Evidence for local production of antibodies to auto and non-self antigens in periodontal disease.

UNLABELLED: Autoimmune mechanisms may contribute to periodontal disease (PD) pathogenesis; autoantibody to collagen type 1 is produced at the periodontal site and local levels are found to be higher than in serum. OBJECTIVES: To find any evidence of autoimmune destruction in diseased periodontal tissues in patients with periodontitis. The study examines the relationship of antibodies to a self antigen collagen Type 1 and antigens from two periodontal pathogens namely Porphyromonas gingivalis (Pg), Actinobacillus actinomycetemcomitans (Aa) and a non-oral bacterium Bacteroides fragilis (Bf) in disease sites and in serum. MATERIALS AND METHODS: Granulomatous tissues from periodontally diseased sites and serum samples were obtained from 13 patients (15 sites) undergoing surgical therapy. Tissues were homogenized at 4 degrees C on Tris saline buffer [1 g (5 ml)-1], homogenate was centrifuged and the resultant supernatants were used in assays. Antibody to collagen and Aa, Pg and Bf in tissue eluates and serum were determined by competitive enzyme linked immunosorbant assay (ELISA) and conventional ELISA respectively using an alkaline phosphatase/p-nitrophenyl phosphate enzyme-substrate system. Sera from age and sex matched healthy subjects and pooled human serum were used as controls. Antibody (Ab) levels in tissues and serum were standardized by concomitant albumin assay. RESULTS: Level of antibodies to collagen type 1 in tissue was significantly higher than in serum (P = 0.0001). Antibody levels in tissue to Pg were significantly higher than in serum (P = 0.0271). Ab levels to both Aa and Bf in tissues and serum were not significantly different from each other. CONCLUSIONS: These findings confirm the process of the local production of antibodies to autoantigen namely collagen type-1 and to bacterial antigens in the granulomatous tissues housed within the periodontal lesions in patients with periodontitis.

The use of irradiated-crosslinked human collagen membrane in guided tissue regeneration.

Irradiated glutaraldehyde-crosslinked human collagen membrane was evaluated for its effects on new attachment formation in clinical trials, using the principle of guided tissue regeneration (GTR). 19 adult periodontitis patients with 52 matched bilateral periodontal defects received scaling and polishing with oral hygiene instruction. The bilateral periodontal defects were treated by reflecting a flap with collagen membrane (test) or flap reflection alone (control). Plaque (P1I) and gingival index (GI) scorings, probing pocket depth (PPD) and probing attachment level (PAL) along with classification of furcation involvement (FI) and bony defects were made at pre- and post surgery (6 weeks, 3 and 6 months). Improvement of P1I and GI scores was seen in both test and control sites following the surgical therapy. Reductions in PPD and PAL were significantly (p less than 0.001) more pronounced at 6 months in the test sites compared to the controls. The 2 Class I furcations in the graft-treated teeth showed complete resolution, while the corresponding furcations in the control teeth showed incomplete closure. The use of human collagen membrane based on the GTR technique for treatment of human periodontal defects provided greater gain of clinical attachment than when flap surgery alone was undertaken.

Freeze-dried, cross-linked bovine type I collagen: analysis of properties.

This study was undertaken to assess the physical and biological properties of freeze-dried cross-linked bovine type I collagen and to assess its potential for use in the guided tissue regeneration method of treatment of periodontal disease in human adult subjects. The modulus of elasticity, swelling ratio, and biodegradation rate were investigated. The collagen sponge was implanted subdermally into Sprague-Dawley rats and a histological study carried out at 2, 7, 21, 35, and 49 days post implantation. Growth of human gingival and periodontal ligament derived fibroblasts on collagen sponge was assessed, as well as the effect of bovine collagen supernatants upon gingival and periodontal fibroblast cultures. The physical properties of the collagen sponge were consistent with good handling qualities and, therefore, it was appropriate for use at a surgical site. The histological study demonstrated a reduction in thickness of the collagen at 21 days; at 35 days there was a hazy appearance of the collagen remnants; and at 49 days the graft material had been completely replaced with fibrous tissues. The in vitro response of human gingival and periodontal fibroblasts to bovine collagen showed that, after 21 days, confluent fibroblast growth was observed around and underneath the sponge. The effect of bovine collagen supernatants upon fibroblasts demonstrated an apparent proliferative effect of the supernatant with both gingival and periodontal ligament fibroblasts. However, the non-parametric Friedman test revealed no significant differences between dilutions or time points. The overall findings provide encouraging evidence of the safety of freeze-dried cross-linked bovine collagen sponge in the surgical treatment of periodontal disease.(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.

Autoimmunity to collagen in adult periodontal disease: immunoglobulin classes in sera and tissue.

The immunoglobulin class distribution of antibody to human collagen type I has been examined in sera and gingival extracts from patients with adult chronic periodontitis. Tissue extracts were made either by simple washing or ultrasonication. With either method, IgG and IgA antibodies to collagen were present in higher concentration in tissue extracts than in autologous serum when adjustment was made for dilution differences. No significant differences were found for IgM antibodies. Antibodies to human collagen type I are usually "natural antibodies" of the IgM class and, therefore, our findings suggest a class switch to IgG in inflamed gingivae, presumably due to prolonged antigenic stimulation.

