Summary Background Microparticles (MPs), small vesicles shed from stimulated cells, permit cross-talk between cells within a particular environment. Their composition is thought to reflect their cell of origin, and differs according to whether they are produced by stimulation or by apoptosis. Whether MP properties vary according to stimulus is not yet known. Methods We studied the characteristics of MPs produced from monocytic THP-1 cells upon stimulation with lipopolysaccharide or a soluble P-selectin chimera, using proteomics, flow cytometry, western blotting, and electron microscopy. Results Utilizing a novel criterion of calcein-AM staining to define MPs, we found that MP populations were similar with respect to size, presence and organization of cytoskeleton, and expression of certain antigens. The MPs shared the same level of procoagulant activity. We found that MPs also have distinct characteristics, depending on stimuli. These include differences in phosphatidylserine expression and expression of proteins from specific subcellular locations such as the mitochondria, and of unique antigens such as leukocyte-associated immunoglobin-like-receptor (LAIR)-1, which was found only upon stimulation with the soluble P-selectin chimera. Conclusion We found that the properties of MPs depend on the stimulus that produced them. This supports the concept that monocytic MPs differentially modulate thrombosis, inflammation and immune regulation according to stimulus.

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Recent success in phase I/II clinical trials (Konstan, M. W.; Davis, P. B.; Wagener, J. S.; Hilliard, K. A.; Stern, R. C.; Milgram, L. J.; Kowalczyk, T. H.; Hyatt, S. L.; Fink, T. L.; Gedeon, C. R.; Oette, S. M.; Payne, J. M.; Muhammad, O.; Ziady, A. G.; Moen, R. C.; Cooper, M. J. Hum. Gene Ther. 2004, 15 (12), 1255-69) has highlighted pegylated poly-L-lysine (C1K30-PEG) as a nonviral gene delivery agent capable of achieving clinically significant gene transfer levels in vivo. This study investigates the potential of a C1K30-PEG gene delivery system for cancer gene therapy and evaluates its mode of cellular entry with the purpose of developing an optimally formulated prototype for tumor cell transfection. C1K30-PEG complexes have a neutral charge and form rod-like and toroid-like nanoparticles. Comparison of the transfection efficiency achieved by C1K30-PEG with other cationic lipid and polymeric vectors demonstrates that C1K30-PEG transfects cells more efficiently than unpegylated poly-L-lysine and compares well to commercially available vectors. In vivo gene delivery by C1K30-PEG nanoparticles to a growing subcutaneous murine tumor was also demonstrated. To determine potential barriers to C1K30-PEG gene delivery, the entry mechanism and intracellular fate of rhodamine labeled complexes were investigated. Using cellular markers to delineate the pathway taken by the complexes upon cellular entry, only minor colocalization was observed with EEA-1, a marker of early endosomes. No colocalization was observed between the complexes and the transferrin receptor, which is a marker for clathrin-coated pits. In addition, complexes were not observed to enter late endosomes/lysosomes. Cellular entry of the complexes was completely inhibited by the macropinocytosis inhibitor, amiloride, indicating that the complexes enter cells via macropinosomes. Such mechanistic studies are an essential step to support future rational design of pegylated poly-L-lysine vectors to improve the efficiency of gene delivery.

Erythrocytes have a defined lifespan in vivo, and the signals that maintain their survival in circulation or trigger their death are unknown. Here, we investigated the control of erythrocyte survival and death in an in vitro culture system where erythrocytes survived for 10 days in serumfree medium in the presence or absence of bovine serum. Death of the cells in culture was correlated with increased exposure of phosphatidylserine and increased levels of intracellular calcium. Cell death could be suppressed by supplementing the medium with human plasma or serum, resulting in a doubling of the lifespan to 20 days. Freshly isolated erythrocytes and cultured erythrocytes were both found to express Bcl-X L and, to a lesser extent, Bak in membrane protein extracts. Treatment of the cells with a Bak-derived BH3 peptide fused to the internalization sequence of the antennapedia protein, which has previously been shown to enter cells by diffusion and antagonize Bcl-X L , resulted in substantial cell death in erythrocyte cultures. BH3-induced death was accompanied by an immediate increase in accumulation of intracellular calcium and could be suppressed by plasma, but not by the caspase inhibitor zVAD. A BH3 peptide mutated at amino acid 78 of full-length Bak required for heterodimerization with Bcl-X L had no effect on cell viability or calcium levels. We conclude that the BH3 peptide accelerates erythrocyte death through antagonization of Bcl-X L . The data suggest that erythrocyte survival is promoted by survival factors in plasma and by membrane- IntroductionCell death and survival in hematopoietic cells is regulated by signals from cell-surface receptors that either promote apoptosis, such as Fas and tumor necrosis factor (TNF), or repress cell death, such as receptors for cytokines and survival factors such as erythropoietin (EPO), interleukin 3, and insulinlike growth factor I (IGF-I). [1][2][3] Ligation of the receptors for Fas/TNF can lead directly to activation of caspases, which can act as effectors of cell death by catalyzing structural changes associated with apoptosis, including cytoskeletal reorganization and DNA fragmentation. Survival factors mediate signals through several kinases, such as phosphoinositol 3 kinase and AKT, ultimately leading to transcriptional events or to regulation of Bcl-2 family proteins. 4 The Bcl-2 family acts as central regulators of cell death in many cells [5][6][7] and is divided into 2 main groups consisting of suppressors of apoptosis, including Bcl-2, Bcl-X L , Bcl-w, and Mcl-1, and potent apoptosis promoters, including Bax, Bak, Bad, and Bid. Bcl-2 and Bcl-X L survival activity can be antagonized by heterodimerization with proteins containing a BH3 domain, which is the essential domain for the binding and proapoptotic activity of Bak, Bax, Bik, and Bid. 8 Bcl-2 and Bcl-X L are expressed in outer mitochondrial membranes where antagonization of their function is associated with rupture of the membrane, release of the proapoptotic factors cytochrome c, and activation of caspas...