Erythrocytes Nanoparticle Delivery: A Boon for Targeting Tumor.

Although nanoparticles (NPs) have many advantages as drug delivery systems, their poor stability in circulation, premature drug release, and nonspecific uptake in non-target organs have prompted biomimetic approaches to camouflage nano vehicles using natural cell membranes. Among them, which are extensively studied in erythrocytes, are the most abundant circulating blood cells. They are specially used for biomimetic coating on artificial NPs due to their excellent properties of good biocompatibility, biodegradability, non-immunogenicity, and long-term blood circulation. Erythrocyte-mimicking nanoparticles (EM-NPs) are prepared by combining nanoparticle cores with naturally derived erythrocyte (red blood cell or RBC) membranes. Compared with conventional nanosystems, EM-NPs hold the preferable characteristics of prolonged blood circulation time and immune evasion. In this review, the biomimetic platform of erythrocyte membrane-coated NPs is described in various aspects, with particular focus placed on the coating mechanism, preparation methods, characterization method, and recent advances in the biomedical applications of EM-NPs concerning cancer and targeted delivery.

Small-molecule α-lipoic acid targets ELK1 to balance human neutrophil and erythrocyte differentiation.

BACKGROUND: Erythroid and myeloid differentiation disorders are commonly occurred in leukemia. Given that the relationship between erythroid and myeloid lineages is still unclear. To find the co-regulators in erythroid and myeloid differentiation might help to find new target for therapy of myeloid leukemia. In hematopoiesis, ALA (alpha lipoic acid) is reported to inhibit neutrophil lineage determination by targeting transcription factor ELK1 in granulocyte-monocyte progenitors via splicing factor SF3B1. However, further exploration is needed to determine whether ELK1 is a common regulatory factor for erythroid and myeloid differentiation. METHODS: In vitro culture of isolated CD34+, CMPs (common myeloid progenitors) and CD34+ CD371- HSPCs (hematopoietic stem progenitor cells) were performed to assay the differentiation potential of monocytes, neutrophils, and erythrocytes. Overexpression lentivirus of long isoform (L-ELK1) or the short isoform (S-ELK1) of ELK1 transduced CD34+ HSPCs were transplanted into NSG mice to assay the human lymphocyte and myeloid differentiation differences 3 months after transplantation. Knocking down of SRSF11, which was high expressed in CD371+GMPs (granulocyte-monocyte progenitors), upregulated by ALA and binding to ELK1-RNA splicing site, was performed to analyze the function in erythroid differentiation derived from CD34+ CD123mid CD38+ CD371- HPCs (hematopoietic progenitor cells). RNA sequencing of L-ELK1 and S-ELK1 overexpressed CD34+ CD123mid CD38+ CD371- HPCs were performed to assay the signals changed by ELK1. RESULTS: Here, we presented new evidence that ALA promoted erythroid differentiation by targeting the transcription factor ELK1 in CD34+ CD371- hematopoietic stem progenitor cells (HSPCs). Overexpression of either the long isoform (L-ELK1) or the short isoform (S-ELK1) of ELK1 inhibited erythroid-cell differentiation, but knockdown of ELK1 did not affect erythroid-cell differentiation. RNAseq analysis of CD34+ CD123mid CD38+ CD371- HPCs showed that L-ELK1 upregulated the expression of genes related to neutrophil activity, phosphorylation, and hypoxia signals, while S-ELK1 mainly regulated hypoxia-related signals. However, most of the genes that were upregulated by L-ELK1 were only moderately upregulated by S-ELK1, which might be due to a lack of serum response factor interaction and regulation domains in S-ELK1 compared to L-ELK1. In summary, the differentiation of neutrophils and erythrocytes might need to rely on the dose of L-ELK1 and S-ELK1 to achieve precise regulation via RNA splicing signals at early lineage commitment. CONCLUSIONS: ALA and ELK1 are found to regulate both human granulopoiesis and erythropoiesis via RNA spliceosome, and ALA-ELK1 signal might be the target of human leukemia therapy.

Erythrocytes membrane fluidity changes induced by adenylyl cyclase cascade activation: study using fluorescence recovery after photobleaching.

