Actinomycetemcomitans Strain Y4 Research Articles

Distinct Signaling Pathways Between Human Macrophages and Primary Gingival Epithelial Cells by Aggregatibacter actinomycetemcomitans.

In aggressive periodontitis, the dysbiotic microbial community in the subgingival crevice, which is abundant in Aggregatibacter actinomycetemcomitans, interacts with extra- and intracellular receptors of host cells, leading to exacerbated inflammation and subsequent tissue destruction. Our goal was to understand the innate immune interactions of A. actinomycetemcomitans with macrophages and human gingival epithelial cells (HGECs) on the signaling cascade involved in inflammasome and inflammatory responses. U937 macrophages and HGECs were co-cultured with A. actinomycetemcomitans strain Y4 and key signaling pathways were analyzed using real-time PCR, Western blotting and cytokine production by ELISA. A. actinomycetemcomitans infection upregulated the transcription of TLR2, TLR4, NOD2 and NLRP3 in U937 macrophages, but not in HGECs. Transcription of IL-1β and IL-18 was upregulated in macrophages and HGECs after 1 h interaction with A. actinomycetemcomitans, but positive regulation persisted only in macrophages, resulting in the presence of IL-1β in macrophage supernatant. Immunoblot data revealed that A. actinomycetemcomitans induced the phosphorylation of AKT and ERK1/2, possibly leading to activation of the NF-κB pathway in macrophages. On the other hand, HGEC signaling induced by A. actinomycetemcomitans was distinct, since AKT and 4EBP1 were phosphorylated after stimulation with A. actinomycetemcomitans, whereas ERK1/2 was not. Furthermore, A. actinomycetemcomitans was able to induce the cleavage of caspase-1 in U937 macrophages in an NRLP3-dependent pathway. Differences in host cell responses, such as those seen between HGECs and macrophages, suggested that survival of A. actinomycetemcomitans in periodontal tissues may be favored by its ability to differentially activate host cells.

Azithromycin enhances phagocytic killing of Aggregatibacter actinomycetemcomitans Y4 by human neutrophils.

Aggregatibacter actinomycetemcomitans resists killing by neutrophils and is inhibited by azithromycin (AZM) and amoxicillin (AMX). AZM actively concentrates inside host cells, whereas AMX enters by diffusion. The present study is conducted to determine whether AZM is more effective than AMX at enhancing phagocytic killing of A. actinomycetemcomitans by neutrophils. Killing assays were conducted in the presence of either 2 μg/mL AZM or 16 μg/mL AMX (equipotent against A. actinomycetemcomitans). Neutrophils were loaded by incubation with the appropriate antibiotic. Opsonized A. actinomycetemcomitans strain Y4 was incubated with the indicated antibiotic alone, with loaded neutrophils and antibiotic, or with control neutrophils (without antibiotic) at multiplicities of infection (MOIs) of 30 and 90 bacteria per neutrophil. Neutrophil incubation with 2 μg/mL AZM yielded an intracellular concentration of 10 μg/mL. At an MOI of 30, neutrophils loaded with AZM failed to kill significantly more bacteria than control neutrophils during the 60- and 90-minute assay periods. At an MOI of 90, neutrophils loaded with AZM killed significantly more bacteria than either AZM alone or control neutrophils during 60- and 90-minute incubations (P < 0.05), and killed significantly more bacteria after 90 minutes than the sum of the killing produced by AZM alone or neutrophils alone. Neutrophils incubated with AMX under identical conditions also killed significantly more bacteria than either AMX alone or control neutrophils, but there was no evidence of synergism between AMX and neutrophils. Neutrophils possess a concentrative transport system for AZM that may enhance killing of A. actinomycetemcomitans. Its effects are most pronounced when neutrophils are greatly outnumbered by bacteria.

