Human oral mucosa cell atlas reveals a stromal-neutrophil axis regulating tissue immunity.

The oral mucosa remains an understudied barrier tissue. This is a site of rich exposure to antigens and commensals, and a tissue susceptible to one of the most prevalent human inflammatory diseases, periodontitis. To aid in understanding tissue-specific pathophysiology, we compile a single-cell transcriptome atlas of human oral mucosa in healthy individuals and patients with periodontitis. We uncover the complex cellular landscape of oral mucosal tissues and identify epithelial and stromal cell populations with inflammatory signatures that promote antimicrobial defenses and neutrophil recruitment. Our findings link exaggerated stromal cell responsiveness with enhanced neutrophil and leukocyte infiltration in periodontitis. Our work provides a resource characterizing the role of tissue stroma in regulating mucosal tissue homeostasis and disease pathogenesis.

Activating the healing process: three-dimensional culture of stem cells in Matrigel for tissue repair.

BACKGROUND: To establish a strategy for stem cell-related tissue regeneration therapy, human gingival mesenchymal stem cells (hGMSCs) were loaded with three-dimensional (3D) bioengineered Matrigel matrix scaffolds in high-cell density microtissues to promote local tissue restoration. METHODS: The biological performance and stemness of hGMSCs under 3D culture conditions were investigated by viability and multidirectional differentiation analyses. A Sprague‒Dawley (SD) rat full-thickness buccal mucosa wound model was established, and hGMSCs/Matrigel were injected into the submucosa of the wound. Autologous stem cell proliferation and wound repair in local tissue were assessed by histomorphometry and immunohistochemical staining. RESULTS: Three-dimensional suspension culture can provide a more natural environment for extensions and contacts between hGMSCs, and the viability and adipogenic differentiation capacity of hGMSCs were significantly enhanced. An animal study showed that hGMSCs/Matrigel significantly accelerated soft tissue repair by promoting autologous stem cell proliferation and enhancing the generation of collagen fibers in local tissue. CONCLUSION: Three-dimensional cell culture with hydrogel scaffolds, such as Matrigel, can effectively improve the biological function and maintain the stemness of stem cells. The therapeutic efficacy of hGMSCs/Matrigel was confirmed, as these cells could effectively stimulate soft tissue repair to promote the healing process by activating the host microenvironment and autologous stem cells.

circMTO1/miR-30c-5p/SOCS3 axis alleviates oral submucous fibrosis through inhibiting fibroblast-myofibroblast transition.

BACKGROUND: circRNAs have been shown to participate in diverse diseases; however, their role in oral submucous fibrosis (OSF), a potentially malignant disorder, remains obscure. Our preliminary experiments detected the expression of circRNA mitochondrial translation optimization 1 homologue (circMTO1) in OSF tissues (n = 20) and normal mucosa tissues (n = 20) collected from Hunan Xiangya Stomatological Hospital, and a significant decrease of circMTO1 expression was showed in OSF tissues. Therefore, we further explored circMTO1 expression in OSF. METHODS: Target molecule expression was detected using RT-qPCR and western blotting. The migration and invasion of buccal mucosal fibroblasts (BMFs) were assessed using wound healing and Transwell assays. The interaction between miR-30c-5p, circMTO1, and SOCS3 was evaluated using dual luciferase, RNA immunoprecipitation (RIP), and RNA pull-down assays. The colocalisation of circMTO1 and miR-30c-5p was observed using fluorescence in situ hybridisation (FISH). RESULTS: circMTO1 and SOCS3 expression decreased, whereas miR-30c-5p expression increased in patients with OSF and arecoline-stimulated BMFs. Overexpression of circMTO1 effectively restrained the fibroblast-myofibroblast transition (FMT), as evidenced by the increase in expression of Coll I, α-SMA, Vimentin, and the weakened migration and invasion functions in BMFs. Mechanistic studies have shown that circMTO1 suppresses FMT by enhancing SOCS3 expression by sponging miR-30c-5p and subsequently inactivating the FAK/PI3K/AKT pathway. FMT induced by SOCS3 silencing was reversed by the FAK inhibitor TAE226 or the PI3K inhibitor LY294002. CONCLUSION: circMTO1/miR-30c-5p/SOCS3 axis regulates FMT in arecoline-treated BMFs via the FAK/PI3K/AKT pathway. Expanding the sample size and in vivo validation could further elucidate their potential as therapeutic targets for OSF.

