Actinobacillus actinomycetemcomitans is recognized as a primary pathogen in localized juvenile periodontitis (LJP). Restriction fragment length polymorphisms (RFLP) within a collection of subgingival plaque isolates of this bacterium were identified and characterized as the first step in understanding the pathogenesis of LJP. Over 800 isolates, from members of 18 families (LJP families) with at least one member with active LJP or a documented history of the disease and one or more siblings, less than 13 years of age, having no clinical evidence of LJP and 32 healthy control subjects, were assigned to one of 13 distinct RFLP groups (II to XIV) by using a previously characterized 4.7-kb DNA probe cloned from the reference strain FDC Y4. Isolates belonging to RFLP groups II, IV, V, and XIII predominated subgingival sites in the subjects. Members of RFLP groups II, IV, VII, VIII, X, and XI were recovered only from LJP family subjects, while group XIII and XIV variants were found exclusively in healthy controls. A synthetic oligonucleotide, homologous to the 5' end of the leukotoxin gene (lktA), and the A. actinomycetemcomitans plasmid, pVT745, were tested for their abilities to subdivide the 13 RFLP groups. The leukotoxin probe specifically identified all RFLP group II variants because of the absence of a HindIII site in the upstream noncoding region of the lkt gene complex. The plasmid probe was not as selective but may be useful for identifying clinical isolates belonging to RFLP group I. The use of these probes for the identification of genetic variants of A. actinomycetemcomitans that may be preferentially colonize diseased and healthy subjects will facilitate the study of the role of this important pathogen in periodontal diseases.
The periodontal pathogen Actinobacillus actinomycetemcomitans produces a leukotoxin that is considered a primary virulence factor in localized juvenile Periodontitis (LJP). Select strains of the bacterium contain a 530-bp deletion in the promoter region of the leukotoxin gene operon which results in enhanced transcription of the leukotoxin. DNA hybridization and polymerase chain reaction (PCR) were used to examine genetic variants of A. actinomycetemcomitans in 24 LJP-susceptible children from 21 families having a history of the disease and 34 control children from non-LJP families. A significant association was found between the detection of variants that had a deletion in the leukotoxin promoter region, indicative of a high level expression leukotoxin genotype, and conversion from a healthy periodontal status to disease. Subjects harboring A. actinomycetemcomitans of this genotype were more likely to convert to LJP than those subjects who had variants containing the full length leukotoxin promoter region (odds ratio = 22.50, 95% C.I.). These findings support the concept that highly virulent strains or clonal types of periodontal pathogens play a major role in the initiation of periodontal disease in susceptible hosts.
Periodontitis; juvenile/epidemiology; Actinobacillus actinomycetemcomitans; leukotoxin; epidemiology; polymerase chain reaction; polymorphism; restriction fragment length
The present study was performed to estimate the observed frequencies of the immunoglobulin heavy-chain (Gm) and light-chain (Km) allotypes among patients with early-onset periodontitis (EOP) and their effect on the IgG2 subclass responses against Actinobacillus actinomycetemcomitans Y4 and Porphyromonas gingivalis 381, respectively. Sixty-nine EOP patients, including 11 with localized juvenile periodontitis (LJP), 19 who had LJP, 15 with LJP-rapidly progressing periodontitis (RPP), and 24 with RPP, were examined for the Gm and Km allotypes by a hemagglutination inhibition test. Levels of immunoglobulin G2 (IgG2) antibodies against the two organisms were determined by enzyme-linked immunosorbent assay. Fifty race- and age-matched, periodontally healthy subjects were also included as a control group. The observed frequencies of the Gm haplotype afnb and Km(1) were significantly higher in the RPP and LJP groups, respectively. The G2m(n)+ group of those with RPP and the Km(1)+ group of those with LJP had significantly higher levels of IgG2 antibodies to A. actinomycetemcomitans and P. gingivalis, respectively. The results indicate that linkage disequilibrium of the G2m(n) locus in RPP patients or the Km(1) locus in LJP patients may be associated with high IgG2 antibody responses to the respective bacteria. It was reasoned that the IgG2 antibody responses are associated with the immunoglobulin allotypes. The function of IgG2 antibodies in their reaction to different bacterial antigens may be interpreted as either protective or nonprotective in the two different types of EOP (i.e., LJP and RPP).
