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The indigenous flora acts as a deterrent to the establishment of some pathogenic species. We propose that advances in oral health research will lead to control of oral infections by altering the indigenous microflora to create a specific pathogen-free human. Investigations of important endogenous and exogenous factors which affect the oral flora and the interactions among these parameters, in health and disease, will have to be undertaken for this goal to be achieved. Several approaches to produce a specific pathogen-free human include: (1) introduction of individual or collective moieties which inhibit detrimental interactions on a genetic and molecular level; (2) genetic modification of salivary flow and protein composition by use of transgenic techniques; (3) therapeutic replacement with altered bacterial strains; (4) alteration of host immune responses to produce specific isotype immunity at the most appropriate time in the ontogeny of the oral environment; (5) production of isotype and/or antigen-specific regulatory molecules at the most appropriate time in development ; (6) use of synthetic vaccines; (7) genetic alteration or replacement of cells with defective protective capabilities; and (8) use of anti-idiotype vaccines.

An in vitro assay is described using saliva-treated bovine enamel slabs for determining the potential of chemotherapeutic agents to adsorb to tooth surfaces and act against plaque-forming bacteria. Chlorhexidine was found to inhibit the formation of in vitro plaque by Actinomyces viscosus, A naeslundii, Streptococcus mutans and S sanguis. Actinobolin was found to have marked antibacterial properties but limited adsorptive qualities.

The present report reviews data implicating Actinobacillus actinomycetemcomitans in the etiology of human periodontal disease. Recent data are also presented relative to: (1) serological studies of this microorganism using monoclonal antibodies and the serodiagnosis of A. actinomycetemcomitans infections; (2) characterization of the serotype antigens; (3) studies of the serotype distribution of A. actinomycetemcomitans in extra-oral infections; and (4) examination of the correlation between A. actinomycetemcomitans colony morphology and fimbriae.

The lymphocyte blastogenic response in humans to oral bacterial antigens was investigated during resolution and recurrence of naturally occurring gingivitis. The following conclusions were drawn: 1. Actinomyces and Fusobacterium and the host responses they evoke, are associated with gingival inflammation. 2. The magnitude of the blastogenic response to these antigens is directly related to the severity of gingival inflammation. 3. Scaling may cause a transient rise in the blastogenic response. 4. Conservative therapy not only improves the clinical state of gingival health, but also interrupts a possible pathogenic mechanism of periodontal disease.

The effects of repeated subgingival irrigation on Actinobacillus actinomycetemcomitans was examined. 24 periodontal pockets harboring A. actinomycetemcomitans in 3 juvenile and 4 adult periodontitis patients were studied. The protocol included bi-weekly subgingival irrigation with hydrogen peroxide of the periodontal sites until the micro-organism was no longer detected by selective culture, or for 6 months. A. actinomycetemcomitans was gradually suppressed to below detection following the irrigation regime and could no longer be detected in 46% of the sites at completion of the irrigation protocol. The sites were microbiologically re-examined 5 months after cessation of the irrigation regime. A. actinomycetemcomitans re-occurred in only 2 of the sites from which it had originally been suppressed below detection. The results indicate: (1) that the irrigation regime tested has some potential to suppress A. actinomycetemcomitans in periodontal pockets; (2) that the effect of the irrigation protocol generally lasted for 5 months; (3) that the reduction rate of A. actinomycetemcomitans to below detectable levels seems related to the initial number of cultivable bacteria from the periodontal pocket.

The gene encoding the fimbrial subunit protein of Bacteroides gingivalis 381, fimbrilin, has been cloned and sequenced. The gene was present as a single copy on the bacterial chromosome, and the codon usage in the gene conformed closely to that expected for an abundant protein. The predicted size of the mature protein was 35,924 daltons, and the secretory form may have had a 10-amino-acid, hydrophilic leader sequence similar to the leader sequences of the MePhe fimbriae family. The protein sequence had no marked similarity to known fimbrial sequences, and no homologous sequences could be found in other black-pigmented Bacteroides species, suggesting that fimbrillin represents a class of fimbrial subunit protein of limited distribution.

The hemagglutinating factor (hemagglutinin) of Bacteroides gingivalis was prepared from the supernatant of a 5-day diffusate broth culture by ammonium sulfate precipitation and column chromatography with a hydrophobic column of Phenyl-Sepharose CL-4B, DEAE-Sephadex A-50, and Sephadex G-100 gel filtration. The hemagglutinating activity of the preparation was 53.3 times higher than that of ammonium sulfate precipitate. In electron microphotographs, hemagglutinin appears to have a vesicle or tubelike structure. The hemagglutinating activity of intact cells was completely destroyed by heating at 100 degrees C for 10 min, but the activity of extracted hemagglutinin was heat stable. The activity of hemagglutinin was inhibited by L-arginine and L-lysine and partially inhibited by phospholipase D, but it was not affected by proteolytic enzymes, neuraminidase, hyaluronidase, lipase, phospholipase A and C, or sugars. The B. gingivalis hemagglutinin appeared to be comprised mainly of a 40,000-molecular-weight material. The Fab fragment of immunoglobulin G prepared from rabbit antiserum to whole cells of B. gingivalis and monoclonal antibody against the hemagglutinin bound to the cell surface and inhibited the hemagglutinating activity of both the cells and the purified hemagglutinin.

