Biomarkers in Human Peripheral Blood Mononuclear Cells: The State of the Art in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease, characterized by the progressive loss of lower motor neurons, weakness and muscle atrophy. ALS lacks an effective cure and diagnosis is often made by exclusion. Thus, it is imperative to search for biomarkers. Biomarkers can help in understanding ALS pathomechanisms, identification of targets for treatment and development of effective therapies. Peripheral blood mononuclear cells (PBMCs) represent a valid source for biomarkers compared to cerebrospinal fluid, as they are simple to collect, and to plasma, because of the possibility of detecting lower expressed proteins. They are a reliable model for patients’ stratification. This review provides an overview on PBMCs as a potential source of biomarkers in ALS. We focused on altered RNA metabolism (coding/non-coding RNA), including RNA processing, mRNA stabilization, transport and translation regulation. We addressed protein abnormalities (aggregation, misfolding and modifications); specifically, we highlighted that SOD1 appears to be the most characterizing protein in ALS. Finally, we emphasized the correlation between biological parameters and disease phenotypes, as regards prognosis, severity and clinical features. In conclusion, even though further studies are needed to standardize the use of PBMCs as a tool for biomarker investigation, they represent a promising approach in ALS research.     

Suitability of the In Vitro Cytokinesis-Block Micronucleus Test for Genotoxicity Assessment of TiO2 Nanoparticles on SH-SY5Y Cells

Standard toxicity tests might not be fully adequate for evaluating nanomaterials since their unique features are also responsible for unexpected interactions. The in vitro cytokinesis-block micronucleus (CBMN) test is recommended for genotoxicity testing, but cytochalasin-B (Cyt-B) may interfere with nanoparticles (NP), leading to inaccurate results. Our objective was to determine whether Cyt-B could interfere with MN induction by TiO₂ NP in human SH-SY5Y cells, as assessed by CBMN test. Cells were treated for 6 or 24 h, according to three treatment options: co-treatment with Cyt-B, post-treatment, and delayed co-treatment. Influence of Cyt-B on TiO₂ NP cellular uptake and MN induction as evaluated by flow cytometry (FCMN) were also assessed. TiO₂ NP were significantly internalized by cells, both in the absence and presence of Cyt-B, indicating that this chemical does not interfere with NP uptake. Dose-dependent increases in MN rates were observed in CBMN test after co-treatment. However, FCMN assay only showed a positive response when Cyt-B was added simultaneously with TiO₂ NP, suggesting that Cyt-B might alter CBMN assay results. No differences were observed in the comparisons between the treatment options assessed, suggesting they are not adequate alternatives to avoid Cyt-B interference in the specific conditions tested.     

The Relevance of Physico-Chemical Properties and Protein Corona for Evaluation of Nanoparticles Immunotoxicity—In Vitro Correlation Analysis on THP-1 Macrophages

Alongside physiochemical properties (PCP), it has been suggested that the protein corona of nanoparticles (NPs) plays a crucial role in the response of immune cells to NPs. However, due to the great variety of NPs, target cells, and exposure protocols, there is still no clear relationship between PCP, protein corona composition, and the immunotoxicity of NPs. In this study, we correlated PCP and the protein corona composition of NPs to the THP-1 macrophage response, focusing on selected toxicological endpoints: cell viability, reactive oxygen species (ROS), and cytokine secretion. We analyzed seven commonly used engineered NPs (SiO₂, silver, and TiO₂) and magnetic NPs. We show that with the exception of silver NPs, all of the tested TiO₂ types and SiO₂ exhibited moderate toxicities and a transient inflammatory response that was observed as an increase in ROS, IL-8, and/or IL-1β cytokine secretion. We observed a strong correlation between the size of the NPs in media and IL-1β secretion. The induction of IL-1β secretion was completely blunted in NLR family pyrin domain containing 3 (NLRP3) knockout THP-1 cells, indicating activation of the inflammasome. The correlations analysis also implicated the association of specific NP corona proteins with the induction of cytokine secretion. This study provides new insights toward a better understanding of the relationships between PCP, protein corona, and the inflammatory response of macrophages for different engineered NPs, to which we are exposed on a daily basis.     

