Bone Marrow Cell Suspensions Research Articles

Validation of an Automated, Scalable Comprehensive Genomic Profiling Assay for Hematologic Malignancies

Introduction: The optimal diagnosis, prognosis, and treatment selection for hematologic disorders requires the assessment of somatic mutations across a subset of clinically relevant genes. Here we present the clinical validation results of a targeted next generation sequencing panel comprising all exons of 141 clinically relevant genes for myeloid and lymphoid malignancies. Methods: The panel was designed to include genes (n=141) based of clinical guidelines for myeloid diseases including acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and myeloproliferative neoplasms (MPN), and lymphoid diseases including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and some lymphoma subtypes. The panel was validated to detect single nucleotide variants (SNVs), insertions/deletions (indels), FLT3 internal tandem duplications ( FLT3-ITDs), and sex. Gene level copy number alterations (CNAs) were validated for 11 genes and 5 sub-gene level CNAs in IKZF1, KMT2A, RUNX1, TET2, and TP53, and were selected based on clinical utility and frequency observed in a population of >10,000 microarray samples with confirmed myeloid or lymphoid indications. Custom hybridization probes (Twist Biosciences) were designed to interrogate all coding exons of the 141 genes using genomic libraries created from 250 ng of gDNA extracted from peripheral blood, bone marrow, or flow cytometry cell suspensions, followed by hybrid capture, and sequencing on Illumina DNA sequencers. Data analysis was performed using an in-house analysis pipeline to detect variants and perform rational filtering of technical artifacts. Validation studies were performed in a CAP/CLIA accredited clinical laboratory. Precision and concordance studies were performed using previously characterized clinical samples. SNVs, indels and FLT3-ITDs were orthogonally confirmed by an NGS-based laboratory developed test (ArcherDx) or by Sanger sequencing. CNAs were confirmed by digital multiplexed ligation-dependent probe amplification (dMLPA; MRC Holland). Results: Assay precision was determined using 15 previously characterized clinical samples with 36 SNVs with a variant allele frequency (VAF) range of 5.7-91.4%, 11 indels with a VAF range of 6.2-79.7%, one 24bp FLT3-ITD with a VAF of 30.2%, 17 deletions with a copy number (CN) range of 0.53-0.83, and 7 gains with CN range of 1.32-1.66. Three replicates of each sample were assessed for precision with 100% concordance observed for SNVs (36/36), indels (11/11), FLT3-ITD (1/1), CNAs (24/24), and gender (15/15). Inter-assay precision was assessed using multiple operators, instruments, reagent lots, and sample barcodes with the clinical samples. Interim analysis of concordance performed on 130 clinical samples demonstrated a positive percent agreement (PPA) of 99.7% for SNVs (334/335 in 114 clinical samples), 97.7% for indels (146/151 in 85 clinical samples), 98.7% for CNAs (30/30 in 17 clinical samples), and 96.7% for sex (127/130 in 130 clinical samples). Dilution series of both cell lines and clinical samples that will be used to determine analytical sensitivity are underway. Conclusions: This study highlights the clinical validation of a 141 gene NGS assay for the detection of clinically informative genomic alterations in hematologic malignancies. The detection of CNAs by NGS may be used to additionally confirm or monitor abnormalities previously detected by cytogenetics/microarray or inform the need for additional testing. Results from this validation study, when complete, will include at least 480 samples plus orthogonal testing. These data describe the performance of an assay to enable a comprehensive evaluation of genomic alterations using a single sample, further facilitating the use of broad NGS assays in hematologic malignancies.

Effect of Glycodelin on the Cytokine Profile of Rats during Allogeneic Bone Marrow Cell Transplantation

The protein glycodelin (PP14, PAEP) is associated with pregnancy and exhibits pronounced immunotropic properties. We studied the effect of glycodelin on the cytokine profile of blood serum during transplantation a suspension of allogeneic red bone marrow cells to Wistar rats. Recombinant glycodelin was administered to the animals 4 times (14 μg each time). Against the background of bone marrow cell transplantation, the levels of proinflammatory (IL-1α, IL-1β, and IL-18) and regulatory (IL-7, IL-12) cytokines and CSF (M-CSF) increased in blood serum, which indicates a systemic inflammatory response to the allograft. Glycodelin administration against the background of bone marrow cell allotransplantation led to a significant decrease in the proinflammatory cytokine IL-17A on day 21 of the experiment; the concentrations of the other cytokines remained unchanged. In general, glycodelin can suppress the level of IL-17A, a marker of graft rejection, which opens perspectives for its further investigation as a potential drug.

