Differential expression analysis identifies a prognostically significant extracellular matrix-enriched gene signature in hyaluronan-positive clear cell renal cell carcinoma.

Hyaluronan (HA) accumulation in clear cell renal cell carcinoma (ccRCC) is associated with poor prognosis; however, its biology and role in tumorigenesis are unknown. RNA sequencing of 48 HA-positive and 48 HA-negative formalin-fixed paraffin-embedded (FFPE) samples was performed to identify differentially expressed genes (DEG). The DEGs were subjected to pathway and gene enrichment analyses. The Cancer Genome Atlas Kidney Renal Clear Cell Carcinoma (TCGA-KIRC) data and DEGs were used for the cluster analysis. In total, 129 DEGs were identified. HA-positive tumors exhibited enhanced expression of genes related to extracellular matrix (ECM) organization and ECM receptor interaction pathways. Gene set enrichment analysis showed that epithelial-mesenchymal transition-associated genes were highly enriched in the HA-positive phenotype. A protein-protein interaction network was constructed, and 17 hub genes were discovered. Heatmap analysis of TCGA-KIRC data identified two prognostic clusters corresponding to HA-positive and HA-negative phenotypes. These clusters were used to verify the expression levels and conduct survival analysis of the hub genes, 11 of which were linked to poor prognosis. These findings enhance our understanding of hyaluronan in ccRCC.

Concordance of Fathers and Mothers in the Assessment of Their 5-Year-Old Child's Dental Fear.

The aim of this study was to evaluate the concordance of parents' assessments of their child's dental fear. Cross-sectional secondary analysis used data from the multidisciplinary FinnBrain Birth Cohort Study. Child dental fear was assessed at age 5 with the Finnish translation of the modified Children's Fear Survey Schedule Dental Subscale (CFSS-M) by both fathers (n = 588) and mothers (n = 1100). Reply alternatives were from 1 = not afraid to 5 = very afraid and 6 = no experience coded as missing and 1. In total, 514 mother-father pairs were eligible for the analyses. Descriptive statistics, percentage agreement and Cohen's Kappa coefficients were used in the analyses. The concordance of parents' assessments was poor (Kappa range 0.072-0.258). The majority of parents replied "No Experience" to items related to invasive treatment or being unable to breathe. Thus, coding of this reply alternative had a significant impact on the mean values of the child's fear. When assessing the fear of a five-year-old child, it might not be safe to rely only on one parent's assessment, and whether or not the child has experience with the question asked should also be considered.

Translatome profiling reveals Itih4 as a novel smooth muscle cell-specific gene in atherosclerosis.

AIMS: Vascular smooth muscle cells (SMCs) and their derivatives are key contributors to the development of atherosclerosis. However, studying changes in SMC gene expression in heterogeneous vascular tissues is challenging due to the technical limitations and high cost associated with current approaches. In this paper, we apply Translating Ribosome Affinity Purification sequencing (TRAP-Seq) to profile SMC-specific gene expression directly from tissue. METHODS AND RESULTS: To facilitate SMC-specific translatome analysis, we generated SMCTRAP mice, a transgenic mouse line expressing EGFP-tagged ribosomal protein L10a (EGFP-L10a) under the control of the SMC-specific αSMA promoter. These mice were further crossed with the atherosclerosis model Ldlr-/-, ApoB100/100 to generate SMCTRAP-AS mice and used to profile atherosclerosis-associated SMCs in thoracic aorta samples of 15-month-old SMCTRAP and SMCTRAP-AS mice. Our analysis of SMCTRAP-AS mice showed that EGFP-L10a expression was localized to SMCs in various tissues, including the aortic wall and plaque. The TRAP fraction demonstrated high enrichment of known SMC-specific genes, confirming the specificity of our approach. We identified several genes, including Cemip, Lum, Mfge8, Spp1, and Serpina3, that are known to be involved in atherosclerosis-induced gene expression. Moreover, we identified several novel genes not previously linked to SMCs in atherosclerosis, such as Anxa4, Cd276, Itih4, Myof, Pcdh11x, Rab31, Serpinb6b, Slc35e4, Slc8a3, and Spink5. Among them, we confirmed the SMC-specific expression of Itih4 in atherosclerotic lesions using immunofluorescence staining of mouse aortic roots and spatial transcriptomics of human carotid arteries. Furthermore, our more detailed analysis of Itih4 showed its link to coronary artery disease (CAD) through the colocalization of GWAS, splice-QTL, and protein-QTL signals. CONCLUSIONS: We generated a SMC-specific TRAP mouse line to study atherosclerosis and identified Itih4 as a novel SMC-expressed gene in atherosclerotic plaques, warranting further investigation of its putative function in extracellular matrix stability and genetic evidence of causality.