Appropriate immunization with whole cell vaccines of S mutans appear to induce antibodies that inhibit implantation of S mutans on tooth surfaces and associated dental caries. To better understand the mechanisms by which vaccination prevents S mutans implantation and dental caries, and to prepare antigens whose effectiveness and safety can be tested in animal models of caries, we set out to purify and chemically characterize the CAG of S mutans. The CAG of S mutans strain 6715 was prepared by extracting cells with potasssium hydroxide at 100 C. After neutralization and extensive washing, the water-insoluble product was characterized by a battery of chemical analyses and found to be a relatively pure glucan. The CAG was weakly immunogenic in rabbits when administered in Freund's complete adjuvant. In monkeys (M irus) immunized via the parotid duct with an aqueous solution of CAG, a definite but weak serum IgG, IgM, IgA, and salivary IgA antibody response was observed. Absorption experiments showed that the CAG induced antibodies that cross reacted with Sephadex G-25 and others that reacted with unique determinants on CAG. Retention of native antigenic determinants through the purification procedures was verified by the observations that antiserums to CAG reacted with whole cells of S mutans and by the fact that antiserums to S mutans cells reacted with CAG.

The topical application of hydrogen peroxide (H2O2) and sodium bicarbonate (NaHC03), individually and in combination, has been used empirically in the treatment of periodontal diseases. In this study, we examined both minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of these disinfectants individually and in combination against selected facultative, Gram-negative oral bacteria in a microtiter dilution assay. The bacteria studied included Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Eikenella corrodens, and Capnocytophaga gingivalis. These bacteria exhibited MBC (one hr) values ranging from 75 μ mol/L to greater than 10 mmol/L and MIC from 2O2. The tested bacteria exhibited MIC values for NaHCO3 of from 23 to 182 mmol/L, and the MBC (one hr) exceeded 728 mmol/L for most of the strains examined. At sublethal (sub-MIC) concentrations, sodium bicarbonate antagonized the ability of H2O2 to inhibit bacterial growth in MIC assays, but sublethal concentrations of H2O 2 had no effect on the MIC values of NaHCO3. Lethal concentrations of H2O2 and NaHC03 exhibited synergistic antimicrobial activity in combination in one-hour bactericidal assays. Since the bactericidal properties of these antimicrobial agents are synergistic, we conclude that it may be rational to use them in combination to treat certain forms of periodontal disease. Also, lower and perhaps safer concentrations of H2O2 can be used in combination with NaHC03 when oxidative antimicrobial chemotherapy is indicated.

Actinobacillus actinomycetemcomitans is a Gram-negative oral microorganism, which has been implicated in the etiology of localized juvenile periodontitis and in severe medical infections such as bacterial endocarditis. This study evaluated the ability of periodontal probes to transmit A actinomycetemcomitans from juvenile periodontitis lesions to healthy gingival sulci in the same patient. Localized juvenile periodontitis patients exhibiting first molar and incisor alveolar bone loss and with large numbers of A actinomycetemcomitans in deep periodontal pockets were included in this study. A periodontal probe was inserted into periodontal pockets of 6 mm or greater depth. The probe was then placed into a healthy gingival sulcus of 3 mm or less, in the same subject. Fifty-five transfers by probing were made and A actinomycetemcomitans in both the donor and recipient sites was assessed by a selective culture technique. The results indicate that periodontal probes can become contaminated with A actinomycetemcomitans from juvenile periodontitis lesions during routine dental examinations and can transfer this microorganism from infected to previously uninfected sites. However, A actinomycetemcomitans inoculated into the healthy gingival sulci did not permanently colonize these sites since the organisms were eliminated within 3 weeks.

Bacterial specificity in human periodontal diseases suggests the possibility of diagnosing and treating periodontitis as specific infections and using microbiological diagnostic means to evaluate the efficacy of periodontal therapy. In a series of clinical trials, the usefulness of topical antimicrobial agents in combination with surgical and non-surgical conventional periodontal therapy were tested. The healing result was estimated by monitoring probing attachment levels (PAL). The usefulness of clinical and microbiological parameters to evaluate post-treatment healing result was tested. 9 of the patients exhibiting sites with recurrent periodontal disease were then evaluated for clinical and microbiological parameters to define accurate means to differentiate between active and inactive periodontal disease. The results showed that the frequency of periodontal lesions with significant loss of PAL after treatment was less in patients treated with antimicrobial agent. Specific microbiological parameters showed stronger correlation than clinical parameters with gain and/or loss of PAL post-treatment. Thus Actinobacillus actinomycetemcomitans and Bacteroides gingivalis occurred in periodontal lesions with progressing disease after treatment, but were rarely detected in samples from pockets of the same depths which did not exhibit further loss of PAL over a study period of 1 year. This study points to the usefulness of topical antimicrobial agent as an adjunct to mechanical subgingival debridement in the treatment of periodontitis in adults. The results also indicate the utility of diagnostic microbiology in the assessment of periodontal disease activity post-treatment.

