Novel LysM motifs for antigen display on lactobacilli for mucosal immunization.

We characterized two LysM domains of Limosilactobacillus fermentum, belonging to proteins Acglu (GenBank: KPH22907.1) and Pgb (GenBank: KPH22047.1) and bacterium like particles (BLP) derived from the immunomodulatory strain Lacticaseibacillus rhamnosus IBL027 (BLPs027) as an antigen display platform. The fluorescence protein Venus fused to the novel LysM domains could bind to the peptidoglycan shell of lactobacilli and resisted harsh conditions such as high NaCl and urea concentrations. Acglu with five LysM domains was a better anchor than Pgb baring only one domain. Six-week-old BALB/c mice were nasally immunized with the complex Venus-Acglu-BLPs027 at days 0, 14 and 28. The levels of specific serum IgG, IgG1 and IgG2a and the levels of total immunoglobulins (IgT) and IgA in broncho-alveolar lavage (BAL) were evaluated ten days after the last boosting. Venus-Acglu-BLPs027, nasally administered, significantly increased specific BAL IgT and IgA, and serum IgG levels. In addition, spleen cells of mice immunized with Venus-Acglu-BLPs027 secreted TNF-α, IFN-γ and IL-4 when stimulated ex vivo in a dose-dependent manner. We constructed a Gateway compatible destination vector to easily fuse the selected LysM domain to proteins of interest for antigen display to develop mucosal subunit vaccines.

Improvement of Myelopoiesis in Cyclophosphamide-Immunosuppressed Mice by Oral Administration of Viable or Non-Viable Lactobacillus Strains.

Myelosuppression is the major dose-limiting toxicity of cancer chemotherapy. There have been many attempts to find new strategies that reduce myelosuppression. The dietary supplementation with lactic acid bacteria (LAB) improved respiratory innate immune response and the resistance against respiratory pathogens in immunosupressed hosts. Although LAB viability is an important factor in achieving optimal protective effects, non-viable LAB are capable of stimulating immunity. In this work, we studied the ability of oral preventive administration of viable and non-viable Lactobacillus rhamnosus CRL1505 or L. plantarum CRL1506 (Lr05, Lr05NV, Lp06V or Lp06NV, respectively) to minimize myelosuppressive and immunosuppressive effects derived from chemotherapy. Cyclophosphamide (Cy) impaired steady-state myelopoiesis in lactobacilli-treated and untreated control mice. Lr05V, Lr05NV and Lp06V treatments were the most effective to induce the early recovery of bone marrow (BM) tissue architecture, leukocytes, myeloid, pool mitotic and post-mitotic, peroxidase positive, and Gr-1Low/High cells in BM. We selected the CRL1505 strain for being the one capable of maintaining its myelopoiesis-enhancing properties in its non-viable form. Although the CRL1505 treatments do not modify the Cy ability to induce apoptosis, both increased the incorporation of BrdU in BM cells. Consequently, Lr05NV and Lr05V treatments were able to promote early recovery of LSK cells (Lin-Sca-1+c-Kit+ cells), multipotent progenitors (Lin-Sca-1+c-Kit+CD34+ cells), and myeloid cells (Gr-1+Ly6G+Ly6C- cells) with respect to the untreated Cy control. In addition, these treatments were able to increase the frequency of IL17A-producing innate lymphoid cells in the intestinal lamina propria (IL-17A+RORγt+CD4-NKp46+ cells) after Cy injection. These results were correlated with an increase in the IL-17A serum levels, a GM-CSF high expression and a CXCL12 lower expression in BM. Therefore, both Lr05V and Lr05NV treatments are able to activate beneficially the IL-17A/GM-CSF axis and accelerate the recovery of Cy-induced immunosuppression by increasing BM myeloid precursors. We demonstrated for the first time the beneficial effect of CRL1505 strain on myelopoiesis affected by a chemotherapeutic drug. Furthermore, Lr05NV could be a good and safe resource for reducing chemotherapy-induced leukopenia. The results are a starting point for future research and open up broad prospects for future applications of the immunobiotics.