Oncogenic KEAP1 mutations activate TRAF2-NFκB signaling to prevent apoptosis in lung cancer cells.

The Kelch-like ECH-associated protein 1 (KEAP1) - Nuclear factor erythroid 2 -related factor 2 (NRF2) pathway is the major transcriptional stress response system in cells against oxidative and electrophilic stress. NRF2 is frequently constitutively active in many cancers, rendering the cells resistant to chemo- and radiotherapy. Loss-of-function (LOF) mutations in the repressor protein KEAP1 are common in non-small cell lung cancer, particularly adenocarcinoma. While the mutations can occur throughout the gene, they are enriched in certain areas, indicating that these may have unique functional importance. In this study, we show that in the GSEA analysis of TCGA lung adenocarcinoma RNA-seq data, the KEAP1 mutations in R320 and R470 were associated with enhanced Tumor Necrosis Factor alpha (TNFα) - Nuclear Factor kappa subunit B (NFκB) signaling as well as MYC and MTORC1 pathways. To address the functional role of these hotspot mutations, affinity purification and mass spectrometry (AP-MS) analysis of wild type (wt) KEAP1 and its mutation forms, R320Q and R470C were employed to interrogate differences in the protein interactome. We identified TNF receptor associated factor 2 (TRAF2) as a putative protein interaction partner. Both mutant KEAP1 forms showed increased interaction with TRAF2 and other anti-apoptotic proteins, suggesting that apoptosis signalling could be affected by the protein interactions. A549 lung adenocarcinoma cells overexpressing mutant KEAP1 showed high TRAF2-mediated NFκB activity and increased protection against apoptosis, XIAP being one of the key proteins involved in anti-apoptotic signalling. To conclude, KEAP1 R320Q and R470C and its interaction with TRAF2 leads to activation of NFκB pathway, thereby protecting against apoptosis.

A practical prognostic peripheral blood-based risk model for the evaluation of the likelihood of a response and survival of metastatic cancer patients treated with immune checkpoint inhibitors.

BACKGROUND: Less than half of unselected metastatic cancer patients benefit from the immune checkpoint inhibitor (ICI) therapy. Systemic cancer-related inflammation may influence the efficacy of ICIs and thus, systemic inflammatory markers could have prognostic and/or predictive potential in ICI therapy. Here, we aimed to identify a combination of inflammation-related laboratory parameters to establish a practical prognostic risk model for the pretreatment evaluation of a response and survival of ICI-treated patients with different types of metastatic cancers. METHODS: The study-cohort consisted of a real-world patient population receiving ICIs for metastatic cancers of different origins (n = 158). Laboratory parameters determined before the initiation of the ICI treatment were retrospectively collected. Six inflammation-related parameters i.e., elevated values of neutrophils, platelets, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and lactate dehydrogenase (LDH), and the presence of anemia, were each scored with one point, giving 0-6 risk points for each patient. The patients with information of all these six parameters (n = 109) were then stratified into low-risk (0-3 points) and high-risk (4-6 points) groups. The overall response rate (ORR), overall survival (OS), and progression-free survival (PFS) according to the risk scores were determined. RESULTS: The risk model was strongly associated with the outcome of the patients. The ORR to ICI treatment in the high-risk group was 30.3% in comparison to 53.9% in the low-risk group (p = 0.023). The medians for OS were 10.0 months and 27.3 months, respectively (p < 0.001), and the corresponding medians for PFS were 3.9 months and 6.3 months (p = 0.002). The risk group remained as a significant prognostic factor for both OS (HR 3.04, 95% CI 1.64-5.64, p < 0.001) and PFS (HR 1.79, 95% CI 1.04-3.06, p = 0.035) in the Cox multivariate analyses. CONCLUSIONS: We propose a readily feasible, practical risk model consisted of six inflammation-related laboratory parameters as a tool for outcome prediction in metastatic cancer patients treated with ICIs. The risk model was strongly associated with the outcome of the patients in terms of all the evaluated indicators i.e., ORR, OS and PFS. Yet, further studies are needed to validate the risk model.