P1414: MARROW ADIPOCYTE-ENRICHED NICHE HOSTS AND INFLUENCES A FRACTION OF HEMATOPOIETIC PROGENITORS IN ADULT HIP BONE

Background: The hematopoietic process is tightly regulated by the bone marrow (BM) microenvironment. Emerging studies recognized a regulatory role of mouse BM adipose tissue (BMAT) on self-renewal and differentiation of hematopoietic stem cells (HSC) and progenitors, while results on human BMAT are still scarce. Aims: Although hip arthroplasty-derived BM serves as an available resource for non-malignant hematopoietic cell research, the BMAT component of this BM and its’ impact on hematopoietic progenitors is not yet revealed. Therefore, our aims were to explore the contribution of human BMAT to the maintenance of hematopoietic progenitors. Methods: BM samples of acetabular bones were obtained from patients undergoing hip arthroplasty (n=13, 65.7±12.5 years, Ethical approval 187/18). Centrifugation was applied to separate the fatty layer containing BMAT from the BM cell suspension. The BM fraction was used for isolation of BM mononuclear (BMC) and expansion of BM mesenchymal stromal cells (BM-MSC). Digestion of BMAT was applied to separate the non-adipocytic cells of BMAT from mature adipocytes, expand them, and to investigate their functions and transcriptional activity by RNA sequencing. Multiparametric flow cytometry was applied to explore surface marker expression. Hematopoietic activity was investigated by short-term colony assays of cells ex vivo and in vitro upon co-culture with BM-MSC or sub-cultured BMAT adipocytes. Results are presented as mean ±SD and statistical significance was estimated by paired tests. Results: We found that the total colony forming cell number in BMC (0.147±0.068) was significantly higher than in BMAT-associated cells (0.0684±0.062%, p=0.0002, n=11). The strongest reduction within BMAT was observed in colony forming unit (CFU)-macrophages and CFU-granulocyte-erythroid-macrophage/monocyte-megakaryocytes. The content of CD45+CD34+ cells was higher in BMC (0.901±1.009%) than in BMAT (0.357±0.505%), while interestingly no differences were found in CD33+ cell frequencies. Both, expanded BMAT and sub-cultured BMAT adipocytes showed stronger adipogenic potential than BM-MSC. When compared to BM-MSC, sub-cultured BMAT adipocytes showed weaker potential to support growth of committed hematopoietic progenitors within BMC ex vivo, favorizing the growth of early and late erythroid progenitors at the expense of other hematopoietic cells. Transcriptomic analyses showed that HSC niche factors (Angptl4, Wnt5b, Il7, Dkk3), but also LepR were upregulated in BM-MSC compared to expanded BMAT cells. Summary/Conclusion: Our findings indicate a yet unrevealed participation of the BMAT niche in compartmentalization of adult hip bone hematopoietic progenitors by hosting a set of hematopoietic cells and potentially, BMAT progenitors in the close proximity of BM adipocytes. Since a behavior of BM populations is dependent on local microenvironmental context and bone region, obtained results might reflect pathologic settings in arthroplasty patients. Further studies have to address the potential coupling of marrow adipogenesis and hematopoietic progenitor maintenance during aging and diseases.

Clonal analysis of bone marrow stromal cells in multiple exostotic chondrodysplasia and systemic osteoporosis: features of cell cloning efficiency

We studied the clonal features of bone marrow stromal cells obtained by biopsy of the iliac wing of patients with systemic skeletal diseases: multiple exostotic chondrodysplasia (16 patients) and systemic osteoporosis (24 patients). The proliferative potential of cells was investigated, the main indicator of which is the efficiency of cloning. We analyzed the induction-morphogenetic relationship between the efficiency of cloning and biological inducers contained in the autologous and added suspension of bone marrow cells (the so-called feeder). The rapid growth and proliferation of exostoses in multiple exostotic chondrodysplasia is apparently due to both the increased proliferative activity of the cultured cells themselves, and (especially) the increase in the growth-stimulating effect of the autologous "feeder", which distinguishes fast-growing exostoses from the so-called calm ones, in which the proliferative activity itself cells are increased (but to a lesser extent), and no enhancement of the effect of the autofeed is detected. In systemic osteoporosis, a sharp decrease in the efficiency of cloning of the target cells themselves has been established, which is probably associated with a weakening of their colony-forming properties and / or a decrease in their number in the explanted suspension.

