Bacterial-mediated DNA delivery to tumour associated phagocytic cells.

Phagocytic cells including macrophages, dendritic cells and neutrophils are now recognised as playing a negative role in many disease settings including cancer. In particular, macrophages are known to play a pathophysiological role in multiple diseases and present a valid and ubiquitous therapeutic target. The technology to target these phagocytic cells in situ, both selectively and efficiently, is required in order to translate novel therapeutic modalities into clinical reality. We present a novel delivery strategy using non-pathogenic bacteria to effect gene delivery specifically to tumour-associated phagocytic cells. Non-invasive bacteria lack the ability to actively enter host cells, except for phagocytic cells. We exploit this natural property to effect 'passive transfection' of tumour-associated phagocytic cells following direct administration of transgene-loaded bacteria to tumour regions. Using an in vitro-differentiated human monocyte cell line and two in vivo mouse models (an ovarian cancer ascites and a solid colon tumour model) proof of delivery is demonstrated with bacteria carrying reporter constructs. The results confirm that the delivery strategy is specific for phagocytic cells and that the bacterial vector itself recruits more phagocytic cells to the tumour. While proof of delivery to phagocytic cells is demonstrated in vivo for solid and ascites tumour models, this strategy may be applied to other settings, including non-cancer related disease.

Enhancement of electroporation facilitated immunogene therapy via T-reg depletion.

Regulatory T cells (T-regs) can negatively impact tumor antigen-specific immune responses after infiltration into tumor tissue. However, depletion of T-regs can facilitate enhanced anti-tumor responses, thus augmenting the potential for immunotherapies. Here we focus on treating a highly aggressive form of cancer using a murine melanoma model with a poor prognosis. We utilize a combination of T-reg depletion and immunotherapy plasmid DNA delivered into the B16F10 melanoma tumor model via electroporation. Plasmids encoding murine granulocyte macrophage colony-stimulating factor and human B71 were transfected with electroporation into the tumor and transient elimination of T-regs was achieved with CD25-depleting antibodies (PC61). The combinational treatment effectively depleted T-regs compared to the untreated tumor and significantly reduced lung metastases. The combination treatment was not effective in increasing the survival, but only effective in suppression of metastases. These results indicate the potential for combining T-reg depletion with immunotherapy-based gene electrotransfer to decrease systemic metastasis and potentially enhance survival.

Peak B endorphin concentration in cerebrospinal fluid: reduced in chronic pain patients and increased during the placebo response.

The level of an endogenous opioid (peak B endorphin) was measured in chromatographically fractionated cerebrospinal fluid (CSF) sampled from two groups of chronic pain patients before and after intrathecal saline (placebo) injection. As assessed by a verbal rating scale, one group reported no change in their level of pain (non-responders, NR; n = 6) while the other group reported complete or greater than 50% pain relief (placebo responders, PR; n = 14). We find, as has been reported previously, that initial peak B levels were lower (by 50%) in these chronic pain patients' CSF than in CSF from pain-free (PF) normal controls (P less than 0.001, t-test). Peak B levels measured from CSF of the NR group undergoing this procedure did not change (P greater than 0.4, paired t-test). In contrast, a significant 2.3-fold increase was measured in the CSF peak B level of the PR group (P less than 0.05, paired t-test). This is the first direct evidence that a CSF opioid is correlated with placebo pain relief in chronic pain patients. Peak B is a potent analgesic substance when administered by the intracerebroventricular route in mice and its level is related to the patients' pain status in a presumably causal manner.

Partial characterization of a novel endogenous opioid in human cerebrospinal fluid.

Human cerebrospinal fluid (CSF) contains many uncharacterized endogenous opioids, in addition to the known enkephalins, endorphins, and dynorphins. These opioids may be separated by gel filtration chromatography and identified by radioreceptor assay for opioid activity. One region of the chromatographic elution profile, designated "Peak B" has previously been shown to be related to the pain status of chronic pain patients. We now report that human Peak B isolated from the CSF of pain-free elective surgery patients is present at a typical concentration equivalent in activity to 1.4 pmol of morphine sulfate per ml of CSF measured by radioreceptor assay. At a dose of 0.06 and 0.12 pmol morphine sulfate equivalents of CSF (MSE), injected into the cerebroventricular system of the mouse, Peak B produced an antinociceptive effect, the intensity and duration of which was dose-dependent and which was antagonized by naloxone. The mouse vas deferens (MVD) preparation was inhibited by Peak B in a manner that was sensitive to antagonism by naloxone only at low (less than 1.0 microM) but not at higher (greater than 6.0 microM) concentrations of the antagonist. Peak B activity in the MVD assay was unaffected by treatment with trypsin or alpha-chymotrypsin.

