Bifidobacterium animalis subsp lactis HN019 presents antimicrobial potential against periodontopathogens and modulates the immunological response of oral mucosa in periodontitis patients.

OBJECTIVE: To evaluate the effects of Bifidobacterium animalis subsp. lactis HN019 (HN019) on clinical periodontal parameters (plaque accumulation and gingival bleeding), on immunocompetence of gingival tissues [expression of beta-defensin (BD)-3, toll-like receptor 4 (TLR4), cluster of differentiation(CD)-57 and CD-4], and on immunological properties of saliva (IgA levels) in non-surgical periodontal therapy in generalized chronic periodontitis (GCP) patients. Adhesion to buccal epithelial cells (BEC) and the antimicrobial properties of HN019 were also investigated. MATERIALS AND METHODS: Thirty patients were recruited and monitored clinically at baseline (before scaling and root planing-SRP) and after 30 and 90 days. Patients were randomly assigned to Test (SRP+Probiotic, n = 15) or Control (SRP+Placebo, n = 15) group. Probiotic lozenges were used for 30 days. Gingival tissues and saliva were immunologically analyzed. The adhesion of HN019 with or without Porphyromonas gingivalis in BEC and its antimicrobial properties were investigated in in vitro assays. Data were statistically analyzed (p<0.05). RESULTS: Test group presented lower plaque index (30 days) and lower marginal gingival bleeding (90 days) when compared with Control group. Higher BD-3, TLR4 and CD-4 expressions were observed in gingival tissues in Test group than in Control group. HN019 reduced the adhesion of P. gingivalis to BEC and showed antimicrobial potential against periodontopathogens. CONCLUSION: Immunological and antimicrobial properties of B. lactis HN019 make it a potential probiotic to be used in non-surgical periodontal therapy of patients with GCP. CLINICAL RELEVANCE: B. lactis HN019 may be a potential probiotic to improve the effects of non-surgical periodontal therapy. Name of the registry and registration number (ClinicalTrials.gov): "Effects of probiotic therapy in the treatment of periodontitis"-NCT03408548.

Anti-adhesion methods as novel therapeutics for bacterial infections.

Anti-adhesion therapies for bacterial infections offer an alternative to antibiotics, with those therapies bacteria are not killed but are prevented from causing harm to a host by inhibiting adherence to host cells and tissues, a prerequisite for the majority of infectious diseases. The mechanisms of these potential therapeutic agents include inhibition of adhesins and their host receptors, vaccination with adhesins or analogs, use of probiotics and dietary supplements that interfere with receptor-adhesin interactions, subminimal inhibitory concentrations of antibiotics and manipulation of hydrophobic interactions. Once developed, these drugs will contribute to the arsenal for fighting infectious disease in the future, potentially subverting antibiotic resistance.

Vaccination of dairy cows with recombinant Streptococcus uberis adhesion molecule induces antibodies that reduce adherence to and internalization of S. uberis into bovine mammary epithelial cells.

Streptococcus uberis is an important environmental mastitis pathogen that causes subclinical and clinical mastitis in lactating and nonlactating cows and heifers throughout the world. Previous work from our laboratory suggests that S. uberis adhesion molecule (SUAM) is involved in S. uberis pathogenesis and may be an excellent target for vaccine development. The objective of this study was to evaluate the antibody response of cattle vaccinated with recombinant SUAM (rSUAM). Uninfected primiparous dairy cows (n=30) in late lactation were divided randomly into three groups of 10 cows each: control, 200 µg rSUAM, and 400 µg rSUAM. Cows in groups vaccinated with 200 µg and 400 µg rSUAM received an emulsion containing adjuvant, phosphate-buffered saline (PBS) and affinity purified rSUAM. Cows in the control group received an emulsion containing adjuvant and PBS. Cows were vaccinated subcutaneously in the neck region at drying off (D-0), 28 d after drying off (D+28) and within 7 d after calving. Serum was collected at D-0, D+28, at calving (C-0), calving vaccination (CV), and during early lactation (CV+14). Serum antibody responses were measured by an ELISA against rSUAM. Following the first vaccination a significant increase in anti-rSUAM antibodies was detected at D+28 in cows from groups vaccinated with 200 µg and 400 µg rSUAM when compared to the control group. This increase in anti-rSUAM antibodies continued following the second immunization at D+28; reaching the highest levels in the post-parturient sampling period (C0), after which antibodies appeared to plateau. S. uberis UT888 pretreated with several dilutions of heat-inactivated serum from cows vaccinated with rSUAM, affinity purified antibodies against rSUAM, and to a 17 amino acid long peptide from the N terminus of SUAM (pep-SUAM) were co-cultured with bovine mammary epithelial cells and adherence to and internalization of S. uberis into epithelial cells was measured. Compared to untreated controls, opsonization of two strains of S. uberis with sera from cows vaccinated with rSUAM, with affinity purified rSUAM antibodies, or with affinity purified pep-SUAM antibodies significantly reduced adherence to and internalization of this pathogen into bovine mammary epithelial cells. In conclusion, subcutaneous vaccination of dairy cows with rSUAM during physiological transitions of the mammary gland either from or to a state of active milk synthesis induced antibodies in serum and milk and these antibodies reduced adherence to and internalization of S. uberis into mammary epithelial cells under in vitro conditions. SUAM appears to be an excellent candidate for vaccine development.

