The extracellular release of IL‐1β by cultured peripheral blood monocytes from 26 periodontitis p... more The extracellular release of IL‐1β by cultured peripheral blood monocytes from 26 periodontitis patients and 26 control subjects was measured by radioimmunoassay. Unstimulated monocytes from periodontitis patients released significantly more IL‐1β than controls during 24 h of culture; there was a wide variation in the amount of IL‐1β released (0.45–13.00 ng/ml per 106 cells) which did not correlate with either the degree of bone loss or pocket formation observed clinically. When stimulated with lipopolysaccharide (LPS; Actinobacillus actinomycetemcomitans; 5 μg/ml) monocytes from periodontitis patients produced significantly more IL‐1β than those from control subjects. Monocyte culture supernatants from another 10 periodontitis patients and 10 control subjects were also assayed for both IL‐1β and TNF‐α by enzyme‐linked immunosorbent assays. Spontaneous and LPS‐stimulated (Bacteroides gingivalis; 5 μ/ ml) IL‐1β release were again significantly higher for periodontitis patients. TNF‐α was detected in the periodontitis cultures (0–765 pg/ml per 106 cells), but the mean value was not significantly different from controls. LPS‐stimulated TNF‐α release, however, was significantly higher than for control subjects, and there was a strong correlation between spontaneous IL‐1β and TNF‐α release by monocytes from the periodontitis group. Measurement of interferon‐γ (IFN‐γ) in lymphocyte cultures from these patients by immunoradiometric assay showed that IFN‐γ levels in periodontitis cultures were consistently low, but not significantly so when compared to controls; both groups responded equally to concanavalin‐A (5 μg/ml). Although the precise roles of IL‐1β and TNF‐α in periodontitis remain unclear, these data provide evidence that both cytokines may participate in the pathogenesis of the disease.
Connective tissue remodeling is essential for normal growth and development, and many diseases ha... more Connective tissue remodeling is essential for normal growth and development, and many diseases have long been associated with the breakdown of the collagenous matrix of bone, cartilage, and related tissues. Recent work has established that members of the family of matrix metalloproteinases (MMPs) are key enzymes in matrix degradation. They function at neutral pH and can digest synergistically all the matrix macromolecules. Biochemical and cloning studies indicate that there are three major groups, collagenases, gelatinases, and stromelysins. Naturally occurring inhibitors, TIMPs (Tissue Inhibitors of MetalloProteinases), are important controlling factors in the actions of MMPs, and tissue destruction in disease processes often correlates with an imbalance of MMPs over TIMPs. The major inhibitor is TIMP-1 (or TIMP), a 30-kDa glycoprotein that is synthesized by most cells. The expression of MMPs and TIMPs by cells is regulated by many cytokines (particularly interleukin-1, IL-1), growth factors, and hormones, some of which are specific to cell type and others that are ubiquitous (e.g., transforming growth factor beta, TGF-beta). One way in which pathogenic organisms might mediate tissue degradation in periodontal diseases is through the ability of cell wall antigens to stimulate cytokine production by circulating mononuclear cells. These would then induce MMP synthesis by resident gingival cells, thereby initiating degradative events. Direct in vivo evidence for the source of collagenase and other MMPs in periodontal tissues is limited. By using specific polyclonal antibodies and indirect immunofluorescence, we could demonstrate the presence of collagenase, stromelysin-1, gelatinase A, and TIMP in human gingival biopsy specimens.(ABSTRACT TRUNCATED AT 250 WORDS)
Biochimica Et Biophysica Acta - General Subjects, Nov 11, 1994
Matrix proteins were extracted from bovine cortical bone with EDTA/Tris-HCl under non-dissociativ... more Matrix proteins were extracted from bovine cortical bone with EDTA/Tris-HCl under non-dissociative conditions at neutral pH. Four distinct bone resorptive proteins with molecular masses of 14, 25, 29 and 40 kDa were purified and partially characterized using an in vitro neonatal mouse calvarial assay and a growth factor assay using BALB/c/3T3 cells. The 14 kDa protein was purified by anion exchange chromatography (Mono Q) and gel filtration (Superdex 75HR) using FPLC (fast protein liquid chromatography); this factor stimulated the proliferation of MCF-7 human breast cancer cells, a bioassay which is specific for the insulin-like growth factors (IGFs). The 25, 29 and 40 kDa proteins were purified by sequential chromatography as follows: anion-exchange (Mono Q), heparin-Sepharose, hydroxyapatite, concanavalin A-Sepharose, phenyl-Superose, reversed phase high performance liquid chromatography (HPLC) and sodium dodecylsulfate polyacrylamide gelelectrophoresis (SDS-PAGE). The 25 kDa protein was identified as TGF-beta by its inhibitory effect on the proliferation of mink lung cells. The 40 kDa protein enhanced the formation of multinucleate tartrate-resistant acid phosphatase positive cells in a murine bone marrow differentiation assay, but was without effect in an isolated osteoclast assay and had no growth factor activity; this protein is likely to be a colony stimulating factor. The 29 kDa protein was also without growth factor activity; it was, however, able to stimulate bone resorption in the isolated osteoclast assay, suggesting a direct action in osteoclast function. The 29 and 40 kDa proteins may be osteoblast gene products that have been sequestrated by the bone matrix in a similar fashion to TGF-beta and the IGFs. This is the first report of proteins isolated from bone matrix which directly stimulate osteoclast differentiation and activity.
