Courtney M. Lappas

A2A Adenosine Receptor Induction Inhibits IFN-γ Production in Murine CD4+ T Cells

Incubation of purified C57BL/6 murine CD4+ T lymphocytes with anti-CD3 mAb serves as a model of TCR-mediated activation and results in increased IFN-γ production and cell surface expression of CD25 and CD69. We demonstrate here that signaling through the TCR causes a rapid (4-h) 5-fold increase in A2A adenosine receptor (AR) mRNA, which is correlated with a significant increase in the efficacy of A2AAR-mediated cAMP accumulation in these cells. A2AAR activation reduces TCR-mediated production of IFN-γ by 98% with a potency order of 4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxytetrahydrofuran-2-yl)-9H-purin-2-yl]prop-2-ynyl}cyclohexanecarboxylic acid methyl ester (ATL146e; EC50 = 0.19 ± 0.03 nM) > 4-{3-[6-amino-9-(5-cyclopropyl-carbamoyl-3,4-dihydroxytetrahydrofuran-2-yl)-9H-purin-2-yl]prop-2-ynyl}piperidine-1-carboxylic acid methyl ester (ATL313; 0.43 ± 0.06 nM) > 5′-N-ethylcarboxamidoadenosine (3.5 ± 0.77 nM) > 2-[4-(2-carboxyethyl)phenethylamino]-5′-N-ethylcarboxamidoadenosine (CGS21680; 7.2 ± 1.4 nM) ≫ N6-cyclohexyladenosine (110 ± 33 nM) > 2-chloro-N6-(3-iodobenzyl)-5′-N-methylcarboxamide (390 ± 160 nM), similar to the potency order to compete for radioligand binding to the recombinant murine A2AAR but not the A3AR. The selective A2AAR antagonist, 4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM241385), inhibits the effect of ATL146e with a pA2 of 0.34 nM and also inhibits the effects of N6-cyclohexyl-adenosine and 2-chloro-N6-(3-iodobenzyl)-5′-N-methylcarboxamide. In CD4+ T cells derived from A2AAR−/− and A2AAR+/− mice, the IFN-γ release response to ATL146e is reduced by 100 and 50%, respectively, indicative of a gene dose effect. The response of T cells to the phosphodiesterase inhibitor, 4-(3′-cyclopentyloxy-4′-methoxyphenyl)-2-pyrrolidone (rolipram), is not affected by A2AAR deletion. We conclude that the rapid induction of the A2AAR mRNA in T cells provides a mechanism for limiting T cell activation and secondary macrophage activation in inflamed tissues.

Adenosine A2A receptor activation limits graft-versus-host disease after allogenic hematopoietic stem cell transplantation.

GVHD is a major barrier to broader use of allogenic HSCT for nonmalignancy clinical applications such as the treatment of primary immunodeficiencies and hemoglobinopathies. We show in a murine model of C57BL/6J (H2‐kb) → B6D2F1/J (H2‐kb/d) acute GVHD that when initiated 2 days before transplant, the activation of the adenosine A2AR with the selective agonist ATL146e inhibits the weight loss and mortality associated with disease progression. Furthermore, circulating levels of proinflammatory cytokines and chemokines, including IFN‐γ, IL‐6, CCL2, KC, and G‐CSF, are reduced significantly by 14‐day ATL146e treatment. The up‐regulation of CD25, CD69, and CD40L expression by donor CD4+ and CD8+ T cells is inhibited by A2AR activation; fewer CD3+ T cells are found in the liver, skin, and colon of ATL146e‐treated mice as compared with vehicle‐treated controls; and associated tissue injury is lessened. The delayed administration of ATL146e, beginning 9 days after HSCT, reverses GVHD‐associated body weight loss successfully, and improvement is sustained for the duration of treatment. We conclude that the selective activation of the A2AR has therapeutic potential in the prevention and treatment of acute GVHD.