Bone marrow mesenchymal stem cells attenuate pyroptosis lipopolysaccharide-induced renal injury rats

To investigate the effect and mechanism of bone marrow mesenchymal stem cell (BMSC) on pyroptosis of rats with kidney injury. Bone marrow of 4-5 week-old female Sprague-Dawley (SD) rats was isolated in vitro and BMSC was obtained. The third generations of BMSC were used to further experiments. Fifteen 6 week-old SD rats were cluster-randomized divided into control group, kidney injury group and BMSC group (5 rats in each group). Rats in kidney injury group were injected with lipopolysaccharide (LPS) 1 mg/kg via tail vein; the control group was given the same amount of normal saline. BMSC group was injected with 0.5 mL BMSC (including 2×106 BMSC) via tail vein after modeling; the kidney injury group received the same amount of normal saline. On day 3 after these injections, serum creatinine (SCr) was detected by picric acid method, and blood urea nitrogen (BUN) was detected by diacetyl monoxime. The levels of cystatin C (Cys C), interleukins (IL-1β and IL-18) in blood were detected by enzyme-linked immunosorbent assay (ELISA). The rats were then sacrificed and their kidneys were removed for subsequent detection. The mRNA expression levels of NOD-like receptor protein 3 (NLRP3) and cysteinyl aspartate-specific protease-1 (caspase-1) of kidney were detected by quantificational real-time quantitative polymerase chain reaction (qRT-PCR). The protein expression levels of NLRP3 and caspase-1 of kidney were detected by Western blotting. In vitro, after bone marrow cell suspension was cultured for 24 hours, a large number of round adherent cells and suspended cells appeared in each culture flask. After 4-5 days of culture, a large number of long spindle cells adhered to the wall, and there were still obvious impurity cells. After trypsin digestion and passage to the third generation, the long spindle adherent cells grew mainly in the culture flask and were basically purified as BMSC. In vivo, compared with the control group, the levels of SCr, BUN, Cys C, IL-1β and IL-18 in kidney injury group were increased [SCr (μmol/L): 85.22±2.29 vs. 21.80±0.59, BUN (mmol/L): 11.50±0.64 vs. 5.86±0.83, Cys C (mg/L): 0.13±0.01 vs. 0.11±0.02, IL-1β (ng/L): 31.49±1.42 vs. 4.74±0.49, IL-18 (ng/L): 29.01±1.95 vs. 1.52±0.03, all P < 0.05]. The mRNA and protein expression levels of NLRP3, caspase-1 were significantly increased [NLRP3 mRNA (2-ΔΔCt): 3.635±0.296 vs. 1.000±0.002, caspase-1 mRNA (2-ΔΔCt): 4.020±0.228 vs. 1.001±0.003; NLRP3 protein (NLRP3/β-actin): 1.560±0.868 vs. 0.902±0.036, caspase-1 protein (caspase-1/β-actin): 1.392±0.097 vs. 0.895±0.046, all P < 0.05]. Compared with kidney injury group, the levels of SCr, BUN, IL-1β and IL-18 in BMSC group were significantly decreased [SCr (μmol/L): 51.64±3.84 vs. 85.22±2.29, BUN (mmol/L): 9.90±0.46 vs. 11.50±0.64, IL-1β (ng/L): 24.20±1.45 vs. 31.49±1.42, IL-18 (ng/L): 12.97±1.25 vs. 29.01±1.95, all P < 0.05]. The mRNA and protein expression levels of NLRP3, caspase-1 were significantly decreased [NLRP3 mRNA (2-ΔΔCt): 1.488±0.136 vs. 3.635±0.296, caspase-1 mRNA (2-ΔΔCt): 1.643±0.143 vs. 4.020±0.228; NLRP3 protein (NLRP3/β-actin): 1.227±0.053 vs. 1.560±0.868, caspase-1 protein (caspase-1/β-actin): 1.159±0.107 vs. 1.392±0.097, all P < 0.05]. In vivo, BMSC may attenuate pyroptosis in LPS-induced kidney injury rats.

