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RESEARCH REPORT |
Clinical Research Center for Periodontal Disease, Virginia Commonwealth University, School of Dentistry, PO Box 980566, Richmond, VA 23298-0566, USA;
* corresponding author, haschenk{at}vcu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: antibody • cardiolipin • periodontitis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Anti-phospholipid antibodies comprise a class of autoantibodies found in 1-5% of the systemically healthy population (Petri, 2000). Elevated levels of these antibodies can be found in several conditions, including a variety of infectious diseases, and are a hallmark of the Antiphospholipid Syndrome (APS). APS is present in about 30 to 40% of patients with SLE, although there are individuals with the primary form of APS who do not have SLE. The major clinical symptoms of APS include recurrent venous or arterial thrombosis and fetal loss, and patients with APS may demonstrate premature atherosclerosis (Levine et al., 2002).
The pathogenesis of APS is related to the prothrombotic activity of some anti-phospholipid antibodies (Levine et al., 2002). Among the major groups of antibodies detected in APS patients are ß-2-glycoprotein I-dependent anti-cardiolipin (anti-CL), anti-ß-2-glycoprotein I (anti-ß2GPI), and lupus anticoagulant (LA). ß2GPI is a 50-kDa plasma phospholipid-binding protein that functions as a natural anticoagulant (Kandiah and Krilis, 1994). Immunoassays that measure pathogenic anti-CL require the incorporation of ß2GPI bound to CL for detection of anti-CL autoantibodies that promote procoagulant activity (Galli et al., 1990; McNeil et al., 1990). Such antibodies are characteristic of patients with APS. Autoantibodies directed at ß2GPI may also be directly detected in immunoassays that omit CL, though these subsets of anti-CL and anti-ß2GPI may not be identical (Hojnik et al., 1994). Anti-ß2GPI immunoassays have high specificity for the APS, while antibodies that bind directly to CL are also present in patients with syphilis or other infectious diseases. LA antibodies, also characteristic of patients with APS, are associated with thrombo-embolic events and are detected in clotting assays in which they prolong clotting (despite their clear association with in vivo thrombosis) (Levine et al., 2002). In summary, this group of antibodies is heterogeneous, and clinical tests for APS usually involved multiple assays to detect autoimmune anti-CL, anti-ß2GPI, and LA.
Recent studies strongly implicate bacterial and viral infections in the etiology of APS due to induction of cross-reactive anti-CL autoantibodies. Blank et al.(2002) identified a hexapeptide (TLRVYK) sequence in ß2GPI that is recognized by some anti-ß2GPI monoclonal antibodies. Mice immunized with microbial pathogens such as Hemophilus influenzae or Neisseria gonorrheae with homologous sequences related to TLRVYK produced cross-reactive anti-ß2GPI that induced APS-like symptoms when subsequently purified and passively infused into mice. Thus, bacterial infections could lead to production of pathogenic anti-CL and be responsible for a subset of cases of APS.
The remarkable similarity between the hallmark symptoms of APS and the reported systemic sequellae of periodontal infections, as well as the likely infectious origin of the anti-CL antibodies, suggested that patients with periodontitis may have elevated anti-CL antibodies that could partly explain the pathogenesis of these systemic disorders. To assess the possibility that periodontal infections could induce production of autoantibodies to phospholipids, we quantitated a series of antiphospholipid antibodies in sera from patients with a variety of periodontal diagnoses.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The subjects were categorized by diagnostic group as follows:
Laboratory Methods
All antibodies were assessed by ELISA. For determination of IgG and IgM anti-CL and IgG anti-ß2GPI, we utilized the following kits from Pharmacia Diagnostics (Kalamazoo, MI, USA): Varelisa Cardiolipin IgG Antibodies (Cat. No. 15596), Varelisa IgM Cardiolipin Antibodies (Cat. No. 15696), and Varelisa ß2-Glycoprotein 1 (IgG) Antibodies (Cat. No. 18796). For each assay, a positive test was defined as greater than 15 units/mL according to the manufacturer.
Statistical Analyses
Continuous response variables were compared by ANOVA followed by Tukey’s post hoc test. Categorical response variables were compared by a chi-square test. The relationship between the clinical and demographic variables and anti-CL results was assessed by logistic regression. We first screened the variables one at a time to determine their relationship to anti-CL and then used a multiple logistic regression model to determine the significant predictors. Significance was determined at alpha = 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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depicts the characteristics of the subject population utilized in this study. The groups varied with respect to age, gender, race, smoking habits, and mean attachment loss, due in part to the nature of their periodontal disease categories.
