Background
The innate immune response (IMR) is critical for the oral mucosa due to their continuous exposure to various oral pathogens. Keratinocytes play important role in IMR. Therefore, to date, ...keratinocytes from different sources have been used as in vitro research model for the study of IMR. However, current keratinocyte research models are hampered by the limited supply, patients' dependency and batch to batch variation. Therefore, in this study, we demonstrated the use of human embryonic stem cells (hESCs) derived keratinocytes (H9‐Kert) as an alternative research model for the study of IMR.
Methods
The expression kinetics of toll‐like receptor (TLR) 2, TLR 4, interleukin (IL) ‐6, IL‐8, inducible nitric oxide synthase (iNOS) and tumour necrosis factor‐alpha (TNF‐α), in H9‐Kert and immortalized human keratinocyte cell line (HaCaT) were analysed at mRNA levels by both reverse transcription polymerase chain reaction (RT‐PCR) and quantitative real‐time RT‐PCR. The activation of the inflammatory transcription factor nuclear factor kappa‐b (NFĸB) was assayed in these cells by transiently transfecting the cells with NFĸB reporter plasmid. Activation of NFĸB following treatment with heat‐killed Porphyromonas gingivalis (P. gingivalis), an oral pathogen, was determined by assaying for the reporter, secreted alkaline phosphatase activity.
Results
The expression of TLRs, cytokines and activation of NFĸB following bacterial stimulation showed in both H9‐Kert and the widely used HaCaT keratinocyte cell line was similar.
Conclusion
Overall, our results support the potential application of hESCs as an alternative limitless cell source for primary keratinocytes which can be used as consistent and dependable research tool with minimum variations and no donor's dependency.
Periodontal disease is associated with changes in the composition of the oral microflora, where health-associated oral streptococci decrease while Gram-negative anaerobes predominate in disease. A ...key feature of periodontal disease-associated anaerobes is their ability to produce hydrogen sulfide (H2S) abundantly as a by-product of anaerobic metabolism. So far, H2S has been reported to be either cytoprotective or cytotoxic by modulating bacterial antioxidant defense systems. Although oral anaerobes produce large amounts of H2S, the potential effects of H2S on oral streptococci are currently unknown. The aim of this study was to determine the effects of H2S on the survival and biofilm formation of oral streptococci. The growth and biofilm formation of Streptococcus mitis and Streptococcus oralis were inhibited by H2S. However, H2S did not significantly affect the growth of Streptococcus gordonii or Streptococcus sanguinis. The differential susceptibility of oral streptococci to H2S was attributed to differences in the intracellular concentrations of reduced glutathione (GSH). In the absence of GSH, H2S elicited its toxicity through an iron-dependent mechanism. Collectively, our results showed that H2S exerts antimicrobial effects on certain oral streptococci, potentially contributing to the decrease in health-associated plaque microflora.
Gingival connective tissue and its vasculature play a crucial role in the host's immune response against the periodontal microbiome and serve as a bridge between the oral and systemic environments. ...However, there is a lack of representative models that mimic the complex features of vascularized gingival connective tissue and its interaction with the periodontal microbiome, hindering our understanding of periodontal health and disease. Towards this pursuit, we present the characterization of vascularized gingival connective tissue equivalents (CTEs) as a model to study the interactions between oral biofilm colonizers and gingival tissues in healthy and diseased states. Whole-mount immunolabeling and label-free confocal reflectance microscopy of human fibrin-based matrix embedded with gingival fibroblasts and microvascular endothelial cells demonstrated the generation of bi-cellular vascularized gingival CTEs. Next, we investigated the response of the vascularized gingival CTEs to early, intermediate, and late oral biofilm colonizers. Despite colonization, the early colonizers did not elicit any significant change in the production of the cytokines and chemokines by the CTEs representative of the commensal and homeostatic state. In contrast, intermediate and late colonizers representing a transition to a diseased state exhibited connective tissue and vascular invasion, and elicited a differential immune response accompanied by increased monocyte migration. The culture supernatants produced by the vascularized gingival CTEs in response to early and intermediate colonizers polarized macrophages towards an immunomodulatory M2-like phenotype which activates and protects the host, while the late colonizers polarized towards a pro-inflammatory M1-like phenotype. Lastly,
analysis showed a high strength of associations between the proteins and transcripts investigated with periodontitis and vascular diseases. In conclusion, the vascularized gingival CTEs provide a biomimetic
platform to study host-microbiome interactions and innate immune response in periodontal health and diseased states, which potentially paves the way toward the development and assessment of novel periodontal therapeutics.
