V. P. B. Parachuru

Toll-Like Receptors and Cancer, Particularly Oral Squamous Cell Carcinoma

It is becoming increasingly apparent that the tumor microenvironment plays an important role in the progression of cancer. The microenvironment may promote tumor cell survival and proliferation or, alternatively may induce tumor cell apoptosis. Toll-like receptors (TLRs) are transmembrane proteins, expressed on immune cells and epithelial cells, that recognize exogenous and endogenous macromolecules. Once activated, they initiate signaling pathways leading to the release of cytokines and chemokines, which recruit immune cells inducing further cytokine production, the production of angiogenic mediators and growth factors, all of which may influence tumor progression. This paper examines the actions of TLRs in carcinogenesis with particular emphasis on their role in oral squamous cell carcinoma.

Downregulation of toll-like receptor-mediated signalling pathways in oral lichen planus.

OBJECTIVE The objective of this study was to investigate the expression of Toll-like receptors (TLR) and TLR-associated signalling pathway genes in oral lichen planus (OLP). METHODS Initially, immunohistochemistry was used to determine TLR expression in 12 formalin-fixed archival OLP tissues with 12 non-specifically inflamed oral tissues as controls. RNA was isolated from further fresh samples of OLP and non-specifically inflamed oral tissue controls (n = 6 for both groups) and used in qRT(2)-PCR focused arrays to determine the expression of TLRs and associated signalling pathway genes. Genes with a statistical significance of ±two-fold regulation (FR) and a P-value < 0.05 were considered as significantly regulated. RESULTS Significantly more TLR4(+) cells were present in the inflammatory infiltrate in OLP compared with the control tissues (P < 0.05). There was no statistically significant difference in the numbers of TLR2(+) and TLR8(+) cells between the groups. TLR3 was significantly downregulated in OLP (P < 0.01). TLR8 was upregulated in OLP, but the difference between the groups was not statistically significant. The TLR-mediated signalling-associated protein genes MyD88 and TIRAP were significantly downregulated (P < 0.01 and P < 0.05), as were IRAK1 (P < 0.05), MAPK8 (P < 0.01), MAP3K1 (P < 0.05), MAP4K4 (P < 0.05), REL (P < 0.01) and RELA (P < 0.01). Stress proteins HMGB1 and the heat shock protein D1 were significantly downregulated in OLP (P < 0.01). CONCLUSION These findings suggest a downregulation of TLR-mediated signalling pathways in OLP lesions.

Forkhead box-P3+ regulatory T cells and toll-like receptor 2 co-expression in oral squamous cell carcinoma

BACKGROUND The function of forkhead box-P3 (FoxP3) regulatory T cells (Treg) and toll-like receptor (TLR)2 protein in the oral cancer microenvironment is not fully understood, but evidence from other malignancies suggests it is likely they are involved with tumour development and progression. The aim of this study was to investigate the distribution of FoxP3+cells, TLR2+ cells and double-labelled FoxP3+TLR2+ immune cells in oral squamous cell carcinoma (OSCC), using immunohistochemistry (IHC) and immunofluorescence (IF). METHODS 25 archival cases of OSCC were immunostained with anti-FoxP3 and anti-TLR2 antibodies. Inflamed hyperplastic oral mucosal tissues were used as controls. The proportion of single-labelled, double-labelled and negative cells was determined. RESULTS A higher frequency of double-labelled FoxP3+TLR2+ Tregs was observed within the immune cells of OSCC compared to inflamed controls using IHC (p<0.05). Cell-to-cell contact between single-stained TLR2+ cells and FoxP3+ cells was noted. Double IF studies validated demonstration of co-expression of FoxP3+/TLR2+ immune cells in OSCC. CONCLUSION The presence of FoxP3+TLR2+ cells within the OSCC microenvironment may represent a dendritic cell-dependent pathway capable of inhibiting Treg suppressive activity, potentially enhancing the anti-tumour response. Modulation of TLR2-Treg interactions should be further explored to determine if they have a role in the therapeutic management of OSCC.