The cell envelope of Actinobacillus actinomycetemcomitans includes a number of outer membrane proteins (OMPs) which appear to be important targets for immunoglobulin G (IgG) antibodies in sera from localized juvenile periodontitis (LJP) patients. In this study, we examined the subclass distribution of IgG antibodies reactive to the 16.6- and 29-kDa OMPs of A. actinomycetemcomitans in sera from LJP patients and periodontally healthy individuals. Antibody responses were determined in a quantitative enzyme-linked immunosorbent assay that employed human IgG subclass-restricted monoclonal antibodies. High-titer LJP sera (93% black; geometric mean titer, 32,673) were found to contain significantly elevated levels of IgG1, IgG2, and IgG3 antibodies to the 29-kDa OMP of A. actinomycetemcomitans, compared with those of low-titer LJP sera (mean titer, 1,421) and sera from periodontally healthy, race-matched control subjects. The concentration of IgG2 antibody to this protein was greater than or equal to the corresponding IgG1 concentration in 7 of 14 high-titer sera, although mean IgG1 and IgG2 concentrations were not significantly different. The concentrations of IgG1 and IgG2 antibodies to the 16.6-kDa protein were also significantly elevated in LJP sera, although of considerably lesser magnitude than that observed for the 29-kDa protein. The IgG2 response to the 29-kDa protein could not be attributed to the presence of IgG2 antibodies to lipopolysaccharide contaminants or to Fc-binding activity, nor does this molecule appear to be a glycoprotein. Hence, LJP subjects produce IgG2 antibodies, as well as IgG1 and IgG3 antibodies, directed to at least one of the major OMPs of A. actinomycetemcomitans.
Sera from patients with localized juvenile periodontitis (LJP) often contain markedly elevated immunoglobulin G (IgG) antibody titers to serospecific determinants of the lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans. The objective of the present study was to define the subclass distribution of the IgG antibody response of LJP patients to this key cell envelope antigen. IgG subclass antibody responses to A. actinomycetemcomitans LPS were quantified in an enzyme-linked immunosorbent assay with human IgG subclass-restricted monoclonal antibodies. Serum antibody concentrations were calculated by heterologous interpolation of a dose-response curve constructed by using human-mouse chimeric antibodies. Sixteen of 17 LJP serum samples tested contained significantly greater concentrations of IgG2 than IgG1 antibodies reactive toward A. actinomycetemcomitans LPS. Geometric mean antibody concentrations of IgG1 and IgG2 were 7.8 and 136.5 micrograms/ml, respectively, among LJP patients with elevated IgG titers to LPS (94% of whom were black). However, both IgG1 and IgG2 antibody concentrations were significantly greater than the corresponding values obtained from sera from LJP patients with low IgG titers to LPS. Among LJP patients with elevated IgG titers to A. actinomycetemcomitans LPS, serum IgG2 concentration and total IgG concentration were also significantly elevated compared with both low-titered LJP sera and sera from periodontally healthy race-matched controls. The results of this study indicate that the humoral response of a predominantly black population of LJP patients to A. actinomycetemcomitans includes the production of LPS-reactive IgG antibodies which are primarily of the IgG2 subclass.
Polymorphonuclear leukocyte (PMNL) chemotaxis studies of 32 patients with localized juvenile periodontitis (periodontosis or LJP), 10 adult patients with a history of LJP (post-LJP), 8 patients with generalized juvenile periodontitis (GJP), and 23 adults with moderate to severe periodontitis were performed: (i) to determine the prevalence of a PMNL chemotaxis defect in a large group of LJP patients; (ii) to study PMNL chemotaxis in patients with other forms of severe periodontal disease; and (iii) to determine if the PMNL chemotaxis defect seen in LJP patients is a cell-associated defect or is mediated by humoral factors. The effect of periodontal treatment on PMNL chemotaxis was studied in nine LJP patients. The chemotactic response was measured with the Boyden chamber procedure, and patient's peripheral PMNL were compared with those of control subjects, using endotoxin-activated serum, bacterial factor, N-formylmethionyl-leucylphenylalanine, and leukocyte-derived chemotactic factor as the standard chemoattractants. Based upon statistical analysis of chemotaxis assays, most carried out on at least two and often three or more separate occasions, 26 of 32 LJP patients, 7 of 10 post-LJP patients, and 5 of 8 GJP patients exhibited cellular defects of chemotaxis, whereas only 2 of 23 of the patients with adult periodontitis exhibited depressed chemotaxis. Elevated PMNL chemotaxis was occasionally found in subjects with juvenile periodontitis (2 of 32 with LJP and two of eight with GJP); however, it was found in a significant number (10 of 23) of patients with adult periodontitis. In eight of nine LJP patients, depressed PMNL chemotaxis was observed before and after periodontal therapy. The results indicate that the PMNL chemotaxis defect observed in juvenile periodontitis is due to a cell-associated defect of long duration. These studies suggest that the PMNL plays a major protective role against periodontal infection and that the cellular chemotactic defects and may predispose subjects to LJP.
