lnterleukin-1β and phenytoin reduce α1 (1) procollagen mRNA expression in human gingival fibroblasts

Effects of and interactions between interleukin-1β (IL-1 β) and phenytoin (PHT) on α1 (I) procollagen gene and protein expression in human gingival fibroblasts and its relation to prostaglandin E₂ (PGE₂) formation were studied. IL-1β (300 pg/ ml) reduced the steady-state level of αl(I) procollagen mRNA by 50% and decreased the amount of procollagen I by 35%. PHT (10 μg/ml) reduced the level of α1(I) procollagen mRNA by 40% but the amount of procollagen I in the medium was unchanged. In combination with IL-1β, PHT potentiated the inhibitory effect of IL-1β on αl(I) procollagen mRNA level that was accompanied by an increased PGE₂ formation. Preincubation with indomethacin (10^-6m) partially reduced the inhibitory effect of IL-1β as well as of IL-1β in combination with PHT on the mRNA level of αl(I) procollagen. The inhibitory effect of PHT was unaffected by indomethacin treatment. Addition of exogenous PGE₂ (≥10 nm) dose-dependently reduced steady-state level of α1(I) procollagen mRNA as well as the amount of procollagen 1. The study indicates that IL-1 reduces the expression of αl(I) procollagen mRNA in human gingival fibroblasts partly by a prostaglandin endoperoxide (PGH) synthase-mediated pathway and partly by a PGH-synthase independent pathway, whereas PHT reduces α1(I) procollagen gene expression by a PGH-synthase independent pathway. The potentiation of the inhibitory effect of IL-1 induced by PHT was mediated mainly by a PGH-synthase dependent pathway.     

Interleukin-1α produced in human gingival fibroblasts induces several activities related to the progression of periodontitis by direct contact

Previous observations suggest that interleukin-1 (IL-1) may play an important role in the progression of periodontitis. In the present study, we investigated whether a cell-associated IL-1α (CAIL-lα) produced in human gingival fibroblasts (HGF) induces biological activities related to the progression of periodontitis. HGF were treated with recombinant human IL-1β (rhIL-1β) for 12 h. After that, the cell layers of HGF were washed 3 times with fresh medium and were then fixed with 1% paraformaldehyde. The fixed cell layers of HGF were used for assays for bone resorbing activity, prostaglandin E₂ (PGE₂) production and collagenase activity. Fixed cell layers of HGF treated with rhIL-1β enhanced not only calcium release from BALB/c mouse calvaria but also PGE₂ production and collagenase activity in HGF and human periodontal ligament fibroblasts (HPLF) cultured on the fixed cell layers. These activities were neutralized by treatment with monoclonal mouse anti-human IL-1α antibody, but monoclonal mouse antihuman IL-1β antibody showed no effects on these activities. The induction of these activities by fixed cell layers of HGF required direct contact between the fixed cell layers and the calvaria, HGF, or HPLF. These results suggest that CAIL-1α produced in HGF treated with rhIL-1β induces bone resorbing activity, PGE₂ production and collagenase activity in the target cells by direct contact; CAIL-lα may play an important role in the progression of periodontitis.     

Prostaglandin E2 and I2 regulate intercellular adhesion molecule-1 expression in interleukin-1 beta-stimulated human gingival fibroblasts

The present study investigated the effect of prostaglandin (PG) E₂ and PGI₂ on intercellular adhesion molecule-1 (ICAM-1) expression in interleukin-1β (IL-1β)-stimulated human gingival fibroblasts (HGF). IL-1β potently induced ICAM-1 expression in HGF and indomethacin, a cyclooxygenase inhibitor, enhanced ICAM-1 expression in the cells. These data showed that endogenous PGs generated by HGF stimulated with IL-1β downregulated ICAM-1 expression. IL-1β significantly increased the levels of PGE₂ and, to a lesser extent, those of 6-keto-PGF_1α (a stable metabolite of PGI₂) in the culture media of HGF. Indomethacin completely inhibited the production of PGE₂ and 6-keto-PGF_1α in IL-1β-stimulated HGF. Exogenous PGE₂ and carbacyclin (a stable derivative of PGI₂) in the presence of indomethacin dose-dependently suppressed ICAM-1 expression in IL-1β-challenged HGF. Since PGE₂ and PGI₂ are known to elevate intracellular cyclic AMP (cAMP) levels, we examined the effect of dibutyryl cAMP, a cAMP analogue, and isobutylmethylxanthine, a phosphodiesterase inhibitor, on ICAM-1 expression. Both agents downregulated ICAM-1 expression in IL-1β-stimulated HGF. These results suggest that PGE₂ and PGI₂ downregulate ICAM-1 expression in IL-1β-stimulated HGF through a cAMP-dependent mechanism and that intracellular cAMP elevation in HGF may control inflammatory and immune responses in periodontal disease.     

