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1.
J Dairy Sci ; 102(12): 10772-10778, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31629525

RESUMO

Colostrum plays an important role in initiating the development of the intestinal barrier in newborn mammals. Given its bioactivity, there is much interest in the potential use of bovine colostrum to improve human gastrointestinal health throughout the life span. There is evidence that bovine colostrum is effective at improving small intestinal barrier integrity and some indication that it may alter colonic motility. However, for colostrum to be used as a product to improve intestinal health, it needs to be bioactive after processing. The aim of this study was to determine whether industrial processing of bovine colostrum affects its ability to improve small intestinal barrier integrity or alter distal colon motility. Three colostrum sample types were compared; raw whole colostrum powder (WCP), raw skim colostrum powder (SCP), and industrially produced colostrum milk protein concentrate (CMPC). To determine whether these colostrum powders had different effects on small intestinal barrier integrity, their effects on the transepithelial electrical resistance across an in vitro intestinal epithelial layer (Caco-2 cells) were measured, both with and without a challenge from the proinflammatory cytokine tumor necrosis factor-α. These results showed that CMPC enhanced transepithelial electrical resistance across unchallenged epithelial cell layers, whereas the raw colostrum samples, WCP and SCP, did not have an effect. The colostrum samples were also compared to determine how they affect contractility in the distal colon isolated from the rat. Skim colostrum powder was the only sample to act directly on colonic tissue to modulate motility, increasing the amplitude of contractions. The results show that bovine colostrum is able to improve small intestinal barrier integrity and alter colon motility, and they implicate different components. The barrier integrity enhancement was apparent only in the industrial CMPC, which may have been due to the increase in protein concentration or the release of small peptides as a result of processing. The ability to alter colon motility was present in SCP but absent in WCP, again implying that an increase in protein concentration is responsible for the effect. However, this effect was not apparent for the industrially processed CMPC, suggesting denaturation or degradation of the active component. The beneficial effect of colostrum on small intestinal barrier integrity was present after processing, confirming that it is feasible to industrially produce an active product for gut health.


Assuntos
Colostro , Mucosa Intestinal/efeitos dos fármacos , Proteínas do Leite/farmacologia , Animais , Células CACO-2 , Bovinos , Humanos , Proteínas do Leite/metabolismo , Ratos , Fator de Necrose Tumoral alfa/metabolismo
2.
PLoS One ; 13(1): e0190839, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29304106

RESUMO

Appropriate intestinal barrier maturation is essential for absorbing nutrients and preventing pathogens and toxins from entering the body. Compared to breast-fed infants, formula-fed infants are more susceptible to barrier dysfunction-associated illnesses. In infant formula dairy lipids are usually replaced with plant lipids. We hypothesised that dairy complex lipids improve in vitro intestinal epithelial barrier integrity. We tested milkfat high in conjugated linoleic acid, beta serum (SureStart™Lipid100), beta serum concentrate (BSC) and a ganglioside-rich fraction (G600). Using Caco-2 cells as a model of the human small intestinal epithelium, we analysed the effects of the ingredients on trans-epithelial electrical resistance (TEER), mannitol flux, and tight junction protein co-localisation. BSC induced a dose-dependent improvement in TEER across unchallenged cell layers, maintained the co-localisation of tight junction proteins in TNFα-challenged cells with increased permeability, and mitigated the TEER-reducing effects of lipopolysaccharide (LPS). G600 also increased TEER across healthy and LPS-challenged cells, but it did not alter the co-location of tight junction proteins in TNFα-challenged cells. SureStart™Lipid100 had similar TEER-increasing effects to BSC when added at twice the concentration (similar lipid concentration). Ultimately, this research aims to contribute to the development of infant formulas supplemented with dairy complex lipids that support infant intestinal barrier maturation.


Assuntos
Intestino Delgado/efeitos dos fármacos , Lipídeos/farmacologia , Animais , Transporte Biológico , Células CACO-2 , Bovinos , Indústria de Laticínios , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/fisiologia , Intestino Delgado/fisiologia , Permeabilidade/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo
3.
Cell Microbiol ; 20(2)2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29112296

RESUMO

Inappropriate activation of intestinal innate immune receptors, such as toll-like receptors (TLRs), by pathogenic bacteria is linked to chronic inflammation. In contrast, a "tonic" level of TLR activation by commensal bacteria is required for intestinal homeostasis. A technical challenge when studying this activation in vitro is the co-culturing of oxygen-requiring mammalian cells with obligate anaerobic commensal bacteria. To overcome this, we used a novel apical anaerobic co-culture system to successfully adapt a TLR activation assay to be conducted in conditions optimised for both cell types. Live Faecalibacterium prausnitzii, an abundant obligate anaerobe of the colonic microbiota, induced higher TLR2 and TLR2/6 activation than the dead bacterium. This enhanced TLR induction by live F. prausnitzii, which until now has not previously been described, may contribute to maintenance of gastrointestinal homeostasis. This highlights the importance of using physiologically relevant co-culture systems to decipher the mechanisms of action of live obligate anaerobes.


