Fibrin, Bone Marrow Cells and macrophages interactively modulate cardiomyoblast fate.

Interactions between macrophages, cardiac cells and the extracellular matrix are crucial for cardiac repair following myocardial infarction (MI). The paracrine effects of cell-based treatments of MI might modulate these interactions and impact cardiac repair. The immunomodulatory capacity of the therapeutic cells is therefore of interest and could be modulated by the use of biomaterials. We first showed that bone marrow cells (BMC) associated with fibrin could treat MI. Then, we interrogated the influence of fibrin, as a biologically active scaffold, on the secretome of BMC and the impact of their association on macrophage fate and cardiomyoblast proliferation. Methods: In vivo, two weeks post-MI, rats were treated with epicardial implantation of BMC and fibrin or sham-operated. High-resolution echocardiography was performed to evaluate the heart function and structure changes after 4 weeeks. Histology and immunostaining were performed on harvested hearts. In vitro, BMC were first primed with fibrin. Second, non-polarized macrophages were differentiated toward either pro-inflammatory or anti-inflammatory phenotypes and stimulated with the conditioned medium of fibrin-primed BMC (F-BMC). Proteomic, cytokine levels quantification, and RT-PCR were performed. EdU incorporation and real-time cell analysis assessed cell proliferation. Results: The epicardial implantation of fibrin and BMC reduced the loss of cardiac function induced by MI, increased wall thickness and prevented the fibrotic scar expansion. After 4 and 12 weeks, the infarct content of CD68+ and CD206+ was similar in control and treated animals. In vitro, we showed that fibrin profoundly influenced the gene expression and the secretome of BMC, simultaneously upregulating both pro- and anti-inflammatory mediators. Furthermore, the conditioned medium from F-BMC significantly increased the proliferation of macrophages in a subsets dependent manner and modulated their gene expression and cytokines secretion. For instance, F-BMC significantly downregulated the expression of Nos2, Il6 and Ccl2/Mcp1 while Arg1, Tgfb and IL10 were upregulated. Interestingly, macrophages educated by F-BMC increased cardiomyoblast proliferation. In conclusion, our study provides evidence that BMC/fibrin-based treatment lowered the infarct extent and improved cardiac function. The macrophage content was unmodified when measured at a chronic stage. Nevertheless, acutely and in vitro, the F-BMC secretome promotes an anti-inflammatory response that stimulates cardiac cell growth. Finally, our study emphases the acute impact of F-BMC educated macrophages on cardiac cell fate.

Single-Cell RNA-Seq of Bone Marrow Cells in Aplastic Anemia

Aplastic anemia (AA) is an autoimmune disease characterized by peripheral blood pancytopenia and bone marrow failure. Recently, a research study verified bone marrow failure of AA patients resulting from hematopoietic stem and progenitor cell (HSPC) attack by active T cells. Nonetheless, whether B cells, as one of the important immune cells, destruct the hematopoiesis is still unclear. Here, a large-scale single-cell transcriptomic sequencing of 20,000 bone marrow cells from AA patients and healthy donors was performed. A total of 17 clusters and differentially expressed genes were identified in each cluster relative to other clusters, which were considered potential marker genes in each cluster. The top differentially expressed genes in HSPCs (S100A8, RETN, and TNFAIP3), monocytes (CXCL8, JUN, and IL1B), and neutrophils and granulocytes (CXCL8, NFKBIA, and MT-CYB) were related to immune and inflammatory injury. Then, the B-cell receptor (BCR) diversities and pairing frequencies of V and J genes were analyzed. The highest pairing frequencies in AA patients were IGHV3-20-IGKJ2, IGHV3-20-IGKJ4, and IGHV3-20-IGHLJ2. Meanwhile, there were 3 V genes, including IGHV3-7, IGHV3-33, and IGLV2-11, with elevated expression in B cells from AA patients. Cell type–specific ligand–receptor was further identified in B-cell interaction with hematopoietic cells in the bone marrow. The changed ligand–receptor pairs involved antigen presentation, inflammation, apoptosis, and proliferation of B cells. These data showed the transcriptomic landscape of hematopoiesis in AA at single-cell resolution, providing new insights into hematopoiesis failure related with aberrance of B cells, and provide available targets of treatment for AA.

