Bronwyn M. Kivell

Inefficient presentation of tumor-derived antigen by tumor-infiltrating dendritic cells

BackgroundTransplantable B16 melanoma is widely used as a tumor model to investigate tumor immunity. We wished to characterize the leukocyte populations infiltrating B16 melanoma tumors, and the functional properties of tumor-infiltrating dendritic cells (TIDC).Materials and methodsWe used the B16 melanoma cell line expressing ovalbumin protein (OVA) to investigate the phenotype and T cell stimulatory capacity of TIDC.ResultsThe majority of leukocytes in B16 melanoma were macrophages, which colocalized with TIDCs, B and T cells to the peripheral area of the tumor. Both myeloid and plasmacytoid DC populations were present within tumors. Most of these DCs appeared immature, but about a third expressed a mature phenotype. TIDCs did not present tumor-derived antigen, as they were unable to induce the proliferation of tumor-specific CD4+ and CD8+ T cells in vitro unless in the presence of specific peptides. Some presentation of tumor-derived antigen could be demonstrated in the tumor-draining lymph node using in vivo proliferation assays. However, while proliferation of CD8+ T cells was reproducibly demonstrated, no proliferation of CD4+ T cells was observed.ConclusionIn summary, our data suggest that DCs in tumors have limited antigen-presenting function. Inefficient antigen presentation extends to the tumor-draining lymph node, and may affect the generation of antitumor immune responses.

Kappa opioids and the modulation of pain

Background and rationalePain is a complex sensory experience, involving cognitive factors, environment (setting, society, and culture), experience, and gender and is modulated significantly by the central nervous system (CNS). The mechanisms by which opioid analgesics work are understood, but this class of drugs is not ideal as either an analgesic or anti-hyperalgesic. Accordingly, considerable effort continues to be directed at improved understanding of nociceptor function and development of selective analgesics that do not have the unwanted effects associated with opioid analgesics.ObjectiveThe purpose of this paper is to provide a review of the role of KOP receptors in the modulation of pain and highlight several chemotypes currently being explored as peripherally restricted KOP ligands.ResultsA growing body of literature has shown that KOP receptors are implicated in a variety of behavioral pain models. Several different classes of peripherally restricted peptidic and nonpeptidic KOP agonists have been identified and show utility in treating painful conditions.ConclusionThe pharmacological profile of KOP agonists in visceral pain models suggest that peripherally restricted KOP agonists are potentially useful for a variety of peripheral pain states. Further, clinical investigation of peripherally restricted KOP agonists will help to clarify the painful conditions where KOP agonists will be most effective.

Effect of kappa-opioid receptor agonists U69593, U50488H, spiradoline and salvinorin A on cocaine-induced drug-seeking in rats.

Our previous work indicated that pretreatment with the selective kappa-opioid receptor (KOPr) agonist, U69593, attenuated the ability of priming injections of cocaine to reinstate extinguished cocaine-seeking behavior. The present study expanded these initial tests to include other traditional KOPr agonists, U50488H, spiradoline (SPR), and salvinorin A (Sal A), an active constituent of the plant Salvia divinorum. Following acquisition and stabilization of cocaine self-administration, cocaine-produced drug-seeking was measured. This test was conducted in a single day and comprised an initial phase of self-administration, followed by a phase of extinguished responding. The final phase examined reinstatement of extinguished cocaine self-administration followed by a priming injection of cocaine (20.0mg/kg, intraperitoneal (I.P.)) in combination with the various KOPr agonists. Cocaine-induced drug-seeking was attenuated by pretreatment with U69593 (0.3mg/kg, subcutaneous (S.C.)), U50488H (30.0mg/kg, I.P.), SPR (1.0, 3.0mg/kg, I.P.) and Sal A (0.3, 1.0mg/kg, I.P.). Sal A (0.3, 1.0mg/kg, I.P.) had no effect on operant responding to obtain sucrose reinforcement or on cocaine-induced hyperactivity. These findings show that Sal A, like other traditional KOPr agonists attenuates cocaine-induced drug-seeking behavior.

