Activity Of Bone Marrow Cells Research Articles

Preliminary Results of the Oral CD73 Inhibitor, Oric-533, in Relapsed/Refractory Multiple Myeloma (RRMM)

Background: Although recently available immune-based approaches in multiple myeloma produce high response rates, curative outcomes have not yet been achieved, which highlights the need for new agents that further activate the immune system without increasing toxicity. ORIC-533 is a highly potent and selective, orally bioavailable, small molecule inhibitor of CD73, an enzyme expressed by various cell types, including immune cells and which catalyzes the generation of adenosine from adenosine monophosphate (AMP) (Allard 2019). Adenosine itself is highly immunosuppressive, diminishing activity of a range of immune cell types, including T, NK, and dendritic cells (Young 2014). By blocking adenosine production from AMP via inhibition of CD73, ORIC-533 has demonstrated the ability to restore and enhance immune function. Multiple myeloma (MM) patient bone marrow (BM) contains multiple cell types that also express CD73, including immune cells, cancer-associated fibroblasts, mesenchymal stem cells, and endothelial cells (Allard 2019). Moreover, elevated adenosine levels in the MM BM niche correlate with disease progression (Horenstein 2016; Yang 2020). We have previously observed that CD73-mediated adenosine activity suppresses the cytolytic antitumor immune function in the MM BM milieu (Ray 2022). ORIC-533 treatment reduced adenosine production, overcame this immune suppression with evidence of restored DC-mediated priming and activated cytotoxic CD8 + T-cell function, and significantly enhanced immune cell cytolytic behavior against RRMM tumor cells in autologous patient ex vivo experiments (Junttila 2022), making ORIC-533 a potential immunotherapy for patients with MM. Methods: ORIC-533-01 (NCT05227144) is an ongoing i3+3 Phase 1b dose escalation study in patients with RRMM. The primary objectives are to determine single agent safety/tolerability and pharmacokinetics (PK) of ORIC-533 to select the RP2D. Pharmacodynamic assessments include measurement of CD73 functional activity in serum and BM, as well as enumeration of immune cell subsets and activation state in blood and BM. Patients are also evaluated for clinical efficacy using IMWG criteria. Results: As of 23 June 2023, 17 patients received ORIC-533 once daily across 4 dose levels (400 mg to 1600 mg). All patients were triple-class refractory, 88% were penta-refractory, and 59% also received prior anti-BCMA/CD3 bispecific therapy or anti-BCMA CAR-T therapy. Overall, ORIC-533 was very well tolerated, with 5 patients experiencing a total of 10 treatment-related adverse events (TRAEs). Fatigue was the only G3 AE and the only TRAE seen in more than 1 patient (1 event each of G3 and G2); all other events seen in 1 patient each, were G1 or G2 in severity, and of no specific system organ class. No dose limiting toxicities, no Grade ≥4 TRAEs, and no treatment-related serious adverse events have been observed. PK showed increased exposure with dose with a plasma half-life of ~24 hrs. Soluble CD73 enzymatic activity was nearly completely inhibited by C1D15 in serum of all patients across all dose levels, while the largest relative suppression in surface CD73 activity in BM cells was observed at the highest dose level. There was also evidence for increases in the abundance of circulating NK and CD8+ T cells after 1 treatment cycle, with preliminary evidence of enhanced CD8+ T-cell activation at the highest dose levels tested in both peripheral blood and BM. In addition, early evidence of single agent clinical activity was observed. Conclusions: ORIC-533 has demonstrated an acceptable safety profile and preliminary evidence of immune activation in a heavily pretreated RRMM patient population. Enrollment is ongoing and updated data will be presented.

Study of the Mutagenic Effect of the Betamide Drug

The tested Betamide drug is an amide of betulonic acid, obtained as a result of chemical modification of the triterpenoid betulin isolated from birch bark. The drug has antioxidant, hepatoprotective, anti-inflammatory, antimetastatic and antitumor activity. The drug is supposed to be used in the treatment of cancer. Experimental studies were carried out on male and female C57/BL/6 mice free from specific pathogens (SPF). The drug was administered once intragastrically at doses of 2000 mg/kg, 1000 mg/kg and 500 mg/kg. It turned out that the maximum dose of 2000 mg/kg in males causes a decrease in the mitotic activity of bone marrow cells. It has been established that the drug in none of the studied doses has the ability to induce mutations in somatic cells. The maximum dose studied produced a small antimutagenic effect. In males, it is expressed in a reduced proportion of cells with multiple aberrations. For females treated with Betamide at a dose of 2000 mg/kg, there was a significant decrease in the total number of cells with disorders compared to the control.

