21 results on '"Trudy J. Milne"'
Search Results
2. Thein vitroeffect of VEGF receptor inhibition on primary alveolar osteoblast nodule formation
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Sobia Zafar, Katy I. McLaughlin, Trudy J. Milne, Mary P. Cullinan, Dawn E. Coates, Diogo Godoy Zanicotti, and Gregory J. Seymour
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Vascular Endothelial Growth Factor A ,Angiogenesis ,medicine.medical_treatment ,Immunofluorescence ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Osteogenesis ,medicine ,Humans ,030212 general & internal medicine ,General Dentistry ,Osteoblasts ,medicine.diagnostic_test ,Cell Differentiation ,Osteoblast ,030206 dentistry ,Bisphosphonate ,In vitro ,Vascular endothelial growth factor ,Receptors, Vascular Endothelial Growth Factor ,medicine.anatomical_structure ,chemistry ,Cell culture ,Intramembranous ossification ,cardiovascular system ,Cancer research - Abstract
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.
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- 2020
3. Differential behaviour and gene expression in 3D cultures of femoral‐ and calvarial‐derived human osteoblasts under a cyclic compressive mechanical load
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Richard D. Cannon, Mauro Farella, Murray C. Meikle, Trudy J. Milne, Yana Itskovich, and Dawn E. Coates
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musculoskeletal diseases ,Osteoblasts ,Mechanical load ,food.ingredient ,Strain (chemistry) ,Chemistry ,Gene Expression ,Osteoblast ,Bone morphogenetic protein 2 ,Gelatin ,Cell biology ,Durapatite ,medicine.anatomical_structure ,food ,Gene expression ,medicine ,Humans ,Femur ,Stress, Mechanical ,Viability assay ,General Dentistry ,Endochondral ossification - Abstract
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.
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- 2021
4. A TaqMan™-based quantitative PCR screening assay for the probiotic Streptococcus salivarius K12 based on the specific detection of its megaplasmid-associated salivaricin B locus
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Nicholas C. K. Heng, Deepti Krishnan, John D. F. Hale, Peter Reid, Julian Crane, Trudy J. Milne, and John R. Tagg
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Microbiology (medical) ,Specific detection ,Locus (genetics) ,Biology ,Real-Time Polymerase Chain Reaction ,Microbiology ,Streptococcus salivarius ,law.invention ,03 medical and health sciences ,Probiotic ,Bacteriocin ,Bacterial Proteins ,law ,TaqMan ,Humans ,Molecular Biology ,Gene ,030304 developmental biology ,0303 health sciences ,030306 microbiology ,Probiotics ,biology.organism_classification ,Bacterial Load ,Real-time polymerase chain reaction ,bacteria ,Plasmids - Abstract
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.
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- 2019
5. Expression of IL33 and IL35 in oral lichen planus
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V. P. B. Parachuru, Gregory J. Seymour, L. R. Javvadi, Trudy J. Milne, and Alison M. Rich
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Adult ,Male ,0301 basic medicine ,Biopsy ,CD3 ,medicine.medical_treatment ,Fluorescent Antibody Technique ,Connective tissue ,Dermatology ,Immunofluorescence ,T-Lymphocytes, Regulatory ,03 medical and health sciences ,0302 clinical medicine ,stomatognathic system ,Gene expression ,Humans ,Medicine ,Aged ,Aged, 80 and over ,medicine.diagnostic_test ,biology ,business.industry ,Interleukins ,Mouth Mucosa ,EBI3 ,General Medicine ,Middle Aged ,Interleukin-33 ,medicine.disease ,stomatognathic diseases ,030104 developmental biology ,medicine.anatomical_structure ,Cytokine ,Gene Expression Regulation ,Immunology ,biology.protein ,Immunohistochemistry ,Female ,Oral lichen planus ,business ,Lichen Planus, Oral ,030215 immunology - Abstract
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
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- 2018
6. Effects of environmental tobacco smoke on the oral health of preschool children
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Dawn E. Coates, Trudy J. Milne, N. N. b. Hasmun, Bernadette K. Drummond, Alison M Meldrum, and Mary P. Cullinan
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Male ,Saliva ,Oral Health ,Dental Caries ,Oral health ,Tobacco smoke ,03 medical and health sciences ,Gingivitis ,0302 clinical medicine ,Surveys and Questionnaires ,Environmental health ,Humans ,Medicine ,Dentistry (miscellaneous) ,030212 general & internal medicine ,Child ,Respiratory Tract Infections ,Pregnancy ,Enamel paint ,business.industry ,Dental health ,Infant ,030206 dentistry ,medicine.disease ,Immunoglobulin A ,Otitis Media ,stomatognathic diseases ,Breast Feeding ,Case-Control Studies ,Child, Preschool ,visual_art ,Pediatrics, Perinatology and Child Health ,visual_art.visual_art_medium ,Gestation ,Female ,Tobacco Smoke Pollution ,medicine.symptom ,business ,New Zealand - Abstract
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.
