Bone Marrow Formation Research Articles

Abstract 1171: Genotoxic preventive potential of ethanol leaves extract of Annona muricata on N-Nitroso-N-ethylurea pro-leukaemia carcinogen in mice by bone marrow micronucleus assay

Abstract Background: Genotoxicity which is an important indicator of DNA damage, repair and cell rejuvenation has been a predisposing factor to cancer development evidenced by bone marrow suppression in leukemia and studies have proven the effect of several agents inclusive of natural products to induce, prevent and treat genotoxicity through experimental models and clinical trials. Aim:The genotoxic preventive potential of Annona muricata ethanol leaves extract on N-nitroso-N-ethylurea (NEU) pro-leukemia carcinogen in mice model using micronuclei formation in bone marrow was assessed. Materials and Methods: 48 mice weighing 18-24g were randomly divided into six groups of eight mice each comprising of the; mice intravenously administered 20mg/kg of NEU 48 hourly 3 times (A); 80mg/kg of NEU 48 hourly 3 times (B); negative control fed with feed and water only (C); 0.2ml (0.1g/ml) ethanolic leaves extract of Annona muricata for 3 weeks prior to administration of low dose of NEU (D); 0.2ml (0.1g/ml) ethanolic extract of Annona muricata for 3 weeks prior to administration of high dose of NEU (E); and Annona muricata (ethanolic leaves extract) and commercial diet for adverse/toxicity group (F). After six weeks of last exposure, animals were ethically sacrificed, bone marrow harvested, smeared, stained using MayGrumwald and number of micronucleus polychromatic erythrocyte (MNPCEs) were determined microscopically.Result: Groups exposed to various dosage of the NEU yielded significantly increased MNPCEs with group B producing higher MNPCEs than A. The groups treated with extract displayed a significant reduction in the MNPCEs despite the prior exposure concentrations of NEU. The adverse group displayed no difference in MNPCEs when compared with the negative control (Table 1). Conclusion: The extract displayed profound capacity to prevent genotoxicity thereby alleviating leukemia condition and it is well tolerated. Week Number Groups (Mean±Standard deviation of MNPCE) A B C D E F 2 14.13±0.56 50.45±2.31 4.52±0.34 6.15±0.49 17.11±1.11 4.53±0.45 3 15.01±0.18 53.77±1.95 4.61±0.67 7.85±0.98 20.34±2.34 4.62±0.23 4 16.11±0.22 56.95±2.01 4.91±0.88 8.01±1.01 25.11±1.67 5.63±0.45 5 19.64±1.68 68.23±1.56 4.82±0.75 10.07±0.67 30.54±2.34 5.92±0.67 Citation Format: Olufemi Emmanuel Akanni, Eunice Oluwaseyi Bamisaye, Sunday Anthony Fashina, Fatai Adesina Abdulraheem, Solomon Olufemi Fadiora. Genotoxic preventive potential of ethanol leaves extract of Annona muricata on N-Nitroso-N-ethylurea pro-leukaemia carcinogen in mice by bone marrow micronucleus assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1171.

Histological Evaluation of Bone Regeneration Using Hydroxyapatite Based Bone Substitute Derived from Antler: An Animal Study.

Guided bone regeneration (GBR) is considered as a prerequisite in some cases of implant dentistry. For this purpose, bone materials are commonly used. Calcium compounds and Ca-P based materials like hydroxyapatite (HA, Ca10 (PO4)6(OH)2), due to their similarity with the human bone, can be used as graft materials for bone regeneration. This study aimed to evaluate biocompatibility of antler xenograft and compare the osteoconduction effects of antler xenograft with Cerabone in regeneration of calvarium bony defects of rabbits. Five defects with a diameter of 6 mm and a depth of 3 mm were prepared in the calvarium of four rabbits. Thereafter, two defects were randomly grafted with antler xenograft, two defects were filled with Cerabone, and one defect remained as the untreated group. Histological evaluations, including measuring percentage of new regenerated bone and the amounts of osteoblast, osteoclast, and osteocyte cells, were also performed. To do statistical analyses, paired t-test, chi-square, and Fisher tests were applied. The percentage of new bone formation was significantly higher in antler xenograft (73.33%) and in Cerabone (48.91%) compared to the untreated group (18.91%). The amounts of osteocytes and osteoblasts were obtained as 3.52 ± 0.17 and 2.41 ± 0.24 in the Antler xenograft and as 2.57 ± 0.29 and 2.31 ± 0.32 in the Cerabone group, respectively. Bone marrow formation were significantly higher in antler xenograft (6.66 ± 5.34) and Cerabone (1.99 ± 3.17) compared to the untreated group. According to this pilot study, results of using antler xenograft as an osteoconductive materials in regeneration of rabbit calvarial defects are comparable with Cerabone. Although more clinical studies are needed.