In this study, fluorescence recovery after photobleaching (FRAP) experiments were performed on RBC labeled by lipophilic fluorescent dye CM-DiI to evaluate the role of adenylyl cyclase cascade activation in changes of lateral diffusion of erythrocytes membrane lipids. Stimulation of adrenergic receptors with epinephrine (adrenaline) or metaproterenol led to the significant acceleration of the FRAP recovery, thus indicating an elevated membrane fluidity. The effect of the stimulation of protein kinase A with membrane-permeable analog of cAMP followed the same trend but was less significant. The observed effects are assumed to be driven by increased mobility of phospholipids resulting from the weakened interaction between the intermembrane proteins and RBC cytoskeleton due to activation of adenylyl cyclase signaling cascade.

Hemocompatibility studies in nanotoxicology: Hemolysis or eryptosis? (A review).

Hemocompatibility evaluation is an important step in nanotoxicological studies. It is generally accepted that nanomaterials promote lysis of erythrocytes, blood clotting, alter phagocytosis, and upregulate pro-inflammatory cytokines. However, there are no standardized guidelines for testing nanomaterials hemocompatibility despite the fact that nanomaterials enter the bloodstream and interact with blood cells. In this review, the current knowledge on the ability of nanomaterials to induce distinct cell death modalities of erythrocytes is highlighted primarily focusing on hemolysis and eryptosis. This review aims to summarize the molecular mechanisms underlying erythrotoxicity of nanomaterials and critically compare the sensitivity and efficiency of hemolysis or eryptosis assays for nanomaterials blood compatibility testing. The list of eryptosis-inducing nanomaterials is growing, but it is still difficult to generalize how physico-chemical properties of nanoparticles affect eryptosis degree and molecular mechanisms involved. Thus, another aim of this review is to raise the awareness of eryptosis as a nanotoxicological tool to encourage the corresponding studies. It is worthwhile to consider adding eryptosis to in vitro nanomaterials hemocompatibility testing protocols and guidelines.

Fatty acids characterisation by GC-MS, antiglycation effect at multiple stages and protection of erythrocytes cells from oxidative damage induced by glycation of albumin of Opuntia ficus-indica (L.) Mill seed oil cultivated in Eastern Morocco: Experimental and computational approaches.

ETHNOPHARMACOLOGICAL RELEVANCE: Opuntia ficus-indica (L.) Mill is frequently observed in the Moroccan traditional medicinal system, where these approaches are employed to mitigate the onset of diabetes and the subsequent complications it may entail. AIM OF THE STUDY: The aim of this research was to examine the effectiveness of Opuntia ficus-indica seed oil in preventing diabetic complications. Specifically, the study assessed its ability to counteract glycation at various stages, protected red blood cells from the harmful effects of glycated albumin, and inhibited pancreatic lipase digestive enzymes to understand its potential antihyperglycemic properties. Additionally, the study aimed to identify the chemical components responsible for these effects, evaluate antioxidant and anti-inflammatory properties, and conduct computational investigations such as molecular docking. MATERIALS AND METHODS: The assessement of Opuntia ficus-indica seed oil antiglycation properties involved co-incubating the extract oil with a bovine serum albumin-glucose glycation model. The study investigated various stages of glycation, incorporating fructosamine (inceptive stage), protein carbonyls (intermediate stage), and AGEs (late stage). Additionally, measurement of ß-amyloid aggregation of albumin was performed using Congo red, which is specific to amyloid structures. Additionally, the evaluation of oil's safeguarding effect on erythrocytes against toxicity induced by glycated albumin included the measurement of erythrocyte hemolysis, lipid peroxidation, reduced glutathione. The fatty acid of Opuntia ficus-indica seed oil were analyzed using Gas Chromatography-Mass Spectrometry (GC-MS). The in vitro evaluation of antihyperglycemic activity involved the use of pancreatic lipase enzyme, while the assessement of antioxidant capability was carried out through the utilization of the ABTS and FRAP methods. The in vitro assessement of the denaturation of albumin activity was also conducted. In conjunction with the experimental outcomes, computational investigations were undertaken, specifically employing ADMET (absorption, distribution, metabolism, excretion, and toxicity) analysis. Furthermore, molecular docking was utilized to predict antioxidant and antiglycation mechanisms based on protein targets. RESULTS: In vitro glycation assays, Opuntia ficus-indica seed oil displayed targeted inhibitory effects at multiple distinct stages. Within erythrocytes, in addition to mitigating hemolysis and lipid peroxidation induced by glycated albumin. GC-MS investigation revealed a richness of fatty acids and the most abundant compounds are Linoleic acid (36.59%), Palmitic acid (20.84%) and Oleic acid (19.33%) respectively. The findings of antioxidant ability showed a remarkable activity on FRAP and ABTS radicals. This oil showed a pronounced inhibitory impact (p < 0.001) on pancreatic lipase enzyme. It also exerted a notibale inhibition of albumin denaturation, in vitro. CONCLUSION: The identified results were supported by the abundant compounds of fatty acids unveiled through GC-MS analysis, along with the computational investigation and molecular docking.