Mobile phone specific radiation disturbs cytokinesis and causes cell death but not acute chromosomal damage in buccal cells: Results of a controlled human intervention study.

Several human studies indicate that mobile phone specific electromagnetic fields may cause cancer in humans but the underlying molecular mechanisms are currently not known. Studies concerning chromosomal damage (which is causally related to cancer induction) are controversial and those addressing this issue in mobile phone users are based on the use of questionnaires to assess the exposure. We realized the first human intervention trial in which chromosomal damage and acute toxic effects were studied under controlled conditions. The participants were exposed via headsets at one randomly assigned side of the head to low and high doses of a UMTS signal (n = 20, to 0.1 W/kg and n = 21 to 1.6 W/kg Specific Absorption Rate) for 2 h on 5 consecutive days. Before and three weeks after the exposure, buccal cells were collected from both cheeks and micronuclei (MN, which are formed as a consequence of structural and numerical chromosomal aberrations) and other nuclear anomalies reflecting mitotic disturbance and acute cytotoxic effects were scored. We found no evidence for induction of MN and of nuclear buds which are caused by gene amplifications, but a significant increase of binucleated cells which are formed as a consequence of disturbed cell divisions, and of karyolitic cells, which are indicative for cell death. No such effects were seen in cells from the less exposed side. Our findings indicate that mobile phone specific high frequency electromagnetic fields do not cause acute chromosomal damage in oral mucosa cells under the present experimental conditions. However, we found clear evidence for disturbance of the cell cycle and cytotoxicity. These effects may play a causal role in the induction of adverse long term health effects in humans.

The effect of resolvin D1n-3 DPA on primary oral epithelial cell migration in vitro.

Specialized pro-resolving lipid mediators (SPMs) are known for their anti-inflammatory and pro-resolving actions. The aim of the present study was to find new functions of the SPM resolvin D1n-3 DPA (RvD1n-3 DPA) on oral epithelial cells. As a starting point, we used a dataset obtained by RNA high-throughput sequencing of oral epithelial cells exposed to TNF-α and RvD1n-3 DPA versus TNF-α alone. GOrilla enrichment analysis showed that the actin cytoskeleton was significantly overrepresented after adjustment for multiple hypothesis testing. As actin, amongst others, is closely related to cell migration, we then explored whether RvD1n-3 DPA can modulate oral epithelial cell migration. To this end, we used an in vitro cell migration model, including TNF-α treatment, to mimic an inflammatory cell state. The analysis revealed that RvD1n-3 DPA increased oral epithelial cell migration in the presence but not in the absence of TNF-α. Addition of RvD1n-3 DPA also induced F actin accumulation around the cell nucleus, indicating that RvD1n-3 DPA potentially can mediate processes of intracellular transport. This indicates that this lipid mediator may be a promising therapeutic candidate in oral mucosal wound healing.

Oral mucosa - an examination map for confocal laser endomicroscopy within the oral cavity: an experimental clinical study.

OBJECTIVES: Confocal laser endomicroscopy (CLE) is an optical method that enables microscopic visualization of oral mucosa. Previous studies have shown that it is possible to differentiate between physiological and malignant oral mucosa. However, differences in mucosal architecture were not taken into account. The objective was to map the different oral mucosal morphologies and to establish a "CLE map" of physiological mucosa as baseline for further application of this powerful technology. MATERIALS AND METHODS: The CLE database consisted of 27 patients. The following spots were examined: (1) upper lip (intraoral) (2) alveolar ridge (3) lateral tongue (4) floor of the mouth (5) hard palate (6) intercalary line. All sequences were examined by two CLE experts for morphological differences and video quality. RESULTS: Analysis revealed clear differences in image quality and possibility of depicting tissue morphologies between the various localizations of oral mucosa: imaging of the alveolar ridge and hard palate showed visually most discriminative tissue morphology. Labial mucosa was also visualized well using CLE. Here, typical morphological features such as uniform cells with regular intercellular gaps and vessels could be clearly depicted. Image generation and evaluation was particularly difficult in the area of the buccal mucosa, the lateral tongue and the floor of the mouth. CONCLUSION: A physiological "CLE map" for the entire oral cavity could be created for the first time. CLINICAL RELEVANCE: This will make it possible to take into account the existing physiological morphological features when differentiating between normal mucosa and oral squamous cell carcinoma in future work.

