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Hovedbudskap Digital patologi gir nye muligheter for undervisning i oral patologi Histopatologiske vevssnitt kan vises uten bruk av mikroskop Studentene kan laste opp vevssnittene på egen datamaskin Gir bedre muligheter for integrert undervisning med kliniske fag som oral kirurgi og oral medisin

Background Tumor immune infiltrate has been explored in oral squamous cell carcinoma (OSCC), but studies on simultaneous characterization of multiple immune cell subtypes separately in stromal and intraepithelial tumor compartments are limited. Objectives We aimed to investigate the immune cell infiltrate in OSCC by using immunohistochemistry (IHC) for a panel of inflammatory cells in stromal and epithelial tumor compartments for a better characterization of the tumors. Methods Thirty-six OSCC lesions and nine normal oral mucosa (NOM) samples from patients attending Khartoum Dental Teaching Hospital, Sudan were investigated for presence of tumor infiltrating lymphocytes, tumor-associated macrophages, tumor-associated neutrophils, and PD-L1 positive cells in the inflammatory infiltrate by single and double IHC. Digital quantitative analysis (Aperio Technologies Inc.) was performed separately for stromal and epithelial compartments. Results OSCC cases displayed a higher inflammatory infiltrate in the associated stroma, but not in the epithelial compartment when compared to NOM. The immunosuppressive type of inflammatory infiltrate, that is, T regulatory cells (FoxP3+ cells) was identified to be significantly higher in the epithelial compartment of tumors with advanced clinical state. An immunoscore developed by combining intraepithelial FoxP3+ and CD4+ cells was found significantly higher in lesions from elderly patients, localized at toombak dipping-related sites, poorly differentiated OSCCs, or with loco-regional lymph node spreading. Conclusions Despite heavy immune cell infiltration in tumor-associated stroma, the majority of OSCCs in this cohort displayed a low intraepithelial immune infiltration. An immunoscore based on combined CD4 and FoxP3 intraepithelial expression may serve as an indicator of advanced tumor progression and should be further investigated for its use as potential prognostic biomarker in OSCC. publishedVersion

Hovedbudskap Bruken av snus er økende i Norge, særlig blant yngre mennesker Kasuspresentasjonene viser mukogingivale forandringer og gingivale retraksjoner som ofte er observert hos pasienter. Det er viktig å være klar over de orale forandringene som kan skje i forbindelse med bruken av snus og anbefale pasientene å slutte.

Tissue engineering for fibrocartilage regeneration using mesenchymal stromal cells (MSC) and biomaterial scaffolds is emerging as a promising strategy, but inhibiting vascularization to prevent endochondral ossification is important to develop stable implants. The objective of this study was to investigate the effect of angiostatin on inhibition of angiogenesis and promotion of chondrogenesis by collagen scaffolds with or without MSC implanted subcutaneously in rats. One scaffold from the following groups was implanted in each animal: Collagen scaffolds only, scaffolds functionalized with angiostatin, scaffolds loaded with MSC and scaffolds functionalized with angiostatin and loaded with MSC. The various scaffolds were harvested after 2 and 8 weeks for histological analysis, Real-time quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence quantification. Results demonstrated significantly decreased expression of inflammatory (interleukin 1 alpha and beta) and angiogenic genes (platelet and endothelial cell adhesion molecule 1) in scaffolds functionalized with angiostatin after 2 weeks in vivo. Histologically, after 8 weeks, the scaffolds with angiostatin had less inflammatory cells and more collagen matrix formation, but no fibrocartilage formation was detected. Thus, although angiostatin suppressed angiogenesis, it did not stimulate ectopic chondrogenesis in tissue engineered constructs in vivo.

The cemented Spectron EF stem in combination with the cemented non-crosslinked Reflection All-Poly cup showed a high rate of mid-term aseptic loosening. However, the failure mechanisms are not fully known. We assessed the inflammatory tissue reactions and wear particles in periprosthetic tissues, implant wear and blood metal ion levels in 28 patients with failed implants. Histological analysis showed a macrophage pre-dominant pattern with randomly distributed lymphocytes, with various amounts of neutrophils and giant cells. The number of different cell types in the tissue samples from patients in the cup group and in the stem group was similar. Wear particles, mainly ZrO2, CoCrMo, and polyethylene particles of different sizes and shapes, were associated with macrophages/giant cells, and total particle load/mm2 was higher in cases of stem loosening. The Spectron EF stems were heavily worn, abraded, and polished. Stem abrasion correlated with metal ion concentrations in blood. The median polyethylene wear rate of the Reflection cups was 0.23 mm/year. The high proximal roughness of the Spectron EF stem resulted in excessive cement wear during loosening. The resulting inflammatory tissue responses to the degradation products both from the cup and the stem led to massive osteolysis and subsequent implant loosening. publishedVersion

BackgroundRecent studies have shown aberrant expression of micro-RNAs in cancer-associated fibroblasts (CAFs). This study aimed to investigate miR-138 dysregulation in CAFs in oral squamous cell carcinoma (OSCC) and its effects on their phenotype and invasion of adjacent OSCC cells.MethodsExpression of miR-138 was first investigated in OSCC lesions (n = 53) and OSCC-derived CAFs (n = 15). MiR-138 mimics and inhibitors were used to functionally investigate the role of miR-138 on CAF phenotype and the resulting change in their ability to support OSCC invasion.ResultsExpression of miR-138 showed marked heterogeneity in both OSCC tissues and cultured fibroblasts. Ectopic miR-138 expression reduced fibroblasts’ motility and collagen contraction ability and suppressed invasion of suprajacent OSCC cells, while its inhibition resulted in the opposite outcome. Transcript and protein examination after modulation of miR-138 expression showed changes in CAF phenotype-specific molecules, focal adhesion kinase axis, and TGFβ1 signaling pathway.ConclusionsDespite its heterogeneous expression, miR-138 in OSCC-derived CAFs exhibits a tumor-suppressive function.

