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The use of quantitative real-time reverse transcriptase PCR (qRT

Growing adipose-derived stem cells (ADSC) in serum-free conditions is important as it represents a way of expanding multipotent cells in a clinical grade medium. Most cultured ADSC are expanded and tested in serum-containing media, which can pose significant health risks if these cells were used in clinical applications. Moreover, cells grown in serum-free conditions behave very different than those cultured in serum-containing media. Here, we present a technique to culture adipose-derived stem cells in serum-free conditions. The methods described in this chapter were optimized for ovine ADSC. The appropriate optimization should be done for other cell lines.

Resorption of alveolar bone after tooth extraction is a common problem often requiring bone grafting. The success of the grafting procedures is dependent on multiple factors including the presence of growth factors. This is the first in vivo study to investigate the role of the pleiotrophin family of cytokines in alveolar bone regeneration. This research investigated the role of the pleiotrophin-midkine (PTN-MDK) axis during osteogenesis, with and without a grafting material, after tooth extraction in a sheep model.Thirty Romney-cross ewes were anesthetized, and all premolar teeth on the right side were extracted. The sockets were randomized to controls sites with no treatment and test sites with Bio-Oss® graft material and Bio-Gide® membrane. Samples were harvested after sacrificing animals 4, 8, and 16 weeks post-grafting (n = 10 per time-point). Tissue for qRTWithin the healing sockets, high expression of genes for PTN, MDK, NOTCH2, and ALK was found at all time-points and in both grafted and non-grafted sites, while PTPRZ1 was only expressed at low levels. The relative gene expression of the PTN family of cytokines was not statistically different at the three time-points between test and control groups (p .05). Immunohistochemistry found PTN and MDK in association with new bone, NOTCH2 in the connective tissue, and PTPRZ1 and ALK in association with cuboidal osteoblasts involved in bone formation.The PTN-MDK axis was highly expressed in both non-grafted and grafted sockets during osteogenesis in a sheep model of alveolar bone regeneration with no evidence that grafting significantly affected expression. The activation of NOTCH2 and PTPRZ1 receptors may be important during bone regeneration in vivo. The discovery of the PTN-MDK axis as important during alveolar bone regeneration is novel and opens up new avenues of research into these stably expressed highly active cytokines. Growth factor supplementation with PTN and/or MDK during healing may be an approach for enhanced regeneration or to initiate healing where delayed.

Background: Vascular endothelial growth factor (VEGF) is a master regulator and is required for the effective coupling of angiogenesis and osteogenesis supporting both skeletal development and postnatal bone repair. A direct role for VEGF in intramembranous‐derived osteoblast growth and differentiation is not clear. We investigated the expression of primary alveolar osteoblast VEGF receptors and the subsequent effects on mineralisation and nodule formation in vitro following VEGFR inhibition. Methods: Primary human alveolar osteoblasts (HAOBs) were cultured either in the presence of VEGF receptor inhibitors, exogenous VEGF or the bisphosphonate, zoledronic acid. VEGF, VEGFR1 and VEGFR2 mRNA expression and nodule formation following 21 days of culture. VEGFR1 protein expression was examined using immunofluorescence after 48 hrs. Results: The HAOBs expressed high levels of VEGF and VEGFR1 protein but VEGFR2 was not detected. The VEGFR1/2 inhibitors, ZM306416 and KRN633, lead to a dose dependent decrease in mineralisation. Treatment with zoledronic acid showed no difference in HAOB VEGF receptor expression. Conclusion: VEGF/VEGFR1 pathway appears to be important for intramembranous‐ derived osteoblast differentiation and maturation in vitro.

The aim of the study was to compare the response of calvarial and femoral osteoblasts cultured in a 3D hydrogel environment to cyclic compressive mechanical loading. Human foetal femoral and calvarial osteoblasts were encapsulated in a semi-synthetic thiol-modified hyaluronan gelatin polyethylene glycol diacrylate (PEGDA) cross-linked HyStemC hydrogel. Constructs were subjected to a cyclic compressive strain of 33.4 kPa force every second for 5 s every hour for 6 h per day using FlexCell BioPress culture plates and compared to non-compressed constructs. Cell viability, mineralisation, and morphological changes were observed over 21 days. BMP2, ALP, COL1A1, COL2A1, and OCN gene expression levels were quantified. Encapsulated osteoblast numbers increased and formed hydroxyapatite over a 21-day period. Cell viability decreased under a cyclical strain when compared to cells under no strain. Femoral osteoblasts under strain expressed increased levels of BMP2 (53.9-fold) and COL1A1 (5.1-fold) mRNA compared to no strain constructs. Surprisingly, no BMP2 mRNA was detected in calvarial osteoblasts. Osteoblasts derived from endochondral (femoral) and intra-membranous (calvarial) processes behaved differently in 3D-constructs. We therefore recommend that site-specific osteoblasts be used for future bone engineering and bone replacement materials and further research undertaken to elucidate how site-specific osteoblasts respond to cyclic compressive loads.

In the fungal pathogen Aspergillus fumigatus, resistance to azole antifungals is often linked to mutations in CYP51A, a gene that encodes the azole antifungal drug target lanosterol 14α-demethylase. The aim of this study was to investigate whether similar changes could be associated with azole resistance in a Malaysian Fusarium solani species complex (FSSC) isolate collection. Most (11 of 15) clinical FSSC isolates were Neocosmospora keratoplastica and the majority (6 of 10) of environmental isolates were Neocosmospora suttoniana strains. All 25 FSSC isolates had high minimum inhibitory concentrations (MICs) for itraconazole and posaconazole, low MICs for amphotericin B, and various (1 to >32 mg/l) voriconazole susceptibilities. There was a tight association between a 23 bp CYP51A promoter deletion and high (>32 mg/l) voriconazole MICs; of 19 FSSC strains sequenced, nine isolates had voriconazole MICs > 32 mg/l, and they all contained the 23 bp CYP51A promoter deletion, although it was absent in the ten remaining isolates with low (≤12 mg/l) voriconazole MICs. Surprisingly, this association between voriconazole resistance and the 23 bp CYP51A promoter deletion held true across species boundaries. It was randomly distributed within and across species boundaries and both types of FSSC isolates were found among environmental and clinical isolates. Three randomly selected N. keratoplastica isolates with low (≤8 mg/l) voriconazole MICs had significantly lower (1.3–7.5 times) CYP51A mRNA expression levels than three randomly selected N. keratoplastica isolates with high (>32 mg/l) voriconazole MICs. CYP51A expression levels, however, were equally strongly induced (~6,500-fold) by voriconazole in two representative strains reaching levels, after 80 min of induction, that were comparable to those of CYP51B. Our results suggest that FSSC isolates with high voriconazole MICs have a 23 bp CYP51A promoter deletion that provides a potentially useful marker for voriconazole resistance in FSSC isolates. Early detection of possible voriconazole resistance is critical for choosing the correct treatment option for patients with invasive fusariosis.

