Exploring the Link between Hydrodynamic Size and Immunoglobulins of Circulating Immune Complexes in Rheumatoid Arthritis Patients.

The function of immune complexes in rheumatoid arthritis (RA) is related to their composition and size. Using dynamic light scattering (DLS), we investigated the link between the RA circulating immune complex (CIC) particles' size and the CIC immunoglobulin level. In this study, 30 RA patients and 30 healthy individuals were included. IgA, IgG, and IgM were found in all analyzed CICs, but more IgA and IgG were found in RA than in control CICs. In both control and RA CICs, DLS detected 50 particles that differed in size and clustered around two size groups: with a 7.5-164 nm radius and with a 342-1718 nm radius. An increased level of IgA in RA CICs, compared to control ones, was associated with more than 50% of CIC particles. In RA, compared to the control, a higher number of CICs with 28.2 nm, 531 nm, 712 nm, and 1718 nm particles and a lower number of CICs with 78.8 nm particles were detected. This particle distribution pattern did not reflect the changes in the CIC immunoglobulin level. Thus, RA elevated CIC IgA was linked with all these particles (except the 1718 nm particle), the IgM increase was linked with 43.8 nm and 712 nm particles, and the IgG increase was linked with the 712 nm particle only. This study provides the very first data on the association between CIC particles' size, CIC immunoglobulin level, and RA. It opens the possibility that the size of CICs determined by DLS can be used as a criterion in RA diagnosis or monitoring after a large-scale study confirmation.

Dynamic light scattering analysis of immune complexes in sera of rheumatoid arthritis patients.

The size of circulating immune complexes (CICs) in rheumatoid arthritis (RA) could be an emerging criterion in disease diagnosis. This study analyzed size and electrokinetic potential of CICs from RA patients, healthy young adults, and RA patients age-matched controls aiming to establish their unique CIC features. Pooled CIC of 30 RA patients, 30 young adults, and 30 RA group's age-matched controls (middle-aged and oldеr healthy adults), and in vitro IgG aggregates from pooled sera of 300 healthy volunteers were tested using dynamic light scattering (DLS). Size distribution of CIC in healthy young adults exhibited high polydispersity. RA CIC patients and their age-matched control showed distinctly narrower size distributions compared with young adults. In these groups, particles clustered around two well-defined peaks. Particles of peak 1 were 36.1 ± 6.8 nm in RA age-matched control, and 30.8 ± 4.2 nm in RA patients. Particles of peak 2 of the RA age-matched control's CIC was 251.7 ± 41.2 nm, while RA CIC contained larger particles (359.9 ± 50.5 nm). The lower zeta potential of RA CIC, compared to control, indicated a disease-related decrease in colloidal stability. DLS identified RA-specific, but also age-specific distribution of CIC size and opened possibility of becoming a method for CIC size analysis in IC-mediated diseases.

Interactions of ultrashort laser pulses with hemoglobin: Photophysical aspects and potential applications.

Hemoglobin (Hb), a life-sustaining and highly abundant erythrocyte protein, is not readily fluorescent. A few studies have already reported Two-Photon Excited Fluorescence (TPEF) of Hb, however, the mechanisms through which Hb becomes fluorescent upon interaction with ultrashort laser pulses are not completely understood. Here, we characterized photophysically this interaction on Hb thin film and erythrocytes using fluorescence spectroscopy upon single-photon/two-photon absorption, and UV-VIS single-photon absorption spectroscopy. A gradual increase of the fluorescence intensity, ending up with saturation, is observed upon prolonged exposure of Hb thin layer and erythrocytes to ultrashort laser pulses at 730 nm. When compared to protoporphyrin IX (PpIX) and oxidized Hb by H2O2, TPEF spectra from a thin Hb film and erythrocytes showed good mutual agreement, broad peaking at 550 nm, supporting hemoglobin undergoes degradation and that same fluorescent specie(s) originating from the heme moiety are generated. The uniform square shaped patterns of the fluorescent photoproduct exhibited the same level of the fluorescence intensity even after 12 weeks from the formation, indicating high photoproduct stability. We finally demonstrated the full potential of the formed Hb photoproduct with TPEF scanning microscopy towards spatiotemporally controlled micropatterning in HTF and single human erythrocyte labelling and tracking in the whole blood.

