Search Results (25)

Omega-6 polyunsaturated fats (ω-6 PUFAs) are vital for many physiological functions, but their impact on cardiovascular disease (CVD) risk is controversial. Eryptosis alters blood viscosity by providing a procoagulant surface and leads to anemia, which is a recognized risk factor for CVD. This study examines the toxic mechanisms of adrenic acid (ADR), an ω-6 PUFA enriched in inflammatory and oxidative conditions, in red blood cells (RBCs). Purified RBCs were prepared from healthy volunteers and treated with 10–100 μM of ADR for 24 h at 37 °C under various physiological conditions. Eryptotic markers were studied through flow cytometry including Ca²⁺ (Fluo4/AM), loss of volume (forward scatter), phosphatidylserine (PS) exposure (annexin-V-FITC), and oxidative stress (H₂DCFDA). Moreover, hemolytic markers were measured by colorimetric methods, whereas cellular morphology was visualized using a scanning electron microscope. ADR led to significant Ca²⁺ elevation, cell shrinkage and schistocyte formation, PS externalization, hemolysis, and oxidative stress. While guanosine, heparin, and NSC 23766 prevented eryptosis and hemolysis, melatonin, ATP, adenine, and L-NAME only prevented eryptosis. Conversely, mannitol and urea exacerbated eryptosis, whereas caffeine, mannitol, and urea under Ca²⁺ deprivation and membrane potential dissipation aggravated hemolysis. ADR induces erythrocyte membrane injury and eryptosis through Ca²⁺ elevation, oxidative stress, and metabolic exhaustion subject to inhibition by the Rac1 GTPase/NOS/COX pathway. Altogether, these findings present a novel mechanistic link between lipid dysregulation and RBC dysfunction which may improve dietary strategies to prevent and manage CVD. Full article

Background/Objectives: The strategy of targeting endotoxins and circulating histones to alleviate excessive inflammation and tissue damage has been proposed as an important immunoregulatory strategy against sepsis. However, the development of a multifunctional hemoperfusion adsorber that simultaneously removes endotoxins and histones remains an unmet clinical need in sepsis management. Methods: We synthesized chitosan-cellulose composite (CSCE) microspheres utilizing phase inversion technology, while heparin-grafted chitosan-cellulose composite (CSCEHEP) microspheres were developed by grafting heparin onto CSCE microspheres through the carbodiimide coupling method. In our experimental design, we allocated healthy New Zealand rabbits to four distinct groups: a healthy control group, a lipopolysaccharides (LPS) group, a CSCE group, and a CSCEHEP group. Following the administration of LPS for 12 h, septic rabbits underwent extracorporeal hemoperfusion with either CSCE or CSCEHEP microspheres for a duration of 6 h, notably without the inclusion of heparin in the blood circuits. Post-hemoperfusion, we conducted an analysis of thrombus formation and total protein adsorption on the column. Concurrently, blood samples were collected from the venous side to evaluate inflammatory cytokine concentrations, liver and kidney function levels, LPS levels, the histone presence, and to perform histopathological assessments of liver and kidney injury. Results: Our in vivo experiments demonstrated that CSCEHEP microspheres for extracorporeal circulation could achieve a 6 h hemoperfusion session in septic rabbits without the need for continuous anticoagulation with heparin. A CSCEHEP column turns into a very light-red color (almost the original white) and light contamination or clotting was observed after the 6 h hemoperfusion. Moreover, CSCEHEP microspheres effectively reduced the concentration levels of leukocyte, serum IL-6 and TNF-α, mitigated pathological damage to the liver and kidneys, and removed over 56.7% of LPS and nearly 58.6% of histone H3 from the blood of septic rabbits during hemoperfusion. Conclusions: Hemoperfusion utilizing CSCEHEP microspheres exhibits excellent self-anticoagulation capabilities, remarkable anti-inflammatory performance, efficient endotoxin adsorption and histone antagonism properties, rendering it both effective and safe for use in septic rabbits. Full article

