Immunomodulatory effect of probiotic exopolysaccharides in a porcine in vitro co-culture model mimicking the intestinal environment on ETEC infection.

The aim of this study was to evaluate the immunomodulatory effect of EPS-L26 isolated from the probiotic strain Lactobacillus (Limosilactobacillus) reuteri L26 Biocenol™, in a model of infection with an enterotoxigenic E. coli (ETEC) by establishing monocultures consisting of the IPEC-J2 cell line or monocyte-derived dendritic cells (moDCs) and creating a 3D model of cell co-cultures established with IPEC-J2 cells and moDCs. The immunomodulatory and immunoprotective potential of used EPS-L26 was confirmed in monocultures in an experimental group of pretreated cells, where our study showed that pretreatment of cells with EPS-L26 and subsequent exposure to infection resulted in significantly down-regulated mRNA levels of genes encoding inflammatory cytokines compared to ETEC challenge in single cell cultures (in IPEC-J2, decreased mRNA levels for TNF-α, IL-6, IL-1ß, IL-12p35; in moDCs, decreased mRNA levels for IL-1ß). Similar to monocultures, we also demonstrated the immunostimulatory potential of the ETEC strain in the co-culture model on directly treated IPEC-J2 cells cultivated on insert chambers (apical compartment) and also on indirectly treated moDCs cultivated in the lower chamber (basolateral compartment), however in the co-culture model the expression of inflammatory cytokines was attenuated at the mRNA level compared to monocultures. Pretreatment of the cells on the insert chambers pointed to the immunoprotective properties of EPS-L26, manifested by decreased mRNA levels in both cell lines compared to ETEC challenge (in IPEC-J2 decreased mRNA levels for IL-12p35; in moDCs decreased mRNA levels for IL-1ß, IL-6). Our results suggest intercellular communication via humoral signals derived from IPEC-J2 cells by influencing the gene expression of indirectly treated moDC cells located in the basolateral compartment.

Influence of standard culture conditions and effect of oleoresin from the microalga Haematococcus pluvialis on splenic cells from healthy Balb/c mice - a pilot study.

In this work, we used splenocytes from healthy mice to study the effects of the two most commonly used cell culture media (A, B) with different compositions of redox reagents. The incubation of cells for 24 h resulted in a significant decrease in viability and metabolic activity of splenocytes, and the negative effects of incubation in medium B were more pronounced. In standard conditions, oxidative stress in cells was manifested by reduced mitochondrial potential, and this effect correlated with the transition of 58.3% of cells to the early stage of apoptosis under reducing conditions of medium A and up to 66.1% of cells under super-reducing conditions in medium B, suggesting altered cell physiology. High levels of ROS/RNS activated transcription factor Nrf2, superoxide dismutase 1, and catalase. The higher mRNA levels of these genes were under the conditions of medium B, whose super-reducing environment in combination with the environment of conventional incubators proved to be less suitable for the cells compared to medium A. Treatment of the cells with a lower concentration (10 µg/ml) of oleoresin obtained from the microalga H. pluvialis partially eliminated the negative effects of cultivation. Higher concentration of oleoresin (40 µg/ml) was slightly cytotoxic, due to the significant antioxidant effect of astaxanthin, the main bioactive component of the extract, which eliminated most of the ROS/RNS acting as signalling molecules. This study shows that the standard culture conditions do not reflect the physiological in vivo cell conditions; therefore, they are not generally suitable for incubation of all cell types.

The role of primed and non-primed MSC-derived conditioned media in neuroregeneration.