Effects of 200cH medications on mice bone marrow cells and macrophages

Paracelsus once wrote: "All things are poison and nothing is without poison, only the dose permits something not to be poisonous." Latter Hahnemann formulated the law of similars, preparations which cause certain symptoms in healthy individuals if given in diluted form to patients exhibiting similar symptoms will cure it. Highly diluted natural complexes prepared according to Hahnemann’s ancient techniques may represent a new form of immunomodulatory therapy. The lack of scientific research with highly diluted products led us to investigate the in vivo and in vitro actions of commonly used medications. Here we describe the results of experimental studies aimed at verifying the effects of Mercurius solubilis, Atropa Belladonna, Lachesis muta and Bryonia alba. All medications were at 200cH dilution. Animals were maintained for 7 days and were allowed to drink the medications, which were prepared in a way that the final dilution and agitation (200cH) was performed in drinking water. The medication bottle was changed and sucussed every afternoon. Co-culture of non treated mice bone marrow cells and in vitro treated peritoneal macrophages were also performed. After animal treatment the bone marrow cells were immunophenotyped with hematopoietic lineage markers on a flow cytometer. We have determined CD11b levels on bone marrow cells after culture and co-culture with treated macrophages and these macrophages were processed to scanning electron microscopy. We have observed by morphological changes that macrophages were activated after all treatments. Mercurius solubilis treated mice showed an increase in CD3 expression and in CD11b on nonadherent bone marrow cells after co-culture with in vitro treatment. Atropa Belladonna increased CD45R and decreased Ly-6G expression on bone marrow cells after animal treatment. Lachesis muta increased CD3, CD45R and, CD11c expression and decreased CD11b ex vivo and in nonadherent cells from co-culture. Bryonia alba increased Ly-6G, CD11c and CD11b expression ex vivo and when in co-culture CD11b was increased in adherent cells as well as decreased in nonadherent cells. With these results we have demonstrated that highly diluted medications act on immune cells activating macrophages, and changing the expression profile of hematopoietic lineage markers. Highly diluted medications are less toxic and cheaper than other commonly used medications and based on our observations, it is therefore conceivable that this medications which are able to act on bone marrow and immune cells may have a potential therapeutic use in clinical applications in diseases were the immune system is affected and also as regenerative medicine as it may allow proliferation and differentiation of progenitor cells.

Analysis of the Appearance of Micronuclei in the Erythrocytes and Activity of Bone Marrow Cells Proliferation after the Prolonged Low Dose Fast Neutrons Irradiation of Mice

Purpose: To analyze the level of cytogenetic damage and the activity of bone marrow cells proliferation in C57BL/6 mice after prolonged fast neutrons low dose irradiation at 10–500 mGy. Material and methods: Male C57BL/6 mice at the age of 7–8 and 16 weeks were used in the experiments. Irradiation was carried out on an OR-M installation in the field of fast neutrons and gamma quanta using five Pu(α,n)Be radionuclide sources with a high fast neutron yield at a dose rate of 2.13 mGy/h. The frequency of polychromatophilic (PCE) and normochromic (NCE) erythrocytes with micronuclei (MN) and the ratio of PCE and NCE were analyzed using light microscopy after cytochemical staining of the bone marrow cells of control and irradiated mice. The proliferation activity of bone marrow cells was determined by the number of Ki-67+-cells. The parameters of the cell cycle and the level of apoptosis were studied after DNA staining with DAPI using flow cytometry. Statistical processing of the results was carried out according to the Student’s method using the computer program Origin. Results: It was found that prolonged irradiation of mice with fast neutrons at a low dose rate (2.13 mGy/h) at doses from 10 to 500 mGy after 24 h led to statistically significant increase in the frequency of PCE with MN at all studied doses. No dose dependence of this parameter was observed in the studied range. The increase in the frequency of PCE with MN at a dose of 500 mGy was prolonged and persisted for at least 72 h. A significant increase in the frequency of NCE with MN 24 h after irradiation was found only at a dose of 500 mGy, which persisted up to 48 h. At this dose, there was also a decrease in the number of nucleated cells in the bone marrow 24 – 72 h after exposure, a decrease in the number of Ki-67+-cells 24 h after irradiation of mice, a block of the cell cycle in the G2/M phase, and a decrease of cells in the G0/G1 phase, but after 48 h, there were no disturbances in the cell cycle. Conclusion: It has been shown that after a single total prolonged irradiation of mice at low doses (10–500 mGy), when analyzing the frequency of PCE with MN, cytogenetic damage is recorded in the bone marrow, which indicates the genetic danger of exposure to even such low levels of fast neutron irradiation. A decrease in Ki67+ cells and cell cycle arrest at the G2/M phase were found only after irradiation of mice at a dose of 500 mGy and only 24 h after exposure, while the number of nucleated cells in the bone marrow at this dose was reduced, at least to 72 h.