A Histone Deacetylase (HDAC) Inhibitor with Pleiotropic In Vitro Anti-Toxoplasma and Anti-Plasmodium Activities Controls Acute and Chronic Toxoplasma Infection in Mice

Toxoplasmosis is a highly prevalent human disease, and virulent strains of this parasite emerge from wild biotopes. Here, we report on the potential of a histone deacetylase (HDAC) inhibitor we previously synthesized, named JF363, to act in vitro against a large panel of Toxoplasma strains, as well as against the liver and blood stages of Plasmodium parasites, the causative agents of malaria. In vivo administration of the drug significantly increases the survival of mice during the acute phase of infection by T. gondii, thus delaying its spreading. We further provide evidence of the compound’s efficiency in controlling the formation of cysts in the brain of T. gondii-infected mice. A convincing docking of the JF363 compound in the active site of the five annotated ME49 T. gondii HDACs was performed by extensive sequence–structure comparison modeling. The resulting complexes show a similar mode of binding in the five paralogous structures and a quite similar prediction of affinities in the micromolar range. Altogether, these results pave the way for further development of this compound to treat acute and chronic toxoplasmosis. It also shows promise for the future development of anti-Plasmodium therapeutic interventions.     

New In Vitro Model of Oxidative Stress: Human Prostate Cells Injured with 2,2-diphenyl-1-picrylhydrazyl (DPPH) for the Screening of Antioxidants

The antioxidant activity of natural compounds consists in their ability to modulate gene and protein expression, thus inducing an integrated cell protective response and repair processes against oxidative stress. New screening tools and methodologies are crucial for the actual requirement of new products with antioxidant activity to boost endogenous oxidative stress responsive pathways, Reactive Oxygen Species (ROS) metabolism and immune system activity, preserving human health and wellness. In this study, we performed and tested an integrated oxidative stress analysis, using DPPH assay and PNT2 cells injured with DPPH. We firstly investigated the mechanism of action of the oxidising agent (DPPH) on PNT2 cells, studying the variation in cell viability, oxidative stress genes, inflammatory mediator and ROS levels. The results reveal that DPPH activated ROS production and release of Prostaglandin E₂ in PNT2 at low and intermediate doses, while cells switched from survival to cell death signals at high doses of the oxidising agent. This new in vitro oxidative stress model was validated by using Trolox, β-carotene and total extract of the green microalga Testraselmis suecica. Only the T. suecica extract can completely counteract DPPH-induced injury, since its chemical complexity demonstrated a multilevel protecting and neutralising effect against oxidative stress in PNT2.     

Accumulation of Cytochrome b558 at the Plasma Membrane: Hallmark of Oxidative Stress in Phagocytic Cells

Neutrophils play a very key role in the human immune defense against pathogenic infections. The predominant players in this role during the activation of neutrophils are the release of cytotoxic agents stored in the granules and secretory vesicles and the massive production of reactive oxygen species (ROS) initiated by the enzyme NADPH oxidase. In addition, in living organisms, cells are continuously exposed to endogenous (inflammations, elevated neutrophil presence in the vicinity) and exogenous ROS at low and moderate levels (travels by plane, radiotherapy, space irradiation, blood banking, etc.). To study these effects, we used ROS induced by gamma radiation from low (0.2 Gy) to high (25 Gy) dose levels on PLB-985 cells from a myeloid cell line differentiated to neutrophil-like cells that are considered a good alternative to neutrophils. We determined a much longer lifetime of PLB-985 cells than that of neutrophils, which, as expected, decreased by increasing the irradiation dose. In the absence of any secondary stimulus, a very low production of ROS is detected with no significant difference between irradiated and non-irradiated cells. However, in phagocytosing cells, irradiation doses above 2 Gy enhanced oxidative burst in PLB-985 cells. Whatever the irradiation dose, NADPH oxidase devoid of its cytosolic regulatory units is observed at the plasma membrane in irradiated PLB-985 cells. This result is different from that observed for irradiated neutrophils in which irradiation also induced a translocation of regulatory subunits suggesting that the signal transduction mechanism or pathway operate differently in both cells.     