Морфофункціональні характеристики гемопоетичних клітин-попередників кісткового мозку у гуманізованій культуральній системі in vivo

Diffusion capsules implanted in the abdominal cavity of the mouse recipient are one of the variants of the humanized model of "human-animal" systems. The aim of our study was to determine the optimal parameters for the cultivation of hematopoietic progenitor cells in gel diffusion chambers and their functional activity of bone marrow cells from different locations. To achieve this goal, the method of cultivation in gel diffusion chambers in vivo combined with the method of colony formation in semi-liquid agar, cytological research methods and light microscopy were used. The optimal conditions of cultivation and kinetic indicators of cell aggregate formation by bone marrow precursors of patients without hematological pathology in semi-liquid medium were established in vivo in 16 days of cultivation. We determined that the most suitable term for taking the results of experiments is at 12th day of cultivation. The number of cells that was determined to be optimal for further studies was 5x10(5) cells in 1 ml, or 1x10(5) cells per chamber. Our results suggest that regardless of the place of collection of biological material in humans, the formation of colony in the culture of diffusion chambers is maintained at nearly the same level. The CFU activity for the sternal puncture was 36.5 ± 2.3, the CFU activity for bone marrow from the rib fragment was 38.5 ± 4.8, the CFU activity for trepan biopsy from the anterior upper part of the iliac crest was 35.5 ± 6.8 and the CFU activity from the posterior upper part of the iliac crest was 37.3 ± 5.5. These observations are important for evaluation of functional activity and further use of stem cells from different parts of bone tissue in the case of transplantation. This is further supported by the proliferative potential of colony formation and the index of cell maturation in clones. The obtained data deepen our understanding of the functioning of the hematopoietic stem cells in the human body under normal conditions.

АНАЛИЗ ПОЯВЛЕНИЯ МИКРОЯДЕР В ЭРИТРОЦИТАХ И АКТИВНОСТИ ПРОЛИФЕРАЦИИ КЛЕТОК КОСТНОГО МОЗГА ПОСЛЕ ПРОЛОНГИРОВАННОГО ОБЛУЧЕНИЯ МЫШЕЙ БЫСТРЫМИ НЕЙТРОНАМИ В НИЗКИХ ДОЗАХ

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.

Short-Term Reloading After Prolonged Unloading Ensures Restoration of Stromal but Not Hematopoietic Precursor Activity in Tibia Bone Marrow of C57Bl/6N Mice.

Bone and muscle tissues are mostly susceptible to different kinds of hypodynamia, including real and simulated microgravity (sμg). To evaluate the effect of sμg on bone marrow (BM), male C57Bl/6N mice were divided into three groups: vivarium control (VC), 30-day hindlimb suspension (HS), and subsequent 12-h short-term support reloading (RL). The effects on BM total mononucleated cells (MNCs) as well as stromal and hematopoietic progenitors from murine tibia were studied. The number of BM MNCs, immunophenotype, proliferation, colony-forming units (CFUs), differentiation and secretory activity of hematopoietic and stromal BM cells were determined. HS led to a twofold decrease in MNCs, alteration of surface molecule expression profiles, suppression of proliferative activity of BM cells, and change of soluble mediators' levels. The stromal compartment was characterized by a decrease of CFU of fibroblasts and suppression of spontaneous osteo-commitment after HS. Among the hematopoietic precursors, a decrease in the total number of CFUs was found mainly at the expense of suppression of CFU-GM and CFU-GEMM. After RL, restoration of the stromal precursor's functional activity to control levels and overabundance of paracrine mediator's production were detected, whereas the complete recovery of hematopoietic precursor's activity did not occur. These data demonstrate the fast functional reaction of the stromal compartment on restoration of loading support.

