Aline Carvalho Batista

Tumor-associated macrophages and the profile of inflammatory cytokines in oral squamous cell carcinoma

OBJECTIVE To evaluate and characterize macrophage populations (M1/M2) in the tumor microenvironment of oral cavity squamous cell carcinoma (OCSCC). The relationship between macrophages and clinicopathological factors, such as survival data, lymph node metastasis, tumoral proliferation, and WHO histological grading are also analyzed. MATERIALS AND METHODS The samples consisted of surgically excised specimens from patients with non-metastatic and metastatic OCSCC and normal oral mucosa (control). Immunohistochemistry, flow cytometry, and qRT-PCR were used to evaluate macrophage populations and the expression of pro- (IL-12, IL-23, and INF-γ) and anti-inflammatory (IL-10 and TGF-β) cytokines. The level required for statistical significance was defined as p<0.05. RESULTS The data showed a predominance of M2 phenotype (high percentage of IL-10(+)TGF-β(+)) macrophages in the tumor microenvironment of OCSCC. A higher percentage of macrophages expressing TGF-β was seen in the OCSCC group when compared with healthy individuals. The assessment of mRNA expression also presented a greater expression of anti-inflammatory cytokines TGFβ and IL10 in OCSCC when compared with the control group. The percentage of macrophages, demonstrated by immunohistochemistry, was significantly higher in the metastatic OCSCC group than in the non-metastatic and control groups. The log-rank test also showed that the mean survival time for patients with high levels of macrophages was less (44 months) when compared with patients with a low percentage of such cells (93 months). CONCLUSION A predominance of the M2 phenotype in the tumor microenvironment of OCSCC could contribute to local immunosuppression, via TGF-β production, and consequently greater lymph node involvement and reduced patient survival time.

Comparative immunohistochemical study of the presence of mast cells in apical granulomas and periapical cysts: possible role of mast cells in the course of human periapical lesions

Cells other than macrophages and lymphocytes have recently been shown capable of producing cytokines and mediators. Among these are mast cells, a cell population now recognized for its immunoregulatory properties. Little is known about the complex interactions between cells, cytokines, and other inflammatory elements in periapical lesions. The objective of this investigation was to determine the immunohistochemical pattern of expression of mast cells tryptase in periapical lesions based on study of 20 apical granulomas and 20 periapical cysts. Microscopic analysis revealed mast cells to be present in greater numbers in periapical cysts than in apical granulomas, and in cysts were more numerous in regions of active inflammation. Mast cells tended to be more common in the peripheral regions of both periapical lesions, and were often found in close proximity to lymphocytes. These findings lead us to propose a functional relationship between these two cell populations that may facilitate elicitation of an immune response contributory to the pathogenesis of periapical lesions.

Differential infiltration of CD8+ and NK cells in lip and oral cavity squamous cell carcinoma.

BACKGROUND CD8+ and natural killer (NK) cells have been considered the most effective cells in the combat of cancer, contributing to better prognosis and longer survival. METHODS The aim of this study was to evaluate the population of CD8+ and NK cells, by immunohistochemistry, in samples of oral cavity squamous cell carcinoma (OCSCC) and lip squamous cell carcinoma (LSCC), leukoplakia, actinic cheilitis, and healthy oral mucosa (control). The relationship of CD8+ and NK cells with survival data, lymph node metastasis, tumor size, and proliferative index was also evaluated. RESULTS The number of peritumoral and intratumoral CD8+ and NK cells was significantly higher in LSCC, when compared with control, pre-malignant lesions, and OCSCC. A higher proportion of peritumoral CD8+ cells demonstrated correlation with a lower neoplastic proliferative index. Moreover, patients with OCSCC with a high density of peritumoral CD8+ cells showed a tendency towards a longer survival time. CONCLUSIONS The differential CD8+ and NK cells infiltration in oral SCC might reflect a distinctive tumor microenvironment with a favorable local cytotoxic immune response against neoplastic cells.

Involvement of CXCL12 and CXCR4 in Lymph Node Metastases and Development of Oral Squamous Cell Carcinomas

The chemokine stromal cell-derived factor (SDF-1/CXCL12) and its specific receptor, CXCR4, have been implicated in the regulation of tumor growth and organ-specific spread. The aim of this study was to determine the expression of CXCL12 and CXCR4 in samples obtained from primary squamous cell carcinoma (SCC) of the oral cavity (OCSCC) and of the lip (LSCC) and in metastatic and non-metastatic lymph node tissues. The relationship of CXCL12/CXCR4 with clinical and microscopic parameters was also evaluated. The analysis of mRNA expression revealed a higher expression of CXCR4 in oral SCC compared with healthy oral mucosa (p = 0.006). The density of CXCR4+ cells was higher in parenchyma of OCSCC with lymph node metastases than in LSCC. With regard to the stroma, OCSCC showed a greater CXCR4+ and CXCL12+ cell percentage in relation to LSCC. Furthermore, the density of CXCL12+ and CXCR4+ nodal cells was higher in metastatic than non-metastatic lymph nodes in the same patients. Considering clinical and microscopic parameters, we found a positive association between the percentages of CXCL12+ and CXCR4+ stromal cells and the tumor proliferation index. Our findings suggest that the CXCL12/CXCR4 system may play a role in tumor cell spread to lymph nodes and also in neoplastic development.

