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Chaperones are molecules found in all cells and are critical in stabilization of synthesized proteins, in repair/removal of defective proteins, and as immunodominant antigens in innate and adaptive immunity. Subjects with gingivitis colonized by the oral pathogen Porphyromonas gingivalis previously demonstrated levels of anti-human chaperone Hsp90 that were highest in individuals with the best oral health. We hypothesized that similar antibodies to pathogen chaperones might be protective in periodontitis. This study examined the relationship between antibodies to P. gingivalis HtpG and clinical statuses of healthy and periodontitis-susceptible subjects. We measured the humoral responses (immunoglobulin G [IgG], IgA, and IgM) to peptides of a unique insert (P18) found in Bacteroidaceae HtpG by using a high-throughput, quantitative fluorescence enzyme-linked immunosorbent assay. Indeed, higher levels of IgG class anti- P. gingivalis HtpG P18 peptide ( P < 0.05) and P18α, consisting of the N-terminal 16 amino acids of P18 ( P < 0.05), were associated with better oral health; these results were opposite of those found with anti- P. gingivalis whole-cell antibodies and levels of the bacterium in the subgingival biofilm. When we examined the same sera for IgA and IgM class antibodies, we found no significant relationship to subject clinical status. The relationship between anti-P18 levels and clinical populations and individual subjects was found to be improved when we normalized the anti-P18α values to those for anti-P18γ (the central 16 amino acids of P18). That same ratio correlated with the improvement in tissue attachment gain after treatment ( P < 0.05). We suggest that anti- P. gingivalis HtpG P18α antibodies are protective in periodontal disease and may have prognostic value for guidance of individual patient treatment.

Background Bacterocins are antimicrobial peptides produced by bacteria with a relatively narrow range of activity against closely related organisms. Subtilosin A is a bacteriocin produced by Bacillus subtilis that has activity against Listeria monocytogenes, which might indicate antimicrobial activity unusual for bacteriocins. Objectives To examine the antimicrobial activity and factors affecting the activity of subtilosin A against a range of potentially pathogenic bacteria. Methods The peptide was purified from cultures of B. subtilis and the MIC determined for 18 species of bacteria using a microdilution methodology. The extent of capsule formation was determined using microscopic examination of cells mounted in India ink. Protease mutants of a susceptible bacteria and mild heat shock were used to examine the effect of environmental stress on subtilosin A activity. Results Subtilosin A proved to have antimicrobial activity against a wide range of bacteria including Gram-positive and Gram-negative bacteria and both aerobes and anaerobes. The peptide was less effective against capsulated forms of two Gram-negative bacteria than the non-capsulated strains of either. Heat shock but not protease activity also altered the effectiveness of the bacteriocin. Conclusions Subtilosin A has limited antimicrobial activity against a number of human pathogens which, combined with its relative ineffectiveness against some capsulated pathogens, may limit its usefulness as a human therapeutic.

Background/aims: The etiologic relationship between periodontitis and Porphyromonas gingivalis is attributed to the ability of the organism to express a variety of virulence factors, many of which are cell surface components including lipopolysaccharide and arginine-specific cysteine proteases (Arg-gingipains, RgpA, and RgpB). P. gingivalis responds to the stress of rapid elevation in temperature by activating a set of genes to produce heat shock proteins that mediate the effects of sudden changes in environmental temperatures by repairing or eliminating cellular proteins denatured by that stress. Methods: We used restriction fragment differential display (RFDD) to identify and measure the genes expressed by surrogates of environmental stresses, heat and oxidative stress. The results were then confirmed using quantitative reverse-transcription polymerase chain reaction. Results: We selected 16 genes differentially induced from over 800 total expression fragments on the RFDD gels for further characterization. With primers designed from those fragments we found that a + 5°C heat shock caused a statistically significant increase in expression compared 12 of 18 untreated genes tested. The exposure of P. gingivalis to atmospheric oxygen resulted in statistically significant increases in five of the target genes. These genes are likely involved in transport and synthesis of components of the lipopolysaccharide biosynthetic pathway important in anchoring the Arg-gingipains required for virulence-related activities. Conclusion: These results emphasize the need for studies to measure the coordinated responses of bacteria like P. gingivalis which use a multitude of interrelated metabolic activities to survive the environmental hazards of the infection process.

Earlier studies suggested that specific immunoreactive domains of the prokaryotic homologue of Hsp90, HtpG, might contribute to the virulence of the periodontal pathogen, Porphyromonas gingivalis (Pg) [J. Periodontol. 70 (1999) 1185]. Since serum antibodies to this protein appeared to be associated with oral health, we developed a rapid epitope-mapping system that could be tailored to detect antibodies against specific immunoreactive regions of the Pg HtpG protein. This paper describes the use of Caulobacter crescentus (Cc) and the creation of a Cc RsaA fusion protein library that defined specific regions of the Pg HtpG protein. The fusion protein library was used to identify immunoreactive regions in the Pg HtpG dominant in patient and control sera. The development of methods to rapidly localize dominant immunoreactive regions in protein antigens may prove useful for the development of screening tests, vaccines and therapeutics in periodontal and other infectious diseases.

Our previous reports implicated the Hsp90 homologue (HtpG) of Porphyromonas gingivalis (Pg) in its virulence in periodontal disease. We investigated the role of the HtpG stress protein in the virulence of Pg. This report describes the (i) expression of a recombinant Pg HtpG (rHtpG), (ii) generation and characterization of a polyclonal rabbit anti-Pg rHtpG antiserum, and (iii) construction of a Pg htpG isogenic mutant and evaluation of the growth, adherence and invasion properties compared to the wild-type parental strain. The disruption of the htpG gene did not significantly affect growth, and had no effect on Pg adherence to and invasion of cultured human cells.

Human prostate cancers (PCa) express great variability in their ability to metastasize to bone. The identification of molecules associated with aggressive phenotypes will help to define PCa subsets and will ultimately lead to better treatment strategies. The chemokine stromal-derived factor-1 (SDF-1 or CXCL12) and its receptor CXCR4 are now known to modulate the migration and survival of an increasing array of normal and malignant cell types including breast, pancreatic cancers, glioblastomas, and others. The present investigation extends our previous investigations by determining the expression of CXCR4 and CXCL12 in humans using high-density tissue microarrays constructed from clinical samples obtained from a cohort of over 600 patients. These data demonstrate that CXCR4 protein expression is significantly elevated in localized and metastastic cancers. At the RNA level, human PCa tumors also express CXCR4 and message, but overall, they were not significantly different suggesting post-transcriptional regulation of the receptor plays a major role in regulating protein expression. Similar observations were made for CXCL12 message, but in this case more CXCL12 message was expressed by metastastic lesions as compared to normal tissues. PCa cell lines also express CXCL12 mRNA, and regulate mRNA expression in response to CXCL12 and secrete biologically active protein. Furthermore, neutralizing antibody to CXCL12 decreased the proliferation of bone homing LNCaP C4-2B and PC3 metastastic tumor cells. These investigations provide important new information pertaining to the molecular basis of how tumors may 'home' to bone, and the mechanisms that may account for their growth in selected end organs.