Patients with localized juvenile periodontitis (LJP) often have high titers of antibody reactive with the serotype-specific immunodominant carbohydrate antigen of Actinobacillus actinomycetemcomitans serotype b. The vast majority of this A. actinomycetemcomitans serotype b-specific antibody is immunoglobulin G2 (IgG2). The present study was undertaken to determine whether the overall total levels of IgG2 in the sera of LJP patients are elevated. LJP patients and nonperiodontitis (NP) controls matched for age, race (black and white), and gender were studied. Additional controls included patients with adult periodontitis (AP) and patients similar in age to LJP patients but with the more-severe, generalized form of early-onset periodontitis (SP). Sera from over 700 periodontally characterized subjects were examined by using radial immunodiffusion to quantitate IgG2 as well as IgG1, -3, and -4, which were included for comparison. Serum IgG2 levels increased with age, and this was most dramatic around puberty. Black subjects in all periodontal groups had nearly 1 mg more IgG2 per ml than their white counterparts. Serum IgG2 levels were elevated (about 30 to 40%) in LJP patients of both races compared with their age- and race-matched NP controls (P < 0.01). In contrast, SP patients and AP patients had IgG2 levels comparable to their age- and race-matched NP controls. No other IgG subclass concentration correlated with periodontal diagnosis except for IgG3, which was elevated in white LJP patients. We reason that the high levels of serum IgG2 in LJP may be helpful in localizing periodontal destruction.
The phylogeny of 20 Actinobacillus actinomycetemcomitans strains isolated from patients with localized juvenile periodontitis (LJP) was investigated by using partial sequence analysis of 16S rRNA genes, arbitrarily primed PCR (AP-PCR), and four additional PCR assays that amplified polymorphic regions in the leukotoxin (lkt), cytolethal distending toxin (cdt), major fimbrial subunit (flp-1), and serotype-specific O polysaccharide gene clusters. Our analysis also included four strains isolated from healthy subjects and nine reference strains. We found that A. actinomycetemcomitans strains comprised three major phylogenetic lineages. One lineage consisted of serotype b strains, a second lineage consisted of serotype c strains, and a third lineage consisted of serotype a, d, e, and f strains. 16S rRNA sequences within each lineage were highly conserved (<1% base substitutions), whereas sequences between lineages were exceptionally divergent (1.9 to 5.0% substitutions). Two strains exhibited 16S rRNA sequences that were even more distantly related to those of the three major lineages (2.7 to 6.7% substitutions), indicating that additional minor lineages or variants exist. The distribution of 16S rRNA sequences and lkt, cdt, flp-1, and AP-PCR genotypes was consistent with a clonal population structure, with little evidence of assortative recombination between strains of different serotypes. Strains from all three major lineages were recovered from LJP patients, suggesting that phylogenetically diverse strains of A. actinomycetemcomitans carry pathogenic potential.
Localized juvenile periodontitis (LJP) runs in families, and a predisposition to develop disease appears to be inherited in an autosomal dominant fashion. Patients with LJP have elevated levels of serum immunoglobulin G2 (IgG2), and this is most striking in black LJP patients. We hypothesized that the markedly elevated serum IgG2 levels related to LJP status and race may be attributable to a fundamental difference in the response of black LJP leukocytes. To test this possibility, leukocytes from black LJP patients, black non-periodontitis (NP) controls, and white NP controls were cultured with a nonspecific mitogen (pokeweed mitogen) which stimulates immunoglobulin production. The levels of IgG2 produced were measured using an enzyme-linked immunosorbent assay. The results revealed that the serum IgG2 level differences among black LJP patients and white and black NP subjects were reproducible in peripheral blood leukocytes in vitro. Analysis revealed that B cells from the LJP patients appeared to be predisposed to produce high levels of IgG2. Further analysis supported the concept that the high IgG2 responses of B cells from black LJP patients were regulated by monocytes. Replacing the monocytes in cultures from white NP subjects with LJP monocytes from black patients resulted in production of IgG2 at levels that were comparable with those produced by the LJP B cells from black patients. In short, B cells from black LJP patients produce elevated levels of IgG2 in vitro, and at least part of this elevation appears to be attributable to regulation via the LJP monocytes.
Actinobacillus actinomycetemcomitans strains with enhanced levels of production of leukotoxin are characterized by a 530-bp deletion from the promoter region of the leukotoxin gene operon. Previous isolates with this deletion constituted a single clone belonging to serotype b, although they displayed minor differences among each other. We have analyzed the geographic dissemination of this clone by examining 326 A. actinomycetemcomitans isolates from healthy and periodontally diseased individuals as well as from patients with different types of extraoral infections originating from countries worldwide. A total of 38 isolates, all belonging to the same clone, showed the 530-bp deletion. Comparison of a 440-bp sequence from the promoter region of the leukotoxin gene operon from 10 of these strains revealed complete identity, which indicates that the deletion originates from a single mutational event. This particular clone was exclusively associated with localized juvenile periodontitis (LJP). In at least 12 of 28 families from which the clone was isolated, more than one family member had LJP. Notably, all the subjects carrying this clone had a genetic affiliation with the African population. These observations suggest that juvenile periodontitis in some adolescents with an African origin is associated with a disseminating clone of A. actinomycetemcomitans.
