Proinflammatory cytokine levels in hyperlipidemic patients with periodontitis after periodontal treatment

Oral Diseases (2012) 18 , 299–306 Objective: The aim of this study was to evaluate the effects of periodontal treatment on serum and gingival crevicular fluid (GCF) proinflammatory cytokine levels in hyperlipidemic patients with periodontitis. Materials and Methods: Fifty‐two patients with hyperlipidemia and periodontitis and 28 systemically healthy controls with periodontitis (C) were included in the study. Hyperlipidemic groups were divided into two groups as suggested diet (HD) and prescribed statin (HS). The clinical periodontal parameters, fasting venous blood, and GCF samples were obtained, and serum tumor necrosis factor‐alpha (TNF‐α), interleukin (IL) 1‐beta, and IL‐6 levels were evaluated at baseline and at 3 months follow‐up (3MFU) after the completion of the non‐surgical periodontal treatment that included scaling and root planning. Results: Percentage of bleeding on probing was significantly higher in the HS group than both the HD and C groups. In the HD and HS groups, there were significant decreases in serum IL‐6 and GCF TNF‐α levels between the 3MFU and baseline. A significant decrease was also found in GCF IL‐6 at the end of the study period in the HS group. Conclusion: The combination of the periodontal therapy and antilipemic treatment may provide beneficial effects on the metabolic and inflammatory control of hyperlipidemia.     

Delayed osteoblast differentiation and altered inflammatory response around implants placed in incisor sockets of type 2 diabetic rats

Objective: Central to the process of osseointegration is the recruitment of mesenchymal progenitor cells to the healing site, their proliferation and differentiation to bone synthesising osteoblasts. The process is under the control of pro‐inflammatory cytokines and growth factors. The aim of this study was to monitor these key stages of osseointegration and the signalling milieu during bone healing around implants placed in healthy and diabetic bone. Methods: Implants were placed into the sockets of incisors extracted from the mandibles of normal Wistar and diabetic Goto‐Kakizaki rats. Mandibles 1–12 weeks post‐insertion of the implant were examined by histochemistry and immunocytochemistry to localise the presence of Stro‐1‐ positive mesenchymal progenitor cells, proliferating cellular nuclear antigen proliferative cells, osteopontin and osteocalcin, macrophages, pro‐inflammatory cytokines interleukin (IL)‐1β, IL‐6, tumour necrosis factor (TNF)‐α and tumour growth factor (TGF)‐β1. Image analysis provided a semi‐quantification of positively expressing cells. Results: Histological staining identified a delay in the formation of mineralised bone around implants placed in diabetic animals. Within the diabetic bone, the migration of Stro‐1 mesenchymal cells in the healing tissue appeared to be unaffected. However, in the diabetic healing bone, the onset of cell proliferation and osteoblast differentiation were delayed and subsequently prolonged compared with normal bone. Similar patterns of change were observed in diabetic bone for the presence of IL‐1β, TNF‐α, macrophages and TGF‐β1. Conclusion: The observed alterations in the extracellular presence of pro‐inflammatory cytokines, macrophages and growth factors within diabetic tissues that correlate to changes in the signalling milieu, may affect the proliferation and differentiation of mesenchymal progenitor cells in the osseointegration process. To cite this article:
Colombo JS, Balani D, Sloan AJ, St Crean J, Okazaki J, Waddington RJ. Delayed osteoblast differentiation and altered inflammatory response around implants placed in incisor sockets of type 2 diabetic rats
Clin. Oral Impl. Res 22 , 2011; 578–586
doi: 10.1111/j.1600‐0501.2010.01992.x     

In vitro engineering of cartilage: effects of serum substitutes, TGF-beta, and IL-1alpha

