Calciprotein Particles Cause Physiologically Significant Pro-Inflammatory Response in Endothelial Cells and Systemic Circulation.

Calciprotein particles (CPPs) represent an inherent mineral buffering system responsible for the scavenging of excessive Ca2+ and PO43- ions in order to prevent extraskeletal calcification, although contributing to the development of endothelial dysfunction during the circulation in the bloodstream. Here, we performed label-free proteomic profiling to identify the functional consequences of CPP internalisation by endothelial cells (ECs) and found molecular signatures of significant disturbances in mitochondrial and lysosomal physiology, including oxidative stress, vacuolar acidification, accelerated proteolysis, Ca2+ cytosolic elevation, and mitochondrial outer membrane permeabilisation. Incubation of intact ECs with conditioned medium from CPP-treated ECs caused their pro-inflammatory activation manifested by vascular cell adhesion molecule 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1) upregulation and elevated release of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1/ C-C motif ligand 2 (MCP-1/CCL2). Among the blood cells, monocytes were exclusively responsible for CPP internalisation. As compared to the co-incubation of donor blood with CPPs in the flow culture system, intravenous administration of CPPs to Wistar rats caused a considerably higher production of chemokines, indicating the major role of monocytes in CPP-triggered inflammation. Upregulation of sICAM-1 and IL-8 also suggested a notable contribution of endothelial dysfunction to systemic inflammatory response after CPP injections. Collectively, our results demonstrate the pathophysiological significance of CPPs and highlight the need for the development of anti-CPP therapies.

Advantages of Fibrin Polymerization Method without the Use of Exogenous Thrombin for Vascular Tissue Engineering Applications.

Fibrin is widely used in vascular tissue engineering. Typically, fibrin polymerization is initiated by adding exogenous thrombin. In this study, we proposed a protocol for the preparation of completely autologous fibrin without the use of endogenous thrombin and compared the properties of the prepared fibrin matrix with that obtained by the traditional method. Fibrinogen was obtained by ethanol precipitation followed by fibrin polymerization by adding either exogenous thrombin and calcium chloride (ExThr), or only calcium chloride (EnThr). We examined the structure, mechanical properties, thrombogenicity, degradation rate and cytocompatibility of fibrin matrices. Factor XIII (FXIII) quantitative assay was performed by ELISA, and FXIII activity was assessed by SDS-PAGE detection of γ-γ cross-links. The results show that network structure of EnThr fibrin was characterized by thinner fibers. The EnThr fibrin matrices had higher strength, stiffness and resistance to proteolytic degradation compared to ExThr fibrin. EnThr fibrin matrices exhibited less thrombogenicity in vitro than ExThr, and retained high cytocompatibility. Thus, the proposed approach has several advantages over the traditional method, namely the fabrication of a completely autologous coating material that has better mechanical properties, higher resistance to proteolysis and lower thrombogenicity.

Biomaterials Based on Carbon Nanotube Nanocomposites of Poly(styrene-b-isobutylene-b-styrene): The Effect of Nanotube Content on the Mechanical Properties, Biocompatibility and Hemocompatibility.

Nanocomposites based on poly(styrene-block-isobutylene-block-styrene) (SIBS) and single-walled carbon nanotubes (CNTs) were prepared and characterized in terms of tensile strength as well as bio- and hemocompatibility. It was shown that modification of CNTs using dodecylamine (DDA), featured by a long non-polar alkane chain, provided much better dispersion of nanotubes in SIBS as compared to unmodified CNTs. As a result of such modification, the tensile strength of the nanocomposite based on SIBS with low molecular weight (Mn = 40,000 g mol-1) containing 4% of functionalized CNTs was increased up to 5.51 ± 0.50 MPa in comparison with composites with unmodified CNTs (3.81 ± 0.11 MPa). However, the addition of CNTs had no significant effect on SIBS with high molecular weight (Mn~70,000 g mol-1) with ultimate tensile stress of pure polymer of 11.62 MPa and 14.45 MPa in case of its modification with 1 wt% of CNT-DDA. Enhanced biocompatibility of nanocomposites as compared to neat SIBS has been demonstrated in experiment with EA.hy 926 cells. However, the platelet aggregation observed at high CNT concentrations can cause thrombosis. Therefore, SIBS with higher molecular weight (Mn~70,000 g mol-1) reinforced by 1-2 wt% of CNTs is the most promising material for the development of cardiovascular implants such as heart valve prostheses.

