Genetic aspects of immunoglobulins and cyclophilin A in milk as potential indicators of mastitis resistance in Holstein cows.

Mastitis is one of the most frequent and costly diseases affecting dairy cattle. Natural antibodies (immunoglobulins) and cyclophilin A (CyPA), the most abundant member of the family of peptidyl prolyl cis/trans isomerases, in milk may serve as indicators of mastitis resistance in dairy cattle. However, genetic information for CyPA is not available, and knowledge on the genetic and nongenetic relationships between these immune-related traits and somatic cell score (SCS) and milk yield in dairy cattle is sparse. Therefore, we aimed to comprehensively evaluate whether immune-related traits consisting of 5 Ig classes (IgG, IgG1, IgG2, IgA, and IgM) and CyPA in the test-day milk of Holstein cows can be used as genetic indicators of mastitis resistance by evaluating the genetic and nongenetic relationships with SCS in milk. The nongenetic factors affecting immune-related traits and the effects of these traits on SCS were evaluated. Furthermore, the genetic parameters of immune-related traits according to health status and genetic relationships under different SCS environments were estimated. All immune-related traits were significantly associated with SCS and directly proportional. Additionally, evaluation using a classification tree revealed that IgA, IgG2, and IgG were associated with SCS levels. Genetic factor analyses indicated that heritability estimates were low for CyPA (0.08) but moderate for IgG (0.37), IgA (0.44), and IgM (0.44), with positive genetic correlations among Ig (0.25-0.96). We also evaluated the differences in milk yield and SCS of cows between the low and high groups according to their sires' estimated breeding value for immune-related traits. In the high group, IgA had a significantly lower SCS in milk at 7 to 30 d compared with that in the low group. Furthermore, the Ig in milk had high positive genetic correlations between healthy and infected conditions (0.82-0.99), suggesting that Ig in milk under healthy conditions could interact with those under infected conditions, owing to the genetic ability based on the level of Ig in milk. Thus, Ig in milk are potential indicators for the genetic selection of mastitis resistance. However, because only the relationship between immune-related traits and SCS was investigated in this study, further study on the relationship between clinical mastitis and Ig in milk is needed before Ig can be used as an indicator of mastitis resistance.

Sharing of Moonlighting Proteins Mediates the Symbiotic Relationship among Intestinal Commensals.

The human gastrointestinal tract is inhabited by trillions of symbiotic bacteria that form a complex ecological community and influence human physiology. Symbiotic nutrient sharing and nutrient competition are the most studied relationships in gut commensals, whereas the interactions underlying homeostasis and community maintenance are not fully understood. Here, we provide insights into a new symbiotic relationship wherein the sharing of secreted cytoplasmic proteins, called "moonlighting proteins," between two heterologous bacterial strains (Bifidobacterium longum and Bacteroides thetaiotaomicron) was observed to affect the adhesion of bacteria to mucins. B. longum and B. thetaiotaomicron were cocultured using a membrane-filter system, and in this system the cocultured B. thetaiotaomicron cells showed greater adhesion to mucins compared to that shown by monoculture cells. Proteomic analysis showed the presence of 13 B. longum-derived cytoplasmic proteins on the surface of B. thetaiotaomicron. Moreover, incubation of B. thetaiotaomicron with the recombinant proteins GroEL and elongation factor Tu (EF-Tu)-two well-known mucin-adhesive moonlighting proteins of B. longum-led to an increase in the adhesion of B. thetaiotaomicron to mucins, a result attributed to the localization of these proteins on the B. thetaiotaomicron cell surface. Furthermore, the recombinant EF-Tu and GroEL proteins were observed to bind to the cell surface of several other bacterial species; however, the binding was species dependent. The present findings indicate a symbiotic relationship mediated by the sharing of moonlighting proteins among specific strains of B. longum and B. thetaiotaomicron. IMPORTANCE The adhesion of intestinal bacteria to the mucus layer is an important colonization strategy in the gut environment. Generally, the bacterial adhesion process is a characteristic feature of the individual cell surface-associated adhesion factors secreted by a particular bacterium. In this study, coculture experiments between Bifidobacterium and Bacteroides show that the secreted moonlighting proteins adhere to the cell surface of coexisting bacteria and alter the adhesiveness of the bacteria to mucins. This finding indicates that the moonlighting proteins act as adhesion factors for not only homologous strains but also for coexisting heterologous strains. The presence of a coexisting bacterium in the environment can significantly alter the mucin-adhesive properties of another bacterium. The findings from this study contribute to a better understanding of the colonization properties of gut bacteria through the discovery of a new symbiotic relationship between them.