Terpene Lactucopicrin Limits Macrophage Foam Cell Formation by a Reduction of Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 in Lipid Rafts.

Sustained inflammation promotes macrophage foam cell formation by promoting cholesterol influx and impairing cholesterol efflux. Terpene lactucopicrin, affluent in vegetables of the Asteraceae family (e.g., chicory, curly escarole, and lettuce) can inhibit atherogenesis in mice. However, it remains unknown whether and how lactucopicrin regulates macrophage foam cell formation. Lactucopicrin at physiologically reachable concentrations inhibits oxidized low-density lipoprotein (oxLDL)-induced foam cell formation in inflammatory mouse bone marrow derived macrophages established by 50 pg mL-1 of LPS, reachable level in patients with metabolic endotoxemia. This effect is not due to modulation of cholesterol efflux, but reliant on a reduction in lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1)-mediated cholesterol influx. Mechanistically, lactucopicrin does not affect LOX-1 expression, cellular oxidative stress, and exocytosis, known mechanisms regulating LOX-1 function in cholesterol influx. Strikingly, lactucopicrin selectively decreases LOX-1 content in lipid rafts, an effect responsible for the lactucopicrin effect on cholesterol influx. Moreover, ApoE-/- mice fed a high fat diet supplemented with lactucopicrin for 12 weeks display fewer macrophage foam cells within atherosclerotic plaques relative to the control mice. Lactucopicrin limits macrophage foam cell formation through a reduction of LOX-1 distribution in lipid rafts, thus contributing to its atheroprotective effect.

CHD5 inhibits metastasis of neuroblastoma

CHD5, a tumor suppressor at 1p36, is frequently lost or silenced in poor prognosis neuroblastoma (NB) and many adult cancers. The role of CHD5 in metastasis is unknown. We confirm that low expression of CHD5 is associated with stage 4 NB. Forced expression of CHD5 in NB cell lines with 1p loss inhibited key aspects of the metastatic cascade in vitro: anchorage-independent growth, migration, and invasion. In vivo, formation of bone marrow and liver metastases developing from intravenously injected NB cells was delayed and decreased by forced CHD5 expression. Genome-wide mRNA sequencing revealed reduction of genes and gene sets associated with metastasis when CHD5 was overexpressed. Known metastasis-suppressing genes preferentially upregulated in CHD5-overexpressing NB cells included PLCL1. In patient NB, low expression of PLCL1was associated with metastatic disease and poor survival. Knockdown of PLCL1 and of p53 in IMR5 NB cells overexpressing CHD5 reversed CHD5-induced inhibition of invasion and migration in vitro. In summary, CHD5 is a metastasis suppressor in NB.

Whole-Genome CRISPR Screening Identifies N-Glycosylation As an Essential Pathway and a Potential Novel Therapeutic Target in CALR-Mutant MPN

