Ex vivo peripheral blood mononuclear cell response to R848 in children after supplementation with the probiotic Lactobacillus acidophilus NCFM/Bifidobacterium lactis Bi-07.

Several studies have demonstrated a decrease in upper respiratory infection (URI) frequency and severity in subjects taking probiotic supplements. We hypothesised beneficial effects of probiotics on viral URI in children are due to modulation of inflammatory innate immune responses. We tested this hypothesis, providing children with a probiotic combination of Lactobacillus acidophilus/Bidfidobacterium animalis ssp. lactis Bi-07 (NCFM/Bi-07) and measuring levels of cytokines in response to stimulation of peripheral blood mononuclear cells (PBMCs) to toll-like receptor (TLR) 7/8 agonist resiquimod (R848). In this open label study, 21 (2 dropouts) children received probiotic containing 5×109 cfu each of NCFM/(Bi-07) daily for 30 days. Whole blood was taken from each subject at study entry and 30 days for culture of PBMCs. PBMCs stimulated with resiquimod (R848) or unstimulated were incubated and a panel of immune markers was measured. There was a significant decrease in the net (stimulated-null) level of myeloid progenitor inhibitory factor 1 (MPIF-1) (mean decrease 0.1 ng/ml, 95% confidence interval 0.01-0.24, P=0.032) following probiotic supplementation. The change in immune marker levels after supplementation, when analysed together with respect to expected inflammatory/anti-inflammatory effects, was increased for interleukin (IL)-10 and decreased for MPIF-1, IL-8, interferon gamma induced protein 10, macrophage inflammatory protein 3 alpha (MIP-3α) and E-selectin (P=0.01). Adverse events were mild. In conclusion, supplementation with this probiotic combination was safe and resulted in significant modulation of PBMC limited immune response to TLR7/8 agonist R848 and in levels of MPIF-1 and MIP-3α. The anti-inflammatory effect may be one mechanism by which probiotics modulate the immune system however further study is needed.

Importance of the gastrointestinal life cycle of Bacillus for probiotic functionality.

Bacillus spp. are widely used in animal production for their probiotic properties. In many animal species, feed supplementation with specific Bacillus strains can provide numerous benefits including improvement in digestibility, the gut microbiota and immune modulation, and growth performance. Bacilli are fed to animals as spores that can sustain the harsh feed processing and long storage. However, the spores are metabolically quiescent and it is widely accepted that probiotics should be in a metabolically active state to perform certain probiotic functions like secretion of antimicrobial compounds and enzymes, synthesis of short chain fatty acids, and competition for essential nutrients. These functions should become active in the host gastrointestinal tract (GIT) soon after digestion of spores in order to contribute to microbiota and host metabolism. Considering that bacterial spores are metabolically dormant and many health benefits are provided by vegetative cells, it is of particular interest to discuss the life cycle of Bacillus in animal GIT. This review aims to capture the main characteristics of spores and vegetative cells and to discuss the latest knowledge in the life cycle of beneficial Bacillus in various intestinal environments. Furthermore, we review how the life cycle may influence probiotic functions of Bacillus and their benefits for human and animal health.

Effect of probiotic on innate inflammatory response and viral shedding in experimental rhinovirus infection - a randomised controlled trial.

Ingestion of probiotics appears to have modest effects on the incidence of viral respiratory infection. The mechanism of these effects is not clear; however, there is evidence from animal models that the probiotic may have an effect on innate immune responses to pathogens. The purpose of this randomised, placebo-controlled study was to determine the effect of administration of Bifidobacterium animalis subspecies lactis Bl-04 on innate and adaptive host responses to experimental rhinovirus challenge. The effect on the response of chemokine (C-X-C motif) ligand 8 (CXCL8) to rhinovirus infection was defined as the primary endpoint for the study. 152 seronegative volunteers who had been supplemented for 28 days, 73 with probiotic and 79 with placebo, were challenged with RV-A39. Supplement or placebo administration was then continued for five days during collection of specimens for assessment of host response, infection, and symptoms. 58 probiotic and 57 placebo-supplemented volunteers met protocol-defined criteria for analysis. Probiotic resulted in higher nasal lavage CXCL8 on day 0 prior to virus challenge (90 vs 58 pg/ml, respectively, P=0.04, ANCOVA). The CXCL8 response to rhinovirus infection in nasal lavage was significantly reduced in the probiotic treated group (P=0.03, ANCOVA). Probiotic was also associated with a reduction in nasal lavage virus titre and the proportion of subjects shedding virus in nasal secretions (76% in the probiotic group, 91% in the placebo group, P=0.04, Fisher Exact test). The administration of probiotic did not influence lower respiratory inflammation (assessed by exhaled nitric oxide), subjective symptom scores, or infection rate. This study demonstrates that ingestion of Bl-04 may have an effect on the baseline state of innate immunity in the nose and on the subsequent response of the human host to rhinovirus infection. Clinicaltrials.gov registry number: NCT01669603.