Periodontitis is considered a consequence of a pathogenic microbial infection at the periodontal site and host susceptibility factors. Periodontal research supports the association of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, and Bacteroides forsythus, and periodontitis; however causality has not been demonstrated. In pursuit of the etiology of periodontitis, we hypothesized that the intracellular bacteria, Chlamydia trachomatis, may play a role. As a first step, a cross-sectional study of dental school clinic patients with established periodontitis were assessed for the presence of C. trachomatis in the oral cavity, and in particular from the lining epithelium of periodontal sites. C. trachomatis was detected using a direct fluorescent monoclonal antibody (DFA) in oral specimens from 7% (6/87) of the patients. Four patients tested positive in specimens from the lining epithelium of diseased periodontal sites, one patient tested positive in healthy periodontal sites, and one patient tested positive in the general mucosal specimen. In conclusion, this study provides preliminary evidence of C. trachomatis in the periodontal sites. Planned studies include the use of a more precise periodontal epithelial cell collection device, the newer nucleic acid amplification techniques to detect C. trachomatis, and additional populations to determine the association of C. trachomatis and periodontitis.

Porphyromonas gingivalis is implicated in the etiology of periodontal disease. Associations between microbial virulence and stress protein expression have been identified in other infections. For example, Hsp90 homologues in several microbial species have been shown to contribute to virulence. We previously reported that P. gingivalis possessed an Hsp90 homologue (HtpG) which cross-reacts with human Hsp90. In addition, we found that elevated levels of serum antibody to Hsp90 stress protein in individuals colonized with this microorganism were associated with periodontal health. However, the role of HtpG in P. gingivalis has not been explored. Therefore, we cloned the htpG gene and investigated the characteristics of HtpG localization and expression in P. gingivalis. htpG exists as a single gene of 2,052 bp from which a single message encoding a mature protein of approximately 68 kDa is transcribed. Western blot analysis revealed that the 68-kDa polypeptide was stress inducible and that a major band at 44 kDa and a minor band at 40 kDa were present at constitutive levels. Cellular localization studies revealed that the 44- and 40-kDa species were associated with membrane and vesicle fractions, while the 68-kDa polypeptide was localized to the cytosolic fractions.

Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans are closely associated with the onset and severity of adult periodontal disease. However, little is known regarding the colonization by, and host antibody response to, these microorganisms in children.Plaque and sera were obtained from 40 healthy children, 2 to 18 years old. Gingival health was assessed by the periodontal disease index (PDI), papillary bleeding score (BS) and the modified total papillary margin attachment index (M-PMA). P. gingivalis and A. actinomycetemcomitans in plaque samples were detected by slot immunoblotting (SIB). Serum antibody levels against these microorganisms were evaluated using ELISA.More than 60% of the children had detectable levels of P. gingivalis in their plaque. Those having detectable levels had more gingival inflammation than those having none; however, these differences were significant only in children over the age of 12 years (PDI, BS). In contrast, while 75% of the children had detectable A. actinomycetemcomitans, there were significant differences in gingival inflammation associated with colonization in children from 3 to 7 years of age (PDI) and over 12 years of age (M-PMA). Serum antibody levels to P. gingivalis were inversely correlated with gingival inflammation in all age groups, while A. actinomycetemcomitans titers were positively correlated with gingival inflammation only in the children over 12 years. No significant relationship between the presence of either A. actinomycetemcomitans or P. gingivalis and antibodies to them was found.Our findings show that P. gingivalis and A. actinomycetemcomitans are readily detected as early as 3 years of age and that their presence is associated with the onset and severity of gingivitis.

We previously reported an association between elevated serum antibody titers to the 90-kDa human heat shock protein (Hsp90), periodontal health and colonization by Porphyromonas gingivalis. In this study, we examined the cellular localization of the Hsp90 homologue of P. gingivalis. Cultures of P. gingivalis were heat-stressed (45 degrees C) and examined for localization of the Hsp90 homologue. Heat stress induced a 4-5-fold increase in anti-Hsp90 antibody reactivity over that of the unstressed controls. Western blot analysis revealed two bands (44 and 68 kDa) that reacted with anti-Hsp90 antibodies. The 68-kDa band was heat-inducible, while the 44-kDa band was not. Immunogold staining revealed that the Hsp90 homologue localized principally to the membrane and extracellular vesicles. Subcellular fractionation confirmed that the Hsp90 homologue was primarily membrane-associated.

There is evidence that microbial heat shock (stress) proteins (Hsp) are immunodominant antigens of many microorganisms. Immunity to these proteins has been shown in non-oral infections to contribute to protection. This study was undertaken to assess the relationship(s) between immunity to human and microbial heat shock proteins, periodontal disease status, and colonization by periodontal disease-associated microorganisms.Subgingival plaque and blood samples obtained from 198 patients during an earlier clinical study were examined for the presence of specific periodontal disease-associated microorganisms and antibodies to selected human and microbial heat shock proteins (Hsp70, Hsp90, DnaK, and GroEL). Particle concentration immunofluorescence assay (PCFIA) was used to detect anti-Hsp antibodies and slot immunoblot assay (SIB) was used to detect subgingival plaque species. Regression models were used to examine the contribution of age, gender, gingival index, probing depth, attachment loss, calculus index, plaque index, and microbial colonization to the anti-Hsp antibody concentrations.Our studies demonstrated that, when evaluated by ANOVA, patients with higher anti-Hsp (Hsp90, DnaK, and GroEL) antibody concentrations tended to have significantly (Por =0.05) healthier periodontal tissues. This was most obvious when the relationship between mean probing depths and antibody concentrations were studied. For Hsp90 antibodies, 2 variables (probing depth and P. gingivalis concentration) were found to have significant contributions (R = 0.293, P0.0002). The equation derived from the regression model was y = 12558-2070*PD +1842*PG. This confirmed the inverse relationship with probing depth and the positive relationship with colonization by P. gingivalis. Attempts to model the other stress protein antibodies were not successful.We believe that the present observations reflect the presence of protective anti-Hsp antibodies, rather than simply the presence of the microorganism in the gingival sulcus. The clinical significance of these observations lies in the potential of identifying patients who are at risk for developing periodontal disease based on their inability to mount an immune response to specific Hsp or Hsp epitopes, as well as the development of vaccines based on Hsp epitopes.

