Expression of CD163, interleukin‐10, and interferon‐gamma in oral squamous cell carcinoma: mutual relationships and prognostic implications

Tumor‐associated macrophages (TAMs) and their associated inflammatory cytokines represent the major inflammatory component of the stroma of many tumors and can affect prognosis in the case of neoplasms. The objective of this study was to determine the prognostic significance of CD163⁺ cells, interleukin‐10 (IL‐10), and interferon‐gamma (IFN‐γ) in oral lesions associated with oral squamous cell carcinoma (OSCC). The levels of CD163, IFN‐γ, and IL‐10 in the tissue samples of 240 patients with OSCC and 58 patients with other oral lesions were assessed by immunohistochemistry. Individuals with low IFN‐γ levels, high IL‐10 levels, and low CD163 levels were of special concern with respect to OSCC progression. We found that high levels of CD163, or a combination of low IFN‐γ levels, high IL‐10 levels, and low CD163 levels, were associated with poorer overall survival (OS). CD163⁺ cells provide better predictive power for OS in comparison with traditional markers, such as clinical stage and lymph node metastasis. Therefore, CD163⁺ cells may be effective prognostic predictors of OSCC. IL‐10 may also indicate poor outcomes when IFN‐γ secretion is low and the cells are CD163^?.     

Regulatory T cells and M2-polarized tumour-associated macrophages are associated with the oncogenesis and progression of oral squamous cell carcinoma

Regulatory T cells (Tregs) and tumour-associated macrophages (TAMs) contribute to the tumour microenvironment by inhibiting anti-tumour immune responses. This study was performed to investigate the roles of Tregs and TAMs in oral squamous cell carcinoma (OSCC) and oral epithelial precursor lesions (OEPL). The expression of Treg markers CD25 and FoxP3 and TAM markers CD163 and CD204 was investigated in 82 OSCC and 45 OEPL specimens, and their associations with clinicopathological parameters were analyzed. Correlations were found among CD25, FoxP3, CD163, and CD204 levels (P < 0.001), and these targets were up-regulated in OSCC compared to OEPL (P < 0.001). In OSCC, infiltration of Tregs and/or M2 TAMs was associated with sex and clinicopathological features, such as tumour size, nodal metastasis, tissue differentiation, stromal reaction, invasive behaviour, and invasive depth. In OEPL, CD25, FoxP3, CD163, and CD204 immunoreactivities were significantly associated with sex, postoperative recurrence, and cancerization to OSCC. This study is novel in showing that the infiltration of Tregs and M2 TAMs is significantly associated with the progression of premalignant lesions to OSCC. This suggests that these cells represent prognostic biomarkers for premalignant lesion progression and that immunotherapeutic approaches to control Treg/M2 TAM numbers could protect against progression to malignancy.     

Cancer-associated fibroblasts and CD163-positive macrophages in oral squamous cell carcinoma: their clinicopathological and prognostic significance

J Oral Pathol Med (2012) 41 : 444–451 Background: Stromal cells are believed to affect cancer invasion and metastasis. The purpose of this study was to evaluate the distribution of cancer‐associated fibroblasts (CAFs) and the incidence of tumor‐associated macrophages (TAMs) in oral squamous cell carcinoma (OSCC), focusing on clinicopathological factors and patient prognosis, as well as cancer invasion. Methods: The study included 108 patients with OSCC. Anti‐α‐smooth muscle actin, CD68, and CD163 antibodies were used to identify CAFs and TAMs. CAFs were divided into 4 grades on the basis of staining intensity: negative (0), scanty (1), focal (2), and abundant (3). The most intensive areas of macrophage concentration in each tumor invasive stroma were also evaluated. Results: The cancer specimens were divided into Grade 0/1, Grade 2, and Grade 3 on the basis of CAF grade. In addition, they were divided into low‐ and high‐grade groups on the basis of the number of CD68‐positive and CD163‐positive macrophages. The latter were significantly increased in the Grade 2 CAF group compared to the Grade 0/1 group (P = 0.009). Kaplan–Meier and multivariate survival analyses revealed that Grade 2 CAFs (P = 0.003) and high CD163‐positive macrophage levels (P = 0.007) significantly correlated with a poor outcome in patients with OSCC, and that a high CD163‐positive macrophage level was a significant and an independent prognostic factor (P = 0.045). Conclusions: Cancer‐associated fibroblasts and CD163‐positive macrophages may be potential prognostic predictors of OSCC.     

