Dysbiosis of oral microbiota and its association with salivary immunological biomarkers in autoimmune liver disease.

The gut microbiota has recently been recognized to play a role in the pathogenesis of autoimmune liver disease (AILD), mainly primary biliary cholangitis (PBC) and autoimmune hepatitis (AIH). This study aimed to analyze and compare the composition of the oral microbiota of 56 patients with AILD and 15 healthy controls (HCs) and to evaluate its association with salivary immunological biomarkers and gut microbiota. The subjects included 39 patients with PBC and 17 patients with AIH diagnosed at our hospital. The control population comprised 15 matched HCs. Salivary and fecal samples were collected for analysis of the microbiome by terminal restriction fragment length polymorphism of 16S rDNA. Correlations between immunological biomarkers measured by Bio-Plex assay (Bio-Rad) and the oral microbiomes of patients with PBC and AIH were assessed. Patients with AIH showed a significant increase in Veillonella with a concurrent decrease in Streptococcus in the oral microbiota compared with the HCs. Patients with PBC showed significant increases in Eubacterium and Veillonella and a significant decrease in Fusobacterium in the oral microbiota compared with the HCs. Immunological biomarker analysis showed elevated levels of inflammatory cytokines (IL-1ß, IFN-γ, TNF-α, IL-8) and immunoglobulin A in the saliva of patients with AILD. The relative abundance of Veillonella was positively correlated with the levels of IL-1ß, IL-8 and immunoglobulin A in saliva and the relative abundance of Lactobacillales in feces. Dysbiosis of the oral microbiota is associated with inflammatory responses and reflects changes in the gut microbiota of patients with AILD. Dysbiosis may play an important role in the pathogenesis of AILD.

Relationship between serologic markers of periodontal bacteria and metabolic syndrome and its components.

BACKGROUND: Periodontitis is a result of a complex biologic alteration of the periodontal microenvironment and a distributional shift of key periodontal pathogens. Metabolic syndrome (MetS), a complex cluster of cardiovascular risk factors, has been linked to periodontal diseases; however, the contribution of periodontal bacteria to systemic conditions remains unclear. METHODS: The study population comprised 7,848 United States adults who participated in an interview, underwent a clinical oral-health examination, and had serum immunoglobulin G titers measured against 19 periodontal bacteria as part of the third National Health and Nutritional Examination Survey. The z-score antibody titers were clustered into four mutually exclusive groups and named after Socransky's classification of periodontal bacteria (Orange-Red, Red-Green, Yellow-Orange, and Orange-Blue). Survey logistic regression was used to investigate the independent associations between the cluster scores, and MetS and each component, including hypertension, hypertriglyceridemia, low high-density lipoprotein cholesterol, central obesity, and elevated fasting glucose. RESULTS: The Orange-Red cluster score (that included Porphyromonas gingivalis and Prevotella spp.) was positively associated (odds ratio [OR] = 1.067, 95% confidence interval [CI] = 1.02 to 1.12) and the Orange-Blue cluster score (which included Actinomyces naeslundii and Eubacterium nodatum) was inversely associated (OR = 0.93, 95% CI = 0.88 to 0.97) with elevated fasting glucose (≥ 110 mg/dL) after adjustment for clusters and potential confounders. Neither MetS nor its other remaining MetS components were associated with a particular cluster score. CONCLUSIONS: The associations between specific antibody clusters (Orange-Red and Orange-Blue) against periodontal bacteria and elevated plasma glucose were in qualitatively opposite directions after multivariable adjustment in a large, adult population. The periodontal bacterial profile was not found to be associated with metabolic control other than a very moderate association with elevated plasma glucose.

Gut microbiology: fitting into the intestinal neighbourhood.

Microbes inhabiting the gut affect our health in profound and unexpected ways: new studies now show that these effects depend on synergistic and competitive interactions between the bacteria, which are influenced by diet.

Comparison of type 1 and type 2 cytokine production by mononuclear cells cultured with streptococcus mutans and selected other caries bacteria.