In Silico Comparative Genomic Analysis Revealed a Highly Conserved Proteolytic System in Lactobacillus delbrueckii.

Lactobacillus delbrueckii, the type species of the genus Lactobacillus, is widely recognized as the primary starter culture in the dairy industry due to its proteolytic activity, which enables it to growth in milk. In this study, a comprehensive genomic analysis of the proteolytic system was conducted on L. delbrueckii strains. The analysis included 27 genomes of L. delbrueckii, with a specific focus on the key enzyme involved in this system, the cell envelope-associated proteinase (CEP). The amino acid sequences, as well as the protein-structure prediction of the CEPs, were compared. Additionally, syntenic analysis of the genomic locus related to the CEPs revealed high conservation in L. delbrueckii subsp. bulgaricus strains, while L. delbrueckii subsp. lactis strains exhibited greater variability, including the presence of insertion sequences, deletions, and rearrangements. Finally, the CEP promoter region and putative regulatory elements responsible for controlling the expression of the proteolytic system in lactobacilli were investigated. Our genomic analysis and in silico characterization of the CEPs contribute to our understanding of proteolytic activity and the potential applications of these lactic acid bacteria in the dairy industry. Further research in this area will expand our knowledge and potential practical uses of these findings.

Hypermucoviscous Carbapenem-Resistant Klebsiella pneumoniae ST25 Infect Human Intestinal Epithelial Cells and Induce Moderate Inflammation.

Klebsiella pneumoniae is an opportunistic pathogen that can produce moderate and severe infections in immunosuppressed hosts. In recent years, an increase in the isolation of hypermucoviscous carbapenem-resistant K. pneumoniae with sequence type 25 (ST25) in hospitals in Norwest Argentina was observed. This work aimed to study the virulence and inflammatory potential of two K. pneumoniae ST25 strains (LABACER01 and LABACER27) in the intestinal mucosa. The human intestinal Caco-2 cells were infected with the K. pneumoniae ST25 strains, and their adhesion and invasion rates and changes in the expression of tight junction and inflammatory factors genes were evaluated. ST25 strains were able to adhere and invade Caco-2 cells, reducing their viability. Furthermore, both strains reduced the expression of tight junction proteins (occludin, ZO-1, and claudin-5), altered permeability, and increased the expression of TGF-ß and TLL1 and the inflammatory factors (COX-2, iNOS, MCP-1, IL-6, IL-8, and TNF-α) in Caco-2 cells. The inflammatory response induced by LABACER01 and LABACER27 was significantly lower than the one produced by LPS or other intestinal pathogens, including K. pneumoniae NTUH-K2044. No differences in virulence and inflammatory potential were found between LABACER01 and LABACER27. In line with these findings, no major differences between the strains were found when the comparative genomic analysis of virulence factors associated with intestinal infection/colonization was performed. This work is the first to demonstrate that hypermucoviscous carbapenem-resistant K. pneumoniae ST25 infects human intestinal epithelial cells and induces moderate inflammation.

Lacticaseibacillus rhamnosus CRL1505 Peptidoglycan Modulates the Inflammation-Coagulation Response Triggered by Poly(I:C) in the Respiratory Tract.