Actinobacillus actinomycetemcomitans is a facultative gram-negative coccobacillus associated with periodontal disease and nonoral infections. This organism is resistant to serum bactericidal mechanisms but is nevertheless killed by human neutrophils under aerobic and anaerobic conditions. Most of the killing attributable to oxidative mechanisms is inhibited by sodium cyanide, which suggests that the myeloperoxidase-hydrogen peroxide-chloride (MPO-H2O2-Cl-) system may be a key factor in the oxidative killing process. In this report, we examine whether the isolated MPO-H2O2-Cl- system is bactericidal against A. actinomycetemcomitans. We found that three major chromatographic forms of MPO were capable of killing A. actinomycetemcomitans at sublethal concentrations of H2O2 and that both catalase-positive and catalase-negative strains of this organism were sensitive to killing by the MPO-H2O2-Cl- system. We conclude that the isolated MPO-H2O2-Cl- system is bactericidal for A. actinomycetemcomitans independent of other neutrophil granule constituents and may be an important component of the oxygen-dependent bactericidal activity of the neutrophil with respect to this periodontopathic organism.

We assayed chlorhexidine and a series of its analogues, in which the chlorophenyl terminal substituents were replaced with alkyl chains, for their in vitro antimicrobial activity against the Gram-negative and Gram-positive oral bacteria. Changes in antimicrobial activity were correlated with changes in agent structure for identification of structural criteria which may be important in the optimization of agent activity. Chlorhexidine showed substantial antimicrobial activity against the Gram-negative as well as the Gram-positive oral bacteria. The alkyl agents were comparable with chlorhexidine in their activity against Bacteroides gingivalis and Bacteroides intermedius, black-pigmented Gram-negative obligate anaerobes associated with periodontal disease in adults. Alkyl agents alexidine, heptihexidine (1,6-bis-n-heptylbiguanidohexane), hexoctidine (1,8-bis-n-hexylbiguanidooctane), and hexhexidine (1,6-bis-n -hexylbiguanidohexane), as well as chlorhexidine, were active against Actinobacillus actinomycetemcomitans, a Gram-negative organism associated with localized juvenile periodontitis. Hexidecidine (1,10-bis-n-hexylbiguanidodecane) and heptoctidine (1,8-bis-n-heptylbiguanidooctane) were more active, and hexhexidine was as active as chlorhexidine against Fusobacterium nucleatum, also associated with periodontal disease. Seven of the agents were more active than chlorhexidine against Actinomyces species. All test agents were active against Streptococcus mutans, a Gram- positive coccus associated with dental caries. Hexidecidine had activity equal to that of chlorhexidine when evaluated against the entire battery of organisms. Analysis of structure-activity relationships revealed that alkyl chains could replace chlorophenyl groups with retention or improvement of antimicrobial activity. Agents with lower lipophilicity were generally more active than those with higher lipophilicity. Within an isolipophilic series, activity increased as methylene bridge length increased, with a requirement for a bridge of at least six carbons for activity against most bacteria. Agents with branched terminal groups were more active than agents of the same bridge length but with unbranched terminal groups. The alkyl bisbiguanides, agents which do not contain chlorophenyl moieties, have activity against the oral flora comparable with that of chlorhexidine. Of the alkyl agents evaluated in this study, hexidecidine, hexhexidine, and alexidine offer the most promise for use in control of oral diseases.

Myeloperoxidase (MPO) is an enzyme found in the azurophil granules of neutrophils. Cation-exchange chromatography on carboxymethyl-cellulose previously has been used to demonstrate the heterogeneity of the peroxidase enzymes isolated from human neutrophils. In this study, fast protein liquid chromatography (FPLC) was used to separate and purify three major (I, II, and III) and three minor (IIa, IIIa, IIIb) forms of MPO from isolated neutrophil granules. Purity was confirmed by polyacrylamide gel electrophoresis in the presence of cetyltrimethylammonium bromide (CETAB-PAGE), by crossed immunoelectrophoresis, and by spectral characteristics. All three major forms were indistinguishable by immunodiffusion against rabbit antiserum, scanning spectrophotometry, and amino acid composition. They differed in their elution from a cation-exchange resin, inhibition by 3-amino-1,2,4-triazole, migration rate in CETAB-PAGE, and subunit molecular weight. Subunit molecular weight was examined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). All three major forms appeared to consist of heavy (H), intermediate (M), and light (L) peptides. The M peptide appeared to be derived from the H subunit. All L subunits exhibited a molecular weight of 14,500. The molecular weights for the H subunits varied, and were 60,000, 59,000, and 57,000 for MPO I, II, and III, respectively. The molecular weights for the M peptides were 44,100, 43,000, and 42,000 for MPO I, II, and III, respectively. The treatment of neutrophils, granules, and extracts with protease inhibitors and sodium azide did not block the appearance of three major forms of MPO. Thus, neither protease activity nor MPO autooxidation during extraction and purification procedures is responsible for the appearance of multiple chromatographic forms of MPO derived from human neutrophils.

Capnocytophaga is a newly described genus of gram-negative bacteria which can cause serious oral and extraoral infections in the susceptible host. In the present study, sensitivity of Capnocytophaga spp. to the bactericidal properties of human serum was investigated. Laboratory strains representative of Capnocytophaga sputigena, C. ochracea, and C. gingivalis and seven oral isolates of Capnocytophaga spp. obtained in primary culture were determined to be sensitive to killing by pooled normal serum. In contrast, little or no killing of Capnocytophaga spp. was observed when these organisms were incubated in the presence of hypogammaglobulinemic serum despite evidence for alternative pathway activation. However, hypogammaglobulinemic serum could be reconstituted to bactericidal activity by the addition of the immunoglobulin M fraction of normal serum. Capnocytophaga spp. failed to activate the classical pathway in hypogammaglobulinemic serum, thus ruling out an antibody-independent mechanism of killing. In contrast, good correlation was observed between serum-mediated killing and antibody-dependent classical pathway consumption. These results indicate that complement in the presence of bactericidal antibody may be an important determinant of host resistance to intra- and extraoral infections caused by Capnocytophaga spp.