Cyclosporin A upregulates prostaglandin E2 production in human gingival fibroblasts challenged with tumor necrosis factor alpha in vitro

The effect of Cyclosporin A (CsA) on prostaglandin E₂ (PGE₂) production in human gingival fibroblasts challenged with tumor necrosis factor alpha (TNF-α) was studied. TNF-α (1-100 ng/ml) dose-dependently stimulated PGE₂; formation in 24 h cultures. CsA (1-100 ng/ml) did not induce PGE₂; formation itself but potentiated TNF-α induced PGE; formation in gingival fibroblasts in a manner dependent on the concentrations of both CsA and TNF-α. TNF-α (10 ng/ml) stimulated the release of [³H]-arachidonic acid (A.A) from prelabelled fibroblasts that was potentiated by CsA (100 ng/ml). Addition of exogenous unlabelled AA (5-20 μM/ml) to the cells resulted in enhanced PGE₂: formation that was not potentiated by CsA (100 ng/mi). Furthermore. CsA (100 ng/ml) did not further increase the level of cyclooxygenase-2 mRNA induced by TNF-α (10 ng/ml). although PGE₂ formation was enhanced. The results indicate that CsA and TNF-α act in concert on PGE₂ formation in gingival fibroblasts. which may be of importance in the pathogenesis of gingival overgrowth induced by the drug.     

Production of inflammatory mediators and cytokines by human gingival fibroblasts following bacterial challenge

Bacteria can indirectly affect the course of periodontal diseases by activating host cells to produce and release inflammatory mediators and cytokines. These mediators and cytokines, manifest potent proinflammatory and catabolic activity and may play key roles in local amplification of the immune response as well as in periodontal tissue breakdown. This study tested the effect of Actinobacillus actinomycetemecomitans (Aa) and Campylobacter rectus (Cr) challenge on PGE₂, IL-1β, IL-6 and IL-8 production by human gingival fibroblasts (HGF). Contact-inhibited HGF were prepared and formalin-killed bacterial cells ( Aa JP2, ATCC 29523 & 33384 and Cr ATCC 33238) at 10⁶-10⁹ were added to the HGF. Culture supernatants were collected at varying time intervals and analyzed for cytokine and mediator content. All concentrations of Aa JP2 and Cr ATCC 33238 suppressed IL-1β production up to approximately 50% during the initial 3-12-h period. No bacterial concentration tested was able to increase IL-1β production above the maximum basal levels. Both bacterial species stimulated production of IL-6 and IL-8. Aa JP2 did not affect PGE₂ levels significantly, whereas Cr ATCC 33238 was stimulatory only at the highest concentration tested (10⁹). There were no significant differences among the three Aa strains with respect to IL-1β production. However, Aa ATCC 29523 and ATCC 33384 were less capable of stimulating IL-6 secretion and more efficient in stimulating. IL-8 production than Aa JP2. In general. Cr was the most potent enhancer of cyto-kine and mediator production by HGF. In conclusion, Aa and Cr are capable of amplifying the local immune response and promoting periodontal tissue inflammation by stimulating HGF to secrete mainly IL-6 and IL-8.     