Facilitating the gut brain axis by probiotic bacteria to modulate neuroimmune response on lead exposed zebra fish models

Unravelling the efficacy of gut biome has a major impact on health. An unbalanced microbiome composition is linked to many common illnesses such as gut dysbiosis, mental deformities and immunological imbalance. An optimistic influence on the gut biome can be made by consumingprobiotics. This would stimulate neuroprotection and immunomodulation intended by heavy metals pollution. Lead is a major source of neurotoxin that can induce neural deformities. Lactobacillusspecies isolated from curd were characterized to confirm its specificity. Zebra fish was reared at standard conditions and preclinical assessment on the intensity of induced neurotoxin lead was performed. The embryo toxic assay, immunomodulation effects and animal behavioural models endorsed the consequence of neurotoxicity. Different concentrations of bacterial isolate with standard antidepressant was considered for analysing the vigour of toxicity and its influence on cognitive behaviour by novel tank diving method. The restrain in the animal behaviour was also conferred by all the test samples with a decreased bottom dwelling time which was authenticated with haematology and histopathological studies. The alterations in morphology of the lymphocytes were balanced by the treated test samples. This study paves a twofold potential of probiotic as neuroprotectant and immune modulator against heavy metal toxicity.

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.

S-Layer Glycoprotein From Lactobacillus kefiri Exerts Its Immunostimulatory Activity Through Glycan Recognition by Mincle.

The development of new subunit vaccines has promoted the rational design of adjuvants able to induce a strong T-cell activation by targeting specific immune receptors. The S-layer is a (glyco)-proteinaceous envelope constituted by subunits that self-assemble to form a two-dimensional lattice that covers the surface of different species of Bacteria and Archaea. Due to their ability to self-assemble in solution, they are attractive tools to be used as antigen/hapten carriers or adjuvants. Recently, we have demonstrated that S-layer glycoprotein from Lactobacillus kefiri CIDCA 8348 (SLP-8348) enhanced the LPS-induced response on macrophages in a Ca2+-dependent manner, but the receptors involved in these immunomodulatory properties remain unknown. Therefore, we aim to determine the C-type lectin receptors (CLRs) recognizing this bacterial surface glycoprotein as well as to investigate the role of glycans in both the immunogenicity and adjuvant capacity of SLP-8348. Here, using a mild periodate oxidation protocol, we showed that loss of SLP-8348 glycan integrity impairs the cell-mediated immune response against the protein. Moreover, our data indicate that the adjuvant capacity of SLP-8348 is also dependent of the biological activity of the SLP-8348 glycans. In order to evaluate the CLRs involved in the interaction with SLP-8348 an ELISA-based method using CLR-hFc fusion proteins showed that SLP-8348 interacts with different CLRs such as Mincle, SingR3, and hDC-SIGN. Using BMDCs derived from CLR-deficient mice, we show that SLP-8348 uptake is dependent of Mincle. Furthermore, we demonstrate that the SLP-8348-induced activation of BMDCs as well as its adjuvant capacity relies on the presence of Mincle and its signaling adaptor CARD9 on BMDCs, since SLP-8348-activated BMDCs from Mincle-/- or CARD9-/- mice were not capable to enhance OVA-specific response in CD4+ T cells purified from OT-II mice. These findings significantly contribute to the understanding of the role of glycans in the immunomodulation elicited by bacterial SLPs and generate a great opportunity in the search for new adjuvants derived from non-pathogenic microorganisms.

Effect of inoculants combining Lactobacillus buchneri strain LN40177 in different strata of the silo

