Trimming conditions for DADA2 analysis in QIIME2 platform

Accurate identification of microbes facilitates the prediction, prevention, and treatment of human diseases. To increase the accuracy of microbiome data analysis, a long region of the 16S rRNA is commonly sequenced via paired-end sequencing. In paired-end sequencing, a sufficient length of overlapping region is required for effective joining of the reads, and high-quality sequencing reads are needed at the overlapping region. Trimming sequences at the reads distal to a point where sequencing quality drops below a specific threshold enhance the joining process. In this study, we examined the effect of trimming conditions on the number of reads that remained after quality control and chimera removal in the Illumina paired-end reads of the V3–V4 hypervariable region. We also examined the alpha diversity and taxa assigned by each trimming condition. Optimum quality trimming increased the number of good reads and assigned more number of operational taxonomy units. The pre-analysis trimming step has a great influence on further microbiome analysis, and optimized trimming conditions should be applied for Divisive Amplicon Denoising Algorithm 2 analysis in QIIME2 platform.

Trimming conditions for DADA2 analysis in QIIME2 platform

Accurate identification of microbes facilitates the prediction, prevention, and treatment of human diseases. To increase the accuracy of microbiome data analysis, a long region of the 16S rRNA is commonly sequenced via paired-end sequencing. In paired-end sequencing, a sufficient length of overlapping region is required for effective joining of the reads, and high-quality sequencing reads are needed at the overlapping region. Trimming sequences at the reads distal to a point where sequencing quality drops below a specific threshold enhance the joining process. In this study, we examined the effect of trimming conditions on the number of reads that remained after quality control and chimera removal in the Illumina paired-end reads of the V3–V4 hypervariable region. We also examined the alpha diversity and taxa assigned by each trimming condition. Optimum quality trimming increased the number of good reads and assigned more number of operational taxonomy units. The pre-analysis trimming step has a great influence on further microbiome analysis, and optimized trimming conditions should be applied for Divisive Amplicon Denoising Algorithm 2 analysis in QIIME2 platform.

Practical considerations for the study of the oral microbiome

In the oral cavity, complex microbial community is shaped by various host and environmental factors. Extensive literature describing the oral microbiome in the context of oral health and disease is available. Advances in DNA sequencing technologies and data analysis have drastically improved the analysis of the oral microbiome. For microbiome study, bacterial 16S ribosomal RNA gene amplification and sequencing is often employed owing to the cost-effective and fast nature of the method. In this review, practical considerations for performing a microbiome study, including experimental design, molecular analysis technology, and general data analysis, will be discussed.

Inhibitory effects of Coptis chinensis extract on the growth and biofilm formation of Streptococcus mutans and Streptococcus sobrinus

Streptococcus mutans and Streptococcus sobrinus play important roles in dental caries. Coptis chinensis is a natural product with antimicrobial activity against enterobacteria; however, its effects on oral streptococci are still unknown.Therefore, the effects of C. chinensis on the growth and biofilm formation of the representative cariogenic bacteria S. mutans and S. sobrinus were investigated for the possible use of C. chinensis as an anticaries agent. The C.chinensis extract was diluted with sterile distilled water, and 0.1–2.5% of the extract was used in the experiment. The effects of the C. chinensis extract on the growth and glucan formation of S. mutans and S. sobrinus were measured by viable cell counting and spectrophotometry at 650 nm absorbance, respectively. Crystal violet staining was also carried out to confirm the C. chinensis extract’s inhibitory effect on biofilm formation. The C. chinensis extract significantly inhibited the growth of S. mutans and S. sobrinus at concentrations of ≥ 0.3% as compared with the control group. The viable cell count of colonies decreased by 1.7-fold and 1.2-fold at 2.5% and 1.25%, respectively, compared with the control group. The biofilm formation of S. mutans and S. sobrinus was inhibited by > 20-fold at C.chinensis extract concentrations of ≥ 1.25% as compared with the control group. In summary, the C. chinensis extract inhibited the growth and biofilm and glucan formation of S. mutans and S. sobrinus. Therefore, C. chinensis might be a potential candidate for controlling dental caries.