A feature of pulpal immune responses is the predominance of type 1 cytokine mRNA under shallow caries and a mixed (type 1/type 2) profile under deep caries. These results prompted an examination of the cytokine profiles induced by bacteria in shallow caries (Streptococcus mutans and Actinomyces viscosus) and deep caries (Lactobacillus casei, Pseudoramibacter alactolyticus, and Prevotella intermedia). All isolates induced interferon-gamma and interleukin-10, whereas interleukin-4 and interleukin-2 titers were low to undetectable. S. mutans was the most potent and persistent interferon-gamma inducer. Differences in interleukin-10 were apparent at low doses but were less dramatic, with L. casei the dominant producer. S. mutans induced substantially more interferon-gamma than interleukin-10 over all doses and time points, suggesting strong type 1 polarization. P. alactolyticus induced significantly more interleukin-10 than interferon-gamma at higher concentrations, suggesting polarization toward type 2. A similar amount of interferon-gamma and interleukin-10 induced by L. casei, A. viscosus, and P. intermedia reflected a mixed profile. A better understanding of pulpal immune response to caries bacteria may enable us to develop an immune system-based pulp therapy in the future.

[The prevalence of Actinobaculum suis in boars of breeding herds in the Omsk region (Russian Federation) by indirect immunofluorescence technique]. / Untersuchung zur Verbreitung von Actinobaculum suis unter den Ebern in den Schweinezuchtbetrieben des Gebiets Omsk (Russische Föderation) unter Verwendung der indirekten Immunofluoreszenz-Technik.

Actinobaculum suis (Corynebacterium suis, Eubacterium suis, Actinomyces suis) was detected in the preputial diverticulum of 64,8% of 162 boars investigated in 8 districts of the region Omsk (Russian Federation) by indirect immunofluorescent technique. Until yet no informations were available about the prevalence of Actinobaculum (A.) suis in swine herds of the Russian Federation. The study shows that A. suis, as a main aetiological factor of cystitis and pyelonephritis in sows, is widely spread among the boars of the region Omsk. Prevalence of A. suis was not influenced by housing conditions, age or breed of investigated boars. Indirect immunofluorescent technique for detection of A. suis provides a good method for screening investigations with high numbers of samples.

Evaluation of serological markers to differentiate between ulcerative colitis and Crohn's disease: pANCA, ASCA and agglutinating antibodies to anaerobic coccoid rods.

BACKGROUND: Accurate diagnosis of inflammatory bowel disease, in particular the differentiation between ulcerative colitis and Crohn's disease, is important for treatment and prognosis. Several serological markers have been used as non-invasive diagnostic tools in inflammatory bowel disease patients both to differentiate ulcerative colitis from Crohn's disease and to define patient subgroups. AIM: To evaluate the diagnostic accuracy of three serological tests in differentiating ulcerative colitis from Crohn's disease by single or combined use. METHODS: Sera from 51 patients with clinically well-defined ulcerative colitis and 50 patients with clinically well-defined Crohn's disease were analysed. Detection assays for the presence of perinuclear anti-neutrophil cytoplasmatic antibodies (pANCA), antibodies against (ASCA) and serum agglutinating antibodies to anaerobic coccoid rods were studied. Sensitivity, specificity, predictive values and likelihood ratios of each of these serological tests were determined. RESULTS: In supporting the diagnosis of ulcerative colitis, the sensitivity and specificity of the pANCA test were 63% and 86%, respectively. The ASCA test (immunoglobulin A or immunoglobulin G positive) for diagnosing Crohn's disease had a sensitivity of 72% and a specificity of 82%. The sensitivity of antibodies to anaerobic coccoid rods in diagnosing Crohn's disease was 52%, whereas specificity was 90%. A combination of pANCA-positive and ASCA-negative results in the case of ulcerative colitis showed a sensitivity and specificity of 51% and 94%, respectively. However, for ASCA-positive and pANCA-negative results in the case of Crohn's disease, sensitivity was 64% and specificity was 94%. The combination of all three tests increased positive predictive value and specificity to 100% for both ulcerative colitis and Crohn's disease. In Crohn's disease patients, positive pANCA was correlated with colonic involvement. No correlation was found between the presence of any of these antibodies and disease activity, duration and behaviour or medical treatment. CONCLUSIONS: The value of these serological tests in differentiating ulcerative colitis from Crohn's disease is limited when used separately but, by combining two or more tests, the positive predictive value and specificity can be improved substantially. These tests might be of help in studying disease heterogeneity, and may contribute to defining various subgroups of patients with different pathogeneses.