To develop a model to investigate a potential relationship between mechanical strain, cell responses, and endoplasmic reticulum stress in periodontal ligament (PDL) cells, primary PDL cell cultures were obtained from extracted premolars. Cells were cultured in hydrogel and subjected to 24 h of static mechanical strain, resulting in 18% dimensional substrate elongation. Cell viability, caspase-3/7 activity, and mRNA levels for 28 genes, including unfolded protein response (UPR)-related and mechanically responsive genes, serving as positive controls for stress induction, were examined. Compared with unstrained cultures, no difference in caspase activity was observed; however, viability responses differed between cell lines. Multiple UPR-related genes were differentially upregulated, with marginal statistical significance, including cAMP responsive element binding protein 3 like 3 (CREB3L3) (mean fold-regulation = 1.91), an adenosine monophosphate-dependent transcription factor with roles in UPR activation and the acute inflammatory response; and the pro-apoptotic UPR gene, endoplasmic reticulum to nucleus signaling 2 (ERN2) (mean fold-regulation = 4.01). The observed effect on cell viability following strain with no change in caspase activity suggests that reduction in viability may be mediated via caspase-3/7-independent mechanisms. Three-dimensional mechanical strain PDL cell culture models offer a method to study the role of endoplasmic reticulum stress and UPR, and provide a framework and potential UPR targets for future investigations.

In order to assess the colonization efficacy of the oral probiotic Streptococcus salivarius K12, a rapid method for specific detection and enumeration of the strain was developed. Here, we describe a two-step TaqMan™ quantitative PCR assay using primer-probe combinations targeting genes of the locus encoding the lantibiotic bacteriocin salivaricin B.

Oral lichen planus (OLP) is a complex immunological disorder, mediated in part by the release of cytokines by activated T-cells. Recently, the role of novel cytokines including IL33 and IL35 has been described in various chronic inflammatory diseases. IL33, a member of the IL-1 superfamily of cytokines, functions as an ‘alarmin’ released after cell necrosis to alert the immune system to tissue damage or stress. IL35, a member of IL12 cytokine family, is produced by regulatory T-cells and suppresses the immune response. The expression of IL33 and IL35 is yet to be investigated in OLP. The aim of this study was to determine the presence and topographical distribution of IL33 and IL35 in OLP using immunohistochemistry and quantitative real-time reverse transcriptase polymerase chain reaction (qPCR). For IHC, formalin-fixed paraffin-embedded archival specimens of OLP (n = 10) and a non-specific inflammatory (NSI) control group (n = 9) were used. A double-labelling immunofluorescence technique was used to determine the expression of IL33 and IL35 on CD3+ T-cells. In addition, 12 fresh tissue samples (OLP n = 6 and NSI controls n = 6) were used to determine the gene expression of IL33 and EBI3 (one chain of the dimeric IL35). Quantitative and qualitative analysis was performed with statistical significance set at p

Background and objective T cells are known to play a pivotal role in periodontal disease; however, less is known about the T-helper subsets of regulatory T cells (Tregs) and Th17 cells. The aim of this study was to investigate the cell types expressing FoxP3 and interleukin (IL)-17A within periodontal disease tissues and to determine gene and protein expression profiles associated with periodontitis. Material and methods A total of 10 healthy/gingivitis and 10 chronic periodontitis tissues were investigated. Immunohistochemistry and immunofluorescence techniques were used to identify the FoxP3 and IL17-positive cells and to determine the cell types respectively. Gene expression was determined using semi-quantitative polymerase chain reaction array technology that allowed the analysis of 84 pathway-focused genes known to be associated with Tregs and Th17 cells. Transforming growth factor (TGF)-β1, IL10 and IL17A protein levels were determined using enzyme-linked immunosorbent assay. Results Double immunofluorescence labeling revealed that all FoxP3+ cells were CD4+ , while IL17+ cells were neither CD4+ nor CD8+ but were tryptase+ , suggestive of mast cells. More FoxP3+ cells than IL17+ cells were found in all the tissues examined and overall there were few IL17+ cells. Statistically significant increases in gene expression were found for STAT5A, STAT3, SOCS1, TGFβ1 and IL10 in the chronic periodontitis specimens predominantly infiltrated with B cells and plasma cells when compared with healthy/gingivitis specimens predominantly infiltrated with T cells. Protein analysis demonstrated higher levels of the TGFβ1 and IL10 cytokines in periodontitis tissues and in B-cell and plasma cell predominant gingival tissues than in healthy/gingivitis tissues and T-cell predominant gingival tissues. IL17A gene and protein expression was not detected in any of the tissues. Conclusion Based on the findings of this study, we suggest that the source of low levels of IL17A in periodontal tissues is mast cells not Th17 cells and that Tregs may have a more prominent role in the pathogenesis of periodontal disease than Th17 cells.