Cytokines in crevicular fluid and orthodontic tooth movement

This review aimed to evaluate studies on cytokines in the gingival crevicular fluid (GCF) during orthodontic treatment, summarizing the regulation patterns of the most commonly studied cytokines and exploring their clinical implications. To achieve this, a number of key databases were searched using MESH terms and free text terms. An additional search was made by reference tracking. The procedures suggested by the QUOROM statement were followed. Data from the included studies were extracted into orthodontic mechanics, GCF sampling/handling methods, and cytokine measurements. From the 85 relevant studies identified, 23 studies could be included. Common drawbacks consisted mainly of inadequacies in the study design (e.g. short duration and small number of study subjects). The most consistent result was a peak of cytokine levels at 24 h. Associations existed between prostaglandin E₂ (PGE₂) and interleukin-1β (IL-1β) and pain, velocity of tooth movement, and treatment mechanics. Interleukin-1β and PGE₂ showed different patterns of up-regulation, with IL-1β being more responsive to mechanical stress and PGE₂ more responsive to synergistic regulation of IL-1β and mechanical force. The results might be taken to support, at the cellular level, the use of light continuous forces for orthodontic treatment.     

Expression of cyclooxygenase-1 and -2 in IL-1β-induced synovitis of the temporomandibular joint

Background:  In this study, we analyzed the gene expression profile of fibroblast-like synoviocyte (FLS) cultures from the temporomandibular joint (TMJ) to identify candidate genes associated with intracapsular pathologic conditions of TMJ. Cyclooxygenase (COX)-2 was one of the genes in FLS upregulated following stimulation by interleukin (IL)-1β, a cytokine thought to play a key role in several pathological conditions. This study investigated the expression of COX-1 and COX-2 in cultured human FLS and rat TMJ synovium following stimulation with IL-1β.
Methods:  RNA was isolated from human FLS after IL-1β treatment. COX-1 and -2 expression was examined using a GeneChip and real-time polymerase chain reaction. Prostaglandin E₂ (PGE₂) levels in conditioned media from FLS were measured using enzyme-linked immunosorbent assay. Synovial tissues from TMJs of IL-1β-injected rats were examined for COX-1 and COX-2 expression by immunohistochemical staining.
Results:  Following treatment of FLS with IL-1β, expression of the COX-2 gene increased up to 8 h and peaked at 4 h, whereas COX-1 expression did not change. Stimulation with IL-1β increased the level of PGE₂ in conditioned media of cultured FLS in a time-dependent manner up to 48 h. Immunohistochemistry showed a strong positive staining for COX-2 in the lining and sub-lining synovial tissues of the TMJ of IL-1β-injected rats. In contrast, staining for COX-1 was the same in synovial tissues with and without IL-1β injection.
Conclusion:  These data suggest that COX-2 expression stimulated by IL-1β stimulates the production of PGE₂ in FLS and plays important roles in the progression of inflammation in TMJ.     

Response of human gingival fibroblasts to prostaglandins

The effects of various prostaglandins (PGs) on the functions of human gingival fibroblasts (Gin-1 cells; ATCC CRL 1292) were examined by phase-contrast microscopy, cell-counting and radioautographic experiments. Tested PGs were PGA₁, PGA₂, PGB₁, PGB₂, PGD₂, PGE₁, PGE₂, PGF₁α, PGF₂α, PGI₂, 6-keto-PGF₁α, 9α-11α-methanoepoxy-PGF₂α, and thromboxane (TX) B₂. PGA₁, and PGD₂ at 30 μM caused morphological deformation of Gin-1 cells. All the PGs tested at 30 μM suppressed the proliferation of Gin-1 cells in the logarithmic growth phase. Furthermore, all the PGs tested at 10 μM suppressed DNA synthesis, collagen synthesis, and noncollagenous protein synthesis in confluent Gin-1 cells, while exerting no effect on GAG synthesis. The concentrations of PGs used are beyond those found in healthy gingiva. However, in periodontitis the local concentrations of some PGs within the gingiva are expected to be extremely elevated beyond the physiological level. These results suggest that PGs may play an important role as a negative regulator in metabolism and some pathologic gingival conditions by suppressing the functions of gingival fibroblasts.     

