Bone Marrow Cell Cultures Research Articles

Generation and characterization of chicken monocyte-derived dendritic cells

IntroductionDendritic cells (DCs) play a crucial role in orchestrating immune responses by bridging innate and adaptive immunity. In vitro generation of DCs from mouse and human tissues such as bone marrow and peripheral blood monocytes, has been widely used to study their immunological functions. In chicken, DCs have mainly been derived from bone marrow cell cultures, with limited characterization from blood monocytes.MethodsThe present study takes advantage of newly available chicken immunological tools to further characterize chicken monocyte-derived dendritic cells (MoDCs), focusing on their phenotype, and functions, including antigen capture and T-cell stimulation, and response to live Newcastle disease virus (NDV) stimulation.ResultsAdherent chicken PBMCs were cultured with recombinant chicken granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), for 5 days, leading to the upregulation of putative CD11c and MHCII, markers of DC differentiation. Subsequent stimulation with lipopolysaccharide (LPS) or 24 h triggered phenotypic maturation of MoDCs, characterized by the increased surface expression of MHCII and co-stimulatory molecules CD80 and CD40, and elevated IL-12p40 secretion. This maturation reduced endocytic capacity but enhanced the allogenic stimulatory activity of the chicken MoDCs. Upon NDV stimulation for 6 h, MoDCs upregulated antiviral pathways, including retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), melanoma differentiation-associated protein 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2), alongside increased production of type I interferons (IFNs), and the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), IL-1β, and IL-6. However, these responses were downregulated after 24 hours.ConclusionThese findings provide a comprehensive characterization of chicken MoDCs and suggest their potential as a model for studying host-pathogen interactions.

Hippo Signaling Pathway Involvement in Osteopotential Regulation of Murine Bone Marrow Cells Under Simulated Microgravity.

The development of osteopenia is one of the most noticeable manifestations of the adverse effects of space factors on crew members. The Hippo signaling pathway has been shown to play a central role in regulating the functional activity of cells through their response to mechanical stimuli. In the present study, the components of the Hippo pathway and the protective properties of osteodifferentiation inducers were investigated under simulated microgravity (smg) using a heterotypic bone marrow cell culture model, which allows for the maintenance of the close interaction between the stromal and hematopoietic compartments, present in vivo and of great importance for both the fate of osteoprogenitors and hematopoiesis. After 14 days of smg, the osteopotential and osteodifferentiation of bone marrow stromal progenitor cells, the expression of Hippo cascade genes and the immunocytochemical status of the adherent fraction of bone marrow cells, as well as the paracrine profile in the conditioned medium and the localization of Yap1 and Runx2 in mechanosensitive cells of the bone marrow were obtained. Simulated microgravity negatively affects stromal and hematopoietic cells when interacting in a heterotypic murine bone marrow cell culture. This is evidenced by the decrease in cell proliferation and osteopotential. Changes in the production of pleiotropic cytokines IL-6, GROβ and MCP-1 were revealed. Fourteen days of simulated microgravity induced a decrease in the nuclear translocation of Yap1 and the transcription factor Runx2 in the stromal cells of the intact group. Exposure to osteogenic induction conditions partially compensated for the negative effect of simulated microgravity. The data obtained will be crucial for understanding the effects of spaceflight on osteoprogenitor cell growth and differentiation via Hippo-Yap signaling.

Spatio-temporal clustering of African swine fever virus (Asfarviridae: Asfivirus) circulating in the Kaliningrad region based on three genome markers.

The rapid spread of African swine fever in the Kaliningrad region makes it necessary to use the methods of molecular epidemiology to determine the dynamics and direction of ASF spread in this region of Russia. The aim of the study was to determine single nucleotide polymorphisms within molecular markers K145R, O174L and MGF 505-5R of ASFVs isolated in Kaliningrad region and to study the circulating of the pathogen in European countries by subgenotyping and spatio-temporal clustering analysis. Blood samples from living domestic pigs and organs from dead domestic pigs and wild boars, collected in the Kaliningrad region between 2017 and 2022 were used. Virus isolation was carried out in porcine bone-marrow primary cell culture. Amplicons of genome markers were amplified by PCR with electrophoretic detection and subsequent extraction of fragments from agarose gel. Sequencing was performed using the Sanger method. The circulation of two genetic clusters of ASFV isolates on the territory of the Kaliningrad has been established: epidemic (K145R-III, MGF 505-5R-II, O174L-I - 94.3% of the studied isolates) and sporadic (K145R-II, MGF 505-5R-II, O174L-I - 5.7%). The broaden molecular genetic surveillance of ASFV isolates based on sequencing of genome markers is necessary in the countries of the Eurasian continent to perform a more detailed analysis of ASF spread between countries and within regions.

