Methods in molecular biology (Clifton, N.J.), 2016
The fungus Candida albicans and bacterium Staphylococcus aureus can coexist in polymicrobial biof... more The fungus Candida albicans and bacterium Staphylococcus aureus can coexist in polymicrobial biofilms. S. aureus attaches strongly to hyphae, but not to the yeast form, of C. albicans with important consequences for virulence. Hyphae-associated S. aureus is less susceptible to antibiotic treatment. Furthermore, co-inoculation of C. albicans and S. aureus causes more severe and widespread infection than either microorganism alone. In this chapter, a basic in vitro model for studying the interaction between C. albicans hyphae and S. aureus is presented, which makes use of a fluorescently labeled S. aureus strain. Furthermore, two protocols are described that allow investigation of the effect of C. albicans and S. aureus interaction on antibiotic susceptibility or on interactions with the host. The latter focuses on phagocytosis of C. albicans-adhered S. aureus by macrophages. The protocols presented here may serve as a starting point to study the interaction of C. albicans with variou...
The inflammatory cytokine tumor necrosis factor-alpha (TNF-α) is elevated in inflamed periodontal... more The inflammatory cytokine tumor necrosis factor-alpha (TNF-α) is elevated in inflamed periodontal tissues and may contribute to periodontitis progression. TNF-α stimulates formation and activity of osteoclasts, the cells that are recruited in periodontitis, that cause alveolar bone degradation and subsequent tooth loss. We previously showed that TNF-α is elevated in co-cultures of periodontal ligament fibroblast (PDLF) and peripheral blood mononuclear cells (PBMC). Hence, TNF-α could be a determining factor in osteoclast formation in these cultures, as osteoclasts are formed despite the fact that prototypical osteoclast differentiation factor receptor activator of nuclear factor kappa-B ligand is outnumbered at least 100-fold by its inhibitor osteoprotegerin in these cultures. To assess the role of TNF-α in periodontitis-associated osteoclast formation in vitro, osteoclast formation was analyzed in the presence of the anti-TNF-α therapeutic agent infliximab in two culture systems: (...
Dental plaque is a multispecies biofilm in the oral cavity that significantly influences oral hea... more Dental plaque is a multispecies biofilm in the oral cavity that significantly influences oral health. The presence of the oral anaerobic pathogen Porphyromonas gingivalis is an important determinant in the development of periodontitis. Direct and indirect interactions between P. gingivalis and the host play a major role in disease development. Transcriptome analysis recently revealed that P. gingivalis gene-expression is regulated by LuxS in both an AI-2-dependent and an AI-2 independent manner. However, little is known about the role of LuxS-signaling in P. gingivalis-host interactions. Here, we investigated the effect of a luxS mutation on the ability of P. gingivalis to induce an inflammatory response in human oral cells in vitro. Primary periodontal ligament (PDL) fibroblasts were challenged with P. gingivalis ΔluxS or the wild-type parental strain and gene-expression of pro-inflammatory mediators IL-1β, IL-6 and MCP-1 was determined by real-time PCR. The ability of P. gingivalis ΔluxS to induce an inflammatory response was severely impaired in PDL-fibroblasts. This phenotype could be restored by providing of LuxS in trans, but not by addition of the AI-2 precursor DPD. A similar phenomenon was observed in a previous transcriptome study showing that expression of PGN_0482 was reduced in the luxS mutant independently of AI-2. We therefore also analyzed the effect of a mutation in PGN_0482, which encodes an immuno-reactive, putative outer-membrane protein. Similar to P. gingivalis ΔluxS, the P. gingivalis Δ0482 mutant had an impaired ability to induce an inflammatory response in PDL fibroblasts. LuxS thus appears to influence the pro-inflammatory responses of host cells to P. gingivalis, likely through regulation of PGN_0482.
