Immunoglobulin isotype distribution of locally produced autoantibodies to collagen type I in adult periodontitis

Production of antibodies to collagen type I was analyzed by means of an enzyme-linked immunospot (ELISPOT) assay in patients with chronic adult periodontitis (AP) before and after periodontal hygiene treatment. Anti-collagen type I antibody-secreting cells were found among mononuclear cells enzymatically eluted from inflamed gingiva in 9 of 15 patients with untreated AP and in 4 of 14 hygiene-treated patients with a varied isotype distribution. A notably high prevalence of IgG and IgM isotypes was observed for the anti-collagen antibodies in untreated patients. With wide variation, chronic AP was characterized by a high frequency of spontaneous IgG and low numbers of IgA and IgM-producing cells. Periodontal hygiene treatment significantly reduced the number of IgA and IgM-secreting cells. Although AP is not an autoimmune disease in the accepted sense, our results indicate that local autoimmune reactions to collagen type I are common in untreated AP, implying an interplay between periodontal infection and autoimmunity.     

Caries development in children in relation to the presence of mutans streptococci in dental plaque and of serum antibodies against whole cells and protein antigen I/II of Streptococcus mutans

Twenty-eight children, aged 0.8-3.8 years, were studied with respect to the presence of mutans streptococci (MS) in dental plaque and the amount and avidity of specific serum IgG and IgM antibodies against whole cells and streptococcal protein antigen I/II (SA I/II) of Streptococcus mutans. The presence or absence of MS in dental plaque at the median age of 1.9 years predicted the caries development during a 2.7-year follow-up period with a sensitivity and specificity of 73 and 92%, respectively. The caries predictive value of a positive finding of MS at this age was as high as 92%. Almost all children had detectable amounts of serum IgG and IgM antibodies against whole cells and SA I/II of S. mutans, irrespective of the presence of detectable levels of this bacterium in dental plaque. These antibodies increased with age. The antibody levels did not differ significantly between children who were MS-negative or MS-positive. However, in MS-free children as well as in the whole study group, total specific and high-avidity antibodies of IgG class against whole cells correlated positively with antibodies against SA I/II. In MS-infected children such an association was observed for IgM but not for IgG antibodies. This different serum antibody profile in MS-negative and -positive children may be related to the mode of immunization with S. mutans.     

Detection of serum antibodies against cytomegalovirus, varicella zoster virus and human herpesvirus 6 in patients with recurrent aphthous stomatitis

There has recently been renewed interest in the possible role of viruses in recurrent aphthous stomatitis (RAS). In this study, sera from 22 patients with RAS, 24 patients with oral lichen planus (OLP) and 15 healthy controls were screened for IgG and IgM class antibodies to human cytomegalovirus (HCMV), varicella zoster virus (VZV) and human herpesvirus 6 (HHV-6). Commercially available ELISA and immunofluorescence kits were employed. There were no significant differences in the prevalence of IgG antibodies to HCMV, VZV or HHV-6 among the three patient groups. Similarly, there were no significant differences between the prevalence of HCMV and VZV IgM antibodies among RAS patients and controls. However, specific HHV-6 IgM was detected in 21 (95%) of the RAS patients and 17 (71%) of the lichen planus patients compared with 8 (53%) of the healthy controls. This difference between RAS patients and controls was statistically significant (P<0.01). These results do not support an aetiological role for HCMV or VZV in RAS but suggest possible involvement of HHV-6.     

Autoimmunity to collagen in adult periodontal disease

Although it has been documented that exogenous antigens of microbial origin are involved in the induction of the local inflammatory responses in human adult periodontitis (AP), endogeneous antigens may contribute to the chronicity of this common disease. In this study, we used the enzyme-linked immunospot (ELISPOT) test to enumerate antibody-secreting cells to human collagen Types 1-VI by cells isolated from the gingivae and peripheral blood of AP patients. Analyses of dissociated cells from gingivae of 39 AP patients revealed the presence of high numbers of cells that secrete antibodies to Type I collagen, and to a lesser extent, Type III. Although the majority of such cells produced specific antibodies of the IgG class, IgA- and IgM- anti-collagen -secreting cells were also detected. When compared to the total antibody-producing cells, the numbers of cells forming specific antibodies to collagen Type I were surprisingly high. In contrast, anti-collagen antibody-producing cells were rarely detected in the peripheral blood of patients with adult periodontal disease and only low levels of anti-collagen antibodies were present in the serum. The finding of local production of anti-collagen antibodies in AP suggests that autoimmunity may contribute to the pathogenesis of this common disease.     