Nicotine effects on PGE2 and IL-1β release by LPS-treated human monocytes

Cigarette smoking is a major risk factor in the development and further progression of periodontitis. However, little is known regarding the pathogenesis of smoking-related periodontal diseases. The purpose of this study was to examine the effects of nicotine, alone and in combination with lipopolysaccharide (LPS), on monocyte secretion of bone-resorbing factors, PGE₂ and IL-1β. Peripheral blood monocytes (PBM) were isolated by counterflow centrifugal elutriation from 15 healthy, non-smoking donors. PBM were incubated for 24 h in RPMI 1640 containing nicotine (0, 50 μg/ml, I μg/ml, 10 μg/ml and 100 μg/ml) with or without 10 μg/ml Porphyromonas gingivalis LPS or Escherichia coli LPS. Culture supernatants were assayed for PGE₂ and IL-1β by ELISA. None of the nicotine preparations resulted in significant PBM secretion of PGE₂ and IL-1β above that of unstimulated cultures. However, PGE₂ release was potentiated 1.7-fold by the combination of P. gingivalis LPS and 10 μg/ml nicotine relative to P. gingivalis LPS alone (p<0.05, one-way ANOVA). Prostaglandin E₃ release also was potentiated 3.5-fold by P. gingivalis LPS and 100 μg/ml nicotine relative to P. gingivalis LPS alone (p<0.00001, one-way ANOVA) and 3.1-fold by E. coli LPS and 100 μg/ml nicotine relative to E. coli LPS alone (p<0.00001, I. one-way ANOVA). IL-1β secretion was lower for either LPS plus 100 μg/ml nicotine relative to LPS alone, although not significantly. These data demonstrate upregulation of LPS-mediated monocyte secretion of PGE₂ by nicotine and suggest a potential role for nicotine in periodontal disease pathogenesis.     

Prostaglandin E2 and interleukin-1 concentrations in nicotine-exposed oral keratinocyte cultures

Oral keratinocytes are the first cells in contact with tobacco components and are capable of producing various inflammatory mediators, including PGE2 and IL-1. The purpose of this study was to examine PGE2 and IL-1 concentrations in nicotine-exposed oral keratinocyte cultures. Gingival keratinocyte cultures were established from healthy gingival tissues obtained from 7 subjects. Cultures were divided into 4 groups exposed to serum free medium (control), 0.1 μ m , 10 μ m or 1 mM nicotine for 4, 24 or 48 h. Using enzyme-linked immunosorbent assays, PGE₂ and IL-1α were quantified in culture supernatants; IL-1 α and β were also measured in lysed cells. A repeated measures analysis of variance was used to identify significant differences over time and treatment. Nicotine exposure did not significantly alter PGE2 levels at any given time period; however, PGE ₂ quantities declined significantly (p=0.0001) over time. At both 24 and 48 h, IL-1 α concentrations in lysates from 1 mM nicotine-exposed cells were significantly (p < 0.01) greater than those for all other treatments. Interleukin-1α quantities also declined significantly (p=0.037) over time in the cultures. Interleukin-1β concentrations were elevated, albeit not significantly, in the 1 mM treated cells at 24 and 48 h. Cell viability, mass and counts were not affected by nicotine treatment; these parameters increased significantly (p < 0.005) over time. In summary, nicotine treatment significantly increased IL-1 a concentrations in cultured keratinocytes; however, PGE₂ synthesis was not altered. Elevated IL-1 production by keratinocytes may have implications in tobacco-induced lesions, given the central role IL-1 plays in tissue response to injury.     

Levels of prostaglandin E2, thromboxane, and prostacyclin in periodontal tissues

Prostaglandin E₂ (PGE₂), thromboxane A₂ (TXA₂), and prostacyclin (PGI₂) are naturally occurring metabolites of arachidonic acid which have been associated with inflammation. To assess the relative importance of these mediators in periodontal diseases, the levels of all three were measured by radioimmunoassay in human tissues removed during surgery. TXA₂ and PGI₂ were determined as their stable hydrolysis products thromboxane B₂ (TXB₂) and 6-keto-prostaglandin F_1α (6-K-PGF_1α), respectively. Tissues from periodontal pockets were separated into superficial (n = 8) and deep (n = 22) samples. Noninflamed gingival samples (n = 3) were taken from distal wedges with no clinical evidence of periodontal disease. Most of the samples taken from deep sites (73%) had measurable PGE₂ with a mean level of 122 pg/mg. Half of these samples also had measurable TXB₂ which correlated positively with levels of PGE₂. Half of the superficial gingival samples had detectable levels of PGE₂, and none had detectable TXB₂. 6-K-PGF_1α was found in virtually all samples but at somewhat lower levels in noninflamed tissue. Neither PGE₂ nor TXB₂ were detected in noninflamed samples.     

