Porphyromonas gingivalis induce apoptosis in human gingival epithelial cells through a gingipain-dependent mechanism

<p>Abstract</p> <p>Background</p> <p>The oral pathogen <it>Porphyromonas gingivalis </it>has been shown to modulate apoptosis in different cell types, but its effect on epithelial cells remains unclear.</p> <p>Results</p> <p>We demonstrate that primary human gingival epithelial cells (HGECs) challenged with live <it>P. gingivalis </it>for 24 hours exhibit apoptosis, and we characterize this by M30 epitope detection, caspase-3 activity, DNA fragmentation and Annexin-V staining. Live bacteria strongly upregulated intrinsic and extrinsic apoptotic pathways. Pro-apoptotic molecules such as caspase-3, -8, -9, Bid and Bax were upregulated after 24 hours. The anti-apoptotic Bcl-2 was also upregulated, but this was not sufficient to ensure cell survival. The main <it>P. gingivalis </it>proteases arginine and lysine gingipains are necessary and sufficient to induce host cell apoptosis. Thus, live <it>P. gingivalis </it>can invoke gingival epithelial cell apoptosis in a time and dose dependent manner with significant apoptosis occurring between 12 and 24 hours of challenge via a gingipain-dependent mechanism.</p> <p>Conclusion</p> <p>The present study provides evidence that live, but not heat-killed, <it>P. gingivalis </it>can induce apoptosis after 24 hours of challenge in primary human gingival epithelial cells. Either arginine or lysine gingipains are necessary and sufficient factors in <it>P. gingivalis </it>elicited apoptosis.</p

In vitro modeling of host-parasite interactions: the 'subgingival' biofilm challenge of primary human epithelial cells

BACKGROUND: Microbial biofilms are known to cause an increasing number of chronic inflammatory and infectious conditions. A classical example is chronic periodontal disease, a condition initiated by the subgingival dental plaque biofilm on gingival epithelial tissues. We describe here a new model that permits the examination of interactions between the bacterial biofilm and host cells in general. We use primary human gingival epithelial cells (HGEC) and an in vitro grown biofilm, comprising nine frequently studied and representative subgingival plaque bacteria. RESULTS: We describe the growth of a mature 'subgingival' in vitro biofilm, its composition during development, its ability to adapt to aerobic conditions and how we expose in vitro a HGEC monolayer to this biofilm.Challenging the host derived HGEC with the biofilm invoked apoptosis in the epithelial cells, triggered release of pro-inflammatory cytokines and in parallel induced rapid degradation of the cytokines by biofilm-generated enzymes. CONCLUSION: We developed an experimental in vitro model to study processes taking place in the gingival crevice during the initiation of inflammation. The new model takes into account that the microbial challenge derives from a biofilm community and not from planktonically cultured bacterial strains. It will facilitate easily the introduction of additional host cells such as neutrophils for future biofilm:host cell challenge studies. Our methodology may generate particular interest, as it should be widely applicable to other biofilm-related chronic inflammatory diseases

Periapical Microsurgery: The Effect of Root Dentinal Defects on Short- and Long-term Outcome

The purpose of this prospective clinical study is to evaluate the clinical outcome of endodontic microsurgery on roots exhibiting the presence or absence of dentinal defects at one year and three-year follow up period

Sphingosine Kinase-1 Is Required for Toll Mediated β-Defensin 2 Induction in Human Oral Keratinocytes

Host defense against invading pathogens is triggered by various receptors including toll-like receptors (TLRs). Activation of TLRs is a pivotal step in the initiation of innate, inflammatory, and antimicrobial defense mechanisms. Human beta-defensin 2 (HBD-2) is a cationic antimicrobial peptide secreted upon gram-negative bacterial perturbation in many cells. Stimulation of various TLRs has been shown to induce HBD-2 in oral keratinocytes, yet the underlying cellular mechanisms of this induction are poorly understood.Here we demonstrate that HBD-2 induction is mediated by the Sphingosine kinase-1 (Sphk-1) and augmented by the inhibition of Glycogen Synthase Kinase-3beta (GSK-3beta) via the Phosphoinositide 3-kinase (PI3K) dependent pathway. HBD-2 secretion was dose dependently inhibited by a pharmacological inhibitor of Sphk-1. Interestingly, inhibition of GSK-3beta by SB 216763 or by RNA interference, augmented HBD-2 induction. Overexpression of Sphk-1 with concomitant inhibition of GSK-3beta enhanced the induction of beta-defensin-2 in oral keratinocytes. Ectopic expression of constitutively active GSK-3beta (S9A) abrogated HBD-2 whereas kinase inactive GSK-3beta (R85A) induced higher amounts of HBD-2.These data implicate Sphk-1 in HBD-2 regulation in oral keratinocytes which also involves the activation of PI3K, AKT, GSK-3beta and ERK 1/2. Thus we reveal the intricate relationship and pathways of toll-signaling molecules regulating HBD-2 which may have therapeutic potential