Lacticaseibacillus rhamnosus CRL1505 beneficially modulates the inflammation-coagulation response during respiratory viral infections. This study evaluated the capacity of the peptidoglycan obtained from the CRL1505 strain (PG-Lr1505) to modulate the immuno-coagulative response triggered by the viral pathogen-associated molecular pattern poly(I:C) in the respiratory tract. Adult BALB/c mice were nasally treated with PG-Lr1505 for two days. Treated and untreated control mice were then nasally challenged with poly(I:C). Mice received three doses of poly(I:C) with a 24 h rest period between each administration. The immuno-coagulative response was studied after the last administration of poly(I:C). The challenge with poly(I:C) significantly increased blood and respiratory pro-inflammatory mediators, decreased prothrombin activity (PT), and increased von Willebrand factor (vWF) levels in plasma. Furthermore, tissue factor (TF), tissue factor pathway inhibitor (TFPI), and thrombomodulin (TM) expressions were increased in the lungs. PG-Lr1505-treated mice showed significant modulation of hemostatic parameters in plasma (PT in %, Control = 71.3 ± 3.8, PG-Lr1505 = 94.0 ± 4.0, p < 0.01) and lungs. Moreover, PG-Lr1505-treated mice demonstrated reduced TF in F4/80 cells from lungs, higher pro-inflammatory mediators, and increased IL-10 compared to poly(I:C) control mice (IL-10 in pg/mL, Control = 379.1 ± 12.1, PG-Lr1505 = 483.9 ± 11.3, p < 0.0001). These changes induced by PG-Lr1505 correlated with a significant reduction in lung tissue damage. Complementary in vitro studies using Raw 264.7 cells confirmed the beneficial effect of PG-Lr1505 on poly(I:C)-induced inflammation, since increased IL-10 expression, as well as reduced damage, production of inflammatory mediators, and hemostatic parameter expressions were observed. In addition, protease-activated receptor-1 (PAR1) activation in lungs and Raw 264.7 cells was observed after TLR3 stimulation, which was differentially modulated by PG-Lr1505. The peptidoglycan from L. rhamnosus CRL1505 is able to regulate inflammation, the procoagulant state, and PAR1 activation in mice and macrophages in the context of the activation of TLR3 signaling pathways, contributing to a beneficial modulation of inflammation-hemostasis crosstalk.

Immunobiotic Ligilactobacillus salivarius FFIG58 Confers Long-Term Protection against Streptococcus pneumoniae.

Previously, we isolated potentially probiotic Ligilactobacillus salivarius strains from the intestines of wakame-fed pigs. The strains were characterized based on their ability to modulate the innate immune responses triggered by the activation of Toll-like receptor (TLR)-3 or TLR4 signaling pathways in intestinal mucosa. In this work, we aimed to evaluate whether nasally administered L. salivarius strains are capable of modulating the innate immune response in the respiratory tract and conferring long-term protection against the respiratory pathogen Streptococcus pneumoniae. Infant mice (3-weeks-old) were nasally primed with L. salivarius strains and then stimulated with the TLR3 agonist poly(I:C). Five or thirty days after the last poly(I:C) administration mice were infected with pneumococci. Among the strains evaluated, L. salivarius FFIG58 had a remarkable ability to enhance the protection against the secondary pneumococcal infection by modulating the respiratory immune response. L. salivarius FFIG58 improved the ability of alveolar macrophages to produce interleukin (IL)-6, interferon (IFN)-γ, IFN-ß, tumor necrosis factor (TNF)-α, IL-27, chemokine C-C motif ligand 2 (CCL2), chemokine C-X-C motif ligand 2 (CXCL2), and CXCL10 in response to pneumococcal challenge. Furthermore, results showed that the nasal priming of infant mice with the FFIG58 strain protected the animals against secondary infection until 30 days after stimulation with poly(I:C), raising the possibility of using nasally administered immunobiotics to stimulate trained immunity in the respiratory tract.

Genomic and Immunological Characterization of Hypermucoviscous Carbapenem-Resistant Klebsiella pneumoniae ST25 Isolates from Northwest Argentina.

In recent years, an increase in the prevalence hypermucoviscous carbapenem-resistant Klebsiella pneumoniae with sequence type 25 (ST25) was detected in hospitals of Tucuman (Northwest Argentina). In this work, the virulence and the innate immune response to two K. pneumoniae ST25 strains (LABACER 01 and LABACER 27) were evaluated in a murine model after a respiratory challenge. In addition, comparative genomics was performed with K. pneumoniae LABACER01 and LABACER27 to analyze genes associated with virulence. Both LABACER01 and LABACER27 were detected in the lungs of infected mice two days after the nasal challenge, with LABACER01 counts significantly higher than those of LABACER27. Only LABACER01 was detected in hemocultures. Lactate dehydrogenase (LDH) and albumin levels in bronchoalveolar lavage (BAL) samples were significantly higher in mice challenged with LABACER01 than in LABACER27-infected animals, indicating greater lung tissue damage. Both strains increased the levels of neutrophils, macrophages, TNF-α, IL-1ß, IL-6, KC, MCP-1, IFN-γ, and IL-17 in the respiratory tract and blood, with the effect of LABACER01 more marked than that of LABACER27. In contrast, LABACER27 induced higher levels of IL-10 in the respiratory tract than LABACER01. Genomic analysis revealed that K. pneumoniae LABACER01 and LABACER27 possess virulence factors found in other strains that have been shown to be hypervirulent, including genes required for enterobactin (entABCDEF) and salmochelin (iroDE) biosynthesis. In both strains, the genes of toxin-antitoxin systems, as well as regulators of the expression of virulence factors and adhesion genes were also detected. Studies on the genetic potential of multiresistant K. pneumoniae strains as well as their cellular and molecular interactions with the host are of fundamental importance to assess the association of certain virulence factors with the intensity of the inflammatory response. In this sense, this work explored the virulence profile based on genomic and in vivo studies of hypermucoviscous carbapenem-resistant K. pneumoniae ST25 strains, expanding the knowledge of the biology of the emerging ST25 clone in Argentina.