Assuntos
Faecalibacterium prausnitzii/patogenicidade , Mucosa Intestinal/metabolismo , Intestinos/microbiologia , Receptor 2 Toll-Like/metabolismo , Receptor 6 Toll-Like/metabolismo , Animais , Bactérias Anaeróbias/patogenicidade , Linhagem Celular , Técnicas de Cocultura/métodos , Células HEK293 , Homeostase/fisiologia , Humanos , Mamíferos/metabolismo
4.
Nutrients ; 9(12)2017 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-29231875

RESUMO

Appropriate intestinal barrier maturation during infancy largely depends on colonization with commensal bacteria. Faecalibacterium prausnitzii is an abundant obligate anaerobe that colonizes during weaning and is thought to maintain colonic health throughout life. We previously showed that F. prausnitzii induced Toll-like receptor 2 (TLR2) activation, which is linked to enhanced tight junction formation. Therefore, we hypothesized that F. prausnitzii enhances barrier integrity, an important factor in appropriate intestinal barrier maturation. In order to test metabolically active bacteria, we used a novel apical anaerobic co-culture system that allows the survival of both obligate anaerobic bacteria and oxygen-requiring intestinal epithelial cells (Caco-2). The first aim was to optimize the culture medium to enable growth and active metabolism of F. prausnitzii while maintaining the viability and barrier integrity, as measured by trans-epithelial electrical resistance (TEER), of the Caco-2 cells. This was achieved by supplementing the apical cell culture medium with bacterial culture medium. The second aim was to test the effect of F. prausnitzii on TEER across Caco-2 cell layers. Live F. prausnitzii did not improve TEER, which indicates that its benefits are not via altering tight junction integrity. The optimization of the novel dual-environment co-culturing system performed in this research will enable the investigation of new probiotics originating from indigenous beneficial bacteria.


Assuntos
Bactérias Anaeróbias/imunologia , Faecalibacterium prausnitzii/imunologia , Imunossenescência/fisiologia , Mucosa Intestinal/imunologia , Intestino Grosso/imunologia , Células CACO-2 , Técnicas de Cocultura/métodos , Faecalibacterium prausnitzii/crescimento & desenvolvimento , Humanos , Mucosa Intestinal/microbiologia , Intestino Grosso/microbiologia
5.
J Agric Food Chem ; 65(28): 5632-5638, 2017 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-28627169

RESUMO

This study investigates the influence of tomato and feijoa juices as fermentable carriers of Lactobacillus plantarum (LP DSM20205) on the ability of the bacterium to improve intestinal barrier function using the trans-epithelial electrical resistance (TEER) assay in an apical anaerobic model. The survival of LP DSM20205 in different fruit juices during in vitro digestion, its adhesion capacity, and potential cytotoxic effect on Caco-2 cells were also studied. The results showed that carrier fruit juices have a significant influence on LP DSM20205 growth, survival during in vitro digestion, adhesion capacity, and TEER. All fermented samples were not cytotoxic to Caco-2 cells. The fermented tomato juice showed the largest improvement to intestinal barrier integrity. The digested fermented juices did not increase TEER, although the LP DSM20205 in these samples adhered well. Therefore, LP DSM20205 has the potential to be used as a probiotic in the production of fermented tomato and feijoa juices.


Assuntos
Sucos de Frutas e Vegetais/microbiologia , Intestinos/microbiologia , Lactobacillus plantarum/metabolismo , Probióticos/metabolismo , Células CACO-2 , Feijoa/microbiologia , Fermentação , Humanos , Mucosa Intestinal/metabolismo , Lactobacillus plantarum/crescimento & desenvolvimento , Lycopersicon esculentum/microbiologia
6.
Front Neurosci ; 11: 20, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28184185