Localized juvenile periodontitis (LJP) is characterized by severe, early-onset, molar and incisor bone loss; neutrophil chemotaxis disorders; and a high prevalence of Actinobacillus actinomycetemcomitans infection. LJP is further characterized by significant familial aggregation of the disease. Recent work in our laboratory has demonstrated the selective depletion of a surface glycoprotein of 110,000 Mr (GP110) from LJP neutrophils by using surface labeling with [14C]formaldehyde and autofluorography. The function of GP110 is unknown; however, it does not appear to be a chemotactic factor receptor. Rather, it is bound by a monoclonal antibody (NCD-1) that recognizes a neutrophil differentiation antigen and which itself alters neutrophil chemotactic and secreting functions. To quantify GP110 on LJP and normal neutrophils, fluorescein-labeled NCD-1 was bound to neutrophils and the amount of fluorescence was evaluated by using cytofluorography. Our results indicate that there is a quantifiable reduction (40%) of GP110 on the surface of LJP and GJP neutrophils, compared with controls. Other patients with neutrophil defects express normal quantities of GP110, suggesting disease specificity. Our data suggest that GP110 may be a useful disease marker for LJP and may provide a useful probe for the study of neutrophil chemotactic function and dysfunction.
A high-molecular-weight polysaccharide-containing antigen was isolated from a phenol-water extract of Actinobacillus actinomycetemcomitans ATCC 43718 (formerly Y4) by gel permeation chromatography in lipopolysaccharide (LPS)-disaggregating buffer. The polysaccharide antigen formed a precipitin band with rabbit serotype b-specific antiserum but not with rabbit antisera to serotype a or c. Electroblotted serotype b antigen was probed with serum from a patient with localized juvenile periodontitis (LJP), resulting in a diffuse "smear" in the upper region of the lane. By utilizing an enzyme-linked immunosorbent assay, it was demonstrated that the geometric mean immunoglobulin G antibody titer to the serotype b polysaccharide was significantly higher in sera from LJP patients than in sera from periodontally healthy individuals. Moreover, LJP antibody titers to the serotype b polysaccharide exhibited age-dependent variation. Double immunodiffusion analysis revealed that the serotype b antigen formed a line of identity with low-molecular-weight LPS following reaction with serotype b-specific antiserum. Incubation of LJP serum in the presence of a lipid-free polysaccharide moiety obtained by mild acid hydrolysis of LPS from A. actinomycetemcomitans Y4 markedly reduced immunoglobulin G titer to the serotype b antigen. In contrast, solubilized lipid A was only weakly inhibitory. The results of this study indicate that the serotype b-specific determinant of A. actinomycetemcomitans resides in the polysaccharide moiety of LPS and represents a major target for immunoglobulin G antibody in serum of LJP subjects colonized by this organism.
Sera of localized juvenile periodontitis (LJP) patients colonized by Actinobacillus actinomycetemcomitans serotype b often contain markedly elevated levels of immunoglobulin G (IgG) antibodies to serospecific determinants in the O polysaccharide of lipopolysaccharide (LPS), as well as to outer membrane proteins of this species. IgG antibodies in LJP sera are known to opsonize A. actinomycetemcomitans for subsequent phagocytosis and killing by human neutrophils. The objective of this study was to determine whether outer membrane proteins or serospecific determinants in LPS are the primary target for opsonic IgG antibodies in LJP sera. An A. actinomycetemcomitans serotype b O-polysaccharide affinity column was constructed and subsequently used to purify LPS-specific IgG antibodies from LJP serum. The affinity-purified anti-LPS IgG antibodies were enriched in content of IgG2 (66.2%, compared with 37.0% in the total IgG fraction) and were immunospecific for A. actinomycetemcomitans serotype b LPS. In an opsonophagocytic assay using neutrophils from donors who were homozygous for the H131 allotype of Fcy receptor IIa (CD32), it was found that LPS-specific IgG antibodies exhibited substantially greater opsonic activity toward A. actinomycetemcomitans serotype b than an LJP IgG fraction that was depleted of LPS-reactive antibodies but contained antibodies against outer membrane proteins of this species. The results of this study indicate that serospecific determinants in the O polysaccharide of A. actinomycetemcomitans serotype b are a principal target for opsonic antibodies in sera of LJP subjects.