HovedbudskapPyogent (telangiektatisk) granulom (PG) og granuloma gravidarum (GG) er lesjoner som oftest oppstår på grunn av lokale irritamenter eller kroniske traumer.De fleste finnes på marginale gingiva, men de kan være lokalisert til alle intraorale slimhinner.De er som regel røde av farge, med delvis gul overflate. Det gule representerer fibrindekket ulcerasjon.Klinisk kan lesjonene lett forveksles med perifert kjempecellegranulom og perifert ossifiserende fibrom, og derfor er histologisk undersøkelse er et absolutt krav.Behandlingen er kirurgisk excisjon.

Hovedbudskap Metastaser til kjever og munnlimhinne forekommer sjelden De fleste metastasene til «vårt» område viser seg oftest intraossøst i underkjeven og kan sees på røntgen som radiolucenser /osteolyse De forekommer meget sent i en kreftsykdom Ett av de viktigste symptomene er anestesi av underleppe/hake Hvor ofte slik diagnose stilles, var ukjent for Kreftregisteret

Simple Summary In addition to the transformation of epithelial cells, dysfunction of stroma is crucial in carcinogenesis; cancer-associated stroma can regulate the phenotype of cancer cells and thereby influence the clinical outcome. Our study aimed to investigate the correlation of stromal miR-204 with progression of oral squamous cell carcinoma (OSCC) and assert its clinical utility. We first established a chromogen-based method that combined immunohistochemistry and in situ hybridization for exact delimitation of stroma from the tumor islands and concomitant visualization of miRs, and have developed a guide to digital miR quantification using the publicly available tool ImageJ and the licensed software Aperio ImageScope. We have then applied the method for investigating stromal miR-204 as a putative prognostic biomarker on an OSCC cohort and identified expression of miR204 in the stroma at tumor front as an independent prognostic biomarker for this disease. Abstract Micro-RNAs (miRs) are emerging as important players in carcinogenesis. Their stromal expression has been less investigated in part due to lack of methods to accurately differentiate between tumor compartments. This study aimed to establish a robust method for dual visualization of miR and protein (pan-cytokeratin) by combining chromogen-based in situ hybridization (ISH) and immunohistochemistry (IHC), and to apply it to investigate stromal expression of miR204 as a putative prognostic biomarker in oral squamous cell carcinoma (OSCC). Four different combinations of methods were tested and ImageJ and Aperio ImageScope were used to quantify miR expression. All four dual ISH-IHC methods tested were comparable to single ISH in terms of positive pixel area percentage or integrated optical density of miRs staining. Based on technical simplicity, one of the methods was chosen for further investigation of miR204 on a cohort of human papilloma virus (HPV)-negative primary OSCC (n = 169). MiR204 stromal expression at tumor front predicted recurrence-free survival (p = 0.032) and overall survival (p = 0.036). Multivariate Cox regression further confirmed it as an independent prognostic biomarker in OSCC. This study provides a methodological platform for integrative biomarker studies based on simultaneous detection and quantification of miRs and/or protein and reveals stromal miR204 as a prognostic biomarker in OSCC.

Background: Oral squamous cell carcinoma (OSCC) is increasing at an alarming rate particularly in low-income countries. This urges for research into noninvasive, user-friendly diagnostic tools that can be used in limited-resource settings. This study aims to test and validate the feasibility of e-nose technology for detecting OSCC in the limited-resource settings of the Sudanese population. Methods: Two e-nose devices (Aeonose™, eNose Company, Zutphen, The Netherlands) were used to collect breath samples from OSCC (n = 49) and control (n = 35) patients. Patients were divided into a training group for building an artificial neural network (ANN) model and a blinded control group for model validation. The Statistical Package for the Social Sciences (SPSS) software was used for the analysis of baseline characteristics and regression. Aethena proprietary software was used for data analysis using artificial neural networks based on patterns of volatile organic compounds. Results: A diagnostic accuracy of 81% was observed, with 88% sensitivity and 71% specificity. Conclusions: This study demonstrates that e-nose is an efficient tool for OSCC detection in limited-resource settings, where it offers a valuable cost-effective strategy to tackle the burden posed by OSCC. publishedVersion

Hovedbudskap Perifert ossifiserende fibrom (POF) er en lesjon som oftest er lokalisert til marginale gingiva Utveksten kan klinisk være vanskelig å skille fra pyogent (telangiektatisk) granulom og perifert kjempecellegranulom Vanskeligheten ligger i at bendannelsen ikke fremkommer på et vanlig intraoralt røntgenbilde Lesjonens farge kan være rød med tendens til ulcerasjon eller ha rosa farge som normal gingiva Lesjonen har residivtendens og bør kontrolleres hos egen tannlege

