Host intestinal epithelial cells (IEC) present at the gastrointestinal interface are exposed to pathogenic and non-pathogenic bacteria and their products. Certain probiotic lactic acid bacteria (LAB) have been associated with a range of host-immune modulatory activities including down-regulation of pro-inflammatory gene expression and cytokine production by IEC, with growing evidence suggesting that these bacteria secrete bioactive molecules with immunomodulatory activity. The aim of this study was to determine whether two lactobacilli with immunomodulatory activity [Lactobacillus rhamnosus R0011 (Lr) and Lactobacillus helveticus R0389 (Lh)], produce soluble mediators able to influence IEC responses to Pattern Recognition Receptor (PRR) ligands and pro-inflammatory cytokines [Tumor Necrosis Factor α (TNFα), Interleukin-1β (IL-1β)], signals inducing IEC chemokine production during infection. To this end, the effects of cell-free supernatants (CFS) from Lr and Lh on IEC production of the pro-inflammatory chemokines interleukin (IL)-8 and cytokine-induced neutrophil chemoattractant 1 (CINC-1) induced by a range of host- or pathogen-derived pro-inflammatory stimuli were determined, and the impact on human HT-29 IEC and a primary IEC line (rat IEC-6) was compared. The Lr-CFS and Lh-CFS did not significantly modulate basal IL-8 production from HT-29 IECs or CINC-1 production from IEC-6 cells. However, both Lr-CFS and Lh-CFS significantly down-regulated IL-8 production from HT-29 IECs challenged with varied PRR ligands. Lr-CFS and Lh-CFS had differential effects on PRR-induced CINC-1 production by rat IEC-6 IECs, with no significant down-regulation of CINC-1 observed from IEC-6 IECs cultured with Lh-CFS. Further analysis of the Lr-CFS revealed down-regulation of IL-8 production induced by the pro-inflammatory cytokines IL-1β and TNFα Preliminary characterization of the bioactive constituent(s) of the Lr-CFS indicates that it is resistant to treatment with DNase, RNase, and an acidic protease, but is sensitive to alterations in pH. Taken together, these results indicate that these lactobacilli secrete bioactive molecules of low molecular weight that may modulate host innate immune activity through interactions with IEC.
Lactic acid bacteria (LAB) are used as starter cultures in the production of fermented dairy products and have the potential to confer bioactivity relevant to cardiovascular health, as they possess extensive proteolytic systems that liberate small bioactive peptides from larger milk proteins. Certain casein-derived peptides released by various LAB strains during fermentation have been shown to reduce hypertension and to modulate the immune system. We investigated the growth and peptide production of 2 LAB strains, Lactobacillus helveticus R0389 and Lactocaseibacillus rhamnosus R0011, their immunomodulatory activities, as well as their abilities to inhibit the angiotensin-converting enzyme (ACE). Peptide fractions collected from the cell-free supernatant of both medium-grown and milk fermentation cultures were assessed for ACE-inhibitory activity and their effects on the production of proinflammatory and regulatory cytokines by human THP-1 monocytes. Cultures were grown in medium, with or without supplementation with 0.1% casein, or in 3.25% milk fermented with each LAB strain. Casein supplementation increased the growth rate of both LAB strains, and significantly increased ACE-inhibitory activity of peptide fractions collected from both L. helveticus R0389 and L. rhamnosus R0011 cultures grown for 12 h. Fermentation peptide fractions of L. rhamnosus R0011 showed comparable ACE-inhibitory activity to known ACE inhibiting peptides Val-Pro-Pro and Ile-Pro-Pro (up to 79% inhibition) with a significant difference between culture peptide fractions and acidified and nonacidified control fractions collected after 6 d of fermentation. Many milk and casein-derived peptides reported in previous studies have been identified as part of a larger bioactive fraction. We synthesized a group of these peptides to individually assess both ACE-inhibi-tory and immunomodulatory activity. The known ACE inhibitors Val-Pro-Pro and Ile-Pro-Pro showed similar ACE inhibition to previously published results, while also inducing the production of the regulatory cytokine IL-10 by monocytes in the presence and absence of a proinflammatory stimulant. These synthesized peptides could also induce the production of nitric oxide (NO), a potent vasodilator, in human endothelial cell cultures. Investigating the relationships among these bioactive properties could improve the use of probiotic organisms and their secreted products in the food industry.
Fermented dairy products have become attractive functional foods for the delivery of probiotics and their biologically active metabolites. The aim of this study was to examine the immunomodulatory activity of milk fermented with the probiotic lactic acid bacterium Lactobacillus rhamnosus R0011 (LrF) on macrophages challenged with lipopolysaccharide (LPS), a potent pro-inflammatory stimulus. To this end, human THP-1 or U937 monocytes were differentiated into resting macrophages then stimulated with LPS and co-incubated with the LrF or with milk controls. Levels of pro-inflammatory and immunoregulatory cytokines were determined by enzyme-linked immunosorbent assays. Culturing of LPS-stimulated U937 macrophages with either the whole or filtered LrF resulted in an increase in Interleukin (IL)-1Ra production relative to the negative control. THP-1 macrophages cultured with the LrF demonstrated an increase in LPS-induced IL-10 and IL-1β production, while production of LPS-induced IL-6, sCD54, IL-8, IL-1β, TNF-α, IL-12p70 and Transforming Growth Factor-β (TGF-β) was unaffected. Further, the LrF induced the expression of DC-SIGN and CD206 , markers of immunoregulatory M2 macrophage polarization, in LPS-challenged THP-1 macrophages. Taken together, milk fermented with L. rhamnosus R0011 increased regulatory cytokine production from LPS-challenged U937 and THP-1 macrophages, while simultaneously up-regulating the production of IL-1β and expression of DC-SIGN and CD206 , a profile characteristic of polarization into the immunoregulatory M2 macrophage phenotype.
The Medical Laboratory Sciences profession in Canada is undergoing a transformation in education whereby students can now earn an undergraduate degree alongside a professional technologist's certification. To accomplish both goals, the undergraduate program must focus simultaneously on the performance of clinical diagnostic testing in the laboratory and the underlying basic science principles and mechanisms. This paper is a reflective analysis of the strategies and approaches to enhance student exposure to foundational science in the context of the competency profile for the profession. We also share our guiding principles in course design and delivery, as well as our lessons learned. In conclusion, each course delivered within the program must take a coordinated approach to underscore integration of basic science into clinical concepts to most effectively enhance clinical knowledge and skills.
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