The aim of this study was to assess by means of an ELISA technique, the occurrence of 3 putative periodontopathogens, Porphyromonas gingivalis, Bacteroides forsythus, and Treponema denticola, in 3 clinically-defined adult periodontal conditions. Thirty systemically-healthy subjects were selected and grouped into 3 categories according to their periodontal health: 1) 10 periodontitis subjects (PS), having moderate adult chronic periodontitis; 2) 10 untreated gingivitis subjects (UGS), exhibiting no signs of periodontal destruction but presenting with clinical signs of mild gingivitis; and, 3) 10 treated gingivitis subjects (TGS), having the same clinical status as UGS, but who received a thorough prophylaxis treatment within the past 7 to 14 days prior to the baseline examination. A total of 60 samples were collected subgingivally from the six Ramfjord teeth per subject in each group and ELISA analysis was carried out to give a semiquantitative estimate of P. gingivalis. B. forsythus, and T. denticola. The immunologic detection method suggested the presence of antigens of P. gingivalis, B. forsythus, and T. denticola in subjects from each of the 3 groups. When a global analysis for the 3 disease groups was performed at one time, statistically significant differences were found among the ELISA scores of the 3 bacterial species. For example, comparisons of the ELISA scores showed that the concentrations of P. gingivalis differed significantly when comparing TGS to UGS and PS, but not when examining UGS/PS. The ELISA scores for B. forsythus were significantly different between TGS and PS. Mean concentrations of T. denticola were significantly different when comparing PS to TGS or UGS, whereas no difference was found between the latter categories. Within the limited scope of this study, the concentration of antigens detectable from putative periodontopathogens like P. gingivalis, B. forsythus, and T. denticola differed among the 3 diseased groups, with periodontitis subjects often showing the greatest level of antigens. Thus, it is reasonable to expect that, when using sensitive immunological detection methods, antigens of suspected periodontal pathogens can be found irrespective of the individual's clinical status. However, while detectable in the periodontal sites, the concentrations of these microorganisms are most likely to be above the threshold necessary to induce clinically-significant disease. Studies with larger sample size and standardized antigens are necessary to determine if the groups we found not to differ, were, in fact, different.

There are few objective assays for studies of the epidemiology of periodontal diseases. The PerioScan is an assay capable of detecting three periodontal pathogens, namely T. denticola, P. gingivalis, and B. forsythus, which have been associated with adult periodontitis. The PerioScan was tested in a sample of 301 Brazilians. Clinical indices--bleeding, probing depth, gingival index, and periodontal index--were recorded from four sites in each subject. Subgingival plaque samples were collected from those sites and placed on the PerioScan card. Color results were scored in the field after 15 minutes. The plaque samples were screened with polyclonal antibodies for the three species by an ELISA system. The PerioScan, when compared with the ELISA system, yields a sensitivity of 91 percent, specificity of 89 percent, and an accuracy of 90 percent. When the PerioScan was compared to clinical indices, there was a high sensitivity (at least 93%) and a low specificity (no less than 47%), with an accuracy of at least 61 percent.

The relative avidity and titer of antibodies representing the 4 immunoglobulin G (IgG) subclasses (IgG1-4) reactive with Porphyromonas gingivalis, P. gingivalis-lipopolysaccharide (-LPS), streptokinase (SK) and tetanus toxoid (TT) in the sera of patients having adult periodontitis and of healthy controls were measured. Patient antibody titers to P. gingivalis and P. gingivalis-LPS were found to be significantly elevated for IgG, IgG1 (no P. gingivalis-LPS antibodies) and IgG2. The predominant antibody response to P. gingivalis and P. gingivalis-LPS occurred in the IgG2 subclass. When the relative avidity of the antibodies to P. gingivalis and P. gingivalis-LPS were examined, no significant differences between control and patient sera could be identified. However, anti-P. gingivalis and P. gingivalis-LPS antibodies were found to possess significantly lower relative avidity than either SK or TT antibodies. The IgG1 subclass antibodies to P. gingivalis, SK and TT all appeared to be of high relative avidity. In contrast, anti-P. gingivalis and P. gingivalis-LPS of the IgG2 subclass were of significantly lower relative avidity. Since the predominant humoral response to P. gingivalis occurs in the IgG2 subclass, the low relative avidity of these antibodies predominates in measurements of whole serum activity.

Most forms of periodontal disease are associated with the presence or overgrowth of anaerobic species that could include Treponema denticola, Porphyromonas gingivalis, and Bacteroides forsythus among others. These three organisms are among the few cultivable plaque species that can hydrolyze the synthetic trypsin substrate benzoyl-DL-arginine-naphthylamide (BANA). In turn, BANA hydrolysis by the plaque can be associated with periodontal morbidity and with the presence of these three BANA-positive organisms in the plaque. In this investigation, the results of the BANA test, which simultaneously detects one or more of these organisms, were compared with the detection of these organisms by (i) highly specific antibodies to P. gingivalis, T. denticola, and B. forsythus; (ii) whole genomic DNA probes to P. gingivalis and T. denticola; and (iii) culturing or microscopic procedures. The BANA test, the DNA probes, and an enzyme-linked immunosorbent assay or an indirect immunofluorescence assay procedure exhibited high sensitivities, i.e., 90 ot 96%, and high accuracies, i.e., 83 to 92%, in their ability to detect combinations of these organisms in over 200 subgingival plaque samples taken from the most periodontally diseased sites in 67 patients. This indicated that if P. gingivalis, T. denticola, and B. forsythus are appropriate marker organisms for an anaerobic periodontal infection, then the three detection methods are equally accurate in their ability to diagnose this infection. The same statement could not be made for the culturing approach, where accuracies of 50 to 62% were observed.

The mouths of young children become colonized by a variety of bacteria, but there have been only a few studies that have sought the presence of periodontopathic species in this population. Almost all of these studies used culturing techniques rather than the newer detection methodologies for various periodontopathogens. Studies in adults have shown that Treponema denticola and Porphyromonas (Bacteroides) gingivalis can be detected in dental plaque by use of the BANA and ELISA diagnostic tests. In the present study, plaque samples from four subgingival sites in each of 157 children (aged from two to 18 years) were tested for BANA hydrolysis with a BANA reagent card, and for T. denticola and P. gingivalis with an ELISA assay. Anaerobic periodontopathogens hydrolyzing the BANA substrate were found to be present in at least one of four plaque samples in 88 children (56%). T. denticola and/or P. gingivalis were detected by ELISA in at least one plaque sample in each of 135 children (86%). This study shows that children are widely colonized by these micro-organisms. A higher proportion of Black children than Caucasian children was colonized by these BANA-positive organisms. Also, children having a parent with a documented history of periodontal disease were more likely to be BANA-positive than were children of parents with unknown periodontal status.

Background Chaperones are ubiquitous conserved proteins critical in stabilization of new proteins, repair/removal of defective proteins and immunodominant antigens in innate and adaptive immunity. Periodontal disease is a chronic inflammatory infection associated with infection by Porphyromonas gingivalis that culminates in the destruction of the supporting structures of the teeth. We previously reported studies of serum antibodies reactive with the human chaperone Hsp90 in gingivitis, a reversible form of gingival disease confined to the oral soft tissues. In those studies, antibodies were at their highest levels in subjects with the best oral health. We hypothesized that antibodies to the HSP90 homologue of P. gingivalis (HtpG) might be associated with protection/resistance against destructive periodontitis. Methodology/Principal Findings ELISA assays using cloned HtpG and peptide antigens confirmed gingivitis subjects colonized with P. gingivalis had higher serum levels of anti-HtpG and, concomitantly, lower levels of attachment loss. Additionally, serum antibody levels to P. gingivalis HtpG protein were higher in healthy subjects compared to patients with either chronic or aggressive periodontitis. We found a negative association between tooth attachment loss and anti-P. gingivalis HtpG (p = 0.043) but not anti-Fusobacterium nucleatum (an oral opportunistic commensal) HtpG levels. Furthermore, response to periodontal therapy was more successful in subjects having higher levels of anti-P. gingivalis HtpG before treatment (p = 0.018). There was no similar relationship to anti-F. nucleatum HtpG levels. Similar results were obtained when these experiments were repeated with a synthetic peptide of a region of P. gingivalis HtpG. Conclusions/Significance Our results suggest: 1) anti-P. gingivalis HtpG antibodies are protective and therefore predict health periodontitis-susceptable patients; 2) may augment the host defence to periodontitis and 3) a unique peptide of P. gingivalis HtpG offers significant potential as an effective diagnostic target and vaccine candidate. These results are compatible with a novel immune control mechanism unrelated to direct binding of bacteria.