Simple Summary This study aimed at exploring the modulations of p53 in cell toxicity induced by an extract of the herb khat, a natural stimulant used by millions of people. We previously reported that khat-extract induced cell death by affecting mitochondrial function and the receptor- and mitochondria-mediated cell death pathways, in leukemic cell lines and cells of the oral cavity, in vitro. We included the cancer therapeutic camptothecin, which induces apoptosis in various cancer cell lines. By studying modulations of p53 full-length protein and p53 β/γ isoforms following exposure to khat-extract and camptothecin, we wished to elucidate differences and similarities resulting from the treatments using MOLM-13 and MV-4-11 leukemic cell lines. Our results demonstrate that molecular effects of the cytotoxic treatments resulted in different p53 isoforms patterns and post-translational modifications. We suggest that analysis of p53 modulations could be useful in the search for new chemical probes and experimental cancer therapeutics. Abstract Khat (Catha edulis (Vahl) Forssk. ex Endl.) is habitually used as a natural stimulant by millions of people, but is associated with adverse effects on gastrointestinal, cardiovascular and central neural systems. At the cellular level khat toxicity involves p53 induction and cell cycle arrest, decreased mitochondrial function and activation of receptor- and mitochondria-mediated cell death pathways. In this study we have examined an extract of khat for induction of p53 post-translational modifications (PTMs) and the functional role of p53 in khat-mediated cell death. Khat was shown to induce phosphorylation and acetylation of p53 in both the khat-sensitive MOLM-13 and the khat-resistant MV-4-11 cell line, but accumulation of the full-length p53 isoform was only observed in the khat sensitive cell line. Small molecule inhibitors of p38 MAP kinase sensitized MV-4-11 cells for khat-treatment without concomitant stabilization of p53. Experiments using a p53 knock-down cell line and murine p53 knock-out bone marrow cells indicated that p53 was redundant in khat-mediated cell death in vitro. We suggest that analysis of isoform patterns and p53 PTMs are useful for elucidation of biological effects of complex plant extracts, and that p53 protein analysis is particularly useful in the search for new chemical probes and experimental cancer therapeutics.

Hovedbudskap Perifert kjempecellegranulom (PKCG) er lokalisert til marginale gingiva Lesjonen kan være vanskelig klinisk å skille fra pyogent (teleangiektatisk) granulom og perifert ossifiserende fibrom En blålig farge kan være til hjelp i diagnostiseringen Der er residivtendens slik at postoperative kontroller er påkrevet, vanligvis hos egen tannlege Lesjonene kan forekomme ved implantater og er ofte forbundet med bennedbrytning slik at implantatet kan gå tapt

Helicobacter pylori (HP) infection is an established causative agent for gastric cancer. Although the oral cavity is a part of the gastrointestinal system, the presence and possible causative role of HP in oral squamous cell carcinoma (OSCC) is a subject of controversy. Therefore, the current study aimed to investigate HP infection in two cohorts of OSCC patients with different demographic characteristics, lifestyles and habitual risk factors. A total of 242 formalin-fixed paraffin-embedded OSCC specimens from two different patient cohorts (Norway, n = 171 and Nepal, n = 71) were used to examine HP using immunohistochemistry (IHC) and quantitative polymerase chain reaction (qPCR). Two different HP specific genes (23S rRNA and ureA) were used for TaqMan-based qPCR, and for subsequent verification using HP specific RIDAGENE HP kit and SYBR Green based qPCR. All of the OSCC specimens from both cohorts were found to be negative for HP infection with IHC and qPCR, although the positive control specimens tested positive. Our findings suggest that HP is absent in the examined OSCC cohorts, irrespective of race, lifestyle and habitual risk factors. This indicates that, in contrast to gastric cancer, HP is an unlikely contributing factor for OSCC pathogenesis.

Background - We previously showed a tumor‐suppressive function of S100A14 in oral squamous cell carcinoma (OSCC). This study aimed to examine the prognostic significance and differentiation‐related function of S100A14 in OSCC. Methods - S100A14 expression was examined in 170 OSCCs from Norwegian and Nepalese populations using immunohistochemistry. Pro‐differentiation function was investigated by overexpressing and silencing S100A14 expression in OSCC‐derived cells. External transcriptomic datasets were used to validate association between S100A14 and differentiation markers in OSCC. Result - Loss of S100A14 expression at the invading tumor fronts significantly correlated with poor differentiation and reduced 10‐years survival of OSCC‐patients. Multivariate Cox analysis identified S100A14 to be an independent prognostic factor. Modulation of S100A14 expression in OSCC‐derived cells positively correlated with the expression of differentiation markers. Analysis of external datasets supported the pro‐differentiation function of S100A14. Conclusion - These results indicate that S100A14 is a pro‐differentiation protein and its expression might be useful as a prognostic marker in OSCC.

Background - Numerous studies have been presented on histological grading of oral squamous cell carcinomas (OSCC) for predicting survival, but uncertainty of their usefulness rises due to discordances of results. A scoring system should be robust and well validated, and intra‐ and interrater agreement can be used as a tool to visualize the strength of reproducibility. Methods - Here, we present an intra‐ and inter‐observer study on evaluation of OSCC using some of the most common histopathological parameters. The observers were from different Norwegian university hospitals, and calibration to ensure accuracy was first performed. Percentage of the agreement was calculated for the score made by the individual observer at different times, as well as between pairs of observers. Results - The evaluation made by the same observer at two different time points (intrarater) correlated better than observations made by different participants (interrater). In an attempt to increase the rate of agreement, many of the parameters were either dichotomized into simply low‐ and high grade, or to a three‐tier system when more than three options in the original design. This increased the concurrence with 15.4% for the intrarater and with 23% for the interrater comparisons. Conclusion - High agreement for histopathological parameters can be difficult to obtain on hematoxylin and eosin staining in scoring systems with many options. A simpler system might be more advantageous to achieve higher degree of reproducibility.