Introduction: With growing significance in nervous system repair, mesenchymal stem cell-derived conditioned media (MSCCM) have been used in cell-free therapies in regenerative medicine. However, the immunomodulatory and neuroregenerative effects of MSCCM and the influence of priming on these effects are still poorly understood. Methods: In this study, by various methods focused on cell viability, proliferation, neuron-like differentiation, neurite outgrowth, cell migration and regrowth, we demonstrated that MSCCM derived from adipose tissue (AT-MSCCM) and amniotic membrane (AM-MSCCM) had different effects on SH-SY5Y cells. Results and discussion: AT-MSCCM was found to have a higher proliferative capacity and the ability to impact neurite outgrowth during differentiation, while AM-MSCCM showed more pronounced immunomodulatory activity, migration, and re-growth of SH-SY5Y cells in the scratch model. Furthermore, priming of MSC with pro-inflammatory cytokine (IFN-γ) resulted in different proteomic profiles of conditioned media from both sources, which had the highest effect on SH-SY5Y proliferation and neurite outgrowth in terms of the length of neurites (pAT-MSCCM) compared to the control group (DMEM). Altogether, our results highlight the potential of primed and non-primed MSCCM as a therapeutic tool for neurodegenerative diseases, although some differences must be considered.

Silver(I) pyrrole- and furan-2-carboxylate complexes - From their design and characterization to antimicrobial, anticancer activity, lipophilicity and SAR.

Two silver(I) complexes with biologically relevant heterocyclic ligands, pyrrole and furan-2- carboxylic acid, were synthesized and their composition was confirmed using elemental, spectral, thermal and structural analyses. The {[Ag(Py2c)]}n (AgPy2c, Py2c = pyrrole-2-carboxylate) and {[Ag(Fu2c)]}n (AgFu2c, Fu2c = furan-2-carboxylate) solubility and stability in biological test stock solution were confirmed by 1H NMR spectroscopy. The X-ray analysis has enabled us to determine typical argentophilic interactions and bridging carboxylate coordination mode of both ligands. Potentiometric data analysis by BSTAC program resulted in the determination of the stability constant of only one species, i.e., the ML (M = Ag+, L = Fu2c-), log ßML = 0.59 ± 0.04. Antimicrobial and anticancer tests were performed against selected microorganisms and cell lines with new silver(I) complexes and compared with AgSD (silver(I) sulfadiazine) and cisplatin. From their microbial toxicity point of view, selectivity was determined against lactobacilli (AgPy2c is 8× more effective against S. aureus and E. coli and AgFu2c is 8× more effective against E. coli and 4× against S. aureus). AgFu2c significant anticancer activity was determined against Jurkat cell lines (IC50 = 8.00 µM) and was similar to cisPt (IC50 = 6.3 µM) similarly to its selectivity (SI (AgFu2c) = 7.3, SI (cisPt) = 6.4, SI = selectivity index). In addition, cell cycle arrest was observed already in the Sub-G0 phase during a flow cytometry experiment. To evaluate the AgPy2c and AgFu2c bioavailability we also discuss their Lipinski's Rule of Five.

Antibacterial activity, quality and stability study of creams with new potential silver(I) complexes and in vivo case report.

The aim of this study was to evaluate the antibacterial activity, quality and stability of creams (at 1 % concentration) prepared with synthesized silver(I) complexes: [Ag(Nam)2]NO3·H2O ( AgNam), [Ag2(HGly)2]n(NO3)2n (AgGly) (Nam - nicotin-amide, Gly - glycine) and silver(I) sulfadiazine (AgSD), which is commercially available. Antibacterial activity was evaluated by agar well diffusion method and in in vivo case. The pure silver(I) complexes as well as all three tested creams loaded with AgGly, AgSD and AgNam showed antibacterial potential. Moreover, the creams loaded with AgGly and AgNam showed higher antibacterial effects against S. aureus and B. subtilis than the cream loaded with AgSD. In terms of appearance, all cream samples were opaque and odourless, and no phase separation was observed. Creams were soluble in water (o/w emulsions) and they had a pseudoplastic behaviour. The pH of the creams was in the range of 4.87-5.75. No visible changes were observed in the case of commercially used AgSD cream during one month testing period at conditions -16 ± 1 °C; 6 ± 1 °C and 56 % relative humidity; 20 ± 1 °C and 58 % relative humidity and 40 ± 1 °C and 75 % relative humidity. However, creams containing AgGly and AgNam changed their colour depending on the tested conditions.

Development and evaluation of a fish feed mixture containing the probiotic Lactiplantibacillus plantarum prepared using an innovative pellet coating method.