An &amp;lt;i&amp;gt;in vitro&amp;lt;/i&amp;gt; study of antimicrobial agents incorporated into interim restorative materials

Aim: To test the hypothesis that antimicrobial agents can be incorporated into bis-acryl resin and that the material will inhibit bacterial growth in vitro. Materials and Methods: 0.5%, 1% and 2% triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol) and 0.05% 0.01% and 0.15% cetylpyridinium chloride were incorporated into bis-acryl resins and molded into 1 mm × 10 mm discs. The discs were placed on bacterial culture media inoculated with Aggregatibacter actinomycetemcomitans strain Y4, Porphyromonas gingivalis strain SUNYaB A7A1-28 or Streptococcus gordonii strain 10558, incubated and examined for growth inhibition. Results: Triclosan-containing bisacryl resin discs produced dose-dependent zones of growth inhibition for A. actinomycetemcomitans but not S. gordonii or P. gingivalis. Cetylpyridinium chloride-containing discs produced dose-dependent zones of growth inhibition for Porphyromonas gingivalis and Streptococcus gordonii but not A. actinomycetemcomitans. Conclusion: Triclosan and cetylpyridinium chloride can be incorporated into bis-acryl resin and can inhibit the growth of plaque bacteria in a dose dependent manner. Bis-acryl resin containing antimicrobial agents may inhibit in vivo plaque accumulation and gingival inflammation.

Effect of eel galectin AJL-1 on periodontopathic bacterial biofilm formation and their lipopolysaccharide-mediated inflammatory cytokine induction

Porphyromonas gingivalis, Prevotella intermedia and Aggregatibacter actinomycetemcomitans, infectious pathogenic bacteria found in oral biofilm, cause periodontal disease. The inhibitory effect of AJL-1, a galectin present in the skin mucus of the Japanese eel Anguilla japonica, on biofilm formation by each of these strains was investigated by staining adherent bacteria on culture plates with crystal violet. An ATP bioluminescence assay was used to determine whether inhibition of biofilm formation was due to the bactericidal activity of AJL-1. The effect of AJL-1 on cytokine induction in human umbilical vascular endothelial cells (HUVECs) by lipopolysaccharide (LPS) isolated from A. actinomycetemcomitans was also investigated by enzyme-linked immunosorbent assay (ELISA). AJL-1 significantly inhibited biofilm formation by A. actinomycetemcomitans strains Y4, ATCC 29523 and ATCC 29524 but not by any strain of P. gingivalis or P. intermedia, and showed no bactericidal activity against A. actinomycetemcomitans strains. AJL-1 markedly suppressed interleukin (IL)-6 and IL-8 induction in HUVECs by LPS from A. actinomycetemcomitans strains Y4 and ATCC 29523. These observations indicate that AJL-1 is an effective inhibitor of biofilm formation by A. actinomycetemcomitans as well as of inflammatory cytokine induction in HUVECs by LPS. These finding indicate that AJL-1 may be of therapeutic value in A. actinomycetemcomitans-associated periodontal diseases.

Evidence that the cytolethal distending toxin locus was once part of a genomic island in the periodontal pathogen Aggregatibacter (Actinobacillus) actinomycetemcomitans strain Y4

The authors have previously shown that the periodontal pathogen Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans Y4 contains an operon for a genotoxin known as the cytolethal distending toxin (Cdt). The cdt locus in strain Y4 is flanked by remnants of heterologous plasmid and integrase sequences. In this study, the DNA sequence immediately downstream from the cdt locus on the Y4 chromosome was examined. The extended sequence contained a region that had all the characteristics of a typical bacterial pathogenicity or genomic island. The genomic island (GIY4-1) was approximately 22 kb long, was flanked by a bacteriophage attachment (att) sequence and contained a full-length integrase/resolvase gene (xerD). A total of 22 complete and partial ORFs represented putative DNA replication/DNA binding/conjugation proteins as well as hypothetical proteins. GIY4-1 was most closely related to putative genomic islands in Haemophilus ducreyi 35000HP and Haemophilus influenzae 86-028NP and to a chromosomal region in Haemophilus somnus 129PT. GIY4-1 was not present in HK1651, which was used as the prototype strain for genomic sequencing of A. actinomycetemcomitans. Several sequences in GIY4-1 were homologous to ORFs found on the A. actinomycetemcomitans plasmid pVT745. None of the identified ORFs in GIY4-1 appeared to encode potential virulence genes. However, several unique observations supported the possibility that the cdt locus of A. actinomycetemcomitans Y4 was originally contained within the genomic island.

