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The conditions of aquatic environments have a great influence on the microbiota of several animals, many of which are a potential source of microorganisms of biotechnological interest. In this study, bacterial strains isolated from aquatic environments were bioprospected to determine their probiotic profile and antimicrobial effect against fish and food pathogens. Two isolates, identified via 16S rRNA sequencing as Lactococcus lactis (L1 and L2) and one as Enterococcus faecium 135 (EF), produced a bacteriocin-like antimicrobial substance (BLIS), active against Listeria monocytogenes, Salmonella Choleraesuis and Salmonella Typhimurium. Antimicrobial activity of BLIS was reduced when exposed to high temperatures and proteolytic enzymes (trypsin, pepsin, papain and pancreatin). All strains were sensitive to 7 types of antibiotics (vancomycin, clindamycin, streptomycin, gentamicin, chloramphenicol, rifampicin and ampicillin), exhibited a high rate of adherence to Caco-2 cells and expressed no hemolysin and gelatinase virulence factors. EF showed some resistance at pH 2.5 and 3.0, and L2/EF showed higher resistance to the action of bile salts. Finally, the presence of bacteriocin genes encoding for proteins, including Nisin (L1 and L2), Enterocin A, B, P, and Mundticin KS (EF) was detected. The molecular and physiological evidence suggests that the bacterial isolates in this study could be used as natural antimicrobial agents and may be considered safe for probiotic application.

The demand for animal protein for human consumption has been risen exponentially. Modern animal production practices are associated with the regular use of antibiotics, potentially increasing the emerging multi-resistant bacteria, which may have a negative impact on public health. In poultry production, substances capable of maximizing the animals’ performance and displaying an antimicrobial activity against pathogens are very well desirable features. Probiotic can be an efficient solution for such a task. In the present work, lactic acid bacteria (LAB) were isolated from chicken cecum and screened for their antagonistic effect towards many pathogens. Their capacity of producing the B-complex vitamins folate and riboflavin were also evaluated. From 314 isolates, three (C43, C175 and C195) produced Bacteriocin-Like Inhibitory Substances (BLIS) against Staphylococcus aureus (inhibition zones of 18.9, 21.5, 19.5 mm, respectively) and also inhibited the growth of Salmonella Heidelberg. The isolate C43 was identified as Enterococcus faecium , while C173 and C195 were both identified as Lactococcus lactis subsp. lactis . Moreover, the isolates L. lactis subsp. lactis strains C173 and C195 demonstrated high potential to be used as probiotic in poultry feed, in addition to their advantage of producing folate (58.0 and 595.5 ng/mL, respectively) and riboflavin (223.3 and 175.0 ng/mL, respectively).

Glutamine (GLN) is the most abundant free amino acid in the body, and is considered as a conditionally essential amino acid under stress conditions, acting as an important modulator of the immune response. We here investigated the role of exogenous GLN treatment on leukocyte migration after the onset of endotoxemia and the intracellular mechanisms of GLN actions on neutrophils. Two in vivo models of endotoxemia caused by lipopolysaccharide of Escherichia coli (LPS) injection were carried out in male outbred Balb/C mice 2–3 months old, as follow: (1) LPS (50 μg/kg) was intravenously injected 1 h prior to intravenous injection of GLN (0.75 mg/kg) and samples were collected 2 h later to investigate the role of GLN on the acute lung inflammation; (2) LPS (1 mg/kg) was intraperitoneally injected 1 h prior to intravenous injection of GLN (0.75 mg/kg) and samples were collected 18 h later to measure the effects of GLN on local and later phases of inflammation in the peritoneum. Results showed that GLN administration reduced the number of neutrophils in the inflamed lungs, partially recovery of the reduced number of leukocytes in the blood; reduced adhesion molecules on lung endothelium and on circulating neutrophils. Moreover, GLN treatment diminished the number of neutrophils, levels of chemotactic cytokine CXCL2 in the inflamed peritoneum, and neutrophils collected from the peritoneum of GLN-treated mice presented lower levels of Rho, Rac, and JNK. Together, our data show novel mechanisms involved in the actions of GLN on neutrophils migration.