RESUMO

Attention is increasingly being focussed on probiotics as potential agents to restore or improve gastrointestinal (GI) transit. Determining mechanism of action would support robust health claims. The probiotic bacterium Bifidobacterium lactis HN019 reduces transit time, but its mechanisms of action and effects on motility patterns are poorly understood. The aim of this study was to investigate changes in GI motility induced by an extract of HN019 on distinct patterns of colonic motility in isolated rat large intestine, compared with a known promotility modulator, prucalopride. The large intestines from male Sprague Dawley rats (3-6 months) were perfused with Kreb's buffer at 37°C in an oxygenated tissue bath. Isometric force transducers recorded changes in circular muscle activity at four independent locations assessing contractile propagation between the proximal colon and the rectum. HN019 extract was perfused through the tissue bath and differences in tension and frequency quantified relative to pre-treatment controls. Prucalopride (1 µM) increased the frequency of propagating contractions (by 75 ± 26%) in the majority of preparations studied (10/12), concurrently decreasing the frequency of non-propagating contractions (by 50 ± 11%). HN019 extract had no effect on contractile activity during exposure (n = 8). However, following wash out, contraction amplitude of propagating contractions increased (by 55 ± 18%) in the distal colon, while the frequency of non-propagating proximal contractions decreased by 57 ± 7%. The prokinetic action of prucalopride increased the frequency of synchronous contractions along the length of colon, likely explaining increased colonic rate of transit in vivo. HN019 extract modified motility patterns in a different manner by promoting propagating contractile amplitude and inhibiting non-propagations, also demonstrating prokinetic activity consistent with the reduction of constipation by B. lactis HN019 in humans.

7.
J Dairy Sci ; 100(2): 886-891, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27939537

RESUMO

Bovine lactoferrin is an important milk protein with many health-promoting properties, including improving intestinal barrier integrity. Dysfunction of this barrier, commonly referred to as "leaky gut," has been linked to inflammatory and autoimmune diseases. With some processing techniques, lactoferrin isolated from milk collected at the start of the milking season (early lactation) may have lower purity than that isolated from milk collected during the rest of the milking season (mid-lactation) and could result in differences in bioactivity based on the stage of lactation. We compared reversed-phase HPLC chromatographs of early-lactation and mid-lactation preparations and found that both had large chromatograph peaks at the time predicted for lactoferrin. The notable difference between the 2 chromatographs was a much larger peak in the early-lactation lactoferrin sample that was determined to be angiogenin. Angiogenin was first identified due to its ability to induce new blood vessel formation, but is now known to be involved in numerous physiological processes. Then, we compared the effects of early-lactation and mid-lactation lactoferrin preparations in 2 bioassays: trans-epithelial electrical resistance (TEER), a measure of intestinal barrier integrity, and peripheral blood mononuclear cell cytokine secretion, a measure of immune-stimulatory properties. We found that early-lactation lactoferrin increased TEER across Caco-2 cell layers compared with control from 10 to 48 h, mid-lactation lactoferrin did not alter TEER. We also found that early-lactation lactoferrin reduced the amount of IL-8 produced by peripheral blood mononuclear cells (compared with those treated with control medium) to a greater extent than mid-lactation lactoferrin. A pro-inflammatory chemokine, IL-8 is also known to decrease barrier function. These results suggest that the decrease in IL-8 production in the presence of early-lactation lactoferrin may be the mechanism by which it increases TEER. The anti-inflammatory effect of early-lactation lactoferrin may be related to the presence of angiogenin, which is known to suppress inflammatory responses. This work indicates that products rich in angiogenin may have intestinal health benefits, and further work to investigate this is warranted.


Assuntos
Células CACO-2 , Lactoferrina , Animais , Bovinos , Feminino , Humanos , Lactação , Leucócitos Mononucleares/efeitos dos fármacos , Leite/química
8.
Dev Comp Immunol ; 68: 21-25, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27845173

RESUMO

Secretory IgA (SIgA) from milk contributes to early colonization and maintenance of commensal/symbiotic bacteria in the gut, as well as providing defence against pathogens. SIgA binds bacteria using specific antigenic sites or non-specifically via its glycans attached to α-heavy-chain and secretory component. In our study, we tested the hypothesis that human and bovine SIgA have similar innate-binding activity for bacteria. SIgAs, isolated from human and bovine milk, were incubated with a selection of commensal, pathogenic and probiotic bacteria. Using flow cytometry, we measured numbers of bacteria binding SIgA and their level of SIgA binding. The percentage of bacteria bound by human and bovine SIgA varied from 30 to 90% depending on bacterial species and strains, but was remarkably consistent between human and bovine SIgA. The level of SIgA binding per bacterial cell was lower for those bacteria that had a higher percentage of SIgA-bound bacteria, and higher for those bacteria that had lower percentage of SIgA-bound bacteria. Overall, human and bovine SIgA interacted with bacteria in a comparable way. This contributes to longer term research about the potential benefits of bovine SIgA for human consumers.