Evaluation of clumping factor A binding region A in a subunit vaccine against Staphylococcus aureus-induced mastitis in mice.

The present study evaluated the potential of recombinant binding region A of clumping factor A (rClfA-A) to be an effective component of a vaccine against mastitis induced by Staphylococcus aureus in the mouse. rClfA-A and inactivated S. aureus were each emulsified in Freund's adjuvant, mineral oil adjuvant, and Seppic adjuvant; phosphate-buffered saline was used as a control. Seven groups of 12 mice each were immunized intraperitoneally three times at 2-week intervals. The titers of IgG and subtypes thereof (IgG1 and IgG2a) in the rClfA-A-immunized group were more than 1,000-fold higher than those in the killed-bacteria-immunized group (P < 0.01). Of the three adjuvants used, mineral oil adjuvant induced the highest antibody levels for both antigens (P < 0.001). Furthermore, the anti-rClfA-A antibody capacities for bacterial adhesion and opsonizing phagocytosis were significantly greater in the rClfA-A-immunized group than in the killed-bacteria-immunized group (P < 0.05). Lactating mice immunized with either rClfA-A or inactivated vaccine were challenged with S. aureus via the intramammary route. The numbers of bacteria recovered from the murine mammary glands 24 h after inoculation were significantly lower in the rClfA-A group than in the killed-bacteria-immunized group (P < 0.001). Histologic examination of the mammary glands showed that rClfA-A immunization effectively preserved tissue integrity. Thus, rClfA-A emulsified in an oil adjuvant provides strong immune protection against S. aureus-induced mastitis in the mouse.

Effect of specific colostral antibodies and selected lactobacilli on the adhesion of Helicobacter pylori on AGS cells and the Helicobacter-induced IL-8 production.

Helicobacter pylori infection is the most common cause of gastritis, gastric ulcer and adenocarcinoma. It has proven difficult to cure because of its capability to develop strains resistant to antibiotics. The effect of three strains of lactic acid bacteria (LAB) and bovine colostral preparations on the adhesion of H. pylori NCTC 11637 on gastric adenocarcinoma (AGS) cells and on the interleukin (IL)-8 production was studied. Before infection, H. pylori were pretreated with Lactobacillus plantarum MLBPL1, Lactobacillus rhamnosus GG, Lactococcus lactis, or with a colostral preparation with or without specific H. pylori antibodies. The relative number of H. pylori adhered on AGS cells was determined by urease test. IL-8 produced by the cells was studied by enzyme-linked immunosorbent assay. Colostral preparations with and without specific antibodies reduced the adhesion of H. pylori on AGS cells in a dose-dependent manner. Live LAB at a concentration of 10(10) CFU/ml reduced the adhesion by approximately 50% (P < 0.05). After the infection of AGS cells by H. pylori, the IL-8 level rose up to about 10-fold (5500 +/- 1600 pg/ml). Pretreatment of H. pylori with colostral preparations or high concentrations of LAB prevented this IL-8 rise. Similar effect was seen with live and heat-killed LAB, the live LAB being more effective. Heat-killed LAB at a concentration of 10(10) CFU/ml rose the IL-8 level of non-infected cells significantly. Suppression of IL-8 production by LAB or colostral products could have a suppressive effect on inflammation in Helicobacter infection.

Secretory immunoglobulin A obtained from pooled human colostrum and milk for oral passive immunization.

