Mechanism of the Immunomodulatory Effect of the Combination of Live Bifidobacterium, Lactobacillus, Enterococcus, and Bacillus on Immunocompromised Rats.

Immunodeficiency is a very common condition in suboptimal health status and during the development or treatment of many diseases. Recently, probiotics have become an important means for immune regulation. The present study aimed to investigate the mechanism of the immunomodulatory effect of a combination of live Bifidobacterium, Lactobacillus, Enterococcus, and Bacillus (CBLEB), which is a drug used by approximately 10 million patients every year, on cyclophosphamide-immunosuppressed rats. Cyclophosphamide (40 mg/kg) was intraperitoneally injected to induce immunosuppression in a rat model on days 1, 2, 3, and 10. Starting from day 4, the rats were continuously gavaged with CBLEB solution for 15 days. The samples were collected to determine routine blood test parameters, liver and kidney functions, serum cytokine levels, gut microbiota, fecal and serum metabolomes, transcriptomes, and histopathological features. The results indicated that CBLEB treatment reduced cyclophosphamide-induced death, weight loss, and damage to the gut, liver, spleen, and lungs and eliminated a cyclophosphamide-induced increase in the mean hemoglobin content and GGT, M-CSF, and MIP-3α levels and a decrease in the red blood cell distribution width and total protein and creatinine levels in the blood. Additionally, CBLEB corrected cyclophosphamide-induced dysbiosis of the gut microbiota and eliminated all cyclophosphamide-induced alterations at the phylum level in rat feces, including the enrichment in Proteobacteria, Fusobacteriota, and Actinobacteriota and depletion of Spirochaetota and Cyanobacteria. Furthermore, CBLEB treatment alleviated cyclophosphamide-induced alterations in the whole fecal metabolome profile, including enrichment in 1-heptadecanol, succinic acid, hexadecane-1,2-diol, nonadecanoic acid, and pentadecanoic acid and depletion of benzenepropanoic acid and hexane. CBLEB treatment also alleviated cyclophosphamide-induced enrichment in serum D-lyxose and depletion of serum succinic acid, D-galactose, L-5-oxoproline, L-alanine, and malic acid. The results of transcriptome analysis indicated that the mechanism of the effect of CBLEB was related to the induction of recovery of cyclophosphamide-altered carbohydrate metabolism and signal transduction. In conclusion, the present study provides an experimental basis and comprehensive analysis of application of CBLEB for the treatment of immunodeficiency.

Cervicovaginal microbiota and metabolome predict preterm birth risk in an ethnically diverse cohort.

The syndrome of spontaneous preterm birth (sPTB) presents a challenge to mechanistic understanding, effective risk stratification, and clinical management. Individual associations between sPTB, self-reported ethnic ancestry, vaginal microbiota, metabolome, and innate immune response are known but not fully understood, and knowledge has yet to impact clinical practice. Here, we used multi-data type integration and composite statistical models to gain insight into sPTB risk by exploring the cervicovaginal environment of an ethnically heterogenous pregnant population (n = 346 women; n = 60 sPTB < 37 weeks' gestation, including n = 27 sPTB < 34 weeks). Analysis of cervicovaginal samples (10-15+6 weeks) identified potentially novel interactions between risk of sPTB and microbiota, metabolite, and maternal host defense molecules. Statistical modeling identified a composite of metabolites (leucine, tyrosine, aspartate, lactate, betaine, acetate, and Ca2+) associated with risk of sPTB < 37 weeks (AUC 0.752). A combination of glucose, aspartate, Ca2+, Lactobacillus crispatus, and L. acidophilus relative abundance identified risk of early sPTB < 34 weeks (AUC 0.758), improved by stratification by ethnicity (AUC 0.835). Increased relative abundance of L. acidophilus appeared protective against sPTB < 34 weeks. By using cervicovaginal fluid samples, we demonstrate the potential of multi-data type integration for developing composite models toward understanding the contribution of the vaginal environment to risk of sPTB.

Effect of exopolysaccharides from cariogenic bacteria on human gingival fibroblasts.