Distinctive clinical and microscopic features of squamous cell carcinoma of oral cavity and lip

OBJECTIVE This study was undertaken to gain insight into the distinctive features of squamous cell carcinoma of the lip (LSCC) and oral cavity (OCSCC). STUDY DESIGN A total of 37 cases of LSCC and 54 of OCSCC were compared regarding clinical and microscopic findings. RESULTS Predominance of head and neck TNM stages (HNTNM) T1-T2 in LSCC and T3-T4 in OCSCC was observed. A significant percentage (33.3%) of patients with OCSCC at HNTNM T1-T2 presented cervical lymph node metastasis and 18.75% died, whereas lymph node metastasis was absent and no patient died with LSCC at HNTNM T1-T2. LSCC demonstrated a higher number of cases with intense peritumoral inflammatory infiltrate compared with OCSCC. The analysis of the proliferative index demonstrated a significantly higher percentage of PCNA+ and Ki-67+ cells in OCSCC compared with LSCC. CONCLUSION Our results suggest that OCSCC and LSCC demonstrate distinct clinical and microscopic characteristics that reflect different biologic behavior and prognosis.

Investigation of Ehrlich ascites tumor cell death mechanisms induced by Synadenium umbellatum Pax.

ETHNOPHARMACOLOGICAL RELEVANCE Synadenium umbellatum Pax. is widely found in South America and empirically used in Brazil for the treatment of several diseases, mainly cancer. The aim of the study was to investigate cell death mechanisms induced by Synadenium umbellatum Pax. using Ehrlich ascites tumor (EAT) cells, as well as the myelotoxicity potential of this plant. MATERIALS AND METHODS S. umbellatum cytotoxicity was evaluated in EAT cells by trypan blue exclusion and MTT reduction test and the mechanisms involved in EAT cell death were investigated by light and fluorescence microscopy, flow cytometry and immunocytochemistry. Investigation of S. umbellatum myelotoxicity was performed by clonogenic assay of colony forming unit- granulocyte macrophage (CFU-GM). RESULTS AND CONCLUSION Our results demonstrated that S. umbellatum decreased the viability of EAT cells using both methods. Morphological analyses revealed that S. umbellatum-treatment induced EAT cell death by apoptotic pathway. We demonstrated the occurrence of reactive oxygen species (ROS) overgeneration, increased intracellular Ca(2+) concentration, alteration in mitochondrial membrane potential, phosphatydylserine externalization, and activation of caspases 3, 8, and 9. However, S. umbellatum produced myelotoxicity in bone marrow cells in a concentration-dependent manner. In comparison to EAT cells, the effects of S. umbellatum in bone marrow cells were 8-fold lower. Taken together, our results showed that S. umbellatum induced apoptosis in EAT cells at several levels and seems more toxic to tumor cells than to normal bone marrow cells.

Expression of bone resorption regulators (RANK, RANKL, and OPG) in odontogenic tumors.

OBJECTIVE To investigate the expression of bone resorption regulators (receptor activator of nuclear factor kappa B [RANK], RANK ligand [RANKL], and osteoprotegerin [OPG]) in calcifying cystic odontogenic tumor (CCOT), adenomatoid odontogenic tumor (AOT), calcifying epithelial odontogenic tumor (CEOT), odontogenic myxoma (OM), and ameloblastic fibroma (AF). STUDY DESIGN The expression of these mediators was evaluated by means of immunohistochemistry. RESULTS All specimens demonstrated positive immunoreactivity to RANK, RANKL, and OPG. The quantification of these mediators in epithelium revealed a similar pattern of expression for RANKL and OPG in CCOT, AOT, CEOT, and AF. With regard to stromal/mesenchymal cells, the majority of AOT and CCOT cases showed a higher content of OPG than RANKL, whereas CEOT, OM, and especially AF had a tendency to present a greater content of RANKL than OPG. CONCLUSION Our data indicate that the CCOT, AOT, CEOT, OM, and AF cell constituents express key regulators of bone metabolism that might locally modulate tumor-associated bone resorption.

