Activation of TLR4 of Mesenchymal Stem Cells Enhances the Regenerative Properties of Their Secretomes.

Mesenchymal stem cells (MSC), like macrophages, can be polarized in vitro. In particular, activation of type 4 Toll-like receptor in MSC leads to the appearance of the so-called "proinflammatory" MSC phenotype (MSC1). We showed that secretome (conditioned media) of MSC1 can affect the wound healing processes: promote healing and modulate exudative inflammation and subsequent fibroplastic processes in the damaged area. These effects of secretomes of polarized MSC were superior to those of intact MSC.

Phenotypic Features of Mesenchymal Stem Cell Subpopulations Obtained under the Influence of Various Toll-Like Receptors Ligands.

The paper characterizes phenotypic features of subpopulations of human adipose tissue mesenchymal stem cells (MSC) resulting from exposure to Toll-like receptors ligands. MSC1 and MSC2 phenotypes were induced by exposure to LPS and polyinosinic-polycytidylic acid, respectively. Different exposures to Toll-like receptors ligands, short-term (3 h) and long-term (24 h), were studied. The cytokine profile of cells with the MSC2 phenotype differed depending on the duration of exposure to the inductor, while the cytokine profile of cells with the MSC1 phenotype remained stable. Morphometric features of mesenchymal stem cells with the MSC1 and MSC2 phenotypes, as well as differences in the IDO gene expression were identified. These differences can be used to distinguish between these cell types.

A Cell Model of Human Small Intestinal Wall Based on Genetically Modified Caco-2 Cells.

We propose a cell model of the human small intestinal wall based on genetically modified Caco-2 cells that allows visualization and quantitative assessment of activation of NF-κB factor and related intracellular pathway by using fluorescence microscopy. A dose-dependent increase in fluorescence intensity of the obtained cells in response to TNFα exposure in concentrations of 1-100 ng/ml was demonstrated. It was found that this parameter correlates with a decrease in the transepithelial resistance of the cell monolayer in response to TNFα and can be used to assess the toxic effects of substances on epithelial cells of the human small intestine.

[Proteome of the human HaCaT keratinocytes: Identification of the oxidative stress proteins after sodium dodecyl sulpfate exposur].

Oxidative stress is a universal response of the skin cell damage of various origins. Sodium dodecyl sulfate (SDS, sodium lauryl sulfate) is an anionic surfactant commonly used as an emulsifying detergent in household cleaners. Sodium dodecyl sulfate is the reference compound for testing toxicity on cellular skin models. The effect of sodium dodecyl sulfate in sub toxic dose 25 µg/mL during 48 h on the protein profile of human keratinocytes HaCaT was studied by tandem mass spectrometry with electrospray ionization. In total, 1064 proteins were found in immortalized human keratinocytes HaCaT, of which about 80% were identified by two or more peptides. The change of the 217 proteins content was revealed, among them 39 according to Gene Ontology are associated with oxidative stress. It has been found that sodium dodecyl sulfate leads to a decrease in the number of proteins/peptides containing carboxymethylated and/or carboxyethylated lysine. We concluded about the promising of the cells redox-balance analysis at testing chemicals in the doses, which do not lead to a decrease in their viability. Possible involvement of sodium dodecyl sulfate in the development of cutaneous neoplasia is discussed.

Sodium Dodecyl Sulfate Cytotoxicity towards HaCaT Keratinocytes: Comparative Analysis of Methods for Evaluation of Cell Viability.

Viability of keratinocytes of HaCaT immortalized line incubated with sodium dodecyl sulfate for 3 min, 1 and 48 h, was studied by light microscopy, MTT test, and neutral red absorption test. The IC50 values were determined for each of the studied lengths of exposure. HaCaT cells exhibited a dose-dependent decrease of viability under the effect of sodium dodecyl sulfate, proportional to the length of exposure. The values measured by different methods (MTT test and neutral red absorption test) varied, the differences were determined by the duration of exposure to sodium dodecyl sulfate. The dispersion of values for 1 and 48 h exposure, obtained by MTT method, was greater than of the values obtained by neutral red absorption test.

Changes in the Proteome of HaCaT Keratinocytes Induced by Cytotoxic Substance Triton X-100.

Changes in the proteome of keratinocytes of immortalized HaCaT line exposed to cytotoxic substance Triton X-100 in concentrations of 12.5 and 25 µg/ml were studied by liquid chromatography combined with mass spectrometry. The appearance of proteins involved in the regulation of mitosis, RNA stability, and catabolic processes were detected; the number of apoptosis-associated proteins increased, while the number of proteins involved in differentiation and energy metabolism of keratinocytes decreased.

Effect of oxidized dextran on reparative regeneration of the skin after burn injury.

