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Poor prognosis associated with the dysregulated expression of activin A in a number of malignancies has been related to with numerous aspects of tumorigenesis, including angiogenesis. The present study investigated the prognostic significance of activin A immunoexpression in blood vessels and cancer cells in a number of oral squamous cell carcinoma (OSCC) cases and applied in vitro strategies to determine the impact of activin A on angiogenesis. In a cohort of 95 patients with OSCC, immunoexpression of activin A in both blood vessels and tumor cells was quantified and the association with clinicopathological parameters and survival was analyzed. Effects of activin A on the tube formation, proliferation and migration of human umbilical vein endothelial cells (HUVECs) were evaluated in gain-of-function (treatment with recombinant activin A) or loss-of-function [treatment with activin A-antagonist follistatin or by stable transfection with short hairpin RNA (shRNA) targeting activin A] conditions. Conditioned medium from an OSCC cell line with shRNA-mediated depletion of activin A was also tested. The profile of pro- and anti-angiogenic factors regulated by activin A was assessed with a human angiogenesis quantitative PCR (qPCR) array. Vascular endothelial growth factor A (VEGFA) and its major isoforms were evaluated by reverse transcription-qPCR and ELISA. Activin A expression in blood vessels demonstrated an independent prognostic value in the multivariate analysis with a hazard ratio of 2.47 [95% confidence interval (CI), 1.30-4.71; P=0.006) for disease-specific survival and 2.09 (95% CI, 1.07-4.08l: P=0.03) for disease-free survival. Activin A significantly increased tubular formation of HUVECs concomitantly with an increase in proliferation. This effect was validated by reduced proliferation and tubular formation of HUVECs following inhibition of activin A by follistatin or shRNA, as well as by treatment of HUVECs with conditioned medium from activin A-depleted OSCC cells. Activin A-knockdown increased the migration of HUVECs. In addition, activin A stimulated the phosphorylation of SMAD2/3 and the expression and production of total VEGFA, significantly enhancing the expression of its pro-angiogenic isoform 121. The present fndings suggest that activin A is a predictor of the prognosis of patients with OSCC, and provide evidence that activin A, in an autocrine and paracrine manner, may contribute to OSCC angiogenesis through differential expression of the isoform 121 of VEGFA.

Chronic periodontitis (CP) is an infectious inflammatory disease that affects tooth-supporting structures and in which dental plaque bacteria, immune mechanisms and genetic predisposition play important roles. Interleukin 4 (IL-4) is a key anti-inflammatory cytokine with relevant action in imbalances in inflamed periodontal tissue. Individuals carrying the TCI/CCI genotype (S-haplotype) of the IL-4 gene are 5 times more susceptible to CP, whereas the CTI/TTD genotype (P-haplotype) confers protection against CP. Compared with the S-haplotype, subjects with the P-haplotype produce higher levels of the IL-4 protein after non-surgical periodontal therapy. The present in vitro study aimed to investigate the functionality of IL-4 haplotypes in immune cells to obtain insight into the influence of these genetic variations in regulating immune responses to CP-associated bacteria. Peripheral blood was collected from 6 subjects carrying each haplotype, and their immune cells were challenged with periodontopathogens to compare responses of the different haplotypes with regard to gene expression, protein secretion and the immunophenotype of T helper responses. We found higher IL-4 mRNA and protein levels in the P-haplotype, which also presented higher levels of anti-inflammatory cytokines. In contrast, cells from S-haplotype subjects responded with higher levels of pro-inflammatory cytokines. S-haplotype individuals exhibited significantly greater polarization toward the Th1 phenotype, whereas the P-haplotype was associated with an attenuated response to periodontopathogens, with suggestive skewing toward Th2/M2 phenotypes. In conclusion, IL-4 genetic variations associated with susceptibility to or protection against chronic periodontitis are directly associated with influencing the response of immune cells to periodontopathogens.

