Measurements of Aperture Averaging on Bit-Error-Rate

We report on measurements made at the Shuttle Landing Facility (SLF) runway at Kennedy Space Center of receiver aperture averaging effects on a propagating optical Gaussian beam wave over a propagation path of 1,000 in. A commercially available instrument with both transmit and receive apertures was used to transmit a modulated laser beam operating at 1550 nm through a transmit aperture of 2.54 cm. An identical model of the same instrument was used as a receiver with a single aperture that was varied in size up to 20 cm to measure the effect of receiver aperture averaging on Bit Error Rate. Simultaneous measurements were also made with a scintillometer instrument and local weather station instruments to characterize atmospheric conditions along the propagation path during the experiments

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes

Efavirenz promotes β-secretase expression and increased Aβ1-40,42 via oxidative stress and reduced microglial phagocytosis: implications for HIV associated neurocognitive disorders (HAND).

Efavirenz (EFV) is among the most commonly used antiretroviral drugs globally, causes neurological symptoms that interfere with adherence and reduce tolerability, and may have central nervous system (CNS) effects that contribute in part to HIV associated neurocognitive disorders (HAND) in patients on combination antiretroviral therapy (cART). Thus we evaluated a commonly used EFV containing regimen: EFV/zidovudine (AZT)/lamivudine (3TC) in murine N2a cells transfected with the human "Swedish" mutant form of amyloid precursor protein (SweAPP N2a cells) to assess for promotion of amyloid-beta (Aβ) production. Treatment with EFV or the EFV containing regimen generated significantly increased soluble amyloid beta (Aβ), and promoted increased β-secretase-1 (BACE-1) expression while 3TC, AZT, or, vehicle control did not significantly alter these endpoints. Further, EFV or the EFV containing regimen promoted significantly more mitochondrial stress in SweAPP N2a cells as compared to 3TC, AZT, or vehicle control. We next tested the EFV containing regimen in Aβ - producing Tg2576 mice combined or singly using clinically relevant doses. EFV or the EFV containing regimen promoted significantly more BACE-1 expression and soluble Aβ generation while 3TC, AZT, or vehicle control did not. Finally, microglial Aβ phagocytosis was significantly reduced by EFV or the EFV containing regimen but not by AZT, 3TC, or vehicle control alone. These data suggest the majority of Aβ promoting effects of this cART regimen are dependent upon EFV as it promotes both increased production, and decreased clearance of Aβ peptide

EFV or EFV/3TC/AZT treatment promotes Aβ generation in cultured neuronal cells <i>via</i> BACE-1 activation <i>in vitro.</i>

<p>Aβ species were analyzed in cell lysates from SweAPP N2a cells (<b>A</b>) by ELISA. Data are represented as the mean ± of a percentage of Aβ peptides secreted 24 h after 3TC, AZT, EFV, or 3TC/EFV/AZT administration, relative fold over control (PBS treated). Significant increases in Aβ were observed in EFV or EFV/3TC/AZT treated cells were observed compared to control (***<i>P</i><0.001 and **<i>P<0.05</i> respectively by ANOVA). (<b>B</b>) Western blot (6E10 antibody) of conditioned media shows increased oligomeric Aβ species vs. s-APP-α (control) in the EFV or EFV/3TC/AZT treated cells (***<i>P</i><0.001 and **<i>P<0.05</i> respectively). (<b>D</b>) BACE-1 expression in cultured media revealed significant differences between EFV or EFV/3TC/AZT treated cells compared to untreated control (***<i>P</i><0.001). β-actin is used for the internal loading control. Results are representative of three independent experiments.</p

EFV/3TC/AZT inhibits microglial phagocytosis of Aβ_1-42 peptide.