In adult Wistar rats, a 3B degree skin burn was modeled and treated by daily application of 5% aqueous solution of oxidized dextran (molecular weight of 60 kDa) on the wound surface. In animals treated with oxidized dextran, neutrophil count in the connective tissue adjacent to the wound increased by day 5 and then decreased by day 21 after burn infliction; proliferation of fibroblasts was observed later than in untreated animals, in whom inflammation run a subacute course. Oxidized dextran increased the content of macrophages in the wound and surrounding connective tissue from days 14 to 21 after burn infliction and promoted effective and complete healing of the skin defect. Regeneration was realized mainly due to proliferation of keratinocytes at the wound edges and was completed by 7 days earlier than in untreated animals, in whom the area of injury by day 21 decreased by only 2 times (vs. 10 times in treated rats).

Structural and functional peculiarities of mast cells in undifferentiated connective tissue dysplasia.

We performed an immunomorphological study of mast cells from undamaged skin in women with phenotypical evidence of undifferentiated connective tissue dysplasia syndrome, patients of cosmetological clinics. It was found that the numerical density of mast cells containing chymase granules in this condition 1.7-fold surpassed the corresponding parameter in patients without signs of connective tissue dysplasia syndrome, which probably was a result of compensatory and adaptive reaction aimed at activation of the synthesis of the connective tissue extracellular matrix components. It was hypothesized that increased content of chymase-positive mast cells in the skin of patients with connective tissue dysplasia syndrome contributed to the formation of associated arterial hypertension.

Morphofunctional characteristics of the immune system in CBA and C57Bl/6g mice.

We studied peculiarities of morphofunctional organization of the immune system in C57Bl/6g and CBA mice differing by their susceptibility to various types of infectious agents. The revealed differences in the structure of lymphoid organs, T lymphocyte subpopulation ratio and their differentiation into Th1/Th2 cells after mitogen stimulation drove us to a conclusion on genetically determined regularities in the development of the immune response in these animal strains.

Protein dataset of immortalized keratinocyte HaCaT cells and normal human keratinocytes.

Learning of the molecular mechanisms of the pathological processes development in the normal human keratinocytes (NHK) are difficult. Immortalized keratinocytes HaCaT are often used as an analogue of NHK since they have a number of advantages over the latter - they do not require the presence of growth and differentiation factors in the medium, have unlimited potential for proliferation, demonstrate stable phenotype regardless of the number of passages [1]. Taking into account the properties and characteristics of the HaCaT line, these cells can be considered as a promising experimental model for research of various physiological processes occurring in human keratinocytes. However, to understand the limitations of such an experimental model, a detailed comparative characterization of HaCaT and NHK is required, which can be obtained by carrying out its proteomic analysis. In this article we present datasets obtained through the high-throughput shotgun proteomics analysis of normal human keratinocytes and immortalized HaCaT keratinocytes. As a protocol for proteomic profiling of cells, we used the approach of obtaining LC-MS / MS measurements followed by their processing with Progenesis LC-MS software (Nonlinear Dynamics Ltd.). The mzML files were deposited to the Mendeley Data.

[Comparative study of the human keratinocytes proteome of the HaCaT line: identification of proteins encoded by genes of 18 chromosomes under the influence of detergents]. / Sravnitel'noe issledovanie proteoma kletok HaCaT keratinotsitov cheloveka: identifikatsiia belkov, kodiruemykh genami 18 khromosomy pri vozdeistvii detergentov.

Using electrospray ionization tandem mass spectrometry, a comparative analysis of the HaCaT keratinocyte proteins encoded by the 18th chromosome was performed before and after exposure to sodium dodecyl sulfate (25 mg/ml) and to Triton X-100 (12.5 mg/ml) in a subtoxic dose for 48 hours. Proteins were identified using the SearchGUI platform (X!Tandem and MS-GF+ search engines). In total, 1284 proteins were found in immortalized human HaCaT keratinocytes and about 75% of them were identified by two or more peptides. Were identified, that 26 proteins were encoded by genes of chromosome 18. Among these proteins, 17 were common for control cells and HaCaT cells treated with SDS. Proteins MARE2 and CTIF were identified only in control keratinocytes. Seven identified proteins encoded by genes of chromosome 18 were found only in detergent-treated keratinocytes: LMAN1, NDUV2, SPB3, VPS4B, KDSR, ROCK1 and RHG28.

Proteome dataset of mouse macrophage cell line infected with tick-borne encephalitis virus.

We report the proteomic datasets on the mouse macrophage cell line PMJ2R infected with tick-borne encephalitis virus (TBEV) for two and six days. Data were acquired using shotgun ultra-high resolution mass spectrometry. Peptide identifications were done using the Mascot version 2.4 (Matrix Science), and quantification was performed by a label-free approach. Protein profiles of early (two days) and late (six days) stages of infection were compared between each other and the respective control samples. Protein profiles of infected and control samples differed in the number of identified proteins and their relative abundances. Proteins detected in the TBEV-infected cells were involved in various processes related to the infection, including defense response against the virus, regulation of viral process, negative regulation of viral genome replication, RNA binding, or innate immune response. Also, proteins specific for the early and late stages of infection were identified.

Structural peculiarities of epidermal barrier in individuals with syndrome of undifferentiated connective tissue dysplasia.