Porphyromonas gingivalis is an oral pathogen strongly associated with destruction of the tooth-su... more Porphyromonas gingivalis is an oral pathogen strongly associated with destruction of the tooth-supporting tissues in human periodontitis. Gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDLF) are functionally different cell types in the periodontium that can participate in the host immune response in periodontitis. This study aimed to investigate the effects of viable P. gingivalis on the expression of genes associated with inflammation and bone degradation by these fibroblast subsets. Primary human GF and PDLF from six healthy donors were challenged in vitro with viable P. gingivalis W83 for 6 h. Gene expression of inflammatory cytokines in GF and PDLF was analyzed using real-time PCR, and protein expression was analyzed using ELISA. Viable P. gingivalis induced a strong in vitro inflammatory response in both GF and PDLF. We found increased gene expression of interleukin (IL)-1beta, IL-6, IL-8, tumor necrosis factor-alpha, monocyte chemotactic protein-1 and regulated upon activation, normal T-cell expressed and secreted (RANTES). Macrophage colony-stimulating factor was induced and the expression of osteoprotegerin was decreased in GF, but not in PDLF. In nonchallenged cells, a higher level of expression of IL-6 was observed in GF than in PDLF. Between individual donors there was large heterogeneity in responsiveness to P. gingivalis. Also, in each individual, either GF or PDLF was more responsive to P. gingivalis. Considerable heterogeneity in responsiveness to P. gingivalis exists both between GF and PDLF and between individuals, which may be crucial determinants for the susceptibility to develop periodontitis.
To assess inflammatory reactions of fibroblasts in the pathophysiology of peri-implantitis, we co... more To assess inflammatory reactions of fibroblasts in the pathophysiology of peri-implantitis, we compared the pro-inflammatory and matrix-degrading responses of gingival and granulation tissue fibroblasts from periodontally healthy controls, peri-implantitis, and periodontitis lesions to an in vitro challenge with Porphyromonas gingivalis. Fibroblasts from periodontally healthy, peri-implantitis and periodontitis donors were challenged with viable P. gingivalis. The inflammatory reactions of fibroblasts were analyzed before and after 6 h P. gingivalis challenge, and 2.5 and 18 h after removal of the challenge. Gene expression and induction of pro-inflammatory mediators, and matrix metalloproteinases (MMPs) were assessed by real-time polymerase chain reaction. Protein expression was measured by enzyme-linked immunosorbent assay. Non-challenged fibroblasts from peri-implantitis and periodontitis lesions expressed higher levels of interleukin (IL)-1β, IL-8, and monocyte chemotactic protein (MCP)-1 than fibroblasts from periodontally healthy individuals. The P. gingivalis challenge induced expression of IL-1β, IL-8, IL-6, MCP-1, and MMP-1 in periodontitis and peri-implantitis fibroblasts, but not in fibroblasts from periodontally healthy individuals. MMP-8 expression was higher in non-challenged peri-implantitis fibroblasts than in fibroblasts from periodontally healthy individuals. However, the P. gingivalis challenge downregulated MMP-8 gene expression in peri-implantitis fibroblasts. After removal of the P. gingivalis challenge, peri-implantitis fibroblasts sustained higher induction of IL-1β, MCP-1, and MMP-1 compared to periodontitis fibroblasts. Fibroblasts from peri-implantitis and periodontitis lesions gave a more pronounced inflammatory response to the P. gingivalis challenge than fibroblasts from healthy donors. They may therefore be involved in the development of inflammation in peri-implantitis and periodontitis. Moreover, the sustained upregulation of inflammatory mediators and MMP-1 in peri-implantitis fibroblasts may play a role in the pathogenesis of peri-implantitis.
Titanium wear particles have been found in peri-implant tissues, but their role in the pathogenes... more Titanium wear particles have been found in peri-implant tissues, but their role in the pathogenesis of peri-implantitis remains unclear. We aimed to determine the in vitro inflammatory responses of peri-implant granulation tissue fibroblasts (PIGFs) to titanium particles alone and in the presence of viable Porphyromonas gingivalis. Peri-implant granulation tissue fibroblasts were challenged either with TiO2 particles, P. gingivalis or a combination of TiO2 particles and P. gingivalis. Gene expression and protein production of pro-inflammatory mediators by PIGFs were measured with PCR and ELISA, respectively. Higher doses of TiO2 were toxic to PIGFs and in sub-toxic doses, TiO2 caused an increase in gene expression of tumour necrosis factor (TNF)-A and increased protein production of TNF-α, interleukin (IL)-6 and IL-8. A challenge with P. gingivalis alone induced gene expression of TNF-A, IL-1β, IL-6 and IL-8. A combined challenge with TiO2 and P. gingivalis caused a stronger increase in gene expression of TNF-A and protein production of TNF-α and MCP-1 than P. gingivalis alone. TiO2 particles and P. gingivalis, individually, can induce pro-inflammatory responses in PIGFs. Furthermore, TiO2 particles and viable P. gingivalis further enhance gene expression and production of TNF-α by PIGFs. Therefore, Ti wear particles in the peri-implant tissues in combination with P. gingivalis infection may contribute to the pathogenesis of peri-implantitis by enhancing the inflammation in peri-implant tissues.
