Immunohistochemical analysis of interleukin‐1α, its type I receptor and antagonist in keratocystic odontogenic tumors

Background: Interleukin‐1α (IL‐1α) is thought to play a crucial role in the growth of keratocystic odontogenic tumors (KCOTs) in the jaw. The function of IL‐1α is regulated by the local levels of IL‐1α, its receptor and receptor antagonist (IL‐1Ra) in tissues. In this study, the expression of these proteins was investigated both before and after marsupialization in KCOTs. Methods: The expression of IL‐1α, IL‐1 receptor type I (IL‐1RI) and IL‐1Ra was detected immunohistochemically in 10 specimens of KCOTs. Results: IL‐1α was intensively expressed throughout the epithelium in all cases, while mild expression of IL‐1α was detected in the subepithelial layer endothelial cells and fibroblasts. Mild or intensive immunoreactivity for IL‐1RI was also observed in the epithelial cells in all cases, and in the endothelial cells and fibroblasts in five cases respectively. The expression of IL‐1Ra was detected in the epithelial cells in five cases, and in the endothelial cells and fibroblasts in three cases. After marsupialization, the immunoreactivity for IL‐1α and IL‐1RI in the epithelial cells decreased, while the immunoreactivity for IL‐1Ra in the epithelial cells increased. However, the immunoreactivity for IL‐1RI and IL‐1Ra in endothelial cells and fibroblasts did not change significantly. Conclusion: The effects of IL‐1α on the epithelial cells might be downregulated after marsupialization by changing the expression levels of IL‐1α, IL‐1RI and IL‐1Ra in the epithelium of KCOTs.     

Local Minocycline Effect on Inflammation and Clinical Attachment During Periodontal Maintenance: Randomized Clinical Trial

Background: Minocycline microspheres (MMs) are being used to treat residual inflamed periodontal pockets during periodontal maintenance therapy (PMT), but evidence for efficacy from randomized clinical trials is lacking. The purpose of this study is to evaluate the effect of MMs plus scaling and root planing (SRP) on these sites. Methods: Sixty patients with chronic periodontitis on 6‐month PMT intervals to be followed for 1 year were randomized (51 completed the study) into two statistically similar groups, SRP + MM (aged 66.8 years) and SRP alone (aged 67 years), to treat a ≥5 mm posterior interproximal pocket during PMT with a history of bleeding on probing (BOP). Group treatments were applied to the site at baseline and 6 months. Clinical attachment levels (CALs; primary outcome), probing depths (PDs), plaque, and BOP also were recorded at baseline and 6 and 12 months. In addition, gingival crevicular fluid was analyzed for an inflammation index ratio of interleukin (IL)‐1β/IL‐1 receptor antagonist (ra) using enzyme‐linked immunosorbent assays. Results: All clinical parameters improved significantly (P <0.005) from baseline in both groups with no differences between groups at any time point. CAL decreased 17% (0.9 ± 0.8 mm) and 13% (0.7 ± 0.9 mm) in SRP + MM and 11% (0.7 ± 1.1 mm) and 21% (1.2 ± 0.9 mm) in SRP at 6 and 12 months, respectively. The odds of having BOP decreased 90% (down to 38% of patients) and 95% (26%) in SRP + MM and 82% (42%) and 82% (41%) in SRP at 6 and 12 months, respectively. IL‐1β/IL‐1ra decreased a significant 61% (P = 0.009) only in SRP + MM at 6 months. Conclusions: SRP of inflamed moderate pockets during 6‐month PMT, with or without MMs, improves CALs, along with PDs and BOP over a 1‐year period. The use of MMs did not result in an additional benefit over SRP alone.     

Immunohistochemical study of interleukin-1beta and interleukin-1 receptor antagonist in an antigen-induced arthritis of the rabbit temporomandibular joint.

