Lactiplantibacillus plantarum ST-III and Lacticaseibacillus rhamnosus KF7 Enhance the Intestinal Epithelial Barrier in a Dual-Environment In Vitro Co-Culture Model.

Intestinal barrier hyperpermeability, which is characterised by impaired tight junction proteins, is associated with a variety of gastrointestinal and systemic diseases. Therefore, maintaining intestinal barrier integrity is considered one of the effective strategies to reduce the risk of such disorders. This study aims to investigate the potential benefits of two probiotic strains (Lactiplantibacillus plantarum ST-III and Lacticaseibacillus rhamnosus KF7) on intestinal barrier function by using a physiologically relevant in vitro model of the intestinal epithelium. Our results demonstrate that both strains increased transepithelial electrical resistance, a measure of intestinal barrier integrity. Immunolocalisation studies indicated that this improvement in barrier function was not due to changes in the co-localisation of the tight junction (TJ) proteins ZO-1 and occludin. However, we observed several modifications in TJ-related genes in response to the probiotics, including the upregulation of transmembrane and cytosolic TJ proteins, as well as TJ signalling proteins. Gene expression modulation was strain- and time-dependent, with a greater number of differentially expressed genes and higher fold-change being observed in the L. plantarum ST-III group and at the latter timepoint. Further studies to investigate how the observed gene expression changes can lead to enhanced barrier function will aid in the development of probiotic foods to help improve intestinal barrier function.

Effects of bovine whey protein on exercise-induced gut permeability in healthy adults: a randomised controlled trial.

PURPOSE: Intestinal permeability is a critical component of gut barrier function. Barrier dysfunction can be triggered by certain stressors such as exercise, and if left unmanaged can lead to local and systemic disorders. The aim of this study was to investigate the effects of a specific whey protein fraction in alleviating exercise-induced gut permeability as assessed by recovery of lactulose/rhamnose (L/R) and lactulose/mannitol (L/M) urinary probes. METHODS: Eight males and eight females (aged 18-50) completed two arms of a double-blind, placebo-controlled, crossover study. For each arm participants performed two baseline intestinal permeability assessments, following which they consumed the treatment (2 g/day of milk powder containing 200 mg of whey protein) or placebo (2 g/day of milk powder) for 14 days, before performing a post-exercise permeability assessment. The exercise protocol involved a 20-min run at 80% of maximal oxygen uptake on a 1% incline. RESULTS: Mixed model analysis revealed an increase in L/R (23%; P < 0.001) and L/M (20%; P < 0.01) recovery following exercise. However, there was no treatment or treatment × exercise effect. CONCLUSION: The exercise protocol utilised in our study induces gut permeability. However, consuming whey protein, at the dose and timing prescribed, is not able to mitigate this effect.

Biotransformation of Rutin in In Vitro Porcine Ileal and Colonic Fermentation Models.

Quercetin, a polyphenol antioxidant, is widely distributed in food in the form of glycoside rutin, which is not readily absorbed in the gastrointestinal tract. The microbiota of the colon is known to biotransform rutin, generating quercetin aglycones that can be absorbed. We investigated the role of the ileal and colonic microbiota in rutin biotransformation using established in vitro fermentation models. Overall, a higher rate of rutin biotransformation was observed during colonic fermentation compared with ileal fermentation. The colonic microbiome showed higher potential for rutin conversion to quercetin through an increased abundance of α-rhamnosidase- and ß-glucosidase-encoding genes compared to the ileal microbiome. Nonetheless, rutin metabolism occurred rapidly during ileal fermentation (∼20% rutin disappearance after 1 h). The appearance of quercetin varied depending on the ileal inoculum and correlated with an increased abundance of Firmicutes, suggesting that quercetin absorption could be improved via modulation of the ileal microbiota.

A bioactive bovine whey protein extract improves intestinal barrier function in vitro.

The human intestine plays an important role as a barrier against the ingress of pathogens and other harmful antigens. Accordingly, proper regulation of the intestinal barrier is essential for optimal health. Intestinal barrier function is regulated in part by the interactions between dietary compounds and the intestinal immune system. Bioactive whey proteins from bovine milk (such as lactoferrin, lactoperoxidase, and immunoglobulins) are known to exert a range of physiological functions, including modulation of the immune system, and thus have the potential to regulate intestinal barrier function. While the effects of individual whey proteins on intestinal barrier function have been studied to some extent, less is known about the potentially synergistic properties of whey protein mixtures. Here we investigated the effects of a bioactive bovine whey protein (BWP) extract containing all whey proteins with an isoelectric point >6.8 on intestinal barrier function in vitro. Intestinal epithelial cell (Caco-2) monolayers were treated with BWP before measuring the barrier integrity over 48 h by means of trans-epithelial electrical resistance (TEER). Treatment of epithelial monolayers with 1 mg/mL BWP resulted in an increase in TEER compared with untreated epithelial monolayers. To determine whether BWP could mitigate immune-mediated intestinal barrier dysfunction, we challenged differentiated Caco-2 cell monolayers with tumor necrosis factor α (TNFα) to obtain an in vitro model of a "leaky" intestinal epithelium. The TNFα challenge led to a decrease in TEER over time across untreated control monolayers, indicating a loss of barrier function. This loss of barrier function was mitigated in monolayers treated with 1 mg/mL BWP, but not monolayers treated with the equivalent amount of lactoferrin present in 1 mg/mL BWP. These data suggest that naturally co-occurring bioactive proteins together may enhance intestinal barrier integrity and protect against inflammation-induced barrier dysfunction to a greater extent than lactoferrin alone. Further work is required to determine the key proteins and protein combinations within BWP, and the mechanisms through which BWP modulates intestinal barrier function.