ABSTRACT Objectives: Porphyromonas gingivalis is strongly associated with periodontitis. A major ... more ABSTRACT Objectives: Porphyromonas gingivalis is strongly associated with periodontitis. A major clinical symptom of periodontitis is alveolar bone loss due to resorptive activity by osteoclasts. Gingiva fibroblasts (GF) respond in vitro to P. gingivalis by producing pro-inflammatory factors that can play a role in osteoclast formation. Also, P. gingivalis-factors may influence osteoclast formation directly. To investigate if P. gingivalis influences osteoclast formation in vitro, we analyzed the effects of conditioned-medium from P. gingivalis alone, or from human GF challenged with P. gingivalis, on the formation of TRACP-positive multinucleated cells (TRACP+MNCs). Methods: Conditioned-medium from viable or heat-inactivated P. gingivalis alone (Pg-conditioned-medium), or conditioned-medium from human GF (6 healthy donors) challenged for 6h with viable or heat-inactivated P. gingivalis (GF-Pg-conditioned-medium), was added (conc.1:2) to human peripheral blood mononuclear cells containing osteoclast-precursors, and refreshed every 3 days. After 21 days, cells were stained for TRACP. The number of TRACP+MNCs (>3 nuclei) per mm2 was determined as a measure for osteoclast formation. Results: With viable bacteria, Pg-conditioned-medium stimulated the formation of TRACP+MNCs (206% increase over medium alone, p=0.0374). GF-Pg-conditioned-medium also stimulated the formation of TRACP+MNCs (199% increase over medium from only GF, p=0.0058), similar to Pg-conditioned medium, suggesting no additional effect of GF. However, Pg-conditioned-medium from dead bacteria had no effect on the formation of TRACP+MNCs, whereas GF-Pgdead-conditioned-medium did (40% increase over medium from only GF, p=0.0294), indicating that P. gingivalis stimulated GF to produce factors that had an additional effect. We assume that viable bacteria degraded GF-produced factors in GF-Pg-conditioned medium, but at the same time released factors that stimulated the formation of TRACP+MNCs. Conclusion: P. gingivalis stimulates the formation of TRACP+MNCs in vitro in two ways. Directly, by bacterial factors secreted into the medium by viable bacteria, or indirectly, by GF-factors produced in response to P. gingivalis.
Methods in molecular biology (Clifton, N.J.), 2016
The fungus Candida albicans and bacterium Staphylococcus aureus can coexist in polymicrobial biof... more The fungus Candida albicans and bacterium Staphylococcus aureus can coexist in polymicrobial biofilms. S. aureus attaches strongly to hyphae, but not to the yeast form, of C. albicans with important consequences for virulence. Hyphae-associated S. aureus is less susceptible to antibiotic treatment. Furthermore, co-inoculation of C. albicans and S. aureus causes more severe and widespread infection than either microorganism alone. In this chapter, a basic in vitro model for studying the interaction between C. albicans hyphae and S. aureus is presented, which makes use of a fluorescently labeled S. aureus strain. Furthermore, two protocols are described that allow investigation of the effect of C. albicans and S. aureus interaction on antibiotic susceptibility or on interactions with the host. The latter focuses on phagocytosis of C. albicans-adhered S. aureus by macrophages. The protocols presented here may serve as a starting point to study the interaction of C. albicans with variou...
The inflammatory cytokine tumor necrosis factor-alpha (TNF-α) is elevated in inflamed periodontal... more The inflammatory cytokine tumor necrosis factor-alpha (TNF-α) is elevated in inflamed periodontal tissues and may contribute to periodontitis progression. TNF-α stimulates formation and activity of osteoclasts, the cells that are recruited in periodontitis, that cause alveolar bone degradation and subsequent tooth loss. We previously showed that TNF-α is elevated in co-cultures of periodontal ligament fibroblast (PDLF) and peripheral blood mononuclear cells (PBMC). Hence, TNF-α could be a determining factor in osteoclast formation in these cultures, as osteoclasts are formed despite the fact that prototypical osteoclast differentiation factor receptor activator of nuclear factor kappa-B ligand is outnumbered at least 100-fold by its inhibitor osteoprotegerin in these cultures. To assess the role of TNF-α in periodontitis-associated osteoclast formation in vitro, osteoclast formation was analyzed in the presence of the anti-TNF-α therapeutic agent infliximab in two culture systems: (...