Bacterial biofilm (dental plaque) plays a key role in caries etiopathogenesis and chronic periodontitis in humans. Dental plaque formation is determined by exopolysaccharides (EPSs) produced by cariogenic and periopathogenic bacteria. The most frequent cariogenic bacteria include oral streptococci (in particular S. mutans) and lactobacilli (most frequently L. acidophilus). In turn, the dominant periopathogen in periodontitis is Porphyromonas gingivalis. Development of dental caries is often accompanied with gingivitis constituting the mildest form of periodontal disease. Basic cellular components of the gingiva tissue are fibroblasts the damage of which determines the progression of chronic periodontitis. Due to insufficient knowledge of the direct effect of dental plaque on metabolic activity of the fibroblasts, this work analyses the effect of EPSs produced by S. mutans and L. acidophilus strains (H2O2-producing and H2O2-not producing) on ATP levels in human gingival fibroblasts (HGF-1) and their viability. EPSs produced in 48-hours bacterial cultures were isolated by precipitation method and quantitatively determined by phenol - sulphuric acid assay. ATP levels in HGF-1 were evaluated using a luminescence test, and cell viability was estimated using fluorescence test. The tests have proven that EPS from S. mutans did not affect the levels of ATP in HGF-1. Whereas EPS derived from L. acidophilus strains, irrespective of the tested strain, significantly increased ATP levels in HGF-1. The analysed EPSs did not affect the viability of cells. The tests presented in this work show that EPSs from cariogenic bacteria have no cytotoxic effect on HGF-1. At the same time, the results provide new data indicating that EPSs from selected oral lactobacilli may have stimulating effect on the synthesis of ATP in gingival fibroblasts which increases their energetic potential and takes a protective effect.

S-layer associated proteins contribute to the adhesive and immunomodulatory properties of Lactobacillus acidophilus NCFM.

BACKGROUND: Surface layers (S-layers) are two-dimensional crystalline arrays of repeating proteinaceous subunits that form the outermost layer of many bacterial cell envelopes. Within the Lactobacillus genus, S-layer presence is frequently associated with probiotic-relevant properties such as improved adherence to host epithelial cells and modulation of the immune response. However, recent studies have demonstrated that certain S-layer functions may be supplemented by a novel subset of proteins embedded within its lattice, termed S-layer associated proteins (SLAPs). In the following study, four Lactobacillus acidophilus NCFM SLAPs (LBA0046, LBA0864, LBA1426, and LBA1539) were selected for in silico and phenotypic assessment. RESULTS: Despite lacking any sequence similarity or catalytic domains that may indicate function, the genes encoding the four proteins of interest were shown to be unique to S-layer-forming, host-adapted lactobacilli species. Likewise, their corresponding deletion mutants exhibited broad, host-relevant phenotypes including decreased inflammatory profiles and reduced adherence to Caco-2 intestinal cells, extracellular matrices, and mucin in vitro. CONCLUSIONS: Overall, the data presented in this study collectively links several previously uncharacterized extracellular proteins to roles in the underlying host adaptive mechanisms of L. acidophilus.

Immune enhancing effects of Lactobacillus acidophilus on Newcastle disease vaccination in chickens.

INTRODUCTION AND PURPOSE: Despite the various vaccination programs for protection against New Castle disease, it remains an important threat to the poultry industry. The ability of the probiotic bacteria to improve the immune system in both animals and humans supports their use as immune adjuvants for vaccination. The aim of the present study was to examine the effects of Lactobacillus acidophilus in ND vaccination. MATERIALS AND METHODS: A total of 170 one day old chicks were divided in 5 groups. In groups A, B and C chicks were received L. acidophilus (5 × 109, 3 × 109 and 2 × 109) and also vaccinated with inactivated and attenuated ND vaccines. In group D, chicks only vaccinated without bacterial inoculation and group E was negative control with neither vaccine nor bacteria. Then IgG and HI NDV antibody titers were measured in all tested groups. RESULTS: IgG and HI NDV antibody levels were significantly higher in Lactobacillus treated groups especially in group A with 5 × 109 bacteria, than only vaccinated and negative control groups. Also antibody levels against NDV increased during the vaccination period especially in probiotic treated groups. CONCLUSION: In conclusion, L. acidophilus can use for improving immunogenicity of NDV vaccination programs.

Modulation of Toll-like receptor-mediated innate immunity in bovine intestinal epithelial cells by lactic acid bacteria isolated from feedlot cattle.