Colony-forming potential of murine bone marrow-derived progenitor cells in experimental neurodegenerative pathology and under the impact of melatonin in vivo or in vitro

Cell therapy is a promising direction in the treatment of Parkinson’s disease/parkinsonism and multiple sclerosis, the effectiveness of which can be increased with the help of biologically active factors, in particular melatonin. The purpose of the study: to investigate the colony-forming ability of stromal and hematopoietic cells-progenitors of the bone marrow of mice with experimentally induced neurodegenerative pathology and reveal the possibility to change it under the influence of melatonin in vivo or in vitro. Materials and methods: In adult (6-7 months) male FVB/N (haplotype H-2q) and 129/Sv (haplotype H-2b) mice, the parkinsonism model was reproduced by a single subcutaneous injection of the neurotoxin 1-methyl-4-phenyl -1,2,3,6-tetrahydropyridine (MPTP) at a dose of 30 mg/kg 129/Sv mice also received the neurotoxin cuprizone daily with food (at the rate of 0.2 % of the mass of the pure substance from the daily feed) for three weeks (multiple sclerosis model). Melatonin was administered to the 129/Sv mice intraperitoneally at a dose of 1 mg/kg, daily, at 17.00, from the 8th day of receiving cuprizone, or was added in vitro to the culture of bone marrow cells of mice of both strains with parkinsonism at a dose of 0.1 μg/0.1 mL. In the bone marrow, the absolute number of nucleated cells was counted, the relative content of CD4+ lymphocytes was determined by flow cytometry, and the number of colony-forming precursor cells for fibroblasts (CFU-F) and for granulocyte-macrophages (CFU-GM) in culture in vitro was estimated. Results. In the bone marrow of 129/Sv mice with a model of multiple sclerosis, the number of nucleated cells, CFU-GM and CD4+ cells decrease, while under the influence of exogenous melatonin applied in vivo, the values of these indicators increase to the level of intact animals. After the administration of MPTP in the bone marrow of FVB/N mice, in contrast to 129/Sv mice, the number of nucleated cells and CFU-F decreases. After the incubation with melatonin of bone marrow cells of 129/Sv mice with a model of parkinsonism, an increase in the amount of CFU-F was observed compared to control values, and there were no changes in the indicator values in FVB/N mice. Conclusions. In the bone marrow of mice with the MPTP-model of parkinsonism, a decrease in the absolute amount of CFU-F was found, and in mice with the cuprizone model of multiple sclerosis – CFU-GM. The application of melatonin in vitro increases the colony-forming potential of progenitor cells for fibroblasts in the bone marrow of mice with the MPTP model of parkinsonism. The use of melatonin in vivo increases the colony-forming ability of progenitor cells for granulocyte-macrophages and the relative content of CD4+ cells in the bone marrow of mice with the cuprizone model of multiple sclerosis. Changes in the amount of CFU-F in the bone marrow in parkinsonism and under the influence of melatonin in vitro depend on the H-2 haplotype mice, which can be the basis for the development of personalized cell therapy for this pathology.

Vitamin A enhanced periosteal osteoclastogenesis is associated with increased number of tissue-derived macrophages/osteoclast progenitors

A deleterious effect of elevated levels of vitamin A on bone health has been reported in numerous clinical studies. Mechanistic studies in rodents have shown that numbers of periosteal osteoclasts are increased, while endocortical osteoclasts are simultaneously decreased by vitamin A treatment. These observations indicate that osteoclastogenesis on the endocortical and periosteal surfaces of bone is differentially controlled by vitamin A. The present study investigated the in vitro and in vivo effect of all-trans retinoic acid (ATRA), the active metabolite of vitamin A, on periosteal osteoclast progenitors. Mouse calvarial bone cells were cultured in media containing ATRA, with or without the osteoclastogenic cytokine RANKL, on plastic dishes or bone discs. Whereas ATRA did not stimulate osteoclast formation alone, the compound robustly potentiated the formation of RANKL-induced bone resorbing osteoclasts. This effect was due to stimulation by ATRA (EC50 ∼3nM) on the numbers of macrophages/osteoclast progenitors in the bone cell cultures, as assessed by mRNA and protein expression of several macrophage and osteoclast progenitor cell markers, such as M-CSF receptor, RANK, F4/80 and CD11b, as well as by FACS-analysis of CD11b+/F480+/Gr1- cells. The stimulation of macrophage numbers in the periosteal cell cultures was not mediated by increased M-CSF or IL-34. In contrast, ATRA did not enhance macrophages in bone marrow cell cultures. Importantly, ATRA treatment upregulated the mRNA expression of several macrophage-related genes also in the periosteum of tibia in adult mice. These observations demonstrate a novel mechanism by which vitamin A enhances osteoclast formation specifically on periosteal surfaces.