Regulatory T-cells and IL17A+ cells infiltrate oral lichen planus lesions

Oral lichen planus (OLP) is a complex immunological disorder, mediated in part by the release of cytokines from activated T-cells. Of late, two closely related T-helper (Th) cell subsets; regulatory T-cells (Tregs; FoxP3(+)) and Th17 cells (IL17(+)) have been described in various chronic inflammatory diseases. The aim of this study was to determine the expression of FoxP3 and IL17 in OLP using immunohistochemistry (IHC) and quantitative real-time reverse transcriptase polymerase chain reaction (qPCR). For IHC, formalin fixed, paraffin embedded archival specimens, an OLP group (n=10) and a non-specific inflammatory (NSI) control group (n=9) were used. In addition, 12 fresh tissue samples were used to determine gene expression of FoxP3 and IL17. Significantly more FoxP3(+) cells were present in OLP than in NSI. IL17(+) cells were significantly more frequent in the control tissues than in OLP. The gene expression experiments revealed a significantly higher expression of FoxP3 in OLP when compared to the controls. IL17 gene expression was not different between the groups. Double labelling immunofluorescence indicated co-localisation of IL17 with tryptase(+) mast cells. These findings suggest FoxP3(+) Tregs have a more prominent role in the pathogenesis of OLP when compared to IL17(+)cells.

FoxP3+ regulatory T cells, interleukin 17 and mast cells in chronic inflammatory periodontal disease

BACKGROUND AND OBJECTIVE T cells are known to play a pivotal role in periodontal disease; however, less is known about the T-helper subsets of regulatory T cells (Tregs) and Th17 cells. The aim of this study was to investigate the cell types expressing FoxP3 and interleukin (IL)-17A within periodontal disease tissues and to determine gene and protein expression profiles associated with periodontitis. MATERIAL AND METHODS A total of 10 healthy/gingivitis and 10 chronic periodontitis tissues were investigated. Immunohistochemistry and immunofluorescence techniques were used to identify the FoxP3 and IL17-positive cells and to determine the cell types respectively. Gene expression was determined using semi-quantitative polymerase chain reaction array technology that allowed the analysis of 84 pathway-focused genes known to be associated with Tregs and Th17 cells. Transforming growth factor (TGF)-β1, IL10 and IL17A protein levels were determined using enzyme-linked immunosorbent assay. RESULTS Double immunofluorescence labeling revealed that all FoxP3+ cells were CD4+ , while IL17+ cells were neither CD4+ nor CD8+ but were tryptase+ , suggestive of mast cells. More FoxP3+ cells than IL17+ cells were found in all the tissues examined and overall there were few IL17+ cells. Statistically significant increases in gene expression were found for STAT5A, STAT3, SOCS1, TGFβ1 and IL10 in the chronic periodontitis specimens predominantly infiltrated with B cells and plasma cells when compared with healthy/gingivitis specimens predominantly infiltrated with T cells. Protein analysis demonstrated higher levels of the TGFβ1 and IL10 cytokines in periodontitis tissues and in B-cell and plasma cell predominant gingival tissues than in healthy/gingivitis tissues and T-cell predominant gingival tissues. IL17A gene and protein expression was not detected in any of the tissues. CONCLUSION Based on the findings of this study, we suggest that the source of low levels of IL17A in periodontal tissues is mast cells not Th17 cells and that Tregs may have a more prominent role in the pathogenesis of periodontal disease than Th17 cells.

The nexus between periodontics and oral pathology

A wide variety of lesions may arise from the oral mucosa, fibrous connective tissue, bone and cementum of the periodontium. The commonest pathology occurs as a result of bacterial infection and is very well known to dentists and periodontists, but rarer conditions present as gingival pathology. The pathogenesis of these conditions ranges from genetic to traumatic to immunological to neoplastic, and includes benign, malignant and metastatic lesions. This paper outlines some of these conditions and describes how the periodontist and oral pathologist can work together using a framework, and how with careful consideration of the clinical features and the use of appropriate special tests, including obtaining an adequate tissue specimen, a timely and accurate diagnosis can be obtained.

Becoming a ‘pharmaceutical person’: Medication use trajectories from age 26 to 38 in a representative birth cohort from Dunedin, New Zealand

Despite the abundance of medications available for human consumption, and frequent concerns about increasing medicalization or pharmaceuticalization of everyday life, there is little research investigating medicines-use in young and middle-aged populations and discussing the implications of young people using increasing numbers of medicines and becoming pharmaceutical users over time. We use data from a New Zealand longitudinal study to examine changes in self-reported medication use by a complete birth cohort of young adults. Details of medications taken during the previous two weeks at age 38 are compared to similar data collected at ages 32 and 26, and by gender. Major drug categories are examined. General use profiles and medicine-types are considered in light of our interest in understanding the formation of the young and middle-aging ‘pharmaceutical person’ – where one’s embodied experience is frequently and normally mediated by pharmaceutical interventions having documented benefit/risk outcomes.