This study investigated the association between the prevalence of oral health problems (caries, gingivitis, mucosal pigmentation and enamel defects in one to 5 year-old children exposed and not exposed to environmental tobacco smoke before and/or after birth. Exposure to environmental tobacco smoke (ETS) in childhood may have significant health effects. A structured questionnaire was used to collect data on a child’s current and previous illnesses, oral health behaviours, dietary habits, parental smoking behaviours and parents’ dental history. The intraoral examination recorded dental caries (dmfs), enamel defects, gingival health, melanin pigmentation and soft tissue health. Stimulated saliva was collected. Total sIgA levels were quantified using indirect competitive ELISA with a SalimetricsTM kit. The 44 children (aged 15–69 months) recruited were divided into two groups: ETS and non-ETS (control). There were 22 children in each: 16 who were exposed to ETS during and after gestation were identified as the ETSB subgroup. Participants exposed to ETS were more likely to have had upper respiratory tract and middle ear infections during the neonatal period and had higher mean dmft, mean dmfs, mean percent of surfaces with demarcated opacities and mean GI than the non-ETS participants. The children exposed to ETS before and after birth had the highest occurrence of enamel opacities showed a higher risk for dental caries even though more children in this group used the recommended fluoride toothpaste (1000 ppm fluoride). Mothers who smoked either never breastfed their children or breastfed their children for less than the recommended period of 6 months. Children exposed to ETS were shown to have higher mean total sIgA (μg/ml) than the children in the control group. Associations between ETS exposure before and after gestation and oral health, including salivary changes in young children were shown in the present study. Dental health professionals should include a question about household smoking in children’s dental histories, which would allow opportunities to discuss the impact of smoking on child oral health. Longitudinal oral health studies should include a history of maternal smoking during pregnancy and afterwards.

OBJECTIVE To establish how oral bacteria are related to cough sensitivity and pneumonia in a clinical stroke population. BACKGROUND Stroke patients are at risk of colonisation by respiratory pathogens due, in part, to sudden discontinuation of effective oral hygiene. When combined with reduced cough reflex sensitivity, aspiration of contaminated oropharyngeal contents and can lead to pneumonia. Relationships between oral bacteria, cough sensitivity and pneumonia have not been established. MATERIALS AND METHODS A total of 102 patients with acute stroke underwent saliva sampling and cough reflex testing at admission to hospital, discharge and one month. A qPCR assay compared levels of bacteria in saliva. Pneumonia events were recorded. RESULTS Relative levels of bacteria were lowest at admission to hospital (6.04 × 10-6 ). There was a slight (non-significant) increase in bacterial levels at discharge (1.69 × 10-2 , P = .73). By one month, bacterial levels had significantly increased (9.17 × 10-2 ) relative to admission [P

Background: Osteonecrosis of the jaws is recognised as a serious complication for patients receiving bisphosphonates. The anti-angiogenic effects of bisphosphonates have been implicated in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ). The purpose of this study was to determine the effects of zoledronic acid on cultured human gingival fibroblasts in relation to the modulation of genes associated with angiogenic regulation.Methods: Primary cultures of fibroblasts were developed from gingival tissues excised during crown lengthening surgery from three patients. Cells were cultured with and without 30 mu M zoledronic acid for 6, 12 and 24h and cellular proliferation and migration investigated using CellTiter-Blue and scratch wound assays, respectively. Gene expression was determined using semi-quantitative PCR array technology that allowed the analysis of 84 pathway-focused genes known to be important in the regulation of angiogenesis.Results: Zoledronic acid increased the proliferation of the gingival fibroblasts in a dose dependent manner with 12 and 24 h of exposure. Scratch wounding of the human gingival fibroblasts and treatment with increasing doses and time exposure to zoledronic acid (ZA) inhibited their migration. Statistically significant increases in gene expression were found for RHOB, VEGFA, CD55 and BMP2 (p

Objectives: The condylar cartilage is a key site of growth and development of the mandible. The aim of this research was to determine the mRNA expression levels of a number of chondrogenic and osteogenic regulatory factors in the condylar cartilage of the postnatal rat. Materials and methods: Condyles were extracted from 40 rats aged 4, 10, 21 or 90 days with 10 rats assigned to each age group. The condyles from one rat from each age group was fixed and decalcified in 10% EDTA for histology. Using cryogenic grinding combined with QIAzol reagent total RNA was purified from pooled samples collected for each age group. Each pool contained six condyles (N = 3). mRNA expression levels for 28 genes were determined using qPCR. Results: Histological analysis revealed distinct morphological differences in the condyle tissue of the 4, 10, 21 and 90 day old postnatal rats. Expression of all examined genes was detected. High levels of mRNA for Alpl, Bglap, Col1a1, Col2a1, Runx2, Sox9 and Sp7 but not Msx1 were detected. Fgf1 and Fgf2 were expressed at a similar level. No significant difference (defined as ± fold-regulation > 2 and P < 0.05) in the gene mRNA expression levels was found when days 10, 21 or 90 were compared to day 4. Conclusions: Apparent morphological changes of the rat condylar cartilage are not reflected in a change in the expression levels of the chondrogenic and osteogenic regulatory factor mRNA investigated in this study.

Objective The objective of this study was to investigate the expression of Toll-like receptors (TLR) and TLR-associated signalling pathway genes in oral lichen planus (OLP). Methods Initially, immunohistochemistry was used to determine TLR expression in 12 formalin-fixed archival OLP tissues with 12 non-specifically inflamed oral tissues as controls. RNA was isolated from further fresh samples of OLP and non-specifically inflamed oral tissue controls (n = 6 for both groups) and used in qRT2-PCR focused arrays to determine the expression of TLRs and associated signalling pathway genes. Genes with a statistical significance of ±two-fold regulation (FR) and a P-value

Background and objective The salivary transcriptome may present as a readily available and non-invasive source of potential biomarkers. The development of chronic periodontitis is determined by individual patient susceptibility; hence, the aim of this study was to determine the potential of the salivary transcriptome as a biomarker of disease susceptibility using chronic periodontitis as an example. Material and methods Using an Oragene® RNA kit, the total RNA was purified from the saliva of 10 patients with chronic periodontitis and 10 patients without chronic periodontitis. The quantity and quality of the total RNA was determined, and a measure of gene expression via cDNA was undertaken using the Affymetrix microarray system. The microarray profiling result was further validated by real-time quantitative polymerase chain reaction. Results Spectrophotometric analysis showed the total RNA purified from each participant ranged from 0.92 μg/500 μL to 62.85 μg/500 μL. There was great variability in the quantity of total RNA obtained from the 2 groups in the study with a mean of 10.21 ± 12.71 μg/500 μL for the periodontitis group and 15.97 ± 23.47 μg/500 μL for the control group. Further the RNA purity (based on the A260 /A280 ratio) for the majority of participants (9 periodontitis and 6 controls) were within the acceptable limits for downstream analysis (2.0 ± 0.1). The study samples, showed 2 distinct bands at 23S (3800 bp) and 16S (1500 bp) characteristic of bacterial rRNA. Preliminary microarray analysis was performed for 4 samples (P2, P6, H5 and H9). The percentage of genes present in each of the 4 samples was not consistent with about 1.8%-18.7% of genes being detected. Quantitative real-time polymerase chain reaction confirmed that the total RNA purified from each sample was mainly bacterial RNA (Uni 16S) with minimal human mRNA. Conclusion This study showed that minimal amounts of human RNA were able to be isolated from the saliva of patients with periodontitis as well as controls. Further work is required to enhance the extraction process of human mRNA from saliva if the salivary transcriptome is to be used in determining individual patient susceptibility.