In Vitro Antimicrobial Efficacy of Tobramycin Against Staphylococcus aureus Biofilms in Combination With or Without DNase I and/or Dispersin B: A Preliminary Investigation.

Staphylococcus aureus in biofilms is highly resistant to the treatment with antibiotics, to which the planktonic cells are susceptible. This is likely to be due to the biofilm creating a protective barrier that prevents antibiotics from accessing the live pathogens buried in the biofilm. S. aureus biofilms consist of an extracellular matrix comprising, but not limited to, extracellular bacterial DNA (eDNA) and poly-ß-1, 6-N-acetyl-d-glucosamine (PNAG). Our study revealed that despite inferiority of dispersin B (an enzyme that degrades PNAG) to DNase I that cleaves eDNA, in dispersing the biofilm of S. aureus, both enzymes were equally efficient in enhancing the antibacterial efficiency of tobramycin, a relatively narrow-spectrum antibiotic against infections caused by gram-positive and gram-negative pathogens, including S. aureus, used in this investigation. However, a combination of these two biofilm-degrading enzymes was found to be significantly less effective in enhancing the antimicrobial efficacy of tobramycin than the individual application of the enzymes. These findings indicate that combinations of different biofilm-degrading enzymes may compromise the antimicrobial efficacy of antibiotics and need to be carefully assessed in vitro before being used for treating medical devices or in pharmaceutical formulations for use in the treatment of chronic ear or respiratory infections.

Dissecting the regulation of bile-induced biofilm formation in Staphylococcus aureus.

Aspiration of bile into the cystic fibrosis (CF) lung has emerged as a prognostic factor for reduced microbial lung biodiversity and the establishment of often fatal, chronic pathogen infections. Staphylococcus aureus is one of the earliest pathogens detected in the lungs of children with CF, and once established as a chronic infection, strategies for its eradication become limited. Several lung pathogens are stimulated to produce biofilms in vitro in the presence of bile. In this study, we further investigated the effects of bile on S. aureus biofilm formation. Most clinical S. aureus strains and the laboratory strain RN4220 were stimulated to form biofilms with sub-inhibitory concentrations of bovine bile. Additionally, we observed bile-induced sensitivity to aminoglycosides, which we exploited in a bursa aurealis transposon screen to isolate mutants reduced in aminoglycoside sensitivity and augmented in bile-induced biofilm formation. We identified five mutants that exhibited hypersensitivity to bile with respect to bile-induced biofilm formation, three of which carried transposon insertions within gene clusters involved in wall teichoic acid (WTA) biosynthesis or transport. Strain TM4 carried an insertion between the divergently oriented tagH and tagG genes, which encode the putative WTA membrane translocation apparatus. Ectopic expression of tagG in TM4 restored a wild-type bile-induced biofilm response, suggesting that reduced translocation of WTA in TM4 induced sensitivity to bile and enhanced the bile-induced biofilm formation response. We propose that WTA may be important for protecting S. aureus against exposure to bile and that bile-induced biofilm formation may be an evolved response to protect cells from bile-induced cell lysis.

Human oral isolate Lactobacillus fermentum AGR1487 induces a pro-inflammatory response in germ-free rat colons.

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.

Evaluation of protease resistance and toxicity of amyloid-like food fibrils from whey, soy, kidney bean, and egg white.

The structural properties of amyloid fibrils combined with their highly functional surface chemistry make them an attractive new food ingredient, for example as highly effective gelling agents. However, the toxic role of amyloid fibrils in disease may cause some concern about their food safety because it has not been established unequivocally if consumption of food fibrils poses a health risk to consumers. Here we present a study of amyloid-like fibrils from whey, kidney bean, soy bean, and egg white to partially address this concern. Fibrils showed varied resistance to proteolytic digestion in vitro by either Proteinase K, pepsin or pancreatin. The toxicity of mature fibrils was measured in vitro and compared to native protein, early-stage-fibrillar protein, and sonicated fibrils in two immortalised human cancer cell lines, Caco-2 and Hec-1a. There was no reduction in the viability of either Caco-2 or Hec-1a cells after treatment with a fibril concentration of up to 0.25 mg/mL.

Live Faecalibacterium prausnitzii in an apical anaerobic model of the intestinal epithelial barrier.

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.

Cryopreservation of monocytes or differentiated immature DCs leads to an altered cytokine response to TLR agonists and microbial stimulation.

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.

Regulation of tight junction permeability by intestinal bacteria and dietary components.

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.