Is micronucleus assay in oral cells suitable biomarker for evaluating the risk of carcinogenesis in gas station attendants? A systematic review.

This study aimed to evaluate all studies which used the micronucleus assay using oral cells in the attempt to understand whether such technique is efficient in evaluating genotoxicity in gas station attendants. Full manuscripts from 16 studies were carefully selected by the authors. Our results demonstrate that continuous exposure to derivatives of petroleum may lead to genotoxic effects since all studies demonstrated positive findings (16 out of 16) and 11 of them had a strong or moderate final rating. In summary, our results reveal that gas station attendants are occupationally exposed to genotoxic agents and that the micronucleus assay in oral mucosa is indeed an effective method to evaluate genotoxicity in this specific case. Such findings are very important for protecting these professionals who are continuously exposed to chemicals for long periods.

Buccal Mesenchymal Stromal Cells as a Source of Osseointegration of Titanium Implants.

We studied the interaction of human buccal mesenchymal stem cells (MSCs) and osteoblasts differentiated from them with the surface of titanium samples. MSCs were isolated by enzymatic method from buccal fat pads. The obtained cell culture was presented by MSCs, which was confirmed by flow cytometry and differentiation into adipocytes and osteoblasts. Culturing of buccal MSCs on titanium samples was accompanied by an increase in the number of cells for 15 days and the formation of a developed network of F-actin fibers in the cells. The viability of buccal MSCs decreased by 8 days, but was restored by 15 days. Culturing of osteoblasts obtained as a result of buccal MSC differentiation on the surface of titanium samples was accompanied by a decrease in their viability and proliferation. Thus, MSCs from buccal fat pads can be used to coat implants to improve osseointegration during bone reconstruction in craniofacial surgery and dentistry. To improve the integration of osteoblasts, modification of the surface of titanium samples is required.

The PedBE clock accurately estimates DNA methylation age in pediatric buccal cells.

The development of biological markers of aging has primarily focused on adult samples. Epigenetic clocks are a promising tool for measuring biological age that show impressive accuracy across most tissues and age ranges. In adults, deviations from the DNA methylation (DNAm) age prediction are correlated with several age-related phenotypes, such as mortality and frailty. In children, however, fewer such associations have been made, possibly because DNAm changes are more dynamic in pediatric populations as compared to adults. To address this gap, we aimed to develop a highly accurate, noninvasive, biological measure of age specific to pediatric samples using buccal epithelial cell DNAm. We gathered 1,721 genome-wide DNAm profiles from 11 different cohorts of typically developing individuals aged 0 to 20 y old. Elastic net penalized regression was used to select 94 CpG sites from a training dataset (n = 1,032), with performance assessed in a separate test dataset (n = 689). DNAm at these 94 CpG sites was highly predictive of age in the test cohort (median absolute error = 0.35 y). The Pediatric-Buccal-Epigenetic (PedBE) clock was characterized in additional cohorts, showcasing the accuracy in longitudinal data, the performance in nonbuccal tissues and adult age ranges, and the association with obstetric outcomes. The PedBE tool for measuring biological age in children might help in understanding the environmental and contextual factors that shape the DNA methylome during child development, and how it, in turn, might relate to child health and disease.

IL-1α released from oral epithelial cells upon candidalysin exposure initiates an early innate epithelial response.

Candida albicans is a commensal fungus that predominantly resides on mucosal surfaces and can cause lethal systemic infection when the host defense is compromised. Candidalysin is a cytolytic peptide toxin produced by C. albicans hyphae that is essential for mucosal tissue damage and is believed to contribute to the establishment of systemic infection and mortality. Candidalysin is also required for the epithelial innate response in which proinflammatory cytokines and chemokines are produced and neutrophil recruitment is initiated. It was recently reported that epidermal growth factor receptor (EGFR) was essential for the candidalysin-triggered epithelial response. The present study identified IL-1α as another component of candidalysin-mediated initial epithelial activation. We found that human oral epithelial cells released IL-1α rapidly after candidalysin exposure. Blockade of IL-1α/IL-1 receptor (IL-1R) signaling in candidalysin-exposed cells resulted in decreased phosphorylation of IκBα, decreased induction of IκBζ and decreased production of granulocyte-macrophage colony-stimulating factor and IL-8. Expression of c-Fos, which is induced downstream of EGFR signaling in candidalysin-treated cells, is less affected by IL-1R blockade. Inversely, blockade of EGFR signaling does not affect candidalysin-mediated phosphorylation of IκBα and induction of IκBζ, suggesting that independent signaling pathways contribute to the induction of NF-κB and c-Fos downstream of the candidalysin pore formation site. Consistently, antibody inhibition of both EGFR and IL-1R enhanced the suppressive effect of cytokine production in candidalysin-treated cells. Thus, we identified the immediate release of IL-1α and its synergistic role with EGFR ligands on the initial activation of oral epithelial cells in response to candidalysin.

