Expression of cell‐surface heparan sulfate proteoglycans in human cyclosporin‐induced gingival overgrowth

Gnoatto, Nelson; Lotufo, Roberto Fraga Moreira; Matsuda, Monique; Penna, V.; Marquezini, Mônica V.

doi:10.1111/j.1600-0765.2007.00981.x

Cited by 14 publications

(10 citation statements)

References 43 publications

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“…One day before incubation with C. pneumoniae, siRNA or scrambled RNA was incubated with RNAiMAX trans-fection reagent (Invitrogen) according to the manufacturer's instructions. Gene silencing of the NLRP3 gene was confirmed by reverse transcription (RT)-PCR using primers specific for the NLRP3 gene (forward, 5=-AGC CAC GCT AAT GAT CGA CT-3=; reverse, 5=-CAG GCT CAG AAT GCT CAT CA-3=) (26) and the GAPDH gene (24).…”

Section: Methodsmentioning

confidence: 99%

“…cDNA was synthesized with random primers in ReverTra Ace quantitative PCR reverse transcription (qPCR RT) master mix (Toyobo, Osaka, Japan). qPCR was performed with primers specific for IL-1␤ (forward, 5=-ACA GAT GAA GTG CTC CTT CCA-3=; reverse, 5=-GTC GGA GAT TCG TAG CTG GAT-3=), for IL-8 (forward, 5=-CTG CGC CAA CAC AGA AAT TA-3=; reverse, 5=-ATT GCA TCT GGC AAC CCT AC-3=), for tumor necrosis factor alpha (TNF-␣) (forward, 5=-CCC CAG GGA CCT CTC TCT AA-3=; reverse, 5=-TGA GGT ACA GGC CCT CTG AT-3=) (23), and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (forward, 5=-AAC GGG AAG CTC ACT GGC ATG-3=; reverse, 5=-TCC ACC ACC CTG TTG CTG TAG-3=) (24). The PCR conditions consisted of 5 min of denaturation at 95°C, followed by 40 cycles, each of 30 s of denaturation at 95°C, 30 s of annealing at 60°C, and 45 s of extension at 72°C.…”

Section: Methodsmentioning

confidence: 99%

“…Whole DNA was also extracted from stimulated or unstimulated cells using a High Pure PCR template preparation kit (Roche) according to the manufacturer's instructions. Extracted DNA was used for qPCR with pairs of primers specific for C. pneumoniae 16S rRNA (forward, 5=-GGT CTC AAC CCC ATC CGT GTC GG-3=; reverse, 5=-TGC GGA AAG CTG TAT TTC TAC AGT T-3=) (25) and host cellular GAPDH gene (24). The thermal cycling conditions were 95°C for 5 min, followed by 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 45 s. The amount of amplified DNA of the Chlamydia 16S rRNA gene was normalized to that of the GAPDH gene.…”

Section: Methodsmentioning

confidence: 99%

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Synergistic Costimulatory Effect of Chlamydia pneumoniae with Carbon Nanoparticles on NLRP3 Inflammasome-Mediated Interleukin-1β Secretion in Macrophages