Intraperitoneal immune cell responses to Eubacterium saphenum in mice.

Oral asaccharolytic Eubacterium saphenum, which are newly isolated gram-positive rods and one of the predominant microorganisms in human periodontal pockets, were injected intraperitoneally in mice to elucidate their pathogenicity in periodontal diseases. Infiltrating immune cells in the peritoneal exudate were quantitated and intracellular T cell (CD4+/CD8+/gammadelta+) production of cytokines IL-4 and IFN-gamma which are related to cellular and humoral immunity, respectively, was determined. Neutrophils appeared first in peritoneal exudates, followed by macrophages and lymphocytes, after the injection of either E. saphenum or Porphyromonas gingivalis. Intracellular IL-4+ and IFN-gamma+ gammadelta T cells were detected in the exudates after the injection of E. saphenum (4.6 +/- 0.8% and 10.1 +/- 1.4%, respectively) and P. gingivalis (5.3 +/- 1.6% and 10.1 +/- 2.1%, respectively). The intracellular production of IL-4/IFN-gamma in CD4+/CD8+ T cells was rather low indicating that the main response was from gammadelta T cells which initiated the immune reactions in mouse peritoneal cavities after injection of E. saphenum or P. gingivalis. Serum IgG and IgM levels were elevated in animals injected with E. saphenum and similarly with P. gingivalis. The present study showed that with slight differences, similar modes of cell response and cytokine and Ig production were observed after intraperitoneal injection of both E. saphenum and P. gingivalis, indicating that E. saphenum may play just as important a role in periodontal diseases as P. gingivalis.

What determines arthritogenicity of bacterial cell wall? A study on Eubacterium cell wall-induced arthritis.

OBJECTIVE: To study what determines the arthritogenicity of the bacterial cell wall (CW) using Eubacterium CW-induced arthritis in the rat. METHODS: Eubacterium aerofaciens, previously reported as arthritogenic, and E. limosum and E. alactolyticum, known as non-arthritogenic, were used. Gas chromatography-mass spectrometry (GC-MS) was applied to analyse the chemical composition of the bacterial cell wall. Cellular immune response was measured by concanavalin A (Con A) stimulation and FACScan analysis. Also, serum antibodies against the injected cell wall were determined. RESULTS: Unexpectedly, from the two strains of E. aerofaciens used only one proved to be arthritogenic (with a CW inducing chronic arthritis after a single intraperitoneal injection), even though these two strains were 100% identical by 16S rDNA analysis. CW of the other E. aerofaciens strain induced only transient acute arthritis; CW of E. limosum and E. alactolyticum induced weak signs of acute arthritis. Based on the GC-MS analysis and on the results published previously, putative structures of peptidoglycan (PG) in the four CW preparations are presented. It is apparent that the presence of lysine in position 3 of the PG stem peptide contributes to arthritogenicity but is alone not decisive. Both strains of E. aerofaciens were immunosuppressive, when tested by Con A response at 2 weeks after CW injection. Such an immunosuppression was not observed after injection of CW from E. limosum or E. alactolyticum. FACScan analysis for six T cell markers and studies on serum antibody responses did not reveal any differences in the effect of the four bacterial strains used. CONCLUSIONS: The results obtained suggest that the chemical structure of PG present in the bacterial CW is decisive in determining arthritogenicity/non-arthritogenicity. Therefore, from two bacterial strains belonging to normal human intestinal flora and 100% identical by 16S rDNA analysis, one proved to be arthritogenic and the other non-arthritogenic.

Tissue distribution and persistence of arthritogenic and non-arthritogenic Eubacterium cell walls.