The novel cytotoxic polybisphosphonate osteodex decreases bone resorption by enhancing cell death of mature osteoclasts without affecting osteoclastogenesis of RANKL-stimulated mouse bone marrow macrophages

SummaryIt has previously been demonstrated that the polybisphosphonate osteodex (ODX) inhibits bone resorption in organ-cultured mouse calvarial bone. In this study, we further investigate the effects by ODX on osteoclast differentiation, formation, and function in several different bone organ and cell cultures. Zoledronic acid (ZOL) was used for comparison. In retinoid-stimulated mouse calvarial organ cultures, ODX and ZOL significantly reduced the numbers of periosteal osteoclasts without affecting Tnfsf11 or Tnfrsf11b mRNA expression. ODX and ZOL also drastically reduced the numbers of osteoclasts in cell cultures isolated from the calvarial bone and in vitamin D3–stimulated mouse crude bone marrow cell cultures. These data suggest that ODX can inhibit osteoclast formation by inhibiting the differentiation of osteoclast progenitor cells or by directly targeting mature osteoclasts. We therefore assessed if osteoclast formation in purified bone marrow macrophage cultures stimulated by RANKL was inhibited by ODX and ZOL and found that the initial formation of mature osteoclasts was not affected, but that the bisphosphonates enhanced cell death of mature osteoclasts. In agreement with these findings, ODX and ZOL did not affect the mRNA expression of the osteoclastic genes Acp5 and Ctsk and the osteoclastogenic transcription factor Nfatc1. When bone marrow macrophages were incubated on bone slices, ODX and ZOL inhibited RANKL-stimulated bone resorption. In conclusion, ODX does not inhibit osteoclast formation but inhibits osteoclastic bone resorption by decreasing osteoclast numbers through enhanced cell death of mature osteoclasts.

Small-molecule inhibition of kinesin KIF18A reveals a mitotic vulnerability enriched in chromosomally unstable cancers

Chromosomal instability (CIN) is a hallmark of cancer, caused by persistent errors in chromosome segregation during mitosis. Aggressive cancers like high-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC) have a high frequency of CIN and TP53 mutations. Here, we show that inhibitors of the KIF18A motor protein activate the mitotic checkpoint and selectively kill chromosomally unstable cancer cells. Sensitivity to KIF18A inhibition is enriched in TP53-mutant HGSOC and TNBC cell lines with CIN features, including in a subset of CCNE1-amplified, CDK4–CDK6-inhibitor-resistant and BRCA1-altered cell line models. Our KIF18A inhibitors have minimal detrimental effects on human bone marrow cells in culture, distinct from other anti-mitotic agents. In mice, inhibition of KIF18A leads to robust anti-cancer effects with tumor regression observed in human HGSOC and TNBC models at well-tolerated doses. Collectively, our results provide a rational therapeutic strategy for selective targeting of CIN cancers via KIF18A inhibition.

Emodin suppresses adipogenesis of bone marrow derived mesenchymal stem cells from aplastic anemia via increasing TRIB3 expression

BackgroundIncreasing evidence indicate that enhanced adipogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs) could contribute to the adiposity alteration in marrow microenvironment of aplastic anemia (AA). Identifying small molecule drugs with role in inhibiting adipogenesis of BM-MSCs may represent a novel direction in AA therapy by improving BM-MSCs mediated marrow microenvironment. MethodsFor the purpose, we isolated AA BM-MSCs through whole bone marrow cell culture, evaluated a series of small molecule drugs using the in vitro adipogenic differentiation model of BM-MSCs, and finally focused on emodin, a natural anthraquinone derivative. Subsequently, we systematically investigated the molecular mechanism of emodin in attenuating adipogenic process by means of microarray profiling, bioinformatics analysis and lentivirus-mediated functional studies and rescue assay. ResultsWe found that emodin presented significantly suppressive effect on the in vitro adipogenic differentiation of AA BM-MSCs. Further mechanistic investigation revealed that emodin could increase the expression of Tribbles homolog 3 (TRIB3) which exhibited remarkably decreased expression in AA BM-MSCs compared with the normal counterparts and was subsequently demonstrated as a negative regulator in adipogenesis of AA BM-MSCs. Besides, TRIB3 depletion alleviated the suppressive effect of emodin on the adipogenic differentiation of AA BM-MSCs. ConclusionOur findings propose that emodin mediated TRIB3 up-regulation alleviates the adipogenic capacity of AA BM-MSCs, and emodin could serve as a potential therapeutic regimen for AA therapy.

Loss of Anti-Tumor Efficacy by Polyamine Blocking Therapy in GCN2 Null Mice.