Introduction: Due to the intensification of fish farming and the associated spread of antimicrobial resistance among animals and humans, it is necessary to discover new alternatives in the therapy and prophylaxis of diseases. Probiotics appear to be promising candidates because of their ability to stimulate immune responses and suppress the growth of pathogens. Methods: The aim of this study was to prepare fish feed mixtures with various compositions and, based on their physical characteristics (sphericity, flow rate, density, hardness, friability, and loss on drying), choose the most suitable one for coating with the selected probiotic strain Lactobacillus plantarum R2 Biocenol™ CCM 8674 (new nom. Lactiplantibacillus plantarum). The probiotic strain was examined through sequence analysis for the presence of plantaricin- related genes. An invented coating technology based on a dry coating with colloidal silica followed by starch hydrogel containing L. plantarum was applied to pellets and tested for the viability of probiotics during an 11-month period at different temperatures (4°C and 22°C). The release kinetics of probiotics in artificial gastric juice and in water (pH = 2 and pH = 7) were also determined. Chemical and nutritional analyses were conducted for comparison of the quality of the control and coated pellets. Results and discussion: The results showed a gradual and sufficient release of probiotics for a 24-hour period, from 104 CFU at 10 mi up to 106 at the end of measurement in both environments. The number of living probiotic bacteria was stable during the whole storage period at 4°C (108), and no significant decrease in living probiotic bacteria was observed. Sanger sequencing revealed the presence of plantaricin A and plantaricin EF. Chemical analysis revealed an increase in multiple nutrients compared to the uncoated cores. These findings disclose that the invented coating method with a selected probiotic strain improved nutrient composition and did not worsen any of the physical characteristics of pellets. Applied probiotics are also gradually released into the environment and have a high survival rate when stored at 4°C for a long period of time. The outputs of this study confirm the potential of prepared and tested probiotic fish mixtures for future use in in vivo experiments and in fish farms for the prevention of infectious diseases.

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.

Astaxanthin Extract from Haematococcus pluvialis and Its Fractions of Astaxanthin Mono- and Diesters Obtained by CCC Show Differential Antioxidant and Cytoprotective Effects on Naïve-Mouse Spleen Cells.

Carotenoids are the most abundant lipid-soluble phytochemicals and are used as dietary supplements to protect against diseases caused by oxidative stress. Astaxanthin, a xanthophyll carotenoid, is a very potent antioxidant with numerous beneficial effects on cellular functions and signaling pathways. In this study, using spleen cells from healthy Balb/c mice, we report the bio-functional effects of an astaxanthin-rich extract (EXT) prepared from the microalga Haematococcus pluvialis and its astaxanthin monoesters-rich fraction (ME) and astaxanthin diesters-rich fraction (DE) obtained by fractionation of EXT using countercurrent chromatography (CCC). After incubation under standard culture conditions (humidity, 37 °C, 5% CO2, atmospheric oxygen), the viability of untreated splenocytes, as determined by the trypan blue exclusion assay, the MTT assay, and the neutral red assay, decreases to approximately 75% after 24 h compared with naïve splenocytes. This effect correlated with the decrease in mitochondrial membrane potential and the transition of ~59% of cells to the early stage of apoptosis, as well as with the decreased ROS production, indicating that hyperoxia in cell-culture deteriorates cell functions. They are restored or stimulated by co-cultivation with EXT, ME, and DE up to 10 µg/mL in the order EXT > DE > ME, suggesting that esterification increases bioavailability to cells in vitro. ROS and H2O2 concentrations reflect mRNA transcriptional activity of Nrf2, superoxide dismutase 1 (SOD1), catalase, and glutathione peroxidase 1, as well as SOD-mediated ROS conversion, whereas they inversely correlate with iNOS-mediated NO production. The highest-tested concentration of EXT, ME, and DE (40 µg/mL) is detrimental to cells, probably because of the overwhelming scavenging activity of astaxanthin and its esters for the reactive oxygen/nitrogen species required for cellular functions and signal transduction at low physiological concentrations. In this study, we demonstrate that differential activities of ME and DE contribute to the final antioxidant and cytoprotective effects of astaxanthin extract, which is beneficial in preventing a wide range of ROS-induced adverse effects, with DE being more effective. In addition, the selection of physioxia-like conditions for pharmacological research is highlighted.

