cells and stimulate cytokine expression. This effect of tomatine, in vitro digested tomatine or in vitro digested tomatine with tomato matrix differs slightly, probably due to variations of bioactivity or bioavailability of the tomatine. The results lead to the hypothesis that tomatine acts as hormetic compound that can induce beneficial or risk toxic effects whether used in low or high dose.
Assuntos
Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes/efeitos dos fármacos , Intestinos/citologia , Tomatina/farmacologia , Células CACO-2 , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Intestinos/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Modelos Biológicos , Estrutura Molecular , Receptores de LDL/genética , Junções Íntimas/genética , Tomatina/químicaRESUMO
Berries are generally considered beneficial to health. This health-promoting potential has mainly been ascribed to berries' phytochemical and vitamin content, and little attention has been paid to the potential benefits of berries for the digestive tract, despite this being the first point of contact. In vivo studies that described the health effects of berries on individual parts of the digestive tract (ie, the mouth, esophagus, stomach, small and large intestine, microbiome, and immune system) were reviewed. Immune effects were included because a large part of the immune system is located in the intestine. Beneficial health effects were mainly observed for whole berry extracts, not individual berry components. These effects ranged from support of the immune system and beneficial microbiota to reduction in the number and size of premalignant and malignant lesions. These results demonstrate the potency of berries and suggest berries can serve as a strong adjuvant to established treatments or therapies for a variety of gastrointestinal and immune-related illnesses.
Assuntos
Fenômenos Fisiológicos do Sistema Digestório/efeitos dos fármacos , Frutas , Sistema Imunitário , Compostos Fitoquímicos , Dieta , Humanos , Sistema Imunitário/efeitos dos fármacos , Sistema Imunitário/fisiologiaRESUMO
Intestinal epithelial cells, like Caco-2, are commonly used to study the interaction between food, other luminal factors and the host, often supported by microarray analysis to study the changes in gene expression as a result of the exposure. However, no compiled dataset for Caco-2 has ever been initiated and Caco-2-dedicated gene expression networks are barely available. Here, 341 Caco-2-specific microarray samples were collected from public databases and from in-house experiments pertaining to Caco-2 cells exposed to pathogens, probiotics and several food compounds. Using these datasets, a gene functional association network specific for Caco-2 was generated containing 8937 nodes 129711 edges. Two in silico methods, a modified version of biclustering and the new Differential Expression Correlation Analysis, were developed to identify Caco-2-specific gene targets within a pathway of interest. These methods were subsequently applied to the AhR and Nrf2 signalling pathways and altered expression of the predicted target genes was validated by qPCR in Caco-2 cells exposed to coffee extracts, known to activate both AhR and Nrf2 pathways. The datasets and in silico method(s) to identify and predict responsive target genes can be used to more efficiently design experiments to study Caco-2/intestinal epithelial-relevant biological processes.
Assuntos
Genes Reporter , Análise em Microsséries , Transdução de Sinais/genética , Algoritmos , Células CACO-2 , Análise por Conglomerados , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Especificidade de Órgãos/genéticaRESUMO
Modulation of inflammation-related immune response on THP-1 macrophages of protein hydrolysates derived from tilapia mince, casein and pea protein, were investigated. The protein substrates were hydrolyzed by Virgibacillus halodenitrificans SK1-3-7 proteinase. The degree of hydrolysis (DH) of casein was observed to be the highest throughout the course of hydrolysis. When challenging THP-1 macrophages, tilapia mince hydrolysate (TMH) enhanced innate immunity through induction of IL-1ß and COX-2 expression. Anti-inflammatory activity was observed in casein hydrolysate (CH) and pea protein hydrolysate (PPH) by attenuating lipopolysaccharide- (LPS) induced pro-inflammatory gene expression in THP-1 macrophages. CH suppressed IL-1ß, IL-6, IL-8, TNF-α and COX-2, while PPH reduced LPS-induced IL-6 and TNF-α responses. In addition, CH and PPH showed stronger suppression of LPS-induced pro-inflammatory gene expression compared with non-hydrolyzed casein and pea protein. These results suggest that TMH, CH and PPH prepared from V. halodenitrificans SK1-3-7 proteinase are potential functional food ingredients with immunomodulatory activity.
