Melbourne Dental School - Research Publications
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ItemThe Potential of Calcium Phosphate Nanoparticles as Adjuvants and Vaccine Delivery Vehicles(FRONTIERS MEDIA SA, 2021-12-22)Vaccination is one of the most efficacious and cost-effective ways to protect people from infectious diseases and potentially cancer. The shift in vaccine design from disrupted whole pathogens to subunit antigens has brought attention on to vaccine delivery materials. For the last two decades, nanotechnology-based vaccines have attracted considerable attention as delivery vehicles and adjuvants to enhance immunogenicity, exemplified with the current COVID vaccines. The nanoparticle vaccines display unique features in protecting antigens from degradation, controlled antigen release and longer persisting immune response. Due to their size, shape and surface charge, they can be outstanding adjuvants to achieve various immunological effects. With the safety and biodegradable benefit of calcium phosphate nanoparticles (CaP NPs), they are an efficient carrier for vaccine design and adjuvants. Several research groups have studied CaP NPs in the field of vaccination with great advances. Although there are several reports on the overview of CaP NPs, they are limited to the application in biomedicine, drug delivery, bone regeneration and the methodologies of CaP NPs synthesis. Hence, we summarised the basic properties of CaP NPs and the recent vaccine development of CaP NPs in this review.
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ItemA Systematic Review of MicroRNA Signatures Associated with the Progression of Leukoplakia with and without Epithelial Dysplasia(MDPI, 2021-12)Oral cancer is a significant public health issue, being the eighth most common cancer worldwide with over 300,000 cases diagnosed annually. Early diagnosis and adequate management of oral potentially malignant disorders (OPMDs) before transformation into oral squamous cell carcinoma (OSCC) is critical to reduce deaths, morbidity, and to improve overall prognosis. MicroRNAs (miRNAs) are small noncoding RNAs involved in the post-transcriptional regulation of protein expression and implicated in the control of numerous cellular pathways and impacting physiological, developmental, and pathological processes. Dysregulation of miRNAs has been reported in many cancers and has been demonstrated to play a critical role in cancer initiation, progression, apoptosis, invasion and metastasis. This systematic review provides a comprehensive summary of the prevailing literature on miRNA signatures in OPMDs, specifically leukoplakia with or without oral epithelial dysplasia, and their utility in predicting malignant transformation into OSCC. Eighteen articles describing 73 unique and differentially expressed microRNAs met the criteria for inclusion in this review. We reviewed the characteristics and methodology for each of these studies and assessed the sensitivity and specificity of the studied miRNAs in predicting malignant transformation. This systematic review highlights the significant interest in miRNAs and their tremendous potential as prognostic markers for predicting the malignant transformation of OPMDs into OSCC.
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ItemOral keratinocytes synthesize CTACK: A new insight into the pathophysiology of the oral mucosa(WILEY, 2018-02)The skin-associated chemokine CTACK plays a key role in many inflammatory conditions and could be instrumental in the pathophysiology of tissue-specific immunological diseases such as oral lichen planus (OLP). In this study, we investigated, by RT-PCR, ELISA, chemotaxis assays, and fluorescence-activated cell sorting (FACS), the production of CTACK in oral keratinocytes, its expression in tissues from normal and OLP patients, and its role in T-cell recruitment.CTACK was produced by the oral epithelium, and it affects chemotaxis of memory CLA+ cells to the oral epithelium. CTACK mRNA was expressed constitutively in primary oral epithelium and was increased during pro-inflammatory IFN-γ treatment. We found a constitutive production of CTACK at a protein level in oral primary cells that increased after IFN-γ treatment. Moreover, we confirmed that CTACK attracts memory T cells and those T cells that express CLA above the level of basal migration.