CXCL8 (interlukin 8, IL-8) has a diverse spectrum of biological activities including T cell, neutrophil and basophil chemotactic properties. It is produced by a wide variety of cell types and plays a significant role in the initiation of the acute inflammatory response. During inflammation, CXCL8 attracts and activates leukocytes at the site of infection leading to leukocyte infiltration, which can lead to tissue damage. Porphyromonas gingivalis, an aetiological agent of periodontitis, induces production of CXCL8 from several types of cells via its LPS and outer membrane proteins. Bacterial chaperones elicit a strong pro-inflammatory response in cells of the innate immune system. In P. gingivalis the htpG gene codes for the homologue of human Hsp90, a chaperone that associates with transcription factors, hormone receptors and protein kinases, affecting signal transduction pathways. CXCL8 mRNA and CXCL8 protein production was induced in monocytic/human microvascular vein endothelial cells treated with P. gingivalis cells or rHtpG protein. Blocking of receptors CD91 and TLR4 reduced the production of CXCL8 by rHtpG either using receptor-specific antibody or by siRNA silencing. Pre-incubation of P. gingivalis rHtpG preparations with human anti-HtpG significantly inhibited CXCL8 production. A P. gingivalis HtpG disruption mutant also induced less CXCL8 mRNA and protein. These results suggest that P. gingivalis HtpG might be involved in CXCL8-mediated immunopathogenesis.

Induction of resistance of oral anaerobes to the effects of human β-defensin 1 (hβD-1) to hβD-4 was investigated by pretreating cells with either sublethal levels of defensins or environmental factors, followed by a challenge with lethal levels of defensins. Cultures ofPorphyromonas gingivaliswere (i) pretreated with defensins at 1 ng/ml, (ii) heated to 42°C (heat stress), (iii) exposed to normal atmosphere (oxidative stress), or (iv) exposed to 1 mM hydrogen peroxide (peroxide stress). Samples (10 μl) were distributed among the wells of sterile 384-well plates containing hβD-1 to -4 (100 μg/ml). Plates were incubated at 37°C for 36 h in an anaerobe chamber. Growth inhibition was determined by a system that measures the total nucleic acid of a sample with a DNA binding dye. The MICs of the four defensins forP. gingivaliswere 3 to 12 μg/ml. We found that sublethal levels of the defensins and heat and peroxide stress, but not oxidative stress, induced resistance to 100 μg of defensin per ml inP. gingivalis. Resistance induced by sublethal levels of hβD-2 lasted 90 min, and the resistance induced by each defensin was effective against the other three. Multiple strains exposed to hβD-2 all evidenced resistance induction. Defensin resistance is vital to the pathogenic potential of several human pathogens. This is the first report describing the induction of defensin resistance in the oral periodontal pathogenP. gingivalis. Such resistance may have an effect on the ability of oral pathogens to persist in the mouth and to withstand innate human immunity.

An etiological relationship between periodontitis, a significant oral health problem, and the anaerobe Porphyromonas gingivalis may be related to the expression of a variety of putative virulence factors. The objective of the experiments described here was to develop a quantitative reverse transcription polymerase chain reaction (QRT-PCR) method to examine P. gingivalis gene expression in human dental plaque from periodontitis subjects. PCR primers and probes for six target genes representing putative virulence factors were chosen and evaluated in vitro for specificity. A potential cross-reactivity level of only 10 copies/10(7) whole genomic equivalents was occasionally observed with non-P. gingivalis microbes. P. gingivalis cells stressed in vitro by a 5 degrees C temperature increase showed a rapid rise in the mRNA associated with the molecular chaperons (htpG, dnaK, groEL), SOD (sodA) and gingipain (rgp-1) genes. We examined the stability of bacterial RNA in plaque specimens and found no significant difference in the amount of RNA obtained before or after storage 3 months in a stabilizing buffer (p=0.786, t-test). Sixty-five percent of plaque samples obtained from two clinical locations contained P. gingivalis; there was a mean level of gene expression (fold increase) for all samples tested for groEL, dnaK, htpG, sodA, PG1431 and rgp-1 of 0.84+/-2.03 to 7.85+/-10.0. ANOVA showed that the levels of stress gene transcription for dnaK and htpG were significantly elevated (p0.05) at diseased sites; groEL gene transcription approached statistically significant elevation (p=0.059). We found correlations between probing depth and increased transcription of groEL, htpG and rgp-1 and between attachment loss and htpG. When sorted by disease status, we detected correlations between disease status and elevated expression of dnaK and htpG.

Gingival inflammation in Down's syndrome children (DS) develops earlier and is more rapid and extensive than in non-DS children. Abnormalities in host response to the oral flora have been proposed as etiological factors of this gingival inflammation. However, the relationship between gingivitis and the host response to oral microorganisms in DS by age has not been determined. The objective of this study was to clarify this relationship. Sera were obtained from 75 DS subjects (aged 2 to 18 years) and their gingival health assessed using a modified PMA Index (M-PMA). Antibody titers to Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Treponema denticola (Td), Fusobacterium nucleatum (Fn), Selenomonas sputigena (Sel), Actinobacillus actinomycetemcomitans (Aa), and Streptococcus mitis (Mi) were determined using the micro-ELISA. DS subjects under 4 years old were found to have significantly more gingival inflammation than did normal children the same age. A significant positive correlation (r = 0.548, P < 0.0001) existed in the relationship between M-PMA score and plaque score for subjects in the G1 age group (deciduous dentition). At G1, the average antibody titers to Aa, Mi, and Fn exceeded those of the normal adult reference serum pool. In addition, IgG antibody titers to Pg, Aa, Fn, Sel, and Mi correlated significantly with the M-PMA scores in the G1 age group. There was a correlation between age (2 to 18 years) and these antibody titers. IgG antibody titers to Pg, Aa, Sel, and Mi increased significantly with increasing M-PMA score. Furthermore, the IgG antibody titers to Pg were higher (P < 0.05) in the most extensive disease group compared to the DS no-disease group. The IgG antibody titers to Pg at G3 (early puberty) were significantly higher when compared to G1 (preschool children). The IgM antibody titers to Aa at G3 were higher (P < 0.05) when compared to G1. This study suggests that colonization by Aa and Fn are closely associated with the onset of gingival inflammation in DS patients under 5 years old. Colonization by Pg, Aa, Sel, and Mi in DS appears to be associated with gingivitis at puberty.

