Human gut-derived commensal suppresses generation of T-cell response to gliadin in humanized mice by modulating gut microbiota.

The human intestinal tract is colonized by a large number of diverse microorganisms that play various important physiologic functions. In inflammatory gut diseases including celiac disease (CeD), a dysbiotic state of microbiome has been observed. Interestingly, this perturbed microbiome is normalized towards eubiosis in patients showing recovery after treatment. The treatment has been observed to increase the abundance of beneficial microbes in comparison to non-treated patients. In this study, we investigated the effect of Prevotella histicola or Prevotella melaninogenica, isolated from the duodenum of a treated CeD patient, on the induction and maintenance of oral tolerance to gliadin, a CeD associated subgroup of gluten proteins, in NOD.DQ8.ABo transgenic mice. Conventionally raised mice on a gluten free diet were orally gavaged with bacteria before and after injection with pepsin trypsin digested gliadin (PTD-gliadin). P. histicola suppressed the cellular response to gliadin, whereas P. melaninogenica failed to suppress an immune response against gliadin. Interestingly, tolerance to gliadin in NOD.DQ8.ABo mice may be associated with gut microbiota as mice gavaged with P melaninogenica harbored a different microbial diversity as compared to P. histicola treated mice. This study provides experimental evidence that gut microbes like P. histicola from treated patients can suppress the immune response against gliadin epitopes.

Suppression of Inflammatory Arthritis by Human Gut-Derived Prevotella histicola in Humanized Mice.

OBJECTIVE: The gut microbiome regulates host immune homeostasis. Rheumatoid arthritis (RA) is associated with intestinal dysbiosis. This study was undertaken to test the ability of a human gut-derived commensal to modulate immune response and treat arthritis in a humanized mouse model. METHODS: We isolated a commensal bacterium, Prevotella histicola, that is native to the human gut and has systemic immune effects when administered enterally. Arthritis-susceptible HLA-DQ8 mice were immunized with type II collagen and treated with P histicola. Disease incidence, onset, and severity were monitored. Changes in gut epithelial proteins and immune response as well as systemic cellular and humoral immune responses were studied in treated mice. RESULTS: When treated with P histicola in prophylactic or therapeutic protocols, DQ8 mice exhibited significantly decreased incidence and severity of arthritis compared to controls. The microbial mucosal modulation of arthritis was dependent on regulation by CD103+ dendritic cells and myeloid suppressors (CD11b+Gr-1+ cells) and by generation of Treg cells (CD4+CD25+FoxP3+) in the gut, resulting in suppression of antigen-specific Th17 responses and increased transcription of interleukin-10. Treatment with P histicola led to reduced intestinal permeability by increasing expression of enzymes that produce antimicrobial peptides as well as tight junction proteins (zonula occludens 1 and occludin). However, the innate immune response via Toll-like receptor 4 (TLR-4) and TLR-9 was not affected in treated mice. CONCLUSION: Our results demonstrate that enteral exposure to P histicola suppresses arthritis via mucosal regulation. P histicola is a unique commensal that can be explored as a novel therapy for RA and may have few or no side effects.

[The use of solid-phase immunoenzyme analysis for the diagnosis of a suppurative infection of the maxillofacial area caused by Bacteroides melaninogenicus and Staphylococcus aureus]. / Primenenie tverdofaznogo immunofermentnogo analiza dlia diagnostiki gnoinoi infektsii cheliustno-litsevoi oblasti, vyzvannoi Bacteroides melaninogenicus i Staphylococcus aureus.

In 186 patients with odontogenic infection the level of antibodies to B.melaninogenicus polysaccharide antigen, teichoic acid and S.aureus polysaccharide was determined in the enzyme-linked immunosorbent assay (ELISA). The positive values of ELISA indices were found to coincide with the data of bacteriological study in more than 90% of cases. The role of B.melaninogenicus and S.aureus associations in the development of purulent infections was confirmed with the use of ELISA in 34.2% of the patients for S.aureus and in 50% of the patients for B.melaninogenicus and bacteriologically in 31.4% and 46.9% of the patients respectively.

[The development of a test system for determining antibodies to Bacteroides melaninogenicus using solid-phase immunoenzyme analysis]. / Razrabotka test-sistemy dlia opredeleniia antitel k Bacteroides melaninogenicus s pomoshch'iu tverdofaznogo immunofermentnogo analiza.

A test system for enzyme immunoassay of antibodies to Bacteroides melaninogenicus has been developed. The authors describe a method for the preparation of the protein and polysaccharide antigens with the use of ultrasonic, lysozyme, and beta-naphthol treatment. A total of 105 blood serum samples, 46 samples of gingival blood and gingival fluid from patients with maxillofacial phlegmons and periodontitis were examined, making use of the 1 +/- 3 omega index (from the values in healthy donors). The results evidence statistically significant difference in antibody levels of patients from whom B. melaninogenicus were isolated and of those with the negative results of bacteriologic analysis. The titers of antibodies to B. melaninogenicus in the blood and gingival fluid of patients with periodontitis were in good correlation.