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. Subjects positive for these antibodies were those with either IgG or IgM levels greater than 15 units of antibody. Those subjects who were anti-CL-positive demonstrated significantly greater pocket depth and attachment loss than those who were negative. Additionally, patients who were former or current smokers were more likely to be positive for these antibodies. There were no differences noted in age, sex, or race between subjects positive and those negative for anti-CL.
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). Patients with generalized periodontitis had a significantly greater prevalence of IgG or IgM antibody (p = 0.0033). The prevalence in CP patients was 16.2%, while the prevalence in GAgP was 19.3%. In contrast, the overall prevalence of patients with positive tests for IgG or IgM antibodies in the healthy control group was 6.79% and that in the LAgP was 3.2%. The IgG and IgM assays by themselves approached statistical significance.
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We performed stepwise logistic regression analysis to determine which demographic, clinical, or diagnostic variables (including those variables shown in Table 2
) best predicted a positive (IgG or IgM) anti-CL test (Table 4). The significant variables included only periodontal diagnoses (LR chi-square = 13.7, df = 3, p = 0.0033).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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ß2GPI is a plasma protein that binds to negatively charged phospholipids and is thought to provide a protective homeostatic mechanism preventing pathological prothrombotic reactions initiated by platelets or endothelial cells (Kandiah and Krilis, 1994). The pathogenesis of APS remains controversial. Pathogenic autoantibodies reactive with phospholipids which target ß2GPI are thought to cause arterial and venous thrombosis and fetal loss by interfering with homeostatic ß2GPI function. These antibodies are hypothesized to cause disease by several different mechanisms, including activation of endothelial cells, inducement of oxidant-mediated injury due to their reactivity with oxidized LDL, or interference with the natural anticoagulant function of ß2GPI (Levine et al., 2002). Our data do not address the pathogenicity or function of the anti-CL found in periodontitis patients. However, ß2GPI-dependent anti-CL are frequently pathogenic, and such antibodies may be induced by certain bacterial and viral pathogens. On the other hand, our failure to detect anti-ß2GPI does not necessarily indicate a lack of pathogenic anti-CL; for example, several studies have demonstrated the relative insensitivity (but high specificity) of the anti-ß2GPI immunoassay in detection of patients with verifiable APS, while the anti-CL assay is more sensitive (Forastiero et al., 1996; Sanmarco et al., 1997; Helbert et al., 2001). We have assessed the levels of antibodies reactive with cardiolipin itself (in the absence of ß2GP1) and found that this assay does not distinguish among the periodontal diagnostic groups. We interpret this observation as indicating that the antibody specificity of the anti-CL in the sera from periodontitis subjects is ß2GP1-dependent, but somewhat different from that required for binding to ß2GP1 alone. Additional examination of these antibodies from patients with periodontitis is needed to determine their pathogenic potential.
It is noteworthy that an increased frequency of anti-CL was seen in CP and GAgP patients compared with LAgP patients, even when age was included in the model as a covariate. Analysis of our data indicates that patients with elevated anti-CL have greater mean attachment loss and increased pocket depth. Thus, it appears that the presence, in some patients, of forms of periodontitis demonstrating greater extent and severity of disease and inflammation may lead to the production of anti-CL in those patients. Alternatively, differences in the bacterial flora between localized and generalized disease forms could also explain the lack of anti-CL in LAgP patients.
Analysis of these data suggests that a subset of patients with generalized periodontitis produces elevated anti-CL antibodies during the course of chronic periodontal infection. Such antibodies at moderate but abnormal levels could be pathogenic and participate in the induction of the systemic sequellae of periodontitis. It has been proposed that increased incidence of stroke occurs in periodontitis patients (Joshipura, 2002), an observation consistent with the prothrombotic activity of pathogenic anti-CL. Additionally, it has been reported that adverse pregnancy outcomes are more common when the mother has severe generalized periodontitis (Jeffcoat et al., 2001; Offenbacher et al., 2001). The observation that fetal loss and impaired fetal growth are characteristic outcomes of APS is consistent with this characteristic of women with periodontitis. Finally, the known cross-reactivity of anti-CL with some anti-oxLDL antibodies, along with our unpublished observation that the levels of anti-CL and anti-oxLDL are highly correlated in the sera of the subjects reported upon in this study, indicates that these antibodies could also play a role in modifying the development of atherosclerosis. Additional studies are required to examine these possibilities.
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ACKNOWLEDGMENTS |
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Received May 28, 2003; Last revision August 26, 2003; Accepted September 8, 2003
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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