Extracellular Hemoglobin: Modulation of Cellular Functions and Pathophysiological Effects.

Hemoglobin is essential for maintaining cellular bioenergetic homeostasis through its ability to bind and transport oxygen to the tissues. Besides its ability to transport oxygen, hemoglobin within erythrocytes plays an important role in cellular signaling and modulation of the inflammatory response either directly by binding gas molecules (NO, CO, and CO2) or indirectly by acting as their source. Once hemoglobin reaches the extracellular environment, it acquires several secondary functions affecting surrounding cells and tissues. By modulating the cell functions, this macromolecule becomes involved in the etiology and pathophysiology of various diseases. The up-to-date results disclose the impact of extracellular hemoglobin on (i) redox status, (ii) inflammatory state of cells, (iii) proliferation and chemotaxis, (iv) mitochondrial dynamic, (v) chemoresistance and (vi) differentiation. This review pays special attention to applied biomedical research and the use of non-vertebrate and vertebrate extracellular hemoglobin as a promising candidate for hemoglobin-based oxygen carriers, as well as cell culture medium additive. Although recent experimental settings have some limitations, they provide additional insight into the modulatory activity of extracellular hemoglobin in various cellular microenvironments, such as stem or tumor cells niches.

The effects of incubation media on the assessment of the shape of human erythrocytes by flow cytometry: a contribution to mathematical data interpretation to enable wider application of the method.

Flow cytometry (FC) analysis of erythrocyte shape and related biomechanical properties, such as osmotic fragility, have not moved from a research tool to regular clinical testing. The main reason is existing evidence that various pre-analytical factors influence the mathematical interpretation of the data obtained. With an aim to contribute to the standardization and broaden the use of FC for human erythrocyte shape assessment, freshly prepared peripheral blood erythrocytes isolated from healthy donors were incubated in iso and hypo-osmotic solutions (pure saline, saline with potassium and calcium, and phosphate buffered saline) and examined by FC using values of forward scatter (FSC) and side scatter (SSC). Kurtosis, skewness, Pearson's second skewness coefficient of dissymmetry (PCD), and spherical index, calculated from FSC distributions, were used for the erythrocyte shape evaluation. In all isotonic media FSC distribution and FSC-based morphology parameters showed huge inter-individual and inter-medium variation. With decreasing osmolality, in all media and samples, the size of the erythrocytes increased, and swelling index and kurtosis decreased. However, changes in skewness and PCD were influenced by the medium used and the sample tested. Compared to FSC, SSC signal in isotonic and its change in hypotonic media showed lower inter-individual variation and was not influenced by the type of medium. We propose a spherical index and kurtosis as FSC-based indicators of erythrocyte shape. As more resistant to the influence of the preanalytical treatment, SSC data appeared to be unfairly neglected for the assessment of erythrocyte shape, in comparison to the usually employed FSC data.

Insight into the Biological Activity of Hennosides-Glucosides Isolated from Lawsonia inermis (henna): Could They Be Regarded as Active Constituents Instead.

