Takashi Sasano

Identification of IL-18 and Th17 Cells in Salivary Glands of Patients with Sjögren's Syndrome, and Amplification of IL-17-Mediated Secretion of Inflammatory Cytokines from Salivary Gland Cells by IL-18

IL-18 is a proinflammatory cytokine and plays an important pathogenic role in inflammatory and autoimmune disorders. IL-17 is also a proinflammatory cytokine and IL-17-secreting Th17 cells are involved in autoimmunity. However, the pathological roles of IL-18 and Th17 cells in Sjögren’s syndrome (SS) remain to be elucidated. This study showed that the expression of IL-18 was detected in acinar cells, intraducts, and CD68+ macrophages in salivary glands of SS patients, but not in those of healthy subjects or patients with chronic graft-vs-host disease, by immunohistochemistry, and immunoblot analysis revealed that 24-kDa precursor form of IL-18 (proIL-18) and 18-kDa mature IL-18 were detected in SS salivary glands. The majority of the infiltrating cells in the salivary glands of SS patients were CD4+ T cells, and CD8+ T cells were infiltrated to a lesser extent. The predominant expression of IL-17 was found in infiltrating CD4+ T cells, whereas a small number of infiltrating CD8+ T cells expressed IL-17. Human salivary gland HSY and acinar AZA3 cells constitutively expressed proIL-18 and caspase-1, and a calcium ionophore A23187 induced the secretion of IL-18 from the cells. HSY and AZA3 cells expressed IL-18R and IL-17R on the cell surface, and IL-18 amplified the secretion of IL-6 and IL-8 that were induced by low amounts of IL-17. Primary salivary gland cells from normal subjects partially confirmed these findings. These results suggest that IL-18 and Th17 cells detected in the salivary glands in SS patients are associated with the pathogenesis of SS in the salivary glands.

FGF-2 Stimulates Periodontal Regeneration Results of a Multi-center Randomized Clinical Trial

The efficacy of the local application of recombinant human fibroblast growth factor-2 (FGF-2) in periodontal regeneration has been investigated. In this study, a randomized, double-blind, placebo-controlled clinical trial was conducted in 253 adult patients with periodontitis. Modified Widman periodontal surgery was performed, during which 200 µL of the investigational formulation containing 0% (vehicle alone), 0.2%, 0.3%, or 0.4% FGF-2 was administered to 2- or 3-walled vertical bone defects. Each dose of FGF-2 showed significant superiority over vehicle alone (p < 0.01) for the percentage of bone fill at 36 wks after administration, and the percentage peaked in the 0.3% FGF-2 group. No significant differences among groups were observed in clinical attachment regained, scoring approximately 2 mm. No clinical safety problems, including an abnormal increase in alveolar bone or ankylosis, were identified. These results strongly suggest that topical application of FGF-2 can be efficacious in the regeneration of human periodontal tissue that has been destroyed by periodontitis.

Periodontal tissue regeneration using fibroblast growth factor -2:Randomized controlled phase II clinical trial

Background The options for medical use of signaling molecules as stimulators of tissue regeneration are currently limited. Preclinical evidence suggests that fibroblast growth factor (FGF)-2 can promote periodontal regeneration. This study aimed to clarify the activity of FGF-2 in stimulating regeneration of periodontal tissue lost by periodontitis and to evaluate the safety of such stimulation. Methodology/Principal Findings We used recombinant human FGF-2 with 3% hydroxypropylcellulose (HPC) as vehicle and conducted a randomized double-blinded controlled trial involving 13 facilities. Subjects comprised 74 patients displaying a 2- or 3-walled vertical bone defect as measured ≥3 mm apical to the bone crest. Patients were randomly assigned to 4 groups: Group P, given HPC with no FGF-2; Group L, given HPC containing 0.03% FGF-2; Group M, given HPC containing 0.1% FGF-2; and Group H, given HPC containing 0.3% FGF-2. Each patient underwent flap operation during which we administered 200 µL of the appropriate investigational drug to the bone defect. Before and for 36 weeks following administration, patients underwent periodontal tissue inspections and standardized radiography of the region under investigation. As a result, a significant difference (p = 0.021) in rate of increase in alveolar bone height was identified between Group P (23.92%) and Group H (58.62%) at 36 weeks. The linear increase in alveolar bone height at 36 weeks in Group P and H was 0.95 mm and 1.85 mm, respectively (p = 0.132). No serious adverse events attributable to the investigational drug were identified. Conclusions Although no statistically significant differences were noted for gains in clinical attachment level and alveolar bone gain for FGF-2 groups versus Group P, the significant difference in rate of increase in alveolar bone height (p = 0.021) between Groups P and H at 36 weeks suggests that some efficacy could be expected from FGF-2 in stimulating regeneration of periodontal tissue in patients with periodontitis. Trial Registration ClinicalTrials.gov NCT00514657

