Osteogenic Commitment of MSC Is Enhanced after Interaction with Umbilical Cord Blood Mononuclear Cells In Vitro.

The effectiveness of stroma-dependent expansion of hematopoietic cells ex vivo may depend on the level of commitment of multipotent mesenchymal stromal cells (MSC). Markers of MSC osteodifferentiation and the level of soluble hematopoiesis regulators were determined during their interaction with umbilical cord blood mononuclears. After 72-h co-culturing, an increase in the expression of ALPL and alkaline phosphatase activity was revealed. In conditioned medium of co-cultures, the levels of osteopontin and osteoprotegerin were elevated and the levels of osteocalcin and sclerostin were reduced. Co-culturing of umbilical cord blood mononuclears with osteocommitted MSC was accompanied by more pronounced increase in the concentration of both positive (GM-CSF and G-CSF) and negative (IP-10, MIP-1α, and MCP-3) regulators of hematopoiesis. Thus, umbilical cord blood mononuclears induced the formation of early osteogenic progenitor phenotype in MSC ex vivo, providing the microenvironmental conditions necessary to support hematopoiesis. Preliminary osteocommitted MSC were more sensitive to the effect of umbilical cord blood mononuclears.

Differential Expression of Bipotent Commitment-Related Genes in Multipotent Mesenchymal Stromal Cells at Different O2 Levels.

The transcriptomic profile associated with osteo- and adipogenic differentiation in growth-arrested multipotent mesenchymal stromal cells (MSCs) from human adipose tissue was analyzed in vitro at 20% (standard laboratory) and 5% (tissue-related) O2 levels. Compared with day 7, at 5% O2 on day 14 spontaneous upregulation of osteo- (RUNX2, SP7, BGLAP, and SPP1) and adipogenic differentiation (CEBPA, PPARG, and ADIPOQ) genes in MSCs was observed (p < 0.05). Thus, upon expansion under tissue-related O2, MSCs demonstrated a bipotent transcriptomic profile, which may contribute to the improvement of their hematopoiesis-supportive function.

Evaluation of committed and primitive cord blood progenitors after expansion on adipose stromal cells.

Umbilical cord blood mononuclear fraction is a valuable source of hematopoietic stem and progenitor cells (CB HSPCs). The rarity of this population is a serious limitation of its application in cell therapy. Ex vivo expansion enables to significantly amplify the number of hematopoietic precursors of different commitment. Here, we expand CB MNCs in co-culture with human adipose tissue-derived stromal cells (ASCs) to enrich HSPCs and describe phenotypic features of newly formed hematopoietic populations. The CD34+-HSPCs demonstrated 6-fold enrichment with 9000 CFUs per 50 × 103 HSPCs on average. A part of the floating HSPCs were bearing lineage markers, while others were primitive precursors (CD133-/CD34+). Among ASC-associated HSPCs, two subsets of cord blood-borne cells were revealed: СD90+/СD45- and СD90+/СD45+. The proportion of CD3+/CD8+ and NK-T as well as CD25+ and HLA-DR+ Т cells among СD90+/СD45- cells was significantly higher compared to MNCs and floating HSPCs. More than 80% of CD45+/СD90+ HSPCs were identified as late primitive precursors (CD133-/CD34+). Thus, CB MNC expansion in the presence of ASCs provides the generation of both lineage committed lymphoid progenitors and CD34+/CD133- primitive HSPCs. Substantially enriched with primitive precursors, ASC-associated HSPCs could be considered as a perspective tool for a long-term restoration of hematopoiesis in various hematologic disorders.

Effect of 30-Day Hindlimb Unloading and Hypergravity on Bone Marrow Stromal Progenitors in C57Bl/6N Mice.

We studied the effect of 30-day hindlimb unloading and subsequent simulated hypergravity on the cellularity and proliferative, clonogenic, and differentiation potential of bone marrow stromal progenitors in mice. Clonogenic and differentiation activity of stromal cells decreased after unloading; proliferative and differentiation activity of bone marrow stromal progenitors increased after hypergravity simulation. Our findings demonstrated negative effect of unloading on functional activity of mouse bone marrow stromal progenitors. Short-term hypergravity after unloading produced a stimulating effect on the bone marrow stromal progenitors.

[PROFILE OF THE MARROW-DERIVED STROMAL PRECURSORS POPULATION IN C57BL/6N MICE FLOWN ON BIOSATELLITE BION-M1].

