Colonic inflammation accompanies an increase of ß-catenin signaling and Lachnospiraceae/Streptococcaceae bacteria in the hind gut of high-fat diet-fed mice.

Consumption of an obesigenic/high-fat diet (HFD) is associated with a high colon cancer risk and may alter the gut microbiota. To test the hypothesis that long-term high-fat (HF) feeding accelerates inflammatory process and changes gut microbiome composition, C57BL/6 mice were fed HFD (45% energy) or a low-fat (LF) diet (10% energy) for 36 weeks. At the end of the study, body weights in the HF group were 35% greater than those in the LF group. These changes were associated with dramatic increases in body fat composition, inflammatory cell infiltration, inducible nitric oxide synthase protein concentration and cell proliferation marker (Ki67) in ileum and colon. Similarly, ß-catenin expression was increased in colon (but not ileum). Consistent with gut inflammation phenotype, we also found that plasma leptin, interleukin 6 and tumor necrosis factor α concentrations were also elevated in mice fed the HFD, indicative of chronic inflammation. Fecal DNA was extracted and the V1-V3 hypervariable region of the microbial 16S rRNA gene was amplified using primers suitable for 454 pyrosequencing. Compared to the LF group, the HF group had high proportions of bacteria from the family Lachnospiraceae/Streptococcaceae, which is known to be involved in the development of metabolic disorders, diabetes and colon cancer. Taken together, our data demonstrate, for the first time, that long-term HF consumption not only increases inflammatory status but also accompanies an increase of colonic ß-catenin signaling and Lachnospiraceae/Streptococcaceae bacteria in the hind gut of C57BL/6 mice.

Intestinal inflammation modulates expression of the iron-regulating hormone hepcidin depending on erythropoietic activity and the commensal microbiota.

States of chronic inflammation such as inflammatory bowel disease are often associated with dysregulated iron metabolism and the consequent development of an anemia that is caused by maldistribution of iron. Abnormally elevated expression of the hormone hepcidin, the central regulator of systemic iron homeostasis, has been implicated in these abnormalities. However, the mechanisms that regulate hepcidin expression in conditions such as inflammatory bowel disease are not completely understood. To clarify this issue, we studied hepcidin expression in mouse models of colitis. We found that dextran sulfate sodium-induced colitis inhibited hepcidin expression in wild-type mice but upregulated it in IL-10-deficient animals. We identified two mechanisms contributing to this difference. Firstly, erythropoietic activity, as indicated by serum erythropoietin concentrations and splenic erythropoiesis, was higher in the wild-type mice, and pharmacologic inhibition of erythropoiesis prevented colitis-associated hepcidin downregulation in these animals. Secondly, the IL-10 knockout mice had higher expression of multiple inflammatory genes in the liver, including several controlled by STAT3, a key regulator of hepcidin. The results of cohousing and fecal transplantation experiments indicated that the microbiota was involved in modulating the expression of hepcidin and other STAT3-dependent hepatic genes in the context of intestinal inflammation. Our observations thus demonstrate the importance of erythropoietic activity and the microbiota in influencing hepcidin expression during colitis and provide insight into the dysregulated iron homeostasis seen in inflammatory diseases.

Relationship between mucosa-associated microbiota and inflammatory parameters in the ileal pouch after restorative proctocolectomy for ulcerative colitis.

BACKGROUND: Our aim was to assess the relationship between the ileal-pouch microbiota and inflammatory parameters in patients operated on for ulcerative colitis. METHODS: In this cross-sectional study, 32 consecutive outpatients returning for follow-up endoscopy were recruited. Pouch biopsies were obtained during endoscopy for culture of bacteria adherent to the mucosa, histology, and analysis of local inflammation (IL-1ß, IL-6, and TNFα by immunometric assay; and toll-like receptor [TLR] 2 and 4 mRNA by quantitative real-time PCR). Fecal samples were collected for analysis of lactoferrin by ELISA. RESULTS: Granulocyte and monocyte mucosal infiltration correlated directly with mucosal Bacteriodiaceae spp. counts. Clostridiaceae spp. counts showed a direct correlation with mucosal ulceration and number of daily stools. In patients with pouchitis, Enterococcaceae spp. counts were less than in healthy patients. Enterobacteriaceae spp., Streptococcaceae spp. and Enterococcaceae spp. counts correlated inversely with immune cell infiltration. TLR-2 and TLR-4 mRNA, and mucosal levels of IL-1ß levels all correlated directly with Veilonella spp. counts. CONCLUSION: Bacteriodaceae spp. and, Clostridiaceae spp. may be associated with inflammation of the pouch mucosa. Conversely, Enterococcaceae spp., and possibly Enterobacteriaceae spp. and Streptococcaceae spp., may have an active role in maintaining immunologic homeostasis within the pouch mucosa.