Extracts of parotid glands taken from rhesus monkeys at various times after infection with mumps virus were assayed for the ability to inhibit the migration of guinea pig peritoneal macrophages. Inhibitory activity was found in extracts of glands removed 2 or 7 days after infection. Separation of a pool of these extracts by means of Sephadex G-100 chromatography yielded two active fractions, one containing substances of molecular weight between 45,000 and 65,000, and the second containing substances of approximately 12,000 molecular weight. The larger molecules occur in the same fraction in which migration inhibitory substances generated by virus-infected tissue cultures of nonlymphoid cells are found. Extracts of parotid glands from immune animals challenged with mumps virus were also analyzed for migration inhibitory activity. Positive extracts separated on Sephadex G-100 showed a band of activity in fractions covering molecular weights from 5000 to 30,000 corresponding to those obtained with human MIF produced by antigen-stimulated lymphocytes. Thus it appears that factors with MIF-like activity may be generated in vivo by both immunologic and nonimmunologic means.

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

Localized juvenile periodontitis (LJP) is characterized by severe, early onset alveolar bone loss, localized to the first molars and incisors and a high prevalence of infection with Actinobacillus actinomycetemcomitans and associated neutrophil functional abnormalities. Due to the frequent occurrence of this condition in families, it was the purpose of this investigation to determine the association of neutrophil chemotaxis abnormalities and clinical periodontal disease in families with LJP. Twenty-two families were studied in which the proband was selected based upon presentation of LJP. All siblings were examined for the presence of LJP and neutrophil chemotaxis was measured on all subjects. The results indicate that there is a high association of LJP and neutrophil chemotaxis disorders and that this association is consistent along family lines. Specially, in families in which the proband exhibits a neutrophil chemotaxis disorder, all affected siblings (LJP) also exhibit depressed neutrophil chemotaxis, whereas, non-affected siblings beyond the age of puberty have normal neutrophil chemotaxis. On the other hand, in families where the proband (LJP) exhibits normal chemotaxis, both normal and affected siblings exhibit normal chemotaxis. The finding among families with LJP that some exhibit neutrophil chemotactic depression and others exhibit normal neutrophils suggests heterogeneity of LJP. Accordingly, it is proposed that there is a syndrome of familial localized juvenile periodontitis with depressed neutrophil chemotaxis and another form of familial localized juvenile periodontitis with normal neutrophil chemotaxis. The chemotaxis disorder and LJP occur in nearly one-half of the siblings in families with LJP. which is consistent with a dominant trait. however, multigenerational studies are necessary to determine the mode of inheritance. Hence, neutrophil chemotaxis is a disease marker which can be used in genetic studies of familial LJP with depressed neutrophil chemotaxis. Furthermore, prepubertal siblings often exhibit defective neutrophil chemotaxis in the families in which the proband exhibits such a defect. Since the prepubertal siblings are not affected by clinically detectable LJP, the finding of neutrophil chemotactic depression in these children further suggests that the neutrophil chemotactic defect is genetic in origin, precedes and may predispose to localized juvenile periodontitis.

In the present study the function of the neutrophil, which exerts a major role in protection against bacterial infections, was assessed in patients with a family history of diabetes mellitus and severe periodontitis. The neutrophil chemotactic function of two groups of patients was compared, both exhibited severe periodontitis. One group had a family history of diabetes and is therefore classified as individuals with potential for abnormal glucose tolerance (Pot-AGT Group). The other group had comparable levels of severe periodontal disease but did not have a family history of diabetes. Neither group showed evidence of systemic disease as assessed by an extensive medical history and a battery of clinical laboratory tests including CBC, SMA-12, prothrombin time, urinalysis and glycosylated hemoglobin levels. The peripheral blood neutrophil chemotactic response of patients in both groups was measured using Boyden chambers charged with the standard chemotactic agents; endotoxin activated serum, E. coli cultu...

Data from 27 subjects with localized juvenile periodontitis were used to compute estimates of the intraclass correlation coefficient for initial periodontal pocket depth measurements and total subgingival Actinobacillus actinomycetemcomitans counts, and for changes from pre- to post-treatment in periodontal pocket depth, probing attachment level and Actinobacillus actinomycetemcomitans counts. The estimates for the clinical parameters were considerably higher than those found in a similar study of adult periodontitis. On the average, the estimates for the intraclass correlation coefficients for Actinobacillus actinomycetemcomitans counts did not differ markedly from the corresponding estimates for the clinical parameters. Although the results of this study indicate that the major component of variability in parameters commonly used in studies of periodontal disease can be attributed to site-specific factors, sites within subjects cannot be considered statistically independent. For the analysis of data arising from studies of periodontal disease, it is recommended that statistical techniques, like the nested mixed effects analysis of variance model be employed, which account for this dependence. These techniques will allow meaningful biological questions concerning site-specific phenomenon to be answered. However, changes in clinical and microbiological parameters, when therapeutic measures are employed on localized juvenile periodontitis patients, are often dramatic enough to allow informal data presentation.

