Synthesis of conjugates of (aR,7S)-colchicine with monoterpenoids and investigation of their biological activity.

Conjugates of the natural alkaloid (aR,7S)-colchicine with bicyclic monoterpenoids and their derivatives were synthesized for the first time. Molecular docking of the synthesized agents in the active site of the main viral protease of the SARS-CoV-2 virus was carried out. The cytotoxic properties of the agents against different cell lines and the ability to inhibit the main viral protease 3CLPro were studied.

Investigation of the Distribution of Magnetic Nanoparticles in Tumor Tissues by the Method of Scanning Magnetic Force Nanotomography.

The development of effective biomedical technologies using magnetic nanoparticles (MNPs) for the tasks of oncotherapy and nanodiagnostics requires the development and implementation of new methods for the analysis of micro- and nanoscale distributions of MNPs in the volume of cells and tissues. The paper presents a new approach to three-dimensional analysis of MNP distributions - scanning magnetic force nanotomography as applied to the study of tumor tissues. Correlative reconstruction of MNP distributions and nanostructure features of the studied tissues made it possible to quantitatively estimate the parameters of three-dimensional distributions of composite nanoparticles based on silicon and iron oxide obtained by femtosecond laser ablation and injected intravenously and intratumorally into tumor tissue samples of B16/F1 mouse melanoma. The developed technology based on the principles of scanning probe nanotomography is applicable for studying the features of three-dimensional micro- and nanoscale distributions of magnetic nanoparticles in biomaterials, cells and tissues of various types.

Effect of Intranasal Administration of Multipotent Mesenchymal Stromal Cell Exosomes on Memory of Mice in Alzheimer's Disease Model.

We studied the effect of intranasal administration of exosomes obtained by culturing of multipotent mesenchymal stromal cells (MMSC) isolated from the Wharton's jelly of the human umbilical cord on spatial memory of olfactory bulbectomized mice demonstrating the basic signs of a sporadic form of Alzheimer's disease. Intranasal administration of isolated exosomes expressing typical markers CD9, CD63 and CD81 improved spatial memory in bulbectomized animals, which manifested in a significant increase in the number of visits to the target sector and the time spent there in comparison with indifferent sectors. After administration, labeled exosomes were found in the hippocampus and neocortex, the structures playing an important role in learning and memory processes and affected by Alzheimer's disease. The advantages of exosomes in comparison with MMSC are their small size, low immunogenicity, and inability to cause cell transformation together with high therapeutic efficacy.

Fusion with an albumin-binding domain improves pharmacokinetics of an αvß3-integrin binding fibronectin scaffold protein.

Fusion with an albumin-binding domain (ABD) of streptococcal protein G represents a popular approach for half-life extension of small protein therapeutics in the organism. To increase the circulation time of engineered αvß3-integrin-binding protein (JCL) based on the 10th human fibronectin type III domain (10 Fn3), we have constructed several fusions with ABD with different orientations of the partner proteins and linker length. The recombinant proteins were expressed in Escherichia coli cells and purified by nickel-affinity chromatography. All fusion proteins bound human serum albumin (HSA) in ELISA assay; however, fusions with longer linkers demonstrated better performance. Interaction of ABD-L15 -JCL and JCL-L14 -ABD with HSA was confirmed by analytical size exclusion chromatography and pull-down assays. Surprisingly, the thermal stability of ABD-L15 -JCL was dramatically decreased in comparison with JCL and JCL-L14 -ABD proteins. Pharmacokinetic studies revealed that JCL-L14 -ABD circulated in murine blood about 10 times longer than ABD-L15 -JCL and 960 times longer than JCL. Biodistribution studies of JCL-L14 -ABD in mice revealed its increased level in blood and a decreased accumulation in liver and kidneys in comparison with JCL. Obtained results demonstrate the utility of the fusion with ABD for half-life extension of the binding proteins based on 10 Fn3.

Myths, reality and future of mesenchymal stem cell therapy.

Mesenchymal stem cell (MSC) therapy represents an alternative approach for tissue regeneration and inflammation control. In spite of a huge amount of preclinical data that has been accumulated on the therapeutic properties of MSCs, there are many conflicting results, possibly due to differences in the properties of MSCs obtained from different sources or underestimated mechanisms of MSC in vivo behavior. This review consolidates the in vivo effects of MSC therapy, discusses the fate of MSCs after intravascular and local delivery and proposes possible trends in MSC therapy.

[Immuno-PCR Assay for Quantitation of Antibodies to Epstein-Barr Virus].