Serum-free culture of rat post-natal and fetal brainstem neurons.

Serum-free medium is essential for cell culture studies in which complete control of the environment is required. Primary culture of post-natal brainstem neurons in defined medium has not been described in the literature, and successful culture of primary brainstem neurons is typically restricted to embryonic ages E14-E18. This study describes a method for culture of fetal and post-natal brainstem neurons using a serum-free culture medium. The culture system is based on Neurobasal medium supplemented with antioxidant-rich B27. Media and supplements are commercially available products from Life Technologies. Neuron survival was optimized by replacing glutamine with GlutaMaxI, by matching osmolality with neuronal age, and by using Hibernate medium to increase neuron survival during tissue dissociation. Fetal E14, E16, E20, and post-natal P3 and P6 cultures were examined after 4, 7, and 9 days in culture. Neuron and glial cells present in the cultures were identified using immunocytochemistry with antibodies raised against microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP), respectively. Fetal E14 cultures had more bipolar neurons than multipolar neurons compared with developmentally older P6 cultures. Early fetal cultures had a higher percentage of neurons than late fetal and early post-natal cultures. Neuron survival was similar between 4 and 9 days in culture for all age groups tested. This is the first reliable, defined culture medium that supports brainstem neurons from late fetal and early post-natal stages of the rat for up to 6 days post-partum.

Method for serum-free culture of late fetal and early postnatal rat brainstem neurons

Primary culture of postnatal brainstem neurons in defined medium has not been described in the literature. Successful primary culture of brainstem neurons is typically restricted to embryonic ages E14-E18. This study describes a method for culture of late fetal and early postnatal brainstem neurons using a serum-free culture medium. The culture system is based on Neurobasal medium supplemented with antioxidant-rich B27 (Life Technologies). Neuron survival was optimized by replacing glutamine with GlutaMaxI, by matching osmolality with neuronal age, and by using Hibernate medium to increase neuron survival during tissue dissociation. This paper describes the first reliable method for culturing brainstem neurons from late fetal and early postnatal stages of the rat for up to 6 days postpartum.

Synthesis and κ-opioid receptor activity of furan-substituted salvinorin A analogues.

The neoclerodane diterpene salvinorin A, found in the leaves of Salvia divinorum, is a potent κ-opioid receptor agonist, making it an attractive scaffold for development into a treatment for substance abuse. Although several successful semisynthetic studies have been performed to elucidate structure–activity relationships, the lack of analogues with substitutions to the furan ring of salvinorin A has prevented a thorough understanding of its role in binding to the κ-opioid receptor. Herein we report the synthesis of several salvinorin A derivatives with modified furan rings. Evaluation of these compounds in a functional assay indicated that sterically less demanding substitutions are preferred, suggesting the furan ring is bound in a congested portion of the binding pocket. The most potent of the analogues successfully reduced drug-seeking behavior in an animal model of drug-relapse without producing the sedation observed with other κ-opioid agonists.

Peloruside- and laulimalide-resistant human ovarian carcinoma cells have βI-tubulin mutations and altered expression of βII- and βIII-tubulin isotypes