ВЛИЯНИЕ ПИЩЕВОЙ ДОБАВКИ E407А НА МЕТАБОЛИЧЕСКУЮ АКТИВНОСТЬ КЛЕТОК РАЗЛИЧНЫХ КЛЕТОЧНЫХ КУЛЬТУР

Objectives. To study the effects of various concentrations of the food additive E407a (semi-refined carrageenan) on the metabolic activity of fetal liver cells, splenocytes, and bone marrow cells. Material and methods. Fetal liver, splenocytes and bone marrow cell cultures were incubated with the food additive E407a at concentrations varying from 0 mg/ml to 10 mg/ml for 24 hours (n=8). To analyze the effects of this food additive on the metabolic activity of cells, a colorimetric MTT assay was used. It is based on the ability of viable, metabolically active cells to convert 3 (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide into formazan. The data were statistically processed using Kruskal-Wallis and Dunn’s criteria. Results. The bone marrow cell culture was found to be the most sensitive to carrageenan. More than a twofold statistically significant (p<0.0001) increase in the metabolic activity of bone marrow cells was observed when using E407a from 200 μg/ml and above. The metabolic activity of splenocytes increased approximately 1.5 times and over (p<0.0001) when using carrageenans at the concentration of 500 μg/ml and higher. Fetal liver cells turned out to be the most resistant to the direct toxic effect of the food additive E407a. Conclusions. The food additive E407a is cytotoxic to bone marrow cells and splenocytes at concentrations of 200 μg/ml and 500 μg/ml, respectively.

METHODS FOR STUDYING AND ASSESSING CYTOGENETIC CHANGES IN BONE MARROW CELLS OF LABORATORY ANIMALS RECEIVING GENE-MODIFIED

The aim was to study cytogenetic changes in bone marrow cells of laboratory animals that received and did not receive a genetically modified (GM) product (GM-soy) in a comparative aspect. It was found that in the group of white outbred rats that received GM-soy, cytogenetic changes were found in the cells of the red bone marrow, expressed in partial inhibition of cell proliferation due to the cytotoxic effect. They also had polyploidy (5.35%) and aneuploidy (5.35%) in 10.7% of cases. In addition, this group contained cells with mitotic pathology - chromosome scattering, chromosome pulverization, impaired spiralization and despiralization of chromosomes, premature spiralization of chromosomes, delayed mitosis at the prophase stage. In the group of animals that did not receive soy with and without GM in the cells, there were metaphase plates with a normal karyotype. The mitotic activity of bone marrow cells in the control group was higher (MI - 9%) than in the first (MI - 3%) and the second group (MI - 5%).

Comprehensive Subchronic Inhalation Toxicity Assessment of an Indoor School Air Mixture of PCBs.

Few in vivo inhalation studies have explored the toxicity of environmentally relevant mixtures of polychlorinated biphenyls (PCBs). The manufacture of industrial PCBs was banned in 1978, but PCBs continue to be formed in industrial and consumer products. Schools represent a significant source of airborne exposures to legacy and nonlegacy PCBs, placing children at risk. To evaluate the impact of these exposures, we generated an airborne mixture of PCBs, called the School Air Mixture (SAM), to match the profile of an older school from our adolescent cohort study. Female Sprague-Dawley rats were exposed either to SAM or filtered air in nose-only exposure systems, 4 h/day for 4 weeks. Congener-specific air and tissue PCB profiles were assessed using gas chromatography with tandem mass spectrometry (GC-MS/MS). PCB exposures recapitulated the target school air profile with a similarity coefficient, cos θ of 0.83. PCB inhalation yielded μg/g ∑209 PCB levels in tissues. Neurobehavioral testing demonstrated a modest effect on spatial learning and memory in SAM-exposed rats. PCB exposure induced oxidative stress in the liver and lungs, affected the maturational stages of hematopoietic stem cells, reduced telomerase activity in bone marrow cells, and altered the gut microbiota. This is the first study to emulate PCB exposures in a school and comprehensively evaluate toxicity.

The importance of bone marrow and the immune system in driving increases in blood pressure and sympathetic nerve activity in hypertension.

