Wnt3a signaling induces murine dental follicle cells to differentiate into cementoblastic/osteoblastic cells via an osterix-dependent pathway.

BACKGROUND AND OBJECTIVE: Dental follicle cells, putative progenitor cells for cementoblasts, osteoblasts and periodontal ligament cells, interplay with Hertwig's epithelial root sheath (HERS) cells during tooth root formation, in which HERS is considered to have an inductive role in initiating cementogenesis by epithelial-mesenchymal interaction. However, the specific mechanisms controlling the cementoblast/osteoblast differentiation of dental follicle cells are not fully understood. Canonical Wnt signaling has been implicated in increased bone formation by controlling mesenchymal stem cell or osteoblastic cell functions. This study examined the possible expression of canonical Wnt ligand in HERS and the role of Wnt signaling during the cementoblast/osteoblast differentiation of dental follicle cells. MATERIAL AND METHODS: The expression of Wnt3a, a representative canonical Wnt ligand, in HERS was assessed by immunohistochemistry. The differentiation and function of immortalized murine dental follicle cells were evaluated by measuring alkaline phosphatase (ALP, Alpl) activity and osteogenic gene expression. RESULTS: We identified the expression of Wnt3a in HERS during mouse tooth root development by immunohistochemistry as well as in cultured human epithelial rest cells of Malassez by real-time polymerase chain reaction, while no expression of Wnt3a was detected in cultured dental mesenchymal cells. Exposure of immortalized murine dental follicle cells to Wnt3a-induced ALP activity as well as expression of the Alpl gene. Pretreatment of cells with Dickkopf-1, a potent canonical Wnt antagonist, markedly attenuated the effect of Wnt3a on ALP expression. Furthermore, Wnt3a induced transcriptional activity of runt-related transcription factor 2 (Runx2) and expression of osterix at gene and/or protein levels. Treatment with osterix-small interfering RNA significantly inhibited Wnt3a-induced ALP expression at gene and protein levels. CONCLUSION: These findings suggest that HERS has a potential role in stimulating cementoblast/osteoblast differentiation of dental follicle cells via the Wnt/ß-catenin signaling pathway.

Calcification during bone healing in a standardised rat calvarial defect assessed by micro-CT and SEM-EDX.

OBJECTIVE: The study was designed to investigate the process of calcification during bone healing in a standardized rat calvarial bone defect model, measured by bone mineral density and the concentrations and distributions of calcium, phosphorus and carbon in the bone matrix. MATERIALS AND METHODS: A standard defect was made on the parietal bone of 12-week-old rats under anaesthesia. The rats were fixed in weeks 1, 2, 4 and 8,and the calvaria were resected and examined with microcomputed tomography, then frozen and sectioned for histology and analysed with energy-dispersive X-ray spectroscopy (EDX). Parietal bone of 12-week-old control rats was processed similarly. RESULTS: The mineral density of healing bone increased with time. The healing bone became thicker and denser with time in histology. The distributions of Ca and P expanded over the bone matrix, whereas that of C became localised and complemented that of C and P. The Ca/P concentration ratio increased, whereas the C/Ca and C/P ratios decreased in the healing bone matrix. CONCLUSION: Healing bone is immaturely calcified initially and proceeds calcification gradually, that is, as the bone volume increases, mineral increases in density and matures in quality, while organic components decrease.

Transportation of sublingual antigens across sublingual ductal epithelial cells to the ductal antigen-presenting cells in mice.

BACKGROUND: Sublingual immunotherapy (SLIT) has proven to be safe and efficient for the treatment of type I allergies. However, the mechanisms underlying allergen transportation within the sublingual compartment, the localization of antigens, and the identities of the cells responsible for this immunization remain incompletely understood. OBJECTIVE: In this study, we focused on the sublingual ductal system and analysed the localization and transportation of antigens after their sublingual application. METHODS: In mice given adjuvant-free antigens sublingually, tissues were removed at 0, 0.5, 1, or 2 h after the application and subjected to immunohistochemistry. Cells isolated from the sublingual duct and mucosa were analysed by flow cytometry. RESULTS: Substantial immunoreactivity to ovalbumin (OVA) was evident in sublingual ductal epithelial cells at 30 min and 1 h after sublingual administration of OVA, but it had disappeared at 2 h. The ductal epithelial cells incorporated not only OVA, but also particulate antigens such as latex or silica beads and microbes. MHC class II (MHCII)(+) antigen-presenting cells (APCs) were located around the sublingual ductal system, and MHCII(+) cells were co-localized with, and around, antigen-incorporated sublingual duct cells. CD11b(+) CD11c(-) cells were present among CD45(+) MHCII(+) cells at greater frequency in the sublingual duct than in the sublingual mucosa, and they were the main contributors to the incorporation of OVA in vitro. CONCLUSIONS AND CLINICAL RELEVANCE: This study reveals that sublingual antigens can be transported across sublingual ductal epithelial cells to the ductal APCs. If the system is the same in humans as in mice, the ductal APCs may prove to be important target cells for SLIT.