The oral mucosa has an essential role in protecting against physical, microbial, and chemical harm. Compromise of this barrier triggers a wound healing response. Key events in this response such as immune infiltration, re-epithelialization, and stroma remodeling are coordinated by cytokines that promote cellular migration, invasion, and proliferation. Cytokine-mediated cellular invasion and migration are also essential features in cancer dissemination. Therefore, exploration of cytokines that regulate each stage of oral wound healing will provide insights about cytokines that are exploited by oral squamous cell carcinoma (SCC) to promote tumor development and progression. This will aid in identifying potential therapeutic targets to constrain SCC recurrence and increase patient survival. In this review, we discuss cytokines that overlap in oral wounds and SCC, emphasizing how these cytokines promote cancer progression.

ObjectivesStudies have documented the anti-inflammatory effects of spices, which may be related to treatment of chronic diseases. The purpose of this study was to evaluate the influence of curcumin and piperine and their association on experimental periodontal repair in rats.Materials and methodsPeriodontitis was induced via the installation of a ligature around the first molar. After 15 days, the ligatures were removed, and the rats were separated into groups (12 animals per group): (i) curcumin, (ii) piperine, (iii) curcumin+piperine, (iv) corn oil vehicle, and (v) control group (animals had ligature-induced periodontitis but were not treated). The compounds were administered daily, for 15 days by oral gavage. Animals were euthanized at 5 and 15 days, and hemimaxillae and gingival tissues were harvested. Bone repair was assessed by μCT (microcomputer tomography). Histological sections were stained with hematoxylin/eosin (H/E) for the assessment of cellular infiltrate or picrosirius red for quantification of collagen content, and subjected to immunohistochemistry for detecting NF-ĸB. Gingival tissues were used to evaluate levels of TGF-β and IL-10 (ELISA).ResultsCurcumin and piperine increased the TGF-β level, significantly improved the collagen repair, and decreased the cellularity and activation of NF-ĸB in the periodontal tissues, but only curcumin caused a significant increase in early bone repair.ConclusionCurcumin and piperine promoted a substantive effect on tissue repair; however, there was not synergistic effect of compounds administered in combination.Clinical relevanceCurcumin and piperine stimulates the tissue repair and may be potential candidates for the treatment of periodontal disease.

Background Oral squamous cell carcinoma (SCC) is associated with oral microbial dysbiosis. In this unique study, we compared pre- to post-treatment salivary microbiome in patients with SCC by 16S rRNA gene sequencing and examined how microbiome changes correlated with the expression of an anti-microbial protein. Results Treatment of SCC was associated with a reduction in overall bacterial richness and diversity. There were significant changes in the microbial community structure, including a decrease in the abundance of Porphyromonaceae and Prevotellaceae and an increase in Lactobacillaceae . There were also significant changes in the microbial community structure before and after treatment with chemoradiotherapy, but not with surgery alone. In patients treated with chemoradiotherapy alone, several bacterial populations were differentially abundant between responders and non-responders before and after therapy. Microbiome changes were associated with a change in the expression of DMBT1, an anti-microbial protein in human saliva. Additionally, we found that salivary DMBT1, which increases after treatment, could serve as a post-treatment salivary biomarker that links to microbial changes. Specifically, post-treatment increases in human salivary DMBT1 correlated with increased abundance of Gemella spp., Pasteurellaceae spp . , Lactobacillus spp., and Oribacterium spp . This is the first longitudinal study to investigate treatment-associated changes (chemoradiotherapy and surgery) in the oral microbiome in patients with SCC along with changes in expression of an anti-microbial protein in saliva. Conclusions The composition of the oral microbiota may predict treatment responses; salivary DMBT1 may have a role in modulating the oral microbiome in patients with SCC. Graphical Abstract After completion of treatment, 6 months after diagnosis, patients had a less diverse and less rich oral microbiome. Leptotrichia was a highly prevalent bacteria genus associated with disease. Expression of DMBT1 was higher after treatment and associated with microbiome changes, the most prominent genus being Gemella -6tcCpfJtMWsEpEDBUWdC3 Video Abstract

