Marine Polysaccharides Carrageenans Enhance Eryptosis and Alter Lipid Order of Cell Membranes in Erythrocytes.

Aim In the current study, hemocompatibility of three major commercially available types of carrageenans (ι, κ and λ) was investigated focusing on eryptosis. MATERIALS AND METHODS: Carrageenans of ι-, κ- and λ-types were incubated with washed erythrocytes (hematocrit 0.4%) at 0-1-5-10 g/L for either 24 h or 48 h. Incubation was followed by flow cytometry-based quantitative analysis of eryptosis parameters, including cell volume, cell membrane scrambling and reactive oxygen species (ROS) production, lipid peroxidation markers and confocal microscopy-based evaluation of intracellular Ca2+ levels, assessment of lipid order in cell membranes and the glutathione antioxidant system. Confocal microscopy was used to assess carrageenan cellular internalization using rhodamine B isothiocyanate-conjugated carrageenans. RESULTS: All three types of carrageenans were found to trigger eryptosis. Pro-eryptotic properties were type-dependent and λ-carrageenan had the strongest impact inducing phosphatidylserine membrane asymmetry, changes in cell volume, Ca2+ signaling and oxidative stress characterized by ROS overproduction, activation of lipid peroxidation and severe glutathione system depletion. Eryptosis induction by carrageenans does not require their uptake by erythrocytes. Changes in physicochemical properties of cell membrane were also type-dependent. No carrageenan-induced generation of superoxide and hydroxyl radicals was observed in cell-free milieu. CONCLUSIONS: Our findings suggest that ι-, κ- and λ-types trigger eryptosis in a type-dependent manner and indicate that carrageenans can be further investigated as potential eryptosis-regulating therapeutic agents.

The effect of human PBMCs immobilization on their Аß42 aggregates-dependent proinflammatory state on a cellular model of Alzheimer's disease.

The leading pathological mechanisms of Alzheimer's disease are amyloidosis and inflammation. The presented work was aimed to study the effect of human peripheral blood mononuclear cells (hPBMcs) cells-matrix adhesion on their pro-inflammatory state in vitro. Although direct interaction of Аß42 to PBMC is not a cellular model of Alzheimer's disease, PBMCs may serve as test cells to detect Аß42-dependent molecular effects in monitoring disease progression. Peripheral blood mononuclear cells (PBMCs) are used to assess changes in cytokines released in response to diseases or Alzheimer's disease-specific cytotoxic molecules such as Aß42. The effect of recombinant amyloid ß-peptide rАß42 on the concentration of endogenous amyloid ß-peptide Aß40 and pro-inflammatory cytokines TNFα and IL-1ß in human peripheral blood mononuclear cells that were cultured in suspension and immobilized in alginate microcarriers for 24 h were investigated. The localization and accumulation of Aß40 and rAß42 peptides in cells, as well as quantitative determination of the concentration of Aß40 peptide, TNFα and IL-1ß cytokines, was performed by intravital fluorescence imaging. The results were qualitatively similar for both cell models. It was determined that the content of TNFα and Aß40 in the absence of rAß42 in the incubation medium did not change for 24 h after incubation, and the content of IL-1ß was lower compared to the cells that were not incubated. Incubation of cells in vitro with exogenous rAß42 led to an increase in the intracellular content of TNFα and Aß40, and no accumulation of IL-1ß in cells was observed. The accumulation of Aß40 in the cytoplasm was accompanied by the aggregation of rAß42 on the outer surface of the cell plasma membrane. It was shown that the basic levels of indicators and the intensity of the response of immobilized cells to an exogenous stimulus were significantly greater than those of cells in suspension. To explore whether non-neuronal cells effects in alginate microcarriers were cell-matrix adhesion mediated, we tested the effect of blocking ß1 integrins on proamyloidogenic and proinflammation cellular state. Immobilization within alginate hydrogels after incubation with the ß1 integrins blocking antibodies showed a remarkable inhibition of TNFα and Aß40 accumulation in rAß42-treated cells. It can be concluded that activation of signal transduction and synthesizing activity of a portion of mononuclear cells of human peripheral blood is possible (can significantly increase) in the presence of cell-matrix adhesion.

Rare-earth orthovanadate nanoparticles trigger Ca2+-dependent eryptosis.

Introduction. Rare-earth orthovanadate nanoparticles (ReVO4:Eu3+, Re = Gd, Y or La) are promising agents for photodynamic therapy of cancer due to their modifiable redox properties. However, their toxicity limits their application.Objective. The aim of this research was to elucidate pro-eryptotic effects of GdVO4:Eu3+and LaVO4:Eu3+nanoparticles with identification of underlying mechanisms of eryptosis induction and to determine their pharmacological potential in eryptosis-related diseases.Methods. Blood samples (n= 9) were incubated for 24 h with 0-10-20-40-80 mg l-1GdVO4:Eu3+or LaVO4:Eu3+nanoparticles, washed and used to prepare erythrocyte suspensions to analyze the cell membrane scrambling (annexin-V-FITC staining), cell shrinkage (forward scatter signaling), reactive oxygen species (ROS) generation through 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) staining and intracellular Ca2+levels via FLUO4 AM staining by flow cytometry. Internalization of europium-enabled luminescent GdVO4:Eu3+and LaVO4:Eu3+nanoparticles was assessed by confocal laser scanning microscopy.Results.Both nanoparticles triggered eryptosis at concentrations of 80 mg l-1. ROS-mediated mechanisms were not involved in rare-earth orthovanadate nanoparticles-induced eryptosis. Elevated cytosolic Ca2+concentrations were revealed even at subtoxic concentrations of nanoparticles. LaVO4:Eu3+nanoparticles increased intracellular calcium levels in a more pronounced way compared with GdVO4:Eu3+nanoparticles. Our data disclose that the small-sized (15 nm) GdVO4:Eu3+nanoparticles were internalized after a 24 h incubation, while the large-sized (∼30 nm) LaVO4:Eu3+nanoparticles were localized preferentially around erythrocytes.Conclusions.Both internalized GdVO4:Eu3+and non-internalized LaVO4:Eu3+nanoparticles (80 mg l-1) promote eryptosis of erythrocytes after a 24 h exposurein vitrovia Ca2+signaling without involvement of oxidative stress. Eryptosis is a promising model for assessing nanotoxicity.

