Phenotypic and Functional Characteristics of Th17 (CD4+IL17A+) Cells in Human Oral Squamous Cell Carcinoma and Its Clinical Relevance.

Recent studies have suggested an important role of T helper 17 (Th17) cells in tumor biology however, their phenotypic and functional aspects are poorly understood in context with oral cancer. We therefore, investigated the various phenotypic and functional markers of Th17 cells elucidating their relevance in oral squamous cell carcinoma (OSCC). Multi-color flow cytometry (FACs) was used to analyze the frequency and different markers of circulating Th17 cells ex vivo in peripheral blood mono-nuclear cells (PBMCs) from 69 OSCC patients and 35 healthy controls. Percent Mean ± SEM of different types of cells were compared between the two groups using Mann-Whitney U test. We found significantly (p < 0.0001) increased frequency of Th17 cells in patients as compared to controls. These cells were found to express CCR6 profoundly but not CXCR4, CD62L, and CCR7 as chemokine receptors. Additionally, it expressed HLA-DR, CD69, and CD25 moderately but CD28 and CD161 highly. The cytokine profiling revealed 3 subsets namely Th17/1 (IL17A+IFNγ+), Th17/inflammatory (IL17A+IL8+), and Th17/2 (IL17A+IL4+) which were found to be elevated in patients as compared to controls. The early stage patients had a shift toward Th17/1 type and vice versa. Our results suggest that Th17 cells may have effector immune functions in oral cancer immunity through CCR6, CD161, HLA-DR, CD69, CD28 receptors and inducing Th17/1 type of cells expressing polyfunctional antitumor IFNγ cytokine. Thus, novel immune-boosting regimens based on enhancement of Th17 cells in oral cancer patients may provide therapeutic benefits in them.

Operculina turpethum extract inhibits growth and proliferation by inhibiting NF-κB, COX-2 and cyclin D1 and induces apoptosis by up regulating P53 in oral cancer cells.

OBJECTIVES: Herbal drugs are popularly emerging as complementary and alternative medicines in cancer patients because of their cost effectiveness and minimal side-effects. The extract of Operculina turpethum (OT) is known to have antipyretic, anti-inflammatory and purgative properties. Since it is popularly known have antiinflammatory activity, we investigated its anti-tumor activity on four oral squamous cell carcinoma cell lines (OSCC) namely, (SCC-4, KB, SCC-9 and SCC-25). DESIGN: Antitumor activities of Operculina turpathum extract (OTE) was investigated by MTT and clonogenic assay, effect on cell cycle and apoptosis induction by Annexin-V/propidium iodide (PI) staining and flow cytometry and invasive potential of the tumor was determined by matrigel assay. The expression of various proteins involved in these mechanisms was analysed by western blotting. RESULTS: OTE specifically inhibited the growth and colony formation of OSCC cells in a dose-dependent manner via inhibiting NF-κB and its downstream target COX-2. It further arrested cell cycle at G0/G1 phase by inhibiting cyclin-D1 and induced early apoptosis by up-regulating P53 in OSCC cells. It also limits the invasion capacity of OSCC cells by up to 55-60%. CONCLUSIONS: OTE shows antitumor activities in OSCC cells by inhibiting NF-κB, COX-2 and cyclin D1 and upregulation of p53 expression. It may be developed as a safe and promising alternative chemopreventive/chemotherapeutic agent for oral cancer.

Inter-relation of Th1, Th2, Th17 and Treg cytokines in oral cancer patients and their clinical significance.

