cytochalasin-b and Periodontitis

Resistin is an adipocytokine that induces insulin resistance and is predominantly expressed in adipocytes and peripheral blood mononuclear cells. Resistin expression increases in inflammatory diseases as well as diabetes mellitus, and is upregulated by bacterial pathogens and proinflammatory cytokines. The aim of this study was to identify resistin in human gingival crevicular fluid, to compare the resistin levels in gingival crevicular fluid between subjects with and without periodontitis and diabetes mellitus and to investigate the regulation of resistin release from human neutrophils by Porphyromonas gingivalis lipopolysaccharide (P-LPS).. Gingival crevicular fluid samples were collected from patients with chronic periodontitis (n = 24), patients with diabetes mellitus-related periodontitis (n = 18) and healthy subjects (n = 21). Resistin in gingival crevicular fluid was determined using western blot analysis and an ELISA kit. The glycated hemoglobin (HbA(1c)) value was obtained from patients with diabetes mellitus-related periodontitis by a medical interview. Human neutrophils were cultured with P-LPS (0-1000 ng/mL), or incubated with inhibitors of actin or microtubule polymerization in the absence or presence of P-LPS. The medium and cellular fractions were used for determination of resistin by ELISA.. The resistin level in gingival crevicular fluid from patients with periodontitis or diabetes mellitus-related periodontitis was significantly higher than that of healthy subjects. The resistin level in gingival crevicular fluid was correlated with gingival index score, but not blood HbA(1c) value. The P-LPS increased resistin release from human neutrophils, and its induction was decreased by actin polymerization inhibitors.. We show, for the first time, the presence of resistin in gingival crevicular fluid. A high resistin level in gingival crevicular fluid samples from periodontitis patients may to some extent be related to P-LPS-induced resistin release from neutrophils.

Topics: Actin Capping Proteins; Adult; Aged; Cell Culture Techniques; Chronic Periodontitis; Colchicine; Cytochalasin B; Cytochalasin D; Diabetes Complications; Female; Gingival Crevicular Fluid; Glycated Hemoglobin; Humans; Lipopolysaccharides; Male; Middle Aged; Neutrophils; Nocodazole; Nucleic Acid Synthesis Inhibitors; Periodontal Index; Periodontal Pocket; Periodontitis; Porphyromonas gingivalis; Resistin; Tubulin Modulators

Release of potent lysosomal enzymes by degranulation of polymorphonuclear leukocytes (PMNs) in host gingiva may contribute significantly to tissue destruction and the pathogenesis of periodontal disease. A pilot study established that peripheral blood PMNs from humans with rapidly progressive periodontitis (RPP) contained significantly increased amounts of intracellular lysosomal beta-glucuronidase as compared to healthy controls. This investigation gained insight into the question: are the increased levels of beta-glucuronidase in persons with RPP an a priori genetically determined PMN characteristic, or a reactive phenomenon induced by the periodontal disease process during granulopoiesis? Twelve healthy controls and twelve otherwise healthy individuals with RPP participated in a repeated measures design to T0 (initial, baseline), T1 (four weeks after disease control therapy), and T2 (two months later). At each visit clinical indices (GI, pocket depths, GCF flow, plaque index) were performed and peripheral blood obtained. PMNs were isolated and suspended as 5 x 10(6) cells in 2.0 ml of HBSS. PMN suspensions were tested for total intracellular beta-glucuronidase, degranulation induced by 1 x 10(-6)M and 5 x 10(-7) M FMLP challenges, and unchallenged for non-specific enzyme release. PMNs from individuals with RPP contained significantly higher absolute amounts of beta-glucuronidase and released greater absolute amounts at FMLP challenge at T0, T1, and T2 compared to controls. No relationship was found between any of the clinical indices and beta-glucuronidase levels and no pattern was discovered relating to the repeated measures over time. We conclude that RPP peripheral blood PMNs contain elevated levels of beta-glucuronidase that are not induced by the periodontal disease process.