Assessment of the plasma/serum IgG test to screen for periodontitis.

Chronic periodontitis is a silent infectious disease prevalent worldwide and affects lifestyle-related diseases. Therefore, efficient screening of patients is essential for general health. This study was performed to evaluate prospectively the diagnostic utility of a blood IgG antibody titer test against periodontal pathogens. Oral examination was performed, and IgG titers against periodontal pathogens were measured by ELISA in 1,387 individuals. The cut-off value of the IgG titer was determined in receiver operating characteristic curve analysis, and changes in periodontal clinical parameters and IgG titers by periodontal treatment were evaluated. The relationships between IgG titers and severity of periodontitis were analyzed. The best cut-off value of IgG titer against Porphyromonas gingivalis for screening periodontitis was 1.682. Both clinical parameters and IgG titers decreased significantly under periodontal treatment. IgG titers of periodontitis patients were significantly higher than those of healthy controls, especially in those with sites of probing pocket depth over 4 mm. Multiplied cut-off values were useful to select patients with severe periodontitis. A blood IgG antibody titer test for Porphyromonas gingivalis is useful to screen hitherto chronic periodontitis patients.

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.

Micro-CT analysis of alveolar bone healing using a rat experimental model of critical-size defects.

OBJECTIVE: This study was designed to establish a rat model of a critical size alveolar bone defect. MATERIALS AND METHODS: Standardized buccal or mesiobuccal alveolar bone defects were made around the right first mandibular molar of 12-week-old rats, and the left was used as a control. Alveolar bone healing was examined quantitatively by three-dimensional micro-computed tomographic imaging. Bone matrix production of osteoblasts and osteocytes during repair of alveolar bone defects was examined with in situ hybridization for type I collagen. RESULTS: Buccal defects were repaired significantly and the volume decreased by 88.3% in week 24, whereas mesiobuccal defects were repaired little. Osteoblasts and osteocytes expressed type I collagen in both defects in week 3 but showed little expression by week 6 and thereafter, leaving the mesiobuccal defects largely unrepaired. CONCLUSION: The mesiobuccal defect is a critical-size defect that is not ultimately repaired with bone. It may be an appropriate experimental model for investigating the effectiveness of bone regenerative agents in human alveolar bone loss.

Porphyromonas gingivalis fimbriae induce unique dendritic cell subsets via Toll-like receptor 2.

BACKGROUND AND OBJECTIVE: Dendritic cells (DCs) play a critical role in the activation of T cells as well as in shaping immune responses. We have reported previously that Porphyromonas gingivalis lipopolysaccharides (Pg LPS) induced a CD14(+)CD16(+) DC subset with a weak immuno-stimulatory activity. In contrast, Escherichia coli LPS (Ec LPS) induced fully matured DCs with strong immunostimulatory activities. Since Pg LPS as well as Pg fimbriae have been indicated to work as Toll-like receptor (TLR) 2 ligands, we speculate that the TLR usage of bacterial antigens may be critical for DC maturation. MATERIAL AND METHODS: We investigated the effect of Pg fimbriae on the phenotype and function of human peripheral blood DCs in comparison with a TLR2 ligand, peptidoglycan, and a TLR4 ligand, Ec LPS. RESULTS: Flow cytometry revealed that Pg fimbriae and peptidoglycan but not Ec LPS induced CD14 and CD16 expression on peripheral blood DCs (CD14(-)CD16(-)). A monoclonal antibody against TLR2 abrogated this induction, but an antibody against TLR4 had no effect. Dendritic cells stimulated with Pg fimbriae had a weaker capability to induce allogenic T cell proliferation and exhibited a weaker production of interleukin-8 and regulated upon activation, normal T cell expressed and secreted (RANTES) than DCs stimulated with Ec LPS. CONCLUSION: These results indicate that different TLR usage affects mature DC phenotype and function and is thus crucial to the regulation of immunity to the pathogen.