Immunoglobulins in milk from cows immunized with oral strains of Actinomyces, Prevotella, Porphyromonas, and Fusobacterium.

Immunization of pregnant cows with bacteria leads to the presence of high concentrations of specific antibodies in colostrum and milk. A total of 14 cows was immunized with single strains of heat-killed oral bacteria or pools of strains of Actinomyces, Porphyromonas, Prevotella, and Fusobacterium. Two cows were treated with adjuvant alone. The mean percentages of IgG1, IgG2, IgM, and IgA in all of the milks were 83.8, 3.8, 9.3, and 3.1, respectively. ELISA and whole cell agglutination assays demonstrated high titers in the milks from the cows immunized with either individual strains or the bacterial pools. The highest titers determined by ELISA belonged to the IgG1 isotype and in several milks were 64-fold greater than titers in milk from cows treated with adjuvant alone. The concentrations of all antibodies and the titers determined by ELISA and whole cell agglutination assays markedly decreased from the first to the sixth milkings. The functional specificity of the antibodies was demonstrated by agglutination tests against a wide range of bacteria including members of Actinomyces, Fusobacterium, Porphyromonas, Prevotella, Streptococcus, Eubacterium, Propionibacterium, Peptostreptococcus, Bacteroides, Actinobacillus, Haemophilus, Capnocytophaga, and Wolinella. Minimal cross-reactions with bacteria in other genera were observed with all of the milks. High-titer milk preparations have been obtained from immunized cows, and the capacity of the bovine antibodies to agglutinate target bacteria indicates their potential usefulness in oral passive immunization studies.

[Relationship between serum antibody to Bacteroides gingivalis and clinical parameters in periodontitis patients].

Prevalence and proportion of black-pigmented Bacteroides species (BPB) in supragingival and subgingival plaque were determined in ten adult periodontitis patients. Serum antibody against Bacteroides gingivalis (Bg) of these patients were tested using ELISA. Clinical parameters (PI, GI, PD, AL) were collected prior to blood withdrawn. Results showed that BPB were detected in all patients. Mean serum anti-Bg IgG level was significantly greater in the patient group than that in healthy control group. Although the sample size was too small to show statistical difference, there was a trend showing the sera anti-Bg IgG level tended to be greater in accordance with the increase of disease severity and BPB%.

Purification and characterization of an immunoglobulin A1 protease from Bacteroides melaninogenicus.

Attention has recently been focused on bacterial proteases with the capacity to cleave immunoglobulin A (IgA proteases) as possible pathogenic factors in bacterial meningitis, gonorrhoea, and destructive periodontal disease. Here, we describe a method for the rapid purification of a specific IgA1 protease from Bacteroides melaninogenicus. The IgA1 protease was purified 6,172-fold with a yield of 9% by ammonium sulfate precipitation, DEAE-ion exchange chromatography, and separation on a preparative TSK-G 3000SWG high-pressure gel permeation chromatography column. The enzyme was specific for human IgA1 and cleaved a prolyl-seryl peptide bond in the hinge region of the alpha 1 chain between residues 223 and 224. The molecular weight of the enzyme was 62,000, the isoelectric point was 5.0, and the Km was 3.4 X 10(-6). The enzyme was active over a broad pH range and had maximal activity at pH 5.0. B. melaninogenicus IgA1 protease was classified as a thiol protease on the basis of its inhibition by traditional protease inhibitors and the fact that it was active only under reducing conditions.

An improved method for assessing the incorporation of labeled precursors into DNA by human mononuclear cells.

The blastogenic responsiveness of activated lymphoid cells is usually assessed in vitro by measuring the incorporation of radioactive thymidine or iododeoxyuridine, a thymidine analog, into DNA. The accuracy of this method is compromised by the presence in activated and unactivated lymphocytes and in some of the substances used to activate them, of degradative enzymes which compete with DNA synthetase, the incorporation efficiency of exogenous precursor is inherently low. We have done studies aimed at improving both the efficiency and the accuracy of the assay system by selectively inhibiting the enzymes responsible for thymidylate synthesis de novo and DNA precursor degradation. Culture conditions were investigated and potential inhibitors were tested using human peripheral blood mononuclear cells activated with phytohemagglutinin. Nucleoside-degrading activity of mammalian and bacterial cells is due largely to nucleoside phosphorylases, enzymes that require orthophosphate for activity. We partly inhibited DNA precursor degradation by lowering the phosphate concentration in the culture medium and lowering the pH, thereby reducing the orthophosphate concentration. To reduce precursor degradation further, we tested several potential nucleoside phosphorylase and thymidylate synthetase inhibitors at various concentrations. Our data show that the addition of 1 mM fluorouracil and 1 mM deoxyuridine to the culture medium largely prevents degradation of radioactive thymidine and iododeoxyuridine without unduly compromising the DNA-labeling efficiency of cells activated with mitogens or bacterial homogenates. Under these conditions, label incorporation increases linearly as the number of blast cells or the labeling time increases.

Bacterial IgG and IgM antibody titers in acute necrotizing ulcerative gingivitis.