Tissue biopsies are very precious. A method that allows the isolation of a high quality and quantity of genomic DNA, total RNA, and total protein from a single biopsy that is suitable for downstream applications (e.g., DNA methylation analysis, quantitative PCR, and gel electrophoresis techniques) is very desirable. The method described here combines a tissue stabilization reagent combined with a spin-column method for the simultaneous purification of gingival tissue DNA, RNA, and protein. The genomic DNA is then used for quantitative analysis of DNA methylation using real-time PCR (the qAMP method). Subsequent data analysis is very straightforward using online software. Future analyses may include the RNA transcript analysis as well as protein expression levels of genes identified as differentially methylated.

The use of quantitative real-time reverse transcriptase PCR (qRT

The use of quantitative real-time reverse transcriptase PCR (qRT2-PCR) for the identification of differentially regulated genes is a powerful technology. The protocol presented here uses qRT2-PCR gene arrays to investigate the regulation of 84 angiogenic related genes in human primary alveolar osteoblasts following treatment with the bisphosphonate, zoledronic acid (ZA), and geranylgeraniol (GGOH). GGOH has potential as a therapeutic agent for Bisphosphate-Related Osteonecrosis of the Jaw (BRONJ), a serious side-effect resulting from the treatment for metastatic cancer (Zafar et al., J Oral Pathol Med 43:711-721, 2014; Ruggiero, Ann NY Acad Sci 1218:38-46, 2011). The isolation of the primary osteoblast cells follows the methods previously described (Dillon et al., Methods Mol Biol 816:3-18, 2012) with a new RNA extraction technique described fully. The method highlights the importance of obtaining high-quality RNA which is DNA-free. Relative levels of gene expression are normalized against selected housekeeping genes (HKG) and a number of examples of how fold regulation (2(-Delta Delta Cq)) and gene expression level (2(-Delta Cq)) data can be presented are given.

Tissue biopsies are very precious. A method that allows the isolation of a high quality and quantity of genomic DNA, total RNA, and total protein from a single biopsy that is suitable for downstream applications (e.g., DNA methylation analysis, quantitative PCR, and gel electrophoresis techniques) is very desirable. The method described here combines a tissue stabilization reagent combined with a spin-column method for the simultaneous purification of gingival tissue DNA, RNA, and protein. The genomic DNA is then used for quantitative analysis of DNA methylation using real-time PCR (the qAMP method). Subsequent data analysis is very straightforward using online software. Future analyses may include the RNA transcript analysis as well as protein expression levels of genes identified as differentially methylated.

Background Interleukin (IL)-17 is a pro-inflammatory cytokine with pro- and antitumour effects. The aim of this study was to investigate the presence and potential sources of IL-17 in oral squamous cell carcinoma (OSCC). Methods Immunohistochemistry was used to label and compare IL-17+ cells in the tissue sections of OSCC and inflammatory controls (IC), n = 14 for both. In OSCC, the comparison was made between the number of IL-17+ cells in the tumoral islands (TI), tumour–stroma interface (TS) and more distant stroma (DS). Cells expressing IL-17 were identified using double-labelling immunofluorescence and examined using laser scanning microscopy. The production of IL-17 from tumour cells was determined in the culture supernatants of OSCC cell lines, SCC4, SCC15 and SCC25, using sandwich ELISA. Results Significantly more IL-17+ cells were observed in OSCC compared with IC (Mann–Whitney, P 0.05). However, the TI had significantly fewer IL-17+ cells than the combined stroma (both TS and DS together, Mann–Whitney, P < 0.01). Laser scanning microscopy revealed helper T cells, cytotoxic T cells, macrophages and mast cells co-expressed IL-17. ELISA experiments did not detect IL-17 in the supernatants of OSCC cell lines. Conclusions Although the tumour cells themselves did not express IL-17, a range of cell types did, suggesting multiple cellular sources for IL-17 in OSCC. The spatial distribution of IL-17+ cells suggests specific interactions with cells within the tumour microenvironment, implying that IL-17+ cells are likely to play a role in the pathogenesis of OSCC.

Background.A loss of mucosal tolerance to the resident microbiome has been postulated in the aetiopathogenesis of spondyloarthritis, thus the purpose of these studies was to investigate microbial communities that colonise the oral cavity of patients with axial spondyloarthritis (AxSpA) and to compare these with microbial profiles of a matched healthy population.Methods.Thirty-nine participants, 17 patients with AxSpA and 22 age and gender-matched disease-free controls were recruited to the study. For patients with AxSpA, disease activity was assessed using the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). All participants underwent a detailed dental examination to assess oral health, including the presence of periodontal disease assessed using probing pocket depth (PPD). Plaque samples were obtained and their bacterial populations were profiled using Ion Torrent sequencing of the V6 region of the 16S rRNA gene.Results.Patients with AxSpA had active disease (BASDAI 4.1 ± 2.1 [mean ± SD]), and a significantly greater prevalence of periodontitis (PPD ≥ 4 mm at ≥4 sites) than controls. Bacterial communities did not differ between the two groups with multiple metrics ofαandβdiversity considered. Analysis of operational taxonomic units (OTUs) and higher levels of taxonomic assignment did not provide strong evidence of any single taxa associated with AxSpA in the subgingival plaque.Discussion.Although 16S rRNA gene sequencing did not identify specific bacterial profiles associated with AxSpA, there remains the potential for the microbiota to exert functional and metabolic influences in the oral cavity which could be involved in the pathogenesis of AxSpA.

