Design of magnetic polyplexes taken up efficiently by dendritic cell for enhanced DNA vaccine delivery.

Dendritic cells (DC) targeting vaccines require high efficiency for uptake, followed by DC activation and maturation. We used magnetic vectors comprising polyethylenimine (PEI)-coated superparamagnetic iron oxide nanoparticles, with hyaluronic acid (HA) of different molecular weights (<10 and 900 kDa) to reduce cytotoxicity and to facilitate endocytosis of particles into DCs via specific surface receptors. DNA encoding Plasmodium yoelii merozoite surface protein 1-19 and a plasmid encoding yellow fluorescent gene were added to the magnetic complexes with various % charge ratios of HA: PEI. The presence of magnetic fields significantly enhanced DC transfection and maturation. Vectors containing a high-molecular-weight HA with 100% charge ratio of HA: PEI yielded a better transfection efficiency than others. This phenomenon was attributed to their longer molecular chains and higher mucoadhesive properties aiding DNA condensation and stability. Insights gained should improve the design of more effective DNA vaccine delivery systems.

Myeloid derived suppressor cells and their role in diseases.

Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of myeloid progenitors that can play a major role in tumour development and chronic inflammation. The importance of the suppressive function ofMDSCs was first suggested by studies involving cancer patients and cancer-bearing mice. In addition, recent studies have demonstrated that MDSCs can also be involved in many other pathological conditions. MDSCs have unique ways of abrogatingan immune response in addition to those utilised by other immune-suppressive cell types, for example via the induction of arginase-1 and consequent upregulation in reactive oxygen species (ROS) production. Due to their heterogeneity,they further can express a variety of lineage markers, which overlap with other myeloid cell types such as Gr1,CD11b, MHCIIlo, Ly6C and Ly6G, making it difficult to identify them by surface phenotype alone. The disparity between mouse and human MDSCs further complicates the identification of these elusive cell populations. In this review, we will summarise the recent updates on the methods for eliciting and studying different MDSC subsets, including newly proposed surface phenotypes, as well as insights into how their function is being characterised in both mice and humans. In addition, exciting new discoveries suggesting their involvement across a number of different pathological settings, such as sepsis, autoimmunity and Leishmaniasis, will be discussed.

A simple method allowing DIC imaging in conjunction with confocal microscopy.

Current optical methods to collect Nomarski differential interference contrast (DIC) or phase images with a transmitted light detector (TLD) in conjunction with confocal laser scanning microscopy (CLSM) can be technically challenging and inefficient. We describe for the first time a simple method that combines the use of the commercial product QPm (Iatia, Melbourne Australia) with brightfield images collected with the TLD of a CLSM, generating DIC, phase, Zernike phase, dark-field or Hoffman modulation contrast images. The brightfield images may be collected at the same time as the confocal images. This method also allows the calculation of contrast-enhanced images from archival data. The technique described here allows for the creation of contrast-enhanced images such as DIC or phase, without compromising the intensity or quality of confocal images collected simultaneously. Provided the confocal microscope is equipped with a motorized z-drive and a TLD, no hardware or optical modifications are required. The contrast-enhanced images are calculated with software using the quantitative phase-amplitude microscopy technique (Barone-Nugent et al., 2002). This technique, being far simpler during image collection, allows the microscopist to concentrate on their confocal imaging and experimental procedures. Unlike conventional DIC, this technique may be used to calculate DIC images when cells are imaged through plastic, and without the use of expensive strain-free objective lenses.

Tracking membrane and secretory immunoglobulin alpha heavy chain mRNA variation during B-cell differentiation by real-time quantitative polymerase chain reaction.

Primary transcripts for all Ig heavy chain isotypes are alternatively processed to encode either secreted or membrane forms of the same antibody and, in plasma cells, a shift towards the secreted form occurs. In principle, measuring the relative quantities of secreted and membrane forms for a particular isotype could monitor B-cell plasmacytoid differentiation. Ratios of alpha heavy chain mRNA secreted (alphas) to membrane (alpham) form were assessed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR; TaqMan) using an IgA plasma cell line (NCI-H929), a surface IgA+ line (Dakiki) and human tonsillar B cells. While NCI-H929 cells showed the highest alphas: alpham ratio as expected, alphas mRNA predominated for all unstimulated B cells and Dakiki cells. Treatment of B cells and Dakiki cells with IL-2 and IL-10 resulted in a further progression towards the alphas form, correlating with increased human plasma cell antigen-1 (HPC1) mRNA levels. However, alpha mRNA processing and HPC1 expression were independently regulated, as IFN-gamma treatment suppressed HPC1 levels while increasing alphas: alpham ratios. Cytokine-mediated increases in the alphas: alpham ratio resulted from strongly enhanced levels of alphas with relatively constant alpham values. Differentiation-related changes in mRNA processing can thus be tracked by automated quantitative PCR.