Peloruside A and laulimalide are potent microtubule-stabilizing natural products with a mechanism of action similar to that of paclitaxel. However, the binding site of peloruside A and laulimalide on tubulin remains poorly understood. Drug resistance in anticancer treatment is a serious problem. We developed peloruside A- and laulimalide-resistant cell lines by selecting 1A9 human ovarian carcinoma cells that were able to grow in the presence of one of these agents. The 1A9-laulimalide resistant cells (L4) were 39-fold resistant to the selecting agent and 39-fold cross-resistant to peloruside A, whereas the 1A9-peloruside A resistant cells (R1) were 6-fold resistant to the selecting agent while they remained sensitive to laulimalide. Neither cell line showed resistance to paclitaxel or other drugs that bind to the taxoid site on β-tubulin nor was there resistance to microtubule-destabilizing drugs. The resistant cells exhibited impaired peloruside A/laulimalide-induced tubulin polymerization and impaired mitotic arrest. Tubulin mutations were found in the βI-tubulin isotype, R306H or R306C for L4 and A296T for R1 cells. This is the first cell-based evidence to support a β-tubulin–binding site for peloruside A and laulimalide. To determine whether the different resistance phenotypes of the cells were attributable to any other tubulin alterations, the β-tubulin isotype composition of the cells was examined. Increased expression of βII- and βIII-tubulin was observed in L4 cells only. These results provide insight into how alterations in tubulin lead to unique resistance profiles for two drugs, peloruside A and laulimalide, that have a similar mode of action. Mol Cancer Ther; 10(8); 1419–29. ©2011 AACR.

Estimating cross-price elasticity of e-cigarettes using a simulated demand procedure.

INTRODUCTION Our goal was to measure the cross-price elasticity of electronic cigarettes (e-cigarettes) and simulated demand for tobacco cigarettes both in the presence and absence of e-cigarette availability. METHOD A sample of New Zealand smokers (N = 210) completed a Cigarette Purchase Task to indicate their demand for tobacco at a range of prices. They sampled an e-cigarette and rated it and their own-brand tobacco for favorability, and indicated how many e-cigarettes and regular cigarettes they would purchase at 0.5×, 1×, and 2× the current market price for regular cigarettes, assuming that the price of e-cigarettes remained constant. RESULTS Cross-price elasticity for e-cigarettes was estimated as 0.16, and was significantly positive, indicating that e-cigarettes were partially substitutable for regular cigarettes. Simulated demand for regular cigarettes at current market prices decreased by 42.8% when e-cigarettes were available, and e-cigarettes were rated 81% as favorably as own-brand tobacco. However when cigarettes cost 2× the current market price, significantly more smokers said they would quit (50.2%) if e-cigarettes were not available than if they were available (30.0%). CONCLUSION Results show that e-cigarettes are potentially substitutable for regular cigarettes and their availability will reduce tobacco consumption. However, e-cigarettes may discourage smokers from quitting entirely as cigarette price increases, so policy makers should consider maintaining a constant relative price differential between e-cigarettes and tobacco cigarettes.

Pharmacology and anti-addiction effects of the novel κ opioid receptor agonist Mesyl Sal B, a potent and long-acting analogue of salvinorin A.

Acute activation of κ opioid (KOP) receptors results in anticocaine‐like effects, but adverse effects, such as dysphoria, aversion, sedation and depression, limit their clinical development. Salvinorin A, isolated from the plant Salvia divinorum, and its semi‐synthetic analogues have been shown to have potent KOP receptor agonist activity and may induce a unique response with similar anticocaine addiction effects as the classic KOP receptor agonists, but with a different side effect profile.

Predicting decreases in smoking with a cigarette purchase task: evidence from an excise tax rise in New Zealand

Background Tobacco excise taxes are known to be effective in reducing smoking at the population level, but less research has examined how individual smokers respond to changes in tax policy. We ask whether price elasticities for individual smokers, derived from simulated demand curves obtained with a cigarette purchase task (CPT), can predict changes in smoking after a tax increase. Method Smokers (N=357) were recruited from four New Zealand cities and interviewed before and after a 10% tobacco excise tax increase. Results Simulated demand curves from the CPT were curvilinear and well described by an exponential model. Smokers reported significant reductions in cigarettes/day and addiction scores at Wave 2 (n=226). Local elasticities derived from the demand curves significantly predicted decreases in cigarettes/day after controlling for covariates. Conclusions Elasticities from simulated demand curves can predict decreases in consumption for individual smokers after an excise tax increase. Understanding individual differences in tobacco demand curves may help to predict how different groups of smokers will respond to price increases.