Protective effects of fatty acid amide hydrolase inhibition in UVB-activated microglia.

Neuroinflammation is a hallmark of several neurodegenerative disorders that has been extensively studied in recent years. Microglia, the primary immune cells of the central nervous system (CNS), are key players in this physiological process, demonstrating a remarkable adaptability in responding to various stimuli in the eye and the brain. Within the complex network of neuroinflammatory signals, the fatty acid N-ethanolamines, in particular N-arachidonylethanolamine (anandamide, AEA), emerged as crucial regulators of microglial activity under both physiological and pathological states. In this study, we interrogated for the first time the impact of the signaling of these bioactive lipids on microglial cell responses to a sub-lethal acute UVB radiation, a physical stressor responsible of microglia reactivity in either the retina or the brain. To this end, we developed an in vitro model using mouse microglial BV-2 cells. Upon 24 h of UVB exposure, BV-2 cells showed elevated oxidative stress markers and, cyclooxygenase (COX-2) expression, enhanced phagocytic and chemotactic activities, along with an altered immune profiling. Notably, UVB exposure led to a selective increase in expression and activity of fatty acid amide hydrolase (FAAH), the main enzyme responsible for degradation of fatty acid ethanolamides. Pharmacological FAAH inhibition via URB597 counteracted the effects of UVB exposure, decreasing tumor necrosis factor α (TNF-α) and nitric oxide (NO) release and reverting reactive oxidative species (ROS), interleukin-1ß (IL-1ß), and interleukin-10 (IL-10) levels to the control levels. Our findings support the potential of enhanced fatty acid amide signaling in mitigating UVB-induced cellular damage, paving the way to further exploration of these lipids in light-induced immune responses.

Oxidative Stress, Cytotoxic and Inflammatory Effects of Azoles Combinatorial Mixtures in Sertoli TM4 Cells.

Triazole and imidazole fungicides are an emerging class of contaminants with an increasing and ubiquitous presence in the environment. In mammals, their reproductive toxicity has been reported. Concerning male reproduction, a combinatorial activity of tebuconazole (TEB; triazole fungicide) and econazole (ECO; imidazole compound) in inducing mitochondrial impairment, energy depletion, cell cycle arrest, and the sequential activation of autophagy and apoptosis in Sertoli TM4 cells (SCs) has recently been demonstrated. Given the strict relationship between mitochondrial activity and reactive oxygen species (ROS), and the causative role of oxidative stress (OS) in male reproductive dysfunction, the individual and combined potential of TEB and ECO in inducing redox status alterations and OS was investigated. Furthermore, considering the impact of cyclooxygenase (COX)-2 and tumor necrosis factor-alpha (TNF-α) in modulating male fertility, protein expression levels were assessed. In the present study, we demonstrate that azoles-induced cytotoxicity is associated with a significant increase in ROS production, a drastic reduction in superoxide dismutase (SOD) and GSH-S-transferase activity levels, and a marked increase in the levels of oxidized (GSSG) glutathione. Exposure to azoles also induced COX-2 expression and increased TNF-α production. Furthermore, pre-treatment with N-acetylcysteine (NAC) mitigates ROS accumulation, attenuates COX-2 expression and TNF-α production, and rescues SCs from azole-induced apoptosis, suggesting a ROS-dependent molecular mechanism underlying the azole-induced cytotoxicity.

Microglial Endocannabinoid Signalling in AD.

Chronic inflammation in Alzheimer's disease (AD) has been recently identified as a major contributor to disease pathogenesis. Once activated, microglial cells, which are brain-resident immune cells, exert several key actions, including phagocytosis, chemotaxis, and the release of pro- or anti-inflammatory mediators, which could have opposite effects on brain homeostasis, depending on the stage of disease and the particular phenotype of microglial cells. The endocannabinoids (eCBs) are pleiotropic bioactive lipids increasingly recognized for their essential roles in regulating microglial activity both under normal and AD-driven pathological conditions. Here, we review the current literature regarding the involvement of this signalling system in modulating microglial phenotypes and activity in the context of homeostasis and AD-related neurodegeneration.

Effects of dietary iodine supplement on sheep milk and cheese.