Background: In vitro blood circulation loop systems are utilized to assess the hemocompatibility and performance of medical devices that come into contact with blood, in accordance with the international standards ASTM F1830 and ASTM F1841. However, a method for evaluating the specific type of anticoagulant and the blood characteristics of each animal species is necessary to ensure consistent and reliable results. Methods: Blood was collected from healthy rabbits, pigs, rhesus monkeys, and cynomolgus monkeys to evaluate whole blood preserved in anticoagulants (ACD-A, CPDA-1, and heparin). For each sample, red blood cells were monitored over time, and their morphological characteristics were documented. Results: The morphological grade of erythrocytes gradually decreased over time. Significant differences were observed based on the type of anticoagulant used in the experiment, and variations were noted among different animal species. Conclusions: The hemocompatibility of in vitro blood circulation loop systems may vary depending on the animal species. Observing erythrocyte morphology can serve as a control measure to ensure reproducible results. Full article

We established a real-time Förster resonance energy transfer (FRET) based assay to evaluate targeted drug delivery using polymeric micelles. Red fluorescent protein (RFP)-expressing E. coli cells were used as a test system to monitor the delivery of drug-fluorophore such as curcumin and umbelliferones (MUmb and AMC) encapsulated in the polymeric micellar formulations. The efficiency of the drug delivery was quantified using the FRET efficiency, measured as the degree of energy transfer from the drug to the RFP. FRET efficiency directly provides the determination of the delivery efficacy, offering a versatile platform adaptable to various drugs and cell types. We used polymer micelles as a carrier for targeted delivery of fluorescent drugs to bacterial cells expressing RFP. The physicochemical characterization of the interaction between the drugs and the micelles including spectral properties, and the solubility and binding constants, were determined. We revealed a stronger affinity of MUmb for heparin-based micelles (K_d~10⁻⁵ M) compared to chitosan-based micelles (K_d~10⁻⁴ M), underscoring the influence of polymer composition on drug loading efficiency. For micelles containing MUmb, a FRET efficiency significantly exceeds (by three times) the efficiency for non-micellar MUmb, which have minimal penetration into bacterial cells. The most noticeable effect was observed with the use of the micellar curcumin providing pronounced activation of the RPF fluorescence signal, due to the interaction with curcumins (fluorophore-donor). Curcumin delivery using Chit5-OA micelle resulted in a 115% increase in RFP fluorescence intensity, and Hep-LA showed a significant seven-fold increase. These results highlight the significant effect of micellar composition on the effectiveness of drug delivery. In addition, we have developed a visual platform designed to evaluate the effectiveness of a pharmaceutical product through the visualization of the fluorescence of a bacterial culture on a Petri dish. This method allows us to quickly and accurately assess the penetration of a drug into bacteria, or those located inside other cells, such as macrophages, where the intercellular latent forms of the infection are located. Micellar formulations show enhanced antibacterial activity compared to free drugs, and formulations with Hep-OA micelles demonstrate the most significant reduction in E. coli viability. Synergistic effects were observed when combining curcumin and MUmb with moxifloxacin, resulting in a remarkable 40–50% increase in efficacy. The presented approach, based on the FRET test system with RFP expressed in the bacterial cells, establishes a powerful platform for development and optimizing targeted drug delivery systems. Full article

