Possible role of apoptosis in the pathogenesis and clinical evolution of radicular cyst: an immunohistochemical study.

AIM: To investigate whether the apoptotic cascade is activated through the extrinsic pathway in epithelial lining and connective tissue of radicular cysts. METHODOLOGY: Fifteen radicular cysts were fixed in formalin, embedded in paraffin wax and processed for immunohistochemistry to evaluate the expression of polyclonal antibodies against Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), DR5 and caspase-3. Immunocomplexes were treated with the secondary antibodies and finally detected using the avidin-biotin-peroxidase complex. Immunoreactivity was visualized by development with 3,3'-diaminobenzidine. Data were analysed using the Mann-Whitney U-test; P < 0.05 was considered significant. RESULTS: The three antibodies were detected in connective tissue fibroblasts of all radicular cysts; TRAIL and DR5 immunoexpression was significantly greater (P < 0.05) compared with that of caspase-3. The three antibodies were also expressed in almost all epithelial layers and in endothelial cells of newly formed vessels. CONCLUSION: The involvement of apoptosis in the pathogenesis of radicular cysts, demonstrated by the immunoexpression patterns of TRAIL, DR5 and caspase-3 in lining epithelium and connective tissue, may explain their bland clinical aggressiveness and slow, benign evolution.

Quantitative antibody-free LC-MS/MS analysis of sTRAIL in sputum and saliva at the sub-ng/mL level.

Soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) induces apoptosis via the extrinsic death receptor pathway and may be a biomarker in the pathogenesis of a broad range of diseases. To investigate the role of sTRAIL in asthma, we developed a quantitative LC-MS/MS method with a lower limit of quantitation (LLOQ) of ≈3pM in induced sputum (174pg/mL) and saliva (198pg/mL) without the use of antibodies. sTRAIL was enriched by immobilized metal affinity chromatography (IMAC) solid-phase extraction (SPE) followed by tryptic digestion and subsequent enrichment of a signature peptide by strong cation exchange (SCX) SPE. The method was validated with respect to stability, accuracy and precision using the standard addition approach and fully metabolically (15)N-labelled hrTRAIL as internal standard. Our results indicate that it is possible to quantify cytokines like sTRAIL at the pM level by LC-MS/MS without the use of antibodies, which has, to our knowledge, never been shown before.

Sex differences in inflammatory and apoptotic signaling molecules in normal and diseased human gingiva.

BACKGROUND: The purpose of this study is to determine whether sex dimorphism exists in the expression of inflammatory and apoptotic mediators in gingiva obtained from normal and diseased sites of periodontal disease. METHODS: Gingival papillae were obtained from individuals (56 males and 62 females) who required extraction of adjacent teeth. Gingival samples were grouped by adjacent sulcus depth: 1 to 3 mm (normal), 3 mm with bleeding on probing (slight disease), 3 to 6 mm (moderate disease), and >6 mm (severe disease). The tissue concentrations of cysteine-requiring aspartate-directed protease 3 (caspase-3), interleukin-2, tumor necrosis factor-related apoptosis-inducing ligand, Fas ligand, p38α mitogen-activated protein kinase, extracellular signal-related kinase 1/2, and survivin were determined by enzyme-linked immunosorbent assay. These mediator concentrations, age of donor, sex of donor, and gingival sulcular depth were the outcome variables. Data were compared by factorial analysis of variance, post hoc Tukey, and Pearson correlation test. P <0.05 was used to indicate significant differences among the outcome variables. RESULTS: The mean gingival sulcular depth was significantly greater in male than in female groups (P <0.05). The majority of the tested mediators were significantly correlated with both sex and sulcular depth and with caspase-3 (P <0.05). The concentration of caspase-3 in female gingiva at all diseased sites was significantly greater than in gingiva derived from male sites (P <0.05). CONCLUSIONS: These data suggest sex dimorphism in the presence of gingival apoptosis at sites of periodontal disease, with females having the highest incidence of apoptosis. Because apoptosis clears inflammatory cells and promotes healing, this phenomenon could provide a mechanism for sex dimorphism for the incidence of periodontal disease.

Profiling biomarkers in gingival crevicular fluid using multiplex bead immunoassay.

OBJECTIVE: Biomarkers in gingival crevicular fluid (GCF) have been investigated; however, measurements were limited by the small sample volume available. The aim of this study was to determine the levels of 40 different cytokines and chemokines in GCF samples. DESIGN: Eleven patients with generalised chronic periodontitis participating in a supportive periodontal therapy programme with remaining probing pocket depths (PDs) of >5mm were enrolled. One healthy and two diseased sites were sampled in each subject. Forty biomarkers in GCF were examined using a multiplex bead immunoassay. Porphyromonas gingivalis from the diseased sites was quantified by real-time polymerase chain reaction. RESULTS: Twenty-six biomarkers were detected in the GCF samples using the multiplex bead immunoassay. The levels of nine biomarkers were significantly different between the diseased and healthy sites after adjustment with Bonferroni's correction. The level of 26 biomarkers in diseased sites was compared between bleeding on probing (BOP)-positive and BOP-negative sites. Interleukin (IL)-1ß and interferon-inducible protein (IP)-10 levels were significantly higher in BOP-positive diseased sites than BOP-negative diseased sites after adjustment for multiple comparisons (IL-1ß, p=0.0007, IP-10; p=0.0009). In addition, the levels of IL-1ß in GCF were found to be strongly correlated with the P. gingivalis ratio (r=0.646, p=0.0012). CONCLUSION: IL-1ß levels in GCF correlate with the PDs, BOP and the presence of P. gingivalis in subgingival plaque. Multiplex bead assays can be useful in GCF studies. These findings can help in identifying new diagnostic methods in the diagnosis of periodontal disease.