Peripheral Blood Lymphocyte Subsets (CD4+, CD8+ T Cells,NK Cells) in Patients with Cardiovascular and Neurological Complications after Carotid Endarterectomy

Background: The aim of the study was to evaluate the differences in the circulating immune cells’ subgroups after the atherosclerotic plaque removal in patients presenting with postoperative complications as compared to the patients without complications after carotid endarterectomy (CEA). Methods: Patients with significant carotid atherosclerosis (n = 124, age range: 44 to 87 years) who underwent CEA were enrolled in a prospective study. The immunology study using flow cytometry was performed to determine the percentages of peripheral blood T cells (CD4⁺, CD8⁺, Treg—CD4⁺/CD25⁺) and NK (natural killer) cells before and after the procedure. The data were expressed as the percentage of total lymphocytes ± the standard error of mean. Results: The mean percentage of lymphocytes (61.54% ± 17.50% vs. 71.82% ± 9.68%, p = 0.030) and CD4 T lymphocytes (T helper, 38.13% ± 13.78% vs. 48.39% ± 10.24%, p = 0.027) was significantly lower six hours after CEA in patients with postoperative 30-day cardiovascular and neurological complications as compared to the group without complications. On the other hand the mean NK level in the group with complications was significantly higher (21.61% ± 9.00% vs. 15.80% ± 9.31%, p = 0.048). Conclusions: The results of this study suggest that after carotid endarterectomy the percentages of circulating immune cells subsets differ in patients with and without postoperative complications.     

Cytotoxicity,Mitochondrial Functionality,and Redox Status of Human Conjunctival Cells after Short and Chronic Exposure to Preservative-Free Bimatoprost 0.03% and 0.01%: An In Vitro Comparative Study

Prostaglandin analogues (PGAs), including bimatoprost (BIM), are generally the first-line therapy for glaucoma due to their greater efficacy, safety, and convenience of use. Commercial solutions of preservative-free BIM (BIM 0.03% and 0.01%) are already available, although their topical application may result in ocular discomfort. This study aimed to evaluate the in vitro effects of preservative-free BIM 0.03% vs. 0.01% in the human conjunctival epithelial (HCE) cell line. Our results showed that long-term exposure to BIM 0.03% ensues a significant decrease in cell proliferation and viability. Furthermore, these events were associated with cell cycle arrest, apoptosis, and alterations of ΔΨ_m. BIM 0.01% does not exhibit cytotoxicity, and no negative influence on conjunctival cell growth and viability or mitochondrial activity has been observed. Short-time exposure also demonstrates the ability of BIM 0.03% to trigger reactive oxygen species (ROS) production and mitochondrial hyperpolarisation. An in silico drug network interaction was also performed to explore known and predicted interactions of BIM with proteins potentially involved in mitochondrial membrane potential dissipation. Our findings overall strongly reveal better cellular tolerability of BIM 0.01% vs. BIM 0.03% in HCE cells.     

Fabrication and Dielectric,Mechanical, and Thermal Properties of Low-Density Polyethylene (LDPE) Composites Containing Surface-Passivated Silicon (Si/SiO2 Core/Shell Nanoparticles)

Composites of low-density polyethylene containing between 1 and 5?wt% of Si/SiO₂ core/shell nanoparticles were prepared by ball milling method. The thermal, mechanical, and dielectric properties of composites were investigated in terms of composition, frequency, and temperature. The results showed that the dielectric permittivity increased smoothly with a rise of Si/SiO₂ particle. The dielectric permittivity and loss decreases and increases with temperature, respectively. The resistance of composites to erosion due to partial discharge was significantly improved by adding nanoparticles. The results have demonstrated that ball milling was an effective method for producing relatively homogeneous nanocomposite up to 4?wt% Si/SiO₂.     