Autoimmunity to collagen in adult periodontal disease

Although it has been documented that exogenous antigens of microbial origin are involved in the induction of the local inflammatory responses in human adult periodontitis (AP), endogenous antigens may contribute to the chronicity of this common disease. In this study, we used the enzyme-linked immunospot (ELISPOT) test to enumerate antibody-secreting cells to human collagen Types I-VI by cells isolated from the gingivae and peripheral blood of AP patients. Analyses of dissociated cells from gingivae of 39 AP patients revealed the presence of high numbers of cells that secrete antibodies to Type I collagen, and to a lesser extent, Type III. Although the majority of such cells produced specific antibodies of the IgG class, IgA- and IgM- anti-collagen -secreting cells were also detected. When compared to the total antibody-producing cells, the numbers of cells forming specific antibodies to collagen Type I were surprisingly high. In contrast, anti-collagen antibody-producing cells were rarely detected in the peripheral blood of patients with adult periodontal disease and only low levels of anti-collagen antibodies were present in the serum. The finding of local production of anti-collagen antibodies in AP suggests that autoimmunity may contribute to the pathogenesis of this common disease.     

Comparative immunogenicity and protective effect against dental caries of a low (3800) and a high (185,000) molecular weight protein in rhesus monkeys (Macaca mulatta)

The immunogenicity and protective effect of two peptides derived from the human oral bacterium Streptococcus mutans (serotype c) was examined. Furthermore, the effect of immunization was examined in monkeys previously given fluoride in their diet and which had developed a low incidence of dental caries when offered a human type of diet containing about 15 per cent sucrose. The 3800 peptide streptococcal antigen (SA) has two major antigenic determinants, similar to those in the 185,000 SA I/II. Immunization with 10 (or 1) micrograms of the 3800 SA, made up in an aluminium-hydroxide adjuvant, induced a consistent increase in serum IgG, IgM and IgA antibodies to SA I/II throughout the period of investigation. Salivary-IgA antibodies were only slightly raised. Sequential examination up to 76 weeks showed a significantly lower incidence of dental caries and a lower proportion of Strep. mutans in the immunized compared with sham-immunized, control monkeys. Thus immunization with the 185,000 or 3800 SA can almost completely prevent dental caries in rhesus monkeys which otherwise develop a low incidence of caries.     

Antibodies to Streptococcus mutans and immunoglobulin levels in children with dental caries

An immunological investigation of rampant and non-rampant caries was carried out in children aged 2.5–5.5 yr. Serum IgG, IgA and IgM classes of antibodies to Streptococcus mutans were detected and significant negative correlations were found between the DMFS and the IgG:IgA or IgG:IgM ratios. Immunological response against dental caries appears to be associated with the proportion of IgG to IgA and IgM classes of antibodies to Strep. mutans. Serum IgM, unlike IgG or IgA, concentrations showed a significant positive correlation with the DMFS and IgM concentration may be a measure of antigenic load in the child. The study emphasizes the importance of antibody class and age of evaluation of the immune responses in the mechanism of protection against dental caries.     

Serum antibody responses to Streptococcus mutans antigens in humans systemically infected with oral streptococci

Sera from patients with subacute bacterial endocarditis (SBE) due to Streptococcus mutans or other oral streptococci and from normal subjects were assayed by enzyme-linked immunosorbent assay for antibodies to defined S. mutans antigens. Antibodies of IgG and IgA isotypes to Ag I/II and Ag III were greatly elevated in S. mutans-SBE sera, and the IgA antibodies in 3 sera included both polymeric and monomeric forms. Elevated IgM and IgG anti-lipoteichoic acid and IgG and IgA anti-serotype c polysaccharide antibodies were also found. The sera of 4 of 6 patients infected with other oral streptococci also displayed antibodies to S. mutans Ag I/II. Sera of 3 patients infected with Streptococcus mitis or Streptococcus oralis, but none of the S. mutans-infected cases, showed elevated antibodies to human heart sarcolemma, and all SBE sera had elevated rheumatoid factor. These results suggest that the known surface protein antigens of S. mutans are immunodominant in humans, and are not likely to be heart cross-reactive.     

