Essential Oil from Eucalyptus globulus (Labill.) Activates Complement Receptor-Mediated Phagocytosis and Stimulates Podosome Formation in Human Monocyte-Derived Macrophages

Eucalyptus essential oil and its major constituent eucalyptol are extensively employed in the cosmetic, food, and pharmaceutical industries and their clinical use has recently expanded worldwide as an adjuvant in the treatment of infective and inflammatory diseases. We previously demonstrated that essential oil from Eucalyptus globulus (Labill.) (EO) stimulates in vitro the phagocytic activity of human monocyte-derived macrophages and counteracts the myelotoxicity induced by the chemotherapeutic 5-fluorouracil in immunocompetent rats. Here we characterize some mechanistic aspects underlying the immunostimulatory ability exerted by EO on macrophages. The internalization of fluorescent beads, fluorescent zymosan BioParticles, or apoptotic cancer cells was evaluated by confocal microscopy. Pro-inflammatory cytokine and chemokine release was determined by flow cytometry using the BD cytometric bead array. Receptor involvement in EO-stimulated phagocytosis was assessed using complement- or IgG-opsonized zymosan particles. The localization and expression of podosome components was analyzed by confocal microscopy and western blot. The main results demonstrated that: EO-induced activation of a macrophage is ascribable to its major component eucalyptol, as recently demonstrated for other cells of innate immunity; EO implements pathogen internalization and clearance by stimulating the complement receptor-mediated phagocytosis; EO stimulates podosome formation and increases the expression of podosome components. These results confirm that EO extract is a potent activator of innate cell-mediated immunity and thereby increase the scientific evidence supporting an additional property of this plant extract besides the known antiseptic and anti-inflammatory properties

N-acetyl-l-cysteine fosters inactivation and transfer to endolysosomes of c-Src

The non-receptor-protein tyrosine kinase c-Src is overexpressed and activated in a large number of human cancers, in which it is associated with tumor development and progression. Canonical regulation takes place by means of an alternative phosphorylation of tyrosine residues -- Tyr419 for activation and Tyr530 for inactivation. An independent redox regulation mechanism, involving cysteine residues, has also been proposed, in which oxidation activates the enzyme. Here we present a kinetic analysis of the effect of N-acetyl-l-cysteine (NAC) on c-Src, demonstrating that reduction reverts the oxidation-driven activation. In cancer cells, we show that NAC treatment produces an increase in specifically labeled reduced thiols of c-Src cysteines, thus confirming a redox transition. In addition to a decrease in Tyr419 phosphorylation, this leads to a massive shift of c-Src from plasma membranes -- where its active form is located -- to endolysosomal compartments. With the objective of deciphering the complex issue of c-Src regulation and of devising new strategies to revert its activation in cancers, redox regulation thus emerges as a promising area for study

One site on the apoB-100 specifically binds 17-beta-estradiol and regulates the overall structure of LDL.

The major protein component (apoB-100) of low-density lipoprotein (LDL) is known as a multipotential molecule the several functional regions of which can all be affected by key structural modifications driven by specific domains. Based on our previous report on structural and conformational modifications of apoB-100 in the presence of 17-beta-estradiol (E2), we characterized the interaction between E2 and the apoB-100 and further explored the induced alterations in terms of the structural arrangement of the whole LDL particle. We report evidence for the existence on apoB-100 of a single specific and saturable binding site for E2, the occupancy of which modifies the overall structure of the protein, inducing an increase in the alpha-helix fraction. As a consequence, the structure of the LDL particle is deeply perturbed, with a change in the arrangement of both the outer shell and lipid core and an overall volume shrinkage. The evidence of a regulation of apoB-100 structure by a physiological ligand opens new perspectives in the study of the biological addressing of the LDL particle and suggests a novel rationale in the search for mechanisms underlying the beneficial role of E2 in decreasing the risk of early lesions in atherosclerosis

A Promise in the Treatment of Endometriosis: An Observational Cohort Study on Ovarian Endometrioma Reduction by N-Acetylcysteine

Urged by the unmet medical needs in endometriosis treatment, often with undesirable side effects, and encouraged by N-acetylcysteine (NAC) efficacy in an animal model of endometriosis and by the virtual absence of toxicity of this natural compound, we performed an observational cohort study on ovarian endometriosis. NAC treatment or no treatment was offered to 92 consecutive Italian women referred to our university hospital with ultrasound confirmed diagnosis of ovarian endometriosis and scheduled to undergo laparoscopy 3 months later. According to patients acceptance or refusal, NAC-treated and untreated groups finally comprised 73 and 72 endometriomas, respectively. After 3 months, within NAC-treated patients cyst mean diameter was slightly reduced (-1.5 mm) versus a significant increase (+6.6 mm) in untreated patients (P=0.001). Particularly, during NAC treatment, more cysts reduced and fewer cysts increased their size. Our results are better than those reported after hormonal treatments. Twenty-four NAC-treated patients—versus 1 within controls—cancelled scheduled laparoscopy due to cysts decrease/disappearance and/or relevant pain reduction (21 cases) or pregnancy (1 case). Eight pregnancies occurred in NAC-treated patients and 6 in untreated patients. We can conclude that NAC actually represents a simple effective treatment for endometriosis, without side effects, and a suitable approach for women desiring a pregnancy

Antenna-enhanced mid-infrared detection of extracellular vesicles derived from human cancer cell cultures

Background: Extracellular Vesicles (EVs) are sub-micrometer lipid-bound particles released by most cell types. They are considered a promising source of cancer biomarkers for liquid biopsy and personalized medicine due to their specific molecular cargo, which provides biochemical information on the state of parent cells. Despite this potential, EVs translation process in the diagnostic practice is still at its birth, and the development of novel medical devices for their detection and characterization is highly required. Results: In this study, we demonstrate mid-infrared plasmonic nanoantenna arrays designed to detect, in the liquid and dry phase, the specific vibrational absorption signal of EVs simultaneously with the unspecific refractive index sensing signal. For this purpose, EVs are immobilized on the gold nanoantenna surface by immunocapture, allowing us to select specific EV sub-populations and get rid of contaminants. A wet sample-handling technique relying on hydrophobicity contrast enables effortless reflectance measurements with a Fourier-transform infrared (FTIR) spectro-microscope in the wavelength range between 10 and 3 µm. In a proof-of-principle experiment carried out on EVs released from human colorectal adenocarcinoma (CRC) cells, the protein absorption bands (amide-I and amide-II between 5.9 and 6.4 µm) increase sharply within minutes when the EV solution is introduced in the fluidic chamber, indicating sensitivity to the EV proteins. A refractive index sensing curve is simultaneously provided by our sensor in the form of the redshift of a sharp spectral edge at wavelengths around 5 µm, where no vibrational absorption of organic molecules takes place: this permits to extract of the dynamics of EV capture by antibodies from the overall molecular layer deposition dynamics, which is typically measured by commercial surface plasmon resonance sensors. Additionally, the described metasurface is exploited to compare the spectral response of EVs derived from cancer cells with increasing invasiveness and metastatic potential, suggesting that the average secondary structure content in EVs can be correlated with cell malignancy. Conclusions: Thanks to the high protein sensitivity and the possibility to work with small sample volumes-two key features for ultrasensitive detection of extracellular vesicles- our lab-on-chip can positively impact the development of novel laboratory medicine methods for the molecular characterization of EVs