The addition of dexamethasone in membranes for guided bone regeneration is promising due to its dual effect: (1) anti-inflammatory action and (2) induction of osteogenesis in host stem cells. Electrospun fiber coating with dexamethasone using the layer-by-layer (LBL) technique offers an interesting alternative for the gradual release of the drug, aiming for enhanced osteodifferentiation activity. This study aimed to develop synthetic poly-L-lactide (PLLA) membranes with dexamethasone incorporated into the fibers or coated on their surface, and to evaluate the drug release rate, as well as the material’s ability to promote proliferation, osteoconduction, and osteodifferentiation of human periodontal ligament stem cells (hPDLSCs). PLLA membranes were produced by electrospinning. Dexamethasone was incorporated using three techniques: (A) electrospinning of a co-solution of PLLA with 2.5 w/w% dexamethasone; (B) deposition of four layers on the PLLA membrane using alternating solutions of chitosan and heparin/dexamethasone; (C) deposition of 10 layers on the PLLA membrane using the same solutions. hPDLSC proliferation was measured via CCK-8 at 1, 7, 14, and 21 days. Cellular differentiation was assessed by alkaline phosphatase activity (7 days) and alizarin red staining (21 days) in clonogenic and osteogenic media (ODM). Data were analyzed using one or two-way ANOVA and Tukey test. Electrospun membranes with dexamethasone and those with 4 layers showed immediate drug release within 24 h, whereas 10 layers exhibited gradual release over 14 days. Cumulative drug release was higher for electrospun membranes at 1 and 7 days, similar to 10 layers at 14 and 21 days. The 4 LBL membrane promoted lower hPDLSC proliferation compared to the 10 LBL and electrospun membranes at 21 days but showed increased extracellular matrix mineralization in osteogenic media. No significant differences in alkaline phosphatase expression were observed between materials. Therefore, the addition of dexamethasone in 10 layers, combined with heparin, enables gradual drug release. However, lower drug release in the first 24 h by four LBL membranes improved the material’s osteogenesis properties. None of the materials improved the osteodifferentiation in the clonogenic medium. Full article

Anticancer drugs cause anemia in patients through eryptosis and hemolysis. We thus studied the in vitro toxicity of galangin (GAL) in red blood cells (RBCs). RBCs were exposed to 50–500 μM of GAL and analyzed for markers of eryptosis and hemolysis. Ca²⁺ nucleation, phosphatidylserine (PS) externalization, oxidative stress, and cell size were detected via fluorescence-activated cell sorting using Fluo4/AM, annexin-V-FITC, 2′,7′-dichlorodihydrofluorescein diacetate, and forward scatter (FSC), respectively. Acetylcholinesterase (AChE) activity was measured via Ellman’s assay and ultrastructural morphology was examined via scanning electron microscopy. Membrane rupture and extracellular hemoglobin, aspartate transaminase (AST), and lactate dehydrogenase (LDH) were assessed via colorimetric methods. Distinct experiments were carried out to identify protective agents and signaling pathways using small-molecule inhibitors. GAL triggered sucrose-sensitive hemolysis with AST and LDH leakage, increased annexin-V-FITC and Fluo4 fluorescence, and decreased FSC and AChE activity which was associated with the formation of granulated echinocytes. Ca²⁺ omission and energy replenishment with glucose, adenine, and guanosine blunted PS externalization and preserved cellular volume. Moreover, caffeine, Trolox, heparin, and uric acid had similar ameliorative effects. Hemolysis was abrogated via caffeine, Trolox, heparin, mannitol, lactate, melatonin, and PEG 8000. Notably, co-treatment of cells with GAL and staurosporin, D4476, or acetylsalicylic acid prevented PS externalization whereas only the presence of SB203580 and NSC23766 rescued the cells from GAL-induced hemolysis. Ca²⁺ nucleation and metabolic collapse mediated by PKC/CK1α/COX/p38/Rac1 drive GAL-induced eryptosis and hemolysis. These novel findings carry ramifications for the clinical prospects of GAL in anticancer therapy. Full article

Background: This study was conducted to confirm the reliability of an in vitro mechanically induced hemolysis test (ISO 10993-4:2017), which is essential for ensuring the safety of blood pumps. Methods: For appropriate anticoagulant selection, porcine blood was prepared in anticoagulant citrate dextrose solution A (ACD-A), heparin, and citrate phosphate dextrose adenine (CPDA-1), respectively, according to the ASTM F1830 standard. Anticoagulant-treated porcine and bovine blood were circulated in a mock circulatory loop (MCL) for 6 h to observe the rate of plasma-free hemoglobin (pfHb) and RBCs with morphological integrity. Results: A morphological loss of red blood cells (RBCs) was observed over time. While there were differences in morphological loss depending on the anticoagulant, no consistent trend could be identified. The pfHb concentration was significantly higher in bovine than in porcine blood. Conversely, the number of RBCs with morphological integrity decreased over time in both, but the ratio of RBCs with morphological integrity was similar across all timepoints. Conclusions: The percentage of RBCs with morphological integrity can be used as a reliable indicator for the interpretation of mechanically induced hemolysis results in different blood types. Furthermore, the reliability of the in vitro mechanically induced hemolysis test (ISO 10993-4:2017) was assessed. Full article