Intelligent berberine-loaded erythrocytes attenuated inflammatory cytokine productions in macrophages.

Erythrocytes are impressive tools for drug delivery, especially to macrophages. Therefore, berberine was loaded into erythrocytes using both hypotonic pre-swelling and endocytosis methods to target macrophages. Physicochemical and kinetic parameters of the resulting carrier cells, such as drug loading/release kinetics, osmotic fragility, and hematological indices, were determined. Drug loading was optimized for the study using Taguchi experimental design and lab experiments. Loaded erythrocytes were targeted to macrophages using ZnCl2 and bis-sulfosuccinimidyl-suberate, and targeting was evaluated using flow cytometry and Wright-Giemsa staining. Differentiated macrophages were stimulated with lipopolysaccharide, and the inflammatory profiles of macrophages were evaluated using ELISA, western blotting, and real-time PCR. Findings indicated that the endocytosis method is preferred due to its low impact on the erythrocyte's structural integrity. Maximum loading achieved (1386.68 ± 22.43 µg/ml) at 1500 µg/ml berberine treatment at 37 °C for 2 h. Berberine successfully inhibited NF-κB translation in macrophages, and inflammatory response markers such as IL-1ß, IL-8, IL-23, and TNF-α were decreased by approximately ninefold, sixfold, twofold, eightfold, and twofold, respectively, compared to the LPS-treated macrophages. It was concluded that berberine-loaded erythrocytes can effectively target macrophages and modulate the inflammatory response.

Proportions of trans fatty acids in erythrocyte of Canadian adults before the prohibition of partially hydrogenated oils in foods: results from the Canadian Health Measures Survey 2012-2015.

BACKGROUND: The partially hydrogenated oil (PHO) prohibition came into effect in Canada in September 2018 to reduce the intakes of total trans fatty acids (t-TFAs) and industrially produced TFAs (i-TFAs). OBJECTIVES: We aimed to estimate the red blood cell (RBC) proportions of t-TFA (primary objective) and total 18:1 TFA (secondary objective) of adults in Canada before the PHO prohibition and to identify the population subgroups at risk of higher TFA intakes. METHODS: We pooled data from 4025 adult participants of the cross-sectional Canadian Health Measures Survey cycles 3 and 4 (2012-2015). We estimated mean proportions, relative to total fatty acids (FAs), of RBC t-TFA and 18:1 TFA and their associations with sociodemographic, health, and lifestyle characteristics using multiple linear regression models. RESULTS: The nonadjusted mean RBC proportions of t-TFA and total 18:1 TFA were 0.59% (95% CI: 0.54, 0.63) and 0.27% (95% CI: 0.25, 0.29), respectively. In the adjusted models, the same participant characteristics were associated with t-TFA and 18:1 TFA but differences were generally smaller for 18:1 TFA than for t-TFA. Race, BMI, and alcohol intake were independently associated with RBC t-TFA and 18:1 TFA. Asian and Black participants had lower RBC t-TFA (-0.05% and -0.10% of total FA, respectively) than White participants. Obesity and high risk alcohol drinking were associated with slightly lower (≤0.06%) t-TFA proportions than lower adiposity and alcohol intake concentrations, respectively. CONCLUSIONS: Pre-PHO prohibition in food in Canada, t-TFA proportions were relatively low compared with a proposed threshold of 1% of total RBC FAs, over which cardiovascular disease risk may be higher. Previous voluntary initiatives to reduce i-TFA in the food supply may explain these relatively low RBC t-TFA concentrations. Some population subgroups had higher baseline RBC TFA than other subgroups, but the physiological implications of these small differences, at relatively low baseline RBC TFA proportions, remain to be determined.

