Lactobacillus casei CRL431 modulates hemostatic activation induced by protein malnourishment and pneumococcal respiratory infection.

Previously, we demonstrated that Lactobacillus casei CRL431, a well-known immunomodulatory bacterium, beneficially regulates coagulation activation, fibrin formation in lung, and the pro-inflammatory state induced by protein malnourishment and pneumococcal infection. In this study, we deepen in the understanding of the mechanisms involved in the immunoregulatory activity of L. casei CRL431 during a nutritional repletion process by evaluating (a) platelet and endothelial activation, (b) tissue factor (TF) expression, and (c) protease-activated receptor (PAR) activation in an experimental bacterial respiratory infection model in malnourished mice. Our findings demonstrate for the first time that the repletion diet supplemented with L. casei CRL431 was effective to normalize platelet counts in blood, modulate platelet activation and their recruitment into the lung, and regulate local and systemic TF expression and endothelial activation, which were affected by malnourishment. Streptococcus pneumoniae challenge induced local and systemic increase of platelet counts, PARs activation, P-selectin and TF expression, as well as endothelial activation in both well-nourished and malnourished mice. Malnourished animals evidenced the highest alterations of the parameters evaluated while the mice fed with the probiotic bacterium had similar behavior to normal controls but with lower PAR activation in lung. These results demonstrate that supplementation of repletion diet with L. casei CRL431 is effective to modulate alterations induced by malnourishment and pneumococcal infection, restraining coagulation activation, the inflammatory process, and lung damage. These observations contribute to set the basis for the application of probiotic functional foods to modulate the inflammation-hemostasis interactions altered by malnourishment or bacterial respiratory infections. KEY POINTS: • Pneumococcal infection increases pro-coagulant state induced by protein malnourishment. • Repletion with L. casei CRL431 modulates platelet, TF, and endothelial activation. • L. casei CRL431 improves immune-coagulative response in protein malnourishment.

CCR8 leads to eosinophil migration and regulates neutrophil migration in murine allergic enteritis.

Allergic enteritis (AE) is a gastrointestinal form of food allergy. This study aimed to elucidate cellular and molecular mechanisms of AE using a murine model. To induce AE, BALB/c wild type (WT) mice received intraperitoneal sensitization with ovalbumin (an egg white allergen) plus ALUM and feeding an egg white (EW) diet. Microarray analysis showed enhanced gene expression of CC chemokine receptor (CCR) 8 and its ligand, chemokine CC motif ligand (CCL) 1 in the inflamed jejunum. Histological and FACS analysis showed that CCR8 knock out (KO) mice exhibited slightly less inflammatory features, reduced eosinophil accumulation but accelerated neutrophil accumulation in the jejunums, when compared to WT mice. The concentrations of an eosinophil chemoattractant CCL11 (eotaxin-1), but not of IL-5, were reduced in intestinal homogenates of CCR8KO mice, suggesting an indirect involvement of CCR8 in eosinophil accumulation in AE sites by inducing CCL11 expression. The potential of CCR8 antagonists to treat allergic asthma has been discussed. However, our results suggest that CCR8 blockade may promote neutrophil accumulation in the inflamed intestinal tissues, and not be a suitable therapeutic target for AE, despite the potential to reduce eosinophil accumulation. This study advances our knowledge to establish effective anti-inflammatory strategies in AE treatment.

Characterization of folate production and probiotic potential of Streptococcus gallolyticus subsp. macedonicus CRL415.

Mandatory fortification of foods with folic acid is being questioned by many scientists principally because of the potential adverse secondary effects associated with their excessive consumption. It has been shown that selected strains of lactic acid bacteria (LAB) are able to produce natural forms of folate and these could be included in foods to prevent deficiencies without causing adverse effects. The aim of this study was to evaluate folate production and fol gene expression by Streptococcus gallolyticus subsp. macedonicus (S. macedonicus) CRL415 under different growth conditions in vitro and to assess its potential probiotic application. Results showed that glucose as the principal carbon source, and incubation at 42 °C under controlled pH conditions (6.0) increased folate production, fol gene expression, and growth of S. macedonicus CRL415. This strain was able to produce elevated folate concentrations during milk fermentation without the need of prolonged incubation times and was able to resist conditions simulating the gastrointestinal tract. In addition, S. macedonicus was susceptible to all required antibiotics, and had a good adhesion level to intestinal cells in vitro, making it a promising candidate for biotechnological application as functional starter cultures in the dairy industry.

