Impact of culture medium on the interpretation of qRT-PCR data in HepG2 incubated with lactobacilli.

Recently, an increasing number of studies have investigated the mechanism of action of lactobacilli in the treatment of non-alcoholic fatty liver disease. Using four computational tools (NormFinder, geNorm, Delta Ct, and BestKeeper), six potential reference genes (RGs) were analyzed in the human liver cell line HepG2 cultivated 24 h in the presence of two strains of heat-killed lactobacilli, Limosilactobacillus reuteri E and Lactiplantibacillus plantarum KG4, respectively, in different cultivation media [Dulbecco´s Modified Eagle´s Medium (DMEM) high glucose or Roswell Park Memorial Institute (RPMI)]. The analysis revealed that the suitability of RG was similar between the two lactobacilli but quite different between the two media. The commonly used RGs, 18S rRNA and glyceraldehyde-3-phosphate dehydrogenase were the most unstable in DMEM high glucose. Normalization of the mRNA expression of the target gene encoding sterol regulatory element-binding protein 1c (SREBP-1c) to different RGs resulted in different expression profiles. This demonstrates that validation of candidate RGs under specific experimental conditions is crucial for the correct interpretation of quantitative polymerase chain reaction data. In addition, the choice of media has a profound impact on the effect of lactobacilli on lipogenesis at the gene expression level, as shown by the transcription factor SREBP-1c.

Mucin pre-cultivated Lactobacillus reuteri E shows enhanced adhesion and increases mucin expression in HT-29 cells.

Adhesion of probiotic bacteria to the mucus layer lining the gastrointestinal tract is necessary for its effective colonisation and specific therapeutic effects. Enrichment of growth medium with mucin might stimulate bacterial adhesion, probably by increasing the expression of surface structures responsible for bacteria-gut epithelia and/or mucus interactions. The aim of this study was to determine if pre-cultivation of potentially probiotic strain Lactobacillus reuteri E (LRE) with mucin stimulates its adherence to colon cell line HT-29 and if the increased adhesion modulates mucin expression in these cells. The mucin-producing HT-29 cell line was co-cultivated for 2 h with LRE grown in MRS broth or MRS broth enriched with pig gastric mucin (LRE + M). The adherence ability of LRE was evaluated microscopically and by plate counting. The relative gene expression was measured by qPCR. Pre-cultivation of LRE in mucin enriched medium significantly increased its adhesion to 14 days HT-29 in comparison with LRE by both methods (28.64% vs. 23.83%, evaluated microscopically, and 14.31 ± 3.95 × 106 CFU ml-1 vs. 8.54 ± 0.43 × 106 CFU ml-1, evaluated by plate counting). MUC2, MUC5AC, and IL-10 were significantly upregulated after co-cultivation with LRE + M in comparison to LRE and control group (lactobacilli-free HT-29). Obtained results suggest that pre-cultivation of lactobacilli with mucin may not only stimulate their adhesion abilities but also promote their effectiveness to modulate the pathways involved in the pathophysiology of some diseases, e.g., with defective mucin synthesis in ulcerative colitis or colorectal cancer.

[Potential immunomodulatory and antiinflammatory mechanisms of probiotics]. / Predpokladané mechanizmy imunomodulacného a protizápalového pôsobenia probiotík.

UNLABELLED: The number of preclinical and clinical studies showing efficacy of probiotics in the treatment and prophylaxis of certain diseases, e.g. diarrhoea of various origin, irritable bowel syndrome, inflammatory bowel disease, allergies, hypercholesterolemia, bacterial vaginosis, and colorectal cancer, is increasing. These health benefits are often species and strain specific. This article provides an overview of available knowledge about the supposed mechanisms of probiotic microorganisms action focusing in particular on the interaction between probiotic and host cells. One of the results of this interaction is induction of pro- or anti-inflammatory immune response in the macroorganism. Detailed knowledge of the signalling pathways involved in the communication between bacterial and human cells can find application in the selection of optimal probiotics for the targeted treatment of selected diseases. Additional possibilities for their use in clinical practice are provided by genetic manipulation of probiotic microorganisms. KEY WORDS: probiotics inflammation signalling pathways immunomodulation genetic manipulation.

Isolation and identification of new lactobacilli from goatling stomach and investigation of reuterin production in Lactobacillus reuteri strains.

Five new strains of lactobacilli isolated from goatling's stomach were identified by molecular-biological approaches. Profiles of fermentable saccharides, Gram staining, and cell morphology were also determined. They were identified as Lactobacillus reuteri (strains KO4b, KO4m, KO5) and as Lactobacillus plantarum (strains KG1z, KG4). In DNA samples of all newly isolated L. reuteri strains as well as in L. reuteri E (Lreu E; originated from lamb), the part of gldC gene, coding large subunit of glycerol dehydratase, that is necessary for 3-hydroxypropionaldehyde (3-HPA; reuterin) production, was amplified using two designed primer sets. However, the 3-HPA production was revealed only in the strain Lreu E. It produced five- or ten-fold lower amount of 3-HPA in comparison with probiotic L. reuteri ATCC 55730 in aerobic or anaerobic conditions, respectively. Moreover, Lreu E completely lost its production ability after ca. five passages in MRS medium. The co-incubation of Lreu E, but not other L. reuteri isolates, with Escherichia coli re-induced 3-HPA production. In the case of L. reuteri ATCC 55730, the 3-HPA production increased more than four times after co-incubation with E. coli.