Several cationic, mixed and amphoteric surfactants were tested for their antimicrobial activity and ability to inhibit the formation of in vitro plaque by oral microorganisms. All had antimicrobial activity against Actinomyces viscosus. Actinomyces naeslundii and Streptococcus mutans. Cationic surfactants were comparable to chlorhexidine in antimicrobial activity but were less effective in inhibiting plaque formation. Amphoteric surfactants were less effective than other detergents in antibacterial activity and had very limited capacity for the inhibition of plaque formation. Comparison of drug structure provides evidence that surfactant substantivity to saliva-coated enamel is a cation active process. Saliva was found to have an antagonistic effect on the activity of cetylpyridinium chloride but not on Triburon.

Supernatants obtained from sonication of human and macaque dental plaque contained components which, when added to homologous peripheral blood lymphocytes and simultaneously cultured in vitro with suboptimal amounts of mitogen, caused a synergistic effect upon the synthesis of DNA by the lymphocytes. The component in human plaque was heat stable, predominantly non-dialysable and present exclusively in the void volume fraction after Sephadex G-150 column chromatography. A proposed mechanism for the synergism is that among the many cells non-specifically stimulated to divide in culture by the mitogen are cells which were specifically sensitized to plaque antigens; the number of these cells therefore increases and the synergism observed occurs as these sensitized cells become further stimulated by plaque antigens.

The present results showed that maarked and long-lasting changes in the subgingival microflora associated with periodontal disease could be achieved by a single course of periodontal treatment. Immediately following therapy, the total number of subgingival organisms decreased 10- to 100-fold and the proportions of cultivable Gram negative organisms and anaerobic organisms generally decreased 3- to 4-fold or more. After treatment, most periodontal pockets were populated by a scant microflora predominated by facultative Actinomyces and Streptococcus species. The kinetics of the subgingival bacterial recolonization revealed that the total cell counts and the proportions of spirochetes and Capnocytophaga species did not reach their pretreatment levels even after 6 months. Other Gram negative anaerobic species returned to pretreatment proportions after 3 to 6 months. Several Gram positive species exhibited higher posttreatment than pretreatment proportions throughout the 6 months study. The microbiological shifts paralleled significant changes in the clinical status of the periodontal tissues. Following therapy, the periodontal pocket depths decreased generally 1 to 4 mm, the gingival inflammatory index, the gingival fluid flow, and the suppurative index were generally lower, and nine of 33 test pockets examined showed apposition of alveolar bone. The microbiological and clinical changes described were exhibited by two patients treated with periodontal scaling and root planing alone and by two patients treated with the adjunctive use of systemic tetracycline therapy. In two other patients, mechanical periodontal therapy only slightly reduced the total number of subgingival organisms and the proportions of spirochetes and other Gram negative anaerobic rods. A shift in the subgingival microbial composition was achieved in these two patients after tetracycline therapy. The following model for treatment of periodontal disease is proposed: (1) Conventional therapy including thorough periodontal scaling and root planing; (2) Monitoring the subgingival flora and the clinical course; and (3) Use of antimicrobial therapy in refractory cases. Further studies are needed to develop means for rapid identification of refractory patients, and to determine the optimal antimicrobial agent, the optimal route of administration, and the optimal dosage regime.

1. Gingival fluid from severely inflamed periodontal tissue represents a 15 to 30% dilution of serum with respect to the proteins studied, with the exception of C3 and in some subjects, C4. 2. A marked decrease in C3 levels is found in most, if not all, gingival fluids, and a marked decrease of C4 levels is found in some gingival fluids. This suggests that complement might be activated during periodontal inflammation. 3. The concentrations of proteins in the gingival fluid are not related to their molecular weight.

Since Actinobacillus actinomycetemcomitans appears to be a key etiologic agent in localized juvenile periodontitis, this study determined the effectiveness of different treatment modalities in suppressing A. actinomycetemcomitans in localized juvenile periodontitis lesions. A total of 25 deep periodontal lesions from 7 patients with localized juvenile periodontitis were included in the study. The test periodontal lesions either received scaling and root planing alone, scaling and root planing together with soft tissue curettage, or modified Widman flap surgery. Subgingival A. actinomycetemcomitans were enumerated using selective culturing. Clinical measurements included changes in probing periodontal attachment level, probing periodontal pocket depth, gingival index, plaque index, and digital subtraction of standardized serial radiographs. The microbiological and clinical effects of treatment were monitored over a period of 16 weeks. All periodontal lesions studied demonstrated high numbers of A. actinomycetemcomitans prior to treatment. Scaling and root planing alone did not markedly change the subgingival A. actinomycetemcomitans counts, nor any of the clinical parameters studied. In contrast, soft tissue curettage as well as modified Widman flap surgery suppressed A. actinomycetemcomitans to undetectable levels immediately after therapy in more than 80% of the lesions studied. A total of 5 periodontal lesions exhibited gain of probing periodontal attachment after subgingival curettage or Widman flap treatment; 3 of these sites revealed no detectable A. actinomycetemcomitans, and the remaining 2 sites harbored only low levels of A. actinomycetemcomitans. 5 periodontal lesions which lost probing attachment after treatment all demonstrated high numbers of subgingival A. actinomycetemcomitans. Changes in alveolar bone, assessed by digital subtraction of serial radiographs, correlated with changes in probing periodontal attachment level, confirming the clinical results. The present study revealed a close relationship between post-treatment A. actinomycetemcomitans levels and the clinical response to treatment, which supports the concept that A. actinomycetemcomitans is an important organism in the etiology of localized juvenile periodontitis. This study also showed that a substantial suppression of subgingival A. actinomycetemcomitans cannot be achieved by periodontal scaling and root planing alone, but can be accomplished by surgical removal of periodontal tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