Inflammatory cues enhance TGFß activation by distinct subsets of human intestinal dendritic cells via integrin αvß8.

Regulation of intestinal T-cell responses is crucial for immune homeostasis and prevention of inflammatory bowel disease (IBD). A vital cytokine in regulating intestinal T cells is transforming growth factor-ß (TGFß), which is secreted by cells as a latent complex that requires activation to function. However, how TGFß activation is regulated in the human intestine, and how such pathways are altered in IBD is completely unknown. Here we show that a key activator of TGFß, integrin αvß8, is highly expressed on human intestinal dendritic cells (DCs), specifically on the CD1c+ but not the CD141+ intestinal DC subset. Expression was significantly upregulated on intestinal DC from IBD patients, indicating that inflammatory signals may upregulate expression of this key TGFß-activating molecule. Indeed, we found that the Toll-like receptor 4 ligand lipopolysaccharide upregulates integrin αvß8 expression and TGFß activation by human DC. We also show that DC expression of integrin αvß8 enhanced induction of FOXP3 in CD4+ T cells, suggesting functional importance of integrin αvß8 expression by human DC. These results show that microbial signals enhance the TGFß-activating ability of human DC via regulation of integrin αvß8 expression, and that intestinal inflammation may drive this pathway in patients with IBD.

Effects of probiotic supplementation over 5 months on routine haematology and clinical chemistry measures in healthy active adults.

Use of probiotic-containing foods and probiotic supplements is increasing; however, few studies document safety and tolerability in conjunction with defined clinical end points. This paper reports the effects of 150 days of supplementation with either a single- (Bifidobacterium animalis subsp. lactis Bl-04) or a double-strain (Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp. lactis Bi-07) probiotic on routine haematology and clinical chemistry measures in healthy active adults. Pre- to post-intervention changes in laboratory measures were determined and compared between supplement and placebo groups. Overall there were few differences in routine haematology and clinical chemistry measures between supplement and placebo groups post-intervention. Exceptions included plasma calcium (P=0.03) and urea (P=0.015); however, observed changes were small and within assay-specific laboratory reference ranges. These data provide evidence supporting the use of these probiotic supplements over a period of 5 months in healthy active adults without obvious safety or tolerability issues.

Microbes bind complement inhibitor factor H via a common site.

To cause infections microbes need to evade host defense systems, one of these being the evolutionarily old and important arm of innate immunity, the alternative pathway of complement. It can attack all kinds of targets and is tightly controlled in plasma and on host cells by plasma complement regulator factor H (FH). FH binds simultaneously to host cell surface structures such as heparin or glycosaminoglycans via domain 20 and to the main complement opsonin C3b via domain 19. Many pathogenic microbes protect themselves from complement by recruiting host FH. We analyzed how and why different microbes bind FH via domains 19-20 (FH19-20). We used a selection of FH19-20 point mutants to reveal the binding sites of several microbial proteins and whole microbes (Haemophilus influenzae, Bordetella pertussis, Pseudomonas aeruginosa, Streptococcus pneumonia, Candida albicans, Borrelia burgdorferi, and Borrelia hermsii). We show that all studied microbes use the same binding region located on one side of domain 20. Binding of FH to the microbial proteins was inhibited with heparin showing that the common microbial binding site overlaps with the heparin site needed for efficient binding of FH to host cells. Surprisingly, the microbial proteins enhanced binding of FH19-20 to C3b and down-regulation of complement activation. We show that this is caused by formation of a tripartite complex between the microbial protein, FH, and C3b. In this study we reveal that seven microbes representing different phyla utilize a common binding site on the domain 20 of FH for complement evasion. Binding via this site not only mimics the glycosaminoglycans of the host cells, but also enhances function of FH on the microbial surfaces via the novel mechanism of tripartite complex formation. This is a unique example of convergent evolution resulting in enhanced immune evasion of important pathogens via utilization of a "superevasion site."