Passive immunization is useful in cases of immunodeficiencies or infectious diseases, but usually seric gammaglobulin or hyperimmune sera are administered parenterally, providing good systemic immunization, though with low protection of the mucosal surfaces. The oral administration of secretory antibodies, especially surface immunoglobulin (SIg)A, which is perfectly adapted to the mucosal environment, would therefore, be preferable. The aim of the present study was to obtain a SIgA preparation from pooled human colostrum and milk, which should maintain the essential properties of the antibodies suitable for clinical oral administration. IgA preparations were obtained from colostrum and milk pools by salt precipitation. The final products were evaluated in terms of yield and purity, as well as antibody activity to bacterial antigens and toxins and inhibitory activity of bacterial adhesion to epithelial cells. The best yield and purity were achieved when the colostrum pool was used as a source of IgA; the final product showed very few bands of protein contamination. The IgA preparations preserved the antibody reactivity against various microbial antigens, well comparable with the reactivity exhibited by the original milk and colostrum pools. SIgA preparations were able to inhibit greatly the adhesion of enteropathogenic Escherichia coli to Hep-2 cells and the invasion of enteroinvasive E. coli. These promising results show the feasibility of obtaining SIgA suitable for oral use, which may in future be administered to immunodeficient patients with gastrointestinal manifestations, from human colostrum and milk.

Differential immunogenicity of a DNA vaccine containing the Streptococcus mutans wall-associated protein A gene versus that containing a truncated derivative antigen A lacking in the hydrophobic carboxyterminal region.

Two plasmid DNA constructs were obtained by cloning separately into the eukaryotic expression vector pcDNA3.1/V5-His-TOPO the wall-associated protein A (wapA) gene of Streptococcus mutans GS-5 or its truncated derivative antigen A (agA) gene encoding a known candidate antigen for dental caries vaccine. The immunogenicity of the two constructs, designated pcDNA-wapA and pcDNA-agA, was compared by intranasal immunization of two groups of mice using the cationic DMRIE-C (1,2-dimyristyloxypropyl-3-dimethylhydroxy ethyl ammonium bromide-cholesterol) as an adjuvant. Immunization with pcDNA-wapA or pcDNA- agA resulted in specific salivary IgA and systemic IgG antibodies to the target antigens after two doses given at 3-week intervals. Higher salivary IgA level was observed in the mice immunized with the pcDNA-wapA vaccine compared to those immunized with the pcDNA-agA vaccine. Furthermore, anti-WapA antibody inhibited S. mutans sucrose-dependent adherence suggesting a potential protection against S. mutans colonization of the tooth, while anti-AgA had no significant effect. Indeed, prediction and analysis of protein epitopes showed that WapA contains highly promiscuous MHC-II binding motifs in addition to those found in AgA. Immunodot assay confirmed that WapA bound biotin-labeled dextran, whereas AgA did not. These data indicated that full-length WapA is a better candidate vaccine antigen than the soluble AgA, which is truncated in the hydrophobic membrane and wall-spanning region.

Colostrum from healthy Brazilian women inhibits adhesion and contains IgA antibodies reactive with Shiga toxin-producing Escherichia coli.

UNLABELLED: Although Shiga toxin-producing Escherichia coli (STEC) has been isolated in Brazil, severe manifestations of the infection, such as haemorrhagic colitis and haemolytic-uraemic syndrome, are extremely rare in our population. Enteropathogenic Escherichia coli (EPEC) is the main aetiological agent of acute infantile diarrhoea in Brazil. There are many similarities between STEC and EPEC, such as the ability to produce attaching and effacing (A/E) lesions and some virulence-associated factors. Our aim was to investigate the presence of anti-STEC antibodies in healthy people living in an EPEC endemic area. Colostrum samples collected from 51 women living in low socio-economic conditions were analysed. Two STEC strains: O111:H- (Stx1) and O157:H7 (Stx2), and one EPEC strain (O111:H-) were used in the bacterial adhesion assays to HEp-2 cells, in the Stx1 and Stx2 cytotoxicity assays on Vero cells, in immunoblotting and in ELISA assays. All the samples strongly inhibited the adhesion of the three strains and contained SIgA antibodies reactive with antigens of EPEC O111:H-, STEC O111:H- and STEC O157:H7, mainly STEC and EPEC 94 kDa adhesin intimin. High titres of anti-LPS O111 antibodies were found in many samples. Nevertheless, the cytotoxic effect of both Stx1 and Stx2 on Vero cells was not neutralised by any sample. CONCLUSION: Our results suggest that Brazilian people may be exposed to Shiga toxin-producing Escherichia colimore frequently than previously thought or alternatively there may be a cross reactive immunity between enteropathogenic Escherichia coliand Shiga toxin-producing Escherichia coli.

[In vitro evaluation of influence of medicinal plant decoctions and antibacterial antibodies to bifidobacteria on bifidobacterium adhesion]. / Otsenka vliianiia otvarov lekarstvennykh rastenii i protivobakterial'nykh antitel k bifidobakteriiam na ikh adgeziiu in vitro.