Application of Lactobacillus reuteri B1/1 (Limosilactobacillus reuteri) Improves Immunological Profile of the Non-Carcinogenic Porcine-Derived Enterocytes.

In our previous studies, Lactobacillus reuteri B1/1, which was renamed Limosilactobacillus reuteri (L. reuteri), was able to modulate the production of pro-inflammatory cytokines and other components of the innate immune response in vitro and in vivo. In this study, we evaluated the effect of Lactobacillus reuteri B1/1 in two concentrations (1 × 107 and 1 × 109 CFU) on the metabolic activity, adherence ability and relative gene expression of pro-inflammatory interleukins (IL-1ß, IL-6, IL-8, IL-18), lumican and olfactomedin 4 produced by non-carcinogenic porcine-derived enterocytes (CLAB). CLAB cells were cultured in a 12-well cell culture plate at a concentration of 4 × 105 cells/well in DMEM medium in a controlled humidified atmosphere for 48 h. A 1 mL volume of each probiotic bacterial suspension was added to the CLAB cells. Plates were incubated for 2 h and 4 h. Our results revealed that L. reuteri B1/1 was able to adhere to CLAB cells in sufficient numbers in both concentrations. In particular, the concentration of 109L. reuteri B1/1 allowed to modulate the gene expression of pro-inflammatory cytokines, as well as to increase the metabolic activity of the cells. In addition, administration of L. reuteri B1/1 in both concentrations significantly stimulated gene expression for both proteins in the CLAB cell line after 4 h of incubation.

Impact of Canine Amniotic Mesenchymal Stem Cell Conditioned Media on the Wound Healing Process: In Vitro and In Vivo Study.

The aim of this study was to provide a beneficial treatment effect of mesenchymal stem cell products derived from the canine amniotic membrane (AM-MSC) on the complicated wound healing process in dogs. AM-MSCs were characterized in terms of morphology, phenotypic profile, and multilineage differentiation potential. The in vitro study of the effect of canine amniotic mesenchymal stem cell conditioned media (AMMSC-CM) on a primary skin fibroblast cell culture scratch assay showed a decrease in the measured scratch area of about 66.39% against the negative control (Dulbecco's Modified Eagle's Medium-32.55%) and the positive control (Dulbecco's Modified Eagle's Medium supplemented with FGF2, N2, B27, and EGF-82.077%) after 72 h treatment. In the experimental study, seven dogs with complicated nonhealing wounds were treated with a combination of antibiotics, NSAIDs, and local AMMSC-CM application. After 15 days of therapy, we observed a 98.47% reduction in the wound surface area as opposed to 57.135% in the control group treated by conventional therapy based on debridement of necrotic tissue, antibiotic therapy, pain management, and change of wound dressing.

Humic Substances as a Feed Supplement and the Benefits of Produced Chicken Meat.

Humic substances with a high proportion of humic acids (more than 40%) have been classified by the European Commission as feed materials that can be used in animal nutrition since 2013. A protective effect on the intestinal mucosa, as well as anti-inflammatory, adsorptive and antimicrobial properties, were recorded. Nutrient absorption, nutritional status and the immune response in chickens supplemented with HSs were significantly improved. HSs have the ability to enhance protein digestion as well as the utilization of calcium and trace elements. They are known to improve feed digestibility as a result of maintaining an optimal pH in the gut, leading to lower levels of nitrogen excretion and less odor in the husbandry environment. HSs not only increase digestibility and result in greater utilization of the feed ration but also improve the overall quality of the meat produced. They increase the protein content and reduce the fat content in breast muscles. They also contribute to improving the sensory characteristics of the meat produced. Their antioxidant properties improve the oxidative stability of meat during storage. The influence of HSs on fatty acid composition may be one of the reasons that meat has a more beneficial effect on the health of consumers.

Preparation and Evaluation of a Dosage Form for Individualized Administration of Lyophilized Probiotics.

