Characterization of the evolution of immune phenotype during the development and progression of squamous cell carcinoma of the head and neck.

While studies have indicated that squamous cell carcinoma of the head and neck (HNSCC) is associated with immune suppression, these studies did not analyze the immune response at the dysplastic stage. The present study utilized a mouse model of 4-nitroquinoline 1-oxide-induced oral carcinogenesis to examine the alterations in immune phenotype at the premalignant and malignant stages of HNSCC. Cervical lymph nodes of HNSCC-bearing mice were found to contain a greater number of cells, including a greater number of conventional (Tconv) and regulatory (Treg) T cells, compared to cervical lymph nodes of control and premalignant lesion-bearing mice, though the Tconv cells appear to be less proliferative and the Treg cells appear to be less suppressive at the HNSCC stage. Premalignant lesion-bearing mouse lymph nodes consist of a greater percentage of Tconv cells expressing markers for activation, memory, and exhaustion compared to both control and HNSCC-bearing mice. Also, lymph nodes' cells from both premalignant lesion-bearing and HNSCC-bearing mice include increased levels of Th1, Tc1, and Th17 cells, with no differences in levels of Th2 cells, compared to control mice. The data show that while there is the expected increase in immunosuppressive Tregs in lymph nodes when HNSCC is present, there is also an unexpected increase in immune populations usually associated with a beneficial antitumor response, including Tconv cells and Th1 and Tc1 cells. In addition, the results demonstrate that the premalignant stage of HNSCC development is associated with a robust immune response involving an increase in inflammatory Th1, Tc1, and Th17 cells.

Immunological modulation by 1α,25-dihydroxyvitamin D3 in patients with squamous cell carcinoma of the head and neck.

Prior studies showing that treatment of head and neck squamous cell carcinoma (HNSCC) patients with 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] stimulated intratumoral immune infiltration were extended to analysis of cytokine profiles in the periphery and in oral tissues. Most prominent was the disparity between cytokine levels in plasma and in either pathologically normal oral tissue or HNSCC tissue from patients that were untreated or treated with 1,25(OH)(2)D(3). Levels of IL-6 and IL-10, but not IL-2, IFN-γ or TNF-α, tended to be increased in the plasma of HNSCC patients and 1,25(OH)(2)D(3) further increased plasma levels of all of these cytokines. While these cytokines tended to be increased in HNSCC tissue, 1,25(OH)(2)D(3) resulted in variable cytokine responses that showed a general tendency toward further increased levels. Levels of IL-8 and VEGF were increased in plasma and tissue of untreated HNSCC patients, and were further increased in plasma, but not in tissues, of patients treated with 1,25(OH)(2)D(3). Levels of IL-1α and IL-1ß were similar in plasma of controls and HNSCC patients, but were increased in HNSCC tissues. In contrast to that seen in plasma where 1,25(OH)(2)D(3) increased levels of IL-1α and IL-1ß, this was not seen in tissue following 1,25(OH)(2)D(3) treatment. These results show a discordant relationship between systemic and intratumoral cytokine profiles and suggest a tendency of 1,25(OH)(2)D(3)to increase a multitude of cytokines within tumor tissue.

Matrix metalloproteinase-8 expression in periodontal tissues surgically removed from diabetic and non-diabetic patients with periodontal disease.

BACKGROUND: Although it is known that periodontal matrix metalloproteinase-8 (MMP-8) expression is associated with periodontal disease, the information concerning the periodontal MMP-8 expression in diabetic patients with periodontal disease is insufficient. MATERIALS AND METHODS: Periodontal tissue specimens were collected from seven patients without periodontal disease and diabetes (Group 1), 15 patients with periodontal disease alone (Group 2) and 10 patients with both periodontal disease and diabetes (Group 3). The frozen sections were prepared and MMP-8 protein expression was detected using immunohistochemistry and quantified. For in vitro study, human U937 mononuclear cells were pre-exposed to normal or high glucose and then treated with lipopolysaccharide (LPS). RESULTS: The nonparametric Kruskal-Wallis test showed that the difference in MMP-8 protein levels among the three groups were statistically significant (p = 0.003). Nonparametric analysis using Jonckheere-Terpstra test showed a tendency of increase in periodontal MMP-8 levels across Group 1 to Group 2 to Group 3 (p = 0.0002). In vitro studies showed that high glucose and LPS had a synergistic effect on MMP-8 expression. CONCLUSION: Our current study showed an increasing trend in MMP-8 protein expression levels across patients without both periodontal disease and diabetes, patients with periodontal disease alone and patients with both diseases.

DPP-4 (CD26) inhibitor alogliptin inhibits atherosclerosis in diabetic apolipoprotein E-deficient mice.

Dipeptidyl peptidase-4 (DPP-4 or CD26) inhibitors, a new class of antidiabetic compounds, are effective in the treatment of hyperglycemia. Because atherosclerosis-related cardiovascular diseases are the major complications of diabetes, it is important to determine the effect of DPP-4 inhibitors on atherosclerosis. In this study, nondiabetic and diabetic apolipoprotein E-deficient mice were treated with DPP-4 inhibitor alogliptin for 24 weeks, and atherosclerotic lesions in aortic origins were examined. Results showed that diabetes significantly increased atherosclerotic lesions, but alogliptin treatment reduced atherosclerotic lesions in diabetic mice. Metabolic studies showed that diabetes increased plasma glucose and that alogliptin treatment reduced glucose. Furthermore, immunohistochemistry study showed that diabetes increased interleukin-6 (IL-6) and IL-1ß protein expression in atherosclerotic plaques, but alogliptin treatment attenuated diabetes-augmented IL-6 and IL-1ß expression. In consistence with the observations from the mouse models, our in vitro studies showed that alogliptin-inhibited toll-like receptor 4 (TLR-4)-mediated upregulation of IL-6, IL-1ß, and other proinflammatory cytokines by mononuclear cells. Taken together, our findings showed that alogliptin-inhibited atherosclerosis in diabetic apolipoprotein E-deficient mice and that the actions of alogliptin on both glucose and inflammation may contribute to the inhibition.