The work reported in this paper addresses the iodine nutritional deficiency that still affects a large number of people. For this purpose, we analyzed the possibility to use, as iodine vehicle, a hard typical ewe cheese, called Canestrato d'Abruzzo, derived from milk of ewes fed with an iodine-fortified diet. Both in the milk and the cheese of these animals, the iodine level was higher than that measured in sheep with a normal diet. An increase in the lactoferrin and iron content was evident in the whey derived from milk of the iodine group. Furthermore, in derived cheese, the caseins seemed more efficiently transformed in small peptides making the product more digestible and, for this reason, particularly suitable for feeding the elderly. In conclusion, the dairy products obtained from ewes fed with iodine diet contain more bioactive compounds so that they represent a useful food to prevent iodine and iron deficiency in lamb and humans.

Antipseudomonal and Immunomodulatory Properties of Esc Peptides: Promising Features for Treatment of Chronic Infectious Diseases and Inflammation.

Persistent infections, such as those provoked by the Gram-negative bacterium Pseudomonas aeruginosa in the lungs of cystic fibrosis (CF) patients, can induce inflammation with lung tissue damage and progressive alteration of respiratory function. Therefore, compounds having both antimicrobial and immunomodulatory activities are certainly of great advantage in fighting infectious diseases and chronic inflammation. We recently demonstrated the potent antipseudomonal efficacy of the antimicrobial peptide (AMP) Esc(1-21) and its diastereomer Esc(1-21)-1c, namely Esc peptides. Here, we confirmed this antimicrobial activity by reporting on the peptides' ability to kill P. aeruginosa once internalized into alveolar epithelial cells. Furthermore, by means of enzyme-linked immunosorbent assay and Western blot analyses, we investigated the peptides' ability to detoxify the bacterial lipopolysaccharide (LPS) by studying their effects on the secretion of the pro-inflammatory cytokine IL-6 as well as on the expression of cyclooxygenase-2 from macrophages activated by P. aeruginosa LPS. In addition, by a modified scratch assay we showed that both AMPs are able to stimulate the closure of a gap produced in alveolar epithelial cells when cell migration is inhibited by concentrations of Pseudomonas LPS that mimic lung infection conditions, suggesting a peptide-induced airway wound repair. Overall, these results have highlighted the two Esc peptides as valuable candidates for the development of new multifunctional therapeutics for treatment of chronic infectious disease and inflammation, as found in CF patients.

Antibacterial and Anti-Inflammatory Activity of an Antimicrobial Peptide Synthesized with D Amino Acids.

The peptide SET-M33 is a molecule synthesized in tetra-branched form which is being developed as a new antibiotic against Gram-negative bacteria. Its isomeric form with D amino acids instead of the L version (SET-M33D) is also able to kill Gram-positive bacteria because of its higher resistance to bacterial proteases (Falciani et al., PLoS ONE, 2012, 7, e46259). Here we report the strong in vitro activity of SET-M33D (MIC range 0.7-6.0 µM) against multiresistant pathogens of clinical interest, including Gram-positives Staphylococcus aureus, Staphylococcus saprophyticus, and Enterococcus faecalis, and various Gram-negative enterobacteriaceae. SET-M33D antibacterial activity is also confirmed in vivo against a MRSA strain of S. aureus with doses perfectly compatible with clinical use (5 and 2.5 mg/Kg). Moreover, SET-M33D strongly neutralized lipopolysaccharide (LPS) and lipoteichoic acid (LTA), thus exerting a strong anti-inflammatory effect, reducing expression of cytokines, enzymes, and transcription factors (TNF-α, IL6, COX-2, KC, MIP-1, IP10, iNOS, NF-κB) involved in the onset and evolution of the inflammatory process. These results, along with in vitro and in vivo toxicity data and the low frequency of resistance selection reported here, make SET-M33D a strong candidate for the development of a new broad spectrum antibiotic.

Bronchial epithelium repair by Esculentin-1a-derived antimicrobial peptides: involvement of metalloproteinase-9 and interleukin-8, and evaluation of peptides' immunogenicity.