Nasal immunization with recombinant chimeric pneumococcal protein and cell wall from immunobiotic bacteria improve resistance of infant mice to Streptococcus pneumoniae infection.

Respiratory tract infections and invasive disease caused by Streptococcus pneumoniae in high-risk groups are a major global health problem. Available human vaccines have reduced immunogenicity and low immunological memory in these populations, as well as high cost as a public health strategy in poor communities. In addition, no single pneumococcal protein antigen has been able to elicit protection comparable to that achieved using protein-polysaccharide conjugate vaccines. In this context, chimeric pneumococcal proteins raise as potential good vaccine candidates because of their simplicity of production and reduced cost. The aim of this work was to study whether the nasal immunization of infant mice with the recombinant chimeric pneumococcal protein (PSFP) was able to improve resistance to S. pneumoniae, and whether the immunomodulatory strain Lactobacillus rhamnosus CRL1505 or its cell wall (CW1505) could be used as effective mucosal adjuvants. Our results showed that the nasal immunization with PSPF improved pneumococcal-specific IgA and IgG levels in broncho-alveolar lavage (BAL), reduced lung bacterial counts, and avoided dissemination of pneumococci into the blood. Of interest, immunization with PSPF elicited cross-protective immunity against different pneumococcal serotypes. It was also observed that the nasal immunization of infant mice with PSPF+CW1505 significantly increased the production of pneumococcal-specific IgA and IgG in BAL, as well as IgM and IgG in serum when compared with PSPF alone. PSPF+CW1505 immunization also improved the reduction of pneumococcal lung colonization and its dissemination in to the bloodstream when compared to PSPF alone. Our results suggest that immunization with PSPF together with the cell wall of the immunomodulatory strain L. rhamnosus CRL1505 as a mucosal adjuvant could be an interesting alternative to improve protection against pneumococcal infection in children.

Targeting of Immune Cells by Dual TLR2/7 Ligands Suppresses Features of Allergic Th2 Immune Responses in Mice.

BACKGROUND: TLR ligands can promote Th1-biased immune responses, mimicking potent stimuli of viruses and bacteria. AIM: To investigate the adjuvant properties of dual TLR2/7 ligands compared to those of the mixture of both single ligands. METHODS: Dual TLR2/7 ligands: CL401, CL413, and CL531, including CL264 (TLR7-ligand) and Pam2CysK4 (TLR2-ligand), were used. Immune-modulatory capacity of the dual ligands with the individual ligands alone or as a mixture in mouse BMmDCs, BMmDC:TC cocultures, or BMCMCs was compared and assessed in naïve mice and in a mouse model of OVA-induced intestinal allergy. RESULTS: CL413 and CL531 induced BMmDC-derived IL-10 secretion, suppressed rOVA-induced IL-5 secretion from OVA-specific DO11.10 CD4+ TCs, and induced proinflammatory cytokine secretion in vivo. In contrast, CL401 induced considerably less IL-10 secretion and led to IL-17A production in BMmDC:TC cocultures, but not BMCMC IL-6 secretion, or IL-6 or TNF-α production in vivo. No immune-modulating effects were observed with single ligands. All dual TLR2/7 ligands suppressed DNP-induced IgE-and-Ag-specific mast cell degranulation. Compared to vaccination with OVA, vaccination with the mixture CL531 and OVA, significantly suppressed OVA-specific IgE production in the intestinal allergy model. CONCLUSIONS: Based on beneficial immune-modulating properties, CL413 and CL531 may have utility as potential adjuvants for allergy treatment.

Factors stimulating riboflavin produced by Lactobacillus plantarum CRL 725 grown in a semi-defined medium.

Riboflavin (vitamin B2 ) is one of the B-group water-soluble vitamins and is essential for energy metabolism of the cell. The aim of this study was to determine factors that affect riboflavin production by Lactobacillus (L.) plantarum CRL 725 grown in a semi defined medium and evaluate the expression of its rib genes. The factors found to enhance riboflavin production in this medium were incubation at 30 °C, and the addition of specific medium constituents, such as casamino acids (10 g L-1 ), guanosine (0.04 g L-1 ), and sucrose as carbon source (20 g L-1 ). In these conditions, higher riboflavin concentrations were directly associated with significant increases in the expression of ribA, ribB, and ribC genes. The culture conditions defined in this work and its application to a roseoflavin resistant mutant of L. plantarum allowed for a sixfold increase in riboflavin concentrations in our semi-defined medium which were also significantly higher than those obtained previously using the same strain to ferment soymilk. These conditions should thus be evaluated to increase vitamin production in fermented foods.