Inhibitory effects of Coptis chinensis extract on the growth and biofilm formation of Streptococcus mutans and Streptococcus sobrinus

Streptococcus mutans and Streptococcus sobrinus play important roles in dental caries. Coptis chinensis is a natural product with antimicrobial activity against enterobacteria; however, its effects on oral streptococci are still unknown.Therefore, the effects of C. chinensis on the growth and biofilm formation of the representative cariogenic bacteria S. mutans and S. sobrinus were investigated for the possible use of C. chinensis as an anticaries agent. The C.chinensis extract was diluted with sterile distilled water, and 0.1–2.5% of the extract was used in the experiment. The effects of the C. chinensis extract on the growth and glucan formation of S. mutans and S. sobrinus were measured by viable cell counting and spectrophotometry at 650 nm absorbance, respectively. Crystal violet staining was also carried out to confirm the C. chinensis extract’s inhibitory effect on biofilm formation. The C. chinensis extract significantly inhibited the growth of S. mutans and S. sobrinus at concentrations of ≥ 0.3% as compared with the control group. The viable cell count of colonies decreased by 1.7-fold and 1.2-fold at 2.5% and 1.25%, respectively, compared with the control group. The biofilm formation of S. mutans and S. sobrinus was inhibited by > 20-fold at C.chinensis extract concentrations of ≥ 1.25% as compared with the control group. In summary, the C. chinensis extract inhibited the growth and biofilm and glucan formation of S. mutans and S. sobrinus. Therefore, C. chinensis might be a potential candidate for controlling dental caries.

Xylitol stimulates saliva secretion via muscarinic receptor signaling pathway

Xylitol is well-known to have an anti-caries effect by inhibiting the replication of cariogenic bacteria. In addition, xylitol enhances saliva secretion. However, the precise molecular mechanism of xylitol on saliva secretion is yet to be elucidated. Thus, in this study, we aimed to investigate the stimulatory effect of xylitol on saliva secretion and to further evaluate the involvement of xylitol in muscarinic type 3 receptor (M3R) signaling. For determining these effects, we measured the saliva flow rate following xylitol treatment in healthy individuals and patients with dry mouth. We further tested the effects of xylitol on M3R signaling in human salivary gland (HSG) cells using real-time quantitative reverse-transcriptase polymerase chain reaction, immunoblotting, and immunostaining. Xylitol candy significantly increased the salivary flow rate and intracellular calcium release in HSG cells via the M3R signaling pathway. In addition, the expressions of M3R and aquaporin 5 were induced by xylitol treatment. Lastly, we investigated the distribution of M3R and aquaporin 5 in HSG cells. Xylitol was found to activate M3R, thereby inducing increases in Ca²⁺ concentration. Stimulation of the muscarinic receptor induced by xylitol activated the internalization of M3R and subsequent trafficking of aquaporin 5. Taken together, these findings suggest a molecular mechanism for secretory effects of xylitol on salivary epithelial cells.

Effect of Various Agents on Oral Bacterial Phagocytosis in THP-1 Cells

Phagocytosis is a fundamental process in which phagocytes capture and ingest foreign particles including pathogenic bacteria. Several oral pathogens have anti-phagocytic strategies, which allow them to escape from and survive in phagocytes. Impaired bacteria phagocytosis increases inflammation and contributes to inflammatory diseases. The purpose of this study is to investigate the influences of various agents on oral pathogenic phagocytosis. To determine phagocytosis, Streptococcus mutans, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis were stained with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), and was measured using flowcytometery and confocal microscopy. The influencing factors on phagocytosis were evaluated through the pretreatment of ROS inhibitor (N-acetyl-L-cysteine (NAC)), lysozyme, potassium chloride (KCI) and adenosine triphosphate (ATP) in THP-1 cells. Expression of pro-inflammatory cytokines was determined by enzyme-linked immunosorbent assay (ELISA). The phagocytosis of various bacteria increased in a MOI-dependent manner. Among the tested bacteria, phagocytosis of P. gingivalis showed the highest fluorescent intensity at same infection time. Among the tested inhibitors, the NAC treatment significantly inhibited phagocytosis in all tested bacteria. In addition, NAC treatment indicated a similar pattern under the confocal microscopy. Moreover, NAC treatment significantly increased the bacteria-induced secretion of IL-1β among the tested inhibitors. Taken together, we conclude that the phagocytosis occurs differently depending on each bacterium. Down-regulation by ROS production inhibited phagocytosis and lead increased of oral pathogens-associated inflammation.