Somatic mutations in the ER chaperone calreticulin (CALR) are frequent and disease-initiating in myeloproliferative neoplasms (MPN). Although the mechanism of mutant CALR-induced MPN is known to involve pathogenic binding between mutant CALR and MPL, this insight has not yet been exploited therapeutically. Consequently, a major deficiency is the lack of clonally selective therapeutic agents with curative potential. Hence, we set out to discover and validate unique genetic dependencies for mutant CALR-driven oncogenesis.We first performed a whole-genome CRISPR knockout screen in CALR Δ52 MPL-expressing hematopoietic cells to identify genes that were differentially required for the growth of cytokine-independent, transformed CALR Δ52 cells as compared to control cells. Using gene-set enrichment analyses, we identified the N-glycan biosynthesis, unfolded protein response, and the protein secretion pathways to be amongst the most significantly differentially depleted pathways (FDR q values <0.001, 0.014, and 0.025, respectively) in CALR Δ52 cells. We performed a secondary CRISPR pooled screen focused on significant pathways from the primary screen and confirmed these findings.Strikingly, seven of the top ten hits in both screens were linked to protein N-glycosylation. Four of those genes encode proteins involved in the enzymatic activity of dolichol-phosphate mannose synthase (DPM1, DPM2, DPM3, and MPDU1). This enzyme synthesizes dolichol D-mannosyl phosphate, an essential substrate for protein N-glycosylation. Importantly, these findings from an unbiased whole-genome screen align with prior mechanistic studies demonstrating that both the N-glycosylation sites on MPL and the lectin-binding sites on CALR Δ52 are required for mutant CALR-driven oncogenesis.We next performed single gene CRISPR Cas9 validation studies and found that DPM2 is required for CALR Δ52-mediated transformation, as demonstrated by increased cell death, reduced p-STAT5 and decreased MPL cell-surface levels, when Dpm2 is knocked out. Importantly, cells cultured in cytokine-rich medium were unaffected by DPM2 loss. Upon cytokine withdrawal, a sub-clone of non-edited Dpm2WT CALR Δ52 cells grew out, further demonstrating requirement for DPM2 for the survival of CALR Δ52 cells. Additionally, we observed a >50% reduction in ex vivo myeloid colony formation of murine CalrΔ52 Dpm2 ko bone marrow (BM) compared with CRISPR-Cas9 non-targeting controls, with non-significant effects on CalrWT BM cells.To enable clinical translation, we performed a pharmacological screen targeting pathways significantly depleted in our CRISPR screens. Screening 70 drugs, we found that the N-glycosylation pathway was the only pathway in which all tested compounds preferentially killed CALR Δ52 transformed cells.We then treated primary Calr Δ52/+ mice with a clinical grade N-glycosylation (N-Gi) inhibitor and found platelet counts (Sysmex) to be significantly reduced (vehicle 3x10 6/mL, N-Gi 1x10 6/mL after 18 days, p<.0001). Concordantly, the proportion of megakaryocyte erythrocyte progenitors (MEPs) was significantly reduced in CalrΔ52 BM (p=0.03).We next performed competitive BM transplantation assays using CD45.2 UBC-GFP MxCre CalrΔ52 knockin and CD45.1 mice. We found that mice treated with N-Gi had significantly reduced platelet counts (vehicle 1440x10 6/mL, N-Gi 845x10 6/mL, p=0.005) as well as significantly reduced platelet chimerism (vehicle 55%, N-Gi 27%, p<0.001), indicating a distinct vulnerability of CalrΔ52 over WT cells.Finally, we interrogated RNA-sequencing data from primary human MPN platelets. We found N-glycosylation-related pathways to be significantly upregulated in CALR-mutated platelets (n = 13) compared to healthy control platelets (n = 21), highlighting the relevance of our findings to human MPN.In summary, using unbiased genetic and focused pharmacological screens, we identified the N-glycan biosynthesis pathway as essential for mutant CALR-driven oncogenesis. Using a pre-clinical MPN model, we found that in vivo inhibition of N-glycosylation normalizes key features of MPN and preferentially targets CalrΔ52 over WT cells. These findings have therapeutic implications through inhibiting N-glycosylation alone or in combination with other agents to advance the development of clonally selective therapeutic approaches in CALR-mutant MPN.AEM and JSJ contributed equally. [Display omitted] DisclosuresMullally: Janssen, PharmaEssentia, Constellation and Relay Therapeutics: Consultancy.

Overexpression of Transmembrane TNF Drives Development of Ectopic Lymphoid Structures in the Bone Marrow and B Cell Lineage Alterations in Experimental Spondyloarthritis.

TNF is important in immune-mediated inflammatory diseases, including spondyloarthritis (SpA). Transgenic (tg) mice overexpressing transmembrane TNF (tmTNF) develop features resembling human SpA. Furthermore, both tmTNF tg mice and SpA patients develop ectopic lymphoid aggregates, but it is unclear whether these contribute to pathology. Therefore, we characterized the lymphoid aggregates in detail and studied potential alterations in the B and T cell lineage in tmTNF tg mice. Lymphoid aggregates developed in bone marrow (BM) of vertebrae and near the ankle joints prior to the first SpA features and displayed characteristics of ectopic lymphoid structures (ELS) including presence of B cells, T cells, germinal centers, and high endothelial venules. Detailed flow cytometric analyses demonstrated more germinal center B cells with increased CD80 and CD86 expression, along with significantly more T follicular helper, T follicular regulatory, and T regulatory cells in tmTNF tg BM compared with non-tg controls. Furthermore, tmTNF tg mice exhibited increased IgA serum levels and significantly more IgA+ plasma cells in the BM, whereas IgA+ plasma cells in the gut were not significantly increased. In tmTNF tg × TNF-RI-/- mice, ELS were absent, consistent with reduced disease symptoms, whereas in tmTNF tg × TNF-RII-/- mice, ELS and clinical symptoms were still present. Collectively, these data show that tmTNF overexpression in mice results in osteitis and ELS formation in BM, which may account for the increased serum IgA levels that are also observed in human SpA. These effects are mainly dependent on TNF-RI signaling and may underlie important aspects of SpA pathology.

Diterbutyl phthalate attenuates osteoarthritis in ACLT mice via suppressing ERK/c-fos/NFATc1 pathway, and subsequently inhibiting subchondral osteoclast fusion.

