Alkylamides from Echinacea Modulate Induced Immune Responses in Macrophages

The ability of Echinacea and its components to alter the immune response was examined 'in vitro' in a macrophage cell line under either basal or immunostimulated conditions. Potential immunostimulatory and inflammatory activity was determined using a nuclear transcription factor (NFкB) expression, tumour necrosis factor α (TNFα) and nitric oxide (NO) production as biomarkers. In the absence of alternate stimulation, the only significant effects seen were a decrease in NFкB expression by a 2-ene alkylamide ((2E)-N-isobutylundeca-2-ene-8,10-diynamide (1)) and a decrease in TNFα levels by cichoric acid and an Echinacea alkylamide fraction (EPL AA). When the cells were stimulated by lipopolysaccharide (LPS), inhibition of the increased NFкB expression levels was caused by cichoric acid, an Echinacea preparation (EPL), EPL AA and a 2,4-diene ((2E,4E,8Z,10Z)-N-isobutyldodeca-2,4,8,10-tetraenamide (2)). Increases in TNFα levels were inhibited by cichoric acid, EPL and EPL AA but enhanced by 1 in the presence of LPS, while only EPL AA was able to inhibit the stimulated increases in NO. When using phorbol myristate acetate to stimulate the cells, NFкB and NO levels were unaffected by Echinacea or its components while only cichoric acid and 2 inhibited TNFα levels. Although cichoric acid was found to have an effect, it is probably not an important contributor to the Echinacea modulation of the immune response in vivo, as it is not bioavailable. Echinacea appears to attenuate the response of macrophages to an immune stimulus and its combination of phytochemicals exhibits different pharmacological properties to one or more of the isolated major individual components

Neuronal Glutathione Content and Antioxidant Capacity can be Normalized In Situ by N-acetyl Cysteine Concentrations Attained in Human Cerebrospinal Fluid

N-acetyl cysteine (NAC) supports the synthesis of glutathione (GSH), an essential substrate for fast, enzymatically catalyzed oxidant scavenging and protein repair processes. NAC is entering clinical trials for adrenoleukodystrophy, Parkinson’s disease, schizophrenia, and other disorders in which oxidative stress may contribute to disease progression. However, these trials are hampered by uncertainty about the dose of NAC required to achieve biological effects in human brain. Here we describe an approach to this issue in which mice are used to establish the levels of NAC in cerebrospinal fluid (CSF) required to affect brain neurons. NAC dosing in humans can then be calibrated to achieve these NAC levels in human CSF. The mice were treated with NAC over a range of doses, followed by assessments of neuronal GSH levels and neuronal antioxidant capacity in ex vivo brain slices. Neuronal GSH levels and antioxidant capacity were augmented at NAC doses that produced peak CSF NAC concentrations of ≥50 nM. Oral NAC administration to humans produced CSF concentrations of up to 10 μM, thus demonstrating that oral NAC administration can surpass the levels required for biological activity in brain. Variations of this approach may similarly facilitate and rationalize drug dosing for other agents targeting central nervous system disorders. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13311-015-0404-4) contains supplementary material, which is available to authorized users

Uptake of Fluorescent Iron Oxide Nanoparticles by Oligodendroglial OLN-93 Cells

To investigate the cellular accumulation and intracellular localization of dimercaptosuccinate-coated iron oxide nanoparticles (D-IONPs) in oligodendroglial cells, we have synthesized IONPs that contain the fluorescent dye BODIPY (BP) in their coat (BP-D-IONPs) and have investigated the potential effects of the absence or presence of this dye on the particle uptake by oligodendroglial OLN-93 cells. Fluorescent BP-D-IONPs and non-fluorescent D-IONPs had similar hydrodynamic diameters and -potentials of around 60 nm and -58 mV, respectively, and showed identical colloidal stability in physiological media with increasing particle size and positivation of the -potential in presence of serum. After exposure of oligodendroglial OLN-93 cells to BP-D-IONPs or D-IONPs in the absence of serum, the specific cellular iron content increased strongly to around 1,800 nmol/mg. This strong iron accumulation was lowered for both types of IONPs by around 50 % on exposure of the cells at 4 C and by around 90 % on incubation in presence of 10 % serum. The accumulation of both D-IONPs and BP-D-IONPs in the absence of serum was not affected by endocytosis inhibitors, whereas in the presence of serum inhibitors of clathrin-dependent endocytosis lowered the particle accumulation by around 50 %. These data demonstrate that oligodendroglial cells efficiently accumulate IONPs by an endocytotic process which is strongly affected by the temperature and the presence of serum and that BP-D-IONPs are a reliable tool to monitor by fluorescence microscopy the uptake and cellular fate of D-IONPs

Fluorescent castasterone reveals BRI1 signaling from the plasma membrane

Receptor-mediated endocytosis is an integral part of signal transduction as it mediates signal attenuation and provides spatial and temporal dimensions to signaling events. One of the best-studied leucine-rich repeat receptor-like kinases in plants, BRASSINOSTEROID INSENSITIVE 1 (BRI1), perceives its ligand, the brassinosteroid (BR) hormone, at the cell surface and is constitutively endocytosed. However, the importance of endocytosis for BR signaling remains unclear. Here we developed a bioactive, fluorescent BR analog, Alexa Fluor 647-castasterone (AFCS), and visualized the endocytosis of BRI1-AFCS complexes in living Arabidopsis thaliana cells. Impairment of endocytosis dependent on clathrin and the guanine nucleotide exchange factor for ARF GTPases (ARF-GEF) GNOM enhanced BR signaling by retaining active BRI1-ligand complexes at the plasma membrane. Increasing the trans-Golgi network/early endosome pool of BRI1-BR complexes did not affect BR signaling. Our findings provide what is to our knowledge the first visualization of receptor-ligand complexes in plants and reveal clathrin- and ARF-GEF-dependent endocytic regulation of BR signaling from the plasma membrane