The airway epithelium is seriously damaged upon pulmonary Pseudomonas aeruginosa infection, especially in cystic fibrosis (CF) sufferers. Therefore, the discovery of novel anti-infective agents accelerating healing of infected injured tissues is crucial. The antipseudomonal peptides esculentin-1a(1-21)NH2 and its diastereomer Esc(1-21)-1c (Esc peptides) hold promise in this respect. In fact, they stimulate airway epithelial wound repair, but no mechanistic insights are available. Here we demonstrated that this process occurs through promotion of cell migration by an indirect activation of epidermal growth factor receptor mediated by metalloproteinases. Furthermore, we showed an increased expression of metalloproteinase 9, at both gene and protein levels, in peptide-treated bronchial epithelial cells with a functional or mutated form of CF transmembrane conductance regulator. In addition, the two peptides counteracted the inhibitory effect of Pseudomonas lipopolysaccharide (mimicking an infection condition) on the wound healing activity of the airway epithelium, and they enhanced the production of interleukin-8 from both types of cells. Finally, no immunogenicity was discovered for Esc peptides, suggesting their potential safety for clinical usage. Besides representing a step forward in understanding the molecular mechanism underlying the peptide-induced wound healing activity, these studies have contributed to highlight Esc peptides as valuable therapeutics with multiple functions.

Saffron: A Multitask Neuroprotective Agent for Retinal Degenerative Diseases.

Both age related macular degeneration (AMD) and light induced retinal damage share the common major role played by oxidative stress in the induction/progression of degenerative events. Light damaged (LD) rats have been widely used as a convenient model to gain insight into the mechanisms of degenerative disease, to enucleate relevant steps and to test neuroprotectants. Among them, saffron has been shown to ameliorate degenerative processes and to regulate many genes and protective pathways. Saffron has been also tested in AMD patients. We extended our analysis to a possible additional effect regulated by saffron and compared in AMD patients a pure antioxidant treatment (Lutein/zeaxanthin) with saffron treatment. Methods: Animal model. Sprague-Dawley (SD) adult rats, raised at 5 lux, were exposed to 1000 lux for 24 h and then either immediately sacrificed or placed back at 5 lux for 7 days recovery period. A group of animals was treated with saffron. We performed in the animal model: (1) SDS-PAGE analysis; (2) Western Blotting (3) Enzyme activity assay (4) Immunolabelling; in AMD patients: a longitudinal open-label study 29 (±5) months in two groups of patients: lutein/zeaxanthin (19) and saffron (23) treated. Visual function was tested every 8 months by ERG recordings in addition to clinical examination. Results: Enzymatic activity of MMP-3 is reduced in LD saffron treated retinas and is comparable to control as it is MMP-3 expression. LD treated retinas do not present "rosettes" and microglia activation and migration is highly reduced. Visual function remains stable in saffron treated AMD patients while deteriorates in the lutein/zeaxanthin group. Conclusion: Our results provide evidence of an additional way of action of saffron treatment confirming the complex nature of neuroprotective activities of its chemical components. Accordingly, long term follow-up in AMD patients reveals an added value of saffron supplementation treatment compared to classical antioxidant protocol.

Apoptotic effects of bovine apo-lactoferrin on HeLa tumor cells.

Lactoferrin (Lf), a cationic iron-binding glycoprotein of 80 kDa present in body secretions, is known as a compound with marked antimicrobial activity. In the present study, the apoptotic effect of iron-free bovine lactoferrin (apo-bLf) on human epithelial cancer (HeLa) cells was examined in association with reactive oxygen species and glutathione (GSH) levels. Apoptotic effect of iron-free bovine lactoferrin inhibited the growth of HeLa cells after 48 hours of treatment while the diferric-bLf was ineffective in the concentration range tested (from 1 to 12.5 µM). Western blot analysis showed that key apoptotic regulators including Bax, Bcl-2, Sirt1, Mcl-1, and PARP-1 were modulated by 1.25 µM of apo-bLf. In the same cell line, apo-bLf induced apoptosis together with poly (ADP-ribose) polymerase cleavage, caspase activation, and a significant drop of NAD+ . In addition, apo-bLf-treated HeLa cells showed a marked increase of reactive oxygen species level and a significant GSH depletion. On the whole, apo-bLf triggered apoptosis of HeLa cells upon oxygen radicals burst and GSH decrease.

Effects of azidothymidine on protein kinase C activity and expression in erythroleukemic cell K562 and acute lymphoblastic leukemia cell HSB-2.