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- 2017
7. The association between oral bacteria, the cough reflex and pneumonia in patients with acute stroke and suspected dysphagia
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Maggie-Lee Huckabee, Sarah E. Perry, Trudy J. Milne, and Geoffrey R. Tompkins
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Saliva ,medicine.medical_specialty ,Cough reflex ,Population ,Aspiration pneumonia ,Pneumonia, Aspiration ,03 medical and health sciences ,0302 clinical medicine ,Internal medicine ,Reflex ,medicine ,Humans ,Respiratory system ,education ,General Dentistry ,Stroke ,education.field_of_study ,Bacteria ,business.industry ,030206 dentistry ,Pneumonia ,medicine.disease ,Dysphagia ,Cough ,medicine.symptom ,business ,Deglutition Disorders ,030217 neurology & neurosurgery - Abstract
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
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- 2019
8. The bisphosphonate zoledronic acid regulates key angiogenesis-related genes in primary human gingival fibroblasts
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W.D. Duncan, E.J. Ohlrich, Sobia Zafar, Trudy J. Milne, Dawn E. Coates, Gregory J. Seymour, Mary P. Cullinan, and Y. Zhao
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Vascular Endothelial Growth Factor A ,0301 basic medicine ,Pathology ,medicine.medical_specialty ,Angiogenesis ,RHOB ,medicine.medical_treatment ,Gingiva ,Bone Morphogenetic Protein 2 ,Neovascularization, Physiologic ,Polymerase Chain Reaction ,Zoledronic Acid ,03 medical and health sciences ,0302 clinical medicine ,Cell Movement ,RhoB GTP-Binding Protein ,Gene expression ,medicine ,Humans ,rhoB GTP-Binding Protein ,General Dentistry ,Cells, Cultured ,Chemokine CCL2 ,Cell Proliferation ,Regulation of gene expression ,Bone Density Conservation Agents ,CD55 Antigens ,Diphosphonates ,Interleukin-6 ,business.industry ,Imidazoles ,Cell Biology ,General Medicine ,Fibroblasts ,Bisphosphonate ,Up-Regulation ,Vascular endothelial growth factor A ,030104 developmental biology ,Zoledronic acid ,Gene Expression Regulation ,Otorhinolaryngology ,030220 oncology & carcinogenesis ,Cancer research ,business ,medicine.drug - Abstract
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
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- 2016
9. Downregulation of toll-like receptor-mediated signalling pathways in oral lichen planus
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Fiona A Firth, Trudy J. Milne, Suraya Hani Mohd. Sinon, Alison M. Rich, V. P. B. Parachuru, and Gregory J. Seymour
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Male ,0301 basic medicine ,TIRAP ,Cancer Research ,medicine.medical_specialty ,MAPK8 ,Down-Regulation ,Biology ,Pathology and Forensic Medicine ,03 medical and health sciences ,0302 clinical medicine ,stomatognathic system ,Downregulation and upregulation ,Internal medicine ,medicine ,Humans ,HSP70 Heat-Shock Proteins ,HMGB1 Protein ,Receptor ,Membrane Glycoproteins ,Toll-Like Receptors ,Mouth Mucosa ,Receptors, Interleukin-1 ,Middle Aged ,medicine.disease ,Immunohistochemistry ,Up-Regulation ,stomatognathic diseases ,TLR2 ,030104 developmental biology ,Endocrinology ,Otorhinolaryngology ,030220 oncology & carcinogenesis ,Myeloid Differentiation Factor 88 ,Immunology ,TLR4 ,Periodontics ,Female ,Oral lichen planus ,Oral Surgery ,Lichen Planus, Oral ,Signal Transduction - Abstract
