Heparan Sulfate Glycosaminoglycan Is Predicted to Stabilize Inflammatory Infiltrate Formation and RANKL/OPG Ratio in Severe Periodontitis in Humans.

Since chronically inflamed periodontal tissue exhibits extracellular matrix (ECM) degradation, the possible alternative to standard periodontitis treatment is to restore ECM by supplementing its components, including heparan sulfate glycosaminoglycan (HS GAG). Supplementation of the degraded ECM with synthetic derivatives of HS GAGs has been shown to be effective for periodontal tissue regeneration in experimental animal models of periodontitis. However, the potential of HS GAG supplementation for the treatment of periodontal disease in humans is still unknown. Here, we used a statistical model to investigate the role of HS GAG on inflammatory infiltrate formation and alveolar bone resorption in humans with severe periodontitis. The model was based on data from immunofluorescence staining (IF) of human gingiva samples, and reconstruction of a subset of HS GAG -related proteins from STRING reactome database. According to predictions, increased expression of native HS GAG might stabilize the accumulation of gingival inflammatory infiltrate (represented by the general inflammatory cell marker CD45) and alveolar bone resorption (represented by Receptor Activator of Nuclear ΚΒ ligand (RANKL) and osteoprotegerin (OPG) ratio) but could not restore them to healthy tissue levels. Therefore, supplementation of native HS GAG may be of limited benefits for the treatment of sever periodontitis in humans.

Novel approach for quantification of multiple immunofluorescent signals using histograms and 2D plot profiling of whole-section panoramic images.

We describe a novel approach for quantification and colocalization of immunofluorescence (IF) signals of multiple markers on high-resolution panoramic images of serial histological sections utilizing standard staining techniques and readily available software for image processing and analysis. Human gingiva samples stained with primary antibodies against the common leukocyte antigen CD45 and factors related to heparan sulfate glycosaminoglycans (HS GAG) were used. Expression domains and spatial gradients of IF signals were quantified by histograms and 2D plot profiles, respectively. The importance of histomorphometric profiling of tissue samples and IF signal thresholding is elaborated. This approach to quantification of IF staining utilizes pixel (px) counts and comparison of px grey value (GV) or luminance. No cell counting is applied either to determine the cellular content of a given histological section nor the number of cells positive to the primary antibody of interest. There is no selection of multiple Regions-Of-Interest (ROIs) since the entire histological section is quantified. Although the standard IF staining protocol is applied, the data output enables colocalization of multiple markers (up to 30) from a given histological sample. This can serve as an alternative for colocalization of IF staining of multiple primary antibodies based on repeating cycles of staining of the same histological section since those techniques require non standard staining protocols and sophisticated equipment that can be out of reach for small laboratories in academic settings. Combined with the data from ontological bases, this approach to quantification of IF enables creation of in silico virtual disease models.

Syndecans and Enzymes for Heparan Sulfate Biosynthesis and Modification Differentially Correlate With Presence of Inflammatory Infiltrate in Periodontitis.

Periodontitis is a common degenerative disease initiated by the bacteria in subgingival biofilm. The exposure to bacterial biofilm triggers host inflammatory response whose dysregulation is ultimately responsible for the destruction of hard and soft periodontal tissues resulting in tooth loss. To date, significant effort has been invested in the research of the involvement of host cells and inflammatory mediators in regulation of inflammatory response in periodontitis. Syndecans (Sdcs) belong to a four-member family of heparan sulfate proteoglycans (HSPGs). Sdcs are compound molecules comprised of the core protein to which several heparan sulfate (HS) glycosaminoglycan (GAG) chains are attached. The role of Sdcs in pathogenesis of periodontitis is poorly investigated despite the numerous reports from experimental studies about the critical involvement of these factors in modulation of various aspects of inflammatory response, such as the formation of inflammatory mediators gradients, leukocyte recruitment and extracellular matrix remodeling in resolution of inflammation. Most of these functions of Sdcs are HS-related and, thus, dependent upon the structure of HS. This, in turn, is determined by the combinatorial action of enzymes for biosynthesis and modification of HS such as exostosis (EXTs), sulfotransferases (NDSTs), and heparanase 1 (HPSE1). The data presented in this study clearly indicate that some Sdcs display different expression profiles in healthy and diseased periodontal tissue. Additionally, the differences in expression profiles of HS GAG biosynthesis and modification enzymes (EXTs, NDSTs, and HPSE1) in healthy and diseased periodontal tissue imply that changes in HS GAG content and structure might also take place during periodontitis. Most notably, expression profiles of Sdcs, EXTs, NDSTs, and HPSE1 differentially correlate with the presence of inflammatory infiltrate in healthy and diseased periodontal tissue, which might imply that these factors could also be involved in modulation of inflammatory response in periodontitis.