Mucosal delivery of therapeutic and prophylactic molecules using lactic acid bacteria.

Studies of lactic acid bacteria (LAB) as delivery vehicles have focused mainly on the development of mucosal vaccines, with much effort being devoted to the generation of genetic tools for antigen expression in different bacterial locations. Subsequently, interleukins have been co-expressed with antigens in LAB to enhance the immune response that is raised against the antigen. LAB have also been used as a delivery system for a range of molecules that have different applications, including anti-infectives, therapies for allergic diseases and therapies for gastrointestinal diseases. Now that the first human trial with a Lactococcus strain that expresses recombinant interleukin-10 has been completed, we discuss what we have learnt, what we do not yet understand and what the future holds for therapy and prophylaxis with LAB.

Serological cross-reactivity of environmental isolates of Enterobacter, Escherichia, Stenotrophomonas, and Aerococcus with Shigella spp.-specific antisera.

Using protocols designed for the isolation of Shigella from environmental freshwater samples from different regions of Bangladesh, 11 bacterial strains giving rise to Shigella-like colonies on selective agar plates and showing serological cross-reaction with Shigella-specific antisera were isolated. Phylogenetic analyses revealed that three of the isolates were most closely related to Escherichia coli, four to Enterobacter sp., two to Stenotrophomonas, and two isolates belonged to the Gram-positive genus Aerococcus. The isolates cross-reacted with six different serotypes of Shigella and were, in each case, highly type-specific. Two of the isolates belonging to the Enterobacter and Escherichia genera gave extremely strong cross-reactivity with Shigella dysenteriae and Shigella boydii antisera, respectively. The Aerococcus isolates gave relatively weak but significant cross-reactions with S. dysenteriae. Western blot analysis revealed that a number of antigens from the isolates cross-react with Shigella spp. The results indicate that important Shigella spp. surface antigens are shared by a number of environmental bacteria, which have implications for the use of serological methods in attempts for the detection and recovery of Shigella from aquatic environments.

LTA and LPS mediated activation of protein kinases in the regulation of inflammatory cytokines expression in macrophages.

BACKGROUND: Lipoteichoic acid (LTA) and lipopolysaccharide (LPS), the toxicants from bacteria, are potent inducers of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF) and interleukin-1beta (IL-1). Although LTA is much less reported than that on LPS, LTA is regarded as the gram-positive equivalent to LPS in some aspects. We investigated the LTA-induced signal transduction and biological effects, as well as to compare the effect of LTA with that of LPS. METHODS: Kinase assay, ELISA and RT-PCR were performed to delineate LTA and LPS signaling as well as to determine the secretion and RNA expression of TNF and IL-1. RESULTS: Src, Lyn and MAPKs are involved in LTA and LPS signaling in murine macrophages. Additionally, blockades of PKC, PI3K and p38, respectively, caused significant inhibition of both LTA- and LPS-induced proIL-1/IL-1 and TNF expression. ERK inactivation moderately reduced LTA- and LPS-induced proIL-1/IL-1, but considerably reduced TNF expression. Inhibition of JNK engendered super-induction of IL-1 secretion, but diminished TNF secretion. Strikingly, both IL-1 and TNF protein induction were declined by overexpression of dominant negative form of JNK. CONCLUSIONS: The results clarify the similarity and difference between LTA- and LPS-mediated signal transduction and induction of inflammatory cytokines in macrophages.

Immunogen-dependent quantitative and qualitative differences in phagocytic responses of the circulating hemocytes of the lobster Homarus americanus.

