Adenovirus-mediated gene transfer of pathogen-associated molecular patterns for cancer immunotherapy.

The delivery of stimulatory signals to dendritic cells (DCs) in the tumor microenvironment could be an effective means to break tumor-induced tolerance. The work presented here evaluates the immunostimulatory properties of pathogen-associated molecular patterns (PAMPs), microbial molecules which bind Toll-like receptors and deliver activating signals to immune cells, when expressed in tumor cells using adenoviral (Ad) vectors. In vitro, transduction of A549 tumor cells with Ad vectors expressing either flagellin from Listeria monocytogenes or P40 protein from Klebsiella pneumoniae induced the maturation of human monocyte-derived DCs in co-cultures. In mixed lymphocyte reactions (MLRs), Ad-flagellin and Ad-P40 transduction of tumor cells stimulated lymphocyte proliferation and the secretion of IFN-gamma. In vivo, these vectors were used either as stand-alone immunoadjuvants injected intratumorally or as vaccine adjuvants combined with a tumor antigen-expressing vector. When Ad-PAMPs were administered intratumorally to mice bearing subcutaneous syngeneic B16F0-CAR (cocksackie-adenovirus receptor) melanomas, tumor progression was transiently inhibited by Ad-P40. In a therapeutic vaccine setting, the combination of Ad-MUC1 and Ad-PAMP vectors injected subcutaneously delayed the growth of implanted RenCa-MUC1 tumors and improved tumor rejection when compared with vaccination with Ad-MUC1 alone. These results suggest that Ad-PAMPs could be effective immunoadjuvants for cancer immunotherapy.

Targeted delivery of a suicide gene to human colorectal tumors by a conditionally replicating vaccinia virus.

We have generated a thymidine kinase gene-deleted vaccinia virus (VV) (Copenhagen strain) that expressed the fusion suicide gene FCU1 derived from the yeast cytosine deaminase and uracil phosphoribosyltransferase genes. Intratumoral inoculation of this thymidine kinase gene-deleted VV encoding FCU1 (VV-FCU1) in the presence of systemically administered prodrug 5-fluorocytosine (5-FC) produced statistically significant reductions in the growth of subcutaneous human colon cancer in nude mice compared with thymidine kinase gene-deleted VV treatments or with control 5-fluorouracil alone. A limitation of prodrug therapies has often been the requirement for the direct injection of the virus into relatively large, accessible tumors. Here we demonstrate vector targeting of tumors growing subcutaneously following systemic administration of VV-FCU1. More importantly we also demonstrate that the systemic injection of VV-FCU1 in nude mice bearing orthotopic liver metastasis of a human colon cancer, with concomitant administration of 5-FC, leads to substantial tumor growth retardation. In conclusion, the insertion of the fusion FCU1 suicide gene potentiates the oncolytic efficiency of the thymidine kinase gene-deleted VV and represents a potentially efficient means for gene therapy of distant metastasis from colon and other cancers.

Continuous enzyme-linked fluorometric detection of L-(+)-lactate released from rat brain vesicles under anoxic conditions.

A method is described for the on-line detection of L-(+)-lactate released from brain vesicles under physiological conditions. The principle of L-lactate detection is based on the reversible oxidation of L-lactate catalysed by L-lactate dehydrogenase (LDH, EC 1.1.1.27) employing 3-acetylpyridine-adenine-dinucleotide (APAD) as analogue of NAD according to the reaction: L-lactate + APAD reversible pyruvate + APADH. In practical terms, L-lactate synthesis of vesicles incubated in the presence of LDH and APAD was continuously followed by the fluorescence (490 nm) of APADH excited at 410 nm. Addition of a L-lactate standard (10 mumol/l) enhanced APADH fluorescence with a half-life of 6.0 +/- 0.6 s allowing us to uncover a short-term alteration of L-lactate synthesis. This method was applied to evaluate a prospective change of L-lactate generation caused by the anoxia-induced increase in intravesicular Na+ and Ca2+ concentration ([Na+]i, [Ca2+]i), both fluorometrically determined by SBFI and Fura, respectively. Upon anoxia, [Na+]i and [Ca2+]i increased continuously up to 40 mmol/l Na+ and 900 nmol/l Ca2+ within 400 s. Concurrently, intravesicular NADH ([NADH]i) and basal L-lactate synthesis were enhanced within a few seconds, the latter from 4.2 +/- 1.5 to 15.8 +/- 1.5 nmol L-lactate/min per mg protein. Incubation of vesicles in the presence of 10 mumol/l tetrodotoxin (TTX) suppressed the increase in [Na+]i and [Ca2+]i but failed to influence L-lactate synthesis. The data indicate a continuous Na+ influx via voltage-dependent Na+ channels accompanied by an increase in [Ca2+]i during anoxia which did not affect anaerobic L-lactate synthesis. The method of fluorometric L-lactate determination was confirmed to be suitable for the detection of L-lactate released under physiological conditions from brain vesicles and seems to be applicable to various cell models.

The protective action of tetrodotoxin and (+/-)-kavain on anaerobic glycolysis, ATP content and intracellular Na+ and Ca2+ of anoxic brain vesicles.

Because recent reports point to Na+ channel blockers as protective agents directed against anoxia-induced neuronal damage including protection of anaerobic glycolysis, the influences of tetrodotoxin (TTX) and (+/-)-kavain on anoxic rat brain vesicles were investigated with respect to lactate synthesis, vesicular ATP content and cytosolic free Na+ and Ca2+ ([Na+]i, [Ca2+]i), both of the latter determined fluorometrically employing SBFI and FURA-2, respectively. After anoxia, basal lactate production was increased from 2.9 to 9.8 nmol lactate/min/mg protein. Although lactate synthesis seemed to be stable for at least 45 min of anoxia, as deduced from the linearity of lactate production, the ATP content declined continuously with a half life (tau 1/2) of 14.5 min, indicating that anaerobic glycolysis was insufficient to cover the energy demand of anoxic vesicles. Correspondingly, [Na+]i and [Ca2+]i increased persistently after anoxia by 22.1 mmol/l Na+ and 274.9 nmol/l Ca2+, determined 6.3 min after onset. An additional stimulation of vesicles with veratridine accelerated the drop of ATP (tau 1/2 = 5.1 min) and provoked a massive Na+ overload, which levelled off to 119 mmol/l Na+ within a few minutes. Concomitantly, [Ca2+]i increased linearly with a rate of 355 nmol Ca2+/l/min. Despite the massive perturbation of ion homeostasis, lactate production was unaffected during the first 8 min of veratridine stimulation. However, complete inhibition of lactate synthesis took place 30 min after veratridine was added. The Na+ channel blockers TTX and (+/-)-kavain, if applied before anoxia, preserved vesicular ATP content, diminished anoxia-induced increases in [Na+]i and [Ca2+]i and prevented both the veratridine-induced increases of [Na+]i and [Ca2+]i and the inhibition of lactate production. The data indicate a considerable Na+ influx via voltage-dependent Na+ channels during anoxia, which speeds up the decline in ATP and provokes an increase in [Ca2+]i. A massive Na+ and Ca2+ overload induced by veratridine failed to influence lactate synthesis directly, but initiated its inhibition.