The Mucus Binding Factor Is Not Necessary for Lacticaseibacillus rhamnosus CRL1505 to Exert Its Immunomodulatory Activities in Local and Distal Mucosal Sites.

Both viable and non-viable orally administered Lacticaseibacillus rhamnosus CRL1505 modulate immunity in local (intestine) and distal (respiratory) mucosal sites. So, intestinal adhesion and colonization are not necessary for this probiotic strain to exert its immunomodulatory effects. In this work, a mucus-binding factor knockout CRL1505 strain (ΔmbfCRL1505) was obtained and the lack of binding ability to both intestinal epithelial cells and mucin was demonstrated in vitro. In addition, two sets of in vivo experiments in 6-week-old Balb/c mice were performed to evaluate ΔmbfCRL1505 immunomodulatory activities. (A) Orally administered ΔmbfCRL1505 prior to intraperitoneal injection of the Toll-like receptor 3 (TLR3) agonist poly(I:C) significantly reduced intraepithelial lymphocytes (CD3+NK1.1+CD8αα+) and pro-inflammatory mediators (TNF-α, IL-6 and IL-15) in the intestinal mucosa. (B) Orally administered ΔmbfCRL1505 prior to nasal stimulation with poly(I:C) significantly decreased the levels of the biochemical markers of lung tissue damage. In addition, reduced recruitment of neutrophils and levels of pro-inflammatory mediators (TNF-α, IL-6 and IL-8) as well as increased IFN-ß and IFN-γ in the respiratory mucosa were observed in ΔmbfCRL1505-treated mice when compared to untreated control mice. The immunological changes induced by the ΔmbfCRL1505 strain were not different from those observed for the wild-type CRL1505 strain. Although it is generally accepted that the expression of adhesion factors is necessary for immunobiotics to induce their beneficial effects, it was demonstrated here that the mbf protein is not required for L. rhamnosus CRL1505 to exert its immunomodulatory activities in local and distal mucosal sites. These results are a step forward towards understanding the mechanisms involved in the immunomodulatory capabilities of L. rhamnosus CRL1505.

Transcriptome Modifications in the Porcine Intramuscular Adipocytes during Differentiation and Exogenous Stimulation with TNF-α and Serotonin.

Adipocytes are dynamic cells that have critical functions to maintain body energy homeostasis. Adipocyte physiology is affected by the adipogenic differentiation, cell program, as well as by the exogenous stimulation of biochemical factors, such as serotonin and TNF-α. In this work, we investigated the global transcriptome modifications when porcine intramuscular preadipocyte (PIP) was differentiated into porcine mature adipocyte (pMA). Moreover, we studied transcriptome changes in pMA after stimulation with serotonin or TNF-α by using a microarray approach. Transcriptome analysis revealed that the expression of 270, 261, and 249 genes were modified after differentiation, or after serotonin and TNF-α stimulation, respectively. Expression changes in APP, HNF4A, ESR1, EGR1, SRC, HNF1A, FN1, ALB, STAT3, CBL, CEBPB, AR, FOS, CFTR, PAN2, PTPN6, VDR, PPARG, STAT5A and NCOA3 genes which are enriched in the 'PPAR signaling' and 'insulin resistance' pathways were found in adipocytes during the differentiation process. Dose-dependent serotonin stimulation resulted in a decreased fat accumulation in pMAs. Serotonin-induced differentially expressed genes in pMAs were found to be involved in the significant enrichment of 'GPCR ligand-binding', 'cell chemotaxis', 'blood coagulation and complement', 'metabolism of lipid and lipoproteins', 'regulation of lipid metabolism by PPARA', and 'lipid digestion, mobilization and transport' pathways. TNF-α stimulation also resulted in transcriptome modifications linked with proinflammatory responses in the pMA of intramuscular origin. Our results provide a landscape of transcriptome modifications and their linked-biological pathways in response to adipogenesis, and exogenous stimulation of serotonin- and TNF-α to the pMA of intramuscular origin.

