Quinolactacins revisited: from lactams to imide and beyond

Chemical analysis of a solid phase fermentation of an Australian Penicillium citrinum strain has returned all known examples of a rare class of N-methyl quinolone lactams, quinolactacins A2 (1), B2 (2), C2 (3) and Al (4), together with the new quinolactacins B1 (5), C1 (6), D1 (7) and D2 (8), and the novel derivatives quinolonimide (9) and quinolonic acid (10). Complete stereostructures were assigned to all these compounds by detailed spectroscopic analysis and chemical interconversion. Carefully controlled and monitored decomposition studies have confirmed that quinolactacins readily undergo C-3 epimerization and oxidation, and under appropriate conditions convert to quinolonimide and quinolonic acid. Mechanisms for key transformations are proposed. The decomposition studies suggested that only quinolactacins A2 (1) and B2 (2) are genuine natural products, with all other isolated compounds being decomposition artefacts. Quinolactacins C1 (6), C2 (3), and the racemic mixture of quinolactacins D1/D2 (8/7) all displayed notable cytotoxic activity.

Doom and gloom or a time for optimism: Potential aspirants' views about school leadership - now and for the future

Principal recruitment has attracted national and international attention in recent years (eg. Barty et al, 2005 in Australia; Earley et al, 2002 in the UK; Brooking et al, 2003 in New Zealand; Williams, 2003 in Canada). Importantly, Australian research in both state and non-state schools suggests that potential principal aspirants are less enthusiastic than might be expected in their desire to become principals (D’Arbon et al, 2002; Cranston et al, 2004; Lacey, 2002). Given the importance of ensuring we have quality leaders for our schools in the future, the research reported here (which is on-going) examined the views of potential aspirants (primary and secondary deputy principals) from one large government education system in Australia about the principalship and their intentions in seeking promotion (or otherwise) to such positions and the reasons driving these intentions. Data were collected via the Aspiring Principals Questionnaire (APQ) – especially developed for the study – comprising 38 closed items mainly of a Likert-type format, 5 open-ended items linked to particular closed items allowing participants to add their own suggestions/ideas, expand/elaborate on responses; and 4 further more general open-ended items. A number of system-level policy and practice recommendations have been developed from the findings.

Liposomes based on dimethyldioctadecylammonium promote a depot effect and enhance immunogenicity of soluble antigen

The mechanism behind the immunostimulatory effect obtained with the cationic liposomal vaccine adjuvant DDA:TDB remains unclear. One of the proposed hypotheses is the 'depot effect' in which the liposomal carrier helps to retain the antigen at the injection site thereby increasing the time of vaccine exposure to the immune cells. In the present study we devise a method to quantify the in vivo movement of liposomes and vaccine antigen using the radioisotopes H(3) and I(125) respectively. H(3)-labeled liposomes composed of dimethyldioctadecylammonium bromide (DDA) or an 8:1 molar ratio of DDA and trehalose 6,6-dibehenate (TDB) were administered in combination with I(125)-labeled Ag85B-ESAT-6 antigen, both via intramuscular and subcutaneous injection to mice. Furthermore characterisation of the liposomal system in simulated in vivo conditions was undertaken. Our results show that this dual-labeling technique is functional and reproducible. The administration of Ag85B-ESAT-6 without a liposomal carrier leads to rapid dissemination of the antigen from the site of injection. The administration of Ag85B-ESAT-6 together with either DDA or DDA:TDB liposomes however leads to deposition of the antigen at the injection site with detectable levels still being present 14 days post injection. Neither the incorporation of TDB nor the route of injection had any significant influence on the depot effect of DDA-based liposomes. The presence of TDB in DDA liposomes improves draining of liposomes to the lymph node in addition to increasing monocyte influx to the site of injection as highlighted by the intensive blue colouring of the injection site after pontamine blue staining of phagocytic cells in vivo. Our findings provide conclusive evidence for a cationic liposome-mediated deposition of antigen at the injection site with improved monocyte infiltration.

