Serum antibodies to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in juvenile periodontitis and adult periodontitis (part I)

Recent microbiological studies support the concept that specific gram negative bacteria play a major role in the etiology and pathogenesis of human chronic inflammatory periodontal disease. Actinobacillus actinomycetemcomitans has been isolated frequently from juvenile periodontitis and Porphyromonas gingivalis has been shown to be a prominent species in adult periodontitis in humans. The purpose of this study was to determine levels of the specific antibodies to A.actinomycetemcomitans and P.gingivalis in 17 patients with juvenile and 15 patients with adult periodontitis and 24 healthy subjects. IgG and IgM antibody titers against these antigens were determined by enzyme-linked immunosorbent assay (ELISA). The levels of antibodies against A.actinomycetemcomitans were significantly higher in the juvenile periodontitis compared to the adult periodontitis patients and controls. Anti-P.gingivalis antibodies were elevated in adult periodontitis compared to juvenile periodontitis patients and controls.     

The effects of periodontal therapy on serum antibody levels to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis (part II)

Levels of IgG and IgM antibodies were estimated against Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis were determined by enzyme linked immunosorbent assay (ELISA) in 17 patients with juvenile periodontitis, 15 with adult periodontitis and 24 healthy controls at the beginning of treatment and 3 to 8 months after periodontal therapy. After treatment, antibodies to A. actinomycetemcomitans and P.gingivalis had decreased in patients, but the levels were still significantly higher than in healthy controls. Whether or not an of antibody level against a specific bacteria changes after periodontal treatment is however, still debatable.     

Effects of combined topical metronidazole and mechanical treatment on the subgingival flora in deep periodontal pockets in cuspids and bicuspids

The Effect on the subgingival microflora of a single topical administration of a 95% collagen and 5% metronidazole device in combination with debridement was investigated in 30 adult periodontitis patients in comparison with mechanical treatment alone. For each patient, plaque samples from test and control sites in cuspids and bicuspids were collected for culture and enumeration of total anaerobically cultivable bacteria (TA), black-pigmented anaerobes (BPA), and Actinobacillus actinomycetemcomitans (Aa). Spirochetes and fusiforms were quantified by direct microscopic examination after Giemsa staining. A decrease was observed for all parameters, and a significant difference in comparison with the control group was found for fusiforms. After treatment, a lower number of Aa positive sites were observed in the test group (13/25). These results show that a single application of topical metronidazole seems to be effective as adjunctive antimicrobial treatment in adult periodontitis.     

Opsonization of Actinobacillus actinomycetemcomitans by immunoglobulin G antibodies to the O polysaccharide of lipopolysaccharide

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.     

A clinical evaluation of non-steroidal anti-inflammatory drugs (NSAIDS) as adjuncts in the management of periodontal disease

Several effectors and mediators of inflammation have been identified as principle factors in periodontal disease progression. It has been reported that topical and systemic application of flurbiprofen (non-steroidal anti-inflammatory drug) reduced the rate of alveolar bone loss as well as signs of gingival inflammation. Twenty rapidly progressive as well as juvenile periodontitis patients were included in the present study. Results of the present study showed a significant reduction in pocket depth & gingival index in the experimental group over the control group. Radiographs revealed more bone fill in the experimental group over the control group.     

Molecular characterization of an outer membrane protein of Actinobacillus actinomycetemcomitans belonging to the OmpA family

The major outer membrane protein (OMP) of Actinobacillus actinomycetemcomitans is an OmpA homolog that demonstrates electrophoretic heat modifiability. The gene encoding this protein was isolated from a genomic library of A. actinomycetemcomitans NCTC 9710 by immunoscreening with serum from a patient with localized juvenile periodontitis. Expression of the cloned gene in Escherichia coli and subsequent Western blot analysis revealed a protein with an approximate molecular mass of 34 kDa. The amino acid sequence predicted from the cloned gene demonstrated that the mature protein had a molecular mass of 34,911 Da and significant identity to members of the OmpA family of proteins. We have named the major OMP of A. actinomycetemcomitans Omp34, and its corresponding gene has been named omp34.     