Effect of in vitro aging on Campylobacter rectus lipopolysaccharide-stimulated PGE2 release from human gingival fibroblasts

OBJECTIVE: We examined the influence of in vitro aging on Campylobacter rectus (C. rectus) lipopolysaccharide (LPS)-stimulated prostaglandin (PG) E₂ release from human gingival fibroblasts (HGFs).
MATERIALS AND METHODS: LPS was prepared from C. rectus ATCC33238. HGFs were established from healthy gingival tissue removed from three patients (donors A, B and C), aged 10–12 years. Aging of the cells in culture was determined with increasing population doubling. The cells were cultured until confluence, then stimulated with LPS (1.0 μg ml^-1), and the levels of PGE₂ in the medium were measured after 24 h by radioimmunoassay.
RESULTS: The LPS-stimulated PGE₂ production in each old cell (passage 17–20) was significantly increased to about 1.6–2.6 times than that in the corresponding young cells (passage 5–6). The gene expression of cyclooxygenase-2 mRNA in the old cells was higher than that in the young cells in response to LPS. In the absence of LPS, PGE, production levels in both the young and old cells were very low, and also at the same level. However, there was a higher level of LPS-stimulated PGE₂ production in the young cells from donor C compared to that in the old cells from donor B. The LPS-stimulated PGE₂ production in each young cell from donors A and C was almost equal to that in each old cell from donors B and A, respectively.
CONCLUSIONS: The results suggested that aging in HGFs may be one of the factors that take part in the stimulation of C. rectus LPS-stimulated PGE₂ production in old cells.     

Interleukin-1 stimulates proteoglycan and hyaluronic acid production by human gingival fibroblasts in vitro

The effect of recombinant interleukin 1β [IL-1β] on proteoglycan and hyaluronic acid synthesis by human gingival fibroblasts has been investigated. It was found to stimulate gingival fibroblast proliferation in a dose dependent fashion with the midpoint of this response being in the 10⁻¹¹ mol/L range. At a concentration of 10⁻¹¹ mol/L, IL-1β stimulated proteoglycan synthesis by 40 per cent. Although IL-1β can stimulate cell proliferation and prostaglandin synthesis, its effect on proteoglycan synthesis was independent of these parameters. The kinetics of proteoglycan degradation in the presence or absence of IL-1β was monitored by pulse chase experiments and were found not to differ between treated and untreated cultures. The molecular size and carbohydrate composition of the proteoglycans was not affected by IL-1β. Additional studies revealed the synthesis of hyaluronic acid was also stimulated by IL-1β. As for the proteoglycans, inhibition of cell proliferation did not affect the stimulatory effect of IL-1β. However, blockage of prostaglandin synthesis abolished the stimulatory effect of IL-1β on hyaluronic acid synthesis. The effect of IL-1β on hyaluronic acid synthesis was found to be related to elevated levels of the enzyme hyaluronate synthetase. Molecular size analysis of newly synthesized hyaluronic acid revealed that cells treated with IL-1β synthesized more large molecular mass hyaluronic acid. Taken together, these findings are considered to reflect the ability of gingival fibroblasts to respond to inflammatory mediators in a manner indicative of early tissue repair.     

Serine protease activity is essential for thrombin-induced protein synthesis in cultured human dental pulp cells: modulation roles of prostaglandin E2