Influence of preparation and combustion parameters of coal-water slurries on gas emission chemistry.

The article presents the results of experimental studies performed for the comparative analysis of factors affecting the concentration of the main gas emissions during the combustion of fuel suspensions obtained from coal enrichment waste. The factors influencing the formation of anthropogenic emissions during the flaring of coal-water fuel suspensions were investigated. Experiments were carried out with coal processing waste, both with and without additives in the form of spent mineral oil. The article shows how the concentration of sulfur dioxide, nitrogen dioxide, and hydrocarbons is affected by the parameters of preparation (particle size, solid-liquid phase ratio, additives of used engine oil) and flame combustion (temperature in the combustion chamber, excess air coefficient, and droplet size after spraying) of fuel suspensions. The ranges of parameters were selected at which the best combustion parameters are observed, based on the data obtained: degree of grinding is less than 100 µm, solid part content in the suspension is from 55 to 60%; combustion temperature is in the range from 950 to 1050° C, droplet size when spraying the suspension is less than 300 µm, additive of waste mineral oil is from 3 to 7 wt.%. and air-fuel ratio from 1.2 to 1.3.

Adipokine gene expression in adipocytes isolated from different fat depots of coronary artery disease patients.

To compare DPP4, LCN2, NAMPT, ITLN1, APLN mRNA levels in adipocytes isolated from the biopsies of subcutaneous, epicardial and perivascular fat obtained from 25 patients with coronary artery disease. Gene expression signature was determined by RT-qPCR with hydrolysis probes. We found DPP4 and APLN mRNA was higher expressed only in adipocytes isolated from epicardial adipose tissue compared to the subcutaneous fat. The ITLN1 gene was overexpressed in epicardial adipose tissue compared to both subcutaneous and perivascular tissues. APLN mRNA expression was positively correlated with total and LDL cholesterol plasma level, and DPP4 mRNA expression - with VLDL cholesterol concentration. Thus, adipocytes isolated from different adipose depots are characterised by differential gene expression of adipokines. Epicardial adipose tissue is of particular interest in the context of its function, molecular and genetic mechanisms of regulation of the cardiovascular system and as a therapeutic target for correction of adipose tissue-induced effects on health.

Industrial processing of phosphogypsum into organomineral fertilizer.

The purpose of this study is to solve the problem of industrial waste storage. Waste from the production of mineral fertilizers is considered in this study (as an example-phosphogypsum LLC «Industrial group «Phosphorit¼). Waste storages on a landfill have a significant negative impact on the environment. This fact has been confirmed by studies of wastewater from the dump (drainage water). Phosphogypsum utilization as a mineral component of an organic-mineral mixture without preliminary purification is the most promising approach. In this case, the joint utilization of lignin sludge as an organic component is required. The absence of excess content of toxic elements and the presence of plant nutrients in the required quantities was established as a result of laboratory studies of soil additive. In addition, the authors noted an increased content of stable strontium in phosphogypsum. Localization of strontium in the soil mixture is ensured by fractional application. Strontium is replaced by covalent calcium when the components are absorbed by the plants. The developed soil mixture should be considered as a single-use organomineral additive with prolonged action. Forestry activities, reclamation of disturbed lands, slopes of highways and landfills of solid municipal waste are promising areas of implementation.

Tropism of Extracellular Vesicles and Cell-Derived Nanovesicles to Normal and Cancer Cells: New Perspectives in Tumor-Targeted Nucleic Acid Delivery.