Gingival crevice and gingival crevicular fluid (GCF) flow play a crucial role at the gingiva‐oral microbiome interface which contributes toward maintaining the balance between gingival health and ...periodontal disease. Interstitial flow of GCF strongly impacts the host‐microbiome interactions and tissue responses. However, currently available in vitro preclinical models largely disregard the dynamic nature of gingival crevicular microenvironment, thus limiting the progress in the development of periodontal therapeutics. Here, a proof‐of‐principle “gingival crevice‐on‐chip” microfluidic platform to culture gingival connective tissue equivalent (CTE) under dynamic interstitial fluid flow mimicking the GCF is described. On‐chip co‐culture using oral symbiont (Streptococcus oralis) shows the potential to recapitulate microbial colonization, formation of biofilm‐like structures at the tissue‐microbiome interface, long‐term co‐culture, and bacterial clearance secondary to simulated GCF (s‐GCF) flow. Further, on‐chip exposure of the gingival CTEs to the toll‐like receptor‐2 (TLR‐2) agonist or periodontal pathogen Fusobacterium nucleatum demonstrates the potential to mimic early gingival inflammation. In contrast to direct exposure, the induction of s‐GCF flow toward the bacterial front attenuates the secretion of inflammatory mediators demonstrating the protective effect of GCF flow. This proposed in vitro platform offers the potential to study complex host‐microbe interactions in periodontal disease and the development of periodontal therapeutics under near‐microphysiological conditions.
The authors present a gingival crevice‐on‐chip microfluidic device to model the gingival crevicular microenvironment and unidirectional flow of gingival crevicular fluid. The platform facilitates compartmentalization of host tissue and oral microbiome, and long‐term microbial colonization. Importantly, it enables the simulation of the protective roles of gingival crevicular fluid flow that include bacterial clearance and immune attenuation.
Program in Infectious Diseases, Department of Microbiology, Faculty of Medicine, National University of Singapore, Singapore
Corresponding author: Dr K.P. Song (e-mail: micskp{at}nus.edu.sg ).
...Received 23 Aug. 2000; revised version accepted 11 Dec. 2000.
Abstract
Toxigenic strains of Clostridium difficile produce two large bacterial toxins called toxins A (TcdA) and B (TcdB). tcdA and tcdB genes are located on the pathogenicity locus of C. difficile , a unique characteristic of toxigenic strains of this species. Intergenic to the two toxin genes is tcdE , a small 501-bp open reading frame of unknown function. Expression of the tcdE gene in Escherichia coli caused bacterial cell death. Computational analysis of the amino acid sequence of TcdE revealed structural features that are strikingly similar to a class of bacteriophage proteins called holins. Holins are cytolytic proteins that cause lysis of bacterial hosts to effect the release of progeny phages. Further analysis of the recombinant clone expressing TcdE by transmission electron microscopy confirmed that the site of action of TcdE is on the bacterial cell membrane. The results provide evidence that TcdE is structurally and functionally similar to holin proteins. TcdE may function as a lytic protein to facilitate the release of TcdA and TcdB to the extracellular environment, as these toxins lack signal peptide.
Oral mucositis (OM) is a common complication of cancer therapy, however OM management remains unsatisfactory. There is a growing interest in the therapeutic potential of probiotics in OM due to ...positive findings of its use in intestinal mucositis. This study aimed to determine the efficacy and safety of the probiotic combination Lactobacillus reuteri DSM 17938 and ATCC PTA 5289 strains in chemotherapy-induced OM. Mice were divided into 4 groups. PBS/water and PBS/LR groups comprised of mice injected with PBS intraperitoneally (i.p.), and were given water or the mixture of L. reuteri (LR) DSM 17938 and ATCC PTA 5289 in water respectively. The 5-FU/water and 5-FU/LR groups comprised of mice injected with 5-FU i.p., and were given water or L. reuteri DSM 17938 and ATCC PTA 5289 in water respectively. Histopathological analysis revealed that the oral epithelia of the 5-FU/water and 5-FU/LR groups were thinner compared to PBS/water and PBS/LR groups. However, epithelial damage was significantly reduced in the 5-FU/LR compared to 5-FU/water group. Additionally, the 5-FU/LR group showed reduced oxidative stress and inflammation in the oral mucosa. We further showed that L. reuteri reduced oxidative stress through the nuclear factor E2-related factor-2 (Nrf-2) signalling. There was no evidence of translocation of L. reuteri systemically. This study demonstrated for the first time that L. reuteri protected oral mucosa against damage induced by chemotherapy.