The Association of CD8+ Cytotoxic T Cells and Granzyme B+ Lymphocytes with Immunosuppressive Factors, Tumor Stage and Prognosis in Cutaneous Melanoma.

The immunosuppressive tumor microenvironment (TME) consists of suppressive cells producing a variety of immunomodulatory proteins, such as programmed death ligand 1 (PD-L1) and indoleamine-2,3-dioxygenase (IDO). Although granzyme B (GrB) is known to convey the cytolytic activities of CD8+ cytotoxic lymphocytes, it is also expressed by other cells, such as regulatory T and B cells, for immunosuppressive purposes. The role of GrB+ lymphocytes in melanoma has not been examined extensively. In this study, benign, premalignant, and malignant melanocytic tumors were stained immunohistochemically for CD8 and GrB. PD-L1 was also stained from malignant samples that had accompanying clinicopathological data. The association of CD8+ and GrB+ lymphocytes with PD-L1 expression, tumor stage, prognosis, and previously analyzed immunosuppressive factors were evaluated. Our aim was to obtain a more comprehensive perception of the immunosuppressive TME in melanoma. The results show that both CD8+ and GrB+ lymphocytes were more abundant in pT4 compared to pT1 melanomas, and in lymph node metastases compared to primary melanomas. Surprisingly, a low GrB/CD8 ratio was associated with better recurrence-free survival in primary melanomas, which indicates that GrB+ lymphocytes might represent activated immunosuppressive lymphocytes rather than cytotoxic T cells. In the present study, CD8+ lymphocytes associated positively with both tumor and stromal immune cell PD-L1 and IDO expression. In addition, PD-L1+ tumor and stromal immune cells associated positively with IDO+ stromal immune and melanoma cells. The data suggest that IDO and PD-L1 seem to be key immunosuppressive factors in CD8+ lymphocyte-predominant tumors in CM.

Irradiation Alters the Expression of MUC1, CD44 and Hyaluronan in Oral Mucosal Epithelium.

Purpose: It is well established that cancer cells exploit aberrant synthesis of mucin 1 (MUC1) and hyaluronan (HA) synthesis along with HA's physiological cell surface receptor CD44. However, their role in irradiated oral tissue has not been reported previously. We, therefore, aimed to study MUC1, CD44 and HA immunohistochemically in irradiated oral mucosa and their role in the long-term effects after radiotherapy. Materials and Methods: Oral mucosal biopsies were obtained from healthy subjects as controls and from patients after radiotherapy for head and neck cancer (irradiated group) during dental implant surgery.The presence of MUC1, CD44, and HA in oral mucosa was studied by immunohistochemical methods. The differences in the localization and intensity in the oral epithelium between control and irradiated tissue were analyzed. Results: The staining intensity of MUC1 was confined to the superficial epithelial layer, whereas HA and CD44 were found in the cell membranes in the epithelial basal and intermediate layers of control specimens. In irradiated epithelium, MUC1 staining was distributed throughout all the layers of the oral epithelium, with significant staining in the basal and intermediate layers. Accordingly, HA and CD44 staining extended to involve the superficial cells of the irradiated epithelium. The staining pattern of MUC1 and CD44 showed significant changes in irradiated samples. Conclusions: Our results showed that the staining intensities of MUC1, CD44, and HA were significantly elevated in irradiated tissue compared to controls. MUC1, CD44, and HA are important markers and take part in long-term changes in the oral mucosa after radiotherapy.

