Interleukin-1 beta (IL-1 beta), a potent stimulator of bone resorption, has been implicated in the pathogenesis of periodontal destruction. However, the relationship between cytokines and periodontal disease has not been studied sufficiently to allow definitive conclusions. The aims of this study are to investigate crevicular IL-1 beta and the clinical status of patients with periodontitis and the effect of phase I periodontal therapy on levels of IL-1 beta. For this study, 130 gingival crevicular fluid (GCF) samples were harvested from non-inflamed (15) and diseased sites (115) in 11 patients with periodontitis. The gingival index (GI) and probing depth (PD) of each site was recorded initially and one month after treatment. The amount of IL-1 beta in the GCF was measured by enzyme-linked immunosorbent assay (ELISA) using an antibody specific for this cytokine. Before treatment, IL-1 beta was found in 12 of 15 non-inflamed gingival crevices and in 112 of 115 diseased pockets. The amount of IL-1 beta varied from 4.03 to 511.12 pg/site. The average amount of IL-1 beta from diseased sites was 3-fold greater than that from non-inflamed sites. Both total amount of IL-1 beta and the GCF volume, but not IL-1 beta concentration, were found to be correlated, positively, with GI score and PD. After therapy, 63 sites from 7 patients were re-examined, and the amount of IL-1 beta in 49 of 63 sites was found to have declined. These data suggest that the amount of crevicular IL-1 beta is closely associated with periodontal status. This relationship may be valuable in monitoring periodontal disease activity.

OBJECTIVE:The aim of the present study was to investigate whether interleukin (IL)-1beta in diseased tissues adjacent to periodontal pockets can reflect the degree of inflammation and destruction of these tissues pathologically. BACKGROUND:IL-1beta-dependent mechanisms have been strongly implicated in contributing to inflammation and destruction of bone and attachment loss, which are characteristic features of periodontal disease. This biochemical mediator released during pro-inflammatory processes has not been objectively integrated with clinical and histopathologic features of periodontal disease. METHODS:Periodontitis-affected inflamed tissue and clinically nonaffected healthy gingivae were harvested from 14 periodontal patients, respectively. The severity of tissue inflammation was illustrated by clinical parameters and cellular histologic changes and quantified by histometric assessments. IL-1beta in these extracted specimens was measured with an enzyme-linked immunosorbent assay (ELISA) technique. Pathogenic roles that IL-1beta plays in gingival inflammation and pathologic tissue changes in tissue sections were analyzed statistically. RESULTS:The overall total tissue IL-1beta, tissue concentration of IL-1beta, and percentage of inflammatory cell infiltration (PICI) determined from diseased gingivae were obviously higher than those of controls from both healthy sites of periodontitis and non-periodontitis subjects. With increasing gingival index (GI), plaque index (PlI), and probing depth (PD), there was a marked elevation in total tissue IL-1beta. Total tissue IL-1beta was significantly correlated with GI, PlI, the PICI, and tissue alterations. Polymorphonuclear leukocytes (PMNs) and monocyte-macrophage cells seemed to predominate in heavily infiltrated areas of diseased gingiva. These cell types were confirmed by immunocytochemical localization with either monoclonal mouse antihuman neutrophil elastase antibody or monoclonal mouse antihuman macrophage (CD68) antibody, respectively. Total tissue IL-1beta and the PICI were also elevated in diseased gingivae near deeper PD, while neither total IL-1beta nor tissue concentration was statistically correlated with PD. Thus, correlation analysis indicates that IL-1beta level in inflamed periodontal tissues correlates highly with clinical parameters (GI and PlI) and PICI (the degree of inflammation). CONCLUSIONS:These observations suggest that IL-1beta plays a significant role in the pathogenic mechanisms of periodontal tissue destruction, and that measurement of tissue IL-1beta would be a valuable aid and useful for diagnostic markers of periodontal diseases.