d-Limonene modulates T lymphocyte activity and viability

d-Limonene, a cyclic terpene that is a major component of several plant essential oils, is used widely as an additive in perfumes, soaps, foods and beverages, and has also been shown to possess chemopreventative and chemotherapeutic activity. A limited number of studies have been conducted investigating the effect of d-limonene on immune system function. We show that d-limonene and its metabolites limonene-1-2-diol and perillic acid inhibit the production by CD3(+)CD4(+) T cells of IFN-γ, IL-2, TNF-α, IL-4 and IL-13, and the production by CD3(+)CD8(+) T cells of IFN-γ, IL-2, and TNF-α. Additionally, the upregulation of CD25, CD69 and CD40L by activated T lymphocytes is modulated by d-limonene, limonene-1-2-diol and perillic acid treatment. Furthermore, high concentrations of d-limonene, limonene-1-2-diol and perillic acid induce T lymphocyte cell death. These data suggest that d-limonene possesses immunomodulatory activity that must be considered when utilizing the compound for therapeutic or commercial purposes.

The plant hormone zeatin riboside inhibits T lymphocyte activity via adenosine A2A receptor activation.

Cytokinins are plant hormones that play an integral role in multiple aspects of plant growth and development. The biological functions of cytokinins in mammalian systems are, however, largely uncharacterized. The naturally occurring cytokinin zeatin riboside has recently been demonstrated to activate the mammalian adenosine A2A receptor, which is broadly expressed by various cell types including immune system cells, with the activation of the A2AR playing a role in the regulation of cells involved in both innate and adaptive immunity. We show for the first time that zeatin riboside modulates mammalian immune system activity via an A2AR-dependent mechanism. Specifically, zeatin riboside treatment induces the production of cyclic adenosine monophosphate (cAMP) by T lymphocytes and inhibits the production by CD3+CD4+ T cells of interferon (IFN)-γ, IL-2, tumor-necrosis factor (TNF)-α, IL-4 and IL-13, and the production by CD3+CD8+ T cells of IFN-γ, IL-2 and TNF-α. Additionally, the upregulation of CD25, CD69 and CD40L by activated T lymphocytes is modulated by zeatin riboside. Zeatin riboside treatment also potently inhibits thioglycollate-induced peritoneal leukocytosis. The immunomodulatory activities of zeatin riboside are blocked by co-treatment with the selective A2AR antagonist ZM241385. These data suggest that zeatin riboside possesses therapeutic potential as a mammalian immunomodulatory agent.

The myth of the angry atheist.

ABSTRACT Atheists are often portrayed in the media and elsewhere as angry individuals. Although atheists disagree with the pillar of many religions, namely the existence of a God, it may not necessarily be the case that they are angry individuals. The prevalence and accuracy of angry-atheist perceptions were examined in 7 studies with 1,677 participants from multiple institutions and locations in the United States. Studies 1–3 revealed that people believe atheists are angrier than believers, people in general, and other minority groups, both explicitly and implicitly. Studies 4–7 then examined the accuracy of these beliefs. Belief in God, state anger, and trait anger were assessed in multiple ways and contexts. None of these studies supported the idea that atheists are particularly angry individuals. Rather, these results support the idea that people believe atheists are angry individuals, but they do not appear to be angrier than other individuals in reality.

ZnS nanocrystal cytotoxicity is influenced by capping agent chemical structure and duration of time in suspension

As a result of their characteristic physical and optical properties, including their size, intense fluorescence, broad excitation, narrow emission and resistance to photobleaching, semiconductor nanocrystals are potentially useful for a variety of biological applications including molecular imaging, live‐cell labeling, photodynamic therapy and targeted drug delivery. In this study, zinc sulfide (ZnS) semiconductor nanocrystals were synthesized in the 3 to 4 nm size range with selected capping agents intended to protect the nanocrystal core and increase its biological compatibility. We show that the biocompatibility of ZnS nanocrystals with primary murine splenocytes is influenced by the chemical structure of the outer capping agent on the nanocrystal. Additionally, the cytotoxicity of ZnS nanocrystals increases markedly as a function of time spent in suspension in phosphate‐buffered saline (PBS). These data suggest that the potential therapeutic and/or biological use of ZnS nanocrystals is inherently dependent upon the proper choice of capping agent, as well as the conditions of nanocrystal preparation and storage. Copyright

