Attenuation of radiation‐ and chemoradiation‐induced mucositis using gamma‐d‐glutamyl‐l‐tryptophan (SCV‐07)

Oral Diseases (2010) 16 , 655–660 Objective: To evaluate the efficacy of a novel immunomodulating peptide (SCV‐07) in attenuating the course of radiation‐induced mucositis in an established animal model of oral mucositis (OM). Material and Methods: In three separate experiments, golden Syrian hamsters received either an acute radiation challenge to the buccal mucosa of eight fractionated doses of 7.5 Gy of radiation over a 2‐week‐period, or a combination of acute radiation and cisplatin. In each experiment, animals were treated with varying doses or schedules of SCV‐07 or placebo. OM was scored in a blinded fashion using digital images obtained during the experimental period. Results: We found that SCV‐07 reduced the severity and duration of both acute and fractionated radiation‐induced OM. Similarly, when radiation and chemotherapy were used to induce OM, treatment with SCV‐07 significantly reduced the duration of ulcerative OM. The therapeutic benefit was dependent on both dose and schedule of administration. Conclusion: Taken together, we found SCV‐07 was able to modify the duration and severity of oral mucositis and was dependent on schedule and dose.     

Nicotine modulates gelatinase B (MMP‐9) and epilysin (MMP‐28) expression in reconstituted human oral epithelium

J Oral Pathol Med (2011) 40 : 33–36 Oral epithelial keratinocytes express nicotinic cholinergic receptors which activation modulates keratinocytes differentiation and migration through different metabolic pathways. Matrix metalloproteinases (MMPs) are Zn‐dependent enzyme involved in cell migration. Among them, gelatinase B (MMP‐9) and epilysin (MMP‐28) are two MMPs expressed by human keratinocytes during both wound healing and proliferation. Their expression has been investigated in a reconstituted human oral epithelium (HOE) exposed to nicotine (Nic, 1–50 μM) for 72 h both in the absence and presence of the nicotinic antagonist mecamylamine (Mec), H7, a PKC inhibitor and PD98059, a MAPK inhibitor (PD). At the end of treatment, MMP‐28 expression has been analyzed in epithelium sections using an anti‐MMP‐28 antibody, whereas MMP‐9 presence and activity has been measured in cell‐conditioned medium analyzed by gelatine zymography. The expression of MMP‐9 was reduced by Nic in a dose‐dependent fashion and this effect was antagonized by Mec, H7 and PD. On the other hand, Nic increased the expression of MMP‐28, and this effect was blocked both by H7 and PD, whereas Mec even enforced it. Nic effects on MMP‐9 and MMP‐28 expression by oral keratinocytes were not previously reported and these data suggest MMPs expression mediated by PKC and MAPK as a possible target for Nic toxicity in oral epithelium.     

Toll‐Like Receptor 2 Knockdown Modulates Interleukin (IL)‐6 and IL‐8 but not Stromal Derived Factor‐1 (SDF‐1/CXCL12) in Human Periodontal Ligament and Gingival Fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll‐like receptors (TLRs), recognize pathogen‐associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll‐like receptor 2 (TLR2) and an antagonist or agonist for Toll‐like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)‐6, IL‐8, and stromal derived factor‐1 (SDF‐1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA‐mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL‐6, IL‐8, and CXCL12 mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL‐6 and IL‐8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL‐6 and IL‐8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.     

Biochemical and functional properties of a pyruvate formate‐lyase (PFL)‐activating system in Streptococcus mutans

Streptococcus mutans has an oxygen‐sensitive enzyme, pyruvate formate‐lyase (PFL), which is a key enzyme in anaerobic sugar fermentation. We have shown that S. mutans has an activating system, including a PFL‐activating enzyme (PFL‐activase) and an electron transport system; the latter transfers an electron from NADPH to PFL‐activase, as occurs in Escherichia coli. NADPH was a physiological electron donor for the electron transport system and as little as 0.02 mM NADPH activated over 80% of PFL of S. mutans. The optimum pH of the PFL‐activating system was around 6.8, whereas the optimum of the E. coli system is at alkaline pH. In addition, small dialyzable molecules in cell‐free extracts participated in keeping PFL active in S. mutans. These results suggest that, in dental plaque under anaerobic conditions where sugar supply is often limited or pH frequently falls below neutrality, S. mutans always keeps PFL active through the activating system.     