Osteoarthritis (OA) is the most common arthritis with a rapidly increasing prevalence. Disease progression is irreversible, and there is no curative therapy available. During OA onset, abnormal mechanical loading leads to excessive osteoclastogenesis and bone resorption in subchondral bone, causing a rapid subchondral bone turnover, cyst formation, sclerosis, and finally, articular cartilage degeneration. Moreover, osteoclast-mediated angiogenesis and sensory innervation in subchondral bone result in abnormal vascularization and OA pain. The traditional Chinese medicine Panax notoginseng (PN; Sanqi) has long been used in treatment of bone diseases including osteoporosis, bone fracture, and OA. In this study we established two-dimensional/bone marrow mononuclear cell/cell membrane chromatography/time of flight mass spectrometry (2D/BMMC/CMC/TOFMS) technique and discovered that diterbutyl phthalate (DP) was the active constituent in PN inhibiting osteoclastogenesis. Then we explored the therapeutic effect of DP in an OA mouse model with anterior cruciate ligament transaction (ACLT). After ACLT was conducted, the mice received DP (5 mg·kg-1·d-1, ip) for 8 weeks. Whole knee joint tissues of the right limb were harvested at weeks 2, 4, and 8 for analysis. We showed that DP administration impeded overactivated osteoclastogenesis in subchondral bone and ameliorated articular cartilage deterioration. DP administration blunted aberrant H-type vessel formation in subchondral bone marrow and alleviated OA pain assessed in Von Frey test and thermal plantar test. In RANKL-induced RAW264.7 cells in vitro, DP (20 μM) retarded osteoclastogenesis by suppressing osteoclast fusion through inhibition of the ERK/c-fos/NFATc1 pathway. DP treatment also downregulated the expression of dendritic cell-specific transmembrane protein (DC-STAMP)and d2 isoform of the vacuolar (H+) ATPase V0 domain (Atp6v0d2) in the cells. In conclusion, we demonstrate that DP prevents OA progression by inhibiting abnormal osteoclastogenesis and associated angiogenesis and neurogenesis in subchondral bone.

The IL-17 Receptor IL-17RA Is Expressed By Early Erythroid Progenitors and Stimulates Erythropoiesis

The central regulator of erythropoeitic rate is erythropoietin (Epo). Epo action is augmented by factors that act on early erythroid progenitors upstream of erythroid terminal differentiation: glucocorticoids, Stem Cell Factor and BMP4. Of these, only Epo and glucocorticoids are useful agents clinically, leaving a gap when they are ineffective or contraindicated. The molecular identities of early erythroid progenitors are not well understood, posing an obstacle to the discovery of novel erythropoietic agents. To address this gap, we used single-cell RNA-seq (scRNA-seq) technology to delineate a continuous erythroid trajectory in the mouse, beginning with multi-potential progenitors (MPPs) and ending with the onset of erythroid terminal differentiation (described in Tusi, Wolock et al.). Based on gene expression analysis, we predicted two novel developmental stages within the erythroid trajectory, that we termed Early Erythroid Progenitors (EEP) and Committed Erythroid Progenitors (CEP). We devised a flow cytometric strategy that enriches for these novel progenitor stages, and found that they correspond to cells with BFU-e and CFU-e activity, respectively.Our scRNA-seq work predicted cell surface molecules expressed by EEPs and CEPs, including receptors with known endogenous ligands or drug agonists or antagonists, but no previously documented role in erythropoiesis. The first three such receptors that we tested, Mst1r, Ryk and IL-17RA, all modified adult and/or fetal liver BFU-e and CFU-e colony formation in vitro . In the case of IL-17RA, its IL-17a ligand cooperated synergistically with Epo to expand the number of adult bone marrow CFU-e in vitro, while having no effect on fetal liver CFU-e. Specifically, at the modestly elevated Epo concentration of 0.05 U/ml, addition of IL-17a (10 ng/ml) resulted in a 2.5 fold increase in CFU-e colonies (p<0.001); at levels of Epo that elicit its maximal effect (2 U/ml), addition of IL-17a increased the number of CFU-e colonies by an additional 44% (p=0.002). A dose response analysis showed that EC50 for IL-17a's synergistic action with Epo was 2 ng/ml (60 pM). Furthermore, addition of IL-17a boosted the formation of human bone marrow CFU-e but not BFU-e in vitro .IL-17a had no effect on CFU-e formation in bone marrow derived from IL-17RA-/- mouse, confirming the specificity of this interaction. In work that is ongoing, we found that IL-17a activated intracellular Stat3 and Stat5 signalings in early erythroid progenitors. Taken together, these findings suggest that IL-17a is a novel erythropoietic stimulator, synergizing with Epo to accelerate erythropoietic rate. [Display omitted] DisclosuresKlein:OneCell Bio: Other: Equity Ownership and Membership on an entity's Board of Directors or advisory committees.

Biofabrication of Prevascularised Hypertrophic Cartilage Microtissues for Bone Tissue Engineering.