Inhibitory effect of synthetic histatin 5 on leukotoxin from Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans is a gram-negative bacterium strongly implicated in the pathogenesis of juvenile periodontitis. This periodontal pathogen synthesizes a leukotoxin that destroys human polymorphonuclear leukocytes (PMNs), and this toxin is thought to be responsible for the virulence of A. actinomycetemcomitans. It was therefore of interest to assess whether major virulence factors of periodontal pathogens were neutralized by salivary components. This study focuses on the effect of histatins, components of the nonimmune oral defense system, on leukotoxin activity. Leukotoxin was extracted with polymyxin B from freshly grown anaerobic cultures of A. actinomycetemcomitans strain Y4. PMNs isolated from blood of healthy human volunteers were incubated in a cytotoxicity assay containing PMNs (10(7) cells/ml) and leukotoxin preparation (0-500 microg/ml) in Hanks' balanced salt solution at 37 degrees C for 0-120 min with or without synthetic histatin 5 (0-500 microM). Cytotoxicity was measured by release of lactate dehydrogenase (LDH) at different time intervals. Histatin 5 neutralized the toxic effect of the leukotoxin preparation in a concentration-dependent manner, with an IC(50) value of 150 microM. When PMNs were preincubated with histatin 5 (300 microM), washed and subsequently exposed to leukotoxin, no protective effect was observed. This observation suggests a mechanism of inhibition whereby histatin 5 either directly neutralizes the leukotoxin or interferes with the leukotoxin-PMN interaction. The inhibitory effect of histatin 5 on leukotoxic activity may suggest a new biological function of histatins in the oral cavity as a naturally occurring secondary antibiotic.

Identification of genes involved in biosynthesis of Actinobacillus pleuropneumoniae serotype 1 O-antigen and biological properties of rough mutants

Actinobacillus pleuropneumoniae is an important pathogen of swine. Lipopolysaccharide (LPS) has been identified as the major adhesin of A. pleuropneumoniae and it is involved in adherence to porcine respiratory tract cells. We previously generated seven rough LPS mutants of A. pleuropneumoniae serotype 1 by using a mini-Tn10 transposon mutagenesis system [Rioux S, Galarneau C, Harel J et al. Isolation and characterization of mini-Tn10 lipopolysaccharide mutants of Actinobacillus pleuropneumoniae serotype 1. Can J Microbiol 1999; 45: 1017-1026]. The purpose of the present study was to characterize these mutants in order to learn more about LPS O-antigen biosynthesis genes and their organization in A. pleuropneumoniae, and to determine the surface properties and virulence in pigs of these isogenic mutants. By mini-Tn10 insertions in rough mutants, four putative genes (ORF12, ORF16, ORF17, and ORF18) involved in O-antigen biosynthesis in A. pleuropneumoniae serotype 1 were found within a region of 18 ORFs. This region is homologous to the gene cluster of serotype-specific O-polysaccharide biosynthesis from A. actinomycetemcomitans strain Y4 (serotype b). Two mutants showed homology to a protein with identity to glycosyltransferases (ORF12); two others had the mini-Tn10 insertion localized in genes encoding for two distinct proteins with identity to rhamnosyltransferases (ORF16 and ORF17) and three showed homology to a protein which is known to initiate polysaccharide synthesis (ORF18). These four ORFs were also present in A. pleuropneumoniae serotypes 9 and 11 that express an O-antigen that serologically cross-reacts with serotype 1. Evaluation of some biological properties of rough mutants seems to indicate that the absence of O-chains does not appear to have an influence on the virulence of the bacteria in pigs and on the overall surface hydrophobicity, charge and hemoglobin-binding activity, or on LAL activation. An acapsular mutant was included in the present study in order to compare the influence of O-chains and capsule polysaccharides on different cell surface properties. Our data suggest that capsular polysaccharides and not O-chains polysaccharides have a major influence on surface properties of A. pleuropneumoniae serotype 1 and its virulence in pigs.

Genetic analysis of the gene cluster responsible for synthesis of serotype e-specific polysaccharide antigen in Actinobacillus actinomycetemcomitans

A gene cluster associated with the biosynthesis of the serotype e-specific polysaccharide antigen (SPA) of Actinobacillus actinomycetemcomitans IDH1705 belonging to serotype e was cloned and sequenced. This cluster consisted of 18 open reading frames. Escherichia coli produced the polysaccharide that reacts with the serotype e-specific antiserum when transformed with a plasmid containing the cluster. Comparing the structure of the gene cluster with similar clusters from A. actinomycetemcomitans strains Y4 (serotype b) and NCTC9710 (serotype c) revealed that a 5.3-kb region containing the distal half of one gene and two entire genes in the cluster from strain IDH1705 replaced a 6.2-kb region containing eight genes in the cluster from strain Y4, and a 4.7-kb region containing four genes in the cluster from strain NCTC9710. These results suggest that this region is essential to the antigenic specificity of serotype e A. actinomycetemcomitans.