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
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- 2015
10. Feasibility of the salivary transcriptome as a novel biomarker in determining disease susceptibility
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Trudy J. Milne, M. F. H. Hidayat, Gregory J. Seymour, and Mary P. Cullinan
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0301 basic medicine ,Male ,Saliva ,Gene Expression ,Biology ,Real-Time Polymerase Chain Reaction ,Transcriptome ,03 medical and health sciences ,0302 clinical medicine ,Gene expression ,medicine ,Humans ,RNA, Messenger ,Pathology, Molecular ,Periodontitis ,Messenger RNA ,Bacteria ,Gene Expression Profiling ,RNA ,030206 dentistry ,Ribosomal RNA ,Middle Aged ,medicine.disease ,Microarray Analysis ,Molecular biology ,030104 developmental biology ,Real-time polymerase chain reaction ,Chronic Periodontitis ,Periodontics ,Female ,Disease Susceptibility ,Biomarkers - Abstract
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.
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- 2017
11. A Protocol for the Determination of the Methylation Status of Gingival Tissue DNA at Specific CpG Islands
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Trudy J, Milne
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Epigenomics ,Genome, Human ,Gene Expression Profiling ,Gingiva ,Computational Biology ,Humans ,CpG Islands ,Genomics ,DNA Methylation ,Transcriptome ,Polymerase Chain Reaction ,Epigenesis, Genetic - Abstract
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.
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- 2016
12. Quantitative Real-Time Gene Profiling of Human Alveolar Osteoblasts
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Dawn E, Coates, Sobia, Zafar, and Trudy J, Milne
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Osteoblasts ,Diphosphonates ,Gene Expression Regulation ,Gene Expression Profiling ,Imidazoles ,Computational Biology ,Humans ,Cell Separation ,Real-Time Polymerase Chain Reaction ,Transcriptome ,Zoledronic Acid ,Cells, Cultured - Abstract
The use of quantitative real-time reverse transcriptase PCR (qRT
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- 2016
13. Multiple cells express interleukin 17 in oral squamous cell carcinoma
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Gregory J. Seymour, Trudy J. Milne, Avadhoot Avadhani, Alison M. Rich, and V. P. B. Parachuru
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0301 basic medicine ,Cancer Research ,Cell type ,medicine.medical_treatment ,Enzyme-Linked Immunosorbent Assay ,Biology ,Immunofluorescence ,Pathology and Forensic Medicine ,03 medical and health sciences ,0302 clinical medicine ,Cell Line, Tumor ,medicine ,Cytotoxic T cell ,Humans ,Analysis of Variance ,medicine.diagnostic_test ,Interleukin-17 ,Interleukin ,Molecular biology ,Immunohistochemistry ,stomatognathic diseases ,030104 developmental biology ,Cytokine ,Otorhinolaryngology ,Cell culture ,030220 oncology & carcinogenesis ,Gingival Diseases ,Carcinoma, Squamous Cell ,Periodontics ,Mouth Neoplasms ,Interleukin 17 ,Oral Surgery - Abstract
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.