GCN2 is one of the main sensors of amino acid starvation stress, and its activation in the stressful tumor microenvironment plays a crucial role in tumor survival and progression. We hypothesized that elevated polyamine biosynthesis and subsequent depletion of precursor arginine activates GCN2, thus rewiring metabolism to support tumor cell survival and drive myeloid immunosuppressive function. We sought to determine if the anti-tumor efficacy of a polyamine blocking therapy (PBT) may be mediated by its effect on GCN2. Unlike wild-type mice, PBT treatment in GCN2 knockout mice bearing syngeneic B16.F10 or EG7 tumors resulted in no tumor growth inhibition and no changes in the profile of infiltrating tumor immune cells. Studies with murine bone marrow cell cultures showed that increased polyamine metabolism and subsequent arginine depletion and GCN2 activation played an essential role in the generation and cytoprotective autophagy of myeloid derived suppressor cells (MDSCs) as well as the M2 polarization and survival of macrophages, all of which were inhibited by PBT. In all, our data suggest that polyamine-dependent GCN2 signaling in stromal cells promotes tumor growth and the development of the immunosuppressive tumor microenvironment, and that the PBT anti-tumor effect is mediated, at least in part, by targeting GCN2.

Anti-Siglec-15 Antibody Prevents Marked Bone Loss after Acute Spinal Cord Injury-Induced Immobilization in Rats.

Rapid and extensive sublesional bone loss after spinal cord injury (SCI) is a difficult medical problem that has been refractory to available interventions except the antiresorptive agent denosumab (DMAB). While DMAB has shown some efficacy in inhibiting bone loss, its concurrent inhibition of bone formation limits its use. Sialic acid-binding immunoglobulin-like lectin (Siglec)-15 is expressed on the cell surface of mature osteoclasts. Anti-Siglec-15 antibody (Ab) has been shown to inhibit osteoclast maturation and bone resorption while maintaining osteoblast activity, which is distinct from current antiresorptive agents that inhibit the activity of both osteoclasts and osteoblasts. The goal of the present study is to test a Siglec-15 Ab (NP159) as a new treatment option to prevent bone loss in an acute SCI model. To this end, 4-month-old male Wistar rats underwent complete spinal cord transection and were treated with either vehicle or NP159 at 20 mg/kg once every 2 weeks for 8 weeks. SCI results in significant decreases in bone mineral density (BMD, -18.7%), trabecular bone volume (-43.1%), trabecular connectivity (-59.7%), and bone stiffness (-76.3%) at the distal femur. Treatment with NP159 almost completely prevents the aforementioned deterioration of bone after SCI. Blood and histomorphometric analyses revealed that NP159 is able to greatly inhibit bone resorption while maintaining bone formation after acute SCI. In ex vivo cultures of bone marrow cells, NP159 reduces osteoclastogenesis while increasing osteoblastogenesis. In summary, treatment with NP159 almost fully prevents sublesional loss of BMD and metaphysis trabecular bone volume and preserves bone strength in a rat model of acute SCI. Because of its unique ability to reduce osteoclastogenesis and bone resorption while promoting osteoblastogenesis to maintain bone formation, Siglec-15 Ab may hold greater promise as a therapeutic agent, compared with the exclusively antiresorptive or anabolic agents that are currently used, in mitigating the striking bone loss that occurs after SCI or other conditions associated with severe immobilization. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Heterotypic Cell Culture from Mouse Bone Marrow under Simulated Microgravity: Lessons for Stromal Lineage Functions.

Muscle and skeleton structures are considered most susceptible to negative factors of spaceflights, namely microgravity. Three-dimensional clinorotation is a ground-based simulation of microgravity. It provides an opportunity to elucidate the effects of microgravity at the cellular level. The extracellular matrix (ECM) content, transcriptional profiles of genes encoding ECM and remodelling molecules, and secretory profiles were investigated in a heterotypic primary culture of bone marrow cells after 14 days of 3D clinorotation. Simulated microgravity negatively affected stromal lineage cells, responsible for bone tissue formation. This was evidenced by the reduced ECM volume and stromal cell numbers, including multipotent mesenchymal stromal cells (MSCs). ECM genes encoding proteins responsible for matrix stiffness and cell-ECM contacts were downregulated. In a heterotypic population of bone marrow cells, the upregulation of genes encoding ECM degrading molecules and the formation of a paracrine profile that can stimulate ECM degradation, may be mechanisms of osteodegenerative events that develop in real spaceflight.