What is the topic of this review? This manuscript provides a review of the current understanding of the role of the sympathetic nervous system in regulation of bone marrow-derived immune cells and the effect that the infiltrating bone marrow cells may have on perpetuation of the sympathetic over-activation in hypertension. What advances does it highlight? We highlight the recent advances in understanding of the neuroimmune interactions both peripherally and centrally as they relate to blood pressure control. The sympathetic nervous system (SNS) plays a crucial role in maintaining physiological homeostasis, in part by regulating, integrating and orchestrating processes between many physiological systems, including the immune system. Sympathetic nerves innervate all primary and secondary immune organs, and all cells of the immune system express β-adrenoreceptors. In turn, immune cells can produce cytokines, chemokines and neurotransmitters capable of modulating neuronal activity and, ultimately, SNS activity. Thus, the essential role of the SNS in the regulation of innate and adaptive immune functions is mediated, in part, via β-adrenoreceptor-induced activation of bone marrow cells by noradrenaline. Interestingly, both central and systemic inflammation are well-established hallmarks of hypertension and its co-morbidities, including an inflammatory process involving the transmigration and infiltration of immune cells into tissues. We propose that physiological states that prolong β-adrenoreceptor activation in bone marrow can disrupt neuroimmune homeostasis and impair communication between the immune system and SNS, leading to immune dysregulation, which, in turn, is sustained via a central mechanism involving neuroinflammation.

LIGHT/TNFSF14 regulates estrogen deficiency-induced bone loss.

Bone loss induced by ovariectomy is due to the direct activity on bone cells and mesenchymal cells and to the dysregulated activity of bone marrow cells, including immune cells and stromal cells, but the underlying mechanisms are not completely known. Here, we demonstrate that ovariectomy induces the T-cell co-stimulatory cytokine LIGHT, which stimulates both osteoblastogenesis and osteoclastogenesis by modulating osteoclastogenic cytokine expression, including TNF, osteoprotegerin, and the receptor activator of nuclear factor-κB ligand (RANKL). Predictably, LIGHT-deficient (Tnfsf14-/- ) mice are protected from ovariectomy-dependent bone loss, whereas trabecular bone mass increases in mice deficient in both LIGHT and T and B lymphocytes (Rag -/- Tnfsf14 -/- ) and is associated with an inversion of the TNF and RANKL/OPG ratio. Furthermore, women with postmenopausal osteoporosis display high levels of LIGHT in circulating T cells and monocytes. Taken together, these results indicate that LIGHT mediates bone loss induced by ovariectomy, suggesting that patients with postmenopausal osteoporosis may benefit from LIGHT antagonism. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The alliance between nerve fibers and stem cell populations in bone marrow: life partners in sickness and health.

The bone marrow (BM) is the central hematopoietic organ in adult mammals, with great potential to be used as a tool to improve the efficacy of the body's response to a number of malignancies and stressful conditions. The nervous system emerges as a critical regulatory player of the BM both under homeostatic and pathologic settings, with essential roles in cellular anchorage and egress, stem cell differentiation, and endothelial cell permeability. This review collects the current knowledge on the interplay between the nervous system and the BM cell populations, with a focus on how the nervous system modulates hematopoietic stem and progenitor cell, mesenchymal stromal cell, and endothelial progenitor cell activity in BM. We have also highlighted the pathologies that have been associated with disturbances in the neuronal signaling in BM and discussed if targeting the nervous system, either by modulating the activity of specific neuronal circuits or by pharmacologically leveling the activity of sympathetic and sensorial signaling-responsive cells in BM, is a promising therapeutic approach to tackling pathologies from BM origin.-Leitão, L., Alves, C. J., Sousa, D. M., Neto, E., Conceição, F., Lamghari, M. The alliance between nerve fibers and stem cell populations in bone marrow: life partners in sickness and health.

The comparative study of the effects of Fe2 O3 and TiO2 micro- and nanoparticles on oxidative states of lung and bone marrow tissues and colony stimulating factor secretion.

Nowadays, increased use of nanomaterials in industry and biomedicine poses potential risks to human health and the environment. Studying their possible toxicological effects is therefore of great significance. The present investigation was designed to examine the status of oxidative stress induced by nanoparticles (NPs) of ferric oxide (Fe2 O 3 ) and titanium oxide (TiO 2 ) with their micro-sized counterpart on mouse lung and bone marrow-derived normal tissue cells. We assessed the induction of oxidative stress by measuring its indicators such as antioxidant scavenging activity of superoxide dismutase and catalase as well as malondialdehyde concentration. Moreover, colony formation of bone marrow cells was assayed following induction with colony stimulating factor (CSF) from lung cells. NPs had a more potent stimulatory effect on the oxidative stress status than their micron-sized counterparts. In addition, the highest level of oxidative stress derived from TiO 2 NPs was observed in both tissue types. Cotreatment with NPs and the antioxidant α-tocopherol reduced antioxidant activities and membrane lipid peroxidation (LPO) in the lung cells, but increased CSF-induced colony formation activity of bone marrow cells, suggesting that oxidative stress may be the cause of the cytotoxic effects of NPs. It is concluded that free radicals generated following exposure to NPs resulted in significant oxidative stress in mouse cells, indicated by increased LPO and antioxidant enzyme activity and decreased colony formation.