Administration of a vaccine composed of dendritic cells pulsed with premalignant oral lesion lysate to mice bearing carcinogen-induced premalignant oral lesions stimulates a protective immune response.

The use of dendritic cell (DC) vaccines as treatment for malignancy is complicated by immune evasion tactics often employed by carcinomas such as head and neck squamous cell carcinoma (HNSCC). The present study aims to determine if an immune response can be elicited by administering a DC vaccine during the premalignant stages of HNSCC, prior to development of immune escape. Mice treated with the carcinogen 4-nitroquinoline 1-oxide (4NQO) in drinking water develop premalignant oral lesions that progress to HNSCC. As previous studies demonstrated that premalignant lesions and HNSCC overexpress common tumor antigens, bone marrow-derived DCs were pulsed with premalignant lesion lysate (DCpm) and administered to 4NQO-treated mice exhibiting premalignant lesions. Lesion progression was tracked through endoscopy, which revealed that DCpm vaccination and control vaccination with dendritic cells pulsed with normal tongue epithelium lysate (DCnt) significantly decreased lesion burden at 8weeks. Analysis of lymph node cells revealed that while DCnt vaccination resulted in a rapid increase in total lymphocyte count, levels of activated conventional CD4(+) T cells and Th1, Tc1, Th17, Tc17, and Th2 cells, DCpm vaccination results in a delayed, yet substantial, increase in these immune effector mechanisms. This suggests that dendritic cell vaccination may have a beneficial effect on clinical outcome regardless of type of antigenic stimulation. Also, pulsing DCs with premalignant lysate rather than normal tongue epithelium lysate affects the dendritic cells in a way that delays the immune effector response upon vaccination of premalignant lesion-bearing mice.

Insulin treatment attenuates diabetes-increased atherosclerotic intimal lesions and matrix metalloproteinase 9 expression in apolipoprotein E-deficient mice.

Patients with diabetes mellitus have increased mortality and morbidity of cardiovascular diseases compared with nondiabetic patients. Although clinical studies have shown that effective glycemic control with insulin treatment in patients with type 1 diabetes is associated with reduced cardiovascular events, the underlying mechanisms have not been well understood. In this study, we treated diabetic apolipoprotein E-deficient (apoE-/-) mice with insulin for 20 weeks and studied the effect of insulin treatment on intimal lesion size and matrix metalloproteinase (MMP) 9 expression known to be involved in plaque destabilization. Results showed that insulin treatment, which effectively reduced plasma glucose level in diabetic mice, attenuated diabetes-increased intimal lesion size and significantly inhibited diabetes-increased MMP9 expression, but had no effect on tissue inhibitor of metalloproteinase 1 in atherosclerotic plaques. Furthermore, we observed that insulin treatment did not reduce diabetes-increased macrophage content but inhibited interleukin 6 expression, a stimulator for MMP expression. Taken together, this study has shown for the first time that insulin treatment in diabetic apoE-/- mice changes atherosclerotic lesions and gene expression to a state that favors plaque stability.

DPP-4 (CD26) inhibitor alogliptin inhibits TLR4-mediated ERK activation and ERK-dependent MMP-1 expression by U937 histiocytes.

Dipeptidyl peptidase-4 (DPP-4)/CD26, a cell surface glycoprotein, is expressed by a variety of cells including T cells, B cells, NK cells, and macrophages. Although it has been shown that DPP-4/CD26 is involved in T cell activation, its role in biological functions in macrophages has not been well investigated. In this study, we used alogliptin, a specific inhibitor of DPP 4/CD26, to study the effect of DPP-4/CD26 on the activation of the extracellular signal-regulated kinase (ERK) that plays a critical role in the expression of proinflammatory cytokines and matrix metalloproteinases (MMPs) in U937 histiocytes. Results showed that 1nM of alogliptin inhibited ERK phosphorylation induced by lipopolysaccharide (LPS), a ligand for toll-like receptor (TLR)4, by 91%. Furthermore, results showed that alogliptin inhibited LPS-stimulated MMP-1 expression in a concentration-dependent manner and 1nM of alogliptin inhibited MMP-1 expression by 60%. To confirm the involvement of the ERK pathway in MMP-1 expression by U937 cells, we showed that PD98059, a specific inhibitor for the ERK pathway, blocked LPS-stimulated MMP-1 expression. In addition to MMP-1, our study showed that alogliptin also inhibited MMP-9, -12 and -15, but had no effect on TIMP-1 and -2 expression. Taken together, this study showed for the first time that the inhibition of DPP-4/CD26 by alogliptin suppressed TLR4-mediated ERK activation and ERK-dependent MMP expression by U937 cells, suggesting that DPP-4/CD26 may play an important role in macrophage-mediated inflammation response and tissue remodeling.