A phylogeny of members of the family Taeniidae based on the mitochondrial cox1 and nad1 gene data.

The cestode family Taeniidae consists of 2 genera, Taenia and Echinococcus, which both have been the focus of intensive taxonomic and epidemiological studies because of their zoonotic importance. However, a comprehensive molecular phylogeny of this family has yet to be reconstructed. In this study, 54 isolates representing 9 Taenia species were characterized using DNA sequences in the mitochondrial cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes. Phylogenetic relationships within the family Taeniidae were inferred by combining cox1 and nad1 sequence data of the present and previous studies. In the phylogenetic analysis, the genus Echinococcus was shown to be monophyletic, but Taenia proved to be paraphyletic due to the position of T. mustelae as a probable sister taxon of Echinococcus. This indicates that T. mustelae should form a genus of its own. Taenia ovis krabbei was placed distant from T. ovis ovis, as a sister taxon of T. multiceps, supporting its recognition as a distinct species, T. krabbei. High intraspecific sequence variation within both T. polyacantha and T. taeniaeformis suggests the existence of cryptic sister species.

Molecular characterization of Echinococcus isolates of cervid origin from Finland and Sweden.

The species Echinococcus granulosus is made up of several genotypic strain groups, whose taxonomical classification is still undetermined. Genotypes in the cervid-wolf life-cycle are poorly known, especially in Europe. In this study, 33 Echinococcus isolates from cervids from Finland and Sweden were characterized using mitochondrial ND1 gene sequencing. In addition, phylogenetic analysis of E. granulosus strains using the mitochondrial ATP6, ND1, ND3 and CO1 genes was performed using maximum likelihood, neighbour-joining and maximum parsimony methods. The Finnish and Swedish cervid isolates were found to represent the genotype G10. In the phylogenetic analyses, the camel (G6), pig (G7), cervid (G8) and Fennoscandian cervid (G10) strains clustered in a well-supported monophyletic group. This group differed clearly from the common sheep (G1) and horse (G4, 'E. equinus') strains, but was closely related to the cattle strain (G5, 'E. ortleppi'). Our results support the previous studies suggesting that the genotypes G6-10 should be separated from the species E. granulosus sensu stricto. However, additional morphological studies are needed, and the relationship to the cattle strain ('E. ortleppi') should be thoroughly evaluated before a final decision of the taxonomical status of the G6-10 group can be made.

Molecular genetic characterization of the Fennoscandian cervid strain, a new genotypic group (G10) of Echinococcus granulosus.

The northern biotype of Echinococcus granulosus occurs in North America and northern Eurasia in life-cycles involving cervids. Previously, cervid isolates of E. granulosus from North America have been characterized using molecular genetic techniques as the G8 genotype. In this study, 5 isolates of E. granulosus were collected from 4 reindeer and 1 moose in north-eastern Finland. DNA sequences within regions of mitochondrial cytochrome c oxidase I (COI) and NADH dehydrogenase I (NI)I) genes and the internal transcribed spacer 1 (ITS-1) fragment of the ribosomal DNA were analysed. The mitochondrial nucleotide sequences were identical in all isolates, but high sequence variation was found in the ITS-1 region. Mitochondrial and nuclear sequences of the Finnish cervid E. granulosus and the camel strain (G6) of E. granulosus resembled closely each other. According to phylogenetic analyses, the Finnish isolates have close relationships also with the pig (G7) and cattle (G5) strains. Although some similarities were found with the previously published North American cervid strain (G8), particularly in the NDI sequence and some of the ITS-1 clones, the Finnish E. granulosus form represents a distinct, previously undescribed genotype of E. granulosus. The novel genotype is hereby named as the Fennoscandian cervid strain (G10).