Feeding-Regime-Dependent Intestinal Response of Rainbow Trout after Administration of a Novel Probiotic Feed.

Intensive fish farming is associated with a high level of stress, causing immunosuppression. Immunomodulators of natural origin, such as probiotics or phytoadditives, represent a promising alternative for increasing the immune function of fish. In this study, we tested the autochthonous trout probiotic strain L. plantarum R2 in a newly developed, low-cost application form ensuring the rapid revitalization of bacteria. We tested continuous and cyclic feeding regimes with regard to their effect on the intestinal immune response and microbiota of rainbow trout. We found that during the continuous application of probiotic feed, the immune system adapts to the immunomodulator and there is no substantial stimulation of the intestinal immune response. During the cyclic treatment, after a 3-week break in probiotic feeding and the reintroduction of probiotics, there was a significant stimulation of the gene expression of molecules associated with both cellular and humoral immunity (CD8, TGF-ß, IL8, TLR9), without affecting the gene expression for IL1 and TNF-α. We can conclude that, in aquaculture, this probiotic feed can be used with a continuous application, which does not cause excessive immunostimulation, or with a cyclic application, which provides the opportunity to stimulate the immunity of trout, for example, in periods of stress.

Silybin Showed Higher Cytotoxic, Antiproliferative, and Anti-Inflammatory Activities in the CaCo Cancer Cell Line while Retaining Viability and Proliferation in Normal Intestinal IPEC-1 Cells.

The anticancer potential of silymarin is well known, including its anti-inflammatory as well as antiproliferative effect mediated by influencing the cell cycle, suppression of apoptosis, and inhibition of cell-survival kinases. However, less is known about silybin, the main component of the silymarin complex, where studies indicate its dual effect on the proliferation and immune response of various cell types in a dose-dependent manner. Moreover, there is a lack of studies comparing the effect of silybin on the same type of healthy and tumor cells, especially intestinal ones. Therefore, our study aimed to investigate the concentration-dependent effect of silybin on the normal intestinal porcine epithelial cell line-1 (IPEC-1) and the human epithelial colorectal adenocarcinoma cell line (CaCo-2). The metabolic viability, cell cycle, mitochondrial membrane potential, apoptosis, and the relative gene expression for pro- and anti-inflammatory cytokines were monitored in cells treated with silybin. Silybin stimulates metabolic viability as well as proliferation in IPEC-1 cells, protects the mitochondrial membrane, and thus exerts a cytoprotective effect, and has only a minimal effect on the gene expression of pro-inflammatory cytokines but significantly increases the expression of anti-inflammatory TGF-ß. In contrast, it inhibits metabolic viability in tumor intestinal CaCo-2 cells, has an antiproliferative effect accompanied by increased apoptosis, and significantly reduces the expression of genes for pro-inflammatory interleukins as well as TGF-ß. The antiproliferative and anti-inflammatory effect of silybin on tumor intestinal cells without a negative effect on healthy cells is a prerequisite for its potential use in the adjuvant therapy of colon cancer; however, further studies are necessary.

Biofilm-forming lactic acid bacteria of honey bee origin intended for potential probiotic use.

Scientists around the world are focusing their interest on the use of probiotics in honey bees as an alternative method of prophylaxis against causative agents of both American and European foulbrood. In our study we tested inhibitory activity against Paenibacillus larvae and the biofilm formation activity by various lactic acid bacteria isolated from honey bee guts or fresh pollen samples in the presence of different sugars added to the cultivation media. In addition, we tested the probiotic effect of a newly selected Apilactobacillus kunkeei V18 in an in situ experiment in bee colonies. We found antibacterial activity against P. larvae in four isolates. Biofilm formation activity of varying intensity was noted in six of the seven isolates in the presence of different sugars. The strongest biofilm formation (OD570 ≥ 1) was noted in A. kunkeei V18 in the presence of fructose; moreover, this isolate strongly inhibited the growth of P. larvae under laboratory conditions. Inhibition of P. larvae and Melissococcus plutonius by A. kunkeei V18 in situ was confirmed in a pilot study.

An in vitro selective inhibitory effect of silver(i) aminoacidates against bacteria and intestinal cell lines and elucidation of the mechanism of action by means of DNA binding properties, DNA cleavage and cell cycle arrest.