Effect of a single immersion in cold water below 4 °C on haemorheological properties of blood in healthy men.

Cold water immersion (CWI) involves rapid cooling of the body, which, in healthy individuals, triggers a defence response to an extreme stimulus, to which the body reacts with stress. The aim of the study was to determine the effect of CWI on hemorheological blood indicators. The study group consisted of 13 young males. Blood samples were collected before and after CWI. The assessed parameters included the complete blood count, fibrinogen, hs-C-reactive protein (CRP), proteinogram, and blood rheology factors, such as erythrocyte elongation index (EI), half-time of total aggregation, and aggregation index. Additionally, the effect of reduced temperature on primary human vascular endothelium was investigated in vitro. CWI resulted in the decrease of body temperature to 31.55 ± 2.87 °C. After CWI, neutrophil count and mean corpuscular volume (MCV) were significantly increased in the study group, while lymphocyte count was significantly decreased. Significantly higher levels of total blood protein and albumin concentration were detected after the immersion. Among hemorheological characteristics, erythrocyte EIs at shear stress values ranging from 2.19 to 60.30 Pa were significantly lower after CWI. No significant changes in other rheological, morphological or biochemical parameters were observed. In vitro, human umbilical vein endothelial cells responded to 3 h of temperature decrease to 25 °C with unchanged viability, but increased recruitment of THP-1 monocytic cells and changes in cell morphology were observed. This was the first study to evaluate the effect of single CWI on rheological properties of blood in healthy young men. The results indicate that a single CWI may increase blood protein concentrations and worsen erythrocyte deformability parameters.

Hematin- and Hemin-Induced Spherization and Hemolysis of Human Erythrocytes Are Independent of Extracellular Calcium Concentration.

Pathologies such as malaria, hemorrhagic stroke, sickle cell disease, and thalassemia are characterized by the release of hemoglobin degradation products from damaged RBCs. Hematin (liganded with OH-) and hemin (liganded with Cl-)-are the oxidized forms of heme with toxic properties due to their hydrophobicity and the presence of redox-active Fe3. In the present study, using the original LaSca-TM laser particle analyzer, flow cytometry, and confocal microscopy, we showed that both hematin and hemin induce dose-dependent RBC spherization and hemolysis with ghost formation. Hematin and hemin at nanomolar concentrations increased [Ca2+]i in RBC; however, spherization and hemolysis occurred in the presence and absence of calcium, indicating that both processes are independent of [Ca2+]i. Both compounds triggered acute phosphatidylserine exposure on the membrane surface, reversible after 60 min of incubation. A comparison of hematin and hemin effects on RBCs revealed that hematin is a more reactive toxic metabolite than hemin towards human RBCs. The toxic effects of heme derivatives were reduced and even reversed in the presence of albumin, indicating the presence in RBCs of the own recovery system against the toxic effects of heme derivatives.

Hypoxia alters the upper thermal limits and blood physiology in zebrafish, Danio rerio.

Hypoxic aquatic environments occur more frequently as a result of climate change, thereby exerting challenges on the physiological and metabolic functions of aquatic animals. In this study, a model fish, zebrafish (Danio rerio) was used to observe the climate-induced hypoxic effect on the upper thermal limit (critical thermal maximum; CTmax), hemoglobin, and blood glucose levels, and abnormalities of erythrocytes at cellular and nuclear level. The value of CTmax decreased significantly under hypoxia (39.10 ± 0.96 °C) compared to normoxia (43.70 ± 0.91 °C). At CTmax, hemoglobin levels were much lower (9.33 ± 0.60 g/dL) and blood glucose levels were significantly higher (194.20 ± 11.33 mg/L) under hypoxia than they were under normoxia and at the beginning of the experiment. Increased frequencies of abnormalities in the erythrocytes at both cellular (fusion, twin, elongated, spindle and tear drop shaped) and nuclear (micronucleus, karyopyknosis, binuclei, nuclear degeneration and notched nuclei) levels were also found under hypoxia compared to normoxia. These results suggest that hypoxic conditions significantly alter the temperature tolerance and subsequent physiology in zebrafish. Our findings will aid in the development of effective management techniques for aquatic environments with minimum oxygen availability.