The effect of morphine upon CSF opioids in dogs.

Cerebrospinal fluid opioid activity was measured in dogs before and after morphine administration in order to investigate whether morphine results in an increase in CSF endogenous opioid levels. CSF was fractionated on a gel filtration column, and these fractions were assayed using a radioreceptor assay. Several peaks of opioid activity were seen in dog CSF both before and after morphine treatment. The predominant change in opioid peak patterns was the appearance after morphine treatment of a large peak in the elution position of morphine itself. Radiotracer studies verified that micromolar concentrations of morphine were present in CSF, which is enough morphine for potent binding activity. In addition to the increased activity in the elution position of morphine, several peaks of endogenous opioid activity showed increases after morphine treatment which were statistically significant.

Morphine tolerance is associated with elevated levels of an uncharacterized endorphin (peak B) in mouse brain with no change in 3H-dihydromorphine binding.

A partially characterized mouse brain endorphin was shown to be elevated (p 0.01 U-Test) in ICR strain mice that had been made tolerant but not dependent upon morphine (5 mg/kg sc., given for 32 days). The animals were tolerant to the antinociceptive effect of morphine as judged by the tail immersion assay (48 degrees C) but showed no detectable dependence or withdrawal syndrome effects following the administration of naloxone (writhing, jumping, diarrhea or hypermotility) on day 33. No significant changes were seen in any other mouse brain endorphins (p 0.05 U-Test). Also there was no apparent change in the number of binding properties of 3H-dihydromorphine (3H-DHM) receptors (mu-receptors) in chronic morphine (CM) treated, as compared with chronic saline (CS) treated animals.

Decreased normorphine sensitivity of maternal guinea pig ilea at parturition.

Several approaches have been used by others to explore the possible involvement of endorphins in pregnancy and parturition. We employed the opioid sensitive preparation, the guinea pig ileum, to measure opioid sensitivity in three groups of adult guinea pigs: non-pregnant, near parturition, and a few weeks after parturition. Elevated normorphine EC50s occurred near the time of parturition. At 14-16 days post-partum, the EC50s for normorphine had returned to non-pregnant control levels. These data point to the activation of endorphin systems at the time of parturition.

Effects of beta-endorphin and morphine on the sleep-wakefulness behavior of cats.

The effects of beta-endorphin and of morphine SO4 (0.5 microgram and 2.0 microgram, respectively, injected intraventricularly) upon the sleep-wakefulness behavior of cats were examined. Both agents produced insomnia. Deep slow wave sleep was sharply inhibited, and rapid eye movement (REM) sleep was entirely suppressed. Light slow wave sleep, occurring in brief, isolated episodes, became the most abundant stage of sleep. The nuchal electromyogram was markedly increased after both agents. Naloxone (100 microgram/kg), injected subcutaneously 30 min before beta-endorphin or morphine SO4, entirely reversed these agents' effects on the two stages of slow wave sleep, and antagonized the exaggerated electromyogram. But naloxone did not counteract the REM-suppressant effect of either beta-endorphin or morphine SO4. Total sleep time reverted towards control values after naloxone pretreatment, but not entirely; the difference may be due to the persistent deficit of REM sleep. The data may indicate an involvement of an inner opioid in the regulation of sleep and wakefulness in the cat, and may point to a role for more than one endorphin receptor in the effects of opioids on the states of vigilance in cats.

Binding of adenosine 5'-monophosphate to bovine liver fructose 1,6-bisphosphatase.

Bovine liver fructose 1,6-bisphosphatase bound 4 mol of its allosteric inhibitor AMP per mole of enzyme with half-saturation at 17 mumol/l AMP. The presence of a mixture of positive and negative cooperativity in the binding of AMP to the enzyme was suggested by several procedures for analyzing binding data. In particular, calculation of the intrinsic binding constants for AMP yielded the relationships: K1' less than K2' greater than K3' less than K4', indicating mixed cooperativity.

Synthetic scotophobin in goldfish: specificity and effect on learning.

Synthetic rat scotophobin was injected intracranially into common goldfish (Carassius auratus) which were then trained to avoid light or dark. The substance interacts with the learning process in goldfish in an apparently specific way, facilitating the acquisition of dark avoidance, a task homologous with that acquired by rats from which the natural peptide was isolated, while inhibiting acquisition of light avoidance.