<p>(A) Primary microglia (1×10⁵ cells/well in 24-well tissue culture plates) were treated with aged FITC tagged Aβ_1-42 (50 nM) in complete medium for 60 min with antiretroviral medications (10uM) combined or singly as indicated, or PBS (control). As a control for nonspecifically incorporated Aβ, microglial cells were incubated at 4°C with the same treatment followed by DAPI staining. EFV or 3TC/AZT/EFV inhibited microglia-colocalization by fluorescence microscopy. Green indicates Aβ_1-42 positive; blue indicates microglia nuclei. Addition of heat inactivated HIV-1 Tat yielded similar results as vehicle control (data not shown) (B) Cell supernatants and lysates were analyzed for extracellular (top) and cell associated (bottom) FITC-Aβ using a fluorimeter. Data are represented as the relative fold of mean fluorescence change (mean ± SD), calculated as the mean fluorescence for each sample at 37°C divided by mean fluorescence at 4°C (<i>n</i> = 6 for each condition presented). One-way ANOVA followed by <i>post-hoc</i> comparison showed a significant difference between EFV (***<i>P<</i>0.001) or EFV/3TC/AZT <i>(**P<</i>0.05) but not 3TC or AZT compared to control.</p

EFV/3TC/AZT increases soluble Aβ levels in Tg2576 mice <i>via</i> BACE-1 activation <i>in vivo.</i>

<p>(<b>A</b>) Aβ_{40, 42} peptides were analyzed in brain homogenates from 8 month old Tg2576 mice by ELISA (n = 5 mice for each group). One-way ANOVA followed by <i>post hoc</i> comparison revealed significant differences between control (Tg2576mice treated with PBS) and EFV or EFV/3TC/AZT -treated Tg2576 mice (<i>P<0.001</i> and <i>0.05</i> respectively with n = 5 mice/group). (<b>B</b>) Western Blot of brain homogenates using anti-Aβ_1-17 antibody (6E10) shows total APP and a bands corresponding to soluble Aβ oligomer species. β-actin was an internal control. A <i>t-test</i> revealed significant differences in soluble Aβ species between EFV-treated compared to 3TC/AZT/EFV, 3TC or AZT treated Tg2576 mice (<i>P</i><0.01) (<b>C</b>) BACE-1 expression in brain homogenate of Tg2576 mice significantly was increased in EFV or EFV/3TC/AZT -treated Tg2576 mice (<i>P</i><0.001).</p

Proposed mechanism of EFV neurotoxicity.

<p>Our present work suggests that EFV promotes an increase in Aβ <i>in vitro</i> and <i>in vivo</i> on both the production and clearance fronts <i>via</i> its inhibition of neurnoal MMP resulting in reduced ATP stores and thus a high ROS environment in the CNS. Previous studies indicate such high ROS microenvironments in the CNS promote BACE-1 APP processing and also inhibit microglial Aβ clearance functions. These events in turn all promote production of Aβ species. (*Note: Red arrows = inhibition, Green arrows = promotion).</p

cART treatment of SweAPP N2a cells promotes mitochondrial dysfunction.

<p>(<i>A) ATP levels are reduced in EFV or EFV/3TC/AZT treated SweAPP N2a neuron cells:</i> SweAPP N2a cells were grown with 10 µM of each medication or all three medications combined for 48 h. We found a significant decrease in ATP levels in cells treated with EFV or 3TC/AZT/EFV (***<i>P</i><0.001). <i>(B)MMP is reduced in EFV or EFV/3TC/</i>AZT <i>SweAPP N2a cells:</i> In accord with reduced ATP levels we found a similar reduction in MMP in the EFV or EFV/3TC/AZT treated groups <i>(***</i>P<<i>0.001</i>) <i>(C</i>–<i>F) ROS levels are increased in EFV or EFV/3TC/AZT treated SweAPP N2a cells:</i> EFV-treated primary neuron cells have significantly higher ROS contents (^**<i>P</i><0.001) after incubation for 60 min than untreated primary neuron. (C–E)The average relative fluorescence units of DCFDA in neurons from each treatment group as indicated by the mean ± standard deviations (D, F) The ROS content in the antiretroviral treatment is expressed as % RFU ± standard deviations for each group compared to untreated control primary neuron cells (100%). (<i>*P</i><<i>0.05, *** P</i><<i>0.001</i>).</p