The plaque-derived gram-negative microorganism Y4 identified as a member of the genus Actinobacillus, was tested for a soluble cytotoxic factor(s). Sonication or incubation of viable Y4 microorganisms in distilled water or normal human serum resulted in liberation of a soluble material which was cytotoxic in vitro for human polymorphonuclear leukocytes (PMNs). The Y4 soluble sonic extract was also cytotoxic to human peripheral blood monocytes. However, human lymphocytes, platelets, and fibroblasts, as well as rabbit, rat, and mouse leukocytes and chicken embryo fibroblasts, were not killed by exposure to the Y4 sonic extract. No hemolytic activity was detected in the Y4 sonic extract. Consequently, the factor(s) in the Y4 sonic extract was referred to as Y4 leukotoxin. The Y4 leukotoxin was inactive at 40C, heat sensitive (560C, 30 min), and inactivated by proteases. The cytotoxic effect of Y4 leukotoxin on PMNs was dose, time, and temperature dependent. The leukotoxin did not bind to viable PMNs at 40C but

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Sequences contributing to epitopes recognized by a panel of monoclonal antibodies (mAbs) against the Streptococcus mutans surface protein P1 were delineated by Western blot and enzyme-linked immunosorbent assay using a battery of deletion constructs and recombinant polypeptides. mAbs that recognize complex discontinuous epitopes reconstituted by combining the alanine-rich and proline-rich repeat domains and varying degrees of flanking sequence were identified as well as mAbs that bound epitopes contained within contiguous segments of P1. Cross-reactivity with SspA and SspB from Streptococcus gordonii is also reported. This information enables insight into the structure and function of a streptococcal adhesin and its correlates of protection and furthers our understanding of the immunomodulatory and bacterial-adherence inhibition activities of anti-P1 mAbs.

In the present study we identified a gram-negative anaerobic rod referred to as Y4 which was cytotoxic for human polymorphonuclear leukocytes. Y4 was isolated from dental plaque of a patient with juvenile periodontitis and presented most of the taxonomic characteristics of Actinobacillus species. Under experimental conditions, viable Y4 were cytotoxic for human peripheral blood polymorphonuclear leukocytes in serum-free cultures. Cytotoxicity was dependent on bacterial concentrations and was enhanced in the presence of a fresh or heat-inactivated (56 degrees C, 30 min) autologous serum. Leukotoxicity was independent of phagocytosis. Y4 leukotoxic effect was abolished when bacteria were heat treated (56 degrees C, 30 min) or when incubations were carried out at 4 degrees C instead of at 37 degrees C. The leukotoxicity was monitored by electron microscopy and biochemically by measuring lactate dehydrogenase indicator of cell viability. No cytotoxic effects of Y4 on human mononuclear cells, chicken fibroblasts, or mouse macrophages were detected under the conditions studied. Polymorphonuclear leukocytes may play an important role in the host defense against bacteria in periodontal disease. The cytotoxic effect of Y4 for polymorphonuclear leukocytes presented in this study is the first report of a direct offensive microbial vector in a plaque-derived microorganism and may prove to be relevant in the pathogenesis of juvenile periodontitis.

The aim of the present study was to analyze the relationship between attachment loss and clinical, microbiological, and immunological parameters in a group of 21 human subjects exhibiting poor response to previous periodontal therapy. All had been treated with periodontal surgery, tetracycline, and subsequent maintenance recalls to periodontists who, upon detection of disease progression, referred the subjects to our clinic. In our clinic, each subject received oral hygiene instruction and a thorough subgingival scaling and root planing utilizing as many appointments as necessary. Clinical indices, including gingival index, bleeding on probing, suppuration, plaque index, pocket depth, and duplicate measurements of attachment level from an acrylic stent, were collected at monthly intervals. Probing measurements were performed using the Florida Probe. When significant attachment loss (0.8-1.2 mm) was detected in at least 1 site, a bacterial sample was taken from that site and from a comparably deep, but non-progressing, control site. Microbial samples were enumerated by darkfield microscopy, on selective and non-selective media, and by predominant cultivable technique. Blood samples were also collected to determine antibody levels against potential pathogens. There was no difference in the amount of plaque present in sites gaining or losing attachment, but losing sites exhibited more bleeding and suppuration. 20 of the 21 subjects were tested; of these, 17 exhibited elevated serum antibody against one or more of the following microorganisms: Actinobacillus actinomycetemcomitans, Bacteroides, gingivalis, and Eikenella corrodens. However, few, if any, of the "classical" pathogens were detected in the plaque samples obtained at the time progressive disease was diagnosed. The only exception was Streptococcus intermedius, which occurred in slightly higher numbers in active sites.