A 2-year study of adjunctive minocycline-HCl in Actinobacillus actinomycetemcomitans-associated periodontitis

To study the effects of a step-wise treatment regimen on Actinobacillus actinomycetemcomitans-(Aa) associated periodontitis, 4 clusters among 33 patients harboring the organism were followed during successive periods of systemic minocycline plus mechanical debridement and minocycline plus modified Widman flap treatment. Localized periodontitis was found in 2 clusters, one with 7 localized juvenile periodontitis patients and a 24-year old male with localized destruction and extremely low plaque levels (LJP), and the other consisting of 10 patients with plaque and gingivitis and a wider age range (16 to 54 years, LP). Generalized severe and moderate periodontitis was found in 2 clusters which were further discriminated by severe gingivitis and high levels of supragingival plaque (9 patients, GSP), and mild inflammation and low plaque levels (6 patients, GMP). Mean percentages of Aa, as determined by selective cultivation of microbiota from at least 2 periodontal pockets of 6 mm or more were 63, 16, 33, and 7.8% in the clusters (P < 0.01). Six months after active treatment, Aa was present in 6/9 patients and 50% of sites in GSP, and 3/6 patients and 46% of sites in GMP patients. In contrast, the organism was virtually eliminated by scaling and flap procedures in the localized periodontitis clusters, and did not reappear after 6 months (P < 0.05). Combined antibiotic, mechanical, and surgical therapy resulted in a persistence of 20% of sites with residual probing depth of > or = 4 mm in GMP patients after active therapy. At this point, 3 of the GMP patients and 1 GSP patient left the study. Multiple regression analysis showed a significant influence of log-transformed numbers in Aa in cheek and saliva samples at the end of the study, and cluster on the percent residual number of sites with periodontal probing depth of > or = 7 mm (P < 0.001). The present results suggest that the applied therapy would be appropriate in localized forms of Aa periodontitis, but inappropriate in more severe and generalized forms to predictably eliminate Aa. Controlled long-term studies with larger groups of patients will be needed to establish the difference in treatment response suggested by these studies.     

Epidemiology and transmission of Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans among children and their family members. A report of 4 surveys

The distribution and transmission of Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans in 4 families were studied. The families were included, based on the isolation of P. gingivalis from a young child or adolescent. The probands of these 4 families were: a 5-year old periodontally healthy boy; a 17-year old girl with severe generalized juvenile periodontitis; an 11-year old girl with prepubertal periodontitis; 2 sisters, 5 and 17-years old, with untreated severe periodontitis as a component of the Papillon-Lefèvre syndrome. All members of the 4 families were examined clinically and microbiologically for the presence of P. gingivalis and A. actinomycetemcomitans. Most of the parents appeared to be adult periodontitis patients; the parents of one proband were edentulous. Results showed that in all cases at least one of the parents was positive for P. gingivalis. On the basis of indistinguishable restriction endonuclease patterns (REPs) of P. gingivalis and A. actinomycetemcomitans isolates from parents and their children, and distinct REPs from unrelated individuals, the present study indicates that P. gingivalis and A. actinomycetemcomitans were transmitted between parents and their children.     

Attachment loss and serum antibody levels against autologous and reference strains of Actinobacillus actinomycetemcomitans in untreated localized juvenile periodontitis patients