M1 Macrophages Induce Protumor Inflammation in Melanoma Cells through TNFR-NF-κB Signaling.

The tumor microenvironment, with distinctive cell types and a complex extracellular matrix has a tremendous impact on cancer progression. In this study, we investigated the effects of proinflammatory (M1) and immunosuppressive (M2) macrophages on hyaluronan (HA) matrix formation and inflammatory response in melanoma cells. Proinflammatory factors secreted from M1 macrophages stimulated the formation of a thick pericellular HA matrix in melanoma cells due to upregulation of HA synthase 2 (HAS2). HAS2 silencing reversed the effect of M1 conditioned medium on pericellular HA coat formation, and interestingly, it also partly downregulated the M1 conditioned medium‒induced upregulation of inflammation-related genes (IL1ß, IL6), as did the inhibitors for TNFR and IKKγ. Gene set enrichment analysis revealed that genes related to inflammatory responses and TNF-α signaling via NF-κB are enriched in the M1 conditioned medium‒treated melanoma cells. Moreover, the expression of matrix metalloproteinase 9 and three-dimensional cell invasion were induced in these cells, whereas M2 macrophages had no effect on HA synthesis, inflammatory response, or invasion. Our results indicate that the activation of TNFR-NF-κB signaling in M1 conditioned medium‒treated cells leads to HAS2 upregulation, which associates with a protumor inflammatory and invasive phenotype of melanoma cells.

The role of FoxP3+ regulatory T cells and IDO+ immune and tumor cells in malignant melanoma - an immunohistochemical study.

BACKGROUND: FoxP3+ Regulatory T cells (Tregs) and indoleamine-2,3-dioxygenase (IDO) participate in the formation of an immunosuppressive tumor microenvironment (TME) in malignant cutaneous melanoma (CM). Recent studies have reported that IDO expression correlates with poor prognosis and greater Breslow's depth, but results concerning the role of FoxP3+ Tregs in CM have been controversial. Furthermore, the correlation between IDO and Tregs has not been substantially studied in CM, although IDO is known to be an important regulator of Tregs activity. METHODS: We investigated the associations of FoxP3+ Tregs, IDO+ tumor cells and IDO+ stromal immune cells with tumor stage, prognostic factors and survival in CM. FoxP3 and IDO were immunohistochemically stained from 29 benign and 29 dysplastic nevi, 18 in situ -melanomas, 48 superficial and 62 deep melanomas and 67 lymph node metastases (LNMs) of CM. The number of FoxP3+ Tregs and IDO+ stromal immune cells, and the coverage and intensity of IDO+ tumor cells were analysed. RESULTS: The number of FoxP3+ Tregs and IDO+ stromal immune cells were significantly higher in malignant melanomas compared with benign lesions. The increased expression of IDO in melanoma cells was associated with poor prognostic factors, such as recurrence, nodular growth pattern and increased mitotic count. Furthermore, the expression of IDO in melanoma cells was associated with reduced recurrence-free survival. We further showed that there was a positive correlation between IDO+ tumor cells and FoxP3+ Tregs. CONCLUSIONS: These results indicate that IDO is strongly involved in melanoma progression. FoxP3+ Tregs also seems to contribute to the immunosuppressive TME in CM, but their significance in melanoma progression remains unclear. The positive association of FoxP3+ Tregs with IDO+ melanoma cells, but not with IDO+ stromal immune cells, indicates a complex interaction between IDO and Tregs in CM, which demands further studies.

The Impact of Hyaluronan on Tumor Progression in Cutaneous Melanoma.

