Saponins from Quillaja saponaria Molina: Isolation, Characterization and Ability to Form Immuno Stimulatory Complexes (ISCOMs)

ISCOMs have received much attention as vaccine adjuvants due to their immunostimulatory effects. They are colloidal particles typically comprised of phospholipids, cholesterol and Quil A, a crude mixture of saponins extracted from the bark of Quillaja saponaria Molina. We have previously shown that ISCOMs can be prepared by ether injection wherein an ether solution of phospholipids and cholesterol in a mass ratio of 5:2 is injected into a solution of Quil A at a mass ratio of 7 lipids: 3 Quil A. The aim of this study was firstly to isolate and characterise discrete fractions of Quil A and secondly to investigate which of these fractions were able to form ISCOMs by the method of ether injection. Six fractions of Quil A were isolated by semi-preparative reverse phase high performance liquid chromatography (RP-HPLC) and characterised by analytical HPLC, liquid chromatography tandem mass spectrometry (LC-MS) and the qualitative Liebermann- Burchard and Molisch tests for triterpenoids and carbohydrates respectively. ISCOMs were subsequently prepared from the isolated fractions by the method of ether injection and the resulting preparations characterized by photon correlation spectroscopy (PCS) and negative stain transmission electron microscopy (TEM). The molecular weights of the major compounds in the fractions ranged from ∼1200 to ∼2300 Da; all fractions tested positive for triterpenoids and saccharides and four of the fractions were identified as QS-7, QS-17, QS-18 and QS-21 by analysis (LC-MS and analytical HPLC). Injection of ether solutions of lipids into aqueous solutions of QS-17, QS-18 or QS-21 all resulted in homogeneous ISCOM dispersions. The combination of lipids and QS-7 by ether injection produced lamellae and liposomes as the prominent structures and a minor amount of ISCOMs. The remaining two hydrophilic, low molecular weight fractions of Quil A did not produce ISCOMs, instead liposomes and helical structures predominated in the samples.

Engineering of needle-free physical methods to target epidermal cells for DNA vaccination

A challenge in epidermal DNA vaccination is the efficient and targeted delivery of polynucleotides to immunologically sensitive Langerhans cells. This paper investigates this particular challenge for physical delivery approaches. The skin immunology and material properties are examined in the context of the physical cell targeting requirements of the viable epidermis. Selected current physical cell targeting technologies engineered to meet these needs are examined: needle and syringe; diffusion patches; liquid jet injectors; microneedle arrays/patches; and biolistic particle injection. The operating methods and relative performance of these approaches are discussed, with a comment on potential future developments and technologies. (c) 2005 Elsevier Ltd. All rights reserved.

Numerical analysis of gas and micro-particle interactions in a hand-held shock-tube device

A unique hand-held gene gun is employed for ballistically delivering biomolecules to key cells in the skin and mucosa in the treatment of the major diseases. One of these types of devices, called the Contoured Shock Tube (CST), delivers powdered micro-particles to the skin with a narrow and highly controllable velocity distribution and a nominally uniform spatial distribution. In this paper, we apply a numerical approach to gain new insights in to the behavior of the CST prototype device. The drag correlations proposed by Henderson (1976), Igra and Takayama (1993) and Kurian and Das (1997) were applied to predict the micro-particle transport in a numerically simulated gas flow. Simulated pressure histories agree well with the corresponding static and Pitot pressure measurements, validating the CFD approach. The calculated velocity distributions show a good agreement, with the best prediction from Igra & Takayama correlation (maximum discrepancy of 5%). Key features of the gas dynamics and gas-particle interaction are discussed. Statistic analyses show a tight free-jet particle velocity distribution is achieved (570 +/- 14.7 m/s) for polystyrene particles (39 +/- 1 mu m), representative of a drug payload.

The microbiology of the eye

Infection of the external structures of the eye is one of the commonest types of eye disease worldwide. In addition, although relatively impermeable to microorganisms, infection within the eye can result from trauma, surgery or systemic disease. This article reviews the general biology of viruses, bacteria, fungi and protozoa and the major ocular infections that they cause. In addition, the effectiveness of the various antimicrobial agents in controlling ocular disease is discussed. Because of changes in the normal ocular flora, continuous monitoring of the microbiology of the eye will continue to be important in predicting future types of eye infection. Basic research is also needed into the interactions of microbes at the ocular surface. There is increasing microbial resistance to the antimicrobial agents used to treat ocular infections and hence, new antimicrobial agents will continue to be needed together with new methods of drug delivery to increase the effectiveness of existing antimicrobial agents.