The CFU-F number, proliferative activity and spontaneous differentiation potential of stromal cells derived from the tibia marrow of C57BL/6N mice readapted to the 1-g gravity following a long-term flight on biosatellite Bion-M1 were evaluated. The CFU-F number, proliferative activity and spontaneous adipogenic and osteogenic differentiation of marrow-derived stromal cells from the space flown group were no different from the group of vivarium control. However, the proliferative activity and adhesion properties of the cells were down-regulated on day 7 of readaptation. These results suggest that space flight factors did not impact the stromal differon of the mouse marrow. The decline of stromal cells activity indicates the decompensation of their functions under 1g gravity.

Enrichment of umbilical cord blood mononuclears with hemopoietic precursors in co-culture with mesenchymal stromal cells from human adipose tissue.

We demonstrated the possibility of enrichment of umbilical cord blood mononuclear fraction with early non-differentiated precursors under conditions of co-culturing with mesenchymal stromal cells from the human adipose tissue. It was established that umbilical cord blood mononuclear cells adhered to mesenchymal stromal cell feeder and then proliferate and differentiate into hemopoietic cells. In comparison with the initial umbilical cord blood mononuclear fraction, the cell population obtained after 7-day expansion contained 2-fold more CFU and 33.4 ± 9.5 and 24.2 ± 11.2% CD34(+) and CD133(+) cells, respectively, which corresponds to enrichment of precursor cell population by 148 ± 60. The proposed scheme of expansion of hemopoietic cells from umbilical cord blood is economically expedient and can widely used in biology and medicine.

Changes in levels of gene expression in human aortal intima during atherogenesis.

Changes in the contents of 36 mRNAs species related to lipid turnover, inflammation, metabolism and the action of sex hormones in samples of aortal intima along the "intact tissue - lesions of type I - lesions of type II - lesions of type Va" sequence were analyzed using quantitative PCR. The expression of several mRNAs coding for components of the vesicular transfer and lipid turnover machinery was found to be resistant to atherogenesis or even decline in the course of atherogenesis. Decrease in expression was also recorded for steroid sulfatase, androgen receptor, and low density lipoprotein receptor mRNAs. However, the contents of the majority of other mRNA species increased gradually during disease progression. The earliest changes found as early as in lesions of type I were characteristic for estrogen sulfotransferase, apolipoprotein E, scavenger receptor SR-BI, collagen COL1A2, as well as chemokine CCL18 mRNAs. The contents of several mRNAs in intact tissue and atherosclerotic injuries had gender differences. Additionally, responses of two mRNAs, for aromatase and sterol regulatory element binding protein 2, to atherosclerotic lesion were also sex-differentiated. The contents of the majority of analyzed mRNAs in peripheral blood monocyte-derived macrophages were higher than in intact aorta. The correlations found in atherosclerotic lesions between mRNA species that predominant in macrophages and those expressed at comparable levels in macrophages and intact aorta or mainly in aorta suggest that the observed rise in the content of the majority of mRNAs during atherogenesis is determined by increase in expression in resident cells. The data suggest that the revealed absence of homeostatic regulation of expression of a number of genes associated with vesicular transfer and lipid turnover can serve as one of the reasons for lysosomal function insufficiency that leads to foam cell formation in atheroma. The observed sex differences in expression of a number of mRNAs suggest that estrogens in women perform their atheroprotective effects starting with predisposition to the disease and finishing with advanced stages of the pathologic process.

Effect of sex hormones on levels of mRNAs coding for proteins involved in lipid metabolism in macrophages.

The effects of sex hormones estradiol (E2), testosterone (Te), and 5α-dihydrotestosterone (DT) on cholesterol accumulation induced by modified low density lipoproteins (LDL) in macrophages differentiated from human peripheral blood monocytes and on the levels of mRNAs coding for proteins involved in lipid metabolism have been studied. All three hormones at physiological concentrations (1 nM) are capable of reducing cholesterol accumulation in cells. The treatment of cells with modified and native (not inducing cholesterol accumulation) LDL results in similar alterations in the expression of several mRNAs aimed primarily at homeostatic regulation of lipid metabolism. These alterations depend on the sex of macrophage donors and in some cases are even reversed in cells obtained from male and female donors. The cells not treated with modified LDL have no significant gender differences in the expression of the examined mRNAs. Hormones, either independently or in combination with the modified LDL, influence the levels of some mRNAs, and each hormone shows an individual range of effects. Correlation analysis of changes in mRNA content in the cells showed that the hormones may interfere with coordination of gene expression. Hormone action leads to: (1) reduced coupling of the content of individual mRNAs with their initial levels in the control cells; (2) reduced coupling of different mRNA levels; (3) regrouping of mRNAs between the clusters; and (4) changes in the number of factors that determine the correlation links between mRNAs. The data show that sex hormones may have impact on the level of expression of certain genes and, in particular, on the coordination of gene expression in macrophages.