The influence of CBCT-derived 3D-printed models on endodontic microsurgical treatment planning and confidence of the operator

Aims Use of 3D printed models in Endodontics has been gaining popularity since the technology to create them became more affordable. Currently, there are no studies that evaluate the influence of 3D models on endodontic surgical treatment planning and on operator confidence. Therefore, aims of this study were to: (i) Determine whether the availability of a 3D printed analogue can influence treatment-planning and operator confidence; and, (ii) Assess which factors of operator confidence are influenced, if any. Materials and Methods Endodontists were asked to analyze a pre-selected CBCT scan of an endodontic surgical case and to answer a questionnaire that determined their surgical approach for that case. After 30 days, the same participants were asked to analyze again the same CBCT scan. This time however, a 3D printed model of the scan was made available to the participants and to perform a mock osteotomy on the model. The participants were then asked to respond to the same questionnaire that they responded to 30 days prior to determine if there would be any changes to their treatment plan. A new set of questions were added to the survey to evaluate the influence of the 3D printed model on participants’ confidence in performing endodontic surgery. The responses were statistically analyzed using Chi square test followed by either logistic or ordered regression analysis while adjusting for experience of participant. Adjustment for multiple comparison analysis was done using Bonferroni correction. Statistical significance was set at £0.005. Results Availability of the 3D printed model and the CBCT scan together resulted in statistically significant differences in the participants’ responses to their ability to clearly detect bone landmarks, accurately predict the location of osteotomy, and in determining the following: size of osteotomy, angle of instrumentation, involvement of critical structures in flap reflection and involvement of vital structures during curettage. In addition, the participants’ confidence in performing surgery was significantly higher versus having CBCT scans alone. There were no statistically significant changes with decisions on flap design and extent, visualizing critical structures, lesion size, injury to vital structures during osteotomy, the length of root that could be resected and the number of roots involved. Conclusions The availability of 3D printed models did not alter the participants’ surgical approach, but it significantly improved their confidence for endodontic microsurgery, which can be attributed to better visualization of anatomical structures

The influence of CBCT-derived 3D-printed models on endodontic microsurgical treatment planning and confidence of the operator

Aims Use of 3D printed models in Endodontics has been gaining popularity since the technology to create them became more affordable. Currently, there are no studies that evaluate the influence of 3D models on endodontic surgical treatment planning and on operator confidence. Therefore, aims of this study were to: (i) Determine whether the availability of a 3D printed analogue can influence treatment-planning and operator confidence; and, (ii) Assess which factors of operator confidence are influenced, if any. Materials and Methods Endodontists were asked to analyze a pre-selected CBCT scan of an endodontic surgical case and to answer a questionnaire that determined their surgical approach for that case. After 30 days, the same participants were asked to analyze again the same CBCT scan. This time however, a 3D printed model of the scan was made available to the participants and to perform a mock osteotomy on the model. The participants were then asked to respond to the same questionnaire that they responded to 30 days prior to determine if there would be any changes to their treatment plan. A new set of questions were added to the survey to evaluate the influence of the 3D printed model on participants’ confidence in performing endodontic surgery. The responses were statistically analyzed using Chi square test followed by either logistic or ordered regression analysis while adjusting for experience of participant. Adjustment for multiple comparison analysis was done using Bonferroni correction. Statistical significance was set at £0.005. Results Availability of the 3D printed model and the CBCT scan together resulted in statistically significant differences in the participants’ responses to their ability to clearly detect bone landmarks, accurately predict the location of osteotomy, and in determining the following: size of osteotomy, angle of instrumentation, involvement of critical structures in flap reflection and involvement of vital structures during curettage. In addition, the participants’ confidence in performing surgery was significantly higher versus having CBCT scans alone. There were no statistically significant changes with decisions on flap design and extent, visualizing critical structures, lesion size, injury to vital structures during osteotomy, the length of root that could be resected and the number of roots involved. Conclusions The availability of 3D printed models did not alter the participants’ surgical approach, but it significantly improved their confidence for endodontic microsurgery, which can be attributed to better visualization of anatomical structures