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ItemThe role of human papillomavirus in p16-positive oral cancers(WILEY, 2018-01)BACKGROUND: The aim of this study was to identify the presence and frequency of human papillomavirus (HPV) nucleic acid in p16-positive oral squamous cell carcinomas (OSCCs), to assess whether the virus was transcriptionally active and to assess the utility of p16 overexpression as a surrogate marker for HPV in OSCC. METHODS: Forty-six OSCC patients treated between 2007 and 2011 with available formalin-fixed paraffin-embedded (FFPE) specimens were included. Twenty-three patients were positive for p16 by immunohistochemistry (IHC) and these were matched with 23 patients with p16-negative tumours. Laser capture microdissection of the FFPE OSCC tissues was undertaken to isolate invasive tumour tissue. DNA was extracted and tested for high-risk HPV types using a PCR-ELISA method based on the L1 SPF10 consensus primers, and a real-time PCR method targeting HPV-16 and HPV-18 E6 region. Genotyping of HPV-positive cases was performed using a reverse line blot hybridization assay (Inno-LiPA). RNAScope® (a chromogenic RNA in situ hybridization assay) was utilized to detect E6/E7 mRNA of known high-risk HPV types for detection of transcriptionally active virus. RESULTS: HPV DNA was found in 3 OSCC cases, all of which were p16 IHC-positive. Two cases were genotyped as HPV-16 and one as HPV-33. Only one of the HPV-16 cases was confirmed to harbour transcriptionally active virus via HPV RNA ISH. CONCLUSION: We have shown that the presence of transcriptionally active HPV rarely occurs in OSCC and that p16 is not an appropriate surrogate marker for HPV in OSCC cases. We propose that non-viral mechanisms are responsible for the majority of IHC p16 overexpression in OSCC.
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ItemA panel of microRNAs can be used to determine oral squamous cell carcinoma(WILEY, 2017-11)BACKGROUND: Subjective histopathology is currently used to diagnose oral squamous cell carcinoma (OSCC). We tested if abundances of a panel of microRNA could be an objective OSCC indicator. METHOD: Literature review enabled identification of 10 microRNAs associated with oral and head and neck malignancies. We extracted RNA from formalin-fixed paraffin-embedded biopsies; 20 each with OSCC, dysplasia, or histologically normal epithelium (HNE) and 10 with oral lichen planus (OLP). Relative abundances of microRNAs in HNE and OSCC were determined using reverse transcription and then real-time PCR with global mean normalization. MicroRNAs differentially expressed (test microRNA, T-miR) and non-differentially expressed (normalization microRNA, N-miR) were identified. The raw microRNA Cq data were incorporated in a developed algorithm that output a T-miR expression value (T-miREV) score. Raw Cq data from HNE, OSCC, dysplasia, and OLP samples were then used to test the algorithm scoring and OSCC classification. RESULTS: Four test and normalization microRNAs were identified. Algorithm output of T-mirEV >1 or <-1 indicated high and low OSCC probability score, respectively, and gave 88.9% sensitivity, 100% specificity, and 93.5% accuracy. Grouping high and intermediate T-mirEV scores (T-miREV ≥-1) resulted in sensitivity of 90%, specificity of 65%, and accuracy of 77.5% in OSCC classification. All 20 dysplasias and eight of 10 OLP had T-miREV ≥-1 indicating intermediate to high probability of malignant changes. CONCLUSION: A microRNA panel combined with our algorithm can identify tissue with probable oncogenic changes. IMPACT: The developed algorithm serves as a baseline for prospective trials, which may result in potential clinical utility.
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ItemOral swirl samples - a robust source of microRNA protected by extracellular vesicles(WILEY, 2017-04)BACKGROUND: MicroRNAs are small non-coding RNAs which are dysregulated in disease states, such as oral cancer. Extracellular vesicles, a potential source of microRNA, are found in saliva. OBJECTIVE: To demonstrate that a quantifiable amount of microRNA can be isolated from oral swirl samples. Additionally, we hypothesized that extracellular vesicles may protect contained microRNA from degradation in these samples. METHOD: A polyethylene glycol-based precipitation was used for extracellular vesicle enrichment of oral swirl samples. Comparison was made between samples treated with and without RNase. Further, samples from three subjects were exposed to a range of conditions over 7 days and assessed for presence of microRNA by reverse-transcription quantitative PCR. Extracellular vesicles from samples were identified under transmission electron microscopy. RESULTS: An adequate quantity of microRNA for qPCR analysis was extractable from samples despite exposure to conditions under which degradation of RNA would be expected. CONCLUSION: A technique was developed to isolate an adequate quantity of microRNA for analysis from oral swirl samples. Extracellular vesicle-associated microRNA may be protected from degradation. This technique moves towards chairside application of translational microRNA research in the field of oral cancer prognostics.