Humoral immunity in root caries in an elderly population. I.

IgA, IgG and IgM antibody activity (ELISA Units/ml) to Streptococcus mutans, Actinomyces viscous and Escherichia coli CF8 in serum, parotid saliva and whole saliva was measured using the amplified ELISA (a-ELISA) while the concentration (microgram/ml) of each isotype of immunoglobulin as well as albumin and lactoferrin, was determined using sandwich ELISAs. Selection of suitable reagents from those commercially available was based on specificity tests using purified human immunoglobulin; most polyclonal reagents required further absorption to attain class specificity. Cross-absorption studies indicated the absence of patient antibodies that were cross-reactive among the bacteria studied, except for IgM in some cases. Expression of response in ELISA Units (E.U.) per microgram of immunoglobulin, i.e. specific activity, revealed that IgG specific activity was significantly higher in parotid saliva than in either whole saliva or serum for all bacteria studied; serum and whole saliva did not differ except for the higher specific activity in whole saliva to E. coli. The value of one E.U. was determined using the Comparative Antibody-immunoglobulin Capture Assay (CACA). Using this novel method, we estimated that about 0.05 percent of serum IgA was specific for Streptococcus mutans, 0.008 for Actinomyces viscosus and 0.004 for Escherichia coli CF8. The percentage of specific IgM antibodies was higher than for IgA and IgG. The concentration of IgA anti-Streptococcus mutans, Actinomyces viscosus and Escherichia coli levels are approximately 92 ng/ml, 25 ng/ml and 16 ng/ml in whole saliva and 46 ng/ml, 9.4 ng/ml and 6.3 ng/ml in parotid saliva.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunity to self-antigens in periodontal disease.

Auto-antibody to collagen, previously detected in periodontal disease, may represent either a response to local tissue damage or be the manifestation of a disturbance of the host immune response induced by the periodontal flora and its products. In an effort to distinguish between these two hypotheses, this study was undertaken to determine circulating IgG auto-antibody levels in 41 periodontal-disease patients against 12 self-antigens (salmon DS-DNA, calf SS-DNA, human and bovine thyroglobulin, rabbit proteoglycan, horse myoglobin, bovine myosin, actin, fetuin, human transferrin, cytochrome C, and human Type I collagen) and compare them to those in 21 periodontal disease-free subjects. None of the detected IgG auto-antibody levels were significantly different between periodontal disease and control sera (Mann-Whitney U-test, P greater than 0.05) except for human Type I collagen (P less than 0.05). Fifty-six percent of patients and 38% of controls were "broad responders;" i.e., 50% or more of the auto-antibody levels were higher than the median values of the control group; however, these values were not significantly different using the chi-square test. It was concluded that the destruction of connective tissue components is the primary driving force in the induction of the enhanced auto-antibody response found in periodontal disease. This response is apparently secondary to the primary bacterial infection which remains the major etiologic event.     

Defensin-induced adaptive immunity in mice and its potential in preventing periodontal disease

The severity of periodontal disease is dependent on a combination of host, microbial agent and environmental factors. One strong correlate related to periodontal disease pathogenesis is the immune status of the host. Here we show that human neutrophil peptide (HNP) defensins or human beta-defensins (HBD), co-administered intranasally with the antigen ovalbumin (OVA), induce unique immune responses that if used with microbial antigens may have the potential to hinder the pathogenesis of periodontal disease. C57BL/6 mice were immunized intranasally with phosphate buffered saline (PBS) containing 1 micro g HNP-1, HNP-2, HBD1 or HBD2 with and without 50 microg OVA. At 21 days, isotypes and subclasses of OVA-specific antibodies were determined in saliva, serum, nasal wash, bronchoalveolar lavage fluid, and fecal extracts. OVA-stimulated splenic lymphoid cell cultures from immunized mice were assessed for interferon (IFN)-gamma, Interleukin (IL)-4 and IL-10. In comparison with mice immunized with only OVA, HNP-1 and HBD2 induced significantly higher (P < 0.05) OVA-specific serum IgG, lower, but not significant, serum IgM and significantly lower (P < 0.05) IFN-gamma. In contrast, HNP-2 induced low OVA-specific serum IgG and higher, but not significant, serum IgM. HBD1 induced significantly higher (P < 0.05) OVA-specific serum IgG, higher, but not significant, serum IgM, and significantly higher (P < 0.05) IL-10. The elevated serum IgG subclasses contained IgG1 and IgG2b.     