OBJECTIVES: Cartilage is avascular and relatively homogeneous, making it an attractive tissue for in vitro histogenesis and surgical use in patients. We developed novel platform technologies in order to define the requirements for optimal in vitro chondrogenesis by isolated cells. In this series of studies, we tested alternatives to fetal bovine serum (FBS) and the effects of growth factors on formation of cartilage in 3D porous collagen sponges. DESIGN: We used porous collagen sponges to assess the effects of serum substitutes and exogenous TGF-beta1 and IL-1alpha on chondrocytes (bovine articular chondrocytes, bACs) and on chondroinduced human dermal fibroblasts (hDFs). We determined the effects of low concentrations of FBS and two serum substitutes, Nutridoma and ITS(+3), on cellularity and matrix production. After culture for intervals, sponges were harvested for histological and biochemical measurement of cartilage-specific chondroitin 4-sulfate proteoglycan (C 4-S PG). RESULTS: Cultured bACs showed equivalent growth in Nutridoma (1%) and 10% FBS. Both TGF-beta1 and IL-1alpha significantly stimulated accumulation of C 4-S PG by bACs in 3D porous collagen sponges. Many endogenous growth factors were upregulated in hDFs cultured with chondroinductive DBP. Addition of TGF-beta1 and IL-1alpha for 11 days significantly stimulated accumulation of C 4-S PG by hDFs cultured in DMEM with 1% Nutridoma. CONCLUSION: Porous collagen sponges are supportive of chondrogenesis and of chondroinduction by DBP. Optimization of serum-free culture conditions, including growth factors, matrix components, and mechanical stimuli will expedite translation to wider clinical applications. Use of autogenous dermal fibroblasts pre-cultured with DBP and induced to chondrocytes offers an alternative to autogenous chondrocytes.     

2-Hydroxyethyl methacrylate (HEMA) promotes IgG but not IgM antibody production in vivo in mice

Individuals working in a dental clinic are exposed to 2-hydroxyethyl methacrylate (HEMA). HEMA has been found to have several effects on the immune system, including acting as an adjuvant in mice and stimulating the production of human IgG1 in vitro. In this study we continued to explore the immunomodulatory properties of HEMA in mice. Mice were co-injected subcutaneously with the following: HEMA + ovalbumin (OVA) in bicarbonate buffer, OVA in bicarbonate buffer, HEMA in bicarbonate buffer, or bicarbonate buffer alone. Mice immunized with OVA were killed 2 wk after a booster injection. Mice exposed to HEMA only were killed 6 d after the last injection with HEMA. Serum and spleens were collected. The activities of anti-OVA IgG and anti-OVA IgM were determined using ELISAs, as was the in vitro production of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by splenocytes after 2 d of incubation. Splenocyte proliferation was analyzed using [(3) H]thymidine decomposition. Mice exposed twice to HEMA in vivo had a higher baseline and a higher concanavalin A-stimulated proliferation of splenocytes, and produced less TNF-α in relation to IL-6, compared with controls. Immunization of mice with OVA/HEMA resulted in a higher anti-OVA IgG activity, relative to anti-OVA IgM activity, compared with controls. In conclusion, HEMA has selective effects on cytokine and antibody production in mice.     

Hepatocytes produce tumor necrosis factor-α and interleukin-6 in response to Porphyromonas gingivalis

Takano M, Sugano N, Mochizuki S, Koshi RN, Narukawa TS, Sawamoto Y, Ito K. Hepatocytes produce tumor necrosis factor‐α and interleukin‐6 in response to Porphyromonas gingivalis. J Periodont Res; 2012; 47: 89–94. © 2011 John Wiley & Sons A/S Background and Objective: The liver plays a major role in clearing systemic bacterial infections. In addition, inflammatory cytokines produced in the liver play a critical role in systemic cytokine levels. The aim of this study was to investigate the production of tumor necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6) by hepatocytes in response to periodontal pathogens. Material and Methods: The mouse hepatic carcinoma cell line Hepa‐1.6 and the mouse macrophage‐like cell line RAW 264 were co‐cultured in Transwell insert plates. Cells were stimulated with bacterial extracts prepared from Porphyromonas gingivalis and the induction of TNF‐α and IL‐6 was measured using real‐time PCR and ELISA. Results: After stimulation with bacteria, the induction of TNF‐α and IL‐6 was observed in RAW 264 cells and Hepa‐1.6 cells. Significant reduction of TNF‐α mRNA expression in Hepa‐1.6 cells was observed after treatment with antibody to TNF‐α. Conclusion: The results obtained in the present study show that P. gingivalis extract induces TNF‐α and IL‐6 in an in vitro liver model and that macrophage‐derived TNF‐α mediates the induction of TNF‐α in hepatocytes.     