Immunoregulatory Effects Triggered by Lactic Acid Bacteria Exopolysaccharides: New Insights into Molecular Interactions with Host Cells.

Researchers have demonstrated that lactic acid bacteria (LAB) with immunomodulatory capabilities (immunobiotics) exert their beneficial effects through several molecules, including cell wall, peptidoglycan, and exopolysaccharides (EPS), that are able to interact with specific host cell receptors. EPS from LAB show a wide heterogeneity in its composition, meaning that biological properties depend on the strain and. therefore, only a part of the mechanism of action has been elucidated for these molecules. In this review, we summarize the current knowledge of the health-promoting actions of EPS from LAB with special focus on their immunoregulatory actions. In addition, we describe our studies using porcine intestinal epithelial cells (PIE cells) as a model to evaluate the molecular interactions of EPS from two immunobiotic LAB strains and the host cells. Our studies showed that EPS from immunobiotic LAB have anti-inflammatory capacities in PIE cells since they are able to reduce the production of inflammatory cytokines in cells challenged with the Toll-like receptor (TLR)-4-agonist lipopolysaccharide. The effects of EPS were dependent on TLR2, TLR4, and negative regulators of TLR signaling. We also reported that the radioprotective 105 (RP105)/MD1 complex, a member of the TLR family, is partially involved in the immunoregulatory effects of the EPS from LAB. Our work described, for the first time, that LAB and their EPS reduce inflammation in intestinal epithelial cells in a RP105/MD1-dependent manner. A continuing challenge for the future is to reveal more effector-receptor relationships in immunobiotic-host interactions that contribute to the beneficial effects of these bacteria on mucosal immune homeostasis. A detailed molecular understanding should lead to a more rational use of immunobiotics in general, and their EPS in particular, as efficient prevention and therapies for specific immune-related disorders in humans and animals.

Ancestral Andean grain quinoa as source of lactic acid bacteria capable to degrade phytate and produce B-group vitamins.

The lactic acid bacteria (LAB) microbiota of quinoa grains (QG) and spontaneous sourdough (QSS) was evaluated. Different strains of Lactobacillus (L.) plantarum (7), L. rhamnosus (5), L. sakei (1), Pediococcus (Ped.) pentosaceus (9), Leuconostoc (Leuc.) mesenteroides (1), Enterococcus (E.) casseliflavus (2), E. mundtii (3), E. hirae (1), E. gallinarum (12), Enterococcus sp. (1), and E. hermanniensis (2) were isolated, identified and characterized. Only four strains isolated from QSS and eight strains isolated from QG showed amylolytic activity. L. plantarum CRL 1973 and CRL 1970, L. rhamnosus CRL 1972 and L. sakei CRL 1978 produced elevated concentrations of folate with strain CRL 1973 producing the highest concentration (143±6ng/ml). L. rhamnosus, isolated from QSS, was the LAB species that produced the most elevated concentrations of total riboflavin (>270ng/ml) with strain CRL 1963 producing the highest amounts (360±10ng/ml). Phytase activity, evaluated in forty-four LAB strains from quinoa, was predominantly detected in L. rhamnosus and Enterococci strains with the highest activities observed in E. mundtii CRL 2007 (957±25U/ml) followed by E. casseliflavus CRL 1988 (684±38U/ml), Leuc. mesenteroides CRL 2012 (617±38U/ml) and L. rhamnosus CRL 1983 (606±79U/ml). In conclusion, this study shows that a diverse LAB microbiota is present in quinoa with important properties; these microorganisms could be used as potential starter cultures to increase the nutritional and functional properties of Andean grains based foods.

Draft Genome Sequence of Lactobacillus delbrueckii subsp. bulgaricus CRL871, a Folate-Producing Strain Isolated from a Northwestern Argentinian Yogurt.

Lactobacillus delbrueckii subsp. bulgaricus CRL871 is the first strain of L. delbrueckii subsp. bulgaricus reported as a folate-producing strain. We report the draft genome sequence of L. delbrueckii subsp. bulgaricus CRL871 (2,063,981 bp, G+C content of 49.1%). This strain is of great biotechnological importance to the dairy industry because it constitutes an alternative to folic acid fortification.