- Published
- 2016
14. Regulatory T-cells and IL17A(+) cells infiltrate oral lichen planus lesions
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Alison M. Rich, Gregory J. Seymour, Trudy J. Milne, V. P. B. Parachuru, and L. R. Javvadi
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0301 basic medicine ,Adult ,Male ,Fluorescent Antibody Technique ,Tryptase ,Biology ,Immunofluorescence ,Polymerase Chain Reaction ,T-Lymphocytes, Regulatory ,Pathology and Forensic Medicine ,03 medical and health sciences ,stomatognathic system ,T-Lymphocyte Subsets ,Gene expression ,medicine ,Humans ,Aged ,Aged, 80 and over ,medicine.diagnostic_test ,Interleukin-17 ,FOXP3 ,Middle Aged ,medicine.disease ,Immunohistochemistry ,stomatognathic diseases ,030104 developmental biology ,Immunology ,biology.protein ,Oral lichen planus ,Female ,Interleukin 17 ,IL17A ,Lichen Planus, Oral - Abstract
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.
- Published
- 2015
15. Cultured human periodontal ligament cells constitutively express multiple osteotropic cytokines and growth factors, several of which are responsive to mechanical deformation
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David C. Wescott, Trudy J. Milne, Mark N. Pinkerton, Benjamin J. Gaffey, Kyle T. Beggs, and Murray C. Meikle
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Dental Stress Analysis ,Periodontal Ligament ,medicine.medical_treatment ,Biology ,Interleukin 22 ,Tensile Strength ,medicine ,Humans ,Periodontal fiber ,Growth Substances ,Cell Shape ,Cells, Cultured ,Oligonucleotide Array Sequence Analysis ,Reverse Transcriptase Polymerase Chain Reaction ,Gene Expression Profiling ,Growth factor ,Lymphokine ,Interleukin ,Cell biology ,Cytokine ,Gene Expression Regulation ,RANKL ,Immunology ,biology.protein ,Cytokines ,Periodontics ,Tumor necrosis factor alpha ,Bone Remodeling - Abstract
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
- Published
- 2008
16. Effects of zoledronic acid and geranylgeraniol on the cellular behaviour and gene expression of primary human alveolar osteoblasts
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Dawn E. Coates, Gregory J. Seymour, Bernadette K. Drummond, Sobia Zafar, Mary P. Cullinan, and Trudy J. Milne
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Adult ,Adolescent ,Cell Survival ,Gene Expression ,Apoptosis ,Bioinformatics ,Real-Time Polymerase Chain Reaction ,Zoledronic Acid ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Geranylgeraniol ,Microscopy, Electron, Transmission ,Cell Movement ,Gene expression ,Alveolar Process ,Medicine ,Cytotoxic T cell ,Humans ,Viability assay ,General Dentistry ,Cells, Cultured ,Cell Proliferation ,Osteoblasts ,Bone Density Conservation Agents ,Diphosphonates ,business.industry ,Imidazoles ,030206 dentistry ,Middle Aged ,medicine.disease ,Zoledronic acid ,chemistry ,030220 oncology & carcinogenesis ,Cancer research ,Female ,Mevalonate pathway ,Diterpenes ,business ,Osteonecrosis of the jaw ,Biomarkers ,medicine.drug - Abstract
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.
- Published
- 2015
17. Analysis of P. gingivalis, T. forsythia and S. aureus levels in edentulous mouths prior to and 6 months after placement of one-piece zirconia and titanium implants
- Author
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Trudy J. Milne, Allauddin Siddiqi, Mary P. Cullinan, and Gregory J. Seymour
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0301 basic medicine ,DNA, Bacterial ,Staphylococcus aureus ,Gingiva ,Dentistry ,medicine.disease_cause ,Real-Time Polymerase Chain Reaction ,Microbiology ,03 medical and health sciences ,0302 clinical medicine ,Forsythia ,Tongue ,medicine ,Tannerella forsythia ,Humans ,Porphyromonas gingivalis ,Dental Implants ,Titanium ,biology ,business.industry ,030206 dentistry ,biology.organism_classification ,030104 developmental biology ,Real-time polymerase chain reaction ,medicine.anatomical_structure ,Edentulous Mouths ,Implant ,Zirconium ,Oral Surgery ,Mouth, Edentulous ,business - Abstract
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.