Assuntos
Caseínas/farmacologia , Proteínas de Peixes/farmacologia , Peptídeo Hidrolases/metabolismo , Hidrolisados de Proteína/farmacologia , Virgibacillus/enzimologia , Animais , Anti-Inflamatórios/farmacologia , Células Cultivadas , Ciclídeos , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Regulação da Expressão Gênica , Humanos , Hidrólise , Imunidade Inata , Imunomodulação , Inflamação , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Lipopolissacarídeos/toxicidade , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Pisum sativum/química , Proteínas de Plantas/farmacologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The angiotensin I-converting enzyme (ACE) inhibitory activity of protein hydrolysates from tilapia muscle fractions, namely mince (M), washed mince (WM), and sarcoplasmic protein (SP), were investigated. Each fraction was hydrolyzed by Virgibacillus halodenitrificans SK1-3-7 proteinases for up to 24h. After 8h of hydrolysis, the M hydrolysate (48% degree of hydrolysis (DH)) showed the highest ACE inhibitory activity, with an IC50 value of 0.54mg/ml, while the SP hydrolysate exhibited the lowest DH and ACE inhibition. In vitro gastrointestinal digestion reduced the ACE inhibitory activity of the M hydrolysate but enhanced its transport across Caco-2 cell monolayers. The transported peptides were found to contain 3-4 amino acid residues showing strong ACE inhibition. The novel ACE inhibitory peptide with the highest inhibition was found to be MCS, with an IC50 value of 0.29µM. Therefore, tilapia mince hydrolyzed by V. halodenitrificans proteinases contained ACE inhibitory peptides that are potentially bioavailable.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Proteínas Musculares/farmacologia , Peptídeo Hidrolases/metabolismo , Peptídeos/farmacologia , Hidrolisados de Proteína/farmacologia , Virgibacillus/enzimologia , Animais , Disponibilidade Biológica , Células CACO-2 , Humanos , TilápiaRESUMO
THP-1 is a human leukemia monocytic cell line, which has been extensively used to study monocyte/macrophage functions, mechanisms, signaling pathways, and nutrient and drug transport. This cell line has become a common model to estimate modulation of monocyte and macrophage activities. This review attempts to summarize and discuss recent publications related to the THP-1 cell model. An overview on the biological similarities and dissimilarities between the THP-1 cell line and human peripheral blood mononuclear cell (PBMC) derived-monocytes and macrophages, as well as the advantages and disadvantages of the use of THP-1 cell line, is included. The review summarizes different published co-cultivation studies of THP-1 cells with other cell types, for instance, intestinal cells, adipocytes, T-lymphocytes, platelets, and vascular smooth muscle cells, which can be an option to study cell-cell interaction in vitro and can be an approach to better mimic in vivo conditions. Macrophage polarization is a relatively new topic which gains interest for which the THP-1 cell line also may be relevant. Besides that an overview of newly released commercial THP-1 engineered-reporter cells and THP-1 inflammasome test-cells is also given. Evaluation of recent papers leads to the conclusion that the THP-1 cell line has unique characteristics as a model to investigate/estimate immune-modulating effects of compounds in both activated and resting conditions of the cells. Although the THP-1 response can hint to potential responses that might occur ex vivo or in vivo, these should be, however, validated by in vivo studies to draw more definite conclusions.
Assuntos
Linhagem Celular Tumoral , Imunomodulação , Leucócitos Mononucleares/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Comunicação Celular , Diferenciação Celular , Humanos , Modelos BiológicosRESUMO
Anthocyanins can contribute to human health through preventing a variety of diseases. The uptake of these compounds from food and the parameters determining uptake efficiency within the human body are still poorly understood. Here we have employed a Caco-2 cell based system to investigate the transport of key antioxidant food components from sour cherry (Prunus cerasus L.) across the intestinal epithelial barrier. Anthocyanins and (-)-epicatechin were supplied in three contrasting matrices: fruit, processed fruit cherry juice, and polyphenolic fractions obtained by solid-phase extraction. Results show that both compound types behave differently. Fruit or juice matrices display comparable transport across the epithelial cell layer. The juice supplements sucrose and citric acid, which are regularly added to processed foods, have a positive effect on stability and transport. Polyphenolic fractions display a lower transport efficiency, relative to that of the fruit or juice, indicating the importance of food matrix components for intestinal absorption of polyphenols.