Sera from patients with localized juvenile periodontitis (LJP) often contain markedly elevated levels of immunoglobulin G2 (IgG2) antibodies reactive to cell envelope constituents of Actinobacillus actinomycetemcomitans. The objective of this study was to determine if these IgG2 antibodies are capable of supporting phagocytosis and killing of A. actinomycetemcomitans by human neutrophils. Polyclonal IgG2 antibodies were prepared from high-titer LJP serum by affinity chromatography, yielding a preparation which was > 99% subclass restricted and retained immunoreactivity to A. actinomycetemcomitans antigens. Affinity-purified IgG2 antibodies were evaluated by an in vitro opsonophagocytic assay that employed neutrophils obtained from donors who were homozygous for the H131 allotype of Fc gamma receptor type IIa (CD32), which efficiently binds human IgG2 antibodies. Affinity-purified IgG2 antibodies from LJP serum but not from sera of periodontally healthy individuals promoted phagocytosis and killing of A. actinomycetemcomitans. The expression of IgG2-dependent opsonic activity required the presence of complement. Incubation of A. actinomycetemcomitans with neutrophils in the presence of an optimal concentration of LJP IgG2 (50 micrograms/ml) and 5% hypogammaglobulinemic serum (as a complement source) resulted in a > 1 log10 reduction in bacterial viability within 30 min. The opsonic activity of IgG2 antibodies was found to be comparable to that observed with affinity-purified IgG1 antibodies. Moreover, IgG1 antibodies interacted synergistically with IgG2 antibodies in promoting opsonophagocytosis of A. actinomycetemcomitans. The results of this study indicate that LJP serum contains IgG2 antibodies which, when employed in conjunction with neutrophils that express Fc gamma receptors capable of recognizing this subclass, are opsonic for A. actinomycetemcomitans.
The oral bacterium Actinobacillus actinomycetemcomitans is implicated as a causative agent of localized juvenile periodontitis (LJP). A. actinomycetemcomitans is classified into five serotypes (a to e) corresponding to five structurally and antigenically distinct O polysaccharide (O-PS) components of their respective lipopolysaccharide molecules. Serotype b has been reported to be the dominant serotype isolated from LJP patients. We determined the lipopolysaccharide O-PS structure from A. actinomycetemcomitans CU1000, a strain isolated from a 13-year-old African-American female with LJP which had previously been classified as serotype b. The O-PS of strain CU1000 consisted of a trisaccharide repeating unit composed of l-rhamnose and 2-acetamido-2-deoxy-d-galactose (molar ratio, 2:1) with the structure →2)-α-l-Rhap-(1–3)-2-O-(β-d-GalpNAc)-α-l-Rhap-(1→. O-PS from strain CU1000 was structurally and antigenically distinct from the O-PS molecules of the five known A. actinomycetemcomitans serotypes. Strain CU1000 was mutagenized with transposon IS903φkan, and three mutants that were deficient in O-PS synthesis were isolated. All three transposon insertions mapped to a single 1-kb region on the chromosome. The DNA sequence of a 13.1-kb region surrounding these transposon insertions contained a cluster of 14 open reading frames that was homologous to gene clusters responsible for the synthesis of A. actinomycetemcomitans serotype b, c, and e O-PS antigens. The CU1000 gene cluster contained two genes that were not present in serotype-specific O-PS antigen clusters of the other five known A. actinomycetemcomitans serotypes. These data indicate that strain CU1000 should be assigned to a new A. actinomycetemcomitans serotype, designated serotype f. A PCR assay using serotype-specific PCR primers showed that 3 out of 20 LJP patients surveyed (15%) harbored A. actinomycetemcomitans strains carrying the serotype f gene cluster. The finding of an A. actinomycetemcomitans serotype showing serological cross-reactivity with anti-serotype b-specific antiserum suggests that a reevaluation of strains previously classified as serotype b may be warranted.
The gentle agitation of suspensions of Actinobacillus actinomycetemcomitans serotype a, b, or c in saline resulted in the release of a proteinaceous surface-associated material (SAM) which produced a dose-dependent inhibition of tritiated thymidine incorporation by the osteoblast-like cell line MG63 in culture. This cell line was sensitive to low concentrations of SAM (50% inhibitory concentration, 200 ng/ml for serotype c). Immunoglobulin G antibodies to constituents of the SAM were found in the blood of patients with localized juvenile periodontitis (LJP). Sera from 9 of 16 patients with LJP significantly neutralized the antiproliferative activity of the SAM, while sera from 15 controls, with no evidence of periodontal disease, were unable to neutralize this activity. Neutralization was not directly related to the patient's antibody titer to the whole SAM. Characterization of the antiproliferative activity in the SAM demonstrated that it was not cytotoxic and was heat and trypsin sensitive. The active component separated in a well-defined peak in anion-exchange high-performance liquid chromatography (HPLC) which, when further analyzed by size exclusion HPLC, revealed a single active peak, which had an apparent molecular mass of approximately 8 kDa. The lipopolysaccharide from A. actinomycetemcomitans was only weakly active. SAM from Porphyromonas gingivalis W50 and Eikenella corrodens NCTC 10596 did not exhibit any antiproliferative activity with this cell line, even at concentrations as high as 10 micrograms/ml. This study has shown that SAM from A. actinomycetemcomitans contains a potent antiproliferative protein whose activity can be neutralized by antibodies in the sera from some patients with LJP.