BACKGROUND: In temporomandibular joint (TMJ) arthritis, there is limited knowledge of the relationship between interleukin-1beta (IL-1beta) and interleukin-1 receptor antagonist (IL-1ra), as well as the source of these cytokines. We investigated the development of an antigen-induced arthritis in the rabbit TMJ immunohistochemically. METHODS: Unilateral TMJ arthritis was induced in 32 adult New Zealand White rabbits. From 6 h to 12 weeks after induction of arthritis, topology of IL-1beta and IL-1ra were observed. RESULT: The acute stage of induced arthritis lasted for one week after induction, thereafter it became chronic. In the early phase of the acute stage, infiltrating inflammatory cells, as well as synovial cells, produced IL-1beta and IL-1ra. In the late phase of the acute stage, the main source of these cytokines was subsynovial fibroblasts. In this phase of arthritis, IL-1beta and IL-1ra did not appear to be produced by synovial cells. From the early to intermediate phase of the chronic stage, proliferating synovial cells produced IL-1beta and IL-1ra. In this phase of the arthritis, these cytokines were also observed in a cluster formation in chondrocytes. CONCLUSION: This arthritis model shows a staging of the joint inflammation process with time. IL-1beta and IL-1ra are produced by a certain kind of cells depending on the stage of inflammation.     

Expression of Vascular Endothelial Growth Factor Has a Regulatory Role in Gingival Venules in Experimental Diabetes

Background: In the present study, the possible localization and role of vascular endothelial growth factor receptor type 2 (VEGFR2) in the regulation of gingival venules in a rat model of experimental diabetes are examined. Methods: Six weeks after streptozotocin premedication, Wistar male rats presenting blood sugar levels >20 mmol/L were selected for investigation. The VEGFR2 antagonist ZM323881 [5‐((7‐benzyloxyquinazolin‐4‐yl)amino)‐4‐fluoro‐2‐methylphenol‐hydrochloride] (20 μg/mL) was dripped onto the gingiva between the mandibular incisors. Changes in diameter of the selected gingival venule were measured by vital microscopy combined with digital photography at specified times. Immunohistochemical staining was used to localize VEGFR2. For controls, the same protocol was used on animals with normal blood sugar levels and healthy gingiva. Results: There was a significant difference between the baseline venule diameter of the diabetic and the control groups (47 ± 1 and 28 ± 2 μm, respectively). After 15, 30, and 60 minutes of local application of ZM323881, significant vasoconstriction was observed in the venules of diabetic rats compared with the baseline (81.4% ± 4.6%, 81.8% ± 4.4%, and 80.6% ± 5.1%, respectively). The control group showed no change in the venule diameter. The immunohistochemical analysis showed significantly increased VEGFR2 expression in the mast cells along the venules in the diabetic group, whereas mast cells were rarely found in the control group. Conclusions: The findings suggest that VEGF expression is increased in gingiva in experimentally induced diabetes. After VEGFR2 activation, the mast cell–derived vasodilatory and inflammatory mediators may contribute markedly to the concomitant changes in the microcirculation.     

IL-1 beta, IL-1 receptor antagonist and soluble type II IL-1 receptor in synovial fluid of patients with temporomandibular disorders

Among the members of the interleukin-1 (IL-1) family, IL-1 beta, which is a major agonist, has been detected in synovial fluid (SF) of the temporomandibular joint (TMJ) of patients with temporomandibular joint disorders (TMD). However, there is little knowledge regarding suppressive molecules, such as IL-1 receptor antagonist (IL-1ra) and the soluble form of type II IL-1 receptor (sIL-1RII), in TMD patients. The aim of this study was to investigate the levels of IL-1 beta, IL-1ra and sIL-1RII in the TMJ SF of patients with TMD and their relationship. Fifty-two SF samples from TMD patients and nine samples from asymptomatic volunteers were examined. Detected levels of IL-1 beta and sIL-1RII were significantly higher in the TMD group compared with the volunteer group. There was no significant difference in IL-1ra levels between the TMD and volunteer groups. The IL-1 beta/IL-1ra ratio in the TMD group, however, was higher than that in the volunteer group. In the TMD group, positive correlations were found between IL-1 beta and IL-1ra, IL-1ra and sIL-1RII, and IL-1 beta and sIL-1RII. In addition to increased IL-1 beta, development of TMD may also lead to decreased IL-1ra and increased sIL-1RII in response to increasing IL-1.     