Assuntos
Antocianinas/farmacocinética , Mucosa Intestinal/metabolismo , Prunus/química , Bebidas , Disponibilidade Biológica , Células CACO-2 , Meios de Cultura/química , Meios de Cultura/farmacologia , Células Epiteliais/efeitos dos fármacos , Manipulação de Alimentos , Frutas/química , Humanos , Intestinos/citologia , Intestinos/efeitos dos fármacos , Extração em Fase SólidaRESUMO
Little is known about the polarizing potential of currently used human macrophage cell lines, while a better understanding phenomena can support the prediction of effects in vivo based on in vitro analysis. To test the polarization capability of PMA differentiated-THP-1 macrophages (M0), cells were stimulated with 20 ng ml(-1) IFNγ + 1 µg ml(-1) LPS and 20 ng ml(-1) IL-4, which are known to influence macrophage polarization in vivo and ex vivo into the M1 and M2 state, respectively. Apart from several well-known M1 and M2 markers, also new possible markers for M1 and M2 polarization were analysed in this study. The expression of M1 marker genes was up-regulated in IFNγ + LPS stimulated-M0 THP-1 macrophages. The IL-4 stimulated-M0 THP-1 macrophages expressed M2 cell membrane receptor genes. However, M2 chemokine and their receptor genes were only slightly up-regulated which might be due to the complexity of the secondary cell-cell interaction of the chemokine system. Lipopolysaccharides from E. coli (LPS) and food compounds [lentinan, vitamin D3 (vD3) and the combination of lentinan + vitamin D3 (Len + vD3)] were investigated for their polarizing ability on M0 THP-1 macrophages towards either the M1 or M2 state. LPS (700 ng ml(-1)) was able to skew M0 THP-1 macrophages towards the M1 direction since all analysed M1 marker genes were strongly expressed. Lentinan, vD3 and Len + vD3 did not induce expression of either M1 or M2 markers, indicating no polarizing ability of these compounds. Based on the expression of M1 and M2 marker genes we concluded that THP-1 macrophages could be successfully polarized into either the M1 or M2 state. Therefore, they can be used as a new macrophage polarizing model to estimate the polarizing/switching ability of test food compounds.
Assuntos
Agaricales/química , Escherichia coli/química , Lentinano/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/imunologia , Agaricales/imunologia , Diferenciação Celular , Citocinas/genética , Citocinas/imunologia , Escherichia coli/imunologia , Humanos , Lentinano/imunologia , Lipopolissacarídeos/imunologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacosRESUMO
In this study, two novel FIPs have been identified and characterized. The first is FIP-nha, identified in the ascomycete Nectria haematococca, and as such, FIP-nha would be the first FIP to be identified outside the order of Basidiomycota. The second is LZ-9, an LZ-8 like protein identified in Ganoderma lucidum. Recombinant FIPs (rFIPs) were produced in Pichia pastoris and purified using His-affinity magnetic beads. The bioactive characteristics of FIP-nha and LZ-9 were compared to the well-known FIPs, LZ-8 from G. lucidum and FIP-fve from Flammulina velutipes, which were produced and purified using the same method. The produced rFIPs: rLZ-8, rLZ-9, rFIP-fve and rFIP-nha were investigated for their hemagglutinating activity which revealed that rLZ-8, rLZ-9 and rFIP-nha were able to agglutinate rabbit, mouse and sheep red blood cells while rFIP-fve only agglutinated rabbit red blood cells. None of the rFIPs were able to agglutinate human red blood cells unless the cells were trypsinized. In addition, all rFIPs were studied and compared to several lectins for their effect on Caco-2 intestinal cell layer integrity using transepithelial electrical resistance (TEER) measurement. rLZ-9 appeared to have the highest effect in lowering TEER, similar to one of the tested lectins. Testing of rFIPs for their activation of inflammation-related genes of THP-1 macrophages showed rFIP-fve to be the strongest inducer of pro-inflammatory cytokine transcription. These results indicate that each rFIP has a unique bioactive profile as well as each lectin, creating the basis for further studies to relate structure to biological activity.
