Identification of intracellular oral species within human crevicular epithelial cells from subjects with chronic periodontitis by fluorescence <i>in situ</i> hybridization

Colombo, A. V.; Silva, C. M. Da; Haffajee, A. D.; Colombo, Ana Paula Vieira

doi:10.1111/j.1600-0765.2006.00938.x

Cited by 90 publications

(96 citation statements)

References 28 publications

(49 reference statements)

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“…We also observe P. gingivalis internalized in gingival tissues where overloaded rounded cells coexisted with poorly or non-invaded cells. Studies from Colombo et al (2007) and Rudney et al (2005a, b), in epithelial cells scraped from periodontal pockets and oral mucosa, also reported similar observations. The exposure of host cells to P. gingivalis invasion resulted in an apparent cell rounding, probably due to cleavage of cadherins and integrins by gingipains, as reported by Sheets et al (2005).…”

Section: Discussionsupporting

confidence: 56%

“…The best hybridization conditions were found to be at a temperature of 59 • C for 150 min, which make our FISH protocol as fast as the previously reported for DNA-FISH (Zijnge et al, 2010;Rudney et al, 2001;Colombo et al, 2007;Stoltenberg et al, 1993;Sunde et al, 2003;Rudney et al, 2005a,b;Johnson et al, 2008) and suitable both for clinical, if it proves useful (Colombo et al, 2009;Sayehmiri et al, 2015;Hong et al, 2015), and research purposes.…”

Section: Discussionmentioning

confidence: 55%

“…The PgPNA1007 probe has a higher theoretical performance in P. gingivalis detection compared with the DNA probes used in several previous studies (Rudney et al, 2001;Colombo et al, 2007;Stoltenberg et al, 1993;Sunde et al, 2003;Rudney et al, 2005a,b;Johnson et al, 2008). Its theoretical performance is only comparable to the DNA probe previously developed by Sunde et al (2003).…”

Section: Discussionmentioning

confidence: 79%

“…Some authors have used this technique to observe, in vivo, the spatial distribution of periodontal pathogens in the supra and subgingival biofilm (Zijnge et al, 2010), as well as, their ability to invade host epithelial cells (Rudney et al, 2001;Colombo et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

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Novel strategy to detect and locate periodontal pathogens: The PNA-FISH technique

Mendes

Rocha

Azevedo

et al. 2016

Microbiological Research

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Purpose: We aim to develop peptic nucleic acid (PNA) probes for the identification and localization of Aggregatibacter actinomycetemcomintans and Porphyromonas gingivalis in sub-gingival plaque and gingival biopsies by Fluorescence in situ Hybridization (FISH).Methods: A PNA probe was designed for each microorganism. The PNA-FISH method was optimized to allow simultaneous hybridization of both microorganisms with their probe (PNA-FISH multiplex). After being tested on representative strains of P. gingivalis and A. actinomycetemcomitans, the PNA-FISH method was then adapted to detect microorganisms in the subgingival plaque and gingival samples, collected from patients with severe periodontitis. Results:The best hybridization conditions were found to be 59 • C for 150 min for both probes (PgPNA1007 and AaPNA235). The in silico sensitivity and specificity was both 100% for PgPNA1007 probe and 100% and 99.9% for AaPNA235 probe, respectively. Results on clinical samples showed that the PNA-FISH method was able to detect and discriminate target bacteria in the mixed microbial population of the subgingival plaque and within periodontal tissues.Conclusion: This investigation presents a new highly accurate method for P. gingivalis and A. actino-mycetemcomitans detection and co-location in clinical samples, in just few hours. With this technique we were able to observe spatial distribution of these species within polymicrobial communities in the periodontal pockets and, for the first time with the FISH method, in the organized gingival tissue.2

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Section: Discussionsupporting

confidence: 56%

Section: Discussionmentioning

confidence: 55%

Section: Discussionmentioning

confidence: 79%

Section: Introductionmentioning

confidence: 99%

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Novel strategy to detect and locate periodontal pathogens: The PNA-FISH technique

Mendes

Rocha

Azevedo

et al. 2016

Microbiological Research

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“…While the contribution of T. forsythia in periodontitis has been well established through epidemiological and clinical intervention studies, the mechanisms underlying T. forsythia virulence are poorly understood (Sharma, 2010). T. forsythia has been detected intracellularly in buccal and crevicular epithelium of patients with periodontitis (Colombo et al, 2007;Rudney et al, 2005). Furthermore, T. forsythia has been shown to invade epithelial cells in vitro (Han et al, 2000;Inagaki et al, 2006;Kirschbaum et al, 2010;Sabet et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Tannerella forsythia invasion in oral epithelial cells requires phosphoinositide 3-kinase activation and clathrin-mediated endocytosis

Mishima

Sharma

2011

Microbiology

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Tannerella forsythia, a Gram-negative anaerobe implicated in periodontitis, has been detected within human buccal epithelial cells and shown to invade oral epithelial cells in vitro. We have previously shown that this bacterium triggers host tyrosine kinase-dependent phosphorylation and actin-dependent cytoskeleton reorganization for invasion. On the bacterial side, the leucine-rich repeat cell-surface BspA protein is important for entry. The present study was undertaken to identify host signalling molecules during T. forsythia entry into human oral and cervical epithelial cells. Specifically, the roles of phosphatidylinositol 3-kinase (PI3K), Rho-family GTPases, cholesterol-rich membrane microdomains and the endocytic protein clathrin were investigated. For this purpose, cell lines were pretreated with chemical inhibitors or small interfering RNAs (siRNAs) that target PI3Ks, Rho GTPases, clathrin and cholesterol (a critical component of ‘lipid rafts’), and the resulting effects on T. forsythia uptake were determined. Our studies revealed that T. forsythia entry is dependent on host PI3K signalling, and that purified BspA protein causes activation of this lipid kinase. Bacterial entry also requires the cooperation of host Rac1 GTPase. Finally, our findings indicate an important role for clathrin and cholesterol-rich lipid microdomains in the internalization process

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Contemporary Concepts in the Diagnosis of Periodontal Disease

Dumitrescu

2012

Understanding Periodontal Research

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Identification of intracellular oral species within human crevicular epithelial cells from subjects with chronic periodontitis by fluorescence in situ hybridization

Abstract: Those findings indicate that crevicular and buccal cells present internalized bacteria, regardless of periodontal status. However, higher bacterial loads are detected in cells from subjects with periodontitis.

Cited by 90 publications

References 28 publications

Novel strategy to detect and locate periodontal pathogens: The PNA-FISH technique

Novel strategy to detect and locate periodontal pathogens: The PNA-FISH technique

Tannerella forsythia invasion in oral epithelial cells requires phosphoinositide 3-kinase activation and clathrin-mediated endocytosis

Contemporary Concepts in the Diagnosis of Periodontal Disease

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