Binding of the capsule-like serotype-specific polysaccharide antigen and the lipopolysaccharide from Actinobacillus actinomycetemcomitans to human complement-derived opsonins.

We investigated the molecular mechanism of resistance of Actinobacillus actinomycetemcomitans to complement-dependent chemiluminescence response by human polymorphonuclear leukocytes. Whole cells of serotype b-specific polysaccharide antigen-defective mutants ST2 and ST5 were constructed by inserting transposon Tn916 into A. actinomycetemcomitans strain Y4. These strains induced strong chemiluminescence response by human polymorphonuclear leukocytes and markedly bound to human complement-derived opsonins. In contrast, strain Y4 induced weak chemiluminescence response and weakly bound to complement-derived opsonins. The biosensor analysis revealed that lipopolysaccharide from strain Y4 strongly bound to human C3b, but serotype b-specific polysaccharide antigen did not. The serotype b-specific polysaccharide antigen molecule might sterically hinder the interaction between complement-derived opsonins and lipopolysaccharide to reduce complement-dependent chemiluminescence response by human polymorphonuclear leukocytes.

Serum IgG antibody response to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis: implications for periodontal diagnosis.

The relationship of the serum antibody titer and avidity to the putative periodontal pathogens Actinobacillus actinomycetemcomitans (Aa) strains Y4 and 29523 and Porphyromonas gingivalis (Pg) strain 381 were examined in relation to clinical parameters in 26 gingivitis and 28 periodontitis patients. The relationship of antibody titer and avidity to infection with the homologous organism was also examined in a subset of 30 patients. Antibody titer was determined by an enzyme-linked immunosorbent assay, and antibody avidity was assessed using a dissociation assay. Considering all patients, there was a significant negative correlation between mean probing depth and antibody titer (r=-0.28) and avidity (r=-0.28) to Aa Y4. There was a significant positive correlation of probing depth and antibody titer (r=0.46) and avidity (r=0.46) to Pg. The correlation of antibody titer and avidity to Aa and infection with Aa Y4 (r=-0.32, r=-0.21) and Aa 29523 (r=-0.35, r=-0.39) was negative, while the correlations of titer and avidity to Pg and presence of the organisms was strongly positive (r=0.40, r=0.35). These data indicate that the relationship of serum antibody titer and avidity to clinical parameters of periodontal disease severity and the level of infection with the homologous organism appears to be different for Aa and Pg. The development of an antibody response to Aa appears to protect the individual from infection with the organism. In contrast, the development of an antibody response to Pg was not able to eliminate the infection. These results should be considered when developing a diagnostic strategy for periodontal disease utilizing the humoral immune response.

Role of prostaglandin in the formation of osteoclasts induced by capsular-like polysaccharide antigen of Actinobacillus actinomycetemcomitans strain Y4.

We found no reports that capsular-like polysaccharide antigen purified from Actinobacillus actinomycetemcomitans either induces osteoclastic bone resorption in mouse organ cultures or promotes osteoclast formation in mouse marrow cultures. In contrast, capsular-like polysaccharide antigen purified from A. actinomycetemcomitans strain Y4 induced bone resorption in mouse organ culture. To examine the mechanism of bone resorption induced by A. actinomycetemcomitans, mouse bone marrow cells were cultured with A. actinomycetemcomitans strain Y4 capsular-like polysaccharide antigen. A. actinomycetemcomitans strain Y4 capsular-like polysaccharide antigen stimulated osteoclast-like cell formation in mouse bone marrow cultures. However, the polysaccharide of A. actinomycetemcomitans lipopolysaccharide did not induce the formation of osteoclast-like cells. Indomethacin inhibited osteoclast-like cell formation mediated by A. actinomycetemcomitans strain Y4 capsular-like polysaccharide antigen in a dose-dependent manner. There was a good correlation between the number of osteoclast-like cells formed in the marrow culture and the amount of prostaglandin E2 released into the culture media. When mouse bone marrow cells were cultured with prostaglandin E2 during the culture periods, many osteoclast-like cells were formed. These results indicate that prostaglandin E2 is involved in the mechanism of the formation of osteoclast-like cells mediated by A. actinomycetemcomitans strain Y4 capsular-like polysaccharide antigen. A. actinomycetemcomitans strain Y4 capsular-like polysaccharide antigen may play an important role in inflammatory bone resorption by promoting osteoclast formation in periodontal disease.