Neurokinin-1受体介导P物质对破骨细胞骨吸收功能的影响

目的 　了解P物质对体外培养大鼠破骨细胞骨吸收功能的影响和作用机制,探讨P物质在正畸牙槽骨改建中的调控机制。方法　 通过机械分离新生大鼠四肢长骨方法体外培养破骨细胞,细胞免疫组织化学染色观察NK1受体在大鼠破骨细胞内的表达情况;加入含不同浓度P物质（10-7-10-4 mol/L）和10-5 mol/L P物质受体拮抗剂（NK1受体拮抗剂）的培养液,观察P物质和NK1受体拮抗剂对破骨细胞骨吸收功能的影响。结果 免疫组化染色发现,大
鼠破骨细胞内NK1受体呈强阳性表达,着色位于细胞浆,胞核呈阴性;P物质明显增加破骨细胞的骨吸收陷窝面积（P<0.05）,但未见骨吸收陷窝数目明显增加（P>0.05）。NK1受体拮抗剂抑制了P物质对破骨细胞骨吸收功能的刺激
效应。结论 NK1受体可能介导P物质增强破骨细胞骨吸收功能,从而在正畸牙槽骨改建中发挥局部调节作用。     

Interleukin-1 and IL-1 receptor antagonist in gingival crevicular fluid

BACKGROUND/AIMS: This study aimed to investigate the cytokine IL-1beta and its receptor antagonist IL-1ra in gingival crevicular fluid (GCF), in patients with adult periodontitis. METHOD: A total of 40 GCF samples were harvested from 10 subjects with moderate to severe adult periodontitis and 10 healthy controls. Subjects were selected from both genders, with all the upper anterior teeth present, and with no relevant systemic illness, pregnancy or recent medication. All subjects were non-smokers and had not received any periodontal therapy within the preceding 3 months. Deep bleeding sites, deep non-bleeding sites and healthy sites were investigated in relation to upper anterior teeth. Clinical measurements were recorded for each site, after obtaining a GCF sample. IL-1beta and IL-1ra were quantified using new commercially available ELISA kits (Quantikine), and could be detected in all samples. RESULTS: The mean concentration for IL-1beta was 0.11 (SD 0.14) pg/microl for bleeding periodontitis sites, 0.04 (0.05) pg/microl for non-bleeding periodontitis sites, and 0.01 (0.03) pg/microl for healthy sites (p<0.001). In contrast, the mean concentration for IL-1ra was 6.99 (9.78) pg/microl for healthy sites, 0.59 (0.44) pg/microl for non-bleeding periodontitis sites, and 0.44 (0.36) pg/microl for bleeding periodontitis sites (p<0.001, except for comparisons between bleeding and non-bleeding periodontitis sites, p>0.05). For healthy sites, a strong inverse relationship was found between IL-1beta and IL-1ra levels in GCE. CONCLUSIONS: The results suggest a strong relationship between the severity of adult periodontitis and the increasing GCF levels of IL-1beta and decreasing levels of IL-1ra.     

人白细胞介素-1受体拮抗剂基因转染人颞颌关节软骨细胞的基因表达

目的 研究人白细胞介素-1受体拮抗剂（hIL-1ra）基因转染人颞颌关节软骨细胞的基因表达情况。方法hIL-1ra基因以阳离子脂质体介导转染体外培养的人颞颌关节软骨细胞,描记细胞生长曲线,计算细胞群体倍增时间,并采用酶联免疫吸附法检测瞬时转染和稳定转染细胞中hIL-1ra蛋白的表达。结果 转染细胞与正常细胞的增殖率有一定差异,转染细胞胞浆内和培养基上清液中均有hIL-1ra蛋白的表达。在细胞转染结束后48 h,细胞内hIL-1ra蛋白的表达量最高,稳定转染细胞中基因表达最长时间达72 d以上。结论 经阳离子脂质体介导hIL-1ra基因转染人颞颌关节软骨细胞后,目的基因在细胞内有一定数量的表达。     