Labial mucosal epithelium grafting in an ex vivo human donor cornea model.

The purpose of the study was to establish a simple ex vivo corneal re-epithelization model and study the labial mucosal epithelium grafting as a potential approach for ocular surface reconstruction. Four human donor corneal buttons were overstored in a corneal cold storage solution at 4 °C for 32-52 days. Four labial oral mucosa strips were dissected from four patients during fornix reconstruction after they signed informed consent. The substantia propria was trimmed off, and the resulting graft was sutured near the corneal limbus with running sutures (thus forming the tissue construct). Constructs were cultured under the standard conditions with the anterior corneal side outwards. After 3 weeks of culture, constructs were removed, washed, and fixed. Sections were stained with hematoxylin and eosin (HE), anti-keratins 4, 13, 19, and p63. Nuclei were counterstained with Hoechst. After the cultivation, all constructs were integral with the attached graft and non-loosened sutures. The native cells were absent in all donor corneas. Histological evaluation demonstrated that the labial mucosal grafts were attached to the Bowman's membrane (BM), and its cellular outgrowths were found to be transit from the graft to the BM over the anterior surface in all constructs. Cells expressed mucosal epithelial keratins 4, 13, and 19, and several were p63-positive in nuclei. In the study, a simple ex vivo corneal re-epithelization model was successfully established. The model was potent in studying the labial mucosal epithelium grafting as an option for autologous ocular surface reconstruction in patients with bilateral limbal stem cell deficiency.

SOX2 regulation by hedgehog signaling controls adult lingual epithelium homeostasis.

Adult tongue epithelium is continuously renewed from epithelial progenitor cells, a process that requires hedgehog (HH) signaling. In mice, pharmacological inhibition of the HH pathway causes taste bud loss within a few weeks. Previously, we demonstrated that sonic hedgehog (SHH) overexpression in lingual progenitors induces ectopic taste buds with locally increased SOX2 expression, suggesting that taste bud differentiation depends on SOX2 downstream of HH. To test this, we inhibited HH signaling in mice and observed a rapid decline in Sox2 and SOX2-GFP expression in taste epithelium. Upon conditional deletion of Sox2, differentiation of both taste and non-taste epithelial cells was blocked, and progenitor cell number increased. In contrast to basally restricted proliferation in controls, dividing cells were overabundant and spread to suprabasal epithelial layers in mutants. SOX2 loss in progenitors also led non-cell-autonomously to taste cell apoptosis, dramatically shortening taste cell lifespans. Finally, in tongues with conditional Sox2 deletion and SHH overexpression, ectopic and endogenous taste buds were not detectable; instead, progenitor hyperproliferation expanded throughout the lingual epithelium. In summary, we show that SOX2 functions downstream of HH signaling to regulate lingual epithelium homeostasis.

cG-CAOMECS-clinical-grade cultured autologous oral mucosal epithelial cell sheet.