THE LOCALIZATION OF CD8-POSITIVE CELLS OF THE LYMPH NODES IN DIFFERENT TIMES AFTER THE INJECTION OF THE ALLOGENIC BONE MARROW

The immune system plays an important role in the formation of an adequate immune response during transplantation of allogeneic bone marrow. The aim of the study was to study the localization of CD8-positive cells in the lymph nodes at the early stages after the venous injection of a suspension of allogeneic bone marrow cells. The experiments were carried out on 50 outbred male rats. The animals were divided into three groups: group 1 - intact rats (n=10), group 2 - control animals (n=10), which were injected 3 ml of 0,85% natrium chloride solution; 3rd group - experimental rats (n=30), which were injected into the tail vein with one ml of a cell suspension of bone marrow extracted from the epiphysis of the femur of another rat and mixed with 2 ml of 0,85% natrium chloride solution. Animals were withdrawn from the experiment after 40, 120 and 240 minutes from the start of the experiment. After 40 minutes from the start of allogeneic bone marrow transplantation in the lymph nodes, CD8+ lymphocytes are detected in the expanded subcapsular, paracortical zones, near the vessels, in the cortical sinuses, and in the medulla. After 120 minutes from the start of transplantation, the number of lymphoid nodules with different numbers of CD8+ lymphocytes and different intensity of their color increases. The reproduction of CD8+-lymphocytes occurs under the capsule in the cortex of some lymphoid nodules, the vessels dilate, fragmentation of the medulla occurs with the appearance of new groups of CD8+-lymphocytes. After 240 minutes from the start of allogeneic bone marrow injection, a small number of CD8+ lymphocytes are detected in the subcapsular zone, in the peripheral layer of single lymphoid nodules, along the lymphatic and blood vessels, and as groups in the medulla. There is a fatty degeneration of the medulla of the lymph nodes. Thus, based on the localization and reproduction of CD8+ lymphocytes in the lymph nodes, it can be assumed that the recognition of allogeneic bone marrow antigens occurs in the subcapsular zone, the peripheral layer of lymphoid nodules, and areas of the medulla.

Influence of PSG peptides on the morphofunctional state of the spleen in allogenous bone marrow cell transplantation

It is known that proteins associated with pregnancy provide immune tolerance during pregnancy. One of them is pregnancy-specific β1-glycoprotein (PSG), which has a pronounced immunomodulatory effect. Tetrapeptide fragments (YECE, YQCE, YVCS, and YACS) have been identified in the primary structure of PSG that have immunopharmacological potential. The aim of the study was to evaluate the effect of short peptide fragments of PSG on the morphological and immunohistochemical state of the spleen and its compartments during allogeneic transplantation of a suspension of red bone marrow cells in an in vivo experiment in Wistar rats. It was found that intraperitoneal injection of bone marrow cells in the dynamics of the experiment (35 days) caused splenic hyperplasia with accumulation of lymphoid cells and macrophages in the functional areas of the organ. Administration of short peptide fragments of PSG against the background of allogeneic bone marrow transplantation promotes activation of cells of the immune system in the spleen toward their proliferation (Ki-67) and differentiation, while reducing the content of macrophages (CD68). Thus, short peptide fragments of PSG stabilize proliferative processes and promote the development of adaptive responses in response to an allogeneic graft.

Electrical Conductivity of Bone Marrow Cell Cultures and Their Connection with Proliferative Activity: Age Aspect

Functional activity is a basic concept of biology; however, instrumental methods of its determination are absent. The interrelation between bone marrow cell proliferative activity, as one of the functional activity parameters, the content of calcium, copper, and reactive oxygen species (ROS) in the cells, and the electrical conductivity of the cell culture was studied in young and old Wistar rats aged 3 and 20 months. We determined the electrical conductivity of the bone marrow cell cultures and parameters of bone marrow cells (proliferative activity and calcium, copper and ROS content). The bone marrow cells of old intact animals had higher proliferative activity in the primary culture as compared to that of young animals. The content of the 8 main morphological cell types in the young and old animals were similar, while the lymphocyte content was larger in the old animals. Content of ions in the bone marrow cell cultures of old animals as compared with that in young animals varied as follows: Content of calcium was 75 % higher, copper was 5 times lower, and ROS was 2.5 times lower. It was found that the conductivities of the pure culture medium and of that after the cultivation of the bone marrow cells in the nutrient medium were significantly different. It has been suggested that the electrical conductivity of biological media could be used as an integral characteristic of the ionic composition and structural organization of biological media. HIGHLIGHTS Electrical conductivity of bone marrow cells suspension of young animals was decrease to compared with this indicator of old animals Proliferative activity is correlated with electrical conductivity Electrical conductivity of biological media is influenced both the composition, and macromolecules and their state of aggregation

The Possible Role of Human Mesenchymal Stem Cells on Human Osteoclast Differentiation from CD34+ Bone Marrow Hematopoietic Progenitors.