Successful disease prevention and therapy critically depend on timely diagnosis of infections. Quantitative immuno-PCR (qiPCR) technology improves the sensitivity in the detection of antibodies to pathogens. A qiPCR-based assay was developed to determine IgG antibodies to Epstein-Barr virus (EBV) in the human blood serum. EBV nuclear protein 1 fragment (pEBV) was expressed in Escherichia coli. A synthetic single-stranded deoxyribonucleotide was conjugated to streptavidin, and the conjugate was used to detect рEBV-IgG1-biotin complexes by qiPCR. The IgG1 titers determined by qiPCR were compared to the results of enzyme-linked immunosorbent assay (ELISA). The sensitivity of qiPCR was one order of magnitude higher than that of ELISA. Thus, a highly sensitive qiPCR-based assay was developed to quantitate antibodies specific to the recombinant EBV antigen.

Activation of NK Cells in Mixed Cultures of Wharton's Jelly Mesenchymal Stromal Cells and Peripheral Blood Lymphocytes.

Mesenchymal stromal cells possess immunosuppressive properties that might be used for the therapy of inflammatory diseases of various geneses. The effects of mesenchymal stromal cells depend on their lifetime in the recipient tissues. During heterologous transplantation, mesenchymal stromal cells are eliminated by NK cells. We studied NK cell formation in mixed cultures of Wharton's jelly mesenchymal stromal cells and peripheral blood lymphocytes from an autologous donor. Lymphocytes were activated by a mitogen or IL-2. The lifetime of mesenchymal stromal cells was estimated by MTT test. Cytotoxic activity and phenotype of NK cells were evaluated by flow cytometry. It was found that activation of NK cells depended on IL-2 and was registered on day 2 of incubation with IL-2. In cultures with mitogen-activated lymphocytes, cytotoxicity was observed after 5-6 days. Cytotoxicity of NK correlated with significant decrease in CD16+ and increase in CD56+ NK and with reduction of mesenchymal stromal cell viability. Thus, the main mechanism of elimination of mesenchymal stromal cells is cytotoxicity of NK cells that depended on IL-2 production.

Proliferation of Peripheral Blood Lymphocytes and Mesenchymal Stromal Cells Derived from Wharton's Jelly in Mixed and Membrane-Separated Cultures.

We studied the effect of mesenchymal stromal cells on proliferation of CFSE-stained T cells in mixed and membrane-separated (Transwell) cultures and in 3D culture of mesenchymal stromal cells from Wharton's jelly. The interaction of mesenchymal stromal cells with mitogen-activated peripheral blood lymphocytes from an allogeneic donor was followed by suppression of T-cell proliferation in a wide range of cell proportions. Culturing in the Transwell system showed the absence of suppression assessed by the fraction of proliferating cells and by the cell cycle analysis. In 3D cultures, contact interaction of mesenchymal stromal cells and lymphocytes was demonstrated that led to accumulation of G2/M phase lymphocytes and G0/G1 phase mesenchymal stromal cells. The suppressive effect of mesenchymal stromal cells from Wharton's jelly is mediated by two mechanisms. The effects are realized within 6 days, which suggests that the therapeutic effects of mesenchymal stromal cells persist until their complete elimination from the body.

Cytokine Production in Mixed Cultures of Mesenchymal Stromal Cells from Wharton's Jelly and Peripheral Blood Lymphocytes.

We compared the production of 19 humoral factors in mixed cultures of mesenchymal stromal cells from Wharton's jelly and allogenic peripheral blood lymphocytes. For evaluation of the specificity of immunosuppressive activity of mesenchymal stromal cells, comparative analysis of the production of these humoral factors in mixed cultures of lymphocytes and epithelial BxPC-3 cells was conducted. The production of soluble factors in both mono- and mixed cultures significantly correlated (p<0.05). The maximum production was found for proinflammatory chemokine IP-10 and IFN-γ and anti-inflammatory cytokine IL-10. The major difference of mesenchymal stromal cells from epithelial BxPC-3 cells was 7-fold higher production of IL-10, which can explain the immunosuppressive effect of mesenchymal stromal cells.

Antiproliferative Effects of Mesenchymal Stem Cells and Epithelial Cells on Lymphocytes.

We analyzed the interactions between peripheral blood lymphocytes from heterologous donors with mesenchymal stem cells obtained from the tooth pulp and trophoblast. In mixed cultures, proliferation of both lymphocytes and mesenchymal stem cells was suppressed. Similar suppressive effects were observed in lymphocyte cultures mixed with epithelial cells (hepatocytes HeG2 and renal epithelial cells HEK293). This suppression can be determined by impairment of normal adhesion contacts between cells of different origin.

Interaction of Lymphocytes with Mesenchymal Stem Cells.