Histological, morphometric, and immunohistochemical analysis showed that the specific features of epidermal barrier structure in patients with undifferentiated connective tissue dysplasia syndrome are increases number of keratinocyte rows in the basal and prickly layer as a result of their higher mitotic activity, increased numerical density of Langerhans cells, and suppression of terminal keratinocyte differentiation.

Phagocytosis of hybrid molecular nanosomal compositions containing oxidized dextrans conjugated with isonicotinic acid hydrazide by macrophages.

We studied phagocytic activity of macrophages towards hybrid molecular nanosomal compositions consisting of 150-800-nm nanoliposomes containing oxidized dextrans with a molecular weight of 35 and 60 kDa obtained by chemical ("permanganate") and radiochemical oxidation of dextran conjugated with isonicotinic acid hydrazide (dextrazides, intracellular prolonged antituberculous drugs). Phagocytic activity of macrophages towards hybrid molecular nanosomal compositions containing dextrazides obtained by chemical oxidation of dextrans is higher than activity towards hybrid molecular nanosomal compositions containing dextrazides prepared by radiochemical oxidation and depends on the size of hybrid molecular nanosomal compositions and molecular weight of oxidized dextrans.

Phagocytic activity of macrophages against liposomes with conjugates of oxidized dextrans and isonicotinic acid hydrazide during modeling of phagocytosis disturbances in vitro.

We studied phagocytic activity of macrophages against molecular-liposome hybrid compositions consisting of liposomes (diameter 200-450 nm) containing oxidized dextrans with a molecular weight of 35 or 60 kDa conjugated with the basic antituberculosis preparation isonicotinic acid hydrazide (dextrazides) during modeling of various disturbances of endocytosis function of phagocytic cells in vitro. Preincubation of macrophages with trypsin, colchicine, or sodium azide did not change the parameters of adhesion of molecular-liposome hybrid compositions to macrophages. It was found that preincubation of cells with colchicine or sodium azide reduced parameters of phagocytosis of the molecular-liposome hybrid compositions; this reduction did not depend on the molecular weight of dextrans entering the composition of the molecular-liposome hybrid compositions.

Effects of molecular liposomal hybrid compositions with oxidized dextrans and isonicotinic acid hydrazide on production of granulocytic macrophage colony-stimulating factor by macrophages.

The effects of molecular liposomal hybrid compositions consisting of liposomes (200-450 nm) containing oxidized dextrans (dextranals; 35-60 kDa) conjugated with isonicotinic acid hydrazide (dextrazides), their components, and native dextrans on the production of granulocytic macrophage CSF by peritoneal macrophages were studied in vitro. Dextranals proved to be more potent inductors of granulocytic macrophage CSF than native dextrans. Conjugation of nicotinic acid hydrazide with dextranals did not modify their capacity to stimulate the production of granulocytic macrophage CSF. Liposomes in the molecular liposomal hybrid compositions did not attenuate the dextrazide capacity to stimulate the production of granulocytic macrophage CSF. Molecular liposomal compositions containing 60 kDa dextrazide exhibited the most potent stimulatory effect on macrophage production of granulocytic macrophage CSF.

Immunomorphological study of Langerhans cells in the skin of patients with atopic dermatitis.

Immunomorphological study of Langerhans cells in affected skin of patients with moderate exacerbation and remission of atopic dermatitis revealed an increase in numerical density (compared to healthy volunteers) and change in morphological characteristics and topography of these cells.

In vitro effect of oxidized dextrans on peritoneal cells.

We studied the dependence of in vitro dextran biocompatibility on the method of oxidation of 35-kDa dextran. The biocompatibility of dextran oxidized with potassium permanganate was higher compared to that obtained by radiochemical oxidation. It was related to the formation of peroxide compounds during radiochemical oxidation.

Comparative study of the in vitro effect of nanoliposomes with oxidized dextrans on peritoneal cells.

We studied the in vitro effect of hybrid molecular-nanosomal biocompatible compositions on cultured peritoneal cells. The compositions consisted of oxidized dextrans with a mean molecular weight of 35 and 60 kDa, which were obtained by chemical and radiochemical oxidation of dextran. Hybrid nanoliposomal compositions of chemically oxidized dextran (permanganate method) had greater biocompatibility and tropic activity to macrophages compared to nanoliposomes of radiochemically oxidized dextran.

[Quality control study of engineered skin tissue]. / Metod otsenki kachestva rekonstruirovannogo épidermisa.

OMERO service was used to annotate the cell line HaCaT microscope images by two independent expert groups. The images were obtained in the course of developing tissue-engineered epithelium which consisted of several layers of the keratinocytes. Evaluation of expert opinions was performed by calculation of specificity, sensitivity and accuracy. The best convergence of opinions (91%) was achieved for the confluence of the cell monolayers. Accuracy 70% was observed in determining the extent of cell differentiation after 10 days of incubation. The paper illustrates the usefulness of OMERO service for dynamic cross-validation of quality in the development and standardization of cell preparations.