[Human lymphocyte immunophenotype after interaction with mesenchymal stromal cells].

Human multipotent mesenchymalstromal cells were cocultured for 72h with allogeneic blood-borne mononuclear cells (MNCs) of different maturity (lymphocytes from adult peripheral blood and umbilical cord blood) and functional state (intact and PHA-activated human peripheral blood lymphocytes): After coculture with MMSCs the share ofB-cells among MNCs and cbMNCs decreased. The proportion ofT- and NK cells declined only among cbMNCs (p < 0.05). Only T-NK subpopulation reduced among MNC and cbMNC T-cells. The drop of CD8+ cells was detected in coculture of MMSCs and PHA-MNCs. Activated HLA-DR+ cells declined, CD25+ cells increased compared to monoculture of PHA-MNCs. MMSCs supported MNC, PHA-MNC and cbMNC viability. These results are important for understanding the physiology of the allogeneic cell-to-cell interaction in connection of potential clinical application of allogeneic cell products of blood-borne and stroinal origin.

Immunosuppressive effects of multipotent mesenchymal stromal cells in cultures with different O2 content in the medium.

We studied the effects of multipotent mesenchymal stromal cells isolated from the adipose tissue on proliferation and viability of immunocompetent cells at different concentration of O(2) (5 and 20%) in culture medium. It was shown that co-culturing with multipotent mesenchymal stromal cells 3-fold reduced proliferative index of phytohemagglutinin-activated lymphocytes, while their viability remained unchanged and did not depend on partial oxygen pressure in the medium. These findings suggest that low O(2)concentration in tissues will not affect immunosuppressive properties of multipotent mesenchymal stromal cells, which is very important for their application in regenerative medicine.

Subpopulation composition and activation of T lymphocytes during coculturing with mesenchymal stromal cells in medium with different O(2) content.

The concentration of O(2) during coculturing practically did not affect the subpopulation composition of T lymphocytes (CD3(+)/CD4(+), CD3(+)/CD8(+), CD3(+)/CD16(+)/CD56(+) T cells) under conditions of PHA-induced activation. Coculturing with mesenchymal stromal cells (MSC) led to a significant decrease in the ratio of lymphocytes carrying activation markers (CD3(+)/CD25(+) and CD3(+)/HLA-DR(+)) and increase in the number of CD3(+)/CD16(+)/CD56(+) T cells. The percent of activated HLA-DR(+) T cells in a heterotypic culture with MSC at 5% O(2) was much lower than that observed under normal conditions of culturing (20% O(2)). Our results suggest that antigen presentation by T lymphocytes due to HLA-DR expression can be reduced in the target tissues at low concentration of O(2), while the interaction between allogeneic MSC probably contributes to more significant inhibition of the immune response.

[Dynamics of immunological characteristics and investigation of apoptosis of palatal tonsillar lymphocytes in patients presenting with chronic tonsillitis and treated by conservative therapy].

The enhanced amount of viable lymphocytes and the decreased number of apoptotic cells as well as the reduced levels of IgA and IgM and the elevated concentration of sIgA in lacunar secretion are considered to be the reliable criteria for the efficacy of the conservative treatment of chronic tonsillitis. Combined therapy of this condition including irrigation of the palatal tonsillar lacunae with a miramistin solution, their contact ultrasonic treatment, and application of imudon makes it possible to maintain the optimal ratio of viable to apoptotic lymphocytes during a period of up to 6-7 months.

Сord blood hematopoietic stem cells ex vivo enhance the bipotential commitment of adipose mesenchymal stromal progenitors.