A small-molecule PAI-1 inhibitor prevents bone loss by stimulating bone formation in a murine estrogen deficiency-induced osteoporosis model.

Osteoporosis is a progressive bone disease caused by an imbalance between bone resorption and formation. Recently, plasminogen activator inhibitor‐1 (PAI‐1) was shown to play an important role in bone metabolism using PAI‐1‐deficient mice. In this study, we evaluated the therapeutic benefits of novel, orally available small‐molecule PAI‐1 inhibitor (iPAI‐1) in an estrogen deficiency‐induced osteoporosis model. Eight‐week‐old C57BL/6J female mice were divided into three groups: a sham + vehicle (Sham), ovariectomy + vehicle (OVX + v), and OVX + iPAI‐1 (OVX + i) group. iPAI‐1 was administered orally each day for 6 weeks starting the day after the operation. Six weeks of iPAI‐1 treatment prevented OVX‐induced trabecular bone loss in both the femoral bone and lumbar spine. Bone formation activity was significantly higher in the OVX + i group than in the OVX + v and Sham groups. Unexpectedly, OVX‐induced osteoclastogenesis was partially, but significantly reduced. Fluorescence‐activated cell sorting analyses indicated that the number of bone marrow stromal cells was higher in the OVX + i group than that in the OVX + v group. A colony‐forming unit‐osteoblast assay indicated enhanced mineralized nodule formation activity in bone marrow cells isolated from iPAI‐1‐treated animals. Bone marrow ablation analysis indicated that the remodeled trabecular bone volume was significantly higher in the iPAI‐1‐treated group than that in the control group. In conclusion, our results suggest PAI‐1 blockade via a small‐molecule inhibitor is a new therapeutic approach for the anabolic treatment of postmenopausal osteoporosis.

Hematopoietic activity of rat bone marrow at the influence of salt extract of Hirudo verbana Carena, 1820

The mitotic activity of the bone marrow of non-linear female rats after feeding their offspring was studied and this index was estimated in their offspring in the early stages of postembryonic development – on the 1, 15, 30, 45, 60th day. Females two weeks prior to mating with males and two weeks after were intraperitoneally injected with Hirudo verbana saline extract (5 mg/kg of the weight of an animal), once, every week. In the experimental group of females, the mitotic index significantly increased (by 33.61%); in their offspring, the maximum increase in the mitotic index was observed on the first day (by 50.88%); the increase in the proliferative activity of bone marrow cells was observed at other observation times: on the 30th day (by 25.67%) and on the 60th day (by 18.35%). In the experimental group of animals, all hematopoietic parameters of peripheral blood (the number of leukocytes, erythrocytes, hemoglobin) also increased. An increase in the number of leukocytes in the experimental group of females occurred without significant changes in their relative population ratios in the leukocyte blood formula. In the experimental group of the offspring, the effect of the leech extract on the changes in the differentiation of leukocyte populations was revealed. Thus, in the offspring for the first day the shift of the leukocyte blood formula to the left decreased due to the decrease in cells of innate immunity (granulocytes and monocytes) and increase in adaptive immunity cells (lymphocytes), which indicates an accelerated differentiation of the lymphoid system towards adult animals. Some differences in the differentiation of leukocytes were noted at the onset of sexual maturation (on the 30th day) and at its end (on the 60th day). At these times, a neutrophilic shift occurred to the left, as a consequence of the intension of the congenital link of immunity in the presence of an adequate number of cells of adaptive immunity. Thus, as a result of the study, the stimulating effect of the extract of the medical leech on the mitotic activity of the bone marrow was revealed, both in mature females and in their offspring, which resulted in an increase in the number of leukocytes, erythrocytes, hemoglobin, and color index. In the offspring, during the period of increased stress of morphogenesis, there is an activation of both the congenital and adaptive links of immunity in the experimental group of animals.

