Subantimicrobial dose doxycycline as an adjunct to scaling and root planing: post-treatment effects.

BACKGROUND/OBJECTIVE: Subantimicrobial dose doxycycline (SDD 20 mg bid) plus scaling and root planing (SRP) significantly improved clinical attachment level (CAL) and reduced probing depth (PD) compared with placebo plus SRP in a double-blind, placebo-controlled, multicenter study of patients with adult periodontitis (AP). In a study conducted as a follow-up, the post-treatment effects of SDD were assessed in patients who completed the SRP study. METHODS: The SRP study was a 9-month, active-treatment study and the follow-up was a 3-month, no-treatment study. In the SRP study, tooth sites in qualifying quadrants were scaled and root planed and patients were randomized to receive twice daily SDD 20 mg or placebo. In the follow-up, patients received no study drug; investigators and patients remained blinded to the previous treatment group assignments. Efficacy measures included the change in CAL and PD from baseline values determined at the start of the SRP study in tooth sites stratified by baseline PD (i.e., 0-3 mm, 4-6 mm, > or =7 mm). Safety was evaluated using adverse event data and the results of clinical laboratory tests, oral pathology examinations, and microbiological assessments. RESULTS: Within each disease stratum, the incremental improvements in PD and CAL demonstrated in the SDD group over 9 months of active treatment were maintained through 3 additional months of no treatment. Treatment cessation did not result in an accelerated regression of periodontal health. No differences in the incidence of adverse events (including those related to infection) or laboratory or microbiological parameters were noted between the SDD group and the placebo group. CONCLUSIONS: The administration of SDD 20 mg bid for a period of up to 9 months is not associated with rebound effects or delayed or negative after-effects for a 3-month period after cessation of therapy.

Expression of stromal-derived factor-1 is decreased by IL-1 and TNF and in dermal wound healing.

Stromal-derived factor-1 (SDF-1) is a CXC chemokine that is believed to be constitutively expressed by stromal cells of numerous tissues. In this report, we demonstrate that dermal fibroblasts and vessels of noninflamed tissues express SDF-1. Unexpectedly, we found that expression of SDF-1 is regulated by inflammation. Expression of SDF-1 by primary cultures of human gingival fibroblasts is potently inhibited by activated macrophages via secretion of IL-1alpha and TNF-alpha. Levels of SDF-1 mRNA also decrease in acutely inflamed mouse dermal wounds. We propose that SDF-1 functions as a homeostatic regulator of tissue remodeling, whose expression stabilizes existing dermal architecture.

Treatment with subantimicrobial dose doxycycline improves the efficacy of scaling and root planing in patients with adult periodontitis.

BACKGROUND: In a previous study, subantimicrobial dose doxycycline (SDD) significantly improved clinical parameters associated with periodontal health in patients with adult periodontitis (AP) when used as an adjunct to a maintenance schedule of supragingival scaling and dental prophylaxis. In this double-blind, placebo-controlled, parallel-group, multicenter study, the efficacy and safety of SDD were evaluated in conjunction with scaling and root planing (SRP) in patients with AP. METHODS: Patients (n = 190) received SRP at the baseline visit and were randomized to receive either SDD 20 mg bid or placebo bid for 9 months. Efficacy parameters included the per-patient mean changes in clinical attachment level (CAL) and probing depth (PD) from baseline, the per-patient percentages of tooth sites with attachment loss (AL) > or = 2 mm and > or = 3 mm from baseline, and the per-patient percentage of tooth sites with bleeding on probing. Prior to analysis, tooth sites were stratified by the degree of disease severity evident at baseline RESULTS: In tooth sites with mild to moderate disease and severe disease (n = 183, intent-to-treat population), improvements in CAL and PD were significantly greater with adjunctive SDD than with adjunctive placebo at 3, 6, and 9 months (all P <0.05). In tooth sites with severe disease, the per-patient percentage of sites with AL > or = 2 mm from baseline to month 9 was significantly lower with adjunctive SDD than with adjunctive placebo (P<0.05). Improvements in clinical outcomes occurred without detrimental shifts in the normal periodontal flora or the acquisition of doxycycline resistance or multiantibiotic resistance. SDD was well tolerated, with a low incidence of discontinuations due to adverse events. CONCLUSIONS: The adjunctive use of SDD with SRP is more effective than SRP alone and may represent a new approach in the long-term management of AP.

Tooth-related issues.