Bayesian latent class evaluation of three tests for the screening of subclinical caprine mastitis in Bangladesh.

Routine monitoring for subclinical infection is one of the key mastitis control approaches. However, the accuracy of the most commonly used screening tests has not yet been established. The aim of the present study was therefore to evaluate the accuracy of three screening tests, namely California mastitis test (CMT), white side test (WST), and surf field mastitis test (SFMT) for the screening of subclinical caprine mastitis. A cross-sectional study based on 484 randomly collected milk (242 goats) samples from three districts of Bangladesh was conducted for the screening of subclinical mastitis by the aforementioned tests. The Bayesian latent class model was implemented in WinBUGS to estimate the tests' characteristics and true prevalence of subclinical mastitis. The Bayesian posterior estimates of sensitivities with a 95% credible intervals (CrIs) were 98.60% (95.18-99.95%), 98.28% (94.56-99.92%), and 89.98% (83.39-95.03%), and specificities with 95% CrIs were 99.19% (98.11-99.96%), 99.27% (97.34-99.98%), and 99.28% (97.35-99.98%), respectively for CMT, WST, and SFMT. The true prevalence of subclinical caprine mastitis was estimated to be 43.49% (95% CrI 37.46-48.98%). The positive predictive values (PPV) of the three tests were similar. The serial and parallel interpretation of any test pairs increased the PPV and negative predictive value respectively close to 100%. Based on the simplicity, cost and performance as well WST and SFMT simultaneously could be recommended for the screening of caprine subclinical mastitis in Bangladesh.

Characterization of the immunomodulatory and anti-Helicobacter pylori properties of the human gastric isolate Lactobacillus rhamnosus UCO-25A.

The ability to form biofilms and the potential immunomodulatory properties of the human gastric isolate Lactobacillus rhamnosus UCO-25A were characterized in vitro. It was demonstrated that L. rhamnosus UCO-25A is able to form biofilms on abiotic and cell surfaces, and to modulate the inflammatory response triggered by Helicobacter pylori infection in gastric epithelial cells and THP-1 macrophages. L. rhamnosus UCO-25A exhibited a substantial anti-inflammatory effect in both cell lines and improved IL-10 levels produced by challenged macrophages. Additionally, UCO-25A protected AGS cells against H. pylori infection with a higher pathogen inhibition when a biofilm was formed. Given the importance of inflammation in H. pylori-mediated diseases, the differential modulation of the inflammatory response in the gastric mucosa by an autochthonous strain is an attractive alternative for improving H. pylori eradication and reducing the severity of the diseases that arise from the resulting chronic inflammation.

Immunoregulatory effects triggered by immunobiotic Lactobacillus jensenii TL2937 strain involve efficient phagocytosis in porcine antigen presenting cells.

BACKGROUND: Immunobiotic Lactobacillus jensenii TL2937 modulates porcine mononuclear phagocytes from Peyer's patches (PPMPs) and induces a differential production of pro- and anti-inflammatory cytokines in response to Toll-like receptor (TLR)-4 activation. In view of the important role played by phagocytosis in the activation of antigen presenting cells (APCs), the aim of the present work was to examine the interaction of TL2937 with porcine PPMPs focusing on phagocytosis. In addition, this study aimed to investigate whether the effects of L. jensenii TL2937 in porcine blood monocyte-derived dendritic cells (MoDCs) are similar to those found in PPMPs considering that MoDCs do not recapitulate all functions of mucosal APCs. RESULTS: Studies showed a high ability of porcine CD172a(+) PPMPs to phagocytose L. jensenii TL2937. Interestingly, our results also revealed a reduced capacity of the non-immunomodulatory L. plantarum TL2766 to be phagocytosed by those immune cells. Phagocytosis of L. jensenii TL2937 by porcine PPMPs was partially dependent on TLR2. In addition, we demonstrated that TL2937 strain was able to improve the expression of IL-1ß, IL-12 and IL-10 in immature MoDCs resembling the effect of this immunobiotic bacterium on PPMPs. Moreover, similarly to PPMPs those immunomodulatory effects were related to the higher capacity of TL2937 to be phagocytosed by immature MoDCs. CONCLUSIONS: Microbial recognition in APCs could be effectively mediated through ligand-receptor interactions that then mediate phagocytosis and signaling. For the immunobiotic strain TL2937, TLR2 has a partial role for its interaction with porcine APCs and it is necessary to investigate the role of other receptors. A challenge for future research will be advance in the full understanding of the molecular interactions of immunobiotic L. jensenii TL2937 with porcine APCs that will be crucial for the successful development of functional feeds for the porcine host. This study is a step in that direction.

