Satya N. Das

Disregulated expression of the Th2 cytokine gene in patients with intraoral squamous cell carcinoma.

It has been seen that advanced stage oral squamous cell carcinoma is associated with impaired T‐cell function and higher antibody response. In order to find out if such immune disregulation is associated with alteration of T‐helper (Th) type CD4+ T‐cell phenotype leading to altered cytokine production, we studied the Th‐like cytokine profile in 35 oral squamous cell carcinoma patients and 21 normal controls. Concomitant expression of both Th1 and Th2 cytokine genes was studied by reverse transcription and Polymerase Chain Reaction (PCR) based amplification (RT‐PCR) of mRNA extracted from freshly isolated peripheral blood mononuclear cells (PBMC) using specific primers for Interferon (IFN)‐γ, Interleukin (IL)‐2, IL‐4 and IL‐10. Almost 63% of oral cancer patients showed polarization of a Th‐like cytokine response as compared to 33 % of the normal controls while 66.6% of normal controls showed a predominantly non‐polarized Th0 response. Expression of IFN‐γ and IL‐2 genes was more commonly seen in the early stage of the disease (p < 0.02) whereas majority of advanced stage tumours was associated with enhanced expression of IL‐4 and IL‐10 but not IFN‐γ and IL‐2 genes. Patients with lymphnode metastases and poorly differentiated tumours expressed IL‐4 and IL‐10 more frequently with concomitant suppression of IFN‐γ and IL‐2 genes. It seems therefore, that the development of oral squamous cell carcinoma leads to polarization of cytokine gene expression that is skewed towards the Th1‐like response in the early stage. However, increasing tumour load and lymphnode invasion suppresses Th1 cytokine genes, thus skewing it toward a Th2‐like cytokine response.

Effect of rhythmic breathing (Sudarshan Kriya and Pranayam) on immune functions and tobacco addiction.

Stress, a psychophysiological process, acts through the immune‐neuroendocrine axis and affects cellular processes of body and immune functions, leading to disease states including cancer. Stress is also linked to the habit of tobacco consumption and substance abuse, which in turn also leads to diseases. Sudarshan Kriya (SK) and Pranayam (P), rhythmic breathing processes, are known to reduce stress and improve immune functions. Cancer patients who had completed their standard therapy were studied. SK and P increased natural killer (NK) cells significantly (P <0.001) at 12 and 24 weeks of the practice compared to baseline. Increase in NK cells at 24 weeks was significant (P <0.05) compared to controls. There was no effect on T‐cell subsets after SK and P either in the study group or among controls. SK and P helped to control the tobacco habit in 21% of individuals who were followed up to 6 months of practice. We conclude that the inexpensive and easy to learn and practice breathing processes (SK and P) in this study demonstrated an increase in NK cells and a reduction in tobacco consumption. When confirmed in large and randomized studies, this result could mean that the regular practice of SK and P might reduce the incidence and progression of cancer.

Effect of cyclosporine on fertility in male rats

Abstract The effect of cyclosporine (CsA) on fertility has assumed greater importance with the increasing numbers of pediatric transplantations being performed all over the world. Conflicting reports on the effects of CsA on sex hormones are available. This experimental animal study was designed to examine the effect of CsA on testicular weight, sperm counts, seminiferous tubular diameter (STD), testicular morphology, DNA flowcytometry, sex hormone levels, and fertility in male rats. Those rats who received CsA (20 mg/kg per day) showed significant reductions in testicular weight (P < 0.05), sperm count (P < 0.01), Johnsen score (P < 0.05), STD (P < 0.01), serum testosterone levels (P < 0.05), haploid cell population (P < 0.001) in the testis, and fertility (P < 0.001) compared to those receiving CsA 10 mg/kg per day and control rats. These findings will have an important bearing for children receiving cyclosporine for long periods to guide the physician in optimally adjusting long-term treatment.

Association of TNF-α and TNFR1 promoters and 3′ UTR region of TNFR2 gene polymorphisms with genetic susceptibility to tobacco-related oral carcinoma in Asian Indians

Tobacco-related oral squamous cell carcinoma is a common malignancy in Asian people. It accounts for almost 40% of cancers among Indian men and 3% in the Western world. Smokeless tobacco has been shown to induce tumor necrosis factor-alpha (TNF-alpha), which, along with its receptors, is over-expressed in people with oral carcinoma. Single nucleotide polymorphisms (SNPs) in TNF-alpha and TNF receptor genes may affect their expression and may be a potential determinant of susceptibility to tobacco-related oral carcinomas. We assessed SNPs in TNF-alpha(-308, -238) and TNF receptor 1 (TNFR1; -609) promoters by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and at four sites of TNF receptor 2 gene (TNFR2; exon 9 site 1176; exon 10 sites 1663, 1668 and 1690) by PCR-sequence-specific primers (PCR-SSP) techniques, respectively, in 94 patients and 130 healthy controls. TNF-alpha-308 G allele was significantly lower (Pc=0.004; OR=3.85), whereas A allele was significantly higher (Pc=0.004; OR=0.25) in patients compared with controls. No significant change was observed at -238 promoter site between the two groups. In the case of TNF receptors, both TNFR1 -609 TT (Pc=0.006; OR=15.3) and TNFR2 1690 CT (Pc=0.018; OR=5.6) genotypes were significantly lower in patients compared with controls. It seems that TNF-alpha-308 G/A may be related to susceptibility, whereas -609 TT TNFR1 and 1690 C/T TNFR2 SNPs may be protective to tobacco-related oral squamous cell carcinoma. These SNPs may be useful as a marker for high-risk groups among Asian Indians.