Expression of functional Toll-like receptors and nucleotide-binding oligomerization domain proteins in murine cementoblasts and their upregulation during cell differentiation.

BACKGROUND AND OBJECTIVE: While the primary role of cementoblasts is to synthesize the components of cementum, we have reported that immortalized murine cementoblasts (OCCM-30) express functional Toll-like receptor (TLR)-2 and -4, and these receptors are involved in the alteration of gene expression associated with cementum formation and in the upregulation of osteoclastogenesis-associated molecules, such as receptor activator of nuclear factor-kappaB (NF-kappaB) ligand. We hypothesized that cementoblasts express a wide range of pattern recognition receptors in a manner comparable to osteoblasts, which are known to express various functional TLRs and nucleotide-binding oligomerization domain (NOD) proteins. MATERIAL AND METHODS: Murine cementoblasts and pre-osteoblasts were used. The gene and protein levels of TLRs/NODs were analyzed using real-time polymerase chain reaction and flow cytometry. Interleukin-6 (IL-6) and activated NF-kappaB were measured using enzyme-linked immunosorbent assay. RESULTS: The expressions of TLR-1, -2, -4, -6 and -9, CD14, NOD-1 and -2 were detected in cementoblasts and were upregulated upon differentiation induced by ascorbic acid. Similar patterns were observed in the mouse MC3T3-E1 osteoblast cell line. Synthetic ligands, Pam3CSK4 (TLR-1/2 agonist), Pam2CGDPKHPKSF (TLR-2/6 agonist), lipid A (TLR4 agonist), CpG DNA (TLR-9 agonist), FK565 (NOD1 agonist) and muramyldipeptide (NOD2 agonist), effectively induced NF-kappaB activation in cementoblasts and/or ascorbic acid-treated cementoblasts. Furthermore, these ligands induced IL-6 production in a NF-kappaB-dependent manner in cementoblasts and/or ascorbic acid-treated cementoblasts. CONCLUSION: These results indicate that cementoblasts possess functional TLR and NOD signaling systems and have a similar capacity to osteoblasts in responding to a wide variety of pathogens.

Histamine amplifies immune response of gingival fibroblasts.

Histamine is an important mediator in immune responses, but it is unclear whether periodontal tissues express histamine receptors and are able to respond to histamine. We hypothesized that histamine, inflammatory cytokines, and bacterial components released in inflamed periodontal tissues may be synergistically involved in periodontitis. The present study showed that human gingival fibroblasts mainly express histamine receptor H1R, and responded to histamine to produce interleukin (IL)-8. Stimulation of gingival fibroblasts with tumor necrosis factor-alpha, IL-1alpha, and lipopolysaccharide markedly induced IL-8 production, and the IL-8 production was synergistically augmented in the presence of or pre-treatment with histamine. Selective inhibitors of mitogen-activated protein kinases (MAPKs), nuclear factor (NF)-kappaB, and phospholipase C (PLC) significantly inhibited the synergistic effect. These results indicate that histamine induces IL-8 production from gingival fibroblasts through H1R, and synergistically augments the inflammatory stimuli by amplification of the MAPK and NF-kappaB through H1R-linked PLC. Abbreviations used: HDC, histidine decarboxylase; LPS, lipopolysaccharide; IL, interleukin; TNF, tumor necrosis factor; HR, histamine receptor; PLC, phospholipase C; MAPK, mitogen-activated protein kinase; NF, nuclear factor; ERK, extracellular signal-related kinase; JNK, c-Jun N-terminal kinase; R, receptor; TLR, Toll-like receptor; alpha-MEM, alpha-minimum essential medium; FCS, fetal calf serum; RT-PCR, reverse-transcriptase polymerase chain-reaction; ELISA, enzyme-linked immunosorbent assay; SD, standard deviation; LDH, lactate dehydrogenase.

Cleavage of PDGF receptor on periodontal ligament cells by elastase.