BACKGROUND: Altered cytokine production can lead to immune dysfunction in cancer patients. Hence, we investigated the cytokine balance in oral squamous cell carcinoma (OSCC) patients and their significance in providing new therapeutic insights. METHODS: We quantified Th17 (IL17A), Treg (TGFß1), Th1 (IL2, IFNγ) and Th2 (IL4, IL10) like cytokines in the sera of 78 cases and 39 controls by ELISA. The intracellular expression of these cytokines was analyzed in 10 subjects from each group by flow cytometry. RESULTS: Serum levels of IL17A, TGFß1, IL4 and IL10 were significantly higher while IL2 and IFNγ were relatively lower in patients as compared to controls. TGFß1 (r=0.55), IL4 (r=0.75) and IL10 (r=0.80) significantly (P<0.0001) correlated with disease progression and their elevated levels showed increased odd ratios of approximately 18, 14 and 37, respectively. IL17A appeared as a risk factor (OR=2.21, 95% CI=0.89-5.42) although statistically insignificant. The levels neither correlated with disease progression nor with TGFß1, IL4 and IL10 but showed positive association with IL2 (r=0.51, P<0.0001) and IFNγ (r=0.24). Flow cytometry data also showed similar trend. CONCLUSIONS: We reported a distinct TGFß1 and Th2 (IL4, IL10) polarization with a borderline elevation of IL17A while, a suppression of Th1 (IL2, IFNγ) cytokines in OSCC patients.

Natural killer T cell anergy, co-stimulatory molecules and immunotherapeutic interventions.

Natural killer T (NKT) cells are a unique subset of glycolipid-reactive T lymphocytes that share properties with natural killer (NK) cells. These lymphocytes can produce array of cytokines and chemokines that modulate the immune response, and play a pivotal role in cancer, autoimmunity, infection and inflammation. Owing to these properties, NKT cells have gained attentions for its potential use in antitumor immunotherapies. To date several NKT cell-based clinical trials have been performed in patients with cancer using its potent ligand α-galactosylceramide (α-GalCer). However, inconsistent therapeutic benefit, and inevitable health risks associated with drug dose and NKT cell activation have been observed. α-GalCer-activated NKT cells become anergic and produce both Th1 and Th2 cytokines that may function antagonistically, limiting the desired effector functions. Besides, various co-stimulatory and signaling molecules such as programmed death-1 (PD-1; CD279), casitas B-cell lymphoma-b (Cbl-b) and CARMA1 have been shown to be implicated in the induction of NKT cell anergy. In this review, we discuss the role of such key regulators and their functional mechanisms that may facilitate the development of improved approaches to overcome NKT cell anergy. In addition, we describe the evidences indicating that tailored-ligands can optimally activate NKT cells to obtain desired immune responses.

Single nucleotide polymorphism in hMLH1 promoter and risk of tobacco-related oral carcinoma in high-risk Asian Indians.

hMLH1 is a member of mismatch repair genes (MMR) that plays a crucial role in correcting replication errors, cell cycle arrest, apoptosis and oxidative stress. We explored the risk associated with hMLH1 -93 A>G (rs 1800734) single nucleotide polymorphism (SNP) with the oral squamous cell carcinoma (OSCC) in Asian Indians. We genotyped 242 patients with tobacco-related OSCC and 205 healthy controls by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The frequency of AA genotype was found to be significantly (Pc<0.0006) lower in patients as compared to the controls (21.49% vs. 47.8%) while GG genotype showed significantly higher (Pc<0.0006) prevalence in patients as compared to the healthy controls (41.32% vs. 13.66%). In logistic regression analysis AG (adjusted OR=1.95, 95% CI=0.72-5.26) and GG genotype (adjusted OR=4.5, 95% CI=1.54-13.16, P=0.006) appeared susceptible when compared with the wild-type AA genotype. The allelic distribution showed that variant G allele is significantly higher (Pc<0.0004) in patients and associated with increased risk (adjusted OR=2.36, 95% CI=1.33-4.19, P=0.003) as compared to the wild-type A allele. Altogether, our results suggest that the hMLH1 -93 A>G polymorphism is associated with the higher risk of tobacco-related OSCC in Asian Indians and could be useful in screening population at a higher risk.

Skewed immunological balance between Th17 (CD4(+)IL17A (+)) and Treg (CD4 (+)CD25 (+)FOXP3 (+)) cells in human oral squamous cell carcinoma.