Toll-like Receptors, NOD1, and NOD2 in Oral Epithelial Cells

Oral epithelium might be the first barrier against oral bacteria in periodontal tissue. We hypothesized that oral epithelium is endowed with innate immune receptors for bacterial components, which play roles in host defense against bacterial infection without being accompanied by excessive inflammatory responses. We found clear expression of Toll-like receptor (TLR)4 as well as TLR2, and strong expression of NOD1 and NOD2 in normal oral epithelial tissues by immunohistochemical analysis. We also showed that primary oral epithelial cells in culture expressed these molecules using PCR, flow cytometry, and immunostaining. In inflamed oral epithelium, cell-surface localizations of TLR2 and TLR4 were more clearly observed than in healthy tissue. Upon stimulation with synthetic ligands for these receptors, the expression of β-defensin 2 was markedly up-regulated. These findings indicate that these molecules in oral epithelial cells are functional receptors that induce antibacterial responses.

Infiltrating CD8+ T Cells in Oral Lichen Planus Predominantly Express CCR5 and CXCR3 and Carry Respective Chemokine Ligands RANTES/CCL5 and IP-10/CXCL10 in Their Cytolytic Granules: A Potential Self-Recruiting Mechanism

Lichen planus is a chronic inflammatory disease of the skin and oral mucosa in which the cell-mediated cytotoxicity is regarded as a major mechanism of pathogenesis. To understand its pathophysiology further, the present study examined the in situ expression of chemokines and chemokine receptors in oral lichen planus. Immunohistochemical analysis of 15 cases has consistently revealed that infiltrating CD4(+) and CD8(+) T cells in the submucosa predominantly expressed CCR5 and CXCR3. Furthermore, infiltrating T cells, particularly CD8(+) T cells, were positive for RANTES/CCL5 and IP-10/CXCL10, the ligands of CCR5 and CXCR3, respectively. By immunoelectron microscopy, these chemokines were localized in the cytolytic granules of CD8(+) T cells. Lesional keratinocytes also overexpressed the ligands of CXCR3, namely, MIG/CXCL9, CXCL10, and I-TAC/CXCL11. Our data suggest that the chemokines signaling via CCR5 and CXCR3, which are known to be selectively expressed by type 1 T cells, are predominantly involved in T-cell infiltration of oral lichen planus. Furthermore, the presence of CCL5 and CXCL10 in the cytolytic granules of tissue-infiltrating CD8(+) T cells expressing CCR5 and CXCR3 reveals a potential self-recruiting mechanism involving activated effector cytotoxic T cells.

Chemically synthesized pathogen‐associated molecular patterns increase the expression of peptidoglycan recognition proteins via toll‐like receptors, NOD1 and NOD2 in human oral epithelial cells

Peptidoglycan recognition proteins (PGRPs), a novel family of pattern recognition molecules (PRMs) in innate immunity conserved from insects to mammals, recognize bacterial cell wall peptidoglycan (PGN) and are suggested to act as anti‐bacterial factors. In humans, four kinds of PGRPs (PGRP‐L, ‐Iα, ‐Iβ and ‐S) have been cloned and all four human PGRPs bind PGN. In this study, we examined the possible regulation of the expression of PGRPs in oral epithelial cells upon stimulation with chemically synthesized  pathogen‐associated  molecular patterns (PAMPs) in bacterial cell surface components: Escherichia coli‐type tryacyl lipopeptide (Pam3CSSNA), E. coli‐type lipid A (LA‐15‐PP), diaminopimelic acid containing desmuramyl peptide (γ‐ d‐glutamyl‐meso‐DAP; iE‐DAP), and muramyldipeptide (MDP). These synthetic PAMPs markedly upregulated the mRNA expression of the four PGRPs and cell surface expression of PGRP‐Iα and ‐Iβ, but did not induce either mRNA expression or secretion of inflammatory cytokines, in oral epithelial cells. Suppression of the expression of Toll‐like receptor (TLR)2, TLR4, nucleotide‐binding oligomerization domain (NOD)1 and NOD2 by RNA interference specifically inhibited the upregulation of PGRP mRNA expression induced by Pam3CSSNA, LA‐15‐PP, iE‐DAP and MDP respectively. These PAMPs definitely activated nuclear factor (NF)‐κB in the epithelial cells, and suppression of NF‐κB activation clearly prevented the induction of PGRP mRNA expression induced by these PAMPs in the cells. These findings suggested that bacterial PAMPs induced the expression of PGRPs, but not proinflammatory cytokines, in oral epithelial cells, and the PGRPs might be involved in host defence against bacterial invasion without accompanying inflammatory responses.

Accuracy of intraoral radiography, multidetector helical CT, and limited cone-beam CT for the detection of horizontal tooth root fracture.