Immunohistochemical analysis of CD45RO+ T cells and vascular endothelial growth factor expression in cyclosporin A-induced rat gingival tissue

Background: The aim of this study is to evaluate CD4⁺, CD8⁺, and CD45RO⁺ T cells, and vascular endothelial growth factor (VEGF) expression in cyclosporin A (CsA)–induced rat overgrown gingival tissue during an 8‐week period. Methods: Sixty male Sprague‐Dawley rats weighing 200 to 250 g were used in this study. Mandibular first molars were ligated with 3–0 silk suture. The rats received daily doses of 0.09% NaCl (control group) or 10 mg/kg body weight of CsA (test group) by intraperitoneal injections. Five rats from the control group and 10 rats from the test group were sacrificed at each experimental period (2, 4, 6, and 8 weeks after the beginning of CsA treatment). The specimens were examined immunohistochemically. Results: CD4⁺, CD8⁺, and CD45RO⁺ T cells, and VEGF expression were more prevalent in the CsA‐treated group than in the control group (P <0.05). VEGF was significantly correlated with CD4⁺ T cells, CD4⁺/CD8⁺ ratio, and CD45RO⁺ cells (P <0.05). Conclusion: Based on our findings, we conclude that VEGF, a major regulator of angiogenesis, and CD4⁺, CD8⁺, and CD45RO⁺ memory T cells play a key role in CsA‐induced gingival overgrowth.     

Immunohistochemical detection of Ki‐67 is not associated with tumor‐infiltrating macrophages and cyclooxygenase‐2 in oral squamous cell carcinoma

J Oral Pathol Med (2010) 39 : 565–570 Background: An inflammatory component consisting of cells and chemical mediators may influence the proliferation and dissemination of the oral squamous cell carcinoma (OSCC). In the present study, we evaluated the possible relationship between Ki‐67, tumor‐associated macrophages (TAMs), and COX‐2 in OSCCs. In addition, the immunodetection of these proteins was associated with different histological grades of malignancy, including invasive and in situ tumors. Methods: Twenty‐seven OSCC cases were examined by light microscopy using criteria adopted WHO, and immunohistochemistry for Ki‐67, CD68, and COX‐2 using EnVision System in invasive and in situ lesions. Immunohistochemical detection of these proteins was assessed and scored for COX‐2, and results were compared with their histological grades of malignancy. Results: A correlation between Ki‐67, COX‐2, and CD68 was not found. Histological grade of malignancy (HDM) was associated with the Ki‐67 immunostaining (P = 0.00), but this was not observed regarding both CD68 (P = 0.51) and COX‐2 (P = 0.89). Furthermore, there was a COX‐2 overexpression in 62.96% of the sample, and a high density of TAMs in both OSCCs and in situ carcinomas. Conclusions: Imunolabeling for Ki‐67 was directly correlated with less‐differentiated tumors, suggesting that this marker may contribute to understand the biological behavior of OSCC, and help to distinguish risk groups of OSCC. Furthermore, the lack of correlation between Ki‐67, COX‐2, and CD68 indicates that the latter two markers may play a pivotal role in oral carcinogenesis. However, further studies are needed to clarify their contribution for cell proliferation and tumor differentiation.     