Effect of vesicle size on tissue localization and immunogenicity of liposomal DNA vaccines

The formulation of plasmid DNA (pDNA) in cationic liposomes is a promising strategy to improve the potency of DNA vaccines. In this respect, physicochemical parameters such as liposome size may be important for their efficacy. The aim of the current study was to investigate the effect of vesicle size on the in vivo performance of liposomal pDNA vaccines after subcutaneous vaccination in mice. The tissue distribution of cationic liposomes of two sizes, 500 nm (PDI 0.6) and 140 nm (PDI 0.15), composed of egg PC, DOPE and DOTAP, with encapsulated OVA-encoding pDNA, was studied by using dual radiolabeled pDNA-liposomes. Their potency to elicit cellular and humoral immune responses was investigated upon application in a homologous and heterologous vaccination schedule with 3 week intervals. It was shown that encapsulation of pDNA into cationic lipsomes resulted in deposition at the site of injection, and strongest retention was observed at large vesicle size. The vaccination studies demonstrated a more robust induction of OVA-specific, functional CD8+ T-cells and higher antibody levels upon vaccination with small monodisperse pDNA-liposomes, as compared to large heterodisperse liposomes or naked pDNA. The introduction of a PEG-coating on the small cationic liposomes resulted in enhanced lymphatic drainage, but immune responses were not improved when compared to non-PEGylated liposomes. In conclusion, it was shown that the physicochemical properties of the liposomes are of crucial importance for their performance as pDNA vaccine carrier, and cationic charge and small size are favorable properties for subcutaneous DNA vaccination.

Liposomal vaccine delivery systems

INTRODUCTION: Liposomes remain at the forefront of drug and vaccine design owing to their well-documented abilities to act as delivery vehicles. Nevertheless, the concept of liposomes as delivery vehicles is not a new one, with most works focusing on their use for the delivery of genes and drugs. However, in the last 10 years a significant amount of research has focused on using liposomes as vaccine adjuvants, not only as an antigen delivery vehicle but also as a tool to increase the immunogenicity of peptide and protein antigens. AREAS COVERED: This paper reviews liposomal adjuvants now in vaccine development, with particular emphasis on their adjuvant mechanism and how specific physicochemical characteristics of liposomes affect the immune response. The inclusion of immunomodulators is also discussed, with prominence given to Toll-like receptor ligands. EXPERT OPINION: The use of liposomes as vaccine delivery systems is evolving rapidly owing to the combined increase in technological advances and understanding of the immune system. Liposomes that contain and deliver immunostimulators and antigens are now being developed to target diseases that require stimulation of both humoral and cell-mediated immune responses. The CAF liposomal system, described in detail in this review, is one liposomal model that shows such flexibility.

Microscopy imaging of liposomes:from coverslips to environmental SEM

A plethora of techniques for the imaging of liposomes and other bilayer vesicles are available. However, sample preparation and the technique chosen should be carefully considered in conjunction with the information required. For example, larger vesicles such as multilamellar and giant unilamellar vesicles can be viewed using light microscopy and whilst vesicle confirmation and size prior to additional physical characterisations or more detailed microscopy can be undertaken, the technique is limited in terms of resolution. To consider the options available for visualising liposome-based systems, a wide range of microscopy techniques are described and discussed here: these include light, fluorescence and confocal microscopy and various electron microscopy techniques such as transmission, cryo, freeze fracture and environmental scanning electron microscopy. Their application, advantages and disadvantages are reviewed with regard to their use in analysis of lipid vesicles.

A cationic vaccine adjuvant based on a saturated quaternary ammonium lipid have different in vivo distribution kinetics and display a distinct CD4 T cell-inducing capacity compared to its unsaturated analog