The main advantage of extracellular vesicles (EVs) as a drug carrier system is their low immunogenicity and internalization by mammalian cells. EVs are often considered a cell-specific delivery system, but the production of preparative amounts of EVs for therapeutic applications is challenging due to their laborious isolation and purification procedures. Alternatively, mimetic vesicles prepared from the cellular plasma membrane can be used in the same way as natural EVs. For example, a cytoskeleton-destabilizing agent, such as cytochalasin B, allows the preparation of membrane vesicles by a series of centrifugations. Here, we prepared cytochalasin-B-inducible nanovesicles (CINVs) of various cellular origins and studied their tropism in different mammalian cells. We observed that CINVs derived from human endometrial mesenchymal stem cells exhibited an enhanced affinity to epithelial cancer cells compared to myeloid, lymphoid or neuroblastoma cancer cells. The dendritic cell-derived CINVs were taken up by all studied cell lines with a similar efficiency that differed from the behavior of DC-derived EVs. The ability of cancer cells to internalize CINVs was mainly determined by the properties of recipient cells, and the cellular origin of CINVs was less important. In addition, receptor-mediated interactions were shown to be necessary for the efficient uptake of CINVs. We found that CINVs, derived from late apoptotic/necrotic cells (aCINVs) are internalized by in myelogenous (K562) 10-fold more efficiently than CINVs, and interact much less efficiently with melanocytic (B16) or epithelial (KB-3-1) cancer cells. Finally, we found that CINVs caused a temporal and reversible drop of the rate of cell division, which restored to the level of control cells with a 24 h delay.

Calciprotein Particles Link Disturbed Mineral Homeostasis with Cardiovascular Disease by Causing Endothelial Dysfunction and Vascular Inflammation.

An association between high serum calcium/phosphate and cardiovascular events or death is well-established. However, a mechanistic explanation of this correlation is lacking. Here, we examined the role of calciprotein particles (CPPs), nanoscale bodies forming in the human blood upon its supersaturation with calcium and phosphate, in cardiovascular disease. The serum of patients with coronary artery disease or cerebrovascular disease displayed an increased propensity to form CPPs in combination with elevated ionised calcium as well as reduced albumin levels, altogether indicative of reduced Ca2+-binding capacity. Intravenous administration of CPPs to normolipidemic and normotensive Wistar rats provoked intimal hyperplasia and adventitial/perivascular inflammation in both balloon-injured and intact aortas in the absence of other cardiovascular risk factors. Upon the addition to primary human arterial endothelial cells, CPPs induced lysosome-dependent cell death, promoted the release of pro-inflammatory cytokines, stimulated leukocyte adhesion, and triggered endothelial-to-mesenchymal transition. We concluded that CPPs, which are formed in the blood as a result of altered mineral homeostasis, cause endothelial dysfunction and vascular inflammation, thereby contributing to the development of cardiovascular disease.

Tissue-Engineered Carotid Artery Interposition Grafts Demonstrate High Primary Patency and Promote Vascular Tissue Regeneration in the Ovine Model.

Tissue-engineered vascular graft for the reconstruction of small arteries is still an unmet clinical need, despite the fact that a number of promising prototypes have entered preclinical development. Here we test Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)Poly(ε-caprolactone) 4-mm-diameter vascular grafts equipped with vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and stromal cell-derived factor 1α (SDF-1α) and surface coated with heparin and iloprost (PHBV/PCL[VEGF-bFGF-SDF]Hep/Ilo, n = 8) in a sheep carotid artery interposition model, using biostable vascular prostheses of expanded poly(tetrafluoroethylene) (ePTFE, n = 5) as a control. Primary patency of PHBV/PCL[VEGF-bFGF-SDF]Hep/Ilo grafts was 62.5% (5/8) at 24 h postimplantation and 50% (4/8) at 18 months postimplantation, while all (5/5) ePTFE conduits were occluded within the 24 h after the surgery. At 18 months postimplantation, PHBV/PCL[VEGF-bFGF-SDF]Hep/Ilo grafts were completely resorbed and replaced by the vascular tissue. Regenerated arteries displayed a hierarchical three-layer structure similar to the native blood vessels, being fully endothelialised, highly vascularised and populated by vascular smooth muscle cells and macrophages. The most (4/5, 80%) of the regenerated arteries were free of calcifications but suffered from the aneurysmatic dilation. Therefore, biodegradable PHBV/PCL[VEGF-bFGF-SDF]Hep/Ilo grafts showed better short- and long-term results than bio-stable ePTFE analogues, although these scaffolds must be reinforced for the efficient prevention of aneurysms.