The present study reports the feasibility and successful production of rabbit cG-CAOMECS, designed to reconstruct corneal epithelium of patients with bilateral limbal stem cell deficiency. To produce a safe, chemically defined and FDA compliant cG-CAOMECS, oral mucosal epithelial cells were isolated from a biopsy of rabbit buccal tissue and seeded on a cGMP-certified cell culture surface coated with GMP-grade extracellular matrix. A newly designed clinical-grade medium (KaFa™ medium) was utilized to carry out cell expansion. Detachment and harvesting of the produced cell sheet was accomplished using collagenase treatment. Live cell imaging and morphological analysis techniques were used to examine cell growth. Cells attached onto the surface and self-assembled into colony-forming units (CFUs). Microscopic examination showed that CFUs formed during the first 5 days, and basal monolayer cell sheet formed in less than 10 days. Cells expanded to form a multilayered epithelial cell sheet that was harvested after 17-19 days in culture. Immunostaining and Western blot analyses showed that deltaNp63 was expressed in the basal cells and K3/K12 was expressed in the apical cells, indicating the presence of corneal epithelial-like cells in the produced cell sheet. Adhesion molecules, E-cadherin, beta-catenin, and Cnx43 were also expressed and exhibited the epithelial integrity of the cell sheet. The expression of integrin-beta1 and beta4 confirmed that the collagenase treatment used for detaching and harvesting the cell sheet did not have adverse effects. Our results showed that the utilization of clinical-grade and FDA-approved reagents successfully supported the production of cG-CAMECS.

Zonula occludens-1 demonstrates a unique appearance in buccal mucosa over several layers.

Tight junctions (TJs) firmly seal epithelial cells and are key players in the epithelial barrier. TJs consist of several proteins, including those of the transmembrane claudin family and the scaffold zonula occludens (ZO) family. Epithelial tissues are exposed to different conditions: to air in the stratified epithelium of the skin and to liquids in the monolayer of the intestine. The TJs in stratified oral mucosal epithelium have remained insufficiently elucidated in terms of distributions, appearances and barrier functions of TJ proteins in normal buccal mucosa. We investigated these and ZO-1 and claudin-1 were found to be expressed in the top third and in the bottom three quarters of the mucosal epithelium. ZO-1 in the buccal mucosa was found to have an irregular linear appearance. ZO-1 in the buccal mucosa continuously existed in several layers. Electron microscopy revealed the buccal mucosa to have kissing points. In a biotin permeation assay that sought to investigate inside-outside barrier function, the biotin tracer penetrated several ZO-1 layers but did not pass through all the ZO-1 layers. We found that the oral mucosal cell knockdown of TJP1 or CLDN1 resulted in decreases of TER but no significant change in FITC-dextran leakage. Our results suggest that the distribution and appearance of ZO-1 in the buccal mucosa differ from those in the skin. We were unable to prove barrier function in this study but we did show barrier function against small molecules in vivo and against ions in vitro.

Adult stem cells in endometrial regeneration: Molecular insights and clinical applications.

Endometrial damage is an important cause of female reproductive problems, manifested as menstrual abnormalities, infertility, recurrent pregnancy loss, and other complications. These conditions are collectively termed "Asherman syndrome" (AS) and are typically associated with recurrent induced pregnancy terminations, repeated diagnostic curettage and intrauterine infections. Cancer treatment also has unexpected detrimental side effects on endometrial function in survivors independently of ovarian effects. Endometrial stem cells act in the regeneration of the endometrium and in repair through direct differentiation or paracrine effects. Nonendometrial adult stem cells, such as bone marrow-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells, with autologous and allogenic applications, can also repair injured endometrial tissue in animal models of AS and in human studies. However, there remains a lack of research on the repair of the damaged endometrium after the reversal of tumors, especially endometrial cancers. Here, we review the biological mechanisms of endometrial regeneration, and research progress and challenges for adult stem cell therapy for damaged endometrium, and discuss the potential applications of their use for endometrial repair after cancer remission, especially in endometrial cancers. Successful application of such cells will improve reproductive parameters in patients with AS or cancer. Significance: The endometrium is the fertile ground for embryos, but damage to the endometrium will greatly impair female fertility. Adult stem cells combined with tissue engineering scaffold materials or not have made great progress in repairing the injured endometrium due to benign lesions. However, due to the lack of research on the repair of the damaged endometrium caused by malignant tumors or tumor therapies, the safety and effectiveness of such stem cell-based therapies need to be further explored. This review focuses on the molecular insights and clinical application potential of adult stem cells in endometrial regeneration and discusses the possible challenges or difficulties that need to be overcome in stem cell-based therapies for tumor survivors. The development of adult stem cell-related new programs will help repair damaged endometrium safely and effectively and meet fertility needs in tumor survivors.

Liquid Chromatography-Nanoelectrospray Ionization-High-Resolution Tandem Mass Spectrometry Analysis of Apurinic/Apyrimidinic Sites in Oral Cell DNA of Cigarette Smokers, e-Cigarette Users, and Nonsmokers.