Xylitol Mitigate Neutrophil Inflammatory Response Against Porphyromonas gingivalis Infection

Periodontitis is generally a chronic disorder characterized by breakdown of tooth-supporting tissues, producing dentition loss. Porphyromonas gingivalis (P. gingivalis), a Gram-negative anaerobic rod, is one of the major pathogens associated with periodontitis. Neutrophils are first line defense cells in the oral cavity that play a significant role in inflammatory response. Xylitol is a known anti-caries agent and has anti-inflammatory effects. In this study, we conducted experiments to evaluate anti-inflammatory effects of xylitol on P. gingivalis infected neutrophils for possible usage in prevention and treatment of periodontal infections. P. gingivalis was intraperitoneally injected and peritoneal lavage was collected for cytokine determination. For in vitro study, neutrophils were collected from mouse peritoneal cells after zymosan injection or bone marrow cells. Neutrophils were stimulated with live P. gingivalis and ELISA was used to determine the effect of xylitol on P. gingivalis induced cytokine production. IL-1β, IL-6, TNF-α concentration and neutrophil population in the peritoneal lavage was increased in P. gingivalis-infected mouse. Peritoneal cells infected with live P. gingivalis revealed significantly increased production of IL-1β, IL-6 and TNF-α at multiplicity of infection of 10. Neutrophils from bone marrow and peritoneal lavage revealed increased production of IL-1β, IL-6 and TNF-α. Xylitol significantly mitigated P. gingivalis induced cytokine production in neutrophils. Findings indicate that xylitol is an anti-inflammatory agent in neutrophils infected with live P. gingivalis, that suggests its use in periodontitis management.

Changes in Oral Microbiota in Patients Receiving Radical Concurrent Chemoradiotherapy for The Head and Neck Squamous Cell Carcinoma

Radiotherapy (RT) is a mainstay in the treatment of head and neck squamous cell carcinoma (HNSCC). For locally advanced HCSCC, concurrent chemoradiotherapy (CCRT) benefits HCSCC patients in terms of better survival and loco-regional control. In this study, we evaluated changes in oral microbiota in patients, who received CCRT for head and neck cancer. Oral rinsed samples were weekly collected before and during CCRT and at 4 weeks following treatment from HNSCC patients, who had received 70 Gy of radiation delivered to the primary sites for over 7 weeks and concurrent chemotherapy. Oral microbiota changes in three patients were analyzed by next-generation sequencing using 16S rRNA 454 pyrosequencing. On an average, 15,000 partial 16S rRNA gene sequences were obtained from each sample. All sequences fell into 11 different bacterial phyla. During early CCRT, the microbial diversity gradually decreased. In a patient, who did not receive any antibiotics during the CCRT, Firmicutes and Proteobacteria were the most abundant phylum. During the early CCRT, proteobacteria gradually decreased while Firmicutes increased. During the late CCRT, firmicutes gradually decreased while Bacteroides and Fusobacteria increased. In all the patients, yellow complex showed a gradual decrease, while orange and red complex showed a gradual increase during the CCRT. At 4 weeks after CCRT, the recovery of oral microbiota diversity was limited. During CCRT, there was a gradual increase in major periodontopathogens in association with the deterioration of the oral hygiene. Henceforth, it is proposed that understanding oral microbiota shift should provide better information for the development of effective oral care programs for patients receiving CCRT for HNSCC.

Antibacterial and remineralization effects of orthodontic bonding agents containing bioactive glass / 대한치과교정학회지

OBJECTIVE: The aim of this study was to evaluate the mechanical and biological properties of orthodontic bonding agents containing silver- or zinc-doped bioactive glass (BAG) and determine the antibacterial and remineralization effects of these agents. METHODS: BAG was synthesized using the alkali-mediated solgel method. Orthodontic bonding agents containing BAG were prepared by mixing BAG with flowable resin. Transbond™ XT (TXT) and Charmfil™ Flow (CF) were used as controls. Ion release, cytotoxicity, antibacterial properties, the shear bond strength, and the adhesive remnant index were evaluated. To assess the remineralization properties of BAG, micro-computed tomography was performed after pH cycling. RESULTS: The BAG-containing bonding agents showed no noticeable cytotoxicity and suppressed bacterial growth. When these bonding agents were used, demineralization after pH cycling began approximately 200 to 300 µm away from the bracket. On the other hand, when CF and TXT were used, all surfaces that were not covered by the adhesive were demineralized after pH cycling. CONCLUSIONS: Our findings suggest that orthodontic bonding agents containing silver- or zinc-doped BAG have stronger antibacterial and remineralization effects compared with conventional orthodontic adhesives; thus, they are suitable for use in orthodontic practice.