Human leukocyte elastase, a neutrophil serine protease, is considered to be a potential immunoregulatory protease. Since the PDGF receptor (PDGFR) on periodontal ligament (PDL) cells is a crucial element for various functions, such as wound healing in periodontal tissue, we investigated the effect of elastase on the expression of PDGFR on PDL cells by flow cytometry and Western blotting. We found that PDGFR-alpha disappeared with an increasing dose of elastase, and PDGFR-beta was degraded into several fragments. Elastase degraded both receptors on fixed cells, indicating that the degradation resulted from direct proteolysis on the cell surface. Elastase also then disturbed the phosphorylation of ERK1/2, JNK/SARK, and p38, triggered by PDGF-AA and PDGF-BB, suggesting that elastase inhibited PDGFR-dependent cell activation in PDL cells. These results suggest that elastase may modulate the PDGF-mediated activity of PDL cells during periodontal wound healing.

Detection frequency of periodontitis-associated bacteria by polymerase chain reaction in subgingival and supragingival plaque of periodontitis and healthy subjects.

The aim of this study was to compare the detection frequencies of 25 bacterial species in subgingival and supragingival plaque of 18 untreated periodontitis subjects and 12 periodontally healthy subjects. Genomic DNA was extracted from subgingival and supragingival plaque samples, and bacterial detection was performed by polymerase chain reaction of the 16S rRNA genes. Fourteen bacteria showed no relationship with periodontitis, and 11 of these 14 species were frequently detected (> or =50%) in subgingival plaque in both periodontitis and healthy subjects. Nine bacteria such as Eubacterium saphenum, Prevotella intermedia, and Treponema denticola seemed to be related to periodontitis; their detection frequencies in subgingival plaque samples were higher in periodontitis than in healthy subjects, but these differences were not statistically significant by multiple comparisons (0.002< or =P<0.05). Two species (Mogibacterium timidum and Porphyromonas gingivalis) were detected significantly more frequently in subgingival plaque of periodontitis subjects than of healthy subjects (P<0.002), with P. gingivalis being detected only in periodontitis subjects, suggesting that these two species are closely related to periodontitis. There were no significant differences in the detection frequencies of the 25 bacteria between subgingival and supragingival plaque, suggesting that the bacterial flora of supragingival plaque reflects that of subgingival plaque.

Proteolysis of ICAM-1 on human oral epithelial cells by gingipains.

Cysteine proteinases (gingipains) from Porphyromonas gingivalis are considered key virulence factors of severe periodontitis and host immune evasion. Since expression of intercellular adhesion molecule-1 (ICAM-1) on gingival epithelium is indispensable in polymorphonuclear leukocyte (PMN) migration at the site of periodontitis, we examined the effects of gingipains on the expression of ICAM-1 on human oral epithelial cell lines (KB and HSC-2) by flow cytometry and Western blotting. We found that three purified forms of gingipains efficiently reduced ICAM-1 expression on the cells in a time- and dose-dependent manner. Gingipains reduced the expression on fixed cells and degraded the ICAM-1 in the cell membranes, indicating that the reduction resulted from direct proteolysis. They then disturbed the ICAM-1-dependent adhesion of PMNs to the cells. These results indicate that gingipains cleave ICAM-1 on oral epithelial cells, consequently disrupting PMN-oral epithelial cell interaction, and are involved in immune evasion by the bacterium in periodontal tissues.

Expression of MMP-8 and MMP-13 mRNAs in rat periodontium during tooth eruption.

The present study was designed to investigate mRNA expression of matrix metalloproteinase-8 (MMP-8) and MMP-13 in forming periodontium during tooth eruption in the rat. RT-PCR for the decalcified paraffin sections indicated expression of MMP-8 and MMP-13 in the periodontal tissues. In situ hydridization demonstrated expression of MMP-8 in osteoblasts, osteocytes, periodontal ligament cells, cementoblasts, and cementocytes along with collagen types I and III. In contrast, transcripts of MMP-13 were confined to a small population of osteoblasts and osteocytes in alveolar bone. The results suggested that MMP-8 may be involved in remodeling the periodontium during tooth eruption, and its expression may be coordinated with that of collagen types I and III, whereas the participation of MMP-13 may be rather limited.

In vivo platelet response to lipopolysaccharide in mice: proposed method for evaluating new antiplatelet drugs.