Microbial colonization of barrier materials used in guided tissue regeneration (GTR) is known to adversely affect treatment outcomes. The purpose of this study was to compare the rate at which 11 commonly-occurring oral bacteria species colonize three different barrier materials (collagen, expanded polytetrafluoroethylene, and polylactic acid). The study group consisted of 10 systemically healthy individuals with no history of periodontal disease and absence of antimicrobial therapy within the previous 3 months. In each patient, 4 teeth per quadrant (P1, P2, M1, M2) were selected and 3 teeth were randomly assigned as test teeth while the remaining tooth acted as a control site (i.e., natural colonization of the tooth surface). These teeth were then randomly assigned to receive one of the three barrier types (i.e., each patient received 4 barriers of each type, 1 per quadrant). A 2 x 5 mm piece of barrier material was positioned over the oral surface of the buccal marginal gingiva and secured with an external sling suture. With oral hygiene procedures suspended, one barrier of each type was collected at 1, 3, 7, and 14 days. Slot immunoblot assay demonstrated that all species types (A. actinomycetemcomitans, A. viscosus, B. melaninogenicus, F. nucleatum, P. gingivalis, P. intermedia, S. mutans, S. sanguis, Selenomonas sputigena, T. denticola, and T. vincentii) were present. Semi-quantitative scoring (scale 0 to 3) of slot blot results and analysis by chi-square ratio and Pearson correlation test indicated that while total bacteria adherence increased over time (P < 0.05), the 3 barrier types and the control sites did not differ in numbers or species of colonizing bacteria detected per time point. These results suggest that under these experimental conditions the barrier materials tested do not differ in bacteria adherence or antimicrobial properties.

The development of diagnostic tests for a periodontal infection raises the issue as to what the appropriate reference standard, or "gold standard," should be for the evaluation of a new test. The present research was initiated to compare the ability of several detection methods, i.e., a serial dilution anaerobic culture and/or microscopic procedure, a DNA probe procedure, and immunological reagents using both an enzyme-linked immunosorbent assay and an indirect immunofluorescence assay to detect Treponema denticola, Porphyromonas gingivalis, Bacteroides forsythus, and Actinobacillus actinomycetemcomitans in subgingival plaque samples taken from 204 periodontally diseased tooth sites. The prevalence of the four monitored species varied as a function of both the species and the detection method. Spirochetes were present in 99% of the plaques, whereas A. actinomycetemcomitans was detected at the lowest frequency. The culture method yielded the lowest prevalence values for the three cultivable species. This raised the question as to which results, those obtained by culture or those obtained by the DNA probes and the immunological reagents, were the most reliable. This issue was addressed by looking at the prevalence profile of the monitored organisms, as determined by all the detection methods. If the species was detected by three or four of the detection methods, then it was considered present, whereas if it was absent by three or four of the detection methods, then it was considered absent. This approach showed the DNA probes and immunological reagents to be significantly superior (P less than 0.05) to the culture approach for the detection of P. gingivalis, A. actinomycetemcomitans, and B. forsythus and to be comparable to the microscopic approach in the detection of T. denticola.

A rapid method for qualitative and quantitative detection of specific oral microorganisms from subgingival dental plaque is described. Plaque samples were suspended in phosphate-buffered saline containing protease inhibitors and 0.5% formaldehyde, briefly sonicated to disperse bacterial aggregates, and applied to nitrocellulose membranes in a slot blot manifold. Subsequent incubations with species-specific rabbit antibody and anti-rabbit antibody-alkaline phosphatase conjugate and development with BCIP-NBT substrate resulted in an easily discernible, permanent stain being deposited at the sample application site. Comparison with known concentrations of pure, cultured microorganisms applied to the same membranes permitted qualitative or semiquantitative plaque characterization by visual inspection. Analysis of the blots with a computer-linked flatbed scanner provided quantitative data on microbial content. The reproducibility of the results (standard error of the mean, less than 10%) obtained with slot immunoblotting greatly exceeded that of the results obtained with immunofluorescence analysis (standard error of the mean, greater than 57%). Because it is versatile, rapid, sensitive, reproducible, permanent, and relatively inexpensive, slot immunoblotting lends itself to use in large-scale investigations for the detection and quantitation of specific microbial species.

Relative avidities of antibodies to Porphyromonas (Bacteroides) gingivalis in the sera of 15 patients having adult periodontitis and 15 healthy subjects were evaluated using an ammonium thiocyanate-dissociated ELISA. Graded concentrations of ammonium thiocyanate were added to a single dilution of serum in order to dissociate low avidity antibody binding to P. gingivalis. The concentration of thiocyanate resulting in 50% reduction in binding (absorbance) was termed the ID50 for that serum. When IgG-class antibodies were examined, the ID50 of anti-P. gingivalis antibodies in the sera of patients was significantly elevated (0.96M vs 0.71M; p less than 0.01, Student's t-test). In contrast, when IgM-class antibodies were examined no significant differences in ID50 between patients and controls were found for P. gingivalis (0.54M vs 0.53M). While the ID50 values of patient antibodies were found to be elevated relative to those of healthy controls, comparison with antibodies from rabbits immunized with P. gingivalis and with ID50 values from other human studies suggests that adult humans, in general, produce very low-avidity antibodies to P. gingivalis. It is suggested that the presence of low-avidity antibodies contributes to the pathology associated with periodontal disease.

Treponema denticola, Porphyromonas (Bacteroides) gingivalis, and Bacteroides forsythus are among the anaerobic species frequently associated with adult forms of periodontal disease. These organisms hydrolyze the synthetic peptide benzoyl-DL-arginine-naphthylamide (BANA), and such enzyme activity can be detected in the plaque and related to clinical disease and the presence of spirochetes. In this investigation, the liquid BANA assay was compared with a commercially developed BANA assay which employed a paper format and which could be read after a 15-min incubation. In the paper format, strips of a Whatman filter paper were impregnated with BANA and strips of nitrocellulose paper were impregnated with fast black K salt. Both strips were applied lengthwise across a paper card (3 by 5 in. [7.6 by 12.7 cm]). The BANA strip at the bottom was inoculated with the test sample (pure culture, plaque), folded back so that it contacted the fast black strip, and then incubated for 15 min at 55 degrees C. T. denticola, P. gingivalis, and B. forsythus always gave a positive reaction, whereas 51 other plaque species were always negative. Six Bacteroides and Capnocytophaga species on occasion had weak reactions. The proportional agreement between BANA positiveness and clinical disease was similar for both the liquid and the paper assays. The sensitivity, specificity, and accuracy relative to the clinical standard of the liquid assay were 74, 76, and 77%, respectively, while those of the paper assay were 81, 78, and 80%, respectively. The paper assay was significantly associated with the presence of either T. denticola or P. gingivalis or both in the plaque samples, with a sensitivity of 85%, a specificity of 53%, and an accuracy of 79%. These findings indicate that a rapid paper assay for BANA hydrolysis gives data comparable to those obtained with the liquid BANA assay.