Assuntos
Bactérias/metabolismo , Imunoglobulina A Secretora/metabolismo , Leite/metabolismo , Polissacarídeos/metabolismo , Animais , Bactérias/imunologia , Bovinos , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Imunoglobulina A Secretora/química , Imunoglobulina A Secretora/imunologia , Polissacarídeos/química , Polissacarídeos/imunologia , Ligação Proteica , Especificidade da Espécie , Simbiose
9.
Nutrients ; 8(12)2016 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-27983629

RESUMO

Whey protein concentrate (WPC) and hydrolysate (WPH) are protein ingredients used in sports, medical and pediatric formulations. Concentration and hydrolysis methods vary for whey sourced from cheese and casein co-products. The purpose of this research was to investigate the influence of whey processing methods on in vitro gastrointestinal (GI) health indicators for colonic motility, epithelial barrier integrity and immune modulation. WPCs from casein or cheese processing and WPH (11% or 19% degree of hydrolysis, DH) were compared for their effects on motility in a 1 cm section of isolated rat distal colon in an oxygenated tissue bath. Results showed that WPC decreased motility irrespective of whether it was a by-product of lactic acid or mineral acid casein production, or from cheese production. This indicated that regardless of the preparation methodology, the whey protein contained components that modulate aspects of motility within the distal colon. WPH (11% DH) increased contractile frequency by 27% in a delayed manner and WPH (19% DH) had an immediate effect on contractile properties, increasing tension by 65% and frequency by 131%. Increased motility was associated with increased hydrolysis that may be attributed to the abundance of bioactive peptides. Increased frequency of contractions by WPH (19% DH) was inhibited (by 44%) by naloxone, implicating a potential involvement of opioid receptors in modulation of motility. Trans-epithelial electrical resistance and cytokine expression assays revealed that the WPC proteins studied did not alter intestinal barrier integrity or elicit any discernible immune response.


Assuntos
Colo/efeitos dos fármacos , Motilidade Gastrointestinal/efeitos dos fármacos , Proteínas do Leite/química , Hidrolisados de Proteína/farmacologia , Proteínas do Soro do Leite/farmacologia , Animais , Caseínas , Bovinos , Queijo , Colo/fisiologia , Hidrólise , Ratos , Ratos Sprague-Dawley
10.
Sci Rep ; 6: 20318, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26843130

RESUMO

Lactobacilli are thought to be beneficial for human health, with lactobacilli-associated infections being confined to immune-compromised individuals. However, Lactobacillus fermentum AGR1487 negatively affects barrier integrity in vitro so we hypothesized that it caused a pro-inflammatory response in the host. We compared germ-free rats inoculated with AGR1487 to those inoculated with another L. fermentum strain, AGR1485, which does not affect in vitro barrier integrity. We showed that rats inoculated with AGR1487 had more inflammatory cells in their colon, higher levels of inflammatory biomarkers, and increased colonic gene expression of pro-inflammatory pathways. In addition, our in vitro studies showed that AGR1487 had a greater capacity to activate TLR signaling and induce pro-inflammatory cytokines in immune cells. This study indicates the potential of strains of the same species to differentially elicit inflammatory responses in the host and highlights the importance of strain characterization in probiotic approaches to treat inflammatory disorders.


Assuntos
Lactobacillus fermentum/fisiologia , Boca/microbiologia , Probióticos/administração & dosagem , Animais , Biomarcadores/metabolismo , Colite/etiologia , Colite/metabolismo , Colo/citologia , Colo/microbiologia , Colo/patologia , Citocinas/metabolismo , Expressão Gênica , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Lactobacillus fermentum/isolamento & purificação , Linfócitos/citologia , Linfócitos/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais , Receptores Toll-Like/metabolismo
11.
Microbiologyopen ; 4(4): 541-52, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25943073