Natural antibodies to the surface antigens of bifidobacteria contained in blood sera and determined in the passive hemagglutination test blocked the adhesion of bifidobacteria to human red blood cells in vitro. The decoctions of medicinal plants, often used by pediatricians for therapeutic purposes, were found to have a pronounced inhibiting effect on the adhesion of bifidobacteria after the preliminary treatment of both microorganisms and human red blood cells by these decoctions. Suggestion was made on the possible role of natural human antibodies in changing the contents of the indigenous intestinal microflora; the prolonged use of preparations obtained from medicinal plants could probably produce negative influence on the microbiocenosis of the intestine.

Inhibition of Helicobacter pylori and Helicobacter mustelae binding to lipid receptors by bovine colostrum.

Helicobacter pylori, the etiologic agent of chronic-active gastritis and duodenal ulcers in humans, and Helicobacter mustelae, a gastric pathogen in ferrets, bind to phosphatidylethanolamine (PE), a constituent of host gastric mucosal cells, and to gangliotetraosylceramide (Gg4) and gangliotriaosylceramide (Gg3). The effect of a bovine colostrum concentrate (BCC) on the interaction of H. pylori and H. mustelae to their lipid receptors was examined. BCC blocked attachment of both species to Gg4, Gg3, and PE. Partial inhibition of binding was observed with native bovine and human colostra. BCC lacked detectable antibodies (by immunoblotting) to H. pylori surface proteins (adhesins). However, colostral lipid extracts contained PE and lyso-PE that bound H. pylori in vitro. These results indicate that colostrum can block the binding of Helicobacter species to select lipids and that binding inhibition is conferred, in part, by colostral PE or PE derivatives. Colostral lipids may modulate the interaction of H. pylori and other adhesin-expressing pathogens with their target tissues.

Intervención del flagelo de campylobacter jejuni subsp. jejuni en la adherencia a cultivos celulares: evidencias bacteriológicas e inmunológicas / Intervention of campylobacter campylobacter jejuni subsp. jejuni flagella in the adhesion to cellular cultures: bacteriological and immunological evidences

The participation of the flagella of a virulent strain (O52) of campylobacter jejuni subsp. jejuni in the adhesion to HEp-2 cells and their inhibition by means of homologous polyclonal antibodies, moniclonal antiflagella antibodies and colostral natural antibodies (IgA) was studied. An aflagellated strain (T1) was used as negative control. Adhesion was observed in higher rates with O52 strain (72 percent) than with T1 strain (27,5 percent). Polyclonal, monoclonal and colostral antibodies inhibited O52 strain adhesion in more than 70 percent (p<0,001). T1 strain adhesion was inhibited only by polyclonal and colostral natural antibodies. Our results suggest that the flagella of C. jejuni subsp. jejuni could participate effectively in the adhesion process. However, the inhibition of T1 strain by polyclonal and colostral antibodies suggest the existence of other kinds of adhesins in the bacterial surface

Effect of microwave radiation, pasteurization and lyophilization on the ability of human milk to inhibit Escherichia coli adherence to HEp-2 cells.

The effect of different physical treatments on the ability of colostrum and human milk to inhibit the adherence of enteropathogenic Escherichia coli (EPEC) to human epithelial cells was studied. Pools of colostrum and milk were submitted to microwave radiation, pasteurization or lyophilization, and then tested for the ability to inhibit the adherence of EPEC O111:H- to HEp-2 cells. The inhibitory effect of untreated colostrum and human milk on localized adherence was not significantly modified after exposure to any treatment. The total protein values of colostrum and milk were maintained, but IgA concentration and colostral anti-EPEC IgA were reduced after pasteurization. Nevertheless, the remaining IgA was sufficient to be effective in adhesion inhibition assay. Western blotting assays carried out with EPEC antigens showed that the treated and untreated pools recognize a 94-kDa outer-membrane protein which molecular weight is compatible with intimin, an EPEC adhesin related to bacterial attachment to epithelial cells. These results suggest that the protection of colostrum and milk to infantile diarrhoea due to EPEC remains unalterable after the physical treatments studied.

Active release of bound antibody by Streptococcus mutans.