The objective of this work was to evaluate the chemical composition and rumen disappearance rate of dry matter of corn silages with inoculants combining L. buchneri strain LN40177 in different strata of the silo. The experimental design was a 3x2 randomized complete block design, with three treatments: Control: corn silage without inoculant; 11CFT: corn silage with inoculant which combines L. buchneri strain LN40177 (1.1 x 1011 CFU g-1 ) with L. casei (1.1 x 1011 CFU g-1 ); and 11C33: corn silage with inoculant which combines L. buchneri strain LN40177 (1.1 x 1011 CFU g-1 ) with L. plantarum (1.1 x 1011 CFU g-1 ) and Enterococcus faecium (1 x 1010 CFU g-1 ), associated with two strata of the silo (lower and upper). The silage inoculated with 11C33 presented higher contents of crude protein and NDF and lower hemicellulose content in relation to the control treatment and 11CFT. The use of both inoculants resulted in silages with higher concentrations of soluble nutrients. Lower stratum silage had a higher rumen disappearance rate of dry matter compared to the upper stratum. In general, the combinations of L. buchneri promoted nutritional improvements in corn silage, but in presence of L. casei, there were more outstanding improvements.(AU)

Effect of inoculants combining Lactobacillus buchneri strain LN40177 in different strata of the silo

The objective of this work was to evaluate the chemical composition and rumen disappearance rate of dry matter of corn silages with inoculants combining L. buchneri strain LN40177 in different strata of the silo. The experimental design was a 3x2 randomized complete block design, with three treatments: Control: corn silage without inoculant; 11CFT: corn silage with inoculant which combines L. buchneri strain LN40177 (1.1 x 1011 CFU g-1 ) with L. casei (1.1 x 1011 CFU g-1 ); and 11C33: corn silage with inoculant which combines L. buchneri strain LN40177 (1.1 x 1011 CFU g-1 ) with L. plantarum (1.1 x 1011 CFU g-1 ) and Enterococcus faecium (1 x 1010 CFU g-1 ), associated with two strata of the silo (lower and upper). The silage inoculated with 11C33 presented higher contents of crude protein and NDF and lower hemicellulose content in relation to the control treatment and 11CFT. The use of both inoculants resulted in silages with higher concentrations of soluble nutrients. Lower stratum silage had a higher rumen disappearance rate of dry matter compared to the upper stratum. In general, the combinations of L. buchneri promoted nutritional improvements in corn silage, but in presence of L. casei, there were more outstanding improvements.

Probiotic Lactobacillus Strains Stimulate the Inflammatory Response and Activate Human Macrophages.

Lactobacilli have been shown to promote health functions. In this study, we analyzed the mechanism by which four different strains of probiotics affected innate immunity, such as regulation of ROS, cytokines, phagocytosis, bactericidal activity, signaling by NF-κB pp65, and TLR2 activation. The production of ROS was dependent on the concentration and species of Lactobacillus. The results obtained from the tested strains (Lactobacillus rhamnosus GG, L. rhamnosus KLSD, L. helveticus IMAU70129, and L. casei IMAU60214) showed that strains induced early proinflammatory cytokines such as IL-8,TNF-α, IL-12p70, and IL-6. However, IL-1ß expression was induced only by L. helveticus and L. casei strains (after 24 h stimulation). Phagocytosis and bactericidal activity of macrophages against various pathogens, such as S. aureus, S. typhimurium, and E. coli, were increased by pretreatment with Lactobacillus. The nuclear translocation NF-κB pp65 and TLR2-dependent signaling were also increased by treatment with the probiotics. Taken together, the experiments demonstrate that probiotic strains of Lactobacillus exert early immunostimulatory effects that may be directly linked to the initial inflammation of the response of human macrophages.

Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) stimulates murine macrophages infected with Citrobacter rodentium.

Citrobacter rodentium is a specific murine enteropathogen which causes diarrheal disease characterized by colonic hyperplasia and intestinal inflammation. Recruitment of neutrophils and macrophages constitute a key step to control the infection. Since modulation of the activity of professional phagocytic cells could contribute to improve host´s defences against C. rodentium, we investigated the effect of Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) on the interaction between murine macrophages (RAW 264.7) and C. rodentium. Phagocytosis, surface molecules and inducible nitric oxide synthase (iNOs) expression were determined by flow cytometry. Reactive oxygen species (ROS) were assessed by fluorescence microscopy. The presence of lactobacilli increased phagocytosis of C. rodentium whereas C. rodentium had no effect on lactobacilli internalization. Survival of internalized C. rodentium diminished when strain CIDCA 133 was present. CD-86, MHCII, iNOs expression and nitrite production were increased when C. rodentium and lactobacilli were present even though strain CIDCA 133 alone had no effect. Strain CIDCA 133 led to a strong induction of ROS activity which was not modified by C. rodentium. Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) is able to increase the activation of murine macrophages infected with C. rodentium. The sole presence of lactobacilli is enough to modify some stimulation markers (e.g. ROS induction) whereas other markers require the presence of both bacteria; thus, indicating a synergistic effect.