Vulvar squamous cell carcinoma associated with lichen sclerosus (VLS-VSCC) are rare tumors but with higher recurrence and worse prognosis than other types of VSCC. Lack of experimental models has limited the search for better understanding of the biology and development of treatment modalities. In this study, we isolated and characterized primary cells from VSCC (n = 7) and normal vulvar tissue adjacent to tumor (n = 7). Detailed characterization of the novel spontaneously immortalized cell line, VCC1 revealed a characteristic epithelial morphology in vitro and a well-differentiated keratinizing SCC histology in vivo, closely resembling the tumor of origin. VCC1 expressed higher levels of epithelial-mesenchymal transition markers and higher clonogenic properties as compared to other established non VLS-VSCC cell lines. In vitro 3D organotypic assays and in vivo xenografts revealed a prominent role of cancer-associated fibroblasts in VCC1 invasion and tumor formation. In conclusion, VCC1 mirrored several major VLS-VSCC features and provided a robust experimental tool for further elucidation of VLS-related oncogenesis and drug testing. publishedVersion

Background Oral squamous cell carcinoma (OSCC) is the sixth leading malignancy worldwide. The incidence of OSCC has increased in recent years. Tumor-associated macrophages have been shown to promote tumor progression by influencing tumor invasion, migration, and angiogenesis. So far, no study investigating different biomarkers in cohorts of patients with OSCC from Armenia has been published. Objective The aim of this study was to evaluate the role of tumor-associated macrophages in the prognosis of OSCC and investigate the association between the expression of Ki67 and p53 biomarkers and its relation with clinicopathologic parameters (tumor stage and grade) in a cohort of patients with OSCC from Armenia. Methods Available samples from patients diagnosed with OSCC in 2017/2018 were collected from Armenian hospitals after ethics approval (n = 13). Immunohistochemistry was performed using Visualization System and EnVision Double stain System (DAKO) for Ki67, p53, and CD163/CD68 biomarkers. Stained tissues were scanned and quantified using Image Scope software (Aperio Technologies Inc.) and QuPath v0.2.0-m9 (OSS for DPI analysis). Data were analyzed using SPSS 25.0. Results Intense nuclear expression of p53 was identified in 69.2% cases but not strongly correlated with a high proliferation index (>50% Ki67 positive cells; P = .125) and M2 type macrophages expression (P = .308). In contrast, M2 type macrophages count was correlated with tumor stage (P = .026). Conclusions This preliminary study identified a positive correlation between increased expression of M2 type macrophages and OSCC stage. This should be further investigated in a larger cohort of patients.

Khat (Catha edulis (Vahl) Forssk. ex Endl.) is habitually used as a natural stimulant by millions of people, but is associated with adverse effects on gastrointestinal, cardiovascular and central neural systems. At the cellular level khat toxicity involves p53 induction and cell cycle arrest, decreased mitochondrial function and activation of receptor- and mitochondria-mediated cell death pathways. In this study we have examined an extract of khat for induction of p53 post-translational modifications (PTMs) and the functional role of p53 in khat-mediated cell death. Khat was shown to induce phosphorylation and acetylation of p53 in both the khat-sensitive MOLM-13 and the khat-resistant MV-4-11 cell line, but accumulation of the full-length p53 isoform was only observed in the khat sensitive cell line. Small molecule inhibitors of p38 MAP kinase sensitized MV-4-11 cells for khat-treatment without concomitant stabilization of p53. Experiments using a p53 knock-down cell line and murine p53 knock-out bone marrow cells indicated that p53 was redundant in khat-mediated cell death in vitro. We suggest that analysis of isoform patterns and p53 PTMs are useful for elucidation of biological effects of complex plant extracts, and that p53 protein analysis is particularly useful in the search for new chemical probes and experimental cancer therapeutics. publishedVersion

This study aimed to evaluate the tumorigenic potential of functionalising poly(LLA-co-CL) scaffolds. The copolymer scaffolds were functionalised with nanodiamonds (nDP) or with nDP and physisorbed BMP-2 (nDP-PHY) to enhance osteoinductivity. Culturing early neoplastic dysplastic keratinocytes (DOK\(^{Luc}\)) on nDP modified scaffolds reduced significantly their subsequent sphere formation ability and decreased significantly the cells' proliferation in the supra-basal layers of in vitro 3D oral neoplastic mucosa (3D-OT) when compared to DOK\(^{Luc}\) previously cultured on nDP-PHY scaffolds. Using an in vivo non-invasive environmentally-induced oral carcinogenesis model, nDP scaffolds were observed to reduce bioluminescence intensity of tumours formed by DOK\(^{Luc}\) + carcinoma associated fibroblasts (CAF). nDP modification was also found to promote differentiation of DOK\(^{Luc}\) both in vitro in 3D-OT and in vivo in xenografts formed by DOKLuc alone. The nDP-PHY scaffold had the highest number of invasive tumours formed by DOK\(^{Luc}\) + CAF outside the scaffold area compared to the nDP and control scaffolds. In conclusion, in vitro and in vivo results presented here demonstrate that nDP modified copolymer scaffolds are able to decrease the tumorigenic potential of DOK\(^{Luc}\), while confirming concerns for the therapeutic use of BMP-2 for reconstruction of bone defects in oral cancer patients due to its tumour promoting capabilities.