Three different populations of sulfated polysaccharides can be found in the cell wall of the red alga Botryocladia occidentalis. In a previous work, the structures of the two more sulfated polysaccharides were revised. In this work, NMR-based structural analysis was performed on the least sulfated polysaccharide and its chemically modified derivatives. Results have revealed the presence of both 4-linked α- and 3-linked β-galactose units having the following chemical features: more than half of the total galactose units are not sulfated, the α-units occur primarily as 3,6-anhydrogalactose units either 2-O-methylated or 2-O-sulfated, and the β-galactose units can be 4-O-sulfated or 2,4-O-disulfated. SPR-based results indicated weaker binding of the least sulfated galactan to thrombin, factor Xa, and antithrombin, but stronger binding to heparin cofactor II than unfractionated heparin. This report together with our previous publication completes the structural characterization of the three polysaccharides found in the cell wall of the red alga B. occidentalis and correlates the impact of their composing chemical groups with the levels of interaction with the blood co-factors. Full article

Allylbenzenes (apiol, dillapiol, myristicin and allyltetramethoxybenzene) are individual components of plant essential oils that demonstrate antitumor activity and can enhance the antitumor activity of cytotoxic drugs, such as paclitaxel, doxorubicin, cisplatin, etc. Triphenylphosphine (PPh₃) derivatives of allylbenzenes are two to three orders of magnitude more potent than original allylbenzenes in terms of IC₅₀. The inhibition of efflux pumps has been reported for allylbenzenes, and the PPh₃ moiety is deemed to be responsible for preferential mitochondrial accumulation and the depolarization of mitochondrial membranes. However, due to poor solubility, the practical use of these substances has never been an option. Here, we show that this problem can be solved by using a complex formation with cyclodextrin (CD-based molecular containers) and polyanionic heparin, stabilizing the positive charge of the PPh₃ cation. Such containers can solubilize both allylbenzenes and their PPh₃ derivatives up to 0.4 mM concentration. Furthermore, we have observed that solubilized PPh₃ derivatives indeed work as adjuvants, increasing the antitumor activity of paclitaxel against adenocarcinomic human alveolar basal epithelial cells (A549) by an order of magnitude (in terms of IC50) in addition to being quite powerful cytostatics themselves (IC₅₀ in the range 1–10 µM). Even more importantly, CD-solubilized PPh₃ derivatives show pronounced selectivity, being highly toxic for the A549 tumor cell line and minimally toxic for HEK293T non-tumor cells, red blood cells and sea urchin embryos. Indeed, in many cancers, the mitochondrial membrane is more prone to depolarization compared to normal cells, which probably explains the observed selectivity of our compounds, since PPh₃ derivatives are known to act as mitochondria-targeting agents. According to the MTT test, 100 µM solution of PPh₃ derivatives of allylbenzenes causes the death of up to 85% of A549 cancer cells, while for HEK293T non-cancer cells, only 15–20% of the cells died. The hemolytic index of the studied substances did not exceed 1%, and the thrombogenicity index was < 1.5%. Thus, this study outlines the experimental foundation for developing combined cytostatic medications, where effectiveness and selectivity are achieved through decreased concentration of the primary ingredient and the inclusion of adjuvants, which are safe or practically harmless substances. Full article

This is a case of an eleven-year-old female Sudanese child, a known Sickle Cell Anemia (SCA) patient, who presented with fever, as well as left thigh and leg swelling that was associated with pain and warmness, which was diagnosed as Deep Vein Thrombosis (DVT) of her left lower limb. She had a previous history of admissions to the emergency room, during which she once received blood. The patient was managed by carrying out a basic routine initial laboratory investigation. A Doppler ultrasound scan showed features consistent with DVT. Based on the clinical findings and investigation results, management began by providing the patient with intravenous fluid, analgesia, packed Red Blood Cells (RBCs), intravenous antibiotics, and low-molecular-weight heparin. Further consultations showed that there was no need for vascular surgery or surgical intervention. This case highlights the need for more studies on DVT and Venous Thromboembolism (VTE) complications in children with SCA, so as to develop strategies for diagnosis and management in order to reduce the risk of life-threatening complications of VTE in patients with Sickle Cell Disease SCD. Full article