Proliferative activity of bone marrow cells of rats under the influence of biologically active substances of a medical leech

Hirudotherapy has been used in medicine since time immemorial. The advantage of this type of treatment is that the bite of a leech has a restorative effect on the entire body. The leech bites the skin and injects into the patient’s blood biologically active substances that activate blood microcirculation and promote immunity. The saliva of the leech contains more than 100 substances that contribute to the spectrum of therapeutic effects. But there are no studies on the effect of hirudotherapy on the proliferation of bone marrow cells, which is ontogenetically primary in the development of myeloid and lymphoid tissues. Therefore, it is important to study the mitotic activity of bone marrow of rats at the early stages of postembryonic development against the background of influence by the leech. Female nonlinear rats, two weeks before and two weeks after mating with males, were presented each week to one hungry medical leech weighing 400 mg. The females were examined after feeding their offspring and their offspring were likewise examined on day 1, 15, 30, 45, 60 of the experiment. The animals were kept in vivarium conditions on a standard diet in individual cages. All animals were decapitated under ether anesthesia and the number of leukocytes, the leukocyte blood count, the number of erythrocytes, hemoglobin, and the color index were examined. After that, the femurs were quickly removed and the adjacent muscles separated with a sterile gauze. We cut the epiphyses and cut along the bone. Bone marrow from the femur was flushed with a warm (37 °C) hypotonic 0.9% sodium citrate solution into a sterile centrifuge tube using a Pasteur pipette. The resulting cell suspension was incubated in a hypotonic solution for 10 minutes at 37 °C. The resulting suspension was centrifuged for 5 minutes at 1000 rpm leaving only a small residue of sediment in the test tube. The cells were fixed in methyl alcohol with glacial acetic acid (three parts methyl alcohol and one part acetic acid). 3,000 cells were analyzed, among which we determined those that are in mitosis, and expressed their share. As a result of our studies, the immunomodulating effect of the saliva of the medical leech was revealed, which manifested itself in an increase in the proliferative activity of bone marrow cells, which led to an increase in the number of red blood cells, increased hemoglobin and increase in the number of leukocytes. The color index and the leukocyte blood count were within the normal limits.

Sirtuin-3 Promotes Adipogenesis, Osteoclastogenesis, and Bone Loss in Aging Male Mice.

Sirtuin-3 (Sirt3) is an essential metabolic regulatory enzyme that plays an important role in mitochondrial metabolism, but its role in bone marrow and skeletal homeostasis remains largely unknown. In this study, we hypothesize that increased expression of Sirt3 plays a role in skeletal aging. Using mice that overexpress Sirt3 [i.e., Sirt3 transgenic (Sirt3Tg)], we show that Sirt3 is a positive regulator of adipogenesis and osteoclastogenesis and a negative regulator of skeletal homeostasis. Sirt3Tg mice exhibited more adipocytes in the tibia compared with control mice. Bone marrow stromal cells (BMSCs) from Sirt3Tg mice displayed an enhanced ability to differentiate into adipocytes compared with control BMSCs. We found a 2.5-fold increase in the number of osteoclasts on the bone surface in Sirt3Tg mice compared with control mice (P < 0.03), and increased osteoclastogenesis in vitro. Importantly, Sirt3 activates the mechanistic target of rapamycin (mTOR) pathway to regulate osteoclastogenesis. Sirt3Tg male mice exhibited a significant reduction in cortical thickness at the tibiofibular junction (P < 0.05). In summary, Sirt3 activity in bone marrow cells is associated with increased adipogenesis, increased osteoclastogenesis through activation of mTOR signaling, and reduced bone mass. Interestingly, Sirt3 expression in bone marrow cells increases during aging, suggesting that Sirt3 promotes age-related adipogenesis and osteoclastogenesis associated with bone loss. These findings identify Sirt3 as an important regulator of adipogenesis and skeletal homeostasis in vivo and identify Sirt3 as a potential target for the treatment of osteoporosis.

Genotoxicity of Casiopeina III-Ea in mouse bone marrow cells

Casiopeina III-Ea® (Cas III-Ea®) is a chelated copper complex with antineoplastic activity that is capable of reducing tumor size and inducing antiproliferative and apoptotic effects. However, little is known about its in vivo genotoxic effects. Therefore, this study evaluated two cytogenetic and two proliferative parameters 24 h after the administration of Casiopeina III-Ea® to male CD-1 mice. Three doses of Cas III-Ea® were administered by intraperitoneal injections of 1.69, 3.39 and 6.76 mg/kg (corresponding to 1/8, 1/4 and 1/2 of LD50, respectively). A reduction in the mitotic index (MI) and an increased numbers of cells with structural chromosomal aberrations (SCA) were detected. Additionally, a low but significant increase in the frequency of sister chromatid exchange (SCE) was observed at the highest dose. Changes in the DNA replication index (RI) were not observed. These results indicate that Casiopeina III-Ea® shows cytotoxic and clastogenic activity in bone marrow cells from treated mice.