Tumour infiltrating CD25+ FoxP3+ regulatory T cells (Tregs) relate to tumour grade and stromal inflammation in oral squamous cell carcinoma

J Oral Pathol Med (2011) 40 : 636–642 This research is aimed to quantitate and characterize the subtypes of tumour infiltrating lymphocytes (TILs), in particular the presence of FoxP3+ Tregs in different grades of oral squamous cell carcinoma using monospecific antibody staining of formalin‐fixed, paraffin‐embedded tissue sections. The correlation between tumour grade and the intensity of tumour infiltrating lymphocytes was tried to be tested, to assume a putative linkage between them. Thirty‐four cases of histologically proven primary oral squamous cell carcinoma (OSCC) of different grades of differentiation were assorted to groups 1–3. Three‐micron sections of tissue were cut and captured on electrically charged slides (Vision BioSystem, Mount Waverley, Australia) and stained using monospecific antibody against FoxP3+ Treg phenotype (dilution 1:40, Mouse monoclone No: 236A/E7, Ab 20034, IgG1; Abcam, Cambridge, UK). Automated protocols were employed for staining and scoring of staining intensity using Bond^? system (Vision BioSystem). Significant difference in staining intensities (Tregs) was noted among the histologic grades of tumour, where well‐differentiated OSCC had significantly low expression of FoxP3+ Tregs in comparison with moderately and poorly differentiated tumours. A significant linear correlation was established between tumour grade and the intensity of TILs, where high grade tumours (poor differentiation) were more markedly infiltrated. There was also a significant positive correlation between FoxP3+ Tregs and TILs in cases studied. The correlation of these three variables noted in the study (FoxP3+ Tregs, tumour grade and TILs) and their significance in a meta‐analysis may prove useful in targeting patients with high‐risk neoplasms for more aggressive treatment protocols and management strategies.     

Alveolar Bone Resorption Induced by CD4+CD45RB High‐Density T‐Cell Transfer in Immunocompromised Mice

Background: The aims of this study are to determine whether the antigen‐inexperienced (naive, CD45RB high‐density) T‐cell (CD4⁺CD45RB^High T‐cell) transfer model is associated with alveolar bone resorption, to elucidate the local osteogenic/adipogenic potential of alveolar bone marrow stromal cells (ABCs) from T‐cell–transferred animals, and to investigate the systemic osteogenic potential by transplanting human periodontal ligament stem cells (hPDLSCs) into these animals. Methods: CD4⁺CD45RB^High and CD4⁺CD45RB^Low (antigen‐experienced [memory, CD45RB low‐density]) T cells were sorted and transferred into severe combined immunodeficiency (SCID) mice to induce inflammatory bowel disease–like syndrome (n = 8). hPDLSCs were transplanted into T‐cell–transferred SCID mice to examine ectopic cementum formation 8 weeks after T‐cell transfer. The mandibles and tibias of these mice were retrieved for microcomputed tomography (micro‐CT), histomorphometric analysis, and isolation of ABCs 16 weeks after T‐cell transfer. The in vitro osteogenic and adipogenic potentials of the ABCs were evaluated. Results: Histologic and micro‐CT analysis revealed that the transfer of CD4⁺CD45RB^High T‐cell subset was sufficient for alveolar bone resorption and affected the osteogenic/adipogenic potential of ABCs. Furthermore, it was found that CD4⁺CD45RB^High T‐cell–transferred animals have decreased systemic osteogenic potential, as evidenced using the in vivo ectopic hPDLSC transplantation model. Conclusion: CD4⁺CD45RB^High T‐cell transfer induced both alveolar bone resorption and reduced systemic osteogenic potential, with a concomitant downregulation of the osteogenic potential of ABCs.     

Comparison of gingival and peripheral blood T cells among patients with periodontitis suggests skewing of the gingival T cell antigen receptor V beta repertoire