Antigen Specificity of Serum Antibody in A. actinomycetemcomitans-infected Periodontitis Patients

We hypothesized that serum antibody with selected antigen specificities would relate to infection and disease in the patients and, thus, describe the characteristics of potential protective antibody. This study used serum samples from 24 periodontitis patients with subgingival infection and elevated serum IgG antibody to A. actinomycetemcomitans to define the antigenic specificities of IgG, IgM, IgA, and IgG1-4 antibody to A. actinomycetemcomitans strain Y4 outer membrane antigens (OMA). Uniform IgG antibody (> 70% of the patients) was noted to antigens with M(r) of 65, 38, 29, and 17 kDa. Both IgA and IgM specificities reflected those shown for IgG in each patient. IgG1 and IgG2 antibody reacted with several OMA bands in each patient, while IgG3 antibodies were directed to numerous OMA bands in many patients and represented the most broad-based response. The IgG4 response patterns were limited to a few OMA bands. We noted a prominent occurrence of IgG reactions with OMA bands that were characteristic for individual patients. The frequency of responses to OMA of higher M(r) (i.e., > 80 kDa) and to the 34-, 31-, and 24-kDa antigens was positively related to the total IgG antibody levels. Antibody reactive with OMA bands at 65-, 38-, 29-, 17-, 15-, and 11-kDa antigens was detected in patients with few to many teeth infected with A. actinomycetemcomitans. Furthermore, patients with a high percentage of teeth with > or = 6 mm pockets had a decreased frequency of responses to the high-M(r) antigens (i.e. > 90 kDa) as well as to the 58-kDa antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Membrane protein expression by Actinobacillus actinomycetemcomitans in response to iron availability.

Iron-limited growth conditions under anaerobiosis were established for Actinobacillus actinomycetemcomitans strains Y4, JP2, and 75 by use of the ferrous ion chelator 2,2'-dipyridyl. Growth inhibition was reversible with both ferrous and ferric iron sources. Sarcosyl-insoluble membrane fractions of iron-stressed anaerobic A. actinomycetemcomitans cultures revealed a similar iron-repressible protein of approximately 70 kDa in A. actinomycetemcomitans strains Y4, JP2, and 75. This 70-kDa protein was recognized by serum from localized juvenile periodontitis patients and a periodontally healthy subject. This suggested that the 70-kDa iron-repressible protein may be expressed in vivo. When A. actinomycetemcomitans was grown under aerobic conditions, the ferric iron chelator, ethylenediamine di(o-hydroxyphenylacetic acid) (EDDA) was utilized for growth limitation. EDDA inhibition was reversible in strain Y4 with ferrous and ferric iron sources. An iron-repressible protein of approximately 70 kDa was also noted in iron-stressed aerobic cultures. The 70-kDa protein may be involved in iron transport by A. actinomycetemcomitans. Preliminary experiments were performed to examine potential iron transport systems for A. actinomycetemcomitans. Production of the two most common chemical types of siderophore was not detected in A. actinomycetemcomitans culture supernatants. Iron-starved A. actinomycetemcomitans cells did not bind transferrin or lactoferrin in a dot blot assay.

Humoral IgG antibodies to oral microbiota in a population at risk for root-surface caries.

Mutans streptococci have been strongly implicated in the initiation of dental caries on coronal surfaces. Their role in development of root-surface caries is less clear. The etiologic agents of both types of dental caries are likely to elicit systemic immune responses. The objective of the present study, therefore, was to study the association of clinical variables of disease with humoral IgG antibodies to nine oral micro-organisms in 314 adult subjects, aged 45-65 years, who were at risk for root-surface caries. Antibody activity to Streptococcus mutans strain Ingbritt, S. mutans/S. sobrinus GTFs, S. faecalis strain 19433, Actinomyces viscosus strain WVU 626, Actinomyces naeslundii strain 12, Lactobacillus casei, Actinobacillus actinomycetemcomitans strain Y4, Porphyromonas gingivalis strain 381, Eikenella corrodens strain 1073, and Wolinella recta strain 371 was measured by ELISA. Pearson correlation coefficients among log10 antibody levels within subjects revealed marked positive correlations among subgingival bacteria, generally weak positive correlations among supragingival micro-organisms, and no correlations between elements of the supragingival battery with the subgingival battery. IgG antibody levels to mutans streptococcal antigens were significantly correlated with subject DMF scores (r = 0.23; p less than 0.0001). No significant correlation was seen between DMF scores and antibody to any other supragingival micro-organism tested. Further relationships between levels of S. mutans antibody and individual clinical variables were analyzed by step-wise multiple linear regression, resulting in a model that was highly significant (p = 0.0001), with an r2 = 0.14. Numbers of missing teeth, coronal caries, root-surface caries, and root-surface restorations were each positively associated with antibody levels to mutans streptococci.(ABSTRACT TRUNCATED AT 250 WORDS)