Effect of Lactobacillus sp. isolates supernatant on Escherichia coli O157: H7 enhances the role of organic acids production as a factor for pathogen control / Efeito do sobrenadante de isolados de Lactobacillus sp. sobre Escherichia coli O157: H7 reforça o papel da produção de ácidos orgânicos como fator de controle patogênico

Many attempts have been made to establish the control of foodborne pathogens through Lactobacillus isolates and their metabolism products with success being obtained in several situations. The aim of this study was to investigate the antagonistic effect of eight Lactobacillus isolates, including L. casei subsp. pseudoplantarum, L. plantarum, L. reuteri and L. delbrueckii subsp. delbrueckii, on the pathogenic Escherichia colistrain O157:H7. The inhibitory effect of pure cultures and two pooled cultures supernatants of Lactobacillus on the growth of pathogenic bacteria was evaluated by the spot agar method and by monitoring turbidity. Antimicrobial activity was confirmed for L. reuteri and L. delbrueckii subsp. delbrueckii and for a pool of lactic acid bacteria. The neutralized supernatant of the pool exerted a higher antimicrobial activity than that of the individual strains. Furthermore, D-lactic acid and acetic acid were produced during growth of the Lactobacillus isolates studied.(AU)

Muitas tentativas têm sido feitas para se estabelecer o controle de patógenos de origem alimentar através do uso de estirpes de Lactobacillus e dos seus produtos de metabolismo, com sucesso sendo sucedido em várias situações. O objetivo deste trabalho foi investigar o efeito antagônico do sobrenadante de culturas de oito isolados de Lactobacillus, incluindo L. casei subsp. pseudoplantarum, L. plantarum L. reuteri e L. delbrueckii subsp. delbrueckii, sobre Escherichia coli amostra O157:H7. Os efeitos inibidores de culturas puras e de dois "pools" de cultura de Lactobacillus sobre o crescimento da bactéria foram avaliados através do método de inibição em ágar e através do monitoramento da turbidez da cultura bacteriana. A atividade antimicrobiana foi confirmada para Lactobacillus reuteri e Lactobacillus delbrueckii subsp. delbrueckii e para o "pool" de bactérias acido-láctica. O sobrenadante neutralizado do "pool" de Lactobacillus exerceu uma atividade antimicrobiana mais elevada do que aquela das estirpes individuais. Além disso, ácido D-láctico e ácido acético foram produzidos durante o crescimento dos Lactobacillusestudados.(AU)

Protective effects of lactococci strains delivering either IL-10 protein or cDNA in a TNBS-induced chronic colitis model.

BACKGROUND: Oral treatment with Lactococcus lactis strains secreting the anti-inflammatory cytokine interleukin (IL)-10 has previously shown success as a therapy for inflammatory bowel diseases (IBD). GOALS: Our aim was to compare the protective effects of IL-10, delivered by recombinant lactoccoci using 2 novel expression systems, in a murine colitis model mimicking the relapsing nature of IBD. The first system is based on a Stress-Inducible Controlled Expression system for the production and delivery of heterologous proteins at mucosal surfaces and the second allows the delivery to the host cells of an il-10 cDNA cassette, harbored in a eukaryotic DNA expression vector (pValac). STUDY: Colitis was induced in female BALB/c mice by intrarectal injection of 2,4,6-trinitrobenzenesulphonic acid (TNBS). Mice that recovered received one of the bacteria treatments or saline solution orally during 14 days. Colitis was reactivated 25 days after the first TNBS injection with a second TNBS challenge. Three days after colitis reactivation, cytokine profiles and inflammation in colon samples were evaluated. RESULTS: Animals (N=9) receiving L. lactis strains secreting IL-10 using Stress-Inducible Controlled Expression system or delivering pValac:il-10 plasmid showed lower weight loss (P<0.005), lower damage scores (P<0.005), and immune activation in their large intestines compared with inflamed nontreated mice. CONCLUSIONS: Our results confirm the protective effect of IL-10 delivered either as a protein or as a cDNA in a colitis model mimicking the relapsing nature of IBD and provides a step further in the "proof-of-concept" of genetically engineered bacteria as a valid system to deliver therapeutic molecules at mucosal level.

