IgG subclasses in gingival crevicular fluid from active versus stable periodontal sites.

Since IgG subclasses are common immunoglobulins associated with the periodontium and have different biological characteristics, these subclasses were measured in gingival crevicular fluid (GCF) from periodontally active (greater than or equal to 2 mm clinical attachment loss within three months of sample) versus clinically similar but stable or healthy sites. A sandwich enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies was performed to quantitate IgG subclass and albumin concentrations in serum and interproximal GCF samples from at least one each of the three disease categories from each of 20 periodontal maintenance patients. Although much variability existed among sites, mean IgG1 (p less than 0.05) and IgG4 (p less than 0.01) concentrations were higher in GCF from active periodontitis areas than stable sites, even though both had similar clinical characteristics. When IgG subclass concentrations were adjusted per mg albumin, both IgG1 and IgG4 levels in GCF from active sites were still significantly elevated over stable areas (p less than 0.05). Mean adjusted concentrations in GCF were generally greater than in serum, especially for IgG4 (active site GCF:serum = 24.2:1). GCF IgG4 concentrations may be useful as an indicator of the immunopathological changes which occur in active periodontitis.

In situ lymphocyte subpopulations from active versus stable periodontal sites.

The purpose of this study was to evaluate lymphocyte subset densities and distributions within gingival biopsies from active sites (greater than or equal to 2 mm clinical attachment loss within three months of biopsy) versus clinically similar but stable or healthy sites. Small interproximal gingival biopsies representing at least one of each of the above categories were obtained from each of 20 periodontal maintenance patients. Serial cryostat sections displaying a cross section of the gingiva were labeled with monoclonal antibodies for (1) pan T cells, (2) T cytotoxic/suppressor cells, (3) T helper/inducer cells and (4) pan B cells and were developed using an avidin-biotin-peroxidase system. Lymphocyte populations were enumerated in repeatable fields from the sulcular, middle and oral one-third of each section. Relative proportions of the same lymphocyte subsets were analyzed in peripheral blood samples from the same patients using direct immunofluorescence. Pan B cells were significantly more prevalent in infiltrates from active sites than in stable (P less than 0.05) or healthy (P less than 0.01) sites. The T/B cell ratio was also significantly lower in active than stable biopsies (P less than 0.05), and in active biopsies versus blood (P less than 0.05). The T helper/T suppressor cell ratio did not vary significantly between blood and any gingival tissue disease group or location, but a trend toward lower relative numbers of T helper cells in the sulcular infiltrates of active sites was noted. These results support the premise that active periodontal sites display elevated B cell populations and abnormal immune regulation possibly involving the T helper cell subset.

Induction of spheroplasts in Capnocytophaga ochracea.

A gentle technique for preparing spheroplasts of Capnocytophaga ochracea strain 25 is described. Cells in the exponential phase were washed with 1.0 M-NaCl, agitated in 1.0 M-NaCl for 2 h at 30 degrees C and exposed to lysozyme in a Tris/salts buffer, pH 7.0. This procedure resulted in 98% spheroplast formation with complete removal of the peptidoglycan layer as detected by both phase-contrast and electron microscopy in combination with chemical analysis.

Purification and chemical characterization of an exopolysaccharide isolated from Capnocytophaga ochracea.

Purification and chemical characterization of an immunosuppressive exopolysaccharide from Capnocytophaga ochracea strain 25 are described. This polysaccharide was extracted from spent culture medium by cold ethanol precipitation. Purification was accomplished by trichloroacetic acid and pronase treatments in combination with diethylaminoethyl-Sepharose and concanavalin A-Sepharose chromatography. Purity of the exopolysaccharide was ascertained by polyacrylamide gel electrophoresis using periodic acid--Schiff staining. The exopolysaccharide was free of protein, nucleic acid, and lipopolysaccharide, but contained large amounts of mannose with lesser quantities of glucose, galactose, glucuronic acid, and glucosamine.

Modulation of in vitro human lymphocyte responses by an exopolysaccharide from Capnocytophaga ochracea.

An exopolysaccharide (EP), purified from spent culture fluid previously inoculated with Capnocytophaga ochracea strain 25, suppressed in vitro human peripheral blood lymphocyte responses to the mitogen concanavalin A. EP was suppressive when added to cultures before the mitogen, but enhanced responses when added 24 or 48 hr after the mitogen.

Suppression of murine lymphocyte mitogen responses by exopolysaccharide from Capnocytophaga ochracea.

An extracellular polysaccharide was purified from culture supernatants of Capnocytophaga ochracea 25, a gram-negative bacillus associated with human periodontal disease. The extracellular polysaccharide suppressed in vitro mitogenic responses of murine splenic lymphocytes to concanavalin A and lipopolysaccharide. This suppression wad dose dependent, persisted up to 120 h, and was not caused by direct toxicity of the extracellular polysaccharide.

Effect of nonsterile versus sterile water irrigation with ultrasonic scaling on postoperative bacteremias.

The contribution of nonsterile water irrigation during ultrasonic scaling to postoperative blood stream contamination was evaluated. Thirty human volunteers were subjected to a random split-mouth technique comparing sterile and municipal tap water irrigation with ultrasonic root preparation on contralateral mandibular quadrants. Scaling was performed by a single operator using a premeasured quantity of irrigant. Postoperative blood samples were obtained immediately and cultured aerobically and anaerobically in tryptic soy broth and on BHI agar plates for enumeration of colony forming units. Positive blood cultures were inoculated onto selective media for presumptive identification. The difference in the bacteremia incidence after scaling with sterile water (50%) versus scaling with tap water (53.3%) was not significant. The degree of the bacteremias (less than 1 colony forming unit/ml) was similar between groups. Therefore tap water irrigation used in ultrasonic scaling did not appear to be a significant causative agent in postoperative bacteremias.