颞下颌关节紊乱病关节液中白细胞介素-1受体拮抗剂和白细胞介素-10的检测

探讨白细胞介素 - 1受体拮抗剂 (IL - 1ra)和白细胞介素 - 10 (IL - 10 )在颞下颌关节紊乱病(TMD)病因及发病机理中的作用。方法　采用夹心法酶联免疫吸附测定法 (Sandwich -ELISA)对 31侧TMD患者及 7侧健康志愿者的关节液进行了IL - 1ra和IL - 10的检测。结果　健康志愿者的滑液中不能检测到IL -1ra的存在 ,TMD患者的关节液中IL - 1ra的平均水平为 182 .40± 5 5 .83pg/ml。TMD患者关节液的IL - 1ra水平与健康志愿者滑液中IL - 1ra水平之间的差异具有显著性 (P <0 .0 1)。颞下颌关节结构紊乱患者的IL - 1ra水平为 175 .78± 5 2 .43pg/ml(n =14) ;TMJ骨关节病 (OA)患者的IL - 1ra水平为187.85± 5 9.5 1pg/ml(n =17) ;两者的差异不具有显著性。健康志愿者和TMD患者的滑液中均不能检测到IL - 10的存在。结论　IL - 1ra和IL -10的缺乏或缺失在颞下颌关节紊乱病的病因及发病机理中可能起着重要作用。     

H3 and H3.3 histone mRNA amounts and ratio in oral squamous cell carcinoma and leukoplakia

Histone variants (e.g. H3) play an important role in chromatin structure and gene expression regulation of normal cells. Aims of this study were to: (1) estimate H3 and H3.3 histone mRNA expressions and their ratio in oral squamous cell carcinoma (OSCC) and oral leukoplakia (OL); (2) investigate whether H3 and H3.3 variants could play a role in the pathogenesis of OSCC and OL, also conditionally to HPV infection, age, gender, and main habits (tobacco smoking and alcohol drinking) in human beings studied. Twenty-three cases of OSCC and 20 cases of OL were examined in lesion site (LS) and juxtaposed clinically undamaged site (JUS) by RT-PCR for H3 and H3.3 histone mRNA; 13 healthy oral mucosa samples (HS) were investigated in a single site as controls. HPV DNA presence was investigated in the respective exfoliated oral mucosa cells by nested PCR (nPCR: MY09-MY11/GP5-GP6). The data showed that both H3 and H3.3 histone mRNA crude concentrations are higher in OSCC (LS = 2901 +/- 459 ng of H3; JUS = 2699 +/- 658 ng of H3; LS = 3190 +/- 411 ng of H3.3; JUS = 2596 +/- 755 ng of H3.3) than those in OL (LS = 2095 +/- 349 ng of H3; JUS = 2192 +/- 897 ng of H3; LS = 2076 +/- 911 ng of H3.3; JUS = 1880 +/- 654 ng of H3.3) and in HS (2579 +/- 959 ng of H3; 2300 +/- 758 ng of H3.3), although not reaching any statistical significance. Interestingly, ratio of H3/H3.3 mRNA amounts decrease both in OSCC (0.99) and OL (1.009) vs HS (1.121). No association was found for H3 and H3.3 histone mRNA expressions in OSCC and OL with respect to HPV infection and the social-demographical variables considered (P > 0.2). The overall higher expression of H3.3 in damaged tissues up to the ratio inversion in OSCC especially in HPV+ alcohol drinkers (60.0%) represents the most interesting finding, in consideration of the proven ability of alcohol to act as permeability enhancer of human oral mucosa, to alter the mucosal structure and by this dynamics could favour the penetration through the epithelial layers of HPV.     

白细胞介素-1受体拮抗剂抑制兔颞下颌关节骨关节炎软骨破坏的研究

目的:研究关节腔内注射白细胞介素-1受体拮抗剂对颞下颌关节骨关节炎关节软骨修复的影响。方法:取新西兰大白兔24只,用关节盘部分切除致使穿孔法建立双侧颞下颌关节骨关节炎模型。4周后,在每只动物右侧(实验侧)颞下颌关节腔内一次性注射重组人白细胞介素-1受体拮抗剂50μg,左侧(对照侧)关节腔内注射等量安慰剂。注射后12、24周分别处死12只动物,取双侧颞下颌关节标本进行组织学及RT-PCR检测。结果:双侧关节软骨均呈现出不同程度的骨关节炎病损,对照侧病损更严重。实验侧组织学分数明显高于对照侧(P<0.05)。注射后12和24周时实验侧II型胶原及聚集蛋白聚糖mRNA表达高于对照侧(P<0.05);12周时实验侧关节软骨聚集蛋白聚糖酶mRNA表达低于对照侧(P<0.05),24周时两侧无明显差异(P>0.05)。肿瘤坏死因子-αmRNA表达两侧无明显差异(P>0.05)。结论:颞下颌关节腔内注射IL-Ra能缓解骨关节炎导致的软骨病损,可望成为治疗颞下颌关节骨关节炎的一种新方法。     

