Butyrate induces reactive oxygen species production and affects cell cycle progression in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Short-chain fatty acids, such as butyric acid and propionic acid, are metabolic by-products generated by periodontal microflora such as Porphyromonas gingivalis, and contribute to the pathogenesis of periodontitis. However, the effects of butyrate on the biological activities of gingival fibroblasts (GFs) are not well elucidated. MATERIAL AND METHODS: Human GFs were exposed to various concentrations of butyrate (0.5-16 mm) for 24 h. Viable cells that excluded trypan blue were counted. Cell cycle distribution of GFs was analyzed by propidium iodide-staining flow cytometry. Cellular reactive oxygen species (ROS) production was measured by flow cytometry using 2',7'-dichlorofluorescein (DCF). Total RNA and protein lysates were isolated and subjected to RT-PCR using specific primers or to western blotting using specific antibodies, respectively. RESULTS: Butyrate inhibited the growth of GFs, as indicated by a decrease in the number of viable cells. This event was associated with an induction of G0/G1 and G2/M cell cycle arrest by butyrate (4-16 mm) in GFs. However, no marked apoptosis of GFs was noted in this experimental condition. Butyrate (> 2 mm) inhibited the expression of cdc2, cdc25C and cyclinB1 mRNAs and reduced the levels of Cdc2, Cdc25C and cyclinB1 proteins in GFs, as determined using RT-PCR and western blotting, respectively. This toxic effect of butyrate was associated with the production of ROS. CONCLUSION: These results suggest that butyrate generated by periodontal pathogens may be involved in the pathogenesis of periodontal diseases via the induction of ROS production and the impairment of cell growth, cell cycle progression and expression of cell cycle-related genes in GFs. These events are important in the initiation and prolongation of inflammatory processes in periodontal diseases.

Effects of TGF-beta s on the growth, collagen synthesis and collagen lattice contraction of human dental pulp fibroblasts in vitro.

Transforming growth factor-beta (TGF-beta) is important in regulating the repair and regeneration of damaged dental pulp. For further elucidating the roles of different isoforms of TGF-beta in the healing and inflammatory processes of human dental pulp, we found that TGF-beta1, TGF-beta2 and TGF-beta3 inhibited the growth of two human dental pulp cell strains in vitro by 19-29, 18-25 and 23-26%, respectively, at a concentration of 0.5 ng/ml. TGF-beta also differentially stimulated the collagen synthesis of pulp cells. Collagen synthesis increased by 1 ng/ml of TGF-beta1 and TGF-beta2 by 42 and 51%, respectively. TGF-beta3 (0.1-1 ng/ml) lacked of stimulatory effect on collagen synthesis of pulp cells. Pulp cells have the intrinsic capacity to contract collagen lattice, leading to decreasing of lattice diameter. An 8 h exposure to TGF-beta1 and TGF-beta2 enhanced the pulp cell-populated collagen lattice contraction at concentrations ranging from 0.2 to 3 ng/ml. At similar concentrations, TGF-beta3 lacked of this stimulatory effect. When collagen lattice were detached after 24 h of exposure, TGF-beta1 and TGF-beta2 (0.6-3 ng/ml) induced the pulp cells-populated collagen lattice contraction within 4-8h of gel detachment. These results indicate that TGF-beta-induced collagen lattice contraction is a late cellular event. These in vitro results indicate that effects of TGF-beta isoforms on the growth, collagen synthesis and collagen lattice contraction of pulp cells may play crucial roles in the pathobiological processes of dental pulp.

The effects of extracellular citric acid acidosis on the viability, cellular adhesion capacity and protein synthesis of cultured human gingival fibroblasts.

Root surface demineralization is widely used as an adjunct to periodontal treatment. To clarify the influence of citric acid root conditioning on periodontal wound healing, the effects of citric acid and associated extracellular acidosis on the viability (MTT assay), attachment and protein synthesis ([3H]-proline incorporation into trichloroacetic acid-precipitated proteins) of human gingival fibroblasts (GF) were investigated. A concentration of 47.6 mmol/L of citric acid (pH 2.3) in water led to total cell death within three minutes of incubation. Media containing 23.8 mmol/L and 47.6 mmol/L of citric acid exerted strong cytotoxicity (47 to 90 per cent of cell death) and inhibited protein synthesis (IC50 = 0.28 per cent) of GF within three hours of incubation. Incubation of cells in a medium containing 11.9 mmol/L of citric acid also suppressed the attachment and spreading of fibroblasts on culture plates and Type I collagen, with 58 per cent and 22 per cent of inhibition, respectively. Culture medium supplemented with 11.9, 23.8 and 47.6 mmol/L of citric acid also led to extracellular acidosis by decreasing the pH value from 7.5 to 6.3, 5.2 and 3.8, respectively. In addition, it was confirmed that the toxic effect of media containing citric acid was due to their acidity rather than the citrate content. Most of the citric acid-induced cell death could be prevented by adjusting the pH value of the culture medium to pH 7.5. Sodium citrate, at a concentration of 47.6 mmol/L, also exerted little cytotoxicity. The results suggested that toxicity of citric acid in specific stages of the healing process must be considered prior to its clinical application. Careful management of citric acid in order to avoid contact with tissue or the development of other demineralizing agents is important in enhancing periodontal wound healing.

Contribution of stimulus attributes to errors in duration and distance judgments--a developmental study.

In Matsuda's 1996 study, 4- to 11-yr.-old children (N = 133) watched two cars running on two parallel tracks on a CRT display and judged whether their durations and distances were equal and, if not, which was larger. In the present paper, the relative contributions of the four critical stimulus attributes (whether temporal starting points, temporal stopping points, spatial starting points, and spatial stopping points were the same or different between two cars) to the production of errors were quantitatively estimated based on the data for rates of errors obtained by Matsuda. The present analyses made it possible not only to understand numerically the findings about qualitative characteristics of the critical attributes described by Matsuda, but also to add more detailed findings about them.

Entero-Aggregative- Haemorrhagic Escherichia coli (EAHEC) Serotype O104:H4 -The Evolving "Superbug".

The rare, E.coli strain O104:H4 has been identified as the causative agent of one of the largest ever reported food-borneoutbreaks of gastroenteritis and Hemolytic-uremic syndrome (HUS) in Germany this year. This hypervirulent pathotype possess a unique combination of two pathogens: enterohemorrhagicE.coli (EHEC) and enteroaggregative E.coli (EAEC) strains. The serotype has rarely been described previously in humans and never associated with any earlier large scale EHEC outbreaks. It is now being referred to as the Entero-Aggregative-Haemorrhagic Escherichia coli (EAHEC).Advances in high-throughput sequencing technologies helped in rapid complete genome sequencing of the outbreak strains by different laboratories.Comparison of the genome sequence of the outbreak strain with other diarrhea-associated EAEC serotype O104:H4 indicate that the chromosome of the outbreak strain is most similar to that of an early isolated EAEC strain 55989 and has evolved to become more virulent by the acquisition of a Shiga toxin 2 encoding prophage, a plasmid encoding CTX-M beta-lactamases, and substituting the aggregative adherence fimbria III (AAF/III)with the rarer aggregative adherence fimbria I (AAF/I). The present article reviews the virulent traits ofthe outbreak strain, and also presents an update of the different intervention strategies that are being tested for the treatment of infections by such highly pathogenic strains.