BACKGROUND: Several studies have documented modulation of Th17 and T regulatory (Treg) cells in various human malignancies which may vary with the type and extent of the disease. However, such data in patients with oral cancer is scarce and hence the current study was designed to elaborate the immunological balance between these two T cell subsets in oral cancer. METHODS AND RESULTS: We analyzed various T cell subsets in the peripheral blood of 45 oral squamous cell carcinoma (OSCC) patients and 40 healthy volunteers. We found that, compared with the healthy controls, patients had a significantly (p < 0.0001) higher proportion of both Th17 (CD4(+)IL17A(+)) and Treg (CD4(+)CD25(+)FOXP3(+)) cells, which further showed a reciprocal balance in relation to clinico-pathological parameters in patients. We also detected a circulating CD8(+) subset of these cells in both patients and healthy controls, although the difference between the two groups was statistically insignificant. Higher frequencies of Th17 cells were found in patients with early stages and without lymph node involvement, while an increased prevalence of Tregs was associated with higher clinical stages and lymph node involvement. Moreover, Th17 cells were quantitatively and positively correlated to CD4(+)T and CD8(+)T cells and inversely correlated with Tregs. Contrarily, Tregs showed a negative association with CD4(+)T and CD8(+)T cells. CONCLUSIONS: Our results suggest an increase in Th17/Tregs ratio in early stages and a decrease in this ratio in higher stages of oral cancer. Such counter regulation of Th17 and Tregs may be a significant prognostic factor in oral cancer patients.

Determination of nonlinear absorption and refraction in direct and indirect band gap crystals by Z-scan method.

A systematic investigation on nonlinear optical properties such as three photon absorption (3PA) wavelength dependent of Kerr type nonlinear refraction in direct and indirect band gap crystals has been reported in the present work. The Z-scan measurements are recorded for both ZnO and CdI(2) with femtosecond laser pulses while the wavelength dependent of the Kerr nonlinearity are in agreement with a two band model. The wavelength dependence of the 3PA is determined by [(3E(photon)/E(g))-1](5/2)[(3E(photon)/E(g))](-9) in the case of direct band gap crystal and [(3E(photon)±â„Ω/E(g))-1](5/2)[(3E(photon)±â„Ω/E(g))](-9) in the case of indirect band gap crystal. In the present investigation the value of 3PA in the case of indirect band gap crystal is lower than the direct band gap crystal which is due to the phonon assisted transition. The materials of large band gap with optical nonlinearity and fast response speed should be dominating factor for further photonic devices such as optical limiters, optical switches and optical modulators. The higher order nonlinear optical effects have also been determined in the present study.

Functional genetic variants of TGF-ß1 and risk of tobacco-related oral carcinoma in high-risk Asian Indians.

Transforming growth factor (TGF)-ß1, the most abundant isoform of TGF-ß have been implicated in various stages of carcinogenesis such as epithelial to mesenchymal transition, enhanced expression of metalloproteases, down-regulation of cellular adhesion molecule, increased tumor motility and angiogenesis as well as local and systemic immunosuppression leading to a more aggressive and metastatic behavior. We assessed the association of TGF-ß1 functional genetic polymorphisms at codon 10 (869 T>C) and 25 (915 G>C) of exon 1 in 140 patients with tobacco-related oral squamous cell carcinoma (OSCC) and 120 normal subjects by PCR-RFLP. The frequency of 869 CC genotype and C allele were significantly higher in patients as compared to controls (P(c), 0.024 and 0.0004, respectively) while no significant difference was observed in the frequency of 915 CC genotype and C allele. In logistic regression analysis CC genotype (OR, 3.87; 95% CI, 1.78-8.41) and C allele (OR, 2.20; 95% CI 1.51-3.20) appeared as susceptible while TT genotype and T allele as protective. In addition C(869)-C(915) haplotype with OR of 2.48 at 95% CI, 1.51-4.06 significantly (P=0.0003) increased the risk of tobacco-related OSCC in Asian Indians.