The ability of lactobacilli isolated from feedlot cattle environment to differentially modulate the innate immune response triggered by Toll-like receptors (TLRs) activation in bovine intestinal epithelial (BIE) cells was evaluated. BIE cells were stimulated with Lactobacillus mucosae CRL2069, Lactobacillus acidophilus CRL2074, Lactobacillus fermentum CRL2085 or Lactobacillus rhamnosus CRL2084 and challenged with heat-stable pathogen associated molecular patterns (PAMPs) from enterotoxigenic Escherichia coli (ETEC) to induce the activation of TLR4 or with polyinosinic:polycytidylic acid (poly(I:C)) to activate TLR3. Type I interferons, cytokines, chemokines and negative regulators of TLR signalling were studied by RT-PCR. L. mucosae CRL2069 significantly reduced the expression of interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1 in BIE cells in the context of TLR3 activation. L. mucosae CRL2069 also reduced the expression of tumour necrosis factor-α, IL-ß, MCP-1, and IL-8 in heat-stable ETEC PAMPs-challenged BIE cells. In addition, reduced expressions of IL-6, MCP-1, and IL-8 were found in BIE cells stimulated with L. rhamnosus CRL2084, although its effect was significantly lower than that observed for the CRL2069 strain. The reduced levels of pro-inflammatory factors in BIE cells induced by the CRL2069 and CRL2085 strains was related to their ability of increasing the expression of TLR negative regulators. L. mucosae CRL2069 significantly improved the expression of A20-binding inhibitor of NFκ-B activation 3 (ABIN-3), interleukin-1 receptor-associated kinase M (IRAK-M) and mitogen-activated protein kinase 1 (MKP-1) while L. rhamnosus CRL2084 augmented ABIN-3 expression in BIE cells. The results of this work suggest that among the studied strains, L. mucosae CRL2069 was able to regulate TLR3-mediated innate immune response and showed a remarkable capacity to modulate TLR4-mediated inflammation in BIE cells. The CRL2069 strain induce the up-regulation of three TLR negative regulators that would influence nuclear factor kB and mitogen-activated protein kinases signalling pathways while reducing the expression of pro-inflammatory cytokines and chemokines. Therefore, L. mucosae CRL2069 is an interesting immunobiotic candidate for the protection of the bovine host against TLR-mediated intestinal inflammatory damage.

Modulation effect of Lactobacillus acidophilus KLDS 1.0738 on gut microbiota and TLR4 expression in Beta-lactoglobulin-induced allergic mice model

OBJECTIVES: Β-lactoglobulin (Β-Lg)-sensitized mice model was employed to investigate the correlation between Lactobacillus acidophilus KLDS 1.0738 (Lap KLDS 1.0738) modulating gut microbiota and inducting Toll-like receptors (TLRs) expression. METHODS: The alterations of mice fecal microbiota were analyzed by 16S rRNA gene sequencing. The serum cytokines production and TLR4/NF-κB mRNA expression in the colon tissues were measured by ELISA kit and quantitative RT-PCR, respectively. RESULTS: The results showed that Lap KLDS 1.0738 pretreatment attenuated Β-Lg-induced hypersensitivity, accompanied with a diminished expression of TLR4/NF-κB signaling. Moreover, oral administration of Lap KLDS 1.0738 improved the richness and diversity of fecal microbiota, which was characterized by fewer Proteobacteria phylum and Helicobacteraceae family, and higher Firmicutes phylum and Lachnospiraceae family than allergic group. Notably, TLR4/NF-κB expression was positively correlated with the family of Helicobacteraceae in allergic group, but negatively correlated with the family of Lachnospiraceae, Ruminococcaceae and anti-inflammatory cytokines level. A significant positive correlation was observed between TLR4/NF-κB expression and the production of histamine, total IgE and pro-inflammatory cytokines. CONCLUSIONS: Intake of Lap KLDS 1.0738 can influence the gut bacterial composition, which might result in recognizing TLRs signaling so as to inhibit allergic response