OBJECTIVE: To study the tissue distribution and persistence of arthritogenic and non-arthritogenic Eubacterium cell walls (CWs), using arthritogenic Eubacterium aerofaciens and non-arthritogenic Eubacterium limosum. METHODS: Eubacterium aerofaciens or Eubacterium limosum CW was injected into Lewis rats intraperitoneally. Inflammatory changes in the synovium and periarticular tissues were graded histologically. On days 14, 28 and 56 after the injection, the presence of CW in the liver, spleen, mesenteric lymph nodes and synovium was studied by indirect immunofluorescence. In parallel, CW-derived muramic acid in the liver and spleen was measured by gas chromatography-mass spectrometry. In addition, serum TNF-alpha, IL-1 beta and IL-10 concentrations were determined by ELISA. RESULTS: Systemic injection of Eubacterium aerofaciens CW, but not of Eubacterium limosum CW, resulted in chronic arthritis. Both E. aerofaciens and E. limosum CWs were observed in the liver and spleen at all of the time points studied. In addition, Eubacterium limosum CW was present in non-arthritic synovium on day 14. It was not, however, detected in the synovium or lymph nodes on days 28 and 56, in clear contrast to the rats injected with E. aerofaciens CW. According to the analysis by gas chromatography-mass spectrometry, non-arthritogenic E. limosum CW had accumulated in the liver cells on days 14 and 28 after the injection to a greater extent than arthritogenic E. aerofaciens CW, leading to a lesser distribution in the other organs. A weak trend was observed suggesting that the production of TNF-alpha and IL-1 beta, but not of IL-10, is stimulated better by arthritogenic CW than by non-arthritogenic CW. CONCLUSION: Our results indicate that non-arthritogenic CWs are handled by the rat's defence mechanisms in a different way than arthritogenic CWs. The tissue distribution and persistence of CWs play a role in arthritogenicity, but additional factors must exist to determine why the CWs of certain bacteria are arthritogenic and those of others are not.

Antitumor mechanisms of Eubacterium lentum and its components.

In the present study, some antitumor mechanisms of Eubacterium lentum (TYH-11) and bacterial components having antitumor effects were investigated. E.lentum induced maximum NK cell activity in C3H/He mice on day 1 after injection (90.6% against 33.9% of control at E:T ratio 50:1) and the activity was kept at a level of 48.6% on day 7. Tumoricidal peritoneal macrophages were induced 9 days after E.lentum injection into BALB/c mice (56.2% against 10.1% control at E:T ratio 10:1). Tumoricidal macrophage activity persisted at the same level for at least 11 days. Cytotoxic T lymphocyte (CTL) activity was induced only in tumor bearing mice treated with E.lentum, 4 weeks after tumor inoculation. Antitumor activity was observed in the cell wall (CW) and membrane fractions (CM) of E.lentum. CW induced NK cell activity; the activity was transient while the kinetics of NK activity by CM showed 2 peaks, on day 1 and day 7. Tumoricidal macrophages were induced by CW and the activity level was the same as that induced by whole body, while that induced by CM was at a lower level. Neither CW nor CM induced CTL in tumor bearing mice.

[Serologic examinations for the detection of antibodies against Eubacterium suis in swine]. / Serologische Untersuchungen zum Nachweis von Antikörpern gegen Eubacterium suis beim Schwein.

Determination of antibodies against Eubacterium suis (E. suis) in the serum of pigs was carried out by an indirect immunofluorescence test. Therefore 35 sows with urinary tract infections as well as 43 healthy control sows were investigated. E. suis could be detected in the urine of 19 sows. Serological results were compared with the bacteriological examinations. To reach a specificity of 100% for detection of animals actually infected with E. suis, titres from 1:16++ can be accepted as positive. Therefore sensitivity gave a level of 78.9%. Seroconversion and development of antibody titers were studied on 15 sows, which were experimentally infected with E. suis. Antibodies could be shown 3 weeks after infection at the earliest, they were preferably demonstrated in sows with a haemorrhagic cystitis. No correlation existed between the affection of the kidneys and the development and the level of titres respectively. Serological investigations can turn out negative in spite of E. suis infection, if immunological response still has not taken place or development of antibodies has failed to appear after short-time infection of the bladder.

The gingival immune response to periodontal pathogens in juvenile periodontitis.