IgG AND IgM ANTIBODY TITERS to eight bacterial isolates were measured by indirect immunofluorescence and ELISA in sera from acute necrotizing ulcerative gingivitis (ANUG) patients during the acute phase, from ANUG patients during the convalescent phase, from patients with gingivitis and from subjects with normal gingiva. Subjects were matched with respect to age and sex. Compared to the gingivitis and healthy groups, the ANUG groups exhibited significantly higher IgG and IgM titers to intermediate-sized spirochetes and higher IgG titers to Bacteroides melaninogenicus subsp intermedius. The findings support recent studies showing that these organisms are major bacterial components in ANUG lesions. They also suggest that these bacteria proliferate above-normal levels several weeks or months prior to the clinical onset of ANUG.

Reaction of clinical isolates and typed strains of the family Bacteroidaceae with rabbit anti-Bacteroides sera examined by radial immunodiffusion.

Antisera were produced which were reactive with Bacteroides fragilis, B. gingivalis, or B. melaninogenicus subsp. melaninogenicus. Anti-B. gingivalis serum exhibited strong reactions with strains of homologous species and reacted with only one other species, B. thetaiotaomicron. Antigens shared by organisms of the B. fragilis group and B. melaninogenicus subspecies of the Bacteroidaceae must be considered when serological methods are used for their identification.

Humoral immune response to oral microorganisms in periodontitis.

Serum antibody titers from patients with periodontitis were compared with those from periodontally healthy subjects. With the micro-enzyme-linked immunosorbent assay, immunoglobulin G (IgG), IgA, and IgM antibody titers to isolates of Streptococcus sanguis, Actinomyces viscosus, Bacteroides gingivalis, Bacteroides melaninogenicus subsp. intermedius, Bacteroides gingivalis, Bacteroides melaninogenicus subsp. intermedius, Bacteroides ochraceus, and Fusobacterium nucleation were determined. Antibody titers of the IgG and IgA classes to B. melaninogenicus, B. ochraceus, F. nucleatum, and S. sanguis were found to be significantly higher in the controls than in the patients. No correlations were found with serum IgM titers. These findings indicate that periodonitis may be associated with depressed antibacterial serum antibody titers of the IgG and IgA classes.

Impairment by Bacteroides species of opsonisation and phagocytosis of enterobacteria.

The ability of human polymorphonuclear leucocytes to phagocytose and kill Proteus mirabilis was impaired in vitro when the human serum, used to opsonise the target bacteria, was pretreated with cultures of various Bacteroides species. Live and dead, either heat-killed or clindamycin-treated, bacteroides cells elicited the same phenomenon. When bacteroides-treated serum was used to opsonise different Proteus species, the subsequent uptake of all strains by polymorphonuclear leucocytes was inhibited, whereas bacteroides-treated serum inhibited the uptake of some but not all of the test strains of Escherichia coli. The opsonic activity of untreated human serum was reduced when the classical complement pathway was inhibited by ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetra-acetic acid (EGTA); subsequent treatment with bacteroides did not further reduce the opsonic activity of the serum for P. mirabilis.

Gengivite e periodontite: detecçäo de antígeno e anticorpo pelas técnicas de imunofluorescência e imunoeletroforese em relaçäo ao Bacteroides melaninogenicus subsp. intermedius / Gingivitis and periodontitis: detection of antigen and antibody by immunofluorescence and immunoelectrophoresis in relation about Bacteroides melaninogenicus subsp. intermedius

A doença periodontal inflamatória é considerada como resultante da açäo das bactérias contidas na placa dentária. Uma vez que as bactérias inteiras näo säo encontradas na intimidade dos tecidos periodontais é provável que a doença ocorra devido à difusäo dos seus produtos através do epitélio do sulco gengival, provocando uma resposta inflamatória e imune. Assim, torna-se importante identificar esses produtos, principalmente das bactérias que predominam na placa dental, como é o caso do Bacteroides melaninogenicus e suas subespécies, que tem se distinguido como patógenos importantes na doença periodontal. O propósito deste estudo foi verificar a presença de antígenos solúveis de B. melaninogenicus subsp. intermedius nos tecidos gengivais, bem como de anticorpos séricos para o mesmo microrganismo em portadores de doença periodontal...

Detection of Bacteroides fragilis and Bacteroides melaninogenicus by direct immunofluorescence.

A new diagnostic kit, which contains a polyvalent antiserum for either Bacteroides fragilis or Bacteroides melaninogenicus, was tested for reliability and specificity on 146 clinical samples of different origin. A correlation between the culture and immunofluorescence was observed for B. fragilis in 87.39% of cases and for B. melaninogenicus in 81.48% of cases. When pure cultures were tested, aerobically as well as anaerobically, false-positive reactions were observed with staphylococci and Clostridium ramosum spores. The well-defined morphology of these bacteria and spores allows for the elimination of any diagnostic error. The method is rapid, and the margin for error is limited. The test gives a semiquantitative idea of the number of bacteroides organisms present in the clinical specimens even in the presence of a mixed flora.