Henna is the current name of the dye prepared from the dry leaf powder of Lawsonia inermis (Lythraceae). Several studies have focused on the chemistry and pharmacology of the henna dyeing active compound, lawsone, obtained from the main constituents of leaves, hennosides, during the processing of plant material. However, knowledge regarding the biological activity of hennosides is largely lacking. In this paper, the redox activity of three hennoside isomers is reported. The pro-oxidative activity was confirmed by their ability to induce mild lysis of erythrocytes and to increase the level of methemoglobin at the concentration ≥ 500 µg/mL. The antioxidant activity of hennosides (concentration ≥100 µg/mL) was determined by FRAP and ABTS assays. At concentration of 500 µg/mL, antioxidant activity of hennoside isomers was equivalent to 0.46 ± 0.08, 0.62 ± 0.28 and 0.35 ± 0.03 mM FeSO4 × 7H2O, and 0.15 ± 0.01, 0.30 ± 0.01 and 0.09 ± 0.01 mM Trolox. Hennosides at 100 µg/mL concentration did not influence viability of human breast cancer cell lines MDA231 and MCF-7 and primary human peripheral blood and periodontal ligament-mesenchymal stem cells, but produced a modest increase in concentration of antioxidants in the cell culture supernatants. The evidenced antioxidant and pro-oxidant activities indicate their potential to act as redox balance regulator, which opens up the possibility of using hennosides in commercial phytomedicines.

Structural characteristics of circulating immune complexes in calves with bronchopneumonia: Impact on the quiescent leukocytes.

Calf bronchopneumonia is accompanied by increased level of circulating immune complexes (CIC), and we analysed size, and protein and lipid constituents of these CIC with an attempt to elucidate the connection between the CIC structural properties and their capacity to modulate leukocyte function. CIC of heathy calves (CICH) and calves with naturally occurring bronchopneumonia (CICD) were isolated by PEG precipitation and analysed by electrophoresis and chromatography. The predominant CIC proteins were IgG, albumin, and transferrin. Affinity isolated serum and CIC IgG coprecipitated several proteins, but only 75 and 80 kDa proteins bound CIC IgG, exclusively. 60 and 65 kDa proteins co-precipitated with CICD IgG, unlike CICH IgG. In both CICH and CICD, oleic acid-containing phospholipids predominated. In CICD, the content of oleic and vaccenic acid was higher than in CICH, while myristic, palmitic, stearic, linoleic and arachidonic acid showed lower content. Dynamic light scattering displayed difference in particle size distribution between CICH and CICD; 1280 nm large particles were present only in CICD. The effect of CICH and CICD on mononuclear cells (MNC) and granulocytes was analysed in vitro. CICH and CICD, with slight difference in intensity, stimulate MNC apoptosis, promote cell cycle arrest of unstimulated MNC, and cell cycle progression of PHA stimulated MNC. Both CIC reduced granulocyte apoptosis after 24 h while after 48 h this effect was detected for CICD only. These results indicate that structural differences of CICH and CICD might interfere with the CIC functional capacity, which we consider important for evaluation of CIC immunoregulatory function.

Native bovine hemoglobin reduces differentiation capacity of mesenchymal stromal cells in vitro.

We have tested in vitro effects of hemoglobin from bovine slaughterhouse blood (BHb) on stromal cells of mesodermal origin, with an aim to explore its use as a component of cell culture media. Human peripheral blood mesenchymal stromal cells (PB-MSCs) and three mouse cell lines (ATDC5, MC3T3-E1 and 3T3-L1) were employed to study BHb effects on their growth and migration. The cells multilineage differentiation capacity in the presence of BHb was evaluated after induced differentiation, by histochemical staining and by RT-PCR analysis of the expression of genes specific for chondrogenic, adipogenic and osteogenic lineages. The effects of BHb on the cell proliferation and motility were dependent on both, cell type and BHb concentration (0.1 µM, 1 µM and 10 µM). In the lowest concentration (0.1 µM) BHb showed the least prominent effect on the cell proliferation and migration. In this concentration BHb reduced the differentiation capacity of all tested cells and its effect was dependent of composition of induction medium and the culture period. Obtained data suggest that BHb has the potential to be used as a component of cell culture media through maintaining proliferation and reducing differentiation capacity of mesenchymal stromal cells.

Adipoinductive effect of extracellular matrix involves cytoskeleton changes and SIRT1 activity in adipose tissue stem/stromal cells.