The spontaneous interaction between human papillomavirus type 16 (HPV16) L1 virus-like particles (VLPs) and non-functionalized gold nanoparticles (nfGNPs) interferes with the nfGNPs’ salt-induced aggregation, inhibiting the red–blue color shift in the presence of NaCl. Electron microscopy and competition studies showed that color-shift inhibition is a consequence of direct nfGNP–VLP interaction and, thus, may produce a negative impact on the virus entry cell process. Here, an in vitro infection system based on the HPV16 pseudovirus (PsV) was used to stimulate the natural infection process in vitro. PsVs carry a pseudogenome with a reporter gene, resulting in a fluorescent signal when PsVs infect a cell, allowing quantification of the viral infection process. Aggregation assays showed that nfGNP-treated PsVs also inhibit color shift in the presence of NaCl. High-resolution microscopy confirmed nfGNP–PsV complex formation. In addition, PsVs can interact with silver nanoparticles, suggesting a generalized interaction of metallic nanoparticles with HPV16 capsids. The treatment of PsVs with nfGNPs produced viral infection inhibition at a higher level than heparin, the canonical inhibitor of HPV infection. Thus, nfGNPs can efficiently interfere with the HPV16 cell entry process and may represent a potential active component in prophylactic formulations to reduce the risk of HPV infection. Full article

Cardiovascular diseases (CVD) are a leading cause of mortality worldwide, and dietary habits represent a major risk factor for dyslipidemia; a hallmark of CVD. Saturated fatty acids contribute to CVD by aggravating dyslipidemia, and, in particular, lauric acid (LA) raises circulating cholesterol levels. The role of red blood cells (RBCs) in CVD is increasingly being appreciated, and eryptosis has recently been identified as a novel mechanism in CVD. However, the effect of LA on RBC physiology has not been thoroughly investigated. RBCs were isolated from heparin-anticoagulated whole blood (WB) and exposed to 50–250 μM of LA for 24 h at 37 °C. Hemoglobin was photometrically examined as an indicator of hemolysis, whereas eryptosis was assessed by Annexin V-FITC for phosphatidylserine (PS) exposure, Fluo4/AM for Ca²⁺, light scatter for cellular morphology, H₂DCFDA for oxidative stress, and BODIPY 581/591 C11 for lipid peroxidation. WB was also examined for RBC, leukocyte, and platelet viability and indices. LA caused dose-responsive hemolysis, and Ca²⁺-dependent PS exposure, elevated erythrocyte sedimentation rate (ESR), cytosolic Ca²⁺ overload, cell shrinkage and granularity, oxidative stress, accumulation of lipid peroxides, and stimulation of casein kinase 1α (CK1α). In WB, LA disrupted leukocyte distribution with elevated neutrophil-lymphocyte ratio (NLR) due to selective toxicity to lymphocytes. In conclusion, this report provides the first evidence of the pro-eryptotic potential of LA and associated mechanisms, which informs dietary interventions aimed at CVD prevention and management. Full article

Chronic lymphocytic leukemia (CLL) patients have a greater predisposition to develop autoimmune complications. The most common of them is autoimmune hemolytic anemia (AIHA) with a frequency of 7–10% of cases. Pathogenesis is multifactorial involving humoral, cellular, and innate immunity. CLL B-cells have damaged apoptosis, produce less immunoglobulins, and could be responsible for antigen presentation and releasing inflammatory cytokines. CLL B-cells can act similar to antigen-presenting cells activating self-reactive T helper cells and may induce T-cell subsets imbalance, favoring autoreactive B-cells which produce anti-red blood cells autoantibodies. Treatment is individualized and it depends on the presence and severity of clinical symptoms, disease status, and comorbidities. Corticosteroids are the standardized first-line treatment; second-line treatment comprises rituximab. Patients not responding to corticosteroids and rituximab should be treated with CLL-specific drugs as per current guidelines according to age and comorbidities. New targeted drugs (BTK inhibitors and anti BCL2) are recently used after or together with steroids to manage AIHA. In the case of cold agglutinin disease, rituximab is preferred, because steroids are ineffective. Management must combine supportive therapies, including vitamins; antibiotics and heparin prophylaxis are indicated in order to minimize infectious and thrombotic risk. Full article