The Effects of the COVID-19 Pandemic on Postgraduate Endodontic Programs in the United States

In December 2019, the coronavirus disease 2019 (COVID-19) was first identified as an acute infectious disease in Wuhan, China and subsequently led to an ongoing pandemic. At the onset of the pandemic, dental professionals were understood to face the greatest exposure risk to SARS-Cov-2 due to aerosolization of fluids from the oral cavity and respiratory airways . As a result, dental professionals, including academic institutions and their students and residents halted much of their operations to minimize exposure risks and potentially slow the spread of infection to peers and patients alike. Currently, there is little in the literature that describes the changes that academic institutions have implemented in the face of pandemics. This study will discuss the chronology, modifications, and possible resultant outcomes of COVID-19 related events in respect to Graduate Endodontic programs in the United States

The Effects of the COVID-19 Pandemic on Postgraduate Endodontic Programs in the United States

In December 2019, the coronavirus disease 2019 (COVID-19) was first identified as an acute infectious disease in Wuhan, China and subsequently led to an ongoing pandemic. At the onset of the pandemic, dental professionals were understood to face the greatest exposure risk to SARS-Cov-2 due to aerosolization of fluids from the oral cavity and respiratory airways . As a result, dental professionals, including academic institutions and their students and residents halted much of their operations to minimize exposure risks and potentially slow the spread of infection to peers and patients alike. Currently, there is little in the literature that describes the changes that academic institutions have implemented in the face of pandemics. This study will discuss the chronology, modifications, and possible resultant outcomes of COVID-19 related events in respect to Graduate Endodontic programs in the United States

Predicting the response of the dental pulp to SARS-CoV2 infection: a transcriptome-wide effect cross-analysis

Pulpitis, inflammation of the dental pulp, is a disease that often necessitates emergency dental care. While pulpitis is considered to be a microbial disease primarily caused by bacteria, viruses have also been implicated in its pathogenesis. Here, we determined the expression of the SARS-CoV2 receptor, angiotensin converting enzyme 2 (ACE2) and its associated cellular serine protease TPMRSS2 in the dental pulp under normal and inflamed conditions. Next, we explored the relationship between the SARS-CoV-2/human interactome and genes expressed in pulpitis. Using existing datasets we show that both ACE2 and TPMRSS2 are expressed in the dental pulp and, that their expression does not change under conditions of inflammation. Furthermore, Master Regulator Analysis of the SARS-CoV2/human interactome identified 75 relevant genes whose expression values are either up-regulated or down-regulated in both the human interactome and pulpitis. Our results suggest that the dental pulp is vulnerable to SARS-CoV2 infection and that SARS-CoV-2 infection of the dental pulp may contribute to worse outcomes of pulpitis

Predicting the response of the dental pulp to SARS-CoV2 infection: a transcriptome-wide effect cross-analysis

Pulpitis, inflammation of the dental pulp, is a disease that often necessitates emergency dental care. While pulpitis is considered to be a microbial disease primarily caused by bacteria, viruses have also been implicated in its pathogenesis. Here, we determined the expression of the SARS-CoV2 receptor, angiotensin converting enzyme 2 (ACE2) and its associated cellular serine protease TPMRSS2 in the dental pulp under normal and inflamed conditions. Next, we explored the relationship between the SARS-CoV-2/human interactome and genes expressed in pulpitis. Using existing datasets we show that both ACE2 and TPMRSS2 are expressed in the dental pulp and, that their expression does not change under conditions of inflammation. Furthermore, Master Regulator Analysis of the SARS-CoV2/human interactome identified 75 relevant genes whose expression values are either up-regulated or down-regulated in both the human interactome and pulpitis. Our results suggest that the dental pulp is vulnerable to SARS-CoV2 infection and that SARS-CoV-2 infection of the dental pulp may contribute to worse outcomes of pulpitis