Several conditions exist around teeth that may predispose the periodontium to disease. These situations may occur as a result of the condition or position of teeth or as a result of tooth treatment. In certain cases these tooth-related factors may contribute to the initiation of periodontal disease. While the etiology of periodontal disease is bacterial, factors that enhance bacterial accumulation or allow the ingress of bacteria into the periodontium should be considered in the classification and diagnosis of periodontal diseases. This is because many times these tooth-related issues can cause site-specific problems that require treatment in an otherwise intact periodontium. Several factors related to tooth/root anatomy, restorative, and endodontic considerations have been associated with gingival inflammation, attachment loss, and bone loss. These factors will be reviewed as they relate to their potential to promote damage to the periodontium.

Fibroblasts as sentinel cells. Synthesis of chemokines and regulation of inflammation.

In this commentary, we have suggested that the fibroblast should be considered a sentinel cell. This concept is based on the fibroblast's ability to function both as a structural element and as a vital immunoregulatory cell. In some tissues, these capabilities may be ascribable to subsets of fibroblasts, rather than to some of the general fibroblast populations. The pioneering research of Xia et al, as well as that of others, highlights the need to explore the importance of fibroblasts as playing critical roles in disease. Emerging concepts regarding tissue-specific fibroblasts and fibroblast heterogeneity need to be considered in studies of their biosynthetic capabilities. Of special importance is the recent insight that the NF kappa B/RelB family of transcription proteins have apparently different regulatory roles in fibroblasts and hematopoietic cells. Therefore, with regard to therapeutic strategies targeting molecules such as RelB, caution should be exercised as their interruption may have very different consequences in macrophages compared with fibroblasts. For example, inhibition of RelB in macrophages may well prevent enhanced chemokine expression, whereas in fibroblasts, a critical governor for preventing chemokine expression would be lost. Overall, this could lead to exacerbation of inflammation rather than to an attenuation of the process.

CD40 mediated activation of gingival and periodontal ligament fibroblasts.

CD40 is a 50 kDa transmembrane protein important for regulating B lymphocyte proliferation and differentiation. This novel activation antigen is primarily expressed by hematopoietic cells including B lymphocytes, follicular dendritic cells, and monocytes. Recently, human fibroblasts from a variety of tissues were shown to display CD40; however, its function was unknown. Cellular responses mediated by CD40 are naturally triggered by its counter-receptor, the CD40 ligand, which is displayed on activated T cells, mast cells, eosinophils, basophils, and B lineage cells. This study investigated the functional significance of CD40 expression on periodontal fibroblasts, in the context of periodontal inflammation. The experiments reported herein demonstrate constitutive CD40 expression on cultured periodontal ligament (PDL) and gingival fibroblasts. Interestingly, cells of gingival origin displayed up to 13-fold higher constitutive levels of CD40, versus fibroblasts from PDL. Interferon gamma (IFN gamma) treatment enhanced CD40 expression on PDL and gingival fibroblasts, with up to 61-fold induction of expression. Immunohistochemical staining was used to detect CD40 on fibroblastic cells in both normal and acutely inflamed gingival tissue. Expression of CD40 in inflamed tissue was significantly higher than in uninflamed tissue. Western blot analysis of anti-CD40 triggered cells revealed the induction of tyrosine phosphorylation on a 50 kDa protein in PDL and gingival fibroblasts. These results indicate that CD40 is an active signaling conduit in periodontal fibroblasts. This concept was further substantiated by the fact that CD40 engagement stimulated interleukin 6 (IL-6) production by gingival fibroblasts, but not periodontal ligament fibroblasts. Overall, these results demonstrate that CD40 on periodontal fibroblasts may functionally interact with CD40L-expressing cells. This CD40/CD40L interaction can stimulate fibroblast activation and synthesis of the proinflammatory cytokine IL-6.

Fibroblast heterogeneity in the periodontium and other tissues.

Fibroblasts are the major resident cells which inhabit the periodontal tissues. As such, they are crucial for maintaining the connective tissues which support and anchor the tooth. Little is known of their origins, synthesis of regulatory cytokines and growth factors in health and disease, and importance in soft tissue regeneration. An emerging concept is that fibroblasts are not homogeneous, but instead consist of subsets of cells which can regulate bone marrow-derived cells such as T lymphocytes. Fibroblasts can be separated into subsets on the basis of morphology, size and expression of intermediate filaments as well as collagen subtypes. Differential surface marker expression has also been a key feature to distinguish fibroblast subsets from many tissues. Antigens such as Thy-1, class II MHC, and C1q are among those surface proteins which have been employed successfully to separate fibroblasts. Importantly, these fibroblast subsets are not only antigenically diverse, but also possess distinct functions. Thy 1+ pulmonary fibroblasts can display class II MHC antigens, synthesize IL-1 and can activate T lymphocytes, whereas the Thy 1+ subset is devoid of these functions. Recently, fibroblasts from the human orbit have also been shown to be separable on the basis of Thy 1 surface marker expression. Fibroblasts derived from human gingiva and periodontal ligament also appear to be composed of subsets with a heritable pattern of surface markers which will permit their separation into functional subpopulations. This paper will review findings of fibroblast heterogeneity in periodontal and other tissues. Evidence will be presented for the use of surface markers to delineate functional subsets. The ability to discriminate subsets of fibroblasts will aid in studies of periodontal disease pathogenesis and wound healing.

