Prevention of Biofilm Infections

Biofilms are well-organized, complex microbial communities that are often highly resistant to antimicrobial agents and host defenses. Biofilms are often formed on the surfaces of surgical implants and indwelling catheters. Being extremely resistant to removal, biofilms, once formed, cause numerous complications and often result in persistent infections that require long-term hospitalization for treatment. Until now, preventive measures employing prophylactic antimicrobials that prohibit or restrict biofilm formation have been the only feasible, effective options available, with the constant concomitant threat of antimicrobial resistance. However, the development of chemical agents that specifically act upon the virulence of biofilms, rather than destroying the microorganisms or suppressing their growth, is a promising new approach. Such agents are highly desirable in that they might allow clinicians to prevent the development of antimicrobial resistance. Effective suppression of biofilm formation would dramatically change the way to treat infectious disease. In this literature review, the types of infections associated with biofilms and relevant therapeutic options that have been approved, in use, or under development to treat biofilm infections are discussed, along with novel approaches to biofilm control that may be applicable to the development of future anti-biofilm agents.

Expression of Osteoprotegerin and Osteoclast Differentiation Factor in Human Periodontal Ligament Fibroblast Cells / 대한치주과학회지

Recently, soluble TNF receptor homolog osteoprotegerin (OPG) and its membrane-bound ligand osteoclast differentiation factor (ODF) were found to regulate osteoclast formation and function, and bone metabolism. It is now well established that ODF acts via RANK expressed on hematopoietic osteoclast precursor cells to facilitate their differentiation to osteoclasts, and OPG prevents the formation of osteoclasts by interfering the binding of ODF and RANK. Expression of OPG and ODF was believed to be closely related to the pathogenesis of bone resorption and destruction from osteoporosis, periodontal diseases, malignant bone tumor, and arthritis. The periodontal ligament fibroblasts (PDLF), located between the tooth and tooth socket, has been thought to play an important role in maintaining bone homeostasis of periodontal tissues. However, the exact mechanism by which bone formation and resorption are regulated by PDLF is not well understood. In this study we have prepared primary cultures of human PDLF from periodontium of malaligned tooth extracted due to orthodontic reason, and determined steady state or inflammatory signal-induced OPG and ODF expression using RT-PCR and western blot analysis. OPG and ODF mRNA and protein were expressed constitutively in the PDLF and these expression were slightly increased by osteotropic cytokine IL-1beta. Lipopolysaccharide-treated PDLF showed decrease in OPG mRNA and protein expression, and increase in ODF mRNA and protein expression. These results indicated that PDLF influence the osteoclastogenesis by OPG and ODF expression in the inflammatory situation as well as physiological condition, and thereby pathogenesis of periodontal alveolar bone destruction.

Human Periodontal Ligament Fibroblasts Support the Osteoclastogenesis of RAW264.7 Cells / 대한치주과학회지

The fibroblasts are the principal cells in the periodontal ligament of periodontium. As the periodontal ligament fibroblasts (PDLF) show similar phenotype with osteoblasts, the PDLF are thought to play an important role in alveolar bone remodeling. Cell-to-cell contacted signaling is crucial for osteoclast formation. Recently it has been reported that PDLF enhance the bone resorbing activity of osteoclasts differentiated from hematopoietic preosteoclasts. The aims of this study were to clarify the mechanism of PDLF-induced osteoclastogenesis and 2) whether we can use preosteoclast cell line instead of primary hematopoietic preosteoclast cells for studying the mechanism of PDLF-induced osteoclastogenesis. Osteoclastic differentiation of mouse macrophage cell line RAW264.7 was compared with that of mouse bone marrow-derived M-CSF dependent cell (MDBM), a well-known hematopoietic preosteoclast model, by examining, 1) osteoclast-specific gene expression such as calcitonin receptor, M-CSF receptor (c-fms), cathepsin K, receptor activator nuclear factor kappa B (RANK), 2) generation of TRAP(+) multinucleated cells (MNCs), and 3) generation of resorption pit on the OAASTM plate. RAW264.7 cultured in the medium containing of soluble osteoclast differentiation factor (sODF) showed similar phenotype with MDBM-derived osteoclasts, those are mRNA expression pattern of osteoclast-specific genes, TRAP(+) MNCs generation, and bone resorbing activity. Formation of resorption pits by osteoclastic MNCs differentiated from sODF-treated RAW264.7, was completely blocked by the addition of osteoprotegerin (OPG), a soluble decoy receptor for ODF, to the sODF-containing culture medium. The effects of PDLF on differentiation of RAW264.7 into the TRAP(+) multinucleated osteoclast-like cells were examined using coculture system. PDLF were fixed with paraformaldehyde, followed by coculture with RAW264.7, which induced formation of TRAP(+) MNCs in the absence of additional treatment of sODF. When compared with untreated and fixed PDLF (fPDLF), IL-1beta-treated, or lipopolysaccharide- treated and then fixed PDLF showed two-fold increase in the supporting activity of osteoclastogenesis from RAW264.7 coculture system. There were no TRAP(+) MNCs formation in coculture system of RAW264.7 with PDLF of no fixation. These findings suggested that we can replace the primary hematopoietic preosteoclasts for RAW264.7 cell line for studying the mechanism of PDLF-induced osteoclastogenesis, and we hypothesize that PDLF control osteoclastogenesis through ODF expression which might be enhanced by inflammatory signals.