Tumor necrosis factor-α and interleukin-1β expression pathway induced by Streptococcus mutans in macrophage cell line RAW 264.7

Streptococcus mutans, a major etiological agent of dental caries, frequently causes systemic disease, such as subacute bacterial endocarditis, if it enters the bloodstream. In this study, the production pathways of the proinflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), induced by S. mutans in mouse macrophage were examined using a quantitative real-time polymerase chain reaction and an enzyme-linked immunosorbent assay. The S. mutans stimulated the expression of TNF-α and IL-1β mRNA at a multiplicity of infection of 1 : 100, which increased at 2 and 4 h, respectively, to 24 h. It also induced the production of high levels of the TNF-α and IL-1β proteins, which increased at 2 h and reached a peak at 4 and 24 h, respectively. Nuclear factor-κB (NF-κB) was activated and reached a maximum level 30 min after the S. mutans treatment. The expression of TNF-α and IL-1β mRNA and protein was suppressed by the treatment with pyrrolidine dithiocarbamate, an NF-κB inhibitor. The S. mutans-induced TNF-α expression was suppressed by the presence of SB203580, a p38 mitogen-activated protein (MAP) kinase inhibitor, or SP600125, a Jun N-terminal kinase (JNK) MAP kinase inhibitor. On the other hand, IL-1β expression was inhibited by extracellular signal-regulated kinase (ERK)/p38/JNK MAP kinase inhibitor pretreatment. In addition, TNF-α production was suppressed more in the Toll-like receptor 2(-/-) (TLR2(-/-)) macrophages than in the TLR4(-/-) macrophages, whereas IL-1β production was suppressed more in the TLR4(-/-) macrophages than in the TLR2(-/-) macrophages. These results show that S. mutans stimulates the production of TNF-α and IL-1β in the mouse macrophage cell line, RAW 264.7, by activating ERK/p38/JNK, and NF-κB through TLR2 and TLR4, respectively.     

After-effects of stress on crevicular interleukin-1beta

BACKGROUND: In a previous study, we found stress to increase crevicular interleukin-1beta (Il-1beta) secretion induced by supragingival plaque. While in that study, stress and plaque were presented concomitantly, we now wondered whether a consecutive presentation of these 2 factors would still exert stress effects. METHOD: 39 medical students participated in the study; 18 took part in a major exam while the remaining 21 served as controls. From the day after the last exam, students neglected oral hygiene in 2 antagonistic quadrants for 21 days (experimental gingivitis), while they maintained perfect hygiene at the remaining sites. Crevicular fluid samples were taken at days 0, 5, 8, 15, 18, and 21 of experimental gingivitis. RESULTS: A significant effect of pre-exposure to academic stress on crevicular Il-1beta concentration was found (area under the curve: p=0.042), the effect size, however, being smaller than in our previous study when stress and plaque were presented concomitantly. CONCLUSIONS: It is concluded that pre-exposure to stress may persistently alter the immunological effects of microbial challenge to the periodontium.     