The potential neuroprotective capacity of four different sulfated glycans: Botryocladia occidentalis-derived sulfated galactan (BoSG) (MW > 100 kDa), Lytechinus variegatus-derived sulfated fucan (LvSF) (MW~90 kDa), high-molecular weight dextran sulfate (DxS) (MW 100 kDa), and unfractionated heparin (UFH) (MW~15 kDa), was assessed in response to the HIV-1 proteins, R5-tropic glycoprotein 120 (gp120) and/or trans-activator of transcription (Tat), using primary murine neurons co-cultured with mixed glia. Compared to control-treated cells in which HIV-1 proteins alone or combined were neurotoxic, BoSG was, among the four tested sulfated glycans, the only one capable of showing significant concentration-dependent neuroprotection against Tat and/or gp120, alone or combined. Surface plasmon resonance-based data indicate that BoSG can bind both HIV-1 proteins at nM concentrations with preference for Tat (7.5 × 10⁻⁸ M) over gp120 (3.2 × 10⁻⁷ M) as compared to UFH, which bound gp120 (8.7 × 10⁻⁷ M) over Tat (5.7 × 10⁻⁶ M). Overall, these data support the notion that sulfated glycan extracted from the red alga B. occidentalis, BoSG, can exert neuroprotection against HIV-1 Tat and gp120, potentially via direct molecular interactions. Full article

Anaphylaxis is a severe, acute, life-threatening multisystem allergic reaction resulting from the release of a plethora of mediators from mast cells culminating in serious respiratory, cardiovascular and mucocutaneous manifestations that can be fatal. Medications, foods, latex, exercise, hormones (progesterone), and clonal mast cell disorders may be responsible. More recently, novel syndromes such as delayed reactions to red meat and hereditary alpha tryptasemia have been described. Anaphylaxis manifests as sudden onset urticaria, pruritus, flushing, erythema, angioedema (lips, tongue, airways, periphery), myocardial dysfunction (hypovolemia, distributive or mixed shock and arrhythmias), rhinitis, wheezing and stridor. Vomiting, diarrhea, scrotal edema, uterine cramps, vaginal bleeding, urinary incontinence, dizziness, seizures, confusion, and syncope may occur. The traditional (or classical) pathway is mediated via T cells, Th2 cytokines (such as IL-4 and 5), B cell production of IgE and subsequent crosslinking of the high affinity IgE receptor (FcεRI) on mast cells and basophils by IgE-antigen complexes, culminating in mast cell and basophil degranulation. Degranulation results in the release of preformed mediators (histamine, heparin, tryptase, chymase, carboxypeptidase, cathepsin G and tumor necrosis factor alpha (TNF-α), and of de novo synthesized ones such as lipid mediators (cysteinyl leukotrienes), platelet activating factor (PAF), cytokines and growth factors such as vascular endothelial growth factor (VEGF). Of these, histamine, tryptase, cathepsin G, TNF-α, LTC₄, PAF and VEGF can increase vascular permeability. Recent data suggest that mast cell-derived histamine and PAF can activate nitric oxide production from endothelium and set into motion a signaling cascade that leads to dilatation of blood vessels and dysfunction of the endothelial barrier. The latter, characterized by the opening of adherens junctions, leads to increased capillary permeability and fluid extravasation. These changes contribute to airway edema, hypovolemia, and distributive shock, with potentially fatal consequences. In this review, besides mechanisms (endotypes) underlying IgE-mediated anaphylaxis, we also provide a brief overview of IgG-, complement-, contact system-, cytokine- and mast cell-mediated reactions that can result in phenotypes resembling IgE-mediated anaphylaxis. Such classifications can lead the way to precision medicine approaches to the management of this complex disease. Full article