Hemocompatibility of dextran-graft-polyacrylamide/zinc oxide nanosystems: hemolysis or eryptosis?

Aim. In this study, blood compatibility of ZnO nanoparticles-polymer nanocomplex (D-PAA/ZnONPs(SO42-)) synthesizedin situinto dextran-graft-polyacrylamide (D-PAA) using zinc sulphate as a precursor was tested using hemolysis, osmotic fragility and eryptosis assays.Materials and methods. Dose-dependent ability to induce eryptosis was assessed following 24 h incubation at concentrations of 0-800 mg l-1analyzing hallmarks of eryptosis (cell shrinkage and phosphatidylserine externalization), as well as reactive oxygen species generation. Hemolysis was detected spectrophotometrically based on hemoglobin release following exposure to the D-PAA/ZnONPs(SO42-) nanocomplex. Osmotic fragility test (OFT) involved detection of hemolysis of red blood cells exposed to 0.2% saline solution following incubation with the D-PAA/ZnONPs(SO42-) nanocomplex. Additional incubation of the nanocomplex in the presence or absence of either ascorbic acid or EGTA was used to reveal the implication of oxidative stress- or Ca2+-mediated mechanisms in D-PAA/ZnONPs(SO42-) nanocomplex-induced erythrotoxicity.Results. Hemocompatibility assessment of the D-PAA/ZnONPs(SO42-) nanocomplex revealed that it induced hemolysis and reduced resistance of erythrocytes to osmotic stress at concentrations of above 400 and 200 mg l-1, respectively. Oxidative stress- or Ca2+-mediated mechanisms were not involved in D-PAA/ZnONPs(SO42-) nanocomplex-induced hemolysis. Strikingly, the D-PAA/ZnONPs(SO42-) nanocomplex did not promote cell membrane scrambling, cell shrinkage and oxidative stress in red blood cells following the direct exposure for 24 h. Thus, the D-PAA/ZnONPs(SO42-) nanocomplex did not induce eryptosisin vitro. Eryptosis is generally considered to occur earlier than hemolysis in response to stress in order to prevent hemolytic cell death. Counterintuitively, our data suggest that hemolysis can be triggered by nanomaterials prior to eryptosis indicating that eryptosis and hemolysis assays should be used in combination for testing blood compatibility of nanomaterials.Conclusions. The D-PAA/ZnONPs(SO42-) nanocomplex has a good hemocompatibility profile at low concentrations. Hemocompatibility testing in nanotoxicology should include both eryptosis and hemolysis assays.

THE LEVEL OF REACTIVE OXYGEN SPECIES AS A MARKER OF ASTHMA SEVERITY IN CHILDREN.

OBJECTIVE: The aim of the research was to assess the reactive oxygen species (ROS) levels in granulocytes of patients with asthma. PATIENTS AND METHODS: Materials and methods: The study involved 35 children aged 5 to 17 years. 26 children with persistent asthma, partially controlled course in the period of exacerbation were divided into groups: 1 group - mild asthma (n = 12), group 2 - moderate asthma (n = 7) group 3 - severe asthma (n = 7) and control group included almost healthy children (n = 9). ROS levels in granulocytes were evaluated using BD FACSDiva™. The spirographic complex was used to assess the function of external respiration. RESULTS: Results: The level of ROS in granulocytes of patients with severe asthma was significantly reduced compared with children in the control group and patients with mild and moderate asthma (p1-3 = 0.0003, p2-3 = 0.0017, p c-3 = 0.0150). The concentration of ROS in granulocytes ≤ 285 a.u. was prognostically significant with high specificity and sensitivity with severe asthma. CONCLUSION: Conclusions: The concentration of ROS levels in neutrophils in patients with severe asthma probably reflected the suppression of their products, which suggests the depletion of the reserve capacity of neutrophils. Decreased concentrations of reactive oxygen species in children with asthma can be considered as a possible marker of asthma severity.

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.

Experimental Evaluation of the Impact of Gadolinium Orthovanadate GdVO4:Eu3+ Nanoparticles on the Carrageenan-Induced Intestinal Inflammation.

AIM: To evaluate the effects of orally administered gadolinium orthovanadate GdVO4:Eu3+ nanoparticles (VNPs) on the course of chronic carrageenan-induced intestinal inflammation. METHODS: Samples of small intestinal tissue were collected from four groups of rats (intact, after administration of VNPs, with carrageenaninduced intestinal inflammation, with carrageenan-induced intestinal inflammation orally exposed to VNPs) to assess the intestinal morphology and HSP90α expression. Levels of seromucoid, C-reactive protein, TNF-α, IL-1ß and IL-10 were determined in blood serum. RESULTS: Oral exposure to VNPs was associated with neither elevation of inflammation markers in blood serum nor HSP90α overexpression in the small intestine, i.e. no toxic effects of VNPs were observed. Carrageenan-induced intestinal inflammation was accompanied by higher levels of TNF-α and IL-1ß, as well as HSP90α upregulation in the intestinal mucosa, compared with controls. Administration of VNPs to rats with enteritis did not lead to statistically significant changes in concentrations of circulating pro-inflammatory cytokines with the trend towards their increase. CONCLUSION: No adverse effects were observed in rats orally exposed to VNPs at a dose of 20 µg/kg during two weeks. Using the experimental model of carrageenan-induced enteritis, it was demonstrated that VNPs at the dose used in our study did not affect the course of intestinal inflammation.