Extracellular matrix analysis of nifedipine-induced gingival overgrowth: immunohistochemical distribution

The purpose of this study was to demonstrate the localization of collagen types I, III, IV, V, VI and VII as well as the glycoprotein fibronectin in nifedipine-induced gingival overgrowth. The slices, after the use of indirect immunofluorescence (incubation with antibodies against these extracellular matrix components), showed a diffuse distribution with the anit-types I and III in the stroma and fluorescent staining of the basement membranes of the epithelium, blood vessels and nerves with collagen type IV antibodies. The increased number of vessels was localized near the surface of the lesion. Collagen tyep V - seen as a filamentous - and collagen type VI - as microfibrillar - components were also localized in the tissue, showing completely different patterns of distribution. Collagen type V appeared "crater"-like and type VI displayed a "honeycomb"-shaped structural model. The blood vessels were not stained but the area around their walls demonstrated an intense fluorescence with these antibodies. Collagen type VII showed a characteristic linear staining near to the epithelial basement membrance. In contrast to this, fibronectin localized with a varied intensity in the different areas of the tissues and presented a "could"-like structure. This shows differences between the matrix components in nifedipine-induced hyperplasia and confirms the heterogeneity of the matrix in health and in gingival alterations.     

Immune responses to implanted human collagen graft in rats

Immunity to collagen implants may be mediated by cellular and humoral immune responses. To examine the possibility of such immunological reactivity and crossreactivity to collagen, 39 Sprague-Dawley rats (female, 10 weeks old, approximately 250 g wt) were implanted subcutaneously at thigh sites with crosslinked, freeze-dried human placental type I collagen grafts (4 x 4 x 2 mm) which had been irradiated (520 Gray) or left untreated. Blood was obtained by intracardiac sampling prior to implantation or from normal rats, and at various times afterwards when the animals were sacrificed. The sera from these animals were examined for circulating antibodies to human, bovine and rat tail (type I) collagens by enzyme-linked immunosorbent assay (ELISA). Also, the lymphoblastogenic responses of spleen lymphocytes from the irradiated collagen-implanted animals were assessed in culture by measuring thymidine uptake with autologous and normal rat sera in the presence of human and bovine type I collagens. Implantation of the irradiated and non-irradiated collagen grafts in rats led to a significant increase in the level of circulating antibodies to human collagen. Also antibody to bovine and rat tail collagens was detectable in the animals implanted with irradiated collagen grafts but at a lower level than the human collagen. There was a raised lymphoblastogenic response to both human and bovine collagens. The antibody level and lymphoblastogenesis to the tested collagens gradually decreased towards the end of the post-implantation period.     

Streptococcus cricetus and Streptococcus rattus bind to different segments of collagen molecules

Strains of Streptococcus cricetus and Streptococcus rattus exhibited striking differences in their ability to bind to different types of collagen. For example, S. cricetus AHT bound in highest numbers to hydroxyapatite (HA) treated with human type V collagen, while rat type I collagen was ineffective. In contrast, human type V collagen was least effective in promoting attachment of S. rattus LB-1, while treatment with rat or human type I collagen was effective. Adsorption of both species to human type I collagen-treated HA showed a high correlation with a Langmuir model. Estimates of adsorption parameters indicated there were greater numbers of binding sites with higher affinity for S. rattus LB-1 than for S. cricetus AHT. Treatment of HA with either the alpha 1 (1) or alpha 2 (1) polypeptide chains of collagen was effective in promoting adhesion of S. rattus LB-1 cells. In contrast, the alpha 2 (1) chain was more effective than the alpha 1 (1), chain for S. cricetus AHT. These observations indicate that S. cricetus AHT and S. rattus LB-1 cells bind to different segments of collagen molecules. Adhesion of both species was also promoted by collagen-rich fractions of human dentin.     