We previously found evidence (based on the use of 5HT as a marker) that i.v. injection of a lipopolysaccharide (LPS) into mice induces a rapid accumulation of platelets in liver and lung. Our previous studies lacked measurement of the platelet count itself, but we have now compared the LPS-induced changes in 5HT levels with the change in platelet count. We also examined the effects on the platelet response of some drugs that act on platelets. In mice, sublethal doses of LPS induced parallel decreases in platelets and 5HT in the blood. The 5HT lost from the blood accounted well for the 5HT accumulated in liver and lung. Soon after this accumulation, the levels of platelets and 5HT in the blood recovered in parallel, and these recoveries corresponded well with the decreases in 5HT occurring in liver and lung. Aspirin and dexamethasone were effective at both reducing pulmonary platelet-accumulation and promoting their return to the circulation. By contrast, oestrogen tended to reduce the return of platelets from lung to circulation. Heparin did not inhibit pulmonary platelet-accumulation but it did decrease their return to the circulation. These results suggest that (i) in response to sublethal doses of LPS, platelets translocate into the liver and lung, then return to the circulation; (ii) this platelet response involves mechanisms that can be modified by drugs; and (iii) the use of this platelet response as a tool for drug evaluation might help identify new drugs with therapeutic potential.

Osteoblastic differentiation of periosteum-derived cells is promoted by the physical contact with the bone matrix in vivo.

The periosteum contains osteoprogenitors that differentiate to osteoblasts in bone growth or repair. Our previous studies suggested the hypothesis that the physical contact of the periosteum with the bone matrix is requisite for the differentiation of osteoblasts. To test the hypothesis, the present study was designed to investigate how the contact between the periosteum and the bone matrix influences the osteoblastic differentiation of periosteal cells with establishing a new experimental model in vivo. Differentiation of osteoblasts was assessed by gene expression of type I collagen, osteocalcin and bone sialoprotein using in situ hybridization. A barrier was designed to prevent periosteal cells from contacting the bone matrix using the membrane filter. The membrane filter was inserted surgically between the surface of rat parietal bone and the periosteum after being punched out with pin holes. Periosteal cells were allowed to contact with the bone surface only through the pin holes. The pin hole was filled with cells derived from the periosteum 1 week after inserting the filter. Differentiation of osteoblasts in week 2 and noticeable bone formation in week 3 were identified on the bone surface only under the pin hole but not under the filter. The present study demonstrated that the physical contact with the bone matrix promotes osteoblastic differentiation of periosteum-derived cells in vivo.

Porphyromonas gingivalis-specific IgG subclass antibody levels as immunological risk indicators of periodontal bone loss.

BACKGROUND: It has been well demonstrated a positive association between the magnitude of host antibody response and periodontal disease status. Previous studies also reported that Porphyromonas gingivalis-specific IgG subclass antibodies were elevated in sera from adult periodontitis patients. However, the rôle and the association of these IgG subclass antibodies to the development of periodontal diseases are poorly understood. AIM: The aim of present investigation was to examine the relation of serum IgG subclass antibody levels and alveolar bone loss in treated and untreated periodontitis patients. METHODS: Serum samples were taken from 20 treated and maintained periodontitis patients (SPT patients), 30 untreated patients and 19 periodontally healthy subjects. We determined the IgG subclass antibody titers to P. gingivalis whole cells using an enzyme-linked immunosorbent assay (ELISA). Subgingival plaque samples were taken from the mesio-buccal surface of 6 randomly selected teeth of SPT patients and evaluated for the presence of P. gingivalis by immunofluorescence microscopy. Clinical measurements were also taken including full mouth intraoral radiographs to measure interproximal alveolar bone loss at baseline (BLS1) and at a 5-year recall visit in the SPT patients (BLS2). RESULTS: Our results indicated that both patient groups had detectable levels of IgG1, IgG2, and IgG4. Significantly higher IgG1 was observed in both patient groups compared to the healthy subjects. The untreated patients also exhibited significantly elevated IgG2 response (p<0.05). The mean IgG4 level of the SPT patients was significantly higher compared to the other subject group (p<0.05). A statistically significant positive correlation between IgG2 levels and changes in bone levels (DeltaBLS: BLS2-BLS1) was seen in the SPT patients (p<0.001). SPT patients with high IgG2 and low IgG4 showed greater bone loss than those with low IgG2 and high IgG4 (p<0.05), although the mean prevalence of P. gingivalis in the 2 groups did not differ significantly. CONCLUSION: Our data suggest that the prolonged IgG2 response after periodontal treatment may be indicative of recurrent or persistent periodontal destruction.

The effect of short-term tooth intrusion on human pulpal blood flow measured by laser Doppler flowmetry.