Immunoexpression of TNF-α and TGF-β in central and peripheral giant cell lesions of the jaws

J Oral Pathol Med (2012) 41 : 194–199 Background: Peripheral giant cell lesion (PGCL) is a reactive process associated with a local irritating factor that shows low recurrence after treatment, especially if the irritating factor is eliminated. On the other hand, central giant cell lesion (CGCL) presents a variable clinical behavior ranging from slow and asymptomatic growth without recurrence to rapid, painful and recurrent growth. Our aim was to compare the immunoexpression of tumor necrosis factor‐alpha (TNF‐α) and transforming growth factor‐beta (TGF‐β) in CGCL and PGCL. Methods: Twenty CGCL and 20 PGCL were selected for analysis of the immunoexpression of TNF‐α and TGF‐β in multinucleated giant cells (MGC) and mononucleated cells (MC). Results: The PGCL showed lightly higher expression of TNF‐α than CGCL. In comparison with PGCL, the CGCL showed higher expression of TGF‐β in MC and MGC (P < 0.05) and in total cells (P < 0.05). Significant positive correlation was found between expressions of TGF‐β and TNF‐α in CGCL (P < 0.05). Conclusions: Our results suggest that, in CGCL, coordinated interactions between TGF‐β and TNF‐α may be important for osteoclastogenesis and bone resorption. PGCL occasionally cause bone resorption but to a lower extent, a fact that might be explained by the lower expression of TGF‐β in these lesions.     

TGF-β1 gene polymorphism in renal transplant patients with and without gingival overgrowth

Oral Diseases (2011) 17 , 414–419 Background: The incidence of gingival overgrowth among renal transplant patients treated with cyclosporine A ranges from 13% to 84.6%, and the overgrowth is not only esthetic but also a medical problem. We studied the determination of association between TGF‐β1 (TGFB1) gene polymorphism and gingival overgrowth in kidney transplant patients medicated with cyclosporin A. Methods: Eighty‐four kidney transplant patients with gingival overgrowth and 140 control transplant patients without overgrowth were enrolled into the case control study. TGFB1 polymorphism was determined using the PCR‐RFLP assay for +869T>C in codon 10 and +915G>C in codon 25 as well as TaqMan real‐time PCR assays for promoter ?800G>A and ?509C>T SNPs. Results: In kidney transplant patients suffering from gingival overgrowth, mean score of gingival overgrowth was 1.38 ± 0.60, whereas in control subjects it was 0.0. The patients with gingival overgrowth were characterized by similar distribution of TGFB1 genotypes and allele in comparison to subjects without gingival overgrowth. Among 16 potentially possible haplotypes of TGFB1 gene, only four were observed in the studied sample of kidney transplant patients: G_C_T_G, G_T_C_G, G_C_C_C, and A_C_T_G, with similar frequency in patients with and without gingival overgrowth. Conclusion: No association between the TGFB1 gene polymorphism and gingival overgrowth was revealed in kidney transplant patients administered cyclosporine A.     

Salivary cortisol, 17β-estradiol, progesterone, dehydroepiandrosterone, and α-amylase in patients with burning mouth syndrome

Oral Diseases (2012) 18, 613–620 Objective: The aim of this study was to investigate salivary markers related with burning mouth syndrome (BMS). Materials and Methods: Thirty female patients with BMS and twenty female control subjects were included. Unstimulated (UWS) and stimulated whole saliva samples (SWS) were collected, and their flow rates were determined. Salivary levels of cortisol, 17β‐estradiol, progesterone, dehydroepiandrosterone (DHEA), and enzymatic activity of α‐amylase were determined. Salivary transferrin level was measured to determine the level of blood contamination in saliva samples. Results: The levels of all analytes in UWS were significantly correlated with those of SWS. The levels of 17β‐estradiol, progesterone, and DHEA in UWS were significantly correlated with age. Age‐matched comparisons revealed that the patient group had significantly higher levels of cortisol in UWS and of 17β‐estradiol in SWS. When the patients were divided into older (≥60 years) and younger (<60 years) groups, the older group showed a significantly lower level of progesterone in UWS. There were no significant relationships between treatment efficacy and levels of salivary analytes. Conclusions: In conclusion, patients with BMS showed significantly higher levels of cortisol in UWS and of 17β‐estradiol in SWS compared with controls.     