Probiotics have been used in human and veterinary medicine to increase resistance to pathogens and provide protection against external impacts for many years. Pathogens are often transmitted to humans through animal product consumption. Therefore, it is assumed that probiotics protecting animals may also protect the humans who consume them. Many tested strains of probiotic bacteria can be used for individualized therapy. The recently isolated Lactobacillus plantarum R2 Biocenol™ has proven to be preferential in aquaculture, and potential benefits in humans are expected. A simple oral dosage form should be developed to test this hypothesis by a suitable preparation method, i.e., lyophilization, allowing the bacteria to survive longer. Lyophilizates were formed from silicates (Neusilin® NS2N; US2), cellulose derivates (Avicel® PH-101), and saccharides (inulin; saccharose; modified starch® 1500). They were evaluated for their physicochemical properties (pH leachate, moisture content, water absorption, wetting time, DSC tests, densities, and flow properties); their bacterial viability was determined in conditions including relevant studies over 6 months at 4 °C and scanned under an electron microscope. Lyophilizate composed of Neusilin® NS2N and saccharose appeared to be the most advantageous in terms of viability without any significant decrease. Its physicochemical properties are also suitable for capsule encapsulation, subsequent clinical evaluation, and individualized therapy.

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.

Co-Treatment with Human Leukocyte Extract and Albendazole Stimulates Drug's Efficacy and Th1 Biased Immune Response in Mesocestoides vogae (Cestoda) Infection via Modulation of Transcription Factors, Macrophage Polarization, and Cytokine Profiles.

The model flatworm Mesocestoides vogae proliferating stage of infection elicits immunosuppression in the host. It was used to investigate the effects of human leukocyte extract (DLE) alone and in combination with anthelmintic albendazole (ABZ) on the reduction in peritoneal infection, peritoneal exudate cells (PECs), their adherent counterparts, and peritoneal exudates after the termination of therapy. Balb/c mice were infected with the larvae of M. vogae. PECs and adherent macrophages were studied via flow cytometry, mRNA transcript levels, and immunofluorescence. The cytokine levels were measured via ELISA and larvae were counted. ABZ significantly reduced larval counts (581.2 ± 65, p < 0.001), but the highest reduction was observed after combined treatment with ABZ and DLE (389.2 ± 119, p < 0.001) in comparison with the control. Compared to an infected group, the proportions of CD11b+CD19- myeloid cells with suppressive ability decreased after albendazole (ABZ) in combination with DLE, which was the most effective in the elevation of B cells and CD11b+F4/80mid/highMHCIIhigh macrophages/monocytes (22.2 ± 5.4%). Transcripts of the M2 macrophage markers (arginase 1, FIZZ-1, and Ym-1) were downregulated after DLE and combined therapy but not after ABZ, and the opposite trend was seen for iNOS. This contrasts with reduced ex vivo NO production by LPS-stimulated PECs from DLE and ABZ+DLE groups, where adherent macrophages/monocytes had elevated transcripts of the INF-γ receptor and STAT1 and reduced expression of STAT3, STAT6, and IL-10. Each therapy differentially modulated transcription profiles and concentrations of IFN-γ, TNF-α, IL-12p40, IL-6, IL-10, and TGF-ß cytokines. DLE strongly ameliorated ABZ-induced suppression of INF-γ and IL-12 and preserved downregulation for IL-4, IL-10, and TGF-ß. Epigenetic study on adherent macrophages from infected mice showed that ABZ, ABZ-sulfoxide, and DLE could interact with the mRNA of examined markers in a dose-dependent pattern. Co-administration of DLE with ABZ seemed to augment the drug's larvicidal effect via modulation of immunity. In comparison with ABZ, combined therapy was the most effective in alleviating parasite-induced Th2/Treg/STAT3/STA6 directed immunosuppression by stimulating the Th1 cytokines, M1 macrophage polarization, and activation of the IFNγ/STAT1 signaling pathway.

Beneficial Effect of Faecal Microbiota Transplantation on Mild, Moderate and Severe Dextran Sodium Sulphate-Induced Ulcerative Colitis in a Pseudo Germ-Free Animal Model.

