The participation of tumor residing pericytes in oral squamous cell carcinoma.

Pericytes are perivascular cells related to vessel structure and angiogenesis that can interact with neoplastic cells, interfering with cancer progression and outcomes. This study focused on the characterization of pericytes in oral squamous cell carcinoma (OSCC) using clinical samples and a transgenic mouse model of oral carcinogenesis. Nestin-/NG2+ (type-1) and nestin+/NG2+ (type-2) pericytes were analyzed by direct fluorescence after induction of oral carcinogenesis (4-nitroquinoline-1-oxide). Gene expression of neuron glial antigen-2 (NG2), platelet-derived growth factor receptor beta (PDGFR-ß), and cluster of differentiation 31 (CD31) was examined in human OSCC tissues. The protein expression of von Willebrand factor and NG2 was assessed in oral leukoplakia (i.e., oral potentially malignant disorders) and OSCC samples. Additionally, clinicopathological aspects and survival data were correlated and validated by bioinformatics using The Cancer Genome Atlas (TCGA). Induction of carcinogenesis in mice produced an increase in both NG2+ pericyte subsets. In human OSCC, advanced-stage tumors showed a significant reduction in CD31 mRNA and von Willebrand factor-positive vessels. Low PDGFR-ß expression was related to a shorter disease-free survival time, while NG2 mRNA overexpression was associated with a reduction in overall survival, consistent with the TCGA data. Herein, oral carcinogenesis resulted in an increase in NG2+ pericytes, which negatively affected survival outcomes.

Salivary molecules of bone remodeling and tissue repair after head and neck radiotherapy.

Head and neck radiotherapy causes quantitative and qualitative changes in saliva. The objective of this case-control study was to evaluate the salivary biomarkers associated with bone remodeling and tissue repair in patients submitted to radiotherapy for head and neck cancer treatment, compared with non-irradiated individuals. Total unstimulated saliva was collected for ELISA assay analysis of receptor activator for nuclear factor κ B (RANK) and its ligand (RANK-L), osteoprotegerin, matrix metalloproteinase-9/ tissue inhibitor of metalloproteinase-2, vascular endothelial growth factor, and epidermal growth factor. Statistics were performed, and revealed that salivary RANK (p = 0.0304), RANK-L (p = 0.0005), matrix metalloproteinase-9/ tissue inhibitor of metalloproteinase-2 (p = 0.0067), vascular endothelial growth factor (p = 0.0060), and epidermal growth factor (p < 0.0001) were reduced in patients, compared with the control group. Osteoprotegerin did not differ between the groups (p = 0.3765). Salivary biomarkers did not differ according to radiotherapy completion time (p > 0.05). In conclusion, the lower output of the salivary molecules - essential for bone remodeling and tissue repair - may disrupt tissue homeostasis and play a role in the pathogenesis of the radiotherapy-induced deleterious effects in the oral cavity.

Revisões integrativas: conceitos, planejamento e execução / Integrative reviews: concepts, planning, and execution

Objetivo: Fornecer um guia no formato de checklist para auxiliar pesquisadores na condução de revisões integrativas em Odontologia. Métodos:O guiapara revisões integrativas em Odontologiafoi construído a partir do Preferred Reporting Items for Systematic Reviews and Meta-Analyses (The PRISMA Statement). Resultados:Para o delineamento de revisões integrativas em Odontologia é preciso percorrer etapas distintas: 1. Identificação do tema (elaboração da pergunta de pesquisa); 2. Estabelecimento dos critérios de elegibilidade de estudos; 3. Busca sistematizada em diversas fontes de informação; 4. Coleta de dados; 5. Análise dos dados; 6. Discussão; 7. Apresentação da revisão/síntese do conhecimento. Os erros mais comuns ao realizar uma revisão integrativa estão relacionados à descrição incompleta ou não realização de etapas importantes, tais como: 1. Processo de seleção das evidências; 2. Estratégia de busca reprodutível; 3. Detalhes relacionados à busca, seleção e inclusão de estudos; 4. Aplicação dos critérios de elegibilidade; 5. Processo de extração dos dados (definição clara dos dados a serem extraídos, número de revisores envolvidos); 6. Apresentação do processo de seleção de estudos no formato de fluxograma; 7. Avaliação da qualidade dos estudos; 8. Síntese dos resultados. Conclusão:O guia para revisões integrativas em Odontologia apresenta utilidade na redução de equívocos metodológicos frequentemente observados nesse desenho de estudo, bem como estimula a condução de trabalhos com delineamentos robustos.