This study evaluated the effects of citric acid demineralization and autologous fibronectin application on cell proliferation after mucoperiosteal flap surgery. Three adult rhesus monkeys were used. After flaps were raised, the roots were surgically exposed and planed. Surfaces on the experimental sides were decalcified with citric acid, and after thorough rinsing, the inner aspect of the flaps and the roots were bathed with 1 ml of autologous plasma fibronectin in normal saline (400 micrograms m/ml) and the flaps sutured. Contralateral teeth, acting as controls, were treated only with the surgical procedure. One hour prior to sacrifice, the animals were injected with an intravenous injection of tritiated thymidine (1 microCi/gm body weight). Surgeries were staggered to produce the following time periods: 3, 7, 15, 21 and 28 days. After processing, autoradiographs were obtained for evaluation, and labeled cells were counted in five compartments at 400 x: (1) oral epithelium, (2) crevicular area, (3) supracrestal connective tissue, (4) coronal periodontal membrane and (5) coronal bone marrow. Forty tissue sections per procedure (20 slides per tooth) were counted and means obtained for the three monkeys. Differences between experimental and control values were statistically evaluated for each component, at each time interval, using pairwise t tests. Fibronectin-treated areas showed significantly increased cellular proliferation (P less than 0.01) during the first 2 weeks, affecting mainly all the supracrestal tissues. Histologically, the establishment of a well-organized fibrinous clot at 3 days was noted in these areas. Results show a faster healing after surgery with the use of citric acid and fibronectin. It was concluded that citric acid followed by fibronectin enhanced cellular proliferation.

The level of cellular differentiation of human oral, sulcular, and junctional epithelium was compared by immunohistochemical analysis of cell membrane-associated blood group-specific carbohydrates. Identification of the blood group A-specific carbohydrate and its two immediate precursor substances, type 2 chain H and N-acetyllactosamine, was accomplished by an indirect immunofluorescence technique. Murine monoclonal antibodies reacting specifically with the antigenic determinants of the blood group substances were used as markers. The blood group A substance, indicating the highest level of cellular differentiation, was demonstrated on the cells in the upper layers of the oral epithelium. In the sulcular epithelium, the A substance was present on a few cells only, while type 2 chain H was observed frequently. This indicates an intermediate differentiation level of sulcular epithelium. The type 2 chain H precursor, N-acetyllactosamine, the indicator of the lowest level of cell differentiation among the tested substances, was the only blood group substance detected on the junctional epithelial cells and on the basal cells of the sulcular and oral epithelium. Based upon previous studies of cell renewal and differentiation in oral epithelium, the present results indicate that the variations in distribution of the different blood group substances correspond with the regional rates of cell division and the levels of cellular differentiation. The findings also suggest that the cells in the junctional epithelium differentiate to a level similar to that of basal cells in the oral epithelium.

While fibronectin (FN) has previously been demonstrated to be present in gingival crevicular fluid (GCF), its quality and quantity has not been reported. Since this information is relevant for ongoing studies on the use of FN for gingival reattachment, we performed these measurements and compared plasma levels in healthy subjects, patients with gingivitis and periodontitis, and in patients undergoing maintenance therapy. Plasma and GCF samples were obtained from 4 sites in each subject using a Periotron to permit quantification of samples. FN concentrations were determined in a microELISA using hyperimmune anti-FN antibody. Purified FN served as a reference for quantification. The functional activity of each sample was assessed by examining the natural affinity of FN for gelatin. Subjects with gingivitis and those in maintenance had significantly depressed levels of plasma fibronectin. While little fibronectin could be detected in the GCF of healthy sites regardless of patient category, examination of the most diseased sites in each group revealed that the concentration of FN in the GCF was highest in health and reduced when there was gingival inflammation. In no case was GCF FN found to be biologically active.

Actinomyces viscosus was fractionated into cell wall (PEL) and intracellular supernatant (SUP) fractions following ultrasonication. In vitro lymphocyte transformation induced by each fraction was assessed using the lymphocytes obtained from subjects with minimal periodontal disease. In kinetic experiments, a detectable blastogenic response to both fractions was measured by the fourth day, with a peak at day 7. Nuclease treatment enhanced the immunostimulatory activity of the PEL fraction 5.5-fold over untreated PEL; it had no effect on the activity of SUP. Although pronase treatment had no effect on the PEL fractions, it abrogated the ability of SUP to activate lymphocytes. Fractions of nuclease-treated and untreated SUP were batch-eluted from DEAE-sepharose columns with increasing concentrations of NaCl, producing 3 major stimulatory subfractions which accounted for 75 per cent of the activity of unfractionated SUP. Comparisons of lymphocyte responses to the DEAE-fractionated SUP indicated that not all individuals responded equally to each subfraction. As there was a differential responsiveness to the various antigenic components, detailed evaluation of the antigens of A. viscosus is warranted to define differences in antigen responsiveness in individuals with differing severities of periodontal disease.

Human peripheral blood lymphocytes were cultured for 48 hr in the presence of an ultrasonicate supernatant fraction of Actinomyces viscosus (AV-SUP). After washing to remove AV-SUP, the lymphocytes were cultured in the presence of concanavalin A or pokeweed mitogen for an additional 72 hr. Measurement of [3H]thymidine incorporation into DNA indicated that AV-SUP preculture resulted in an amplified or elevated response to mitogen stimulation compared to mock precultured cells and unstimulated controls. Addition of fresh cells prior to mitogen activation resulted in an additional incremental increase over controls. The modulated cell appeared to reside in the AV-SUP precultured population, since treatment of these cells with mitomycin C prior to coculture with fresh cells totally abrogated the effect. However, mitomycin C treatment of the fresh population had no effect on mitogen-induced amplification. Results of these experiments suggest that mitogen stimulation activates a helper function which allows the precultured cells to respond to AV-SUP stimulation.

Antibodies directed against lysergyl-poly- l -lysine were elicited in rabbits. A ligand binding assay was developed to monitor antibody activity. Results using this assay showed that immunoadsorption selects for the antibody population representing the average affinity in the serum with subsequent loss of high and low affinity subpopulations. Measurements of average intrinsic association constants ( K o ) and heterogeneity indices ( a ) were made with IgG fractions of anti-lysergyl antisera and compared with purified antibody populations after immunoadsorption.

Human peripheral blood lymphocytes (PBL) are activated to become suppressor T cells (S-T-C) by incubation with Concanavalin-A (Con-A). This has become the standard method for evaluation of suppressor function in patients. S-T-C function has been found to be impaired in several autoimmune diseases, including systemic lupus erythematosus (SLE). Using this assay, we have investigated suppressor-cell function in 2 autoimmune disorders, bullous pemphigoid (BP) and pemphigus vulgaris (PV), studying 6 patients from each group. Three patients with active SLE (positive controls), and 11 normal donors (negative controls) were also included. None of these patients had received systemic therapy with the exception of 2 patients with PV who were treated with gold in the past. PBL from these patients were incubated with and without 40 micrograms/ml Con-A for 72 hr to generate suppressor cells. Both groups of PBL were then irradiated wih 1500 r cobalt. Co-cultures were set up in sextuplicate using normal PBL as responders. Responder PBL were stimulated with 0.5, 1.0, and 2.0 micrograms/ml of phytohemagglutin (PHA) and 5.0, 10.0, and 20.0 micrograms/ml of Con-A. Cultures were pulsed on day 3 with 3H-thymidine and harvested on day 4. Data were analyzed using Student's t-test. S-T-C function was found to be significantly impaired in SLE vs normal (p = 0.0316). No statistically significant difference was seen in BP (p = 0.5883) and PV (p = 0.0921) as compared with normals. A defect in suppressor cell function may still be present in patients with PV and BP for the defect may be antigen-specific and therefore remain undetected by the Con-A suppressor assay.