Cyclophilin A is a new M cell marker of bovine intestinal epithelium.

Microfold (M) cells in the follicle-associated epithelium (FAE) of Peyer's patches contribute to the mucosal immune response by the transcytosis of microorganisms. The mechanism by which M cells take up microorganisms, and the functional proteins by which they do this, are not clear. In order to explore one such protein, we developed a 2H5-F3 monoclonal antibody (2H5-F3 mAb) through its binding to bovine M cells, and identified the antibody reactive molecule as cyclophilin A (Cyp-A). The localization patterns of Cyp-A were very similar to the localization pattern of cytokeratin (CK) 18-positive M cells. Cyp-A was identified at the luminal surface of CK18-positive M cells in bovine jejunal and ileal FAE. The membranous localization of Cyp-A in the bovine intestinal cell line (BIE cells) increased as cells differentiated toward M cells, as determined by flow cytometry analysis. Additionally, BIE cells released Cyp-A to the extracellular space and the differentiation of BIE cells to M cells increased the secretion of Cyp-A, as determined by western blotting. Accordingly, Cyp-A may be localized in M cells in the small intestinal epithelium of cattle. The rise of the membranous localization and secretion of Cyp-A by differentiation toward M cells indicates that Cyp-A has an important role in the function of M cells. While Cyp-A of the M cell membrane may contribute to the uptake of viruses with peptidyl-prolyl cis-trans isomerase activity, in the extracellular space Cyp-A may work as a chemokine and contribute to the distribution of immuno-competent cells.

Immunobiotic Lactobacillus strains reduce small intestinal injury induced by intraepithelial lymphocytes after Toll-like receptor 3 activation.

OBJECTIVE: Intestinal intraepithelial lymphocytes (IELs) play critical roles in disrupting epithelial homeostasis after Toll-like receptor (TLR)-3 activation with genomic rotavirus dsRNA or the synthetic dsRNA analog poly(I:C). The capacity of immunobiotic Lactobacillus rhamnosus CRL1505 (Lr1505) or Lactobacillus plantarum CRL1506 (Lp1506) to beneficially modulate IELs response after TLR3 activation was investigated in vivo using a mice model. RESULTS: Intraperitoneal administration of poly(I:C) induced inflammatory-mediated intestinal tissue damage through the increase of inflammatory cells (CD3(+)NK1.1(+), CD3(+)CD8αα(+), CD8αα(+)NKG2D(+)) and pro-inflammatory mediators (TNF-α, IL-1ß, IFN-γ, IL-15, RAE1, IL-8). Increased expression of intestinal TLR3, MDA5, and RIG-I was also observed after poly(I:C) challenge. Treatment with Lr1505 or Lp1506 prior to TLR3 activation significantly reduced the levels of TNF-α, IL-15, RAE1, and increased serum and intestinal IL-10. Moreover, CD3(+)NK1.1(+), CD3(+)CD8αα(+), and CD8αα(+)NKG2D(+) cells were lower in lactobacilli-treated mice when compared to controls. The immunomodulatory capacities of lactobacilli allowed a significant reduction of intestinal tissue damage. CONCLUSIONS: This work demonstrates the reduction of TLR3-mediated intestinal tissue injury by immunobiotic lactobacilli through the modulation of intraepithelial lymphocytes response. It is a step forward in the understanding of the cellular mechanisms involved in the antiviral capabilities of immunobiotic strains.

Inhibitory/suppressive oligodeoxynucleotide nanocapsules as simple oral delivery devices for preventing atopic dermatitis in mice.