Members of the family of matrix metalloproteinases (MMPs) are key enzymes in normal and pathologi... more Members of the family of matrix metalloproteinases (MMPs) are key enzymes in normal and pathological tissue remodelling. They function at neutral pH and can digest synergistically all the macromolecules of the extracellular matrix. Biochemical and cloning studies indicate that there are three major groups: the specific collagenases cleave interstitial collagens; the gelatinases degrade other types of collagen and act synergistically with collagenases by degrading denatured collagens (gelatins); and the stromelysins which have broader specificity and can degrade basement membrane collagens as well as proteoglycans and matrix glycoproteins. Others in the family, but not in the major groups, are matrilysin, metallo-elastase, and several recently cloned membrane-bound metalloproteinases. Naturally occurring inhibitors, TIMPs (tissue inhibitors of metalloproteinases), are important controlling factors in the actions of MMPs, and tissue destruction in disease processes often correlates with an imbalance of MMPs over TIMPs. The relevance of recent molecular research to periodontal diseases is discussed.
The forces that orthodontic appliances apply to the teeth are transmitted through the periodontal... more The forces that orthodontic appliances apply to the teeth are transmitted through the periodontal ligament (PDL) to the supporting alveolar bone, leading to the deposition or resorption of bone, depending upon whether the tissues are exposed to a tensile or compressive mechanical strain. To evaluate the osteogenic potential of PDL cells, we applied a 12% uni-axial cyclic tensile strain to cultured human PDL cells and analyzed the differential expression of 78 genes implicated in osteoblast differentiation and bone metabolism by real-time RT-PCR array technology. Sixteen genes showed statistically significant changes in expression in response to alterations in their mechanical environment, including cell adhesion molecules and collagen fiber types. Genes linked to the osteoblast phenotype that were up-regulated included BMP2, BMP6, ALP, SOX9, MSX1, and VEGFA; those down-regulated included BMP4 and EGF. This study has expanded our knowledge of the transcriptional profile of PDL cells and identified several new mechanoresponsive genes.
2341 MSX1 Sequence Changes in Isolated Hypodontia. M. HARRISON 1 , M. MEIKLE 1 , and S. MALCOLM 2... more 2341 MSX1 Sequence Changes in Isolated Hypodontia. M. HARRISON 1 , M. MEIKLE 1 , and S. MALCOLM 2 , 1 Guy's, Kings' and St Thomas' Dental Institute, London, United Kingdom, 2 Institute of Child Health, London, United Kingdom ...