Effect of intestinal colonisation by two Lactobacillus strains on the immune response of gnotobiotic mice.

The effect of intestinal colonisation on the immune system was investigated in germ-free mice monoassociated with Lactobacillus strains isolated from calf faeces. Single doses of Lactobacillus acidophilus L36 or Lactobacillus salivarius L38 were administered to germ-free mice by intragastric gavage. Ten days later, the mice were euthanised. Gene expression levels of interleukin 5 (IL-5), IL-6, IL-10, IL-12b, IL-17a, gamma interferon (IFN-γ), transforming growth factor beta 1 (TGF-ß1), and tumour necrosis factor alpha (TNF-α) were quantified in segments of the small and large intestines by real time quantitative polymerase chain reaction. All the mice were colonised rapidly after Lactobacillus administration with intestinal counts ranging from 6.53 to 8.26 log cfu/g. L. acidophilus L36 administration increased the expression of cytokines involved with the Th2 (IL-5, IL-6 and TGF-ß1) and Th17 (IL-17a, TNF-α and IL-6) inflammatory response, whereas L. salivarius L38 appeared to stimulate a pattern of less diversified cytokines in the intestine. Intragastric gavage of L. acidophilus L36 and L. salivarius L38 induced similar levels of colonisation in the digestive tracts of germ-free mice but stimulated different immune responses in the intestinal mucosa. The different immunomodulation patterns might facilitate the potential use of these lactobacilli as probiotics to treat distinct pathological conditions, for example protection against Citrobacter rodentium infection by stimulating IL-17 production.

Single or combined effects of Lactobacillus sakei and inulin on growth, non-specific immunity and IgM expression in leopard grouper (Mycteroperca rosacea).

The aim of this study was to evaluate the single or combined effects of Lactobacillus sakei with inulin suitable for immunological in vivo studies in farmed fish. By in vitro assays, L. sakei strain 5-4 showed antibacterial activities against all assayed fish pathogens (except the Vibrio harveyi strain CAIM-1793). L. sakei was able to survive at high fish bile concentrations. Fermentation of the agave inulin resulted in a large increase in number of lactobacilli. For the in vivo study, fish were fed for 8 weeks four practical diets: control diet (control), L. sakei 5-4 (10(7) CFU/g), inulin (1% or 10 g/kg) and L. sakei + inulin (10(7) CFU/g + 10 g/kg). The weight gain showed clearly the synergistic effect of L. sakei 5-4 and inulin at 6 and 8 weeks of treatments. Leopard grouper fed with L. sakei alone or combined with inulin have significantly increased the assayed physiological and humoral immune parameters. By real-time PCR assays, the mRNA transcripts of immunoglobulin M (IgM) were found to be higher expressed in intestine, head kidney, mucus, gill, spleen and skin. Moreover, mRNA expression levels of IgM in head kidney and anterior intestine were measured by real-time PCR. L. sakei 5-4 and L. sakei + inulin supplemented diet up-regulated the expression of IgM at week 4 and 8 in intestine and head kidney, respectively. These results support the idea that the L. sakei 5-4 alone or combined with agave inulin improved growth performance and stimulates the immune system of leopard grouper.

Immunological and microbiologic changes during caries development in young children.