Cigarette smoking is an established risk factor for oral cancer. The health effects of e-cigarettes are still under investigation but may disturb oral cavity homeostasis and cause lung and cardiovascular diseases. Carcinogens and toxicants in tobacco products and e-cigarettes may damage DNA, resulting in the formation of apurinic/apyrimidinic (AP) sites and initiation of the carcinogenic process. In this study, we optimized a liquid chromatography-nanoelectrospray ionization-high-resolution tandem mass spectrometry method to analyze AP sites in buccal cell DNA of 35 nonsmokers, 30 smokers, and 30 e-cigarette users. AP sites in e-cigarette users (median 3.3 per 107 nts) were significantly lower than in smokers (median 5.7 per 107 nts) and nonsmokers (median 6.0 per 107 nts). AP sites in smokers were not significantly different from nonsmokers (p > 0.05). The e-cigarette vaporizing solvents propylene glycol and glycerin were tested and did not protect against AP site formation in in vitro control and carcinogen exposed rat liver homogenates. However, propylene glycol may inhibit bacteria in oral cells, resulting in reduced inflammation and related effects, and reduced AP site levels in e-cigarette user DNA. This is the first study to examine AP site formation in e-cigarette users and to evaluate AP sites in human oral cell DNA.

A prospective, multi-center, open-label, single-arm phase 2b study of autologous adult live cultured buccal epithelial cells (AALBEC) in the treatment of bulbar urethral stricture.

PURPOSE: To evaluate the safety and efficacy of autologous adult live cultured buccal epithelial cells (AALBEC) in treatment and management of bulbar urethral stricture in men. METHODS: This was a prospective, multi-center, open-label, single-arm phase 2b study. A total of 18 male patients with bulbar urethral stricture of at least 1 - 4 cm in length were enrolled in the study. All 16 patients had AALBEC implanted and were included in the safety set. Change in total American Urology Association (AUA) symptom score, urinary flow rates assessed by uroflowmetry and a requirement for surgery after 24 weeks from baseline were determined in patients. Data of treatment efficacy were analyzed. RESULTS: The AUA score at baseline was 21 (3.9) that showed a statistically significant reduction starting from week 2 [8 (4.4), p = 0.0001] which sustained until week 24 [2 (1.2), p = 0.0005]. Overall mean total AUA symptom score was reduced by 90.5% after the treatment. Significant reductions from baseline at week-24 were also observed in voiding time (92.5 (47.3) vs. 51.9 (17.4) s, p = 0.0046) and flow time [86.9 (48.2) vs. 47.9 (19.6) s, p = 0.0052]. All patients showed absence of any significant adverse events. CONCLUSION: Significant improvement was seen in the AUA symptom score and uroflowmetry parameters and no patients required surgery during 24 weeks post-treatment. It can be concluded that AALBEC is a safe and effective treatment for bulbar urethral stricture of 1 - 4 cm length to improve the quality of life and the physiological function of urethra.

Exposure to arsenic at different life-stages and DNA methylation meta-analysis in buccal cells and leukocytes.

BACKGROUND: Arsenic (As) exposure through drinking water is a global public health concern. Epigenetic dysregulation including changes in DNA methylation (DNAm), may be involved in arsenic toxicity. Epigenome-wide association studies (EWAS) of arsenic exposure have been restricted to single populations and comparison across EWAS has been limited by methodological differences. Leveraging data from epidemiological studies conducted in Chile and Bangladesh, we use a harmonized data processing and analysis pipeline and meta-analysis to combine results from four EWAS. METHODS: DNAm was measured among adults in Chile with and without prenatal and early-life As exposure in PBMCs and buccal cells (N = 40, 850K array) and among men in Bangladesh with high and low As exposure in PBMCs (N = 32, 850K array; N = 48, 450K array). Linear models were used to identify differentially methylated positions (DMPs) and differentially variable positions (DVPs) adjusting for age, smoking, cell type, and sex in the Chile cohort. Probes common across EWAS were meta-analyzed using METAL, and differentially methylated and variable regions (DMRs and DVRs, respectively) were identified using comb-p. KEGG pathway analysis was used to understand biological functions of DMPs and DVPs. RESULTS: In a meta-analysis restricted to PBMCs, we identified one DMP and 23 DVPs associated with arsenic exposure; including buccal cells, we identified 3 DMPs and 19 DVPs (FDR < 0.05). Using meta-analyzed results, we identified 11 DMRs and 11 DVRs in PBMC samples, and 16 DMRs and 19 DVRs in PBMC and buccal cell samples. One region annotated to LRRC27 was identified as a DMR and DVR. Arsenic-associated KEGG pathways included lysosome, autophagy, and mTOR signaling, AMPK signaling, and one carbon pool by folate. CONCLUSIONS: Using a two-step process of (1) harmonized data processing and analysis and (2) meta-analysis, we leverage four DNAm datasets from two continents of individuals exposed to high levels of As prenatally and during adulthood to identify DMPs and DVPs associated with arsenic exposure. Our approach suggests that standardizing analytical pipelines can aid in identifying biological meaningful signals.

