Francesco Maximillian Anthony Shelton Agar

Antiphospholipid syndrome is a systemic autoimmune disease characterized by elevated circulating antiphospholipid antibodies and a predisposition to recurrent venous and arterial thrombosis. The underlying pathophysiology of antiphospholipid syndrome remains under active investigation, with endothelial dysfunction recognized as a key contributor to vascular complications. In this study, we showed that serum from antiphospholipid syndrome patients differentially affects distinct endothelial cell types, leading to variations in migration and angiogenic behavior. Using human umbilical vein endothelial cells and human microvascular endothelial cells, we assessed the biological response to serum exposure. Treatment with antiphospholipid syndrome serum was associated with an enhanced angiogenic phenotype in venous endothelium, with a significant increase in two of the four tubulogenesis parameters assessed and a trend towards increased migration, while microvascular endothelium remained largely unaffected. Consistently, metalloproteinase-9 activity was upregulated, with these effects being more pronounced in venous compared to microvascular endothelium. Moreover, caspase-3 activity remained unchanged, suggesting that caspase-3-dependent apoptosis is unlikely to be a major contributor to the endothelial response observed under these conditions. These results enhance our understanding of the pathogenesis and highlight the distinct endothelial responses to the antiphospholipid environment. Further investigation into the underlying mechanisms may facilitate the development of targeted therapeutic strategies to mitigate vascular complications.

Background: Endogenous estradiol/progesterone (E2/P4) regulates neuronal mitochondrial bioenergetics and redox balance. However, the effects of combined oral contraceptive (COC) steroids under physiologically relevant hormonal conditions remain poorly understood. Therefore, this study aims to investigate how COC steroids modulate mitochondrial function within the defined E2/P4 hormonal milieus. Methods: Human SH-SY5Y cells (Cytion) were starved for 48 h in Cytion medium without FBS and with 1% ITS, and then exposed for 48 h to six conditions: vehicle (F0); follicular-like E2/P4 (F1); luteal-like E2/P4 (F2); F1 + dienogest/ethinylestradiol (DNG/EE; F3); F2 + DNG/EE (F4); and DNG/EE alone (F5). The primary endpoints were mitochondrial membrane potential (JC-1 red/green ratio) and ROS (H₂DCFDA/DCF); nitric oxide (DAF-FM) was also recorded. Hoechst 33342 nuclear fluorescence served both as a per-well proxy of cell number and as a proportionality factor for normalization. Results: Follicular- and luteal-like backgrounds were associated with distinct ΔΨm/ROS set-points. The addition of DNG/EE was accompanied by background-dependent shifts, generally characterized by higher DCF signals and lower JC-1 ratios relative to the vehicle, whereas DAF-FM did not reveal statistically robust changes in NO. Hoechst-based normalization preserved these patterns, suggesting that the observed effects likely reflect per-cell functional modulation rather than differences in cell number, although modest, background-dependent variations in proliferation were observed, with DNG/EE associated with a greater increase in ROS under follicular-like conditions and a more pronounced ΔΨm membrane potential in a luteal-like milieu. Conclusions: These findings suggest that neuroendocrine background may influence mitochondrial ΔΨm/ROS states in SH-SY5Y cells, with physiologically inspired E2/P4 milieus potentially shaping baseline conditions onto which COC components exert context-dependent effects at 48 h. These preliminary findings provide a standardized framework for subsequent image-based analyses of mitochondrial function.