Summary Oral lichen planus (OLP) is a complex immunological disorder, mediated in part by the release of cytokines from activated T-cells. Of late, two closely related T-helper (Th) cell subsets; regulatory T-cells (Tregs; FoxP3 + ) and Th17 cells (IL17 + ) have been described in various chronic inflammatory diseases. The aim of this study was to determine the expression of FoxP3 and IL17 in OLP using immunohistochemistry (IHC) and quantitative real-time reverse transcriptase polymerase chain reaction (qPCR). For IHC, formalin fixed, paraffin embedded archival specimens, an OLP group ( n =10) and a non-specific inflammatory (NSI) control group ( n =9) were used. In addition, 12 fresh tissue samples were used to determine gene expression of FoxP3 and IL17. Significantly more FoxP3 + cells were present in OLP than in NSI. IL17 + cells were significantly more frequent in the control tissues than in OLP. The gene expression experiments revealed a significantly higher expression of FoxP3 in OLP when compared to the controls. IL17 gene expression was not different between the groups. Double labelling immunofluorescence indicated co-localisation of IL17 with tryptase + mast cells. These findings suggest FoxP3 + Tregs have a more prominent role in the pathogenesis of OLP when compared to IL17 + cells.

Saminathan A, Vinoth KJ, Wescott DC, Pinkerton MN, Milne TJ, Cao T, Meikle MC. The effect of cyclic mechanical strain on the expression of adhesion-related genes by periodontal ligament cells in two-dimensional culture. J Periodont Res 2012; 47: 212–221. © 2011 John Wiley & Sons A/S Background and Objective: Cell adhesion plays important roles in maintaining the structural integrity of connective tissues and sensing changes in the biomechanical environment of cells. The objective of the present investigation was to extend our understanding of the effect of cyclic mechanical strain on the expression of adhesion-related genes by human periodontal ligament cells. Material and Methods: Cultured periodontal ligament cells were subjected to a cyclic in-plane tensile deformation of 12% for 5 s (0.2 Hz) every 90 s for 6–24 h in a Flexercell FX-4000 Strain Unit. The following parameters were measured: (i) cell viability by the MTT assay; (ii) caspase-3 and -7 activity; and (iii) the expression of 84 genes encoding adhesion-related molecules using real-time RT-PCR microarrays. Results: Mechanical stress reduced the metabolic activity of deformed cells at 6 h, and caspase-3 and -7 activity at 6 and 12 h. Seventy-three genes were detected at critical threshold values

Increases in bone strains above a certain threshold have a positive effect on bone mass, whereas reductions in strain magnitude lead to bone loss and osteopenia; the term 'mechanostat' has been introduced to describe this tissue-level negative feedback mechanism. The mechanobiology of bone and particularly alveolar bone is poorly understood, and whether the mechanostat theory has any relevance to explaining the osseous changes that occur during orthodontic tooth movement remains unclear. To investigate the relationship further, an expansile force of 0.2 N was applied to the maxillary molars of 36, 6-week-old Wistar rats by helical coil springs. The animals were sacrificed at 1, 2, 4, and 8 days and the tissue response analyzed by histological, biochemical, and finite element (FE) methods. Differences between groups were determined by Student's t-test (two-tailed). The appliance produced an increase in the intermolar width averaging 0.5 mm after 8 days. Tetracycline uptake in the control rats suggested a rapid turnover of bone in both the interradicular domain and the bone-periodontal ligament interface. In the experimental group, however, incorporation of tetracycline into the interradicular domain was reduced and conventional histology revealed evidence of bone loss and osteopenia, in both the experimental and a group of sham-treated positive controls (with inactive, annealed springs). Serum alkaline phosphatase declined significantly in both experimental and sham-treated groups over the 8-day time course, indicating decreased bone formation. Serum acid phosphatase also declined, suggesting a concomitant decrease in bone resorption. Three-dimensional FE analysis of the stresses generated in the bone following occlusal (2 N) and orthodontic loading showed that the orthodontic force created a constant loading condition shielding some areas of bone from mechanical stress. Areas of low mechanical stimulation were coincident with sites of bone loss observed histologically, while bone mass was preserved in areas with higher levels of loading. These findings suggest that (1) the osteopenia resulted from stress shielding of the interradicular bone by the appliance, and a consequent reduction in occlusal loading below the critical threshold required for maintaining normal osseous architecture and (2) the mechanostat model can be employed to explain, at least in part, the response of the bone to orthodontic loading.

Background and Objective: A role for cytokines and growth factors in mediating the cellular and molecular events involved in orthodontic tooth movement is well established. The focus to date, however, has been largely on individual mediators, rather than to study cytokines in terms of complex interacting networks. Our objective was to expand our knowledge of the cytokines and growth factors expressed by human periodontal ligament (PDL) cells and to identify new genes that are responsive to mechanical deformation. Material and Methods: Human PDL cells were strained with a cyclic deformation of 12% for 6–24 h, and the differential expression of 79 cytokine and growth factor genes was quantified using real-time RT-PCR arrays. For statistical comparison, t-tests were used with mean critical threshold (CT) values derived from triplicate samples. Results: Forty-one genes were detected at CT values

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a serious complication of bisphosphonate therapy. The mechanism underlying BRONJ pathogenesis is poorly understood. To determine the effects of zoledronic acid (ZA) and geranylgeraniol (GGOH) on the mevalonate pathway (MVP) in osteoblasts generated from the human mandibular alveolar bone in terms of cell viability/proliferation, migration, apoptosis and gene expression. Primary human osteoblasts (HOBs) isolated from the mandibular alveolar bone were phenotyped. HOBs were cultured with or without ZA and GGOH for up to 72 h. Cellular behaviour was examined using a CellTiter-Blue® viability assay, an Ibidi culture-insert migration assay, an Apo-ONE® Homogeneous Caspase-3/7 apoptosis assay and transmission electron microscopy (TEM). Quantitative real-time reverse transcriptase polymerase chain reaction (qRT2-PCR) was used to determine the simultaneous expression of 168 osteogenic and angiogenic genes modulated in the presence of ZA and GGOH. ZA decreased cell viability and migration and induced apoptosis in HOBs. TEM revealed signs of apoptosis in ZA-treated HOBs. However, the co-addition of GGOH ameliorated the effect of ZA and partially restored the cells to the control state. Twenty-eight genes in the osteogenic array and 27 genes in the angiogenic array were significantly regulated in the presence of ZA compared with those in the controls at one or more time points. The cytotoxic effect of ZA on HOBs and its reversal by the addition of GGOH suggests that the effect of ZA on HOBs is mediated via the MVP. The results suggest that GGOH could be used as a possible therapeutic/preventive strategy for BRONJ.