The granulocyte colony-stimulating factor (G-CSF) is a hematopoietic cytokine that has important clinical applications for treating neutropenia. Nartograstim is a recombinant variant of human G-CSF. Nartograstim has been produced in Escherichia coli as inclusion bodies (IB) and presents higher stability and biological activity than the wild type of human G-CSF because of its mutations. We developed a production process of nartograstim in a 10-L bioreactor using auto-induction or chemically defined medium. After cell lysis, centrifugation, IB washing, and IB solubilization, the following three refolding methods were evaluated: diafiltration, dialysis, and direct dilution in two refolding buffers. Western blot and SDS-PAGE confirmed the identity of 18.8-kDa bands as nartograstim in both cultures. The auto-induction medium produced 1.17 g/L and chemically defined medium produced 0.95 g/L. The dilution method yielded the highest percentage of refolding (99%). After refolding, many contaminant proteins precipitated during pH adjustment to 5.2, increasing purity from 50 to 78%. After applying the supernatant to cation exchange chromatography (CEC), nartograstim recovery was low and the purity was 87%. However, when the refolding solution was applied to anion exchange chromatography followed by CEC, 91%–98% purity and 2.2% recovery were obtained. The purification process described in this work can be used to obtain nartograstim with high purity, structural integrity, and the expected biological activity.Key points• Few papers report the final recovery of the purification process from inclusion bodies.• The process developed led to high purity and reasonable recovery compared to literature.• Nartograstim biological activity was demonstrated in mice using a neutropenia model.

Ionizing radiation (IR) is an important medical tool. Despite the effects associated with high-dose radiation during or after treatment, as well as in accidental exposures, the direct or indirect effect of low-dose IR in cells remain poorly documented. IR can affect the tissue microenvironment, including mesenchymal stem cells (MSCs), which have high regenerative and immunomodulatory capacities. This study aimed to investigate the effect of low-dose IR in association with the inflammatory stimuli of TNF-α on the immunomodulatory capacity of MSCs. MSCs were irradiated with a low-dose IR, stimulated with TNF-α, and cultivated in a bystander system with murine spleen cells. The results showed that TNF-R1 is expressed in MSCs and is not affected, even in irradiated MSCs. However, irradiated MSCs produced reduced amounts of IL-6 and increased amounts of IL-10. The levels of PGE2 and NO• in MSCs were also increased when stimulated with TNF-α. Furthermore, conditioned media from irradiated MSCs reduced the proliferation of bystander lymphocytes and reduced the metabolic activity of macrophages. In addition, conditioned media from irradiated MSCs modulated the profile of cytokines in bystander spleen cells (lymphocytes and macrophages), reducing inflammatory and increasing anti-inflammatory cytokines, also increasing Treg cells. In conclusion, low-dose IR in association with an inflammatory stimulus affects the immunomodulatory properties of MSCs. In this way, the immunosuppressive capability of MSCs can be explored for several disease treatments where IR usually part of the context of the treatment. However, a complete understanding of the mechanisms underlying these interactions need further investigation.

•Protein malnutrition leads to leukopenia not reversible by granulocyte-colony stimulating factor.•Protein malnutrition impairs granule-monocytic progenitors production.•Protein malnutrition decreases granulocyte-colony stimulating factor receptor in granule-monocytic progenitors. It is well known that protein malnutrition (PM) states can affect hematopoiesis, leading to severe leukopenia and reduced number of granulocytes, which act as the first line of defense, and are important to the innate immune response. The aim of this study was to elucidate some of the mechanisms involved in the impairment of granulopoiesis in PM. Male C57BL/6 mice were submitted to PM with a low-protein diet containing 2% protein. Control mice were fed a 12% protein-containing diet. Bone marrow histology and the percentage of granulocytic progenitors were evaluated after in vivo granulocyte-colony stimulating factor (G-CSF) stimulus. Cell proliferation, STAT3 signaling, and the expression of G-CSF receptor were evaluated in hematopoietic progenitor cells. Malnourished animals presented with leukopenia associated with reduced number of granulocytes and reduced percentage of granulocytic progenitors; however, no differences were observed in the regulatory granulopoietic cytokine G-CSF. Additionally, the malnourished group presented with impaired response to in vivo G-CSF stimulus compared with control animals. PM was implicated in decreased ability of c-Kit+ cells to differentiate into myeloid progenitor cells and downregulated STAT3 signaling. Furthermore, the malnourished group exhibited reduced expression of G-CSF receptor on granule-monocytic progenitors. This reduced expression was not completely reversible with G-CSF treatment. This study implies that PM promotes intrinsic alterations to hematopoietic precursors, which result in hematologic changes, mainly neutropenia, observed in peripheral blood in PM states.