Chang MC, Lan WH, Chan CP, Lin CP, Hsieh CC, Jeng JH: Serine protease activity is essential for thrombin-induced protein synthesis in cultured human dental pulp cells: modulation roles of prostaglandin E₂. J Oral Pathol Med 1998; 27: 23–9. © Munksgaard, 1998.
Irritations and injuries to the dental pulp usually lead to different degrees of pulpal inflammation. To investigate the roles of thrombin and prostaglandins in the healing and inflammatory processes of dental pulp as well as their effects on pulpal protein synthesis, human dental pulp cell cultures were established and their protein production was measured with or without the presence of exogenous thrombin and prostaglandins. At concentrations of 1–25 U/ml, a-thrombin increased the protein synthesis to 1.4–2.3 fold over the vehicle control. On the contrary, 0.1 (μg/ml of prostaglandin E] (PGE₁ suppressed protein synthesis by 60%. Prostaglandin E₂ (PGE₂) also inhibited protein synthesis with an IC50 of 0.4 ug/ml. The stimulatory effects of thrombin (10 U/ml) can be inhibited by antithrombin III (2 U/ml) (a natural thrombin inhibitor) with heparin (2 U/ml), PPACK (D-Phe-Pro-ArgCH₂Cl) (20–50 ug/ml) (a serine protease inhibitor), and PGE₂ (0.5–1.0 μg/ml). Moreover, TRAP (20–40 μg/ml), a thrombin receptor agonist peptide, also exerted a stimulatory effect (1.21–1.37 fold). In conclusion, thrombin-induced protein synthesis by pulp cells is dependent on proteolytic activity, but not on binding to receptors. Both PGE₁ and PGE₂ exert suppressive effects on protein synthesis, indicating that interactions between thrombin and prostaglandins are important in regulating the inflammation, repair and regeneration of pulp tissue following injury.     

Influence of phenytoin on cytoplasmic free Ca2+ level in human gingival fibroblasts

Abstract— Influence of 5,5-diphenylhydantoin (phenytoin;PHT) on the cytoplasmic free Ca²⁺ concentration, [Ca²⁺]ⁱ, was studied in fura 2 loaded adherent monolayers of human gingival fibroblasts derived from three patients before and after 9 months of PHT therapy. In the patient where gingival overgrowth developed during PHT medication (responder), addition of PHT to gingival fibroblasts derived before PHT medication induced a transient extracellular Ca²⁺ dependent increase in [Ca²⁺]ⁱ. In a non-responder patient, where gingival overgrowth did not develop during the same period of PHT therapy, addition of PHT to gingival fibroblasts derived before the start of medication did not significantly affect [Ca²⁺]_i. Under extracellular Ca²⁺ deficient conditions, addition of PHT to serum-starved fibroblasts derived from the two categories of patients before the medication resulted in an increase in [Ca²⁺]_i. In fibroblasts derived from the responder patient during PHT medication, in contrast to those from the non-responders (n = 2), the basal level of [Ca²⁺]_i was significantly decreased. The results indicate that, in the cases studied, there is a relationship between PHT induced alterations in [Ca²⁺]_i in gingival fibroblasts and the clinical development of gingival overgrowth.     

Measurement of eight prostaglandins in human gingival and periodontal disease using high pressure liquid chromatography and radioimmunoassay

Eight prostaglandins (PG) of biological importance, namely PGE,. PGE₁ 13,14-dihydro-15-keto-PGE, (DHK-PGE₂), PGF_2α, PGF_1α, 13, 14-dihydro-15-keto-PGF_2α (DHK-PGF_2α, 6-keto-PGF_1α, and thromboxane B₂ (TxB₂), were measured in the human gingiva in a healthy state, gingivitis. and periodontitis. High pressure liquid chromatography (HPLC) in combination with radioimmunoassay (RIA) was employed for quantitative analysis. The advantages of HPLC over the classic silicic acid column chromatography of prostaglandins were discussed. All prostaglandins considered were shown to increase during advancing periodontal destruction. The most quantitatively significant endoperoxide metabolites at every stage of inflammation were TxB₂ and 6-keto-PGF_1α followed by PGE₂. DHIK-PGE₂, PGE₁, PGF_1α. PGF_3α, and DHK-PGF_2α in decreasing order.     

The effect of interleukin-1 β on hyaluronic acid synthesized by adult human gingival fibroblasts in vitro