A 46-year-old woman with biopsy-verified pulmonal sarcoidosis presented with submucosal noduli of the lower lip ([figure 1][1]). The noduli had, according to the patient, first appeared 6 months earlier. The lip was tender to traumatic biting and varied in size. The lesion and nearby small

The epithelial rests of Malassez (ERMs) might represent a valuable source of oral epithelial cells with stem cell properties. The purpose of this study was to isolate and characterize cells derived from human ERM, and compare them with cells derived from matched normal oral mucosa (NOM). Matched tissue specimens of the periodontal ligament of extracted tooth and NOM were collected. Cells were isolated in culture, then characterized by immunohistochemistry and flow cytometry for expression of pancytokeratin, ESA, PDGFRB, CD31 and CD44. 3D organotypic cultures were constructed by growing epithelial cells on top of fibroblast-populated collagen gels. Both ERM and NOM-isolated cells expressed the markers of epithelial lineage (ESA and pancytokeratin), and to some extent PDGFR, an indicator of a more mesenchymal phenotype, but not the endothelial cell marker CD31. Cells with epithelial morphology were isolated from periodontium of cervical, middle and apical parts of the root, but contained a significantly lower percentage of ESA and pancytokeratin-positive cells than when isolating cells from NOM (p

Background Microenvironmental cues play a major role in head and neck cancer. Biodegradable scaffolds used for bone regeneration might also act as stimulative cues for head and neck cancer. The purpose of this study was to establish an experimental model for precise and noninvasive evaluation of tumorigenic potential of microenvironmental cues in head and neck cancer. Methods Bioluminescence was chosen to image tumor formation. Early neoplastic oral keratinocyte (DOK) cells were luciferase-transduced (DOKLuc), then tested in nonobese diabetic severe combined immunodeficient IL2rγnull mice either orthotopically (tongue) or subcutaneously for their potential as “screening sensors” for diverse microenvironmental cues. Results Tumors formed after inoculation of DOKLuc were monitored easier by bioluminescence, and bioluminescence was more sensitive in detecting differences between various microenvironmental cues when compared to manual measurements. Development of tumors from DOKLuc grown on scaffolds was also successfully monitored noninvasively by bioluminescence. Conclusion The model presented here is a noninvasive and sensitive model for monitoring the impact of various microenvironmental cues on head and neck cancer in vivo. © 2015 The Authors Head & Neck Published by Wiley Periodicals, Inc. Head Neck, 2015

Background: Although several approaches for identification and isolation of carcinoma cells with tumour initiating properties have been established, enrichment of a population of pure and viable tumour-initiating cells (TICs) is still problematic. This study investigated possibilities to isolate a population of cancer cells with tumour initiating properties based on their adherence properties, rather than expression of defined markers or clonogenicity. Methods: Several human cell lines derived from oral dysplasia and oral squamous cell carcinoma (OSCC), as well as primary cells derived from patients with OSCC were allowed to adhere to collagen IV-coated dishes sequentially. Rapid adherent cells (RAC), middle adherent cells (MAC) and late adherent cells (LAC) were then harvested and further investigated for their morphology, stem cell-like properties and molecular profile while grown in vitro and tongue xenotransplantation in NOD-SCID mice at serial dilutions. Results: RAC showed significantly higher colony forming efficiency (p < 0.05), sphere forming ability, greater migration ability (p < 0.05), exhibited longer G2 phase and displayed higher expression of integrin b1 and other stem-cell related molecules as compared to MAC and LAC. RAC induced tongue tumours in NOD-SCID mice with the highest incidence. These tumours were also bigger and metastasised more frequently in loco-regional lymph nodes than MAC and LAC. Conclusions: These findings prove for the first time that OSCC cells with tumour initiating properties can be enriched based on their rapid adhesiveness to collagen IV. This separation procedure provides a potentially useful tool for isolating TICs in OSCC for further studies on understanding their characteristics and drug-resistant behaviour. publishedVersion

Objective Hyperbaric oxygen therapy (HBOT) has been used to enhance microcirculation and thereby oxygen tension in tissues. The present study aimed to investigate the effect of HBOT on radiation injury in the mandibular area of rats. Study design The left mandibles of rats were irradiated by external radiotherapy (15 Gy every other week for a total of 75 Gy). Four HBOT strategies were used: 2 prophylactic groups receiving HBOT either between each radiation treatment or immediately following terminated radiation treatment, and 2 therapeutic groups receiving HBOT after the latent period of 6 weeks after irradiation either every day (standard HBOT protocol) or 3 days a week for 6 weeks. Tissue samples of the irradiated area were taken from skin, the salivary gland, and the mandible. All tissues were stained with hematoxylin and eosin for morphologic examination. Furthermore, skin samples were stained with CD31 for blood vessel analysis. Results There was no change in blood vessel density or morphology between controls and HBOT tissues after radiation. The dentin of 2 of the 5 rats that received HBOT either normalized or was not affected by irradiation. Conclusions HBOT did not affect radiation injury of the mandibular area in rats within 12 weeks after irradiation.