The incidence of cutaneous melanoma is rapidly increasing worldwide. Cutaneous melanoma is an aggressive type of skin cancer, which originates from malignant transformation of pigment producing melanocytes. The main risk factor for melanoma is ultraviolet (UV) radiation, and thus it often arises from highly sun-exposed skin areas and is characterized by a high mutational burden. In addition to melanoma-associated mutations such as BRAF, NRAS, PTEN and cell cycle regulators, the expansion of melanoma is affected by the extracellular matrix surrounding the tumor together with immune cells. In the early phases of the disease, hyaluronan is the major matrix component in cutaneous melanoma microenvironment. It is a high-molecular weight polysaccharide involved in several physiological and pathological processes. Hyaluronan is involved in the inflammatory reactions associated with UV radiation but its role in melanomagenesis is still unclear. Although abundant hyaluronan surrounds epidermal and dermal cells in normal skin and benign nevi, its content is further elevated in dysplastic lesions and local tumors. At this stage hyaluronan matrix may act as a protective barrier against melanoma progression, or alternatively against immune cell attack. While in advanced melanoma, the content of hyaluronan decreases due to altered synthesis and degradation, and this correlates with poor prognosis. This review focuses on hyaluronan matrix in cutaneous melanoma and how the changes in hyaluronan metabolism affect the progression of melanoma.

Cellular hyaluronan is associated with a poor prognosis in renal cell carcinoma.

PURPOSE: Hyaluronan, a major glycosaminoglycan of the extracellular matrix, can act as an oncogenic component of the tumor microenvironment in many human malignancies. We characterized the hyaluronan content of renal cell carcinomas (RCCs) and investigated its correlations with clinicopathological parameters and patient survival. PATIENTS AND METHODS: This retrospective study included data from 316 patients that had undergone surgery for RCC in Kuopio University Hospital in 2000 to 2013. The hyaluronan content of surgical tumor samples were histochemically stained with a biotinylated hyaluronan-specific affinity probe. The amount of tumor infiltrating lymphocytes was evaluated in each tumor. Kaplan-Meier and univariate and multivariate Cox-regression analyses were performed to estimate the impact of hyaluronan content on overall survival, disease-specific survival, and metastasis-free survival. RESULTS: Detectable cellular hyaluronan was associated with higher tumor grades and the presence of tumor infiltrating lymphocytes. Cellular hyaluronan identified a prognostically unfavourable subgroup among low-grade carcinomas. Multivariate analyses showed that measurable cellular hyaluronan was an independent negative prognostic factor for overall survival (hazard ratio [HR] 1.4; 95% confidence interval [CI]: 1.02-2.0; P = 0.039), Disease-specific survival (HR 2.07; 95% CI: 1.2-3.3; P = 0.002), and metastasis-free survival (HR 2.45; 95% CI: 1.37-4.4; P = 0.003). CONCLUSIONS: Cellular hyaluronan was significantly associated with unfavourable features and a poor prognosis in RCC. Further studies are needed to investigate the biological mechanism underlying hyaluronan accumulation in RCC.

High-Resolution Confocal Fluorescence Imaging of Serine Hydrolase Activity in Cryosections - Application to Glioma Brain Unveils Activity Hotspots Originating from Tumor-Associated Neutrophils.

BACKGROUND: Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. ABPP utilizes cell/tissue proteomes and features the FP-warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Existing ABPP approaches characterize global SH activity based on mobility in gel or MS-based target identification and cannot reveal the identity of the cell-type responsible for an individual SH activity originating from complex proteomes. RESULTS: Here, by using an activity probe with broad reactivity towards the SH family, we advance the ABPP methodology to glioma brain cryosections, enabling for the first time high-resolution confocal fluorescence imaging of global SH activity in the tumor microenvironment. Tumor-associated cell types were identified by extensive immunohistochemistry on activity probe-labeled sections. Tissue-ABPP indicated heightened SH activity in glioma vs. normal brain and unveiled activity hotspots originating from tumor-associated neutrophils (TANs), rather than tumor-associated macrophages (TAMs). Thorough optimization and validation was provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. CONCLUSIONS: Our study advances the ABPP methodology to tissue sections, enabling high-resolution confocal fluorescence imaging of global SH activity in anatomically preserved complex native cellular environment. To achieve global portrait of SH activity throughout the section, a probe with broad reactivity towards the SH family members was employed. As ABPP requires no a priori knowledge of the identity of the target, we envisage no imaginable reason why the presently described approach would not work for sections regardless of species and tissue source.