Interleukin 1 and receptor antagonist levels in gingival crevicular fluid in heavy smokers versus non-smokers

BACKGROUND/AIMS: This study aimed to investigate the concentration of the cytokine interleukin (IL)-1beta and its receptor antagonist IL-1ra in gingival crevicular fluid (GCF) in patients with adult periodontitis who were heavy smokers compared with non-smokers. METHOD: GCF samples were collected from two groups of subjects: smokers and non-smokers. Thirty-nine GCF samples were harvested from 13 subjects with moderate to severe adult periodontitis who were heavy smokers. A further 30 GCF samples were harvested from 10 subjects with moderate to severe adult periodontitis who were non-smokers. Subjects were selected from both genders and none had any relevant systemic illness, were pregnant, had recent medication or had received any periodontal therapy in the preceding 3 months. One deep bleeding site, one deep non-bleeding site and one healthy site were investigated in each subject. Clinical measurements were recorded for each site, after obtaining a GCF sample using a Periopaper strip. IL-1beta and IL-1ra were quantified using new commercially available ELISA kits (Quantikine), and could be detected in all samples. RESULTS: For smokers, the mean concentrations for IL-1beta were 2714.5 (SD 4416.2) pg/ micro L for healthy sites, 37.0 (SD 57.2) pg/ micro L for non-bleeding periodontitis sites and 24.5 (SD 29.2) pg/ micro L for bleeding periodontitis sites. The concentrations of IL-1beta for non-smokers for the same category of sites were 393.8 (SD 867.1), 74.2 (SD 107.0) and 73.1 (SD 61.0) pg/ micro L, respectively. The mean concentrations of IL-1ra for smokers were 5.8 x 10(5) (SD 9.7) pg/ micro L for healthy sites, 2.2 x 10(5) (SD 0.15) pg/ micro L for deep non-bleeding sites and 0.19 x 10(5) (SD 0.07) pg/ micro L for deep bleeding sites. The concentrations for non-smokers were: 4.1 x 10(10) (SD 3.8), 18.1 x 10(5) (SD 20.4) and 3.2 x 10(5) (SD 2.3) pg/ micro L, respectively. Significance levels of P < 0.05 were found for comparisons of healthy vs. deep bleeding and deep non-bleeding sites for IL-1beta and IL-1ra in smokers, before adjustments for multiple testing. However, none of these comparisons reached statistical significance following adjustments for multiple testing. P < 0.05 for the correlation between IL-1beta and IL-1ra at healthy sites in smokers only. Differences in GCF concentrations for IL-1beta in smokers vs. non-smokers were significant for deep bleeding sites only (P < 0.05), the mean concentration of IL-1beta being lower in GCF from smokers vs. non-smokers. All differences in GCF concentrations of IL-1ra reached statistical significance for smokers vs. non-smokers. The mean concentrations of IL-1ra in GCF were lower in smokers compared with non-smokers for all categories of sites. CONCLUSIONS: A decreased concentration of IL-1beta and also IL-1ra was found in GCF from periodontitis sites compared to healthy sites in smokers and in non-smokers, although this did not reach statistical significance following adjustments for multiple testing. For comparisons between heavy smokers and non-smokers, statistically significant differences were found in the GCF concentrations of IL-1beta from deep bleeding sites only. Statistically significant differences were found in the IL-1ra concentrations for smokers vs. non-smokers for all categories of sites.     