Adipose tissue (AT) homeostasis and expansion are dependent on complex crosstalk between resident adipose stromal/stem cells (ASCs) and AT extracellular matrix (ECM). Although adipose tissue ECM (atECM) is one of the key players in the stem cell niche, data on bidirectional interaction of ASCs and atECM are still scarce. Here, we investigated how atECM guides ASCs' differentiation. atECM altered shape and cytoskeleton organization of ASCs without changing their proliferation, ß-galactosidase activity and adhesion. Cytoskeleton modifications occurred due to fostered parallel organization of F-actin and elevated expression of Vimentin in ASCs. After seven-day cultivation, atECM impaired osteogenesis of ASCs, simultaneously decreasing expression of Runx2. In addition, atECM accelerated early adipogenesis concomitantly with altered Vimentin organization in ASCs, slightly increasing PPARγ, while elevated Adiponectin and Vimentin mRNA expression. Early adipogenesis triggered by atECM was followed by upregulated mitochondrial activity and Sirtuin 1 (SIRT1) expression in ASCs. Proadipogenic events induced by atECM were mediated by SIRT1, indicating the supportive role of atECM in adipogenesis-related metabolic state of ASCs. These results provide a closer look at the effects of atECM on ASC physiology and may support the advancement of engineering design in soft tissue reconstruction and fundamental research of AT.

Investigation of metabolic properties and effects of 17ß-carboxamide glucocorticoids on human peripheral blood leukocytes.

The biological activity of three previously synthesized 17ß-carboxamide glucocorticoids (BG, BEG, and MPEA) was tested in vitro on mitogen stimulated and non-stimulated peripheral blood mononuclear cells (MNCs) and granulocytes from human healthy donors, and the results were compared to the conventional glucocorticoid dexamethasone. The tested 17ß-carboxamide glucocorticoids did not induce decreases in MNC viability and proliferation, while modulation of reactive oxygen species (ROS) synthesis in granulocytes was dependent on the cell donor. The obtained results indicate the possibility of avoidance of strong lymphocyte suppression, which is generally recognized during administration of conventional glucocorticoids. Furthermore, the metabolism of the tested derivatives was predicted in silico. The predicted metabolites were synthesized and the in silico results were confirmed by in vitro evaluation of the metabolism of BG, BEG, and MPEA in human serum and in cultures of peripheral blood MNCs. The results of the biological activity and metabolism evaluation and of previous in vivo evaluations of biological activity indicate the soft drug nature of BG, BEG, and MPEA. In order to be fully considered as soft glucocorticoids, further investigations on the toxicity and activity of the formed metabolites are required.

Mapping of hemoglobin in erythrocytes and erythrocyte ghosts using two photon excitation fluorescence microscopy.

The present study describes utilization of two photon excitation fluorescence (2PE) microscopy for visualization of the hemoglobin in human and porcine erythrocytes and their empty membranes (i.e., ghosts). High-quality, label- and fixation-free visualization of hemoglobin was achieved at excitation wavelength 730 nm by detecting visible autofluorescence. Localization in the suspension and spatial distribution (i.e., mapping) of residual hemoglobin in erythrocyte ghosts has been resolved by 2PE. Prior to the 2PE mapping, the presence of residual hemoglobin in the bulk suspension of erythrocyte ghosts was confirmed by cyanmethemoglobin assay. 2PE analysis revealed that the distribution of hemoglobin in intact erythrocytes follows the cells' shape. Two types of erythrocytes, human and porcine, characterized with discocyte and echinocyte morphology, respectively, showed significant differences in hemoglobin distribution. The 2PE images have revealed that despite an extensive washing out procedure after gradual hypotonic hemolysis, a certain amount of hemoglobin localized on the intracellular side always remains bound to the membrane and cannot be eliminated. The obtained results open the possibility to use 2PE microscopy to examine hemoglobin distribution in erythrocytes and estimate the purity level of erythrocyte ghosts in biotechnological processes.

Comparative studies on osmosis based encapsulation of sodium diclofenac in porcine and outdated human erythrocyte ghosts.