Aim: To provide a checklist to assist researchers in conducting integrative reviews in Dentistry. Methods: This guideline for integrative reviews in Dentistry was designed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyzes (The PRISMA Statement). Results: Planning integrative reviews in Dentistry involves different stages: 1. Identification of the theme (elaboration of the research question); 2. Establishment of eligibility criteria; 3. Systematized search in several databases and other data sources; 4. Data collection; 5. Data analysis; 6. Discussion; 7. Report on the review/summary of findings. The most common errors when conducting an integrative review are related to incomplete description or failure to perform important steps, such as: 1. Evidence synthesis; 2. A reproducible search strategy; 3. Details related to studies' search, screening, selection; 4. Clear state eligibility criteria; 5. Data extraction process (clear definition of the data to be extracted, number of reviewers involved); 6. Presentation studies' screening and selection process in a flowchart format; 7. Evaluation of the quality of the studies; 8. Summary of the findings. Conclusion:A guideline for integrative reviews in Dentistry intends to reduce methodological issues frequently observed in this study design, as well as to encourage researchers to conduct studies with a robust design.

Salivary molecules of bone remodeling and tissue repair after head and neck radiotherapy

Abstract Head and neck radiotherapy causes quantitative and qualitative changes in saliva. The objective of this case-control study was to evaluate the salivary biomarkers associated with bone remodeling and tissue repair in patients submitted to radiotherapy for head and neck cancer treatment, compared with non-irradiated individuals. Total unstimulated saliva was collected for ELISA assay analysis of receptor activator for nuclear factor κ B (RANK) and its ligand (RANK-L), osteoprotegerin, matrix metalloproteinase-9/ tissue inhibitor of metalloproteinase-2, vascular endothelial growth factor, and epidermal growth factor. Statistics were performed, and revealed that salivary RANK (p = 0.0304), RANK-L (p = 0.0005), matrix metalloproteinase-9/ tissue inhibitor of metalloproteinase-2 (p = 0.0067), vascular endothelial growth factor (p = 0.0060), and epidermal growth factor (p < 0.0001) were reduced in patients, compared with the control group. Osteoprotegerin did not differ between the groups (p = 0.3765). Salivary biomarkers did not differ according to radiotherapy completion time (p > 0.05). In conclusion, the lower output of the salivary molecules - essential for bone remodeling and tissue repair - may disrupt tissue homeostasis and play a role in the pathogenesis of the radiotherapy-induced deleterious effects in the oral cavity.

Pericyte in Oral Squamous Cell Carcinoma: A Systematic Review.

The microenvironment of oral cancer is highly dynamic and has been proved to affect tumor progression. Pericytes are blood vessels surrounding cells that have recently gained attention for their roles in vascular and cancer biology. The objective of the present study was to survey the scientific literature for conclusive evidence about whether pericytes are part of blood vessels in oral squamous cell carcinoma (OSCC) and their roles in the tumor microenvironment and clinical outcomes. A systematic electronic search was undertaken in Medline Ovid, PubMed, Web of Science, and Scopus. Eligibility criteria were: publications adopting in vivo models of OSCC that included pericyte detection and assessment by pericyte markers (e.g., α-smooth muscle actin, neuron-glial antigen 2 and platelet-derived growth factor receptor-ß). The search yielded seven eligible studies (from 2008 to 2018). The markers most commonly used for pericyte detection were α-smooth muscle actin and neuron-glial antigen 2. The studies reviewed showed the presence of immature vessels exhibiting a reduction of pericyte coverage in OSCC and indicated that anti-cancer therapies could contribute to vessel normalization and pericyte regain. The pericyte population is significantly affected during OSCC development and cancer therapy. While these findings might suggest a role for pericytes in OSCC progression, the limited data available do not allow us to conclude whether they modify the tumor microenvironment and clinical outcome.