Orally administered gadolinium orthovanadate GdVO4:Eu3+ nanoparticles do not affect the hydrophobic region of cell membranes of leukocytes.

AIM: To assess the phospholipid bilayer of white blood cells (WBCs) and the ability of leukocytes to generate reactive oxygen species (ROS) in rats orally exposed to GdVO4:Eu3+ nanoparticle (VNP) solution for 2 weeks by fluorescent probes-ortho-hydroxy derivatives of 2,5-diaryl­1,3­oxazole. METHODS: Steady-state fluorescence spectroscopy, i.e., a study by the environment-sensitive fluorescent probes 2­(2'-OH-phenyl)-5-(4'-phenyl-phenyl)-1,3-oxazole (probe O6O) and 2­(2'-OH-phenyl)-phenanthro[9,10]-1,3-oxazole (probe PH7), and flow cytometry, i.e., analysis of 2',7'-dichlorofluorescein (DCF), a product of a dye 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), fluorescence in CD45+/7-aminoactinomycin D (7-AAD)- cells, were used to evaluate the state of cell membranes and reactive oxygen species (ROS) generation in leukocytes of rats orally exposed to gadolinium orthovanadate nanoparticles(VNPs). RESULTS: No significant changes were detected in the spectra of the fluorescent probes bound to the WBCs from the rats orally exposed to nanoparticles in comparison with the corresponding spectra of the probes bound to the cells from the control group of animals. This indicates that in the case of the rats orally exposed to nanoparticles, no noticeable changes in physicochemical properties (i.e., in the polarity and the proton-donor ability) are observed in the lipid membranes of WBCs in the region where the probes locate. There was no statistically significant difference in the amount of ROShigh viable leukocytes in rats treated with VNPs and control samples. CONCLUSION: Neither changes in the physical and chemical properties of the leukocyte membranes nor in ROS generation by WBCs are detected in the rats orally exposed to VNP solution for 2 weeks.

Fascin overexpression in nasal mucosa in patients with chronic rhinosinusitis with and without nasal polyps / Superexpressão de fascina na mucosa nasal em pacientes com rinossinusite crônica com e sem pólipos nasais

ABSTRACT Objective: The aim of our research is to assess fascin expression in nasal tissues of patients with chronic rhinosinusitis with (CRSwNP) and without (CRSsNP) nasal polyps. Methods: Fascin expression in nasal tissues of 11 CRSwNP patients and 10 CRSsNP patients was immunohistochemically evaluated and compared with control subjects. Results: Fascin was found to be strongly expressed in epithelial cells in polyps in CRSwNP and nasal tissue in CRSsNP. Its strong expression was observed both in lamina propria and nasal epithelial cells in CRSsNP. Fascin overexpression in nasal mucosa in CRSwNP was more pronounced compared with CRSsNP. In addition, proliferating epithelial cells in polyp tissue were weakly immunostained, whereas mature cells expressed much more fascin. Conclusion: CRSwNP and CRSsNP are associated with fascin overexpression, which makes fascin a promising target for therapeutic interventions.

RESUMEN Objetivo: El objetivo de esta investigación fue evaluar la expresión de fascina en los tejidos nasales de pacientes con rinosinusitis crónica con (RSCcPN) y sin pólipos nasales (RSCsPN). Métodos: La expresión de fascina en los tejidos nasales de 11 pacientes con RSCcPN y 10 pacientes con RSCsPN fue analizada por inmunohistoquímica y comparada con los individuos control. Resultados: Fascina fue encontrada por ser fuertemente expresada en células epiteliales de pólipos en la RSCcPN y en tejido nasal en la RSCsPN. Su fuerte expresión fue observada tanto en la lámina propia como en las células epiteliales nasales en la RSCsPN. La sobreexpresión de fascina en la mucosa nasal en la RSCcPN fue más pronunciada en comparación con la RSCsPN. Además, las células epiteliales proliferantes en el tejido del pólipo fueron inmunoteñidas débilmente, mientras las células maduras expresaron mucho más fascina. Conclusión: RSCcPN y RSCsPN están asociadas a la sobreexpresión de fascina, lo que hace la fascina un objetivo prometedor para intervenciones terapéuticas.

RESUMO Objetivo: O objetivo desta pesquisa foi avaliar a expressão de fascina nos tecidos nasais de pacientes com rinossinusite crônica com (RSCcPN) e sem (RSCsPN) pólipos nasais. Métodos: A expressão de fascina nos tecidos nasais de 11 pacientes com RSCcPN e 10 pacientes com RSCsPN foi avaliada imuno-histoquimicamente e comparada com os indivíduos-controle. Resultados: Fascina foi encontrada por ser fortemente expressa em células epiteliais em pólipos na RSCcPN e em tecido nasal na RSCsPN. Sua forte expressão foi observada tanto na lâmina própria quanto nas células epiteliais nasais na RSCsPN. A superexpressão de fascina na mucosa nasal na RSCcPN foi mais pronunciada em comparação com a RSCsPN. Além disso, as células epiteliais em proliferação no tecido do pólipo foram imunocoradas fracamente, enquanto as células maduras expressaram muito mais fascina. Conclusão: RSCcPN e RSCsPN estão associadas à superexpressão de fascina, o que torna a fascina um alvo promissor para intervenções terapêuticas.