We explored the association between caries development, colonization with caries-associated microflora, and immunity as children begin the transition to mixed dentition. Forty children received dental examinations at 3-4 years of age, repeated a year later. Children were grouped into caries-free (n = 23; CF) and caries-active (n = 17; CA ≥3 new lesions on follow-up). Salivary IgA and IgA antibody to Streptococcus mutans virulence epitopes were measured by Luminex assay. Mutans streptococci (MS), lactobacilli and total microorganisms were enumerated on selective media from plaque samples. There was no significant difference in baseline levels of MS or lactobacilli between CF and CA groups. However, both MS and lactobacilli levels were higher at follow-up in the CA group. Furthermore, children with detectable lactobacilli at baseline had significantly higher caries risk. Salivary IgA concentrations increased significantly in both groups during the study. Both CF and CA groups also displayed significant increases in salivary IgA antibody levels to glucosyltransferase, glucan-binding protein (Gbp) and antigen I/II salivary binding region. CF antibody levels to seven peptides associated with domains of biological importance increased at follow-up, in contrast to increases to only three peptides in CA saliva samples. Multivariate modeling showed that a lower baseline level of salivary IgA anti-GbpB was associated with higher caries risk. These data indicate that MS and lactobacilli are associated with caries in this population, that the secretory immune system is undergoing significant maturation during this period, and that the breadth of mucosal IgA response to epitopes of S. mutans virulence components may influence the degree to which these cariogenic microorganisms can cause disease.

Lactic acid bacteria in the prevention of pneumococcal respiratory infection: future opportunities and challenges.

Lactic acid bacteria (LAB) are technologically and commercially important and have various beneficial effects on human health. Several studies have demonstrated that certain LAB strains can exert their beneficial effect on the host through their immunomudulatory activity. Although most research concerning LAB-mediated enhanced immune protection is focused on gastrointestinal tract pathogens, recent studies have centered on whether these immunobiotics might sufficiently stimulate the common mucosal immune system to provide protection to other mucosal sites as well. In this sense, LAB have been used for the development of probiotic foods with the ability to stimulate respiratory immunity, which would increase resistance to infections, even in immunocompromised hosts. On the other hand, the advances in the molecular biology of LAB have enabled the development of recombinant strains expressing antigens from respiratory pathogens that have proved effective to induce protective immunity. In this review we examine the current scientific literature concerning the use of LAB strains to prevent respiratory infections. In particular, we have focused on the works that deal with the capacity of probiotic and recombinant LAB to improve the immune response against Streptococcus pneumoniae. Research from the last decade demonstrates that LAB represent a promising resource for the development of prevention strategies against respiratory infections that could be effective tools for medical application.

Validade e confiabilidade de kits para detecção dos níveis de estreptococos do grupo mutans e lactobacilos na saliva de crianças e adultos / Validity and reliability of kits for salivary detection of mutans streptococci and lactobacilli in infants and adults

Objetivo: Avaliar a validade e a confiabilidade dos resultados obtidos por meio de kits pré-fabricados disponíveis no mercado brasileiro para a detecção e quantificação dos níveis de estreptococos do grupo mutans e lactobacilos na saliva de crianças e adultos. Método: Amostras salivares de 15 crianças (12-24meses) e 14 adultos (17-35 anos) foram coletadas e analisadas quanto aos níveis salivares de estreptococos do grupo mutans e lactobacilos pelo uso dos kits Dentacult I e II (Laborclin) e por método microbiológico convencional, baseado em cultura em meios seletivos. Os resultados de ambos os métodos foram comparados pelo uso dos coeficientes Kappa ponderado (Kp) e de correlação de Spearman. Para a determinação da concordância inter e intraexaminador da leitura dos resultados obtidos com o uso dos kits, os dados foram comparados por meio do Kp. As análises estatísticas foram realizadas utilizando o software Minitab 15 (versão 15.1.0.0.). Foi adotado o nível de significância de 5%. Resultados: Os resultados apresentados pelo kit Dentalcult I apresentaram concordância regular e correlação não-significante com os obtidos com o método microbiológico convencional (rs=0,53; p maior que 0,05). Por outro lado, os resultados obtidos com o kit Dentalcult II apresentaram boa concordância e correlação estatisticamente significante com os do método convencional (rs=0,77; p menor que 0,05). Os kits Dentalcult I e II tiveram confiabilidade de leitura perfeita (Kp=1) em momentos distintos, independentes do examinador. A concordância interexaminadores foi perfeita para o kit Dentalcult II (Kp=1) e ótima para o Dentalcult I (Kp=0,94). Conclusão: O kit Dentalcult II pode ser indicado para a estimativa dos níveis salivares de estreptococos do grupo mutans em substituição aos métodos convencionais.