Lipoplexes formulation and optimisation:in vitro transfection studies reveal no correlation with in vivo vaccination studies

The aim of these studies was to compare the effect of liposome composition on physico-chemical characteristics and transfection efficacy of cationic liposomes both in vitro and in vivo. Comparison between 4 popularly used cationic lipids, showed 3b-N-(dimethylaminoethyl)carbamate (DC-Chol) to promote the highest transfect levels in cells in vitro with levels being at least 6 times higher than those of 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA). 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and dimethyldioctadecylammonium (DDA) and approximately twice as efficient as dipalmitoyl-trimethylammonium-propane (DPTAP). To establish the role of the helper lipid, DC-Chol liposomes were formulated in combination with either 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) or cholesterol (Chol) (1:1 molar ratio) with and without the addition of phosphatidyl choline. The choice of helper lipid incorporated within the bilayer was found to influence the formation of complexes, their resultant structure and their transfection efficiency in vitro, with SUV-DNA complexes containing optimum levels of DOPE giving higher transfection than those containing cholesterol. The inclusion of PC within the formulation also reduced transfection efficiency in vitro. However, when administered in vivo, SUV-DNA complexes composed of PC:Chol:DC-Chol at a molar ratio of 16:8:4 micromole/ml were the most effective at inducing splenocyte proliferation upon exposure to antigen in comparison to control spleens. These results demonstrate that there is no in vitro/in vivo correlation between the transfection efficacy of these liposome formulations and in vitro transfection in the above cell model cannot be taken as a reliable indicator for in vivo efficacy of DNA vaccines.

In vitro characterization of a collagen scaffold enzymatically cross-linked with a tailored elastin-like polymer

Collagen, the main structural component of the extracellular matrix (ECM), provides tensile stiffness to different structures and organs against rupture. However, collagen tissue-engineered implants are hereto still lacking in mechanical strength. Attempts to create stiffer scaffolds have resulted in increased brittleness of the material, reducing the versatility of the original component. The hypothesis behind this research is that the introduction of an elastic element in the scaffold will enhance the mechanical properties of the collagen-based scaffolds, as elastin does in the ECM to prevent irreversible deformation. In this study, an elastin-like polymer (ELP) designed and synthesized using recombinant DNA methodology is used with the view to providing increased proteolytic resistance and increased functionality to the scaffolds by carrying specific sequences for microbial transglutaminase cross-linking, endothelial cell adhesion, and drug delivery. Evaluation of the effects that cross-linking ELP-collagen has on the physicochemical properties of the scaffold such as porosity, presence of cross-linking, thermal behavior, and mechanical strength demonstrated that the introduction of enzymatically resistant covalent bonds between collagen and ELP increases the mechanical strength of the scaffolds in a dose-dependent manner without significantly affecting the porosity or thermal properties of the original scaffold. Importantly, the scaffolds also showed selective behavior, in a dose (ELP)-dependent manner toward human umbilical vein endothelial cells and smooth muscle cells when compared to fibroblasts.

Bacterial resistance to antibiotics:modified target sites

Alteration in the target sites of antibiotics is a common mechanism of resistance. Examples of clinical strains showing resistance can be found for every class of antibiotic, regardless of the mechanism of action. Target site changes often result from spontaneous mutation of a bacterial gene on the chromosome and selection in the presence of the antibiotic. Examples include mutations in RNA polymerase and DNA gyrase, resulting in resistance to the rifamycins and quinolones, respectively. In other cases, acquisition of resistance may involve transfer of resistance genes from other organisms by some form of genetic exchange (conjugation, transduction, or transformation). Examples of these mechanisms include acquisition of the mecA genes encoding methicillin resistance in Staphylococcus aureus and the various van genes in enterococci encoding resistance to glycopeptides. © 2005 Elsevier B.V. All rights reserved.