Immunological data have been suggested to be a potential tool in the diagnosis, classification and monitoring of periodontal diseases. However, the role of circulating antibodies in periodontal patients is poorly understood. Patients suffering from localized juvenile periodontitis (LJP) are often reported to show high titers of serum IgG antibodies against Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans), but several affected patients do not. Most studies use well-known reference strains of the bacterium for testing against the patients' sera. The aim of the present investigation was to study the relationship between serum IgG antibody levels to autologous A. actinomycetemcomitans strains and clinical attachment loss (CAL). In addition, we wanted to assess the patients' serum titers against 4 well-known reference strains of the bacterium as well as their general potential immunoglobulin response. Intravenous blood samples were taken from 23 LJP patients and 10 healthy individuals, and autologous A. actinomycetemcomitans strains were cultured from 18 of the LJP patients. CAL was measured at 4 different sites around all present teeth and assessed as a % of teeth with at least 1 site moderately > or = 2 < 5 mm) or severely (> or = 5 mm) involved. An enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the serum titers of IgG antibodies to A. actinomycetemcomitans antigens. No significant correlation was found between serum IgG antibody titers to autologous strains and CAL. However, there was a trend that low responders had more moderately affected teeth than had high responders and patients with undetectable A. actinomycetemcomitans levels, which is in agreement with a hypothetically protective role of the antibodies. The total counts of immunoglobulin assessed in all participants showed that the predominant class was IgG and the reference group displayed significantly less (p < 0.05) IgG and IgG1 counts than the LJP patients. Both the reaction pattern against reference and autologous strains varied widely. We conclude that the specific antibody response against A. actinomycetemcomitans shows a weak correlation to clinical attachment levels in LJP patients.     

Localized prepubertal periodontitis--nonsurgical treatment of an adolescent patient: a case report

The treatment of localized juvenile periodontitis has been previously described in the literature, utilizing primarily a long-term (2 to 6 week) antibiotic regimen, notably tetracycline. This case report of juvenile periodontitis with extensive bone loss describes a short-term treatment (8 days), using a combination of two antibiotics and mechanical debridement. Clinical treatment included instruction of proper oral hygiene techniques. Initial scaling and root planing were performed to remove supragingival and subgingival accretions, followed by 2-month maintenance recalls. Pre- and postoperative radiographs, taken one year after the treatment, are used to document the evidence of natural bone regeneration. The learning objective of this article is to present an effective method of treatment-a debridement/antibiotic combination, followed by bone regeneration.     

LPS from Actinobacillus actinomycetemcomitans and production of nitric oxide in murine macrophages J774

Nitric oxide (NO) plays a complex role in the modulation of the inflammatory response, having either a pro-inflammatory or a protective role. Actinobacillus actinomycetemcomitans is considered an important etiological agent in localized juvenile periodontitis. We have studied the effect of lipopolysaccharide (LPS) extracted from this periodontopathogenic bacterium on NO synthesis in an in vitro murine macrophage system. LPS from A. actinomycetemcomitans induced a significant production of NO even at concentrations as low as 1 ng/ml, whereas LPS from E. coli had to be added in concentrations of 100 ng/ml to obtain similar effects. Production of NO was blocked by NG-nitro-L-arginine methylester, and pre-treatment of LPS from A. actinomycetemcomitans with polymyxin B abolished the production of NO, while prostaglandin E2 enhanced the synthesis of NO.     

Distribution of a newly described species, Kingella oralis, in the human oral cavity

The oral distribution of Kingella oralis was investigated in 10 periodontally healthy subjects. 11 untreated adult periodontitis patients and 6 untreated localized juvenile periodontitis patients. From each subject, 6-8 each of supra- and subgingival tooth samples, 4 mucosa samples and a saliva sample were examined by culture for the presence of K. oralis. K. oralis was found in at least one oral site in 26 of the 27 study subjects, and in at least one tooth site in each of these 26 positive subjects. Its prevalence in dental plaque ranged from 23% to 59% in different subject groups. The mean percentage of K. oralis in total microbiota in the dental plaque ranged from 0.40% in the periodontally healthy group to 4.60% in localized juvenile periodontitis subjects. The organism was a significant species in a few periodontitis sites, constituting > 5% of the total microbiota.     