Modulation of Cellular Response to Different Parameters of the Rotating Magnetic Field (RMF)—An In Vitro Wound Healing Study

Since the effect of MFs (magnetic fields) on various biological systems has been studied, different results have been obtained from an insignificant effect of weak MFs on the disruption of the circadian clock system. On the other hand, magnetic fields, electromagnetic fields, or electric fields are used in medicine. The presented study was conducted to determine whether a low-frequency RMF (rotating magnetic field) with different field parameters could evoke the cellular response in vitro and is possible to modulate the cellular response. The cellular metabolic activity, ROS and Ca²⁺ concentration levels, wound healing assay, and gene expression analyses were conducted to evaluate the effect of RMF. It was shown that different values of magnetic induction (B) and frequency (f) of RMF evoke a different response of cells, e.g., increase in the general metabolic activity may be associated with the increasing of ROS levels. The lower intracellular Ca²⁺ concentration (for 50 Hz) evoked the inability of cells to wound closure. It can be stated that the subtle balance in the ROS level is crucial in the wound for the effective healing process, and it is possible to modulate the cellular response to the RMF in the context of an in vitro wound healing.     

The Effect of Sera from Children with Obstructive Sleep Apnea Syndrome (OSAS) on Human Cardiomyocytes Differentiated from Human Embryonic Stem Cells

Obstructive sleep apnea syndrome (OSAS) patients suffer from cardiovascular morbidity, which is the leading cause of death in this disease. Based on our previous work with transformed cell lines and primary rat cardiomyocytes, we determined that upon incubation with sera from pediatric OSAS patients, the cell’s morphology changes, NF-κB pathway is activated, and their beating rate and viability decreases. These results suggest an important link between OSAS, systemic inflammatory signals and end-organ cardiovascular diseases. In this work, we confirmed and expanded these observations on a new in vitro system of beating human cardiomyocytes (CM) differentiated from human embryonic stem cells (hES). Our results show that incubation with pediatric OSAS sera, in contrast to sera from healthy children, induces over-expression of NF-κB p50 and p65 subunits, marked reduction in CMs beating rate, contraction amplitude and a strong reduction in intracellular calcium signal. The use of human CM cells derived from embryonic stem cells has not been previously reported in OSAS research. The results further support the hypothesis that NF-κB dependent inflammatory pathways play an important role in the evolution of cardiovascular morbidity in OSAS. This study uncovers a new model to investigate molecular and functional aspects of cardiovascular pathology in OSAS.     

Evaluating Cu(II) Removal From Aqueous Solutions with Response Surface Methodology by Using Novel Synthesized CaCO3 Nanoparticles Prepared in a Colloidal Gas Aphron System

Removal of heavy metals from water and wastewaters has recently gained a great deal of attention due to their serious environmental problems. In this study, novel synthesized calcium carbonate nanoparticles, prepared in a colloidal gas aphron (CGA) system, were used as adsorbents for the removal of Cu²⁺ ions from aqueous solutions under different conditions. A developed pseudo-second-order (PSO) model well described the adsorption kinetics of the process. Langmuir and Freundlich adsorption isotherms have been examined and the maximum adsorption capacity from the Langmuir isotherm equation was found to be 666.67?mg Cu/g adsorbent. The effects of temperature, Cu²⁺ initial concentration, and CaCO₃ dosage on the removal capacity were also investigated using the three-level Box–Behnken experimental design method. The response surface modeling results demonstrated that under certain experimental conditions (i.e., T?=?26°C, [Cu²⁺]?=?200?mg/L, and [CaCO₃]?=?0.5?g/L), maximum removal capacity value (393.52?mg/g) was achieved.     