Increased Expression of Interleukin (IL)‐35 and IL‐17, But Not IL‐27, in Gingival Tissues With Chronic Periodontitis

Background: Interleukin (IL)‐35 plays an important role in immune regulation through the suppression of effector T‐cell populations, including T‐helper 17 (Th17) cells. Although Th17 cells and IL‐17 are involved in the pathogenesis of periodontitis, the level of IL‐35 in inflamed periodontal tissues is unclear. Here, IL‐35, IL‐17, and IL‐27 production/expression in gingival crevicular fluid (GCF) and human gingival tissue were investigated. Methods: GCF samples were collected from buccal (mesial, center, and distal) sites of teeth from patients with chronic periodontitis (CP) and healthy controls and were analyzed by enzyme‐linked immunosorbent assay for IL‐35 (periodontitis, n = 36; healthy, n = 30) and IL‐17 (periodontitis, n = 16; healthy, n = 13). Gingival tissue, including sulcus/pocket epithelium and underlying connective tissue, was collected from an additional 10 healthy participants and 10 patients with CP and were analyzed by quantitative polymerase chain reaction (qPCR) for Epstein Barr virus‐induced gene 3 (EBI3), IL12A, and IL17A. IL27p28 was also tested by qPCR. Results: IL‐35 and IL‐17 were significantly higher in GCF from patients with periodontitis than healthy participants (P <0.01, P <0.05, respectively). In both healthy participants and those with periodontitis, positive correlations were found among IL‐35 and probing depth and clinical attachment level (CAL) as well as between IL‐17 and CAL. EBI3, IL12A (components of IL‐35), and IL17A messenger RNA expression levels were significantly higher in inflamed gingival tissue than in healthy control tissues (P <0.05). IL27p28 was not detected in any sample, suggesting that IL‐27 is not produced in large quantities in periodontal tissue. Conclusion: IL‐35 and IL‐17, but not IL‐27, may play important roles in the pathogenesis of periodontitis.     

Stimulation of epithelial cell matrix metalloproteinase (MMP‐2, ‐9, ‐13) and interleukin‐8 secretion by fusobacteria

Background/aims: Bacterial pathogens involved in periodontal diseases exert their destructive effects primarily by stimulating the host cells to increase their secretion of proinflammatory cytokines and matrix metalloproteinases (MMPs). This study aimed to determine the epithelial cell matrix metalloproteinase and interleukin‐8 (IL‐8) secretion upon exposure to fusobacteria. Methods: Eight different oral and non‐oral Fusobacterium strains were incubated with HaCaT epithelial cells. Gelatin zymography and Western blot analysis were performed to detect collagenase 3 (MMP‐13), gelatinase A (MMP‐2), gelatinase B (MMP‐9), and IL‐8 secretion by epithelial cells. Results: All Fusobacterium strains, especially Fusobacterium necrophorum ATCC 25286, Fusobacterium nucleatum ATCC 25586, and Fusobacterium varium ATCC 51644, increased MMP‐9 and MMP‐13 secretion. Fusobacterium simiae ATCC 33568, and to a lesser extent F. nucleatum and F. necrophorum, increased epithelial MMP‐2 secretion. F. nucleatum and F. necrophorum also increased IL‐8 secretion. F. varium ATCC 27725, a strain that only weakly stimulated MMP production, strongly increased the IL‐8 production, suggesting that their expression is differently regulated. Conclusion: We conclude that the pathogenic potential of fusobacteria may partly result from their ability to stimulate secretion of MMP‐9, MMP‐13, and IL‐8 from epithelial cells.     

Aloin Inhibits Interleukin (IL)‐1β−Stimulated IL‐8 Production in KB Cells

Background: Interleukin (IL)‐1β, which is elevated in oral diseases including gingivitis, stimulates epithelial cells to produce IL‐8 and perpetuate inflammatory responses. This study investigates stimulatory effects of salivary IL‐1β in IL‐8 production and determines if aloin inhibits IL‐1β?stimulated IL‐8 production in epithelial cells. Methods: Saliva was collected from volunteers to determine IL‐1β and IL‐8 levels. Samples from volunteers were divided into two groups: those with low and those with high IL‐1β levels. KB cells were stimulated with IL‐1β or saliva with or without IL‐1 receptor agonist or specific mitogen‐activated protein kinase (MAPK) inhibitors. IL‐8 production was measured by enzyme‐linked immunosorbent assay (ELISA). MAPK protein expression involved in IL‐1β?induced IL‐8 secretion was detected by Western blot. KB cells were pretreated with aloin, and its effect on IL‐1β?induced IL‐8 production was examined by ELISA and Western blot analysis. Results: Saliva with high IL‐1β strongly stimulated IL‐8 production in KB cells, and IL‐1 receptor agonist significantly inhibited IL‐8 production. Low IL‐1β–containing saliva did not increase IL‐8 production. IL‐1β treatment of KB cells induced activation of MAPK signaling molecules as well as nuclear factor‐kappa B. IL‐1β?induced IL‐8 production was decreased by p38 and extracellular signal‐regulated kinase (ERK) inhibitor treatment. Aloin pretreatment inhibited IL‐1β?induced IL‐8 production in a dose‐dependent manner and inhibited activation of the p38 and ERK signaling pathway. Finally, aloin pretreatment also inhibited saliva‐induced IL‐8 production. Conclusions: Results indicated that IL‐1β in saliva stimulates epithelial cells to produce IL‐8 and that aloin effectively inhibits salivary IL‐1β–induced IL‐8 production by mitigating the p38 and ERK pathway. Therefore, aloin may be a good candidate for modulating oral inflammatory diseases.