Bone tissue engineering (TE) has the potential to transform the treatment of challenging musculoskeletal pathologies. To date, clinical translation of many traditional TE strategies has been impaired by poor vascularisation of the implant. Addressing such challenges has motivated research into developmentally inspired TE strategies, whereby implants mimicking earlier stages of a tissue’s development are engineered in vitro and then implanted in vivo to fully mature into the adult tissue. The goal of this study was to engineer in vitro tissues mimicking the immediate developmental precursor to long bones, specifically a vascularised hypertrophic cartilage template, and to then assess the capacity of such a construct to support endochondral bone formation in vivo. To this end, we first developed a method for the generation of large numbers of hypertrophic cartilage microtissues using a microwell system, and encapsulated these microtissues into a fibrin-based hydrogel capable of supporting vasculogenesis by human umbilical vein endothelial cells (HUVECs). The microwells supported the formation of bone marrow derived stem/stromal cell (BMSC) aggregates and their differentiation toward a hypertrophic cartilage phenotype over 5 weeks of cultivation, as evident by the development of a matrix rich in sulphated glycosaminoglycan (sGAG), collagen types I, II, and X, and calcium. Prevascularisation of these microtissues, undertaken in vitro 1 week prior to implantation, enhanced their capacity to mineralise, with significantly higher levels of mineralised tissue observed within such implants after 4 weeks in vivo within an ectopic murine model for bone formation. It is also possible to integrate such microtissues into 3D bioprinting systems, thereby enabling the bioprinting of scaled-up, patient-specific prevascularised implants. Taken together, these results demonstrate the development of an effective strategy for prevascularising a tissue engineered construct comprised of multiple individual microtissue “building blocks,” which could potentially be used in the treatment of challenging bone defects.

Ionic dissolution products of Cerium-doped bioactive glass nanoparticles promote cellular osteogenic differentiation and extracellular matrix formation of human bone marrow derived mesenchymal stromal cells

Cerium (Ce) is a promising candidate ion for application in bone tissue engineering (BTE) since it reduces the presence of reactive oxygen species. Ce-doped mesoporous bioactive glass nanoparticles (MBGNs) serving as vectors for the local application of Ce already demonstrated stimulating effects on the expression of pro-osteogenic genes in Saos-2 cells. So far, there is no evidence available about the effects of Ce-doped MBGNs on the viability, osteogenic differentiation and the formation of the osseous extracellular matrix (ECM) of primary human bone marrow-derived mesenchymal stromal cells (BMSCs). Therefore, in this study, the biocompatibility of the ionic dissolution products (IDPs) of MBGNs containing increasing concentrations of CeO2 (0.05 MCe-MBGNs, composition in mol%: 86.6SiO2-12.1CaO-1.3CeO2; and 0.2 MCe-MBGNs, composition in mol%: 86.0SiO2-11.8CaO-2.2CeO2) and unmodified MBGNs (composition in mol%: 86SiO2-14CaO) was evaluated using human BMSCs. Eventually, the impact of the MBGNs’ IDPs on the cellular osteogenic differentiation and their ability to build and mature a primitive osseous ECM was assessed. The Ce-doped MBGNs had a positive influence on the viability and stimulated the cellular osteogenic differentiation of human BMSCs evaluated by analyzing the activity of alkaline phosphate as a marker enzyme for osteoblasts in the present setting. Furthermore, the formation and calcification of a primitive osseous ECM was significantly stimulated in the presence of Ce-doped MBGNs in a positive concentration-dependent manner as demonstrated by an elevated presence of collagen and increased ECM calcification. The results of this in-vitro study show that Ce-doped MBGNs are attractive candidates for further application in BTE.

Apoptotic Ablation of Platelets Reduces Atherosclerosis in Mice With Diabetes.

People with diabetes are at a significantly higher risk of cardiovascular disease, in part, due to accelerated atherosclerosis. Diabetic subjects have increased number of platelets that are activated, more reactive, and respond suboptimally to antiplatelet therapies. We hypothesized that reducing platelet numbers by inducing their premature apoptotic death would decrease atherosclerosis. Approach and Results: This was achieved by targeting the antiapoptotic protein Bcl-xL (B-cell lymphoma-extra large; which is essential for platelet viability) via distinct genetic and pharmacological approaches. In the former, we transplanted bone marrow from mice carrying the Tyr15 to Cys loss of function allele of Bcl-x (known as Bcl-xPlt20) or wild-type littermate controls into atherosclerotic-prone Ldlr+/- mice made diabetic with streptozotocin and fed a Western diet. Reduced Bcl-xL function in hematopoietic cells significantly decreased platelet numbers, exclusive of other hematologic changes. This led to a significant reduction in atherosclerotic lesion formation in Bcl-xPlt20 bone marrow transplanted Ldlr+/- mice. To assess the potential therapeutic relevance of reducing platelets in atherosclerosis, we next targeted Bcl-xL with a pharmacological strategy. This was achieved by low-dose administration of the BH3 (B-cell lymphoma-2 homology domain 3) mimetic, ABT-737 triweekly, in diabetic Apoe-/- mice for the final 6 weeks of a 12-week study. ABT-737 normalized platelet numbers along with platelet and leukocyte activation to that of nondiabetic controls, significantly reducing atherosclerosis while promoting a more stable plaque phenotype. These studies suggest that selectively reducing circulating platelets, by targeting Bcl-xL to promote platelet apoptosis, can reduce atherosclerosis and lower cardiovascular disease risk in diabetes. Graphic Abstract: A graphic abstract is available for this article.