Cordycepin Abrogates Methotrexate-Induced Suppression of Osteoclastogenesis

In this study, we have explored the reasons for the poor efficiency of 3′-deoxyadenosine in the treatment of rheumatoid arthritis. The effects of cordycepin and methotrexate on osteoclastogenesis were evaluated using the culture system of rat whole bone marrow cells and immunofluorescence staining with Kat1, a novel cell-surface antigen specifically expressed on rat osteoclasts. The effect of cordycepin on osteoclast formation was evaluated by tartaric acid-resistant phosphatase staining of bone marrow cell culture lines (pre-osteoclast formation lines) in which mesenchymal cells were removed. In the bone marrow culture system for evaluating osteoclastogenesis, methotrexate-induced suppression of osteoclastogenesis was abrogated only by the addition of cordycepin. In the pre-osteoclast formation system, cordycepin (3′-deoxyadenosine) was found to have no significant effect on osteoclast formation. To sum up, cordycepin should be carefully used in some cases of rheumatoid arthritis treated with methotrexate and in the severe stages of rheumatoid arthritis.

The induction of histamine synthesis from rat bone marrow derived mast cells with rat stem cell factor

Abstract Mast cells are the main cells to produce histamine regarded as the main factor to induce allergy. To comprehend the allergy by mast cell activation, The mast cells of bone marrow derived mast cells (BMMC) are usually prepared by culturing bone marrow cells including rodent hematopoietic stem cells, in the presence of both IL-3 and stem cell factor (SCF). However, BMMC was reported not to produce the high levels histamine comparable to mature mast cells in host tissue. In this study, rat BMMC (rBMMC) were cultured in 96 well plate to induce diverse conditions in cell numbers per unit culture media, rat IL-3 and rat SCF to find the conditions to synthesize high level histamine. Cells of rat bone marrow cells were cultured in the cell density of 1.1×10 5/ml (low density, lowD) and 5.5 × 10 5/ml (high D), rat IL-3 was added to the levels of 10 ng/ml (low 3) or 50 ng/ml (high 3), Rat SCF was adjusted to the levels of 12.5 (lows), 25 (midS) and 50 (highS) ng/ml. Histamine included in 5×10 4BMMC were extracted by perchloric acid, n-butanol, and heptane. The levels of histamine were measured by ophthalialdehyde solution and read for fluorometric intensity. The levels at culture day 4 (D 4) were in the range of 12~45 ng. The significant elevation was observed at D 11 in the conditions of lowD high3 (153 ng), however, D 8 in highD high3 (253 ng). The average histamine level was 74 ng in the condition of low Dlow 3, but 112 ng in low Dhigh 3 at D 11. That was 106 ng in high Dlow 3 but 183 ng in high Dhigh 3 at culture D 8. These results indicated that rat IL-3 increased the level of histamine synthesis and shorten the histamine production from undifferentiated hematopoietic stem cells. The induction of histamine synthesis from rat bone marrow derived mast cells with rat stem cell factor

Elucidation of regulatory mechanisms in the activation of dendritic cells by combined stimuli with PRR ligands and sulfated polysaccharide fucoidan

Abstract Fucoidan, a series of natural high-molecular weight sulfated polysaccharides derived from brown algae, has been reported to have various physiological effects such as antitumor, antiviral, and immunomodulatory activities. In a previous study, we demonstrated that fucoidan from Cladosiphon okamuranus (Okinawamozuku) was effectively activate murine macrophage-like cell line RAW264 in synergistic action with dectin-1-stimulating beta-glucan from Saccharomyces cerevisiae. In this study, we further examined their synergistic mode of action of Okinawamozuku-derived fucoidan in cooperate with pathogen stimulation in terms of dendritic cell activation. After myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC) were differentiated by culturing bone marrow cells isolated from C57BL/6J mice with Flt3 ligand, cytokine production upon stimulation with fucoidan and various pathogen components (pattern recognition receptor, PRR ligands) was measured. As the results, fucoidan alone enhanced the production of interferon (IFN)-gamma by the Flt3-L-induced mixed BMDCs, and besides, fucoidan synergistically augmented IFN-gamma production with Pam3CSK4 (bacterial TLR1/TLR2 ligand) or Poly(I:C)/LyoVec (viral RIG-I ligand). On the other hand, their IFN-alpha productions were reinforced with Poly(I:C) (viral TLR3 ligand), ODN1585 (viral TLR9 ligand) and Poly(I:C)/LyoVec, although no synergic enhancement by fucoidan was observed. These results suggested that fucoidan was extremely effective in activating dendritic cells, which play a central role in the regulation of immune function to eliminate infectious pathogens. Supported by grants from JSPS KAKENHI (JP21K05423)