Hematopoietic progenitors differentiate into osteoclasts through the influence of systemic hormones and factors produced within the bone marrow microenvironment. Beyond the differentiation to nonhematopoietic tissues, including bone, cartilage, muscle, of pluripotent mesenchymal stem cells (MSCs) are components of this microenvironment and have been shown to produce extracellular matrix, cytokines and growth factors that regulate the formation, activity, and survival of osteoclasts in vitro. In this study, we sought to establish a reproducible coculture model of human osteoclastogenesis using highly purified human MSCs and CD34+ hematopoietic stem cells (HSCs). Fifty milliliters of bone marrow were harvested from breast cancer patients to whom planning autologous BMT. Bone marrow cell suspensions were centrifuged on Ficoll-Paque to isolate mononuclear cells. The mononuclear cell fraction was cultured in standard medium to obtain MSCs. After 4–5 passage mesenchymal differentiation was determined by morphologically, Vimentin, and CD54 and CD58 positivity. Ostoeogenic differentiaiton of MSCs was induced with an osteogenic supplement (OS) containing dexamethasane, B-glycerophosphate, L-ascorbic acid for 12 days incubation. CD34+ HSCs were purified from bone marrow mononuclear cells by positive selection using CliniMACS. Aliquots of DAPI-stained HSCs were added to monolayers of MSCs or OS-treated MSCs, and the coculture was maintained at 37°C in 5 % CO2 for up to 3 weeks. To assess the effect of vitamin D3 on osteoclast formation 1,25(OH)2D3 was added to the coculture. Human fibroblasts were used as negative control instead of MSCs. To determine the role of cell contact in osteoclast formation, experiments were performed in which HSCs and MSCs were cultured in either the same chamber or in chambers separated by 0.45-μm porous membrane for 3 weeks. After 3 weeks, this coculture system yielded that MSCs promote differentiation of DAPI-stained HSCs into TRAP+ multinucleated cells in the absence of added hormones, cytokines, and growth factors. These cells showed CD14, CD11b, CD11c, CD56 positivity, whereas CD34, CD45, HLA-DR, CD13, CD71, CD42 were negative after 3 weeks of culture. In contrast, human fibroblasts failed to support the formation of MNCs, the majority of HSCs degenerated within 2 weeks of culture. Separation of MSCs and HSCs reduced osteoclast formation by 80%, suggesting that cell contact is important, but not absolutely required, for osteoclast formation. OS-treated MSCs actually inhibited the formation of osteoclasts from HSCs. The inhibitory effect of OS-treated MSCs on osteoclast formation is dictated by the differentiated state of MSCs. Osteoclast formation was increased if the coculture was performed in the presence of 1,25(OH)2D3. The data show that MSCs support multilineage differentiation of hematopoietic cells and suggest that our model provides opportunities to understand the mechanisms by which MCSs regulate human osteoclastogenesis.

The Electrical Conductivity of Bone Marrow Cells Suspension Associated with Proliferative Activity and Depends on the Donor's Age and the Functional Activity of the Liver.

Copper ions play various roles in mammalian cells. Some investigations indicated that copper correlates with liver fibrosis and changes in bone marrow cells. Here, we explored the role of bone marrow cell parameters in copper liver fibrosis development and possible underlying mechanisms. The age-related features of biochemical indicators, bone marrow cell parameters, and electrical conductivity of 3- and 20-month-old male Wistar rats treated with copper II sulfate pentahydrate (CuSO4 × 5H2O) have been investigated. Based on the obtained earlier experimentally data in our laboratory, dose of 1 mg/100 g (0.58 mg/g of liver) was used. Induced liver fibrosis caused a change in the number and ratio of morphotypes of bone marrow cells, calcium and copper content, redox-system activity. These parameters depended of animals' age and presence of liver fibrosis. We also demonstrated that electrical conductivity can be used as integral parameter. In conclusion, the "adaptive response" modifies the emerging reactions of the organism to new external factors.