RESUMO

Lactobacillus fermentum is commonly found in food products, and some strains are known to have beneficial effects on human health. However, our previous research indicated that L. fermentum AGR1487 decreases in vitro intestinal barrier integrity. The hypothesis was that cell surface structures of AGR1487 are responsible for the observed in vitro effect. AGR1487 was compared to another human oral L. fermentum strain, AGR1485, which does not cause the same effect. The examination of phenotypic traits associated with the composition of cell surface structures showed that compared to AGR1485, AGR1487 had a smaller genome, utilized different sugars, and had greater tolerance to acid and bile. The effect of the two strains on intestinal barrier integrity was determined using two independent measures of paracellular permeability of the intestinal epithelial Caco-2 cell line. The transepithelial electrical resistance (TEER) assay specifically measures ion permeability, whereas the mannitol flux assay measures the passage of uncharged molecules. Both live and UV-inactivated AGR1487 decreased TEER across Caco-2 cells implicating the cell surfaces structures in the effect. However, only live AGR1487, and not UV-inactivated AGR1487, increased the rate of passage of mannitol, implying that a secreted component(s) is responsible for this effect. These differences in barrier integrity results are likely due to the TEER and mannitol flux assays measuring different characteristics of the epithelial barrier, and therefore imply that there are multiple mechanisms involved in the effect of AGR1487 on barrier integrity.


Assuntos
Mucosa Intestinal/fisiologia , Lactobacillus fermentum/fisiologia , Permeabilidade , Células CACO-2 , Humanos
12.
Cell Microbiol ; 17(2): 226-40, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25224879

RESUMO

Faecalibacterium prausnitzii, an abundant member of the human commensal microbiota, has been proposed to have a protective role in the intestine. However, it is an obligate anaerobe, difficult to co-culture in viable form with oxygen-requiring intestinal cells. To overcome this limitation, a unique apical anaerobic model of the intestinal barrier, which enabled co-culture of live obligate anaerobes with the human intestinal cell line Caco-2, was developed. Caco-2 cells remained viable and maintained an intact barrier for at least 12 h, consistent with gene expression data, which suggested Caco-2 cells had adapted to survive in an oxygen-reduced atmosphere. Live F. prausnitzii cells, but not ultraviolet (UV)-killed F. prausnitzii, increased the permeability of mannitol across the epithelial barrier. Gene expression analysis showed inflammatory mediators to be expressed at lower amounts in Caco-2 cells exposed to live F. prausnitzii than UV-killed F. prausnitzii, This, consistent with previous reports, implies that live F. prausnitzii produces an anti-inflammatory compound in the culture supernatant, demonstrating the value of a physiologically relevant co-culture system that allows obligate anaerobic bacteria to remain viable.


Assuntos
Clostridium/crescimento & desenvolvimento , Células Epiteliais/microbiologia , Células Epiteliais/fisiologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/fisiologia , Células CACO-2 , Sobrevivência Celular , Técnicas de Cocultura , Perfilação da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Manitol/metabolismo , Modelos Teóricos , Permeabilidade
13.
Nutrients ; 7(1): 45-73, 2014 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-25545102

RESUMO

The human gastrointestinal tract is colonised by trillions of commensal bacteria, most of which are obligate anaerobes residing in the large intestine. Appropriate bacterial colonisation is generally known to be critical for human health. In particular, the development and function of the immune system depends on microbial colonisation, and a regulated cross-talk between commensal bacteria, intestinal epithelial cells and immune cells is required to maintain mucosal immune homeostasis. This homeostasis is disturbed in various inflammatory disorders, such as inflammatory bowel diseases. Several in vitro and in vivo studies indicate a role for Faecalibacterium prausnitzii, Bacteroides thetaiotaomicron, Bacteroides fragilis, Akkermansia muciniphila and segmented filamentous bacteria in maintaining intestinal immune homeostasis. These obligate anaerobes are abundant in the healthy intestine but reduced in several inflammatory diseases, suggesting an association with protective effects on human health. However, knowledge of the mechanisms underlying the effects of obligate anaerobic intestinal bacteria remains limited, in part due to the difficulty of co-culturing obligate anaerobes together with oxygen-requiring human epithelial cells. By using novel dual-environment co-culture models, it will be possible to investigate the effects of the unstudied majority of intestinal microorganisms on the human epithelia. This knowledge will provide opportunities for improving human health and reducing the risk of inflammatory diseases.