Food additive E407a stimulates eryptosis in a dose-dependent manner.

BACKGROUND: Concerns about the biosafety of the common food additive E407a have been raised. It has been demonstrated to induce intestinal inflammation, accompanied by activation of apoptosis, upon oral exposure. Thus, it is of interest to investigate how E407a affects eryptosis, a suicidal cell death mode of red blood cells. OBJECTIVE: To evaluate the effects of semi-refined carrageenan (E407a) on eryptosis. METHODS: Flow cytometry was employed to assess eryptosis in blood exposed to various concentrations of E407a (0 g/L, 1 g/L, 5 g/L, and 10 g/L) during incubation for 24 h by analyzing phosphatidylserine externalization in erythrocytes using annexin V staining and via evaluating reactive oxygen species (ROS) generation using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). In addition, the eryptosis indices mentioned above were determined in rats orally administered E407a at a dose of 140 mg/kg weight for 2 weeks. Confocal scanning laser microscopy was performed to visualize cell membrane scrambling. RESULTS: Oral intake of E407a for 2 weeks by rats was not associated with membrane scrambling in erythrocytes. However, ROS overproduction was observed. Meanwhile, incubation of blood with various concentrations of semi-refined carrageenan resulted in a dose-dependent promotion of eryptosis, evidenced by the enhanced percentage of annexin V-positive erythrocytes and higher mean fluorescence intensity (MFI) values of annexin V-FITC in all erythrocytes. The highest concentration of E407a promotes a statistically significant increase in ROS generation in erythrocytes, suggesting the role of ROS-mediated induction of eryptosis in this case. CONCLUSION: Incubation of blood with the food additive E407a leads to the activation of eryptosis in a dose-dependent manner. ROS-mediated mechanisms are partially responsible for E407a-induced eryptosis.

Semi-refined carrageenan promotes generation of reactive oxygen species in leukocytes of rats upon oral exposure but not in vitro.

AIM: To assess the ability of the common food additive E407a (semi-refined carrageenan) to enter leukocytes in vitro and generate reactive oxygen species (ROS) in leukocytes as a whole and granulocytes in particular, both during incubation and in experimental animals. METHODS: ROS production was assessed in leukocytes incubated with E407a for 2 h at the final concentrations of 5 and 10 g/L using the dye 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), as well as in cells isolated from rats orally exposed to E407a (140 mg/kg of weight) during 2 weeks (n = 8) and control rats (n = 8), by flow cytometry. Carrageenan uptake by leukocytes was estimated by confocal microscopy using incubation of rhodamine B isothiocyanate-labelled carrageenan with leukocyte suspensions. RESULTS: Uptake of carrageenan by viable neutrophils, monocytes, and lymphocytes was confirmed. Oral administration of the food additive E407a was associated with excessive ROS formation by viable leukocytes (CD45+, 7­aminoactinomycin D- cells) and especially in granulocytes. Unexpectedly, a direct impact of semi-refined carrageenan during incubation for 2 h did not affect ROS production in leukocytes, evidenced by statistically insignificant differences in mean fluorescence intensity values of 2',7'-dichlorofluorescein, which is a ROS-sensitive product of intracellular H2DCFDA conversion. Oral intake of E407a and direct exposure of leukocyte suspensions to it decreased the viability of leukocytes. CONCLUSION: Food-grade carrageenan can enter leukocytes without affecting ROS generation as a result of incubation for 2 h with leukocyte suspensions. On the contrary, oral exposure to E407a is accompanied by ROS overproduction by white blood cells, suggesting an indirect mechanism for the stimulation of ROS synthesis in vivo. E407a promotes cell death of leukocytes both in vivo and in vitro.

DNA-Specific DAPI Staining of the Pyrenoid Matrix During its Fission in Dunaliella salina (Dunal) Teodoresco (Chlorophyta).

The algal pyrenoid is a naked phase-separated liquid compartment inside the chloroplast consisting predominantly of densely packaged Rubisco and most often transversed by a system of lipid membranes. The pyrenoid participates in carbon-concentrating mechanisms of algae. During the cell division, the daughter cells of algae acquire the pyrenoids via their assembly or fission, the mechanisms of which are not fully understood. We suppose that the chloroplast nucleoid scaffolds the new pyrenoid like the cyanobacterial nucleoid positions carboxysomes before the cell division. This work was aimed at visualization and localization of the chloroplast DNA relative to the pyrenoid in synchronously dividing cells of Dunaliella salina with DNA-specific fluorescent DAPI stain through the fluorescent confocal microscope. The intense DNA-specific blue DAPI fluorescence was discovered in the pyrenoids matrix under the starch shell in the presumably pre-mitotic cells, during and following the pyrenoid fission. In the interphase cells, the chloroplast DNA localized both in the pyrenoid core and in several small nucleoids on the outer surface of the starch shell around the pyrenoid. The observations were compared with the literature data on the same and other algal species. The spatial pre-requisite exists in D. salina for the chloroplast nucleoid to scaffold the pyrenoid fission. A potential alternative explanation was declared being the algal pyrenoid as the chloroplast genetic center. The theoretical and practical implications of the findings were discussed.