Fluoretec-M is a polyvalent conjugate used in direct fluorescent-antibody staining for identification of the Bacteroides asaccharolyticus-Bacteroides melaninogenicus group. The Fluoretec-M reagent detected all oral and nonoral test strains of B. melaninogaenicus subsp. intermedius, all test strains of B. melaninogenicus subsp. melaninogenicus, and the nonoral strains of B. asaccharolyticus. However, the Fluoretec-M polyvalent reagent and the monovalent conjugates which constitute Fluoretec-M did not detect the oral strains B. asaccharolyticus. The use of Fluoretec-M can therefore generate false-negative results in studies of specimens from oral cavity and from nonoral sites in which an infection with B. asacacharolyticus of oral origin may have taken place. It is suggested that antibodies reactive with the oral antigenic type of B. asaccharolyticus be included in the preparative procedure of the Fluoretec-M reagent.

Various relationships including inhibition or stimulation of growth have been demonstrated among the bacteria present in dental plaque, both in vitro and in vivo. A large number of these relationships involved oral Streptococci. An earlier study found that strains of Streptococcus mitis inhibited the growth of potential periodontopathic microorganisms, such as Actinobacillus actinomycetemcomitans, Capnocytophaga and species of Bacteroides and Fusobacterium. The present investigation showed that this inhibitory effect resulted primarily from lactic acid production.

Polymorphonuclear leukocyte chemotaxis and chemokinesis, in the micropore filter assay, have been evaluated using Regression Line Analysis. The method discriminates between migration rate and the number of migrating cells by statistical comparison of the slope of the regression line and the γ -intercept, respectively. The derivation of this new method of assessment of cell migration is reported. This report also includes critical evaluation of the chemokinesis dose/ response curve and the directional component of chemotaxis. The effect of inhibitors of chemotaxis were evaluated using the new method. Colchicine was found to effect the directional component of chemotaxis, as indicated by decreased rate of locomotion in chemotaxis experiments, and had no effect on random migration and chemokinesis. Cytochalasin B had a general inhibitory effect upon cell locomotion by depressing the rate of random migration and chemotaxis. 1-Tosylamide-2-phenylethyl chloromethylketone (TPCK) had the apparent effect of uncoupling the receptor and effector mechanisms of chemotaxis, or altering the binding of the chemotactic ligand, since random migration was unaffected but the rate of chemotaxis and chemokinesis were depressed. Ionophore A23187 had a generalized inhibitory effect on the cell rate under all three conditions; however, at low concentrations it induced an increase in the number of migrating cells. These studies indicate that the method of regression line analysis is effective in differentiating in vitro the mechanisms of chemotaxis inhibition. The method may be used effectively in the assessment of defective neutrophil chemotaxis in disease states and may prove a useful adjunct to the study of cell locomotory mechanisms.

The lymphocyte blastogenic responses to antigen preparations of bacteria from dental microbial plaque and to other non-oral stimulants were longitudinally assayed in eitht adult subjects using the experimental gingivitis model. During the 28 days of the experimental period, these subjects abstained from all oral hygiene procedures which let to accumulatio of increased amounts of microbial dental plaque and subsequent gingivitis. In general, all subjects demonstrated a significant increase in the lymphoproliferative response to all doses of phytohemagglutinin (PHA) during the experimental period. The subjects were divided into 2 groups based on rate of plaque accumulation. Slow plaque formers exhibited a gradual linear increase in blastogenic response to bacterial antigen preparations throughout the experimental period. In the group characterixed by rapid accumulation of plaque, a marked early increase in blastogenic responses to antigens of Actinomyces viscosus, Actinomyces naeslundii, Bacteroides melaninogenicus, and Treponema denticola was observed. These same subjects, however, demonstrated a reduction in lymphocyte response to the above antigens during the later phase of the experimental period, when gingivitis was most severe. This decrease, in light of the similtaneous increase in PHA response, suggests the existence of a homeostatic mechanism to control the immune response during episodes of increased antigen load.

A sonic extract of Actinobarillus actinomycetemcomitans Y4, a microorganism originally isolated from a patient with juvenile periodontitis, contains a leukotoxin (Aa-leukotoxin) which specifically kills human polymorphonuclear leukocytes (PMNs) and monocytes. In the presence of normal human sera, the toxicity of the leukotoxin is enhanced, whereas sera from patients with juvenile periodontitis neutralize leukotoxic activity. In juvenile penodontitis serum immunoglobulin G was identified as the specific inhibitor of the Aaleukotoxin. The leukotoxin enhancing factor(s) in normal human serum appeared to be a large molecular weight protein which was not immunoglobulin. Pooled sera from rabbits immunized with A. actinomycetemcomitans Y4 sonic extract also neutralized Aaleukotoxic activity while normal rabbit serum enhanced toxicity. The presence of antileukotoxin antibody in sera from individuals with juvenile periodontilis suggests that these people have been immunized with A. actittotnyCetemeomitans microorganisms. Monitoring of this antibody may be a convenient tool in the study of the etiology of juvenile periodontitis.