Branched-chain amino acids (BCAA) are essential for maintaining intestinal mucosal integrity. However, only a few studies have explored the role of BCAA in the modulation of intestinal inflammation. In this study, we investigated in vitro effects of BCAA on the inflammatory response induced by lipopolysaccharide (LPS) (1 µg/mL) in Caco-2 cells. Caco-2 cells were assigned to six groups: control without BCAA (CTL0), normal BCAA (CTL; 0.8 mM leucine, 0.8 mM isoleucine, and 0.8 mM valine); leucine (LEU; 2 mM leucine), isoleucine (ISO; 2 mM isoleucine), valine (VAL; 2 mM valine), and high BCAA (LIV; 2 mM leucine, 2 mM isoleucine, and 2 mM valine). BCAA was added to the culture medium 24 h before LPS stimulation. Our results indicated that BCAA supplementation did not impair cell viability. The amino acids leucine and isoleucine attenuated the synthesis of IL-8 and JNK and NF-kB phosphorylation induced by LPS. Furthermore, neither BCAA supplementation nor LPS treatment modulated the activity of glutathione peroxidase or the intracellular reduced glutathione/oxidized glutathione ratio. Therefore, leucine and isoleucine exert anti-inflammatory effects in Caco-2 cells exposed to LPS by modulating JNK and NF-kB phosphorylation and IL-8 production. Further in vivo studies are required to validate these findings and gather valuable information for potential therapeutic or dietary interventions.

PurposeDietary protein deficiency is common in the elderly, compromising hematopoiesis and the immune response, and may cause a greater susceptibility to infections. Mesenchymal stem cells (MSCs) have immunomodulatory properties and are essential to hematopoiesis. Therefore, this study aimed to investigate, in an aging model subjected to malnutrition due a reduced protein intake, aspects related to the immunomodulatory capacity of MSCs.MethodsMale C57BL/6 mice from young and elderly groups were fed with normoproteic or hypoproteic diets (12% and 2% of protein, respectively) and nutritional, biochemical and hematological parameters were evaluated. MSCs from bone marrow were isolated, characterized and their secretory parameters evaluated, along with gene expression. Additionally, the effects of aging and protein malnutrition on MSC immunomodulatory properties were assessed.ResultsMalnourished mice lost weight and demonstrated anemia, leukopenia, and bone marrow hypoplasia. MSCs from elderly animals from both groups showed reduced CD73 expression and higher senescence rate; also, the malnourished state affected CD73 expression in young animals. The production of IL-1β and IL-6 by MSCs was affected by aging and malnutrition, but the IL-10 production not. Aging also increased the expression of NFκB, reducing the expression of STAT-3. However, MSCs from malnourished groups, regardless of age, showed decreased TGF-β and PGE2 production. Evaluation of the immunomodulatory capacity of MSCs revealed that aging and malnutrition affected, mainly in lymphocytes, the production of IFN-γ and IL-10.ConclusionAging and reduced protein intake are factors that, alone or together, influence the immunomodulatory properties of MSCs and provide basic knowledge that can be further investigated to explore whether MSCs’ therapeutic potential may be affected.

Background and objectives: BCAAs have a unique place in protein synthesis stimulation, serving as energetic substrates for synthesis of intracellular signaling molecules. However, in inflammation and intense catabolic state, such as sepsis and cancer, the supply of these amino acids must be increased to preserve individual's immunocompetence. In vitro studies have demonstrated that immune cells use isoleucine, valine and especially leucine as substrates for production of cytokines and antibodies, besides having a modulating effect on cellular functions, as in nitric oxide synthesis. Also, studies have found that the absence of any of these amino acids in culture medium results in complete suppression of protein synthesis and cell proliferation. The aim of this study was to investigated the effect of BCAAs supplementation on cell viability and nitric oxide production in LPS-stimulated RAW 264.7 macrophages. Methods: Cells were cultured in DMEM (with 2 mM glutamine, 10% FBS and 1% penicillin-streptomycin). After reaching 90% confluence, cell cultures were distributed into five groups: CTL - without supplementation with BCAA; LEU - supplemented with leucine (2 mmol/L); ISO - supplemented with isoleucine (2 mmol L); VAL - supplemented with valine (2 mmol/L) and LIV - supplemented with leucine (2 mmol/L), isoleucine (2 mmol/L) and valine (2 mmol/L). The inflammatory state was induced by LPS (1 μg/ml) for 24 hours and then cell cultures were supplemented with the respective amino acids for another 24 hours. The cell viability assay was performed by MTT test and the indirect nitric oxide was measured by Griess reaction. Results: The LIV group presented higher percentage of cell viability in comparison to CTL group (p <0.05). There was no statistical difference between other groups related to macrophage viability, evidencing that the sinergy among BCAAs is the responsible for increasing cell viability. Regarding production of nitric oxide, there was no significant difference between groups. Conclusions: Supplementation with BCAAs association shows to be more effective in increasing cellular viability of LPS-stimulated cells compared to supplementation with isolated BCAAs. In addition, there was no difference in nitric oxide synthesis by macrophages in any of the supplemented groups.