AIMS: Stroma-dependent ex vivo expansion of hematopoietic stem progenitor cells (HSPCs) is a valid approach for cell therapy needs. Our goal was to verify whether HSPCs can affect stromal cells to optimize their functions during ex vivo expansion. MAIN METHODS: HSPCs from cord blood (cb) were cocultured with growth-arrested adipose mesenchymal stromal cells (MSCs). Commitment-related transcriptional and secretory profiles as well as hematopoiesis-supportive activity of intact and osteo-induced MSCs were examined. KEY FINDINGS: During expansion, cbHSPCs affected the functional state of MSCs, contributing to the formation of early stromal progenitors with a bipotential osteo-adipogenic profile. This was evidenced by the upregulation of certain MSC genes of osteo- and adipodifferentiation (ALPL, RUNX2, BGLAP, CEBPA, ADIPOQ), as well as by elevated alkaline phosphatase activity and altered osteoprotein patterns. Joint paracrine profiles upon coculture were characterized by a balance of "positive" (GM-SCF) and "negative" (IP-10, MIP-1α, MCP-3) myeloid regulators, effectively supporting expansion of both committed and primitive cbHSPCs. Short-term (72 h) osteoinduction prior to coculture resulted in more pronounced shift of the bipotential transcriptomic and osteoprotein profiles. The increased proportions of late primitive CD133-/CD34+cbHSPCs and unipotent CFUs suggested that cbHSPCs after expansion on osteo-MSCs were more committed versus cbHSPCs from coculture with non-differentiated MSCs. SIGNIFICANCE: During ex vivo expansion, cbHSPCs can drive the bipotential osteo-adipogenic commitment of MSCs, providing a specific hematopoiesis-supportive milieu. Short-term preliminary osteo-induction enhanced the development of the bipotential profile, leading to more pronounced functional polarization of cbHSPCs, which may be of interest in an applied context.

Beta-blockers: propranolol, metoprolol, atenolol, pindolol, alprenolol and timolol, manifest atherogenicity on in vitro, ex vivo and in vivo models. Elimination of propranolol atherogenic effects by papaverine.

The addition of the beta-blockers propranolol, metoprolol, atenolol, pindolol, alprenolol and timolol to a culture of peritoneal macrophages or smooth muscle cells induced an increase in the intracellular cholesterol content. Blood serum obtained from a rabbit after a peroral administration of beta-blockers also induced cholesterol accumulation. This property of drug or blood serum obtained after peroral administration is conventionally referred to as atherogenic potential or atherogenicity. Regular administration of propranolol during a 21-day period evoked stable atherogenicity of rabbit blood serum. This was accompanied by stimulation of manifestations of atherosclerosis in the aorta deendothelialized with a balloon catheter. Propranolol increased neointimal thickening, lipid accumulation, an increase in cell number and in the collagen content. In vitro, the combination of propranolol with papaverine eliminated the atherogenic effect of propranolol which manifested itself as stimulation of cholesterol accumulation in cultured cells. Simultaneous peroral administration of propranolol and papaverine prevented the appearance of serum atherogenicity. Papaverine eliminated neointimal thickening, an increase in cell number and in the lipid and collagen contents evoked by propranolol. Papaverine itself had no effect on these parameters. Thus, the atherogenicity of propranolol as well as capacity of papaverine to eliminate beta-blocker atherogenicity revealed in cell culture was confirmed in vivo. We hope that these results may be useful in the development of new drugs and optimization of antiatherosclerotic drug therapy.

Stellate cells of aortic intima: I. Human and rabbit.

Stellate cells hitherto accounted exclusively in the innermost elastic-hyperplastic layer were already reported to inhabit human aortic intima. The present paper shows that most of these cells are situated just beneath the endothelium. Stellate cells also appear in the deendothelialization-induced myointimal thickening of rabbit aorta. In the myointimal thickening these cells were revealed in the direct proximity to the endothelium. A conclusion is available that the previously demonstrated polymorphism of human aortic intimal cells may be reproduced in a simple experimental model, which gives new possibilities for the study of the cellular polymorphism in the vessel wall.

[The structure of the endothelialized and nonendothelialized areas of myointimal thickening of the rabbit aorta]. / Struktura éndotelizirovannoi i neéndotelizirovannoi zon miointimal'nogo utolshcheniia aorty krolika.

De-endothelialization of the abdominal aorta of the rabbit was performed with a balloon catheter. As a result, a partially endothelialized myointimal thickening was formed consisting of smooth myocytes and macrophages. As compared with the zone devoid of the endothelium the endothelialized zone was characterized by less thickness and by the presence of an additional elastic membrane. Great amount of leukocytes and solitary thrombocytes were adhesed just in the place of contact. In the center of nonendothelialized zone no adhesion was observed. In the zone covered by the endothelium smooth myocytes of the synthetic and contractile phenotype were seen with similar frequency. The nonendothelialized zone was presented mainly by smooth myocytes of the synthetic phenotype and macrophages. The myointimal thickening of the rabbit aorta might be considered as an informative model for analysis of myoendothelial interactions.