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Different cytokine patterns induced by Helicobacter pylori and Lactobacillus acidophilus extracts in PBMCs of patients with abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a degenerative inflammatory disease with unknown etiology. AAA is characterized by abdominal aortic dilatation more than 3 cm and is often asymptomatic, but the rupture of aneurysm can lead to death. Age, smoking and male sex are major predisposing factors of AAA. This study compares the effect of Helicobacter (H.) pylori and Lactobacillus (L.) acidophilus on the cytokine profile of PBMCs of 5 men with abdominal aortic aneurysm (AAA) and 5 men with normal/insignificant angiography, CT-Scan and ultrasonography results in the single-culture and in the co-culture with HUVECs. IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17 F, IL-21, IL-22, IFN-γ and TNF-α were measured in culture supernatants using a commercial fluorescent-labeled-bead assay. In general, CagA+ H. pylori-extract induced higher production of IFN-γ, IL-13 and IL-21 by PBMCs. Treatment of patients' PBMCs with CagA+H. pylori-extract induced Th2 cytokines while treatment of controls' PBMCs with CagA+H. pylori-extract increased Th1 cytokines. In the co-culture, however, patients' PBMCs produced Th1 cytokines irrespective of extract treatment, while controls' PBMCs produced Th2 cytokines and decreased IL-10. CagA+ H. pylori- as well as L. acidophilus-extract induced higher levels of IL-9 by controls' PBMCs in co-culture with HUVECs than patients (P = 0.05 and P = 0.01). The cytokine pattern of PBMCs induced by CagA+ H. pylori- and L. acidophilus-extracts in the co-culture with HUVECs shows differences in AAA patients and in comparison to controls. Decreased secretion of IL-9, IL-21 and IL-22 by PBMCs of patients treated with CagA+ H. pylori extract in co-culture, as opposed to non-AAA controls may indicate the active role ECs play in AAA. Simultaneous production of IL-10 and Th1 cytokines in patients and pronounced Th2 cytokines in controls in response to both bacteria may point to the inherent differences between patients and controls, which need further investigation.

Mannan Enhances IL-12 Production by Increasing Bacterial Uptake and Endosomal Degradation in L. acidophilus and S. aureus Stimulated Dendritic Cells.

The mannose receptor (MR) is a C-type lectin involved in endocytosis and with a poorly defined ability to modulate cellular activation. We investigated the effect of mannan treatment prior to stimulation of murine bone marrow-derived dendritic cells with the Gram-positive bacteria Lactobacillus acidophilus NCFM (L. acidophilus) on the induction of Interleukin (IL)-12. Mannan enhanced the IL-12 production induced by L. acidophilus in a dose dependent manner (up to 230% enhancement). Additionally, mannan-enhanced IL-12 induction was also demonstrated with another Gram-positive bacteria, Staphylococcus aureus (S. aureus), while an IL-12 reducing effect was seen on Escherichia coli stimulated cells. Furthermore, the expression of Interferon ß (Ifnb) was increased in cells treated with mannan prior to stimulation with L. acidophilus. The addition of mannan but not of bacteria led to endocytosis of MR, while addition of mannan prior to L. acidophilus or S. aureus resulted in increased endocytosis of bacteria, a faster killing of endocytosed bacteria, and increased reactive oxygen species production. Expression of signaling lymphocytic activation molecule (SLAMF)1 shown previously to be involved in the facilitation of endosomal degradation was upregulated by mannan but not by L. acidophilus and S. aureus. The IL-12 enhancement by mannan but not the IL-12 induced by the bacteria was abrogated by addition of inhibitors of clathrin coated pits (chlorpromazine and monodansylcadaverine). Furthermore, the addition of acid sphingomyelinase, a facilitator of ceramide raft formation, prior to addition of L. acidophilus enhanced the IL-12 production and the endocytosis of bacteria. In summary, our results show that mannan increases the IL-12 production induced by some Gram-positive bacteria through MR-endocytosis, which increases bacterial endocytosis and endosomal killing. The differential effect of MR activation on the IL-12 production induced by Gram-positive and Gram-negative bacteria may influence the immune response toward allergens and other glycoproteins.

Combined dietary supplementation of long chain inulin and Lactobacillus acidophilus W37 supports oral vaccination efficacy against Salmonella Typhimurium in piglets.