Several lines of evidence indicate that the monocytes of subjects with localized juvenile periodontitis (LJP) are functionally distinct from cells of age- and race-matched nonperiodontitis (NP) subjects. Among the abnormalities are the propensity to secrete large amounts of prostaglandin E2 and the induction of immunoglobulin G2 (IgG2) antibodies. The experiments described here were performed to further characterize the LJP monocytes and to determine if these cells mature differently than NP monocytes. When adherent monocytes from LJP subjects were cultured in the presence of human serum, both macrophages and cells with the morphology of immature monocyte-derived dendritic cells (MDDC) were observed. Within 4 days the prevalence of the immature MDDC was approximately twofold higher in LJP cultures than in NP cultures. In addition to their dendritic morphology, these cells were CD11c+ and CD14− or CD14low and stimulated potent autologous mixed leukocyte reactions, consistent with differentiation to the MDDC phenotype. Like LJP monocytes, cultures of MDDC generated with interleukin-4 and granulocyte-macrophage colony-stimulating factor selectively induced IgG2 in cultures of pokeweed mitogen-stimulated NP leukocytes. Together, these data suggest that the monocytes of LJP subjects have a propensity to differentiate into MDDC and that this differentiation may be related to the high levels of IgG2 that are observed in the sera of LJP subjects. As high levels of circulating IgG2 are correlated with less severe disease, the propensity of LJP monocytes to differentiate into MDDC may have important implications for both the host response against oral pathogens and the progression of LJP.
Actinobacillus acrinomycetemcomirans isolates from periodontal pockets were examined for restriction fragment-length polymorphism using a characterized 4.7-kb DNA probe. A total of 6 patterns of RFLP was found in 133 isolates originating from 12 subjects. No relatedness was found between RFLP types and serotypes. Different periodontal sites within the same subject and different individuals within the same family sometimes showed only one type of A. actinomycetemcomitans RFLP. When members among the same family showed 2 RFLP types, children were always infected with the A. acfinomycefemcomitans strains found in at least one of the parents. These findings support the concept of familial spread of A. actinomycetemcomitans. A. actinomycetemcomitans RFLP type B, corresponding to reference strain JP2, seems to be particularly virulent, as indicated from the presence of RFLP type B in 3 subjects who converted from a healthy periodontal state to localized juvenile periodontitis. RFLP type B was not detected in any of the 21 A. acrinomycetemcomitans-infected patients with adult periodontitis. The RFLP method seems to be useful in determining the epidemiology and possibly the potential virulence of periodontal strains of A. actinomycetemcomitans.
juvenile periodontitis; epidemiology; virulence; restriction fragment-length polymorphism; DNA probes; Actinobaccillus actinomycetemcomitans
The promyelocytic HL-60 cell line was examined for susceptibility to leukotoxin from Actinobacillus actinomycetemcomitans. Strains of A. actinomycetemcomitans which caused lysis of human peripheral blood polymorphonuclear leukocytes also lysed HL-60 cells as determined by release of intracellular lactate dehydrogenase. The killing of HL-60 cells by A. actinomycetemcomitans was dose dependent and temperature dependent, reached maximal levels after 45 min of incubation, and was inhibited by rabbit antisera to A. actinomycetemcomitans. Of 100 oral isolates of A. actinomycetemcomitans from 55 subjects, 16% from 11 healthy subjects, 43% from 13 adult periodontitis patients, 75% from 4 insulin-dependent diabetics, 66% from 2 generalized juvenile periodontitis patients, and 55% from 25 localized juvenile periodontitis patients produced leukotoxin. The same subject could harbor both leukotoxin-producing and -nonproducing isolates. The significantly higher proportion of leukotoxin-producing isolates in the disease groups compared with the healthy group is consistent with the hypothesis that leukotoxin from A. actinomycetemcomitans is an important virulence factor in the pathogenesis of certain forms of periodontal disease.