Environmental cadmium exposure perturbs systemic iron homeostasis via hemolysis and inflammation, leading to hepatic ferroptosis in common carp (Cyprinus carpio L.).

Cadmium (Cd) pollution is considered a pressing challenge to eco-environment and public health worldwide. Although it has been well-documented that Cd exhibits various adverse effects on aquatic animals, it is still largely unknown whether and how Cd at environmentally relevant concentrations affects iron metabolism. Here, we studied the effects of environmental Cd exposure (5 and 50 µg/L) on iron homeostasis and possible mechanisms in common carp. The data revealed that Cd elevated serum iron, transferrin saturation and iron deposition in livers and spleens, leading to the disruption of systemic iron homeostasis. Mechanistic investigations substantiated that Cd drove hemolysis by compromising the osmotic fragility and inducing defective morphology of erythrocytes. Cd concurrently exacerbated hepatic inflammatory responses, resulting in the activation of IL6-Stat3 signaling and subsequent hepcidin transcription. Notably, Cd elicited ferroptosis through increased iron burden and oxidative stress in livers. Taken together, our findings provide evidence and mechanistic insight that environmental Cd exposure could undermine iron homeostasis via erythrotoxicity and hepatotoxicity. Further investigation and ecological risk assessment of Cd and other pollutants on metabolism-related effects is warranted, especially under the realistic exposure scenarios.

Blackcurrant press cake by-product: Increased chemical bioaccessibility and reduced antioxidant protection after in vitro simulation of gastrointestinal digestion.

This study describes the bioaccessibility in terms of total phenolic content (TPC) and antioxidant capacity before and after in vitro digestion from blackcurrant press cake extracts (BPC) and the bioactivity in cell culture, human erythrocytes as well as the in silico analysis. Chemical analysis of BPC presented an increase in TPC (270%) and anthocyanins (136%) after in vitro digestion, resulting in an improvement of antioxidant activity (DPPH 112%; FRAP: 153%). This behavior may be related to the highest activity of cyanidin-3-rutinoside, as confirmed by in silico analysis. The digested BPC did not exert cytotoxicity in cells and showed less antioxidant activity against the oxidative damage induced in endothelial cells and human erythrocytes compared to the non-digested extract. The results raise a question about the reliability we should place on results obtained only from crude samples, especially those that will be used to produce foods or nutraceuticals.

Differences in serum and plasma levels of microRNAs and their time-course changes after blood collection.

Background: Serum and plasma are used for measurements of microRNAs (miRNAs) as biomarkers of various diseases. However, no consistent findings have been obtained regarding differences in serum and plasma levels of miRNAs. The purpose of this study was to clarify differences in serum and plasma levels of total miRNAs and their time-course changes after blood collection. Methods: Venous blood was collected from healthy men, and samples were prepared at the time points of 0, 15, 30, 60 and 180 min after blood collection for plasma and after clot formation for serum. Levels of total miRNAs were analyzed by the hybridization method using the 3D-Gene miRNA Oligo chip. Results: About one third of 2632 miRNAs tested showed levels high enough for comparison of serum and plasma levels and for investigation of their time-course changes. Levels of 299 miRNAs at time 0 were significantly different in serum and plasma. Levels of representative platelet-derived miRNAs including miR-185-5p, -22-3p and -320b were significantly higher in plasma than in serum, while levels of representative erythrocyte-derived miRNAs including miR-451a, -486-5p and -92a-3p were not significantly different in serum and plasma. Plasma levels of 173 miRNAs and 6 miRNAs showed significant decreasing and increasing tendencies, respectively, while there were no miRNAs in serum that showed significant time-course changes. Conclusion: The results suggest that careful attention should be paid when comparing serum and plasma levels of miRNAs and that plasma samples should be prepared early after blood collection.

Indicaxanthin prevents eryptosis induced by cigarette smoke extract by interfering with active Fas-mediated signaling.