This chapter contains sections titled: Introduction: Polymerase Chain Reaction Principles of Nucleic Acid Separation CE Methods: Principles and Strategies for Nucleic Acids PCR Applications Conclusion: Future Applications

Concepts and Principles of High Performance Capillary Electrophoresis Franco Tagliaro, Franco TagliaroSearch for more papers by this authorZdeneck Deyl, Zdeneck DeylSearch for more papers by this authorIvan Miks̆ík, Ivan Miks̆íkSearch for more papers by this authorKathi J. Ulfelder, Kathi J. UlfelderSearch for more papers by this author Franco Tagliaro, Franco TagliaroSearch for more papers by this authorZdeneck Deyl, Zdeneck DeylSearch for more papers by this authorIvan Miks̆ík, Ivan Miks̆íkSearch for more papers by this authorKathi J. Ulfelder, Kathi J. UlfelderSearch for more papers by this author Book Editor(s):Edward F. Rossomando, Edward F. Rossomando University of Connecticut Health CenterSearch for more papers by this author First published: 07 April 1998 https://doi.org/10.1002/9780470110591.ch3Citations: 1Book Series:Methods of Biochemical Analysis AboutPDFPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShareShare a linkShare onFacebookTwitterLinked InRedditWechat Summary This chapter contains sections titled: Introduction HPCE: Definition, History, and Literature Basic Components and Operations The Process of Electrophoretic Separation Instrumentation in Detail Separation Efficiency and Resolution Methods Summary Citing Literature HPLC in Enzymatic Analysis RelatedInformation

This chapter contains sections titled: Introduction Technical Aspects of Microdialysis Applications of Microdialysis/HPLC in Enzymatic Analysis Conclusions

This chapter contains sections titled: Introduction Catecholamine Metabolism Proteinase Amino Acid and Peptide Metabolism Polyamine Metabolism Heme Metabolism Carbohydrate Metabolism Steroid Metabolism Purine Metabolism Oxygenations Pterin Metabolism Lipid Metabolism Modification of Proteins and Peptides Vitamin Metabolism Xenobiotic Metabolism Pyrimidine Metabolism Metabolism of Complex Saccharides and Glycoproteins Miscellaneous Nucleic Acid Modification and Expression Summary and Conclusions

A protocol for increasing transcription from plasmid expression vectors is presented. A vector containing chloramphenicol acetyltransferase (CAT) gene was digested leaving the transcription cassette intact. Heat inactivation of restriction enzymes followed by ligation of the digestion products yielded concatemers which migrated as a single band in agarose gel electrophoresis. Mouse fibroblasts transfected with the concatemers gave a CAT activity that was 14-fold greater than that of cells transfected with a similar mass (equimolar gene number) of the native plasmid. The effect was independent of promoter type, restriction enzyme, number of restriction sites and with a noted exception, cell line.

IL-1 beta is one of the cytokines which can induce bone resorption and connective tissue destruction. Previous studies demonstrated that the amount of IL-1 beta in gingival crevicular fluid (GCF) was closely related to the severity of gingival index (GI) and probing depth (PD). To further investigate whether the amount of IL-1 beta in GCF and the tissues adjacent to the periodontal pockets can reflect the degree of inflammation and destruction of these tissues, the GCF and inflamed gingival specimens were harvested from 14 periodontal patients and IL-1 beta in these samples was measured by ELISA. Clinical parameters of the sampled teeth were also recorded. Possible relations among periodontal parameters, GCF or tissue IL-1 beta and the degrees of inflammation in tissue sections were analysed statistically. The results showed that with increasing GI and PD, there was an elevation of GCF and tissue IL-1 beta activity. GCF flow also showed a tendency to increase with a higher percentage of inflammatory infiltrate. The GCF and tissue IL-1 beta activity were significantly correlated with clinical parameters (GI, PD and GCF flow), as well as with the percentage of inflammatory infiltrate and tissue alterations. With the exception of a significantly negative correlation with the extent of inflammation, no obvious relation between the GCF or tissue concentration of IL-1 beta and the state of tissue pathology could be detected. In conclusion, measurements of IL-1 beta activity in GCF or diseased tissues based on the classification of clinical parameters can reflect the degree of inflammation within periodontal disease tissue. These observations indicate that clinical parameters can provide reliable means of evaluation of the severity of periodontal disease, and that IL-1 beta plays a pivotal role in the pathogenic mechanism of periodontal tissue destruction.