Oral microbial dysbiosis linked to worsened periodontal condition in rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint inflammation. Individuals with RA have a higher risk of periodontitis and periodontitis has been linked to RA through the production of enzymes by periodontal pathogens that citrullinate proteins. This linkage is supported by findings that periodontitis is associated with increased RA severity and treatment of periodontitis can improve the symptoms of RA. The possible mechanism for this association is through dysbiosis of the oral microbiota triggered by RA-induced systemic inflammation. We examined the RA status of subjects by measuring the number of tender and swollen joints, anti-citrullinated protein antibody and rheumatoid factor. Periodontal disease status and salivary cytokine levels were measured, and dental plaque analyzed by 16S rRNA high throughput sequencing. RA patients had a higher bacterial load, a more diverse microbiota, an increase in bacterial species associated with periodontal disease, more clinical attachment loss, and increased production of inflammatory mediators including IL-17, IL-2, TNF, and IFN-γ. Furthermore, changes in the oral microbiota were linked to worse RA conditions. Our study provides new insights into the bi-directional relationship between periodontitis and RA and suggest that monitoring the periodontal health of RA patients is particularly important.

Role of atypical chemokine receptor ACKR2 in experimental oral squamous cell carcinogenesis.

BACKGROUND: Chemokines and chemokine receptors are critical in oral tumourigenesis. The atypical chemokine receptor ACKR2 is a scavenger of CC chemokines controlling the availability of these molecules at tumour sites, but the role of ACKR2 in the context of oral carcinogenesis is unexplored. METHODS: In this study, wild-type (WT) and ACKR2 deficient mice (ACKR2-/-) were treated with chemical carcinogen 4-nitroquinoline-1-oxide (4NQO) for induction of oral carcinogenesis. Tongues were collected for macro and microscopic analysis and to evaluate the expression of ACKRs, CC chemokines and its receptors, inflammatory cytokines, angiogenic factors, adhesion molecules and extracellular matrix components. RESULTS: An increased expression of ACKR2 in squamous cell carcinoma (SCC) lesions of 4NQO-treated WT mice was observed. No significant differences were seen in the ACKR1, ACKR3 and ACKR4 mRNA expression comparing SCC lesions from WT and ACKR2-/- treated mice. Significantly higher expression of CCL2, IL-6 and IL-17 was detected in ACKR2-/- treated mice. In contrast, the expression of other CC-chemokines, and receptors, angiogenic factors, adhesion molecules and extracellular matrix components were similarly increased in SCC lesions of both groups. Clinical and histopathological analysis revealed no differences in inflammatory cell recruitment and in the SCC incidence comparing WT and ACKR2-/- treated mice. CONCLUSION: The results suggest that ACKR2 expression regulates inflammation in tumour-microenvironment but the absence of ACKR2 does not impact chemically-induced oral carcinogenesis.

Relevance of CCL3/CCR5 axis in oral carcinogenesis.

The chemokine CCL3 is a chemotactic cytokine crucial for inflammatory cell recruitment in homeostatic and pathological conditions. CCL3 might stimulate cancer progression by promoting leukocyte accumulation, angiogenesis and tumour growth. The expression of CCL3 and its receptors CCR1 and CCR5 was demonstrated in oral squamous cell carcinoma (OSCC), but their role was not defined. Here, the functions of CCL3 were assessed using a model of chemically induced tongue carcinogenesis with 4-nitroquinoline-1-oxide (4NQO). Lineages of OSCC were used to analyse the effects of CCL3 in vitro. The 4NQO-induced lesions exhibited increased expression of CCL3, CCR1 and CCR5. CCL3-/- and CCR5-/- mice presented reduced incidence of tongue tumours compared to wild-type (WT) and CCR1-/- mice. Consistently, attenuated cytomorphological atypia and reduced cell proliferation were observed in lesions of CCL3-/- and CCR5-/- mice. OSCC from CCL3-/- mice exhibited lower infiltration of eosinophils and reduced expression of Egf, Fgf1, Tgf-ß1, Vegfa, Vegfb, Itga-4, Vtn, Mmp-1a, Mmp-2 and Mmp-9 than WT mice. In vitro, CCL3 induced invasion and production of CCL5, IL-6, MMP -2, -8, -9. Blockage of CCL3 in vitro using α-CCL3 or Evasin-1 (a CCL3-binding protein) impaired tumour cell invasion. In conclusion, CCL3/CCR5 axis has pro-tumourigenic effects in oral carcinogenesis. The induction of inflammatory and angiogenic pathways and eosinophils recruitment appear to be the underlying mechanism explaining these effects. These data reveal potential protective effects of CCL3 blockade in oral cancer.