Osteoprotegerin is present on the membrane of osteoclasts isolated from mouse long bones

Osteoprotegerin (OPG), a member of the tumor necrosis factor receptor superfamily, is known to inhibit osteoclastogenesis by acting as a soluble decoy receptor for the receptor activator of NF-kB ligand (RANKL). We report the presence of OPG on the membrane of osteoclasts and the possibility of the direct action of OPG on them. Highly pure osteoclast precursors were isolated from mouse long bones and induced to differentiate into mature osteoclasts by M-CSF and soluble RANKL (sRANKL). The presence of OPG on the membrane of these cells was confirmed by western blotting and immunostaining. Furthermore, sRANKL was found to be bound to the OPG on the osteoclast precursors. These results suggest that OPG might have a new role during the differentiation of osteoclasts beyond its role as a soluble decoy receptor. The mechanism of the existence of OPG on osteoclast precursors remains to be found.

Expression of Antigen Presenting Function-Associated Surface Molecules on Interferon gamma-Treated Gingival Fibroblasts and Periodontal Ligament Fibroblasts / 대한치주과학회지

It is becoming increasingly clear that human gingival fibroblasts(HGF) may play a role in regulating immune responsiveness in inflammatory periodontal lesions. Stimulation of HGF with locally-secreted T cell cytokine IFNgamma induces human leukocyte antigen class II(HLA II) expression on HGF, which is one of the characteristic feature of professional antigen presenting cells(pAPC). However, IFNgamma-treated HGF and other nonprofessional antigen presenting cells(npAPC) are known to be ineffective or less effective antigen presenter to resting T cells. This study, therefore, was undertaken in an effort to elucidate the differences in expression of cell surface molecules between npAPC in periodontal tissues, such as HGF and periodontal ligament fibroblasts(PDLF), and pAPC such as monocytes/macrophages. Using flow cytometry, the levels of cell surface expression of HLA-D, ICAM-1, LFA-3, and B7-1, which are involved in antigen presentation, were determined in HGF, PDLF and human myelomonocytic cell line THP-1. IFNgamma clearly induced HLA-D expression on both of fibroblasts and monocytes dose dependently. However, expression level on monocytes were 4 to 5 times higher than that on fibroblasts, and induction rate was faster in monocytes than in fibroblasts. The levels of ICAM-1 expression on fibroblasts and monocytes were enhanced by IFNgamma in a dose dependent manner. On the other hand, the expression of LFA-3 molecule, which could be detected in fibroblasts and monocytes without cytokine stimulation, was no more enhanced by addition of IFNgamma. B7-1, important costimulatory molecule in T cell activation and proliferation, was not detected on both of fibroblasts and monocytes even when stimulated with IFNgamma, except on monocytes fully differentiated by pretreatment of PMA and treated by IFNgamma. These results suggest that delayed expression of HLA-D and absence of B7-1 on IFNgamma -treated fibroblasts may at least in part be involved in the ineffectiveness of fibroblasts as primary APC. And it is postulated that although periodontal fibroblasts may not serve as primary APC in normal periodontium, sustained expression of HLA II on ubiquitous fibroblasts in inflammatory lesions may perpetuate immune responses and produce chronic inflammation and tissue injury.

Immunochemical study on the Role of beta2 Integrin in the Activation of Monocytes Upon Direct Contact with T Lymphocytes / 대한치주과학회지

The modulation of leukocyte cell surface adhesion molecules may influence the development of cellular events that determine the course of the inflammatory process. Direct interaction between activated T cells and monocytes resulted in a large production of IL-1beta by monocytes. In this reactions, adhesion molecules play an important part, yet the role of them in T-monocytes interaction remain unclear. This study was undertaken in an effort to elucidate, 1) the influence of 1.25(OH)2D3-induced differentiation on the monocyte responsiveness to direct contact with T lymphocytes, and 2) the role of adhesion molecules on the T-monocyte direct interaction. Initially, I observed that direct contact of monocyte cell line THP-1 with stimulated fixed T cell line HuT78 markedly induces IL-1beta production by THP-1. IL-1beta production was higher when THP-1 had been previously exposed to 1.25(OH)2D3 as compared to control, with alpha-1.25(OH)2D3 dose-dependent and exposure time-dependent manner. It was shown that 1.25(OH)2D3 also increased the expression of beta2 integrin adhesion receptor Mac-1(CD11b/CD18) dose- and time- dependently, but did not increase the expression of human leukocyte antigen-D(HLA-D) and intercellular adhesion molecule-1(ICAM-1). The IL-1beta producing activity of THP-1 cells correlated well with the ability to induce the Mac-1 expression on THP-1 surface. Monoclonal antibody raised against relevant cell surface glycoproteins on THP-1 were tested for their ability to block the response of THP-1 to T cells. Antibody to Mac-1 only partially blocked IL-1beta production by THP-1, whereas antibodies to ICAM-1 and HLA-D did not. These data indicate that regulation of Mac-1 expression on THP-1 cells can alter the responsiveness of these cells to contact by activated T cells, however other unknown structures on the THP-1 cells may be involved in this process also.