Adenosine A2A receptor activation limits chronic granulomatous disease-induced hyperinflammation

Chronic granulomatous disease (CGD) is caused by defects in the NADPH oxidase complex and is characterized by an increased susceptibility to infection. Other significant complications of CGD include autoimmunity and non-infectious hyperinflammatory disorders. We show that a gp91(phox) deficiency leads to the development of phenotypically altered T lymphocytes in mice and that this abnormal, hyperactive phenotype can be modulated by activation of the adenosine A(2A) receptor. T cells isolated from CGD mice produce significantly higher levels of the pro-inflammatory cytokines IFN-γ, IL-2, TNF-α, IL-4 and IL-13 than do WT cells after TCR-mediated activation; treatment with the selective adenosine A(2A) receptor agonist, CGS21680, potently inhibits this response. Additionally, the over exuberant inflammatory response elicited by thioglycollate challenge in gp91(phox) deficient mice is attenuated by CGS21680. These data suggest that treatment with A(2A)R agonists may be an effective therapy by which to regulate the immune system hyperactivity that results from a gp91(phox) deficiency.

The Influence of Safety, Efficacy, and Medical Condition Severity on Natural versus Synthetic Drug Preference.

Research indicates that there is a preference for natural v. synthetic products, but the influence of this preference on drug choice in the medical domain is largely unknown. We present 5 studies in which participants were asked to consider a hypothetical situation in which they had a medical issue requiring pharmacological therapy. Participants ( N = 1223) were asked to select a natural, plant-derived, or synthetic drug. In studies 1a and 1b, approximately 79% of participants selected the natural v. synthetic drug, even though the safety and efficacy of the drugs were identical. Furthermore, participants rated the natural drug as safer than the synthetic drug, and as that difference increased, the odds of choosing the natural over synthetic drug increased. In studies 2 and 3, approximately 20% of participants selected the natural drug even when they were informed that it was less safe (study 2) or less effective (study 3) than the synthetic drug. Finally, in study 4, approximately 65% of participants chose a natural over synthetic drug regardless of the severity of a specific medical condition (mild v. severe hypertension), and this choice was predicted by perceived safety and efficacy differences. Overall, these data indicate that there is a bias for natural over synthetic drugs. This bias could have implications for drug choice and usage.

Chapter 8 – Analytical Samples

Although no sample selected for analysis by forensic toxicologists is ideal, each type of sample has benefits and disadvantages associated with its use. Antemortem samples used for toxicological analysis usually are restricted to blood, urine, oral fluid, and hair, whereas for postmortem analyses, although virtually any sample may be available at autopsy, the samples most commonly used are blood, urine, and vitreous humor. In postmortem cases, when the commonly used or preferred samples are not available, or when commonly used samples may not provide the information necessary, a wide assortment of uncommonly or rarely used samples (so-called alternative samples) may be selected for analysis. This chapter provides an overview of the characteristics, handling, advantages, and disadvantages of various analytical samples including blood, urine, breath, vitreous humor, hair, oral fluid, nails, sweat, gastric contents, liver, bile, brain, lung, adipose tissues, bone and bone marrow, skeletal muscle, breast milk, neonatal samples, and nonhuman samples.

Forensic Toxicology Resources

This chapter is a compilation of selected books, journals, online resources, and organizations from which information of direct or ancillary importance to forensic toxicology may be found. Maintaining competence is of obvious importance to forensic toxicologists. This can be achieved by attending symposia, professional meetings, continuing education courses, academic courses, and by reading the relevant literature in forensic toxicology. Fortunately, there is a wealth of resources that may be used to achieve this goal. This chapter provides a description of books, ranging from historical classics to discipline specific volumes, which have become the body of knowledge of forensic toxicology. Refereed journals that publish articles of specific interest to forensic toxicologists, as well as those that publish works of more general forensic interest are described. Additionally, a compilation of web resources and professional organizations devoted to forensic toxicology is included.