Background Cancer-associated fibroblasts (CAFs) were shown to be important for tumour progression in head and neck squamous cell carcinomas (HNSCCs). Their heterogeneity and lack of specific markers is increasingly recognized. Integrin α11 was recently shown to be expressed by CAFs and might serve as a specific CAF marker. Aim To investigate integrin α11 expression and its correlation with the expression of a well-known marker of CAF, alpha smooth muscle actin (α-SMA), in HNSCC. Methods Fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) samples from healthy volunteers (n = 24), oral lichen planus (OLP) (n = 32) and HNSCC (n = 106) were collected together with clinical data after ethical approval. Immunohistochemistry to detect integrin α11 and α-SMA was performed on FF and FFPE samples. qPCR for integrin α11 (ITGA11) and α-SMA (ACTA2) was performed on FF samples. Data were analysed using chi-square test and Kaplan–Meier survival analysis. Results Significantly higher levels of integrin α11 and α-SMA at both protein and mRNA levels were found in HNSCC vs. normal controls and OLP. A strong correlation was found between integrin α11 and α-SMA expression, and double staining showed their colocalization. Both integrin α11 and α-SMA were detected surrounding metastatic islands. Expression of α-SMA at tumour front but not tumour centre correlated with patient survival. Conclusion Integrin α11 was overexpressed in HNSCC stroma and colocalized with α-SMA. Expression of α-SMA at tumour front but not tumour centre had prognostic value for survival, pinpointing the importance of assessing tumour front when evaluating stromal molecules as prognostic biomarkers.

In Scandinavia, as in many European countries, most patients consult their general dentist once a year or more. This gives the dentist a unique opportunity and an obligation to make an early diagnosis of oral diseases, which is beneficial for both the patient and the society. Thus, the dentist must have knowledge of clinical symptoms, local and systemic signs and clinical differential diagnoses to make an accurate diagnosis. The dentist must be competent in selecting appropriate diagnostic tests, for example, tissue biopsy and microbiological samples, and conducting them correctly, as well as in interpreting test results and taking appropriate action accordingly. Furthermore, the dentist must be aware of diseases demanding multidisciplinary cooperation and be able to recognise his/her professional limitation, and to refer to other specialists when required. The dental curriculum changes over time as new approaches, treatments and diagnostic possibilities develop. Likewise, the role of the dentist in the community changes and may vary in different countries. As members of the Scandinavian Fellowship for Oral Pathology and Oral Medicine and subject representatives of oral pathology and oral medicine, we feel obliged to contribute to the discussion of how the guidelines of the dental curriculum support the highest possible standards of dental education. This article is meant to delineate a reasonable standard of oral pathology and oral medicine in the European dental curriculum and to guide subject representatives in curriculum development and planning. We have created an advisory topic list in oral pathology and oral medicine.

J Oral Pathol Med (2010) 39 128–140 Background: The high incidence of oral cancer in Sudan has been associated with the use of toombak, the local type of smokeless tobacco. However, its specific effects on human oral cells are not known. We aimed to investigate the effects of toombak on primary normal human oral keratinocytes, fibroblasts, and a dysplastic oral keratinocytic cell line, and to compare them with the effects induced by Swedish snuff. Method: Aqueous extracts were prepared from moist toombak and Swedish snuff and added in serial dilutions on in vitro monolayer cultured cells. Cell viability, morphology and growth, DNA double-strand breaks (γH2AX staining), expression of phosphatidylserine (Annexin V staining), and cell cycle were assessed after various exposure time periods. Results: Significant decrease in cell number, occurrence of DNA double-strain breaks, morphological and biochemical signs of programmed cell death were detected in all oral cell types exposed to clinically relevant dilutions of toombak extract, although to a lesser extent in normal oral fibroblasts and dysplastic keratinocytes. G2/M-block was also detected in normal oral keratinocytes and fibroblasts exposed to clinically relevant dilutions of toombak extract. Swedish snuff extract had less adverse effects on oral cells, mainly at non-clinically relevant dilutions. Conclusion: This study indicates a potential for toombak, higher than for Swedish snuff, to damage human oral epithelium. Dysplastic oral keratinocytes were less sensitive than their normal counterparts, suggesting that they might have acquired a partially resistant phenotype to toombak-induced cytotoxic effects while still being prone to DNA damage that could lead to further malignant progression.

Khat-chewing has been associated with oral lesions including oral cancer, but the mechanisms leading to their development are not known. We hypothesized that khat interferes with the physiological processes of the oral mucosa, such as cell proliferation and differentiation, and aimed at investigating the effects of khat exposure on in vitro-reconstructed human normal buccal mucosa. Khat decreased cell proliferation, epithelial thickness, and cytokeratin 13 expression, while inducing premature expression of p21Waf1/Cip1, transglutaminases, involucrin, and filaggrin. This suggests that khat is able to induce abnormal differentiation of the buccal epithelium. Khat-induced alterations were accompanied by increased levels of p38 and were reversed by p38 inhibition, pointing to p38 as the key player in this process. The morphological changes described herein mirror the in vivo changes previously described in khat users, and demonstrate for the first time that khat induces pathological alterations in human buccal mucosa, providing evidence that raises concerns about the effects of khat use on oral health.

