l-Arginine plays a central role in the normal function of several organs including the immune system. It is metabolized in macrophages by inducible nitric oxide synthase to produce nitric oxide, important in the cytotoxic mechanisms, and by arginase I (ASE I) and arginase II (ASE II) to synthesize l-ornithine and urea, the first being the precursor for the production of polyamines needed for cell proliferation. l-Arginine availability can modulate T cell function. Human T cells stimulated and cultured in the absence of l-arginine lose the expression of the TCR ζ-chain (CD3ζ) and have an impaired proliferation and a decreased cytokine production. The aim of this work was to test whether activated macrophages could modulate extracellular levels of l-arginine and alter T cell function, and to determine which metabolic pathway was responsible for this event. The results show that macrophages stimulated with IL-4 + IL-13 up-regulate ASE I and cationic amino acid transporter 2B, causing a rapid reduction of extracellular levels of l-arginine and inducing decreased expression of CD3ζ and diminished proliferation in normal T lymphocytes. Competitive inhibitors of ASE I or the addition of excess l-arginine lead to the re-expression of CD3ζ and recovery of T cell proliferation. In contrast, inducible nitric oxide synthase or ASE II failed to significantly reduce the extracellular levels of l-arginine and modulate CD3ζ expression. These results may provide new insights into the mechanisms leading to T cell dysfunction and the down-regulation of CD3ζ in cancer and chronic infectious diseases.

L-Arg plays a central role in the normal function of several organ systems including the immune system. L-Arg can be depleted by arginase I produced by macrophages and hepatocytes in several disease states such as trauma and sepsis and following liver transplantation. The decrease in L-Arg levels induces a profound decrease in T cell function through mechanisms that have remained unclear. The data presented here demonstrate that Jurkat T cells cultured in medium without L-Arg (L-Arg-free RPMI) have a rapid decrease in the expression of the T cell antigen receptor chain (CD3), the principal signal transduction element in this receptor, and a decrease in T cell proliferation. This phenomenon is completely reversed by the replenishment of L-Arg but not other amino acids. These changes are not caused by cell apoptosis; instead, the diminished expression of CD3 protein is paralleled by a decrease in CD3 mRNA. This change in CD3 mRNA expression is not caused by a decrease in the transcription rate but rather by a significantly shorter CD3 mRNA half-life. This mechanism is sensitive to cycloheximide. Therefore, the regulation of L-Arg concentration in the microenvironment could represent an important mechanism to modulate the expression of CD3 and the T cell receptor and consequently of T cell function.L-Arg plays a central role in several functions of the immune system (1-3). It is metabolized in macrophages by two independent enzymatic pathways (4), the inducible nitric-oxide synthetase and arginase I, leading to different effects on the immune system (4 -11). L-Arg is metabolized by inducible nitricoxide synthetase to produce nitric oxide, one of the principal cytotoxic mechanisms in macrophages (12-15). Alternatively, arginase I metabolizes L-Arg to L-ornithine and urea, the first being the precursor for the production of polyamines that are essential for cell proliferation and fibroblast function (5, 6, 11). The depletion of L-Arg by an increased production of arginase I following liver transplantation (16 -18), severe trauma (9, 20), or sepsis (21) coincides with a major decrease in T cell proliferation. Furthermore, the infusion of high doses of L-Arg results in a recovery of T cell function and an increase in the number of CD4 ϩ cells (22-24), suggesting that L-Arg may play an important role in regulating the T cell function by mechanisms that have remained unclear.The T cell receptor chain (CD3) is the principal signal transduction element of the T cell antigen receptor (TCR) (25)(26)(27). A decreased expression of CD3 has been described in T cells from patients with cancer (28 -32), lupus (33), and chronic infectious diseases such as leprosy (34) and tuberculosis (35). The mechanisms mediating the CD3 decrease are poorly understood. We tested the effect of the absence of L-Arg on T cell signal transduction. The results show that Jurkat T cells cultured in tissue culture medium without L-Arg had a rapid decrease in the expression of CD3 but not of other chains of the TCR such as CD3⑀ (36). The absence of L-Arg...

Epidemiologic and preclinical data support the oral-cancer prevention potential of green tea extract (GTE). We randomly assigned patients with high-risk oral premalignant lesions (OPLs) to receive GTE at 500 mg/m2, 750 mg/m2, or 1000 mg/m2 or placebo TID for 12 weeks, evaluating biomarkers in baseline and 12-week biopsies. The OPL clinical response rate was higher in all GTE arms (n=28; 50%) versus placebo (n=11; 18.2%; p=0.09) but did not reach statistical significance. However, the 2 higher-dose GTE arms (58.8% [750, 1000 mg/m2], 36.4% [500 mg/m2], and 18.2%, [placebo], p=0.03) had higher responses, suggesting a dose-response effect. GTE treatment also improved histology (21.4% versus 9.1%, p=0.65), though not statistically significant. GTE was well-tolerated although higher doses increased insomnia/nervousness but produced no grade-4 toxicity. Higher mean baseline stromal VEGF correlated with a clinical (p=0.04) but not histologic response. Baseline scores of other biomarkers (epithelial VEGF, p53, Ki-67, cyclin D1, and p16 promoter methylation) were not associated with a response or survival. Baseline p16 promoter methylation (n=5) was associated with a shorter cancer-free survival. Stromal VEGF and cyclin D1 expression were downregulated in clinically responsive GTE patients and upregulated in non-responsive patients at 12 weeks (versus at baseline). An extended (median 27.5 months) follow-up showed a median time to oral cancer of 46.4 months. GTE may suppress OPLs, in part through reducing angiogenic stimulus (stromal VEGF). Higher doses of GTE may improve short-term (12 week) OPL outcome. The present results support longer-term clinical testing of GTE for oral cancer prevention.

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BACKGROUND:Treatment options for patients with advanced head and neck squamous cell carcinoma (HNSCC) are scarce. This phase 2 study was conducted to evaluate the safety, tolerability, pharmacokinetics, and efficacy of dasatinib in this setting. METHODS: Patients with recurrent and/or metastatic HNSCC after platinum-based therapy were treated with dasatinib either orally or via percutaneous feeding gastrostomy (PFG). Primary endpoints were 12-week progression-free survival (PFS) and objective response rate with a 2-stage design and early withdrawal if the 12-week PFS rate was 20% and no patients had an objective response (OR). Forty-nine serum cytokines and angiogenic factors (CAFs) were analyzed from treated patients. RESULTS: Of the 15 patients enrolled, 12 were evaluable for response, and all patients were evaluable for toxicity. No OR was observed and 2 patients (16.7%) had stable disease (SD) at 8 weeks. The median treatment duration was 59 days, the median time to disease progression was 3.9 weeks, and the median survival was 26 weeks. One patient required a dose reduction, 3 patients required dose interruptions, and 4 patients were hospitalized for toxicity. Dasatinib inhibited c-Src both when administered orally and via PFG. Greater mean drug exposure, decreased half-life, and greater maximum concentration were observed in patients receiving dasatinib via PFG. Eleven baseline CAFs were associated with treatment outcome and 1 CAF, macrophage migration inhibitory factor, was found to be differentially modulated in correlation with SD versus disease progression. CONCLUSIONS: Single-agent dasatinib failed to demonstrate significant activity in patients with advanced HNSCC, despite c-Src inhibition. The toxicity profile was consistent with that reported in other solid tumors, and the drug can be given via PFG tube.