Relationships between the genome and some phenotypical properties of Lactobacillus fermentum CECT 5716, a probiotic strain isolated from human milk.

Lactobacillus fermentum CECT 5716, isolated from human milk, has immunomodulatory, anti-inflammatory, and anti-infectious properties, as revealed by several in vitro and in vivo assays, which suggests a strong potential as a probiotic strain. In this work, some phenotypic properties of L. fermentum CECT 5716 were evaluated, and the genetic basis for the obtained results was searched for in the strain genome. L. fermentum CECT 5716 does not contain plasmids and showed neither bacteriocin nor biogenic amine biosynthesis ability but was able to produce organic acids, glutathione, riboflavin, and folates and to moderately stimulate the maturation of mouse dendritic cells. No prophages could be induced, and the strain was sensitive to all antibiotics proposed by European Food Safety Authority (EFSA) standards, while no transmissible genes potentially involved in antibiotic resistance were detected in its genome. Globally, there was an agreement between the phenotype properties of L. fermentum CECT 5716 and the genetic information contained in its genome.

Applicability of a Lactobacillus amylovorus strain as co-culture for natural folate bio-enrichment of fermented milk.

The ability of 55 strains from different Lactobacillus species to produce folate was investigated. In order to evaluate folic acid productivity, lactobacilli were cultivated in the folate-free culture medium (FACM). Most of the tested strains needed folate for growth. The production and the extent of vitamin accumulation were distinctive features of individual strains. Lactobacillus amylovorus CRL887 was selected for further studies because of its ability to produce significantly higher concentrations of vitamin (81.2 ± 5.4 µg/L). The safety of this newly identified folate producing strain was evaluated through healthy experimental mice. No bacterial translocation was detected in liver and spleen after consumption of CRL887 during 7 days and no undesirable side effects were observed in the animals that received this strain. This strain in co-culture with previously selected folate producing starter cultures (Lactobacillus bulgaricus CRL871, and Streptococcus thermophilus CRL803 and CRL415) yielded a yogurt containing high folate concentrations (263.1 ± 2.4 µg/L); a single portion of which would provide 15% of the recommended dietary allowance. This is the first report where a Lactobacillus amylovorus strain was successfully used as co-culture for natural folate bio-enrichment of fermented milk.

Lactobacillus rhamnosus CRL1505 beneficially modulates the immuno-coagulative response after pneumococcal infection in immunocompromised malnourished mice.

This work evaluated the effect of orally or nasally administered Lactobacillus rhamnosus CRL1505 on the resistance of immunocompromised protein-malnourished mice to pneumococcal infection. In particular, we aimed to gain knowledge of the mechanism involved in the immunomodulatory effect of L. rhamnosus CRL1505 in malnourished hosts by evaluating its impact on the immuno-coagulative response. Malnutrition significantly increased lung tissue damage caused by Streptococcus pneumoniae infection. Lung damage was associated with a deregulated activation of coagulation and an altered inflammatory response. Pneumococcal colonization of lung and bacteremia were significantly reduced (p < 0.05) in malnourished mice receiving the CRL1505 strain. Moreover, mice repleted with supplemental L. rhamnosus CRL1505 showed the least alteration of the alveolar-capillary barrier and cell damage in lungs after the infectious challenge, especially when the CRL1505 strain was administered by nasal route. Besides, mice treated with L. rhamnosus CRL1505 showed an improved respiratory innate immune response and a lower activation of coagulation. The results of this work indicate that L. rhamnosus CRL1505 is able to beneficially modulate the inflammation-coagulation interaction after respiratory infections in malnourished hosts.

Modulation of the immuno-coagulative response in a pneumococcal infection in malnourished mice nasally treated with Lactobacillus casei.