The objective of our study was to develop controlled drug delivery system based on erythrocyte ghosts for amphiphilic compound sodium diclofenac considering the differences between erythrocytes derived from two readily available materials - porcine slaughterhouse and outdated transfusion human blood. Starting erythrocytes, empty erythrocyte ghosts and diclofenac loaded ghosts were compared in terms of the encapsulation efficiency, drug releasing profiles, size distribution, surface charge, conductivity, surface roughness and morphology. The encapsulation of sodium diclofenac was performed by an osmosis based process - gradual hemolysis. During this process sodium diclofenac exerted mild and delayed antihemolytic effect and increased potassium efflux in porcine but not in outdated human erythrocytes. FTIR spectra revealed lack of any membrane lipid disorder and chemical reaction with sodium diclofenac in encapsulated ghosts. Outdated human erythrocyte ghosts with detected nanoscale damages and reduced ability to shrink had encapsulation efficiency of only 8%. On the other hand, porcine erythrocyte ghosts had encapsulation efficiency of 37% and relatively slow drug release rate. More preserved structure and functional properties of porcine erythrocytes related to their superior encapsulation and release performances, define them as more appropriate for the usage in sodium diclofenac encapsulation process.

Biomembranes from slaughterhouse blood erythrocytes as prolonged release systems for dexamethasone sodium phosphate.

The present study investigated preparation of bovine and porcine erythrocyte membranes from slaughterhouse blood as bio-derived materials for delivery of dexamethasone-sodium phosphate (DexP). The obtained biomembranes, i.e., ghosts were characterized in vitro in terms of morphological properties, loading parameters, and release behavior. For the last two, an UHPLC/-HESI-MS/MS based analytical procedure for absolute drug identification and quantification was developed. The results revealed that loading of DexP into both type of ghosts was directly proportional to the increase of drug concentration in the incubation medium, while incubation at 37°C had statistically significant effect on loaded amount of DexP (P < 0.05). The encapsulation efficiency was about fivefold higher in porcine compared to bovine ghosts. Insight into ghosts' surface morphology by field emission-scanning electron microscopy and atomic force microscopy confirmed that besides inevitable effects of osmosis, DexP inclusion itself had no observable additional effect on the morphology of the ghosts carriers. DexP release profiles were dependent on erythrocyte ghost type and amount of residual hemoglobin. However, sustained DexP release was achieved and shown over 3 days from porcine ghosts and 5 days from bovine erythrocyte ghosts. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1046-1055, 2016.

Calcium-Alginate-Inulin Microbeads as Carriers for Aqueous Carqueja Extract.

Carqueja (Pterospartum tridentatum) is an endemic species and various bioactive compounds have been identified in its aqueous extract. The aim of this study was to protect the natural antioxidants from the aqueous extract of carqueja by encapsulation in Ca-alginate microbeads and Ca-alginate microbeads containing 10% and 20% (w/v) of inulin. The microbeads produced by electrostatic extrusion technique had an average diameter from 625 µm to 830 µm depending on the portion of inulin. The sphericity factor of the hydrogel microbeads had values between 0.014 and 0.026, while freeze dried microbeads had irregular shape, especially those with no excipient. The reduction in microbeads size after freeze drying process (expressed as shrinkage factor) ranged from 0.338 (alginate microbeads with 20% (w/v) of inulin) to 0.523 (plain alginate microbeads). The expressed radical scavenging activity against ABTS and DPPH radicals was found to be between 30% and 40% for encapsulated extract, while the fresh extract showed around 47% and 57% of radical scavenging activity for ABTS and DPPH radicals, respectively. The correlation between antioxidant activity and the total phenolic content were found to be positive (in both assay methods, DPPH and ABTS), which indicate that the addition of inulin didn't have influence on antioxidant activity. The presence of inulin reduced stiffness of the hydrogel, and protected bead structure from collapse upon freeze-drying. Alginate-inulin beads are envisaged to be used for delivery of aqueous P. tridentatum extract in functional food products.