Phagocytic responses in circulating hemocytes of the lobster Homarus americanus were measured before and after treatment of lobsters with 2 different immunogens: (1) lipolysaccharide (LPS) or endotoxin from a non-pathogenic Pseudomonas perolens, and (2) a vancomycin/live Gram-positive pathogen (Aerococcus viridans [var.] homari) combination, essentially attenuated cells, shown previously to induce a high degree of resistance to this pathogen. The responses elicited by each of the immunogens were markedly different. Hemocytes drawn from LPS-treated lobsters showed significant, largely non-specific, increases in phagocytic responses over baseline values against sheep red blood cells and an array of test bacteria, with the notable exception of the pathogen. In marked contrast, induction with the vancomycin/live pathogen combination resulted in highly significant and specific increases in phagocytic responses to the pathogen and to the related, (but avirulent) strains of the pathogen, as well as inducing in the lobsters the usual high degree of resistance to the pathogen. These results suggest that quantitative and qualitative variations in phagocytic and resistance levels induced in at least 1 crustacean genus are determined largely by the particular characteristics of the immunogen.

L-ficolin is a pattern recognition molecule specific for acetyl groups.

L-ficolin and H-ficolin are molecules of the innate immune system. Upon recognition of a suitable target they activate the complement system. The ligand recognition structure of ficolins is contained within a fibrinogen-like domain. We examined the selectivity of the ficolins through inhibiting the binding to bacteria or to beads coupled with N-acetylglucosamine. The binding of L-ficolin to Streptococcus pneumoniae 11F and the beads was inhibited by N-acetylated sugars and not by non-acetylated sugars. However, it was also inhibited by other acetylated compounds. Based on this selectivity L-ficolin is not easily defined as a lectin. The binding of H-ficolin to Aerococcus viridans was not inhibited by any of the sugars or other compounds examined. Based on the selectivity of L-ficolin we developed a new purification procedure involving affinity chromatography on N-acetylcysteine-derivatized Sepharose. The column was loaded in the presence of EDTA and high salt, and L-ficolin was eluted by decreasing the salt concentration. Further purification was achieved by ion exchange chromatography.

L-ficolin specifically binds to lipoteichoic acid, a cell wall constituent of Gram-positive bacteria, and activates the lectin pathway of complement.

The lectin pathway of complement is activated when a carbohydrate recognition complex and associated serine proteases binds to the surface of a pathogen. Three recognition subcomponents have been shown to form active initiation complexes: mannan-binding lectin (MBL), L-ficolin, and H-ficolin. The importance of MBL in antimicrobial host defense is well recognized, but the role of the ficolins remains largely undefined. This report shows that L-ficolin specifically binds to lipoteichoic acid (LTA), a cell wall component found in all Gram-positive bacteria. Immobilized LTA from Staphylococcus aureus binds L-ficolin complexes from sera, and these complexes initiate lectin pathway-dependent C4 turnover. C4 activation correlates with serum L-ficolin concentration, but not with serum MBL levels. L-ficolin binding and corresponding levels of C4 turnover were observed on LTA purified from other clinically important bacteria, including Streptococcus pyogenes and Streptococcus agalactiae. None of the LTA preparations bound MBL, H-ficolin, or the classical pathway recognition molecule, C1q.

Induced resistance to infection of lobsters Homarus americanus by Aerococcus viridans (var.) homari, the bacterium causing gaffkemia.

A vaccine composed of steam sterilized (autoclaved) cells of a virulent strain of Aerococcus viridans (var.) homari was effective in protecting lobsters Homarus americanus against gaffkemia. At 15 degrees C the heat-killed vaccines (HKV) at concentrations between 1 and 5 x 10(7) particles kg(-1) lobster body wt induced maximal protection in induction periods ranging from 7 to 11 d. Protection was substantial over the course of a 30 d post-induction trial period. Spring-caught lobsters (i.e. those more fully rehabilitated following ecdysis) gained more protection (LD50 = 1.9 x 10(4)) from the vaccination than did those caught in the late fall-early winter period (lobsters that were not yet fully recovered from ecdysis) (LD50 = 3.2 x 10(3)). The protection offered by the HK vaccine was comparable to that induced by a vaccine produced by incubating the pathogen with low concentrations (2 pg ml(-1)) of the antibiotic vancomycin. The bacterins produced by both methods exhibited similar new properties: (1) agglutination at low titres by lobster hemolymph serum, suggesting an impaired capsule layer, and (2) increased permeability to the large Alcian Blue molecule. With both vaccines, the protection may be a direct result of increased exposure to intact bacterial cell structures by the lobster defences, an exposure which otherwise would be prevented by an intact capsule.