First Experimental Study of the Influence of Extracellular Vesicles Derived from Multipotent Stromal Cells on Osseointegration of Dental Implants.

Herein, the aim was to study the state of the bone tissue adjacent to dental implants after the use of extracellular vesicles derived from multipotent stromal cells (MSC EVs) of bone marrow origin in the experiment. In compliance with the rules of asepsis and antiseptics under general intravenous anesthesia with propofol, the screw dental implants were installed in the proximal condyles of the tibia of outbred rabbits without and with preliminary introduction of 19.2 µg MSC EVs into each bone tissue defect. In 3, 7, and 10 days after the operation, the density of bone tissue adjacent to different parts of the implant using an X-ray unit with densitometer was measured. In addition, the histological examinations of the bone site with the hole from the removed device and the soft tissues from the surface of the proximal tibial condyle in the area of intra-bone implants were made. It was found out that 3 days after implantation with the use of MSC EVs, the bone density was statistically significantly higher by 47.2% than after the same implantation, but without the injection of MSC EVs. It is possible that as a result of the immunomodulatory action of MSC EVs, the activity of inflammation decreases, and, respectively, the degree of vasodilation in bones and leukocyte infiltration of the soft tissues are lower, in comparison with the surgery performed in the control group. The bone fragments formed during implantation are mainly consolidated with each other and with the regenerating bone. Day 10 demonstrated that all animals with the use of MSC EVs had almost complete fusion of the screw device with the bone tissue, whereas after the operation without the application of MSC EVs, the heterogeneous histologic pattern was observed: From almost complete osseointegration of the implant to the absolute absence of contact between the foreign body and the new formed bone. Therefore, the use of MSC EVs during the introduction of dental implants into the proximal condyle of the tibia of rabbits contributes to an increase of the bone tissue density near the device after 3 days and to the achievement of consistently successful osseointegration of implants 10 days after the surgery.

bFGF and SDF-1α Improve In Vivo Performance of VEGF-Incorporating Small-Diameter Vascular Grafts.

Tissue-engineered vascular grafts are widely tested as a promising substitute for both arterial bypass and replacement surgery. We previously demonstrated that incorporation of VEGF into electrospun tubular scaffolds from poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/poly(ε-caprolactone) enhances formation of an endothelial cell monolayer. However, an overdose of VEGF can induce tumor-like vasculature; thereby, other bioactive factors are needed to support VEGF-driven endothelialization and successful recruitment of smooth muscle cells. Utilizing emulsion electrospinning, we fabricated one-layer vascular grafts with either VEGF, bFGF, or SDF-1α, and two-layer vascular grafts with VEGF incorporated into the inner layer and bFGF and SDF-1α incorporated into the outer layer with the following structural evaluation, tensile testing, and in vivo testing using a rat abdominal aorta replacement model. The latter graft prototype showed higher primary patency rate. We found that the two-layer structure improved surface topography and mechanical properties of the grafts. Further, the combination of bFGF, SDF-1α, and VEGF improved endothelialization compared with VEGF alone, while bFGF induced a rapid formation of a smooth muscle cell layer. Taken together, these findings show that the two-layer structure and incorporation of bFGF and SDF-1α into the vascular grafts in combination with VEGF provide a higher primary patency and therefore improved in vivo performance.

Biocompatible Nanocomposites Based on Poly(styrene-block-isobutylene-block-styrene) and Carbon Nanotubes for Biomedical Application.