Khat chewing is widely practiced in Eastern Africa and the Middle East. Khat is genotoxic to cells within the oral mucosa, and several studies have suggested an association between khat use and oral lesions like hyperkeratosis and oral cancer. This study investigated the mechanism of khat-induced cytotoxicity using primary normal human oral keratinocytes (NOK) and fibroblasts (NOF). Khat induced rounding up of cells, plasma membrane blebbing, and condensation of nuclear chromatin within 3-6 h of exposure. The cells also showed externalization of phosphatidylserine and fragmentation of DNA. Morphological and biochemical features were compatible with cell death by apoptosis. Khat also induced an increase in cytosolic reactive oxygen species (ROS) and a depletion of intracellular glutathione (GSH) within 1 h of exposure. Antioxidants reduced ROS generation, GSH depletion and delayed the onset of cytotoxicity in both cell types. Generally, NOF cells were more sensitive to khat-induced cytotoxicity than NOK cells. These effects were elicited at concentrations of khat expected to occur in the oral cavity during khat chewing. In summary, khat induced apoptotic cell death in primary normal oral keratinocytes and fibroblasts by an early effect on mechanisms that regulate oxidative stress.

Khat is a psychostimulant plant used by over 10 million people daily, mainly in eastern Africa and the Middle East. Previous studies have suggested an association between khat use and oral lesions such as hyperkeratosis and oral cancer. This study investigated the effects of an extract of khat on primary normal human oral keratinocytes (NOK) and normal human oral fibroblasts (NOF). Low (sublethal) concentrations of khat inhibited the proliferation of both cell types in a dose-dependent and time-dependent manner. Both NOK and NOF treated with khat accumulated in the G1-phase of the cell cycle and showed increased expression of the stress-sensitive p53 protein after 24 h. Normal human oral keratinocytes showed a profound increase in p16(INK4A) (p16) after 24 h and showed morphological changes suggesting cell differentiation. Normal human oral fibroblasts showed growth inhibition and increased expression of p21(WAF1/CIP1) (p21) within 24 h. The concentrations of khat tested in this study were within the range of those found in the oral cavity of khat chewers. The results show that stress induced by khat modulates the cell cycle in oral keratinocytes and fibroblasts. It is further speculated whether khat could have similar effects in vivo, especially in keratinocytes.

To estimate the point prevalence of primary Sjögren's syndrome (pSS) in two populations, aged 40-44 and 71-74 years, using two sets of classification criteria.The participating individuals were recruited from the Hordaland Health Study (HUSK) conducted during 1997-99. A total of 18 592 individuals born 1953-57 and 3346 individuals born 1925-27 were sent a questionnaire covering various health-related questions, including four questions about sicca symptoms. Among those answering positive to at least one of the four questions, 99 and 90 individuals born 1953-57 and 1925-27, respectively, were examined further. For diagnosis of pSS two classifications were used, the preliminary European criteria from 1993, and the revised European criteria from 1996.By using the two classification criteria from 1993 and 1996, the point prevalences were 0.44% [95% confidence interval (CI) 0.34-0.57] and 0.22% (95% CI 0.15-0.32), respectively, for the population group born 1953-57. The corresponding estimates were 3.39% (95% CI 2.77-4.14) and 1.40% (95% CI 1.02-1.92) for the population born 1925-27.The point prevalence of pSS was approximately seven times higher in the elderly population aged 71-74 years compared to individuals aged 40-44 years, regardless of the classification criteria used.

Sodium lauryl sulphate (SLS) is a common detergent known to cause irritation and inflammatory reactions in skin. SLS is also the most commonly used toothpaste detergent and has been related to intraoral adverse effects. However, its specific biological effects on the oral mucosa (OM) have not yet been identified. The objective of this study was to investigate the putative effects of SLS on human oral epithelium using a novel in vitro reconstructed three-dimensional cell culture model. Reconstructed human OM, generated from primary normal human oral keratinocytes and fibroblasts, was exposed to clinically relevant concentrations of SLS (range 0.015-1.5%). The cultured tissues were evaluated by histomorphometry, immunohistochemistry (Ki-67, epithelial (E)-cadherin, alpha6-, beta1-integrins, cleaved caspase-3) and the TUNEL method. Increased epithelial thickness, enhanced proliferation (Ki-67), a more pronounced expression of E-cadherin throughout all epithelial cell layers and single TUNEL-positive cells in the middle spinous cell layers were observed in cultures exposed to low concentrations (0.015%) of SLS. At exposure to higher SLS concentrations (>or=0.15%), epithelial thickness, cell proliferation and E-cadherin expression gradually decreased and in the central areas of exposed regions, cells detached from each other and underwent cell death. In conclusion, clinically relevant concentrations of SLS have dual effects on reconstituted human OM; although occasional cell death within the epithelium was also observed, the increased epithelial thickness, proliferation and E-cadherin expression induced at lower concentrations might be associated with a protective mucosal response, whereas at higher concentrations a more destructive type of reaction predominated.

Oral lichen planus (OLP) may be associated with a small risk of malignant transformation of the oral mucosa. Using cases which had transformed, and those which had not, this study aimed to evaluate the potential of DNA content, expression of Cyclooxygenase-2 (Cox-2) and of epithelial (E)-cadherin as risk markers in lesions of OLP. We investigated 78 archival biopsies from; (1) 26 OLP patients with at least two biopsies, of whom seven presented OLP with epithelial dysplasia, followed by oral squamous cell carcinoma (OSCC) in five of them, (2) 19 OLP patients with one biopsy taken. Image cytometry for measurement of DNA content and immunohistochemistry for visualisation of Cox-2 and E-cadherin expression were performed. All OLP biopsies investigated were classified as diploid, one OLP with epithelial dysplasia was tetraploid and all OSCC were diploid. Cox-2 was detected in the epithelium of all OLP specimens investigated, as well as in epithelial dysplasias and OSCC. Focal loss of E-cadherin expression was observed in basal keratinocytes in 88% of the OLP specimens investigated, in all epithelial dysplasias and OSCC. In conclusion, neither aneuploidy, Cox-2 expression, nor loss of E-cadherin expression, were significant reliable markers to select OLP lesions at risk for development of OSCC in the present patient material.