Background Salivary agglutinin, also known as deleted in malignant brain tumors 1 (DMBT1), is an anti-microbial protein. DMBT1 is low in saliva from patients with oral squamous cell carcinoma (OSCC) and dramatically increases after treatment, with accompanying microbial changes. While this suggests an association between DMBT1 suppression and changes in the oral microbiota, causation has not been established. DMBT1 is also a tumor suppressor protein; its loss promotes OSCC progression, but its role in OSCC development is unknown. In this study, OSCC development was investigated in a murine carcinogen model that simulates human OSCC. Microbiota were standardized between Dmbt1 knockout ( Dmbt1 −/− ) and wild-type ( Dmbt1 + / + ) mice via interbreeding and co-housing. Saliva was collected at baseline and at 4, 8, 12, 16, and 22 weeks post-carcinogen initiation (stopped at 16 weeks). Tongues were harvested at week 22 for histopathology, and the salivary microbiome was profiled by 16S rRNA sequencing. Microbial diversity metrics and conditional dependence networks assessed community structure, while longitudinal patterns were analyzed using a locally sparse varying coefficient mixed model and functional principal component analysis (fPCA). Results Despite microbiota standardization, Dmbt1 −/− and Dmbt1 + / + displayed differences in microbiome composition based on β-diversity metrics. At endpoint, carcinogen-treated Dmbt1 −/− showed higher OSCC prevalence and more aggressive invasion than Dmbt1 + / + . Several OTUs, including those from Lachnospiraceae, Sphingomonas , Carnobacteriaceae, and Candidatus Saccharibacteria families, demonstrated differential abundance patterns over time, either genotype-specific, diagnosis-specific, or both. Notably, Sphingomonas and Lachnospiraceae exhibited time-dependent abundance differences in mice that developed OSCC. fPCA identified taxa with abundance trajectories that were different between OSCC and precancer and genotype specific. Conclusions Thus, DMBT1 shapes salivary microbiota composition and protects against OSCC development. Dynamic, genotype-specific microbial shifts during carcinogenesis underscore the complex interplay between the oral microbiota and cancer progression. 52sTgPoWwfY7A3YogZ1rT1 Video Abstract Graphical Abstract

Objectives The aim of this study was to evaluate the effect of specific inhibition of MMP-13 on inflammation and inflammatory bone resorption in a murine model of lipopolysaccharide (LPS)-induced periodontitis. Materials and methods Periodontitis was induced in mice by micro-injections of LPS into the gingival tissues adjacent to the palatal surfaces of maxillary molars twice a week for 15 days. Matrix metalloproteinase-13 ( Mmp-13 ) shRNA or a specific biochemical inhibitor were also injected into the same sites in alternating days with the LPS injections. Efficacy of shRNA-mediated silencing of Mmp-13 was verified by quantitative real-time polymerase chain reaction (qPCR) and immunoblot. Bone resorption was assessed by microcomputed tomography (uCT). Histological sections stained with hematoxylin/eosin (H/E) were used in the stereometric analysis of the inflammatory infiltrate. Gingival tissues were used to evaluate expression of Mmp-13 , Il-6 , Tnf-α , Ptgs2 , and Rankl (qPCR). Protein levels of TGF-β and IL-10 in the tissues were determined by enzyme-linked immunosorbent assays (ELISA) or by MMP-13 and p38 immunoblot. Results Silencing Mmp-13 expression reduced bone resorption significantly. Expression of Mmp-13, Il-6, and Tnf-α, as well as the protein levels of IL-6 and TNF-α, was reduced in the animals treated with adenovirus-delivered shRNA; however, these effects were not associated with modulation of p38 MAPK signaling. Interestingly, inhibition Mmp-13 did not affect the severity of inflammatory infiltrate. Conclusions Site-specific inhibition of MMP-13 reduced bone resorption and production of inflammatory mediators associated with periodontal disease. Clinical relevance The results suggest that site-specific inhibition of MMP-13 may be an interesting strategy to modulate inflammation and reduce bone resorption in osteolytic inflammatory diseases.