We studied the interaction of neural stem cells and dental pulp-derived mesenchymal stem cells with lymphocytes from autologous and heterologous donors. Flow cytometry analysis with the use of CFSE-labeled lymphocytes demonstrated an increase in the content of proliferating CD8, CD16 and CD56 cells, but not CD4 cells in cultures of HLA-DR-negative mesenchymal stromal cells from the dental pulp co-cultured with lymphocytes. In neural cultures expressing HLA-DR, all subpopulations of T cells and NK cells were activated. No differences between the autologous and heterologous cultures were revealed.

[Mechanisms of the interaction of chitosan and its derivatives with the cell].

The review summarizes the data on the mechanisms of the interaction of chitosan and other molecules, as well as the drug delivery systems based on them, with mammalian cells. The mechanisms of binding, endocytosis, and the further distribution of chitosan and nanoparticles with different physic-chemical properties in cells remain unclear at the present time.

[Analysis of toxicity and biocompatibility of chitosan derivatives with different physico-chemical properties].

A comparative study of the toxicity and hemocompatibility of chitosan and its derivatives with different acetylation degrees, molecular masses, charges, and hydrophobicity has been performed. It has been shown that only positively charged chitosan derivatives activate platelets and suppress cell proliferation, regardless of the acetylation degree, molecular mass, and hydrophobicity. Chitosan quaternization decreases toxicity at a low degree of substitution and abruptly increases it at a high one. Negatively charged chitosan derivatives were nontoxic and compatible with blood components. It was concluded that the toxicity of chitosan and its derivatives is defined by their charge and solubility at a neutral pH.

Innate immunity: Bacterial cell-wall muramyl peptide targets the conserved transcription factor YB-1.

The bacterial cell wall muramyl dipeptides MDP and glucosaminyl-MDP (GMDP) are powerful immunostimulators but their binding target remains controversial. We previously reported expression cloning of GMDP-binding polypeptides and identification of Y-box protein 1 (YB-1) as their sole target. Here we show specific binding of GMDP to recombinant YB-1 protein and subcellular colocalization of YB-1 and GMDP. GMDP binding to YB-1 upregulated gene expression levels of NF-κB2, a mediator of innate immunity. Furthermore, YB-1 knockdown abolished GMDP-induced Nfkb2 expression. GMDP/YB-1 stimulation led to NF-κB2 cleavage, transport of activated NF-κB2 p52 to the nucleus, and upregulation of NF-κB2-dependent chemokine Cxcr4 gene expression. Therefore, our findings identify YB-1 as new target for muramyl peptide signaling.

Intracellular sorting of differently charged chitosan derivatives and chitosan-based nanoparticles.

Chitosan (Chi) is a biodegradable nontoxic polycation with multiple reactive groups that is easily used to obtain derivatives with a desired charge and hydrophobic properties. The aim of this work was to study the intracellular traffic of positively charged hexanoyl-chitosan (HC) or HC-based nanoparticles (HCNPs) and negatively charged succinoyl-chitosan (SC) and SCNPs in epithelial and macrophage cell lines. By using flow cytometry we demonstrated that positively charged HC adhered to cell membranes quicker and more efficiently than negatively charged SC or NPs. However confocal studies showed that SC and SCNPs penetrated cells much more efficiently than HC while HCNPs did not enter the epithelial cells. Macrophages also phagocyted better negatively charged material but were able to engulf both HC and HCNPs. Upon entering the cells, SC and SCNPs were co-localized with endosomes and lysosomes while HC was found in mitochondria and, to a lesser extent, in lysosomes of epithelial cells. Macrophages, RAW264.7, more efficiently transported all Chi samples to the lysosomal compartment while some positively charged material was still found in mitochondria. Incubation of Chi derivatives and ChiNPs at pH specific to mitochondria (8.0) and lysosomes (4.5) demonstrated the neutralization of Chi charge. We concluded that epithelial cells and, to a lesser extent, macrophages sort charged material to the organelles neutralizing Chi charge.

Expression of duodenase-like protein in epitheliocytes of Brunner's glands in human duodenal mucosa.

A duodenase, a protease structurally related to human cathepsin G, was found earlier in bovine duodenal mucosa. It was demonstrated that under the influence of duodenase an enteropeptidase zymogen is activated in vitro showing the possible participation of duodenase in the cascade of activation of digestive enzymes. To identify a duodenase functional analog in human duodenum, an immunofluorescence study of duodenal mucosa was conducted by confocal microscopy using antibodies to human cathepsin G and to bovine duodenase. The previously unknown place of synthesis and secretion of cathepsin G - Paneth cells located at the bottom of Lieberkuhn crypts - was revealed. Binding of cathepsin G-specific antibodies in a rough endoplasmic reticulum zone and in the cryptal duct was observed. Duodenase-specific immunofluorescence but not that of cathepsin G was found in the epitheliocytes and secretory ducts of Brunner's glands, which are characteristic sites of duodenase biosynthesis in cattle. Binding of CD14-specific antibodies in the Brunner's glands, where the antibodies co-localized with the antibodies to duodenase, was also demonstrated. These data indicate the presence of a protein immunologically similar to duodenase in the human duodenal mucosa. Our study demonstrated the absence of its co-localization with cathepsin G in Brunner's glands.