Topics: Adult; Case-Control Studies; Cell Degranulation; Cytochalasin B; Dental Plaque Index; Female; Follow-Up Studies; Gingival Crevicular Fluid; Glucuronidase; Humans; Lysosomes; Male; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Periodontal Index; Periodontal Pocket; Periodontitis; Periodontium; Pilot Projects

The activity of elastase increases significantly in the gingival sulcus during inflammation. The release of this enzyme from crevicular and peripheral polymorphonuclear leucocytes (PMNs) was studied in seven patients with gingivitis, six with adult periodontitis, seven with rapidly progressive periodontitis and in nine healthy subjects. Peripheral PMNs were isolated from blood and crevicular PMNs from gingival washings. After preincubation with cytochalasin B, the same numbers of crevicular and peripheral cells were incubated either in phosphate-buffered saline (spontaneous release) or in the same buffer containing formyl methionyl-leucyl-phenyl-alanine (stimulated release). Elastase activity was measured in the supernatant by a fluorimetric technique. The results confirm that compared to peripheral PMNs, crevicular cells show a higher spontaneous release of elastase and a lower stimulated release. The activity of elastase released either spontaneously or after stimulation of crevicular cells was significantly lower in the controls as compared with cells obtained from patients with gingivitis or periodontitis. Crevicular PMNs collected from patients with deeper pockets appear to release larger amounts of elastase than those from controls.

Topics: Adult; Blood; Cytochalasin B; Female; Fluorometry; Gingiva; Gingivitis; Humans; Male; Middle Aged; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Pancreatic Elastase; Periodontal Pocket; Periodontitis; Periodontium

Peripheral PMNs were collected from blood, and crevicular PMNs separated by filtration from gingival washings in 13 patients, aged 22-75 y, who had varying degrees of gingivitis and periodontitis. After pre-incubation with cytochalasin B, the same number of crevicular and peripheral cells were incubated either in PBS (with Ca2+ and Mg2+) (spontaneous release) or in the same buffer containing increasing concentrations of FMLP (stimulated release); elastase activity was measured in the supernatant by a fluorometric technique. There was a higher spontaneous release of enzyme from crevicular than from peripheral neutrophils. The average elastase activity in the supernatant of 1 x 10(4) crevicular cells was more than five times higher than that obtained from the same number of peripheral cells. However, stimulated crevicular PMNs liberated smaller amounts of enzyme than did stimulated peripheral PMNs. These results suggest that crevicular PMNs are already releasing elastase, and are consistent with the possibility that lysosomal enzymes contribute to tissue damage during gingivitis and periodontitis.

Topics: Adult; Aged; Blood; Chronic Disease; Cytochalasin B; Gingival Crevicular Fluid; Gingival Pocket; Gingivitis; Humans; Middle Aged; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Pancreatic Elastase; Periodontal Index; Periodontal Pocket; Periodontitis

Direct interaction between Fusobacterium nucleatum and human neutrophils resulted in the secretion of a neutrophil self-regulatory factor(s). The secretion of this factor was bacteria specific, and depended on the integrity of the bacteria cell surface. Factor secretion occurred within 15 min of bacteria--neutrophil interaction. Pre-treatment of neutrophils with cytochalasin B but not sodium fluoride inhibited factor secretion. The factor was sensitive to trypsin and heat treatment. Ultrafiltration experiments showed that it has a molecular weight between 10,000 and 30,000 daltons. Its biologic role may be that of a molecular mediator for the recruitment of resting neutrophils so as to amplify the immunological and inflammatory response.

Topics: Antigens, Bacterial; Bacterial Proteins; Blood Bactericidal Activity; Chemotaxis, Leukocyte; Cytochalasin B; Formaldehyde; Fusobacterium; Hot Temperature; Humans; Kinetics; Molecular Weight; Neutrophils; Periodontitis; Phagocytosis; Sodium Fluoride; Trypsin