et al. 2015

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The obligate intracellular bacterium Chlamydia pneumoniae is not only a causative agent of community-acquired pneumonia but is also associated with a more serious chronic disease, asthma, which might be exacerbated by air pollution containing carbon nanoparticles. Although a detailed mechanism of exacerbation remains unknown, the proinflammatory cytokine interleukin-1␤ (IL-1␤) is a critical player in the pathogenesis of asthma. C. pneumoniae induces IL-1␤ in macrophages via NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome activation and Toll-like receptor 2/4 (TLR2/4) stimulation. Carbon nanoparticles, such as carbon nanotubes (CNTs), can also evoke the NLRP3 inflammasome to trigger IL-1␤ secretion from lipopolysaccharide-primed macrophages. This study assessed whether costimulation of C. pneumoniae with CNTs synergistically enhanced IL-1␤ secretion from macrophages, and determined the molecular mechanism involved. Enhanced IL-1␤ secretion from C. pneumoniae-infected macrophages by CNTs was dose and time dependent. Transmission electron microscopy revealed that C. pneumoniae and CNTs were engulfed concurrently by macrophages. Inhibitors of actin polymerization or caspase-1, a component of the inflammasome, significantly blocked IL-1␤ secretion. Gene silencing using small interfering RNA (siRNA) targeting the NLRP3 gene also abolished IL-1␤ secretion. Other inhibitors (K ؉ efflux inhibitor, cathepsin B inhibitor, and reactive oxygen species-generating inhibitor) also blocked IL-1␤ secretion. Taken together, these findings demonstrated that CNTs synergistically enhanced IL-1␤ secretion from C. pneumoniae-infected macrophages via the NLRP3 inflammasome and caspase-1 activation, providing novel insight into our understanding of how C. pneumoniae infection can exacerbate asthma. Chlamydia pneumoniae is an obligate intracellular bacterium and a causative agent of respiratory tract infections, including community-acquired pneumonia (1, 2). The seroprevalence rates of C. pneumoniae infection, which start to rise relatively early in childhood, are increased by 50% at 20 years of age and subsequently reach 70 to 80% by 60 to 70 years of age (3, 4), suggesting most individuals will have had some exposure to the bacterium in their lifetime. Therefore, C. pneumoniae is likely a ubiquitous pathogen in individuals worldwide (4). The symptoms of pulmonary infection vary considerably according to age from asymptomatic or mild illness to serious pneumonia, especially in pediatric infection (1-3). Regarding this, several studies indicate that bacterial infection might exacerbate pulmonary inflammation and thus is associated with a more serious chronic disease, asthma (3). The proinflammatory cytokine interleukin-1␤ (IL-1␤), which is induced by C. pneumoniae (5-8), is thought to play a critical role in the development of chronic inflammatory disease, although detailed mechanisms remain unclear. Air pollution containing carbon nanoparticles, environmental air contaminants that might be easily inhaled with C. pneu...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Synergistic Costimulatory Effect of Chlamydia pneumoniae with Carbon Nanoparticles on NLRP3 Inflammasome-Mediated Interleukin-1β Secretion in Macrophages

et al. 2015

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“…Reverse transcription of 500-ng total RNA was performed with ReverTraAce qPCR RT Master Mix (Toyobo, Osaka, Japan). Resultant cDNAs were used for PCR amplification with the following primers: ( gapdh ) (forward 5′-AAC GGG AAG CTC ACT GGC ATG-3′; reverse 5′-TCC ACC ACC CTG TTG CTG TAG-3′) [ 28 ], aldolase A (forward 5′-CGC AGA AGG GGT CCT GGT GA-3′; reverse 5′-CAG CTC CTT CTT CTG CTC CGG GGT-3′) [ 29 ], enolase (forward 5′-GAG CTC CGG GAC AAT GAT AA-3′; reverse 5′-CTG TTC CA TCC ATC TCG ATC-3′) [ 30 ], β-actin (forward 5′-GAC CAC ACC TTC TAC AAT GAG-3′; reverse 5′-GCA TAC CCC TCG TAG ATG GG-3′) [ 31 ] and chlamydial 16S rRNA (forward 5′-CGG CGT GGA TGA GGC AT-3′; reverse 5′-TCA GTC CCA GTG TTG GC-3′) [ 32 ]. PCR conditions were 95°C for 10 min, followed by 25–40 cycles of 95°C for 30 s, 55 or 60°C for 30 s and 72°C for 45 s, followed by 72°C for 10 min.…”

Section: Methodsmentioning

confidence: 99%

Chlamydia pneumoniae effector chlamydial outer protein N sequesters fructose bisphosphate aldolase A, providing a benefit to bacterial growth

et al. 2014

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BackgroundPathogenic chlamydiae are obligate intracellular pathogens and have adapted successfully to human cells, causing sexually transmitted diseases or pneumonia. Chlamydial outer protein N (CopN) is likely a critical effector protein secreted by the type III secretion system in chlamydiae, which manipulates host cells. However, the mechanisms of its action remain to be clarified. In this work, we aimed to identify previously unidentified CopN effector target in host cells.ResultsWe first performed a pull-down assay with recombinant glutathione S-transferase (GST) fusion CopN proteins (GST–CpCopN: Chlamydia pneumoniae TW183, GST–CtCopN: Chlamydia trachomatis D/UW-3/CX) as “bait” and soluble lysates obtained from human immortal epithelial HEp-2 cells as “prey”, followed by SDS-PAGE with mass spectroscopy (MS). We found that a host cell protein specifically bound to GST–CpCopN, but not GST–CtCopN. MS revealed the host protein to be fructose bisphosphate aldolase A (aldolase A), which plays a key role in glycolytic metabolism. We also confirmed the role of aldolase A in chlamydia-infected HEp-2 cells by using two distinct experiments for gene knockdown with an siRNA specific to aldolase A transcripts, and for assessment of glycolytic enzyme gene expression levels. As a result, both the numbers of chlamydial inclusion-forming units and RpoD transcripts were increased in the chlamydia-infected aldolase A knockdown cells, as compared with the wild-type HEp-2 cells. Meanwhile, chlamydial infection tended to enhance expression of aldolase A.ConclusionsWe discovered that one of the C. pneumoniae CopN targets is the glycolytic enzyme aldolase A. Sequestering aldolase A may be beneficial to bacterial growth in infected host cells.