BAFF induces CXCR5 expression during B cell differentiation in bone marrow

B cell activating factor (BAFF) plays an important role in antibody production through differentiation and maturation of B cells mainly in secondary lymphoid organs. On the other hand, the role of BAFF in the bone marrow, the primary lymphoid organ of B cell development, has not been well elucidated. Here, effects of BAFF in bone marrow B cell development were examined by using BAFF-deficient mice. When mRNA expression levels of B cell differentiation markers including Cd19, Bcl2, Igμ, Il7r and Cxcr5 were compared between bone marrow of wild-type and BAFF-KO mice, a lower level of Cxcr5 expression was found in the KO mice. Additionally, protein expression of CXCR5 on IgM+ cells in the bone marrow was decreased by BAFF deficiency. In vitro studies also confirmed the effect of BAFF on CXCR5 by IgM+ cells; culturing bone marrow cells from BAFF-KO mice with BAFF in vitro increased the proportion of CXCR5+ cells in IgM+ cells compared with non-treated bone marrow cells. In addition, BAFF synergized with TNF-α and IL-6 to increase the expression of CXCR5+ on IgM+ cells. The BAFF-mediated up-regulation of CXCR5 expression was reproduced by using CD19+ cells purified from BAFF-KO bone marrow cells, suggesting that BAFF directly affects B-lineage cells in bone marrow to promote CXCR5 expression. Together, this study suggests that BAFF has an important role in B cell differentiation in bone marrow by directly inducing CXCR5 expression which affect their migration to secondary lymphoid organs.

Chicken CSF2 and IL-4-, and CSF2-dependent bone marrow cultures differentiate into macrophages over time.

Chicken bone marrow-derived macrophages (BMMΦ) and dendritic cells (BMDC) are utilized as models to study the mononuclear phagocytic system (MPS). A widely used method to generate macrophages and DC in vitro is to culture bone marrow cells in the presence of colony-stimulating factor-1 (CSF1) to differentiate BMMΦ and granulocyte-macrophage-CSF (GM-CSF, CSF2) and interleukin-4 (IL-4) to differentiate BMDC, while CSF2 alone can lead to the development of granulocyte-macrophage-CSF-derived DC (GMDC). However, in chickens, the MPS cell lineages and their functions represented by these cultures are poorly understood. Here, we decipher the phenotypical, functional and transcriptional differences between chicken BMMΦ and BMDC along with examining differences in DC cultures grown in the absence of IL-4 on days 2, 4, 6 and 8 of culture. BMMΦ cultures develop into a morphologically homogenous cell population in contrast to the BMDC and GMDC cultures, which produce morphologically heterogeneous cell cultures. At a phenotypical level, all cultures contained similar cell percentages and expression levels of MHCII, CD11c and CSF1R-transgene, whilst MRC1L-B expression decreased over time in BMMΦ. All cultures were efficiently able to uptake 0.5 µm beads, but poorly phagocytosed 1 µm beads. Little difference was observed in the kinetics of phagosomal acidification across the cultures on each day of analysis. Temporal transcriptomic analysis indicated that all cultures expressed high levels of CSF3R, MERTK, SEPP1, SPI1 and TLR4, genes associated with macrophages in mammals. In contrast, low levels of FLT3, XCR1 and CAMD1, genes associated with DC, were expressed at day 2 in BMDC and GMDC after which expression levels decreased. Collectively, chicken CSF2 + IL-4- and CSF2-dependent BM cultures represent cells of the macrophage lineage rather than inducing conventional DC.

The effects of 1,25(OH)2D3 treatment on metabolic reprogramming and maturation in bone marrow-derived dendritic cells from control and diabetic mice

Activated dendritic cells (DCs) undergo significant metabolic reprogramming, which is characterized by an increase in aerobic glycolysis and a concurrent progressive loss of oxidative phosphorylation. The modulation of metabolic reprogramming is believed to be closely related to the function of DCs. Vitamin D has been reported to inhibit the maturation of DCs. DC dysfunction has been reported in diabetic patients, and hyperglycemia is associated with impaired glycolytic metabolism in immune cells. Therefore, vitamin D and diabetes may affect intracellular metabolism, thereby regulating the activity of DCs. We investigated the effect of in vitro treatment of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on metabolic reprogramming and maturation of bone marrow-derived dendritic cells (BMDCs) from diabetic mouse. Six-week-old male C57BLKS/J-m+/m+ mice (CON) and C57BLKS/J-db/db mice (db/db) were fed with a 10% kcal fat diet for seven weeks. BMDCs were generated by culturing bone marrow cells from the mice with rmGM-CSF (20 ng/mL) in the absence or presence of 10 nM 1,25(OH)2D3. The maturation of BMDCs was induced via lipopolysaccharide (LPS, 50 ng/mL) stimulation for 24 h. LPS stimulation induced iNOS protein expression and decreased the mitochondrial respiration, while increased lactate production and the expression of glycolytic pathway-related genes (Glut1 and Pfkfb3) in BMDCs from both CON and db/db groups. In LPS-stimulated mature BMDCs, 1,25(OH)2D3 treatment decreased the expression of surface markers related to immunostimulatory functions (MHC class II, CD80, CD86, and CD40) and production of IL-12p70 in both CON and db/db groups. While the mRNA level of the gene related to glucose uptake (Glut1) was increased in both groups, lactate production was decreased by 1,25(OH)2D3 treatment. mTORC1 activity was suppressed following 1,25(OH)2D3 treatment. Collectively, our findings confirmed that metabolic reprogramming occurred in BMDCs following LPS stimulation. In vitro 1,25(OH)2D3 treatment induced tolerogenic phenotypes by reducing the expression of surface markers, as well as cytokine production. However, no significant difference was observed regarding the effects of 1,25(OH)2D3 treatment on metabolic conversion and maturation of BMDCs between the control and diabetic mice. Additionally, the decreased aerobic glycolysis induced by the 1,25(OH)2D3 treatment appeared to be associated with the diminished maturation of BMDCs, and mTORC1 appears to play a key role in the 1,25(OH)2D3-mediated regulation of glycolysis.