Metformin decreases hyaluronan synthesis by vascular smooth muscle cells.

Metformin is the first-line drug in the treatment of type 2 diabetes worldwide based on its effectiveness and cardiovascular safety. Currently metformin is increasingly used during pregnancy in women with gestational diabetes mellitus, even if the long-term effects of metformin on offspring are not exactly known. We have previously shown that high glucose concentration increases hyaluronan (HA) production of cultured human vascular smooth muscle cells (VSMC) via stimulating the expression of hyaluronan synthase 2 (HAS2). This offers a potential mechanism whereby hyperglycemia leads to vascular macroangiopathy. In this study, we examined whether gestational metformin use affects HA content in the aortic wall of mouse offspring in vivo. We also examined the effect of metformin on HA synthesis by cultured human VSMCs in vitro. We found that gestational metformin use significantly decreased HA content in the intima-media of mouse offspring aortas. In accordance with this, the synthesis of HA by VSMCs was also significantly decreased in response to treatment with metformin. This decrease in HA synthesis was shown to be due to the reduction of both the expression of HAS2 and the amount of HAS substrates, particularly UDP-N-acetylglucosamine. As shown here, gestational metformin use is capable to program reduced HA content in the vascular wall of the offspring strongly supporting the idea, that metformin possesses long-term vasculoprotective effects.

HAS3-induced extracellular vesicles from melanoma cells stimulate IHH mediated c-Myc upregulation via the hedgehog signaling pathway in target cells.

Intercellular communication is fundamental to the survival and maintenance of all multicellular systems, whereas dysregulation of communication pathways can drive cancer progression. Extracellular vesicles (EVs) are mediators of cell-to-cell communication that regulate a variety of cellular processes involved in tumor progression. Overexpression of a specific plasma membrane enzyme, hyaluronan synthase 3 (HAS3), is one of the factors that can induce EV shedding. HAS3, and particularly its product hyaluronan (HA), are carried by EVs and are known to be associated with the tumorigenic properties of cancer cells. To elucidate the specific effects of cancerous, HAS3-induced EVs on target cells, normal human keratinocytes and melanoma cells were treated with EVs derived from GFP-HAS3 expressing metastatic melanoma cells. We found that the HA receptor CD44 participated in the regulation of EV binding to target cells. Furthermore, GFP-HAS3-positive EVs induced HA secretion, proliferation and invasion of target cells. Our results suggest that HAS3-EVs contains increased quantities of IHH, which activates the target cell hedgehog signaling cascade and leads to the activation of c-Myc and regulation of claspin expression. This signaling of IHH in HAS3-EVs resulted in increased cell proliferation. Claspin immunostaining correlated with HA content in human cutaneous melanocytic lesions, supporting our in vitro findings and suggesting a reciprocal regulation between claspin expression and HA synthesis. This study shows for the first time that EVs originating from HAS3 overexpressing cells carry mitogenic signals that induce proliferation and epithelial-to-mesenchymal transition in target cells. The study also identifies a novel feedback regulation between the hedgehog signaling pathway and HA metabolism in melanoma, mediated by EVs carrying HA and IHH.

Nrf2 and SQSTM1/p62 jointly contribute to mesenchymal transition and invasion in glioblastoma.

Accumulating evidence suggests that constitutively active Nrf2 has a pivotal role in cancer as it induces pro-survival genes that promote cancer cell proliferation and chemoresistance. The mechanisms of Nrf2 dysregulation and functions in cancer have not been fully characterized. Here, we jointly analyzed the Broad-Novartis Cancer Cell Line Encyclopedia (CCLE) and the Cancer Genome Atlas (TCGA) multi-omics data in order to identify cancer types where Nrf2 activation is present. We found that Nrf2 is hyperactivated in a subset of glioblastoma (GBM) patients, whose tumors display a mesenchymal subtype, and uncover several different mechanisms contributing to increased Nrf2 activity. Importantly, we identified a positive feedback loop between SQSTM1/p62 and Nrf2 as a mechanism for activation of the Nrf2 pathway. We also show that autophagy and serine/threonine signaling regulates p62 mediated Keap1 degradation. Our results in glioma cell lines indicate that both Nrf2 and p62 promote proliferation, invasion and mesenchymal transition. Finally, Nrf2 activity was associated with decreased progression free survival in TCGA GBM patient samples, suggesting that treatments have limited efficacy if this transcription factor is overactivated. Overall, our findings place Nrf2 and p62 as the key components of the mesenchymal subtype network, with implications to tumorigenesis and treatment resistance. Thus, Nrf2 activation could be used as a surrogate prognostic marker in mesenchymal subtype GBMs. Furthermore, strategies aiming at either inhibiting Nrf2 or exploiting Nrf2 hyperactivity for targeted gene therapy may provide novel treatment options for this subset of GBM.