Erufosine simultaneously induces apoptosis and autophagy by modulating the Akt-mTOR signaling pathway in oral squamous cell carcinoma.

We investigated the anticancer activity of erufosine in oral squamous carcinoma cell lines in terms of cell proliferation, colony formation, induction of autophagy/apoptosis, cell cycle and mTOR signaling pathway. Erufosine showed dose-dependent cytotoxicity in all cell lines, it induced autophagy as well as apoptosis, G2 cell cycle arrest and modulation of cyclin D1 expression. Further erufosine downregulated the phosphorylation of major components of mTOR pathway, like p-Akt at Ser473 and Thr308 residues, p-Raptor, p-mTOR, p-PRAS40 and its downstream substrates p-p70S6K and p-4EBP1 in a dose-dependent manner. The pre-treatment of tumor cells with p-mTOR siRNA increased cytotoxic effects of erufosine comparable to cisplatin but higher than rapamycin.

Cell surface expression of 70 kDa heat shock protein in human oral dysplasia and squamous cell carcinoma: correlation with clinicopathological features

To understand the role of 70 kDa heat shock protein (HSP70) in the pathogenesis of oral cancer, its expression was studied in human normal, premalignant and malignant oral mucosa. Cell surface expression of HSP70 was assessed in oral tissue specimens by flow cytometric analysis using a mouse monoclonal antibody against HSP70. A significant increase in cell surface expression of HSP70 was observed as the tissue progressed from normal to dysplasia towards carcinoma. Correlation of HSP70 expression with clinicopathological features showed a positive association with the severity of dysplasia. Among the oral tumours assessed, a significantly positive association was observed between HSP70 expression and dedifferentiation of oral squamous cell carcinomas (SCCs). Poorly differentiated tumours showed significantly higher levels of HSP70 expression on the surface of tumour cells, compared with well differentiated carcinomas (P < 0.05). Cell surface expression of HSP70 was observed to be significantly higher in clinically advanced stage tumours (T3/T4), compared with T1/T2 stage tumours (P < 0.05). The variation in cell surface expression of HSP70 in normal, dysplastic and malignant oral lesions suggests that it is differentially expressed during oral tumorigenesis and may play a specific role in the pathogenesis of oral cancer.

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

BackgroundSeveral 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 resultsWe 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.ConclusionsOur 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.

Differential expression of multidrug resistance gene product, P-glycoprotein, in normal, dysplastic and malignant oral mucosa in India.

Multidrug resistance (MDR) in human cancer is often associated with over‐expression of the mdr‐1 gene, which encodes a 170‐kDa transmembrane protein, termed P‐glycoprotein (P‐gp). We evaluated the immunoreactivity of P‐gp in oral tissues at different stages of tumorigenesis in the Indian population by flow cytometry, using the MRK‐16 monoclonal antibody, which recognizes an external epitope of P‐gp. The expression of P‐gp was studied in human oral normal tissues (12 cases), dysplastic lesions (13 cases), primary untreated squamous‐cell carcinomas (12 cases) and recurrent tumors (18 cases). Quantitative flow‐cytometric analysis of P‐gp expression showed a significant increase in P‐gp levels in untreated primary oral tumors (p < 0.01) and in dysplastic lesions (p < 0.05) as compared with normal oral tissues. A marked significant increase in P‐gp expression was observed in recurrent oral carcinomas as compared with normal oral tissues (p < 0.001) and dysplastic lesions (p < 0.01). Among recurrent tumors, a significant increase in the level of P‐gp was observed in T4‐stage tumors as compared with T3‐stage tumors (p < 0.01). We conclude that P‐gp is differentially expressed during oral tumorigenesis, and may be an indicator of the biological behavior of oral malignancies. Int. J. Cancer 74:128–133.

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.

Enumeration of lymphocyte subsets using flow cytometry: Effect of storage before and after staining in a developing country setting

Lymphocyte subset estimations by flow cytometry in population-based studies require transportation of samples from the field site to the laboratory. As samples arrive late in the day they have to wait overnight before being processed. The effect of two possible approaches, sample storage for 24 h before staining and immediate staining with analysis after 24 h and 48 h were evaluated. Two sets of experiments were performed with EDTA (ethylenediamine tetra-acetate) anticoagulated peripheral blood. In the first experiment, after collection, each sample was divided into two portions. One portion was stained at the time of blood collection and the other 24 h later after keeping it at room temperature (38–45°C). In the second experiment, blood samples were stained within 1–2 h. Each sample was analyzed immediately upon completion of staining process and subsequently after 24 h and 48 h of storage at 4°C. Results suggest that blood collected in EDTA can be processed using whole blood lysis method, after storage at room temperature (38–45°C) for 24 h with some but not significant alteration in T-cell subsets. Storage at 4°C after staining for 24 h results in a lesser and insignificant loss of cells or alteration of T-cell subsets and may be the method of choice.