Serum concentration of Hakata antigen, a member of the ficolins, is linked with inhibition of Aerococcus viridans growth.

BACKGROUND: Hakata antigen (Hakata) is a novel serum glycoprotein that consists of collagen- and fibrinogen-like domains, similar to ficolin/p35. Our research suggested that serum Hakata may be a target of a polysaccharide (PSA) produced by Aerococcus viridans. METHODS: A. viridans was incubated with human plasma and Hakata-depleted plasma to examine Hakata binding and growth inhibition of A. viridans through binding with PSA. RESULTS: When A. viridans was mixed with human acid citrate dextrose-one (ACD-A) plasma, it pulled down Hakata complexed with mannose-binding lectin (MBL)-associated serine proteases 1 and 2 (MASP-1 and MASP-2). This complex had the potential for C4 deposition. Serum Hakata circulates as Hakata-MASPs complex in the blood and is proteolytically active. By mixing A. viridans with human plasma, we prepared a Hakata-depleted plasma, deficient in Hakata-MASPs complex. This plasma failed to inhibit A. viridans growth plasma, but does not inhibit Staphylococcus aureus, Yersinia enterocolitica and Escherichia coli. However, a decrease of growth inhibition of A. viridans in Hakata-depleted plasma could be restored by adding a Hakata-MASPs complex preparation in a dose-dependent manner. On the other hand, the Hakata-MASPs complex exhibited strong binding to A. viridans, but not to S. aureus, Y. enterocolitica and E. coli. CONCLUSIONS: The serum concentration of Hakata is linked with growth inhibition of A. viridans upon binding of Hakata via PSA.

Detection of serum thermolabile beta-2 macroglycoprotein (Hakata antigen) by enzyme-linked immunosorbent assay using polysaccharide produced by Aerococcus viridans.

Although a serum thermolabile beta-2 macroglycoprotein (TMG) may play a role in host defense as a lectin, little is known of its related physiological functions, mainly due to a lack of appropriate methods for tracing the functions of TMG. We identified a polysaccharide from Aerococcus viridans, PSA, which reacts with TMG, and based on this finding, we developed an enzyme-linked immunosorbent assay to trace the functions of TMG. Using ethanol precipitation and DEAE-Sepharose and Sephacryl S-400 column chromatographies, we isolated PSA from cultured medium of A. viridans, and it exhibited specific binding against TMG in blood samples. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the isolated PSA showed ladder bands that implied the existence of repeating units composed of D-glucose, N-acetyl-D-glucosamine, D-mannose, and D-xylose, as confirmed by gas chromatography-mass spectrometry. SDS-PAGE and immunochemical analysis, using rabbit anti-TMG antibody, showed that PSA specifically binds solely to intact serum TMG but not to TMG heated at 56 degrees C for 30 min, a condition under which antigenicity is lost. TMG in serum samples bound to PSA in a dose-dependent manner, and this binding was clearly suppressed by addition of PSA. These observations indicate that PSA is a useful adsorbent to TMG and can be used to develop appropriate methods for tracing the functions of TMG.

Penaeidins, antimicrobial peptides with chitin-binding activity, are produced and stored in shrimp granulocytes and released after microbial challenge.

Penaeidins are members of a new family of antimicrobial peptides isolated from a crustacean, which present both Gram-positive antibacterial and antifungal activities. We have studied the localization of synthesis and storage of penaeidins in the shrimp Penaeus vannamei. The distribution of penaeidin transcripts and peptides in various tissues reveals that penaeidins are constitutively synthesized and stored in the shrimp haemocytes. It was shown by immunocytochemistry, at both optical and ultrastructural levels, that the peptides are localized in granulocyte cytoplasmic granules. The expression and localization of penaeidins were further analysed in shrimp subjected to microbial challenge. We found that (1) penaeidin mRNA levels decrease in circulating haemocytes in the first 3 hours following stimulation and (2) an increase in plasma penaeidin concentration occurs after microbial challenge, together with (3) a penaeidin immunoreactivity in cuticular tissue, which can be related to the chitin-binding activity we demonstrate here for penaeidins.

Murine model of recurrent group G streptococcal cellulitis: no evidence of protective immunity.

