It has been over seventeen years since the scientific definition of probiotics was crafted, along with guidelines ensuring the appropriate use of the term. This definition is now used globally, yet ...on a consistent basis scientists, media and industry misrepresent probiotics or make generalized statements that illustrate a misunderstanding of their utility and limitations. The rate of discovery of novel organisms with potentially therapeutic benefit for both human and environmental health is progressing at an unprecedented rate. However, the term "probiotic" is often misapplied to describe any microbe with plausible therapeutic utility in the human host. It is argued that strict compliance to the scientific definition of the term "probiotic" and avoidance of generalizations to the whole field of probiotics based upon studies of one product, will help advance the development and validation of microbial therapies, and applications to improve human health.
The number of live bacterial cells is the most used parameter to assess the quality of finished probiotic products. Plate counting (PC) is the standard method in industry to enumerate cells. ...Application of PC implies critical aspects related to the selection of optimal nutrient media and growth conditions and underestimation of viable but not cultivable (VBNC) cells. Flow-cytometry (FC) is a culture-independent methodology having the potential to selectively enumerate live, damaged, and dead cells representing a powerful tool for in-depth monitoring of probiotic products. We monitored the shelf life of a clinical batch of a synbiotic composition PDS-08 targeting the pediatric population by means of PC and FC according to International Conference on Harmonization (ICH) pharma guidelines testing the Arrhenius model as predictive tool; PC enumeration revealed higher destruction rate than FC suggesting a faster reduction in cultivability than membrane integrity and thus a possible shift of the bacteria into a VBNC status. PDS-08 maintained acidification capability over time, when re-suspended in nutrient medium, even in samples tested sub-optimally for CFU detection (below 1 billion cells/dose). Due to similar kinetics described by the study of metabolic activity and membrane integrity, FC might be suggested as a valid tool for the study of functional stability of a probiotic product.
The human microbiome is a rich factory for metabolite production and emerging data has led to the concept that orally administered microbial strains can synthesize metabolites with neuroactive ...potential. Recent research from ex vivo and murine models suggests translational potential for microbes to regulate anxiety and depression through the gut-brain axis. However, so far, less emphasis has been placed on the selection of specific microbial strains known to produce the required key metabolites and the formulation in which microbial compositions are delivered to the gut. Here, we describe a double-capsule technology to deliver high numbers of metabolically active cells derived from the 24-strain probiotic product SH-DS01 to the gastrointestinal tract, including the small intestine, where immune responses and adsorption of metabolites into the bloodstream occur. Based on its genome sequence, Limosilactobacillus reuteri SD-LRE2-IT was predicted to have the genetic capacity to de novo produce a specific metabolite of interest to brain health, vitamin B12, which could be confirmed in vitro. Taken together, our data conceptualizes the importance of rationally defined microbial strain characterization based on genomics and metabolomics data, combined with carefully designed capsule technology for delivery of live cells and concomitant functionality in and beyond the gut ecosystem.
The increased prevalence of many chronic inflammatory diseases linked to gut epithelial barrier leakiness has prompted us to investigate the role of extensive use of dishwasher detergents, among ...other factors.
We sought to investigate the effects of professional and household dishwashers, and rinse agents, on cytotoxicity, barrier function, transcriptome, and protein expression in gastrointestinal epithelial cells.
Enterocytic liquid-liquid interfaces were established on permeable supports, and direct cellular cytotoxicity, transepithelial electrical resistance, paracellular flux, immunofluorescence staining, RNA-sequencing transcriptome, and targeted proteomics were performed.
The observed detergent toxicity was attributed to exposure to rinse aid in a dose-dependent manner up to 1:20,000 v/v dilution. A disrupted epithelial barrier, particularly by rinse aid, was observed in liquid-liquid interface cultures, organoids, and gut-on-a-chip, demonstrating decreased transepithelial electrical resistance, increased paracellular flux, and irregular and heterogeneous tight junction immunostaining. When individual components of the rinse aid were investigated separately, alcohol ethoxylates elicited a strong toxic and barrier-damaging effect. RNA-sequencing transcriptome and proteomics data revealed upregulation in cell death, signaling and communication, development, metabolism, proliferation, and immune and inflammatory responses of epithelial cells. Interestingly, detergent residue from professional dishwashers demonstrated the remnant of a significant amount of cytotoxic and epithelial barrier–damaging rinse aid remaining on washed and ready-to-use dishware.
The expression of genes involved in cell survival, epithelial barrier, cytokine signaling, and metabolism was altered by rinse aid in concentrations used in professional dishwashers. The alcohol ethoxylates present in the rinse aid were identified as the culprit component causing the epithelial inflammation and barrier damage.
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The epithelial barrier theory links the recent rise in chronic non-communicable diseases, notably autoimmune and allergic disorders, to environmental agents disrupting the epithelial barrier. Global ...pollution and environmental toxic agent exposure have worsened over six decades because of uncontrolled growth, modernization, and industrialization, affecting human health. Introducing new chemicals without any reasonable control of their health effects through these years has led to documented adverse effects, especially on the skin and mucosal epithelial barriers. These substances, such as particulate matter, detergents, surfactants, food emulsifiers, micro- and nano-plastics, diesel exhaust, cigarette smoke, and ozone, have been shown to compromise the epithelial barrier integrity. This disruption is linked to the opening of the tight-junction barriers, inflammation, cell death, oxidative stress, and metabolic regulation. Consideration must be given to the interplay of toxic substances, underlying inflammatory diseases, and medications, especially in affected tissues. This review article discusses the detrimental effect of environmental barrier-damaging compounds on human health and involves cellular and molecular mechanisms.
