Lactobacillus rhamnosus GG inhibits the toxic effects of Staphylococcus aureus on epidermal keratinocytes

Few studies have evaluated the potential benefits of the topical application of probiotic bacteria or material derived from them. We have investigated whether a probiotic bacterium, Lactobacillus rhamnosus GG, can inhibit Staphylococcus aureus infection of human primary keratinocytes in culture. When primary human keratinocytes were exposed to S. aureus, only 25% of the keratinocytes remained viable following 24 h of incubation. However, in the presence of 10(8) CFU/ml of live L. rhamnosus GG, the viability of the infected keratinocytes increased to 57% (P = 0.01). L. rhamnosus GG lysates and spent culture fluid also provided significant protection to keratinocytes, with 65% (P = 0.006) and 57% (P = 0.01) of cells, respectively, being viable following 24 h of incubation. Keratinocyte survival was significantly enhanced regardless of whether the probiotic was applied in the viable form or as cell lysates 2 h before or simultaneously with (P = 0.005) or 12 h after (P = 0.01) S. aureus infection. However, spent culture fluid was protective only if added before or simultaneously with S. aureus. With respect to mechanism, both L. rhamnosus GG lysate and spent culture fluid apparently inhibited adherence of S. aureus to keratinocytes by competitive exclusion, but only viable bacteria or the lysate could displace S. aureus (P = 0.04 and 0.01, respectively). Furthermore, growth of S. aureus was inhibited by either live bacteria or lysate but not spent culture fluid. Together, these data suggest at least two separate activities involved in the protective effects of L. rhamnosus GG against S. aureus, growth inhibition and reduction of bacterial adhesion

Multiple Proteins of Lacticaseibacillus rhamnosus GG Are Involved in the Protection of Keratinocytes From the Toxic Effects of Staphylococcus aureus

We have previously shown that lysates of Lacticaseibacillus rhamnosus GG confer protection to human keratinocytes against Staphylococcus aureus. L. rhamnosus GG inhibits the growth of S. aureus as well as competitively excludes and displaces the pathogen from keratinocytes. In this study, we have specifically investigated the anti-adhesive action. We have tested the hypothesis that this activity is due to quenching of S. aureus binding sites on keratinocytes by molecules within the Lacticaseibacillus lysate. Trypsinisation or heat treatment removed the protective effect of the lysate suggesting the involvement of proteins as effector molecules. Column separation of the lysate and analysis of discrete fractions in adhesion assays identified a fraction of moderate hydrophobicity that possessed all anti-adhesive functions. Immunoblotting demonstrated that this fraction contained the pilus protein, SpaC. Recombinant SpaC inhibited staphylococcal adhesion to keratinocytes in a dose-dependent manner and improved keratinocyte viability following challenge with viable S. aureus. However, SpaC did not confer the full anti-adhesive effects of the LGG lysate and excluded but did not displace S. aureus from keratinocytes. Further purification produced four protein-containing peaks (F1-F4). Of these, F4, which had the greatest column retention time, was the most efficacious in anti-staphylococcal adhesion and keratinocyte viability assays. Identification of proteins by mass spectrometry showed F4 to contain several known "moonlighting proteins"-i.e., with additional activities to the canonical function, including enolase, Triosephosphate isomerase (TPI), Glyceraldehyde 3 phosphate dehydrogenase (G3P) and Elongation factor TU (EF-Tu). Of these, only enolase and TPI inhibited S. aureus adhesion and protected keratinocytes viability in a dose-dependent manner. These data suggest that inhibition of staphylococcal binding by the L. rhamnosus GG lysate is mediated by SpaC and specific moonlight proteins.Peer reviewe

Hyperglycemia-induced oxidative stress and epigenetic regulation of ET-1 gene in endothelial cells

Introduction: Hyperglycemia-induced endothelial dysfunction and the subsequent increase of oxidative stress could lead to aberrant regulation of various genes which are responsible for a range of functions. This study aims to find out how hyperglycemia affect oxidative stress and then the expression and methylation of endothelin 1 (ET-1) gene in in human umbilical vein endothelial cells (HUVEC).Methods: Cells were cultured in growth medium and exposed to low and high glucose concentrations to mimic normal and diabetic condition respectively. Computational analysis were performed using UCSC genome browser and eukaryotic promoter database (EPD). The expression of ET-1 gene was investigated by real time PCR. Cytotoxicity and oxidative stress were determined by MTT and DCFH-DA assays respectively. Promoter methylation was assessed by the bisulfite sequencing method.Results: DCFH-DA assay showed that hyperglycemia can significantly increase the regulation of reactive oxygen species synthesis. The relative expression of ET-1 gene was increased due to exposure to high glucose concentration. MTT assay revealed reduced viability of cells due to the glucose induced damage. Methylation analysis revealed hypomethylation of the promoter of ET-1 however the difference was not significant. Out of 175 CpGs at 25 CpG sites, only 36 CpGs were methylated (20.5% methylation) in cell treated with normal glucose. Upon exposure to high glucose only 30 CpGs were methylated in 175 CpGs at 25 CpG sites (17.1% methylation).Discussion: Our study concludes a significantly high expression of ET-1 gene in response to high glucose exposure in HUVECs. It also reports that hyperglycemic condition leads to elevated oxidative stress. No significant change was found in methylation when cells were treated with high and low glucose concentrations