Routine use of antibiotics in livestock animals strongly contributed to the creation of multidrug-resistant Salmonella Typhimurium strains (STM). Vaccination is an alternative to the use of antibiotics but often suffers from low efficacy. The present study investigated whether long-chain inulin (lcITF) and Lactobacillus acidophilus W37 (LaW37) can support vaccination efficacy against STM and if the interventions influence possible gut microbiota changes. Piglets received daily supplementation until sacrifice. Animals were vaccinated on day 25 after birth, one day after weaning, and were challenged with STM on days 52-54. Dietary intervention with lcITF/LaW37 enhanced vaccination efficacy by 2-fold during challenge and resulted in higher relative abundance of Prevotellaceae and lower relative abundance of Lactobacillaceae in faeces. Although strongest microbial effects were observed post STM challenge on day 55, transient effects of the lcITF/LaW37 intervention were also detected on day 10 after birth, and post-weaning on day 30 where increased relative abundance of faecal lactobacilli was correlated with higher faecal consistency. LcITF treatment increased post-weaning feed efficiency and faecal consistency but did not support vaccination efficacy. Vaccination in immune-immature young animals can be enhanced with functional additives which can simultaneously promote health in an ingredient-dependent fashion.

High Mobility Group Box 1 and TLR4 Signaling Pathway in Gnotobiotic Piglets Colonized/Infected with L. amylovorus, L. mucosae, E. coli Nissle 1917 and S. Typhimurium.

High mobility group box 1 (HMGB1) is a DNA-binding nuclear protein that can be actively secreted by immune cells after different immune stimuli or passively released from cells undergoing necrosis. HMGB1 amplifies inflammation, and its hypersecretion contributes to multiple organ dysfunction syndrome and death. We tested possible immunomodulatory effect of commensal Lactobacillus amylovorus (LA), Lactobacillus mucosae (LM) or probiotic Escherichia coli Nissle 1917 (EcN) in infection of gnotobiotic piglets with Salmonella Typhimurium (ST). Transcription of HMGB1 and Toll-like receptors (TLR) 2, 4, and 9 and receptor for advanced glycation end products (RAGE), TLR4-related molecules (MD-2, CD14, and LBP), and adaptor proteins (MyD88 and TRIF) in the ileum and colon were measured by RT-qPCR. Expression of TLR4 and its related molecules were highly upregulated in the ST-infected intestine, which was suppressed by EcN, but not LA nor LM. In contrast, HMGB1 expression was unaffected by ST infection or commensal/probiotic administration. HMGB1 protein levels in the intestine measured by ELISA were increased in ST-infected piglets, but they were decreased by previous colonization with E. coli Nissle 1917 only. We conclude that the stability of HMGB1 mRNA expression in all piglet groups could show its importance for DNA transcription and physiological cell functions. The presence of HMGB1 protein in the intestinal lumen probably indicates cellular damage.

Nod2 is required for antigen-specific humoral responses against antigens orally delivered using a recombinant Lactobacillus vaccine platform.

Safe and efficacious orally-delivered mucosal vaccine platforms are desperately needed to combat the plethora of mucosally transmitted pathogens. Lactobacillus spp. have emerged as attractive candidates to meet this need and are known to activate the host innate immune response in a species- and strain-specific manner. For selected bacterial isolates and mutants, we investigated the role of key innate immune pathways required for induction of innate and subsequent adaptive immune responses. Co-culture of murine macrophages with L. gasseri (strain NCK1785), L. acidophilus (strain NCFM), or NCFM-derived mutants-NCK2025 and NCK2031-elicited an M2b-like phenotype associated with TH2 skewing and immune regulatory function. For NCFM, this M2b phenotype was dependent on expression of lipoteichoic acid and S layer proteins. Through the use of macrophage genetic knockouts, we identified Toll-like receptor 2 (TLR2), the cytosolic nucleotide-binding oligomerization domain containing 2 (NOD2) receptor, and the inflammasome-associated caspase-1 as contributors to macrophage activation, with NOD2 cooperating with caspase-1 to induce inflammasome derived interleukin (IL)-1ß in a pyroptosis-independent fashion. Finally, utilizing an NCFM-based mucosal vaccine platform with surface expression of human immunodeficiency virus type 1 (HIV-1) Gag or membrane proximal external region (MPER), we demonstrated that NOD2 signaling is required for antigen-specific mucosal and systemic humoral responses. We show that lactobacilli differentially utilize innate immune pathways and highlight NOD2 as a key mediator of macrophage function and antigen-specific humoral responses to a Lactobacillus acidophilus mucosal vaccine platform.