Construction of mutants of Actinobacillus actinomycetemcomitans defective in serotype b-specific polysaccharide antigen by insertion of transposon Tn916.

Mutants of Actinobacillus actinomycetemcomitans strain Y4 defective in the capsular-like serotype b-specific polysaccharide antigen (SPA) were constructed by inserting the transposon Tn916. Southern blot analysis suggested that the transposon was inserted into a variety of different sites on the chromosome. Whole cells from two mutants (strains ST1 and ST2) lacked reactivity with a monoclonal antibody to SPA of A. actinomycetemcomitans Y4 (mAb S5) in enzyme-linked immunosorbent assay, but those from another nine mutants (e.g. strains ST3 and ST5) reacted very weakly with mAb S5. Immunodiffusion tests showed that mAb S5 or rabbit antiserum against whole cells of strain Y4 produced a fused precipitin band with purified SPA and autoclaved extract from strain Y4, but no precipitin band with autoclaved extracts from these four mutants. The hydrolysate of autoclaved extract from strain Y4 contained equal amounts of rhamnose and fucose, component sugars of SPA. The hydrolysates of autoclaved extracts from strains ST1 and ST2 contained a trace amount of rhamnose, but not fucose. Those of autoclaved extracts from strains ST3 and ST5 contained a trace amount of fucose, but not rhamnose. All of these SPA-defective mutants reacted with a mAb to lipopolysaccharide of strain Y4. The cell hydrophobicity of SPA-defective mutants was higher than that of the parent strain. These mutant clones will be useful for analysing the gene complex responsible for the synthesis of SPA of A. actinomycetemcomitans and the regulation of expression of the polysaccharide.

Immunodominant antigens of Actinobacillus actinomycetemcomitans serotype b in early-onset periodontitis patients.

Previous work with Actinobacillus actinomycetemcomitans strain Y4 (serotype b) indicates that the immunodominant antigen in high-responding patients (top 10%, 80% of which were black) is the serotype-specific antigen. In this study we examined the immunodominant antigens of A. actinomycetemcomitans strain Y4 in both black and white patients having a range of antibody titers. We sought to test the hypothesis that the immunodominant antigen in these subjects was the same antigen found in high responders. Seropositive white early-onset periodontitis (EOP) patients were selected from 99 EOP patients. Black subjects were then selected with comparable antibody titers. Double immunodiffusion and competition assays were used to determine whether reactive antibodies were A. actinomycetemcomitans serotype b-specific or whether the response was to serotype a or c. The immunodominant antigens were then determined for the patients reacting specifically to A. actinomycetemcomitans Y4 using limiting dilution analysis on Western blots. The immunodominant antigen for the A. actinomycetemcomitans Y4-specific patients appeared to be the serotype-specific carbohydrate for most subjects (19/20 or 95%, including: 13/14 black and 6/6 white patients). In conclusion, the immunodominant antigen for A. actinomycetemcomitans Y4 was the serotype-specific carbohydrate regardless of antibody titer for both black and white specifically reactive patients.

Salivary IgA Antibodies Against Bacteria Incriminated as Periodontal Pathogens in Kenyan Adolescents: Correlation with Disease Status and Demonstration of Antibody Specificity

Kenyan adolescents, without destructive periodontal disease, possess salivary IgA antibodies to Actinobacillus actinomycetemcomitans strain Y4, Porphyromonas gingivalis strain W83, Prevotella intermedia , Capnocytophaga ochracea and Streptococcus mutans . There were strong correlations between the levels of antibodies to each species tested, to the total antibacterial IgA response and to the levels of the total IgA isotype. The mean antibody levels to all the organisms tested was significantly positively correlated with the Bleeding Index and negatively with the Plaque Index. Reduction of the saliva with periodate decreased the IgA binding to each species tested, suggesting that a part of the specific IgA antibodies may be associated with bacteria binding salivary glycoproteins. The association between the IgA antibodies to each species was unaffected by periodate reduction. Keywords: IgA; Salivary; Bacteria; Periodontal; Antibodies.