Effects of Direct Cell Contact Between Monocytes and Fibroblasts on the Interleukin-6 Production and Cell Proliferation of Human Gingival and Periodontal Ligament Fibroblasts / 대한치주과학회지

In order to reveal immunopathogenesis of periodontal tissue destruction, it is important to clarify the molecular mechanism of trafficking and retention of activated leukocytes, including monocytes/macrophages. Gingival fibroblasts may be involved in the regulation of inflammatory cell accumulation in the extravascular periodontal connective tissues via cytokine production and surface expression of adhesion molecules. In this study, it was investigated the molecular basis for the adhesive interactions between monocytes and fibroblasts such as periodontal ligament fibroblast(PDLF), human gingival fibroblast(HGF), and human dermal fibroblast(HDF). First, it was examined the evidence whether monocyte-fibroblast cell contact may cause signal transduction in fibroblasts. Being directly in contact with fixed human monocyte cell line THP-1, or U937, upregulation of IL-6 production, TNF-alpha mRNA expression and increased cell proliferation could be seen for fibroblasts. IL-6 production induced by monocyte-fibroblast coculture were further increased when fibroblasts had been pretreated with IFN-gamma or IL-1beta, and monocytes with LPS. Next, it was examined the expression of ICAM-1 which has been known to be involved in accumulation and activation of leukocytes in inflammatory diseases such as periodontitis. ICAM-1 was upregulated up to 10-fold on PDLF, HGF, and HDF by exposure to IFN-gamma or IL-1beta. Furthermore, anti-ICAM-1 monoclonal antibody clearly blocked coculture-induced IL-6 production by fibroblasts, suggesting that ICAM-1/beta2 integrin pathway is involved in periodontal fibroblast-monocyte interaction. Overall, these findings provide evidence that periodontal fibroblasts could be involved in the accumulation and retention of monocytes/macrophages in periodontal inflammatory lesion at least in part by ICAM-1 expression. In addition, periodontal fibroblast-monocyte interaction could cause activation signals in fibroblasts intracellularly which result in cytokine production and cell proliferation. Thus, periodontal fibroblasts are speculated to play an important role in immunoregulation and tissue destruction in chronic periodontal diseases by interaction with monocytes/macrophages.

Effects of differentiation/activation factors on the endotoxin-induced expression of MHC class II molecule and integrins, and production of cytokine by monocyte / 대한치주과학회지

1,25-Dihydroxyvitamin D3(1,25(OH)2D3) and interferon-gamma(IFN-gamma) are capable of regulating cells in immune system and are representative differentiation inducer/activator of monocyte/macrophage lineage cells. They may affect the proinflammatory and immune effector functions of monocyte/macrophage activated with immunostimulants such as bacterial endotoxin. The expression of HLA-D, costimulatory adhesion molecules such as ICAM-1(CD54) and Mac-1(CD11b), and secretion of IL-1betawere used to evaluate the influence of 1,25(OH)2D3 and IFN-gammaon human promyelocytic cell line THP-1 cells. IFN-gammamarkedly induces the expression of ICAM-1 and HLA-D, and 1,25(OH)2D3 induces the expression of Mac-1 to a lesser extent. LPS alone markedly induces the expression of ICAM-1 molecule without any increase of HLA-D expression. LPS-induced ICAM-1 expression is synergistically increased by IFN-gammapriming, but fail to increase Mac-1 and HLA-D expression. IFN-gamma, 1,25(OH)2D3, and LPS separately do not induce the secretion of IL-1betaby THP-1, however, IL-1betasecretion is synergistically increased when THP-1 is exposed to the combination of IFN-gammaand LPS. LPS-induced IL-1betaproduction by IFN-gamma-primed cell is much higher when THP-1 have been previously exposed to 1,25(OH)2D3. In conclusion, these results show that expression of MHC class II and costimulatory adhesion molecules, and production of IL-1betaby cells of monocyte/macrophage lineage is highly regulated and monocyte /macrophage differentiation is required for optimal proinflammatory and immune reaction in response to exposure of bacterial endotoxin.