A new method for coupling proteins to plasmid expression vectors is presented. Biotin was covalently attached to a plasmid expression vector containing a chloramphenicol acetyltransferase (CAT) gene. The specific label was one biotin per 100 bp. An electrophoretic mobility shift assay showed that the plasmid was capable of binding multiple streptavidin molecules. When transfected into mouse fibroblasts, the biotinylated plasmid retained 40% of the native plasmid's biological activity, as determined by CAT assay, and was not affected by the binding of streptavidin. The method allows for attachment of any protein to plasmid DNA expression vector while retaining biological function. Hybrid plasmids in which the transcription cassettes were kept free of biotin label were constructed by digesting biotinylated and unbiotinylated plasmids at sites outside the transcription cassette and re-ligating the digestion products. Electron microscopy studies show that the ligation products formed large tangled assemblages of plasmid DNA. When equimolar (with respect to gene number) amounts of these large hybrid biotinylated plasmids were transfected into mouse fibroblasts by means of calcium phosphate precipitation, an increase in CAT expression 25-fold greater than that of original biotinylated plasmid was observed. Slot-blot analysis of total DNA extracted from transfected cells shows that this enhanced activity was not due to increased transfection efficiency. Receptor-mediated delivery could not be shown when a complex comprising biotinylated asialoglycoprotein/streptavidin/biotinylated CAT expression vector was placed in media containing Hep G2 cells.

The production of cytokines has been associated with the biology of tooth movement in animal populations. The purpose of this study was to measure tumor necrosis factor-alpha (TNF) directly in the human gingival sulcus before and after the application of an orthodontic force. To recover TNF from the sulcus, paramagnetic beads, coated with monoclonal antibodies for TNF, were introduced into the gingival sulcus of 50 teeth undergoing orthodontic tooth movement (by two force systems) in 20 patients. Retrieval was performed by a permanent magnetic device designed to fit the periodontal sulcus. The samples were taken before force application (controls), and at a fixed time after force application. The amount of immunoabsorbed TNF was quantified with an immunochemical assay. There was a greater than twofold increase in TNF recoverable from the gingival sulcus after application of orthodontic forces (mean of 12.9 ng vs 30.5 ng). A Student's t test for paired samples demonstrated statistical significance at p < 0.01. We conclude that the quantity of paradental TNF, found in human gingival sulcus, is elevated during tooth movement. The source may be from the adjacent gingiva, but more likely the compressed periodontal ligament and the resorbing bone adjacent to the root surface.

In the present study reverse transcriptase (RT) polymerase chain reaction (PCR) products were generated from the RNA of polio virus. The products of the RT-PCR were analyzed by slab-gel electrophoresis (SGE) on 4% agarose gels, and capillary electrophoresis (CE). CE separations were performed in a coated capillary containing a linear polyacrylamide. Samples were injected hydrodynamically or electrokinetically. Detection of the RT-PCR products on CE was by UV absorbance at (254 nm) or by laser-induced fluorescence (LIF). While SGE resulted in adequate separation of 163 and 97 base pair RT-PCR products, separation of the 97, 71 and 53 base pair products was minimal. CE separations showed baseline resolution for all the above PCR products. Finally, it was possible to quantitate the amount of RT-PCR product by developing a standard curve showing a linear relationship between the amount of RNA used in the RT-PCR and the amount of product formed in the RT-PCR. These results suggest the greater resolution and enhanced sensitivity observed, together with the ease of quantitation, make CE a powerful alternative to SGE for the separation and quantitation of PCR products.

Tumor necrosis factor-alpha (TNF-alpha) has been shown not only to induce the biosynthesis and secretion of collagenase but also to change the organization of cytoskeletal components. In the present study we explore the correlation between the biosynthesis of collagenase (by mRNA hybridization, indirect immunofluorescence and collagenolytic activity), and cytoskeletal reorganization (by rhodamine-phalloidin staining of F-actin) induced in fibroblasts by recombinant TNF (rTNF). In the concentration range of 1-100 ng/ml, rTNF increased extracellular collagenase activity 8-fold and collagenase mRNA 4-fold. In addition, whereas the collagenase mRNA was detected as early as 24 h posttreatment, the appearance of extracellular collagenase activity required 48 h. Using phalloidin to follow the organization of the cytoskeleton we observed that rTNF disrupted the parallel array of stress fibers normally observed in the perinuclear region. In contrast to the time required to affect collagenase synthesis, the effect of rTNF on stress fiber organization occurred as early as 6 h post-treatment. Finally, while the number of cells exhibiting this change increased with increasing concentrations of rTNF, a maximum of about 30% of the cells showed this effect. Interestingly, double staining studies demonstrated that both stress fiber changes and procollagenase production occurred in the same cells. This finding, together with the observation that the cytoskeletal disorganization preceded collagenase gene induction by at least 18 h is consistent with the conclusion that the organizational status of the microfilaments may have a role as a regulator of procollagenase gene expression.