In Vitro Characterization of Sphingosine 1-Phosphate Receptor 1 (S1P1) Expression and Mediated Migration of Primary Human T and B Cells in the Context of Cenerimod,a Novel,Selective S1P1 Receptor Modulator

Cenerimod is a potent, selective sphingosine 1-phosphate receptor 1 (S1P₁) modulator currently investigated in a Phase IIb study in patients with systemic lupus erythematosus (SLE) ({"type":"clinical-trial","attrs":{"text":"NCT03742037","term_id":"NCT03742037"}}NCT03742037). S1P₁ receptor modulators sequester circulating lymphocytes within lymph nodes, thereby reducing pathogenic autoimmune cells (including T and B lymphocytes) in the bloodstream and inflamed tissues, making them an effective therapeutic concept for autoimmune disorders. Although the effect of S1P receptor modulators in reducing circulating lymphocytes is well documented, the precise molecular role of the S1P₁ receptor on these cell types is not fully understood. In this study, the mode of action of cenerimod on human primary lymphocytes in different activation states was investigated focusing on their chemotactic behavior towards S1P in real-time, concomitant to S1P₁ receptor expression and internalization dynamics. Here, we show that cenerimod effectively prevents T and B cell migration in a concentration-dependent manner. Interestingly, while T cell activation led to strong S1P₁ re-expression and enhanced migration; in B cells, an enhanced migration capacity and S1P₁ receptor surface expression was observed in an unstimulated state. Importantly, concomitant treatment with glucocorticoids (GCs), a frequently used treatment for autoimmune disorders, had no impact on the inhibitory activity of cenerimod on lymphocytes.     

Synergistic Activity of Ketoconazole and Miconazole with Prochloraz in Inducing Oxidative Stress,GSH Depletion,Mitochondrial Dysfunction,and Apoptosis in Mouse Sertoli TM4 Cells

Triazole and imidazole fungicides represent an emerging class of pollutants with endocrine-disrupting properties. Concerning mammalian reproduction, a possible causative role of antifungal compounds in inducing toxicity has been reported, although currently, there is little evidence about potential cooperative toxic effects. Toxicant-induced oxidative stress (OS) may be an important mechanism potentially involved in male reproductive dysfunction. Thus, to clarify the molecular mechanism underlying the effects of azoles on male reproduction, the individual and combined potential of fluconazole (FCZ), prochloraz (PCZ), miconazole (MCZ), and ketoconazole (KCZ) in triggering in vitro toxicity, redox status alterations, and OS in mouse TM4 Sertoli cells (SCs) was investigated. In the present study, we demonstrate that KCZ and MCZ, alone or in synergistic combination with PCZ, strongly impair SC functions, and this event is, at least in part, ascribed to OS. In particular, azoles-induced cytotoxicity is associated with growth inhibitory effects, G0/G1 cell cycle arrest, mitochondrial dysfunction, reactive oxygen species (ROS) generation, imbalance of the superoxide dismutase (SOD) specific activity, glutathione (GSH) depletion, and apoptosis. N-acetylcysteine (NAC) inhibits ROS accumulation and rescues SCs from azole-induced apoptosis. PCZ alone exhibits only cytostatic and pro-oxidant properties, while FCZ, either individually or in combination, shows no cytotoxic effects up to 320 µM.     

Association of Polycyclic Aromatic Hydrocarbons (PAHs) with Different Sizes of Atmospheric Particulate in Hisar City and its Health Aspects