The present study investigated the expression of different variable regions of T cell receptor β-chain (Vβ) among functional subsets of T cells, i.e. CD45RO⁺ (activated/memory), CD4⁺ and CD8⁺in gingiva and peripheral blood of patients with periodontitis. Gingival tissue specimens (n= 25) and peripheral blood were procured from 18 patients with periodontitis during periodontal surgery or extraction. Single-cell suspensions of gingival tissues were made by enzymatic digestion. These cells were immunofluorescently labeled with a panel of monoclonal antibodies specific for 18 TCR Vβ regions, in concert with markers for various T cell subsets. The cells were then analyzed with 3-color multivariate flow cytometry. Results demonstrated that a significantly higher proportion of T cells in gingiva expressed Vβ5.2 (0.0005), Vβ6 (0.0007) and Vβ9 (0.003) regions compared to those in peripheral blood. Comparison of CD45RO⁺ (activated/memory) and CD45RO^? (naïve) subsets of gingival T cells revealed differences in the expression of TCR Vβ regions. Vβ5.2 expression was significantly higher among CD45RO⁺ gingival T cells (p= 0.004), whereas Vβ14 expression was elevated among the CD45RO^? subset relative to peripheral blood (p= 0.008). Analysis of TCR Vβ region expression among CD4⁺ and CD8⁺ subsets did not reveal any statistically significant differences between gingiva and peripheral blood, although some Vβ regions approached significance. Collectively, these results demonstrate that the T cell repertoire in the gingival compartment differs significantly from that in the peripheral blood. Furthermore, since the skewing of TCR Vβ was observed among naive, as well as activated/memory T cells, it is likely that both developmental and environmental factors are influential in shaping the gingival TCR repertoire in patients with periodontitis. Elucidation of the cause of the skewed expression of T cell receptors in gingiva can provide insights into the specificity of T cells in periodontitis.     

Macrophages and prognosis of oral squamous cell carcinoma: A systematic review

Oral Squamous Cell Carcinoma (OSCC) presents a tumor microenvironment rich in inflammatory cells. Depending on the stimulus, macrophages can polarize in M1 or M2 profile, where M1 acts as proinflammatory and antitumor, and M2 is anti‐inflammatory and shows protumor activity. Several studies have shown that macrophages are important to the prognosis of patients with different types of cancer. Our aim was to conduct a systematic review to evaluate the role of macrophages in the prognosis of OSCC patients. A search in the Pubmed, Scopus, and ISI Web of Knowledge database was performed, and it was included only studies that evaluated the importance of macrophages in the prognosis of OSCC patients. From initial 286 articles, 14 fully attended the inclusion criteria. In the majority of the articles, it was evaluated only CD68, a panmacrophage marker, or CD163, a M2 marker. Only one article evaluated the M1 marker, CD11c. Besides, 5 articles analyzed the presence of macrophages in different areas of the tumor. Higher concentrations of CD68 and CD163 were associated with worse survival. In conclusion, macrophages are important to OSCC patients’ prognosis; however, it is necessary to address in which tumor region the presence of polarized macrophage is more important to the outcome.     

Small oral squamous cell carcinomas with nodal lymphogenic metastasis show increased infiltration of M2 polarized macrophages – An immunohistochemical analysis

BackgroundIn solid malignancies the influence of immunological parameters – especially of macrophages – on invasiveness, metastatic potential and prognosis has been shown. There are no studies quantitatively analysing the macrophage polarization in oral squamous cell carcinoma (oscc). The aim of this study was to correlate macrophage polarization in the epithelial and stromal compartment of oscc with histopathologic parameters.MethodsT1 and T2 oscc samples (n = 34) were used. Automated immunohistochemical staining detected CD68, CD11c, CD163 and MRC1 positive cells. All samples were completely digitalized using whole slide imaging and the number of stained cells per area was assessed quantitatively.ResultsPrimary tumours with lymphogenic metastasis (N+) showed a significantly (p < 0.05) increased count of CD68, CD11c, CD163 and MRC1 positive cells in the epithelial fraction compared to N0 tumours. The ratio of CD163 positive cells (M2 macrophages) to CD68 positive cells (M1 and M2 macrophages) was significantly (p < 0.05) increased in N+ tumours.ConclusionAn increased macrophage infiltration and an increased M2 polarization in primary oral squamous cell carcinomas with lymphogenic metastasis was shown. Macrophages that migrated into the epithelial tumour fraction seem to be of special biological importance.The results indicate a central role of macrophages in the progression of oscc.     