In this study, we incorporated carbon nanotubes (CNTs) into poly(styrene-block-isobutylene-block-styrene) (SIBS) to investigate the physical characteristics of the resulting nanocomposite and its cytotoxicity to endothelial cells. CNTs were dispersed in chloroform using sonication following the addition of a SIBS solution at different ratios. The resultant nanocomposite films were analyzed by X-ray microtomography, optical and scanning electron microscopy; tensile strength was examined by uniaxial tension testing; hydrophobicity was evaluated using a sessile drop technique; for cytotoxicity analysis, human umbilical vein endothelial cells were cultured on SIBS-CNTs for 3 days. We observed an uneven distribution of CNTs in the polymer matrix with sporadic bundles of interwoven nanotubes. Increasing the CNT content from 0 wt% to 8 wt% led to an increase in the tensile strength of SIBS films from 4.69 to 16.48 MPa. The engineering normal strain significantly decreased in 1 wt% SIBS-CNT films in comparison with the unmodified samples, whereas a further increase in the CNT content did not significantly affect this parameter. The incorporation of CNT into the SIBS matrix resulted in increased hydrophilicity, whereas no cytotoxicity towards endothelial cells was noted. We suggest that SIBS-CNT may become a promising material for the manufacture of implantable devices, such as cardiovascular patches or cusps of the polymer heart valve.

Biodegradable Patches for Arterial Reconstruction Modified with RGD Peptides: Results of an Experimental Study.

Modification by Arg-Gly-Asp (RGD) peptides is a promising approach to improve the biocompatibility of biodegradable vascular patches for arteriotomy. In this study, we evaluated the performance of vascular patches electrospun using a blend of polycaprolactone (PCL) and polyhydroxybutyrate/valerate (PHBV) and additionally modified with RGDK, AhRGD, and c[RGDFK] peptides using 1,6-hexamethylenediamine or 4,7,10-trioxa-1,13-tridecanediamine (TTDDA) linkers. We examined mechanical properties and hemocompatibility of resulting patches before implanting them in rat abdominal aortas to assess their performance in vivo. Patches were explanted 1, 3, 6, and 12 months postoperation followed by histological and immunofluorescence analyses. Patches manufactured from the human internal mammary artery or commercially available KemPeriplas-Neo xenopericardial patches were used as a control. The tensile strength and F max of KemPeriplas-Neo patches were 4- and 16.7-times higher than those made of human internal mammary artery, respectively. Both RGD-modified and unmodified PHBV/PCL patches demonstrated properties similar to a human internal mammary artery patch. Regardless of RGD modification, experimental PHBV/PCL patches displayed fewer lysed red blood cells and resulted in milder platelet aggregation than KemPeriplas-Neo patches. Xenopericardial patches failed to form an endothelial layer in vivo and were prone to calcification. By contrast, TTDDA/RGDK-modified biodegradable patches demonstrated a resistance to calcification. Modification by TTDDA/RGDK and TTDDA/c[RGDFK] facilitated the formation of neovasculature upon the implantation in vivo.

Human Peripheral Blood-Derived Endothelial Colony-Forming Cells Are Highly Similar to Mature Vascular Endothelial Cells yet Demonstrate a Transitional Transcriptomic Signature.

Endothelial colony-forming cells (ECFC) are currently considered as a promising cell population for the pre-endothelialization or pre-vascularization of tissue-engineered constructs, including small-diameter biodegradable vascular grafts. However, the extent of heterogeneity between ECFC and mature vascular endothelial cells (EC) is unclear. Here, we performed a transcriptome-wide study to compare gene expression profiles of ECFC, human coronary artery endothelial cells (HCAEC), and human umbilical vein endothelial cells (HUVEC). Characterization of the abovementioned cell populations was carried out by immunophenotyping, tube formation assay, and evaluation of proliferation capability while global gene expression profiling was conducted by means of RNA-seq. ECFC were similar to HUVEC in terms of immunophenotype (CD31+vWF+KDR+CD146+CD34-CD133-CD45-CD90-) and tube formation activity yet had expectedly higher proliferative potential. HCAEC and HUVEC were generally similar to ECFC with regards to their global gene expression profile; nevertheless, ECFC overexpressed specific markers of all endothelial lineages (NRP2, NOTCH4, LYVE1), in particular lymphatic EC (LYVE1), and had upregulated extracellular matrix and basement membrane genes (COL1A1, COL1A2, COL4A1, COL4A2). Proteomic profiling for endothelial lineage markers and angiogenic molecules generally confirmed RNA-seq results, indicating ECFC as an intermediate population between HCAEC and HUVEC. Therefore, gene expression profile and behavior of ECFC suggest their potential to be applied for a pre-endothelialization of bioartificial vascular grafts, whereas in terms of endothelial hierarchy they differ from HCAEC and HUVEC, having a transitional phenotype.