Using online game-based platforms to improve student performance and engagement in histology teaching.

BACKGROUND: Human morphology is a critical component of dental and medical graduate training. Innovations in basic science teaching methods are needed to keep up with an ever-changing landscape of technology. The purpose of this study was to investigate whether students in a medical and dental histology course would have better grades if they used gaming software Kahoot® and whether gamification effects on learning and enjoyment. METHODS: In an effort to both evoke students' interest and expand their skill retention, an online competition using Kahoot® was implemented for first-year students in 2018 (n = 215) at the University of Eastern Finland. Additionally, closed (160/215) or open-ended (41/215) feedback questions were collected and analyzed. RESULTS: The Kahoot® gamification program was successful and resulted in learning gains. The overall participant satisfaction using Kahoot® was high, with students (124/160) indicating that gamification increased their motivation to learn. The gaming approach seemed to enable the students to overcome individual difficulties (139/160) and to set up collaboration (107/160); furthermore, gamification promoted interest (109/160), and the respondents found the immediate feedback from senior professionals to be positive (146/160). In the open-ended survey, the students (23/41) viewed collaborative team- and gamification-based learning positively. CONCLUSION: This study lends support to the use of gamification in the teaching of histology and may provide a foundation for designing a gamification-integrated curriculum across healthcare disciplines.

Alterations in the expression of EMT-related proteins claudin-1, claudin-4 and claudin-7, E-cadherin, TWIST1 and ZEB1 in oral lichen planus.

BACKGROUND: Oral lichen planus (OLP) is a chronic T-cell-mediated inflammatory disease, which is associated with increased risk of developing oral squamous cell carcinoma. Epithelial-to-mesenchymal transition is a physiological phenomenon occurring during growth and organogenesis, but it has also an important role in tumorigenesis. In the present work, we studied the expression of known epithelial-to-mesenchymal transition markers in oral lichen planus. METHODS: In total, 54 oral lichen planus and 22 control samples were analyzed for epithelial-to-mesenchymal transition markers. Samples were immunohistochemically stained for claudin-1, claudin-4 and claudin-7, cadherin-1 (E-cadherin), Twist-related protein 1 (TWIST1) and zinc finger E-box-binding homeobox 1 (ZEB1). RESULTS: The expression of claudin-1, claudin-4 and E-cadherin was significantly weaker in oral lichen planus epithelium compared to controls (P < 0.001). The quantity of claudin-7-expressing cells (P < 0.001) and claudin-7 staining intensity (P < 0.05) in the stroma was greater in lichen planus than in control samples. TWIST1 and ZEB1 stainings were negative in the epithelium in both lichen planus and controls. The number of TWIST1-expressing cells in the stroma was higher in lichen planus than in controls (P < 0.001). There was a statistically significant difference in ZEB1 staining intensity in the stroma between lichen planus and control samples (P < 0.05). CONCLUSIONS: The data indicate that the expression of claudin-1, claudin-4 and E-cadherin is decreased in oral lichen planus. This may lead to disturbance in epithelial tight junctions, cell-cell connections and epithelial permeability, contributing to oral lichen planus pathogenesis. Based on the present study, the role of TWIST1 and ZEB1 in oral lichen planus remains unclear.

Melanocyte Hyaluronan Coat Fragmentation Enhances the UVB-Induced TLR-4 Receptor Signaling and Expression of Proinflammatory Mediators IL6, IL8, CXCL1, and CXCL10 via NF-κB Activation.