Here, we report a simple and low-cost oral oligodeoxynucleotide (ODN) delivery system targeted to the gut Peyer's patches (PPs). This system requires only Dulbecco's modified eagle's medium, calcium chloride, ODNs, and basic laboratory equipment. ODN nanocapsules (ODNcaps) were directly delivered to the PPs through oral administration and were taken up by macrophages in the PPs, where they induced an immune response. Long-term continuous oral dosing with inhibitory/suppressive ODNcaps (iODNcaps, "iSG3caps" in this study) was evaluated using an atopic dermatitis mouse model to visually monitor disease course. Administration of iSG3caps improved skin lesions and decreased epidermal thickness. Underlying this effect is the ability of iSG3 to bind to and prevent phosphorylation of signal transducer and activator of transcription 6, thereby blocking the interleukin-4 signaling cascade mediated by binding of allergens to type 2 helper T cells. The results of our iSG3cap oral delivery experiments suggest that iSG3 may be useful for treating allergic diseases.

Isolation of lactic acid bacteria from swine milk and characterization of potential probiotic strains with antagonistic effects against swine-associated gastrointestinal pathogens.

Probiotics are usually isolated from the gastrointestinal tract of humans and animals. The search of probiotics in human milk is a recent field of research, as the existence of the human milk microbiome was discovered only about a decade ago. To our knowledge, no reports regarding the potential probiotic effect of bacteria from swine milk have been published. In this work, we isolated several lactic acid bacteria from swine milk and evaluated them for them potential as probiotics. Among the isolated strains, Lactobacillus curvatus TUCO-5E showed antagonistic effects against swine-associated gastrointestinal pathogens. TUCO-5E was able to reduce the growth of enterotoxigenic and enterohemorrhagic Escherichia coli strains as well as pathogenic salmonella. In vitro exclusion and displacement assays in intestinal epithelial cells showed a remarkable antagonistic effect for L. curvatus TUCO-5E against Salmonella sp. strain TUCO-I7 and Salmonella enterica ATCC 13096. Moreover, by using a mouse model of Salmonella infection, we were able to demonstrate that preventative administration of L. curvatus TUCO-5E for 5 consecutive days was capable of decreasing the number of Salmonella enterica serovar Typhimurium in the liver and spleen of treated mice, compared with the controls, and prevented dissemination of the pathogen to the blood stream. Therefore, we have demonstrated here that swine milk is an interesting source of beneficial bacteria. In addition, the results of this work suggest that L. curvatus TUCO-5E is a good candidate to study in vivo the protective effect of probiotics against intestinal infection and damage induced by Salmonella infection in the porcine host.

Nasal priming with immunobiotic Lactobacillus rhamnosus modulates inflammation-coagulation interactions and reduces influenza virus-associated pulmonary damage.

OBJECTIVE: To evaluate the effect of the nasal administration of live and heat-killed Lactobacillus rhamnosus CRL1505 (Lr1505) on immune-coagulative response during influenza virus (IFV) infection to improve survival and reduce lung injury. METHODS: Six-week-old BALB/c mice were treated with live or heat-killed Lr1505 by the nasal route during two consecutive days. Treated and untreated control mice were then nasally challenged with IFV. RESULTS: Both viable and non-viable Lr1505 protected infected mice by reducing pulmonary injury and lung viral loads trough several mechanisms: (a) Inflammatory cytokines were efficiently regulated allowing higher clearance of virus and reduction of inflammatory lung tissue damage, associated to higher levels of the regulatory cytokine IL-10. (b) The antiviral immune response was enhanced with improved levels of type I interferons, CD4(+)IFN-γ(+) lymphocytes, and lung CD11c(+)CD11b(low)CD103(+) and CD11c(+)CD11b(high)CD103(-) dendritic cells. (c) The procoagulant state was reversed mainly by down-regulating tissue factor expression and restoring thrombomodulin levels in lung. The capacity of Lr1505 to improve the outcome of IFV infection would be related to its ability to beneficially modulate lung TLR3-triggered immune response. CONCLUSIONS: Our work is the first to demonstrate the ability of an immunobiotic strain to beneficially modulate inflammation-coagulation interactions during IFV infection. Interestingly, non-viable L. rhamnosus CRL1505 was as effective as the viable strain to beneficially modulate respiratory antiviral immune response.

Oral delivery of Lactococcus lactis that secretes bioactive heme oxygenase-1 alleviates development of acute colitis in mice.