Background: Endogenous estradiol/progesterone (E2/P4) shape neuronal mitochondrial bioenergetics and redox. The mechanisms by which combined oral contraceptive (COC) steroids operate within physiologically relevant hormonal milieus remain poorly defined. Methods: Human SH-SY5Y cells (Cytion) were starved 48 h in Cytion medium without FBS and with 1% ITS, then exposed for 48 h to six conditions: vehicle (F0); follicular-like E2/P4 (F1); luteal-like E2/P4 (F2); F1 + dienogest/ethinylestradiol (DNG/EE; F3); F2 + DNG/EE (F4); and DNG/EE alone (F5). Primary endpoints were mitochondrial membrane potential (JC-1 red/green ratio) and ROS (H₂DCFDA/DCF); nitric oxide (DAF-FM) was also recorded. Hoechst 33342 nuclear fluorescence served both as a per-well proxy of cell number and as a proportionality factor for normalization. Results: Follicular- and luteal-like backgrounds produced distinct ΔΨm/ROS set-points. Superimposing DNG/EE generated background-dependent shifts marked by higher DCF and lower JC-1 ratios versus vehicle; DAF-FM indicated concordant NO changes. Hoechst-based normalization preserved these patterns, indicating per-cell functional modulation rather than cell-number artifacts; proliferation differences were modest and background-dependent. Conclusions: Neuroendocrine background matters: physiologic E2/P4 milieus prime mitochondrial ΔΨm/ROS/NO states in SH-SY5Y, onto which COC components impose con-text-dependent modulation at 48 h. These preliminary findings provide a standardized framework for subsequent image-based mitochondrial phenotyping.

Background: Glyphosate has been extensively used as herbicide since the early 1970s. The daily exposure limit is set at 0.3 mg/kg bw/d in Europe and 1.75 mg/kg bw/d in the USA. Among its derivatives, aminomethylphosphonic acid is the most stable and abundant. Understanding their biological effects then requires reliable methods for quantification in biological samples. Methods: We developed and validated a fast, low-cost, and reliable chromatographic method for determining glyphosate and aminomethylphosphonic acid concentrations. The validation included following parameters: specificity, selectivity, matrix effect, accuracy, precision, calibration performance, limit of quantification, recovery, and stability. Sample extraction employed an anion exchange resin with elution using hydrochloric acid 50.0 mmol/L. For HPLC analysis, analytes were derivatized, separated on a C18 column with a mobile phase of phosphate buffer (0.20 mol/L, pH 3.0) and acetonitrile (85:15), and detected at 240 nm. Results: The method demonstrated high reliability and reproducibility across various matrices. Its performance met all validation criteria, confirming its suitability for quantifying glyphosate and aminomethylphosphonic acid in different biological and experimental setups. Conclusions: This method can offer a practical resource for applications in experimental research, medical diagnostics, quality control, and food safety.

Background: Hydroxyurea (HU), also known as hydroxycarbamide, is an oral ribonucleotide reductase inhibitor. In 1999, the United States Food and Drug Administration (FDA) approved HU for the treatment of sickle cell disease (SCD). Since then, it has become the cornerstone in the management of SCD patients, helping to reduce vaso-occlusive crises, acute chest syndrome, the need for blood transfusions, hospitalizations and mortality. There is considerable variability among individuals in HU pharmacokinetic (Pk) parameters that can influence treatment efficacy and toxicity. The objective of this work is part of a clinical study aimed at investigating HU Pk and determining the optimal sampling time to estimate the Area Under the Curve (AUC) in SCD patients. Methods: HU plasma concentration in 80 patients at various time points (2, 4, 6, 24 h) following a 48-h drug washout was quantified using High-Pressure Liquid Chromatography (HPLC) coupled with an ultraviolet (UV) detection method previously described in the literature and adapted to new conditions with partial modifications. Results: The mean HU administered dose was 19.5 ± 5.1 mg/kg (range: 7.7–37.5 mg/kg). The median AUC quantified in plasma patients was 101.3 mg/L/h (Interquartile Range (IQR): 72.5–355.9) and it was not influenced by the weight-based dose. However, there was a strong positive correlation between AUC and Body Mass Index (BMI) as well as dose per Body Surface Area (BSA). Along with a three-point approach for AUC determination present in the literature, we show results obtained from a four-point sampling strategy, which is more useful and effective for better optimizing dose escalation to the maximum tolerated dose (MTD). Moreover, we observed that most patients achieved the maximum HU plasma concentration two hours after drug administration, regardless of age differences. Conclusions: HU treatment, which represents a milestone in the treatment of SCD due to its ability to reduce disease complications and improve patients’ quality of life, requires careful monitoring to optimize the individual dose for saving potential side effects and/or adverse events.