Transplantation of faecal microbiota (FMT) is generally considered a safe therapeutic procedure with few adverse effects. The main factors that limit the spread of the use of FMT therapy for idiopathic inflammatory bowel disease (IBD) are the necessity of minimising the risk of infection and transfer of another disease. Obtaining the animal model of UC (ulcerative colitis) by exposure to DSS (dextran sodium sulphate) depends on many factors that significantly affect the result. Per os intake of DSS with water is individual for each animal and results in the development of a range of various forms of induced UC. For this reason, the aim of our study was to evaluate the modulation and regenerative effects of FMT on the clinical and histopathological responses and the changes in the bowel microenvironment in pseudo germ-free (PGF) mice of the BALB/c line subjected to chemical induction of mild, moderate and serious forms of UC. The goal was to obtain new data related to the safety and effectiveness of FMT that can contribute to its improved and optimised use. The animals with mild and moderate forms of UC subjected to FMT treatment exhibited lower severity of the disease and markedly lower damage to the colon, including reduced clinical and histological disease index and decreased inflammatory response of colon mucosa. However, FMT treatment failed to achieve the expected therapeutic effect in animals with the serious form of UC activity. The results of our study indicated a potential safety risk involving development of bacteraemia and also translocation of non-pathogenic representatives of bowel microbiota associated with FMT treatment of animals with a diagnosed serious form of UC.

Immunomodulatory Effect of Lactobacillus reuteri (Limosilactobacillus reuteri) and Its Exopolysaccharides Investigated on Epithelial Cell Line IPEC-J2 Challenged with Salmonella Typhimurium.

The gastrointestinal tract is the largest and most complex component of the immune system. Each component influences the production and regulation of cytokines secreted by intestinal epithelial cells. The aim of this study was to see how the probiotic strain Limosilactobacillus reuteri L26 and its exopolysaccharide (EPS) affect porcine intestinal-epithelial cells IPEC-J2 infected with Salmonella Typhimurium. The results revealed that Salmonella infection up-regulated all studied pro-inflammatory cytokines such as TNF-α, IL-8, IL-6 and TLR4, TLR5 signaling pathways, while decreasing the expression of TGF-ß. An immunosuppressive activity was found in EPS-treated wells, since the transcriptional levels of the studied pro-inflammatory cytokines were not increased, and the pretreatment with EPS was even able to attenuate up-regulated pro-inflammatory genes induced by Salmonella infection. However, there was a significant increase in the expression of mRNA levels of IL-8 and TNF-α in L26-treated cells, although this up-regulation was suppressed in the case of pretreatment. The immunoregulatory function of L. reuteri was also confirmed by the increased level of mRNA expression for TGF-ß, a known immunosuppressive mediator. The most relevant finding of this ex vivo study was a case of immunity modulation, where the probiotic strain L. reuteri stimulated the innate immune-cell response which displayed both anti- and pro-inflammatory activities, and modulated the expression of TLRs in the IPEC-J2 cell line. Our findings also revealed that the pretreatment of cells with either EPS or live lactobacilli prior to infection has a suppressive effect on the inflammatory response induced by Salmonella Typhimurium.

A Comparative Study of Canine Mesenchymal Stem Cells Isolated from Different Sources.

In this study, we provide comprehensive analyses of mesenchymal stem cells (MSCs) isolated from three types of canine tissues: bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and amniotic tissue (AM-MSCs). We compare their morphology, phenotype, multilineage potential and proliferation activity. The BM-MSCs and AM-MSCs showed fibroblast-like shapes against the spindle shape of the AT-MSCs. All populations showed strong osteogenic and chondrogenic potential. However, we observed phenotypic differences. The BM-MSCs and AT-MSCs revealed high expression of CD29, CD44, CD90 and CD105 positivity compared to the AM-MSCs, which showed reduced expression of all the analysed CD markers. Similarly, the isolation yield and proliferation varied depending on the source. The highest isolation yield and proliferation were detected in the population of AT-MSCs, while the AM-MSCs showed a high yield of cells, but the lowest proliferation activity, in contrast to the BM-MSCs which had the lowest isolation yield. Thus, the present data provide assumptions for obtaining a homogeneous MSC derived from all three canine tissues for possible applications in veterinary regenerative medicine, while the origin of isolated MSCs must always be taken into account.