- Published
- 2014
18. Zoledronic acid and geranylgeraniol regulate cellular behaviour and angiogenic gene expression in human gingival fibroblasts
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Dawn E. Coates, Gregory J. Seymour, Sobia Zafar, Trudy J. Milne, Mary P. Cullinan, and Bernadette K. Drummond
- Subjects
Adult ,Vascular Endothelial Growth Factor A ,Cancer Research ,Cell Survival ,Cell Culture Techniques ,Gingiva ,Bone Morphogenetic Protein 2 ,Mevalonic Acid ,Neovascularization, Physiologic ,Caspase 3 ,Apoptosis ,Zoledronic Acid ,Epiregulin ,Pathology and Forensic Medicine ,chemistry.chemical_compound ,Geranylgeraniol ,Microscopy, Electron, Transmission ,Polyisoprenyl Phosphates ,medicine ,Humans ,Viability assay ,rhoB GTP-Binding Protein ,Cells, Cultured ,Bone Density Conservation Agents ,Diphosphonates ,Imidazoles ,Interferon-alpha ,Fibroblasts ,Middle Aged ,Farnesol ,Vascular endothelial growth factor A ,Zoledronic acid ,Otorhinolaryngology ,chemistry ,Biochemistry ,Gene Expression Regulation ,Cancer research ,Periodontics ,Female ,Mevalonate pathway ,Oral Surgery ,Diterpenes ,Sesquiterpenes ,medicine.drug ,Signal Transduction - Abstract
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.
- Published
- 2014
19. Forkhead box P3-positive regulatory T-cells and interleukin 17-positive T-helper 17 cells in chronic inflammatory periodontal disease
- Author
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Haizal Mohd Hussaini, Alison M. Rich, Trudy J. Milne, Gregory J. Seymour, Dawn E. Coates, and V. P. B. Parachuru
- Subjects
Male ,Pathology ,medicine.medical_specialty ,CD3 Complex ,CD3 ,T-Lymphocytes ,Antigens, CD19 ,Palatine Tonsil ,Plasma Cells ,Inflammation ,Cell Communication ,T-Lymphocytes, Regulatory ,CD19 ,Immune system ,medicine ,Humans ,Lymphocyte Count ,Cell Size ,B-Lymphocytes ,biology ,Interleukin-17 ,Peripheral tolerance ,FOXP3 ,Forkhead Transcription Factors ,Middle Aged ,medicine.disease ,Chronic periodontitis ,Gingivitis ,biology.protein ,Periodontics ,Th17 Cells ,Female ,Interleukin 17 ,medicine.symptom - Abstract
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
- Published
- 2013
20. Real-time PCR focused-gene array profiling of gingival and periodontal ligament fibroblasts
- Author
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Patty, Chou and Trudy J, Milne
- Subjects
Periodontal Ligament ,Reverse Transcriptase Polymerase Chain Reaction ,Gingiva ,Humans ,Fibroblasts ,Polymerase Chain Reaction ,Cells, Cultured - Abstract
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.
- Published
- 2010
21. Osteogenic gene expression by human periodontal ligament cells under cyclic tension
- Author
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Benjamin J. Gaffey, Kyle T. Beggs, Murray C. Meikle, M. N. Pinkerton, D. C. Wescott, and Trudy J. Milne
- Subjects
0301 basic medicine ,Pathology ,medicine.medical_specialty ,Tooth Movement Techniques ,Periodontal Ligament ,Protein Array Analysis ,Bone morphogenetic protein 2 ,Bone remodeling ,03 medical and health sciences ,0302 clinical medicine ,stomatognathic system ,Osteogenesis ,Tensile Strength ,medicine ,Alveolar Process ,Periodontal fiber ,Humans ,Bicuspid ,General Dentistry ,Dental alveolus ,Cells, Cultured ,Osteoblasts ,Chemistry ,Cell adhesion molecule ,Osteoblast ,030206 dentistry ,Alkaline Phosphatase ,Resorption ,Cell biology ,Bone morphogenetic protein 6 ,030104 developmental biology ,medicine.anatomical_structure ,Gene Expression Regulation ,Bone Morphogenetic Proteins ,Stress, Mechanical ,Transcription Factors - Abstract
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.
- Published
- 2007
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