Recent data indicate that smoking is an important risk factor for the development of periodontitis. Smoking is also known to reduce serum immunoglobulin G (IgG) levels. Interestingly, patients with the localized form of early-onset periodontitis (LJP) have elevated levels of serum IgG2, and those who smoke are not clinically different from nonsmoking LJ subjects. In contrast, patients with the generalized form of early-onset periodontitis (G-EOP) who smoke have more extensive destruction than their nonsmoking counterparts. Given the effects of smoking on EOP and the association of IgG2 with less severe disease, we hypothesized that smoking might reduce serum IgG2 and that this might be most apparent in G-EOP. We therefore examined the effects of smoking on serum IgG subclass concentrations in race-matched groups: LJP, G-EOP, and age-matched periodontally healthy controls (NPs). Smoking status was established from serum cotinine levels, and serum IgG subclass concentrations were determined by using radial immunodiffusion. The data indicated that the effects of smoking were remarkably selective with respect to both IgG subclass and race. Smoking did not appear to have any effect on the concentration of IgG1 or IgG3 in either black or white subjects. In contrast, smoking was associated with depressed serum IgG2 concentrations in both white NP and G-EOP subgroups. Serum IgG2 levels in black subjects did not appear to be depressed by smoking, with the single striking exception of the black G-EOP subgroup which also had depressed serum IgG4 levels. The results here confirm that smoking has effects on serum immunoglobulin levels, but the effects were both race and serum IgG subclass specific. Furthermore, the periodontal diagnosis of EOP subjects appeared to be important, as indicated by the fact that IgG2 and IgG4 levels were reduced in smoking black G-EOP subjects whereas the IgG2 and IgG4 levels in black LJP and NP subjects were not reduced by smoking.
Aggregatibacter actinomycetemcomitans is frequently associated with localized aggressive periodontitis (LAP); however, longitudinal cohort studies relating A. actinomycetemcomitans to initiation of LAP have not been reported. A periodontal assessment was performed on 1,075 primarily African-American and Hispanic schoolchildren, ages 11 to 17 years. Samples were taken from each child for A. actinomycetemcomitans. A cohort of 96 students was established that included a test group of 38 A. actinomycetemcomitans-positive students (36 periodontally healthy and 2 with periodontal pockets) and 58 healthy A. actinomycetemcomitans-negative controls. All clinical and microbiological procedures were repeated at 6-month intervals. Bitewing radiographs were taken annually for definitive diagnosis of LAP. At the initial examination, clinical probing attachment measurements indicated that 1.2% of students had LAP, while 13.7% carried A. actinomycetemcomitans, including 16.7% of African-American and 11% of Hispanic students (P = 0.001, chi-square test). A. actinomycetemcomitans serotypes a, b, and c were equally distributed among African-Americans; Hispanic students harbored predominantly serotype c (P = 0.05, chi-square test). In the longitudinal phase, survival analysis was performed to determine whether A. actinomycetemcomitans-positive as compared to A. actinomycetemcomitans-negative students remained healthy (“survived”) or progressed to disease with attachment loss of >2 mm or bone loss (failed to “survive”). Students without A. actinomycetemcomitans at baseline had a significantly greater chance to remain healthy (survive) compared to the A. actinomycetemcomitans-positive test group (P = 0.0001). Eight of 38 A. actinomycetemcomitans-positive and none of 58 A. actinomycetemcomitans-negative students showed bone loss (P = 0.01). A. actinomycetemcomitans serotype did not appear to influence survival. These findings suggest that detection of A. actinomycetemcomitans in periodontally healthy children can serve as a risk marker for initiation of LAP.
Actinobacillus actinomycetemcomitans is a facultative gram-negative bacterium which has been associated with severe oral and nonoral infections. This study examined its occurrence in the oral cavities of 10 normal juveniles, 11 normal adults, 10 juvenile periodontitis patients, and 12 adult periodontitis patients. Four deep periodontal pockets and two normal periodontal sites were sampled in the diseased patients, and six normal periodontal sites were sampled in the healthy individuals. In all subjects samples were obtained from the cheek, tongue, and saliva. Samples from a total of 172 normal periodontal sites, 83 deep periodontal pockets, 42 cheek mucosae, 42 tongue dorsa, and 42 salivas were examined. Isolation was performed by using a medium for selective isolation of A. actinomycetemcomitans (Trypticase soy agar [BBL Microbiology Systems] supplemented with 10% serum and 75 μg of bacitracin per ml). The carrier rates were 20% for normal juveniles, 36% for normal adults, 50% for adult periodontitis patients, and 90% for juvenile periodontitis patients. A. actinomycetemcomitans was on average recovered in about fivefold-higher numbers from infected deep periodontal pockets than from infected normal subgingival areas. Samples of periodontal pockets generally contained 100-fold-more cells of A. actinomycetemcomitans than did samples of the cheek, tongue, and saliva. A. actinomycetemcomitans is commonly isolated from patients with juvenile periodontitis, often isolated from patients with adult periodontitis, and occasionally isolated from normal juveniles and adults. Its primary oral ecological niche appears to be dental plaque and periodontal pockets.