Antimutagenic Activity of Cassia Auriculata Linn Fractions along with Anticancer Activity in Male Albino Mice

Background: In recent years, there has been a surge in interest in studying plant-derived materials and their impact on DNA. Herbal products include a number of natural substances that may help protect cells against mutagen-induced cell damage. Aim: The purpose of this research was to assess the genotoxic effects of Cassia Auriculata Linn flavonoids (CAF) and Cassia Auriculata Linn saponin (CAS) rich fractions on mouse bone marrow cells utilizing chromosomal aberration test and micronucleus assay. Methodology: The suppressive impact of CAF and CAS on 7, 12-dimethylbenz (α) anthracene (DMBA) and Croton oil induced skin tumor promotion in mice with topical administration twice weekly for 18 weeks is also investigated in this work. Three dosages of 100 and 200 mg/kg body weight were used. Single oral dosages of CAF and CAS Fraction at the three levels did not enhance the number of micronucleate polychromatic erythrocytes in the micronucleus experiment. Result: In mice bone marrow cells, a single oral treatment of CAF and CAS fraction revealed no significant alterations in mitotic indices or chromosomal aberration induction. The clastogenicity of CYP was considerably decreased by pretreatment with CAF and CAS fraction. As a result, it can be stated that CAF and CAS fraction had no genotoxic impact on mouse bone marrow cells. Conclusions: The portions of Cassia Auriculata have been shown to be non-genotoxic and non-clastogenic at the quantities utilized in this investigation. CAF and CAS Fraction might possibly be a promising skin tumor promotion reducing agent, according to this research.

Morphofunctional features of proliferating cells exposed to PSMA peptide

The effect of the synthetic PSMA peptide on dividing cells of laboratory animals was studied. The experiment was carried out on male white laboratory mice of the BALB/c-line. The toxic effect of PSMA peptidi was evaluated at therapeutic (1.4 μg / kg of animal weight or 0.04 μg / animal) and subtoxic (140 μg / kg of animal weight or 4.0 μg / animal) doses. The cytotoxic effect of PSMA peptide on red bone marrow cells and cambial intestinal cells of the of laboratory mice was determined. A decrease in the proliferative activity of the colon crypt cells was revealed upon administration of a subtoxic dose of the PSMA peptide and there were no signs of toxic damage to the red bone marrow cells of animals. Key words: toxicity, proliferation, synthetic peptides, mitotic index, micronucleus test.

Study of the effects of ultra-low intensity electromagnetic fields on biological objects

The object of research is the efficiency of exposure to electromagnetic field (EMF) of ultra-low intensity on biological objects, which is formed by a generator of broadband radiation. The principle of action of the generator is based on formation of electromagnetic radiation induced by periodic pulsed gas discharge in coaxial system of electrodes, which is loaded on a dielectric rod antenna. The method of selection of signals and corresponding equipment, which energy characteristics of radiation correspond to the criterion of non-thermal influence on bioobjects, is developed for obtaining a comparative assessment of influence bioefficiency. The proposed new method for processing experimental data using statistical calculations that meet the requirements for the processing and interpretation of the results. The seeds of wheat and interaction of millimeter range electromagnetic oscillations with bone marrow cells of rats were used as biological objects for investigating the effect of millimeter range electromagnetic oscillations. A biosensory effect was obtained when exposed to broadband radiation of ultra-low intensity, compared to the control sample. A change in the properties of the seeds, in particular, heat resistance, is observed. According to the experimental data, seeds turn out to be less susceptible to heat as a result of their pretreatment with EMF. The biological response is observed to depend on the frequency and time of irradiation. Also, the dependence of the decrease in the number of dead cells on the time of EMF irradiation was experimentally proved. The equation of dependence of selective average proportion of dead cells in rat bone marrow on irradiation time was calculated. Biosensory effect of exposure to broadband ultra-low intensity EMF of the developed emitter was revealed. It was established and statistically proved that the minimum time with the maximum positive effect of exposure to electromagnetic radiation of millimeter range on bone marrow cells of rats is 30 minutes, compared with an unirradiated sample. The results make it possible to evaluate the positive effect of electromagnetic oscillations on biological objects and propose the results of studies for practical use in the development of medical systems.