Specificity of antibodies present in human periapical lesions

Various classes of immunoglobulins have been found in human periapical lesions. The specificity of secreted antibodies against antigens egressing from the root canal system has yet to be thoroughly investigated. The purpose of this study was to test the specificity of antibodies present in human periapical lesions. Human periapical biopsies were removed and cultured as organ culture explants. Antibodies present in the lesions were extracted in the cell culture fluids. A modified enzyme-linked immunosorbent assay was used to determine the presence, type, and concentration of different classes of antibodies against a number of commonly found bacterial species present in the root canal system. The data show the presence of specific antibodies (IgG, IgM, and IgA) against all 16 microorganisms tested. Peptostreptococcus micros, Actinomyces israelii, Staphylococcus intermedius, and Fusobacterium nucleatum produced significantly high levels of IgG antibodies in these lesions.     

Effect of phase I periodontal therapy on anti-cardiolipin antibodies in patients with acute myocardial infarction associated with chronic periodontitis

Background: Serum anti‐cardiolipin (aCL) antibodies are prevalent in patients with periodontal diseases. These increased aCL concentrations were identified to play a modulating role in cardiovascular diseases. The present study aims to explore the effect of phase I periodontal therapy on immunoglobulin (Ig)M and IgG aCL antibodies in patients with acute myocardial infarction (AMI) associated with chronic periodontitis. Methods: A cross‐sectional randomized clinical study was conducted within two groups comprising a sample size of 72 patients (n = 36 each). Group 1 had clinical features of AMI, and group 2 had clinical features of AMI associated with chronic periodontitis. After a thorough clinical and oral examination, the plaque index (PI), gingival index (GI), probing depth (PD), and clinical attachment loss (AL) were recorded. Serum sample collection by venipuncture was done for estimation of serum IgM and IgG aCL concentration by using an enzyme‐linked immunosorbent assay method. In group 2, phase I periodontal therapy was performed, and clinical and biochemical parameters were reanalyzed after 1 month. Results: In group 2, the mean PI, GI, PD, clinical AL, and serum IgM and IgG aCL antibody levels were significantly higher than in group 1 patients. In addition, study results showed significant alterations in concentrations of serum IgM (P = 0.008) and IgG (P <0.001) aCL along with periodontal parameters after phase I periodontal therapy. Conclusion: The phase I periodontal therapy altered levels of serum IgG and IgM aCL antibodies in patients with AMI associated with chronic periodontitis.     

Detection of, and anti-collagen antibody produced by, CD5-positive B cells in inflamed gingival tissues

This study was performed to investigate the frequency and distribution of CD5-positive (CD5+) B cells in inflamed gingival tissues using flow cytometric and immunohistochemical analyses. The ability of CD5+ B cells to produce anti-type I collagen antibody was also examined. CD5+ B cells expressed "low" fluorescence intensity in the peripheral blood of both healthy subjects and patients with adult periodontitis. However, in inflamed gingival tissues the intensity of this surface marker was high. The percentage of B cells bearing CD5 surface marker was statistically higher in gingiva than in peripheral blood obtained from both the patients and healthy subjects. These CD5+ B cells were observed in gingival subepithelial connective tissues from the bottom to the middle of the periodontal pocket. This area showed destruction of collagen fibers and dense cell infiltrations. Anti-collagen IgG antibody level in patients' gingival crevicular fluids (GCF) was higher than that in sera from healthy subjects, and slightly higher than in autologous sera. IgM anti-collagen antibody in GCF was lower than in autologous sera and in sera from healthy subjects. EBV-transformed CD5+ B cells produced considerably more IgM and IgG antibody to collagen than CD5- B cells. Therefore CD5+ B cells may contribute to the pathogenesis of inflamed gingival tissues.     

Salivary antimicrobial proteins and mutans streptococci in tonsillectomized children.