5-Fluorouracil Treatment of CT26 Colon Cancer Is Compromised by Combined Therapy with IMMODIN.

Due to the physiological complexity of the tumour, a single drug therapeutic strategy may not be sufficient for effective treatment. Emerging evidence suggests that combination strategies may be important to achieve more efficient tumour responses. Different immunomodulators are frequently tested to reverse the situation for the purpose of improving immune response and minimizing chemotherapy side effects. Immodin (IM) represents an attractive alternative to complement chemotherapy, which can be used to enhance the immune system after disturbances resulting from the side effects of chemotherapy. In the presented study, a model of CT26 tumor-bearing mice was used to investigate the effect of single IM or its combination with 5-fluorouracil (5-FU) on colon cancer cells. Our results highlight that the beneficial role of IM claimed in previous studies cannot be generalised to all chemotherapeutic drugs, as 5-FU toxicity was not increased. On the contrary, the chemotherapeutic anti-cancer efficacy of 5-FU was greatly compromised when combined with IM. Indeed, the combined treatment was significantly less effective regarding the tumour growth and animal survival, most probably due to the increased number of tumour-associated macrophages, and increased 5-FU cytotoxic effect related to kidneys and the liver.

The adverse effects of synthetic acaricide tau-fluvalinate (tech.) on winter adult honey bees.

Currently several pyrethroids (e.g., flumethrin and tau-fluvalinate) are used in apiculture worldwide as acaricides/miticides. The long half-lives of pyrethroids in synthetic acaricides applied to hive matrices, may adversely affect the health of bee colony. The potentially adverse effects of synthetic acaricide/miticide tau-fluvalinate (tech.) on winter honeybees were assessed in this study (OECD 245 2017). No dose-dependent mortality in in vitro reared winter honeybees was observed after chronic oral 10-day exposure to syrup (50% w/v) spiked with a maximum concentration of 750 µg a.i./kg diet and its 1/10 concentration. The No Observed Effect Concentration is ≥ 750 µg a.i./kg diet. Tau-fluvalinate testing for the sublethal effects on bee immune system showed up-regulated gene expression encoding abaecin, lysozyme, and defensin in both tested groups, however the expression of hymenoptaecin gene was reduced. Moreover, tau-fluvalinate significantly induced levels of DNA damage in exposed bees, which can result in adverse genotoxic effect.

Surviving of production probiotic strains in a selected application form.

Research in probiotics for aquaculture is at an early stage of development and much work is still needed. Lactiplantibacilli belong to the microorganisms most frequently used to prepare the probiotics. The available information is inconclusive, since few experiments with sufficiently robust design have been conducted to permit critical evaluation. The development of probiotics applicable to commercial use in aquaculture is a multistep and multidisciplinary process requiring both empirical and fundamental research, full-scale trials, and an economic assessment of its use. The aim of the study was to prepare a probiotic aquafeed via excipients and subsequently to observe the survival of probiotic bacterial cells in the feed during the nine months storage period at a refrigerator (4 °C) or room temperature (22 °C). The strain Lactobacillus plantarum R2 Biocenol (CCM 8674) (according to the new taxonomy Lactiplantibacillus plantarum), potentially usable as a probiotic in aquaculture, was administered to prepare the aquafeed. Better survival of probiotic bacterial cells was recorded in a samples of pellets A (Aquatex 41 HMD) compared to the samples of probiotic pellets B (Inicio 918-2). Since oxidation of fatty acids in feed affects the nutritional quality of individual feed components, we assume that higher amounts of oil in feed B negatively affected the survival of probiotic bacterial cells. The highest numbers of viable probiotic bacteria cells were recorded at 4 °C storage of probiotic feed samples. The number of lactiplantibacilli dropped from 7.30 log10CFU . g-1 to 5.57 log10CFU . g-1 after the nine months storage period of feed samples A at 4 °C. Temperature is considered as a critical factor influencing probiotic viability and survival during storage period.