Postorthodontic external root resorption is associated with IL1 receptor antagonist gene variations

Oral Diseases (2012) 18 , 198–205 Objective: External apical root resorption (EARR) is a frequent iatrogenic effect of orthodontic treatment. Patients with genetic variants in the interleukin 1 gene have been related to an increased risk of suffering EARR. The objective of this study is to determine whether variants in the interleukin 1 receptor antagonist gene are positively/negatively associated with interleukin 1 gene polymorphisms, particularly in relation to the clinical features of EARR patients. Materials and Methods: Genetic screening of 54 orthodontic patients was performed for three polymorphisms (rs1800587, rs1143634 and rs419598) in the IL1 gene cluster. Subjects were divided according to the presence or absence of EARR of more than 2 mm. The genotype distributions and allelic frequencies were calculated by the chi‐square‐test. Odds ratios (OR) and 95% confidence intervals were also calculated. Results: A highly significant association was found in the comparative analysis of homozygous subjects [1/1(CC)] for the IL1B gene, resulting in an increased risk of suffering postorthodontic EARR (OR: 3.47; P = 0.027; CI: 95%). While no association was found for the IL1A gene (P = 0.097), subjects who were homozygous [1/1(TT)] for the IL1RN gene were more likely to be affected with EARR (OR: 6.75; P = 0.001; CI: 95%). Conclusion: Variations in the interleukin 1 receptor antagonist gene (rs419598) – and not only in the IL1B gene (rs1800587) – are determinants of a predisposition to postorthodontic EARR.     

Interleukin-1 receptor antagonist gene polymorphism and gingivitis in children

AIM: To investigate the role of the polymorphism of a variable numbers of tandem repeats of interleukin-1 receptor antagonist gene (IL-1RN) on gingivitis in children. MATERIALS AND METHODS: A total of 146 Caucasian subjects (98 subjects with gingivitis and 48 controls) aged 8-12 years, were enrolled. Plaque and Calculus Indices were recorded to assess the oral hygiene. Gingival and Bleeding on Probing Indices were used to identify patients with gingivitis. DNA was extracted from epithelial cells of the cheek. Normal polymerase chain reaction was used for IL-1Ra genotyping. RESULTS: A significant association was observed between IL-1Ra gene polymorphism and gingivitis in children (P = 0.008). The IL-1RN*2 allele (A2) was significantly more frequent in controls (37%vs 22% in children with gingivitis). In addition, the carriage of A2 seemed to be protective against gingivitis, and it was more frequent in controls (60%vs 40% in children with gingivitis, P = 0.008). Moreover, multiple logistic regression analysis showed that the association between IL-1Ra gene polymorphism and gingivitis in children remained significant (P = 0.014) regardless of the significant influence of plaque (P = 0.013). CONCLUSION: IL-1Ra gene polymorphisms could have an active role in the pathogenesis of gingivitis in Caucasian children and IL-1RN*2 allele could be a protective marker against gingivitis.     

Gingival crevicular interleukin-1 and interleukin-1 receptor antagonist levels in periodontally healthy and diseased sites

Interleukin-l (IL-1) molecules, IL-lα and IL-lβ are cytokines involved in the acute-phase response against infection and in the pathogenesis of periodontal destruction. Administration of exogenous IL-1 receptor antagonist (IL-1ra) is effective in reducing the inflammatory reactions mediated by IL-1. However, the relationship between these three naturally occurring IL-1 molecules and periodontal diseases has been poorly characterized. We investigated the correlation of gingival crevicular IL-1 molecules and the clinical status of patients with different severities of periodontitis. IL-lα, IL-1β, IL-1ra and the total IL-1/IL-1ra ratio (IL-1 activity index; IL-1AI) were measured in 75 gingival crevicular fluid (GCF) samples from non-inflamed gingiva sites in 2 healthy subjects and diseased sites in 7 patients with several types of periodontitis. IL-lα, IL-1bT and IL-1ra were measured by specific non-cross-reactive enzyme linked immunosorbent assay. The probing depth, gingival index and alveolar bone loss of each site was recorded at the time of GCF sampling. The total amount of IL-lα, IL-1β and the IL-1AI, but not total IL-1ra, were found to be correlated with alveolar bone loss score. Three IL-1 molecules were also measured in the gingival tissue of patients with periodontitis. A similar progressive decrease of the IL-1AI was detected in gingival tissue with periodontitis. These results suggest that the amounts of both crevicular IL-1 and IL-1AI are closely associated with periodontal disease severity.