Mechanisms Underlying the Role of Myeloid-Derived Suppressor Cells in Clinical Diseases: Good or Bad.

Myeloid-derived suppressor cells (MDSCs) have strong immunosuppressive activity and are morphologically similar to conventional monocytes and granulocytes. The development and classification of these cells have, however, been controversial. The activation network of MDSCs is relatively complex, and their mechanism of action is poorly understood, creating an avenue for further research. In recent years, MDSCs have been found to play an important role in immune regulation and in effectively inhibiting the activity of effector lymphocytes. Under certain conditions, particularly in the case of tissue damage or inflammation, MDSCs play a leading role in the immune response of the central nervous system. In cancer, however, this can lead to tumor immune evasion and the development of related diseases. Under cancerous conditions, tumors often alter bone marrow formation, thus affecting progenitor cell differentiation, and ultimately, MDSC accumulation. MDSCs are important contributors to tumor progression and play a key role in promoting tumor growth and metastasis, and even reduce the efficacy of immunotherapy. Currently, a number of studies have demonstrated that MDSCs play a key regulatory role in many clinical diseases. In light of these studies, this review discusses the origin of MDSCs, the mechanisms underlying their activation, their role in a variety of clinical diseases, and their function in immune response regulation.

Peptidylarginine Deiminase Inhibition Prevents Diabetes Development in NOD Mice.

Protein citrullination plays a role in several autoimmune diseases. Its involvement in murine and human type 1 diabetes has recently been recognized through the discovery of antibodies and T-cell reactivity against citrullinated peptides. In the current study, we demonstrate that systemic inhibition of peptidylarginine deiminases (PADs), the enzymes mediating citrullination, through BB-Cl-amidine treatment, prevents diabetes development in NOD mice. This prevention was associated with reduced levels of citrullination in the pancreas, decreased circulating autoantibody titers against citrullinated glucose-regulated protein 78, and reduced spontaneous neutrophil extracellular trap formation of bone marrow–derived neutrophils. Moreover, BB-Cl-amidine treatment induced a shift from Th1 to Th2 cytokines in the serum and an increase in the frequency of regulatory T cells in the blood and spleen. In the pancreas, BB-Cl-amidine treatment preserved insulin production and was associated with a less destructive immune infiltrate characterized by reduced frequencies of effector memory CD4+ T cells and a modest reduction in the frequency of interferon-γ–producing CD4+ and CD8+ T cells. Our results point to a role of citrullination in the pathogenesis of autoimmune diabetes, with PAD inhibition leading to disease prevention through modulation of immune pathways. These findings provide insight in the potential of PAD inhibition for treating autoimmune diseases like type 1 diabetes.

Jabuticaba peel extract modulates adipocyte and osteoblast differentiation of MSCs from healthy and osteoporotic rats.

The jabuticaba peel extract (JPE) contains bioactive compounds that regulate fat metabolism. Because the negative correlation between fat accumulation and bone formation in bone marrow, we hypothesized that JPE inhibits adipocyte as well as favors osteoblast differentiation of mesenchymal stromal cells (MSCs) under healthy and osteoporotic conditions, a disease that display an imbalance between adipocyte and osteoblast differentiation resulting in reduced bone mass. To test these hypotheses, bone marrow MSCs were harvested from healthy and osteoporotic ratsand cultured in adipogenic and osteogenic media with three concentrations of JPE, 0.25, 5 and 10µg/ml, and vehicle (control). After selecting the most efficient concentrations of JPE, we used them to evaluate adipocyte and osteoblast differentiation of MSCs from both sources. We observed that, in general, JPE inhibited adipocyte differentiation of MSCs with more pronounced effects in cells from healthy than osteoporotic rats. In addition, JPE increased osteoblast differentiation, exhibiting a slightly higher osteogenic potential on MSCs from osteoporotic compared to healthy condition. Our results demonstrated that JPE drives MSCs to inhibit adipocyte differentiation and toward osteoblast differentiation under healthy and osteoporotic conditions. These findings pave the way for further translational studies to investigate the therapeutic possibilities of JPE in both prevention and treatment of osteoporosis.

