Storage characteristics of multiple-donor pooled red blood cells compared to single-donor red blood cell units.

BACKGROUND: Each unit of blood donated is processed and stored individually resulting in variability in the amount of red blood cells (RBCs) collected, RBC properties, and the 24-hour posttransfusion RBC survivability. As a result, each unit differs in its ability to deliver oxygen and potentially its effects on the recipient. The goal of this study was to investigate the storage of pooled RBCs from multiple donors in comparison to control standard RBC units. STUDY DESIGN AND METHODS: Two units of irradiated, leukoreduced RBCs of same ABO, D, E, C, and K antigen phenotype were collected from each of five donors using apheresis. One unit from each donor was pooled in a 2-L bag and remaining units were used as controls. After being pooled, RBCs were separated in five bags and stored at 4°C along with the controls. Quality indexes were measured on Days 2, 14, and 28 for all the units. RESULTS: Adenosine triphosphate assays for both pooled and controls showed a slight decrease from Day 2 to Day 28 (pooled/control from 5.22/5.24 to 4.35/4.33 µmol/g hemoglobin [Hb]). 2,3-Diphosphoglycerate was successfully rejuvenated for all RBC units on Day 28 (pooled 11.46 µmol/g Hb; control 11.86 µmol/g Hb). The results showed a nonsignificant difference between pooled and control units, with a general trend of lower standard deviation for pooled units when compared to controls. CONCLUSION: Pooled units have reduced unit-to-unit variability. Future exploration of their immunogenicity is required before using pooled units for transfusion.

In vivo electroporation and non-protein based screening assays to identify antibodies against native protein conformations.

In vivo electroporation has become a gold standard method for DNA immunization. The method assists the DNA entry into cells, results in expression and the display of the native form of antigens to professional cells of the immune system, uses both arms of immune system, has a built-in adjuvant system, is relatively safe, and is cost-effective. However, there are challenges for achieving an optimized reproducible process for eliciting strong humoral responses and for the screening of specific immune responses, in particular, when the aim is to mount humoral responses or to generate monoclonal antibodies via hybridoma technology. Production of monoclonal antibodies demands generation of high numbers of primed B and CD4 T helper cells in lymphoid organs needed for the fusion that traditionally is achieved by a final intravenous antigen injection. The purified antigen is also needed for screening of hundreds of clones obtained upon fusion of splenocytes. Such challenges make DNA vaccination dependent on purified proteins. Here, we have optimized methods for in vivo electroporation, production, and use of cells expressing the antigen and an in-cell Western screening method. These methods resulted in (1) reproducibly mounting robust humoral responses against antigens with different cell localizations, and (2) the ability to screen for antigen eliminating a need for protein/antigen purification. This process includes optimized parameters for in vivo electroporation, the use of transfected cells for final boost, and mild fixation/permeabilization of cells for screening. Using this process, upon two vaccinations via in vivo electroporation (and final boost), monoclonal antibodies against nucleus and cytoplasmic and transmembrane proteins were achieved.

Peptide-conjugated PAMAM dendrimer as a universal DNA vaccine platform to target antigen-presenting cells.

DNA-based vaccines hold promise to outperform conventional antigen-based vaccines by virtue of many unique features. However, DNA vaccines have thus far fallen short of expectations, due in part to poor targeting of professional antigen-presenting cells (APC) and low immunogenicity. In this study, we describe a new platform for effective and selective delivery of DNA to APCs in vivo that offers intrinsic immune-enhancing characteristics. This platform is based on conjugation of fifth generation polyamidoamine (G5-PAMAM) dendrimers, a DNA-loading surface, with MHC class II-targeting peptides that can selectively deliver these dendrimers to APCs under conditions that enhance their immune stimulatory potency. DNA conjugated with this platform efficiently transfected murine and human APCs in vitro. Subcutaneous administration of DNA-peptide-dendrimer complexes in vivo preferentially transfected dendritic cells (DC) in the draining lymph nodes, promoted generation of high affinity T cells, and elicited rejection of established tumors. Taken together, our findings show how PAMAM dendrimer complexes can be used for high transfection efficiency and effective targeting of APCs in vivo, conferring properties essential to generate effective DNA vaccines.