Assuntos
Bactérias Anaeróbias/fisiologia , Intestino Grosso/imunologia , Intestino Grosso/microbiologia , Microbiota , Simbiose , Células Epiteliais/microbiologia , Homeostase , Humanos , Sistema Imunitário/microbiologia , Doenças Inflamatórias Intestinais/microbiologia
14.
PLoS One ; 8(11): e78774, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24244356

RESUMO

Lactobacillus fermentum is found in fermented foods and thought to be harmless. In vivo and clinical studies indicate that some L. fermentum strains have beneficial properties, particularly for gastrointestinal health. However, L. fermentum AGR1487 decreases trans-epithelial electrical resistance (TEER), a measure of intestinal barrier integrity. The hypothesis was that L. fermentum AGR1487 decreases the expression of intestinal cell tight junction genes and proteins, thereby reducing barrier integrity. Transcriptomic and proteomic analyses of Caco-2 cells (model of human intestinal epithelial cells) treated with L. fermentum AGR1487 were used to obtain a global view of the effect of the bacterium on intestinal epithelial cells. Specific functional characteristics by which L. fermentum AGR1487 reduces intestinal barrier integrity were examined using confocal microscopy, cell cycle progression and adherence bioassays. The effects of TEER-enhancing L. fermentum AGR1485 were investigated for comparison. L. fermentum AGR1487 did not alter the expression of Caco-2 cell tight junction genes (compared to L. fermentum AGR1485) and tight junction proteins were not able to be detected. However, L. fermentum AGR1487 increased the expression levels of seven tubulin genes and the abundance of three microtubule-associated proteins, which have been linked to tight junction disassembly. Additionally, Caco-2 cells treated with L. fermentum AGR1487 did not have defined and uniform borders of zona occludens 2 around each cell, unlike control or AGR1485 treated cells. L. fermentum AGR1487 cells were required for the negative effect on barrier integrity (bacterial supernatant did not cause a decrease in TEER), suggesting that a physical interaction may be necessary. Increased adherence of L. fermentum AGR1487 to Caco-2 cells (compared to L. fermentum AGR1485) was likely to facilitate this cell-to-cell interaction. These findings illustrate that bacterial strains of the same species can cause contrasting host responses and suggest that food-safe status should be given to individual strains not species.


Assuntos
Comunicação Celular , Regulação da Expressão Gênica , Mucosa Intestinal , Lactobacillus fermentum , Mucosa Bucal/microbiologia , Junções Íntimas/metabolismo , Adulto , Células CACO-2 , Feminino , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Lactobacillus fermentum/isolamento & purificação , Lactobacillus fermentum/metabolismo , Masculino , Junções Íntimas/microbiologia
15.
PLoS One ; 8(5): e63251, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23700416

RESUMO

In addition to their role in absorption and secretion, epithelial cells play an important role in the protection of the colon mucosa from the resident microbiota and are important for the maintenance of homeostasis. Microarray analysis of intact colon samples is widely used to gain an overview of the cellular pathways and processes that are active in the colon during inflammation. Laser microdissection of colon epithelial cells allows a more targeted analysis of molecular pathways in the mucosa, preceding and during inflammation, with potentially increased sensitivity to changes in specific cell populations. The aim of this study was to investigate the molecular changes that occur in early and late inflammation stages in colon epithelium of a mouse model of inflammatory bowel diseases. Microarray analysis of intact colon samples and microdissected colon epithelial cell samples from interleukin-10 gene deficient and control mice at 6 and 12 weeks of age was undertaken. Results of gene set enrichment analysis showed that more immune-related pathways were identified between interleukin-10 gene deficient and control mice at 6 weeks of age in epithelial cells than intact colon. This suggests that targeting epithelial cells could increase sensitivity for detecting immune changes that occur early in the inflammatory process. However, in the later stages of inflammation, microarray analyses of intact colon and epithelium both provide a similar overview of gene expression changes in the colon mucosa at the pathway level.


Assuntos
Colo/metabolismo , Células Epiteliais/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Interleucina-10/deficiência , Transcriptoma , Animais , Colo/imunologia , Colo/patologia , Células Epiteliais/imunologia , Redes Reguladoras de Genes , Mediadores da Inflamação/metabolismo , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/imunologia , Interleucina-10/genética , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Intestino Delgado/patologia , Microdissecção e Captura a Laser , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos
16.
Mediators Inflamm ; 2013: 237921, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23576850

RESUMO

Lactobacillus species can exert health promoting effects in the gastrointestinal tract (GIT) through many mechanisms, which include pathogen inhibition, maintenance of microbial balance, immunomodulation, and enhancement of the epithelial barrier function. Different species of the genus Lactobacillus can evoke different responses in the host, and not all strains of the same species can be considered beneficial. Strain variations may be related to diversity of the cell surface architecture of lactobacilli and the bacteria's ability to express certain surface components or secrete specific compounds in response to the host environment. Lactobacilli are known to modify their surface structures in response to stress factors such as bile and low pH, and these adaptations may help their survival in the face of harsh environmental conditions encountered in the GIT. In recent years, multiple cell surface-associated molecules have been implicated in the adherence of lactobacilli to the GIT lining, immunomodulation, and protective effects on intestinal epithelial barrier function. Identification of the relevant bacterial ligands and their host receptors is imperative for a better understanding of the mechanisms through which lactobacilli exert their beneficial effects on human health.