Assuntos
Proteínas Fúngicas/farmacologia , Fatores Imunológicos/farmacologia , Lectinas/farmacologia , Sequência de Bases , Células CACO-2 , Linhagem Celular Tumoral , Flammulina , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Hemaglutinação/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Nectria , Proteínas Recombinantes/farmacologia , Reishi , Análise de Sequência de DNARESUMO
SCOPE: We aimed to examine different immunological aspects of ß-glucans derived from different food sources (oat, barley and shiitake) on phorbol myristate acetate (PMA)-differentiated THP-1 macrophages. Commercially purified barley ß-glucan (commercial BG) and lentinan were included to compare ß-glucans from the same origin but different degree of purity and processing. METHODS AND RESULTS: Chemical composition and molecular weight distribution of ß-glucan samples were determined. Inflammation-related gene expression kinetics (IL-1ß, IL-8, nuclear factor kappa B [NF-κB] and IL-10) after 3, 6 and 24 h of stimulation with 100 µg/mL ß-glucan were investigated. All tested ß-glucans mildly upregulated the observed inflammation-related genes with differential gene expression patterns. Similar gene expression kinetics, but different fold induction values, was found for the crude ß-glucan extracts and their corresponding commercial forms. Pre-incubation of THP-1 macrophages with ß-glucans prior to lipopolysaccharide (LPS) exposure decreased the induction of inflammation-related genes compared to LPS treatment. No production of nitric oxide (NO) and hydrogen peroxide (H2O2) was detected in ß-glucan stimulated THP-1 macrophages. Phagocytic activity was not different after stimulation by ß-glucan samples. CONCLUSION: Based on these in vitro analyses, it can be concluded that the analysed ß-glucans have varying levels of immunomodulating properties, which are likely related to structure, molecular weight and compositional characteristic of ß-glucan.
Assuntos
Fatores Imunológicos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , beta-Glucanas/farmacologia , Avena/química , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatografia em Gel , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosídeo Hidrolases/metabolismo , Hordeum/química , Humanos , Peróxido de Hidrogênio/metabolismo , Inflamação/tratamento farmacológico , Inflamação/genética , Lipopolissacarídeos/farmacologia , Óxido Nítrico/metabolismo , Fagocitose/efeitos dos fármacos , Extratos Vegetais/análise , Extratos Vegetais/química , Cogumelos Shiitake/química , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
BACKGROUND: The small intestinal epithelium functions both to absorb nutrients, and to provide a barrier between the outside, luminal, world and the human body. One of the passageways across the intestinal epithelium is paracellular diffusion, which is controlled by the properties of tight junction complexes. We used a differentiated Caco-2 monolayer as a model for small intestinal epithelium to study the effect of crude apple extracts on paracellular permeability. RESULTS: Exposure of crude apple homogenate to the differentiated Caco-2 cells increased the paracellular resistance, determined as trans-epithelial electrical resistance (TEER). This increase was linearly related to the concentration of apple present. The TEER-enhancing effect of apple extract was due to factors mainly present in the cortex, and the induction was not inhibited by protein kinase inhibitors. Apple-induced resistance was accompanied by increased expression of several tight junction related genes, including claudin 4 (CLDN4). CONCLUSION: Crude apple extract induces a higher paracellular resistance in differentiated Caco-2 cells. Future research will determine whether these results can be extrapolated to human small intestinal epithelia.
Assuntos
Claudinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Malus/química , Extratos Vegetais/farmacologia , Células CACO-2 , Claudina-4 , Claudinas/genética , Relação Dose-Resposta a Droga , Frutas/química , Perfilação da Expressão Gênica , Humanos , Extratos Vegetais/administração & dosagem , Extratos Vegetais/química , Junções Íntimas/fisiologiaRESUMO
Activation tagging is a powerful tool to identify new mutants and to obtain information about possible biological functions of the overexpressed genes. The quadruple cauliflower mosaic virus (CaMV) 35S enhancer fragment is a strong enhancer, which is most commonly used for this purpose. However, the constitutive nature of this enhancer may generate lethal mutations or aberrations in different plant organs by the same overexpressed gene. A tissue-specific activation tagging approach may overcome these drawbacks and may also lead more efficiently to the desired phenotype. For this reason the SHATTERPROOF2 (SHP2) promoter fragment was analysed for enhancer activity. The SHP2 gene is involved in dehiscence zone development and expressed during silique development. The aim of the experiments described here was to identify a dehiscence zone specific enhancer that could be used for tissue-specific activation tagging. The chosen SHP2 enhancer fragment was found to be expressed predominantly in the dehiscence zone and showed enhancer activity as well as ectopic expression activity. This activity was not influenced by its orientation towards the promoter and it was still functional at the largest tested distance of 2.0 kb. Based on these results, the SHP2 enhancer fragment can potentially be used in a tissue-specific activation tagging approach to identify new Arabidopsis mutants with an altered dehiscence zone formation.