Lipophilic prodrugs of a triazole-containing colchicine analogue in liposomes: biological effects on human tumor cells.

Colchicine site binders--blockers of tubulin polymerization--are potential antimitotic agents for anticancer therapy. To reduce their systemic toxicity and improve biodistribution, encapsulation in nanosized liposomes may be employed. Liposomes present a convenient means for preparation of injectable formulations of hydrophobic compounds, however colchicine as such is known to leak through the lipid bilayer. In this study, newly synthesized triazole-containing analogues of colchicine and allocolchicine, and their palmitic and oleic esters (lipophilic prodrugs) were tested for anti-proliferative activity and apoptosis-inducing potential. In contrast to colchicine conjugates, whose activities ranged with those of colchicine, allocolchicine derivatives exhibited drastically lower effects and were discarded. Liposomes of about 100 nm in diameter composed of egg phosphatidylcholine--yeast phosphatidylinositol--palmitic or oleic prodrug, 8 : 1: 1, by mol, were prepared by standard extrusion technique and tested in a panel of four human tumor cell lines. Liposome formulations preserved the biological activities of the parent colchicinoid the most towards human epithelial tumor cells. Moreover, liposomal form of the oleoyl bearing colchicinoid inhibited cell proliferation more efficiently than free lipophilic prodrug. Due to substantial loading capacity of the liposomes, the dispersions contain sufficient concentration of the active agent to test wide dose range in experiments on systemic administration to animals.

Susceptibility of mice to invasive aspergillosis correlates with delayed cell influx into the lungs.

Ubiquitous fungus Aspergillus fumigatus (A. fumigatus) is involved in invasive pulmonary aspergillosis (IPA), a frequent infection in immunocompromized patients. Genetic differences are likely to play a role predisposing to IPA. This study was aimed to compare six genetically different mouse strains in their susceptibility to IPA and to determine possible mechanisms involved in the pathogenesis of this infection. Immunosuppressed BALB/c and C57BL/6 mice infected with A. fumigatus conidia were more resistant to IPA than DBA/1, DBA/2, CBA, and A/Sn strains. Phagocytosis of A. fumigatus conidia by blood polymorphonuclear neutrophils (PMN) or bone marrow derived dendritic cells showed no difference between strains. All IPA susceptible strains demonstrated decreased PMN influx into the lungs during infection compared with resistant strains. Flow cytometry analysis of the composition of lung infiltrating cells showed that IPA susceptible mice had a decreased number of phagocytes before the infection. After infection the numbers of Gr-1(+)CD11b(+) PMN cells in the lungs of immunosuppressed mice increased from 10-20% to 50-60% while the percentage of CD11(+)F4/80(+) resident macrophages was unchanged. Among susceptible strains DBA/2 and A/Sn have a defect in C5 component of complement. Injection of normal serum into complement deficient but not into complement sufficient CBA or DBA/1 mice significantly improved their survival. We showed that complement replacement significantly increased PMN homing to the lungs of complement deficient mice. Thus, defect in complement system can predispose to IPA. Our results demonstrated that early influx of PMN into the lungs of mice is important for the resistance to IPA.

Mucoadjuvant properties of lipo- and glycoconjugated derivatives of oligochitosans.

Chitosan, (1-4)-2-amino-2-deoxy-beta-d-glucan, is a deacetylated form of chitin, an abundant biodegradable, positively charged natural polysaccharide. Chitosan is used for antigen delivery through mucosal barrier due to its ability to disrupt tight junctions. Here we produced new water-soluble low-molecular weight chitosan (LMW-Chi) lipid derivatives and compared their ability to stimulate humoral response with the effect of unmodified LMW-Chi or its oligosaccharide derivatives. LMW-Chi effectively penetrated into macrophage-like, lymphoid and epithelial cells. It also stimulated in mice IgG production to model proteins delivered either by subcutaneous or intranasal routes. Adjuvant effect of chitosan derivatives was comparable to or lower than that of unmodified LMW-Chi. Thus, it is possible that adjuvant effect is induced by unmodified glucosamine units of chitosan.