Background Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide, with human papillomavirus (HPV)-related HNSCC rising to concerning levels. Extensive clinical, genetic and epigenetic differences exist between HPV-associated HNSCC and HPV-negative HNSCC, which is often linked to tobacco use. However, 5-hydroxymethylation (5hmC), an oxidative derivative of DNA methylation and its heterogeneity among HNSCC subtypes, has not been studied. Results We characterized genome-wide 5hmC profiles in HNSCC by HPV status and subtype in 18 HPV(+) and 18 HPV(−) well-characterized tumors. Results showed significant genome-wide hyper-5hmC in HPV(−) tumors, with both promoter and enhancer 5hmC able to distinguish meaningful tumor subgroups. We identified specific genes whose differential expression by HPV status is driven by differential hydroxymethylation. CDKN2A (p16), used as a key biomarker for HPV status, exhibited the most extensive hyper-5hmC in HPV(+) tumors, while HPV(−) tumors showed hyper-5hmC in CDH13, TIMP2, MMP2 and other cancer-related genes. Among the previously reported two HPV(+) subtypes, IMU (stronger immune response) and KRT (more keratinization), the IMU subtype revealed hyper-5hmC and up-regulation of genes in cell migration, and hypo-5hmC with down-regulation in keratinization and cell junctions. We experimentally validated our key prediction of higher secreted and intracellular protein levels of the invasion gene MMP2 in HPV(−) oral cavity cell lines. Conclusion Our results implicate 5hmC in driving differences in keratinization, cell junctions and other cancer-related processes among tumor subtypes. We conclude that 5hmC levels are critical for defining tumor characteristics and potentially used to define clinically meaningful cancer patient subgroups.

Human papillomavirus-positive oropharyngeal squamous cell carcinoma (HPV+ OPSCC) is now the leading HPV+ cancer in the United States and United Kingdom. Despite high cure rates, a significant subset of patients have aggressive HPV+ OPSCC that recurs; deciphering the underlying mechanisms will identify biomarkers that delineate patient subgroups for personalized treatment. In a comprehensive investigation using complementary clinically-relevant models of HPV+ OPSCC, we demonstrated that elevated expression of a HPV16 E6 spliced isoform (E6*I) compared to full-length E6 (E6FL), is critical for aggressive invasion. Introduction of splice switching oligonucleotides (SSOs) to inhibit E6FL splicing, effectively mitigated invasion. Mechanistic studies revealed that aggressive invasion occurs via E6*I-induced endocytosis of E-cadherin (ECAD) from the cell membrane, consistent with partial epithelial-to-mesenchymal transition (p-EMT). The clinical relevance of this mechanism was validated in patient cohorts where a reduced ratio of E6FL to all E6 splice variants was associated with high p-EMT signature, lower membrane:cell ECAD, and worse recurrence-free survival. Together, our findings show that low membrane:cell ECAD ratio in pre-treatment biopsies of HPV+ OPSCC, could stratify patients according to risk; patients with a low ratio may not be candidates for treatment de-intensification trials. Importantly, since ECAD detection is by immunohistochemistry, which is widely used even in diagnostic pathology laboratories with limited resources, membrane:cell ECAD ratio could be a clinically scalable biomarker. Targeting E6FL splicing with SSOs should be explored further as a logical strategy for therapeutic intervention in patients with HPV+ OPSCC.

Differential abundance (DA) analysis in microbiome studies has recently been used to uncover a plethora of associations between microbial composition and various health conditions. While current approaches to DA typically apply only to cross-sectional data, many studies feature a longitudinal design to better understand the underlying microbial dynamics. To study DA in longitudinal microbial studies, we introduce a novel varying coefficient mixed-effects model with local sparsity. The proposed method can identify time intervals of significant group differences while accounting for temporal dependence. Specifically, we exploit a penalized kernel smoothing approach for parameter estimation and include a random effect to account for serial correlation. In particular, our method operates effectively regardless of whether sampling times are shared across subjects, accommodating irregular sampling and missing observations. Simulation studies demonstrate the necessity of modeling dependence for precise estimation and support recovery. The application of our method to a longitudinal study of mice oral microbiome during cancer development revealed significant scientific insights that were otherwise not discernible through cross-sectional analyses. An R implementation is available at https://github.com/fontaine618/LSVCMM.