Whole saliva from 53 children who had been tonsillectomized when they were younger than 4 years old was analyzed for selected antimicrobial proteins and oral mutans streptococci 3-4 years after the operation. The results were compared with those from age- and gender-matched control children with no history of tonsillectomy. The salivary analyses comprised both immune (total IgA, IgG and IgM) and selected nonimmune (lactoferrin, myeloperoxidase, salivary peroxidase) antimicrobial proteins. Specific IgA and IgG antibodies against viral antigens (adeno-, cytomegalo-, respiratory syncytial- and Epstein-Barr-viruses) and against Streptococcus mutans cells were quantitated in both groups. The tonsillectomized children had statistically significantly higher concentrations of all immunoglobulin isotypes (P 0.001) as well as of lactoferrin (P less than 0.005), and myeloperoxidase (P less than 0.001) in saliva. However, no differences were found in the numbers of cariogenic mutans streptococci or in the total oral aerobic flora. In line with the streptococcal counts, no differences existed in anti-S. mutans IgA or IgG titers between the groups. Most antibodies against viruses, especially of IgG isotype, were significantly (P less than 0.001) higher in saliva of tonsillectomized children than in that of the controls. The results suggest that, within a long run, the humoral immune status of human saliva is not weakened by tonsillectomy. Also, mainly serum-derived antimicrobial proteins (myeloperoxidase, lactoferrin, IgG) exist in high concentrations in whole saliva after tonsillectomy.     

Evaluation of serum anti-cardiolipin and oxidized low-density lipoprotein levels in chronic periodontitis patients with essential hypertension

BACKGROUND: The aim of the present study was to investigate whether chronic periodontitis caused the elevated levels of anti-cardiolipin antibodies (anti-CL) and oxidized low-density lipoprotein (oxLDL) in subjects with essential hypertension. METHODS: Seventy-two subjects were categorized as healthy controls, subjects with essential hypertension and periodontal health (healthy-hypertension group), subjects with essential hypertension and gingivitis (gingivitis-hypertension group), or subjects with essential hypertension and chronic periodontitis (periodontitis-hypertension group). Individuals with essential hypertension who had been taking antihypertensive medication > or =2 years were included in the present study. The presence of supragingival plaque, bleeding on probing (BOP), probing depth (PD), and clinical attachment level were recorded, and blood samples were collected. Serum immunoglobulin M (IgM) and immunoglobulin G (IgG) anti-CL and oxLDL levels were assessed by enzyme-linked immunosorbent assay. For IgM and IgG anti-CL assays, positive tests were defined as > or =15 IgM phospholipid units and > or =10 IgG phospholipid units, respectively. RESULTS: The mean IgM anti-CL level and the prevalence of subjects positive for IgM anti-CL were significantly higher in the periodontitis-hypertension group compared to the other groups (P = 0.001). No significant differences were observed in the mean IgG anti-CL and oxLDL levels or in the number of subjects positive for IgG anti-CL and positive for IgM or IgG anti-CL among the study groups. The Pearson correlation analysis revealed positive correlations between IgM anti-CL levels and supragingival plaque, BOP, and PD scores. CONCLUSIONS: Chronic periodontitis might play a causal role in the elevated serum levels of anti-CL antibodies in individuals with essential hypertension. These elevated anti-CL levels that are due to chronic periodontitis might contribute to an increased risk for atherosclerosis in individuals with essential hypertension.     

Initial characterization of neutral proteinases from oral spirochetes

Intermediate size oral spirochetes were isolated and cultured from subgingival plaque of periodontitis patients utilizing a membrane separation and rifambin selection technique. Neutral salt extracts of the spirochetes were assayed for proteolytic activity against collagen types I and IV, gelatin, and synthetic elastase and trypsin substrates. Type IV collagen obtained from lens capsule basement membrane was degraded to small peptides by the spirochete proteinases. No marked degradation of type I collagen was found when incubations were performed at 25°C. The extracts were able, however, to activate latent collagenase obtained from human gingival fibroblasts and to degrade further the ¼ and ¾ fragments resulting from the collagenase cleavage. Denatured collagen was also degraded by the extracts. High trypsin-Iike activity and relatively lower elastase-like enzyme activity were also present in spirochete extracts. The results show that oral spirochetes have a potential for degradation of several proteins and that they may, therefore, have an active role in tissue destruction during periodontal disease.