Fibroblast heterogeneity in the healing wound.

Although fibroblasts are traditionally described as static cells providing framework and support for tissues, there is an accumulating body of evidence showing that fibroblasts are a dynamic cell type which exist in functionally and morphologically heterogeneous subpopulations. Fibroblast subsets have been shown to play a critical role in the production and regulation of extracellular matrix components, in wound repair and regeneration, and have been implicated in the pathogenesis of fibrotic conditions. We have reviewed the evidence supporting heterogeneity of fibroblasts from pulmonary, periodontal, and dermal tissues. In addition, we will explore the role fibroblast subpopulations may play in the complex process of wound repair and regeneration.

Comparison between mechanical cleaning and an antimicrobial rinse for the treatment and prevention of interdental gingivitis.

This study compared the efficacy of an antimicrobial mouthrinse (0.12% chlorhexidine gluconate) plus toothbrushing (mouthrinse group), mechanical interdental cleaning plus toothbrushing (mechanical group), and toothbrushing alone (control group), at reducing and preventing interdental gingival inflammation. 92 male subjects were examined for interdental inflammation using the Eastman interdental bleeding index at baseline, then monthly for 3 months after using one of the above oral hygiene regimens. The mechanical cleaning group had significant reductions in bleeding sites compared to baseline at 1 month (56.90% versus 13.17%) that persisted throughout the study (2 months = 6.65%, 3 months = 5.70%). The other regimens showed no significant bleeding reduction at any time point in the study. The mechanical interdental cleaning group showed improvement over baseline at 1 month with the full benefit apparent after 2 months. The effect of location in the mouth on bleeding reduction was also assessed. The % of posterior sites which bled was always higher than anterior sites. Analysis of maxillary versus mandibular, and buccal versus lingual sites showed no significant differences. Additional observations of the data demonstrated that sites which bled at baseline were more likely to stop bleeding in the mechanical cleaning group. Also, sites which did not bleed at baseline were unlikely to bleed subsequently when mechanical cleaning was used. Neither of these observations were true for the other cleaning regimens. These data show that only mechanical interdental plaque removal combined with toothbrushing is effective at reducing or preventing interdental inflammation. This underscores the importance of instituting mechanical interdental cleaning to eliminate interdental inflammation.

CD4-class II major histocompatibility complex interaction does not enhance killing by a class I-restricted CD4+CD8+ cytotoxic T cell clone.

As unusual tumor-specific cytotoxic T lymphocyte (CTL) clone was isolated which expressed both CD4 and CD8 molecules. The target cells for this CTL can be induced to express either class I major histocompatibility complex (MHC) alone (with dimethylsulfoxide) or both class I and class II MHC (with interferon-gamma). Lysis of the tumor target depends on expression of class I MHC molecules, but does not require expression of class II MHC molecules. Furthermore, the lysis of target cells expressing both class I and class II is inhibited only by antibodies to class I (Kd), and not by antibodies to class II, demonstrating that the T cell receptor is class I restricted. We have used this CTL to assess the role of the interaction between CD4 and class II MHC in the absence of a class II-restricted T cell receptor. Our data indicate that CD4-class II interaction does not contribute to recognition by T cells in the absence of binding of the T cell receptor to class II molecules.

Examiner reliability for an invasive gingival bleeding index.

This investigation was undertaken to determine the intra- and inter-examiner reliability of the method of stimulation for bleeding used in the Eastman interdental bleeding index. 26 subjects were examined twice, 1 h apart, by either a single examiner or 2 examiners in each half of their mouths, for the presence bleeding after stimulation with a wooden interdental cleaner. Scores were tabulated and intra- and inter-examiner % agreements and kappa-coefficients calculated. Z-tests were performed on the pairs of agreement statistics to check for significant differences. Overall, intra-examiner agreement statistics were high (91.3% to 93.1% agreement; 0.79 to 0.86 kappa-coefficient). Further breakdowns of the data into facial and lingual sites by arch and location (anterior or posterior) resulted in similar levels of reliability, with no significant differences within examiners. The overall inter-examiner agreement statistics were good (82.8% to 87.6% agreement; 0.62-0.75 kappa coefficient). When inter-examiner data were analyzed at facial or lingual sites by arch and location, a significant difference existed in reliability for mandibular posterior lingual sites, but reliability was high in all other areas. These data demonstrate a high level of reproducibility for this method, which suggests that the Eastman interdental bleeding index is suitable for clinical trials and epidemiologic studies of interdental gingivitis.