Previous studies have shown that Streptococcus mutants is capable of releasing many surface protein antigens, particularly antigen P1. Antigen P1 is immunodominant and has been implicated in adherence of S. mutants to the acquired pellicles. The purpose of this study is to investigate the significance of release of this antigen by the cells. S. mutants NG8 (serotype c) was incubated with an anti-P1 rabbit immunoglobulin G (IgG) or a human colostral IgA which contains natural anti-P1 activity. Results indicated that the bound antibodies were released by the cells in a pH- and time-dependent manner. The optimal pH for release was between 6 and 8, and the release rate reached a plateau in 1 h at 37 degrees C. The release of bound antibodies was considered an active process, since heat-killed cells remained capable of antibody binding but failed to release the antibodies. The release was also dependent on the age of the culture, with early-exponential-phase cells releasing the maximum amount of bound IgG. The released IgG was isolated by polyethylene glycol precipitation and protein A-Sepharose column chromatography and found to be associated with antigen P1, indicating that the antibodies were released together with the antigen in the form of immune complexes. The binding of S. mutans by secretory IgA (SIgA) inhibited the adherence of the cells to salivary agglutinin-coated hydroxylapatite. However, when the SIgA-coated S. mutans was allowed to release the bound antibodies, the inhibitory effect of SIgA on adherence was abrogated. These results suggest that S. mutans is capable of shedding surface-bound antibodies in the form of antibody-antigen immune complexes. Such an action may be a strategy employed by the cells to counter the neutralizing effect of naturally occurring antibodies in the oral cavity.

Inhibition of localized adhesion of enteropathogenic Escherichia coli to HEp-2 cells by immunoglobulin and oligosaccharide fractions of human colostrum and breast milk.

Secretory IgA (sIgA) purified from colostrum and breast milk obtained from 14 women inhibited the localized adherence of an enteropathogenic Escherichia coli (EPEC) to HEp-2 cells. Inhibition decreased as lactation continued even when the concentration of sIgA was maintained constant at 1 mg/ml. sIgA responded to a 94-kDa plasmid-encoded outer membrane protein implicated as the EPEC adherence factor. An oligosaccharide-enriched fraction (OEF) from these samples also inhibited the attachment of this EPEC. Inhibition by OEFs decreased as lactation continued because of a general reduction in oligosaccharide content. Localized adherence of six other EPEC was also inhibited by sIgA and OEF, whereas attachment of isolates with diffuse or aggregative adherence was not inhibited by these fractions. Experiments with purified oligosaccharide fractions revealed that EPEC attach to HEp-2 cells through a carbohydrate-mediated mechanism based on the preferential recognition of fucosylated residues in human milk.

Isolation and characterization of a 180-kiloDalton salivary glycoprotein which mediates the attachment of Actinomyces naeslundii to human buccal epithelial cells.

The adherence of Actinomyces naeslundii to human buccal mucosa is mediated by specific interactions between the bacterial cell surface fimbriae and complementary beta-linked galactoside receptors on the epithelial cell surface. The buccal mucosa and the bacteria that colonize its surface are constantly bathed in saliva. Several salivary components are thought to play an important role in modulating adhesive interactions between oral bacteria and the buccal epithelium. We have observed that pretreatment of isolated buccal epithelial cells (BEC) with human parotid saliva increased the attachment of three different strains of A. naeslundii. By employing affinity chromatography, ion-exchange and high-pressure liquid chromatographic techniques we have isolated a 180 kDa A. naeslundii-binding salivary glycoprotein (An-SPG). This salivary glycoprotein was capable of mediating separate but specific binding interactions with A. naeslundii and BEC. Pretreatment of BEC with increasing amounts of An-SGP resulted in a corresponding increase in the attachment of A. naeslundii. The adherence of A. naeslundii to An-SGP-coated BEC is sensitive to the same inhibitors previously shown to block adherence of A. naeslundii to uncoated BEC, namely lactose- and galactosyl-binding lectins. When a solubilized extract of freshly isolated and washed BEC was reacted on a Western blot with antibodies to An-SGP, a prominent 180 kDa immunoreactive band was detected. Furthermore, the immunoreactive component was demonstrated on the BEC surface when assayed by immunofluorescence using An-SGP-specific antibodies, suggesting that An-SGP or a protein structurally and immunologically identical to the isolated glycoprotein is present on BEC.

Glutamine-supplemented total parenteral nutrition improves gut immune function.

Glutamine has been demonstrated to be an important source of fuel for the gut. The purpose of this study was to evaluate the effect of glutamine-supplemented hyperalimentation on gut immune function. Thirty-six female Fischer rats were randomized into three groups: group 1 (chow) was fed rat chow and water ad libitum, group 2 (total parenteral nutrition) received a standard hyperalimentation formula, and group 3 (total parenteral nutrition-glutamine) received a hyperalimentation solution that contained 2% glutamine. Animals were maintained on their respective diets for 2 weeks and then killed. Mesenteric lymph nodes were harvested for culture, bile was assayed for secretory IgA, and bowel was excised to assay bacterial adherence. Results indicated that glutamine-supplemented total parenteral nutrition protects against bacterial translocation from the gut seen with standard formulas. This effect may be mediated by the secretory IgA immune system.