Adjuvants are often composed of different constituents that can be divided into two groups based on their primary activity: the delivery system which carries and presents the vaccine antigen to antigen-presenting cells, and the immunostimulator that activates and modulates the ensuing immune response. Herein, we have investigated the importance of the delivery system and in particular its physical characteristics by comparing the delivery properties of two lipids which differ only in the degree of saturation of the acyl chains, rendering the liposomes either rigid (DDA, dimethyldioctadecylammonium) or highly fluid (DODA, dimethyldioleoylammonium) at physiological temperature. We show that these delivery systems are remarkably different in their ability to prime a Th1-directed immune response with the rigid DDA-based liposomes inducing a response more than 100 times higher compared to that obtained with the fluid DODA-based liposomes. Upon injection with a vaccine antigen, DDA-based liposomes form a vaccine depot that results in a continuous attraction of antigen-presenting cells that engulf a high amount of adjuvant and are subsequently efficiently activated as measured by an elevated expression of the co-stimulatory molecules CD40 and CD86. In contrast, the fluid DODA-based liposomes are more rapidly removed from the site of injection resulting in a lower up-regulation of co-stimulatory CD40 and CD86 molecules on adjuvant-positive antigen-presenting cells. Additionally, the vaccine antigen is readily dissociated from the DODA-based liposomes leading to a population of antigen-presenting cells that are antigen-positive but adjuvant-negative and consequently are not activated. These studies demonstrate the importance of studying in vivo characteristics of the vaccine components and furthermore show that physicochemical properties of the delivery system have a major impact on the vaccine-induced immune response. © 2012 Elsevier B.V. All rights reserved.

How to do (or not to do) a critical literature review

More and more students are required to perform a critical literature review as part of their undergraduate or postgraduate studies. Whilst most of the latest research methods textbooks advise how to do a literature search, very few cover the literature review. This paper covers two types of review: a critical literature review and a systematic review.

Limitations of the isolated GP-STN network

An in vitro mouse slice preparation from control and MPTP-treated mice in which functional reciprocal GP-STN connectivity is maintained, does not produce oscillatory bursting or synchronous activity neuronal activity. Pharmacological interventions that produce bursting activity do so without concomitant neuronal synchrony, or a requirement for glutamate or GABA transmission. Pre-treatment with MPTP did not alter this behaviour. Thus, we have no evidence that the functionally connected, but isolated, GP — STN network can act as a pacemaker for synchronous correlated activity in the basal ganglia and must conclude that other inputs such as those from cortex and/or striatum are required.

Subthalamic nucleus neurones in slices from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mice show irregular, dopamine-reversible firing pattern changes, but without synchronous activity

The loss of dopamine in idiopathic or animal models of Parkinson's disease induces synchronized low-frequency oscillatory burst-firing in subthalamic nucleus neurones. We sought to establish whether these firing patterns observed in vivo were preserved in slices taken from dopamine-depleted animals, thus establishing a role for the isolated subthalamic-globus pallidus complex in generating the pathological activity. Mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) showed significant reductions of over 90% in levels of dopamine as measured in striatum by high pressure liquid chromatography. Likewise, significant reductions in tyrosine hydroxylase immunostaining within the striatum (>90%) and tyrosine hydroxylase positive cell numbers (65%) in substantia nigra were observed. Compared with slices from intact mice, neurones in slices from MPTP-lesioned mice fired significantly more slowly (mean rate of 4.2 Hz, cf. 7.2 Hz in control) and more irregularly (mean coefficient of variation of inter-spike interval of 94.4%, cf. 37.9% in control). Application of ionotropic glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2-amino-5-phosphonopentanoic acid (AP5) and the GABAA receptor antagonist picrotoxin caused no change in firing pattern. Bath application of dopamine significantly increased cell firing rate and regularized the pattern of activity in cells from slices from both MPTP-treated and control animals. Although the absolute change was more modest in control slices, the maximum dopamine effect in the two groups was comparable. Indeed, when taking into account the basal firing rate, no differences in the sensitivity to dopamine were observed between these two cohorts. Furthermore, pairs of subthalamic nucleus cells showed no correlated activity in slices from either control (21 pairs) or MPTP-treated animals (20 pairs). These results indicate that the isolated but interconnected subthalamic-globus pallidus network is not itself sufficient to generate the aberrant firing patterns in dopamine-depleted animals. More likely, inputs from other regions, such as the cortex, are needed to generate pathological oscillatory activity. © 2006 IBRO.