Platelet augmentation activity of mature leaf juice of Sri Lankan wild type cultivar of Carica papaya L: Insights into potential cellular mechanisms

Ethnopharmacological relevanceCarica papaya L., a common fruit crop of the family Caricaceae and its leaf juice/extract is a traditionally commended preparation against dengue and other thrombocytopenic diseases by many Asian countries. Aim of the studyThe present study posits the potential cellular mechanisms of platelet augmentation activity of mature leaf juice of Sri Lankan wild-type Carica papaya. Materials and methodsC. papaya leaf juice prepared from different cultivar types, maturity of the leaf, agro-climatic region, and preparation methods were orally administered to hydroxyurea-induced thrombocytopenic rats at 0.72 ml/100 g BW dosage to investigate the most potent platelet increasing preparation.The papaya juice doses; low dose (LD-0.18 ml/100 g BW), human equivalent dose (HED-0.36 ml/100 g BW), and high dose (HD-0.72 ml/100 g BW), were administered to thrombocytopenic rats (N = 6/group) daily for three consecutive days and post-treatment plasma levels of interleukin 6 (IL-6), thrombopoietin (TPO), and platelet-activating factor (PAF) were quantified using specific rat ELISA kits. The mature leaf juice of C. papaya induced IL-6 secretion from bone marrow cell (BMC) cultures was quantified using ELISA. The ability of papaya juice to protect the platelet membrane, from the damage caused by the lytic agent was analyzed in vitro using the lactate dehydrogenase (LDH) assay. The effect of the mature leaf juice of C. papaya on secondary hemostasis was investigated using blood coagulation and clot hydrolyzing activity. ResultsThe comparative analysis revealed that the platelet increasing activity of C. papaya leaf did not significantly differ among different types of cultivar, maturity of the leaf, agro-climatic regions and preparation methods (p > 0.05). Both TPO and PAF levels in thrombocytopenic rats diminished when treated with all three doses of the mature leaf juice of C. papaya (p < 0.05), yet IL-6 plasma level was unaltered (p > 0.05). Nevertheless, ex vivo treatment of the mature leaf juice of C. papaya had significantly enhanced IL-6 levels of rat BMC cultures (p < 0.05). Pre-treatment of platelets with the mature leaf juice of C. papaya at different concentrations significantly inhibited LDH leakage from platelets and may have reduced the membrane damage caused by the lytic agent (p < 0.05). Treatment of mature leaf juice of C. papaya also significantly reduced blood clotting time through the extrinsic pathway of the blood coagulation cascade (p < 0.05). Further, prolonged incubation of the plasma clot with different concentrations of the papaya leaf juice revealed dose-dependent hydrolysis of the blood clot, indicating fibrinolysis activity. ConclusionsThe current study exceeded the traditional medicinal claims, and scientifically affirmed the platelet augmentation activity of mature leaf juice of C. papaya. The mechanistic rationale tested herein explicated that the platelet augmentation activity of the papaya leaf juice can be partially attributed to the stimulation of bone marrow megakaryocytes via modulating thrombopoietic cytokines TPO and IL-6, and by inhibiting the secretion of PAF, while reducing the peripheral platelet destruction by stabilizing the platelet membrane. Further, mature leaf juice of C. papaya imparted both pro-coagulation and fibrinolysis activity of secondary hemostasis endorsing its potential against thrombocytopenia.