TNF binding protein of variola virus acts as a TNF antagonist at epicutaneous application.

VARV-CrmB is a TNF binding protein of variola virus. VARV-CrmB protein was previously shown to be active as a TNF-antagonist in a number of in vivo and in vitro models. Here we investigated the epicutaneous effect of recombinant VARV-CrmB protein using an experimental model of muTNFinduced migration of skin leukocytes as well as colony forming activity of bone marrow cells (BMC). Epiсutaneous applications of muTNF enhanced the number of cells migrating from skin flaps of BALB/c mice, whereas subsequent applications of VARV-CrmB protein in 30 min after muTNF, abolished that effect. Epicutaneously applied muTNF influenced the activity of committed hematopoietic progenitors causing a reduction of erythroid (BFUe+CFUe) colonies and increase of granulocyte-macrophage (CFU-GM) colonies in the colony-forming tests. VARV-CrmB, applied in combination with muTNF, demonstrated an ability to reverse this effect, namely, to increase BFUe+CFUe and reduce CFU-GM back to the control levels. Taking together, these data demonstrate the TNF-blocking properties of VARV-CrmB in vivo at epicutaneous applications. As effective TNF antagonist VARV-CrmB protein might be conceded as a beneficial candidate for future research and development of therapeutic approaches in the field of inflammatory skin diseases.

Hematopoietic effect of deer antler extract fermented by Bacillus subtilis on murine marrow cells.

BACKGROUND/OBJECTIVESWe examined the chemical composition and the effect of fermented deer antler on hematopoietic factors in bone marrow cells.MATERIALS/METHODSFor the preparation of fermented deer antler extract (FAB), fermentation was carried out using Bacillus subtilis at 30℃ for 7 days. The hematopoietic effect of FAB was investigated hematopoietic factors in marrow cells.RESULTSThe contents of total sugar, sulfated glycosaminoglycans, and uronic acid and the dry weight gradually increased with fermentation time. The sialic acid content (from 0.14 mg/mL to 0.54 mg/mL) was the highest on the 4th day of fermentation after which it decreased. The proliferating activity of bone marrow cells increased with fermentation times. The levels of various hematopoietic growth factors were determined to verify the beneficial effect of deer antler extract fermented by B. subtilis on hematopoiesis. FAB increased the number of stem cell factors and granulocyte colony-stimulating factor in bone marrow cells. In addition, FAB augmented the burst-forming unit erythroid and total colonies in splenocyte-conditioned medium compared with non-fermented antler extract (NFA). However, FAB did not affect the mRNA levels of erythropoietin, an important factor for erythropoiesis.CONCLUSIONSFAB, like NFA, did not directly affect hematopoiesis, but contributed to hematopoiesis by stimulating the production of hematopoietic factors.

골수세포에 대한 Bordetella bronchiseptica 항원의 자극 효과 및 면역기억반응

(Received: June 26, 2014; Revised: October 8, 2014; Accepted: 13 October, 2014)Abstract : Bone marrow is a hematological and immunological organ that provides multiple immune cells, includingB lymphocytes, and thus plays a critical role in the efficacy of vaccine. We previously demonstrated that Bordetella(B.) bronchiseptica antigen has high immunogenicity in spleen cells, a peripheral immune organ. In this study, weinvestigated the immunogenicity of B. bronchiseptica antigen in bone marrow cells, a central immune organ. B.bronchiseptica antigen increased the cellular activity of bone marrow cells and significantly enhanced the productionof nitric oxide, IL-6, and TNF-α. Bone marrow cells primed with B. bronchiseptica antigen in vivo were harvested andstimulated with the same antigen in vitro. The stimulation of B. bronchiseptica antigen significantly increased the cellularactivity and proliferation rate of the primed cells. B. bronchiseptica antigen also greatly induced the production of antigen-specific antibody in the primed cells. Taken together, the present study demonstrated that B. bronchiseptica antigen canstimulate bone marrow cells, a central immune organ, and recall the immune response of the primed bone marrow cells.Keywords : antigen, Bordetella bronchiseptica, immune memory responses, stimulatory effects, vaccine