Skin is constantly exposed to UVR, the most critical risk factor for melanoma development. Hyaluronan is abundant in the epidermal extracellular matrix and may undergo degradation by UVR. It is hypothesized that an intact hyaluronan coat around the cells protects against various agents including UVR, whereas hyaluronan fragments promote inflammation and tumorigenesis. We investigated whether hyaluronan contributes to the UVB-induced inflammatory responses in primary melanocytes. A single dose of UVB suppressed hyaluronan secretion and the expression of hyaluronan synthases HAS2 and HAS3, the hyaluronan receptor CD44, and the hyaluronidase HYAL2, as well as induced the expression of inflammatory mediators IL6, IL8, CXCL1, and CXCL10. Silencing HAS2 and CD44 partly inhibited the inflammatory response, suggesting that hyaluronan coat is involved in the process. UVB alone caused little changes in the coat, but its removal with hyaluronidase during the recovery from UVB exposure dramatically enhanced the surge of these inflammatory mediators via TLR4, p38, and NF-κB. Interestingly, exogenous hyaluronan fragments did not reproduce the inflammatory effects of hyaluronidase. We hypothesize that the hyaluronan coat on melanocytes is a sensor of tissue injury. Combined with UVB exposure, repeated injuries to the hyaluronan coat could maintain a sustained inflammatory state associated with melanomagenesis.

Activated hyaluronan metabolism in the tumor matrix - Causes and consequences.

Hyaluronan accumulates in the stroma of several solid tumors and promotes their progression. Both enhanced synthesis and fragmentation of hyaluronan are required as a part of this inflammatory process resembling wound healing. Increased expression of the genes of hyaluronan synthases (HAS1-3) are infrequent in human tumors, while posttranslational modifications that activate the HAS enzymes, and glucose shunted to the UDP-sugar substrates HASs, can have crucial contributions to tumor hyaluronan synthesis. The pericellular hyaluronan influences virtually all cell-cell and cell-matrix interactions, controlling migration, proliferation, apoptosis, epithelial to mesenchymal transition, and stem cell functions. The catabolism by hyaluronidases and free radicals appears to be as important as synthesis for the inflammation that promotes tumor growth, since the receptors mediating the signals create specific responses to hyaluronan fragments. Targeting hyaluronan metabolism shows therapeutic efficiency in animal experiments and early clinical trials.

The number and localization of CD68+ and CD163+ macrophages in different stages of cutaneous melanoma.

The role of tumor-associated macrophages (TAMs) in cutaneous melanoma is controversial. TAMs include immunogenic and immunosuppressive subtypes, and have distinct functions according to their microanatomical localization. Our aim was to investigate TAMs in benign, premalignant, and malignant melanocytic lesions to determine possible associations with tumor progression and clinicopathological characteristics. In total, 184 tissue samples, including benign and dysplastic nevi, in-situ melanomas, superficial (Breslow's depth <1 mm), and deep (Breslow's depth >4 mm) invasive melanomas and lymph node metastases, were analyzed for macrophage content. Samples were stained immunohistochemically for CD68 and CD163, representing all TAMs and M2-macrophages, respectively. Macrophages were counted by hotspot analysis, and assessed semiquantitatively from the tumor cell nests and stromal component of malignant cases. CD68+ and CD163+ TAMs were more abundant in invasive melanomas compared with benign nevi. The proportion of TAMs in the tumor nests was higher in deep melanomas and lymph node metastases compared with superficially invasive melanomas. High amounts of CD68+ macrophages in tumor cell nests were associated with recurrence, whereas low CD163+ macrophage proportion in tumor stroma was associated with recurrence and in primary melanomas also with poor overall survival. TAMs seem to promote tumor progression in cutaneous melanoma. In particular, CD68+ TAMs and their abundance in tumor nests were associated with poor prognostic factors. However, the correlation of low stromal CD163+ TAM proportion with a poor prognosis indicates that the role of TAMs depends on their subtype and microanatomical localization.