Immunohistological analysis of Tannerella forsythia‐induced lesions in a murine model

Tannerella forsythia has been implicated as a defined periodontal pathogen. In the present study a mouse model was used to determine the phenotype of leukocytes in the lesions induced by subcutaneous injections of either live (group A) or nonviable (group B) T. forsythia. Control mice (group C) received the vehicle only. Lesions were excised at days 1, 2, 4, and 7. An avidin‐biotin immunoperoxidase method was used to stain infiltrating CD4⁺ and CD8⁺ T cells, CD14⁺ macrophages, CD19⁺ B cells, and neutrophils. Hematoxylin and eosin sections demonstrated lesions with central necrotic cores surrounded by neutrophils, macrophages and lymphocytes in both group A and group B mice. Lesions from control mice exhibited no or only occasional solitary leukocytes. In both groups A and B, neutrophils were the dominant leukocyte in the lesion 1 day after injection, the numbers decreasing over the 7‐day experimental period. There was a relatively low mean percent of CD4⁺ and CD8⁺ T cells in the lesions and, whereas the percent of CD8⁺ T cells remained constant, there was a significant increase in the percent of CD4⁺ T cells at day 7. This increase was more evident in group A mice. The mean percent of CD14⁺ macrophages and CD19⁺ B cells remained low over the experimental period, although there was a significantly higher mean percent of CD19⁺ B cells at day 1. In conclusion, the results showed that immunization of mice with live T. forsythia induced a stronger immune response than nonviable organisms. The inflammatory response presented as a nonspecific immune response with evidence of an adaptive (T‐cell) response by day 7. Unlike Porphyromonas gingivalis, there was no inhibition of neutrophil migration.     

Association of the infiltration of tumor-associated macrophages,expression of Smad7 protein and prognosis in oral squamous cell carcinoma

ObjectiveTo explore the association between Smad7 expression and tumor-associated macrophage (TAM), and their relationship with clinicopathological features and prognosis in patients with oral squamous cell carcinoma (OSCC).MethodsThis study collected cancer tissues from 314 OSCC patients from May 2002 to May 2012 at our hospital. Immunohistochemistry was carried out to detect the density of CD68⁺ cells and Smad7.ResultsThe densities of CD68TF_Mean and CD68TF_Hotspot shared a significant negative correlation with the immunoscore (IS) of Smad7, indicated that Smad7 was evidently increased with the decrease densities of CD68TF_Mean and CD68TF_Hotspot in OSCC tissues. Besides, low differentiation degree together with high TNM, T and N stage of OSCC patients presented decreased densities of CD68TF_Mean and CD68TF_Hotspot but increased expression of Smad7. Kaplan-Meier univariate survival analysis showed that the prognosis of OSCC patients was associated with differentiation degree, clinical stages, Smad7 expression, as well as densities of CD68TF_Mean and CD68TF_Hotspot. Cox regression analysis results demonstrated that N staging, the densities of CD68TF_Mean and CD68TF_Hotspot and Smad7 expression were independent risk factors influencing the survival rate of OSCC patients.ConclusionDecreased densities of CD68TF_Mean and CD68TF_Hotspot were negatively correlated with the increased Smad7 expression in OSCC tissues, both of which linked to clinicopathological features and prognosis of OSCC.     

Organotypic culture of normal,dysplastic and squamous cell carcinoma‐derived oral cell lines reveals loss of spatial regulation of CD44 and p75NTR in malignancy