Protein kinase C is a key molecule in neutrophil signal transduction after receptor stimulation by soluble bioactive molecules. It has been reported that neutrophils from most patients with localized juvenile periodontitis (LJP) do not have a normal response after stimulation with a chemotactic ligand such as N-formylmethionylleucylphenylalanine (FMLP). To further clarify the mechanism of this altered response and to confirm and expand earlier observations, the calcium-dependent protein kinase C activity of neutrophils from patients with LJP was evaluated. Peripheral blood neutrophils from 12 patients and 12 healthy subjects, age, sex, and race matched, were sonicated and subsequently subfractionated by ultracentrifugation into a soluble fraction (cytosol rich) and a particulate fraction (membrane rich). The calcium-dependent protein kinase C activity was evaluated in each fraction by phosphorylation of histone with radiolabeled ATP in the presence or in the absence of phorbol 12-myristate 13-acetate stimulation. Results revealed that the total calcium-dependent protein kinase C activity of neutrophils from patients with LJP and depressed chemotactic migration to FMLP (201.0 +/- 63.6 pmol/min/10(7) cells) was lower than that of neutrophils from healthy subjects (287.6 +/- 55.7 pmol/min/10(7) cells) (P < 0.01). The calcium-dependent protein kinase C activity in neutrophils from patients with LJP exhibited a positive correlation with chemotactic migration to FMLP (P < 0.05). The low activity of calcium-dependent protein kinase C in neutrophils from the patients reflected the low activity in the soluble fraction from the neutrophils. After stimulation with phorbol 12-myristate 13-acetate, the calcium-dependent protein kinase C activity was found to be lower from patients with LJP than from healthy subjects. These results suggest that lower calcium-dependent protein kinase C in neutrophils is a predisposing factor for LJP.
The epithelial cell invasiveness of Actinobacillus actinomycetemcomitans strains of different restriction fragment-length polymorphism (RFLP) groups associated with disease conversion and asymptomatic carrier status in localized juvenile periodontitis was examined. Twenty clinical isolates were studied for their ability to invade KB monolayers, using the quantitative gentamicin killing assay. Five isolates were found to be invasive; five were not invasive; and the other 10 did not invade better than an invasion negative control Haemophilus aphrophilus strain ATCC 19415. Using probe-specific DNA fingerprinting, 11 strains were assigned to RFLP group II (disease–associated); 4 to RFLP type XIII (carrier status-associated); and the others to groups III, IV, V and VII. Eight isolates, all RFLP group II, were leukotoxin producers as determined by PCR amplification of the lkt promoter region. No correlation was found between invasiveness and RFLP group. Leukotoxin production was more associated with noninvasive than invasive strains.
Actinobacillus actinomycetemcomitans; invasion; KB epithelial cell; restriction fragment-length polymorphism; leukotoxin
Actinobacillus actinomycetemcomitans from the human oral cavity was serologically characterized with rabbit antisera to the type strain NCTC 9710; a number of reference strains, including Y4, ATCC 29522, ATCC 29523, ATCC 29524, NCTC 9709; and our own isolates representative of each of 10 biotypes. Using immunoabsorbed antisera, we identified three distinct serotypes by immunodiffusion and indirect immunofluorescence. Serotype a was represented by ATCC 29523 and SUNYaB 75; serotype b was represented by ATCC 29522 and Y4; and serotype c was represented by NCTC 9710 and SUNYaB 67. Indirect immunofluorescence revealed no reaction between the three A. actinomycetemcomitans serotype-specific antisera and 62 strains representing 23 major oral bacterial species. Distinct from the serotype antigens were at least one A. actinomycetemcomitans species common antigen and an antigen shared with other Actinobacillus species, Haemophilus aphrophilus, and Haemophilus paraphrophilus. All serotype a A. actinomycetemcomitans strains failed to ferment xylose, whereas all serotype b organisms fermented xylose. Serotype c included xylose-positive as well as xylose-negative strains. A total of 301 isolates of A. actinomycetemcomitans from the oral cavity of 74 subjects were serologically categorized by indirect immunofluorescence with serotype-specific rabbit antisera. Each patient harbored only one serotype of A. actinomycetemcomitans. Fourteen healthy subjects, five diabetics, and seventeen adult periodontitis patients exhibited serotypes a and b in approximately equal frequency, whereas serotype c was found less frequently. In contrast, in 29 localized juvenile periodontitis patients, the incidence of serotype b was approximately two times higher than that of serotypes a or c, suggesting a particularly high periodontopathic potential of A. actinomycetemcomitans serotype b strains. In subjects infected with A. actinomycetemcomitans, serum antibodies were detected to the serotype antigens, indicating that these antigens may play a role in the pathogenesis of periodontal disease.