[Structure of the neointima after balloon catheter injury of the rabbit aorta in the context of hypercholesterolemia: analysis of endothelial influence on lipid accumulation]. / Struktura neointimy aorty krolika posle povrezhdeniia ballonnym kateterom na fone giperkholesterinemii: analiz vliianiia éndoteliia na nakoplenie lipidov.

Balloon catheter de-endothelialization of abdominal aorta of cholesterol-fed rabbits was performed. As a result, partly endothelialized neointima was formed, consisting of smooth muscle cells and macrophages. Neutral lipids stored more intensely in non-endothelialized part of neointima. Preferential intracellular lipid accumulation in this area was observed while predominantly extracellular lipid accumulation was found in endothelium-covered part of neointima. It is supposed that endothelium inhibits intracellular lipid accumulation and at the same time stimulates an extracellular one, but as a whole it reduces the density of lipid inclusions in neointima.

[Hypotensive effect of long-acting garlic tablets allicor (a double-blind placebo-controlled trial)]. / Gipotenzivnyi éffekt chesnochnykh tabletok prolongirovannogo deistviia allikor (dvoinoe slepoe platsebo-kontroliruemoe issledovanie).

AIM: To evaluate a hypotensive action of long-acting garlic powder tablets allicor in patients with mild or moderate hypertension and to compare allicor effects with those of foreign analog--kwai garlic tablets. MATERIAL AND METHODS: A double-blind, randomized and placebo-controlled study enrolled 85 patients with mild or moderate hypertension. The patients were divided into 4 groups: group 1 received allicor in a dose 600 mg/day, group 2--2400 mg/day, group 3--kwai in a dose 900 mg/day, group 4--placebo. RESULTS: Allicor produced reaction in both systolic and diastolic pressure. An increase of allicor daily dose to 2400 mg does not provide an additional hypotensive effect. Kwai results in only systolic but not diastolic arterial pressure lowering. CONCLUSION: Allicor is more effective than kwai in reduction of diastolic blood pressure. It can be recommended as a hypotensive treatment in mild and moderate arterial hypertension.

[Garlic effectiveness in rheumatoid arthritis]. / Effektivnost' chesnoka pri revmatoidnom artrite.

AIM: To perform of clinical trial of alisate--a garlic preparation produced in Russia. MATERIALS AND METHODS: An open controlled trial of alisate enrolled 30 patients with rheumatoid arthritis (RA). 15 patients with RA of varying clinical form, stage and activity were given alisate in a dose 300 mg (1 tablet) twice a day for 4-6 weeks. 15 control RA patients received conventional antirheumatic therapy. RESULTS: The alisate group achieved a good and partial response in 86.5% of cases. The drug was well tolerated and had no side effects. In control group, some parameters changed for the worse. CONCLUSION: Alisate can be recommended for treatment of RA patients in combined and monotherapy.

[Effect of long-acting garlic tablets "allicor" on the incidence of acute respiratory viral infections in children]. / Vliianie chesnochnykh tabletok prolongirovannogo deistviia "allikor" na zabolevaemost' ostrymi respiratornymivirusnymi infektsiiami u detei.

AIM: To elucidate the prospects administration of allicor (long-releasing garlic tablets) in prevention of acute respiratory diseases (ARD) in children vs benzimidazole (dibazole). MATERIAL AND METHODS: At the first stage, tolerance of allicor (600 mg/day) and its effects on ARD morbidity were investigated in an opened 5-month study in 172 children aged 7-16 years compared to 468 controls. As the second stage, the effects of allicor (300 mg/day) on ASRD morbidity were investigated in a double-blind placebo-controlled randomized 5-month trial in 42 children aged 10-12 years in comparison with 41 placebo-treated children and 73 benzimidazole-treated children. RESULTS: At the first stage of the study allicor was not observed to induce gastrointestinal side effects in children at any dosage while ARD morbidity was reduced 2-4-fold as compared to the controls. At the second stage of the study allicor reduced ARD morbidity 1.7-fold compared to placebo and 2.4-fold vs benzimidazole. There was no significant difference in ARD morbidity between placebo- and benzimidazole-treated groups. Health index in allicor-treated group was 1.5-fold higher as compared either to placebo- or benzimidazole-treated children. CONCLUSION: Thus, the results of this study have demonstrated that allicor is effective for non-specific prevention of acute respiratory infections in children and has no side effects. ARD prevention with benzimidazole appeared ineffective in placebo-controlled study, so the development of new useful and safe preparations is of ultimate importance.