Biochemical characterization of a recombinant Lactobacillus acidophilus strain expressing exogenous FomA protein.

In previous research, to combine the immunogenicity of Fusobacterium nucleatum (F. nucleatum) and the probiotic properties of Lactobacillus acidophilus (L. acidophilus), we constructed a FomA-expressing L. acidophilus strain and assessed its immunogenicity. Our findings indicated that oral administration of the recombinant L. acidophilus strain reduced the risk of periodontal infection by Porphyromonas gingivalis (P. gingivalis) and F. nucleatum. However, because the exogenous FomA is an heterologous protein for the original bacterium, in this study, we assessed whether the biochemical characteristics of the recombinant L. acidophilus strain change due to the expression of the exogenous FomA protein. OBJECTIVES: To test the biochemical characteristics of a recombinant L. acidophilus strain expressing exogenous FomA and assess its antibiotic sensitivity. DESIGNS: We assessed the colony morphology, growth, acid production, and carbohydrate fermentation abilities of the recombinant L. acidophilus strain. In addition, we tested the adhesive ability and antimicrobial activity of the recombinant and assessed its antibiotic sensitivity through a drug susceptibility test. RESULTS: The experimental results showed that the colony and microscopic morphology of the recombinant L. acidophilus strain was consistent with the original strain, and the recombinant strain grew well when cultured under aerobic or anaerobic conditions, exhibiting a growth rate that was identical to that of the standard strain. Similarly, the supernatants of the recombinant L. acidophilus can inhibit the growth of E. coli and P. gingivalis at different concentrations, and the recombinant strain displayed essentially the same drug sensitivity profile as the original L. acidophilus. However, to our surprise, the recombinant strains exhibited a greater adhesion ability than the reference strain. CONCLUSIONS: Our study demonstrated that, in addition to an increased adhesion ability, the recombinant L. acidophilus strain maintained the basic characteristics of the standard strain ATCC 4356, including antibiotic sensitivity. Thus, the recombinant strains have great potential to be utilized as a safe and effective periodontitis vaccine in the future.

Lactobacillus acidophilus Improves Intestinal Inflammation in an Acute Colitis Mouse Model by Regulation of Th17 and Treg Cell Balance and Fibrosis Development.

Disruption of the balance among the microbiota, epithelial cells, and resident immune cells in the intestine is involved in the pathogenesis of inflammatory bowel disease (IBD). Probiotics exert protective effects against IBD, and probiotic commensal Lactobacillus species are common inhabitants of the natural microbiota, especially in the gut. To investigate the effects of Lactobacillus acidophilus on the development of IBD, L. acidophilus was administered orally in mice with dextran sodium sulfate (DSS)-induced colitis. DSS-induced damage and the therapeutic effect of L. acidophilus were investigated. Treatment with L. acidophilus attenuated the severity of DSS-induced colitis. Specifically, it suppressed proinflammatory cytokines such as interleukin (IL)-6, tumor necrosis factor-α, IL-1ß, and IL-17 in the colon tissues, which are produced by T helper (Th) 17 cells. Moreover, in vitro L. acidophilus treatment directly induced T regulatory (Treg) cells and the production of IL-10, whereas the production of IL-17 was suppressed in splenocytes. In addition, we found that L. acidophilus treatment decreased the levels of α-smooth muscle actin, a marker of activated myofibroblasts, and type I collagen compared with control mice. These results suggest that L. acidophilus may be a novel treatment for IBD by modulating the balance between Th17 and Treg cells, as well as fibrosis development.

Probiotics modulate gut microbiota and improve insulin sensitivity in DIO mice.

