Flow Cytometric Measurement of Reactive Oxygen Species to Assess the Effects of Preconditioning Total Body Irradiation on NOG Mice.

BACKGROUND: Preclinical development of new drugs for cancer immunotherapy requires preconditioning total body irradiation (TBI) of mice to be humanized via hematopoietic stem cell transplantation. To assess the effect of preconditioning TBI, we detected the reactive oxygen species (ROS), Annexin V, propidium iodide (PI) level in bone marrow samples by flow cytometer. METHODS: We divided all NOG mice between irradiated (n = 20) and control groups (n = 10) for two time points. Irradiated mice were exposed to 3.5 Gy of radiation. After sacrificing BM samples were collected, the flow cytometric percentage of ROS, Annexin V, and PI markers were investigated on days 2 and 14 after exposure. RESULTS: At the first time point, the level of ROS was higher in the irradiated group than in the control group, and this difference was statistically significant (P < 0.05). Also, at the second time point, the mean differences of all markers in the irradiated group were significantly compared to the control group (P < 0.05). CONCLUSION: Thus, in NOG mice, the measurement of ROS level is helpful to the assessment of preconditioning TBI.

Effects of a new pathogen-reduction technology (Mirasol PRT) on functional aspects of platelet concentrates.

BACKGROUND: Several strategies are being developed to reduce the risk of pathogen transmission associated with platelet (PLT) transfusion. STUDY DESIGN AND METHODS: The impact of a new technology for pathogen reduction based on riboflavin plus illumination (Mirasol PRT, Navigant Biotechnologies, Inc.) at 6.2 and 12.3 J per mL on functional and biochemical characteristics of PLTs was evaluated. PLT concentrates (PCs) obtained by apheresis were treated with Mirasol PRT and stored at 22 degrees C. Modifications in major PLT glycoproteins (GPIbalpha, GPIV, and GPIIb-IIIa), adhesive ligands (von Willebrand factor [VWF], fibrinogen [Fg], and fibronectin), activation antigens (P-selectin and LIMP), and apoptotic markers (annexin V binding and factor [F]Va) were analyzed by flow cytometry. Adhesive and cohesive PLT functions were evaluated with well-established perfusion models. Studies were performed on the preparation day (Day 0) and during PCs storage (Days 3 and 5). RESULTS: Levels of glycoproteins remained stable during storage in PCs treated with 6.2 J per mL pathogen reduction technology (PRT) and similar to those observed in nontreated PCs. When 12.3 J per mL PRT was applied, however, levels of GPIbalpha moderately decreased on Days 3 and 5. VWF, Fg, and FVa were not modified in their expression levels, either by treatment or by storage period. Fibronectin appeared more elevated in all PRT samples. A progressive increase in P-selectin and LIMP expression and in annexin V binding was observed during storage of PRT-treated PCs. Functional studies indicated that 6.2 J per mL Mirasol PRT-treated PLTs preserved adhesive and cohesive functions to levels compatible with those observed in the respective control PCs. CONCLUSION: PLT function was well preserved in PCs treated with 6.2 J per mL Mirasol PRT and stored for 5 days.

Synchrotron radiation diffraction from two-dimensional protein crystals at the air/water interface.

Protein structure determination by classical x-ray crystallography requires three-dimensional crystals that are difficult to obtain for most proteins and especially for membrane proteins. An alternative is to grow two-dimensional (2D) crystals by adsorbing proteins to ligand-lipid monolayers at the surface of water. This confined geometry requires only small amounts of material and offers numerous advantages: self-assembly and ordering over micrometer scales is easier to obtain in two dimensions; although fully hydrated, the crystals are sufficiently rigid to be investigated by various techniques, such as electron crystallography or micromechanical measurements. Here we report structural studies, using grazing incidence synchrotron x-ray diffraction, of three different 2D protein crystals at the air-water interface, namely streptavidine, annexin V, and the transcription factor HupR. Using a set-up of high angular resolution, we observe narrow Bragg reflections showing long-range crystalline order in two dimensions. In the case of streptavidin the angular range of the observed diffraction corresponds to a resolution of 10 A in plane and 14 A normal to the plane. We show that this approach is complementary to electron crystallography but without the need for transfer of the monolayer onto a grid. Moreover, as the 2D crystals are accessible from the buffer solution, the formation and structure of protein complexes can be investigated in situ.