Streptococcus mutans activates the AIM2, NLRP3 and NLRC4 inflammasomes in human THP-1 macrophages / 国际口腔科学杂志·英文版

Streptococcus mutans (S. mutans), a major aetiologic agent of dental caries, is involved in systemic diseases, such as bacterial endocarditis, if it enters the bloodstream through temporary bacteraemia. Interleukin (IL)-1β, a proinflammatory cytokine, is related to the host defences against pathogens, and its synthesis, maturation, and secretion are tightly regulated by the activation of the inflammasome, an inflammatory signalling complex. This study examined the signalling mechanism of IL-1β secretion and the inflammasome pathway induced by S. mutans to explain the molecular mechanism through which systemic infection by oral streptococci can occur. After infection of THP-1 cells with S. mutans, the expression of inflammasome components was detected using various methods. S. mutans induced IL-1β secretion via caspase-1 activation, and S. mutans-induced IL-1β secretion required absent in melanoma (AIM2), NLR family pyrin domain-containing 3 (NLRP3) and NLR family CARD domain-containing 4 (NLRC4) inflammasome activation. In particular, the S. mutans-induced NLRP3 inflammasome was mediated by adenosine triphosphate (ATP) release, potassium depletion and lysosomal damage. Our study provides novel insight into the innate immune response against S. mutans infection.

The Relationship between Mitochondria and NLRP3 Inflammasome

Mitochondria participate in various intracellular metabolic pathways such as generating intracellular ATP, synthesizing several essential molecules, regulating calcium homeostasis, and producing the cell's reactive oxygen species (ROS). Emerging studies have demonstrated newly discovered roles of mitochondria, which participate in the regulation of innate immune responses by modulating NLRP3 inflammasomes. Here, we review the recently proposed pathways to be involved in mitochondria-mediated regulation of inflammasome activation and inflammation: 1) mitochondrial ROS, 2) calcium mobilization, 3) nicotinamide adenine dinucleotide (NAD+) reduction, 4) cardiolipin, 5) mitofusin, 6) mitochondrial DNA, 7) mitochondrial antiviral signaling protein. Furthermore, we highlight the significance of mitophagy as a negative regulator of mitochondrial damage and NLRP3 inflammasome activation, as potentially helpful therapeutic approaches which could potentially address uncontrolled inflammation.

Chracterization of THP-1 Cell Death Induced by Porphyromonas gingivalis Infection

BACKGROUND: Periodontitis is generally a chronic disorder characterized by the breakdown of tooth-supporting tissues. P. gingivalis, a Gram-negative anaerobic rod, is one of the major pathogens associated with periodontitis. Frequently, P. gingivalis infection leads to cell death. However, the correlation between P. gingivalis–induced cell death and periodontal inflammation remains to be elucidated. Among cell deaths, the death of immune cells appears to play a significant role in inflammatory response. Thus, the aim of this study was to examine P. gingivalis–induced cell death, focusing on autophagy and apoptosis in THP-1 cells. METHODS: Human acute monocytic leukemia cell line (THP-1) was used for all experiments. Autophagy induced by P. gingivalis in THP-1 cells was examined by Cyto ID staining. Intracellular autophagic vacuoles were observed by fluorescence microscopy using staining Acridine orange (AO); and 3-methyladenine (3-MA) was used to inhibit autophagy. Total cell death was measured by LDH assay. Cytokine production was measured by an ELISA method. RESULTS: P. gingivalis induced autophagy in an MOI-dependent manner in THP-1 cells, but 3-MA treatment decreased autophagy and increased the apoptotic blebs. P. gingivalis infection did not increase apoptosis compared to the control cells, whereas inhibition of autophagy by 3-MA significantly increased apoptosis in P. gingivalis-infected THP-1 cells. Inhibition of autophagy by 3-MA also increased total cell deaths and inflammatory cytokine production, including IL-1β and TNF-α. CONCLUSION: P. gingivalis induced autophagy in THP-1 cells, but the inhibition of autophagy by 3-MA stimulated apoptosis, leading to increased cell deaths and pro-inflammatory cytokines production. Hence, the modulation of cell deaths may provide a mechanism to fight against invading microorganisms in host cells and could be a promising way to control inflammation.