Assuntos
Trato Gastrointestinal/microbiologia , Lactobacillus/citologia , Lactobacillus/fisiologia , Aderência Bacteriana/fisiologia , Humanos , Lactobacillus/metabolismo , Probióticos
17.
J Immunol Methods ; 373(1-2): 136-42, 2011 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-21878338

RESUMO

Literature on the effects of cryopreservation and thawing of monocytes or monocyte-derived immature dendritic cells (iDCs) on the subsequent functional capacities of the DCs is limited to a few specific maturation stimuli and is focused on applications in clinical immunotherapy. Given the cardinal role of DCs in regulating tolerance and immunity at mucosal surfaces there is a growing interest in understanding the effect of stromal, microbial and probiotic signals on DC function. Therefore our aim was to investigate the effects of cryopreservation on the functional properties of DCs stimulated with bacteria or the bacterial components using a standardized method. Surface markers CD83 and CD86 were expressed at similar levels on iDCs generated from cryopreserved or freshly isolated monocytes. Cryopreservation of iDCs led to slightly decreased expression of CD86 and CD83 compared to freshly generated iDCs prepared from unfrozen cells but this did not affect the capacity of DCs to acquire fully mature characteristics after stimulation. In contrast the cytokine response to lipoteichoic acid and bacterial stimulation was altered by cryopreservation of monocytes or iDCs, particularly for IL-12p70 which was decreased up to 250 fold or not detected. Cryopreservation also decreased TNF-α and IL-1ß production in stimulated iDCs but to a lesser extent than for IL-12p70, depending on the maturation factors used. The amounts of IL-10 produced by stimulated iDCs were increased up to 3.6 fold when iDCs were cryopreserved, but decreased up to 90 fold when generated from cryopreserved monocytes. Immature DCs are often used to investigate the immunomodulatory properties of probiotics and here we show for the first time that cryopreserved monocytes and cryopreserved iDCs have a skewed cytokine response to microbial stimulation. These findings have implications for the methods used in bacterial-DC immune assays and highlight the importance of comparing different cytokines and stimuli in immune cell cryopreservation protocols.


Assuntos
Criopreservação/métodos , Citocinas/imunologia , Células Dendríticas/imunologia , Monócitos/imunologia , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígeno B7-2/imunologia , Antígeno B7-2/metabolismo , Diferenciação Celular/imunologia , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/microbiologia , Citometria de Fluxo , Interações Hospedeiro-Patógeno , Humanos , Imunoensaio/métodos , Imunoglobulinas/imunologia , Imunoglobulinas/metabolismo , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-12/imunologia , Interleucina-12/metabolismo , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Lactobacillus/fisiologia , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/microbiologia , Ácidos Teicoicos/farmacologia , Fatores de Tempo , Receptores Toll-Like/agonistas , Receptores Toll-Like/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo
18.
J Nutr ; 141(5): 769-76, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21430248

RESUMO

The human intestinal epithelium is formed by a single layer of epithelial cells that separates the intestinal lumen from the underlying lamina propria. The space between these cells is sealed by tight junctions (TJ), which regulate the permeability of the intestinal barrier. TJ are complex protein structures comprised of transmembrane proteins, which interact with the actin cytoskeleton via plaque proteins. Signaling pathways involved in the assembly, disassembly, and maintenance of TJ are controlled by a number of signaling molecules, such as protein kinase C, mitogen-activated protein kinases, myosin light chain kinase, and Rho GTPases. The intestinal barrier is a complex environment exposed to many dietary components and many commensal bacteria. Studies have shown that the intestinal bacteria target various intracellular pathways, change the expression and distribution of TJ proteins, and thereby regulate intestinal barrier function. The presence of some commensal and probiotic strains leads to an increase in TJ proteins at the cell boundaries and in some cases prevents or reverses the adverse effects of pathogens. Various dietary components are also known to regulate epithelial permeability by modifying expression and localization of TJ proteins.