Contribution of atypical chemokine receptor 2/ackr2 in bone remodeling.

INTRODUCTION: Bone remodeling is a tightly regulated process influenced by chemokines. ACKR2 is a decoy receptor for CC chemokines functioning as regulator of inflammatory response. In this study we investigated whether the absence of ACKR2 would affect bone phenotype and remodeling induced by mechanical loading. METHODS: An orthodontic appliance was placed between incisors and first molar of ACKR2 deficient (ACKR2-/-) and C57BL6/J (wild-type/WT) mice. Microtomography, histology and qPCR were performed to evaluate bone parameters, orthodontic tooth movement (OTM), bone cells counts and the expression of ACKR2, bone remodeling markers, CC chemokines and chemokines receptors. Bone marrow cells (BMC) from WT and ACKR2-/- mice were differentiated in osteoclasts and osteoblasts for analysis of activity and expression of specific markers. RESULTS: Mechanical stimulus induced ACKR2 production in periodontium. The expression of ACKR2 in vitro was mostly detected in mature osteoclasts and early-differentiated osteoblasts. Although ACKR2-/- mice exhibited regular phenotype in maxillary bone, the amount of OTM, osteoclasts counts and the expression of pro-resorptive markers were increased in this group. In contrast, the number of osteoblasts and related markers were decreased. OTM resulted in augmented expression of CC chemokines and receptors CCR5 and CCR1 in periodontium, which was higher in ACKR2-/- than WT mice. In vitro experiments demonstrated an augmented formation of osteoclasts and diminished differentiation of osteoblasts in ACKR2-/- mice. CONCLUSIONS: These data suggests that ACKR2 functions as a regulator of mechanically-induced bone remodeling by affecting the differentiation and activity of bone cells and the availability of CC chemokines at periodontal microenvironment. Therapeutic strategies based on increase of ACKR2 might be useful to hinder bone loss in inflammatory conditions.

Significance of chemokine and chemokine receptors in head and neck squamous cell carcinoma: A critical review.

Chemokines are small chemotactic proteins that coordinate circulation of immune/inflammatory cells throughout body compartments. Because of this property chemokines and their cell surface receptors are implicated in several physiological and pathological conditions, including cancer. These molecules are expressed by neoplastic or stromal cells and have effects at tumor primary site (e.g. stimulating angiogenesis and tumor cells motility) and lymph nodes (creating a gradient to direct migration of neoplastic cells). In this article we review the current knowledge about the function(s) of chemokines and receptors in squamous cell carcinoma from the oral cavity and head and neck region. Accumulating evidence suggests some chemokine(s) and receptor(s) as potential targets in adjuvant therapies for these malignancies.

Cytokine measurements in gingival crevicular fluid and periodontal ligament: Are they correlated?

INTRODUCTION: Mechanical stress can induce molecular changes in gingival crevicular fluid (GCF) and the periodontal ligament (PDL). It is still not clear whether changes in the PDL and GCF are linked. In this study, we aimed to analyze the expression of cytokines in GCF and PDL after mechanical stress. METHODS: Twenty-three healthy patients were included. The experimental group consisted of premolars subjected to a force of 0.980 N for 1, 3, 7, 14, 21, or 28 days. The contralateral teeth were the controls. GCF and PDL samples were collected at the same time points for analysis of cytokines using the cytometric bead array. RESULTS: Interleukin (IL)-6 (IL-6) production was significantly elevated in the PDL on day 1 after force application. Significantly strong positive correlations between GCF and PDL in experimental group were seen on days 3 (interferon-gamma), 7 (IL-10), 14 (IL-17A), and 28 (IL-17A, tumor necrosis factor-alpha), and significantly strong negative correlation were seen on days 14 (interferon-gamma) and 21 (IL-2, IL-10). CONCLUSIONS: Different patterns of IL-6 expression were seen in the PDL and GCF after mechanical stress. Despite occasional correlations between GCF and PDL, the molecular contributions of the PDL to the GCF changes could not be clearly defined by our model.