Exposure to traffic-related air pollution and changes in exhaled nitric oxide and DNA methylation in arginase and nitric oxide synthase in children with asthma.

BACKGROUND: Traffic-related air pollution (TRAP) has been associated with increased risk of airway inflammation in children with asthma. While epigenetic changes could potentially modulate TRAP-induced inflammatory responses, few studies have assessed the temporal pattern of exposure to TRAP, epigenetic changes and inflammation in children with asthma. Our goal was to test the time-lag patterns of personal exposure to TRAP, airway inflammation (measured as fractional exhaled nitric oxide, FeNO), and DNA methylation in the promoter regions of genes involved in nitric oxide synthesis among children with asthma. METHODS: We measured personal exposure to black carbon (BC) and FeNO for up to 30 days in a panel of children with asthma. We collected 90 buccal cell samples for DNA methylation analysis from 18 children (5 per child). Methylation in promoter regions of nitric oxide synthase (NOS1, NOS2A, NOS3) and arginase (ARG1, ARG2) was assessed by bisulfite pyrosequencing. Linear-mixed effect models were used to test the associations of BC at different lag periods, percent DNA methylation at each site and FeNO level. RESULTS: Exposure to BC was positively associated with FeNO, and negatively associated with DNA methylation in NOS3. We found strongest association between FeNO and BC at lag 0-6 h while strongest associations between methylation at positions 1 and 2 in NOS3 and BC were at lag 13-24 h and lag 0-24 h, respectively. The strengths of associations were attenuated at longer lag periods. No significant associations between exposure to TRAP and methylation levels in other NOS and ARG isoforms were observed. CONCLUSIONS: Exposure to TRAP was associated with higher levels of FeNO and lower levels of DNA methylation in the promoter regions of the NOS3 gene, indicating that DNA methylation of the NOS3 gene could be an important epigenetic mechanism in physiological responses to TRAP in children with asthma.

Acid-electrolyzed functional water-induces Interleukin-1α release from Intracellular Storage Sites in Oral Squamous Cell Carcinoma.

The aim of this study was to examine the acid-electrolyzed functional water (FW)-mediated cytokine release in an oral squamous cell carcinoma-derived cell line (OSCC) following treatment with FW. FW is generated by the electrolysis of a sodium chloride solution and accelerate the burn wound healing. To elucidate the underlying mechanisms, the cytokine/chemokine secretion profile of HSC3 cells was examined using a cytokine array. FW treatment significantly induced interleukin (IL)-1α secretion, which was confirmed by enzyme-linked immunosorbent assay. Subsequently, the HSC3 cells were pre-treated with cycloheximide (CHX) for 1 h prior to FW stimulation to determine whether the augmented IL-1α secretion was due to enhanced protein synthesis. CHX pre-treatment did not affect IL-1α secretion suggesting that the secreted IL-1α might have been derived from intracellular storage sites. The amount of IL-1α in the cell lysate of the FW-treated HSC3 cells was significantly lower than that of the non-treated cells. Immunofluorescence staining using a polyclonal antibody against full-length IL-1α revealed a drastic reduction in IL-1α inside the FW- treated cells. IL-1α is synthesized in its precursor form (pIL-1α) and cleaved to produce pro-piece and mature IL-1α (ppIL-1α and mIL-1α) inside the cells. In the present study, only pIL-1α was detected within the HSC3 cells in its resting state. However, FW stimulation resulted in the release of the 33 kDa and two other smaller forms (about 19 kDa) of the protein. These results indicates that FW treatment induces IL-1α secretion, a typical alarmin, from the intracellular storage in OSCC cells.