Dental plaque is a multispecies biofilm in the oral cavity that significantly influences oral hea... more Dental plaque is a multispecies biofilm in the oral cavity that significantly influences oral health. The presence of the oral anaerobic pathogen Porphyromonas gingivalis is an important determinant in the development of periodontitis. Direct and indirect interactions between P. gingivalis and the host play a major role in disease development. Transcriptome analysis recently revealed that P. gingivalis gene-expression is regulated by LuxS in both an AI-2-dependent and an AI-2 independent manner. However, little is known about the role of LuxS-signaling in P. gingivalis-host interactions. Here, we investigated the effect of a luxS mutation on the ability of P. gingivalis to induce an inflammatory response in human oral cells in vitro. Primary periodontal ligament (PDL) fibroblasts were challenged with P. gingivalis ΔluxS or the wild-type parental strain and gene-expression of pro-inflammatory mediators IL-1β, IL-6 and MCP-1 was determined by real-time PCR. The ability of P. gingivalis ΔluxS to induce an inflammatory response was severely impaired in PDL-fibroblasts. This phenotype could be restored by providing of LuxS in trans, but not by addition of the AI-2 precursor DPD. A similar phenomenon was observed in a previous transcriptome study showing that expression of PGN_0482 was reduced in the luxS mutant independently of AI-2. We therefore also analyzed the effect of a mutation in PGN_0482, which encodes an immuno-reactive, putative outer-membrane protein. Similar to P. gingivalis ΔluxS, the P. gingivalis Δ0482 mutant had an impaired ability to induce an inflammatory response in PDL fibroblasts. LuxS thus appears to influence the pro-inflammatory responses of host cells to P. gingivalis, likely through regulation of PGN_0482.
Porphyromonas gingivalis is an oral pathogen strongly associated with destruction of the tooth-su... more Porphyromonas gingivalis is an oral pathogen strongly associated with destruction of the tooth-supporting tissues in human periodontitis. Gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDLF) are functionally different cell types in the periodontium that can participate in the host immune response in periodontitis. This study aimed to investigate the effects of viable P. gingivalis on the expression of genes associated with inflammation and bone degradation by these fibroblast subsets. Primary human GF and PDLF from six healthy donors were challenged in vitro with viable P. gingivalis W83 for 6 h. Gene expression of inflammatory cytokines in GF and PDLF was analyzed using real-time PCR, and protein expression was analyzed using ELISA. Viable P. gingivalis induced a strong in vitro inflammatory response in both GF and PDLF. We found increased gene expression of interleukin (IL)-1beta, IL-6, IL-8, tumor necrosis factor-alpha, monocyte chemotactic protein-1 and regulated upon activation, normal T-cell expressed and secreted (RANTES). Macrophage colony-stimulating factor was induced and the expression of osteoprotegerin was decreased in GF, but not in PDLF. In nonchallenged cells, a higher level of expression of IL-6 was observed in GF than in PDLF. Between individual donors there was large heterogeneity in responsiveness to P. gingivalis. Also, in each individual, either GF or PDLF was more responsive to P. gingivalis. Considerable heterogeneity in responsiveness to P. gingivalis exists both between GF and PDLF and between individuals, which may be crucial determinants for the susceptibility to develop periodontitis.
To assess inflammatory reactions of fibroblasts in the pathophysiology of peri-implantitis, we co... more To assess inflammatory reactions of fibroblasts in the pathophysiology of peri-implantitis, we compared the pro-inflammatory and matrix-degrading responses of gingival and granulation tissue fibroblasts from periodontally healthy controls, peri-implantitis, and periodontitis lesions to an in vitro challenge with Porphyromonas gingivalis. Fibroblasts from periodontally healthy, peri-implantitis and periodontitis donors were challenged with viable P. gingivalis. The inflammatory reactions of fibroblasts were analyzed before and after 6 h P. gingivalis challenge, and 2.5 and 18 h after removal of the challenge. Gene expression and induction of pro-inflammatory mediators, and matrix metalloproteinases (MMPs) were assessed by real-time polymerase chain reaction. Protein expression was measured by enzyme-linked immunosorbent assay. Non-challenged fibroblasts from peri-implantitis and periodontitis lesions expressed higher levels of interleukin (IL)-1β, IL-8, and monocyte chemotactic protein (MCP)-1 than fibroblasts from periodontally healthy individuals. The P. gingivalis challenge induced expression of IL-1β, IL-8, IL-6, MCP-1, and MMP-1 in periodontitis and peri-implantitis fibroblasts, but not in fibroblasts from periodontally healthy individuals. MMP-8 expression was higher in non-challenged peri-implantitis fibroblasts than in fibroblasts from periodontally healthy individuals. However, the P. gingivalis challenge downregulated MMP-8 gene expression in peri-implantitis fibroblasts. After removal of the P. gingivalis challenge, peri-implantitis fibroblasts sustained higher induction of IL-1β, MCP-1, and MMP-1 compared to periodontitis fibroblasts. Fibroblasts from peri-implantitis and periodontitis lesions gave a more pronounced inflammatory response to the P. gingivalis challenge than fibroblasts from healthy donors. They may therefore be involved in the development of inflammation in peri-implantitis and periodontitis. Moreover, the sustained upregulation of inflammatory mediators and MMP-1 in peri-implantitis fibroblasts may play a role in the pathogenesis of peri-implantitis.