Background Inflammatory periodontal diseases are initiated by microbial biofilms. The reduction of the biofilm is important in the management of the disease. This study compares periodontopathogen levels following the treatment of chronic periodontitis using Er:YAG laser (ERL) debridement and mechanical scaling and root planing (SRP). Methods Using a split-mouth design, two quadrants were randomly allocated for treatment. Two hundred and fifty-two subgingival plaque samples were collected from 21 patients, before treatment (baseline) and at 6 and 12 weeks post-therapy. Multiplex qPCR was used to determine relative levels of Porphyromonas gingivalis (Pg), Treponema denticola (Td), Tannerella forsythensis (Tf), and Aggregatibacter actinomycetemcomitans (Aa). Results Tf and Pg were significantly reduced post-treatment for both ERL and SRP. ERL treatment resulted in a reduction of Td at 12 weeks. Following SRP treatment Aa was significantly reduced at 12 weeks. No statistically significant difference was seen when treatments were compared at 6 and 12 weeks. Conclusions A comparable reduction in the level of the four periodontal pathogens assayed was achieved with Er:YAG laser debridement and mechanical scaling and root planing.

BACKGROUND It has been suggested that completely edentulous patients harbour fewer periodontopathic bacteria compared with dentate patients, due to the removal of the subgingival periodontal environment. However, reappearance of certain microbes has been reported after the placement of implants in these patients. AIM The aim of this study was to determine whether the periodontopathic bacteria Porphyromonas gingivalis and Tannerella forsythia, as well as the non-periodontopathic bacterium, Staphylococcus aureus, emerged in edentulous patients 6 months after placement of one-piece zirconia and titanium implants. MATERIALS AND METHODS Twenty-six patients were included in the study (titanium = 13, zirconia = 13). Microbial samples were collected from the tongue prior to implant placement and 6 months after implant placement from both the tongue and from around the implants. A qRT-PCR assay using SYBR green/ROX chemistry was used for the detection and quantification of rgp, nuc and karilysin single-copy gene of P. gingivalis, T. forsythia and S. aureus, respectively. Positive controls used in the study were pure bacterial gDNA purified from cultures of P. gingivalis and S. aureus, a cloned sequence of the karilysin gene for T. forsythia, a plaque sample positive for P. gingivalis and T. forsythia, and nasal gDNA for S. aureus. RESULTS The results show that prior to implant placement, all three bacterial species were below the lower limit of quantification in all edentulous patients. The samples collected from the tongue and around the implants remained below the lower limit of quantification for each of the three species. However, all positive controls used in the study were detectable in the samples. qPCR standard curves showed correlation coefficients >0.97 and efficiencies >94.5% (slope range -3.19 to -3.46) for each of the SYBR green PCR assays. CONCLUSION The results of this study indicate that the tested organisms did not emerge 6 months after implant placement irrespective of the nature of the implant biomaterial. A further follow-up of at least 2 years post-implantation of these patients is suggested to determine whether there are any changes in the oral microbiota and whether such changes are associated with the development of peri-implant disease.

The mevalonate pathway (MVP) and the anti-angiogenic effect of bisphosphonates have been shown to play a role in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ). This study determined the effect of the bisphosphonate, zoledronic acid and the replenishment of the MVP by geranylgeraniol on human gingival fibroblasts. Cell viability, apoptosis, morphological analysis using transmission electron microscopy, and gene expression for vascular endothelial growth factor A, bone morphogenic protein 2, ras homologue gene family member B, epiregulin and interferon-alpha were conducted. Results showed cellular viability was decreased in the presence of zoledronic acid and the co-addition of zoledronic acid with geranylgeraniol restored cell viability to control levels. Caspase 3/7 was detected in zoledronic-acid-treated cells indicating apoptosis. Transmission electron microscopy revealed dilation of the rough endoplasmic reticulum with zoledronic acid and the appearance of multiple lipid-like vesicles following the addition of geranylgeraniol. Zoledronic acid significantly (P0.05, FR± 2) up-regulated vascular endothelial growth factor A, bone morphogenic protein 2, ras homologue gene family member B and epiregulin at one or more time points but not interferon-alpha. Addition of geranylgeraniol resulted in a reduction in the expression of all five genes compared with zoledronic-acid-treated human gingival fibroblasts. The study concluded geranylgeraniol partially reversed the effects of zoledronic acid in human gingival fibroblasts both at the cellular and genetic levels, suggesting the regulation of these genes is mediated via the mevalonate pathway.