Despite the well-known tendency of cellulitis due to beta-hemolytic streptococci to recur, little is known regarding the mechanisms of human immunity to this infection. We established cellulitis in mice by using a strain of group G streptococcus (1750) originally isolated from the bloodstream of a patient with acute cellulitis. This strain, which has been studied extensively in our laboratory, expresses M protein structurally and functionally analogous to that of group A streptococci, and we have cloned and sequenced the gene encoding this protein (emmMG1). Mice injected with 5 x 10(7) CFU of strain 1750 developed nonlethal necrotic skin and soft tissue infections that healed spontaneously after 14 to 16 days. After healing, the mice were repetitively reinoculated three times with the same challenge dose of 1750. Lesion size did not decrease in severity, size, or time to healing after repetitive challenge. The maximum lesion size and tissue concentration of microorganisms increased between the first and fourth challenges. Pretreatment of 1750 cells with opsonic antisera to MG1 diminished neither the maximum lesion size nor the time course of evolution of the lesions. Thus, in the mouse model used here, there was no evidence of acquired protective immunity to experimentally induced cellulitis.

Effect of intravenous immunoglobulin G on the deposition of immunoglobulin G and C3 onto type III group B streptococcus and Escherichia coli K1.

Seventeen neonates with suspected or proven sepsis received either a 750 mg/kg dose of intravenous immunoglobulin G (IVIG) or placebo. Compared with values in adult serum, the preinfusion serum concentrations of immunoglobulin G (IgG) and complement component C3 were diminished; the concentrations were unaffected by the administration of placebo to nine infants. Fifteen minutes after infusion in the eight IVIG recipients, the serum concentration of IgG increased from 3.66 mg/ml to 16.58 mg/ml but the C3 concentration of 540 micrograms/ml was unaffected. Similarly, a radioimmunoassay revealed that during incubation of bacteria with sera from the neonates, the quantities of IgG and C3 bound to type III group B streptococcus and Escherichia coli O7:K1: NM were low and were unaffected by the infusion of placebo. During incubation of bacteria with the postinfusion sera from the IVIG recipients, the amount of IgG, but not C3, deposited onto the bacteria increased to a level equivalent to that observed in adult serum. Therefore IVIG enhanced the capacity of sera from ill neonates to deposit IgG but not C3 onto bacteria. We speculate that in neonates with sepsis, a diminished capacity to deposit C3 onto bacteria may possibly limit the therapeutic efficacy of IVIG.

Urosepticemia and fatal endocarditis caused by aerococcus-like organisms.

Two cases of invasive infections with aerococcus-like organisms (ALO) are presented: an 81-year-old man with fatal endocarditis and a 63-year-old man with urosepticemia. No antigenic relationship was found between ALO and Aerococcus viridans (NCTC 8251) in crossed immunoelectrophoretic assay.

Differentiation of Gemella haemolysans (Thjøtta and Bøe 1938) Berger 1960, from Streptococcus morbillorum (Prevot 1933) Holdeman and Moore 1974.

Recently, G. haemolysans and S. morbillorum were postulated to be identical organisms, so that consequently their names were synonyms. In the present paper it was demonstrated, that, in spite of many similarities, both species can be differentiated by nitrite reduction, lacking in S. morbillorum, and some further enzymatic activities as well as by antigenic specificity and certain dissimilarities in morphology, growth conditions and their ability to induce beta-hemolysis. S. morbillorum apparently represents a group of strains with divergent properties and might be assigned to the genus Gemella rather than to Streptococcus.

Prevalence of Gemella haemolysans on the pharyngeal mucosa of man.

By culturing pharyngeal swabs from 199 students a carrier rate for Gemella haemolysans of 29.7% was detected. Demonstration of hemolysis depended on blood species and on agar base. Optimum growth was obtained under aerobic conditions in a 10% CO2-enriched atmosphere. The cells divided in two planes which were not regularly at right angles to each other. They appeared to be surrounded by a small capsule. Contrary to earlier descriptions, acid was produced from galactose, acetoin was produced by practically all strains and nitrite was reduced by all strains. Acid production from trehalose and N-acetyl-glucosamine, alkaline and acid phosphatase, C4 and C8 esterase, pyrrolidone arylamidase and phosphoamidase activities were detected. In tube precipitation, antigenic relations between type strain and the new isolates were demonstrated.