Light and electron microscopic evaluation of biocompatibility, resorption and penetration characteristics of human collagen graft material.

This study was initiated to test the biocompatibility, resorption and penetration characteristics of human collagen graft material in vitro and in vivo using light (LM) and electron microscopy (EM). To study this relationship, pieces of glutaraldehyde cross-linked collagen sponges (1 x 1 x 0.5 cm), were: (1) cultured in sterile Petri dishes with human gingival fibroblasts and human periodontal ligament fibroblasts for 2 weeks; (2) implanted in subcutaneous pockets made in both thighs (total 20 sites) of 10 Sprague-Dawley rats for 7-56 days. The behaviour of the growth of the fibroblasts was studied by inverted light microscopy (LM), then tissue culture specimens were studied from without and within using low-temperature scanning electron microscopy (LTSEM). Blocks obtained from the graft sites of the rat were processed for LM and transmission EM. Long-term LM observations showed attachment and random orientation of cells on and around the collagen sponge in culture during the first 48 h. Between 7 and 14 days, the majority of the cells adjacent to the sponge were orientated at right angles to its margin with their long axes approximately parallel to each other. The LTSEM revealed that large numbers of HGF and HPLF grew onto the collagen sponges, but no cellular penetration to the middle of the sponge was seen. LM and TEM of the rat specimens showed a cellular reaction to the collagen graft, as well as slow resorption, and fibroblast invasion of the graft at 6-8 weeks. It was concluded that the human collagen graft was biocompatible with HGF and HPLF, with penetration first observed at 42 days post-implantation. In the in vivo study, the collagen underwent slow resorption over a period of 8 weeks.

Autoimmunity in periodontal disease.

Periodontal disease in characterized by the loss of the normal supporting tissues of the teeth and a humoral and cellular immune response to bacterial antigen of dental plaque which accumulates at the dento-gingival junction. This review considers the evidence for the existence of an autoimmune component of the host immune response, the possible origin of such a response and the way in which such a host response may contribute to the changes observed in the periodontium in the disease.

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.

Development and testing of a human collagen graft material.

Human Type I collagen was extracted from placenta using pepsin and salt fractionation. The collagen was characterized by SDS-PAG electrophoresis dispersed in acidic medium, freeze-dried, and cross-linked in an 0.25% glutaraldehyde solution pH 4.5 for 2 days. After washing for 7 days and freeze drying the resultant collagen sponge was tested with regard to mechanical, physical, enzymatic degradation properties and biological responses. The modulus of elasticity was found to be 289 +/- 10 g/mm2 and the sponge was insoluble in water, buffered saline, or tissue culture medium over a period of 6 weeks with swelling occurring at less than 5% of volume. The sponge had a high fluid binding capacity, amounting to 56 +/- 5 mL tissue culture medium per gram of dry weight. Bacterial collagenase produced slow degradation of the sponge with complete disappearance by 24 h only when high concentrations (200 units enzyme per mg of the collagen sponge) were used. Cytotoxicity studies using human gingival and periodontal ligament fibroblasts revealed less than 5% apparent cytotoxicity or proliferation. Subcutaneous implantation was followed by resorption and vascularization over a period of 6-8 weeks. It was concluded that the collagen sponge prepared from human Type I collagen has potential as a graft material in oral surgical procedures.

Antibody to collagen type I in gingival crevicular fluid.

Gingival crevice fluid (GCF) was collected from inflamed sites in 20 patients before and 6 weeks after treatment. Levels of IgG to human collagen Type I were measured in the GCF and autologous serum using an enzyme-linked immunosorbent assay and compared with levels in control sera. IgG antibody to collagen was detected in GCF at significantly (P less than 0.01) higher levels than in control sera, but these levels were not significantly (P greater than 0.05) different from those in autologous sera. The levels of IgG antibody in GCF and autologous sera did not alter significantly (P greater than 0.05) after treatment. IgG antibody to collagen Type I is present in GCF in the diseased and healing state.

Spontaneous lymphocyte proliferation in severe periodontal disease: role of T and B cells.

Spontaneous proliferation of T cells, B cells and unseparated lymphocytes was studied in patients with severe periodontal disease and control subjects. In the patient group only, spontaneous lymphocyte proliferation was reduced, whereas B cell proliferation was enhanced. The findings offer further support for the existence of a disturbance in immune regulation in patients with severe periodontal disease.

Association between HLA antigens and periodontal disease.

HLA-A, B and DR antigen frequencies were determined in three groups of periodontally diagnosed subjects: 49 patients with rapidly progressive periodontitis, 40 elderly subjects with minimal disease (considered as a resistant group) and 30 young subjects with minimal disease. The relative risk for HLA-A9 (previously reported to be associated with periodontal disease) was 15.5. HLA-A9 was present in 36.7% of the patients and 2.5% of the resistant group. HLA-A10 showed a significantly increased incidence in the resistant group (30.0%) compared to a non-periodontally diagnosed control population (9.0%), and was absent from the patient group. These findings provide additional evidence for the involvement of HLA-A9 in susceptibility to periodontitis, and suggest that A10 may play a role in resistance to the disease.