Influence of glycodelin on morphofunctional state of secondary immune organs in experimental model of allogeneic transplantation

Glycodelin (PP14, PAEP, alpha-2-microglobulin, dimeric glycoprotein with a molecular weight of 42 to 56 kDa) is a marker of reproductive tissue receptivity. Immunoregulatory potential of glycodelin allows us to consider it among key factors that promote maternal immune tolerance to the developing embryo. In general, glycodelin has prospects for use in biomedicine as a biopharmaceutical to treat post-transplant complications. This work aimed to study the effect of glycodelin on morpho-functional state of the secondary organs of immune response in the course of in vivo experiment during allogeneic transplantation of bone marrow cell suspension to Wistar rats. The experiment was performed in white male Wistar rats aged 2-3 months (body mass, 250 g). The animals were kept at the Perm State National Research University animal clinic under GOST 33216-2014 “Rules for working with laboratory rodents and rabbits”. The animals were sacrificed on the 3rd, 7th, 14th, and 21st days according to international rules for working with experimental animals. In the control group, animals (n = 6) were injected once with a suspension of camptothecin-treated bone marrow cells; in experimental group, the animals (n = 12) were injected with a bone marrow cells in combination with glycodelin administered 4 times.We used recombinant glycodelin (MyBioSource, Germany), which was administered to the animals at a concentration corresponding to pregnancy state (0.75 μg/mL). Intraperitoneal injection of bone marrow cells simultaneously with glycodelin over the terms of experiment (21 days) showed that allogeneic transplantation caused hyperplasia of spleen and lymph nodes. In the functional zones of organs, proliferation and differentiation of immune cells developed. Injection of glycodelin at the early stages of experiment (3-7 days) caused an increase in proliferative processes in the organs of both T- and B-dependent immunity. Moreover, there were no signs of inflammation and apoptotic cell death in the organs. Since the 14th day of the experiment, eosinophilic infiltration of the organs was evident, being an indirect positive sign of response to the transplanted cells. By the end of study, differentiation processes dominated over proliferation in the organs. Thus, glycodelin stabilizes proliferative processes and promotes emergence of a new cell generation, thus supporting host response to the transplant. Apparently, glycodelin can participate in development of adaptive mechanism in secondary organs of immune system.

Comparison between three different protocols for isolation and culture of mouse bone marrow derived mesenchymal stem cells

Bone marrow derived mesenchymal stem cells (BM-MSCs) represent a promising source for cell therapy, As they can differentiate into bone, cartilage, fat, tendon and many other organ progenitor cells. Although the culture of MSCs has been studied for over 30 years. The identification of standard protocol for isolation and characterization have yet to be developed. A comparison study was made on three different isolation techniques, which include: direct plating, red blood lysis buffer strategies, and density gradient (DG) method (using two different gradient media: Ficoll and Percoll) to find the optimal isolation and culture condition for adequate amount of MSCs for clinical use.The results demonstrate that direct plating of whole bone marrow (BM) suspension provides a suitable alternative protocol for isolation of BM-MSCs with minimal requirements, which mainly based on the frequent medium change in primary culture. The BM crud cell suspension were isolated by aspiration from albino mice and cultured in fresh complete isolation media (CIM) RPMI-1640/10%FBS. Adherent cells from BM cells suspension were MSCs which then expanded by complete expansion media (CEM) α-MEM/ 10% FBS. By concluding, direct plating of whole bone marrow represent the alternative method for isolating of mesenchymal stem cells from small specimen of bone marrow. By concluding, the present study was designed to investigate the optimal technique for the isolation of BM-MSCs for use in tissue engineering and regenerative medicine.

Galega officinalis extract regulate the diabetes mellitus related violations of proliferation, functions and apoptosis of leukocytes