A gingival explant culture system was utilized to evaluate the reactivity of local immunoglobulins produced by juvenile periodontitis tissue. Gingival explant culture supernatant fluids were screened, via a standardized dot-immunobinding assay, for antibodies reactive to: Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Campylobacter rectus, Eikenella corrodens, Peptostreptococcus micros, Peptostreptococcus anaerobius, Capnocytophaga ochracea, Eubacterium nodatum and Fusobacterium nucleatum and one nonoral microorganism, Porphyromonas asaccharolytica. Of the 75 juvenile periodontitis supernatant fluids tested, the organisms that reacted with the highest numbers of supernatant fluids were E. nodatum (72%) and A. actinomycetemcomitans (49%). More juvenile periodontitis than healthy tissue samples showed supernatant fluid reactivity to P. intermedia, C. ochracea, E. nodatum and P. micros. No significant difference was observed between the juvenile periodontitis group supernatant fluids reactivity and the supernatant fluids of the other periodontal disease groups tested. Cluster analysis revealed the association, as determined by supernatant fluid reactivity, of P. micros and C. ochracea in the juvenile periodontitis group. The data from this investigation are consistent with a hypothesis of multiple possible etiologies of periodontal destruction in juvenile periodontitis and other forms of periodontal diseases.

Detection of intestinal flora-derived bacterial antigen complexes in splenic macrophages of rats.

We studied the presence of bacterial antigens in rat tissues. We produced a monoclonal antibody (MAb 2E9) directed against intestinal flora-derived peptidoglycan-polysaccharide complexes from human and rat feces. With several immunological techniques, the specificity of 2E9 for this bacterial product was demonstrated. Using 2E9 in an immunohistological assay, we were able to show the presence of bacterial products in macrophages in the red pulp of spleens of conventional Lewis rats. However, we found no correlation between the development of the intestinal flora and positive spleen staining with MAb 2E9. The results were confirmed by immunohistology with a previously described MAb 2-4 directed to muramyl dipeptide. Other lymphoid organs did not stain positively with 2E9 and 2-4. Neonatal and young rats showed no staining of the spleen, but positivity could be induced by injecting peptidoglycan-polysaccharide complexes systemically. We conclude that bacterial fragments are present in splenic macrophages of conventional rats.

Immunological specificity of oral Eubacterium species.

Antigens of Eubacterium species including E. alactolyticum, E. brachy, E. nodatum, E. saburreum, E. timidum, E. yurii subsp. yurii and E. yurii subsp. margaretiae, which have been isolated frequently from periodontal pockets and associated with periodontal diseases, were extracted by ultrasonication from whole bacterial cells. Antigens were also prepared from E. aerofaciens, E. lentum and E. rectale, which have been found in intestinal tracts and infected abscesses in human oral cavities. The antigens of the oral Eubacterium species were compared with antigens from E. limosum, the type species of the genus Eubacterium, by using SDS-PAGE and Western immunoblot assays. SDS-PAGE gels stained with Coomassie brilliant blue indicated that no major peptide bands were common among the Eubacterium species examined. The protein profile patterns were distinctly different from each other. Western immunoblotting reactions with rabbit antisera showed that the Eubacterium species could be clearly distinguished serologically, and that the species-specific antigens were peptide components of ultrasonic extracts from the whole bacterial cells. The present study demonstrates that these Eubacterium species show great heterogeneity in their peptide components and immunological reactions, which may be useful for identification of the Eubacterium species from human oral specimens.

Levels of salivary IgA antibodies reactive with bacteria from dental plaque are associated with susceptibility to experimental gingivitis.

Serum IgG, IgA and IgM and salivary IgA antibody levels reactive with extracts from Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Eubacterium saburreum and Streptococcus mutans, were measured by enzyme-linked immunosorbent assay in samples from 12 persons before, during and after experimental gingivitis. The participants refrained from cleaning their teeth until 50% of their gingival units showed bleeding after gentle probing, but not longer than 15 days. Samples were taken from serum and saliva, before and during the period of experimental gingivitis, and up to 8 weeks after the start of the experiment. A pattern with minor fluctuations in specific serum and salivary antibody activities was consistently found in all patients. This indicates that immunoregulatory mechanisms succeed in maintaining unchanged antibody levels when plaque load increases. A subgroup of participants with low mean numbers of bleeding gingival units after plaque accumulation, showed significantly higher salivary IgA antibody levels reactive with S. mutans, A. actinomycetemcomitans and E. saburreum, as compared with the subgroup reaching high bleeding after probing scores (p < 0.05). When 1 person with outlying values (p < 0.05) for P. gingivalis was excluded from the tests, the former group also showed statistically significant higher salivary antibody levels to this bacterial species. High levels of salivary IgA directed against bacteria in dental plaque might thus protect against the development of gingivitis.