Objective: To compare the results of commercially available kits for determination of mutans streptococci (MS) and lactobacilli (LB) salivary levels in infants and adults. Method: Salivary samples of 15 children (aged 12-24 months-old) and 14 adults (aged 17-35 years-old) were collected and evaluated for salivary levels of MS and LB by using the Dentacult I and II kits, respectively (Laborclin) and conventional methods based on culture in selective media. The results of both methods were compared to those obtained by the weighted kappa coefficients (Kp) and Spearman's correlation. Statistical analysis was performed using Minitab software 15 (version 15.1.0.0). It was adopted a significance level of 5%. Results: The data obtained with the Dentalcult I kit showed regular agreement and no significant correlation with the conventional microbiological method (rs=0.53, p greater than 0.05). On the other hand, results provided by the Dentalcult II kit produced good agreement and were statistical significant correlated with those of the conventional method (rs=0.77, p less than 0.05). The kits Dentalcult I and II presented perfect reading reliability (Kp=1) in distinct moments, not -dependent on the examiner. The inter-examiner agreement was considered perfect for the kit Dentalcult II (Kp=1) and satisfactory for Dentalcult I (Kp=0.94). Conclusion: The kit Dentalcult II proved validity and reliability for detecting salivary MS.

Potential roles and clinical utility of prebiotics in newborns, infants, and children: proceedings from a global prebiotic summit meeting, New York City, June 27-28, 2008.

Initial bacterial colonization, including colonization with health-positive bacteria, such as bifidobacteria and lactobacilli, is necessary for the normal development of intestinal innate and adaptive immune defenses. The predominance of beneficial bacteria in the gut microflora of breast-fed infants is thought to be, at least in part, supported by the metabolism of the complex mixture of oligosaccharides present in human breast milk, and a more adult-type intestinal microbiota is found in formula-fed infants. Inadequate gut colonization, dysbiosis, may lead to an increased risk of infectious, allergic, and autoimmune disorders later in life. The addition of appropriate amounts of selected prebiotics to infant formulas can enhance the growth of bifidobacteria or lactobacilli in the colonic microbiota and, thereby, might produce beneficial effects. Among the substrates considered as prebiotics are the oligosaccharides inulin, fructo-oligosaccharides, galacto-oligosaccharides, and lactulose. There are some reports that such prebiotics have beneficial effects on various markers of health. For example, primary prevention trials in infants have provided promising data on prevention of infections and atopic dermatitis. Additional well-designed prospective clinical trials and mechanistic studies are needed to advance knowledge further in this promising field.

Detection of T lymphocytes in intestine of broiler chicks treated with Lactobacillus spp. and challenged with Salmonella enterica serovar Enteritidis.

The expression of immune response in the form of leukocytic infiltrate by CD3+, CD4+, and CD8+ cells in the epithelium and in the intestinal lamina propria of chicks was studied in the present work by means of immunohistochemical reaction. The chicks were treated with Lactobacillus spp. or cecal microflora (CM) and experimentally challenged or not with Salmonella enterica serovar Enteritidis. The 320 birds utilized were divided into 4 groups containing 80 chicks each and submitted to treatments with Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus acidophilus, and CM. Each group was subdivided into 4 subgroups of 20 birds each and classified into a subgroup that did not receive treatment (negative control), subgroup treated, subgroup treated and challenged with Salmonella Enteritidis, and subgroup only challenged with Salmonella Enteritidis (positive control). The results obtained show that the treatment with L. reuteri, L. salivarius, L. acidophilus, or CM and challenged or not with Salmonella Enteritidis determine immune response in the form of leukocytic infiltrate by CD3+ and CD8+ lymphocytes followed by CD4+ in the epithelium and in the lamina propria of the duodenum, jejunum, and cecum of chicks up to 12 d of age. The quantity of CD3+ lymphocytes was significantly higher (P < 0.05) in the intestine of chicks treated with L. acidophilus or CM and challenged or not with Salmonella Enteritidis; however, the higher quantity of CD8+ lymphocytes was in the intestine of chicks treated with CM and challenged with Salmonella Enteritidis. The duodenum was the segment in which the immune response by T cells (CD3+, CD4+, and CD8+) was stimulated with the greatest intensity, followed by, respectively, the jejunum and cecum. The quantity of CD3+ lymphocytes present in the duodenum, jejunum, and cecum increases with the age of chicks, independent of the stimulus determined by treatments or challenge.