OBJECTIVE The accuracies of intraoral radiography (IOR), multidetector helical computerized tomography (MDHCT) at slice thicknesses 0.63 mm and 1.25 mm, and limited cone-beam computerized tomography (LCBCT) were compared for detection of horizontal tooth root fracture. STUDY DESIGN In 7 beagle dogs, 28 maxillary anterior teeth were used, of which 13 had artificially induced horizontal root fracture. The specimens were examined by the above-mentioned 4 modalities. Diagnosis of root fracture was based on direct visualization of radiolucent line in each image by 6 radiologists. RESULTS Sensitivity, negative predictive value, and diagnostic accuracy (true positives + true negatives) for detecting fracture lines in LCBCT (0.96 +/- 0.04, 0.97 +/- 0.03, 0.93 +/- 0.04, respectively) were significantly higher than MDHCT at 0.63 mm (0.76 +/- 0.09, 0.8 +/- 0.05, 0.8 +/- 0.05, respectively), MDHCT at 1.25 mm (0.49 +/- 0.09, 0.66 +/- 0.04, 0.69 +/- 0.05, respectively), and IOR (0.51 +/- 0.18, 0.67 +/- 0.08, 0.69 +/- 0.08, respectively). Specificity and positive predictive value showed no significant intermethod difference among the 4 modalities. CONCLUSION Limited cone-beam CT is more useful than the other 3 radiographic modalities for diagnostic imaging of horizontal tooth root fracture.

Arterial Blood Pressure Regulation of Pulpal Blood Flow as Determined by Laser Doppler

Dynamic changes of pulpal blood flow (PBF) and gingival blood flow (GBF) induced by intra-venous injection of two kinds of vasoactive drugs were observed in dogs by means of Laser Doppler Velocimetry. Intra-venous injection of norepinephrine caused PBF to increase, corresponding to the blood pressure (BP) increase, while GBF decreased. Orciprenaline sulfate caused PBF to decrease parallel to the BP decrease, as compared with a GBF increase. The effects of these vasoactive drugs lasted longer on GBF than on PBF and BP. These results indicate that the regulation of blood flow in the dental pulp is more dependent on systemic blood pressure than on local vasoconstriction or vasodilation.

Accumulation of platelets in the lung and liver and their degranulation following antigen-challenge in sensitized mice.

Mast cells and basophils are believed to trigger allergic reactions and anaphylaxis. They rapidly release histamine (H), a typical mediator of inflammation, in response to antigens. In the mouse, platelets contain much 5‐hydroxytryptamine (5HT), an additional inflammatory mediator, while human platelets contain both H and 5HT. Here, we examined the response of platelets in sensitized mice to antigen challenge. Platelets accumulated in the lung and liver almost immediately after intravenous injection of ovalbumin (OVA), in mice sensitized to it, and platelet degranulation occurred during these reactions. These responses of platelets preceded H release from mast cells and/or basophils, occurred at doses of OVA lower than those inducing H release, and contributed to the signs of shock. We reported previously that intravenous injection into mice of LPS (a membrane constituent of gram‐negative bacteria) induces a similar platelet response (accumulation of platelets in the lung and liver) and shock. Blood that has passed through the body (other than the digestive tract) passes first to the lungs before being recirculated by the heart, and blood that has passed through the digestive tract passes next to the liver. Thus, our findings suggest that in addition to their role in haemostasis, platelets, tiny anuclear cytoplasts, may be important in both innate and acquired immunity, and that the lung and liver may be the fronts at which platelets wage war on pathogens.

Proinflammatory Cytokines Induce Proteinase 3 as Membrane-Bound and Secretory Forms in Human Oral Epithelial Cells and Antibodies to Proteinase 3 Activate the Cells through Protease-Activated Receptor-2

We wish to retract the article titled “Proinflammatory Cytokines Induce Proteinase 3 as Membrane-Bound and Secretory Forms in Human Oral Epithelial Cells and Antibodies to Proteinase 3 Activate the Cells through Protease-Activated Receptor-2,” by Akiko Uehara, Yumiko Sugawara, Takashi Sasano, Haruhiko Takada, and Shunji Sugawara, The Journal of Immunology, 2004, 173: 4179–4189. This retraction follows an investigation by Tohoku University into scientific misconduct. The investigation pointed out the following: 1. Fig. 1A: HSC-2, KB, and HO-1-u-1 are different cell lines, but the same photo is used for their GAPDH. 2. Fig. 7A: The cells were transfected with PR3or Lamin A/C-specific siRNA, which are different, but the patterns of GAPDH are the same. The first author, who conducted these experiments, could not counter the argument by adducing raw data at the investigation, and the investigation recognized them as scientific misconduct. Therefore, we wish to retract the article. We deeply regret these errors and apologize to the scientific community for the need to retract the article.