Cellular immunity to collagen in periodontal disease: role of T, B lymphocytes and adherent cells.

Peripheral blood lymphocytes of patients with periodontal disease and control subjects were fractionated into T enriched and B enriched lymphocyte populations and plastic adherent cells (PACs). T enriched and B enriched lymphocytes were cultured: (a) with human Type I collagen with varying concentrations of PACs and (b) without collagen, but with cells or supernatants from autologous and heterologous PACs which had been previously cultured with and without collagen. In patients with periodontal disease, both T enriched and B enriched blastogenic responses were higher than in control subjects, but B enriched cell responses of patients were highest. The addition of PACs had no significant effect upon the enriched T cell responses to collagen; the B enriched cell responses were enhanced in the presence of PACs, the maximum response occurring with the addition of approximately 10% PACs. T enriched cells did not appear to respond to the collagen co-cultured PACs or PAC supernatants; B enriched cells responded maximally to PACs co-cultured with collagen. Autologous PACs, co-cultured with collagen, induced higher responses than heterologous PACs similarly treated.

The value of systemically administered metronidazole in the modified Widman flap procedure.

This double-blind cross-over study was undertaken to assess the effect of systemically administered metronidazole when used as an adjunct to periodontal surgery for the treatment of moderate and advanced periodontitis. The effect of metronidazole was compared with that of placebo in patients undergoing modified Widman flap procedures in two areas of the same jaw which could be matched for type of tooth and severity of the periodontal disease. Clinical and microbiological parameters were examined prior to surgery and then 7 days, 1 month, and 3 to 6 months, postoperatively. The clinical parameters recorded were pocket depth (PD), Sulcus Bleeding Index (SBI), probing attachment level (PAL), and patients' preference and pain score. Subgingival plaque samples were studied with dark-field microscopy for differential bacterial count. Pocket depths and SBIs were reduced significantly at all stages, in both groups. Probing attachment levels increased at 7 days, to significant levels only in the metronidazole group, subsequently PALs decreased in both groups with no significant differences between the groups. Although the differential bacterial count altered markedly in both groups at all times, only the straight rod count at 1 month was significantly (P less than 0.05) lower in the metronidazole group. Metronidazole with surgery did not exert a significantly greater beneficial effect than placebo with surgery.

Antibody to collagen type I in periodontal disease.

Serum antibody levels to human collagen Type I were measured in 97 patients with periodontal disease (Russell Periodontal Index 1.0-7.0) and 57 control subjects (Periodontal Index less than 1.0) using an enzyme-linked immunosorbent assay, which had been standardized with antisera prepared against human collagen Type I in rabbits. Absorption studies were used to confirm the specificity of antibodies to human Type I collagen. Levels of antibody to Type I collagen detected in patients were higher (P less than 0.001) than in the control subjects.

Cellular and soluble mediator components of the local immune response to dental plaque.

The host immune response to dental plaque products leads to altered levels of activity by several types of cells involved in host defence. Cells of the periodontium which contribute to the supporting tissues of the teeth may be directly damaged by cellular attack, or their activity may be modulated by a range of soluble mediators released by host immune cells. These changes contribute to the modification of the tooth-supporting tissue which is seen in chronic inflammatory periodontal disease.

Impaired lymphocyte responsiveness to phytohaemagglutinin associated with the possession of HLA-B8/DR3.

An examination was made of blastogenic response to phytohaemagglutinin (PHA) in HLA-B8 and/or DR3 positive subjects and B8/DR3 negative individuals. Both B8 and DR3 antigens were associated with a depression of the response at all three doses of PHA used. The possession of both these antigens did not lead to a further depression of the response.

Mixed lymphocyte reaction supernatants exert varied effects upon gingival fibroblasts in vitro.

Supernatants from mixed lymphocyte cultures (MLRs) were tested for modulation of gingival fibroblast growth by both dye and 3H-thymidine uptake assays. Significant levels of inhibition of growth were observed with both assays with 24 and 72 hr MLR supernatants, but stimulation of growth was seen to occur, consistently, with 48 and 96 hr MLR supernatants as assessed by the dye uptake method. Fractionation of 24 hr supernatants by gel-filtration, on Ultrogel ACA 54 yielded fractions with enhanced growth inhibitory activity. The MLR supernatants would appear to contain mediators with opposing growth effects at different times during the culture.

Pressures recorded during periodontal pocket irrigation.

Pressures arising during periodontal pocket irrigation with 5 ml and 20 ml disposable plastic syringes, a peristaltic pump and a Waterpik pulsating irrigator were measured with an in-line transducer and recorder in 11 patients. Three types of needles were used with each device; disposable 15-gauge straight and angled needles and a 15-gauge angled reusable needle. Measurements were made with the needle tips just within, and 3 mm into the pocket. With both the 5 ml and 20 ml syringes and all needles, pressures of 100- to 500-mm mercury were recorded. With the peristaltic pump, pressures of 70- to 340-mm mercury were recorded with all needles. The Waterpik apparatus produced pressures of not more than 60-mm mercury in all cases.