Immunosuppressive mediators of oral squamous cell carcinoma in tumour samples and saliva.

The goal of this study was to compare the salivary concentrations of IL-10, TGF-ß1 and soluble HLA-G (sHLA-G) in patients with oral squamous cell carcinoma (OSCC) to those in healthy individuals (control group), and to correlate the expression of these mediators in saliva with that in the tumour microenvironment. Neoplastic tissue and saliva samples from patients with OSCC (n=22) were analysed by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) respectively. We detected high expression of IL-10 and HLA-G in the tumour microenvironment when compared to healthy oral mucosa samples. Determination of IL-10 salivary concentration enabled us to distinguish patients with OSCC from healthy individuals (P=0.038), which showed correlation with tissue expression of this cytokine. HLA-G salivary release was similar in both groups (P=0.17) and no correlation with tumour expression was observed. TGF-ß1 expression was low or absent in tumours, and salivary concentration was similar between groups. Our results suggest that of the three markers analysed, IL-10 is a potential salivary biomarker. Furthermore, the elevated expression of HLA-G and IL-10 in tumour sites could favour the escape of tumour cells from immune defense mechanisms.

Eosinophil depletion protects mice from tongue squamous cell carcinoma induced by 4-nitroquinoline-1-oxide.

AIMS: Tumor-associated tissue eosinophilia (TATE) has been correlated with prognosis in oral squamous cell carcinoma (OSCC). This study aimed to investigate whether eosinophils depletion affects experimental oral carcinogenesis. METHODS AND RESULTS: BALB/c (wild type - WT) and eosinophil-deficient (Δdb/GATA-1) mice were treated with the carcinogen 4-nitroquinoline-1-oxide (4NQO) in drinking water for 28 weeks. Tongues were collected for histopathological and immunohistochemical analysis, as well as for the evaluation of cytokines/chemokines by ELISA. The tongue SCC induced by 4NQO was associated with a rise in eosinophil numbers. WT-treated group showed a significantly increased incidence of SCC, with higher cytological atypia, in comparison with Δdb/GATA-1 mice. Consistently, the proliferative index was higher in WT compared to the Δdb/GATA-1-treated group. No significant changes in the concentration of CCL3, CCL11 and TNF-α were detected for both groups after 4NQO treatment. CONCLUSIONS: These results suggest that eosinophils might be responsible for the deleterious outcome of experimental tongue carcinogenesis, given that their ablation protects mice from OSCC.

Midkine expression in oral squamous cell carcinoma and leukoplakia.

BACKGROUND: Midkine (MK), a 13-kDa heparin-binding growth factor, is overexpressed in various human cancers. However, its role in the development and progression of oral cavity squamous cell carcinoma (OCSCC) is still unclear. Thus, the aim of this study was to evaluate the expression of MK in samples of OCSCC, leukoplakia, and healthy oral mucosa (control). METHODS: Surgically excised specimens from patients with primary OCSCC (n = 28) were immunostained for MK, Ki-67, PCNA, p53, bcl-2, Bax, and CD31. Besides this, MK expression was also investigated in leukoplakia and normal oral mucosa. The relationship of MK(+) cells with clinical parameters (tumor location, tumor size, lymph node metastasis, and survival) and microscopic parameters (WHO histological grading, intensity of inflammation, proliferation index, apoptosis, and angiogenesis) was also evaluated. RESULTS: The results showed that MK expression was increased in OCSCC in relation to leukoplakia and normal mucosa. Furthermore, MK expression was increased in late-stage tumors (T3/T4) compared with early-stage lesions (T1/T2). MK-positive lesions also showed increased expression of the anti-apoptotic protein bcl-2. CONCLUSION: OCSCC, particularly late-stage tumors, exhibits increased MK expression, which may be involved in tumor progression via upregulation of anti-apoptotic genes, as shown by the augmented bcl-2 positivity in MK-positive tumors.