Characterization of inflammatory cell infiltrate in human dental pulpitis.

INTRODUCTION To evaluate the microscopic characteristics and densities (per mm(2) ) of tryptase(+) mast cells, CD4(+) T helper lymphocytes, CD45RO(+) memory T lymphocytes, foxp3(+) T regulatory lymphocytes, CD20(+) B lymphocytes, CD68(+) macrophages, and CD31(+) blood vessels in human dental pulpitis (n=38) and healthy pulpal tissue (n=6). METHODOLOGY The pulps of 38 human teeth with a clinical diagnosis of irreversible pulpitis were removed by pulpectomy. The pulp tissue was immersed in 10% buffered formalin for evaluation using light microscopy. Tryptase, CD4, CD45RO, foxp3, CD20, CD68, and CD31 expressions were analysed using immunohistochemistry; other microscopic features, such as intensity of inflammatory infiltrate and collagen deposition, were evaluated using haematoxylin and eosin stain. Wilcoxon and Mann-Whitney tests were used for statistical analysis. The significance level was set at α=5%. RESULTS Two microscopic patterns of pulpitis were found: group 1 (G1) (n=15) had an intense inflammatory infiltrate and mild collagen deposition; conversely, group 2 (G2) (n=23) had a scarce inflammatory infiltrate and intense collagen deposition. The numbers of CD68(+) macrophages (P=0.004) and CD20(+) B (P=0.068) lymphocytes and the density of blood vessels (P=0.002) were higher in G1 than in G2. However, a similar number of CD4(+) and CD45RO(+) T lymphocytes was found in both groups (P>0.05). When present, tryptase(+) mast cells were equally distributed in G1 and G2, whereas foxp3(+) T regulatory lymphocytes were detected in 59% and 14% of the samples of G1 and G2. Controls exhibited lower numbers of foxp3, tryptase, CD4, CD45RO, CD68 and CD20 positive cells than G1 and G2. CONCLUSIONS Irreversible pulpitis had distinct microscopic features with important quantitative and qualitative differences in inflammatory cell infiltration.

A comparative study of microvessel density in squamous cell carcinoma of the oral cavity and lip

OBJECTIVES The objective of this study was to comparatively evaluate the density of lymphatic vessels (LVD) and neoformed microvessels (NMVD) in squamous cell carcinoma of the oral cavity (OCSCC) and lip (LSCC). Association between LVD/NMVD and vascular endothelial growth factor (VEGF)-A/-C was also assessed. STUDY DESIGN OCSCC and LSCC were compared with regard to immunoexpression of LVD, NMVD, and vascular endothelial growth factor-A (VEGF)-A/-C. Association between VEGF-A/-C with vascularity was also assessed. Statistical analyses were performed using t test, Pearson χ(2), and Mann-Whitney tests. Statistical significance was accepted at P less than .05. RESULTS The NMVD and VEGF-C expressions were significantly higher in OCSCC compared with LSCC. NMVD was associated with VEGF-C in OCSCC, but not in LSCC. CONCLUSIONS Differences in NMVD and VEGF-C were found between OCSCC and LSCC. Positive association between VEGF-C and NMVD was observed in OCSCC, but not in LSCC, which may be one of the contributing factors that account for the distinctive clinical-biological behavior of these lesions.

Association of CCL2 with Lymph Node Metastasis and Macrophage Infiltration in Oral Cavity and Lip Squamous Cell Carcinoma

Aims/Methods: We investigated the expression of CCL2 by immunohistochemistry in samples of oral cavity squamous cell carcinoma (OCSCC), lip squamous cell carcinoma (LSCC) and metastatic and nonmetastatic lymph nodes. The relationship of CCL2 with survival data was also evaluated. Results: The percentage of CCL2+ cells in tumor parenchyma was similar in OCSCC and LSCC. In stroma, OCSCC showed a higher number of CCL2+ cells when compared with LSCC. In contrast, higher CD68+ macrophage counts were observed in LSCC. Low macrophage counts were significantly related to a greater proliferative index in tumoral cells. The density of CCL2+ nodal cells was significantly higher in metastatic lymph nodes when compared with nonmetastatic lymph nodes. When considering CCL2 in the parenchyma, the mean survival time for the patients with high CCL2+ cell counts was lower than that for patients with low CCL2+ cell counts. In contrast, in stroma, the mean survival time for the patients with high CCL2+ cell percentages was higher than for those with low CCL2+ cell percentages. Conclusion: Our findings suggest a role for CCL2 in the spreading of tumoral cells to the lymph node in OCSCC. Furthermore, the CCL2-independent macrophage infiltration may play a role in determining less aggressive behavior.