Upregulation of immunoreactivity of endothelin-1 and alpha-SMA in PDL microvasculature following acute tooth loading: an immunohistochemical study in the marmoset

Authors – Sims MR, Ashworth JF, Sampson WJ Objectives – To test the hypothesis that a continuous mechanical tooth load would elevate immunoreactivity of endothelin‐1 (ET‐1) and alpha‐smooth muscle actin (α‐SMA) in the periodontal ligament (PDL) microvasculature. Design – A randomized control study employing 1.5 h of loading to first molars. Setting and Sample Population – Orthodontic Research Laboratory, Dental School, Adelaide University. Four young adult, male marmoset monkeys were consecutively anaesthetized and treated. Experimental Variable – An external telescoping frame applied a jaw closing load (120–200 g) transmitted occlusally, via a rubber pad, to randomly assigned mandibular left or right first molars. Contralateral molars were used as controls. Outcome Measure – Undemineralized, midsagittal, mandibular molar slices, ～150 μm thick were immunolabelled with ET‐1 and α‐SMA antibodies and examined in a confocal laser scanning microscope (CLSM) for vascular endothelium and smooth muscle immunolabelling. Results – Three categories of post‐capillary‐sized venule endothelial cell immunolabelling occurred: endothelium labelled solely with ET‐1; endothelium labelled solely with α‐SMA; endothelium labelled with both ET‐1 and α‐SMA. In endothelial cells, the α‐SMA showed a moderate cytoplasmic distribution with dense peripheral concentration. Loading increased arteriole α‐SMA actin labelling. Conclusion – Scattered expression of ET‐1 is the default state in primate PDL endothelial cells. Increased antigenicity of endothelial cells to both ET‐1 and α‐SMA, and of arteriolar smooth muscle to α‐SMA, is a response to shear and compression loads.     

Adhesion of a chondrocytic cell line (USAC) to fibronectin and its regulation by proteoglycan.

BACKGROUND: Chondrocytes produce various extracellular matrices during chondrogenesis. Fibronectin and proteoglycan are major extracellular matrix proteins in cartilage tissue, but the interactions between them are not clear. METHODS: Recently, we succeeded in establishing a cell line (USAC) with phenotypes of chondrocytes from a human osteogenic sarcoma of the mandible. Using this cell line, cell adhesion to fibronectin, the effect of proteoglycan on the cell adhesion and expression of integrin alpha5beta1 were investigated. RESULTS: Cells immediately adhered to fibronectin and then spread. Proteoglycan inhibited cell adhesion to fibronectin dose-dependently, whereas collagen did not. The expression of both mRNAs of alpha5 and beta1 subunits was detected 12 h after treatment with proteoglycan, but the expression of beta1 subunit mRNA had diminished by 24 h after treatment. CONCLUSIONS: These findings suggest that proteoglycan might modulate signal transduction from fibronectin by decreasing the expression of alpha5beta1 integrin.     

Transgenic α-1-antitrypsin secreted into the bloodstream from salivary glands is biologically active

Oral Diseases (2011) 17 , 476–483 Objectives: Salivary glands are potentially a valuable target for gene therapeutics. Herein, we examined the expression and biochemical activity of human alpha‐1‐antitrypsin (hA1AT) produced in rodent submandibular glands after gene transfer. Methods: A serotype 5 adenoviral vector (Ad.hA1AT) was constructed and first characterized by dose response and time course studies using SMIE cells in vitro. hA1AT expression was analysed by ELISA and the biologic activity determined by the inhibition of human neutrophil elastase (hNE) and formation of hA1AT‐hNE complexes. Ad.hA1AT was administered to submandibular glands of rats and mice. The levels and activity of hA1AT were analysed in saliva, serum and gland extracts. Treatment with endoglycosidase H and Peptide N‐Glycosidase F was used to assess N‐linked glycosylation. Results: Transgenic hA1AT, expressed in submandibular glands following Ad.hA1AT administration, was secreted into the bloodstream, N‐glycosylated and biochemically active. Conclusion: After in vivo gene transfer, rodent salivary glands can produce a non‐hormonal, transgenic, secretory glycoprotein exhibiting complex and conformation‐dependent biologic activity.