Surface tensions of normal and pathological granulocytes were determined by (1) adhesion to solid substrates of different surface tensions while suspended in liquid media of different surface tensions, and by (2) measurement of cell-liquid-vapor contact angles obtained with sessile drops of saline water on cell monolayers. The results obtained by the two different methods were in close conformation with one another. With the cell adhesion method some residual leukocyte adhesion still persists even under conditions where there no longer is a van der Waals attraction between cells and solid substrate. At low ionic strength and by the abolishment of all multivalent cations through the admixture of EDTA, that residual cell adhesion virtually disappears (with normal as well as with pathological granulocytes), indicating that the earlier residual cell adhesion did indeed arise from electrostatic interactions mediated by multivalent cations (probably Ca2+). Comparison of the capacities for engulfment and the surface thermodynamics data of normal and pathological granulocytes obtained in this study leads to the novel observation that the phagocytic episode from half to complete engulfment of bacterial particles by granulocytes appears to be the crucial step from the thermodynamic point of view.

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.

Bacteroides gingivalis produces large amounts of proteolytic enzymes which may play a role in its virulence. These enzymes may participate in the tissue destruction of the inflammatory process. In this study, the characteristics of two such enzymes, N-CBz-glycyl-glycyl-arginyl peptidase (N-CBz-Gly-Gly-Arg peptidase) and glycyl-prolyl peptidase (Gly-Pro peptidase) were investigated. The enzymes eluted in different peaks from an anion exchange column. N-CBz-Gly-Gly-Arg peptidase was associated with cells up to 48 h in culture. If cultured longer, it also released in the supernatant. It exhibited optimal activity between pH of 7.0 and 7.5 and was readily inactivated by heat treatment (45°C for 15 min). The enzyme activity was inhibited by p-chloromercuribenzoic acid (PCMB), leupeptin and antipain, suggesting that it is a thiol protease. The B. gingivalis N-CBz-Gly-Gly-Arg peptidase was different from the serum enzyme that digests the same substrate. The serum enzyme was more resistant to heat treatment and was inhibited by diisopro-pylfluorophosphate (DFP). B. gingivalis also produced Gly-Pro peptidase that is released in the supernatant. The enzyme has an optimal pH range between 7.5 and 8.0. The B. gingivalis Gly-Pro peptidase was inhibited by DFP, suggesting that it represents a serine protease. The serum Gly-Pro peptidase did not differ from the bacterial enzyme with respect to its sensitivity to inhibitors; however, they were markedly different in heat sensitivity. The bacterial enzyme was completely inactivated at 60°C for 30 min, whereas the serum enzyme was not inactivated even at 1 h at 60°C.

Periodontal diseases are recognized as bacterial infections, and some forms are associated with specific organisms, such as Actinobacillus actinomycetemcomitans in juvenile periodontitis, and Bacteroides gingivalis and others in adult periodontitis. The source of the periodontal organisms, whether they are part of the indigenous or resident flora and overgrow to become opportunistic oral pathogens, or whether they are exogenous oral pathogens, is important to determine. The chain of periodontal infection, microbial agent(s) and their transmission, and host response are reviewed with respect to the role of A. actinomycetemcomitans in localized juvenile periodontitis and B. gingivalis in adult periodontitis. The present data lead us to hypothesize that some periodontal organisms may be exogenous pathogens.Prevention of periodontal diseases may be influenced by the knowledge of whether various forms are caused by opportunistic organisms or exogenous pathogens. If exogenous pathogens are responsible, prevention can be directed to intercepting transmission, thereby preventing colonization. On the other hand, if the organisms are opportunistic pathogens, prevention might be directed at interfering with initial acquisition of the flora earlier in life, as well as suppressing them to low levels consistent with health. For those exogenous periodontal infections, attempts at eradication and prevention of re-infection are likely to be effective. If the organisms are part of the indigenous flora, there is little hope of complete elimination of the organism.Criteria for distinguishing exogenous periodontal pathogens from opportunistic periodontal pathogens include the prediction that exogenous pathogens would be transient members of the oral flora associated with periodontal disease, likely to be comprised of one or a few clonal types, and intrinsically virulent. In contrast, opportunistic periodontal pathogens would likely be members of the indigenous flora and would overgrow. They would likely be comprised of many clonal types, and have an intrinsically low level of virulence.