Investigation of the structure of lipid A from Actinobacillus actinomycetemcomitans strain Y4 and human clinical isolate PO 1021‐7

The lipopolysaccharides of Actinobacillus actinomycetemcomitans strain Y4 and a human clinical isolate PO 1021-7 were examined by SDS/PAGE, deoxycholate/PAGE and mass spectrometry. PAGE analysis revealed an electrophoretic pattern similar to the SR-type lipopolysaccharide (LPS) of Salmonella. Deoxycholate/PAGE indicated the LPS of A. actinomycetemcomitans to consist of short sugar chains. Chemical analysis revealed the presence of thiobarbituric-acid-positive material (3-deoxy-D-manno-octulosonic acid equivalents) and four neutral sugars: glucose, galactose, D-glycero-D-manno-heptose and L-glycero-D-manno-heptose. Phosphate, glucosamine, glycine, and the fatty acids, 3-hydroxymyristic acid, myristic acid and palmitic acid, comprised the remainder of the molecule. The structure of the free lipid A revealed it to consist of a 1,6-glucosamine disaccharide esterified at C4' by a phosphomonoester. The hydroxyl group at C3 and the amide group of the non-reducing glucosamine were both acylated by 3-myristoylmyristic acid; analogous sites on the reducing glucosamine were acylated by 3-hydroxymyristic acid. Hydroxyl groups at C4 and C6' in the free lipid A were unsubstituted, with C6 being the proposed attachment site of the polysaccharide moiety. Chemical analysis revealed the presence of glycine in the intact LPS; its exact location in the A. actinomycetemcomitans LPS is still to be determined. Both intact LPS and free lipid A were highly lethal to galactosamine-sensitized mice, comparable to that of Salmonella.

Role of interleukin‐1 and prostaglandin in in vitro bone resorption induced by Actinobacillus actinomycetemcomitans lipopolysaccharide

Lipopolysaccharide (Y4 LPS) isolated from Actinobacillus actinomycetemcomitans strain Y4 induced bone resorption in BALB/c mouse calvaria organ culture. The calcium release from LPS-low responsive C3H/HeJ mouse calvaria by Y4 LPS was very low. Indomethacin almost completely inhibited prostaglandin E2 (PGE2) production by Y4 LPS-stimulated BALB/c mouse calvaria, but did not suppress interleukin-1 (IL-1) release from the calvaria, and partially suppressed the bone resorption. Dexamethasone strongly inhibited the PGE2 and IL-1 production by Y4 LPS-stimulated BALB/c mouse calvaria, as well as Y4 LPS-induced bone resorption. Dexamethasone inhibited expression of membrane IL-1 on osteoblastic cells stimulated with Y4 LPS, but indomethacin did not. Furthermore, anti-IL-1 serum partially suppressed the calcium release from Y4 LPS-stimulated BALB/c mouse calvaria. These results suggest that both PGE2 and IL-1 participate in Y4 LPS-induced bone resorption in vitro.

Interaction of gram‐negative periodontal pathogens with retinoic acid‐induced and dimethyl sulfoxide‐induced HL‐60 cells

As a first step toward elucidating the reasons for differences among periodontal pathogens in their cytotoxic effects on HL-60 cells, we used transmission electron microscopy to examine morphological aspects of granulocyte-bacteria interactions. Unopsonized Actinobacillus actinomycetemcomitans strain Y4 and Bacteroides gingivalis ATCC 33277 adhered to, and were phagocytosed by, retinoic acid-induced and dimethyl sulfoxide-induced HL-60 cells. In contrast, there was only minimal interaction between Wolinella recta ATCC 33238 and these induced granulocyte-like cells. Only isolated examples of adherence of W. recta to HL-60 cells were seen. In specimens prepared for routine transmission electron microscopy, ingested W. recta were not observed. In immunogold experiments, phagocytosed W. recta were noted, but only rarely. Opsonization of A. actinomycetemcomitans, B. gingivalis and W. recta with specific antisera appeared to increase their level of interaction with the HL-60 cells. We suggest that the HL-60 cell line may be useful in elucidating structure-function relationships between human neutrophil-like cells and putative periodontopathogens.