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“…DNA was extracted from the cultured cells using the LaboPass™ Tissue Mini kit (Hokkaido System Science). PCR was performed using primer sets specific to chlamydial 16S rDNA (which recognize a wide range of chlamydiae) (sense primer, 5ʹ-GGA CCT TAG CTG GAC TTG ACA TGT-3ʹ; antisense primer, 5ʹ-CCA TGC AGC ACC TGT GTA TCT G-3ʹ) [27] and glyceraldehyde 3-phosphate dehydrogenase (gapdh) (sense primer: 5ʹ-AAC GGG AAG CTC ACT GGC ATG-3ʹ, antisense primer: 5ʹ-TCC ACC AAC CTG TTG CTG TAG-3ʹ) [28]. The number of bacteria per culture was expressed as the ratio of chlamydial 16S rDNA:gapdh.…”

Section: Quantitative (Q) Pcrmentioning

confidence: 99%

Impact of capsaicin, an active component of chili pepper, on pathogenic chlamydial growth ( Chlamydia trachomatis and Chlamydia pneumoniae ) in immortal human epithelial HeLa cells

Yamakawa

Matsuo

Okubo

et al. 2018

Journal of Infection and Chemotherapy

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Chlamydia trachomatis is the leading cause of sexually transmitted infections worldwide. Capsaicin, a component of chili pepper, which can stimulate actin remodeling via capsaicin receptor TRPV1 (transient receptor potential vanilloid 1) and anti-inflammatory effects via PPARγ (peroxisome proliferator-activated receptor-γ) and LXRα (liver X receptor α), is a potential candidate to control chlamydial growth in host cells. We examined whether capsaicin could inhibit C. trachomatis growth in immortal human epithelial HeLa cells. Inclusion forming unit and quantitative PCR assays showed that capsaicin significantly inhibited bacterial growth in cells in a dose-dependent manner, even in the presence of cycloheximide, a eukaryotic protein synthesis inhibitor. Confocal microscopic and transmission electron microscopic observations revealed an obvious decrease in bacterial numbers to inclusions bodies formed in the cells. Although capsaicin can stimulate the apoptosis of cells, no increase in cleaved PARP (poly (ADP-ribose) polymerase), an apoptotic indicator, was observed at a working concentration. All of the drugs tested (capsazepine, a TRPV1 antagonist; 5CPPSS-50, an LXRα inhibitor; and T0070907, a PPARγ inhibitor) had no effect on chlamydial inhibition in the presence of capsaicin. In addition, we also confirmed that capsaicin inhibited Chlamydia pneumoniae growth, indicating a phenomena not specific to C. trachomatis. Thus, we conclude that capsaicin can block chlamydial growth without the requirement of host cell protein synthesis, but by another, yet to be defined, mechanism.

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Expression of cell‐surface heparan sulfate proteoglycans in human cyclosporin‐induced gingival overgrowth

Abstract: Our findings agree with the current concept of cyclosporin A-induced gingival overgrowth and provide new evidence that its noncollagenous extracellular matrix is overexpressed.

Cited by 14 publications

References 43 publications

Synergistic Costimulatory Effect of Chlamydia pneumoniae with Carbon Nanoparticles on NLRP3 Inflammasome-Mediated Interleukin-1β Secretion in Macrophages

Synergistic Costimulatory Effect of Chlamydia pneumoniae with Carbon Nanoparticles on NLRP3 Inflammasome-Mediated Interleukin-1β Secretion in Macrophages

Chlamydia pneumoniae effector chlamydial outer protein N sequesters fructose bisphosphate aldolase A, providing a benefit to bacterial growth

Impact of capsaicin, an active component of chili pepper, on pathogenic chlamydial growth ( Chlamydia trachomatis and Chlamydia pneumoniae ) in immortal human epithelial HeLa cells

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