Mechanism of cytolytic T lymphocyte killing of a low class I-expressing tumor.

Many tumors have been shown to express minimal levels of class I MHC Ag, which makes them more resistant to recognition and lysis by cytolytic T lymphocytes. Line 1, a BALB/c spontaneous lung carcinoma, normally expresses very low levels of class I Ag, but expression can be increased 50-fold by treatment with agents such as DMSO or IFN-gamma. Because class I Ag serve as restricting elements for cytolytic T cell recognition of tumor Ag, we wished to determine if cytotoxic T lymphocytes could play a role in the immune response to this type of class I low, but inducible, tumor. After immunization in vivo and restimulation of splenic cells in vitro we were able to generate T cell clones that lysed line 1 cells induced to express high levels of class I, but did not lyse uninduced, low class I expressing line 1 cells in short term (6-h) 51Cr release assays. Paradoxically, incubation of the T cells with uninduced class I low line 1 cells for a few days resulted in complete destruction of the tumor cells. We demonstrate that the T cells, stimulated by the tumor cells, produce IFN-gamma, which in turn induces class I expression on the line 1 cells making them susceptible to lysis by the T cell clone. This suggests that a positive feedback reaction can occur in generating a response to this and perhaps other inducible tumor cell lines.

Class-I MHC expression in the mouse lung carcinoma, line 1: a model for class-I inducible tumors.

We have examined the expression and biological effects of class-I MHC molecules on the immune response to the line I lung carcinoma. The line I system is of interest because these tumor cells have very low constitutive levels of class-I molecules but can be induced to express levels found on spleen cells, by culturing the cells with agents such as dimethylsulfoxide (DMSO) or interferon gamma (IFN-gamma). This induction is significant immunologically, since induced cells can be lysed very effectively by cytotoxic T lymphocytes (CTL), whereas the uninduced cells cannot. CTL clones that are reactive with line I cells have been generated and used in vitro and in vivo, to examine the interactions of T cells with line I. We have shown that the expression of class I on tumor cells is induced in vivo by IFN-gamma, and that this induction is associated with the ability to reject the tumor. We will also introduce preliminary data concerning the mechanism of induction in which CTL appear to induce class-I MHC both in vitro and in vivo. The results are discussed in terms of a model which may be important generally for class-I inducible tumors.

Alteration of the metastatic potential of line 1 lung carcinoma cells: opposite effects of class I antigen induction by interferons versus DMSO or gene transfection.

Class I antigens are necessary for the recognition of tumor cells by cytotoxic T lymphocytes (CTL). The line 1 lung carcinoma is a spontaneous murine tumor deficient in class I antigen expression. Consistent with this, line 1 cells are highly metastatic in vivo. We investigated whether increasing class I antigen expression on line 1 cells could alter the metastatic potential of these tumor cells using an in vivo lung metastasis model. We used three methods to induce class I antigen expression on line 1 cells: gene transfection, treatment with dimethyl sulfoxide (DMSO), or treatment with interferon (IFN)-beta or -gamma. We found that line 1 cells expressing a transfected class I gene were significantly less metastatic than parental line 1 cells. DMSO-treated line 1 cells also formed significantly fewer metastases than parental line 1 cells. These results indicate that increased class I antigen expression decreases the metastatic potential of line 1 cells in vivo. However, we did not observe a significant decrease in the number of lung metastases in mice receiving line 1 cells treated with IFN-beta or -gamma, despite high levels of class I antigen expression. Thus, increasing class I antigen expression with IFN has an opposite effect on metastasis from class I antigen expression induced by transfection or DMSO. These results show that the method used to increase class I antigen expression is critical in terms of the in vivo effect observed. To investigate a possible mechanism for the differences observed in vivo between these class I expressing cells, we tested whether IFN alters or blocks susceptibility of line 1 cells to immune effector cells. We found IFN treatment increased the ability of line 1 cells to be recognized by CTL but concomitantly decreased the susceptibility of line 1 cells to NK cell lysis by a non-class I antigen-related mechanism. In contrast, transfected or DMSO-treated line 1 cells which were less metastatic in vivo were susceptible to both CTL and NK-mediated lysis. Taken together, these results suggest that immune intervention against metastasizing line 1 cells may involve NK cells and CTL.