Inhibition of Axin1 in osteoblast precursor cells leads to defects in postnatal bone growth through suppressing osteoclast formation

Axin1 is a negative regulator of β-catenin signaling and its role in osteoblast precursor cells remains undefined. In the present studies, we determined changes in postnatal bone growth by deletion of Axin1 in osteoblast precursor cells and analyzed bone growth in newborn and postnatal Axin1Osx mice and found that hypertrophic cartilage area was largely expanded in Axin1Osx KO mice. A larger number of chondrocytes and unabsorbed cartilage matrix were found in the bone marrow cavity of Axin1Osx KO mice. Osteoclast formation in metaphyseal and subchondral bone areas was significantly decreased, demonstrated by decreased TRAP-positive cell numbers, associated with reduction of MMP9- and cathepsin K-positive cell numbers in Axin1Osx KO mice. OPG expression and the ratio of Opg to Rankl were significantly increased in osteoblasts of Axin1Osx KO mice. Osteoclast formation in primary bone marrow derived microphage (BMM) cells was significantly decreased when BMM cells were cultured with conditioned media (CM) collected from osteoblasts derived from Axin1Osx mice compared with BMM cells cultured with CM derived from WT mice. Thus, the loss of Axin1 in osteoblast precursor cells caused increased OPG and the decrease in osteoclast formation, leading to delayed bone growth in postnatal Axin1Osx KO mice.

A case report: renal cystic tumoural calcinosis with ossification and bone marrow formation

BackgroundTumoural calcinosis (TC) is a rare disorder characterized by nonneoplastic amorphous calcium deposition that tends to occur in soft tissues around the large joint. Here, we report a case of cystic TC with ossification and bone marrow formation in the kidney.Case presentationWe report a 63-year-old woman who presented with a complaint of intermittent right lumbar pain for 2 months. Computed tomography (CT) revealed a large cystic lesion on the lateral side of the right kidney, with a circular calcified wall around the lesion, which compressed, deformed and displaced the right kidney. To relieve the symptoms of right lumbar pain, the patient underwent surgical resection of this cystic lesion without partial removal of the renal parenchyma. The pathological results further confirmed the diagnosis of cystic TC with ossification and bone marrow formation in the right kidney. No recurrence was detected 1 year after surgery.ConclusionsThe main differential diagnoses of TC in the kidney are kidney stone, renal tuberculosis, renal cyst with a calcified wall, and tumour. Patients are treated mainly by complete surgical resection of the lesion.

Suppression of Hematopoietic Primitive Cells in Patients with Secondary Failure of Platelet Recovery after Acute Graft-versus-Host Disease.

Secondary failure of platelet recovery (SFPR) can occur after allogeneic hematopoietic stem cell transplantation (alloHSCT), and 20% of cases are related to acute graft-versus-host disease (aGVHD). The underlying mechanisms of this association are unclear, however. The aim of the present study was to investigate the potential mechanisms of SFPR secondary to aGVHD, which may provide a new therapeutic strategy for these patients. A total of 468 patients with malignant hematologic disease who underwent alloHSCT were included. Sixty-six patients developed SFPR after alloHSCT, and in 45 of these 66 patients (68.2%), SFPR was secondary to grade II-IV aGVHD (SFPR/aGVHD). Compared with patients with good graft function (GGF), patients with SFPR had poor overall survival (20.72% versus 88.01%; P < .0001). Grade II-IV aGVHD was identified as an independent risk factor for SFPR in multivariate analysis (hazard ratio, 9.512; P < .0001). We observed reduced erythroid and megakaryocyte colony formation in bone marrow (BM) samples isolated from SFPR/aGVHD patients, consistent with the lower frequency of megakaryocyte and erythrocyte progenitors in BM. Levels of the inflammatory cytokines IL-2R and TNF-R1 were significantly higher in the SFPR/aGVHD group compared with the GGF group (P=.002 and .001, respectively), as were the frequencies of proinflammatory T helper subsets. Furthermore, the pathways that regulate hematopoiesis and immune responses were universally underexpressed in CD34+ cells isolated from SFPR/aGVHD patients. Differentially expressed genes were significantly enriched in the hematopoietic cell lineage pathway and other pathways involved in both immune responses and megakaryopoiesis. In summary, we found that both the immune microenvironment and compromised proliferation of hematopoietic primitive cells contribute to the development of SFPR secondary to aGVHD, and our data provide new insight into the mechanisms of SFPR in the context of aGVHD.

Cylindrospermopsin-Microcystin-LR Combinations May Induce Genotoxic and Histopathological Damage in Rats.

Cylindrospermopsin (CYN) and microcystins (MC) are cyanotoxins that can occur simultaneously in contaminated water and food. CYN/MC-LR mixtures previously investigated in vitro showed an induction of micronucleus (MN) formation only in the presence of the metabolic fraction S9. When this is the case, the European Food Safety Authority recommends a follow up to in vivo testing. Thus, rats were orally exposed to 7.5 + 75, 23.7 + 237, and 75 + 750 μg CYN/MC-LR/kg body weight (b.w.). The MN test in bone marrow was performed, and the standard and modified comet assays were carried out to measure DNA strand breaks or oxidative DNA damage in stomach, liver, and blood cells. The results revealed an increase in MN formation in bone marrow, at all the assayed doses. However, no DNA strand breaks nor oxidative DNA damage were induced, as shown in the comet assays. The histopathological study indicated alterations only in the highest dose group. Liver was the target organ showing fatty degeneration and necrotic hepatocytes in centrilobular areas, as well as a light mononuclear inflammatory periportal infiltrate. Additionally, the stomach had flaking epithelium and mild necrosis of epithelial cells. Therefore, the combined exposure to cyanotoxins may induce genotoxic and histopathological damage in vivo.

