Changes in aqueous immunoglobulin and albumin levels following penetrating keratoplasty.

The feline model of induced rejection of corneal allografts was employed to define the changes in the concentrations of immunoglobulins and albumin in the anterior chamber prior to, and concomitant with the rejection of the transplanted cornea. Fourteen animals received unilateral exchange corneal allografts. Aqueous humor obtained by anterior chamber paracentesis at regular intervals prior to and following the performance of the penetrating keratoplasties was analyzed for IgG, IgM and albumin concentrations using the micro enzyme-linked immunosorbant assay (ELISA). Two patterns of anterior chamber protein modulation were observed. Eight of the animals demonstrated a biphasic pattern in which both immunoglobulin and albumin concentrations were elevated two- to five-fold above presurgical values 14 days postkeratoplasty, returning to preoperative values by day 42. Three to 5 weeks after corneal rejection was induced increases in protein concentrations were observed that correlated with the appearance of clinical signs of rejection. A second, monophasic pattern of anterior chamber protein modulation following keratoplasty was observed in four of the animals. It was distinguishable from the biphasic pattern in that levels did not return to baseline values after the initial rise following keratoplasty until the rejection process was completed. The monophasic response was found to be characteristic of more rapid and vigorous corneal rejection. Examination of albumin to immunoglobulin ratios suggested that all changes in protein levels following keratoplasty were a result of increased influx of serum proteins into the anterior chamber, rather than due to local immunoglobulin synthesis.

Cellular localization of a Hsp90 homologue in Porphyromonas gingivalis.

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.

The relationship between gingivitis and the serum antibodies to the microbiota associated with periodontal disease in children with Down's syndrome.

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.

The relationship between gingivitis and colonization by Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans in children.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Humoral immunity to stress proteins and periodontal disease.

BACKGROUND: 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. METHODS: 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. RESULTS: Our studies demonstrated that, when evaluated by ANOVA, patients with higher anti-Hsp (Hsp90, DnaK, and GroEL) antibody concentrations tended to have significantly (P< or =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, P<0.0002). 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. CONCLUSIONS: 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.

Slot immunoblot assay for detection and quantitation of periodontal disease-associated microorganisms in dental plaque.

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.

Comparison of various detection methods for periodontopathic bacteria: can culture be considered the primary reference standard?

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.