The concentration of polycyclic aromatic hydrocarbons (PAHs) associated with different particulate sizes of suspended particulate matter (SPM) was studied in Hisar city with the help of a MOUDI-NR 10-stage (18 μm to 0.056 μm) cascade impactor. The vehicular-cum-commercial, vehicular, commercial, and institutional/residential locations had the average concentration (ng/g) of 11.39, 10.39/10.22 (NH 10/Bus Terminus), 8.89, and 8.93, respectively. Vehicular emission was the chief source and diesel-vehicle dominated areas represented higher concentration of PAHs associated with coarse fraction. An increase in PAH associated with fine fraction was observed with an increase in vehicular density. Maximum average concentration (11.26 ng/g) was found to be associated in particle range of <0.56–0.32 μm. It was followed by particle range (μm) of <18–10, <1.8–1, <0.18–0.1, <1–0.56, <5.6–3.2, <0.32–0.18, <3.2–1.8 <0.1–0.056, and <10–5.6 with average values (ng/g) of 10.75, 10.35, 10.22, 10.16, 10.06, 9.50, 9.18, 9.18, and 9.00, respectively. Among the PAHs studied, maximum levels were observed for pyrene, followed by Benzo(b)fluoranthene, Benzo(e)pyrene, benzo(k)fluoranthene, fluoranthene, phenanthrene, benzo(ghi)perylene, anthracene, and naphthalene. With respect to the percent fraction of PAHs studied, the vehicular-cum-commercial area represented maximum fraction (22%) followed by vehicular area (21% each for NH-10 and Bus Terminus), institutional/residential area (18%), and commercial area (18%). The isomeric ratios revealed that most of the PAHs originate from combustion of diesel, gasoline, used engine oil, and coal/wood. The association of PAHs with fine fraction of health concern since it can penetrate and get accumulated in deep respiratory regions.     

Kinetic effect of electrolytes on the oligomerization of hydrosulfide into polysulfides and colloidal sulfur with iron(III) trans-1,2-diaminocyclohexanetetraacetic acid in anoxic aqueous solutions

The kinetic electrolyte effect of dissolved NaCl, LiCl and Na₂SO₄ on the oligomerization of the hydrosulfide ion (HS^-) into polysulfides then in colloidal sulfur assisted by iron(III) trans-1,2,-diaminocyclohexanetetraacetate (iron(III)-cdta) complexes was studied in anoxic alkaline solutions at . The three electrolytes have been confirmed to boost the HS^- oligomerization reaction considerably. For example, the HS^- half-life decreased from 16.7 min in demineralized water (pH=10.5) to 12.4 min for the corresponding 0.01 mol/L NaCl solution. It further decreased to 5.1 min for a 0.1 mol/L NaCl solution. The kinetic enhancement factor is attributed to the cationic species (i.e. Na⁺, Li⁺) both affecting the reactive species’ activity coefficients within experimental and calculation accuracy. It improves the initial HS^- and iron(III)-cdta reaction rates, reduces the induction period for elemental sulfur formation which in turn enhance the overall HS^- consumption rate. The HS^- and iron(III)-cdta concentration profiles were also fitted with corresponding rate laws derived from a simplified HS^- oligomerization mechanism and application of kinetic electrolyte constants on the activity coefficient Hückel equation.     

Comparative Study of the Mechanical and Thermal Properties of Polyamide-66 Filled with Commercial and Nano-Mg(OH)2 Particles

In the present work, polyamide-Mg(OH)₂ nanocomposites were prepared via melt intercalation on a twin-screw extruder. Different particle sizes (24, 20, 11 nm) of Mg(OH)₂ were synthesized by in-situ deposition technique and its shape and sizes was confirmed on transmission electron microscope (TEM). Nano-Mg(OH)₂ was added from 1 to 4 wt% in the polyamide. Properties such as tensile strength, elongation at break, hardness, and flame retardency were studied. These results were then compared with commercial Mg(OH)₂-filled composites. There was propounding effect to be observed on properties of polyamide nanocomposites due to uniform dispersion of nano-Mg(OH)₂ and commercial Mg(OH)₂. Moreover, thermal property like thermal degradation was studied on TGA. Extent of dispersion of nano-Mg(OH)₂ was studied along with microcracks generated during tensile testing using AFM. It was found that nano-Mg(OH)₂ is thermally more stable compared to that of commercial Mg(OH)₂. Besides that, T_g and M.T. are studied on DSC.