Assuntos
Dieta , Bactérias Gram-Negativas/fisiologia , Bactérias Gram-Positivas/fisiologia , Mucosa Intestinal/fisiologia , Junções Íntimas/fisiologia , Animais , Permeabilidade da Membrana Celular , Interações Hospedeiro-Patógeno , Humanos , Mucosa Intestinal/microbiologia , Probióticos , Transdução de Sinais
19.
BMC Microbiol ; 10: 316, 2010 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-21143932

RESUMO

BACKGROUND: Intestinal barrier function is important for preserving health, as a compromised barrier allows antigen entry and can induce inflammatory diseases. Probiotic bacteria can play a role in enhancing intestinal barrier function; however, the mechanisms are not fully understood. Existing studies have focused on the ability of probiotics to prevent alterations to tight junctions in disease models, and have been restricted to a few tight junction bridging proteins. No studies have previously investigated the effect of probiotic bacteria on healthy intestinal epithelial cell genes involved in the whole tight junction signalling pathway, including those encoding for bridging, plaque and dual location tight junction proteins. Alteration of tight junction signalling in healthy humans is a potential mechanism that could lead to the strengthening of the intestinal barrier, resulting in limiting the ability of antigens to enter the body and potentially triggering undesirable immune responses. RESULTS: The effect of Lactobacillus plantarum MB452 on tight junction integrity was determined by measuring trans-epithelial electrical resistance (TEER) across Caco-2 cell layers. L. plantarum MB452 caused a dose-dependent TEER increase across Caco-2 cell monolayers compared to control medium. Gene expression was compared in Caco-2 cells untreated or treated with L. plantarum MB452 for 10 hours. Caco-2 cell RNA was hybridised to human oligonucleotide arrays. Data was analysed using linear models and differently expressed genes were examined using pathway analysis tools. Nineteen tight junction-related genes had altered expression levels in response to L. plantarum MB452 (modified-P < 0.05, fold-change > 1.2), including those encoding occludin and its associated plaque proteins that anchor it to the cytoskeleton. L. plantarum MB452 also caused changes in tubulin and proteasome gene expression levels which may be linked to intestinal barrier function. Caco-2 tight junctions were visualised by fluorescent microscopy of immuno-stained occludin, zona occludens (ZO)-1, ZO-2 and cingulin. Caco-2 cells treated with L. plantarum MB452 had higher intensity fluorescence of each of the four tight junction proteins compared to untreated controls. CONCLUSIONS: This research indicates that enhancing the expression of genes involved in tight junction signalling is a possible mechanism by which L. plantarum MB452 improves intestinal barrier function.


Assuntos
Expressão Gênica , Intestinos/fisiologia , Lactobacillus plantarum/fisiologia , Junções Íntimas/metabolismo , Células CACO-2 , Impedância Elétrica , Células Epiteliais/química , Células Epiteliais/microbiologia , Células Epiteliais/fisiologia , Humanos , Intestinos/química , Intestinos/citologia , Intestinos/microbiologia , Transdução de Sinais , Junções Íntimas/química , Junções Íntimas/microbiologia
20.
FEMS Microbiol Lett ; 309(2): 184-92, 2010 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-20618863

RESUMO

The aim of this research was to identify bacterial isolates having the potential to improve intestinal barrier function. Lactobacillus plantarum strains and human oral isolates were screened for their ability to enhance tight junction integrity as measured by the transepithelial electrical resistance (TEER) assay. Eight commercially used probiotics were compared to determine which had the greatest positive effect on TEER, and the best-performing probiotic strain, Lactobacillus rhamnosus HN001, was used as a benchmark to evaluate the isolates. One isolate, L. plantarum DSM 2648, was selected for further study because it increased TEER 135% more than L. rhamnosus HN001. The ability of L. plantarum DSM 2648 to tolerate gastrointestinal conditions and adhere to intestinal cells was determined, and L. plantarum DSM 2648 performed better than L. rhamnosus HN001 in all the assays. Lactobacillus plantarum DSM 2648 was able to reduce the negative effect of Escherichia coli [enteropathogenic E. coli (EPEC)] O127:H6 (E2348/69) on TEER and adherence by as much as 98.75% and 80.18%, respectively, during simultaneous or prior coculture compared with EPEC incubation alone. As yet, the precise mechanism associated with the positive effects exerted by L. plantarum DSM 2648 are unknown, and may influence its use to improve human health and wellness.


Assuntos
Aderência Bacteriana , Intestinos/microbiologia , Lactobacillus plantarum/fisiologia , Probióticos , Células CACO-2 , Impedância Elétrica , Humanos , Intestinos/química , Lactobacillus plantarum/química , Lactobacillus plantarum/genética , Lactobacillus plantarum/isolamento & purificação , Modelos Biológicos , Boca/microbiologia , Probióticos/química , Probióticos/isolamento & purificação
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