Maturation of dendritic cells (DC) is critical for the induction of Ag-specific immunity. Ag-loaded DC matured with LPS, which mediates its effects by binding to Toll-like receptor 4 (TLR4), induce Ag-specific CTL in vitro and in vivo in animal models. However, clinical use of LPS is limited due to potential toxicity. Therefore, we sought to mimic the maturation-inducing effects of LPS on DC by stimulating TLR4-mediated signaling in the absence of exogenous LPS. We developed a constitutively active TLR4 (caTLR4) and demonstrated that transfection of human DC with RNA encoding caTLR4 led to IL-12 and TNF-α secretion. Transfection with caTLR4 RNA also induced a mature DC phenotype. Functionally, transfection of DC with caTLR4 RNA enhanced allostimulation of CD4+ T cells. DC transfected with RNA encoding the MART (Melan-A/MART-1) melanoma Ag were then used to stimulate T cells in vitro. Cotransfection of these DC with caTLR4 RNA enhanced the generation of MART-specific CTL. This CTL activity was superior to that seen when DC maturation was induced using either LPS or a standard mixture of cytokines (TNF-α, IL-6, IL-1β, and PGE2). We conclude that transfection of DC with RNA encoding a functional signaling protein, such as caTLR4, may provide a new tool for studying TLR signaling in DC and may be a promising approach for the induction of DC maturation for tumor immunotherapy.

This study investigated whether the therapeutic index of regional melanoma therapy using parenteral temozolomide could be improved by chemomodulation with O 6 -benzylguanine (O 6 BG), an inhibitor of the DNA repair enzyme O 6 -alkylguanine-DNA alkyltransferase (AGT). Using a nude rat s.c. human melanoma xenograft model of the extremity, tumors were analyzed for AGT level 2 to 3 hours after the i.p. injection of 3.5 to 70.0 mg/kg O 6 BG to inhibit AGT activity. Survival studies were conducted using animals that were treated with a 15-minute isolated limb infusion with 10% DMSO in PBS (control), temozolomide alone, or temozolomide in conjunction with single or multiple doses of i.p. O 6 BG. Tumor volume and toxicity level were monitored every other day. Administration of 3.5 mg/kg O 6 BG depleted tumor AGT activity by 93.5% (P < 0.01). Groups treated with regional temozolomide alone (350 mg/kg), systemic temozolomide with O 6

Purpose: Previous preclinical studies have shown that regional temozolomide therapy via isolated limb infusion is more effective than melphalan, the current drug of choice for regional chemotherapy for advanced extremity melanoma. The aim of this study was to determine whether hyperthermia could further augment the efficacy of temozolomide, an alkylating agent, against melanoma and improve its therapeutic index in a rat model of isolated limb infusion. Experimental Design: Athymic rats bearing s.c. human melanoma xenografts (DM6) in their hind limbs were randomized to a 15-minute isolated limb infusion procedure with or without temozolomide at room temperature, normothermic (37.5jC), or hyperthermic (43jC) conditions. Results: The concomitant administration of hyperthermia during an infusion with temozolomide led to the greatest increase in tumor growth delay, decreased proliferative index, and increased cell death. Isolated limb infusion treatment with a low dose (350 mg/kg) of temozolomide was ineffective at producing tumor growth delay (P = 0.07). Similarly, temozolomide infusion under normothermia yielded minimal tumor growth delay (P = 0.08). In contrast, the combination of hyperthermia plus temozolomide treatment produced marked tumor growth delay of 10.4 days (P = 0.02) with minimal toxicity. The addition of heat to temozolomide treatment yielded the smallest proliferative index (P = 0.001), while markedly increasing the level of apoptosis 48 hours after isolated limb infusion. Conclusion: This study, the first to examine the interaction between hyperthermia and temozolomide, shows a strong, synergistic antitumor effect when hyperthermia is combined with temozolomide for regional treatment of melanoma confined to an extremity. The mechanism of this synergy seems to be through an augmentation, by hyperthermia, of the antiproliferative and proapoptotic effects of temozolomide. Isolated limb perfusion and isolated limb infusion haveemerged as effective forms of treatment for regionally advanced melanoma confined to an extremity. These procedures address all in-transit lesions confined to an extremity in a single procedure by supplying a regional concentration of chemotherapeutic agents at least 10 times higher than that reached after systemic administration, without the systemic side effects.Hyperthermia is widely used in conjunction with isolated limb perfusion and infusion to improve the response to the regional chemotherapy. This is usually done by heating the perfusate (or infusate) to 39jC to 41jC and by applying external warming blankets to prevent heat loss. Hyperthermic isolated limb perfusion was introduced based on in vitro evidence of a synergistic, cytotoxic interaction between heat and melphalan, the alkylating agent that is the current drug of choice for regional therapy of melanoma (1). Melphalan has been extensively studied for its efficacy in hyperthermic isolated limb perfusion, with response rates in the 80% range with 40% to 80% being complete responders (2, 3).However, a recent,...

Such trends suggest that PSCA may prove to be a valuable pancreatic cancer tumor marker. More generally, the technique of qPCR is shown to provide a sensitive method of evaluating markers in cancer patients.