Juvenile diabetics have been shown to have an increased susceptibility to gingivitis and periodontitis following puberty. However, little data are available on changes in the microbial flora that occur at the onset of puberty. This study was performed to determine if antibacterial antibody titers to selected periodontal disease-associated microorganisms might be helpful in revealing changes in plaque flora at the onset and conclusion of puberty. Sera was obtained from 35 subjects (ages 7 to 18 years) selected from a population of insulin-dependent diabetics. The subjects were given a thorough medical examination which included an assessment of sexual maturation and a dental examination which included the recording of onset and magnitude of bleeding according to the papillary bleeding score. Antibody titers to A. naeslundii (AN), B. intermedius (BI), B. gingivalis (BG), F. nucleatum (FN), A. actinomycetemcomitans (AA), C. ochracea (CO) and T. denticola (TD) were determined using the microELISA. Stratification of antibody titers by age groups (less than or equal to 12 years, 12 to 15 years, greater than 15 years) revealed that titers to AN increased significantly (P less than 0.025, ANOVA) and progressively (P less than 0.05, regression analysis) with increasing age. In contrast, the titers to FN were maximal in the under 12 year group and decreased with age (ANOVA, P less than 0.05; regression analysis, P less than 0.05). There were no significant variations in titers observed for the other microorganisms. Stratification by sexual maturity revealed a similar progressive decrease of the titer to FN (ANOVA, P less than 0.05; regression analysis, P less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Serum antibody titers from patients with periodontitis were compared with those from periodontally healthy subjects. With the micro-enzyme-linked immunosorbent assay, immunoglobulin G (IgG), IgA, and IgM antibody titers to isolates of Streptococcus sanguis, Actinomyces viscosus, Bacteroides gingivalis, Bacteroides melaninogenicus subsp. intermedius, Bacteroides gingivalis, Bacteroides melaninogenicus subsp. intermedius, Bacteroides ochraceus, and Fusobacterium nucleation were determined. Antibody titers of the IgG and IgA classes to B. melaninogenicus, B. ochraceus, F. nucleatum, and S. sanguis were found to be significantly higher in the controls than in the patients. No correlations were found with serum IgM titers. These findings indicate that periodonitis may be associated with depressed antibacterial serum antibody titers of the IgG and IgA classes.

This study evaluated the effects of citric acid demineralization and autologous fibronectin application in association with a modified Widman flap in the treatment of periodontitis. The study population comprised 29 patients under treatment for moderate to advanced periodontitis who reached the one-year posttherapy evaluation. After thorough scaling and root planing, a split mouth design was used in which two quadrants were treated by modified Widman flap alone, and the other two randomly assigned quadrants were treated by modified Widman flap combined with citric acid demineralization and autologous fibronectin application. Fibronectin, which had previously been isolated from the patient's own plasma, was applied with a tuberculin syringe on the citric acid demineralized root surfaces and the inner aspect of the flap. After suturing provided good flap adaptation, additional fibronectin was again applied under the flap and external pressure was applied. Patients were clinically evaluated at baseline and at one year. Statistical evaluation of the data using paired t test and Chi-square analysis indicated that both approaches, modified Widman flap alone or in combination with citric acid and fibronectin, significantly reduced probing pocket depth and increased clinical attachment. However, the changes achieved with citric acid and fibronectin were statistically greater than those obtained with the flap alone. Furthermore, the number of sites gaining 2 mm or more of clinical attachment were significantly increased. The results suggest that the use of citric acid and fibronectin holds promise in promoting reattachment after periodontal therapy.

The blood-group isoantigens are macromolecules localized to the plasma membranes of certain epithelial tissues.2,11-15 These substances are not detectable on the epithelium once it has undergone malignant transformation.2,9,13 Results of this investigation have demonstrated that the loss of detectability of the blood-group isoantigens does not appear to be related to a "masking" effect by an increase in surface sialic acid. Using fluorescein-labeled lectins specific for sugar subunits which are components of the blood-group oligosaccharide chain, it was found that the malignant cells and cells of the para basal layer of normal oral epithelium had high levels of N-acetyl-D-glucosamine (GlcNAC), the subterminal sugar residue of the blood-group chain. The basal cells of normal epithelium and a minority of the malignant cells demonstrated levels of D-galactose-N-acetyl-D-galactosamine, which are the most proximal blood-group sugar subunits, as well as subunits of other membrane antigens. Our results indicate that malignant cells seem to be capable of synthesizing the blood-group oligosaccharide chains to the same level as the normal cells of the para basal layer of stratified squamous epithelium. This level is just subjacent to the terminal D-galactose residue of the blood-group precursor chain. Increased or decreased differentiation characteristics of squamous cell carcinomas did not alter the level of blood-group synthesis. However, there may be a correlation between the level of synthesis of these antigens and the ability of the cells to demonstrate motility and to proliferate.

The enzyme-linked immunosorbent assay (ELISA) is used extensively in immunologic research for obtaining quantitative estimates of immunoglobulin concentration in cell culture supernates. Through incorporation of a microcomputer for data acquisition, storage and rapid calculation of results, a substantial reduction in total assay time may be realized. Described here are a set of menu-driven programs written in Basic for the IBM-PC which provide advantages over existing software in simplicity, versatility and accuracy. Hardware requirements are minimal. These programs should encourage greater flexibility in terms of the size and complexity of experimental designs.

Publisher Summary This chapter highlights the current status of a low molecular weight suppressor (LMWS) of lymphocyte proliferation produced by cultured spleen cells and macrophages during unstimulated conditions. Antiproliferative factors released by both adherent spleen and macrophage populations have been implicated as potential regulators of in vivo and in vitro lymphoid cell proliferation. Splenic supernates produce a rapid, progressive decrease in tritiated thymidine incorporation, maximal 10–24 h after exposure to thymocyte target cells. Nonlymphoid cell suspensions obtained from neonatal rat liver, kidney, and intestinal mucosa are unaffected by inhibitor concentrations that produce a 50% inhibition of lymphoid targets. ConA and PHA activation result in similar effects on levels of LMWS released by producing spleen cells. Coexisting with an activatable adherent suppressor T cell, the lymphoid organ would possess a highly specific mechanism to regulate the response of a specific clone in the absence of a specific macrophage antigen recognition factor.

