GTP-dependent and -independent activation of superoxide producing NADPH oxidase in a neutrophil cell-free system

GTP and GTP-gamma-S enhanced several-fold the NADPH-dependent superoxide production induced by sodium dodecyl sulfate in a cell-free system of pig neutrophils consisting of the membrane fraction and two cytosolic fractions separated by gel filtration. The enhanced activity was decreased by the addition of GDP in a dose-dependent manner, but 70% of the activity in the absence of GTP remained even at 1 mM GDP. Only one cytosol fraction besides the membrane fraction was required for the activation in the presence of GTP. The cytosol fraction was analyzed by chromatography on 2',5'-ADP agarose and two components responsible for the GTP-dependent and independent activation were separated. These results suggest that at least two pathways are available for the activation of superoxide production in the cell-free system of pig neutrophils.     

Measurement and analysis of the shape recovery process of each erythrocyte for estimation of its deformability using the microchannel technique: the influence of the softness of the cell membrane and viscosity of the hemoglobin solution inside the cell

This study tried to evaluate the deformability of each erythrocyte by measuring the time constant of shape recovery just after the erythrocytes left the microchannels. We fabricated a microchannel array with a 5μm-square, 100μm-long cross-section on a PDMS sheet. Three different kinds of blood samples were prepared—healthy erythrocytes as a control, artificially membrane-hardened erythrocytes and artificial hemoglobin solution-diluted erythrocytes—to investigate the influence of erythrocyte's mechanical property changes on the time constant of shape recovery. These shape recovery processes were modeled and analyzed by a standard liner solid model. As a result, the temporal variation of the compressive strain of all erythrocytes showed exponential decay with time elapsed like a first order lag system, so the time constant of shape recovery could be calculated from the semi-logarithmic relaxation curve. The stiffer the cell membrane was using glutaraldehyde, the shorter the time constant for relaxation became compared to healthy erythrocytes. The diluted hemoglobin erythrocytes snapped back quicker than healthy ones. In addition, the time constant of healthy blood drawn from females was clearly shorter than that collected from males. However, the time constant of fully hemoglobin substituted erythrocytes was not affected by gender difference. These results indicate that there is not a significant difference in the stiffness of healthy cell membranes regardless of individual and gender differences. On the other hand, the viscosity of the hemoglobin solution inside the cell is one of the significant factors affecting the time constant. Therefore, these results suggest that the deformability of individual erythrocytes can be quantitated by the time constant for relaxation measured by microchannel techniques.     

Generation of Large Numbers of Antigen-Expressing Human Dendritic Cells Using CD14-ML Technology

We previously reported a method to expand human monocytes through lentivirus-mediated introduction of cMYC and BMI1, and we named the monocyte-derived proliferating cells, CD14-ML. CD14-ML differentiated into functional DC (CD14-ML-DC) upon addition of IL-4, resulting in the generation of a large number of DC. One drawback of this method was the extensive donor-dependent variation in proliferation efficiency. In the current study, we found that introduction of BCL2 or LYL1 along with cMYC and BMI1 was beneficial. Using the improved method, we obtained CD14-ML from all samples, regardless of whether the donors were healthy individuals or cancer patients. In vitro stimulation of peripheral blood T cells with CD14-ML-DC that were loaded with cancer antigen-derived peptides led to the establishment of CD4⁺ and CD8⁺ T cell lines that recognized the peptides. Since CD14-ML was propagated for more than 1 month, we could readily conduct genetic modification experiments. To generate CD14-ML-DC that expressed antigenic proteins, we introduced lentiviral antigen-expression vectors and subjected the cells to 2 weeks of culture for drug-selection and expansion. The resulting antigen-expressing CD14-ML-DC successfully induced CD8⁺ T cell lines that were reactive to CMVpp65 or MART1/MelanA, suggesting an application in vaccination therapy. Thus, this improved method enables the generation of a sufficient number of DC for vaccination therapy from a small amount of peripheral blood from cancer patients. Information on T cell epitopes is not necessary in vaccination with cancer antigen-expressing CD14-ML-DC; therefore, all patients, irrespective of HLA type, will benefit from anti-cancer therapy based on this technology.     

A part of the transmembrane domain of pro-TNF can function as a cleavable signal sequence that generates a biologically active secretory form of TNF.

To determine the minimum requirement in the 76-residue leader sequence of pro-tumor necrosis factor (TNF) for membrane translocation across the endoplasmic reticulum (ER) and for the maturation of pro-TNF, we constructed pro-TNF mutants in which a part of the transmembrane domain of pro-TNF was directly linked to the N-terminus of the mature domain, and evaluated their translocational behavior across the ER-membrane and their secretion from the transfected cells. The in vitro translation/translocation assay involving a canine pancreatic microsomal membrane system including a mutant, Delta-75-47, -32-1, revealed that the N-terminal half of the transmembrane domain of pro-TNF consisting of 14 residues functioned as a cleavable signal sequence; it generated a cleaved form of TNF having a molecular mass similar to that of mature TNF. Analysis of the cleavage site by site-directed mutagenesis indicated that the site was inside the leader sequence of this mutant. When the mutant, Delta-75-47, -32-1, was expressed in COS-1 cells, efficient secretion of a biologically active soluble TNF was observed. Further deletion of the hydrophobic domain from this mutant inhibited the translocation, indicating that some extent of hydrophobicity is indispensable for the membrane translocation of the mature domain of TNF. Thus, the N-terminal half of the transmembrane domain of pro-TNF could function as a cleavable signal sequence when linked to the mature domain of TNF, and secretion of a biologically active secretory form of TNF could be achieved with this 14-residue hydrophobic segment. In intact pro-TNF, however, this 14-residue sequence could not function as a cleavable signal sequence during intracellular processing, indicating that the remainder of the 76-residue leader sequence of pro-TNF inhibits the signal peptide cleavage and thus enables the leader sequence to function as a type II signal-anchor sequence that generates a transmembrane form of TNF.