Structural studies of the antigenic polysaccharide of Eubacterium saburreum, strain T19.

The antigenic polysaccharide produced by Eubacterium saburreum, strain T19, contains unusual sugars, including D-glycero-D-galacto-heptose (Hep) and D-fucose (D-Fuc). A repeating unit of the polysaccharide is composed of a linear chain of D-glycero-D-galacto-heptopyranosyl tetrasaccharide as its backbone structure, i.e., -[-->6)-beta-Hep p-(1-->3)-beta-Hep p-(1-]2-->, and a D-fucofuranosyl disaccharide as a branched group, i.e., alpha-D-Fuc f-(1-->2)-alpha-D-Fuc f-(1-->, which is linked to O-4 of one (1--6)-linked D-glycero-D-galacto-heptopyranosyl residue. The polysaccharide also contains O-acetyl groups.

Immunohistology of joint inflammation induced in rats by cell wall fragments of Eubacterium aerofaciens.

After a single intraperitoneal injection of cell wall fragments of Eubacterium aerofaciens, a main resident from the human intestinal flora, an acute arthritis develops within 2 days which is followed by a chronic arthritis that lasts at least 90 days. In an earlier report the histological appearance of the joint inflammation during this period has been described. In this study we investigated in more detail the cell types that are involved in the development of arthritis by using cell-type-specific monoclonal antibodies in an immunohistological assay. In the acute phase of arthritis, T-helper cells appeared in the synovial tissue together with ED1-positive (ED1+) and ED3-positive (ED3+) macrophages. After a temporary decline at day 12 all macrophage subsets, as well as T-helper cells, reappeared or increased again at day 33. Later, in the chronic phase (days 47-90), an increased number of ED1-positive (ED1+) cells in the synovial tissue and a decreased number of ED2-positive (ED2+) cells in the synovial lining was the most prominent finding when compared with control rats. These results indicate that, apart from T lymphocytes, macrophages also play an important role in the development and continuation of chronic arthritis in this model.

Immunochemical and structural characterization of the antigenic polysaccharide from Eubacterium saburreum T18.

An antigenic surface polysaccharide produced by Eubacterium saburreum strain T18, isolated from human dental plaque, was purified from formamide extract of whole cells. Methylation analysis, Smith degradation, optical rotation data and nuclear magnetic resonance spectra demonstrated that the purified antigen was a homopolysaccharide composed of D-glycero-D-galacto-heptose (Hep.) residues. The structure of the repeating unit in the polysaccharide was: -[----6)-[alpha-Hep.furanosyl-(1----4)]-beta-Hep.pyranosyl- (1----6)-[alpha-Hep.furanosyl-(1----2), alpha-Hep.furanosyl-(1----4)]-beta- Hep.pyranosyl-(1-)4----6)-beta-Hep.pyranosyl-(1----. No heptose residues were acetylated. Immunodiffusion reactions in agar gel suggested that the immunodeterminant of the antigenic polysaccharide was D-glycero-D-galacto-heptofuranosyl residues as branched nonreducing terminals.

Serum IgG reactive with oral anaerobic microorganisms associated with infections of endodontic origin.

Numerous species of bacteria have been implicated with infections of endodontic origin. The purpose of this study was to compare the levels of serum IgG antibodies reactive with a panel of 10 oral anaerobic microorganisms implicated in infections of endodontic origin. Serum samples were collected from 4 patient groups that included healthy patients without endodontic or periodontal disease, patients with chronic adult periodontal disease, patients with endodontic disease and patients with combined endodontic-periodontal disease. When Prevotella intermedia was allowed to react with sera from the 4 patient groups, significant pairwise differences were shown between the healthy group and each of the other 3 groups. In addition, there was a significant difference between the periodontal disease group and the combined endodontic-periodontal disease group. When Porphyromonas gingivalis was allowed to react with sera from the 4 patient groups, significant pairwise differences were shown between the healthy group and the periodontal disease group, the healthy group and the combined endodontic-periodontal disease group, the endodontic disease group and the periodontal disease group and the endodontic disease group and the combined endodontic-periodontal disease group. The results of this investigation support other studies that associate P. intermedia with both endodontic disease and chronic adult periodontal disease. The results also support studies that implicate P. gingivalis as a periodontopathogen.