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ItemAntimicrobial activity and regulation of CXCL9 and CXCL10 in oral keratinocytes(WILEY, 2016-10)Chemokine (C-X-C motif) ligand (CXCL)9 and CXCL10 are dysregulated in oral inflammatory conditions, and it is not known if these chemokines target microorganisms that form oral biofilm. The aim of this study was to investigate the antimicrobial activity of CXCL9 and CXCL10 on oral microflora and their expression profiles in oral keratinocytes following exposure to inflammatory and infectious stimuli. Streptococcus sanguinis was used as a model and Escherichia coli as a positive control. The antimicrobial effect of CXCL9/CXCL10 was tested using a radial diffusion assay. mRNA transcripts were isolated from lipopolysaccharide (LPS)-treated and untreated (control) oral keratinocyte cell lines at 2-, 4-, 6-, and 8-h time-points of culture. The CXCL9/10 expression profile in the presence or absence of interferon-γ (IFN-γ) was assessed using semiquantitative PCR. Although both chemokines demonstrated antimicrobial activity, CXCL9 was the most effective chemokine against both S. sanguinis and E coli. mRNA for CXCL10 was expressed in control cells and its production was enhanced at all time-points following stimulation with LPS. Conversely, CXCL9 mRNA was not expressed in control or LPS-stimulated cells. Finally, stimulation with IFN-γ enhanced basal expression of both CXCL9 and CXCL10 in oral keratinocytes. Chemokines derived from oral epithelium, particularly CXCL9, demonstrate antimicrobial properties. Bacterial and inflammatory-stimulated up-regulation of CXCL9/10 could represent a key element in oral bacterial colonization homeostasis and host-defense mechanisms.
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ItemCandida virulence and ethanol-derived acetaldehyde production in oral cancer and non-cancer subjects(WILEY, 2016-11)OBJECTIVES: To compare biofilm-forming ability, hydrolytic enzymes and ethanol-derived acetaldehyde production of oral Candida isolated from the patients with oral cancer and matched non-oral cancer. MATERIAL AND METHODS: Fungal biofilms were grown in RPMI-1640 medium, and biofilm mass and biofilm activity were assessed using crystal violet staining and XTT salt reduction assays, respectively. Phospholipase, proteinase, and esterase production were measured using agar plate method, while fungal acetaldehyde production was assessed via gas chromatography. RESULTS: Candida isolated from patients with oral cancer demonstrated significantly higher biofilm mass (P = 0.031), biofilm metabolic activity (P < 0.001), phospholipase (P = 0.002), and proteinase (P = 0.0159) activity than isolates from patients with non-oral cancer. High ethanol-derived acetaldehyde-producing Candida were more prevalent in patients with oral cancer than non-oral cancer (P = 0.01). In univariate regression analysis, high biofilm mass (P = 0.03) and biofilm metabolic activity (P < 0.001), high phospholipase (P = 0.003), and acetaldehyde production ability (0.01) were significant risk factors for oral cancer; while in the multivariate regression analysis, high biofilm activity (0.01) and phospholipase (P = 0.01) were significantly positive influencing factors on oral cancer. CONCLUSION: These data suggest a significant positive association between the ability of Candida isolates to form biofilms, to produce hydrolytic enzymes, and to metabolize alcohol to acetaldehyde with their ability to promote oral cancer development.
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ItemThe assessment of the robustness of microRNAs from oral cytological scrapings(WILEY, 2017-05)BACKGROUND: Sampling of suspect oral lesions in the general dental clinic may increase early carcinoma detection thus oral cancer survival rates. One means of lesion sampling that is an alternative to incisional biopsy is cytological scraping. MicroRNA alterations are also being explored as a means of diagnosing carcinoma as an alternative to histopathology. METHODS: We obtained cytological scrapings using 10 strokes ('light') or 40 strokes ('heavy') from the buccal mucosa of one healthy subject using a dermatological curette. MicroRNA was isolated from oral cytological scrapings immediately, or the scrapings were stored in buffer or RNA later, at 4°C, room temperature or 36°C, from 1 to 7 days prior to RNA isolation. All scrape comparisons and test conditions were conducted in triplicate. MicroRNAs were measured using qRT-PCR. RESULTS: MicroRNAs can be obtained from cytological scrapings independent of the number of strokes and can be measured using qRT-PCR after storage under all conditions tested. CONCLUSION: MicroRNAs are robust to a wide range of storage conditions that bodes well for use of cytological scrapings to be of use in a clinical setting as a chair side sampling method for suspect oral lesions.