Naturally-occurring IgA antibodies to glycerol-teichoic acid in human saliva. Correlation with caries activity.

Human subjects were examined for caries activity and for naturally-occurring salivary IgA of glycerol-teichoic acid (TA) specificity by an enzyme-linked immunosorbent assay. Subjects free of active caries produced higher levels of anti-TA in their saliva than persons with one or more active carious lesions.

Modulation of murine lymphocyte mitogen responses by glycerol-teichoic acid.

Glycerol-teichoic acid (GTA) showed a modulatory effect on the in vitro response of murine splenocytes to the mitogens concanavalin A (Con A) and lipopolysaccharide (LPS) as measured by incorporation of 3H-thymidine. GTA inhibited the response to Con A when added prior to addition of the mitogen, while addition 24 hr after had no significant effect on the response. The degree of suppression was dose dependent in a range from 0.1-5 microgram GTA/culture. The spleen cell response to LPS was enhanced by GTA when added prior to the mitogen. Peak enhancement occurred at 1-2 microgram GTA/culture, depending on the time of addition. GTA added 24 hr after LPS produced no significant effect on mitogenesis. Addition of GTA alone to spleen cell cultures produced a slight suppression of DNA synthesis and was toxic at 10 microgram/culture if incubated at least 66 hr. GTA is bound to murine spleen cells as indicated by decreased passive hemagglutination inhibition activity of culture supernates.

Immunosuppression of anti-sheep erythrocyte responses by glycerol teichoic acid immune complexes.

Sprague-Dawley rats which produce "naturally occurring" antibodies to glycerol teichoic acid (GTA) displayed immunosuppression of anti-sheep erythrocyte plaque-forming cell and serum antibody responses when a single dose of lipid-free GTA was administered 24 h before immunization. Such suppression was enhanced by administering GTA complexed with anti-GTA immunoglobulin G. Animals fed a GTA-free diet produced no anti-GTA immunoglobulins and failed to show GTA-mediated immunosuppression under similar experimental conditions. However, when those animals were given GTA-anti-GTA complexes, suppression was evident. The results suggested antigenic competition mediated by immune complex-activated suppressor T cells. Lipid-free GTA did not stimulate serum antibody responses under the experimental conditions employed.

Rearing of gnotobiotic rodents by feeding a commercially-prepared infant formula.

A simple, inexpensive method for the rearing of germfree rats was developed. Germfree rats were fed from age 10 days to 40 weeks a diet consisting of commercially prepared Similac infant formula, water, and corn oil. Weight gains and gross appearances were comparable to littermate controls. Antibodies to bacterial teichoic acid were present in all controls while absent in rats fed the Similac diet.

Cyclic appearance of antibody-producing cells in the peripheral blood in response to a chemically-defiend polyglycerophosphate antigen.

Cyclic production of serum antibody and of plaque-forming cells (PFC) of polyglycerophosphate (PGP) specificity was observed in the peripheral blood of rabbits following a single injection of antigen. Individual animals were examined at 4-day intervals up to 28 days post-injection. Direct and indirect PFC displayed initial peaks at 4 days post-injection with an 8-day lag between the first and second peaks and a 12-day lag between the second and third peaks. Serum IgM rose to a peak 4 days after the initial PFC peak and gradually dropped to baseline levels. Serum IgG rose sharply to a peak at 4 days following the initial PFC peak and generally remained at elevated levels for the duration of the experiment. Cycling of background sheep erythrocyte PFC was also observed, but cycles were out of phase with the PGP cycles and with each other.

Environmental origin of natural antibodies to teichoic acid.

In an effort to determine the origin of natural antibodies to teichoic acid, rats were fed a sterile liquid diet free of detectable teichoic acid and virtually free of gram-positive bacteria. Both germ-free and conventional Sprague-Dawley rats raised on this diet failed to produce antibodies to polyglycerophosphate, whereas 100% of their counterparts fed the usual teichoic acid-containing diet did produce these antibodies. The intestinal flora was similar in both groups of animals. When the test animals were immunized intraperitoneally or orally with gram-positive bacteria, 100% displayed immunocompetency by producing significant levels of antibody. These results demonstrate the environmental nature of the antigenic stimulus for these antibodies and suggest the importance of food as the major source of stimulation. The experimental model described here furnishes a valuable tool for studies of immunologic responses where a single known specificity and a controlled system would be advantageous.

Cyclic antibody formation to polyglycerophosphate in normal and injected rats.

Normal adult Sprague-Dawley rats were bled serially over a 30-week period and their sera were examined for antibodies to polyglycerophosphate (PGP) by a standardized passive hemolysis test. Levels of "natural" antibodies to PGP fluctuated during this period with a majority of animals exhibiting pronounced cycling of serum antibody levels, however, the individual cycles were not synchronized with each other. Feeding of radiolabeled Gram-positive bacilli to these animals and examination of lymphoid tissue by liquid scintillation counting revealed that the antigen persisted mainly in the mesenteric lymph nodes. A second group of rats was injected i.v. with radiolabeled Gram-positive bacilli and tissues were examined for plaque-forming cells (PFC) of PGP specificity, and the sera were examined by passive hemolysis. Cycling of both anti-PGP antibodies and PFC became synchronized in the injected animals with peaks of serum antibody evident at 16 and 28 days post-injection and splenic PFC peaks at 4 and 16 days post-injection. Cycling was also observed in the mesenteric lymph nodes and bone marrow Examination of lymphoid tissue from the rats injected i.v. revealed that antigen introduced by this route also perisisted mainly in the mesenteric lymph nodes, This report demonstrates individual cycling of natural responses to environmental antigen and to the same determinant in secondary responses, indicating its importance as a regulatory mechanism.