A physiological mechanism of programmed cell death called eryptosis occurs in aged or damaged red blood cells (RBCs). Dysregulated eryptosis contributes to abnormal microcirculation and prothrombotic risk. Cigarette smoke extract (CSE) induces a p38 MAPK-initiated, Fas-mediated eryptosis, activating the death-inducing signaling complex (DISC). Indicaxanthin (Ind) from cactus pear fruits, is a bioavailable dietary phytochemical in humans and it is able to incorporate into RBCs enhancing their defense against numerous stimuli. This in vitro work shows that Ind, at concentrations that mimic plasma concentrations after a fruit meal, protects erythrocytes from CSE-induced eryptosis. CSE from commercial cigarettes was prepared in aqueous solution using an impinger air sampler and nicotine content was determined. RBCs were treated with CSE for 3 h in the absence or presence of increasing concentrations of Ind (from 1 to 5 µM). Cytofluorimetric measurements indicated that Ind reduced CSE-induced phosphatidylserine externalization and ceramide formation in a concentration-dependent manner. Confocal microscopy visualization and coimmunoprecipitation experiments revealed that Ind prevented both CSE-triggered Fas aggregation and FasL/FADD/caspase 8 recruitment in the membrane, indicating inhibition of DISC assembly. Ind inhibited the phosphorylation of p38 MAPK, caspase-8/caspase-3 cleavage, and caspase-3 activity induced by CSE. Finally, Ind reduced CSE-induced ATP depletion and restored aminophospholipid translocase activity impaired by CSE treatment. In conclusion, Ind concentrations comparable to nutritionally relevant plasma concentrations, can prevent Fas-mediated RBC death signaling induced by CSE, which suggests that dietary intake of cactus pear fruits may limit the deleterious effects of cigarette smoking.

Supplementing Glucose Intake Reverses the Inflammation Induced by a High-Fat Diet by Increasing the Expression of Siglec-E Ligands on Erythrocytes.

Siglec-9/E is a cell surface receptor expressed on immune cells and can be activated by sialoglycan ligands to play an immunosuppressive role. Our previous study showed that increasing the expression of Siglec-9 (the human paralog of mouse Siglec-E) ligands maintains functionally quiescent immune cells in the bloodstream, but the biological effects of Siglec-9 ligand alteration on atherogenesis were not further explored. In the present study, we demonstrated that the atherosclerosis risk factor ox-LDL or a high-fat diet could decrease the expression of Siglec-9/E ligands on erythrocytes. Increased expression of Siglec-E ligands on erythrocytes caused by dietary supplementation with glucose (20% glucose) had anti-inflammatory effects, and the mechanism was associated with glucose intake. In high-fat diet-fed apoE-/- mice, glucose supplementation decreased the area of atherosclerotic lesions and peripheral inflammation. These data suggested that increased systemic inflammation is attenuated by increasing the expression of Siglec-9/E ligands on erythrocytes. Therefore, Siglec-9/E ligands might be valuable targets for atherosclerosis therapy.

Attenuation of Oxidative Stress in Erythrocytes Stored with Vitamin C and l-Carnitine in Additive Solution-7.

Background: Blood transfusion has advanced toward component therapy for specific requirements during trauma and surgery. Oxidative stress is induced in erythrocytes during storage. Hence, antioxidants as additives can be employed to counteract oxidative stress and enhance antioxidant defenses. Therefore, this study investigates the combinatorial effects of vitamin C and l-carnitine on erythrocytes during storage. Methodology: Erythrocyte samples were categorized into control and experimental groups-vitamin C (10 mM) and l-carnitine (10 mM) and stored under blood bank conditions (at 4°C) for 35 days. Hemoglobin (Hb), antioxidant enzymes (superoxide dismutase [SOD], catalase [CAT] and glutathione peroxidase [GPX]), lipid peroxidation products (conjugate dienes and thiobarbituric acid reactive substances [TBARSs]), protein oxidation products, metabolic markers (glucose, lactate dehydrogenase), glutathione (GSH), superoxides, and hemolysis were assessed at weekly intervals. Results: SOD activity increased on day 7 in the controls, whereas it increased on days 7 and 14 in the experimental groups. CAT activity increased on day 35 in both the groups. GPX activity increased on day 7 in the controls. Hb levels decreased on days 14 and 35 in the controls and on day 35 in the experimental groups. Hemolysis increased from day 7 onward in both the groups. Protein oxidation products were maintained throughout the storage. GSH levels increased on day 21 in the controls and on days 14 and 21 in the experimental groups. Superoxides and conjugate dienes decreased from day 14 in both the groups. TBARSs decreased on day 7 in the experimental groups. Conclusion: Vitamin C and l-carnitine have synergistically enhanced the efficacy of stored erythrocytes in terms of Hb, antioxidant enzymes, and lipid peroxidation.

