Analysis of the Expression of Macrophage among Periodontitis Rat Model after Treatment with Graptophyllum Pictum (L.) Griff. Leaves Extract Gel

@#Introduction: Aggressive periodontitis has the characteristics of rapid loss of periodontal tissue and bone destruction resulting in tooth loss. Graptophyllum pictum (L.) Griff. is widely used as herbal medicine in Indonesia. The flavonoid content in Graptophyllum pictum (L.) Griff. is known to have a role as an anti-inflammatory and anti-oxidant. This research aimed to analyze the role of Graptophyllum Pictum (L.) Griff. extract gel on the amount of macrophages as an inflammatory indicator on periodontal tissue of Wistar rats with periodontitis. Methods: Periodontitis was produced in Wistar rats by induced of 2 ml 109 CFU A. actinomycetemcomitans at gingival sulcus of the upper right second molar, afterward were treated with 7.5%, 15%, and 30% Graptophyllum Pictum (L.) Griff. extract gel for 3 days. Gingival tissues were removed for Hematoxylin Eosin staining for histopathological analysis and measurement of the number of macrophages. Results: Graptophyllum Pictum (L.) Griff. extract gel at concentrations of 7.5%, 15%, and 30% could significantly decrease the number of macrophages, but only group with a concentration of 15 and 30% can reduce the number of macrophages to reach an amount equivalent to the level in the negative control group. A concentration of 30% extract gel could reduce the number of macrophage cells more than the other two treatment groups. Conclusion: The concentration of 30% Graptophyllum Pictum (L.) Griff. extract gel was the most effective concentration in decreasing the amount of macrophages.

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.

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.

Detection of putative periodontopathic bacteria in type 1 diabetic and healthy children: A comparative study.

Aim: The aim of this study was to compare and assess the risk of periodontitis due to the presence of four putative periodontopathic bacteria (Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Aggregatibacter actinomycetemcomitans) in type 1 diabetic and healthy children. Materials and Methods: Fifty type 1 diabetic and 50 healthy children in the age group of 7-14 years were recruited for the study. Subgingival plaque samples collected from permanent first molars were subjected to polymerase chain reaction assay to detect 16S rRNA gene of P. gingivalis, T. forsythia, T. denticola and A. actinomycetemcomitans. The data were analyzed using Fisher exact test. The P < 0.05 was considered statistically significant. Results: The prevalence of subgingival periodontal pathogens in diabetic and healthy children was 2% and 4% for P. gingivalis, 34% and 34% for T. denticola, 20% and 18% for A. actinomycetemcomitans and for T. forsythia, 4% and 34%, respectively. Significant statistical difference was not observed with regard to the prevalence of P. gingivalis, T. denticola, and A. actinomycetemcomitans among type 1 diabetic and healthy children (P = 1.00). Conversely, T. forsythia was less prevalent in diabetic children compared to healthy children. Conclusion: Statistical significance was not observed for the prevalence of periodontopathic bacteria in type 1 diabetic subjects. The results of the present study thus reveal the absence of risk of periodontitis by these bacterial species in type 1 diabetic subjects.

Contaminación in vitro de cepillos dentales / In vitro contamination of toothbrushes

Se deierminó que los cepillos dentales mantienen viables y pueden transmitir 3 microorganismos frecuentemente implicados en infecciones orales. Los microorganismos fueron inoculados in vitro en cepillos mantenidos a temperatura ambiente y sometidos a subcultivo desde las 3 horas hasta los 16 días. Inóculos de aproximadamente 5 millones de bacterias/ml de un importante periodontopático, el Actinobacillus actinomycetemcomitans; de un entérico oportunista, el Enterobacter cloacae y una dosis infectiva 50, ID50 de un herpesvirus oral, el Herpes simplex virus tipo 1, (HSV-1) fueron colocados sobre cerdas de los cepillos. A. actinomycetemcomitans y el HSV-1 resultaron viables hasta por 72 horas. E. cloacae fué viable hasta por 16 dias, el tiempo máximo de subcultivo planteado en este estudio (Tabla 1). La viabilidad bacteriana se demostró por subcultivo de los microorganismos en TSBV y la identidad de los microorganismos se determinó por la morfología de la colonia, la catalasa, el MUG, la reacción en cadena de la polimerasa especie/especifico para A. actinomycetemcomitans y pruebas bioquímicas rápidas como subcultivo en McConkey y asimilación de sustratos para E. cloacae. La viabilidad viral se estableció por aparición del efecto citopatogénico en mononocapas de pulmón embrionario humano del inóculo recuperado de los cepillos, pase seriado e lFA indirecta contra HSV-1. Este estudio concluye que los cepillos dentales pueden ser un reservorio y además transmitir importantes patógenos orales entre familiares o individuos.

This study determined that toothbrushes could maintain viable and perhaps transmit to other family member 3 important oral pathogens. The toothbrushes were infected with an approximate inoculum of 5 million bacteria's per ml of Actinobacillus actinomycetemcomitans, and Enterobacter cloacae, respectively and an infective dose 50 (ID 50) of Herpes Simplex type I, (H5V-1 ). These microorganisms were placed directly on the tooth bristles at room temperature and subcultured at different times to establish individual microbial survival rates. Microorganisms were cultured at 3 hours. 24 hours, 96 hours, 5 days, 12 days, and 16 days after the initial toothbrush inoculation. A. actinomycetemcomitans, and HSV-1 resulted viable after 72 hours on toothbrushes. E. cloacae was viable as far as 16 days after the initial inoculation. The microbial viability was determined by subculture in TSBV and the identity of the microorganisms established by the bacterial colony morphology, rapid biochemical tests, and specie-specific polymerase chain reaction for A. actinomycetemcomitans. Viral viability was determined by visualization of the viral induced cytopathic effect on a cultured monolayer of embryonic lung fibroblasts from replicating HSV-1. Positive cultures were confirmed by IFA assay against HSV-L 1. In conclusion this study demonstrated in vitro that toothbrushes could act as a reservoir of microbes and maybe transmit important oral pathogens.