The extracellular release of IL‐1β by cultured peripheral blood monocytes from 26 periodontitis p... more The extracellular release of IL‐1β by cultured peripheral blood monocytes from 26 periodontitis patients and 26 control subjects was measured by radioimmunoassay. Unstimulated monocytes from periodontitis patients released significantly more IL‐1β than controls during 24 h of culture; there was a wide variation in the amount of IL‐1β released (0.45–13.00 ng/ml per 106 cells) which did not correlate with either the degree of bone loss or pocket formation observed clinically. When stimulated with lipopolysaccharide (LPS; Actinobacillus actinomycetemcomitans; 5 μg/ml) monocytes from periodontitis patients produced significantly more IL‐1β than those from control subjects. Monocyte culture supernatants from another 10 periodontitis patients and 10 control subjects were also assayed for both IL‐1β and TNF‐α by enzyme‐linked immunosorbent assays. Spontaneous and LPS‐stimulated (Bacteroides gingivalis; 5 μ/ ml) IL‐1β release were again significantly higher for periodontitis patients. TNF‐α was detected in the periodontitis cultures (0–765 pg/ml per 106 cells), but the mean value was not significantly different from controls. LPS‐stimulated TNF‐α release, however, was significantly higher than for control subjects, and there was a strong correlation between spontaneous IL‐1β and TNF‐α release by monocytes from the periodontitis group. Measurement of interferon‐γ (IFN‐γ) in lymphocyte cultures from these patients by immunoradiometric assay showed that IFN‐γ levels in periodontitis cultures were consistently low, but not significantly so when compared to controls; both groups responded equally to concanavalin‐A (5 μg/ml). Although the precise roles of IL‐1β and TNF‐α in periodontitis remain unclear, these data provide evidence that both cytokines may participate in the pathogenesis of the disease.
Connective tissue remodeling is essential for normal growth and development, and many diseases ha... more Connective tissue remodeling is essential for normal growth and development, and many diseases have long been associated with the breakdown of the collagenous matrix of bone, cartilage, and related tissues. Recent work has established that members of the family of matrix metalloproteinases (MMPs) are key enzymes in matrix degradation. They function at neutral pH and can digest synergistically all the matrix macromolecules. Biochemical and cloning studies indicate that there are three major groups, collagenases, gelatinases, and stromelysins. Naturally occurring inhibitors, TIMPs (Tissue Inhibitors of MetalloProteinases), are important controlling factors in the actions of MMPs, and tissue destruction in disease processes often correlates with an imbalance of MMPs over TIMPs. The major inhibitor is TIMP-1 (or TIMP), a 30-kDa glycoprotein that is synthesized by most cells. The expression of MMPs and TIMPs by cells is regulated by many cytokines (particularly interleukin-1, IL-1), growth factors, and hormones, some of which are specific to cell type and others that are ubiquitous (e.g., transforming growth factor beta, TGF-beta). One way in which pathogenic organisms might mediate tissue degradation in periodontal diseases is through the ability of cell wall antigens to stimulate cytokine production by circulating mononuclear cells. These would then induce MMP synthesis by resident gingival cells, thereby initiating degradative events. Direct in vivo evidence for the source of collagenase and other MMPs in periodontal tissues is limited. By using specific polyclonal antibodies and indirect immunofluorescence, we could demonstrate the presence of collagenase, stromelysin-1, gelatinase A, and TIMP in human gingival biopsy specimens.(ABSTRACT TRUNCATED AT 250 WORDS)
Biochimica Et Biophysica Acta - General Subjects, Nov 11, 1994
Matrix proteins were extracted from bovine cortical bone with EDTA/Tris-HCl under non-dissociativ... more Matrix proteins were extracted from bovine cortical bone with EDTA/Tris-HCl under non-dissociative conditions at neutral pH. Four distinct bone resorptive proteins with molecular masses of 14, 25, 29 and 40 kDa were purified and partially characterized using an in vitro neonatal mouse calvarial assay and a growth factor assay using BALB/c/3T3 cells. The 14 kDa protein was purified by anion exchange chromatography (Mono Q) and gel filtration (Superdex 75HR) using FPLC (fast protein liquid chromatography); this factor stimulated the proliferation of MCF-7 human breast cancer cells, a bioassay which is specific for the insulin-like growth factors (IGFs). The 25, 29 and 40 kDa proteins were purified by sequential chromatography as follows: anion-exchange (Mono Q), heparin-Sepharose, hydroxyapatite, concanavalin A-Sepharose, phenyl-Superose, reversed phase high performance liquid chromatography (HPLC) and sodium dodecylsulfate polyacrylamide gelelectrophoresis (SDS-PAGE). The 25 kDa protein was identified as TGF-beta by its inhibitory effect on the proliferation of mink lung cells. The 40 kDa protein enhanced the formation of multinucleate tartrate-resistant acid phosphatase positive cells in a murine bone marrow differentiation assay, but was without effect in an isolated osteoclast assay and had no growth factor activity; this protein is likely to be a colony stimulating factor. The 29 kDa protein was also without growth factor activity; it was, however, able to stimulate bone resorption in the isolated osteoclast assay, suggesting a direct action in osteoclast function. The 29 and 40 kDa proteins may be osteoblast gene products that have been sequestrated by the bone matrix in a similar fashion to TGF-beta and the IGFs. This is the first report of proteins isolated from bone matrix which directly stimulate osteoclast differentiation and activity.