Gut immune stimulation by non pathogenic Gram(+) and Gram(-) bacteria. Comparison with a probiotic strain.

We analyzed the gut immune stimulation induced by Gram-positive bacteria: non probiotic Lactobacillus acidophilus CRL 1462 and Lactobacillus acidophilus A9; two potentially probiotic strains: L. acidophilus CRL 924 and Lactobacillusdelbrueckii subsp. bulgaricus CRL 423; comparatively with a probiotic strain: Lactobacillus casei CRL 431. We also studied Gram-negative bacteria: Escherichia coli 129 and E. coli 13-7 in BALB/c mice. All the strains increased the number of IgA+ cells. We analyzed the cytokines IFNgamma, TNFalpha, IL-17, IL-12, IL-6 and MIP-1alpha. The Gram(+) strains increased the number of IL-10+ cells. Gram(-) strains did not increase IL-10+ cells, but they increased the number of IL-12+ cells. The probiotic strain increased mainly IFNgamma and TNFalpha. In the study of the receptors TLR-2, TLR-4 and CD-206, we demonstrated that only the probiotic strain increased the number of CD-206+ cells. All the Gram(+) strains increased the number of TLR-2+ cells and the Gram(-) strains of the TLR-4+ cells. The probiotic strain induced the release of IL-6 by a preparation enriched in intestinal epithelial cells (IEC). Gram(+) and Gram(-) bacteria activated different immune receptors and induced a different cytokine profile. The probiotic strain showed a great activity on the immune cells and the enriched population in IEC, activating mainly cells of the innate immune system.

Genetic transformation of novel isolates of chicken Lactobacillus bearing probiotic features for expression of heterologous proteins: a tool to develop live oral vaccines.

BACKGROUND: The use of lactic acid bacteria as vehicles to delivery antigens to immunize animals is a promising issue. When genetically modified, these bacteria can induce a specific local and systemic immune response against selected pathogens. Gastric acid and bile salts tolerance, production of antagonistic substances against pathogenic microorganisms, and adhesive ability to gut epithelium are other important characteristics that make these bacteria useful for oral immunization. RESULTS: Bacteria isolated on de Man, Rogosa and Sharpe medium (MRS) from different gastrointestinal portions of broiler chicks were evaluated for their resistance to artificial gastric acid and bile salts, production of hydrogen peroxide, and cell surface hydrophobicity. Thirty-eight isolates were first typed at species level by PCR amplification of 16S-23S rRNA intergenic spacers using universal primers that anneal within 16S and 23S genes, followed by restriction digestion analyses of PCR amplicons (PCR-ARDRA). An expression cassette was assembled onto the pCR2.1-Topo vector by cloning the promoter, leader peptide, cell wall anchor and terminator sequences derived from the laminin binding S-layer protein gene of L. crispatus strain F5.7 (lbs gene). A sequence encoding the green fluorescent protein (GFP) was inserted as reporter gene, and an erythromycin resistance gene was added as selective marker. All constructs were able to express GFP in the cloning host E. coli XL1-Blue and different Lactobacillus strains as verified by FACS and laser scanning confocal microscopy. CONCLUSION: Lactobacillus isolated from gastrointestinal tract of broiler chickens and selected for probiotic characteristics can be genetically modified by introducing an expression cassette into the lbs locus. The transformed bacteria expressed on its cell wall surface different fluorescent proteins used as reporters of promoter function. It is possible then that similar bacterial model expressing pathogen antigens can be used as live oral vaccines to immunize broilers against infectious diseases.