Novel silver(i) aminoacidate complexes {[Ag(HVal)(H2O)(NO3)]}n (AgVal) and {[Ag3(HAsp)2(NO3)]}n·nH2O (AgAsp) were prepared, investigated and fully characterized by vibrational spectroscopy (mid-IR), elemental analysis, thermogravimetric analysis, X-ray crystallography and mass spectrometry. Their stability in D2O and PBS buffer was verified by time-dependent 1H and 13C NMR measurements. Their in vitro antibacterial activity (against pathogenic Staphylococcus aureus CCM4223, Escherichia coli CCM4787) and that against probiotic bacteria Lactobacillus plantarum CCM7102 and Lactobacillus reuteri (L26) were determined and potential dosing concentration was evaluated. The cytotoxicity of both the complexes against intestinal porcine epithelial (IPEC-1) and human epithelial colorectal adenocarcinoma (CaCo-2) cell lines was determined using the colorimetric MTT assay and against human metastatic melanoma (A2058), human pancreatic adenocarcinoma (PaTu 8902), human cervical adenocarcinoma (HeLa), human colorectal carcinoma (HCT116), human leukaemic T cell lymphoma (Jurkat), and human dermal fibroblasts (HDF) using colorimetric MTS assay. The selectivity index (SI) was identified for intestinal cancer (CaCo-2) and healthy (IPEC-1) cells. The mechanism of action of AgVal and AgAsp was further elucidated and discussed by the study of their binding affinity toward the CT DNA, the ability to cleave the supercoiled form of pUC19 DNA and the ability to influence numbers of cells within each cell cycle.

Effect of Application of Probiotic Pollen Suspension on Immune Response and Gut Microbiota of Honey Bees (Apis mellifera).

Although the use of probiotic bacteria in invertebrates is still rare, scientists have begun to look into their usage in honey bees. The probiotic preparation, based on the autochthonous strain Lactobacillus brevis B50 Biocenol™ (CCM 8618), which was isolated from the digestive tracts of healthy bees, was applied to the bee colonies in the form of a pollen suspension. Its influence on the immune response was determined by monitoring the expression of genes encoding immunologically important molecules in the honey bee intestines. Changes in the intestinal microbiota composition were also studied. The results showed that the probiotic Lact. brevis B50, on a pollen carrier, significantly increased the expression of genes encoding antimicrobial peptides (abaecin, defensin-1) as well as pattern recognition receptors (toll-like receptor, peptidoglycan recognition proteins). Gene expression for the other tested molecules included in Toll and Imd signaling pathways (dorsal, cactus, kenny, relish) significantly changed during the experiment. The positive effect on intestinal microbiota was manifested mainly by a significant increase in the ratio of lactic acid bacteria to enterobacteria. These findings confirm the potential of the tested probiotic preparation to enhance immunity in bee colonies and thus increase their resistance to infectious diseases and stress conditions.

Cytokine response after stimulation of culture cells by zinc and probiotic strain.

Intestinal porcine epithelial cells were used for an in vitro analysis of mRNA expression levels of inflammatory cytokines (IL-8, IL-18) and transcriptional factors (MyD88 and NF-κß). Cells were exposed to inorganic and organic zinc sources (in two different concentrations-50 µmol/L and 100 µmol/L) alone or combined with Lactobacillus reuteri B6/1, which was also applied individually. The total exposure time was 4 h. Quantitative reverse transcriptase PCR was used to determine expression levels of the aforementioned parameters. In general, upregulation was observed; however, a decrease of some mRNA's abundance was also determined. Differences in expression were analysed statistically using ANOVA and Tukey analyses. High relative expression was shown for IL-8, IL-18 and MyD88 in groups treated with 100 µmol/L of inorganic sources of zinc (ZnSO4) (p < 0.05), while groups treated with the organic form did not exhibit significant changes in expression. Also, 50 µmol/L of either zinc source did not significantly modify the transcriptional profile of the cytokines and transcription factors, showing that even inorganic sources, at lower concentrations, do not elicit a significant inflammatory reaction. In summary, supplementation of organic zinc source (Gly-Zn chelate) ensures that IL-8, IL-18, MyD88 and NF-κß expression levels are not positively regulated. In contrast, inorganic sources of zinc (ZnSO4) could induce an inflammatory reaction. However, this response could be dampened if L. reuteri B6/1 is administered, showing the helpful aspect of using probiotics to modulate an inflammatory response. Conclusively, the use Gly-Zn chelate appears as an optimal alternative for Zn administration that does not compromise normal intestinal homeostasis.