The purpose was to examine the effect of brief intrusive forces on human pulpal blood flow (PBF). Laser Doppler flowmetric measurements were made from 17 vital upper left central incisors of 17 participants who had clinically healthy tooth crowns and periodontal tissues. Brief intrusive forces (0.5,1,5 N; duration 20 s) were applied to the incisal edges of the examined teeth, and apical displacement of the teeth and the PBF were measured simultaneously. Recordings were made with and without an opaque rubber dam applied to the examined teeth. Intrusive force significantly reduced PBF flux both with and without the dam (P<0.05, Friedman analysis). The results indicate that transient apical displacement can reduce PBF.

Production of interleukin-8 and nitric oxide in human periapical lesions.

Bacterial infection of the pulp and root canal system leads to the recruitment of immunocompetent cells in the periapex and stimulates inflammatory cell responses to produce a variety of inflammatory mediators. Cytokines, reactive oxygen intermediates, and reactive nitrogen intermediates are frequently found at sites of acute inflammation. In this study, we measured the levels of interleukin (IL)-8 and nitric oxide (NO) in the periapical exudate (PE) from human periapical lesions and investigated the association of these mediators with the clinical symptoms of periapical periodontitis. PE samples were collected from root canals during routine endodontic treatments. The IL-8 concentration was measured by the enzyme-linked immunosorbent assay, and the NO level was measured as nitrite/nitrate concentration assayed by the Griess reaction. Detectable levels of IL-8 and nitrite/nitrate were found in 24 and 19 of 27 PE-samples, respectively. Although PE-IL-8 and nitrite/nitrate concentration showed a broad range, a significantly positive correlation was found between both mediators. Also, significantly higher IL-8 levels were found in PE from lesions that had painful symptoms at the sampling visit. However, there was no relationship between elevated NO levels and clinical symptoms. These results suggest that the up-regulation of IL-8 may have a critical role in the development of the symptoms of periapical disease.

Cleavage of CD14 on human gingival fibroblasts cocultured with activated neutrophils is mediated by human leukocyte elastase resulting in down-regulation of lipopolysaccharide-induced IL-8 production.

Activated polymorphonuclear leukocytes (PMNs) release various types of proteases and express them on the cell surface. The proteases play important roles in PMN-mediated events. In the present study, flow cytometric analysis revealed that CD14 expression on human gingival fibroblasts (HGF) was markedly reduced by PMA-activated PMNs in a coculture system. We found that this reduction was caused by both secreted and cell surface proteases produced by activated PMNs. A protease responsible for the reduction was found to be human leukocyte elastase (HLE) secreted from the activated PMNs by use of various protease inhibitors, although HLE was only partially involved in CD14 reduction caused by cell-bound molecule(s) on fixed PMNs. Analysis with purified HLE revealed a time- and dose-dependent reduction of CD14 on HGF, and complete reduction was observed by 20 microg/ml HLE treatment for 30-60 min, but the other molecules such as CD26, CD59, CD157, and MHC class I on HGF were only slightly reduced. This reduction of CD14 resulted from direct proteolysis by HLE on the cell surface, because HLE reduced CD14 on fixed HGF and also on purified cell membranes. As a result of CD14 proteolysis, IL-8 production by HGF was suppressed when triggered by 10 ng/ml LPS, but not by IL-1alpha, indicating that HLE inhibited a CD14-dependent cell activation. These findings suggested that activated PMNs have a potential negative feedback mechanism for HGF function at the inflammatory site, particularly in periodontal tissues.

Immunobiological activities of a chemically synthesized lipid A of Porphyromonas gingivalis.

A synthetic lipid A of Porphyromonas gingivalis strain 381 (compound PG-381), which is similar to its natural lipid A, demonstrated no or very low endotoxic activities as compared to Escherichia coli-type synthetic lipid A (compound 506). On the other hand, compound PG-381 had stronger hemagglutinating activities on rabbit erythrocytes than compound 506. Compound PG-381 also induced mitogenic responses in spleen cells from lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice, as well as LPS-responsive C3H/HeN mice. The addition of polymyxin B resulted in the inhibition of mitogenic activities, however, compound 506 did not show these capacities. Additionally, compound PG-381 showed a lower level of activity in inducing cytokine production in peritoneal macrophages and gingival fibroblasts from C3H/HeN mice, but not C3H/HeJ mice, in comparison to compound 506. Thus, this study demonstrates that the chemical synthesis of lipid A, mimicking the natural lipid A portion of LPS from P. gingivalis, confirms its low endotoxic potency and immunobiological activity.