Differential susceptibility of macrophage cell lines to Bacillus anthracis-Vollum 1B.

Bacillus anthracis (BA) is a spore forming bacterium and the causative agent of anthrax disease. Macrophages (Mphis) play a central role in anthrax disease. An important step in disease progression is the ability of BA to secrete lethal toxin (LeTx) that kills Mphis. LeTx is a heterodimer composed of protective antigen (PA) and lethal factor (LF). Researchers have shown that Mphi cell lines demonstrate differential susceptibility to purified LeTx; for example RAW264.7 and J774A.1 Mphis are sensitive to LeTx whereas IC-21 Mphis are resistant. Research has also suggested that exogenous factors, including other BA proteins, can influence the activity of LeTx. For this reason, the objective of the current work was to examine if RAW264.7, J774A.1, and IC-21 Mphis demonstrated differential susceptibility when cultured with a LeTx-producing strain of BA. Here, we co-cultured Mphis with LeTx+ Vollum 1B (V1B) spores for >15 h and assayed for Mphi cell death by morphology, trypan blue (TB) staining, neutral red (NR) activity, and lactate dehydrogenase (LDH) activity in the culture media. Following the addition of V1B spores, necrosis (approximately 50% mortality) was observed in RAW264.7 and J774A.1 Mphis at 7.5 and 10 h, respectively. By 15 h, both RAW264.7 and J774A.1 Mphis demonstrated 100% mortality. In contrast, IC-21 Mphis, under identical culture conditions, remained viable (98%) and activated throughout the course of the experiment (>24 h). The mechanism of RAW264.7 cell death appeared to involve LeTx because the V1B-induced cytotoxicity was dose-dependently reversed by the addition of anti-PA antibody to the culture media. These observations suggest there is differential susceptibility of Mphi cell lines to the LeTx+ V1B strain of BA. Further development of this in vitro model may be useful to further characterize the interactions between Mphis and BA spores.

Spectroelectrochemical sensing based on multimode selectivity simultaneously achievable in a single device. 5. Simulation of sensor response for different excitation potential waveforms.

The simulation of the optical response in spectroelectrochemical sensing has been investigated. The sensor consists of a sensing film coated on an optically transparent electrode (OTE). The mode of detection is attenuated total reflection. Only species that partition into the sensing film, undergo electrochemistry at the potentials applied to the OTE, and have changes in their absorbance at the wavelength of light propagated within the glass substrate of the OTE can be sensed. A fundamental question arises regarding the excitation potential waveforms employed to initiate the electrochemical changes observed. Historically, selection has been based solely upon the effectiveness of the waveform to quickly electrolyze any analyte observable by the optical detection method employed. In this report, additional requirements by which the waveform should be selected for use in a remote sensing configuration are discussed. The effectiveness of explicit finite difference simulation as a tool for investigating the applicability of three different excitation potential waveforms (square, triangle, sinusoid) is demonstrated. The simulated response is compared to experimental results obtained from a prototype sensing platform consisting of an indium tin oxide OTE coated with a cation-selective, sol-gel-derived Nafion composite film designed for the detection of a model analyte, tris(2,2'-bipyridyl)ruthenium(II) chloride. Using a diffusion coefficient determined from experimental data (5.8 x 10(-11) cm2 s for 5 x 10(-6) M Ru(bipy)3(2+)), the simulator program was able to accurately predict the magnitude of the absorbance change for each potential waveform (0.497 for square, 0.403 for triangular, and 0.421 for sinusoid), but underestimated the number of cycles required to approach steady state. The simulator program predicted 2 (square), 3 (triangle), and 5 cycles (sinusoid), while 5 (square), 15 (triangle), and 10 (sinusoid) cycles were observed experimentally.