Oral squamous cell carcinomas (OSCC) often arise from dysplastic lesions. The role of cancer stem cells in tumour initiation is widely accepted, yet the potential existence of pre‐cancerous stem cells in dysplastic tissue has received little attention. Cell lines from oral diseases ranging in severity from dysplasia to malignancy provide opportunity to investigate the involvement of stem cells in malignant progression from dysplasia. Stem cells are functionally defined by their ability to generate hierarchical tissue structures in consortium with spatial regulation. Organotypic cultures readily display tissue hierarchy in vitro; hence, in this study, we compared hierarchical expression of stem cell‐associated markers in dermis‐based organotypic cultures of oral epithelial cells from normal tissue (OKF6‐TERT2), mild dysplasia (DOK), severe dysplasia (POE‐9n) and OSCC (PE/CA P J15). Expression of CD44, p75^NTR, CD24 and ALDH was studied in monolayers by flow cytometry and in organotypic cultures by immunohistochemistry. Spatial regulation of CD44 and p75^NTR was evident for organotypic cultures of normal (OKF6‐TERT2) and dysplasia (DOK and POE‐9n) but was lacking for OSCC (PE/CA PJ15)‐derived cells. Spatial regulation of CD24 was not evident. All monolayer cultures exhibited CD44, p75^NTR, CD24 antigens and ALDH activity (ALDEFLUOR^® assay), with a trend towards loss of population heterogeneity that mirrored disease severity. In monolayer, increased FOXA1 and decreased FOXA2 expression correlated with disease severity, but OCT3/4, Sox2 and NANOG did not. We conclude that dermis‐based organotypic cultures give opportunity to investigate the mechanisms that underlie loss of spatial regulation of stem cell markers seen with OSCC‐derived cells.     

Identification of gingipain‐specific I‐Ab‐restricted CD4+ T cells following mucosal colonization with Porphyromonas gingivalis in C57BL/6 mice

Chronic periodontitis is associated with Porphyromonas gingivalis infection. Although virulence factors of P. gingivalis are hypothesized to contribute to the pathogenesis of periodontitis, it is unclear whether the local CD4⁺ T‐cell‐mediated response they elicit prevents or contributes to periodontal bone destruction. We hypothesize that major histocompatibility complex class II I‐A^b‐binding peptides existing in Kgp and RgpA are presented to CD4⁺ T cells during P. gingivalis oral colonization. The protein sequences of gingipains RgpA and Kgp, and OMP40 and OMP41 of P. gingivalis were scanned using an I‐A^b‐binding matrix. From this analysis we identified 53 candidate peptides that had the potential to engage the peptide‐binding groove of the I‐A^b molecule of C57BL/6 mice. An ELISpot‐based screen revealed those peptide‐primed effector/memory CD4⁺ T cells that could be re‐stimulated in vitro with P. gingivalis or the peptide itself to produce interleukin‐17A or interferon‐γ. Two immunodominant peptides, Kgp_467–477 (pKgp) and RgpA_1054–1064/Kgp_1074–1084 (pR/Kgp) were identified and engineered to be displayed on I‐A^b molecular tetramers. Peptide pR/Kgp is conserved across all sequenced P. gingivalis strains. C57BL/6 mice were orally inoculated with P. gingivalis strain 53977 and cervical lymph node cells were stained with phycoerythrin‐conjugated pKgp::I‐A^b and pR/Kgp::I‐A^b tetramers. We found that only pR/Kgp::I‐A^b bound with the desired specificity to gingipain‐specific CD4⁺ T cells. The pR/Kgp::I‐A^b tetramer complex will allow the identification of effector/memory CD4⁺ T cells specific for two virulence factors of P. gingivalis strains associated with periodontal disease.     

Production of IL‐10 and IL‐12 by antigen‐presenting cells in periapical lesions

J Oral Pathol Med (2010) 39 : 690–696 Background: Interferon‐γ (IFN‐γ) plays an important role in the pathogenesis of periapical lesions. Its expression is up‐regulated by interleukin (IL)‐12) and down‐regulated by IL‐10. The aim of this work was to study the cellular source of these cytokines and their mutual interactions in human periapical lesions. Methods: Mononuclear cells, macrophages and dendritic cells were isolated from periapical lesions using plastic adherence and osmotic gradients. Cytokines were measured in culture supernatants by a microbeads fluorescence assay. Phenotypic characteristics of cells were studied by immunocytochemistry, whereas allostimulatory activity of antigen‐presenting cells was tested using a mixed leukocyte reaction. Results: We observed the positive correlations between the levels of IL‐12 and IFN‐γ as well as IL‐12 and IL‐10 in cultures of mononuclear cells. As IL‐10 and IL‐12 are produced by dendritic cells and activated macrophages, we examined their contribution to the production of these cytokines. Macrophages, CD14⁺ adherent cells, produced high levels of IL‐10 and very low levels of IL‐12. In contrast, non‐adherent, strongly HLA‐DR⁺ dendritic cells, potent stimulators of the alloreactive T‐cell response, produced low levels of IL‐10 and moderate levels of IL‐12. Dendritic cells stimulated the production of IFN‐γ by allogeneic CD4⁺ T cells. In contrast, the level of IFN‐γ was significantly decreased and the production of IL‐10 was enhanced by addition of macrophages to the culture system. Conclusion: Our results suggest that a fine balance between the production of IL‐10 and IL‐12 by different antigen‐presenting cells, through IFN‐γ, may control the course of chronic inflammation in periapical lesions.     