Ursodeoxycholic Acid Inhibits Inflammatory Cytokine Expression in THP-1 Cells Infected with Aggregatibacter actinomycetemcomitans

BACKGROUND: Periodontitis is an inflammatory disease characterized by the breakdown of tooth-supporting tissues, leading to tooth loss. Aggregatibacter actinomycetemcomitans are major etiologic bacterium causing aggressive periodontitis. Ursodeoxycholic acid (UDCA), a hydrophilic gall bladder acid, has been used as an effective drug for various diseases related to immunity. The aim of this study was to investigate the effect of UDCA on the inflammatory response induced by A. actinomycetemcomitans. METHODS: A human acute monocytic leukemia cell line (THP-1) was differentiated to macrophage- like cells by treatment with phorbol 12-mystristate 13-acetate (PMA) and used for all experiments. The cytotoxic effect of UDCA was examined by MTT assay. THP-1 cells were pretreated with UDCA for 30 min before A. actinomycetemcomitans infection and the culture supernatant was analyzed for various cytokine production by ELISA. The effect of UDCA on bacterial growth was examined by measuring optical densities using a spectrophotometer. RESULTS: UDCA showed no cytotoxic effect on THP-1 cells, up to 80 µM Ed highlight: Please confirm technical meaning. UDCA pretreatment inhibited the A. actinomycetemcomitans-induced IL-1β, TNF-α, and IL-17A secretion in a dosedependent manner. UDCA also inhibited IL-21 production at 60 µM. The production of IL-12 and IL-4 was not influenced by A. actinomycetemcomitans infection. CONCLUSION: These findings indicate that UDCA inhibits the production of inflammatory cytokines involved in innate and Th17 immune responses in A. actinomycetemcomitans-infected THP-1-derived macrophages, which suggests its possible use for the control of aggressive periodontitis.

Link between Periodontal Disease and Diabetes

Diabetes mellitus (DM) is a complex disease with various systemic and oral complications including periodontitis. Periodontitis is a disease that leads to destruction of the soft and hard tissues of periodontium, which can result in periodontal bone loss and tooth loss. Although the etiology for periodontitis is bacterial plaque, the host immune response also mediates the destruction of periodontal tissues. DM is related to the development, progression and severity of periodontitis. There are several factors potentially contributing to the development of periodontitis in DM patients: 1) altered immune function, 2) hyperglycemia and advanced glycation end products, and 3) altered lipid metabolism. With limited number of studies, the potential mechanisms involved in the development of DM in periodontitis have also been suggested. DM clearly increases the risk of periodontitis and biological mechanisms have been illucidated. Less clear is the impact of periodontitis on the development of DM. It is possible that periodontitis may serve as initiators or propagators of insulin resistance in a way similar to obesity, thereby aggravating glycemic control.

Rutin induces autophagy in cancer cells

Rutin (3,3′,4′,5,7-pentahydroxyflavone-3-rhamnoglucoside) is a bioactive flavonoid from the plant kingdom. Rutin has been studied as potential anticancer agent due to its wide range of pharmacological properties including antioxidative, anti-inflammatory and anticancer. Autophagy is a conserved intracellular catabolic pathway to maintain cell homeostasis by formation of autophagosome. Processing of autophagy involves various molecules including ULK1 protein kinase complex, Beclin-1–Vps34 lipid kinase complex, ATG5, ATG12, and LC3 (light chain 3). Cargo-carried autophagosomes fuse with lysosomes resulting in autophagolysosome to eliminate vesicles and degrade cargo. However, the actions of rutin on autophagy are not clearly understood. In this study, we analyzed the effect of rutin on autophagy and inflammation in cancer cell lines. Interestingly, rutin induced autophagy in leukemia (THP-1), oral (CA9-22), and lung (A549) cell lines. TNF-α, key modulator of inflammation, was upregulated by inhibition of rutin-induced autophagy. Taken together, these data indicated that rutin induced autophagy and consequently suppressed TNF-α production.

