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Udgivelsesdato: 1997-Jul Oral Prevotella and Capnocytophaga species, regularly isolated from periodontal pockets and associated with extraoral infections, secret specific immunoglobulin A1 (IgA1) proteases cleaving human IgA1 in the hinge region into intact Fab and Fc fragments. To investigate whether these enzymes are subject to inhibition in vivo in humans, we tested 34 sera from periodontally diseased and healthy individuals in an enzyme-linked immunosorbent assay for the presence and titers of inhibition of seven Prevotella and Capnocytophaga proteases. All or nearly all of the sera inhibited the IgA1 protease activity of Prevotella buccae, Prevotella oris, and Prevotella loescheii. A minor proportion of the sera inhibited Prevotella buccalis, Prevotella denticola, and Prevotella melaninogenica IgA1 proteases, while no sera inhibited Capnocytophaga ochracea IgA1 protease. All inhibition titers were low, ranging from 5 to 55, with titer being defined as the reciprocal of the dilution of serum causing 50% inhibition of one defined unit of protease activity. No correlation between periodontal disease status and the presence, absence, or titer of inhibition was observed. The nature of the low titers of inhibition in all sera of the IgA1 proteases of P. buccae, P. oris, and P. loescheii was further examined. In size exclusion chromatography, inhibitory activity corresponded to the peak volume of IgA. Additional inhibition of the P. oris IgA1 protease was found in fractions containing both IgA and IgG. Purification of the IgG fractions of five sera by passage of the sera on a protein G column resulted in recovery of inhibitory IgG antibodies against all three IgA1 proteases, with the highest titer being for the P. oris enzyme. These finding indicate that inhibitory activity is associated with enzyme-neutralizing antibodies.

Linkage analysis in separately ascertained families of probands with juvenile myoclonic epilepsy (JME) has previously provided evidence both for and against the existence of a locus (designated "EJM1"), on chromosome 6p, predisposing to a trait defined as either clinical JME, its associated electroencephalographic abnormality, or idiopathic generalized epilepsy. Linkage analysis was performed in 19 families in which a proband and at least one first- or two second-degree relatives have clinical JME. Family members were typed for seven highly polymorphic microsatellite markers on chromosome 6p: D6S260, D6S276, D6S291, D6S271, D6S465, D6S257, and D6S254. Pairwise and multipoint linkage analysis was carried out under the assumptions of autosomal dominant inheritance at 70% and 50% penetrance and autosomal recessive inheritance at 70% and 50% penetrance. No significant evidence in favor of linkage to the clinical trait of JME was obtained for any locus. The region formally excluded (LOD score < -2) by using multipoint analysis varies depending on the assumptions made concerning inheritance parameters and the proportion of linked families, alpha-that is, the degree of locus heterogeneity. Further analysis either classifying all unaffected individuals as unknown or excluding a subset of four families in which pyknoleptic absence seizures were present in one or more individuals did not alter these conclusions.

BACKGROUND: Cellular immunity has been implicated in periodontal destruction for over 25 years. Studies in the 1970s used lymphocyte transformation and lymphokine assays to establish a role for cell-mediated mechanisms in periodontal disease. Immunohistological studies subsequently showed that the formation of gingivitis followed a similar pattern to the formation of a delayed type hypersensitivity reaction. Further functional studies suggested that a T cell/macrophage immunoregulatory imbalance may exist locally in the periodontitis lesion and that this imbalance may be antigen specific. RECENT EVIDENCE: More recently, T cell subsets have been dichotomised on the basis of their cytokine profiles. In general, Th1 cells produce IL-2 and IFN-gamma while Th2 cells produce IL-4, IL-5 and IL-6. The major function of Th1 cells is to mediate delayed type hypersensitivity. In contrast-the major function of Th2 cells is to provide B cell help. HYPOTHESIS: A model for periodontal disease has now been developed based on this functional dichotomy which provides a framework for the study of cytokine profiles in periodontal disease. Early studies in this context have demonstrated a higher proportion of IL-4 producing cells in periodontitis tissues suggesting a role for Th2 cells in the progressive lesion. Clonal studies have shown that the selection of a particular cytokine profile is not antigen dependent and that differences may be due to the host susceptibility although this remains to be determined. CONCLUSION: These emerging data clearly establish a role for cell-mediated mechanisms in the control of periodontal destruction and raise the possibility that in the future cytokine therapy for the treatment of periodontal disease in susceptible subjects may become a viable option.

Porphyromonas gingivalis is generally recognized as a major periodontopathogen such that a study of T cell responses to this organism may help to elucidate immune regulation in periodontal disease. The aim of this study was to examine interleukin (IL)-4, interferon (IFN)-gamma and IL-10 production by P. gingivalis-responsive T cell lines and clones derived from the peripheral blood of two P. gingivalis-infected subjects with different disease expression and from the gingival tissues of one of the P. gingivalis-infected subjects. FACS analysis was used to determine the percentage of T cells staining positive for cytoplasmic IL-4, IFN-gamma and IL-10 and reverse transcriptase polymerase chain reaction (RT-PCR) was performed to determine the presence of mRNA for IL-4 and IFN-gamma in the T cell lines and clones. FACS analysis showed that virtually all the T cell lines and clones contained IL-4- and IFN-gamma-producing T cells. The RT-PCR results generally supported this trend. However, a higher percentage of cells in the clones derived from one subject produced IL-4 while a lower percentage produced IFN-gamma compared with the clones derived from the other subject. FACS analysis also demonstrated that the lines and clones derived from the two subjects showed differences in IL-10 production. This study has demonstrated that there may be differences in IL-4 and IL-10 production by the P. gingivalis responsive lines and clones derived from P. gingivalis-infected subjects with different disease expression. Any relationship to disease however, remains to be determined.

Udgivelsesdato: 1995-Oct Immunoglobulin A1 (IgA1) proteases secreted by oral Prevotella and Capnocytophaga species specifically cleave IgA1 at the same peptide bond in the hinge region, leaving intact monomeric Fab and Fc fragments. Assuming that Prevotella- and Capnocytophaga-induced Fab fragments of IgA1 expose a specific immunogenic neoepitope at the cleavage site, we established an enzyme-linked immunosorbent assay to measure human serum antibodies to this neoepitope as indirect evidence of in vivo activity of Prevotella and Capnocytophaga IgA1 proteases. The assay used a monoclonal antibody with specificity for the neoepitope, and the ability to block binding of the monoclonal antibody to the neoepitope was investigated. Absorption of sera with Prevotella melaninogenica-induced Fab fragments of IgA1 resulted in removal of antibodies blocking binding of the monoclonal antibody, whereas absorption with Fab fragments induced by bacterial IgA1 proteases of other cleavage specificities did not remove blocking antibodies. Consequently, we assume that the antibodies detected had been induced by a neoepitope an the Fab fragment of IgA1 exposed exclusively after cleavage with IgA1 proteases from Prevotella and Capnocytophaga, indicating in vivo activity of these IgA1 proteases. Evidence, though indirect, of in vivo activity of Prevotella and Capnocytophaga IgA1 proteases was present in 42 of 92 sera examined and in a significantly higher proportion of sera from adults with periodontal disease compared with control individuals. No correlation with disease was observed for the juvenile periodontitis groups.