BACKGROUND AND OBJECTIVE The role of two recently identified and closely related T-helper cell subsets - regulatory T-cells [Tregs; forkhead box P3-positive (FOXP3(+) )] and Th17 cells [interleukin-17-positive (IL-17(+) )] - in periodontal disease is yet to be determined. Tregs are essential in maintaining peripheral tolerance and regulating the immune response. Th17 cells play a critical role in several autoimmune diseases, inflammation and host defence. The aim of this study was to determine the presence of FOXP3(+) Tregs and IL-17(+) cells, and their possible spatial interaction, in diseased periodontal tissues. MATERIAL AND METHODS Twenty-nine archival tissues with nonspecific gingival inflammation were grouped based on the intensity (minimally or intensely inflamed) and nature (T-cell predominant or B- and plasma-cell predominant) of the inflammatory infiltrate. Using double-labelling immunohistochemistry, the concomitant presence of FOXP3(+) and IL-17(+) cells was determined and their spatial relationship was established. In addition, the proportions of FOXP3(+) and IL-17(+) cells were compared between the groups. RESULTS Of the 29 gingival specimens investigated, 17 were intensely inflamed (≥ 1000 inflammatory cells per 0.12 mm(2) ) and 12 were minimally inflamed (≤ 600 cells per 0.12 mm(2) ). Based on the percentage of CD19(+) B-cells and plasma cells collectively and CD3(+) T-cells, gingival tissues were also grouped into B- and plasma-cell-predominant gingival tissues (n = 21; 50.7% total B- and plasma cells vs. 19.1% T cells; p

Gramicidin A analogs, labeled with 13C in the backbone carbonyl groups and the C-2 indole carbons of the tryptophan-11 and tryptophan-13 residues, were synthesized using t-Boc-protected amino acids. The purified analogs were incorporated into phosphatidylcholine bilayers at a 1:15 molar ratio and macroscopically aligned between glass coverslips. The orientations of the labeled groups within the channel were investigated using solid-state NMR and the effect of a monovalent ion (Na+) on the orientation of these groups determined. The presence of sodium ions did not perturb the 13C spectra of the tryptophan carbonyl groups. These results contrast with earlier results in which the Leu-10, Leu-12, and Leu-14 carbonyl groups were found to be significantly affected by the presence of sodium ions and imply that the tryptophan carbonyl groups are not directly involved in ion binding. The channel form of gramicidin A has been demonstrated to be the right-handed form of the beta 6.3 helix: consequently, the tryptophan carbonyls would be directed away from the entrance to the channel and take part in internal hydrogen bonding, so that the presence of cations in the channel would have less effect than on the outer leucine residues. Sodium ions also had no effect on the C-2 indole resonance of the tryptophan side chains. However, a small change was observed in Trp-11 when the ether lipid, ditetradecylphosphatidylcholine, was substituted for the ester lipid, dimyristoylphosphatidylcholine, indicating some sensitivity of the gramicidin side chains to the surrounding lipid.

The techniques for the establishment of primary gingival and periodontal ligament fibroblast cultures have been well established for over 30 years. It is only more recently, with the commercial availability of real-time PCR (RT-PCR) gene arrays that the expression profiles of up to 84 genes can be carried out simultaneously. Each focused panel of genes can identify the up- or down-regulation of genes associated with any one of over 100 biological pathways or specific disease states. Fibroblasts for RNA extraction and subsequent gene expression analysis can be collected under various experimental conditions and stored in RNA-preserving solution (e.g., RNAlater) for processing at a later date or extracted immediately. The "gold standard" method for the extraction of RNA from fibroblasts for RT-PCR purposes is the TRIzol reagent method. With the addition of a spin-column clean-up step, any phenol carried over from the TRIzol step is removed, thus ensuring a high yield of quality RNA. The RNA is then reverse transcribed to cDNA and analyzed using the RT-PCR focused-gene arrays. Data analysis is made easy using on-line array analysis software packages.

The techniques for the establishment of primary gingival and periodontal ligament fibroblast cultures have been well established for over 30 years. It is only more recently, with the commercial availability of real-time PCR (RT-PCR) gene arrays that the expression profiles of up to 84 genes can be carried out simultaneously. Each focused panel of genes can identify the up- or down-regulation of genes associated with any one of over 100 biological pathways or specific disease states. Fibroblasts for RNA extraction and subsequent gene expression analysis can be collected under various experimental conditions and stored in RNA-preserving solution (e.g., RNAlater) for processing at a later date or extracted immediately. The "gold standard" method for the extraction of RNA from fibroblasts for RT-PCR purposes is the TRIzol reagent method. With the addition of a spin-column clean-up step, any phenol carried over from the TRIzol step is removed, thus ensuring a high yield of quality RNA. The RNA is then reverse transcribed to cDNA and analyzed using the RT-PCR focused-gene arrays. Data analysis is made easy using on-line array analysis software packages.

Myelin basic protein (MBP) from the Whaler shark (Carcharhinus obscurus) has been purified from acid extracts of a chloroform/methanol pellet from whole brains. The amino acid sequence of the majority of the protein has been determined and compared with the sequences of other MBPs. The shark protein has only 44% homology with the bovine protein, but, in common with other MBPs, it has basic residues distributed throughout the sequence and no extensive segments that are predicted to have an ordered secondary structure in solution. Shark MBP lacks the triproline sequence previously postulated to form a hairpin bend in the molecule. The region containing the putative consensus sequence for encephalitogenicity in the guinea pig contains several substitutions, thus accounting for the lack of activity of the shark protein. Studies of the secondary structure and self‐association have shown that shark MBP possesses solution properties similar to those of the bovine protein, despite the extensive differences in primary structure. Copyright

The forces that orthodontic appliances apply to the teeth are transmitted through the periodontal ligament (PDL) to the supporting alveolar bone, leading to the deposition or resorption of bone, depending upon whether the tissues are exposed to a tensile or compressive mechanical strain. To evaluate the osteogenic potential of PDL cells, we applied a 12% uni-axial cyclic tensile strain to cultured human PDL cells and analyzed the differential expression of 78 genes implicated in osteoblast differentiation and bone metabolism by real-time RT-PCR array technology. Sixteen genes showed statistically significant changes in expression in response to alterations in their mechanical environment, including cell adhesion molecules and collagen fiber types. Genes linked to the osteoblast phenotype that were up-regulated included BMP2, BMP6, ALP, SOX9, MSX1, and VEGFA; those down-regulated included BMP4 and EGF. This study has expanded our knowledge of the transcriptional profile of PDL cells and identified several new mechanoresponsive genes.