Obesity and type 2 diabetes are characterized by subclinical inflammatory process. Changes in composition or modulation of the gut microbiota may play an important role in the obesity-associated inflammatory process. In the current study, we evaluated the effects of probiotics (Lactobacillus rhamnosus, L. acidophilus and Bifidobacterium bifidumi) on gut microbiota, changes in permeability, and insulin sensitivity and signaling in high-fat diet and control animals. More importantly, we investigated the effects of these gut modulations on hypothalamic control of food intake, and insulin and leptin signaling. Swiss mice were submitted to a high-fat diet (HFD) with probiotics or pair-feeding for 5 weeks. Metagenome analyses were performed on DNA samples from mouse feces. Blood was drawn to determine levels of glucose, insulin, LPS, cytokines and GLP-1. Liver, muscle, ileum and hypothalamus tissue proteins were analyzed by Western blotting and real-time polymerase chain reaction. In addition, liver and adipose tissues were analyzed using histology and immunohistochemistry. The HFD induced huge alterations in gut microbiota accompanied by increased intestinal permeability, LPS translocation and systemic low-grade inflammation, resulting in decreased glucose tolerance and hyperphagic behavior. All these obesity-related features were reversed by changes in the gut microbiota profile induced by probiotics. Probiotics also induced an improvement in hypothalamic insulin and leptin resistance. Our data demonstrate that the intestinal microbiome is a key modulator of inflammatory and metabolic pathways in both peripheral and central tissues. These findings shed light on probiotics as an important tool to prevent and treat patients with obesity and insulin resistance.

Immunomodulatory effects and anti-Candida activity of lactobacilli in macrophages and in invertebrate model of Galleria mellonella.

Due to the growing number of multi-resistant Candida spp., adjuvant treatments that may help combat these fungal pathogens are relevant and useful. This study evaluated the immunomodulation and anti-Candida activity of Lactobacillus rhamnosus (LR), Lactobacillus acidophilus and Lactobacillus paracasei suspensions, either single- or multiple-strain, in mouse macrophages (RAW 264.7) and Galleria mellonella (GM). Mouse macrophages were activated by different lactobacilli suspensions and challenged with C. albicans (CA). Tumor necrosis factor (TNF)-α, interleukin IL-1ß, IL-6 and IL-17 production and cell viability were investigated. LR was the best suspension for stimulating all evaluated cytokines and thus was used in subsequent in vivo assays. Two C. albicans clinical strains, CA21 and CA60, were then added to the GM assays to further confirm the results. LR suspension was injected into the larvae 24 h before challenging with CA. Survival curve, CFU per larva and hemocytes were counted. In the GM, the LR suspension increased the survival rate and hemocyte counts and decreased the CFU per larva counts for all groups. Lactobacilli suspensions presented strain-dependent immunomodulation; however, single suspensions showed better results. Anti-Candida activity was demonstrated by decreased Candida counts in the GM with the use of LR.

Probiotics to prevent necrotising enterocolitis and nosocomial infection in very low birth weight preterm infants.

The aim of the study was to determine whether routine probiotic supplementation (RPS) with Lactobacillus rhamnosus GG (LGG) or Lactobacillus acidophilus +Lactobacillus bifidum is associated with reduced risk of necrotising enterocolitis (NEC)≥Stage II in preterm neonates born at ≤32 weeks' gestation. We conducted a retrospective cohort study on the effect of probiotic supplementation in very low birth weight infants in our neonatal unit by comparing two periods: before and after supplementation. The incidence of NEC≥Stage II, late-onset sepsis and all-cause mortality was compared for an equal period 'before' (Period I) and 'after' (Period II) RPS with LGG or L. acidophillus+L. bifidum. Multivariate logistic regression analysis was conducted to adjust for relevant confounders. The study population was composed of 261 neonates (Period I v. II: 134 v. 127) with comparable gestation duration and birth weights. In <32 weeks, we observed a significant reduction in NEC≥Stage II (11·3 v. 4·8 %), late-onset sepsis (16 v. 10·5 %) and mortality (19·4 v. 2·3 %). The benefits in neonates aged ≤27 weeks did not reach statistical significance. RPS with LGG or L. acidophillus+L. bifidum is associated with a reduced risk of NEC≥Stage II, late-onset sepsis and mortality in preterm neonates born at ≤32 weeks' gestation.

Adjuvant treatment with a symbiotic in patients with inflammatory non-allergic rhinitis.