Azidothymidine (AZT) is one of the anti-retroviral drugs currently used for the treatment of HIV-infected patients. Several other effects of the drug have been studied in vitro, such as the alterations of some enzymes, the inhibition of cell proliferation, and the increase of transferrin receptor expression. In this study, we investigated the alterations of protein kinase C (PKC) activity, PKCα and PKCßII expressions and plasmatic membrane fluidity induced by AZT in two cancer cell lines, human chronic myeloid (K562) and human acute lymphoblastic (HSB-2) leukemia cells, respectively. The results showed that both PKC activity and membrane fluidity in HSB-2 cells increased after 24 h of drug incubation. PKCα expression in HSB-2 cells decreased after 48 h of AZT exposure, when the cell growth also decreased. However, in K562 cells, the PKCα and PKCßII expressions enhanced in the presence of the drug when the growth was inhibited. The results indicate that AZT is less effective in inhibiting the growth of acute lymphoblastic HSB-2 leukemia cells than inhibiting that of chronic myeloid K562 cells. In fact, after 24 h exposure, the HSB-2 cell growth decreased less than K562 cell growth.

Isomerization of an antimicrobial peptide broadens antimicrobial spectrum to gram-positive bacterial pathogens.

The branched M33 antimicrobial peptide was previously shown to be very active against Gram-negative bacterial pathogens, including multidrug-resistant strains. In an attempt to produce back-up molecules, we synthesized an M33 peptide isomer consisting of D-aminoacids (M33-D). This isomeric version showed 4 to 16-fold higher activity against Gram-positive pathogens, including Staphylococcus aureus and Staphylococcus epidermidis, than the original peptide, while retaining strong activity against Gram-negative bacteria. The antimicrobial activity of both peptides was influenced by their differential sensitivity to bacterial proteases. The better activity shown by M33-D against S. aureus compared to M33-L was confirmed in biofilm eradication experiments where M33-L showed 12% activity with respect to M33-D, and in vivo models where Balb-c mice infected with S. aureus showed 100% and 0% survival when treated with M33-D and M33-L, respectively. M33-D appears to be an interesting candidate for the development of novel broad-spectrum antimicrobials active against bacterial pathogens of clinical importance.

Lactoferrin derived peptides: mechanisms of action and their perspectives as antimicrobial and antitumoral agents.

Antimicrobial peptides, AMPs, exert their function acting mainly at the membrane level. In the wide AMPs panorama a particular position is occupied by lactoferrin derived peptides. They also possess antiviral, antifungal and antitumor activities and their parent molecules are available in several mammalian fluids and in dairy industries waste.

Zidovudine inhibits protein kinase C activity in human chronic myeloid (K562) cells.

In this paper we show that human erythroleukaemia (K562) cells exhibited a significant inhibition of protein kinase C activity when cells were exposed to 40 micro M zidovudine in a time interval of 5-180 min., whereas prolonged treatment (24 hr) was uneffective. The addition of an excess of thymidine (125:1, mol:mol), in the cell suspension with or without zidovudine fully restored the protein kinase C activity. Interestingly, either in cell homogenates and in commercially purified rat brain protein kinase C, both zidovudine and its monophosphate derivative, caused inhibition that was higher than in intact cells. This inhibition reached a maximal value of 45% when zidovudine or zidovudine monophosphate were incubated with the pure commercial enzyme and in this case the addition of thymidine did not prevent the enzyme inhibition. The conclusions from these data are that either zidovudine or zidovudine monophosphate interact directly with the pure enzyme, causing inhibition, while in intact cells exposed to the drug, zidovudine monophosphate appears to be the main metabolite responsible for protein kinase C inhibition.

Improvements in technical assessment and protocol for EPR evaluation of magnetic fields effects on a radical pair reaction.

The effects of either static or pulsed magnetic fields on the reaction rate of Fremy's salt-ascorbic acid were studied directly by EPR spectroscopy. Radical pair mechanism (RPM) accounts for the magnetic field effects, but the expected amounts are so small that they need to be observed with particular care with EPR technique. The method is based on the resolution of a pair of EPR signals by the addition of a stationary field gradient, where the signals are coming from the exposed and control capillary sample. To this purpose, a suitable device for the gradient generation was used. Others improvements were the strictly keeping of the same boundary temperature condition in the capillary pairs, obtained by a refrigerating system controlled by a thermocouple, and the use of a pair of Helmholtz coils to generate an external high homogeneous magnetic field. By this experimental set up, we found that the magnetic field induce the decrease of the studied radical reaction rate. This EPR approach is a significant alternative to the spectrophotometric one. Moreover, it offers the advantage to detect both the radicals and/or intermediates involved in the reaction.