Investigation of the cytotoxicity, genotoxicity and antioxidant prospects of JM-20 on human blood cells: A multi-target compound with potential therapeutic applications.

JM-20 is a 1,5-benzodiazepine compound fused to a dihydropyridine fraction with different pharmacological properties. However, its potential toxic effects on blood cells have not yet been reported. Thus, the present study aimed to investigate, for the first time, the possible cytotoxicity of JM-20 through cell viability, cell cycle, morphology changes, reactive species (RS) to DCFH-DA, and lipid peroxidation in human leukocytes, its hemolytic effect on human erythrocytes, and its potential DNA genotoxicity using plasmid DNA in vitro. Furthermore, the compound's ability to reduce the DPPH radical was also measured. Human blood was obtained from healthy volunteers (30 ± 10 years old), and the leukocytes or erythrocytes were immediately isolated and treated with different concentrations of JM-20. A cytoprotective effect was exhibited by 10 µM JM-20 against 1 mM tert-butyl hydroperoxide (t-but-OOH) in the leukocytes. However, the highest tested concentrations of the compound (20 and 50 µM) changed the morphology and caused a significant decrease in the cell viability of leukocytes (p < 0.05, in comparison with Control). All tested concentrations of JM-20 also resulted in a significant increase in intracellular RS as measured by DCFH-DA in these cells (p < 0.05, in comparison with Control). On the other hand, the results point out a potent antioxidant effect of JM-20, which was similar to the classical antioxidant α-tocopherol. The IC50 value of JM-20 against the lipid peroxidation induced by (FeII) was 1.051 µM ± 0.21, while the IC50 value of α-tocopherol in this parameter was 1.065 µM ± 0.34. Additionally, 50 and 100 µM JM-20 reduced the DPPH radical in a statistically similar way to the 100 µM α-tocopherol (p < 0.05, in comparison with the control). No significant hemolysis in erythrocytes, no cell cycle changes in leukocytes, and no genotoxic effects in plasmid DNA were induced by JM-20 at any tested concentration. The in silico pharmacokinetic and toxicological properties of JM-20, derivatives, and nifedipine were also studied. Here, our findings demonstrate that JM-20 and its putative metabolites exhibit similar characteristics to nifedipine, and the in vitro and in silico data support the low toxicity of JM-20 to mammals.

Immune Mediators Important for a Protective Secondary Response to Babesia microti.

Babesia microti (B. microti) is a tick-transmitted protozoan parasite that invades red blood cells. It is the primary cause of human babesiosis in the US. The severity of babesiosis caused by B. microti infection can range from asymptomatic to fatal. Risk factors for severe disease include general immune suppression, advanced age (>50) and lack of a spleen. However, severe disease can occur in the absence of any known risk factors. The degree to which tick-transmitted B. microti infection confers protection from subsequent exposure is largely unexplored. This is an important question as both the prevalence and geographic range of tick-transmitted B. microti infection continues to increase and individuals in endemic regions may have multiple exposures over their lifetime. In the current study we used a mouse model to evaluate the degree to which primary infection with B. microti protected against secondary challenge with the same parasite strain. We show that CD4 T cells, and to a lesser extent B cells, contribute to protection. However, mice exhibited significant protection from secondary parasite challenge even in the absence of either CD4 T cells or B cells. The protection mediated by CD4 T cells did not depend on their production of IFN-γ as mice with a targeted gene deletion for the IFN-γ receptor remained fully protected against secondary challenge. Other factors including inducible nitric oxide synthase (iNOS) and the adaptor protein MyD88, important for toll-like receptors, IL-18 and IL-1 signaling, were not important for protection against primary or secondary challenge with B. microti. Thus, our study shows that resolution of primary infection with B. microti results in robust protection against secondary challenge with parasites, at least in the short term. Further studies are needed to evaluate the length of protection and the degree to which protection is impacted by parasite heterogeneity. Although we show an important role for CD4 T cells in protection against secondary challenge, our results suggest that no single aspect of the immune system is solely responsible for adequate protection against secondary challenge with B. microti.