Inflammatory non-allergic rhinitis (INAR) is characterized by the presence of an inflammatory infiltrate and a non-IgE-mediated pathogenesis. This retrospective, controlled, multicentre study investigated whether a symbiotic, containing Lactobacillus acidophilus NCFM, Bifidobacterium lactis, and fructo-oligosaccharides (Pollagen®, Allergy Therapeutics, Italy), prescribed as adjunctive therapy to a standard pharmacological treatment, was able to reduce symptom severity, endoscopic features, and nasal cytology in 93 patients (49 males and 44 females, mean age 36.3±7.1 years) with INAR. The patients were treated with nasal corticosteroid, oral antihistamine, and isotonic saline. At randomization, 52 patients were treated also with symbiotic as adjunctive therapy, whereas the remaining 41 patients served as controls. Treatment lasted for 4 weeks. Patients were visited at baseline, after treatment, and after 4-week follow-up. Adjunctive symbiotic treatment significantly reduced the percentages of patients with symptoms and endoscopic signs, and diminished inflammatory cells. In conclusion, the present study demonstrates that a symbiotic was able, as adjuvant treatment, to significantly improve symptoms, endoscopic feature, and cytology in patients with INAR, and its effect may be long lasting.

Dietary Lactobacillus acidophilus modulated skin mucus protein profile, immune and appetite genes expression in gold fish (Carassius auratus gibelio).

The objective of the present study was to investigate the effect of dietary Lactobacillus acidophilus on skin mucus protein pattern, immune and appetite related genes expression as well as growth performance in gold fish (Carassius auratus gibelio). Three hundred healthy gold fish (2.5 ± 0.05) juveniles were randomly distributed in 12 glass aquariums (400-L; 25 fish per aquaria) and fed experimental diets contain different levels of L. acidophilus (0, 1.5 × 108, 3 × 108 and 6 × 108) for 8 weeks. SDS-PAGE analysis of skin mucus protein profile at the end of the feeding trial revealed differences in protein profile of probiotic fed fish and control group; even three new bands were observed in L. acidophilus treated groups. Furthermore, fish fed 6 × 108 CFU g-1 supplemented diet showed up-regulation of both TNF-1α and TNF-2α gene expression (P < 0.05). Evaluation of appetite related gene expression showed down-regulation of ghrelin in probiotic fed fish compared those of control treatment (P < 0.05). However, administration of different levels of L. acidophilus had no significant effects on growth performance (P > 0.05). These results demonstrated that while no beneficial effects on growth performance, dietary L. acidophilus affects immune and appetite related genes expression as well as skin mucus protein profile.

Plasma membrane Toll-like receptor activation increases bacterial uptake but abrogates endosomal Lactobacillus acidophilus induction of interferon-ß.

Lactobacillus acidophilus induces a potent interferon-ß (IFN-ß) response in dendritic cells (DCs) by a Toll-like receptor 2 (TLR2) -dependent mechanism, in turn leading to strong interleukin-12 (IL-12) production. In the present study, we investigated the involvement of different types of endocytosis in the L. acidophilus-induced IFN-ß and IL-12 responses and how TLR2 or TLR4 ligation by lipopolysaccharide and Pam3/4CSK4 influenced endocytosis of L. acidophilus and the induced IFN-ß and IL-12 production. Lactobacillus acidophilus was endocytosed by constitutive macropinocytosis taking place in the immature cells as well as by spleen tyrosine kinase (Syk) -dependent phagocytosis but without involvement of plasma membrane TLR2. Stimulation with TLR2 or TLR4 ligands increased macropinocytosis in a Syk-independent manner. As a consequence, incubation of DCs with TLR ligands before incubation with L. acidophilus enhanced the uptake of the bacteria. However, in these experimental conditions, induction of IFN-ß and IL-12 was strongly inhibited. As L. acidophilus-induced IFN-ß depends on endocytosis and endosomal degradation before signalling and as TLR stimulation from the plasma membrane leading to increased macropinocytosis abrogates IFN-ß induction we conclude that plasma membrane TLR stimulation leading to increased macropinocytosis decreases endosomal induction of IFN-ß and speculate that this is due to competition between compartments for molecules involved in the signal pathways. In summary, endosomal signalling by L. acidophilus that leads to IFN-ß and IL-12 production is inhibited by TLR stimulation from the plasma membrane.