ABSTRACT
Periodontal disease is associated with changes in the composition of the oral microflora, where health-associated oral streptococci decrease while Gram-negative anaerobes predominate in ...disease. A key feature of periodontal disease-associated anaerobes is their ability to produce hydrogen sulfide (H
2
S) abundantly as a by-product of anaerobic metabolism. So far, H
2
S has been reported to be either cytoprotective or cytotoxic by modulating bacterial antioxidant defense systems. Although oral anaerobes produce large amounts of H
2
S, the potential effects of H
2
S on oral streptococci are currently unknown. The aim of this study was to determine the effects of H
2
S on the survival and biofilm formation of oral streptococci. The growth and biofilm formation of
Streptococcus mitis
and
Streptococcus oralis
were inhibited by H
2
S. However, H
2
S did not significantly affect the growth of
Streptococcus gordonii
or
Streptococcus sanguinis
. The differential susceptibility of oral streptococci to H
2
S was attributed to differences in the intracellular concentrations of reduced glutathione (GSH). In the absence of GSH, H
2
S elicited its toxicity through an iron-dependent mechanism. Collectively, our results showed that H
2
S exerts antimicrobial effects on certain oral streptococci, potentially contributing to the decrease in health-associated plaque microflora.
Periodontal disease is associated with changes in the composition of the oral microflora, where health-associated oral streptococci decrease while Gram-negative anaerobes predominate in disease. A ...key feature of periodontal disease-associated anaerobes is their ability to produce hydrogen sulfide (H...S) abundantly as a by-product of anaerobic metabolism. So far, H...S has been reported to be either cytoprotective or cytotoxic by modulating bacterial antioxidant defense systems. Although oral anaerobes produce large amounts of H...S, the potential effects of H...S on oral streptococci are currently unknown. The aim of this study was to determine the effects of H...S on the survival and biofilm formation of oral streptococci. The growth and biofilm formation of Streptococcus mitis and Streptococcus oralis were inhibited by H...S. However, H...S did not significantly affect the growth of Streptococcus gordonii or Streptococcus sanguinis. The differential susceptibility of oral streptococci to H...S was attributed to differences in the intracellular concentrations of reduced glutathione (GSH). In the absence of GSH, H...S elicited its toxicity through an iron-dependent mechanism. Collectively, our results showed that H...S exerts antimicrobial effects on certain oral streptococci, potentially contributing to the decrease in health-associated plaque microflora. (ProQuest: ... denotes formulae/symbols omitted.)
Ciprofloxacin, amoxicillin, and metronidazole are antibiotics used in regenerative endodontic therapy (RET). Although their antimicrobial properties are well-documented, there is a lack of ...information on the effects of these antibiotics on the immune response by host macrophages and periapical healing. Thus, this study had 2 objectives: (1) to determine the immune response of macrophages to bacterial infection in response to the combination of ciprofloxacin or amoxicillin and metronidazole and (2) using conditioned media produced by these macrophages to simulate the periapical microenvironment, to determine the impact on the expression of extracellular matrix (ECM) components by periodontal fibroblasts.
Macrophages were treated with ciprofloxacin and metronidazole or amoxicillin and metronidazole at 10–1000 μg/mL. The treated macrophages were exposed to lipopolysaccharide, and the pro- and anti-inflammatory cytokines produced were quantified with enzyme-linked immunosorbent assay. Periodontal fibroblasts were treated with conditioned media from these treated macrophages, and the expression of ECM genes was determined by quantitative polymerase chain reaction.
Lipopolysaccharides elicited the production of proinflammatory cytokines interleukin 1 beta and tumor necrosis factor alpha by macrophages, but this was suppressed by ciprofloxacin and metronidazole. Moreover, only conditioned media from macrophages treated with ciprofloxacin and metronidazole rescued microbial-induced down-regulation of ECM genes by periodontal fibroblasts. Specifically, ciprofloxacin was the antibiotic responsible for these observations. In contrast, these effects were not observed with amoxicillin and metronidazole.
Apart from disinfection of the root canal system, the combination of ciprofloxacin and metronidazole also exerts an immunomodulatory effect, which may aid in periapical healing.
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