Background
The rising prevalence of many chronic diseases related to gut barrier dysfunction coincides with the increased global usage of dietary emulsifiers in recent decades. We therefore ...investigated the effect of the frequently used food emulsifiers on cytotoxicity, barrier function, transcriptome alterations, and protein expression in gastrointestinal epithelial cells.
Methods
Human intestinal organoids originating from induced pluripotent stem cells, colon organoid organ‐on‐a‐chip, and liquid–liquid interface cells were cultured in the presence of two common emulsifiers: polysorbate 20 (P20) and polysorbate 80 (P80). The cytotoxicity, transepithelial electrical resistance (TEER), and paracellular‐flux were measured. Immunofluorescence staining of epithelial tight‐junctions (TJ), RNA‐seq transcriptome, and targeted proteomics were performed.
Results
Cells showed lysis in response to P20 and P80 exposure starting at a 0.1% (v/v) concentration across all models. Epithelial barrier disruption correlated with decreased TEER, increased paracellular‐flux and irregular TJ immunostaining. RNA‐seq and targeted proteomics analyses demonstrated upregulation of cell development, signaling, proliferation, apoptosis, inflammatory response, and response to stress at 0.05%, a concentration lower than direct cell toxicity. A proinflammatory response was characterized by the secretion of several cytokines and chemokines, interaction with their receptors, and PI3K‐Akt and MAPK signaling pathways. CXCL5, CXCL10, and VEGFA were upregulated in response to P20 and CXCL1, CXCL8 (IL‐8), CXCL10, LIF in response to P80.
Conclusions
The present study provides direct evidence on the detrimental effects of food emulsifiers P20 and P80 on intestinal epithelial integrity. The underlying mechanism of epithelial barrier disruption was cell death at concentrations between 1% and 0.1%. Even at concentrations lower than 0.1%, these polysorbates induced a proinflammatory response suggesting a detrimental effect on gastrointestinal health.
This study provides new insights into the underlying mechanisms of intestinal epithelial barrier defects in response to commonly used food emulsifiers P20 and P80. We demonstrated that P20 and P80 directly impair barrier integrity of gut epithelial cells and cause molecular toxicity and proinflammation at doses 20 times lower than those currently authorized for use. At RNA transcription and protein levels, development, cell signaling, proliferation, apoptosis, inflammation and response to stress were altered.Abbreviations: CCL, C‐C motif ligand; CXCL, C‐X‐C motif ligand; IL, interleukin; IL22RA1, interleukin 22 receptor subunit alpha 1; iPSC, induced pluripotent stem cell; LAP, latency‐associated peptide; P20/P80, polysorbate 20/polysorbate 80; TEER, transepithelial electrical resistance; TGF‐β, transforming growth factor beta; TNF, tumor necrosis factor; TJs, tight junctions
Background
Epithelial barrier impairment is associated with many skin and mucosal inflammatory disorders. Laundry detergents have been demonstrated to affect epithelial barrier function in vitro ...using air–liquid interface cultures of human epithelial cells.
Methods
Back skin of C57BL/6 mice was treated with two household laundry detergents at several dilutions. Barrier function was assessed by electric impedance spectroscopy (EIS) and transepidermal water loss (TEWL) measurements after the 4 h of treatments with detergents. RNA sequencing (RNA‐seq) and targeted multiplex proteomics analyses in skin biopsy samples were performed. The 6‐h treatment effect of laundry detergent and sodium dodecyl sulfate (SDS) was investigated on ex vivo human skin.
Results
Detergent‐treated skin showed a significant EIS reduction and TEWL increase compared to untreated skin, with a relatively higher sensitivity and dose–response in EIS. The RNA‐seq showed the reduction of the expression of several genes essential for skin barrier integrity, such as tight junctions and adherens junction proteins. In contrast, keratinization, lipid metabolic processes, and epidermal cell differentiation were upregulated. Proteomics analysis showed that the detergents treatment generally downregulated cell adhesion‐related proteins, such as epithelial cell adhesion molecule and contactin‐1, and upregulated proinflammatory proteins, such as interleukin 6 and interleukin 1 beta. Both detergent and SDS led to a significant decrease in EIS values in the ex vivo human skin model.
Conclusion
The present study demonstrated that laundry detergents and its main component, SDS impaired the epidermal barrier in vivo and ex vivo human skin. Daily detergent exposure may cause skin barrier disruption and may contribute to the development of atopic diseases.
Household laundry detergents led to reduced electrical impedance, indicating impaired skin barrier. RNA‐seq and targeted multiplex protein analyses in skin biopsy demonstrated that laundry detergents disrupted several major pathways involved in the skin barrier and induced inflammation. Laundry detergents and SDS caused barrier damage in ex vivo human skin.Abbreviations: ACVRL1, activin A receptor like type; CCL, C‐C motif chemokine ligand; CLSTN2, calsyntenin‐2; CNTN1, contactin‐1; EPCAM, epithelial cell adhesion molecule; FASTL3, follistatin‐related protein 3; IL1B, interleukin 1 beta; IL6, interleukin 6; PBS, phosphate‐buffered saline; RNA‐seq, RNA sequencing; SDS, sodium dodecyl sulfate
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