Apoptosis and the expression of Fas receptor (Fas) and Fas ligand (FasL) were studied in 8 samples of normal oral mucosa (OM) and in 19 oral squamous cell carcinomas (OSCC) by immunohistochemistry and the TUNEL method. Fas was detected in less than 2% of cells in OSCC compared with 84.3+/-9.0% of cells in the basal layer in OM. FasL was found to be highly expressed in poorly differentiated lesions (90.9+/-3.6%), and in cells of both the basal (88.3+/-4.3%) and central (85.3+/-5.7%) parts of moderately differentiated lesions, whereas in well-differentiated (WD) lesions expression was considerably lower in both basal (42.7+/-4.1%) and central (11.5+/-2.4%) parts. In normal OM FasL was primarily detected in cells of the basal layer, but in a high proportion of cells (84.9+/-4.3%). Apoptotic cell death was greater in OSCC (1.6+/-0.6%) than in OM (0.6+/-0.2%, P

Background Altered expression of S100A16 has been reported in human cancers, but its biological role in tumorigenesis is not fully understood. This study aimed to investigate the clinical significance and functional role of S100A16 in oral squamous cell carcinoma (OSCC) suppression. Methods S100A16 mRNA and/or protein levels were examined by quantitative RT-PCR and immunohistochemistry in whole- and laser microdissected-specimens of normal human oral mucosa (NHOM, n = 65), oral dysplastic lesions (ODL, n = 21), OSCCs (n = 132) and positive cervical nodes (n = 17). S100A16 protein expression in OSCC was examined for correlations with clinicopathological variables and patient survival. S100A16 was over-expressed and knocked-down in OSCC-derived (CaLH3 and H357) cells by employing retroviral constructs to investigate its effects on cell proliferation, sphere formation and three dimensional (3D)-organotypic invasive abilities in vitro and tumorigenesis in a mouse xenograft model. Results Both S100A16 mRNA and protein levels were found to be progressively down-regulated from NHOM to ODL and OSCC. Low S100A16 protein levels in OSCC significantly correlated with reduced 10-year overall survival and poor tumor differentiation. Analysis of two external OSCC microarray datasets showed a positive correlation between the mRNA expression levels of S100A16 and keratinocyte differentiation markers. CaLH3 and H357 cell fractions enriched for differentiated cells either by lack of adherence to collagen IV or FACS sorting for low p75NTR expression expressed significantly higher S100A16 mRNA levels than the subpopulations enriched for less differentiated cells. Corroborating these findings, retroviral mediated S100A16 over-expression and knock-down in CaLH3 and H357 cells led to respective up- and down-regulation of differentiation markers. In vitro functional studies showed significant reduction in cell proliferation, sphere formation and 3D-invasive abilities of CaLH3 and H357 cells upon S100A16 over-expression. These functional effects were associated with concomitant down-regulation of self-renewal (Bmi-1 and Oct 4A) and invasion related (MMP1 and MMP9) molecules. S100A16 over-expression also suppressed tumorigenesis of H357 cells in a mouse xenograft model and the resulting tumor xenografts displayed features/expression of increased differentiation and reduced proliferation/self-renewal. Conclusions These results indicate that S100A16 is a differentiation promoting protein and might function as a tumor suppressor in OSCC. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1622-1) contains supplementary material, which is available to authorized users.

Background Radiation technology focuses on delivering the radiation as precisely as possible to the tumor, nonetheless both acute and long-term damage to surrounding normal tissue may develop. Injuries to the surrounding normal tissue after radiotherapy of head and neck cancer are difficult to manage. An animal model is needed to elucidate good treatment modalities. The aim of this study was to establish a rat model where a certain radiation dose gives reproducible tissue reactions in the mandibular area corresponding to injuries obtained in humans. Method The left mandible of male Sprague Dawley rats was irradiated by external radiotherapy (single fraction 15 Gy, total dose 75 Gy) every second week five times. Endpoint was six weeks after last radiation treatment, and the test group was compared to non-irradiated controls. Morphological alterations of the soft tissues, bone and tooth formation, as well as alterations of salivation, vascularity and collagen content were assessed. An unpaired, non-parametric Mann–Whitney test was used to compare the statistical differences between the groups. Results Analysis of the soft tissues and mandible within the radiation field revealed severe unilateral alopecia and dermatitis of the skin, extensive inflammation of the submandibular gland with loss of serous secretory cells, hyperkeratinization and dense connective fiber bundles of the gingival tissue, and disturbed tooth development with necrosis of the pulp. Production of saliva and the vascularity of the soft tissues were significantly reduced. Furthermore, the collagen fibril diameter was larger and the collagen network denser compared to non-irradiated control rats. Conclusion We have established an animal model of radiation injury demonstrating physiological and histological changes corresponding to human radiation injuries, which can be used for future therapeutic evaluations. publishedVersion