28 K in squamous metaplasia of the bladder in patients with spinal cord injury

Actinobacillus actinomycetemcomitans has been strongly implicated in the etiology of localized juvenile periodontitis. Techniques used in the identification of this periodontal pathogen include cultural, biochemical, immunological and DNA hybridization analysis. In this study, we report the use of polymerase chain reaction (PCR) to amplify unique sequences of A. actinomycetemcomitans. Specific oligonucleotide primers LKT2 and LKT3 were designed to hybridize to the A. actinomycetemcomitans lktA gene, which encodes leukotoxin, a putative A. actinomycetemcomitans virulence factor. The LKT2 and LKT3 primers amplified lktA-specific sequences from all 12 A. actinomycetemcomitans strains tested. In another set of experiments, 13 other bacterial species, most of which are normal residents of the oral cavity, were tested with these primers. These PCR amplifications also contained 2 additional primers, RRN4 and RRN5, which served as positive controls; RRN4 and RRN5 were designed to amplify specific sequences of eubacterial 16S ribosomal DNA (rDNA). PCR amplifications of all bacterial species tested, including A. actinomycetemcomitans, yielded 16S rDNA-specific DNA fragments. Furthermore, each bacterial species tested, with the exception of A. actinomycetemcomitans, failed to amplify lktA sequences. The LKT and RRN primers were used in further PCR experiments to detect A. actinomycetemcomitans directly from gingival fluid samples. The results clearly demonstrate the simplicity, rapidity, specificity and accuracy of the LKT primers in the identification of A. actinomycetemcomitans.     

Differential features of transglottic carcinoma

Actinobacillus actinomycetemcomitans is considered to be an aetiological agent in various forms of periodontitis, with serotype b-specific carbohydrate being the immunodominant antigen of A. actinomycetemcomitans Y4 in high-responder patients. Lipopolysaccharide (LPS) of the organism may also be an important antigen. The purpose of the present study was to clarify the importance of LPS as an antigen of A. actinomycetemcomitans. Twenty patients who had high antibody titres to strain Y4 were selected, and the reactivity of their sera with LPS was determined by ELISA and Western blotting. Two groups of patients were observed: group 1 had high IgG titres only to serotype b strain, whereas group 2 had high IgG titres to serotypes a, b and c strains. The results of adsorption tests showed that anti-A. actinomycetemcomitans Y4 antibody in group 1 patients mostly consisted of antibody reactive with the serotype b-specific carbohydrate, whereas the antibody in group 2 patients mostly consisted of antibody reactive with the LPS of all serotypes. These data show that anti-LPS antibody is present and predominant in anti-A. actinomycetemcomitans Y4 antibody from some high-responder patients, and indicate an important role for LPS as an antigen in the humoral immune response to the organism.     

Diagnostic utility of specific microbiological markers for periodontal diseases

Specific detection of marker organisms Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans with an immunoassay provided 2 types of useful information directly into private clinical practice: 1) persistence of P. gingivalis in patients undergoing regular treatment allowed rapid identification of pockets requiring further treatment without waiting for measurable progression of lesions and 2) presence of A. actinomycetemcomitans in adults at any stage of diagnosis or treatment identified patients who may prove to have difficult-to-manage periodontitis. We made these findings in 253 patients (234 in specialist periodontal practices [F-ME 55; MHM 179] and 19 in general dental practice [EWM]). The search for useful diagnostic markers overlaps only partly with the search for periodontal pathogens. The P. gingivalis marker and the A. actinomycetemcomitans marker identify 2 different patterns of infection that appear to reflect 2 different underlying problems. Demonstration of pocket-dependent infection with P. gingivalis in treated patients provides an outcome marker for sites not converting to marker-negative sites at detection levels of the immunoassay. This information facilitates selection of sites and patients requiring adjustment of treatment regimens. Detection of A. actinomycetemcomitans in adult patients is significantly associated with periodontitis characterized as refractory. Positive identification of A. actinomycetemcomitans with the immunoassay supports clinical decision-making by drawing attention to adult patients who require closer monitoring and intensive persistent treatment. Successful application of immunoassay detection of microbiological markers is based on continuous patient monitoring to support clinical decisions; it does not replace careful clinical judgment.     