Cytochalasin-B-Inducible Nanovesicle Mimics of Natural Extracellular Vesicles That Are Capable of Nucleic Acid Transfer.

Extracellular vesicles provide cell-to-cell communication and have great potential for use as therapeutic carriers. This study was aimed at the development of an extracellular vesicle-based system for nucleic acid delivery. Three types of nanovesicles were assayed as oligonucleotide carriers: mesenchymal stem cell-derived extracellular vesicles and mimics prepared either by cell treatment with cytochalasin B or by vesicle generation from plasma membrane. Nanovesicles were loaded with a DNA oligonucleotide by freezing/thawing, sonication, or permeabilization with saponin. Oligonucleotide delivery was assayed using HEK293 cells. Extracellular vesicles and mimics were characterized by a similar oligonucleotide loading level but different efficiency of oligonucleotide delivery. Cytochalasin-B-inducible nanovesicles exhibited the highest level of oligonucleotide accumulation in HEK293 cells and a loading capacity of 0.44 ± 0.05 pmol/µg. The loaded oligonucleotide was mostly protected from nuclease action.

Adipocytes Directly Affect Coronary Artery Disease Pathogenesis via Induction of Adipokine and Cytokine Imbalances.

This study aimed to investigate the adipokine and cytokine profiles of adipocytes from epicardial and subcutaneous adipose tissues in interconnection with the visceral adipose tissue area and the biochemical and clinical characteristics of patients with coronary artery disease. We assessed 84 patients with coronary artery disease (65 men, 19 women) and divided them into two groups based on the presence of visceral obesity. We sampled epicardial and subcutaneous adipose tissues from the patients with visceral obesity. We then cultured the adipocytes and evaluated their adipokine profiles and pro-inflammatory activity. Results show that the mRNA expression of adiponectin in cultures of epicardial adipocytes from patients with and without visceral obesity was lower than that in subcutaneous adipocytes. Moreover, adiponectin mRNA expression in cultures of subcutaneous and epicardial adipocytes from patients with visceral obesity was lower than that in patients without obesity. For leptin, the reverse pattern was observed, with expression higher in cultures of epicardial adipocytes than in subcutaneous adipocytes and higher in epicardial adipocytes from patients with visceral obesity than in those from subjects without visceral obesity. In addition, in epicardial adipocytes, increased expression of proinflammatory cytokine genes (IL6, TNF) was observed compared with that in subcutaneous adipocytes. In contrast, expression of IL10 was higher in cultures of subcutaneous adipocytes than in epicardial adipocytes. The epicardial adipose tissue area was associated with the presence of higher levels of leptin and TNF-α within adipocytes and serum, increased lipid and carbohydrate metabolism. Coronary artery disease, in the context of the status of epicardial adipocytes, can be characterized as "metabolic inflammation," suggesting the direct involvement of adipocytes in pathogenesis through the development of adipokine imbalances and activation of proinflammatory processes.

Biocompatibility of Small-Diameter Vascular Grafts in Different Modes of RGD Modification.