Serum Cytokine Levels As Critical Parameters in Early Diagnosis of Disease Progression in COVID-19: A Pilot Study

Abstract Background: The severity of Coronavirus disease 2019 (COVID-19) has been proposed to be associated with cytokine dysregulation. A significant number of patients become serious and need intensive care in hospitals. Methods: The concentrations of cytokines interleukin (IL-6, IL-10) and tumor necrosis factor (TNF) were estimated using enzyme-linked immunosorbent assay (ELISA) in serum samples of 60 adult patients infected with SARS-CoV-2 along with 50 healthy controls of the same age. The mean age of the subjects was 50-52 years and included an equal number of males and females. The patients were further grouped as severe (38 patients) and non-severe cases (22 patients). Results: The mean serum cytokine levels were significantly higher in the COVID-19 patients than in the healthy controls. IL-6 was excessively elevated in comparison to IL-10 and TNF. Comparative analysis of severe versus non-severe cases revealed only slight alterations in the cytokine levels: IL-6 being the most elevated in severe cases. The concentration of the liver enzyme ALT was higher than AST in both severe and non-severe cases. The mean concentration of serum electrolytes (Na, K, and Ca) did not vary much between the patients and healthy controls. Conclusion: There was a significant positive correlation between the levels of cytokines serum biomarkers in COVID-19 patients. It may be suggested that early detection of cytokines, especially IL-6 and serum biomarkers can help predict disease prognosis and severity in COVID-19 patients

Lactobacillus rhamnosus GG Lysate Increases Re-Epithelialization of Keratinocyte Scratch Assays by Promoting Migration

A limited number of studies have investigated the potential of probiotics to promote wound healing in the digestive tract. The aim of the current investigation was to determine whether probiotic bacteria or their extracts could be beneficial in cutaneous wound healing. A keratinocyte monolayer scratch assay was used to assess re-epithelialization; which comprises keratinocyte proliferation and migration. Primary human keratinocyte monolayers were scratched then exposed to lysates of Lactobacillus (L) rhamnosus GG, L. reuteri, L. plantarum or L. fermentum. Re-epithelialization of treated monolayers was compared to that of untreated controls. Lysates of L. rhamnosus GG and L. reuteri significantly increased the rate of re-epithelialization, with L. rhamnosus GG being the most efficacious. L. reuteri increased keratinocyte proliferation while L. rhamnosus GG lysate significantly increased proliferation and migration. Microarray analysis of L. rhamnosus GG treated scratches showed increased expression of multiple genes including the chemokine CXCL2 and its receptor CXCR2. These are involved in normal wound healing where they stimulate keratinocyte proliferation and/or migration. Increased protein expression of both CXCL2 and CXCR2 were confirmed by ELISA and immunoblotting. These data demonstrate that L. rhamnosus GG lysate accelerates re-epithelialization of keratinocyte scratch assays, potentially via chemokine receptor pairs that induce keratinocyte migration

The beneficial effect of Adansonia digitata products success to modulate lipid profiles and inhibit LDL oxidation in-vitro: An associational study

BackgroundThere is a growing interest in medicinal plants in recent years due to their many therapeutic benefits and low side effects. Among the medicinal plants is the African Adansonia digitata (baobab) that has edible fruit. In the current study, the effect of A. digitata juice consumption on the lipid profile was investigated. In addition, inhibition of the oxidation of low-density lipoprotein cholesterol (LDL-C) in-vitro by A. digitata essential oil (EO) was also investigated. MethodsIn this cohort study, a total of 70 subjects of A. digitata users (AD group, 42 male and 28 female) and 70 non A. digitata users (Non-AD group, 44 male and 26 female) were recruited to participate in this study. We evaluated lipid profile, HbA1c, liver/kidney functions, and phytosterol contents in fasting blood samples of all participants.ResultsThe present findings illustrated significantly lower levels of total cholesterol, triglycerides, and LDL in the AD group compared to Non-AD (p < 0.01). In addition, essential oil of A. digitata inhibited LDL oxidation in-vitro as shown by the significant decreases in the formation of malonaldehyde (MDA), protein carbonyl (PC), and lipid hydroperoxide (LHP) (P<0.05). No significant changes in fasting blood glucose, HbA1c, HDL, kidney function, and liver function enzymes between the two groups were detected (P>0.05). ConclusionThe juice of A. digitata has hypolipidemic and antioxidative effects and might be beneficial for the management of lipid levels in the body