We studied the systemic effects of the intranasal administration of Lactobacillus casei on the immuno-coagulative response in pneumoccocal infection in immunocompromised mice. Weaned mice consumed a protein-free diet (PFD) for 21 days and were therefore malnourished. Malnourished mice were fed a balanced conventional diet (BCD) for 7 days (BCD group) or a BCD for 7 days with nasal administration of viable L. casei on days 6 and 7 (BCD+LcN group). The malnourished control mice (MNC) received a PFD, whereas the well-nourished control mice (WNC) continually consumed a BCD. At the end of the treatment period, the mice were infected with Streptococcus pneumoniae. At different times after infection, we analysed the following parameters: global coagulation system, activation of coagulation, coagulation inhibitors, platelet count, leukocyte count and myeloperoxidase (MPO) activity, total proteins, albumin and acute phase proteins (APPs). The MNC group showed greater impairment in the coagulation tests and an increase in the positive APPs. These parameters were normalized by the L. casei treatment. However, the number of leukocytes, decreased by malnutrition, was improved only by the administration of L. casei. After infection, the BCD+LcN group showed similar results to those of the WNC group for most of the haemostatic parameters. The BCD+LcN group did not show significant variations in the prothrombin time or in the level of anticoagulant protein C, but showed higher levels of fibrinogen, platelets, albumin, leukocytes and MPO activity compared with the different experimental groups. The intranasal administration of L. casei was effective in modulating the pro-inflammatory aspects of coagulation without affecting coagulation itself.

Production of natural folates by lactic acid bacteria starter cultures isolated from artisanal Argentinean yogurts.

Folate is a B-group vitamin that cannot be synthesized by humans and must be obtained exogenously. Although some species of lactic acid bacteria (LAB) can produce folates, little is known about the production of this vitamin by yogurt starter cultures. Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains were isolated from artisanal Argentinean yogurts and were grown in folate-free culture medium (FACM) and nonfat milk after which intracellular and extracellular folate production were evaluated. From the initial 92 isolated LAB strains, 4 L. delbrueckii subsp. bulgaricus and 32 S. thermophilus were able to grow in the absence of folate. Lactobacillus delbrueckii subsp. bulgaricus CRL 863 and S. thermophilus CRL 415 and CRL 803 produced the highest extracellular folate levels (from 22.3 to 135 µg/L) in FACM. In nonfat milk, these strains were able to increase the initial folate concentrations by almost 190%. This is the first report where native strains of L. delbrueckii subsp. bulgaricus were shown to produce natural folate. The LAB strains identified in this study could be used in developing novel fermented products bio-enriched in natural folates that could in turn be used as an alternative to fortification with the controversial synthetic chemical folic acid.

Inflammation-hemostasis relationship in infected malnourished mice: modulatory effect of Lactobacillus casei CRL 431.

OBJECTIVE AND DESIGN: The coagulation system is considered part of the defense machinery, but its excessive activation can lead to additional damage. We studied the effects of oral administration of Lactobacillus casei CRL 431--a probiotic bacterium--on the activation of coagulation and the relationship with inflammatory parameters during a respiratory infection in malnourished mice. MATERIALS AND METHODS: Malnourished Swiss albino mice were nourished with a balanced commercial diet (BCD) for 7 days or BCD with L. casei for the last 2 days (BCD + Lc). BCD, BCD + Lc, malnourished (MNC) and well-nourished controls (WNC) were infected with Streptococcus pneumoniae. Blood and bronchoalveolar lavage samples were obtained at different times post-infection. RESULTS AND CONCLUSIONS: Malnutrition altered most of the evaluated parameters before and after infection. The repletion diet with supplemental L. casei was the most effective in limiting coagulation activation and normalizing coagulation inhibition mechanisms. These findings will help develop further strategies to reduce the damaging effects of clotting and enhance its beneficial contribution to immune reactions.

Coagulation activation in an experimental pneumonia model in malnourished mice.

Malnutrition induces a decrease in immunity that affects the ability of the organism to deal with an infectious challenge. The clotting system is considered a branch of immunity and its activation is important in the pathogenesis of an infectious disease. This work was conducted to determine coagulation modifications in malnourished hosts before and during infection. Weaned mice were malnourished via a protein-free diet. Well-nourished control mice (WNC) consumed a balanced conventional diet. Malnourished mice (MN) and WNC were challenged intranasally with Streptococcus pneumoniae. Blood, bronchoalveolar lavages (BAL), and lung samples were taken at different times post infection. The results were that MN showed altered hemostatic tests and fibrin(ogen) deposits in the lung. Thus, an increase in thrombin-antithrombin complexes (TATc) in plasma and BAL was observed. In the MN group, infection induced a rise in TATc in plasma and BAL and increased plasma fibrinogen and fibrin(ogen) deposits in the lung. A decrease in activated protein C and antithrombin in BAL and an early decrease followed by an increase in plasma Factor VIII were also observed. Thus, malnourishment induced a procoagulant state increased by infection. This is the first work that presents results of an exhaustive study of coagulation in malnourished hosts before and during an infection.