PB1914: CYTOGENETIC FINDINGS IN 18 CHILDREN WITH MYELODYSPLASTIC SYNDROMES BY KARYOTYPIC AND FISH ANALYSIS

Background: Pediatric myelodysplastic syndromes (MDS) are rare disorders characterized by hematopoietic cell dysfunction, an increased risk of AML and a poor prognosis. Although it is known that cytogenetic abnormalities are seen in approximately half of children diagnosed with MDS, their detailed cytogenetic profile has not been elucidated yet due to the rarity of the disease. Aims: We tried to detect chromosomal abnormalities in pediatric patients with MDS, including submicroscopic aberrations such as gene alterations associated with the disease and possible malignant transformation by karyotypic and molecular cytogenetic analysis (fluorescence in situ hybridization, FISH) to contribute to the elucidation of the cytogenetic profile of pediatric MDS and the investigation of the pathogenesis of the disease. Methods: In this study 18 children with MDS are included. Karyotypic analysis was performed on 24h, 48h and 72h bone marrow (BM) cultures using GTG-banding. Karyotypic analysis was carried out on 25 at least metaphases. FISH analysis was performed on cultured BM cells using IVD probes for centromere (CEP) of chromosomes 7 and 8 and the chromosomal regions 7q22, 7q31, 8q24(MYC), 11q22.3(ATM), 11q23(MLL), and 17p13(TP53). For each sample at least 200 interphase cells (iFISH) and 5 metaphases (mFISH) were analyzed to increase sensitivity. Normal cut-off was estimated at 3%. Results: The sex ratio was 1,25 (10 M/8 F) and the mean age was 9,42 years (range 0,4y - 19y). Eleven patients had RCC, 5 MDS-EB and 2 t-MDS. Normal karyotypes were detected in 10 patients (55,6%), 6 patients with RCC, 3 with MDS-EB and 1 with t-MDS. Abnormal karyotypes were found in 8 patients (44,4%), 4 with RCC, 2 with MDS-EB and 1 with t-MDS. Monosomy 7 was observed in 2/18 patients (>70% of metaphases), +8 in 2/18 patients (5-6.6% of metaphases), inv(17)(p11.2q21) in 1/18 patients (18.75% of metaphases), complex karyotypes in 2/18 patients (31.3-73.2% of metaphases including abnormalities of 1q, 2q, 4q, 5q, 16q, 18 and Xp), in one patient after SCT in the transformation of MDS into s-AML and in the other at diagnosis with t-MDS) and chromatid and chromosome breaks in 1/18 patients, (8,6-9,6% of metaphases) in 2 different BM samples taken with an interval of 3 months, which were also found in his brother’s BM karyotype. FISH revealed -7 in 2/18 patients confirming their karyotypes, loss of one p53 allele in 3/18 patients at low frequencies (6,5%, 4,5%, 3,5%) all with hypocellular RCC and transfusion-dependency that underwent SCT, +8 in 2/18 (in 8% and 2,7% of cells) confirming trisomy 8 in their karyotypes and loss of one MLL allele and MLL rearrangement in a patient that was transformed to s-AML in 4,4% of cells. A normal pattern of hybridization was observed for the above probes in the rest cases and for MYC and ATM genes in all the examined cases. Summary/Conclusion: Karyotypic aberrations were detected in 44,4% of children including -7, +8, inv(17), complex karyotypes and chromatid and chromosome breaks, revealing that other chromosome aberrations except -7/del(7q) and trisomy 8 are characterized this disease. Clonal chromosome abnormalities can be detected at low frequencies in the karyotypes of pediatric MDS patients so except from iFISH, mFISH is also needed especially in cases with small clones or submicroscopic translocations. The low frequency of submicroscopic aberrations such as p53 deletions observed in 3 cases of hypocellular RCC must be confirmed in larger studies by molecular analysis as they may play a potential role in MDS pathogenic evolution.

Flt3L, LIF, and IL-10 combination promotes the selective in vitro development of ESAMlow cDC2B from murine bone marrow.

The development of two conventional dendritic cells (DC) subsets (cDC1 and cDC2) and the plasmacytoid DC (pDC) in vivo and in cultures of bone marrow (BM) cells is mediated by the growth factor Flt3L. However, little is known about the factors that direct the development of the individual DC subsets. Here, we describe the selective in vitro generation of murine ESAMlow CD103- XCR1- CD172a+ CD11b+ cDC2 from BM by treatment with a combination of Flt3L, LIF, and IL-10 (collectively named as FL10). FL10 promotes common dendritic cell progenitors (CDP) proliferation in the cultures, similar to Flt3L and CDP sorted and cultured in FL10 generate exclusively cDC2. These cDC2 express the transcription factors Irf4, Klf4, and Notch2, and their growth is reduced using BM from Irf4-/- mice, but the expression of Batf3 and Tcf4 is low. Functionally they respond to TLR3, TLR4, and TLR9 signals by upregulation of the surface maturation markers MHC II, CD80, CD86, and CD40, while they poorly secrete proinflammatory cytokines. Peptide presentation to TCR transgenic OT-II cells induced proliferation and IFN-γ production that was similar to GM-CSF-generated BM-DC and higher than Flt3L-generated DC. Together, our data support that FL10 culture of BM cells selectively promotes CDP-derived ESAMlow cDC2 (cDC2B) development and survival in vitro.