Titer and subclass distribution of serum IgG antibody reactive with Actinobacillus actinomycetemcomitans in localized juvenile periodontitis

Most patients with localized juvenile periodontitis (LJP) manifest serum IgG antibodies specifically reactive with antigens of Actinobacillus actinomycetemcomitans serotype b (Aa-b). Whether these antibodies are protective, destructive, or irrelevant to the progress of the disease remains unclear. We report results of studies aimed at assessing the subclass IgG responses in 35 LJP patients and 35 periodontally normal control subjects using well-characterized monoclonal antibody subclass reagents in an enzyme-linked immunosorbent assay. Our data show that the mean value for total IgG reactive with antigens of Aa-b was more than sevenfold higher for patients than for normal control sera (2349.6 micrograms/ml for patients vs 332.2 micrograms/ml for controls). Individual patients and control subjects were classified as high- or low-titer, using twice the median value for total anti-Aa-b IgG in control sera as the cutoff. Of 35 patients, 26 (74%) were high-titer, and 9 (26%) were low-titer. This compares to 5 normal control subjects (14%) high-titer and 30 (86%) low-titer. IgG2 accounted for the major quantitative response in both patients and control subjects. Indeed, the mean IgG2 values for both concentration and percentage of total specific IgG were greater than the combined values for specific anti-Aa-b IgG1, IgG3, and IgG4. Of the 26 high-titer sera, IgG2 predominated in 24, with IgG1 and IgG3 predominating in 1 each; IgG2 predominated in only 2 of the low-titer sera.     

Serum antibody opsonic activity against Actinobacillus actinomycetemcomitans in human periodontal diseases

Actinobacillus actinomycetemcomitans is frequently associated with severe periodontitis. Many periodontitis patients have elevated levels of serum IgG antibodies to A. actinomycetemcomitans, but the role of these antibodies is unknown. This study evaluated the functional capacity of anti-A. actinomycetemcomitans IgG antibody to enhance phagocytosis of A. actinomycetemcomitans by polymorphonuclear leukocytes. Chemoluminescence assays were done using sera from 64 subjects, 61 of whom had severe periodontitis; results were compared with the subject's anti-A. actinomycetemcomitans IgG titer and avidity. There was a strong correlation between chemoluminescence and antibody log titer (P < .00001) and a weak correlation between chemoluminescence and antibody avidity (P < .05). The results support the hypothesis that anti-A. actinomycetemcomitans IgG antibodies are important in promoting phagocytosis and killing of A. actinomycetemcomitans. Subjects who develop high levels of highly avid antibodies against A. actinomycetemcomitans may have greater resistance to continued or repeated infection by this pathogen.     

Follow-up of two cases of Papillon-Lefèvre syndrome and presentation of two new cases

Periodontitis resulting from Papillon-Lefèvre Syndrome has been known to cause early loss of primary dentition with subsequent involvement of the permanent dentition. In this study, two Papillon-Lefèvre Syndrome patients were followed for 3 years after initial treatment and improvement of their periodontal condition. In addition, two new cases of Papillon-Lefèvre Syndrome are presented. The follow-up treatment of the first two patients included monitoring the oral hygiene and performing ultrasonic scaling. Their present clinical appearance is very satisfactory. The periodontal condition of the third (new) patient was brought under control by extracting the involved deciduous teeth under antibiotic coverage, and by scaling and root planing the already erupted permanent teeth as well as by maintaining a high standard of oral hygiene. In the fourth case, all permanent teeth had erupted and the periodontium had already been severely destroyed. Actinobacillus actinomycetemcomitans was not detected by microbiologic examination after the periodontal conditions improved, except in the fourth case. Western blot analysis showed that the three first three patients had positive antibody response to the same antigens of Actinobacillus actinomycetemcomitans. Phagocytosis by polymorphonuclear neutrophil leukocytes) had not decreased, but the expression of surface receptors of polymorphonuclear neutrophil leukocytes was within the normal limits.