Modification with Arg-Gly-Asp (RGD) peptides is a promising approach to improve biocompatibility of small-calibre vascular grafts but it is unknown how different RGD sequence composition impacts graft performance. Here we manufactured 1.5 mm poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/poly(ε-caprolactone) grafts modified by distinct linear or cyclic RGD peptides immobilized by short or long amine linker arms. Modified vascular prostheses were tested in vitro to assess their mechanical properties, hemocompatibility, thrombogenicity and endothelialisation. We also implanted these grafts into rat abdominal aortas with the following histological examination at 1 and 3 months to evaluate their primary patency, cellular composition and detect possible calcification. Our results demonstrated that all modes of RGD modification reduce ultimate tensile strength of the grafts. Modification of prostheses does not cause haemolysis upon the contact with modified grafts, yet all the RGD-treated grafts display a tendency to promote platelet aggregation in comparison with unmodified counterparts. In vivo findings identify that cyclic Arg-Gly-Asp-Phe-Lys peptide in combination with trioxa-1,13-tridecanediamine linker group substantially improve graft biocompatibility. To conclude, here we for the first time compared synthetic small-diameter vascular prostheses with different modes of RGD modification. We suggest our graft modification regimen as enhancing graft performance and thus recommend it for future use in tissue engineering.

Post-exposure administration of chimeric antibody protects mice against European, Siberian, and Far-Eastern subtypes of tick-borne encephalitis virus.

Tick-borne encephalitis virus (TBEV) is the most important tick-transmitted pathogen. It belongs to the Flaviviridae family and causes severe human neuroinfections. In this study, protective efficacy of the chimeric antibody chFVN145 was examined in mice infected with strains belonging to the Far-Eastern, European, and Siberian subtypes of TBEV, and the antibody showed clear therapeutic efficacy when it was administered once one, two, or three days after infection. The efficacy was independent of the TBEV strain used to infect the mice; however, the survival rate of the mice was dependent on the dose of TBEV and of the antibody. No enhancement of TBEV infection was observed when the mice were treated with non-protective doses of chFVN145. Using a panel of recombinant fragments of the TBEV glycoprotein E, the neutralizing epitope for chFVN145 was localized in domain III of the TBEV glycoprotein E, in a region between amino acid residues 301 and 359. In addition, three potential sites responsible for binding with chFVN145 were determined using peptide phage display libraries, and 3D modeling demonstrated that the sites do not contact the fusion loop and, hence, their binding with chFVN145 does not result in increased attachment of TBEV to target cells.

Endovascular Interventions Permit Isolation of Endothelial Colony-Forming Cells from Peripheral Blood.

BACKGROUND: Isolation of endothelial colony-forming cells (ECFCs) is difficult due to the extremely low concentration of their precursors in the peripheral blood (PB). We hypothesized that mechanical injury to the arterial wall during percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG) may increase the release of circulating ECFC precursors and induce their growth in vitro. METHODS: PB samples from patients with coronary artery disease were collected before, immediately after, and 24 h after the surgery in the CABG group. In the PCI group, PB was isolated before, immediately after the insertion of the catheter, immediately after balloon angioplasty, and 24 h after the PCI. A mononuclear fraction of PB was isolated and differentiated into ECFCs with the following immunophenotyping and evaluation of angiogenic properties. RESULTS: The obtained cultures corresponded to the phenotype and tube forming potential consistent with ECFCs. The isolation of ECFCs in the PCI group was successful in 75% of cases (six out of eight patients) after catheter insertion and in 87.5% (seven out of eight patients) after the balloon inflation and stent deployment. These cultures had high/medium proliferative activity in contrast to those obtained before or 24 h after the intervention. CONCLUSIONS: Mechanical injury during PCI increases the release of ECFC precursors to the PB and, hence, the efficacy of ECFC isolation.

Modifications in routine protocol of RNA isolation can improve quality of RNA purified from adipocytes.

Adipose tissue is of interest in the context of its role in the pathogenesis of cardiovascular diseases. Modern experimental techniques require a well-purified RNA, but all the routine protocols for RNA extraction have a number of limitations in case of fatty tissues. Here we described a modified protocol for RNA extraction from human adipocytes based on routine column method. Suggested modifications optimized the sample preparation, lysis and washing lead to enhance RNA purity. We conclude that the current protocol for total RNA purification from adipocytes allows extracting a high-quality RNA devoid of fatty acids, organic solvents and salts contamination.