Fab (3·5 S ) fragments were derived from IgG fractions of anti-dinitrophenyl and anti-lysergil antisera by papain digestion to reduce avidity forces due to polyvalency. The Fab fractions were immunoadsorbed to obtain active purified fragments. Ligand binding properties of the purified Fab fragments were measured by equilibrium dialysis and were compared to the properties of the original unadsorbed fractions. Purified Fab fragments possessed higher average intrinsic association constants ( K o ) than intact IgG antibodies from which they were derived. Purified anti-dinitrophenyl Fab fragments exhibited a K o of 3·5 × 10 6 M −1 compared to 8·7 × 10 5 M −1 for the unadsorbed Fab and IgG fractions. Anti-lysergyl Fab fragments exhibited a K o of 1·7 × 10 6 M −1 compared to 1·4 × 10 5 M −1 for its unadsorbed Fab and IgG fractions. It is propsed that this increase in K o for the adsorbed Fab fragments is a result of the loss of avidity or functional affinity.

Coculture of human peripheral blood lymphocyte suspensions, previously pulsed with either pokeweed mitogen (PWM) or an Actinomyces viscosus ultrasonicate supernatant fraction (AV), resulted in a blastogenic response that was greater than the sum of the responses of the two independently cultured lymphocyte suspensions. Selective cobalt-51 irradiation of the precultured lymphocytes prior to coculture revealed that PWM was inducing a helper activity which facilitated the blastogenic response to the poorly mitogenic AV. Subsequent experiments revealed that both B- and T-enriched lymphocyte populations were capable of providing such help, however, the B-cell help appeared to require T-cell interaction. As few as one PWM-activated helper cell per 150 AV-pulsed cells was sufficient to provide measureable help. In the presence of PWM helper cells, both B and T cells gave strong blastogenic responses to AV fractions. Results of this study suggest that lymphocytes can respond to poorly mitogenic substances from bacteria if helper cell activity is provided by a second, unrelated lymphocyte stimulant.

The purpose of this study was to evaluate the effect of fibronectin application on healing of replanted teeth. Three rhesus monkeys were used. Maxillary and mandibular incisors and premolars were extracted and replanted. Teeth were extracted with forceps and placed in saline solution. After 5 minutes, each tooth was returned to its socket and immobilized by interproximal acid-etch splints, which were removed after 1 week. Of the forty-eight teeth replanted, twenty-four control teeth were replanted as described. On each of the remaining teeth, the root surface and the inner walls of the socket were bathed with 1 ml of fibronectin in saline solution (400 μg/ml) during the 5-minute interval between booth extraction and its replantation. Replanted teeth and animal killings were scheduled to provide observations 1, 3, 7, 14, 28, and 45 days after replantation. Each monkey received an intravenous injection of tritiated thymidine, 1 μCi/g of body weight, 1 hour before it was killed. Tissue specimens were processed for histologic and autoradiographic evaluation following standard procedures. For each of the six points of time, four pairs of contralateral teeth were available for evaluation; four teeth were treated with fibronectin and four without it. The findings of this study indicate that fibronectin use resulted in enhanced healing by (1) early replacement of the fibrin clot, (2) increased connective tissue cell proliferation, (3) reduction of the inflammatory response, and (4) inhibition of both cementum resorption and dentoalveolar ankylosis.

Human peripheral blood lymphocytes were cultured in vitro with lipopolysaccharide (LPS) for 48 h. After washing, stimulation with a concanavalin A (ConA) or pokeweed mitogen (PWM) resulted in a synergistic blastogenic response that was greater than the sum of the independently stimulated control cultures. Addition of fresh autologous lymphocytes after LPS preculture produced an additional increment of synergy. The nature of the responding and helping effects was determined by coculturing irradiated or nonirradiated lymphocyte suspensions that had been precultured with either LPS or ConA/PWM. Such studies indicated that amplification was the result of a mitogen-activated helper activity, which facilitated the blastogenic response to LPS. Experiments with lymphocytes resolved into T- and B-cell-enriched fractions indicated that the LPS-responsive cells were of the B type and that the help was provided by a mitogen-activated T-cell population. These studies indicated that LPS can induce human B-cell blastogenesis; however, a helper function must be provided by a T-cell subpopulation. This helper activity is inducible by pretreatment of the T cells with plant lectins.

The purpose of this histologic and histometric study was to examine the effect of citric acid and increasing concentrations of fibronectin on new connective tissue attachment following periodontal flap surgery. Full thickness, mucoperiosteal flaps were elevated in six healthy mongrel dogs. Two to 3 mm of alveolar bone were removed along the buccal aspect of the teeth in the mandible and into the interproximal areas of each surgical site. Cementum was removed from the exposed root surfaces and reference notches were inscribed into the roots at the margin of the recontoured bone. Citric acid, pH 1.0, was applied to the instrumented root surfaces for 3 minutes and rinsed with sterile saline. Both the root surfaces and the inner surface of the flap were then bathed in either sterile saline or increasing concentrations (0.38, 0.75 and 1.5 mg/ml saline) or exogenous fibronectin. All the flaps were returned to their original preoperative positions, secured using figure 8 sutures with 4-0 braided silk and allowed to heal. Two dogs per time-point were sacrificed at 1, 2 and 6 weeks after surgery. Block specimens of the surgical sites were demineralized and serial sections prepared for histologic and histometric evaluations. Histologically, tissue sections were examined for: (1) epithelial proliferation and attachment, (2) periodontal fiber organization and maturation, (3) inflammatory cell types, (4) presence or absence of new cementum deposition and (5) degree of vascularity of the tissues. Histometric measurements were taken: (1) from the root surface notch to the apical extent of the junctional epithelium and (2) from the apical extent of the junctional epithelium to the free gingival margin.(ABSTRACT TRUNCATED AT 250 WORDS)

Background/aims: Treponema denticola outer membrane proteins are postulated to have key roles in microbe–host interactions in periodontitis. Because there are no reports of in vivo expression of these putative virulence factors, we examined several T. denticola strains to determine whether sera from human subjects recognized specific T. denticola outer membrane proteins. Methods: Soluble extracts were prepared from exponential phase cultures of T. denticola strains representing three serotypes, from defined T. denticola mutants defective in Msp (major surface protein) or PrtP lipoprotein protease complex (CTLP; dentilisin), and Escherichia coli strains expressing distinctly different T. denticola Msp. Extracts were subjected to Western immunoassays using archived human serum samples. Results: Human serum antibodies (immunoglobulin G class) recognized multiple protein bands in T. denticola strains. In the parent strain ATCC 35405, these included bands at 72-, 53-, 40-, and 30-kDa. Bands corresponding to Msp and the PrtP protease complex proteins were absent in isogenic msp and protease complex mutants, respectively. Individual human sera showed specificity for one or more Msp types. Conclusions: This is the first definitive report of human serum antibody responses to specific T. denticola antigens. T. denticola Msp and the proteins comprising the PrtP lipoprotein protease complex are expressed in vivo and are immunogenic in humans. Human antibody recognition of Msp exhibits strain specificity and is consistent with strain serotyping. These results demonstrate the utility of T. denticola isogenic mutants in characterizing host immune responses to periodontal pathogens.