BACKGROUND: Mucosal delivery of therapeutic proteins using genetically modified strains of lactic acid bacteria (gmLAB) is being investigated as a new therapeutic strategy. METHODS: We developed a strain of gmLAB, Lactococcus lactis NZ9000 (NZ-HO), which secretes the anti-inflammatory molecule recombinant mouse heme oxygenase-1 (rmHO-1). The effects of short-term continuous oral dosing with NZ-HO were evaluated in mice with dextran sulfate sodium (DSS)-induced acute colitis as a model of inflammatory bowel diseases (IBD). RESULTS: We identified the secretion of rmHO-1 by NZ-HO. rmHO-1 was biologically active as determined with spectroscopy. Viable NZ-HO was directly delivered to the colon via oral administration, and rmHO-1 was secreted onto the colonic mucosa in mice. Acute colitis in mice was induced by free drinking of 3 % DSS in water and was accompanied by an increase in the disease activity index score and histopathological changes. Daily oral administration of NZ-HO significantly improved these colitis-associated symptoms. In addition, NZ-HO significantly increased production of the anti-inflammatory cytokine interleukin (IL)-10 and decreased the expression of pro-inflammatory cytokines such as IL-1α and IL-6 in the colon compared to a vector control strain. CONCLUSIONS: Oral administration of NZ-HO alleviates DSS-induced acute colitis in mice. Our results suggest that NZ-HO may be a useful mucosal therapeutic agent for treating IBD.

Extracellular cyclophilin A possesses chemotaxic activity in cattle.

Cyclophilin A (CyPA) was originally discovered in bovine thymocytes as a cytosolic binding protein of the immunosuppressive drug cyclosporine A. Recent studies have revealed that in mice and humans, CyPA is secreted from cells in injured or infected tissues and plays a role in recruiting inflammatory cells in those tissues. Here we found that in cattle abundant level of extracellular CyPA was observed in tissues with inflammation. To aid in investigating the role of extracellular CyPA in cattle, we generated recombinant bovine CyPA (rbCyPA) and tested its biological activity as an inflammatory mediator. When bovine peripheral blood cells were treated with rbCyPA in vitro, we observed that rbCyPA reacts with the membranous surface of granulocytes, monocytes and lymphocytes. Chemotaxis analysis showed that the granulocytes migrate toward rbCyPA and the migration is inhibited by pre-treatment with an anti-bovine CyPA antibody. These results indicate that, as for mice and humans, extracellular CyPA possesses chemotactic activity to recruit inflammatory cells (e.g., granulocytes) in cattle, and could thus be a potential therapeutic target for the treatment of inflammation.

Immunobiotic Lactobacillus jensenii as immune-health promoting factor to improve growth performance and productivity in post-weaning pigs.

BACKGROUND: Immunoregulatory probiotics (immunobiotics) have been proposed to improve piglets' immune system to avoid intestinal infections and reduce unproductive inflammation after weaning. Previously, it was demonstrated that Lactobacillus jensenii TL2937 (LjTL2937) attenuated the inflammatory response triggered by activation of Toll-like receptor 4 (TLR-4) in porcine intestinal epithelial (PIE) cells and antigen presenting cells (APCs) from porcine Peyer's patches (PP). OBJECTIVE: In view of the critical importance of PIE-APCs interactions in the regulation of intestinal immune responses, we aimed to examine the effect of LjTL2937 on activation patterns of APCs from swine PPs in co-cultures with PIE cells. In addition, we investigated whether LjTL2937 was able to beneficially modulate intestinal immunity of piglets after weaning to improve immune-health status. RESULTS: Stimulation of PIE-APCs co-cultures with LjTL2937 increased the expression of MHC-II, CD80/86, IL-10, and Bcl-3 in CD172a+CD11R1- and CD172a+CD11R1high APCs. In addition, the TL2937 strain caused the upregulation of three negative regulators of TLR4 in PIE cells: MKP-1, Bcl-3 and A20. These changes significantly reduced the inflammatory response triggered by TLR4 activation in PIE-APCs co-cultures. The in vivo experiments using castrated male piglets (crossbreeding (LWD) with Landrace (L), Large Yorkshire (W) and Duroc (D))of 3 weeks of age demonstrated that feeding with LjTL2937 significantly reduced blood complement activity and C reactive protein concentrations while no changes were observed in blood leukocytes, ratio of granulocytes to lymphocyte numbers, macrophages' activity and antibody levels. In addition, treatment with LjTL2937 significantly improved growth performance and productivity, and increased carcass quality. CONCLUSIONS: We demonstrated that the use of immunobiotics strains like LjTL2937, as supplemental additives for piglets feedings, could be used as a strategy to maintain and improve intestinal homeostasis; that is important for the development of the pig and for health and performance throughout the productive life of the animal.