Dynamics of hematological parameters, macromorphological and radiological results of reparative osteogenesis in rabbits for the use of platelet concentrates and hydroxyapatite ceramics

The results of the dynamics of the morphological parameters of the blood of a rabbit with model defects of the opening of the spongy and compact bone tissue at diff erent periods of reparative osteogenesis are presented. Formed 4 groups of rabbits. Bone lesions in the fi rst experimental group were fi lled with injectable platelet-rich fi brin, in the second - platelet-rich fi brin, in the third - a combination of platelet-rich fi brin, and hydroxyapatite with β-tricalcium phosphate.In the control group, the defects remained healed under a blood clot. All animals were in the same conditions of feeding and keeping, had unlimited access to water. During the study, rabbits were clinically observed. Blood was taken for morphological examination before surgery and on the 3rd, 7th, 14th, 21st, 42nd day. Animals were taken from the experiment on the 14th, 21st and 42nd days, an X-ray examination was carried out, bone tissue samples were taken. It was established that trauma of bone tissue leads to a number of reactions of the body aimed at restoring the damaged area. Against the background of the general picture of the obtained morphological results, the level of platelets signifi cantly changes compared to the physiological norm, while the number of red blood cells and white blood cells does not go beyond it. There is a slight increase in hemoglobin levels, especially in the experimental groups on the twenty-fi rst and forty-second day. Signifi cant changes in the number of red blood cells, white blood cells, platelets and hemoglobin concentration in the experimental groups were noted compared with the control group at diff erent periods of reparative osteogenesis.The use of various types of platelet concentrates aff ects the overall reaction of the body. On radiographs of the radial bones (compact bone tissue) on the twenty-fi rst day in the second and third experimental groups, where fi brin enriched with platelets and its combination with hydroxyapatite materials with β-tricalcium phosphate were used to replace bone defects, the infl ammatory reaction was manifested to a lesser extent. There was no signifi cant formation of bone callus compared with the fi rst, in which model defects were fi lled with injectable platelet-rich fi brin and the control group. Each of the types of platelet concentrates and their combination with hydroxyapatite ceramics have a diff erent eff ect on the restoration of bone defects and is accompanied by the appearance of a number of reactions, both local and general. According to the degree of intensity of this process, they can be placed in the following sequence: i-PRF ˂ PRF ˂ PRF+GT. Macromorphologically and radiologically signifi cant diff erences were not detected for diff erent types of bone tissue due to the use of each of the substances that concentrates platelets. The combination of hydroxyapatite granules with β-tricalcium phosphate and platelet-rich fi brin proved to be the best option for repairing the damaged area. Its use provides a signifi cantly smaller manifestation of the local infl ammatory reaction and causes the formation of optimal bone marrow. Key words: PRF, i-PRF, granules, centrifuges, growth factors, bone marrow.

Experimental evaluation of the efficiency of chitosan matrixes under conditions of modeling of bone defect in vivo (preliminary message)

Background. Despite the wide range of studies, the development of osteoplastic material, which has not only osteoconductive but also osteoinductive properties, remains an extremely topical issue in modern medical materials science. This work is devoted to experimental evaluation of the effectiveness of synthetic osteoplastic composite material based on chitosan and hydroxyapatite.&#x0D; Aim. This study aimed to determine the effects of spongy implants based on chitosan and its composite with hydroxyapatite nanoparticles in an amount of 50 wt. % on early osteogenesis in the area of the through defect of the ileum.&#x0D; Materials and methods. The studied materials were sponge implants based on chitosan and its composite with hydroxyapatite nanoparticles in an amount of 50 wt. %. Comparison groups include those without implant placement and those with replacement with commercial Reprobone osteoplastic material. Materials were implanted into the zone of the through defect of the ileum of rabbits for a period of 28 days.&#x0D; Results. A high rate of resorption of materials based on chitosan in bone tissue and active growth of reticulofibrotic bone tissue along the edges of the defect was established, and the formation of cartilaginous islands and bone marrow was recorded in the group of chitosan implants with hydroxyapatite. The aseptic effect was observed with the use of implants made of chitosan and hydroxyapatite.&#x0D; Conclusions. The data obtained allow us to argue about the osteoconductivity of the studied materials and the prospects for further development in this direction.