Members of the family of matrix metalloproteinases (MMPs) are key enzymes in normal and pathologi... more Members of the family of matrix metalloproteinases (MMPs) are key enzymes in normal and pathological tissue remodelling. They function at neutral pH and can digest synergistically all the macromolecules of the extracellular matrix. Biochemical and cloning studies indicate that there are three major groups: the specific collagenases cleave interstitial collagens; the gelatinases degrade other types of collagen and act synergistically with collagenases by degrading denatured collagens (gelatins); and the stromelysins which have broader specificity and can degrade basement membrane collagens as well as proteoglycans and matrix glycoproteins. Others in the family, but not in the major groups, are matrilysin, metallo-elastase, and several recently cloned membrane-bound metalloproteinases. Naturally occurring inhibitors, TIMPs (tissue inhibitors of metalloproteinases), are important controlling factors in the actions of MMPs, and tissue destruction in disease processes often correlates with an imbalance of MMPs over TIMPs. The relevance of recent molecular research to periodontal diseases is discussed.
The forces that orthodontic appliances apply to the teeth are transmitted through the periodontal... more The forces that orthodontic appliances apply to the teeth are transmitted through the periodontal ligament (PDL) to the supporting alveolar bone, leading to the deposition or resorption of bone, depending upon whether the tissues are exposed to a tensile or compressive mechanical strain. To evaluate the osteogenic potential of PDL cells, we applied a 12% uni-axial cyclic tensile strain to cultured human PDL cells and analyzed the differential expression of 78 genes implicated in osteoblast differentiation and bone metabolism by real-time RT-PCR array technology. Sixteen genes showed statistically significant changes in expression in response to alterations in their mechanical environment, including cell adhesion molecules and collagen fiber types. Genes linked to the osteoblast phenotype that were up-regulated included BMP2, BMP6, ALP, SOX9, MSX1, and VEGFA; those down-regulated included BMP4 and EGF. This study has expanded our knowledge of the transcriptional profile of PDL cells and identified several new mechanoresponsive genes.
2341 MSX1 Sequence Changes in Isolated Hypodontia. M. HARRISON 1 , M. MEIKLE 1 , and S. MALCOLM 2... more 2341 MSX1 Sequence Changes in Isolated Hypodontia. M. HARRISON 1 , M. MEIKLE 1 , and S. MALCOLM 2 , 1 Guy's, Kings' and St Thomas' Dental Institute, London, United Kingdom, 2 Institute of Child Health, London, United Kingdom ...
Mechanical strain is an important determinant of bone mass and architecture, and the aim of this ... more Mechanical strain is an important determinant of bone mass and architecture, and the aim of this investigation was to further understand the role of the cell–cell signaling molecules, IL-1β, TNF-α, and IL-6 in the mechanobiology of bone. Mouse calvarial osteoblasts in mono- layer culture were subjected to a cyclic out-of-plane deformation of 0.69% for 6 s, every 90 s for 2–48 h, and the levels of each cytokine plus their downstream targets RANKL and OPG measured in culture supernatants by ELISAs. Mouse osteoblasts constitutively synthesized IL-1β, TNF-α, and IL-6, the production of which was significantly up-regulated in all three by cyclic mechanical strain. RANKL and OPG were also constitutively synthesized; mechan- ical deformation however, resulted in a down-regulation of RANKL and an up-regulation OPG synthesis. We next tested whether the immunoreactive RANKL and OPG were bio- logically active in an isolated osteoclast resorption pit assay – this showed that culture supernatants from mechanically deformed cells significantly inhibited osteoclast-mediated resorptive activity across the 48 h time-course.These findings are counterintuitive, because IL-1β, TNF-α, and IL-6 have well-established reputations as bone resorptive agents. Never- theless, they are pleiotropic molecules with multiple biological activities, underlining the complexity of the biological response of osteoblasts to mechanical deformation, and the need to understand cell–cell signaling in terms of cytokine networks. It is also important to recognize that osteoblasts cultured in vitro are deprived of the mechanical stimuli to which they are exposed in vivo – in other words, the cells are in a physiological default state that in the intact skeleton leads to decreased bone strains below the critical threshold required to maintain normal bone structure.
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