A large body of research implicates Bacteroides gingivalis in the etiology of adult periodontitis, however, the application of this information to clinical diagnosis and treatment has been hampered by the need for a simple, rapid, and reliable means of detecting this microorganism. In the present study, indirect immunofluorescence microscopy using species specific, polyclonal antisera and a monoclonal antibody was evaluated in the clinical identification and quantitation of B. gingivalis in human subgingival dental plaque. One hundred and twenty subgingival plaque samples were obtained from predetermined sites by means of sterile paper points from 20 human subjects including 10 adult periodontitis patients and 10 periodontally normal subjects. The proportions of cultivable B. gingivalis in each sample were determined following anaerobic culture on nonselective blood agar media and selective media containing kanamycin. These results were then compared to quantitative estimates of B. gingivalis by indirect immunofluorescent microscopic evaluation of heat-fixed plaque smears. Using both immunofluorescence microscopy and bacterial culture, the present study confirms the importance of B. gingivalis in adult periodontitis previously described by culture. The organism was cultivable from 70% of the adult periodontitis patients but not from any of the normal adults. In contrast, indirect immunofluorescence microscopy detected the organism in up to 40% of the subgingival sites in 100% of the adult periodontitis patients as well as four sites in the periodontally normal subjects. The sensitivity of indirect immunofluorescence microscopy compared to culture ranged from 91 to 100% while the specificity varied from 87 to 89%. The numbers of B. gingivalis identified by immunofluorescence microscopy were directly proportional to the severity of periodontal disease as measured by clinical indices. Linear regression analysis of the immunofluorescent data suggests that the probability of a subject having adult periodontitis approaches 100% when B. gingivalis comprises 9% or more of the subgingival microflora. The present study indicates that indirect immunofluorescence microscopy using speciesspecific serodiagnostic reagents is useful in the clinical detection of B. gingivalis in human subgingival dental plaque. The data also suggests that certain cases of adult periodontitis can be diagnosed solely on the basis of this laboratory test.

A series of 55 salicylamides, including 3,5-dibromo-, 5-n-alkyl-, and 5-n-acylsalicyloyl derivatives of various anilines, heterocyclic amines, benzylamines, and alkylamines, was synthesized and evaluated for in vitro antibacterial activity against Actinomyces viscosus, an adherent oral microorganism implicated in periodontal disease. The in vitro minimum inhibitory concentrations of 15 4'-bromosalicylanilides were found to correlate (r = 0.92) with estimated log D values. Several nonhalogenated salicylanilides, such as 5-n-hexyl- (40) and 5-n-decanoyl-4'-nitrosalicylanilide (47), were found to exhibit higher levels of in vitro antibacterial activity against a number of Actinomycetes than did tribromsalan (1) or fluorophene (2).

Serological techniques were used to assess the relationships among the following species: Actinobacillus equuli, Actinobacillus lignieresii, Actinobacillus pleuropneumoniae, “Actinobacillus seminis”, Actinobacillus suis, Actinobacillus actinomycetemcomitans Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus segnis, Haemophilus parainfluenzae, Haemophilus influenzae, Haemophilus parahaemolyticus, Haemophilus aegyptius, and Gardnerella vaginalis. Deoxyribonucleic acid-deoxyribonucleic acid hybridization analyses of A. actinomycetemcomitans, H. segnis, H. aphrophilus, and H. paraphrophilus showed that these four species have measurable deoxyribonucleic acid relatedness of 28% or greater, consistent with their placement in a single genus. These bacterial species also demonstrated common antigens by immunodiffusion. In view of these genetic and serological relationships and previous inclusion of X- and V-factor-independent bacterial species in the genus Haemophilus, reassignment of A. actinomycetemcomitans to the genus as Haemophilus actinomycetemcomitans comb. nov. is proposed.

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.

1. C3 activation products C3c and C3d can be demonstrated in gingival pocket fluid from patients with severe periodontitis. 2. Quantitatively, C3d levels in GF are comparable to levels attained consequent to immune-complex and tryptic activation of human serum. 3. The alternate pathway of complement activation occurs in the periodontal pocket, as demonstrated by conversion of C3 proactivator (Factor B) to C3-activator Bb. 4. C4 is present in an altered form in some, but not all, GF's. This form is electrophoretically similar to that generated from serum by C1s and several proteolytic enzymes.

Nineteen rhesus monkeys were inoculated with mumps virus by retrograde ductal instillation into the parotid gland. Evidence of infection was obtained in all instances. Virus was isolated from buccal swabbings and parotid biopsies for 1 week after inoculation. A vigorous serum-neutralizing antibody response occurred within 3 weeks, and there was marked monocytic infiltration of the parotid stroma. The monocytic infiltrate comprised as much as 60% of the total gland volume 1 week after infection. The predominant inflammatory cells were non-immunoglobulin-containing mononuclear cells resembling lymphocytes. Plasma cells containing immunoglobulin G (IgG), IgA, IgM, and IgE increased in numbers in the gland after infection, the greatest increase occurring in IgG-containing cells. Neutralizing antibodies and interferon were found in extracts prepared from the infected glands. Neutralizing activity was highest in samples taken 3 weeks after infection but was detectable in samples taken as soon as 36 to 48 h after infection. Interferon activity was detected in significant amounts 36 to 48 h after infection. Challenge of previously infected animals resulted in an increase in the monocytic infiltrate as well as an increase in numbers of immunoglobulin-containing plasma cells. However, reinfection did not occur as evidenced by the inability to culture shed virus after challenge. This model should be useful for in vivo study of biochemical mediators which evoke inflammatory cell infiltration and which may be significant both in protection and in tissue damage.

Using a recently developed assay for glucosyltransferase activity based on 14 C-glucose incorporation into an alcohol-insoluble polysaccharide, we were able to study inhibition by antibody of this enzyme activity. Rabbit antibody was relatively specific for the strain of Streptoccus mutans from which the enzyme was obtained. Absorption studies showed that neither removal of antibodies directed to dextran nor absorption with intact bacteria offset the enzyme-inhibitory capacity of these sera, whereas absorption with partially purified enzyme did result in removal of the inhibitory capacity.