Background: Interleukin 17 (IL17) is a proinflammatory cytokine with increased expression in some cancers. It has been demonstrated to exhibit both pro- and anti-tumor effects. Our group has previously demonstrated expression of IL17 from multiple cells in oral squamous cell carcinoma (OSCC) such that the aim of the current study was to examine the role of IL17 in OSCC progression. Methods: The cytoplasmic SEFIR sequence of the transmembrane IL17 receptor (IL17R) was detected in formalin Fixed Paraffin Embedded (FFPE) OSCC tissues (n = 14) using immunohistochemistry. Soluble IL17R was detected in the cell culture supernatants of three OSCC cell lines (SCC4, SCC15 and SCC25) using a sandwich ELISA. Human recombinant IL17 over a range of concentrations (0, 10, 50, 100 ng/mL) was used to stimulate the three OSCC cell lines.Proliferation at 0, 24, 48, 72 hours was then assayed by cell titer blue and invasion at 48 hours using a QCM ECMatrix cell invasion assay. The data were analyzed using unpaired Student's t test and ANOVA as appropriate using GraphPad Prism 6 software. Results:Cytoplasmic expression of transmembrane IL17R was detected in all FFPE OSCC tissues. Soluble IL17R was detected in the cell culture supernatants of all three OSCC cell lines and its concentration increased in a time dependent manner. SCC25 had significantly higher concentration of soluble IL17R at 72 hours than SCC4 and SCC15. The rate of proliferation of all three OSCC cell lines was not affected with the addition of recombinant IL17. However, IL17 promoted the in vitro invasion of SCC25 and SCC15 in a dose dependent manner. At the highest concentration of IL17 (100ng/mL), the SCC25 cells showed significant invasion compared to control cells (p Conclusions: This study is the first to demonstrate in vitro association between IL17 and possible invasion of OSCC. The exact mechanism by which IL17 enhances invasion and thereby tumor progression in vivo remains to be elucidated and could involve a number of different mechanisms including cell adhesion and extracellular matrix proteins. Citation Format: Avadhoot Avadhani, Trudy Milne, Gregory Seymour, Alison Rich. Interleukin 17 promotes tumor progression in oral squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4151. doi:10.1158/1538-7445.AM2015-4151

The pathogenesis-related (PR) protein superfamily is widely distributed in the animal, plant, and fungal kingdoms and is implicated in human brain tumor growth and plant pathogenesis. The precise biological activity of PR proteins, however, has remained elusive. Here we report the characterization, cloning and structural homology modeling of Tex31 from the venom duct of Conus textile. Tex31 was isolated to >95% purity by activity-guided fractionation using a para-nitroanilide substrate based on the putative cleavage site residues found in the propeptide precursor of conotoxin TxVIA. Tex31 requires four residues including a leucine N-terminal of the cleavage site for efficient substrate processing. The sequence of Tex31 was determined using two degenerate PCR primers designed from N-terminal and tryptic digest Edman sequences. A BLAST search revealed that Tex31 was a member of the PR protein superfamily and most closely related to the CRISP family of mammalian proteins that have a cysteine-rich C-terminal tail. A homology model constructed from two PR proteins revealed that the likely catalytic residues in Tex31 fall within a structurally conserved domain found in PR proteins. Thus, it is possible that other PR proteins may also be substrate-specific proteases.

Background A wealth of data supports a role for the microbiome in the pathogenesis of spondyloarthtritis (SpA). To date, studies have concentrated on the intestinal tract, which harbours the largest and most diverse microbiota. However, the oral microbiota is also notable in its diversity and has recently been the focus of interest as a potential factor in the pathogenesis of rheumatoid arthritis. Recent studies have demonstrated a higher prevalence of periodontitis in patients with ankylosing spondylitis, raising the possibility that exposure to the oral microbiome through a compromised mucosal barrier may be a factor in the pathogenesis of SpA. Objectives To investigate the oral cavity as a site of chronic inflammation in axial spondyloarthritis (AxSpA) and to compare profiles of microbial communities which colonise the oral cavity in health and AxSpA. Methods Thirty nine participants, 17 with AxSpA and 22 age and gender matched, disease free controls, were recruited. Disease activity in patients with AxSpA was assessed using the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and CRP. All participants underwent a detailed dental examination including assessment of probing pocket depth (PPD), clinical attachment loss (CAL), bleeding on probing (BOP), plaque index, oral mucosal conditions, and caries status. Plaque samples were obtained and their bacterial populations were profiled using Ion Torrent sequencing of the V6 region of the 16S rRNA gene. Results Patients with AxSpA had active disease (BASDAI Mean ± SD 4.1±2.1) and had significantly greater prevalence of both periodontitis (PPD≥4mm at ≥4 sites) and a higher plaque index than controls (P=0.008 and P=0.006 respectively). Analysis of bacterial communities showed no difference between AxSpA patients and healthy controls, either in their overall within sample organism diversity or in community structure between groups. However, analysis at the level of operational taxonomic units (OTU) demonstrated a significant association between AxSpA and several bacteria including Campylobacter spp. and Actinomycetes spp . Additionally, certain OTUs were present in higher relative abundances in patients with active AxSpA (see Table). Conclusions Patients with AxSpA have a higher prevalence of periodontitis. Analysis of plaque communities suggests that certain bacteria are more prevalent in the periodontal tissues of patients with AxSpA and associates with active disease. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3406

Introduction GRP78 has a critical role in the attenuation of cellular stress and recovery from injury. It is involved in the folding and assembly of proteins in the endoplasmic reticulum (ER) and is induced in times of ER stress. Russell bodies (RB) are dilated ER cisternae containing condensed immunoglobulins and are thought to arise from failure of ER quality control mechanisms. Purpose To investigate the relationship between Russell bodies and ER stress in periodontal inflammation. Methods Formalin fixed paraffin embedded (FFPE) specimens were retrieved and categorised into three groups: inflamed period-ontal tissues with RB (Group 1), inflamed tissues without RB (Group 2), and lightly inflamed control tissue (Group 3). Serial sections were prepared and stained with H&E, PAS, methyl green pyronin and anti-GRP78. The number of RB and the distribution and types of cells staining positively with GRP78 were analysed. Results Of the inflammatory cells in Groups 1 and 2 which were GRP78+, most were plasma cells. In the less inflamed Group 3 tissues lymphocytes were also GRP78+. Inflamed tissues had statistically significant more apoptotic/degenerate bodies than control tissues. There was no statistically significant difference between GRP78 staining and the presence or absence of RB in inflamed tissues and control tissues. Conclusion Inflammation, rather than RB, correlated positively with GRP78 immunostaining.