The effect of recombinant interleukin-1β (IL-lβ) on hyaluronic acid synthesis by human gingival fibroblasts was studied. IL-1bT caused a dose-dependent increase in the incorporation of (³lucosamine into hyaluronic acid. The ³⁵S/35H ratios of labeled macromolecules did not change regardless of the presence or absence of TL-lβ and indicates stimulation of hyaluronic acid synthesis. Inhibition of cell proliferation by hydroxyurea caused an increase in hyaluronic acid synthesis. The effect of IL-1β on hyaluronic acid synthesis in the presence of hydroxyurea was increased over untreated and IL-lβ-treated controls, but equivalent to the hydroxyurea-treated controls. Thus the effect of IL-1β on hyaluronic acid synthesis may be independent of cell proliferation. Furthermore, inhibition of prostaglandin E2 synthesis by indomethacin abolished the effect of IL-1β on hyaluronic acid synthesis. Inhibition of new protein synthesis by cycloheximide negated the effect of IL-β on hyaluronic acid synthesis. This may be related to inhibition of new hyaluronate synthetase synthesis, since IL-1β stimulated the level of hyaluronate synthetase activity. Sepharose CL-2B chromatography revealed that most of the newly synthesized hyaluronic acid was of large molecular size. The cells exposed to IL-1β retained more large molecular size hyaluronic acid in their cell layer environment than did the control cells. These responses by fibroblasts to IL-1β may be indicative of early tissue repair.     

Automated enzyme immunoassay to measure prostaglandin E2 in gingival crevicular fluid

An automated enzyme immunoassay (EIA) to measure prostaglandin E₂ (PGE₂) in gingival crevicular fluid (GCF) of humans and dogs was developed as an indicator of periodontal disease. GCF is noninvasively collected on Periopaper strips and the PGE₂ is extracted by a simple method. Samples containing 10-500 pg/ml PGE² can be measured. A commercially available kit is used to perform the competitive EIA in microtiter plates. In the EIA, rabbit anti PGE₂ antisera binds to either the PGE₂ in the sample or to the acetylcholinesterase-linked PGE₂. The assay is automated using the Biomek 1000 workstation, resulting in day-to-day variability of less than 5% CV. Dog models of chronic and ligature-induced periodontitis were used to demonstrate that increased GCF PGE₂, as measured by our assay, correlates with increased pocket depth and gingival bleeding scores.     

Role of interleukin-1 and prostaglandin in in vitro bone resorption induced by Actinobacillus actinomycetemcomitans lipopolysaccharide

Lipopolysaccharide (Y4 LPS) isolated from Actinobacillus actinomycetemcomitans strain Y4 induced bone resorption in BALB/c mouse calvaria organ culture. The calcium release from LPS-low responsive C3H/HeJ mouse calvaria by Y4 LPS was very low. Indomethacin almost completely inhibited prostaglandin E₂ (PGE₂) production by Y4 LPS-stimulated BALB/c mouse calvaria, but did not suppress interleukin-1 (IL-1) release from the calvaria, and partially suppressed the bone resorption. Dexamethasone strongly inhibited the PGE₂ and IL-1 production by Y4 LPS-stimulated BALB/c mouse calvaria. as well as Y4 LPS-induced bone resorption. Dexamethasone inhibited expression of membrane IL-1 on osteoblastic cells stimulated with Y4 LPS, but indomethacin did not. Furthermore, anti-IL-1 serum partially suppressed the calcium release from Y4 LPS-stimulated BALB/c mouse calvaria. These results suggest that both PGE₂ and IL-1 participate in Y4 LPS-induced bone resorption in vitro .     

Interleukin-1β+3953 allele 2: association with disease status in adult periodontitis

Abstract. Adult periodontitis is a complex multifactorial disease whose etiology is not well defined. The pro-inflammatory and bone resorptive properties of interleukin-1 beta (IL-1β) strongly suggest a role for this cytokine in the pathogenesis of periodontal disease. In the study reported here, the frequency of IL-1β⁺³⁹⁵³ genotypes including allele 2 of the IL-1β⁺³⁹⁵³ restriction fragment length bi-allelic polymorphism was significantly increased in patients with advanced adult periodontitis compared to those with early and moderate disease. Furthermore, allele 2 was associated with increased production of IL-1β by activated peripheral blood polymorphonuclear cells of patients with advanced disease, although this increase failed to reach statistical significance. Finally, the data obtained revealed significant linkage disequilibrium between allele 2 of the IL-1β⁺³⁹⁵³ polymorphism and allele 2 of the bi-allelic IL-1α⁻⁸⁸⁹ polymorphism in both patients and orally healthy controls. These findings provide new insight into the possible role of IL-1α and β gene polymorphisms in the susceptibility to adult periodontitis.