Titanium wear particles have been found in peri-implant tissues, but their role in the pathogenes... more Titanium wear particles have been found in peri-implant tissues, but their role in the pathogenesis of peri-implantitis remains unclear. We aimed to determine the in vitro inflammatory responses of peri-implant granulation tissue fibroblasts (PIGFs) to titanium particles alone and in the presence of viable Porphyromonas gingivalis. Peri-implant granulation tissue fibroblasts were challenged either with TiO2 particles, P. gingivalis or a combination of TiO2 particles and P. gingivalis. Gene expression and protein production of pro-inflammatory mediators by PIGFs were measured with PCR and ELISA, respectively. Higher doses of TiO2 were toxic to PIGFs and in sub-toxic doses, TiO2 caused an increase in gene expression of tumour necrosis factor (TNF)-A and increased protein production of TNF-α, interleukin (IL)-6 and IL-8. A challenge with P. gingivalis alone induced gene expression of TNF-A, IL-1β, IL-6 and IL-8. A combined challenge with TiO2 and P. gingivalis caused a stronger increase in gene expression of TNF-A and protein production of TNF-α and MCP-1 than P. gingivalis alone. TiO2 particles and P. gingivalis, individually, can induce pro-inflammatory responses in PIGFs. Furthermore, TiO2 particles and viable P. gingivalis further enhance gene expression and production of TNF-α by PIGFs. Therefore, Ti wear particles in the peri-implant tissues in combination with P. gingivalis infection may contribute to the pathogenesis of peri-implantitis by enhancing the inflammation in peri-implant tissues.
ABSTRACT Objectives: Porphyromonas gingivalis is strongly associated with periodontitis. A major ... more ABSTRACT Objectives: Porphyromonas gingivalis is strongly associated with periodontitis. A major clinical symptom of periodontitis is alveolar bone loss due to resorptive activity by osteoclasts. Gingiva fibroblasts (GF) respond in vitro to P. gingivalis by producing pro-inflammatory factors that can play a role in osteoclast formation. Also, P. gingivalis-factors may influence osteoclast formation directly. To investigate if P. gingivalis influences osteoclast formation in vitro, we analyzed the effects of conditioned-medium from P. gingivalis alone, or from human GF challenged with P. gingivalis, on the formation of TRACP-positive multinucleated cells (TRACP+MNCs). Methods: Conditioned-medium from viable or heat-inactivated P. gingivalis alone (Pg-conditioned-medium), or conditioned-medium from human GF (6 healthy donors) challenged for 6h with viable or heat-inactivated P. gingivalis (GF-Pg-conditioned-medium), was added (conc.1:2) to human peripheral blood mononuclear cells containing osteoclast-precursors, and refreshed every 3 days. After 21 days, cells were stained for TRACP. The number of TRACP+MNCs (>3 nuclei) per mm2 was determined as a measure for osteoclast formation. Results: With viable bacteria, Pg-conditioned-medium stimulated the formation of TRACP+MNCs (206% increase over medium alone, p=0.0374). GF-Pg-conditioned-medium also stimulated the formation of TRACP+MNCs (199% increase over medium from only GF, p=0.0058), similar to Pg-conditioned medium, suggesting no additional effect of GF. However, Pg-conditioned-medium from dead bacteria had no effect on the formation of TRACP+MNCs, whereas GF-Pgdead-conditioned-medium did (40% increase over medium from only GF, p=0.0294), indicating that P. gingivalis stimulated GF to produce factors that had an additional effect. We assume that viable bacteria degraded GF-produced factors in GF-Pg-conditioned medium, but at the same time released factors that stimulated the formation of TRACP+MNCs. Conclusion: P. gingivalis stimulates the formation of TRACP+MNCs in vitro in two ways. Directly, by bacterial factors secreted into the medium by viable bacteria, or indirectly, by GF-factors produced in response to P. gingivalis.
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Papers by Nina Scheres