Expression of IL33 and IL35 in oral lichen planus

Oral lichen planus (OLP) is a complex immunological disorder, mediated in part by the release of cytokines by activated T-cells. Recently, the role of novel cytokines including IL33 and IL35 has been described in various chronic inflammatory diseases. IL33, a member of the IL-1 superfamily of cytokines, functions as an ‘alarmin’ released after cell necrosis to alert the immune system to tissue damage or stress. IL35, a member of IL12 cytokine family, is produced by regulatory T-cells and suppresses the immune response. The expression of IL33 and IL35 is yet to be investigated in OLP. The aim of this study was to determine the presence and topographical distribution of IL33 and IL35 in OLP using immunohistochemistry and quantitative real-time reverse transcriptase polymerase chain reaction (qPCR). For IHC, formalin-fixed paraffin-embedded archival specimens of OLP (n = 10) and a non-specific inflammatory (NSI) control group (n = 9) were used. A double-labelling immunofluorescence technique was used to determine the expression of IL33 and IL35 on CD3+ T-cells. In addition, 12 fresh tissue samples (OLP n = 6 and NSI controls n = 6) were used to determine the gene expression of IL33 and EBI3 (one chain of the dimeric IL35). Quantitative and qualitative analysis was performed with statistical significance set at p < 0.05. IHC showed positive immunostaining with IL33 and IL35 in both OLP and NSI. Comparison of the numbers of IL33+ and IL35+ cells in OLP and NSI did not show any significant difference. In OLP, there were significantly more IL33+ cells in the deeper connective tissue region than at the epithelial–connective tissue interface. Interestingly, all IL35+ cells observed in both OLP and NSI tissues showed ovoid/plasmacytoid morphology. Double-labelling immunofluorescence showed that IL33 and IL35 expression was not localized within CD3+ T-cells. The gene expression experiments showed significantly higher expression of EBI3 (fold regulation 14.02) in OLP when compared to the inflammatory controls. IL33 gene expression was not different between the groups. However, within the OLP tissues, there was a significantly higher expression of IL33 than EBI3. Our data demonstrate the expression of IL33 and IL35 in OLP lesions. Further studies are needed to understand the functional role of these cytokines in OLP pathogenesis.

Multiple cells express interleukin 17 in oral squamous cell carcinoma.

BACKGROUND Interleukin (IL)-17 is a pro-inflammatory cytokine with pro- and antitumour effects. The aim of this study was to investigate the presence and potential sources of IL-17 in oral squamous cell carcinoma (OSCC). METHODS Immunohistochemistry was used to label and compare IL-17+ cells in the tissue sections of OSCC and inflammatory controls (IC), n = 14 for both. In OSCC, the comparison was made between the number of IL-17+ cells in the tumoral islands (TI), tumour-stroma interface (TS) and more distant stroma (DS). Cells expressing IL-17 were identified using double-labelling immunofluorescence and examined using laser scanning microscopy. The production of IL-17 from tumour cells was determined in the culture supernatants of OSCC cell lines, SCC4, SCC15 and SCC25, using sandwich ELISA. RESULTS Significantly more IL-17+ cells were observed in OSCC compared with IC (Mann-Whitney, P < 0.0001). In OSCC, the numbers of IL-17+ cells were not significantly different in three compartments, TI, TS and DS (one-way ANOVA, P > 0.05). However, the TI had significantly fewer IL-17+ cells than the combined stroma (both TS and DS together, Mann-Whitney, P < 0.01). Laser scanning microscopy revealed helper T cells, cytotoxic T cells, macrophages and mast cells co-expressed IL-17. ELISA experiments did not detect IL-17 in the supernatants of OSCC cell lines. CONCLUSIONS Although the tumour cells themselves did not express IL-17, a range of cell types did, suggesting multiple cellular sources for IL-17 in OSCC. The spatial distribution of IL-17+ cells suggests specific interactions with cells within the tumour microenvironment, implying that IL-17+ cells are likely to play a role in the pathogenesis of OSCC.