Association of macrophages with angiogenesis in oral verrucous and squamous cell carcinomas

J Oral Pathol Med (2010) 39 : 559–564 Background: Tumor‐associated macrophages (TAMs) are a major cellular component of human cancers, yet there is still no consensus as to their role in cancer growth and angiogenesis. Methods: The association between TAMs and angiogenesis was investigated in formalin‐fixed, paraffin‐embedded archival material of oral squamous cell carcinoma (OSCC) and oral verrucous carcinoma (OVC). TAMs shown by immunohistochemistry for CD68 and microvessels demonstrated by immunohistochemistry for CD31 were quantified using an image analyzer computer system. Results: TAMs were observed in all studied specimens. The area percentage of CD68 immunoreactivity and microvessel density (MVD) were significantly lower in OVC compared with the different grades of OSCC (P = 0.0009), (P = 0.0045). Both parameters increased in high‐grade malignancy of OSCC. Linear regression analysis revealed a positive correlation between the area percentage of CD68 immunoreactivity and the MVD in the studied tumors. Conclusions: Increased TAMs is associated with angiogenesis and higher histopathological grades in oral cancer.     

Kinetics of Th17‐related cytokine expression in experimentally induced rat periapical lesions

Th17‐related cytokines are essential factors in various pathological states, including inflammatory bone destruction. This study investigated the contribution of Th17‐related cytokines to the progress of experimentally induced rat periapical lesions. Periapical pathoses were induced by unsealed exposure of the pulp chamber of the lower first molars. A variety of immunocompetent cells, including CD68⁺ macrophages, Ia antigen⁺ cells and TCRαβ⁺ T cells, were observed in the lesions. The expression levels of Th17‐related cytokines, IL‐17 and IL‐23, and of pro‐inflammatory cytokines, IL‐1β and IL‐6, were significantly increased at 14 days (expansion stage) compared with normal periapical tissues. The expression levels of Foxp3, a regulatory T cell (Treg)‐related gene, and of IL‐10, an anti‐inflammatory cytokine, were higher at 28 days (chronic stage) than at 14 days. These findings suggest that Th17‐related cytokines may be primary contributors to the initiation of periapical bone destruction, and that lesion expansion may be regulated by anti‐inflammatory mediators.     

Interleukin‐2, interleukin‐6 and T regulatory cells in peripheral blood of patients with Behçet’s disease and recurrent aphthous ulcerations

J Oral Pathol Med (2012) 41 : 73–79 Background: One of the factors involved in the pathogenesis of Behçet disease (BD) and recurrent aphthous ulcerations (RAU) is a cell‐mediated immune response in which several cytokines (interleukin‐2, interleukin‐6) and T regulatory cell (T reg cell) population seem to play a major role. The aim of this study was to measured the interleukin‐2 (IL‐2), interleukin‐6 (IL‐6) levels and analysis of CD4⁺ CD25⁺ Foxp‐3⁺ Treg cells in peripheral blood from patients with BD and RAU. In addition; we also analysed peripheral blood from healthy subjects for comparison. Methods: Thirty patients (15 men and 15 women) with BD, 30 patients (12 men and 18 women) with RAU and 15 healthy control subjects (nine men and six women) participated in the study. Analysis of CD4⁺ CD25⁺ Foxp‐3⁺ Treg cells, IL‐2 and IL‐6 levels have been measured in flow cytometry. Results: No statistical differences were observed in the serum levels of IL‐2 and IL‐6 between BD and RAU patients, and healthy subjects. Although there were no statistical differences in the number of CD4⁺ CD25⁺ Foxp‐3⁺ cells between groups, there were statistically significant differences in the number of CD4⁺ CD25^bright Treg cells. CD4⁺ CD25^bright Treg cells were significantly increased in BD and RAU patients compared to healthy subjects. Statistical analysis revealed no difference according to the number of CD4⁺ CD25^bright cells between BD and RAU patients. Conclusions: These results indicate that CD4⁺ CD25^bright T regulatory cells may be contributing factor in the pathogenesis of BD and RAU.     

