ZnO nanoparticles (nZnO) are commonly used in nanotechnology applications despite their demonstrated cytotoxicity against multiple cell types. This underscores the significant need to determine the physicochemical properties that influence nZnO cytotoxicity. In this study, we analyzed six similarly sized nZnO formulations, along with SiO₂-coated nZnO, bulk ZnO and ZnSO₄ as controls. Four of the nZnO samples were synthesized using various wet chemical methods, while three employed high-temperature flame spray pyrolysis (FSP) techniques. X-ray diffraction and optical analysis demonstrated the lattice parameters and electron band gap of the seven nZnO formulations were similar. However, zeta potential measures, hydrodynamic size, photocatalytic rate constants, dissolution potential, reactive oxygen species (ROS) production and, more importantly, the cytotoxicity of the variously synthesized nZnO towards Jurkat leukemic and primary CD4⁺ T cells displayed major differences. Surface structure analysis using FTIR, X-ray photoelectron spectroscopies (XPS) and dynamic light scattering (DLS) revealed significant differences in the surface-bound chemical groups and the agglomeration tendencies of the samples. The wet chemical nZnO, with higher cationic surface charge, faster photocatalytic rates, increased extracellular dissolution and ROS generation demonstrated greater cytotoxicity towards both cell types than those made with FSP techniques. Furthermore, principal component analysis (PCA) suggests that the synthesis procedure employed influences which physicochemical properties contribute more to the cytotoxic response. These results suggest that the synthesis approach results in unique surface chemistries and can be a determinant of cellular cytotoxicity and oxidative stress responses.

Strict regulation of T cell function is imperative to control adaptive immunity, and dysregulation of T cell activation can contribute to infectious and autoimmune diseases. Vasoactive intestinal peptide receptor-1 (VPAC-1), an anti-inflammatory G-protein coupled receptor, has been reported to be downregulated during T cell activation. However, the regulatory mechanisms controlling the expression of VPAC-1 in T cells are not well understood. Therefore, mouse splenic CD4 T cells were treated in complete media+/-anti-CD3 for 24h, total RNA isolated and VPAC-1 levels measured by qPCR. Surprisingly, we discovered that T cells incubated in complete media steadily upregulated VPAC-1 mRNA levels over time (24h). Importantly, CD4 T cells isolated from blood also showed elevated VPAC-1 expression compared to splenic T cells. Collectively, these data support that the vascular environment positively influences VPAC-1 mRNA expression that is negatively regulated by TCR signaling. This research was supported by a national service award (1KO1 DK064828) to G.D., the Center for Protease Research (2P20RR015566), and INBRE (P20 RR016741).

Pore-forming toxins are alluring tools for delivering biologically-active, impermeable cargoes to intracellular environments by introducing large conductance pathways into cell membranes. However, the lack of regulation often leads to the dissipation of electrical and chemical gradients, which might significantly affect the viability of cells under scrutiny. To mitigate these problems, we explored the use of lysenin channels to reversibly control the barrier function of natural and artificial lipid membrane systems by controlling the lysenin’s transport properties. We employed artificial membranes and electrophysiology measurements in order to identify the influence of labels and media on the lysenin channel’s conductance. Two cell culture models: Jurkat cells in suspension and adherent ATDC5 cells were utilized to demonstrate that lysenin channels may provide temporary cytosol access to membrane non-permeant propidium iodide and phalloidin. Permeability and cell viability were assessed by fluorescence spectroscopy and microscopy. Membrane resealing by chitosan or specific media addition proved to be an effective way of maintaining cellular viability. In addition, we loaded non-permeant dyes into liposomes via lysenin channels by controlling their conducting state with multivalent metal cations. The improved control over membrane permeability might prove fruitful for a large variety of biological or biomedical applications that require only temporary, non-destructive access to the inner environment enclosed by natural and artificial membranes.

This study investigates the signaling pathways that regulate mRNA expression of vasoactive intestinal peptide receptor‐1 (VPACR‐1) in murine, primary CD4 T cells. VPACR‐1 is an anti‐proliferative, G‐protein coupled receptor that is highly expressed on naïve T cells, but is downregulated upon activation. To date, the signaling molecules involved in mediating VPACR‐1 downregulation have yet to be identified. Splenic CD4 T cells isolated from C57BL/6 mice by magnetic beads (Miltenyi) were pretreated with several pharmacological inhibitors and activated with plate bound anti‐CD3 for 24 hrs. Total RNA was isolated, and VPACR‐1 mRNA levels were measured by qPCR. Our results show that the JNK MAPK pathway downstream from src kinases Fyn/Lck is involved in TCR‐induced VPACR‐1 downregulation. Preliminarily, the adaptor molecule, Zap70, and the JNK upstream G‐protein signaling molecule, Rac1, also appear to downregulate VPACR‐1. Surprisingly, we did not collect evidence to support other MAPK pathways, such as Ras→PKC→ERK and p38, as having roles in downregulating VPACR‐1. Furthermore, two other common T cell signaling molecules, calcineurin and PI3‐K, were not statistically involved in VPACR‐1 regulation. By studying the signaling pathways that regulate VPACR‐1, we can gain greater insight into the role of this receptor in infectious disease, autoimmunity and T cell biology. 1KO1 DK064828, COBRE‐ 2P20RR015566

Presently, Leukemia affects about 250,000 people in America and it is expected that 45,000 new cases will be diagnosed in 2009. We have previously published that Ikaros (IK), a tumor suppressor, downregulates vasoactive intestinal receptor 1 (VPAC1), an anti‐proliferative G protein coupled receptor, mediated by IK engagement to VPAC1. Currently, it is not known how post‐translational modifications, such as phosphorylation, regulate IK DNA binding activity. Therefore, we hypothesized that inhibition of casein kinase II, CKII, known to phosphorylate IK, would shift the equilibrium to favor its dephosphorylated IK state, causing greater DNA binding and repression of VPAC1. To test this, HuT‐78 cells were cultured in the presence and absence of 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazol (DRB) for 0, 12, and 24 h. The levels of VPAC1 mRNA expression were measured by TaqMan qPCR. After treatment with DRB, the levels of VPAC1 in the cells decreased from 0 to 12 h by about 70%, but were restored after 24 hours. These results support that the hypophosphorylated IK pool is most likely responsible for transcriptional regulation of VPAC1. Overall, DRB downregulates VPAC1 expression by inhibiting the phosphorylation of IK through the inhibition of CKII. Funding: NIH‐K01 1K01DK064828 and COBRE 2P20RR05566.