BackgroundAn impaired leukocytes function is the factor causing the susceptibility of patients with diabetes mellitus to infections. The outmost importance for the understanding of the immunological processes involved in diabetes pathogenesis is to give the characteritics of the immunological profile and changes therein, during the course of desease. Long-used in folk medicine to treat diabetes Galega officinalis L. has been chosen for the correction of the immune system dysfunction.MethodsThe experiments were conducted on male Wistar rats. Fractionation of bone marrow cells suspension was performed in a three-layer ficoll–sodium amidotrizoate density gradient. The lymphocytic-granulocytic cells proliferative activity was studied using enzyme immunoassay with 5-bromo-2′-deoxyuridine (BrdU). For staining of bone marrow preparations May-Gruenwald-Romanowsky-Giemsa (Pappenheim) method was used. To evaluate the content of cationic proteins and myeloperoxidase in neutrophilic leukocytes cytochemical studies were performed. Content of tumor necrosis factor alpha was carried out by immuno-enzymatic analysis. Lymphocytes apoptosis was examined by fluorescent analysis using annexin V.ResultsDiabetes mellitus development was accompanied with violation of neutrophils and lymphocytes proliferation, increased activity of myeloperoxidase and enhanced apoptosis process. Administration of Galega officinalis extract under the condition of diabetes promotes the restoration of neutrophils bone marrow pool and the reduction of lymphoblasts number and causes inhibition of the lymphocytes apoptosis process.ConclusionsInvestigated medicine has a pronounced immunocorrective effect under the conditions of diabetes mellitus and can become the basis for creating a new generation of antidiabetic drugs.

BON E TISSUE ENGINEER ING BA SED ON BONE MARROW I N BLOOD CLOT LOADED ON MINER AL MATRIX CARRIER: E XPERIMENTAL STUDY IN SUBCUTANEOUS MICE MODEL

Repair of bone defects can be supported by appropriate biomaterials, cells and regulatory signals, which is the basic principle of bone tissue engineering. The components of bone marrow and blood clot come in close contact with bone defects after injury. Since these structures represent the source of different cell types and regulatory signals, we wanted to examine their effect on osteoreparatory process in ectopic implants. Subcutaneous implantation was performed on male Balb/c mice. Bio-Oss® biomaterial in the function of mineral matrix carrier was loaded with blood diluted with bone marrow cell suspension (MMB-type implants) or only with blood diluted with saline (MB-type implants, control). Implant extraction was performed 2, 8 and 12 weeks after implantation. MMB-type implants were characterized with more pronounced beginning of resorption in comparison with MB-type implants. High cell density of connective tissue was also preserved during the 12-weeks observation period in MMB-type implants, as well as good vascularity, which was most pronounced 8 weeks after implantation. Osteoblast-like cells and osteon-like structures could be observed. These results suggest that bone marrow in synergy with blood clot might have a stimulating effect on osteoreparation process.

Nitro-oleic acid regulates growth factor-induced differentiation of bone marrow-derived macrophages

Many diseases accompanied by chronic inflammation are connected with dysregulated activation of macrophage subpopulations. Recently, we reported that nitro-fatty acids (NO2-FAs), products of metabolic and inflammatory reactions of nitric oxide and nitrite, modulate macrophage and other immune cell functions. Bone marrow cell suspensions were isolated from mice and supplemented with macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) in combination with NO2-OA for different times. RAW 264.7 macrophages were used for short-term (1–5min) experiments. We discovered that NO2-OA reduces cell numbers, cell colony formation, and proliferation of macrophages differentiated with colony-stimulating factors (CSFs), all in the absence of toxicity. In a case of GM-CSF-induced bone marrow-derived macrophages (BMMs), NO2-OA acts via downregulation of signal transducer and activator of transcription 5 and extracellular signal-regulated kinase (ERK) activation. In the case of M-CSF-induced BMMs, NO2-OA decreases activation of M-CSFR and activation of related PI3K and ERK. Additionally, NO2-OA also attenuates activation of BMMs. In aggregate, we demonstrate that NO2-OA regulates the process of macrophage differentiation and that NO2-FAs represent a promising therapeutic tool in the treatment of inflammatory pathologies linked with increased accumulation of macrophages in inflamed tissues.

Flow cytometric isolation method of mesenchymal stem cells from murine bone marrow cell suspension

Flow cytometric isolation method of mesenchymal stem cells from murine bone marrow cell suspension

Characteristics of Lymphoproliferative Disorders with More Than One Aberrant Cell Population as Detected by 10-Color Flow Cytometry.