Production of interleukin‐8 in vitro by mononuclear cells isolated from human periapical lesions

Introduction: Interleukin‐8 (IL‐8) is an important mediator of inflammation. However, little is known about its production in chronic dental periapical lesions and this was the main aim of this work. Methods: Inflammatory cells were isolated from clinically different periapical lesions and analyzed by morphological criteria. The mononuclear cells were isolated, phenotypically analyzed by immunocytochemistry and cultivated in vitro. IL‐8 was measured in culture supernatants of these periapical lesion mononuclear cells (PL‐MNC) using a microbeads fluorescence assay. Results: We found a relatively high production of IL‐8 in 19 out of 21 periapical lesions included in the study. The level of IL‐8 and the proportion of neutrophil granulocytes were significantly higher in the group of symptomatic lesions, compared to the asymptomatic lesions, but there was no statistically significant correlation between these parameters. According to the predominance of CD3⁺ T cells and Ig⁺/CD19⁺ B cells and plasma cells, lesions were divided into T‐type and B‐type lesions, respectively. The levels of IL‐8 were significantly higher in the culture supernatants of PL‐MNC in the T‐type lesions and were positively correlated with the proportion of macrophages/dendritic cells (CD11c⁺ cells) and CD4⁺ T cells. Such a correlation was not shown in B‐type lesions. Conclusion: These results suggest that PL‐MNC are a significant source of IL‐8, which is probably an important chemokine for the migration and function of different cell types at the site of chronic inflammation.     

Areca nut extracts enhance the development of CD11b+Gr‐1+ cells with the characteristics of myeloid‐derived suppressor cells in antigen‐stimulated mice

J Oral Pathol Med (2011) 40 : 769–777 Background: Areca quid chewing is an etiological factor contributing to the development of oral cancer and pre‐cancers, whose pathophysiology has been linked to inflammation and immune deterioration. Myeloid‐derived suppressor cells (MDSC) play a key role in the regulation of immunity under certain pathological conditions, such as inflammation and cancer. As areca nut extracts (ANE) have been reported to induce a proinflammatory effect in antigen‐stimulated mice, we hypothesized that ANE might enhance the development of MDSC. Methods: Ovalbumin (OVA)‐sensitized BALB/c mice were daily administered with ANE (5–50 mg/kg), polyphenol‐enriched ANE (PANE; 25 mg/kg) or arecoline (5 mg/kg) by intraperitoneal injection for 10 doses. The mouse footpads were then subcutaneously challenged with OVA to induce local inflammatory responses. Results: ANE and PANE treatment significantly increased the spleen index and the population of CD11b⁺Gr‐1⁺ cells in the spleen and peripheral blood, whereas arecoline was inactive. In addition, ANE and PANE treatment enhanced the expression of cytokines and enzymes associated with the immunosuppressive function of MDSC, including IL‐10, arginase‐I and iNOS in splenic CD11b⁺ cells. Concordantly, ANE and PANE treatment augmented the infiltration of Gr‐1⁺IL‐10⁺ cells in the footpads challenged with OVA. Conclusions: Our results suggested that areca nut constituents, in particular, polyphenols enhanced the development of myeloid‐derived suppressor cells in vivo, which may be a critical mechanism linking inflammation and the compromised immunity reported to be associated with the pathophysiology of areca‐related oral diseases.