Expression of Inflammasome Complex Following Various Oral Bacterial Infection in THP-1 Cells

Interleukin-1b (IL-1β), a proinflammatory cytokine, regulates the innate immune responses against bacterial infection. Mature IL-1β is produced from pro-IL-1β by activated caspase-1, which in turn is activated by the inflammasome complex formation. In this study, we compared the inflammasome mRNA expression induced by S. sanguinis, S. oralis, F. nucleatum and P. intermedia. Among the tested bacteria, S. sanguinis induced the highest IL-1β secretion. S. oralis, F. nucleatum and P. intermedia induced very weak IL-1β secretion. S. sanguinis mostly induced the NLRP3 mRNA expressions. Although F. nucleatum did not induce high IL-1β secretion, it induced high expression levels of AIM2, NLRP2, and NLRP3. No specific inflammasomes were induced by S. oralis and P.intermedia. Studying the inflammasome complex activation induced by oral bacteria may thus enhance our understanding of the pathogenesis of oral diseases.

Contribution of Periodontal Disease in Atherosclerosis

Periodontitis is a disease that leads to destruction of the soft and hard tissues of periodontium, which can result in periodontal bone loss and tooth loss in severe cases. Atherosclerosis is a disease characterized by artery wall thickening as a result of invasion and accumulation of foam cells. Epidemiologic studies have suggested the association with periodontitis and atherosclerosis. Periodontopathogens are frequently found in atheroma plaque. The possible mechanisms for systemic dissemination of oral bacteria have been suggested: 1) direct translocation of bacteria from dental plaque to systemic circulation through transcellular mechanism or by physical perturbations of the gingiva, 2) indirect dissemination to distant sites via survival in immune cells including macrophages and dendritic cells. There are several mechanisms by which oral bacteria may contribute to atherosclerosis development: 1) activation of innate immune response, 2) mediators activated by oral bacteria and 3) involvement of cytokines and heat shock proteins from oral bacteria. Thus, better understanding the role of periodontitis in atherosclerosis may be the key to improve the prevention and treatment of atherosclerosis.

Performance Comparison of Benchtop Next-generation Sequencing Systems

With fast development and wide applications of next generation sequencing (NGS), genomic sequence information is within reach to various research fields. Three benchtop NGS instruments are now available. The 454 GS Junior (Roche), Ion PGM (Life Technologies) and MiSeq (Illumina) are laser-printer sized and offer modest set-up and running costs. By reviewing 2 studies that compared the performance of these instruments, the major characteristics of each benchtop platforms are compared to enable direct comparisons. The 454 GS Junior generated the longest reads and most contiguous assemblies but had the lowest throughput. The Ion Torrent PGM had the highest throughput and fastest run time. The MiSeq had the highest throughput per run and lowest error rates. The Ion Torrent PGM and 454 GS Junior both produced homopolymer-associated indel errors. Although all the platforms allow multiplexing of samples, details of experimental design, library preparation and data analysis may constrain the options. The features of the platforms provide opportunities both to conduct groundbreaking studies and to waste money. Thus, careful considerations should be made before purchasing or using any of them.

Nuclear Factor-kappaB Dependent Induction of TNF-alpha and IL-1beta by the Aggregatibacter actinomycetemcomitans Lipopolysaccharide in RAW 264.7 Cells

Aggregatibacter actinomycetemcomitans is an important pathogen in the development of localized aggressive periodontitis. Lipopolysaccharide (LPS) is a virulent factor of periodontal pathogens that contributes to alveolar bone loss and connective tissue degradation in periodontal disease. Our present study was designed to investigate the cytokine expression and signaling pathways regulated by A. actinomycetemcomitans LPS (Aa LPS). Cytokine gene expression profiling in RAW 264.7 cells was performed by microarray analyses. The cytokine mRNA and protein levels and related signaling pathways induced by Aa LPS were measured by RT-PCR, ELISA and western blotting. Microarray results showed that Aa LPS strongly induced the expression of NF-kappaB, NF-kappaB-related genes, inflammatory cytokines, TNF-alpha and IL-1beta in RAW 264.7 cells. NF-kappaB inhibitor pretreatment significantly reduced the levels of TNF-alpha and IL-1beta mRNA and protein. In addition, the Aa LPS-induced TNF-alpha and IL-1beta expression was inhibited by p38/JNK MAP kinase inhibitor pretreatment. These results show that Aa LPS stimulates TNF-alpha and IL-1beta expression through NF-kappaB and p38/JNK activation in RAW 264.7 cells, suggesting the essential role of this pathway in the pathogenesis of localized aggressive periodontitis.