We have evaluated the frequency of lymphoproliferative disorders with more than one aberrant population of monotypic B-cells detected during routine hematopathological diagnostics. 2600 samples peripheral (blood, bone marrow, fine-needle aspirate, lymph node, and pleural fluid cell suspensions) were analyzed using a 10-color B-cell panel and a 10-color T-cell panel. A 10-color plasma cell/lymphoplasmacytic panel was performed when appropriate. 790/2600 samples (30%) showed at least one aberrant B-cell population and 27(1%) showed an aberrant T-cell population. 41/790 samples (5.1%) showed two aberrant B-cell populations. Thirteen patients had two B-cell populations with different surface immunoglobulin restriction (one kappa+ and one lambda+), most with B-cell chronic lymphocytic leukemia-related phenotype. Five cases showed two B-cell populations with the same light chain restriction but distinctly different immunophenotypes. In 23 cases, two populations had the same light chain restriction and differed by expression of one or 2 markers, thus, a possibility of intraclonal differentiation could not be excluded. Cases with possible intraclonal differentiation had a significantly higher proportion of aberrant B-cells than those with two coexisting aberrant B-cell populations (49.9% vs. 27.7%, p = 0.008). In only one sample one population of clonal B-cell and one clonal T-cell population with large granular lymphocyte related phenotype were found. Using our panels 5.1% of cases with lymphoproliferative disorder-associated aberrant findings show two aberrant (clonal) lymphoid and/or plasma cell populations. © 2016 International Clinical Cytometry Society.

Application of retrograde dissection method for isolation of bone marrow cells from rat femurs and tibiae

Currently, there is no practical and efficient method for the isolation of bone marrow cells (BMCs) from rat femurs and tibiae. Here, we attempted to develop a rapid, simple, effective, and non-contaminating method for the isolation of BMCs from rat femurs and tibiae. Rat femurs and tibiae were dissected from the ankle to the hip joint; subsequently, a three-step "locate-slide-twist" procedure was performed using scissors and forceps to remove the femurs and tibiae completely, from the surrounding musculature. The bones were flushed with phosphate-buffered saline to harvest BMCs. The femurs and tibiae were dissected in 1.8 ± 0.6 min, and the BMC suspension preparation time was 13.1 ± 2.3 min. The bone marrow cavities did not incur any fractures or injuries during the isolation. Culture of harvested BMCs for 72 h led to a significant increase in cell number from 4.4 ± 0.3 x 106 to 6.9 ± 0.7 x 10(6) (P < 0.01) with no significant decrease in viability (98.1 ± 0.6% vs 96.2 ± 1.1%; P > 0.05). Microscopic examination of the isolated BMCs after the 72-h incubation period revealed the no-microbial or muscle cell contamination. Furthermore, flow cytometry revealed that cultured BMCs (72-h culture) grew well. Here, we have reported a rapid, simple, effective, and non-contaminating method for the isolation of BMCs from rat femurs and tibiae by using retrograde dissection. This method can be used to harvest a large number of viable BMCs without the risk of contamination from muscle and connective tissues.

Electrogenesis of end-plates of mdx mice diaphragm: effect of cell therapy

Disturbances of muscle electrogenesis are observed in a number of muscle pathologies, particularly, they are found in the mdx mice, which are a laboratory model of Duchenne myodystrophy. This myodystrophy develops due to mutations in the gene of the dystrophin protein, which controls the synthesis of this protein in the cytoskeleton. The effectiveness of therapy for such myodystrophy by methods of cellular and genetic engineering has not been studied with regard to the muscle membrane electrogenesis. In this study, two months old mdx mice were irradiated by X-ray at a dose of 3 Gy and injected intravenously by wild type bone marrow cells suspension from long bones of C57Bl/6 mice. Four months after such non-myeloablative bone marrow cells transplantation the recovery of resting membrane potentials as well as parameters of miniature end-plate potentials of mdx mice diaphragm muscle was observed. Our data show the effectiveness of the replacement of the mutant bone marrow with the bone marrow of wild type in myodystrophy caused by the deficiency in dystrophin protein synthesis. Refs 20. Figs 3. Table 1.

Cytoprotective Effect of Afobazole and Its Main Metabolite M-11.

Cell damage depending on activity of quinone reductase 2 (MT3 receptor) was simulated in experiments on bone marrow cell suspension and assessed by menadione-induced DNA breaks measured by comet assay. We analyzed the protective effect of afobazole interacting with MT1, MT3, σ1 receptors, and monoamine oxidase A and its main metabolite M11 that specifi cally binds to MT3 receptors. Both compounds reduced the level of menadione-induced DNA damage (afobazole was effective in lower concentrations in comparison with M-11). Conclusion was made on the contribution of MT3 receptors to the protective effect of afobazole, but the observed concentration differences indicate possible contribution of other targets of anxiolytic drug to the protective mechanisms.